Betaine Deficiency in Maize1 - NCBI

Plant Physiol. (1991) 95, 1113-1119 0032-0889/91/95/11 13/07/$01 .00/0

Received for publication October 1, 1990 Accepted December 11, 1990

Betaine Deficiency in Maize1 Complementation Tests and Metabolic Basis Claudia Lerma, Patrick J. Rich, Grace C. Ju, Wen-Ju Yang, Andrew D. Hanson*, and David Rhodes Departamento de Bioquimica, CINVESTAV, Instituto Polit6cnico Nacional, Apdo. Postal 14-740, 07000 Mexico D.F., M6xico (C.L.); Center for Plant Environmental Stress Physiology, Department of Horticulture, Purdue University, West Lafayette, Indiana 47907 (P.J.R., G.C.J., W.-J.Y, D.R.); and MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824 (A.D.H.) ABSTRACT

several heterotic groups (3), and so represent considerable genetic diversity (5). Genetic analyses of betaine deficiency in a sweetcorn cross (18) and a Corn Belt dent cross (17) have indicated in both cases that a recessive allele of a single nuclear gene is responsible. As it has not been established whether betaine deficiency in these and other genotypes is conditioned by the same gene or by distinct genes, one aim of the present research was to resolve this issue. Complementation tests were therefore conducted on sources of betaine deficiency representative of those described previously (3, 19), together with some novel sources reported here for the first time. Betainedeficient genotypes were also crossed with betaine-positive types to confirm that deficiency is in all cases recessive. In the course of screening public Corn Belt dent germplasm for betaine deficiency, one population (P77) was found to be a mixture of betaine-positive and -deficient plants. This prompted us to develop related lines with and without betaine by selfing homozygous betaine-positive and -deficient individuals. These related lines were used to investigate the metabolic basis of betaine deficiency by comparing their ability to oxidize supplied choline and betaine aldehyde to betaine. The metabolic block was also probed by measuring the endogenous choline and betaine aldehyde pool sizes in various betaine-positive and -deficient genotypes.

Maize (Zea mays L.) is a betaine-accumulating species, but certain maize genotypes lack betaine almost completely; a single recessive gene has been implicated as the cause of this deficiency (D Rhodes, PJ Rich [1988] Plant Physiol 88: 102-108). This study was undertaken to determine whether betaine deficiency in diverse maize germplasm is conditioned by the same genetic locus, and to define the biochemical lesion(s) involved. Complementation tests indicated that all 13 deficient genotypes tested shared a common locus. One maize population (P77) was found to be segregating for betaine deficiency, and true breeding individuals were used to produce related lines with and without betaine. Leaf tissue of both betaine-positive and betaine-deficient lines readily converted supplied betaine aldehyde to betaine, but only the betaine-containing line was able to oxidize supplied choline to betaine. This locates the lesion in betaine-deficient plants at the choline betaine aldehyde step of betaine synthesis. Consistent with this location, betaine-deficient plants were shown to have no detectable endogenous pool of betaine

aldehyde.

Osmotic adjustment, which results from the accumulation of solutes within cells, can favor plant growth and survival under dry or saline conditions (reviews: 12, 14, 23). Betaine (glycinebetaine) accumulation in response to drought or salinity stress is proposed to play an important role in osmotic adjustment in members of the Chenopodiaceae and Gramineae (6, 22) by functioning as a compatible or protective osmolyte in the cytoplasm and/or chloroplast (10, 13, 20). Considered as a species, maize (Zea mays L.) has a moderate capacity for betaine accumulation, with maximum reported levels in the range 5 to 10 ,umol/g fresh weight (3, 8, 17-19). However, certain maize genotypes lack betaine almost completely; these include sweetcorn (18) and Corn Belt dents from

MATERIALS AND METHODS Production and Evaluation of Plant Material The crosses of maize (Zea mays) listed in Tables I and II were made in the 1988 and 1989 growing seasons in West Lafayette, IN. The parents were (a) a representative subset of the public inbreds shown previously to be betaine positive or deficient (3); (b) 729-13F3, a betaine-deficient F3 family derived from hybrid 1146 x 1506 (17); (c) 86W-9S2, a betainedeficient selection (see text) from population P77 (P77 was obtained in 1983 from Dr. A. J. Pryor, CSIRO, Canberra); (d) 86M-9S2, a selection from the betaine-deficient Gdhl null mutant 82-137S (82-137S was from Dr. A. J. Pryor); (e) the betaine-deficient sweetcorn hybrid 2708 (19); (f) a betainedeficient selection (RSA) from the popcorn 'Red South American' ('Red South American' was obtained from Dr. J. L. Bennetzen, Purdue University). All parents except e and f were Corn Belt dent types. The hybrids were evaluated for

