PHT1;5 Repressed by ANT Mediates Pi Acquisition and Distribution under Low Pi and Salinity in Salt Cress.

Salinity and phosphate (Pi) starvation are the most common abiotic stresses that threaten crop productivity. Salt cress (Eutrema salsugineum) displays good tolerance to both salinity and Pi limitation. Previously, we found several Phosphate Transporter (PHT) genes in salt cress upregulated under salinity. Here, EsPHT1;5 induced by both low Pi (LP) and salinity was further characterized. Overexpression of EsPHT1;5 in salt cress enhanced plant tolerance to LP and salinity, while the knock-down lines exhibited growth retardation. The analysis of phosphorus (P) content and shoot/root ratio of total P in EsPHT1;5-overexpressing salt cress seedlings and the knock-down lines as well as arsenate uptake assays suggested the role of EsPHT1;5 in Pi acquisition and root-shoot translocation under Pi limitation. In addition, overexpression of EsPHT1;5 driven by the native promoter in salt cress enhanced Pi mobilization from rosettes to siliques upon a long-term salt treatment. Particularly, the promoter of EsPHT1;5 outperformed that of AtPHT1;5 in driving gene expression under salinity. We further identified a transcription factor EsANT, which negatively regulated EsPHT1;5 expression and plant tolerance to LP and salinity. Taken together, EsPHT1;5 plays an integral role in Pi acquisition and distribution in plant response to LP and salt stress. Further, EsANT may be involved in the cross-talk between Pi starvation and salinity signaling pathways. This work provides further insight into the mechanism underlying high P use efficiency in salt cress in its natural habitat, and evidence for a link between Pi and salt signaling.

SbYS1 and SbWRKY72 regulate Cd tolerance and accumulation in sweet sorghum.

MAIN CONCLUSION: SbYS1 and its upstream transcription factor SbWRKY72 were involved in Cd tolerance and accumulation and are valuable for developing sweet sorghum germplasm with high-Cd tolerance or accumulation ability through genetic manipulation. Cadmium (Cd) is highly toxic and can severely affect human health. Sweet sorghum, as an energy crop, shows great potential in extracting cadmium from Cd-contaminated soils. However, its molecular mechanisms of Cd-tolerance and -accumulation remain largely unknown. Here, we isolated a YSL family gene SbYS1 from the sweet sorghum genotype with high Cd accumulation ability and the expression of SbYS1 in roots was induced by cadmium. GUS staining experiment exhibited that SbYS1 was expressed in the epidermis and parenchyma tissues of roots. Further subcellular localization analysis suggested that SbYS1 was localized in the endoplasmic reticulum and plasma membrane. Yeast transformed with SbYS1 exhibited a sensitive phenotype compared to the control when exposed to Cd-NA (chelates of cadmium and nicotianamine), indicating that SbYS1 may absorb cadmium in the form of Cd-NA. Arabidopsis overexpressing SbYS1 had a longer root length and accumulated less Cd in roots and shoots. SbWRKY72 bound to the promoter of SbYS1 and negatively regulated the expression of SbYS1. Transgenic Arabidopsis of SbWRKY72 showed higher sensitivity to cadmium and increased cadmium accumulation in roots. Our results provide references for improving the phytoremediation efficiency of sweet sorghum by genetic manipulation in the future.

Cell size and xylem differentiation regulating genes from Salicornia europaea contribute to plant salt tolerance.

Cell wall is involved in plant growth and plays pivotal roles in plant adaptation to environmental stresses. Cell wall remodelling may be crucial to salt adaptation in the euhalophyte Salicornia europaea. However, the mechanism underlying this process is still unclear. Here, full-length transcriptome indicated cell wall-related genes were comprehensively regulated under salinity. The morphology and cell wall components in S. europaea shoot were largely modified under salinity. Through the weighted gene co-expression network analysis, SeXTH2 encoding xyloglucan endotransglucosylase/hydrolases, and two SeLACs encoding laccases were focused. Meanwhile, SeEXPB was focused according to expansin activity and the expression profiling. Function analysis in Arabidopsis validated the functions of these genes in enhancing salt tolerance. SeXTH2 and SeEXPB overexpression led to larger cells and leaves with hemicellulose and pectin content alteration. SeLAC1 and SeLAC2 overexpression led to more xylem vessels, increased secondary cell wall thickness and lignin content. Notably, SeXTH2 transgenic rice exhibited enhanced salt tolerance and higher grain yield. Altogether, these genes may function in the succulence and lignification process in S. europaea. This work throws light on the regulatory mechanism of cell wall remodelling in S. europaea under salinity and provides potential strategies for improving crop salt tolerance and yields.

