Metabolism of (±) 2-[14C]-abscisic acid in Lactuca sativa achenes

Achenes ofLactuca sativa L. cv. Grand Rapids were treated with (±) 2-[¹⁴C]-abscisic acid (ABA) at 10⁵ - or 2-10⁶ M for 6, 12, 24, 48 or 96 h in darkness at 24°C. They were then extracted in 80% ethanol. Two acidic diethyl ether phases which contained the free acids and the acids released after mild alkaline hydrolysis respectively, were analyzed as well as the radioactivity which remained in the final aqueous phase. For treatment durations between 6 and 96 h, the major part of the radioactivity was found in the free phase, in the form of ABA. For treatment durations up to 48 h, no radioactivity was detected at the Rf of phaseic acid or dihydrophaseic acid (free and hydrolysed phases). After 96 h culture on 10⁵ M ABA, dihydrophaseic acid was present, but only in very small quantities. Two ABA metabolites were detected. One was characterized as β-d -glucopyranosyl abscisate since its Rf was the same as that of an authentic sample in three different solvent systems and also since it released ABA on mild alkaline hydrolysis. It increased steadily with time and represented the main metabolite. The other metabolite found in the aqueous phase after mild alkaline hydrolysis and extraction with ether at pH 3 was a very polar compound, resistant to alkaline hydrolysis in the presence of concentrated ammonia and to methylation. It was, however, metabolized by apple embryo, yielding essentially dihydrophaseic acid and an ester which released dihydrophaseic acid on mild alkaline hydrolysis. These results indicate that under the conditions tried, the metabolism of [¹⁴C]-ABA by lettuce achenes leads almost exclusively to the formation of conjugates, oxidative metabolism of ABA being almost non-existent. Separate analysis of the integuments and of the endosperm plus embryo after culture of whole achenes for 48 h in the presence of 10⁵ M [¹⁴C]-ABA showed that ABA metabolism occurred only in the endospermembryo tissue.     

蔗糖调控培养对胡萝卜体细胞胚内源ABA水平的效应

利用酶联免疫吸附测定（ＥＬＩＳＡ）技术,分析了经不同浓度蔗糖调控的胡萝卜（ＤａｕｃａｓｃａｒｏｔａＬ．）体细胞胚及胚性器官的内源ＡＢＡ水平。结果表明,随着胚的生长发育,内源ＡＢＡ含量呈上升趋势,到子叶胚时,达到最高值。在胚的早期发育阶段,不同处理之间,ＡＢＡ水平只有较小的差异,而当子叶胚开始膨大时,这种差异则较明显。一旦将调控胚解调控,体细胞胚内源ＡＢＡ含量则明显下降。在两个月内,调控胚及胚性器官的ＡＢＡ含量基本维持不变。表明蔗糖浓度可导致体胚内源ＡＢＡ水平的变化,但这种影响依发育时期而异;高浓度的ＡＢＡ水平有利于胚性状态的维持。与此同时,比较研究了外源ＡＢＡ处理效应与高浓度蔗糖处理的差异,结果表明,蔗糖的调控效应与ＡＢＡ的调控相似又不尽相同。总之,在蔗糖调控胡萝卜体细胞胚发育的信号传递网络中ＡＢＡ可能是一个重要的中介信号因子。     

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.     

苹果果实组织脱落酸结合位点的亚细胞定位

在苹果（ＭａｌｕｓｐｕｍｉｌａＭｉｌ）果实细胞的可溶性组分中存在ＡＢＡ特异结合位点,这些位点能与ＡＢＡ形成稳定的３ＨＡＢＡ蛋白结合复合物,而微粒体部分未表现出３ＨＡＢＡ结合活性。可溶性组分对３ＨＡＢＡ的结合效率与结合稳定性需要活体组织完整细胞的存在。ＡＢＡ共价交联物ＡＢＡＬＹＣＨ,具有与ＡＢＡ相同的抑制红苋菜（ＡｍａｒａｎｔｈｕｓｔｒｉｃｏｌｏｒＬ．）种子萌发的生物活性,能有效地竞争抑制果实组织圆片对３ＨＡＢＡ的结合。通过ＡＢＡＬＹＣＨ对苹果果实组织圆片及果实组织游离细胞的荧光染色表明,未成熟果实的细胞外周被特异性地染色。在ＢＳＡ存在的情况下,ＡＢＡＬＹＣＨ复合物被转运进细胞,形成密集的强烈荧光团。结果表明,ＡＢＡＬＹＣＨ与３ＨＡＢＡ的竞争性结合发生在细胞质膜水平。     

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.     

