The novel galactosyl transferase-like (SbGalT) gene from Salicornia brachiata maintains photosynthesis and enhances abiotic stress tolerance in transgenic tobacco.

We hereby report in planta function characterization of a novel galactosyl transferase-like (SbGalT) gene from Salicornia brachiata for enhanced abiotic stress tolerance. The SbGalT gene had an open reading frame of 1563 bp. The ectopic expression of SbGalT gene in tobacco improved the seed germination, seedling growth, biomass accumulation and potassium/sodium ratio under salt and osmotic stress. The SbGalT over-expression delayed stress-induced senescence, pigment break-down and ion induced cytotoxicity in tobacco. Higher contents of organic solutes and potassium under stress maintained the osmotic homeostasis and relative water content in tobacco. Higher activity of antioxidant enzymes under stress in transgenic tobacco curtailed the accumulation of reactive oxygen species (ROS) and maintained the membrane integrity. The chlorophyll a fluorescence transient indicated no effects of the imposed strengths of stress on basal state of photosystem (PS) I in transgenic tobacco over-expressing the SbGalT gene. Due to improved membrane integrity, the transgenic tobacco exhibited improved photosynthesis, stomatal conductance, intercellular CO2, transpiration, maximum quantum yield and operating efficiency of PSII, electron transport, photochemical and non-photochemical quenching. In agreement with photosynthesis, physiological health, tolerance index and growth parameters, transgenic tobacco accumulated higher contents of sugar, starch, amino acid, polyphenol and proline under stress conditions. The multivariate data analysis exhibited significant statistical distinctions among osmotic adjustment, physiological health and growth, and photosynthetic responses in control and SbGalT transgenic tobacco under stress conditions. The results strongly indicated novel SbGalT gene as a potential candidate for developing the smart agriculture.

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.

Salinity relief aniline induced oxidative stress in Suaeda salsa: Activities of antioxidative enzyme and EPR measurements.

Wastewater from printing and dyeing processes often contains aniline and high salinity, which are hazardous to aquatic species. Glycophytic plants cannot survive under high-salinity conditions, whereas halophytes grow well in such an environment. In this study, we investigated the influence of NaCl on the antioxidant level in Suaeda salsa affected by aniline stress. The seedlings showed various growth toxicity effects under different concentrations of aniline. The results showed that the effect of the aniline was more severe for the root growth compared to that for the shoot growth. Aniline exposure significantly increased the total free radicals and ·OH radicals in the plants. Suaeda salsa exposure to aniline caused oxidative stress by altering the superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activity, which resulted in the overproduction of H2O2 and the inducement of lipid peroxidation. Analysis revealed that the malondialdehyde (MDA) content was enhanced after aniline exposure and that the chlorophyll content was significantly decreased. The results showed that aniline induced the production of free radicals and reactive oxygen species (ROS), and changed the antioxidant defense system. This ultimately resulted in oxidative damage in S. salsa; however, it was found that moderate salinity could mitigate the effects. In conclusion, salinity may alleviate the growth inhibition caused by aniline by regulating the antioxidant capacity of S. salsa.

Salinity-induced alterations in plant growth, antioxidant enzyme activities, and lead transportation and accumulation in Suaeda salsa: implications for phytoremediation.