Supported by U.S. National Science Foundation grant INT8814927, by U.S. Department of Energy contract No. DE-AC0276ERO-1338, by grants to C. L. from CINVESTAV del IPN and CONACYT of Mexico, and to D. R. from the Corporation for Science and Technology in Indiana. P. J. R. was supported by a fellowship from the McKnight Foundation. Purdue University Agricultural Experiment Station journal article No. 12185.

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LERMA ET AL.

betaine content under rainfed field conditions in West Lafayette during the summers of 1989 and 1990, as indicated in the text. For these field evaluations, young expanding leaves of three individuals of each hybrid were sampled (1-1.5 g fresh weight per sample) at 9 weeks after planting. Initial field studies with population P77 were conducted during the summer of 1986 in West Lafayette, IN (see ref. 17 for 1986 field trial details). Samples (1-1.5 g fresh weight per sample) were taken from mature, expanded leaves of 11 individual plants, and the plants were then selfed. Field evaluations of SI progeny of these selections were conducted in the summer of 1987 (see ref. 3 for details of 1987 trial), sampling from young, expanding leaves (1-1.5 g fresh weight per sample). S2 populations were produced in the summer of 1988 from selections of the SI populations (see ref. 3 for details of 1988 trial). S2 populations were evaluated for betaine level under field conditions in West Lafayette, IN, the summer of 1989, as for the hybrids described above. Certain populations (including P77, Si, and S2 populations derived from P77 selections, and the progenitors of P77; inbreds B14 and R168) were also evaluated under greenhouse conditions. The original P77 population was grown in the Purdue University Horticulture Department greenhouse under well-irrigated conditions as described previously (17). Plants were sampled 8 weeks after planting from mature, expanded leaves, as for the field-grown P77 population. Each plant sampled was selfed. The SI and S2 populations derived from P77 selections, and inbreds B14 and R168 (and their Rpd (2) derivatives), were initially established for 3 weeks in the absence of salinization, and were then salinized gradually to a final NaCl concentration of 150 mm by increasing the salt concentration from 0 to 50 mm (weeks 3-4), 50 to 100 mm (weeks 4-5) and finally from 100 to 150 mm (weeks 5-6) (18); young expanding leaves of individual plants were sampled at 6 weeks. Tests with a range of betaine-positive and -deficient inbreds and hybrids established that betaine titers of salinized plants in the greenhouse were highly correlated with those observed in the field (r2 = 0.959, n = 21), so that results of the two evaluation methods can be compared directly. The inbreds R168 and B14 (and their Rpd (2) derivatives) were obtained from Dr. W. Pedersen (University of Illinois); these were tested for betaine content but not included in crosses. Extraction and Determination of Betaine

Leaf samples from field or greenhouse plants were extracted in 10 mL methanol, and methanol extracts were phaseseparated with chloroform and water as described previously (17, 19). After evaporating the aqueous phase to dryness in an air stream, samples were dissolved in 2 mL of water and then processed via Dowex-1 (OH-) and Dowex-50 (H+) ion exchange resins (17, 19). Betaine in the Dowex-50 eluate was determined either by FAB-MS2 using a 2H9- or 2Hg-'Nglycinebetaine internal standard (3, 17, 19) or by the spectrophotometric periodide method as given by Ladyman et al. 2

Abbreviations: FAB-MS, fast atom bombardment mass spectrometry; amu, atomic mass units; DCI-MS, desorption chemical ionization mass spectrometry; m/z, mass/charge ratio.

Plant Physiol. Vol. 95, 1991

(11), as indicated in the text. The methods agreed well (r2 = 0.85, n = 16).