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.     

An Immunogold Microscopic Study on the Effects of Water Stress on ABA Localization and Contents in Roots of Vicia faba

ABA localization in roots of Vicia faba L. was studied using immunogold microscopy. In cells of promeristem gold particles were mainly localized in the nuclei. In cells of ground meristem and cortex of the front part of elongation zone, some gold particles were found in cytoplasm near. the plasmalemma. Substantial amounts of gold particles were observed in cells of vascular cylinder especially in apoplast of vascular tissue. Cells of middle elongation zone and root hair zone were also labelled by many gold particles. In cells of the primary meristem and the front part of elongation zone, water stress could lead to acute increase of the gold particle density, and also in the cells of the elongation and root hair zone. The distribution of ABA in subcellular level and its relationship with transportation were discussed in the text. and the results provided evidence for ABA as a root-to-shoot transporting stress signal.     

蚕豆保卫细胞原生质体质膜ABA结合蛋白与气孔对ABA敏感性的关系

在测定蚕豆(Vicia faba L.)保卫细胞原生质体质膜ABA结合蛋白的解离常数(Kd)和最大结合容量的基础上,进一步研究了几种因子(pH、光)对Kd和最大结合容量的影响。结果显示:pH并不改变结合蛋白的Kd值,而仅影响每个原生质体结合的分子数;光、暗处理的结果表明,光可使结合蛋白的Kd值增大,每个原生质体结合的ABA分子数减少,而暗处理可使结合蛋白的Kd值减小及每个原生质体结合的分子数增多,说明黑暗可提高气孔保卫细胞对ABA的敏感性,而光可以降低气孔保卫细胞对ABA的敏感性。因此,光、暗可以通过调节ABA结合蛋白的Kd值来调节气孔对ABA的敏感性。     

用胶体金免疫电镜技术研究水分胁迫对蚕豆根中ABA分布与含量的影响

用胶体金免疫电镜技术对蚕豆 ( Vicia faba L.)根中 ABA的定位结果表明 ,在原分生组织金颗粒主要分布在细胞核。在基本分生组织或伸长区前部皮层细胞的质膜附近有较多的金颗粒标记 ,维管柱特别是维管组织的质外体中发现有大量金颗粒标记。伸长区中部及根毛区细胞也有较多的金颗粒标记。水分胁迫可导致初生分生组织或伸长区前部细胞金颗粒标记密度大增 ,伸长区中部及根毛区细胞的金颗粒密度也明显增加。对根中 ABA在超微结构水平上的分布及其与运输的关系进行了讨论 ,为 ABA有可能作为由根向地上部分传递的逆境信息提供了进一步的证据。     

赤霉素与脱落酸对番茄种子萌发中细胞周期的调控

利用细胞流检仪检测番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．） ＧＡ－缺陷型、ＡＢＡ－缺陷型和相应的正常品种（野生型）成熟种子胚根尖细胞倍性水平时发现：ＧＡ－缺陷型和野生型种子绝大多数细胞ＤＮＡ 水平为２Ｃ,而ＡＢＡ－缺陷型种子则含有较多的４Ｃ细胞。在标准发芽条件下,ＡＢＡ－缺陷型和野生型种子浸种１ ｄ 后胚根尖细胞ＤＮＡ 开始复制,随后胚根突破种皮而发芽。然而ＧＡ－缺陷型种子除非加入外源ＧＡ,否则既不发生细胞ＤＮＡ 复制,也不发芽。这说明内源ＧＡ 是启动番茄种子胚根尖细胞ＤＮＡ 复制的关键因素,同时也说明番茄根尖细胞ＤＮＡ 复制是种子发芽的必要条件。实验证明：ＡＢＡ 不抑制细胞ＤＮＡ 合成,但阻止Ｇ２ 细胞进入到Ｍ 期。外源ＡＢＡ处理野生型种子与渗控处理结果相似,可以大幅度提高胚根尖４Ｃ／２Ｃ细胞的比例,但抑制种子的最终发芽     

