Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis.

Exogenous abscisic acid (ABA) induced the alcohol dehydrogenase gene (Adh) in Arabidopsis roots. Both the G-box-1 element and the GT/GC motifs (anaerobic response element) were required for Adh inducibility. Measurement of endogenous ABA levels during stress treatment showed that ABA levels increased during dehydration treatment but not following exposure to either hypoxia or low temperature. Arabidopsis ABA mutants (aba1 and abi2) displayed reduced Adh mRNA induction levels following either dehydration treatment or exogenous application of ABA. Low-oxygen response was slightly increased in the aba1 mutant but was unchanged in abi2. Low-temperature response was unaffected in both aba1 and abi2 mutants. Our results indicate that, although induction of the Adh gene by ABA, dehydration, and low temperature required the same cis-acting promoter elements, their regulatory pathways were at least partially separated in a combined dehydration/ABA pathway and an ABA-independent low-temperature pathway. These pathways were in turn independent of a third signal transduction pathway leading to low-oxygen response, which did not involve either ABA or the G-box-1 promoter element.     

Regulation of alcohol dehydrogenase gene expression in barley aleurone by gibberellin and abscisic acid

The expression of the Adh1 gene (alcohol dehydrogenase, EC 1.1.1.1) was studied in the aleurone layer of barley ( Hordeum vulgare cv. Himalaya). Expression increased markedly during grain development at the levels of activity, enzyme protein and mRNA. mRNA content, but not enzyme activity, could be increased further by exogenous abscisic acid (ABA) when isolated, de-embryonated developing grains were pre-treated with gibberellic acid (GA₃) or fluridone. In isolated mature aleurone layers incubated with exogenous hormones, ADH mRNA was strongly up-regulated by ABA and down-regulated by GA₃ within 6 h. With ABA, this increase in mRNA was followed by an increase in ADH protein and activity, peaking at 18 h. With GA₃, the decrease in mRNA was accompanied by simultaneous decreases in protein and activity. In general, GA₃ counteracted the effect of ABA and vice versa. In the aleurone of germinating grain, ADH activity decayed in a distal direction from the embryo, consistent with down-regulation by gibberellin(s) diffusing from it. It was concluded that ADH gene expression in the aleurone of the intact grain is regulated by an ABA/gibberellin interaction.     

Characterization of a cDNA from Arabidopsis thaliana encoding a potential thiol protease whose expression is induced independently by wilting and abscisic acid

The sequence and expression characteristics are described of a wilt-inducible gene in Arabidopsis thaliana. A 1494 encodes a potential thiol protease whose mRNA accumulates rapidly in shoot tissue upon the loss of turgor. A1494 mRNA levels peaked after ca. 4 h and declined thereafter. Dehydration also induced rapid biosynthesis of the phytohormone abscisic acid (ABA), which continued for at least 9 h. Exogenous ABA induced the accumulation of A1494 mRNA, with kinetics similar to those after wilting. Rehydration of wilted shoots led to a rapid decline in the content of both ABA and A1494 mRNA. Wilting and ABA independently induced A1494 expression as evidenced by the effects of ABA and wilting on the ABA-deficient aba-1 and ABA-insensitive abi-1 and abi-3 genotypes. A1494 mRNA was not detectable in aba-1 shoots but accumulated rapidly after either wilting or ABA treatment, whereas the shoot ABA content was increased only by ABA treatment. ABA had no effect on A1494 mRNA levels in the abi-1 and abi-3 mutants but wilting did result in enhanced A1494 expression. Heat shock had only a minor effect on A1494 mRNA levels, whereas exposure to low temperature resulted in substantial accumulation of A1494 mRNA in wild-type shoots. However, this latter response, unlike that to drought, was mediated exclusively via ABA synthesis as demonstrated by the lack of A1494 mRNA accumulation in cold-treated aba-1 shoots.     

