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Crop Science 43:162-169 (2003)
© 2003 Crop Science Society of America

CROP PHYSIOLOGY & METABOLISM

Genotypic Variation for Glycinebetaine in Sorghum

Wen-Ju Yanga, Patrick J. Richa, John D. Axtellb, Karl V. Woodc, Connie C. Bonhamd, Gebisa Ejeta, Michael V. Mickelbart*,e and David Rhodesa

a Dep. of Horticulture and Landscape Architecture, Purdue Univ., West Lafayette, IN 47907
b Dep. of Agronomy, Purdue Univ., West Lafayette, IN 47907
c Dep. of Chemistry, Purdue Univ., West Lafayette, IN 47907
d Dep. of Biochemistry, Purdue Univ., West Lafayette, IN 47907
e Soil, Plant, and Ecological Sciences Division, Lincoln University, Canterbury, New Zealand

* Corresponding author (mickelbm{at}lincoln.ac.nz)


   ABSTRACT
TOP
 NOTES
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Glycinebetaine (GB) accumulation has been suggested to be an adaptive response to several abiotic environmental stresses. Genetic and metabolic studies of GB accumulation in maize (Zea mays L.) indicate that recessive alleles of a single locus are responsible for the phenotype of GB nonaccumulation. The present study was undertaken to determine whether a similar genetically determined range of GB levels exists in the related C4 species Sorghum bicolor (L.) Moench. In a preliminary analysis of 240 sorghum genotypes, sampled at the postflowering stage, total quaternary ammonium compound (QAC) levels in the betaine fraction of the flag leaves were found to range from as low as 0.1 µmol g-1 FW to as much as 33 µmol g-1 FW. Stable isotope dilution desorption chemical ionization mass spectrometry of six genotypes with high QAC levels and five genotypes with low QAC levels confirmed that this variation could be attributed almost exclusively to genetic variability for GB level. GB-nonaccumulating sorghum genotypes were confirmed to be GB-non-accumulating in a second year of field-testing, and in greenhouse studies under salinized and non-salinized conditions. GB levels increased with seedling age and/or salinization in GB-accumulating genotypes. Also, GB levels were highest in the youngest leaves of GB-accumulating sorghum genotypes. This work shows that GB is the major QAC in sorghum, that genetic differences in GB accumulation exist in sorghum as they do in maize, and that the level of GB in GB-accumulating lines is developmentally and environmentally regulated. A list of GB levels of publicly available lines of sorghum is also provided.

Abbreviations: DCI-MS, desorption chemical ionization mass spectrometry • FAB-MS, fast atom bombardment mass spectrometry • m/z, mass/charge ratio • QAC, quaternary ammonium compound • GB, glycinebetaine • g FW, grams fresh weight • GB:AA, glycinebetaine:total amino acids ratio • RWC, relative water content


   INTRODUCTION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE BREEDING of crop plants for environmental stress tolerance has been difficult and slow. The quantitative nature of stress tolerance and the problems associated with developing appropriate and replicable testing environments make it difficult to distinguish stress-tolerant lines from sensitive lines. One approach to a better understanding of plant stress tolerance is to isolate those characteristics that are proposed to contribute to stress tolerance and to determine their relative importance. Only then can focused breeding approaches be developed.

Accumulation of solutes, either actively or passively, is an important adaptation mechanism for plants in response to osmotic stress. Cellular dehydration is a general consequence of osmotic stresses, including water deficit and high salinity levels. In response to this condition, many organisms synthesize solutes that either help retain water within cells or protect cellular components from injury caused by dehydration. One such solute, glycinebetaine (GB) has been shown to act in both capacities (Incharoensakdi et al., 1986; Robinson and Jones, 1986).

