Root epidermis-specific expression of a phosphate transporter TaPT2 enhances the growth of transgenic Arabidopsis under Pi-replete and Pi-depleted conditions.

Although attempts to improve the phosphate (Pi) uptake and use efficiency by constitutively overexpressing phosphate transporters have resulted in enhanced Pi or total phosphorous contents, growth promotion by Pi acquisition was observed in only a few cases. This study examined the effect of the tissue-specific overexpression of phosphate transporter on Pi acquisition and plant growth. We cloned cDNA for a wheat phosphate transporter, TaPT2, using PCR and confirmed its Pi transport activity in Arabidopsis suspension cells. The overexpression of TaPT2 by the Arabidopsis Shaker family inward rectifying potassium channel 1 (AKT1) promoter, dominantly expressed in root epidermal cells, resulted in increased root and shoot growth of transgenic Arabidopsis under Pi-replete and Pi-depleted conditions. However, their Pi and total P contents did not increase. The overexpression of TaPT2 by the constitutive promoter, actin8 (ACT8), increased shoot total P contents in transgenic plants, but did not promote their growth. These results suggested that enhanced Pi uptake in root epidermal cells is suitable as a driving force for Pi transport from roots to shoots, improving subsequent Pi use in shoots. Thus, the root epidermal cell-specific expression of TaPT2 may be a simple and promising strategy for enhancing plant Pi uptake and efficiency.

Salt tolerance and regulation of Na+, K+, and proline contents in different wild turfgrasses under salt stress.

Turfgrasses show a wide range of salinity tolerance. In this study, twenty wild turfgrasses were collected from coastal regions in Japan, and their species; evolutionary lineage; salt tolerance levels; shoot and root K+, Na+, and proline contents; and amounts of ions secreted from their salt glands were determined. Among them, eighteen turfgrass species were determined based on the internal transcribed spacer 1 sequences. All collected wild turfgrasses were identified as halophytes and were divided into two salt-tolerant levels. They maintained the shoot relative water contents and suppressed excess Na+ accumulation in their shoots and roots and K+ content homeostasis compared with rice, resulting in the maintenance of a higher K+/Na+ ratio under salt stress. These characteristics must be part of the salt tolerance mechanisms. Among the four turfgrasses with salt glands, three selectively secreted Na+ from their salt glands; however, interestingly, one secreted K+ over Na+, although it still maintained a K+/Na+ ratio comparable to that of the other turfgrasses. A significant amount of proline synthesis was observed in most of the turfgrasses in response to salt stress, and the proline content was highly correlated with the salt tolerance, suggesting its key role in the salt tolerance mechanisms. These wild turfgrasses with such diverse ion control mechanisms and proline synthesis profiles are useful materials for investigating the salt tolerant mechanisms and breeding salt tolerant turfgrasses.

Comparative analysis of various root active promoters by evaluation of GUS expression in transgenic Arabidopsis.

To prepare various root active promoters for expressing transgenes and prevent gene silencing caused by the repeated use of the same promoter, the expression characteristics of various root active promoters were comparatively evaluated using GUS as a reporter gene. The high-affinity potassium transporter (HKT1;1), the Shaker family potassium ion channel (SKOR), the Shaker family inward rectifying potassium channel (AKT1), the major facilitator superfamily protein (MFS1), and the senescence associated gene 14 (SAG14) promoter from Arabidopsis (Arabidopsis thaliana) were used, and for comparison, four additional constitutive or green tissue specific promoters in the expression vectors were also employed. As the Gateway cloning technology provided by Invitrogen can offer high efficiency and cloning reliability, and easy manipulation of fusion constructs in vitro, our expression vectors are based on binary (destination) vectors compatible with this cloning technique. These destination vectors are also advantageous for stable expression of the transgene, as the heat shock protein terminator is utilized. The AtHKT1;1, SKOR, AKT1, MFS1 and SAG14 promoters were all active in roots but showed slightly different tissue specificities: AtHKT1;1, SKOR, and MFS1 were dominantly active in vascular bundle tissue, while AtHKT1;1 and MFS1- but not SKOR, AKT1, and SAG14-were active in root tips. SKOR showed the strongest root-specificity, and SAG14 showed the highest activity among the five root active promoters. The activity of MFS was developmentally regulated. These destination vectors are now available to express multiple transgenes in transgenic plants, especially in roots.

