A PHYSIOLOGICAL STUDY OF THE DIFFERENTlAL RESPONSE OF NAVY BEANS (PHASEOLUS. VULGARIS L.) TO ZINC Thesis for the Oegree of M. S. MICHEGAR. SYATE UNWERSFFY RONALD GENE SHELLENBERGER 1970 M. ........................... g, Illlllflllljllzllflglfllwflflfl”WIN w £5833“: University \ IIN‘DING av ‘7 HMS & SINS’ 800K BINDERY INC. uamw smozns sullen-r. mm '11!" ' ABSTRACT A PHYSIOLOGICAL STUDY OF THE DIFFERENTIAL RESPONSE OF NAVY BEANS (PHASEOLUS VULGARIS L.) TO ZINC BY Ronald Gene Shellenberger In 1965 two varieties of navy beans were reported to respond differentially to low and high levels of zinc (Zn). Saginaw was tolerant and Sanilac sensitive. There- fore, these varieties have been utilized in physiological studies to gain an understanding of the role of Zn in plants. The parameters measured in this study were: (1) a comparison of the dry weight accumulation between the two varieties at several levels of Zn supply; (2) comparative nutrient accumulation in various anatomical parts; and (3) an electron probe analysis of specific tissues. The results of dry weight determinations provided quantitative confirmation of what was previously observed; 1 Ronald Gene Shellenberger namely that Saginaw was tolerant and Sanilac was sensitive to both deficient and excess Zn concentrations. In addi- tion the dry weight data indicated that Saginaw grew better at the 0 Zn supply than at the .005 ppm Zn treat— ment. Elemental analysis in the various plant parts in- dicated that the severity of Zn deficiency symptoms is not reflected by the Zn concentration in the tissue, but is likely due to increased phosphorous (P) accumulation, translocation, and to the abnormal P/Zn ratio. Electron microprobe analysis showed a fluctuation in the calcium (Ca)/P and potassium (K)/P ratios with re- spect to various tissues, but appeared to complement each other in root and stem tissues. Further indications were that Ca and K appeared to be affected differentially in. their rates of translocation. Calcium was affected most drastically when the Zn concentration in the growth medium was varied. Its rate of translocation was greatly affected. Another occurrence disclosed by microprobe anal- ysis was a differential accumulation of elements at the 2 Ronald Gene Shellenberger point of juncture between a lateral and main root. Sag- inaw accumulated K, P, Ca, and Zn in this area while Sanilac indicated no accumulation. A PHYSIOLOGICAL STUDY OF THE DIFFERENTIAL RESPONSE OF NAVY BEANS (PHASEOLUS VULGARIS L.) TO ZINC BY Ronald Gene Shellenberger A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Crop and Soil Sciences 1970 ACKNOWLEDGMENTS The author wishes to express his appreciation to Dr. M“ W. Adams for his help in preparing this manuscript and for his intellectual stimulation and guidance. Dr. H. P. Rasmussen and V. E. Shull were very instru- mental in providing the assistance required for perform- ing the electron microprobe analysis. In connection with these studies, I would also like to acknowledge the fi- nancial assistance obtained from A. E. C. Contract No. AT(ll-l)-888. Special gratitude is extended to the author's wife, Marcia, for her unselfish contributions to this work. ii TABLE OF CONTENTS ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . LIST OF TABLES. . . . . . . . . . . . . . . . . . . L I ST QF F IGURE S O O O O O O O I O O. O O O O O O O 0 INTRODUCTION. . . . . . . . . . . . . . . . . . . . REVIEW OF LITEMTURE O O 0 O O O O O O 0 O O O O O I. Morphological Effects of Zn Deficiency. . . II. Zn Deficiency in Navy Beans (Phaseolus' vulgaris L.) in Michigan. . . . . . . . . III. Physiological Studies Conducted with Beans. IV. P-zn InteraCtion. o o o o o o o o o o o o o METHODSOFPROCEDURE................ I. Tissue Preparation for MicrOprobe Analysis. Paraffin. . . . . . . . . . . . . . . . , Freeze Dry. . . . . . . . . . . . . . . . Cryostat. . . . . . . . . . . . . . . . . RESULTS AND DISCUSSION. . . . . . . . . . . . . . . Section on Gross Tissue Analysis. . . . . . . I. Low Zinc Treatments . . . . . . . . . A, Deficiency Symptoms . . . . . . B. Dry Wt. Accumulation. . . . . . C. P and Zn Accumulation . . . . . iii Page ii vii m-bcn 10 14 16 17 18 29 29 29 29 37 43 TABLE OF CONTENTS (Cont.) Page II. Reaction to a Toxic Zn Treatment. . . 62 A. P, Zn, and Dry Wt. Accumula- tion. 0 O O O O O O O O O O O 62 Section on Microanalysis. . . . . . . . . . . . 69 SUMMARY . . . . . . . . . . . . . . . . . . , . . . 95 REFERENCES 0 O O O I O O O O O O O O O O O O O O O O 97 APPENDIX. . . . . . . . . . . . . . . . . . . . . . 102 iv Table LIST OF TABLES Comparisons of the elemental concentrations in Saginaw and Sanilac root vascular tis— sue prepared by three different technics (electron micrOprobe analysis). . . . . . . Comparisons of the elemental concentrations in Saginaw and Sanilac transition zone tissue prepared by three different tech- nics (electron micr0probe analysis) . . . . Comparisons of the elemental concentrations in Saginaw and Sanilac stem prepared by three different technics (electron micro- probe analysis) . . . . . . . . . . . . . Comparison of dry wt. (grams) of Saginaw and Sanilac plants harvested 7 weeks after emergence . . . . . . . . . . . . . . . . . Comparison of tOp/root ratios of Saginaw and Sanilac at various Zn concentrations. . . . Comparison of dry wt. (in grams) of Saginaw and Sanilac plants grown at several Zn concentrations and harvested at five weeks after emergence . . . . . . . . . . . . . . Percent dry wt. (dry wt./fresh wt.) in grams, of plants grown at several Zn concentra— tions and harvested at five weeks after emergence . . . . . . . . . . . . . . . . . Page 25 25 26 37 38 41 42 LIST OF TABLES (Cont.) Table Page 8. Comparison of the iron concentration (micro- grams/g tissue) in root, stem, petiole, and leaf of Saginaw and Sanilac plants, grown at several Zn concentrations. . . . . 44 9. Comparison of the potassium concentration (mg/g tissue) in root, stem, petiole, and leaf of Saginaw and Sanilac beans, grown at several Zn concentrations. . . . . . . . 45 10. Comparison of the calcium concentration (mg/ 9 tissue) in root, stem, petiole, and leaf of Saginaw and Sanilac beans, grown at sev- eral Zn concentrations. . . . . . . . . . . 46 11. Comparison of Zn accumulation (mg Zn/total tissue) in the root, stem, petiole, and leaf of Saginaw and Sanilac beans, grown at several Zn concentrations. . . . . . . . 47 12. Comparison of the Zn concentration (micro- grams Zn/g tissue) in the root, stem, petiole, and leaf tissue of Saginaw and Sanilac bean plants . . . . . . . . . . . . 48 513. Comparison of P concentration (mg P/g tissue) in the root, stem, petiole, and leaf of Saginaw and Sanilac beans . . . . . . . . . 51 14. Comparison of the P/Zn concentration ratio in the root, stem, petiole, and leaf tissue of Saginaw and Sanilac bean plants . 58 15. Percentage increase in the Zn concentration for different tissues between .5 and 5 ppm Zn treatments . . . . . . . . . . . . . . . 63 vi LIST OF FIGURES Figure 1. Scan line concentration record showing comparison of calcium concentration in Sanilac stem prepared by three different technics (electron microprobe analysis). . 2. Scan line concentration record showing com- parison of potassium concentration in Sanilac stem prepared by three different technics (electron micrOprobe analysis). . 3. Scan line concentration record showing com- parison of phosphorus concentration in Sanilac stem prepared by three different technics (electron microprobe analysis). . 4. Sample current oscillogram and X-ray oscil- lograms of Sanilac stem sectioned on the cryostat . . . . . . . . . . . . . . . . . 5. Comparison of Saginaw and Sanilac bean plants grown at a 0 level of Zn (photo— graphed at 7 weeks). . . . . . . . . . . . 6. Comparison of Saginaw and Sanilac bean plants grown at a .005 ppm Zn concentra- tion (photographed at 7 weeks) . . . . . . 7. Comparison of Saginaw and Sanilac bean plants grown at a .05 ppm Zn concentra— tion (photographed at 7 weeks) . . . . . . 8. Comparison of Saginaw and Sanilac bean plants grown at a .5 ppm Zn concentration (photographed at 7 weeks). . . . . . . . . vii Page 21 22 23 24 31 32 33 34 LIST OF FIGURES (Cont.) Figure Page 9. Comparison of Saginaw and Sanilac bean plants grown at a 5 ppm Zn concentration (photographed at 7 weeks). . . . . . . . . 35 10. Comparison of the growth curves between Saginaw and Sanilac over a range of five Zn treatments (grams dry wt.). . . . . . . 39 11. Comparison of the P concentration between Saginaw and Sanilac root, stem, petiole, and leaf tissue at the 0 Zn treatment (mg P/g tissue). . . . . . . . . . . . . . . . 52 12. Comparison of the P concentration between Saginaw and Sanilac bean leaves under five Zn treatments (mg P/g tissue) . . . . 53 13. Comparison of the top/root P concentration ratio between Saginaw and Sanilac over a range of five Zn treatments (mg P/g tissue). . . . . . . . . . . . . . . . . . 54 14. Comparison of the P/Zn ratio in the leaf tissue of Saginaw and Sanilac bean plants. 59 15. Comparison of the P concentration between Saginaw and Sanilac root, stem, petiole. and leaf tissue at the 5 ppm Zn treat- ment (mg P/g tissue) . . . . . . . . . . . 67 16. Comparison of the Zn concentration between Saginaw and Sanilac root, stem, petiole, and leaf tissue at the 5 ppm 2n treatment (mg Zn/g tissue) . . . . . . . . . . . . . 68 17. Comparison of the Ca/P ratio in the root tissue of Saginaw and Sanilac over a range of four Zn treatments (electron microprobe analysis) . . . . . . . . . . . 71 viii LIST OF FIGURES (Cont.) Figure Page 18. Comparison of the K/P ratio in the root tissue of Saginaw and Sanilac over a range of four Zn treatments (electron microprobe analysis) . . . . . . . . . . . 72 19. Comparison of the Ca/P ratio in the phloem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). 75 20. Comparison of the Ca/P ratio in the xylem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron micrOprobe analysis). 76 21. Comparison of the K/P ratio in the phloem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). 78 22. Comparison of the K/P ratio in the xylem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). 79 23. Comparison of the Ca/P ratio in the xylem, phloem, and mesophyll tissues of the leaf between Saginaw and Sanilac grown over a range of four Zn concentrations (electron microprobe analysis) . . . . . . . . . . . 82 24. Comparison of the K/P ratio in the xylem, phloem, and mesophyll tissues of the leaf between Saginaw and Sanilac grown over a range of four Zn concentrations (electron microprobe analysis) . . . . . . . . . . . 83 ix LIST OF FIGURES (Cont.) Figure 25. 26. 27. 28.. 29. 