Compartmental distribution and redistribution of abscisic acid in intact leaves

Using a computer model written for whole leaves (Slovik et al. 1992, Planta 187, 14–25) we present in this paper calculations of abscisic acid (ABA) redistribution among different leaf tissues and their compartments in relation to stomatal regulation under drought stress. The model calculations are based on experimental data and biophysical laws. They yield the following results and postulates: (i) Under stress, compartmental pH-shifts come about as a consequence of the inhibition of the pH component of proton-motive forces at the plasmalemma. There is a decrease of net proton fluxes by about 8.6 nmol · s^–1 · m^–2. (ii) Using stress-induced pH-shifts we demonstrate how stress intensities can be quantified on a molecular basis. (iii) As the weak acid ABA is the only phytohormone which behaves in vivo and in vitro ideally according to the Henderson-Hasselbalch equation, pH-shifts induce a complicated redistribution amongst compartments in the model leaf. (iv) The final accumulation of ABA in guard-cell walls is intensive: up to 16.1-fold compared with only up to 3.4-fold in the guard-cell cytosol. We propose that the binding site of the guard-cell ABA receptor faces the apoplasm. (v) A twoto three-fold ABA accumulation in guard-cell walls is sufficient to induce closure of stomata. (vi) The minimum time lag until stomata start to close is 1–5 min; it depends on the stress intensity and on the guard-cell sensitivity to ABA: the more moderate the stress is, the later stomata start to close or they do not close at all. (vii) In the short term, there is almost no influence of the velocity of pH-shifts on the velocity of the ABA redistribution, (viii) Six hours after the termination of stress there is still an ABA concentration 1.4-fold the initial level in the guard-cell cytosol (delay of ABA relaxation, aftereffect), (ix) The observed induction of net ABA synthesis after onset of stress may be explained by a decrease in cytosolic ABA degradation. About 1 h after onset of stress the model leaf would start to synthesise ABA (and its conjugates) automatically, (x) This ABA net synthesis serves to inform roots via an increased ABA concentration in the phloem sap. The stress-induced ABA redistribution is per se not sufficient to feed the ploem sap with ABA. (xi) The primary target membrane of stress is the plasmalemma, not thylakoids. (xii) The effective stress sensor, which induces the proposed signal chain finally leading to stomatal closure, is located in epidermal cells. Mesophyll cells are not capable of creating a significant ABA signal to guard cells if the epidermal plasmalemma conductance to undissociated molecular species of ABA (HABA) is indeed higher than the plasmalemma conductance of the mesophyll (plasmodesmata open), (xiii) All model conclusions which can be compared with independent experimental data quantitatively fit to them. We conclude that the basic experimental data of the model are consistent. A stress-induced ABA redistribution in the leaf lamina elicits stomatal closure.Abbreviations ABA abscisic acid - CON vacuolar ABA conjugates We are grateful to Prof. U. Heber (Lehrstuhl Botanik I, University of Würzburg, FRG) for stimulating discussions. This work has been performed within the research program of the Sonderforschungsbereich 251 (TP 3 and 4) of the University of Würzburg. It has been also supported by the Fonds der Chemischen Industrie.     

Sugar uptake into strawberry fruit is stimulated by abscisic acid and indoleacetic acid

