In“! ~ AA‘ .f _‘ A «I “ “‘“N LEERAWY Rfiefitigan flit-”ate University l ,3 ——v— T Hig‘s w—v— ‘- This is to certify that the dissertation entitled The adaptive significance of glycinebetaine accumulation in water or salt stressed barley. presented by Rebecca Grumet has been accepted towards fulfillment of the requirements for PILn- degree in We Mag/6am Date Aj‘v‘f 2/;35 MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 ‘}V1ESI.J RETURNING MATERIALS: Place in book drop to LJBRARJES remove this checkout from w your record. FINES will be charged if BEER—is returned after the date stamped below. THE ADAPTIVE SIGNIFICANCE OF GLYCINEBETAINE ACCUMULATION IN WATER- OR SALT-STRESSED BARLEY BY Rebecca Grumet A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1985 ABSTRACT THE ADAPTIVE SIGNIFICANCE OF GLYCINEBETAINE ACCUMULATION IN WATER- OR SALT-STRESSED BARLEY BY Rebecca Grumet Betaine (glycinebetaine) accumulates in certain plants during osmotic stress and may be central to cytoplasmic osmoregulation. To test the effect of altered betaine levels on response to stress, I deve10ped two barley (Hordeum vulgare L.) isopopulations differing in betaine content. First, inheritance of betaine level was studied. Neither maternal nor cytoplasmic effects were significant for four pairs of reciprocal crosses. General combining ability accounted for 74.6% of the genetic effects from an incomplete 13 X 13 diallel. Generation mean analysis showed that betaine level was a predominantly additive trait. Narrow-sense heritabililty values were 0.53 and 0.63 from midparent-offspring regression and generation mean analysis, respectively. Since the betaine trait in barley is nuclear and highly heritable, an iSOpOpulation approach was possible. To minimize linkage effects the isopopulation procedure included several parents and two rounds of crossing. The two isOpOpulations had significantly different betaine Rebecca Grumet levels in unstressed conditions (22.8 vs. 32.4 umol°g-l dry weight). Several morphological and developmental characters indicated that the populations were otherwise comparable. When the isopopulations were salinized, 300 mM NaCl caused an 8-fold increase in betaine and a 5 bar drOp in solute potential (we). The absolute difference in betaine between the populations did not change with salinization. Although selected only for betaine level, high betaine isopopulations and parents maintained a 1 bar lower ws at all salt levels. Betaine level was perfectly coordinated with $8 (r2=0.99); all genotypes fell on the same line. These observations are readily explained if: betaine accumulation is a mandatory component of osmoregulation in barley, and osmoregulation as a whole is under genetic control. . The isopopulations were compared for response to water deficits in greenhouse trials. The low betaine-high ws population had a higher rate of leaf production, and in optimal environments accumulated up to 352 more above-ground dry matter. When water stressed, the dry matter differences disappeared. The populations did not differ for water use efficiency or assimilate partitioning to the leaves. Although selection for high betaine-low ws produced a more stable population with respect to water stress (regression response technique, b-O.84), growth in Optimal environments was reduced. ACKNOWLEDGMENTS I would like to thank my former and current committee members, Dr. James Asher, Dr. Robert Herner, Dr. Amy Iezzoni, Dr. Tom Isleib, and Dr. James Hancock for their encouragement, assistance, and advice. I especially want to thank the chairman of my committee, Dr. David Dilley, for his constant support and many efforts on my behalf. Most of all, I must thank my major professor, Dr. Andrew Hanson, for all his help - it would not be possible to have had a better or more concerned advisor. . Thanks also go to Dr. C. Cress for statistical advice, Elliot Light and the greenhouse staff for their helpfulness, Barbara Meier and Kurt Stepnitz for graphics and photography, Beckie Oswalt for thesis assembly, and the peOple in the lab over the years for their helpfulness and camaraderie, and for making it so pleasant to be there every day. Finally, I would like to thank Katrina Cornish and Jocelyn Ozga whose friendship has meant so much to me, and most of all, I thank Jim for his love. ii LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION TABLE OF CONTENTS Osmoregulation and Compatible Solutes . . . Osmoregulation in Plants . . . Evidence that Betaine is a Compatible, Cytoplasmic Osmoticum . . . . . Potential Agricultural Significance of Betaine Accumulation . . . . . . . . . Testing Adaptive Value Using a Physiological- Genetic Approach . . . . . . . Literature Cited CHAPTER I GENETIC CONTROL OF GLYCINEBETAINE LEVEL 1.1 Abstract 0 O O O O O O O O O O O O O O O O 102 IntrOduction e o o o o o o o o o o o o o o 1.3 Materials and Methods . . . . . . . . . . . 1.3.1 Selection of Parental Genotypes . . 1.3.2 Plant culture 0 O O O O O O I O O 0 1.3.3 Betaine Assay O O C O O O O O O O 0 1.3.4 Test for Maternal and Cytoplasmic Inheritance . . . . . . . . 1.3.5 Partial Diallel Cross and Analysis . 1.3.6 Generation Mean Analysis . . . . . . 1.3.7 Heritability Estimates . . . . . . . 1.4 Results 0 O O O O O O O O O O O O O O O O 0 1.4.1 Maternal and CytOplasmic Inheritance 1.4.2 Partial Diallel Analysis - Estimation Of GCA and SCA O O I O O I O O O O 0 1.4.3 Generation Mean Analysis- Estimation of Genetic Effects . . . . . . . . . 1.4.4 Heritability Estimates . . . . . . . iii page vi vii viii 10 12 15 20 21 23 24 24 25 27 27 28 29 29 29 31 34 35 1.6 Discussion . . . . . . . . . . . . Literature Cited . . . . . . . . . CHAPTER II GENETIC EVIDENCE FOR AN OSMOREGULATORY FUNCTION OF GLYCINEBETAINE ACCUMULATION IN BARLEY AbstraCt O O O O O O O O O O O O 0 Introduction . . . . . . . . . . . Materials and Methods . . . . . . . 2.3.1 Isopopulation Development . 2.3.2 Salt Stress Experiments with Isopopulations and Parent Mixes 2.3.3 Characterization of Individual Parental Genotypes . . . . . 2.3.4 Statistical Analysis . . . . . 2.3.5. Exogenous Betaine Experiment . Results . . . . . . . . . . . . . . . 2.4.1 Characterization of the Parents 2.4.2 ISOpOpulation Development . . 2.4.3 Responses of the IsopOpulations to Salt Stress . . . . . . . . Discussion . . . . . . . . . . . . Literature Cited . . . . . . . . . CHAPTER III GROWTH STUDIES OF TWO BARLEY ISOPOPULATIONS DIFFERING IN GLYCINEBETAINE LEVEL AND OSMOREGULATION Abstract . . . . . . . . . . . . . Introduction . . . . . . . . . . . Materials and Methods . . . . . . . Results and Discussion . . . . . . Literature Cited . . . . . . . . . iv page 38 41 43 44 46 46 SO 53 53 54 55 55 6O 66 68 75 78 79 80 82 94 page DISCUSSION What type of variability was found and at what level did selection act? . . . . . . . . . . . . 96 Was the type of variability found constrained by the method of selection? . . . . . . . . . . . 99 Are there inherent limitations of natural variability for physiological-genetic studies? . 101 Literature Cited 0 I O O O O O O O O O O O O O O 103 APPENDIX C O O O O O O O O O O O O O O O O O O O O O O 104 Table LIST OF TABLES Betaine content of four pairs of reciprocal F1 crosses and their parents. Analysis of variance and orthogonal com- parisons for betaine content of four pairs of reciprocal crosses and their parents. Analysis of variance for GCA and SCA of betaine content and relative contributions of GCA and SCA effects. Generation mean analysis of Proctor, Arimar, and their F1’ F2, and two backcross generations for betaine content. Mean values : standard deviations for several characteristics of the selected F3 families forming the isOpopulations. Effect of applied betaine on unstressed barley plants. Average plant weight at mid-anthesis (43 DAP) for the well-watered iso- populations in competitive and non- competitive plantings. vi page 30 32 35 37 67 69 86_ Figure 10 11 12 LIST OF FIGURES Betaine levels (umol°g-1 dry wt) of the 13 parents and 68 F1 hybrids used in the partial diallel. Distribution of betaine content in Proctor, Arimar, and their F1, F2, and two backcross generations. The modified isopopulation development procedure. Betaine levels (A), solute potentials (B), and mg dry weight/g fresh weight (C) for the 13 parents used to initiate the iSOpOpulations. Growth responses: fresh weight (A,B), dry weight (C,D) and mg dry weight/g fresh weight (E,F), of the parent mixes and iSOpOpulations to increasing salt. Osmotic potentials of the parent mixes (A) and isopopulations (B) in response to increasing salt. Betaine level of the parent mixes (A) and isopOpulations (B) in response to salt stress. Distribution of betaine levels during the iSOpOpulation deve10pment procedure. Betaine level vs. solute potential for the isOpOpulations and parent mixes (inset). Yield at mid-anthesis (total above ground dry matter) of the high betaine-low w pOpulation or low betaine-high W papalation vs. pot mean yield. 8 Rate of leaf production for the two isopopulations in a competitive fall planting. Possible relationship of observed differences between the isopopulations. vii page 33 36 48 57 59 61 62 64 71 84 88 98 AOV BC .CI CV DAP df GCA LSD PPFD rep R.H. RWC SCA SD SE SS USDA WUE uE LIST OF ABBREVIATIONS analysis of variance backcross cereal introduction cultivar days after planting degrees of freedom general combining ability heritability least significant difference probability level photosynthetic photon flux density replicate relative humidity relative water content specific combining ability standard deviation standard error sum of squares United States Department of Agriculture water use efficiency microEinsteins solute potential viii INTRODUCTION Betaine (glycinebetaine) accumulates in several plant species in response to water- or salt-stress and is hypothesized to be central to osmoregulation by acting as a compatible, cytOplasmic solute (Wyn Jones et al., 1977). A good deal of circumstantial evidence supports this view (Wyn Jones and Storey, 1981; Hanson and Hitz, 1982), but the effect of differing levels lfiflilfl has never been tested directly. For this dissertation I set out to test the effect of genetically altered betaine level on the response of barley plants to water- or salt-stress by creating two iSOpOpulations differing primarily in betaine level. Osmoregulation and Compatible Salutes. Osmoregulation, the active accumulation of solutes within the cell in response to a reduction in external water potential, is an important adaptive response that enables an organism to establish equilibrium between the osmotic strength of the cell and that of the environment (Brown, 1964; Yancey et al., 1982). The solutes accumulated may either be salts that are readily absorbed from the environment, organic solutes that are synthesized in response to osmotic stress, or some combination of the two (Flowers et al., 1977; Jeffries, 1981). Because proteins are susceptible to conformational changes in response to ionic influences, most macromolecular function is extremely sensitive to the cellular ionic environment (Yancey et al., 1982). From an evolutionary perspective there are two possible adaptive responses to the conflicting demands of osmotic equilibrium and biochemical activity (Yancey and Somero, 1979): (a) The evolution of enzymes capable of functioning in high salt conditions, or (b) the evolution of a concentrated intracellular environment that is compatible with biochemical activity. Examples of both sorts of adaptations exist in nature, but the latter is more common. The evolution of different sets of enzymes seems to be limited to the extremely halophilic bacteria (Yancey et al., 1982). When various citrate cycle enzymes were isolated from several bacterial species and studied igfvitro, they were found to have NaCl Optima of 1-4 M (Brown, 1964). This is in direct contrast to inhibition by NaCl at concentrations greater than 200 mM for enzymes from most other organisms (Flowers at al., 1977; Brown, 1964). Amino acid sequence data showed there to be extensive substitution in the enzymes from the haIOphilic bacteria (Brown, 1964). Thus these bacteria have evolved a rather drastic form of adaptation requiring the modification of hundreds, or possibly thousands, of proteins (Yancey et al., 1979). Alteration of the cellular ionic environment is more commonly observed. In most systems the enzymes from salt-tolerant and salt-sensitive species do not differ iE-vitro (Flowers et al., 1977). Many species actively exclude inorganic salts and, instead, achieve osmotic balance with low molecular weight, compatible, organic solutes (Munns et al., 1982; Flowers et al., 1977). The concept of compatible solutes was introduced by Brown and Simpson (1972) to describe molecules that protect enzymes against inhibition in conditions of low water potential. In fact, there is evidence to suggest that there has been stringent evolutionary selection for the solute composition of cells (Jeffries, 1981; Yancey et al., 1979). Throughout nature the major osmolytes for water- or salt-stressed eukaryotes have been restricted to a few classes of low molecular weight organic compounds including: polyols, reducing sugars, amino acids, urea“ and methylamines (Jeffries, 1981; Yancey et al., 1979). Timasheff et a1. (1982) studied the relation of protein hydration and structural stability and found that all structure-stabilizing solutes were preferentially excluded from the domain of the protein molecule. Protein self-association was promoted by molecules such as sucrose, glycerol, hexylene glycol, and amino acids and was linked to preferential hydration of the protein. The Opposite was true for denaturants, these all interacted preferentially with functional groups of the proteins and induced unfolding. The accumulation of urea in many salt-water fish is surprising because it is a destabilizing solute, but Yancey and Somero (1979) found that it was always accompanied by the accumulation of stabilizing methylamines and amino acids in a constant 2:1 ratio (urea: methylamines and amino acids). At these relative concentrations the methylamines and amino acids were found to counteract the destabilizing effect of urea on the melting temperature of ribonuclease and the renaturation of lactate dehydrogenase. This implies that both the type and relative concentrations of solutes are important aspects of osmoregulation. Osmoregulation in Plants. In the past decade a good deal of experimental work with higher plants has shown osmoregulation to be a mechanism of adaptation to both drought- and salt-stress (Hsiao et al., 1976; Turner and Jones, 1980; Morgan, 1984). The water relations of a plant cell may be summarized as ww - $8 + up, where ww’ w and up, are water, solute, and 8, turgor potentials, respectively (Boyer, 1985). Since the water potential of pure water is defined to be zero and water moves down a water potential gradient, ww’ and $8, are negative terms; vp is positive. A drop in solute potential caused by the accumulation of solutes will promote water movement into the cell. This process may enable a plant to maintain turgor, and thereby growth, in environments of low external water potential (Hsiao et al., 1976; Turner and Jones, 1980; Morgan, 1984a). To maintain turgor, any solute may be used, and studies have shown that despite known toxicity, Na+ ions can be major contributors to decreased solute potential in several plant species (Munns et al., 1982). However, reports comparing enzymes isolated from salt-tolerant and salt-sensitive plant species have failed to show inevitro differences among the enzymes. Greenway and Osmond (1972b) studied malate dehydrogenase, aspartate transaminase, glucose-6-phosphate dehydrogenase, and isocitrate dehydrogenase from the halophytes, Atriplex spongiosa and Salicornia australis and from the glycophyte Phaseolus vulgaris. NaCl concentrations in excess of 200 mM were inhibitory to enzymes from all three species; the sensitivity existed whether or not the plants had been growing in high salt conditions. Mannitol, however, did not inhibit the enzymes and so the inhibition was not attributable to low osmotic potential per se. Similarly, Ben Amotz and Avron (1972) studied the halophilic alga Dunaliella which accumulates high levels of NaCl within the cell and