Phosphatidylserine Synthase from Salicornia europaea Is Involved in Plant Salt Tolerance by Regulating Plasma Membrane Stability.

Salinity-induced lipid alterations have been reported in many plant species; however, how lipid biosynthesis and metabolism are regulated and how lipids work in plant salt tolerance are much less studied. Here, a constitutively much higher phosphatidylserine (PS) content in the plasma membrane (PM) was found in the euhalophyte Salicornia europaea than in Arabidopsis. A gene encoding PS synthase (PSS) was subsequently isolated from S. europaea, named SePSS, which was induced by salinity. Multiple alignments and phylogenetic analysis suggested that SePSS belongs to a base exchange-type PSS, which localises to the endoplasmic reticulum. Knockdown of SePSS in S. europaea suspension cells resulted in reduced PS content, decreased cell survival rate, and increased PM depolarization and K+ efflux under 400 or 800 mM NaCl. By contrast, the upregulation of SePSS leads to increased PS and phosphatidylethanolamine levels and enhanced salt tolerance in Arabidopsis, along with a lower accumulation of reactive oxygen species, less membrane injury, less PM depolarization and higher K+/Na+ in the transgenic lines than in wild-type (WT). These results suggest a positive correlation between PS levels and plant salt tolerance, and that SePSS participates in plant salt tolerance by regulating PS levels, hence PM potential and permeability, which help maintain ion homeostasis. Our work provides a potential strategy for improving plant growth under multiple stresses.

Comparative analysis of sRNAs, degradome and transcriptomics in sweet sorghum reveals the regulatory roles of miRNAs in Cd accumulation and tolerance.

MAIN CONCLUSION: Key miRNAs including sbi-miR169p/q, sbi-miR171g/j, sbi-miR172a/c/d, sbi-miR172e, sbi-miR319a/b, sbi-miR396a/b, miR408, sbi-miR5384, sbi-miR5565e and nov_23 were identified to function in the regulation of Cd accumulation and tolerance. As an energy plant, sweet sorghum shows great potential in the phytoremediation of Cd-contaminated soils. However, few studies have focused on the regulatory roles of miRNAs and their targets under Cd stress. In this study, comparative analysis of sRNAs, degradome and transcriptomics was conducted in high-Cd accumulation (H18) and low-Cd accumulation (L69) genotypes of sweet sorghum. A total of 38 conserved and 23 novel miRNAs with differential expressions were identified under Cd stress or between H18 and L69, and 114 target genes of 41 miRNAs were validated. Furthermore, 25 miRNA-mRNA pairs exhibited negatively correlated expression profiles and sbi-miR172e together with its target might participate in the distinct Cd tolerance between H18 and L69 as well as sbi-miR172a/c/d. Additionally, two groups of them: miR169p/q-nov_23 and miR408 were focused through the co-expression analysis, which might be involved in Cd uptake and tolerance by regulating their targets associated with transmembrane transportation, cytoskeleton activity, cell wall construction and ROS (reactive oxygen species) homeostasis. Further experiments exhibited that cell wall components of H18 and L69 were different when exposed to cadmium, which might be regulated by miR169p/q, miR171g/j, miR319a/b, miR396a/b, miR5384 and miR5565e through their targets. Through this study, we aim to reveal the potential miRNAs involved in sweet sorghum in response to Cd stress and provide references for developing high-Cd accumulation or high Cd-resistant germplasm of sweet sorghum that can be used in phytoremediation.

Variation of PHT families adapts salt cress to phosphate limitation under salinity.

