A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana

A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress.     

Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression

In plants, a cis-acting element, DRE/CRT, is involved in ABA-independent gene expression in response to dehydration and low-temperature stress. To understand signal transduction pathways from perception of the dehydration stress signal to gene expression, we characterized a gene family for DRE/CRT-binding proteins DREB2A and DREB2B in Arabidopsis thaliana. Northern analysis showed that both genes are induced by dehydration and high-salt stress. Organ-specific northern analysis with gene-specific probes showed that these genes are strongly induced in roots by high-salt stress and in stems and roots by dehydration stress. The DREB2A gene is located on chromosome 5, and DREB2B on chromosome 3. We screened an Arabidopsis genomic DNA library with cDNA fragments of DREB2A and DREB2B as probes, and isolated DNA fragments that contained 5-flanking regions of these genes. Sequence analysis showed that both genes are interrupted by a single intron at identical positions in their leader sequence. Several conserved sequences were found in the promoter regions of both genes. The -glucuronidase (GUS) reporter gene driven by the DREB2 promoters was induced by dehydration and high-salt stress in transgenic Arabidopsis plants.     

Mercury-induced genes in Arabidopsis thaliana: identification of induced genes upon long-term mercuric ion exposure

Mercuric‐ion‐induced gene expression was studied in Arabidopsis thaliana Columbia wild type. Rosettes of plants grown for 21 d on agar medium supplemented with 20, 30 and 40 µm HgCl₂ were pooled and used to isolate cDNAs of induced genes by suppression subtractive hybridization. Of the 576 clones isolated initially, 31 turned out to be mercury‐induced by Northern hybridization. However, kinetic studies using cDNA arrays clearly showed that seven genes were exclusively mercuric‐ion‐induced, 14 were induced by mercury but also affected by a diurnal rhythm, and 10 clones were only modulated by the day–night cycle. The expression levels of the metal‐induced genes increased from 1·5‐fold to 10‐fold. Functional classification resulted in genes encoding proteins for the photosynthetic apparatus and for the antioxidative system. In addition, unexpected genes, whose connection to mercury ion stress is not evident, were identified.     

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1‐related protein kinase 2.10 in response to osmotic stress

SNF1‐related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA‐dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA‐non‐activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S‐segment is involved in the regulation of dehydrin subcellular localization in response to stress.     

Origin and evolution of genes related to ABA metabolism and its signaling pathways

Since plants cannot move to avoid stress, they have sophisticated acclimation mechanisms against a variety of abiotic stresses. The phytohormone abscisic acid (ABA) plays essential roles in abiotic stress tolerances in land plants. Therefore, it is interesting to address the evolutionary origins of ABA metabolism and its signaling pathways in land plants. Here, we focused on 48 ABA-related Arabidopsis thaliana genes with 11 protein functions, and generated 11 orthologous clusters of ABA-related genes from A. thaliana, Arabidopsis lyrata, Populus trichocarpa, Oryza sativa, Selaginella moellendorffii, and Physcomitrella patens. Phylogenetic analyses suggested that the common ancestor of these six species possessed most of the key protein functions of ABA-related genes. In two species (A. thaliana and O. sativa), duplicate genes related to ABA signaling pathways contribute to the expression variation in different organs or stress responses. In particular, there is significant expansion of gene families related to ABA in evolutionary periods associated with morphological divergence. Taken together, these results suggest that expansion of the gene families related to ABA signaling pathways may have contributed to the sophisticated stress tolerance mechanisms of higher land plants.     