has a high NaCl requirement for $271$12 photosynthesis. The requirement for high salt, however, was not observed for photosynthesis and enzyme activity infli££25 in fact, salt was inhibitory. They further reported that the requirement for salt 32-1132 could be replaced by glucose or glycine indicating that the NaCl had a non-specific osmoregulatory function. The apparent contradiction of high internal salt levels and the inhibitory effects of NaCl on enzyme function led both groups of investigators to suggest that intracellular compartmentation may play a important role in osmoregulation for plants. Mature plant cells are highly vacuolated and so it is possible that potentially toxic levels of inorganic ions may be tolerated by sequestration in the vacuole (Flowers at al., 1977; Hellebust, 1976). Several experimental approaches have indicated non-uniform distribution of ions within the cell. X-ray dispersive spectra (Jeschke and Stelter, 1976) and electron probe x-ray microanalysis (Pitman et al., 1981) indicated preferential accumulation of Na in the vacoule. The K/Na ratio was approximately 1 in the vacuole and 10 in the cytoplasm. X-ray microanalysis of the intertidal alga Porphyra umbilicalis also showed Na levels to be low in the cytOplasm and high in the vacuoles (Wiencke, et al., 1983). The differential distribution of ions within the cell presumably requires active transport across the tonoplast (Jeffries, 1981; Kirst and Bisson, 1982). The tonOplast is unable to withstand hydrostatic pressure differences (Jeffries, 1981) and so it is necessary to maintain osmotic balance between the vacuolar and cytOplasmic compartments. This led Wyn Jones et a1. (1977) to suggest that in plant cells, compatible solutes serve as cytoplasmic osmolytes and thereby maintain both cytoplasmic, biochemical function, and osmotic balance within the cell. Wyn Jones and Gorham (1983) note that many low molecular weight organic compounds that accumulate in osmotically,stressed plant cells (e.g. glycerol, proline, and betaine), are not present in sufficient quantity to be osmotically important unless they are selectively accumulated within the cytoplasm. Thus plant cells may utilize both inorganic and organic solutes for osmoregulation (Munns et al., 1982). The ability to tolerate large quantities of Na+ within the cell varies among species and is thought to be one differentiating feature between halophytes and glycophytes (Greenway and Munns, 1980; Munns et al., 1982). Other solutes that can make a major contribution to osmoregulation in expanded leaves include sugars, sugar alcohols, amino acids, organic acids, K+ and Cl- (Morgan, 1984; Ford and Wilson, 1981). In summary, the importance of osmoregulation for all forms of life is increasingly recognized (Strom et al., 1983). It should be noted, though, that direct evidence for a biological role of individual compatible solutes in plant or animal cells is very limited; evidence has yet to be' produced verifying that accumulation of these compounds is adaptive and not a reflection of impaired metabolism (Strom et al., 1983; Yancey et al., 1982; Hanson and Grumet, 1985). Strom et al. (1983) emphasize that hard experimental evidence for the function of these compounds in plant cells should be of highest priority. Evidence that Betaine is a Compatible, Cytoplasmic Osmoticum. Quaternary ammonium compounds such as betaine are found in many microbial, plant, and animal species (Mackay et al., 1984; Yancey et al., 1982; Wyn Jones and Storey, 1981). Within the plant kingdom betaine accumulates in members of the Gramineae, Chenopodiaceae, Amaranthaceae, and Compositae in response to water- or salt-stress (Wyn Jones and Storey, 1981). Betaine is hypothesized to function as a compatible, cytoplasmic osmoticum. Ecological and physiological evidence supports an osmoregulatory role for betaine in plants: the species that accumulate the most betaine are generally halophytes (Wyn Jones and Storey, 1981) the amount of betaine accumulated is directly prOportional to the external stress level (Hanson and Wyse, 1982; Coughlan and Wyn Jones 1982; Storey and Wyn Jones, 1978) and steady accumulation of betaine occurs in barley and trapical pasture grasses during long-term water deficits in the field (Hitz et a1” 1982; Ford and Wilson, 1981). To determine whether or not betaine is compatible with biochemical function, many investigators have tested the effect of betaine on enzymatic activity and various metabolic processes infilitrg. Betaine at 0.5 M was shown not to inhibit the activity of malate dehydrogenase, glutamate oxaloacetate transaminase, and pyruvate kinase (Pollard and Wyn Jones, 1979). Paleg et al. (1981) found that thermal inactivation of several enzymes was reduced in a concentration dependent manner if the enzymes were heated in the presence of betaine. Paleg et a1. (1984) also tested the effect of betaine on polyethylene glycol induced precipitation of glutamine synthase and found the effectiveness of 1 M betaine in inhibiting precipitation to vary with pH. Nash et al. (1982) studied the effects of betaine on the thermostability of enzymes in intact, isolated organelles. They reported that at physiological levels, (20 mM) there was no effect, but at 0.5 M betaine, heat inactivation of NAD isocitrate dehydrogenase and malate dehydrogenase was retarded in intact mitochondria. This level of betaine had no inhibitory effect on these enzymes from lysed mitochondria. High betaine concentrations ( 0.5 or 1 M) were also found to be compatible with inevitro polysome stabililty (Brady et al., 1984) and protein synthesis (Gibson et al., 1984). Larkum and Wyn Jones (1979) found betaine to be superior to sorbitol for the stability of isolated 10 chloroplasts. A range of betaine levels (10 - 500 mM) also had protective effects on bacteria growing in high salt conditions (LeRudulier and Valentine, 1982). Thus betaine has the properties of a compatible solute. Support for a cytOplasmic location of betaine in plants comes from electron microsc0py studies (Hall et al., 1978) and studies with isolated vacuoles (Leigh et. a1, 1981) which indicate a disproportionate amount of betaine is located in the cytoplasm relative to the vacuole. All of this evidence is consistent with the hypothesis that betaine functions as a compatible, cytoplasmic osmoticum; it accumulates during stress, is probably localized in the cytoplasm, and is non-toxic. It has been suggested that betaine accumulation is an adaptive response to osmotic stress (Wyn Jones et al., 1977). It should be noted, though, that this evidence is circumstantial; there is not yet direct evidence for the function of betaine in plant cells $272l22’ nor has the effect of directly altering physiological levels of betaine ig-vivo been examined. Potential Aggicultural Signficance of Betaine Accumulation. The possibility that increased betaine level is an adaptive response to water- or salt-stress has agricultural implications. Lack of water and/or poor quality water are major factors limiting crap productivity throughout the world (Boyer, 1982). Forty percent of the land surface is 11 estimated to be arid or semi-arid (Fisher and Turner, 1978), and additional acreage is being continually lost to salinization as a result of high fertilizer use and poor quality irrigation water (Flowers at al., 1977). But if world food production is to continue to meet expanding demand, it will almost certainly be necessary to cultivate increasingly marginal land (Lewis and Christiansen, 1981). Although the need to breed for stress-resistant craps has long been recognized (Gauss, 1910), it is becoming increasingly important. A focus of plant breeders, physiologists, and more recently molecular biologists, has been to identify specific traits that confer stress resistance. The goal is to enhance their expression in crop plants, or transfer them to other cultivars or species (Blum, 1979; LeRedulier, et al., 1984; Quarrie, 1980; Rick, 1978). Betaine accumulation is one stress resistance candidate that has been investigated in recent years (LeRudulier et al., 1984; Hanson and Grumet, 1985; Wyn Jones and Gorham, 1983). A potentially adaptive biochemical trait such as betaine accumulation is of particular interest because metabolic traits are probably the most suitable for emerging molecular genetic technologies (Hanson et al., 1985; Hanson and Grumet, 1985). On the other hand, metabolic traits have not yet been exploited to any great extent. This may, in large part, be due to the difficulty in extrapolating from changes on a 12 biochemical level to effects on whole plant growth, and ultimately, crop performance (Hanson and Grumet, 1985). As Reitz (1974) cautions, many traits have been correlated with plant stress responses, but it is a long step from correlation to establishing a determining influence in stress resistance. Furthermore, traits that confer stress resistance, or enhance survival, are not necessarily the same as those that increase yield (Atsmon, 1973). With regard to betaine accumulation, many important questions remain unanswered. For example: will increasing the level of betaine in a crop species that normally accumulates moderate amounts of betaine enhance stress resistance? Will introducing betaine into a species that does not normally accumulate this compound increase its stress resistance? Will altering betaine levels independently of other aspects of osmoregulation affect the solute balance within the cell? Will increasing betaine have an effect on productivity in favorable environments? Testing Adaptive Value Using a Physiological-Genetic Approach. One of the best methods to begin to answer the above sorts of questions is to ”measure the worth” of a trait by isogenic analysis (Eslick and Hockett, 1974; Reitz, 1974; Quarrie, 1980). The aim is to produce genotypes that differ in known genetic ways for use in physiological studies. 13 This sort of approach has recently been used by Morgan (1984b) to study osmoregulation and Quarrie and Henderson (1982) to study the role of ABA in drought resistance. I therefore set out to test the effect of altered betaine levels using a physiological-genetic approach. I utilized‘naturally occurring variability for betaine content (Ladyman et al., 1983) to develop two barley isopopulations that differed in betaine level but were otherwise genetically comparable. Because betaine accumulation is a metabolic trait and possibly overshadowed by other morphological or phenological characters that confer stress resistance, special care was taken when developing the isOpOpulations to minimize potentially confounding effects of linkage. The two populations were studied for growth patterns and osmoregulatory behavior in well-watered, salt-stressed and water-stressed environments. In assessing the usefulness of a potentially adaptive trait, it is also important to determine inheritance of the trait (Townley-Smith and Hurd, 1979; Blum, 1979). In conjunction with isopOpulation development, I also performed genetic analyses of betaine level in barley. The dissertation is divided into three chapters: (1) genetic control of glycinebetaine level in barley, (2) genetic evidence for an osmoregulatory function of glycinebetaine accumulation, and (3) growth studies of two 14 barley isopopulations differing in betaine level and osmoregulation. 15 Literature Cited Atsmon, D. 1973. Breeding for drought resistance in field craps. p. 157-176. In R. Moav (ed.) Agricultural Genetics: Selected T0pics. John Wiley and Sons, New York. Ben-Amotz, A. and M. Avron. 1972. Photosynthetic activities of the ha10philic alga Dunaliella parva. Plant Physiol. 49:240-243. Blum, A. 1979. Genetic improvement of drought resistance in crap plants. A case for sorghum. p.429-445. In H. Mussel and R. C. Staples (ed.) Stress Physiology in Crop Plants. John Wiley and Sons, New York. Boyer, J.S. 1982. Plant productivity and environment. Science 218:443-448. Boyer, J.S. 1985. Water transport. Ann. Rev. Plant Physiol. 36:473-516. . Brady, C.J., T.S. Gibson, E.W.R. 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Betaine accumulation: metabolic pathways and genetics. p.71-92. In J.L. Key and T. Kosuge (ed.) Cellular and Molecular Biology of Plant Stress. UCLA Symposia on Cellular and Molecular Biology. Vol. 22. Alan R. Liss, New York. Hanson, A.D. and W.D. Hitz. 1982. Metabolic responses of meSOphytes to plant water deficits. Ann. Rev. Plant PhYBiOlo 333163-203. Hanson, A.D., N.E. Hoffman and C. Samper. 1985. Identifying and manipulating metabolic stress resistance traits. Hort Science (in press) ’ Hanson, A.D. and R. Wyse. 1982. Biosynthesis, translocation, and accumulation of betaine in sugarbeet and its progenitors in relation to salinity. Plant Physiol. 70:1191-1198. ‘Hellebust, J.A. 1976. Osmoregulation. Ann. Rev. Plant Physiol. 27:485-505. Hitz, W.D., J.A.R. Ladyman and A.D. Hanson. 1982. Betaine synthesis and accumulation in barley during field water stress. Crop Sci. 22:47-54. Hsiao, T.C., E. Acevedo, E. Fereres, D.W. Henderson. 1976. Stress metabolism. Water stress, growth, and osmotic adjustment. Phil. Trans. R. Soc. Lond. Ser. B. 273:479-500. . H1 3L 17 Jeffries, R.L. 1981. Osmotic adjustment and the response of halaphytic plants to salinity. BioScience 31:42-46. Jeschke, W.D. and W. Stelter. 1976. Measurement of longitudinal ion profiles in single roots of Hordeum and Atriplex by use of flameless atomic absorption spectroscapy. Planta 128:107-112. Rirst, 6.0. and M.A. Bisson. 1982. Vacuolar and cytoplasmic pH, ion composition, and turgor pressure in Lamprothamnium as a function of external pH. Planta 155:287-295. Ladyman, J.A.R., R.M. Ditz, R. Grumet and A.D. Hanson. 1983. Genotypic variation for glycinebetaine accumulation by cultivated and wild barley in relation to water stress. Crop Sci. 23:465-468. Larkum, A.W.D. and R.G. Wyn Jones. 1979. Carbon dioxide fixation by chlorOplasts isolated in glycinebetaine. Planta 145:393-394. Leigh, R.A., N. Ahmad and R.G. Wyn Jones. 1981. Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153:34-41. LeRudulier, D., A.R. Strom, A.M. Dandekar, L.T. Smith, and R.G. Valentine. 1984. Molecular biology of osmoregulation. Science 224:1064-1068. LeRudulier, D. and R.G. Valentine. 1982. Genetic engineering in agriculture: osmoregulation. TIBS 7:431-433. Lewis, C.F. and M.N. Christiansen. 1981. Breeding plants for stress environments. p.151-178. 13 K.J. Frey (ed.) Plant Breeding 11. Iowa State Univ. Press. Ames, Iowa. Mackay, M.A., R.S. Norton and L.J. Borowitzka. 1984. Organic osmoregulatory solutes in cyanobacteria. J. Gen. Microbiol. 130:2177-2192. Morgan, J.M. 1984a. Osmoregulation and water stress in higher plants. Ann. Rev. Plant Physiol. 35:299-319. Morgan, J.M. 1984b. Osmoregulation as a selection criterion for drought tolerance in wheat. Aust. J. Agric. Res. 34:607-614. Munns, R., H. Greenway and G.O. Rirst. 1982. Halotolerant eukaryotes. p.59-135. lg O.E. Lange, C.B. Osmond, H Zeiger (ed.) Vol. 12C. Physiological Plant Ecology. Encyclopedia of Plant Physiology. Springer-Verlag, Heidelberg. 18 Nash, D., L.G. Paleg and J.T. Wiskich. 1982. Effect of proline, betaine and some other solutes on the heat stability of mitochondrial enzymes. Aust. J. Plant Physiol. 9:47-57. Paleg, L.G., T.J. Douglas, A. van Daal, D.B. Keech. 1981. Praline, betaine and other organic solutes protect enzymes against heat inactivation. Aust. J. Plant Physiol. 8:107-114. Paleg, L.G., G.R. Stewart, and J.W. Bradbeer. 1984. Praline and glycinebetaine influence protein salvation. Plant PhYSiOlo 79 :974’978 e Pitman, M.G., A. Lauchli and R. Stelzer. 1981. Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis. Plant Physiol. 68:673-679. Pollard, A. and R.G. Wyn Jones. 1979. Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta 144:291-298. Quarrie, S.A. 1980. Cereal yields and drought resistance. Nature 285:612-613. Quarrie, S.A. and I.E. Henderson. 1982. Biparental inheritance of drought-induced accumulation of abscisic acid in wheat and pearl millet. Annals of Botany 49:265-269. Rick, C.M. 1978. The tomato. Sci. Amer. 239:76-87. Reitz, L.P. 1974. Breeding for more efficient water use - is it real or a mirage? Agric. Meterol. 14:3-11. Storey, R. and R.G. Wyn Jones. 