A single nucleotide substitution in TaHKT1;5-D controls shoot Na+ accumulation in bread wheat

Improving salinity tolerance in the most widely cultivated cereal, bread wheat (Triticum aestivum L.), is essential to increase grain yields on saline agricultural lands. A Portuguese landrace, Mocho de Espiga Branca accumulates up to sixfold greater leaf and sheath sodium (Na⁺) than two Australian cultivars, Gladius and Scout, under salt stress in hydroponics. Despite high leaf and sheath Na⁺ concentrations, Mocho de Espiga Branca maintained similar salinity tolerance compared to Gladius and Scout. A naturally occurring single nucleotide substitution was identified in the gene encoding a major Na⁺ transporter TaHKT1;5-D in Mocho de Espiga Branca, which resulted in a L190P amino acid residue variation. This variant prevents Mocho de Espiga Branca from retrieving Na⁺ from the root xylem leading to a high shoot Na⁺ concentration. The identification of the tissue-tolerant Mocho de Espiga Branca will accelerate the development of more elite salt-tolerant bread wheat cultivars.     

Cell-specific localization of Na+ in roots of durum wheat and possible control points for salt exclusion

Sodium exclusion from leaves is an important mechanism for salt tolerance in durum wheat. To characterize possible control points for Na(+) exclusion, quantitative cryo-analytical scanning electron microscopy was used to determine cell-specific ion profiles across roots of two durum wheat genotypes with contrasting rates of Na(+) transport from root to shoot grown in 50 mm NaCl. The Na(+) concentration in Line 149 (low transport genotype) declined across the cortex, being highest in the epidermal and sub-epidermal cells (48 mm) and lowest in the inner cortical cells (22 mm). Na(+) was high in the pericycle (85 mm) and low in the xylem parenchyma (34 mm). The Na(+) profile in Tamaroi (high transport genotype) had a similar trend but with a high concentration (130 mm) in the xylem parenchyma. The K(+) profiles were generally inverse to those of Na(+). Chloride was only detected in the epidermis. These data suggest that the epidermal and cortical cells removed most of the Na(+) and Cl(-) from the transpiration stream before it reached the endodermis, and that the endodermis is not the control point for salt uptake by the plant. The pericycle as well as the xylem parenchyma may be important in the control of net Na(+) loading of the xylem.     

The Na+ transporter AtHKT1;1 controls retrieval of Na+ from the xylem in Arabidopsis

HKT-type transporters appear to play key roles in Na(+) accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na(+) into roots, recirculate Na(+) in the phloem and control root : shoot allocation of Na(+). We tested these hypotheses using (22)Na(+) flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na(+) and retrieval of Na(+) from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K(+) transport and the hkt1;1 mutant showed altered net K(+) accumulation, (86)Rb(+) uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K(+); however, HKT1;1 knockout did not alter K(+) or (86)Rb(+) accumulation in sos1.     

Enhanced salt tolerance mediated by AtHKT1 transporter-induced Na unloading from xylem vessels to xylem parenchyma cells

AtHKT1 is a sodium (Na+) transporter that functions in mediating tolerance to salt stress. To investigate the membrane targeting of AtHKT1 and its expression at the translational level, antibodies were generated against peptides corresponding to the first pore of AtHKT1. Immunoelectron microscopy studies using anti-AtHKT1 antibodies demonstrate that AtHKT1 is targeted to the plasma membrane in xylem parenchyma cells in leaves. AtHKT1 expression in xylem parenchyma cells was also confirmed by AtHKT1 promoter-GUS reporter gene analyses. Interestingly, AtHKT1 disruption alleles caused large increases in the Na+ content of the xylem sap and conversely reduced the Na+ content of the phloem sap. The athkt1 mutant alleles had a smaller and inverse influence on the potassium (K+) content compared with the Na+ content of the xylem, suggesting that K+ transport may be indirectly affected. The expression of AtHKT1 was modulated not only by the concentrations of Na+ and K+ but also by the osmolality of non-ionic compounds. These findings show that AtHKT1 selectively unloads sodium directly from xylem vessels to xylem parenchyma cells. AtHKT1 mediates osmolality balance between xylem vessels and xylem parenchyma cells under saline conditions. Thus AtHKT1 reduces the sodium content in xylem vessels and leaves, thereby playing a central role in protecting plant leaves from salinity stress.     

