Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.     

An Ipomoea batatas Iron-Sulfur Cluster Scaffold Protein Gene,IbNFU1, Is Involved in Salt Tolerance

Iron-sulfur cluster biosynthesis involving the nitrogen fixation (Nif) proteins has been proposed as a general mechanism acting in various organisms. NifU-like protein may play an important role in protecting plants against abiotic and biotic stresses. An iron-sulfur cluster scaffold protein gene, IbNFU1, was isolated from a salt-tolerant sweetpotato (Ipomoea batatas (L.) Lam.) line LM79 in our previous study, but its role in sweetpotato stress tolerance was not investigated. In the present study, the IbNFU1 gene was introduced into a salt-sensitive sweetpotato cv. Lizixiang to characterize its function in salt tolerance. The IbNFU1-overexpressing sweetpotato plants exhibited significantly higher salt tolerance compared with the wild-type. Proline and reduced ascorbate content were significantly increased, whereas malonaldehyde (MDA) content was significantly decreased in the transgenic plants. The activities of superoxide dismutase (SOD) and photosynthesis were significantly enhanced in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbNFU1 up-regulated pyrroline-5-carboxylate synthase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that IbNFU1gene is involved in sweetpotato salt tolerance and enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system.     

Exogenous ethanol treatment alleviates oxidative damage of Arabidopsis thaliana under conditions of high-light stress

Abiotic stresses, such as high light and salinity, are major factors that limit crop productivity and sustainability worldwide. Chemical priming is a promising strategy for improving the abiotic stress tolerance of plants. Recently, we discovered that ethanol enhances high-salinity stress tolerance in Arabidopsis thaliana and rice by detoxifying reactive oxygen species (ROS). However, the effect of ethanol on other abiotic stress responses is unclear. Therefore, we investigated the effect of ethanol on the high-light stress response. Measurement of chlorophyll fluorescence showed that ethanol mitigates photoinhibition under high-light stress. Staining with 3,3′-diaminobenzidine (DAB) showed that the accumulation of hydrogen peroxide (H₂O₂) was inhibited by ethanol under high-light stress conditions in A. thaliana. We found that ethanol increased the gene expressions and enzymatic activities of antioxidative enzymes, including ASCORBATE PEROXIDASE1 (AtAPX1), Catalase (AtCAT1 and AtCAT2). Moreover, the expression of flavonoid biosynthetic genes and anthocyanin contents were upregulated by ethanol treatment during exposure to high-light stress. These results imply that ethanol alleviates oxidative damage from high-light stress in A. thaliana by suppressing ROS accumulation. Our findings support the hypothesis that ethanol improves tolerance to multiple stresses in field-grown crops.     

Arabidopsis thaliana Metallothionein, AtMT2a, Mediates ROS Balance during Oxidative Stress

Cold stress has been shown to induce the production of reactive oxygen species (ROS), which can elicit a potentially damaging oxidative burden on cellular metabolism. Here, the expression of a metallothionein gene (AtMT2a) was upregulated under low temperature and hydrogen peroxide (H₂O₂) stresses. The Arabidopsis T-DNA insertion mutant, mt2a, exhibited more sensitivity to cold stress compared to WT plants during the seed germination, and H₂O₂ levels in mt2a mutant were higher than that in WT plants during the cold stress. Synthetic GFP fused to AtMT2a was observed to be localized in cytosol. These results indicated that AtMT2a functions in tolerance against cold stress by mediating the ROS balance in the cytosol. Interestingly, mRNA level of AtMT2a was increased in seedlings of Arabidopsis cat2 mutant after cold treatment compared to WT seedlings, and overexpression of AtMT2a in cat2 could improve CAT activity under chilling stress. Moreover, the enzymatic activity of CAT in mt2a was higher than that in WT plants after cold treatment, suggesting that AtMT2a and CAT might complement each other in antioxidative process potentially in Arabidopsis. Taken together, our results provided a novel insight into the relationship between MTs and antioxidative enzymes in the ROS-scavenging system in plants.     

