Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes.     

Arabidopsis mutants deregulated in RCI2A expression reveal new signaling pathways in abiotic stress responses

To uncover new pathways involved in low-temperature signal transduction, we screened for mutants altered in cold-induced expression of RCI2A, an Arabidopsis gene that is not a member of the CBF/DREB1 regulon and is induced not only by low temperature but also by abscisic acid (ABA), dehydration (DH) and NaCl. This was accomplished by generating a line of Arabidopsis carrying a transgene consisting of the RCI2A promoter fused to the firefly luciferase coding sequence. A number of mutants showing low or high RCI2A expression in response to low temperature were identified. These mutants also displayed deregulated RCI2A expression in response to ABA, DH or NaCl. Interestingly, however, they were not altered in stress-induced expression of RD29A, a CBF/DREB1-target gene, suggesting that the mutations affect signaling intermediates of CBF/DREB1-independent regulatory pathways. Several mutants showed alterations in their tolerance to freezing, DH or salt stress, as well as in their ABA sensitivity, which indicates that the signaling intermediates defined by the corresponding mutations play an important role in Arabidopsis tolerance to abiotic stresses. Based on the mutants identified, we discuss the involvement of CBF/DREB1-independent pathways in modulating stress signaling.     

Characterization of a Novel Plantain Asr Gene, MpAsr,that is Regulated in Response to Infection of Fusarium oxysporum f.sp. cubense and Abiotic Stresses

Asr (abscisic acid, stress, ripening induced) genes are typically upregulated by a wide range of factors, including drought, cold, salt, abscisic acid (ABA) and injury; in addition to plant responses to developmental and environmental signals. We isolated an Asr gene, MpAsr, from a suppression subtractive hybridization (SSH) cDNA library of cold induced plantain (Musa paradisiaca) leaves. MpAsr expression was upregulated in Fusarium oxysporum f. sp. cubense infected plantain leaves, peels and roots, suggesting that MpAsr plays a role in plantain pathogen response. In addition, a 581-bp putative promoter region of MpAsr was isolated via genome walking and cis-elements involved in abiotic stress and pathogen-related responses were detected in this same region. Furthermore, the MpAsr promoter demonstrated positive activity and inducibility in tobacco under F. oxysporum f. sp. cubense infection and ABA, cold, dehydration and high salt concentration treatments. Interestingly, transgenic Arabidopsis plants overexpressing MpAsr exhibited higher drought tolerance, but showed no significant decreased sensitivity to F. oxysporum f. sp. cubense. These results suggest that MpAsr might be involved in plant responses to both abiotic stress and pathogen attack.     

The phosphatidylcholine-hydrolysing phospholipase C NPC4 plays a role in response of Arabidopsis roots to salt stress

Phosphatidylcholine-hydrolysing phospholipase C, also known as non-specific phospholipase C (NPC), is a new member of the plant phospholipase family that reacts to environmental stresses such as phosphate deficiency and aluminium toxicity, and has a role in root development and brassinolide signalling. Expression of NPC4, one of the six NPC genes in Arabidopsis, was highly induced by NaCl. Maximum expression was observed from 3?h to 6?h after the salt treatment and was dependent on salt concentration. Results of histochemical analysis of P(NPC4):GUS plants showed the localization of salt-induced expression in root tips. On the biochemical level, increased NPC enzyme activity, indicated by accumulation of diacylglycerol, was observed as early as after 30?min of salt treatment of Arabidopsis seedlings. Phenotype analysis of NPC4 knockout plants showed increased sensitivity to salinity as compared with wild-type plants. Under salt stress npc4 plants had shorter roots, lower fresh weight, and reduced seed germination. Expression levels of abscisic acid-related genes ABI1, ABI2, RAB18, PP2CA, and SOT12 were substantially reduced in salt-treated npc4 plants. These observations demonstrate a role for NPC4 in the response of Arabidopsis to salt stress.     