The potassium transporter OsHAK21 functions in the maintenance of ion homeostasis and tolerance to salt stress in rice

The intracellular potassium (K⁺) homeostasis, which is crucial for plant survival in saline environments, is modulated by K⁺ channels and transporters. Some members of the high‐affinity K⁺ transporter (HAK) family are believed to function in the regulation of plant salt tolerance, but the physiological mechanisms remain unclear. Here, we report a significant inducement of OsHAK21 expression by high‐salinity treatment and provide genetic evidence of the involvement of OsHAK21 in rice salt tolerance. Disruption of OsHAK21 rendered plants sensitive to salt stress. Compared with the wild type, oshak21 accumulated less K⁺ and considerably more Na⁺ in both shoots and roots, and had a significantly lower K⁺ net uptake rate but higher Na⁺ uptake rate. Our analyses of subcellular localizations and expression patterns showed that OsHAK21 was localized in the plasma membrane and expressed in xylem parenchyma and individual endodermal cells (putative passage cells). Further functional characterizations of OsHAK21 in K⁺ uptake‐deficient yeast and Arabidopsis revealed that OsHAK21 possesses K⁺ transporter activity. These results demonstrate that OsHAK21 may mediate K⁺ absorption by the plasma membrane and play crucial roles in the maintenance of the Na⁺/K⁺ homeostasis in rice under salt stress.     

Comparative mapping of HKT genes in wheat, barley, and rice, key determinants of Na+ transport, and salt tolerance

Salt tolerance of plants depends on HKT transporters (High-affinityK⁺ Transporter), which mediate Na⁺-specific transport or Na⁺-K⁺co-transport. Gene sequences closely related to rice HKT geneswere isolated from hexaploid bread wheat (Triticum aestivum)or barley (Hordeum vulgare) for genomic DNA southern hybridizationanalysis. HKT gene sequences were mapped on chromosomal armsof wheat and barley using wheat chromosome substitution linesand barley–wheat chromosome addition lines. In addition,HKT gene members in the wild diploid wheat ancestors, T. monococcum(A^m genome), T. urartu (A^u genome), and Ae. tauschii (D^t genome)were investigated. Variation in copy number for individual HKTgene members was observed between the barley, wheat, and ricegenomes, and between the different wheat genomes. HKT2;1/2-like,HKT2;3/4-like, HKT1;1/2-like, HKT1;3-like, HKT1;4-like, andHKT1;5-like genes were mapped to the wheat–barley chromosomegroups 7, 7, 2, 6, 2, and 4, respectively. Chromosomal regionscontaining HKT genes were syntenic between wheat and rice exceptfor the chromosome regions containing the HKT1;5-like gene.Potential roles of HKT genes in Na⁺ transport in rice, wheat,and barley are discussed. Determination of the chromosome locationsof HKT genes provides a framework for future physiological andgenetic studies investigating the relationships between HKTgenes and salt tolerance in wheat and barley. Key words: Barley, comparative mapping, HKT, rice, salt tolerance, sodium transport, wheat     

Variations in green plant regeneration response from anthers of indica rice and their hybrids with japonica cv. Taipei 309

The green plant regeneration ability from anthers of BR-7, a high yielding indica cultivar, Binnatoa (BA), a salt tolerant indica land race and IR-43 was tested in N6, M8, He2 and R2 media. The response was calculated on the basis of number of anthers producing green plants. The number of green plants per responding anther was also recorded. The response of BR-7 and BA was poor compared to the indica cultivar IR-43 in three of the media that were tested. In N6 medium, green plant regeneration of BA and BR-7 was respectively 10-fold and 100-fold less than the japonica cultivar Taipei 309 (T-309). No anther-derived green regenerant was obtained from another salt tolerant indica land race, Rajashail (RAJ). The N6 medium was selected to test green plant regeneration frequency from anthers obtained from the F1 crosses of T-309 × BR-7, T-309 × BA, T-309 × RAJ and T-309 × BR-7 AC regenerants backcrossed with BA. Our objective was to combine the salt tolerant trait of BA and the high yield of BR-7 in a single line. The intermediate crossing step with T-309 was performed to increase the green plant regenerability of the anthers. All F1 progeny from the crosses with T-309 showed significantly increased callus induction compared to the indica parent although the values were lower than the midparent means. Green plant regeneration compared to their respective indica parents either increased or decreased but never approached the level of T-309. This revised version was published online in June 2006 with corrections to the Cover Date.     

