Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Growth and Water Relations of Wilty Mutants of Tomato (Lycopersicon esculentum Mill.)

In contrast to some previous reports on the growth of the ABA-deficientwilty mutants of tomato, growth was at least as rapid in themutants as in the wild type, as long as an adequate plant waterstatus was maintained by growing the plants under mist. Moreover,shoot extension was greater and the rate of leaf productionmore rapid in the mutants. Stomatal changes in response to environmentand to time in the light-dark cycle were generally similar inboth wilty mutants and the wild type, though the wild-type weregenerally more closed. Grafting experiments confirmed that thegenotype of the shoot was dominant in determining stomatal aperture,though wild-type rootstocks could cause a slight reduction inthe stomatal conductance of mutant leaves. The effect on plantwater relations of draughting only part of the root system wasinvestigated in a ‘split-root’ experiment. Withholdingwater from only part of the root system was found to lower significantlythe mean leaf water potential, even though the potential evaporationrate was kept very small. Key words: Abscisic acid, stomata, tomato     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant II. Water relations, stomatal conductance, abscisic acid content in leaves and xylem sap of plants subjected to water deficiency

The response of w-1, a wilty sunflower (Helianthus annuus L.)mutant, to water stress is described in comparison with thecontrol line (W-1). Detached leaves of w-1 strongly dehydratedduring the first 30 min without significant changes in leafconductance, whereas W-1 responded rapidly to water loss byreducing stomatal aperture. After 2 h stress ABA increased slightlyin w-1, while W-1 leaves showed a 20-fold increase. When waterstress was imposed to potted plants by water withholding, w-1quickly dehydrated, and lost turgor, while W-1 maintained positiveturgor values for a longer period. Wild-type plants respondedto small changes in leaf water potential by accumulating ABAand by closing stomata, whereas in the mutant significant changesin ABA content and in stomatal conductance were found only atvery low water potentials. In another experiment in which waterwas withheld under high relative humidity, when soil water contentstarted to decrease W-1 rapidly closed stomata in the absenceof any change in leaf water status and the reduction in conductancewas paralleled by a rise in xylem sap ABA concentration. Bycontrast the mutant started to accumulate ABA in the xylem sapand to close stomata when soil water content and leaf waterpotential were dramatically reduced. The low endogenous ABAlevels and the inability to synthesize the hormone rapidly eitherin the leaves or in the roots seem to be responsible for thehigh sensitivity of w-1 to water stress. Key words: ABA, Helianthus annuus L, water relations, stomatal conductance, drought, wilty mutant     

The Physiology of a Wilty Pea: Abscisic Acid Production under Water Stress

‘Wilty’ (JI 1069), a mutant of Pisum sativum, hasbeen examined for its ability to produce abscisic acid (ABA)under water stress. ABA was measured using combined gas chromatography-massspectrometry and multiple-ion-monitoring employing a deuteratedinternal standard. In intact droughted plants, ‘Wilty’produced less ABA than a non-wilty line (JI 1194) and maximumproduction was delayed. Detached leaves of the wilty mutantlost significantly more water than control leaves but did notshow an increase in ABA content. Non-stressed mutant materialfrom both intact plants and isolated leaves contained less ABAthan control tissue. Key words: Pea, Wilty mutant, Abscisic acid     

Wild barley<Emphasis Type="Italic"> eibi1</Emphasis> mutation identifies a gene essential for leaf water conservation

Drought is a major abiotic stress that limits plant growth and crop productivity. A spontaneous wilty mutant (eibi1) hypersensitive to drought was identified from wild barley (Hordeum spontaneum Koch). eibi1 showed the highest relative water loss rate among the known wilty mutants, which indicates that eibi1 is one of the most drought-sensitive mutants. eibi1 had the same abscisic acid (ABA) level, the same ability to accumulate stress-induced ABA, and the same stomatal movement in response to light, dark, drought, and exogenous ABA as the wild type, revealing that eibi1 was neither an ABA-deficient nor an ABA-insensitive mutant. The eibi1 leaves had a larger chlorophyll efflux rate in 80% ethanol than the wild-type leaves; and the transpiration rate of eibi1 was more closely related to chlorophyll efflux rate than to stomatal density, demonstrating that the cuticle of eibi1 was defective. eibi1 will be a promising candidate to study the actual barrier layer in the cuticle that limits water loss of the plant. Exogenous ABA reduced leaf length growth in eibi1 more than in the wild type, implying an interaction on plant growth of ABA signal transduction and the eibi1 product. One may infer that the eibi1 product may reverse the growth inhibition induced by ABA.Abbreviation ABA Abscisic acid     