Halophytes have been considered promising candidates for accumulating heavy metals from saline soils; however, little information has been given on plant physiological responses and heavy metal transportation and accumulation in halophytes that grow in heavy metal-polluted saline soils. This study hypothesized that salinity or heavy metals could induce alterations in plant growth, antioxidant enzyme activities and accumulation and transportation of heavy metals or sodium (Na) in Suaeda salsa. Pot experiments were conducted to test the above hypothesis. Lead (Pb) was selected as the representative heavy metal, and NaCl was added to simulate the Pb-polluted saline soil. The results showed that 0.5% NaCl addition alleviated the inhibition of plant growth under moderate Pb stress (35 and 100 mg kg-1 Pb levels), while the phytotoxicity on plants was magnified by 1.0% NaCl addition. NaCl weakened the oxidative stress in Pb-treated plants by increasing the activity levels of antioxidative enzymes (dismutase (SOD), peroxidase (POD) and catalase (CAT)). At all Pb levels, as the NaCl addition increased, significant increases were observed in the concentration of Na. The 100 mg kg-1 Pb induced a greater increase in Na concentrations than the 35 mg kg-1 Pb did, while the latter induced a greater increase than the 300 mg kg-1 Pb did. NaCl improved Pb translocation factor and its accumulation in Suaeda salsa under Pb stress, indicating that NaCl improves Pb uptake and translocation from roots to shoots and enhances the phytoextraction of Pb. Compared with the 0.1% NaCl treatment, the 0.5 and 1.0% NaCl treatments increased the concentrations of bioavailable Pb in the rhizosphere by 15.0-19.2 and 28.6-35.1%, respectively, indicating the contribution of salinity in producing more available Pb for plant uptake. Moderate salinity may be profitable for Pb transportation and accumulation in plants when there are positive effects on plant growth, antioxidant enzyme activities and Pb availability. These facts suggest that the halophyte Suaeda salsa may be exploited to remediate heavy metal-contaminated saline soils.

Impact of short-term extreme temperature events on physiological performance of Salicornia ramosissima J. Woods under optimal and sub-optimal saline conditions.

Increasing extreme temperature climatic events could exert an important effect on plant photosynthetic performance, which could be modulated by the co-occurrence with other environmental factors, such as salinity, in estuarine ecosystems. Therefore, a mesocosm experiment was designed to assess the impact of temperature events for three days (13/5 °C, 25/13 °C and 40/28 °C) in combination with two NaCl concentrations (171 and 1050 mM NaCl) on the physiological performance of Salicornia ramosissima. Extreme temperature events had a negative impact on S. ramosissima photosynthetic efficiency, this effect being more marked with cold wave at both salinities, compared with heat wave, even in presence of NaCl excess. This differential thermotolerance in the photosynthetic apparatus was ascribed to the greater integrity and functioning of its photosynthetic pathway at high temperature, as indicated by constant gs, Vc,max values at optimal salinity and the higher values of those parameters and gm recorded in combination with NaCl excess. Moreover, S. ramosissima was able to upregulate the energy sink capacity of its photochemical apparatus at elevated temperature and salinity by a greater energy excess dissipation capacity. This could have contributed to reducing the risk of oxidative stress, along with the recorded higher capacity for antioxidant enzyme activity modulation under these conditions.

[Effects of Sediment Burial and Exogenous Cd Input on Biomass Allocation and Antioxidative Enzyme Activities of Suaeda salsa in the Coastal Wetland of the Yellow River Delta].

The Yellow River Delta has been facing the threat of functional degradation during the recent years. The Water-Sediment Regulation Project not only supplements abundant freshwater, but also alters the sediment burial and heavy metal levels, which affects vegetation growth. Thus, we selected the pioneer species Suaeda salsa, to study the effects of different sediment burial depths (0, 3, 6, 12 cm) and exogenous Cd inputs (0, 0.5, 1.0, 1.5 mg·kg-1) on biomass allocation and activities of antioxidative enzymes in the coastal wetlands of the Yellow River delta. The results showed that a shallow or moderate burial depth had a stimulatory effect on chlorophyll content, while an excessive burial depth inhibited the growth of Suaeda salsa and chlorophyll content. With increasing Cd input, chlorophyll content and dry mass decreased. At a lower Cd input and moderate burial depth, activities of CAT and SOD increased, and at high levels, SOD activities decreased, while activities of CAT at a 12 cm burial depth and 1.0 mg·kg-1, 1.5 mg·kg-1 Cd input were higher than those for the control (62.66% and 58.56%). CAT activities reached high values (15.76 U·mg-1) at a high Cd input (1.5 mg·kg-1) and burial depth (12 cm). Analysis of variance showed that Cd input had a significant effect on protein content, and CAT and SOD activities, and sediment burial depth had a significant effect on the protein content and SOD activities. Interaction between Cd input and sediment burial depth had a significant effect on CAT and SOD activities (P<0.05). These results demonstrated that sediment burial depth and Cd input had a great influence on the growth of Suaeda salsa, and to some extent, Suaeda salsa could change its biomass allocation and antioxidative enzyme activities to adapt to severe environments.

