A tomato cDNA inducible by salt stress and abscisic acid: nucleotide sequence and expression pattern

We have characterized a new tomato cDNA, TAS14, inducible by salt stress and abscisic acid (ABA). Its nucleotide sequence predicts an open reading frame coding for a highly hydrophilic and glycine-rich (23.8%) protein of 130 amino acids. Southern blot analysis of tomato DNA suggests that there is one TAS14 structural gene per haploid genome. TAS14 mRNA accumulates in tomato seedlings upon treatment with NaCl, ABA or mannitol. It is also induced in roots, stems and leaves of hydroponically grown tomato plants treated with NaCl or ABA. TAS14 mRNA is not induced by other stress conditions such as cold and wounding. The sequence of the predicted TAS14 protein shows four structural domains similar to the rice RAB21, cotton LEA D11 and barley and maize dehydrin genes.     

Lanthanide ions are agonists of transient gene expression in rice protoplasts and act in synergy with ABA to increase Em gene expression

Previous work has shown that in rice suspension cells, NaCl at 0.4 M can induce Em gene expression and act synergistically with ABA, possibly by potentiating the ABA response pathway through a rate-limiting intermediate (R.M. Bostock and R.S. Quatrano (1992) Plant Physiol., 98, 1356–1363). Since calcium is an intermediate in ABA regulation of stomatal closure, we tested the effect of calcium changes on ABA-inducible Em gene expression in transiently transformed rice protoplasts. We show that calcium is required for ABA-inducible Em-GUS expression and can act in synergy with ABA. The trivalent ions lanthanum, gadolinium, and aluminum, which are known to interact with calcium- and other signaling pathways, can act at sub-millimolar concentrations to increase GUS reporter gene expression driven by several promoters in transiently transformed rice protoplasts. This effect is not specific for the ABA-inducible Em promoter, but is synergistic with ABA. The lanthanum synergy with ABA does not require calcium. In rice suspension cells, lanthanum alone does not induce Em gene expression, in contrast to transiently transformed protoplasts, yet can act synergistically with ABA to effectively increase the sensitivity to ABA greater than tenfold. Trivalent ions may be a useful tool to study the regulation of gene expression. The possible effects of trivalent ions on ABA signal transduction and gene expression are discussed.     

Expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl

Reactive oxygen species are thought to play an important role in NaCl stress. Therefore, the expression patterns of the gene family encoding the H(2)O(2)-scavenging enzyme ascorbate peroxidase (APx; EC1.11.1.11) were analysed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Applying semi-quantitative RT-PCR, the mRNA levels were quantified for two cytosolic (OsAPx1 and OsAPx2), two peroxisomal (OsAPx3 and OsAPx4), and four chloroplastic (OsAPx5, OsAPx6, OsAPx7, and OsAPx8) isoforms identified in the rice genome. NaCl at 150 mM and 200 mM increased the expression of OsAPx8 and the activities of APx, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, OsAPx6, and OsAPx7 in rice roots. However, NaCl at 300 mM up-regulated OsAPx8 expression, increased APx activity, and down-regulated OsAPx7 expression, but had no effect on the expression of OsAPx1, OsAPx2, OsAPx3, OsAPx4, OsAPx5, and OsAPx6. The accumulation of abscisic acid (ABA) in response to NaCl was observed in rice roots. Exogenously applied ABA also specifically enhanced the expression of OsAPx8 in rice roots. The accumulation of ABA in rice roots in response to NaCl was inhibited by fluridone (Flu), an inhibitor of carotenoid biosynthesis. Flu treatment also suppressed NaCl-enhanced OsAPx8 expression and APx activity. The effect of Flu on the expression of OsAPx8 and increase in APx activity was reversed by the application of ABA. It appears that NaCl-enhanced expression of OsAPx8 in rice roots is mediated through an accumulation of ABA. Evidence is provided to show that Na(+) but not Cl(-) is required for enhancing OsAPx8 expression, APx activity, and ABA accumulation in rice roots treated with NaCl. H(2)O(2) treatment resulted in an enhancement of OsAPx8 induction but no accumulation of ABA. Diphenylene iodonium treatment, which is known to inhibit NaCl-induced accumulation of H(2)O(2) in rice roots, did not suppress OsAPx8 induction and ABA accumulation by NaCl. It appears that H(2)O(2) is not involved in the regulation of NaCl-induced OsAPx8 expression in rice roots.     

