A rice really interesting new gene H2‐type E3 ligase,OsSIRH2‐14, enhances salinity tolerance via ubiquitin/26S proteasome‐mediated degradation of salt‐related proteins

Salinity is a deleterious abiotic stress factor that affects growth, productivity, and physiology of crop plants. Strategies for improving salinity tolerance in plants are critical for crop breeding programmes. Here, we characterized the rice (Oryza sativa) really interesting new gene (RING) H2‐type E3 ligase, OsSIRH2‐14 (previously named OsRFPH2‐14), which plays a positive role in salinity tolerance by regulating salt‐related proteins including an HKT‐type Na⁺ transporter (OsHKT2;1). OsSIRH2‐14 expression was induced in root and shoot tissues treated with NaCl. The OsSIRH2‐14‐EYFP fusion protein was predominately expressed in the cytoplasm, Golgi, and plasma membrane of rice protoplasts. In vitro pull‐down assays and bimolecular fluorescence complementation assays revealed that OsSIRH2‐14 interacts with salt‐related proteins, including OsHKT2;1. OsSIRH2‐14 E3 ligase regulates OsHKT2;1 via the 26S proteasome system under high NaCl concentrations but not under normal conditions. Compared with wild type plants, OsSIRH2‐14‐overexpressing rice plants showed significantly enhanced salinity tolerance and reduced Na⁺ accumulation in the aerial shoot and root tissues. These results suggest that the OsSIRH2‐14 RING E3 ligase positively regulates the salinity stress response by modulating the stability of salt‐related proteins.     

Improved Salinity Tolerance of Rice Through Cell Type-Specific Expression of AtHKT1;1

Previously, cell type-specific expression of AtHKT1;1, a sodium transporter, improved sodium (Na⁺) exclusion and salinity tolerance in Arabidopsis. In the current work, AtHKT1;1, was expressed specifically in the root cortical and epidermal cells of an Arabidopsis GAL4-GFP enhancer trap line. These transgenic plants were found to have significantly improved Na⁺ exclusion under conditions of salinity stress. The feasibility of a similar biotechnological approach in crop plants was explored using a GAL4-GFP enhancer trap rice line to drive expression of AtHKT1;1 specifically in the root cortex. Compared with the background GAL4-GFP line, the rice plants expressing AtHKT1;1 had a higher fresh weight under salinity stress, which was related to a lower concentration of Na⁺ in the shoots. The root-to-shoot transport of ²²Na⁺ was also decreased and was correlated with an upregulation of OsHKT1;5, the native transporter responsible for Na⁺ retrieval from the transpiration stream. Interestingly, in the transgenic Arabidopsis plants overexpressing AtHKT1;1 in the cortex and epidermis, the native AtHKT1;1 gene responsible for Na⁺ retrieval from the transpiration stream, was also upregulated. Extra Na⁺ retrieved from the xylem was stored in the outer root cells and was correlated with a significant increase in expression of the vacuolar pyrophosphatases (in Arabidopsis and rice) the activity of which would be necessary to move the additional stored Na⁺ into the vacuoles of these cells. This work presents an important step in the development of abiotic stress tolerance in crop plants via targeted changes in mineral transport.     

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.     

New allelic variants found in key rice salt‐tolerance genes: an association study

Salt stress is a complex physiological trait affecting plants by limiting growth and productivity. Rice, one of the most important food crops, is rated as salt‐sensitive. High‐throughput screening methods are required to exploit novel sources of genetic variation in rice and further improve salinity tolerance in breeding programmes. To search for genotypic differences related to salt stress, we genotyped 392 rice accessions by EcoTILLING. We targeted five key salt‐related genes involved in mechanisms such as Na⁺/K⁺ ratio equilibrium, signalling cascade and stress protection, and we found 40 new allelic variants in coding sequences. By performing association analyses using both general and mixed linear models, we identified 11 significant SNPs related to salinity. We further evaluated the putative consequences of these SNPs at the protein level using bioinformatic tools. Amongst the five nonsynonymous SNPs significantly associated with salt‐stress traits, we found a T67K mutation that may cause the destabilization of one transmembrane domain in OsHKT1;5, and a P140A alteration that significantly increases the probability of OsHKT1;5 phosphorylation. The K24E mutation can putatively affect SalT interaction with other proteins thus impacting its function. Our results have uncovered allelic variants affecting salinity tolerance that may be important in breeding.     

