THESL“ 0—169 "7"“ hie». “A... ~ A 1‘. Univ“ rim W- -‘ 3:. This is to certifg that the thesis entitled MECHANISM OF NAPHTHALENEACETIC ACID ACCUMULATION BY SOYBEAN (GLYCINE fl MERR.) LEAF CELLS WITH SPECIAL EMPHASIS 0N TISSUE PREPARATION AND VIABILITY presented bg MARK LEE BRENNER has been accepted towards fulfillment of the requirements for Mdegree im Afd’éé- Date if 36 (DA L122 2 ‘* «My! .9 x1. hr 'r' the mech lation by lea being devoted tissue prepara Iedium that 0; '35 formulated ABSTRACT MECHANISM OF NAPHTHALENEACETIC ACID ACCUMULATION BY SOYBEAN (GLYCINE MAX MERR.) LEAF CELLS WITH SPECIAL EMPHASIS ON TISSUE PREPARATION AND VIABILITY BY Mark Lee Brenner The mechanism of naphthaleneacetic acid (NAA) accumu— lation by leaf cells was studied with special consideration being devoted first to the improvement of methods of tissue preparation and cell isolation. A new treating medium that optimized maximum leaf tissue metabolic activity was formulated using neo-tetrazolium reduction as a quanti— tative index. Leaf strips, which were cut cross—sectionally 250 pm thick from a 25 mm wide piece of tissue, were used for development of a medium and as a basis of comparison to the several different preparations of isolated cells. Enzymatically isolated leaf cells exhibited approximately 50% of the viability of the leaf strip tissue, while mechanically isolated cells had minimal activity. Viability comparisons were based on oxygen exchange rates in the I - ”I 1 I I leaf ;.-,. lation by the backgramd of to isolated as compound that acetyl asparta Iminant role Mark Lee Brenner light and dark, NAA and PO43* uptake, l4C02vfixation (total, and incorporation into protein), l4C—acetate catabolism, tetrazolium reduction, and light and electron microscopic examinations. Polarographic analysis of light and dark oxygen exchange rates provided a rapid and'reliable index of leaf tissue viability. The mechanism of NAA accumu— lation by the leaf strips was examined first to provide a background of NAA accumulation and a basis for comparison to isolated cells. NAA was rapidly metabolized into a compound that chromatographed identical to naphthalene— acetyl aspartate (NAAsp) and this compound appeared to play a dominant role in NAA accumulation. Once an internal con— centration of about 100 pmoles of NAA/10 mg dry weight of tissue was established, the uptake rate increased rapidly. This surge was dependent upon the interaction of time and external NAA concentration. By use of metabolic inhibitors and substrates it appeared that an enzyme (likely L-aspartic acid—N—acylase) was being either activated or synthesized. Results with low temperature and metabolic inhibitors Supported the concept that NAA uptake required metabolic energy. Light had a slight depressing effect on uptake. The conjugated NAA was bound tightly within the tissue. NAA uPtake by enzymatically isolated leaf cells was also found related to formation of NAAsp. Unlike leaf strips, the Mark Lee Brenner conjugating enzyme appeared to be functioning within the cells at the time the uptake studies were initiated, so that only limited metabolic control could be demonstrated. 1“ Parti MECHANISM OF NAPHTHALENEACETIC ACID ACCUMULATION BY SOYBEAN (GLYCINE MAX MERR.) LEAF CELLS WITH SPECIAL EMPHASIS ON TISSUE PREPARATION AND VIABILITY BY Mark Lee Brenner A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 "III 1+1 With since thank Dr. M. J. counsel, and un most grate ful t 542.745 7—/~7° ACKNOWLEDGMENTS With sincere appreciation, the author wishes to thank Dr. M. J. Bukovac for his perceptive guidance, counsel, and unselfish willingness to assist. I am also most grateful to the members of my Guidance Committee: Drs. A. A. DeHertogh, A. L. Kenworthy, N. C. Leeling, and H. M. Sell. The suggestions by and laboratory fadilities of Drs. D. R. Dilley and H. P. Rasmussen were most appreci- ated. I also wish to thank Mr. R. Bednarz for his generous assistance conducting the electron microscope examinations. This investigation was supported in part by Public thalth Service Grant CC 00246 and by the Food and Drug Administration Grant F D 00223. To my wife, Ruth, I wish to express my sincere gratitude for her patience, encouragement, and support. ii LIST 01" TABLES' LIST 02 FIGURES LIST OF APPENDI INTRODUCTION‘ ' ' LITERATURE REVI I. uptake-w A. Intr B. Cell c. Pass D. ACti 11.Foliar Upta A. Leaf B. Meth C. Spec III.Auxin Uptak A. EXpe B. Fact . Isolation C A. Meth B. Meth c. Gene Cell EXPERIMENTAL. . . I. Hypocotyl E A. Gron B. Prep C. Gene D. Esta l. 2. III 3. Dete the] 1. El TABLE OF CONTENTS 'LIST OF TABLES. on...-noon...-...-oooooo-nooacooou-uto LIST OF FIGURES ........ . ..... LIST OF APPENDIx....................; INTRODUCTION..... lcooaooooI-oocoooooovqo ooooooo one-no- LITERATURE REVIEW ....... . ....... ............ ..... ... I. Uptake........... ............. ........ ...... . ..... A. Introduction....................... ...... .. B. Cell Structure............................. C. Passive Uptake ..... . ........ ............... D. Active Uptake.. ..... ....................... II. Foliar Uptake ......... .......... ...... ...... ..... . A. Leaf Structure as Related to Uptake. ..... .. 'B. Methods of Studying Foliar Penetration..... C. Special Aspects of Foliar Uptake ....... .... III. Auxin Uptake ............. . ...... .... .............. A. Experimental Techniques............. ....... B. Factors Affecting Auxin Uptake...... ....... IV. Isolation of Leaf Cells for Auxin Uptake Studies. . A. Methods of Isolation................. ...... B. Methods of Assessing Viability.......... ... C. General Methods Used for Handling Plant Cells and their Organelles......... ...... .. EXPERIMENTAL. ................................. ..... .. 1- Hypocotyl Section Studies.............. ........... A. Growing of Plants........ ............... ... B. Preparation of Hypocotyl Sections.......... C. General Uptake Methods ....... ....... ....... D. Establishment of Experimental Parameters... 1. Determination of Self-Absorption ..... 2. Determination of Optimum Section Length........ ......... . .......... ... 3. Effect of Section Position........... E. Determination of Factors Affecting Naph— thaleneacetic Acid Uptake.. ............... . 1. Effect of Bacteria on the Hypocotyl System........................ once... iii Page vii xii XV 35 39 39 39 4O 41 43 43 43 46 50 50 3. 4. 5. II. Development Assessment 1 A. Grow: B. Prep; C. Assa D. Esta] with l. 2. 3- Impn Usint Page 2. Time-Course Studies of Naphthalene— acetic Acid Uptake................... 55 3. Methods for Killing Tissue........... 55 4. Methods of Infiltrating Tissue....... 59 5. Effect of Composition of Treatment Media................................ 63 II. Development of a Leaf Strip Technique and an Assessment of Tissue Viability.................... 66 A. Growing of Plants.......................... 66 B. Preparation of Leaf Strips................. 68 C. Assay of Viability by Tetrazolium Reduction 70 D. Establishment of Experimental Parameters with 2,3,5-Triphenyl Tetrazolium Chloride.. 71 1. Determination of Light Absorption Curve............. ...... ............. 71 2. Determination of Optimum 2,3,5- Triphenyl Tetrazolium Chloride Con- centration........................... 72 3. Refinement of Leaf Strip Preparation. 72 4. Modification of Treatment Media and Conditions............ ......... . ..... 77 E. Improvement of Leaf Strip Technique Using Nee-Tetrazolium Chloride..... ..... ... 85 1. Determination of Light AbSorption Curve ..... ..................... ...... 85 2. Determination of Optimum Neo—Tetra— zolium Chloride Concentration........ 86 3. Determination of Optimum Strip Width. 86 F. Refinement of Treatment Media... ...... ..... 91 1. Effect of pH and Buffers.... ......... 91 2. Effect of Various Enzyme Stabilizers. 103 3. Effect of Mannitol. ...... ............ 107 G. Time-Course of Neo—Tetrazolium Chloride Reduction ...... . ...... ......... ............ 107 H. Further Assessment of the Leaf Tissue Viability.. ..... ............ ........ ....... 107 1. Measurement of Protein Synthesis ..... 107 2. Measurement of Oxygen Exchange Rates. 114 I. Interaction of Neo-Tetrazolium and Naph- thaleneacetic Acid... .......... ...... ...... 116 III. Development of a Cell Isolation Technique and Viability Comparisons to Leaf Strips...... ..... .. 119 A. Methods of Mechanical Isolation of Leaf Cells. ....... .................. ............ 119 B. Refinement of the Mechanical Isolation Technique.... ..... ............. ........... . 126 C. Comparisons of Mechanically Isolated Cells to Leaf Strips.. ........... ....... ...... ... 131 D. Method of Enzymatic Cell Isolation. ........ 134 E. Comparisons among Mechanically and Enzy— matically Isolated Leaf Cells and Leaf Strips—Metabolic Studies........ ..... . ..... 138 iv F. Comp mati Stri l. 2. G. Fina Proc IV. Uptake and Leaf Strips A. Gene Acid 1. 2. 3. B Meta Upta l. 2. 3. C Meta I Stri ‘ Make of N ISOlated Le A. Gene Upta l. 2. 3 Meta Upta l. 2. 3. ISIUSSION. II Assessmem' . Development In r11155116. _ . . . 'Mphthalené 2‘ HYpo ‘ Leaf c. 1801 F. Comparisons among Mechanically and Enzy— matically Isolated Leaf Cells and Leaf Strips-Microscopic Studies. ....... ... ..... . 1. Light Microscope Studies....,........ 2. Electron Microscope Studies.......... G. Final Modification of Enzymatic Isolation Procedure for Leaf Cells. ...... ............ IV. Uptake and Metabolism of Naphthaleneacetic Acid by Leaf Strips ..... ........ ..... ...... ....... ........ A. General Parameters of Naphthaleneacetic Acid Uptake ......... ... ..... .... ...... ..... l. Time-Course............... ..... ...... 2. Effect of Naphthaleneacetic Acid Concentration..... ........... ....... 3. Loss of Accumulated Naphthaleneacetic Acid from Tissue and Determination of Free Space........................... B. Metabolic Aspects of Naphthaleneacetic Acid Uptake... ........ . ..... ......... ........ ... 1. Effect of Temperature................ 2. Effect of Light on Naphthaleneacetic Acid Uptake ....................... 3. The Effect of Metabolic Inhibitors on Naphthaleneacetic Acid Uptake ..... C. Metabolism of Naphthaleneacetic Acid by Leaf Strips .................. ...... ........... V. Uptake of Naphthaleneacetic Acid by Enzymatically Isolated Leaf Cells ...... . ........................ A. General Aspects of Naphthaleneacetic Acid Uptake................... ......... . ....... 1. Time— Course and Concentration Study... 2. Loss of Accumulated Naphthaleneacetic Acid ...... ....... ..................... B. Metabolic Aspects of Naphthaleneacetic Acid Uptake ...... ............. .......... . ....... 1. Effect of Light ..... . ......... . ...... 2. Effect of Temperature.... ......... 3. Metabolism of Naphthaleneacetic Acid. DISCUSSION ......... .. ................. . .............. I. Assessment of Tissue Viability ............. . ..... . II. Development of Techniques to Handle Soybean Leaf Tissue ............... ... ....... . ...... ....... III. Naphthaleneacetic Acid Uptake...... ............ ... A. Hypocotyl Tissue ..... . ............... . ..... B. Leaf Strip Tissue..... ........ .... ......... C. Isolated Cells.......... ....... ....... ..... SUMMARY..... ............ . ........ .. ........... . ....... v Page 152 152 161 169 173 173 173 176 179 186 186 188 189 195 210 210 210 213 215 215 215 215 223 223 228 235 235 236 247 248 Page .....I.............IIOOIOODIOO.Q.IOOO 250 ...-IOOOICCOCUOIQ.......C.ICOOOOOOIOOIIOOIQOIII .269 hue I. Effect of of 4 mate Level of syst of c Effect of: upta sevei phen 10‘ 11. LIST 7 OF TABLES Table 1. Effect of position on the hypocotyl as a source of 4 mm sections for experimental material ...... ................... ....... .. 2. Level of bacteria after 4 hr in the hypocotyl system treated with several concentrations of chloramphenicol..... ..... . ..... . ...... . 3. Effect of bacteria on naphthaleneacetic acid uptake by hypocotyl sections treated with several concentrations of D—threo chloram— phenicol ..... ........... ....... . .......... 4. Effect of bacteria on oxygen uptake at 30 C by hypocotyl sections treated with several concentrations of chloramphenicol......... 5. Effect of several methods of killing hypocotyl sections on naphthaleneacetic acid uptake. 6. Effect of infiltration of the medium containing varied concentrations of sodium azide into hypocotyl sections prior to naphtha- leneacetic acid uptake.. .............. .... 1 Effect of various substrates on naphthalene- ‘ acetic acid uptake by hypocotyl sections.. 8. Effect of varying concentrations of mannitol on naphthaleneacetic acid uptake by hypo— cotyl sections ...... ......... ............. 9. Effect of 2,3,5—tripheny1 tetrazolium chloride concentration on its reduction by leaf strips.. ...... . ....... ........... ......... Effect of pretreatment temperature and leaf strip size on 2,3,5—triphenyl tetrazolium chloride reduction... ........... ......... Effect of age of leaf used as a source of leaf strips on 2,3,5-triphenyl tetrazolium chloride reduction ........... ...... ....... Vii Page 49 53 54 56 60 64 65 67 75 76 78 MMe M. Effect of init 2,3, redu B. Effect of M. Effect of tetr stri M. Effect of 201i M. Effect of meta redu 17- Effect of PYrr redu M. Effect of tetr stri N- Modified ' Distribut 30 m Table 12. Effect of mannitol concentration during the initial wash of leaf strips prior to 2,3,5-tripheny1 tetrazolium chloride reduction. ....... . .......... ...... ........ 13. Effect of leaf strip width on oxygen uptake.... 14. Effect of several buffers at pH 6.6 on neo- tetrazolium chloride reduction by leaf strips.................................... 15. Effect of Ficoll and Dextran-40 on neo—tetra- zolium chloride reduction by leaf strips.. 16. Effect of polyvinyl pyrrolidone and potassium metabisulfite on neo—tetrazolium chloride reduction by leaf strips......... ......... 17. Effect of bovine serum albumin and polyvinyl pyrrolidone on neo-tetrazolium chloride reduction by leaf strips........ .......... 18. Effect of mannitol concentration on neo— tetrazolium chloride reduction by leaf strips in the refined medium ..... ......... 19. Modified treatment medium ...... ........... 20. Distribution of 14C02 in leaf strips after 30 min of fixation in either light or dark 21. Effect of infiltration of the treatment medium into leaf strips on oxygen exchange rates in dark and light..... ...... ... ...... ..... 22. Time-course of the effect of naphthaleneacetic acid on nee-tetrazolium reduction by leaf strips. ....... ... .......... ...... ......... 23. lime—course of naphthaleneacetic acid uptake 24. 25. by leaf strips as affected by neo—tetra— zolium chloride............... ...... ...... Oxygen exchange rates in dark and light of four separate mechanically isolated leaf cell preparations in time....... .......... Cbmparison of the effect of several media on oxygen exchange rates of mechanically isolated leaf cells in dark and light..... viii Page 80 94 100 104 105 106 108 109 113 115 117 118 127 129 the %. Effect of mech and N. Compariso isol naph tetr and N. Effect of leaf dark 0. Time-cour diff in d m. Compariso 36. 35. Table 26. Effect of pH on oxygen exchange rates of mechanically isolated leaf cells in dark and light ..... .... ........ . ...... ......... 27. Comparison of leaf strips to mechanically isolated leaf cells by oxygen uptake, naphthaleneacetic acid uptake, neo- tetrf olium reduction, phosphorus uptake and C02 fixation................... ..... 28. Effect of cell density of enzymatically isolated leaf cells on oxygen exchange rates in dark and 1ight............... ...... ....... 29. Time4course of oxygen exchange rates of several different types of leaf cell preparations in dark and light.... ..... .. ..... . ........ 30. Comparison by oxygen exchange rates in dark and light of leaf strips and cells iso- lated by either mechanical or enzymatic procedures..................... ...... ..... 31. Comparison by 14CO fixation of leaf strips and cells isolated by either mechanical or enzymatic procedures ............ .... ...... 32. Comparison by 14C02 incorporation into protein of leaf strips and cells isolated by either mechanical or enzymatic procedures........ 33. Comparison by naphthaleneacetic acid uptake and neo—tetrazolium chloride reduction of leaf strips and cells isolated by either mechanical or enzymatic procedures ........ 34. Comparison by measurement of chlorophyll and protein content of leaf strips and cells isolated by either mechanical or enzy— matic procedures .......................... comparison by oxygen exchange rates in dark and light of leaf strips and cells isolated by either mechanical or enzymatic pro— cedures ........... . ....................... comparison by phosphorus uptake of leaf strips and cells isolated by either mechanical or enzymatic procedures ......... . ......... ix Page 130 133 137 139 141 142 144 145 147 148 150 Table N. aal m. Compariso leaf mech Compariso stri ical Compariso and or e Analysis of e and Effect of on i . Effect of leaf upta from lene. Effect of acid 010 Value. 0n 0 in d. 48. 49. Table V Page 37. Comparison by acetate-2-14C catabolism of leaf strips and cells isolated by either mechanical or enzymatic procedures........ 151 38. Comparison by oxygen exchange rates of leaf strips and cells isolated by either mechan— ical or enzymatic procedures.............. 153 39. Comparison by phosphorus uptake of leaf strips and cells isolated by either mechanical or enzymatic procedures ....... . ......... .. 153 40. Analysis of stability of oxygen exchange rates of enzymatically isolated cells in dark and light .......... . ...... . .............. 172 41. Effect of naphthaleneacetic acid concentration on its own uptake by leaf strips.......... 180 42. Effect of successive washings on the amount of naphthaleneacetic acid retained by leaf strips after naphthaleneacetic acid uptake... .......... . ......... . ............ 181 43. Time—course of naphthaleneacetic acid efflux from leaf strips labeled with naphtha— leneacetic acid ............ .......... ..... 185 44. Effect of temperature on naphthaleneacetic acid uptake by leaf strips in the dark.... 187 45. 910 values for naphthaleneacetic acid uptake.... 187 46. Effect of naphthaleneacetic acid pretreatment on oxygen exchange rates of leaf strips in dark and light .............. . .......... 192 41 Effect of several metabolic inhibitors on naphthaleneacetic acid uptake by leaf strips ............. . ................ . ..... 193 Effect of 3—(4—chlorophenyl)-1,l-dimethylurea on oxygen exchange rates of leaf strips in dark and light ....... .... ....... . ...... 194 Effect of malonic acid and cycloheximide on oxygen exchange rates of leaf strips in dark and light ..... ...... ........ . ..... ... 196 'thal mati fl. Effect of acid cell E. 010 value 54. Time-com: by e affe Table Page 50. Effect of malonic acid and cycloheximide individually and in combination, on naphthaleneacetic acid uptake by leaf strips.................................... 197 51. Effect of washing with selected media on naph— ' thaleneacetic acid retention by enzy- matically isolated leaf cells prelabeled with naphthaleneacetic acid. ...... . ....... 214 52. Effect of temperature on naphthaleneacetic acid uptake by enzymatically isolated leaf cells..... ..... ............. ........ ...... 218 53. Qlo values for naphthaleneacetic acid uptake by enzymatically isolated leaf cells...... 218 54. Time-course of naphthaleneacetic acid uptake by enzymatically isolated leaf cells as affected by several treatments............ 220 xi Figure l. Self-adso acid labe 2. Effect of thal 3. Time-coui by l 4. Time-con: by 1 met? 5' Light abs leaf tetI tri< 6' Effect 01 ligl chl( LIST OF FIGURES Figure l. 2. 3. lo. 11. Self—adsorption curve of 14 C—naphthaleneacetic acid in varying amounts of uniformly labeled homogenized hypocotyl tissue...... Effect of hypocotyl section length on naph- thaleneacetic acid uptake.... ...... . ...... Time—course of naphthaleneacetic acid uptake by hypocotyl sections ............ ......... Time-course of naphthaleneacetic acid uptake by hypocotyl sections killed by several methods.... ....... ... ........... . ......... Light absorption curves of xylene extracts of leaf strips and reduced 2,3,5-triphenyl tetrazolium chloride with and without trichloroacetic acid...... ........ . ....... Effect of sodium succinate concentration and light on 2,3,5-triphenyl tetrazolium chloride reduction by leaf strips.... ..... Time—course of 2,3,5-tripheny1 tetrazolium chloride reduction by leaf strips ......... Light absorption curves of xylene extract of non—treated leaf strips and reduced neo— tetrazolium chloride.... .................. Effect of neo—tetrazolium chloride concentra— tion on its reduction by leaf strips ...... Effect of leaf strip width on neo-tetrazolium chloride reduction............ nnnnnnnnnnnn Effect of pH on nee—tetrazolium chloride reduction by leaf strips... xii Page 45 48 58 62 74 82 84 88 90 93 97 Figure n. Effect of redi Tris B. Effect 01 zoli pH \ M. Time-con: duct B. Schematic tech M. Density-g N. Photomicx stri Figure 12. Effect of pH on nee—tetrazolium chloride reduction by leaf strips using 0.01 g . Tris—maleate buffer....................... 13. 'Effect of HEPES and MES buffers on nee—tetra- zolium reduction by leaf strips at several pH values.......... ..... .... l4. Time—course of neoetetrazolium chloride re- duction by leaf strips in light and dark.. 15. Schematic diagram of mechanical isolation technique for leaf cells.................. 16. Density-gradient separation of leaf cells...... 17. Photomicrograph of a cross—section of a leaf strip viewed under bright light........... 18. Mechanically isolated cells.... ........ . ....... 19. Enzymatically isolated cells............. ...... 20. Cytoplasmic streaming in enzymatically isolated cells as shown by time—sequence photo- micrographs ........ .... ............... .... 21. Electron micrograph of an enzymatically iso- lated cell compared to a cell in a leaf strip ......... .................. .......... 22. Ultrastructure of viable enzymatically isolated cells .................. . .......... ........ 21 Ultrastructure of damaged enzymatically iso- lated cells..... ....... ........ ........... 24. Time—course of naphthaleneacetic acid uptake by leaf strips in the light..... .......... 25. Effect of naphthaleneacetic acid concentration on its own uptake by leaf strips ...... .... 26. Short-time course of naphthaleneacetic acid uptake by leaf strips at 5 C in the dark.. 21 ?8. Time—course of naphthaleneacetic acid uptake in light and dark by leaf strips.. ........... Radiochromatogram traces of authentic and ex— tracted naphthaleneacetic acid...... ...... xiii Page 99 102 111 121 125 155 157 160 163 166 168 171 175 178 184 191 200 34. Trace of nap lea Figure 29. Radiochromatograph traces used to determine the form of accumulated naphthaleneacetic acid from treatments listed in Table 50... 30. Time—course of l4C—naphthaleneacetic acid up— take by leaf strips as affected by several treatments................................ 31. Radiochromatograph traces used to determine the form of accumulated naphthaleneacetic acid from treatments shown in Figure 30... 32. The uptake of naphthaleneacetic acid by enzy— matically isolated leaf cells as affected by concentration and time... ............ .. 33. Time-course of naphthaleneacetic acid uptake by light, dark, and temperature........... 34. Trace of radiochromatogram of accumulated naphthaleneacetic acid from isolated leaf cells after 2 hr of uptake...... ..... xix! Page 204 207 209 212 217 :m' 9.19.6 OJ 59211 .9955 " ‘ '1 T m1} ‘4 91541110511 bsifilmmiaas .1 e , T"! I) . ;- ‘ j .. g 7401‘5- : "T311 hIl‘S #7" sink?" :1! 39.381 .-.,. -. . . I .. ... WANT". A i a; _ ' '0'. ....... 4 I“ 5 “f “'1‘ ‘ ' ' 'JOI $53 . If " if ., 5 .. . DJbMO Ad ‘ 0.1 .598” .2an 7- q _ 1‘ ' , 3 ' .» Ouillfl‘l“ ’ .39.?6514' .-. 1 1.. I > I ' I .. . -.‘...-. k 5 I 7‘ ..h. _ ,‘pgfij :,-; r 7 .. 1 '7 '7’ JY'N’. . .. . ‘ swim." I - - . k ‘ . .1 ‘3'". —'~ I" r I t,- . j ... ', I J I - I - I: ' . 9m pr"; 390.513 dqarpoxmomsorbm? .9: A “‘H'C'ii-I‘Lfi J» fable Ill. Effect 0 tri by 12. Effect 0 red 13. Liquid S M- The upta mat by l’igure M, Sample c aci LIST OF APPENDIX Table Page A1. Effect of mannitol concentration of 2,3,5- triphenyl tetrazolium chloride reduction by leaf strips................ ......... ... 269 A2. Effect of dialyzed Ficoll on neo-tetrazolium reduction by leaf strips.................. 270 A3. Liquid scintillation cocktail used ..... . ...... . 271 A4. The uptake of naphthaleneacetic acid by enzy- matically isolated leaf cells as affected by concentration and time................. 272 Figure Page Al. Sample calculation for final naphthaleneacetic acid internal concentration ...... . ........ 273 XV Biologi laden agricu omtinnes to ' foliar sprays dependent upo actively grow 'leaf penetrau and perhaps t substances are The objective faliar uptake zodel canpounc' Iequlators. Several n “f leaf cells Price, 1967). tically examir he viability (“Why and De INTRODUCTION Biologically active compounds play a major role in modern agricultural practices today and their importance continues to grow. Many of these materials are applied as foliar sprays, and their effectiveness is often totally dependent upon the amount of material that penetrates into actively growing tissue. As Audus (1967) has stated that, "leaf penetration is fraught with technical difficulties“, and perhaps the greatest lack of knowledge is how growth substances are accumulated in the leaf parenchyma tissue. The objective of this thesis is to study the mechanisms of foliar uptake of d-naphthaleneacetic acid (NAA) used as a model compound from the larger class of auxin type growth regulators. Several methods have been reported for the isolation of leaf cells (Jyung _3 al., 1965b; Takebe g: al., 1968; Price, 1967). Unfortunately none of these workers cri- tically examined the viability of their cell preparations. The viability of these cell preparations has been questioned (Jacoby and Dagan, 1967) and a more reliable intact system 2 (Smith and Epstein, 1967) has been recommended by Jacoby and Dagan (1967). Recognizing the desirability of a system using viable isolated leaf cells, a considerable effort has been directed toward the refinement of the methods of cell isolation. Special emphasis was directed to making 'an accurate assessment of the viability of the isolated cell preparation comparing it to an intact leaf system. NAA uptake by stem sections has been briefly studied to establish some of the basic parameters that can be used for studying NAA accumulation by leaf tissue. Considerable detail was given to uptake of NAA by leaf strips and leaf cells. I. Uptake A. Intru Studies . into living t this thesis i Inner or att they have rev. the basic asp Plant tissue, the ensuing st ce115- Monogr i1956) covered ilimal cells, 311993 a a_l. and Wallace (] rechanisms the ii a brief nor. Ens and how mnort. Sh LITERATURE REVIEW I. Uptake A. Introduction Studies on the mechanisms of molecular and ion uptake into living tissue have been voluminous. The intent of this thesis is not to review these works in a detailed manner or attempt to answer the many unsolved problems they have revealed. Rather, it is the purpose to summarize the basic aspects of uptake mechanisms, particularly in plant tissue, as a point of reference and terminology for the ensuing study of auxin uptake by leaf tissue and isolated cellsfi Monographs written by Stein (1967) and Troshin (1966) covered general aspects of penetration, mainly for animal cells, emphasizing problems of membrane permeability. Briggs _§_al. (1961), Sutcliffe (1962), Jennings (1963), and Wallace (1963), have written monographs on uptake Emchanisms that are common to plant cells. Robertson (1968) in a brief monograph, emphasized charge separation in plant cells and how it relates to metabolic energetics and ion transport. Shorter reviews on uptake by plant cells have been presente leggett (1968 The simp been into pas isms imply th expenditure 0 involves the . move a molecu (Ussing, 1949 B. Cell ; The varil bEtter Unders‘ 4 been presented by hrisz (1963), Brouwer (1965), and Leggett (1968). The simplest subdivision of uptake mechanisms has been into passive and active components. Passive mechan— isms imply those processes that can occur without the expenditure of metabolic energy, while active uptake involves the direct expenditure of metabolic energy to move a molecule against an electro—chemical potential (Ussing, 1949). B. Cell Structure The various barriers discussed in this thesis are better understood by first describing the structure of the plant cell with particular reference to its membranes. , The cell is bound externally by a fibrous structure of cellulose intermixed with pectins and hemicellulose called the primary wall (Esau, 1965). The fibral net—like structure has no specific orientation and has many openings in the order of 100 g, which means the wall will allow the passage, with little resistance, of small molecules (Briggs §£__l., 1961). Covering the primary wall is a layer of pectinaceous material called the middle lamella, which cements adjacent cells together (Esau, 1965). In older, woody cells, a secondary wall composed of lignin develops under the primary wall. vacuole bound Between the t cmtaining va all of the ce‘ 5 Within the wall structure lies the external limiting membrane of the cell, the plasmalemma. This is the first true membranous structure a penetrating molecule must traverse. Most mature parenchymous cells contain a large vacuole bounded by a membrane called the tonoplast. Between the tonoplast and the plasmalemma is the cytoplasm, containing various organelles that carry on practically all of the cell's life sustaining functions. For a review of the structure and function of cellular organelles see Pridham, 1968. The endoplasmic reticulum appears as a continuous membrane structure within the cytoplasm. It establishes many zones or areas within the cytoplasm restricting the various organelles to limited paths during cytoplasmic streaming (Northcote, 1968). The general structure of these membranes, all double layers, is currently (Branton. 