Increase in Salt Tolerance of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> by <Emphasis Type="Italic">TaDi19</Emphasis>

The gene expression profile chip of salt-resistant wheat mutant RH8706-49 under salt stress was investigated. The overall length of the cDNA sequence of the probe was obtained using electronic cloning and RT-PCR. An unknown gene induced by salt was obtained, cloned, and named TaDi19 (Triticum aestivum drought-induced protein). No related report or research on the protein is available. qPCR analysis showed that gene expression was induced by many stresses, such as salt. Arabidopsis thaliana was genetically transferred using the overexpressing gene, which increased its salt tolerance. After salt stress, the transgenic plant demonstrated better physiological indicators (higher Ca²⁺ and lower Na⁺) than those of the wild-type plant. Results of non-invasive micro-test technology indicate that TaDi19-overexpressing A. thaliana significantly effluxed Na⁺ after salt treatment, whereas the wild-type plant influxed Na⁺. Chelating extracellular Ca²⁺ resulted in insignificant differences in salt tolerance between overexpressing and wild-type A. thaliana. Subcellular localization showed that the gene encoding protein was mainly located in the cell membrane and nucleus. TaDi19 was overexpressed in wild-type A. thaliana, and the transgenic lines were more salt-tolerant than the control A. thaliana. Thus, the wheat gene TaDi19 could increase the salt tolerance of A. thaliana.     

<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.     

Salt tolerance of two perennial grass <Emphasis Type="Italic">Brachypodium sylvaticum</Emphasis> accessions

Key message

We studied the salt stress tolerance of two accessions isolated from different areas of the world (Norway and Tunisia) and characterized the mechanism(s) regulating salt stress in Brachypodium sylvaticum Osl1 and Ain1.

Abstract

Perennial grasses are widely grown in different parts of the world as an important feedstock for renewable energy. Their perennial nature that reduces management practices and use of energy and agrochemicals give these biomass crops advantages when dealing with modern agriculture challenges such as soil erosion, increase in salinized marginal lands and the runoff of nutrients. Brachypodium sylvaticum is a perennial grass that was recently suggested as a suitable model for the study of biomass plant production and renewable energy. However, its plasticity to abiotic stress is not yet clear. We studied the salt stress tolerance of two accessions isolated from different areas of the world and characterized the mechanism(s) regulating salt stress in B. sylvaticum Osl1, originated from Oslo, Norway and Ain1, originated from Ain-Durham, Tunisia. Osl1 limited sodium transport from root to shoot, maintaining a better K/Na homeostasis and preventing toxicity damage in the shoot. This was accompanied by higher expression of HKT8 and SOS1 transporters in Osl1 as compared to Ain1. In addition, Osl1 salt tolerance was accompanied by higher abundance of the vacuolar proton pump pyrophosphatase and Na⁺/H⁺ antiporters (NHXs) leading to a better vacuolar pH homeostasis, efficient compartmentation of Na⁺ in the root vacuoles and salt tolerance. Although preliminary, our results further support previous results highlighting the role of Na⁺ transport systems in plant salt tolerance. The identification of salt tolerant and sensitive B. sylvaticum accessions can provide an experimental system for the study of the mechanisms and regulatory networks associated with stress tolerance in perennials grass.

     

Water sources and water-use efficiency of desert plants in different habitats in Dunhuang,NW China

To understand habitat associated differences in desert plant water-use patterns, water stable oxygen isotope composition was used to determine water source and leaf carbon isotope composition (δ ¹³C) was used to estimate long-term water-use efficiency in three typical habitats (saline land, sandy land and Gobi) in Dunhuang. The primary findings are: (1) in the three habitats, plant species used mainly deep soil water (>120 cm), except for Kalidium foliatum in the saline land, which relied primarily on 0–40 cm soil water; (2) in the saline land and Gobi habitat, Alhagi sparsifolia had the most negative foliar δ ¹³C; in the sandy land, Elaeagnus angustifolia leaf was enriched in ¹³C than the other three species in 2011, but no species differences in foliar δ ¹³C was observed among the four species in 2012; (3) common species (Tamarix ramosissima and A. sparsifolia) may alter their water sources to cope with habitat differences associated changes in soil water availability with deeper water sources were used in the Gobi habitat with lower soil water content (SWC) compared to in the saline land and sandy land; (4) we detected significant habitat differences in foliar δ ¹³C in A. sparsifolia which may have resulted from differences in SWC and soil electrical conductivity. However, no habitat differences in foliar δ ¹³C were observed in T. ramosissima, which may attribute to the strong genetic control in T. ramosissima or the ability to access stable deep soil water. Overall, the results suggest that extremely arid climate, root distribution and soil properties worked together to determine plant water uptake in Dunhuang area.     