Salt cress (Eutrema salsugineum) presents relatively high phosphate (Pi) use efficiency cy in its natural habitat. Phosphate Transporters (PHTs) play critical roles in Pi acquisition and homeostasis. Here, a comparative study of PHT families between salt cress and Arabidopsis was performed. A total of 27 putative PHT genes were identified in E. salsugineum genome. Notably, seven tandem genes encoding PHT1;3 were found, and function analysis in Arabidopsis indicated at least six EsPHT1;3s participated in Pi uptake. Meanwhile, different expression profiles of PHT genes between the two species under Pi limitation and salt stress were documented. Most PHT1 genes were down-regulated in Arabidopsis while up-regulated in salt cress under salinity, among which EsPHT1;9 was further characterized. EsPHT1;9 was involved in root-to-shoot Pi translocation. Particularly, the promoter of EsPHT1;9 outperformed that of AtPHT1;9 in promoting Pi translocation, K+ /Na+ ratio, thereby salt tolerance. Through cis-element analysis, we identified a bZIP transcription factor EsABF5 negatively regulating EsPHT1;9 and plant tolerance to low-Pi and salt stress. Altogether, more copies and divergent transcriptional regulation of PHT genes contribute to salt cress adaptation to the co-occurrence of salinity and Pi limitation, which add our knowledge on the evolutionary and molecular component of multistress- tolerance of this species.

Comparative transcriptome combined with morpho-physiological analyses revealed key factors for differential cadmium accumulation in two contrasting sweet sorghum genotypes.

Cadmium (Cd) is a widespread soil contaminant threatening human health. As an ideal energy plant, sweet sorghum (Sorghum bicolor (L.) Moench) has great potential in phytoremediation of Cd-polluted soils, although the molecular mechanisms are largely unknown. In this study, key factors responsible for differential Cd accumulation between two contrasting sweet sorghum genotypes (high-Cd accumulation one H18, and low-Cd accumulation one L69) were investigated. H18 exhibited a much higher ability of Cd uptake and translocation than L69. Furthermore, Cd uptake through symplasmic pathway and Cd concentrations in xylem sap were both higher in H18 than those in L69. Root anatomy observation found the endodermal apoplasmic barriers were much stronger in L69, which may restrict the Cd loading into xylem. The molecular mechanisms underlying these morpho-physiological traits were further dissected by comparative transcriptome analysis. Many genes involved in cell wall modification and heavy metal transport were found to be Cd-responsive DEGs and/or DEGs between these two genotypes. KEGG pathway analysis found phenylpropanoid biosynthesis pathway was over-represented, indicating this pathway may play important roles in differential Cd accumulation between two genotypes. Based on these results, a schematic representation of main processes involved in differential Cd uptake and translocation in H18 and L69 is proposed, which suggests that higher Cd accumulation in H18 depends on a multilevel coordination of efficient Cd uptake and transport, including efficient root uptake and xylem loading, less root cell wall binding, and weaker endodermal apoplasmic barriers.

The V-ATPase subunit A is essential for salt tolerance through participating in vacuolar Na+ compartmentalization in Salicornia europaea.

MAIN CONCLUSION: The V-ATPase subunit A participates in vacuolar Na + compartmentalization in Salicornia europaea regulating V-ATPase and V-PPase activities. Na+ sequestration into the vacuole is an efficient strategy in response to salinity in many halophytes. However, it is not yet fully understood how this process is achieved. Particularly, the role of vacuolar H+-ATPase (V-ATPase) in this process is controversial. Our previous proteomic investigation in the euhalophyte Salicornia europaea L. found a significant increase of the abundance of V-ATPase subunit A under salinity. Here, the gene encoding this subunit named SeVHA-A was characterized, and its role in salt tolerance was demonstrated by RNAi directed downregulation in suspension-cultured cells of S. europaea. The transcripts of genes encoding vacuolar H+-PPase (V-PPase) and vacuolar Na+/H+ antiporter (SeNHX1) also decreased significantly in the RNAi cells. Knockdown of SeVHA-A resulted in a reduction in both V-ATPase and vacuolar H+-PPase (V-PPase) activities. Accordingly, the SeVHA-A-RNAi cells showed increased vacuolar pH and decreased cell viability under different NaCl concentrations. Further Na+ staining showed the reduced vacuolar Na+ sequestration in RNAi cells. Taken together, our results evidenced that SeVHA-A participates in vacuolar Na+ sequestration regulating V-ATPase and V-PPase activities and thereby vacuolar pH in S. europaea. The possible mechanisms underlying the reduction of vacuolar V-PPase activity in SeVHA-A-RNAi cells were also discussed.

Establishment of a gene function analysis system for the euhalophyte Salicornia europaea L.