2H3-Betaine Aldehyde Feeding Experiments Greenhouse-grown plants of betaine-deficient (86W-8152) and betaine-containing (86W-8JS2) selections from population P77 were used. Plants had been previously exposed to salinity stress (150 mm NaCl) as described above, for determination ofbetaine levels at 6 weeks after planting. Following screening of the populations for betaine level, plants were returned to normal irrigation with nutrient medium lacking NaCl (17) for a further 2 weeks. Leaf discs (23 mm diameter) from expanded leaves from five individual plants of each genotype were bulked and vacuum-infiltrated with 1.6 mM 2H3-betaine aldehyde chloride and then incubated for up to 5 h under fluorescent lights (19). Betaine pools were extracted at various time intervals and purified by ion exchange chromatography as described above. Quantification of 2H3- and 2Ho-betaine was by FAB-MS of n-butyl esters at m/z 177 and 174, respectively, relative to a 2Hg-betaine internal standard atm/z 183 (19).

[14C]Choline Feeding Experiments [14C]Choline (55 mCi/mmol) was supplied by Amersham; analyses before and after treatment with alkaline H202 (7) showed that the levels of ['4C]betaine and [14C]betaine aldehyde contaminants were very low (0.08 and 0.21%, respectively). Plants of betaine-deficient and betaine-positive selections from population P77 were used, in addition to plants from CIMMYT Drought Tolerant Population Cycle 3. The latter population, formed from tropical and temperate germplasm from dry areas which performed well under drought, had been shown to contain high levels of betaine. These materials were grown under irrigation in the field at Tlaltizapan, Morelos, Mexico (19°N, 940 m elevation) during the 1988/1989 dry season, and used for experiments at 11 to 15 weeks after planting. Two plants of each genotype were uprooted in the morning and kept with their roots in water until the leaf blades to be labeled were harvested. The blades were cut under water, placed with their bases in 20-mL vials containing 2 ,uCi of [14C]choline in 2 mL of water or 5 mm KCI, and then exposed to full sun for 6 h, replacing the water or KCI solution in the vials as it was drawn down by transpiration. At the end of the experiment, the blades were separated into three parts of equal length; each part was then cut into 1-cm sections and stored in 20 mL methanol at -15°C or below. Tests showed that the basal part retained most of the label; only this part was analyzed further, as follows. Chloroform (0.42 volume) and water (0.08 volume) were added to the methanol containing the leaf tissue, which was then ground with sand. The residue was extracted with a further 10 mL of methanol/chloroform/water (12:5:1, v/v); the extracts were pooled and separated into aqueous and organic phases by adding 0.25 volume of chloroform and 0.38 volume of water. An aliquot of the aqueous phase was taken for scintillation counting, and the remainder was evaporated to dryness at 50°C in a stream of N2. One-third ofthis aqueous extract was redissolved in water, and fractionated on a two-

BETAINE DEFICIENCY IN MAIZE

column series (mixed-bed Dowex- l/Biorex-70, followed by Dowex-50), as described previously (8). After washing the columns with water, the betaine-containing fraction was eluted from Dowex-50 with 4 N NH40H, and evaporated to dryness in an air stream under an infrared lamp. Aliquots of this fraction were used to determine '4C-incorporation into betaine; analysis by high voltage electrophoresis on ITLC SA plates (Gelman) in 1.5 N formic acid (9) confirmed that the only labeled product present comigrated with betaine. Measurements of Choline and Betaine Pools by DCI-MS

For the genotypes used for ['4C]choline feeding experiments, the endogenous pools of choline and betaine were estimated by isotope dilution/DCI-MS, using a Finnigan 4000 GC/MS with Incos Data System (San Jose, CA) and a Vacumetrics (Ventura, CA) desorption chemical ionization probe. The samples (containing 1000 nmol 2Hg-choline and 492 nmol 2Hg-betaine as internal standards) were placed on the rhenium filament wire of the probe from solution (methanol solvent) and evaporated prior to desorption and mass analysis. The sample was desorbed by passing 3 A of current (rise time 7.5 s) through the filament. The reagent gas was isobutane, at an ion source gauge pressure of 0.4 torr. The ion source temperature was maintained at 250°C. Cationic fractions eluted from Dowex-50 (H+) with 2.5 N HCI were evaporated to dryness and the residue was extracted in acetonitrile:methanol (20: 1, v/v). The acetonitrile:methanol extracts (essentially free of inorganic salts) were evaporated to dryness for DCI-MS analysis. Choline was determined directly, and betaine after derivatization to the n-butyl ester. For choline measurements, the mass range scanned was 60 to 180 amu; 2Ho- and 2Hg-choline yield strong signals at m/z 90 and 96, respectively, corresponding to loss of a single methyl group from the quaternary ammonium moiety. Choline was quan-

