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Salt stress in the rice field is one of the most common abiotic stresses, reducing crop productivity, especially at reproductive stage, which is very sensitive to salt stress. The aim of this investigation was to study mRNA-related Na+ uptake/translocation and Na+ enrichment in the cellular level, leading to physiological changes, growth characteristics, and yield attributes in FL530 [salt-tolerant genotype; carrying SKC1 (in relation to high-affinity potassium transporters controlling Na+ and K+ translocation) and qSt1b (linking to salt injury score) QTLs] and KDML105 (salt-sensitive cultivar; lacking both QTLs) parental lines and 221–48 (carrying SKC1 and qSt1b QTLs) derived from BILs (backcross introgression lines) at 50% flowering of rice, under 150-mM NaCl until harvesting process. The upregulation of OsHKT1;5 (mediating Na+ exclusion into xylem parenchyma cells) and OsNHX1 (Na+/H+ exchanger to secrete Na+ into vacuole) and downregulation of OsHKT2;1 and OsHKT2;2 (mediating Na+ restriction in the roots, leaf sheath and older leaves) in cvs. FL530 and 221–48 (+ SKC1; + qSt1b) under salt stress were observed. It restricted Na+ level in flag leaf, thereby preventing salt toxicity, as indicated by maintenance of photon yield of PSII (ΦPSII), net photosynthetic rate (Pn), transpiration rate (E) and overall growth performances. In contrast, Na+ enrichment in flag leaf of cv. KDML105 (−SKC1;−qSt1b) caused the reduction in ΦPSII by 30.5% over the control, leading to the reduction in Pn by 62.3%, in seed sterility by 88.2%, and yield loss by 85.1%. Moreover, the negative relationships between Na+ enrichment in flag leaf, physiological changes, and yield traits in rice crop grown under salt stress were demonstrated. Based on this investigation, rice genotype 221–48 was found to possess salt-tolerant traits at reproductive stage and thus could prove to be a potential candidate for future breeding programs.

Being a salt-sensitive crop, the growth, development and productivity of rice is severely affected by saline soil. In the present study, Na+ homeostasis, physiological and morphological responses in both the root and leaf tissues of rice (Oryza sativa) seedlings of backcross introgression lines of KDML105 (KD; −/−) × FL530 (+/+) with or without SKC1 and qSt1b QTLs grown under 200 mM NaCl were investigated. Expression levels of OsHKT1;5 in FL530 (+/+) were very low, causing to low Na+ in both the root and leaf tissues, whereas it was up-regulated in KD (−/−), relating to Na+ enrichment. Expression levels of OsHKT2;1 in rice lines, 221-44 (+/+) and 221-48 (+/+) were down-regulated, thus limiting Na+ xylem upload, and store Na+ in the root tissues. OsSOS1 expression level in the leaves of 221-44 (+/+) and 221-58 (+/−) was up-regulated in relation to Na+ efflux. Na+ secretion into vacuoles via OsNHX1 in the leaf tissues was evidently demonstrated in 221-48 (+/+) as a major defense mechanism adopted by the plants to maintain photosynthetic abilities and growth performances, when plants were subjected to salt stress. In addition, the high Fv/Fm ratio in the leaves of 221-58 (+/−) was associated with increased expression level of OsNHX3, in comparison to KD (−/−) seedlings grown under salt stress (diminution by 22.8% over control). Moreover, reduction on growth characters and physiological parameters, viz., shoot fresh weight, shoot height, number of leaves, leaf area, Pn, and accumulation of Na+ in the roots under salt stress were applied as effective indices; thereby classifying FL530 (+/+) and 221-48 (+/+) as salt-tolerant, and 221-44 (+/+), 221-58 (+/−), KD (−/−), 221-3 (+/−), and 221-54 (−/−) as salt-susceptible.

Sugarcane is a sugar-producing crop widely grown in tropical regions in over 120 countries of the world. Salt-affected soil is one of the most significant abiotic constraints that inhibit growth and crop productivity, and, consequently, reduce sucrose concentration in the stalk. The present study investigated vacuolar ion homeostasis, Na+ accumulation, and physiological and morphological adaptations under salt stress in two different sugarcane genotypes (salt-tolerant K88-92 and salt-sensitive K92-80) under greenhouse conditions. Na+ was rapidly absorbed by the root tissues of both sugarcane genotypes within 3-7 days of 150 mM NaCl treatment, as confirmed by the results of CoroNa Green fluorescence staining. In addition, the rate of Na+ translocation from roots to shoots was evidently reduced, leading to lower amount of Na+ in the leaf tissues. At the cellular level, expression of ShNHX1 (vacuolar Na+/H+ antiporter), ShV-PPase (vacuolar H+-pyrophosphatase), and ShV-ATPase (vacuolar H+-ATPase) was upregulated in salt-stressed plants for the compartmentation of Na+ into the vacuoles of root cells. Interestingly, sucrose, glucose, and fructose in root tissues of salt-stressed sugarcane cv. K88-92 were increased by 10.61, 5.58, and 1.81 folds, respectively, over the control. Total soluble sugars in the roots and free proline in the leaves of sugarcane cv. K88-92 (salt-tolerant) were enriched by 3.08 and 1.99 folds, respectively, when plants were exposed to 150 mM NaCl, leading to maintain better photosynthetic abilities, net photosynthetic rate (Pn), stomatal conductance (gs), transpiration rate (E), and water use efficiency (WUE) in sugarcane cv. K88-92 than those in cv. K92-80. The study concludes that Na+ compartmentation in the root tissue acts as a major defense mechanism in sugarcane, especially in salt-tolerant genotype.

