Purification and characterization of a barley aleurone abscisic acid-binding protein

A protein designated ABAP1 and encoded by a novel gene (GenBank accession number AF127388) was purified and shown to specifically bind abscisic acid (ABA). ABAP1 protein is a 472-amino acid polypeptide containing a WW protein interaction domain and is induced by ABA in barley aleurone layers. Polyclonal antiidiotypic antibodies (AB2) cross-reacted with purified ABAP1 and with a corresponding 52-kDa protein associated with membrane fractions of ABA-treated barley aleurones. ABAP1 genes were detected in diverse monocot and dicot species, including wheat, tobacco, alfalfa, garden pea, and oilseed rape. The recombinant ABAP1 protein optimally bound (3)H-(+)-ABA at neutral pH. Denatured ABAP1 protein did not bind (3)H-(+)-ABA, nor did bovine serum albumin. The maximum specific binding as shown by Scatchard plot analysis was 0.8 mol of ABA mol(-1) protein with a linear function of r(2) = 0.94, an indication of one ABA-binding site with a dissociation constant (K(d)) of 28 x 10(-9) m. ABA binding in aleurone plasma membranes showed a maximum binding capacity of 330 nmol of ABA g(-1) protein with a K(d) of 26.5 x 10(-9) m. The similarities in the dissociation constants for ABA binding of the recombinant protein and that of the plasma membranes suggest that the protein within the plasma membrane fraction is the native form of ABAP1. The stereospecificity of ABAP1 was established by the incapability of ABA analogs and metabolites, including (-)-ABA, trans-ABA, phaseic acid, dihydrophaseic acid, and (+)-abscisic acid-glucose ester, to displace (3)H-(+)-ABA bound to ABAP1. However, two ABA precursors, (+)-ABA aldehyde and (+)-ABA alcohol, were able to displace (3)H-(+)-ABA, an indication that the structural requirement of ABAP1 at the C-1 position is not strict. Our data show that ABAP1 exerts high binding affinity for ABA. The interaction is reversible, follows saturation kinetics, and has stereospecificity, thus meeting the criteria for an ABA-binding protein.     

Biotin-labeled abscisic acid as a probe for investigating abscisic acid binding sites on plasma membranes of barley aleurone protoplasts

Plant hormone abscisic acid (ABA) plays important roles in dormancy and stress responses, but its binding sites have not yet been fully elucidated. In this report, we suggest the utility of biotin-labeled abscisic acid (bioABA) as a probe to investigate ABA-binding sites on the plasma membrane of barley aleurone protoplasts. BioABA was approximately 100 times less effective than ABA in inhibiting expression of gibberellin-inducible alpha-amylase and in inducing expression of a reporter gene fused to the dehydrin promoter. To ascertain that bioABA could bind to ABA-binding sites on the plasma membrane, we used fluorescence flow cytometry to measure the fluorescence intensity of aleurone protoplasts treated with a combination of bioABA and fluorescence-labeled streptavidin. Addition of bioABA increased the fluorescence of aleurone protoplasts in a concentration-dependent manner, but addition of non-active bioABA derivatives did not. Furthermore, the increase in fluorescence intensity observed upon addition of bioABA was eliminated by co-treatment with excess ABA, but it was not eliminated by co-treatment with other plant hormones. These results suggest that bioABA binds to ABA-binding sites, and that bioABA should be a valuable probe for investigating ABA-binding sites on the plasma membrane.     

Purification and identification of a 42-kilodalton abscisic acid-specific-binding protein from epidermis of broad bean leaves

Purification of abscisic acid (ABA)-binding proteins is considered to constitute a major step toward isolating ABA receptors. We report here that an ABA-binding protein was for the first time, to our knowledge, purified from the epidermis of broad bean (Vicia faba) leaves via affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing electrophoresis, and isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis two-dimensional electrophoresis of the purified ABA-binding protein all identified a single protein band with a molecular mass of 42 kD and an isoelectric point 4.86. The Scatchard plot for the purified protein showed a linear function with a maximum binding activity of 0.87 mol mol(-1) protein and an equilibrium dissociation constant of 21 nM, indicating that the purified protein may be a monomeric one, possessing one binding site. The ABA-binding protein was enriched more than 300-fold with a yield of 14%. (-)ABA and trans-ABA were substantially incapable of displacing (3)H-(+/-)ABA bound to the ABA-binding protein, and (+/-)ABA was less effective than (+)ABA in the competition. These findings allow establishment of the stereospecificity of the 42-kD protein and suggest its ABA receptor nature. Pretreatment of the guard cell protoplasts of broad bean leaves with the monoclonal antibody raised against the 42-kD protein significantly decreased the ABA specific-induced phospholipase D activity in a dose-dependent manner. This physiological significance provides more clear evidence for the potential ABA-receptor nature of the 42-kD protein.     

