Movement of Abscisic Acid into the Apoplast in Response to Water Stress in Xanthium strumarium L

The effect of water stress on the redistribution of abcisic acid (ABA) in mature leaves of Xanthium strumarium L. was investigated using a pressure dehydration technique. In both turgid and stressed leaves, the ABA in the xylem exudate, the `apoplastic' ABA, increased before `bulk leaf' stress-induced ABA accumulation began. In the initially turgid leaves, the ABA level remained constant in both the apoplast and the leaf as a whole until wilting symptoms appeared. Following turgor loss, sufficient quantities of ABA moved into the apoplast to stimulate stomatal closure. Thus, the initial increase of apoplastic ABA may be relevant to the rapid stomatal closure seen in stressed leaves before their bulk leaf ABA levels rise.

Following recovery from water stress, elevated levels of ABA remained in the apoplast after the bulk leaf contents had returned to their prestress values. This apoplastic ABA may retard stomatal reopening during the initial recovery period.

     

The Modified Stomata of the Floral Nectary of Vicia faba L.

A comparison of developmental features and physiological responses between the modified stomata (MS) of the floral nectary and the stomata of leaves of Vicia faba L. has revealed several significant differences. In mature tissues, the frequency per unit area of MS is three times that of foliar stomata, and when only the distal quarter of nectary projections is considered, it is twelve fold higher. The walls of guard cells (GCs) of MS near the pore were four to five times thinner than those of their foliar counterparts. Average pore widths of the nectary MS remained constant throughout the day and night, contrary to those of leaves; maximal foliar apertures were similar to average pore widths of MS of nectaries. Experiments with plasmolytica demonstrated a higher osmotic potential in nectary MS, and that sister GCs of the nectary only occasionally closed their pores by movements, even when they were plasmolysed, whereas GCs of leaves were found to be plasmolysed only after their pores had shut. Abscisic acid (ABA) was always detected naturally in floral nectar. The results of experiments involving K⁺-localization and the secretion of large quantities of ABA in nectar suggested that GCs of the MS lack operational systems involving K⁺-influx and ABA-receptor sites. All information gained is consistent with the earlier conclusion that the MS do not have a regulatory role in nectar secretion by flowers of V. faba.     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Reversal of Abscisic Acid-Induced Stomatal Closure by trans-Cinnamic and p-Coumaric Acid

Abscisic acid (ABA)-induced increase in stomatal diffusive resistance (SDR) in excised leaves of bean (Phaseolus vulgaris L. cv Pencil Pod) and maize (Zea mays L. cv Golden Bantam) is inhibited by low concentrations of trans-cinnamic acid (TCA) (1 micromolar) and p-coumaric acid (PCA) (10 micromolar) when given together with ABA (10 micromolar) in the transpiration stream through the cut end of the petiole or leaf blade. A concentration effect is observed both in the ABA action and its reversal by phenolic acids. Leaves having attained a high diffusive resistance in ABA solution recover rapidly when transferred to water. ABA (10 micromolar) induced closure of the stomata in onion, Allium cepa L. and Vicia faba epidermal peels. This is associated with loss of K⁺ from guard cells. In the presence of TCA (10 micromolar) and PCA (10 micromolar) K⁺ is retained in the guard cells with open stomata. The dark closure of stomata is also inhibited by TCA and PCA. It is suggested that these phenolic acids may inhibit the ABA effect by competing with or acting on some ABA-specific site, probably located on the plasma membrane, regulating flux of K⁺ ions. A weak association of ABA with the plasma membrane is envisaged because of the rapid recovery obtained upon transferral of the leaves to water.     

Effect of Abscisic Acid on Cell-Wall Metabolism in Guard Cells of Vicia faba L.

