Plant developmental biology is associated with various gene regulatory pathways involved in diffe... more Plant developmental biology is associated with various gene regulatory pathways involved in different phases of their life cycle. In course of development, growth and differentiation of different organs in plants are regulated by specific sets of gene expression. With the advances in genomic and bioinformatic techniques, particularly high-throughput sequencing technology, many transcriptional units with no protein-coding potential have been discovered. Previously thought to be the dark matters of genome, long non-coding RNAs (lncRNAs) are gradually gaining importance as crucial players in gene regulation during different developmental phases. Some lncRNAs, showing complementarity to microRNAs (miRNAs), are used as endogenous target mimics of specific miRNA family. A number of lncRNAs can also act as natural antisense transcripts to attenuate the expression of coding genes. Although lncRNA-mediated regulations have extensively been studied in animals, plant lncRNA research is still in its initial phase. The present review highlights the regulatory mechanism and different physiological aspects of lncRNAs in plant development. In plants, lncRNAs are found to be associated with a number of plant developmental functions such as lateral root development, vernalization, photomorphogenesis, pollen development, fiber development and nodulation. Understanding these potent roles of lncRNAs in plant development can further provide novel tools for crop improvement programs in future.
bioRxiv (Cold Spring Harbor Laboratory), Mar 27, 2023
Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. Howe... more Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid (β-ODAP) has restricted its cultivation. Although various cultivars with low β-ODAP
bioRxiv (Cold Spring Harbor Laboratory), Apr 27, 2020
While the role of R40 family of lectin proteins in osmotic stress has long been known, their mech... more While the role of R40 family of lectin proteins in osmotic stress has long been known, their mechanism of stress tolerance has not yet been explored. Among them, the expression of Osr40g3 was most strongly induced in response to salt stress. Here, we report that Osr40g3 plays a dual role in regulating salinity tolerance as well as seed development in rice. The overexpression of Osr40g3 under control of a constitutive promoter significantly improved salinity tolerance but impaired seed development in transgenic rice. Remarkably, transgenic Arabidopsis ectopically expressing Osr40g3 exhibited enhanced salinity tolerance but produced seeds normally. It was further confirmed that this Osr40g3-mediated impairment in seed development was due to pollen sterility in the transgenic rice while the pistils remained unaffected. The gene further displayed a precise tissue-specific expression pattern which was essential for proper seed development in rice. The constitutive overexpression led to the interaction of Osr40g3 with OsGF14e in the stamens affecting its protein stability. This interaction further interfered with the gibberellic acid metabolism thus leading to pollen sterility. This interaction was, however, highly specific due to the structural distinctiveness of OsGF14e making the phenomenon rice-specific. Nevertheless, in salinity stressed leaves, Osr40g3 interacted with OsEG45 or its ortholog thus leading to salinity tolerance in both rice and Arabidopsis.
bioRxiv (Cold Spring Harbor Laboratory), Nov 4, 2022
Phosphorus is a macronutrient that regulates a wide range of physiological processes, including p... more Phosphorus is a macronutrient that regulates a wide range of physiological processes, including plant growth and development. The scarcity of bioavailable inorganic phosphate (Pi) is often a constraint for plant growth in most agricultural ecosystems. Under Pi-limited conditions, plants increase Pi acquisition or alter Pi distribution within tissues by activating different Pi transporters and Pi starvation-induced genes. Glutathione (GSH), on the other hand, is known to play a critical role in regulating iron and zinc homeostasis in Arabidopsis. However, how it regulates Pi homeostasis under Pi-limited conditions remains unexplored. We found that the two GSH-deficient mutants, cad2-1, and pad2-1, were hypersensitive to Pi starvation. Under Pi-sufficient condition, the root Pi content was lower in the mutants but significantly higher in the shoot over the Col-0 plants. The pattern was reversed under Pideficient condition. Interestingly, among the different Pi transporters analyzed, the AtPHT1;5 was significantly down-regulated in the two GSH-deficient mutants. Treatment with exogenous GSH could alter Pi accumulation in Col-0 and the pht1;5 complementation lines but not in the pht1;5 mutant. This observation confirmed the role of GSH in Pi reallocation during Pi starvation via the AtPHT1;5 transporter. Further, promoter analysis demonstrated that GSH regulates the transcription of the AtPHT1;5 gene. The Pi-responsive transcription factor (TF), AtWRKY75 was identified to interact with the AtPHT1;5 promoter, and Pi deficiency or GSH treatment enhanced its binding affinity. Together our investigation delineates that GSH regulates the AtPHT1;5 transporter via the AtWRKY75 TF to modulate Pi distribution under altered Pi conditions. .
Several signaling molecules critically regulate the physiological responses in plants. Among them... more Several signaling molecules critically regulate the physiological responses in plants. Among them, miRNAs, generally 21-24 nucleotides long, are widely distributed in different plant species and play as key signaling intermediates in diverse physiological responses. The mature miRNAs are synthesized from MIR genes by RNA polymerase II and processed by Dicerlike (DCL) protein family members associated with some accessory protein molecules. The processed miRNAs are transported to the cytoplasm from the nucleus by specific group of transporters and incorporated into RNA-induced silencing complex (RISC) for specific mRNA cleavage. MicroRNAs can suppress the diverse gene expression, depending on the sequence complementarity of the target transcript except of its own gene. Besides, miRNAs can modulate the gene expression by DNA methylation and translational inhibition of the target transcript. Different classes of DCLs and Argonaute proteins (AGOs) help the miRNAs-mediated gene silencing mechanism in plants.
While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involve... more While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involvement in modulating subcellular Fe homeostasis remained elusive. In this study, we report that the GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant down-regulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4 and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe content in vacuoles and lower in chloroplasts compared with Col-0 under Fe limited condition. Exogenous GSH treatment could enhance the chloroplast Fe content in Col-0 plants but failed to do so in the nramp3nramp4 double mutant demonstrating the role of GSH in modulating subcellular Fe homeostasis. Pharmacological experiments, mutant analysis and promoter assay revealed that this regulation involved transcriptional activation of the transporter genes by a GSH-GSNO module. The Fe responsive bHLH transcription factors (TFs), AtbHLH2...
Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. Howe... more Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid (β-ODAP) has restricted its cultivation. Although various cultivars with low β-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal β-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the β-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the ...
Phosphorus is a macronutrient that regulates a wide range of physiological processes, including p... more Phosphorus is a macronutrient that regulates a wide range of physiological processes, including plant growth and development. The scarcity of bioavailable phosphate is often a constraint for plant growth in most agricultural ecosystems. Under phosphate limited conditions, plants increase phosphate acquisition or alter its distribution within tissues by activating different phosphate transporters and phosphate starvation-induced genes. Glutathione (GSH), on the other hand, is known to play a critical role in regulating iron and zinc homeostasis in Arabidopsis. However, how it regulates phosphate homeostasis under phosphate limited conditions remains unexplored. We found that the two GSH-deficient mutants,cad2-1, andpad2-1, were hypersensitive to phosphate starvation. Under phosphate-sufficient condition, the root phosphate content was lower in the mutants but significantly higher in the shoot over the Col-0 plants. The pattern was reversed under phosphate-deficient condition. Interes...
With the changing climatic conditions, drought has become one of the most threatening abiotic str... more With the changing climatic conditions, drought has become one of the most threatening abiotic stress factors that adversely affect rice cultivation and productivity. Although the involvement of the jacalin domain-containing protein, OsSalT, has been reported in drought and salinity tolerance, its functional mechanism still remains largely unexplored. In this study, expression of the OsSalT gene was found to be positively correlated with the drought tolerance potential with its higher transcript abundance in the tolerant indica rice cultivar, Vandana and lower abundance in the susceptible cultivar, MTU1010. Moreover, the ectopic expression of OsSalT in tobacco imparted drought stress tolerance in the transgenic lines. The transgenic lines exhibited significantly improved growth and higher osmolyte accumulation over the wild-type (WT) plants together with the induction in the OsSalT expression under drought stress. Fascinatingly, the yeast two-hybrid and bimolecular fluorescence compl...
While the role of R40 family of lectin proteins in osmotic stress was already reported, their mec... more While the role of R40 family of lectin proteins in osmotic stress was already reported, their mechanism of stress tolerance still remained elusive. Among them, the expression of Osr40g3 was most strongly induced in response to salinity. Here, we explored the mechanism of Osr40g3-mediated salinity tolerance in rice. The overexpression of Osr40g3 under control of a constitutive promoter significantly improved salinity tolerance but displayed pollen sterility and impaired seed development. The gene further displayed precise tissue-specific expression which was essential for proper seed development in rice. In salinity-stressed leaves, Osr40g3 interacted with OsEG45 leading to salinity tolerance. Overexpression of OsEG45 significantly improved salinity tolerance while the OsEG45 silencing lines displayed severe sensitivity thus confirming its role in imparting salinity tolerance. On the other hand, the constitutive overexpression of Osr40g3 led to its interaction with OsGF14e in stamens...
Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated wit... more 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.
The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing... more The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants. Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds.
Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial bl... more 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.
Micronutrient deficiency is one of the major nutritional problem in the population of developing ... more Micronutrient deficiency is one of the major nutritional problem in the population of developing countries. Rice, a staple food contains very low amount of minerals and vitamins in the consumable milled seeds. In mature rice seed, external aleurone layer and embryo accumulates maximum amount of minerals, which are removed during milling leaving behind very low nutrient content in the edible seeds. Therefore, to increase the nutritional components in consumable rice grains, biotechnological strategy have been employed. Rice ferritin gene (Osfer2) has been cloned and overexpressed in Pusa Sugandhi II under the control of the endosperm specific promoter OsglutelinA2. The resulting transgenic milled seeds of Pusa sugandhi II showed up to 7.8 fold of ferritin overexpression and 2.09 fold of iron increment. In addition, phytic acid functions as antinutrient by chelating divalent metals such as iron, zinc that reduces mineral nutrition in cereal grains. The biosynthesis of phytic acid involves a complex metabolic pathway where myo-inositol phosphate synthase (MIPS) and inositol phosphate kinase-1 (IPK1) play the vital role. Aleurone layer specific downregulation of phytic acid biosynthesis through RNAi mediated silencing of mips and ipk gene expression has been employed for indirect enrichment of mineral in transgenic rice seed endosperms. Moreover, it has been noted that the transgenic plants possess no significant differences in morphological as well as other agronomic characters with those of non-transgenic plants. For accumulation of carotenoids (pro-vitaminA) in rice endosperm, phytoene synthase (psy) gene cloned from rice variety has been used together with phytoene desaturase to transform rice variety where both the genes were driven by endosperm-specific glutalin promoter. We are aiming to develop low phytate, low lipoxygenase rice with enhanced iron, zinc and β-carotene enriched nutrition rice.
Phytic acid (InsP 6) is considered to be the major source of phosphorus and inositol phosphates i... more Phytic acid (InsP 6) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP 6 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 T 4 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.
Plant developmental biology is associated with various gene regulatory pathways involved in diffe... more Plant developmental biology is associated with various gene regulatory pathways involved in different phases of their life cycle. In course of development, growth and differentiation of different organs in plants are regulated by specific sets of gene expression. With the advances in genomic and bioinformatic techniques, particularly high-throughput sequencing technology, many transcriptional units with no protein-coding potential have been discovered. Previously thought to be the dark matters of genome, long non-coding RNAs (lncRNAs) are gradually gaining importance as crucial players in gene regulation during different developmental phases. Some lncRNAs, showing complementarity to microRNAs (miRNAs), are used as endogenous target mimics of specific miRNA family. A number of lncRNAs can also act as natural antisense transcripts to attenuate the expression of coding genes. Although lncRNA-mediated regulations have extensively been studied in animals, plant lncRNA research is still in its initial phase. The present review highlights the regulatory mechanism and different physiological aspects of lncRNAs in plant development. In plants, lncRNAs are found to be associated with a number of plant developmental functions such as lateral root development, vernalization, photomorphogenesis, pollen development, fiber development and nodulation. Understanding these potent roles of lncRNAs in plant development can further provide novel tools for crop improvement programs in future.
bioRxiv (Cold Spring Harbor Laboratory), Mar 27, 2023
Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. Howe... more Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid (β-ODAP) has restricted its cultivation. Although various cultivars with low β-ODAP
bioRxiv (Cold Spring Harbor Laboratory), Apr 27, 2020
While the role of R40 family of lectin proteins in osmotic stress has long been known, their mech... more While the role of R40 family of lectin proteins in osmotic stress has long been known, their mechanism of stress tolerance has not yet been explored. Among them, the expression of Osr40g3 was most strongly induced in response to salt stress. Here, we report that Osr40g3 plays a dual role in regulating salinity tolerance as well as seed development in rice. The overexpression of Osr40g3 under control of a constitutive promoter significantly improved salinity tolerance but impaired seed development in transgenic rice. Remarkably, transgenic Arabidopsis ectopically expressing Osr40g3 exhibited enhanced salinity tolerance but produced seeds normally. It was further confirmed that this Osr40g3-mediated impairment in seed development was due to pollen sterility in the transgenic rice while the pistils remained unaffected. The gene further displayed a precise tissue-specific expression pattern which was essential for proper seed development in rice. The constitutive overexpression led to the interaction of Osr40g3 with OsGF14e in the stamens affecting its protein stability. This interaction further interfered with the gibberellic acid metabolism thus leading to pollen sterility. This interaction was, however, highly specific due to the structural distinctiveness of OsGF14e making the phenomenon rice-specific. Nevertheless, in salinity stressed leaves, Osr40g3 interacted with OsEG45 or its ortholog thus leading to salinity tolerance in both rice and Arabidopsis.
bioRxiv (Cold Spring Harbor Laboratory), Nov 4, 2022
Phosphorus is a macronutrient that regulates a wide range of physiological processes, including p... more Phosphorus is a macronutrient that regulates a wide range of physiological processes, including plant growth and development. The scarcity of bioavailable inorganic phosphate (Pi) is often a constraint for plant growth in most agricultural ecosystems. Under Pi-limited conditions, plants increase Pi acquisition or alter Pi distribution within tissues by activating different Pi transporters and Pi starvation-induced genes. Glutathione (GSH), on the other hand, is known to play a critical role in regulating iron and zinc homeostasis in Arabidopsis. However, how it regulates Pi homeostasis under Pi-limited conditions remains unexplored. We found that the two GSH-deficient mutants, cad2-1, and pad2-1, were hypersensitive to Pi starvation. Under Pi-sufficient condition, the root Pi content was lower in the mutants but significantly higher in the shoot over the Col-0 plants. The pattern was reversed under Pideficient condition. Interestingly, among the different Pi transporters analyzed, the AtPHT1;5 was significantly down-regulated in the two GSH-deficient mutants. Treatment with exogenous GSH could alter Pi accumulation in Col-0 and the pht1;5 complementation lines but not in the pht1;5 mutant. This observation confirmed the role of GSH in Pi reallocation during Pi starvation via the AtPHT1;5 transporter. Further, promoter analysis demonstrated that GSH regulates the transcription of the AtPHT1;5 gene. The Pi-responsive transcription factor (TF), AtWRKY75 was identified to interact with the AtPHT1;5 promoter, and Pi deficiency or GSH treatment enhanced its binding affinity. Together our investigation delineates that GSH regulates the AtPHT1;5 transporter via the AtWRKY75 TF to modulate Pi distribution under altered Pi conditions. .
Several signaling molecules critically regulate the physiological responses in plants. Among them... more Several signaling molecules critically regulate the physiological responses in plants. Among them, miRNAs, generally 21-24 nucleotides long, are widely distributed in different plant species and play as key signaling intermediates in diverse physiological responses. The mature miRNAs are synthesized from MIR genes by RNA polymerase II and processed by Dicerlike (DCL) protein family members associated with some accessory protein molecules. The processed miRNAs are transported to the cytoplasm from the nucleus by specific group of transporters and incorporated into RNA-induced silencing complex (RISC) for specific mRNA cleavage. MicroRNAs can suppress the diverse gene expression, depending on the sequence complementarity of the target transcript except of its own gene. Besides, miRNAs can modulate the gene expression by DNA methylation and translational inhibition of the target transcript. Different classes of DCLs and Argonaute proteins (AGOs) help the miRNAs-mediated gene silencing mechanism in plants.
While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involve... more While glutathione (GSH) is known to regulate iron (Fe) deficiency response in plants, its involvement in modulating subcellular Fe homeostasis remained elusive. In this study, we report that the GSH depleted mutants, cad2-1 and pad2-1 displayed increased sensitivity to Fe deficiency with significant down-regulation of the vacuolar Fe exporters, AtNRAMP3 and AtNRAMP4 and the chloroplast Fe importer, AtPIC1. Moreover, the pad2-1 mutant accumulated higher Fe content in vacuoles and lower in chloroplasts compared with Col-0 under Fe limited condition. Exogenous GSH treatment could enhance the chloroplast Fe content in Col-0 plants but failed to do so in the nramp3nramp4 double mutant demonstrating the role of GSH in modulating subcellular Fe homeostasis. Pharmacological experiments, mutant analysis and promoter assay revealed that this regulation involved transcriptional activation of the transporter genes by a GSH-GSNO module. The Fe responsive bHLH transcription factors (TFs), AtbHLH2...
Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. Howe... more Grass pea seeds are consumed as food in several South Asian and Sub-Saharan African nations. However, the presence of the neurotoxic compound N-oxalyl-L-diamino propionic acid (β-ODAP) has restricted its cultivation. Although various cultivars with low β-ODAP levels have been developed, their cultivation is still limited due to the risk of neurolathyrism from long-term grass pea seed ingestion. In this study, we employed the CRISPR-Cas9-mediated gene editing technique to generate grass pea seeds with zero or minimal β-ODAP levels. We targeted the BAHD-AT3 gene that encodes a key enzyme in the β-ODAP biosynthesis pathway. We developed bahd-at3 knock-out lines using three gRNAs targeting different regions of this gene and characterized them. Cas9-free independent lines from each event carrying the desired on-target mutation were selected and backcrossed twice with the wild-type to eliminate any off-target mutation present therein. Various agronomical parameters were analyzed from the ...
Phosphorus is a macronutrient that regulates a wide range of physiological processes, including p... more Phosphorus is a macronutrient that regulates a wide range of physiological processes, including plant growth and development. The scarcity of bioavailable phosphate is often a constraint for plant growth in most agricultural ecosystems. Under phosphate limited conditions, plants increase phosphate acquisition or alter its distribution within tissues by activating different phosphate transporters and phosphate starvation-induced genes. Glutathione (GSH), on the other hand, is known to play a critical role in regulating iron and zinc homeostasis in Arabidopsis. However, how it regulates phosphate homeostasis under phosphate limited conditions remains unexplored. We found that the two GSH-deficient mutants,cad2-1, andpad2-1, were hypersensitive to phosphate starvation. Under phosphate-sufficient condition, the root phosphate content was lower in the mutants but significantly higher in the shoot over the Col-0 plants. The pattern was reversed under phosphate-deficient condition. Interes...
With the changing climatic conditions, drought has become one of the most threatening abiotic str... more With the changing climatic conditions, drought has become one of the most threatening abiotic stress factors that adversely affect rice cultivation and productivity. Although the involvement of the jacalin domain-containing protein, OsSalT, has been reported in drought and salinity tolerance, its functional mechanism still remains largely unexplored. In this study, expression of the OsSalT gene was found to be positively correlated with the drought tolerance potential with its higher transcript abundance in the tolerant indica rice cultivar, Vandana and lower abundance in the susceptible cultivar, MTU1010. Moreover, the ectopic expression of OsSalT in tobacco imparted drought stress tolerance in the transgenic lines. The transgenic lines exhibited significantly improved growth and higher osmolyte accumulation over the wild-type (WT) plants together with the induction in the OsSalT expression under drought stress. Fascinatingly, the yeast two-hybrid and bimolecular fluorescence compl...
While the role of R40 family of lectin proteins in osmotic stress was already reported, their mec... more While the role of R40 family of lectin proteins in osmotic stress was already reported, their mechanism of stress tolerance still remained elusive. Among them, the expression of Osr40g3 was most strongly induced in response to salinity. Here, we explored the mechanism of Osr40g3-mediated salinity tolerance in rice. The overexpression of Osr40g3 under control of a constitutive promoter significantly improved salinity tolerance but displayed pollen sterility and impaired seed development. The gene further displayed precise tissue-specific expression which was essential for proper seed development in rice. In salinity-stressed leaves, Osr40g3 interacted with OsEG45 leading to salinity tolerance. Overexpression of OsEG45 significantly improved salinity tolerance while the OsEG45 silencing lines displayed severe sensitivity thus confirming its role in imparting salinity tolerance. On the other hand, the constitutive overexpression of Osr40g3 led to its interaction with OsGF14e in stamens...
Vitamin A deficiency (VAD) is the leading cause of blindness among children and is associated wit... more 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.
The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing... more The present study highlights the molecular regulation of iron transport in soyFER1-overexpressing transgenic rice. Accumulation of iron in three different seed developmental stages, milk, dough, and mature, has been examined. The transgenic seeds of the milk stage showed significant augmentation of iron and zinc levels compared with wild-type seeds, and similar results were observed throughout the dough and mature stages. To investigate the regulation of iron transport, the role of miRNAs was studied in roots of transgenic rice. Sequencing of small RNA libraries revealed 153 known and 41 novel miRNAs in roots. Among them, 59 known and 14 novel miRNAs were found to be significantly expressed. miR166, miR399, and miR408 were identified as playing a vital role in iron uptake in roots of transgenic plants. Most importantly, four putative novel miRNAs, namely miR11, miR26, miR30, and miR31, were found to be down-regulated in roots of transgenic plants. For all these four novel miRNAs, natural resistance-associated macrophage protein 4 (NRAMP4), encoding a metal transporter, was predicted as a target gene. It is hypothesized that the NRAMP4 transporter is activated in roots of transgenic plants due to the lower abundance of its corresponding putative novel miRNAs. The relative transcript level of the NRAMP4 transcript was increased from 0.107 in the wild type to 65.24 and 55.39 in transgenic plants, which demonstrates the elevated amount of iron transport in transgenic plants. In addition, up-regulation of OsYSL15, OsFRO2, and OsIRT1 in roots also facilitates iron loading in transgenic seeds.
Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial bl... more 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.
Micronutrient deficiency is one of the major nutritional problem in the population of developing ... more Micronutrient deficiency is one of the major nutritional problem in the population of developing countries. Rice, a staple food contains very low amount of minerals and vitamins in the consumable milled seeds. In mature rice seed, external aleurone layer and embryo accumulates maximum amount of minerals, which are removed during milling leaving behind very low nutrient content in the edible seeds. Therefore, to increase the nutritional components in consumable rice grains, biotechnological strategy have been employed. Rice ferritin gene (Osfer2) has been cloned and overexpressed in Pusa Sugandhi II under the control of the endosperm specific promoter OsglutelinA2. The resulting transgenic milled seeds of Pusa sugandhi II showed up to 7.8 fold of ferritin overexpression and 2.09 fold of iron increment. In addition, phytic acid functions as antinutrient by chelating divalent metals such as iron, zinc that reduces mineral nutrition in cereal grains. The biosynthesis of phytic acid involves a complex metabolic pathway where myo-inositol phosphate synthase (MIPS) and inositol phosphate kinase-1 (IPK1) play the vital role. Aleurone layer specific downregulation of phytic acid biosynthesis through RNAi mediated silencing of mips and ipk gene expression has been employed for indirect enrichment of mineral in transgenic rice seed endosperms. Moreover, it has been noted that the transgenic plants possess no significant differences in morphological as well as other agronomic characters with those of non-transgenic plants. For accumulation of carotenoids (pro-vitaminA) in rice endosperm, phytoene synthase (psy) gene cloned from rice variety has been used together with phytoene desaturase to transform rice variety where both the genes were driven by endosperm-specific glutalin promoter. We are aiming to develop low phytate, low lipoxygenase rice with enhanced iron, zinc and β-carotene enriched nutrition rice.
Phytic acid (InsP 6) is considered to be the major source of phosphorus and inositol phosphates i... more Phytic acid (InsP 6) is considered to be the major source of phosphorus and inositol phosphates in most cereal grains. However, InsP 6 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 T 4 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.
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Papers by Soumitra Paul