Salt stress in the rice field is one of the most common abiotic stresses, reducing crop productivity, especially at reproductive stage, which is very sensitive to salt stress. The aim of this investigation was to study mRNA-related Na

Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C15 -C19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. Recently, two universal but mutually exclusive hydrocarbon production pathways in Cyanobacteria were discovered. In this study, we constructed a deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942. The mutant was incapable to produce alkanes and exhibited normal growth phenotype at low salinity. But, the mutant became salt sensitive. Overexpression of alkane synthesis genes from halotolerant Aphanothece halophytica in Synechococcus PCC7942 restored the growth defect. The alkane synthesis gene from halotolerant cyanobacterium A. halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results indicate the requirement of alkanes for salt tolerance, and the alkane synthesis genes from A. halophytica could be a promising candidate for future biofuel application. Significance and impact of the study Cyanobacteria have been attracting great interest in the research area of biofuel production. All Cyanobacteria contain C15 -C19 hydrocarbons, but physiological roles of hydrocarbons remain to be clarified. In this study, it was found that the deletion mutant of alkane synthesis genes in fresh water cyanobacterium Synechococcus elongates PCC 7942 was incapable to produce alkanes and salt sensitive. The alkane synthesis gene from halotolerant cyanobacterium Aphanothece halophytica was salt induced and produced a significant amount of alkanes at high salinity. These results demonstrate the alkane synthesis genes from A. halophytica could be a promising candidate for future biofuel application.

Betaine (trimethylglycine) is an important compatible solute that accumulates in response to abiotic stresses such as drought and salinity. Biosynthetic pathways of betaine have been extensively studied, but it remains to be clarified on algae. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems. Here we show that the genome sequence of Thalassiosira suggests the presence of two biosynthetic pathways for betaine, via three step methylation of glycine and via two step oxidation of choline. The choline oxidation via choline dehydrogenase was suggested and its sequential characteristics were analyzed. A candidate gene TpORF1 for glycine methylation encodes a protein consisted of 574 amino acids with two putative tandem repeat methyltransferase domains. The TpORF1 was expressed in E. coli, and the purified protein was shown to synthesize betaine via three step methylation of glycine and designated as TpGSDMT. The proteins containing C-terminal half or N-terminal half were expressed in E. coli and exhibited the methyl transferase activities with different substrate specificity for glycine, sarcosine and dimethylglycine. Upregulation of TpGSDMT transcription and betaine levels were observed at high salinity, suggesting the importance of TpGSDMT for salt tolerance in T. pseudonana cells.

Raffinose (sucrosylgalactoside oligosaccharide) is a water soluble carbohydrate and accumulates in response to abiotic stresses in plants. Plant raffinose synthases are poorly characterized, and the genes involved in raffinose biosynthesis are unknown in sugar beet. Here, we report the isolation of two genes encoding raffinose synthase (BvRS1 and BvRS2) as well as a gene encoding galactinol synthase (BvGolS1) from sugar beet. BvRS1 and BvRS2 show high homologies to Arabidopsis raffinose synthase AtRS5. BvRS1 and BvGolS1 were expressed in Escherichia coli. Crude extracts showed the activities of raffinose synthase and galactinol synthase. The K m values of BvRS1 for galactinol and sucrose and the K m values of BvGolS1 for UDP-galactose and myo-inositol were determined. The expression levels of BvRS1 were significantly higher than that of BvRS2. The mRNA for BvRS1 was rapidly induced by cold stress whereas the mRNA for BvRS2 was slowly induced by cold and salt stresses. These data suggest that BvRS1 and BvRS2 encode raffinose synthase genes responsible to cold and salt stress, respectively.

The present study investigated the significance of serine biosynthetic genes for salt stress in sugar beet (Beta vulgaris). We isolated a total of four genes, two each encoding D-3-phosphoglycerate dehydrogenase (BvPGDHa and BvPGDHb) and serine hydroxymethyl transferase (BvSHMTa and BvSHMTb). mRNA transcriptional expression for BvPGDHa was significantly enhanced under salt stress conditions in both leaves and roots of sugar beet, whereas it was reduced for BvPGDHb. On the other hand, BvSHMTa was expressed transiently in leaves and roots under salt stress, whereas expression level of BvSHMTb was not altered. PGDH activity was high in storage root. After salt stress, PGDH activity was increased in leaf, petiole, and root. Recombinant proteins were expressed in Escherichia coli. The K m values for 3-phosphoglycerate in PGDHa and PGDHb were 1.38 and 2.92 mM, respectively. The findings suggest that BvPGDHa and BvSHMTa play an important role during salt stress in sugar beet.

Mycosporine-like amino acids (MAAs) are a group of natural sunscreen compounds that possess highly photoprotective properties. The most commonly found MAAs in marine organisms is shinorine, porphyra-334, and mycosporine-glycine. However, the halophilic species accumulate mycosporine-2-glycine (M2G) as the major MAA. In this study, we have investigated the protective effect of M2G against oxidative stress. In vitro radical scavenging activity revealed that M2G exhibited a significant inhibition with scavenging concentration (SC) 50 value of 22±1.4μM. In vivo analysis using the human melanoma A375 and a control cell line (NHSF) showed that M2G at low concentration (i.e. micromolar range) protected the cells against the oxidative stress (H2O2)-induced cell death. Comet assay to assess total DNA strand breaks demonstrated that M2G was not genotoxic and protected against the DNA damage by H2O2 treatment at the same level as ascorbic acid. To our knowledge, this is the first evidence demonstrating potential protective role of the natural sunscreen compound M2G against oxidative stress-induced DNA damage in human cell lines. The potent antioxidant activity combined with DNA protection ability of M2G may support its endorsement as a potential natural sunscreen with antioxidant property. These findings provide important clues for possible use of M2G in pharmaceutical and biotechnological applications.