Changes in sugar uptake into strawberry fruits with maturation and the hormonal effect on uptake mechanisms, though important to fruit development, are not known. Therefore, the kinetics of sugar uptake into strawberry ( Fragaria x ananassa Duch cv. Nyoho) fruit tissue and the effects of abscisic acid (ABA) and indoleacetic acid (LAA) on the mechanism of uptake were investigated at 25 and 35 days after pollination (DAP). Uptake of ¹⁴C-sugar was measured over the concentration range of 2 to 30 m M. Uptake kinetics showed a biphasic response to increasing external concentration of ¹⁴C-sugars, and indicated the presence of P -chlorormercuribenzenesulfonic acid (PCMBS)-sensitive and PCMBS-insensitive uptake. The K_m value for each sugar was in the range of 10 to 20 m M. Stage of development had no effect on K_m. but V_max for glucose decreased with maturation. Further, sucrose was not taken up through a PC-MBS-sensitive transport at 35 DAP. ABA, especially 10 μ M , at 25 DAP stimulated uptake of all sugars, mostly through enhanced PCMBS-insensitive uptake but not PC-MBS-sensitive uptake. In contrast to ABA, stimulation of sugar uptake by IAA was most effective at 1 μ M . The PCMBS-insensitive uptake of each sugar was also stimulated by IAA. Further, the PCMBS-sensitive uptake of glucose was enhanced. The developmental change of PCMBS-sensitive sugar uptake and the effect of ABA and IAA on uptake mechanism in this study are considered to be important in influencing the development and enlargement of fruits.     

Changes in phenolic acids and abscisic acid in sugar pine seed coats during stratification

The phenolic acids and abscisic acid (ABA) of sugar pine ( Pinus lambertiana Dougl.) seeds coats, separated by high-pressure liquid chromatography, were analyzed during 90 days stratification of the seeds. Although levels of seed coat phenolic acids and ABA declined significantly during, stratification, this decrease did not appear to be responsible for the loss of dormancy due to stratification. Lack of improved germination following washing, cracking, or removal of the seed coats, plus additional evidence, did not support a significant role for the seed coat in the dormancy of sugar pine seeds.     

Changes in phenolic acids and abscisic acid in sugar pine embryos and megagametophytes during stratification

The phenolic acids and abscisic acid (ABA) of sugar pine ( Pinus lambertiana Dougl.) embryos and megagametophytes, separated by high-pressure liquid chromatography, were analyzed during 90 days stratification of the seeds. The phenolic acids occurred mainly as glycosides. Following hydrolysis, the majority of phenolics present could be identified as common benzoic and ciranamic acid derivatives. Levels of phenolic acids were relatively low in dormant seeds, but increased substantially in the embryos during stratification at 5°C, particularly cinnamic acid, p -coumaric acid, ferulic acid, and one unknown. This active synthesis during stratification did not support an inhibitory function for phenolic acids. During stratification at 5°C, changes in ABA levels in both tissues followed a triphasic pattern, with no loss during the first 30 days, a significant decrease the second 30 days, and a lesser decrease the last 30 days. Loss of ABA from moist seeds at 25°C occurred three times as rapidly, so that by 30 days the ABA level of these seeds was equivalent to that of seeds stratified 90 days at 5°C; however, dormancy was not alleviated at 25°C. Application of exogenous ABA (10⁻⁷ to 10⁻⁴M) to stratified seeds did not significantly reduce germination. Together, the above results did not support a primary role for ABA in the maintenance of dormancy in sugar pines.
A correlated increase in phenylpropanoid metabolism and respiratory capacity with increased germinability during stratification suggests that loss of dormancy may be more closely dependent on increased levels of growth promoters or shifts in metabolic pathways.     

Synthesis and biological activity of abscisic acid esters

16 ABA esters including 11 new compounds were prepared by two different esterification routes. All the structures of ABA esters were confirmed by ¹H NMR, ¹³C NMR and HRMS. Their biological activity and hydrolysis stability were investigated. Fortunately, there were 15 and 9 compounds which displayed much better or nearly the same inhibition activity for rice seedling growth and Arabidopsis thaliana seed germination compared to ABA, respectively. Especially, compounds 2d and 2g showed better biological activities than ABA in the three tests. Moreover, we found that chemical hydrolysis ability of the esters in vitro had little relationship to their biological activity.     

