Review of wheat improvement for waterlogging tolerance in Australia and India: the importance of anaerobiosis and element toxicities associated with different soils.

BACKGROUND AND AIMS: The lack of knowledge about key traits in field environments is a major constraint to germplasm improvement and crop management because waterlogging-prone environments are highly diverse and complex, and the mechanisms of tolerance to waterlogging include a large range of traits. A model is proposed that waterlogging tolerance is a product of tolerance to anaerobiosis and high microelement concentrations. This is further evaluated with the aim of prioritizing traits required for waterlogging tolerance of wheat in the field. METHODS: Waterlogging tolerance mechanisms of wheat are evaluated in a range of diverse environments through a review of past research in Australia and India; this includes selected soils and plant data, including plant growth under waterlogged and drained conditions in different environments. Measurements focus on changes in redox potential and concentrations of diverse elements in soils and plants during waterlogging. KEY RESULTS: (a) Waterlogging tolerance of wheat in one location often does not relate to another, and (b) element toxicities are often a major constraint in waterlogged environments. Important element toxicities in different soils during waterlogging include Mn, Fe, Na, Al and B. This is the first time that Al and B toxicities have been indicated for wheat in waterlogged soils in India. These results support and extend the well-known interactions of salinity/Na and waterlogging/hypoxia tolerance. CONCLUSIONS: Diverse element toxicities (or deficiencies) that are exacerbated during waterlogging are proposed as a major reason why waterlogging tolerance at one site is often not replicated at another. Recommendations for germplasm improvement for waterlogging tolerance include use of inductively coupled plasma analyses of soils and plants.

Ecophysiology of Acer rubrum seedlings from contrasting hydrologic habitats: growth, gas exchange, tissue water relations, abscisic acid and carbon isotope discrimination.

Eight red maple (Acer rubrum L.) provenances, four each from wet and dry sites, were grown under the same conditions and their physiological responses to soil water availability investigated. Under well-watered conditions, seedlings of wet-site provenances grew faster and had consistently higher net photosynthesis, leaf conductance, maximum carboxylation rate, maximum rate of coupled photosynthetic electron transport, apparent quantum use efficiency, light-saturated photosynthesis and dark respiration than seedlings of dry-site provenances. Under conditions of low soil water availability, only dry-site provenances responded with decreased osmotic potential at full hydration and at the turgor loss point; however, provenances from wet sites showed a smaller reduction in absolute growth rate, a greater reduction in gas exchange and a greater increase in abscisic acid concentrations than dry-site provenances.

Water deficit inhibits cell division and expression of transcripts involved in cell proliferation and endoreduplication in maize endosperm.

Water deficit at the early post-pollination stage in cereal grains decreases endosperm cell division and, in turn, decreases the capacity for storage material accumulation. Post-mitotic replication of nuclear DNA (endoreduplication) may also play a role in stress effects. To gain a better understanding of the extent to which cell proliferation and endoreduplication are affected by water deficit, nuclear numbers and size were examined in endosperms of maize (Zea mays L.) by flow cytometry and the transcript levels of genes which have recognized roles in the cell cycle were quantified. Water deficit from 5-13 d after pollination (DAP) decreased the rate of endosperm cell division by 90% and inhibited [3H]-thymidine incorporation into DNA from 9-13 DAP. The proportion of nuclei engaging in endoreduplication and nuclear DNA content increased steadily from 9-13 DAP in controls, but water deficit initially increased the proportion of endoreduplicating nuclei at 9 DAP, then halted further entry into endoreduplication and S-phase cycling from 9-13 DAP. Transcript levels of alpha-tubulin, and the S-phase gene products histone H3 and PCNA were not affected by water deficit until 13 DAP, whereas those of ZmCdc2, a cyclin dependent kinase (CDK) with regulatory roles in mitosis, were inhibited substantially from 9-13 DAP. Cell proliferation and associated processes were inhibited at initial stages of the stress episode, whereas endoreduplication and associated S-phase processes were not inhibited until the stress was more advanced. It was concluded that endosperm mitosis has greater sensitivity than endoreduplication to water deficit.

Regulation of alcoholic fermentation in coleoptiles of two rice cultivars differing in tolerance to anoxia.

