A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

<Emphasis Type="Italic">SlHDA5</Emphasis>, a Tomato Histone Deacetylase Gene,Is Involved in Responding to Salt,Drought, and ABA

Histone deacetylation catalyzed by histone deacetylases is an important type of histone modification. Histone deacetylases affect various processes of plant development and involve in responding to hormones and biotic and abiotic stresses. Here, we report a tomato PRD3/HDA1 histone deacetylase gene, SlHDA5, which is expressed ubiquitously in different tissues and development stages. Expression profiles in hormone treatments showed that SlHDA5 was induced by abscisic acid (ABA) and methyl jasmonate (MeJA). Seedlings growth of SlHDA5-RNAi lines were more inhibited on the medium containing salt compared with wild type (WT). Under salt stress, chlorophyll in mature leaves degraded earlier in transgenic leaves than that in WT, and transgenic plants displayed wilting earlier and more severe than WT. After drought treatment, transgenic plants wilted and dehydrated earlier than WT, which was confirmed by lower water and chlorophyll content, and higher malondialdehyde (MDA) content in transgenic plants manifesting that the tolerance of transgenic plants to drought receded. Under the treatment of ABA, root length of transgenic seedlings was more strongly repressed by contrast with WT, suggesting repression of SlHDA5 increased seedling sensibility to ABA. Our study indicated that silencing of SlHDA5 resulted in decreasing tolerance to salt, drought, and ABA.     

Tolerance to soil water stress by <Emphasis Type="Italic">Oryza sativa</Emphasis> cv. IR20 was improved by expression of <Emphasis Type="Italic">Wsi18</Emphasis> gene locus from <Emphasis Type="Italic">Oryza nivara</Emphasis>

Wild rice genotypes are rich in genetic diversity. This has potential to improve agronomic rice by allele mining for superior traits. Late embryogenesis abundant (LEA) proteins are often associated with desiccation tolerance and stress signalling. In the present study, a group 3 LEA gene, Wsi18 from the wild rice Oryza nivara was expressed under its own inducible promoter element in stress susceptible cultivated indica rice (cv. IR20). The resulting transgenic plants cultivated in a greenhouse showed enhanced tolerance to soil water deficit. Transgenic plants had higher grain yield, plant survival rate, and shoot relative water content compared to wild type (WT) IR20. Cell membrane stability index, proline and soluble sugar content were also greater in transgenic than WT plants under water stress. These results demonstrate the potential for improving SWS tolerance in agronomically important rice cultivar by incorporating Wsi18 gene from a wild rice O. nivara.     

A Novel Little Membrane Protein Confers Salt Tolerance in Rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

Plasma membrane proteins play critical roles in sensing and responding abiotic and biotic stresses in plants. In the present study, we characterized a previously unknown gene stress associated little protein 1 (SALP1) encoding a plasma membrane protein. SALP1, a small and plant-specific membrane protein, contains only 74 amino acid residues. SALP1 was constitutively expressed in various rice tissues while highly expressed in roots, leaf blade, and immature panicles. Expression analysis indicated that SALP1 was induced by various abiotic stresses and abscisic acid (ABA). Subcellular localization assay indicated that SALP1 was localized on plasma membrane in rice protoplast cells. Overexpressing of SALP1 in rice improved salt tolerance through increasing free proline contents and the expression level of OsP5CS gene, and balancing ion contents under salt stress. Moreover, SALP1 transgenic rice showed reduced sensitivity to ABA treatment, and expression level of SALP1 is not altered by ABI5-like 1 protein. Conclusively, SALP1, a novel membrane protein, is involved in salt tolerance through an ABA-independent signaling pathway in rice.     

