Activity, but not Expression, of Soluble and Cell Wall-Bound Acid Invertases Is Induced by Abscisic Acid in Developing Apple Fruit

The present experiment, involving both the in vivo injection of abscislc acid （ABA） Into apple （Malus domestica Brohk.） fruits and the in vivo Incubation of fruit tissues in ABA-contalnlng medium, revealed that ABA activates both soluble and cell wall-bound acid invertases. Immunoblottlng and enzyme-linked Immunosorbent assays showed that this ABA-induced acid invertase activation is Independent of the amount of enzyme present. The acid Invertase activation induced by ABA is dependent on medium pH, time course, ABA dose, living tissue and developmental stage. Two isomers of cls-（＋）-ABA, （-）-ABA and trans- ABA, had no effect on acid invertases, showing that ABA-induced acid invertase activation is specific to physiologically active cis-（＋）ABA. Protein kinase inhlbltors K252a and H7 as well as acid phosphatase Increased the ABA-Induced effects. These data indicate that ABA specifically activates both soluble and cell wall-bound acid Invertases by a posttranslational mechanism probably Involving reversible protein phosphorylatlon, and this may be one of the mechanisms by which ABA Is Involved In regulating fruit development.     

Mitogen-activated protein kinase is involved in abscisic acid-induced antioxidant defense and acts downstream of reactive oxygen species production in leaves of maize plants

The role of mitogen-activated protein kinase (MAPK) in abscisic acid (ABA)-induced antioxidant defense was investigated in leaves of maize (Zea mays) plants. Treatments with ABA or H(2)O(2) induced the activation of a 46-kD MAPK and enhanced the expression of the antioxidant genes CAT1, cAPX, and GR1 and the total activities of the antioxidant enzymes catalase, ascorbate peroxidase, glutathione reductase, and superoxide dismutase. Such enhancements were blocked by pretreatment with several MAPK kinase inhibitors and reactive oxygen species inhibitors or scavengers. Pretreatment with MAPK kinase inhibitors also substantially arrested the ABA-induced H(2)O(2) production after 2 h of ABA treatment, but did not affect the levels of H(2)O(2) within 1 h of ABA treatment. Pretreatment with several inhibitors of protein tyrosine phosphatase, which is believed to be a negative regulator of MAPK, only slightly prevented the ABA-induced H(2)O(2) production, but did not affect the ABA-induced MAPK activation and ABA-enhanced antioxidant defense systems. These results clearly suggest that MAPK but not protein tyrosine phosphatase is involved in the ABA-induced antioxidant defense, and a cross talk between H(2)O(2) production and MAPK activation plays a pivotal role in the ABA signaling. ABA-induced H(2)O(2) production activates MAPK, which in turn induces the expression and the activities of antioxidant enzymes. The activation of MAPK also enhances the H(2)O(2) production, forming a positive feedback loop.     

蛋白激酶组在玉米叶片ABA和H202诱导抗氧化防护中的作用

蛋白磷酸化在植物细胞脱落酸（ABA）介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和ca^2＋依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa,52kDa,49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用,而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

Abscisic acid stimulates a calcium-dependent protein kinase in grape berry

It has been demonstrated that calcium plays a central role in mediating abscisic acid (ABA) signaling, but many of the Ca2+-binding sensory proteins as the components of the ABA-signaling pathway remain to be elucidated. Here we identified, characterized, and purified a 58-kD ABA-stimulated calcium-dependent protein kinase from the mesocarp of grape berries (Vitis vinifera x Vitis labrusca), designated ACPK1 (for ABA-stimulated calcium-dependent protein kinase1). ABA stimulates ACPK1 in a dose-dependent manner, and the ACPK1 expression and enzyme activities alter accordantly with the endogenous ABA concentrations during fruit development. The ABA-induced ACPK1 stimulation appears to be transient with a rapid effect in 15 min but also with a slow and steady state of induction after 60 min. ABA acts on ACPK1 indirectly and dependently on in vivo state of the tissues. Two inactive ABA isomers, (-)-2-cis, 4-trans-ABA and 2-trans, 4-trans-(+/-)-ABA, are ineffective for inducing ACPK1 stimulation, revealing that the ABA-induced effect is stereo specific to physiological active (+)-2-cis, 4-trans-ABA. The other phytohormones such as auxin indoleacetic acid, gibberellic acid, synthetic cytokinin N-benzyl-6-aminopurine, and brassinolide are also ineffective in this ACPK1 stimulation. Based on sequencing of the two-dimensional electrophoresis-purified ACPK1, we cloned the ACPK1 gene. The ACPK1 is expressed specifically in grape berry covering a fleshy portion and seeds, and in a developmental stage-dependent manner. We further showed that ACPK1 is localized in both plasma membranes and chloroplasts/plastids and positively regulates plasma membrane H+-ATPase in vitro, suggesting that ACPK1 may be involved in the ABA-signaling pathway.     

