A novel rice calmodulin-like gene, <Emphasis Type="Italic">OsMSR2,</Emphasis> enhances drought and salt tolerance and increases ABA sensitivity in Arabidopsis

Many abiotic stimuli, such as drought and salt stresses, elicit changes in intracellular calcium levels that serve to convey information and activate adaptive responses. Ca²⁺ signals are perceived by different Ca²⁺ sensors, and calmodulin (CaM) is one of the best-characterized Ca²⁺ sensors in eukaryotes. Calmodulin-like (CML) proteins also exist in plants, but their functions at the physiological and molecular levels are largely unknown. In this report, we present data on OsMSR2 (Oryza sativa L. Multi-Stress-Responsive gene 2), a novel calmodulin-like protein gene isolated from rice Pei’ai 64S (Oryza sativa L.). Expression of OsMSR2 was strongly up-regulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice, as revealed by both microarray and quantitative real-time RT-PCR analyses. Analysis of the recombinant OsMSR2 protein demonstrated its potential ability to bind Ca²⁺ in vitro. Expression of OsMSR2 conferred enhanced tolerance to high salt and drought in Arabidopsis (Arabidopsis thaliana) accompanied by altered expression of stress/ABA-responsive genes. Transgenic plants also exhibited hypersensitivity to ABA during the seed germination and post-germination stages. The results suggest that expression of OsMSR2 modulated salt and drought tolerance in Arabidopsis through ABA-mediated pathways.     

Functional analyses of a putative plasma membrane Na+/H+ antiporter gene isolated from salt tolerant Helianthus tuberosus

Jerusalem artichokes (Helianthus tuberosus L.) can tolerate relatively higher salinity, drought and heat stress. In this paper, we report the cloning of a Salt Overly Sensitive 1 (SOS1) gene encoding a plasma membrane Na⁺/H⁺ antiporter from a highly salt-tolerant genotype of H. tuberosus, NY1, named HtSOS1 and characterization of its function in yeast and rice. The amino acid sequence of HtSOS1 showed 83.4 % identity with the previously isolated SOS1 gene from the Chrysanthemum crassum. The mRNA level in the leaves of H. tuberosus was significantly up-regulated by presence of high concentrations of NaCl. Localization analysis using rice protoplast expression showed that the protein encoded by HtSOS1 was located in the plasma membrane. HtSOS1 partially suppressed the salt sensitive phenotypes of a salt sensitive yeast strain. In comparison with wild type (Oryza sativa L., ssp. Japonica. cv. Nipponbare), the transgenic rice expressed with HtSOS1 could exclude more Na⁺ and accumulate more K⁺. Expression of HtSOS1 decreased Na⁺ content much larger in the shoot than in the roots, resulting in more water content in the transgenic rice than WT. These data suggested that HtSOS1 may be useful in transgenic approaches to improving the salinity tolerance of glycophyte.     

Polyamine oxidase 3 is involved in salt tolerance at the germination stage in rice

Soil salinity inhibits seed germination and reduces seedling survival rate,resulting in significant yield reductions in crops.Here,we report the identification of a polyamine oxidase,OsPAO3,conferring salt tolerance at the germination stage in rice(Oryza sativa L.),through map-based cloning approach.OsPAO3 is up-regulated under salt stress at the germination stage and highly expressed in various organs.Overexpression of OsPAO3 increases activity of polyamine oxidases,enhancing the polyamine cont...     

ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.     

<Emphasis Type="Italic">OsLEA3</Emphasis>, a late embryogenesis abundant protein gene from rice,confers tolerance to water deficit and salt stress to transgenic rice

OsLEA3 is a late embryogenesis abundant group 3 protein. The OsLEA3 gene located on chromosome 5 of rice (Oryza sativa L.) includes one intron and two exons and encodes a protein of 200 amino acid residues. Expression analysis revealed that OsLEA3 was induced by water deficit and salt stress. Overexpression of the OsLEA3 gene in the transgenic rice plants allowed us to test the role of the OsLEA3 protein in stress tolerance. The accumulation of the OsLEA3 protein in the vegetative tissues of transgenic rice plants enhanced their tolerance to water deficit and salt stress. These results demonstrate a role for the OsLEA3 protein in stress protection and suggest the potential of the OsLEA3 gene for genetic engineering of stress tolerance.     