30. Comparison of the Ca/K ratio in the root tissue between Saginaw and Sanilac over a range of four Zn treatments (electron microprobe analysis) . . . . . . . . . Comparison of the Ca/K ratio in the xylem tissue of the stem between Saginaw and Sanilac over a range of four Zn treat- ments (electron microprobe analysis) . Comparison of the Ca/K ratio in the phloem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). Oscillograms and elemental concentration scan of Saginaw root, at lateral, for the 5 ppm Zn treatment (electron microprobe analysis). . . . . . . . . . . . . . . Oscillograms and elemental concentration scan of Sanilac root, at lateral, at the 5 ppm Zn treatment (electron microprobe anal- ysis). . . . . . . . . . . . . . . . . X-ray oscillograms of Saginaw root, at late eral, grown at the 5 ppm Zn treatment (compare to oscillogram on figure 27). Page 86 91 92 INTRODUCTION Several instances of varietal differences have been reported in ion accumulation and translocation in beans. However, the mechanisms involved in differential accumulation are not understood. Plant varieties that respond differentially offer a means of studying ion accumulation and mode of action by their comparative physiology. Ellis (1965) reported Zn deficiency in navy beans (Phageolus vulgaris L.) in Michigan. Since then, much work has been done in trying to elucidate the possible mechanism or mechanisms involved in the differential sus- ceptibility of two bean varieties (Saginaw and Sanilac). Sanilac, a bush bean, is sensitive to low and high Zn while Saginaw, a vine, exhibits a high degree of toler- ance at both levels. Results obtained in the present study do not answer the basic questions, but provide some information necessary for the initiation of more critical research. Two areas of investigation were pursued: (1) a comparison of the dry weight accumulation between the two varieties under several levels of Zn supply: (2) a gross tissue analysis of various elements at both low and high Zn levels with an attempt to localize any interaction or uptake mechanism to a specific plant part (root, stem, petiole, or leaves); (3) a form of micro-analysis with the use of the electron micrOprobe to determine elemental localization within specific plant tissues (xylem, phloem, or mesophyll). REVIEW OF LITERATURE I. Morphological Effects of Zn Deficiency Reed (21) and Hewitt (11) discussed the morpho- logical effects of Zn deficiency. They found that sev- eral species of plants were dwarfed at an early stage and eventually ceased growth. In addition the leaves were curved downward, chlorotic, involuted, and often necrotic. The disrupted metabolism in the leaves was associated with a scarcity of plastids, production of melanotic material, and the presence of calcium oxalate crystals. These symptoms were usually found to affect the older leaves first. Stunting was due to a lack of internodal elongation. Total yield was drastically rev duced due to delayed maturity. II, Zinc Deficiency in Navy Beans (ghaseglgs vulgaris L.) in Michigan Ellis (9) reported in 1965 that Sanilac navy beans produced yields ranging from 450 lbs. per acre 3 without Zn to 2,340 lbs. per acre with addition of Zn. The Saginaw variety produced 1,080 lbs. per acre without Zn and 2,100 lbs. per acre with Zn. This experimentation was conducted on calcareous soil in Michigan, It was also discovered that a P-Zn relationship existed. Navy beans fertilized with 0, 87, 174, 348, and 696 lbs. of P per acre yielded, respectively, 1,850, 1,450, 1.190, l;000,.and 530 lbs. per acre. But when 4 lbs. of Zn per acre was added to all treatments yields averaged 2,240 lbs. per acre. III, Physiological Studies Conducted With Beans Polson (18), in a physiological study of the difw ferential response of the Saginaw and Sanilac varieties, found by reciprocal grafting experiments that the geno- type of the scion determined tolerance to high Zn. In his micronutrient studies the differential tolerance to high Zn was not due to differential Zn uptake. At low Zn he found that Sanilac accumulated more Zn, but translocated less to the tops than did Saginaw. This difference in distribution was associated with a greater release of Zn into the vascular tissue as inferred from higher concentrations of Zn in the exudate of the Saginaw variety. Ambler and Brown (1) and Pauli (17) con- ducted studies on P-Zn interaction. Iron (Fe) and P were found to aggravate Zn deficiency. Differential absorption of Fe and P by Saginaw and Sanilac was thought to be the cause of the differential susceptibility to low Zn. San- ilac took up more Fe and P than Saginaw in a growth medium relatively low in Zn. Calcium carbonate was found to decrease the trans- location of Zn, and increase the translocation of P, from roots to leaves. Zinc transport to leaves of Sanilac was reduced 90%.when CaCO was added. High P treatment with— 3 out CaCO3 increased the Zn concentration in all.p1ant parts. Veits (25) found in Red Mexican beans that anal- ysis of plants with different levels of Zn supply, as judged by deficiency symptoms and fertilization experi- ments, showed that all vegetative parts had a wide range of Zn concentration. Deficiency symptoms were manifest at 20 ppm Zn or less in the tops. IV. P:Zn Interaction Millikan (16) studied the P-Zn interaction in sub- terranean clover. In Zn deficient plants a greater per- centage of the total Zn was in the roots. Also in Zn deficient plants the 65 Zn concentration was highest in tissues exhibiting Zn deficiency symptoms. Plants showing "little leaf" symptoms had P/Zn ratios greater than 400 in their tops. Subterranean clover also showed no increase in Zn concentration in the tOp between the O and .005 ppm Zn treatments when the P concentration was at 3 ppm. Sim- ilarly P decreased in the tops with increased Zn at all P levels. However, P concentration was not related to a lower Zn concentration in the taps. Plants grown at 30 to 60 ppm P showed reduced growth which was associated with high P concentrations in the tops. In this regard, Rossiter (22) found that subterranean clover showing in- cipient P toxicity symptoms contained a P concentration of 1.43%. Millikan (15), working with subterranean clover, found a highly significant variety x P interaction effect on growth at adequate Zn levels, which indicated that P requirements of the EdenhOpe variety were considerably greater than for Clare. By contrast, tolerance to high levels of Zn in the growth medium was greater in Clare than in Edenhope. No relationships were found between Zn concentration and the occurrence of Zn deficiency symptoms. Further, when the Zn level in the substrate was insufficient for normal growth, increased P caused reduced growth and increased severity of Zn deficiency, whereas at sufficient Zn levels increased P was benefi- cial to growth. The results indicated that Zn was essen— tial to P utilization by the plant. The reduced growth induced by high P and low Zn was not due to a decrease in Zn concentration resulting from high P treatment, but was the result of a high P/Zn ratio. Millikan (15) found that the minimum P/Zn ratio for the occurrence of severe Zn deficiency was 300 for 97 day old plants, but the minimum changed with the age of plants. Halim (10) found differences in Zn and P levels in corn but these were not associated with Zn deficiency. Lines of corn susceptible to Zn deficiency showed a re- duction in dry weight of leaves under high P treatment while resistant lines showed no such reduction. This indicates a close relationship between Zn and P in re- gard to the develOpment of deficiency symptoms. Boawn (6) found that, in potatoes, increasing the supply of P induced a growth disorder that could be eliminated by increasing the Zn supply. This disorder was attributed to the develOpment of a P/Zn ratio of greater than 400. Watanabe (26) found that a P/Zn con- centration ratio of greater than 300 resulted in a slight depression in yield in pinto beans. Biddulph (2) postulated that the passage of ions through tissue rich in P was interferred with and much of the Fe was precipitated along the conductive tissue and that the principal effect of high P on the appearance of chlorosis was explainable on this basis. Some reports (4, 5, and 8) indicated that phos- phate has an adverse effect on Zn absorption or utiliza- tion. Boawn (7), however, showed that the plant can obtain adequate Zn from Zn3(P04)2. "It is unlikely that Zn would ever become a limiting nutrient while a free precipitate of Zn3(PO4)2 exists in the soil." He also indicated that a precipitate of Zn3(PO4)2 or other Zn complexes of the root might exist. He found 10 times more Zn in acid—washed roots of both bean and corn roots than in tops. The reports here all indicate a general antago- nism between P and Zn in their uptake and accumulation in the plant. METHODS OF PROCEDURE Two varieties of navy beans (Phaseolus vulgaris L.), Saginaw and Sanilac, were grown in 9 in. porcelain pots lined with polyethylene bags. The pots were filled with acid—washed (2% oxalic acid in 2N HCl) silica sand #4098. The sand was flushed with deionized water until the pH approached 6.5. Prior to sowing, the pots were flushed with a phosphate buffered macronutrient solution to stabilize the pH at 6.5. The plants were grown in a modified Hoagland's solution containing 40 ppm P and Zn concentrations of 0, .005, .05, .5, and 5 ppm. The concentrations of the macronutrient solution were as follows: Chemical Quantity Ca(NO3)2 - 4H20 2.268 grams/liter KNO3 1.054 grams/liter MgSO4 - 7H20 .464 grams/liter KCl .055 grams/liter (NH4)2HPO4 .077 grams/liter KH2P04 .079 grams/liter 10 11 Reagent grade chemicals were used with deionized water to make up stock solutions. The pH of the nutrient solution was maintained at 6.5 by the use of both mono- basic and dibasic phosphate. The micronutrients were according to Hoagland with the exception of the varying Zn concentrations. Upon emergence the cotyledons were removed in order to eliminate them as a Zn source. Concomitantly the selective Zn treatments were introduced. Each pot was watered with 400 ml. of nutrient solution every other day and with 200 ml. of deionized water on alternate days. The pots were well drained so no waterlogging could occur. The plants were grown in a greenhouse under na- tural light supplemented by 900 ft. candles of artificial light. The photo period was 14 hours and the temperature ranged from 70°-80°F. All the experiments were conducted in the same fashion up to the point of harvest. Three experiments were conducted: (1) gross tis- sue analysis; (2) electron micrOprobe analysis; and (3) an experiment where plants were grown beyond the sampling date for chemical analysis in order to permit an expansion of the weight differences between the different treatments. 