<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.     

OsTSD2‐mediated cell wall modification affects ion homeostasis and salt tolerance

Salt stress is a major environmental threat to meeting the food demands of an increasing global population. The identification and exploitation of salt adaption mechanisms in plants are therefore vital for crop breeding. We here define the rice mutant (sstm1) whose salt sensitivity was unambiguously assigned to a single T‐DNA insertion through segregational analysis following backcrossing to the wild type line. Insertion was within OsTSD2, which encoded a pectin methyltransferase. The sstm1 and allelic mutants, collectively known as tsd2, displayed higher content of Na⁺ and lower level of K⁺ in the shoot, which is likely to lead to reduced salt tolerance. Molecular analysis revealed reduced expression of the genes maintaining K⁺/Na⁺ homeostasis in tsd2, including OsHKT1;5, OsSOS1, and OsKAT1. Furthermore, OsTSD2 influenced ion distribution between the hull and the rice seed, which could improve food safety with heavy metal pollution. Amino acid levels tended to be increased in tsd2 mutants, implicating a role of pectin in the regulation of metabolism. Taken together, we have demonstrated an important facet of salt tolerance, which implicated OsTSD2‐mediated cell wall pectin modification as a key component that could be widely applied in crop science.     

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na⁺ and K⁺ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na⁺ transport or Na⁺ and K⁺ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na⁺/K⁺ selectivities of the K⁺-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K⁺ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na⁺ uptake, but little Rb⁺ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na⁺-K⁺ cotransport in plant cells such that extracellular K⁺ stimulated OsHKT2;2-mediated Na⁺ influx and vice versa. Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediated Na⁺ influx into plant cells without adding extracellular K⁺. This study shows that the Na⁺/K⁺ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K⁺ and Ca²⁺ down-regulated OsHKT2;1-mediated Na⁺ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.Intracellular Na⁺ and K⁺ homeostasis play vital roles in growth and development of higher plants (Clarkson and Hanson, 1980). Low cytosolic Na⁺ and high K⁺/Na⁺ ratios aid in maintaining an osmotic and biochemical equilibrium in plant cells. Na⁺ and K⁺ influx and efflux across membranes require the function of transmembrane Na⁺ and K⁺ transporters/channels. Several Na⁺-permeable transporters have been characterized in plants (Zhu, 2001; Horie and Schroeder, 2004; Apse and Blumwald, 2007). Na⁺/H⁺ antiporters mediate sequestration of Na⁺ into vacuoles under salt stress conditions in plants (Blumwald and Poole, 1985, 1987; Sze et al., 1999). Na⁺ (cation)/H⁺ antiporters are encoded by six AtNHX genes in Arabidopsis (Arabidopsis thaliana; Apse et al., 1999; Gaxiola et al., 1999; Yokoi et al., 2002; Aharon et al., 2003). A distinct Na⁺/H⁺ antiporter, Salt Overly Sensitive1, mediates Na⁺/H⁺ exchange at the plasma membrane and mediates cellular Na⁺ extrusion (Shi et al., 2000, 2002; Zhu, 2001; Ward et al., 2003). Electrophysiological analyses reveal that voltage-independent channels, also named nonselective cation channels, mediate Na⁺ influx into roots under high external Na⁺ concentrations (Amtmann et al., 1997; Tyerman et al., 1997; Buschmann et al., 2000; Davenport and Tester, 2000); however, the underlying genes remain unknown.Potassium is the most abundant cation in plants and an essential nutrient for plant growth. The Arabidopsis genome includes 13 genes encoding KUP/HAK/KT transporters (Quintero and Blatt, 1997; Santa-María et al., 1997; Fu and Luan, 1998; Kim et al., 1998), and 17 