Sugarcane is a sugar-producing crop widely grown in tropical regions in over 120 countries of the world. Salt-affected soil is one of the most significant abiotic constraints that inhibit growth and crop productivity, and, consequently, reduce sucrose concentration in the stalk. The present study investigated vacuolar ion homeostasis, Na

Among abiotic stresses, salt stress adversely affects growth and development in rice. Contrasting salt tolerant (CSR27), and salt sensitive (MI48) rice varieties provided information on an array of genes that may contribute for salt tolerance of rice. Earlier studies on transcriptome and proteome profiling led to the identification of salt stress-induced serine hydroxymethyltransferase-3 (SHMT3) gene. In the present study, the SHMT3 gene was isolated from salt-tolerant (CSR27) rice. OsSHMT3 exhibited salinity-stress induced accentuated and differential expression levels in different tissues of rice. OsSHMT3 was overexpressed in Escherichia coli and assayed for enzymatic activity and modeling protein structure. Further, Arabidopsis transgenic plants overexpressing OsSHMT3 exhibited tolerance toward salt stress. Comparative analyses of OsSHMT3 vis a vis wild type by ionomic, transcriptomic, and metabolic profiling, protein expression and analysis of various traits revealed a pivotal role of OsSHMT3 in conferring tolerance toward salt stress. The gene can further be used in developing gene-based markers for salt stress to be employed in marker assisted breeding programs. HIGHLIGHTS simple - The study provides information on mechanistic details of serine hydroxymethyl transferase gene for its salt tolerance in rice. Keywords: rice, Arabidopsis, salinity, serine, glycine, aquaporins Introduction Rice (Oryza sativa) is a staple food for ∼90% of the Asian population (Mohanty et al., 2013). Production of rice in 2011–2014 was 495.63 million tons (MT) (FAO, 2014 1). The global population is projected to increase by 25% by 2050 (United Nations World Population Prospects, 2017 2). Therefore, there is a need for a commensurate increase in rice production to meet the ever-increasing demands of the growing population (FAOSTAT, 2009 3). Although India has the largest acreage under rice [44.5 million ha (Mha)], the average productivity (2.5 tons ha-1) is abysmally low compared with China (Singh et al., 2016). This low productivity could be attributed to harsh conditions in different agroclimatic regions of India under which rice is cultivated and often subjected to various abiotic stresses (salinity, drought, flood, and nutrient deficiencies). In India, a large proportion of agriculture land (6.73 Mha) is affected by high saline content (Singh et al., 2016). Salinity adversely affects growth and development, particularly during seedling and flowering stages, and consequently the yield potential of rice (Krishnamurthy et al., 2016; Zeng et al., 2018; Morton et al., 2019). The salinity-tolerant plants have evolved an array of adaptive strategies (exclusion, compartmentation, and secretion of Na+) to mitigate the toxic effects of salinity stress (Arzani and Ashraf, 2016; Zeng et al., 2018). Potassium (K) and calcium (Ca) signaling also exert significant influences toward conferring tolerance toward salinity stress in rice (Shabala and Cuin, 2008; Frouin et al., 2018). Reactive oxygen species (ROS) and osmolytes (choline, glycine betaine, sugars, etc.) have also been implicated in conferring variable levels of tolerance toward salt stress (Hanson et al., 2000). Complete sequencing of rice genome (IRGSP, 2005) has expedited the process of deciphering the molecular entities, which play pivotal roles in conferring tolerance to salt stress. Availability of salt-tolerant and salt-sensitive rice varieties and state-of-the-art omics technologies further provided the fillip in this endeavor. Serine hydroxymethyltransferase (SHMT), a pyridoxal phosphate-dependent enzyme, plays a pivotal role in cellular one-carbon pathways by catalyzing the reversible conversions of L-serine to glycine and tetrahydrofolate to 5,10-methylenetetrahydrofolate in lower and higher organisms (Schirch and Szebenyi, 2005). Structurally conserved SHMT has been implicated in various roles across lower and higher organisms (Moreno et al., 2005; Voll et al., 2006). In plants, SHMT activity has been detected in different organelles (mitochondria, cytosol, plastids, and nuclei) suggesting their diverse roles in metabolic pathways (Besson et al., 1995; Neuburger et al., 1996; Zhang et al., 2010). In rice, the SHMT family comprises five members (OsSHMT1-5). OsSHMT1, encoding the largest subunit of SHMT and an ortholog of Arabidopsis thaliana SHM1, was identified by employing enhancer trapping and the characterization of chlorophyll-deficient mutant (oscdm1) (Wu et al., 2015), photorespiratory mutant osshm1 and map-based cloning (Wang et al., 2015). In Arabidopsis also, the mutation in AtSHM1 (At4g37930) caused an aberration in mitochondrial SHMT activity and exhibited a lethal photorespiratory phenotype during growth at ambient CO2 (Somerville and Ogren, 1981; Voll et al., 2006). These studies suggested conserved function of SHMT1 in photorespiration in taxonomically diverse rice and Arabidopsis. Interestingly, in soybean, SHMT plays a pivotal role in cyst nematode (SCN) resistance (Liu et al., 2012; Kandoth et al., 2017). In another study, overexpression of ApSHMT from halotolerant cyanobacteria Aphanothece halophytica in Escherichia coli (E. coli) triggered a higher accumulation of glycine betaine due to elevated levels of precursors, choline and serine, and consequently augmented tolerance toward salinity-stress (Waditee-Sirisattha et al., 2012). These studies thus suggested roles of SHMT not only in photorespiration but also in other metabolic pathways in different plant species. The recombinant inbred line (RIL) population of salt-tolerant (CSR27) and salt-sensitive (MI48) rice genotypes were screened for their extreme tolerance and sensitivity toward salt stress, and bulk segregant analysis together with a gene expression study led to the identification of an array of differentially regulated salt stress responsive genes including OsSHMT3 (Pandit et al., 2010) using bulk tolerant (BT) and bulk sensitive (BS) RILs. In a subsequent study, these BT and BS populations were also analyzed for their proteomic profiling, which revealed high expression of several proteins including serine hydroxymethyltransferase-3 (OsSHMT3) in the former (Mishra et al., 2016). Overexpression of salt-tolerance-related genes as well as stress-inducible transcription factors has led to the transgenic plants with enhanced salt tolerance (Arzani and Ashraf, 2016). Overexpression of OsCYP94C2, (gene from Cyt450 family) and C-terminal centrin-like domain (OsCCD1) conferred tolerance toward salt stress (Kurotani et al., 2015; Jing et al., 2016). In this context, it is intriguing whether overexpression of OsSHMT3 would elicit any tolerance toward salt stress. Here, we examined the role of SHMT in conferring salt stress tolerance by mediating biosynthetic pathway of glycine to serine interconversion and synthesis of amino acids. In the present study the OsSHMT3 was amplified from salt-tolerant rice. OsSHMT3 was overexpressed in E. coli and assayed for enzymatic activity and modeling protein structure. Further, transgenic Arabidopsis overexpressing OsSHMT3 (OEs) lines were tested for their tolerance toward salt stress. Comprehensive and comparative analyses of the wild-types and OEs for their ionomic, transcriptomic and metabolic profiles, protein expression and different traits were employed to study the role of OsSHMT3 in conferring tolerance toward salt stress in Arabidopsis.