1969) considered to be a bimolecular leaflet system with a layer of lipid bound on either side with protein (PLP), as proposed by Davson and Danielli (1943). These membranes could have specific areas of specialized structure to allow for various required functions (Branton, 1969). This hypothesis is in agree- ment with the sub—unit theory for membranes, but would hunt their occurrence to specific areas of the cell's respective membranes. Northcote (1968) has reviewed the plant tissue: 1)) ions retai tissue that m surrounding m can he remove cell wall and 6 various plant cell membranes with special emphasis on the chloroplast membrane, which has a definite sub—unit matrix. Stadelmann (1969) concurs with this concept. C. PassiVe Uptake: Brouwer (1965), among others, has specified that passive uptake occurs in the following two fractions for plant tissue: a) ions held in water free space, and b) ions retained in Donnan free space. Water free space is that volume in the plant cell or tissue that may be easily penetrated by molecules from the surrounding medium. The molecules are in equilibrium and can be removed easily by washing with distilled water. The cell wall and intercellular spaces are thought to be the prime location for water free space (Briggs g: gl., 1961). Donnan free space, believed to be mainly located in the wall and outer edge of cytoplasm of the plant cell (Briggs g: _l., 1961 and Sutcliffe, 1962), is that volume where freely diffusible ions are retained by electrostatic charges. The pectins, with their free carboxyl groups are Often neutralized by calcium and magnesium. Dainty and Hope (1959) believed that these would be excellent sites for such ion exchange reactions. The total number of molecules retained by a cell in the above two categories represents collectively the apparent Several uptake procee mesa include equilibrium at Facilitated d ism for passi Diffusio 0f one compon Of a molecule Chiuses slighi d“fusion. thl Elecules mov; Extansive 1y 8. 7 free space, A.F.S. (Briggs §§_§ll, 1961). Brouwer (1965) suggested the existence of a third fraction that is also passively accumulated. Namely, these are molecules that would accumulate within the cell plasma- lemma. Several mechanisms that could explain how passive uptake proceeds have been reviewed by Sutcliffe (1962). These include diffusion, mass flow, ion exchange, Donnan equilibrium adsorption, and chemical combination. Facilitated diffusion can also be a very important mechan— ism for passive uptake (Jennings, 1963, and Stein, 1967). Diffusion as expressed by Fick's law is the movement of one component randomly within another. When speaking of a molecule passing through a membrane, the terminology changes slightly to factors of permeability instead of diffusion, thereby inferring a given barrier for the molecules moving through. This is an area that has been extensively studied. Stein (1967), in his treatise, gave muexcellent review for animal tissue, while Stadelmann (1969) reviewed the same field for plant tissue. Milborrow and Williams (1968) reexamined Collander's (1954) perme— ability data using regression analysis and concluded that his equation for permeability of a non—electrolyte was basically correct if one includes a quadratic component for ether: water partition sys mmbrane (Nit Of a molecule 3‘ Jenninss (: qaqlo 0f a] 8 the partition coefficient. This means that permeability is dependent on a 10910 function of the partition coefficient of the molecule in a quadratic expression minus‘a log function for the molecular weight. Most of Collander's observed data were fitted to such an equation with a 95% confidence level, providing they first rejected all molecules that were possibly metabolically transported or complexed. They agreed with Collander that olive oil: water, rather than ether: water was the best approximation for an artificial partition system with which to match the data of the plant membrane (Nitella cells). This meant that the permeability of a molecule through a plant membrane is dependent mainly on a complex function of the molecule‘s partition coefficient into the membrane divided by a function of the molecule's molecular weight. Facilitated diffusion is a non—metabolic selective movement of molecules across a cell membrane, but not against a concentration gradient (Miller, 1960). Facili— tated diffusion, as proposed by Danielli (1952) and reviewed by Jennings (1963), denotes that the penetrating molecule has to acquire a certain kinetic energy to pass into and thanback out of the respective membrane. Thermal agitation would be one source of this energy. This results in a 010 of about 2 to 3. the surface 1962) . Shorts a highe iSutcliffe, 1 Finally, “chanism Whe ”Things. 19 E“Change mech 9 The movement of one type of molecule along with the mass of a second type, is mass flow (Sutcliffe, 1962). The second type is driven by a physical force such as gravity, diffusion, shows a higher Q10 than the simple adsorption system (Sutcliffe, 1962). Finally, ion exchange, as mentioned earlier, is the nechanigm whereby molecules of like charge exchanged (Jennings, 1963). D. Active Uptake compared to the knowledge on passive mechanisms, little Epstein and Epstein one of the b that is oper to its actua for Ea" and concentration e"lune-mechan 1“! or great 11) transport lorii and Lat 3‘76 also fall They pastulat “me mechani Eelch and Eps healizes bOt‘ Mstioned th iepleteq root mini-5311113; fawn that Ca 10 molecules move across an electro-chemical gradient with the energy being derived metabolically. One of the more commonly held concepts implicates various enzymatic—like carrier mechanisms (Sutcliffe, 1962). Epstein and Rains (1965), Rains and Epstein (1965), and Epstein (1966) have described what is considered as one of the best examples of a specific carrier mechanism that is operative in plant root tissue. Without eluding to its actual nature, they have described a dual mechanism for Na+ and K+ uptake using enzyme kinetics. At low ion concentrations only K+ is transported (a high affinity enzyme—mechanism I), while at ionic concentrations of 1 mM or greater, a second mechanism (low affinity—mechanism II) transports, with less specificity, either K& or Na+. Torii and Laties (1966) (for a review cf. Laties, 1969), have also found the same dual mechanism in the corn root. They postulated that mechanism I lies in the plasmalemma while mechanism II probably functions in the tonoplast. Wehflland Epstein (1969) have presented kinetic data that loealizes both mechanisms in the plasmalemma. Pitman (1967) qUestioned these results, since data were obtained on salt depleted roots. Leggett (1969) also questioned the Universality of Ca2+ in the treating medium, when it is now known that Ca2+ reduces passive permeability of membranes and sometime: 1965). The abm group and the on where the offered a fir. remains unres Sutcliff relatively hi ion accumulat 11 and sometimes enhances metabolic ion uptake (Handley g; _1., 1965). The above difference of opinion between Epstein's group and that of Laties is representative of the controversy on where the permeation barrier exists. Jennings (1963) offered a fine diSCussion on this subject, but the problem remains unresolved. Sutcliffe (1960) reported that chloramphenicol at relatively high concentrations (1 to 2 mg/ml) retarded net ion accumulation by beet and carrot root sections. Similar results were reported for root tissue (Jacoby and Sutcliffe, 1962; Peaud—Lenoel and Gourmay—Margerie, 1962; Uhler and Russell, 1963) and several photosynthetic tissues (Parthier, 1965; Jyung g: al. 1965a; Brenner and Maynard, 1966). All concluded that there was a possible direct linkage of protein synthesis with ion uptake. As Sutcliffe (1962) hypothesized, the mechanism of protein turnover (hydrolysis and resynthesis) could provide many sites for ions to be bound and subsequently released. Several of the above authors (Uhler and Russell, 1963; Peaud—Lenoel and Gourmay— Margerie, 1962) reported that chloramphenicol at high con— centrations did not affect respiration. Since most of the above-mentioned studies were carried out under relatively sterile conditions, the effect of bacteria has been ruled out. Hanson 2 phenicol (0.8 phosphorylati while phosphc was not affec cally would 11 MacDonal phenicol (the and L-threo-c 12 Hanson and Hodges (1963) reported that chloram- phenicol (0.8 mg/ml, 2.7 mg) uncoupled oxidative phosphorylation of corn shoot mitochondria. ‘They found that while phosphorylation was always reduced, oxygen consumption was not affected. Thus, following respiration manometri— cally would not reveal the chloramphenicol effect. MacDonald gt__1. (1968), studying D—threo—chloram- phenicol (the only active isomer in bacterial systems) and L-threo‘chloramphenicol, found that both inhibited ion uptake at low concentrations (0.03 mg/ml, 0.1 mg). Using higher concentrations (0.6 mg/m1,2 mu), D—threo- chloramphenicol was more active than the L—threo isomer in inhibiting protein synthesis. They hypothesized that the two isomers, at low concentrations, could be acting preferentially on the ATP—ase enzyme system of the cell. At higher concentrations the D—threo-chloramphenicol would inhibit protein synthesis while the L-threo would uncouple oxidative phosphorylation. Recently, Ellis (1969) reported that the D—threo isomer (0.15 mg/ml, 0.5 mg) specifically inhibited protein synthesis in chloroplast (suggesting that chloroplast ribosomes are similar to bacterial ribosomes) while being without effect on cytoplasmic ribosomes. The L—threo— dfloramphenicol was inactive on both chloroplast and mitochondria chloramphenic of a direct 1 especially ir Skou (IS erythrocytes , cell membrane C911 while pt hidrolysis 5} Concentration iide acceptan tissue. The every 2-}(t Wi of only 4 kca Merged, ATP 13 mitochondria preparations. 'These results with chloramphenicol cast considerable doubt on the assumption of a direct linkage of protein synthesis.with ion uptake, especially in chloroplast—free tissue. Skou (1964, 1965) proposed, based on work with erythrocytes, that an ATPase enzyme system functions in the cell membrane to catalyze the transfer of Na+ out of the cell while pumping Kl into the cell. The activity of the hydrolysis system would be regulated by the respective concentrations of Na+ and K+. This hypothesis has gained wide acceptance (for a review cf. Glynn, 1968) for animal tissue. The kinetics show that 3—Na+ are transferred for every 2—K+ with the hydrolysis of l—ATP leaving a surplus of only 4 kcal. If the ionic balance were artifically reversed, ATP could be synthesized. Also, Glynn (1968) reported that oligomycin and the cardiac glycoside, ouabain, were both potent inhibitors of the process of pumping Na+ for K+. A convincing argument is presented by Robertson (1968) that charge separation is the basic mechanism by which energy was transferred within the plant cell. Charge Separation is the mechanism, proposed by Mitchell (for review cf. Mitchell, 1966), where protons are transferred, without a high energy intermediate, to form ATP or drive Iobertso: have been sho ‘ oxidative or At present, h! 14 ion transport. A gradient of protons could drive a reversible ATPase enzyme to form ATP from ADP and Pi. More importantly, the reverse of this reaction could re— lease energy to transport ions. Robertson (1964) reviewed how such energy relations have been shown to transport ions when coupled with either oxidative or photosynthetic phosphorylation in plant cells. At present, however, only indirect evidence is available that an ATPase system is responsible for ion movements through the plasmalemma. Chang and Bandurski (1964) reported ATPase activity in cell wall preparations of corn roots. Hall (1969) also demonstrated histochemically that ATPase was associated with the cell wall, especially near the plasmalemma. Hall noted that ATPase activity was enhanced with 2 mM Ca(NO3)2. Hodges (1966) reported that oligomycin rapidly and preferentially inhibited K+ trans— port into the roots of oat, rye grass, and pea without affecting respiration. These root tissues were found insensitive to ouabain. The latter substance, however, inhibited Na+ efflux (movement out of the tissue) from carrot slices, but had no effect on K+ influx (movement into the tissue) (Cram, 1968). Evidence is accumulating that an ATPase system, probably slightly different than in annals, might be one of the important means by which plant cells regulal Another root tissue 1 findings Shoo transaminati< binding sites This means t1 15 cells regulate the accumulation of ions. Another type of metabolic accumulation mechanism in root tissue has been described by Hiatt (1968). His findings show that organic acid synthesis, co-ordinated with transamination to form amino acids, would be a source of binding sites to accumulate cations and anions simultaneously. This means that metabolic energy would be involved only for the synthesis of the respective acCeptor molecules for ionic association to create and maintain a directed chemical gradient. Actually, uptake itself would be a diffusion process. At high external ion concentrations, additional neutral salts could still enter the cell by the Donnan phenomena, even though the level of receptor sites are not increased within the cell. This system could be comparable to Epstein's carrier system (mechanism II), but the involvement of membrane carriers is not necessary. In Hiatt‘s concept the membrane functions only for specific molecular selectivity and retention of organic and amino acids within the cell. II. Foliar Uptake A. Leaf Structure as Related to Uptake Hull (1964) gave a detailed description of leaf structure in relation to foliar penetration. Numerous shorter revie Currier and I and Wittwer, The abou as being cove structure. I of long chain polar and non The cuticle i penetrating n 1964: Hull, 1 the cuticular thel’ Occur in 1956)_ Theh to the low pe 19597 Hull, 1 l6 shorter reviews are also available (van Overbeek, 1956; Currier and Dybing, 1959; Crafts, 1964; Foy, 1964; JYung and Wittwer, 1965; Sargent, 1965; and Franke, 1967). The above authors described the leaf's outer surface as being covered with a cuticle, a non-living membraneous structure. -It is composed of a matrix of oxidized polymers of long chain fatty acids and alcohols. They impart both polar and non—polar properties to the cuticle (Foy, 1964). The cuticle is considered the most limiting barrier to penetrating molecules (Currier and Dybing, 1959; Crafts, 1964; Hull, 1964). Waxes are often found deposited on both the cuticular surface and within the matrix. On the surface they occur in the form of platelets and rodlets (van Overbeek, 1956). The hydrophobic nature of the waxes contributes to the low permeability of the cuticle (Currier and Dybing, 1959; Hull, 1964; Norris and Bukovac, 1968). A layer of pectin joins the cuticle to the subtending epidermal cells (Norris and Bukovac, 1968). The epidermal cells are the first living cells a penetrating molecule encounters. These cells are relatively large and are free of chloroplast. Stomata are generally found within the epidermis. Guard cells (containing dfloroplasts) border the stomatal opening, and regulate the aperture width (Esau, 1965). Although the stomata are open pores, ' 1964). Surf; into the sub: Hull, 1964), presence of i (Currier and B. Methi Many te< Uptake. (me 17 open pores, their internal cavity is bound by cuticle (Hull, 1964). Surface tension restricts penetration of liquids into the substomatal chamber (Currier and Dybing, 1959; Hull, 1964), but some researchers have shown that the presence of a surfactant will permit stomatal penetration (Currier and Dybing, 1959; Crafts, 1964; Greene, 1969). B. Methods of Studying Foliar Penetration Many techniques have been employed in studying foliar uptake. One of the simplest methods is applying the chemical to the whole aerial portion of the plant (Hauser, 1955). With such a system, however, it is difficult to distinguish between uptake and transport (Audus, 1967). Day (1952), using direct leaf application of micro—droplets of auxin solutions and measuring uptake by growth response, separated translocation from uptake by removing leaves at various intervals. Pallas (1960) and Middleton and Sanderson (1965) applied radio—labeled chemicals to leaves miintact plants and found uptake was dependent upon humidity and light. Jyung and Wittwer (1964) develOped a leaf immersion technique minimizing the effect of humidity. Several excised intact leaf systems have been devised to remove the effect of translocation from leaf uptake. Inckwill and Lloyd—Jones (1962) used a micro—droplet system, Biswas e; 3;. (1962) utilized agar blocks containing l4C herbicides o (1964a) appl attached to developed a translocatio Since t rate limitin been made to method for t' in the Uptak in Various t Ariz. 1963; 18 herbicides on the leaf surfaces, and Kaminura and Goodman (1964a) applied solutions in small glass cylinders attached to the leaf. Sargent and Blackman (1962, 1965) developed a glass vial technique and further reduced the translocation effect by use of leaf disks. Since the cuticular membrane may be considered as the rate limiting barrier for foliar uptake, attempts have been made to circumvent the cuticle in uptake studies. One method for the determination of the role of the leaf cells in the uptake process was to float pieces of leaf tissue in various treatment solutions (Kylin, 1960a, 1960b, 1960c; Ariz, 1963; and Hardwick and Woolhouse, 1968). Smith and Epstein (1964) reported that leaf strip width was a limiting factor and recommended that when such a system was used, the optimum strip width should be 300 um. This technique has been employed for many successful studies on the role of leaf cells in foliar uptake (Rains, 1967, 1968; Bowen, 1968, 1969; Osmond, 1968). Another approach has been to separate the cuticle and cells from the leaf and study them separately (Yamada, EL a1. —_ I 1965; Jyung e: 1., 1965a, 1965b; Kannan and Wittwer, 1967: Price, 1967; Bukovac and Norris, 1966; Norris and Bukovac, 1968, 1969; Wittwer et _1,, 1967; Gabbott and Larman, 1968). The use of isolated leaf cells should be an ideal approach for uptake. Hon (Jacoby and the cells on C. Spec The uni penetration CUticular st Effects, as m05t often b butcliffe' l 19 approach for studying the metabolic aspects of foliar uptake. However, this technique has been criticized (Jacoby and Dagan, 1967) for the very low viability of the cells compared to the leaf strip technique. C. Special Aspects of Foliar Uptake The unique role of the cuticle as a barrier to foliar penetration was not fully appreciated until isolated cuticular studies were reported. For example, temperature effects, as measured by Q10 values greater than 1.2, have most often been associated with metabolic activity Sutcliffe, 1962; Sufi, 1963; Jyung and Wittwer, 1965). Sargent and Blackman (1965) reported 910 coefficients as high as 4.3 for 2,4—D penetration into leaf disks, while coefficients of 5.6 and 3.6 have been determined by Norris and Bukovac (1969) and Gabbott and Larman (1968), respective— ly, for penetration of other organic compounds through isolated cuticle membranes. Thus, the cuticle can con— tribute significantly, if not totally, to high tempera— ture coefficients of foliar penetration. Penetration differs significantly depending upon which side of the leaf the material is applied. Kaminura and Goodman (1964), and Sargent and Blackman (1962) found Greater penetration rates through the lower surface. Sargent and Blackman (1965) suggested that a higher guard cell to suri isolated cut higher rates They suggest wax structur In mime enhance foli and Wittwer, The light ef Photosynthet intensities GOOdman, 196 Rains ( was indePend stomatal PEI] He Cone luded 20 cell to surface ratio was an important aspect. Using isolated cuticle, Norris and Bukovac (1968) found similar higher rates for penetration through the lower surface. They suggested these findings might be due to less organized wax structure (birefringent wax) in that surface. In numerous_studies, light has been reported to enhance foliar uptake_(Kylin, 1960a; Arisz, 1963; Jyung and Wittwer, 1964; Sargent and Blackman, 1965, 1969;). The light effect has been attributed to the production of photosynthetic metabolites, especially when high light intensities are required (Arisz and Sol, 1956; Kaminura and Goodman, 1964b). Rains (1967, 1968) has shown that the light effect was independent of both oxidative phosphorylation and stomatal penetration for K+ accumulation by leaf strips. He concluded that light supplied energy for uptake either via ATP or possibly direct coupling to electron transport. This latter idea is considered one type of charge separation supported by Robertson (1968). It should be noted that even though leaf tissue is photosynthetic, light does not always enhance active uptakes; Bowen (1969) has reported light had no effect for Cu2+, Zn2+ and Mn2+ uptake by leaf strips. 21 III. Auxin Uptake A. Experimental Techniques Two basic sampling methods have been used for almost all auxin uptake studies by plant tissue. The first system measured loss of auxin from the treatment solution. This technique was mainly used before isotopes were available and was dependent on colorimetric assay analysis for auxin detection (Sutter, 1944; Reinhold, 1954). As Reinhold pointed out, this method required the use of high auxin concentration (above 1x10"4 M 2,4—dichlorophenoxyacetic acid [2,4—D]) to permit accurate detection. Sabnis and Audus (1967a) have reported a spectrofluorometric analysis procedure that is sensitive for physiological concentrations of auxin (1x10'8 M to 5x10"5 M 3-indoleacetic acid [IAA]). This first detection method, as originally used, was inadequate, requiring high concentrations of auxin that were far above physiological concentrations. The work of Epstein‘s group (1966) indicated the possibility that working at a high concentration might mean one could actually be studying a mechanism that may not normally be Operative. Also, high auxin concentrations have been usually considered toxic to the tissue (Housley, 1961). The spectrofluormetric analysis, although much more sensitive than the colorimetric technique, was found to be 22 misleading under certain conditions, probably due to leakage of fluorescent plant material (Sabnis and Audus, 1967a). I The second sampling method, now the basic practice for all Work in this field, is to determine net accumu— lation of auxin in the tissue by using radio—labeled auxins (e.g. Johnson and Bonner, 1956; Wedding and Erickson, 1957; Thimann and Wardlaw, 1963; Luckwill and Lloyd-Jones, 1962; Sargent and Blackman, 1962, 1965; Jenner e; 1., 1968a, 1968b; Saunders e; _1., 1965a, 1965b). Two additional, less accurate assay techniques have been applied to determine auxin uptake. The first assay, which resulted in the stimulation of growth (e.g. Day, 1952), has not been very accurate since not all accumulated auxin influences growth (Andreae, 1967). The other procedure involved radioautography (e.g. Yamaguchi and Crafts, 1958) and this method gave only a qualitative determination of uptake. Tissue size has been an important factor in facili— tating optimum uptake of auxin. Reinhold (1954) and Jenner e: _1. (1968b) have reported that optimum accumu- lation occurred into their smallest prepared sections, 5 and 2 mm respectivelY- B. Fact A time- nost auxin L' uptake in ti rate follows have been ol Bonner, 1956 23 B. Factors Affecting Auxin Uptake A time—course of accumulation has been included in most auxin uptake studies. Sutter (1944) observed that uptake in time is usually characterized by an initial rapid rate followed by a second slower phase. Similar results have been observed by others (Reinhold, 1954; Johnson and Bonner, 1956; Wedding and Erickson, 1957; Poole and Thimann, 1964; Saunders 2; a1., 1965a, 1965b; Sabnis and Audus, 1967a, 1967b; Jenner 2E a1., 1968a). Though the two phases of uptake have been easily recognized, their specific signi— ficance has been subjected to considerable discussion. Sutter (1944) considered both to be manifestations of passive accumulation. Reinhold (1954), as well as several others, suggested that the first phase represented a passive uptake component, while the second phase was likely an active uptake component. Saunders et g1, (1965b) noted that the observed two-phase auxin uptake was followed by a third phase of net auxin loss, and that this last phase could be associated with a change in tissue properties independent of an auxin effect. From these data, Saunders §£.ei. (1965b) categorized auxin uptake into two components: Type I uptake where auxin was accumulated and later lost from the tissue (free space) and Type II uptake where auxin was bound irreversibly within the tissue. They found that strong auxins lation, while A single when the acct labeled auxin has been repi auxin concem nest of the ; tun-phase up‘ having an up- A linea to its uptak 24 strong auxins were mainly associated with Type I accumu— lation, while weak auxins were by the Type II mechanism. A single phase of l4C-auxin uptake has been observed when the accumulating tissue was pretreated with non— labebd auxin (Zenk, 1962). A three—phase uptake of IAA has been reported by Poole and Thimann (1964). Using an auxin concentration lower (lxlO"8 M) than that used for most of the above-mentioned reports, they noted a similar two-phase uptake that was then followed by a third phase having an uptake rate faster than the second. A linear relationship of external auxin concentration to its uptake by plant tissue has been reported by Johnson and Bonner (1956), Wedding and Erickson (1957), Sabnis and Audus (1967a, 1967b), and Jenner _p _1. (1968a, 1968b). Poole and Thimann (1964) have observed a similar linear relationship and established that the uptake ratio showed that the internal concentration was higher than the external concentration. Efflux of accumulated auxin has been shown to consist only of auxin that was held in a free, non-bound form (Andreae, 1967). Jenner §£.§l- (1968a) reported that a constant amount of auxin was released after varied periods 0f prelabelinq. Poole and Thimann (1964) observed that almost all the auxin (IAA) accumulated in a 30—minute uptake peril concluded t1 free space. loss for 2,1 volume as t] 19 to 22%. lation by Q value of 30'; 25 uptake period could be removed by subsequent washing. They concluded that this fraction represented accumulation into free space. Johnson and Bonner (1956) found a comparable loss for 2,4-D after a 30—minute wash and calculated this volume as the free space fraction in Avena coleoptiles as 19 to 22%. Sabnis and Audus (1967a), studying IAA accumu- lation by ESQ mesocotyl sections, reported a free space value of 30%. The effect of low pH has been shown by Currier and Dybing (1959), Sargent and Blackman (1962), Poole and Thimann (1964) to enhance auxin uptake. Bukovac and Norris (1966) reported that at pH values below the pK (4.2) of NAA, binding to leaf surfaces was significantly increased. Simmon and Beevers (1952) proposed that activity of weak organic acid was associated with the amount of acid in the undissociated form. Wedding and Erickson (1957) derived a model system to match their data for 2,4—D penetrability into Chlorella. They concluded that the Chlorella was 800 to 1000 times more permeable to the undissociated molecule compared to its anion. Reinhold 0954) determined that a change in the passive accumulation rate occurred one pH unit above the pK of the auxin being studied (IAA). She therefore concluded that auxin anions were accumulated at a higher rate than the uncharged molecule. concentrati: Temper; haVe been r and Blackman (Johnson am mm min has b1 4-3 (Sargem 26 Accumulation of auxin against a concentration gradient has been one criterion often used as evidence for active uptake. Johnson and Bonner (1956) and Poole and Thimann (1964) reported that for 2,4—D and IAA, respectively, net auxin accumulation exceeded the external concentration. Such a criterion should only be considered proof of active uptake when it has been established that the auxin is held in a free, non-bound form in excess of the external auxin concentration (Zenk, 1962). Temperature coefficients (Q10) of auxin uptake rates have been reported ranging from 1.67 for Chlorella (Wedding and Blackman, 1961) to 2.00 for coleoptiles of Avena (Johnson and Bonner, 1956) and hypocotyls for Gossypium hirsutum (Saunders _pta1., 1965b). Foliar penetration of auxin has been found to exhibit Q10 coefficients as high as 4.3 (Sargent and Blackman, 1965; Greene, 1969). As discussed earlier, these high Q10 values might be attributed to limiting cuticular penetration (Norris and Bukovac, 1969). Light has been shown to stimulate the rate of auxin uptake by Chlorella (Wedding and Blackman 1961), leaf disks (Sargent and Blackman, 1965; Greene, 1969) and green pea stem sections (Thimann and Wardlaw, 1963). These reports inferred that the light effect was due to the pro— duction of photosynthetic products to thereby enhance us the Ingnitu . Dinitropheno lation, has I 50%. dependi: andhudus, 1‘ 1956). Pota Oordes, 1966' accumulation As Andr Ietabolism o accumulation Occurred, an than it wouli lie remainin pith IraSpar lhdreae and lPlate to me lsselstein ( “957) repor 27 respiration. Wedding and Blackman (1961) effectively demonstrated this by showing that dark—treated Chlorella in the presence of citrate accumulate more 2,4-D than th0se alga cells treated only with light. The use of metabolic inhibitors has helped to resolve the magnitude of the active uptake component for auxin. Dinitrophenol (DNP), an uncoupler of oxidative phosphory— lation, has been shown to inhibit uptake of auxins by 20 to 80%, depending upon the duration of the experiment (Sabnis and Audus, 1967a; Jenner e; al., 1968a; Johnson and Bonner, 1956). Potassium cyanide, a respiratory poison (Mahler and Cordes, 1966), has been shown to also inhibit auxin accumulation (Johnson and Bonner, 1956). As Andreae (1967) summarized for pea root tissue, root metabolism of auxin can have a pronounced effect on net accumulation. For example, if decarboxylation of IAA occurred, and one were following carboxyl 14C—labeled IAA, then it would appear as a net loss of auxin from the tissue. The remaining free IAA within the tissue was conjugated with L—aspartate to form indoleacetylaspartate (IAAsp) (Andreae and Good, 1955, 1957). The relationship of uPtake to metabolism was described by Andreae and van Ysselstein (1960a, 1960b) and Andreae e; a1. (1961). Andreae (1967) reported that although free IAA is found in the roots r magnitude of! Dinitrophenol (n lation, has been 80%, depending u] and Audus, 1967a 1956). Potassiui Cordes, 1966), ha acciuuulation (J01 As Andreae Ietabolism of an: acciuoulation. F: occurred, and on: then it would ap] lle remaining fr: ‘fl'th L-aspartate [Andreae and 600' "hike to metabo llsselstein (1960. “957) reported 27 respiration. Wedding and Blackman (1961) effectively demonstrated this by showing that dark—treated Chlorella in the presence of citrate accumulate more 2,4-D than those alga cells treated only with light. The use of metabolic inhibitors has helped to resolve the magnitude of the active uptake component for