Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.     

Brassinosteroid signaling positively regulates abscisic acid biosynthesis in response to chilling stress in tomato

Brassinosteroids (BRs) and abscisic acid (ABA) are essential regulators of plant growth and stress tolerance. Although the antagonistic interaction of BRs and ABA is proposed to ensure the balance between growth and defense in model plants, the crosstalk between BRs and ABA in response to chilling in tomato (Solanum lycopersicum), a warm-climate horticultural crop, is unclear. Here, we determined that overexpression of the BR biosynthesis gene DWARF (DWF) or the key BR signaling gene BRASSINAZOLE-RESISTANT1 (BZR1) increases ABA levels in response to chilling stress via positively regulating the expression of the ABA biosynthesis gene 9-CIS-EPOXYCAROTENOID DIOXYGENASE1 (NCED1). BR-induced chilling tolerance was mostly dependent on ABA biosynthesis. Chilling stress or high BR levels decreased the abundance of BRASSINOSTEROID-INSENSITIVE2 (BIN2), a negative regulator of BR signaling. Moreover, we observed that chilling stress increases BR levels and results in the accumulation of BZR1. BIN2 negatively regulated both the accumulation of BZR1 protein and chilling tolerance by suppressing ABA biosynthesis. Our results demonstrate that BR signaling positively regulates chilling tolerance via ABA biosynthesis in tomato. The study has implications in production of warm-climate crops in horticulture.     

A novel inhibitor of 9-cis-epoxycarotenoid dioxygenase in abscisic acid biosynthesis in higher plants

Abscisic acid (ABA) is a major regulator in the adaptation of plants to environmental stresses, plant growth, and development. In higher plants, the ABA biosynthesis pathway involves the oxidative cleavage of 9-cis-epoxycarotenoids, which may be the key regulatory step in the pathway catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). We developed a new inhibitor of ABA biosynthesis targeting NCED and named it abamine (ABA biosynthesis inhibitor with an amine moiety). Abamine is a competitive inhibitor of NCED, with a Ki of 38.8 microm. In 0.4 m mannitol solution, which mimics the effects of osmotic stress, abamine both inhibited stomatal closure in spinach (Spinacia oleracea) leaves, which was restored by coapplication of ABA, and increased luminescence intensity in transgenic Arabidopsis containing the RD29B promoter-luciferase fusion. The ABA content of plants in 0.4 m mannitol was increased approximately 16-fold as compared with that of controls, whereas 50 to 100 microm abamine inhibited about 50% of this ABA accumulation in both spinach leaves and Arabidopsis. Abamine-treated Arabidopsis was more sensitive to drought stress and showed a significant decrease in drought tolerance than untreated Arabidopsis. These results suggest that abamine is a novel ABA biosynthesis inhibitor that targets the enzyme catalyzing oxidative cleavage of 9-cis-epoxycarotenoids. To test the effect of abamine on plants other than Arabidopsis, it was applied to cress (Lepidium sativum) plants. Abamine enhanced radicle elongation in cress seeds, which could be due to a decrease in the ABA content of abamine-treated plants. Thus, it is possible to think that abamine should enable us to elucidate the functions of ABA in cells or plants and to find new mutants involved in ABA signaling.     

Production of Polyamines Is Enhanced by Endogenous Abscisic Acid in Maize Seedlings Subjected to Salt Stress

It is known that salt stress and exogenously applied abscisic acid （ABA） can enhance the polyamine content in plants and that salt stress itself can lead to an increase in endogenous ABA production. In the present study, the relationships between salt-induced ABA and polyamine accumulation were inves- tigated using ABA-deficient mutant （vp5/vp5） maize （Zea mays L.） seedlings and ABA and polyamine biosynthesis inhibitors. The results show that reduced endogenous ABA levels, as a result of either the mutation or by using a chemical inhibitor （sodium tungstate）, also reduced the accumulation of polyamines in salt-stressed leaves of maize seedlings. The polyamine synthesis inhibitors D-arginine and α- difluoromethylornithine also reduced the polyamine content of the leaves of maize seedling under salt stress. Both ABA and polyamine enhanced the dry weight accumulation of salt-stressed seedlings and also increased the activities of the two dominant tonoplast membrane enzymes, H^＋-ATPase and H^＋-PPase, when plants were under salt stress. The results suggest that salt stress induces an increase in endogenous ABA levels, which then enhances polyamine synthesis. Such responses may increase a plant＇s tolerance to salt.     

