MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped be10w. ESTINATION or THE APOPLASTIC POTASSIUM CONTENT or LEAFLET LANINA TISSUE or THE W NUTANT or m m L. BY Jean Marie Long A THESIS Submitted to Michigan State University in partial fulfill-ent of the require-ents for the degree of MASTER OF SCIENCE I Department of Horticulture 1987 ABSTRACT ESTIMATION OF THE APOPLASTIC POTASSIUM CONTENT OF LEAFLET LAHINA TISSUE OF THE ABQENIEQH,HUTANT OF £1§Qfl_fifilllflfl,L. By Jean Marie Long The apoplastic K+ content of leaflet lamina tissue of the Anggntggl mutant of Eiggn,§§11111, L. was estimated by eluting K+ at 1° C into a 5 mM CaClz solution bathing an area of tissue with abaxial epidermis removed. The elution time-course curve was interpreted as indicating an initial rapid diffusion from the apoplast followed by a slower, constant net rate of efflux across the plasmalemna. Extrapolation of the constant efflux rate to zero tine was used to estimate apoplastic K+ content. When plants were grown at 2 and 10 m! K+, estimates were 88 and 142 ug K+ 1, respectively. Assuming an apoplastic solution volume gfw' of 0.1 ml gfw", these correspond to concentrations of 23 and 36 mH, respectively. Apoplastic K+ represented approximately 2% of tissue K+. Estimated K+ bound by fixed negative charges in the cell wall during elution was 0.1 ueq gfw", or 4 to 11% of apoplastic KI. ACKNOWLEDGMENTS I wish to thank the members of my thesis committee, Drs. James A. Flore, Robert C. Herner and Stanley L. Flegler, for their helpful comments and suggestions. I am particularly grateful to Dr. Irvin E. Hidders, my major professor, for his constant support, endless supply of ideas and assistance with some of the experiments. I also thank the staff of the Center for Electron Optics for their suggestions and encouragement. I owe utmost thanks and a number of past-due dinners to my husband, Dave, who patiently guided me into the Computer Age. Without his help I might still be writing this. ii TABLE OF CONTENTS Page LIST OF TABLES ......................................... vi LIST OF FIGURES .... ..... . ..... ............... ........ .. vii INTRODUCTION ..... ...... ...... ..... ......... ....... ..... 1 LITERATURE REVIEW ................................ ...... 6 Functions and Movement of Potassium in Plants ........ 6 Characteristics of the Apoplast ...................... 9 Apoplastic Transport ........... ...... _................ ii Free-Space Studies ................................... i7 Determinations of Apoplastic Solute Contents ......... 24 MATERIALS AND METHODS . ...................... .. ......... 29 Selection of Plant Material .......................... 29 Plant Culture ...... ..... ... ..... ...........r......... 31 Elution Procedure .................................... 37 Special Considerations for the Elution Procedure ..... 41 Effects of sealant . ...... . ................ . ........ 4i Vacuum infiltration ................................ 42 Sampling technique .... ........ ..................... 42 Isolation of Cell Nalls .............................. 44 Movement of Elution Solution into Tissue ......,...... 44 Determination of Free-Space Characteristics .......... 47 Collection and Analysis of Xylem Sap ................. 50 Determination of Tissue K+ and Rb+ Contents .......... 51 iii Solution Uptake through the Petiole ................ .. Pulse-Chase Experi.ent ......0.0.00...OOOOOOOOOOOOOOOO Glassware Washing Procedures ..... .................... Statistical Analysis ......... .......... . ........... .. RESULTS AND DISCUSSION ................ ......... ........ Characteristics of the Elution Curve .... ........ ..... Principles of Ion Fluxes ............................. Interaction of Elution Solution with Tissue .......... Effects of ions in solution ......... ............... Effect of pH of the elution solution ............... Infiltration of tissue by elution solution ......... Analysis of Components of the Elution Curve ...... .... Temperature effects ................................ Effects of altering apoplastic K+ concentration .... Effects of extra- and intracellular KI concentrations .... ............................. . Estimation of Apoplastic K+ Content and concentration ......... ...... ............ ........ .0... Estimation of apoplastic K+ content using the elutlon curve .0... ...... ......OOOOOO0.00000 000000000 Conversion of apoplastic K+ content to concentration ....................... .............. . Comparison with previous estimates ................. Comparison of xylem-sap and estimated apoplastic K+ concentrations ....................... Free-Space Characteristics of Argentgnl_beaflets ..... Estimation of XI Bound by the Donnan Phase ........... Elution Sensitivity to Changes in Xylem-Sap K+ concentration .......................... ...... ..... iv 90 99 104 109 110 113 117 119 Page Pulse-Chase Experiment ............................... 125 Plants Grown at Two KI Levels ........................ 131 SUMMARY AND CONCLUSIONS ................................ 136 APPENDIX: X-RAY MICROANALYSIS OF FREEZE-DRIED LEAFLETLAHINATISSUE...............OOOOOOCOOOOOOOOOOO.14o Introduction ......................................... 140 Saaple Preparation Methods .......................... . 141 Materials and Methods ................................ 144 Results and Discussion .. ..... ........................ 149 Conclusions ....... ....... ............................ 157 LITERATURE CITED OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO ‘59 Table LIST OF TABLES Potassium removed by elution fron Arggn&333_leaf- lets at 1° C in 24 hours plus that renaining in the eluted volume of tissue compared with total K contents of analogous but noneluted tissue volumes from the opposite half of each leaflet. Results of two experiments are shown. Values are means of six replications per experiment t Standard errorBOO00......O.......OOOOOOOOOOOOOOOOOO Apoplastic K+ content and concentration of leaflets from plants grown at 4.25 mM K as estimated from various elution experiments. Values are means of six replications per experi- ment 1 standard errors............................. Free-space characteristics of fully expanded A;ggn131n,leaflet lamina tissue.................... Estimates of K+ bound in Donnan phase in equi- librium with elution solution and as a percent of apoplastic K+ content for experiments included in Table zOOOOOOOOOOOOOOOOOO0.000000000000000000000 Steady-state Rb+ elution rates and estinated apoplastic contents of A:ggn1g1.,leaflets that had taken up 1, 10 or 100 mM RbCl solution for 0.5, 3 or 22 hours. Values are means of six replications 1 standard errors..................... Summary of experimental results when excised Agggn1g3.,leaves took up a pulse of 50 mM RbCl solution and Rb+ was subsequently eluted fron leaflets after a delay of 10, 30 or 180 minutes. Values are means of six replications 1 standard errorSOOOOOOOOOOOOO0.0.0.0.........OOOOOOOOOOOOOOOO Summary of experimental results for leaflets of Argentgng plants grown in sand and supplied with nutrient solution containing 2 or 10 mM K Values are means of six replications 1 standard errorSOOOOOOOOOO0.0......OOOOOOOOOOOO0.000.000...O. Results of X-ray microanalysis of cell wall in freeze-dried Argentggl leaflet tissue ..... .. ....... vi Page 77 108 114 118 121 128 133 150 Figure LIST OF FIGURES Page Scanning electron nicrographs of leaf spongy mesophyll (200K) revealed when abaxial epidermis was renoved fron (a) Argentgnl_leaflet and (b) tomato leaf................................... Effects of removal of abaxial epidermis on ion diffusion at 1° C into and gat of Apggntgnl_leaf- let tissue. (a) Uptake of Cl- -labeled 5 mM CaClz solution by discs with nd without epidermis. Rates (peeledi 2. 64x10’ , and unpeeled, 1. 38x10”2 ml gfw min ) significantly different at 5% level. (b) Elution of KI from peeled and unpeeled leaflets. Constant +rates l(peeled, 1. 72, and unpeeled, 0.15 ug KI gfw"l Iin ) significantly different at 1% level. Points on both plots are means of six replications t representative stand- ard errors except where smaller than symbol....... Elution of KI at 1° C into unbuffered (pH 5.0) and buffered (pH 7.0) 5 mM CaCIz solutions. Constan rates (pH 5.0, i. 68, and pH 7. 0, 1. 50 ug K: gfw min ) and Y intercepts (81.5 and 84.2 ug KI gfw' I, respectively) not significantly different. Points are means of six replications t representative standard errors................................... Potassium (°°RbI) and c1' (35c1') taken up at 1° c by peeled leaflet tissue from KCl bathing solu- tions of various concentrations. Difference curve, obtained by subtracting Cl' curve from KI curve, represents KI bound in DFS................. Potassium elution at 1° C into 5 mM CaClz and 5 mM CaSO4 solutions and deionized water. Constant rates CaCl , 2.26, CaSO , 1.58, and H20, 1. 38 ug K gfw min" ) not signifIcantIy different. Y intercep s for CaCIz and CaSO4 (96. 6 and 99.4 ug KI gfw' , respectively) not significantly different. That for H O (83. 7 ug KI gfw significantly differen from both at 5% level. Points are means of six replications t repre- sentative standard errors......................... vii 32 34 43 49 67 Figure Page 6. Potassium elution at 1° C into 0. 5, 5 and 50 mM CaClz solutions. Constant rates for 0. 5 IM and 5 mM (2.79 and 2.12 ug KI gwa ninI , respec- tively) significantly different at 5% level. That for 50 mM (0.84 ug K gwa minI ) significantly different from both at 1% level. Y intercepts for 0. 5 mM and 50 mM (131.4 and 69. 6 ug KI gwa 1, respectively) significantly di feren at 5% level. That for 5 mM (88. 9 ug KI gwa minI ) not sig- nificantly different from either. Points are means of six replications t representative standard errors.................................. 69 7. Potassiun elution at 1° C into 5 mM CaClz and 10 NM RbCl (plus 0.5 mM CaCl ) solutions. Con- stan rate (CaCl , 0.73, an RbCl, 1.24 ug x+ gwa minI ) signIficantly different at 1% level. Y intercepts (70.0 and 69.0, respectively) not significantly different. Points are means of six replications t representative standard errors.... 71 a. Infiltration of 3°ClI-labeled 5 mM oac12 solu- tion at 1° C into only the peeled tissue within the cylinder (disc) and into the entire half leaflet to whic cylinder was attached. Rates (disc, 3.4 x10I , and half leaflet, 2.12x10I ml gfw minI ) not significantly different. Points are means of six replications t representative standard errors.................................. 75 9. Potassium elution at 20° and 1° C. onsta t rates (20°, 2. 04, and 1°, 0.27 ug x+ gwa minI ) sig- nificantly different at 1 level. Y intercepts (87. 6 and 63. 4 ug KI gwa , respectively) not significantly different. Points are means of six replications t standard errors................... 79 10. Twenty-four hour potassium elution at 20° and 1° C. Elution rates at one hour (20°, 1. 55, and 1°, 0.33 ug KI gwa min )_and at 1eight hours (0.088 and 0.28 ug KI gwa minI 1, re- spectively) significantly different at 5% level. Rates at four hours (0.26 and 0.28 ug KI gwa minI , respectively) not significantly dif- ferent. Points are means of six replications t representative standard errors................... 83 V111 Figure Page 11. Potassiun elution from frozen/thawed leaflets at 20° and 1° C. Rates at _five minutes (20°, 673, and 1°, 597 ug wfw 1, 20 minutes (113 and 89.1 ug KI gwa einI , respectively)l and 40 minutes (41. 7 and 30. 3 ug KI gwa minI , respectively) not significantly different. Points are means of six replications 1 repre- sentative standard errors......................... 85 12. Potassium elution at 1° C from leaflet tissue with and without cells ruptured by abrasion. Rates at five a nutes (abraded, 94. 4, nonabraded, 10. 2 ug KI lgwa minI 1) significantly different at 1% level , but constant rates (2. 82 and 2. 83 ug KI gwa min, respectively) not significantly different. Points are means of six replications 1 representative standard errors.................. 87 13. Potassium elution at 1° C from leaflet discs, previously rinsed for 20 minutes in 5 mM CaClz solution, and from rinsed and unrinsed peeled tissue of intact leaflets. Constant rates for leaf ets ( insed, 3. 62, and unrinsed, 4.89 ug KI gwa minI ) not significantly d1 feren . That for rinsed discs (10. 5 ug KI gwa minI,) sig- nificantly different from both at 18 level. Points are means of six replications 1 repre- sentative standard errors......................... 89 14. Effects of single versus repeated sampling of solution eluting a given leaflet on (a) K concentration in the solution over time and (b) time course of KI elution. ~Points on both plots are means of six replications 1 repre- sentative standard errors......................... 93 15. Appearance in Agggntggl, leaflets of 86RbI- labeled 10 mM KCl solution taken up at 25° C by excised leaves through petioles . Points are means of two replications......................... 96 16. Elution of RbI at 1° c from leaflets after excised leaves had taken up 20 mM RbCl solution through petioles for 15 or 90 minutes at 25° C. Rates for first 10 minutes and constant rates (given in text) significantly different at 11 level. Points are neans of six replications 1 representative standard errors except where smaller than symbol............................... 98 ix Figure Page 17. Typical curve for KI elution from Azggntgg; leaf- let lamina tissue at 1° C and difference curve obtained by subtracting out constant cell efflux rate. Y intercept of linear extrapolations is estimate of apoplastic KI content................. 102 18. Rubidium elution at 1° C from leaflets after excised leaves had taken up 1 mM RbCl solution through petioles for 0.5, 3 or 22 hours at 25° C. Results in Table 5. Points are neans of six replications 1 representative standard errors except where smaller than synbol ............ . ..... 122 19. Rubidium elution at 1° C from leaflets after excised leaves had taken up 1, 10 or 100 mM RbCl solutions through petioles for three hours at 25° C. Results in Table 5. Points are means of six replications 1 representative standard errors............................................ 124 20. Rubidiun elution at 1° C from leaflets after excised leaves had taken up a six-minute pulse of 50 mM RbCl solution through petioles at 25° C. Delays between pulse and start of elution were 10, 30 and 180 minutes. Results in Table 6. Points are means of six replications 1 representative standard errors ..... ... ............. .............. 127 21. Potassium elution at 1° C from leaflets of plants grown at 2 or 10 mM KI. Results in Table 7. Points are means of six replications 1 representative standard errors except where smaller than symbol.....-......................... 132 22. Positioning of leaflet in specimen holder and area cut out before freezing in liquid propane ..... .... 