1978. Salt stress and comparative physiology in the Gramineae. III. Effect of salinity upon ion relations and glycinebetaine and proline levels in Spartina townsendii. Aust. J. Plant Physiol. 5 3817-8290 Strom, A.R., D. LeRudulier, M.W. Jacowec, R.C. Bunnell, and R.C. Valentine. 1983. Osmoregulatory genes and osmoprotective compounds. p.39-59. In T. Rosuge, C.P. Meredith, and A. Hallaender (ed.) Genetic Engineering of Plants - An Agricultural Perspective. Plenum Press, New York. 19 Timasheff, S.N., T. Arakawa, H. Inoue, K. Gekko, M.J. Gorbunoff, J.C. Lee, G.C. Na, E.P. Pittz, and V. Prakesh. 1982. The role of salvation in protein structure stability and unfolding. p.48-50. In F. Franks and S.F. Mathias (ed.) Biophysics of Water. John Wiley and Sons, New York. Turner, N.C. and M.M. Jones. 1980. Turgor maintenance by osmotic adjustment. p.87-104. In N.C. Turner and P.J. Kramer (ed.) Adaptation of Plants to Water and High Temperature Stress. John Wiley and Sons, New York. Townley-Smith, T.F. and E.A. Hurd. 1979. Testing and selecting for drought resistance in wheat. p.447-464 In H. Mussel and R.C. Staples (ed.) Stress Physiology in Crop Plants. John Wiley and Sons, New York. Wiencke, C., R. Stelzer, and A. Lauchli. 1983. Ion compartmentation in Porphyra umbilicalis determined by electron probe x-ray microanalysis. Planta 159:336-341. Wyn Jones, R.G. and J. Gorham. 1983. Aspects of salt and drought tolerance in higher plants. p.355-370. In T. Kosuge, C.P. Meredith and A. Hollaender. (ed.) Genetic Engineering of Plants - An Agricultural Perspective. Plenum Press, New York. Wyn Jones, R.G. and R. Storey. 1981. Betaines. p.171-204. In L.G. Paleg and D. ASpinall (ed.) Physiology and Biochemistry of Drought Resistance. Academic Press, Sydney. Wyn Jones, R.G., R. Storey, R.A. Leigh, N. Ahmad and A. Pollard. 1977. A hypothesis on cytoplasmic osmoregulation. p.126-136. 13 E. Marre and O. Ciferri (ed.) Regulation of cell membrane activities in plants. Elsevier Press, Amsterdam. Yancey, P.H., M.E. Clark, S.C. Hand, R.D. Bowlus and G.N. Somero. 1982. Living with water stress: evolution of osmolyte systems. Science 217:1212-1222. Yancey, P.H. and G.N. Somero. 1979. Counteraction of urea destabilization of protein structure by methylamine osmoregulatory compounds in Elasmobranch fish. Biochem J. 183:317-323. CHAPTER I GENETIC CONTROL OF GLYCINEBETAINE LEVEL IN BARLEY 1.1 Abstract The accumulation of betaine (glycinebetaine, N,N,N-trimethylglycine) in water- or salt-stressed barley (Hordeum vulgare L.) plants may be a metabolic adaptation to stress. We investigated the inheritance of betaine level using genotypes varying in betaine content. Shoots of unstressed plants grown in growth chambers were assayed for betaine at the three-leaf stage. Analyses of four pairs of reciprocal crosses indicated that neither maternal nor cytoplasmic effects were significant but that there may be some dominance of the law betaine trait. An incomplete diallel made with eight low- and five high-betaine parents indicated that general combining ability accounted for the majority (74.6%) of genetic effects, suggesting a largely additive trait. Additive, dominance, and epistatic components of heritability were estimated by generation mean analysis of the parental, F1, F2, and backcross 20 21 generations of a single high- X law-betaine cross. This analysis also showed that betaine accumulation was a predominantly additive trait. Narrow-sense heritability values for the trait were 0.53 and 0.63, from mid-parent- offspring regression and generation mean analysis, respectively. The level of betaine accumulation in barley is a nuclear, predominantly additive trait of relatively high narrow-sense heritability and so evaluation of the adaptive significance of betaine in water- and salt-stressed plants using an isopopulation approach should be possible. 1.2 Introduction Betaine (glycinebetaine, NLNLN-trimethylglycine) accumulates in chenopods and several grasses in response to water- or salt-stress. In recent years, much circumstantial physiological and biochemical evidence has supported the view that this is a metabolic adaptation to stress (Wyn Jones and .Storey, 1981; Hanson and Hitz, 1982). The amount of betaine accumulated by various salt-tolerant plants is proportional to the external salt concentration (Hanson and Wyse, 1982; Coughlan and Wyn Jones, 1980; Storey and Wyn Jones, 1978, 1979), and steady accumulation of betaine occurs during long-term water deficits in the field with barley (Hordeum vulgare L.) and tropical pasture grasses (Hitz et al., 1982; Ford and Wilson, 1981). Wyn Jones et al. (1977) have proposedthat betaine acts as a non-toxic, and possibly 22 protective, cytoplasmic osmoticum in stressed plants. Some experimental evidence exists for a predominantly cytoplasmic location of betaine (Hall et al., 1978; Leigh et al., 1981) and for protective effects of betaine in Xl££2 (Larkum and Wyn Jones, 1979; Pollard and Wyn Jones, 1979; Jolivet et al., 1982). Moreover, applied betaine protected various bacteria against the inhibitory effects of high salt concentrations (Strom et al., 1983). Although these observations are consistent with an adaptive role for betaine during water- and salt-stress, they do not eliminate the possibility that betaine accumulation in plants is the result of stress injury. To test the adaptive value of stress-induced betaine accumulation, and to evaluate the possibility of breeding for high betaine levels, it is desirable to develop genetically comparable lines differing in betaine content. Ladyman et al. (1983) showed significant genetic variability for betaine content among barley genotypes. Accordingly, we are developing barley isop0pulations differing primarily in betaine content for evaluation under water- and salt-stress. Although stress conditions cause a large increase (3-10 fold) in betaine content, the relative betaine contents of a range of°genotypes were similar whether measured in young, well-watered plants grown in controlled environments or in older plants under water-stress conditions in the field (Ladyman et al., 1983). It can therefore be considered valid to evaluate the genetic potential for betaine accumulation by testing young, 23 unstressed plants grown in controlled conditions. To determine the suitability Of a trait for physiological-genetic study, as well as its potential usefulness in plant breeding, knowing its made of inheritance is useful. In this study three questions were asked about the betaine trait as measured in young, unstressed plants: Are the genes controlling betaine level nuclear or cytoplasmic? Are the genetic effects additive, dominant, or epistatic? How heritable is the trait? 1.3 Materials and Methods Four experiments were used to answer the questions above. (1) The significance of maternal and cytoplasmic effects was tested using four pairs of reciprocal high- X low-betaine F1 crosses and their parents. (2) A partial diallel cross of 13 parents (eight low-betaine accumulators and five high-betaine accumulators) was made, and relative contributions of general (GCA) vs. specific combining abilities (SCA) estimated. (3) Generation mean analysis was performed to determine additive, dominance, and epistatic components of gene action using a high-betaine parent, a low-betaine parent, the F1, the F2, and two backcross generations. (4) Heritability estimates were made using midparent-offspring regression, ratios of genetic variances, and the separation of environmental from genetic variance among sets of diverse genotypes. ‘24 1.3.1 Selection of Parental Genotypes Selection of barley genotypes used as parents for the inheritance studies was based on the following criteria: (1) high- or low-betaine content as measured under water-stressed and nonstressed conditions in the growth chamber and field (Ladyman et al., 1983), (2) compatibility of flowering dates for crossing, and (3) ease of working with cultivated (rather than wild) accessions. [Accessions of both Hordeum vulgare and its wild relative, H. spontaneum C. Koch, were evaluated (Ladyman et al., 1983); since the range in betaine content for both groups was very similar, H. vulgare accessions were chosen]. Seeds were obtained from the USDA Small Grains Collection (Beltsville, Md.); all were spring barleys. Cereal Introduction (CI) numbers of the low-betaine genotypes were: 709, 5199, 9309, 10064, 11806 (cv. 'Proctor'), 12456, 13057, and 15293; the high-betaine genotypes were 3480, 6577, 10138, 13626 (cv. 'Arimar'), and 14936. 1.3.2 Plant Culture Seeds were stratified on moist filter paper in the dark at 4 C for 1 week; they were then kept in darkness at room temperature for 1.day to allow radicle emergence before planting in a mix of peat, sand, and loam (1:2:1). The plants for crossing were grown in the greenhouse and watered with half-strength Hoagland's solution. Powdery mildew (Erzsiphe graminis DC. f. sp. hordei Em. Marshal) was controlled with sulfur pots, aphids (Macrasipnum avenae 25 Fabricius) by occasional spraying with Pirimore [2-(dimethylamino)-5,6-dimethyl-4-pyrimidinyl dimethylcarbamate, 502 wettable powder, ICI Americas Inc., Wilmington, Del]. Plants analyzed for betaine content were grown in the soil mix given above in controlled environment chambers (16-hour day, 21 C, R.H. 702; 8-hour night, 16 C, R.H. 852) and were watered daily with half-strength Hoagland's solution. 1.3.3 Betaine Assay Shoots were harvested 14-17 days after planting, frozen in liquid nitrogen, freeze-dried, and ground in a Wiley Mill to mesh size 40. Betaine content of the shoots (umol betaine°g dry wt‘l) was measured using a modification of the spectrophotometric assay of Wall et al. (1960); an ion exchange step was added to eliminate interfering compounds, and the periodide precipitate was rinsed to lower blank readings. Accurately weighed 30-50 mg subsamples were extracted in'5 ml of water for 30 min or 1 h at 100 C. A 4-m1 portion of the aqueous leaf extract (or 4 m1 of water containing 50-600 ug betaine standards) was shaken with 0.2 ml of a slurry of the H+ form of AG-50 resin (Bio-Rad Laboratories, Richmond, CA; 2 vol settled resin: 1 vol water) to bind betaine and other cations. The supernatant was aspirated off the settled resin and discarded, and the resin was washed twice with 2 ml of water. Betaine was eluted from the resin with 3 ml 4N NH4OH, which was drawn off and evaporated to dryness under an infrared lamp in a 26 stream of air. The dried eluate was taken up in 0.6 ml of 1 N H2304 of which 0.5 ml was transferred to a 1-ml vial (ReactiVials, Pierce Chemical Company, Rockford, IL) and cooled to 4 C.‘ Then 0.2 ml of cold KI-12 reagent (Wall et al., 1960) was added, and the contents of the vial were mixed and left overnight at 4 C to allow crystallization of the periodide. The vials were centrifuged at 4 C in a clinical centrifuge for 5 min, the supernatant was drawn off using a Pasteur pipet with a very fine tip, taking care not to disturb the pellet. The pellet was then rinsed with 0.2 m1 cold 1 N H2804, recentrifuged at 4 C, and the supernatant was drawn off as above. The periodide in the pellet was dissolved by agitating with 0.2 ml dichloroethane; in plant samples, a light brown residue remained after the periodide had dissolved. A 30 ul aliquot of the periodide solution was diluted with a further 4 ml dichloroethane, and absorbance at 365 nm was read. With standards, the relationship between absorbance (y) and betaine (x) was linear in the range 50-600 ug (typical standard curve with triplicate samples: y - 0.0019x - 0.039, r2 - 0.98), and spikes of betaine (200 ug) added to barley shoot samples were recovered quantitatively. The spectrophotometric assay (y) was checked against the pyrolysis - gas chromatography assay (x) (Hitz and Hanson, 1980) by using both methods to analyze 10 barley shoot samples spanning a range of betaine levels; agreement between methods was very good (y - 1.28x - 6.47, 1'2 - 0099). 27 1.3.4 Test for Maternal and Cytoplasmic Inheritance Four pairs of reciprocal Fl crosses were made using CI 3480 and CI 14936 (high betaine) and CI 13057 and CI 15293 (low betaine) as parents in a high vs. low mating design. The eight F1 hybrids and their four parents were grown in a randomized, complete block design with nine replicates; each block was a 20.5-cm pot divided into 12 sections. Reciprocal differences were tested using orthogonal contrasts. Degree of dominance was assigned to the betaine trait if the F1 was significantly different from the midparent value (LSD0.05); partial dominance was assigned if the F1 value was intermediate between the low parent and midparent value, complete dominance was assigned if the F1 was not significantly different from the low parent. 1.3.5 Partial Diallel Cross and Analysis Crosses were made using all 13 selected genotypes (8 low in betaine, 5 high) as parents. Seeds were obtained for 68 of the 78 possible F1 combinations (Fig. 1). Reciprocal crosses were not included. The 13 parents and 68 F1 hybrids were grown for betaine analysis in a 9 X 9 triple lattice design. Each block was a 16-cm pot containing nine entries. Adjusted means from the analysis of variance of the lattice design were used to partition the adjusted genotypic SS into components for GCA and SCA. The partitioning was performed according to Griffing's (1956) Model I, method 2, using multiple regression. The SCA effects for missing F1 28 hybrids were set equal to zero for purposes of analysis. (Estimates and t-tests of SCA effects for other crosses showed this to be reasonable.) The relative contribution of GCA to total genetic variation was calculated as the ratio of SS GCA to SS entries. A similar statistic was calculated for SCA. 1.3.6 Generation Mean Analysis The F1, F2, and two backcross generations of a cross between CI 11806 'Proctor' (P, low betaine) and CI 13626 'Arimar' (A, high betaine) were made. The F1 population tested for betaine content included equal numbers of P X A and A X P; the F2 and backcross generations were derived from P X A F1 plants. Because the large, segregating generations could not be blocked, pots were rotated within the growth chamber daily. The generations were grown in four groups at different times in the growth chamber: parents with Fl's, parents with Fz's, parents with the backcross to Proctor, and parents with the backcross to Arimar. Generation mean analysis was performed using weighted least squares regression (Mather and Jinks, 1977; Rowe and Alexander, 1980). The adequacy of models containing environmental, additive, dominance, epistatic, and genotype X environmental interaction effects was evaluated using chi-square tests. [r—- g: 31 ti 29 1.3.7 Heritability Estimates Narrow sense heritability was estimated using the midparent-offspring regression value for the 68 F1 hybrids (Falconer, 1981). Narrow-sense heritability was also calculated from generation variances as h - [20%2 - (ogcl + ' agcz )l/o%2 using the generations derived from the Proctor X Arimar cross (Warner, 1952). Broad-sense heritability was estimated by separating genetic from environmental variation in replicated trials. The data used for this estimate were those reported by Ladyman et al. (1983) for 145 diverse Ha vulgare and H. spontaneum genotypes entered in replicated screening trials. The plants were grouped into three experiments, each containing 49 genotypes; check plants of Proctor and Arimar were included in each trial. Estimates of genetic and environmental components of variance were calculated for each experiment using the expected mean square for treatments [MS(T) - a: + rag] and the expected mean square for error [MS(E) -o: ]. Heritability was calculated 2 as h - 02/(0 + 02) (Allard, 1960). 