Expression of a salt-induced protein (SALT) in suspension-cultured cells and leaves of rice following exposure to fungal elicitor and phytohormones

Phytohormones are essential signal compounds in the regulation of stress-related and defense-related genes. However, there is no clear evidence for any effect of these signal molecules and biotic elicitors on the regulation of the SALT gene in suspension-cultured rice cells. We characterized the expression of a SALT gene following treatment with fungal elicitor, phytohormones, cycloheximide, and inhibitors of protein kinase/phosphatases. SALT expression was up-regulated following treatment with a fungal elicitor, jasmonic acid (JA), abscisic acid (ABA), and NaCl. However, salicylic acid (SA) alone or in combination with one of the other elicitors not only strongly inhibited SALT gene expression but also exhibited an antagonistic effect in suspension cells and leaves. Cycloheximide inhibited SALT accumulation in suspension cells and in leaves, but the inhibitors of protein kinase/phosphatase did not. Immunolocalization revealed that SALT protein was present in xylem parenchyma cells of vascular bundles in the major and minor leaf veins.     

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.     

Gene cloning and expression of cytosolic glutathione reductase in rice (Oryza sativa L.)

We have isolated a cDNA (RGRC2) encoding glutathione reductase (GR) from rice (Oryza sativa L.). The comparison of deduced amino acid sequences from RGRC2 and other plant GR cDNAs indicated that RGRC2 encodes a putative cytosolic isoform. The recombinant RGRC2 protein had enzymatic properties comparable to those of GR from rice embryo. Subcellular fractionation showed that the RGRC2 protein is localized primarily in cytosol. mRNA and protein of RGRC2 were observed mainly in roots and calli but little in leaf tissues. Southern blot analysis showed that the RGRC2 gene exists as a single copy gene. Here, we have also isolated a genomic clone completely corresponding to RGRC2. The RGRC2 gene is split into 16 exons spread about 7.4 kb of chromosomal DNA, with coding sequence beginning in the 2nd exon and ending in the 16th exon. From the presence of two ABA-responsive elements in the 5'-flanking region of RGRC2, we examined the expression in rice seedlings treated with ABA and the ABA-related environmental stresses, chilling, drought and salinity. The expression of RGRC2 was strongly induced by all these treatments. We suggest that the expression of the rice cytosolic GR gene is regulated via ABA-mediated signal transduction pathway under environmental stresses.     

Induction of RAB18 gene expression and activation of K+ outward rectifying channels depend on an extracellular perception of ABA in Arabidopsis thaliana suspension cells

Important progress has been made regarding the characterization of the ABA signalling components using genetic and molecular approaches (Leung and Giraudat, 1998). However, we do not yet know the mechanism of ABA perception. Conflicting results concerning the site of ABA perception have been published. The prevailing view is that since ABA controls many responses, different sites of perception for ABA might exist. In order to establish the cellular localisation of the ABA receptors in Arabidopsis thaliana suspension cells, we developed two physiological tests based upon the capacity of impermeant ABA-BSA conjugate to mimic permeant free ABA effects. We show that purified ABA-BSA conjugate is able to trigger RAB18 gene expression and that this response is strictly due to the natural (+)-ABA enantiomer. The rate of RAB18 gene expression was independent of the level of ABA uptake by the cells. Using the voltage-clamp technique we show that ABA-BSA, similarly to ABA, evokes a membrane depolarization and activates time- and voltage-dependent outward rectifying currents (ORC). We demonstrate that these ORC are due to a K+ efflux as assessed by tail currents and specific inhibition by both tetraethylammonium (TEA) and Ba2+. These observations provide evidence in favour of an extracellular site for ABA perception.     