Endogenous abscisic acid and wheat germ agglutinin content in wheat grains during development

Abscisic acid (ABA) and wheat germ agglutinin content of immature wheat grains and embryos was determined by immunoassay throughout the development of a field-grown wheat crop ( Triticum aestivum cv. Timmo). Wheat germ agglutinin accumulation in the embryo was not preceded by an increase in endogenous abscisic acid amount or concentration in either embryos or grains. At a later stage in development the endogenous concentration of abscisic acid in both embryos and grains was found to be two orders of magnitude lower than the endogenous levels required to inhibit precocious germination and promote wheat germ agglutinin accumulation in excised embryos cultured in vitro. These findings are discussed in the context of the control of embryo development in vivo by both ABA and the water status of the grain and embryo.     

Proteomic analysis of phosphoproteins regulated by abscisic acid in rice leaves

Abscisic acid (ABA) is a hormone that regulates plant development and adaptation to environmental stresses. Protein phosphorylation has been recognized as an important mechanism for ABA signaling. However, the target phosphoproteins regulated by ABA are still largely unknown. Here, we report the identification of ABA-regulated phosphoproteins in rice using proteomic approaches. Six ABA-regulated phosphoproteins were identified as G protein beta subunit-like protein, ascorbate peroxidase, manganese superoxide dismutase, triosephosphate isomerase, putative Ca²⁺/H⁺ antiporter regulator protein, and glyoxysomal malate dehydrogenase. These results provide new insight into the regulatory mechanism for some ABA signaling proteins and implicate several previously unrecognized proteins in ABA action.     

Endogenous levels of free and conjugated forms of auxin, cytokinins and abscisic acid during seed development in Douglas fir

Endogenous levels of free and conjugated forms of three classes of planthormones were quantified at various stages of megagametophyte development inDouglas fir. Megagametophytes were excised weekly from 8–16 weeks pastpollination (WPP), a period encompassing the central cell to the earlymaturation stage of seed development. The hormones indole-3 acetic acid (IAA),indole-3-aspartate (IAAsp), zeatin (Z), zeatin riboside (ZR), isopentenyladenine(iP), isopentenyladenosine (iPA), abscisic acid (ABA) and abscisic acid glucoseester (ABA-GE) were extracted, purified, fractionated by high- performanceliquid chromatography (HPLC), and then quantified using an enzyme-linkedimmunosorbent assay (ELISA) method. Z levels ranged from 0–25ng/g dry weight (DW) and were highest in megagametophytes at thecentral cell stage (8 WPP). During embryogenesis, Z levels peakedduring week 13. In contrast, the ZR conjugate was not detected over the periodstudied. The iP content of megagametophytes increased at 10 and 13WPP, while the iPA concentration increased at 13 WPP.Prior to fertilisation, IAA was highest in megagametophytes at 9WPP. During embryogenesis, the major IAA accumulations occurred at11, 13 and 15 WPP, the concentration ranging from 0–0.43g/g DW. IAAsp concentrations reached their highest level duringembryogenesis at 14 WPP. ABA content increased at 11 and 13WPP, with a concentration range of 0.1–13 g/gDW. In contrast, ABA-GE levels were relatively constant over the 9-weekperiod analyzed. The endogenous levels of plant hormones varied withmegagametophyte development and were associated with morphological changes.     

Conjugates of abscisic acid, brassinosteroids, ethylene, gibberellins, and jasmonates

Phytohormones, including auxins, abscisic acid, brassinosteroids, cytokinins, ethylene, gibberellins, and jasmonates, are involved in all aspects of plant growth, and developmental processes as well as environmental responses. However, our understanding of hormonal homeostasis is far from complete. Phytohormone conjugation is considered as a part of the mechanism to control cellular levels of these compounds. Active phytohormones are changed into multiple forms by acylation, esterification or glycosylation, for example. It seems that conjugated compounds could serve as pool of inactive phytohormones that can be converted to active forms by de-conjugation reactions. Some conjugates are thought to be temporary storage forms, from which free active hormones can be released after hydrolysis. It is also believed that conjugation serves functions, such as irreversible inactivation, transport, compartmentalization, and protection against degradation. The nature of abscisic acid, brassinosteroid, ethylene, gibberellin, and jasmonate conjugates is discussed.     

