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THE ACCUMULATION AND METABOLISM OF GLYCINEBETAINE BY BARLEY IN RELATION TO WATER STRESS By Juanita Ann Ria Ladyman A DISSERTATION Submitted to Michigan State University in partial fulfiiiment of the requriements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Piant Pathoiogy 1982 'H’ :5 //7’t ABSTRACT THE ACCUMULATION AND METABOLISM OF GLYCINEBETAINE BY BARLEY IN RELATION TO WATER STRESS By Juanita Ann Ria Ladyman The objectives of this research were: (1) To test the hypo- thesis that in barley the betaine level of shoot organs may be an index of the stress history of the plant; this could have application in crop breeding and management; (2) To survey genetic diversity for betaine levels in Hordeum vulgare and fl. spontaneum to determine if physiological-genetic studies to evaluate the purported adaptive role of betaine accumulation would be feasible; (3) To seek evidence bearing on the adaptive value of betaine accumulation. Radiochemical tracer experiments and chemical analyses with both laboratory- and field-grown plants demonstrated that: (l) Betaine was a metabolic end—product accumulated during water-stress by young leaves as a result 0f.ifl.§i£! synthesis and translocation via the phloem; (2) Betaine levels in the uppermost leaves were significantly correlated with an integrated value, based on water potential measure- ments, of the seasonal plant water stress, irrespective of plant N— status; (3) During grain filling, betaine partitioned neither like total-nitrogen nor like dry matter and remained predominantly in Juanita Ann Ria Ladyman vegetative tissue. Although these findings support use of betaine level in the uppermost leaves as a stress index, this use cannot be recommended because: (a) The difference in betaine level between irrigated and non-irrigated crops was quite small and variable; (b) Betaine accumulation occurred no earlier than readily-detected morphological changes. Genetic variability for betaine accumulation was found among genotypes in laboratory trials with seedlings and in field studies with mature plants. The betaine level of the uppermost leaves of non-irrigated, mature, field-grown plants was significantly corre- lated with the betaine level of laboratory—grown, well-watered seed- lings. Apparently, in field-grown plants betaine levels were not related to values of solute potential measured when the leaves were harvested. Seedlings fed betaine via the root showed slightly reduced leaf area; this probably explained the slowed rate of soil-water depletion and retarded wilting of betaine-treated plants during water stress. High internal levels of betaine in well-watered seedlings depressed [2-]4C]ethanolamine incorporation into both betaine and phosphatidyl choline, suggesting that betaine synthesis is subject to retroinhibition. Results from autoradiographic studies at the light microscope level indicated that in stressed-rewatered seedlings, betaine was dis- tributed equally between vacuolar and cyt0plasmic compartments. DEDICATION Oats, peas, beans and barley grow Oats, peas, beans, and barley grow Will you or I or anyone know How oats, peas, beans and barley grow? Anonymous To Midge and Syd Ladyman and to my sister, Tonia ii ACKNOWLEDGMENTS To thank and express my appreciation to all the pe0ple who have helped me throughout my graduate career at M.S.U. would double the length of this dissertation. Especial thanks go to the faculty, post-docs, and graduate students of the D.0.E. Plant Research Labora- tory for their advice, assistance and moral support. Particular thanks goes to Ms. Kimberly Ditz, not only for her considerable tech- nical assistance in screening genotypes of barley for betaine levels, but also for her friendship and support. I thank my parents, and my sister Tonia, for their unflagging interest in all I do, and for their assistance and understanding, especially while on "vacation" with me in Michigan. I greatly appreciate all the efforts made on my behalf by Dr. Charles Arntzen and Dr. Anton Lang. My thanks also go to Dr. S. K. Ries for his advice on, and land used for, my field experiments; to Dr. C. Cress for his invaluable statistical advice; and to Dr. T. Giddings for his instruction on sectioning and staining procedures. I wish to thank my academic advisor, Dr. Jan Zeevaart and the members of my committee--Drs. Wayne Adams, Norman Good, and Gene Safir, for their constructive advice and encouragement. Last, but by no means least, I thank my dissertation advisor, Dr. Andrew Hanson for his guidance, patience, and general support throughout my sojourn in Michigan. 111' TABLE OF CONTENTS Page LIST OF TABLES . LIST OF FIGURES LIST OF APPENDICES LIST OF ABBREVIATIONS INTRODUCTION . . . . . . . . . . . . . . . . l Nomenclature, Properties, and Natural Occurrence of Betaine . . l Physiological Functions of Betaine . 4 Use of Indicators of Water Stress in Plant Breeding 6 Potential Use of Metabolic Responses to Stress in Plant Breeding . 7 Betaine Accumulation as an Index of Stress History . 9 The Objectives of the Research Reported in This Dissertation . . . . . . . . . 9 Chapter I. THE TRANSLOCATION AND METABOLISM OF GLYCINEBETAINE BY BARLEY PLANTS GROWN IN CONTROLLED ENVIRONMENTS . 10 1.1 Introduction . . . . . . . . . . . 10 1.2 Materials and Methods . . . . . 11 1.2.1 Plant Material and Stress Regimes . . . 11 1.2.2 Betaine Extraction and Determination . . 12 1.2.3 Labeled Compounds and Mode of Application to Plants . . . . 13 1.2.4 Extraction and Separation of Labeled Metabolites . . . . 15 1.2.5 Detection and Determination of Radio— activity . . . . 16 1.2.6 Translocation of Labeled Compounds . . . 16 1.3 Results . . . . 21 1.3.1 Betaine Levels During Stress and Rewater- ing Cycles . . . . . . . . . . . 21 iv Chapter II. III. 1.3.2 Fate of [Methyl- 4C]Betaine Applied to Mature Leaves . . . 1.3.3 Phloem Translocation of [Methyl-14C]- Betaine . . 1.3.4 Metabolism of [Methle4CJBetaine Aldehyde by Leaf Disks . . 1.3.5 Translocation of 14C- Labeled Compounds in Mature Plants . . . . . . . 1.4 Discussion THE ACCUMULATION AND METABOLISM OF GLYCINEBEIAINE IN FIELD- GROWN BARLEY 2.1 Introduction . 2. 2 Materials and Methods 2.2.1 P1ant Material 2 Field Plots . .3 Sampling Times and Measurements Taken in the Field . 4 Estimation of Cumulative Stress Expe- rienced by Plants 5 Chemical Analyses .6 Radioactive Labeling Experiments 2.3 1t ts and Discussion . . . . 1 1979 Pilot Field Experiment 2.3.2 1980 Field Experiment GENETIC VARIATION FOR BETAINE LEVELS IN CULTIVATED AND WILD BARLEY . . . 3.1 Introduction . 3. 2 Materials and Methods 3. 2.1 Plant Material . .2 Betaine Determination . . 3 Water Potential and Solute Potential Measurements . . . 4 Controlled Environment Tests . 5 Field Experiment. 1 1 3.3 e u ts 3 2 3 2 3.2. 3.2. R s . . . . . . . . . . . . . 3. 3. Results of Controlled Environment Tests 3.3.2 Results of Field Study. 3. 4 Discussion . Page 26 28 36 37 37 103 103 105 105 105 106 106 108 111 111 118 122 Chapter IV. THE EFFECT OF EXOGENOUS BETAINE ON GROWTH AND BETAINE SYNTHESIS IN BARLEY SEEDLINGS . . . . 4.1 4. 2 4.3 4. 4 Introduction . Materials and Methods . . . 4.2.1 Plant Material and Seed Sterilization Procedures . . . . . . . Media . P1ant Culture Conditions Incubation with [14C]Compounds Localization, Extraction and Separation of Labeled Metabolites . Betaine Determination . . Statistical Analysis of Results . C fi 01 0 (Do 0 DOWN“) NNNN baa-1:4: Jana-4:4: d—‘NO‘ 0'1th Effects of Exogenous Betaine 6n Seedling Performance during4 Stress and Rewatering. 4. 3. 2 Metabolism of [2 4C]Ethanolamine Discussion . . V. SUBCELLULAR LOCALIZATION OF [METHYL-14CJBETAINE BY MICROAUTORADIOGRAPHY . . . . . 5.1 5.2 5.3 CONCLUSION APPENDICES BIBLIOGRAPHY Introduction . Materials and Methods 5.2.1 Plant Material . Growing Conditions and Stress Regimes . Labeled Compounds and Mode of Applica- tion . . . Preparation for Microautoradiography Microautoradiography and Section Stain- ing . . . . . Results and Discussion NM MN: 0101 0101 01-h (JON vi Page 126 126 129 129 130 131 136 137 139 139 140 140 148 151 157 157 160 160 160 161 161 163 164 169 173 197 Table 10. 11. 12. LIST OF TABLES Distribution of 14C among organs of barley 3 days after supplying 8.5 nmol of [methyl-14 C]betaine (specific radioactivity 59 uCi/umol) to the blade of the second leaf . . Translocation velocities for 14C-labeled compounds applied to the second leaves of barley seedlings Movement of applied [6, 6' (n)- 3H]sucrose and [methyl- 14C]betaine from blades to sheaths of the second leaves of soil- -grown barley seedlings . . Approximate dates of appearance during 1979 of common pests of barley and control measures adopted Yield, yield components, and selected growth charac- teristics of the Proctor crops in 1979 Betaine concentration of selected organs during the growing season for Proctor (1979) . . Concentration of betaine in (flag plus flag- -1) leaf blades of Bankuti Korai at 42 DAP . Harvest indices of dry weight, nitrogen and betaine of Proctor barley at maturity (1979) a-Amino-N (A) and betaine-N content (B) of selected organs of Proctor (1979) at various dates after planting . . Total -N (including NO;- -N) content of selected organs of Proctor barley (19 9) at various dates after planting NO§-N level in the shoot vegetative tissue of Proctor barley (1979) at various dates after planting Yield, yield components and selected growth character- istics of the Proctor crop (A) and Arimar crop (B) of 1980.. . . . . . . . . . . vii Page 27 29 33 54 63 71 71 73 74 75 75 79 Table Page 13. The average leaf diffusive resistance of the youngest mature leaf blade over the growing season (1980) . . 80 14. Nitrogen content (as % dry weight) of the total shoot of Proctor (1980) at anthesis . . . . . 84 15. The average betaine content and dimensions of the flag- 1 leaf blade of N- fertilized Proctor barley 71 DAP (1980) . . . . . . . . . 85 16. Betaine level in the youngest organs of Proctor barley . . . . . . . . . . . . . 90 17. Betaine level of the youngest organs of Arimar barley shoots at 3 dates after planting . . . . . . . . 91 18. Distribution of [methyl-”C]betaine in shoots of non- irrigated Proctor barley in 1980 (Experiment 1) . . . 93 19. Distribution of 14C- -activity in selected organs of non- irrigated Proctor barley (1981) after [14C]betaine (0.75 uCi) was fed to the flag-1. leaf blade 62 DAP (Experiment 3). . . . 100 20. Designation and origin of barley cultivars tested for betaine accumulation in non-irrigated field condi- tions . . . . . . . . . . . . . . . . . 109 21. Variation in w and Vs in the youngest mature leaf of Arimar and Proctor Barley . . . . . . . . . . 111 22. Shoot betaine concentrations of Proctor and Arimar in laboratory trials A, B, and C . . . . . . . . . 115 23. The relationship between the betaine concentration in the shoots of water-stressed (y) and 1n well-watered (x) seedlings of barley in 3 laboratory screening trials . 116 24. Summary of results of experiments in which seedlings were grown from excised embryos . . . . . . . . 141 25. Selected growth characteristics of betaine-treated and untreated barley seedlings, measured after stress in experiment 4 and 5 . . . . . . . . . . . . 145 viii Table Page 26. Incorporation of 14C-activity (nCi) into betaine and phosphatidylcholinetn/the second leaf blade of barley seedlings having various internal levels of betaine . 150 27. The number of silver grains/10 pm2 in the vacuolar and cytoplasmic compartments of mesophyll cells and in the surrounding extra-cellular spaces in the first leaf blade of a stressed-rewatered seedling tiller . . . 166 8.1 Betaine level in shoots of irrigated and water-stressed seedlings of genotypes included in laboratory Trial A . 181 B.2 Betaine level in shoots of irrigated and water-stressed seedlings of genotypes included in laboratory Trial B . 183 3.3 Betaine level in shoots of irrigated and water-stressed seedlings of genotypes included in laboratory Trial C . 185 B.4 Betaine concentration and solute potential of the youngest mature leaf, or flag- -1 leaf, for thirteen cul- tivars of H. vulgare at three dates during the growing season (1981) . . . . . . . . . 187 .l B 5 medium (Gamborg and Wetter, 1975) . . . . . . 189 Murashige and Skoog medium (Murashige and Skoog, 1962). 191 Modified Wick's medium (Steidl, 1976) . . . . . . 192 Hoagland's medium (full strength) . . . . . . . 193 Tests for bacterial contamination . . . . . . . 194 common 01-wa .1 Composition of Spurr's resin used for microautoradio— graphic studies in Chapter I (Spurr, 1969) . . . . 196 ix Figure 10. 11. 12. LIST OF FIGURES Apparatus used to detect any evolution of 14C02 and volatile £14C1bases by barley seedlings supplied with [methyl-1 C]betaine Diagrams to show the translocation paths and the posi- tions of Geiger-Mfiller (G-M) probes 1 and 2, for studies of the translocation of radioactively labeled compounds in seedlings (A) and mature plants (B) . Betaine levels in shoots (If soil-grown barley plants subjected to two cycles of stress-rewatering The distribution of betaine amongst various shoot organs in relation to water-stress history Representative data for translocation of applied [Methyl-14C]betaine and [14C]sucrose, fed in sequence, from blade to sheath in the second leaf of barley Exudation of [14C]betaine from the cut end of the second leaf of barley . . . Representative data for translocation of 14C- labeled assimilates (A), [14C]sucrose (B) and [14C]betaine (C) from f1ag-1eaf sheath to spike in mature barley plants . . . . . Specification sketches of the manual rain-out shelters used in 1980 . . . . . . Photographs of the rainout shelters in fine (A) (1980) and rainy (B) weather (1979) . . . Plan of twinwall trickle irrigation installation used in 1980 . Trends in above ground dry matter accumulation (A) and tillering (B) of Proctor barley (1979) The trends through the growing season of leaf diffusive resistance (r sec/cm) of the youngest mature leaves of Proctor (1979) . . . . . . . . . Page 17 19 22 24 30 35 38 47 49 53 61 62 Figure Page 13. Photographs of Proctor barley crops at ear emergence. A and C: Non-irrigated crops in 1979 and 1980, respec- tively; B and D: Irrigated crops in 1979 and 1980 . respectively . . . . . . . . . . . . . . 64 14. Stage of growth, and trends in water potential of the blade of the youngest mature leaf, or flag- 1 leaf, of Bankuti Korai planted July 19,1979 . . . . . 67 15. Betaine level of the whole shoot, excluding spike tissue (A) and the developing spike (B) for each irrigation and nitrogen treatment of Proctor barley (1979) . . . . . . . . . . . . . . . . 69 16. Betaine level of the two youngest mature leaf blades of N- fertilized (A) and N- unfertilized (B) Proctor barley (1980).. . . . . . 81 17. Betaine concentration of the two youngest mature leaf blades of Arimar barley (1980) . . . . . . . . 83 18. The relationship between the betaine concentration of the youngest mature leaf blades and the IS experienced by Proctor and Arimar barley crops (1980) . . . . 87 19. Autoradiograph of a Proctor barley plant fed [14C]- betaine to flag-2 leaf blade 54 DAP . . . . . . 95 20. Autoradiographs of barley4 plants fed 1 uCi [14C]- betaine (A, B) or 50 uCi 4C02 (C,D) at 22 (A,C) or 41 (B, 0) DAP . . . . . . . . . . . 97 21. Frequency distributions for betaine concentration in cultivated and wild barley genotypes entered in 3 laboratory trials (A, B, C) . . . . . . . . . 113 22. Frequency distributions for 13 barley cultivars: A, betaine concentration; B, p; C, Vs . . . . . . . 119 23. The structures of betaine and CCC . . . . . . . 127 24. Schemes describing chronological order of culture and stress events for seedlings grown from embryo culture . . . . . . . . . . . . . . . . 132 25. Culture conditions for embryo (A) and intact seeds in semi-sterile (B) and sterile (C) conditions.. . . . 134 26. Experiment 4. The effect of betaine pretreatment on the development of wilting symptoms during water- withholding . . . . . . . . . . . . . . 143 xi Figure 27. 28. 29. 30. A.l Experiment 5. Representative data describing pot water loss during a 12-day period of water-withhold- ing from barley seedlings that had been pre-treated with 160 pmol betaine or untreated . . . A schematic representation of the major pathway believed to operate in the biosynthesis of betaine from ethanolamine in barley . Relationship between endogenous betaine level and [14C]ethanolamine (nmol/leaf) conversion to betaine and phosphatidylcholine (PC) when [2-14C]ethanolamine was fed to the third leaf blade of well—watered seedlings . . . . . . . . . . Photographs of transverse sections through the first leaf of the first tiller of a stressed- rewatered barley plant supplied with [14C]betaine via a leaf on the main culm . . . . Oxidation of choline xii Page 147 149 152 166 175 LIST OF APPENDICES Appendix A. Synthesis of [methyl-3H]betaine, [methyl-14C]- betaine, and [methyl-14C1betaine aldehyde B. Wild and cultivated barley genotypes screened in laboratory and field trials . . C. Media used in sterile embryo and intact seed culture . D. Resin formula used in microautoradiographic studies xiii Page 174 179 188 195 ABA CCC Ci DAP GLC G-M min PC TLC TLE U.S.D.A. wt LIST OF ABBREVIATIONS Abscisic acid (2-chloroethyl)trimethyl ammonium chloride Curie Days after planting Gas-liquid chromatography Geiger-MUller Hour Minute Phosphatidyl choline Thin-layer chromatography Thin-layer electrophoresis United States Department of Agriculture Weight Water potential Solute potential xiv INTRODUCTION Nomenclature, Properties, and Natural Occurrence of’Betaine Onium compounds have been defined as "substances that are formed by an addition reaction, in the course of which some atom increases its valence by one unit, and in doing so, increases its formal charge algebraically by one unit" (Wheeland,l953). This broad definition includes aliphatic quaternary ammonium compounds, e.g., choline and fully N-methylated amino acids (classed as "betaines"), heterocyclic compounds that contain a quaternary nitrogen in the ring structure, e.g., thiamine and the curare alkaloids, and sul- phonium compounds, e.g., S-adenosyl methionine and dimethylpropiothe- tin (Cantoni, 1960). Glycine betaine, or N,N,N trimethylglycine [(CH3)3+NCH2COO'], is structurally the simplest of the class of aliphatic compounds termed betaines and in biological literature, is generally referred to as betaine. Betaine is highly water soluble (160 g/100 ml) and sparingly soluble in ether (Stecher, 1968). At 0.2 molar concentration, betaine has an activity coefficient (y) close to unity (y = 1.07); y increases nearly 2-fold with a lO-fold increase in concentration (y = 1.95 at 2 molar concentration) (Smith and Smith, 1940). Betaine is excluded from the hydration water of some polymers (London et a1., 1967) including proteins (Wyn Jones and Pollard, 1981). Betaine is a L I (11 . «’1‘ ‘11.. zwitterion with only one titratable group, the carboxyl, which has a pka of 1.85 at 25°C; the positive charge on the N is fixed (Wyn Jones and Storey, 1981). Hence, at physiological pH betaine has no net charge and no buffering capacity. Betaine is widely distributed throughout the animal and plant kingdoms. Betaine has been reported in bacteria (Schieh, 1965), fungi (Kfing, 1914), crustaceans (Carr, 1978), molluscs (Yasumoto.et a1., 1978), pisces (Carr, 1978; Bowlus and Somero, 1979), and terrestial vertebrates (Cantoni, 1960; Greenberg, 1969). In higher plants, betaine appears to be present in significant amounts (>10 pmol/g dry wt) only in certain taxa; thus it is found in vegetative tissues among at least some members of the Chenopodiaceae, Gramineae, Leguminosae, and Amaranthaceae families (Wyn Jones and Storey, 1981). Betaine has also been reported to be present in the juice of citrus fruits (family Rutaceae; Lewis, 1966). In animals and micro-organisms considerable research has been conducted on the oxidation of choline to betaine (e.g., Mann and Quastel, 1937; Mann et a1., 1938; Williams, 1952; Schieh, 1965; Tyler, 1977). The intermediate of the oxidation, betaine aldehyde, is quite labile and does not usually accumulate in vivg (De Ridder and Van Dam, 1973). In animals and micro-organisms choline is derived by sequential N-methylations of ethanolamine, the product of serine decarboxylation. The methylation steps involve both gg £919 synthesis of a methyl group from one-carbon precursors (Sakami and Welch, 1950) and transfer of a methyl group from a methyl donor ,1»!- wt m- Jul 1... 'II ‘R- '4- (I. ¢_1._ (I) C/‘D 2 As; i 1"- nl .