Changes in Kinetic Characteristics of the ABA Binding Protein(s) of the Grapevine Fruit During Its Developmental Process

By using the micro-volume radio-ligand binding essay (MRLB), the changes in the kinetic characteristics of the ABA binding protein (s) of the Kyoho grapevine (V/t/s vinifera × V. labrusca) fruits during the different stages of fruit development have been studied. The changes in the berry volume growth, concentration of sugar, organic acids and ABA in fruit mesocarp have been determined especially for the studies of ABA-binding protein. The dissociation constant (Kd) and ABA-binding maximum volumes (Bronx)were determined by the Scatchard plots for the ABA-binding protein (s) in microsomes of the fruits. They were: Kd =17.5, 50. 0, 6.3, 13.3 nmol/L: Bmax=98.6, 523.0, 41.6, 85. 4 pmol/g protein respectively for the fruit developmental phase Ⅰ , Ⅱ, veraison and phase Ⅲ. The Scatchard plots showed a rectilinear function for all of the developmental phases including veraison, which suggests in the fruit microsomes at the same developmental phase, the existence of either one kind of the same or several different kinds of ABA-binding sites of identical or similar kinetic characteristics and of high affinity. In different developmental stages, however, changes of the protein at the ABA-binding sites might occur. The binding affinity of ABA-binding protein (s) for ABA was showed to be higher at veraison stage than in other developmental phases and this binding affinity increased nearly by 10 times from the phase Ⅱ to veraison, whereas the concentration (Bmax) of the ABA-binding protein (s) decreased to the minimum at veraison. The reason why such low concentration of ABA at veraison is capable to trigger the onset of fruit ripening might be due to the increase of the affinity of ABA-binding protein (s) for ABA at this time. The possible functions of the ABA-binding protein (s) for fruit development during the different developmental stages were discussed, and it is suggested that the protein (s) detected could be the putative receptor (s) or carrier (s) for the action of this plant hormone in grapevine.     

Abscisic acid-specific binding sites in the flesh of developing apple fruit

Abscisic acid (ABA) specific-binding sites localized in the cytosol were identified and characterized in the flesh of developing apple (Malus pumila L. cv. Starkrimon) fruit. ABA binding activity was scarcely detectable in the microsomes but high ABA binding activity in the cytosolic fraction was detected. The ABA-binding sites possessed a protein nature with both active serine residues and thiol-groups of cysteine residues in their functional binding sites. ABA binding was shown to be saturable, reversible and of high affinity. A Scatchard plot provided evidence for two different ABA binding proteins, one with higher affinity (K(d)=2.3 nM) and the other with lower affinity (K(d)=58.8 nM). Phaseic acid, trans-ABA and (-)-ABA had essentially no affinity for the binding proteins, indicating their stereo-specificity to bind physiologically active cis-(+)-ABA. The time-course, pH- and temperature-dependence of the ABA-binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.     

Active oxygen and cell death in cereal aleurone cells

The cereal aleurone layer is a secretory tissue whose function is regulated by gibberellic acid (GA) and abscisic acid (ABA). Aleurone cells lack functional chloroplasts, thus excluding photosynthesis as a source of active oxygen species (AOS) in cell death. Incubation of barley aleurone layers or protoplasts in GA initiated the cell death programme, but incubation in ABA delays programmed cell death (PCD). Light, especially blue and UV-A light, and H(2)O(2) accelerate PCD of GA-treated aleurone cells, but ABA-treated aleurone cells are refractory to light and H(2)O(2) and are not killed. It was shown that light elevated intracellular H(2)O(2), and that the rise in H(2)O(2) was greater in GA-treated cells compared to cells in ABA. Experiments with antioxidants show that PCD in aleurone is probably regulated by AOS. The sensitivity of GA-treated aleurone to light and H(2)O(2) is a result of lowered amounts of enzymes that metabolize AOS. mRNAs encoding catalase, ascorbate peroxidase and superoxide dismutase are all reduced during 6-18 h of incubation in GA, but these mRNAs were present in higher amounts in cells incubated in ABA. The amounts of protein and enzyme activities encoded by these mRNAs were also dramatically reduced in GA-treated cells. Aleurone cells store and metabolize neutral lipids via the glyoxylate cycle in response to GA, and glyoxysomes are one potential source of AOS in the GA-treated cells. Mitochondria are another potential source of AOS in GA-treated cells. AOS generated by these organelles bring about membrane rupture and cell death.     