146 23. Portion of X-ray spectrum for cell wall of freeze- dried A;ggntggn_leaflet lamina tissue correspond- ing to analysis 1a in Table 8..... ..... ........... 151 INTRODUCTION Potassium (KI) is considered the most important inorganic solute in higher plants (Lauchli and Pfluger, 1978; Marschner, 1983). Its roles in the production of cell osmotic potential (Mengel and Arneke, 1982), enzyme activation (Evans and Hildes, 1971) and electrical charge balancing (Smith and Raven, 1976) have aaior implications in plant growth, water use, metabolisn, assimilate transport and pH regulation. Numerous studies have been conducted to determine the total KI content of whole plants as well as of individual organs, tissues, cells and subcellular organelles. In contrast, the extracellular, or apoplastic, KI content of plant tissues has received relatively little attention. There are several reasons why the extracellular KI of leaves in particular is of interest. Apoplastic KI may have specific functions in leaves. In some species, for example, it seems to be involved in one or more phases of phloem loading (Giaquinta, 1983). It is not known whether extracellular KI levels normally occurring in leaves play a regulatory role in this process (Do-an and Geiger, 1979). If so, these levels nay affect carbohydrate partitioning within the plant and, thus, quality and yield. 2 Apoplastic KI nay also have enzyne-activation and pH- regulating roles. Knowledge of the extracellular KI content of leaves would be helpful in delineating the relative importance of intra- and extracellular paths of KI transport between cells or tissues. To date, such studies have largely concerned roots, where ion movement from the soil solution to conducting tissue has been of interest. It is equally important to understand KI transport in the leaf with regard to overall ion distribution as well as that required for specific processes such as salt excretion, changes in guard- cell turgor pressure during stonatal movement, and turgor- mediated leaf novements. The relationship between extracellular KI and regulation of total leaf content is of considerable interest. An optimum leaf KI status is inportant to plant growth and productivity. Yet the facility of KI retranslocation within the plant, particularly from older to developing tissues (Lauchli, i972b), often leads to a decline in the KI content of nature leaves (Naughman and Bellamy, 1981). This may occur to the point of leaf KI deficiency, as during fruit development on some tomato cultivars (Lingle and Lorenz, 1969; Hidders and Lorenz, 1982b). The existence of evidence against a significant loss in the ability of leaf cells to take up x+ (Hidders and Lorenz, 1983a) suggests that availability of the ion for uptake say be an important factor. 3 Robinson (1971) and Pitman (1975) proposed similar models for the regulation of leaf ion content in which the apoplastic and symplastic (intracellular) compartments are considered to be interdependent. According to these models, the symplastic ion concentration is the net result of fluxes through plasmodesmata and across the plasma membrane, the latter being dependent in part on the content of the apoplast. The apoplastic concentration, in turn, is the net result of import and export through vascular tissues, cell uptake and efflux, and ion exchange at the cell wall. The influence of extracellular KI concentration on cell uptake has been demonstrated by Nidders and Lorenz (1983a and b), who found KI fluxes in tomato leaf cells to depend on external KI concentration. The ability to determine how the extracellular ion concentration changes during plant ontogeny or under varying growing conditions may therefore be essential to understanding and potentially altering changes in leaf KI status. Determination of extracellular KI content presents special problems for conventional methods of compartmental analysis. In leaves the problens are compounded by the presence of the cuticular barrier to diffusion. Kinetic analysis of radioactive tracer efflux ’from leaf tissue (discussed in Walker and Pitman, 1976) has required cutting the tissue as well as extensive presoaking to load the tracer, making the method unsuitable for quantitative determinations. 4 Ion loss during precipitation techniques (Harvey 1; al., 1979) and the low resolution of autoradiography for 42KI (Flowers and Lauchli, 1983) limit their use for extracellular KI quantitation. Several workers (Etherton, 1968; Lauchli, 1972b; Flowers and Lauchli, 1983) have cited the difficulty in manipulation of ion-sensitive microelectrodes as a limiting factor for analyzing compartments as small as the cell wall, where apoplastic KI is thought to be located. In view of the above, the objective of the present study was to develop and evaluate alternate methods for estimating extracellular leaf lamina KI content. The research focused on two methods. The first method considered was kinetic analysis of the elution of endogenous KI from leaflet lamina tissue of the Argentggl mutant of algal, 3331111,L. A modification of previously used efflux techniques, it does not require cutting or presoaking of the tissue. It is an indirect method requiring little sample preparation and generally available instrumentation, and could be expected to yield information on overall apoplastic KI content. The second method evaluated was X-ray microanalysis of freeze-dried Argentgnn, leaflet tissue. The potential for good spatial resolution and quantification of elemental content make X-ray microanalysis suitable for use in compartmental KI analysis (Lauchli, 1972a). It is a direct method requiring rigorous sample preparation and highly 5 sophisticated instrumentation. However, it has the capability of determining extracellular KI concentration at specific locations in the leaf. The first method is described in the body of this paper. The second, which proved less successful, is discussed in the appendix. LITERATURE REVIEW W Potassium is an essential nutrient for higher plants, required for normal growth and development (Mengel and Kirkby, 1982). Though not a structural constituent of tissues or metabolites (Lauchli and Pfluger, 1978) it may account for up to 8% of the dry matter of higher plants, its concentration exceeding that of any other cation (Evans and Sorger, 1966). This high content attests to potassium’s roles in numerous physiological processes. As the principal osmotically active solute in plants, KI is required for attaining the turgor responsible for cell expansion during growth (Zimmerman, 1978; Mengel and Arneke, 1982). Potassium fluxes are also involved in the changes in motor-organ turgor causing nyctinastic leaf movements in several legume species (Satter and Galston, 1971; Kiyosawa and Tanaka, 1976; Campbell gt aL., 1981). Considerable work has established a central role for KI in the turgor-mediated opening and closing of stomates (Humble and Raschke, 19713 Raschke and Fellows, 1971; Edwards and Bowling, i984), and therefore in the regulation of the plant's overall water status (Brag, 1972; Hsiao, 1976). 7 Numerous enzymes are activated by KI, including a starch synthase (Hawker gt_al,, 1974), pyruvate kinase, and some of those required for protein and nucleotide synthesis (Evans and Sorger, 1966; Suelter, 19701. A KI-stimulated ATPase located at the plasmalemma appears to be involved in proton pumping and transport of other ions across the membrane (Hodges, 1976). Potassium balances organic and inorganic anion charges in long-distance transport in the phloem (Marschner, 1983) and xylem (Kirkby and Armstrong, 1980). It serves as a counterion in active HI transport across membranes, which may have implications in overall regulation of intracellular pH (Smith and Raven, 1976). This role seems to be important in both oxidative (Kirk and Hanson, 1973) and photosynthetic (Steineck and Haeder, 1978) phosphorylation where proton gradients across mitochondrial and thylakoid membranes, respectively, may drive ATP formation (Mitchell, 1966). Potassium has additional roles in photosynthesis. Its promotion of the synthesis of RuBP carboxylase (Steineck and Haeder, 1978) required for C02 reduction, as well as its role in stomatal movement, affect the rate of diffusion of 002 to chloroplasts in the leaf mesophyll (Peoples and Koch, 1979; O'Toole g1,gl., 1980: Moorby and Besford, 1983). Potassium may also be required in chloroplasts for chlorophyll synthesis (Marschner and Possingham, 1975) and to maintain the structure of grana stacks of the thylakoid membrane (Penny g1,31,, 1976). There is considerable evidence that KI promotes the translocation of photosynthates (Hawker gt_aL., 19743 Mengel and Viro, 1974). Its high phloem-sap concentration (Hall and Baker, 1972) contributes to the osmotic potential in sieve tubes, maintaining turgor at low sucrose concentrations (Giaquinta, 1983) and contributing to osmotically generated phloem movement (Moorby and Besford, 1983: Vreugdenhil, 1985). Mengel and Haeder (1977) reported a positive relationship between KI supply and phloem loading in 313131;. The extracellular KI content in particular seems to influence one or more aspects of loading from the apoplast thought to occur in some species. In 31;; yglgagig, for example, sugars seem to be actively loaded into the phloem from the apoplast, having been released from the symplast in the vicinity of the phloem (Giaquinta, 19763 1983). Several workers (Hawker g3, 31., 1974; Doman and Geiger, 1979; Peel and Rogers, '1982) have reported stimulated sucrose efflux from mesophyll cells into the apoplast with increased external KI concentration. The work of Malek and Baker (1977) suggests that in Risingg, extracellular KI is directly involved in phloem loading from the apoplast. These workers and Hutchings (1978) have proposed that apoplastic KI is pumped into the companion cell-sieve element complex as protons are pumped out, the resulting proton gradient driving a proton-sucrose cotransport into the phloem. 9 The availability of KI for its many functions is enhanced by its high mobility in the plant. Potassium moves readily in both phloem and xylem, allowing it to freely circulate among organs (Lauchli and Pfluger, 1978). Its principal direction of transport is toward meristematic tissue which may be supplied to a large extent with potassium retranslocated from older plant parts (Greenway and Pitman, 1965). Fruit are particularly strong sinks for KI. Hocking and Pate (1978) reported retranslocation of up to 731. of leaflet 10 to fruit in m; similarly, Hidders and Lorenz (1979, 1982b) found considerable KI redistribution from vegetative tissue to fruit in tomato. WW Potassium's high mobility is in part the result of its moving readily between the two major compartments of plant tissue, the symplast and apoplast (Lauchli and Pfluger, 1978). The symplast consists of the cytoplasmic continuum bounded by the plasmalemma, with intercellular connections in the form of plasmodesmata (Crafts and Crisp, 1971). Conversely, the apoplast is that portion of plant tissue lying outside the plasmalemma and consisting mainly of the cell wall continuum and intercellular space (Esau, 1977). Mature, non-living xylem vessel elements and tracheids are also considered part of the apoplast (Esau, 1977). The cell wall consists of a cellulose framework embedded in a matrix of hemicelluloses, pectins and proteins 10 (Northcote, 1972; Haynes, 1980). The spaces between cellulose microfibrils, the units of wall structure (Esau, 1977), are large enough to accommodate water molecules as well as many solutes (Gaff gL_ 1L., 1964; Franke, 1967; Lauchli, 1976). While the size of these spaces is reduced by the matrix substances, water and ions are able to migrate in cell walls unless diffusion is restricted by lignification or suberization (Haynes, 1980). The apoplast therefore represents a transport pathway parallel to the symplast in plants (Neatherly, 1970). Pectic substances of the cell wall matrix are of particular interest in relation to ion transport in the apoplast. Pectic acid, a polymer of alpha-D- polygalacturonic acid, has free carboxyl groups which can provide fixed negative charges (Briggs g1,§L., 1961; Luttge and Higinbotham, 1979). With the exception of protons, divalent cations are bound to these negative sites in preference to monovalent cations (Haynes, 1980). Proteins in the cell wall, which provide additional negative charges, may also have a small number of positively charged sites (Lauchli, 1976; Luttge and Higinbotham, 1979). The binding of ions to these fixed charges is significant in that it modifies ionic concentrations near the plasmalemma, which may ultimately influence ion transport and accumulation (Lauchli, 1976; Haynes, 1980; Greenleaf g1_al,, 1980). Besides serving as a transport pathway, the apoplast is the site of physiological processes (Preston, 1979). Due to 11 the presence of solutes and enzymes in the cell wall, biosynthetic and other reactions may occur there (Northcote, 1972). For example, sucrose hydrolysis in the apoplast may be a prerequisite for sugar transport in many plant tissues (Giaquinta, 1980). The apoplast is in communication with the symplast via the plasmalemma, and so is affected by cell processes (Preston, 1979). Its composition and content are determined in part by cell metabolism and seem to be maintained at a certain equilibrium with cell contents (Kursanov, 1984). MW There has long been interest in the plant apoplast as a transport pathway for water and ions. As ~early as 1928, Scott and Priestley proposed that the soil solution diffuses into roots via the cell walls. In a review in 1951, Robertson suggested that ions move passively through the root along a concentration gradient in cell walls and water- filled intercellular spaces. Jacobson gt, 1L. (1958) and Brouwer (1959) demonstrated the existence of a barrier between the root medium and xylem vessels, precluding an entirely extracellular pathway and requiring ion uptake across the plasmalemma. This barrier was further elucidated by Robards and Robb (1972), who studied ion movement in the apoplast of barley roots using an electron-dense stain. Except in the root tip, ion movement through cell walls was restricted by the 12 endodermis. Similar results were reported by Nagahashi g1, aL. (1974) for corn roots. Peterson :1, 1;. (1986) used lanthanum (La3I) as an electron-dense marker of apoplastic solute movement in corn and barley roots. Within the. root meristem solutes were found to move to the stele via the apoplast. Several studies of apoplastic ion transport in roots have focused specifically on the movement of KI. Vakhmistrov (1967) studied the ability of various root cells to absorb KI and concluded that the extracellular solution might serve as a source for its uptake. The root apoplast does not seem to be significant in KI