Changes in redox regulation during transition from C3 to single cell C4 photosynthesis in Bienertia sinuspersici.

Bienertia sinuspersici performs single cell C4 photosynthesis without Kranz anatomy. Peripheral and central cytoplasmic compartments in a single chlorenchyma cell act as mesophyll cells and bundle sheath cells. Development of this specialized mechanism is gradual during plant development. Young leaves perform C3 photosynthesis, while mature leaves have complete C4 cycle. The aim of this work was to investigate changes in redox regulation and antioxidant defence during transition from C3 to single cell C4 photosynthesis in B. sinuspersici leaves. First, we confirmed gradual development of C4 with protein blot and qRT-PCR analysis of C4 enzymes. After this activities and isoenzymes of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and H2O2 and TBARS and glutathione pool and redox status (GSH/GSSG) were determined in young, developing and mature leaves during transition from C3 to single cell C4 photosynthesis. Activities of SOD, APX and POX decrease, while GR and DHAR were increased. However, most striking results were the changes in isoenzyme patterns of SOD, CAT and GR which were gradual through transition to C4 photosynthesis.

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.

Molecular phylogeny and forms of photosynthesis in tribe Salsoleae (Chenopodiaceae).

While many C4 lineages have Kranz anatomy around individual veins, Salsoleae have evolved the Salsoloid Kranz anatomy where a continuous dual layer of chlorenchyma cells encloses the vascular and water-storage tissue. With the aim of elucidating the evolution of C4 photosynthesis in Salsoleae, a broadly sampled molecular phylogeny and anatomical survey was conducted, together with biochemical, microscopic, and physiological analyses of selected photosynthetic types. From analyses of photosynthetic phenotypes, a model for evolution of this form of C4 was compared with models for evolution of Kranz anatomy around individual veins. A functionally C3 proto-Kranz phenotype (Proto-Kranz Sympegmoid) and intermediates with a photorespiratory pump (Kranz-like Sympegmoid and Kranz-like Salsoloid types) are considered crucial transitional steps towards C4 development. The molecular phylogeny provides evidence for C3 being the ancestral photosynthetic pathway but there is no phylogenetic evidence for the ancestry of C3-C4 intermediacy with respect to C4 in Salsoleae. Traits considered advantageous in arid conditions, such as annual life form, central sclerenchyma in leaves, and reduction of surface area, evolved repeatedly in Salsoleae. The recurrent evolution of a green stem cortex taking over photosynthesis in C4 clades of Salsoleae concurrent with leaf reduction was probably favoured by the higher productivity of the C4 cycle.

In planta Transformed Cumin (Cuminum cyminum L.) Plants, Overexpressing the SbNHX1 Gene Showed Enhanced Salt Endurance.

Cumin is an annual, herbaceous, medicinal, aromatic, spice glycophyte that contains diverse applications as a food and flavoring additive, and therapeutic agents. An efficient, less time consuming, Agrobacterium-mediated, a tissue culture-independent in planta genetic transformation method was established for the first time using cumin seeds. The SbNHX1 gene, cloned from an extreme halophyte Salicornia brachiata was transformed in cumin using optimized in planta transformation method. The SbNHX1 gene encodes a vacuolar Na+/H+ antiporter and is involved in the compartmentalization of excess Na+ ions into the vacuole and maintenance of ion homeostasis Transgenic cumin plants were confirmed by PCR using gene (SbNHX1, uidA and hptII) specific primers. The single gene integration event and overexpression of the gene were confirmed by Southern hybridization and competitive RT-PCR, respectively. Transgenic lines L3 and L13 showed high expression of the SbNHX1 gene compared to L6 whereas moderate expression was detected in L5 and L10 transgenic lines. Transgenic lines (L3, L5, L10 and L13), overexpressing the SbNHX1 gene, showed higher photosynthetic pigments (chlorophyll a, b and carotenoid), and lower electrolytic leakage, lipid peroxidation (MDA content) and proline content as compared to wild type plants under salinity stress. Though transgenic lines were also affected by salinity stress but performed better compared to WT plants. The ectopic expression of the SbNHX1 gene confirmed enhanced salinity stress tolerance in cumin as compared to wild type plants under stress condition. The present study is the first report of engineering salt tolerance in cumin, so far and the plant may be utilized for the cultivation in saline areas.

