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.     

Ectopic expression of wheat expansin gene TaEXPA2 improved the salt tolerance of transgenic tobacco by regulating Na+/K+ and antioxidant competence

High salinity is one of the most serious environmental stresses that limit crop growth. Expansins are cell wall proteins that regulate plant development and abiotic stress tolerance by mediating cell wall expansion. We studied the function of a wheat expansin gene, TaEXPA2, in salt stress tolerance by overexpressing it in tobacco. Overexpression of TaEXPA2 enhanced the salt stress tolerance of transgenic tobacco plants as indicated by the presence of higher germination rates, longer root length, more lateral roots, higher survival rates and more green leaves under salt stress than in the wild type (WT). Further, when leaf disks of WT plants were incubated in cell wall protein extracts from the transgenic tobacco plants, their chlorophyll content was higher under salt stress, and this improvement from TaEXPA2 overexpression in transgenic tobacco was inhibited by TaEXPA2 protein antibody. The water status of transgenic tobacco plants was improved, perhaps by the accumulation of osmolytes such as proline and soluble sugar. TaEXPA2‐overexpressing tobacco lines exhibited lower Na⁺ but higher K⁺ accumulation than WT plants. Antioxidant competence increased in the transgenic plants because of the increased activity of antioxidant enzymes. TaEXPA2 protein abundance in wheat was induced by NaCl, and ABA signaling was involved. Gene expression regulation was involved in the enhanced salt stress tolerance of the TaEXPA2 transgenic plants. Our results suggest that TaEXPA2 overexpression confers salt stress tolerance on the transgenic plants, and this is associated with improved water status, Na⁺/K⁺ homeostasis, and antioxidant competence. ABA signaling participates in TaEXPA2‐regulated salt stress tolerance.     

Silicon‐mediated changes in polyamines participate in silicon‐induced salt tolerance in Sorghum bicolor L.

Silicon (Si) is generally considered a beneficial element for the growth of higher plants, especially under stress conditions, but the mechanisms remain unclear. Here, we tested the hypothesis that Si improves salt tolerance through mediating important metabolism processes rather than acting as a mere mechanical barrier. Seedlings of sorghum (Sorghum bicolor L.) growing in hydroponic culture were treated with NaCl (100 mm ) combined with or without Si (0.83 mm ). The result showed that supplemental Si enhanced sorghum salt tolerance by decreasing Na⁺ accumulation. Simultaneously, polyamine (PA) levels were increased and ethylene precursor (1‐aminocyclopropane‐1‐carboxylic acid: ACC) concentrations were decreased. Several key PA synthesis genes were up‐regulated by Si under salt stress. To further confirm the role of PA in Si‐mediated salt tolerance, seedlings were exposed to spermidine (Spd) or a PA synthesis inhibitor (dicyclohexylammonium sulphate, DCHA) combined with salt and Si. Exogenous Spd showed similar effects as Si under salt stress whereas exogenous DCHA eliminated Si‐enhanced salt tolerance and the beneficial effect of Si in decreasing Na⁺ accumulation. These results indicate that PAs and ACC are involved in Si‐induced salt tolerance in sorghum and provide evidence that Si plays an active role in mediating salt tolerance.     

蛋白质组学研究揭示的植物根盐胁迫响应机制

植物根是感知外界盐胁迫信号的首要器官。近年来,人们利用高通量的差异表达蛋白质组学技术,分析了水稻(Oryzasativa)、拟南芥(Arabidopsis thaliana)、大豆(Glycine max)、大麦(Hordeum vulgare)、小麦(Triticum aestivum)、木榄(Bruguieragymnorhiza)和匍匐翦股颖(Agrostis stolonifera)等植物根应答盐胁迫过程中蛋白质组的动态变化特征。通过整合植物根响应盐胁迫蛋白质组学研究结果,揭示了植物根部响应盐胁迫的多种调节机制,包括:利用多种信号通路与蛋白质磷酸化/去磷酸化感知并传递盐胁迫信号;通过膜蛋白与转运蛋白调节离子吸收/外排与区室化;通过抗氧化酶系统活性清除活性氧,并通过合成多种渗透调节物质与防御物质减轻细胞受到的伤害;通过改变参与糖类与能量代谢相关酶的表达调节能量代谢水平;通过细胞骨架动态重塑保持正常的细胞结构、物质运输与信息传递;通过转录、翻译与翻译后调控调节各种蛋白质的动态变化与相互作用;通过调控各种基础代谢与次生代谢水平保持细胞结构与代谢状态正常。     