genes have been identified encoding this family of transporters in rice (Oryza sativa ‘Nipponbare’; Bañuelos et al., 2002). Several KUP/HAK/KT transporters have been characterized as mediating K⁺ uptake across the plasma membrane of plant cells (Rigas et al., 2001; Bañuelos et al., 2002; Gierth et al., 2005).Ionic balance, especially the Na⁺/K⁺ ratio, is a key factor of salt tolerance in plants (Niu et al., 1995; Maathuis and Amtmann, 1999; Shabala, 2000; Mäser et al., 2002a; Tester and Davenport, 2003; Horie et al., 2006; Apse and Blumwald, 2007; Chen et al., 2007; Gierth and Mäser, 2007). Salinity stress is a major problem for agricultural productivity of crops worldwide (Greenway and Munns, 1980; Zhu, 2001). The Arabidopsis AtHKT1;1 transporter plays a key role in salt tolerance of plants by mediating Na⁺ exclusion from leaves (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005; Rus et al., 2006; Davenport et al., 2007; Horie et al., 2009). athkt1;1 mutations cause leaf chlorosis and elevated Na⁺ accumulation in leaves under salt stress conditions in Arabidopsis (Mäser et al., 2002a; Berthomieu et al., 2003; Gong et al., 2004; Sunarpi et al., 2005). AtHKT1;1 and its homolog in rice, OsHKT1;5 (SKC1), mediate leaf Na⁺ exclusion by removing Na⁺ from the xylem sap to protect plants from salinity stress (Ren et al., 2005; Sunarpi et al., 2005; Horie et al., 2006, 2009; Davenport et al., 2007).The land plant HKT gene family is divided into two classes based on their nucleic acid sequences and protein structures (Mäser et al., 2002b; Platten et al., 2006). Class 1 HKT transporters have a Ser residue at a selectivity filter position in the first pore loop, which is replaced by a Gly in all but one known class 2 HKT transporter (Horie et al., 2001; Mäser et al., 2002b; Garciadeblás et al., 2003). While the Arabidopsis genome includes only one HKT gene, AtHKT1;1 (Uozumi et al., 2000), seven full-length OsHKT genes were found in the japonica rice cv Nipponbare genome (Garciadeblás et al., 2003). Members of class 1 HKT transporters, AtHKT1;1 and SKC1/OsHKT1;5, have a relatively higher Na⁺-to-K⁺ selectivity in Xenopus laevis oocytes and yeast than class 2 HKT transporters (Uozumi et al., 2000; Horie et al., 2001; Mäser et al., 2002b; Ren et al., 2005). The first identified plant HKT transporter, TaHKT2;1 from wheat (Triticum aestivum), is a class 2 HKT transporter (Schachtman and Schroeder, 1994). TaHKT2;1 was found to mediate Na⁺-K⁺ cotransport and Na⁺ influx at high Na⁺ concentrations in heterologous expression systems (Rubio et al., 1995, 1999; Gassmann et al., 1996; Mäser et al., 2002b). Thus, class 1 HKT transporters have been characterized as Na⁺-preferring transporters with a smaller K⁺ permeability (Fairbairn et al., 2000; Uozumi et al., 2000; Su et al., 2003; Jabnoune et al., 2009), whereas class 2 HKT transporters function as Na⁺-K⁺ cotransporters or channels (Gassmann et al., 1996; Corratgé et al., 2007). In addition, at millimolar Na⁺ concentrations, class 2 HKT transporters were found to mediate Na⁺ influx, without adding external K⁺ in Xenopus oocytes and yeast (Rubio et al., 1995, 1999; Gassmann et al., 1996; Horie et al., 2001). However, the differential cation transport selectivities of the two types of HKT transporters have not yet been analyzed and compared in plant cells.A study of the barley (Hordeum vulgare) and wheat class 2 transporters has suggested that the transport properties of HvHKT2;1 and TaHKT2;1 expressed in yeast are variable, depending on the constructs from which the transporter is expressed, and have led to questioning of the K⁺ transport activity of HKT transporters characterized in Xenopus oocytes and yeast (Haro et al., 2005). It was further proposed that the 5′ translation initiation of HKT proteins in yeast at nonconventional (non-ATG) sites affects the transporter