Re-export and metabolism of xylem-delivered ABA in attached maize leaves under different transpirational fluxes and xylem ABA concentrations

By feeding radioactive³H-ABA into attached maize leaves, there-export and metabolism of xylem-delivered ABA and their relationshipswith xylem ABA transpirational fluxes and concentrations wereinvestigated. ABA entering leaves in the transpirational streamwas re-exported out of leaves slowly. Within 24 h the proportionof fed radioactivity that was re-exported was less than 45%.When different concentrations of ³H-ABA (100 nM versus 500 nM)was fed, no difference between the two concentrations was foundin their rates of re-export of the fed radioactivity duringthe first 5 h. After 5 h, very little fed radioactivity wasre-exported in leaves that were fed with 100 nM ³H-ABA, whileleaves that were fed with 500 nM ³H-ABA continued to re-exportsuch that the final proportion remaining in leaves after 24h was less as a result, suggesting a concentration-stimulatedre-export. When ³H-ABA was fed at two different transpirationrates which were induced by different air humidity, a 4-folddifference in transpirational fluxes did not produce any differencein terms of re-exportation of fed radioactivity. The rate ofcatabol-ism of xylem-fed ³H-ABA in the attached leaves was muchfaster than that of re-export. On average fed ³H-ABA had a half-lifeof 2.2 h and only 8% remained unmodified after 24 h of incubation,suggesting that re-exported radioactivity might not be the intactform of ABA at all. Using the parameters obtained from the feeding experiment, wecalculated that in a real soil-drying situation the possiblemaximum amount of xylem-delivered ABA that could accumulatein leaves during a day. It was found that the proportion ofdaily accumulated ABA was only 5% of the leaf ABA in well-wateredplants. In soil-dried plants the maximum amount of daily accumulationby xylem ABA could reach 20% of the leaf ABA at the beginningof soil drying, but it soon declined to about 5% again. Thedeclined contribution was mainly due to a reduced transpirationand an increased total leaf ABA as a result of aggravated leafwater deficit. A tight relationship between leaf conductanceand the accumulation of xylem-delivered ABA was not found. Key words: Abscisic acid, ABA, ABA export, ABA metabolism, xylem-delivered ABA, maize     

Comparison of exportation and metabolism of xylem-delivered ABA in maize leaves at different water status and xylem sap pH

³H-ABA was introduced into the xylem stream of maize ( Zea mays}) leaves on intact plants by incubation of a semi-attached flap of the sheath in solutions. The relative contribution of exportation and metabolism to the fate of xylem-delivered ABA was assessed in leaves which were either kept at different water potentials through soil drying treatments or subjected to different xylem pHs (pH 7.4 vs. pH 5.5) through a phosphate buffer in the feeding solutions. Xylem-delivered ABA was rapidly metabolised in well-watered leaves with a half-life of 2.19 h in the relatively mature leaves used in this study. Re-exportation of xylem-delivered ABA from leaves was much slower than metabolism. It took 24 h for half of the fed radioactivity to disappear from the well-watered leaves, and very possibly this radioactivity was in the form of metabolites of fed ³H-ABA. Although soil drying usually increases the output of ABA through phloem as reported in previous studies, it greatly reduced the re-exportation of xylem-fed ABA and/or its metabolites. Metabolism was also significantly reduced by the treatment of soil drying (half-life extended from 2.19 to 3.63 h), although the magnitude of change was much less than that of exportation. Manipulation of the pH in the feeding solution also had its effect on the re-exportation. A shift of pH from 5.5 to 7.4 reduced the rate of disappearance of the total radioactivity fed into the attached leaves, but showed no significant effect on the rate of ABA metabolism. It was concluded that it was the ABA metabolism, rather than a re-exportation from leaves, which was mainly responsible for the disposal of the ABA signal from the xylem and therefore preventing an accumulation in leaves. Water stress and pH increase of xylem sap would increase the time of such ABA's presence in the leaves. Since xylem-imported ABA is unlikely to be re-exported from leaves in its intact form, we believe a recycling of ABA from xylem to phloem through leaves plays only a minor role.     

Effects of ABA in 'Isolated' Guard Cells of Commelina communis L.

The effects of 2 x 10^–3 M ABA on ion fluxes in isolatedguard cells of Commelina communis L. have been studied, using⁸⁶RbCl and K⁸²Br, in epidermal strips in which all cells otherthan guard cells have been killed by treatment at low pH. Theeffect of ABA on influx is small, if present, and the majoreffect is a marked transient stimulation of the efflux of both⁸⁶Rb and ⁸²Br at the plasmalemma; there is also an increasein the flux of ⁸²Br from vacuole to cytoplasm. The stimulationis transient, and the cells do not simply become more leaky.The results are not consistent with previous speculations onthe mechanism by which ABA reduces aperture.     