Salt stress reveals differential physiological,biochemical and molecular responses in <Emphasis Type="Italic">T. monococcum</Emphasis> and <Emphasis Type="Italic">T. durum</Emphasis> wheat genotypes

Salt stress responses implicate a complex mechanism and differ from plant species to another. In this study, we analyzed the physiological, biochemical and molecular responses to salt stress of the diploid wheat (T. monococcum) and compared to the tetraploid wheat (T. durum). Our results showed that the diploid wheat cultivar (cv. Turkey) is relatively tolerant to different salt stress conditions than the tetraploid wheat cultivar (cv. Om Rabia3). This tolerance was manifested by significant germination, plant growth and uptake of water generating cell turgor and development. Moreover, total chlorophyll content was higher in the diploid wheat than that in the tetraploid wheat. The Na⁺ content in leaf blade of the cv. Om Rabia3 was significantly higher than that of the cv. Turkey, suggesting that the diploid cultivar accumulates less toxic sodium in the photosynthetic tissues. This mechanism could be explained by the recirculation of the toxic ions Na⁺ into the xylem sap by SOS1 protein, which coordinates with HKT-like proteins to reduce the accumulation of Na⁺ ions in leaf blade. Interestingly, the expression of the three genes SOS1, HKT and NHX was enhanced under salinity especially in leaf blade of the cv. Turkey. Moreover, this wheat cultivar induced the antioxidative enzymes CAT and SOD activity more efficiently than the other cultivar.     

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K<Superscript>+</Superscript> and Na<Superscript>+</Superscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis> root cell

While the role of the vacuolar NHX Na⁺/H⁺ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na⁺ and K⁺ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na⁺/H⁺ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na⁺ and K⁺ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na⁺ into root cell vacuoles and also promoted high cytosolic and vacuolar K⁺ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na⁺ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na⁺ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na⁺ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na⁺ sequestration in the vacuole and K⁺ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.     

Significance of Na<Superscript>+</Superscript> and K<Superscript>+</Superscript> for Sustained Hydration of Organ Tissues in Ecologically Distinct Halophytes of the Family Chenopodiaceae

The contents of Na⁺, K⁺, water, and dry matter were measured in leaves and roots of euhalophytes Salicornia europaea L. and Climacoptera lanata (Pall.) Botsch featuring succulent and xeromorphic cell structures, respectively, as well as in saltbush Atriplex micrantha C.A. Mey, a halophyte having bladder-like salt glands on their leaves. All three species were able to accumulate Na⁺ in their tissues. The Na⁺ content in organs increased with elevation of NaCl concentration in the substrate, the concentrations of Na⁺ being higher in leaves than in roots. When these halophytes were grown on a NaCl-free substrate, a trend toward K⁺ accumulation was observed and was better pronounced in leaves than in roots. Particularly high K⁺ concentrations were accumulated in Salicornia leaves. There were no principal differences in the partitioning of Na⁺ and K⁺ between organs of three halophyte species representing different ecological groups. At all substrate concentrations of NaCl, the total content of Na⁺ and K⁺ in leaves was higher than in roots. This distribution pattern persisted in Atriplex possessing salt glands, as well as in euhalophytes Salicornia and Climacoptera. The physiological significance of such universal pattern of ion accumulation and distribution among organs in halophytes is related to the necessity of water absorption by roots, its transport to shoots, and maintenance of sufficient cell water content in all organs under high soil salinity.     

Physiological adaptation and gene expression analysis of <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> under salt stress

Casuarina equisetifolia is widely planted in coastal areas of tropical and subtropical regions as windbreaks or to stabilize dunes against wind erosion due to its high salt tolerance and nitrogen-fixing ability. To investigate the mechanisms responsible for its salt tolerance, we examined growth, mineral composition, expression of genes for sodium (Na⁺) and potassium (K⁺) transport proteins, and antioxidant responses under NaCl treatments. Increasing NaCl concentrations inhibited lateral root elongation and decreased plant height, length of internodes, and numbers of branches and twigs. The Na⁺ content significantly increased whereas the K⁺ content significantly decreased in both shoots and roots with increasing external NaCl concentration, resulting in a significant increase in Na⁺/K⁺ ratio. Most of the Na⁺/H⁺ antiporter genes (NHXs) were obviously upregulated in roots after 24 and 168 h of salt stress, and NHX7 was especially induced after 168 h. Almost all salt overly sensitive (SOS) genes were induced after 168-h treatment. Additionally, activities of superoxide dismutase, glutathione peroxidase, and catalase were significantly changed in shoots and roots under salt stress. Hence, we conclude that salinity tolerance of C. equisetifolia mainly relied on sequestering excess Na⁺ into vacuoles and on induced expression of NHX and SOS genes in roots and thus the maintenance of sufficient K⁺ content in shoots.     