To investigate regulation of anaerobic carbohydrate catabolism in anoxia-tolerant plant tissue, rate of alcoholic fermentation and maximum catalytic activities of four key enzymes were assessed in coleoptiles of two rice cultivars that differ in tolerance to anoxia. The enzymes were ATP-dependent phosphofructokinase (PFK), pyrophosphate-dependent phosphofructokinase (PFP), pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH). During anoxia, rates of coleoptile elongation and ethanol synthesis were faster in the more tolerant variety Calrose than in IR22. Calrose coleoptiles, in contrast to IR22, also showed a sustained Pasteur effect, with the estimated rate of glycolysis during anoxia being 1.4-1.7-fold faster than that of aerobic coleoptiles. In Calrose after 5 d anoxia, maximum catalytic activities of crude enzyme extracts were (in mumol substrate g-1 fresh weight min.-1) 170-240 for ADH, 4-6 for PDC and PFP and 0.4-0.7 for PFK. During anoxia, activity per coleoptile of all four enzymes increased 3-5.5-fold, suggesting that PFK, and PFP, like PDC and ADH, are synthesised in anoxic rice coleoptiles. Enzyme activities, on a fresh weight basis, were lower in IR22 than in Calrose. In vivo activities of PDC and PFK in anoxic coleoptiles from both cultivars were calculated using in vitro activities, estimated substrate levels, cytoplasmic pH, and S0.5 (the substrate level at which 0.5Vmax is reached, without inferring Michaelis-Menten kinetics). Data indicated that potential carbon flux through PFK, rather than through PDC, more closely approximated rates of alcoholic fermentation. That PFK is an important site of regulation was supported further for Calrose coleoptiles by a decrease in the concentration of its substrate pool (F-6-P + G-6-P) following the onset of anoxia. By contrast, in IR22, there was little evidence for control by PFK, consistent with recent evidence that suggests substrate supply limits alcoholic fermentation in this cultivar.

Regulation of endoreduplication in maize (Zea mays L.) endosperm. Isolation of a novel B1-type cyclin and its quantitative analysis.

To investigate the involvement of cyclin in mitotic and endoreduplicative cell cycle control, we have isolated a mitotic cyclin clone from a maize endosperm cDNA library. The deduced amino acid sequence of this clone identifies a novel B1-type cyclin with distinctly different sequence in regions with putative involvement in intracellular localization. This cyclin, designated Zeama;CycB 1;3 (CycZme1), was shown by RNA gel blots and quantitative RT-PCR to be specific for tissues engaging in cell proliferation. It accumulated in metaphase-arrested cells and declined rapidly upon release into G1 phase. During the transition from mitosis to endoreduplication in maize endosperm, CycZme1 transcript declined precipitously while transcripts associated with S phase (histone-H3 and PCNA) and multiple phases of the cell cycle (Cdc2, alpha-tubulin) remained at moderate to high levels. We conclude that CycZme1 down-regulation is involved in the cellular transition to endoreduplication.

Alternative splicing of cyclin transcripts in maize endosperm.

Cyclins are the regulatory subunits of cyclin-dependent protein kinases. In investigations of the expression of a cyclin gene during maize endosperm development, we detected a cyclin transcript with a 63-bp deletion in the region encoding the conserved 'cyclin box' where cyclin interacts with p34cdc2, the catalytic domain of the cyclin-dependent protein kinase. Analysis of cDNA and genomic sequences, and other observations, indicated that the deletion was caused by alternative splicing of a retained intron in the normally spliced transcript. Whereas the normally spliced cyclin RNA was mitotically functional, as indicated by its ability to promote maturation of Xenopus oocytes, the alternatively spliced transcript was unable to promote maturation. In addition to maize endosperm, the alternatively spliced cyclin was detected in apical meristem, mature leaf, root tip and mature root.

Role of Auxin in Maize Endosperm Development (Timing of Nuclear DNA Endoreduplication, Zein Expression, and Cytokinin).

The timing of developmental events and regulatory roles of auxin were examined in maize (Zea mays L.) endosperms. Zeatin, zeatin riboside, and indole-3-acetic acid (IAA) levels were determined by enzyme-linked immunosorbent (ELISA). Zeatin and zeatin riboside increased to maximal concentrations at an early stage (9 d after pollination [DAP]), corresponding to the stage when cell division rate was maximal. In contrast, IAA concentration was low at 9 DAP and abruptly increased from 9 to 11 DAP, thus creating a sharp decline in the cytokinin to auxin ratio. Coincident with the increase in IAA was an increase in DNA content per nucleus, attributed to postmitotic DNA replication via endoreduplication. Exogenous application of 2,4-dichlorophenoxyacetic acid (2,4-D) at 5 or 7 DAP hastened the time course of DNA accumulation per nucleus and increased the average nuclear diameter, whereas 2-(para-chlorophenoxy)isobutyric acid delayed such development. Exogenously applied 2,4-D hastened the accumulation of the zein polypeptides of apparent molecular masses of 12, 14, and 16 kD and the expression of mRNA hybridizing with a zein DNA probe. We conclude that an abrupt increase in auxin induces cellular differentiation events in endosperm, including endoredupliction and expression of particular zein storage proteins.