auxin. Dinitrophenol (DNP), an uncoupler of oxidative phosphory— lation, has been shown to inhibit uptake of auxins by 20 to 80%, depending upon the duration of the experiment (Sabnis and Audus, 1967a; Jenner e£_§1., 1968a; Johnson and Bonner, 1956). Potassium cyanide, a respiratory poison (Mahler and Cordes, 1966), has been shown to also inhibit auxin accumulation (Johnson and Bonner, 1956). As Andreae (1967) summarized for pea root tissue, root metabolism of auxin can have a pronounced effect on net accumulation. For example, if decarboxylation of IAA occurred, and one were following carboxyl 14C—labeled IAA, then it would appear as a net loss of auxin from the tissue. The remaining free IAA within the tissue was conjugated with L—aspartate to form indoleacetylaspartate (IAAsp) (Andreae and Good, 1955, 1957). The relationship of uPtake to metabolism was described by Andreae and van Ysselstein (1960a, 1960b) and Andreae e: l. (1961). Andreae ”967) reported that although free IAA is found in the roots acetylaapartic not occur. "-" could be found uptake, report most of the significant am Andreae ( in pea root sec lated that a 2, sections. Klambt (19 Ccnjugation pro (InGlu). Zenk “NPIex (NAGlu) glucose complex decreased with Thomas et 3;. ( ethnically h olhectic acids 28 in amounts above the external concentration, it was con— verted almost totally to IAAsp within several hours and further accumulation was only in the form of the conjugate. Zenk (1962) found that pea epicotyl sections formed a similar aspartyl conjugate with NAA termed naphthyl— acetylaspartic acid (NAAsp), but that decarboxylation did not occur. Zenk's data showed a 4—hr lag before NAAsp could be found while Andreae (1967), also studying NAA uptake, reported a 6—hr lag. Both noted that after 16 hr most of the NAA was complexed. However, a small but significant amount did remain in the free form. Andreae (1967) did not find any metabolism of 2,4—D in pea root sections, but Jenner _§_a1. (1968a) specu— lated that a 2,4—D metabolite was formed by AXEEE mesocotyl sections. Klambt (1961) and Zenk (1961) each reported a second conjugation product of IAA to be indoleacetyl—B—D glucose (IAGlu). Zenk (1962) also identified an NAA glucose complex (NAGlu). Zenk's (1962, 1963) data showed this glucose complex to be an earlier metabolic product that decreased with time as the aspartyl conjugate increaSed. Thomas SE a1. (1963, 1964) found that Avena stem sections metabolically hydroxylated some of the chlorinated phen- Oxyacetic acids which had then accumulated as their induction of acid could be? benzoic acid the induction example of in- anyone. The physi llgate has bee for most of ac: sidered relatii 1957: Hertel ar Auxins, es lOund to protei nucleic acids ( IV- Isolation o A. Methods Racusen ant SePartition proc: insisted of gr: 29 respective glycosides. Formation of the aspartyl complex with NAA was the result of an inductive process, dependent upon protein synthesis (Zenk, 1962). Venis (1964) found that IAA induction of the conjugation of benzoic acid with aspartic acid could be inhibited by blocking RNA synthesis, so that ybenzoic acid was not conjugated. Sudi (1966) suggested that the induction of either the NAA or IAA conjugate was an example of induced enzyme formation, possibly an allosteric enzyme. The physiological significance of the aspartyl con— jugate has been described as a detoxified auxin product for most of accumulated auxin (Andreae, 1967). It is con— sidered relatively immobile within plant tissue (Veen, 1966, 1967; Hertel and Flory, 1968; Eschrich, 1968). Auxins, especially IAA, have been reported to occur bound to proteins (Zenk, 1963; Winter and Thimann, 1966) and nucleic acids (Kaur—Sawhney g§_§l,, 1967; Fellenberg, 1969). IV. Isolation of Leaf Cells for Auxin Uptake Studies A. Methods of Isolation Racusen and Aronoff (1953) reported a mechanical separation procedure to isolate leaf cells. Their method consisted of grinding soybean leaves in a mortar with a pestle I“ although amin this techniqu by homogenizin one minute and described by E several separa containing 1.0 buffer at pH 7 cells capable and respiration activity, but . assumed that h: Racnsen and Art llithod still p: t0 intact leave Ball and .1 were suitable “my found it Peanut leaves, 30 in 0.25 g sucrose and a phosphate buffer solution at pH 6.8. Cells were separated by filtration and centrifugation. Cells isolated in this manner had one-fifth the photo— synthetic capacity of intact leaves as measured by 14C02 fixation. None of the carbon fixed was recovered in protein, although amino acids were labeled. Price (1967) modified this technique to obtain greater quantities of leaf cells by homogenizing soybean leaves in a high—speed blender for one minute and then recovering cells by the procedure described by Racusen and Aronoff (1953). Price tested several separation media and found that a nutrient solution containing 1.0 mg EDTA, 0.40 g sucrose and 0.05 g tricine buffer at pH 7.2 to be sufficient for the separation of cells capable of exhibiting protein synthesis, photosynthesis, and respiration. Price observed that these cells had low activity, but did not compare them to intact tissue. He assumed that his experiments were in a similar range as Racusen and Aronoff's (1953). Price emphasized that this method still permitted qualitative comparison of the cells to intact leaves. Ball and Joshi (1965) reported that few plant species Were suitable for mechanical separation of intact leaf cells. They found it was possible to tease a few cells free from Peanut leaves, after which the cells were capable of sir pectinase from applied the er cells that wen JYlmggt a1. 1 prepared tobac Gabbott and La g 11,, isolat accumulation . relatively low Offer a compar (1965b) were a “Ptake studies PBCtinase prep; leaf cells thai “11118. Macero: 1967b) is main] tuned above, a Values of 5.0 (Zaitlin and C Both enz 31 dividing when cultured for several days on a complete tissue medium. Chayen (1952) described an enzymatic separation procedure for the isolation of root cells using a fungal pectinase from Penicillium digitatum. Zaitlin (1959), applied the enzymatic procedure to isolate tobacco leaf cells that were capable of supporting virus multiplication. Jyung _E _l, (1965b) modified Zaitlin's procedure and prepared tobacco leaf cell isolates for ion uptake studies. Gabbott and Larman (1968), using the technique of Jyung e: al., isolated leaf cells from tobacco to follow atrazine accumulatiOn. The latter two research groups each reported relatively low respiration rates for the cells but did not offer a comparison to intact leaf tissue. Only Jyung t El- (l965b) were able to show any metabolic involvement in their uptake studies. Takebe g: 3;. (1968), using a different pectinase preparation called Macerozyme, isolated tobacco leaf cells that supported the growth of tobacco mOSaic virus. Macerozyme (characterized by Suzuki g; al., 1967a, 1967b) is mainly polygalacturonase like the pectinase men— tioned above, but its optimum activity occurred at pH Values of 5.0 and 6.5, compared to 4.5 and 6.0 for pectinase (Zaitlin and Coltrin, 1964). Both enzyme preparations are contaminated with small mannitol haf cells d Unfortun . “fitabolic acti '30 that Of the drawn on how r “9113 were to . an effort to In using the tech] them to leaf 51 “1e technique n resPirntory aci respmtively, ( 32 amounts of proteases (Zaitlin and Coltin, 1964; Suzuki 2: 1., 1967a). Chayen (1952), aware of this, used 1% peptone to dilute the protease activity. Jyung g; _l. (1965b) used a lower concentration, 0.2% peptone, for the same purpose. Takebe .£.§i- (1968) reported that high mannitol concentrations (0.6 to 0.8 M) combined with a low molecular weight dextran sulfate salt adequately protected leaf cells during isolation. Unfortunately, none of the above reports compared the metabolic activity of the enzymatically isolated leaf cells to that of their parent tissue, so no conclusions can be drawn on how representative enzymatically isolated leaf cells were to intact tissue. Jacoby and Dagan (1967), in an effort to resolve this point, isolated bean leaf cells using the techniques of Jyung _p _l. (1965b) and compared them to leaf strips of the same tissue prepared according to the technique of Smith and Epstein (1964). They found that respiratory activity and Na+ accumulation was 7.3% and 1.5%, respectively, of leaf strip tissue. They observed that only cells not completely separated, stained with neutral red. Jyung sag al’ (1965) noted that ion uptake by the isolated Cells was only 3% of that of intact leaves. B. Methods of Assessing Cell Viability Thus, as ideal as isolated cells appear to be, no L'! catim can be nmda _e_t_ 5.1; , as being in a with organell distinct charJ Vital std been reported heiner and Les distinction is 'l‘roshin (1966) cells often st should also be qualitative as one. hutofluor been reported (oppenheimer a the vacuole, c 33 procedure is currently satisfactory for uptake studies. One reason little progress has been made may be the difficulty of applying reliable tests for cell viability. The appearance of isolated cells under high magnifi— cation can be a very critical assay (Hongladrom, 1964; Honda 23 _l., 1966). They described the living leaf cell as being in a dynamic state of protoplaSmic streaming with organelles such as the chloroplasts having very distinct characteristics compared to dead cells. Vital staining, particularly with neutral red, has been reported as a reliable assay for viability (Oppen— heimer and Leshem, 1966), particularly when a definite distinction is to be made betWeen living and dead tissue. Troshin (1966) has criticized such an assay, since damaged cells often stain more intensely than non—damaged ones. It should also be recognized that this technique provides a qualitative assessment of viability and not a quantitative one. Autofluorescence, as a similar qualitative assay, has been reported as a valid criterion of life in plant tissue (Oppenheimer and Jacoby, 1961). The blue fluorescence of the vacuole, caused by ultraviolet light radiation, was used as the indicator of viability. Unfortunately, the test has seldom been successful in green tissue containing respmaes of that bacterial tissue respira measuring resp free of bacter The abili 111/Racusen and of isolated 1e protein contai more critical Tetrazoliu Metal dehydrc used successful qualitative has I3aVies and Seam tetrazolium chl quantified by e and then measur interference of he meChanism o 34 chloroplasts, for their red fluorescence masked the blue fluorescence of the vacuole. The red fluorescence.was seldom lost in death (Oppenheimer and Jacoby, 1961). Respiration has been one of the most often measured responses of living tissue. Hallaway (1968) indicated that bacterial contamination can often offset the true tissue respiration by a factor of 10 to 100. Thus, when measuring respiration, it is essential that the system be free of bacteria. The ability to fix l4C02 has successfully been used by Racusen and Aronoff (1953) to compare metabolic activity of isolated leaf cells to intact tissue. The synthesis of protein containing fixed 14C from photosynthesis may be a more critical test for viability (Davies and Cooking, 1967). Tetrazolium reduction, an in vivo metabolic assay for general dehydrogenase activity (Pearse, 1960) has been used successfully to determine tissue viability on a qualitative basis (e.g. Currier and van der Zweep, 1956). Davies and Seaman (1968) reported that the triphenyl tetrazolium chloride (TTC) reaction could be colorimetrically quantified by extracting the reduced dye with ethylacetate and then measuring its absorbance at 485 nm to avoid interference of the Elodea pigment simultaneously extracted. The mechanism of tetrazolium reduction has been shown to be preparing and h considerable re unicellular or for subcellular for intact cell for one type of (Hallaway, 1965 evaluated again group of them, One of the 0rginnelle work, Once (Hallaway, this point by pr Procedures. The “5 further docu s“Zillntically is 35 coupled to electron transport via NADH and NADPH diaphorase (Farber §:_gl., 1956) rather than a specific dehydrogenase. C. General Methods Used for Handling Plant Cells and their Organelles .* Although little work has been reported on methods of 'preparing and handling fresh preparations of leaf cells, considerable research has been done on isolation of subcellular organelles. Some of the general techniques used for subcellular work would be expected to be applicable for intact cell systems.) However, the optimum preparation for one type of organelle may be detrimental to another (Hallaway, 1965), so a suggested preparation must be evaluated again on the total system, the intact cell, or a group of them, like in leaf strips. One of the basic principles, that has evolved from organelle work, was that the best methods were the milder ones (Hallaway, 1965, 1968). Hulme _E _l. (1964a) documented this point by preparing mitochondria by various grinding procedures. They reported that a roller—mill they develOped yielded the most active mitochondria compared to others isolated by homogenation in either a blender or a mortar with Pestle. The sensitivity of plant cells to mechanical shock was further documented by Ruesink and Thimann (1963), who enzymatically isolated protoplasts and noticed that even v1" {1 _ lire W- van also dis. .. that water, a1» V111 often rupt between 0.2 and to prevent prot. ivery detailed ”1’th osmotic he found 0.36 tc Mal osmotic p The mainten walkway. 1968 ) heir best metho us to carefully a"no“ medium M use a buffer we“ The use M a Cllmbersom 111W °Ptimum ac 36 low speed centrifugation would inactivate their prepara- tions. D'Alessio and Trim (1968) found that by using cell wall lysing enzymes, they could minimize mechanical shock and were thereby successful in isolating large numbers of leaf cell nuclei. The importance of the medium for organelle activity was also discussed by Hallaway (1965, 1968). She reported that water, alone, is seldom used as its hypotonic stress will often rupture the membranes. For this reason, sucrose between 0.2 and 0.4 M has been used as an osmotic medium to prevent protein denaturation. Stadelmann (1966) gave a very detailed description for the determination of the optimum osmotic condition for plant cells. For Hedra leaves, he found 0.36 to 0.43 M sucrose to be equivalent to its normal Osmotic potential. The maintenance of the prOper pH also has been stressed (Hallaway, 1968). Romani e; al. (1969) described that their best method for isolation of pear fruit mitochondria was to carefully monitor and adjust the pH of the prep— aration medium during the separation procedure. They did not use a buffer because of the detrimental effects of the buffer. The use of Good's (1966) buffers might simplify such a cumbersome practice (Romani g; 1., 1969), and yet allow optimum activity (Good_g§ l., 1966). at lower sumo; leaf cells. A stabiliz rim to he ben (Hallavtay, 1968 Plrrolidone (P Iellol‘ted to be ‘ 3““ Rowan. 1967 Hongladrom “16 incorporatic their treating n “W found that mm and Dextr ”138 medium comm dined Wide accr [9581- Kuehl (1 Such as N—octanc Zing iSOlated ni‘ Tissue cult :I'eatment media 37 Bovine serum albumin (BSA) has been shown to be very effective in maintaining the activity of isolated mitochondria (Jones e: a1., 1964; Sarkissian and Srivastava, 1968). Hongladrom (1964) and Honda 33 ;l. (l966) found that BSA at lower concentrations stabilized micro-preparations of leaf cells. A stabilizer of phenolic compounds also has been shown to be beneficial if included in the isolating medium (Hallaway, 1968). Hulme _E _l. (1964b) found polyvinyl pyrrolidone (PVP) effective, while metabisulfite has been reported to be more effective (Anderson, 1968; Anderson and Rowan, 19677 Stokes g; _l., 1968). Hongladrom (1964) and Honda _§__l. (1966) found that the incorporation of high molecular weight polymers in their treating medium stabilized microleaf strip preparations. They found that a very specific ratio of the polymers Ficoll and Dextran with sucrose and BSA gave optimum results. This medium commonly referred to as the "Honda medium", has gained wide acceptance for organelle isolation (Hallaway, 1968). Kuehl (1964) observed that other special molecules such as N—octanol and gum arabic are effective in stabili— zing isolated nuclei. Tissue culture media are another important class of treatment media (e.g. Murashige and Skoog, 1962; Lamport, ‘ and. division 0 1. E ' cell populatio 38 1964). These media have been developed to permit long—term experiments on the growth and division from a small amount of tissue prepared under optimum conditions. Such a technique does not provide fOr the optimum recovery of all the initial material, but rather the promotion of growth and division of the healthiest cells within the initial cell population. I. Hypocotyl s = Soybean, e was chosen as t ease with which (Price, 1967) t the leaves. Initial st cotyl sections Ieters necessa an optimum medir A. Growing Approximate ”m on 2 inches Wen flats. 'l iemliculite and filled with tap ishibition, thus subilequent dail} me grown in a EXPERIMENTAL I. Hypocotyl Section Studies Soybean, Glycine max Merr. var. ChippeWa or Hawkeye, was chosen as the source of plant material due to the ease with which the plants could be grown and the report (Price, 1967) that cells could be mechanically isolated from the leaves. Initial studies were conducted with soybean hypo- cotyl sections to establish the basic experimental para— meters necessary to follow auxin uptake and to establish an optimum medium for maximum tissue activity. A. Growing of Plants Approximately 35 g of Chippewa soybean seeds were sown on 2 inches of vermiculite contained in 12x18x4—inch wooden flats. The seeds were covered with 1/2 inch of vermiculite and the flats were placed for l/2 hour in a pan filled with tap water. This last step assured uniform imbibition, thus yielding a uniform population of seedlings. Subsequent daily watering was by subirrigation. The plants were grown in a growth chamber equipped with cool white and 39 1' 20 C, ... Hays after 5 ntely 9 cm lon still tightly B. Prepara Seedlings roots immersed experimental us Washed for 5 mi distilled water 0f six hypocotyl me slice was ma Paraffin served [“0 sets of twc “198 um below t three immediate] “"9 placed in f Him, to be ac ifitims to coné “Stet! of 0.35 a ”finite. and a 4O incandescent lamps such that the seedlings were illuminated for 12 hr daily with 1200 ft-c at the surface of the vermiculite. The day and night temperatures were maintained at 25 and 20 C, respectively. Seedlings were harvested 7 days after sowing, when their hypocotyls were approxi— mately 9 cm long, cotyledons unfolded and the primary leaves still tightly folded. B. Preparation of Hypocotyl Sections Seedlings were transferred to the laboratory and their roots immersed in water for not more than 1 hr before experimental use. Prior to sectioning, the seedlings were washed for 5 min under running tap water, followed by a distilled water rinse. Two 4—mm sections were cut from each of six hypocotyls simultaneously, unless otherwise noted. The slice was made with three mounted razor blades and paraffin served as a cutting base. A total of four sections (two sets of two) were cut from each hypocotyl, the first one 8 mm below the cotyledonary node and the following three immediately proximal. After cutting, the sections were placed in filter (0.45 um pore) sterilized treatment medium. to be accumulated with sufficient additional sections to conduct the ensuing experiment. The medium con— Sisted of 0.35 M mannitol,0.0l E Tris—HCl, 0.01 g sodium succinate, and adjusted to a pH of 7.4. After a l/2—hr x? within a 1/2~' plugs . c. Genera The 25 m1 sections and 7 incubated in a Scientific Co. 25.0 i 0.25 c . fluorescent 1i- 14C~Carb01p ‘- (Tracerlab, Wa] ' POlmd, because ,“After a Hit p! final concentr: to give a fina. letivity 16 11c, centration was calibrated wit aiitiition of th 41 wash in the medium, the sections were collected in a Bfichner funnel (No. 2A), rinsed with fresh media, and divided into groups of 12 sections each. Each group was then placed in 25 ml Erlenmeyer flasks containing the treat— ment medium. The flasks were stoppered with urethane foam plugs. C. General Uptake Methods The 25 ml Erlenmeyer flasks containing the hypocotyl sections and 7 ml of the respective treatment media were incubated in a gyrorotory water bath shaker (New Brunswick Scientific Co., Metabolyte-G77). The bath was held at 25.0 i 0.25 C and illuminated with 500 ft—c cool—white fluorescent light. The flasks were agitated at 200 rpm within a l/Z—inch orbit. l4C—Carboxyl labeled a-naphthaleneacetic acid (14C—NAA) (Tracerlab, Waltham, Mass.) was chosen as the test com— pound, because of its stability and physiological activity. After a l—hr pretreatment period, 1.0 ml of l4C—NAA, 8x final concentration, was added to the respective flasks to give a final concentration of leO'6 Q NAA, specific activity 16 Uc/umole. The l4C-NAA stock solution con— centration was checked using liquid scintillation counting calibrated with an internal standard of l4C-toluene. The addition of the 14C~NAA was staggered to allow sufficient Iii ‘ ”I 5933:3110: 939w snows-.13 sri: .mijlbsm void «1 they 1 timefor; ace -~ - - . - a» a r 1 ~. 4;": Isndjfifi ' Loin. .smsn dawn $.31“ 19?. » ~ ~ “1"" r ‘ x “Uptake Pe . , ... L‘ J. ' i "' ‘ ) D J ‘ removal of th s, . ,. t ,. , . . , . ,, Tile sections Hashes, all wi water was rapi syringe. The washe oven dried at mg. The weigh ethanol in a 1 (Thomas Co. , P quantitatively steel planchets This procedure statistica finance. Comp 42 time for accurate handling of the appropriate treatments. The uptake period was terminated after 3 hr by the rapid removal of the labeled treating solution using suction. The sections were quickly rinsed, followed by two 5—min washes, all with Sud of deionized distilled water. The wash water was rapidly administered using an automatic filling syringe. The washed sections were placed in l-inch planchets, oven dried at 70 C for 12 hr, and weighed to within $0.1 mg. The weighed sections were homogenized with absolute ethanol in a lO-ml Thomas—Ten Broeck glass tissue grinder (Thomas Co., Philadelphia, Pa.). The homogenates were quantitatively transferred onto 2X5/l6—inch ringed stainless steel planchets and the ethanol removed by evaporation. This procedure yielded a uniform distribution of the hypocotyl tissue within the planchet. Radioactivity was measured by using a low background proportional counter (Beckman Low Beta II). All samples were measured for at least 2000 counts (4.25% chance of error). Corrections were made for background, efficiency and sample weight. All mathematical transformations were done as a single step Operation on a desktop computer (Olivetti Programa). Statistical evaluation was achieved by analysis of variance. Comparison of means were made by the Tukey 1. To: amount of g - manner descri placed in a 2 material for procedure was divided into ‘ weighed. Thu: hypocotyl tiss As shown aSignificant lass was plate dlied soybean iithout signi 43 wprocedure (Steel and Torrie, 1960). Standard errors of the mean (SE) are given when the data were representative of non-homogeneous populations. D. Estanlishment of Experimental Parameters 1. Determination of Self—Absorption To facilitate the uniform labeling of a large amount of hypocotyl tissue, 10 mm sections were cut in the manner described above. Fifty of these sections were placed in a 25 ml Erlenmeyer flask. This represented material for one of four replications. The standard uptake procedure was used, except the dried labeled sections were divided into groups of varying numbers before being weighed. Thus, varying amounts (mass) of uniformly labeled hypocotyl tissue were plated onto 2-inch planchets. As shown in Figure l, self—absorption did not become 2 of tissue a significant factor until more than 1.3 mg/cm mass was plated in a planchet. Therefore, up to 20 mg of dried soybean tissue could be plated in a 2-inch planchet without significant self—absorption. 2. Determination of Optimum Section Length Hypocotyl sections of varying length (2.0, 4.0, 6.0 and 8.0 mm) were prepared in the manner previously described. To maintain a constant mass of tissue per treat— ment, sufficient sections of the respective length sections 44 I400 I200 I000 on O O tic Self—absorption curve of l4C—napht?al::::g:é acid in varying amounts of uniform y homogenized hypocotyl tissue. a) O Figure l. ”A DIOAC‘TIV/TY - 09m 435 O O RA DIOACT/V/TY - cpm I400 I200 IOOO 800 O) O 400 . 200 0.0 45 0.5 |.O DENSITY- mg /cm |.5 2 2.0 46 were used to give a total length of sections_equal to 48 mm. uptake period). A section length of 4 mm was found to be optimum size for maximum NAA accumulation (Figure 2). Sodium azide fresh weight and section length were measured before the samples were dried and assayed for 14C. Due to the complexity of this experi— ment, each replication was staggered by 45 min. To avoid the flat of seedlings was brotht to the laboratory and plants were removed as needed. 47 Figure 2. Effect of hypocotyl section length on naph— thaleneacetic acid uptake. Uptake was in the presence or abSence of NaN3 (1x10-3 5) . UPmKE—pmolos/IO mg do! wt 48- TREA TED NON - LENGTH-mm 2 rLII... . _ _ {to w. m m w No w o 5 4 4 3 3 2 2 a... be as 0\\uo\oEQImk§lb iill|lll| I. . H. I Table 1. Effeq of 4 Position Fr 49 Table 1. Effect of position on the hypocotyl as a source of 4 mm sections for experimental material. Position Fr wt1 Dry wt2 NAA accumulated“2 Non- NaN33 _ treated (1x10"3 gl— (mg) (mg) (pmoles/10 mg dry wt) 1 108.4b 9.5a 426a 198a 2 106.4b 8.1b 445a 185a 3 110.5b 7.7b 430a 190a 4 109.5b 7.3b 452a 203a 5 113.7ab 7.5b 444a 216a 5 ll7.4a 7.8b 400a 230a W 1Means within a column followed by different letters are significantly different at P=0.05. 2Values comparing NaN3 and non-treated are significantly different for all positions at P=0.05. 3Present for entire experiment (pretreatment and uptake period). (at the end 01 significantly. differed with and basal posi value of posit cell division, Probably refle thickening. T pesitions show sidered the 0p E- DEterm Acid U Having de the 0543 Of hYpI the hYPOCOtyl : 50 (at the end of the 4—hr experiment), did not elongate. significantly. However, both fresh weight and dry weight differed with sampling position, especially for the apical and basal poSitions on the hypocotyl. The high dry weight value of position one probably reflected the zone of cell division, and the high fresh weight of position six probably reflected the area of cell enlargement and wall 'thickening. The sections from the four intermediate positions showed least variation and were therefore con— sidered the optimum zone as a source of hypocotyl sections. E. Determination of Factors Affecting Naphthaleneacetic Acid Uptake Having determined those factors that directly affected the use of hypocotyl tissue, attention was focused on how the hypocotyl system could be used to elucidate the mechanism of NAA uptake and the development of techniques for use on leaf tissue. 1. Effect of Bacteria on the Hypocotyl System A factorial design was used to study the effect of bacterial contamination on NAA uptake by hypocotyl tissue. Three levels of D—threo—chloramphenicol (O, 10, and 250 ug/ml) were imposed on three levels of bacterial con— taminated media.. NAA, oxygen uptake and bacterial levels were determined for each treatment. The high by inoculating of media from bacteria. The the standard ( same as the st seedlings were After 4 h flask, serial Plated on Sepa The bacterial of garden Soil 51 The highest level of contamination was established by inoculating the standard uptake preparation with 1 ml of media from a similar preparation contaminated with I bacteria. The intermediate level was established from the standard uptake preparation. The low level was the same as the standard preparation except that the intact seedlings were washed with 1.0% (v/v) NaClO for 2 min. After 4 hr, a 1.0 m1 sample was taken from each flask, serial dilutions prepared with 0.85% NaCl, and plated on separate media for bacterial or fungal culture. The bacterial medium was prepared by autoclaving 1.0 kg of garden soil with 1.0 liter of water and 0.5 g CaCO3. The resulting suspension was filtered until clear, and then to every 100 ml of solution 2.0 g agar, 1.0 9 glucose, 1.5 g KZHPO4 was added. The pH was adjusted to 6.8. The fungal media consisted of 22 g of dehydrated potatoes (Pillsbury—Hungry Jack's) steamed 5 min in 1.0 liter of distilled water. This solution was filtered through cotton and filter paper (Whatman No. 1) and then made up to 1.0 liter. The following substances were added to the solution before it was divided into 200 ml lots for final autoclaving: 20 9 glucose and 1 ml NPX (bacteriacide). The two freshly antoclaved media were stored 2 hr at 43 C before being used for respective plating in sterile dispOSable Petri dishes. 52 Inoculation with bacteria increased markedly the bacterial population while surface sterilization lowered the bacterial level normally found (Table 2). Chloram— phenicol only seemed to lower the high level of bacteria contamination. No fungal contamination was observed. For the-NAA uptake experiments all hypocotyl sections were given the standard wash, so it can be assumed that most of the bacteria Were removed from the tissue before weighing and radioassay. The presence of bacteria in the experimental system had little effect on NAA accumulation, although surface sterilization decreased NAA uptake (Table 3). Chloram— phenicol at a level of 10 ug/ml had no effect, but at a concentration of 250 ng/ml significantly stimulated accumulation. During the uptake period, two replications of all treatments were assayed for oxygen consumption using standard Warburg manometric techniques (Umbreit _§ a1., 1964). Twenty-four sections were used in 4.0 ml of treat- ment medium and 0,5 m1 of 10% (w/V) KOH held in one of the two side arms of the flask (no centerwell). The bath temperature was 30 C and the unit was kept covered with a black cloth between the two 1—hr readings. The flasks were shaken at 120 orbits per minute. Table 2. Leve syst ch10 ChloramPhenico Concn —53 Table 2. Level of bacteria after 4 hr in the hypocotyl system treated with several concentrations of chloramphenicol. Chloramphenicol Bacteria level concn Contaminated1 Standard2 Sterilized3 (US/m1) (bacteria/ml) 0 4x107 2x103 1x102 10 2x107 5x102 1x102 250 2x106 4x103 1x102 Ww— 1Contaminated=medium inoculated with bacteria. 2Standard=filter sterilized medium with water washed hypocotyl sections. 