12 The two experiments for the chemical analysis of tissue were harvested as the earliest signs of differentiation occurred in order to eliminate the use of necrotic tissue. Four seeds were planted in those pots used in experiments for chemical determinations and two seeds per pot for dry weight determination. The dry weight experiment was replicated twice and the chemical analysis once. In the dry weight experiment plants were har- vested at 7 weeks, this being after pod development had begun in some treatments. The plants were harvested and separated into roots, stems, petioles, and leaves after which they were dried at 70°C for four days in a forced air oven. Dry weights were then taken. Plants for gross tissue analysis were harvested five weeks from the date of emergence. The four plants were pooled for analysis. The plants were separated into roots, stems, petioles (including pulvinus) and leaves. The roots were washed in water to remove the sand. They were subsequently rinsed five times in deionized water. Both fresh weights and dry weights were taken. These 13 various plant parts were ground in a Wiley mill (20 mesh) with stainless steel parts to prevent contamination. Sample preparation followed that of Polson (1967). One half gram samples were used when available. Weights were recorded to correct the resultive analysis values. The samples were dry-ashed in porcelain crucibles at 550°C for 24 hours in a muffle furnace. The temperature was raised gradually to prevent ignition of the plant mate- rial. The ash was dissolved in 5 ml of 2N HCl and left for 5 hours after which it was filtered through a Whatman no. 2 filter paper. Nine rinses of 5 ml each were made with deionized—distilled water; including the crucible, the filter paper, and the funnel, the final volume being 50 ml at .2N HCl. The resulting solution was analyzed for Zn, Fe, K, and P. Zinc, Fe, Ca, and K were analyzed with a Perkin—Elmer model 303 atomic absorption spectro- photometer. Calcium was determined in a 1% lanthanum oxide solution in 2N HCl. This eliminates the problem of interference from magnesium. The technic used for P analysis was a colorimetric determination using an ammon- ium vanadate reaction as described by Jackson (1958). l4 Readings for P were made on a Beckman model DU spectro- photometer at 440 millimicrons. Dilutions of one to one were necessary to obtain a concentration that would fall into the proper range of absorption. Within the range of 28 to 78 on the atomic ab— sorption instrument, absorption had to be converted to absorbence to obtain linearity but from 0 to 18 absorp- tion was linear. Concentrations were determined from a standard curve. These values were then converted to either micrograms or milligrams/gram dry weight of tissue by the following equation. volume ofg§olution . X Concentration of solution sample weight The preceding analysis was replicated twice and an average of the two were taken. I. Tiggue Prepagation for Microgrobe Analysis Recently the electron microprobe X-ray analyzer has been successfully applied to the analysis of biolog- ical samples (Rasmussen 1968 and Ingram and Hogben 1957). 15 The probe, as it is often called, uses a beam of accel— erated electrons to excite a sample, causing it to emit X-rays whose wavelengths are characteristic of a given element. A spectrometer, with a diffraction crystal is used to select the wavelength, and the intensity of the emission is measured with conventional X-ray detectors. This being proportional to the quantity of the element excited. The advantages of the probe over other methods of microanalysis are that; one can detect any element of atomic number greater than five, the sample may be in various forms, the element can be detected in place, and the amount of the element can be as low as 10"15 grams (Ingram and Hogben 1957). Since the application of the electron microprobe X-ray analyzer to plant tissue, one of the greatest dif- ficulties has been the preparation of plant material in such a way as to avoid- movement of the element or ele- ments to be analyzed (Rasmussen 1968). Preliminary to the actual experiment a study in technic was conducted to determine the method of tissue preparation best suited for retaining elemental 16 localization within the plant tissue. Distribution and relative concentrations of 3 elements (Ca, K, and P), under various tissue preparation technics, was estab- lished by electron micrOprobe analysis. Plants for this study were grown as previously described but only the plants (both Saginaw and Sanilac) grown at the .05 ppm level of Zn were used. The three tissue preparation technics tested were the Paraffin (Sass 1958), Freeze-Dry, and the Cryostat (Rasmussen et a1. 1968) technics. A description of these particular technics as used in this study follows. Paraffin Tissue samples for the Paraffin technic were fixed in formalin, alcohol, and acetic acid (FAA): Ethyl alcohol (95%) ............... 50cc. Glacial acetic acid ............... 5cc. Formaldehyde (27—40%) ............. 10cc. Water .................... . ........ 35cc. Tissue was then carried through dehydration and infiltra- tion steps (Sass 1958): l7 1 1 7' fi Grade 95%.Ethyl Absolute Tertiary Water No. alcohol ethyl alcohol butyl alcohol 1 50 10 40 2 50 20 30 3 50 35 15 4 50 50 5 25 75 (Sass 1958) Several changes of 100% tertiary butyl alcohol (TBA) were added to this series followed by 2 changes of a mixture of 1/2 TBA and paraffin oil. The tissue was embedded in Tissuemat (M. P. 60°C) (Fisher Scientific Co.) following three changes in the same. Sections were cut at 15 mi- crons on a rotary microtome and affixed to a 1" diameter carbon disk with Haupt's adhesive. with xylene. Freeze-Dry Paraffin was removed Fresh tissue was plunged into liquid nitrogen cooledzisopentane. The use of isOpentane eliminates the 18 formation of gas bubbles around the tissue which would result if dropped directly into liquid nitrogen. Fol- lowing freezing, the tissue was immediately transferred to a vacuum gas-flow freeze drying apparatus evacuated to 1 cm. and held at -70°C for three days. Under these conditions dry air was circulated around the tissue for a period of three days, thus facilitating an even drying of the sample. Maintaining the vacuum, the tissue was then submerged into degassed molten paraffin and infil- trated for three days. This was accomplished by raising the temperature of the apparatus to 60 degrees. This melts the paraffin that had been supporting the tissue up to this point. The tissue was sectioned on a rotary microtome at 15 microns and affixed to a carbon disk as in the paraffin method. Paraffin was again removed with xylene. Cryostat Tissue for crystat sectioning was frozen on dry ice and stored at -25°F until sectioning. For section- ing, the tissue was kept frozen as much as possible, al- though some thawing may have occurred during transfer to 19 the cutting medium. The tissue was frozen in Optimum Cutting-Temperature Compound (OCT —15 to -30 degrees C) (Fisher Scientific Company). Sections were cut at a temperature of ~15 degrees C and at a thickness of 16 microns and then placed on warm (25 degrees C) polished carbon disks. The cutting compound also served as a mounting medium so sections were then ready for analysis. Sections of root, transition zone, and stem of Saginaw and Sanilc were fixed and further prepared by the three technics previously described. To insure homogen- eity of tissue between technics, one centimeter long sec— tions of stem and transition zone were split longitudi- nally, resulting in four identical tissue sections, with one segment used for each technic as demonstrated in the following diagram. Roots were not divided in the same manner but the tissue for each technic was sampled from the same root system. Figures 1, 2, and 3 represent the relative con- centration of Ca, K, and P with respect to each method of preparation in the variety Sanilac. The position of the scan is represented on the respective reverse sample current oscillogram. The oscillogram for Sanilac stem Y x End view of one centimeter long whole stem or transition zone section showing division. freeze-dry cryostat paraffin is displayed in Figure 4. The y axis of Figures 1, 2, and 3 represent the intensity along the x axis which is marked on the reverse sample current oscillogram, the distance in microns. The analysis was determined over 300 microns in all scans, running from right to left on the oscillogram scan line and left to right on the x, y plots (Figures 1, 2, and 3). The sample current oscil- logram has accompanying X-ray oscillograms for the var- ious elements analyzed (Figure 4). Tables 1, 2, and 3 represent the relative concentration of each element under the various treatments. Planometric measurements 21 Fig. l.—-Scan line concentration plot showing comparisons of calcium concentration in Sanilac stem.prepared by three different technics. (electron microprobe analysis) 300 a ' 1 o . o s \\ - . 3 : O 2 I :A o : "/\ : ) \ o. 0. \ : I. \ //\ o. ). \00“'.. I O \ x’I’ / \\ .' ' -‘-—---’ I F‘ / \—--ao-. VI .' o. . O t ‘90 0 microns of tissue scanned 300 --cryostat ----paraffin °'°°freeze-dry 22 Fig. 2.--Scan line concentration plot showing comparison of potassium concentration in Sanilac stem prepared by three different technics. (electron microprobe analysis) 1000 6 Q) m \ W ‘5 O O. O1;:'_"..:'.:'—'v'—'-'MT,T.T.T.?.042-:uflxflvt.fi‘;';';';L'L'L'L'g" 0 microns of tissue scanned 300 -—-cryostat ----paraffin °°°°freeze-dry 23 Fig. 3.--Scan line concentration plot showing comparison of phosphorus concentration in Sanilac stem prepared by three different technics. (electron microprobe analysis) 100 3 A m I I ‘\ 3 \ g A / \ o I \ I \ I A \ II \ . /I\\ o\\ I \ I \\ b./.. ‘.0 L." 'oJ .',\. . ‘0....... I \_._, . . «.0000. \l '0.e.b---;‘.......o. 0 ll 0 microns of tissue scanned 300 -—-cryostat ----paraffin °---freeze—dry 24 Fig. 4.-—Sample current oscillogram and X-ray oscillogram of Sanilac stem sectioned on the cryostat. nu ma x-sunw PHO§DHOROUS POTASSIUM 25 TABLE l.-—Comparisons of the elemental concentrations in Saginaw root vascular tissue prepared by three different technics (electron micrOprobe analysis). ‘— I;- Y 4 —. Saginaw Sanilac P K Ca P K Ca Freeze-dry 5.8 9.4 8.6 1.7 9.8 15.2 Cryostat 4.8 112.4 14.6 10.5 69.5 24.5 Paraffin 1.6 16.4 10.0 5.2 14.8 23.7 relative concentrations/30 microns of scanned tissue TABLE 2.--Comparisons of the elemental concentrations in Saginaw and Sanilac transition zone tissue prepared by three different teChnics (electron micrOprobe'analySis) Saginaw Sanilac P i KI Ca P K Ca Freeze-dry -— -- -- 3.6 12.0 19.8 Cryostat 16.6 366.0 60.0 1.0 270.0 30.6 Paraffin 2.8 9.4 9.6 6.2 14.0 19.9 _—_r relative concentrations/30 microns of scanned tissue 26 TABLE 3.--Comparisons of the elemental concentrations in Saginaw and Sanilac stem prepared by three different tech- nics (electron micrOprobe analysis). Saginaw Sanilac P K Ca P K Ca Freeze-dry -- -- -- 7.3 15.3 25.0 Cryostat 9.2 90.0 75.6 12.2 117.0 38.4 Paraffin 7.4 15.4 33.5 8.7 12.2 19.6 in 7‘1' relative concentrations/30 microns of scanned tissue were made in sq. cm. by measuring the area under the curve. These values are the average in Sq. cm./30 microns of tissue scanned. Working conditions for all data gathered were 25 Kv accelerating potential with .022 micro amperes sample current. This study suggests the cryostat technic as the superior method. Although better quality sections were attained by the paraffin method, the least amount of dis— placement and/br washing out of the elements occurred in the cryostat sections. 