Alterations in Water Status,Endogenous Abscisic Acid Content,and Expression of rab18 Gene during the Development of Freezing Tolerance in Arabidopsis thaliana

Treatments as diverse as exposure to low temperature (LT), exogenous abscisic acid (ABA), or drought resulted in a 4 to 5[deg]C increase in freezing tolerance of the annual herbaceous plant Arabidopsis thaliana. To correlate the increase in freezing tolerance with the physiological changes that occur in response to these treatments, we studied the alterations in water status, endogenous ABA levels, and accumulation of rab18 (V. Lang and E.T. Palva [1992] Plant Mol Biol 20: 951-962) mRNA. Exposure to LT and exogenous ABA caused only a minor decline in total water potential ([psi]w), in contrast to a dramatic decrease in [psi]w during drought stress. Similarly, the endogenous ABA levels were only slightly and transiently increased in LT-treated plants in contrast to a massive increase in ABA levels in drought-stressed plants. The expression of the ABA-responsive rab18 gene was low during the LT treatment but could be induced to high levels by exogenous ABA and drought stress. Taken together, these results suggest that the moderate increases in freezing tolerance of A. thaliana might be achieved by different mechanisms. However, ABA-deficient and ABA-insensitive mutants of A. thaliana have impaired freezing tolerance, suggesting that ABA is, at least indirectly, required for the development of full freezing tolerance.     

Signaling events in the hypoxic induction of alcohol dehydrogenase gene in Arabidopsis

Expression of the alcohol dehydrogenase gene (ADH) of Arabidopsis is induced during hypoxia. Because many plants increase their ethylene production in response to hypoxic stress, we examined in this report whether ethylene is involved in the hypoxic induction of ADH in Arabidopsis. We found that the hypoxic induction of ADH can be partially inhibited by aminooxy acetic acid, an inhibitor of ethylene biosynthesis. This partial inhibition can be reversed by the addition of 1-aminocyclopropane-1-carboxylic acid, a direct precursor of ethylene. In addition, the hypoxic induction of the ADH gene is also reduced in etr1-1 and ein2-1, two ethylene insensitive mutants in ethylene-signaling pathways, whereas the addition of exogenous ethylene or an increase in cellular ethylene alone does not induce ADH under normoxic conditions. Kinetic analyses of ADH mRNA accumulation indicated that an ethylene signal is required for the induction of ADH during later stages of hypoxia. Therefore, we conclude that ethylene is needed, but not sufficient for, the induction of ADH in Arabidopsis during hypoxia.     

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.     

Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh

Summary Exogenously applied abscisic acid (ABA) induced frost hardening of Arabidopsis thaliana (L.) Heynh. The freezing tolerance of A. thaliana plantlets treated with ABA (15 mg/l) at a non-acclimating temperature (20 °C) appeared to increase even more rapidly than following a low temperature (4 °C) acclimation. Analysis of in vivo-labelled soluble proteins by two-dimensional gel electrophoresis revealed several low temperature — or ABA — induced proteins, which where not produced in non-acclimated plants. A subset of these proteins was induced by both low temperature and ABA treatments, suggesting that they might be directly involved in the frost hardening process in A. thaliana.     

A distinctive class of spermidine synthase is involved in chilling response in rice

A cDNA for a putative 42 kD spermidine synthase (OsSPDS2) was cloned from rice. The deduced OsSPDS2 sequence showed highest similarity with Arabidopsis AtSPDS3. Phylogenetic analysis revealed that OsSPDS2 and AtSPDS3 form a distinctive subclass in the spermidine synthase family in plants. OsSPDS2 mRNA accumulated in roots during long term exposure to chilling temperature (12 degrees C). In contrast, no such induction of the paralogous OsSPDS1 was observed during the chilling treatment. ABA treatment up-regulated OsSPDS2, whereas salt stress did not change OsSPDS2 levels significantly. Data suggested a distinct function of OsSPDS2 in chilling response in rice.     

Separate signal pathways regulate the expression of a low-temperature-induced gene in Arabidopsis thaliana (L.) Heynh.

A cDNA clone corresponding to a novel low-temperature-induced Arabidopsis thaliana gene, named lti140, was employed for studies of the environmental signals and the signal pathways involved in cold-induced gene expression. The single-copy lti140 gene encodes a 140 kDa cold acclimation-related polypeptide. The lti140 mRNA accumulates rapidly in both leaves and roots when plants are subject to low temperature or water stress or are treated with the plant hormone abscisic acid (ABA), but not by heat-shock treatment. The low-temperature induction of lti140 is not mediated by ABA, as shown by normal induction of the lti140 mRNA in both ABA-deficient and ABA-insensitive mutants and after treatment with the ABA biosynthesis inhibitor fluridone. The effects of low temperature and exogenously added ABA are not cumulative suggesting that these two pathways converge. The induction by ABA is abolished in the ABA-insensitive mutant abi-1 indicating that the abi-1 mutation defines a component in the ABA response pathway. Accumulation of the lti140 mRNA in plants exposed to water stress was somewhat reduced by treatment with fluridone and in the ABA-insensitive mutant abi-1 suggesting that the water stress induction of lti140 could be partly mediated by ABA. It is concluded that three separate but converging signal pathways regulate the expression of the lti140 gene.     