A novel <Emphasis Type="Italic">TaSST</Emphasis> gene from wheat contributes to enhanced resistance to salt stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

In this research, through the analyzing of the Triticum aestivum salt-tolerant mutant gene expression profile, under salt stress. A brand new gene with unknown functions induced by salt was cloned. The cloned gene was named Triticum aestivum salt stress protein (TaSST). GenBank accession number of TaSST is ACH97119. Quantitative polymerase chain reaction (qPCR) results exhibited that the expression TaSST was induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG). TaSST could improve salt tolerance of Arabidopsis-overexpressed TaSST. After salt stress, physiological indexes of transgenic Arabidopsis were better compared with WT (wild-type) plants. TaSST was mainly located in the cytomembrane. qPCR analyzed the expression levels of nine tolerance-related genes of Arabidopsis in TaSST-overexpressing Arabidopsis. Results showed that the expression levels of SOS3, SOS2, KIN2, and COR15a significantly increased, whereas the expression of the five other genes showed no obvious change. OsI_01272, the homologous gene of TaSST in rice, was interfered using RNA interference (RNAi) technique. RNAi plants became more sensitive to salt than control plants. Thus, we speculate that TaSST can improve plant salt tolerance.     

A Leucine-Rich Repeat Receptor-like Kinase from the Antarctic Moss <Emphasis Type="Italic">Pohlia nutans</Emphasis> Confers Salinity and ABA Stress Tolerance

Plant leucine-rich repeats receptor-like kinases (LRR-RLKs) play key roles in plant growth, development, and responses to environmental stresses. However, the functions of LRR-RLKs in bryophytes are still not well documented. Here, a putative LRR-RLK gene, PnLRR-RLK, was cloned and characterized from the Antarctic moss Pohlia nutans. Phylogenetic analysis revealed that PnLRR-RLK protein was clustered with the Arabidopsis thaliana LRR XI family proteins. Subcellular localization analysis of PnLRR-RLK revealed that it was mainly localized on plasma membrane. The expression of PnLRR-RLK was induced by mock high salinity, cold, drought, and exogenously supplied abscisic acid (ABA) and methyl jasmonate (MeJA). Meanwhile, the overexpression of PnLRR-RLK showed an increased tolerance of transgenic Arabidopsis to salt and ABA stresses than that of the wild type (WT) plants. Furthermore, the expression levels of several salt tolerance genes (AtHKT1, AtSOS3, AtP5CS1, and AtADH1) and an ABA negatively regulating gene AtABI1 were significantly increased in transgenic plants. Meanwhile, the expression levels of ABA biosynthesis genes (AtNCED3, AtABA1, and AtAAO3) and ABA early response genes (AtMYB2, AtRD22, AtRD29A, and AtDREB2A) were decreased in transgenic Arabidopsis after salt stress treatment. Therefore, these results suggested that PnLRR-RLK might involve in regulating salt stress-related and ABA-dependent signaling pathway, thereby contribute to the salinity tolerance of the Antarctic moss P. nutans.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

<Emphasis Type="Italic">OsNHX2</Emphasis>, an Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene,can enhance salt tolerance in rice plants through more effective accumulation of toxic Na<Superscript>+</Superscript> in leaf mesophyll and bundle sheath cells

The Na⁺/H⁺ antiporters play an important role in salt tolerance in plants. However, the functions of OsNHXs in rice except OsNHX1 have not been well studied. Using the gain- and loss-of-function strategies, we studied the potential role of OsNHX2 in salt tolerance in rice. Overexpression of OsNHX2 (OsNHX2-OE) in rice showed the significant tolerance to salt stress than wild-type plants and OsNHX2 knockdown transgenic plants (OsNHX2-KD). Under salt treatments of 300-mM NaCl for 5 days, the plant fresh weights, relative water percentages, shoot heights, Na⁺ contents, K⁺ contents, and K⁺/Na⁺ ratios in leaves of OsNHX2-OE transgenic plants were higher than those in wild-type plants, while no differences were detected in roots. K⁺/Na⁺ ratios in rice leaf mesophyll cells and bundle sheath cells were higher in OsNHX2-OE transgenic plants than in wild-type plants and OsNHX2-KD transgenic plants. Our data indicate that OsNHX2 plays an important role in salt stress based on leaf mesophyll cells and bundle sheath cells and can be served in genetically engineering crop plants with enhanced salt tolerance.     