Improving crop salt tolerance using transgenic approaches: An update and physiological analysis

Salinization of land is likely to increase due to climate change with impact on agricultural production. Since most species used as crops are sensitive to salinity, improvement of salt tolerance is needed to maintain global food production. This review summarises successes and failures of transgenic approaches in improving salt tolerance in crop species. A conceptual model of coordinated physiological mechanisms in roots and shoots required for salt tolerance is presented. Transgenic plants overexpressing genes of key proteins contributing to Na⁺ ‘exclusion’ (PM-ATPases with SOS1 antiporter, and HKT1 transporter) and Na⁺ compartmentation in vacuoles (V-H⁺ATPase and V-H⁺PPase with NHX antiporter), as well as two proteins potentially involved in alleviating water deficit during salt stress (aquaporins and dehydrins), were evaluated. Of the 51 transformations, with gene(s) involved in Na⁺ ‘exclusion’ or Na⁺ vacuolar compartmentation that contained quantitative data on growth and include a non-saline control, 48 showed improvements in salt tolerance (less impact on plant mass) of transgenic plants, but with only two tested in field conditions. Of these 51 transformations, 26 involved crop species. Tissue ion concentrations were altered, but not always in the same way. Although glasshouse data are promising, field studies are required to assess crop salinity tolerance.     

Cloning and Functional Characterization of a Novel Glutathione <Emphasis Type="Italic">S</Emphasis>-Transferase Gene from <Emphasis Type="Italic">Limonium bicolor</Emphasis>

Plant glutathione S-transferases (GSTs) are involved in protecting plants against both diverse biotic and abiotic stresses. In the present study, a novel GST gene (LbGST1) was cloned from Limonium bicolor (Bunge) Kuntze (Plumbaginaceae). To characterize its function in salt tolerance, tobacco lines transformed with LbGST1 were generated. Compared with wild-type (WT) tobacco, transgenic plants overexpressing LbGST1 exhibited both GST and glutathione peroxidase activities. Moreover, superoxide dismutase, peroxidase (POD), and catalase activities in transgenic plants were significantly higher than those in WT plants, particularly when grown under conditions of salt stress. Similarly, levels of proline in transgenic plants were also higher than those in WT plants grown under NaCl stress conditions. Whereas, Malondialdehyde contents in transgenic plants were lower than those in WT plants under NaCl conditions. Furthermore, Na⁺ content in transgenic plants was lower than that in WT plants under these stress conditions. Subcellular localization analysis revealed that the LbGST1 protein was localized in the nucleus. These results suggested that overexpression of LbGST1 gene can affect many physiological processes associated with plant salt tolerance. Therefore, we hypothesize that LbGST1 gene can mediate many physiological pathways that enhance stress resistance in plants.     

The effect of NaCl on proline accumulation in potato seedlings and calli

The effects of salt stress were studied on the accumulation and metabolism of proline and its correlation with Na⁺ and K⁺ content in shoots and callus tissue of four potato cultivars, viz., Agria, Kennebec (relatively salt tolerant), Diamant and Ajax (relatively salt sensitive). Na⁺ and proline contents increased in all cultivars under salt stress. However, K⁺ and protein contents decreased in response to NaCl treatments. The activities of enzymes involved in proline metabolism, Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) increased and decreased, respectively, in response to elevated NaCl concentrations. The changes of P5CS and ProDH activities in more salt sensitive cultivars (Diamant, Ajax) were more than those in the tolerant ones. Then the stimulation of synthesis in combination with a partially increase of protein proteolysis, a decrease in proline utilization and inhibition of oxidation resulted in high proline contents in seedlings and calli under salt stress. In callus tissue, reduced growth and cell size may be partially responsible for high proline accumulation in response to high NaCl levels. However, although the basic proline contents in the seedlings of more salt tolerant cultivars were higher than the sensitive ones, a clear relationship was not generally observed between accumulation of proline and salt tolerance in potato.     

