Mapping of QTLs Controlling Na+, K+ and CI− Ion Concentrations in Salt Tolerant Indica Rice Variety CSR27

Soil salinity and sodicity are major constraints to rice production in about twenty per cent of the irrigated crop land. Inbuilt genetic tolerance to salinity is the most economical and environmentally sustainable way to solve this problem. A mapping population of 200 F₂ plants and their corresponding F₃ families, derived from a cross between a salt tolerant indica rice variety CSR27 and a salt sensitive variety MI48 were used to map OTLs for salt tolerance. Seventeen different parameters, including seedling salt injury score, Na⁺, K⁺, CI^? concentrations and Na⁺/K⁺ ratio in leaf and stem tissues at vegetative and reproductive stages were mapped. A framework linkage map was constructed using 79 SSR and EST markers distributed over the twelve rice chromosomes at an average interval of 20.7cM and total map distance of 1634.5 cM. Twenty five major OTLs, each explaining more than ten per cent of the trait phenotypic variance, were mapped on chromosomes 1, 2, 3 and 8. These included one OTL for seedling salt injury score, nine for Na⁺ concentration, three for K⁺ concentration and four for Cl^? concentration in leaf and stem tissues at vegetative and reproductive stages. The Na⁺/K⁺ ratio, an important ion balancing parameter for the salt tolerance, was controlled by eight OTLs explaining phenotypic variance in the range of 42.88–52.63%. Four OTL intervals were robust with major effect and having OTLs for multiple salt tolerance parameters that might be governed by common or tightly linked genes. One major OTL for multiple salt tolerance parameters on chromosome 8 and three major OTLs for CI^? ion concentration are novel for this study. The OTLs identified here will serve as a base for fine mapping, gene tagging and marker assisted selection for salt tolerance in rice.     

Disruption of <Emphasis Type="Italic">RCI2A</Emphasis> leads to over-accumulation of Na<Superscript>+</Superscript> and increased salt sensitivity in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> plants

For plant salt tolerance, it is important to regulate the uptake and accumulation of Na⁺ ions. The yeast pmp3 mutant which lacks PMP3 gene accumulates excess Na⁺ ions in the cell and shows increased Na⁺ sensitivity. Although the function of PMP3 is not fully understood, it is proposed that PMP3 contributes to the restriction of Na⁺ uptake and consequently salt tolerance in yeasts. In this paper, we have investigated whether the lack of RCI2A gene, homologous to PMP3 gene, causes a salt sensitive phenotype in Arabidopsis (Arabidopsis thaliana (L.) Heynh.) plants; and to thereby indicate the physiological role of RCI2A in higher plants. Two T-DNA insertional mutants of RCI2A were identified. Although the growth of rci2a mutants was comparable with that of wild type under normal conditions, high NaCl treatment caused increased accumulation of Na⁺ and more reduction of the growth of roots and shoots of rci2a mutants than that of wild type. Undifferentiated callus cultures regenerated from rci2a mutants also accumulated more Na⁺ than that from wild type under high NaCl treatment. Furthermore, when wild-type and rci2a plants were treated with NaCl, NaNO₃, Na₂SO₄, KCl, KNO₃, K₂SO₄ or LiCl, the rci2a mutants showed more reduction of shoot growth than wild type. Under treatments of tetramethylammonium chloride, CaCl₂, MgCl₂, mannitol or sorbitol, the growth reduction was comparable between wild-type and rci2a plants. These results suggested that RCI2A plays a role directly or indirectly for avoiding over-accumulation of excess Na⁺ and K⁺ ions in plants, and contributes to salt tolerance.     