Effects of cotton rootstock on endogenous cytokinins and abscisic acid in xylem sap and leaves in relation to leaf senescence

Leaf senescence varies greatly among cotton cultivars, possiblydue to their root characteristics, particularly the root-sourcedcytokinins and abscisic acid (ABA). Early-senescence (K1) andlate-senescence (K2) lines, were reciprocally or self-graftedto examine the effects of rootstock on leaf senescence and endogenoushormones in both leaves and xylem sap. The results indicatethat the graft of K1 scion onto K2 rootstock (K1/K2) alleviatedleaf senescence with enhanced photosynthetic (Pn) rate, increasedlevels of chlorophyll (Chl) and total soluble protein (TSP),concurrently with reduced malondialdehyde (MDA) contents inthe fourth leaf on the main-stem. The graft of K2 scion ontoK1 rootstock enhanced leaf senescence with reduced Pn, Chl,and TSP, and increased MDA, compared with their respective self-graftedcontrol plants (K1/K1 and K2/K2). Reciprocally grafted plantsdiffered significantly from their self-grafted control plantsin levels of zeatin and its riboside (Z+ZR), isopentenyl andits adenine (iP+iPA), and ABA, but not in those of dihydrozeatinand its riboside (DHZ+DHZR) in leaves in late season, whichwas consistent with variations in leaf senescence between reciprocallyand self-grafted plants. The results suggest that leaf senescenceis closely associated with reduced accumulation of Z+ZR, andiP+iPA rather than DHZ+DHZR, or enhanced ABA in leaves of cotton.Genotypic variation in leaf senescence may result from the differencein root characteristics, particularly in Z+ZR, iP+iPA, and ABAwhich are regulated by the root system directly or indirectly. Key words: Abscisic acid, cotton, cytokinins, grafting, leaf senescence Received 23 October 2007; Revised 17 January 2008 Accepted 23 January 2008     

Stimulation by abscisic acid of RNA synthesis in discs from healthy and tobacco mosaic virus-infected tobacco leaves

Uptake of abscisic acid from the culture medium by discs of healthy and tobacco mosaic virus-infected tobacco leaves was measured. Small (two to five-fold) increases in abscisic acid concentration in discs caused increases in rates of [³H]uridine and [³H]adenine incorporation into total nucleic acid, virus RNA and host ribosomal RNA. Net accumulation of virus RNA was also enhanced by abscisic acid. This evidence for stimulation of RNA synthesis is compared with previous reports showing inhibition of RNA synthesis in other tissues. It is suggested that the increase in endogenous abscisic acid caused by tobacco mosaic virus infection may be at least partly responsible for observed increases in rates of RNA synthesis after infection.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

The effects of two abscisic acid analogues, WL19224 and WL19377, on stomatal closure

The effect on stomatal closure by ABA and its analogues, WL19224 and WL19377 was investigated. The rate of closure showed a sigmoid curve when various concentrations of ABA were applied. A concentration of 10^-9 M ABA was the threshold for stomatal closure; maximal closure occurred at higher concentrations (from 10^-6 M to 10^-3 M). Use of the analogue WL19224 resulted in similar closure responses. However, ABA was more effective at lower concentrations. For example, at 10^-3 M of either WL19224 and ABA, stomata closed to 2.2 μm and about 3 μm, respectively. In contrast, applications of the ABA analogue WL19377 had no effect on stomatal closure. In fact, at concentrations of WL19377 higher than 10^-4 M, stomata were stimulated to open, to about 10% of their initial size. Likewise, applications of WL19377 along with ABA, inhibited ABA-induced stomatal closure. This inhibition was linearly related to the concentrations of the compounds applied. In conclusion, the structural requirements for biological activity of ABA and its analogues cannot be considered individually, but must be assessed for their roles as part of an entire functional group. Although compounds may have similar structures, their ability to control certain physiological activities may be quite different.     