Effects of abscisic acid on the contents of polyamines and proline in common bean plants under salt stress

The effects of ABA treatment on the contents of polyamines (PAs) and proline (Pro) in the glycophyte Phaseolus vulgaris L. during plant adaptation to salt stress were studied. Two-week-old common bean seedlings grown in the phytotron chamber on the Jonson nutrient medium were subjected to salinity for 6 days by one-time NaCl addition to medium up to final concentrations of 50 and 100 mM. During first three days of salinity, the root system was daily treated with ABA (1, 5, 10, or 50 μM) for 30 min. Salt stress (100 mM NaCl) elevated the level of endogenous ABA, increased the content of Pro 14-fold, reduced sharply the content of free PAs (putrescine, spermidine, spermine, and cadaverine), and the accumulation of 1,3-diaminopropan, a product of oxidation of high-molecular PAs. Common bean plant treatment with 1 μM ABA weakened the adverse effects of salt stress (100 mM NaCl), which was manifested in the maintenance of plant growth, stimulation of chlorophyll (a and b) and carotenoid accumulation, a stabilization of water and Na⁺ balance. Seedling treatment with ABA suppressed NaCl-induced Pro and intracellular ABA accumulation and restored the levels of putrescine and spermidine. The content of spermine in the leaves of plants subjected to salt stress and treated with ABA was approximately threefold higher than in control plants, whereas the content of cadaverine increased under similar conditions more than fivefold. Simultaneously, the contents of 1,3-diaminopropan and malondialdehyde as well as activity of superoxide dismutase were reduced, which indicates a weakening of oxidative stress, one of the possible causes of defensive ABA effects against salt stress. In addition, the suppression by exogenous ABA of Pro accumulation and stimulation of PA content under salt stress confirm indirectly our hypothesis that ABA is involved in the coordinated regulation of two biosynthetic pathways, Pro and PA formation, which use a common precursor, glutamate, and play an important protective role during stress in plants.     

Identification and functional roles of CaDIN1 in abscisic acid signaling and drought sensitivity

Plants frequently face challenges caused by various abiotic stresses, including drought, and have evolved defense mechanisms to counteract the deleterious effects of these stresses. The phytohormone abscisic acid (ABA) is involved in signal transduction pathways that mediate defense responses of plants to abiotic stress. Here, we report a new function of the CaDIN1 protein in defense responses to abiotic stress. The CaDIN1 gene was strongly induced in pepper leaves exposed to ABA, NaCl, and drought stresses. CaDIN1 proteins share high sequence homology with other known DIN1 proteins and are localized in chloroplasts. We generated CaDIN1-silenced peppers and overexpressing transgenic Arabidopsis plants and evaluated their response to ABA and drought stress. Virus-induced gene silencing of CaDIN1 in pepper plants conferred enhanced tolerance to drought stress, which was accompanied by low levels of lipid peroxidation in dehydrated leaves. CaDIN1-overexpressing transgenic plants exhibited reduced sensitivity to ABA during seed germination and seedling stages. Transgenic plants were more vulnerable to drought than that by the wild-type plants because of decreased expression of ABA responsive stress-related genes and reduced stomatal closure in response to ABA. Together, these results suggest that CaDIN1 modulates drought sensitivity through ABA-mediated cell signaling.     

Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses

Drought and salt stress tolerance of Arabidopsis (Arabidopsis thaliana) plants increased following treatment with the nonprotein amino acid beta-aminobutyric acid (BABA), known as an inducer of resistance against infection of plants by numerous pathogens. BABA-pretreated plants showed earlier and higher expression of the salicylic acid-dependent PR-1 and PR-5 and the abscisic acid (ABA)-dependent RAB-18 and RD-29A genes following salt and drought stress. However, non-expressor of pathogenesis-related genes 1 and constitutive expressor of pathogenesis-related genes 1 mutants as well as transgenic NahG plants, all affected in the salicylic acid signal transduction pathway, still showed increased salt and drought tolerance after BABA treatment. On the contrary, the ABA deficient 1 and ABA insensitive 4 mutants, both impaired in the ABA-signaling pathway, could not be protected by BABA application. Our data demonstrate that BABA-induced water stress tolerance is based on enhanced ABA accumulation resulting in accelerated stress gene expression and stomatal closure. Here, we show a possibility to increase plant tolerance for these abiotic stresses through effective priming of the preexisting defense pathways without resorting to genetic alterations.     