RETRACTED ARTICLE: Analysis of the physiological mechanism of salt-tolerant transgenic rice carrying a vacuolar Na+/H+ antiporter gene from Suaeda salsa

Salt stress is one of the most serious factors limiting the productivity of agricultural crops. Increasing evidence has demonstrated that vacuolar Na⁺/H⁺ antiporters play a crucial role in plant salt tolerance. In the present study, we expressed the Suaeda salsa vacuolar Na⁺/H⁺ antiporter SsNHX1 in transgenic rice to investigate whether this can increase the salt tolerance of rice, and to study how overexpression of this gene affected other salt-tolerant mechanisms. It was found that transgenic rice plants showed markedly enhanced tolerance to salt stress and to water deprivation compared with non-transgenic controls upon salt stress imposition under outdoor conditions. Measurements of ion levels indicated that K⁺, Ca²⁺ and Mg²⁺ contents were all higher in transgenic plants than in non-transformed controls. Furthermore, shoot V-ATPase hydrolytic activity was dramatically increased in transgenics compared to that of non-transformed controls under salt stress conditions. Physiological analysis also showed that the photosynthetic activity of the transformed plants was higher whereas the same plants had reduced reactive oxygen species generation. In addition, the soluble sugar content increased in the transgenics compared with that in non-transgenics. These results imply that up-regulation of a vacuolar Na⁺/H⁺ antiporter gene in transgenic rice might cause pleiotropic up-regulation of other salt-resistance-related mechanisms to improve salt tolerance.Fengyun Zhao and Zenglan Wang contributed equally to this work.     

Salt tolerance in rice in vitro: Implication of accumulation of Na+, K+ and proline

The implication of accumulation of both inorganic (Na⁺, K⁺) and organic (proline) solutes were evaluated in unadapted and NaCl-adapted callus of a salt-sensitive (Basmati 370) and a salt-tolerant (SR-26B) cultivar of rice (Oryza sativa L.) after a NaCl shock. Accumulation of Na⁺,K⁺ and/or proline in callus was co relatable and the relative presence of these components in tissues after shock treatment was found to be important factors to support differential regrowth capacities of the shock treated calluses. Presence or retention of K⁺ in rice callus was a key factor for salt tolerance as it was observed to be positively correlated with growth in both the varieties. The results indicated that K⁺ was the first candidate to counteract the negative water potential of outside milieu, while proline was probably the last metabolic device that rice calluses opted for when exposed to salt stress.     

<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.     

Molecular and functional analyses of rice NHX-type Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter genes

We previously cloned a vacuolar Na⁺/H⁺ antiporter gene (OsNHX1) from rice (Oryza sativa). Here we identified four additional NHX-type antiporter genes in rice (OsNHX2 through OsNHX5) and performed molecular and functional analyses of those genes. The exon–intron structure of the OsNHX genes and the phylogenetic tree of the OsNHX proteins suggest that the OsNHX proteins are categorized into two subgroups (OsNHX1 through OsNHX4 and OsNHX5). OsNHX1, OsNHX2, OsNHX3, and OsNHX5 can suppress the Na⁺, Li⁺, and hygromycin sensitivity of yeast nhx1 mutants and their sensitivity to a high K⁺ concentration. The expression of OsNHX1, OsNHX2, OsNHX3, and OsNHX5 is regulated differently in rice tissues and is increased by salt stress, hyperosmotic stress, and ABA. When we studied the expression of β-glucuronidase (GUS) driven by either the OsNHX1 or the OsNHX5 promoter, we observed activity in the stele, the emerging part of lateral roots, the vascular bundle, the water pore, and the basal part of seedling shoots with both promoters. In addition, each promoter had a unique expression pattern. OsNHX1 promoter–GUS activity only was localized to the guard cells and trichome, whereas OsNHX5 promoter–GUS activity only was localized to the root tip and pollen grains. Our results suggest that the members of this gene family play important roles in the compartmentalization into vacuoles of the Na⁺ and K⁺ that accumulate in the cytoplasm and that the differential regulation of antiporter gene expression in different rice tissues may be an important factor determining salt tolerance in rice.     