Binding of abscisic acid to human LANCL2

The phytohormone abscisic acid (ABA) is the central regulator of abiotic stress in plants and plays important roles during plant growth and development. In animal cells, ABA was shown to be an endogenous hormone, acting as a stress signal and stimulating cell functions involved in inflammatory responses and in insulin release. Recently, we demonstrated that Lanthionine synthetase component C-like protein 2 (LANCL2) is required for ABA binding to the plasmamembrane of granulocytes and for the activation of the signaling pathway triggered by ABA in human granulocytes and in rat insulinoma cells. In order to investigate whether ABA activates LANCL2 via direct interaction, we performed specific binding studies on human LANCL2 recombinant protein using different experimental approaches (saturation binding, scintillation proximity assays, dot blot experiments and affinity chromatography). Altogether, results indicate that human recombinant LANCL2 binds ABA directly and provide the first demonstration of ABA binding to a mammalian ABA receptor.     

Effect of low-temperature stress on abscisic acid,jasmonates, and polyamines in apples

The effect of low temperatures on polyamines, jasmonates, abscisic acid (ABA), and antioxidant activities was investigated in apple fruitlets. Although endogenous ABA concentrations were not significantly different between untreated control fruit kept at −2°C and those kept at 20°C, endogenous jasmonic acid (JA), putrescine, and spermidin concentrations at −2°C were generally higher than those at 20°C. Endogenous ABA concentrations increased in n-propyl dihydrojasmonate (PDJ)—or spermine-treated fruit in comparison to the untreated control at 20 and −2°C. The applications of PDJ or spermine decreased low-temperature injuries such as splitting and spotting in fruit. Although the IC₅₀ of 1,1-diphenil-2-pycrylhydrazyl (DPPH)-radical scavenging activities was not significantly different among the treatments, the IC₅₀ of O₂ ⁻-scavenging activities in PDJ-treated or Spm-treated fruit at 5 days after the low-temperature treatment was lower than in the untreated control at 20 and −2°C. The expression of MdCHS increased in Spm-treated fruit. The concentrations of ascorbic acid, catechin, chlorogenic acid, epi-catechin, and phloridzin in Spm-treated fruit were higher than in the untreated control at −2 or 20°C. These facts suggest that ABA, jasmonates and polyamines may be associated with low-temperature stress tolerance in apple fruitlets.     

Changes in abscisic acid and phenols during flower development in two diverse species of rose

Changes in endogenous abscisic acid (ABA) and phenols were determined in petals of two diverse species of rose, viz., Rosa damascena Mill and Rosa bourboniana Desport during flower development. A progressive increase in free ABA was observed during flower development till full bloom in both the species with higher content of free ABA in Rosa damascena. While bound ABA level increased in Rosa damascena till stage 6, in Rosa bourboniana it continued to increase till full bloom. Acidic phenols increased slowly in both the species till stage 4, but sharply afterwards and no significant differences were noticed during full bloom period. Bound phenols content was higher in Rosa damascena during full bloom period. The significance of these changes in relation to flowering in the two diverse species of rose is discussed.     

