ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat

In plants, metabolism of the plant hormone (+)- S -abscisic acid (ABA) occurs by oxidation at the 8'-carbon on the ABA ring, producing (+)-8'-hydroxy-ABA (8'OH-ABA), which undergoes ring closure to phaseic acid (PA), or at the 7'-carbon producing 7'-hydroxy-ABA (7'OH-ABA). New methods for synthesixing and stabilizing 8'OH-ABA have enabled us for the first time to compare the biological activities of the ABA metabolites. 8'OH-ABA. PA and 7'OH-ABA. Activities were determined in wheat ( Triticum aestivum , cvs Brevor and Clarks Cream) using optically pure natural enantiomers of ABA. 8'OH-ABA, PA, and 7'OH-ABA. Comparison of wheat embryo germination inhibitory activity showed that only the metabolite 7'OH-ABA had significant activity (10 to 40% of effective ABA activity) as a germination inhibitor of wheat embryos. The metabolites had differential effects on ABA-responsive gene expression. Both 8'OH-ABA and PA were effective inducers of an ABA-responsive LEA (late embryogenesis abundant) gene, wheat group 3 LEA , in seedling roots, suggesting that ABA metabolites can maintain and prolong LEA gene expression. The metabolites had only a slight effect on accumulation of an ABA-responsive protein kinase ( PKABA1 ) mRNA or pMA1951 . These results are consistent with the idea that there are several control points in the ABA metabolic pathway and indicate that assessment of ABA signalling responses should include consideration of the biological activities of ABA metabolites.     

ABA调控种子发育的研究进展

种子发育是一个复杂的生物学过程，受各种遗传和外界因素的调节，显著影响农作物特别是禾谷类作物的种子活力和产量与质量。脱落酸(ABA)是调控种子发育和萌发最重要的植物激素之一，其活性水平、信号转导及其LAFL网络在种子发育包括胚胎发生和成熟过程的调控中起关键作用。该文主要综述了近年来ABA调控种子发育的研究取得的重要进展，包括ABA代谢和信号转导对种子发育的调控，ABA与种子成熟转录因子(AFL-B3、FUS3、ABI3、LEC2等)的作用，以及ABA在种子发育中的作用机制，并提出了需要进一步研究的科学问题，为深入理解种子发育的分子机制提供参考，从而提高种子的活力、产量和质量。     

GC-MS identification of abscisic acid and abscisic acid metabolites in seed of Vigna unguiculata

Abscisic acid, phaseic acid and 4′-dihydrophaseic acid were identified by GC-MS of derivatized (Me, MeTMSi) extracts from immature fruits of Vigna unguiculata. The fruits also contained some other ABA-related compounds, one of which might be epi-4′-dihydrophaseic acid while another was tentatively identified as 6′-hydroxymethylabscisic acid.     

Response of French bean cultivars to water deficits: Changes in endogenous hormones, proline and chlorophyll

Effects of water stress at different stages of plant growth on leaf relative water content (RWC), osmotic potential (Ψos) and changes in contents of chlorophyll, abscisic acid (ABA), zeatin riboside (t-ZR), ethylene and proline in six cultivars of French bean (Phaseolus vulgaris L.) were studied. Under water stress, Ψos and RWC were highest in cv. Contender and lowest in cvs. IIHR-909 and Sel-2. The increase in contents of ABA and proline was marked in cv. Contender followed by cv. UPF-626. Decrease in t-ZR and chlorophyll contents was prominent in cv. IIHR-909. Ethylene production surged in all the cultivars under 4- and 8-d stress and declined under 12-d stress. This revised version was published online in August 2006 with corrections to the Cover Date.     

