Promoter Analysis for Three Types of EUL-Related Rice Lectins in Transgenic Arabidopsis

In this study, the promoter activity for three types of Euonymus-related lectins (EUL) from rice, further referred to as OrysaEULS2, OrysaEULS3, and OrysaEULD1A was analyzed. In silico promoter analyses showed that the EUL promoters from rice contain next to the typical promoter elements some motifs that are considered to be stress-responsive elements. Furthermore, Arabidopsis thaliana plants were transformed with a promoter::β-glucuronidase (GUS) construct for each of the proteins under study. Subsequently, one-insertion homozygous lines were selected and analyzed for GUS activity. Experiments were performed under normal growth conditions or after application of different stress conditions, in particular treatments with 150 mM NaCl, 100 mM mannitol, and 100 μM abscisic acid (ABA) for 24 h. GUS activity was detected with the OrysaEULS3 and OrysaEULD1A promoters especially in the cotyledons and the young true leaves, respectively, but not with the OrysaEULS2 promoter. The activity of OrysaEULS3 and OrysaEULD1A promoters was increased after ABA and mannitol treatments but decreased after NaCl treatment. We hypothesize that the Euonymus-related rice proteins have a role in sensing and responding to external stresses as well as in the growth of the plant.     

OsLEA3-2, an Abiotic Stress Induced Gene of Rice Plays a Key Role in Salt and Drought Tolerance

Late embryogenesis abundant (LEA) proteins are involved in tolerance to drought, cold and high salinity in many different organisms. In this report, a LEA protein producing full-length gene OsLEA3-2 was identified in rice (Oryza sativa) using the Rapid Amplification of cDNA Ends (RACE) method. OsLEA3-2 was found to be only expressed in the embryo and can be induced by abiotic stresses. The coding protein localizes to the nucleus and overexpression of OsLEA3-2 in yeast improved growth performance compared with control under salt- and osmotic-stress conditions. OsLEA3-2 was also inserted into pHB vector and overexpressed in Arabidopsis and rice. The transgenic Arabidopsis seedlings showed better growth on MS media supplemented with 150 mM mannitol or 100 mM NaCl as compared with wild type plants. The transgenic rice also showed significantly stronger growth performance than control under salinity or osmotic stress conditions and were able to recover after 20 days of drought stress. In vitro analysis showed that OsLEA3-2 was able to protect LDH from aggregation on freezing and inactivation on desiccation. These results indicated that OsLEA3-2 plays an important role in tolerance to abiotic stresses.     

Over-expression of a <Emphasis Type="Italic">LEA</Emphasis> gene in rice improves drought resistance under the field conditions

Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T₁ families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T₁ families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T₁ generation, were tested in the field for drought resistance in the T₂ and T₃ generations and in the PVC pipes for drought tolerance in the T₂ generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T₂ and T₃ families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol.     

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.     

Function of the wheat TaSIP gene in enhancing drought and salt tolerance in transgenic Arabidopsis and rice

Microarray analysis of a salt-tolerant wheat mutant identified a gene of unknown function that was induced by exposure to high levels of salt and subsequently denoted TaSIP (Triticum aestivum salt-induced protein). Quantitative PCR analysis revealed that TaSIP expression was induced not only by salt, but also by drought, abscisic acid (ABA), and other environmental stress factors. Transgenic rice plants that expressed an RNA interference construct specific for a rice gene homologous to TaSIP was more susceptible to salt stress than wild-type rice plants. Subcellular localization studies showed that the TaSIP localized to the cell membrane. Under conditions of salt and drought stress, transgenic Arabidopsis plants that overexpressed TaSIP showed superior physiological properties compared with control plants, including lower Na⁺ content and upregulation of several stress resistance genes. Staining of transgenic tissues with β-glucuronidase (GUS) failed to indicate tissue-specific activity of the full-length TaSIP promoter. Quantitative analysis of GUS fluorescence in transgenic plants treated with ABA or salt stress revealed that the region 1,176–1,410 bp from the start codon contained an ABA-responsive element and that the region 579–1,176 bp from the start codon upstream of the exon contained a salt-stress-responsive element. Based on these results, we conclude that the key part of the TaSIP gene is the region of its promoter involved in salt tolerance.     

ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.     

Expression of cold and drought regulatory protein (CcCDR) of pigeonpea imparts enhanced tolerance to major abiotic stresses in transgenic rice plants

Main conclusion

Transgenic rice expressing pigeonpea Cc CDR conferred high-level tolerance to different abiotic stresses. The multiple stress tolerance observed in CcCDR -transgenic lines is attributed to the modulation of ABA-dependent and-independent signalling-pathway genes.