Regulation of cation transporter genes by the arbuscular mycorrhizal symbiosis in rice plants subjected to salinity suggests improved salt tolerance due to reduced Na<Superscript>+</Superscript> root-to-shoot distribution

Rice is a salt-sensitive crop whose productivity is strongly reduced by salinity around the world. Plants growing in saline soils are subjected to the toxicity of specific ions such as sodium, which damage cell organelles and disrupt metabolism. Plants have evolved biochemical and molecular mechanisms to cope with the negative effects of salinity. These include the regulation of genes with a role in the uptake, transport or compartmentation of Na⁺ and/or K⁺. Studies have shown that the arbuscular mycorrhizal (AM) symbiosis alleviates salt stress in several host plant species. However, despite the abundant literature showing mitigation of ionic imbalance by the AM symbiosis, the molecular mechanisms involved are barely explored. The objective of this study was to elucidate the effects of the AM symbiosis on the expression of several well-known rice transporters involved in Na⁺/K⁺ homeostasis and measure Na⁺ and K⁺ contents and their ratios in different plant tissues. Results showed that OsNHX3, OsSOS1, OsHKT2;1 and OsHKT1;5 genes were considerably upregulated in AM plants under saline conditions as compared to non-AM plants. Results suggest that the AM symbiosis favours Na⁺ extrusion from the cytoplasm, its sequestration into the vacuole, the unloading of Na⁺ from the xylem and its recirculation from photosynthetic organs to roots. As a result, there is a decrease of Na⁺ root-to-shoot distribution and an increase of Na⁺ accumulation in rice roots which seems to enhance the plant tolerance to salinity and allows AM rice plants to maintain their growing processes under salt conditions.     

Overexpression of AeNHX1, a root-specific vacuolar Na+/H+ antiporter from Agropyron elongatum, confers salt tolerance to Arabidopsis and Festuca plants

Agropyron elongatum, a species in grass family, has a strong tolerance to salt stress. To study the molecular mechanism of Agropyron elongatum in salt tolerance, we isolated a homolog of Na⁺/H⁺ antiporters from the root tissues of Agropyron plants. Sequence analysis revealed that this gene encodes a putative vacuolar Na⁺/H⁺ antiporter and was designated as AeNHX1. The AeNHX1–GFP fusion protein was clearly targeted to the vacuolar membrane in a transient transfection assay. Northern analysis indicated that AeNHX1 was expressed in a root-specific manner. Expression of AeNHX1 in yeast Na⁺/H⁺ antiporter mutants showed function complementation. Further, overexpression of AeNHX1 promoted salt tolerance of Arabidopsis plants, and improved osmotic adjustment and photosynthesis which might be responsible for normal development of transgenic plants under salt stress. Similarly, AeNHX1 also functioned in transgenic Festuca plants. The results suggest that this gene might function in the roots of Agropyron plants, and its expression is involved in the improvement of salt tolerance.     

The endophyte Serendipita indica reduces the sodium content of Arabidopsis plants exposed to salt stress: fungal ENA ATPases are expressed and regulated at high pH and during plant co-cultivation in salinity

Serendipita indica (formerly Piriformospora indica) is an endophytic fungus that colonizes plant roots producing a beneficial effect on plant growth and development under optimal and suboptimal conditions provoked by any biotic or abiotic stress, such as salt stress. Salinity induces osmotic and ionic imbalances in plants, mainly by altering the Na⁺ and K⁺ contents. However, the mechanism by which Serendipita improves plant growth has yet to be elucidated. Previous works suggest that this fungus improves the plant osmotic state but not much is known about whether it participates in readjustment of the ionic imbalance in plants. Here, we report that co-cultivation with Serendipita reduces the Na⁺ content of Arabidopsis plants under saline conditions. Additionally, we describe the functional characterization of the two Serendipita ENA ATPases, which are homologous to the main proteins involved in the salt tolerance of other fungi. Their heterologous expression in salt-sensitive yeast mutants shows that SiENA1 is involved in Na⁺ and K⁺ efflux, while SiENA5 seems to only be involved in Na⁺ detoxification. Both are induced and might have a relevant function at alkaline pH, condition in which there are few chlamydospores in the mycelium, as well as during co-cultivation with plants exposed to saline conditions.     

Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.)

Trifoliate orange (Poncirus trifoliata L. Raf.), a rootstock widely used for citrus species, is salt-sensitive. Worldwide, salinity is a major abiotic stress affecting citrus growth and yield. Glycinebetaine (GB) is an important osmoprotectant involved in responses to salt stress. However, current evidence regarding the effect of salt stress on GB accumulation in trifoliate orange is limited, and the GB synthesis gene has not yet been shown to confer enhanced salt stress tolerance to this species in a transgenic context. In the current study, we first examined the change in GB level of trifoliate orange seedlings exposed to salt stress, and found that salt increased endogenous GB level in a concentration-dependent manner. A betaine aldehyde dehydrogenase gene (AhBADH) cloned from Atriplex hortensis was introduced into the trifoliate orange by means of Agrobacterium-mediated transformation. RT-PCR analysis on three selected transgenic lines showed that the AhBADH gene was overexpressed in each of them. GB levels in these lines were also higher than those in untransformed wild-type (WT) plants. In the transgenic lines, exposure to 200 mM NaCl resulted in significantly less serious leaf burning and defoliation, lower MDA accumulation, and higher chlorophyll contents than those in the WT plants. Moreover, when exposed to salt, shoots of transgenic plants contained lower levels of Na⁺ and Cl⁻, higher levels of K⁺, and a higher K/Na ratio, while the same was true for the roots in most cases. Taken together, the data suggest that overexpression of the AhBADH gene in transgenic trifoliate orange enhanced salt stress tolerance. This may be correlated with the low levels of lipid peroxidation, protection of the photosynthetic machinery, and increase in K⁺ uptake.     