Choline is a vital metabolite in plant and synthesized from phosphocholine by phosphocholine phosphatase. The Arabidopsis At1g17710 was identified as the first plant gene encoding the phosphatase for both phosphoethanolamine and phosphocholine (PECP) with much higher catalytic efficiency (>10-fold) for former. In betaine accumulating plants, choline is further required for betaine synthesis. In this report, we found three putative PECP genes in sugar beet, betaine accumulating plants. Two genes encode the proteins of 274 amino acid residues and designated as BvPECP1S and BvPECP2S. Another gene encodes the 331 amino acid protein (BvPECP2L) consisted of BvPECP2S with extra C-terminal amino acid. Enzymatic assays of BvPECP1S revealed that BvPECP1S exhibited the phosphatase activity for both phosphoethanolamine and phosphocholine with higher affinity (>1.8-fold) and catalytic efficiency (>2.64-fold) for phosphocholine. BvPECP2L exhibited low activity. RT-PCR experiments for BvPECP1S showed the increased expression in young leaf and root tip under salt-stress whereas the increased expression in all organs under phosphate deficiency. The expression level of BvPECP2L in salt stressed young leaf and root tip was induced by phosphate deficient. Physiological roles of BvPECP1S and BvPECP2L for the betaine synthesis were discussed.

Aminotransferases catalyze the reversible pyridoxal phosphate-dependent transfer of amino groups from amino acids to oxo acids and play important roles for the balance between carbon and nitrogen metabolism. In this report, four aminotransferases (Ap1-Ap4) from a halotolerant cyanobacterium Aphanothece halophytica were examined. The results revealed that Ap1 and Ap2 exhibited the aspartate:2-oxoglutarate aminotransferase (AspAT) activity whereas Ap2 catalyzed further aminotransferase activities with alanine (AlaAT) and LL-diaminopimelate (an intermediate for the synthesis of Lys/peptidoglycan) as amino donors. Ap4 exhibited bifunctional aminotransferase with phosphoserine (PSAT) and glycine (GGAT) as amino donors. No activity was observed for Ap3. We identified third gene encoding phosphoserine phosphatase (PSP) in phosphorylate serine biosynthetic pathway. The levels of mRNA for Ap2 and ApMurE encoding UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase were increased after salt stress. These results suggest the link among photorespiratory metabolite (serine, glycine, glyoxylate), phosphorylate serine biosynthetic pathway and aspartate metabolism via aminotransferases for the synthesis of peptidoglycan and betaine under salt stress conditions.

Proline, glycinebetaine, and trehalose function as compatible solutes and are upregulated in plants under abiotic stress. The uptake and inter-organ transport in plants are largely unknown. We review the current information on the transport of these osmoprotectants under abiotic stress. Proline metabolism involves several subcellular compartments. Proline concentrations are regulated by the interplay of biosynthesis, degradation, and transport processes. Among the proline transporter proteins, both general amino acid permeases and selective compatible solute transporters were identified. The review summarized our current knowledge on proline transport under abiotic stress conditions. Trehalose is a nonreducing disaccharide formed by two glucose molecules. Sugar transporters have essential roles in the appropriate distribution of carbohydrates throughout the plants. Trehalose transporter has been poorly characterized because it is difficult to predict the characteristics of sugar transporters based solely on the amino acid sequences. Transport properties of exogenous applied trehalose for abiotic stress tolerance have been discussed. Glycinebetaine is synthesized by two-step oxidations of choline with enzymes choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH). Different biosynthetic pathways among monocot and dicot plants were discussed. Expression and substrate specificity of betaine/proline transporters from various plants were compared. Exogenous application of glycinebetaine to plants under stress conditions improved abiotic stress tolerance and gained some attentions. Further application using the important plants both in laboratory and field will contribute to increase the crop production under stress environments.

Serine hydroxymethyltransferase (SHMT) catalyzes the conversion of serine to glycine and provides activated one-carbon units required for synthesis of nucleic acids, proteins and numerous biological compounds. SHMT is involved in photorespiratory pathway of oxygenic photosynthetic organisms. Accumulating evidence revealed that SHMT plays vital role for abiotic stresses such as low CO2 and high salinity in plants, but its role in cyanobacteria remains to be clarified. In this study, we examined to overexpress the SHMT from halotolerant cyanobacterium Aphanothece halophytica in freshwater cyanobacterium, Synechococcus elongatus PCC7942. The transformed cells did not show an obvious phenotype under non-stress condition, but exhibited more tolerance to salinity than the control cells harboring vector only under high salinity. Elevated levels of enzymes in phosphorylated serine biosynthetic pathway and photorespiration pathway were observed in the transformed cells. Glycine level was also increased in the transformed cells. Physiological roles of SHMT for salt tolerance were discussed.

Dimethylsulfoniopropionate (DMSP) is one of the most abundant molecules on earth and plays a pivotal role in the marine sulfur cycle. DMSP is believed to be synthesized from methionine by a four-step reaction pathway in marine algae. The genes responsible for biosynthesis of DMSP remain unidentified. A diatom Thalassiosira pseudonana CCMP1335 is an important component of marine ecosystems and contributes greatly to the world's primary production. In this study, through genome search, in vivo activity and functional studies of cDNA products, a gene encoding Thalassiosira methyltransferase (TpMMT) which catalyzes the key step of DMSP synthesis formation of 4-methylthio-2-hydroxybutyrate (DMSHB) from 4-methylthio-2-oxobutyrate (MTHB), was identified. The amino acid sequence of TpMMT was homologous to the methyltransferase from Phaeodactylum tricornutum CCAP 1055/1, but not the recently identified bacterium gene. High salinity and nitrogen limitation stresses caused the increase of DMSP content and TpMMT protein in Thalassiosira. In addition to TpMMT, the enzyme activities for the first three steps could be detected and enhanced under high salinity, suggesting the importance of four-step DMSP synthetic pathway in Thalassiosira.