Table I. Betaine Levels in F1 Hybrids between Betaine-Deficient and Betaine-Positive Maize Genotypes Mean Betaine Levela nmol/g fresh wt

Cross

Al 88(_)b A641 (-) A656(-)

A554(+)b

4696 (693) 2643 (290) 2501 (188) 5710 (499) x A632(+) 729-13F3(-) x 2708(-) 5631 (516) A632(+) 4500(278) x Oh43(-) B73(+) x MS71 (+) 1746C (334) 86W-9S2(-) x N6(-) 2423C (1 14) A632(+) x WF9(+) 1 922C (188) H84(-) x 86M-9S2(-) 2895C (237) W64A(+) 221 9C (182) x RSA(-) H49(+) x H95(-) 3352 (115) A632(+) 1999 (113) x B37(-) B73(+) aBetaine determined by the periodide method on field grown material. All values are the means of three individual plants. Standard b Betaine-deficient types are errors are shown in parentheses. c Determined in designated by (-), betaine-positive types by (+). the 1990 growing season; all other determinations were in the 1989 growing season. x

x A554(+) x A554(+)

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Table II. Betaine Levels in F1 Hybrids among Betaine-Deficient Maize Genotypes Cross

Mean Betaine Levela nmol/g fresh wt

50 (17) A188 x A641 38 (16) A188 x A656 Al 88 x 729-13F3 20 (7) 55 (21) Al88 x 2708 Al 88 x Oh43 29b (4) 34b (6) Al 88 x 86W-9S2 30b (7) A188 x N6 17(9) N6xH84 41 (7) H84 x 86M-9S2 35 (14) H84 x RSA 29 (11) H84 x H95 14 (6) H95 x B37 a Betaine determined by the periodide method on field grown material. All values are the means of three individual plants. Standard b Determined in the 1990 growerrors are shown in parentheses. ing season; all other determinations were in the 1989 growing season.

tified from the ratio of ion intensities at m/z 90:96. For betaine determinations, the mass range scanned was 100 to 200 amu; 2Ho- and 2H9-betaine n-butyl esters yield strong signals at m/z 160 and 166, respectively, again resulting from loss of one methyl group of the quaternary ammonium moiety. Betaine was quantified from the ion ratios at m/z 160:166. Values were converted from a leaf area to a fresh wt basis using a conversion factor of 18 mg/cm2 (17). Measurements of Endogenous Betaine Aldehyde Pools Eight genotypes were chosen from CIMMYT drought screening nurseries grown at Tlaltizapan during the 1988/ 1989 dry season. These genotypes were chosen to represent a range of betaine levels; they included germplasm from temperate and tropical sources. Temperate materials were: F2 populations from Pioneer hybrids 3358 and 3184 (betaine deficient) and Nebraska NS(FS)CT-F-Family 8101-81 (betaine positive). Tropical materials (all betaine positive) were: Tamaulipas 44, Coahila 54, Puebla 209 x Pool 34, Pool Sequia x Tuxpefio Nuevo Leon Gpo. 2, and Pool Sequia x Tabloncillo Sinaloa 31. Plots were sampled at 11 weeks after planting (near anthesis). Thirty-six 2.4-cm discs were harvested from young, fully expanded leaves of each entry under both irrigated and nonirrigated regimes, and stored in 15 mL of methanol. Sample extraction and betaine assay by FABMS were as above. Betaine aldehyde was determined by FABMS as the di-n-butyl acetal derivative (19). Values were converted from a leaf area to a fresh weight basis as above. RESULTS AND DISCUSSION Crosses between Betaine-Deficient and Betaine-Positive Genotypes The 13 betaine-deficient types included in this study were crossed to various betaine-positive wild types. The resulting hybrids were all betaine positive (Table I), with betaine contents approximately 30- to 200-fold higher than those typical

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of deficient types (ref. 3; see also Table II). Confirming this, 17 further hybrids produced by crossing the betaine-deficient genotypes to one or more additional betaine-positive types were also found to be betaine-positive (data not shown). These data demonstrate that betaine deficiency is a recessive condition in all 13 genotypes tested, so that complementation tests for allelism can be applied. Complementation Tests

