Dependence of Abscisic Acid Accumulation in Leaves of Pearl Millet (Pennisetum americanum[L.] Leeke) on Rate of Development of Water Stress

Henson, I. E. 1985. Dependence of abscisic acid accumulationin leaves of pearl millet {Pennisetum americanum [L.] Leeke)on rate of development of water stress.—J. exp. Bot. 36:1232-1239. The amount of abscisic acid (ABA) which accumulated in attachedleaves of pearl millet (Pennisetum americanum [L.] Leeke) inresponse to water stress was found to depend on the rate atwhich the stress developed, as well as on the intensity of stress.In young, pot-grown plants, the increase in leaf ABA concentration([ABA]) was linearly related to the fall in water () and turgor(_p) potentials. However, the amount by which [ABA] increasedper unit change in or _p, was greater at high than at low ratesof stress development. Evidence is presented which suggeststhat the same effect may occur under field conditions and contributeto differences in ABA accumulation between plants grown in potsand those grown in the Held. Key words: Pennisetum americanum, abscisic acid, water stress, rate of stress development     

Solute Accumulation and Growth in Plants of Pearl Millet (Pennisetum americanum [L.] Leeke) Exposed to Abscisic Acid or Water Stress

Henson, I. E. 1985. Solute accumulation and growth in plantsof pearl millet (Pennisetum americanum [L.] Leeke) exposed toabscisic acid or water stress.—J. exp. Bot. 36: 1889–1899.Experiments were conducted to investigate whether abscisic acid(ABA) elicits the accumulation of solutes and lowering of osmotic(solute) potential (₂) which occurs in leaves of pearl millet(Pennisetum americanum [L.] Leeke) exposed to water stress.When (?)–ABA was injected into the base of the shoot of15–d–old plants, ₂ of the fifth leaf was reducedsignificantly below controls 27–72 h after treatment.The reductions, however, were small (< 0.10 MPa) and wereaccompanied by a significant inhibition of shoot growth. Incontrast, no significant reduction in ₂ or in growth occurredwhen the hormone was introduced directly into leaf five viaits mid–rib. ABA concentrations in leaf five were highshortly after direct injection, but declined to control levelswithin 48 h. Injecting ABA into the shoot base resulted in lowerleaf five ABA concentrations. Hence, the ABA concentration inthe leaf was not the most critical factor for its effect on₂. ABA also reduced ₂ of shoots when applied to seedlings 48h or 72 h after sowing via the roots. As with older plants,the effects of ABA on ₂ were small ( 0–2 MPa) and wereaccompanied by inhibition of shoot growth. A water stress treatmentand an ABA treatment were compared. Although both treatmentsresulted in a similar degree of growth inhibition, the stresstreatment was much more effective than was ABA in reducing ₂. Key words: Pennisetum americanum [L.], pearl millet, abscisic acid, water stress, osmotic     

Leaf Abscisic Acid Content and Recovery from Water Stress in Pearl Millet (Pennisetum americanum [L.] Leeke)1

Water potential () and abscisic acid (ABA) content were measuredin leaves of drought-stressed, field-grown plants of pearl millet(Pennisetum americanum [L.] Leeke) during rehydration, initiatedeither in response to a diurnal night-time reduction in evaporativedemand, or to irrigation. Overnight rehydration, manifestedas a substantial increase in , was not accompanied by any reductionin ABA levels. In contrast, an increase in following irrigationresulted in an appreciable reduction in ABA content. Such reductionwas, initially, only partial when field plants were rewateredat dusk, but was rapid and complete within 3 h when irrigationwas applied mid-afternoon. The possibility that light or temperature changes might haveprevented loss of ABA during night-time rehydration was investigatedin pot experiments. At similar air temperatures, young pot-grownplants rehydrated more rapidly, and ABA levels fell more quickly,in darkness than in light. The onset of rehydration and lossof ABA in darkness were delayed by low (20 ?C) compared withhigh (28 ?C) temperature, though after an initial lag, ratesof both processes at 20 ?C were similar to rates at 28 ?C. Neitherlight nor temperature affected the relationship between ABAcontent and *. Key words: Abscisic acid, Rehydration, Pennisetum americanum     

Diurnal Changes in Endogenous Abscisic Acid in Leaves of Pearl Millet (Pennisetum americanum (L. ) Leeke) under Field Conditions