3Sterilized=filter sterilized medium with 1.0% (v/v) NaHClO washed hypocotyl sections. Table 3. Effe upta. seve phen, E Chloramphenico: concn 54 Table 3. Effect of bacteria on naphtheleneacetic acid uptake by hypocotyl sections treated with several concentrations of D—threo chloram- phenicol. Chloramphenicol NAA accumulated1 concn - Contaminated2 Standard3 Sterilized" Mean5 (pg/ml) ‘ (pmoles NAA/10 mg dry wt) 0 687 742 683 702a 10 732 813 706 750a 250 840 876 785 833b Means 750ab 810a 724b 1At the end of the uptake period all tissue samples were washed twice to remove non—bound NAA and bacteria. 2Contaminated=medium inoculated with bacteria. 3Standard=fi1ter sterilized medium with water washed hypocotyl sections. l’Sterilized=fi1ter sterilized medium with 1% (v/v) NaHClO washed hypocotyl sections. 5Means followed by different letters are significantly different at P=0.05. Interaction of concentration vs. bacteria level not Significant. inoculated trs decreased (Tab lIIIPOcotyl resyy 55 Unlike the minimal effect of bacteria on NAA uptake. the presence of bacteria increased dramatically oxygen consumption of the inoculated treatments (Table 4). Chloramphenicol lowered oxygen consumption only in the inoculated treatments where the bacterial level was also decreased (Table 2). Surface sterilization further reduced hypocotyl respiration. 2. Time—Course Studies of Naphthaleneacetic Acid Uptake Two separate experiments were conducted on different days to determine an optimum sampling time for future experiments. The standard uptake procedure was followed except for time. As seen in Figure 3A, uptake proceeded at a relatively steady rate for the entire period (5-180 min.). A second experiment, plotted in Figure 3B, revealed that the initial steady uptake rate continued for about the first 15 hr, followed by a second slower phase Of uptake for the next 20 hr. The level of NAA accumu— lation was very different between the two experiments. However, marked variation between experiments was frequently Observed. The variation within an experiment was usually quite small. 3. Methods for Killing Tissue A prime objective of this study was to work with Ivy/m1) 56 Table 4. Effect of bacteria on oxygen uptake at 30 C by hypocotyl sections treated with several concen— trations of chloramphenicol. Chloramphenicol Oxygen uptake1 concn Contaminated2 Standard3 Sterilized“ (Hg/ml) (ml/100 mg dry wt/hr) 0 1516 581 398 10 1019 646 490 250 808 732 463 lOxygen uptake (manometric) values represent measure— ment of the total system (tissue and bacteria) for each treatment. 2Contaminated=medium inoculated with bacteria. 3Standard=filter sterilized medium with water washed hypocotyl sections. l‘Sterilized=filter sterilized medium with 1.0% (v/vy NaHClO washed hypocotyl sections. 57 l000 800 600 400 200 UPTAKE—pmoles //0 mg dry Wt Figure 3. Time—course of naphthaleneacetic acid uptake by hypocotyl sections. A. Time-course of 5-300 minutes (Study A)- I000 B. Time—course of 1-36 hours (Study B)- 800 $00 400 UPTAKE —pma/oa//0mg dry wr 58 IOOO 800 600 0 200 .3 be as S \ 235a .. mean .3 300 240 80 20 60 TIME-min IOOO O 0 0 W 0 0 m 8 6 4. a! Ace as 0\\ 33:5: Mkcfins TIME-hr l V killed by pla ledium either ice methanol 00$ 59 leaf tissue, particularly leaf cells. Unfortunately, some preparations might have questionable viability. As a check, development of a dead control that could be applied as a base was desirable. Hypocotyl sections were killed by placing the tissue, floating in the standard Vmedium either in a boiling water bath for 2 min or a dry— ice methanol bath until the tissue and media were frozen solid and then thawed slowly. The data presented in Table 5 established that there was no significant difference between boiling or freezing as a method of killing the tissue. The NaN3 treatment, which effectively reduced the amount of NAA accumulated, had little effect on tissue already killed by freeze— thawing. To examine why the NaN3 treatment significantly reduced more uptake compared to the killed treatments, part of the experiment was repeated to follow uptake on a time basis. As shown in Figure 4, the frozen—thawed treatment permitted a greater initial surge of NAA accumulation compared to the NaN3 treatment, but in time the net accumu- lation of the two approached each other, while the none treated control continued to accumulate NAA at a steady rate. 4. Methods of Infiltrating Tissue Preliminary experiments suggested that high levels Table 5. Eff. sec 60 I Table 5. Effect of several methods of killing hypocotyl sections on naphthaleneacetic acid uptake. Treatment NAA accumulated‘ (pmoles/10 mg dry wt) Non—treated 968a NaN3 (lxlO‘3 g) 2, 384d Boiled3 587C Frozen—thawed3 612bc FroZen-thawed3+ NaN3 (1x10"3 M)2 647b 1Means followed by different letters are significantly different at P=0.05. 2Present for entire experiment. aPretreatment. Figure 4. 61 Time—course of naphthaleneacetic acid uptake by hypocotyl sections killed by several methods. Tissue preparations were either non-treated frozen-thawed (pretreatment), or treated Wlth NaN3 (1x10'3 E) for the entire experiment. 800 N o 0 an O O or o O UPTAKE —pmolas //0 mg dry wt cut 4:. O O O O m o O 23 C) 62 o NON-TREATED A FROZEN 800 THAWED 700 0 O O O 0 O O O 0 0 6 5 4. 3 2 a... be as oigaosqnmtanos TIME-hr contai without NW3 a vacuum was Infiltration! 5. Will Variable for II“Elia were u: 63 of NaN3 were required to inhibit NAA uptake. One explana— tion was lack of penetration of the inhibitor and perhaps this problem could be overcome by infiltration. While flasks containing tissue segments and medium with or without NaN3 were being agitated in the water bath shaker, a vacuum was drawn for 10 min and then slowly released. Infiltration had no affect on NAA accumulation (Table 6). 5. Effect of Composition of Treatment Media When a component of the treatment medium was the variable for an experiment, the various respective treatment media were used during the pretreatment, wash and uptake portions of the experiment. The experimental media were prepared by making up the basic medium less the variable ycomponent.> The corresponding treatments consisted of the experimental medium plus the variable component. The pH of the media was always determined and adjusted if necessary. As detailed in Table 7, several substrates, all at 20 mM concentration, were examined for their effective— ness in enhancing NAA uptake. Sodium succinate was the most Promotive, followed by sodium acetate. Sodium citrate and glucose had little effect while sucrose depressed Uptake. An important factor of the medium was its osmotic strength. The results of using mannitol as an osmoticum Table 6. Effl van upn 64 Table 6. Effect of infiltration of the medium containing varied concentrations of sodium azide into hypo- cotyl sections prior to naphthaleneacetic acid uptake. Treatment Infiltrated NAA accumulated1 (pmoles/10 mg dry wt) Non—treated — ' 389b Non—treated + 388b NaN3 (1x10‘3 392 — 218d NaN3 (1x10"3 392 + 228d NaN3 (1x10‘4 Myz - 323c NaN3 (ixio‘4 MH + 315C Frozen—thawed3 - 430a Frozen—thawed3 + 434a 1Means followed by different letters are significantly different at P=0.05. 2Present for entire experiment. 3Pretreatment-no NaN3 present. Table 7. Eff aci 65 Table 7. Effect of various substrates on naphthaleneacetic acid uptake by hypocotyl sections. Medium1 Substrate NAA accumulated2 (20 mg) (pmoles/10 mg dry wt) 1 ————— 234cd 2 ————— 266C 2 sodium acetate 312b 2 sodium succinate 352a 2 sodium citrate 212de 2 glucose 230d 2 sucrose l90e lMedium 1=o.01 M Tris—HCl adjusted to pH 7.4. Medium 2=0.0l M Tris—HCl 0.55 M mannitol and adjusted to pH 7.4. 2MEans followed by_different letters are significantly different at P=0.05. are shown in depressive t1 concentratior II. Developme of Tissue A more dj leaf cells wz technique of however, to : Viability an< know Viabil: A. Grow; SOybean material, as 66 are shown in Table 8. A general, but inconsistent, depressive trend on NAA uptake was observed as the mannitol concentration was_raised. II. Development of a Leaf Strip Technique and an Assessment of Tissue Viability A more direct approach to the study of NAA uptake by leaf cells was a refinement of Smith and Epstein's (1964) technique of preparing leaf strips. It was essential, however, to first develop a quantitative assessment of tissue viability and thus be able to examine uptake on tissue of known viability. A. Growing of Plants Soybean, cv Hawkeye, was used for the source of leaf material, as it was found to produce more vigorous plants in greenhouse culture than Chippewa. Seven to eight seeds were sown l/2—inch deep in a sterilized mixture of soil: sand (3:1) in a 4—inch peat pot. Plants were grown in the greenhouse with a day, night temperature of 25 C and Of 18 C respectively. Photoperiod was maintained at 14 hr and a minimum light intensity of 1200 ft—c at the soil Surface was established during the light phase. When the primary leaves were unfolding, all but the fOUr most vigorous plants were removed from each pot. Table 8. 67 Table 8. Effect of varying concentrations of mannitol on naphthaleneacetic acid uptake by hypocotyl sections.1 Concnl NAA accumulated2 (P4) 0. .000 .200 .250 .275 .300 .325 .350 375 (pmoles/10 mg dry wt) 484a 434a 367bc 326d 362bcd 4lObC 378bcd 342Cd w 1All treatments were present for entire exper— iment and contained 0.01 M Tris-HCl, 20 mg sodium succinate and adjusted to pH 7.4. 2Means followed by different letters are Significantly different at P=0.05. Plants were mercial fert Insect pests B. Prep Fully e 3rd node bel fully immers The leaves w before use . . These 12 strips were then cut in half and stacked together with the last freshly CUt edges directly on top of each other (center edge over 0 enter edge), Thus, a block of 24 pieces of 25 mm wide strips ¥ 1 was'made an compact sty was mounted and section: cyrostat ra: ment Co., N4 tissue bloc] to cut the : The front p; while the be suPPOrt. A tWither . f 69 was'made and then placed between two pieces of fine pore compact styrofoam. The styrofoam—enclosed block of tissue was mounted on a Spencer table microtome (Thomas 7125) and sectioned with a razor blade (blue steel) held in a cyrostat razor blade holder (IEC—3351; International Equip— ment Co., Needham Heights, Mass.). The styrofoam—held tissue block was positioned such that it was only necessary to cut the front piece of styrofoam when cutting the block. The front piece of styrofoam was as thin as possible (2 mm) while the back piece was thicker (5 mm) to offer greater support. A small elastic band helped keep the tissue block together. The razor blade was changed frequently to allow clean cuts through the tissue. Whenever a blade was changed, a check slice was made to realign the blade to the block. The appropriate thickness was dialed before each slice. Four consecutive slices, yielding 96 strips, 250 um X 25 mm, represented the amount of leaf material for each treatment. Unless otherwise noted, sufficient leaf strips were cut from one tissue block to supply all the leaf strips for an entire replication. This method permitted the benefit of working with a complete block design to allow statistical removal of tissue variance. As the strips were cut, they were immediately placed in 7 ml of filter sterilized treatment medium within a 25 ml Erlen: 0.30 l_l mam l. % (w/v) ; serum album When all thi cut, the met any sloughe. replaced wi‘ An imp( technique w; the leaf st: generally e1 70 25 ml Erlenmeyer flask. The initial medium consisted of 0.30 g mannitol; 0.01 M Tris-HCl; 0.004 g sodium succinate; 1.0% (w/v) polvinyl pyrrolidone—k30 (PVP); 50 ug/ml bovine serum albumin, fraction V (BSA); and the pH adjusted to 7.4. When all the leaf strips for a particular experiment were cut, the media from the respective treatments containing any sloughed—off plant material was removed by suction and replaced with fresh media. An important variation of Smith and Epstein's (1964) technique was the omission of the "tea bag” to handle the leaf strips. It was found that the strips (25 mm long) generally entwined so they could be handled en masse with forceps. The massed strips could be placed on one side of the Erlenmeyer flask where they would adhere to the wall; then the flask was tilted in the opposite direction permitting the media to be withdrawn by suction. C. Assay of Viability by Tetrazolium Reduction Initial studies consisted of a modification of the technique of Davies and Seaman (1968). The dye 2,3,5— triphenyl tetrazolium chloride (TTC) was used at 2.0 mg/ml unless otherwise noted. It was administered to the experi— mental system in a manner identical to that described for l4C—NAA in the hypocotyl experiments. The treatment con— ditions were the same except for the previously described different me was terminat taining the addition of until it was tatively tra 10ml volume 10min) the Trichloroace 71 different medium (Exp. II.B.). After 3 hr, the experiment was terminated by rapid withdrawal of the medium con— taining the non-reduced dye by suction, followed by the addition of xylene. The tissue was stored in the dark until it was homogenized. The homogenate was quanti— tatively transferred into centrifuge tubes and diluted to 10 ml volume with xylene. After centrifugation (2000 x19; 10 min) the supernatant was decanted into test tubes. Trichloroacetic acid (TCA) was added (approximately 1 g crystaline) to each tube and absorption was measured (535 nm) immediately thereafter on a Beckman DU Spectrophoto— meter. The spectrophotometer was standardized against a xylene blank. Samples were stored in the dark to minimize photodestruction of the reduced tetrazolium. Dry weights were determined on parallel samples of similarly treated tissue, and were measured on a macro— analytical balance (Mettler B—5). D. Establishment of Experimental Parameters with 2,3,5—Triphenyl Tetrazolium Chloride 1. Determination of Light Absorption Curve An absorption curve was determined on either xylene eXtracted leaf tissue or chemically reduced tetrazolium with a DU—spectroPhotometer at 5 nm increments standardized against a xylene blank. TTC was reduced in a dilute HCl solution co suiting for xylene. Th had conside TCA resulted as intensif treatment 0 mum from 48. 72 solution containing metallic zinc fillings. The re— sulting formizan compound was then partitioned into xylene. The absorption spectra of the two preparations had considerable overlap (Figure 5A). The additiOn of TCA resulted in a shift in the absorption maxima as well as intensification of the TTC absorption. The TCA treatment of the reduced TTC shifted the absorption maxi— mum from 485 to 535 nm (Figure 5B). 2. Determination of Optimum 2,3,5—Triphenyl Tetrazolium Chloride Concentration The effect of TTC concentration was examined to determine the optimum level for reduction by leaf strips. The lowest concentration, 2 mg/ml, was equally effective as 5 and 10 mg/ml, whereas 20 mg/ml slightly depressed reduction (Figure 6). Since tetrazolium salts have been shown to uncouple oxidative phosphorylation (Palmer and Kalina, 1966), 2 mg/ml of TTC was chosen for all further experiments. 3. Refinement of Leaf Strip Preparation The effect of a low temperature pretreatment wash on minimizing the wound response from the cutting process, especially for thin slices, was next established. The data presented in Table 10 show that the low temperature slightly depressed reduction. The frozen—thawed pretreatment data served as a control equivalent to dead tissue (dead control). Furthermore, the thin slices (125 and 225 um) were less Figure 5. 73 Light absorption curves of xylene extracts of leaf strips and reduced 2,3,5—tripheny1 tetra- zolium chloride (TTC) with and without trichloro— acetic acid. A. Xylene extract of TTC-treated leaf strips compared to reduced TTC. B. Xylene extract of TTC—treated leaf strips and reduced TTC, both in the presence of 20% trichloroacetic acid (TCA). O A BSORBA NCE .0 a: 9 4: 02 09 b p (D A as afield NC: 0 'a: o & 74 1.4 l.?. l ’ REDUCED TTC IN XYLE/VE 0.6 A BSORBANCE 9 A XYLENE EXTRACT 0F LEAF STRIPS .0 N 0.0 400 450 500 WAVELENGTH-0m REDUCED TTC IN XYLENE + TCA A BSORBANCE XYLENE EXTRACT 0F LEAF STRIPS + TCA 350 400 450 500 550 600 650 700 WAVELENGTH-mu Table 75 Table 9. Effect of 2,3,5—triphenyl tetrazolium chloride concentration on its reduc- tion by leaf strips.1 TTC concn TTC reduced2 (mg/ml) ' (A-535 nm) 0 1120 2 230ab 5 250a 10 236ab 20 l88b 1Leaf strips were 1000 pm thick; each replicated treatment was cut from a separate leaf. 2Means followed by different letters are significantly different at P=0.05. Table 10. Leaf strip width (um) 76 Table 10. Effect of pretreatment temperature and leaf strip size on 2,3,5-triphenyl tetrazolium chloride reduction. Leaf strip TTC reduced width Wash temperature 0 C 23 C Frozen— thawed (um) (A—535 nm/lO mg dry wt) 125 . 0.057 0.068 0.006 225 0.099 0.120 0.023 1000 0.321 0.366 0.012 Mean 0.1511 0.1841 0.0142 1Wash temperature means are significantly different at P=0.lO by orthogonal comparison. 2Freeze-thawing is significantly different from wash treatments at P=0.01. active thal The p: of materia. ment, and ‘ between re] was variab: ability to logical age considerabj trend for :‘ 77 active than 1000 um slices. The preliminary experiments, to this point, consisted of material from single leaves for each replicated treat- ment, and the resulting data showed considerable variation between replications of a given treatment. One explanation was variability in leaf age of the material on its inherent ability to reduce the TTC. When leaves of various physio— logical ages were sampled for the TTC reduction capacity, considerable variation was found (Table 11). A definite trend for increasing capacity to reduce TTC was apparent with age. The 2nd and 3rd leaves were chosen as the source of future experimental material because of similar activity. 4. Modification of Treatment Media and Conditions The importance of the proper osmotic strength of the treatment medium has been considered to be one of the most important factors in developing a suitable medium (Hallaway, 1968). In a preliminary study, no significant effect of Osmotic strength on TTC was observed using a mannitol concentration range from 0.20 g to 0.35 g (Appendix— Table A1). However, all levels of mannitol resulted in significantly more TTC reduction than did the 0.5 mg CaSO4 base medium recommended by Smith and Epstein (1964). There- fore. a wider range of mannitol concentration was employed only during the initial wash of the cut sections. This Table 11. POSlthI‘L OI Wotan ISt‘unfOlde nd‘expc‘md: 78 Table 11. Effect of age of leaf used as a source of leaf strips on 2,3,5—triphenyl tetrazolium chloride reduction. Position of leaf in relation TTC reduced‘ to shoot apex __ (A—535 nm) lst-unfolded 0.486d 2nd-expanding 0.625c 3rd—expanding 0.655c 4th—expanded 0.867b Sth—expanded 1.027a 7th-expanded—senescent 0.51lcd 4th*expanded—picked three days earlier 0.644c 3rd—expanded—frozen-thawed 0.105e 1Means followed by different letters are significantly different at P=0.05. experimen' hypothesi: period mig released 1 use of a 1 compared t Therefore, in media w Resul Suecinate lation of 79 experiment was designed to examine Takebe's _£__l, (1968) hypothesis that high osmotic strength during this critical period might minimize the uptake of detrimental material released from damaged cells. Data in Table lZflshow that the use of a hypertonic or hypotonic media were detrimental as compared to an isotonic solution (0.25 g) for TTC reduction. Therefore, all of the following experiments were conducted in media with 0.25 E mannitol. Results with hypocotyl sections suggested that sodium succinate (20 mg) was an effective substrate for stimu— lation of l4C—NAA uptake. For leaf strips a lower con— centration (8 mg) was optimum for TTC reduction when expdsed to light (Figure 6). Also, it was apparent that light, peg gs, plays an important role in TTC reduction by leaf strips. An attempt was made to separate uptake of TTC from subsequent reduction by preloading the tissue with TTC and then following reduction under varying conditions. A time-course of TTC reduction is illustrated in Figure 7, where treatments were removed from the TTC and allowed to incubate in TTC—free medium until the end of the experiment. Consistently less net reduced TTC was found in samples removed from the TTC medium and allowed to incubate in TTC—free medium compared to when first removed from the Table 12 . ManIli‘COl c 80 Table 12. Effect of mannitol concentration during the initial wash of leaf strips prior to 2,3,5— triphenyl tetrazolium chloride reduction. Mannitol concnl'2 TTC reduced Strip width (um) 500 1000 Mean3 (fl) (A-535 nm) 0.10 0.397 0.492 0.444bc 0.25 0.462 0.581 0.521a 0.45 0.408 0.468 0.451b 0.60 0.387 0.459 0.423d 0.75 0.421 0.439 0.429cd . 1Pretreatment wash medium=treating medium except variable mannitol concentration. 2Treatment medium during reduction=0.25 M mannitol, 0.01 M Tris-HCl, 4 mM sodium succinate, 10 mg7ml PVP 50 ug7ml BSA and adjusted to pH 7.4. 3Means followed by different letters are significantly different at P=0.05. Interaction of size and mannitol concentration is not significant. Figure 6. 81 Effect of sodium succinate light on 2,3,5—triphenyl te reduction by leaf strips. Leaf strips were 1000 um. The interaction of 8 mg sodium succinate with light and dark 15 significant at P=0.05 by orthogonal comparison. T TC REDUCED -A 535 O O) 0.5 O A 82 2 . CONCENTRA 4 . TlON-mM Figure 7. 83 0.4 h ll V I . a 0.3 Time—course of 2,3,5—triphenyl tetrazolium g chloride reduction by leaf strips. 3 Dashed line represents treatments that were E removed from medium containing TTC and incubated in standard medium until the sixth hour of the 0 experiment. L k 0.2 0.0 A 84 o—--o TISSUE REMOVED FROM TTC; FIXED AT TIME SHOWN o-—O TISSUE REMOVED FROM TTC, INCUBATED UNTIL 6th hr; THEN FIXED 0.4 A535 0.3 TTC REDUCED - 0.2 TIME-hr TTC mediu E.I T The to light stable un tetrazoli‘ COIN wa. was used ; 85 TTC medium. E. Improvement of Leaf Strip Technique Using Neo— Tetrazolium Chloride The net loss of reduced TTC within the tissue exposed to light indicated the need for a tetrazolium compound stable under these conditions. The usefulness of neo— tetrazolium chloride (NTC) (Nutrititional Biochemical Corp.) was next established. Unless otherwise noted, NTC was used at 0.25 mg/ml. The staining procedure was identical to that used for TTC. However, because of low water solubility, NTC was first dissolved in 2 ml 50% ethanol and then the aqueous medium solution was added. The ethanol was then removed by partial flash evaporation. Finally, the resultant NTC solution was made to volume with the treatment medium. At concentrations in excess of 2.0 mg/ml, NTC precipitated out of solution. The assay for the reduced form of NTC was the same as the method for the reduced TTC with the exception that the absorbance was measured directly at 565 nm in a spectro— photometer (Bausch—Lomb Spectronic 20) after centri- fugation of the xylene homogenate. 1. Determination of Light Absorption Curve NTC was chemically reduced and partitioned into xylene. the metho was found between t' tissue ex the use 0 the reduc. NTC (Figuj effECtiVe lower. A the leVel 86 xylene. The light absorption curves were determined by the methods described for the reduced TTC. The use of TCA was found unnecessary, for there was a distinct separation between the absorption curves of the reduced NTC and the tissue extract (Figure 8). This wide separation permitted the use of a colorimeter to determine the absorbance of the reduced NTC at 560 nm. 2. Determination of Optimum Neo—Tetrazolium Chloride Concentration The optimum concentration of NTC was 0.25 mg/ml NTC (Figure 9). The 0.10 mg/ml was significantly less effective while concentrations above 0.50 mg/ml also were lower. A dead control (frozen—thawed) treatment indicated the level of reductive capacity of dead tissue was nominal. 3. Determination of Optimum Strip Width Smith and Epstein (1964) reported that the leaf strips 300 pm in width were optimum for potassium uptake studies. Because of possible light destruction of reduced TTC, the experiments with the TTC may have been misleading in identifying optimum strip width. Narrow strips would have more exposure to light and therefore may have lost more Of their reduced TTC. Also, the previous experiments with NTC revealed the outer edges of the leaf strips stained more intensely. For these reasons, the leaf strip width Figure 8. 87 Light absorption curve non-treated leaf strip zolium chloride. 5 of xylene extract of s and reduced neo—tetra— IA ABSOREANCE ABSORBANCE 88 |.4 XYLENE EXTRACT 0F LEAF STRIPS I.2 REDUCED NTC IN XYLENE I .O _ 0.8 0.6 0.4 0.2 0 .O 350 400 450 500 550 600 650 700 WAVELENGTH- nm Figure 9. 89 Effect of neo—tetrazolium chloride concentration on its reduction by leaf strips. NTC (l mg.ml) reduction by dead control: absorbance of 0.070. Leaf strips were 1000 um wide. 560 IV 7'6 REDUCED —,4 o N N TC REDUCED -A 560 90 0.4 0.3 O N O. I O 0 Ll—J_L_____I___———_J 0.00 0.25 0.50 LOO 2.00 0J0 , CON CENTRATION'mg lml experime stained sections sizes in width fo minimum Figure 10. 92 Effect of leaf strip width 0 chloride reduction. 11 neo— tetrazolium 0.4 k i S m 0.3 E 9 \ o e In e “4 c a e l o 0'2 I. 2 93 0.4 //0 mg dry wt .0 o: 560 engetraZollum I NTC REDUCED-.4 .0 N 0'0 750 I000 o 250 500 1500 STRIP WIDTH-pm d Table 13 . E Number of strips Table 13. 94 Effect of leaf strip width on oxygen uptake. Number of 20 20 20 160 48 Strip width (um) 250 500 1000 250 500 Dry wt (mg) 2.9 5.9 12.2 23.3 13.9 Oxygen uptake“2 strips ___ ---- (ul 02 per hr) (per total wt) 10.5d 23.6c 46.2b 85.2a 54.8b (per 100 mg dry wt) 371a 419a 378a 368a 394a lMeans within a column followed by different letters are significantly different at P=0.05. 2Manometric at 30 C. hydrogen ion pH leveled a during the N concluded thé capacity and 95 NTC either in the same media (freshly changed) or media containing Tris—HCl adjusted to pH 7.4. Figure ll depicts that a significant trend toward pH 6.4 was the optimum hydrogen ion concentration. Because treatments at the same pH leveled stained similarly, irrespective of the pH during the NTC treatment (details in Figure legend), it was concluded that the pH effect was mainly on tissue reduction capacity and not on the uptake of the NTC. This meant that all subsequent pH experiments could be conducted in one step with NTC uptake and reduction, the various pH treatments occurring simultaneously. The same pH range was re—examined more critically using the same buffer. This more detailed study indicated that maximum NTC reduction occurred at pH 6.6 (Figure 12). Good _£.§i- (1966) reported several new hydrogen buffers that would be better suited for biological systems than Tris and similar buffers. As detailed in Table 14, there was no difference between the buffers at a concentra— tion of 0.01 m, but at 0.05 g HEPES appeared superior. Using HEPES and MES buffers, the pH range was re—examined for optimum reduction. As illustrated in Figure 13, the two buffers complimented each other with pH 6.7 being optimum for NTC reduction. Subsequent media was made up with HEPES (0.01 g) as the buffer adjusted to pH 6.7 96 0.30 p N (’l 560 Figure 11. Effect of pH on neo-tetrazolium chloride reduction by leaf strips. All samples were pretreated 2 hr in Tris- maleate buffer at the various pH levels. The values on the solid line represent the same treatments in fresh media with NTC. The points on the dashed line represent treatments from the various pretreatments that were all then incubated in Tris-HCl buffer at pH 7.4 with NTC. Check on NTC reduction by frozen—thawed leaf strip tissue at pH 7.4 in Tris—HCl had an absor- bance of 0.032. ‘ A/TC‘.?£zuamszr—A g) N O 560 NTC REDUCED -A 0.25 0.20 O.I5 0.I0 0.ool.__.____.*J 5.4 6.4 97 7.4 8.4 Figure 12. Effect of pH on nee-tetrazolium chloride. reduction by leaf strips using 0.01 g Tris- maleate buffer. 0.30 0.25 A560 ”Tc REDUCED — _O N O um chloride_ 9 0.01 M Tris- NTC REDUCED-A 0.35 560 99 0. 30 0.25 0.20 0.I5 O.|0 0.0 0L|____|___|___L__L—l——L——J——-|—-LT Table 14. Buffer Tris-maleat. RH21’04-K le MOPS " 100 Table 14. Effect of several buffers at pH 6.6 on neo— tetrazolium chloride reduction by leaf strips. Buffer NTC reduced1 __ Buffer concn: 0.01 M 0.05M Mean (A—560 nm) Tris—maleate2 0.207ab 0.114c 0.l6lc _ '3 . KH2P04 KZHPO4 0.225a 0.178b 0 202abc MOPS“ 0.236a 0.197abc 0.217ab MES5 0.218ab 0.154bC 0.186bC HEPES6 0.243a 0.242a 0.242a —...____——___________—_——————————-——'———-—— 1Means within a column followed by different letters are significantly different at P=0.05. 2Tris-(hydroxymethyl aminomethane), pKa 8.3; maleate— PKa 6.15. 3pK 6.8 at 20 c. l'MoiflfpholinoprOpane sulfonic acid, pK 7.2 at 20 C. 52‘(N—morpholino) ethanesulfonic aci , pKa 6.15 at 20 C. 6N-2—hydroxyethylpiperazine—N—2—ethanesulfonic ac1d, PKa 7.55 at 20 C. 101 0.6 0.5 I l e 1 0 ‘0 I Q t 0.4 b Figure 13. Effect of HEPES and MES buffers on neo—tetra; Q zolium reduction by leaf strips at several p E values. 0 L k 0.3 I 0.2 tetra‘ NTC REDUCED -A 560 0.6 0.5 .0 A O 01 0.2 o.oh—J—I——J—I—I} 7.5 8.0 5.5 MES 6.0 102 6.5 7.0 HEPES 103 wi th KOH . 2. Effect of Various Enzyme Stabilizers A series of experiments were conducted to determine the effect of several enzyme stabilizers on preserving metabolic activity of intact leaf strips. The high molecular weight polymers Ficoll and Dextran— 40 (Pharmacia, Uppsala, Sweden) were first evaluated using a factorial design. Ficoll had a very negative effect on NTC reduction while Dextran—4O had no effect (Table 15). The inhibition by Ficoll was partially attributed to a dialyzable inhibitor (Appendix, Table A—2). Both polyvinyl pyrrolidone-k30 (PVP) and potassium metabisulfite (K28205) significantly enhanced the leaf tissue reduction of NTC when used at up to 5.0 mg/ml and up to 0.4 mg, respectively (Table 16). Because higher concentrations of K28205 were more detrimental than those of PVP, 1.0 mg/ml (0.1% w/v) PVP was chosen for subsequent experiments. The factorial effect of bovine serum albumin (BSA) and PVP is recorded in Table 17. BSA (50 ug/ml) was beneficial, while PVP had no effect in this experiment. The 50 ug/ml BSA was confirmed as an optimum level for subsequent leaf Strip work. Ficoll 104 Table 15. Effect of Ficoll and Dextran—40 on neo—tetra— zolium chloride reduction by leaf strips.l Ficoll NTC reduced concn Dextran concn— O 1.0 2.5 Mean2 (g/100 ml): (g/100 ml) (A-560 nm)‘ 0 0.558 0.458 0.565 0.536a 2.5 0.420 0.405 0.387 0.404b 5.0 0.341 0.356 0.316 0.3360 Mean2 0.440b 0.415b 0.423b 1Basic treatment medium=0.25 M mannitol, 0.01 M HEPES- KOHI8 mM sodium succinate and adjusted to pH 6.7. ~ 2Means followed by different letters are significantly different at P=0.05. Interaction of Ficoll and Dextran—40 was not significant. Table 16. *4 PVP K \ (mg/m1) (1 1.0 - 5.0 _ 10.0 _, 50.0 -. “““ 0 _____ 0‘ """ 1 ..... 