- O compound (Stekol et a1., 1958). The decarboxylation and methylation steps probably occur when the bases are in the phospholipid-bound form in membranes (Kates and Marshall, 1975; Hanson and Hitz, 1982). Enzymes of the biosynthetic pathway of betaine are mainly membrane- bound (Williams, 1952; Van den Bosch, 1974), although a cytoplasmic betaine aldehyde oxidase has been implicated (Christiansen and Daniel, 1953; Rothschild and Guzman Barron, 1954)- In barley, beet and spinach plants, betaine is also synthe- sized via three sequential N-methylations of the decarboxylation product of serine rather than by methylation of glycine (Delwiche and Bregoff, 1958; Hanson and Nelsen, 1978; Hanson and Scott, 1980). In contrast to animal and microbial systems, in barley, sugar beet, and spinach (Hitz et a1., 1981; Coughlan and Wyn Jones, 1982; Hanson and Wyse, 1982) water-soluble intermediates (perhaps phosphoryl base derivatives) appear to be involved in betaine biosynthesis. In sugar beet, phosphorylcholine is the direct precursor of the choline destined for oxidation to betaine (Hanson and Wyse, 1982). In barley, release of free choline from phosphorylcholine is indirect, via phosphatidyl choline (Hitz et a1., 1981). It is not yet known whether physiological significance attaches to the difference between the two pathways. Betaine can be metabolized by microbes and animals (Trier, 1931; Greenberg, 1969). In animals betaine can act as a donor of methyl groups (Du Vigneaud et a1., 1946; Muntz, 1950; Greenberg, 1969; Tyler, 1977); betaine-homocysteine methyltransferases have been demonstrated in the liver of a wide variety of vertebrates but not in plants or microbes (Ericson, 1960). A clinical application for the methylating capacity CH betaine has been proposed in the treatment of benzene poisoning. After administration of betaine and choline to mammals that had been exposed to high levels of benzene, benzene levels in the blood were lowered asairesult of benzene methylation to toluene, a less toxic hydrocarbon (Brair, 1977). Physiological Functions of Betaine Betaine can apparently serve several functions among animals and microbes. Betaine acts as a feeding stimulus to some crustaceans, amphibians (bullfrog), and fish (Carr, 1978; Takagi et a1., 1978). In the cells of some marine invertebrates betaine is present in suffi- cient concentrations to be a significant osmoticum (Schoffeniels, 1976). In studies with marine invertebrates and elasmobranch fish betaine has been found to offset the perturbing effects of urea and inorganic ions, e.g., NaCl, on in 315:9 enzyme activity (Bowlus and Somero, 1979; Yancy and Somero, 1980). However, the concentration of betaine in the muscle tissue of elasmobranch fish has rarely been accurately measured; in shark, concentrations appear to be variable (ll-100 mM) but under most circumstances the contribution of betaine to osmotic regulation seems unlikely to be large (Yancey and Somero, 1980). In moderately halophilic bacteria, exogenous betaine had a stabilizing effect on respiration, a membrane-associated process, in the presence of a high concentration of salt (Shkedy-Vinkler and Avi-Dor, 1975). The functions of betaine in higher plants are not well under- stood. Early reports indicated that betaine had weak methyl donor activity in tobacco and barley (Scribney and Kirkwood, 1954; Byerrum et a1., 1956) but these experiments were subject to artifacts arising from microbial contamination. More recent experiments have indicated that betaine is a metabolic end product in wheat and barley (Bowman and Rohringer, 1970; Ahmad and Wyn Jones, 1979). Some halOphytes contain high levels of betaine (> 100 pmol/g dry wt) which rise further in response to salinity (Storey and Wyn Jones, 1975). Betaine also accumulates in some mesophytes in response to both water and salt stress (Hanson and Nelsen, 1978; Wyn Jones and Storey,1978). Because of the ecological link between high betaine levels and salt tolerance, an adaptive role for its presence and sub- sequent accumulation during stress has been proposed (Wyn Jones et al., 1977b). One possible function of betaine is that it acts as a cytoplasmic osmoticum (Wyn Jones et al., 1977a). The results from three types of experiments lend support to this hypothesis: (1) Betaine was found in low concentrations in intact vacuoles isolated from red beet roots (Wyn Jones et al., 1977a; Leigh et a1., 1981); (2) In the leaves of Suaeda maritima, betaine was judged to be local- ized in the cytoplasm by a histochemical staining technique (Hall et a1., 1978); (3) Ig_yitrg studies showed that relatively high con- centrations (1 M) of betaine were not toxic-~and, in the presence of salt were slightly protective-~to cytoplasmic enzymes from plants as well as from animals (Pollard and Wyn Jones, 1979; Paleg et a1., 1981). Results from experiments in which exogenous betaine was fed to plants in order to study the role of betaine accumulation under stress have been inconclusive due, in part, to microbial contamination (Wyn Jones et a1., 1974). If genetic variability for betaine levels exists within or among inter-fertile species, physiological-genetic studies would be a means of determining if betaine accumulation was beneficial to plants. Correlating genotype performance of mesophytic crops under stress with a specific metabolic response has been attempted by Larqué-Saavedra and Wain; (1976) and Quarrie (1980b) for abscisic acid accumulation, and by Richards (1978) and Hanson et a1. (1977) for proline accumulation. Use of Indicators of Water Stress in Plant Breeding One aim of many breeding programs is to develop varieties that yield well under both adverse conditions such as water stress and favorable conditions. Instead of selecting for yield itself, various other criteria have been, or could potentially be, used in breeding programs to identify such stress-adapted genotypes (Moss et a1., 1974). Criteria of this sort fall into two categories: Criteria based on characteristics believed to confer adaptation to stress, and criteria that are essentially stress symptoms. Characteristics with adaptive value would be selected for; those symptomatic of the stress would be selected against. The general concept of characters symptomatic of stress includes the specific cases of stress—induced phenological, morphological, physiological and metabolic plant responses as indica- tors of the cumulative stress experienced by the plant. For example, one can envisage selecting against characters that indicate that the plant under drought had been unable to maintain a high internal water- status. Indeed, phenological, morphological, and physiological symp- toms of plant stress have been successfully used as selection cri- tera iri this way (Hurd, 1974; O'Toole and Chang, 1978; Atsmon, 1979; Hall et a1., 1979). The disadvantage with many phenological and mor- phological symptoms is that they are usually evident only after con- siderable stress has been experienced. Most physiological indicators of plant water stress (e.g. water potential, solute potential, and leaf diffusive resistance) are based on dynamic plant characteristics which at any instant in time are unlikely to represent an integrated value of the performance of the plant. Therefore, many time consuming measurements throughout the growing season must be made to gain accurate information in order to judge the overall fitness of a geno- type to droughty environments. Potential Use of Metabolic Responses to Stress in Plant Breeding Selection procedures based on metabolic responses to stress are not currently used, mainly because plant metabolism under stress is poorly understood (Boyer and McPherson, 1975; Hanson, 1980; Quarrie, 1980a; Elmore and McMichael, 1981). The levels of two metabolites, namely proline and abscisic acid, that increase in response to water-stress have been proposed as being suitable indicators of the fitness of a genotype to droughty environments. It was proposed, for wheat, that high levels of proline (Singh et a1., 1972) but low levels of ABA (Quarrie, 1978) should be positive selection criteria. When using a metabolic, or in fact, any other indicator of plant water stress, an important consideration is that the selection cri- teria may be affected tn/ environmental conditions that are unrelated to water stress; this would confound selection of any sort. For example, mineral stress in certain cases affects proline accumulation (Elmore and McMichael, 1981). Like many physiological parameters, levels of proline and ABA are likely to be closely related only to the stress that the plant is experiencing at about the time of measurement. Because both proline and ABA are subject to rapid catabolism after relief of stress, the level of neither metabolite could reflect the cumulative internal stress experienced by a plant subjected to episodic stress (Zeevaart, 1980; Boggess et a1., 1976a; Boggess et al., 1976b; Boggess and Stewart, 1976). Thus, an ideal metabolic index of cumulative stress would be one that was: (1) An accurate indication of the stress experienced prior to measurement; (2) Reproducible among gentoypes experiencing the same duration and severity of internal stress; (3) Unaffected by environmental conditions other than the specific one (viz. water availability) under investigation; (4) Readily measured. g. Hfillr M111 Mun- liq alv \ 9‘ D (I) 11- (1- Betaine Accumulation as an Index of Stress History Because in wheat and barley, betaine appeared to be a meta- bolic end product (Bowman and Rohringer, 1970; Ahmad and Wyn Jones, 1979) which accumulated in response to water stress, Hanson and Nelsen (1978) proposed that the betaine content of a plant may reflect its internal-stress history and could thus be used as a cumulative stress index. Such an index could have applications in irrigation management as well as in plant breeding. In a plant- breeding context, selection could be practiced against high betaine levels. Note, however, that should betaine accumulation be found to be adaptive, then the advisability of breeding specifically either against or for high betaine accumulation should be re-evaluated. The Objectives of the Research Reported in This Dissertation This study of the metabolism, translocation, and genetic diversity of betaine accumulation in barley was undertaken in order to: (1) Test the hypothesis that the betaine concentration of certain plant organs could be used as an index of the cumulative stress experienced by the plant (Chapters I and II); (2) Determine whether a physiological-genetic study of the function of betaine accumulation under water-stress would be feasible (ChapterIII); (3) Attempt to obtain evidence bearing on the postulated adaptive role of betaine accumulation by barley (Chapters IV and V). CHAPTER I THE TRANSLOCATION AND METABOLISM OF GLYCINEBETAINE BY BARLEY PLANTS GROWN IN CONTROLLED ENVIRONMENTS 1.1 Introduction The betaine accumulation induced by water stress is a result of de novo synthesis from l-C and 2-C fragments (Hanson and Nelsen, 1978; Hanson and Scott, 1980). Preliminary studies (Hanson et a1., 1978) and two published reports (Ahmad and Wyn Jones, 1979; Bowman and Rohringer, 1970) indicated that betaine, once synthesized, was not further metabolized and was probably mobile within the plant. One conflicting report from Dekhuijzen and Vonk (1974) concluded that the [14CJbetaine synthesized in wheat from supplied [14C]- (2-chloroethyl)trimethylammonium chloride (CCC) underwent degrada- tion to glycine. However, in reaching that conclusion, the authors ignored a more likely route of 14C entry to glycine, namely refixation in the light of the ‘4 C02 generated from degradation of the labeled CCC. This study of the metabolism and mobility of betaine within barley plants grown in controlled environments was the first step in determining whether the betaine concentration of either the whole shoot, or organs thereof, could be used as a cumulative index of the internal water stress experienced by the plant. 10 11 1.2 Materials and Methods 1.2.1 P1ant Material and Stress Regimes Seeds (caryopses) of spring barley (Hordeum vulgare L. cv. Proctor, CI 11806) were obtained originally from the U.S.D.A. Small Grains Collection, Beltsville, Md., and were subsequently multiplied each year in field plots at Michigan State University. Plants were grown to the three-leaf stage (17-22 days old) in plastic pots (12 cm high, 7 cm diameter) in either a soil mixture (peat:loam:sand, 1:1:2 v/v) or perlite (Chem Rock Corp., Nashville, Tenn.), in a controlled environment chamber under l6-h days (photosynthetically active radia- tion, 5 mW cm'z; day/night temperature 22/16°C; day/night relative humidity 70/85%) (Tully et a1., 1979). The plants (six per pot) were watered on alternate days with half-strength Hoagland's nutrient medium. One day before each experiment started, plants were routinely thinned to four per pot. Mature plants (12-13 weeks old) used to investigate the translocation of betaine to the spike (Fig. 2B), were grown in 15 cm clay plots in a soil mixture (peat:loam:sand, 1:1:2 v/v) and thinned at the four-leaf stage to two per pot. They were grown under supple- mental light (16-h day) in a greenhouse (day/night temperature, 22/16°C; relative humidity 70 to 80%). Plants were watered with half-strength Hoagland's solution daily. Plants grown in perlite were osmotically stressed with a solution of polyethylene glycol (PEG) 6000 (Union Carbide Chemicals and Plastics Institute, W. Va.) in half-strength Hoagland's nutrient 12 medium, osmotic potential about -19 bars (Hanson et a1., 1977). Plants grown in soil were stressed by withholding water for up to 3 days. In some experiments, stress was relieved by washing PEG from perlite as described by Hanson et a1. (1977), or by rewatering soil. For the experiment of Figure 3 (page 25),soil-grown plants were subjected to two cycles of water withholding (both of 3 days) each followed by a 2-day period of daily watering. Control plants in this experiment were thinned to three per pot to redUce interplant com- petition which caused premature leaf senescence toward the end of the experiment when there were four plants per pot; the control plants were watered daily. Shoot water potential (w) was measured on the second leaf blade with a pressure chamber (PMS Instrument Co., Corvallis,0r.). To reduce water loss as the measurement was being taken, the blade was wrapped in damp absorbent paper (Meiri et a1., 1975; Ritchie, 1975). Leaf diffusive resistance was measured with a diffusion porometer (Model LI-65, Li—Cor Inc., Lincoln, Nb.). 1.2.2 Betaine Extraction and Determination Whole shoots, or their component organs were freeze-dried and weighed. Tissue, having a dry weight greater than 30 mg was ground in a Thomas-Wiley intermediate mill to pass through mesh 40. Subsamples (15-20 mg) of the milled tissue, or whole organs (15-30 mg), were extracted with 5 ml of water at 100°C for 30 minutes and centrifuged to clear. The betaine in the clear supernatant was 13 determined either by: (l)pyrolysis-GLC after ion-exchange purifica- tion (Hitz and Hanson, 1980); or (2) separating the constituents of the supernatant by thin layer electrophoresis on pre-coated 0.1mm cellu- lose glass-backed plates (Cat. No. 5757; E. Merck, Darmstadt, FRG) with sodium tetraborate buffer (70 mM, pH 9.3) at 2 kV for 10 minutes. The betaine was identified on the plates by spraying with Dragendorff's reagent. Estimates of the amounts of betaine present in spots were made by comparing the size and intensity of the spots with a range of similarly prepared standards (range 5-30 pg). This semi-quantitative method was used for preliminary measurements of betaine content in meristematic tissue. 1.2.3 Labeled Compounds and Mode of Application to Plants [6,6'(n)-3H]Sucrose (15.5 Ci/mmol) and [U-14C]sucrose (625 pCi/umol) were purchased from Amersham Corp., Arlington Heights, 111. [Methyl-14C]Betaine of high specific activity was prepared from [methyl-14C]choline (59 uCi/umol; Amersham Corp.) by oxidation with acidified potassium permanganate solution, according to Lintzel and Fomin (1931). [Methyl-14C]Betaine aldehyde of high specific activity was prepared from [methyl-14C]choline (59 uCi/pmol) using fresh rat- liver mitochondria prepared as described by Williams (1952). (For details of synthesis, purification, and yields, see Appendix A). For studies of metabolism using whole plants, [methyl-14C]- betaine and [methyl-14C]betaine aldehyde were used carrier free (i.e., at specific activity of 59 uCi/umol). When investigating extracellular 14 oxidation of betaine aldehyde, 30 leaf disks (5 mm diameter, 0.2 g fresh wt) were incubated on a shaker at room temperature with [methyl-14C]betaine aldehyde (0.2 pCi, 0.2 pCi/mmol) in potassium- phosphate buffer (20 mM, pH 7). Aliquots (20 pl) were taken from the incubation medium at lS-min intervals for the first hour, then at hourly intervals. At the end of 6 h the leaf disks were rinsed twice in potassium-phosphate buffer (1 ml), blotted dry and extracted in cold 0.5N formic acid (Hanson and Scott, 1980). For translocation studies with [methyl-14C]betaine, [methyl- 14C]choline, L-[U-14C]glutamic acid (282 pCi/pmol; Amersham Corp.) and [U-14C]sucrose, radiochemicals were diluted with sufficient unlabeled compound to give a total dose of 0.5 pmollplant; specific activities ranged from 10 to 20 pCi/pmol. For studies of both metabolism and translocation, labeled com- pounds were fed in a 5—pl droplet of 20 mM potassium-phosphate buffer (pH 7) to the second leaf blade (seedlings) 10 cm up from the ligule or to the flag leaf sheath (mature plants) 5 cm below the ligule through a carborundum-abraded spot of 5-6 mm diameter (Housley et a1., 1977). After the droplet had been taken up, a second 5-pl droplet containing only buffer was applied. When the second droplet had been absorbed, the spot was covered with parafilm to minimize dessication of the abraded area. 14 Gaseous 14CO2 was generated by mixing 20 pl Na C03 (10 pCi, 60 mCi/mmol; Amersham Corp.) with a 200-p1 drop of lactic acid (43%, v/v) in a lO-ml syringe barrel with the plunger at the 10-m1 mark. 14 The total 10 ml volume of CO2 was injected into the leaf-feeding 15 chamber. The chambers were glass tubes (1.5 cm diam x 10.5 cm) with a serum cap at one end; the top 10 cm of leaf blade was inserted into the other end which was then plugged around the leaf with cotton wool over which was stretched parafilm. The 14CO2 was administered for 15 minutes. 1.2.4 Extraction and Separation of Labeled Metabolites When [methyl-14C1betaine was fed, extraction was by the methanol-chloroform-water (MCW) procedure described by Hanson et a1. (1977). When [methyl-14C]betaine aldehyde was fed, extraction was in cold 0.5 N formic acid, to minimize degradation of the [14C]- betaine aldehyde (Hanson and Scott, 1980). The aqueous fraction of MCW extracts and the formic acid extracts were evaporated to dryness in a stream of N2, redissolved in H20, and separated by the three- cOlumn ion-exchange procedure described by Hitz and Hanson (1980). Column fractions eluted from AG-50 and Bio-Rex 70 were further analyzed by thin-layer electrophoresis (TLE) and thin-layer chromatography (TLC). TLE systems were: on pre-coated 0.1 mm glass-backed cellu- lose plates (Cat. No. 5757; E. Merck) with either sodium tetraborate buffer (70 mM, pH 9.3) or formic acid (1.5 N) at 2 kV for 6-12 min; on "ITLC" plates (Gelman Instrument Co., Ann Arbor, Mi.) with formic acid (1.5 N) at 2 kV for 16 min. The TLC system was: pre-coated 0.25 mm Silica Gel-G plastic-backed plates (Brinkman Instruments, Westbury, N.Y.) developed for 90 min in methanol-acetone-conc. HCl (90:10:4 v/v). 16 1.2.5 Detection and Determination of Radioactivity Labeled compounds separated by TLE and TLC were located by autoradiography using SB-5 X-ray film (Eastman-Kodak, Rochester, N.Y.); radioactive zones were eluted as described in Hanson and Tully (1979). Radioactivity in such eluates and in other soluble samples was determined by scintillation counting. Radioactivity in the insoluble fractions remaining after MCW extraction, and in freeze-dried samples of whole tissue, was also determined by scin- tillation counting after combustion in a Packard Tricarb sample oxidizer, model 306 (Packard Instrument Corp., Downers Grove, 111.). In the experiment of Table l, in which [methyl-14C]betaine was fed, plants were enclosed for 9 h in sealed 20-1 glass tanks through which humidified air was passed at a flow rate of 5 l/h (Fig. 1). The enclosure was on the second day of the 3-day experi- mental period. After passage through the tanks, the gas streams were bubbled through traps for volatile bases (0.1 N HCl) and for CO2 (Carbosorb. II; Packard Instrument Corp.). The traps were checked for radioactivity after 9 h. A second COZ-trap (saturated barium hydroxide) was included after the Carbosorb II; absence of any pre- cipitate of BaCO3 at the end of the experiment confirmed complete absorption of CO2 by Carbosorb 11. 1.2.6 Translocation of Labeled Compounds These experiments were carried out under a bank of four 20-W fluorescent tubes (FZOTIZ Agro-Lite; Westinghouse, Pittsburgh, Pa.), l7 .mcwmamamuep-—aguwsu ;u_z cop—naam mmcwpcmmm xupcmn an mommnnuepu m—pumpo> can 8eF we cowuzpo>m ace uuwuuu on com: maumcman< .P .mwm m.— m>>O.—. mun—(n. ASEO 3 fl) 9 is: I? .Eoo \ || LEOQVxI. iEom.v\l 1 .Omwsuahdm. d mmOm .Z_.O. £030 .Omc<0 .UI 18 photosynthetic fluence about 9.9 mw cm-2 at plant level (PSI Model 65 Radiometer; Yellow Springs Instrument Co., Yellow Springs, Ohio). This relatively low light level was used to reduce further desicca- tion of the stressed plants during the experimental period. Both the amount of labeled compounds applied (0.5 pmol/leaf) and the abrasion method used to enhance their uptake into the leaf were based on tech- niques devel0ped for studying translocation of sugars and amino acids (e.g., Housley et al., 1977; Yamaguchi and Islam, 1967). Before each experiment, diffusive resistance was measured on the fed leaf to confirm that stomata were open. To follow translocation of labeled compounds, two Geiger- Mfiller (G-M) tubes were used. In studies with seedlings (Fig. 2A) a mask of aluminum sheet (thickness 0.2 mm) with a slot (20 x 5 mm) was placed on the second leaf blade centered 5 cm up from the ligule and another such mask was centered 5 cm down the sheath from the ligule (Hanson and Tully, l979). In studies investigating the translocation of betaine to the spike (Fig. 28), a mask similar to the ones used in seedling experiments was centered on the flag leaf sheath l cm above its node. Arrival of radioactivity was monitored at the masked- off ZO-mm segments of both seedlings and mature plants with G-M tubes (Model D34; Dosimeter Corp., Ohio) and at the spike of the mature plant withainancake" G-M probe (Model HP-ZlO; Eberline Instrument Co., Sante Fe, N.M.), the window of which covered the whole spike. The G-M tubes were connected to an Eberline radiation monitor which, in turn, was connected to a custom-made digital Counter (Tully et al., 1979). Fig. 2. 19 ABRADEDSPOT-—‘R | u \ G-M1:=1> ‘ / SPOT ,§~~{ 4‘ CM 2 -b ‘°°"‘ \ I :5 ABRADED F---4 PATHLENGTH Diagrams to show the translocation paths and the positions of Geiger-Maller (G-M) probes l and 2, for studies of the translocation of radioactively labeled compounds in seedlings (A) and mature plants (8). Arrival of 14C-activity was monitored at G-M probes l and 2 after the radioactive compound had been administered. 20 14 At all G-M probes the front of C-activity was taken to have arrived when the count was significantly (p §_0.05) higher than the 14 long-term background count rate. The velocity of C-label movement was calculated from the pathlength that separated the probes and the 14C-front took to travel between the probes. time the Leaves were heat-girdled by directing a stream of hot air (approx. 60°C) from a commercial hair dryer to a l-cm section of sheath situated 2 cm below the ligule. The xylem remained intact because the blades remained fully turgid for at least 8 h after girdling. For phloem exudation experiments, attached second leaves were fed with [14C]substrates as above, and the second leaf sheath was then cut under water, 6 cm below the ligule, one h after the radio- active front had passed the second G-M tube. The cut end of the sheath was re-cut under water and then placed in a conical vial con- taining 200 pl ethylenediamine tetraacetate (EDTA) 5 mM,pH 7 (King and Zeevaart, 1974). The mouth of the vial was sealed around the sheath with parafilm to prevent evaporation of the collection medium. The EDTA was changed every 2 h. After 6 h the blade and sheath were separated, out into 5-mm segments and washed in l mH unlabeled com- pound (betaine or betaine plus choline, respectively) for 5 minutes. The blade and sheath were then frozen in liquid nitrogen and extracted separately. Blade and sheath extracts and exudate were analyzed by TLE using glass-backed cellulose plates with sodium tetraborate buffer (70 mM, pH 9.3) at 2 kv for 9 min as described in Section l.2.4. 21 l.3 Results l.3.l Betaine Levels During Stress and Rewatering Cycles The betaine concentration in shoot tissue rose during the first and second stress episodes, and decreased during the rewater- ing periods (Fig. 3). The decline in betaine concentration upon rewatering was the consequence of the dilution by dry weight added as growth resumed, not of net degradation of betaine because the total betaine content per shoot did not decrease during the first rewater- ing period (Fig. 3 inset). Figure 4 confirms that the total quantity of betaine per shoot did not fall upon rewatering, and shows further that the distribution of betaine among shoot organs changed markedly upon stress relief. At the end of the stress period, most of the betaine (82% of the total, i.e., 5.0 umol) was present in the mature leaves (l, 2, and 3). Two days after rewatering, only 2.7 umol remained in these leaves, while the betaine contents of the expanding leaf 4, the spindle and the tillers had risen markedly (from a com- bined total of about l.l umol to about 4.2 pmol). Analysis of shoots from well-watered plants which were 2 days younger than, but compara- ble in size to, the rewatered plants showed that the well-watered shoots contained only about one-fourth as much betaine; the distribu- tion of betaine among shoot organs of well-watered plants was similar to that of stressed-rewatered plants. The stress-induced increase in betaine concentration was greatest in apices (§_8 mm in length), for it rose about 6-fold, from about 17 pmol/g dry wt in well-watered Plants to about l00 umol/g dry wt in stressed seedlings. Fig. 3. 