The plant specific DREPP proteins have been shown to bind Ca2+ and regulate the N-myristoylation signaling and microtubule polymerization in Arabidopsis thaliana. The information about DREPP proteins in other plants is, however, scarce. In the present study, we isolated the DREPP gene from a halophytic grass, Sporobolus virginicus, and tested whether the gene was involved in alkaline salt stress responses. The SvDREPP1 was cloned from S. virginicus by RACE methods. The isolated gene showed high homology to DREPP homologs from C4 grasses, Setaria italica, and Panicum hallii as well as rice (OsDREPP1). The encoded protein contained 202 amino acid residues. It was expressed in E. coli, and its biochemical properties were studied. It was observed that SvDREPP1 was not only Ca2+-binding protein, but also bind to calmodulin and microtubules. The SvDREPP1 mRNA expression in plants grown under alkaline salt stress was upregulated by 3.5 times over the control in leaf tissues after 48-h treatment, whereas it was increased for 6.0 times in the root tissues at 36 h. The data suggests the importance of SvDREPP1 in regulating alkali salt stress responses in the leaf tissues.

Membrane proteins of amino acid–polyamine–organocation (APC) superfamily transport amino acids and amines across membranes and play important roles in the regulation of cellular processes. The alanine or glycine: cation symporter (AGCS) family belongs to APC superfamily and is found in prokaryotes, but its substrate specificity remains to be clarified. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica has two putative ApagcS genes. The deduced amino acid sequence of one of genes, ApagcS1, exhibited high homology to Pseudomonas AgcS. The ApagcS1 gene was expressed in Escherichia coli JW4166 which is deficient in glycine uptake. Kinetics studies in JW4166 revealed that ApAgcS1 is a sodium-dependent glycine transporter. Competition experiments showed the significant inhibition by glutamine, asparagine, and glycine. The level of mRNA for ApagcS1 was induced by NaCl and nitrogen-deficient stresses. Uptake of glutamine by ApAgcS1 was also observed. Based on these data, the physiological role of ApAgcS1 was discussed.

To survive under halophilic environments, halophilic microorganisms must have developed the special systems such as synthesis of osmoprotectant and sunscreen molecules. Osmoprotectants are small molecules that act as osmolytes and help organisms survive under extreme saline conditions. Examples of compatible solutes include betaines, amino acids, dimethylsulfoniopropionate, and sugars. These molecules accumulate in cells and balance the osmotic difference between the cell's surroundings and the cytosol. Compatible solutes have also been shown to play a protective role by maintaining enzyme activity under abiotic stress conditions. Their specific action is unknown but is thought that they are preferentially excluded from the proteins interface due to their propensity to form water structures. Here, we summarize recent progress on the research of osmoprotectant and sunscreen molecules in halophilic algae/cyanobacteria. Their possible biotechnological application in the field of green energy, biomedical research, and various biochemical industries were described.

Nipa palm ( Nypa fruticans ) is the only palm that grows in mangrove vegetation. We investigated the effect of salt stress on the growth and physiology of 6-month-old seedlings of Nipa palm exposed to different degrees of salt stress (as NaCl) in pot culture. The overall growth performance of Nipa palm was unaffected by mild salt stress (8.9−16.6 dS m −1 ), whereas seedlings grown under severe salt stress (EC = 57.2 dS m −1 ) had lower chlorophyll content and fluorescence, reduced net photosynthesis and transpiration, which resulted in reduced growth of the plants. Na + contents in leaf, petiole, and root tissues increased considerably under salt stress, depending upon the NaCl levels in the soil solution. Under salt-stress K + content declined, whereas Ca 2+ content increased somewhat, in parallel to Na + . Free proline accumulated in plants growing under high salt stress (EC = 57.2 dS m −1 ). In contrast, soluble sugars were enriched under intermediate levels of salt stress (EC = 16.6 dS m −1 ). The results obtained in the present study suggest that, based on ecophysiological data, N. fruticans is a species best adapted to grow in mangrove coastal areas with moderate only salt load, and circumscribing quite well the actual areas of occurrence of this palm in the gradient from seawater habitats to inland sites. © 2014 Elsevier GmbH

Mycosporine-like amino acids (MAAs) are valuable molecules that are the basis for important photoprotective constituents. Here we report molecular analysis of mycosporine-like amino acid biosynthetic genes from the halotolerant cyanobacterium Aphanothece halophytica , which can survive at high salinity and alkaline pH. This extremophile was found to have a unique MAA core (4-deoxygadusol)-synthesizing gene separated from three other genes. In vivo analysis showed accumulation of the mycosporine-2-glycine but not shinorine or mycosporine-glycine. Mycosporine-2-glycine accumulation was stimulated more under the stress condition of high salinity than UV-B radiation. The Aphanothece MAA biosynthetic genes also manifested a strong transcript level response to salt stress. Furthermore, the transformed Escherichia coli and Synechococcus strains expressing four putative Aphanothece MAA genes under the control of a native promoter were found to be capable of synthesizing mycosporine-2-glycine. The accumulation level of mycosporine-2-glycine was again higher under the high-salinity condition. In the transformed E. coli cells, its level was approximately 85.2 ± 0.7 μmol/g (dry weight). Successful production of a large amount of mycosporine in these cells provides a new opportunity in the search for an alternative natural sunscreen compound source.