27 Potassium, a relatively mobile element, was almost totally lost in both the paraffin and freeze-dry methods. With the cryostat method, however, potassium was retained, as much as ten fold more as noted in Figure 2. On the other hand, calcium was relatively stable under the two technics illustrated by Figure 1. Although calcium was relatively stable, compared to potassium, there was marked reduction in both the freeze-dry and the paraffin methods of preparation. This confirms that, although the majority of calcium is bound, there is a substantial amount that is not. It seems that to achieve an accurate localization of an element within the tissue, a technic must be em- ployed which does not allow the tissue to come in contact with a liquid medium, causing the element to be leached out. This seems to be what the cryostat technic avoids. Most likely during the infiltration period of paraffin and freeze—dry technics leaching of the elements occurred. Leaching may also occur when paraffin is dis— solved away from the tissue with xylene. The analysis of P (Figure 3) showed very little difference among the three technics. 28 Based on the results of this study the cryostat technic was elected for sample preparation in the micro- analysis section of this thesis. Cryostat sections were prepared from samples selected from the root, stem (nodal area) and the leaf. One scan was made on each root sec- tion except on the 5 ppm Zn treatment where several scans were made. In the stem section one scan was made in the phloem and one in the xylem tissue. In the leaves scans were made in the xylem, phloem, and mesophyll. The treat— ments sampled were the O, .005, .05, and 5 ppm Zn treat— ments. RESULT S AND D ISCUSS ION Section on Gross Tissue Analysis I. Low Zinc Treatments A. ngficiencv Symptoms Zinc deficiency symptoms first appeared in San- ilac at 4.5 weeks after emergence at the 0 level of Zn. The development of a pale green region in the interveinal area of the older leaves was the first symptom observed. This area later developed into a yellow and, at the most severe stages of deficiency, a brown necrotic area. San— ilac grown at .005 ppm Zn began to show deficiency symp- toms at 5 weeks. Saginaw did not develop deficiency symptoms until 5 weeks. These symptoms did not develOp in the 0 but in— stead in the .005 ppm Zn treatment. Symptoms on Saginaw at 0 Zn did not develop until 5.5 weeks after emergence. The Zn deficiency symptoms in Saginaw at 0 Zn were less severe at all times than those of Saginaw at .005 ppm Zn, 29 30 a phenomenon observed in several different growth exper- iments. This was the reverse for Sanilac at these two treatments. Photographs of Saginaw and Sanilac grown at all Zn concentrations are represented in Figures 5, 6, 7, 8, and 9. Here is a situation in which a plant variety (Saginaw) exhibits a more normal response to the lower of two levels of an essential element when both levels are at deficient concentrations. Sanilac responds better at the higher concentration, as one would expect. This was a very unusual and interesting result. It deserves some comment and speculation as to what could have caused this response. Zinc is known to function as a co-factor for sev- eral plant enzymes (Reed 1946). It is possible that at .005 ppm Zn the concentration is just below a threshold level which must necessarily be reached for sustained normal metabolism. If some such threshold effect is in- volved here and the required concentration is approached but not satisfied there may be induction or activation of an enzyme system which subsequently cannot be sustained as a continuously functioning system due to insufficient 31 fluxes: 5 us omnmsumouonmv .GN mo Ho>oH o e um 8.5.3 353% soon :4di usagm one 343 tandem—em mo comfiuoxwaoolnd .mwm 32 30.00: h as ponmmumouonmv .cowumuucoosoo cu 8mm moo. e an enema nuceam Son 923 03.3mm one 553 Sawmmm mo somwuomfioollé .uflh 33 Amxoo3 h um wonmuumouonmv .cowueuusoocoo an and mo. e um csoum undead coma Azcmv oeHficdm use Aummv sandmmm mo comwummaooll.h .mHm 34 A9303 h on oonmsumouonmv .cowusnucoosoo on Sun m. e an 55.3 nus—3m Swen 32mg oeaficmm use 3.23 Ecwmom uo somHHeQEOUIId .mwm 35 e um cacao. 353m soon 3on: b no confidumouosmv . dawusuusoocoo 9N Sum m 329 coin—em e5 Adamo awesome no 8339836 .3...— ‘1‘)- 36 co—factor. However, if such a "partial reaction" were complete enough to initiate feedback inhibition result- ing in the blockage of any alternate pathways it might cause just such a differential response as observed at these two Zn levels. Speculating further, one might note that this would not be the situation at the 0 level of Zn because the Zn concentration would never approach the threshold concentration: therefore, there would be no induction of the target enzyme system and consequently no hindrance to the formation of an alternate pathway. Such an alter- 'nate pathway may be beneficial to the plant but obviously not adequate to sustain normal growth. An explanation of this phenomenon will require much more extensive research than undertaken here. This study is only an effort to disclose some clue to the mechanism responsible for the differential susceptibility to low Zn in these two varieties. Further research is necessary to determine the affect of Zn on the activity of various enzymes. 37 B. Dry Weight Agggmulation Dry weight accumulation for the plants harvested seven weeks after emergence is summarized in Table 4. Both varieties reach their maximum dry weight at .5.ppm Zn. Sanilac grows somewhat better than Saginaw at this Zn level but Saginaw grows better than Sanilac at the low Zn levels. TABLE 4.--Comparison of dry wt. (grams) of Saginaw and Sanilac plants harvested 7 weeks after emergence. Zn cons . in growth root stem ‘ leaf pods plant total medium (ppm) petiole top 0.0 5.8 6.4 11.2 0.20 17.8 23.6 0.005 4.2 5.6 9.8 -- 15.4 19.6 SAGINAW 0.05 6.0 7.8 12.6 1.9 22.3 28.3 0.5 5.7 8.8 14.3 3.2 26.3 32.0 5.0 4.3 7.4 13.2 2.0 22.6 26.9 0.0 3.1 5.4 7.4 -- 12.8 15.9 0.005 3.9 5.9 9.8 0.9 16.7 20.5 SANILAC 0.05 6.4 8.1 14.6 4.1 26.8 33.2 0.5 7.7 9.3 15.6 4.9 29.8 37.5 500 006 006 2.0 -" 206 302 38 In support of the order of deficiency development, Saginaw weighed 20%.more at the 0 Zn level than at the .005 ppm Zn level. This relationship, as before, was just the opposite for Sanilac. The high degree of tolerance in Saginaw to very low Zn, as Opposed to the response of San- ilac, was clearly evident at the 0 Zn level. The growth curves for these two varieties, grown over a range of Zn concentrations, is given in Figure 10 and is broken down into a top/root ratio in Table 5. The ratio for Saginaw is lower than for Sanilac at low Zn levels while at intermediate and high levels the ratio in Saginaw is higher than in Sanilac. TABLE 5.—-Comparison of top/root ratios of Saginaw and Sanilac at various Zn concentrations. W Zn concentration in growth medium (ppm) SAGINAW SANILAC 0.0 3.0 4.1 0.005 3.7 4.3 0.05 3.7 3_5 °~5 4.6 3.9 5.0 5.3 4.1 grams dry wt. 40 35 30 25 15 10 39 Fig. 10.--Comparison of the growth curves between Saginaw and Sanilac over a range of five Zn treatments (grams dry wt.). L. vet .r" .\ i- ’/’ \ I a" s / ‘0 I _ I / +‘ / \ / \\ ’ \\ / \ I ' «v r 1 I, II, J 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (DEM) ----Saginaw -—-Sanilac 40 The root tissue of Sanilac is about half the weight of the root tissue of Saginaw at the 0 Zn level, while the tOps are very similar. This indicates that at this treatment the roots of Sanilac are less vigorous than the t0p. Plants used for elemental analysis were harvested at five weeks with the idea in mind that any differences detected would be most accurate at the earliest possible stage of differential response. The differences in dry weight are less at this stage but the differential plant response is still apparent (Table 6). The dry weight to fresh weight comparison (dry weight/fresh weight) (Table 7) revealed that the percent dry weight of Sanilac tops was 15%.below normal at .005 ppm and 12%.below normal at 0 Zn, using plants grown at either .05 or .5 ppm Zn as normal. Saginaw maintained the normal ratio at .005 ppm Zn but dropped at 0 Zn to 23%.below that of normal Saginaw. This indicated that the percentage increase in fresh weight of Saginaw at 0 Zn over Saginaw .005 ppm Zn was even greater than the percentage dry weight increase. 41 TABLE 6.--Comparison of dry wt. (in grams) of Saginaw and Sanilac plants grown at several Zn concentrations and harvested at five weeks after emergence. Zn cone. in growth root stem petiole leaf plant total medium top (ppm) 0.0 3.4 2.4 0.9 6.9 10.2 13.6 0.005 3.2 2.4 0.9 6.7 10.0 13.2 SAGINAW 0.05 3.1 2.8 0.9 8.3 12.0 15.1 0.5 2.8 2.6 0.9 8.3 11.8 14.6 5.0 1.5 2.3 0.8 7.6 10.7 12.2 0.0 2.4 2.5 0.7 6.2 9.4 11.8 0.005 4.4 2.7 0.6 6.0 9.3 13.7 SANILAC 0.05 3.2 3.0 1.0 7.8 11.8 15.0 0.5 2.5 3.1 0.9 8.4 12.4 14.9 42 TABLE 7.--Percent dry weight of Saginaw and Sanilac bean plants grown at several Zn concentrations and harvested at five weeks after emergence. Zn conc. in growth root stem petiole leaf plant medium tOp (ppm) 0.0 3 4 12.1 6.2 8.7 7.2 0.005 3.4 12.4 6.7 8.5 9.4 SAGINAW 0.05 3.3 12.5 5.9 8.7 9.4 0 5 4 O 12.2 5.7 9 1 9.2 5 0 2 4 11.5 5 3 9 2 9.1 O 0 3 1 12.9 6 5 9.7 9.7 0.005 4.2 13.1 6.3 9.8 9.4 SANILAC 0.05 3.3 15.3 7.5 10.5 11.0 0.5 3.4 13.8 6.9 10.1 10.5 5.0 1.9 9.1 8.6 5.8 13.9 43 C. g_and Zn Accumulation Iron, K, and Ca concentrations did not exhibit large differences in respect to the Zn supply (Tables 8, 9, and 10). Phosphorous and Zn, however, did show large differences and, in particular, P content correlated well with the growth curves (Figures 10 and 12) as well as the appearance of deficienCy and toxicity Symptoms in both varieties. The accumulation of these two plant nutrients will be treated in detail. At the 0 level of Zn, Saginaw accumulated twice as much Zn as Sanilac on a total plant basis, with the greatest difference in the leaf tissue (Table 11). Trans- port of Zn from root to top is also higher in Saginaw. However, the differences are less pronounced on a weight basis (Table 12). This confirms Polson's (1967) finding with 65Zn uptake. With a 48-hour uptake period, he found that Saginaw took up more Zn and translocated higher per— centages of Zn into leaves than Sanilac. At 0 Zn Sanilac had a high concentration of Zn in the petiole relative to both the stem and leaf tissue (Table 12). However, this build-up of Zn in the petiole did not occur in plants grown at .005 ppm Zn. 44 TABLE 8.-—Comparison of the iron concentration (micro— grams/g tissue) in root, stem, petiole, and leaf of Sag- inaw and Sanilac plants, grown at several Zn concentra- tions. Zn conc. I I in growth root stem petiole leaf medium (ppm) 0.0 545.5 47.5 129.9 191.8 0.005 412.8 37.6 119.7 145.3 SAGINAW 0.05 319.8 24.7 53.4 171.2 0.5 491.6 62.9 102.5 155.7 5.0 464.8 81.8 87.1 177.1 0.0 306.6 44.1 75.7 188.1 0.005 549.3 56.0 128.4 173.1 SANILAC 0.05 311.3 25.3 12.9 100.7 0.5 315.5 62.9 133.9 223.3 5.0 220.4 112.3 160.0 159.2 45 TABLE 9.