Two cysteine proteinase inhibitors (cystatins) from Arabidopsis thaliana, designated AtCYSa and AtCYSb, were characterized. Recombinant GST-AtCYSa and GST-AtCYSb were expressed in Escherichia coli and purified. They inhibit the catalytic activity of papain, which is generally taken as evidence for cysteine proteinase inhibitor function. Northern blot analyses showed that the expressions of AtCYSa and AtCYSb gene in Arabidopsis cells and seedlings were strongly induced by multiple abiotic stresses from high salt, drought, oxidant, and cold. Interestingly, the promoter region of AtCYSa gene contains a dehydration-responsive element (DRE) and an abscisic acid (ABA)-responsive element (ABRE), which identifies it as a DREB1A and AREB target gene. Under normal conditions, AtCYSa was expressed in 35S: DREB1A and 35S: AREB1 plants at a higher level than in WT plants, while AtCYSa gene was expressed in 35S: DREB2A plants at the same level as in WT plants. Under stress conditions (salt, drought and cold), AtCYSa was expressed more in all three transgenic plants than in WT plants. Over-expression of AtCYSa and AtCYSb in transgenic yeast and Arabidopsis plants increased the resistance to high salt, drought, oxidative, and cold stresses. Taken together, these data raise the possibility of using AtCYSa and AtCYSb to genetically improve environmental stresses tolerance in plants.
Abscisic acid is a stress-related phytohormone that has roles in dehydration and rehydration. In Arabidopsis (Arabidopsis thaliana), two genes that inactivate abscisic acid, CYP707A1 and CYP707A3, are rapidly up-regulated upon rehydration. The factors that regulate CYP707A1/3 are not well characterized. We expressed a bZIP protein, VIP1, as a green fluorescent protein fusion protein in Arabidopsis and found that the nuclear localization of VIP1 was enhanced within 10 min after rehydration. A yeast one-hybrid assay revealed that the amino-terminal region of VIP1 has transcriptional activation potential. In a transient reporter assay using Arabidopsis protoplasts, VIP1 enhanced the promoter activities of CYP707A1/3. In gel shift and chromatin immunoprecipitation analyses, VIP1 directly bound to DNA fragments of the CYP707A1/3 promoters. Transgenic plants expressing VIP1-green fluorescent protein were found to overexpress CYP707A1/3 mRNAs. The time course of nuclearcytoplasmic shuttling of VIP1 was consistent with the time courses of the expression of CYP707A1/3. These results suggest that VIP1 functions as a regulator of osmosensory signaling in Arabidopsis.
VIP1 is a bZIP protein in Arabidopsis thaliana. VIP1 accumulates in the nucleus under hypo-osmotic conditions and interacts with the promoters of hypo-osmolarity-responsive genes, CYP707A1 and CYP707A3 (CYP707A1/3), but neither overexpression of VIP1 nor truncation of its DNA-binding region affects the expression of CYP707A3 in vivo, raising the possibility that VIP and other proteins are functionally redundant. Here we show further analyses on VIP1 and its close homologs, namely, Arabidopsis group I bZIP proteins. The patterns of the signals of the GFP-fused group I bZIP proteins were similar in onion and Arabidopsis cells, suggesting that they have similar subcellular localization. In a yeast one-hybrid assay, the group I bZIP proteins caused reporter gene activation in the yeast reporter strain. VIP1 and other group I bZIP proteins showed positive results in a yeast two-hybrid assay and a bimolecular fluorescence complementation assay, suggesting that they physically interact. These results support the idea that they have somewhat similar functions. By gel shift assays, VIP1-binding sequences in the CYP707A1/3 promoters were confirmed to be AGCTGT/G. Their presence in the promoters of the genes that respond to hypo-osmotic conditions was evaluated using previously published microarray data. Interestingly, a significantly higher proportion of the promoters of the genes that were up-regulated by rehydration treatment and/or submergence treatment (treatment by a hypotonic solution) and a significantly lower proportion of the promoters of the genes that were down-regulated by such treatment shared AGCTGT/G. To further assess the physiological role of VIP1, constitutively nuclear-localized variants of VIP1 were generated. When overexpressed in Arabidopsis, some of them as well as VIP1 caused growth retardation under a mannitol-stressed condition, where VIP1 is localized mainly in the cytoplasm. This raises the possibility that the expression of VIP1 itself rather than its nuclear localization is responsible for regulating the mannitol responses.
In order to determine the different roles of rice (Oryza sativa L.) cytosolic ascorbate peroxidases (OsAPXa and OsAPXb, GenBank accession nos. D45423 and AB053297, respectively) under salt stress, transgenic Arabidopsis plants over-expressing OsAPXa or OsAPXb were generated, and they all exhibited increased tolerance to salt stress compared to wild-type plants. Moreover, transgenic lines over-expressing OsAPXb showed higher salt tolerance than OsAPXa transgenic lines as indicated by root length and total chlorophyll content. In addition to ascorbate peroxidase (APX) activity, antioxidant enzyme activities of catalase (CAT), superoxide dismutase (SOD) and glutathione reductase (GR), which are also involved in the salt tolerance process, and the content of H2O2 were also assayed in both transgenic and wild-type plants. The results showed that the overproduction of OsAPXb enhanced and maintained APX activity to a much higher degree than OsAPXa in transgenic Arabidopsis during treatment with different concentrations of NaCl, enhanced the active oxygen scavenging system, and protected plants from salt stress by equilibrating H2O2 metabolism. Our findings suggest that the rice cytosolic OsAPXb gene has a more functional role than OsAPXa in the improvement of salt tolerance in transgenic plants.
A high-affinity K+ transporter PutHKT2;1 cDNA was isolated from the salt-tolerant plant Puccinellia tenuiflora. Expression of PutHKT2;1 was induced by both 300 mM NaCl and K+-starvation stress in roots, but only slightly regulated by those stresses in shoots. PutHKT2;1 transcript levels in 300 mM NaCl were doubled by the depletion of potassium. Yeast transformed with PutHKT2;1, like those transformed with PhaHKT2;1 from salt-tolerant reed plants (Phragmites australis), (i) were able to take up K+ in low K+ concentration medium or in the presence of NaCl, and (ii) were permeable to Na+. This suggests that PutHKT2;1 has a high affinity K+-Na+ symport function in yeast. Arabidopsis over-expressing PutHKT2;1 showed increased sensitivities to Na+, K+, and Li+, while Arabidopsis over-expressing OsHKT2;1 from rice (Oryza sativa) showed increased sensitivity only to Na+. In contrast to OsHKT2;1, which functions in Na+-uptake at low external K+ concentrations, PutHKT2;1 functions in Na+-uptake at higher external K+ concentrations. These results show that the modes of action of PutHKT2;1 in transgenic yeast and Arabidopsis differ from the mode of action of the closely related OsHKT2;1 transporter.
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