Function of the wheat TaSIP gene in enhancing drought and salt tolerance in transgenic Arabidopsis and rice

Microarray analysis of a salt-tolerant wheat mutant identified a gene of unknown function that was induced by exposure to high levels of salt and subsequently denoted TaSIP (Triticum aestivum salt-induced protein). Quantitative PCR analysis revealed that TaSIP expression was induced not only by salt, but also by drought, abscisic acid (ABA), and other environmental stress factors. Transgenic rice plants that expressed an RNA interference construct specific for a rice gene homologous to TaSIP was more susceptible to salt stress than wild-type rice plants. Subcellular localization studies showed that the TaSIP localized to the cell membrane. Under conditions of salt and drought stress, transgenic Arabidopsis plants that overexpressed TaSIP showed superior physiological properties compared with control plants, including lower Na⁺ content and upregulation of several stress resistance genes. Staining of transgenic tissues with β-glucuronidase (GUS) failed to indicate tissue-specific activity of the full-length TaSIP promoter. Quantitative analysis of GUS fluorescence in transgenic plants treated with ABA or salt stress revealed that the region 1,176–1,410 bp from the start codon contained an ABA-responsive element and that the region 579–1,176 bp from the start codon upstream of the exon contained a salt-stress-responsive element. Based on these results, we conclude that the key part of the TaSIP gene is the region of its promoter involved in salt tolerance.     

Expression of the Arabidopsis vacuolar H+-pyrophosphatase gene AVP1 in peanut to improve drought and salt tolerance

The Arabidopsis gene AVP1 encodes an H⁺-pyrophosphatase that functions as a proton pump at the vacuolar membranes, generating a proton gradient across vacuolar membranes, which serves as the driving force for many secondary transporters on vacuolar membranes such as Na⁺/H⁺-antiporters. Overexpression of AVP1 could improve drought tolerance and salt tolerance in transgenic plants, suggesting a possible way in improving drought and salt tolerance in crops. The AVP1 was therefore introduced into peanut by Agrobacterium-mediated transformation. Analysis of AVP1-expressing peanut indicated that AVP1-overexpression in peanut could improve both drought and salt tolerance in greenhouse and growth chamber conditions, as AVP1-overexpressing peanuts produced more biomass and maintained higher photosynthetic rates under both drought and salt conditions. In the field, AVP1-overexpressing peanuts also outperformed wild-type plants by having higher photosynthetic rates and producing higher yields under low irrigation conditions.     

Overexpression of the Arabidopsis AtEm6 gene enhances salt tolerance in transgenic rice cell lines

The Arabidopsis thaliana late embryogenesis abundant gene AtEm6 is required for normal seed development and for buffering the rate of dehydration during the latter stages of seed maturation. However, its function in salt stress tolerance is not fully understood. In this investigation, cell suspension cultures of three plant species rice (Oryza sativa L.), cotton (Gossypium hirsutum L.), and white pine (Pinus strobes L.) were transformed using Agrobacterium tumefaciens strain LBA4404 harboring pBI-AtEm6. Integration of the AtEm6 gene into the genome of rice, cotton, and white pine has been confirmed by polymerase chain reaction, Southern blotting, and northern blotting analyses. Three transgenic cell lines from each of O. sativa, G. hirsutum, and P. strobus were used to analyze salt stress tolerance conferred by the overexpression of the AtEm6 gene. Our results demonstrated that expression of the AtEm6 gene enhanced salt tolerance in transgenic cell lines. A decrease in lipid peroxidation and an increment in antioxidant enzymes ascorbate peroxidase, glutathione reductase and superoxide dismutase activities were observed in the transgenic cell lines, compared to the non- transgenic control. In rice, AtEM6 increased expression of Ca²⁺-dependent protein kinase genes OsCPK6, OsCPK9, OsCPK10, OsCPK19, OsCPK25, and OsCPK26 under treatment of salt. These results suggested that overexpression of the AtEM6 gene in transgenic cell lines improved salt stress tolerance by regulating expression of Ca²⁺-dependent protein kinase genes. Overexpression of the AtEM6 gene could be an alternative choice for engineering plant abiotic stress tolerance.     

A cytosolic NADP-malic enzyme gene from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) confers salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

NADP-malic enzyme (NADP-ME, EC 1.1.1.40) functions in many different pathways in plant and may be involved in plant defense such as wound and UV-B radiation. Here, expression of the gene encoding cytosolic NADP-ME (cytoNADP-ME, GenBank Accession No. AY444338) in rice (Oryza sativa L.) seedlings was induced by salt stress (NaCl). NADP-ME activities in leaves and roots of rice also increased in response to NaCl. Transgenic Arabidopsis plants over-expressing rice cytoNADP-ME had a greater salt tolerance at the seedling stage than wild-type plants in MS medium-supplemented with different levels of NaCl. Cytosolic NADPH/NADP⁺ concentration ratio of transgenic plants was higher than those of wild-type plants. These results suggest that rice cytoNADP-ME confers salt tolerance in transgenic Arabidopsis seedlings.     