Modulation of Calmodulin mRNA and Protein Levels in Barley Aleurone

Changes in calmodulin (CaM) mRNA and protein were investigated in aleurone layers of barley (Hordeum vulgare L. cv Himalaya) incubated in the presence and absence of calcium, gibberellic acid (GA3), and abscisic acid (ABA). CaM mRNA levels increased rapidly and transiently following incubation of aleurone layers in H2O, CaCl2, or GA3. The increase in CaM mRNA was prevented by ABA. This increase in CaM mRNA was brought about by physical stimulation during removal of the starchy endosperm from the aleurone layer. CaM protein levels did not increase in response to physical stimulation. Only incubation in GA3 plus CaCl2 brought about a rapid increase in CaM protein levels in the aleurone cell. ABA reduced the level of CaM protein below that found at the beginning of the incubation period. The rise in CaM protein preceded increases in the synthesis and secretion of [alpha]-amylase. Immunocytochemistry with monoclonal antibodies to carrot and mung bean CaM was used to localize CaM in aleurone protoplasts. Monoclonal antibodies to tubulin and polyclonal antibodies to tonoplast intrinsic protein and malate synthase were used as controls. CaM was localized to the nucleus, the vacuolar membrane, and the cytosol, but was not associated with microtubules.     

Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone

Gibberellins (GAs) initiate a series of events that culminate in programmed cell death, whereas abscisic acid (ABA) prevents this process. Reactive oxygen species (ROS) are key elements in aleurone programmed cell death. Incubation of barley (Hordeum vulgare) aleurone layers in H2O2 causes rapid death of all cells in GA- but not ABA-treated layers. Sensitivity to H2O2 in GA-treated aleurone cells results from a decreased ability to metabolize ROS. The amounts and activities of ROS scavenging enzymes, including catalase (CAT), ascorbate peroxidase, and superoxide dismutase are strongly down-regulated in aleurone layers treated with GA. CAT activity, protein, and Cat2 mRNA decline rapidly following exposure of aleurone layers to GA. In ABA-treated layers, on the other hand, the amount and activity of CAT and Cat2 mRNA increases. Incubation in ABA maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA brings about a rapid reduction in the amounts of these enzymes. These data imply that GA-treated cells loose their ability to scavenge ROS and that this loss ultimately results in oxidative damage and cell death. ABA-treated cells, on the other hand, maintain their ability to scavenge ROS and remain viable.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H_2O_2的产生(简报)

逆境下,植物细胞内ABA含量急剧增加,同时植物也可通过一些酶代谢反应积累活性氧,如H_2O_2,O_2~-。ABA作为逆境信号对气孔运动的显著调节作用已被诸多实验所证实,但关于其对气孔运动调节的细节还知之甚少。H_2O_2作为氧化信号分子在植物抗病信号转导中已得到广泛研究,但H_2O_2是否介导保卫细胞的气孔运动还缺乏直接的证据。我们已初步发现H_2O_2可参与外源ABA诱     

NO可能作为H2O2的下游信号介导ABA诱导的蚕豆气孔关闭

ABA、H2O2和硝普钠(SNP)均能诱导蚕豆气孔关闭.NO的清除剂c-PTIO可以减轻由ABA或H2O2所诱导的蚕豆气孔关闭的程度,而过氧化氢酶(CAT)则不能减轻NO诱导的气孔关闭程度.激光共聚焦显微检测结果显示,10μmo1/L的ABA处理后,胞内H2O2的产生速率明显高于NO的产生速率;CAT几乎可完全抑制ABA所诱导的DAF的荧光增加;外源H2O2能显著诱导胞内DAF的荧光增加;c-PTIO对ABA诱导的DCF荧光略有促进作用,但外源SNP不能诱导胞内DCF荧光增加.这些结果表明,在ABA诱导气孔关闭过程中,H2O2可能在NO的上游起作用并受NO的负反馈调节.     

