Dipak Gayen

Development of Low Phytate Rice by RNAi Mediated Seed-Specific Silencing of Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase Gene (IPK1)

Phytic acid (InsP6) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP6 is not utilized efficiently by monogastric animals due to lack of phytase enzyme. Furthermore, due to its ability to chelate mineral cations, phytic acid is considered to be an antinutrient that renders these minerals unavailable for absorption. In view of these facts, reducing the phytic acid content in cereal grains is a desired goal for the genetic improvement of several crops. In the present study, we report the RNAi-mediated seed-specific silencing (using the Oleosin18 promoter) of the IPK1 gene, which catalyzes the last step of phytic acid biosynthesis in rice. The presence of the transgene cassette in the resulting transgenic plants was confirmed by molecular analysis, indicating the stable integration of the transgene. The subsequent T4 transgenic seeds revealed 3.85-fold down-regulation in IPK1 transcripts, which correlated to a significant reduction in phytate levels and a concomitant increase in the amount of inorganic phosphate (Pi). The low-phytate rice seeds also accumulated 1.8-fold more iron in the endosperm due to the decreased phytic acid levels. No negative effects were observed on seed germination or in any of the agronomic traits examined. The results provide evidence that silencing of IPK1 gene can mediate a substantial reduction in seed phytate levels without hampering the growth and development of transgenic rice plants.

Overexpression of Rab16A gene in indica rice variety for generating enhanced salt tolerance

We report here the overexpression of Rab16A full length gene (promoter + ORF), from the salt-tolerant indica rice Pokkali, in the salt-susceptible indica rice variety Khitish, via particle bombardment. Molecular analysis of the transgenics revealed stable integration of the transgene upto T2 generation. High level of expression of the transgene (driven by its own stress-inducible promoter), as well as the protein, was detectable in the leaves under simulated salinity stress (250 mM NaCl, 24 h); the expression level being higher than wild type (WT) plants. The Rab16A transcript also increased gradually with seed maturity, with its maximal accumulation at 30 d after pollination (DAP) i.e., fully matured seeds, explaining the protective role of Rab16A gene during seed maturation. Enhanced tolerance to salinity was observed in the plants transformed with Rab16A. The superior physiological performances of the transgenics under salt treatment were also reflected in lesser shoot or root length inhibition, reduced chlorophyll damages, lesser accumulation of Na+ and reduced loss of K+, increased proline content as compared with the WT plants. All these results indicated that the overproduction of RAB16A protein in the transgenics enable them to display enhanced tolerance to salinity stress with improved physiological traits. Our work demonstrates the profound potential of Group 2 LEA proteins (to which RAB16A belongs to) in conferring stress tolerance in crop plants through their genetic manipulation.

Comparative analysis of nutritional compositions of transgenic high iron rice with its non-transgenic counterpart.

Iron is an essential micronutrient for human nutrition and polished rice contains very low amount of iron. Rice with high iron content in seed endosperm has been developed by insertion of soybean ferritin gene under the control of the endosperm specific glutelin promoter into the genome of indica rice line IR68144. The nutritional composition of the brown and milled rice grain has been compared with that of the non-transgenic rice of the same variety. In this study, the nutritional components, as well as the anti-nutrient levels, were measured. Our studies established that apart from the increased level of iron and zinc in transgenic seeds, the nutritional quality of both the brown and milled rice grains from the transgenic line was substantially equivalent to that of the non-transgenic rice plants. The result clearly shows that the measured amounts of the nutritional components are well within the range of values reported for other commercial lines.

Molecular breeding of Osfer2 gene to increase iron nutrition in rice grain

Rice being a staple food, contains little iron in the edible grain. To increase the iron nutrition in rice grains, our present study highlights the first time development of high iron rice grain by exploring the endosperm specific overexpression of endogenous ferritin gene. The gene has been cloned from rice and overexpressed under the control of endosperm specific GlutelinA2 (OsGluA 2) promoter. After genetic transformation of aromatic indica rice cultivar, Pusa-sugandhi II, the milled seeds of resulting T 3 transgenics exhibited 7.8-fold of ferritin overexpression, which contributed to 2.09- and 1.37-fold of iron and zinc accumulation respectively. T 3 seeds demonstrated endosperm specific localization of iron that confirms the tissue specific activity of GluA2 promoter. Transgenic and non-transgenic plants showed no difference in their agronomic traits. Our study suggested that overexpression of rice endogenous ferritin gene is a step ahead toward cisgenic approach and can act as an effective tool for iron biofortification.

RNAi mediated down regulation of myo-inositol-3-phosphate synthase to generate low phytate rice

BackgroundPhytic acid (InsP6) is considered as the major source of phosphorus and inositol phosphates in cereal grains. Reduction of phytic acid level in cereal grains is desirable in view of its antinutrient properties to maximize mineral bioavailability and minimize the load of phosphorus waste management. We report here RNAi mediated seed-specific silencing of myo-inositol-3-phosphate synthase (MIPS) gene catalyzing the first step of phytic acid biosynthesis in rice. Moreover, we also studied the possible implications of MIPS silencing on myo-inositol and related metabolism, since, first step of phytic acid biosynthesis is also the rate limiting step of myo-inositol synthesis, catalyzed by MIPS.ResultsThe resulting transgenic rice plants (T3) showed a 4.59 fold down regulation in MIPS gene expression, which corresponds to a significant decrease in phytate levels and a simultaneous increment in the amount of inorganic phosphate in the seeds. A diminution in the myo-inositol content of transgenic plants was also observed due to disruption of the first step of phytic acid biosynthetic pathway, which further reduced the level of ascorbate and altered abscisic acid (ABA) sensitivity of the transgenic plants. In addition, our results shows that in the transgenic plants, the lower phytate levels has led to an increment of divalent cations, of which a 1.6 fold increase in the iron concentration in milled rice seeds was noteworthy. This increase could be attributed to reduced chelation of divalent metal (iron) cations, which may correlate to higher iron bioavailability in the endosperm of rice grains.ConclusionThe present study evidently suggests that seed-specific silencing of MIPS in transgenic rice plants can yield substantial reduction in levels of phytic acid along with an increase in inorganic phosphate content. However, it was also demonstrated that the low phytate seeds had an undesirable diminution in levels of myo-inositol and ascorbate, which probably led to sensitiveness of seeds to abscisic acid during germination. Therefore, it is suggested that though MIPS is the prime target for generation of low phytate transgenic plants, down-regulation of MIPS can have detrimental effect on myo-inositol synthesis and related pathways which are involved in key plant metabolism.

Down-regulation of lipoxygenase gene reduces degradation of carotenoids of golden rice during storage

AbstractMain conclusionDown-regulation of lipoxygenase enzyme activity reduces degradation of carotenoids of bio-fortified rice seeds which would be an effective tool to reduce huge post-harvest and economic losses of bio-fortified rice seeds during storage. Bio-fortified provitamin A-enriched rice line (golden rice) expressing higher amounts of β-carotene in the rice endosperm provides vitamin A for human health. However, it is already reported that degradation of carotenoids during storage is a major problem. The gene responsible for degradation of carotenoids during storage has remained largely unexplored till now. In our previous study, it has been shown that r9-LOX1 gene is responsible for rice seed quality deterioration. In the present study, we attempted to investigate if r9-LOX1 gene has any role in degradation of carotenoids in rice seeds during storage. To establish our hypothesis, the endogenous lipoxygenase (LOX) activity of high-carotenoid golden indica rice seed was silenced by RNAi technology using aleurone layer and embryo-specific Oleosin-18 promoter. To check the storage stability, LOX enzyme down-regulated high-carotenoid T3 transgenic rice seeds were subjected to artificial aging treatment. The results obtained from biochemical assays (MDA, ROS) also indicated that after artificial aging, the deterioration of LOX-RNAi lines was considerably lower compared to β-carotene-enriched transgenic rice which had higher LOX activity in comparison to LOX-RNAi lines. Furthermore, it was also observed by HPLC analysis that down-regulation of LOX gene activity decreases co-oxidation of β-carotene in LOX-RNAi golden rice seeds as compared to the β-carotene-enriched transgenic rice, after artificial aging treatment. Therefore, our study substantially establishes and verifies that LOX is a key enzyme for catalyzing co-oxidation of β-carotene and has a significant role in deterioration of β-carotene levels in the carotenoid-enriched golden rice.

Dissecting root proteome of transgenic rice cultivars unravels metabolic alterations and accumulation of novel stress responsive proteins under drought stress

Generation of drought tolerant rice plants by overexpressing Arabidopsis DREB1A is a significant development for abiotic stress research. However, the metabolic network regulated in the drought tolerant transgenic rice plants is poorly understood. In this research study, we have demonstrated the comparative proteome analysis between the roots of wild type and transgenic DREB1A overexpressing homozygous plants under drought stress condition. After 7d of dehydration stress at reproductive stage, the plants were re-watered for 24h. The roots were collected separately from wild type and transgenic plants grown under water, drought stress and re-watering conditions and total proteins were analyzed by two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry (MS). Among the large number of differentially accumulated spots, 30, 27 and 20 spots were successfully identified as differentially expressed proteins in three different conditions respectively. The major class of identified proteins belongs to carbohydrate and energy metabolism category while stress and defense related proteins are especially up-accumulated under drought stress in both the plants. A novel protein, R40C1 was reported to be up-accumulated in roots of transgenic plants which may play an important role in generation of drought tolerant plants. Protein-protein interaction helps to identify the network of drought stress signaling pathways.

Comparative nutritional compositions and proteomics analysis of transgenic Xa21 rice seeds compared to conventional rice

Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial blight diseases caused by Xanthomonas oryzae pv. oryzae (Xoo). However, identification of unintended modifications, owing to the genetic modification, is an important aspect of transgenic crop safety assessment. In this study, the nutritional compositions of seeds from transgenic rice plants expressing the Xa21 gene were compared against non-transgenic rice seeds. In addition, to detect any changes in protein translation levels as a result of Xa21 gene expression, rice seed proteome analyses were also performed by two-dimensional gel electrophoresis. No significant differences were found in the nutritional compositions (proximate components, amino acids, minerals, vitamins and anti-nutrients) of the transgenic and non-transgenic rice seeds. Although gel electrophoresis identified 11 proteins that were differentially expressed between the transgenic and non-transgenic seed, only one of these (with a 20-fold up-regulation in the transgenic seed) shows nutrient reservoir activity. No new toxins or allergens were detected in the transgenic seeds.

Analysis of high iron rice lines reveals new miRNAs that target iron transporters in roots

Highlight Novel miRNAs regulating NRAMP4 activity in root induce iron accumulation in ferritin-overexpressing rice grains during the milk stage of seed development. Enhancement of YSL15, IRT2, and FRO2 facilitates this process.

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 β-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.