PH as a stress signal

The pH of the xylem sap of plants experiencing a range of environmental conditions can increase by over a whole pH unit. This results in an increased ABA concentration in the apoplast adjacent to the stomatal guard cells in the leaf epidermis, by reducing the ability of the mesophyll and epidermal symplast to sequester ABA away from this compartment. As a result the guard cell ABA receptors become activated and the stomata close, enabling the plant to retain water. Were it not for the low concentration of ABA ubiquitous to all land plants, the increase in the pH of the apoplast adjacent to the guard cell would induce stomatal widening, and cause excessive water loss. Not only does ABA prevent this potentially harmful phenomenon, but it also converts the pH increase to a signal which can bring about plant protection.     

GA and ABA Regulation on the Cell Cycle in Germination of Tomato Seeds

Flow cytometric determination of ploidy levels in embryos of GA-deficient, ABA-deficient mutant and isogenic wild type tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds revealed that, large amount of 2C DNA signals existed both in wild type and GA-deficient mutant seeds, showing that most cells had arrested in the cell cycle at presynthesis Gl, whereas a relative amount of 4C proportion which is a sign of seed germination was found in ABA-deficient mutant seeds, indicating that endogenous ABA play a role in regulating the switch from development to germination in seeds. DNA replication was stimulated 1 d after the seed was imbibed in water and a visible germination occurred subsequently either in wild type GA-deficient mutant seeds. But it was not the case for ABA-deficient mutant seeds unless an exogenous GA was supplemented. This demonstrated that DNA replication in embryo root tips cells was subjected to be a compulsory factor for seed germination, whereas endogenous GA triggered DNA synthesis. It was evident that exogenous ABA could inhibit seed germination not by suppressing DNA synthesis but by bloking the route leading to mitosis since a great amount of 4C proportion was found in the germinating wild type and GA-deficient mutant seeds in the ABA solution when visible ger mination did not occur. Finally a simple mode of hormonal regulation on cell cycle in high plants was hypothesized.     

Abscisic acid perception and signaling transduction in strawberry: A model for non-climacteric fruit ripening

On basis of fruit differential respiration and ethylene effects, climacteric and non-climacteric fruits have been classically defined. Over the past decades, the molecular mechanisms of climacteric fruit ripening were abundantly described and found to focus on ethylene perception and signaling transduction. In contrast, until our most recent breakthroughs, much progress has been made toward understanding the signaling perception and transduction mechanisms for abscisic acid (ABA) in strawberry, a model for non-climacteric fruit ripening. Our reports not only have provided several lines of strong evidences for ABA-regulated ripening of strawberry fruit, but also have demonstrated that homology proteins of Arabidopsis ABA receptors, including PYR/PYL/RCAR and ABAR/CHLH, act as positive regulators of ripening in response to ABA. These receptors also trigger a set of ABA downstream signaling components, and determine significant changes in the expression levels of both sugar and pigment metabolism-related genes that are closely associated with ripening. Soluble sugars, especially sucrose, may act as a signal molecular to trigger ABA accumulation through an enzymatic action of 9-cis-epoxycarotenoid dioxygenase 1 (FaNCED1). This mini-review offers an overview of these processes and also outlines the possible, molecular mechanisms for ABA in the regulation of strawberry fruit ripening through the ABA receptors.     

Abscisic acid relationships in the chill-related dormancy mechanism in apple seeds

Abscisic acid (ABA) levels in seeds from three cultivars of apple (Malus domestica Borkh.) which have substantially different chilling requirements were investigated by gas chromatography mass-spectrometry selected ion monitoring (GCMS-SIM) during stratification. The ABA content of dormant unchilled seeds was similar in the three cultivars, suggesting no relationship between the chilling requirement of those seeds and their ABA status. That chilling is not related to ABA changes during stratification was confirmed by warm (20°C) and cold (5°C) stratification experiments. ABA content dropped rapidly and nearly identically under both temperature regimes, but only cold stratification promoted germination. The decline in ABA during stratification was due in large part to leaching from the seed coat and nucellar membrane; the ABA content of the embryo remained nearly constant. The radicle in intact seeds stratified at 5°C began growing 20–30 days after the ABA in the seed coat and nucellar membrane had nearly disappeared. Radicle growth did not occur in unchilled seeds, even though ABA had leached from them as well. It is possible that the leaching of ABA from the seed allows certain promotive forces to develop, but if so, these can develop only at chilling temperatures. Studies were also conducted on 2-trans ABA relationships to apple seed dormancy, but no association was evident.Report No. 12, Department of Fruit and Vegetable Science, Cornell University.     