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant: III. Phenotypic interaction in reciprocal grafts from wilty mutant and wild-type plants

The present study was conducted to evaluate phenotypic interactionin reciprocal grafts between wilty (w-1) sunflower mutant andnormal (W-1) plants. The w-1 genotype is a ‘leaky’ABA-deficient mutant, characterized by high stomatal conductance,in both light and dark conditions, and high transpiration rate. In well-watered conditions, mutant scions grafted on to normalrootstock (w-1/W-1) showed higher leaf relative water content,leaf water potential and ABA levels than those of control grafts(w-1/w-1). In addition, detached leaves of w-1/W-1 exhibitedlower water loss than w-1/w-1 grafts, while mutant rootstockdid not affect the transpiration rate of detached W-1 leaves.When drought stress was imposed to potted plants by withholdingwater, the mutant scions grafted on to normal roots showed apartial phenotypic reversion. A rapid stomatal closure and arise in ABA levels in response to a small decrease in leaf waterpotential was observed. By contrast, in w-1/w-1 grafts significantreductions in stomatal conductance and ABA accumulation weredetected only in conjunction with a severe water deficit. W-1scions on mutant stocks (W-1/w-1) maintained the normal phenotypeof control wild-type grafts (W1/W-1). Key words: ABA, grafting, Helianthus annuus, stomatal conductance, water relations, wilty mutant     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant: I. Abscisic acid content, light-dark changes in the stomatal conductance and genetic analysis

The present study was conducted to characterize geneticallyand physiologically a spontaneous mutation in sunflower whichconfers a wilty phenotype. The wilting condition of the mutantis due to abnormal stomatal behaviour. The mutant stomata resistclosure in darkness. This abnormality is associated with lowlevels of endogenous abscisic acid (ABA). By artificially elevatingthe ABA content of the mutant plants by spray treatments with10^– and 10^– M solutions it proved possible to effecta phenotypic reversion of the mutant. It has, therefore, beenproposed that the primary effect of this spontaneous mutationis to reduce the level of ABA. The genetic analysis has shownthat the willy phenotype is due to recessive nuclear mutationat a single locus. Key words: ABA, Helianthus annuus L, stomatal conductance, wilty mutant     

Abscisic Acid and C10 Dicarboxylic Acids in Wilty Tomato Mutants

Linforth, R. S. T., Taylor, I. B. and Hedden, P. 1987. Abscisicacid and C10 dicarboxylic acids in wilty tomato mutants.—J.exp. Bot. 38: 1734–1740. The concentration of C₁₀ dicarboxylic acids in wilty tomatomutants was investigated. Three of the genotypes studied (flacca,sitiens and the double mutant homozygote flacca/sitiens) werefound to have higher concentrations of 2,7-dimethyl-2,4-octadienedioicacid (ODA) than the isogenic normal form. In contrast, the othergenotypes (notabilisand the double mutant homozygotes notabilis/flaccaand notabilis/sitiens) were found to have lower concentrationsof ODA than the isogenic normal form. The concentration of ODAin flacca plants was increased by water stress and reduced byexogenously applied abscisic acid (ABA). A second structurallyrelated compound, 2,7-dimethyl-4-octenedioic acid (OEA) wasalso quantified, but it showed no clear genotype-related pattern. The concentration of ABA in the wilty tomato mutants was alsoinvestigated. As expected in the light of previously publishedresults, it was reduced in the single mutants relative to theisogenic control plants. In the double mutant flacca/sitiensABA levels were similar to those of the single mutant sitiens.However, in the two double mutants notabilis/flacca and notabilis/sitiensABA was substantially lower than those in any other genotypeinvestigated. Key words: Abscisic acid, 2,7-dimethyl-2,4-octadienedioic acid, 2,7-dimethyl-4-octenedioc acid, tomato, wilty mutants     

Changes in Stomatal Conductance in Intact Ageing Wheat Leaves in Response to Abscisic Acid

