Structural determinants crucial to the RNA chaperone activity of glycine-rich RNA-binding proteins 4 and 7 in Arabidopsis thaliana during the cold adaptation process

Although glycine-rich RNA-binding proteins (GRPs) have been determined to function as RNA chaperones during the cold adaptation process, the structural features relevant to this RNA chaperone activity remain largely unknown. To uncover which structural determinants are necessary for RNA chaperone activity of GRPs, the importance of the N-terminal RNA recognition motif (RRM) and the C-terminal glycine-rich domains of two Arabidopsis thaliana GRPs (AtGRP4 harbouring no RNA chaperone activity and AtGRP7 harbouring RNA chaperone activity) was assessed via domain swapping and mutation analyses. The results of domain swapping and deletion experiments showed that the domain sequences encompassing the N-terminal RRM of GRPs were found to be crucial to the ability to complement cold-sensitive Escherichia coli mutant cells under cold stress, RNA melting ability, and freezing tolerance ability in the grp7 loss-of-function Arabidopsis mutant. In particular, the N-terminal 24 amino acid extension of AtGRP4 impedes the RNA chaperone activity. Collectively, these results reveal that domain sequences and overall folding of GRPs governed by a specific modular arrangement of RRM and glycine-rich sequences are critical to the RNA chaperone activity of GRPs during the cold adaptation process in cells.     

Cold shock domain proteins and glycine-rich RNA-binding proteins from Arabidopsis thaliana can promote the cold adaptation process in Escherichia coli

Despite the fact that cold shock domain proteins (CSDPs) and glycine-rich RNA-binding proteins (GRPs) have been implicated to play a role during the cold adaptation process, their importance and function in eukaryotes, including plants, are largely unknown. To understand the functional role of plant CSDPs and GRPs in the cold response, two CSDPs (CSDP1 and CSDP2) and three GRPs (GRP2, GRP4 and GRP7) from Arabidopsis thaliana were investigated. Heterologous expression of CSDP1 or GRP7 complemented the cold sensitivity of BX04 mutant Escherichia coli that lack four cold shock proteins (CSPs) and is highly sensitive to cold stress, and resulted in better survival rate than control cells during incubation at low temperature. In contrast, CSDP2 and GRP4 had very little ability. Selective evolution of ligand by exponential enrichment (SELEX) revealed that GRP7 does not recognize specific RNAs but binds preferentially to G-rich RNA sequences. CSDP1 and GRP7 had DNA melting activity, and enhanced RNase activity. In contrast, CSDP2 and GRP4 had no DNA melting activity and did not enhance RNAase activity. Together, these results indicate that CSDPs and GRPs help E.coli grow and survive better during cold shock, and strongly imply that CSDP1 and GRP7 exhibit RNA chaperone activity during the cold adaptation process.     

A zinc finger-containing glycine-rich RNA-binding protein, atRZ-1a, has a negative impact on seed germination and seedling growth of Arabidopsis thaliana under salt or drought stress conditions

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to changing environmental conditions, the reports demonstrating their biological roles are severely limited. Here, we examined the functional roles of a zinc finger-containing GRP, designated atRZ-1a, in Arabidopsis thaliana under drought or salt stress conditions. Transgenic Arabidopsis plants overexpressing atRZ-1a displayed retarded germination and seedling growth compared with the wild-type plants under salt or dehydration stress conditions. In contrast, the loss-of-function mutants of atRZ-1a germinated earlier and grew faster than the wild-type plants under the same stress conditions. Germination of the transgenic plants and mutant lines was influenced by the addition of ABA or glucose, implying that atRZ-1a affects germination in an ABA-dependent way. H(2)O(2) was accumulated at higher levels in the transgenic plants compared with the wild-type plants under stress conditions. The expression of several germination-responsive genes was modulated by atRZ-1a, and proteome analysis revealed that the expression of different classes of genes, including those involved in reactive oxygen species homeostasis and functions, was affected by atRZ-1a under dehydration or salt stress conditions. Taken together, these results suggest that atRZ-1a has a negative impact on seed germination and seedling growth of Arabidopsis under salt or dehydration stress conditions, and imply that atRZ-1a exerts its function by modulating the expression of several genes under stress conditions.     

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.     