--Comparison of the potassium concentration (mg/g tissue) in root, stem, petiole, and leaf of Saginaw and Sanilac beans, grown at several Zn concentrations. —_mY Zn conc. in growth root stem petiole leaf medium .(ppm) 0.0 70.6 38.9 75.7 31.7 0.005 70.4 37.7 73.3 30.2 SAGINAW 0.05 83.2 51.2 98.4 33.7 0.5 63.4 48.9 102.0 35.4 5.0 60.8 58.3 105.8 35.0 0.0 70.6 38.9 75.7 31.7 0.005 71.9 34.1 56.8 33.4 SANILAC 0.05 74.0 36.3 88.1 39.7 0.5 68.7 42.5 85.5 34.4 5.0 59.2 36.5 43.0 32.4 46 TABLE lO.--Comparison of the calcium concentration (mg/g: tissue) in root, stem, petiole, and leaf of Saginaw and Sanilac beans, grown at several Zn concentrations. . . . . . . 1‘ I Zn cone. in growth root stem petiole leaf medium (ppm) 0.0 52.8 55.0 130.9 192.2 0.005 40.7 71.3 132.9 202.6 SAGINAW 0.05 70.9 56.4 7120.7 203.1 0.5 81.7 61.7 .114.7 181.7 5.0 65.0 . 70.3 88.6 172.4 0.0 46.1 70.7 127.9 185.4 0.005 48.1 73.9 119.3 231.2 SANILAC 0.05 71.2 53.6 110.2 191.0 0.5 84.0 58.2 102.5 183.8 5.0 40.0 76.8 82.2 83.7 TABLE 11.--Comparison of Zn accumulation (mg Zn/total 42‘ tissue) in the root, stem, petiole, and leaf of Saginaw and Sanilac beans, grown at several Zn concentrations. Iva—— 1" Zn conc. in QFOWth root stem petiole leaf plant total medium t0ps (ppm) 0.0 8.76 0.13 0.05 0.58 0.76 9.52 0.005 5.52 0.07 0.03 0.50 0.60 6.12 SAGINAW 0.05 4.06 0.10 0.06 70.37 0.53 4.59 0.5 5.57 0.18 0.17 0.60 0.85 6.42 5.0 5.48 1.20 0.55 7.49 9.24 14.72 0.0 4.78 0.07 0.04 0.21 0.32 5.10 0.005 5.38 0.05 0.02 0.24 0.31 5.69 SANILAC 0.05 4.10 0.09 0.05 0.29 0.43 4.53 0.5 4.65 2.18 0.11 0.92 3.21 7.86 5.0‘ 3.63 0.83 0.69 3.28 4.80 8.43 48 TABLE l2.--Comparison of the Zn concentration (micrograms Zn/g tissue) in the root, stem, petiole, and leaf tissue of Saginaw and Sanilac bean plants. L T r __r -_ Zn conc. in QFOWth root stem petiole leaf plant medium tOps (ppm) 0.0 2576.0 53.8 57.2 68.7 75.0 0.005 1725.6 29.1 37.6 74.5 60.0 sacrum 0.05 1310.6 36.3 68.1 44.4 44.0 0.5 1989.5 69.3 81.1 71.7 72.0 5.0 3650.7 524.5 690.7 985.5 864.0 0.0 1993.0 27.4 50.3 33.2 34.0 0.005 1221.0 18.4 28.9 40.6 33.0 SANILAC 0.05 1281.8 28.4 53.4 36.6 26.0 0.5 1859.4 703.6 127.6 109.8 259.0 5.0 7253.6 1377.9 2316.4 763.3 923.0 fl 49 These data indicate that the Zn concentration alone in leaf tissue or plant tops cannot be the basis for Zn deficiency. This is because the Zn level in the leaf or plant tap is equal to or greater at the 0 Zn level than that of plants grown at normal levels of Zn, with total uptake being less at the normal level for both varieties. Similar observations were made by Ambler and Brown (1969). This phenomenon where plants accumulate more Zn at low levels than at higher ones deserves further com- ment. If a certain level of Zn is needed at a particular time in ontogeny and that level is not reached, the plant makes out with an alternate, less efficient, and slower process leading to abnormal or much retarded growth. Meanwhile Zn is accumulating slowly in the tissue but the timing is now too late for it to have much influence on growth. The plant's concern with Zn has been side-lined. Another process is functioning. So Zn accumulates as growth proceeds at a slow pace and the plant seems in- capable of going back and making use of the accumulated 50 Zn. This results in a higher Zn concentration in these tissues, even when showing Zn deficiency symptoms. Phosphorous analysis showed that at the 0 Zn level Sanilac contained 22%.more P than Saginaw in the plant tOp, Saginaw contained 26%.more P than Sanilac in the root, and Sanilac contained 15%.more for the total plant on a weight basis (Table 13). Thus there must have been a more rapid uptake and translocation of P in San— ilac than in Saginaw at this Zn level (Figure 11). This does not occur when plants are grown at nor— mal nutrient levels. Both varieties contained abnormally high levels of P at low Zn levels with Sanilac having the higher concentration (Figure 12). The P concentration of Saginaw tops at the 0 level of Zn is 33% greater than at normal Zn and the P concentration in Sanilac teps is 93% greater. Thus, based on the P concentration at the normal level of Zn, there is a large percentage difference in P accumulation between the two varieties. This confirms the findings of Ambler and Brown (1969) in regard to in- creased P accumulation in these two varieties; they also found an increase of Fe in Sanilac. The data here, how- ever, indicate a higher concentration of Fe in Saginaw at 51 TABLE 13.——Comparison of P concentration (mg P/g tissue) in the root, stem, petiole, and leaf of Saginaw and San- ilac beans. (I Zn conc. in growth root stem petiole leaf plant total medium tops (ppm) 0.0 6.8 2.8 4.8 10.1 7.9 736 0.005 6.2 3.3 4.7 11.0 8.6 .8.0 SAGINAW 0.05 6.3 2.9 3.3 7.3 5.9 6.0 0.5 8.1 2.3 3.2 5.1 4.3 5.1 5.0 6.7 3.4 3.6 5.9 5.2 5.4 0.0 5.4 3.9 5.5 12.4 9.6 8.8 0.005 5.7 4.4 5.6 10.7 8.5 7.7 SANILAC 0.05 6.5 3.0 4.0 5.9 5.0 5.3 0.5 7.9 3.1 3.4 4.6 4.7 4.8 5.0 7.7 5.1 4.8 2.5 2.9 3.4 52 Fig. ll.--Comparison of the P concentration between Saginaw and Sanilac root, stem, petiole, and leaf tissue at the 0 Zn treatment (mg P/g tissue). .2L- 1- o l .J_ .1 root stem petiole leaf plant tissue ----Saginaw ——Sanilac 53 Fig. 12.-—Comparison of the P concentration between Saginaw and Sanilac bean leaves under five Zn treatments (mg P/g tissue). mg P/g tissue 2 1 l 1 - I o 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----Saginaw --Sanilac P concentration ratio of top/root Fig. l3.--Comparison of the top/root P concentration ratio between 1.6 1.5 1.4 1.2 1.0 54 Saginaw and Sanilac over a range of five Zn treatments (mg P/g tissue). ----Saginaw -—-—Sanilac l I I 0.005 0.05 0.5 Zn concentration in growth medium (ppm) J- 55 the 0 level of Zn and a higher concentration in Sanilac at the .005 ppm level of Zn (Table 8). There is an inverse relationship between Zn con- centration in the growth medium and P concentration in the leaf tissue for Sanilac (figure 12). A curve for the P concentration in the leaf tissue for both varieties corresponds inversely with the growth curve (Figures 10 and 12), with the exception of the toxic (5 ppm) level of Zn in Sanilac. The P concentration in Saginaw leaves and/or t0ps was lower at the 0 level of Zn than at the .005 ppm level, while the reverse was true for Sanilac. These differences in P accumulation are great enough to be a real factor or at least an indication of a basic difference between Saginaw and Sanilac that may determine their differential susceptibility to Zn defi- ciency. The possibility of actual P toxicity cannot be eliminated. Rossiter (1952) found that subterranean clover showing incipient P toxicity symptoms contained a P concentration of 1.43%“ The P concentration in the leaves of Sanilac at 0 Zn is 1.24%w Biddulph et a1. (1952), in studying the uptake of P by bean plants, found that 56 A concentration of approximately 6 mg. of P/gram of dry matter in trifoliate leaves is attained from solutions at 5 X 10‘5 M P and this value is sufficient for continued growth of leaves. The corresponding combined stem and petiole concen- tration is approximately 2 mg. P/gram dry matter when the leaves are adequately supplied. The stems and petioles will however, build up to 4 mg. P/gram dry matter as more P is made avail- able. The concentration in cordate leaves cor— respondingly rises from 3 to 7 mg. P/gram dry matter. It is this additional accumulation of P beyond the concentration adequate for leaf growth and stem extension, which causes dis- turbances in the metabolic use of the other ions, particularly iron. The passage of such ions through tissue rich in P is interfered with and much of the Fe is precipitated along the conductive tissues. The principal effect of high P concentration on the development of chlorosis is explainable on this basis. It is interesting to note in the light of these observations that Sanilac, at all Zn levels and in both petiole and stem, has higher P levels than Saginaw. This is not so in the leaf tissue; in fact with the exception of the 0 Zn level, the reverse is true. Boawn et a1. (1964) found that increasing the supply of P induced a growth disorder that could be eliminated by an in— creased supply of Zn. Neither the development of the deficiency nor the elimination of the deficiency was associated with the changes in the concentration of Zn in the stem and leaf tissues. High concentration of P in the tissues resulted in high P/Zn concentration ratios which appear to offer a better 57 explanation of the metabolic upset. Healthy plants tended to have P/Zn ratios less than 400, whereas in deficient plants the ratio was generally greater than 400. The severity of Zn deficiency symptoms appeared to be associated with the P/Zn ratio and not to the Zn concen- tration alone in the tissues. At the 0 Zn level there was little difference in the P/Zn ratio in root tissue between Saginaw and Sanilac, while the difference in the tops was about 180%. The P/Zn ratios in the root were 2.6 and 2.7 respectively for Sag- inaw and Sanilac, and in the leaf tissue 373 for Sanilac and 147 for Saginaw (Table 14). This suggests that the major difference is not in uptake but rather in translo- cation. The Zn concentration in Saginaw tops was higher and the P concentration was lower than for Sanilac. The P/Zn ratio for Saginaw leaf tissue remained unchanged at 0, .005, and .05 ppm Zn, while in Sanilac there was a direct relationship between Zn level and the P/Zn ratio in the tissue (Figure 14). This suggests that the roots may be involved in determining the differential response at low Zn. For Sanilac the P/Zn ratio decreased to 161 at .05 ppm Zn and dropped further to 42 at the 58 TABLE 14.--Comparison of the P/Zn concentration ratio in the root, stem, petiole and leaf tissue of Saginaw and Sanilac bean plants. Zn sons. in growth root stem petiole leaf plant total medium top (ppm) 0.0 2.6 52.0 83.9 147.0 98.3 8.9 0.005 3.6 113.4 125.0 147.7 136.4 13.5 SAGINAW 0.05 4.8 79.9 48.5 164.4 90.0 13.6 0.5 4.1 33.2 39.5 71.1 48.2 8.5 5.0 1.8 6.5 5.3 6.0 5.9 3.4 0.0 2.7 142.3 109.3 373.5 198.2 13.6 0.005 4.7 239.1 193.8 263.5 230.0 20.2 SANILAC 0.05 5.1 105.6 74.9 161.2 107.5 13.9 0.5 4.2 4.4 26.6 41.9 11.8 6.8 5.0 1.1 3.7 2.1 3.3 2.8 1.7 59 i Fig. l4.--Comparison of the P/Zn ratio in the leaf tissue of Saginaw and Sanilac bean plants. 300- 200- C: 4v " ’ \ Q .- ’ ’ \ 0' 1' ”””” "" \ \ \ \ \ ioo- ‘\ \ \ \ ----Saginaw \ \ ‘\ -——-Sanilac \. ‘- 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) 6O .5 ppm level. As the P/Zn ratio in Sanilac leaves de- creased from 0 to .005 ppm Zn, the P/Zn ratios in the stem, petiole, and root increased; this is due to both a decrease in Zn and an increase in the P concentration. At the .005 ppm Zn treatment Sanilac stem and petiole had a lower Zn and a higher P concentration than any other treatment. The extremely low Zn concentration and high P concentration in the stem and petiole at the .005 ppm Zn treatment could be an indication of the incipient development of a response in Sanilac similar to that of Saginaw at 0 and .005 Zn treatments. This is also appar- ent in the P/Zn ratio (Table 14). The P/Zn ratio in the leaves of Saginaw remained the same at O and .005 ppm Zn whereas the ratio in the stem, petiole, and root increased. Hence the same trend prevailed for both varieties with the exception of Sag- inaw leaf tissue which maintained a favorable P/Zn ratio at both low Zn levels. To correlate the P/Zn ratio to the more healthy response of Saginaw at 0 than at .005 treatment we must look to the stem, root, and petiole, not the leaf. These tissues all have a considerably smaller P/Zn ratio at the 0 treatment. 