Molecular characterization and expression analysis of the Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchanger gene family in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

One important mechanism plants use to cope with salinity is keeping the cytosolic Na⁺ concentration low by sequestering Na⁺ in vacuoles, a process facilitated by Na⁺/H⁺ exchangers (NHX). There are eight NHX genes (NHX1 through NHX8) identified and characterized in Arabidopsis thaliana. Bioinformatics analyses of the known Arabidopsis genes enabled us to identify six Medicago truncatula NHX genes (MtNHX1, MtNHX2, MtNHX3, MtNHX4, MtNHX6, and MtNHX7). Twelve transmembrane domains and an amiloride binding site were conserved in five out of six MtNHX proteins. Phylogenetic analysis involving A. thaliana, Glycine max, Phaseolus vulgaris, and M. truncatula revealed that each individual MtNHX class (class I: MtNHX1 through 4; class II: MtNHX6; class III: MtNHX7) falls under a separate clade. In a salinity-stress experiment, M. truncatula exhibited ~?20% reduction in biomass. In the salinity treatment, sodium contents increased by 178 and 75% in leaves and roots, respectively, and Cl^? contents increased by 152 and 162%, respectively. Na⁺ exclusion may be responsible for the relatively smaller increase in Na⁺ concentration in roots under salt stress as compared to Cl^?. Decline in tissue K⁺ concentration under salinity was not surprising as some antiporters play an important role in transporting both Na⁺ and K ⁺ . MtNHX1, MtNHX6, and MtNHX7 display high expression in roots and leaves. MtNHX3, MtNHX6, and MtNHX7 were induced in roots under salinity stress. Expression analysis results indicate that sequestering Na⁺ into vacuoles may not be the principal component trait of the salt tolerance mechanism in M. truncatula and other component traits may be pivotal.     

An Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter gene from wheat plays an important role in stress tolerance

A vacuole Na⁺/H⁺ antiporter gene TaNHX2 was obtained by screening the wheat cDNA library and by the 5′-RACE method. The expression of TaNHX2 was induced in roots and leaves by treatment with NaCl, polyethylene glycol (PEG), cold and abscisic acid (ABA). When expressed in a yeast mutant (Δnhx1), TaNHX2 suppressed the salt sensitivity of the mutant, which was deficient in vacuolar Na⁺/H⁺ antiporter, and caused partial recovery of growth of Δnhx1 in NaCl and LiC1 media. The survival rate of yeast cells was improved by overexpressing the TaNHX2 gene under NaCl, KCl, sorbitol and freezing stresses when compared with the control. The results imply that TaNHX2 might play an important role in salt and osmotic stress tolerance in plant cells.     

Soil pH and electrical conductivity are key edaphic factors shaping bacterial communities of greenhouse soils in Korea

Soil microorganisms play an essential role in soil ecosystem processes such as organic matter decomposition, nutrient cycling, and plant nutrient availability. The land use for greenhouse cultivation has been increasing continuously, which involves an intensive input of agricultural materials to enhance productivity; however, relatively little is known about bacterial communities in greenhouse soils. To assess the effects of environmental factors on the soil bacterial diversity and community composition, a total of 187 greenhouse soil samples collected across Korea were subjected to bacterial 16S rRNA gene pyrosequencing analysis. A total of 11,865 operational taxonomic units at a 97% similarity cutoff level were detected from 847,560 sequences. Among nine soil factors evaluated; pH, electrical conductivity (EC), exchangeable cations (Ca²⁺, Mg²⁺, Na⁺, and K⁺), available P₂O₅, organic matter, and NO₃-N, soil pH was most strongly correlated with bacterial richness (polynomial regression, pH: R² = 0.1683, P < 0.001) and diversity (pH: R² = 0.1765, P < 0.001). Community dissimilarities (Bray-Curtis distance) were positively correlated with Euclidean distance for pH and EC (Mantel test, pH: r = 0.2672, P < 0.001; EC: r = 0.1473, P < 0.001). Among dominant phyla (> 1%), the relative abundances of Proteobacteria, Gemmatimonadetes, Acidobacteria, Bacteroidetes, Chloroflexi, and Planctomycetes were also more strongly correlated with pH and EC values, compared with other soil cation contents, such as Ca²⁺, Mg²⁺, Na⁺, and K⁺. Our results suggest that, despite the heterogeneity of various environmental variables, the bacterial communities of the intensively cultivated greenhouse soils were particularly influenced by soil pH and EC. These findings therefore shed light on the soil microbial ecology of greenhouse cultivation, which should be helpful for devising effective management strategies to enhance soil microbial diversity and improving crop productivity.