Role of Protein Kinases in Abscisic Acid and H2O2 Induced Antioxidant Defense in Maize

Protein phosphorylation plays a central role in mediating abscisic acid (ABA) signaling transduction in plant cells, whereas many of the sensory proteins involving in ABA signaling pathway remain unclear. Here, using a modified in vitro kinase assay, our results showed that ABA and H2O2 induced a rapid activation of total protein kinases and calcium dependent protein kinases in the leaves of maize seedlings. However, ABA-induced activation of protein kinases was inhibited by reactive oxygen species (ROS) inhibitors or scavengers. Protein kinase inhibitors decelerated not only the ABA and H2O2 -induced kinase activity but also ABA or H2O2-induced antioxidant enzyme activity. Protein phosphorylation caused by ABA and H2O2 preceded ABA or H2O2 -induced antioxidant defense obviously. Using in-gel kinase assays, our results showed that several protein kinases with molecular masses of 66kDa, 52kDa, 49kDa and 35kDa respectively might mediate ABA and H2O2-induced antioxidant defense. And the 66kDa and 49kDa protein kinases may act downstream of ROS, and the 52kDa and 35kDa protein kinases may act between ABA and ROS in ABA-induced antioxidant defensive signaling.     

蛋白激酶组在玉米叶片ABA和H2O2诱导抗氧化防护中的作用

蛋白磷酸化在植物细胞脱落酸（ABA）介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和Ca2+依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa, 52kDa, 49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用, 而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

Differential abscisic acid regulation of guard cell slow anion channels in Arabidopsis wild-type and abi1 and abi2 mutants.

Abscisic acid (ABA) regulates vital physiological responses, and a number of events in the ABA signaling cascade remain to be identified. To allow quantitative analysis of genetic signaling mutants, patch-clamp experiments were developed and performed with the previously inaccessible Arabidopsis guard cells from the wild type and ABA-insensitive (abi) mutants. Slow anion channels have been proposed to play a rate-limiting role in ABA-induced stomatal closing. We now directly demonstrate that ABA strongly activates slow anion channels in wild-type guard cells. Furthermore, ABA-induced anion channel activation and stomatal closing were suppressed by protein phosphatase inhibitors. In abi1-1 and abi2-1 mutant guard cells, ABA activation of slow anion channels and ABA-induced stomatal closing were abolished. These impairments in ABA signaling were partially rescued by kinase inhibitors in abi1 but not in abi2 guard cells. These data provide cell biological evidence that the abi2 locus disrupts early ABA signaling, that abi1 and abi2 affect ABA signaling at different steps in the cascade, and that protein kinases act as negative regulators of ABA signaling in Arabidopsis. New models for ABA signaling pathways and roles for abi1, abi2, and protein kinases and phosphatases are discussed.     

Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts

Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.     

蛋白激酶组在玉米叶片ABA和H_2O_2诱导抗氧化防护中的作用(英文)

蛋白磷酸化在植物细胞脱落酸(ABA)介导的信号转导中起重要作用。然而,很多参与ABA信号途径的蛋白元件仍不清楚。使用改进的体外激酶试验方法的研究结果表明,在玉米叶片中,ABA和H2O2能够快速活化蛋白激酶总活性和Ca2+依赖型蛋白激酶总活性;ABA诱导的蛋白激酶总活性增加可以被活性氧的抑制剂和清除剂抑制,蛋白激酶抑制剂不仅可以降低ABA和H2O2诱导的激酶活性增加,而且也可以弱化它们对抗氧化防护酶活性的诱导作用;ABA和H2O2引发的蛋白磷酸化作用显著居先于它们诱导的抗氧化防护作用。使用凝胶激酶试验方法进行研究发现,一组分子量分别为66kDa,52kDa,49kDa和35kDa的蛋白激酶可能介导了ABA和H2O2诱导的抗氧化防护反应,并且66kDa和49kDa的蛋白激酶可能在ROS的下游起作用,而52kDa和35kDa的蛋白激酶可能在ABA和ROS的下游起作用。     

