Accumulation of SALT protein in rice plants as a response to environmental stresses

Germination and subsequent hydroponic growth under salt stress (100 mmol/L NaCl) triggered an accumulation of six major stress proteins and resulted in a growth arrest of young seedlings of rice (Oryza sativa L.) cv. Bura Rata. Based on two-dimensional electrophoretic resolution, partial amino acid sequencing and immunodetection techniques, four of the salt stress-induced polypeptides were identified as LEA proteins. Under all experimental conditions wherein seedlings exhibited superior halotolerance, salt stress-induced LEA proteins were expressed at low levels. In contrast, accumulation of LEA proteins was found associated with growth arrest. When returned to non-saline media, seedlings stressed with salt for four days recovered immediately. Longer exposure to 100 mmol/L NaCl, however, progressively delayed recovery and reduced the number of seedlings which could recover from salt stress. Recovery from salt stress was consistently accompanied by degradation of the salt stress-induced LEA proteins. The results of this study show that LEA proteins accumulate during the salinity-triggered growth arrest of young Bura Rata seedlings and are mobilised during the recovery of seedlings from salinity stress.

Source: Journal of Plant Physiology (2003) vol. 160, p. 1165-1174

Development of transgenic rice plants overexpressing the Arabidopsis H+/Ca2+ antiporter CAX1 gene

The gene of the Arabidopsis thaliana H+/Ca2+ transporter, CAX1 (cation exchanger 1) was introduced into Japonica cultivars of rice (Ilpumbyeo) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice callus. The introduction of the CAX1 gene was also proven by PCR using CAX1-specific oligonucleotide primers in regenerated plants. Stable integration and expression of the CAX1 gene in T0 plants and T1 progeny were confirmed by DNA hybridization, Northern blot analysis, and luminescent analysis.

Source: Plant Cell Rep. (2005) vol. 23, p. 678-682

Inheritance and expression of the cry1Abgene in Bt ( Bacillus thuringiensis) transgenic rice

The inheritance and expression patterns of the cry1Ab gene were studied in the progenies derived from different Bt (Bacillus thuringiensis) transgenic japonica rice lines under field conditions. Both Mendelian and distorted segregation ratios were observed in some selfed and crossed F2 populations. Crosses between japonica intrasubspecies had no significant effect on the segregation ratios of the cry1Ab gene, but crossing between japonica and indica inter-subspecies led to distorted segregation of the cry1Ab gene in the F2 population. Field-release experiments indicated that the cry1Ab gene was stably transmitted in an intact manner via successive sexual generations, and the concentration of the CrylAb protein was kept quantitatively stable up to the R6 generation. The cry1Ab gene, driven by the maize ubiquitin promoter, displayed certain kinds of spatial and temporal expression patterns under field conditions. The content of the CrylAb protein varied in different tissues of the main stems, the primary tillers and the secondary tillers. Higher levels of the CrylAb protein were found in the stems, leaves and leaf sheaths than in the roots, while the lowest level was detected in grains at the maturation stage. The content of the Cry1Ab protein in the leaves peaked at the booting stage and was lowest at the heading stage. Furthermore, the CrylAb content of cry1Ab expression in different tissues of transgenic rice varied individually with temperature.

Source: Theoretical and Applied Genetics (2002) vol. 104, p. 727-734

Scavenging of reactive oxygen species in NaCl-stressed rice (Oryza sativa L.)—differential response in salt-tolerant and sensitive varieties

Salinity stress affects the metabolism of plant cells leading to severe crop damage and loss of productivity. Oxidative stress is one consequence of salinity that may be responsible for much of the damage. We investigated the immediate responses (enzymatic and non-enzymatic) to salinity-induced oxidative stress in two major rice (Oryza sativa L.) cultivars, salt sensitive Pusa Basmati I (PB) and salt-tolerant Pokkali (PK). Seedlings of both cultivars were subjected to NaCI stress (100-300 mM) for 42 h. Under NaCI stress, the salt-tolerant cv. PK showed higher activity of the ROS scavenging enzyme, catalase (CAT) and enhanced levels of antioxidants like ascorbate (ASC) and glutathione (GSH), than the sensitive cv. PB. Although superoxide dismutase (SOD) activity was lower in cv. PK, it showed lesser extent of membrane damage (lipid peroxidation) and lower levels of H2O2 than cv. PB under stress. The high levels of catalase activity indicate efficient scavenging of H2O2, which is produced more by non-enzymatic means than via SOD in cv. PK. These data indicate that concerted action of both enzymatic and non-enzymatic ROS scavenging machineries is vital to overcome salinity-induced oxidative stress in rice.

Source: Plant science (2003) vol. 165,p. 1411-1418

Agrobacterium-mediated transformation of Indica rice genotypes: an assessment of factors affecting the transformation efficiency

An efficient Agrobacterium-mediated method for transformation of popular Bangladeshi Indica rice genotypes has been developed. Mature embryo-derived calluses as well as immature embryos were used as the target material. Transgenic plant production frequency was higher using the immature embryos than mature embryo-derived calluses. However, 3-week-old mature embryo-derived calluses served as an excellent starting material. The super-binary vector (pTOK233) was generally more effective than the binary vector (pC1301-Xa21mSS) particularly with recalcitrant Bangladeshi genotypes such as BR22. However, transformation of the Japonica cultivar Taipei-309 was equally effective with either plasmid. Inclusion of acetosyringone (200M) in co-cultivation media proved essential for successful transformation and the optimum co-cultivation period found was to be 3days. A large number of morphologically normal, fertile transgenic plants were obtained which expressed gus as determined by histochemical staining. Integration of the hpt gene into the genome of transgenic plants was confirmed by molecular analysis. Mendelian inheritance of transgenes (hpt and gus gene) was observed in T1 progeny.