Vacuolar Ca2+-transporters could play an important role for salt tolerance in rice (Oryza sativa L.) root. Here, we compared the expression profiles of putative vacuolar cation/H+ exchanger (CAX) and calmodulin-regulated autoinhibited Ca2+-ATPase (ACA) in rice roots of salt tolerant cv. Pokkali and salt sensitive cv. IR29. In addition to five putative vacuolar CAX genes in the rice genome, a new CAX gene (OsCAX4) has been annotated. In the present study, we isolated the OsCAX4 gene and showed that its encoded protein possesses a unique transmembrane structure and is potentially involved in transporting not only Ca2+ but also Mn2+ and Cu2+. These six OsCAX genes differed in their mRNA expression pattern in roots of tolerant versus sensitive rice cultivars exposed to salt stress. For example, OsCAX4 showed abundant expression in IR29 (sensitive) upon prolonged salt stress. The mRNA expression profile of four putative vacuolar Ca2+-ATPases (OsACA4-7) was also examined. Under control conditions, the mRNA levels of OsACA4, OsACA5, and OsACA7 were relatively high and similar among IR29 and Pokkali. Upon salt stress, only OsACA4 showed first a decrease in its expression in Pokkali (tolerant), followed by a significant increase. Based on these results, a role of vacuolar Ca2+ transporter for salt tolerance in rice root was discussed.

The aim of this investigation was to enhance overall growth, yield attributes as well as physio-biochemical adaptive strategies by exogenous foliar application of glycine betaine (GlyBet) in two rice varieties against water deficit stress under greenhouse conditions. Rice crop cvs. RD43 (low amylose content) and SPR1 (high amylose content) grown in clay pots containing garden soil until booting stage were chosen as the test plant material, sprayed by 0 (control) or 100 mM GlyBet and subsequently subjected to: MWD (mild water deficit by 8 d water withholding, 24.80% SWC, Soil water content) or SWD (severe water deficit by 14 d water withholding, 13.63% SWC) or WW (well-watered conditions or control). Free proline content in cv. RD43 was rapidly increased in relation to the degree of water deficit and suppressed by exogenous GlyBet, while free proline in cv. SPR1 was lower than cv. RD43. Overall growth performances and yield traits in both cultivars under MWD were maintained by exogenous application of GlyBet, however, these parameters declined under SWD even after the GlyBet application. Degradation of photosynthetic pigments and chlorophyll fluorescence in pretreated GlyBet plants under SWD were prevented, resulting in elevated net photosynthetic rate (Pn). Interestingly, Pn was very sensitive parameter that sharply declined under SWD in both RD43 and SPR1 genotypes. Positive relationships between physio-morphological and biochemical changes in rice genotypes were demonstrated with high correlation co-efficiency. Based on the key results, it is concluded that foliar GlyBet application may play an important role in drought-tolerant enhancement in rice crops.

We studied the responses of a halophilic cyanobacterium Aphanothece halophytica at surplus (normal composition of growth medium containing 125 µM PO43−), sufficient (the minimum concentration supporting optimal growth, 22 µM PO43−) and deficient (no external supply of Pi) concentrations of inorganic phosphate (Pi). The cyanobacterium was able to grow well in Pi-deficient conditions until the end of incubation (14 days), though at a marginally reduced rate. The cellular P-quota in Pi-surplus cells at the end of incubation was 2.7 times that of their initial P-quota (0.75 µmol mg protein−1), and remained fairly high (0.442 µmol mg protein−1) even in Pi-deficient medium. However, cultures growing in Pi-sufficient medium (22 µM PO43−), upon transfer to Pi-deficient medium, exhibited a rapid decline in cellular P level. Furthermore, cells growing in Pi-surplus medium showed a rapid efflux of P into the external medium. Aphanothece halophytica exhibited a biphasic phosphate transport system involving both high-...

Soluble carbohydrates play a key role as osmolytes and significantly contribute in salt defence mechanism, especially in halophyte species. The objective of this study is to investigate the transcriptional expression of starch-related genes, sugar profile and physiological performances of two contrasting rice genotypes, Pokkali (salt tolerant) and IR29 (salt sensitive), in response to salt stress. Total soluble sugars, glucose and fructose levels in the flag leaf of salt-stressed Pokkali rice were enhanced relative to soluble starch accumulation in plants exposed to EC = 13.25 dS m(-1) (salt stress) for 3 days. In Pokkali, the net photosynthetic rate and starch metabolism may play a key role as energy resources under salt stress. In contrast, photosynthetic performance, indicated by photosynthetic pigment levels and chlorophyll fluorescence parameters, in salt-stressed IR29 was significantly reduced, leading to delayed starch biosynthesis. The reduction in photosynthetic ability and lack of defence mechanisms in IR29 caused growth inhibition and yield loss. Soluble starch and soluble sugar enrichment in Pokkali rice may function alternatively as osmotic adjustment in salt defence mechanism and strengthen carbon energy reserves, greater survival prospects under salt stress and enhanced productivity.

It has been shown that marine algae produce the compatible solute dimethylsulfoniopropionate (DMSP) from methionine (Met) via four enzymatic reactions in which the third step, synthesis of 4-dimethylsulfonio-2-hydroxy-butyrate (DMSHB) from 4-methylthio-2-hydroxybutyrate (MTHB), is the committing step. However, regulation of the biosynthetic pathways and transport properties of DMSP is largely unknown. Here, the effects of sulfur and sodium concentrations on the uptake and synthesis of DMSHB and DMSP were examined in a sterile mutant of Ulva pertusa Kjellm. Sulfur deficiency increased the activity of the sulfur assimilation enzyme O-acetyl serine sulfhydrylase but decreased the MTHB S-methyltransferase activity, suggesting the preferential utilization of sulfur atoms for Met metabolites other than DMSP. Uptake of DMSP and DMSHB was enhanced by S deficiency. High salinity enhanced the MTHB S-methyltransferase activity as well as the uptake of DMSHB. The MTHB S-methyltransferase activity was inhibited by its product DMSP. These data demonstrate the importance of MTHB S-methyltransferase activity and uptake of DMSHB for the regulation of DMSP.