Endosperm cell division in maize kernels cultured at three levels of water potential.

The influence of osmoticum treatments on early kernel development of maize (Zea mays L.) was studied using an in vitro culture method. Kernels with subtending cob sections were placed in culture at 5 days after pollination. Sucrose (0.29, 0.44, or 0.58 molar) and sorbitol (0, 0.15, or 0.29 molar) were used to obtain six media with water potentials of -1.1, -1.6, or -2.0 megapascals. Kernel water potential declined in correspondence with the water potential of the medium; however, fresh weight growth was not significantly inhibited from 5 to 12 days after pollination. In stress treatments with media water potentials of -1.6 or -2.0 megapascals, endosperm tissue accumulated water and solutes from 10 and 12 days after pollination at a rate similar to or greater than that of the control (-1.1 megapascals). In contrast, endosperm cell division was inhibited in all treatments relative to control. At 10 days after pollination, endosperm sucrose concentration was greater in two of the -2.0 megapascal treatments with 0.44 or 0.58 molar media sucrose compared to control kernels cultured in 0.29 molar sucrose at -1.1 megapascals. Significant increases in abscisic acid content per gram of fresh weight were detected in two -2.0 megapascal treatments (0.29 molar sucrose plus 0.29 molar sorbitol and 0.58 molar sucrose) at 10 days after pollination. We conclude that in cultured maize kernels, endosperm cell division was more responsive than fresh weight accumulation to low water potential treatments. Data were consistent with mechanisms involving abscisic acid or lowered tissue water potential, or an interaction of the two factors.

Water deficit induces abscisic Acid accumulation in endosperm of maize viviparous mutants.

To determine whether abscisic acid (ABA) accumulation in endosperms of water-limited maize (Zea mays L.) plants is from synthesis in maternal plant organs or from intraendosperm synthesis, plants heterozygous for viviparous (vp) genes were self-pollinated to create endosperm genotypes capable (+/-/-; +/+/-; +/+/+) or incapable (-/-/-) of carotenoid and ABA synthesis. The mutants vp2, vp5, and vp7, each in W22 inbred background, were utilized. Both in wild-type endosperms capable of ABA synthesis and in mutants incapable of ABA synthesis, ABA concentrations at 15 days after pollination were substantially increased in response to plant water deficit. We conclude that ABA synthesis in maternal organs was the source of ABA that accumulated in endosperms in response to plant water deficit.

Influence of water deficit on maize endosperm development : enzyme activities and RNA transcripts of starch and zein synthesis, abscisic Acid, and cell division.

In maize (Zea mays L.), drought during the post-pollination stage decreases kernel growth and often leads to grain yield losses. Kernels in the apical region of the ear are more severely affected than basally positioned kernels. We hypothesized that water deficit during early endosperm development might inhibit kernel growth by decreasing endosperm cell division, and that this response might be mediated by changes in endosperm abscisic acid (ABA) levels. Greenhouse-grown maize, cultivar Pioneer 3925, was subjected to water limitation from 1 to 15 days after pollination (DAP), spanning the period of endosperm cell division and induction of storage product accumulation. Water deficit decreased the number of endosperm nuclei during the treatment period; the most substantial effect was in the apical region of ears. Correspondingly, endosperm fresh weight, starch accumulation and dry mass at maturity were decreased by water limitation. Abscisic acid concentrations in endosperm were quantified by enzyme-linked immunosorbent assay. Water deficit increased ABA concentration in apical-region endosperm by four-fold compared to controls. ABA concentrations were also increased in middle and basal regions of the ear, but to a lesser extent. Two key enzymes in the starch synthesis pathway, sucrose synthase and granule-bound ADP-glucose starch synthase, and zein, the major storage protein in maize endosperm, were studied as markers of storage product synthesis. Water deficit did not affect sucrose synthase enzyme activity or RNA transcript abundance relative to total RNA. However, ADP-glucose starch synthase activity and RNA transcript abundance decreased slightly in apical-region endosperm of water-limited plants by 15 DAP, compared with well-watered controls. In contrast to starch, there was no treatment effect on the accumulation of zein, evaluated at either the polypeptide or RNA level. We conclude that under the conditions tested, the establishment of starch and zein synthetic potential in endosperm was only slightly affected by plant water deficit during the early phase of kernel growth, and that capacity for growth and starch accumulation was affected by the extent to which cell division was inhibited. Based on correlative changes in ABA concentration and cell division we suggest that ABA may play a role in inhibiting endosperm cell division during water limitation.