Na+ Transporter SvHKT1;1 from a Halophytic Turf Grass Is Specifically Upregulated by High Na+ Concentration and Regulates Shoot Na+ Concentration.

We characterized an Na+ transporter SvHKT1;1 from a halophytic turf grass, Sporobolus virginicus. SvHKT1;1 mediated inward and outward Na+ transport in Xenopus laevis oocytes and did not complement K+ transporter-defective mutant yeast. SvHKT1;1 did not complement athkt1;1 mutant Arabidopsis, suggesting its distinguishable function from other typical HKT1 transporters. The transcript was abundant in the shoots compared with the roots in S. virginicus and was upregulated by severe salt stress (500 mM NaCl), but not by lower stress. SvHKT1;1-expressing Arabidopsis lines showed higher shoot Na+ concentrations and lower salt tolerance than wild type (WT) plants under nonstress and salt stress conditions and showed higher Na+ uptake rate in roots at the early stage of salt treatment. These results suggested that constitutive expression of SvHKT1;1 enhanced Na+ uptake in root epidermal cells, followed by increased Na+ transport to shoots, which led to reduced salt tolerance. However, Na+ concentrations in phloem sap of the SvHKT1;1 lines were higher than those in WT plants under salt stress. Based on this result, together with the induction of the SvHKT1;1 transcription under high salinity stress, it was suggested that SvHKT1;1 plays a role in preventing excess shoot Na+ accumulation in S. virginicus.

Comparative Functional Analysis of Class II Potassium Transporters, SvHKT2;1, SvHKT2;2, and HvHKT2;1, on Ionic Transport and Salt Tolerance in Transgenic Arabidopsis.

Class II high-affinity potassium transporters (HKT2s) mediate Na+-K+ cotransport and Na+/K+ homeostasis under K+-starved or saline conditions. Their functions have been studied in yeast and X. laevis oocytes; however, little is known about their respective properties in plant cells. In this study, we characterized the Na+ and K+ transport properties of SvHKT2;1, SvHKT2;2 and HvHKT2;1 in Arabidopsis under different ionic conditions. The differences were detected in shoot K+ accumulation and root K+ uptake under salt stress conditions, K+ accumulation in roots and phloem sap under K+-starved conditions, and shoot and root Na+ accumulation under K+-starved conditions among the HKT2s transgenic lines and WT plants. These results indicate the diverse ionic transport properties of these HKT2s in plant cells, which could not be detected using yeast or X. laevis oocytes. Furthermore, Arabidopsis expressing HKT2s showed reduced salt tolerance, while over-expression of HvHKT2;1 in barley, which has the ability to sequestrate Na+, showed enhanced salt tolerance by accumulating Na+ in the shoots. These results suggest that the coordinated enhancement of Na+ accumulation and sequestration mechanisms in shoots could be a promising strategy to confer salt tolerance to glycophytes.

The HKT Transporter Gene from Arabidopsis, AtHKT1;1, Is Dominantly Expressed in Shoot Vascular Tissue and Root Tips and Is Mild Salt Stress-Responsive.

The Arabidopsis high-affinity K+ transporter (AtHKT1;1) plays roles in salt tolerance by unloading Na+ from the root xylem to the xylem parenchyma cells and/or uploading Na+ from the shoot/leaf xylem to the xylem parenchyma cells. To use this promoter for the molecular breeding of salt-tolerant plants, I evaluated the expression profile of the AtHKT1;1 promoter in detail. Approximately 1.1 kbp of sequence upstream from the start codon of AtHKT1;1 was polymerase chain reaction (PCR)-amplified, fused to the ß-glucuronidase (GUS) gene, and introduced into Arabidopsis. The resultant transformants were evaluated under nonstressed and salt-stress conditions at the seedling and reproductive stages. Histochemical analysis showed that GUS activity was detected in vascular bundle tissue in roots, hypocotyls, petioles, leaves, and petals, and in root tips. GUS enzyme activity in shoots tended to be higher than that in roots at both stages. After treatment with 50 mM NaCl for 24 h, GUS transcription levels and GUS enzyme activity were enhanced in transgenic lines. These results indicate that the AtHKT1;1 promoter isolated in this study could be useful in expressing transgenes specifically in vascular tissue and root tips, and in a mild salt-stress-responsive manner. The data provide novel insights into the functions of AtHKT1;1.