4 “““ 8 \ l align: 1v 01:21:32: 105 Table 16. Effect of polyvinyl pyrrolidone (PVP) and potassium metabisulfite (K S O ) on neo- tetrazolium chloride reducgignsby leaf strips.1 PVP K25204 NTC reduced2 (mg/m1) (mM) (A-560 nm) —————————— 0.435b 1.0 ————— . 0.528a 5.0 ----- 0.534a 10.0 ————— 0.345b 50.0 ————— 0.189c ————— 0.1 0.515a ————— 0.4 0.590a ————— 1.0 0.258c ————— 4.0 0.182c ----- 8.0 0.125c 1Treatment medium=0.25 M mannitol, 0.01 M HEPES—KOH, 8 mM sodium succinate and adjusted to pH 6.7. _ 2Means followed by different letters are significant— ly different at P=0.05. BSA concn 106 Table 17. Effect of bovine serum albumin (BSA) and poly- vinyl pyrrolidone (PVP) on nee—tetrazolium chlor- ide reduction by leaf strips. BSA concn NTC reduced PVP concn (mg/ml): 0 2.5 Mean1 (pg/ml) (A-560 nm) 0 0.534 0.531 0.533b 50 0.638 0.620 0.629a 100 0.510 0.533 0.522b 500 0.548 0.539 0.544b 1000 0.419 0.473 0.465c Mean1 0.543b 0.539b 1Means followed by different letters are significantly different at P=0.05. Interaction of BSA and PVP was not significant. I Hi .- -.; _*'-.._.g. fa—r t_1:_ ' - I the effect reinvestiga the range a optimal (Ta to the refi' Having esta centration medium had 107 3. Effect of Mannitol Having significantly refined the original medium, the effect of mannitol concentration as an osmoticum was reinvestigated. The optimum NTC reduction occurred within the range of 0.20—0.40 M, with 0.25 M mannitol being optimal (Table 18). The CaSO4—based medium was inferior to the refined medium for NTC reduction\(Table 18). Having established 0.25 M mannitol as a suitable con- centration for future experiments, the final modified medium had the composition listed in Table 19. G. Time—Course of Neo—Tetrazolium Chloride Reduction A time-course of NTC reduction was followed in both light and dark to establish the role of light in the reduction process.- As detailed in Figure 14, light was required only for the high level of reduction while suffi— cient NTC accumulation occurred in either the light or dark. In contrast to the TTC experiment, sufficient NTC accumu— lated during the first hour to supply the tissue with enough dye for additional reduction for the following 2 hr. H. Further Assessment of the Leaf Tissue Viability 1. Measurement of Protein Synthesis Davies and Cocking (1967) reported that green tissue would incorporate l4C into protein following fixation 0f l4C02 via photosynthesis. Testing the leaf strip ..... 108 Table 18. Effect of mannitol concentration on neo—tetrazolium chloride reduction by leaf strips in the refined medium. Mannitol concn1 NTC reduced2 (M) (A-560 nm) 0.00 0.459c 0.10 0.504bc 0.15 0.490bc 0.20 0.518abc 0.25 0.593a 0.30 0.519abc 0.35 0.565ab 0.40 0.535abc 0.25 in the dark 0.108e 3 0.198d ----- 0.5 mM CaSO4 1Treatment medium=variable concn mannitol, 0.01 M HEPES-KOH 8 mM sodium succinate, 1 mg/ml PVP, 50 ug/ml BSA and adjusted to pH 6.7. 2Means followed by different letters are significantly different at P=0.05. 3In place of treating medium, but including 8 mM sodium succinate. Table 19. Modified treatment medium.1 ComEonent Concn Mannitol 0.25 E HEPES—KOH 0.01 g Sodium succinate 0.008 g PVP—k30 1.0 mg/ml BSA ' 50.0 ug/ml 1Final solution adjusted to pH 6.7. Figure 14. 110 Time-course of neo-tetrazolium chloride reduction by leaf strips in light and dark. A solid line to the left of a point indi- cates that NTC was present in the medium until the sample was taken. A dashed line to the left of a point indicates that the medium containing NTC was replaced by fresh medium free of NTC at the last point on that line which is conf nected to a solid line. An open circle indi- cates that the staining was done in the light (500 ft—c) from the last point. Conversely, a solid circle indicates that the staining was done in the dark from the last point. NTC REDUCED - A560 9 A560 NTC REDUCED - 111 ——-o LIGHT+NTC --° LIGHT-NTC -—o DARK + NTC ---O DARK- NTC m1 Erlenmej After 30 m1 ment mediul twice and ‘: suffiCientx 0f samples? l4c fixed V h°m°9enizei Davies and 112 preparation for its ability to incorporate l4C into protein from l4C02 was considered a critical test of its viability. Using the standard treatment conditions, 10 no l4C—bicarbonate (NaHl4CO3) was added to each 25 of sodium ml Erlenmeyer flask which was then tightly stoppered. After 30 min in either light (500 ft—c) or dark the treat— ment medium was withdrawn, the leaf strips were washed twice and resuspended, all with 5 ml distilled water. Sufficient HCl was added to adjust the pH to 2. One set of samples was homogenized in 80% (V/v) ethanol and total 14C fixed was determined. A second set of samples was homogenized in 80% (v/v) ethanol and fractionated using Davies and Cocking's (1967) method. High speed centri— fugation (48,000 x g) for 30 min was necessary to separate fractions 8—13 (Table 20). f 14C were fixed in the Considerable quantities 0 light (Table 20). Fractions 1 and 2, representing sugars and amino acids,were most highly labeled. The next largest labeled fraction was the perchloric acid solubilized material representing starch. Fractions l4 and 15, con— taining protein, were also heavily labeled. The low activity recovered from ethanol washes between major fractions showed the various fractions were distinct entities. - ”I Farr i ,l— Fraction number 113 Table 20. Distribution of 14CO in leaf strips after 30 min of fixation in eithef light or dark. Fraction Solvent l4CO2 fixed number __ Light Dark (500 ft—c) (Cpm) (%) (Cpm) PM Total 36,000 100.0 1,000- 100.0 1 80% Ethanol 9,984 27.7 486 48.6 2 80% Ethanol 1,599 4.4 70 7.0 3 20% Trichloroacetic acid 960 2.7 19 1.9 4 20% Trichloroacetic acid 230 0.6 3 0.3 5 95% Ethanol 25 0.1 3 0.3 6 100% Ethanol 22 0.1 2 0.2 7—9 Chloroform, Chloroform- Ether (l:1), Ether 610 1.7 11 1.1 10 1.0 E Perchloric acid 7,428 20.0 165 16.5 11 100% Ethanol 461 1.3 5 0.5 12—13 Ethanol-Ether (1:1), Ether 97 0.3 2 0,2 14 0.25 E Sodium Hydrox— ide 60 C 3,976 11.4 143 14.3 15 1.0 ENaOH 978 2.7 47 4.7 16 Residue 882 2.4 10 1.0 Unaccounted 9.000 25-0 34 3.4 W ...—“9.4 -< were deter Instrument stirrer as water bath a custom-b Johnson, J on a Sarge The e distilled ElmPlificat PrOVided s than 0‘05 114 2. Measurement of Oxygen Exchange Rates Oxygen exchange rates in both light and dark were determined with an oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, Ohio, Model 53) and stirrer assembly connected to a Temp-unit (Oxford Lab) water bath at 25 i 0.1 C. The electrode was attached to a custom—built Johnson polarographic amplifier (George J. Johnson, Jr., Baltimore, Maryland). The output was recorded on a Sargent SR linear 1—100 mv strip chart recorder. The electrode was standardized in air—saturated distilled water and then bucked down, such that 10x amplification of the remaining signal could be used. This provided sufficient sensitivity so that a change of greater than 0.05 ul Oz/min could be detected in 6.0 ml of solu— tion. For measurement of net oxygen production in the light (the Hill reaction coupled to photosynthesis minus respiration), the leaf strips were illuminated (approx. 1000 ft—c) with an American Optical microscope lamp (A.O. 653) at a 7.5 v setting. Data are reported in Table 21 from an initial experi— ment where an oxygen exchange assay was used to measure the effect of infiltration of the medium. Infiltration Significantly reduced oxygen production, but had little effect on dark respiration. Pressure d infiltrati (mm) 760 (cont: 115 Table 21. Effect of infiltration of the treatment medium into leaf strips on oxygen exchange rates in dark and light. Pressure during Net oxygen exchange1 infiltration Dark Light uptake2 production3 (mm) (pl/100 mg dry wt per hr) 760 (control) —240a 234a 500 -240a l47b 350 —251a 106b 100 —243a ll3b W 1Means followed by different letters are significantly different at P=0.05. . 2Negative numbers indicate oxygen consumption. 3Positive numbers indicate oxygen production. of using 1 during NA] 'Ille l outlined studies W1 medium wi‘: each with at pH 6.7‘ 116 I. Interaction of Neo—Tetrazolium and Naphthaleneacetic Acid Experiments were designed to establish the feasibility of using NTC to monitor leaf strip viability simultaneously during NAA uptake experiments. The NAA uptake was studied by using the procedures outlined for hypocotyl studies (Exp. I.C). The uptake studies were terminated by rapidly removing the labeled medium with suction and washing the tissue twice for 5 min each with 5 m1 of 0.01 M phosphate buffer (KZHPO4-KH2PO4) at pH 6.7. The tissue was stored at 4 C (not more than 12 hr), homogenized in xylene, and centrifuged for 10 min at 2000 x g. The supernatant was removed and the reduced NTC measured. The respective xylene solutions were then transferred to 2—inch planchets and dried. The 14C in the xylene supernatant, as well as that in the xylene insoluble material, was determined by the methods described for the hYpocotyl sections (Exp. I.C.). Data transformations and statistical analysis were also identical. As recorded in Table 22, 5x10'6 M NAA had no effect On the amount of NTC reduced. However, NTC markedly reduced NAA accumulation (Table 23). Furthermore, most of the NAA accumulated in the NTC-free treatment was in the xylene insoluble fraction and suggested that it was tightly bound Table 22. 117 Table 22. Time—course of the effect of naphthaleneacetic acid on neo-tetrazolium reduction by leaf strips. Treatment NTC reducedl NAA2 NTC3 Time: 1 hr 3 hr 6 hr (A-560 mm) + ‘ — 0.168d 0.17ld 0.151d - + 0.226c 0.349b 0.478a + + 0.284c 0.393b 0.494a 1Means followed by different letters are significantly different at P=0.05. 2NAA~concn=5xlO'6 M 3NTC concn=0.l mg/ml Table 23. Fraction XYlene so] ‘NTC +NTC is inhi xYlene in: ‘NTC +NTC % inh: 118 Table 23. Time-course of naphthaleneacetic acid (lxlO‘6 M) uptake by leaf strips as affected by neo- tetrazolium chloride. Fraction NAA accumulated1 Time: 1 hr 3 hr . 6 hr (pmoles/10 mg dry wt) Xylene soluble —NTC 265de 358d 611a +NTC 257de 276de 300de % inhibition 3% 22% 51% Xylene insoluble -NTC 481cde 1569b 3086a +NTC 510cde 502cde 737C % inhibition -6% 68% 76% Total uptake "NTC 746C 1927b 3697a +NTC 767C 778C 1037c % inhibition —3% 60% 72% W lMeans within or between the two fractions, and within the totals followed by different letters are Significantly different at P=0.05. results were obtained using the mechanical isolation pro— cedure described by Price (1967). The following modi— fications of the above two approaches, enzymatic and mechanical separations, were made in developing an isolated cell preparation. Simultaneously, cell viability was monitored. A. Methods of Mechanical Isolation of Leaf Cells SOybean plants were grown and the freshly picked leaves washed using procedures described for leaf strips (EXP. II.A). Cell separation was done at 4 C. As dia— grammatically represented in Figure 15, 25 g of the leaflets, Petioles removed, were homogenized by passing through a rOller mill in groups of two or three. The rollers were flushed with 5—10 ml of medium with each group of Figure 15. Schematic diagram of mechanical isolation €£ technique for leaf cells. ical isolation leaflets. The was poure deep with and cell trapped i possible milk filt (Figure 1 small del: omitted j 122 leaflets.l The leaf homogenate collected in a plastic container was poured into a Bfichner funnel (No. 2A), filled 3—4 cm deep with glass beads (3 mm diameter). Almost all tissue and cell clumps larger than one or two cells were usually trapped in the glass beads. An extra filtration step was possible by passing the suspension through a commercial milk filter (15 cm diameter) supported in a Bfichner funnel (Figure 15). This step permitted only single cells and small debris (fragmented cells) to pass, but was usually omitted for it was too time—consuming and many viable cells were lost. The cell suspension, usually slightly less than 600 ml in volume and containing broken cell debris, was then divided into four 150 m1 polycarbonate centrifuge tubes. After 5 min at 75 x g on a horizontal rotor centrifuge (Sorvall, Norwalk, Conn. GLC—I), the debris was carefully decanted and the sedimented cells massed into one 150 m1 centrifuge tube. The cells were resuspended in 100 ml of medium, and washed a minimum of four times using the 1The roller mill was reported by Hulme g; a1. (1964) to be the best means of homogenizing tissue to yield mito— chondria with high respiratory control values. The roller mill's design was described in a personal communication by J. D. Jones to D. R. Dilley of the Michigan State University, Department of Horticulture. same cent clear. ’1 yield) a i A 0. diluted 1 123 same centrifugation procedure until the supernatant was clear. The washed cells were resuspended in medium to yield a final volume of 100 ml. A 0.5 ml sample of the cell suspension was serially diluted loo—fold and the cell density determined using a Spencer bright—line phase-microscopy hemocytometer (Improved Neubauer type). Based on preliminary experiments that established a relationship of 100,000 cells to yield about 1 mg dry weight, the concentrated cell suspension was appropriately diluted to provide a final cell density of 1.0 mg dry weight of cells per m1 unless otherwise noted. For all experiments, four to six 5—ml samples of the final suspension were collected On tared filter disks (2.1 cm No. 554), washed three times with 5 m1 of distilled water, dried at 70 C for 24 hr and weighed to $0.05 mg on the macro—analytical balance. A further modification of the cell separation technique revealed that a given cell population could be partially fractionated by sedimentation at 200 x g_in a linear density gradient of either Ficoll (Figure 16A) or Dextran— 40 (Figure 16B). The fractionation on the Ficoll gradient demonstrated that the population of a small number of the recovered lighter weight mesophyll cells were composed of a high proportion of spongy cells, while the heavier cells Figure 16. 124 Separation of leaf ce gradient. 115 on a Dextran—40 1157. Han] 207’ Fin 31 “em eaf cells. a Ficoll gradient. a Dextran—40 125 Total Clumps Palisade spongy Cells ' 1257, licoll 20 0 31.2 667. 29 0 347. 667: 36 0 397. 61% 89 1 49% 512 283 24 72% 28% 690 68 667. 347. 430 39 797. 217, 241 36 73% 277. 68 18 757. 257. 207. Ficoll 158 37 757. 252 2024 222(112) 707. 30% B Total Fells Clumps Palisade Spongy 57. Dextran 56 l 507. 50% 80 l 527. 48% 110 3 65% 357. 98 4 59% 4l7. 118 6 70% 307. 125 5 667 347. 194 9 667. 347 193 ll 66% 34% 189 18 72% 287, 10% Dextran 3970 650 79% 21% 5183 7080“.) 757» 257 were mai‘ populati cells of 126 were mainly palisade cells. Tabulation of the total population of cells showed the ratio of palisade to spongy cells of about 3:1. B. Refinement of the Mechanical Isolation Technique Approximately 80 min were required to prepare cells by the mechanical isolation method for one replication of leaf material. Therefore, approximately 4 hr of work were required to prepare material for four replications for each day's experiments. As shown in Table 24, when four such leaf isolations were consecutively prepared, they were quite similar as indexed by oxygen exchange. Unless otherwise noted, all oxygen determinations were made by polarography, as described for leaf strips (Exp. II.G.2.). Samples for the time—course measurements (for the oxygen exchange measurements) were withdrawn from the respective treat— ments incubating in the illuminated shaking water bath (Exp. I.C.). The results presented in Table 24 were interpreted to mean that one large cell preparation would be statistically valid as a source of cells to be used in replicated experiments. Accordingly, one common isolation was made for all cells used to establish the usual repli— cations (four) for each treatment. Due to the success with polarographic analysis of OXygen exchange rates, two additional experiments were Table 24. 127 Table 24. Oxygen exchange rates in dark and light of four separate mechanically isolated leaf cell prepara- .tions in time. ' Preparation Net oxygen exchange1 number 1 hr 4 hr 8 hr Dark Light Dark Light Dark Light (pl/100 mg dry wt per hr) 1 —137bc 314a -107c 221a -200ab 301a 2 ~141bc 273a -150b 184a ~220a 255a 3 ~138bc 171a —137c 188a -173ab 198a 4, —1300 127a -lZlC 251a -162abc 164a 1Light means or dark means among all time samples followed by different letters are significantly different at P=0.05. conduct for the sented in seve to the : might i cells. medium ability Incubat Capabil 128 conducted to determine if the treatment medium was Optimum for the isolated cells. Oxygen exchange rates are pre— sented in Table 25 for equal masses of cells resuspended in several different media to examine if certain additions to the media, such as components of tissue culture media, might increase or stabilize the metabolic activity of the cells. The cell preparation incubated in the standard 'medium (Table 19) in the dark showed that its photosynthetic ability did increase with incubation time (Table 25). Incubation in the light further enhanced the photosynthetic capability but the additibn of nutrients severely reduced photosynthesis and dark respiration. The addition of the high molecular weight polymers Dextran—4O and Ficoll had little effect on the system; thus they were not used in further studies. The effect of pH was re—examined. The most dramatic effect of pH occurred after the first 1 hr where cells incubated at pH 6.6 had the highest photosynthetic activity (Table 26). Proceeding from the 3—hr to the 8—hr sample, the activity of all treatments increased with the greatest activity exhibited by the samples held at pH 6.6 and 7.6. At the end of the 8—hr incubation, the pH values of the media were checked. A distinct shift toward pH 6.6 was noted for all treatments. After the pH levels of the Table 25 Medium1 129 Table 25. Comparison of the effect of several media on oxygen exchange rates of mechanically isolated leaf cells in dark and light. :__ _._.______.—___——_.‘—_..___..A_=rw. __.____. __ __ 3:: Medium1 Net oxygen exchange2 Sample time:1 3 hr 5 hr 7 hr 9 hr (ml/100 mg dry wt per hr) dark -184il3 ~184i6 -178125 “213128 13 in dark light 391:22 458t12 550i20 630i108 dark —194i23 ~217i31 —255r8 —294116 13 in light light 55118 626110 7761109 830:54 dark —2oo:1 —188i20 ~150t19 -158t4 2” in light light 269i53 285150 511127 627115 dark -l64i6 ~15118 —93i8 —12216 35 in light light 135114 234:21 283il 69:0 dark -2llie ~250116 ~225i20 —312112 46 in light light 52014 62010 772:40 595:15 1Cells in respective media stored in water bath shaker at 25 C in 250 rpm. A sample of the suspension was tested at the indicated time. 2SE given. 3Medium 1=o.25 M mannitol, 0.01 g HEPES-KOH, 8 mM sodium succinate, 1 mg/ml PVP, 50 ug/ml BSA and adjsuted to pH 6.7. l*Medium 2=Medium l + inorganic nutrients (Murashige + Skoog, 1962). SMedium 3=Medium l and organic nutrients and vitamins (not hormones) (Murashige and Skoog, 1962). 6Medium 4=Medium l and 5 g/100 ml Ficoll + 2.5 g/100 ml Dextran-40. Table 2 ll Sample time (hr) 203 130 Table 26. Effect of pH (0. 01 M HEPES and 0. 01 M MES buffers) on oxygen exchange rates of mechanically isolated leaf cells in dark and light. Net oxygen exchange1 Sample Buffer: MES MES HEPES HEPES HEPES time pH: 5.6 6.6 6.6 7.6 8.6 (hr) (ml/100 mg dry wt per hr) dark -378illl —351i1 ~348i50 —442:lO5 —455i144 1 light —292:38 479il6 325117 216i37 172139 dark V-351i32 -351i21 ~398i66 -322i34 «322:34 3 light 244i54 661183 624128 460:6 460:6 dark -398i79 —470i37 —464i23 —373i27 —360150 6 light 308i32 563i57 481i39 768i31 353i24 dark -425i43 ~568i3l ~614i94 —97li82 —500i122 8 light 289i135 702i124 892i50 926i0 599i22 (5.8)2 (6.9)2 (6.6)2 (7.2)2 (7.9)2 3 dark ~998i75 —lO75i4l —1l72illl —lO74121 —8211110 20 light -393i32 ~403ill ~354illl -642i116 -576i67 (0.901“ [1.401“ [1.501” [1.081“ [0.14014 lSE given. 2Final pH of medium had shifted to indicated pH. 3Cell suspensions were washed once in fresh media con— taining the respective buffers at the appropriate pH values. ”Absorbance of 560 nm of 0.10 mg/ml NTC reduced (1 hr) by the leaf cells in the respective treatments. respecti' incubate buffer a at the 2 dark at contamin showed a the abil 0f reduc 11110 the 131 respective treatments were readjusted, the cells were incubated an additional 12 hr. Only the sample in HEPES buffer at pH 6.6 maintained a high photosynthetic activity at the 20—hr sampling. The high oxygen consumption in the dark at the 20-hr sampling Was likely due to bacterial contamination. An important secondary experiment on the 8—hr sample showed a definite similarity between oxygen production and ‘ the ability of the cells to reduce NTC. The high values of reduced NTC reflect the ease of penetration of NTC into the cells. C. Comparisons of Mechanically Isolated Cells to Leaf Strips A simultaneous multiple comparison of a single preparation of the mechanically isolated leaf cells with leaf strips (4 replicates) was conducted, by starting with identical leaf material. All treatments were made in 8 m1 (final volume) in 25 ml Erlenmeyer flasks, with the exception for the 02 uptake determinations. The latter measurements were followed manometrically as described for the hypocotyl sections (Exp. I.E.l). NTC (0.1 mg/ml), based on the results in Table 23, was used as an inhibitor in the uptake studies. The techniques and conditions for NAA uptake were similar to those used for the hypocotyl sections (Exp. I.C.), except the celI medium 1 medium 1 phOSpha1 the same transfel 132 the cell suspensions were separated from the labeled medium by centrifugation at 500 x g for 5 min. After the medium was decanted, the cells were washed twice in 5 m1 of phosphate buffer (KH2PO4—K2HPO4, 0.01 M) at pH 6.7 using the same centrifugation system. The washed cells were transferred to 2-inch planchets and radioactivity determined. Phosphorus uptake was followed in the same manner as for l4C-NAA. Carrier—free H332PO4 (Tracerlab, Waltham, Mass.) was added to K2HPO4 to give a final concentration of 5x10“5 M and approximately 0.1 uc 32? per treatment. Exact calibration of the specific activity was determined simultaneously with the rest of the uptake portion of the experiment. l4C02 fixation was carried out as previously described for the leaf strips (Exp. II.4.1.), except 1 uc/flask of NaHl4CO3 (S.A. 2.0 uc/umole) was added. Total l4C02 fixed was measured. NTC reduction was determined on the NTC treatment used to inhibit the NAA uptake. After the cells were washed, the absorbance of the supernatant of the xylene homogenate from the treated tissue was measured at 560 nm. The supernatant was then added to the respective planchets already containing the residue of the xylene extraction. As detailed in Table 27, the leaf strips accumulated Table 27 Treatmel 0 upta] -NTC +NTC5 -NTC +NTC5 me5 re +NTC5 P046 Up ‘NTC +NTcs 133 Table 27. Comparison of leaf strips to mechanically isolated leaf cells by oxygen uptake, naph— thaleneacetic acid uptake, neo-tetrazolium reduction, phosphorus uptake and 14C09 fixation. Treatment Leaf stripsl Mechanically1 isolated cells 02 uptake2 ( N ) 10 mg dry wt/hr ~NTC 352a 167C +NTC5 297b 91d NAA 3 uptake " -NTC +NTC5 NTC5 reduced” +NTC5 P046 uptake” -NTC +NTC5 l4c027 fixed -NTC pmoles ) (10 mg dry wt 2432a 640C 1408b 451C (thaw-a???) 0.415b ( nmoles 1.097a 10 mg dry wt) 72.8a 6.6c 17.5b 4.2c (__L__.. ) 10 mg dry wt 7106b 8594a 1Treatment means within a determination followed by different letters are significantly different at P=0.05. 2Mean of five l-hr readings at 30 C measured mano— metrically. 3NAA concn=lx10_6 M. ”3-hr treatment under 500 ft-c light. 5NTC concn=0.l m 6PO concn=5x10‘ 71/2-hr treatment under 500 ft-c light, 1 uc/flask l4C02. g/ml. signifi the NTC strips was not the cel NTC tha Th mechani not exh Enzymat aPProac ‘—'—_'l 134 significantly more NAA and PO4‘, especially when using the NTC inhibition as a base line for the system. The strips also had a higher respiration rate though the NTC was not as inhibitory to this determination. Interestingly, the cells fixed significantly more 14C02 and reduced more NTC than did a comparable weight of leaf strips. D. Method of Enzymatic Cell Isolation The last sequence of experiments demonstrated that mechanical leaf cell isolation might be practical, but did not exhibit metabolic activity comparable to intact tissue. Enzymatic separation was then evaluated as an alternate approach. Growing and plant preparation procedures were similar to those described for leaf strips (Exp. II.A.). Approxi— mately 15 leaflets at a time were stacked, rolled on their longitudinal axis and thinly sliced (3—4 mm) with a hand— held razor blade. The stack of leaf slices was then cut in the opposite direction and yielded tissue pieces about 4 mm x 4 mm in size. After a sufficient amount (60 to 80 g) of leaves were sliced, the tissue pieces were thoroughly washed with treating medium (Table 19) until all signs of broken cells were removed. The tissue pieces were then placed in ten times their fresh weight (600 to 800 ml) of fresh medium containing 1.0% (w/v) Macerozyme (a crude prepara' Gosha L- peptone rotor k: Sorvall a voltal covered The con 0.5 C) (photo- water j the cel filtrat 4_———fl 135 preparation of endo—polygalacturonase donated by Kanematsa— Gosha Ltd., Tokyo, Japan) unless otherwise noted and 0.5% peptone. This suspension was stirred at 300 rpm with a rotor knife assembly driven by an Omni—mixer motor (I. Sorvall, Norwalk, Conn.) with its power controlled through a voltage regulator (33 v need). The knife blades were covered with latex tubing to minimize cutting of the tissue. The container was partially submerged in a water bath (25 i 0.5 C) illuminated with 1000 ft—c of incandescent light (photo-flood 300W lamps cooled in a plexiglass walled water jacket mounted over the water bath). After 5 hr, the cells were recovered from the tissue suspension by filtration and centrifugation as previously described (Exp. III.A.). Suspension density was established by using a hemo— cytometer and also by weighing aliquots on tared filter disks. The weighing method proved to be a more reliable method to estimate final weights of cell suspensions isolated enzymatically, since variable amounts of cuticle pieces were present, that affected the final weight but were not accounted for in the counting procedure. The initial weights determined after oven drying for 1 hr at 70 C were used to calculate the appropriate cell density dilutions to yield 1 mg dry weight/ml. Final cell density 24 hr a 0F the oxy (Macerc yield 5 respira whereas cell de the lin TV were re n 136 density determinations were made on aliquots dried for 24 hr at 70 C from the diluted cell suspensions. Optimum cell to medium ratio was determined using the oxygen electrode. A uniform batch of enzymatically (Macerozyme) isolated cells was diluted sequentially to yield several different population densities. Dark respiration was independent of cell density (Table 28), whereas light driven oxygen production was optimum at a cell density of 1.0 mg dry weight/ml or less. Light was the limiting factor for the optimum oxygen production. Two, apparently different, pectinase preparations were reported to have been successfully used to isolate tobacco leaf cells. Jyung g: l. (1965b) used pectinase from Nutritional Biochemical Corp. This enzyme had a pH Optimum of 4.5 and was contaminated with several other hydrolytic enzymes not fully characterized (personal communication from Nutritional Biochemical Corp., Cleveland, Ohio). Takebe g3 El. (1968) used Macerozyme, a fungal endo-polygalacturonase preparation with pH Optima of 5.0 and 6.5. Macerozyme was also contaminated with several hydrolytic enzymes, which have been characterized by Suzuki e_t__l. (1967a, 1967b). While separate lots of leaflets were being treated with the two respective enzymes, a third group of leaflets 137 Table 28. Effect of cell density of enzymatically isolated leaf cells on oxygen exchange rates in dark and light. Cell densityl Net oxygen exchange2 Dark Light (mg dry wt/ml) (pl/100 mg dry wt/hr) 0.5 —209a 868a 1.0 ~212a 830a 1.8 ~207a 740a 2.7 -205a 569b 5.3 —229a 307C 11.0 mg dry wt contained approximately 40,000 cells. 2Means within a column followed by different letters are significantly different at P=0.05. was use isolate lations Th isolate method (Table Maceroz respira the isc more ac continu 138 was used for the mechanical isolation of cells. The isolated cells were washed and appropriate cell popu— 1ations established by the methods previously described. There was a dramatic difference between the mechanically isolated cells and those isolated by either enzymatic method for their ability to produce oxygen in the light (Table 29). This measurement also established that Macerozyme was slightly better than pectinase. Dark respiration measurements showed little difference among the isolation methods. Because Macerozyme provided slightly more active cells, it was selected over pectinase for continued evaluation. E. Comparisons Among Mechanically and Enzymatically Isolated Leaf Cells and Leaf Strips-Metabolic Studies. Since the enzymatically isolated cells seemed to be photosynthetically more active than those isolated by the mechanical procedure, a more complete comparison including leaf strips was performed. To cover a broad spectrum of assays, the comparisons were done over a period of 3 days, each being indicated by an experiment number. For the purpose of continuity, polarographic analyses of oxygen exchange rates were measured on all three days (Experiments 1 to 3). At the start of each experiment a common source Of leaf material was utilized, and the experiment was 139 Table 29. Time—course of oxygen exchange rates of several different types of leaf cell preparations in dark and light. Preparation , Time1 Net oxygen exchange2 Dark Light (hr) (HI/100 mg dry wt/hr) Mechanically isolated 1 267130 267110 8 309151 77119 10 325136 -238133 243 216116 —103120 Enzymatically isolated (Macerozyme) 1 44411 730113 8 396130 960128 10 497119 830156 20 4791110 5671120 243 396164 -l810 Enzymatically isolated (pectinase) 1 433121 1001172 8 5781100 7841227 10 361111 598142 20 227121 258141 243 366125 64139 W 1Samples incubated in water bath shaker 25 C, 250 rpm, 500 ft—c light. 2SE given. 