Cell-wall synthesis in guard cells of Vicia faba L. was examinedusing sonicated epidermal strips incubated with [¹⁴C]glucose.The cell walls of the guard cells incorporated [¹⁴C]glucoseat a lower level in the dark than in the light. Stomatal aperturein the epidermal strips was reduced by application of 1 µmabscisic acid (ABA) in the light but not in the dark. The ABAtreatment reduced the incorporation of [¹⁴C]glucose into thecell walls especially in the light. Fractionation of the labeledcell-wall components revealed that ABA inhibited the synthesisof pectic substances and cellulose, but did not affect hemicellulosesynthesis. Microautoradiographs of the cell-wall fraction ofthe epidermal strips showed that a large amount of radioactivitywas distributed at both ends of the guard cells in the absenceof ABA and that removal of pectic substances from the cell-wallfraction resulted in uniform distribution of the radioactivityin the cell walls of the guard cells. These results indicatedthat the synthesis of pectic substances was active at both endsof the guard cells and was inhibited by ABA. Measurement ofspecific activities of neutral sugars in the guard-cell wallsshowed that polymers composed of galactose underwent activeturnover and that synthesis of glucans was inhibited by ABA.These results revealed a strong correlation between the stomatalmovement and the synthesis of pectic substances and cellulosein the guard cells, suggesting that the cell-wall metabolismin the guard cells may play a role in the regulation of stomatalmovement. (Received October 9, 1987; Accepted March 9, 1988)     

Relationship between Stress-Induced ABA and Proline Accumulations and ABA-Induced Proline Accumulation in Excised Barley Leaves

When excised second leaves from 2-week-old barley (Hordeum vulgare var Larker) plants were incubated in a wilted condition, abscisic acid (ABA) levels increased to 0.6 nanomole per gram fresh weight at 4 hours then declined to about 0.3 nanomole per gram fresh weight and remained at that level until rehydrated. Proline levels began to increase at about 4 hours and continued to increase as long as the ABA levels were 0.3 nanomole per gram fresh weight or greater. Upon rehydration, proline levels declined when the ABA levels fell below 0.3 nanomole per gram fresh weight.

Proline accumulation was induced in turgid barley leaves by ABA addition. When the amount of ABA added to leaves was varied, it was observed that a level of 0.3 nanomole ABA per gram fresh weight for a period of about 2 hours was required before proline accumulation was induced. However, the rate of proline accumulation was slower in ABA-treated leaves than in wilted leaves at comparable ABA levels. Thus, the threshold level of ABA for proline accumulation appeared to be similar for wilted leaves where ABA increased endogenously and for turgid leaves where ABA was added exogenously. However, the rate of proline accumulation was more dependent on ABA levels in turgid leaves to which ABA was added exogenously than in wilted leaves.

Salt-induced proline accumulation was not preceded by increases in ABA levels comparable to those observed in wilted leaves. Levels of less than 0.2 nanomole ABA per gram fresh weight were measured 1 hour after exposure to salt and they declined rapidly to the control level by 3 hours. Proline accumulation commenced at about 9 hours. Thus, ABA accumulation did not appear to be involved in salt-induced proline accumulation.

     

Abscisic acid introduced into the transpiration stream accumulates in the guard-cell apoplast and causes stomatal closure

Petioles of water‐sufficient intact Vicia faba L. plants were infused with 1 µm abscisic acid (ABA) to simulate the import of root‐source ABA. This protocol permitted quantitative ABA delivery, up to 300 pmol ABA over 60 min, to the leaf without ambiguities associated with perturbations in plant–water status. The ABA concentrations in whole‐leaf samples and in apoplastic sap increased with the amount infused; ABA degradation was not detected. The ABA concentration in apoplastic sap was consistent with uptake of imported ABA into the leaf symplast, but this interpretation is qualified. Our focus was quantitative cellular compartmentation of imported ABA in guard cells. Unlike when leaves are stressed, the guard‐cell symplast ABA content did not increase because of ABA infusion (P = 0·48; 3·0 ± 0·5 versus 4·0 ± 1·2 fg guard‐cell‐pair^?1). However, the guard‐cell apoplast ABA content increased linearly (R² = 0·98) from ?0·2 ± 0·5 to 3·1 ± 1·3 fg guard‐cell‐pair^?1 (≈ 3·1 µm ) and was inversely related to leaf conductance (R² = 0·82). Apparently, xylem ABA accumulates in the guard‐cell wall as a result of evaporation of the apoplast solution. This mechanism provides for integrating transpiration rate and ABA concentration in the xylem solution.     