Stable transgenic plants expressing Cajanus cajan cold and drought regulatory protein encoding gene (CcCDR), under the control of CaMV35S and rd29A promoters, have been generated in indica rice. Different transgenic lines of CcCDR, when subjected to drought, salt, and cold stresses, exhibited higher seed germination, seedling survival rates, shoot length, root length, and enhanced plant biomass when compared with the untransformed control plants. Furthermore, transgenic plants disclosed higher leaf chlorophyll content, proline, reducing sugars, SOD, and catalase activities, besides lower levels of MDA. Localization studies revealed that the CcCDR-GFP fusion protein was mainly present in the nucleus of transformed cells of rice. The CcCDR transgenics were found hypersensitive to abscisic acid (ABA) and showed reduced seed germination rates as compared to that of control plants. When the transgenic plants were exposed to drought and salt stresses at vegetative and reproductive stages, they revealed larger panicles and higher number of filled grains compared to the untransformed control plants. Under similar stress conditions, the expression levels of P5CS, bZIP, DREB, OsLEA3, and CIPK genes, involved in ABA-dependent and-independent signal transduction pathways, were found higher in the transgenic plants than the control plants. The overall results amply demonstrate that the transgenic rice expressing CcCDR bestows high-level tolerance to drought, salt, and cold stress conditions. Accordingly, the CcCDR might be deployed as a promising candidate gene for improving the multiple stress tolerance of diverse crop plants.

     

Developing Fiber Specific Promoter-Reporter Transgenic Lines to Study the Effect of Abiotic Stresses on Fiber Development in Cotton

Cotton is one of the most important cash crops in US agricultural industry. Environmental stresses, such as drought, high temperature and combination of both, not only reduce the overall growth of cotton plants, but also greatly decrease cotton lint yield and fiber quality. The impact of environmental stresses on fiber development is poorly understood due to technical difficulties associated with the study of developing fiber tissues and lack of genetic materials to study fiber development. To address this important question and provide the need for scientific community, we have generated transgenic cotton lines harboring cotton fiber specific promoter (CFSP)-reporter constructs from six cotton fiber specific genes (Expansin, E6, Rac13, CelA1, LTP, and Fb late), representing genes that are expressed at different stages of fiber development. Individual CFSP::GUS or CFSP::GFP construct was introduced into Coker 312 via Agrobacterium mediated transformation. Transgenic cotton lines were evaluated phenotypically and screened for the presence of selectable marker, reporter gene expression, and insertion numbers. Quantitative analysis showed that the patterns of GUS reporter gene activity during fiber development in transgenic cotton lines were similar to those of the native genes. Greenhouse drought and heat stress study showed a correlation between the decrease in promoter activities and decrease in fiber length, increase in micronaire and changes in other fiber quality traits in transgenic lines grown under stressed condition. These newly developed materials provide new molecular tools for studying the effects of abiotic stresses on fiber development and may be used in study of cotton fiber development genes and eventually in the genetic manipulation of fiber quality.     

The overexpression of OsNAC9 alters the root architecture of rice plants enhancing drought resistance and grain yield under field conditions

Drought conditions limit agricultural production by preventing crops from reaching their genetically predetermined maximum yields. Here, we present the results of field evaluations of rice overexpressing OsNAC9, a member of the rice NAC domain family. Root‐specific (RCc3) and constitutive (GOS2) promoters were used to overexpress OsNAC9 and produced the transgenic RCc3:OsNAC9 and GOS2:OsNAC9 plants. Field evaluations over two cultivating seasons showed that grain yields of the RCc3:OsNAC9 and the GOS2:OsNAC9 plants were increased by 13%–18% and 13%–32% under normal conditions, respectively. Under drought conditions, RCc3:OsNAC9 plants showed an increased grain yield of 28%–72%, whilst the GOS2:OsNAC9 plants remained unchanged. Both transgenic lines exhibited altered root architecture involving an enlarged stele and aerenchyma. The aerenchyma of RCc3:OsNAC9 roots was enlarged to a greater extent than those of GOS2:OsNAC9 and non‐transgenic (NT) roots, suggesting the importance of this phenotype for enhanced drought resistance. Microarray experiments identified 40 up‐regulated genes by more than threefold (P < 0.01) in the roots of both transgenic lines. These included 9‐cis‐epoxycarotenoid dioxygenase, an ABA biosynthesis gene, calcium‐transporting ATPase, a component of the Ca²⁺ signalling pathway involved in cortical cell death and aerenchyma formation, cinnamoyl CoA reductase 1, a gene involved in lignin biosynthesis, and wall‐associated kinases¸ genes involved in cell elongation and morphogenesis. Interestingly, O‐methyltransferase, a gene necessary for barrier formation, was specifically up‐regulated only in the RCc3:OsNAC9 roots. Such up‐regulated genes that are commonly and specifically up‐regulated in OsNAC9 transgenic roots may account for the altered root architecture conferring increased drought resistance phenotype.