Light and abscisic acid signalling are integrated by MIZ1 gene expression and regulate hydrotropic response in roots of Arabidopsis thaliana

Plant roots undergo tropic growth in response to environmental cues, and each tropic response is affected by several environmental stimuli. Even its importance, molecular regulation of hydrotropism has not been largely uncovered. Tropic responses including hydrotropism were impacted by other environmental signal. We found that hydrotropism was reduced in dark-grown seedling. Moreover, we found that the expression of MIZ1, an essential gene for hydrotropism, was regulated by light signal. From our genetic analysis, phytochrome A (phyA)-, phyB- and HY5-mediated blue-light signalling play curial roles in light-mediated induction of MIZ1 and hydrotropism. In addition, we found that abscisic acid (ABA) also induced MIZ1 expression. ABA treatment could recover weak hydrotropism and MIZ1 expression level of hy5, and ABA synthesis inhibitor, abamineSG, further reduced hydrotropic curvature of hy5. In contrast, ABA treatment did not affect ahydrotropic phenotype of miz1. These results suggest that ABA signalling regulates MIZ1 expression independently from light signalling. Our results demonstrate that environmental signals, such as light and stresses mediated by ABA signalling, are integrated into MIZ1 expression and thus regulate hydrotropism. These machineries will allow plants to acquire sufficient amounts of water.     

Somatic embryogenesis induced by the simple application of abscisic acid to carrot (Daucus carota L.) seedlings in culture

Seedlings of carrot (Daucus carota L. cv. Red Cored Chantenay) formed somatic embryos when cultured on medium containing abscisic acid (ABA) as the sole source of growth regulator. The number of embryos per number of seedlings changed depending on the concentration of ABA added to the medium, with a maximum embryo number at 1 × 10⁻⁴M ABA. Seedling age was critical for response to exogenous ABA; no seedling with a hypocotyl longer than 3.0 cm was able to form an embryo. Removal of shoot apices from seedlings completely inhibited the embryogenesis induced by application of exogenous ABA, suggesting that the action of ABA requires some substance(s) that is translocated basipetally from shoot apices through hypocotyls. Histologically, somatic embryos shared common epidermal cells and differentiated not through the formation of embryogenic cell clumps, but directly from epidermal cells. These morphological traits are distinct from those of embryogenesis via formation of embryogenic cell clumps, which has been found in embryogenic carrot cultures established using 2,4-dichlorophenoxyacetic acid or other auxins. These results suggest that ABA acts as a signal substance in stress-induced carrot seedling somatic embryogenesis. Received: 22 April 2000 / Accepted: 8 June 2000     

A Preliminary Study on Neutral and Acid Inhibitors in the Seed of Kalopanax scptemlobus

The method of extraction, isolation, purification and identification of the neutral and acid inhibitors in the seeds of Kalopanax Septemlobus was given. It is proved that the neutral inhibitor is coumarin, the acid inhibitor is abscisic acid (ABA) by means of paper chromatograph, thin layer chromatograph, high performance liquid chromatograph (HPLC), color reaction and bioassay. The neutral inhibitor can strongly inhibite not only the seed germination of Brassica Chinensis and the radical elongation, but also that of the seed of Kalopanax septemlobus. The study showed that ABA and coumarin exist in the seed coat, endosperm and pericarp of the Kalopanax Septemlobus seed. Both ABA and coumarin can transport from seed cover (pericarp, seed coat)to the interior (endosperm, embryo) as the seeds were stored in refrigerator. In addition, the different results of extracting neutral inhibitor with water, methanol, and alcohol were compared in this paper.