Characteristics of Chlorophyll Fluorescence and Antioxidant-Oxidant Balance in <Emphasis Type="Italic">PEPC</Emphasis> and <Emphasis Type="Italic">PPDK</Emphasis> Transgenic Rice under Aluminum Stress

Aluminum is one of the most important heavy metals inducing stress during plant growth and development. In this study, transgenic rice (Oryza sativa L., cv. Kitaake) plants expressing the maize C₄PEPC and PPDK genes were evaluated for aluminum tolerance. A 4.3 and 19.1 folds increase of PPDK and PEPC activities in transgenic rice produced increases in root exudation of oxalate, malate, and citrate (1.20, 1.41, and 1.65 times, respectively) compared to untransformed (WT) plants. Transgenic rice had enhanced aluminum tolerance compared to WT based on chlorophyll fluorescence and chlorophyll levels. Transgenic plants under aluminum stress also had decreased lipid membrane oxidative damage and higher levels of ROS-scavenging enzyme activity. The PEPC and PPDK genes play an important role in aluminum stress tolerance by increasing the effluxes of organic acids.     

Overexpressing osa-miR171c decreases salt stress tolerance in rice

The miRNA171 family is one of the well-conserved miRNA families, and its role under stresses is not known except its expression on genome-wide expression analyses. osa-miR171c was induced by high concentration of salt (150 mM NaCl). A rice dh mutant with osa-miR171c overexpression triggered by a T-DNA insertion, significantly decreased salt tolerance at the stages of germination and seedling. This phenotype was confirmed by osa-miR171c overexpression transgenic rice. Compared with wild-type (WT), dh mutant reduced amounts of free proline and increased the water loss rate after salt treatment. Stomatal density in the leaf epidermis of dh mutant also increased. Moreover, dh mutant increased sensitivity to ABA treatment. Several stressresponsive genes were down-regulated in dh mutant than in WT under salty stress. These results indicate that osamiR171c is involved in modulating physiological changes, stomatal development, ABA-dependent pathways and expression of stress-related genes; thereby, it possibly contributes to salty tolerance.     

Overexpression of <Emphasis Type="Italic">ThGSTZ1</Emphasis> from <Emphasis Type="Italic">Tamarix hispida</Emphasis> improves tolerance to exogenous ABA and methyl viologen

Key message

Molecular analysis of a zeta subfamily GST gene from T. hispida involved in ABA and methyl viologen tolerance in transgenic Arabidopsis and Tamarix.

Abstract

Glutathione S-transferase (GST) genes are important for the improvement of plant abiotic stress tolerance, and our previous study demonstrated that the ThGSTZ1 gene from Tamarix hispida improves plant salt and drought tolerance. To further understand the role of ThGSTZ1 in the response of plants to abscisic acid (ABA) and oxidative stress, three ThGSTZ1-overexpressing transgenic Arabidopsis thaliana lines were analyzed in the current study. The results showed that the transgenic lines exhibited higher biomass accumulation, higher activities of GST and other protective enzymes, and less reactive oxygen species (ROS) and cell damage than wild-type (WT) plants under ABA and methyl viologen (MV) stress. In addition, the analysis of a transgenic T. hispida line transiently expressing ThGSTZ1 confirmed these results. The activities of GST, glutathione peroxidase, and superoxide dismutase were markedly higher in the ThGSTZ1-overexpressing lines compared with the control lines under both ABA and MV treatments, and the transgenic lines also exhibited a lower degree of electrolyte leakage (EL) and a decreased H₂O₂ content. All these results suggested that ThGSTZ1 can also improve plant ABA and oxidation tolerance by regulating ROS metabolism and that ThGSTZ1 represents an excellent candidate gene for molecular breeding to increase plant stress tolerance.