Expression of Arabidopsis DREB1C improves survival, growth, and yield of upland New Rice for Africa (NERICA) under drought

Dehydration-responsive-element-binding protein 1 genes have important roles in response to stress. To improve the drought tolerance of an upland rice cultivar NERICA1, we introduced Arabidopsis AtDREB1C or rice OsDREB1B driven by a stress-inducible rice lip9 promoter. Plants of some transgenic lines survived better than non-transgenic plants under severe drought. AtDREB1C transgenic plants had higher dry weights than non-transgenic plants when grown under moderate drought until the late vegetative growth stage. On the other hand, OsDREB1B transgenic plants had lower dry weights than non-transgenic plants under the same condition. Similar results were obtained under osmotic stress. The AtDREB1C transgenic plants headed earlier, had a larger sink capacity, and had more filled grains than non-transgenic plants. These results suggest that AtDREB1C expressed in NERICA1 improves not only survival under severe drought, but also growth and yield under moderate drought.     

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.     

A DESD-box helicase functions in salinity stress tolerance by improving photosynthesis and antioxidant machinery in rice (Oryza sativa L. cv. PB1)

The exact mechanism of helicase-mediated salinity tolerance is not yet understood. We have isolated a DESD-box containing cDNA from Pisum sativum (Pea) and named it as PDH45. It is a unique member of DEAD-box helicase family; containing DESD instead of DEAD/H. PDH45 overexpression driven by constitutive cauliflower mosaic virus-35S promoter in rice transgenic [Oryza sativa L. cv. Pusa Basmati 1 (PB1)] plants confers salinity tolerance by improving the photosynthesis and antioxidant machinery. The Na⁺ ion concentration and oxidative stress parameters in leaves of the NaCl (0, 100 or 200 mM) treated PDH45 overexpressing T₁ transgenic lines were lower as compared to wild type (WT) rice plants under similar conditions. The 200 mM NaCl significantly reduced the leaf area, plant dry mass, net photosynthetic rate (P_N), stomatal conductance (gs), intercellular CO₂ (Ci), chlorophyll (Chl) content in WT plants as compared to the transgenics. The T₁ transgenics exhibited higher glutathione (GSH) and ascorbate (AsA) contents under salinity stress. The activities of antioxidant enzymes viz. superoxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and glutathione reductase (GR) were significantly higher in transgenics; suggesting the existence of an efficient antioxidant defence system to cope with salinity induced-oxidative damage. Yeast two-hybrid assay indicated that the PDH45 protein interacts with Cu/Zn SOD, adenosine-5′-phosphosulfate-kinase, cysteine proteinase and eIF(4G), thus confirming the involvement of ROS scavenging machinery in the transgenic plants to provide salt tolerance. Furthermore, the T₂ transgenics were also able to grow, flower, and set viable seeds under continuous salinity stress of 200 mM NaCl. This study provides insights into the mechanism of PDH45 mediated salinity stress tolerance by controlling the generation of stress induced reactive oxygen species (ROS) and also by protecting the photosynthetic machinery through a strengthened antioxidant system.     

Production of secondary metabolites from cell and organ cultures: strategies and approaches for biomass improvement and metabolite accumulation

Pyrroline-5-carboxylate reductase (P5CR) lies at the converging point of the glutamate and ornithine pathways and is the last and critical enzyme in proline biosynthesis. In the present study, a P5CR gene, named IbP5CR, was isolated from salt-tolerant sweetpotato line ND98. Expression of IbP5CR was up-regulated in sweetpotato under salt stress. The IbP5CR-overexpressing sweetpotato (cv. Kokei No. 14) plants exhibited significantly higher salt tolerance compared with the wild-type. Proline content and superoxide dismutase and photosynthetic activities were significantly increased, whereas malonaldehyde content was significantly decreased in the transgenic plants. H₂O₂ was also found to be significantly less accumulated in the transgenic plants than in the wild-type. Overexpression of IbP5CR up-regulated pyrroline-5-carboxylate synthase gene and down-regulated proline dehydrogenase and P5C dehydrogenase genes under salt stress. The systemic up-regulation of reactive oxygen species (ROS) scavenging genes was found in the transgenic plants under salt stress. These findings suggest that overexpression of IbP5CR increases proline accumulation, which enhances salt tolerance of the transgenic sweetpotato plants by regulating osmotic balance, protecting membrane integrity and photosynthesis and activating ROS scavenging system. This study indicates that IbP5CR gene has the potential to be used for improving salt tolerance of plants.     