Transcriptomic analysis of Aegilops tauschii during long-term salinity stress.

Aegilops tauschii is the diploid progenitor of the bread wheat D-genome. It originated from Iran and is a source of abiotic stress tolerance genes. However, little is known about the molecular events of salinity tolerance in Ae. tauschii. This study investigates the leaf transcriptional changes associated with long-term salt stress. Total RNA extracted from leaf tissues of control and salt-treated samples was sequenced using the Illumina technology, and more than 98 million high-quality reads were assembled into 255,446 unigenes with an average length of 1398 bp and an N50 of 2269 bp. Functional annotation of the unigenes showed that 93,742 (36.69%) had at least a significant BLAST hit in the SwissProt database, while 174,079 (68.14%) showed significant similarity to proteins in the NCBI nr database. Differential expression analysis identified 4506 salt stress-responsive unigenes. Bioinformatic analysis of the differentially expressed unigenes (DEUs) revealed a number of biological processes and pathways involved in the establishment of ion homeostasis, signaling processes, carbohydrate metabolism, and post-translational modifications. Fine regulation of starch and sucrose content may be important features involved in salt tolerance in Ae. tauschii. Moreover, 82% of DEUs mapped to the D-subgenome, including known QTL for salt tolerance, and these DEUs showed similar salt stress responses in other accessions of Ae. tauschii. These results could provide fundamental insight into the regulatory process underlying salt tolerance in Ae. tauschii and wheat and facilitate identification of genes involved in their salt tolerance mechanisms.

Functional screening of salt tolerance genes from a halophyte Sporobolus virginicus and transcriptomic and metabolomic analysis of salt tolerant plants expressing glycine-rich RNA-binding protein.

Sporobolus virginicus is a halophytic C4 grass found worldwide, from tropical to warm temperate regions. One Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a three-fold higher concentration than the salinity of sea water. To identify the key genes involved in the regulation of salt tolerance in S. virginicus, we produced 3500 independent transgenic Arabidopsis lines expressing random cDNA from S. virginicus and screened 10 lines which showed enhanced salt tolerance compared with the wild type in a medium containing 150 mM NaCl. Among the selected lines, two contained cDNA coding glycine-rich RNA-binding proteins (SvGRP1 and SvGRP2). This is the first reports on the function of GRPs from halophytes in salt tolerance though reports have shown GRPs are involved in diverse biological and biochemical processes including salt tolerance in Arabidopsis and some other glycophytes. Transcriptomic analysis and GO enrichment analysis of SvGRP1-expressing Arabidopsis under salt stress revealed upregulation of polyol and downregulation of glucosinolate and indole acetic acid biosynthesis/metabolic pathways. Metabolomic analysis of the SvGRP1-transformant suggested that the increase in 3-aminoppropanoic acid, citramalic acid, and isocitric acid content was associated with enhanced salt tolerance. These findings could provide novel insight into the roles of GRPs in plant salt tolerance.

High-Affinity K+ Transporters from a Halophyte, Sporobolus virginicus, Mediate Both K+ and Na+ Transport in Transgenic Arabidopsis, X. laevis Oocytes and Yeast.

Class II high-affinity potassium transporters (HKTs) have been proposed to mediate Na+-K+ co-transport in plants, as well as Na+ and K+ homeostasis under K+-starved and saline environments. We identified class II HKTs, namely SvHKT2;1 and SvHKT2;2 (SvHKTs), from the halophytic turf grass, Sporobolus virginicus. SvHKT2;2 expression in S. virginicus was up-regulated by NaCl treatment, while SvHKT2;1 expression was assumed to be up-regulated by K+ starvation and down-regulated by NaCl treatment. Localization analysis revealed SvHKTs predominantly targeted the plasma membrane. SvHKTs complemented K+ uptake deficiency in mutant yeast, and showed both inward and outward K+ and Na+ transport activity in Xenopus laevis oocytes. When constitutively expressed in Arabidopsis, SvHKTs mediated K+ and Na+ accumulation in shoots under K+-starved conditions, and the K+ concentration in xylem saps of transformants was also higher than in those of wild-type plants. These results suggest transporter-enhanced K+ and Na+ uploading to the xylem from xylem parenchyma cells. Together, our data demonstrate that SvHKTs mediate both outward and inward K+ and Na+ transport in X. laevis oocytes, and possibly in plant and yeast cells, depending on the ionic conditions.