Glycinebetaine is thought to play an important role in plant adaptation to saline and arid environments (Wyn Jones and Gorham, 1983; Grumet and Hanson, 1986; Gage and Rathinasabapathi, 1999). Genes determining GB accumulation are thus of considerable interest in plant breeding for stress environments (Grumet and Hanson, 1986). Many cereal crops accumulate GB, although some, notably rice (Oryza sativa L.), do not. Glycinebetaine-nonaccumulating genotypes of both sorghum and maize have been identified, although in sorghum, lines that do not accumulate GB are rare (Yang, 1990). The relative concentrations of GB vary both among and within species. The levels of GB found in sorghum are as much as 10-fold higher than those observed in maize (Mickelbart, unpublished data), and within maize GB-accumulating lines, a wide range of relative levels exists.

In previous studies, we identified significant genotypic variation for the accumulation of this QAC in maize and identified genotypes that are GB-nonaccumulating (Rhodes et al., 1987; Rhodes and Rich, 1988; Brunk et al., 1989; Rhodes et al., 1989; Lerma et al., 1991). GB nonaccumulation appears to be caused by recessive alleles of a single locus in maize (Rhodes and Rich, 1988; Rhodes et al., 1989) that results in an inability to convert choline to betaine aldehyde, the first committed step in the synthesis of GB (Lerma et al., 1991). At least 13 diverse GB-nonaccumulating maize genotypes are allelic with respect to this locus (Lerma et al., 1991). Near-isogenic maize lines that accumulate GB are more salt tolerant than their GB-nonaccumulating sister lines (Saneoka et al., 1995), further supporting the suggestion that GB accumulation is an important stress tolerance mechanism in plants.

GB is synthesized in plants from serine via ethanolamine, choline, and betaine aldehyde (Hanson and Scott 1980). Although other pathways may exist (such as direct N-methylation of glycine), the pathway from choline to GB is the only one that has been identified in GB-accumulating plant species to date (Weretilnyk et al., 1989). Choline monooxygenase (CMO), which catalyzes the oxidation of choline to betaine aldehyde, and betaine aldehyde dehydrogenase (BADH), which oxidizes betaine aldehyde to GB, are both induced under osmotic stress (Hanson et al., 1985; Ishitani et al., 1995; Rathinasabapathi et al., 1997).

Since GB accumulates in sorghum in response to salinity stress (Grieve and Maas, 1984), it was of interest to determine the range and extent of variability for this trait among diverse genotypes of this species. We tested the hypotheses that GB represents the major QAC in sorghum, as it does in maize, and that this QAC is genetically and environmentally regulated. In this paper, we also report on the identification and preliminary biochemical characterization of several GB-nonaccumulating sorghum genotypes.


   MATERIALS AND METHODS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Field Studies and Sampling Procedures
A total of 240 sorghum genotypes were initially screened for QAC level during the summer of 1989 (planting date 1 April 1989) under rainfed conditions at the Purdue University Agronomy farm in West Lafayette, IN. The soil type is a silty clay loam and adequate rainfall during the trials eliminated the potential for drought. Samples were taken from the flag leaf of five individual plants of each genotype (sampling date 9–10 Aug. 1990). All of the genotypes were at the postflowering stage at sampling. Leaves were excised from plants selected at random from the center row of three-row plots. A representative subsample of the leaf tissue bulked from the five individual leaves (1–1.5 g FW) was taken from the leaf lamina (excluding midrib). Leaf tissue was then extracted by immersion in preweighed vials containing 10 mL methanol at the field site (one sample per genotype).

In the 1990 growing season, samples were taken (on 28 Aug. 1990) from representative high-QAC and low-QAC sorghum genotypes grown under rainfed conditions in early- and late-planted trials. In the early-planted trial (planting date 31 May 1990), genotypes were grown in three-row plots, and all genotypes were at the postflowering stage at sampling. The flag leaf (F) and the four leaves below the flag leaf (F-1–F-4) were excised from two representative plants of each genotype that were chosen at random from the center row. A sample (1–1.5 g FW) of the leaf lamina of each plant was extracted in preweighed vials containing 10 mL methanol. Additional samples were taken from the flag leaves of five other plants of each genotype in the early-planted trial, from the center row of each plot. Samples were also taken from the flag leaves of five plants of each genotype grown in single-row plots in the late-planted trial (planting date 13 June 1990). Although at a slightly earlier stage of growth, again all genotypes were at the post-flowering stage at sampling.

