Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress

AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na⁺ content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na⁺ homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.     

The β-Lactam Resistance Protein Blr,a Small Membrane Polypeptide,Is a Component of the Escherichia coli Cell Division Machinery

In Escherichia coli, cell division is performed by a multimolecular machinery called the divisome, made of 10 essential proteins and more than 20 accessory proteins. Through a bacterial two-hybrid library screen, we identified the E. coli β-lactam resistance protein Blr, a short membrane polypeptide of 41 residues, as an interacting partner of the essential cell division protein FtsL. In addition to FtsL, Blr was found to associate with several other divisomal proteins, including FtsI, FtsK, FtsN, FtsQ, FtsW, and YmgF. Using fluorescently tagged Blr, we showed that this peptide localizes to the division septum and that its colocalization requires the presence of the late division protein FtsN. Although Blr is not essential, previous studies have shown that the inactivation of the blr gene increased the sensitivity of bacteria to β-lactam antibiotics or their resistance to cell envelope stress. Here, we found that Blr, when overproduced, restores the viability of E. coli ftsQ1(Ts) cells, carrying a thermosensitive allele of the ftsQ gene, during growth under low-osmotic-strength conditions (e.g., in synthetic media or in Luria-Bertani broth without NaCl). In contrast, the inactivation of blr increases the osmosensitivity of ftsQ1(Ts) cells, and blr ftsQ1 double mutants exhibit filamentous growth in LB broth even at a moderate salt concentration (0.5% NaCl) compared to parental ftsQ1(Ts) cells. Altogether, our results suggest that the small membrane polypeptide Blr is a novel component of the E. coli cell division apparatus involved in the stabilization of the divisome under certain stress conditions.     

Ectopic Expression of Plant RNA Chaperone Offering Multiple Stress Tolerance in <Emphasis Type="Italic">E. coli</Emphasis>

Members of the plant glycine-rich RNA-binding proteins (GR-RBPs) family have been reported in flowering, development, circadian rhythms, biotic and abiotic stresses. Particularly, GR-RBPs are reported to function as RNA chaperones, promoting growth and acclimation during cold shock. It is indispensable to further question the efficacy and mechanism of GR-RBPs under various environmental strains. Monitoring the expression of stress-regulated proteins under stress conditions has been a beneficial strategy to study their functional roles. In an effort to elucidate the NtGR-RBP1 function, stress markers such as salinity, drought, low temperature and heat stresses were studied. The NtGR-RBP1 gene was expressed in E. coli followed by the exposure to stress conditions. Recombinant E. coli expressing NtGR-RBP1 were more tolerant to stresses, e.g., salinity, drought, cold and heat shock. Recombinants exhibited higher growth rates compared to control in spot assays. The tolerance was further confirmed by monitoring the growth in liquid culture assays. Cells expressing NtGR-RBP1 under salt (500 mM NaCl), drought (20% PEG), cold (4 and 20 °C) and heat stresses (50 °C) had enhanced growing ability and better endurance. Our study supports the notion that the protective role of NtGR-RBP1 may contribute to growth and survival during diverse environmental stresses.     

Heterologous Expression of Two Jatropha Aquaporins Imparts Drought and Salt Tolerance and Improves Seed Viability in Transgenic Arabidopsis thaliana

Drought and high salinity are environmental conditions that cause adverse effects on the growth and productivity of crops. Aquaporins are small integral membrane proteins that belong to the family of the major intrinsic proteins (MIPs), with members in animals, plants and microbes, where they facilitate the transport of water and/or small neutral solutes thereby affecting water balance. In this study we characterized two aquaporin genes namely, plasma membrane intrinsic protein (PIP2;7) and tonoplast intrinsic protein TIP1;3 from Jatropha curcas that are localised to the plasma membrane and vacuole respectively. Transgenic Arabidopsis thaliana lines over-expressing JcPIP2;7 and JcTIP1;3 under a constitutive promoter show improved germination under high salt and mannitol compared to control seeds. These transgenic plants also show increased root length under abiotic stress conditions compared to wild type Col-0 plants. Transgenic lines exposed to drought conditions by withholding water for 20 days, were able to withstand water stress and attained normal growth after re-watering unlike control plants which could not survive. Transgenic lines also had better seed yield than control under salt stress. Importantly, seed viability of transgenic plants grown under high salt concentration was 35%-45% compared to less than 5% for control seeds obtained from plants growing under salt stress. The effect of JcPIP2;7 and JcTIP1;3 on improving germination and seed viability in drought and salinity make these important candidates for genetic manipulation of plants for growth in saline soils.     