Phylogenetic and functional analysis of Arabidopsis RCI2 genes

Six new Arabidopsis thaliana genes (AtRCI2C-H) have been identified that show high homology to AtRCI2A and AtRCI2B. Sequence comparisons revealed that AtRCI2-related genes are widely spread among very different organisms, including other plant species, prokaryotes, fungi, and simply organized animals, and are also organized in gene families. Most RCI2 genes show a similar exon-intron organization, which indicates that they have been structurally conserved during evolution, and encode small, highly hydrophobic proteins containing two putative transmembrane domains. Consistently, the majority of AtRCI2 proteins localize in the plasma membrane. RCI2 proteins exhibit an elevated level of sequence similarity and seem to have evolved from a common ancestor. In spite of their high similarity, conserved subcellular localization, and common origin, experimental evidence is presented suggesting that different RCI2 proteins may have distinct functional roles. Thus, as previously demonstrated for AtRCI2A and AtRCI2B, the newly identified AtRCI2 genes (AtRCI2C-H) are differentially regulated in Arabidopsis organs and in response to abiotic stresses and ABA treatment. Furthermore, only the AtRCI2 proteins that do not contain the C-terminal hydrophilic tail (i.e. AtRCI2A-C and AtRCI2H) are able to complement for the loss of the yeast AtRCI2-related gene PMP3. On the basis of these results, different aspects on the evolution and roles of RCI2 genes are discussed.     

Three zinc‐finger RNA‐binding proteins in cabbage (Brassica rapa) play diverse roles in seed germination and plant growth under normal and abiotic stress conditions

Despite the increasing understanding of the stress‐responsive roles of zinc‐finger RNA‐binding proteins (RZs) in several plant species, such as Arabidopsis thaliana, wheat (Triticum aestivum) and rice (Oryza sativa), the functions of RZs in cabbage (Brassica rapa) have not yet been elucidated. In this study, the functional roles of the three RZ family members present in the cabbage genome, designated as BrRZ1, BrRZ2 and BrRZ3, were investigated in transgenic Arabidopsis under normal and environmental stress conditions. Subcellular localization analysis revealed that all BrRZ proteins were exclusively localized in the nucleus. The expression levels of each BrRZ were markedly increased by cold, drought or salt stress and by abscisic acid (ABA) treatment. Expression of BrRZ3 in Arabidopsis retarded seed germination and stem growth and reduced seed yield of Arabidopsis plants under normal growth conditions. Germination of BrRZ2‐ or BrRZ3‐expressing Arabidopsis seeds was delayed compared with that of wild‐type seeds under dehydration or salt stress conditions and cold stress conditions, respectively. Seedling growth of BrRZ3‐expressing transgenic Arabidopsis plants was significantly inhibited under salt, dehydration or cold stress conditions. Notably, seedling growth of all three BrRZ‐expressing transgenic Arabidopsis plants was inhibited upon ABA treatment. Importantly, all BrRZs possessed RNA chaperone activity. Taken together, these results indicate that the three cabbage BrRZs harboring RNA chaperone activity play diverse roles in seed germination and seedling growth of plants under abiotic stress conditions as well as in the presence of ABA.     

A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress.

Two genes, rd29A and rd29B, which are closely located on the Arabidopsis genome, are differentially induced under conditions of dehydration, low temperature, high salt, or treatment with exogenous abscisic acid (ABA). It appears that rd29A has at least two cis-acting elements, one involved in the ABA-associated response to dehydration and the other induced by changes in osmotic potential, and that rd29B contains at least one cis-acting element that is involved in ABA-responsive, slow induction. We analyzed the rd29A promoter in both transgenic Arabidopsis and tobacco and identified a novel cis-acting, dehydration-responsive element (DRE) containing 9 bp, TACCGACAT, that is involved in the first rapid response of rd29A to conditions of dehydration or high salt. DRE is also involved in the induction by low temperature but does not function in the ABA-responsive, slow expression of rd29A. Nuclear proteins that specifically bind to DRE were detected in Arabidopsis plants under either high-salt or normal conditions. Different cis-acting elements seem to function in the two-step induction of rd29A and in the slow induction of rd29B under conditions of dehydration, high salt, or low temperature.     

Overexpression of RCI2A decreases Na+ uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants

We have investigated whether the overexpression of RCI2A gene causes an enhanced salt-tolerant phenotype in Arabidopsis thaliana . Although the growth of RCI2A -overexpressing transgenic plants was comparable with that of wild type under normal conditions, high salinity treatment caused decreased accumulation of Na⁺ and ameliorated suppression of the shoot growth of transgenic plants than that of wild type. Under high salinity treatment, the chlorophyll content of the shoots of wild-type plants significantly decreased compared with transgenic plants. The increases of malondialdehyde (MDA) and of H₂O₂ production caused by high salinity were greater in the shoots of wild type than in that of transgenic plants. These results suggest that overexpression of RCI2A can alleviate salinity-induced growth suppression and photooxidative damages via reducing Na⁺ uptake into the shoots.