Abscisic Acid inhibition of endosperm cell division in cultured maize kernels.

The response of developing maize (Zea mays L.) endosperm to elevated levels of abscisic acid (ABA) was investigated. Maize kernels and subtending cob sections were excised at 5 days after pollination (DAP) and placed in culture with or without 90 micromolar (+/-)-ABA in the medium. A decreased number of cells per endosperm was observed at 10 DAP (and later sampling times) in kernels cultured in medium containing ABA from 5 DAP, and in kernels transferred at 8 DAP to medium containing ABA, but not in kernels transferred at 11 DAP to medium containing ABA. The number of starch granules per endosperm was decreased in some treatments, but the reduction, when apparent, was comparable to the decreased number of endosperm cells. The effect on endosperm fresh weight was slight, transient, and appeared to be secondary to the effect on cell number. Mature endosperm dry weight was reduced when kernels were cultured continuously in medium containing ABA. Endosperm (+)-ABA content of kernels cultured in 0, 3, 10, 30, 100, or 300 micromolar (+/-)-ABA was measured at 10 DAP by indirect ELISA using a monoclonal antibody. Content of (+)-ABA in endosperms correlated negatively (R = -0.92) with endosperm cell number. On the basis of these studies we propose that during early kernel development, elevated levels of ABA decrease the rate of cell division in maize endosperm which, in turn, could limit the storage capacity of the kernel.

Gibberellic Acid Regulates Cell Wall Extensibility in Wheat (Triticum aestivum L.).

Mutations (Rht genes) blocking sensitivity to gibberellic acid (GA) were used to examine phytohormone mediated cell wall expansion in wheat (Triticum aestivum L.). Irreversible extensibility of immature leaf segments, as determined by stress/strain (instron) measurements, declined with Rht gene dose. Exogenous GA(3) significantly increased wall extensibility in the nonmutant controls but had no effect on the near-isogenic GA-insensitive genotypes. Furthermore, ancymidol, an inhibitor of gibberellin biosynthesis, diminished wall extensibility in the nonmutant control. Extensibility of immature segments was highly correlated with mature leaf sheath length (R = +0.95). The results indicate that wall yielding properties of expanding wheat leaves are associated with leaf cell expansion potential and that GA is involved in the determination of those properties.

Carbon Dioxide and Light Responses of Photosynthesis in Cowpea and Pigeonpea during Water Deficit and Recovery.

Greenhouse-grown pigeonpea (Cajanus cajan, [L.] Millsp.; cultivar UW-10) and cowpea (Vigna unguiculata, [L.] Walp.; cultivar California No. 5) were well-watered (control) or subjected to low water potential by withholding water to compare their modes of adaptation to water-limited conditions. Leaf CO(2) exchange rate (CER), leaf diffusive conductance to CO(2) (g(l)), and CO(2) concentration in the leaf intercellular air space (C(i)) were determined at various CO(2) concentrations and photon flux densities (PFD) of photosynthetically active radiation (400 to 700 nanometer). In cowpea, g(l) declined to less than 15% of controls and total water potential (psi(w)) at midafternoon declined to -0.8 megapascal after 5 days of withholding water, whereas g(l) in pigeonpea was about 40% of controls even though midafternoon psi(w) was -1.9 megapascal. After 8 days of withholding water, psi(w) at midafternoon declined to -0.9 and -2.4 megapascals in cowpea and pigeonpea, respectively. The solute component of water potential (psi(s)) decreased substantially less in cowpea than pigeonpea. Photosynthetic CER at saturation photon flux density (PFD) and ambient external CO(2) concentration (360 microliters per liter) on day 5 of withholding decreased by 83 and 55% in cowpea and pigeonpea, respectively. When measured at external, CO(2) concentration in bulk air of 360 microliters per liter, the CER of cowpea had fully recovered to control levels 3 days after rewatering; however, at 970 microliters per liter the PFD-saturated CERs of both species were substantially lower than in controls, indicating residual impairment. In stressed plants of both species the CER responses to C(i) from 250 to 600 microliters per liter indicated that a substantial nonstomatal inhibition of CER had occurred. Although the sensitivity of g(l) to water limitation in cowpea suggested a dehydration avoidance response, parallel measurements of CER at various C(i) and PFD indicated that photosynthetic activity of cowpea mesophyll was substantially inhibited by the water-limited treatment.

