Glycine-rich RNA-binding protein 7 affects abiotic stress responses by regulating stomata opening and closing in Arabidopsis thaliana

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana , on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.     

Nucleotide sequence of psbQ gene for 16-kDa protein of oxygen-evolving complex from Arabidopsis thaliana and regulation of its expression.

The psbQ gene encoding a 16-kDa polypeptide of the oxygen-evolving complex of photosystem II has been isolated from Arabidopsis thaliana and characterized. The gene consists of a 28 nucleotide long leader sequence, two introns and three exons encoding a 223-amino-acid precursor polypeptide. The first 75 amino acids act as a transit peptide for the translocation of the polypeptide into the thylakoid lumen. Expression studies show that the gene is light-inducible and expresses only in green tissues with high steady-state mRNA levels in leaves. Using this gene as a probe, restriction fragment length polymorphism between two ecotypes, Columbia and Estland, has also been detected.     

Enhancer-mediated reporter gene expression in Arabidopsis thaliana: a forward genetic screen

Gene expression is controlled and regulated by interactions between cis-regulatory DNA elements (CREs) and regulatory proteins. Enhancers are one of the most important classes of CREs in eukaryotes. Eukaryotic genes, especially those related to development or responses to environmental cues, are often regulated by multiple enhancers in different tissues and/or at different developmental stages. Remarkably, little is known about the molecular mechanisms by which enhancers regulate gene expression in plants. We identified a distal enhancer, CREβ, which regulates the expression of AtDGK7, which encodes a diacylglycerol kinase in Arabidopsis. We developed a transgenic line containing the luciferase reporter gene (LUC) driven by CREβ fused with a minimal cauliflower mosaic virus (CaMV) 35S promoter. The CREβ enhancer was shown to play a role in the response to osmotic pressure of the LUC reporter gene. A forward genetic screen pipeline based on the transgenic line was established to generate mutations associated with altered expression of the LUC reporter gene. We identified a suite of mutants with variable LUC expression levels as well as different segregation patterns of the mutations in populations. We demonstrate that this pipeline will allow us to identify trans-regulatory factors associated with CREβ function as well as those acting in the regulation of the endogenous AtDGK7 gene.     

The Function of Circadian RNA‐Binding Proteins and Their cis‐Acting Elements in Microalgae

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin‐binding protein (LBP) is controlled at the translational level. Thereby, a clock‐controlled RNA‐binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG‐repeat, which is situated in the lbp 3^′ UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock‐controlled RNA‐binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3^′ UTRs of several mRNAs and recognizes them all via a common cis‐acting element, composed of at least seven UG‐repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO₂ metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.     

Cloning and expression analysis of a gene that shows developmental regulation upon tuberization in potato

An Arabidopsis cDNA clone encoding a DNA-binding protein, RAP-1, was isolated by southwestern screening of an Escherichia coli cDNA expression library. The protein contains a bHLH DNA-binding domain and is homologous to R proteins, regulating anthocyanin biosynthesis. RAP-1 binds to the sequence CACNTG. It is encoded by a single gene, which is expressed to high levels in root and stem and to low levels in leaf and flower. No expression could be detected in siliques. Rap-1 does not correspond to one of the known loci involved in anthocyanin biosynthesis, since it is located at a different map position. In contrast to the maize R protein Lc, RAP-1 did not induce anthocyanin biosynthesis in pea cotyledons. Thus, RAP-1 is a novel member of the bHLH class of DNA-binding proteins.     

Gene expression and genetic mapping analyses of a perennial ryegrass glycine-rich RNA-binding protein gene suggest a role in cold adaptation

A perennial ryegrass cDNA clone encoding a putative glycine-rich RNA binding protein (LpGRP1) was isolated from a cDNA library constructed from crown tissues of cold-treated plants. The deduced polypeptide sequence consists of 107 amino acids with a single N-terminal RNA recognition motif (RRM) and a single C-terminal glycine-rich domain. The sequence showed extensive homology to glycine-rich RNA binding proteins previously identified in other plant species. LpGRP1-specific genomic DNA sequence was isolated by an inverse PCR amplification. A single intron which shows conserved locations in plant genes was detected between the sequence motifs encoding RNP-1 and RNP-2 consensus protein domains. A significant increase in the mRNA level of LpGRP1 was detected in root, crown and leaf tissues during the treatment of plants at 4°C, through which freezing tolerance is attained. The increase in the mRNA level was prominent at least 2 h after the commencement of the cold treatment, and persisted for at least 1 week. Changes in mRNA level induced by cold treatment were more obvious than those due to treatments with abscisic acid (ABA) and drought. The LpGRP1 protein was found to localise in the nucleus in onion epidermal cells, suggesting that it may be involved in pre-mRNA processing. The LpGRP1 gene locus was mapped to linkage group 2. Possible roles for the LpGRP1 protein in adaptation to cold environments are discussed.     

拟南芥WUSCHEL蛋白的原核表达、亲和纯化和多克隆抗体制备

本研究构建了带有His标签的拟南芥(Arabidopsis thaliana)WUSCHEL基因原核表达载体pET-31b(+)-WUS-His(6),优化了大肠杆菌(Escherichia coli)诱导表达体系,将亲和层析纯化后的WUS融合蛋白,经尿素梯度透析复性溶解,免疫新西兰大白兔,成功制备了WUS蛋白多克隆抗体。通过琼脂糖免疫扩散检测确定了抗血清效价和特异性,并以斑点杂交和Western blotting检验其灵敏性。结果表明,成功构建的拟南芥WUS原核表达载体,在E.coli中以0.5mmol/L异丙基-β-D-硫代半乳糖苷(IPTG)28°C诱导表达10h后,融合蛋白得到高水平表达,亲和纯化后目标蛋白纯度达96%以上,所制备的多克隆抗体具有较高特异性和灵敏性,可用来检测纳克级蛋白抗原。     

Chromosomal location and expression of the single-copy gene encoding high-mobility-group protein HMG-I/Y in Arabidopsis thaliana

A cDNA encoding the HMG-I/Y protein from Arabidopsis thaliana has been isolated and characterised by nucleotide sequencing. The 903 bp cDNA contains a 612 bp open reading frame encoding a protein of 204 amino acid residues showing homology to HMG-I/Y proteins from other plant species. The protein contains four copies of the AT-hook motif which is involved in binding A/T-rich DNA. Southern blotting showed that the HMG-I/Y gene was present in a single copy in the Arabidopsis genome. The gene was localised to the top of chromosome 1 by RFLP analysis of F₈ recombinant inbred lines. Northern blotting showed that the gene was expressed in all organs examined, with the highest expression in flowers and developing siliques.