Overexpression of Alternative Oxidase Gene Confers Aluminum Tolerance by Altering the Respiratory Capacity and the Response to Oxidative Stress in Tobacco Cells

Aluminum (Al) stress represses mitochondrial respiration and produces reactive oxygen species (ROS) in plants. Mitochondrial alternative oxidase (AOX) uncouples respiration from mitochondrial ATP production and may improve plant performance under Al stress by preventing excess accumulation of ROS. We tested respiratory changes and ROS production in isolated mitochondria and whole cell of tobacco (SL, ALT 301) under Al stress. Higher capacities of AOX pathways relative to cytochrome pathways were observed in both isolated mitochondria and whole cells of ALT301 under Al stress. AOX1 when studied showed higher AOX1 expression in ALT 301 than SL cells under stress. In order to study the function of tobacco AOX gene under Al stress, we produced transformed tobacco cell lines by introducing NtAOX1 expressed under the control of the cauliflower mosaic virus (CaMV) 35 S promoter in sensitive (SL) Nicotiana tabacum L. cell lines. The enhancement of endogenous AOX1 expression and AOX protein with or without Al stress was in the order of transformed tobacco cell lines > ALT301 > wild type (SL). A decreased respiratory inhibition and reduced ROS production with a better growth capability were the significant features that characterized AOX1 transformed cell lines under Al stress. These results demonstrated that AOX plays a critical role in Al stress tolerance with an enhanced respiratory capacity, reducing mitochondrial oxidative stress burden and improving the growth capability in tobacco cells.     

Overproduction of the Membrane-bound Receptor-like Protein Kinase 1, RPK1, Enhances Abiotic Stress Tolerance in Arabidopsis

RPK1 (receptor-like protein kinase 1) localizes to the plasma membrane and functions as a regulator of abscisic acid (ABA) signaling in Arabidopsis. In our current study, we investigated the effect of RPK1 disruption and overproduction upon plant responses to drought stress. Transgenic Arabidopsis overexpressing the RPK1 protein showed increased ABA sensitivity in their root growth and stomatal closure and also displayed less transpirational water loss. In contrast, a mutant lacking RPK1 function, rpk1-1, was found to be resistant to ABA during these processes and showed increased water loss. RPK1 overproduction in these transgenic plants thus increased their tolerance to drought stress. We performed microarray analysis of RPK1 transgenic plants and observed enhanced expression of several stress-responsive genes, such as Cor15a, Cor15b, and rd29A, in addition to H₂O₂-responsive genes. Consistently, the expression levels of ABA/stress-responsive genes in rpk1-1 had decreased compared with wild type. The results suggest that the overproduction of RPK1 enhances both the ABA and drought stress signaling pathways. Furthermore, the leaves of the rpk1-1 plants exhibit higher sensitivity to oxidative stress upon ABA-pretreatment, whereas transgenic plants overproducing RPK1 manifest increased tolerance to this stress. Our current data suggest therefore that RPK1 overproduction controls reactive oxygen species homeostasis and enhances both water and oxidative stress tolerance in Arabidopsis.     

Overexpression of a GST gene (ThGSTZ1) from Tamarix hispida improves drought and salinity tolerance by enhancing the ability to scavenge reactive oxygen species

Although plant glutathione transferase (GST) genes are reported to be involved in responses to abiotic stress, few GST genes have been functionally characterized in woody halophytes. In the present study, a GST gene from Tamarix hispida, designated ThGSTZ1, was cloned and functionally characterized. Expression of ThGSTZ1 was downregulated by drought and salinity stress, and abscisic acid. Transgenic Arabidopsis thaliana plants with constitutive expression of ThGSTZ1 showed increased survival rates under drought and salinity stress. These transgenic Arabidopsis plants exhibited increased levels of GST, glutathione peroxidase, superoxide dismutase and peroxidase activity, along with decreased malondialdehyde content, electrolyte leakage rates and reactive oxygen species (ROS) levels under salt and drought stress conditions. Transgenic T. hispida that transiently overexpressed ThGSTZ1 showed increased GST and GPX activities under NaCl and mannitol treatments, as well as improved ROS scavenging ability. These results suggest that ThGSTZ1 can improve drought and salinity tolerance in plants by enhancing their ROS scavenging ability. Therefore, ThGSTZ1 represents a candidate gene with potential applications for molecular breeding to increase stress tolerance in plants.     