Increased tolerance to salt stress in the phosphate-accumulating <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants <Emphasis Type="Italic">siz1</Emphasis> and <Emphasis Type="Italic">pho2</Emphasis>

High salinity is an environmental factor that inhibits plant growth and development, leading to large losses in crop yields. We report here that mutations in SIZ1 or PHO2, which cause more accumulation of phosphate compared with the wild type, enhance tolerance to salt stress. The siz1 and pho2 mutations reduce the uptake and accumulation of Na⁺. These mutations are also able to suppress the Na⁺ hypersensitivity of the sos3-1 mutant, and genetic analyses suggest that SIZ1 and SOS3 or PHO2 and SOS3 have an additive effect on the response to salt stress. Furthermore, the siz1 mutation cannot suppress the Li⁺ hypersensitivity of the sos3-1 mutant. These results indicate that the phosphate-accumulating mutants siz1 and pho2 reduce the uptake and accumulation of Na⁺, leading to enhanced salt tolerance, and that, genetically, SIZ1 and PHO2 are likely independent of SOS3-dependent salt signaling.     

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.     

高盐生境中乌拉尔甘草盐离子分泌与积存格局的研究

采用植物水培方法,以乌拉尔甘草为研究材料,用不同浓度(0、80、160、320mmol·L~(-1))NaCl溶液胁迫处理乌拉尔甘草幼苗3周后,分析其叶片表面盐离子(K~+、Ca~(2+)、Na+)分泌速率的差异,并采集盐化低地草甸重盐土生境中2年生乌拉尔甘草植株,应用ICP-AES测定其不同部位(根、根状茎、茎、老叶和幼叶)中的盐离子(K~+、Na~+、Ga~(2+)、Mg~(2+))含量,探究盐离子在乌拉尔甘草叶片上的分泌格局以及盐离子在植株体内的积存格局,为完善甘草耐盐机理的研究提供依据。结果显示:(1)随着盐胁迫浓度的升高,乌拉尔甘草叶片上K~+、Ca~(2+)、Na+的分泌速率均呈增加趋势,且Na~+的分泌速率远远大于Ca~(2+)和K+的分泌速率。(2)在乌拉尔甘草各部位中,K+的积存量从大到小依次为:幼叶根根状茎茎老叶;Na~+在各个部位的积存量都十分有限,且无论地下部分还是地上部分,差异均不大;Ca~(2+)积存量由大到小依次为:老叶幼叶茎根状茎根,且老叶中Ca~(2+)的积存量显著高于其它部位。研究认为,在重盐碱地生境中,K+主要积存在幼叶中,Ga~(2+)主要积存在老叶中,植株体内各个部位Na~+的积存量很低,乌拉尔甘草表现出明显的拒Na现象;叶片分泌的主要盐离子为Na~+;乌拉尔甘草通过泌盐的方式将Na+排出体外,从而有效降低Na~+在体内的积存,这是其能够在重盐碱地生存生长的重要原因。     

QTL Analysis of Na+ and K+ Concentrations in Roots and Shoots under Different Levels of NaCl Stress in Rice (Oryza sativa L.)

The key to plant survival under NaCl salt stress is maintaining a low Na⁺ level or Na⁺/K⁺ ratio in the cells. A population of recombinant inbred lines (RILs, F_2∶9) derived from a cross between the salt-tolerant japonica rice variety Jiucaiqing and the salt-sensitive indica variety IR26, was used to determine Na⁺ and K⁺ concentrations in the roots and shoots under three different NaCl stress conditions (0, 100 and 120 mM NaCl). A total of nine additive QTLs were identified by QTL Cartographer program using single-environment phenotypic values, whereas eight additive QTLs were identified by QTL IciMapping program. Among these additive QTLs, five were identified by both programs. Epistatic QTLs and QTL-by-environment interactions were detected by QTLNetwork program in the joint analyses of multi-environment phenotypic values, and one additive QTL and nine epistatic QTLs were identified. There were three epistatic QTLs identified for Na⁺ in roots (RNC), three additive QTLs and two epistatic QTLs identified for Na⁺ in shoots (SNC), four additive QTLs identified for K⁺ in roots (RKC), four additive QTLs and three epistatic QTLs identified for K⁺ in shoots (SKC) and one additive QTL and one epistatic QTL for salt tolerance rating (STR). The phenotypic variation explained by each additive, epistatic QTL and QTL×environment interaction ranged from 8.5 to 18.9%, 0.5 to 5.3% and 0.7 to 7.5%, respectively. By comparing the chromosomal positions of these additive QTLs with those previously identified, five additive QTLs, qSNC9, qSKC1, qSKC9, qRKC4 and qSTR7, might represent novel salt tolerance loci. The identification of salt tolerance in selected RILs showed that a major QTL qSNC11 played a significant role in rice salt tolerance, and could be used to improve salt tolerance of commercial rice varieties with marker-assisted selection (MAS) approach.     