Comparative whole genome re-sequencing analysis in upland New Rice for Africa: insights into the breeding history and respective genome compositions.

BACKGROUND: Increasing rice demand is one of the consequences of the steadily improving socio-economic status of the African countries. New Rice for Africa (NERICA), which are interspecific hybrids between Asian and African rice varieties, are one of successful breeding products utilizing biodiversity across the two different rice crop species. Upland NERICA varieties (NU) exhibit agronomic traits of value for the harsh eco-geography, including shorter duration, higher yield and stress tolerance, compared to local African varieties. However, the molecular basis of the traits in NU varieties is largely unknown. RESULTS: Whole genome re-sequencing was performed of four NU lines (3, 4, 5, and 7) and for the parental Oryza sativa WAB56-104 and Oryza glaberrima CG14. The k-mer analysis predicted large genomes for the four NU lines, most likely inherited from WAB56-104. Approximately 3.1, 0.10, and 0.40 million single nucleotide polymorphisms, multi nucleotide polymorphisms, and short insertions/deletions were mined between the parental lines, respectively. Integrated analysis with another four NU lines (1, 2, 8, and 9) showed that the ratios of the donor CG14 allelic sites in the NU lines ranged from 1.3 to 9.8%. High resolution graphical genotype indicated genome-level similarities and common genetic events during the breeding process: five xyloglucan fucosyltransferase from O. glaberrima were introgressed in common. Segregation of genic segments revealed potential causal genes for some agronomic traits including grain shattering, awnness, susceptibility to bacterial leaf bright, and salt tolerance. Analysis of unmapped sequences against the reference cultivar Nipponbare indicated existence of unique genes for pathogen and abiotic stress resistance in the NU varieties. CONCLUSIONS: The results provide understanding of NU genomes for rice improvement for Africa reinforcing local capacity for food security and insights into molecular events in breeding of interspecific hybrid crops.

Plant signaling networks involving Ca(2+) and Rboh/Nox-mediated ROS production under salinity stress.

Salinity stress, which induces both ionic and osmotic damage, impairs plant growth and causes severe reductions in crop yield. Plants are equipped with defense responses against salinity stress such as regulation of ion transport including Na(+) and K(+), accumulation of compatible solutes and stress-related gene expression. The initial Ca(2+) influx mediated by plasma membrane ion channels has been suggested to be crucial for the adaptive signaling. NADPH oxidase (Nox)-mediated production of reactive oxygen species (ROS) has also been suggested to play crucial roles in regulating adaptation to salinity stress in several plant species including halophytes. Respiratory burst oxidase homolog (Rboh) proteins show the ROS-producing Nox activity, which are synergistically activated by the binding of Ca(2+) to EF-hand motifs as well as Ca(2+)-dependent phosphorylation. We herein review molecular identity, structural features and roles of the Ca(2+)-permeable channels involved in early salinity and osmotic signaling, and comparatively discuss the interrelationships among spatiotemporal dynamic changes in cytosolic concentrations of free Ca(2+), Rboh-mediated ROS production, and downstream signaling events during salinity adaptation in planta.

Comprehensive analysis of transcriptome response to salinity stress in the halophytic turf grass Sporobolus virginicus.