Cloning and characterization of a novel betaine aldehyde dehydrogenase gene from Suaeda corniculata.

Glycine betaine is an important quaternary ammonium compound that is produced in response to several abiotic stresses in many organisms. The synthesis of glycine betaine requires the catalysis of betaine aldehyde dehydrogenase (BADH), which can convert betaine aldehyde into glycine betaine in plants, especially in halotolerant plants. In this study, we isolated the full-length cDNA of BADH from Suaeda corniculata (ScBADH) using reverse transcriptase-polymerase chain reaction and rapid amplification of cDNA ends. Next, we analyzed the expression profile of ScBADH using real-time PCR. The results showed that ScBADH expression was induced in the roots, stems, and leaves of S. corniculata seedlings under salt and drought stress. Next, ScBADH was overexpressed in Arabidopsis, resulting in the transgenic plants exhibiting enhanced tolerance over wild-type plants under salt and drought stress. We then analyzed the levels of glycine betaine and proline, as well as superoxide dismutase (SOD) activity, during salt stress in WT and transgenic Arabidopsis. The results indicated that overexpression of ScBADH produced more glycine betaine and proline, and increased SOD activity under NaCl treatment. Our results suggest that ScBADH might be a positive regulator in plants during the response to NaCl.

Functional Characterization of the Tau Class Glutathione-S-Transferases Gene (SbGSTU) Promoter of Salicornia brachiata under Salinity and Osmotic Stress.

Reactive oxygen or nitrogen species are generated in the plant cell during the extreme stress condition, which produces toxic compounds after reacting with the organic molecules. The glutathione-S-transferase (GST) enzymes play a significant role to detoxify these toxins and help in excretion or sequestration of them. In the present study, we have cloned 1023 bp long promoter region of tau class GST from an extreme halophyte Salicornia brachiata and functionally characterized using the transgenic approach in tobacco. Computational analysis revealed the presence of abiotic stress responsive cis-elements like ABRE, MYB, MYC, GATA, GT1 etc., phytohormones, pathogen and wound responsive motifs. Three 5'-deletion constructs of 730 (GP2), 509 (GP3) and 348 bp (GP4) were made from 1023 (GP1) promoter fragment and used for tobacco transformation. The single event transgenic plants showed notable GUS reporter protein expression in the leaf tissues of control as well as treated plants. The expression level of the GUS gradually decreases from GP1 to GP4 in leaf tissues, whereas the highest level of expression was detected with the GP2 construct in root and stem under control condition. The GUS expression was found higher in leaves and stems of salinity or osmotic stress treated transgenic plants than that of the control plants, but, lower in roots. An efficient expression level of GUS in transgenic plants suggests that this promoter can be used for both constitutive as well as stress inducible expression of gene(s). And this property, make it as a potential candidate to be used as an alternative promoter for crop genetic engineering.

H(+)-pyrophosphatase from Salicornia europaea enhances tolerance to low phosphate under salinity in Arabidopsis.