22 Betaine levels in shoots of soil-grown barley plants subjected to two cycles of stress-rewatering. Main Figure shows betaine concentration of stressed- rewatered (+) and well-watered control (---A ----) shoots during the l2-day experiment. Initial shoot dry weight (control and stressed plants): 148 mg. Final shoot dry weights: control plants, 620 mg; stressed plants, 330 mg. Values of shoot water potential (u) at end of each stress period are indicated. Inset shows betaine content of stressed- rewatered shoots on a per-shoot basis for the first 6 days only. Beyond 6 days betaine content increased further during the second period of water-withholding, and did not fall significantly upon rewatering at 9 days (not shown). The bar indicates the least signifi- cant difference (LSD) at the 5% probability level and is appropriate for comparing values vertically. BETAINE( pmol/g DRYWT) 23 50 40 3O 20 1O BETAINE ( pmol/PLANT) I 2 4 6 ‘1’ = -30 BARS REWATER \P = -20 BARS ¢ REWATER ¢ WITHHOLD WATER 1 l- .- .. —- C" -------- ‘- a- a. " .I— -— ’ LSD _ 0.05 2 4 6 8 10 DAYS Fig. 4. 24 The distribution of betaine amongst various shoot organs in relation to water-stress history. The areas of the circles are proportional to the betaine content per plant. Sectors of the circles represent the distribution of betaine among organs; numbers in, or adjacent to, sectors give the percentage of total betaine present in each organ. The term "spindle" refers to the apical meristem plus leaf 5 and all younger leaves. Total betaine was 6.l umol per stressed plant, 6.9 umol per rewatered plant and 2.6 umol per well-watered plant. Average shoot dry weights: stressed plants, 126 mg; rewatered plants, 226 mg; well-watered plants 254 mg. 25 WELL WATERED 54% LEAF STRESSED 3 DAYS 13% LEAF 4 2.6% SPINDLE 2.3% TILLERS 82% LEAF 1: 2 '3 14.5% TILLERS 4.5% SPINDLE STRESSED 3 DAYS REWATERED 2 DAYS 32% LEAF 4 39°c LEAF 1-2-3 8% SPINDLE 21 °a TILLERS 26 1.3.2 Fate of [Methyl-14C]Betaine Applied to Mature Leaves The results of the previous experiment indicated that upon rewatering, betaine is not retained by the mature leaves in which it accumulated. Betaine could either be transferred to the actively- growing regions of the shoot, without net destruction occurring, or it could be simultaneously degraded in mature leaves and synthesized in growing tissues upon stress relief. To decide between these alternatives, [methyl-14CJbetaine (8.5 nmol) was fed to the second leaf blade of plants given one of three different treatments (well- watered; continuously stressed; stressed and rewatered). Plants were then incubated for 3 days and dissected for analysis. Relative to the endogenous betaine contents of both well-watered and stressed second leaves (about 0.4 and l.2 pmol, respectively) 8.5 nmol betaine is a tracer quantity. The added radioactivity was recovered quantitatively from the various plant organs in all three treatments; release of 14 14 coz C-labeled volatile bases were negligible in all cases. The l4 and distribution of C among the various plant organs is summarized in Table l; the distribution pattern was consistent with the resUlts of the previous experiment. The rewatered plants mobilized the added label extensively, and generally had larger amounts of 14C in their actively growing regions than did the well-watered controls of the continuously stressed plants, which mobilized it least. In no case was the radioactivity recovered in the organs in any form other than betaine itself. There was thus no detectable metabolic 27 TABLE 1. Distribution of 14C among organs of barley 3 days after supplying 8.5 nmol of [methyl-14CJbetaine (specific radioactivity 59 uCi/umol) to the blade of the second leaf. Plants were grown in perlite; well-watered con- trol plants were irrigated with nutrient solution daily before and after [140 betaine application. Continuously stressed plants were irrigated with polyethylene glycol 6000 (PEG) solution for 2 days before [ C]betaine application and also thereafter; stressed-rewatered plants were irrigated with PEG solution for 2 days before [14C]betaine application and rewatered with nutrient solution immediately following application of the [14C]betaine. Mean recovery of 14C was 97%, based on all three treatments (2 replicates per treatment). Distribution (%) of 14C Recovered Organ Well-watered Continuous Stress- Controls Stress rewater Leaf 2, blade 6l.8 97.5 38.5 Leaf 2, sheath 4.2 0.8 6.7 Leaf l 1.3 0.06 l.6 Leaf 3 5.3 0.2 10.6 Leaf 4 l4.3 0.l l6.9 Spindie§ 2.0 0.6 2.3 Lower crown 0.6 0.6 0.3 Tillers 5.8 0.06 8.0 Roots 4.7 0.04 l5.l §Spindle = apical meristem f leaf 5 and all younger leaves. 28 degradation of betaine over a 3-day period. The separatory system used would have permitted detection of the following possible meta- bolites of [methyl-14C]betaine: trimethylamine, dimethylamine, mono- methylamine, N,N-dimethylglycine, sarcosine, choline, betaine aldehyde, trimethylamine-N-oxide, phosphorylcholine, trigonelline, hordenine and gramine. 1.3.3 Phloem Translocation of [Methyl4T4CjBetaine The patterns of endogenous betaine redistribution and [14C]- betaine mobilization described above were consistent with transport of betaine in the phloem. To test this, four types of experiments were conducted with seedling plants. In the first type, [methyl14CJbetaine (0.5 pmol, l0 pCi/umol) was applied to second leaves of well-watered plants, plants stressed for 1 day only, and stressed-rewatered plants; the movement of 14C to the sheath was then monitored (Fig. 5). Phloem translocation would be expected to be active not only in well-watered and rewatered plants, but also in the plants stressed for l day only, because Tully et al. 14C-photosynthate and nitrogenous (1979) showed that the movement of compounds from the second leaf of barley seedlings did not cease until plants had been wilted for 2 days at which time leaf water potential had reached -18 bars. For all treatments, the [14CJbetaine front (Bl’ 82, Fig. 5) traversed the l0-cm distance between the 2 Geiger- MUller probes at about 0.25 cm/min (Fig. 5, Table 2). The count rate at each probe increased linearly over seven hours. Heat-girdling Fig. 5. 29 Representative data for translocation of applied [Methyl-14C]betaine and [14C]sucrose, fed in sequence, from blade to sheath in the second leaf of barley. Points 8] and 82 indicate times at which the [14C]- betaine front was first detected at Geiger-MUller (G-M) probes l and 2, respectively; points S] and 52 are when the [U-14C]sucrose front was first detected. The respec- tive rates of betaine and sucrose transport were calcu- ‘ lated by dividing the path length (l0 cm) by either (82—81) or (Sg-S ). Broken lines depict the linear patterns of [ 4C1betaine arrival found to continue for at least7h in similar experiments where [U-14C]sucrose was not added. The amounts of [14C]betaine and [14C]- sucrose were 0.5 nmol/leaf, at specific activities of 10 uCi/pmol and 20 uCi/umol, respectively. 14c-Acnvnv (CPM X102) 30 b .l BETAINE SUCROSE \ L l 4 1 1 l 0 40 so 120 160 200 240 TIME FROM [14C]BETAINE APPLICATION (MIN) 31 TABLE 2. Translocation velocities for 14C—labeled compounds agplied to the second leaves of barley seedlings. [Methyl-1 C]- Betaineand [U-14C]sucrose were applied to the second leaf blades, as shown in Figure 2. The values for well-watered plants are the means i standard errors for 5 experiments. For stressed and rewatered plants, stress was imposed by polyethylene glycol 6000 in one pair of experiments, (+PEG), and by withholding water in the other (-H20). Velocity of 14C trans- Methods . Treatment of Stress bgrs port (cm/min) Imp051tion Betaine Sucrose Nell-watered —- -2 0.24:0.03 0.28i0.02 Stressed l day +PEG -10 0.l9 0.22 -H20 -9 0.27 0.25 Stressed 2 days- +PEG -2 0.27 0.29 rewatered 1 day -H20 -2 0.31 0.35 32 the sheath completely prevented radioactivity reaching the lower probe, and caused 14C to accumulate just above the girdle. [14C]- Glutamate (0.5 pmol, 10 uCi/umol), which is recognized as being phloem-mobile (Pate, l976) was fed to a comparable heat-girdled plant. As in the case when [14CJbetaine was fed, radioactivity did not pass the girdle and accumulated imnediately above it. Although these data are consistent with phloem movement, the velocity is in the lower part of the typical range (see reviews by Canny, l973, pp. 205-207; Hardlaw, 1974). To confirm that the observed velocity indeed reflected phloem transport, an "internal standard" of [U-14C]sucrose was fed through the same abraded spot as soon as the [methyl-14C]betaine reached the second probe. Arrival of the [14C]sucrose at probes l and 2 was detected by sudden sharp increases in count rate (5], $2 in Fig. 5). The velocities of sucrose movement between probes l and 2 estimated in this way were similar to those estimated for betaine (Table 2). In the second type of translocation experiment, a mixture of [6,6'(n)-3H]sucrose (0.5 nmol, 153 to 286 uCi/umol) and [‘40]- betaine (0.5 pmol; lO pCi/umol) was applied to the second leaf blade of rewatered and well-watered plants. After a short (60-90 min) period to permit translocation, the 3H/MC ratio in the conduct- 3H and 14C activities ing path (sheath tissue) was determined. Both had qualitatively similar distribution patterns down the sheath. The 3H/MC ratio on the upper part of the sheath was the same as that of the applied mixture (Table 3), although the ratio tended to become about 50% higher than that of the fed mixture in the lower TABLE 3. 33 Movement of applied [6,6'(n)-3H]sucrose and [methyl-14C]- betaine from blades to sheaths of the second leaves of soil-grown barley seedlings. A droplet of feeding solu- tion containing both [3H]sucrose (0.5 pmol) and [14C]betaine (0.5 nmol) was applied to leaves of well- watered and stressed--rewatered plants. When radioactivity first arrived at a Geiger-MUller tube centered 2 cm below the ligule, a 4-cm section of sheath tissue (which included the tissue directl beneath the probe, plus that about l cm above and below it was harvested. This section was cut into 5-mm pieces and freeze-dried; each piece was assayed for 3H and 14C after combustion. Treatment 3H/Mc Ratio Feeding Solution Sheath Tissueg Hell-watered l6.0 15.8:0.2 Stressed 2 days--rewatered 1 day 12.4 l3.0:0.4 Stressed 2 days--rewatered l day 28.5 30.5i1.0 §Mean i standard error for eight 5 mm pieces. 34 part of the sheath, i.e., as the leading edges of the radioactive fronts were approached (data not shown). These results are quite consistent with the transport of [14C]betaine, like that of [3H]- sucrose, in the phloem but indicate that applied sucrose may be loaded into the phloem more rapidly than applied betaine, at least in the first minutes after application. In the third type of translocation experiment, [14C]betaine was sought in phloem exudate. When [methyl-14CJbetaine (5 pCi, lO uCl/um01) was fed to the second leaf blade of a well-watered seedling, betaine was the only labeled compound found in either the exudate collected over 6 h, the blade or the sheath. The exudation rate was constant for 5 h (Fig. 5) which is consistent with the results of Figure 5. Transpiration reduced the volume of the exudation solution by 23% over the 6-h experiment period. From the data of Figure 6, a minimum estimate of the betaine exported from well-watered leaves is 17 pmol/h; it should be noted that this rate is only about 0.l% of the in 3119 rate of net betaine export from the second leaf blade of stressed- rewatered seedlings (Fig. 4). To confirm that endogenously-synthesized betaine, like exogen- ous betaine, could be loaded into and transported by the phloem, a fourth type of experiment was carried out, in which precursors of betaine were fed to leaf blades. [Methyle14CJCholine (0.5 pmol, 20 uCi/pmol) was fed to the second leaf blade of a well-watered seedling and the radioactive products in the exudate examined at the end of 5 h. As well as betaine, which accounted for l4% of the 35 O 1 *1 l I <9 -30 an: uI ’,.o—-—-—-o gm 0’” 20 z": u. q (3“ < :5 3 O7I- (O E; -I1o gqu I.“ X: P' u: o " 0 ‘3 -° *2 3 < c, (tsu- ES £5 - MI E :5 (13'- uI .3. t9 _ v: 02.: (L1 - l l l o 2 4 6 HOURS AFTER EXUDATION BEGAN Fig. 6. Exudation of [14C]betaine from the cut end of the second leaf of barley. [Methyl-14CJBetaine (5 nmol; lO pCi/umol) was applied to an abraded spot on the second leaf blade about 2 h before exudation period began. Exudation medium was changed every 2 h. Inset graph describes the percentage of exudation media taken up by the plant during 2-h periods. 36 radioactivity in the exudate, choline, phosphorylcholine and an unidentified compound were labeled in the blade, sheath and exudate. The unidentified compound was not Dragendorff—positive and ran imme- diately behind betaine on the TLE plate. In a second precursor- feeding experiment, a tracer quantity (l4 nmol) of the immediate precursor of betaine, [methyl-14CJbetaine aldehyde, was applied to second leaf blades of well-watered seedlings. When ‘4 C-activity was detected at the second probe (about l30 min after label application), the sheath was harvested, freeze-dried and extracted. Essentially all (>96%) of the 14C was in the form of betaine. 1.3.4 Metabolism of [Methyl14gjf Betaine Aldehyde by Leaf Disks Because the preceding experiment, and those of Hanson and Scott (1980) showed that betaine aldehyde was oxidized to betaine very actively by leaf tissues, it seemed possible that betaine alde- hyde was not oxidized intracellularly to betaine, but was a product of extracellular, nonspecific oxidase activity. To test this, leaf disks from the second leaf blade of a 2l-day old seedling were incubated for 6 h with [methyl-14C]betaine aldehyde (0.2 uCi, 0.2 uCi/ mmol). Betaine aldehyde was the only 14C-labeled compound found at the end of the experiment in the incubation medium, and in the buffer used to rinse the disks. 0f the betaine aldehyde supplied, 44% was 14 taken up by the leaf disks; of the C taken up by disks, 32% was converted to betaine. 37 l.3.5 Translocation of 14C- Labeled Compounds in Mature Plants 14c0200 pCi), [U-14C]sucrose (10 uCi, 20 uCi/umol), and [methyl-14CJbetaine (10 uCi, 20 uCi/umol) were administered to the flag leaves of well-watered plants of uniform size about 1 week after anthesis (Fig. 7). The experiment with 14C02 confirmed that the plants were assimilating 002 and transporting assimilates normally; the 14C02 assimilates from the flag leaf reached the spike after 2.2 h. When [U-14C]sucrose was fed through an abraded area on the flag leaf sheath, the 14C-activity reached the spike 3.9 h after applica- tion, and required 1.5 h to transverse the pathlength between probes l and 2 (Fig. 7B). Betaine was supplied to the flag leaf sheath in exactly the same manner as [14C]sucrose but it was 6 days before measurable radioactivity reached the spike. The spike, flag leaf sheath, and a 4-cm segment of culm immediately below the spike were extracted in MCW and analyzed by TLE and TLC. Radioactivity was recovered only in betaine. Thus, under laboratory conditions, the spike did not appear to act as a sink for betaine in well-watered plants (Fig. 7C). l.4 Discussion During episodes of water stress betaine accumulated in expand- ing leaves of barley but was not appreciably degraded by these or any other organs before, during, or upon relief of stress. After relief of stress the betaine was exported from the expanded leaves to the young, actively growing regions of the shoot. This is similar Fig. 7. 38 Representative data for translocation of 14C-labeled assimilates (A), [14C]sucrose (B) and [14C]betaine (C) from flag-leaf sheath to spike in mature barley plants. Inset in frame (A) describes position of Geiger-MUller (G-M) probes on plant. Points R1 and R2 indicate times at which radioactivity was first detected at G-M probes l and 2 respectively. When ‘4C02 was fed the output was monitored from G-M probe 2 only. “c-ACTIVITY (CPM) “c-ACTIVITV (0 PM) 1“(z-ACTIVITv (CPM) fi 1 T 1 fl 1 V I ‘1 V Y A " - .- 600 F " ' v \g 0— G-M 2 400 F' fi/ - G M 1 -D 2 _ I - o 2 4 s 8 10 HOURS AFTER 1"co, APPLICATION I 1 T T W V V I’ll—U f I T 400 e a GM 2 J I t ( ‘ 200 - R1 82 /- - G-M1 ’ J 1 n 1 1 ill 1 4 J 4 2 4 6 ’ 26 20 HOURS AFTER (“c1 SUCROSE APPLICATION V I 1 TfiJlT I j v #% T 1 300 - ‘ C .. c M 2 d 100 :‘/ r‘c—i—. II-u-n-u-II I -r LII-"fr: 'x n r ' -' 94°C M 2 A ’152 154 HOURS AFTER (“c1 BETAINE APPLICATION PM“ “MIL-a 40 to the behavior of another simple quaternary ammonium compound, CCC, which preferentially accumulates in the meristematic tissue of winter barley (Belzile et al., l972). This observation raises the question: Has betaine, like many other natural and synthetic quaternary ammonium compounds, appreciable growth-regulating activity (Karanov, l979)? This question is addressed in Chapter IV of this dissertation. The export of betaine from Older leaves most probably occurs in the phloem. In young vegetative barley plants the youngest leaves were the major sinks for both betaine and sucrose. However, although the spike readily imported supplied sucrose, it did not appear to be a dominant sink for betaine in well-watered, mature plants during grain-fill. In the mature plant the longer period of time to elapse 14 between detection of measurable C-activity in each G-M tube for [14C]betaine as compared to [14C]sucrose was probably due to [14C]- betaine arriving at the spike in quantities below the threshold of detection, and not that betaine was translocated at a slower velocity than sucrose. Slow translocation of betaine to the spike is consis- tent with reports that wheat grains accumulate small quantities of betaine during development (Wyn Jones and Storey, 1981); the highest concentrations of betaine in wheat grains occur in the embryo and aleurone layer (Chittenden et al., l978). In this study the betaine concentration in the apices was higher than in any other organ of the water-stressed vegetative shoot. This Observation may bear indirectly upon the subcellular compartmentation of betaine; G’Oring et al. (l978) suggested that BEIGE Indir ~- I~I ‘- J (I) 41 high concentrations of a metabolite in cytoplasm-rich cells, com- pared to its concentration in highly vacuolated cells, provides indirect evidence for cytoplasmic localization. Two additional points emerge from the results of this study regarding the sites of synthesis and the translocation of betaine in well-watered plants. The first point: Betaine concentration in shoots of well-watered plants remained quite steady (at about l5 nmol/g dry wt) as dry weight increased about 3-fold (Fig. 3). Betaine must therefore be synthesized continuously during growth. This synthesis probably occurs at least in part in the mature leaves because mature leaves of well-watered plants can convert various 14C-precursors to betaine at low rates (Hanson and Nelson, 1978; Hanson and Scott, 1980). The second point: Since the betaine content of individual mature leaves (e.g., blade of the second leaf) remains fairly constant (Hanson and Nelsen, l978), any betaine synthesized in such leaves must be exported from them. The results contain some direct experi- mental support for such a continuous export of betaine from mature leaves of unstressed plants. Exogenously supplied [14C]betaine, and betaine endogenously synthesized from either [14C]betaine aldehyde or [14C]choline, moved from the second leaves, apparently in the phloem. An element of spatial separation between sites of synthesis and accumulation is quite Often shown with secondary plant products, which include nitrogenous bases like betaines (see discussions in Floss et al., l974; McKey, 1974). That betaine is both metabolically inert and phloem-mobile lends support to its promise as an indicator of the cumulative water 42 stress experienced by a barley crop (Hanson and Nelsen, 1978). The most appropriate organs to sample for analysis in further tests of this concept appear to be the youngest leaves. ICC n» “t"l '10 0'. ”fl CHAPTER II THE ACCUMULATION AND METABOLISM OF GLYCINEBETAINE IN FIELD-GROWN BARLEY 2.1 Introduction Whereas considerable research has been conducted on the accumulation of betaine in barley plants grown in controlled environ- ments (e.g., Hanson and Nelsen, l978; Wyn Jones and Storey, 1978), no data are available on the accumulation of betaine in field-grown barley. Indeed, there are very few studies that include results on betaine synthesis and accumulation in any mesophytic plants in the field. Among these few studies are those on the relationship between betaine accumulation and rust infection in wheat (Bowman and Rohringer, l969), and on the contribution of betaine to osmotic adjustment dur- ing water stress in tropical pasture crops (Ford and Wilson, l98l). Laboratory work (Chapter I; Hanson and Nelsen, I978; Ahmad and Wyn Jones, l979) indicated that the betaine that accumulates during stress is not readily degraded upon stress relief. This Obser- vation led to the hypothesis that the betaine concentration in a plant may reflect its previous stress history (Hanson and Nelsen, l978), and so serve as index of the cumulative internal stress suf- fered by the plant. Indices that distinguish the most adapted culti- vars (in this context, those cultivars producing high yields under 43 44 stress) are much sought after as selection criteria in crop breeding programs (Fischer, 1970; Wilson, l976). It is desirable that such indices be integrated measures or predictors of plant performance because many single parameters, for example, those that reflect water status, may differ between adapted cultivars that have adopted different strategies to cope with the same environment (Hanson and Nelsen, l980). Numerous indices of overall adaptation to water stress have been proposed, but their practical value has generally not been adequately demonstrated (Petinov, 1955; Hall et al. l979). In most cases measurement of proposed indices is very time consuming, e.g. measurements of average relative water content and related parameters (Dedio, 1975), transpiration productivity (Petinov, l965) and stomatal function (Jones, 1980). Summarizing, the prospect of some plant character (measured either during or at the end of the grow- ing season) that sums or integrates the internal stress experienced by the plant prior to measurement is a very attractive one. Hiler et al. (l972) introduced the concept of a cumulative stress index that was a "measure of the degree and duration of plant water defi- cit" when investigating the sensitivity of southern peas to plant water deficits. Similarly, an integrated stress index (IS) based on shoot water potential measurements, was recently applied to relate the depression of seed yield to the cumulative stress experienced in cowpea (Shouse et al., l98l). Seedlings subjected to short-term water deficits under labora- tory conditions do not necessarily respond to stress in the same way 45 as a mature plant growing in dryland conditions. Hence the objective of this research was to evaluate, under field conditions, the hypothe- sis that the betaine concentration in the shoot, or organs thereof, of barley is a reliable index of the cumulative internal stress expe- rienced by the plant. To accept betaine concentration as such an index, the following criteria must be met: (l) Betaine concentration, at all stages of growth, must be positively and highly correlated with the cumulative stress experienced by the plant; (2) The differ- ence in betaine concentration between water-stressed and irrigated crops should be evident before morphological symptoms of stress develop; (3) There should be no significant differences for betaine concentration between cultivars given the same degree of internal stress; (4) Differences in nitrogen (N) fertilization should not, .pgr_§g, significantly alter the betaine concentration of the tissue. The fourth consideration, that N-fertilization must not be critical, is important as soil-N fertility frequently varies across a field under normal environmental conditions and the concentrations of individual nitrogenous compounds are frequently affected by N-availability (Waller and Nowacki, 1978; Elmore and McMichael, l98l). Thus the following questions were addressed in this study: l. Is betaine metabolically inert in field-grown plants? 