selectivities of HKT transporters (Haro et al., 2005), although direct evidence for this has not yet been presented. However, recent research has shown a K⁺ permeability of OsHKT2;1 but not of OsHKT1;1 and OsHKT1;3 in Xenopus oocytes. These three OsHKT transporters show overlapping and also distinctive expression patterns in rice (Jabnoune et al., 2009).The report of Haro et al. (2005) has opened a central question addressed in this study: are the Na⁺/K⁺ transport selectivities of plant HKT transporters characterized in heterologous systems of physiological relevance in plant cells, or do they exhibit strong differences in the cation transport selectivities in these nonplant versus plant systems? To address this question, we analyzed the Na⁺/K⁺ transport selectivities of the OsHKT2;1 and OsHKT2;2 transporters expressed in cultured tobacco (Nicotiana tabacum ‘Bright-Yellow 2’ [BY2]) cells. OsHKT2;1 and OsHKT2;2 are two highly homologous HKT transporters from indica rice cv Pokkali, sharing 91% amino acid and 93% cDNA sequence identity (Horie et al., 2001). OsHKT2;1 mediates mainly Na⁺ uptake, which correlates with the presence of a Ser residue in the first pore loop of OsHKT2;1 (Horie et al., 2001, 2007; Mäser et al., 2002b; Garciadeblás et al., 2003). In contrast, OsHKT2;2 mediates Na⁺-K⁺ cotransport in Xenopus oocytes and yeast (Horie et al., 2001). Furthermore, at millimolar Na⁺ concentrations, OsHKT2;2 mediates Na⁺ influx in the absence of added K⁺ (Horie et al., 2001). Recent research on oshkt2;1 loss-of-function mutant alleles has revealed that OsHKT2;1 from japonica rice mediates a large Na⁺ influx component into K⁺-starved roots, thus compensating for lack of K⁺ availability (Horie et al., 2007). But the detailed Na⁺/K⁺ selectivities of Gly-containing, predicted K⁺-transporting class 2 HKT transporters have not yet been analyzed in plant cells.Here, we have generated stable OsHKT2;1- and OsHKT2;2-expressing tobacco BY2 cell lines and characterized the cell lines by ion content measurements and tracer influx studies to directly analyze unidirectional fluxes (Epstein et al., 1963). These analyses showed that OsHKT2;1 exhibits Na⁺ uptake activity in plant BY2 cells in the absence of added K⁺, but little K⁺ (Rb⁺), influx activity. In contrast, OsHKT2;2 was found to function as a Na⁺-K⁺ cotransporter/channel in plant BY2 cells, showing K⁺-stimulated Na⁺ influx and Na⁺-stimulated K⁺ (Rb⁺) influx. The differential K⁺ selectivities of the two OsHKT2 transporters were consistently reproduced by voltage clamp experiments using Xenopus oocytes here, as reported previously (Horie et al., 2001). OsHKT2;2 was also found to mediate K⁺-independent Na⁺ influx at millimolar external Na⁺ concentrations. These findings demonstrate that the cation selectivities of OsHKT2;1 and OsHKT2;2 in plant cells are consistent with past findings obtained from heterologous expression analyses under similar ionic conditions (Horie et al., 2001; Garciadeblás et al., 2003; Tholema et al., 2005). Furthermore, the shift in OsHKT2;2 Na⁺-K⁺ selectivity depending on ionic editions is consistent with the model that HKT transporters/channels are multi-ion pores (Gassmann et al., 1996; Corratgé et al., 2007). Classical studies of ion channels have shown that ion channels, in which multiple ions can occupy the pore at the same time, can change their relative selectivities depending on the ionic conditions (Hille, 2001). Moreover, the presence of external K⁺ and Ca²⁺ was found here to down-regulate OsHKT2;1-mediated Na⁺ influx both in tobacco BY2 cells and in rice roots. The inhibitory effect of external K⁺ on OsHKT2;1-mediated Na⁺ influx into intact rice roots, however, showed a distinct difference in comparison with that of BY2 cells, which indicates a possible posttranslational regulation of OsHKT2;1 in K⁺-starved rice roots.     