Salt Tolerance in Aquatic Macrophytes: Ionic Relation and Interaction

Effects of seawater salinity (SWS) and pure NaCl on the intracellular contents of Na⁺, K⁺, Mg²⁺, Ca²⁺, chlorophylls (Chl) and carotenoids (Car) were studied in three submerged aquatic macrophytes, Hydrilla verticillata, Najas indica and Najas gramenia, which differed in their tolerance to salinity. NaCl resulted in significant increase in Chl/Car ratio in the salt-sensitive H. verticillata and moderately salt-tolerant N. indica, but not in the salt-tolerant N. gramenia. SWS treatment did not result in any significant change in the ratio. The intracellular content of Na⁺ increased significantly in all the test plants upon exposure to both NaCl and SWS. The content of K⁺ decreased significantly in these plants upon salinity treatment, except in N. gramenia. The contents of Ca²⁺ and Mg²⁺ decreased significantly upon NaCl treatment and remained unchanged or increased upon SWS treatment. No relationship between salt tolerance and K⁺/Na⁺ ratio was observed. The maintenance of a minimal level of K⁺ was observed to be the most probable requirement of salt tolerance in aquatic macrophytes.     

Evaluation of salinity tolerance and analysis of allelic function of <Emphasis Type="Italic">HvHKT1</Emphasis> and <Emphasis Type="Italic">HvHKT2</Emphasis> in Tibetan wild barley

Tibetan wild barley is rich in genetic diversity with potential allelic variation useful for salinity-tolerant improvement of the crop. The objectives of this study were to evaluate salinity tolerance and analysis of the allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Salinity tolerance of 189 Tibetan wild barley accessions was evaluated in terms of reduced dry biomass under salinity stress. In addition, Na⁺ and K⁺ concentrations of 48 representative accessions differing in salinity tolerance were determined. Furthermore, the allelic and functional diversity of HvHKT1 and HvHKT2 was determined by association analysis as well as gene expression assay. There was a wide variation among wild barley genotypes in salt tolerance, with some accessions being higher in tolerance than cultivated barley CM 72, and salinity tolerance was significantly associated with K⁺/Na⁺ ratio. Association analysis revealed that HvHKT1 and HvHKT2 mainly control Na⁺ and K⁺ transporting under salinity stress, respectively, which was validated by further analysis of gene expression. The present results indicated that Tibetan wild barley offers elite alleles of HvHKT1 and HvHKT2 conferring salinity tolerance.     

Salinity-induced ion flux patterns from the excised roots of <Emphasis Type="Italic">Arabidopsis sos</Emphasis> mutants

The SOS signal-transduction pathway is known to be important for ion homeostasis and salt tolerance in plants. However, there is a lack of in planta electrophysiological data about how the changes in signalling and ion transport activity are integrated at the cellular and tissue level. In this study, using the non-invasive ion flux MIFE technique, we compared net K⁺, H⁺ and Na⁺ fluxes from elongation and mature root zones of Arabidopsis wild type Columbia and sos mutants. Our results can be summarised as follows: (1) SOS mutations affect the function of the entire root, not just the root apex; (2) SOS signalling pathway is highly branched; (3) Na⁺ effects on SOS1 may by-pass the SOS2/SOS3 complex in the root apex; (4) SOS mutation affects H⁺ transport even in the absence of salt stress; (5) SOS1 mutation affects intracellular K⁺ homeostasis with a plasma membrane depolarisation-activated outward-rectifying K⁺ channel being a likely target; (6) H⁺ pump also may be a target of SOS signalling. We provide an improved model of SOS signalling and discuss physiological mechanisms underlying salt stress perception and signalling in plants. Our work shows that in planta studies are essential for understanding the functional genomics of plant salt tolerance.     

Physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage

Salinity tolerance levels and physiological changes were evaluated for twelve rice cultivars, including four white rice and eight black glutinous rice cultivars, during their seedling stage in response to salinity stress at 100 mM NaCl. All the rice cultivars evaluated showed an apparent decrease in growth characteristics and chlorophyll accumulation under salinity stress. By contrast an increase in proline, hydrogen peroxide, peroxidase (POX) activity and anthocyanins were observed for all cultivars. The K⁺/Na⁺ ratios evaluated for all rice cultivars were noted to be highly correlated with the salinity scores thus indicating that the K⁺/Na⁺ ratio serves as a reliable indicator of salt stress tolerance in rice. Principal component analysis (PCA) based on physiological salt tolerance indexes could clearly distinguish rice cultivars into 4 salt tolerance clusters. Noteworthy, in comparison to the salt-sensitive ones, rice cultivars that possessed higher degrees of salt tolerance displayed more enhanced activity of catalase (CAT), a smaller increase in anthocyanin, hydrogen peroxide and proline content but a smaller drop in the K⁺/Na⁺ ratio and chlorophyll accumulation.     

Enhancement of the salt tolerance of Triticum turgidum L. by the Kna1 locus transferred from the Triticum aestivum L. chromosome 4D by homoeologous recombination

Durum wheat, Triticum turgidum L. (2n= 4x=28, genome formula AABB) is inferior to bread wheat, T. aestivum L. (2n=6x=42, genome formula AABBDD), in the ability to exclude Na⁺ under salt strees, in the ratio of the accumulated K⁺ to Na⁺ in the leaves under salt stress, and in tolerance of salt stress. Previous work showed that chromosome 4D has a major effect on Na⁺ and K⁺ accumulation in the leaves of bread wheat. The 4D chromosome was recombined with chromosome 4B in the genetic background of durum wheat. The recombinants showed that Na⁺ exclusion and enhanced K⁺/Na⁺ ratio in the shoots were controlled by a single locus, Kna1, in the long arm of chromosome 4D. The recombinant families were grown in the field under non-saline conditions and two levels of salinity to determine whether Kna1 confers salt tolerance. Under salt stress, the Kna1 families had higher K⁺/Na⁺ ratios in the flag leaves and higher yields of grain and biomass than the Kna1 ^- families and the parental cultivars. Kna1 is, therefore, one of the factors responsible for the higher salt tolerance of bread wheat relative to durum wheat. The present work provides conceptual evidence that tolerance of salt stress can be transferred between species in the tribe Triticeae.     

SbHKT1;4, a member of the high-affinity potassium transporter gene family from Sorghum bicolor,functions to maintain optimal Na＋/K＋ balance under Na＋ stress

In halophytic plants, the high-affinity potassium transporter HKT gene family can selectively uptake K＋ in the presence of toxic concentrations of Na＋. This has so far not been well examined in glycophytic crops. Here, we report the characterization of SbHKTI;4, a member of the HKT gene family from Sorghum bicolor. Upon Na＋ stress, SbHKT1;4 expression was more strongly upregulated in salt-tolerant sorghum accession, correlating with a better balanced Na＋/ K＋ ratio and enhanced plant growth. Heterogeneous expression analyses in mutants of Saccharomyces cerevisiae and Arabidopsis thaliana indicated that overexpressing SbHKT1;4 resulted in hypersensitivity to Na＋ stress, and such hypersensitivity could be alleviated with the supply of elevated levels of K＋, implicating that SbHKT1;4 may mediate K＋ uptake in the presence of excessive Na＋. Further electrophysiological evidence demonstrated that SbHKT1;4 could transport Na＋ and K＋ when expressed in Xenopus laevis oocytes. The relevance of the finding that SbHKTI;4 functions to maintain optimal Na＋/K＋ balance under Na＋ stress to the breeding of salt-tolerant glycophytic crops is discussed.