A study of potassium gradients in the epidermis of intact leaves of Commelina communis L. in relation to stomatal opening

Summary Activated phytochrome lowers the free amino acid pools of all plants investigated by about 15%. The action of red light is preferentially directed to Asp, Glu and Phe. Exogenously supplied Leu-U-¹⁴C is incorporated more quickly into protein of red-light-treated samples compared with dark controls. In contrast, red light decreases the amount of Asp-U-¹⁴C incorporated into protein, but increases the amount of ¹⁴CO₂ respired after feeding with Asp-U-¹⁴C. Red light has no effect on the amount of ¹⁴CO₂ respired after feeding with Leu-U-¹⁴C. Red-light-mediated stimulation of incorporation of Leu-U-¹⁴C into protein occurred within 15 min, well before the red-light-mediated increase in ¹⁴CO₂ production following feeding with Asp-U-¹⁴C could be detected.Abbreviations R red light - FR far-red light - R+FR red immediately followed by far-red light - D dark control - TCA tricarboxylic acid cycle For amino acids as stated in Biochem. J. 126, 773–780 (1972).     

Initiation of the synthesis of 'stress' ABA by (+)-[(2) H(6) ]ABA infiltrated into leaves of Commelina communis

The 'fettered' fraction of abscisic acid (ABA) that is held within the chloroplasts of unwilted bean and Commelina communis leaves is released when the leaves wilt and it is this 'free' ABA that is now proposed to cause the stomata to close within 2 or 3 min, well before the rise in total ABA can be detected. The large increase in 'stress' ABA begins 2-3 h later. The fettered ABA in a centrifuged homogenate is released by hyperosmotic solutions of mannitol (0.8 M) and NaCl (0.4 M). Dilute solution of halothane (10 mM) and colchicine (1 mM), the detergent sodium dodecyl sulphate (1 mM) the herbicide 2,4-d (0.1 mM) and dithiothreitol (0.01 mM) also caused ABA to be released. Zeatin (0.01 mM), cumene hydroperoxide (0.01 mM) and CaCl(2) (1 mM) had negligible effects. It was postulated that the ABA released from the chloroplasts by wilting could be the signal that initiates the synthesis of the dioxygenase and other enzymes necessary to produce the rise (up to 40-fold) in the amount of stress ABA that is seen 2-3 h later. To test this hypothesis, a solution of (+)-[(2) H(6) ]ABA was vacuum infiltrated into unwilted Commelina leaves to mimic the rise in ABA caused by wilting and gas chromatography/mass spectrometry of the ABA in the extract after 3 h showed that concentrations of (+)-[(2) H(6) ]ABA of up to 0.3 μM stimulated synthesis of endogenous [(1) H]ABA by 15-fold in the unwilted leaves. A 0.5 μM solution blocked the increase in the amount of ABA formed and also reduced the amount of ABA formed in response to a 0.8 M mannitol solution.     

Variation in stomatal characteristics over the lower surface of Commelina communis leaves

Abstract. The silicone rubber impression technique was used to measure stomatal apertures in 9 mm² sampling areas covering the entire lower surface of leaves of Commelina communis L. The data were analysed using a computer program which produced 'iso-aperture' contours illustrating local differences in mean stomatal aperture. Little consistency was seen in the iso-aperture patterns among sampling times, although the stomata were always relatively closed at the leaf tip and base. When stomata in the middle of the lamina were open, those near to the leaf margin tended to be relatively closed. In places, gradients of mean stomatal aperture were as high as 1 μm mm⁻¹. Measurements along a transect across the lower epidermis revealed no correlation of stomatal aperture with the presence of major veins in the mesophyll tissue. Variation in guard-cell size and stomatal frequency on the lower leaf surface was also analysed. The guard cells were smallest and the stomata more frequent near to the leaf margins. The significance of the results is discussed in relation to measurements of leaf conductance and models of stomatal function.     