Molecular characterization of soybean GmDjp1 encoding a type III J-protein induced by abiotic stress

Heat stress severely affects plant growth and development causing crop loss worldwide. Classical type I DnaJ proteins (also called as J-proteins, J-domain proteins or HSP40 proteins) function as molecular co-chaperones for the HSP70 proteins. In this study, we have cloned and characterized a novel gene GmDjp1 (G lycine m ax DnaJ protein 1) encoding a type III J-protein of which function has not been identified in plant. Deduced amino acid sequences of GmDjp1 show the highest homology with a J-protein from Medicago truncatula legume plant (83 %) and with Arabidopsis thaliana type III J-class proteins, atDjC53 (77 %) and atDjC32 (50 %). DNA blot analysis revealed that GmDjp1 exists as a 2-copy gene in soybean genome. GmDjp1 mRNA was induced by a broad spectrum of abiotic stresses, including wounding, heat-shock, dehydration, cold or high-salinity stress, suggesting its role in the signaling events in the abiotic stress-related defense response. Subcellular localization studies demonstrated that the GmDjp1-GFP fusion protein was localized in the nucleus. Differential RNA expression of GmDjp1 by heat-shock stress inspired us to test heat-shock tolerance of GmDjp1in E. coli. Heterologous expression of GmDjp1 conferred tolerance to high temperature stress in E. coli. This report provides strong evidence that GmDjp1 may play a critical role during heat-shock stress in cell.     

Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant

Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. ³⁵S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.     

Study of model systems to test the potential function of Artemia group 1 late embryogenesis abundant (LEA) proteins

Embryos of the brine shrimp, Artemia franciscana, are genetically programmed to develop either ovoviparously or oviparously depending on environmental conditions. Shortly upon their release from the female, oviparous embryos enter diapause during which time they undergo major metabolic rate depression while simultaneously synthesize proteins that permit them to tolerate a wide range of stressful environmental events including prolonged periods of desiccation, freezing, and anoxia. Among the known stress-related proteins that accumulate in embryos entering diapause are the late embryogenesis abundant (LEA) proteins. This large group of intrinsically disordered proteins has been proposed to act as molecular shields or chaperones of macromolecules which are otherwise intolerant to harsh conditions associated with diapause. In this research, we used two model systems to study the potential function of the group 1 LEA proteins from Artemia. Expression of the Artemia group 1 gene (AfrLEA-1) in Escherichia coli inhibited growth in proportion to the number of 20-mer amino acid motifs expressed. As well, clones of E. coli, transformed with the AfrLEA-1 gene, expressed multiple bands of LEA proteins, either intrinsically or upon induction with isopropyl-β-thiogalactoside (IPTG), in a vector-specific manner. Expression of AfrLEA-1 in E. coli did not overcome the inhibitory effects of high concentrations of NaCl and KCl but modulated growth inhibition resulting from high concentrations of sorbitol in the growth medium. In contrast, expression of the AfrLEA-1 gene in Saccharomyces cerevisiae did not alter the growth kinetics or permit yeast to tolerate high concentrations of NaCl, KCl, or sorbitol. However, expression of AfrLEA-1 in yeast improved its tolerance to drying (desiccation) and freezing. Under our experimental conditions, both E. coli and S. cerevisiae appear to be potentially suitable hosts to study the function of Artemia group 1 LEA proteins under environmentally stressful conditions.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-015-0647-3) contains supplementary material, which is available to authorized users.     