61 It appears that the P concentration correlated better than the Zn concentration with the severity of Zn deficiency symptoms. What causes the high P and the high P/Zn ratio cannot be resolved here. The only deduction that can be made with respect to P—Zn interaction is that decreasing Zn concentration in the growth medium results in an increasing P concentration in the plant t0ps and causes an increase in the P/Zn ratio in Sanilac. Millikan (1963) found that the minimum P/Zn ratio in relation to the occurrence of Zn Deficiency was about 300 for 97 day old subterranean clover plants, however, the minimum ratio varied with the age of the plants. The key here is that there is a differential up- take and transport of P and this is correlated with the differential susceptibility of these two varieties to Zn deficiency and to the Zn concentration in the growth medium. It should be emphasized, however, that the in— ternal buildup of P does not in itself constitute Zn de- ficiency although it does reflect the development of the symptoms. It is apparent that further research on a time course basis is necessary to clarify the P-Zn relation- ship and the differential reSponse of these two varieties 62 to this relationship. The same basic design could be used with several samplings for analysis at various time intervals during growth. This would give an insight into the P-Zn interaction against the dynamic background of a "growing plant." More critical studies need to be done to deter- mine the activity of various enzymes in relation to the Zn concentration in the growth medium and the Zn require- ments of the two varieties. II. Reaction to a Toxic Zn Concentration A. P, Znyfiand_2£y Weight Accumulation Between 0.5 and 5.0 ppm Zn there is a decrease of 92% in dry weight for Sanilac and only 13% for Saginaw. This indicates a high degree of tolerance to high Zn levels for Saginaw as compared with Sanilac. Sanilac, at the 5 ppm treatment, contains twice as much Zn in the root tissue as Saginaw (Table 12). Similarly there was more Zn in the stem and petiole, how- ever, it had less in the leaves. At the high Zn level 63 there was a relationship between both the Zn and the P concentration in the tissue and the development of tox- icity symptoms (Tables 12 and 13). A comparison of the percentage increase in Zn concentration between tissues from .5 ppm (where both plants grow relatively well) and 5 ppm Zn treatments in- dicated which tissue might be most responsible for the differential response at 5 ppm Zn (Table 15). TABLE 15.--Percentage increase in the Zn concentration for different tissues between .5 and 5 ppm Zn treat— ments. (based on data in Table 12) MW Tissue SAGINAW SANILAC root 88 345 stem 650 96 petiole 752 1215 leaves 1270 600 total 2760 2756 The argument for comparing these varieties on the basis of ratios or percentages of one treatment to another is as follows: It is not always meaningful to deal in 64 absolute values when comparing two different plant sys- tems or two treatments within one system. Plants have different nutritional requirements and a different bal— ance exists among these requirements. It helps to com- pare them on the basis of a ratio of the response in treatment 1 to treatment 2 for one plant system to a ratio of the response in treatment 1 to treatment 2 of the other plant system. This kind of relationship can also be very mis- leading, for example, the Sanilac stem doesn't show a big increase because at .5 ppm Zn its stem has already accumulated a larger quantity of Zn than the Saginaw stem (Table 12). However, this relationship continues to be valuable as long as it is kept in perspective. The San- ilac stem was already "loaded" with Zn at the .5 ppm Zn level, yet the plant continued to grow well. In Table 15 a considerable difference can be seen between Saginaw and Sanilac as a function of the differential response from one treatment to another. This indicates much more than if we simply consider the absolute values (Table 12). From the growth curve (Figure 10) it is clear that these 65 two varieties have differentiated markedly in growth re- sponse between the .5 and 5 ppm Zn treatments. In Table 12 this differentiation between Saginaw and Sanilac is reflected as Zn accumulation in plant tissues. These values show the difference between the two varieties to be similar in all tissues except the leaves. But, with respect to the percentage increase in the tissues (Table 15), it is seen that the values between the two varieties were inverse to each other for each tissue. It is most interesting that the totals of per- cent increase for both varieties turned out to be nearly . the same. We might conclude from this that the increased absorption is at the same rate for each variety at this high Zn level and that the only limiting factor was the extent of the root system, since these values were based on grams dry weight. From the distribution pattern of Zn, expressed as a percent increase from one treatment.to another,.it: would appear that the reason for these differences lies in a differential type of binding and/or transport of Zn from one tissue to another. This was most evident in the large accumulation of Zn in the petiole (Tables 12 and 15). 66 The concentration of Zn and P on a dry weight basis (Tables 12 and 13) showed Sanilac to have twice the Zn of Saginaw in the root and several times more Zn in the stem and petiole. Saginaw had the higher concen- tration of Zn in the leaf tissue. The same pattern ex- isted for P except that the difference in the leaf tissue was much greater: Saginaw leaves contained more than twice as much P as Sanilac. Saginaw maintained its transport of P to the leaf but Sanilac did not at the 5 ppm Zn level (Figure 15). The movement of Zn from petiole to leaf was drastically reduced in both varieties (Figure 16). There was a large accumulation of Zn in the petiole of Sanilac. In compar- ing Figures 15 and 16 we see what appears to be a buildup of Zn and P in the petiole of Sanilac: whereas in Saginaw this condition is not apparent. This supports the strong indications, presented in Table 15, that there is a dif- ferential in Zn binding and/6r transport between these two varieties. Whether or not this is a causitive factor or only a reflection of Zn toxicity cannot be determined here. mg P/g tissue 67 Fig. 15.--Comparison of the P concentration between Saginaw and Sanilac root, stem, petiole, and leaf tissue at the 5 ppm Zn treatment (mg P/g tissue). llr' 2h 1)- 0 | I .J root stem petiole leaf plant tissue ---—Saginaw -——Sanilac mg Zn/g tissue 68 Fig. l6.--Comparison of the Zn concentration between Saginaw and Sanilac root, stem, petiole, and leaf tissue at the 5 ppm Zn treatment (mg Zn/g tissue). root stem petiole plant tissue ----Saginaw --Sanilac leaf 69 There is a differential degree of P-Zn interaction reflected in the petiole-leaf equilibrium of these two varieties, however, further research is necessary to de— termine how this originates. This corraborates the find- ing of Polson (1967) that the differential response at 5 ppm Zn was traceable to the plant top rather than the root, shown by reciprocal grafting. Section on Microanalysis Comparisons in electron microprobe analysis were made on the basis of ratios. A ratio between those ele— ments analyzed for in one scan were compared to a ratio between the same elements analyzed for in another scan. The relative concentrations of elements, as such, were not compared between scans. The reason the elemental concentrations were not compared between different scans was because of the lack of homogeneity among homologous plant tissues when con- sidered ultramicroanalytically. For example in a 300 micron—long scan, which analyzes a volume of about 600 70 cubic microns, 10 cell walls may be traversed in one in— stance while in another only 6. It would be a misrepre- sentation to interpret the resulting elemental concentra- tions as being from representative quantities of tissue. A great many of these discrepancies can be eliminated by the use of ratios. These ratios were derived from values obtained by making planometric measurements of the area under the curve plotted by the x, y, recorder as described in the materials and methods section. The ratios of main con- cern here were Ca/P and K/P. A Zn ratio was not calcu- lated because of the low concentration of this element. Scans made in the root tissue were across the vascular tissue and include both xylem and phloem. Fig- ures l7 and 18 summarize the results obtained after cal— culating both Ca/P and K/P ratios. In the Saginaw variety the two ratios appeared to oscillate from one Zn level to the next. There was also an inverse relationship between the Ca/P and K/P ratios; as the Ca/P ratio increased, the K/P ratio de- creased. This did not occur in Sanilac where both 71 Fig. l7.--Comparison of the Ca/P ratios in the root tissue of Saginaw and Sanilac over a range of four Zn treatments (electron microprobe analysis). 40r 35L 30- I / l 25- / / Q / 8 / V . - ; .fi 0 .4 I l J o 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----Saginaw -——Sanilac 72 Fig. 18.--Comparison of the K/P ratios in the root tissue of Saginaw and Sanilac over a range of four Zn treatments (electron microprobe analysis). 0 1 n I 1 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) --—-Saginaw -—Sanilac 73 ratios had the same general trend, although the K/P ratio was larger than the Ca/P ratio in all treatments. Hewitt (1951) believed that Ca and K act in a complementary fashion toward one another in maintaining ‘cell organization, hydration, and permeability, thus in- directly influencing many cellular processes or systems. It appeared here that there was also some intricate bal— ancing relationship between Ca and K as expressed in these two ratios. At the normal Zn treatment the Ca/P ratio for both varieties was about 2.5 whereas at the 5 ppm Zn treatment the ratio increased to 30.0 in Saginaw but re— mained low in Sanilac. At first glance this would appear to be adverse for Saginaw, but Saginaw is tolerant to 5 ppm Zn. What happened was that the K/P ratio dropped, in the Saginaw variety, from 42 to 5 and, ostensibly, appeared to compensate for the high Ca/P ratio. The K/P ratio also dropped in Sanilac, a drop that was not offset by a high Ca/P ratio; consequently, an imbalance devel— oped. A similar thing happened at the 0 Zn level only the compensation was reversed. The Ca/P ratio went down in Saginaw as the K/P ratio went up. Actually, at the 74 0 Zn treatment, both ratios in Saginaw were the same as at the normal (.05 ppm) Zn level. In summary, in Saginaw the two ratios compensated for each other at various Zn levels and in Sanilac the two ratios fluctuated the same and no compensation occurred. Compensation is spoken of here using the ratios at the .05 Zn treatment as a norm. In the stem the Ca/P ratio in both the xylem and phloem was again the same for both varieties at the .05 ppm Zn treatment although considerably lower than the root (Figures 19 and 20). At the 5 ppm Zn level the Ca/P ratio in the phloem of the stem rose 20—fold in Sanilac while in Saginaw it remained unchanged. Both varieties underwent a slight increase in the xylem. This would indicate, as the ratio is increased in the phloem of San- ilac, and maintained unchanged in the xylem that somewhere there was a reduction in the Ca/P ratio in Sanilac. In the root tissue (Figure 17) the Ca/P ratio in Sanilac was reduced (compared to Saginaw) enough to compensate for the increase in the stem phloem tissue. This compensa- tion suggests that under toxic Zn conditions Sanilac transported a much greater amount of calcium to the stem phloem tissue than Saginaw. 