Regulation of Mg,K-ATPase Activity in Guard Cells of Commelina communis by Phytochrome and ABA

The microsomal fraction obtained from guard cell protoplastswas assayed for ATPase activity at pH 6.5. Triton X-100 didnot affect this stimulation of activity of ATPase by K⁺ up to5 mM, but the detergent abolished the stimulatory effect ofK⁺ at higher concentrations. The ATPase activity was inhibitedby N,N-dicyclohexylcarbodiimide and ABA. Irradiation with redlight enhanced the ATPase activity more than did irradiationwith far-red light. ABA and irradiation with red or far-redlight were effective only in the presence of K⁺. These resultssuggest the possibility that the ATPase activity is modulatedonly indirectly by light and ABA. (Received January 9, 1989; Accepted July 10, 1989)     

Malate metabolism in isolated epidermis of Commelina communis L. in relation to stomatal functioning

Epidermal strips with closed stomata were exposed to malic acid labelled with ¹⁴C either uniformly or in 4-C only. During incubation with [U-¹⁴C]malate, radioactivity appeared in products of the tricarboxylic-acid cycle and in transamination products within 10 min, in sugars after 2 h. Hardly any radioactivity was found in sugars if [4-¹⁴C]malate had been offered. This difference in the degree of labelling of sugars indicates that gluconeogenesis can occur in epidermal tissue, involving the decarboxylation of malate. Epidermis incubated with labelled malate was hydrolyzed after extraction with aqueous ethanol. The hydrolysate contained glucose as the only radioactive product, indicating that starch had been formed from malate. Microautoradiograms were black above stomatal complexes, showing that the latter were sites of starch formation. In order to follow the fate of malate during stomatal closure, malate was labelled in guard cells by exposing epidermes with open stomata to ¹⁴CO₂ and then initiating stomatal closure. Of the radioactive fixation products of CO₂ only malate was released into the water on which the epidermal samples floated; the epidermal strips retained some of the malate and all of its metabolites. In the case of rapid stomatal closure initiated by abscisic acid and completed within 5 min, 63% of the radioactivity was in the malate released, 22% in the malate retained, the remainder in aspartate, glutamate, and citrate. We conclude that during stomatal closing guard cells can dispose of malate by release, gluconeogenesis, and consumption in the tricarboxylic-acid cycle.Abbreviations ABA abscisic acid - NAD nicotinamide adenine dinucleotide - NADP nicotinamide adenine dinucleotide phosphate - PEP phosphoenolpyruvate     

Active chloride transport in the leaf epidermis of Commelina communis in relation to stomatal activity

Summary A chloride selective micro-electrode has been used to determine vacuolar chloride concentrations in individual cells of the leaf epidermis of Commelina communis. When the stomata were open a gradient in chloride concentration across the stomatal complex was observed with the highest concentration in the guard cells. On stomatal closure the chloride gradient was reversed. Calculation of the driving forces on chloride indicated that active transport of chloride was occurring during both stomatal opening and closure. This transport appeared to be energetically independent of the transport of potassium. These results are discussed in relation to the behaviour of other anions during stomatal movements.     

Changes in starch and glucose levels in the epidermis of Commelina communis in relation to stomatal movements

Abstract Starch levels in Commelina communis epidermis, were shown to be higher in the dark than in the light, from plants kept under an imposed photoperiodic regime. These changes were also inversely correlated with diffusive conductance (1/diffusive resistance, 1 /R) of the leaves used. Even after advancing the photoperiod by 7 h the changes in starch levels continued for at least 1 d and were still inversely correlated with 1/R. Corresponding changes in glucose levels were measured in the same tissue and were found to correlate with 1/R at the end of the dark period and mid photoperiod, but not at the beginning of the dark period. These changes are discussed in relation to possible mechanisms of stomatal opening and closing.     

Guard Cell Metabolism in Epidermis of Commelina communis L. During Stomatal Opening and Closing

The rates of CO² incorporation into the epidermis of C. communiswere linear and were similar during the completion of opening(2 h) and closing (1 h) movements of stomata. The kinetics of¹⁴C turnover between metabolites and the rates of ‘leakage’of metabolites were determined for opening and closing movements.When stomata were opening there was a slow turnover of ¹⁴C frommalate chiefly into sugars. Upon stomatal closure ¹⁴C was initiallymainly in sugars, malate, and sugar phosphates. Thereafter,there was a slight loss of label from sugar phosphates witha corresponding increase in malate. Starch became labelled duringopening and closing movements. Rates of incorporation of CO₂found in the ‘leakage’ fraction were greatest whenstomata were opening. Of the labelled compounds Most‘from the tissue, malate was the most highly labelled whetherstomata were opening or closing. Although interpretation of the turnover patterns is difficultwithout knowledge of pool sizes for the metabolites it is suggestedthat a pool of sugars exists within the guard cells, which havefairly direct and reversible access to carbon from starch andmalate. The implications of loss of malate from guard cellsduring stomatal opening and closing are discussed.     

Stomatal Responses to Applied ABA and CO2 in Epidermis Detached from Well-Watered and Water-Stressed Plants of Commelina communis L.