A novel Glycine soja tonoplast intrinsic protein gene responds to abiotic stress and depresses salt and dehydration tolerance in transgenic Arabidopsis thaliana

Tonoplast intrinsic protein (TIP) is a subfamily of the aquaporin (AQP), also known as major intrinsic protein (MIP) family, and regulates water movement across vacuolar membranes. Some reports have implied that TIP genes are associated with plant tolerance to some abiotic stresses that cause water loss, such as drought and high salinity. In our previous work, we found that an expressed sequence tag (EST) representing a TIP gene in our Glycine soja EST library was inducible by abiotic stresses. This TIP was subsequently isolated from G. soja with cDNA library screening, EST assembly and PCR, and named as GsTIP2;1. The expression patterns of GsTIP2;1 in G. soja under low temperature, salt and dehydration stress were different in leaves and roots. Though GsTIP2;1 is a stress-induced gene, overexpression of GsTIP2;1 in Arabidopsis thaliana depressed tolerance to salt and dehydration stress, but did not affect seedling growth under cold or favorable conditions. Higher dehydration speed was detected in Arabidopsis plants overexpressing GsTIP2;1, implying GsTIP2;1 might mediate stress sensitivity by enhancing water loss in the plant. Such a result is not identical to previous reports, providing some new information about the relationship between TIP and plant abiotic stress tolerance.     

Comparative Functional Analysis of Wheat (Triticum aestivum) Zinc Finger-Containing Glycine-Rich RNA-Binding Proteins in Response to Abiotic Stresses

Although the functional roles of zinc finger-containing glycine-rich RNA-binding proteins (RZs) have been characterized in several plant species, including Arabidopsis thaliana and rice (Oryza sativa), the physiological functions of RZs in wheat (Triticum aestivum) remain largely unknown. Here, the functional roles of the three wheat RZ family members, named TaRZ1, TaRZ2, and TaRZ3, were investigated using transgenic Arabidopsis plants under various abiotic stress conditions. Expression of TaRZs was markedly regulated by salt, dehydration, or cold stress. The TaRZ1 and TaRZ3 proteins were localized to the nucleus, whereas the TaRZ2 protein was localized to the nucleus, endoplasmic reticulum, and cytoplasm. Germination of all three TaRZ-expressing transgenic Arabidopsis seeds was retarded compared with that of wild-type seeds under salt stress conditions, whereas germination of TaRZ2- or TaRZ3-expressing transgenic Arabidopsis seeds was retarded under dehydration stress conditions. Seedling growth of TaRZ1-expressing transgenic plants was severely inhibited under cold or salt stress conditions, and seedling growth of TaRZ2-expressing plants was inhibited under salt stress conditions. By contrast, expression of TaRZ3 did not affect seedling growth of transgenic plants under any of the stress conditions. In addition, expression of TaRZ2 conferred freeze tolerance in Arabidopsis. Taken together, these results suggest that different TaRZ family members play various roles in seed germination, seedling growth, and freeze tolerance in plants under abiotic stress.     

In vivo analysis of the overlapping functions of DnaK and trigger factor

Trigger factor (TF) is a ribosome-bound protein that combines catalysis of peptidyl-prolyl isomerization and chaperone-like activities in Escherichia coli. TF was shown to cooperate with the DnaK (Hsp70) chaperone machinery in the folding of newly synthesized proteins, and the double deletion of the corresponding genes (tig and dnaK) exhibited synthetic lethality. We used a detailed genetic approach to characterize various aspects of this functional cooperation in vivo. Surprisingly, we showed that under specific growth conditions, one can delete both dnaK and tig, indicating that bacterial survival can be maintained in the absence of these two major cytosolic chaperones. The strain lacking both DnaK and TF exhibits a very narrow temperature range of growth and a high level of aggregated proteins when compared to either of the single mutants. We found that, in the absence of DnaK, both the N-terminal ribosome-binding domain and the C-terminal domain of unknown function are essential for TF chaperone activity. In contrast, the central PPIase domain is dispensable. Taken together, our data indicate that under certain conditions, folding of newly synthesized proteins in E. coli is not totally dependent on an interaction with either TF and/or DnaK, and suggest that additional chaperones may be involved in this essential process.     

Agitation Down-Regulates Immunoglobulin Binding Protein EibG Expression in Shiga Toxin-Producing Escherichia coli (STEC)

Shiga toxin (Stx)-producing Escherichia coli (STEC) carrying eibG synthesize Escherichia coli immunoglobulin binding protein (EibG). EibG nonspecifically binds to immunoglobulins and tends to aggregate in multimers but is poorly expressed in wild-type strains. To study synthesis of the proteins and their regulation in the pathogens, we identified natural growth conditions that increased EibG synthesis. EibG proteins as well as corresponding mRNA were highly expressed under static growth conditions while shearing stress created by agitation during growth repressed protein synthesis. Further regulation effects were driven by reduced oxygen tension, and pH up-regulated EibG expression, but to a lesser extent than growth conditions while decreased temperature down-regulated EibG. Bacteria with increased EibG expression during static growth conditions showed a distinct phenotype with chain formation and biofilm generation, which disappeared with motion. High and low EibG expression was reversible indicating a process with up- and down-regulation of the protein expression. Our findings indicate that shear stress represses EibG expression and might reduce bacterial attachments to cells and surfaces.     