The turf grass Sporobolus virginicus is halophyte and has high salinity tolerance. To investigate the molecular basis of its remarkable tolerance, we performed Illumina high-throughput RNA sequencing on roots and shoots of a S. virginicus genotype under normal and saline conditions. The 130 million short reads were assembled into 444,242 unigenes. A comparative analysis of the transcriptome with rice and Arabidopsis transcriptome revealed six turf grass-specific unigenes encoding transcription factors. Interestingly, all of them showed root specific expression and five of them encode bZIP type transcription factors. Another remarkable transcriptional feature of S. virginicus was activation of specific pathways under salinity stress. Pathway enrichment analysis suggested transcriptional activation of amino acid, pyruvate, and phospholipid metabolism. Up-regulation of several unigenes, previously shown to respond to salt stress in other halophytes was also observed. Gene Ontology enrichment analysis revealed that unigenes assigned as proteins in response to water stress, such as dehydrin and aquaporin, and transporters such as cation, amino acid, and citrate transporters, and H(+)-ATPase, were up-regulated in both shoots and roots under salinity. A correspondence analysis of the enriched pathways in turf grass cells, but not in rice cells, revealed two groups of unigenes similarly up-regulated in the turf grass in response to salt stress; one of the groups, showing excessive up-regulation under salinity, included unigenes homologos to salinity responsive genes in other halophytes. Thus, the present study identified candidate genes involved in salt tolerance of S. virginicus. This genetic resource should be valuable for understanding the mechanisms underlying high salt tolerance in S. virginicus. This information can also provide insight into salt tolerance in other halophytes.

Growth and physiological adaptation of whole plants and cultured cells from a halophyte turf grass under salt stress.

Understanding the mechanisms used by halophytic members of the Poaceae to cope with salt stress will contribute to the knowledge necessary to genetically engineer salt-tolerant crops. In this study, we identified a genotype of Sporobolus virginicus, a halophytic turf grass collected in Japan, and investigated its growth rate, ion concentration and secretion, and proline concentration in comparison with the reported properties of genotypes collected from the USA, South Africa and Egypt. Surprisingly, the Japanese genotype showed a salinity tolerance up to 1.5 M NaCl, a 3-fold higher concentration than seawater salinity. Shoot growth was stimulated by 100 mM NaCl and root growth was stimulated at salinities of up to 1 M NaCl. Accumulation of Na(+) and CI(-) in shoots and roots was rapidly elevated by salinity stress but did not exceed the levels required for osmotic adjustment, due in part to ion secretion by salt glands, which are present in genotypes of S. virginicus. However, the Japanese genotypes accumulated K(+) to a higher level than other genotypes, resulting in a relatively high K(+)/Na(+) ratio even under salinity stress. An increase in proline concentration was observed that was proportional to the NaCl concentration in the culture solution and might partially account for osmotic adjustment in the shoots. We also generated and characterized cultured cells of S. virginicus. In 500 mM NaCl, the cultured cells showed an enhanced growth compared with cultured cells of rice. The concentration of Na(+) and CI(-) in the cultured cells in 300-500 mM NaCl was lower than in 100 mM NaCl. Cultured cells of S. virginicus accumulated proline to higher levels than rice cells cultured under salinity stress. The active regulation of Na(+), Cl(-) and K(+) influx/efflux and proline accumulation might be involved in salt tolerance mechanisms at the cellular level as well as in planta.

Involvement of elevated proline accumulation in enhanced osmotic stress tolerance in Arabidopsis conferred by chimeric repressor gene silencing technology.

Arabidopsis plants transformed with a chimeric repressor for 6 transcription factors (TFs), including ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23, that were converted by Chimeric REpressor gene Silencing Technology (CRES-T), show elevated salt and osmotic stress tolerance compared with wild type (WT) plants. However, the roles of TFs in salt and osmotic signaling remain largely unknown. Their hyper-osmotic stress tolerance was evaluated using 3 criteria: germination rate, root length, and rate of seedlings with visible cotyledons at the germination stage. All CRES-T lines tested exhibited better performance than WT, at least for one criterion under stress conditions. Under 600 mM mannitol stress, 3-week-old CRES-T lines accumulated proline, which is a major compatible solute involved in osmoregulation, at higher levels than WT. Expression levels of the delta 1-pyrroline-5-carboxylate synthase gene in CRES-T lines were similar to or lower than those in WT. In contrast, expression of the proline dehydrogenase (PHD) gene in DREB26-SRDX was significantly downregulated and that in ADA2b-SRDX and AtGeBP-SRDX was also rather downregulated compared with that in WT. Although plants at different stages were used for stress tolerance test and proline measurement in this study, we previously reported that 4 out of the 6 CRES-T lines showed better growth than WT after 4 weeks of incubation under 400 mM mannitol. These results suggest that proline accumulation caused by PHD gene suppression may be involved in enhanced osmotic stress tolerance in the CRES-T lines, and that these TFs may be involved in regulating proline metabolism in Arabidopsis.

