Contribution of <Emphasis Type="Italic">Eutrema salsugineum</Emphasis> Cold Shock Domain Structure to the Interaction with RNA

Plant cold shock domain proteins (CSDPs) are DNA/RNA-binding proteins. CSDPs contain the conserved cold shock domain (CSD) in the N-terminal part and a varying number of the CCHC-type zinc finger (ZnF) motifs alternating with glycine-rich regions in the C-terminus. CSDPs exhibit RNA chaperone and RNA-melting activities due to their non-specific interaction with RNA. At the same time, there are reasons to believe that CSDPs also interact with specific RNA targets. In the present study, we used three recombinant CSDPs from the saltwater cress plant (Eutrema salsugineum)-EsCSDP1, EsCSDP2, EsCSDP3 with 6, 2, and 7 ZnF motifs, respectively, and showed that their nonspecific interaction with RNA is determined by their C-terminal fragments. All three proteins exhibited high affinity to the single-stranded regions over four nucleotides long within RNA oligonucleotides. The presence of guanine in the single-or double-stranded regions was crucial for the interaction with CSDPs. Complementation test using E. coli BX04 cells lacking four cold shock protein genes (ΔcspA, ΔcspB, ΔcspE, ΔcspG) revealed that the specific binding of plant CSDPs with RNA is determined by CSD.     

Functional annotation of a novel toxin–antitoxin system Xn-RelT of <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>; a combined in silico and in vitro approach

Toxin–antitoxin (TA) complexes play an important role in stress responses and programmed cell death in bacteria. The RelB-RelE toxin antitoxin system is well studied in Escherichia coli. In this study, we used combined in silico and in vitro approaches to study a novel Xn-RelT toxin from Xenorhabdus nematophila bearing its own antitoxin Xn-RelAT—a RelB homolog of E. coli. The structure for this toxin–antitoxin pair is yet unknown. We generated homology-based models of X. nematophila RelT toxin and antitoxin. The deduced models were further characterized for protein–nucleic acid, protein–protein interactions and gene ontology. A detrimental effect of recombinant Xn-RelT on host E. coli was determined through endogenous toxicity assay. When expressed from a isopropyl β-d-1-thiogalactopyranoside-regulated LacZ promoter, Xn-RelT toxin showed a toxic effect on E. coli cells. These observations imply that the conditional cooperativity governing the Xn-RelT TA operon in X. nematophila plays an important role in stress management and programmed cell death.     

Identification of a drought responsive gene encoding a nuclear protein involved in drought and freezing stress tolerance in Arabidopsis

Plants have developed adaptive strategies to survive under different abiotic stressors. To identify new components involved in abiotic stress tolerance, we screened unannotated expressed sequence tags (ESTs) and evaluated their cold or drought response in Arabidopsis. We identified a drought response gene (DRG) encoding a 39.5-kDa polypeptide. This protein was expressed specifically in siliques and was induced by drought stress in most tissues. When a DRG-GFP construct was introduced into Arabidopsis protoplasts, GFP signals were detected only in the nucleus. The drg mutant plant was more sensitive to mannitol-induced osmotic stress in agar plates and to drought or freezing stress in soil than the wild-type. Activating the DRG restored the normal sensitivity of drg mutants to abiotic stressors. No differences in drought or freezing tolerance were observed between the wild-type and transgenic plants overexpressing the DRG. When DRG was expressed in a cold-sensitive Escherichia coli strain BX04, the transformed bacteria grew faster than the untransformed BXO4 cells under cold stress. These results demonstrate that DRG is a nuclear protein induced by abiotic stresses and it is required for drought and freezing tolerance in Arabidopsis.     

Diverse roles of glycine-rich RNA-binding protein 7 in the response of camelina (<Emphasis Type="Italic">Camelina sativa</Emphasis>) to abiotic stress

Camelina sativa L. is an oilseed crop used as a potential low-cost biofuel resource. Despite the economic and agricultural benefits of this crop, studies demonstrating the physiological and genetic response of camelina to changing environmental conditions are limited. In this study, three stress-responsive glycine-rich RNA-binding proteins (GRPs) in camelina—named CsGRP7a, CsGRP7b, and CsGRP7c—were isolated, and their functional roles in stress responses were characterized. The three CsGRP7 genes had similar nucleotide and deduced amino acid sequences, and contained an N-terminal RNA-recognition motif and a C-terminal glycine-rich region. The CsGRP7 genes were ubiquitously expressed in all plant tissues, and CsGRP7 proteins were localized to both the cytoplasm and the nucleus. The expression of CsGRP7 genes was markedly upregulated by cold stress, whereas their expression was only slightly affected by salt or dehydration stress. Analysis of CsGRP7a-expressing transgenic Arabidopsis thaliana and camelina plants revealed that CsGRP7a plays a positive role in cold stress tolerance, but a negative role in salt or drought stress tolerance. All three CsGRP7s harbored RNA chaperone activity. Collectively, these data indicate that the stress-responsive CsGRP7s harbor RNA chaperone activity and play different roles in the plant response to abiotic stresses.     