The role of raffinose in the cold acclimation response of Arabidopsis thaliana

In many plants raffinose family oligosaccharides are accumulated during cold acclimation. The contribution of raffinose accumulation to freezing tolerance is not clear. Here, we investigated whether synthesis of raffinose is an essential component for acquiring frost tolerance. We created transgenic lines of Arabidopsis thaliana accessions Columbia-0 and Cape Verde Islands constitutively overexpressing a galactinol synthase (GS) gene from cucumber. GS overexpressing lines contained up to 20 times as much raffinose as the respective wild-type under non-acclimated conditions and up to 2.3 times more after 14 days of cold acclimation at 4 degrees C. Furthermore, we used a mutant carrying a knockout of the endogenous raffinose synthase (RS) gene. Raffinose was completely absent in this mutant. However, neither the freezing tolerance of non-acclimated leaves, nor their ability to cold acclimate were influenced in the RS mutant or in the GS overexpressing lines. We conclude that raffinose is not essential for basic freezing tolerance or for cold acclimation of A. thaliana.     

A small zinc finger thylakoid protein plays a role in maintenance of photosystem II in Arabidopsis thaliana

This work identifies LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), a Zn finger protein that shows disulfide isomerase activity, interacts with the photosystem II (PSII) core complex, and may act in repair of photodamaged PSII complexes. Two mutants of an unannotated small Zn finger containing a thylakoid membrane protein of Arabidopsis thaliana (At1g75690; LQY1) were found to have a lower quantum yield of PSII photochemistry and reduced PSII electron transport rate following high-light treatment. The mutants dissipate more excess excitation energy via nonphotochemical pathways than wild type, and they also display elevated accumulation of reactive oxygen species under high light. After high-light treatment, the mutants have less PSII-light-harvesting complex II supercomplex than wild-type plants. Analysis of thylakoid membrane protein complexes showed that wild-type LQY1 protein comigrates with the PSII core monomer and the CP43-less PSII monomer (a marker for ongoing PSII repair and reassembly). PSII repair and reassembly involve the breakage and formation of disulfide bonds among PSII proteins. Interestingly, the recombinant LQY1 protein demonstrates a protein disulfide isomerase activity. LQY1 is more abundant in stroma-exposed thylakoids, where key steps of PSII repair and reassembly take place. The absence of the LQY1 protein accelerates turnover and synthesis of PSII reaction center protein D1. These results suggest that the LQY1 protein may be involved in maintaining PSII activity under high light by regulating repair and reassembly of PSII complexes.     

The C-terminal zinc finger domain of Arabidopsis cold shock domain proteins is important for RNA chaperone activity during cold adaptation

Among the four cold shock domain proteins (CSDPs) identified in Arabidopsis thaliana, it has recently been shown that CSDP1 harboring seven CCHC-type zinc fingers, but not CSDP2 harboring two CCHC-type zinc fingers, function as a RNA chaperone during cold adaptation. However, the structural features relevant to this differing RNA chaperone activity between CSDP1 and CSDP2 remain largely unknown. To determine which structural features are necessary for the RNA chaperone activity of the CSDPs, the importance of the N-terminal cold shock domain (CSD) and the C-terminal zinc finger glycine-rich domains of CSDP1 and CSDP2 were assessed. The results of sequence motif-swapping and deletion experiments showed that, although the CSD itself harbored RNA chaperone activity, the number and length of the zinc finger glycine-rich domains of CSDPs were crucial to the full activity of the RNA chaperones. The C-terminal domain itself of CSDP1, harboring seven CCHC-type zinc fingers, also has RNA chaperone activity. The RNA chaperone activity and nuclei acid-binding property of the native and chimeric proteins were closely correlated with each other. Collectively, these results indicate that a specific modular arrangement of the CSD and the zinc finger domain determines both the RNA chaperone activity and nucleic acid-binding property of CSDPs; this, in turn, contributes to enhanced cold tolerance in plants as well as in bacteria.     