Seed dormancy and ABA metabolism in Arabidopsis and barley: the role of ABA 8'-hydroxylase

We have investigated the relationship between seed dormancy and abscisic acid (ABA) metabolism in the monocot barley and the dicot Arabidopsis. Whether dormant (D) or non-dormant (ND), dry seed of Arabidopsis and embryos of dry barley grains all had similarly high levels of ABA. ABA levels decreased rapidly upon imbibition, although they fell further in ND than in D. Gene expression profiles were determined in Arabidopsis for key ABA biosynthetic [the 9-cis epoxycarotenoid dioxygenasegene family] and ABA catabolic [the ABA 8'-hydroxylase gene family (CYP707A)] genes. Of these, only the AtCYP707A2 gene was differentially expressed between D and ND seeds, being expressed to a much higher level in ND seeds. Similarly, a barley CYP707 homologue, (HvABA8'OH-1) was expressed to a much higher level in embryos from ND grains than from D grains. Consistent with this, in situ hybridization studies showed HvABA8'OH-1 mRNA expression was stronger in embryos from ND grains. Surprisingly, the signal was confined in the coleorhiza, suggesting that this tissue plays a key role in dormancy release. Constitutive expression of a CYP707A gene in transgenic Arabidopsis resulted in decreased ABA content in mature dry seeds and a much shorter after-ripening period to overcome dormancy. Conversely, mutating the CYP707A2 gene resulted in seeds that required longer after-ripening to break dormancy. Our results point to a pivotal role for the ABA 8'-hydroxylase gene in controlling dormancy and that the action of this enzyme may be confined to a particular organ as in the coleorhiza of cereals.     

Wounding of potato tubers induces increases in ABA biosynthesis and catabolism and alters expression of ABA metabolic genes

The effects of physical wounding on ABA biosynthesis and catabolism and expression of genes encoding key ABA metabolic enzymes were determined in potato tubers. An increase in ABA and ABA metabolite content was observed 48 h after wounding and remained elevated through 96 h. Wounding induced dramatic increases in the expression of the ABA metabolic genes encoding zeaxanthin epoxidase (ZEP), 9-cis-epoxycarotenoid dioxygenase (NCED), and ABA-8′-hydroxylase. Although the patterns of wound-induced expression of individual genes varied, increased gene expression was observed within 3 h of wounding and remained elevated through 96 h. An apparent correlation between expression of the gene encoding ZEP and the increase in ABA content suggested that the wound-induced increase in ABA biosynthesis was regulated by both substrate availability and increased NCED activity. Suppression of wound-induced jasmonic acid accumulation by rinsing the wounded tissue with water did not inhibit the subsequent increase in ABA content. Exogenous ethylene completely suppressed the wound-induced increase in ABA content and dramatically reduced wound-induced up-regulation of ABA metabolic genes. This study is the first to identify the molecular bases for increased ABA accumulation following physical trauma in potato tubers and highlights the complex physiological interactions between various wound-induced hormones.     

外源ABA对藜麦种子萌发及ABA相关基因表达的影响

穗发芽会导致藜麦籽粒产量和品质严重受损,而脱落酸(ABA)与种子萌发和休眠密切相关。该研究以易穗发芽的‘大黑藜3’(BBQ3)、‘白藜6’(WQ6)和抗穗发芽的‘小黑藜1’(SBQ1)和‘白藜4’(WQ4)为试验材料,在种子萌发3 d内施加不同浓度ABA和氟啶酮(FL),探究ABA和FL对两种粒色藜麦种子萌发、ABA含量以及ABA合成基因NCED、裂解基因8′OH和信号转导基因ABI3表达的影响。结果表明：(1)各藜麦种子萌发均受到各浓度外源ABA抑制,并以25μmol·L^-1浓度效果最佳,同时受到外源低浓度FL促进,且施用2.5μmol·L^-1浓度即可;外源ABA和FL处理的SBQ1和WQ4发芽率降低和升高幅度分别小于BBQ3和WQ6。(2)对照组SBQ1和WQ4种子内源ABA含量分别显著高于BBQ3和WQ6;外源ABA处理可显著提高藜麦种子内源ABA含量,且WQ4显著高于WQ6,种皮略厚的SBQ1内源ABA含量显著低于BBQ3;外源FL处理下4份材料种子内源ABA含量均有所下降,但只有SBQ1达到显著水平。(3)对照组中,SBQ1和WQ4...     

CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T-DNA insertional cyp707a3-1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3-1 mutant accumulated a higher amount of stress-induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA-inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.     

Effect of morphactin on certain plant growth substances in bean roots

The effect of morphactin (methyl-2-chloro-9-hydroxyfluorene-9-carboxylate) on the content of several plant growth substances in bean roots was determined. Beans (Phaseolus vulgaris L. cv. Spartan) were soaked in aqueous solutions of morphactin, 1 x 10^-4, 1 x 10^-5, and 1 x 10^-6M and grown in moist vermiculite. As controls were used beans grown in water-moistened vermiculite either intact or having the root tips removed (decapped). The roots, morphactin-treated, controls, and the decapped ones were analyzed for indol-3-yl acetic acid (IAA), indol-3-yl acrylic acid (IAcA), indol-3-yl pyruvic acid (IPyA), indol-3-yl lactic acid (1LA), abscisic acid (ABA), gibberellins GA₁, GA₃, GA₄, and GA₉ using gas-liquid chromatographic methods. Morphactin, while affecting the geotropical responses, changed also the growth substance content of roots. IAA, ABA, GA₁, and GA₉ contents decreased, IPyA, IAeA, GA₃, and GA₄ contents were not affected and ILA content increased slightly with increasing dosages of morphactin. Growth substance pattern of decapped roots resembled that of the roots treated with the highest dose, 1 x 10^-4M, of morphactin.     

Ethylene promotes submergence-induced expression of OsABA8ox1, a gene that encodes ABA 8'-hydroxylase in rice

A rapid decrease of the plant hormone ABA under submergence is thought to be a prerequisite for the enhanced elongation of submerged shoots of rice (Oryza sativa L.). Here, we report that the level of phaseic acid (PA), an oxidized form of ABA, increased with decreasing ABA level during submergence. The oxidation of ABA to PA is catalyzed by ABA 8'-hydroxylase, which is possibly encoded by three genes (OsABA8ox1, -2 and -3) in rice. The ABA 8'-hydroxylase activity was confirmed in microsomes from yeast expressing OsABA8ox1. OsABA8ox1-green fluorescent protein (GFP) fusion protein in onion cells was localized to the endoplasmic reticulum. The mRNA level of OsABA8ox1, but not the mRNA levels of other OsABA8ox genes, increased dramatically within 1 h after submergence. On the other hand, the mRNA levels of genes involved in ABA biosynthesis (OsZEP and OsNCEDs) decreased after 1-2 h of submergence. Treatment of aerobic seedlings with ethylene and its precursor, 1-aminocyclopropane-1-carboxylate (ACC), rapidly induced the expression of OsABA8ox1, but the ethylene treatment did not strongly affect the expression of ABA biosynthetic genes. Moreover, pre-treatment with 1-methylcyclopropene (1-MCP), a potent inhibitor of ethylene action, partially suppressed induction of OsABA8ox1 expression under submergence. The ABA level was found to be negatively correlated with OsABA8ox1 expression under ACC or 1-MCP treatment. Together, these results indicate that the rapid decrease in ABA levels in submerged rice shoots is controlled partly by ethylene-induced expression of OsABA8ox1 and partly by ethylene-independent suppression of genes involved in the biosynthesis of ABA.     

Abscinazole-E1, a novel chemical tool for exploring the role of ABA 8'-hydroxylase CYP707A

We developed abscinazole-E1 (Abz-E1), a specific inhibitor of abscisic acid (ABA) 8′-hydroxylase (CYP707A). This inhibitor was designed and synthesized as an enlarged analogue of uniconazole (UNI), a well-known plant growth retardant, which inhibits a gibberellin biosynthetic enzyme (ent-kaurene oxidase, CYP701A) as well as CYP707A. Our results showed that Abz-E1 functions as a potent inhibitor of CYP707A and a poor inhibitor of CYP701A both in vitro and in vivo. Abz-E1 application to plants resulted in improved desiccation tolerance and an increase in endogenous ABA.     