2. To what extent and in which organs does betaine accumu- late? 3. At what time during the growing season does betaine accumulate? 46 4. Are there significant differences in betaine concen- tration between cultivars with the same stress history? 5. Is betaine accumulation significantly depressed by low soil-N? 2.2 Materials and Methods 2.2.l Plant Material The spring barley cultivars "Proctor" CI 11806, "Bankuti Korai" CI 12972, and "Arimar" CI 13626 were used. Proctor was planted on April 13, 1979, April 17, 1980, and April 22, 1981; Bankuti Korai was planted on July 19, 1979, and Arimar on July 25, 1980. Seed was treated before planting with Vitavax-200 (2.5 g/kg; Uniroyal Chem., Naugatauk, Ct.). 2.2.2 Field Plots In 1979 and 1980, two plots (irrigated, I; non-irrigated, N1) and in 1980, also a third plot (water-stressed/rewatered; RW) were laid out; in 1981 two NI plots were laid out. In all three years the plots were on a Spinks sandy, mixed mesic soil (Psammentic Hapludalfs); plot size 2.1 m x 3.6 m in 1979, 2.1 x 5.7 m in 1980 and 1981. All plots were protected from rain by shelters that each comprised a permanent wood frame (average height about 1.4 m) equipped with mov- able sheets of clear, flexible plastic (Loretex, A. H. Hummert Seed Co., St. Louis, MO.). The frames were covered with the Loretex sheets at night and during rain, and were uncovered at all other times. Figure 8 gives detailed specifications of the shelters; views of the shelters are shown in the photographs of Figure 9. At all Fig. 8. 47 Specification sketches of the manual rain-out shelters used in 1980. (A) 3-dimensional view; (B) side view. Inset of frame A describes the attachment of the nylon cord to Loretex sheet; at the free end of the sheet a wooden slat is affixed through which are drilled holes. The nylon cord is secured through each hole by a knot. 48 ROLL LORETEX FIBRE ON ALUMINUM PIPE \ . Q ‘Q ~ ~. .- .. Q NYLON CORDS To FACILITATE ROLL- INO LORETEx UP \\ /‘ "I \ I Q lI H" 5cm MESH-CHAIN LINK fi \ FENCING TAUT OVER \ TOP TO SUPPORT L 2.1m J‘o LORETEx SHEET. OR (Hui NYLON CORD Is ROLLED. W=WOODEN BARS TO WHICH END OF LORETEX SHEETS ARE SECURED. \' - AND DOWN "\ § “ ./ 1 \ ,/ by \\ / U H=HANDLE ON ALUMINUM PIPE ON WHICH LORETEX A. E LORETEx COVER FLUSH WITH WOODEN FRAME ’31:? 1' SECURE DY ELASTIC cows 0 g - END OF LORETEx COVER 0 --.._ ~ _ SECURED BY WOODEN - =~ - -- ROD .. C.‘- - __ " '1 ‘ i J / O NYLON CORD LOBETEX SHEET 43.4cm / SUPPORT POST LORETEX ON SIDES ROLLED TO 31 cm ABOVE GROUND AND SECURED BY TENT CABLES 49 Fig. 9. Photographs of the rainout shelters in fine(A) (1980) and rainy (8) weather (1979). The side cover of the plot on the left (B) is rolled up for the photograph. 50 51 times, the sides and top of the shelters were covered by netting (mesh size = 2 cm) placed underneath the plastic to prevent crop damage by birds and mammals. 0n the west side of each shelter (not shown in Fig. 8) snow fence was erected as a precaution against storm damage. To ensure adequate ventilation at all times, the plastic covers were never secured nearer than 30 cm to the ground. Black plastic covered the shelters for 10 days after seeding at the second plantings of each year (Bankuti Korai and Arimar) to reduce heat injury on emergence. Soil pH was measured before each planting and sufficient lime was routinely applied to raise soil pH to about 7.0. For the crops of Proctor (1979, 1980) and Bankuti Korai the plots beneath the shelters were divided in half, and each received a preplant fertili- zation of 56 kg P/ha and 52 kg K/ha. One half of each plot received 64 kg N/ha, the other half was not N-fertilized. The Arimar crop was not subdivided with respect to nitrogen fertilization and each plot received a preplant fertilization of 64 kg N/ha, 56 kg P/ha, and 52 kg K/ha. In 1981 preplant fertilization for the Proctor crop was 52 kg N/ha, 46 kg K/ha and 7 kg Mn/ha. Prior to fertilization, all plots received a preplant irrigation (at least 15 cm) to bring the soil to field capacity. Seed was planted (40 seeds/m) in 12 rows spaced 17.8 cm apart at rates calculated to give stands of 230 plants/m2. In all plots, twin-wall drip-irrigation lines (4 mil, 10.2 cm outlet spacing; Chapin Watermatics Inc., Watertown, N.Y.) were laid between rows after emergence, and a single irrigation (about 1-2 cm water) was given. (For details of the irrigation 52 system, see Fig. 10.) Thereafter, N-I plots received either a single irrigation at about 5 weeks after planting (Proctor, 1979, 2 cm; Proctor, 1980, 5 cm) or no irrigation. I plots were irrigated once (1979) or twice (1980) per week, with sufficient water to replace calculated evapotranspiration losses. [Water lost from an open pan of water is closely related ‘UD evapotranspiration; when there is full plant cover evapotranspiration is about 85% of open pan evapo- ration (Vitosh, 1977). Evapotranspiration losses at all stages in the growing season were estimated by summing daily open pan evapora- tion values (cm lost/day; supplied by C. Vandenbrink, MSU, East Lansing, Mi.) between irrigation days and multiplying the total by 0.85.] RW plots (Proctor) were irrigated at 50 and 68 (anthesis) days after planting (DAP) with 4.6 cm water; RW plots (Arimar) were given a single irrigation of 4.6 cm water at 41 DAP (anthesis). After 68 (Proctor) or 41 (Arimar) DAP, RW plots were irrigated as frequently as I plots. Table 4 summarizes the pest control measures that were taken. 2.2.3 Sampling Times and Measure- ments Taken in the Field Leaf water potential (0), leaf diffusive resistance (r), and leaf temperature (t), were measured weekly (1979) or twice weekly (1980) between noon and 3 p.m. E.S.T. on the blade of either the youngest mature leaf, or the flag-1 leaf. (The uppermost leaf of each mature tiller is termed the flag leaf; the leaf immediately below 53 4 L N: ‘2 3 :1 r IIIIII I'll" P A. 2.1m - HFIZJM -F N GATE VALVE 1‘ WATER FROM PUMP Fig. 10. Plan of twinwall trickle irrigation installation used in 1980. 54 TABLE 4. Approximate dates of appearance during 1979 of common pests of barley and control measures adopted. 83E5r2:k§ Pest Control Measure+ gay 12 Aphids and/or Greenbugs Orthene 755 (0.7 kg/ha) un Aug 10 May 27 Powdery mildew Benomyl (1.7 kg/ha) Aug 15 (Erysjphe graminis) Jun 15 Hessian fly Orthene 753 (0.7 kg/ha) (Mayetiola destructor) [Especially directed into spindle] Aug 30 Rust Plantvax (2.3 kg/ha) (Puccinia hordei) 1981 §These dates were appr°XImately (i4 days) the same in 1980 and +Sprayed at weekly intervals until the pest was no longer evident. the flag leaf is referred to, in this dissertation, as the flag-1 leaf). Leaf water potential was measured with a pressure chamber (Model 1000 PMS;Instrument Co., Corvallis, 0r.). Leaf diffusive resis- tance and leaf temperature were determined on the upper leaf surface with a Lambda Model LI-65 Autoporometer (Li-Cor Inst. Co., Lincoln, Nb.). [The Li-Cor diffusive resistance meter uses a lithium chloride coated sensor, partially enclosed by a plastic cup. When the sensor is placed on a leaf, electrical conductivity of the lithium chloride increases as water vapor, which diffuses from the leaf, is absorbed. The conductivity increases at a rate proportional to the rate of 55 diffusion of water vapor into the cup. The time (AT) taken for the increase in conductivity between 2 preselected values is recorded. At the same time, leaf temperature is measured by a bead-thermistor that is fixed between the LiCl sensor. The AT value is then converted to standard temperature (25°C) using standard conversion factors. The corrected value of AT, AT25C’ is used to calculate leaf diffusive resistance from a calibration curve of AT25C vs. diffusive resistance (sec/cm).] To confirm that the plants under the shelters behaved as a typical field crop, plant height and fertile tiller number/m2 were measured during grain filling. Grain number/spike and grain weight were measured at harvest; grain yield was estimated as the product of the three yield components (Grafius, 1964). In 1979 whole plants of Proctor were harvested at weekly intervals and divided into spindle, or spike, and remaining vegetative tissue, for determination of betaine content (and in some weeks, nitrogen content). At 70 DAP additional flag and flag-l leaf blades were harvested and pooled for betaine and nitrogen determination. To confirm that water-stress induces betaine accumulation in Bankuti Korai the betaine in the pooled (flag plus flag-l) leaf blades was assayed at mid-grain fill. In 1980 the two youngest mature leaf blades and the youngest organs (the spindle early in the growing season, the spike late in the growing season) were harvested at weekly intervals for betaine determination. At 71 DAP (Proctor) or 49 DAP (Arimar), when individual 56 spikes weighed more than 100 mg, they were subdivided; for each spike separate betaine determinations were made on the awns and remainder of the spike, which comprised the rachis, caryopses and glumes. At anthesis, anthers from Proctor were also collected for betaine determination. All samples were taken from the central 8 rows of the plots, or subplots. 2.2.4 Estimation of Cumulative Stress Experienced by Plants The 0 of the blade of the youngest mature leaf, or flag-1 leaf, was used as the basis for calculating an index of integrated water stress (IS) as defined by Turk and Hall (1980). Measurements of m were made at weekly intervals in 1979, and every 3-4 days in 1980; m values were assumed to be representative for these periods. The integrated plant water stress (IS) for each treatment IS 11 €- 0. cf was estimated as the area above the 0 vs. time curve (Figs. 14, 16 and 17 lower frames) by a trapezoidal approximation method; Htrend" lines were judged by eye. To give IS units of "bar-weeks" the area above the curve was divided by the area of l bar-week. In 1979 IS was calculated for Proctor from 28 DAP (t]) to 77 DAP (t2). In 1980 IS was calculated from either 29 DAP (t1 for all treatments Of Proctor) or 21 DAP (t1 for all treatments of Arimar) 57 to the date of maximum betaine level in the two youngest mature leaves (t2); t2 = 78 DAP for I and NI treatments of Proctor, t2 = 85 DAP for RW treatments of Proctor, and t2 = 56 DAP for all treatments Of Arimar. 2.2.5 Chemical Analyses For betaine determination, plant material [2 replicates (1979) or 5 replicates (1980) from each treatment] was transported on ice to the laboratory, frozen in liquid N2, and freeze-dried. The dried organs, divided as described in Section 2.2.3, were weighed and ground in a Micro-Wiley mill to pass a 40-mesh screen. An accu- rately weighed subsample of milled material (about 20 mg) from each plant was extracted with 5 ml of H20 at 100°C for l h and centrifuged to clear. Betaine in the cleared extract was determined by pyrolysis- GLC after ion-exchange purification (Hitz and Hanson, 1980). Because phosphorylcholine can, in some circumstances, appear in the betaine- containing fraction of the ion-exchange procedure, a number of selected plant extracts, from both I and NI treatments, containing 200(k;betaine were analyzed by thin layer electrophoresis [0.1 mm- thick cellulose glass-backed plates (Cat. No. 5757; E. Merck) 2 kv, 10 min, 1.5 N formic acid]. Semi-quantitative estimates of the phosphorylcholine present were made by comparing Dragendorff—reagent sprayed plates with similarly treated standard plates on which had been loaded betaine (200 ug) and phosphorylcholine (5-50 pg, respec- tively); 10 pg phosphorylcholine was easily discernible. As no phosphorylcholine was found in the betaine fraction by this method, 58 it was concluded that there was §_4 umol phosphorylcholine/g dry wt in NI plants and §_2 umol phosphorylcholine/g dry wt in I plants. To extract grains, an additional step before ion-exchange was taken to remove soluble polysaccharides. After reducing the cleared supernatant under a stream of N2 (at about 80°C) to a 2-ml volume, ethanol (100%, 4 ml) was added, mixed thoroughly; the mixture was then left to stand for l h at 4°C to precipitate polysaccharide. After centrifugation the supernatant was dried in a stream of N2 (at about 80°C) and redissolved in 2 m1 H20. The betaine could then be determined as described above. A 0.5-nmol "spike" of [14C]betaine was added to each of 3 grain samples. At the end of the extraction 14C and ion-exchange procedure 3 96% of applied 14C was recovered; was found only in the form of betaine after thin layer electrophoretic analysis (Section 1.2.4). Kjeldahl-N was determined on selected organs of Proctor [2 (1979) or 3 (1980) replicates per treatment] at selected sampling dates. Tissue was frozen, dried and ground as for betaine determina- tion. Duplicate subsamples (50 mg) were analyzed by the method of Cataldo et a1. (1974), except that nag-N was reduced by a 1-h pre- digestion with 3% salicylic acid in H2S04 (conc., 2 m1) prior to the Kjeldahl procedure to ensure total tissue-N was measured (L. Schrader, personal communication). For Proctor (1979), NOS-N was determined on the vegetative shoot tissue 56, 63, and 85 DAP by the method of Cataldo et al. (1975), and a-amino-N was determined on Vegetative shoot tissue 63 DAP and on 59 spike tissue 70 DAP, using the method of Rosen (1957). For each treatment the analysis was carried out with two replicates, each of which consisted of the pooled organs from 2 plants. 2.2.6 Radioactive Labeling_ Experiments [Methyl-14C]Betaine (59 uCi/pmol) was prepared by the oxida- 14C]choline as described in Appendix A. The leaf tion of [methyl- blade that was to be fed was trimmed to about 10 cm, and a 3-ul drop- let of K+-phosphate buffer (20 mM, pH 7) containing [methyl-14C]— betaine (0.75 or 1 uCi) was applied to the cut end. After droplet absorption was complete (15-25 min), four 2-u1 droplets of water were fed sequentially at 10—15 minute intervals; other details of this method are given by Hanson and Scott (1980). 14CO2 was generated from Na[]4C]bicarbonate (3.75 pmol; 40 mCi/mmol, Amersham Corp., Ill.) and 43% lactic acid, and admin- istered to the trimmed leaf blade as described in Section 1.2.3. For experiments with Proctor on the translocation and metabolism of betaine (experiment 1, Table 18), [14C]betaine was fed to various leaves 46-61 DAP. At 74 DAP the whole shoots were harvested and freeze-dried. In experiment 3, flag-l leaf blades were fed 62 DAP and harvested either 74 or 85 DAP. In experiment'l,shoots (except that one used in Fig. 19) were dissected before freeze-drying; in experiment 3 (Table 19) dissection of shoots was performed after 14 freeze-drying and subsequent to localizing the C-activity by auto- radiography on SB-5 X-ray film (Eastman-Kodak). The amount and 60 chemical form of the 14 C present in various organs was determined by extracting the organs or,irIcase of the culm, 20 mg subsamples in duplicate, in MCW as described in section 1.2.4. The aqueous frac- tion was analyzed by ion-exchange chromatography on 1.5 ml columns AG- 50 (H+) followed by AG-l (formate) (Atkins and Canvin, 1971). The AG-l column was sequentially eluted with formic acid (6N, 10 ml) and then HCl (2N, 8 ml). The AG-SO column was eluted with ammonium hydroxide (4N, 8m1). [14C]Betaine in the AG-50 eluate was identified by co-migration with authentic unlabeled betaine using the TLE and TLC methods described in Section 1.2.4. For experiments with Arimar (experiment 2) that investigated translocation during developing 14 water stress in the NI plants, various leaves were fed either C0 2 or [14C]betaine 33 or 41 DAP. Whole shoots were harvested 55 DAP and 14 freeze-dried intact. The C-activity was localized in the shoots by autoradiography using SB-5 X-ray film (Eastman Kodak). 2.3 Results and Discussion 2.3.1 1979 Pilot Field Experiment The field study conducted in 1979 was a pilot project designed to guide detailed planning for the second year of field experimen- tation. 2.3.1.1 Physical and physiological parameters of the crops. Early-season Proctor Crop: The values for the yield components (Table 5) and the trends in dry matter accumulation, tillering habit (Fig. 11) and leaf diffusive resistance (Fig. 12) for the Proctor Fig. 11. DRY WEIGHT (g/m 2) 1200 .0 o C O ENDC) (SCH) 44C") IZCMD TILLER NO./ PLANT 61 ‘ ”\\ ./. . §~¢62->-8=93 ;_- O ’ Q'HJ #4 l 1 A l "Foal DAYs AFTER PLANTING 9W5 Trends in above ground dry matter accumulation (A) and tillering (B) of Proctor barley (1979). 62 #% T j I l 3 - _ A ‘__—A I/’ A ‘E‘ 6 ' , o _ [I A B 5’ 0 4 " I g I- ” I- , A 2 _ ,’ ‘I - (’--.-::.————-t3._--‘3--..<. () ._1a; ‘T 1 IL I 48 58 68 78 DAYS AFTER PLANTING Fig. 12. The trends through the growing season of leaf diffusive resistance (r;sec/cm) of the youngest mature leaves of Proctor (1979). Symbols as in Figure 12. 63 TABLE 5. Yield, yield components, and selected growth character- istics of the Proctor crops in 1979. Treatment Parameter I : +N I : -N NI : +N NI : -N Mature plant ht (cm) 90 85 40 35 Days to 50% anthesis 69 69 69 69 Fertile tillers/m2 393 264 79 75 Grain nO./spike 26 25 12 16 Grain wt. (mg) 36 35 31 34 Grain yield (kg/ha) 3530 2270 290 410 crop in 1979 were all in accordance with the expected performance of a normal barley crop subjected to I and NI conditions (Briggs, 1978; Legg et al., 1979). Leaf temperature on any sampling day was not significantly different between treatments. Both tillering and dry weight increase (Fig. 11) were sensitive to water deficit (Fig. 13; Aspinall et a1., 1964). As the season advanced, w declined steadily in thelLIcrop and, by 10 days before anthesis, leaf resistance was consistently higher than in the I crop. The water stress developed slowly (Fig. 15C) so that changes in growth pattern occurred that were typical for dryland crops (Fig. 11; Fig. 13). The yield of the irrigated crop (at about 3.5 tons/ha) compared favorably to the aver- age yield for spring barley of 1979 for the State of Michigan(about 2.9 tons/ha; R. Morrison, Agricultural Extension Office, Mason, Mi.). 64 Fig. 13. Photographs cfi’ Proctor barley crops at ear emergence. A and C: Non-irrigated crops in 1979 and 1980, respectively B and D: Irrigated crops in 1979 and 1980, respectively. 65 n 2:5, 9 53,554." , .‘ ' ' . l. 4 . ill 1. “f; It ' . ,I , it w I “innuengi. .,:‘>;IIIHI$::Hmu 1 v... a. O, . 66 The extensive reduction in tiller number/m2, and the consequent low yield, of the NI crOp was not caused by a failure in plant establ- lishment; it was due to rodent damage to the NI plants in the later part of the growing season. Above-ground dry matter accumulation, which probably reflected leaf area development, was lower in the N-deficient crop which sug- gested that the rate of soil-water depletion would be less than in the N-fertilized crop. Late-Season Crop: The growing season in Michigan for spring barley is between March or April and June or July, but to fully exploit the summer months for field experimentation, a trial crop of barley, cv. Bankuti Korai, was planted in July. Bankuti Korai is an early maturing cultivar and thus could complete its life cycle within the remaining three months of late summer. Water potential measurements at different stages of growth were recorded (Fig. 14) to use as a guide for management of water-stress development in later summer plantings in future years. For both early- and late-sown crops morphological differences between the plants grown under different levels of N-fertilization were evident--the N-deficient plants having typically a shorter stature, fewer tillers per plant and lighter-green colored leaves than the N-fertilized plants. 2.3.1.2 Results of chemical analyses. As 0 declined, the betaine concentration in the vegetative tissue of the NI, N-fertilized 67 DAYS AFTER PLANTING O 10 2O 30 4O 50 o r 7 T I I ] Q\\- 9 ON 1 o r \ . .. \ O A \A S A \ ~L 2o *- \ _ i \ \* LEAF 30 L. EMERGENCE YML=3 ANTHESIS A SENESCENCE _‘ I i l GRAIN FILL l C: ~4» l l l l I Fig. 14. Stage of growth, and trends in water potential (0) of the blade ofthe youngest mature leaf (YML), or flag-l leaf, of Bankuti Korai planted July 19, 1979. Symbols as in Figure 11. - 68 crop increased to a maximum of 50 nmol/g dry wt at about one week after anthesis (Fig. 15A). However, the betaine concentration in the vegetative shoot tissue of NI, N-deficient plants remained fairly constant at values similar to those of I plants which had consistently high 0 values (Fig. 15A). Such a depression of betaine accumulation by N-deficiency was not unexpected. In the converse situation when N-fertilization was applied to the halophytic grass Spartina alterni- jlgra that grows normally in low N, high salt conditions the betaine levels increased (Cavalieri and Huang, 1981), indicating a link between soil-N status and betaine synthesis. In the N-fertilized, N-I crop the betaine concentration of the vegetative shoot tissues appeared to decline about two weeks after anthesis. Assuming this drop in betaine level after anthesis to be a real effect, it could have been due either to degradation or to translocation to roots or spikes. The highest concentration of betaine in Proctor barley shoots was observed in the (flag plus flag-l) leaf blades (Table 6) where soil-N availability had little effect on betaine accumulation. The betaine concentration in the (flag plus flag-1) leaf blades of Bankuti Korai was also high--of similar magnitude to Proctor--under NI conditions at both levels of N-fertilization (Table 7). In contrast to whole-shoot tissue, the betaine concentration of the spike tissue rose in both N-fertilized, and N-deficient, NI plants (Fig. 158). Betaine, as a nitrogenous compound, did not appear to parti- tion either like total-N or like dry matter (see respective harvest Fig. 15. 69 Betaine level of the whole shoot. excluding spike tissue (A) and the developing spike (B) for each irrigation and nitrogen treatment of Proctor barley (1979). The bottom frame (C) indicates trends in leaf water poten- tial (w) for each irrigation and nitrogen regime. Symbols as in Fig 11. BETAINE (nmol/g dry wt) 1/’ I-bars) 70 Elk [air 1 I I r 50 A. I \ 40 - ’I \ I I Ai” A ,o’“ 2° ” A“ ""’ N 8‘ ’.OA"-°.3'-O’o-O‘°-’A-O ~ 0-: . 2.-- --- -.-o.. 10 — ” 9 o ‘0 O 0 YFF l.‘ I —: i i : I 50 .. 8' ‘....-..__ ‘ A"" - I A 40 h I” ./A\ I ./ \.‘ ’ .I 4‘ ~ 30 — ’I A, z I, .’° 2° "' 8 '1’ o .0 I A o o 10 — ° 0 «41-8 --II-- \ 1O 1- 9“\'\ e\ /8\° \\.A 0/8 20 - \“\.\ \K. \. ‘ 3° ‘- \\§‘. A 0‘ ‘ A 40 [- c. K.-._._._.