Cloning and characterization of four <Emphasis Type="Italic">TpSnRK2s</Emphasis> from dwarf Polish wheat

Protein phosphorylation/dephosphorylation is a major signalling event induced by abiotic stresses in plants. Sucrose nonfermenting 1-related protein kinase 2 (SnRK2) plays important roles in response to osmotic stress. In the present study, four SnRK2s, TpSnRK2.1/3/7/8, were cloned and characterized from Triticum polonicum L. (dwarf Polish wheat, DPW, AABB). All of these were individually located on 2AL, 1AL, 2AL, and 5BL. Two spliced isoforms of TpSnRK2.8 (TpSnRK2.8a and TpSnRK2.8b) were observed. TpSnRK2.1 and TpSnRK2.3 were classified into the group II; TpSnRK2.7 was classified into the group I; and TpSnRK2.8a/b were classified into the group III. Expression patterns revealed that TpSnRK2.1 responded to cold, NaCl, polyethylene glycol (PEG), and abscisic acid (ABA) in both roots and leaves; TpSnRK2.3 was strongly regulated by cold, NaCl, and ABA in both roots and leaves, and by PEG in roots; TpSnRK2.7 was induced by NaCl and PEG in roots, but was not activated by ABA; and TpSnRK2.8s were significantly activated by cold, NaCl, PEG, and ABA in both roots and leaves. From the above results, we inferred that TpSnRK2.1/3/8 may participate in the responses to environmental stresses in ABA-dependent signal transduction pathway but TpSnRK2.7 is possibly involved in responses to environmental stresses in a non-ABA-dependent manner. They play important roles in specific tissues under different stresses.     

Overexpression of calmodulin gene fragment from Antarctic notothenioid fish improves chilling tolerance in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

Calmodulin (CaM) is a highly conserved calcium sensor protein associated with chilling tolerance in living organisms. It has four EF-hand domains for binding of four Ca²⁺, two of them located in the N-terminus, and the other two in the C-terminus. A notothenioid CaM gene fragment (CaMm), which only codes for N-terminus of CaM (with two EF-hand domains), was introduced into Nicotiana benthamiana. Effects of its overexpression on chilling tolerance in plants were explored. During 4?C or 0?C chilling treatment, both CaMm and CaM transgenic plants showed higher PSII maximum quantum yield, actual quantum yield, and soluble protein content, lower electrolyte leakage and malondialdehyde content than that of the control. The changes in these physiological indices were comparable between the CaMm and CaM transgenic plants during the treatments. These results indicate that the N-terminus of calmodulin is likely the key functional domain involved in the adaptive response to cold stress.     

Effect of lowered incubation temperatures on nucleic acid and protein synthesis by a mesophilic and a psychrophilic bacterium

The authors studied changes in the synthesis of nucleic acids (RNA, DNA) and protein by a mesophilic strain ofEscherichia coli B and a psychrophilic strain ofPseudomonas fluorescens at a low incubation temperature giving tenfold prolongation of the generation time. It was found that lowering the incubation temperature was followed by an increase in the intracellular nucleic acid content during the lag phase and the phase of accelerated growth, in which maximum nucleic acid (NA) values were reached. As a result, the total NA level in the cell also remained relatively high during further proliferation, when the increase in NA (particularly RNA) slows down at low incubation temperatures. Proteosynthesis, however, fell in the mesophilic culture. The smaller effect of a lowered temperature on DNA biosynthesis was manifested specifically in the lag phase ofEscherichia coli, in which disproportion developed between the amount of DNA (which was synthesized at a relatively higher rate) and RNA; this was afterwards equalized by a temporary break in DNA production. Pronounced differences in the given types of biosynthesis were found only in the mesophilic culture, while at suboptimal temperatures the metabolism of the psychrophilic strain slowed down but no marked changes occurred.     

Genome-wide identification and abiotic stress responses of <Emphasis Type="Italic">DGK</Emphasis> gene family in maize

Diacylglycerol kinase (DGK) is a kind of phosphokinase that catalyzes the formation of signaling molecule phosphatidic acid. In this study, seven maize (Zea mays) DGK gene family members were identified by an exploration of maize genome via multiple online databases, and designated as ZmDGK1-7, respectively. The proteins encoded by ZmDGKs ranged from 487 to 716 amino acids, and had a molecular weight (MWs) between 54.6 and 80.2 kDa. Phylogenetic analysis revealed that ZmDGKs grouped into three clusters as described for known plant DGK families: Cluster I was composed of three maize DGKs, ZmDGK1, ZmDGK4 and ZmDGK5, cluster II contained ZmDGK6, and the isoforms ZmDGK2, ZmDGK3 and ZmDGK7 fell into cluster III. ZmDGK proteins featured the typical functional domains, while all seven ZmDGKs have a conserved catalytic domain DGKc, only the cluster I ZmDGKs have the DAG/PE binding domain. Most ZmDGK genes showed ubiquitous expression profiles at various developmental stages, while a high relative expression was observed at the tasseling stage. ZmDGK genes exhibited differential expression patterns in response to abiotic stresses including cold, salinity and drought, and all ZmDGK genes were found obviously up-regulated by cold. The distinct roles of ZmDGKs in cold response was also supported by the finding that an accumulation of DGK products–PA under low temperature. This study will help to better understand the roles of DGKs in the development and abiotic stress responses in major crops.     