3Sample stored at 5 C in dark, not stirred. complet Me and pht respira interme highest m-chlox 140 completed on the day of cell isolation. Mechanically isolated cells had lower dark respiration and phOtosynthetic rates than leaf strips (Table 30). Dark respiration rates of enzymatically isolated cells were intermediate, while their photosynthetic capacity was highest. Cells from all three could be uncoupled with m-chloro—carbonyl cyanide phenylhydrazone (Cl—CCP). The effect of Cl—CCP could be observed almost instantaneously. l4C02 fixation was studied using the techniques previously described (Exp. II.H.1.). The experiment was repeated twice, once 2 hr after each tissue preparation and again after 24 hr. Carbon fixation by leaf strips and enzymatically isolated cells was not significantly different at either sampling time, though both lost efficiency with time (Table 31). The mechanically isolated cells had about half the capacity of the strips for the 2—hr sample and then dramatically lost their capacity during the storage period. A parallel set of samples was labeled similarly with 14C02 at the 2-hr measurement. The experiment was terminated by the addition of dilute HCl and the tissue was immediately frozen. The tissue was fractionated by placing leaf strips (homogenized in 80% [v/v] ethanol) and the isolated cells on respective teflon membrane filters (5 um pore size, Leaf st Mechani isolat 141 Table 30. Comparison by oxygen exchange rates in dark and light of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 1). Tissue Sample Net oxygen exchangel ______ preparation time Dark Light Dark+ Light+ Cl-CCP2 C1-CCP2 (hr) (pl/100 mg dry wt/hr) Leaf strips 1 —315150 206116 —524 -lll 2 ~333131 182124 —560 -56 4 —32518 17511 —452 ~79 6 -397132 12711 —532 ~79 243 —32011 7118 -——— -——— Mechanically isolated cells 1 —llO12 18312 —207 —l74 2 —lll16 107110 —174 ~152 4 ~10414 8412 —118 -l36 6 —105110 8416 —174 —119 243 ~112121 2711 —146 —108 Enzymatically isolated cells 1 -l94114 117117 —296 —l9l 2 —l751l3 479149 —296 -181 4 ~224122 25318.5 -3l8 -212 6 —235115 35712 -365 -232 243 ~20912 273110 -222 —l48 lSE given. 2leO‘6 M m—Cl—CCP added 1 min before measurement. _ 3Only sample stored at 5 C in dark; both cell prepara— tions were stirred. Table 3 142 Table 31. Comparison by 14C02 fixation of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 1). l4 Tissue preparation CO2 fixed“2 Time after preparation: 2 hr 24 hr Change in time (cpm/lO mg dry wt) Leaf strips 5809a 3683bc —35% Mechanically isolated cells 2920d 407e —86% Enzymatically isolated cells 5460a 4610b —15% 1Means followed by different letters are significantly different at P=0.05. 230—min fixation period, 1.0 uc/flask, SA 2.0 uc/umole. 45 mm d: solvent: NaOH so. proport: given iv tissue ; followe. l4C02 i: less th. cells. Us for the Strips folIOWe inhibit 143 45 mm diam. Millipore Co.) and stepwise extracted with the solvents listed in Table 20 (10 ml each), except for the NaOH solutions. The 14C in the residue was determined by proportional planchet counting. Net recovered counts are given in Table 32 based on initial weight of the respective tissue preparations. The leaf strips were most efficient followed by the enzymatically isolated cells in incorporating l4C02 into protein. The mechanically isolated cells were less than half as efficient as the enzymatically isolated cells. Using the same experimental technigues described for the comparison of mechanically isolated cells to leaf strips (Exp. III.C.), NAA uptake and NTC reduction were followed for the three cell preparations. NTC significantly inhibited NAA uptake by leaf strips, but dramatically enhanced accumulation by either isolated cell preparation, particularly the mechanically isolated cells (Table 33). Extraction of reduced NTC from isolated cells was found to be more difficult than from leaf strips. By first extracting the tissue with 100% ethanol to remove plant pigments, acetone effectively extracted the reduced NTC. Unlike acetone's reductive effect on TTC, NTC is not reduced by this solvent, though the reduced NTC becomes unstable in light. Therefore, the acetone extracts were Table 3 Tissue Leaf st 144 Table 32. Comparison by 14C02 incorporation into protein of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 1).1 Tissue preparation 14C in protein fractionz'3 (cpm/lO mg dry wt) Leaf strips 1550a Mechanically isolated cells 465C Enzymatically isolated cells 1075b 1Parallel sample of 2 hr l4CO fixation study—Table 31. 2Means followed by different letters are significantly different at P=0.05. 3Protein fraction represents material left on filter after separate washing with solvents listed in Table 20. 145 Table 33. Comparison by naphthaleneacetic acid uptake and neo-tetrazolium chloride reduction of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 1). Tissue NAA NTC preparation accumulated”2 reduced“3 (pmoles/10 mg (A—560 nm dry wt) 10 mg dry wt) Leaf strips —NTC 899C 0.085d +NTC3 387d 0.155b net change 0.070 Mechanically isolated cells —NTC 303d 0.112c +NTC3 1940a 0.279a net change 0.167 Enzymatically isolated cells —NTC 328d 0.070d +NTC3 1262b 0.158b net change 0.088 1Means within a column followed by different letters are significantly different at P=0.05. 2NAA concn=5xlO"6 M. 3NTC concn=0.0l mg/ml. stored at 560 two cel isolate leaf ti 146 stored in the dark until the NTC absorbance was measured at 560 nm. As found with the NAA uptake (Table 33), the two cell preparations, particularly the mechanically isolated cells, reduced more NTC than an equal quantity of leaf tissue. Chlorophyll content was determined by the method of Arnon (1949). Protein was measured by micro—Kjeldahl digestion of acetone—extracted residue followed by steam distillation into boric acid and subsequent titration (Hawk, 1965). Analysis of chlorophyll and protein contents (Table 34) indicated that, on a weight basis, decreasing quantities of chlorophyll and protein were found in the mechanically isolated cells followed by enzymatically isolated cells and then the leaf strips, respectively. By contrast, chlorophyll content was less in isolated cells than in leaf strips when expressed in terms of protein present (Table 34). Polarographic analysis in Experiment 2 (Table 35) indicated the results in Experiment 2 were similar to those Of Experiment 1 (Table 29), except that the level of activity for the mechanically isolated cells was lower even though it was measured at the beginning of the experiment. Using the same techniques of the experiment described for data in Table 27, 32PO4_ uptake was examined. Both NTC 147 Table 34. Comparison by measurement of chlorophyll and pro- tein content of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 1). ' Tissue Chlorophyll1 Protein1 Chlorophylll'z preparation protein (us/10 mg (mg/10 mg (pg/mg) dry wt) dry wt) Leaf strips 47.7c 1.61c 29.8a Mechanically isolated cells 73.8a 2.96a 24.9b Enzymatically isolated cells 55.5b 2.15b 25.6b 1Means within a column followed by different letters are significantly different at P=0.05. 2Chlorophyll per unit of protein. 148 Table 35. Comparison by oxygen exchange rates in dark and light of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 2). Tissue preparation Net oxygen exchange“2 Dark Light (ml/100 mg dry wt/hr) Leaf strips -315122 172120 Mechanically isolated cells ~93118 -44114 Enzymatically isolated cells -l70125 287122 1SE given. 2Tissue analyzed 2 hr after preparation. and C1 the no signif uptake was ab approx cells. 149 and Cl-CCP were used as inhibitors in conjunction with the non—treated control. The inhibitors reduced uptake significantly for all tissue preparations (Table 36). The uptake in the non-treated control for the leaf strips was about twice that of enzymatically isolated cells and approximately 20 times that of the mechanically isolated cells. The measurement of catabolism of a key metabolite was used as another criterion for tissue viability. Meta- 14C was followed at 25 C in the dark in bolism of acetate— a radiorespirometer as described by wang (1962). Millipore filtered (0.45 um pore size) air was pumped through each flask at 60 cc/min to transfer the liberated l4C02 into the trapping solution of ethanol—ethanolamine (1:1 v/v). Acetate—2—14C was added to the tissue preparations after a 1/2—hr pretreatment. Hourly samples were collected after the first lS—min sample was discarded. 14C was determined on the collected samples, properly transferred and diluted, with a liquid scintillation counter (4g BBOT/l toluene). Counting efficiency was determined by internal standardi— zation with l4C—toluene. The l4C02 evolution data (Table 37) showed that leaf strips and enzymatically isolated cells had similar capacities to metabolize acetate while mechanically isolated cells were significantly less active. The rate of Table Tissue 150 Table 36. Comparison by phosphorus uptake of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 2). Tissue preparation P02” accumulated"2 Non- NTC3 Cl-CCP“ treated (nmoles/10 mg dry wt) Leaf strips 51.43a 1.980 17.15b Mechanically isolated cells 1.84a . 0.80c 1.31b Enzymatically isolated cells 21.9a 1.59b 1.59b 1Means within a row followed by different letters are significangly different at P=0.05. 2P04 ’ concn=5x10‘5 M. 3NTC concn=0.l mg/ml. “Cl—CCP concn=1x10'6 M. Table 37. 151 Table 37. Comparison by acetate-2—14C catabolism of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 2). Tissue preparation l4CO2 liberatedlr2 Sample time: lst hr 2nd hr 3rd hr (cpm/lO mg dry wt/hr) Leaf strips 3799c 6649b 9706a Mechanically isolated cells 749d 2185cd 2758c Enzymatically isolated cells 3500c 5720b 9460a lMeans followed by different letters are significantly different at P=0.05. 2Acetate concn=0.l mM, specific activity 1.0 uc/lOO nmoles. catabolism Becaus activity (E (Tables 24; of tissue p isolated ce activity. F. Com Isc Stu _——————————--IIIIIIIIIIIIIIIlIlllIIIII-IIIIIIIIIIIIIIIIIIFT 152 catabolism increased with time for all preparations. Because the mechanically isolated cells had very low activity (Experiment 2) compared to earlier experiments (Tables 24, 25), a further comparison on a third set of tissue preparations was carried out. The mechanically isolated cells again had (Table 38, Table 39) very low activity. F. Comparisons Among Mechanically and Enzymatically Isolated Leaf Cells and Leaf Strips—Microscopic Studies. 1. Light Microscope Studies A Wild M20 research microscope, equipped with both bright field and phase contrast objective lens, was used. Photomicrographs (35 mm) were taken with the aid of a Wild photoautomatic exposure control unit. A leaf strip (50 pm) is shown in cross—section in Figure 17. Two rows of tightly packed palisade cells stand over the more loosely arranged spongy mesophyll cells. The highly vacuolated epidermal cells are banded on the leaf exterior by a cuticle. Though not shown in this photograph, the leaf strips, used for experimental purposes, contained several vascular bundles. The surface of the leaf was characterized by a large number of trichomes. Typical examples of mechanically isolated leaf cells are shown in oil phase contrast photomicrographs of Figure 18. Table 38. Tissue prep Leaf strips Mechanical 1‘ Enzymatical \ 15E 91 Table 39 . 153 Table 38. Comparison by oxygen exchange rates of leaf strips and cells isolated by either mechanical or enzymatic procedures (Experiment 3). ( Tissue preparation Net oxygen exchange1 Dark Light (ul/lOO mg dry wt/hr) Leaf strips -362172 175127 Mechanically isolated cells ~125118 80137 Enzymatically isolated cells -309134 610170 1SE given. Table 39. Comparison by phosphorus uptake of leaf strips and ‘ cells isolated either by mechanical or enzymatic procedures (Experiment 3). Tissue preparation Phosphorus accumulated1 Non- NTC2 Cl—CCP3 treated (nmole/lO mg dry wt) Leaf strips 27.72a 1.48b 2.99b Mechanically isolated cells 3.47a 0.80b 0.53b Enzymatically isolated cells 8.7la 2.35b 2.77b 1Means within a row followed by different letters are Significantly different at P=0.05. 2NTC concn=0.l mg/ml. 3Cl-CCP concn=lx10~6 M. 154 Figure 17. Photomicrograph of a cross—section of a leaf strip (50 mm thick) viewed under bright light- 155 section of a leaf under bright light- ' Figure 18. 156 Mechanically isolated cells. All photomicrographs taken with oil phase contrast. A. Spongy mesophyll cell. Note distinct grana within chloroplasts. B. Palisade mesophyll cell. Note microbodies and mitochondria. C. Palisade mesophyll cell—focused on vacuole. D. Palisade mesophyll cell of C focused on plane between chloroplasts and plasma membrane. Cell size VE cells to as cells were : were highly (4 to 5 um .‘ With critic; occasionallj were isolat sun, starch within the seen in mos bOdies (Fre 158 Cell size varied from about 20x20 um for spongy mesophyll cells to as large as 20x50 pm for palisade cells. Epidermal cells Were seldom recovered by this technique. All cells were highly vacuolated with saucer—shaped chloroplasts (4 to 5 pm in diameter) oriented along the cell membrane. With critical focusing, the grana within the chlorOplast occasionally became distinct (Figure 18A). When cells were isolated from leaves harvested after 6 hr of natural sun, starch gains (not shown) were also readily apparent within the chloroplasts. Distinct small bodies can be seen in most of the isolated cells. They could be micro— bodies (Fredrick and NeWComb, 1969), spherosomes, peroxi— somes and/or mitochondria. They appeared dark or opaque (Figure 18B) and most often were observed in cytoplasm between the cell membrane and the vacuole. This is illus- trated in the photomicrographs (Figures 18C,D) of the same cell focused on the central vacuole side of the chloro— plasts and on the plasma membrane side of the chloroplasts, respectively. It should be stressed that not all mechanically isolated cells were as intact as those illustrated in Figure 18. About 45% of the cells showed some degree of disruption. A representative sample of enzymatically isolated cells is shown in Figure 19A. The general description 159 Figure 19. Enzymatically isolated cells. All photomicrographs taken from bright field. I. A. Low magnification of typical cell popula- ( tion. Note pieces of cuticle (C). B.‘ High magnification of viable palisade mesophyll cell. i C. High magnification image of damaged SPONSY mesophyll cell. . l i 4 i : of the cel isolated c appeared t present in that appea plasm was enzymatica 161 of the cells is very similar to that of the mechanically isolated cells. The only difference between preparations appeared to be a larger number of cuticular fragments present in the enzymatically isolated preparation. Cells that appear darker were considered dead since their proto— plasm was coagulated (Figure 19C). A typical living enzymatically isolated cell is illustrated in Figure 19B. Cytoplasmic streaming was observed in many of the highly vacuolated cells, that had distinct chloroplasts, isolated by either enzymatic or mechanical procedures. Only Brownian movement could be seen in cells where the proto— plasm was coagulated. Protoplasmic streaming is portrayed by the sequence of photomicrographs in Figure 20. The organelles often move in a slow short zig-zag pattern, but they definitely follow distinct paths that can be followed during a period of several minutes. Their movement appeared to be restricted to areas between the chloroplasts and the plasma membrane. These microbodies were not seen in the vacuole. Only by photographic comparisons can the chloro— plasts be Observed to be moving. None of the organelles were found going through pleomorphic changes in structure. 2. Electron Microscope Studies Cells obtained by various tissue preparations were further examined using a Hitachi HU—ll electron microscope Figure 20. 162 ymatically isolated Cytoplasmic streaming in enz tomicrographs. cells as shown by time—sequence pho re taken approxi- Bright field photomicrographs we which was taken mately every 3 min except for F 30 min after E. ollow its point to see ' here microbodies and pOSSlbly The organelles seem mitochondria are moving. D: to follow a discreet path as shown in B, C, E. In time even the chloroplasts show some movement. Note chloroplast in F. 163 (1 ( ( operated at Tissue sampl described by modification dehydration (L K B ultra stain (5 mir of mechanics It can only light micro: Electr< of represen' Strips cell; electron mi. Trains pres 164 operated at an electron accelerating voltage of 75 kV. Tissue samples were fixed and imbedded in Epon 812 as described by Valdovinos 2p g;. (1968) with the slight modification of uranyl staining during the ethanol dehydration sequence. Sections were cut on a glass knife (L K B ultramicrotome). Lead citrate was used as a final stain (5 min). Through an unfortunate error, the samples of mechanically isolated cells were irrevocably damaged. It can only be assumed that the similarities noted in the light microscope work extend to the fine structure level. Electron—photomicrographs (taken on Kodak EIP Plates) of representative enzymatically isolated cells and leaf strips cells revealed a great deal of similarity. The electron microscope study revealed a high number of starch grains present in cells from both preparations. Furthermore, the organelles observed streaming in the cells with the light microscope were likely both microbodies (spheresomes, lysosomes or peroxisomes) (Frederick and Newcomb, 1969) and mitochondria. The l um diameter of the mitochondria noted in Figure 21A means it was just at the limit of resolution Of the light microscope. The mitochondria found in the leaf strips (Figure 21B) had several forms. Higher magnification of other viable enzymatically isolated cells (Figure 22) showed the ultrastructure of the Figure 21. 165 Electron micrograph of an enzymatically isolated cell compared to a cell in a leaf strip. A. Enzymatically isolated cell. Examples of a starch grain (S) within a chloroplast, mitochondria (Mw) and microbody (Mb) are indicated by arrows. B. Leaf strip cell. Examples of a starch grain (S) within a chloroplast and mitochondria are shown with arrows. enzymatically isolated a leaf strip. cell. Examples of bin a chloroplast. microbody (Mb) are f a starch gran ples o dria st and mitochon Figure 22. 167 Ultrastructure of viable enzymatically isolated cells. A. Enzymatically isolated cell. Note distinct grana within chloroplasts, mitochondria Uhfl and microbody (Mb). The partially hydro— lyzed cell wall (Cw) is also shown. B. Enzymatically isolated cell. Cell wall (CW) is indicated. various 0 wall of s (Figure 2 nations, < the ultra. isolated . plasts wi‘ randomly 1 G. F. f( 169 various organelles to have distinct membranes. The cell wall of some of the cells appeared to be partially hydrolyzed (Figure 22A). As found in the light microscopic exami- nations, damaged or disrupted cells were also apparent at the ultrastructure level. Typically damaged enzymatically isolated cells (Figure 23) were characterized by chloro— plasts with ruptured membranes. Starch grains were found randomly distributed within the cells. G. Final Modification of Enzymatic Isolation Procedure for Leaf Cells The several multiple comparisons of tissue prepara— tions all indicated that both cell isolation procedures resulted in tissue of considerably lower viability than that of the leaf strips. However, cells isolated by the enzymatic technique were of sufficient activity and stability to warrant a further refinement of the method and evaluation of their usefulness for uptake studies. Because of the time required for cell isolation, an experiment was designed to determine if cells isolated the evening before could be used the following day for experi— mental studies. The isolated cells had good activity that remained relatively constant for the 6—hr time period in which they were studied (Table 40). Because of this success, all the following enzymatic isolations were performed the 170 Ultrastructure of damaged enzymatically isolated cells. Figure 23. A. Damaged enzymatically isolated cell. B. Another example of damaged enzymatically isolated cell. Table 40. Time at 2 (hr) 1/2 172 Table 40. Analysis of stability of oxygen exchange rates of enzymatically isolated cells in dark and light. Time at 25 C1 Net oxygen exchange2 Dark Light (hr) (pl/100 mg dry wt/hr) 0 ~-258135 275125 1/2 I —26719 250143 1 —335135 27511 2 -327118 241135 3 —l961ll 24111 4 —21519 18119 5 —284126 207117 6 ~2931l7 25019 W 1Cells isolated 12 hr earlier, stored fully diluted on stirrer in dark at 5 C. At the end of the storage period the cells were washed twice in filter sterilized medium and then transferred at 0 hr to 25 C at 200 ft-c light on a stirrer. Samples measured for 02 exchanged at indicated times. 2SE given. evening be Iv. Uptake Strips Leaf previously used for e listed in per 25 ml same as th except the 16 110/ umol unless ott and the ne finalized leaf strip ethanol We quantitatj by leaf 51 during the 173 evening before initiating experiments. IV. Uptake and Metabolism of Na phthaleneacetic Acid by Leaf Strips Leaf strips (250 um) were prepared in the same manner as previously described (Exp. II.B) and 48 such strips were used for each replicated treatment. The treatment medium listed in Table 19 was made up to a final volume of 5 ml per 25 ml Erlenmeyer flask. Uptake conditions were the same as those used for the hypocotyl sections (Exp. I.C.) except the NAA concentration was 1x10"6 M, specific activity 16 uc/umole and the uptake period was extended to 4 hr unless otherwise noted. The experiments were terminated and the necessary procedures were used to produce the finalized data as described for the viability studies with leaf strips (Exp. II.I). The only exception was that 100% ethanol was used in place of xylene and the homogenate was quantitatively transferred to 2—inch planchets. A. General Parameters of Naphthaleneacetic Acid Uptake l. Time—Course An extended time—course of NAA (lxlO‘6 fl) uptake by leaf strips is reported in Figure 24. Frequent samplings during the early portion of the time study (Figure 24A) revealed an initial 1.5 hr low rate of uptake that was then Figure 24. Time-course of naphthaleneacetic acid uptake by leaf strips in the light. A. First 6.0 hr of uptake. B. Complete 24—hr time-cours (frozen—thawed) check at e. Dead—control 12 hr accumulated 51 pmoles NAA/10 mg dry wt. 2800 3 2400 x K I: Q 2000 s 9 \ |600 U) a 0 s 1200 Q I m x 800 E a 400 o 7000 6000 %00 UPTAKE —pma/os//0 my dry Art on O O o I UPTAKE-pmoles //0 mg dry W 2800 N A O o 2000 l600 |200 800 400 7000 6000 8 8 01 O O O N O O O 175 I2 TIME-hr 16 20 24 followed 1 high rate 12 hr and terminate of the hi1 maintainel experimen prepared ‘ concentra 176 followed by a surge—like higher rate of accumulation. The high rate of uptake dropped off to a much lower rate by 12 hr and continued until 24 hr when the experiment was terminated. 2. Effect of Naphthaleneacetic Acid Concentration The 4-hr time was chosen to establish the nature of the high rate of uptake, and the light intensity was maintained at 800 ft—c for this and all subsequent experiments. NAA concentrations, less than lxlO—5 g, were prepared by dilution of the l4C-NAA stock solution. For concentration of 1x10‘5 M to 5x10‘4 M, l4C—NAA was mixed with non—labeled NAA to yield solutions of known specific activity and thereby maintaining a relatively constant level of l4C—NAA for this concentration range. Comparable tissue pretreated for 30 min at 60 C served as a dead control. 14C content of dead tissue was linear with increasing NAA concentration (Figure 25), while in the non—treated tissue, there was a slight jump in rate of accumulation at about 5xlO‘7 fl and then dropped off as the concentration was further increased. It is important to note that the level of NAA in the non—treated tissue is larger, by an order of magnitude, than that of the heated tissue. The results illustrated in Figure 25 can be transformed mathematically into values here termed accumulation ratiOS Figure 25. 177 Effect of naphthaleneacetic acid concentration on its own uptake by leaf strips. 3 Leaf strips were either non—treated or . heated to 60 C for 30 min prior to the experi— ment. UPTAKE-pmo/as/IO mg dry wt 1‘ 178 ic acid concentratlon strips. er non-treated or \ prior to the exp HEATED: 60 c UPTAKE-pma/as/IO mg dry wt lo- 8 '0'? I‘D-'6 '0-5 '0-4 '0.3 CONCENTRATION _ M which ca concentr concentr The fina forming weight) the dry of the t concentr 0f accuIr tent (8C sample c —————————---IIIIIIIIIIlllllIlIllIIIllllllllllllllllllllgfipf' 179 which can help derive the rate of uptake for each treatment concentration. The accumulation ratio is the final internal concentration divided by the initial external concentration. The final internal concentration was determined by trans— forming the accumulated value of NAA (pmoles/10 mg dry weight) into a concentration value based on the fact the dry weight of the leaf strip represented 20.1% (w/w) of the tissue's fresh weight. Therefore, the internal NAA concentration was derived by dividing the number of moles of accumulated NAA by the volume of the tissue water con— tent (80% fresh weight). See Figure A—l (Appendix) for a sample calculation. The NAA accumulation ratios (Table 41) by the non— treated leaf strips showed almost a doubling in rate, going from 1x10"7 M to 5x10"7 M external NAA concentration. As the external concentration was raised to 5x10‘5 g, the rate of uptake dramatically dropped, but it did not exhibit saturation even at 5x10"4 fl NAA. The accumulation ratios of the heated tissue revealed a lower rate of sorption that was relatively independent of concentration. 3. Loss of Accumulated Naphthaleneacetic Acid from Tissue and Determination of Free Space The amount of accumulated NAA freely lost to fresh media is reported in Table 42. Rapid successive washes of Table 41 Treating 1x10‘8 M 5x10“8 [LS 1x10‘7 [3 5le7 M lxlO'6 M 5X10‘6 M 180 Table 41. Effect of naphthaleneacetic acid concentration on its own uptake by leaf strips. Treating concn 1x10“8 5 5x10‘8 3 ixio‘7 g 5x10'7 M 1x10“6 M 5x10‘6 M 1x10"5 fl 5xio"5 I: leO—4 M 5x10‘4 g Accumulation ratiol’ Non—treated 22.5:4.2 20.0i2.9 23.0:2.7 40.0i2.8 46.714.2 37.0t3.3 35.0:4.5 15.8i0.6 12.7i1.2 10.8i0.7 2 Heated to 60 C3 1.25i0.02 0.88i0.25 0.75i0.08 0.60i0.02 0.53i0.02 0.50:0.02 0.5510.02 0.54t0.02 O.55i0.03 0.72i0.05 1SE given. 2Calculated final internal NAA concn/initial external concn. Actual per cent of NAA removed from medium=80% of accumulation ratio values. 330—min pretreatment. (KH Sig] 181 Table 42. Effect of successive washings (2 min) on the amount of naphthaleneacetic acid retained by leaf strips after naphthaleneacetic acid1 uptake. Wash numberz’3 NAA retained” (pmoles/10 mg dry wt) 0 976a l 910ab 2 738C 3 788bc 4 698C 5 737C 1NAA concn=lxlO_6 M. 2Wash medium=0.0l fl phosphate buffer (KH2P94-I'K2HPO4) pH 6.6. . Time for each wash=2 min. l"Means followed by different letters are significantly different at P=0.05. tissue S-ml wa not fir all lea to 2361 Th! compoun( assessi Results treating labeling measuren 182 tissue preloaded with NAA indicated that a minimum of two S—ml washes was required to remove that fraction of NAA not firmly bound by the tissue. Two washes were used for all leaf strip experiments. The fraction removed was equal to 236 pmoles/10 mg dry weight. The determination of the initial rate of uptake of a compound into tissue at low temperatures is another way of assessing the amount of free space (Briggs g; a1., 1961). Results of a study are reported in Figure 26 where the NAA treating concentration was raised to 5x10“6 M to facilitate labeling, during the very short sampling time, for accurate measurements. One set of samples was collected but not washed, while a second set was collected and washed twice before analysis for NAA. Extrapolation back to zero time for the non-washed samples gave a value of about 590 pmoles/10 mg dry weight, while the washed had 100 pmoles/10 mg dry weight. NAA efflux from the leaf strips is detailed in Table 43 where leaf strips were labeled in the dark with NAA. Because of the large size of the experiment, two water baths were required to label sufficient material for use in the efflux study. Unfortunately the temperature of one of the baths had shifted during the labeling portion of the experi— ment, but was corrected in time for the efflux study. As 183 leneacetic acid Short—time course of naphtha t 5 C in (5x10‘6 fl) uptake by leaf strips a the dark. Figure 26. set of treatments was hile the other set hosphate buffer Following uptake, one assayed without washing w was washed twice with 0.01 g p (KH2P04-K2HPO4) at pH 6.7. UPTAKE—pmoles //0 mg dry wt 1thaleneacetic and E strips at 5 C 1n e set of treatments was other set while the hate buffer 01 g phosp 7. UPTAKE-pmoles /IO mg dry wt I200 IOOO 800 (D o o V 400 200 I0 184 ”07' WASHED WASHED 2° 30 TIME-min Table 4 185 Table 43. Time-course of naphthaleneacetic acid efflux from leaf strips labeled with naphthaleneacetic acid.“2 Time NAA retained3 Group I" Group II" Wash Medium NAA5 Cl-CCPS Ci—CCP6 treatment: only + NAA5 (hr) ,‘ (pmoles/10 mg dry wt) 0.0 1184a 1184a 913a 913a 0.5 1121 933 872 875 1.0 1013 990 867 890 2.0 994 919 841 881 4.0 1052 1094 881 819 Mean7 1022b 978b 860a 894a 1Labeling NAA concn—1x10 6 M, 4 hr in dark. 2All tissue washed twice before efflux experiment com— menced. 3Means within Group I or II followed by different letters are significantly different at P=0.05. Interaction of wash vs time was not significant. 1‘All tissue within a group were labeled uniformly but Group I had been labeled at a higher temperature (25 C) than Group II (23 C), and consequently the two groups must be considered independentlg of each other. SNAA wash concn=1x10 6Cl CCP wash concn=lx10 :5 M. 7Mean of 0. 5, 1.0, 2. 0 and 4. O-hr washes. account? group c inhibit Presenc detecte medium. 186 apparent in the O—hr sample (Table 43), the material from the two baths had different initial levels of NAA and, therefore, the results of the two groups were not compared statistically. Within the first group, efflux was compared from tissue in medium alone to that in medium with 10x the labeling NAA concentration. No difference between these two treatments was noted. although a small, but significant, release of NAA was observed. The released NAA was totally accounted for in the wash medium. Efflux from the second group of tissue was examined in the presence of the inhibitor Cl-CCP with and without the 10x NAA. In the presence of the inhibitor no significant loss of NAA was detected irrespective of the addition of NAA in the wash medium. B. Metabolic Aspects of Naphthaleneacetic Acid Uptake 1. Effect of Temperature NAA uptake by leaf strip tissue held at 5, 15 and 25 C was examined on a time basis. Tissue was equili- brated at each temperature for 40 min at pH 6.7 in the standard medium. Tissue heated to 60 C followed by place- ment in fresh medium at pH 6.7 was used as a dead control at each temperature. A second set of dead controls was placed in fresh medium at pH 3.7 (no buffer). As shown in Table 44, a significant temperature effect Table 44 Treatmer Non-tree Dead cor Dead cor ____—————--IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIii’ 187 Table 44. Effect of temperature on naphthaleneacetic acid uptake by leaf strips in the dark. Treatment NAA accumulatedl 5 c 15 c y 25 c Time-hr: 2.0 4.0 2.0 4.0 2.0 4.0 (pmoles/10 mg dry wt) Non—treated pH 6.73 73ef 99de 105de 206C 272b 715a Dead controlsz 6.73 18f 22f 23f 26f 29f 25f Dead controlsz 3.73 123de 107de lOOde 109de 152cd lOOde 1Means followed by different letters are significantly different at P=0.05. 