Diurnal variation in leaf abscisic acid (ABA) content was investigatedin pearl millet (Pennisetum americanum (L. ) Leeke) growingin the field in the semi-arid tropics and subjected to varyingdegrees of water stress. There was a two- to three-fold change in ABA content duringthe photoperiod in three groups of ‘severely’ stressedplants of the genotype BJ 104. Maximum ABA occurred mid-morning(1030 h). ABA levels then declined to a minimum at 1500 h. Changesin ABA content of ‘moderately’ stressed and fullyirrigated plants were smaller, but still significant. Though,when averaged over the day, levels of ABA of the five groupswere positively related to the degree of water stress, relationshipsbetween ABA concentration and total water () or turgor (_p) potentialsvaried considerably with time of sampling. Within groups, changesin ABA contents during the day were not always accounted forby changes in or _p. Temporal changes in leaf ABA content similar to those foundin BJ 104, and largely unrelated to , were observed in the genotypesSerere 39 and B282 in a subsequent year. Leaf ABA content of droughted plants (BJ 104) did not declineappreciably overnight despite a marked increase in . However,a large reduction in ABA content with increase in did occurfollowing heavy rainfall. Diurnal changes in stomatal conductance (g₁) of BJ 104 couldnot be simply accounted for by temporal changes in total leafABA content, even when allowance was made for effects of irradianceand other environmental variables on g₁. It is suggested thatthe sensitivity of stomata to ABA, or accessibility of the hormoneto the stomatal complex, changes during the day.     

Stomatal Responses of Pearl Millet (Pennisetum americanum (L.) Leeke) Genotypes, in Relation to Abscisic Acid and Water Stress

Stomatal responses to water stress and to applied (±)-abscisicacid (ABA) were examined in genotypes of pearl millet (Pennisetumamericanum (L.) Leeke) known to differ in amounts of endogenousABA accumulating during drought. In both a pot and a field experiment,Serere 39, a genotype with a high capacity to accumulate ABA,showed a higher stomatal sensitivity to water stress than didthe ‘low’ ABA accumulator, BJ 104. In the fieldexperiment, a third genotype, B282, accumulating least amountsof ABA, also had the lowest stomatal sensitivity to water stress. There were no significant differences between these genotypesin stomatal response to applied (±)-ABA, or in the relationshipsbetween leaf conductance and levels of endogenous ABA. It isconcluded that the differences in accumulation of endogenousABA by these genotypes of pearl millet are of functional significance,and that endogenous ABA generated during a water stress whichdevelops over days or weeks mediates stomatal responses to suchstress.     

Genotypic Variation in Pearl Millet (Pennisetum americanum (L.) Leeke), in the Ability to Accumulate Abscisic Acid in Response to Water Stress

Variation between genotypes in the ability to accumulate abscisicacid (ABA) in response to water stress was investigated in pearlmillet (Pennisetum americanum (L.) Leeke). Using a detachedleaf test a more than four-fold variation in accumulation capacitywas observed amongst a set of 16 genotypes grown in a controlledenvironment. Two genotypes which contrasted in their accumulationcapacity, BJ 104 and Serere 39 (the latter accumulating mostABA), maintained the difference over a range of leaf water contentsand potentials. Some of the genotypes were grown in the field in the semi-aridtropics with and without irrigation, and sampled for ABA content.In two experiments, substantial genotypic variation in ABA accumulationwas observed, which could not be attributed to differences inleaf water potential (). In a field experiment comparing threegenotypes (Serere 39, BJ 104, and B282), differences in ABAaccumulation were also shown to be largely independent of genotypicdifferences in turgor potential (_p). For a set of six of the genotypes, the amounts of ABA accumulatingin leaves of intact, droughted plants, in the field, when adjustedfor differences in , were found to be significantly (P<0.05)correlated with amounts of ABA accumulated in detached, water-stressedleaves. It is concluded that the detached leaf test adequatelyreflects the ability of pearl millet genotypes to accumulateABA under field conditions.     