The 13 betaine-deficient types were intermated such that each was tested directly or indirectly for complementation with all others (Table II). Thus, F1 hybrids between Al 88 and seven other genotypes including N6 were all betaine deficient, showing that alleles of the same gene are involved. The hybrid N6 x H84 was deficient, establishing that the betaine deficiency gene in H84 is also allelic with that of A188. Last, hybrids between H84, 86M-9S2, RSA, and H95 were again deficient, as was H95 x B37, showing that these sources share the same deficiency gene as all the others. Consistent with this, when the betaine-deficient types were crossed in 18 additional combinations, all of the hybrids were betaine deficient (data not shown). Betaine Deficiency in Population P77 source of betaine deficiency tested above (86W-9S2) selection from a population (P77) which included betaine-positive and -deficient individuals (Table III). As P77

One

was a

Table IV. Oxidation of 2H3-Betaine Aldehyde to Betaine by Maize Leaf Discs 2H3-Betaine 2HO-Betaine Genotype

(endogenous) Oh

Oh

3h

nmo//g fresh Wtb

579 313 30 10,116 86W-8JS2 (+)C 618 299 9 29 86W-81S2 (_)C a Time of incubation in 1.6 mM 2H3-betaine aideb Determined by FAB-MS. c Betaine-deficient types are hyde. designated by (-), betaine-positive types by (+); see Table IlIl for origin of these lines.

appeared to be morphologically and developmentally uniform, it was of interest to study its pedigree and population structure with a view to developing related lines differing in betaine for biochemical studies. P77 was originally derived from IR 11 and was maintained by a combination of sibbing and selfing yearly between 1972/73 and 1983 (personal communication, Dr. A. J. Pryor, CSIRO, Canberra). IRl 1 was an F, hybrid constructed in 1969 by Dr. A. L. Hooker from derivatives of the inbreds R168 and B14 into both of which the Rpidt21 rust resistance gene had been introduced by backcrossing (personal communication, Dr. A. L. Hooker, formerly Professor of Plant Pathology and Genetics, University of Illinois). When the inbreds R168 and B14 (and their RpId(2) derivatives) were tested for betaine under salinized conditions

Table ll. Distribution of Betaine Content in the Maize Population P77 and Its Selfed Progeny Betaine Classd Screening Leaf Type Population/ 5 6 7 8 9 10 2 3 4 1 Selectiona Assayedb Environmentc

11

no. of individuals in each class

1A 0 7c 3F 0 0 0 0 0 0 0 F ME, FAB 0 00 0 0 1B 1 2 4DE oQ G/NS ME, Per 9G oQ 0 0 0 0 0 0 0 F 0 A86W-9S1 YE, FAB 6 5H 0 00 00 0 F 0 0 0 YE, FAB F86W-5S, 0 0 0 0 0 0 0 F 0 6 0 0 YE, Per G86W-9S2 0 0 0 2 2 0 0 F 0 0 0 0 YE, Per H86W-5S2 0 00 0 0 0 0 0 9 00 G/SS YE, Per G86W-9S2 0 0 0 1 3 2 4 0 0 00 G/SS YE, Per H86W-5S2 0 0 000 1 0 1 11 0 0 0 F YE, Per c86W-8S, 0 0 0 0 0 0 0 14 0 0 0 G/SS YE, Per '86W-81S2 1 5 3 1 3 3 1 0 0 0 0 G/SS YE, Per J86W-8JS2 0 0 0 0 0 0 0 0 18 0 0 G/SS YE, Per B86W-12S, 1 0 3 5 1 0 2 0 4 1 1 G/SS D86W-13S1 YE, Per 0 0 3 4 1 0 0 1 9 00 G/SS E86W-14S1 YE, Per a Individuals (A-F) from the original population (P77, redesignated 86W) were selected and selfed, and the resulting S1 populations were evaluated. Individuals (GJ) from these Si populations were further b Initial studies on the original population selfed and the resulting S2 populations were evaluated. (P77) were performed on mature, expanded (ME) leaves. Following the finding that betaine levels tend to reach their maximum in young, expanding (YE) leaves (18), all betaine assays of selfed progeny were performed on YE leaves. Betaine quantification was performed by either FAB-MS (FAB) or by the C The screening environment was of three types: F, field; G/ periodide (Per) method, as indicated. NS, greenhouse with no imposed salinity or water stress; or G/SS, greenhouse with 150 mm NaCI d Betaine classes are as follows (,umol/g fresh wt): Class 1, 8.0. P77 (86W) P77 (86W)