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.     

Effect of leaf water status and xylem pH on metabolism of xylem-transported abscisic acid

Metabolism and distribution of xylem-fed ABA were investigated in leaves of maize (Zea mays) and Commelina communis when water stress and xylem pH manipulation were applied. ³H-ABA was fed to excised leaves via the transpiration stream. Water stress was applied through either a previous soil-drying before leaves were excised, or a quick dehydration after leaves were fed with ABA. Xylem-delivered ABA was metabolised rapidly in the leaves (half-life 0.7 h and 1.02 h for maize and Commelina respectively), but a previous soil-drying or a post-feeding dehydration significantly extended the half-life of fed ABA in both species. In the first few hours after ABA was fed into the detached leaves, percentages of applied ABA remaining unmodified were always higher in leaves which received water stress treatments than in control leaves. However the percentage decreased to below the control levels several hours later in leaves which received a previous soil-drying treatment prior to excision, but had then been rehydrated by the xylem-feeding process itself. One possible explanation for this could be a changed pattern of compartmentalisation for xylem-carried ABA. A post-feeding dehydration treatment also changed the distribution of xylem-fed ABA within the leaves: more ABA was found in the epidermis of Commelina leaves which had been dehydrated rapidly after ABA had been fed, compared to the controls. The levels of xylem-delivered ABA remaining unmodified increased as the pH of the feeding solution increased from 5 to 8. The results support the hypothesis that water stress and a putative stress-induced xylem pH change may modify stomatal sensitivity to ABA by changing the actual ABA content of the leaf epidermis.     

ABA分解代谢及其代谢关键酶——8'-羟化酶

脱落酸(ABA)在植物的生长发育和环境胁迫响应等过程中具有重要作用。ABA合成与分解代谢的动态平衡共同调控植物内源ABA水平。ABA8′位甲基羟基化途径是高等植物内源ABA代谢的主要途径;8′-羟化酶是该代谢途径的关键酶,属于P450酶系。生物化学和基因组学研究表明,拟南芥CYP707A家族基因编码8′-羟化酶,该基因家族广泛存在于高等植物中,调控植物内源ABA代谢,介导ABA相关的生理生化过程。本文综述了ABA分解代谢的基本途径,详细概述了ABA8′位甲基羟基化途径及该代谢途径的关键酶8′-羟化酶。同时介绍了8′-羟化酶编码基因-CYP707A家族基因的生物学特征和功能。     

Effects of ABA and CaCl2 on GABA accumulation in fava bean germinating under hypoxia-NaCl stress

Effects of exogenous abscisic acid (ABA) and CaCl₂ on γ-aminobutyric acid (GABA) accumulation of germinated fava bean under hypoxia-NaCl stress were investigated. Exogenous ABA resulted in the enhancement of glutamate decarboxylase (GAD) and diamine oxidase (DAO) activity as well as GABA content in cotyledon and shoot. CaCl₂ increased both enzyme activities in shoot and GABA content in cotyledon and shoot. ABA downregulated GAD expression in cotyledon and radicle, while upregulated that in shoot; it also upregulated DAO expression in each organ. CaCl₂ upregulated GAD expression in cotyledon, while downregulated that in radicle. However, it upregulated DAO expression in shoot, downregulated that in radicle. ABA inhibitor fluridon and ethylenediaminetetraacetic acid inhibited GAD and DAO activities significantly so that inhibited GABA accumulation through reducing ABA biosynthesis and chelating Ca²⁺, respectively. However, they upregulated GAD and DAO expression in varying degrees. These results indicate that ABA and Ca²⁺ participate in GABA biosynthesis in fava bean during germination under hypoxia-NaCl stress.     