GB Purification and Assay
Methanol extracts were stored at 4°C in the dark and then phase separated with chloroform and water as described previously (Rhodes et al., 1987; Rhodes and Rich, 1988; Brunk et al., 1989; Rhodes et al., 1989). The upper (aqueous) phase was concentrated to dryness under an air stream. In the 1989 field samples, total QAC levels in the betaine fraction were initially determined on half the sample by a spectrophotometric periodide assay (Ladyman et al., 1983; Lerma et al., 1991), following purification of the betaine fraction by Dowex-1-OH- and Dowex-50-H+ ion exchange chromatography as described previously for maize (Rhodes et al., 1987; Rhodes and Rich, 1988; Brunk et al., 1989; Rhodes et al., 1989). Selected samples from the 1989 field studies were additionally analyzed by desorption chemical ionization mass spectrometry (DCI-MS) of n-butyl-betaine esters by means of 2H9–GB as an internal standard added to a known aliquot of the remaining sample after phase separation (Lerma et al., 1991). In DCI-MS applications, GB was quantified from the ratio of ions at m/z 160: m/z 166. These ions correspond to the [M+ - CH3 + H] and [M+ - C2H3 + H] fragment ions of GB-n-butyl ester ([M+] = m/z 174) and 2H9–GB-n-butyl esters ([M+] = m/z 183), respectively. The fragment ions result from thermal loss of a single methyl group from the quaternary ammonium moiety (Lerma et al., 1991). The ion at m/z 180 in the DCI mass spectra was attributed to a fragment ion of trigonelline-n-butyl ester; [M+] = m/z 194; [M+ - CH3 + H] = m/z 180. Total amino acid levels in the samples analyzed for GB by DCI-MS were determined by gas chromatography of N-heptafluorobutyryl isobutyl amino acid esters (with {alpha}-aminobutyrate as an internal standard) as described previously for maize (Rhodes et al., 1989).

The majority of the samples taken during the 1990 growing season were analyzed for QAC by the periodide assay (Ladyman et al., 1983; Lerma et al., 1991), except that one flag leaf sample of each genotype was analyzed for GB and trigonelline by stable isotope dilution fast atom bombardment mass spectrometry (FAB-MS) of n-butyl betaine esters as described previously (Lerma et al., 1991). Betaine was quantified from the ratio of the molecular cations of GB-n-butyl ester ([M+] = m/z 183). Trigonelline was quantified from the ratio of the molecular cation of trigonelline-n-butyl ester ([M+] = m/z 194), and the 2H9–GB-n-butyl ester internal standard ([M+] = m/z 183) (Rhodes et al., 1989).

Greenhouse Screens
Two greenhouse studies involving growth of sorghum genotypes under nonsalinized and salinized conditions were conducted essentially as described for maize (Rhodes et al., 1989; Lerma et al., 1991). The first study was designed to determine the effect of salinization on GB accumulation in a number of GB-nonaccumulating or GB-accumulating lines and on GB accumulation in various organs of representative GB-nonaccumulating (IS2319) and GB-accumulating (P932296) lines. Plants were grown in 14.4-cm-diam pots in Scott's MetroMix (Scott's Horticultural Products, Inc., Marysville, CA). Mean greenhouse daytime/nighttime temperatures were 27/21°C. Natural lighting was not supplemented. Plants were watered with a commercial water-soluble fertilizer (Peters Professional 13-2-13, Scott's Horticultural Products, Inc., Marysville, CA) at a nitrogen rate of 100 µg g-1 supplied primarily as ammonium nitrate.