- -b [IA—{#1 1 l J l I 28 42 49 5° 63 70 77 DAYS AFTER PLANTING 71 TABLE 6. Betaine concentration of selected organs during the growing season for Proctor (l979). Betaine Concentration Shoot Tissue I:+N I:-N NI:+N NI:-N nmol/g dry wt Spindle (leaves 5+ younger) 48 DAP 17 19 27 27 Youngest mature leaf blades 56 DAP 41 33 115 49 (Flag + flag-l) leaf blades 70 DAP 58 34 155 101 Grain at maturity 8 6 9 8 Straw§ at maturity 24 17 72 26 §Straw comprises chaff + vegetative shoot. TABLE 7. Concentration of betaine in (flag plus flag-l) leaf blades of Bankuti Korai at 42 DAP Treatment Betaine (nmol/g dry wt) I : +N 35 I : -N 40 NI : +N 173 NI : -N 100 LSDO.05 = 31 72 indices, Table 8). The distribution of betaine at maturity suggested that it remained in the leaves and stems of the maturing crop. It is interesting to note that in the straw of N-fertilized, NI plants 10% of the total-N was in betaine (Table 9B). In agreement with other reports (e.g. Halse et a1., 1969) the percentage of N in dry matter was similar in plants grown with and without N-fertilization (Table 10). There was, however, far less N in the N-deficient crop because it had less above-ground dry matter accumulation than the N-fertilized crop (Fig. 11). The slightly higher N content (as % dry weight), especially in the grain, of the NI plants as compared to I plants is commonly observed (Day et al., 1978) and can be explained by depressed photosynthate production and accumulation in NI plants. In all treatments, Nog-N (Table 11) was a small proportion of the total-N in the shoot (cf Table 10). No consistent accumulation of Nog-N occurred in the NI plants, although N03 is known to accumu- late in the stems of some water stressed crops (Hanway and Englehorn, 1958). The proportion of total-N in the form of free amino acids (a-amino-N) in vegetative and spike tissues was somewhat higher in NI plants (Table 9), but this effect of water stress was minor as might be expected from laboratory data on amino acid content of barley seedlings subjected to rapidly-induced water stress (Singh et al.,l973;. Tully et al., 1979). Betaine-N (2.2% total-N) approached the contribution to total N of a-amino-N (3.8% total-N) in NI, N-fertilized plants at anthesis. This was not true in N-deficient 73 TABLE 8. Harvest indices of dry weight, nitrogen and betaine of Proctor barley at maturity (l979). Treatment HI§ NHI BHI I:+N 0.25 0.5 0.1 I:-N 0.25 0.41 0.1 NI:+N 0.1 0.22 0.1 NI:-N 0.13 0.39 0.1 Grain Yield § _ HI,Harvest Index ' Total above-ground dry matter Total-N in grain Total-N in total above-ground dry matter NHI, nitrogen harvest index = Betaine in grain Betaine in total above-ground dry matter BHI, betaine harvest index = 74 TABLE EL a-Amino-N (A) and betaine-N content (8) of selected organs of Proctor(1979)at various dates after planting A. a-Amino-N as % Total-N a-Amino-N DAP Organ Treatment 1 : +N I : -N NI : +N NI : -N % Total-N 63 Vegetative tissue 3.0 3.4 3.8 6.2 70 Spike 14.5 15.0 16.2 14.5 B. Betaine-N as % Total-N Betaine -N DAP Organ Treatment I : +N I : -N NI : +N NI : -N % Total-N 63 Shoot (excluding spike) 1.2 1.1 2.2 1.2 70 Spike 1.9 1.5 3.6 3 2 70 Flag + Flag-1 leaf blades 2.3 1.8 4.5 4.0 84 Spike 0.9 1.0 3 2.4 84 Main culm (excluding spike) 2.6 1.2 .7 2.2 96 Grain 0.6 0.5 0.4 0.4 96 Straw 4.5 2.7 10.0, 5.3 75 TABLE 10. Total-N (including NO§-N) content of selected organs of Proctor barley (1979) at various dates after planting. Nitrogen Content DAP Organ Treatment I:+N I:-N NI:+N NI:-N % dry wt. 63 Shoot (excluding spike) 2.0 1.8 2.1 1.7 70 Spike 1.8 1.5 2.1 1.8 Flag + flag-l leaf blades 4.3 3.3 4.2 3.3 84 Spike 1.9 1.1 2.5 1.8 Shoot (excluding spike) 1.0 1.7 1.3 1.2 90 Grain 2.1 1.7 3.0 2.8 Straw 0.7 0.8 1.0 0.6 TABLE 11. NOE-N level in the shoot vegetative tissue of Proctor barley (1979) at various dates after planting. NOS-N Level DAP Treatment I:+N I:-N NI:+N NI:-N % dry wt. 56 0.27 0.13 0.09 0.14 63 0.09 0.10 0.15 0.09 84 0.11 0.09 0.07 0.09 76 plants where'HIfact,.with the exception of the (flag plus flag-l) leaf blades, betaine-N (expressed as % total-N) in all the organs sampled tended to be lower than in N-fertilized plants. The amino acid-N content of the spike represented a much larger proportion of the total-N than did betaine (Table 9). Comparison of data for betaine in the spike at 1 week after anthesis (70 DAP), and in the grain after harvest (96 DAP) (Table 9B) shows that the contribution of betaine to total grain-N was far lower than to total spike-N. This might be explained in one or more of the following ways: (1) Betaine was metabolized by spike tissue; (2) Betaine ceased to be translocated to the spike later in the grow- ing season and so its relative contribution to total-N decreased as grain protein levels rose; (3) Betaine was largely localized in awn and/or rachis tissue. In order to use betaine as a cumulative stress index there must be a strong correlation between an integrated value of the stress experienced by the plant and the betaine level of either the whole shoot or organs thereof. The whole vegetative shoot of N- deficient plants did not accumulate betaine. However, the spike and youngest leaves did accumulate betaine regardless of soil-N level; regression of betaine level in spike tissue on IS, calculated from the weekly measurements of m (Fig. 14; see Section 2.2.4 for defini- tion) showed a significant correlation between the two parameters (r = 0.74**, N-fertilized; r = 0.79**, unfertilized). This result encouraged further testing of the hypothesis that the betaine level 77 of certain organs could be used as an index of the cumulative stress experienced by a crop. 2.3.2.3 Conclusions from the preliminary 1979 field study. It proved possible to simulate dryland farming conditions in Michigan using manual rain-out shelters. The shelters cihi not adversely affect the performance of the I or NI crops. Symptoms of N-deficiency in plants grown without N-fertilization were evident. N-deficiency severely depressed betaine accumulation in whole shoots, but only moderately lowered betaine accumulation in the spike and youngest leaves. In addition, it was unclear whether the lowered betaine levels in the unfertilized NI crop was a consequence of a higher water status, resulting from slower depletion of soil water by a thinner stand. Betaine contributed up to 4.5% to the total-N in NI (flag plus flag-l) leaf blades and up to 10% total-N in the straw at harvest. Betaine appeared to remain predominantly in vegetative tissue, and not to be massively translocated to the main sinks (grains) of the maturing plant. The influence of N-fertilization on betaine accumulation in whole shoots was too great for the betaine concentration in this tissue to be considered further for practical use as an index of cumulative stress. The most likely organs to sample to further test the hypothesis appeared to be the spike (possibly specifically the awns) and the youngest mature leaves. 78 2.3.2 1980 Field Experiment 2.3.2.1 Physical andyphysiological parameters of the crops. Both the Proctor and Arimar barley crops developed and matured satis- factorily (Table 12). The water-stress experienced by the NI crops were less severe than in 1979 and thence leaf firing and tiller die- back were less pronounced (Fig. 13). Leaf w declined in NI periods but after rewatering rose to values similar to those recorded in the I crop. Leaf diffusive resistance of the NI crop was consistently higher than that of the I crOp. Leaf diffusive resistance of the RW crop increased during stress, but fell after irrigation (see Table 13 for average values of leaf resistance for each treatment). Yields of Proctor were higher than in 1979 and this was due to an increase in tiller number/m2; grain weight was similar in all treatments in both years. This observation agrees with the report of Gallagher et al. (1975) that the grain weight of Proctor remains stable (29-41 mg/grain) under various environmental conditions. The yield of the RW crop was low in comparison to the I crop. In the RW crop, after rewatering, there was a proliferation of small tillers which did not set grain. In CV. Proctor, differences due to N-fertilization were dis- cernible under all three irrigation regimes, being especially pro- nounced in NI and RW plots. All N-deficient plants were character- istically 3-8 cm shorter than the corresponding N-fertilized plants with fewer tillers and paler green leaves. 79 TABLE 12. Yield, yield components and selected growth characteris- tics of the Proctor crop (A) and Arimar crop (B) of 1980 A. Proctor 1980 Treatment Parameter '6 § I:+N I:-N NI:+N NI:-N RW:+N RW:-N' Mature Plant Height(cm) 90 85 50 45 50 40 Days to 50% anthesis 62 62 62 62 62 62 Fertile tiller/m2 562 376 261 205 330 195 Grain no./spike 24.2 19.8 15.6 15.6 18.4 14.4 Grain wt (mg) 36.6 31.2 29.3 31.2 36.3 31.3 Grain yield (kg/ha) 5000 2320 1200 1000 2210 890 B. Arimar Treatment Parameter g I:+N NI:+N RW:+N Days to 50% anthesis 40 40 40’ Fertile tiller no./m2 523 162 253 Grain no./spike 26.6 13.4 27.0 Grain wt (mg) 43.2 38.4 34.7 Grain yield (kg/ha) 5870 800 1290 §Small tillers that sprouted after each rewatering were not included in the observations of this table. 80 TABLE 13. The average leaf diffusive resistance of the youngest mature leaf blade over the growing season (1980). Leaf Diffusive Resistance Treatment Proctor§ Arimar sec/cm I 7 7 NI 30 28 RN Stressed period 13 23 Irrigated period 5 9 §Since no significant differences existed between nitrogen treatments, values were pooled for Proctor. 2.3.2.2 Results from chemical analyses. The total-N (includ- ing NOS-N) content of the whole shoot of Proctor plants was measured at anthesis as an indicator of the N-status of the crops (Table 14). The values were lower, but comparable to those of the previous year (Table 11). The irrigated plants had consistently higher 0 values and lower, relatively constant, betaine levels than the NI plants (Fig. 16 and 17). In Proctor, betaine accumulated in the (flag plus flag-l) leaf blades of both N-fertilized and N-unfertilized plants that had been either continuously or periodically stressed (Fig. 16). The slightly lower levels of betaine in NI, N-deficient plants than in NI, N-fertilized plants may be explained by the former. maintaining, on average, a higher water status during the season. 81 TABLE 14. Nitrogen content (as % dry weight) of the total shoot of Proctor (1980) at anthesis. Total-Nitrogen Content§ N-Treatment Irrigation Regime 1 NI RN % dry wt. +N 1.3a 1.8b 1.7b -N l.2a 1-33 1'23 5Data are means of 3 replicates. Means followed by different letters were significantly different (p = 0.05) according to Duncan's Multiple Range Test. The betaine concentration in the (flag plus flag-l) leaf blades of the N-deficient, RW plants appeared to reach a plateau, whereas that in the N-fertilized, RW plants continued to increase until about 3 weeks after flowering. The decline in betaine concentration in the (flag plus flag-l) leaf blades began about 1 week before yellowing, suggesting that betaine synthesis ceased before senescence began, and that net export of betaine from the blade,or betaine degradation, began late in the growing season. Approximate calculations in which it was assumed (a) that the spike was a sink for betaine translocation from (flag plus flag-l) leaves, and (b) that there was no jg_§itu synthesis of betaine in spikes between 77-85 DAP, suggested only 20-25% of the betaine lost during this period from mature (flag Plus flag-l) leaf blades was attributable to translocation to the Fig. 16. 82 Betaine level of the two youngest mature leaf blades of N—fertilized (A) and N-unfertilized (B) Proctor barley (1980). Lower frames indicate the trends of water potential (0) measured on the youngest mature leaf, or the flag-l leaf. Trend lines are hand-drawn. Vertical arrows in lower frame indicate dates of rewatering of RW plots. Symbols for treatments in the lower frames correspond to the symbols described in the frames immediately above. 83 023.243 Kuhn: m>11...”- 92 The contribution of the awns to total spike weight decreased from about 43% at 64 DAP to about 7% at 85 DAP. Thus the difference between the contribution of betaine to total-N in the spike at 70 DAP and the grain at maturity (Table 9) can be explained by preferential localization (on a dry wt basis) of betaine in awn tissue; the con- tribution of betaine to total-N fell as protein accumulated in the grain during development. Betaine has been identified in wheat anthers as a substance stimulating the growth of Fusarium graminearum (Strange et al., 1974). Anthers, which were shedding pollen, were collected from I and NI Proctor plots. Anthers from all treatments had high levels of betaine (average about 100 umol/g dry wt) with no significant differences between treatments. 2.3.2.2 Translocation and metabolism of betaine. Laboratory experiments of a few days' duration (Chapter I) indicated that betaine was not degraded by barley seedlings and that it was transported in the phloem from older to younger organs. Radiochemical experiments in the field sought firstly to evaluate the contribution that trans- location made to betaine accumulation in the (flag and flag-1) leaf blades, and secondly, to investigate the decline in betaine concentra- tion in the flag-l leaf blade two weeks after anthesis (see Fig. 16). In the first experiment, [14C]betaine was applied to various Proctor leaves on three dates after planting. At harvest, the only 14C-labeled compound found in any organ was betaine (Table 18). The 14 recovery of C was low, especially at the earlier planting dates. Roots could not be harvested, but because in laboratory experiments 93 .mcmmso _Fm cH .mcwmpmnmue_u we Ego» mg» cw mm: o .mcwucmpq LOOGm mxwu «A we; even pmm>gmz vp wzu we xmmA .mmpmuwpamg mwgga mo memos wgm mumo+ .Ogmwm mm: s_=u Ewes Ema Conan: Lump mumsm>smz pm mcmmgo uoozn acoe< :owuznwspmwoiocp mama mcwummm AP Damswcmgxmv oma_ e2 »a_sen Lapuosa eauamwssw-eee La mpoaem e2 aewapanmue_-_xepaea to eoLC=a_sam_o .m_ MAmae 94 roots imported up to 15% of the applied betaine after 3 days (Section ‘40 in the field 1.3), it is probable that incomplete recovery of reflects slow downward translocation of betaine to roots. However, loss of 14C02 and other volatile metabolites cannot be ruled out as explanations for the low 14C-recovery. The applied [14C]betaine was extensively exported from the lower two leaves fed (flag-4 and flag-3) and in these cases the main sinks in the shoot were tillers, and leaves above those fed. In contrast, much of the fed [14C]betaine was retained by the flag-2 leaf, and little ‘4 C migrated upwards (Table 18). These results confirm that applied betaine can be translocated from mature to expanding leaves, but indicate that the import of betaine into the (flag-l plus flag) leaf blades was probably only a minor term in their total betaine accumulation. The diminished translocation of betaine from the flag-2 leaf blade might have been caused by a general depression of translocation during the developing water-stress. Figure 19 illustrates the trans- location of [14C]betaine fed through the flag-2 leaf of Proctor 54 DAP (experiment 1). This autoradiograph serves to orient the reader so autoradiographs of experiment 2 are clear. Results (Fig. 20) from 14 feeding [14C]betaine and C02 to the flag-1 leaf blades of Arimar clearly demonstrate that the failure of betaine to be exported did not reflectaigeneralized failure of translocation. Thus, 14002- assimilates and [14C]betaine fed early in the season (Fig. 20A, C) were extensively translocated around the plant but, whereas Fig. 19. 95 Autoradiograph of a Proctor barley plant fed [14C]betaine to flag-2 leaf blade 54 DAP. Harvest at 75 DAP. At harvest, before autoradiography, main culm was cut at the flag leaf node. 96 FLAG LEAF-I FLAG LEAF Mmefi Fig. 20. 97 Autoradiographs of barley plants fed 1 pCi [14C]betaine (A,B)or 50 uCi 14co2 (0.0) at 22 (A,C) or 41 (8,0) DAP. Harvest at 55 DAP. [Experiment 2]. At harvest the main culm was cut at the flag leaf node. Key to letters: f = flag leaf; 5 = spike; f- immediately below flag; t = l = leaf fed leaf. 98 99 14COZ-assimilates were exported from the flag-l leaf later in the season (Fig. 200), applied [14C]betaine was largely retained by this leaf (Fig. 20B). The flag-l leaf blades of the NI plants of the third experi- ment (Table 19) were fed [14C]betaine 62 DAP in 1981 and then were harvested after 12 days (74 DAP) and 23 days (85 DAP), respectively. Autoradiographs of the whole plant prior to extraction indicated 14 considerably more C-activity was in the blades of plants of the early, than in those of the late harvest. Plants of the late harvest had correspondingly more radioactivity in the sheath and culm. Little 14C-activity (< 2.5%) was found in any form other than betaine in the 14C-distribution resembled, in four parts of the plant analyzed and a general way, that of the endogenous betaine at the two dates (Table 19). This result, that betaine moves out of the blade and into other vegetative organs, explains, to a large extent, the drop in betaine levels observed in the (flag plus flag-l) leaf blades after anthesis (Fig. 16). The above results indicate that synthesis of betaine 1 situ is the major source of the betaine accumulated by the (flag plus flag~l) leaf blades, and that in Proctor, betaine is exported from the flag-l leaf blade late in the season to be relocated in the sheath of this leaf and in the culm. The implication of these results is that export, and possibly import, of betaine is regulated in some specific way so that betaine does not simply follow the translocation patterns of other nitrogen compounds or of COZ-assimilates. 100 TABLE 19. Distribution of 14C-activity in selected organs of non- irrigated Proctor barley (1981) after [14C]betaine (0.75 pCi) was fed to the flag-1 leaf blade 62 DAP (Experiment 3). Betaine 14C§ Harvest Organ Time (DAP) pmol/ pmol/ % nCi/ g dry wt organ applied organ 74 Flag-l blade 93 1-8 80 602 Flag-l sheath 50 2.3 2 l6 Culm 2nd internode 17 0.6 0 0 85 Flag-1 blade 59 0.9 35 266 Flag-l Sheath 69 2.9 12 96 Culm 2nd Internode 89 4.1 9 70 Flag leaf sheath 83 4.7 2 12 l4 §397.5% c was in the form of betaine. 101 2.3.2.4 Conclusions from the 1980 field study. The accumu- lation of betaine in the (flag plus flag-l) leaf blades of NI barley is not a shock response to rapidly imposed water deficit but occurs gradually, as w declines, at a time when the plants are making morpho- logical changes in response to their water-stressed condition. Betaine accumulation apparently ceased well before the onset of senes- cence, judged from incipient yellowing. Within the mature plant betaine is not metabolically labile and can be translocated, probably in the phloem. Although betaine is translocated in such mature plants, it does not follow the partitioning pattern of total plant N and photosynthate, and remains primarily in the leaves and culm; some betaine does accumulate in the awns. The concentration of betaine in the grain is always low (< 10 nmol/g dry wt) which agrees with values obtained for wheat grains(Chittenden et al., 1978). Circumstantial evidence from this work, and from the radio- chemical experiments of Hitz et al. (1982), indicates that the accumu- lation of betaine in the (flag plus flag—l) leaf blades is predomi— nantly due to synthesis in 515g and not to translocation from other organs. Unlike betaine accumulation in the shoot as a whole, which is severely depressed by low soil-N, betaine accumulation in the two youngest mature leaves is largely unaffected by N-fertilization. Betaine concentration of the youngest mature leaf pair was correlated (r a 0.9**) with the cumulative degree of stress, expressed as bar-weeks, experienced by the plant. The criteria that were set in order to judge whether the betaine content of the (flag plus flag-l) 102 leaf blades could be used as an index of the cumulative stress expe- rienced by the plant, as described in the Introduction, were largely met. However, betaine concentration could not be recommended as such an index for the following reasons: (1) The difference in betaine level between I and NI plants was comparatively small (approximately 2- to 3-fold). In addition, the levels among I and NI plants were too variable to give an accurate estimation of the cumulative stress experienced by the plants unless many samples were taken; (2) The increase in betaine concentration accompanied, but did not precede, morphological changes in response to water stress. The latter, in any practical situation, are easier to detect and to measure; (3) From studying just two cultivars of barley, genetic variability for basal and stressinducedlewels of betaine was implicated; this could con- found interpretation of results in a breeding program. The timing, the extent, and the site of betaine accumulation (viz. in the two leaves that contribute most to grainfill), are all consistent with betaine accumulation having an adaptive role under stressed conditions. CHAPTER III GENETIC VARIATION FOR BETAINE LEVELS IN CULTIVATED AND WILD BARLEY 3.1 Introduction Halophytes are known to show osmotic adjustment when sub- jected to saline conditions (Flowers et al., 1977); this permits maintenance of turgor and continued water flux through the plant. Many halophytes, especially those from the families Chenopodiaceae and Gramineae, have high shoot concentrations of betaine (100-300 nmol/g dry wt) even when grown in non-saline environments; betaine levels increase further (to 300 - 1000 nmol/g dry wt) in response to salinity (Storey and Wyn Jones, 1975; Wyn Jones et al., 1977b). In such halophytes it has been suggested that betaine accumulation is an adaptive response to salinization. Betaine is believed to act as a nontoxic, or "compatible" osmoticum in the cytoplasm, balancing the accumulation of potentially toxic salts in the vacuole (Wyn Jones et al., 1977a; Hall et al., 1978; Leigh et al., 1981). Some meSOphytes of the above-mentioned families, (e.g., the grasses,barley and rhodes grass)also accumulate betaine in resonse to salt stress, but generally to lower maximum levels than their halophytic relatives (Wyn Jones et al., 1977b). Recent investigations have demonstrated that many meSOphytic crops are able to lower solute potentials by accumulating solutes when 103 rye gra bar f. L 8C gr: 801 et tr,- GET 104 subjected to water stress, i.e., show osmotic adjustment (Morgan, 1977; Jones and Turner, 1978; Turner and Jones, 1980; Wilson et al., 1980). Betaine has been found to accumulate in response to water stress in several mesophytes, notably the cereals barley, wheat, and rye (Hitz and Hanson, 1980), the grasses, green panic and buffel grass (Ford and Wilson, 1980) and spinach (Pan et al., 1981); In barley and wheat, betaine is apparently a metabolic end product accumulated by stressed young leaves as a result of enhanced jn_situ synthesis and translocation from older leaves (Bowman and Rohringer, 1970; Hitz et al., 1982; Section 1.3 and 2.3). In one cultivar of barley grown under non-irrigated conditions in the field, betaine levels in the uppermost leaves reached 200 umol/g dry wt (Section 2.3) which approaches the concentrations observed in some halophytes (Wyn Jones et al., 1977b; Ahmad et a1., 1981). An adaptive role for betaine accumulation in water stressed cereals has been postulated essentially by extrapolation from (a) the association of betaine with ecological halotolerance (Wyn Jones et al., 1977b) and (b) from the protection afforded by betaine to bacteria growing in NaCl (Shkedy-Vinkler and Avi-Dor, 1975). There is now some evidence from field studies of barley in dry environments (Hitz et al., 1982) that is consistent with an adaptive role for betaine accumulation. One approach to assessing the adaptive worth of a metabolic trait, such as betaine accumulation, is to perform physiological- genetic studies similar to those undertaken to evaluate the adaptive m f\_) ) /.. 