不同类型盐生植物适应盐胁迫的生理生长机制及Na+逆向转运研究进展

盐生植物是指能在离子浓度至少200 mmol/L以上的生境中生长并完成生活史的植物。盐生植物可分为稀盐盐生植物、泌盐盐生植物、拒盐盐生植物三类。本文从生长形态、生理和分子3个方面总结三类盐生植物响应盐胁迫的不同策略及研究进展,发现盐生植物在分子水平上主要通过Na⁺转运蛋白和为其提供能量的两类基因应对体内过高Na⁺,这可能是引起盐生植物生理和生长形态异于非盐生植物的重要因素。其中稀盐盐生植物主要通过液泡离子区隔化应对盐胁迫,并表现出肉质化生长形态;泌盐盐生植物通过将体内盐分排出体外应对盐胁迫,并进化出特有的生理结构——盐腺或盐囊泡;拒盐盐生植物通过将盐离子积累在皮层细胞液泡和根部木质部薄壁细胞中减少向上运输Na⁺,同时根部多栓质化减少Na⁺吸收。本综述旨在为今后研究盐生植物及其耐盐机制提供相关依据,为植物耐盐分子育种奠定基础。     

Establishment of stable NaCl-resistant rice plant lines from anther culture: distribution pattern of K+/Na+ in callus and plant cells

 Soil salinity markedly suppresses the growth of rice (Oryza sativa L.). We established rice anther culture to select for rice callus lines adapted to NaCl stress and regenerated plant progenies resistant to a NaCl stress of E.C. 16–18 mS. When exposed to NaCl, NaCl-adapted rice calli lost K⁺ and accumulated little Na⁺. Conversely, plant cells lost relatively little K⁺ and accumulated Na⁺. It is plausible that, NaCl-resistant mechanisms are different at callus and plant level. The stable NaCl-resistant lines produced have potential use in elucidating the molecular mechanisms behind NaCl resistance in rice and in rice breeding. Received: 27 February 1997 / Accepted: 4 April 1997     

Biochemical and immunochemical determination of (Na+ + K+)-ATPase content in subcellular fractions prepared from the avian salt gland: evidence for enzyme heterogeneity

Subcellular membrane fractions were prepared from the salt glands of osmotically-stressed ducklings. Two fractions were characterized biochemically with respect to (Na+ + K+)-ATPase, alkaline phosphodiesterase I, succinate dehydrogenase, esterase, and galactosyltransferase activities and immunochemically with respect to (Na+ + K+)-ATPase. The ratios of the estimates of the (Na+ + K+)-ATPase contents obtained biochemically and immunochemically from the two fractions differed by more than 2 X. The results are consistent with the presence of at least two molecular species of (Na+ + K+)-ATPase, unevenly distributed between the two fractions.     

Differences in shoot Na+ accumulation between two tomato species are due to differences in ion affinity of HKT1;2

HKT1 has been shown to be essential in Na⁺ homeostasis in plants. In this paper, we report the analysis of Na⁺ accumulation in different plant organs of two tomato species with contrasting salt tolerances: Solanum lycopersicum and Solanum pennellii. Furthermore, we relate these differences in Na⁺ accumulation between the two species to the differences in HKT1;2 transport kinetics and HKT1;2 expression. S. lycopersicum showed “Na⁺ excluder” behaviour, whereas S. pennellii showed “Na⁺ includer” behaviour. SlHKT1;2 expression, in contrast to SpHKT1;2 expression showed a significant effect of NaCl treatment, especially stems had a high increase in SlHKT1;2 expression. SlHKT1;2 promoter-GUS reporter gene analysis showed that SlHKT1;2 is expressed in the vasculature surrounding the roots and shoots of transformed Arabidopsis plants. In this paper, we present HKT1;2 protein sequences of both tomato species and provide evidence that both SlHKT1;2 and SpHKT1;2 are Na⁺ transporters. Our kinetic studies showed that SpHKT1;2, in comparison with SlHKT1;2, had a lower affinity for Na⁺. This low affinity of SpHKT1;2 correlated with higher xylem Na⁺ and higher accumulation of Na⁺ in stems and leaves of S. pennellii. Our findings demonstrate the importance of the understanding of transport characteristics of HKT1;2 transporters to improve the understanding of Na⁺ homeostasis in plants.     