For the first greenhouse experiment, nonaccumulating and accumulating lines were randomly assigned to positions on the bench. Because of the number of samples, single samples were taken for each line and each organ. However, GB accumulation trends within the plants are evident, even with no replication. Plants were established for 4 wk in the absence of salinization by irrigation with nutrient medium (Rhodes and Rich, 1988) lacking NaCl, and were either maintained under these conditions until 7 wk after planting (nonsalinized), or were salinized beginning at 4 wk. Salinization was applied in a stepwise manner (50 mM M NaCl during Week 4–5, 100 mM NaCl during Week 5–6, and 150 mM NaCl during Week 6–7). Samples of leaf tissue were taken from young expanding leaves of individual plants of several sorghum genotypes as described for the field samples (above). In the case of IS2319 and P932296, individual plants were dissected into constitutive organs [Leaf 1 (oldest leaf) to Leaf 10 (youngest leaf), stalk, and root], and a tissue sample (1–1.5 g FW) of each organ was then extracted in 10 mL methanol. Total QAC levels of the betaine fractions of these greenhouse-grown plants were determined by the periodide assay. Relative water contents of young expanding leaves were determined as described previously for maize (Rhodes et al., 1989).

A second greenhouse study was conducted to evaluate the levels of GB accumulated under various salinity regimes and at different stages of seedling development. Plants were grown in 14.4-cm-diam pots in the greenhouse for 21, 28, 35, 42, or 49 d and were watered with 100 mM NaCl for 0, 7, or 14 d before the harvest of leaves for GB determination. Plants were laid out on the greenhouse bench as a completely randomized design with three replications per line and treatment. Control plants were watered with a nutrient solution (Rhodes and Rich, 1988) lacking NaCl and salinized plants were watered with the nutrient solution ammended with 100 mM NaCl. Each age–salt level treatment combination was maintained in an individual pot with approximately five plants per pot. Leaf samples (0.5–1 g FW) were taken from young, expanding leaves of five plants per treatment and were extracted in preweighed vials containing 10 mL methanol. Total QAC levels of the betaine fractions of these plants were determined by the periodide assay.


   RESULTS AND DISCUSSION
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
CONCLUSIONS
 REFERENCES
 
Betaine Levels in Diverse Sorghum Genotypes
The frequency distribution of the total QAC levels in the betaine fraction of the flag leaves of 240 sorghum genotypes from samples taken during the 1989 growing season is presented in Figure 1. Each value is based on a single analysis of a sample of five leaves bulked from five individual plants of each genotype from the center row of a three-row plot. Individual QAC levels of all tested genotypes are presented in Table 1. Total QAC levels ranged from as low as 0.1 µmol g FW-1 to over 33 µmol g FW-1. The maximum recorded values for GB in maize are in the range of 10 to 16 µmol g FW-1 (Lerma et al., 1991). Certain sorghum genotypes appear to have a much higher capacity for total QAC accumulation than the highest GB accumulating maize genotypes so far identified. Sorghum genotypes that are apparently GB-nonaccumulating were also identified. Eight genotypes of sorghum were found that exhibited total QAC levels of <1.0 µmol g-1 FW (Fig. 1). While the observed GB levels may be affected to some extent by field variability, other data from our lab indicates tight genetic control of GB synthesis in sorghum (publication in preparation). Furthermore, there were no signs of water stress in any of the lines at the time of sample collection.



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  		Fig. 1. Frequency distribution of total quaternary ammonium compound (QAC) levels (determined by the periodide assay) from the flag leaves of 240 sorghum genotypes in the 1989 growing season. Individual QAC levels for all 240 genotypes tested are presented in Table 1.

 

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  		Table 1. Cultivars within each quaternary ammonium compound (QAC) level class (QAC level of the betaine fractions of the flag leaf) of diverse field-grown sorghum cultivars at the post-flowering stage. Graphical representation presented in Fig. 1.