150011 9035 I 105 role of proline in barley (Hanson et a1., 1977) and rapeseed (Richards and Thurling, 1979) and of abscisic acid in wheat (Quarrie, l980b). The objective of this research was to evaluate the genetic variability for betaine accumulation in cultivated barley and its progenitor, Hordeum spontaneum, and so determine if such a physiological-genetic approach would be feasible. The investigation was limited to_H. vulgare and H. spontaneum as crosses involving these two species have been highly successful with no pronounced sterility or abnormal segrega- tion ratios (Price, 1968); among the other species of the genus Hordeum a considerable degree of incompatibility exists and inter- specific crosses resulting in fertile hybrids are rare (Price, 1968). 3.2 Materials and Methods 3.2.1 Plant Material Seed for both field and laboratory tests was obtained from the USDA Small Grains Collection (Beltsville, Md.). CI 13626 (cv. Arimar), CI 11806 (cv. Proctor), and CI 11509 (cv. Excelsior), origi- nally from the USDA collection, were multiplied in 1979 at sites in E. Lansing, Mi. Seed for laboratory studies was used untreated. Seed was treated with Vitavax-200 (2.5 g/kg, Uniroyal,, Naugatuck, Ct.) before planting in field trials. 3.2.2 Betaine Determination All plant material was freeze-dried. Whole shoots of laboratory-grown plants were weighed and ground in a Wiley Mill to pass mesh size 40. Accurately weighed subsamples (18-21 mg) were extr :11 act mat pot rm ES 111. 106 extracted by boiling in 5 ml water for l h and standing overnight at 4°C. Flag-l leaf blades from field-grown plants were individually weighed and extracted without grinding. Betaine was estimated using the 2-column ion exchange/gas chromatography-pyrolysis procedure described by Hitz and Hanson (1980). 3.2.3 Water Potential (0) and Solute Potential (05) Measurements In laboratory trials 0 was measured on the blade of the second leaf and in field studies on the blade of either the youngest mature leaf or the flag-l leaf, using a pressure chamber (PMS Instrument Co., Corvallis, 0r.). To reduce water loss during the measurements, blades were wrapped in damp paper towels. For ms measurements, duplicate or triplicate 8-mm disks were cut from the mid-blade position of leaves, wrapped in foil, frozen in liquid N2 and then thawed. A Wescor HR- 33T dewpoint micro-voltmeter was used to estimate 05 on the frozen- thawed disks. It should be noted that Us estimated in this way is actually more accurately described as the sum of solute potential and matric potential. However, it is reasonable to assume that matric potential is small (5_-2 bars) in relation to ms, and that it does not differ markedly among the cultivars tested, so that the over- estimation of ms was a minor and consistent effect across cultivars. 3.2.4 Controlled Environment Tests Growth chamber conditions for all tests were: l6-h day, PAR 5 mW cm'z, 21°C, relative humidity 70%; 8-h night, 16°C, relative humidity 85%. The plants were watered on alternate days with con two the re; eff wat Han ent 1101‘ My Inc] In] 107 half-strength Hoagland's nutrient medium. The statistical design for all trials was a partially balanced (3 replicates) 7 x 7 lattice square (Cochran and Cox, 1957). The replications were conducted sequentially, not concurrently, due to limited space in the growth chamber. A total of 147 entries, in 3 sets of 49, were screened for betaine concentration in whole shoot tissue in 3 independent trials (A, B, C). Within each trial, genotypes were assessed for betaine concentration under well-watered and water-stressed conditions in two sub-trials which were conducted concurrently. In each replicate, the 49 genotypes were planted in completely different groups of 7; the groups Of 7 in irrigated and water-stressed sub-trials of each replicate were the same. This design was adopted to reduce the effects of genotypic differences in pot water-depletion and plant water status that occur as stress develops (Quarrie and Jones, 1979; Hanson and Hitz, 1982), by ensuring that each genotype had a differ- ent set of neighbors in each replicate. Trial A comprised 28 genotypes of H. spontaneum and 20 culti- vars of H. vulgare selected for their purported adaptation to diverse environmental conditions (high salt, high altitude, high temperature, dry or moist conditions). Trial 8 included 42 genotypes of H. spontaneum and 7 cultivars of H. vulgare chosen from diverse geogra- phical locations. Trial C comprised 48 cultivars of H. vulgare from 32 countries. Both CI 11806 (Proctor) and CI 13626 (Arimar) were included as checks in each trial to enable inter-trial comparisons. In Trials A and C CV. Proctor was entered twice. 108 Unglazed clay pots (15 cm diam.) were divided into 7 sectors marked off with plastic strips. In Trials A and 8, seed was strati- fied in darkness at 4°C on moist filter paper for one week in order to break dormancy, and transferred at room temperature for 3 days to allow germination. Sprouting seeds were then planted in a soil mix- ture (peat:loam:sand; 1:1:2 v/v), placing 3 seeds Of one genotype per sector. In Trial C, 3 seeds per sector were planted directly into the soil mixture. In all trials, CI 11509 (Excelsior) was planted in the center of each pot; the water status of Excelsior served as a biological "benchmark" for the severity of water stress. After 8 days the seedlings were thinned to one per sector. The well-watered seed- lings were harvested 16 days after planting (DAP; 4-leaf stage), at which time water-withholding from the pots of the water-stressed trial began. The water-stressed plants were harvested 22 DAP; they had been wilted for 4 to 5 days. 3.2.5 Field Experiment The 8 cultivars with the lowest and the 8 cultivars with the highest betaine concentration after stress in laboratory Trial C (Table 20) were chosen to assess the variability for betaine accumu- lation among genotypes under non-irrigated conditions in the field. Growth habit and length of growing season of the cultivars were unknown and so to ensure that at least 6-10 cultivars would be developmentally similar, and hence comparable with respect to water status and to betaine level, 16 cultivars were planted. In fact, it was necessary to omit only three of the cultivars (CI 11806, 109 TABLE 20. Designation and origin of barley cultivars tested for betaine accumulation in non-irrigated field conditions. CI Number§ Provenance Classl High-Betaine 14936 Ethiopia l 1114 Peru 2 6577 Afghanistan 2 3480 Syria 1 11961 Morocco 2 13626 USA 1 Low-Betaine 10064 Ecuador 1 2318 India 1 10138 Afghanistan 1 10359 Indonesia 1 709 USSR 3 13057 USSR 3 9309 China 2 § Cultivars chosen were the 6 highest and 7 lowest accumulators under stress in laboratory trial C; ranges for betaine concentra- tions were: dry wt. +The class designation refers to the betaine accumulation pattern in the flag-l leaf blade under field conditions. 6 highest, 76-83 nmol/g dry wt; 7 lowest, 51-54 nmol/g 110 C1 1610, and CI 742) because they were markedly later in maturity; CI 742 remained vegetative. Preplant fertilization was 52 kg/ha N, 46 kg/ha K and 7 kg/ha Mn in accordance with results from soil analysis. Seed was planted on April 23, 1981, under 2 rain-out shelters (2.1 m x 5.7 m; as described in Section 2.2.1) in E. Lansing, Mi., on a Spinks sandy mixed mesic soil (Psammentic-Hapludalfs). The land under each shelter was divided into two 2.6 m blocks separated by a path. Each block was then subdivided into two sec- tions which were also separated by a path. In each section there were 12 l-m rows, 17.8 cm apart; seed was planted at a density of 50 per meter of row. Each cultivar was entered once per block in a design in which no two cultivars were adjacent in more than one block. Two guard rows of barley were planted around the plots in each shelter. No irrigation was given after seedlings were estab- lished. Water potential and 05 are known to undergo diurnal variation (Hsiao et al., 1976). Sampling all 4 blocks required about 3 h and so to ascertain whether 05 would vary more during the morning or early afternoon,q)and 05 measurements were taken on the blades of the youngest mature leaf (5 replicates) of Proctor and Arimar 47 DAP at 9 a.m., 1 p.m. and 3:30 p.m. E.S.T. The differences between Us at l p.m. and 3:30 p.m. were less than during the morning (Table 21); hence, samples were taken in the early afternoon. Water potential, as expected, varied throughout the day. 111 TABLE 21. Variation in w and w and Proctor Barley s in the youngest mature leaf of Arimar Cultivar§ Time Of Proctor Arimar Day 4 4,1 w 4, bars 9:00 a.m. -4.6 -ll.0 -8.5 -l3.0 1:00 p.m. -9.3 -12.0 -l4.0 -l4.2 3:30 p.m. -6.2 -12.5 -10.1 -l4.5 L500.05 2.9‘ 0.5 2.7 1.1 §5 replicates at each date. TTriplicate samples on each leaf. Blocks were sampled at 41, 59, and 63 DAP. Four replicates, one from each block, were taken at each sampling date; all entries in each block were harvested together. Water potential measurements were made on the youngest mature leaf blade at 41 DAP; and on the flag-l leaf blade at 59 and 63 DAP. The same leaves were then transported on ice to the laboratory for ms measurements, followed by freeze-drying for betaine assay. 3.3 Results 3.3.1 Results of Controlled Environment Tests In all three trials, there was statistically significant variation irI betaine concentration amongst genotypes both before 112 and after water-stress (Fig. 21); for details of genotypes included in each trial, and their average pre- and post-stress shoot betaine concentrations, see Appendix B. Insofar as environmental conditions were very similar for all cultivars and trials, a genetic component in the cultivar variance is implied (Hanson and Hitz, 1982). Under well-watered conditions, the betaine concentrations within the cultivars Proctor and Arimar were not significantly dif- ferent in all three trials (Table 22) so that it was reasonable for purposes of comparison to combine the results of all 3 well-watered trials (Fig. 21, uppermost frame). The severity of water-stress varied between trials, and there were significant differences in betaine levels within the cultivars Proctor and Arimar between trials (Table 22); the highest betaine levels were recorded in trial C, the lowest in trial A. Such differences between water-stressed trials might be anticipated as betaine accumulation in barley seedlings is proportional in a general way to the severity and duration of stress in the range -10 to -20 bars (Wyn Jones and Storey, 1978; Hanson and Nelsen, 1978; Hanson and Scott, 1980). These differences in betaine levels between trials for both Proctor and Arimar were consistent with the 0 data for the benchmark cultivar,, Excelsior. In trial C, the benchmark 0 value (-27 bars) was lower than trial A (-16 bars). As direct comparison between water-stressed trials is therefore pre- cluded, results from each trial are shown separately (Fig. 21, lower frames). In analyzing the primary data of Figure 21, two questions were asked: Fig. 21. 113 Frequency distributions for betaine concentration in cultivated and wild barley genotypes entered in 3 labora- tory trials (A, B, C). The uppermost frame summarizes data for the well-watered seedlings from all 3 trials. The lower 3 frames are data for water-stressed seedlings in the individual trials. Average 0 values of the benchmark plants (cv. Excelsior)in the trials are indi- cated. The vertical arrows show the means, and the horizontal bars indicate the LSD0 05 for the population sampled. One L500 05 value has been given in the upper- most frame as values were the same among well-watered trials. NUMBER OF GENOTYPES 40 T 20 _ WELL WATERED W ' ‘3 BARS A 5 l TRIAL — ID I ‘16 BARS TRIAL ~0- - h- 1_L Tfi A4 W ' ’22 BARS 6'1 1 E5 1 TREAL LSD L 1 T T ‘I 1 fi 0.05 III-‘27 BARS .1 1 IO 11 ,_Lll 30507090110 BETAINE LEVEL (nmol/g dry wt.) 115 TABLE 22. Shoot betaine concentrations of Proctor and Arimar in laboratory trials A, B, and C. Shoot Betaine Concentration§ Cultivar Treatment Trial A B C nmol/g dry wt Proctor Well-watered 11.7a 16.0a 18.0a Well-stressed 44.3Cd 52.0d 52.7d Arimar Well-watered 25.7ab 33.3bc 34.3bc Water-stressed 49.7d 67.0e 83.7f §Mean of 3 replicates. Values followed by the same letter are not significantly different (p < 0.05) according to Duncan's Multiple Range Test; comparisons made between and within columns. 1. Is the betaine level in water-stressed seedlings (y) related to the betaine level under well-watered conditions (x)? If so, additional screening for high- and low-accumulators might be based solely on trials with unstressed seedlings. Betaine levels in water-stressed seedlings were significantly correlated with the betaine levels of well-watered seedlings (Table 23); this relation- ship between the pre- and post-stress betaine levels was stronger in the two trials in which the water-stressed sub-trials reached the lowest 0 values (Table 23). The data did not permit a distinction to be drawn between the accumulation of the same amount of betaine by all genotypes and the accumulation of various amounts of betaine proportional to the basal level characteristic of the genotype 116 .mpmwsu .m:u_>wucw :_ mmaxuocmm macaw Ammmgum msowmn cowpmgucmo -cou mzwmpmn I mmmsum LmDLm cowumsucmocou mcmmumnv Lo m=Fm> mmmLm>m n mmmmsucH mmosm><+ .mpmwcu Fmacw>wccw mmmsum msowmg.:p»umsucmocou wcwmumm n > cw mmazuocmm mcosmfl mmmspm swpmm :o_amsu:mucoo mcwwpmmvhtowapm> mmmsm>m owumm mouse wuumamms ._o.o n a new mo.o n a an mucmuwmmcmwm mumuwucmw«.« o.“ w N.Nm .e.o w m.~ 4446.0 m_.mm + xop._ n A u m.~ w m.mm m.o w N.N eeNA.o mm.- + xNN.F n a m F.o H a.mm _~.o w m.~ «m~.o m.Pv + xmm.o n x < 8m w mmmmgucH um n opumm “copupwmmou +mmmsm>< . mummpm>< :o_wmmeLou comumacm cowmmmgmmm paws» .mpowsa mcwcmwsum Agoumsoamp m cm xm_smn Lo mmcwpcmmm Axv cmgmumzippmz new Aav vmmmmspmigmumz Lo muoogm ecu cw cowumcucmucoo mcwmumn msu cmmZuma awzmcowumpmg Och .mN m4mcho 60L. croLm mm:..uoom co. m+co>o mmoL+m vac 0L:+.:o .0 Lance .oo_mo.o:oL;u mc.n_Lumov mosozum .vN .m_u .+=o eo.ccoo ocox mo.u:+m ac.eoo. o:.e~.0cog+o_ov.uw_ 30.x: +0 as.» . .co_+:_0m +eo.c+:c uo+uo+cou m+ooL no zoom no 2:33 o... 8396 9.9. mac-:63 835.5% :08 mu m toe—.598 c. “cc—n+3 0... 93096 9.0.. «+09. :02: 5 vote“. 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During stress periods, aluminum foil, fitting closely around seedling culms, covered the soil surface. At the end of the stress period some plants were rewatered for up to three weeks to determine if pretreatment with betaine was beneficial to recovery after water stress. 4.2.3.l Embryos. Ten-ml aliquots of media were dispensed into 75-m1 boiling tubes (2 cm diam.); agar was predissolved by autoclaving media for 5 nfin (121°C). Tubes were capped with a wad of cotton wool which was covered by aluminum foil (Fig. 25A) and autoclaved (121°C; 20 min). The embryos (l per tube) were set gently on the agar surface. In order to reduce the amount of light to reach the roots the bottom of the tubes were wrapped with aluminum foil. After the embryos were left 3 days in the dark (25°C), either water alone (0.5 ml) or betaine (OH’) solution (15.3 or 153 pmol betaine/0.5 ml HZO/tube) that had been sterilized by Millipore filter (mesh size 0.22 pm; Millipore Corp. Bedford, Ma.) was added. At this time tubes were transferred to the light (16-h day; day/night tempera- ture 22/16°C). After 10 days when plantlets were at the 2-1eaf stage, they were individually transplanted into perlite (pot size, 7. cm diam. x 12 cm; 1 seedling/pot) and kept in the light as before (day/ night relative humidity 70/85%). At the time of transplantation the agar media were tested for microbial contamination. 134 COTTON WOOL (FOIL CAP) A' —— SEEDLING U—-AGAR SPONGE STOPPER B. &_ \ LIQUID MEDIUM @— cor'rou WOOL (FOIL CAP) V FILTER PAPER LIQUID MEDIUM Fig. 25. Culture conditions for embryo (A) and intaCt seeds in semi- -sterile (B) and steri1e(C) conditions. 135 4.2.3.2 Intact seeds under semi-sterile conditions. Equip- ment and media were sterilized by autoclaving (121°C, 20 min). Betaine was not degraded upon autoclaving as shown by TLC and TLE analysis of sterilized media which had been supplemented with [14C]- betaine (Section 1.4). Sterilized seeds were germinated on sterile filter paper for 3 days in the dark (25°C). The germinated seeds were individually placed between the 2 halves of a sponge stopper (0.5 cm thick, cut vertically to generate the 2 halves) and fitted into the top of 36-ml glass vials (Fig. 258) into which had been measured 20 ml of quarter-strength Hoagland's solution. To prevent light reaching the roots, aluminum foil was wrapped around the vials. The seedlings were left in the growth chamber (16-h day, day/night temperature 22/ l6°C, relative humidity 70/85%) for 4 days. Then, still with seed secured by sponge, they were placed in lO-ml beakers which contained either water (3 ml) or betaine (OHI) solution (27 mM, 3 ml). After 2 days, 2 to 2.5 ml of liquid had been taken up by the seedlings which were then transplanted into a soil mix (Section 1.2.1), one seedling per pot (7 cm diam. x 12 cm). Aliquots (0.3-l ml) were taken from all root media and tested for microbial contamination. Seedlings were grown in the greenhouse under supplemental light (16-h day, day/night temperature 22/l6°C, relative humidity 70 to 80%). 4.2.3.3 Intact seeds under sterile conditions. Half-strength Hoagland's solution (10 ml), plus either betaine (40 mM) in K+-phos- phate buffer (2 ml; pH 7, 20 mM) or K+-phosphate buffer alone was measured into 75-ml boiling tubes (2 cm diam.). A circle of filter paper (Whatman #4; 2 cm diam.) was positioned about 0.5 cm above the 136 liquid surface (Fig. 25C). The intact seed was placed in a perfora- tion (:_3 mm diam.) in the center of the filter paper. The tubes were closed with a wad of cotton wool and covered by aluminum foil. In order to reduce the amount of light reaching the roots, aluminum foil was wrapped around the bottom of the tubes. After 2 days in the dark at 25°C the tubes were transferred to the growth chamber (16-h day, day/night temperature 22/l6°C). After 17 days the seed- lings were transplanted into a soil mtx(Sectionl.2.l) in pots (7 cm diam. x 12 cm; 1 seedling/pot) and transferred to the greenhouse under supplemental light (16-h day, day/night temperature 22/l6°C, relative humidity 70 to 80%). Before transplantation, nutrient media (l—ml aliquots) were tested for microbial contamination. 4.2.4 Incubation with [14C]Compounds In all experiments, [methyl-14CJbetaine (9.7 uCi/pmol; radio- chemical purity > 99%; Amersham Corp., Ill.) was used to spike repre- sentative tubes of root feeding media that contained unlabeled betaine; 0.5 uCi of [MCJbetaine was added per tube. Uptake of betaine by seedlings was estimated from either 14C-activity in the 14C-activity remaining in the root feeding solution, assum- plant or ing: (1) There were no isotope effects; (2) Betaine was not degraded to volatile [14C]products. [2-14C]Ethanolamine (44 pCi/pmol; radiochemical purity > 98.4%; Amersham Corp., 111.) was used to investigate the effect of varying endogenous betaine levels on betaine synthesis in well- watered seedlings. The fully expanded, third-leaf blade was cut 137 ID cm up from the ligule. After waiting 5 min to check that gutta- tion was not occurring from the cut end, the cut end was bevelled and a l-pmol dose of [2-14C]ethanolamine (l.2 pCi/pmol in experiment 6; 0.74 uCi/pmol in experiment 7) was fed to the underside of the cut end in a 3-ul droplet of K+-phosphate buffer (pH 7, 20 mM). For further details of feeding procedure, see Hanson and Scott (1980). The experiment was terminated 9 h after the radiochemical was applied by detaching the leaf blade plus the uppermost stem por— tion of leaf sheath from the seedling. The leaf tissue was weighed and frozen in liquid N2. 4.2.5 Localization, Extraction and Separation of Labeled Metabolites [Methyl-14C]Betaine used as a "spike" in the root medium was localized in selected seedlings by autoradiography on SB-5 X-ray film (Eastman-Kodak, Rochester, N.Y.). The betaine in selected plants and in each root-feeding solution was confirmed to be undegraded by TLC and TLE analysis using the procedures described in Section 1.2.4. In experiments with [2-14C]ethanolamine, the frozen leaf blade, plus the uppermost 5-cm portion of leaf sheath was ground to a fine powder, boiled in 2 ml of isopropyl alcohol for 5 min, and then stored under N2 at -20°C until further processing. Thereafter, extrac- tion, separation, and determination of 14C-labeled metabolites was essentially by the same ion-exchange/TLC-TLE procedures described by Hitz et al. (1981). The isopropanol phase was removed from the insoluble residue after centrifugation and the residue was then 138 further extracted by grinding with 2 ml chloroform-methanol (2:1, v/v) followed by centrifugation and removal of the solvent. This step was repeated. The chloroform-methanol extracts were combined with the isopropanol extract and partitioned once against CaClz (1.2 ml, 20 mM) and twice against 1.2 ml aliquots from the upper phase from a chloroform:isopropanol:methanol:20 mM CaCl2 (22:16:10:10, v/v) mixture. The washed chloroform—alcohol phase was diluted with 6 ml chloroform and an aqueous layer which separated out was aspirated off. The chloroform phase was dried over anhydrous Na2S04 and then reduced to a small volume (about 0.3 ml) in a N2 stream at 40 to 45°C. For isolation of water-soluble metabolites, the combined aqueous washes from the chloroform-alcohol phase were added back to the remaining isoluble residue and heated to 60°C for about 10 min, centrifuged to clear, and evaporated to dryness in a stream of N2 at about 70°C. The aqueous phase was separated by passage through three ion- exchange columns (1.5 ml each) arranged in sequence; AG-l (OH'), BioRex 7o (H+), and AG-SO (H+). The Ag-50 column was eluted with 6 ml NH40H (4N). AG-50 column eluates were reduced to dryness at about 80°C in a stream of N2. The dried Ag-SO eluate was redissolved in water (100 pl). [14C]Betaine was separated from phosphorylcholine in the AG-50 eluate by TLE [on ITLC (Gelman Instruments), in 1.5 N formic acid at 2kv for 10 min. at 4°C]. Phospholipids in the chloroform-alcohol phase were separated by TLC on Silica Gel-G (Brinkmann) in a one-dimensional, two solvent 139 system. Samples equivalent to 0.25 leaf-segments were applied to TLC plates which were developed first in acetonezpetroleum ether (3:1, v/v) followed by development in chloroform:methanol:glacial acetic acidzwater (85:15:10:3.5, v/v). 14C-labeled metabolites were located on TLC or TLE plates by autoradiography. For betaine the 14C-activity in TLE zones was 14c in TLC determined as described by Hanson and Scott (1980). zones was scraped into scintillation vials and shaken with 0.2 ml chloroformzmethanol:ammonia (65:25:5 v/v) to which 10 ml dioxane based scintillant was then added. 4.2.6 Betaine Determination In some plants of experiments 4 and 5 and in [14C]ethanolamine- labeling experiments the betaine concentration of shoot and leaf tissue was determined by the ion exchange/pyrolysis chromatography method as described by Hitz and Hanson (1980). In the ‘4 C-labeling experiments unlabeled blades comparable to those fed were fresh- and dry-weighed so that betaine concentra- tion as "nmol/g fresh wt" of labeled plants could be converted to "pmol/g dry wt" [dry wt = 9.6 (i0.5)% fresh wt]. 4.2.7 Statistical Analysis of Results Where two treatments were involved,means and standard devia- tions of selected parameters were calculated and compared by Students "t" test. Where 3 or more treatments were compared, significance of results was tested by analysis of variance. 