Evidence of a Role for Abscisic Acid in Mediating Stomatal Closure Induced by Obstructing Translocation from Leaves of Pearl Millet (Pennisetum americanum [L.] Leeke)

Application of a heat girdle near the base of the lamina ofthe fifth, fully expanded leaf of young pearl millet (Pennisetumamericanum [L.] Leeke) plants resulted in a decrease in solutepotential, an increase in leaf dry matter content, and a declinein stomatal conductance and in the rate of CO₂ assimilation.Total water potential was largely unaffected by girdling whileturgor potential increased as a consequence of the decreasein solute potential. Abscisic acid (ABA) content of the leaf increased 5 to 6-foldwithin 1 h of girdling, then declined equally rapidly beforeincreasing again at a slower rate. The decline in conductance was correlated with both the decreasein solute potential and the increase in ABA. To determine whichof these factors could be controlling conductance, girdled leaveswere exposed either to 14 h of continuous light or to a similarperiod of darkness followed by a brief light treatment to allowstomata to open. Girdling reduced conductance equally followingdarkness or light but solute accumulation occurred only in thelight. ABA accumulated in girdled leaves in both darkness andlight. Simultaneous measurements of conductance and CO₂ assimilationshowed that intercellular CO₂ concentration did not increasefollowing girdling. It was concluded that the decrease in conductancein millet leaves after girdling was most probably mediated bythe increase in ABA content. Key words: Leaf girdling, Solute accumulation, Stomatal conductance, Abscisic acid; Pennisetum americanum     

An Association between Flowering and Reduced Stomatal Sensitivity to Water Stress in Pearl Millet [Pennisetum americanum (L.) Leeke]

Stomatal sensitivity to water stress was investigated in pearlmillet [Pennisetum americanum (L.) Leeke] in relation to stageof plant development, leaf water status and ABA content by samplingplants at midday. For the same leaf water potential (), droughtedplants with emerged panicles were found to have a greater leafconductance (g_L), indicative of greater stomatal opening, thanplants sampled prior to panicle emergence. The difference betweensuch flowering (F) and non-flowering (NF) plants in at stomatalclosure was estimated to be at least 0.6 MPa. This differencewas considered unlikely to be the result of differential bulkleaf osmotic adjustment, and for most samples from both F andNF plants, bulk leaf turgor potential (_p) was estimated to bezero. Stomatal closure in NF plants was associated in two genotypes(BJ 104 and line 112) with higher leaf ABA levels. Differencesin ABA levels between F and NF plants were, however, smalleror absent in genotypes Serere 39 and B282. These genotypes wereat lower than BJ 104 and line 112 when sampled and showed smallerdifferences between F and NF plants in conductance. Lower ABA levels in F plants are ascribed either to effectsof leaf ageing or to effects of flowering on ABA content ofthe leaf. Significant differences in g_L in the absence of differencesin ABA content are taken to imply changes in stomatal sensitivityto the hormone or in its access to the stomatal complex. Pennisetum americanum (L.) Leeke, pearl millet, flowering, stomata, water stress, abscisic acid     

The Effect of Flowering on Stomatal Response to Water Stress in Pearl Millet (Pennisetum americanum [L.] Leeke)

Stomata on upper leaves of drought-stressed pearl millet (Pennisetumamericanum [L.] Leeke) crops were more open in flowering (F)than in pre-flowering (PF) plants. This was not due to differencesin leaf water potential (). Stomata of PF plants closed when fell to about –1.7 MPa, while on F plants stomata closedonly when approached –2.3 MPa. Osmotic adjustment did not account for these differences asrelations between turgor potential (_P) and were similar inF and PF plants. While stomata of PF plants closed as W becamezero, in F plants stomata remained open even after bulk leafturgor was lost. Differences between F and PF plants were not explained by differencesin age of leaves sampled. However, leaves of water-stressedPF plants had higher levels of abscisic acid (ABA) than leavesof F plants, despite similarities in water status. From theseresults and from relationships between g_L and stage of panicledevelopment, it is concluded that the tendency of stomata toremain open despite water stress and loss of bulk leaf _P isrelated to the presence of an emerged panicle. Hypotheses whichaccount for this effect are discussed. Key words: Pennisetum americanum [L.] Leeke, Pearl millet, Flowering, Stomata, Water stress, Abscisic acid     

Light-Induced Chloroplast [alpha]-Amylase in Pearl Millet (Pennisetum americanum)

In pearl millet (Pennisetum americanum) seedlings light induces the appearance of a leaf [alpha]-amylase isozyme. The leaf [alpha]-amylase isozyme was present in enriched amounts in isolated chloroplast but it could not be detected in isolated etioplasts. The chloroplast [alpha]-amylase was present in both mesophyll and bundle-sheath chloroplasts. Preliminary characterization indicated that molecular properties of chloroplast [alpha]-amylase were like those of a typical [alpha]-amylase. The plastidic [alpha]-amylase had a molecular mass of 46 kD, pH optimum of 6.2, required Ca2+ for activity and thermostability, but lost activity in the presence of ethylenediaminetetracetate. Plastidic [alpha]-amylase activity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis could be renatured in situ by Triton X-100. Western blot analysis demonstrated that this protein was antigenically similar to a maize seed [alpha]-amylase. In vivo [35S]methionine labeling of bundle-sheath strands isolated from light-grown leaves followed by immunoprecipitation revealed that bundlesheath strands synthesized plastidic [alpha]-amylase de novo.     