Identification of Chimeric Repressors that Confer Salt and Osmotic Stress Tolerance in Arabidopsis.

We produced transgenic Arabidopsis plants that express chimeric genes for transcription factors converted to dominant repressors, using Chimeric REpressor gene-Silencing Technology (CRES-T), and evaluated the salt tolerance of each line. The seeds of the CRES-T lines for ADA2b, Msantd, DDF1, DREB26, AtGeBP, and ATHB23 exhibited higher germination rates than Wild type (WT) and developed rosette plants under up to 200 mM NaCl or 400 mM mannitol. WT plants did not grow under these conditions. In these CRES-T lines, the expression patterns of stress-related genes such as RD29A, RD22, DREB1A, and P5CS differed from those in WT plants, suggesting the involvement of the six transcription factors identified here in the stress response pathways regulated by the products of these stress-related genes. Our results demonstrate additional proof that CRES-T is a superior tool for revealing the function of transcription factors.

Chronic stress enhances calcium mobilization and glutamate exocytosis in cerebrocortical synaptosomes from mice.

OBJECTIVES: Our previous study showed that acute restraint stress enhances depolarization-induced increases in intrasynaptosomal free calcium (Ca(2+)) concentration ([Ca(2+)](i)) and Ca(2+)-dependent glutamate release in mouse cerebrocortical nerve terminals (synaptosomes). In the present study, we investigated the effects of chronic stress on [Ca(2+)](i) and glutamate release in cerebrocortical synaptosomes from mice. METHODS: Male ddY strain mice were randomly assigned to one of two experimental groups: control group and chronic stressed group. Mice in the chronic stressed group were subjected to immobilization stress for 2 hours daily for a period of 21 days. [Ca(2+)](i) and glutamate release in cerebrocortical synaptosomes isolated from the mice were determined by fura-2 fluorescence assay and enzyme-linked fluorometric assay, respectively. RESULTS: Chronic stress caused a significant increase in resting [Ca(2+)](i) and significantly enhanced the ability of the depolarizing agents K(+) and 4-aminopyridine (4-AP) to increase [Ca(2+)](i). It also brought about a significant increase in spontaneous (unstimulated) glutamate release and significantly enhanced K(+)- and 4-AP-evoked Ca(2+)-dependent glutamate release. Synaptosomes were more sensitive to the depolarizing agents at lower concentrations following chronic stress than after acute stress. The pretreatment of synaptosomes with a combination of omega-agatoxin IVA (a P-type Ca(2+) channel blocker) and omega-conotoxin GVIA (an N-type Ca(2+) channel blocker) completely suppressed the enhancements of [Ca(2+)](i) and Ca(2+)-dependent glutamate release in chronic stressed mice. DISCUSSION: These results indicate that chronic stress enhances depolarization-evoked glutamate release by increasing [Ca(2+)](i) via stimulation of Ca(2+) entry through P- and N-type Ca(2+) channels, and that chronic stress increases the sensitivity to depolarizing agents.

Usefulness of intravascular ultrasonography monitoring of coil embolization for traumatic direct carotid-cavernous fistula-case report-.

A 61-year-old woman was admitted for head injury after a traffic accident. Two months later, she developed abducens nerve palsy, chemosis, and pulsatile tinnitus. Right internal carotid angiography demonstrated a high flow direct carotid-cavernous fistula (CCF) at the C(5) portion with reflux into the superficial and deep sylvian veins, superior ophthalmic vein, superior petrosal sinus, and inferior petrosal sinus. Intravascular ultrasonography (IVUS) revealed a large fistula at the C(5) portion of the internal carotid artery (ICA). Coil embolization via transarterial and transvenous approaches under IVUS monitoring was performed. During the procedure, IVUS accurately detected protrusion of a coil into the parent ICA, and the parent artery could be preserved. IVUS monitoring is useful for embolization of direct CCF with coils.