transport, however. Van Iren and Van der Sluiis (1980) used autoradiography to localize potassium in barley roots. In mature roots, KI uptake seemed to be at the epidermis or hypodermis, the apoplastic pathway having little significance in radial transport. Similar results were reported by Chino (1981), who used X-ray microanalysis to determine KI distribution in the root cortex. Potassium was found mainly within cells, suggesting symplastic movement. Plant tissues other than roots have also been studied with respect to extracellular transport. Priestley (1929) suggested that cell walls and intercellular spaces may be of importance in transport to the shoot apical meristem prior to differentiation of the vascular system. Hylmo (1953) studied changes in CaClz uptake by intact pea plants at varying transpiration rates and concluded that the external 13 solution passes by mass flow through the plant apoplast coupled with movement of water alone through vacuoles. In a recent study Erwee and Goodwin (1985) used fluorescent dyes to determine the extent of symplastic transport in various tissues of the aquatic plant Egnggb ggnga. They reported barriers in the symplast between some cells connected by plasmodesmata. Their model for transport in the whole plant consists of a large number of symplast domains between which apoplastic transport may be necessary. Studies of leaf tissue suggest the apoplast may be of considerable significance in water . transport in some species. Levitt (1956) calculated that the rate.of water movement from xylem endings through mesophyll cells to evaporative surfaces would be five orders of magnitude slower than the transpiration rate. On these grounds he concluded that water in the transpiration stream moves through leaf cell walls. Gaff and Carr (1961) studied water movement in Eucalyptus leaves. They estimated that as much as 40% of the water content of the turgid leaf could be in the cell wall. On this basis the wall was proposed as the main path for extrafascicular movement of water, possibly serving as a buffer to water loss from the protoplast. In a review of plant water relations, Weatherly (1970) proposed that mesophyll cell walls represent the major pathway for water in leaves. The work of Burbano g1,g1. (1976), who used a stain and light microscopy to study water 14 movement in cotton leaves, supports this hypothesis. In grasses, however, apoplastic water movement seems to be restricted to areas surrounding secondary veins due to the presence of a suberized bundle sheath surrounding major veins (Crowdy and Tanton 1970; O'Brien and Carr, 1970). These findings were more recently corroborated by Canny and McCully (1986) by means of a stain detected by electron microscopy in freeze-substituted, resin-embedded corn leaf tissue. There may also be varying degrees of apoplastic transport of ions and other solutes in leaves. Kylin (1960) proposed that such transport involves both symplastic and apoplastic routes. This concept was supported by the work of Gunning and Pate (1969), who studied transfer cells in leaves. The primary role of these cells was proposed to be in solute exchange between xylem and phloem and their surrounding tissues. The extensive wall ingrowths characteristic of transfer cells were thought to provide an extracytoplasmic compartment from which solutes may be absorbed by cells. Differences among families in number and types of leaf transfer cells suggest the relative importance of apoplastic transport may also vary (Pate and Gunning, 1969). Using precipitation of AgCl, Van Steveninck and Chenoweth (1972) examined ClI transport in the mesophyll of barley seedlings. They reported primarily symplastic 15 movement of the ion, but concluded that apoplastic transport may occur in certain areas of the cell wall. Thompson g1, 31. (1973) delineated an extracellular continuum for ion movement in ALLLRLEK leaves using La3I as an electron-dense tracer. Lanthanum was found to be distributed in walls throughout the mesophyll; however, since La3I did not cross the plasmalemma, this demonstrated only that ions can move in the leaf apoplast and not the extent to which this occurs for those that can be taken up by cells. Campbell g1,aL. (1974) used a similar technique to demonstrate an apoplastic pathway to salt glands in several halophytes. Anatomical changes in the sunflower leaf during development were recorded by Fagerberg and Culpepper (1984). They reported a decrease with leaf age in the extent of the apoplastic path relative to the volume of tissue supplied, suggesting cell walls may be more important in water and ion transport in young leaves than in mature ones. Evert g1,11. (1985) used ion precipitation to delineate a transport pathway in the transpiration stream of the corn leaf. Ions moved readily from vessels into the apoplast of phloem and bundle-sheath cells, but further ion movement was restricted by suberized bundle sheath walls. A pressure-dehydration technique was used by Jachetta g1_a1. (i986a) to determine the distribution of l4C-labeled atrazine and glyphosate between the apoplast and symplast of sunflower leaves to help elucidate the transport pathways of 16 these herbicides. In another recent study the mode of transport of solutes in leaves of Lnglg§_ tnlgglgn, was investigated by Madore gt, aL. (1986) using liposome- encapsulated fluorescent dye injected into cells. These workers reported no apparent barrier to symplastic movement from mesophyll to minor veins that might necessitate apoplastic transport. The extent of apoplastic ion movement to specific leaf tissues has received some attention. K-ray microanalysis was used by Campbell g1,al, (1981) to study the path of KI and 01' migration between opposite sides of the motor organ of the legume Sangng§,during turgor-mediated leaf movement. Results based on ion distribution between the protoplast and cell wall indicated an apoplastic path. In a related study, however, Satter g1, a1. (1982) reported a barrier_ to apoplastic diffusion within the pulvinus, necessitating a partially symplastic pathway. Hsiao (1976) reported guard cells of some plants are able to take up KI from an external medium, suggesting the apoplast may be a source of potassium for stomatal opening. Edwards and Bowling (1984) measured extracellular potassium activity across the stomatal complex of Izgdggggntlg, The large electrical gradients observed between cells suggested little direct continuity between cells of the complex. These workers reported a large increase in KI activity in the guard-cell wall upon stomatal closure, indicating that potassium may move out into the apoplast. 17 Erwee £5.11. (1985) examined cell-to-cell communication in leaves of anngllng, gyang3,with fluorescent dyes. Dye injected into epidermal cells rarely moved into guard cells, where ion fluxes were proposed to occur via the apoplast. W In addition to studies of ion movement within the apoplast, considerable work has investigated that compartment as intermediary for ion movement between cells and an external solution under experimental conditions. Briggs and Robertson (1948) examined the uptake of various substances by potato tuber discs. Small, nonpolar molecules were found to freely penetrate the entire volume, while electrolytes and large molecules readily entered only part of the disc. Since the ions in this freely accessible phase diffused out of the discs into water, diffusion was assumed to be the principal mechanism for their entrance. These workers proposed the existence of a volume lying outside the vacuole whose contents readily exchange with an external solution. The first reported measurements of the regions of the root accessible by free diffusion were by Hope and Stevens (1952), who studied the reversible diffusion of KCl between bean root tips and an aqueous solution. They estimated that 13% of the root volume was occupied by this easily accessible phase. This volume was termed 'Apparent Free Space“ (AFS) since the nature of ionic interactions between 18 the measuring solution and the compartment was not known, allowing only an estimate of its volume. Most of the AFS was believedi to lie within the protoplasm, where these investigators proposed the existence of immobile anions constituting a Donnan phase. . Stiles and Skelding (1940) found the time course of MnZI uptake by carrot root tissue to be characterized by an initial period of rapid uptake followed by a much slower phase. Epstein and Leggett (1954) proposed two modes of ion uptake by roots corresponding to such flux rates. In the first, termed exchange adsorption, roots act as cation exchangers. This uptake, which is not linear with time, reaches equilibrium in about 30 minutes, involves readily exchangeable ions, does not require energy, and is not selective. In the second phase, active transport, ions are essentially not exchangeable. This uptake is linear with time, does not reach rapid equilibrium, requires energy and is selective. Epstein (1956) concluded that ion penetration into the 'free' spaces of plant tissue, the first mode of uptake, is a prerequisite for active transport, the second mode. The model was later amended to include an initial diffusion phase to exchange sites (Epstein, 1962). Briggs (1957) reviewed the concept of free space. The AFS was described as consisting of a 'Water Free Space' (WFS) where the ionic concentration equals that of the surrounding solution, and a ”Donnan Free Space“ (DFS) where the ionic distribution is controlled by the fixed negative 19 charges of cell walls. The characteristic initial rapid phase of uptake was said to represent ion movement into the APS and the slower phase, accumulation by the 'Osmotic Volume.“ Consistent with their earlier report, Briggs and Robertson (1957) proposed that free space includes the intracellular volume outside the tonoplast as well as cell walls and injected intercellular spaces. In contrast, Levitt (1957) argued that only the cell walls are available for diffusion into roots since the protoplasm is surrounded by a differentially permeable membrane which restricts free diffusion. Levitt's point of view was later corroborated by efflux studies by Pitman (1963) and Cram (1968) which indicated that the cytoplasm and vacuole are phases in series with the free space, the plasmalemma and tonoplast acting as boundaries to ion movement. There is now general agreement that the terms apoplast and free space are synonymous (Luttge and Higinbotham, 1979). Early reports of free-space measurements for roots are fairly consistent. Hope and Stevens (1952) determined an AFS volume of 13% for bean roots, while a volume of 8 to 25% was estimated for roots of beans and corn by Bernstein and Nieman (1960). For wheat roots, Butler (1953) reported an AFS of 24 to 34% and later (Butler, 1959), 18 to 20%. Pitman (i965a) estimated an AFS volume in barley roots of 20 to 25%, while the cation exchange capacity of the DPS was 2 meq kgII fresh weight. 20 Briggs g1, 11. (1958) studied radioactive tracer uptake by beet root discs. WFS was found to occupy 20% of the disc tissue, 15% attributable to cut cells and the remainder, to intercellular spaces. The amount of nondiffusible anions in the DPS was determined to be 10 to 14 meq kgIl in a volume of about 2% of the root, for a concentration of 560 meq III. Briggs g1, 1L. (1961) extensively discussed estimation of free-space characteristics and the significant influence of experimental conditions on such determinations. This influence was demonstrated in a recent study by Shane and Flood (1985). Using mannitol along with substances of high molecular weight that did not penetrate the root, they corrected for the contribution of surface film to the measurement of AFS of barley roots. Their resulting AFS estimate was only 4.5 to 5.1% of root volume. Leaf tissue has also been the subject of free-space investigations. Kylin (1957) demonstrated the presence of an 'outer space” in the leaf of Vallisngzigw an aquatic plant. Efflux of labeled sulphate from that tissue was characterized by rapid followed by slow, continuous rates, as found for roots. The AFS was estimated to be about 8% of leaf volume. Kylin and Hylmo (1957) reported similar results for wheat shoot tissue, leading Kylin (1960) to conclude that AFS is a property of green tissues as well as roots and storage tissue. Kylin (1960) reported WFS volumes of 4 to 5% for leaves of gnagggla,and 15 to 18% for wheat. Crowdy and Tanton (1970) studied the location of free space 21 in wheat leaves using precipitation of lead chelate and found it to be localized within cell walls. The free space was estimated to occupy only 3 to 5% of the tissue. In another study of Valllsngnlg, Winter (1961) reported a Donnan phase for leaves which, like that in roots, is located in the cell wall and contains cations removable by exchange. Mecklenburg :1, 1L. (1966) also reported an exchangeable cation pool in the free space of leaves, while Van Steveninck and Chenoweth (1972) found ClI to be partially excluded from the cell wall of barley leaves, supporting the existence of a cation exchange system there. Using ion uptake by Atzlnlgx_leaf slices, Osmond (1968) measured an AFS volume of 20% and estimated the amount of exchange sites in the DPS at 15 ueq g fresh weightIl (gfw). Uptake was thought to be first into an exchangeable free- space fraction and then into a nonexchangeable compartment. On this basis it was suggested that ions transported to the leaf by the vascular system are delivered to the free space, from which they are absorbed by adjacent cells. An examination of KI uptake by corn leaf slices (Smith and Epstein, 1964a) revealed kinetics and selectivity resembling those previously reported for roots, suggesting that ion carriers and their mode of operation are similar for leaves and roots. These workers (Smith and Epstein, 1964b) proposed that leaf free space may play the same role in cellular ion uptake as that of roots. They questioned 22 whether selectivity of the plant is due to mechanisms in the roots only, or in shoot cells as well. Pitman g1,aL. (1974a) used uptake of 86RbI and 36ClI to determine free-space characteristics of barley leaf slices. In support of previous findings, they reported that leaf free space could be considered to consist of WFS and DFS as in storage tissue and roots. The rapidly exchanging WFS was said to include cut cells, injected intercellular spaces and surface films of solution, while a more slowly exchanging inner component corresponded to the DPS. WFS was estimated to be 0.21 ml gwaI. The DFS contained exchange sites equivalent to 3.6 ueq gwa1 in a volume of 0.013 ml gwa1, for a concentration of 280 ueq mlI‘. The pKa of the fixed anions was confirmed to be 2.8. The above authors discussed extrapolation to the intact plant of free-space determinations made on cut tissue. Since