Effect of ABA on the contents of proline, polyamines, and cytokinins in the common ice plants under salt stress

The effects of ABA treatment on the contents of proline, polyamines (PA), and cytokinins (CK) in the facultative halophyte the common ice plant (Mesembryanthemum crystallinum L.) subjected to salt stress were studied. Plants grown in the phytotron chamber on Jonson nutrient medium for 6 weeks were subjected to 6-day-long salinity by a single NaCl adding to medium. During first three days of salinity, half plants of each treatment were placed for 30 min on nutrient medium containing 0, 100, or 300 mM NaCl plus ABA in the final concentration of 1 μM. Salinity reduced biomass accumulation and water and chlorophyll contents in plants. This was accompanied by the increase in the levels of MDA, proline, and sodium ions. ABA treatment of salt-stressed plants favored biomass accumulation and photosynthetic pigment protection, reduced the intensity of oxidative stress and the level of NaCl-induced proline accumulation. ABA treatment increased the contents of putrescine (Put) and spermidine (Spd) in the leaves and roots of control plants (not subjected to salt stress), reduced the losses of Put in the leaves and roots and Spd in the roots in the presence of 100 mM NaCl, and suppressed cadaverine (Cad) accumulation in the roots in the presence of 300 mM NaCl. In the presence of NaCl, ABA reduced the contents of zeatin and zeatin riboside and increased the level of zeatin-O-glucoside in the roots and isopentenyladenosine and isopentenyladenine in the leaves. Thus, ABA protective action under salinity can be realized through the weakening of oxidative stress (a decrease in MDA content) and the regulation of PA, proline, and CK metabolism, which has a great significance in plant adaptation to injurious factors.     

Jasmonic acid interacts with abscisic acid to regulate plant responses to water stress conditions

Phytohormones are key players in signaling environmental stress conditions. Hormone profiling together with proline accumulation were studied in leaves and roots of different mutant lines of Arabidopsis. Regulation of proline accumulation in this system seems complex and JA-deficient (jar1-1) and JA-insensitive (jai1) lines accumulating high levels of proline despite their very low ABA levels seems to discard an ABA-dependent response. However, the pattern of proline accumulation in jai1 seedlings parallels that of ABA. Under stress conditions, there is an opposite pattern of ABA accumulation in roots of jar1-1/coi1-16 (in which ABA only slightly increase) and jai1 (in which ABA increase is even higher than in WT plants). This also makes JA-ABA crosstalk complex and discards any lineal pathway that could explain this hormonal interaction.     

Control of abscisic acid catabolism and abscisic acid homeostasis is important for reproductive stage stress tolerance in cereals

Drought stress at the reproductive stage causes pollen sterility and grain loss in wheat (Triticum aestivum). Drought stress induces abscisic acid (ABA) biosynthesis genes in anthers and ABA accumulation in spikes of drought-sensitive wheat varieties. In contrast, drought-tolerant wheat accumulates lower ABA levels, which correlates with lower ABA biosynthesis and higher ABA catabolic gene expression (ABA 8'-hydroxylase). Wheat TaABA8'OH1 deletion lines accumulate higher spike ABA levels and are more drought sensitive. ABA treatment of the spike mimics the effect of drought, causing high levels of sterility. ABA treatment represses the anther cell wall invertase gene TaIVR1, and drought-tolerant lines appeared to be more sensitive to the effect of ABA. Drought-induced sterility shows similarity to cold-induced sterility in rice (Oryza sativa). In cold-stressed rice, the rate of ABA accumulation was similar in cold-sensitive and cold-tolerant lines during the first 8 h of cold treatment, but in the tolerant line, ABA catabolism reduced ABA levels between 8 and 16 h of cold treatment. The ABA biosynthesis gene encoding 9-cis-epoxycarotenoid dioxygenase in anthers is mainly expressed in parenchyma cells surrounding the vascular bundle of the anther. Transgenic rice lines expressing the wheat TaABA8'OH1 gene under the control of the OsG6B tapetum-specific promoter resulted in reduced anther ABA levels under cold conditions. The transgenic lines showed that anther sink strength (OsINV4) was maintained under cold conditions and that this correlated with improved cold stress tolerance. Our data indicate that ABA and ABA 8'-hydroxylase play an important role in controlling anther ABA homeostasis and reproductive stage abiotic stress tolerance in cereals.     