盐胁迫对4树种叶片中K^＋和Na^＋的影响及其耐盐能力的评价

通过盆栽试验,对我国南方银杏（Ginkgo biloba L.)、侧柏[Patycladus orentalis(L.)Franco]、火炬松（Pinus taeda L.)和剌槐(Robinia Pseudoacacia L.)4造林树种苗木叶片中K^ 、N^ 浓度浓度和N^ ／K^ 比进行测定,盐处理水平为0.0％（CK）、0.1%、0.3%和0.5％。随着盐浓度的提高,各树种叶片中Na^ 浓度和N^ ／K^ 有逐渐升高的趋势,但K^ 浓度变化较小;随着盐胁迫时间的推移,各树种叶片中Na^ 、K^ 浓度和Na^ /K^ 比都没有明显的变化规律。方差分析和多层比较表明,侧柏、火炬松和剌槐叶片中Na^ 、K^ 浓度和N^ ／K^ 比在4组处理间的差异均达显著或极显著水平。4树种中剌槐和侧柏的耐盐能力最强,银杏次之,火炬松最弱。     

NaCl胁迫下棉花体内 Na~+ 、K~+分布与耐盐性

采用盐化土壤方法 ,选择苗期耐盐性较强的陆地棉品种枝棉 3号和中棉所 1 9及耐盐性较弱的品种泗棉 2号和苏棉 1 2号 ,研究了盐胁迫下棉苗体内 Na+、K+的运输和分配与耐盐性的关系。结果表明 ,耐盐品种根系具有一定的截留 Na+作用。棉花地上部盐分器官水平上的区域化分布特征明显 :2 0 0 mmol/L Na Cl胁迫下 ,枝棉 3号叶片中的 Na+含量显著低于泗棉 2号 ,茎及叶柄中的 Na+含量显著高于泗棉 2号 ;棉株地上部茎、叶柄、叶片中的 Na+含量分别由下而上逐渐减小 ,相同节位的茎、叶柄中的 Na+含量大于叶片 ,枝棉 3号更显著。1 0 0 mmol/L和 1 50 mmol/L Na Cl胁迫下 ,枝棉 3号和中棉所 1 9K+/Na+显著高于泗棉 2号和苏棉 1 2号。Na+在茎和叶柄中滞留和积累 ,根中的 K+向地上部选择性运输 ,以维持叶片中较高的 K+/Na+,是棉花耐盐性的一个重要特点     

SUB1A‐dependent and ‐independent mechanisms are involved in the flooding tolerance of wild rice species

Crop tolerance to flooding is an important agronomic trait. Although rice (Oryza sativa) is considered a flood‐tolerant crop, only limited cultivars display tolerance to prolonged submergence, which is largely attributed to the presence of the SUB1A gene. Wild Oryza species have the potential to unveil adaptive mechanisms and shed light on the basis of submergence tolerance traits. In this study, we screened 109 Oryza genotypes belonging to different rice genome groups for flooding tolerance. Oryza nivara and Oryza rufipogon accessions, belonging to the A‐genome group, together with Oryza sativa, showed a wide range of submergence responses, and the tolerance‐related SUB1A‐1 and the intolerance‐related SUB1A‐2 alleles were found in tolerant and sensitive accessions, respectively. Flooding‐tolerant accessions of Oryza rhizomatis and Oryza eichingeri, belonging to the C‐genome group, were also identified. Interestingly, SUB1A was absent in these species, which possess a SUB1 orthologue with high similarity to O. sativa SUB1C. The expression patterns of submergence‐induced genes in these rice genotypes indicated limited induction of anaerobic genes, with classical anaerobic proteins poorly induced in O. rhizomatis under submergence. The results indicated that SUB1A‐1 is not essential to confer submergence tolerance in the wild rice genotypes belonging to the C‐genome group, which show instead a SUB1A‐independent response to submergence.     

Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

<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.     