Increasing soil salinity threatens crop productivity worldwide. High soil salinity is usually accompanied by the low availability of many mineral nutrients. Here, we investigated the potential role that the H(+)- PPase could play in optimizing P use efficiency under salinity in plants. Transgenic Arabidopsis plants overexpressing either SeVP1 or SeVP2 from Salicornia europaea outperformed the wild-types under low phosphate (Pi) as well as low Pi plus salt conditions. Our results suggested that H(+)-PPase could increase external Pi acquisition through promoting root development and upregulating phosphate transporters, thus to protect plants from Pi limiting stress. This study provides a potential strategy for improving crop yields challenged by the co-occurrence of abiotic stresses.

Characterization of the superoxide dismutase genes of the halophyte Suaeda maritima in Japan and Egypt.

KEY MESSAGE: Suaeda maritima varieties native to Japan and Egypt were cultured under aseptic conditions. The varieties differed in genetic distance but exhibited similar expression profiles of superoxide dismutase isozyme genes. The expression characteristics of superoxide dismutase (SOD; EC 1.15.1.1) isozyme genes from halophytic Suaeda marit ima plants native to Japan and Egypt were analyzed using young plants grown under aseptic conditions. A phylogenetic tree based on internal transcribed spacer sequences suggested that Egyptian S. maritima is related to European and India S. maritima, while Japanese S. maritima belongs to a separate clade. An in-gel SOD activity staining assay revealed that leaves from both the Egyptian and Japanese varieties showed high levels of CuZn-SOD and Fe-SOD activity, but no Mn-SOD activity; conversely, stems from both varieties showed Mn-SOD activity as well as other SOD isozyme activities. In Japanese S. maritima leaves, SOD activity was increased by incubation in growth medium containing 400 mM NaCl, while Egyptian S. maritima leaves showed elevated SOD activity in the absence of high salt. Genes encoding Mn-SOD and Fe-SOD were isolated from both plant types. RT-PCR analysis revealed that all SOD isozyme-encoding genes were expressed at the same levels in leaves from both plant types grown in normal or high-salt medium. In contrast, the expression of genes encoding choline monooxygenase and betaine aldehyde dehydrogenase, which are involved in betacyanin biosynthesis, was increased in high-salt medium. In leaves of Japanese S. maritima plants, Fe deficiency without high salt exposure preferentially decreased Fe-SOD activity. On the other hand, Fe deficiency with high salt exposure decreased not only Fe-SOD activity but also CuZn-SOD activity, suggesting that Fe availability is involved in the up-regulation of SOD isozymes mediating salt tolerance.

An assessment of the capacity for phosphoenolpyruvate carboxykinase to contribute to C4 photosynthesis.

Three C4 acid decarboxylases, phosphoenolpyruvate carboxykinase (PEPCK), NADP-malic enzyme (NADP-ME), and NAD-malic enzyme (NAD-ME) were recruited from C3 plants to support C4 photosynthesis. In Poaceae, there are established lineages having PEPCK type species, and some NADP-ME lineages in which PEPCK contributes to C4. Besides family Poaceae, recently PEPCK has been reported to function in C4 photosynthesis in eudicot species including Cleome gynandra (Cleomaceae), Trianthema portulacastrum and Zaleya pentandra (Aizoaceae). We evaluated PEPCK by enzyme assay and western blots in representatives of Poaceae, Aizoaceae, Cleomaceae, and Chenopodiaceae compared to that in the PEPCK type C4 grass Spartina anglica. Eragrostis nutans was identified as the first NAD-ME type C4 grass having substantial amounts of PEPCK. In the eudicots, including C. gynandra, Cleome angustifolia, T. portulacastrum, Z. pentandra, and nine C4 members of family Chenopodiaceae (which has the most C4 species and diversity in forms among eudicot families), amounts of PEPCK were generally very low (barely detectable up to 4% of that in S. anglica). Based on these results, C4 species can be classified biochemically according to the dominant decarboxylase recruited for C4 function; and, Poaceae remains the only family in which PEPCK is known to have a significant role in C4 photosynthesis.

Positive selection of Kranz and non-Kranz C4 phosphoenolpyruvate carboxylase amino acids in Suaedoideae (Chenopodiaceae).