Drought and salinity are the major factors that decrease crop yield. Organisms thriving in osmotic stress environments need adaptive mechanisms for adjusting their intracellular environment to external osmotic stress conditions. One such mechanism, to prevent water loss from the cells is to accumulate large amounts of low molecular weight organic compatible solutes such as proline, betaine and polyols to balance internal osmolarity of the cells. Accumulation of compatible solutes can be achieved by enhanced synthesis and/or reduced catabolism. Certain plants synthesize betaine in chloroplasts via a two-step oxidation of choline and betaine accumulation is associated with enhanced stress tolerance. Many important crop plants have low levels of betaine or none at all. Hence, betaine biosynthetic pathway is a target for metabolic engineering to enhance stress tolerance in crops. Introduction of betaine synthesis pathway into betaine non-accumulating plants has often improved stress tolerance. However, betaine levels of the engineered plants were generally low. To further enhance the betaine accumulation levels, we need to diagnose factors limitng betaine accumulation in engineered plants. Here we discuss recent progress on metabolic engineering of choline precursors for abiotic stress tolerance in plants.

Serine hydroxymethyltransferase (SHMT) is a key enzyme in cellular one-carbon pathway and has been studied in many living organisms from bacteria to higher plants and mammals. However, biochemical and molecular characterization of SHMT from photoautotrophic microorganisms remains a challenge. Here, we isolated the SHMT gene from a halotolerant cyanobacterium Aphanothece halophytica (ApSHMT) and expressed it in Escherichia coli. Purified recombinant ApSHMT protein exhibited catalytic reactions for dl-threo-3-phenylserine as well as for l-serine. Catalytic reaction for l-serine was strongly inhibited by NaCl, but not to that level with glycine betaine. Overexpression of ApSHMT in E. coli resulted in the increased accumulation of glycine and serine. Choline and glycine betaine levels were also significantly increased. Under high salinity, the growth rate of ApSHMT-expressing cells was faster compared to its respective control. High salinity also strongly induced the transcript level of ApSHMT in A. halophytica. Our results indicate the importance of a novel pathway; salt-induced ApSHMT increased the level of glycine betaine via serine and choline and conferred the tolerance to salinity stress.

The aim of this investigation was to comparatively examine the physio-biochemical responses of oil palm seedlings to mannitol- and PEG-induced iso-osmotic stresses. The water content of osmotically stressed oil palm seedlings decreased, but the proline content and the electrolyte leakage of the seedlings increased with decreasing water potential (Ψw). However, the responses varied with the strength of osmotic stress and type of osmotic agent. Relative electrolyte leakage (REL) was negatively correlated to chlorophyll content in the osmotically stressed leaves. Chlorophyll a (Chla), chlorophyll b (Chlb), total carotenoids (Cx+c) and total chlorophyll (TC) in the seedlings were significantly reduced by osmotic stress, subsequently reducing maximum quantum yield of PSII (Fv/Fm) and photon yield of PSII (Φpsii), thereby lowering net-photosynthetic rate (Pn) and inhibiting growth. Physio-biochemical parameters, including REL, Fv/Fm, and Φpsii in oil palm seedlings were reduced more greatly by PEG-induced osmotic stress than by mannitol-induced stress. A deterioration in morphological characters, including leaf chlorosis, leaf burn, and green leaf area reduction were demonstrated in oil palm seedlings under osmotic stress induced by either mannitol or PEG. However, the toxic symptoms in oil palm seedlings under PEG-induced stress were severer than in those under mannitol-induced iso-osmotic stress, especially under severe osmotic stress.

Summary •Peptidases are known to play key roles in multiple biological processes in all living organisms. In higher plants, the vast majority of putative aminopeptidases remain uncharacterized. •In this study, we performed functional and expression analyses of the Arabidopsis LAP2 through cDNA cloning, isolation of T-DNA insertional mutants, characterization of the enzymatic activity, characterization of gene expression and transcriptomics and metabolomics analyses of the mutants. •Loss of function of LAP2, one of the 28 aminopeptidases in Arabidopsis, reduced vegetative growth, accelerated leaf senescence and rendered plants more sensitive to various stresses. LAP2 is highly expressed in the leaf vascular tissue and the quiescent center region. Integration of global gene expression and metabolite analyses suggest that LAP2 controlled intracellular amino acid turnover. The mutant maintained free leucine by up-regulating key genes for leucine biosynthesis. However, this influenced the flux of glutamate strikingly. As a result, γ-aminobutyric acid, a metabolite that is derived from glutamate, was diminished in the mutant. Decrements in these nitrogen-rich compounds are associated with morphological alterations and stress sensitivity of the mutant. •The results indicate that LAP2 is indeed an enzymatically active aminopeptidase and plays key roles in senescence, stress response and amino acid turnover.

It has been shown that marine algae produce the compatible solute dimethylsulfoniopropionate (DMSP) from methionine (Met) via four enzymatic reactions in which the third step, synthesis of 4-dimethylsulfonio-2-hydroxy-butyrate (DMSHB) from 4-methylthio-2-hydroxybutyrate (MTHB), is the committing step. However, regulation of the biosynthetic pathways and transport properties of DMSP is largely unknown. Here, the effects of sulfur and sodium concentrations on the uptake and synthesis of DMSHB and DMSP were examined in a sterile mutant of Ulva pertusa Kjellm. Sulfur deficiency increased the activity of the sulfur assimilation enzyme O-acetyl serine sulfhydrylase but decreased the MTHB S-methyltransferase activity, suggesting the preferential utilization of sulfur atoms for Met metabolites other than DMSP. Uptake of DMSP and DMSHB was enhanced by S deficiency. High salinity enhanced the MTHB S-methyltransferase activity as well as the uptake of DMSHB. The MTHB S-methyltransferase activity was inhibited by its product DMSP. These data demonstrate the importance of MTHB S-methyltransferase activity and uptake of DMSHB for the regulation of DMSP.

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and affects various physiological processes in bacterial, plant and animal cells. In this study, a dnaK gene from a halotolerant cyanobacterium, Aphanothece halophytica, was introduced into Eustoma grandiflorum. The ApdnaK transformed plants could grow at a similar rate with the control plants expressing the vector alone. Although exposure to low temperature is required for the bolting of control Eustoma, it was not required for the ApdnaK transformant. Under normal growth conditions, the glutathione content in the ApdnaK transformants was higher than that of control plants. In the transformant, the pistil is not evident and consequently, no seed formation. The results suggest the involvement of molecular chaperone for the bolting of Eustoma.