Overexpression of an Arabidopsis β-glucosidase gene enhances drought resistance with dwarf phenotype in creeping bentgrass

An Arabidopsis β-glucosidase, AtBG1 is known to hydrolyze glucose-conjugated, biologically inactive abscisic acid (ABA) to produce active ABA, which increases the level of ABA in plants. Since an increase of ABA in plants confers tolerance against abiotic stress such as drought, we introduced the pCAMBIA3301 vector harboring the AtBG1 gene into creeping bentgrass through Agrobacterium-mediated transformation. After transformation, putative transgenic plants were selected using the BASTA resistance assay at a concentration of 0.8?%. Genomic integration of the AtBG1 gene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by Northern blot and Western blot analyses. Interestingly, the transgenic bentgrass plants overexpressing AtBG1 had a dwarf phenotype with reduced growth rates when compared to wild-type creeping bentgrass. In addition, the transgenic plants accumulated higher ABA levels and displayed enhanced drought tolerance. These results suggest that the expression of AtBG1 in plants induces the accumulation of higher ABA levels, which results in the formation of dwarf creeping bentgrass and enhances the survival in water-limiting environments. Key message We used an Arabidopsis β-glucosidase AtBG1 to engineer a crop with elevated active ABA levels, and developed transgenic creeping bentgrass with enhanced drought tolerance and dwarf phenotype.     

Abscisic Acid Metabolism in Relation to Water Stress and Leaf Age in Xanthium strumarium

Intact plants of Xanthium strumarium L. were subjected to a water stress-recovery cycle. As the stress took effect, leaf growth ceased and stomatal resistance increased. The mature leaves then wilted, followed by the half expanded ones. Water, solute, and pressure potentials fell steadily in all leaves during the rest of the stress period. After 3 days, the young leaves lost turgor and the plants were rewatered. All the leaves rapidly regained turgor and the younger ones recommenced elongation. Stomatal resistance declined, but several days elapsed before pre-stress values were attained.

Abscisic acid (ABA) and phaseic acid (PA) levels rose in all the leaves after the mature ones wilted. ABA-glucose ester (ABA-GE) levels increased to a lesser extent, and the young leaves contained little of this conjugate. PA leveled off in the older leaves during the last 24 hours of stress, and ABA levels declined slightly. The young leaves accumulated ABA and PA throughout the stress period and during the 14-hour period immediately following rewatering. The ABA and PA contents, expressed per unit dry weight, were highest in the young leaves. Upon rewatering, large quantities of PA appeared in the mature leaves as ABA levels fell to the pre-stress level within 14 hours. In the half expanded and young leaves, it took several days to reach pre-stress ABA values. ABA-GE synthesis ceased in the mature leaves, once the stress was relieved, but continued in the half expanded and young leaves for 2 days.

Mature leaves, when detached and stressed, accumulated an amount of ABA similar to that in leaves on the intact plant. In contrast, detached and stressed young leaves produced little ABA. Detached mature leaves, and to a lesser extent the half expanded ones, rapidly catabolized ABA to PA and ABA-GE, but the young leaves did not. Studies with radioactive (±)-ABA indicated that in young leaves the conversion of ABA to PA took place at a much lower rate than in mature ones. Leaves of all ages rapidly conjugated PA to PA-glucose ester. Furthermore, when half expanded leaves were stressed on the intact plant, their rate of ABA catabolism was enhanced, an effect not observed in the young leaves.

In conclusion, young leaves on intact Xanthium plants produce little stress-induced ABA themselves, but due to import and a low rate of catabolism accumulate more ABA and PA than mature leaves.

     

Role of abscisic acid in cadmium tolerance of rice (Oryza sativa L.) seedlings

Changes in abscisic acid (ABA) contents in Cd-treated rice (Oryza sativa L.) seedlings of two cultivars were investigated. On treatment with CdCl2, the ABA content rapidly increased in the leaves and roots of Cd-tolerant cultivar (cv. Tainung 67, TNG67) but not in the Cd-sensitive cultivar (cv. Taichung Native 1, TN1). The reduction of transpiration rate of TN1 caused by Cd was less than that of TNG67. Exogenous application of ABA reduced transpiration rate, decreased Cd content, and enhanced Cd tolerance of TN1 seedlings. Exogenous application of the ABA biosynthesis inhibitor, fluridone, reduced ABA accumulation, increased transpiration rate and Cd content, and decreased Cd tolerance of TNG67 seedlings. Fluridone effect on Cd toxicity of TNG67 seedlings was reversed by the application of ABA. The roles of endogenous ABA in Cd tolerance of rice seedlings are discussed and suggested.     