Renal responses to chronic cold exposure

The aim of this study was to assess our hypothesis that the release of antidiuretic hormone (ADH), the renal concentrating response to ADH, or both is decreased by prolonged cold exposure. Six groups (n = 6/group) of rats were used. Three groups were exposed to cold (5 degrees C), whilethe remaining three groups were kept at room temperature (25 degrees C). It was found that urine osmolality decreased significantly and serum osmolality increased significantly during cold exposure. The ratio of water/food intake was not affected by prolonged cold exposure. However, prolonged cold exposure increased the ratio of urine output/food intake in the cold-exposed rats, indicating that more urine flow is required by the cold-exposed rats to excrete the osmotic substance at a given food intake. The difference between water intake and urine output decreased significantly in the cold-exposed rats. Thus, prolonged cold exposure increases water loss from excretion. Renal concentrating responses to 24-h dehydration and Pitressin were decreased significantly in the cold-exposed rats. Plasma ADH levels remained unchanged, but renal ADH receptor (V2 receptor) mRNA was decreased significantly in the cold-exposed rats. The results strongly support the conclusion that cold exposure increases excretive water loss, and this may be due to suppression of renal V2 receptors rather than inhibition of ADH release.     

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.     

Abscisic acid deficiency antagonizes high-temperature inhibition of disease resistance through enhancing nuclear accumulation of resistance proteins SNC1 and RPS4 in Arabidopsis

Plant defense responses to pathogens are influenced by abiotic factors, including temperature. Elevated temperatures often inhibit the activities of disease resistance proteins and the defense responses they mediate. A mutant screen with an Arabidopsis thaliana temperature-sensitive autoimmune mutant bonzai1 revealed that the abscisic acid (ABA)-deficient mutant aba2 enhances resistance mediated by the resistance (R) gene suppressor of npr1-1 constitutive1 (SNC1) at high temperature. ABA deficiency promoted nuclear accumulation of SNC1, which was essential for it to function at low and high temperatures. Furthermore, the effect of ABA deficiency on SNC1 protein accumulation is independent of salicylic acid, whose effects are often antagonized by ABA. ABA deficiency also promotes the activity and nuclear localization of R protein resistance to Pseudomonas syringae4 at higher temperature, suggesting that the effect of ABA on R protein localization and nuclear activity is rather broad. By contrast, mutations that confer ABA insensitivity did not promote defense responses at high temperature, suggesting either tissue specificity of ABA signaling or a role of ABA in defense regulation independent of the core ABA signaling machinery. Taken together, this study reveals a new intersection between ABA and disease resistance through R protein localization and provides further evidence of antagonism between abiotic and biotic responses.     

Analysis of the Arabidopsis cell suspension phosphoproteome in response to short-term low temperature and abscisic acid treatment

To shed light on the early protein phosphorylation events involved in plant cell signaling in response to environmental stresses, we studied changes in the phosphorylation status of the Arabidopsis cell suspension proteome after short-term low temperature and abscisic acid (ABA) treatment. We used radioactive pulse-labeling of Arabidopsis cell suspension cultures and two-dimensional (2-D) gel electrophoresis to identify proteins that are differentially phosphorylated in response to these treatments. Changes in the phosphorylation levels of several proteins were detected in response to short-term (5 min or less) cold (4°C) and chilling (12°C) stress and ABA treatment, and we observed that some of these changes were common between these treatments. In addition, we used Pro-Q Diamond phosphoprotein gel stain to study the steady-state protein phosphorylation status under the same treatments. We demonstrated that Pro-Q Diamond effectively stained phosphorylated proteins, however, the overall Pro-Q Diamond 2-D gel staining pattern of proteins extracted from low-temperature and ABA-treated cells was not consistent with the gel patterns obtained by in vivo radioactive labeling of phosphoproteins. These in vivo pulsed-labeling experiments demonstrate that the Arabidopsis phosphoproteome is dynamic in response to short-term low temperature and ABA treatment, and thus represents a strategy for the identification of signaling proteins that could be utilized in the production of chilling or freeze tolerant crop varieties.     

Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.