 
Six high-QAC and five low-QAC sorghum genotypes identified in the initial screen were further analyzed by performing a duplicate periodide assay and by using a more specific assay for GB on the same sample, employing stable isotope dilution DCI-MS with 2H9–GB as an internal standard on the remaining sample (Lerma et al., 1991). Fair agreement between the mean total QAC values (based on duplicate periodide assays) and GB levels (as determined by DCI-MS) was obtained (r = 0.981) (Table 2). Of the five low-QAC genotypes found in the initial screen and analyzed specifically for GB level by DCI-MS, all exhibited GB levels of <0.25 µmol g-1 FW. The periodide assay tended to slightly overestimate GB level in the low-QAC genotypes. The small discrepancy between GB levels determined by DCI-MS and total QAC levels determined by the periodide assay for the low-QAC genotypes may be in part attributable to the presence of trigonelline in the betaine fractions of sorghum flag leaf tissue (Table 2). Among the six high-QAC genotypes identified in the initial screen, the rank order of GB levels (as determined by DCI-MS) was similar to that determined for total QAC levels by the periodide assay in the same samples (Table 2). These DCI-MS studies reveal that the major QAC in the betaine fraction of sorghum leaf tissue is GB, and that variation in total QAC levels (as determined by the periodide assay) can be taken as a valid indicator of GB titer. Grieve and Maas (1984) also concluded that GB is the major QAC accumulated by sorghum. The periodide assay is more convenient for screening numerous plant samples without undue sacrifice in accuracy in comparison to a more specific spectrophotometric assay for GB such as analyzing the p-bromophenacyl ester (Grieve and Maas, 1984), or analysis by plasma desorption mass spectroscopy (Mickelbart et al., 1999).


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  		Table 2. Comparison of total quaternary ammonium compound (QAC), glycinebetaine, and trigonelline levels, and glycinebetaine (GB): total amino acid (AA) ratios of the flag leaf tissue of six high-QAC and five low-QAC sorghum genotypes.

 
In maize, GB nonaccumulation is not associated with a general nonaccumulation of nitrogenous solutes. Thus, GB nonaccumulation is associated with extremely low GB: total amino acid (GB:AA) ratios (Rhodes and Rich, 1988; Rhodes et al., 1989). Typical GB:AA ratios for GB-accumulating inbreds of maize are in the range of 0.2 to 0.5. Typical GB:AA ratios for GB-non-accumulating lines of maize are in the range from 0.001 to 0.005 (Rhodes and Rich, 1988; Rhodes et al., 1989). GB:AA ratios of the six high-QAC sorghum genotypes ranged from 2.0 to 5.1 (Table 2), which is several-fold greater than those typical of GB-containing maize genotypes. GB:AA ratios of the five low-QAC sorghum genotypes range from 0.013 to 0.034 (Table 2). This >300-fold range of GB:AA ratios in sorghum is clearly largely attributable to genotypic variation for GB.

Effects of Salinity on Betaine Accumulation
It has been reported that salinity stress markedly induces GB accumulation in sorghum (Grieve and Maas, 1984). It was therefore of interest to determine GB levels of several GB-nonaccumulating and GB-accumulating sorghum genotypes under both salinized and nonsalinized conditions. In maize it has been established that the phenotype of GB nonaccumulation is maintained under salinity stress (Rhodes et al., 1989; Lerma et al., 1991). GB-accumulating maize genotypes exhibit a four- to fivefold accumulation of GB primarily in young expanding leaves in response to salinization to 150 mM NaCl (Rhodes et al., 1989). This accumulation of GB in GB-accumulating maize genotypes occurs in proportion to a decline in leaf relative water content (RWC) induced by salinity stress (Rhodes et al., 1989).

Total QAC levels detected in the betaine fractions of young expanding leaves of three GB-accumulating and three GB-nonaccumulating sorghum genotypes are presented in Table 3. Total QAC content was increased by more than fivefold in response to 150 mM NaCl only in the GB-accumulating genotypes. Salinity was associated with a reduction in leaf RWC in all genotypes tested, although reductions in RWC were slight. In one GB-accumulating genotype tested, P932296, there was a pronounced gradient of total QAC concentration within the plant, with the highest concentrations detected in young expanding leaves in salinized plants (Table 4), similar to reported patterns for maize (Rhodes et al., 1989). A betaine-nonaccumulating genotype, IS2319, showed no accumulation of total QACs in the betaine fraction of any organ in either the non-salinized or salinized environment. Total QAC levels in the betaine fraction never exceeded 0.076 µmol g-1 FW in any organ of IS2319 under either salinized or nonsalinized conditions. Root tissues of both GB-accumulating and GB-nonaccumulating sorghum genotypes contained low GB levels under both control and salinized conditions. This is similar to previously reported data on GB accumulationin maize (Rhodes et al., 1989), but contrary to published reports on GB accumulation in wheat (Triticum aestivum L.) seedlings (Krishnamurthy and Bhagwat, 1990).