Trisomic categories in pearl millet (Pennisetum americanum (L.) Leeke)

Summary In pearl millet [Pennisetum americanum (L.) Leeke], in the open pollinated and crossed progenies of autotriploids, desynaptics and translocation heterozygotes, two primary trisomics, one each of secondary and tertiary trisomics, two primary trisomics with interchanges, two interchange secondary trisomics, and three interchange tertiary trisomics were located. These categories were determined on the basis of chromosomal associations formed at meiosis. In one other trisomic, its category, whether tertiary or interchange trisomy, could not be determined. Some of these categories, like the secondary trisomy and interchange tertiary trisomy, are reported for the first time.     

Uptake,Translocation, Distribution and Persistence of 14C-Metalaxyl in Pearl Millet (Pennisetum americanum) [L.] Leeke)1

Time course absorption and desorption of metalaxyl by seeds of pearl millet was analysed by following chemical kinetics equations. Uptake of metalaxyl through roots, leaves and seed, its translocation and distribution in different plant parts and persistence following seed application were studied in pearl millet using ¹⁴C-metalaxyl. Both uptake and efflux of metalaxyl by pearl millet seeds were complex and compartmentalized. Distribution inside the seed was not uniform. A major part of applied fungicide remained within the treated plant part, particularly after seed and foliar applications. Metalaxyl was ambimobile inside the plant and was found to get accumulated at apex and margins of leaf blade. No metalaxyl could be detected in grains, harvested from plants grown from metalaxyl treated seeds.     

Osmotic Adjustment to Water Stress in Pearl Millet (Pennisetum americanum (L. ) Leeke) in a Controlled Environment

A pressure-volume (P-V) and an expressed sap (cryoscopic) techniquewere compared for assessing osmotic adjustment to water stressby pearl millet (Pennisetum americanum (L. ) Leeke) plants grownin a controlled environment cabinet. For leaf water potentials( ) above the point of zero turgor, there was good agreementbetween estimates of solute potential ( _s)and turgor ( _p) obtainedby the two methods. Reductions in pre-dawn leaf to –1.8 MPa over 5–6d resulted in net solute accumulation as indicated by a fallin s at full hydration of about 0.3 MPa. The degree of osmoticadjustment increased linearly with the decrease in pre-dawn. Adjustment in cv. BJ 104 was significantly (P < 0.05) lessduring a second drought than during a first, and cv. Serere39 was significantly (P < 0.05) less able to adjust osmoticallythan BJ 104. Adjustment was greater in leaves which were undergoing extensiongrowth during the drought than in leaves already fully extendedbefore drought started. Much of the adjustment was lost within24 h following rewatering, the loss being most complete in theolder, fully extended leaves.     

Effect of the Duration of the Vegetative Phase on Shoot Growth, Development and Yield in Pearl Millet (Pennisetum americanum (L.) Leeke)

Craufurd, P. Q. and Bidinger, F. R. 1988. Effect of the durationof the vegetative phase on shoot growth, development and yieldin pearl millet (Pennisetum americanum (L.) Leeke).–J.exp. Bot. 38: 124–139 The duration of the vegetative phase (DVP) in millet, whichis the major cause of variation in the crop duration, has markedeffects on the number of productive tillers per plant and onmainshoot (MS) and tiller grain yield. Daylength extensionswere used to vary the DVP and the effect on factors affectingpanicle (tiller) number per plant and panicle yield examinedin millet hybrid 841A x J104, grown in the field at Hyderabad,India. Tiller appearance, shoot leaf appearance and leaf area,and stem and panicle growth, in both MS and primary tillers(PTs), were monitored at frequent intervals over the season.At maturity grain yield per shoot was measured The concept of thermal time was used to describe shoot development.The rates of tiller appearance and shoot leaf appearance werelinearly related to thermal time and were not affected by DVPtreatments. The duration of the growth phase from panicle initiationto flowering (GS2) and from flowering to maturity (GS3) was320 and 390 degree days (°Cd), respectively. There was nodifference in rates of leaf or tiller appearance or developmentbetween MS and PTs. Tiller appearance, tiller leaf appearanceand tiller apical development all ceased at the same time inthe later initiated PTs, approximately 550 °Cd from sowing,shortly after rapid stem growth had begun. Tillers that didnot survive were all vegetative or in the early stages of reproductivedevelopment at this time The rate of accumulation of dry matter per plant was similarin all DVP treatments, but in the longer DVP treatments a greaterproportion of the dry matter was partitioned to the MS. Mainshootstem and panicle growth rates were increased by a longer DVP,as was grain yield on the MS, and these were related to increasedMS leaf area. Concurrently, growth rates and yields in laterinitiated tillers were reduced in relation to their leaf areas.Stem growth rate was proportionately increased more than paniclegrowth rate in the longer DVP treatments and this, combinedwith a longer duration of stem growth, resulted in greater stemdry matter at maturity and, therefore, in reduced harvest index.     