A novel cold-regulated gene from Phlox subulata, PsCor413im1, enhances low temperature tolerance in Arabidopsis

Low temperature stress adversely affects plant growth, development, and crop productivity. Analysis of the function of genes in the response of plants to low temperature stress is essential for understanding the mechanism of chilling and freezing tolerance. In this study, PsCor413im1, a novel cold-regulated gene isolated from Phlox subulata, was transferred to Arabidopsis to investigate its function under low temperature stress. Real-time quantitative PCR analysis revealed that PsCor413im1 expression was induced by cold and abscisic acid. Subcellular localization revealed that PsCor413im1-GFP fusion protein was localized to the periphery of the chloroplast, consistent with the localization of chloroplast inner membrane protein AtCor413im1, indicating that PsCor413im1 is a chloroplast membrane protein. Furthermore, the N-terminal of PsCor413im1 was determined to be necessary for its localization. Compared to the wild-type plants, transgenic plants showed higher germination and survival rates under cold and freezing stress. Moreover, the expression of AtCor15 in transgenic plants was higher than that in the wild-type plants under cold stress. Taken together, our results suggest that the overexpression of PsCor413im1 enhances low temperature tolerance in Arabidopsis.     

Reconstitution of translation from Thermus thermophilus reveals a minimal set of components sufficient for protein synthesis at high temperatures and functional conservation of modern and ancient translation components

Thermus thermophilus is a thermophilic model organism distantly related to the mesophilic model organism E. coli. We reconstituted protein translation of Thermus thermophilus in vitro from purified ribosomes, transfer ribonucleic acids (tRNAs) and 33 recombinant proteins. This reconstituted system was fully functional, capable of translating natural messenger RNA (mRNA) into active full-length proteins at temperatures up to 65°C and with yields up to 60 μg/ml. Surprisingly, the synthesis of active proteins also occurred at 37°C, a temperature well below the minimal growth temperature for T. thermophilus. A polyamine was required, with tetraamine being most effective, for translation at both high and low temperatures. Using such a defined in vitro system, we demonstrated a minimal set of components that are sufficient for synthesizing active proteins at high temperatures, the functional compatibility of key translation components between T. thermophilus and E. coli, and the functional conservation of a number of resurrected ancient elongation factors. This work sets the stage for future experiments that apply abundant structural information to biochemical characterization of protein translation and folding in T. thermophilus. Because it contains significantly reduced nucleases and proteases, this reconstituted thermostable cell-free protein synthesis system may enable in vitro engineering of proteins with improved thermostability.     

Overexpression of SmLEA enhances salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza

Salinity and drought are important abiotic stresses limiting plant growth and development. Late embryogenesis abundant (LEA) proteins are a group of proteins associated with tolerance to water-related stress. We previously cloned an LEA gene, SmLEA, from Salvia miltiorrhiza Bunge. Phylogenetic analysis indicated that SmLEA belongs to Group LEA14, which is involved in the dehydration response. To determine its function in detail, we have now overexpressed SmLEA in Escherichia coli and S. miltiorrhiza. The logarithmic increase in accumulations of SmLEA proteins in E. coli occurred earlier under salinity than under standard conditions. SmLEA-transformed S. miltiorrhiza plants also showed faster root elongation and a lower malondialdehyde concentration than the empty vector control plants did when cultured on MS media supplemented with 60 mM NaCl or 150 mM mannitol. Moreover, SmLEA-overexpressing transgenics experienced a less rapid rate of water loss. Under either salinity or drought, overexpressing plants had greater superoxide dismutase activity and a higher glutathione concentration. These results suggest that SmLEA may be useful in efforts to improve drought and salinity tolerance in S. miltiorrhiza. Our data also provide a good foundation for further studies into the stress resistance mechanism and molecular breeding of this valuable medicinal plant.