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  		Table 3. Total quaternary ammonium compound (QAC) levels in the betaine fraction of young expanding leaves of sorghum genotypes, and leaf relative water contents (RWC) observed under nonsalinized and salinized greenhouse conditions at the preflowering stage.

 

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  		Table 4. Total quaternary ammonium compound (QAC){dagger} levels of betaine fractions of different plant organs of a high-betaine (P932296) and a betaine-non-accumulating (IS2319) sorghum genotype under nonsalinized (0 mM NaCl) and salinized (150 mM NaCl) greenhouse conditions.

 
Total QAC levels in sorghum seedlings of various ages exposed to salinity (100 mM) for 0 (control), 7, or 14 d are presented in Fig. 2. The periodide assay was used to determine total QACs. In all cases, GB-accumulating plants salinized for 7 or 14 d had higher QAC levels than non-salinized plants while QAC levels of the GB-nonaccumulating line were never more than 1 µmol g-1 FW under any salinity regime. Salinity level (Storey and Wyn Jones, 1977) and length of exposure to salinization (Saneoka et al., 1999) have been shown to increase GB accumulation in other GB-accumulating species. There also appeared to be a developmental stage effect on GB accumulation. The accumulation of QACs in response to salinization was greater with increasing plant age. Nonsalinized GB-accumulating plants had similar QAC levels regardless of age, except in 49-d-old plants. This may reflect GB accumulation integrated over time, or may represent a developmental shift in accumulation capacity.



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  		Fig. 2. Total quaternary ammonium compound (QAC) levels ( ± SE mean) in betaine fraction of young expanding leaves of sorghum genotypes IS2319 (open bars) and P932296 (solid bars). Plants were grown for a total of 21, 28, 35, 42, or 49 d (bottom row, x axis) and were salinized with 100 mM NaCl for 0, 7, or 14 d (top row, x axis) before leaves were harvested for QAC isolation and quantification. Total QAC levels were determined in three individual plants by the colorimetric periodide assay after purification of the betaine fraction from plants.

 
Confirmation of Betaine Nonaccumulation
The above studies confirm that representative GB-non-accumulating and GB-accumulating genotypes of sorghum, initially observed at post-flowering stages in the field, maintain the same phenotype at seedling and pre-flowering stages under control or salinized conditions. Therefore, environmental and/or developmental variables do not appear to be the sole determinants of the greater than 300-fold variation in GB level between sorghum genotypes (Table 2).

To confirm that genotype is a significant determinant of observed differences in betaine levels of field-grown sorghum shown in Table 2, the same 11 sorghum genotypes were evaluated for QAC content in the 1990 growing season in trials planted at different dates (early and late; see Materials and Methods). At least five representative plants of each genotype were sampled from each trial. In the early-planted trial, samples were taken from the flag leaves of five plants, four of which were analyzed for total QAC levels in the betaine fraction by the periodide assay, and one of which was analyzed by stable isotope dilution FAB-MS specifically for GB and trigonelline. Two additional plants were sampled from the flag leaf (F) and the four leaves immediately below the flag leaf (F-1–F-4) in the early-planted trial, and were analyzed for total QAC content of the betaine fraction by the periodide assay. Previously identified GB-accumulating genotypes exhibited high total QAC levels in the betaine fraction of flag leaves in both the early-planted and late-planted trials (Table 5). Previously identified low-GB genotypes exhibited low total QAC levels (<1 µmol g-1 FW) in the betaine fraction of the flag leaf in both the early- and late-planted trials. There was generally good agreement between the periodide assay and the more specific FAB-MS method of GB quantification, although it should be emphasized that these FAB-MS determinants were on single plants of each genotype. Notably, genotypes exhibiting low total QAC levels all exhibited low levels of GB as determined by FAB-MS. Again, trigonelline was detected at trace levels in all 11 genotypes (Table 5), and this may account for the tendency of the periodide assay for total QACs to slightly overestimate GB levels in the GB-non-accumulating genotypes.