Effect of increased temperature in apical regions of maize ears on starch-synthesis enzymes and accumulation of sugars and starch.

Apical florets of maize (Zea mays L.) ears differentiate later than basal florets and form kernels which have lower dry matter accumulation rates. The purpose of this study was to determine whether increasing the temperature of apical kernels during the dry matter accumulation period would alter the difference in growth rate between apical and basal kernels. Apical regions of field-grown maize (cultivar Cornell 175) ears were heated to 25 +/- 3 degrees C from 7 days after pollination to maturity (tip-heated ears) and compared with unheated ears (control). In controls, apical-kernel endosperm had 24% smaller dry weight at maturity, lower concentration of sucrose, and lower activity of ADP-Glc starch synthase than basal-kernel endosperm, whereas ADP-Glc-pyrophosphorylase (ADPG-PPase) activities were similar. In tip-heated ears apical-kernel endosperm had the same growth rate and final weight as basal-kernel endosperm and apical kernels had higher sucrose concentrations, higher ADP-Glc starch synthase activity, and similar ADPG-PPase activity. Total grain weight per ear was not increased by tip-heating because the increase in size of apical kernels was partially offset by a slight decrease in size of the basal- and middle-position kernels. Tip-heating hastened some of the developmental events in apical kernels. ADPG-PPase and ADP-Glc starch synthase activities reached peak levels and starch concentration began rising earlier in apical kernels. However, tip-heating did not shorten the period of starch accumulation in apical kernels. The results indicate that the lower growth rate and smaller size of apical kernels are not solely determined by differences in prepollination floret development.

Enzyme activities of starch and sucrose pathways and growth of apical and Basal maize kernels.

Apical kernels of maize (Zea mays L.) ears have smaller size and lower growth rates than basal kernels. To improve our understanding of this difference, the developmental patterns of starch-synthesis-pathway enzyme activities and accumulation of sugars and starch was determined in apical- and basal-kernel endosperm of greenhouse-grown maize (cultivar Cornell 175) plants. Plants were synchronously pollinated, kernels were sampled from apical and basal ear positions throughout kernel development, and enzyme activities were measured in crude preparations. Several factors were correlated with the higher dry matter accumulation rate and larger mature kernel size of basal-kernel endosperm. During the period of cell expansion (7 to 19 days after pollination), the activity of insoluble (acid) invertase and sucose concentration in endosperm of basal kernels exceeded that in apical kernels. Soluble (alkaline) invertase was also high during this stage but was the same in endosperm of basal and apical kernels, while glucose concentration was higher in apical-kernel endosperm. During the period of maximal starch synthesis, the activities of sucrose synthase, ADP-Glc-pyrophosphorylase, and insoluble (granule-bound) ADP-Glc-starch synthase were higher in endosperm of basal than apical kernels. Soluble ADP-Glc-starch synthase, which was maximal during the early stage before starch accumulated, was the same in endosperm from apical and basal kernels. It appeared that differences in metabolic potential between apical and basal kernels were established at an early stage in kernel development.

Reserve carbohydrate in maize stem : [C]glucose and [C]sucrose uptake characteristics.

Maize (Zea mays L.) stem is thought to function alternately as a net importing and net exporting organ during ontogeny, depending on whole plant photosynthetic source and sink status. The [(14)C]sucrose and [(14)C]glucose uptake capacity of stem tissues was investigated to increase our understanding of the transport factors which may influence sink status.Uptake from solutions containing up to 200 millimolar radiolabeled sugar showed that d-glucose uptake consisted of saturable and nonsaturable components, while sucrose uptake was primarily nonsaturable during the kernel-fill stages. l-Glucose uptake lacked the saturable component but both d and l isomers apparently had similar slopes for the nonsaturable component. Uptake was sensitive to inhibitors and temperature, and was increased slightly by lowered pH.The seasonal chronology for saturable uptake by isolated vascular bundles and associated pith revealed highest rates between anthesis and early kernel growth, corresponding with the stage when net sugar accumulation rates were highest. For isolated pith, the rates increased at the final stages of plant development.The rate of labeled l-glucose movement from vascular bundles into pith in isolated stem segments was greater at the silking stage than at later developmental stages, suggesting a lower resistance to diffusive transport from vascular bundles into pith at silking. Studies with stem plus ear explants showed that the capability for sugar transport from pith to vascular bundles and for phloem loading and export from the stem region was present throughout the developmental period from early kernel fill (milk) to late kernel fill (dent).