Stereoselectivity in the biosynthetic conversion of xanthoxin into abscisic acid

All stereoisomers of xanthoxin (XAN) and abscisic aldehyde (ABA-aldehyde) were prepared from (R) and (S)-4-hydroxy--cyclogeraniol via asymmetric epoxidation. Their stomatal closure activities were measured on epidermal strips of Commelina communis L. Natural (S)-ABA-aldehyde showed strong activity comparable to that of (S)-abscisic acid (ABA). Natural (1S, 2R, 4S)XAN and (1S, 2R, 4R)-epi-XAN also induced stomatal closure at high concentrations. On the other hand, unnatural (1R)-enantiomers of XAN, epi-XAN, and ABA-aldehyde were not effective. To further examine the Stereoselectivity on the biosynthetic pathway to ABA, deuterium-labeled substrates were prepared and fed to Lycopersicon esculentum Mill, under non-stressed or water-stressed conditions. Substantial incorporations into ABA were observed in the cases of natural (1S, 2R, 4S)-XAN, (1S, 2R, 4R)-epi-XAN and both enantiomers of ABA-aldehyde, leading to the following conclusions. The negligible effect of unnatural (1R)-enantiomers of XAN, epi-XAN and ABA-aldehyde can be explained by their own biological inactivity and/or their conversion to inactive (R)-ABA. Even in the isolated epidermal strips, putative aldehyde oxidase activity is apparently sufficient to convert ABA-aldehyde to ABA while the activity of XAN dehydrogenase seems very weak. The stereochemistry of the 1, 2-epoxide is very important for the XAN-dehydrogenase while this enzyme is less selective regarding the 4-hydrdxyl group of XAN and converts both (1S, 2R, 4S)-XAN and (1S, 2R, 4R)-epi-XAN to (S)-ABA-aldehyde. Abscisic aldehyde oxidase can nonstereoselectively convert both (S) and (R)-ABA-aldehyde to biologically active (S) and inactive (R)-ABA, respectively.Abbreviations ABA abscisic acid - ABA-aldehyde abscisic aldehyde - DET diethyl tartrate - epi-XAN xanthoxin epimer - FCC flash column chromatography - GC-EI-MS gas chromatography-electron impact-mass spectrometry - MeABA abscisic acid methyl ester - IR infrared - NMR nuclear magnetic resonance - PCC pyridinium chlorochromate - THF tetrahydrofuran - XAN xanthoxin The authors are very grateful to Mr J.K. Heald (Department of Biological Sciences, University of Wales, Aberystwyth, UK) and Dr. R. Horgan for carrying out GC-EI-MS analyses and advice, respectively.This work was supported by the Japan Society for the Promotion of Science (Fellowship for Young Japanese Researcher No. 0040672).     

Contrasting photo- and thermoperiod-induced changes in abscisic acid and lipid contents in leaves of mungbean seedlings

Seedlings of Phaseolus aureus ROXB were grown under 12/12 h light/dark cycles with the light period at 32.5°C and darkness at 10°C (normal conditions N) or with light at 10°C and darkness at 32.5°C (inverse conditions, I). I-conditions affected the level of chlorophyll and carotenoids (very low), monogalactosyldiacylgycerol (low) and phosphatidylinositol (high) in the leaves. Leaves of I-seedlings showed a sharp and durable decline of relative water content during the low temperature phase. For the N-seedlings, loss of water was restricted to the end of this period. The loss of water was accompanied by visible symptoms of wilting at specific times of day. Although the pigment content remained nearly unchanged, ABA content of leaves of both N-and I-seedlings increased during water stress. Upon return to the warm period, ABA level continued to increase after the leaves had regained turgor, this 'after stress'increase being more pronounced in the leaves of I-seedlings. Exogenous application of ABA induced a slight increase in the content of phospholipids in N- and I-leaves and a decrease in free fatty acids, whereas monogalactosyldiacylglycerol content was significantly reduced in N-leaves after application of ABA. Upon transfer of I-plants to 20°C for 12 h during the light period, pigment and chloroplastic lipid content increased rapidly whereas upon a further exposure to 10°C in light, pigments and especially monogalactosyldiacylglycerol were lost. The control of pigment and lipid metabolism and the role of ABA during chilling stress are discussed.     

Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit,leaves and phloem exudate

Abscisic acid (ABA) was identified by combined gas liquid chromatography-mass spectrometry in sieve-tube exudate collected from the cut stylar ends of white lupin fruit. Water stress caused an increase in ABA levels in leaf, seed and pod tissues and phloem exudate. When compared with levels in extracts of these tissues, the concentration of ABA in sieve-tube sap was very high. It is suggested that ABA is actively transported out of mature leaves in the phloem and this finding is discussed in terms of the ABA balance of the plant.Abbreviations ABA abscisic acid - GLC gas liquid chromatography