Plasma membrane depolarization induced by abscisic acid in Arabidopsis suspension cells involves reduction of proton pumping in addition to anion channel activation, which are both Ca2+ dependent

In Arabidopsis suspension cells a rapid plasma membrane depolarization is triggered by abscisic acid (ABA). Activation of anion channels was shown to be a component leading to this ABA-induced plasma membrane depolarization. Using experiments employing combined voltage clamping, continuous measurement of extracellular pH, we examined whether plasma membrane H(+)-ATPases could also be involved in the depolarization. We found that ABA causes simultaneously cell depolarization and medium alkalinization, the second effect being abolished when ABA is added in the presence of H+ pump inhibitors. Inhibition of the proton pump by ABA is thus a second component leading to the plasma membrane depolarization. The ABA-induced depolarization is therefore the result of two different processes: activation of anion channels and inhibition of H(+)-ATPases. These two processes are independent because impairing one did not suppress the depolarization. Both processes are however dependent on the [Ca2+]cyt increase induced by ABA since increase in [Ca(2+)](cyt) enhanced anion channels and impaired H(+)-ATPases.     

Abscisic acid activates ATPase in developing apple fruit especially in fruit phloem cells

Abscisic acid (ABA) improves the sink strength by promoting the phloem unloading and regulating the assimilate metabolism in the economic sink organs of crops, although its mechanism remains unknown. The present experiment, using the techniques of the in vivo injection of ABA into the intact apple fruit attached to a growing apple tree and the in vivo incubation of the fruit tissue in the ABA‐contained medium, showed that ABA strongly activated the fruit ATPase especially P‐ATPase, of which the activity was doubled by ABA treatment. This ATPase activation was shown to be in vivo tissue‐dependent. The ABA‐induced P‐ATPase activation was fruit developmental stage‐, ABA dose‐, medium pH‐ and incubation time‐dependent. Physiological active (+)ABA was shown more effective to stimulate P‐ATPase activity than (+/–)ABA, and two ABA analogues (–)ABA and trans‐ABA, had no effect on P‐ATPase activation, indicating that only physiologically active cis(+)ABA can induce the enzyme activation, and so the ABA‐induced effects are stereospecific. The protein synthesis inhibitor cycloheximide was shown to have no effect on P‐ATPase activation by ABA, suggesting that synthesis of new proteins was not involved in the enzyme activation. The cytochemical assay revealed that P‐ATPase was activated by ABA in both the phloem and its surrounding flesh parenchyma cells, and that the most strongly P‐ATPase activation was observed in the plasma membrane of sieve element/companion cell complex. These data suggest that the improvement of phloem unloading by ABA previously reported in this fruit as in other crop sink organs may be attributed, at least partly, to the ABA‐induced ATPase activation especially in phloem cells.     

Effect of K-252a and abscisic acid on the efflux of citrate from soybean roots

The Al-induced release of organic acid has been suggested as an important mechanism for Al resistance in plants. In this study, the effect of K-252a and abscisic acid (ABA) on the efflux of citrate was investigated in soybean (Glycine max L.) roots. Al initiated citrate efflux from the root apices 30 min after the addition of Al. The Al-triggered efflux of citrate was sensitive to metabolic inhibitors and anion channel inhibitors. Pretreatment or treatment with K-252a, an inhibitor of protein kinase, severely inhibited the Al-induced efflux of citrate accompanying an increase in Al accumulation and intensified Al-induced root growth inhibition. Al-treatment increased the endogenous level of abscisic acid (ABA) in soybean roots in a dose- and time-dependent manner, while K-252a failed to inhibit the Al-induced increase in endogenous ABA. Exogenous application of ABA increased the activity of citrate synthase (EC 4.1.3.7) by 26.2%, and decreased Al accumulation by 32.3%, respectively. ABA-induced increases in citrate efflux and root elongation were suppressed by K-252a, while ABA could not reverse the K-252a effects. Taken together, these results suggest that ABA is probably involved in the early response, after which K-252a-sensitive protein kinases play a key step in regulating the activity of an anion channel, through which citrate is released from the apical cells of soybean roots.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