Abscisic Acid Metabolism in Water-stressed Bean Leaves

Phaseic acid (PA) and dihydrophaseic acid (DPA) are the major metabolites observed when (S)-2-¹⁴C-abscisic acid (ABA) is fed to 14-day excised primary bean leaves (Phaseolus vulgaris L. cv. Red Kidney). The distribution of ¹⁴C in leaves which were wilted after feeding ABA appears to be the same as that observed in unwilted leaves. A reduction in the relative specific radioactivities of the two metabolites after wilting, compared with the specific radioactivities measured in unwilted plants, indicated that these metabolites continue to be formed endogenously after wilting. Estimates of the endogenous ABA levels showed that they rose from 0.04 μg to approximately 0.5 μg/g fresh weight within 4 hours after the beginning of a 10% wilt and remained at that level during a subsequent 20 hours of wilt. In unwilted leaves, the levels of PA and DPA were 5 times and 20 times higher than that of ABA, respectively. Both PA and DPA levels rose throughout the wilt period. PA rose from 0.20 μg to 1.0 μg and DPA from 0.8 μg to over 3 μg/g fresh weight. From these data, we calculated the rate of ABA synthesis to be at least 0.15 μg/hr.g fresh weight during this period. We have interpreted these results to mean that in wilted leaves an elevated level of ABA is maintained because the rate of synthesis and metabolism are both elevated and approximately equal.     

Stomatal Response to Water Stress and its Relationship to Bulk Leaf Water Status and Osmotic Adjustment in Pearl Millet (Pennisetum americanum [L.] Leeke)

The water potential () at which stomata completed closure (_8Lmin)was determined for pearl millet (Pennisetum americanum [L.]Leeke) at two growth stages by monitoring changes in leaf conductance(g_L) and following shoot detachment. Leaf water status wasevaluated concurrently using a pressure-volume (P-V) technique. In a pot experiment with young vegetative plants, _8Lmin closelyapproximated to the estimated at zero turgor (_u) both for controland for drought-conditioned plants which had osmotically adjusted.However, for penultimate leaves of field-grown flowering plants,_8Lmin was found to be 0.61 (irrigated plants) and 0.87 (droughtedplants) MPa below _u. In drought-stressed field-grown plants,osmotic adjustment (characterized by a decrease in solute (osmotic)potential (_s ) at both full hydration and zero turgor) was insufficientto maintain a positive bulk leaf turgor potential (_p) once had declined to below about -1.5 MPa. It is suggested that localizedadjustment by the stomatal complex in response to environmentaldifferences, leaf ageing and/or ontogenetic change, is responsiblefor the uncoupling of stomatal from bulk leaf water status. Key words: Stomata, Water stress, Pennisetum americanum     

Analysis of meiosis and non-random bivalent formation in desynaptic mutants of Pennisetum americanum (L.) Leeke

Meiotic behaviour of induced desynaptic mutants of Pennisetum americanum (L.) Leeke in general was described.The frequency distribution of bivalents in induced desynaptic mutants was compared with that expected on the basis of the binomial series. The deviation from the binomial distribution was tested as to its conformity with models based on intra-and intercellular differences in bivalent formation. It is suggested that in these desynaptic mutants of Pennisetum americanum bivalent formation is non-random, which is largely due to the result of intracellular differences in chromosome behaviour regarding their requirements for chiasma formation.     

Plant Growth Substances Produced by Azospirillum brasilense and Their Effect on the Growth of Pearl Millet (Pennisetum americanum L.)