Epidermal strips from either well-watered or water-stressedplants of Commelina communis L. were subjected to a range ofABA concentrations (10^–6–10^–3 mol m^–3)in the presence (330 parts 10^–6 in air) or virtual absence(3 parts 10–6 in air) of CO₂. The stomatal response toCO₂ was greater in epidermis from water-stressed plants, althoughthere was a distinct CO₂ response in epidermis from well-wateredplants. Additions of ABA via the incubation medium had littleeffect on the relative CO₂ response. Stomata responded to ABAboth in the presence and virtual absence of CO₂, but the relativeresponse to ABA was greatest in the high CO₂ treatment. Whenwell-watered plants were sprayed with a 10^–1 mol m^–3ABA solution 1 d prior to use, the stomatal response of detachedepidermis to both CO₂ and ABA was very similar to that of epidermisdetached from water-stressed leaves. It is hypothesized thata prolonged exposure to ABA is necessary before there is anymodification of the CO₂ response of stomata.     

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.

     

Metabolism of 3- and 4-phosphorylated phosphatidylinositols in stomatal guard cells of Commelina communis L.

Within the plant kingdom the stomatal guard cell is presented as a model system of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-mediated signal transduction. Despite this it is only recently that the phosphoinositide components of animal signal transduction pathways have been identified in stomatal guard cells. Interestingly, stomatal guard cells contain both 3- and 4-phosphorylated phosphatidylinositols though their relative contributions to signalling remain undefined. An appraisal of the routes of synthesis and rates of turnover of these phosphatidylinositols would appear timely as the in vivo biosynthesis of these components is a much neglected facet of the phosphoinositide-mediated signalling paradigm as purported to apply to plants. A non-equilibrium [³²P]P_i labelling strategy and enzymic and chemical dissection of labelled phosphatidylinositols have been used to address not only the route of synthesis but also the rates of turnover of phosphatidylinositols in stomatal guard cells of Commelina communis L. The specific activity of the ATP pool of isolated guard cells was found to increase over a 4 h period when labelled from [³²P]P_i. In separate experiments, isolated guard cells were labelled over a 40–240 min period, their lipids extracted, deacylated and resolved by HPLC. Glycerophosphoinositol phosphate (GroPInsP) and glycerophosphoinositol bisphosphate (GroPInsP₂) peaks were desalted and enzymically cleaved with alkaline phosphatase and human erythrocyte ghosts, respectively. The monoester phosphate in phosphatidylinositol 4-monophosphate (PtdIns4P) accounted for 90–97% of the [³²P]P_i label while the 4- and 5-monoester phosphates of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] accounted for typically 39% and 61% respectively. Therefore, the evidence is consistent with synthesis of PtdIns(4,5)P₂ by successive 4- and 5-phosphorylation of phosphatidylinositol (PtdIns). This study therefore represents the first report of the pathway of the synthesis of 4- and 5-phosphorylated phosphatidylinositols in a single defined hormone-responsive plant cell type. The monoester phosphate in phosphatidylinositol 3-monophosphate (PtdIns3P) accounted for 83–95% of the ³²P label. It was not possible, however, to determine the route of synthesis of phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P₂] owing to the rapid attainment of equilibrium between the 3- and 4-monoester phosphates of PtdIns(3,4)P₂, each containing approximately 50% of the label at just 40 min of labelling. Turnover of PtdIns3P was quicker than that of PtdIns4P. Similarly, turnover of PtdIns(3,4)P₂ was quicker than that of PtdIns(4,5)P₂, and in mass terms PtdIns(3,4)P₂ appeared to predominate over PtdIns(4,5)P₂. By analogy with animal systems, in which signalling molecules such as PtdIns(4,5)P₂ show considerable basal turnover, the evidence presented is consistent with signalling roles for PtdIns3P and PtdIns(3,4)P₂ in addition to those previously indicated for PtdIns(4,5)P₂ in stomatal guard cells.     