RNAi-Directed Downregulation of Vacuolar H+ -ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice

Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H⁺-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H⁺-ATPase activity and an enhancement of plasma membrane H⁺-ATPase activity, thereby increasing the concentrations of extracellular H⁺ and intracellular K⁺ and Na⁺ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H⁺-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.     

Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH₄ ⁺ and Na⁺ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.     

Ectopic expression of PgRab7 in rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200 mM NaCl) and drought stress (up to 12 days after withdrawing water). The protection against salt and drought stress may be by regulating Na⁺ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89–96 % of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33–53 % decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.     

Heterologous expression of a halophilic archaeon manganese superoxide dismutase enhances salt tolerance in transgenic rice

In order to investigate new gene resource for enhancing rice tolerance to salt stress, manganese superoxide dismutase gene from halophilic archaeon (Natrinema altunense sp.) (NaMnSOD) was isolated and introduced into Oryza sativa L. cv. Nipponbare by Agrobacterium-mediated transformation. The transformants (L1 and L2) showed some NaMnSOD expression and increased total SOD and CAT activity, which contributed to higher efficiency of ROS elimination under salt stress. The levels of superoxide anion radicals (O ₂ ^·? ) and hydrogen peroxide (H₂O₂) were significantly decreased. In addition, they exhibited higher levels of photosynthesis, whereas lower relative ion leakage and MDA content compared to wild-type plants. Therefore, transgenic seedlings were phenotypically healthier, and heterologous expression of NaMnSOD could improve rice salt tolerance.     

K+ Efflux and Retention in Response to NaCl Stress Do Not Predict Salt Tolerance in Contrasting Genotypes of Rice (Oryza sativa L.)

Sudden elevations in external sodium chloride (NaCl) accelerate potassium (K⁺) efflux across the plasma membrane of plant root cells. It has been proposed that the extent of this acceleration can predict salt tolerance among contrasting cultivars. However, this proposal has not been considered in the context of plant nutritional history, nor has it been explored in rice (Oryza sativa L.), which stands among the world’s most important and salt-sensitive crop species. Using efflux analysis with ⁴²K, coupled with growth and tissue K⁺ analyses, we examined the short- and long-term effects of NaCl exposure to plant performance within a nutritional matrix that significantly altered tissue-K⁺ set points in three rice cultivars that differ in salt tolerance: IR29 (sensitive), IR72 (moderate), and Pokkali (tolerant). We show that total short-term K⁺ release from roots in response to NaCl stress is small (no more than 26% over 45 min) in rice. Despite strong varietal differences, the extent of efflux is shown to be a poor predictor of plant performance on long-term NaCl stress. In fact, no measure of K⁺ status was found to correlate with plant performance among cultivars either in the presence or absence of NaCl stress. By contrast, shoot Na⁺ accumulation showed the strongest correlation (a negative one) with biomass, under long-term salinity. Pharmacological evidence suggests that NaCl-induced K⁺ efflux is a result of membrane disintegrity, possibly as result of osmotic shock, and not due to ion-channel mediation. Taken together, we conclude that, in rice, K⁺ status (including efflux) is a poor predictor of salt tolerance and overall plant performance and, instead, shoot Na⁺ accumulation is the key factor in performance decline on NaCl stress.     

Overexpression of AtNHX5 improves tolerance to both salt and water stress in rice (Oryza sativa L.)

Overexpression of NHX genes has been previously shown to improve salt tolerance of transgenic plants. In this study, transgenic rice plants overexpressing AtNHX5 showed not only high salt tolerance, but also high drought tolerance. Measurements of ion levels indicated that Na⁺ and K⁺ contents were all higher in AtNHX5 overexpressing shoots than in wild type (WT) shoots in high saline conditions. After exposure to water deficiency and salt stress, the WT plants all died, while the AtNHX5 overexpressing rice plants had a higher survival rate, dry weight, leaf water content, and leaf chlorophyll contents, accumulated more proline, and had less membrane damage than the WT plants. In addition, seeds of both transgenic and WT plants germinated on 1/2 MS medium supplemented with 250 mM mannitol, but overexpression of AtNHX5 improved the shoot growth of the seedlings. Taken together, the results indicate that AtNHX5 gene could enhance the tolerance of rice plants to multiple environmental stresses by promoting the accumulation of more effective osmolytes (ions or proline) to counter the osmotic stress caused by abiotic factors.