Diacylglycerol pyrophosphate is a second messenger of abscisic acid signaling in Arabidopsis thaliana suspension cells

In plants, the importance of phospholipid signaling in responses to environmental stresses is becoming well documented. The involvement of phospholipids in abscisic acid (ABA) responses is also established. In a previous study, we demonstrated that the stimulation of phospholipase D (PLD) activity and plasma membrane anion currents by ABA were both required for RAB18 expression in Arabidopsis thaliana suspension cells. In this study, we show that the total lipids extracted from ABA-treated cells mimic ABA in activating plasmalemma anion currents and induction of RAB18 expression. Moreover, ABA evokes within 5 min a transient 1.7-fold increase in phosphatidic acid (PA) followed by a sevenfold increase in diacylglycerol pyrophosphate (DGPP) at 20 min. PA activated plasmalemma anion currents but was incapable of triggering RAB18 expression. By contrast, DGPP mimicked ABA on anion currents and was also able to stimulate RAB18 expression. Here we show the role of DGPP as phospholipid second messenger in ABA signaling.     

NaCl-induced expression of glutathione reductase in roots of rice (Oryza sativa L.) seedlings is mediated through hydrogen peroxide but not abscisic acid

Reactive oxygen species (ROS) play an important role in NaCl stress. Plants tolerant to NaCl stress may evolve certain strategies to remove these ROS, thus reducing their toxic effects. Therefore, the expression patterns of the gene family encoding glutathione reductase (GR, EC 1.6.4.2) were analyzed in roots of etiolated rice (Oryza sativa L.) seedlings in response to NaCl stress. Semi-quantitative RT-PCR was applied to quantify the mRNA levels for one cytosolic (OsGR2) and two chloroplastic (OsGR1 and OsGR3) isoforms of glutathione reductase identified in the rice genome. The expression of OsGR2 and OsGR3 but not OsGR1 was increased in rice roots treated with 150 mM NaCl. The Rab16A is an abscisic acid (ABA)-responsive rice gene. Increasing concentrations of ABA, from 1 to 12 μM, progressively increased the expression of OsRab16A in rice roots. In the present study, the ABA level was judged by the expression of OsRab16A in rice roots. Treatment with 150 mM NaCl induced the expression of OsRab16A, and the expression increased with increasing concentrations of ABA, which suggests that ABA may be involved in this response in rice roots. In fact, exogenous application of ABA enhanced the expression of OsGR2 and OsGR3 in rice roots. On inhibiting ABA accumulation with sodium tungstate (Tu), an inhibitor of ABA biosynthesis, the expression of OsGR2 and OsGR3 was still induced by NaCl; therefore, NaCl-triggered expression of OsGR2 and OsGR3 in rice roots is not mediated by accumulation of ABA. However, NaCl treatment could induce H₂O₂ production in rice roots, and H₂O₂ treatment resulted in enhanced OsGR2 and OsGR3 induction. On inhibiting the NaCl-induced accumulation of H₂O₂ with diphenylene iodonium, the expression of OsGR2 and OsGR3 was also suppressed. Moreover, the increase in H₂O₂ level was prior to the induction of OsGR2 and OsGR3 in NaCl-treated rice roots. Thus, H₂O₂, but not ABA, is involved in regulation of OsGR2 and OsGR3 expression in NaCl-treated rice roots.     

Okadaic acid, a protein phosphatase inhibitor, blocks calcium changes, gene expression, and cell death induced by gibberellin in wheat aleurone cells.