Azospirillum brasilense, a nitrogen-fixing bacterium found in the rhizosphere of various grass species, was investigated to establish the effect on plant growth of growth substances produced by the bacteria. Thin-layer chromatography, high-pressure liquid chromatography, and bioassay were used to separate and identify plant growth substances produced by the bacteria in liquid culture. Indole acetic acid and indole lactic acid were produced by A. brasilense from tryptophan. Indole acetic acid production increased with increasing tryptophan concentration from 1 to 100 μg/ml. Indole acetic acid concentration also increased with the age of the culture until bacteria reached the stationary phase. Shaking favored the production of indole acetic acid, especially in a medium containing nitrogen. A small but biologically significant amount of gibberellin was detected in the culture medium. Also at least three cytokinin-like substances, equivalent to about 0.001 μg of kinetin per ml, were present. The morphology of pearl millet roots changed when plants in solution culture were inoculated. The number of lateral roots was increased, and all lateral roots were densely covered with root hairs. Experiments with pure plant hormones showed that gibberellin causes increased production of lateral roots. Cytokinin stimulated root hair formation, but reduced lateral root production and elongation of the main root. Combinations of indole acetic acid, gibberellin, and kinetin produced changes in root morphology of pearl millet similar to those produced by inoculation with A. brasilense.     

Collection and evaluation of Pearl Millet (Pennisetum americanum) Germplasm from Ghana

An expedition to Ghana was undertaken during August 1981 to collect mainly the early-maturing pearl millet, Pennisetum americanum. The collection team travelled extensively in most of the pearl millet-growing areas of the eastern and northern provinces of Ghana. The mission was planned to coincide with harvesting so that early-maturing landraces could be obtained from farmers’ fields. Seed samples of late-maturing pearl millet were also obtained from local markets. Early-maturing pearl millet is traditionally intercropped with groundnut (Arachis hypogaea), sorghum (Sorghum bicolor) or late-maturing pearl millet. Pearl millet grain is used in several traditional food preparations: thick porridge called tô, a thin, fermented porridge calledkoko, and a deep-fried pancake calledmarsa. Landrace populations grown by the farmers were mixtures of several types. The material collected varied considerably for shapes, sizes and colors of spikes and grains. Of the 284 samples collected, 227 were grown in a uniform nursery at Patancheru: they flowered in 39–140 days, grew 120–315 cm tall, spikes were short (6–53 cm) and conical, grains were large, globular and gray with starchy endosperm. The samples belong to race globosum and serve as a good source of genes for earliness and large-grain size.     

Betaine Accumulation and [C]Formate Metabolism in Water-stressed Barley Leaves

Barley (Hordeum vulgare L.) plants at the three-leaf stage were water-stressed by flooding the rooting medium with polyethylene glycol 6000 with an osmotic potential of −19 bars, or by withholding water. While leaf water potential fell and leaf kill progressed, the betaine (trimethylglycine) content of the second leaf blade rose from about 0.4 micromole to about 1.5 micromoles in 4 days. The time course of betaine accumulation resembled that of proline accumulation. Choline levels in unstressed second leaf blades were low (<0.1 micromole per blade) and remained low during water stress. Upon relief of stress, betaine-like proline—remained at a high concentration in drought-killed leaf zones, but betaine did not disappear as rapidly as proline from viable leaf tissue during recovery.

When [methyl-¹⁴C]choline was applied to second leaf blades of intact plants in the growth chamber, water-stressed plants metabolized 5 to 10 times more ¹⁴C label to betaine than control plants during 22 hours. When infiltrated with tracer quantities of [¹⁴C]formate and incubated for various times in darkness or light, segments cut from water-stressed leaf blades incorporated about 2- to 10-fold more ¹⁴C into betaine than did segments from unstressed leaves. In segments from stressed leaves incubated with [¹⁴C]formate for about 18 hours in darkness, betaine was always the principal ¹⁴C-labeled soluble metabolite. This ¹⁴C label was located exclusively in the N-methyl groups of betaine, demonstrating that reducing equivalents were available in stressed leaves for the reductive steps of methyl group biosynthesis from formate. Incorporation of ¹⁴C from formate into choline was also increased in stressed leaf tissue, but choline was not a major product formed from [¹⁴C]formate.

These results are consistent with a net de novo synthesis of betaine from 1- and 2-carbon precursors during water stress, and indicate that the betaine so accumulated may be a metabolically inert end product.