Metabolic Regulation of Carotenoid-Enriched Golden Rice Line.

Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated with high risk of maternal mortality. In order to enhance the bioavailability of vitamin A, high carotenoid transgenic golden rice has been developed by manipulating enzymes, such as phytoene synthase (psy) and phytoene desaturase (crtI). In this study, proteome and metabolite analyses were carried out to comprehend metabolic regulation and adaptation of transgenic golden rice after the manipulation of endosperm specific carotenoid pathways. The main alteration was observed in carbohydrate metabolism pathways of the transgenic seeds. The 2D based proteomic studies demonstrated that carbohydrate metabolism-related enzymes, such as pullulanase, UDP-glucose pyrophosphorylase, and glucose-1-phosphate adenylyltransferase, were primarily up-regulated in transgenic rice seeds. In addition, the enzyme PPDK was also elevated in transgenic seeds thus enhancing pyruvate biosynthesis, which is the precursor in the carotenoids biosynthetic pathway. GC-MS based metabolite profiling demonstrated an increase in the levels of glyceric acid, fructo-furanose, and galactose, while decrease in galactonic acid and gentiobiose in the transgenic rice compared to WT. It is noteworthy to mention that the carotenoid content, especially ß-carotene level in transgenic rice (4.3 µg/g) was significantly enhanced. The present study highlights the metabolic adaptation process of a transgenic golden rice line (homozygous T4 progeny of SKBR-244) after enhancing carotenoid biosynthesis. The presented information would be helpful in the development of crops enriched in carotenoids by expressing metabolic flux of pyruvate biosynthesis.

Rice oxalate oxidase gene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice.

Rice sheath blight, caused by the necrotrophic fungus Rhizoctonia solani, is one of the most devastating and intractable diseases of rice, leading to a significant reduction in rice productivity worldwide. In this article, in order to examine sheath blight resistance, we report the generation of transgenic rice lines overexpressing the rice oxalate oxidase 4 (Osoxo4) gene in a green tissue-specific manner which breaks down oxalic acid (OA), the pathogenesis factor secreted by R. solani. Transgenic plants showed higher enzyme activity of oxalate oxidase (OxO) than nontransgenic control plants, which was visualized by histochemical assays and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Transgenic rice leaves were more tolerant than control rice leaves to exogenous OA. Transgenic plants showed a higher level of expression of other defence-related genes in response to pathogen infection. More importantly, transgenic plants exhibited significantly enhanced durable resistance to R. solani. The overexpression of Osoxo4 in rice did not show any detrimental phenotypic or agronomic effect. Our findings indicate that rice OxO can be utilized effectively in plant genetic manipulation for sheath blight resistance, and possibly for resistance to other diseases caused by necrotrophic fungi, especially those that secrete OA. This is the first report of the expression of defence genes in rice in a green tissue-specific manner for sheath blight resistance.

Inducibility of three salinity/abscisic acid-regulated promoters in transgenic rice with gusA reporter gene.

The present study evaluates the pattern of stress inducibility of one natural promoter (from rice Rab16A) and two synthetically designed promoters, viz., 4X ABRE (abscisic acid-responsive element, having four tandem repeats of ABRE) and 2X ABRC (abscisic acid-responsive complex, having two tandem repeats of ABRE and two copies of coupling elements), in response to varying concentrations of NaCl and abscisic acid (ABA). Each promoter, independently linked to gusA (that encodes ß glucuronidase, GUS), was introduced into rice (cv. Khitish) through particle bombardment. The T(2) progenies showed integration of gusA in their genome. The accumulation of gusA transcript, driven by each promoter in T(2) transgenics, increased with increasing salt/ABA concentration, with ABA being the better activator of each promoter. Induction in GUS expression, driven by different promoters, was noted on exogenous salt/ABA treatments in a concentration-dependent manner. The maximum induction was observed with 2X ABRC promoter. All the three promoters could drive stress-inducible GUS expression in both vegetative and floral organs. However, prominent GUS expression was noted in the whole seed (both embryo and aleurone layer of endosperm) only by 2X ABRC, whereas it was localized only in the embryo for the other two promoters. Thus, our observation characterizes three efficient salinity/ABA-inducible promoters that have the potentiality in crop biotechnology to drive transgene expression for stress tolerance, whenever abiotic stress is encountered.

Comparative physiological and molecular responses of a common aromatic indica rice cultivar to high salinity with non-aromatic indica rice cultivars.

In an attempt to understand the molecular basis of salt-stress response in the aromatic rice Gobindobhog, a comprehensive analysis encompassing physiological or biochemical assays and gene expression studies under high salt (200 mM NaCl) supply regimes were initiated and compared with a salt-sensitive (M-1-48) and salt-tolerant (Nonabokra) rice. The detrimental effects of salinity stress were the most pronounced in Gobindobhog, as reflected by the maximally increased root to shoot ratio, the highest chlorophyll degeneration, the highest foliar concentration of Na(+) ions and peroxide content, with their maximum increment after salt treatment. The amplification of oxidative damages was further stimulated by the accumulation of putrescine and lipid peroxidation-derived toxic degradation products (increased malondialdehyde and lipoxygenase activity), which were comparable in M-1-48 and Gobindobhog. Antioxidants like anthocyanin and particularly cysteine and the osmolytes like reducing sugar, proline and polyamines (spermidine and spermine) showed the highest level in Nonabokra. While the inhibition of catalase activity occurred in all the varieties following salt-stress, the maximum induction in guaiacol peroxidase activity, elevated cysteine and proline levels in Gobindobhog probably constituted the detoxification mechanism obligatory for its survival. Intensification of the aroma content with salt treatment was markedly noted in Gobindobhog. A very low abundance of Rab16A/SamDC transcript and the corresponding proteins were observed both in M-1-48 and Gobindobhog, induced only after salt-stress, whereas they were constitutively expressed in Nonabokra. Thus, our data reflect Gobindobhog as a salt-sensitive cultivar, susceptible to high-stress-induced growth-inhibition, ion imbalances, membrane/oxidative damages with lower expression of stress-tolerant genes.