The cereal aleurone functions during germination by secreting hydrolases, mainly alpha-amylase, into the starchy endosperm. Multiple signal transduction pathways exist in cereal aleurone cells that enable them to modulate hydrolase production in response to both hormonal and environmental stimuli. Gibberellic acid (GA) promotes hydrolase production, whereas abscisic acid (ABA), hypoxia, and osmotic stress reduce amylase production. In an effort to identify the components of transduction pathways in aleurone cells, we have investigated the effect of okadaic acid (OA), a protein phosphatase inhibitor, on stimulus-response coupling for GA, ABA, and hypoxia. We found that OA (100 nM) completely inhibited all the GA responses that we measured, from rapid changes in cytosolic Ca2+ through changes in gene expression and accelerated cell death. OA (100 nM) partially inhibited ABA responses, as measured by changes in the level of PHAV1, a cDNA for an ABA-induced mRNA in barley. In contrast, OA had no effect on the response to hypoxia, as measured by changes in cytosolic Ca2+ and by changes in enzyme activity and RNA levels of alcohol dehydrogenase. Our data indicate that OA-sensitive protein phosphatases act early in the transduction pathway of GA but are not involved in the response to hypoxia. These data provide a basis for a model of multiple transduction pathways in which the level of cytosolic Ca2+ is a key point of convergence controlling changes in stimulus-response coupling.     

Gene expression associated with water-stress adaptation of rice cells and identification of two genes as hsp 70 and ubiquitin

We have isolated polyethylene glycol (PEG)-adapted rice cells that can proliferate in a medium with 20% PEG as well as in a medium with 1% NaCl. The adapted-cells overproduce a set of proteins whose roles may be associated with water-stress adaptation. To isolate genes encoding these proteins, we differentially screened a cDNA library and obtained 5 cDNA clones which showed preferential hybridization to mRNA of adapted cells. The present paper describes the pattern of expression and the sequence analysis of these 5 genes. Sequence analysis of partial cDNAs indicates that two genes encode the 70 kDa heat shock protein and the ubiquitin but the identities of the other 3 are not known. The expression of all 5 genes fluctuates slightly during the growth cycle of the PEG-adapted cells grown in the control or in the PEG-containing medium. In contrast, gene expression in the parental cells fluctuates to a much greater extent and always begins with an enhanced expression during the first day after subculture. Sub-lethal concentrations of PEG or NaCl have no immediate effect on gene expression in parental cells but NaCl may have a long term effect in enhancing the expression of two genes after 5 days. Abscisic acid (ABA) has no effect on the expression of 4 genes but suppresses the expression of one gene. The roles of these genes in water-stress adaptation of plant cells are discussed.     

Trivalent ions activate abscisic acid-inducible promoters through an ABI1-dependent pathway in rice protoplasts

The plant hormone abscisic acid (ABA) mediates many vital processes in plant growth and development, including seed dormancy, cell division, water use efficiency, and adaptation to drought, salinity, chilling, pathogen attack, and UV light. Our understanding of ABA signal transduction is fragmentary and would benefit from specific and facile probes of the process. Protoplasts from rice (Oryza sativa L. cv IR54) embryonic suspension cultures cotransformed with effector plasmids encoding the maize (Zea mays) VIVIPAROUS1 cDNA and/or the Arabidopsis dominant negative mutant (abi1-1) ABA-insensitive cDNA demonstrated genetic interactions of VIVIPAROUS1 and abi1-1 in transactivation of the ABA-inducible HVA1 promoter from barley (Hordeum vulgare), suggesting the mechanisms of these effectors are conserved among monocots and dicots. Trivalent ions have been shown to act as an effector of gene expression in plants and animals, although the mechanism of action is unknown. We show in two complementary transient ABA-inducible gene expression assays (beta-glucuronidase and luciferase enzymatic activities and quantitative flow cytometry of green fluorescent protein) that trivalent ions specifically interact with an ABI1-dependent ABA-signaling pathway leading to gene expression. Trivalent ions mimic ABA effects on gene expression and may be a useful tool to study ABA signaling.