Identification of a dihydrophaseic Acid aldopyranoside from soybean tissue.

A previously unidentified abscisic acid metabolite has been isolated and characterized. (+/-)-[2-(14)C]Abscisic acid was incubated in intact soybean leaves and pods; the radiolabeled metabolite was purified by high performance liquid chromatography with on-line scintillation spectrometry detection. Gas chromatography-mass spectrometry was used to obtain spectra of the acetylated and methyl esterified derivatives. The data were consistent with a proposed dihydrophaseic acid-aldopyranoside identity. Conjugation through the 4'-hydroxyl of dihydrophaseic acid is suggested.

Abscisic Acid Translocation and Metabolism in Soybeans following Depodding and Petiole Girdling Treatments.

It was found earlier that depodding and girdling treatments which obstruct translocation, result in increased leaf AbA levels and partial stomatal closure. In the present work (+/-) [2-(14)C]abscisic acid (AbA) was introduced into leaves and the mass, and radioactivity of AbA and AbA-metabolites were analyzed following translocation obstruction to determine whether the increased AbA was due to higher rates of synthesis, or lower rates of catabolism or export. The (+/-) [2-(14)C]AbA was introduced into soybean (Merr.) leaves by injection into the petiole region. AbA and AbA-metabolites (phaseic acid [PA], dihydrophaseic acid [DPA], AbA-conjugate, and an unknown metabolite) were separated with preparative high performance liquid chromatography. Methyl esters of AbA (free and that released after hydrolysis of AbA-conjugate), PA and DPA were determined with gas chromatography using electron capture detection.The level of AbA in leaf blades increased after girdling or depodding as was found previously. Accompanying this was an increase in PA in girdled leaves; but no discernible trends in the levels of DPA and AbA-conjugate were evident. The (+/-) [2-(14)C]AbA specific radioactivities declined similarly for all treatments, indicating that these treatments did not increase the rate of AbA synthesis.Export of injected (+/-)[2-(14)C]AbA from leaves was substantial. After girdling or depodding, this export was obstructed, as evidenced by the lack of decline in leaf blade radioactivity or lack of increase in pod radioactivity following these treatments. The higher AbA levels, which were observed in leaf blades of girdled and depodded plants, could be attributed largely to the translocation obstruction.

Effect of obstructed translocation on leaf abscisic Acid, and associated stomatal closure and photosynthesis decline.

Pod removal or petiole girdling, which causes obstruction of translocation, was found in our previous study to cause reduced rates of photosynthesis in soybean leaves due to stomatal closure. The purpose of this research was to determine the involvement of photoassimilate accumulation and leaf abscisic acid (ABA) levels in the mechanism of stomatal closure induced by such treatments.Leaf glucose and sucrose levels increased during the initial 12-hour period after depodding or petiole girdling. Starch, which represents a much larger pool of leaf carbohydrate, was not perceptibly increased above control levels during the 12-hour posttreatment period.When leaflets were exposed to nonphotosynthetic environments (shading or CO(2)-free air) for a 24-hour period after the translocation-obstructing treatments were applied and then returned to normal light or CO(2) concentration, stomatal diffusive conductivity was reduced 65% and 85% with depodding and girdling, respectively. These reductions were comparable to those previously observed without an intervening nonphotosynthetic exposure, thus indicating that photosynthate accumulations were not necessary for the observed response.Free and bound ABA (released on alkaline hydrolysis) were determined by gas liquid chromatography with electron capture detection following preparative high performance liquid chromatography. Free ABA in monitored leaves increased almost 10-fold 48 hours after complete depodding and 25-fold 24 hours after petiole girdling of such leaves. By 3 hours after treatment, in a time course study, free ABA had increased 2-fold above control values in depodded and 5-fold in girdled leaves. Leaf concentrations of bound ABA did not appear to be related to the treatment effects on stomata.Thus, the translocation-obstructing treatments cause an increased level of ABA by a mechanism not involving accumulation of photoassimilate. Increased leaf ABA levels, which were independent of water stress or leaf water potential, appear to be involved in the stomatal closure response. It is suggested that the mechanism of increased leaf ABA levels following translocation-obstruction may be due to an interference with normal translocation of ABA out of leaves.