Improved Tolerance of <Emphasis Type="Italic">Escherichia coli</Emphasis> to Propionic Acid by Overexpression of Sigma Factor RpoS

Propionic acid (PA) is an economically important compound, but large-scale microbial production of PA confronts obstacle such as acid stress on microbial cells. Here, we show that overexpressing sigma factor RpoS improves the acid tolerance of Escherichia coli. Four genes including rpoS, fur, pgi and dnaK (encoding RNA polymerase sigma factor, ferric uptake regulator, phosphoglucoisomerase, and chaperone, respectively) were independently overexpressed in E. coli. The recombinant E. coli overexpressing rpoS showed the highest PA tolerance. This strain could grow in M9 medium at pH 4.62, whereas wild type E. coli survived only at pHs above 5.12. Moreover, in the shake-flask cultivation, the E. coli strain overexpressing rpoS grew faster than wild type. Notably, the minimum inhibitory concentration of PA for this recombinant strain was 7.81 mg/mL, which was 2-fold higher in comparison with wild type. Overall these results indicated that overexpression of sigma factor rpoS significantly enhanced E. coli tolerance to PA.     

Bioinformatic and expression analyses on carotenoid dioxygenase genes in fruit development and abiotic stress responses in <Emphasis Type="Italic">Fragaria vesca</Emphasis>

Carotenoid dioxygenases, including 9-cis-epoxycarotenoid dioxygenases (NCEDs) and carotenoid cleavage dioxygenases (CCDs), can selectively cleave carotenoids into various apocarotenoid products that play important roles in fleshy fruit development and abiotic stress response. In this study, we identified 12 carotenoid dioxygenase genes in diploid strawberry Fragaria vesca, and explored their evolution with orthologous genes from nine other species. Phylogenetic analyses suggested that the NCED and CCDL groups moderately expanded during their evolution, whereas gene numbers of the CCD1, CCD4, CCD7, and CCD8 groups maintained conserved. We characterized the expression profiles of FveNCED and FveCCD genes during flower and fruit development, and in response to several abiotic stresses. FveNCED1 expression positively responded to osmotic, cold, and heat stresses, whereas FveNCED2 was only induced under cold stress. In contrast, FveNCED2 was the unique gene highly and continuously increasing in receptacle during fruit ripening, which co-occurred with the increase in endogenous abscisic acid (ABA) content previously reported in octoploid strawberry. The differential expression patterns suggested that FveNCED1 and FveNCED2 were key genes for ABA biosynthesis in abiotic stress responses and fruit ripening, respectively. FveCCD1 exhibited the highest expression in most stages of flower and fruit development, while the other FveCCDs were expressed in a subset of stages and tissues. Our study suggests distinct functions of FveNCED and FveCCD genes in fruit development and stress responses and lays a foundation for future study to understand the roles of these genes and their metabolites, including ABA and other apocarotenoid products, in the growth and development of strawberry.     

The opposite roles of <Emphasis Type="Italic">OsmiR408</Emphasis> in cold and drought stress responses in <Emphasis Type="Italic">Oryza sativa</Emphasis>

MiR408 is a conserved miRNA family in plants. Although AtmiR408 is generally regarded as participating in stress responses, it still remains obscure whether OsmiR408 modulates tolerance to environmental stress. In the current study, expression of Pre-OsmiR408 and OsmiR408 was found to be induced by cold stress, but repressed by drought stress in the rice cultivar “Kongyu 131”. By comparing the wild type and OsmiR408 transgenic lines, we found that OsmiR408 overexpression conferred enhanced cold tolerance at both the early seedling stage and the young seedling stage. On the other hand, the OsmiR408 transgenic lines exhibited decreased drought tolerance, which is further verified by greater water loss. We also predicted the putative target genes of OsmiR408 and verified the decreased expression of seven targets in OsmiR408 transgenic lines, including four phytocyanins and three atypical target genes. Among them, Os09g29390, a phytocyanin gene, and Os01g53880, an auxin responsive Aux/IAA gene, were down-regulated by cold treatment, which is opposite to the cold-induced expression of OsmiR408. Taken together, our results suggest opposite roles of OsmiR408 in plant responses to cold and drought stresses.