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.     

Effect of the vacuolar Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> antiporter transgene in a rice landrace and a commercial rice cultivar after its insertion by crossing

The vacuolar Na⁺/H⁺ antiporter is known to alleviate saline stress by sequestering Na⁺ in both wild-type Arabidopsis and rice and when over-expressed in many transgenic plants. Here we report on the effect of the NHX1 transgene on the salt tolerance properties it confers to a rice landrace and a commercial cultivar suitable for the dry winter season, but which suffers loss due to seasonal stresses, particularly in the coastal areas. The Nipponbare Na⁺/H⁺ antiporter 1.9 kb cDNA was cloned into pCAMBIA1305.1 under the control of the CaMV35S promoter and transformed into tissue-culture-responsive rice landrace Binnatoa (BA). The best-expressing transgenic line at T₂ was found to be significantly tolerant at the seedling stage and was advanced to T₃. The transgene was then transferred to the tissue-culture recalcitrant farmer-popular commercial rice genotype, BRRIdhan 28 (BR28) by crossing. The data generated both from semi-quantitative RT-PCR and western blot hybridization revealed that the transgene showed similar expression in the crossbred BR28 plants and BA transgenic line. Comparative stress tolerance tests, however, revealed that the BR28 crossbred lines were significantly less tolerant than its transgenic parent BA at both seedling and reproductive stages. A single successful transgenic event may therefore not show the same performance in the recipient genetic background, if introgressed by crossing.     

Unlocking Allelic Diversity for Sustainable Development of Salinity Stress Tolerance in Rice

Rice is a major cereal crop, negatively impacted by soil-salinity, both in terms of plant growth as well as productivity. Salinity tolerant rice varieties have been developed using conventional breeding approaches, however, there has been limited success which is primarily due to the complexity of the trait, low yield, variable salt stress response and availability of genetic resources. Furthermore, the narrow genetic base is a hindrance for further improvement of the rice varieties. Therefore, there is a greater need to screen available donor germplasm in rice for salinity tolerance related genes and traits. In this regard, genomics based techniques are useful for exploring new gene resources and QTLs. In rice, the vast allelic diversity existing in the wild and cultivated germplasm needs to be explored for improving salt tolerance. In the present review, we provide an overview of the allelic diversity in the Quantitative Trait Loci (QTLs) like Saltol, qGR6.2, qSE3 and RNC4 as well as genes like OsHKT1;1, SKC1 (OsHKT1;5/HKT8) and OsSTL1 (salt tolerance level 1 gene) related to salt tolerance in rice. We have also discussed approaches for developing salt-tolerant cultivars by utilizing the effective QTLs or genes/alleles in rice.     

Relative salinity tolerance of rice cultivars native to North East India: a physiological,biochemical and molecular perspective

Salinity is the second most prevalent abiotic stress faced by plants, and rice is not an exception. Through this study, it has been tried upon, to study the relative salinity tolerance of eight local varieties of North East India. Preliminary screening was based on their dose- and time-dependent physiological responses to salinity stress. Among the cultivars, Tampha was found to be relatively more tolerant, whereas MSE9 the most sensitive. To further ascertain their tolerance capacity, MDA and H₂O₂ content was determined, which also confirmed the tolerance level of the two cultivars. Histochemical assays for root plasma membrane integrity and leaf and root H₂O₂ and O₂ ^? content also showed more damage in Tampha in comparison to MSE9. Finally, gene expression analysis for Na⁺/K⁺ co-transporters, OsHKT2;1, OsHKT2;3 and OsHKT2;4, was performed to observe how the expression level of these transporters varies with the tolerance capacity of these two cultivars in leaves and roots under different time frames. The study reveals Tampha to be the most tolerant and MSE9 the most sensitive when compared to the other six screened cultivars for salinity stress.     

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.     

A rice jacalin‐related mannose‐binding lectin gene,OsJRL, enhances Escherichia coli viability under high salinity stress and improves salinity tolerance of rice