KEY MESSAGE: A Salicornia europaea L. in vitro cell transformation system was developed and further applied to SeNHX1 function investigation. The exploration of salt-tolerant genes from halophyte has seriously been limited by the lack of self-dependent transformation system. Here, an Agrobacterium tumefaciens-mediated in vitro cell transformation system of euhalophyte Salicornia europaea L. was developed. Calli derived from hypocotyl of S. europaea were co-cultured for 3 days with Agrobacterium at OD600 ranging from 1.0 to 1.5 and then selected with 25 mg/L hygromycin (Hyg). The transformed cells were identified from Hyg positive calli by GUS assay and qRT-PCR, and the transformation efficiency was up to 74.4%. The practicality of this system was further tested via genetic manipulation of S. europaea Na+/H+ antiporter 1 (SeNHX1) gene by creating the overexpressing, silencing, and empty vector cells. Survival ratio and Na+ distribution under salt treatment showed obvious differences in SeNHX1-overexpressing, -silencing, and empty vector cells, indicating the feasibility of this system to analyze gene function. This investigation is enlightening for studies in other non-model plants lacking of self-dependent transformation system.

Identification for the capability of Cd-tolerance, accumulation and translocation of 96 sorghum genotypes.

Cadmium (Cd) pollution is a worldwide environmental problem which heavily threatens human health and food security. Sorghum, as one of the most promising energy crop, has been considered to be the source of high-quality feedstock for ethanol fuel. Ninety-six sorghum genotypes were investigated under hydroponic conditions to compare their capabilities of Cd-tolerance, accumulation and translocation for their potential in remediation of Cd contamination. Different genotypes varied largely in the tolerance to Cd stress with tolerance indexes ranked from 0.107 to 0.933. Great difference was also found in Cd uptake and accumulation with concentrations ranging from 19.0 to 202.4mg/kg in shoots and 277.0-898.3mg/kg in roots. The total amounts of Cd ranked from 6.1 to 25.8µg per plant and the highest translocation factor was over 4 times higher than the lowest one. The correlation analysis demonstrated that Cd concentration in shoot reflected the ability of Cd translocation and tolerance of sorghum, and the path coefficient analysis indicated that root biomass could be taken as a biomarker to evaluate Cd extraction ability of sorghum. The results in this study can facilitate the restoring of Cd contaminated areas by sorghum.

High-throughput deep sequencing reveals that microRNAs play important roles in salt tolerance of euhalophyte Salicornia europaea.

BACKGROUND: microRNAs (miRNAs) are implicated in plant development processes and play pivotal roles in plant adaptation to environmental stresses. Salicornia europaea, a salt mash euhalophyte, is a suitable model plant to study salt adaptation mechanisms. S. europaea is also a vegetable, forage, and oilseed that can be used for saline land reclamation and biofuel precursor production on marginal lands. Despite its importance, no miRNA has been identified from S. europaea thus far. RESULTS: Deep sequencing was performed to investigate small RNA transcriptome of S. europaea. Two hundred and ten conserved miRNAs comprising 51 families and 31 novel miRNAs (including seven miRNA star sequences) belonging to 30 families were identified. About half (13 out of 31) of the novel miRNAs were only detected in salt-treated samples. The expression of 43 conserved and 13 novel miRNAs significantly changed in response to salinity. In addition, 53 conserved and 13 novel miRNAs were differentially expressed between the shoots and roots. Furthermore, 306 and 195 S. europaea unigenes were predicted to be targets of 41 conserved and 29 novel miRNA families, respectively. These targets encoded a wide range of proteins, and genes involved in transcription regulation constituted the largest category. Four of these genes encoding laccase, F-box family protein, SAC3/GANP family protein, and NADPH cytochrome P-450 reductase were validated using 5'-RACE. CONCLUSIONS: Our results indicate that specific miRNAs are tightly regulated by salinity in the shoots and/or roots of S. europaea, which may play important roles in salt tolerance of this euhalophyte. The S. europaea salt-responsive miRNAs and miRNAs that target transcription factors, nucleotide binding site-leucine-rich repeat proteins and enzymes involved in lignin biosynthesis as well as carbon and nitrogen metabolism may be applied in genetic engineering of crops with high stress tolerance, and genetic modification of biofuel crops with high biomass and regulatable lignin biosynthesis.