Accumulation and leakage of abscisic acid during embryo development and seed dormancy in wheat

Seed dormancy develops latein embryogenesis after a period of potential prematuregermination and has been associated with levels ofabscisic acid (ABA) in, and sensitivity to, ABA ofembryos. In wheat (Triticum aestivum L.)embryos, there are two peaks in levels of ABA duringdevelopment: the first occurs 25 days afterpollination (DAP) and the second from 35 to 40 DAP. The first peak of ABA appears to be associated withthe development of the embryo's sensitivity to ABAsince such sensitivity was altered in seeds on earsthat were incubated in a solution of ABA from 15 and20 DAP. In the embryos of Kitakei wheat, a line thatexhibits dormancy, the second peak, at around 35 DAP,was more prolonged in comparison to Chihoku, anon-dormant line. The results support the proposedinvolvement of ABA in the formation and maintenance ofseed dormancy during middle and late embryogenesis. When developing embryos were incubated in water,embryonic ABA leaked out from the embryos, inparticular between 30 and 40 DAP. Prematuregermination observed between 30 and 40 DAP might berelated to such leakage of ABA from embryos.     

The relations of stomatal closure and reopening to xylem ABA concentration and leaf water potential during soil drying and rewatering

Two tropical tree species, Acacia confusa and Leucaena leucocephala, were used to study the relationships among stomatal conductance, xylem ABA concentration and leaf water potential during a soil drying and rewatering cycle. Stomatal conductance of both A. confusa and L. leucocephala steadily decreased with the decreases in soil water content and pre-dawn leaf water potential. Upon rewatering, soil water content and pre-dawn leaf water potential rapidly returned to the control levels, whereas the reopening of stomata showed an obvious lag time. The length of this lag time was highly dependent not only upon the degree of water stress but also on plant species. The more severe the water stress, the longer the lag time. When A. confusa and L. leucocephala plants were exposed to the same degree of water stress (around –2.0 MPa in pre-dawn leaf water potential), the stomata of A. confusa reopened to the control level 6 days after rewatering. However, it took L. leucocephala about 14 days to reopen fully. A very similar response of leaf photosynthesis to soil water deficit was also observed for both species. Soil drying resulted in a significant increase in leaf and xylem ABA concentrations in both species. The more severe the water stress, the higher the leaf and xylem ABA concentrations. Both leaf ABA and xylem ABA returned to the control level following relief from water deficit and preceded the full recovery of stomata, suggesting that the lag phase of stomatal reopening was not controlled by leaf and/or xylem ABA. In contrast to drying the whole root system, drying half of the root system did not change the leaf water relations, but caused a significant increase in xylem ABA concentration, which could fully explain the decrease of stomatal conductance. After rewatering, the stomatal conductance of plants in which half of the roots were dried recovered more rapidly than those of whole-root dried plants, indicating that the leaf water deficit that occurred during the drying period was related to the post-stress stomatal inhibition. These results indicated that the decrease in stomatal conductance caused by water deficit was closely related to the increase in xylem ABA, but xylem ABA could not fully explain the reopening of stomata after relief of water stress, neither did the leaf ABA. Some unknown physiological and/or morphological processes in the guard cells may be related to the recovery process.     

The use of abscisic acid analogues to analyse the substrate selectivity of UGT71B6, a UDP-glycosyltransferase of Arabidopsis thaliana

This study analyses the activity of an Arabidopsis thaliana UDP-glycosyltransferase, UGT71B6 (71B6), towards abscisic acid (ABA) and its structural analogues. The enzyme preferentially glucosylated ABA and not its catabolites. The requirement for a specific chiral configuration of (+)-ABA was demonstrated through the use of analogues with the chiral centre changed or removed. The enzyme was able to accommodate extra bulk around the double bond of the ABA ring but not alterations to the 8'- and 9'-methyl groups. Interestingly, the ketone of ABA was not required for glucosylation. Bioactive analogues, resistant to 8'-hydroxylation, were also poor substrates for conjugation by UGT71B6. This suggests the compounds may be resistant to both pathways of ABA inactivation and may, therefore, prove to be useful agrochemicals for field applications.