cutting does not destroy cell wall, DFS measurements using slices were thought to be valid estimates for the intact plant. The WFS volume, however, was considered to be different in the intact plant which would lack cut cells and free water in intercellular spaces. In the intact leaf WFS was felt to be limited to a portion of the cell wall. In a review soon thereafter Pitman (1975) discussed the role of the free space in the regulation of leaf ion content and proposed the model presented earlier. He indicated that export from the leaf competes with cell uptake for ions in the free space and questioned whether the rate of influx to 23 cells is regulated by their vacuolar content or only by the availability of ions in the free space. Smith and Fox (1975) determined the free-space characteristics of Qitzns_leaf slices using efflux of 36ClI and 22NaI and reported a WFS of 0.2 ml gwal. Exchangeable cations in the DPS were 20 to 25 ueq gwaI, considerably higher than the amount reported for barley leaves. These workers speculated that the leaf DPS might act as an extracellular cation reservoir, the significance of which would depend on the pH of the free space and changes in uronic acid levels in cell walls due to age or nutrition. Widders and Lorenz (1983b) determined free-space characteristics of tomato leaf slices using uptake of 36ClI and 86RbI. WFS volume, which was used to evaluate the integrity of the tissue, was estimated as 0.25 ml gwaI. There were 6.8 ueq gwa1 fixed anions in the Donnan phase volume of 0.012 ml gwa1 for an effective concentration of 550 ueq mlIl. A number of studies have more specifically concerned the fixed charges of the cell wall. Dainty and Hope (1959), working with the alga Chang, determined that DFS is located in the cell wall. In a related study, the indiffusible anions in the DPS were found to be proportional to wall thickness (Dainty g1,al,, 1960). Pitman (1965b) determined that exchange sites in the DPS arise from bound uronic acids in the cell wall. 24 A metabolism-linked binding of some cations in the DPS was reported by Ighe and Pettersson (1974), while Luttge and Higinbotham (1979) suggested Donnan exchange in the free space may be indirectly metabolically controlled by maintenance of structure. Pettersson (1966) and Persson (1969) proposed that exchange adsorption near the plasmalemma in the DPS may be the initial step in active ion uptake, forming the pool from which ions are accumulated. Kesseler (1980) examined the hypothesis that the preferential metabolic accumulation of KI by many algae may be enhanced by the preferential adsorption of the ion by the cell wall. In Valgnia, KI was found to be enriched by such selective adsorption. It was proposed that by exchange of selectively adsorbed KI ions with protons liberated during cell metabolism, uptake of KI into cells might be facilitated. QetenaInatI2na_e1_An2nlastls_fieluts_§2ntenta. A number of studies have been conducted to determine overall solute content of the apoplast, but quantitation of specific ions has been limited. Klepper and Kaufmann (1966) measured the osmotic potentials of leaf guttation fluid and xylem exudate from the stem and petiole of tomato. The guttation fluid was found to be dilute compared to the exudate, suggesting that the xylem solution becomes depleted by cell uptake as it passes through the stem and leaf. 25 In a study of extracellular solute concentration as reflected by freezing-point depression, Scholander £1,1L. (1966) collected fluid from leaves of several species using a pressure bomb. Solute concentrations were reported to be low in spite of possible contamination from damaged cells. Oertli (1968) determined the osmotic potential of extracellular fluid from barley leaves grown under varying salt regimes. Salt accumulation in the apoplast was more pronounced under saline conditions and was attributed to the limited capacity of cells to take up salts. Robinson and Smith (1970) studied uptake of ClI by Qit;ng,leaf slices in relation to external concentration. Their results suggested that an increase in cell uptake from the apoplast with increasing external concentration might contribute to the osmotic adjustment of leaf tissues under conditions of high salinity. They speculated that only if salt input to the extracellular compartment exceeded uptake would the osmotic pressure of the apoplast become high enough to cause salt damage. Cosgrove and Cleland (1983) determined the overall solute concentration in the apoplast of herbaceous stem tissue using perfusion techniques. The apopolast of growing stem tissue contained a significant concentration of osmotically active solutes, about 25% of which were inorganic electrolytes. This was attributed to a high solute requirement for maintaining cell turgor as well as a high rate of transpirational water loss from cell walls. The 26 osmotic pressure of the free space ranged from 2.9 bars at the apex to 1.8 bars at the base. The significant osmotic pressure in the wall was said to offer an explanation for negative water potentials in nontranspiring plants. Total concentrations of apoplastic solutes in sunflower leaves were determined by Jachetta g1_a1. (1986b) using the osmolarity of sap fractions expressed over small increments of pressure. The sap concentration of the cell-wall/minor- vein fraction was approximately 8 MO kgII. Jacobson (1971) analyzed extracellular fluid collected by centrifugation from Venus flytrap as a guide to formulating a physiological perfusion fluid compatible with isolated tissue. The estimated cation and anion concentrations in the free space were 27.9 and 16.5 meq III, respectively, with an estimated KI concentration , of 6.4 meq III. A perfusion solution formulated on the basis of these results proved useful in maintaining isolated cells and studying responses of the trap to extracellular ion concentration. Bernstein (1971) sought a direct method for determining solute potential in leaf cell walls. Vacuum perfusion was used to draw water through discs cut from amphistomatous leaves of several species, and successive fractions of the perfusate were collected. Based on the excess solute content in the first as compared to later fractions, the calculated total concentration of solutes originally present 27 in the cell walls ranged from 2 to 10 meq III. Extracellular KI concentrations of 1 to 5 mM were reported. An indirect approach to determining extracellular leaf KI concentration was taken by Pitman g1_al. (1974b), who studied KCl uptake by barley leaf slices from solutions of varying concentrations. These workers reported that cells appear to be in equilibrium with an apoplastic concentration of 5 mM KI (as KCl) and hypothesized an equivalent concentration in the intact leaf. More recently, Widders and Lorenz (1982a) reported a decrease in the KI concentration of tomato xylem sap from 12 to 5 mM during plant development and speculated this might contribute to a reduction in extracellular leaf KI concentration. In a related study to estimate this concentration, these investigators (Widders and Lorenz, 1983a) reported zero net KI flux for tomato leaf slices at external concentrations between 1.0 and 3.5 mM KI. They proposed that a decline in free-space concentration below this level might lead to net efflux from leaf cells. X-ray microanalysis was used by Harvey g1,g1, (1981) to determine ion compartmentation in Sgag§§,.§zltilg,leaves. Estimates were made of NaI, KI and ClI concentrations in intracellular compartments as well as in cell walls. These workers reported KI concentrations of 13 to 17 mM for the cell-walllintercellular-space. This paper was reported to be the first presentation of direct, absolute measurements of ion concentrations in may represent plant tissue. a landmark 28 these compartments. As such, it in compartmental ion analysis in MATERIALS AND METHODS W The cuticle covering leaves is a relatively impermeable barrier to ion exchange with an external solution (Northcote, 1972). This restriction of free diffusion in the intact leaf precludes attainment of a rapid equilibrium between the tissue and solution (Luttge and Higinbotham, 1979) and has imposed difficulties on the study of ion relations in leaf tissue. As a result, special techniques have been required to expose interior leaf surfaces to exogenous solutions. Some workers have used leaf slices to study ion fluxes (e.g. Smith and Epstein, 1964a and b; Pitman g;_a1,, 1974a and b; Smith and Fox, 1975; Widders and Lorenz, 1983a and b). Although freshly cut slices seem to retain valid uptake characteristics (Pitman g1, 31., 1974b), there are serious problems due to tissue damage. Pitman g1, al, (1974a) reported that 30% of the cells in 1-mm leaf slices may be damaged by cutting. Similarly, leaf discs are subject to considerable cell damage during preparation (Smith and Epstein, 1964b; Osmond, 1968). Cut tissue is not suitable for quantitative estimation of extracellular ion content due to ion leakage from cut cells (Robinson, 1971) and because resulting biochemical 29 30 changes in the tissue may affect the permeability of cell membranes still intact (Smith and Robinson, 1971; Ehwald g1 11., 1980). A technique that increases access to the leaf apoplast is removal of the epidermis and, with it, the cuticle. Morrod (1974) used epidermis-free tobacco leaf discs to study cell membrane permeability, while Delrot g1_al, (1983) collected sugars from the apoplast of peeled Vlgia, {aha leaves. The reported advantages to removal of the leaf epidermis are elimination of the cuticular barrier, an increase in the area for penetration of an external solution, and reduction of the diffusion pathway to the mesophyll (Morrod, 1974). The leaflets of Argentg33, a mutant of the garden pea (Elgnl_§§;1ynl,L.), have an epidermis that is easily removed by peeling. Hoch g1_al. (1980) reported large intercellular spaces between the epidermal layers and underlying mesophyll cells, apparently due to a weakened middle lamella, which give the leaflets a silvery appearance. These investigators found epidermal strips from A;ggn1gnn,to be largely free of mesophyll cell-wall fragments. In other respects, there are no apparent differences between mutant and normal plants (Marx, 1982). Peeled Argentggj_ and tomato lamina tissues were prepared for examination by scanning electron microscopy by fixation in 4% glutaraldehyde, dehydration in a graded ethanol series, critical-point drying and gold coating. 31 Scanning electron micrographs revealed negligible damage to underlying mesophyll cells when either abaxial (Figure 1a) or adaxial epidermis was removed from A;ggn;ggn,leaflets. Damage observed was even less than to Vigig, taha,leaves, which are often used for epidermal peels. In contrast, many cells were ruptured when the lower epidermis was removed from tomato leaves (Figure 1b). Azggntgnn,was therefore considered an ideal plant for the elution procedure used in this study. The effects of eliminating the cuticular barrier to diffusion are demonstrated in Figure 2. 36ClI-labeled CaClz solution moved into peeled leaflet discs at a significantly higher rate than for unpeeled discs (Figure 2a), in spite of the extensive cut edge available for diffusion. Figure 2b reveals the much more rapid elution of KI from peeled than from unpeeled areas of intact Axggnignn,leaflets. The methods used in both experiments are described later in this section. W Seeds collected from A;ggn1ggn,plants were sown in a sterile peat-based growing medium (Baccto Grower’s Medium; Michigan Peat Co., Houston, Tex.) in 16-oz opaque plastic drinking cups with drainage holes. Soaking seeds in water overnight before sowing reduced germination time by one to two days. Batches of approximately thirty plants were Figure 1. 32 Scanning electron micrographs of leaf spongy mesophyll (200K) revealed when abaxial epidermis was removed from (a) Argentggl leaflet and (b) tomato leaf. Figure 2. 33 Effects of removal of abaxial epidermis on ion diffusion at 1° C into and 032 of leaflet tissue. (a) Uptake of Cl-labeled 5 mM CaClz solution by discs with and without epidermis. Rates (peeled, 2.64x10I3, and unpeeled, 1.38x10I3 ml gwa1 minII) sig- nificantly different at 5% level. (b) Elution of KI from peeled and unpeeled leaflets. Constant ra es (peeled, 1.72, and unpeeled, 0.15 ug KI gwa minI ) significantly different at 1% level. Points on both plots are means of six replications 1 representative standard errors except where smaller than symbol. 34 a *‘T‘ o 204 Ex . $1. 151 x: 0.9 J ‘1’ 5 10- 2:6 '28 .. 8i.— 0) 5i E . V e Peeled 0 Un eeled 0+ f r ' F r l' r r TP r 0 1O 20 3O 4O 50 b Uptake Period (min) /T'\ 200-4 . Peeled +4 o Unpeeled .C a .9 g 160— _C d ‘8 “L; 120- ?) . U) 3 80- IU - Q) .0.) .2 40- LLJ .1. .. X 4. 