α-淀粉酶在盐酸胍和碳酸胍变性中有关胍基作用的研究

利用紫外差谱、荧光光谱和园二色谱法对比地研究了α-淀粉酶盐酸胍和碳酸胍变性,分析了两种胍变性明显差异的原因。通过等同的胍基浓度下,α-淀粉酶两种胍变性的构象变化与活性关系的实验,表明同等摩尔浓度的两种胍盐变性能力上的明显差异并不主要是由于它们胍基含量上的不同。将盐酸胍从中性pH(6.5)调至碱性pH(10.4),其变性能力大增,紫外差谱与碳酸胍变性相似,出现了290nm的正肩和296nm的正峰,与此同时,酶的荧光强度大大降低,大部分酶活性丧失。由此推论,两种胍变性能力的明显差异的重要原因之一是在碱性介质中胍基的变性能力明显增强,并分析了其增强的原因。     

葡萄果实中脱落酸结合蛋白的存在及其性质

葡萄果实微粒体上存在高亲和力的脱落酸（ＡＢＡ）结合位点,这些位点与ＡＢＡ的结合具有饱和性,高亲和力及低容量,胰蛋白酶或ＤＴＴ处理可以使该位点的特异结合活性下降约９０％,表明此结合位点是一种蛋白质,故称为ＡＢＡ结合蛋白,它含有维系蛋白质特定构象的二硫键,该蛋白与ＡＢＡ反应的最适ｐＨ为６．０,说明与配基结合部位可能存在带有正电荷的氨基酸残基,结合活性在２５℃高于０℃,结合反应达到动态平衡需要３０ｍｉｎ,３０ｍｉｎ以后结合活性随时间延长而下降。该蛋白与ＡＢＡ结合反应的平衡解离常数为１７．５ｎｍｏｌ／Ｌ,最大结合容量（Ｂｍａｘ）为９８．４ｆｍｏｌ／ｍｇｐｒｏｔｅｉｎ。     

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line, BCL1

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. [3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAE-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [3H]TA-receptor complexes to chromatin-cellulose extracted with 0-8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocorticoid receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6-8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.     

Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and with double stranded DNA

Caffeine (CAF) inhibits the intercalation of acridine orange (AO) into cellular DNA. Optical absorption and fluorescence spectroscopy were employed to determine the molecular interactions of AO with itself, with CAF, and with double stranded herring sperm DNA (dsDNA). AO dimerization was observed at concentrations >2 micromol. The sharp increase in fluorescence (lambda(em)=530 nm) at 5 micromol of AO was attributed to AO multimer formation. From 0.5 to 5.0 micromol, the AO self-association binding constant (K(assoc)) was determined to be 38620 mol(-1), however, the presence of 150 mmol NaCl increased K(assoc) to 118000 mol(-1) attributed to the charge neutralization. The K(assoc) for AO with CAF was confirmed to be 256 mol(-1). K(assoc) for the binding of AO with 20 micromol DNA ranged from, 32000 mol(-1) at 2 micromol AO, to approximately 3700 mol(-1) at 10 micromol AO, in the absence of NaCl. This AO concentration dependency of K(assoc) value with DNA was attributed to AO intercalation into dsDNA at high dsDNA/AO ratios, and electrostatic binding of AO to dsDNA at low AO ratios. The findings provide information used to explain fluorescence intensity values at lambda(em) at 530 nm from studies that combine AO, caffeine, and dsDNA.     

共聚焦显微技术研究ABA诱导蚕豆气孔保卫细胞H2O2的产生（简报）

The methods of confocal laser scanning microscopy (CLSM) and microinjection were used to study ABA-induced H2O2 in guard cells (Vicia faba), which were labeled with H2O2 specific probe-2, 7-dichlorofluorescin diacetate(H2DCFDA). The results indicated 100 U/mL catalase (CAT) could inhibit partly stomatal closure induced by ABA. 10(-3) mmol/L ABA could significantly induce H2O2 production in chloroplast in guard cells of Vicia faba following microinjection, and 100 U/mL CAT could partly abolish the effects following simultaneous microinjection of ABA and CAT. These suggest that H2O2 is possibly involved in ABA signaling leading to stomatal closure.     