Ectopic expression of PgRab7 in rice plants (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) results in differential tolerance at the vegetative and seed setting stage during salinity and drought stress

In this work, we have overexpressed a vesicle trafficking protein, Rab7, from a stress-tolerant plant, Pennisetum glaucum, in a high-yielding but stress-sensitive rice variety Pusa Basmati-1 (PB-1). The transgenic rice plants were tested for tolerance against salinity and drought stress. The transgenic plants showed considerable tolerance at the vegetative stage against both salinity (200 mM NaCl) and drought stress (up to 12 days after withdrawing water). The protection against salt and drought stress may be by regulating Na⁺ ion homeostasis, as the transgenic plants showed altered expression of multiple transporter genes, including OsNHX1, OsNHX2, OsSOS1, OsVHA, and OsGLRs. In addition, decreased generation and maintenance of lesser reactive oxygen species (ROS), with maintenance of chloroplast grana and photosynthetic machinery was observed. When evaluated for reproductive growth, 89–96 % of seed setting was maintained in transgenic plants during drought stress; however, under salt stress, a 33–53 % decrease in seed setting was observed. These results indicate that PgRab7 overexpression in rice confers differential tolerance at the seed setting stage during salinity and drought stress and could be a favored target for raising drought-tolerant crops.     

Overexpression of Rat Neurons Nitric Oxide Synthase in Rice Enhances Drought and Salt Tolerance

Nitric oxide (NO) has been shown to play an important role in the plant response to biotic and abiotic stresses in Arabidopsis mutants with lower or higher levels of endogenous NO. The exogenous application of NO donors or scavengers has also suggested an important role for NO in plant defense against environmental stress. In this study, rice plants under drought and high salinity conditions showed increased nitric oxide synthase (NOS) activity and NO levels. Overexpression of rat neuronal NO synthase (nNOS) in rice increased both NOS activity and NO accumulation, resulting in improved tolerance of the transgenic plants to both drought and salt stresses. nNOS-overexpressing plants exhibited stronger water-holding capability, higher proline accumulation, less lipid peroxidation and reduced electrolyte leakage under drought and salt conditions than wild rice. Moreover, nNOS-overexpressing plants accumulated less H₂O₂, due to the observed up-regulation of OsCATA, OsCATB and OsPOX1. In agreement, the activities of CAT and POX were higher in transgenic rice than wild type. Additionally, the expression of six tested stress-responsive genes including OsDREB2A, OsDREB2B, OsSNAC1, OsSNAC2, OsLEA3 and OsRD29A, in nNOS-overexpressing plants was higher than that in the wild type under drought and high salinity conditions. Taken together, our results suggest that nNOS overexpression suppresses the stress-enhanced electrolyte leakage, lipid peroxidation and H₂O₂ accumulation, and promotes proline accumulation and the expression of stress-responsive genes under stress conditions, thereby promoting increased tolerance to drought and salt stresses.     

Cytosolic APx knockdown indicates an ambiguous redox responses in rice

Ascorbate peroxidases (APX, EC 1.1.11.1) are class I heme-peroxidases, which catalyze the conversion of H₂O₂ into H₂O, using ascorbate as a specific electron donor. Previously, the presence of eight Apx genes was identified in the nuclear genome of rice (Oryza sativa), encoding isoforms that are located in different sub-cellular compartments. Herein, the generation of rice transgenic plants silenced for either both or each one of the cytosolic Apx1 and Apx2 genes was carried out in order to investigate the importance of cytosolic Apx isoforms on plant development and on plant stress responses. Transgenic double Apx1/2-silenced plants exhibited normal development, even though these plants showed a global reduction of Apx activity which strongly impacts the whole antioxidant system regulation. Apx1/2-silenced plants also showed increased H₂O₂ accumulation under control and stress situations and presented higher tolerance to toxic concentration of aluminum when compared to wild type plants. On the other hand, silencing OsApx1 and OsApx2 genes individually resulted in strong effect on plant development producing semi-dwarf phenotype. These results suggested that the double silencing of cytosolic OsApx genes induced compensatory antioxidant mechanisms in rice while single knockdown of these genes did not, which resulted in the impairing of normal plant development.