0 i; G + ‘I r T i ' r r l O 10 20 30 4O 50 '60 Figure 2. Elution Time (min) 35 started at two-week intervals to provide a constant supply of plant material. The growing medium was watered every third day until emergence. Thereafter, soluble fertilizer of 20-20-20 formulation (Peter’s Soluble Plant Food; W. R. Grace and Co., Fogelsville, Pa.), mixed at a rate of 1 g 1'1 and providing 4.25 mM KI, was applied every other day to run- through. Plants were grown in a controlled-temperature room at a constant temperature of 15 1 2° C and average relative humidity of approximately 70%. A 15-hour photoperiod was provided by 400-watt mercury vapor lamps (model SON-T 400; Philips Electronics, Ltd., Bloomfield, N. J.) suspended 1 m above the bench. The average light intensity at plant level was 400 uE mI2 sIl. Powdery mildew was found to be a persistent problem, so plants were sprayed to runoff when two true leaves had formed with Bayleton 50% wettable powder (Mobay Chemical Corp., Kansas City, Mo.) mixed at the recommended rate of 0.15 g III. One spraying per batch of plants was found to be sufficient. As an additional precaution, fertilizer solution was applied directly to the medium to avoid wetting the foliage. Plants emerged five to seven days after sowing. Approximately two weeks after emergence, non-mutant (those Lacking a silvery appearance) and stunted plants were removed. The remaining plants were supported with bamboo 36 stakes and branches were pinched off to maintain single stems. Plants were used four to six weeks after emergence, by which time they had developed nine to ten nodes. For the sand-culture experiment, presoaked Azggnngl seeds were sown in the same containers as above in a medium- grade white silica sand moistened with deionized water. Temperature, relative humidity and light conditions were the same as described above. No additional water was supplied until several days after emergence, when seedlings were watered with a one- fourth strength modified Hoagland's solution supplying 1.5 mM KI as described below. The sand surface was then covered with a layer of cheesecloth and i-mm plastic beads to prevent algal growth. Ten days after emergence undesirable plants were removed as above. The remaining plants were randomly assigned to receive full-strength modified Hoagland’s solution supplying either 2 mM (low) or 10 mM (high) KI. As sources of macronutrients, the low-KI solution contained 2 mM KNO3, 4 mM Ca(NO3)2, 2 mM NaH2P04, 1 mM M9804 and 3 mM NH4N03. For the high-KI solution,. KN03 concentration was increased to 6 mM, NH4N03 decreased to 1 mM, and 2 mM K2804 added. Both solutions included a full complement of micronutrients (Epstein, 1972). The one-fourth strength solution was a dilution of the high-KI formula minus K2804. Plants were watered twice weekly until run-through with one of the above solutions. Approximately five weeks after 37 emergence, when plants were at the same stage as above, they were used in experiments. W Plants were randomly selected and transported to the laboratory just prior to each experiment. Recently fully expanded leaflets from leaves at node seven or eight were used. Since the first two nodes of pea stems produce only trifid bracts (Hayward, 1967), the above correspond to the fifth and sixth true leaves. Lower leaves were commonly too wrinkled to allow easy removal of the epidermis. One leaflet was removed from each of six plants per treatment. The leaflets were rinsed with deionized water and immediately placed on a plastic weigh boat atop slushy ice. The abaxial epidermis was removed from an area near the base of each leaflet approximately 1.5 cm wide and extending from the midrib to one margin. Removal was accomplished by carefully slipping one point of sharpened forceps below the epidermis, grasping a portion of epidermis and gently peeling it away parallel to lateral veins. Peels were begun at the midrib or margin to avoid puncturing tissue in the center of the peeled area. After all leaflets were peeled, a glass cylinder 0.8 cm high, cut from glass tubing of 1-cm internal diameter and polished on one end, was attached to the center of the peeled area of each leaflet in a method similar to that described by Greene and Bukovac (1971) for leaf discs. 38 The cylinders were attached using 100% silicone rubber (white General Purpose Sealant; Dow Corning Corp., Midland, Mich.). The sealant was spread in a very thin layer on a clean plastic weigh boat, the polished end of a cylinder placed on it and the cylinder rotated to achieve even coverage. The cylinder was then gently pressed onto a leaflet so that the entire circumference was sealed. For experiments at 1° C, leaflets were transferred to individual weigh boats on the slush for elution. The elution solution was prechilled in a freezer until a thin ice layer had formed and kept on ice during the experiment. For experiments at higher temperature, the elution solution was equilibrated to that temperature and weigh boats containing leaflets were placed in a constant-temperature water bath during the experiment. In most cases the solution was 5 mM CaClz prepared with the monohydrate form of the salt and having a pH of 5.5. At 10-second intervals, 0.5 ml of solution was put into each cylinder using an autopipet. After predetermined periods (typically 2, 5, 10, 15, 20, 30, 40 and 60 minutes), a 200-ul sample was withdrawn from each cylinder with an autopipet at the same intervals and in the same order as the solution had been put in. Before removing a sample, solution was drawn into the pipet tip and released back into the cylinder to equilibrate the tip with the solution and mix the contents of the cylinder. Care was taken not to damage tissue within the cylinder while sampling. As soon 39 as all samples at a given time had been collected, 200 ul of fresh solution were pipetted into each cylinder. Each sample was put in a 4-ml plastic vial containing a diluting solution. For KI analysis, this solution contained sufficient CsI (as CsCl, to suppress KI ionization in the flame) and HCl to yield concentrations of 1000 ppm CsI and 1% HCl after adding the sample; for RbI, the diluted sample was 2000 ppm KI (as KCl) and 1% HCI (Sotera g1_al,, 1979 and 1981). The amount of dilution necessary depended on the nature of the experiment, but typical dilution factors were four for KI and three for RbI. Whenever possible, experiments were conducted under a laminar flow hood. When samples were not being collected, leaflets and sample vials were kept covered to reduce evaporation and contamination. If it was not possible to analyze samples immediately, they were stored covered in a refrigerator no longer than overnight. Samples were analyzed for KI or RbI using an Instrumentation Laboratory (Andover, Mass.) Video 12 atomic absorption/emission spectrophotometer in emission mode. Standard operating conditions (Sotera g1,§L., 1979) were modified to accommodate the small (generally (1 ml) sample size. Adjustments were made following observation of peak formation by the output signal during sample analysis. On the basis of the time over which maximum peak height was reached and sustained, an aspiration rate of 2.5 ml minII, a two-second delay between start of aspiration and readout 40 and a three-second analysis time were determined. Under these conditions it was possible to obtain two consecutive readings from a sample as small as 0.6 ml. The mean of two such readings was recorded for each sample. . Standards were prepared to contain the same concentrations of CsI or KI and HCl as the diluted samples. When RbI was present in samples analyzed for KI, readings were greatly enhanced. It was therefore necessary that RbI be included in those standards at the proper concentration.. Readings in ppm were corrected according to the dilution factor and converted to ug KI or RbI. The total amount of ion eluted from a leaflet by each sampling time was calculated by adding the amount removed in all earlier samples to the current content of the cylinder. The means of these values per gram fresh weight for all replications were plotted against time to produce an elution curve. Fresh weight of the eluted leaflet volume was determined after each experiment was run. Since the eluted tissue was known to have taken up solution, fresh weight was recorded for a peeled disc of the same. diameter as the cylinder, but cut from an analogous position on the opposite half of the leaflet. The weights of such discs from both halves of a leaflet were found to be nearly identical. Discs were not cut before elution since intracellular KI would have been released from out cells into the apoplast. The fresh weight of intact leaflet tissue changed very little over an hour-long period if kept ice 'cold. For 41 elutions over a longer term or at higher temperature, when weight did change, dry/fresh weight ratios of tissue from opposite, untreated leaflets were used to calculate fresh weights of dried eluted tissue. In experiments involving elution from leaflet discs, a disc of the same diameter as a cylinder was cut from each leaflet using a cork borer. The disc was held with a dab of silicone rubber to the bottom of a plastic test-tube cap approximately 1.5 cm in diameter and 2 cm deep, and each cap was placed on a weigh boat. Other procedures were the same as above except that 2 ml of solution were pipetted over each disc at the beginning of the experiment. anss1al_flnns1dsratI2na_f2n.1hs.fllu112n_£zessdune. E11g§t§_gj__§g§lant, The silicone rubber sealant used in this study had no apparent phytotoxic effect and was found not to be a source of KI. It was, however, reported by the manufacturer to release acetic acid upon curing, which was found to reduce the pH of solution placed in a cylinder. By applying only a small amount of sealant to the cylinder, the pH change was kept at about one-half pH unit, resulting in a pH of approximately 5.0 when 5 mM CaClz was used. The pH of bathing solutions used in ion-flux studies is commonly in the range of 5.0 to 7.0 (e.g. Jackson and Edwards, 1966, 5.0; Smith and Epstein, 1964b, 5.2-5.8; Pitman, 1969, and Bernstein, 1971, 5.5; Doman and Geiger, 42 1979, 6.5). When 5 mM CaClz was buffered with CaCO3, there was not a significant difference between elution curves at pH's of 5.0 and 7.0 (Figure 3). Since all solutions used fell within this range even after exposure to the sealant, they were not buffered. The implications of elution solution pH are considered in the Results and Discussion section. Vaggnn_1n1111natign, Subjection of leaf tissue to a vacuum helps to attain complete infiltration by an external solution (MacDonald and Macklon, 1972; MacNicol g1, {11, 1973). In the present study, however, injection of solution into air spaces was not desirable since it could impede gaseous diffusion (MacDonald, 1975) and disrupt normal cell ion fluxes (Cosgrove and Cleland, 1983). In addition, vacuum treatment was found to promote leakage of the elution solution from cylinders. For these reasons vacuum infiltration was not used. aannling_1g§hnigng, The removal of aliquots of elution solution from a cylinder without replacement severely limited sample size and number. The entire volume of solution could not be collected due to the likelihood of damaging tissue with the pipet tip while removing the final samples. In addition, there was some drop in pH and the KI concentration of the solution was found to increase to an undesirable extent over the period of an experiment. By replacing the solution removed in sampling, any number of 200-ul samples could be taken with little change in pH or KI concentration, or danger of rupturing cells. KI Eluted (ug-g fresh weight") Figure 3. 43 e pHiio 0 pH 71) r ' r 10 20 30 40 50 60 T r I l t I 1 l I Elution Time (min) Elution of KI at 1° C into unbuffered (pH 5.0) and buffered (pH 7.0) 5 mM CaClz solutions. 'Constan rates (pH 5.0, 1.68, and pH 7.0, 1.50 ug KI gwa minI) and Y intercepts (81.5 and 84.2 ug KI gwaI, respectively) not significantly different. Points are means of six replications 1 representative standard errors. 44 While this method increased the possibility of volume changes due to pipetting error, it was found that with careful, uniform pipetting such changes were less than 2%. The rate of evaporative loss from cylinders during elution was determined gravimetrically. Such loss was less than 1% of the original volume over a 60-minute period, but was considerably higher during long-term elutions. In these cases the volume of solution replacing samples was adjusted to maintain a more uniform volume. Iaslallsn_21_£sll_flall§. Cell walls were isolated from Arggntgnl, leaflets by homogenization and centrifugation as described by Bernstein (1971). After the final washing, however, the cell-wall preparation was not filtered but rather transferred to tared crucibles, dried at 90° C for 24 hours, and weighed. M21sxsnt_2f_ElN1l2n_§211112n_1n12_xlasns. Leaflets of the same age and position as those used for elution were randomly selected for determination of the volume of bathing solution moving into peeled leaflet tissue. Over the course of the experiment sixteen leaflets for each of six replications were required; since all could not be treated simultaneously, they were excised and prepared as needed. Each leaflet was cut in half lengthwise and the portion without midrib discarded to reduce the 45 volume of tissue analyzed. The remaining half was prepared for elution at 1° C as described above. One-half ml of cold 5 mM oac12 labeled with 3501' (as H3°Cl; New England Nuclear) was placed in each cylinder. The specific activity of the solution was 0.08 uCi mlII, and the change in ClI concentration due to labeling was calculated to be less than 1%. After 2.5, 5, 7.5, 10, 15, 20, 35 and 50 minutes the solution was poured out of two cylinders per replication. For one sample per replication, the entire half leaflet was prepared for counting; for the other, only the tissue covered by the cylinder. Since some leaflet tissue adhered to the cylinder upon removal, both the half leaflet or disc and cylinder were blotted and placed in a 20-ml plastic scintillation vial. Vials were placed in a -20° C freezer overnight. To extract the 36ClI, 2 ml of deionized water were added to each vial. The loosely capped vials were then partially submerged in a water bath which was gently boiled for 10 minutes. After samples had cooled, 14 m1 of scintillation cocktail (Safety-Solve; Research Products International Corp., Mt. Prospect, III.) were added to each vial and samples allowed to sit several hours. Standards, which contained 1 ml of labeled solution, a cylinder and leaflet tissue comparable to that in the samples, were treated in the same way. Samples were counted for 10 minutes on an LKB Instruments (Rockville, Md.) model 1211 Rackbeta liquid 46 scintillation counter. The radioactivity of each sample was converted to milliliters of solution by dividing net counts per minute by the mean radioactivity of the standards, assuming the specific activity of solution within the tissue was the same as that in the bathing solution. The volume of solution that had moved into the tissue was then plotted over time. To test the effect of removal of the epidermis on movement of solution into the tissue, a disc approximately 1 cm in diameter was cut from each of six peeled and six unpeeled leaflets. Each disc was placed on the bottom of a plastic test-tube cap for elution as previously described. Two ml of cold 5 mM CaClz labeled as above with 3601' were pipetted into each cap. After 5, 10, i5, 20, 35 and 50 minutes the solution was poured off one disc per replication. The disc was removed, lightly blotted, and placed in a scintillation vial. Samples were prepared and counted as described above. The amount of solution in the tissue was plotted over time for both peeled and unpeeled discs. I To test for quenching that might result from release of pigments during sample processing, quantities of the labeled CaClz solution ranging from 0.1 to 2.0 ml were placed in scintillation vials along with deionized water to make 2.0 ml and a peeled leaflet disc. Vials were prepared for analysis and counted in the same way as samples. When the radioactivity detected was plotted against 'quantity of 47 labeled solution (amount of isotope), they were found to be directly proportional within the lower range of the plot, where all samples fell. It was therefore not deemed necessary to adjust results for quenching. WW For determination of free-space characteristics of A;ggn;ggn,leaflet laminar tissue, solutions of 0.5, 1, 3, 5, 10 and 20 mM KCl, all of which were also 0.1 mM CaSO4, were labeled with either °°RbI (as 86xbc11 or 35c1' (as H3501) (both from New England Nuclear) to specific activities of 0.1 and 0.08 uCi mlIl, respectively. Leaflets of the same age as those used in elution experiments were randomly excised from five-week-old plants and prepared for elution in the usual manner at 1° C. Over the course of the experiment, 0.5 ml of each of the twelve labeled solutions was placed in the cylinders of four leaflets. After 5, 10, 20 and 40 minutes each solution was poured off one leaflet and the cylinder removed. The area covered by the cylinder was cut out with a razor blade, blotted and placed in a scintillation vial along with the blotted cylinder. Samples were placed in a freezer overnight. Then 2.0 ml of deionized water were added to each vial, which was loosely capped and placed in a gently boiling water bath for 10 minutes. The 3601' samples were further prepared and counted as previously described. Each 86RbI sample received 48 10.0 ml of 2.5 mM ANDA solution (7-amino-i,3-naphtralene disulfonic acid monosodium salt; Eastman Kodak Co., Rochester, N.