Effect of Abscisic Acid on Glucose Metabolism in Guard Cells of Vicia faba L.

The effects of abscisic acid (ABA) on the formation of osmoticallyactive solutes and on cell wall synthesis in guard cells wereexamined using sonicated abaxial epidermal strips of Vicia fabaL. incubated with ¹⁴C-glucose at pH 4 and 6. Radioactivity wasincorporated mainly into malate,sucrose, starch and cell-wallfractions. ¹⁴C- Glucose uptake by the guard cells was reducedwhen 1 µm ABA was added. Malate formation, which was moreactive at pH 6 than at 4, was inhibited by ABA at pH 6, butnot at pH 4. Conversion of ¹⁴C-glucose into ¹⁴C-sucrose wasstimulated by ABA at both pH values. Release of radio activesolutes (composed mainly of glucose and malate)from the guardcells into the medium was more active at pH 6 than at pH 4.ABA stimulated there lease at both pH values. Turnover of starchwas more remarkable when the pH value was 6. ABA inhibited thesynthesis of starch, but did not affect its degradation. Cell-wallsynthesis inthe guard cells was also inhibited by ABA, the inhibitionrate being greater at pH 4 than at pH 6.These results suggestthat ABA may have two different actions on stomatal movement:to changethe metabolic activities in the guard cells so as tolower the concentration of osmotically active solutes, and tochange the mechanical properties of cell walls by modulatingcell-wall metabolism. (Received September 7, 1987; Accepted November 30, 1987)     

Low sink-induced stomatal closure alters photosynthetic rates of source leaves in beans as dependent on H2O2 and ABA accumulation in guard cells

Low sink demand provided by pod removal and stem girdling of beans (Vicia faba, cv. Daqingshan) (-Sink) induced a significantly lower net photosynthetic rate (P _n), stomatal conductance (g _s), internal CO₂ concentration (C _i), and transpiration rate (E) compared with pod and root sink retention (CK). This depression in P _n was due to stomatal limitation. Low sink demand of -Sink plants resulted in a higher leaf sucrose content, but a lower sucrose content in guard cells. Moreover, the significant accumulation of H₂O₂ and ABA were observed in both leaves and guard cells of -Sink plants. The most intensive electron dense deposit of cerium perhydroxides, produced by H₂O₂ reaction with cerium chloride, was present in the cell walls, especially the dorsal walls of guard cells. Immunogold electron-microscopy localization of ABA showed that ABA was distributed in ventral walls of guard cells and the intercellular space of mesophyll cells of -Sink leaves in contrast to CK plants. Application of exogenous sucrose to isolated bean leaves increased H₂O₂ and ABA contents. H₂O₂ and ABA in leaves was likely generated by two independently regulated pathways, each affected by the high sucrose concentration induced by low sink demand. Increased sucrose in leaves in response to low sink demand may have caused the increase of H₂O₂ and ABA, and their accumulation in mesophyll cells and guard cells was likely the primary cause for stomatal closure under low sink demand.     

Stress-related levels of abscisic acid in guard cell protoplasts of Vicia faba L.

Levels of abscisis acid (ABA) were determined in isolated guard cell (GCP) and mesophyll cell (MCP) protoplasts of Vicia faba L. in relation to water stress. Incubation of GCP and MCP in 0.4 M or 0.8 M mannitol resulted in an average increase in the level of free abscisic acid (ABA) in the cells of 34% (GCP) and 38% (MCP) within 15–60 min. It is concluded that guard cell protoplasts form ABA in response to osmotic stress.Abbreviations ABA abscisic acid - BHT butylated hydroxytoluene - GCP guard cell protoplasts - MCP mesophyll cell protoplasts - MES [2-(N-morpholino)-ethanesulfonic acid] - TLC thin layer chromatography Part 20 in the series, Use of Immunoassay in Plant Science     

Water Relations of Cotton Plants under Nitrogen Deficiency: V. Environmental Control of Abscisic Acid Accumulation and Stomatal Sensitivity to Abscisic Acid