140 4.3 Results 4.3.1 Effects of Exogenous Betaine on Seedling Performance during Stress and Rewatering Observations of the seedlings during and after water stress evaluated the effect cfi’ high internal levels of betaine on the following plant characters: (1) The time wilting first became evident; (2) The extent of leaf firing (the percentage of the total area of the fully expanded leaf blades that was fired); (3) Leaf blade area, or leaf length; (4) Tillering upon rewatering after stress. Unstressed barley seedlings (l7-26 days old) grown in con- trolled environments have shoot betaine levels of about 14 umol/g dry wt; after water is withheld for 7 days levels increase up to about 54 pmol/g dry wt. 4.3.1.1 Stress experiments with seedlings from embryo culture. Seedlings raised from excised embryos were variable in size. The uptake of betaine by these seedlings was also variable (Table 24); as far as possible comparisons were made between groups of seedlings of comparable size. Results from experiments l-3 that used seedlings transplanted into perlite from embryo culture are summarized in Table 24. In each experiment an equal number of untreated plants (controls) were sub- jected to the same culture conditions and stress periods as betaine— treated seedlings. [14C]Betaine label taken up by the treated seed- lings was distributed between root and shoot; in both organs 141 .Amo.c n a. ammh mace: «papa—a: m.cuu::a o» mcwuLOuua acmgouw.n apucuu_m.:m.m m. manguuocu :_:u_3 .muaaugma »:a Low gouge. acmgom»_u a an umxop.ou mm=.o> .mu:uu.u.=m_mico= mouau.u:. m: upc.o u a an oucuu.».cm.m mouuuwvc. *l .m:..ouazac .uumo ace—a Lon mum—.pu uo .ozu .uog.~ we: was. mmom.n wea— nuvcuaxo a..:w mg» Co mace .auou one we ammucoucua «nu moumupvcp was.» none mou4+ .ms om. . om. cmozuma «Lo: vomoas_ no: «macaw ocomoa mac..voom Co mugm.ux use. 3... u .955”. I u v33... £2.33 $22333...» 6: m m “N « mm . a.m. mum .me 8.. u .eeeeeu .ew. . eeeaee. .e=.uee m m.emm. u —cLu:ou .aoo— u umuomLaI—osn mm— "can. u umuomgui—osn a.m. .e=.eee a..e.: H.5. eemee..e.aeem ”3.. eaa.e..ae. c. m. N. a .m c.nm. .meee.a .eeeeee ea..a ; a .aeea we...) muea.a ae.aee.-ae.a.em o. o. aN e we a.m. m..e.3 n ..v u .ogucou .— n umuameu .3...1 nm. .N a eeeaeee-.eea a.m. ...ea.a\aee.... .az .m: m... a .ocucou .—... u uoauugui.o53 mm. .p... u umummcuI—oe: m.m. "ANEuV mace ouopn mimno. .ae.m~ u .ogucou .uo. u nouomcu .oe: mm. .amo a vmuomgui.o31 m.m— .+.u. ems.» amen Com. m mm m. H mm a.mm. .mm:..voom concocui.osn mm. ace .ocueou can. eoea. mane ~ .aeea we...z hue..eeem eauaaee-.eea a.m. m m. . e mm m... m..e.z N am. v u .ocucou .w.~ n umuouch . u:u.axm.~...u .02 .m: am.me u .ocucou .um..m u cognac» "Au“ +vmc.m coco use. .msouaesm u..3 mo uwmco :. mucmsuomcu comrade mmucmcmuupv oz o. ¢. — H mm a.m. m: . acmpa mm m ago—m \.051 um..aa=m a \.oe: acme mama IIIIIIIII awed: m=o_uc>comao .o .oz «saga: va..aa:m -.Lmaxu mo:_auam nouus_umu o=_oumm .wc.oumn um» mace—a mo guess: as. a. .ozam m.ocu:ou we guess: a www.casou 355.33.. :< .moxcaem umfluxm so: .59.... 9.03 35.33 5...: F. 35.5.33... we 3.3.9. .3 .2253 .3 ~35 142 14C-activity was only in the form of betaine [recovery of 14C in the six seedlings analyzed was 91.5 (i 3.5)%]. From these experiments, it was possible to conclude only that betaine was not toxic when supplied externally, and that pre- treatment with betaine tended to delay the first signs of wilting. 4.3.1.2 Stress experiments using plants from intact seed culture. In order to overcome the problems of variability for size and vigor among seedlings grown from embryo culture, intact seeds were used in two later experiments. A preliminary experiment (experi- ment 4) using semi-sterile conditions was completed before an experi- ment using sterile intact seed culture was conducted. In experiment 11 plants (7 replicates) exposed to 80‘umol betaine (27 mM; shoot betaine concentration,109 i 30 pmol/g dry wt) wilted about 24 h ajtgr_the untreated plants (Fig. 26). Pot weights and leaf-3 blade areas were measured at the beginning and end of the stress period. At the end of the stress period, 4 plants were randomly selected and their shoot dry weights, leaf-3 and leaf-4 blade areas were measured (Table 25). The remaining 3 plants in each treatment were rewatered for 2 weeks to observe differences in recovery. At the end of the stress period, betaine-treated plants had a smaller leaf-4 blade area and had lost significantly less water (calculated from differences in pot weights at the beginning and at the end of the stress period; Table 25). These results are consistent, in thatéismaller transpiring suface is likely to be associated with slower soil moisture depletion. Differences in pot weights should Fig. 26. 143 Experiment 4. The effect of betaine pretreatment on the development of wilting symptoms during water-withholding. Photograph shows that the seedlings pretreated with betaine (shoot betaine level before stress ~ 109 pmol/g dry wt) were less severely wilted than untreated controls (shoot betaine level before stress 2 l4 pmol/g dry wt) 5 days after irrigation was stopped. Seedlings were grown from intact seeds using semi-sterile culture condi- tions. 144: 145 TABLE 25. Selected growth characteristics of betaine-treated and untreated barley seedlings, measured after stress in experiments 4 and 5. Experi- Organ Parameter Untreated Betaine ment Treated ** 4 --- Total H 0 lost (9) 97.0 88.0 Total Shoot Dry wei ht (mg) 418.0 409.0 Leaf-3 blade Area (cmz) 13.0 11.2, Leaf-4 blade Area (cm?) 21.3 18.9 Leaf blades: % fired ** l + 2 + 3 + 4 60.0 33.0 Total H 0 lost (9) 115.0 l10.0 5 "' 2 8 6 8 0 Leaf'B bIade 15.6 14.] Leaf-4 blade ’ ' * Leaf blades: % fired 77.0 65.0 * 1 + 2 + 3 + 4 *** ’ Indicates significance at p = 0.05 and p = 0.01, respec- tively. tistically non-significant. Unless indicated, differences between treatments were sta- 146 closely reflect the quantity of water transpired as growth would be minimal during stress. No differences in recovery from stress were evident between treatments. The experiment was repeated (experiment 5), but using sterile conditions when supplying betaine (160 nmol) to the seedling shoots, and imposing a longer stress period. As observed previously, the betaine-treated plants (shoot betaine concentration x 150 pmol/g dry wt) exhibited retarded (z 20 h) wilting as compared to control plants. Although the total water lost (calculated from the difference in pot weights at the beginning and end of the stress period) over the 12 days were not significantly different between the two treatments, the pattern of water loss could account for the delayed signs of stress (Fig. 27). The betaine-treated plants depleted the pot water supply at a slower rate than untreated plants. The leaf blades of betaine-treated plants were smaller than those of untreated plants but the difference was not statistically significant in this experi- ment. The blade of leaf-5 was fully emerged in both betaine-treated and untreated plants. However, during the stress period there was more blade expansion (growth) of leaf-5 in betaine-treated plants. This greater expansion of leaf-5 of betaine-treated plants compared to untreated plants is consistent with the former remaining turgid for a longer period of time as growth is very sensitive to loss of tur- gor (Hsiao et al., 1976). Stress induced leaf-firing (leaves 1, 2,3, and 4)was significantly less in treated than in control plants (Table 25). 147 .aee Lee a:..eeem ece we: ecegh .Aev emeeegue: Lo .0. e:_euea Fee: on. new: eeueecuiece came em; ease .meuee..eeg e. mo:..eeem xe.gee seem m:.e.e;;ewzigeuez we eewgee xeeim. e m:..=e mme. seen: you m:_e_cemee meme e>wueucemeceea .m acespgeexm .mm .m.. 3.63 ms... Nw o w w o v N o _ II. . d - . a u . q _ q _ .Ie AU 1 r H dll... 1 1110: Z i I O . .1 . O r a a .. ¢ on S L. o . o 111. 62.5.2. ”.0 . 6 r 206 5...“. .. . I W .m. 1+ 1 ncdnml— H mu 1 0.? W o .3529. 101 m .. .P 533.... 19.. cures .. ( 39.5.3 _ p p . p . . b . . . . L L 148 One plant, selected randomly from each of experiments 4 and 5 was extracted and analyzed to confirm absence of [14C]betaine meta- bolism. [14C]Betaine was distributed between shoot and root, 4:1. No 14C-label was in any form other than betaine. To determine if betaine-treated plants may have had a smaller leaf area at the beginning of the stress period, the leaf blade areas of leaf-l and leaf-2 were measured in a separate experiment using seedlings grown from intact seeds under sterile culture conditions. The second leaf blades of betaine-treated plants were significantly (p = 0.05) smaller (2 = 5.8 cm2 for 15 replicates) than those of 2 untreated plants (x = 6.6 cm for 11 replicates). 4.3.2 Metabolism of [2-IHC]Ethanolamine The major pathway of betaine biosynthesis that is believed to operate in barley (Hitz et al., 1981) is schematically described in Figure 28. [Minor pathways that may contribute to the synthesis of betaine could involve N-methylation reactions occurring either on the free bases, on nucleotide derivatives of the bases, or on phosphatidyl derivatives of the bases (Hitz et al., 1981). These alternative pathways were not shown in Figure 28.] The point at which the supplied [14C]ethanolamine is likely to join the endogenous precursors to betaine is indicated. The incorporation of 14C-labeliW‘cm1[2-14C]ethanolamine into betaine and phosphatidyl choline (PC) at various internal levels of betaine are given in Table 26. Since the pool sizes of other QAC 1449 .28. ...a S 5.... cat... .23.. e. 2.5.22.6 so... 2:38 .0 33:233.. 9.... c. 30.58 0+ ego—.3 >353 3?... of Co co_+o+comoceoc Stigma < 2:30.. 822.3 2:28 o... .96 1| Ill. 1e81~612+1n6 III ozeiyeiznnze all. Siroifoizhnze . z? n . rm- . n5 I :e I ma 8@. 2:06 .m@. 2.8.22.8 .o n. . o... .05 3.5.335... .COcemoE ..e.+oeamoea 0 me o x1... Slalelfeumzelzfilnxe Aimixilxl 6-alel~..e..~:el=~.. 2e 9 _ = 0 n8 o » ae.5.oeo..$.e.....~. .QN .m.L 150 TABLE 26. Incorporation of 14C-activity (nCi) into betaine and phosphatidylcholine by the second leaf blade of barley seedlings having various internal levels of betaine. T4C-content§ Betaine Experiment Level Betaine PC 3T314% nCi/(blade plus sheath segment) 6 15 4.6 22.0 15 4.6 14.0 22 4.8 16.0 65+ 2.3 13.0 76* 2.9 14.0 95* 1.9 8.3 7 8 4.1 14.5 12 2.3 18.7 15 3.2 10.6 42* 1.8 5.8 48+ 1 5 6.3 63+ 3.1 7.4 54‘r 2.4 6.1 69+ 2.3 6.2 73+ 0 61 3.6 77* 1.9 6.1 85+ 2 3 3.8 §[2-14C]ethanolamine supplied 1 nmol/leaf. Specific radio- activity (nCi/pmol): Experiment A = 1202, B = 737. Incubation = 9 h. +Indicates seedlings treated with 100 pmol betaine. 151 intermediates between ethanolamine and betaine are small (Hitz et al., 1981), PC and betaine are the major products of the betaine bio- synthesis pathway after periods of several hours. Increasing the internal levels of betaine tended to depress synthesis of both [14CJPC and [14C]betaine. The relationship between betaine level and [14C]PC and [14C]betaine synthesis are clearly shown by the scatter- plots in Figure 29. As the slopes of the two regression lines in Figure 29 are similar, it would appear that the rates of synthesis of both PC and betaine were depressed to about the same extent. In view of the general growth inhibitory effect of QACs, this result could be ascribed to betaine causing a general reduction in metabolic activities, in which case the observed effects would be non-specific. However, this trivial explanation seems unlikely, as in the studies of the effect of betaine on growth, only small differences between betaine-treated and untreated seedlings were observed. Such small differences in growth cannot be used to explain the 2- to 3—fold reductions in the rates of [14C]PC and [14C]betaine synthesis. As there is no net synthesis of PC after feeding ethanolamine to turgid barley seedlings (Hitz et al., 1981), the lower incorporation of 14 C-label into PC in the presence of elevated levels of betaine probably reflects a diminished turnover of the base moiety of PC. 4.4 Discussion Exogenous application of betaine led to elevated internal betaine levels, and appeared to retard development of symptoms of water stress, i.e., wilting, in barley seedlings. This phenomenon Fig. 29. 152 Relationship between endogenous betaine level and [14C]ethanolamine (nmol/leaf) conversion to betaine and phosphatidylcholine (PC) when [2-14C]ethanolamine was fed to the third leaf blade of well-watered seedlings. Treated (A) and untreated (A) plants of experiment 6; treated (o) and untreated (0) plants of experiment 7. The relationships could be described by the linear regression equations: Ethanolamine (nmol) incorporated into PC = -O.13 (betaine level) + 17.83; r = O.66.** Ethanolamine (nmol) incorporated into betaine = -.0.03 (betaine level) + 4.33; r = 0.79.** 153 =0.66" f O =05... 2.5mm 2. 352002002. 2.250225 .03. 25.0 . b p o. o. 5 O 20.0 - :65... 0... 2. 352062002. m2..2<..02<2..m.03. 5.0 ” 100 80 60 40 20 BETAINE LEVEL (pmollg dry wt) 154 was apparently due to slower soil-water depletion by betaine-treated plants which, in turn, was probably a result of the latter having a slightly smaller transpiring surface. Observations by Wheeler (l969) on Phaseolus vulgaris lend some support to the notion that betaine may act as a mild growth retardant; betaineinhibited enlargement of leaf disks from E. vulgaris over a 24-h period, although there was no decrease in leaf area when betaine was supplied, via a soil drench, to the intact plant. The relatively large contribution that betaine makes to total nitrogen efflux from the aleurone layer of wheat grains during germina- tion led Chittenden et al. (l978) to suggest that betaine was an important source of nitrogen for the germinating seedling. This is unlikely to be correct because degradation of betaine was not observed during barley grain germination in any of the experiments reported here. An increase in the endogenous betaine concentration of leaf 14C-label from [2-‘4c1- blades tended to depress the incorporation of ethanolamine into both PC and betaine. Accepting that this depres- sive effect is specific to betaine synthesis and does not apply to cellular metabolism in general, one may suggest that the regula- tory steps in betaine accumulation can be located at one or more of the following stages: (1) Choline moiety turnover at the phospholipid level; (2) Choline transfer between water-soluble and lipid inter- mediates; (3) N-methylation. These alternatives are described schematically by the dashed or dotted lines in Figure 30. 155 .1-----------------------. I E C) :3 .. v G . ©Lm®C —>F©__}(HOL INF—HRH. Fig. 30. A schematic diagram indicating the possible regulatory steps in betaine accumulation. A rationalization for the regulatory step in the synthesis of betaine occurring at PC turnover (indicated by the dashed line in Fig. 30) may be formulated thus: It is known that water stress may induce membrane deterioration that could be detrimental to cellu- lar metabolism (Gaff, 1980). Possibly one facet of deterioration in membrane structure is an increase in the turnover rate (i.e., break- down and resynthesis) of PC in membranes. Since betaine has been indicated as having a stabilizing effect on membrane functions, (Rafaeli-Eshkol and Avi-Dor, 1968; Schobert, l977), it can be envisaged that a mechanism for regulating PC turnover is to con- vert a fragment (viz. choline) that is a consequence of undesir- able membrane turnover, to a protective substance (viz. betaine) that, by its very accumulation, cuts off the source of its pre- cursor (i.e., choline from PC breakdown). This explanation has bearing on the physiological significance of betaine accumulation by water-stressed barley. Although it is not reasonable to infer from the results of experiments with well-watered seedlings that the syn- thesis of PC and betaine is subject to retroinhibition in stressed 156 plants grown in the field, results from field studies are consistent with feedback regulation of betaine synthesis in mature, stressed barley (Hitz et al., 1982). Since betaine was administered to the seedlings early in their development, it is reasonable to believe that the exogenous betaine had joined the subcellular site of the endogenous betaine by the time that [14C]ethanolamine was fed. The results thus lend support to the hypothesis that at least some betaine is located in the cytoplasm; it seems unlikely a metabolite stored in the vacuole could interfere with a cyt0plasmic activity, viz. betaine synthesis. Because of the long-term nature of the experiment, it was impossible to distinguish between inhibition of enzyme activity or enzyme syn- thesis as explanation of the observed retroinhibition. CHAPTER V SUBCELLULAR LOCALIZATION 0F [METHYL-14C]BETAINE BY MICROAUTORADIOGRAPHY 5.1 Introduction Betaine has been suggested to be an osmotic effector in both halophytes and mesophytic crops (Wyn Jones, 1979). Data collected in the field for water-stressed barley (cv. Arimar) permit a crude estimation of the potential contribution that betaine accumu- lation could make to the osmotic adjustment in the flag-l leaf blades. The maximum difference between betaine levels in the flag-l leaf blade of irrigated and non-irrigated barley grown in the field was l25 pmol/g dry wt, or about 44 mM on a plant-water basis. The difference in solute potential between non-irrigated and irrigated leaves was about l0 bars. Thus if the accumulated betaine were distributed throughout the cell, its contribution (about l bar) to the drop in solute poten- tial would be relatively small. However, were betaine to be localized in the cytoplasm (assumed to be about l0% of cell volume) betaine accumulation could account for all the dr0p in solute potential. Thus, crucial to the hypothesis that betaine is a significant osmotic effector is that it be localized in the cyt0plasm. Knowledge about the subcellular distribution of enzymes and metabolites is essential to understanding many aspects of cellular 157 158 metabolism (e.g., Emes and Fowler, l979). Three approaches to dis- tinguish between vacuolar and cytoplasmic localization are available: (l) Isolation of intact vacuoles and determining the presence or absence of the metabolite (Heck et al., l981; Leigh et al., 198l; Martinoia, 1980); (2) Histochemical localization of the metabolite using specific staining procedures (Jensen, 1962); (3) Microauto- ratiographic procedures that localize radio-labeled metabolites (Jensen, l962; Evans and Callow, 1978). In order to obtain evidence of the subcellular compartmenta- tion of betaine, intact vacuoles from red beet have been isolated (Wyn Jones et al., 1977a; Leigh et al., 198l). These experiments indicated that the levels of betaine were variable, but were always higher in the cytoplasm than in the vacuole. Both nitrate (Martinoia, 1980) and proteinases (Heck et al., l98l) have been successfully identified as vacuolar constituents in barley by isolating vacuoles from turgid primary leaves. Objections to using the procedure of vacuole isolation to identify the subcellular localization of betaine in barley include: (l) The subcellular localization is of greatest interest in tissues subjected to moderate long-term stress. Vacuoles could not be isolated from such tissues without the superimposition of osmotic shock. (2) Betaine may leak from vacuoles during isola- tion; (3) The potential problem associated with assaying an unrepre- sentative sample of vacuoles due to low yields of vacuoles relative to the fresh weight of whole tissue. Hall et al. (1978) investigated the subcellular distribution of betaine in leaf cells of Suadeda maritima by using a histochemical 159 procedure that was based on the fact that quaternary ammonium com- pounds (QAC) form colored complexes with platinum halogenates. The iodoplatinate solution that was used in their study reacts with any quaternary ammonium compound, e.g., choline (Dierichs and Inczedy— Marcsek, 1976) and thus was not specific to betaine. Although leakage of betaine from the tissues did not appear to be a problem in this histochemical study, it would appear from the aqueous and alcohol incubation procedures used that loss or displacement of betaine can not be completely ignored when interpreting the results. Using cryofixation and freeze substitution procedures, water- and alcohol-soluble 14C-labeled compounds have been successfully localized in leaves and maize kernels at the light-and electron microscope level (Fisher and Housely, 1972; Felker and Shannon, 1980). In an intact barley plant, older leaves are known to export applied [14C]betaine to younger organs (Section 1.3 and 2.3). It is reasonable to assume that the [14C]betaine in the young organ will then enter the pool of endogenous betaine. This study sought to identify the subcellular site of [methle4C]betaine accumulation in the fully expanded younger leaves of barley after supplying [methyl-14C1betaine to the second leaf blade of turgid 3-week old seedlings using microautoradiographic techniques at the light microscope level. 160 5.2 Materials and Methods 5.2.1 Plant Material Barley seed, originally from the USDA Small Grains Collection (Beltsville, Md.) was multiplied in East Lansing, Mi. 5.2.2 Growing Conditions and Stress Regimes In the experiment where [methyl-14C]betaine was fed,seedlings were grown in a soil mix (sand:peat:loam, 2:1:1) in plastic pots (12 cm x 7 cm diam.) in a controlled environment chamber under 16-h days (PAR 5 mW cm-z ; day/night temperature 22/16°C; day/night relative humidity 70/85%). The seedlings (6/pot) were irrigated on alternate days with half-strength Hoagland's solution. One day before the experiment began [19 days after planting,(DAPfl seedlings were thinned 14C]Betaine was to 4 per pot and the seedlings irrigated. [Methyl- fed to the second leaf blade of a seedling 20 DAP and water was then withheld from the plant. After 3 days the plants were rewatered for 4 days. At the end of the rewatering period the potted seedlings were taken to the laboratory for cryofixation of selected organs. In experiments using 14C02, plants were grown in the green- house under supplemental light (16-h day; day/night temperature 22/16°C; relative humidity 70 to 80%) in a soil mix [peatzloamzsand, 1:1:2; fertilized with NH4SO4 (0.5 g) and P205 (0.1 9)] in large, well-drained plastic pipes (l m long x 16 cm diam.). Immediately before seeds (7/pot) were planted, sufficient half-strength Hoagland's solution was applied to the soil to completely fill the profile. 161 Seedlings were irrigated with half-strength Hoagland's solution alternate days until seedlings were at the 2-leaf stage. The seed- lings were then thinned to l per pot and water was withheld. The plants matured and successfully completed grainffill using only stored water. 5.2.3 Labeled Compounds and Mode of Application [Methyl-14C]Betaine (7 pCi, 59 pCi/pmol; prepared from [methyl-14C1choline as described in Appendix A) was fed in a 3 pl-drop- let of K+-phosphate buffer (20 mM, pH 7) to the cut end of the second leaf blade as described in Section 2.2.6 (Hanson and Scott, 1980). Water was withheld from the plant for 4 days. The plant was then rewatered for 2 days after which it was taken from the growth chamber to the laboratory for preparation for microautoradiography. At 64 DAP the flag-l leaf blade was placed in a feeding cham- ber (Section 1.2.3) and exposed for 15 min to 14 C02 (1.25 pCi,~60uCi/ pmol), generated as described in Section 1.2.3. After 3 h, the leaf sheath was cut at the node of insertion, and the leaf was quickly transported on ice from the greenhouse to the laboratory for pre- paration for microautoradiography. 