Temperature-regulated expression of a glycine-rich RNA-binding protein in the halotolerant alga Dunaliella salina

Acclimation of the halotolerant alga Dunaliella salina to low temperature induced the accumulation of a 12.4 kDa protein (DsGRP-1) and reduction of a 13.1 kDa protein (DsGRP-2). DsGRP-1 and DsGRP-2 are boiling-stable proteins that are localised in the cytoplasm, as revealed by sub-cellular fractionation and by immuno-localisation. The proteins were partially purified and their corresponding genes were cloned. The predicted sequences are homologous to Glycine-Rich RNA-binding Proteins (GRPs) from plants and cyanobacteria. The nucleotide sequences of grp1 and grp2 differ in a short insert encoding 9 amino acids in the glycine-rich domain of DsGRP-2. grp2 contains a single intron at position 179 indicating that DsGRP-1 and DsGRP-2 are not derived from alternative splicing of a common gene. The level of grp mRNA increased at 7 degrees C and was rapidly depressed at 24 degrees C. Analysis of binding to ribonucleotide homopolymers revealed that DsGRP-1 and DsGRP-2 bind preferentially to poly-G and to poly-U indicating that they are RNA-binding proteins. It is proposed that DsGRP-1 and DsGRP-2 are encoded by distinct genes which are differentially regulated by temperature.     

Subcellular reprogramming of metabolism during cold acclimation in Arabidopsis thaliana

Metabolite changes in plant leaves during exposure to low temperatures involve re‐allocation of a large number of metabolites between sub‐cellular compartments. Therefore, metabolite determination at the whole cell level may be insufficient for interpretation of the functional significance of cellular compounds. To investigate the cold‐induced metabolite dynamics at the level of individual sub‐cellular compartments, an integrative platform was developed that combines quantitative metabolite profiling by gas chromatography coupled to mass spectrometry (GC‐MS) with the non‐aqueous fractionation technique allowing separation of cytosol, vacuole and the plastidial compartment. Two mutants of Arabidopsis thaliana representing antipodes in the diversion of carbohydrate metabolism between sucrose and starch were compared to Col‐0 wildtype before and after cold acclimation to investigate interactions of cold acclimation with subcellular re‐programming of metabolism. A multivariate analysis of the data set revealed dominant effects of compartmentation on metabolite concentrations that were modulated by environmental condition and genetic determinants. While for both, the starchless mutant of plastidial phospho‐gluco mutase (pgm) and a mutant defective in sucrose‐phosphate synthase A1, metabolic constraints, especially at low temperature, could be uncovered based on subcellularly resolved metabolite profiles, only pgm had lowered freezing tolerance. Metabolic profiles of pgm point to redox imbalance as a possible reason for reduced cold acclimation capacity.     

The CCCH zinc finger protein gene AtZFP1 improves salt resistance in Arabidopsis thaliana

The CCCH type zinc finger proteins are a super family involved in many aspects of plant growth and development. In this study, we investigated the response of one CCCH type zinc finger protein AtZFP1 (At2g25900) to salt stress in Arabidopsis. The expression of AtZFP1 was upregulated by salt stress. Compared to transgenic strains, the germination rate, emerging rate of cotyledons and root length of wild plants were significantly lower under NaCl treatments, while the inhibitory effect was significantly severe in T-DNA insertion mutant strains. At germination stage, it was mainly osmotic stress when treated with NaCl. Relative to wild plants, overexpression strains maintained a higher K⁺, K⁺/Na⁺, chlorophyll and proline content, and lower Na⁺ and MDA content. Quantitative real-time PCR analysis revealed that the expression of stress related marker genes KIN1, RD29B and RD22 increased more significantly in transgenic strains by salt stress. Overexpression of AtZFP1 also enhanced oxidative and osmotic stress tolerance which was determined by measuring the expression of a set of antioxidant genes, osmotic stress genes and ion transport protein genes such as SOS1, AtP5CS1 and AtGSTU5. Overall, our results suggest that overexpression of AtZFP1 enhanced salt tolerance by maintaining ionic balance and limiting oxidative and osmotic stress.     

Single-stranded nucleic acid binding in Arabidopsis thaliana cold shock protein is cold shock domain dependent

Cold shock proteins (CSPs) are ancient nucleic acid-binding proteins and well conserved from bacteria to animals as well as plants. In prokaryotes, CSPs possess a single cold shock domain (CSD) while animal CSPs, flanked by N- and C-terminal domains, are commonly named Y-box proteins. Interestingly, the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal CSD. The CSPs have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid-binding affinities of whole CSP as well as independent domains, so that role of each individual domain may be revealed in Arabidopsis thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology-based approach and docking was done with different nucleic acid types.