• Salinity, which is one of the most common abiotic stresses, may severely affect plant productivity and quality. Although plant lectins are thought to play important roles in plant defense signaling during pathogen attack, little is known about the contribution of plant lectins to stress resistance.
• We cloned and functionally characterized a rice jacalin‐related mannose‐binding lectin gene, OsJRL, from rice ‘Nipponbare’. We analyzed the expression patterns of OsJRL under various stress conditions in rice. Furthermore, we overexpressed OsJRL in Escherichia coli and rice.
• The cDNA of OsJRL contained a 438 bp open reading frame, which encodes a polypeptide of 145 amino acids. OsJRL was localized in the nucleus and cytoplasm. Real time PCR analyses revealed that OsJRL expression showed tissue specificity in rice and was upregulated under diverse stresses, namely salt, drought, cold, heat and abscisic acid treatments. Overexpression of OsJRL in E. coli enhanced cell viability and dramatically improved tolerance of high salinity. Overexpression of OsJRL in rice also enhanced salinity tolerance and increased the expression levels of a number of stress‐related genes, including three LEA (late embryogenesis abundant proteins) genes (OsLEA19a, OsLEA23 and OsLEA24), three Na⁺ transporter genes (OsHKT1;3, OsHKT1;4 and OsHKT1;5) and two DREB genes (OsDREB1A and OsDREB2B).
• Based on these results, we suggest that OsJRL plays an important role in cell protection and stress signal transduction.

     

Diversity in Expression Patterns and Functional Properties in the Rice HKT Transporter Family

Plant growth under low K⁺ availability or salt stress requires tight control of K⁺ and Na⁺ uptake, long-distance transport, and accumulation. The family of membrane transporters named HKT (for High-Affinity K⁺ Transporters), permeable either to K⁺ and Na⁺ or to Na⁺ only, is thought to play major roles in these functions. Whereas Arabidopsis (Arabidopsis thaliana) possesses a single HKT transporter, involved in Na⁺ transport in vascular tissues, a larger number of HKT transporters are present in rice (Oryza sativa) as well as in other monocots. Here, we report on the expression patterns and functional properties of three rice HKT transporters, OsHKT1;1, OsHKT1;3, and OsHKT2;1. In situ hybridization experiments revealed overlapping but distinctive and complex expression patterns, wider than expected for such a transporter type, including vascular tissues and root periphery but also new locations, such as osmocontractile leaf bulliform cells (involved in leaf folding). Functional analyses in Xenopus laevis oocytes revealed striking diversity. OsHKT1;1 and OsHKT1;3, shown to be permeable to Na⁺ only, are strongly different in terms of affinity for this cation and direction of transport (inward only or reversible). OsHKT2;1 displays diverse permeation modes, Na⁺-K⁺ symport, Na⁺ uniport, or inhibited states, depending on external Na⁺ and K⁺ concentrations within the physiological concentration range. The whole set of data indicates that HKT transporters fulfill distinctive roles at the whole plant level in rice, each system playing diverse roles in different cell types. Such a large diversity within the HKT transporter family might be central to the regulation of K⁺ and Na⁺ accumulation in monocots.Although it is not clear what levels of Na⁺ are toxic in the plant cell cytosol and actually unacceptable in vivo, the hypothesis that this cation must be excluded from the cytoplasm is widely accepted. The most abundant inorganic cation in the cytosol is K⁺, in plant as in animal cells. This cation has probably been selected during evolution because it is less chaotropic than Na⁺ (i.e. more compatible with protein structure even at high concentrations; Clarkson and Hanson, 1980). Its selection might also be due to the fact that in primitive cells, which originated in environmental conditions (seawater) where Na⁺ was more abundant than K⁺, a straightforward process to energize the cell membrane was to accumulate the less abundant cation and to exclude the most abundant one.In the cell, K⁺ plays a role in basic functions, such as regulation of cell membrane polarization, electrical neutralization of anionic groups, and osmoregulation. Concerning the latter function, K⁺ uptake or release is the usual way through which plant cells control their water potential and turgor. Although toxic at high concentrations, Na⁺ can be used as osmoticum and substituted for K⁺, mainly in the vacuole, when the plant is facing low K⁺ conditions and Na⁺ is available in the soil solution. This use of Na⁺, however, requires a tight regulation of K⁺ and Na⁺ transport and compartmentalization that becomes crucial in conditions of high Na⁺ concentrations in the soil solution. Control of Na⁺ and K⁺ uptake, long-distance transport in the xylem and phloem vasculatures, accumulation in aerial parts, and compartmentalization at the cellular and tissue levels have actually been shown to be essential in plant adaptation to salt stress (Greenway and Munns, 1980; Flowers, 1985; Hasegawa et al., 2000; Mühling and Läuchli, 2002). Thus, accumulation of Na⁺ as osmoticum during K⁺ shortage or plant adaptation to salt stress requires integration at the whole plant level of Na⁺ and K⁺ membrane transport system activities (Apse et al., 1999; Shi et al., 2002; Qi and Spalding, 2004; Ren et al., 2005; Maathuis, 2006; Pardo et al., 2006; Horie et al., 2007).This report concerns transport systems named HKT upon first identification (for High-Affinity K⁺ Transporters) that are active at the plasma membrane and permeable to either K⁺ and Na⁺ or to Na⁺ only (Schachtman and Schroeder, 1994; Rodríguez-Navarro and Rubio, 2006). Several members of the HKT family have already been shown, by genetic approaches, to play important roles in plant salt tolerance (Berthomieu et al., 2003; Ren et al., 2005; Huang et al., 2006; Byrt et al., 2007) or growth in conditions of K⁺ shortage (Horie et al., 2007). In Arabidopsis (Arabidopsis thaliana), the HKT family comprises a single member, AtHKT1;1, which is permeable to Na⁺ only (Uozumi et al., 2000) and contributes to Na⁺ removal from the ascending xylem sap and recirculation from the leaves to the roots via the phloem vasculature (Berthomieu et al., 2003; Sunarpi et al., 2005). Interestingly, the HKT family comprises a much larger number of members in rice (Oryza sativa), with seven to nine genes depending on the cultivar (Garciadeblás et al., 2003). In line with previous reports using rice as a model species to decipher the roles that HKT transporters can play in the plant, we have analyzed the expression patterns of three rice HKT genes, OsHKT2;1, OsHKT1;1, and OsHKT1;3, and investigated the functional properties of these transporters after heterologous expression, revealing new patterns of expression for HKT transporters and striking functional diversity.     