In subfamily Suaedoideae, four independent gains of C4 photosynthesis are proposed, which includes two parallel origins of Kranz anatomy (sections Salsina and Schoberia) and two independent origins of single-cell C4 anatomy (Bienertia and Suaeda aralocaspica). Additional phylogenetic support for this hypothesis was generated from sequence data of the C-terminal portion of the phosphoenolpyruvate carboxylase (PEPC) gene used in C4 photosynthesis (ppc-1) in combination with previous sequence data. ppc-1 sequence was generated for 20 species in Suaedoideae and two outgroup Salsola species that included all types of C4 anatomies as well as two types of C3 anatomies. A branch-site test for positively selected codons was performed using the software package PAML. From labelling of the four branches where C4 is hypothesized to have developed (foreground branches), residue 733 (maize numbering) was identified to be under positive selection with a posterior probability >0.99 and residue 868 at the >0.95 interval using Bayes empirical Bayes (BEB). When labelling all the branches within C4 clades, the branch-site test identified 13 codons to be under selection with a posterior probability >0.95 by BEB; this is discussed considering current information on functional residues. The signature C4 substitution of an alanine for a serine at position 780 in the C-terminal end (which is considered a major determinant of affinity for PEP) was only found in four of the C4 species sampled, while eight of the C4 species and all the C3 species have an alanine residue; indicating that this substitution is not a requirement for C4 function.

Over-expression of the peroxisomal ascorbate peroxidase (SbpAPX) gene cloned from halophyte Salicornia brachiata confers salt and drought stress tolerance in transgenic tobacco.

Salicornia brachiata Roxb., an extreme halophyte, is a naturally adapted higher plant model for additional gene resources to engineer salt tolerance in plants. Ascorbate peroxidase (APX) plays a key role in protecting plants against oxidative stress and thus confers abiotic stress tolerance. A full-length SbpAPX cDNA, encoding peroxisomal ascorbate peroxidase, was cloned from S. brachiata. The open reading frame encodes for a polypeptide of 287 amino acid residues (31.3-kDa protein). The deduced amino acid sequence of the SbpAPX gene showed characteristic peroxisomal targeting sequences (RKRAI) and a C-terminal hydrophobic region of 39 amino acid residues containing a transmembrane domain (TMD) of 23 amino acid residues. Northern blot analysis showed elevated SbpAPX transcript in response to salt, cold, abscisic acid and salicylic acid stress treatments. The SbpAPX gene was transformed to tobacco for their functional validation under stresses. Transgenic plants over-expressing SbpAPX gene showed enhanced salt and drought stress tolerance compared to wild-type plants. Transgenic plants showed enhanced vegetative growth and germination rate both under normal and stressed conditions. Present study revealed that the SbpAPX gene is a potential candidate, which not only confers abiotic stress tolerance to plants but also seems to be involved in plant growth.

The transcriptional regulatory mechanism of the peroxisomal ascorbate peroxidase (pAPX) gene cloned from an extreme halophyte, Salicornia brachiata.

Peroxisomal ascorbate peroxidase detoxifies H2O2 leaching out from peroxisomes into the cytoplasm. The present study describes transcript expression and cis-regulation of the SbpAPX gene cloned from an extreme halophyte, Salicornia brachiata, in the steady state and under different stresses. About 2-fold elevated transcript expression was found in salt- and drought-treated shoots at 12 h compared with control, while 1.9-fold increased expression was observed under heat treatment. In roots, the transcript level was down-regulated at 2 h, thereafter increasing with the time of exposure and reaching a maximum at the control level. The SbpAPX promoter has characteristic cis-regulatory ABA-dependent abiotic stress-responsive elements. The full-length promoter (1,024 bp, PP1) and deletion constructs -838 (PP2), -697 (PP3), -433 (PP4) and -185 bp (PP5) were fused with the GUS (ß-glucuronidase) gene and transformed into tobacco for functional validation. Expression of GUS increased significantly in transgenic plants under stress. Quantitative expression analysis of GUS in T1 plants revealed that promoter PP5 is efficient for gene expression. In planta transient expression further suggested that the promoter PP5 contains efficient stress-inducible elements. A steep decline in GUS expression in PP3, and thereafter an elevated expression in PP4 and PP5, suggested the presence of a repressor element between -696 and -433 bp, while an enhancer element was predicted between -838 and -697 bp. Further, transient expression analyses and electrophoretic mobility shift assay revealed that the core sequence of cis-acting motifs ATAA and CCTCAA function as enhancer and repressor binding sites, respectively. Based on the study, a model is proposed for the cis-regulation of the SbpAPX gene. The present study provides a useful insight for understanding gene expression regulation in a halophyte with or without stress. Furthermore, potential stress-responsive promoter-driven expression of introgressed gene(s) can be used for engineering crops with enhanced stress tolerance.