MAPK级联途径参与ABA信号转导调节的植物生长发育过程

植物激素ABA参与调控植物生长发育和生理代谢以及多种胁迫应答过程,促分裂原活化蛋白激酶（MAPK）级联途径应答于多种生物和非生物胁迫,广泛参与调控植物的生长发育。MAPK级联途径与ABA信号转导协同作用参与调控植物种子萌发、气孔运动和生长发育,本文主要归纳了植物中受ABA调控激活的MAPK级联途径成员,阐述了它们参与ABA信号转导调控植物生理反应和生长发育的过程,并对MAPK级联途径与ABA信号转导的研究方向作出了展望,指出对MAPK下游底物的筛选是完善MAPK级联途径的重要组成部分。     

Age-dependent action of an ABA-inducible receptor kinase, RPK1, as a positive regulator of senescence in Arabidopsis leaves

Leaf senescence, which constitutes the final stage of leaf development, involves programmed cell death and is intricately regulated by various internal and environmental signals that are incorporated with age-related information. ABA plays diverse and important physiological roles in plants, and is involved in various developmental events and stress responses. ABA has long been regarded as a positive regulator of leaf senescence. However, the cellular mediators of ABA-induced senescence have not been identified. We sought to understand the ABA-induced senescence signaling process in Arabidopsis by examining the function of an ABA- and age-induced gene, RPK1, which encodes a membrane-bound, leucine-rich repeat-containing receptor kinase (receptor protein kinase 1). Loss-of-function mutants in RPK1 were significantly delayed in age-dependent senescence. Furthermore, rpk1 mutants exhibited reduced sensitivity to ABA-induced senescence but little change to jasmonic acid- or ethylene-induced senescence. RPK1 thus mediates ABA-induced leaf senescence as well as age-induced leaf senescence. Conditional overexpression of RPK1 at the mature stage clearly accelerated senescence and cell death, whereas induction of RPK1 at an early developmental stage retarded growth without triggering senescence symptoms. Therefore, RPK1 plays different roles at different stages of development. Consistently, exogenously applied ABA affected leaf senescence in old leaves but not in young leaves. The results, together, showed that membrane-bound RPK1 functions in ABA-dependent leaf senescence. Furthermore, the effect of ABA and ABA-inducible RPK1 on leaf senescence is dependent on the age of the plant, which in part explains the mechanism of functional diversification of ABA action.     

Disruption of a guard cell-expressed protein phosphatase 2A regulatory subunit,RCN1, confers abscisic acid insensitivity in Arabidopsis

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell-expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca(2+) increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling.     

Factors affecting 'Hass' avocado fruit size: Carbohydrate, abscisic acid and isoprenoid metabolism in normal and phenotypically small fruit

Carbohydrate and abscisic acid (ABA) metabolism were investigated in normal and phenotypically small 'Hass' avocado ( Persea americana Mill.) fruit in an attempt to link alterations in sugar and ABA content with changes in 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) activity and fruit size. The small-fruit phenotype was characterized by reduced seed HMGR activity, increased seed insoluble acid invertase ( β - d -fructofuranosidase, EC 3.2.1.26), decreased sucrose synthase (SS; UDP- d -glucose: d -fructose-2- α -glucosyl-transferase, EC 2.4.1.13) activity, decreased sucrose content, and increased glucose as a proportion of the total soluble sugar. Sucrose phosphate synthase (SPS; UDP- d -glucose: d -fructose 6-phosphate 2- α - d -glucosyltransferase, EC 2.4.1.14) activity was unaffected in seed but reduced in mesocarp of the small fruit. In addition, the small-fruit variant displayed enhanced respiration and both seed and mesocarp tissue showed increased ABA metabolism. Applied ABA caused an increase in insoluble acid invertase activity in seed tissue of normal fruit while mevastatin reduced HMGR activity in this tissue, caused sucrose depletion and increased the proportion of glucose from 5 to 57% of total soluble sugars. Exogenous glucose suppressed HMGR activity in seed tissue whereas in mesocarp tissue, HMGR activity was reduced to 38% of the control after 6 h but enhanced by 46% by 48 h. Glucose increased ABA biosynthesis and turnover in competent tissues. These results suggest that ABA turnover is mediated, in part, by carbohydrate content and composition which also affects HMGR activity. It is proposed that sugar and ABA signals act in concert to modulate expression and/or activity of HMGR in the control of 'Hass' avocado fruit growth and final fruit size.