Virus-induced gene silencing reveals control of reactive oxygen species accumulation and salt tolerance in tomato by γ-aminobutyric acid metabolic pathway.

γ-Aminobutyric acid (GABA) accumulates in many plant species in response to environmental stress. However, the physiological function of GABA or its metabolic pathway (GABA shunt) in plants remains largely unclear. Here, the genes, including glutamate decarboxylases (SlGADs), GABA transaminases (SlGABA-Ts) and succinic semialdehyde dehydrogenase (SlSSADH), controlling three steps of the metabolic pathway of GABA, were studied through virus-induced gene silencing approach in tomato. Silencing of SlGADs (GABA biosynthetic genes) and SlGABA-Ts (GABA catabolic genes) led to increased accumulation of reactive oxygen species (ROS) as well as salt sensitivity under 200 mm NaCl treatment. Targeted quantitative analysis of metabolites revealed that GABA decreased and increased in the SlGADs- and SlGABA-Ts-silenced plants, respectively, whereas succinate (the final product of GABA metabolism) decreased in both silenced plants. Contrarily, SlSSADH-silenced plants, also defective in GABA degradation process, showed dwarf phenotype, curled leaves and enhanced accumulation of ROS in normal conditions, suggesting the involvement of a bypath for succinic semialdehyde catabolism to γ-hydroxybutyrate as reported previously in Arabidopsis, were less sensitive to salt stress. These results suggest that GABA shunt is involved in salt tolerance of tomato, probably by affecting the homeostasis of metabolites such as succinate and γ-hydroxybutyrate and subsequent ROS accumulation under salt stress.

H(+) -pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat.

High salinity and nitrogen (N) deficiency in soil are two key factors limiting crop productivity, and they usually occur simultaneously. Here we firstly found that H(+) -PPase is involved in salt-stimulated NO3 (-) uptake in the euhalophyte Salicornia europaea. Then, two genes (named SeVP1 and SeVP2) encoding H(+) -PPase from S. europaea were characterized. The expression of SeVP1 and SeVP2 was induced by salt stress and N starvation. Both SeVP1 or SeVP2 transgenic Arabidopsis and wheat plants outperformed the wild types (WTs) when high salt and low N occur simultaneously. The transgenic Arabidopsis plants maintained higher K(+) /Na(+) ratio in leaves and exhibited increased NO3 (-) uptake, inorganic pyrophosphate-dependent vacuolar nitrate efflux and assimilation capacity under this double stresses. Furthermore, they had more soluble sugars in shoots and roots and less starch accumulation in shoots than WT. These performances can be explained by the up-regulated expression of ion, nitrate and sugar transporter genes in transgenic plants. Taken together, our results suggest that up-regulation of H(+) -PPase favours the transport of photosynthates to root, which could promote root growth and integrate N and carbon metabolism in plant. This work provides potential strategies for improving crop yields challenged by increasing soil salinization and shrinking farmland.

Comparative proteomics of root plasma membrane proteins reveals the involvement of calcium signalling in NaCl-facilitated nitrate uptake in Salicornia europaea.

Improving crop nitrogen (N) use efficiency under salinity is essential for the development of sustainable agriculture in marginal lands. Salicornia europaea is a succulent euhalophyte that can survive under high salinity and N-deficient habitat conditions, implying that a special N assimilation mechanism may exist in this plant. In this study, phenotypic and physiological changes of S. europaea were investigated under different nitrate and NaCl levels. The results showed that NaCl had a synergetic effect with nitrate on the growth of S. europaea. In addition, the shoot nitrate concentration and nitrate uptake rate of S. europaea were increased by NaCl treatment under both low N and high N conditions, suggesting that nitrate uptake in S. europaea was NaCl facilitated. Comparative proteomic analysis of root plasma membrane (PM) proteins revealed 81 proteins, whose abundance changed significantly in response to NaCl and nitrate. These proteins are involved in metabolism, cell signalling, transport, protein folding, membrane trafficking, and cell structure. Among them, eight proteins were calcium signalling components, and the accumulation of seven of the above-mentioned proteins was significantly elevated by NaCl treatment. Furthermore, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) was significantly elevated in S. europaea under NaCl treatment. The application of the Ca(2+) channel blocker LaCl3 not only caused a decrease in nitrate uptake rate, but also attenuated the promoting effects of NaCl on nitrate uptake rates. Based on these results, a possible regulatory network of NaCl-facilitated nitrate uptake in S. europaea focusing on the involvement of Ca(2+) signalling was proposed.