Acid-Induced Stomatal Opening in Commelina communis and Vicia faba

The effects of H^$ and fusicoccin (FC) on stomatal opening inthe dark were investigated using epidermal strips of Commelinacommunis and Vicia faba cv. Ryosai Issun. Citrate-phosphatebuffer induced maximal opening of stomata at pH 3.0 when testedover the range of 2.7 to 5.0. HCl at 1 mM also induced stomatalopening without appreciable accumulation of K^$ in the guardcells. After 4 hr treatment with 10 µM FC, stomata openedwith concomitant accumulation of K^$ in the guard cells, although1–2 hr treatment caused opening without concomitant K^$increase. These results suggest that stomatal opening can be caused bysalt accumulation and/or changes of the physicochemical conditionsin the cell wall of the guard cells due to high acidity. ¹ Present address: Biological Laboratory, Faculty of Education,Nagasaki University, Nagasski 852, Japan. (Received April 30, 1982; Accepted July 17, 1982)     

Dynamic analysis of ABA accumulation in relation to the rate of ABA catabolism in maize tissues under water deficit

The plant hormone abscisic acid (ABA) accumulates in plant tissues which experience water deficit (stress ABA). This study analysed its accumulation as a function of both synthesis and catabolism in maize tissues. By following the disappearance of the stress ABA when ABA synthesis was blocked by nordihydroguaiaretic acid (NDGA), the rate of the catabolism of stress ABA was determined. When compared with the catabolic rate of baseline (non-stress) ABA, stress ABA showed a catabolic rate >11 times higher. With such an elevated catabolic rate, it is proposed that the xanthophyll precursor pool may not be able to sustain the ABA accumulation, and such a proposition has been substantiated by further experiments where fluridone is used to limit the availability of upstream ABA precursors. When fluridone was used, stress ABA accumulation could only be sustained for a few hours, i.e. approximately 5 h for leaf and 1 h for root tissues. In detached roots, stress ABA accumulation could not be sustained even if fluridone was not used, suggesting that stress ABA accumulation in root systems requires the continuous import of ABA precursors from the shoots. Such an assumption was substantiated by the observation that defoliation or shading significantly reduced ABA accumulation in intact roots. The present study suggests that ABA catabolism is rapid enough to play an important role in the regulation of ABA accumulation.     

Effect of the Mesophyll on Stomatal Opening in Commelina communis

The effect of a number of factors on the opening of stomatain the intact leaf and in the isolated leaf epidermis of Commelinacommunishas been investigated. Stomata in the intact leaf opened widein the light and closed rapidly on transfer to the dark. Theywere also sensitive to CO₂. In contrast, stomata in isolatedepidermis floated on an incubation solution containing 100 molm^–3KCl responded neither to light nor CO₂. They opened as widelyas those in the intact leaf when treated with fusicoccin. Stomata in isolated epidermis opened almost as wide as thosein the intact leaf when they were incubated with isolatedmesophyllcells in the light. The solution in which the mesophyll cellswere incubated was separated by centrifugation. Themedium fromcells previously incubated in the light caused the stomata inisolated epidermis to open but that from cells kept inthe darkhad no effect. A similar effect was observed when isolated chloroplastswere incubated with the isolated epidermis.However, the supernatantfrom the chloroplast suspension had no significant effect onstomatal opening. These results indicate that the mesophyll plays an importantrole in stomatal opening in the light. The mesophyll appearstoproduce in the light, but not in the dark, a soluble compoundwhich moves to the guard cells to bring about stomatal opening.Theexperiments with isolated chloroplasts suggest that this substanceis a product of photosynthesis. Key words: Commelina communis, stomata, light, mesophyll     

Validation and characterization of a major QTL affecting leaf ABA concentration in maize

A previous study conducted on a maize (Zea mays L.) mapping population derived from Os420 × IABO78 identified a quantitative trait locus (QTL) for leaf-abscisic acid concentration (L-ABA) on chromosome 2 (bin 2.04). In order to validate this QTL, we analyzed with RFLP markers 16 F₄ lines obtained by divergent selection for L-ABA from the same source. Three RFLPs mapping near bin 2.04 showed skewed allelic frequencies; the L-ABA increasing allele (+) was more frequent within the eight lines selected for high L-ABA, while the decreasing allele (–) was more frequent within the eight lines selected for low L-ABA. To characterize more accurately the direct and associated effects of this QTL, near-isogenic lines were developed by molecular marker-assisted back-crossing; four backcross-derived lines were homozygous (+/+) at the QTL and four were (–/–). A pair of near-isogenic hybrids (+/+) and (–/–) at the QTL were also produced. These materials were field tested under water-stressed and well-watered conditions. Across water regimes, the four (+/+) lines averaged a significantly higher mean value than the four (–/–) lines for L-ABA (494 vs. 396 ng ABA g^–1 DW) and a significantly lower mean value for relative water content (90.6 vs. 92.0%). The (+/+) hybrid exceeded the (–/–) for L-ABA (476 vs. 325 ng g^–1 DW) and was less affected by root lodging (44.6 vs. 66.1%). Our results validate the presence of a major QTL for L-ABA on bin 2.04 and indicate that the QTL also affects root traits and relative water content.