Suboptimal N nutrition increased the water potential for stomatal closure in water stressed cotton (Gossypium hirsutum L.) leaves. This increased sensitivity to water stress had two components, increased accumulation of abscisic acid (ABA) and increased apparent stomatal sensitivity to ABA. Low N increased the threshold water potentials for stomatal closure and ABA accumulation by about 4 bars and 2 bars, respectively. Low N also greatly increased stomatal response to low concentrations of exogenous ABA applied to excised leaves through the transpiration stream. In low N leaves, kinetin decreased stomatal response to ABA to the level observed with high N leaves. Kinetin by itself had little effect on stomata, nor did it alter stomatal response to ABA in high N leaves. The results suggest a cytokinin-ABA balance which is altered by suboptimal N nutrition to favor stomatal closure during stress.

Ambient temperature and N nutrition interacted to alter stomatal response to water stress. Stress-induced ABA accumulation and apparent stomatal sensitivity to ABA were independently affected. The effects of each treatment, and their interaction, could be explained as the net result of changes in both accumulation and apparent sensitivity. Although the results document environmental control of stomatal response to ABA, either altered partitioning of ABA between active and inactive pools, or altered sensitivity of the guard cells, could account for the data.

     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.     

A simple method forIn Situ hybridization to RNA in guard cells ofVicia Faba L.: The expression of aquaporins in guard cells

Because the epidermis ofV. faba L. leaves easily can be peeled into strips of one cell layer, we developed a simple method ofin situ hybridization using epidermal peels as a substitute for paraffin, resin and cryosections. Our method sufficiently detected the expression of broad bean aquaporin 1 in guard cells. RT-PCR revealed higher expression of aquaporins (AQPs) in guard cells compared to other leaf cell types; this indicates the importance of AQP for bulk water flow across guard cell membranes and, therefore, for stomatal movements.     

Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns

Previous labeling studies of abscisic acid (ABA) with ¹⁸O₂ have been mainly conducted with water-stressed leaves. In this study, ¹⁸O incorporation into ABA of stressed leaves of various species was compared with ¹⁸O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), ¹⁸O was most abundant in the carboxyl group, whereas incorporation of a second and third ¹⁸O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in ¹⁸O₂. ABA from turgid bean leaves showed significant ¹⁸O incorporation, again with highest ¹⁸O enrichment in the carboxyl group. The ¹⁸O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high ¹⁸O enrichment in the carboxyl group, also much additional ¹⁸O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more ¹⁸O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1′-¹⁸OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of ¹⁸O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange ¹⁸O with water. On the basis of ¹⁸O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.     

High Levels of Malic Enzyme Activities in Vicia faba L. Epidermal Tissue

Specific activities of NADP-malic enzyme, NAD-malic enzyme, phosphoenolpyruvate carboxykinase and pyruvate, orthophosphate dikinase in various cells of Vicia faba L. leaflets were determined. Expressed on dry weight, chlorophyll or protein basis, the averages for NADP- and NAD-malic enzyme specific activities were higher in guard cells than in photosynthetic parenchyma cells. Malic enzyme-specific activities were also high in epidermal cells. Phosphoenolypyruvate carboxykinase activity was not detected in Vicia leaf extracts or guard cells; the assay techniques were validated by mixed Vicia-Brachiaria leaf extraction and assays on nanogram samples of Brachiaria bundlesheath cells. It was inferred from these data that guard cell malate depletion is by decarboxylation to pyruvate in the epidermal layer, but how the various epidermal cells interact remains obscure.     