Knockout of AtDjB1, a J‐domain protein from Arabidopsis thaliana,alters plant responses to osmotic stress and abscisic acid

AtDjB1 is a member of the Arabidopsis thaliana J‐protein family. AtDjB1 is targeted to the mitochondria and plays a crucial role in A. thaliana heat and oxidative stress resistance. Herein, the role of AtDjB1 in adapting to saline and drought stress was studied in A. thaliana. AtDjB1 expression was induced through salinity, dehydration and abscisic acid (ABA) in young seedlings. Reverse genetic analyses indicate that AtDjB1 is a negative regulator in plant osmotic stress tolerance. Further, AtDjB1 knockout mutant plants (atj1‐1) exhibited greater ABA sensitivity compared with the wild‐type (WT) plants and the mutant lines with a rescued AtDjB1 gene. AtDjB1 gene knockout also altered the expression of several ABA‐responsive genes, which suggests that AtDjB1 is involved in osmotic stress tolerance through its effects on ABA signaling pathways. Moreover, atj1‐1 plants exhibited higher glucose levels and greater glucose sensitivity in the post‐germination development stage. Applying glucose promoted an ABA response in seedlings, and the promotion was more evident in atj1‐1 than WT seedlings. Taken together, higher glucose levels in atj1‐1 plants are likely responsible for the greater ABA sensitivity and increased osmotic stress tolerance.     

厚藤脱水素基因IpDHN启动子IpDHN-Pro的克隆和调控转录活性分析

为了解厚藤(Ipomoea pes-caprae)脱水素基因IpDHN (GenBank登录号:KX426069)启动子的转录活性和对非生物胁迫和植物激素ABA的响应,通过染色体步移法克隆了IpDHN的上游启动子序列IpDHN-Pro,长度为974 bp。构建IpDHN-Pro调控下GUS转基因载体,转化拟南芥(Arabidopsis thaliana)植株获得IpDHN-Pro::GUS转基因植株并进行GUS染色,验证IpDHN-Pro启动转录活性以及在氯化钠、甘露醇、ABA处理后拟南芥GUS基因表达变化。结果表明,扩增获得的IpDHN-Pro序列包含多个顺式作用元件,包括1个ABRE、3个Myb转录因子结合位点、富含TC的重复序列以及Skn-1基序等。转基因拟南芥GUS染色及qRT-PCR表明该序列可驱动GUS基因在拟南芥稳定表达,且表达受高盐、渗透压及ABA的诱导。这表明IpDHN-Pro是一个盐旱、ABA诱导的启动子序列,可应用于相关的植物抗逆遗传工程研究。     

Two amidophosphoribosyltransferase genes of Arabidopsis thaliana expressed in different organs

Amidophosphoribosyltransferase (ATase: EC 2.4.2.14) is a key enzyme in the pathway of purine nucleotide biosynthesis. We have identified several cDNA clones whose amino acid sequences exhibit similarity with the known ATases in a cDNA library of young floral buds of Arabidopsis thaliana. The cDNA clones are derived from two genes homologous with each other. These cDNAs represent the first plant representatives of ATase gene. Structural comparison with ATases of other organisms has revealed that the two genes encode [4Fe-4S] cluster-dependent ATases. Northern blot analysis showed that expression level of the genes is different in three organs; one gene is expressed in flowers and roots, while the other gene is mainly expressed in leaves.     

蒙古沙冬青AmRD22基因的克隆及耐逆功能研究

脱水应答蛋白22(RD22)属于植物特有的BURP蛋白家族中的一个亚族,与耐逆性关系密切。该研究从中国西北荒漠区特有的强耐逆植物蒙古沙冬青克隆到一个RD22基因(AmRD22)的全长cDNA,并对其编码蛋白、表达模式和耐逆功能进行了研究。结果表明：(1)AmRD22蛋白(360 aa)的初级结构中含有RD22亚族共有的4个结构域,预测其定位于细胞壁;在功能已知的RD22蛋白中,AmRD22与大豆GmRD22的进化关系最近。(2)在室内培养的蒙古沙冬青幼苗中,AmRD22的表达受失水、高盐、低温和ABA胁迫的诱导显著上调,其中失水和低温胁迫诱导其上调幅度较大;在野外生长的蒙古沙冬青植株嫩叶中,其表达量从中秋至隆冬远高于其他季节。(3)转AmRD22基因拟南芥的耐盐性显著提高且Na⁺含量降低,其耐旱性也有较明显的改善且在种子萌发早期对外源ABA的敏感性降低,但耐冷性和耐冻性无明显变化。     

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.