Glycine betaine involvement in freezing tolerance and water stress in Arabidopsis thaliana

Levels of endogenous glycine betaine in the leaves were measured in response to cold acclimation, water stress and exogenous ABA application in Arabidopsis thaliana. The endogenous glycine betaine level in the leaves increased sharply during cold acclimation treatment as plants gained freezing tolerance. When glycine betaine (10 mM) was applied exogenously to the plants as a foliar spray, the freezing tolerance increased from -3.1 to -4.5 degrees C. In addition, when ABA (1 mM) was applied exogenously, the endogenous glycine betaine level and the freezing tolerance in the leaves increased. However, the increase in the leaf glycine betaine level induced by ABA was only about half of that by the cold acclimation treatment. Furthermore, when plants were subjected to water stress (leaf water potential of approximately -1.6 MPa), the endogenous leaf glycine betaine level increased by about 18-fold over that in the control plants. Water stress lead to significant increase in the freezing tolerance, which was slightly less than that induced by the cold acclimation treatment. The results suggest that glycine betaine is involved in the induction of freezing tolerance in response to cold acclimation, ABA, and water stress in Arabidopsis plants.     

Transport and accumulation rates of abscisic acid and aldehyde oxidase activity in Pisum sativum L. in response to suboptimal growth conditions.

Pea plants (Pisum sativum L.) grown initially in nutrient solutions with adequate nitrogen supply (4 mM NO3-) were transferred to solutions containing salt (50 or 100 mM NaCl), ammonium (4 mM) or a low nitrogen supply (0.4 mM NO3-). No changes of abscisic acid (ABA) content were found in roots of stressed pea plants 9 d after the beginning of the treatments; however, accumulation of ABA in the leaves was observed. Old leaves accumulated ABA to a higher extent than young leaves. Accumulation of ABA in leaves of ammonium-fed plants and plants grown under low nitrogen supply occurred in the absence of both increased ABA xylem loading rate and enhanced aldehyde oxidase (AO, EC 1.2.3.1) activity in roots. Enhanced leaf AO activity was observed in all treatments, with the highest increase in old leaves. Among the three AO isoforms (AO-1, AO-2 and AO-3) detected in extracts of pea leaves, the lowest one AO-3 (highest mobility in the gel) correlated with ABA production and showed the highest increment in response to the treatments. The increase of AO activity detected in leaves after 2 weeks of stress application was less prominent than after 9 d, suggesting a transient enhancement of ABA production following the onset of stress. An increase of ABA xylem loading rate as well as AO root activity 4 d and 9 d after application of the treatments was observed only in salt-treated plants followed by a decrease after 14 d in 100 mM NaCl. Decreased cytokinin (trans-zeatin riboside) delivery rate into the xylem sap was observed in all treatments. The role of abscisic acid and cytokinins as positive and negative growth signals, as well as the involvement of root-generated ABA on ABA accumulation in leaves is discussed.     

Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor

Metabolism of abscisic acid (ABA) was studied after wilting and upon recovery from water stress in individual, detached leaves of Phaseolus vulgaris L. (red kidney bean). Loss of turgor was correlated with accumulation of ABA and its metabolites, resulting in a 10-fold increase in the level of phaseic acid (PA) and a doubling of the level of conjugated ABA. The level of conjugated ABA in turgid leaves was no higher than that of the free acid. These results indicate that accumulation of ABA in wilted leaves resulted from a stimulation of ABA synthesis, rather than from a release from a conjugated form or from inhibition of the metabolism of ABA. The rate of synthesis of ABA was at its maximum between 2.5 and 5 h after turgor was lost, and slackened there-after. In wilted leaves, the rate of conversion of ABA to PA climbed steadly until it matched the rate of synthesis, after about 7.5 h. Upon rehydration of sections from wilted leaves, the rate of synthesis of ABA dropped close to zero within about 3 h, while the rate of conversion to PA accelerated. Formation of PA was two to four times faster than in sections maintained in the wilted condition; it reached a rate sufficient to convert almost one-half of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. The role of turgor in the stimulation of the conversion of ABA to PA was investigated. When leaves that had been wilted for 5 h were rehydrated to different degrees, the amount of ABA which disappeared, or that of PA which accumulated during the next 3 h, did not depend linearly on the water potential of the rehydrated leaf. Rather, re-establishment of the slightest positive turgor was sufficient to result in maximum stimulation of conversion of ABA to PA.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - PA phaseic acid - _leaf leaf water potential - osmotic pressure