8 8 e 1.4 Results 1.4.1 Maternal and Cytoplasmic Inheritance Comparisons among means for betaine content of the genotypes used in the maternal and cytoplasmic inheritance study are listed in Table 1. The analysis of variance of these data showed highly significant differences for betaine 30 Table 1. Betaine content of four pairs of reciprocal F1 crosses and their parents Genotype Betaine contentl u mol'g dry wt"1 Parents CI 3480 (A) 31.2 a CI 14936 (B) 28.4 a CI 13057 (C) 19.2 bc CI 15293 (D) 14.3 d El crosses Midparent value low parent A X C 16.2 cd complete 25.2 C X A 17.1 bcd complete A X D 18.5 .bc partial 22.8 D X A 20.8 b none B X C 18.1 bcd complete 23.8 C X B 16.9 bcd complete B X D 17.0 bcd complete 21.4 D X B 19.2 bc none +Each value is the mean of 9 replicates, SE - 1.45. LSD0.05- 4.08 LSD0.01' 5.42. Means followed by the same letters are not significantly different by LSD0.05. f: 31 content among genotypes (Table 2). The division of genotypic variance into orthogonal contrasts indicated that the I majority of variation was due to the difference between the high- and low-betaine parents. When the members of the four pairs of reciprocal crosses were contrasted with each other, no significant differences were noted, nor was a significant difference detected when the set of F1 crosses with a high betaine female parent was contrasted with those with a low-betaine female parent. However, the F1 values, were generally lower than would be predicted from midparent values (Table 1). 1.4.2 Partial Diallel Analysis - Estimation of GCA and SCA Betaine contents of the 13 parents and their 68 F1 hybrids are shown in Figure 1. The range of betaine levels in the hybrids (range - 9.5 to 26.8 u mol°g dry wt'l) resembled that of the parents (range - 9.1 to 28.9 u mol'g dry wt‘l). High-betaine X high-betaine crosses generally gave high Fl's and low X low crosses generally gave low Fl's, although there were cases where the F1 means were significantly different from their midparent values (Fig. 1). Two of the low- X low-betaine crosses were higher than their respective midparent values (LSDO.05). When those Fl's and their parents were retested in a separate replicated trial for possible complementation, no significant differences were found among the parental and F1 genotypes. Significant variation for betaine content was detected 32 Table 2. Analysis of variance and orthogonal comparisons for betaine content of four pairs of reciprocal Fl crosses and their parents. Source df Mean squares Genotype 11 225.1** High- vs. low parents 1 1501.5** (A x c) vs. (c x 10* 1 4.3 (A X D) vs. (D X A) 1 24.5 (B X C) vs. (C X B) 1 6.1 (B X D) vs. (D X B) 1 21.6 High females vs. low females 1 21.1 Error 87 19.0 **Significant at the 0.01 probability level. +Parents A,B,C, and D were CI nos. 3480, 14936, 13057 and 15293, respectively. Parents A and B are high in betaine content, C and D are low (Table 1). 33 . 43183893588822 mmcufiamugflmfiuo AEETNHQ—d 3§§om 435mg .3... 358.. 5.8.825 so... 8.. as“! 22. 22...... 2.52.22. at-.. 0 P 50¢ 0.0“ . o 3 fir 3.3333: 320... 33. and... o6... " . 2.3 7.2. so... owes. . .352. so a. * 2:23 0523 suing 2:23 @533 33... “00°F?” 0 . o o P o P o 3.02.. sateen. s; o. co. _ 3!... a... w o... o... .d. 2:. Knesset... 3. a... tu. am. noon. seesaw. .o. «a. 2:7: as on a: some a... 11.2%.... n... o. as .._ as no v .. 33. o... *4... 92—...«6 a... o6. s... 33.13. 0.... a... ad. 3.7.... «.6. ..~.. 5.: a... a... A. 3.0 PM... 3.. a... as. 3.7.... 3... .6. 3...— o.o._sq.o. .6 z z o z a a: z a o 094$ @OJ 9(0 VFO ‘6'le 100v? 0‘6 00? 004. 009 000 0060 00! O a v v0 0 V l 04.9 (9 f0». fOOO 34 '- among the genotypes entered in the partial diallel (Table 3). Bath GCA and SCA effects were significant; the relative contributions to total genetic effects were 74.6% and 25.4% respectively. 1.4.3 Generation Mean Analysis - Estimation of Genetic Effects The distribution of betaine content for the six generations analyzed showed that a simple additive-dominance model was not sufficient to describe the betaine trait (Fig. 2, Table 4). The additive-dominance model was significantly improved by including epistatic effects, but not by including genotype X environment interactions. The linear regression coefficients for the genetic component of a model containing environmental, additive, dominance and epistatic effects (model 5) showed that when tested individually, only the coefficient for additive gene action was significantly different from zero. It may be noted, however, that the distribution of betaine content in the F2 generation is skewed to the low end (Fig. 2). 1.4.4 Heritability Estimates Agreement was noted among heritability estimates based on the three groups of genotypes tested and the three different methods used. The narrow-sense estimate based on midparent-offspring regression was 0.53 while the narrow-sense estimate based on generation variances was 0.63. The broad-sense heritability calculated from the replicated trial data of Ladyman et a1. (1983) was 0.53. 35 Table 3. Analysis of variance for GCA and SCA of betaine content and relative contributions of GCA and SCA effects. Contribution Mean to genetic Source df squares effects Entries 80 45.5** General combining ability (GCA) 12 226** 74.62 Specific combining ability (SCA) 68 11.8** 25.4% Error 136 5.67 **Significant at the 0.01 probability level. 36 Parents 1:! Proctor N =26 a: Arimar N = 24 O i Proctor = 1 1 .0 O i Arlmar=21.6 F (Proctor x Arimar 8 l .. 1 Arimar x Proctor) 4.. [.1 m or F,=18.5 N=20 Gun/k . , y .T ......... T F2 3 40: (Proctor x Arimar) 5 32.. or F2=15.7 N=1ss :3 t 0' 24-- 2 .. u_ 16‘ 8. Owl/TA. . A Backcrosses Dacpmm N=55 chArlmar "=56 . *- BCP=16.2 .. 0 i acA=24.s 12-~ , .. / 8... J. / 4" / ,/,,,/ , OJ 9.. , @éfi/W. 81,216 20 24 28 32 36 . -1 pmol Betaine -g dry wt Figure 2. .Distributim of betaine omtait in Proctor, Arimar, and their F1, F2, and two backcross generatims. 37 Table 4. Generation mean analysis of Proctor, Arimar, and their F1, F2, and two backcross generations for betaine content. I. Sequential modeling, weighted least squares regression. Z Variability Model Description df X2 accounted for 1. Environment 8 (0.001 46.9 2. Environment + additive 7 (0.005 95.6 3. Environment + additive + dominance 6 (0.005 95.9 4. Environment + additive + dominance + genotype X environment interaction 3 (0.005 97.0 5. Environment + additive 3 0.203 99.0 + dominance + epistatic II. Genetic effects estimated from model 5. Effect Regression coefficient : S.E. t Additive -4.94 i 0.58 -8.47** Dominance 15.21 i 6.56 2.32 Epistatic: A X A 15.40 1 6.48 2.38 A X D 33.49 1 12.27 2.73 DX D -6008 i 5086 “1004 **Significantly different from zero at the 0.01 probability level by a t-test with 3 df. 38 1.5 Discussion Four pairs of reciprocal F1 high- X low- and low- X high- betaine crosses were used to test for maternal and cytoplasmic inheritance of the betaine trait. Because no significant reciprocal differences were observed among the Fl's, we conclude that maternal and cytoplasmic effects do not influence the betaine trait in these genotypes and that inheritance of betaine content is likely to be controlled by nuclear genes. In the partial diallel, GCA effects were larger than SCA effects for the genotypes entered. GCA effects were approximately 752 of the total genetic effects, indicating that the betaine trait in these genotypes is primarily attributable to additive genetic effects. Consistent with an additive trait was the lack of overall transgressive variation and the lack of complementation among the diallel Fl's. Additive genetic effects were also the most important contributor to the genetic model obtained by generation mean analysis. Environmental and additive effects accounted for 95.61 of the total variability and the estimation of genetic effects showed that additive genetic effects were the only individually significant factor. We conclude from the generation mean analysis and the partial diallel analysis that betaine content is predominantly an additive trait. Although additive genetic effects are more important than non-additive effects for betaine accumulation, the 39 presence of significant SCA effects, and the occurrence of many Fl's in the partial diallel whose betaine levels were below the midparent values implicate some dominance effects. That the F1 values obtained from the reciprocal high X low crosses were generally lower than the midparent values is also consistent with dominance of the low betaine character, as is the skewed distribution of betaine in the F2 generation from the Proctor X Arimar cross (Fig. 2). Heritability estimates suggest that variation for betaine content in barley has a relatively large genetic component (>502). When evaluating the heritability estimate based on midparent-offspring regression, the following considerations should be taken into account. Firstly, heritability estimates are based on the assumption that the parents are a random sample from the population at large (Falconer, 1981), but in this experiment, parental lines had been chosen for high- or low-betaine content. It has been demonstrated, however, that although selection of parents may reduce precision, it does not bias the estimate from midparent-F1 regression (Falconer, 1981). Secondly, because parents and offspring were grown in the same environment, there is a genotype X environment bias which may inflate the estimate of heritability (Casler, 1982). Despite these reservations, the midparent-offspring value (0.53 i 0.08) was consistent with the other two heritability estimates and had a low standard error. 40 A more important consideration relevant to all three heritability values, is that because any heritability estimate is a ratio of genotypic and environmental variance, high values may be obtained by a large genetic component, by a low environmental component, or both. The plants used in these experiments were grown in controlled environment chambers, so it is reasonable to assume that the heritability estimates are higher than would be found in field trials. We therefore conclude that 50-602 is an upper limit for the heritability of betaine content in barley. We note that the attributes of the betaine trait (nuclear, predominantly additive, high heritability) make it amenable to plant breeding efforts--should physiological-genetic studies establish its adaptive worth. 41 1.6 Literature Cited Allard, R.W. 1960. Principles of plant breeding. John Wiley & Sons, Inc. New York. 485 p. Casler, M.D. 1982. Genotype X environment interaction bias to parent-offspring regression heritability estimates. Crop Sci. 22:540-542. Coughlan, S.J., and R.G. Wyn Jones. 1980. Some responses of Spinacea oleracea to salt stress. J. Exp. Bot. 31:883-893. Falconer, D.S. 1981. Introduction to quantitative genetics. Longman, New York. 340 p. Ford, C.W., and J.R. Wilson. 1981. Changes in the level of solutes during osmotic adjustment to water stress in leaves of four tropical pasture species. Aust. J. Plant Physiol. Griffing, B. 1956. Concept of general and specific combining ability in relation to diallel crossing systems. Aust. J. 31010 8C1. 9:463-4930 Hall, J.L., D.M.R. Harvey, and T.J. Flowers. 1978. Evidence for the cytoplasmic localization of betaine in leaf cells in Suaeda maritime. Planta 140:59-62. Hanson, A.D., and W.D. Hitz. 1982. Metabolic responses of mesophytes to plant water deficits. Annu. Rev. Plant Phy31010 33:163’2030 Hanson, A.D., and R. Wyse. 1982. Biosynthesis, translocation, and accumulation of betaine in sugarbeet and its progenitors in relation to salinity. Plant Physiol. 70:1191-1198. Hitz, W.D. and A.D. Hanson. 1980. Determination of glycinebetaine by pyrolysis - gas chromatography in cereals and grasses. Phytochemistry 19:2371-2374. Hitz, W.D., J.A.R. Ladyman, and A.D. Hanson. 1982. Betaine synthesis and accumulation in barley during field water-stress. Crop Sci. 22:47-54. Jolivet, Y., F. Larher, and J. Hamelin. 1982. Osmoregulation in halophytic higher plants: The protective effect of glycinebetaine against the heat destabilization of membranes. Plant Sci. Lett. 25:193-201. Ladyman, J.A.R., R.H. Ditz, R. Grumet, and A.D. Hanson. 1983. Genotypic variation for glycinebetaine accumulation by cultivated and wild barley in relation to water stress. Crop Sci. 23: 465-468. 42 Larkum, A.W.D., and R.G. Wyn Jones. 1979. Carbon dioxide fixation by chloroplasts isolated in glycinebetaine. Planta 145:393-394. Leigh, R.A., N. Ahmad, and R.G. Wyn Jones. 1981. Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153:34-41. Mather, R., and J.L. Jinks. 1977. Introduction to biometrical genetics. Cornell Univ. Press, Ithaca, N.Y. 231 p. Pollard, A., and R.G. Wyn Jones. 1979. Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta 144:291-298. Rowe, R.H., and W.L. Alexander. 1980. Computations for estimating the genetic parameters in joint-scaling tests. Crop Sci. 20:109-110. Storey, R., and R.G. Wyn Jones. 1978. Salt stress and comparative physiology in the Gramineae. III. Effect of salinity upon ion relations and glycinebetaine and proline levels in Spartina townsendii. Aust. J. Plant Physiol. 5:817-829. Storey, R., and R.G. Wyn Jones. 1979. Response of Atriplex s on iosa and Suaeda monoica to salinity. Plant Physiol. 63 3156-162. Strom, A.R., D. LeRudulier, M.W. Jacowec, R.C. Bunnell, and R.C. Valentine. 1983. Osmoregulatory genes and osmOpmotective compounds. p. 39-59. I3 T. Rosuge, C.P. Meredith, and A. Hollaender (ed.) Genetic engineering of plants - An agricultural perspective. Plenum Press, New York. wall, JOSO, DOD. Christianson, Ron Dimler, and FOR. Senti. 1960. Spectrophotometric determination of betaines and other' quaternary nitrogen compounds as their periodides. Anal. Chem. 32:870-874. Warner, J.N. 1952. A method for estimating heritability. Aston. Jo 44:427‘4300 Wyn Jonees, R.G., and R. Storey. 1981. Betaines. p. 171-204. 12 L-(;. Paleg and D. Aspinall (ed.) Physiology and biochemistry of drought resistance. Academic Press, Sydneqr. Wyn JOnes, R.G., R. Storey, R.A. Leigh, N. Ahmad. and A- Pollard. 1977. A hypothesis on cytoplasmic osmoregulation. p. 126‘136. In E. Marre and O. Ciferri (ed.) Regulation of celJl membrzhe activities in plants. Elsevier Press, Ansterdam. CHAPTER II GENETIC EVIDENCE FOR AN OSMOREGULATORY FUNCTION OF GLYCINEBETAINE ACCUMULATION IN BARLEY 2.1 Abstract Betaine (glycinebetaine) accumulates in several plant families in response to water- or salt-stress. Although betaine is hypothesized to be central to cyt0plasmic osmoregulation, there is no direct evidence for this. We therefore genetically altered betaine level in barley by creating two isOpopulations differing significantly in mean betaine level in unstressed condtions (22.8 and 32.4 umol- g.1 dry wt, respectively). To minimize linkage effects, the isopopulation procedure included several parents and two rounds of crossing. Measurements of various morphological and developmental characters indicated that the two populations were otherwise genetically comparable. Response to salinization (from 0 - 300 mM) of the high- and low-betaine isopopulations was compared with that of the high- and low-betaine parents. NaCl at 300 mM caused an 8- 10-fold increase in betaine level in the two populations and a 5 bar drop in solute potential (we). The difference in 43 44 betaine level between the two papulations was constant with salinization. Although selected only for differing betaine level, the parents and 130populations differed also for 1%; the high betaine genotypes maintained a dg 1 bar lower than the low betaine genotypes at all salt levels. Furthermore, in both papulations and parents, betaine level was linearly related to \% (rZ-O.99) implying co-ordinate regulation of the two traits. These observations are most readily explained if betaine accumulation is a mandatory component of osmoregulation in barley. 