The characteristics of ABA-induced changes in the fluxes ofCO₂ and water vapour from whole leaves of spring wheat (Triticumaestivum cv. Wembley) were examined. Aqueous solutions of ABAwere supplied via the transpiration stream to intact leavesof different ages mounted within a gas exchange cuvette. ABA caused a reduction in stomatal conductance (g) that wasproportional to the concentration in the solution fed to theleaf. For the maintenance of a reduction in g there was a requirementfor a continual supply of ABA. At concentrations greater than10^–2 mol m^–3 ABA reduced g by at least 50% of thecontrol value, while 1.0 mol m^–3 closed stomata within2 h. Concentrations as low as 10^–3 mol m^–3 produceda 20% reduction in g. As leaves aged they became less responsiveto applied ABA. The possibility that the stomatal response may change aftera leaf has previously experienced a pulse of ABA was exploredby repeating the exposure of a leaf to 10^–2 mol m^–3ABA. The first pulse of ABA produced a greater reduction ing than a subsequent exposure the following day. This declinein response of g to ABA on repeated exposure was maintainedwith leaves of different ages. The characteristics of the stomatal response to ABA are discussedin the context of what is known about the location of receptorsfor the hormone. It seems likely that a failure to respond toABA that has previously accumulated in the guard cells shouldbe viewed by means of maximizing the sensitivity to the currentsupply of ABA. It is suggested that the smaller response ofthe stomata of older leaves to ABA makes them more susceptibleto water stress, so that they can act as sensors for decliningwater potentials to give early protection to younger, metabolicallyactive leaves. Key words: Abscisic acid, leaf age, stomatal conductance, Triticum aestivum     

Osmotic Stress Temporarily Reverses the Inhibitions of Photosynthesis and Stomatal Conductance by Abscisic Acid--Evidence that Abscisic Acid Induces a Localized Closure of Stomata in Intact, Detached Leaves

Ward, D. A. and Drake, B. G. 1988. Osmotic stress temporarilyreverses the inhibitions of photosynthesis and stomatal conductanceby abscisic acid—evidence that abscisic acid induces alocalized closure of stomata in intact, detached leaves.—J.exp. Bot 39: 147–155. The influence of osmotic stress on whole leaf gas exchange wasmonitored in detached leaves of Glycine max supplied with anexogenous concentration (10^–5 mol dm^–3) of ±abscisicacid (ABA) sufficient to inhibit net photosynthesis and stomatalconductance by 60% and 70%, respectively, under a saturatingirradiance and normal air. Raising the osmotic (sorbitol) concentrationof the ABA solutions feeding leaves elicited rapid and synchronousreversals of the ABA-dependent inhibitions of net photosynthesisand conductance. These reversals reached a peak simultaneously,after which photosynthesis and conductance declined. The magnitudeof the transient stimulations at peak height was dependent uponthe sorbitol concentration of the ABA feeding solution, althoughthe time-course of the transients (half time, 4–6 min)was similar for the different osmotic concentrations applied.Irrespective of transient size the relative changes of photosynthesisand conductance were comparable; consequently the calculatedpartial pressure of CO₂ in the substomatal space (Ci) remainedrelatively constant during the transient phase. In contrastto the ABA-treated leaves, elevating the osmotic concentrationof the distilled water supply feeding control leaves stimulatedconductance to a much greater relative extent than photosynthesis.The co-stimulations of photosynthesis and conductance inducedin ABA-treated leaves by osmotic shock were not due to a restrictionin the transpirational uptake of ABA and occurred irrespectiveof the source osmoticum applied. These data are consistent with the hypothesis that the ABA-dependentinhibition of photosynthesis at constant Ci is an artifact causedby the spatially heterogeneous closure of stomata in responseto ABA. Alternative explanations for the responses are, however,considered. Key words: Abscisic acid, photosynthesis, osmotic stress, Glycine max, stomatal conductance     

Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata

Transgenic tobacco ( Nicotiana tabacum L.) plants ubiquitously accumulating a single-chain variable-fragment (scFv) antibody against abscisic acid (ABA) to high concentrations in the endoplasmic reticulum (RA plants) show a wilty phenotype. High stomatal conductance and loss of CO(2) and light dependence of stomatal conductance are typical features of these plants. ABA was applied to these plants either via the petioles or by daily spraying over several weeks in order to normalise the phenotype. During the long-term experiments, scFv protein concentrations, total and (calculated) free ABA contents, and stomatal conductance and its dependence on CO(2) concentration and light intensity were monitored. The wilty phenotype of transgenic plants could not be normalised by short-term treatment with ABA via the petioles. Only a daily long-term treatment during plant development normalised the physiological behaviour completely. Scanning electron microscopy of stomata showed morphological changes in RA plants compared with wild-type plants that, for structural reasons, prevented regular stomatal movements. After long-term treatment with ABA this defect could be completely eliminated. Guard-cell-specific expression of the anti-ABA scFv did not cause any changes in physiological behaviour compared to the wild type. In addition, mesophyll-specific expression starting in leaves that were already fully differentiated resulted in normal phenotypes, too. We conclude that changes in distribution and availability of ABA in the cells of developing leaves of RA plants cause the development of structural features in stomata that prevent normal function.     