A Bowman-Birk type protease inhibitor is involved in the tolerance to salt stress in wheat

A salt-responsive gene WRSI5 was characterized from salt-tolerant cultivar Shanrong No. 3 (SR3), an introgression line via asymmetric somatic hybrid between Triticum aestivum L. cv. Jinan177 (JN177) and Thinopyrum ponticum Podp. The peptide encoded by WRSI5 contains a Bowman-Birk domain sharing a high level of sequence identity to monocotyledonous protease inhibitors. When expressed in vitro , the WRSI5 gene product exhibited trypsin, but not chymotrypsin inhibition. The expression level of WRSI5 was increased in SR3 roots exposed to salt, drought or oxidative stress. In situ hybridization showed that it is induced in the endodermal cells of the mature region of the SR3 root tip, with no signal detectable in the corresponding region of the salt-susceptible cultivar JN177. SR3 has a higher selectivity for K⁺ over Na⁺, and therefore limits the transport of Na⁺ from the root to the shoot. When overexpressed in Arabidopsis thaliana , WRSI5 improves the ability of seedlings to grow on a medium containing 150 m m NaCl. We suggest that WRSI5 plays an important role in regulating the plant growth rate or long-distance Na⁺ transport in SR3 plants exposed to salt stress.     

High salt‐treatment‐induced Na+ extrusion and low salt‐treatment‐induced Na+ accumulation in suspension‐cultured cells of the mangrove plant,Bruguiera sexangula

A suspension‐cultured cell strain of the mangrove plant (Bruguiera sexangula) was established from a callus culture and maintained in an amino acid medium in the absence of NaCl. NaCl non‐adapted cells were transferred to media containing 0–200 mm NaCl. The initial growth rate decreased gradually with increasing salt concentrations. However, at up to 150 mm NaCl, cell number growth at the highest point was almost the same as that at lower salt concentrations. Cells even continued to grow in the presence of 200 mm NaCl. Cells incubated in a medium containing 50 mm NaCl for 3 weeks accumulated Na⁺, while those incubated in 150 mm NaCl for 2 d showed only a transient increase in Na⁺ and Cl^– concentrations. In the latter treatment, the intracellular concentration of Na⁺ returned to the original low level within 2 weeks. It took a longer time for Cl^– to return to its original level. As a result, the Na⁺ and Cl^– concentrations in cells cultured with 50 mm NaCl were much larger than those in cells cultured with 150 mm NaCl. The intracellular distribution of ions after transfer to the medium containing 150 mm NaCl was analysed by isolating the vacuoles. Treatment with amiloride, an inhibitor of the Na⁺/H⁺ antiporter, suppressed the recovery of Na⁺ to the original level in the cells. Treatment with 150 mm NaCl for 3 d stimulated the activities of both the vanadate‐dependent H⁺‐ATPase and the Na⁺/H⁺ antiporter in the plasma membrane fraction.     

Responses of photosynthesis, chlorophyll fluorescence and ROS-scavenging systems to salt stress during seedling and reproductive stages in rice

BACKGROUND AND AIMS: Salinity is a widespread soil problem limiting productivity of cereal crops worldwide. Rice is particularly sensitive to salt stress during the seedling stage, with consequent poor crop establishment, as well as during reproduction where salinity can severely disrupt grain formation and yield. Tolerance at the seedling stage is weakly associated with tolerance during reproduction. Physiological responses to salinity were evaluated for contrasting genotypes, during the seedling and reproductive stages. METHODS: Three rice genotypes differing in their tolerance of salinity were evaluated in a set of greenhouse experiments under salt stress during both seedling stage and reproduction. KEY RESULTS: Photosynthetic CO2 fixation, stomatal conductance (gs) and transpiration decreased substantially because of salt stress, but with greater reduction in the sensitive cultivar IR29. The tolerant lines IR651 and IR632 had more responsive stomata that tended to close faster during the first few hours of stress, followed by partial recovery after a brief period of acclimation. However, in the sensitive line, gs continued to decrease for longer duration and with no recovery afterward. Chlorophyll fluorescence measurements revealed that non-photochemical quenching increased, whereas the electron transport rate decreased under salt stress. Salt-tolerant cultivars exhibited much lower lipid peroxidation, maintained elevated levels of reduced ascorbic acid and showed increased activities of the enzymes involved in the reactive oxygen scavenging system during both developmental stages. CONCLUSIONS: Upregulation of the anti-oxidant system appears to play a role in salt tolerance of rice, with tolerant genotypes also maintaining relatively higher photosynthetic function; during both the vegetative and reproductive stages.