2.2 Introduction Betaine (glycinebetaine, N,N,N-trimethylglycine) accumulates in‘a number of plant families in response to water- or salt-stress and is hypothesized to function as a non-toxic or compatible cytoplasmic osmoticum (Wyn Jones et al., 1977). Several lines of evidence support the idea that betaine has an osmoregulatory function: many haIOphytes accumulate betaine (Wyn Jones and Storey, 1981); the amount of betaine accumulated by various salt-tolerant plants is directly proportional to the external salt concentration (Hanson and Wyse, 1982; Coughlan and Wyn Jones, 1982; Storey and Wyn Jones, 1978); and steady accumulation of betaine coincides with a decline in solute potential during 45 long-term water deficit in the field in barley and tropical pasture grasses (Hitz et al., 1982; Ford and Wilson, 1981). A cytoplasmic location for betaine is supported by electron microscopy studies (Hall et al., 1978) and analyses of isolated vacuoles (Leigh et al., 1981), which indicate a disproportionate amount of betaine is in the cytoplasm relative to the vacuole. Experiments with isolated chloroplasts (Larkum and Wyn Jones, 1979), enzymes (Pollard and Wyn Jones, 1979; Paleg et al., 1981), and ribosomes (Brady et al., 1984) confirm that betaine is compatible with metabolic functions. Also, exogenous betaine has been shown to protect bacteria grown in high salt conditions (LeRudulier et al., 1984). Although this evidence is consistent with the hypothesis that betaine acts as a cytoplasmic osmoticum, it is circumstantial. There is not yet direct evidence for any such beneficial function of betaine in plant cells, nor can the possibility that betaine accumulation is an injury response be eliminated (Strom et al., 1983; Hanson and Grumet, 1984). We therefore chose a physiological-genetic approach to investigate the effect of altered betaine levels on the response of barley (Hordeum vulgare L.) plants to osmotic stress. In this work, we used naturally occurring variability for betaine level within the barley gene pool (Ladyman et al., 1983) to select several high- and low-betaine 46 genotypes. Since the betaine trait in barley is nuclear and highly heritable (Grumet et al., 1985), these genotypes were then used to create two isopOpulations differing in betaine level. IsopOpulations have the advantages over isogenic lines that they can be used for multigenic traits and require a relatively small number of generations to create (Quizenberry, 1982; Burton,1968). Salt stress experiments provide an opportunity to measure osmoregulation in defined, steady state conditions and so the parental genotypes and high- and low-betaine isopopulations were tested for their response to salinity. In this paper we describe the process of isopOpulation development and present results of salt stress trials. 2.3 Materials and Methods 2.3.1 IsoPopulation development. In the simplest case isopopulation deve10pment involves divergent selection among F2 progeny of high- X low-betaine crosses for high- or low-betaine individuals that are then used to establish isopopulations (Burton, 1968; Eslick and Hockett, 1974). Ideally, all non-betaine traits are segregating randomly, but in practice, linkage associations between betaine and other stress-related, non-betaine traits could bias performance of the isopopulations (Burton, 1968; Eslick and Hockett, 1974; Rasmusson and Gengenbach, 1983). Because betaine accumulation is a metabolic trait, it could 47 be overshadowed by morphological or phenological characaters that confer stress resistance. We therefore took additional steps to minimize potentially confounding effects of linkage: (a) Several high and several low parents of diverse origins were crossed in order to increase the range of phenotypes for all non-betaine traits and to vary the combinations of alleles at linked loci; (b) A second round of crossing was included to further mix the parental genotypes and break linkages. The isOpopulation development procedure is summarized in Figure 3. Since betaine content of young, unstressed plants can be used to predict genetic potential for betaine accumulation during stress (Ladyman et al., 1983), all screening for betaine content was of 2- to 3-week old plants grown in well-watered conditions in the growth chamber. Plant growth conditions for crossing and screening were as in Grumet et al. (1985). Betaine content of the shoots was measured using the periodide assay described in Chapter 1. Criteria for the choice of parents were: (1) High or low betaine content as measured in replicated trials in well-watered and water-stressed conditions in the growth chamber and field (Ladyman et al., 1983); (2) Compatibililty of flowering dates for crossing. Seventy accessions of g. vulgare and 269 of its wild relative g. spontaneum C. Koch were evaluated (Ladyman et al., 1983); since the range in betaine contents for both groups was very similar, only H. 48 PROCEDURE FOR MODIFIED ISOPOPULATION DEVELOPMENT LPOPULATION OF BARLEY GILTIVARS AM) HQBQEIJM W RELATIVES 1 (1) Select high and low parental lines , I _ I Dow PARENTS—I I HIGH PARENTS I L ’ I * I (2) Cross ln diallel DALLEL-DERIVED F1 POPULATION? (3) Select blah and low genotypes l I _ _ j Biwrwsuxu LHIGHF1's(l-l III-fl L l ' (4) Cross tactorlally FACTOPIAL-oeawea F1 POPULATION (L x L) x (H x H) (5) s... to 9,. LP; POPuumofl (6) Select high and low individuals LLOW F; 's I LI-IIOH F2 'iI (7) Sell to F3 r LLOW Fa's I LHIGH F3 '3 I (8) Verify betaine levels and ellmlnate poor lines r '4“ Low POPULATION I Ij‘ HIGH POPUIJITIOIU (9) Repllcated stress tests Figure 3. The undified isopopulation deveth procedure. 49 vulgare genotypes were chosen. Eight low betaine accessions (CI# 709, 5199, 9309, 10064, 11806, 12456, 13057, 15293) and five high betaine accessions (CI# 3480, 6577, 10138, 14936) were used to initiate the populations; all were spring barleys. Seed was obtained from the USDA Small Grains Collection (Beltsville, MD) and multiplied in the greenhouse. All possible crosses among the 13 parents were attempted. Seed was obtained for 68 of the possible 78 hybrid combinations. The 68 F1 genotypes and 13 parental genotypes were grown in a 9 X 9 triple lattice design and analyzed for betaine content. Each block was a 16 cm diam. pot containing 9 entries. The seven hybrids highest in betaine, and the seven lowest, were selected for the second, factorial round of crossing. Thirty-eight of the 49 possible double-cross hybrid combinations were successful. One plant per hybrid was selfed to give the double-cross F 2 pOpulation. Ten individuals per double cross F family (380 2 total) were screened for betaine; the tap and bottom 102 were selected. The selected F2 individuals were selfed to form F3 families. To enable screening, the 76 high or low F3 families were split into 9 planting sets. Each pot contained one representative of each family in a given set. Five replicates of each set were grown in a random design. The five F3 plants per family were bulked at harvest and 50 analyzed for betaine. The number of high and low families was cut approximately in half. Distribution of betaine levels was checked in the F4 generation using seed derived from 30 plants per selected F family. Seed from the F 3 4 families were randomly assigned into 5 planting sets and grown in a randomized design with 3 replicates. 2.3.2 Salt stress experiments with isopopulations and parent mixes. Plant material andgggowth conditions. Parent or population mixes were prepared with an equal number of seeds per high or low parental genotype or F4 family, respectively. The stratification and growth chamber conditions were as previously described (Grumet et al., 1985) with a PPFD of ca. 150 uE m-z sec-l. A split plot design with 5 replicates was used. The main effect, salt level (a, 100, 200, 300 mM NaCl in 502 Hoagland's solution), was assigned using a randomized complete block design; the isopopulations or parent mixes were the subtreatment. Clay pots (21 cm diam.) filled with vermiculite were marked in half; 5 high or 5 low plants were grown in each half. The plants were irrigated daily for 10 days with 502 Hoagland's solution, then daily with stepwise increases of 50 mM NaCl every second day. Each pot was supplied with 1 liter/day to allow for leaching of salts. They were maintained at their respective salt levels until 8 days after the highest level was reached at which time they were harvested. 51 Growth measurements and betaine determinations. Shoot fresh weight of the 5 plants per sample was measured immediately at harvest. One youngest, fully expanded leaf per sample was removed for solute potential (1%) measurements (see below). The remaining shoot material was reweighed, frozen in liquid N and freeze-dried. Dry weights were corrected 2. for the removed leaf. The dried samples were ground in a Wiley mill to mesh size 40 for betaine and ash weight determinations. For comparisons with osmotic potential, betaine levels were expressed on a fresh weight basis using the appropriate fresh weight/dry weight ratios. Ash content was determined by incinerating 400-mg samples in ceramic crucibles at 480 C for 8 h. In one experiment root dry weights were estimated as follows. The roots were freed from the vermiculite by washing with water, frozen in liquid N oven dried at 70 C 2. for 2 d, and weighed. The samples were ground and ashed as described above to correct for trapped vermiculite. Salute potential measurements. To distinguish solute accumulation from passive concentration, all PS measurements were corrected to 1002 relative water content (RWC) using a similar method to that of Ludlow et al. (1983). One youngest, fully expanded leaf blade per sample was put into a test tube with 5 ml distilled H20, covered with a plastic bag and kept in a dark cabinet overnight at 20 C to hydrate before sampling for $8. One 5 mm diameter 52 punch was removed approximately 5 cm from the base of the blade, sealed in a foil envelope, and frozen in liquid N2. Tests showed that betaine levels of the portion of the shoot sampled for I% were representative of the shoot as a whole (data not shown). The disks were stored at -20 C in a sealed container for up to one week before 1% determination. NO trend indicating dehydration during storage was observed when d; values of replicates determined throughout the week were compared. Two more punches, one on each side of the first, were removed and pooled with the other reps of a given treatment (10 punches/treatment). They were fresh-weighed and then floated on distilled H O for 3-4 h, 2 blotted and reweighed (floated weight), and then dried in an oven for 1 d at 70 C to Obtain dry weight. A correction factor (Barrs, 1968) [(fresh weight - dry weight)/(floated weight - dry weight)] was used to adjust 1% to 1002 RWC; all samples used for Us measurements were at or above 902 RWC. A Wescor HR-33T Dewpoint Microvoltmeter equipped with C-52 sample chambers was used to determine PS. Leaf disks were thawed just before placing in the sample chambers. After equilibration (1 h), 2 or 3 consecutive readings/sample were taken at 15-min intervals using the dewpoint made. All chambers were checked regularly to be sure they read within 52 of the predicted value of a 0.61 M sucrose standard. 53 2.3.3 Characterization Of individual parental genotypes. A randomized complete block design with 4 or 6 replicates was used for the 13 genotypes. Each 21 cm diam. clay pot contained 1 plant/genotype planted in vermiculite; the shoots were harvested at 17 or 27 d. Fresh weight and 1% were determined as above. Because each sample consisted 'of only one plant, the leaf used for I; measurement was frozen in liquid N after removing the punches and pooled 2 with the rest of the sample for dry weight and betaine measurements. 2.3.4 Statistical analyses. The salt stress experiments with the isopopulations and the parent mixes were repeated 3 times. The first experiment with the parent mixes had a maximum salt level of 200 mM NaCl; all other experiments included a 300 mM NaCl treatment. Pooled analyses of variance were calculated using the data from all 3 experiments for the isopopulations and for the two experiments including 300 mM NaCl for the parent mixes. There were no significant treatment X experiment interactions. DeterminatiOn of 1% of the individual parental genotypes was repeated 3 times. The dry weight/fresh weight ratios were measured in two of these experiments. The pooled data for the lg and ratios were analyzed using an unweighted analysis of variance. 54 2.3.5 Exoggnous betaine experiments. Seeds (CI# 11806, multiplied Mesa, A2, 1978) were dehusked and surface sterilized by soaking with continuous stirring for 7 min in 702 ethanol and 10 min in 5.252 (w/v) Na-hypochlorite with 0.52 (v/v) Tween 20 (Sigma Chemical Co., St. Loius, MO.). All subsequent work was done in sterile conditions until the plants were transplanted into vermiculite. The seeds were rinsed with distilled H20 and germinated on wet filter paper in Petri dishes in the dark at 20 C for 3 d. Twenty-two germinated seedlings were placed between halves of sponge stappers, inserted into 25-ml vials containing 10 ml of 502 Hoagland's solution, and transferred to the growth chamber for 4 d. 450 umol of betaine then was added to 9 of the vials to give ca. 50 mM betaine solution. The plants were returned to the growth chamber for 4 d (2-leaf stage) at which time volume uptake was noted (average uptake in the control and betaine treated samples was 4.3 and 3 ml, respectively). The plants were removed and the solution checked for contamination by plating 100 ul subsamples on nutrient broth agar. Plants from contaminated vials were discarded. The plants were individually transplanted into vermiculite in 250 ml pots, kept in the growth chamber, and watered daily with 502 Hoagland's solution for 18 d (5 leaf stage). Any plants with morphological abnormalities were 55 discarded at this stage. Fresh and dry weights, 1%, and betaine were determined as above for the individual parental genotypes. 2.4 Results 2.4.1 Characterization of the pgrents. In unstressed conditions, the high and low betaine parents differed approximately two-fold in mean betaine level (Fig. 4a). They did not differ with respect to average values and ranges for the following morphological and phenological characters: plant height, leaf color and shape, stem color, number of rows/head, head length and shape, and heading date. However, the high betaine genotypes had systematically lower 1% and higher dry weight/fresh weight ratios (Fig. 4 b,c). High- and low-betaine parents were tested for their response to salt stress as high or low mixtures in which all genotypes were equally represented. Plants in all stress treatments were turgid and showed no injury symptoms. Salinization inhibited growth as measured by shoot fresh and dry weight (Fig. 5 a,c) to a similar extent in both the high and low parent mixes. In both mixes, salinization increased the dry weight/fresh weight ratio (Fig. 5e); the ca. 5 mg dry wt/g fresh wt difference between highs and lows remained constant with increasing salt. Figure 4. 56 Betaine levels (A), solute potentials (B), and mg dry weight/g fresh weight (C) for the 13 parental genotypes used to initiate the isopopulations. B. Data are the mean of 4 reps. Data are pooled from 3 experiments (4 or 6 reps/experiment). The difference between the highs and laws is significant by AOV (P=0.0l). Data are pooled from two experiments (4 and 6 reps/experiment). The difference between highs and laws is significant by AOV (P=0.0l). No low betaine parent had a significantly lower Us or higher dry weight/fresh weight ratio than any high betaine parent by LSD (P80.05). Frequency 57 an S 6- 4.. 1D 2.. -/12 16 20 24 A. umol Betaine-g dry wt'1 .. LSD=1.0 4:» . 2a -12 —11.2 -1o.4 3. Vs (bars) at 100% ch 4d LSD=6.4 2. \3 76 84 92 100 C. mg dry wtlfresh wt Figure 4 Figure 5. 58 Growth responses: fresh weight (A,B), dry weight (C,D), and mg dry weight/g fresh weight (E,F), of the parent mixes and isopopulations to increasing salt. Each sample consists of 5 plants. Each point is the pooled data from 3 experiments (5 reps/ experiment). Salt level had a highly significant effect (AOV; P=0.0l) on all 3 parameters. The highs and laws were only significantly different for mg dry weight/g fresh weight (AOV; P=0.0l). g fresh wtlsample ug dry wtlg fresh wt 9 dry wtlsample Parents 59 lsopopulations 30-n 0—-o LOWS ,_ O--o HIGHS 60 As expected, salinization also caused a decrease in 1% at 1002 RWC in both groups of parents (Fig. 6a). This osmotic adjustment was linear (r2-0.99), but partial. There was an approximately 1.7 bar drop/100 mM NaCl vs. a predicted 4.4 bar drop based on osmotic potential of the salt solutions (Wyn Jones and Gorham, 1983). In an experiment where 1% was measured using non-rehydrated leaves (not shown) there was a total drop in 1% of 4.3 bars/100 mM NaCl for both highs and lows; the difference between highs and lows persisted. There was a highly significant difference (AOV; P-0.01) between the high and low parent mixes for 1% (Fig. 6a). The high betaine genotypes maintained a consistently lower ‘% at all salt levels by approximately 1 bar. The total adjustment with respect to salt (ca. 5 bars with 300 mM NaCl) was equivalent for the two groups. Betaine levels rose in salinized plants (Fig. 7a); the absolute difference between the high and low betaine groups persisted at all salt concentrations but the relative difference decreased. 