‘ Y.) as a wavelength shifter (Lauchli, 1969). Samples were allowed to sit for several hours before being counted for 10 minutes on the instrument described earlier. Calculations to determine the volume of water free space (WFS), the amount of fixed anions in the Donnan phase (AI), volume of the Donnan phase (VD) and the effective concentration of fixed anions ([AII) were as per Briggs g; 31. (1958) and Pitman gt,al. (1974a) and as discussed in Briggs gg,aL. (1961). WFS was considered to be the mean volume of KCl solution at all concentrations taken up per gram fresh weight, calculated on the basis of 36ClI activity of the tissue after 40 minutes. The free-space contents of KI (8°RbI) and ClI (3°C1I) were estimated for each KCl concentration at each sampling time. This was done by separately plotting ueq gwa‘ of each ion in the tissue over time, regressing lines through the points for each KCl concentration, and extrapolating to zero time to find the Y-axis intercepts. Plots of these free-space contents against KCl concentration yielded curves of different shape for KI and ClI (Figure 4). The KI curve represents total cation content, i.e. both that free in solution and that bound in the DPS. Since only a small portion of the fixed charges of the cell wall are positive (Lauchli, 1976;. Luttge and 49 4 n K' J 0 Difference a Q. d A Cl- DA I C ”E 3- c .x 0'1 J Oe— A I— ; d j S " ' ..C — a) 2- w—(D , u:o cofiunam an» ac coflumoa ummCHH any you cofiumuucmocoo cofiuaacm oomum>ummum .N manmh CH umpsaocfi mucmeHnmaxm Hoe acmucoo +x owummaaoam co acmouma m mm ocm cowuaflom cesusflm cud: sswunfiafiscm CH mmmca cmccoo CH ocaon +x +uo mmumeHumw .e mamas 119 It should be noted that the above estimates for KI bound in the Donnan phase are not completely accurate since the solution used for elution was 5 mH CaZI while that used to establish the difference curve was only 0.1 m! CaZI. Pitman gt, gt, (1974a) reported that KI bound in the DFS of barley leaf slices decreased with increasing CazI concentration of the bathing solution. The present estimates may not be indicative of bound KI in the intact leaflet since a considerable amount of the ion might have been removed during elution by exchange with Ca2I. It is difficult to predict the degree to which the DFS binds KI under normal conditions without knowing the cations present in the extracellular solution and their concentrations. Epstein (1972) proposed that cation exchange at the cell well of roots is probably not significant for highly sqoluble monovalent cations such as KI. Further study is required to determine whether or not this is true for leaves. ., '- ..» . .-.. . ., .. I .. . - .. If the elution method is to be of use in determining changes in apoplastic ion concentration, it must be sensitive enough to detect such changes under varying conditions of ion delivery to the leaf. To evaluate this sensitivity, excised leaves were allowed to take up through the petioles 1, 10 or 100 mH RbCl for 0.5, 3 or 22 hours. 120 The efflux rates for the 20- to 60-minute elution period and estimated apoplastic RbI contents for the various treatments are presented in Table 5. Figure 18 presents elution curves after uptake of 1 mH RbCl for the three periods, while the curves in Figure 19 are for the three RbCl concentrations after a three-hour uptake period. There was considerable variability in results among replicate leaflets of the same treatment, suggesting that leaves differed in their resistance to xylem transport and therefore did not take up solution at the same rate. Such resistance could be attributed to xylem blockage by air bubbles or stomate closure due to stress. It is significant that extracellular RbI was detectable after only a 30-minute uptake of the 1 mH solution. This suggests that the elution method is quite sensitive, particularly since KI concentrations in the xylem solution are typically higher, often greater than 10 mH (Robson and Pitman, 1983). The method was able to clearly differentiate extracellular RbI concentrations resulting from the treatments. For a given RbCl concentration, the estimated apoplastic RbI content increased with uptake time. The net efflux rate followed the same trend, consistent with an increasing intracellular concentration. These values also increased with increasing RbCl concentration at a single uptake time, suggesting that extracellular ion concentration 121 Table 5. Steady-state Rb+ elution rates and estimated apoplastic contents of Argenteum leaflets from excised leaves that had taken up 1, 10 or 100 mM RbCl solution for 0.5, 3 or 22 hours. Values are means of six replications i standard errors. RbCl Uptake Steady-state Estimated Rb+ content concentration period Rb elution rate of apoplast (mM) (h) (ug-gfw-1 min-1) (ug-gfw-l) 1 0.5 0.059 i 0.024 3.87 i 1.19 3.0 0.283 1 0.076 17.9 i 1.4 22.0 0.915 i 0.366 65.4 i 21.8 10 0.5 0.751 3 0.111 124 i 9 3.0 5.73 1 0.85 486 i 51 22.0 90.5 i 19.9 2,370 i 510 100 0.5 6.29 i 0.64 831 i 80 3.0 47.3 i 4.6 4,340 i 160 22.0 836 i 151 67,800 i 10,500 if 8Slope of the linear portion of the elution curve, from 20 to 60 minutes. 122 120.4 s 22 hours a 3hours A L g ‘ a 0.5 hour '03 100— 3i {3 . Q) 80" " l u... U) . 50- U) D .. v "o 40— (D +’ II £2 L0 20- 4. _D D: O O ~5- 4'0 50 so Elution Time (min) 0 —-l O N O (:1 0 Figure 18. Rubidium elution at 1° C from leaflets after excised leaves had taken up 1 mH RbCl solution ' through petioles for 0.5, 3 or 22 hours at 25° 0. Results in Table 5. Points are means of six replications t representative standard errors except where smaller than symbol. Figure 19. 123 Rubidium elution at 1° C from leaflets after excised leaves had taken up i, 10 or 100 mu RbCl solutions through petioles for three hours at 25° C. Results in Table 5. Points are means of six replications t representative standard errors. 124 Rb” Eluted (ug-g fresh weight ") m 10 new 0 5“ . 1 T T I 1 T v r 1 r (x 100) 3 l a 100 mM Rb' 0 . . I I ‘l' I I T 0 1'0 20 30 40 5'0 60 Figure 19. Elution Time (min) 125 does significantly affect the rate of uptake and accumulation into the symplast. It should be noted that the extent of leaflet ion accumulation seen in this experiment may not represent that in the intact plant. Since phloem continuity, and therefore function, had been disrupted, RbI may have accumulated to a greater extent than it would have in intact tissue, where one would expect extracellular ion concentration to eventually stabilize. W A pulse-chase type experiment was conducted as previously described to follow the movement of a uniform amount of RbI into Azggntgtn, leaflets over time. Preliminary rinse/elution experiments were conducted to determine the appropriate lengths of time for the uptake and chase periods. After a six-minute pulse of RbCl and a 10- minute chase, RbI could easily be rinsed from peeled leaflet tissue, suggesting it was largely extracellular. With longer pulse and/or chase periods, cell uptake appeared to become increasingly significant. An uptake period of six minutes was therefore chosen to produce the RbI pulse. A 50 mH RbCl solution was used to assure detection of RbI by elution after the short pulse time. When a water chase was used, there was considerable diffusion of RbI from the petiole back into the water. This was reduced by using a 50 mH K01 chase solution, but some 126 loss of RbI still occurred. This loss was minimized by using a uniform chase period of only 10 minutes, as described, after which all leaflets were excised to promote retention of ions that had been transported to the lamina. Variation in time for ion distribution within the lamina was achieved by waiting 0, 20 or 170 additional minutes before initiating the elution procedure. The total delay between the pulse and elution was therefore 10, 30 or 180 minutes. Figure 20 presents six-hour elution curves for the three delay periods, with the results summarized in Table 6. Samples were also collected after 24 hours. Total RbI in eluted leaflets was estimated by adding the amount removed by elution to that remaining in the entire leaflet. These calculated values were comparable to total RbI in the opposite, noneluted leaflets. Since the elution kinetics differed from those typically seen for endogenous KI, the usual method for estimating extracellular ion content was not appropriate. The method used was that already described in connection with tracer efflux studies. The natural logarithm of RbI remaining in the tissue was plotted over time, a line regressed through the points from 30 to 120 minutes ~and extrapolated to zero time. The Y intercept value was then subtracted from total RbI present before elution to arrive at the estimate of extracellular RbI content. The 30 to 120 minute portions of the log plots were essentially linear for all three treatments. 127 m 10 minutes I 30 minutes a 180 minutes q‘ 1004 ...—J _C .4 .9? (D ; 80—J ;: - 8 .g; 60— m .1 U) 3, 404 13 m . “:3 E] 20 l :2. 1, 0: O 0 Figure 20. I 1 tr 1 60 120 t T I I 150 240 300 350 Elution Time (min) Rubidium elution at 1° C from leaflets after excised leaves had taken up a six-minute pulse of 50 mH RbCl solution through petioles at 25° C. Delays between pulse and start of elution were 10, 30 and 180 minutes. Results in Table 6. Points are means of six replications t representative standard errors. 128 .0C0000000 >00C00000C000 00C 000000 .0000 0 >0 A40 00>00 xm 00 A440 00>0H x0 00 00000 0000 5000 0:0000000 >00C00000C00m C50000 0 C0000; 0C0020 0N H 0N0 440.0 M 0.0 440.0 M 0.00 4000.0 H FPN.0 400.0 N 00.0 00 H Pmn :05 000 mm 0 00— +0.0 0 n.00 +0.0 H 0.00 000.0 « mm0.0 00.0 a 00.0 m0 0 men CHE 0m 00 a 0N0 +4m.m H 0.00 ++M.m u 0.00 4000.0 0 000.0 040m.0 H mw.< 00 a can C05 00 A01300.030 +00 00000 00 001300.03v A01C05 01300.000 A01CHE 01200.0:V A01300.0:V C000300 0 «N CH 0:00000 mm 00000000 00 0000 C000300 +00 0000 C000300 +00 +00 0000000 0C0 00000: 600:00 +bm 0mm0acc< 0:00:00 +00 ; 4-. :05 60-6 00060 ceasaea +00 00060 00000 .000000 0000C00m u 0C0000000000 x00 00 0:005 000 00:00> .0003C0s 000 00 0m .00 00 >0000 0 00000 00000000 5000 000300 >00C0300m0sm 003 +00 0C0 C0000000 0000 :5 0m 00 00030 0 a: x000 m0>000 5000:0000 00000x0 :00: 0000000 000C0EH00ax0 0o >0meeam .0 00000 129 With an increase in delay from 10 to 180 minutes, there was a significant decrease in elution rate for the first 20 minutes, but a significant increase in rate for one to four hours. The period from 20 to 60 minutes was one of transition where average rates were approximately equal. As the delay period increased, there was a significant decrease in the estimated extracellular RbI content. At four, six and 24 hours, however, the total amounts of RbI eluted were not significantly different for the three treatments. The initial rapid elution of RbI followed by a slow rate of efflux observed after a 10-minute delay‘ suggests that the RbI ions were still localized principally in an extracellular compartment and consequently diffused readily into the elution solution. In contrast, the i80-minute curve is characterized by a lengthy steady elution rate which has been associated in the present study with long- term efflux from cells. This curve most closely resembles those obtained for KI elution. However, the steady-state efflux rate (0.21 ug gwal minII) is only about 10% of that typically seen for KI. While RbI taken up by leaflets in this experiment represented about 0.2% of leaflet dry weight, KI content normally ranged from 4 to 6%: therefore, the low accumulation of RbI in the lamina tissue most likely was responsible for the lower efflux rate. Extracellular RbI represented 17.6%, 11.5% and 4.7% of total RbI for the 10-, 30- and 180-minute treatments, respectively (Table 6). These percentages may be somewhat 130 low for the lamina tissue alone since they were calculated using total RbI of entire leaflets in which the midrib may have had a higher content than the remainder of the tissue. Nevertheless, these values support the hypothesis that the apoplastic ionic content was progressively reduced by cell uptake over time. From KI elutions, extracellular KI was calculated to be approximately 2% of the total in the eluted tissue. Even after the i80-minute delay, therefore, the extracellular ion content seems to constitute a larger portion of the total than in the intact leaf. Apparently there was not sufficient time for cells to accumulate the ion to the same extent as in the intact leaf. This is consistent with the relatively lower constant efflux rate for RbI as compared to KI discussed above. Essentially the same amount of RbI had been eluted by the end of the experiment for all three treatments but the elution kinetics varied, seemingly according to compartmentation of the ion in the tissue. These results suggest that with an increase in the time period between the RbI pulse and elution, more of the ion had been taken up by cells, leaving progressively less in the apoplast. Thus, in the intact plant, a significant amount of ions being transported to the leaf lamina via the xylem stream may first diffuse into an apoplastic region of the tissue prior to being taken up and accumulated within individual cells. This hypothesis is supported by the finding of Jachetta gt 131 gt. (1986b) that under conditions of darkness and greatly reduced transpiration, leaf cells can deplete the cell wall of solutes almost entirely. W300 To allow evaluation of the elution method under conditions of differing ionic levels in intact plants, Anggntgtn,plants were grown as described by sand culture and supplied with nutrient solution containing either 2 or 10 mM KI. Potassium concentrations in the soil solution generally range from 0.2 to 10 mH (Van Steveninck, 1962). Leaflets from both treatments appeared morphologically similar. All leaflets, however, were smaller than those of plants grown in the peat-based medium. The sand retained considerable moisture which may have restricted root growth and, consequently, that of the shoot. Figure 21 presents four-hour elution curves for leaflets from high-KI (10 mH) and low-KI (2 mH) plants, with results summarized in Table 7. Total laminar KI in the tissue eluted, steady-state elution rates, estimated apoplastic KI contents and xylem-sap KI concentrations were all significantly higher for the high-KI treatment. Assuming an extracellular solution volume of 0.1 ml gwaI, the calculated apoplastic solution concentrations were 36.2 and 22.65 mH KI for high- and low-KI treatments, respectively. Since the corresponding. xylem-sap concentrations were 14.2 and 5.4 mM, an increase in sap 132 800 O) O O l 200— K” Eluted (ug-g fresh weight") 8 C? Figure 21. a TOInM K’ m 21nM K‘ fl I 1 1 I l I I ' 150 200 240 l 40 80 120 r Elution Time (min) Potassium elution at 1° C from leaflets of plants grown at 2 or 10 m! KI. Results in Table 7. Points are means of six replications t representative standard errors except where smaller than symbol. 