Saline-induced changes of epicuticular waxy layer on the Puccinellia tenuiflora and Oryza sativa leave surfaces

Background

The epicuticular waxy layer of plant leaves enhances the extreme environmental stress tolerance. However, the relationship between waxy layer and saline tolerance was not established well. The epicuticular waxy layer of rice (Oryza sativa L.) was studied under the NaHCO₃ stresses. In addition, strong saline tolerance Puccinellia tenuiflora was chosen for comparative studies.

Results

Scanning electron microscope (SEM) images showed that there were significant changes in waxy morphologies of the rice epicuticular surfaces, while no remarkable changes in those of P. tenuiflora epicuticular surfaces. The NaHCO₃-induced morphological changes of the rice epicuticular surfaces appeared as enlarged silica cells, swollen corns-shapes and leaked salt columns under high stress. Energy dispersive X-ray (EDX) spectroscopic profiles supported that the changes were caused by significant increment and localization of [Na⁺] and [Cl⁻] in the shoot. Atomic absorption spectra showed that [Na⁺]_shoot/[Na⁺]_root for P. tenuiflora maintained stable as the saline stress increased, but that for rice increased significantly.

Conclusion

In rice, NaHCO₃ stress induced localization and accumulation of [Na⁺] and [Cl⁻] appeared as the enlarged silica cells (MSC), the swollen corns (S-C), and the leaked columns (C), while no significant changes in P. tenuiflora.     

The calcium transporter ANNEXIN1 mediates cold‐induced calcium signaling and freezing tolerance in plants

The transient elevation of cytosolic free calcium concentration ([Ca²⁺]_cyt) induced by cold stress is a well‐established phenomenon; however, the underlying mechanism remains elusive. Here, we report that the Ca²⁺‐permeable transporter ANNEXIN1 (AtANN1) mediates cold‐triggered Ca²⁺ influx and freezing tolerance in Arabidopsis thaliana. The loss of function of AtANN1 substantially impaired freezing tolerance, reducing the cold‐induced [Ca²⁺]_cyt increase and upregulation of the cold‐responsive CBF and COR genes. Further analysis showed that the OST1/SnRK2.6 kinase interacted with and phosphorylated AtANN1, which consequently enhanced its Ca²⁺ transport activity, thereby potentiating Ca²⁺ signaling. Consistent with these results and freezing sensitivity of ost1 mutants, the cold‐induced [Ca²⁺]_cyt elevation in the ost1‐3 mutant was reduced. Genetic analysis indicated that AtANN1 acts downstream of OST1 in responses to cold stress. Our data thus uncover a cascade linking OST1‐AtANN1 to cold‐induced Ca²⁺ signal generation, which activates the cold response and consequently enhances freezing tolerance in Arabidopsis.     