5.2.4 Preparation for Microauto- radiography The procedure used was based on methods reported by Fisher and Housely (1972) and Felker and Shannon (1980). All steps in the procedure described were conducted in a dry box through which a 162 continuous stream of dry N2 was passed (relative humidity < 10%). A l-cm2 segment of the flag-l leaf blade from 14COZ-treated plants, or a l—cm2 segment from the first expanded leaf of the first tiller (defined by Briggs, 1978, p. 17) from the [methyl-14C]betaine treated seedling, was quickly cut into small (about 1 mmz) pieces of tissue which were plunged into liquid Freon-12 held at its freezing point with liquid N2. The frozen specimens were rapidly transferred into vials of cold (< -40°C) propylene oxide (20 m1); propylene oxide was kept cold by placing the vials in en1 acetone/dry ice bath. Prior to use,the propylene oxide was thoroughly dried for 3_two weeks over a molecular sieve (propylene oxidezmolecular seive 5:1 v/v, 8-12 mesh beads; Davison Chemical, Baltimore, Md.). Typically only 4-8 small pieces of tissue were substituted in the same vial to ensure a high ratio of solvent to tissue. The tissue was then left for 3 weeks in a plastic box filled with CaSO4 (Hammond Drierite Co., Xenia, Ohio) at -lOO°C for freeze substitution in propylene oxide. At the end of three weeks the tissue was allowed to warm slowly to room temperature; tissue was kept for periods of 12 h at -49°C, -20°C, -lO°C, -4°C and then in ice at room temperature for 16 h. The slow warm up procedure was a precaution against trapping air bubbles in the tissue. When the vials were at room temperature the propylene oxide used for substitution was changed for fresh,dry lDrOpylene oxide. After a further 2 h in the fresh pr0p1yene oxide at room temperature the tissue was then slowly (10% increments of 163 Spurr's medium into propylene oxide at 45 min intervals) infiltrated with Spurr's resin (Appendix D; Spurr, 1969). The tissue was left for two 8-h periods in 100% Spurr resin before final polymerization overnight at 70°C. Sections (1Lm1th1Ck) were cut with a diamond knife on an ultramicrotome. Sections were fixed to microscope slides which had been pretreated by dipping them in an aqueous solution containing 1% gelatin (w/v) and 1% potassium chromium sulphate (w/v). The sections were transferred to the slides in a drop of ethylene glycol and dried in a desiccator at room temperature. 5.2.5 Microautoradiography and Section Staining Microautoradiographs were prepared by dipping the slides in Kodak NTB-Z (Eastman-Kodak Co., N.Y.) nuclear track emulsion after it was melted at 40°C in a water bath. Slides were stored at 4°C in light-tight boxes. After exposure of 16 days the slides were developed by dipping them sequentially in: Microdol-X developer (7 min; Eastman-Kodak), acetic acid (1%, l min), fixer (7 min; East- man Kodak), H20 (10 min). DevelOper and fixer were prepared according to manufacturer's instructions. Dipping and development of slides was conducted in total darkness. Some sections were stained with toluidine blue [filtrate from toluidine blue (1 9) plus Na borate (l g) in 100 ml H20] and not treated with the emulsion, for use in interpreting the pattern of exposed silver grains on the microautoradiographs. 164 5.3 Results and Discussion Results from autoradiography were interpreted in terms of the hypothesis that betaine might be largely confined to the cytoplasm. Thus, the question asked was: What pr0portion of silver grains present over the vacuolar compartment could not be accounted for by 14C-activity in the cyt0plasm? Tissue integrity was satisfactory after the substituting and 14C-activity leached out of the tissue into embedding procedure; 5 5% the substituting solvent prior to embedding the tissue pieces in resin. The cross-sectional area of cells of the flag-l leaf blade of stressed, mature plants grown in the greenhouse was much smaller (about 150-300 pmz) than that of cells of the seedling leaf blades (about 500-800 pmz). As the track of a 14 C B-particle can be expected to be about 2-3 pm (personal communication, R. Hahn, Eastman-Kodak, N.Y.) with the thickness of emulsion used in this study, the larger cell size of seedlings made it possible to ascribe the silver grains to either vacuolar, cytoplasmic or extracellular compartments; the smaller size of the flag-1 leaf cells precluded such an analysis. The preliminary 14002 feeding experiment served only to confirm that the substituting/embedding/autoradiographic procedures could be completed successfully; the stained sections indicated that a high percentage of cell volume (30-50%) was cytoplasm. Microautoradiographs of sections (Fig. 318) were matched with adjacent sections (Fig. 31A) that had been stained with toluidine blue; 165 thus the cyt0plasm and extra-cellular spaces could be delineated. That part of the total vacuolar area unlikely to contain silver grains exposed by B-particles emitted from [14C]betaine localized in the cyto- plasm was defined by drawing a line 2-3 pm within the internal peri- meter of the cytoplasm. The numbers of grains/unit area were not sig- nificantly different between the areas so defined; 14 C-activity attributable to extracellular spaces was significantly lower (Table 27) which indicates the silver grains over the vacuole are not arti- facts of the procedure used. Thus, in stressed-rewatered seedlings, it appears that betaine is not preferentially localized in the cyto- plasm. Since the seedlings were stressed and rewatered prior to freeze-substitution, the possibility must be recognized that con- tinuously stressed tissues might show a different distribution of betaine. Fig. 30. 166 Photographs of transverse sections through the first leaf of the first tiller of a stressed- rewatered barley plant supplied with [14C]betaine via a leaf on the main culm. (A) stained with toluidine blue. (B) Microauto- radiograph of a section adjacent to that in (A). The epidermal cells on the lower leaf surface on the micro- autoradiograph are folded; a reference point is marked by a large vertical arrow. 14C- -activity was about 2 pCi/ml (Ml a plant water basis. In the lower frame the numerous discrete black particles are the silver grains. For clarity five of such grains (one outside the section, which would be considered background, and four inside the section) are indicated by small arrows. TABLE 27. 168 The number of silver grains/10 pm2 in the vacuolar and cytoplasmic compartments of mesophyll cells and in the surrounding extra-cellular spaces in the first leaf blade of a stressed-rewatered seedling tiller. The grain number described is corrected for a background of 11 grains/10 pm2- Grains were counted on photographs 3; sections; magnification at the microscope level was 0 x. Number of Number grains/10 me/compartment Cells Extra-cellular:Vacuolar:Cytoplasmic 7 O 5 l4 l8 7 12 33 ll 8 17 18 7 4 21 20 4.7 13.8 21.3 = 10.6) CONCLUSION The objectives of this study on the metabolism and accumula- tion of betaine by barley were: (1) To evaluate the hypothesis that the level of betaine in certain organs would reflect the stress history of the plant; (2) To investigate the feasibility of physiological- genetic studies to test the hypothesis that betaine accumulation under water stress was of adaptive value; (3) To obtain evidence bearing on the adaptive value of betaine accumulation in barley. Studies with seedlings, grown in controlled environments, and mature plants, grown in the field, demonstrated that betaine was an inert metabolic end product that accumulated via translocation and _i_n situ synthesis in the youngest vegetative organs. In seedlings, betaine was shown to be transported in the phloem. In field-grown plants, betaine accumulated in the flag and flag-l leaves and during grain filling did not partition either like other nitrogenous com- pounds or like dry matter. The developing spikes of field-grown plants were not strong sinks for [14C]betaine applied to flag leaves; this was in accordance with results from laboratory experiments on phloem translocation in mature plants. Betaine could represent up to 10% of the total-N in NI, N-fertilized vegetative shoot tissue at grain-maturity. Low soil-N depressed betaine accumulation in total shoot tissue but did not significantly affect betaine 169 170 accumulation in the youngest leaves. The betaine level in the young- est mature leaves was significantly correlated with an integrated value, based on w measurements, of the stress experienced by the plant prior to the measurement of betaine. The accumulation of betaine in field grown plants occurred slowly during the growing season at a time when the plant was making morphological changes in response to the stress; betaine accumulation is thus not a short- term shock response. Genetic variability for betaine accumulation was found among genotypes of H. vulgare and H. spontaneum in trials with seedlings grown in controlled environments, and among cultivars of H. vulgare grown under non-irrigated conditions in the field. The range in betaine levels among seedlings grown in controlled envirbnments was 3-4 fold under well-watered conditions, and 2-3 fold under water- stressed conditions. In the field, the difference among cultivars for maximum levels of betaine in the flag-l leaf blades was about 4—fold; apparently betaine levels were not related to the instantane- ous values of us. The extent of variability identified in this study was only barely sufficient for physiological-genetic studies, but was large enough to encourage one to believe that greater variability exists among species within the primary gene pool of cultivated barley. The relatively limited extent of betaine accumulation by water-stressed, field-grown barley and the indications of genetic variability for its levels under well-watered and non-irrigated conditions make the betaine level of any shoot organ of barley 171 unsatisfactory as a practical index of the cumulative stress expe- rienced by a crop. Under conditions of a limited water supply pretreatment of seedlings with exogenous betaine tended to delay wilting which was a result of a reduction in the rate of soil-water depletion. This phenomenon was ascribed to betaine causing a slight reduction in leaf area. When [2-14C]ethanolamine was applied to the second leaf blade of well—watered barley seedlings, high, but physiological, internal levels of betaine tended to depress the synthesis of [14C]- betaine and its precursor, [14C]phosphatidyl choline. These results suggested that a feedback regulatory system in betaine synthesis operates in well-watered seedlings. This observation led to specula- tion on the physiological significance of betaine accumulation; possi- bly betaine accumulation mitigates an undesirable acceleration of PC turnover, which may be an aspect of membrane deterioration, during stress. A reduction in the rate of betaine synthesis that was con- sistent with retroinhibition was noted in studies with NI, field- grown barley (Hitz et al., 1981). Results from autoradiographic studies at the light microsc0pe level showed that betaine was distributed about equally between the cytoplasm and the vacuole of mesophyll cells of turgid barley leaves. As betaine represents up to 4.5% of total-N in metabolically active, water—stressed leaf blades, but does not serve as a source of either re-usable nitrogen or energy, it is difficult to believe that 172 betaine accumulation is an incidental response to stress. The slow, but progressive, accumulation of betaine throughout a droughty growing season, the site of betaine accumulation (viz. in the young- est mature leaves which are most important to grain fill), and the effect of exogenous betaine on growth and regulation of PC turnover, are circumstantial evidence consistent with the notion that betaine accumulation has adaptive value. However, there remains the possi- bility that betaine accumulation is an 'evolutionary relic', in that its function is not recognized because it is no longer needed in the conditions in which barley is grown today. APPENDICES 173 APPENDIX A SYNTHESIS OF [METHYL-3H]BETAINE, [METHYL-14CJBETAINE, AND [METHYL-14CJBETAINE ALDEHYDE 174 APPENDIX A SYNTHESIS OF [METHYL-3HJBETAINE, [METHYL-14CJBETAINE, AND [METHYL-14CJBETAINE ALDEHYDE A.l Introduction The oxidation of choline to betaine proceeds via the aldehyde (Fig. A-l). [Methyl-14C]Betaine was prepared chemically and [methyl-14C]betaine aldehyde was biosynthesized using a rat liver mitochondrial preparation. CH3 CH3 CH I l I 3 + + + CH3 — ii — CH2 — CHZOH —0 CH3 — 1'1 —CH2—CH0 —0 CH3 — T — CH2 — COOH CH3 CH3 CH3 Figure A-l. Oxidation of Choline. A.2 Materials and Methods A.2.1 Synthesis of [Methyl-3H1 Betaine and [Methyl-14C]- Betaine (after Lintzel and Fomin, 1931) [Methyl-14C]Choline chloride (100-200 pCi, 59 uCi/pmol) or [methyl-3H]choline chloride (1 mCi, 15 mCi/mmol), KMnO4 (1 mg) and cone. H2S04 (2 pl) in water (200 pl) were mixed throughly and heated for l h in a boiling water bath. The solution was centrifuged for 5 minutes; 175 176 the supernatant was carefully removed and the pellet washed 3 times with warm water (3 x 0.5 ml). The final pellet was discarded and all the supernatants were combined and passed in sequence through 3 columns (each of 1.5 m1 volume): AG-l (OH'), BioRex 7o (H+), AG-SO (H1). The AG-SO column was eluted with ammonium hydroxide (4N, 8 m1) and the eluate dried at about 80°C under a stream of N2. The eluate was redissolved in water. Radiochemical purity of [methyl-14C]betaine (>95%) was con- firmed by the TLC and TLE systems described in Section 1.2.4. The percent recovery of label in betaine was 74-90% when [methy-14C] choline was the precursor and 45% when [methyl-3H]choline was the precursor. Specific radioactivity has taken as that of the original 14 [14c1choiine (59 pCi/pmol for c; 15 mCi/mmol for 3H). A.2.2 Synthesis of [Methyl-14CJBetaine Aldehyde A.2.2.l Preparation of mitochondria following a similar procedure to Williams (1952). The liver of a freshly killed male rat (Sprague-Dawley strain) was weighed, chilled, cut into small pieces and added to ice cold isotonic sucrose solution (0.25M; 50 ml). The preparation was homogenized for 3 min in a Virtis Hi-Speed "23" Homogenizer (Virtis Co., Gardiner, N.Y.). The suspension was then poured into an ice-cold Potter homogenizer with a loose fitting pestle and further gently homogenized until the liver tissue was no longer discernible. The resulting brei was centrifuged at 600 g (10 min) in a refrigerated centrifuge. The 600 g supernatant was then 177 centrifuged at 8500-9000 9 (10 min). This 8500-9000 9 supernatant was discarded and the pellet was rinsed twice in ice-cold sucrose solution (0.25 M, 2 x 20 ml), and re-suspended in 7.0 ml ice cold sucrose solution (0.251%);the protein concentration of the final solution was estimated by Bradford's reagent (Bradford, 1976). The suspension was further centrifuged at 8500 g and the pellet resuspended in sufficient ice cold K+-phosphate buffer (25 mM; pH 7.8) to give a concentration of 8.3 mg protein/ml. During the preparation of the mitochondria all vessels and media were maintained at approximately 2-4°C. A.2.2.2 Oxidation of choline following a similaréprocedure to Kensler and Langemann (1951). The reaction mix comprised [methyl-14C]choline (100 pCi, 59 pCi/pmol; Amersham Corp., 111.), CaCl2 (1 mM), cytochrome-C (1.6 x 10'5 M; Horse heart-type XIII acid modified; Sigma Chemical Co., Mo.) and mitochondria (2.5 mg protein) in 300 pl K+-phosphate buffer (25 mM; pH 7.8). The preparation was sparged with 02 and incubated for 2 h at 37°C. The reaction mix was then centrifuged and the supernatant passed sequentially through three columns (each of 5 ml volume): AG-l (OH'), BioRex 70 (H+), AG-SO (H+). The BioRex 70 column was eluted with HCl (0.5 N, 40 ml). This eluate was evaporated to dryness overnight under an infra-red lamp and ventilated by a fan, and then redissolved in water (150 pl). Betaine aldehyde was separated from choline by TLE on Whatman #3MM paper (27 x 5 cm strips) in 1.5 N formic acid at 2 kv for 20 min. 178 After the band corresponding to betaine aldehyde was localized by autoradiography (l-h exposure) on SB-5 X-ray film (Eastman Kodak, N.Y.) it was cut out and the betaine aldehyde eluted from the paper with water (4 ml) overnight. The elute was dried down under a stream of N2 (~60°C) and redissolved in water (1 ml). Radiochemical purity (>95%) was confirmed by the TLC and TLE systems described in Section 1.2.4. The percent recovery of label in [14C]betaine aldehyde was 48%. There are negligible amounts of free betaine aldehyde in rat liver mitochondria (De Ridder and Van Dam, 1973). The choline in the mitochondrial preparation was estimated to be §_160 nmol (Colbeau et al., 1971), thus the specific activity of the [methyl-14C]betaine aldehyde was taken to be between 54 to 59 pCi/pmol. APPENDIX B WILD AND CULTIVATED BARLEY GENOTYPES SCREENED IN LABORATORY AND FIELD TRIALS 179 APPENDIX B WILD AND CULTIVATED BARLEY GENOTYPES SCREENED IN LABORATORY AND FIELD TRIALS For laboratory screening trials, genotypes of H, vulgare and .H. spontaneum are entered by CI (H, vulgare) or PI number, or where no such numbers have been assigned, by provenance (H, spontaneum). Number and provenance designations were made by the USDA Small Grains Collection (Beltsville, Md.). The average Shoot betaine level (3 replicates) of irrigated and water-stressed seedlings are given. In field trials cultivars of H. vulgare are entered by CI number. Average betaine level and ms of the youngest mature leaf, or flag-1 leaf, for each cultivar at each sampling date are indicated. 180 181 TABLE B.l Betaine level in shoots of irrigated and water-stressed seedlings of genotypes included in Laboratory Trial A. Betaine Level Genotype Irrigated Water-Stressed MM 11 mol/g dry wt 3625 16 46 6566 21 42 6939 19 40 7055 18 45 7243 18 51 8093 15 53 10083 21 55 10146 12 41 10149 14 45 10501 19 40 10870 15 53 10975 13 45 10979 12 47 10996 18 52 11009 15 49 11037 16 46 11806 19 49 13626 26 51 15289 12 48 15480 14 42 PI Number 284742 19 67 284749 23 61 296912 25 54 391071 24 56 391074 22 51 391080 25 40 391081 19 45 391124 18 55 391131 19 49 391133 21 46 391136 25 47 391126 21 48 Provenance Mt. Meron Israel 24 57 Mt. Meron Israel 23 49 Mt. Meron Israel 29 59 Mt. Meron Israel 25 56 182 TABLE B-l Continued. Betaine Level Genotype Irrigated Water-Stressed pmol/g dry wt Mt. Hermon Israel 25 56 Mt. Hermon Israel 17 50 Mt. Hermon Israel 19 44 Mt. Hermon Israel 19 57 Bor Mashash Israel 19 45 Bor Mashash Israel 20 49 Bor Mashash Israel 19 44 Bor Mashash Israel 22 43 Bor Mashash Israel 22 49 Wadi Qilt Israel 25 53 Wadi Qilt Israel 25 45 183 TABLE B.2 Betaine level in shoots of irrigated and water stressed seedlings of genotypes included in laboratory Trial 8. Betaine Level Genotype Irrigated Water-Stressed CI Number pmollg dry Wt 3455 25 67 9201 23 52 10161 25 62 10180 34 82 11806 18 51 13060 29 57 13626 34 65 PI Number 211041 38 84 212305 35 89 220341 38 87 220523 27 71 220664 36 66 227019 36 70 235639 39 86 244776 40 74 244777 31 76 244778 40 79 245739 31 67 249983 24 62 253573 32 66 254894 39 80 268244 34 66 282572 29 59 282576 32 77 282577 34 74 282595 32 72 282592 37 67 282616 23 53 296910 28 60 361678 24 56 370759 31 74 354929 36 72 354934 27 69 354936 31 62 354938 32 75 354941 35 63 354947 25 61 354949 29 68 391000 34 66 184 TABLE B.2 Continued. Betaine Level PI Number Irrigated Water-Stressed pmol/g dry wt-—-————— 391001 30 72 391002 31 60 391003 27 56 391007 31 58 391009 32 62 391012 36 68 391013 33 61 391014 24 59 391015 29 60 391019 20 57 185 TABLE B.3 Betaine level in shoots of irrigated and water-stressed seedlings of genotypes included in laboratory Trial C. Betaine Level CI Number Irrigated Water-Stressed pmol/g dry wt 709 25 51 742 31 84 1114 25 76 1610 36 78 1624 22 59 1641 26 65 2318 22 54 2380 33 62 3480 33 82 5199 21 56 6576 28 61 6577 34 81 6952 32 71 7341 24 73 8044 24 64 8834 30 67 9203 30 62 9309 19 51 9366 30 63 10064 18 54 10138 18 53 10152 23 63 10171 21 70 10333 21 57 10359 26 53 10371 34 60 10500 29 65 10807 20 64 10840 30 67 11197 27 67 11211 26 63 11271 25 59 11622 29 58 11719 27 70 11806 18 54 11832 26 65 11961 27 81 12083 31 66 12456 32 73 13057 23 53 13452 29 68 13626 35 84 TABLE 8.3 Continued. 186 Betaine Level CI Number Irrigated Water-Stressed pmollg dry wt 14935 30 66 14936 34 76 15293 19 55 366465 21 55 382211 39 69 382673 31 62 187 TABLE B.4 Betaine concentration and solute potential (05) of the youngest mature leaf, or flag-l leaf, for thirteen cultivars of H. vulgare at three dates during the growing season (1981) Days after Planting CI Number Betaine Level ws 41 59 63 41 59 63 ———pmol/g dry wt-——~— -————— (-) bars 709 60 64 64 16 19 20 1114 51 86 66 16 20 18 2318 67 93 110 15 18 22 3480 72 87 96 18 19 20 6577 54 92 84 17 17 19 9309 57 68 62 14 20 19 10064 50 59 68 13 15 18 10138 53 60 116 16 18 21 10359 56 65 71 17 20 19 11961 51 76 72 17 21 19 13057 39 36 37 15 18 18 13626 70 107 62 16 20 19 14936 76 104 110 14 18 19 APPENDIX C MEDIA USED IN STERILE EMBRYO AND INTACT SEED CULTURE 188 APPENDIX C MEDIA USED IN STERILE EMBRYO AND INTACT-SEED CULTURE TABLE C.l B-5 medium (Gamborg and Wetter, 1975). B 5 Major g/l Medium§ KNO3 2.5 (NH4)2 504 0.13 M9504 - 7H20 0.25 CaCl2 - 2 H20 0.15 Sucrose 20 B 5 Minor mg/l Medium MnS04fl . H20 100 H3803 30 Na2M004 - 2H20 2.5 CuSO4 - 5 H20 0.25 CoCl2 . 6 H20 0.25 KI 7.5 189 190 TABLE C.l Continued. NaZEDTA 37.4 FeSO4 - 7 H20 27.8 Thiamine HCl 10'0 Pyridoxine HCl 1.0 Nicotinic acid 1.0 Inositol 100.0 §Adjust complete medium to pH 6 with KOH and HCl. 191 TABLE C.2 Murashige and Skoog medium (Murashige and Skoog, 1962) § Major Elements mg/l Medium NH4NO3 1650 KNO3 1900 MgSO4 - 7 H20 370 KH2P04 170 Nae - EDTA 37.3 FeSO4 - 7 H20 27.8 Minor Elements H3803 6.2 MnSO4 - 4 H20 22.3 ZnSO4 - 4 H20 8.6 KI 0.83 Na2M004 - 2 H20 0.25 CuSO4 - 5 H20 0.025 CoClZ - 6 H2 0.025 Nicotinic Acid 0.5 Pyridoxine HCl 0.5 Thiamine HCl 0.1 Glycine 2.0 Myo-Inositol 100.0 g/l medium Sucrose 30.0 §CompIete medium adjusted to pH 6.0 with KOH and HCl. 192 TABLE C.3 Modified Wick medium (Steidl, 1976).- MAJOR MINERALS g/liter Medium§ NaZSO4 0.8 Ca(N03) - 4H20 0.58 Mgso4 - 7 H20+ 0.33 KNO3 0.08 KCl 0.005 MICRONUTRIENTS mg/liter medium MnSO4 0.45 ZnSO4 .6 H3803 .00375 KI .03 Glycine 0 Thiamine HCl OOONOQ’OOO _a Ca-pantothenate 5 C0504 . 5 H20 .025 NaMoO4 .025 Sequestrene (sodium ferric # diethylenetriamine pentaacetate) 16.0 g/liter medium Sucrose 20.0 §Adjust complete medium to pH 5.4 with KOH and HCl. +Dissolve separately. 3+ #Sequestrene, 13% Fe in form of NaFe , Ciba Geigy. 193 TABLE C.4. Hoagland's medium (full strength). Minerals mg/l Medium§ Major Ca(N03)2. . 4H20 590.0 Sequestrene (NaFe 13%) 77.0 KH2P04 68.4 KNO3 253.3 Mgso4 - 7H20 125.5 Minor ZnSO4 ° 7 H20 0.1 MnSO4 ° H20 0.8 -5 H3803 1.5 -5 M003 ° 2H20 1 x 10 §Adjust complete medium to pH 5.2 with KOH and H01. 194 TABLE C.5 Tests for bacterial contamination Media Constituents g/l Media i Yeast extract 10 Bacto - Peptone 20 119804 - 7 H20 20 Agar 10 ii Bacto-tryptone 10 NaCl 5 D-glucose 10 Agar 15 pH 7 iii Bacto-tryptone lO Yeast extract 5 NaCl 10 Agar 15 APPENDIX D RESIN FORMULA USED IN MICROAUTO- RADIOGRAPHIC STUDIES 195 TABLE D.l. Composition of Spurr's resin used for microautoradio- graphic studies in Chapter I (Spurr, 1969). Component§ ;;:mu§;$5tic 9 NSA (Nonenyl succinic anhydride) 26.0 ERL-4206 (Vinyl cyclohexene dioxide) 10.0 DER-4206 (Diglycidyl ether of polypropylene glycol) 6.0 DMAE (Dimethylaminoethanol) 0.4 §The constituents must be weighed into a beaker for mixing in the order described in the table. 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