The Influence of Prior Water Stress and Abscisic Acid Foliar Spraying on Stomatal Responses to CO2, IAA, ABA, and Calcium in Leaves of Solanum melongena

The influence of a water stress or foliar ABA spraying pretreatmenton stomatal responses to water loss, exogenous ABA, IAA, Ca²⁺,and CO₂ were studied using excised leaves of Solanum melongena.Both pretreatments increased stomatal sensitivity of water loss,in the presence and absence of CO₂, but decreased stomatal sensitivityto exogenous ABA. CO₂ greatly reduced the effect of exogenouslyapplied ABA. IAA decreased leaf diffusion resistance for controland ABA sprayed leaves, but did not influence the LDR of previouslywater-stressed leaves. CA²⁺ did not influence LDR of any leavesof any treatments. Key words: Water stress, stomatal response, pretreatments     

Water relations and abscisic acid levels of watermelon as affected by rooting volume restriction

Rooting volume restriction (RVR) reduces shoot growth of plantsprovided with sufficient water or nutrients. The effects ofRVR on water status, abscisic acid (ABA) levels in leaves, roots,or xylem sap from detopped plants of watermelon [Citrullus lanatus(Thunb.) Matsum. and Nakai ‘StarBrite’] seedlingswere evaluated with five rooting volumes (18, 26, 36, 46, or80 cm3). Shoot water potential increased with increasing rootingvolume, with no difference between plants from 18 and 26 cm³cells or between plants from 36 and 46 cm³ cells. Stomatal conductancewas not consistently affected by RVR; at 10 and 20 DAE, stomatalconductance in plants grown in 36 cm³ cells was higher thanthat of plants grown in any other cell volume. Severe RVR (18and/or 26 cm³) tended to produce plants with higher ABA levelsin roots (15 DAE only), xylem sap (all dates), and leaves (5and 10 DAE). Plants grown in 18 and 26 cm³ cells had higherroot ABA levels than those from 46 and 80 cm³ cells at 15 DAE.Plants grown in 18 cm³ cells had the highest xylem sap ABA levelat all dates, but ABA levels did not differ among plants grownin the other cell volumes. Plants grown in 18 cm³ cells at 5DAE and 18 and 26 cm³ cells at 10 DAE also had higher leaf ABAlevels than those from other rooting volumes. The results suggestthat ABA may act as a signal for reduced growth of plants underRVR conditions. Key words: Abscisic acid, ABA, root signals, root volume restriction, water relations     

Abscisic Acid and the Regulation of Water Loss in Plantlets of Brassica oleracea L. var. botrytisRegenerated Through Apical Meristem Culture

Water loss was studied in regenerated plantlets of Brassicaoleracea var. botrytis cv. Currawong derived through apicalmeristem culture. Hardening of plantlets was eliminated by asingle application of a polyvinyl resin (S600) sprayed immediatelyafter transplanting. Plantlets sprayed with S600 had highercuticular resistances than unsprayed plantlets; this treatmenthad no effect on stomatal resistance. Leaves formed during theculture period showed very little wax formation and using markedleaves it was found that only reduced levels of wax formed onthese leaves even after transplanting. New leaves formed aftertransplanting, showed typical wax formation compared to seedgrown plants. Abscisic acid (ABA) at 10^–4 M applied as a leaf sprayto transplants did not cause a substantial increase in stomatalresistance in leaves which had been initiated during the cultureperiod. Leaves of seed-grown plants as well as leaves of plantletsformed after transplanting did respond to a leaf spray of ABAat 10^–4 M by a large increase in stomatal resistance. Relative concentrations of K, Na, Ca, P, S and Mg in guard cellswere calculated for each leaf type by X-ray micro-probe analysis.K/Na values decreased in the order: seedling > leaves formedafter transplanting > leaves intiated during culture. A highpositive correlation was also found between K/Na and K/P forthe three leaf types. K:Mg and K:Ca ratios for leaves formedduring culture were low in comparison to the values obtainedfor leaves formed after transplanting and seedlings for whichthe values were similar. Brassica oleracea var. botrytis, cauliflower, regenerated plantlets, meristem culture, stomatal resistance, water loss, abscisic acid, X-ray micro-probe analysis     