2Pretreatment 40 min by heating to 60 C. 3Uptake medium pH. Table 45. Q10 values for naphthaleneacetic acid uptake by leaf strips. Treatment Q10 uptake 5 c to 15 c 15 c to 25 c 2-hr 4—hr A 2-hr 4-hr A time time Non-treated pH 6.72 1.4 2.1 —-——3 2.6 3.5 4.4 Dead controi1 pH 6.72 1.3 1.2 ----3 1.2 1.1 -~--3 Dead control1 pH 3.72 0.8 1.0 -—--3 1.5 0.9 ——-—3 ‘Pretreatment 40 min by heating to 60 C. 2Uptake medium pH. 3No statistical change in time. was ap at pH I Howeve: the pKz dead cc ‘to 3.5 dead ti in upta then t} as 4.4 other t uptake treatme 188 was apparent only in non-treated tissue. The dead control at pH 6.7 contained less than 30 pmoles/10 mg after 4 hr. However, a similar dead control at pH 3.7, which is below the pKa of 4.2, accumulated over five—fold more than the dead control held at pH 6.7. Calculated Q10 values of 1.4 ‘to 3.5 were obtained for living tissue and 0.8 to 1.5 for dead tissue irrespective of pH (Table 45). If the change in uptake from 2.0 to 4.0 hr is considered a rate value, then the non—treated tissue exhibited a Q10 value as high as 4.4 in the 15 to 25 C temperature range. None of the other treatments could be analyzed in this manner since uptake did not change in time during the temperature treatments. 2. Effect of Light on Naphthaleneacetic Acid Uptake To study the effect of light on NAA uptake, a photo—flood lamp bank (Exp. III.D) was used to provide a light intensity of 2000 ft—c. The temperature of the water bath was carefully regulated at 25.0 C. A sufficient number of leaves was harvested initially for two consecutive experi- ments. The first light study was on freshly harvested leaves. and the second on leaves aged for 2 days by storing in low light (200 ft—c) with their petioles immersed in distilled water. Statistical analysis of the data (only . However, the light significantly reduced the amount of NAA accumulated (Figure 27). Pretreatment with NAA (10‘6 M to 10‘5 M) had no effect on dark 02 respiration (Table 46), while in the light, it significantly reduced 02 production. 3. The Effect of Metabolic Inhibitors on Naphthaleneacetic Acid Uptake As detailed in Table 47, the action of several metabolic inhibitors, each at 3 different concentrations, was examined to determine their effect on NAA accumulation. At the lower light intensity (800 ft-c), uptake was not depressed as compared to a dark control. NaN3 partially inhibited accumulation at concentrations of 5x10‘5 M or higher, while C1—CCP, at lxlO‘6 M, alm0st totally inhibited NAA uptake. The photosynthetic inhibitor 3-(4-chloro— phenyl)—l,l—dimethylurea (monuron, CMU), which inhibited OXygen production (Table 48) had no significant effect on NAA uptake by leaf strips in the light (Table 47). Cyclo— heximide, an inhibitor of plant protein synthesis (Morris, 1966, 1967; Ellis, 1969), at concentrations of lxlO‘5 M to 1x1074 M inhibited NAA uptake by more than 50%, but had no Figure 27. 190 Time-course of naphthaleneacetic acid uptake in light and dark by leaf strips. Light intensity=2000 ft—c. The points represent values from two experiments; one set from freshly harvested leaves and the other from the same group of leaves aged under low light intensity for two days. The interaction of leaf age and light on NAA uptake was not ._ significant, while the effect of light signlfl' cantly reduced uptake with time (P=0.05). UPTAKE—pmoles //0 mg dry wf 9; c» A UPTAKE-pmoles //0 mg dry w! 2000 4000 01 O O O IOOO 191 Table A “ Treatme 192 Table 46. Effect of naphthaleneacetic acid pretreatment on oxygen exchange rates of leaf strips in dark and light. Treatment Net oxygen exchange1 Dark Light (pl/100 mg dry wt/hr) Control —297a 276a NAA (ixio'6 1_4_)2 -3o7a l8lb NAA (1x10-5 MW -3l7a l9lb 1Means within a column followed by different letters are significantly different at P=0.05. 22—hr pretreatment. Table Treatm Non-tr Non-tr NaN3 ( NaN3 ( 193 Table 47.‘ Effect of several metabolic inhibitors on naph— thaleneacetic acid uptake by leaf strips.1r2 Treatment NAA accumulated (pmoles/10 mg dry wt) Non—treated-light 1037 Non—treated-dark 949 NaN3 (1x10‘5 M) 997 NaN3 (5x10~5 M) 7513 NaN3 (1x10-4 M) 6003 c1—ccp (5x10’7 M) 393 C1—CCP (lxlO’6 M) 303 Cl-CCP (5x10‘6 M) 223 CMU (1x10‘6 M) 1054 CMU (5xio-6 M) 1094 CMU (1x10‘5 M) 1065 Cycloheximide (lxlO‘5 M) 4843 Cycloheximide (leO‘5 M) 4153 Cycloheximide (1x10‘4 M) 3893 Chloramphenicol (1X10-4 M) 1149 Chloramphenicol (5x10‘4 5’ 13173 Chloramphenicol (lxlO‘3 M) 14343 Malonic acid (5x10‘4 M) 1138 Malonic acid (1x10‘3 M) 13473,~ Malonic acid (5x10‘3 M) 17973,. lAll time treatments were present during the entire experimental time period including a l-hr pretreatment. zAll treatments were conducted in light (800 ft-c) unless otherwise noted. 3These treatment means are significantly different from the light control at P=0.05. uThese treatment means are significantly different from each other at P=0.05. l Table Treatm 194 Table 48. Effect of 3-(4-chlorophenyl)—l,l-dimethylurea (CMU) on oxygen exchange rates of leaf strips in dark and light. Treatment Net oxygen exchangel Dark Light (pl/100 mg dry wt/hr) Non-treated —263i29 263129 CMU (1x10'6 14.) ~315146 ~123112 CMU (5x10-6 M) —310:22 435133 CMU <1x10-5 M) -375:29 —189ill lSE given. effect observ and hi uptake higher Malona studie to upt critic heximi uptake SO as 195 effect on respiration (Table 49). Greater uptake was Observed in the presence of chloramphenicol at 5xlO’4 M and higher (Table 47). Malonic acid also stimulated NAA uptake with its effect becoming more significant with higher concentrations (lxlO‘3 M_to 5x10“3 M) RTable 47). Malonate (5x10‘3 M) had no effect on respiration (Table 49). C. Metabolism of Naphthaleneacetic Acid by Leaf Strips The results of the malonic acid and cycloheximide studies inferred that the metabolism of NAA may be related to uptake. This hypothesis was investigated by more critically assessing the effect of malonate and cyclo— heximide both independently and in combination on NAA uptake. Sufficient tissue was included in each treatment so as to determine NAA uptake and the form in which NAA was present. These data (Table 50) confirmed the stimulatory effect of malonate and the inhibitory effect of cycloheximide on NAA uptake. The addition of malonate to the cycloheximide treatment partially overcame the effect of cycloheximide but uptake was still significantly less than for the non— treated control (Table 50). For analysis of the form of the accumulated NAA, the leaf strip tissue was homogenized (see Exp. II.C.) in 10 ml of 80% (v/v) ethanol and quantitatively transferred Table Treatm Non-tn 196 Table 49. Effect of malonic acid and cycloheximide on oxygen exchange rates of leaf strips in dark and light. Treatment Net oxygen exchange1 Dark Light ____ (ml/100 mg dry wt/hr) Non—treated -292 147 Malonic acid (5x10'3 94) —315 144 Cycloheximide (5x10‘5 39 -322 133 1No significant difference between means within a column at P=0.05. 197 Table 50. Effect of malonic acid and cyclohexi— mide individually and in combination, on naphthaleneacetic acid uptake by leaf strips.1 Treatment NAA accumulated2 (pmoles/10 mg dry wt) Non—treated 828b Malonic acid (5x10‘3 M) 1273a Cycloheximide (5x10‘5 91) 357d Malonic acid (5x10"3 M) + Cycloheximide (5x10-5 M) 536C 1All treatments were present for the entire experimental time including a pretreatment (1 hr). zMeans followed by different letters are significantly different at P=0.05. Plates 198 to 15 ml centrifuge tubes. After standing for 24 hr, the sample was sedimented at 2000 x g for 10 min. The supernatant, decanted into a 150 x 16 mm test tube, was condensed to 0.1 m1 on a flash evaporator (Buchler Instruments, New York). The tissue residue was washed twice with 5 m1 of 80% (v/V) ethanol, processed as just described and combined with the first extract. The concentrated ethanol extract was streaked in a 3—cm band on a 5 cm x 20 cm thin-layer plate coated with silica gel G 250 pm thick. The chromatograms were developed for 15 cm (ascending) in isopropanol : ammonia : water; 8:1:1 (Solvent 1) unless otherwise noted. The thin—layer plates were scanned for radioactivity using a chromato— graphic scanner (Actigraph I, Nuclear, Chicago) that con— sisted of a 2n gas flow detector connected to a chart recorder through a linear ratemeter (Nuclear Chicago). The thin—layer plates were advanced under the detector at a rate of 12 inches per hr. The micromil window of the detector exposed to the plates was limited by a colli- meter set at 3 mm. Trace of a radiochromatogram of the extracted NAA from the leaf strip tissue is shown in Figure 28A. A compariSOn Of authentic (Figure 28C) NAA (Rf 0.6) and that extracted from leaf tissue (Rf 0.2) were not identical (Figure 28). Figure 28. 199 ochromatogram traces of authentic and haleneacetic acid. AA chromatographed ammonia: Radi extracted napht Extract of accumulated N in Solvent 1 (isopropanol: water; 8:1:1). A. graphed B. Extract of accumulated NAA chromato etic in Solvent 2 (n—butanol: glacial ac acid: water; 521:2.2). n Solvent 1. C. Authentic NAA chromatographed i D. Authentic NAA chromatographed in Solvent 2- ent l) purified in hydrolyzed (l N HCl. graphed in Solvent E. Major peak from A (Solv Solvent 2 system, then 60 C, 1 hr) and rechromato 1. F. Major peak from E hydrolyzed (2 N HCl, 100 0; 4hr) and rechromatographed in Solvent 1. 1000 500 IOOO RAD/OACT/V/Tficpm I000 500 2 0 0 IOOO 1000 B 500 0 '000 I000 D 500 500 3000 Rf:::O{F %.0 OJ 0.2 0.3 0.4 0.5 0.6 07 0.8 0.9 1.00.00 0| 0.2 0.3 0.4 05 06 0.7 08 09 10 RAD/0.4677 V/Tchm 201 This fact is further verified by a comparison of chroma— tograms of tissue extract and authentic NAA run in n—butanol : glacial acetic acid . water: 5:1:2.2 (Solvent 2). Thus, the accumulated NAA chromatographed quite differently from authentic NAA in two different solvent systems. To identify the NAA metabolite, the major labeled peak (Figure 28A) was eluted in 80% ethanol and rechroma- tographed in Solvent 2. The major peak in Solvent 2 (Rf 0.7) was identical to that shown in Figure 28B. This peak was eluted again in 80% ethanol and concentrated to 1.0 ml and hydrolyzed for 1 hr at 60 C in 1 M HCl. The HCl—ethanol solution was evaporated until all traces of acid fumes were removed. The residue was resuspended in 80% ethanol and chromatographed in Solvent 1. Figure 28E shows the presence of a small peak comparable to NAA; however, it appeared that only a portion of the labeled compound was hydrolyzed. Therefore, the major peak at Rf 0.2 was again eluted, concentrated, and hydrolyzed in 2 M HCl at 100 C for 4 hr. Upon chromatography in Solvent 1, a strong peak occurred at Rf 0.6 and the original peak at Rf 0.2 was missing (Figure 28F). ‘The new peak appeared at the same Rf as authentic NAA. Spraying the chromatogram with ninhydrin yielded a positive spot at Rf 0.05 which 202 corresponded to authentic aspartic acid in a separate series of experiments. For this reason, it was believed that NAA was metabolized to NAA acyl—aspartic acid (NAAsp). Chromatographic analysis of the remaining treatments of the experiment detailed in Table 50 showed that the malonate stimulated the formation of the NAA complex (Figure 29B). The labeled material extracted from the cycloheximide treatment was of insufficient quantity to obtain any significant chromatographic peaks so it was not illustrated in Figure 29. However, significant activity was detected in the malonate plus cycloheximide treatment and this activity corresponded in Rf to authentic NAA (Figure 29C). Chromatographic analysis implicated the involvement of aspartic acid during NAA accumulation. A preliminary experiment revealed that pretreatment with aspartic acid (lxlO‘3 M) enhanced NAA uptake. The non-treated tissue accumulated 851 pmoles NAA/10 mg dry weight while the aspartic acid—treated tissue accumulated 1161 pmoles NAA (significantly different at P=0.05). To further examine the metabolic aspects of NAA accumulation, a detailed time—course experiment was con— ducted to determine if the surge in uptake noted in the earlier time-course study (Figure 24) was related to the Figure 29. 203 Radiochromatograph traces used to determine the form of accumulated naphthaleneacetic acid from treatments listed in Table 50. A. Extract of non-treated tissue chromato- graphed in solvent 1. B. Extract of malonate (5x10—3 M) treated tismm chromatographed in solvent 1. C. Extract of malonate (5x10‘3 M) plus cyclohex- imide (5x10‘ 5 M) treatment chromatographed in solvent 1. 1Note—extract of cycloheximide treatment had no significant peaks, so trace is not illustrated. 204 ‘ used to determine 3 naphthaleneacetic acid 0 Table 50.1 g ‘3 x ed tissue chromato- 'Q A E (5}(10‘3 M) treated tissué ‘ olvent 1. g (leO’3 M) plus cyclohex Q: O 0.0 O.) 0.2 0.3 0.4 0.5 0.6 0.7 0.8 09 IO Rf 205 metabolism of absorbed NAA. As shown in Figure 30, pre— treatment of the tissue with non—labeled NAA predisposed the tissue to accumulate NAA at a faster rate. Treatment of the tissue with aspartic acid (1x10"'3 M), during the entire experimental period, which included a l-hr pre— treatment, did not affect the timing of the surge but did enhance (though not quite significantly) the rate of the uptake. Cycloheximide (5x10‘5 M) combined with or without aspartic acid (1x10"3 M) eliminated the surge of NAA uptake. The form of accumulated NAA after 2 hr and 6 hr was determined by eluting the NAA from planchets and analyzing by radiochromatographic procedures previously described. Pretreatment with non—labeled NAA (Figure 31B) enhanced the amount of the NAA complex running at Rf 0.2 compared to the non—pretreated tissue (Figure 31A). Cycloheximide, irrespective of the presence (Figure 31C) or absence (Figure 31E) of aspartic acid, resulted in about equal activity at both Rf 0.2 (where the complex occurred) and 0.6 (where authentic NAA occurred) for the 2—hr sample. By the 6—hr sample all the activity, though still very limited, was found at Rf 0.2. The 2—hr aspartic acid treatment had a small, but significant, amount of activity that corre— sponded to free NAA along with a major peak at Rf 0.2 (Figure 31D). In the 6—hr sample almOSt all of the activity Figure 30. 206 Time—course of l4C-naphthaleneacetic acid (lxlO‘6 M) uptake by leaf strips as affected by several treatments. The experiment included a 1-hr pretrgatment with medium only, non—labeled NAA (1x10 _ 5 ' acid (1x10‘3 10: cycloheximide (5x10‘ M), and as artic acid (1x10’3 M) with cycloheximide pretreatment 11 flasks such ts became the treatments for NAA uptake. The one exception was that the non- labeled NAA treatment medium was removed and replaced by l4C—NAA in fresh medium. Points represent means of three experiments with two replications for each experiment. Points lying between or on two adjacent dashed lines are not fferent at P=0.05 as determined loglo transfor- mations of all observatlons. The antilog values 207 A»NAA ° ASPARTATE ' CYCLOHEXIMIDE ' CYCLOHEXIMIDE + ASPARTATE Lleneacetic acid strips as affected ed a l-hr pretrgatment ' M), eled NAA (1x10 _ 6.)] imide (5x19 _, cyclohex yclohexinde Q; UPTAKE-pmoles/IO mg dry wt Figure 31. 208 Radiochromatograph traces used to determine . the form of accumulated naphthaleneacetic ac1d from treatments shown in Figure 30. Traces represent samples from 2—hr and 6—hr measurements of all treatments chromatographed in Solvent 1. A. Non-treated tissue. B. Non-labeled NAA pretreated tissue. C. Cycloheximide (leo—5 M) for entire experi- mental period. D. Aspartic acid (lxlO'3 M) for entire experi- mental period. E. Cycloheximide plus aspartic acid for entire experimental period. RADIOACT/V/T)’ - cpm 209 2 HOURS 6 HOURS IOOO 4000 55' T) 200%: I } IOOO 4000 B . 500 2000 o 1000 4000C C 500 2000 0 IO 00 4000D D 500 2000 o 1000 4000 E 2000 NAA Nv-A—Ae o (00 0| 0.2 0.3 04 0.5 0.6 0.7 08 0.9 IO 00 0.| 0.2 0.3 0.4 0.5 0.6 0.7 0.8 09 ID ' Acid by Enzymatically Isolated Leaf Cells Soybean leaf cells were isolated the evening before each experiment by following the procedures previously developed (Exp. III.D., III.G.). l4C—NAA concentration was A detailed time-course and concentration study of NAA uptake was simultaneously conducted on one large preparation of enzymatically isolated cells. The standard from each flask. As was done earlier, the experiment was staggered in sets of replications. Examination of the uptake data (Figure 32A) showed that when expressed on a log versus log basis, there was a linear relationship between concentration and uptake. Figure 211 32. The uptake of naphthaleneacetic acid by enzy- matically isolated leaf cells as affected by concentration and time. A. The uptake of NAA as affected by its own concentration. Each line represents a different time sample. The standard errors of the means are no larger than the plotted points for the respective observations. (Values and SE given in Appendix Table A-4L B. Time—course of uptake of NAA at two NAA concentrations. Standard error of the memm were plotted when larger than plotted points. eneacet f cells as affect as affected by its own ' tsa e represen : Ch 111The standard error. 212 IOOO IOO SAMPLE TIME 0 6.00- hr l0 0 3.00- hr A 2.00-hr X LOO—hr + 0.50- hr v 0.25-hr UPTAKE-pmoles/IO mg dry wt le0‘7 2xl0'7 leO‘6 2110’6 1,110-5 1.5100'5 5100’5 nuc‘4 CONCENTRATION-M 400 OJ 0 O air/o"s M NAA 200 Ion—G M NAA IOO UPTAKE-pmoles/IO mg dry wt ———————-—---IIIIIllllIIIlIIIIIIIIIiIIIIIIIIIIIIIIIIIIIII'IIIIEEEEq 213 This concentration response was linear for all times sampled. Two typical time—course studies are shown in Figure 328. Initial uptake was rapid followed by a second slower phase of accumulation. 2. Loss of Accumulated Naphthaleneacetic Acid A large mass of enzymatically isolated leaf cells was uniformly labeled with NAA (lx10‘6 14.) for 4 hr and then divided into three groups of equal size. This provided sufficient material within each group for the different wash treatments, each replicated four times. The phosphate buffer (KH2P04—K2HPO4, 0.1 M, pH 6.7) based wash media consisted of three levels of non—labeled NAA (none, 1x10'5 M and 1x10“4 M). Three successive washes with any of these three media were required to remove a significant amount of the accumulated NAA (Table 51). Because there was no significant interaction between the wash media and the number of washes, only the overall means were compared statistically. All three wash media were effective in reducing the retained level of NAA compared to the non— washed sample by about 16%. However, only the NAA, 100x of the labeling concentration, was effective in further lowering the level of retained NAA. 214 Table 51. Effect of washing with selected media on naphthaleneacetic acid retention by enzy— matically isolated leaf cells prelabeled with naphthaleneacetic acid.1 Wash treatment2 NAA retained Wash medium: A3 Bl+ D5 mean6 (pmoles/10 mg dry wt) No wash 448 ——- -—- -—— 448a One 5—min wash 423 415 425 421ab Two S—min washes 408 427 421 419a Three 5—min washes 395 378 341 37lbc Three S-min washes + one 15—min wash 387 373 336 366bc Three 5-min washes + one 30—min wash 378 363 331 357C Three 5—min washes + one 60—min wash 369 357 318 347C Mean6 448a 394b 386b 352C 1NAA concn= 1x10 6 M; for 4- hr uptake period. 2All washings were done at 5 C. 3Wash medium AFC. 01 M phosphate buffer (a H 2904- K HPO4) pH 6. 7. _5 Was medium B=wash medium A+1x10 M non—labeled NAA. 5Wash medium C=wash medium A+1x10 4 M non—labeled NAA. 6Means within a row or column followed by different letters are significantly different at P=0.05. Interaction of wash time and wash media was not significant. 215 B. Metabolic Aspects of Naphthaleneacetic Acid Uptake 1. Effect of Light The effect of light on NAA uptake was established using the same experimental conditions as used for the leaf strip study (Exp. IV.B.). Several intermediate light intensities were also examined along with the effect of light on tissue held at low temperatures. Light at all intensities studied at 25 C significantly reduced NAA uptake (Figure 33). At the low temperature, light had no effect, though uptake in either light or dark was dramatic— ally reduced. 2. Effect of Temperature An identical experimental design as used for studying the effect of temperature on NAA uptake by leaf strips (Exp. IV. 8.1.) was utilized for enzymatically isolated cells. Temperature affected NAA accumulation only by viable cells. This temperature effect can be expressed as a Q value of 2.4 to 2.5 (Table 53). Lowering 10 the pH of the medium to 3.6 for the dead control tissue enhanced sorption over three—fold, compared to dead cells at pH 6.7 (Table 52). The Q10 values for the sorption into the dead cells ranged from 1.1 to 1.4 (Table 53). 3. Metabolism of Naphthaleneacetic Acid Analysis of the involvement of NAA metabolism as part of the accumulation process was evaluated using the same techniques applied to leaf strips (Exp. III.C.). Figure 33. Time-course of naphthaleneacetic acid uptake by enzymatically isolated leaf cells as affect- ed by light, dark, and temperature. The effect of light compared to dark signifi-t cantly (P=0.05) reduced uptake at 25 C but no at 5 C. - ' take ,aleneacetlc ac1d up fest- ated leaf cells as af d temperature. ompared to dark signifi- ed uptake at 25 C but nc‘. UPTAKE-pmoles/IO mg dry wt 217 DARK 25c' LIGHT (500 ft-c) 25 c. LIGHT (IOOO ft-c) 25 c LlGHT(2000ff-C) 25 C 600 0--0 DARK 5C LIGHT(2000 ff-C) 5 C 500 400 300 200 IOO «———e 218 Table 52. Effect of temperature on naphthaleneacetic acid uptake by enzymatically isolated leaf cells. Treatment NAA accumulated1 5 C 15 C ' 25 C Time—hr: 2.0 4.0 2.0 4.0 2.0 4.0 (pmoles/10 mg dry wt) Non—treated pH 6.73 89f 136e 286d 402c 562b 844a Dead—control2 pH 6.73 319 35g 39g 42g 40g 41g Dead—control2 pH 3.73 91f 104f 129e l43e l43e l32e 1Means followed by different letters are significantly different at P=0.05. 2Pretreatment—40 min by heating to 60 C. 3Uptake medium pH. Table 53. Qlo values for naphthaleneacetic acid uptake by enzymatically isolated leaf cells. Treatment Q10 uptake 5 C to 15 C 15 to 25 C____ 2 hr 4 hr A 2 hr 4 hr A - time time Non—treated pH 6.72 3.2 3.0 2.5 2.0 2.1 2.4 Dead-control1 pH 6.72 1.2 1.2 ——-3 1.0 1.0 -—-3 Dead—control1 pH 3.72 1.4 1.3 ——-3 1.1 1.0 ——-3 W 1Pretreatment—4O min by heating to 60 C. 2Uptake medium pH. _ 3No statistical change in tlme. <— 219 Pretreatment with non—labeled NAA had no significant effect (Table 54). Aspartic acid (1x10.3 M) slightly increased NAA uptake, compared to the nonetreated control. Cycloheximide depressed uptake only in the absence of aspartic acid. None of the treatment effects varied during the times sampled and no surge in uptake was found. Chromatographic analysis of extracts of cells from treat— ments after 2 hr indicated that essentially all of the NAA accumulated by the cells was complexed (Rf 0.2). A typical radiochromatograph trace of the 2-hr sample (pool of two replications) is illustrated in Figure 34. 220 Table 54. Time—course of naphthaleneacetic acid uptake by enzymatically isolated leaf cells as affected by several treatments. Treatment NAA accumulated Time—hr: 0.5 1.0 2.0 3.0 4.0 6.0 Mean1 (pmoles/10 mg dry wt) Non-treated2 187 369 483 689 879 905 585bc NAA3 188 370 491 750 905 929 605bc Cycloheximide“ 182 356 417 732 872 849 568C Aspartic acid5 209 420 461 843 980 1005 653a Aspartic acid5+ cycloheximide” 184 411 462 749 953 926 614ab ___—___________——__———-——-——-———————————— 1Means followed by different letters are significantly different at P=0.05. Interaction of time and treatment was not significant. 2Medium only. —6 3Non-labeled NAA (1x10 M) 1—hr pretreatment, removed before l4C—NAA was added in frEsh medium. . l‘Cycloheximide (5x10‘5 M) present for entire experl— mental period (included l-hr pretreatment). . ‘ 5Aspartic acid (lxlO‘3 M) present for entlre experi- mental period (included l-hr pretreatment). Figure 34. Trace of radiochromatogram of accumulated naphthaleneacetic acid from isolated leaf cells after 2 hr of uptake. . 222 IOOO RAD/0A6 T/V/ rr- cpm 8 O NAA O0.0 0.l 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 LO Rf DISCUSSION I. Assessment of Tissue Viability The primary objective of this thesis was to study the mechanism of NAA accumulation by leaf cells. Since previous studies (Jyung et _i,, 1965b; Takebe g; 1., 1968; Kannan and Wittwer, 1967; Price, 1967) did not consider the viability of the isolated cells in comparison to intact tissue, this aspect was first examined. Several criteria had to be established. Once acceptable criteria were established, they were used in developing techniques to obtain leaf tissue strips and isolated leaf cells of maximum viability. The experiments with hypocotyl tissue were designed to give a first approximation of the general factors involved in NAA accumulation by soybean tissue. It was initially proposed that thOSe factors that would maximize NAA accumulation would also be optimum for tissue viability. The results of the tissue comparison experiments did prove that high levels of NAA accumulation occurred only in viable tissue (Table 27,33). However, as this was not known when the hypocotyl experiments were conducted, only a 223 224 limited number of tests were conducted to avoid developing a system based on a possible artifact. One of the more important facts established by the hypocotyl work was that bacterial contamination could inflate oxygen consumption values (Table 4). This meant that if several tissue preparations were compared with different levels of bacterial contamination, the interpretation of the respiration data would be very difficult. Hallaway (1968) previously reported that such bacterial contamination could easily offset true respiration rates. The addition of chloramphenicol (D—threo—isomer) to hypocotyl segments contaminated with bacteria was done to establish if the antibiotic would reduce the effect of the bacteria on the experimental system (as suggested by Wilson, 1966; Leaver and Edelman, 1965). Chloramphenicol only at a high level (250 ug/ml) reduced the number and effect of bacteria (Table 2), but oxygen consumption was still considerably above the standard preparation (Table 4). Thus, measurements of viability based on oxygen consumptiOn were easily influenced by bacteria and therefore were not suitable as an index in developing an experimental techniqUe for handling isolated leaf cells. Chloramphenicol (not a bactericide [Brock, 1961]) only reduced the level of bacteria population but did not eliminate them. Since 225 chloramphenicol did not provide adequate control of bacterial contamination, it was not an adequate tool. The high level of chloramphenicol also enhanced NAA accumu— lation (Table 3), a response apparently independent of bacterial contamination. _This enhancement was also observed with leaf strips (Table 47). Bacteria removed from the system before metabolic measurements minimized the effect of contamination (Table 3). The presence of bacteria, even though not contained in the plant material being analyzed, might have had a profound effect on the system. Bacteria releasing a permease is an example (Nissen, 1968). The best practiCe was to use conditions as sterile as possible (Wilson, 1966) and minimize the duration of the experiments. Surface sterilization with NaHClO was detrimental to the tissue. The establishment of a dead control to serve as a base line for metabolic comparisons proved to be an important aid for measurements of viability. The action of azide was as a metabolic inhibitor since it did not alter the high NAA binding capacity of dead hypocotyl tissue, while it markedly reduced the NAA accumulation in living tissue (Table 5). The minimal reductive capacity of the dead leaf strip tissue for tetrazolium salts demonstrated the sensitivity of the assay for living tissue. 226 The development of a quantitative tetrazolium assay for tissue viability played a major role in identifying the optimum treatment conditions for the leaf strips. The use of tetrazolium salts to measure reductive capacity has been described as an in vivo assay except for the presence of the actual tetrazolium (Pearse, 1960). The fact that most of the NTC did not penetrate deeply into the wide leaf strip tissue made its initial application ideal, since NTC was concentrated in those parts of the tissue with maximum exposure to the medium and therefore probably most affected by the treating medium. This meant that the NTC would measure mainly the responses of those cells affected by the medium, rather than assessing the entire leaf strip. The simplicity of the assay gave additional merit to the technique since numerous treatments could be conducted simultaneously to permit multiple comparisons of the treat~ ment conditions. The tetrazolium assay also had several deficiences that must be recognized before this procedure can be considered valid. The light sensitivity of the reduced TTC limits its use to experiments where light (Table 7) or tissue size (Table lO)(which would impose different degrees of shading) is not a variable. The poor penetration of NTC implies that factors affecting tissue permeability, such as the 227 different methods of tissue preparation (Tables 27 and 33), may easily lead to false interpretations of tissue viability. Future NTC viability assays, when used in conjunction with other viability factors, might best be approached by first preloading the tissue with NTC and then exposing the NTC-treated tissue to the various treatments. The toxic effect of the tetrazolium salts, due to their potent uncoupling activity (Palmer and Kalina, 1966, 1968; Clark g; 1., 1965) can be detrimental if viability studies are prolonged. Extended experiments would likely result in an underestimation of cell viability. This effect was apparent in the concentration experiments (Table 9, Figure 9). One of the more sensitive assays for viability, adapted in this study, was the polarographic electrode analysis of oxygen exchange rates. The ability to rapidly measure light driven net oxygen evolution and respiratory oxygen consumption, almost simultaneously on a single small sample of tissue, was advantageous. The photo— synthetic dependent oxygen evolution appeared to be the most reliable indicator of overall tissue viability. As demonstrated in the multiple comparison experiments on the several leaf tissue preparations, those tissue preparations that exhibited high oxygen evolution rates were most effecient in metabolizing acetate (Table 37), accumulating P02- 228 (Tables 36, 39) NAA (Table 33), and fixing l4C02 (Tables 31, 32). These last several assays were used to establish a broad range of metabolic functions.’ (The initial lag Observed in the acetate catabolism was typical of such ‘experiments [Doyle and wang, 1960]). II. Development of Techniques to Handle Soybean Leaf Tissue Preliminary experiments established that cells isolated following several suggested pr0cedures (Jyung g; _l., 1965b; Takebe 2; al., 19687 Price, 1967) were of low viability. It was impossible to determine what aspects of the procedure led to a loss in viability because of the complexity of the isolation technique. An unsuitable treat— ment medium, severe mechanical damage to the cells during isolation or cellular disconnection from the simplast system of the leaf may be critical factors. As Hallaway (1965, 1968) reviewed, the correct type of treatment medium was critical in order to sustain active metabolic activity of isolated organelles. Hongladrom (1964) and Honda §£.§l- (1966) noted that a very special medium was necessary to retain cytoplasmic streaming when handling even intact micro—leaf strips. The pH and choice of a buffer system had the most dramatic effect on tissue viability as indexed with NTC 229 (Figures 11, 12, 13; Table 14). The polarographic oxygen respiration analysis revealed the same effect on mechani— cally isolated leaf cells (Table 26). These data sub— stantiate the findings of Romani _p _l. (1969) that pH must be regulated within a very narrow range. The pH optimum of 6.7 for soybean tissue was the same as for pear fruit mitochondria (Romani g: _l., 1969). Kim and Bidwell (1968) reported a similar optimum for incorporation of l4C—glucose into root tissue. The various buffers studied (Table 14) did not exhibit toxic effects below 0.05 g. The use of this higher level was done to simulate a long term exposure at a lower level. Under these conditions the Tris-maleate buffer was most toxic. Such results were consistent with the recent re- port that showed Tris buffer to inhibit the Hill reaction (Yamashita and Butler, 1969). Another disadvantage of the Tris buffer was that the pK shifted 2-3 times faster with changes in temperature than did most of the other buffers (Good et al., 1966). Thus, if a treatment medium was adjusted to pH 7.4 at room temperature (25 C) and then was used to isolate cells at 4 C, the pH would have shifted to pH 8.0. This higher pH would have been less desirable and was probably one reason why cells isolated in the 230 preliminary studies were of low viability. The cold wash treatment of leaf strips (Table 10) documented this detri— mental effect of low temperature where the pH had likely shifted to pH 8.0. HEPES buffer was particularly non—toxic and is in agreement with the work of Good _£_§l. (1966). The MES buffer was equally effective (Figure 13, Table 26). Contrary to several reports (for reviews cf. Hallaway, 1965, 1968), osmotic pressure only slightly affected leaf strip viability. The results Of experiments on NAA uptake by hypocotyl sections (Table 8) demonstrated a negative relationship between mannitol concentration and NAA accumu- lation. This might be explained by the fact that it is more difficult for tissue under hypertonic stress to accumulate material. The effect of mannitol concentration on NTC reduction (Table 18) showed only a general trend toward a 0.25 g optimum, but a broad range of 0.20 to 0.40 g was found to be acceptable for almost the same response. The hypothesis of the benefit of handling the tissue under hypertonic conditions, when the leaf tissue was being exposed to detrimental materials (Takebe _3 a1., 1968), was found to be unsuitable in that the high mannitol concentra— tions required (0.45 M and above) damaged the leaf strip tissue (Table 12). This was one of the better experiments 231 with tetrazolium because the staining was carried out independent of the treatment conditions. These data also revealed that incubating the tissue under hypotonic con— ditions resulted in tissue injury. To avoid starving the excised tissue preparations, the value of added substrate was established. Sodium succinate was an effective substrate for hypocotyl sections (Table 7), while sucrose, on the other hand, actually depressed the accumulation of NAA. It is possible that the use of sucrose in the preliminary studies contributed to the observed low viability. The optimum succinate con— centration (0.008 E, Figure 6) for leaf strip tissue was found to be much lower than that used for hypocotyl tissue (0.02 M). Interestingly the succinate was only stimu— latory in light (Figure 6), and a similar response with intact leaf tissue was observed by Dyar (1953). Sucrose has been reported to enhance leaf disk growth only in the light (Dale, 1966). The high molecular weight polymers Ficoll and Dextran~ 40 along with BSA as stabilizers have been found useful in some excised tissue media (Hongladrom, 1964; Honda _§ al., 1966). The NTC reduction assay (Table 17) revealed that only the BSA (50 ug/ml) had any beneficial effect on viability of soybean tissue while Dextran-4O (Table 15) was 232 without effect and the Ficoll depressed tissue activity. There was no effect by either Ficoll or Dextran on oxygen exchange rates (Table 25) and thus they Were not utilized. If Ficoll is used, one should first dialyze it to remove a toxic component. These polymers might be utilized as a practical aid in cell separations (Figure 16). The beneficial effect of BSA was consistent with results of Jones §£__l. (1964) and Sarkissian and Srivastava (1968). The addition of stabilizers of phenolic compounds also proved helpful. Little difference was noted between PVP and metabisulfite (Table 16). The inhibitory effect of metabisulfite above 1.0 mg was surprising, since Stokes _E._l- (1968) reported that this concentration was the minimum necessary to recover active potato tuber mitochondria. The optimum PVP concentration was also about 5 to 10 fold less than that used by Hulme g; l. (1964a, 1964b) for apple fruit and rose flower mitochondria. The use of Ca2+ in the treatment medium was intentionally avoided, as the role that Ca2+ plays in membrane perme~ ability is as yet uncertain (Leggett, 1968; Handler g; gl., 1965: van Stevenick, 1965). The addition of plant hormones was also intentionally avoided so as not to confound ensuing auxin uptake studies. The development of a treatment medium appropriate for 233 soybean leaf tissue suggested that further failure to isolate viable leaf cells could then be attributed to either the effect of mechanical shock during isolation or to the loss of the symplastic system (Arisz, 1963). Cells isolated by the enzymatic procedure were consistently more active than those isolated mechanically (Table 29 to 39). Apparently the roller mill was too harsh on the leaf tissue. However, the roller mill has been shown as the gentlest method for tissue homogenization (Hulme _3 al., 1964a). Jagendorf and Wildman (1954) noted that damaged tobacco leaf nuclei resulted from harsh mechanical separation. The enzymatic cell separation, though slower and contaminated with damaging enzymes, was sufficiently mild to permit the release of relatively active leaf cells. Pectinase enzymes have aided in isolation of plant cell nuclei by making the isolation procedure milder (D'Alessio and Trim, 1968). Even though the enzymatically isolated cells were far superior to the mechanically isolated cells, they were still much less active than a comparable cell population in leaf strips. A visual analysis of the cells from a typical isolation revealed that the protoplasm was coagulated in almost half of them.- Thus, approximately half of the enzymatically isolated cells were living and 234 the total cell population exhibited about one-half the metabolic activity of the leaf strips. It is tempting to conclude that viability of isolated leaf cells was dependent upon mild handling of the cells in an appropriate treatment medium and that the symplastic system was unnecessary for short term metabolic studies. Though the isolated cells were not as active as intact leaf strip tissue, they were capable of many metabolic functions associated with living cells and absent in preparations previously reported. The transformation of photosynthetically fixed l4C02 into protein (Table 31) indicated a considerable improvement over the data of Racusen and Aronoff (1953) where there was no incorporation into protein. The relatively high respiration rates approached those of intact leaf tissue compared to the very low rates of oxygen consumption reported by Jyung g3 al. (1965b), Gabbott and Larman (1968), and Jacoby and Dagan (1967). The consistently higher photosynthetic oxygen production rates of the enzymatically isolated leaf cells, compared to leaf strips, were probably due to a more even distribution of the tissue mass in the treatment medium. This would have permitted the isolated cells to be exposed to more light. The addition of Cl—CCP, an uncoupler of both 235 photosynthetic and oxidative phosphorylation (Heytler, 1963), eliminated the light effect on oxygen production and enhanced the dark respiration rate. Therefore one can conclude that respiration was coupled in the leaf strips and isolated cells. III. Naphthaleneacetic Acid Uptake A. Hypocotyl Tissue Uptake studies using hypocotyl sections were con— sidered preliminary to the subsequent leaf strip and isolated cell experiments. Results obtained with hypocotyl tissue indicated that tissue size was a critical factor. Unlike the reports of Reinhold (1954) and Jenner _£__l. (1968b), maximum uptake occurred with the 4-mm rather than 2—mm sections, the smallest prepared. It is likely that the difference between the 4—mm and the 2—mm sections was due to a wound response in the 2-mm sections since uptake in the presence of NaN3 was uniform for all tissue sizes. Another important point was the relatively minor effect of bacteria on NAA uptake, providing bacteria were removed from the tissue before radioassay. MacDonald (1967) found that bacterial contamination reduced ion uptake into beet tissue. Possibly, the bacteria had only a limited capacity to accumulate NAA and therefore their effect was 236 minimal. Andreae and van Ysselstein (1960b) reported that bacteria did not reduce IAA uptake in pea tissue, but did reduce the level of IAA in the medium. The frozen—thawed treatments (Table 5, Figure 4) revealed that the tissue membranes Were rate limiting for NAA uptake into tissue treated with NaN3. The freeze— thawing process likely destroyed membrane integrity per— mitting rapid initial entry of NAA, but the total sorptive capacity was not changed. The addition of either succinate or acetate sig— nificantly enhanced NAA uptake. These data, along with inhibition with NaN3 and frozen—thawed tissue, all indicated that tissue metabolism was at least partially involved in the accumulation of NAA. B. Leaf Strip Tissue Since the isolated cell preparations demonstrated con— siderably less metabolic activity than leaf strip tissue, the uptake data for the two preparations will be discussed separately. The last several experiments described for the leaf strip tissue revealed the metabolic formation of an NAA conjugate. The synthesis of a complex is likely the major factor controlling the accumulation of exogenous NAA. Thin» layer chromatographic separations of the extracted compound 237 established a major peak at Rf's (0.2 Solvent 1, 0.6 Solvent 2) corresponding to NAAsp (Zenk, 1962; Veen, 1966, 1967; Sudi, 1964). Venis and Stoessl (1969) have recently reported on the recharacterization of a compound that, chromatographed like benzoylaspartic acid, was actually benzoylmalic acid. They did not find any such new compound for NAA. In this thesis study, the enhancement of the uptake rate by feeding aspartic acid to the tissue (Figure 30) along with the results of the hydrolysis of the complex strongly suggest that the complex was NAAsp. The slight shoulder occurring at Rf 0.28 might be a second compound, but no attempt was made to identify it. There are many similarities between NAAsp and IAAsp in their formation and characterization (Zenk, 1962; Andreae, 1967; Sudi, 1964, 1966; Veen, 1966, 1967). The metabolic conjugation involving other auxins is not as well defined. 2,4—D has been shown to induce the formation of NAAsp and IAAsp (Zenk, 1962; Sudi, 1966), but the actual aspartic acid conjugate of 2,4—D has not been isolated. Several of the phenoxyacetic acid auxins, including 2,4—D, t.§l-, 1964) as a complex with have been isolated (Thomas glucose (e.g. 1-0—2,4—dichlorophenoxyacetyl glucose). A glucose conjugate has also been recovered for NAA and IAA (Klambt, 1961; Zenk, 1962, 1963; Veen, 1966, 1967). The 238 amount of the auxins recovered conjugated with glucose was usually small compared to that of acyl-aspartate. For 2,4—D metabolism, the amount recovered bound is very small (Andreae, 1967). This implies that not all data from 2,4—D uptake should be directly compared to NAA and IAA data. Luckwill and Lloyd—Jones (1962) showed that NAA was metabolized by apple leaf tissue into a water soluble neutral compound and an acidic one. This thesis reports for the first time the significance of a NAAsp—like compound as a major metabolite of NAA in leaves. Zenk (1963) found IAAsp to be formed in leaf tissue, but most of the reports on the formation of the auxin conjugates are based on stem and root tissue (Zenk, 1962; Sudi, 1964, 1966; Andreae, 1967; Andreae and Good, 1955, 1957; Andreae and van Ysselstein, 1960a, 1960b; Veen, 1966, 1967; Veen and Jacobs, 1969). The surge in uptake noted in the time—course study (Figure 24) and later confirmed in the inhibitor study (Figures 30, 31) can be explained by the induction or activation of the enzyme forming the aspartic acid—NAA con— jugate. Zenk (1962) observed a similar surge with pea shoot tissue in uptake after a 4—hr lag and attributed the lag to induction of an enzyme he termed L-aspartic 239 acid—N—acylase. Venis (1964) found that actinomycin—D or puromycin treatments nullified the formation of a similar conjugate, benzoyl aspartic acid. He concluded that the auxin induced a true g; novo synthesis of the enzyme system (gracylaspartate synthetase) involved in the formation of the complex. In this study the cycloheximide inhibition of the surge (Figure 30) suggests that protein synthesis may be involved in formation of the complex. Since addition of aspartic acid to the cycloheximide treatment did not release the inhibition, while aspartic acid added alone was stimulatory, it can be concluded that protein synthesis was required for conjugation of the two components (NAA and aspartic acid) and not for synthesis of a special pool of aspartic acid. Pretreatment with non—labeled NAA showed that it was possible to precondition the tissue to respond to the labeled NAA sooner and thus eliminate the surge (Zenk, 1962, 1964; Sudi, 1964). Since some con— jugated NAA was recovered from the cycloheximide treated tissue, it is impossible to conclude if g; novo synthesis or activation of the enzyme occurred. The surge in uptake might also be explained as a direct effect of auxin on membrane permeability. IAA has been found to induce beet root tissue to lose K+ (van Stevenick, 240 1965), while 2,4—D enhanced penetrability of sucrose into 1., 1959). The presence of non— Chlorella (Wedding g: labeled NAA on the receiver—side of an isolated cuticle membrane has been shown to enhance the movement of labeled NAA through the cuticle into the receiver solution (Norris and Bukovac, 1969). Although such a phenomenon is possible, it is unlikely to be a major factor in the experiments with leaf tissue since cycloheximide treatments that reduced conjugation also removed the surge. If the auxin acted directly on the membrane, then the surge should have been independent of the cycloheximide inhibition. The increased rate of NAA accumulation at leO’7 M compared to lower concentrations (Figure 25, Table 41) indicated a possible concentration dependency for the induction of conjugate formation. Examination of the data (Figures 24, 25, 27, 30) revealed that the surge usually occurred after about 100 pmoles/10 mg dry weight was accumu— lated. Sudi (1966) and Veen (1967) each reported a distinct optimum concentration for formation of the complex. As found in this study, Veen (1967) observed a time and concentration interaction that showed high (10"4 M) NAA concentrations induce the formation of the complex sooner than did the lower (10’5 fl) concentrations. It is likely that a critical internal concentration must first be attained 241 before induction takes place. Light has been shown to enhance auxin uptake in several studies (Greene, 1969; Thimann and Wardlaw, 1963; Sargent and Blackman, 1965, 1969). In contrast to reports (Sargent and Blackman, 1965, 1969) of light dependency for the surge in auxin uptake (2,4—D), light actually depressed NAA uptake by leaf strips (Figure 27). CMU, a photosynthesis inhibitor (Good, 1961), effectively blocked oxygen production in the light (Table 48), but did not affect NAA uptake in the light (Figure 27). Since sub— strate (succinate) was present during the experiment, the lack of an additive light effect indicated that direct coupling of photosynthetic energy (as Rains [1968] reported for K+ accumulation) was not involved in NAA uptake. Assuming that aspartic acid is one of the key metabolites necessary for high levels of NAA uptake, its independence on light for synthesis (Ashton gpl_l., 1961; Zweig and Ashton, 1962; Walker, 1962) might be another reason for the lack of a positive light effect. Fang §E__l. (1959) have shown that the formation of IAAsp is independent of light. The negative effect of light on uptake might also be related to light enhancement of RNase (Bagi and Farkas, 1967) which may be activated by the cutting of leaf strips. They reported that high light intensities, like those used 242 (Figure 27), would affect the RNase system much more than lOWer intensity light (800 ft-c) used in other ex— periments where light did not have a depressive effect (Table 47). Y i ‘ An uncoupler of oxidative and photosynthetic electron transport, m—Cl—CCP, inhibited NAA accumulation (Table 47) equal to the level recovered in the dead controls (Table 44). The level (35 pmoles/10 mg dry weight) found in the dead controls would be slightly less than the external NAA concentration (see Appendix Figure Al for sample cal— culations). Thus, there was a distinct dependency on meta— bolic energy to drive the accumulation mechanism. High levels of NaN3 (10’4 fl) only partially inhibited NAA uptake. The 33% inhibition was similar to the report of Sargent and Blackman (1969). From these studies, it was impossible to determine if free NAA was accumulated against a concentration gradient. The chromatographic separations in all (Figures 29a, 29b, 31) but one case (Figure 29c) showed that no free NAA was extracted at levels above 40 pmoles/10 mg dry weight, which would be equal to the external concentration. In the other treatments, metabolized NAA was recovered in sub- stantial levels even from the 2—hr sample. Thus, the question as to whether free NAA accumulated against a con- centration gradient will have to be left unanswered. However, if it did accumulate against a concentration gradient, it 243 would have been only by a very small amount. Zenk (1962) showed that free NAA did accumulate to twice the treatment medium concentration, while Andreae and van Ysselstein ' ‘ l (1960) found IAA to exceed the concentration gradient by lO—fold. Malonic acid, a possible competitive inhibitor of succinic dehydrogenase (Mahler and Cordes, 1966), actually stimulated NAA uptake. This might be explained in several ways. First, since malonate did not depress respiration (Table 49), it possibly acted as a substrate (Young and Shannon, 1959) or it may have enhanced succinate dehydrogenase activity (tha et a1., 1969). Swets and Wedding (1964) suggested that inhibition of the Krebs cycle enhanced 2,4—D uptake by Chlorella. The stimulatory effect of high levels of chloram— phenicol (above 5x10"4 fl) observed for both the hypocotyl and leaf strip tissue is similar to the data of Sargent and Blackman (1969). One explanation might be that the chloramphenicol, which selectively acts on protein synthesis in chloroplast (Ellis, 1969), inhibited the synthesis of RNase that was activated during the cutting of the leaf strips (Bagi and Farkas, 1967). Since Q10 values for NAA uptake of above 2.0 were only possible for living tissue (Table 45), it should be 244 assumed that the temperature dependency is another expression of the involvement of metabolic energy in the uptake process. Similar temperature effects have been reported for auxin uptake by leaf tissue (Luckwill and Lloyd—Jones, 1962; Sargent and Blackman, 1962; Greene, 1969), but they did not resolve the difference between metabolic and cuticular components. The cuticular com— ponent, likely the most limiting barrier in their system, could be contributing significantly to high Q10 uptake values (Norris and Bukovac, 1969). The very limited amount of NAA lost by efflux from the tissue was likely due to most of the accumulated NAA being immobilized as NAAsp. Andreae (1967) reported that after long treatments (24 hr) with NAA, the complexed NAA could not leak out of the tissue. It has been noted generally that complexed auxins like IAAsp and NAAsp are not translocated out of the tissue in which they are metabolized (Veen, 1966; Veen and Jacobs, 1969; Hertel and Flory, 19687 Goldsmith, 1968; Eschrich, 1968). The formation of the NAA conjugate seemed to be the major metabolic aspect of the NAA accumulation process in the leaf strip tissue. This type of mechanism might be similar to the electrostatic association uptake process that Hiatt (1968) has proposed for ion accumulation by roots. 245 This would mean that the NAA would enter the tissue by a passive mechanism, like diffusion, and would be irreversibly bound as the conjugate, establishing a gradient. Recalling I x l the pronounced sensitivity of NAA uptake to m—Cl—CCP, metae bolic energy was likely involved in either the formation of the conjugate or the "pumping" of the NAA to the site of electrostatic association. An ATPase system would be a possible mechanism for an active NAA pump. Low temperature had its greatest effect by negating the surge in uptake, not uptake above the NAA concentration gradient (Table 44). Thus it is likely that the conjugate forming system has a metabolic energy requirement. Further studies are needed to determine the energy requirement for enzyme synthesis or activation. Since the pH of the medium dramatically affected tissue viability, the effect of pH on NAA uptake was not examined on living tissue. This was necessary to avoid the interaction of the two variables. Andreae (1967) reported that the conjugate formation of NAA by pea roots was reduced greatly by lowering the pH of the medium from 5.3 to 4.6. In this study the effect of pH was examined on sorption by the dead control tissue where it was found that lowering the pH to 3.7 caused a 5—fold increase in sorption compared to pH 6.7. This demonstrated the greater sorption of NAA 246 by the dead leaf tissue when the auxin is in an undissociated form. Poole and Thimann (1964) noted a 5-fold increase in IAA uptake going from pH 7.5 to 4.5. l The almost total recovery of the NAA in the efflux experiment confirms the many reports that NAA is not decarboxylated by plant tissue (Luckwill and Lloyd—Jones, 1962; Zenk, 1962; Veen, 1966; Andreae, 1967). The difference in amount of NAA retained between the washed and non—washed tissue from the short—time course study was about 500 pmoles/10 mg dry weight of leaf strip tissue. The calculated volume, that a 5x10"6 fl NAA solution would occupy to give an equimolar concentration (see appendix for sample of calculation, Figure A—l), was about 100 pl which was about 2.5x the water content (or volume) of the tissue. A 250% value for free space is impossible, so some special factor must be involved that did not affect similar measurements for stem sections (Johnson and Bonner, 1956: Sabnis and Audus, 1967a) where it was found that free space was equal to between 21% and 30%. The tremendous surface area of the finely cut leaf strips might be one factor. Another might be weak binding (like hydrogen bonding) that would allow the NAA to con— centrate on the surface of the tissue. 247 C. Isolated Cells The chromatographic analysis of the accumulated NAA from the cycloheximide treated isolated cells (Table 54) demonstrated that the conjugate—forming enzyme system was likely operating from the beginning of the experiment. The minimal response to the several treatments made this point apparent. The lack of a surge in uptake during the time~course and concentration experiment (Table 32) can also be explained by the fact that the conjugate—forming enzyme was functioning before the experiment commenced. The results of the effect of light, temperature and pH were consistent with those of the leaf strip experiments and, therefore, need not be discussed further. The fact that the NAA conjugating enzyme appeared to be operating at the time the NAA uptake studies had com- menced does not mean that one should not consider their use. It is quite possible that the very nature of the cells being totally isolated, which facilitated a very rapid initial uptake, induced the enzyme system before the surge could be detected or effected by other treatments. It is also pos— sible that the mechanism for auxin uptake by isolated leaf cells might be slightly different from intact tissue. It should be recognized that further improvements on the cell isolation might be required before data from the two systems will correspond. Certainly the modified technique for leaf cell isolation reported in this thesis has been a significant step toward this goal but further refinement would most likely be beneficial. SUMMARY Several techniques were developed to assess tissue and cell viability. Quantitative measurement of tetra— zolium reduction permitted the formulation of a treating medium suitable for maximum soybean leaf tissue metabolic activity. Polarographic measurement of oxygen exchange rates in the light and dark proved to be a reliable method for assessing tissue viability. Using these monitoring methods, an improved technique was developed to enzymatically isolate viable leaf cells and it is described. A multiple comparison, using several viability assays, revealed that the enzymatically isolated leaf cells were about 50% less active than intact leaf strips while cells mechanically isolated exhibited minimal metabolic activity. NAA uptake by soybean hypocotyl tissue was studied to establish some of the basic experimental factors. Bacteria had no effect, while succinate enhanced NAA uptake. NAA uptake by leaf strip tissue was characterized by the following: 248 A minimum of 100 pmoles NAA/10 mg dry weight of tissue was required before the surge occurred. 3. Chromatographic separation of the compound showed that it corresponded to NAAsp. 4. Cycloheximide inhibited uptake and formation of the conjugate while aspartic acid enhanced‘both. 5. Light had a slight negative effect on NAA uptake and an inhibitor of photosynthesis had no effect on uptake in the light. 6. The uncoupler m—C1~CCP blocked NAA uptake above the concentration gradient, revealing a dependency on metabolic energy for uptake. 7. Temperature coefficients above 2.0 (only for living tissue) further substantiated the involvement of a metabolic factor for NAA uptake. 8. Only limited amounts of NAA were lost by efflux from the tissue. Extensive retention of NAA (that fraction not removed by washing with distilled water) made estimation of free space impossible. Sorption of undissociated NAA was 5—fold greater than NAA in its dissociated form. 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Effect of mannitol concentration on 2,3,5— triphenyl tetrazolium chloride reduction by leaf strips.1 TTC reduced Strip width (um) Mannitol concn 500 1000 Mean (M) (A—535 nm) 0.203 0.315 0.392 0.393a 0.25 0.293 0.401 0.347a 0.30 0.308 0.341 0.325a 0.35 0.292 0.354 0.343a 0.30 (frozen- thawed) “ ———— 0.095 0.095c --——0.5 mM CaSO45 0.231 --—— 0.23lb 1The various media were used for entire experiment. 2Means followed by different letters are significantly different at P=0.05. 3Treatment medium=mannitol, 0.01 M Tris—HCl, 4 mM socium succinate, 10 mg/ml PVP, 50 pg/ml BSA and adjusted to pH 7.4. “Pretreatment. 5In place of treatment medium but including 4 mM sodium succinate. 270 Table A2. Effect of dialyzed Ficoll on neo—tetrazolium reduction by leaf strips. Treatment NTC reduced1 (A—560 nm) Non—treated 0.305a Ficoll (5 g/100 ml) 0.205b Dialyzed Ficoll (5 g/100 ml) 0.276ab Dialyzate from Ficoll (5 g/100 ml) 0.247ab NaCl (1x10'3 5) 0.326a NaCl (1x10~3 M) + dialyzed Ficoll 0.213b _______—_______—_______'___.—__———————————————- 1Means followed by different letters are significantly different at P=0.05. 271 Table A3. Liquid scintillation cocktail used. Chemical ggl PPOl 4.0 Popop2 0.2 Naphthalene 60.0- Absolute methanol 100 m1 Ethylene glycol 100 ml 1,4—Dioxane made tonOOO ml 12,3—diphenyloxazole 21,4—bis-2-(5—phenyloxozolyl)—benzene 272 Table A4. The uptake of naphthaleneacetic acid by enzy— matically isolated leaf cells as affected by concentration and time. NAA Accumulated NAA1 concn (min) Sample time: 15 30 60 120 180 360 (M) (pmoles/10 mg dry wt) 1x10“7 6.510.3 1010.7 1610.5 2210.4 2410.9 2911.0 2x10—7 1210.7 1811.3 3012.4 3811.8 4312.7 5411.5 leO—6 4412.0 6612.0 10814.1 14814.9 17118.9 229113.0 2XlO-6 7113.2 10615.1 16817.8 2331ll.5 24619.6 373128.9 1X10_5 200112 29516 428112 605111 733113 1067130 1.5XlO'5259113 346116 547118 726126 906138 1368159 5x10_5 806172 888137 1310169 17461127 20651116 34531284 lxlO‘4 1369154 1855181 22121116 29611269 36001201 55451460 _______________________________________________._____________ 1SE given. 273 Figure Al. Sample calculation for final naphthaleneacetic acid internal concentration. 1. Start with the value=100 pmoles/10 mg dry wt. 2. Convert dry wt to aqueous volume 10 mg dry wt=50 mg fr wt Therefore 10 mg dry wt=40 mg H20 40 mg H20=40 ul1 3. Substitute aqueous volume for dry wt 100 pmoles/10 mg dry wt=100 pmoles/40 1 4- Simplify 100 pmoles/40 ul to 2.5 pmoles/U1 -6 5. Finally, 2.5 pmoles/ul=2.5u moles/l=2.5x10 M 1Calculating the volume occupied by the tissue 96 strips x14OU x 250“ x 25000U gives a calculated final volume of 42.0U1 4“‘imiimlmaluminum?“