2.4.2 Isopopulation development. Distribution of betaine contents measured in unstressed conditions throughout isOpopulation develOpment is shown in Figure 8. There was no transgressive variation in any generation. The seven highest and seven lowest diallel-derived hybrids (8b, shaded) were selected for the We at 100% RWC (bars) 61 A. Parents 8. lsopopulations -10-' .. -12-» . -14... d -16-. “ . H LOWS _ ‘_ o---«o Highs ‘ 18}, I ’f r I, T. A, Tr : 1 t j 0 200 O 200 . mM NaCl Figure6. Solutepotmtialsoftheparmtnnmsmmd isopqmlatims(B)inrespmsetosaltstress. Dataarepooledresults of3expednmts (5 reps/experimt). The salt mid gantype effects were higily signific-it (AOV; P-0.0l). 62 A. Parents 8. lsopopulations H LOWS o -- ' umol betaine-9'1 fresh wt F3 A j 1 .1! qt- 0 260 o {(30 ' mM NaCl Figural Betdmlevelsoftlnparmtndms (A)and isopopulatims (B)inrespmsetosa1tstress. DatampooledresultsofBamedmts (5 reps/Wt). nasaltmdgantypeeffectsmhigxly sigaiflcmt (ADV; M.Dl). 63 Figure 8. Distribution of betaine levels during the isopopulation development procedure. 64 A Selected cultivate 20--B Diallel F, population Factorial-derived double-cross Fa population 48» >. v 8 m 32" a a. u 9 u. 1am , fl. \‘ :\\\ i‘ Selected F, families " ' E F. isopopulations l e 14 22 30 38 46 umol Betaine-g dry wt" 65 second round of crossing; they differed two-fold in mean 1 betaine level (21.6 and 9.7 umol-g- dry wt, respectively). Pedigrees of the selected Fl's indicated that all 7 of the highest Fl's were derived from high X high crosses and that 5 of the 7 lowest Fl's were derived from low X low crosses. To force a mixture of the high and low genetic backgrounds the selected F hybrids were crosssed l factorially (only highs X lows and lows X highs). The double-cross F2 individuals were screened for betaine (Fig. 8c) and the lowest and highest 10% selected (Fig. 8c, shaded). These formed two distinct groups with a two-fold difference in mean betaine level (i lows - 9.0 umol betaine- g.1 dry wt and i highs - 23.9 umol-g"l dry wt). Each of the 13 parents was represented in the pedigrees of both groups. The high and low F2 individuals were selfed and the resulting F families tested for betaine (Fig. 8d). The 3 number of P families carried to the F4 generation was cut 3 in half (Fig. 8d, shaded) to reduce misclassification errors that occur at the F generation due to environmental effects 2 or heterozygosity. The mean betaine contents of the selected F families were 17.3 and 31.4 umol-g-l dry wt, 3 respectively. All 13 of the parents were represented among the selected low F families; 12 of the 13 parents were 3 represented among the selected high F3 families. The selected F3 selected families were selfed to the F4 generation. 66 The mean betaine values of the high- and low-betaine F 1 4 families were 22.8 and 32.4 umol-g- dry wt, respectively; this difference was highly significant (t-test; P=0.01). Root/shoot ratio, shoot ash content, seed yield, heading 'date, plant height, head length, number of tillers, seed weight and percent germination varied within each population .but the mean values and ranges were the same for the two populations (Table 5). Several qualitative characters, stem color, susceptibility to mildew and aphids, onset of senescence of older leaves, and leaf angle, were also not noticeably different between the two populations. 2.4.3 Responses of the isopopulations to salt stress. Although plants were selected only for betaine level, the process of isOpOpulation development did not eliminate differences in ms and dry weight/fresh weight ratio between the highs and lows. The high betaine isopopulation had significantly greater dry weight per unit fresh weight (P-0.01), and at all salt levels, a consistently lower W8 by approximately 1 bar (P-0.01). Thus the isopOpulations responded to salinity in a very similar way to the parent mixes (Fig. 5 b,d,f; 6b; 7b). One of several possible explanations for the failure to eliminate the association between betaine and we would be that betaine level per se, governs ws' To test this idea, barley seedlings (low betaine CI! 11806) were preloaded with betaine to determine whether raising betaine level would 67 Table 5. Mean values i standard deviation for several characteristics of the selected F3 families forming the isopopulations. 1211; may; as: Betaine level (umol-g dry wt-1) 32.4 i 4.8** 22.8 i 4.5** Root/shoot ratio (unstressed) 0.18 i 0.05 0.16 i 0.02 Root/shoot ratio (300 mM NaCl) 0.26 i 0.05 0.24 i 0.02 2 Ash weight (unstressed) 16.9 i 0.8 15.4 i 2.5 ZAsh weight (300 mM NaCl) 16.5 i 0.5 16.4 i 3.1 # tillers (at 3 weeks) 3 i 0.8 3 i 0.8 Age at flowering (days) 53 i 4 55 i 4 Flag leaf width (cm) 1.3 i 0.3 1.2 i 0.2 Mature plant height (cm) 65 i 10 68 i 8 Head length (cm) 5.9 i 1.4 6.5 i 1.4 Seed yeild (g opot-l) 13.6 i 4.5 16.2 i -4.7 Seed weight (mg) 43.4 i 5.6 42.1 i 6.3 7: germination 86 i 12 83 i 14 **highs and lows significantly different (t-test; P-0.01). 68 lower 1% in unstressed conditions. Although the shoot betaine level was raised from 1.3 to 5.5 umol-g.l fresh wt, us was unaffected. There was also no significant effect of preloading betaine on fresh weight, dry weight and dry weight/fresh weight ratio (Table 6). 2.5 Discussion The two groups of parental genotypes were equally variable for several morphological and phenological characteristics showing that a range of non-betaine alleles was introduced into the isOpopulations in differing combinations. If these traits may be considered representative of less easily measured characters, bias due to non-betaine traits should have been minimized at the outset. Notable exceptions to the random distribution, however, were we and dry weight/fresh weight ratio. For both traits the high betaine genotypes had a phenotype generally associated with more stressed environments. The 1-2 umol-g.l fresh wt difference in betaine level between the highs and lows is far too small to account for either the 1 bar difference in we, or the 5 mg dry wt/g fresh wt difference. Note, though, that a 5 mg dry wt/g fresh wt difference is consistent with a 1 bar difference in we. If the difference in osmoregulation was acheived either completely with inorganic ions, e.g. KCl, or completely with organic compounds with an average molecular weight of 200, 69 A~o.oum mumouluv maouucoo one souw ucmuomwav zaucmofimacwam pmumouu mcwmummss .m.m H moumofiaaou u no e mo memos one mama .mmma vmvcmaxm zaazw ummmcaox mnu mo cowuwmoa upmanpwe ecu Eouw mxmwu wood mafia: ma muoosm maoza one moan: vooaauoumv mum3 mam>m~ mcwmumm «H o: ,No.o H 2.0 2.0..." on; c.o H n57 «sooJH om.m 9:33 + ma” odfi fio.o H o~.o o~.o fl om.~ «.0 H H.o—i -.oHu.~m.~ Houucou w\w=w w. w. when as swoon w\Hoa: us :mouw \ua hue uswfiws hue unwfioa swoon ma. Ho>wa magnum; uooaummuh .mucmaa hoaumn pmmmouumos :0 weapons pmfiaaam mo uomwmm .0 manna 70 the predicted dry weight difference between the two groups would be approximately 2 and 8 mg/g fresh wt, respectively. The observed 5 mg difference falls comfortably within this range. Pedigree analysis and evaluation of several morphological and develOpmental characters during and after the isopOpulation development process indicated that the 13 parental genotypes were successfully mixed to produce two genetically comparable populations differing primarily in betaine level. It is striking, though, that despite two hybridization steps, selection only on the basis of betaine level, and theoretical random assortment of all non-betaine traits, the high betaine pOpulation had lower osmotic potentials and higher dry weight/fresh weight ratios. The inability to dissociate differences in betaine from differences in % is even more evident when betaine level is plotted against % (Fig. 9). There is an excellent linear relationship (r2 - 0.99; P<0.001) in which both high and low genotypes fall on the same line. Three possible explanations for the inability to disrupt this association are: (1) Betaine itself governs g; (2) Linkage between gene(s) for betaine and gene(s) for osmoregulation; or (3) Indirect selection for osmoregulatory genes with pleiotrOpic effects. The first possibility, that betaine regulates 1%, is unlikely since preloading unstressed barley plants with 71 ,umol Betaine-g-1 fresh wt ES A A A A A 7’, i -18 Figure9. r I v ‘ -1;6 —14 -12 ‘l’s at 100% RWC Betaine level vs. solute potential for the isopopulatims and parent mates (inset). *** significant at P<0.00l. Data are pooled frun 3 experiments (5 reps/experiment). 72 betaine did not alter lg. According to the relationship shown in Figure 9, the 4 umol-g.1 fresh wt increase in betaine level acheived in the experiment of Table 6 should have lowered ws by approximately 2 bars; a drop of 0.9 bar or greater would have been detected in this experiment (LSD; P-0.05). The interpretation of this experiment is, of course, contingent upon the assumption that supplied betaine was distributed among and within shoot cells in the same way as endogenously produced betaine. This assumption was checked [14] by autoradiography of C-betaine fed tissues which showed the [14] C-betaine to be evenly distributed along the length of leaf blades. It is also supported by results showing that supplied betaine behaves like endogenous betaine in that it is readily loaded into the phloem and translocated from mature leaves to growing organs (Ladyman et al., 1980). The second possibility, failure to break tight linkages during isopopulation development, cannot be discarded on genetic grounds alone (Burton, 1968; Eslick and Hockett, 1974). However, physiological considerations argue strongly against this explanation. Osmoregulation involves coordinated changes in many processes (Morgan, 1984a) and so it is necessary to either postulate close linkage of many different traits that contribute to osmotic adjustment, or alternatively, to suppose that there exists a single gene with major effects on osmoregulation closely linked to a 73 betaine gene. Even if these genes were linked, this would still not fully explain the observed behavior of the isopOpulations. Betaine level was always perfectly coordinated with total lg. Thus, it would also be necessary ,to invoke coordinate regulation of the betaine and osmoregulatory genes. A simpler explanation for our results is that the high- and low-betaine parents did not differ genetically for betaine accumulation per se, but for osmoregulation as a whole, and that betaine levels are controlled by osmoregulatory genes with pleiotropic effects. In this case, differences in betaine would act as a marker for genetic differences in osmoregulation. This possibility, indirect selection for osmoregulatory genes, is supported by the demonstration of such genes in wheat (Morgan, 1984b) and Arabidopsis (Langridge, 1958). Although a lower ‘% might cause an increase in betaine levels via a direct effect on the biosynthetic enzymes, this seems unlikely as such effects have not been observed in $2712532 studies of betaine biosynthesis in spinach (Hanson et al., 1985; Pan et al., 1981). On the other hand, our results can be very readily explained in light of Wyn Jones' hypothesis that betaine is a cytosolic osmolyte (Wyn Jones et al.,1977). In this case, betaine accumulation would be one of several coordinately regulated components of osmoregulation. 74 As genetic analyses of the betaine trait in barley show it to be under nuclear control and highly heritable (Grumet et al., 1985) these conclusions may also be extended to osmotic behavior. Furthermore, the difference in solute potential between the two populations has two implications bearing on osmoregulation. First, because the two populations had equal total dry matter production in all conditions, it is unlikey that the decreased ws of the high betaine-low ws population arose from a back up of unused assimilates resulting from decreased growth. Second, in accordance with observations by McCree et al., (1984) and Schwarz and Gale (1981), since the two pOpulations acheived equal dry matter production in the set of environments tested, the metabolic cost of osmoregulation at a l-bar lower W8 was either minimal, or offset by an increase in assimilation. In conclusion, the inability to genetically dissociate low we frOm high betaine level, and the almost perfect correlation between W8 and betaine content regardless of salt level or genotype, is the first genetic evidence that betaine accumulation in higher plants has an osmoregulatory function. 75 2.6 Literature Cited Barrs, H.D. 1968. Determination of water deficits in plant tissues. p. 236- 268. In T. T. Kozlowski (ed.) Water Deficits and Plant Growth. Vol I. Academic Press, New York. Brady, C.J., T.S. Gibson, E.W.R. Barlow, J. Speirs, and R.G. Wyn Jones. 1984. Salt tolerance in plants. I. Ions, compatible organic solutes, and the stability of plant ribosomes. Plant, Cell and Environ. 7:571-578. Burton, C.W., J.B. Gunnels, and R.S. Lowrey. 1968. Yield and quality of early and late maturing, near-isogenic populations of pearl millet. Crop Sci. 8:431-434. Coughlan, S.J. and R.G. Wyn Jones. 1980. Some responses of Spinacia oleracea to salt stress. J. Exp. Bot. 31:883-893. Eslick, R.F. and F.A. Hockett. 1974. Genetic engineering as key to water use efficiency. Agric. Meterol. 14:13-23. Ford, C.W. and J.R. Wilson. 1981. Changes in the level of solutes during osmotic adjustment to water stress in four tropical pasture species. Aust. J. Plant Physiol. 8:77-91. Grumet, R., T.G. Isleib, and A.D. Hanson. 1985. Genetic control of glycinebetaine level in barley. Crop Sci. 25:618-622. Hall, J. L., D. M. R. Harvey and T. J. Flowers. 1978. Evidence for the cytoplasmic localization of betaine in leaf cells in Suaeda maritima. Planta 140: 59- 62. Hanson, A.D. and R. Grumet. 1985. Betaine accumulation: metabolic pathways and genetics. p.7l-92. ;g_J.L. Key and T. Kosuge (ed.) Cellular and Molecular Biology of Plant Stress. UCLA Symposia on Cellular and Molecular Biology. Vol. 22. Alan R. Liss, New York. Hanson, A.D., A.M. May, R. Grumet, J. Bode, G.C. Jamieson and D. Rhodes. 1985. Betaine synthesis in chenopods; localization in chloroplasts. Proc. Natl. Acad. Sci. USA. 82:3678-3682. Hanson, A.D. and R. Wyse. 1982. Biosynthesis, translocation, and accumulation of betaine in sugarbeet and its progenitors in relation to salinity. Plant Physiol. 70:1191-1198. 76 Hitz, W.D., J.A.R. Ladyman and A.D. Hanson. 1982. Betaine synthesis and accumulation in barley during field water stress. Crop Sci. 22:47-54. Ladyman, J.A.R., K.M. Ditz, R. Grumet, and A.D. Hanson. 1983. Genotypic variation for glycinebetaine accumulation by cultivated and wild barley in relation to water stress. Crop Sci. 23:465-468. Ladyman, J.A.R., W.D. Hitz, and A.D. Hanson. 1980. Translocation and metabolism of glycinebetaine in barley plants in relation to water stress. Planta 150:191-196 Langridge, J. 1958. An osmotic mutant of Arabidopsis thaliana. Aust. J. Biol. Sci. 11:457-470. Larkum, A.W.D. and R.G. Wyn Jones. 1979. Carbon dioxide fixation by chloroplasts isolated in glycinebetaine. Planta 145:393-394. Leigh, R.A., N. Ahmad, and R.G. Wyn Jones. 1981. Assessment of glycinebetaine and proline compartmentation by analysis of isolated beet vacuoles. Planta 153:34-41. LeRudulier, D., A.R. Strom, A.M. Dandekar, L.T. Smith, and R.C. Valentine. 1984. Molecular biology of osmoregulation. Science 224:1064-1068. Ludlow, M.M., A.C.P. Chu, R.J. Clements and R.G. Kerslake. 1983. Adaptation of species of Centrosema to water stress. Anate Je Plant PhYSiOle 10:119-130e McCree, R.J., C.B. Kallsen, and 8.6. Richardson. 1984. Carbon balance of sorghum plants during osmotic adjustment to water stress. Plant Physiol. 76:898-902. Morgan, J.M. 1984a. Osmoregulation and water stress in higher plants. Ann. Rev. Plant Physiol. 35:299-319. Morgan, J.M. 1984b. Osmoregulation as a selection criterion for drought tolerance in wheat. Aust. J. Agric. Res. 34:607-614. Paleg, L.G., T.J. Douglas, A. van Daal, and D.B. Keech. 1981. Proline, betaine and other organic solutes protect enzymes against heat inactivation. Aust. J. Plant Physiol. 8:107-114. Pan, Se, ReAe Korean, Ce Yu, and AeHeCe Huange 19810 Bataine accumulation and betaine aldehyde dehydrogenase in spinach leaves. Plant Physiol. 