Soumitra Paul

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.

miRNA regulation of nutrient homeostasis in plants.

Small RNAs including micro RNAs (miRNA) play an indispensable role in cell signaling mechanisms. Generally, miRNAs that are 20–24 nucleotides long bind to specific complementary transcripts, attenuating gene expression at the post-transcriptional level or via translational inhibition. In plants, miRNAs have emerged as the principal regulator of various stress responses, including low nutrient availability. It has been reported that miRNAs are vital for maintaining nutrient homeostasis in plants by regulating the expression of transporters that are involved in nutrient uptake and mobilization. The present review highlights the role of various miRNAs in several macro- or micronutrient deficiencies in plants. Understanding the regulation of different transporters by miRNAs will aid in elucidating the underlying molecular signal transduction mechanisms during nutritional stress. Recent findings regarding nutrient related-miRNAs and their gene regulation machinery may delineate a novel platform for improving the nutritional status of cereal grains or crop biofortification programs in the future.

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.

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.

Loading and bioavailability of iron in cereal grains

Cereal plants take up iron from the soil via a phytosiderophore-mediated chelation system. Following root absorption, iron is transported through the xylem and phloem of the plant with the help of a variety of efflux and influx transporters belonging to the Zrt Irt-like protein (ZIP) and yellow stripe-like (YSL) protein families. Iron-regulated transporter1, a member of the ZIP family, mobilises ferrous [Fe(II)] ions, while several YSL family members such as YSL2, YSL15 and YSL18 can transport both ferric [Fe(II)] and ferrous [F`III)] ions into developing grains via chelation with mugineic acid or its derivatives. The iron is accumulated largely in the outer aleurone layer and embryo of the grains, which are removed during milling, leaving behind consumable endosperm that contains a very low amount of iron. This review highlights the uptake, transport and loading mechanisms for iron in cereal grains and provides an overview of strategies adopted for developing highly iron-enriched grains.

Development of an Iron-enriched High-yieldings Indica Rice Cultivar by Introgression of A High-iron Trait from Transgenic Iron-biofortified Rice

Low level of iron in staple food crops is one reason for the predominance of iron-deficiency anemia in developing countries. Most of the iron in rice grains accumulates in the outer aleurone layer and embryo, which are removed during milling, and the edible endosperm contains very low amounts of iron. In an effort to increase iron nutrition, we report here the transgene introgression of a high-iron trait into a high-yielding indica rice cultivar. The ferritin gene from soybean (soyfer1) was introduced into rice plants through interbreeding between soybean ferritin-overexpressing transgenic IR68144 and the high-yielding cultivar Swarna. The stable integration of the soyfer1 gene was confirmed in the BC2F4 generation, and the hybrid seeds showed 2.6-fold soybean ferritin gene expression over the recurrent parent Swarna. The hybrid milled seeds revealed a 2.54-fold increase in iron and 1.54-fold increase in zinc compared to Swarna. Agronomic data and an SSR marker analysis of the hybrid rice plants were taken into account for NIL character identification.

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.