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...     

Genome-wide association mapping of salinity tolerance in rice (Oryza sativa)

Salinity tolerance in rice is highly desirable to sustain production in areas rendered saline due to various reasons. It is a complex quantitative trait having different components, which can be dissected effectively by genome-wide association study (GWAS). Here, we implemented GWAS to identify loci controlling salinity tolerance in rice. A custom-designed array based on 6,000 single nucleotide polymorphisms (SNPs) in as many stress-responsive genes, distributed at an average physical interval of <100 kb on 12 rice chromosomes, was used to genotype 220 rice accessions using Infinium high-throughput assay. Genetic association was analysed with 12 different traits recorded on these accessions under field conditions at reproductive stage. We identified 20 SNPs (loci) significantly associated with Na⁺/K⁺ ratio, and 44 SNPs with other traits observed under stress condition. The loci identified for various salinity indices through GWAS explained 5–18% of the phenotypic variance. The region harbouring Saltol, a major quantitative trait loci (QTLs) on chromosome 1 in rice, which is known to control salinity tolerance at seedling stage, was detected as a major association with Na⁺/K⁺ ratio measured at reproductive stage in our study. In addition to Saltol, we also found GWAS peaks representing new QTLs on chromosomes 4, 6 and 7. The current association mapping panel contained mostly indica accessions that can serve as source of novel salt tolerance genes and alleles. The gene-based SNP array used in this study was found cost-effective and efficient in unveiling genomic regions/candidate genes regulating salinity stress tolerance in rice.     

Exploration of relationships between physiological parameters and growth performance of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) seedlings under salinity stress using multivariate analysis

Knowledge of relationships between physiological parameters and growth performance of seedlings and respective genotypic differences would permit selection of salt tolerance at early growth stages. The goals of this study were to investigate the relationships between physiological parameters and growth performance and quantify the respective genotypic differences using multivariate analysis.. Plants of thirty-one genotypes were grown in sand tanks in a greenhouse and irrigated with Yoshida nutrient solution. Two salinity treatments were imposed at 0.9 dSm^–1 (control) and 6.4 dSm^–1 with sodium chloride and calcium chloride (~ 6: 1 molar ratio). Seedlings were sampled 34 days after planting (7^th to 8^th leaf stage). The characters of Na⁺, K⁺, Ca²⁺, K-Na selectivity (SK,Na) and Na-Ca selectivity (S_Na,Ca) were measured as physiological parameters. The characters of tiller number, leaf area, plant height and shoot dry weight were measured as growth performance. Under salinity stress, S_K,Na increased whereas S_Na,Ca decreased compared to the controls. Canonical correlation analysis indicates a strong relationship between physiological parameters and growth performance. Tiller number is a desirable parameter among the growth parameters analyzed to predict seedling growth under salinity stress. Genotypes grouped into four clusters based on ion contents and ion selectivity using Wards minimum-variance cluster analysis. S_K,Na and shoot Na⁺ content contributed the most to the cluster formation. Similarly, genotypes grouped into four clusters based on growth performance. Ge notypes were classified into three categories based on ion cluster rankings: Category 1 with high S_K,Na and low shoot Na⁺ content; Category 2 with intermediate S_K,Na and shoot Na⁺ content; Category 3 with low S_K,Na and high shoot Na⁺ content. The classification of the genotypes into Categories 1 and 3 based on their high or low S_K,Na was generally consistent with their growth performance under salt stress. In contrast, ion selectivity was a less dominant mechanism controlling salt tolerance in Category 2 with intermediate S_K,Na. It was concluded that ion selectivity was a relatively dominant mechanism controlling salt tolerance among rice genotypes although multiple mechanisms may be involved under moderate salt stress. The results also provide the first example of the effectiveness of cluster analysis for physiological responses to salinity stress.