67:1105-1108. 77 Pollard, A. and R.G. Wyn Jones. 1979. Enzyme activities in concentrated solutions of glycinebetaine and other solutes. Planta 144:291-298. Quizenberry, J.E.. 1982. Breeding for drought resistance and plant water use efficiency. p.193-212. 12 M.N. Christiansen and C.F. Lewis (ed.) Breeding Plants for Less Favorable Environments. John Wiley and Sons, New York. Rasmusson, D.C. and 3.6. Gengenbach. 1983. Breeding for physiological traits. p.231-254. 13 D.R. Wood, K.M. Rawal, and N.M. Wood (ed.) Crop Breeding. Amer. Soc. of Agron., Inc. Madison, Wisc. Schwarz, M. and J. Gale. 1981. Maintenance respiration and carbon balance of plants at low levels of sodium chloride salinity. J. Exp. Bot. 32:933-941. Strom, A.R., D. LeRudulier, M.W. Jacowec, R.C. Bunnell, and R.C. Valentine. 1983. Osmoregulatory genes and osmoprotective compounds. p.39-59. £2_T. Kosuge, C.P. Meredith, and A. Hollaender (ed.) Genetic Engineering of Plants - An Agricultural Perspective. Plenum Press, New York. ‘ Storey, R. and R.G. Wyn Jones. 1978. Salt stress and comparative physiology in the Gramineae. III. Effect of salinity upon ion relations and glycinebetaine and proline levels in Spartina townsendii. Aust. J. Plant Physiol. 5:817-829. Wyn Jones, R.G. and J. Gorham. 1983. Osmoregulation. p.35-58 lg O.E. Lange, C.B. Osmond, H. Zeiger (ed.) Vol. 12C. Physiological Plant Ecology. Encyclopedia of Plant Physiology. Springer-Verlag, Heidelberg. Wyn Jones, R.G. and R. Storey. 1981. Betaines. p. 171-204. lg L.G. Paleg and D. ASpinall (ed.) Physiology and Biochemistry of Drought Resistance. Academic Press, New York. Wyn Jones, R.G., R. Storey, R.A. Leigh, N. Ahmad and A. Pollard. 1977. A hypothesis on cytoplasmic osmoregulation. p. 126-136. In E. Marre and 0. Ciferri (ed.) Regulation of Cell Membrane Activities in Plants. Elsevier Press, Amsterdam. CHAPTER III GROWTH STUDIES OF TWO BARLEY ISOPOPULATIONS DIFFERING IN GLYCINEBETAINE LEVEL AND OSMOREGULATION 3.1 Abstract Betaine (glycinebetaine) is thought to act as a cytosolic osmolyte in water- or salt-stressed plants and so high betaine levels have been suggested to be of adaptive value during osmotic stress. We previously developed two barley isopopulations differing in betaine level and solute potential (ws) (Grumet and Hanson). In this work, the isopopulations were compared for growth responses to water stress in greenhouse trials. The low betaine-high ws population had a higher rate of leaf production, and in Optimal environmental conditions (adequate water, high irradiance, warm temperatures) accumulated up to 35% greater total above-ground dry matter production than the high betaine-low we population. The difference in dry matter production disappeared in less favorable environments. The behavior of the populations could not be accounted for by differences in partitioning of dry matter to the leaves or in water use efficiency. Thus, although selection for high 78 79 betaine-low Ws resulted in a population with more stability in water stressed environments as judged by regression analysis (31 environments, b=0.84), it also reduced growth in Optimal environments. 3.2 Introduction Plants subjected to environments of decreasing external_ water potential are often able to maintain turgor by the process of osmotic adjustment (Hsiao et al., 1976; Morgan, 1984). Osmotic adjustment is the active accumulation of solutes within the cell (Turner and Jones, 1980) and betaine, which accumulates in response to osmotic stress in many grasses and chenopods, is thought to contribute to this process by acting as a non-toxic cytoplasmic osmoticum (Wyn Jones et al., 1977). Two barley (Hordeum vulgare L.) isopopulations differing primarily in betaine level and solute potential (we) were previously developed and characterized for their response to salt stress in controlled environment conditions (Grumet and Hanson). Although the two populations differed consistently for betaine level and $8 at all salt levels, no differences in growth were observed between the two populations. In this investigation we sought to determine the effect of differences in we and betaine on plant growth in response to relatively long-term water stress. The isopopulations are highly suited to this type of study because they are 80 genetically comparable for traits other than ws and betaine level (Grumet and Hanson). Greenhouse experiments were used to measure total above—ground dry matter production at mid-anthesis in well-watered and water-stressed conditions, water use efficiency, specific leaf weight, and rate of leaf production for the two iSOpOpulations. 3.3 Materials and Methods Greenhouse water stress experiments. Greenhouse water stress experiments were performed using large, 40-1 plastic pots (36 cm diam.) with drainage holes and a planting density of 300 plants/m2 (30 plants/pot). Seeds were prepared as described in Grumet et a1. (1985) and planted in alternate rows (5 plants/row) of the two iSOpOpulations in a soil mix of peat:sand:loam, 1:2:1. The water holding capacity of the soil (3 H20/g dry soil) was 35%. The seedlings were irrigated daily with 50% Hoagland's solution for 10-12 days before stress regimes were imposed; at this time the pots were mulched with ca. 2 cm of Perlite to limit evaporation. Environments ranging from well-watered (irrigated daily with 502 Hoagland's solution to field capacity) to severely water-stressed were established. In Experiment 1, stressed plants were either surface irrigated with 0.5, 0.75, 1, 2, 3, or 4 1 every 3 d, or allowed to wilt before rewatering to field capacity (wilt-rewater cycles were 7-11 days). In 81 Experiments 2 and 3, stressed plants were subsurface irrigated by supplying 50% Hoagland's solution through drip irrigation lines (Chapin Watermatics Inc., Watertown, NY) into three 10-12 cm deep plastic tubes (2.8 cm diameter) per pot at a rate of 20-60 ml/min. The tubes were embedded in pea gravel to facilitate percolation. Watering levels were: once per week until run-through, 3 l/week, or wilt-rewater. In Experiment 4 all plants were well-watered. All water stress experiments were performed between mid-April and late September. Daily high temperatures at canOpy level ranged from 24 to 43 C; nighttime lows ranged from 14 to 20 C. Supplemental lighting was provided with high intensity Na lamps for 16 h/d. On a sunny day, mid-day PPFD at canopy height was 1500-2000 uE In.2 sec-1. The mean percent sunny days in the 4 experiments was 66%. Powdery mildew and aphids were controlled as in Grumet et al. (1985). Above-ground dry matter was harvested at mid-anthesis by row in order to separate the two populations. The plants were frozen in liquid N2 and oven dried at 60 C. The dry weight data was analyzed by the regression-response technique (Finlay and Wilkinson, 1963) using pot mean yield as an indicator of the yield potential of the environment. Water use efficiency. Conditions for the water use efficiency (WUE) experiments were as above, except for the following changes. Three, 2 cm long, plastic tubes (1 cm 82 diam.) were inserted into drainage holes and secured with silicone. The pots were placed onto wooden platforms with a 1-1 tray underneath. A measured amount of 50% Hoagland's solution was supplied each day to allow for 50-1000 ml run-off. Daily water use was determined by ml supplied - ml of run-off after 24 h (complete run-through took ca. 20 h). Non-competitive, single population plantings were used (30 plants/pot); WUE was calculated as total above-ground dry matter production at mid-anthesis (g)/ kg water supplied. WUE experiments were done in the spring and fall (April and October plantings). Daily high and nighttime low temperatures for the October experiment were 24-40 C and 16-24 C, respectively; the percent sunny days was 45%. Specific leaf weight. To determine specific leaf weight (mg dry wt/cmz), 10 youngest, fully expanded leaves from main tillers were removed from each pot in the October experiment. Leaf areas were estimated by weighing cut-out photocopies of the leaf blades. Leaf weight was determined after oven drying at 60 C. 3.4 Results and Discussion The two isopopulations were studied for growth responses to a range of irrigation levels in the greenhouse in the spring and summer (Fig. 10). The populations were planted in alternate rows to force competition and thereby maximize differences between them. Using the regression Figure 10. 83 Yield at mid-anthesis (total above-ground dry matter) of the high betaine-low w population or the low betaine-high w population vs. pot mean yield. S S n = 31 environments The difference in slope for the two popula- tions is highly significant (t=6.75***). Yield of lsopopulation (g/15 plants) 84 O 140-L . Low Betaine-High W5 0 High Betaine-Low Ws 120" 'v’ ’I ’l 100 4» ° 0 yL="2.1+1.14X 0 3°“ r2= 0.988 g .. 0 ’ 60 ”/0 I" 40.. " yH= 4.1+0.84x r2= 0.983 20-1 .. n=31 A A A J A A A A J I ' I 20 40 60 80 :100'180; Pot Mean Yield (9/15 plants) HIGH LOW STRESS -— ENVIRONMENTAL INDEX -- STRESS Figure 10 85 response technique to analyze yield in the 31 greenhouse "environments", the high betaine—low ws population showed a more stable response to increasing water stress (b=0.84) than did the low betaine-high 08 population (b=1.14). The difference in stability, however, was due to growth differences in the favorable rather than stressed conditions. At mid-anthesis the low betaine-high ws population 628 more total above-ground dry matter than did the high betaine-low W8 pOpulation when the two populations were grown in competitive plantings in well-watered, Optimal conditions (high irradiance, warm temperatures). This difference disappeared when growth conditions were less favorable, either by reduced water or in fall plantings (shorter, cloudier days) (Table 7). The absence of growth differences between the iSOpopulations in the earlier, salt stress experiments (Grumet and Hanson) was probably due to sub-Optimal conditions in the growth chamber (e.g. PPFD of ca. 200 uE m-2 sec"1 in the growth chamber vs. 1500-2000 uE m-2 sec”1 for the greenhouse on sunny days). The difference between the isopopulations was also not apparent in non-competitive plantings in either optimal or sub-optimal conditions (Table 7). The low betaine-high we population produced leaves faster than the high betaine-low $8 population in well-watered environments (Fig. 11). The difference was 86 Table 7: Average plant weight at mid-anthesis (43 DAP) for the well—watered isopopulations in competitive and non- competitive plantings. Average plant weight (g) Planting Planting Low betaine- High betaine- arrangement time high is low is competitive springl 8.3 _+_1.5 6.1 i 0.9 * fall 2.1 10.6 1.9 _+_0.4 non-competitive spring2 7.5 :_0.5 6.9 :_0.6 fwfi Lsimz L8102 * significantly different from the low betaine-high is population (AOV; P=0.05). mean of 4 reps :_S E ; 15 plants/rep mean of 3 reps : S.E.; 30 plants/rep mean of 4 reps :_S E ; 30 plants/rep “ND—I Figure 11. 87 Rate of leaf production for the two iso- populations in a competitive fall planting. Each point is the mean of 4 reps; 15 plants/rep. The difference between the populations is significant (AOV; P=0.05). Mean Number Leaves/Plant 88 20- 16- 14~ 12- 1‘5 18 2E1 9'4 57 80 DAP Figure 11 89 significant in the fall (AOV;P=0.05) and highly significant in the spring (AOV; P=0.0l) for both competitive and non-competitive plantings. The consistently observed difference in rate of leaf production, like the differences in betaine and Us, suggests that it was a genetic effect largely independent of environment. It is likely that the difference in the rate of leaf production would enhance dry matter gain of the low betaine-high $8 pOpulation when growing in competition by giving these plants an advantage in light interception that is compounded throughout the growing season. The competitive advantage would likely be most important in conditions where total growth rate is not limited by other environmental factors. The convergence of the curves for dry matter production as water stress increased (Fig. 10) is noteworthy. Possible explantions based on competition for light and for water were considered. Competition for light when water is not limiting could arise from differences between the iSOpOpulations for dry matter partitioning between photosynthetic and non-photosynthetic structures, e.g. root/shoot partitioning, specific leaf weight, or leaf/stem production. Previous studies indicated that the two populations did not differ for root/shoot partitioning (Grumet and Hanson) and the present work showed that the isopopulations also did not differ for specific leaf weight (values for the low betaine-high ws and high betaine-low Us 90 population were 2.5 and 2.6 mg/cmz, respectively). Although leaf to stem ratio was not quantified, there were no evident differences in growth habit between the iSOpOpulations. A difference in water use efficiency is also unlikely to account for the observed behavior of the iSOpOpulations. In well-watered environments, the two populations had equal water use efficiencies (2.8 g dry matter/kg H20). However, if the populations maintained their ms differences in response to water stress as they did when salinized (Grumet and Hanson), the lower solute potentials of the high betaine-low ws pOpulation may have enabled these plants to extract an additional increment of water at lower soil water potentials (Turner and Jones, 1980). This would allow for additional growth by the high betaine-low ms population in the water-limited environments thereby narrowing the gap between the populations. Thus the advantages of the high betaine-low ms genotypes may outweigh disadvantages as the stress level increases. Although a distinct crossover point where the high betaine-low ms population outgrew the low betaine-high m8 pOpulation was not observed in these experiments, there may be conditions where the high betaine-low ms pOpulation would perform better. Jensen (1981) reports, that despite consistent trends, the crossover points of regression response curves for maize genotypes with respect to drought stress vary greatly with the set of environments. It should 91 also be emphasized that the greenhouse barley trials measured total above-ground dry matter production, and not grain yield. The cause of the difference between the isopopulations for dry matter production in Optimal environments is unclear. While it is possible that the difference in growth is a result of differences in ms or betaine, it may also be due to effects of linked genes, or it may be caused by highly pleiotropic genes which also influence osmoregulation, betaine level, and dry weight/fresh weight ratio (Simmonds, 1979). Although the latter two possibilities cannot be ruled out, it is interesting to Speculate about the mechanistic basis by which differences in ms or betaine could cause differences in growth. Lower solute potentials or higher betaine levels may be metabolically expensive in terms of carbon use for maintenance processes. McCree et al. (1984) and Schwarz and Gale (1981), however, studied the energy demand of osmoregulation and considered it to be trivial. The failure to observe dry matter differences between the isopopulations in light-limited, well-watered conditions, where the relative cost of osmoregulation as a portion of total carbon available should be higher than in optimal conditions, supports these observations. It is also possible that in the highest yielding environments nitrogen became a limiting factor. Since 92 betaine is a nitrogen containing molecule, the high betaine genotypes could be more affected than the low betaine genotypes. However, as for the costs of osmoregulation, the difference in betaine nitrogen between the isopOpulations is a very small fraction of the total nitrogen demand. Given 1 total N = 10-20 mg-g‘ dry wt (Janick et al., 1974), and the betaine difference between the highs and lows is 10-20 umol- g.1 dry wt (Grumet and Hanson), or ca. 0.2 mg N'g“l dry wt, the difference in betaine N is only 1-2% of total N. Another explanation for the difference in growth rate might be that metabolic function and cell growth at low ms are still in some way impaired, even when the toxic osmolytes are excluded from the cytOplasm. Or perhaps, the populations differ primarily in cell wall prOperties so that the threshhold turgor for wall expansion is altered. Osmoregulation and growth would, in turn, be affected. It is interesting that Quisenberry et al. (1984) also observed a similar apparent penalty of low solute potential on shoot dry matter production for several cotton lines. Although the lines that had lower solute potentials had higher turgor pressures, there was a strong, negative correlation between low solute potential and growth (shoot dry matter production). They concluded that selection for enhanced osmotic adjustment would result in decreased growth. 