Effect of drought stress on superoxide dismutase activity in two species of Haloxylon aphyllum and Haloxylon persicum.

Superoxide dismutase activity changes were studied at different periodic tensions using of spectrophotometric measurement of decline in NitroBlue Tetrazolium reduction to Blue Formazan at 560 nm in Haloxylon aphyllum and Haloxylon persicum. The aim of this study was to investigate the changes of superoxide dismutase in applied drought stress in two species of Haloxylon. The results showed that the effect of drought stress on increase in superoxide dismutase activity was significant (p < 0.01) in two haloxylon species. Drought increased enzyme activity at severe tensions. When two haloxylon species were placed under 7 and 14 days no-watering treatments (mild tensions), the enzyme activity was more than its activity in control treatment and less than one in 21 and 28 days no-watering treatments (severe tensions). The enzyme activity in branchlets of Haloxylon aphyllum under 21 and 28 days no-watering treatments was 20.2 and 29.5% more than its activity under control treatment respectively. This activity in Haloxylon persicum was 21.6 and 31.4% more than its activity under control treatment, respectively. With the advent of drought, superoxide dismutase activity increased in two species of haloxylon. The increasing of superoxide dismutase activity in time of dryness advent in Haloxylon aphyllum was more than Haloxylon persicum, which can be raised as an acceptable factor and vindicator in being more resistant of Haloxylon aphyllum to environmental drought.

Heterologous expression of a plant RelA-SpoT homologue results in increased stress tolerance in Saccharomyces cerevisiae by accumulation of the bacterial alarmone ppGpp.

The bacterial alarmone ppGpp is present only in bacteria and the chloroplasts of plants, but not in mammalian cells or eukaryotic micro-organisms such as yeasts and fungi. The importance of the ppGpp signalling system in eukaryotes has therefore been largely overlooked. Here, we demonstrated that heterologous expression of a relA-spoT homologue (Sj-RSH) isolated from the halophilic plant Suaeda japonica in the yeast Saccharomyces cerevisiae results in accumulation of ppGpp, accompanied by enhancement of tolerance against various stress stimuli, such as osmotic stress, ethanol, hydrogen peroxide, high temperature and freezing. Unlike bacterial ppGpp accumulation, ppGpp was accumulated in the early growth phase but not in the late growth phase. Moreover, nutritional downshift resulted in a decrease in ppGpp level, suggesting that the observed Sj-RSH activity to synthesize ppGpp is not starvation-dependent, contrary to our expectations based on bacteria. Accumulated ppGpp was found to be present solely in the cytosolic fraction and not in the mitochondrial fraction, perhaps reflecting the ribosome-independent ppGpp synthesis in S. cerevisiae cells. Unlike bacterial inosine monophosphate (IMP) dehydrogenases, the IMP dehydrogenase of S. cerevisiae was insensitive to ppGpp. Microarray analysis showed that ppGpp accumulation gave rise to marked changes in gene expression, with both upregulation and downregulation, including changes in mitochondrial gene expression. The most prominent upregulation (38-fold) was detected in the hypothetical gene YBR072C-A of unknown function, followed by many other known stress-responsive genes. S. cerevisiae may provide new opportunities to uncover and analyse the ppGpp signalling system in eukaryotic cells.