It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.

Suitable methods for the conservation and reconstruction of eelgrass populations are needed because of decreasing eelgrass populations. For this, it is important to understand the reproduction mechanism of eelgrass populations at a molecular level as well as at a macro level. In the present study, we analyzed the dynamic changes in eelgrass populations in Ise–Mikawa Bay by monitoring their distribution from 1993 to 2005 and by genetic analysis using microsatellite polymorphism. The distribution of eelgrass populations in the eastern part of Ise Bay showed a periodic shift with a cycle of 4 years, that is, eelgrass populations appeared on the bar trough, gradually moved to the inshore side of the habitat every year, and disappeared in the fourth year. Microsatellite analysis revealed that the eelgrass populations in Ise–Mikawa Bay have high genetic diversity. The eelgrass populations in Ise Bay and those in Mikawa Bay showed genetic differentiation and genetic connectivity. The importance of upstream parent populations, seed transportation among local populations and genetic diversity were discussed for the sustainability of eelgrass populations.

Since sweetness is one of the most important qualities of many fruits, and since sugars are translocated from leaves to fruits, the present study investigates photosynthetic activity, activity of sugar metabolizing enzymes, sugar content in leaves and fruits and endogenous levels of hydrogen peroxide in leaves of melon plants treated with various dilutions of hydrogen peroxide, a nonspecific signaling molecule in abiotic stress. For this purpose, 4-month-old melon plants were treated with various concentrations (50mM) of hydrogen peroxide by applying 300 mL per day to the soil of potted plants. The treatments resulted in increased fructose, glucose, sucrose and starch in the leaves and fruits. The most effective concentration of hydrogen peroxide was 20mM. During the day, soluble sugars in leaves were highest at 12:00 h and starch at 15:00 h. Furthermore, the peroxide treatment increased the photosynthetic activity and the activities of chloroplastic and cytosolic fructose-1,6-bisphosphatase, sucrose phosphate synthase and invertases. Thus, our data show that exogenous hydrogen peroxide, applied to the soil, can increase the soluble sugar content of melon fruits.

Electron transfer (ET) through and between proteins is a fundamental biological process. The activation energy for an ET reaction depends upon the Gibbs energy change upon ET (DeltaG(0)) and the reorganization energy. Here, we characterized ET from Pseudomonas aeruginosa cytochrome c(551) (PA) and its designed mutants to cupredoxins, Silene pratensis plastocyanin (PC) and Acidithiobacillus ferrooxidans rusticyanin (RC), through measurement of pseudo-first-order ET rate constants (k(obs)). The influence of the DeltaG (0) value for ET from PA to PC or RC on the k(obs) value was examined using a series of designed PA proteins exhibiting a variety of E (m) values, which afford the DeltaG (0) variation range of 58-399 meV. The plots of the k(obs) values obtained against the DeltaG(0) values for both PA-PC and PA-RC redox pairs could be fitted well with a single Marcus equation. We have shown that the ET activity of cytochrome c can be controlled by tuning the E(m) value of the protein through the substitution of amino acid residues located in hydrophobic-core regions relatively far from the redox center. These findings provide novel insights into the molecular design of cytochrome c, which could be utilized for controlling its ET activity by means of protein engineering.

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Previously, we showed that overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica (ApDnaK) enhances tolerance to abiotic stresses such as high salinity and high temperature in tobacco plants. Here, we tested the transformation of poplar (Populus alba) with ApDnaK for enhancing the growth of transformed poplar plants. Under control growth conditions, transgenic poplar plants exhibited similar growth rates with the wild-type plants during young seedlings under low light intensity, whereas they showed faster growth, larger plant size, and higher cellulose contents when poplar plants were grown under high light intensity. Transgenic young poplar plants exhibited more rapid recovery from the stresses of high salinity, drought, and low temperature compared with those of the wild type plants when poplar plants were grown under low light intensity. These results suggest that ApDnaK could be useful to enhance the growth rate as well as to increase the stress tolerance.

The DnaK/Hsp70 family is a molecular chaperone that binds non-native states of other proteins, and concerns to various physiological processes in the bacterial, plant and animal cells. Transgenic tobacco plants expressing a molecular chaperone DnaK from a halotolerant cyanobacterium Aphanothece halophytica show enhanced seed yields as well as enhanced tolerances for salt and heat stresses. High-temperature treatment during the reproductive stage decreased total dry weight of seeds in both transgenic and wild-type tobacco, but more severely in the wild-type. Transgenic tobacco plants exhibited higher activities of ascorbate peroxidase and catalase than wild-type plants. Similar results were obtained for salt stress during the reproductive stage. Transgenic rice plants expressing ApDnaK was also constructed. Transgenic rice plants exhibited the enhanced activities for Calvin-cycle enzymes, and showed faster growth and higher seed yield compared with the wild-type rice under normal growth conditions. Transgenic rice plants also showed enhanced tolerance for high temperature and salt stress compared with the wild-type rice. These results suggest a relation of increased folding activity with enhanced stress tolerance, increased seed yield, and total plant biomass.

Rice (Oryza sativa) is sensitive to salt stresses and cannot survive under low salt conditions, such as 50 mM NaCl. In an attempt to improve salt tolerance of rice, we introduced katE, a catalase gene of Escherichia coli, into japonica rice cultivar, Nipponbare. The resultant transgenic rice plants constitutively expressing katE were able to grow for more than 14 days in the presence of 250 mM NaCl, and were able to form flower and produce seeds in the presence of 100 mM NaCl. Catalase activity in the transgenic rice plants was 1.5- to 2.5-fold higher than non-transgenic rice plants. Our results clearly indicate that simple genetic modification of rice to express E. coli-derived catalase can efficiently increase its tolerance against salt stresses. The transformant presented here is one of the most salt-tolerant rice plants created by molecular breeding so far.