93 The connection between reduced growth potential in optimal environments and stress-resistance traits has been frequently observed (Blum, 1979; Begg and Turner, 1976; Jensen, 1981) and ecological studies have suggested that slow growth itself may be a mechanism of adaptation to stressful environments (Parsons, 1968). This view has been extended to agricultural systems where it has been demonstrated for grain crOps growing on stored soil moisture, that reduced growth may confer an advantage by conserving more water for the grain-filling period (Richards, 1983; Passioura, 1972). In contrast to the reduced shoot dry matter production observed for the low ws genotypes in this study and by Quisenberry et al. (1984), Morgan (1984) found no deleterious effects of low 08 on the grain yield of F4 lines of wheat differing in osmoregulation. Data for total biomass were not reported. It will therefore be very interesting to see how the barley is0populations compare for grain yield when tested in the field. Multi-site dryland and irrigated trials with our barley iSOpOpulations are currently being performed by Dr. Albrechtsen at Utah State University. '94 3.5. Literature Cited Begg, J.E. and N.C. Turner. 1976. CrOp water deficits. Adv. Agron. 28:161-217. Blum, A. 1979. Genetic improvement of drought resistance in crop plants. A case for sorghum. p.429-445. In H. Mussel and R.C. Staples (ed.) Stress Physiology in Crop Plants. John Wiley and Sons, New York. Finlay, K.W. and G.N. Wilkinson. 1963. The analysis of adaptation in a plant breeding program. Aust. J. Agric. ReSO 143742-7540 Grumet, R. and A.D. Hanson. (in preparation) Genetic evidence for an osmoregulatory function of glycinebetaine accumulation in barley. Grumet, R.G., T.G. Isleib, and A.D. Hanson. 1985. Genetic control of glycinebetaine level in barley. Crop Sci. 25:618-622. Hsiao, T.C., E. Acevedo, E. Fereres, and D.W. Henderson. 1976. Water stress, growth, and osmotic adjustment. Phil. Trans. R. Soc. Lond. 273:479-500. J801Ck, Jo, R.W. SChery, FOWO WOOdS, and VeWe Ruttane 1974. Plant Science - An Introduction to World Crops. W.H. Freeman and Co., San Francisco. Jensen, 8.0. 1981. Breeding plants for stress environments - Discussion. p.167-168. 13 R.J. Frey (ed.) Plant Breeding 11. Iowa State Univ. Press, Ames, Iowa. McCree, R.J., C.E. Kallsen, and 8.0. Richardson. 1984. Carbon balance of sorghum plants during osmotic adjustment to water stress. Plant Physiol. 76:898-902. Meyer, R.F. and J.S. Boyer. 1981. Osmoregulation, solute distribution, and growth in soybean seedlings having low water potentials. Planta 151:482-489. Morgan, J.M. 1984a. Osmoregulation and water stress in higher plants. Ann. Rev. Plant Physiol. 35:299-348. 95 Morgan, J.M. 1984b. Osmoregulation as a selection criterion for drought tolerance in wheat. Aust. J. Agric. Res. 34:607-614. Parsons, R.F. 1968. The significance of growth rate comparisons for plant ecology. Amer; Naturalist 102:595-597. Passioura, J.B. 1972. The effect of root geometry on the yield of wheat growing on stored water. Aust. J. Agric. Res. 23:745-752. - Quisenberry, J.E., G.B. Cartwright, and B.L. McMichael. 1984. Genetic relationship between turgor maintenance and growth in cotton germplasm. Crop Sci. 24:470-483. Richards, R.A. 1983. Manipulation of leaf area and its effect on grain yield in droughted wheat. Aust. J. Agric. Schwarz, M. and J. Gale. 1981. Maintenance respiration and carbon balance of plants at low levels of sodium chloride salinitYO Je EXPO Bot. 32:933-941e Simmonds, N.W. 1979. Priciples of Crop Improvement. Longman Group Limited, New York. Turner, N.C. and M.M. Jones. 1980. Turgor maintenance by osmotic adjustment: a review and evaluation. p.87-104 lg N.C. Turner and P.J. Kramer (ed.) Adaptation of Plants to Water and High Temperature Stress. John Wiley and Sons, New York. Wilson, J.R. and M.M. Ludlow. 1983. Time trends of solute accumulation and the influence of potassium fertilizer on osmotic adjustment of water-stressed leaves of three tropical pasture grasses. Aust. J. Plant Physiol. 10:523-537. Wyn Jones, R.G., R. Storey, R.A. Leigh, N. Ahmad, and A. Pollard. 1977. A hypothesis on cytoplasmic osmoregulation. p. 126-136. 13 E. Marre and 0. Ciferri (ed.) Regulation of Cell Membrane Activities in Plants. Elsevier Press, Amsterdam. DISCUSSION The objective of this research was to assess the adaptive significance of stress-induced betaine accumulation by determining the potential for, and effect of, genetically altering betaine levels in barley. Genetic studies and iSOpOpulation development showed that the betaine trait in barley is nuclear and highly heritable, and that it is possible to select and breed for lines with differing betaine levels. The results of salt- and water-stress trials, however, raise general issues about the types of differences that were found, the method by which they were obtained, and if there are inherent constraints imposed upon such a study by using only natural variability. Each issue will be dealt with in turn. (a) What type of variability was found, and at what level did selection act? The salinization and growth studies showed that the differences in betaine content were associated with differences in several possibly interrelated traits: solute potential (ms), dry weight/fresh weight ratio, total dry matter production in Optimal environments, and rate of leaf 96 97 production. In general, high betaine genotypes resembled mildly stressed plants. This leads to speculation about the organizational level at which selection occurred (Fig. 12). In light of the observed relationship between ms and betaine with response to salt stress in the iSOpopulations and parents, and the failure of applied betaine to cause changes in ws or the dry weight/fresh weight ratio, it is unlikely that selection was at the level of genes coding for enzymes in the betaine biosynthetic pathway (Level C). It is possible that selection was for gene(s) with major effects on osmoregulation (Level B), or alternatively, selection may have acted at an even higher level, for some sort of highly pleiotropic gene(s) that confer the phenotype of a mildly stressed plant (Level A). If selection was at the level of osmoregulation, the selected genes could either be regulatory (i.e. for coordination of the interdependent osmotically related traits), or structural (e.g. for differences in cell wall components so that different turgor pressures are required for cell expansion). Differences in growth could either result from differences in ms, or alternatively, be controlled by genes closely linked to the 08 genes. Selection for highly pleiotropic gene(s) is also not unreasonable; plant breeders have Often found that the traits they select for have pleiotropic effects (Simmonds, 1979). It is possible that the high betaine-low ms 98 .hee_ee.=eee ago me» e_ onenet_e use» me_ete . .copaom—om mo m—O>m_ opapmmon acmmmcnoc mp_otu umpOt_u .m=o_um_=aoaom_ we» consume mooemcmemwu cm>comno do aw:m=o_ucpmc m_n_mmoa .NH mc=m_m =o_ue_=mmcosmo do mueoeoneoo utonmemnu =o_=m -m_mmgu:»m- _eau_>_u:_ no mm_a5mxm :oH upcomto t m:_cuam u h cowuoauocq coupes xto s :o_uo=coca . . e_eet nes_nz emete\eee_nz see A _o>o_ mu=_Om _euop _ copes—asaoun _ > eeeeee’ «mean T 33:33 me: _om_ m aowqoefigm I“ , 1 P =_mnmtuc lee. 4h 00.... mm_ do nasal. . —u_nm= suzocm acoam_mmt muocum_ < t _m>on 99 genotypes have a limit imposed upon their growth rate that is not either a direct cause or consequence of the differences in 1% and betaine. It is of interest that several other studies have indicated that reduced yield potential in optimal environments is associated with stress resistance traits (Jensen, 1981; Blum, 1979; Begg and Turner, 1976). Since it has been suggested that slow growth, per se, is an adaptation to adverse environments (Parsons, 1968), differences in betaine may be part of a larger complex of stress resistance characters that are under the control of highly pleiotropic genes. (b) Was the type of variability found constrained by the method of selection? The results of the salt stress experiments indicated that selection for high or low betaine content in well-watered conditions did not provide genotypes that produced different amounts of betaine in response to stress, i.e. there were no genotype X environment interactions. Instead, the absolute difference in betaine content between the highs and lows was constant at all salt concentrations; at high salt the relative difference between the two groups was quite modest. It is possible that selection for betaine content in stressed conditions, or for the difference in betaine level between stressed and unstressed conditions, would uncover 100 variability for response to stress. It should be noted that although all screening during isopopulation develOpment was done with well-watered plants, the parental genotypes used to intiate the iSOpOpulations were selected on the basis of betaine level in both water-stressed and well-watered trials. Since the parental genotypes also did not differ in response to salinization, the additional labor required for screening using stress trials is probably not justifiable. Furthermore, among the genotypes tested by Ladyman et a1. (1983), the selected parents represented the full range (25-50 umol'g-l dry wt) for absolute increase of betaine in response to water stress. This suggests, that at least among the genotypes screened by Ladyman et al., there was not a great deal of variability for response to stress. The stressed/unstressed betaine ratios of the selected parental genotypes ranged from 2.0 - 3.2. There were, however, a few accessions screened by Ladyman et al. (1983) with somewhat more extreme ratios (1.6 - 3.8). If verified, these lines may be of interest for future work. They may be cases where the association between high betaine and low ms is broken, or alternatively, the genotypes with a very low stressed/unstressed betaine ratio may fail to osmoregulate. Either result could provide further information about the osmoregulatory role of betaine. 101 (c) Are there inherent limitations of natural variability for physiological-genetic studies? There was only a 2-3 fold range in betaine content in unstressed conditions among the H. vulgare and fl. spontaneum genotypes tested; stress did not increase the difference. This relatively narrow range, the absence of betaine nulls, and the lack of transgressive variation in crossing studies, all suggest that variation for betaine level is for some reason, constrained. This is consistent with an adaptive trait. For traits that are subject to selection pressure there is a range of levels that confer adaptedness. To vary a great deal in either direction from that range would have negative effects, and the levels found within successful genotypes fall within the adapted range (Simmonds, 1979). In contrast, a non-adaptive trait, or one that is not subject to selection pressure, would be more likely to vary widely (Hartl, 1980). In future work additional variation might be achieved by isolating betaine-deficient or betaine-overproducing mutants by conventional means, or by altering betaine synthesis using molecular genetic techniques. The use of a betaine null could answer the question: does eliminating betaine have a neutral or deleterious effect? If it were deleterious, this would support the view that normal levels of betaine are of adaptive value. A betaine overproducer 102 could be used to ask: would increasing the levels of betaine have positive or negative effects? The answer to this question is likely to vary with the environment. Another limitation to using natural variability is that, although the isopopulations provided useful insight into the relationship between betaine and ms, and possibly other growth phenomena, it was not possible to test the effect of differing betaine levels per se. The alternative approaches of isolating mutants or molecular genetic manipulation, might provide a better system for addressing questions about the betaine trait itself. An equally important question, however, is: is it desirable to separate differences in betaine from differences in ms? The evidence that betaine is a protective cytOplasmic osmolyte has become increasingly convincing in recent years, but there is no evidence that betaine levels are a limiting factor in osmoregulation. In fact, the results presented in this dissertation imply that the level of betaine is a closely regulated component of Us in balance with other contributing solutes. If adaptation is Of interest, a more relevant question for crop improvement may be: are differences in $8 desirable? It is only recently that investigators have begun to examine the effects of osmoregulatory differences within a species (Morgan, 1984; Quisenberry, 1984); the results to date are promising. 103 Literature Cited Begg, J.E. and N.C. Turner. 1976. CrOp water deficits. Adv. Agron. 28:161-217. Blum, A. 1979. Genetic improvement of drought resistance in crOp plants. A case for sorghum. p.429-445. l3 H. Mussels and R.C. Staples (ed.) Stress Physiology in CrOp Plants. John Wiley and Sons, New York. Hartl, D.L. 1980. Principles of Population Genetics. Sinauer Associates, Inc., Sunderland, MA. Ladyman, J.A.R., K.M. Ditz, R. Grumet, and A.D. Hanson. 1983. Genotypic variation for glycinbetaine accumulation by cultivated and wild barley in relation to water stress. Crop Sci. 23:465-468. Jensen, S.D. 1981. Breeding plants for stress environments - Discussion. p.168-168. 13 K.J. Frey (ed.) Plant Breeding II. Iowa State University Press, Ames, Iowa. Morgan, J.M. 1984. Osmoregulation as a selection criterion for drought tolerance in wheat. Aust; J. Agric. Res. 34:607-614. Parsons, R.F. 1968. The significance of growth rate comparisons for plant ecology. Amer. Naturalist 102:595-597. Quisenberry, J.E., G.B. Cartwright, and B.L. McMichael. 1984. Genetic relationship between turgor maintenance and growth in cotton germplasm. Crop Sci. 24:479-483. Simmonds, N.W. 1979. Principles of Crop Improvment. Longman Group Ltd., New York. APPENDIX 104 APPENDIX Protocol for colorimetric betaine assay. (For written description, see Chapter 1 p.25-26 ) freeze-dry sample, grind to mesh size 40 (Wiley Mill) oven dry at 70 C weigh 30-50 mg sample into small testtube + 5 ml H O, vortex boil 30- 0 min * cool (at least 1 hr., 4 C) l repellet supernatant remove 4 ml + + 200 ul AG-SO (H ) slurry (2:1 resin:water) (begin standards) vortex, let settle Ci, s—iaspirate supernatant resin wash with 2 ml H 0 vortex, let sett e ;— ; aspirate supernatant resin + 3 ml 4 N NH40H vortex and let settle l fresin supernatant remove to 30 ml beaker * dry with infared lamp and fan (ca. 3 h) redissolve in 0.6 ml 1 N H280 remove 0.5 ml to 1 ml reaction vials, chill 1 h, 4 C + 0.2 ml cold KI-12 reagent cover with parafilm, vortex gently * refrigerate overnight 105 centrifuge (in the cold) 5 % aspirate supernatant pellet (with fine-tipped glass needle) + 0.2 ml 1 N cold H2504 centrifuge at 4 C c pellet + 200 ul dichloroethane dissolve thoroughly (mechanically disrupt pellet) —$ aspirate supernatant 30 ul to small testtube + 4 ml dichloroethane read in spectrophotometer at 365 nm * stOpping points in the protocol Notes: 1. KI-I2 reagent: 15.7 g I2 + 20.0 g KI into 100 ml H shake to dissolve store at 4 C 20 2. dichloroethane: reagent grade 3. AG-SO H+ Resin: BioRad Laboratories 2:1 settled resin:H20, vol/vol 4. calculation of umol betaine/g dry weight from standard curve of betaine-HCl (y-mx+b): (absorbance - b)/m x 0.8 x 1/153.6 umol/ug x 103 mg dry wtlsample 5. range of standards for unstressed samples: 50 - 200 ug 6. reagent blanks have no absorbance at 365 nm 7. soak vials in chromic acid after each use