In Vivo Tissue-dependent Abscisic Acid Specific-binding Proteins with High Affinity in Cytosol of Developing Apple Fruits (in English)

The in vivo highly tissue-dependent abscisic acid (ABA) specific-binding sites localized in cytosol were identified and characterized in the flesh of developing apple ( Malus pumila L. cv. Starkrimon) fruits. ABA binding activity was scarcely detectable in the microsomes and the cytosolic fraction isolated from the freshly harvested fruits via an in vitro ABA binding incubation of the subcellular fractions. If, however, instead that the subcellular fractions were in vitro incubated in H-ABA binding medium, the flesh tissue discs were directly in vivo incubated in H-ABA binding medium, a high ABA binding activity to the cytosolic fraction isolated from these tissue discs was detected. The in vivo ABA binding capacity of the cytosolic fraction was lost if the tissue discs had been pretreated with boiling water, indicating that the ABA binding needs a living state of tissue. The in vivo tissue-dependent binding sites were shown to possess protein nature with both active serine residua and thiol-group of cysteine residua in their functional binding center. The ABA binding of the in vivo tissue-dependent ABA binding sites to the cytosolic fraction was shown to be saturable, reversible, and of high affinity. The scatchard plotting gave evidence of two different classes of ABA binding proteins, one with a higher affinity ( Kd =2.9 nmol/L) and the other with lower affinity ( Kd =71.4 nmol/L). Phaseic acid, 2- trans -4- trans -ABA or cis-trans -(－)-ABA had substantially no affinity to the binding proteins, indicating their stereo-specificity to bind physiologically active ABA. The time course, pH- and temperature-dependence of the in vivo tissue-dependent binding proteins were determined. It is hypothesized that the detected ABA-binding proteins may be putative ABA-receptors that mediate ABA signals during fruit development.     

植物ABA受体及其介导的信号转导通路

ABA是调控植物体生长发育和响应外界应激的重要植物激素之一。近年来, ABA受体的筛选和鉴定取得了突破性进展, 为植物中ABA信号转导通路的阐明奠定了重要基础。该文主要综述了ABA-binding protein/H subunit of Mgchelatase (ABAR/CHLH)、G protein-coupled receptor 2 (GCR2)、GPCR-type G protein 1/2 (GTG1/2)和pyrabactin resistant/PYR-like/regulatory component of ABA (PYR/PYL/RCAR)被报道为ABA受体的研究历程, 重点介绍了以ABAR/CHLH PYR/PYL/RCAR为受体的ABA信号转导通路模型的构建, 旨在为ABA受体及其信号转导通路的相关研究提供参考。     

糖、GA3及ABA对印度娃儿藤(Tylophora indica(Burm.f.)Merrill)体细胞胚发生的影响

从印度娃儿藤节间外植体获取愈伤组织,分析了糖、赤霉素(GA3)及脱落酸(ABA)对愈伤组织形成体细胞的影响。实验证明,含4μmol/L2,4-二氯苯氧乙酸(2,4-D)的MS培养基是获得具有成胚功能的愈伤组织的最佳培养基。在含有6μmol/L激动素(Kn)的MS培养基上,高达69%的愈伤组织分化为体细胞胚,平均单位外植体(每克愈伤组织)产胚25个。在6μmol/LKn存在的条件下,分析了蔗糖、葡糖糖对胚产生的影响,不同的糖及不同糖浓度对体细胞胚的发生影响很大。6μmol/L Kn与200mmol/L蔗糖处理胚胎发生率最大(71%),单位外植体生成49个胚。然而葡萄糖与Kn、或者葡糖糖、蔗糖与Kn三者加在一起则降低成胚率及产胚数。一定浓度的GA3和ABA能促进体细胞胚的产生。在含200mmol/L蔗糖的培养基中加10μmol/LGA3胚的生成率为98%,单位外植体产胚51个。在含200mmol/L蔗糖的培养基中加2μmol/L ABA能显著增加体细胞胚的量,该培养基上每外植体平均生成44个胚,产率为95%。本研究显示,含200mmol/L蔗糖的培养基中分别加入6μmol/L Kn、10μmol/L GA3或者2μmol/L ABA能显著提高印度娃儿藤体细胞胚发生率,而单独的葡萄糖或葡糖糖和蔗糖则有抑制作用。得到的胚均能正常发育并分化为植株。