133 .0020 050000000 000:00000cs00 0a: .040 0m>00 00 .0440 0s>00 00 0a 0:0000000 000cae000c000 0:0020 .01300 05 0.0 00 05000> C0000000 0000000000 :0 0:0500000 .0000C05 00N 00 0N .0>000 C000000 000 00 C000000 000C00 00 000000 4* m2 4 . 0+ . 4* 4* 0.0000 1.. 0.0 0 N.00 NN.0 0 N0.0 0.0 0 N.0m 0.00 0 0.000 00.0 0 00.N mmN 0 0000 25 00 0.0 0 0.0 0N.0 0 00.N m.N 0 0.NN N.0_ 0 0.00 00.0 0 00.0 0N0 0 N000 :5 N 0250 +x 00000 0250 001300.000 001:05 0-200.000 001300.000 0+00 0+00 000 500>x 00 +0C00000 00 000000000 00 00000000 00 00000 C000000 +0 +0 0:0500 C0000000 +x 0000000000 0 +x0 000050000 0:00:00 +x 000001 >0000m+ 00000 0:000002 .000000 0000:000 0 0:0000000000 x00 00 0:005 000 00000> +x :5 00 00 N 0:0:000C00 :0000000 0:00000: 0002 00000000 0:0 0:00 :0 C3000 00:000 5000:0000 00 00000000 000 0000000 000C050000x0 00 >00550m .0 00000 134 concentration along the xylem path or in the leaf apoplast would have been required to result in the above values. The K+ content of lamina tissue from high-K+ leaflets was approximately twice that of low-K+ tissue. Since the estimated apoplastic K+ content was 1.6 times as high, it represented a lower percentage of total K+ in leaflets grown at to mH K* (1.82%) than at 2 mH x+ (2.31%). These values are not, however, significantly different. These results are in agreement with previous findings that the K+ concentrations of xylem sap and plant tissue do not change in direct proportion to that supplied to roots. The concentration of solution bathing the root is not necessarily reflected in the xylem-sap concentration since uptake rate varies with external concentration (Pitman, 1975). Conti and Geiger (1982) reported that when K+ supplied to roots of sugar beet was increased from 2 to 10 IN, the corresponding increase in leaf K+ content was only two to three fold. According to these workers, at 2 mM K+ leaf export and import rates were similar so K+ did not accumulate. At the higher concentration, however, xylem import increased to a greater extent than phloem export and there was accumulation of the ion. Further examination of the relationship between ion inport and export rates, apoplastic ion concentration and cell uptake might aid in identifying those factors responsible for changes in leaf K* content. The ability of the elution method to detect changes in apoplastic K+ in the 135 intact plant suggests that it might be useful in such studies. 136 SUMMARY AND CONCLUSIONs Leaflets of the Argentgnl mutant of filann,ga1iygn_L. were found to be suitable material for estimating apoplastic K+ content by elution since the abaxial epidermis may be removed without apparent injury to underlying mesophyll cells. This allows free diffusional access to the mesophyll apoplast with considerably less danger of contamination by intracellular contents than when leaf discs or slices are used. The 60-minute time course for elution of K+ from peeled Argenteum leaflet lamina tissue is characterized by a rapid initial elution rate followed by a slower, steady rate after about 20 minutes. Evaluation of the elution curve suggests that the rapid phase represents a period of ion diffusion from .the apoplast as well as equilibration of that compartment with the bathing solution. The constant rate is thought to represent net efflux out of cells, which appears to begin at some point during the rapid elution phase. Extrapolation of the linear portion of the curve to zero time seems to be a valid means of estimating the original apoplastic K+ content. It is equivalent to subtracting K+ thought to be of intracellular origin from the total eluted. A better understanding of cell efflux rates during the first 20 minutes of elution might make such 137 estimates more precise. Log transformation of data, as done in tracer efflux studies, is not thought to be necessary for compartmental analysis when eluting endogenous ions from leaf tissue. Estimates of apoplastic K+ content ranged from 39 to 97 ug gfw" for plants grown at 4.25 mM K+. When plants were grown at 2 and 10 mM K+, estimates were 88 and 142 ug K+ gfw", respectively; in both cases, apoplastic K+ was found to represent approximately 2% of total laminar K+. Concentrations calculated based on an estimated apoplastic solution volume of 0.1 ml gfw'1 were 10 to 25 mM for plants grown at 4.25 mH K+, and 23 and 36 mM for those grown at 2 and 10 mH K+, respectively. These values are higher than those from previous studies, which may be due to experimental conditions or underestimation of extracellular solution volume in the present case. There is a need to better define the volume of extracellular solution so that more exact concentrations may be determined. The elution method allows the determination of changes in apoplastic K+ contents corresponding to xylem-sap concentrations typically found in plants. It may also be used to detect differences in ion compartmentation within leaf tissue. On this basis, ion movement into the leaf from the xylem seems to be first into an extracellular compartment from which it is progressively taken up by cells. 138 Free-space characteristics of A;ggn;gun,leaflet lamina tissue differed in some respects from past reports. In particular, the lower WFS estimate may be attributable to reduced tissue damage as compared to previously used methods. Estimated K+ bound by negative charges in the cell-wall Donnan phase during elution was approximately 0.1 ueq gfw", or 4 to iii of apoplastic K+. Knowledge of the extent of K+ binding in the intact leaf would help define the role of the DPS in transport of the ion. The method presented in this study is limited in that it is not applicable to most other species, where removal of the epidermis results in severe tearing of mesophyll cells. In addition, it is not suitable for estimating apoplastic K+ near particular cells since only an overall estimate is obtained. Nevertheless, the elution method may be useful in future studies. While the determination of absolute apoplastic ion concentration by elution needs refinement, the method seems appropriate for comparing relative concentrations under varying conditions. A;ggn§gnn,could therefore be used as a model for the study of leaf apoplastic concentrations of K* and other ions. The elution method might serve as an overall estimator, complementary to a detection method with greater resolution, such as X-ray microanalysis. Using these methods, it would be of interest to look at changes in apoplastic ion concentration during leaf and plant ontogeny, with changes in xylem import and phloem 139 export rates, and under varying growing conditions. Hatters that might be addressed include leaf extracellular ion concentration in relation to cell fluxes and regulation of intracellular concentration; .effects of apoplastic ion concentration on specific physiological functions in the leaf; the importance of extracellular transport in general ion distribution within the leaf or in movement to specific sites; and the role of the Donnan phase in ion transport and cell uptake in the leaf. Knowledge gained in these areas would contribute greatly to our understanding of leaf ion relations and their role in determining plant productivity. APPENDI X APPENDIX X-RAY MICROANALYSIS 0F FREEZE-DRIED LEAFLET LAMINA TISSUE mm The development of X-ray microanalysis as an analytical tool in conjunction with electron microscopy, allowing identification and analysis of very small volumes of tissue, has increased the potential for refining our knowledge of the distribution and concentration of ions in plant tissue. X-rays, produced when primary electrons- of the beam generated by an electron microscope displace electrons of atoms in a sample (Marshall, 1980c), may be attributed to a particular element by their energy or wavelength (Flowers and Lauchli, 1983). Since the intensity of radiation is directly proportional to the concentration of the assayed element, this method is useful for quantitative as well as qualitative analysis (Marshall, 1980c). There seems to be general agreement that for X-ray analysis of biological materials, sample preparation is the limiting factor (Chandler, 1979; 'Morgan, 1979; flowers and Lauchli, 1983). This is particularly true for plant tissue, with its high water content (Echlin and Saubermann, 1977: Morgan, 1980). The purpose of this study was to evaluate 140 141 frozen sectioning followed by freeze-drying as a method of preparing leaf tissue for X-ray microanalysis of K+ in the apoplast. W935. The two principal criteria for evaluating sample preparation for ion localization by X-ray microanalysis are that elements be retained essentially Ln, sign, and that morphological detail be sufficient for clear identification of the structures to be analyzed (Chandler, 1979). The difficulty lies in satisfying both criteria in the same sample. The progress made in this regard for biological tissue has been reviewed by Lauchli (1972a), Van Steveninck and Van Steveninck (1978), Morgan (1980) and Spurr (i980). Conventional sample preparation methods for electron microscopy involving fixation and dehydration in liquids may result in retention of only a small percentage of soluble ions (Hall g;,al,, 1974; Lott g1_al,, 1978; Morgan, 1980). Alternate methods have therefore been developed specifically for X-ray microanalysis, the primary method of fixation being rapid freezing at liquid-nitrogen temperature (around -180° C). This has the advantages of stopping physiological processes, diminishing movement of elements, stabilizing cell structure and providing mechanical strength for sectioning (Seveus, 1980). The simplest technique involves analysis of the tissue in the frozen-hydrated state, either bulk-fractured or 142 sectioned (Markhart and Lauchli, 1982). This method requires the use of a cold stage to maintain the sample at low temperature, an apparatus not available for the present study. Drawbacks to analyzing bulk frozen tissue are that the large mass fraction of water reduces the signal-to- background ratio of X-rays generated (VanSteveninck and Van Steveninck, i978) and spatial resolution is poor (Chandler, 1979; Marshall, i980a). These problems are reduced by analyzing thin sections of frozen tissue (Barbi, 1979; Satter g1, 31,, 1982). In both sample types, however, a localized rise in temperature due to electron irradiation may result in ion redistribution (Marshall, 1980b). In freeze substitution, the ice in rapidly frozen tissue is slowly replaced at low temperature by a dehydration agent such as acetone or acrolein in diethyl ether, and the sample is then embedded in a plastic medium (Marshall, 1980d). Embedded tissue is generally more easily sectioned than frozen (Marshall, 1980d)~ and this method allows good preservation of morphological detail (Morgan, 1980). It is said to retain Ln,g1;n,a large percentage of mobile ions as long as the process is totally anhydrous (Pallaghy, 1973; Harvey g1, 31,, 1976; Marshall, iSBOd). There is, however, danger of ion redistribution if the substituting fluid takes up water from the atmosphere or the embedding medium is not properly polymerized (Morgan, 1980: Markhart and Lauchli, i982). Exogenous ions may be 143 introduced in both of these steps (Morgan, 1980), and even anhydrous solvents have been reported to alter X-ray spectra (Marshall, 1980d). Appleton (1977) reported considerable loss or redistribution of elements by freeze substitution. In freeze-drying, ice is removed from the frozen sectioned or bulk tissue by physical dehydration at atmospheric pressure or under vacuum (Morgan, 1980). This method has the advantage of not- requiring the use of solvents, so that maintenance of chemical integrity is theoretically possible (Morgan, 1980). Appleton (1978) reported better resolution in freeze-dried than frozen tissue due to reduction of electron scatter caused by water. While ions cannot be analyzed in solution, they seem to remain compartmentalized within and outside of cells by precipitating onto the nearest structure (Gupta :1, 11., 1976). A maior disadvantage has been poorer structural preservation than with other methods (Morgan, 1980). All of the above methods have been used to prepare plant tissue for ion localization by X-ray analysis, as reviewed by Spurr (1980). The freeze-drying method used in the present study was based on that of Satter g1,aL. (1982). That study was selected as a model because its objective was specifically to analyze for apoplastic K+ and C1“ in the 33.3ngg,pulvinus. The integrity of both protoplasts and cell walls, spatial resolution and elemental compartmentation were reported to be adequately maintained in freeze-dried tissue. :44 11W Plants of the A;ggn1ggn,mutant of Elgnl,aa;113.,L. were grown as described for the general elution experiments and leaflets used at the same stage of development. Prior to freezing the tissue, freshly broken glass knives were prepared. Several layers of masking tape were placed on the back of each knife 2 mm below the cutting edge to support a grid (Appleton, 1978). In addition, a 'shelf' of tape was added further down to keep grids from sliding down the back of the knife. A knife was inserted at a 4° angle into the holder within the cryobowl of a Sorvall-Christensen FTS/LTC-z frozen thin sectioner with low-temperature controller mounted on a Sorvall Porter-Blum MT-z ultramicrotome (Ivan Sorvall, Inc., Norwalk, Conn.). The coolant was vaporized liquid nitrogen. The bowl, knife and specimen chuck holder were then allowed to cool down and equilibrate to -80° C (measured at the chuck-holder assembly) for approximately 20 minutes before sectioning was begun. During the last five minutes of cooling the motor advancing the specimen was turned on to insure temperature equilibration. All tools used during freezing and sectioning were prechilled for several minutes in liquid nitrogen or in the cryobowl. Liquid propane (~190° C), the freezing medium, was prepared by allowing propane gas from a cylinder to condense in a metal container sitting in liquid nitrogen in a Dewar flask. 