Effects of 5-aminolevulinic acid on nitrogen metabolism and ion distribution of watermelon seedlings under salt stress

The effects of foliar spray application of 5-aminolevulinic acid (ALA) on the growth, nitrogen metabolism, and ion distribution of salt-stressed watermelon (Citrullus lanatus (Thunb.) Matsum. and Nakai) seedlings were investigated. Supplementation of the nutrient solution with 100 mM NaCl significantly reduced leaf and root biomass of watermelon plants. Foliar application of 1.25 mM ALA significantly alleviated the inhibition of plant growth under salt stress. Salinity induced significant accumulation of nitrate, ammonium, and soluble protein and a significant decrease in the activities of nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH) in watermelon plants. However, ALA significantly increased the activities of NR, GS, GOGAT, and GDH, but decreased the ammonium content and NiR activity. In addition, salt stress resulted in significant accumulation of Na⁺ and Cl^? in plants, but decreased the contents of K⁺ and Mg²⁺. Application of ALA alleviated the salt stress-induced ion toxicity, and increased the contents of K⁺ and Mg²⁺. ALA also increased soluble protein and proline contents in salt-stressed watermelon plants. These results indicated that application of ALA alleviated the accumulation of Na⁺ and Cl^? in salt-stressed watermelon plants, especially through regulating nitrogen metabolism and ion distribution, which were associated with an improvement in plant growth.     

Effects of silicon on enzyme activity and sodium,potassium and calcium concentration in barley under salt stress

Two contrasting barley (Hordeum vulgare L.) cultivars: Kepin No.7 (salt sensitive), and Jian 4 (salt tolerant) were grown in a hydroponics system containing 120 mol m^-3 NaCl only and 120 mol m^-3 NaCl with 1.0 mol m^-3 Si (as potassium silicate). Compared with the plants treated with salt alone, superoxide dismutase (SOD) activity in plant leaves and H⁺-ATPase activity in plant roots increased, and malondialdehyde (MDA) concentration in plant leaves decreased significantly for both cultivars when treated with salt and Si. The addition of Si was also found to reduce sodium but increase potassium concentrations in shoots and roots of salt-stressed barley. Sodium uptake and transport into shoots from roots was greatly inhibited by added Si under salt stress conditions. However, Si addition exhibited little effect on calcium concentrations in shoots of salt-stressed barley. Thus, Si-enhanced salt tolerance is attributed to selective uptake and transport of potassium and sodium by plants. The results of the present study suggest that Si is involved in the metabolic or physiological changes in plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparative effects of osmotic-, salt- and alkali stress on growth,photosynthesis, and osmotic adjustment of cotton plants

In this study, cotton seedlings were subjected to osmotic-, salt- and alkali stresses. The growth, photosynthesis, inorganic ions, and organic acids in the stressed seedlings were measured, to compare the mechanisms by which plants adapt to these stresses and attempt to probe the mechanisms by which plants adapt to high pH stress. Our results indicated that, at high stress intensity, both osmotic and alkali stresses showed a stronger injurious effect on growth and photosynthesis than salt stress. Cotton accumulated large amount of Na+ under salt and alkali stresses, but not under osmotic stress. In addition, the reductions of K⁺, NO₃ ⁻, and H₂PO₄ ⁻ under osmotic stress were much greater than those under salt stress with increasing stress intensity. The lack of inorganic ions limited water uptake and was the main reason for the higher injury from osmotic-compared to salt stress on cotton. Compared with salt- and alkali stresses, the most dramatic response to osmotic stress was the accumulation of soluble sugars as the main organic osmolytes. In addition, we found that organic acid metabolism adjustment may play different roles under different types of stress. Under alkali stress, organic acids might play an important role in maintaining ion balance of cotton; however, under osmotic stress, malate might play an important osmotic role.     

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.