5 ha

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Table V. Oxidation of [14C]Choline to Betaine by Detached Maize Leaves Tracer [14C]choline (2 glCi/leaf) was introduced via the transpiration stream into detached leaves during 6 h in full sunlight. The lowest one-third of the leaf, which contained most of the label, was separated from the remainder for analysis. The 14C data are means for duplicate leaves. 14C Content Pool Size Leaf Type Choline Genotype Betaine8 Aqueous BetaineCholine

nmol/g fresh wt

Drought pool Cycle 3

6490b

1 220b

Beanl

~~~~~extract

nCi

Expanding Expanded

1049 1065

70.6 64.2

Selections 571 od 760 960d 10.6 86W-5S2 (+)C Expanded 715 0.7 1420' 50d 86W-9S2 (.)c Expanded a Radioactivity in betaine corrected for 0.08% [14C]betaine present in the [14C]choline supplied, taking b Determined on the 14C content of the aqueous extract as an estimate of total [14C]choline uptake. c Betaine-deficient types are designated upper leaves from the plants used for [14C]choline labeling. d Determined on pooled samples by (-), betaine-positive types by (+); see Table IlIl for origin of lines. from the upper leaves of plants comparable to those labeled with [14C]choline. Because the 2Hg-choline intemal standard was added after ion exchange chromatography, choline values were corrected for losses of choline prior to adding the standard, found to average 30%.

in the greenhouse, they were found to be respectively betainedeficient (R168 = 55 ± 5 nmol/g fresh weight; Rl68-Rpid(2) = 89 ± 10 nmol/g fresh weight) and betaine-positive (B14 = 9135 ± 537 nmol/g fresh weight; Bl4-Rp1d(2) = 8176 ± 928 nmol/g fresh weight). Each of these values represents the mean (± standard error) of 5 individuals of each genotype. This information allows two inferences. First, as P77 is a small population that has been inbred with some selection it is expected to show relatively little genetic variance for most characters and to contain many closely related individuals (1). Second, assuming betaine was an essentially unselected trait, P77 would be expected to show segregation for betaine. The results of selfing selected individuals from the P77 population were consistent with the second inference (Table III). True breeding betaine-positive and -deficient plants were found, as well as three individuals whose progeny segregated for betaine. Selections from the SI progeny of two true breeding plants, 86W-9 (deficient) and 86W-5 (positive) (Table III), were themselves selfed and seeds bulked to give the selections denoted 86W-9S2 and 86W-5S2. These showed no obvious differences in morphology or development, consistent with expectations for plants with similar genetic backgrounds. The selections 86W-5S2 and 86W-9S2 have given rise to exclusively betaine-positive and betaine-deficient S3 progeny, respectively (not shown). These selections were used for the ['4C]choline precursor feeding studies below. A pair of lines independently derived from the P77 population (86W-8IS2 [deficient] and 86W-8JS2 [positive]; see Table III) were used for the 2H3betaine aldehyde feeding studies below. These lines, along with betaine-positive and -deficient sweetcorn material (18), will also be useful in developing a set of near-isogenic inbreds and heterotic hybrids differing in betaine, for evaluation of the physiological and agronomic significance of betaine deficiency.

Oxidation of 2H3-Betaine Aldehyde

Leaf discs of typical betaine-positive and deficient selections of P77 grown under salinized greenhouse conditions oxidized 2H3-betaine aldehyde to betaine at similar rates (Table IV), confirming an earlier result obtained with sweetcorn hybrids (19). Taking the endogenous (2Ho) betaine content of 10 ,umol/g fresh weight from Table IV, a maximum relative leaf growth rate of 0.15 d-' (4), and assuming that all betaine synthesis occurs in leaf cells (7, 9), the endogenous rate of betaine synthesis can be estimated at c63 nmol/g.h. The observed rates of 2H3-betaine aldehyde oxidation (- 120 nmol/ h * g) are therefore clearly adequate to account for endogenous betaine synthesis. Oxidation of [14C]Choline

Earlier studies failed to demonstrate conversion of supplied choline to betaine by maize leaf tissue (19). However, the genotypes used were not grown in a stress environment and were quite low in betaine (