75 Fig. l9.--Comparison of the Ca/P ratio in the phloem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). 10(- Ca/P h I -5 he re 0 0.005 0.05 0.5 Zn concentration in growth medium.(ppm) ----Saginaw -—Sanilac J l L .J 76 Fig. 20.--Comparison of the Ca/P ratio in the xylem tissue of the stem between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). . L fl n I 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium.(ppm) -q--Saginaw —-—6anilac 77 Saginaw did not transport a large amount of Ca from the root to the shoot phloem tissue; it showed a similar phenomenon, however, except that K was involved instead of Ca. Saginaw showed a dramatic increase in the K/P ratio in both the xylem and phloem tissue of the stem at the 5 ppm Zn treatment (Figures 21 and 22). This was commensurate with the decrease in the K/P ratio in the root vascular tissue. The limiting factor in the rapid transport of K from root to shoot in Saginaw ap- peared to be a function of root uptake while the rapid transport of Ca in Sanilac appeared to be a function of the movement from xylem to phloem. The reason for making this assumption was that the K/? ratio in Saginaw at the high Zn level was high in both xylem and phloem while the Ca/P ratio in Sanilac was actually higher in the xylem than in the phloem. Further indications that adverse Zn concentrations in the growth medium were upsetting the nutrient balance in Sanilac were apparent at the O and .005 ppm Zn treat- ments. At the .05 ppm Zn treatment the amount of trans— port of K, as indicated by the K/P ratio in Figures 18 78 Pig. 21.--Comparison of the K/P ratio in the phloem tissue of the stem.between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprdbe analysis). 30 f I 25r- I I I I I I 20 L / m I I 9 / I I 15*- . / I I I I 10r' / ’.~ I 4’ \-‘~ I I' ‘~ ’ z'l' ‘~ I a' ‘-\~ I Sp’ q 0 1 .L IV j 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----saginaw --Sanilac 79 Fig. 22.-~Comparison of. the K/P ratio in the xylem tissue of the stem beWeen Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). O I L l w— fir w o ' ' 0.005 V 0.05 0.5 Zn concentration in growth medium (ppm) ----Saginaw ----Sanilac 80 and 21, was greater in Sanilac while at .005 Zn this re- lationship between the two varieties was reversed and the ratios were less under all circumstances. At the 0 Zn level the ratios were again reversed in both root and stem, and approached conditions as they existed at the normal level . The Ca/P and K/? ratios in stem tissue in Saginaw remained relatively stable at the two low Zn levels i (Figures 19. 20, 21. and 22). In all instances the .005 Zn treatment was slightly higher than the 0 and the .05 treatments, which were about the same for both tissues and ratios. There was a similar lack of fluctuation in stem xylem tissue of Sanilac for both ratios: however, the ratios for both low Zn levels were higher than those at normal Zn. . In Sanilac stem phloem tissue there was con- siderable fluctuation between both the Ca/P and the K/P ratios (Figures 19 and 20). At the .005 ppm Zn concen- tration the Ca/P ratio increased to a level 30-fold over that at normal Zn. Simultaneously the K/P ratio in the same tissue dropped, from 15 at normal Zn. to 0 at .005 ppm Zn. At the 0 Zn treatment the Ca/P ratio decreased 81 to only 15 times the normal ratio as the K/P ratio moved back up to almost double the normal level. It is difficult to explain the variations that are observed, even if, indeed the data are part of a con- sistent pattern. It can only be said that by providing either high or low amounts of Zn great fluctuations are brought about, in both stem and root tissues. in the Ca/? and K/P ratios. In addition. these fluctuations vary greatly between xylem and phloem. Root and stem ratios in the vascular tissue were commensurate with establish- ing greater or lesser degrees of transport of either Ca or K within one plant under conditions of a given treat- ment. There was much less variation of the Ca/P and K/P ratios in the leaf tissue than in either the root or stem conducting tissues. The two concentration ratios in the xylem, phloem. and mesophyll cells (for both varieties) were summarized in Figures 23 and 24. The most apparent development was the low Ca/P and K/? ratios in the leaves in both varieties and in all three tissues at the .005 ppm Zn treatment. 82 Fig. 23.--Comparison of the Ca/P ratio in the xylem, phloem, and mesophyll tissues of the leaf betwoen Saginaw and Sanilac. grown over a range of four Zn concentrations (electron micro- probe analysis). 50 " Saginaw ,. ’ 40;. )r Ca/P \ \ 30... l 20)- ‘\ I 10)- i ‘ - . oeeeeeeOO“"... O .0...o........I...'......"'Ti"'f..........I I 60 )" , " ~ - 50 - Sanilac I ~ ._ 40L.\ Ca/P I \ 30- 20 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----phloem —-xylem e e e emesophyll 83 Fig. 24.--Comparieon of the K/P ratio in the xylem, phloem, and mesophyll tissues of the leaf between Saginaw and Sanilac grown over a range of four Zn Concentrations (electron microprobe analysis). 20,. 15!- Saginaw ____.....---0 )~"” 10- ," _“ .eeeeeeeeeeeeeeeoeeoeeooeooeoo. o " 1 _1 l 1 O 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----phloem --xylem -°-°mesophyll 30 F 25 _ Sanilac 20 15 10 000'... 5 G J 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----phloem -—-—xylem °°'°mesophyll 84 In noting the changing values of the ratios as we progress from .005 ppm Zn toward the 0 Zn treatment both ratios in all tissues increased (with the exception of the Saginaw mesophyll tissue which actually decreased in both Ca/P and K/P). The nutrient condition of all tissues approached the state that existed at the normal Zn treat- ment. At the excess Zn treatment the greatest deviation from the normal Zn level occurred in Sanilac. It showed a decrease of the Ca/P ratio in the mesOphyll tissue with an accompanying increase in the K/P ratio. It also ac- quired a higher Ca/P and K/P ratio in the xylem tissue along with a lowering of these ratios in the phloem tissue. It should be considered here that the differences in Sanilac and Saginaw could have been a reflection of the fact that Sanilac was (in 3 of 5 Zn levels) a very "disturbed" plant, whereas Saginaw was somewhat more normal across all Zn levels. The question is; were the changes in Sanilac mere artifacts due to the upset in growth and metabolism induced by Zn deficiency or excess. or were these changes part of the reason for the upset? 85 When dealing with ratios as has been done here it is difficult to determine exactly which particular element to identify as being most responsible for the change or which more strongly reflected the change in the physiolog- ical response of the plant. In an attempt to resolve this difference a further ratio was calculated. Ca/K ratios in the root and stem conducting tissue were used as a means of cross—referencing both Ca and K between two dif— ferent ratios. It is of interest to determine which of the previous ratios (Ca/P and K/P) are most like the third ratio (Ca/K). This should afford some indication of which element (either Ca or K) is most influential in determin- ing the direction of these ratios. An absolute determina- tion cannot be obtained here, only an indication. The results of the Ca/K ratios are illustrated in Figures 25, 26, and 27. When these figures are compared to the K/P ratios (Figures 18, 22, and 21, respectively) there appears to be less correlation than when they are compared to the Ca/P ratios (Figures 17, 20, and 19, respectively). This could indicate that Ca plays the most dominant role in determining the trend of the ratios and consequently would most strongly reflect the changes 86 Fig. 25.--Comparison of the Ca/K ratio in the root tissue between Saginaw and Sanilac over a range of four Zn treatments (elec- tron microprobe analysis). 10‘? L 1 1 l 0 0.005 0.05 0.5 5.0 Zn concentration in growth medium (ppm) ----Saginaw ——Sani lac Ca/K 87 Fig. 26.--Comparison of the Ca/K ratio in the xylem tissue of the stem between Saginaw and Sanilac over a range of four Zn treatments (electron microprobe analysis). 1 10 -9dL I ‘1 I 0 0.005 0.05 0.5 Zn concentration in growth medium (PPm) ----Saginaw --Sanilac l4 1LT Ca/K 88 Fig. 27.--Comparison of the Ca/K ratio in the phloem tissue of the stem.between Saginaw and Sanilac grown over a range of four Zn treatments (electron microprobe analysis). 10 “'20 b \ J l J l O 0.005 0.05 0.5 5.0 Zn concentration in growth medium (PPm) ----Saginaw --Sanilac 89 in the physiological response of the plant. There was no correlation to the Ca/K ratio with either the Ca/P or the K/P ratios in the leaf tissue. It must be kept in mind here that the Ca/K ratio must be compared to the K/P ratio as its reciprocal. It is difficult, however, to determine how valid these values are. There were no replications of the analysis and there is no evidence to suggest just how consistent a ratio between two elements is, throughout all of a particular tissue. This technic for plant analysis is very new and much more intensive research is necessary to establish adequate criteria for the inter— pretation of the data. It was discovered during the microprobe analysis of Saginaw that at the juncture of a lateral root with the mother root in the 5 ppm Zn treatment, there existed a high concentration of all the elements for which anal- yses were made. This phenomenon was not found in the Sanilac variety nor for the other Zn treatments of either variety. Since lateral root primordia originate at the pericycle, they must eventually break through the cortex 90 and epidermis of the mother root. This leaves a "wound" at this juncture extending from the epidermis to the stele of the mother root. It is in the "wound area" that high elemental concentrations were found. Could there be ex- creted, at the wound region or surfaces, an abundance of organic acids or molecules with negative charges to bind cations? Figures 28 and 29 illustrate the vicinity of this area for Saginaw and Sanilac respectively. One reverse sample current oscillogram shows a low magnification of the general area of the root section where the lateral root protrudes from the mother root. This oscillogram is purely for orientation purposes. The other reverse sample current oscillogram is a higher magnification of the spe— cific area of the elemental concentration. The trace line indicates the vicinity of the scan that was made to deter- mine the relative concentrations of the elements. These relative concentrations are indicated on the plot below of which the total distance, left to right, represents the total distance, left to right, of the trace line on the reverse sample current oscillogram. Figure 30 rep- resents x—ray oscillograms for each element analyzed for 91 Fig. 28.