Abscisic Acid accumulation in spinach leaf slices in the presence of penetrating and nonpenetrating solutes

Abscisic acid (ABA) accumulated in detached, wilted leaves of spinach (Spinacia oleracea L. cv Savoy Hybrid 612) and reached a maximum level within 3 to 4 hours. The increase in ABA over that found in detached turgid leaves was approximately 10-fold. The effects of water stress could be mimicked by the use of thin slices of spinach leaves incubated in the presence of 0.6 molar mannitol, a compound which causes plasmolysis (loss of turgor). About equal amounts of ABA were found both in the leaf slices and in detached leaves, whereas 2 to 4 times more ABA accumulated in the medium than in the slices. When spinach leaf slices were incubated with ethylene glycol, a compound which rapidly penetrates the cell membrane causing a decrease in the osmotic potential of the tissue and only transient loss of turgor, no ABA accumulated. Ethylene glycol was not inhibitory with respect to ABA accumulation. Spinach leaf slices incubated in both ethylene glycol and mannitol had ABA levels similar to those found when slices were incubated with mannitol alone. Increases similar to those found with mannitol also occurred when Aquacide III, a highly purified form of polyethylene glycol, was used. Aquacide III causes cytorrhysis, a situation similar to that found in wilted leaves. Thus, it appears that loss of turgor is essential for ABA accumulation.

When spinach leaf slices were incubated with solutes which are supposed to disturb membrane integrity (KHSO₃, 2-propanol, or KCl) no increase in ABA was observed. These data indicate that, with respect to the accumulation of ABA, mannitol caused a physical stress (loss of turgor) rather than a chemical stress (membrane damage).

     

Violaxanthin is an abscisic Acid precursor in water-stressed dark-grown bean leaves

The leaves of dark-grown bean (Phaseolus vulgaris L.) seedlings accumulate considerably lower quantities of xanthophylls and carotenes than do leaves of light-grown seedlings, but they synthesize at least comparable amounts of abscisic acid (ABA) and its metabolites when water stressed. We observed a 1:1 relationship on a molar basis between the reduction in levels of violaxanthin, 9′-cis-neoxanthin, and 9-cis-violaxanthin and the accumulation of ABA, phaseic acid, and dihydrophaseic acid, when leaves from dark-grown plants were stressed for 7 hours. Early in the stress period, reductions in xanthophylls were greater than the accumulation of ABA and its metabolites, suggesting the accumulation of an intermediate which was subsequently converted to ABA. Leaves which were detached, but not stressed, did not accumulate ABA nor were their xanthophyll levels reduced. Leaves from plants that had been sprayed with cycloheximide did not accumulate ABA when stressed, nor were their xanthophyll levels reduced significantly. Incubation of dark-grown stressed leaves in an ¹⁸O₂-containing atmosphere resulted in the synthesis of ABA with levels of ¹⁸O in the carboxyl group that were virtually identical to those observed in light-grown leaves. The results of these experiments indicate that violaxanthin is an ABA precursor in stressed dark-grown leaves, and they are used to suggest several possible pathways from violaxanthin to ABA.     

Stomatal closure in response to xanthoxin and abscisic acid

Summary The stomata of detached leaves of Commelina communis L., Hordeum vulgare L., Zea mays L., Vicia faba L., Phaseolus vulgaris L. and Xanthium strumarium L. closed when xanthoxin (XAN) was added to the transpiration stream. XAN was approximately half as active as (+)-abscisic acid (ABA) at an equivalent concentration. XAN, like ABA, sensitized stomata of Xanthium strumarium to CO₂. In contrast to ABA, XAN was ineffective in closing stomata of isolated epidermal strips of C. communis or V. faba. This may be because XAN added to the transpiration stream is converted to ABA during passage from the xylem to the epidermis.Abbreviations ABA Abscisic acid - XAN xanthoxin     

The Intracellular Location of Abscisic Acid in Stressed and Non-Stressed Leaf Tissue

The intra-cellular location of ABA was investigated in relation to its sites of synthesis. Chloroplasts were isolated from stressed and non-stressed spinach leaves and their ABA content determined. Virtually all of the ABA from non-stressed leaves was contained in the chloroplasts compared with only a small fraction of ABA isolated from stressed leaves. Chloroplasts prepared from turgid leaves and subsequently lysed in vitro retained most of their ABA and phaseic acid (PA) complement but this was removed with organic solvents. While the possibility of extra-chloroplastic synthesis cannot be discounted the data indicate that stress-induced ABA synthesis occurs in the chloroplast and that the ABA readily migrates from there to other parts of the plant.