Transcriptome profiling of the mangrove plant Bruguiera gymnorhiza and identification of salt tolerance genes by Agrobacterium functional screening.

To identify key genes in the regulation of salt tolerance in the mangrove plant Bruguiera gymnorhiza, transcriptome profiling in the lateral and main roots under conditions of salt stress was performed. Statistical analysis revealed that 175 and 403 of 11,997 genes shoewd significantly increased high expression in the lateral and main roots respectively. One hundred and sixty genes were up-regulated in both types of roots in the early time period, 1 to 12 h after salt treatment. Expression vectors for 28 selected salt responsive genes were constructed and transformed in Agrobacterium tumefaciens, and then screened for salt tolerance. A. tumefaciens transformed with genes for lipid transfer, zinc finger, and ankyrin repeat proteins showed enhanced salt tolerance. Transgenic Arabidopsis plants expressing these three genes also exhibited increased tolerance to NaCl. These results indicate that Agrobacterium functional screening is an effective supplemental method of pre-screening genes involved in abiotic stress tolerance.

Proteomic analysis of salt-responsive proteins in the mangrove plant, Bruguiera gymnorhiza.

To identify key proteins in the regulation of salt tolerance in the mangrove plant Bruguiera gymnorhiza, proteome analysis of samples grown under conditions of salt stress was performed. Comparative two-dimensional electrophoresis revealed that two, three and one protein were differentially expressed in the main root, lateral root and leaf, respectively, in response to salt stress. Among these, three proteins were identified by internal peptide sequence analysis: fructose-1,6-bisphosphate (FBP) aldolase and a novel protein in the main root, and osmotin in the lateral root. These results suggest that FBP aldolase and osmotin play roles in salt tolerance mechanisms common to both glycophytes and mangrove plants. Osmotin was abundant at early time points following salt treatment and seems to play a role in initial osmotic adaptation in lateral roots of B. gymnorhiza under salt stress, but does not contribute towards adaptation to prolonged or continuous exposure to salt stress. The amounts of these proteins were not correlated with those of the respective mRNAs, as determined by microarray analysis. A novel salt-responsive protein, not previously detected by expressed sequence tag analysis or transcriptome analysis, was also identified in this proteomic approach, and may provide insight into the salt tolerance mechanism of the mangrove plant. This is the first report of proteome analysis with detailed analysis of main and lateral roots of mangrove plants under salt stress conditions.

Transcriptional and physiological study of the response of Burma mangrove (Bruguiera gymnorhiza) to salt and osmotic stress.

We investigated the transcriptional response of Burma mangrove (Bruguiera gymnorhiza) to high salinity (salt stress; 500 mM NaCl) and hyperosmotic stress (osmotic stress; 1 M sorbitol) by microarray analysis. ANOVA (P < 0.05) and significant analysis of microarray (SAM; FDR < 5%) revealed that 865 of 11,997 genes showed significant differential expression under salt and osmotic stress. Scatter plot analysis revealed that the expression level of genes changed at 6 h after salt stress treatment, but recovered at 24 h, while the change at 6 h after osmotic stress treatment diverged at 24 h. Hierarchical clustering of the 865 genes showed that expression profiles under salt stress were distinctly different from those under osmotic stress. Comparison of gene ontology (GO) categories of differentially expressed genes under the stress conditions revealed that the adaptation of Burma mangrove to salt stress was accompanied by the up-regulation of genes categorized for "cell communication," "signal transduction," "lipid metabolic process," "photosynthesis," "multicellular organismal development," and "transport," and by down-regulation of genes categorized for "catabolic process." Burma mangrove maintained its leaf water potential and recovered from its photosynthesis rate that declined temporarily under salt stress, but not under osmotic stress. These results demonstrated a fundamental difference between the response to salt and osmotic stress. Ion and sugar content analysis suggested that salt tolerance of Burma mangrove might be attributed to their ability to accumulate high concentrations of Na+ and Cl(-), even under non-stressed conditions; to uptake additional Na+ and Cl(-) for use as osmolytes; and to maintain K+ homeostasis under salt stress.