--Oxcillograms and elemental concentration scan of Saginaw root, at lateral, for the 5 ppm Zn treatment. (electron microprobe analysis) 40x 330x7 ---- Ca - Scale I0.000 — K - Scale l.OOO --------- Zn ' Scale 300 ----- P - Scale I,OOO Gag“; intensity 92 Fig. 29.--Oscillograms and elemental concentration scan of Sanilac (electron micro- root, at lateral, at the 5 ppm Zn treatment. probe analysis) K - Scale 300 " Zn ' Scale IOO O m h 0 a n a C . _ _ . -‘xuamemufi , 93 Fig. 30.--x-ray oscillograms of Saginaw root, at lateral, grown at the 5 ppm Zn treatment. (compare to oscillogram on figure 27) potassium phosphorous calcium . zinc 94 in Saginaw. These oscillograms correspond to the high magnification oscillogram in Figure 28. This analysis of this juncture was repeated in a different experiment with all conditions the same as be- fore. The same accumulation of nutrients occurred in the Saginaw variety and again the Sanilac variety did not have such an accumulation. However, the ratios of the relative concentrations of the elements of the first ex- periment, when compared to the ratios of the second ex- periment, were entirely different. The occurrence of a concentration of nutrients in this area of the root was first discovered by Rasmus- sen (1968). He found this to be the point of entrance of aluminum in corn which resulted in aluminum toxicity. It is not yet clear what significance this might have, if any, to the differential susceptibility of these two varieties to excess levels of Zn. SUMMARY The results presented in this thesis suggest that the Zn concentration pg5_§g_in the plant tissue is not responsible for the development of Zn deficiency symptoms, at least the Zn concentration as disclosed by gross tissue analysis. The Zn concentration could, of course, be a re- flection of the differential susceptibility of the two varieties to Zn deficiency since Saginaw leaves do con- tain more Zn than Sanilac leaves. However, it appears once Zn is present in minimal amounts that the degree of P translocation from root to shoot is more nearly respon- sible for the onset of deficiency symptoms (Figure 13). It seems that an adequate supply of Zn in the surroundv ing nutrient medium is essential to adequate P utiliza— tion. Although the effect of deficient quantities of Zn in the nutrient medium is not reflected by the Zn con- centration in the tissues it is reflected in the P con— centration, the P/Zn ratio and in P translocation. 95 96 The effect of Zn at the high concentration can be more closely associated with the Zn concentration in the plant tissues. Sanilac accumulated particularly high quantities of Zn in the stem and petiole (highest in petiole). Saginaw also accumulated much Zn in these re- gions but considerably less than Sanilac. The high Zn level was also associated with a low P concentration in the leaf tissue of Sanilac. Thus, there appeared to be a Zn-P antagonism at both high and low Zn concentrations. Electron micrOprobe analysis showed Zn to affect calcium and potassium translocation differentially for the two bean varieties. Indications are that the Ca translocation was affected to a greater extent. All com- parisons were made in the form of ratios because there were no means of assuring that equal volumes of tissue were analyzed between treatments. Additional micrOprobe analysis showed there to be a large accumulation of nutrients at the juncture of a lateral and main root of the Saginaw variety. Possibly this occurrence could be explained anatomically or physio- logically or both. The differential between the two var- ieties is very great in this respect. REFERENCES 1. 2. 7. REFERENCES Ambler, J. E. and J. C. Brown. 1969. Cause of dif- ferential susceptibility to zinc deficiency in two varieties of navy beans (Phaseolus vulgaris L.). Agronomy Journal 61:41—43. Biddulph, 0. and C. G. Woodbridge. The uptake of phosphorous by bean plants with particular refer- ence to the effects of iron. Plant physiology 33:293-299. Biddulph, 0., Susann Biddulph, R. Cory and H. Doontz. 1958. Circulation patterns for phosphorous, sul- phur and calcium in the bean plant. Plant phys- iology 33:293-299. Bingham, Frank T., and James P. Martin. 1956. Ef- fects of soil phosphorous on growth and minor element nutrition of citrus. Soil Sci. Soc. Amer. Proc. 20:382-385. Bingham, F. T., and M. J. Gerber. 1960. Solubility and availability of micronutrients in relation to phosphorous fertilization. Soil Sci. Soc. Amer. Proc. 24:209-213. Boawn, Louis C. and G. F. Leggett. 1964. Phosphorous and zinc concentration in Russet Burbank potato tissues in relation to development of zinc defi- ciency symptoms. Soil Sci. Amer. Proc. 28:229- 232. Boawn, L. C., Frank G. Viets, Jr., and Carl L. Craw- ford. 1954. Effect of phosphate fertilizers on zinc nutrition of field beans. Soil Science 78: l-7. 98 10. ll. 12. 13. 14. 15. 16. 99 Burleson, C. A., A. D. Dacus, and C. J. Gerard. 1961. The effect of phosphorous fertilization on zinc nutrition of several irrigated crops. Soil Sci. Soc. Amer. Proc. 25:365-368. Ellis, B. G. 1965. Zinc deficiency (a symposium) response and susceptibility. Crops and Soils. 18:10-13. Halim, A. H., C. E. wassom and R. Ellis Jr. 1968. Zinc deficiency symptoms and zinc and phosphorous interactions in several strains of corn (E22.mays L.). Agronomy Journal 60:267-271. Hewitt, E. J. 1951. The role of mineral elements in plant nutrition. Ann. Rev. Plant physiol. 2:25- 52. Ingram, M. J. and C. A. M” Hogben. 1957. Electrolyte analysis of biological fluids with the electron microprobe. Anal. Biochem. 18:54-57. Jackson, M. L. 1958. Soil Chemical Analysis. Prentice-Hall, Englewood Cliffs, N. J. 151-154. Juday, W., J. Melton, G. Leesman, B. Ellis, and J. Davis. 1964. Zinc fertilization of pea beans, corn and sugar beets in 1964. Mich. Agr. Exp. Sta. Farm Sci. Res. Rep. 33:1-8. Millikan C. R. 1963. Effects of different levels of zinc and phosphorous on the growth of subter- ranean clover (Trifolium subterraneum L.). Aust. J. Res. 14:180-205. ‘ Millikan, C. R., B. C. Hanger, and E. N. Bjarnason. 1968. Effect of phosphorous and zinc levels in the substrate on 5zinc distribution in subter- ranean clover and flax. Aust. J. Biol. Sci. 21: 619-640. 17. 18. 19. 20. 21. 22. 23. 24. 25. 100 Pauli.A. W., Roscoe Ellis, Jr., and J. C. Moser. 1968. Zinc uptake and translocation as influ- enced by phosphorous and calcium carbonate. Agronomy Journal 60:394-396. Polson, D. E. 1968. A physiologic-genetic study of the differential response of navy beans (Phaseolus vulgaris L.) to zinc. Ph.D. Thesis, Mich. State Univ. Rasmussen, H. P. 1968. Entry and distribution of aluminum in Zg§_may : The mode of entry and distribution of aluminum in §g3_mays: Electron microprobe X-ray analysis. Planta (Ber1.) 81: 28-37. Rasmussen, H. P., V. E. Shull, and H. T. Dryer. 1968. The determination of element localization in plant tissue with the microprobe. Develop. in Applied Spectroscopy. 6:29-42. Reed, Howard S. 1946. Effects of zinc deficiency on phosphate metabolism of the tomato plant. Am. J. Botany 33:778-784. Rossiter, R. C. 1951. Phosphorous toxicity in sub- terranean clover and oats grown on muchea sand, and the modifying effects of lime and nitrate— nitrogen. Aust. J. Agric. Res. 3:227-243. Sass, J. E. 1958. Botanical Microtechnigue. Iowa State Univ. Press, Ames, Iowa. Sharma, K. C., B. A. Drantz, A. L. Brown, and James Quick. 1968. Interaction of zinc and phosphorous in top and root of corn and tomato. Agronomy Journal 60:453-456. Viets, F. G. Jr., L. C. Boawn, and C. L. Crawford. 1954. Zinc content of plants in relation to de— ficiency symptoms and yield. Plant Physiology 29:76-79. 101 26. watanabe, F. S., W. L. Lindsay, and S. R. Olsen, 1965. Nutrient balance involving phosphorous, iron, and zinc. Soil Sci. Soc. Zmer. Proc. 29: 562—565. APPEND IX 103 Comparisons of the elemental concentrations in Saginaw and (electron microprobe anal- Sanilac root vascular tissue. ysis) W Zn cone. in growth P K Ca medium (ppm) 0.0 4 162 95 0.005 14 251 158 SAGINAW 0.05 18 756 120 5.0 10 52 149 0.0 11 223 290 0.005 2 66 77 SANILAC 0.05 18 455 183 5.0 27 250 500 relative concentration/30 Y—f microns of scanned tissue. 104 Comparisons of the elemental concentrations in Saginaw and Sanilac stem xylem.and phloem tissue. (electron micro— probe analysis) Ca P K Zn conc. in growth medium Ph Xy Ph Xy Ph .Xy (ppm) 0.0 72 25 65 62 286 467 0.005 115 65 107 57 881 500 SAGINAW 0.05 10 5 27 20 130 115 5.0 .12 24 39 16 1050 514 0.0 32 20 7 11 187 186 0.005 8 43 0 27 1 450 SANILAC 0.05 5 13 23 43 356 510 5.0 534 50 52 36 550 330 a w—r r~v relative concentration/30 microns of scanned tissue 105 Comparison of the phosphorous concentration in Saginaw and Sanilac leaf tissues. (electron microprobe analysis) fl Zn conc. ave in in growth meso— ' total . ~ phloem xylem vascular medium phyll . (ave.) tissue (ppm) 0.0 77 18 19 18.5 38 0.005 31 63 57 60.0 50 SAGINAW 0.05 19 21 10 15.5 17 5.0 10 14 24 19.0 16 0.0 7 19 17 18.0 14 0.005 105 30 69 49.5 98 SANILAC 0.05 8 12 13 12.5 11 5.0 27 33 14 23.0 25 relative concentration/30 microns of scanned tissue 106 Comparisons of the potassium concentration in Saginaw and Sanilac leaf tissues. (electron microprobe analysis) Zn conc. ave in in growth meso- ' total . phloem xylem vascular medium phyll . (ave.) tissue (ppm) 0.0 32 129 124 127 95 0.005 41 200 233 216 290 SAGINAW 0.05 41 250 70 160 120 5.0 24 191 222 207 149 0.0 47 500 323 412 290 0.005 131 13 87 50 77 SANILAC 0.05 45 280 225 247 183 5.0 230 800 470 635 500 1., relative concentration/30 microns of scanned tissue 107 Comparisons of the calcium concentration in Saginaw and Sanilac leaf tissues. (electron micrOprobe analysis) Zn conc. ave in in growth meso- ' total . phloem xylem vascular medium phyll . (ave.) tissue (ppm) 0.0 170 450 121 285 247 0.005 118 920 225 572 421 SAGINAW 0.05 12 900 11 455 308 5.0 52 1250 203 746 502 0.0 130 850 209 530 396 0.005 525 225 310 268 353 SANILAC 0.05 137 714 192 453 348 5.0 148 1583 225 904 652 relative concentration/30 microns of scanned tissue HICHIGQN STQTE UNIV. LIBRQRIES l I" ll U H llllllll i 31293000851018