Source: Plant Cell, Tissue and Organ Culture (2005) vol. 82, p. 45-55

Stress-inducible synthesis of proline in transgenic rice confers faster growth under stress conditions than that with constitutive synthesis

Proline accumulation has been shown to correlate with tolerance to drought and salt stresses in plants. Our goal was to compare the growth rate of transgenic rice plants in which the expression of a mothbean Δ1-pyrroline-5-carboxylate synthetase (p5cs) cDNA was driven separately with a constitutive and a stress-inducible promoter. We found that both constitutive expression and stress-inducible expression of the p5cs cDNA in transgenic rice have led to the accumulation of p5cs mRNA and proline. Third-generation (R2) transgenic rice seedlings showed significantly higher tolerance to stress produced by high levels of NaCl or water deficiency as judged by faster growth of shoots and roots in comparison with non-transformed plants. However, stress-inducible expression of the P5CS transgene showed significant advantages over the constitutive expression in increasing the biomass production of transgenic rice grown in soil under stress conditions.

Source: Plant Science (2004) Vol. 166, p. 941-948

Expression of ascorbate peroxidase and glutathione reductase in roots of rice seedlings in response to NaCl and H2O2

The accumulation of H2O2 by NaCl was observed in the roots of rice seedlings. Treatment with NaCl caused an increase in the activities of ascorbate peroxidase (APX) and glutathione reductase (GR) and the expression of OsAPX and OsGR in rice roots. Exogenously applied H2O2 also enhanced the activities of APX and GR and the expression of OsAPX and OsGR in rice roots. The accumulation of H2O2 in rice roots in response to NaCl was inhibited by the NADPH oxidase inhibitors, diphenyleneiodonium chloride (DPI) and imidazole (IMD). However, DPI, IMD, and dimethylthiourea, a H2O2 trap, did not reduce NaCl-enhanced activities of APX and GR and expression of OsAPX and OsGR. It appears that H2O2 is not involved in the regulation of NaCl-induced APX and GR activities and OsAPX and OsGR expression in rice roots.

Source: Journal of plant physiology (2005) vol. 162, p. 291-299

Over-expression of ζ glutathione S-transferase in transgenic rice enhances germination and growth at low temperature

To develop a rice cultivar that would be suitable for direct-seedingcultivation in cooler temperate regions, we generated transgenic rice plants inwhich a rice encoding a -class glutathioneS-transferase (GST) under the control of a maize ubiquitinpromoter. GSTs have been suggested to be responsible for tolerance to variousstresses such as cold, salt and drought by detoxification of xenobioticcompounds and reactive oxygen species. A total of 87 R0 transgenicrice plants harboring a chimeric GST gene were generatedusing Agrobacterium mediated transformation. ThreeR2 lines homozygous for the transgene were assayed for GST activityand had higher GST and glutathione peroxidase activities thannon-transformants.Seedlings of the transgenic lines demonstrated greatly enhanced germination andgrowth rates at low temperature grown under submergence. The GST transgeniclines should be useful for breeding rice cultivars suitable for direct-seedingcultivation in cooler temperate regions.

Source: Molecular Breeding (2002) vol. 9, p. 93-101

Optimising the tissue culture conditions for high efficiency transformation of indica rice

Establishment of high efficiency Agrobacterium-mediated transformation techniques has greatly accelerated the widespread application of transformation in japonica rice. However, transformation in indica rice remains difficult. In this study, we identify two new media for subculture and differentiation, the two major steps in the tissue culture process for transformation. These media were tested using four cultivars representing very different germplasms of indica rice. The results show that the new media significantly improved the growth rate and quality of the calli, and also increased the differentiation rate for all four cultivars tested. Use of these modified media in transformation experiments also greatly improved the transformation efficiency of all four indica cultivars.

Source: Plant Cell Rep. (2005) vol. 23, p. 540-547

Variability in expression of insecticidal Cry1Ab gene in Indica Basmati rice

The expression of an insecticidal gene cry1Ab, under three different promoters was studied in leaves, stem and panicles to determine organ-specificity in Basmati rice. Enhanced resistance against two Lepidopteran insects, stem borer (Scirpophaga incertulas) and leaf folder (Cnaphalocrocis medinalis) was observed. The result of western hybridization and insect bioassays demonstrated that all these promoters express the cry1Ab gene at similar levels in leaves and panicles. The cry1Ab gene was expressed in stems at 0.05% of the total protein under the control of the PEPC promoter alone or in combination with the pollen-specific promoter. On the other hand it was expressed at 0.15% under the control of the ubiquitin promoter. Southern blot hybridization of these plants indicated integration of the complete plant transcriptional unit at multiple insertion sites. These results demonstrated that a specific promoter could be used to limit the expression of cry1Ab gene in the desired parts of Basmati rice plants.

Source: Euphytica (2002) vol. 128, p. 121-128