Na⁺/H⁺ exchanger 1 participates in tobacco disease defence against Phytophthora parasitica var. nicotianae by affecting vacuolar pH and priming the antioxidative system.

Despite the importance of NHX1 (Na(+)/H(+) exchanger 1) in plant salt tolerance, little is known about its other functions. In this study, intriguingly, it was found that NHX1 participated in plant disease defence against Phytophthora parasitica var. nicotianae (Ppn) in Nicotiana benthamiana. NbNHX1 was originally isolated from N. benthamiana, and characterized. The subcellular localization of NbNHX1 with its C-terminus fused with green fluorescent protein indicated that NbNHX1 localized primarily to the tonoplast. Tobacco rattle virus-induced NbNHX1 silencing led to reduced H(+) efflux from the vacuole to cytoplasts, and decreased Ppn resistance in N. benthamiana. After attack by Ppn, NbNHX1-silenced plants exhibited impaired ability to scavenge reactive oxidative species (ROS) induced by the pathogen. Pea early browning virus-mediated ectopic expression of SeNHX1 (from Salicornia europaea) or AtNHX1 (from Arabidopsis thaliana) both conferred enhanced Ppn resistance to N. benthamiana, with a lower H2O2 concentration after Ppn inoculation. Further investigation of the role of NHX1 demonstrated that transient overexpression of NbNHX1 improved the vacuolar pH and cellular ROS level in N. benthamiana, which was coupled with an enlarged NAD(P) (H) pool and higher expression of ROS-responsive genes. In contrast, NbNHX1 silencing led to a lower pH in the vacuole and a lower cellular ROS level in N. benthamiana, which was coupled with a decreased NAD(P) (H) pool and decreased expression of ROS-responsive genes. These results suggest that NHX1 is involved in plant disease defence; and regulation of vacuolar pH by NHX1, affecting the cellular oxidation state, primes the antioxidative system which is associated with Ppn resistance in tobacco.

Chloroplast-targeted Hsp90 plays essential roles in plastid development and embryogenesis in Arabidopsis possibly linking with VIPP1.

The Arabidopsis genome contains seven members of Hsp90. Mutations in plastid AtHsp90.5 were reported to cause defects in chloroplast development and embryogenesis. However, the exact function of plastid AtHsp90.5 has not yet been defined. In this study, albino seedlings were found among AtHsp90.5 transformed Arabidopsis, which were revealed to be AtHsp90.5 co-suppressed plants. The accumulation of photosynthetic super-complexes in the albinos was decreased, and expression of genes involved in photosynthesis was significantly down-regulated. AtHsp90.5 T-DNA insertion mutants were embryo-lethal with embryo arrested at the heart stage. Further investigation showed AtHsp90.5 expression was up-regulated in the siliques at 4 days post anthesis (DPA). Confocal microscopy proved AtHsp90.5 was located in the chloroplasts. Plastid development in the AtHsp90.5 mutants and co-suppressed plants was seriously impaired, and few thylakoid membranes were observed, indicating the involvement of AtHsp90.5 in chloroplast biogenesis. AtHsp90.5 was found to interact with vesicle-inducing protein in plastids 1 (VIPP1) by bimolecular fluorescence complementation system. The ratio between VIPP1 oligomers and monomers in AtHsp90.5 co-suppressed plants drastically shifted toward the oligomeric state. Our study confirmed that AtHsp90.5 is vital for chloroplast biogenesis and embryogenesis. Further evidence also suggested that AtHsp90.5 may help in the disassembly of VIPP1 for thylakoid membrane formation and/or maintenance.

Coordination of carbon fixation and nitrogen metabolism in Salicornia europaea under salinity: Comparative proteomic analysis on chloroplast proteins.