Exogenous ABA Increases Yield in Field-Grown Wheat with Moderate Water Restriction

Water stress is one of the most important environmental factors that regulate a plant’s growth and development. In agronomic practice the effects of water stress are translated into low yield and/or reduced quality. Abscisic acid (ABA) sprays (1 mM) were applied to wheat plants at different phenological stages and the effects on several physiological variables and on yield were evaluated under field conditions at different water regimes. Studies were conducted in the field across three consecutive winter–spring seasons. ABA treatments were applied at the beginning of shoot enlargement and repeated at anthesis. Exogenous ABA increased shoot dry weight and maintained a high concentration of photosynthetic pigments for a longer period of time during grain growth and maturation. Although ABA applications increased stomatal closure immediately after its application, the longer-term effect was to allow for a greater ostiolar opening of the stomatal pore which resulted in increased conductance of gases and water vapor. ABA also improved the transport of photoassimilates from the leaves and stem to the developing grains, that is, it effectively increased the sink strength of the grains. This correlated with a yield increase without significantly changing the protein quality in the grains. Thus, elevated ABA levels from exogenous application or genetic selection could help improve agricultural production of grains in arid areas where irrigation is not possible.     

The Role of Endogenous Abscisic Acid in the Response of Plants to Stress

When a continuous stream of warm air (38°C) was directedon to the leaves of dwarf bean seedlings they wilted and thengradually regained turgor. This process of adaptation was accompaniedby an increasing abscisic acid (ABA) level in the leaves andan increase in leaf resistance (R_L). It is suggested that theleaf-water deficit induced by the warm-air treatment causedthe increase in ABA level and that the latter was responsiblefor stimulating stomatal closure, enabling the plants to regainfull turgor. A similar type of adaptation, brought about byan increased level of ABA in the leaves, is believed to occurin tomato, dwarf bean, and wheat plants when they are flooded.Predictably, in rice, a species adapted to a flooded environment,seedlings showed no increase in ABA level as a result of flooding. It is proposed that adaptation may involve the formation ofan equilibrium between ABA and its conjugate form (i. e. theglucose ester). The ABA-conjugate was observed to disperse slowlyfrom leaves recovering from a water deficit and therefore itmay act as a metabolic ‘back-stop’, enabling the‘free’ ABA level to remain high for a period evenwhen the leaves have regained turgor. Abscisic acid appears to be responsible for alleviating theeffects of water stress in plants, making it possible for plantsto pass through periods of stress with little harm.     

Stomatal action directly feeds back on leaf turgor: new insights into the regulation of the plant water status from non‐invasive pressure probe measurements

Uptake of CO₂ by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard‐cell anion release channel SLOW ANION CHANNEL‐ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard‐cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non‐invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long‐term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild‐type plants responded to CO₂, light, humidity, ozone and abscisic acid (ABA) in a guard cell‐specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild‐type plants, leaves from well‐watered ost1 plants exposed to a dry atmosphere wilted after light‐induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.     

Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana.

Abscisic acid (ABA)-deficient mutants in a variety of species have been identified by screening for precocious germination and a wilty phenotype. Mutants at two new loci, aba2 and aba3, have recently been isolated in Arabidopsis thaliana (L.) Hynh. (K.M. Léon-Kloosterziel, M. Alvarez-Gil, G.J. Ruijs, S.E. Jacobsen, N.E. Olszewski, S.H. Schwartz, J.A.D. Zeevaart, M. Koornneef [1996] Plant J 10: 655-661), and the biochemical characterization of these mutants is presented here. Protein extracts from aba2 and aba3 plants displayed a greatly reduced ability to convert xanthoxin to ABA relative to the wild type. The next putative intermediate in ABA synthesis, ABA-aldehyde, was efficiently converted to ABA by extracts from aba2 but not by extracts from aba3 plants. This indicates that the aba2 mutant is blocked in the conversion of xanthoxin to ABA-aldehyde and that aba3 is impaired in the conversion of ABA-aldehyde to ABA. Extracts from the aba3 mutant also lacked additional activities that require a molybdenum cofactor (Moco). Nitrate reductase utilizes a Moco but its activity was unaffected in extracts from aba3 plants. Moco hydroxylases in animals require a desulfo moiety of the cofactor. A sulfido ligand can be added to the Moco by treatment with Na2S and dithionite. Treatment of aba3 extracts with Na2S restored ABA-aldehyde oxidase activity. Therefore, the genetic lesion in aba3 appears to be in the introduction of S into the Moco.