145 A leaflet was gently clamped in a copper specimen holder at an area of the margin near the base of the leaflet (Figure 22). This positioning was so that the area analyzed would roughly correspond to that used in the elution studies. A drop of 50% sucrose solution was placed in the holder before inserting the leaflet to help support the tissue after freezing. The mounted leaflet was rapidly cut using a razor blade to leave a triangular piece of tissue approximately 3 mm high (Figure 22) and immediately plunged into the liquid propane so the holder was completely submerged. After 15 seconds the sample was transferred to liquid nitrogen and carefully fractured using chilled forceps until a triangular piece of tissue approximately 1 mm high was left. The specimen and holder were then transferred in liquid nitrogen to the cryobowl and mounted in the chuck holder with the specimen perpendicular to the knife edge. The specimen was trimmed by cutting i-um sections until a smooth cutting surface appeared. Before sectioning was begun, a 2 x 1 mm oval copper slotted grid (Ernest P. Fullam, Inc., Schenectady, M. Y.) was placed on the tape support below the knife edge to chill. The grid had been coated on one side with 0.5% Formvar (Fullam) to provide a film support over the slot for the sections. In this way the grid composition would not interfere with analysis (Lauchli, 1972a). 146 triangle of tissue /./A\~\ cut before freezing /' \. brass plate pressed (223) against leaflet by screw copper specimen holder vi Figure 22. Positioning of leaflet in specimen holder and area cut out before freezing in liquid propane. 147 Sections 0.3 um thick were cut at a temperature of -80° C and cutting speed of 0.75 mm sec'l. Frost was removed from the cut surface using a chilled eyelash brush. Sectioning seemed to be most successful when the bowl was covered and the microtome allowed to run undisturbed for one minute or more. If the knife edge became dull, the knife was replaced with one that had chilled in the cryobowl for at least 20 minutes. When a number of sections had collected at the knife edge, they were moved to a grid using a chilled eyelash brush. This procedure was most easily accomplished under conditions of low humidity and low static electricity. With the grid still on the knife, the sections were flattened and pressed gently onto the Formvar using the flat end of a chilled, polished copper rod 2 mm in diameter. The grid was picked up on the bent end of small metal spatula and placed on a 9-mm unpolished carbon planchet (Fullam) which had been cooled in the cyrobowl. A second cold planchet was placed on top to keep the specimen flat and help insulate it from temperature change during transfer to the freezer. When all grids for a given session were complete, they were transferred to a -80° C freezer for drying. To maintain low temperature during the transfer, the planchet- grid sandwiches were placed over liquid nitrogen on a perforated metal plate mounted in a metal cylinder. A cardboard grid held the planchets in place. The cylinder 148 and samples were transferred to a styrofoam-lined metal can containing P205 as desiccant. The can had been allowed to cool in the freezer at least 30 minutes. After 24 hours at -80° C, the can containing samples was transferred in a styrofoam cooler to a freezer at -25° C, where it remained for an additional 24 hours. It was then allowed to come to room temperature before opening. For mounting, a carbon planchet was fixed atop a 9 x 4.5 mm aluminum stub (Pullam) using an adhesive tab (M. E. Taylor Engineering, Wheaton, Md.). Grids were attached to the planchet with a small amount of Television Tube Koat (G. C. Electronics, Rockford, Ill.). They were then carbon coated using a Ladd Research Industries (Burlington, Vt.) vacuum evaporator. One 3/16 point carbon rod was used to coat eight samples at a time, providing a fairly heavy coating. Carbon, which does not contribute significantly to background, makes the sample conductive to minimize heating and local charge buildup (Echlin and Saubermann, i977) and protects against humidity (Appleton, 1977). Samples were stored in a desiccator at atmospheric pressure over P205. ' Samples were subjected to energy-dispersive X-ray microanalysis using a Tracor Northern (Middleton, Wis.) TM- 2000 X-ray analyzer mounted on a JEOL JSM-35C scanning electron microscope (Japan Electron Optical Laboratories, Tokyo) operating in spot mode. An X-ray spectrum was collected from cell wall for 100 seconds (dead time 20-30t) 149 using an accelerating voltage of 15 KeV (Lauchli g1_aL., 1970) and beam current of 240 picoamps. Specimen tilt was 0° from horizontal, and takeoff angle was 40°. Spectral peaks for K‘, 01', P and S were identified by their K. lines and counts above background recorded. Although K+ was of principal interest, data for the other elements were collected to compare relative contents. W Although the above steps were carefully carried out, most of the grids prepared were not suitable for analysis. In some cases, nothing remained on the grid by the time it was ready to be analyzed; in others, what was there was not identifiable. In no case was a complete section obtained, but scattered groups of cells were occasionally visible. Table 8 presents results for four samples in which cell wall could be distinguished, while Figure 23 illustrates a typical X-ray spectrum. Since it is not reasonable to draw conclusions based on so few samples, these results are presented only for general interest. As indicated by the X-ray spectrum (Figure 23), background was quite high relative to peak height for CI' and 8. Some coolants have been reported to cause Cl' contamination, but this generally is not a problem with propane (Markhart and Lauchli, 1982). The Formvar film (Table 8) may have been contaminated during application. The higher i(+ and P counts for analysis 2a as compared to 2b 150 Table 8. Results of X-ray microanalysis of cell wall in freeze-dried Argenteum leaflet lamina tissue. a Net Counts Analysis R? Cl‘ S P 1a 6719 471 894 3058 1b 6698 351 962 1713 Formvar film supporting above sample 189 60 207 126 23 11133 13A ‘1963 3302 2b 8976 158 1864 1802 3 5368 378 411 , . 1225 a 6850 402 840 1689 aAnalyses designated a and b are of different points on the cell wall of the same cell. Samples from four separate leaflets were analyzed. 151 12-- K 3 .. o 8 P .9 3.5 E U Cl 0 I I I l 1 1 I 1 I O I 2 3 4 Energy (KeV) Figure 23. Portion of X-ray spectrum for cell wall of . freeze-dried Argentggn leaflet lanina tissue corresponding to analysis la in Table 8. 152 from the same cell may be due to inadvertent analysis of a portion of plasmalemma. The consistency in the relative amounts of the four elements detected and the fact that K+ concentration in the cell wall seems to be well within the range of detection suggest that with some modification this method may be useful. Factors that might have affected the results of this study are briefly considered below. Freeze fixation acceptable for X-ray analysis requires a rapid cooling rate (Morgan, 1980). Since biological tissues exhibit poor thermal conductivity (Seveus, 1978), it is recommended that only a small volume of tissue (i mm3) be frozen (Appleton, 1977). It is often necessary, however, to compromise between wound effects and rapid freezing (Van Steveninck and Van Steveninck, 1978). This is particularly true for highly mobile ions like K‘, which can quickly diffuse to neighboring tissue (Marshall, 1972). In the present study, to minimize the possibility of analyzing tissue in which the apoplast had been contaminated with intracellular K+, a somewhat larger piece than desired was frozen and subsequently reduced in size for sectioning. To minimize K+ movement, only uncut tissue was exposed to the sucrose solution, and the time from cutting to freezing was no more than two seconds. The use of a larger piece of tissue may, however, have reduced the freezing rate. The advantage of rapid freezing is that it minimizes the formation of ice crystals which can 153 rupture membranes and displace unfrozen solution, enriched in solutes (Nobel, 1975: Morgan, i980). The presence of ice crystals may also make sectioning of frozen tissue more difficult (Marshall, 1980b). Vitrification, the freezing of water without ice- crystal formation, can occur if cooling is rapid enough; but even under ideal conditions it may only occur in the outer i0 um of a sample since the loss of heat inside that volume is too slow (Marshall, 1980a). Azggnlgnl_ leaflets are approximately 125 um thick, so that even under ideal freezing conditions ice crystals probably would have formed in the inner tissue. In addition, crystallization of vitrified ice may occur with a temperature rise (Marshall, i980a).' Temperatures below -60° C are thought necessary to prevent ice crystal formation (Morgan, 1980). Liquid propane is reported to provide a faster cooling rate than other coolants commonly used (e.g. liquid or slushy nitrogen and freon-22) (Costello, 1980). Although liquid propane and liquid nitrogen are at essentially the same temperature, formation of an insulating layer of gas around the specimen in the latter results in poor cooling (Marshall, i980d). Marshall (i980d) reported that regions relatively free of ice-crystal damage have been obtained using liquid propane. The use of cryoprotectants (e.g. gylcerol) has been reported to provide good structural preservation, but may result in ion movement across membranes (Appleton, i978). 154 Even well frozen plant tissue seems to pose problems for sectioning. Echlin g1,gL, (1982) observed that cutting thin sections of such tissue is a 'difficult and troublesome procedure.“ They attributed this difficulty to the high water content of vacuoles relative to cell walls, resulting in a discontinuous material when frozen. The presence of large intercellular air spaces in leaves compounds the problem. A light micrograph of the agngngg,pulvinus studied by Satter g1,gL. (1982) reveals it to be more uniform and compact than leaf tissue, perhaps making it easier to section. 1 In spite of the difficulties, sectioning offers certain advantages. One is that the surface analyzed is smoother than that resulting when tissue is fractured (Echlin 11,gL., 1982; Marshall, 1980a). Irregular specimen topography can result in loss of resolution, lower detection efficiency and generation of spurious signals (Hess, 1980). Differential drying of structures may itself result .in an irregular surface, but sectioning can minimize this effect (Appleton, 1978). As indicated, improved spatial resolution is obtained with sections. In bulk specimens which are too thick to allow the transmission of electrons, the electron beam penetrates in a teardrop form so that x-rays are generated from a volume larger than the surface feature focused on (Barbi, 1979; Morgan,1980). Echlin g1,gL, (1982) reported 155 spatial resolution of 2 to 8 um in bulk specimens versus 0.05 to 0.2 um in sections 0.1 to 1.5 um thick. Even if primary electrons can pass through a bulk specimen, they lose energy, lowering the efficiency of X-ray production (Marshall, i980c). Lower elemental concentrations (down to around 100 ppm) may therefore be detected in thin sections (Van Steveninck and Van Steveninck, 1978). Echlin and Saubermann (1977) recommended a section thickness of 0.2 to 2 um to attain good morphological information, sufficient material for analysis and adequate spacial resolution. Sections were cut at 0.3 um in the present study because tissue appeared to fracture at greater thickness and to be merely scraped off at lesser thickness. Similarly, a sectioning temperature of -80° C was used in the present study because it was the lowest temperature at which the tissue appeared not to fracture. This is slightly lower than the -70° C at which Satter g;,aL, (1982) sectioned. Cutting at very low temperatures ((-100° C) may cause fracturing rather than true sectioning (Seveus, 1980), resulting in uneven section surface and thickness. Marshall (1980b) indicted that while sectioning below -100° C avoids recrystallization and thawing, sections cut at -80° C are more uniform in thickness. He questioned whether there is sufficient thawing at the higher temperature to cause redistribution of diffusible elements. 156 Dempsey and Bullivant (1976) and Appleton (1977) reported no ice recrystallization or significant ion redistribution in biological tissue at -70° to -80° C, the lowest temperatures at which Appleton (1977) felt true sections were obtained. Others, however, recommend lower temperatures. Modson and Marshall (1970) suggested cutting at or below -100° C. Seveus (1980) has recommended a specimen temperature of -140° C with the knife and bowl at -100° C. It is likely that the optimun cutting temperature varies with tissue type and therefore must be individually determined. There was some loss of samples during transfer to the grid due to electrostatic forces and air movement in the cryobowl. Even after being pressed against the Formvar film, samples did not adhere well. Some may have stuck to the carbon planchet placed atop the grid, a problem that might have been aggravated by a temperature increase while samples were transferred to the freezer. Like the sectioning temperature, that used for freeze- drying (-80° C) was somewhat lower than in the method of Satter gt_ aL. (1982). It is possible that ice recrystallization occurred at this temperature, though Ingram :3, al, (1972) and Morgan (1980) have reported successful freeze drying below -60° C. Appleton (1977, 1978) recommended slow drying at atmospheric pressure since surface-tension forces of rapid sublimation under vacuum might disrupt cells. Campbell :1, 15? a1, (1981) reported that drying under vacuum disrupted plant tissue structure and so dried specimens at atmospheric pressure over phosphorus pentoxide, a desiccant with a strong affinity for water. Gradual warming has been recommended so tissue does not suffer thermal shock (Appleton, 1978). 92391331232. The freeze-drying method of sample preparation used in this study appears to have merit, but requires modification if reliable results are to be obtained. A freezing method that allows vitrification of a greater volume of tissue is desirable. In addition, a lower sectioning and freeze- drying temperature than used in this study may be required to avoid ice recrystallization. It is important that utmost care be used to avoid any rise in temperature when samples are transferred between preparation steps. Freeze-substitution merits further investigation since it may allow improved sectioning and retention of morphological detail. If substitution and embedding can be accomplished without ion distribution, this method may prove preferable to freeze-drying. 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