Halophyte, like Salicornia europaea, could make full use of marginal saline land for carbon fixation. How the photosynthesis of S. europaea is regulated under high salinity implicates a significant aspect to exploit this pioneer plant in future. Measurement of photosynthesis parameters demonstrated the reduction of photosynthesis for the 0 and 800 mM NaCl treated plants are more likely due to non-stomatal limitation, which might be caused by changes in the enzymes associated with photosynthesis. Different salinity induced ultrastructure changes other than photosynthetic apparatus damage, suggesting the photosynthesis of S. europaea might be affected via biochemical regulation. Comparative proteomics analysis of chloroplast proteins by 2-D gel electrophoresis reproducibly detected 90 differentially expressed proteins, among which 66 proteins were identified by nanoLC MS/MS. Further study of thylakoid membrane proteins by Blue-Native PAGE proved the increase in abundance of light reaction proteins under salinity. Analysis of gene expression patterns of 12 selected proteins provides evidence for the correlations between transcription and proteomics data. Based on our results, a putative model of photosynthesis regulatory network figured out proper coordination of carbon fixation and nitrogen metabolism in chloroplast of S. europaea under salinity, which provided subcellular level insight into salt tolerance mechanism in S. europaea.

Transformation of beta-lycopene cyclase genes from Salicornia europaea and Arabidopsis conferred salt tolerance in Arabidopsis and tobacco.

Inhibition of lycopene cyclization decreased the salt tolerance of the euhalophyte Salicornia europaea L. We isolated a ß-lycopene cyclase gene SeLCY from S. europaea and transformed it into Arabidopsis with stable expression. Transgenic Arabidopsis on post-germination exhibited enhanced tolerance to oxidative and salt stress. After 8 and 21 d recovery from 200 mM NaCl treatment, transgenic lines had a higher survival ratio than wild-type (WT) plants. Three-week-old transgenic plants treated with 200 mM NaCl showed better growth than the WT with higher photosystem activity and less H(2)O(2) accumulation. Determination of endogenous pigments of Arabidopsis treated with 200 mM NaCl for 0, 2 or 4 d demonstrated that the transgenic plants retained higher contents of carotenoids than the WT. Furthermore, to compare the difference between SeLCY and AtLCY from Arabidopsis, we used viral vector mediating ectopic expression of SeLCY and AtLCY in Nicotiana benthamiana. Although LCY genes transformation increased the salt tolerance in tobacco, there is no significant difference between SeLCY- and AtLCY-transformed plants. These findings indicate that SeLCY transgenic Arabidopsis improved salt tolerance by increasing synthesis of carotenoids, which impairs reactive oxygen species and protects the photosynthesis system under salt stress, and as a single gene, SeLCY functionally showed no advantage for salt tolerance improvement compared with AtLCY.

Modified noninvasive microtest electrophysiological technology for vacuolar H(+) flux detection.

This paper describes a modified noninvasive microtest electrophysiological technology (NMT) for vacuolar H(+) flux detection. In this NMT system, the vacuole isolation procedure and buffer slope were modified, and the measuring errors from small spherical geometry were corrected. The trends in changes of vacuolar H(+) flux (ΔH(+) flux) after ATP or PP(i) supply calculated by NMT were consistent with the activities of V-ATPase and PPase measured by traditional methods. These findings indicate that our modified NMT is an appropriate method for vacuolar H(+) flux detection.

Overexpression of Thellungiella halophila H(+)-PPase (TsVP) in cotton enhances drought stress resistance of plants.

An H(+)-PPase gene, TsVP from Thellungiella halophila, was transferred into two cotton (Gossypium hirsutum) varieties (Lumianyan19 and Lumianyan 21) and southern and northern blotting analysis showed the foreign gene was integrated into the cotton genome and expressed. The measurement of isolated vacuolar membrane vesicles demonstrated that the transgenic plants had higher V-H(+)-PPase activity compared with wild-type plants (WT). Overexpressing TsVP in cotton improved shoot and root growth, and transgenic plants were much more resistant to osmotic/drought stress than the WT. Under drought stress conditions, transgenic plants had higher chlorophyll content, improved photosynthesis, higher relative water content of leaves and less cell membrane damage than WT. We ascribe these properties to improved root development and the lower solute potential resulting from higher solute content such as soluble sugars and free amino acids in the transgenic plants. In this study, the average seed cotton yields of transgenic plants from Lumianyan 19 and Lumianyan 21 were significantly increased compared with those of WT after exposing to drought stress for 21 days at flowering stage. The average seed cotton yields were 51 and 40% higher than in their WT counterparts, respectively. This study benefits efforts to improve cotton yields in arid and semiarid regions.