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  		Table 5. Mean total quaternary ammonium compound (QAC) levels in the betaine fraction of the flag leaves of 11 sorghum genotypes observed at the post-flowering stage during the 1990 growing season in early- and late-planted trials, and glycinebetaine and trigonelline levels of single samples from the early-planted trial.

 
There was up to a two- to three-fold seasonal variation in the betaine level of high-betaine genotypes between the 1990 (Table 5) and 1989 (Table 2) growing seasons. The overall correlation coefficient between flag leaf total QAC levels observed in the 1989 and 1990 (early-planted trial) growing seasons for these 11 genotypes was r = 0.980. The slope of this relationship was 0.411, however, indicating an average 2.5-fold lower betaine level in 1990 versus 1989, among high-betaine genotypes. Because GB accumulation is induced by osmotic stress (Ladyman et al., 1983; Grieve and Maas, 1984; Grumet and Hanson, 1986; Rhodes et al., 1989), this seasonal difference may be a function of differences in water availability, which would be dependent upon rainfall patterns in the different growing seasons. GB levels of maize inbreds have also been shown to vary between growing seasons (Brunk et al., 1989).

Total QAC level of the betaine fraction of the flag leaves and four leaves immediately below the flag leaf for the 11 genotypes evaluated in the 1990 early-planted trial are presented in Table 6. In the GB-accumulating genotypes, the flag leaf tended to exhibit the highest total QAC levels. Low-GB genotypes exhibited low total QAC levels in all leaves sampled. These observations validate the use of the flag leaf at the post-flowering stage for routine screening of sorghum genotypes for GB titer, as initially employed in Fig. 1 and Table 2. The results presented in Tables 4 and 5 confirm that in a second year of field testing the low-betaine phenotype is maintained, but suggest that among GB-accumulating genotypes, there can be marked differences in gradients of GB within the plant. Certain GB-containing genotypes (e.g., IS6451 and IS9335) exhibit less than a 50% decline in GB level from the flag leaf to the fourth leaf below the flag leaf, whereas certain other GB-accumulating genotypes (e.g., P932296) exhibit an approximately 10-fold decline in GB concentration from the flag leaf to the fourth leaf below the flag leaf (Fig. 2).


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  		Table 6. Total quaternary ammonium compound (QAC) levels (determined by the periodide assay) of the flag leaves (F) and the four leaves (F-1 to F-4) immediately below the flag leaf in 11 sorghum genotypes (early-planted trial, 1990).

 

   CONCLUSIONS
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
REFERENCES
 
Several sorghum genotypes have been shown to exhibit extremely low GB levels in a range of environments (field and greenhouse, salinized, and nonsalinized) and at different developmental stages (seedling and pre- and postflowering). This phenotype of betaine nonaccumulation in sorghum strongly resembles the phenotype of betaine nonaccumulation in maize (Rhodes et al., 1987; Rhodes and Rich, 1988; Rhodes et al., 1989; Brunk et al., 1989; Lerma et al., 1991);. Preliminary genetic studies suggest that a recessive allele of a single locus is the cause of this nonaccumulation in one GB-nonaccumulating sorghum genotype, IS2319 (Grote et al., 1994), but it remains to be tested whether the various sources of GB nonaccumulation in sorghum so far identified are allelic. The precise metabolic basis of the GB-nonaccumulation phenotype of sorghum also remains to be elucidated. An understanding of the metabolic and genetic basis of this genetic variation in GB level in sorghum should assist in devising breeding strategies to develop near-isogenic lines differing solely for the GB trait. These lines could then be used to test the contribution of this trait to salt and drought tolerance.


   NOTES
TOP
 NOTES
ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 
This work was supported by a grant from the USDA (grant #90-37280-5721), by Purdue University Agricultural Experiment Station funds, and by a USAID program support grant (DAN-5061-G-SS-9040). P.J.R. was supported by a fellowship from the McKnight Foundation.

Received for publication December 3, 2001.


   REFERENCES
TOP
 NOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 CONCLUSIONS
 REFERENCES
 




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The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome