Browsing by Author "Shashi Pandey Rai"

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An improved thin cell layer culture system for efficient clonal propagation and in vitro withanolide production in a medicinal plant Withania coagulans Dunal
(Elsevier B.V., 2018) Deepika Tripathi; Krishna Kumar Rai; Sanjay Kumar Rai; Shashi Pandey Rai
In vitro micropropagation based on transverse thin cell layer (tTCL), which utilizes a very small number of cells or tissues, has now emerged as a powerful tool in plant tissue differentiation and regeneration. In this study, intense, fast rate shoot multiplication has been achieved utilizing thin cell layer explants for endangered pharmaceutical plant Withania coagulans Dunal (family Solanaceae) having high hypoglycemic potential. To develop an efficient protocol for rapid in vitro propagation of W. coagulans stem node, shoot apical meristem (SAM), and tTCL were used as explants and inoculated in different strength of MS medium. The tTCL explant showed high frequency of shoot regeneration and was also affected by the concentration of plant growth regulators. The full strength solid MS medium was optimal for shoot regeneration (31.94 ± 1.39%) and the highest percent of shoots (93.05 ± 2.77) were observed in MS medium fortified with 2.0 mg l−1 BAP and 0.5 mg l−1 NAA. In contrast to tTCL, SAM cultured on the same medium showed significantly lower multiplication rate with only 1.16 ± 0.08 average shoots per responsive explants. The best rooting of the regenerated shoots was achieved in half strength liquid MS medium supplemented with 2 mg l−1 IBA. Plants were successfully transferred to the field after acclimatization with a survival rate of 90.6%. Monomorphic nature of ISSR and RAPD markers in this study confirmed the genetic fidelity of the in vitro raised tTCL clones. Quantification of withanolides content through high performance liquid chromatography (HPLC) showed 1.4 fold increases in withaferin A content and 1.6 fold withanolide A content in acclimatized field grown plants compared to field grown (wild type) plants. In acclimatized field grown plants the over-expression of SQS gene further attested the improved withanolide production in tTCL micropropagated plants having valuable anti-diabetic potential. The current study reveals the fast rate, cost effective micropropagation and conservation of W. coagulans through tTCL technique for its use as anti-diabetic agent at commercial level. © 2018 Elsevier B.V.
An overview of biotechnological interventions and abiotic elicitors on biomass and withanolide biosynthesis in Withania somnifera (L.) Dunal
(Elsevier B.V., 2023) Lakee Sharma; Bipin Maurya; Shashi Pandey Rai
Withania somnifera is a solanaceous plant species having immense therapeutic potential and commercial values, attributed by pharmacologically active secondary metabolites viz. withanolide and withaferin. The ethanopharmacological use of this plant has been reported since very ancient time and currently, drugs derived from it have been extensively used to cure cancer, sterility, neural and brain disorders. Moreover, withaferin A is extensively used as an anti-cancer agent. Due to extensive harvesting, insufficient withanolide content, and increasing global demand, there is urgent need for in-vitro propagation and conservation techniques in W. somnifera. Biotechnological approaches like micropropagation, hairy root culture in bioreactors and shake flasks, suspension cultures, etc are being used for scaling-up the production of biomass and its pharmaceutical compounds. In addition, abiotic stresses/elicitors also proved to be a promising approach for enhanced synthesis of withanolides. Recently, the emergence of “Omics” era has opened new insights for the engineering of withanolide biosynthetic pathway by metabolic engineering, metabolomics, genetic transformations, gene over-expression, silencing, bioinformatics, and gene mining strategies. However, there are some limitations in genomic data availability, thus there is a strong need of more research for better elucidation of secondary metabolite biosynthesis in W. somnifera. The presented review entails the research works performed for enhanced biomass production and to scale-up the synthesis of withanolides by employing biotechnological tools, abiotic elicitors for growth and enhanced metabolism in W. somnifera. Additionally, this compilation also discusses about update on W. somnifera sequence data in bioinformatic repository sites and their futuristic use for its improvement. © 2023 Elsevier B.V.
An overview on miRNA-encoded peptides in plant biology research
(Academic Press Inc., 2021) Ankita Yadav; Indraneel Sanyal; Shashi Pandey Rai; Charu Lata
MicroRNAs (miRNAs) are short (21–23 nt) regulatory RNA molecules present in plants and animals which are known for regulating the mRNA target gene expression either by cleavage or translational repression. With the advancements in miRNAs research in plants towards their biogenesis and applications has directed the recent discovery of pri-miRNAs encoding functional peptides or microRNA peptides (miPEPs). These miPEPs are encoded by 5′ of pri-miRs containing short ORFs (miORFs). miPEPs are known to enhance the activity of their associated miRNAs by increasing their accumulation and hence downregulating the target genes. Since miPEPs are very specific for each miRNA, they are considered as novel and effective tools for improving traits of interest for plant growth promotion and plant-microbe interaction. Entire peptidome research is the need of the hour. This review thus summarizes recent advancements in miPEP research and its applications as a technology with important agronomical implications with miRNAs augmentation. © 2021 Elsevier Inc.
Arsenic-induced changes in morphological, physiological, and biochemical attributes and artemisinin biosynthesis in Artemisia annua, an antimalarial plant
(2011) Rashmi Rai; Sarita Pandey; Shashi Pandey Rai
Present study is the first to explore physiological, biochemical and molecular changes in the medicinal plant Artemisia annua under arsenic (As) stress. A. annua grown hydroponically in a nutrient solution was spiked with increasing doses of As (0, 1,500, 3,000 and 4,500 μg l -1) for 7 days. Plants accumulated As in a dose dependent manner with bioconcentration factor 13.4 and translocation factor 0.97. While a similar trend of As accumulation was observed under soil culture experiments, the transfer factor went up to 2.1, depicting high efficiency of As translocation from roots to shoots by A. annua. Plants raised in 0-3,000 μg l -1 As containing nutrient solution registered increase in root length, biomass, and carotenoid contents without any visual toxicity symptoms. A dose dependent increase in the activities of enzymes such as superoxide dismutase, ascorbate peroxidase, glutathione reductase and guaiacol peroxidase followed by a gradual decline at higher concentrations suggested their role in alleviating oxidative stress. Significant increase in the levels of thiols, GSH, and pcs gene transcript up to 3,000 μg l -1 As attested their roles in As detoxification. Enhanced artemisinin production (an antimalarial compound) under As stress and upregulation of the transcripts (measured by RT-PCR) of the genes HMGR, FDS, ADS, and CYP71AV1 involved in artemisinin biosynthesis reaffirmed induction of artemisinin biosynthesis in A. annua under As stress. The results of the present study vividly suggested that A. annua has considerable As tolerance, and thus can be successfully cultivated in As contaminated fields. © 2011 Springer Science+Business Media, LLC.
Biotechnological strategies for enhancing heavy metal tolerance in neglected and underutilized legume crops: A comprehensive review
(Academic Press, 2021) Krishna Kumar Rai; Neha Pandey; Ram Prasad Meena; Shashi Pandey Rai
Contamination of agricultural land and water by heavy metals due to rapid industrialization and urbanization including various natural processes have become one of the major constraints to crop growth and productivity. Several studies have reported that to counteract heavy metal stress, plants should be able to maneuver various physiological, biochemical and molecular processes to improve their growth and development under heavy metal stress. With the advent of modern biotechnological tools and techniques it is now possible to tailor legume and other plants overexpressing stress-induced genes, transcription factors, proteins, and metabolites that are directly involved in heavy metal stress tolerance. This review provides an in-depth overview of various biotechnological approaches and/or strategies that can be used for enhancing detoxification of the heavy metals by stimulating phytoremediation processes. Synthetic biology tools involved in the engineering of legume and other crop plants against heavy metal stress tolerance are also discussed herewith some pioneering examples where synthetic biology tools that have been used to modify plants for specific traits. Also, CRISPR based genetic engineering of plants, including their role in modulating the expression of several genes/ transcription factors in the improvement of abiotic stress tolerance and phytoremediation ability using knockdown and knockout strategies has also been critically discussed. © 2020 The Authors
Cultivar specific variations in antioxidative defense system, genome and proteome of two tropical rice cultivars against ambient and elevated ozone
(Academic Press, 2015) Abhijit Sarkar; Aditya Abha Singh; Shashi Bhushan Agrawal; Altaf Ahmad; Shashi Pandey Rai
For the past few decades continuous increase in the levels of tropospheric ozone (O3) concentrations is posing to be a threat for agricultural productivity. Two high yielding tropical rice cultivars (Malviya dhan 36 and Shivani) were evaluated against different concentrations of O3 under field conditions. Experimental design included filtered chambers, non-filtered chambers having ambient O3 and 10 and 20ppb elevated O3 above the ambient. Study was conducted to assess differential response if any in induction of antioxidative defense system, genome stability, leaf proteome, yield and quality of the product in both the test cultivars. Superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), ascorbate peroxidase (APX), and glutathione reductase (GR) were induced under ambient and elevated levels of O3. Native polyacrylamide gel electrophoresis (PAGE) of SOD, CAT and POD also displayed increased enzymatic activity along with associated alterations in specific isoforms. Ascorbic acid, thiols and phenolics were also stimulated at ambient and elevated O3. Structural alterations in DNA of rice plants due to O3 affecting its genome template stability (GTS) was examined using RAPD technique. 2-D PAGE revealed 25 differential spots in Malviya dhan 36 and 36 spots in Shivani after O3 treatment with reductions in RuBisCO subunits. Reductions in yield and change in the quality of grains were also noticed. © 2015 Elsevier Inc.
Downregulation of γECS gene affects antioxidant activity and free radical scavenging system during pod development and maturation in Lablab perpureus L
(Elsevier Ltd, 2017) Krishna Kumar Rai; Nagendra Rai; Shashi Pandey Rai
Fruit development and maturation has been recognized as an oxidative phenomenon, which results in the generation of reactive oxygen species (ROSs). Plants cells contain several antioxidants which help to retained these ROS at low level by indulging them to enter in signalling pathways. The present work describes, the changes occurring in the components of enzymatic antioxidant defence systems during pod development and maturation in two hyacinth bean varieties viz. VRSEM-3 and VRSEM-8, which differ in their pod qualities. The experiment was conducted in randomized block design with three replications and pod samples were collected at the following day post anthesis (DPA) stages: immature (15 DPA), mature (25 DPA) and over mature (40 DPA). Activities of catalase (CAT) and ascorbate peroxidase (APX) reduced up to 1.5/2.0 folds, respectively, during the subsequent stages of pod maturation, indicating a higher accumulation of reactive oxygen species (ROSs) at the latter stages. Glutathione reductase (GR) and superoxide dismutase (SOD) activities decreased progressively during pod development in VRSEM-3 which continued towards the end of maturation as pods turn to the over-mature stage. The role of glutathione-mediated free-radical scavenging was also investigated at transcriptional level, as a plausible system for coping with oxidative stress. The transcript level of γECS (glutamylcysteine synthetase) gene remained relatively constant at the immature and mature stages, whereas it sharply decreased about 15–30 folds at the over-mature stage in both the varieties, which correlated well with the subsequent decrease in the levels of enzymatic antioxidants. Hence, the present study confirmed that the glutathione mediated free radical scavenging system was down-regulated at over-mature stage, conceivably in response to the growing oxidative stress resulting from the inflation of reactive oxygen species, increased solid content and concomitant with a decline in enzymatic and non-enzymatic antioxidants towards the end of maturation. © 2017
Effect of elevated ozone on the antioxidant response, genomic stability, DNA methylation pattern and yield in three species of Abelmoschus having different ploidy levels
(Springer Science and Business Media Deutschland GmbH, 2023) Priyanka Singh; Naushad Ansari; Shashi Pandey Rai; Madhoolika Agrawal; Shashi Bhushan Agrawal
The majority of polyploids can withstand many stresses better than their monoploid counterparts; however, there is no proven mechanism that can fully explain the level of tolerance at the biochemical and molecular levels. Here, we make an effort to provide an explanation for this intriguing but perplexing issue using the antioxidant responses, genomic stability, DNA methylation pattern and yield in relation to ploidy level under the elevated level of ozone in Abelmoschus cytotypes. The outcome of this study inferred that the elevated ozone causes an increase in reactive oxygen species leading to enhanced lipid peroxidation, DNA damage and DNA de-methylation in all the Abelmoschus cytotypes. The monoploid cytotype of Abelmoschus, that is Abelmoschus moschatus L., experienced the highest oxidative stress under elevated O3, resulting in maximum DNA damage and DNA de-methylation leading to the maximum reduction in yield. While the diploid (Abelmoschus esculentus L.) and triploid (Abelmoschus caillei A. Chev.) cytotypes of Abelmoschus with lower oxidative stress result in lesser DNA damage and DNA de-methylation which ultimately leads to lower yield reduction. The result of this experiment explicitly revealed that polyploidy confers better adaptability in the case of Abelmoschus cytotypes under ozone stress. This study can further be used as a base to understand the mechanism behind the ploidy-induced stress tolerance in other plants mediated by gene dosage effect. © 2023, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
Green and cost effective synthesis of silver nanoparticles from endangered medicinal plant Withania coagulans and their potential biomedical properties
(Elsevier Ltd, 2019) Deepika Tripathi; Arusha Modi; Gopeshwar Narayan; Shashi Pandey Rai
Silver nanoparticles (AgNPs) have great potential for their mechanistic role in biomedical researches. Recently, green biosynthetic approaches have been received much attention in plant science for nanoparticles production. Therefore, in the present study AgNPs have been synthesized utilizing in-vitro grown leaf extract of anti-diabetic medicinal plant Withania coagulans Dunal by the reduction of silver nitrate solution. W. coagulans synthesized silver nanoparticles (WcAgNPs) were characterized by UV–visible spectroscopy, scanning electron microscopy, energy dispersive X-ray analysis, transmission electron microscopy, X-ray powder diffraction and Fourier transform Infra-Red spectroscopy. All cumulative results showed that WcAgNPs were ~14 nm in size having spherical shape with face centered cubic structure. High performance liquid chromatography confirmed the involvement of withanolides in AgNPs synthesis as a reducing/capping agent. Synthesized WcAgNPs showed greater antioxidative potential when compared with W. coagulans leaf extract. WcAgNPs have efficient antimicrobial potential and suppresses the growth of both gram positive and gram negative bacteria. In our finding we also observed cytotoxicity of WcAgNPs against SiHa (cervical cancerous, hyper-triploid) cell lines and apoptosis in SiHa cells after 48 hour incubation with 13.74 μg ml −1 (IC 50 ) concentration of WcAgNPs. As results suggested, this is the first report which explain that W. coagulans leaf extract have potential as bio-reducing agent for synthesis of silver nanoparticles, which can be exploited as anti-oxidant, antimicrobial and anti-cancerous agent and depicting an effective way for utilizing bioactive resources in restoration of medicinal properties of this plant with high efficacy. © 2019
Herbal cures for psoriasis, an autoimmune disorder
(Nova Science Publishers, Inc., 2018) Shashi Pandey Rai; Deepika Tripathi; Neha Pandey; Anjana Kumari; Krishna Kumar Rai; Apoorva; Sanjay Kumar Rai
Psoriasis is a persistent, long-lasting (chronic), immune-mediated inflammatory skin problem that changes the life cycle of skin cells There are several factors that contribute to the development and/or aggravation of the disease. The causes of psoriasis pathogenesis are generally a complex interaction among genetic, epigenetic, immunological, and environmental components that involve T cells (T lymphocytes) of the immune system which migrates to the dermis and trigger the release of several cytokines like interleukins (IL), tumor necrosis factor (TNF), and interferons (IF), thereby causing inflammation and rapid production of keratinocytes. Psoriasis also develops due to various stresses like oxidative stress, caused as a result of reactive oxygen species (ROS); psychological stress; and skin injuries. Lipoxygenases (LOXs), a class of enzymes producing inflammatory mediators, are also known to govern psoriasis pathogenesis. Excessive use of certain drugs such as lithium salt, beta blockers and the antimalarial drug chloroquininine can aggravate the disease as well. The bioactive compounds present in medicinal plants have remarkable antioxidant properties required for the protection of the cells against the damaging effects of ROS and LOXs. Thus, they prevent the body against the adverse effects caused by oxidative stress and/or inflammatory responses. These plants are therefore capable of preventing and/or curing pathological conditions of skin and inflammatory diseases. In this chapter, the plant extracts/phytocompounds that have efficient antioxidant, anti-inflammatory, and, immuno-modulatory properties (the significant attributes essential for combating psoriasis), and can be further exploited as potent drug candidates for curing psoriasis were described to prospect them for drug discovery. © 2018 Nova Science Publishers, Inc.
Influence of salicylic acid elicitation on secondary metabolites and biomass production in in-vitro cultured withania coagulans (l.) dunal
(Plant Archives, 2019) Bipin Maurya; Krishna Kumar Rai; Neha Pandey; Lakee Sharma; Niraj Kumar Goswami; Shashi Pandey Rai
Withania coagulans is an important medicinal plant possessing several secondary metabolites collectively termed as withanolides. Salicylic acid is widely known to stimulate plant growth and production under different conditions. Therefore, the present study was conducted to determine how SA regulates secondary metabolites and biomass production in tissue culture raised seedlings of W. coagulans. Among all the four treatments, exogenously supplemented salicylic acid at 150 and 200 μM concentrations exhibited maximum potential to modulate growth and physiological processes of the seedlings compared to 50 and 100 μM treatments. Exogenous application of SA at 150 and 200 μM significantly improved photosynthetic pigment contents, increased biomass, enhanced secondary metabolite contents viz., phenol, proline, and anthocyanin and strengthen antioxidative defense system for scavenging reactive oxygen species. Findings of the present study illustrated that exogenous supplementation of salicylic acid-regulated the mRNA level of genes involved in secondary metabolite biosynthetic pathway thus enhancing the production of the secondary metabolite, photosynthetic efficiency, improved plant growth and increased plant biomass of tissue culture raised seedlings of W. coagulans. © 2019 Plant Archives. All rights reserved.
Interactive role of salicylic acid and nitric oxide on transcriptional reprogramming for high temperature tolerance in lablab purpureus L.: Structural and functional insights using computational approaches
(Elsevier B.V., 2020) Krishna Kumar Rai; Nagendra Rai; Mohd Aamir; Deepika Tripathi; Shashi Pandey Rai
Salicylic acid (SA) and nitric oxide (NO) are considered as putative plant growth regulators that are involved in the regulation of an array of plant's growth and developmental functions under environmental fluctuations when applied at lower concentrations. The possible involvement of NO in SA induced attenuation of high temperature (HT) induced oxidative stress in plants is however, still vague and need to be explored. Therefore, the present study aimed to investigates the biochemical and physiological changes induced by foliar spray of SA and NO combinations to ameliorate HT induced oxidative stress in Lablab purpureus L. Foliar application of combined SA and NO significantly improved relative water content (27.8 %), photosynthetic pigment content (67.2 %), membrane stability (45 %), proline content (1.0 %), expression of enzymatic antioxidants (7.1–18 %) along with pod yield (1.0 %). Heat Shock Factors (HSFs) play crucial roles in plants abiotic stress tolerance, however there structural and functional classifications in L. purpureus L. is still unknown. So, In-silico approach was also used for functional characterization and homology modelling of HSFs in L. purpureus. The experimental findings depicted that combine effect of SA and NO enhances tolerance in HT stressed L. purpureus L. plants by regulating physiological functions, antioxidants, expression and regulation of stress-responsive genes via transcriptional regulation of heat shock factor. © 2020 Elsevier B.V.
Investigating the impact of high temperature on growth and yield of Lablab purpureus L. inbred lines using integrated phenotypical, physiological, biochemical and molecular approaches
(Springer Verlag, 2018) Krishna Kumar Rai; Nagendra Rai; Shashi Pandey Rai
A continuous increase in global mean temperature has become a major challenge for present and future agricultural productivity at worldwide. Therefore, present study was conducted to assess the impact of high temperature (HT) stress on seven Lablab purpureus L. inbred lines along with two parents viz, Arka Vijay and Kashi Khushal as check, to evaluate any genotypic variations exist among inbred lines for HT stress tolerance. The results show that HT induced higher levels of foliar injury in all inbred lines that significantly increased membrane damage, declined photosynthetic pigment contents and redox metabolites which ultimately led to the reduction in their growth and yield. However, among all the inbred lines used, three inbred lines viz, VRBSEM-1, VRBSEM-3 and VRBSEM-15 showed comparatively enhanced enzymatic and non-enzymatic antioxidants with increased secondary metabolites content as well as improved growth and yield related traits, thereby confirming that these inbred lines were efficiently utilizing reactive oxygen species as signalling molecule which regulated their growth and developmental process under HT stress. DNA fingerprinting analysis through ISSR and RAPD markers also established the genetic relationships among the inbred lines that coincides with above physiological and biochemical characterization, and grouped VRBSEM-1 & VRBSEM-3 and VRBSEM-9 & VRBSEM-15 inbred lines into one group and VRBSEM-8, VRBSEM-10, VRBSEM-14 into another group. The experimental findings clearly depict that all the cultivars of L. purpureus L. showed differential response to HT stress. Inbred lines with enhanced biochemical and physiological traits can be crossed to high yielding agronomical elite materials which can help plant breeders in selecting resistant cultivars for the area experiencing high temperature during the cropping season. © 2018, Indian Society for Plant Physiology.
microRNA 166: an evolutionarily conserved stress biomarker in land plants targeting HD-ZIP family
(Springer, 2021) Ankita Yadav; Sanoj Kumar; Rita Verma; Charu Lata; Indraneel Sanyal; Shashi Pandey Rai
MicroRNAs (miRNAs) are significant class of noncoding RNAs having analytical investigating and modulatory roles in various signaling mechanisms in plants related to growth, development and environmental stress. Conserved miRNAs are an affirmation of land plants evolution and adaptation. They are a proof of indispensable roles of endogenous gene modulators that mediate plant survival on land. Out of such conserved miRNA families, is one core miRNA known as miR166 that is highly conserved among land plants. This particular miRNA is known to primarily target HD ZIP-III transcription factors. miR166 has roles in various developmental processes, as well as regulatory roles against biotic and abiotic stresses in major crop plants. Major developmental roles indirectly modulated by miR166 include shoot apical meristem and vascular differentiation, leaf and root development. In terms of abiotic stress, it has decisive regulatory roles under drought, salinity, and temperature along with biotic stress management. miR166 and its target genes are also known for their beneficial synergy with microorganisms in leguminous crops in relation to lateral roots and nodule development. Hence it is important to study the roles of miR166 in different crop plants to understand its defensive roles against environmental stresses and improve plant productivity by reprogramming several gene functions at molecular levels. This review is hence a summary of different regulatory roles of miR166 with its target HD-ZIP III and its modulatory and fine tuning against different environmental stresses in various plants. © 2021, Prof. H.S. Srivastava Foundation for Science and Society.
miRNAs and Plant-Pathogen Interactions
(CRC Press, 2024) Bipin Maurya; Vishnu Mishra; Shashi Pandey Rai
Food security and scarcity are one of the major challenges and main issues of the world. However, agriculture augmentation promotes plant stress via biotic and abiotic stresses. Plant pathogen is imposing serious biotic stresses which impacts severe crop losses including growth, plant yield, and productivity. Globally, 16% of yield is affected due to pathogen stress. MicroRNAs (miRNAs) are a well-characterized group of endogenously produced small interfering RNAs known for regulating their complementary both at the transcriptional and post-translational levels. The roles of miRNAs have been deciphered for their implication in a variety of responses such as immunity, disease resistance, physiology, and development by modulating several molecular and phytohormonal crosstalks during plant-pathogen interactions. Besides miRNAs encoded by the plants, pathogens also modulate host genes by encoding miRNAs to manipulate plant cells and facilitate the entry and hijacking of the plant cell. In this chapter, we summarize the advancement in the area of plant miRNAs, their roles, and the regulation of their respective targets under biotic stresses induced via plant-pathogenic interactions. Here, we also documented the role of plant miRNAs in immunity and disease resistance. The current chapter will provide the advancement in the area of miRNAs mediated gene regulation/silencing during plant-pathogen interactions along with novel techniques such as CRISPR/Cas, target mimic, and resistant target that will help us in the generation of pathogen-resistant crops which could provide immunity against pathogens. © 2025 selection and editorial matter, Peerzada Yasir Yousuf, Peerzada Arshid Shabir, and Khalid Rehman Hakeem; individual chapters, the contributors.
Overexpression of miR166 in Response to Root Rhizobacteria Enhances Drought Adaptive Efficacy by Targeting HD-ZIP III Family Genes in Chickpea
(Springer Science and Business Media Deutschland GmbH, 2024) Ankita Yadav; Sanoj Kumar; Rita Verma; Shiv Narayan; Uma Gupta; Charu Lata; Shashi Pandey Rai; Indraneel Sanyal
Using the transgenic approach, the current study investigated the tripartite interaction of miRNA166, Plant Growth Promoting Rhizobacteria (PGPR), and chickpea crops in response to drought. miR166, an evolutionarily conserved small RNA, was cloned and transformed in a homologous manner. This Car-miR166 is reported in our previous research to have drought-enduring roles in response to microbial candidates. A Pseudomonas putida strain RA (MTCC5279) is used as a PGPR for the whole study. The overexpressed lines generated using tissue-culture practice were functionally validated with physiological parameters studied using Li-Cor 6400XT, including photosynthesis rate, transpiration rate, water-use efficiency, and electron transport rate. We also studied the relative water content of the overexpressed lines in comparison to treated control plants. In biochemical methods, we studied the accumulation of proline, superoxide dismutase, peroxidase, catalase, H2O2 and lipid peroxidation levels. miR166 has its target as ATHB15 (Homeobox-leucine zipper protein-15) validated using 5’ RNA Ligase-Mediated Rapid Amplification of cDNA Ends (RLM-RACE) experiment. At the molecular levels, we carried out the stem-loop quantitative real-time (qRT) PCR analysis of miR166 and the expression analysis of ATHB15 in transgenic lines. As per our study, the results reported that the transgenic lines showed a positive interaction of miR166 with PGPR, resulting in drought stress mitigation and better plant survival in harsh drought conditions. In conclusion, the physiology, biochemistry, and molecular expression levels of Car-miR166 (Cicer arietinum L.) in transgenic lines in response to PGPR support enhanced growth and development in response to PGPR in transgenic lines under drought. © The Author(s) under exclusive licence to Sociedad Chilena de la Ciencia del Suelo 2024.
Overexpression of PGPR responsive chickpea miRNA166 targeting ATHB15 for drought stress mitigation
(Springer Science and Business Media B.V., 2023) Ankita Yadav; Sanoj Kumar; Rita Verma; Shiv Narayan; Ram Jatan; Charu Lata; Shashi Pandey Rai; Pramod A. Shirke; Indraneel Sanyal
Water limitation creates drought-like situations and constrains the life cycle of crop plants by modulating their biological processes at physiological, biochemical, and molecular levels. The microbial measures, including plant growth-promoting rhizobacteria (PGPR), could be used in plant adaptation. These PGPR escape water scarcity conditions and relates to plants by modulating several microRNAs in plant stress responses. The present study relates the beneficial role of PGPR (Pseudomonas putida-RA) responsive Car-miR166 of chickpea in drought mitigation with phytohormonal crosstalk in transgenic Arabidopsis lines. The transgenic lines showed an increased percentage of seed germination in comparison to treated control plants with highest germination rate in T2 (90%) and highest root length was observed in drought treated inoculated T1 lines (29%) under 300 mM of mannitol. The various physiological parameters including photosynthesis rate, transpiration rate, water-use efficiency and stomatal conductance were also better along with lower electrolyte leakage and higher relative water content in treated transgenic lines under inoculated conditions. The biochemical parameters including enzymatic and non-enzymatic antioxidants were improved in transgenic lines with less membrane damage and the highest accumulation of proline in T2 lines under RA inoculation and drought stress in comparison to treated control. The miR166 in drought-treated inoculated plants was highly upregulated (≥ 4) log2 fold change in T3 whereas the target was highly downregulated (≥ -2) log2 fold change in T2. Overall, our results concluded that RA-responsive Car-miR166 plays beneficial stress-mitigating roles under drought in transgenic plants, suggesting its crucial role in crop enhancement in response to PGPR. © 2023, The Author(s), under exclusive licence to Springer Nature B.V.
Plant miRNAs: Biogenesis, Mode of Action, and Their Role
(CRC Press, 2024) Bipin Maurya; Lakee Sharma; Nidhi Rai; Vishnu Mishra; Ashish Kumar; Shashi Pandey Rai
MicroRNA (miRNAs) are a highly conserved, small, non-coding, single-stranded, endogenous class of RNA molecules, which are ~ 21–23 bp long nucleotide sequences mainly present in animals, plants, and some viruses. In the case of plants, miRNAs are well recognized as essential genetic tools for improving crop productivity by regulating various gene expression processes including modification, translational inhibition, or translational repression. The miRNA genes are transcribed by DNA-dependent RNA polymerase II, and the resulting transcript is modified during transcription to form the main transcript. The multiple protein complexes DCL1 (DICER LIKE1), C2H2 Zink finger protein, SERRATE (SE), double-stranded RNA binding protein, and G-patch structural protein (TGH) recognize and cleave these pri-miRNAs, which have partially complementary paired hairpin structures, to produce the precursor of miRNA. The HEN1 protein (methyltransferase), which is essential for the modification and stabilization of double-stranded miRNA, also methylates the miRNA. The advancement of knowledge related to biogenesis of miRNAs and their mode of action has disclosed their involvement in various regulatory processes. There are two well-known vital modes of action of miRNA at the post-transcriptional level, one is transcript degradation and another is translational repression. The 5’ UTR, coding regions, and gene promoters of their target genes have all been observed to interact with miRNA. There are many unanswered problems regarding miRNA synthesis and its method of action, particularly in relation to transcriptional control and other facets of RNA metabolism including splicing. The present book chapter summarizes the current knowledge about miRNA biogenesis and related molecular advancement along with their mode of action in plant morphogenesis processes. © 2025 selection and editorial matter, Peerzada Yasir Yousuf, Peerzada Arshid Shabir, and Khalid Rehman Hakeem; individual chapters, the contributors.
Prediction and validation of DREB transcription factors for salt tolerance in Solanum lycopersicum L.: An integrated experimental and computational approach
(Elsevier B.V., 2019) Krishna Kumar Rai; Nagendra Rai; Shashi Pandey Rai
The dehydration responsive element binding (DREB)transcription factors (TFs)have been intensely reported to regulate plant growth and defence response under stress condition. In this study, we have investigated impact of salt stress (400 mM)at vegetative stage in tomato hybrids on (1)several yield and related components (2)relative water content, membrane stability (3)enzymatic activity and gene expression levels of stress responsive genesand since in tomato, little is known about its functional binding motifs, protein-protein interactions and core amino acid residues involved in the regulation of its expression under stress condition (4)we also used in silico approach to structurally and functionally characterize tomato DREB1 protein in response to salt stress. Salt stress imposed at vegetative stage caused significant reduction in relative water content, chlorophyll content, proline content, expression of stress responsive genes and enhanced membrane damage in all the hybrids. However, hybrids viz., VRTH-1754 and VRTH-1755 showed remarkable tolerance to salt stress as they showed low membrane damage, increased proline content and enhanced activity of antioxidant enzyme along with expression of stress responsive genes. Phylogenetic foot-printing, multiple sequence alignment, and motif analysis of S. lycopersicum DREB1 (SlDREB1)reveal remarkable similarities with its wild homologue showing monophyletic origin with S. pimpinellifolium and close relation with S. pennelli and S. tuberosum. Additionally, DNA-protein interaction study revealed that the SlDREB1 protein binds to Dehydration Responsive Element (DRE)DNA element through conserved KYRG region of AP2/ERF domain with flanking sequences viz., Tyr49, Gly50, Pro51, Cys52, and Arg54.Furthermore, gene ontology (GO)analysis predicted the most significant subcellular localization of SlDREB1 protein to the chloroplast (32.9%)followed by nucleus (28.9%)and cytoplasm (17.6%)revealing that SlDREB1 proteins were mainly involved in ethylene mediated signalling pathway, transcription initiation and defence response. © 2019 Elsevier B.V.
Prospects for Abiotic Stress Tolerance in Crops Utilizing Phyto- and Bio-Stimulants
(Frontiers Media S.A., 2021) Nidhi Rai; Shashi Pandey Rai; Birinchi Kumar Sarma
Environmental stressors such as salinity, drought, high temperature, high rainfall, etc. have already demonstrated the negative impacts on plant growth and development and thereby limiting productivity of the crops. Therefore, in the time to come, more sustainable efforts are required in agricultural practices to ensure food production and security under such adverse environmental conditions. A most promising and eco-friendly way to achieve this goal would be to apply biostimulants to address the environmental concerns. Non-microbial biostimulants such as humic substances (HA), protein hydrolysate, plant-based products and seaweed extracts (SWE), etc. and/or microbial inoculants comprising of plant growth-promoting microbes such as arbuscular mycorrhizal fungi (AMF), fluorescent and non-fluorescent Pseudomonas, Trichoderma spp., Bacillus spp. etc. have tremendous potentiality to enhance plant growth, flowering, crop productivity, nutrient use efficiency (NUE) and translocation, as well as enhancing tolerance to a wide range of abiotic stresses by modifying physiological, biological and biochemical processes of the crop-plants. Similarly, application techniques and timing are also important to achieve the desired results. In this article we discussed the prospects of using seaweed, microbial, and plant-based biostimulants either individually or in combination for managing environmental stresses to achieve food security in a sustainable way. Particular attention was given to the modifications that take place in plant's physiology under adverse environmental conditions and how different biostimulants re-program the host's physiology to withstand such stresses. Additionally, we also discussed how application of biostimulants can overcome the issue of nutrient deficiency in agricultural lands and improve their use efficiency by crop plants. Copyright © 2021 Rai, Rai and Sarma.