Browsing by Author "Upadhyay, Santosh Kumar"

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Compendium of plant-specific CRISPR vectors and their technical advantages
(MDPI AG, 2021) Alok, Anshu; Chauhan, Hanny; Upadhyay, Santosh Kumar; Pandey, Ashutosh; Kumar, Jitendra; Singh, Kashmir
CRISPR/Cas mediated genome editing is a revolutionary approach for manipulating the plant genome. However, the success of this technology is highly dependent on selection of a specific vector and the other components. A plant-specific CRISPR/Cas vector usually consists of a Cas gene, target-specific gRNA, leader sequence, selectable marker gene, precise promoters, and other accessories. It has always been challenging to select the specific vector for each study due to a lack of comprehensive information on CRISPR vectors in one place. Herein, we have discussed every technical aspect of various important elements that will be highly useful in vector selection and efficient editing of the desired plant genome. Various factors such as the promoter regulating the expression of Cas and gRNA, gRNA size, Cas variants, multicistronic gRNA, and vector backbone, etc. influence transformation and editing frequency. For example, the use of polycistronic tRNA-gRNA, and Csy4-gRNA has been documented to enhance the editing efficiency. Similarly, the selection of an efficient selectable marker is also a very important factor. Information on the availability of numerous variants of Cas endonucleases, such as Cas9, Cas12a, Cas12b, Casɸ, and CasMINI, etc., with diverse recognition specificities further broadens the scope of editing. The development of chimeric proteins such as Cas fused to cytosine or adenosine deaminase domain and modified reverse transcriptase using protein engineering enabled base and prime editing, respectively. In addition, the newly discovered Casɸ and CasMINI would increase the scope of genetic engineering in plants by being smaller Cas variants. All advancements would contribute to the development of various tools required for gene editing, targeted gene insertion, transcriptional activation/suppression, multiplexing, prime editing, base editing, and gene tagging. This review will serve as an encyclopedia for plant-specific CRISPR vectors and will be useful for researchers.
Expression of TaNCL2-A ameliorates cadmium toxicity by increasing calcium and enzymatic antioxidants activities in arabidopsis
(Elsevier B.V., 2023) Shumayla; Tyagi, Shivi; Sharma, Yashraaj; Madhu; Sharma, Alok; Pandey, Ashutosh; Singh, Kashmir; Upadhyay, Santosh Kumar
Cadmium (Cd) is a heavy metal that occurs naturally in the environment and is toxic to both animals and plants. The impact of Cd toxicity is shown to be reduced by the exogenous application of calcium (Ca) in crop plants. The sodium/calcium exchanger-like (NCL) protein is involved in Ca enrichment in the cytoplasm by transporting it from the vacuole in the exchange of cytosolic sodium (Na). However, it has not been utilized to ameliorate the Cd toxicity, to date. An elevated expression of TaNCL2-A gene in the root and shoot tissues of bread wheat seedlings, and a higher growth rate of recombinant yeast cells, suggested its role in Cd stress response. The TaNCL2-A expressing transgenic Arabidopsis lines exhibited significant Cd tolerance with increased Ca (∼10-fold) accumulation. The proline content and antioxidant enzymes activities were increased while oxidative stress-related molecules such as H2O2 and MDA were reduced in the transgenic lines. In addition, the growth and yield parameters of transgenic lines such as seed germination rate, root length, leaf biomass, leaf area index, rosette diameter, leaf length and width, and silique count, along with various physiological indicators like chlorophyll, carotenoid, and relative water contents were also improved in comparison to the control plants. Further, the transgenic lines exhibited significant salinity and osmotic stress tolerance, as well. Taken together, these results suggested that the TaNCL2-A could mitigate Cd toxicity along with salinity and osmotic stress. This gene may also be utilized for phytoremediation and Cd sequestration in future studies.
Molecular characterization revealed the role of thaumatin-like proteins of bread wheat in stress response
(Frontiers Media S.A., 2022) Sharma, Alok; Sharma, Himanshu; Rajput, Ruchika; Pandey, Ashutosh; Upadhyay, Santosh Kumar
Thaumatin-like proteins (TLPs) are related to pathogenesis-related-5 (PR-5) family and involved in stress response. Herein, a total of 93 TLP genes were identified in the genome of Triticum aestivum. Further, we identified 26, 27, 39, and 37 TLP genes in the Brachypodium distachyon, Oryza sativa, Sorghum bicolor, and Zea mays genomes for comparative characterization, respectively. They could be grouped into small and long TLPs with conserved thaumatin signature motif. Tightly clustered genes exhibited conserved gene and protein structure. The physicochemical analyses suggested significant differences between small and long TLPs. Evolutionary analyses suggested the role of duplication events and purifying selection in the expansion of the TLP gene family. Expression analyses revealed the possible roles of TLPs in plant development and abiotic and fungal stress response. Recombinant expression of TaTLP2-B in Saccharomyces cerevisiae provided significant tolerance against cold, heat, osmotic, and salt stresses. The results depicted the importance of TLPs in cereal crops that would be highly useful in future crop improvement programs.
Molecular characterization, evolutionary analysis, and expression profiling of BOR genes in important cereals
(MDPI AG, 2022) Sharma, Himanshu; Sharma, Alok; Rajput, Ruchika; Sidhu, Sukhjeet; Dhillon, Harpal; Verma, Praveen Chandra; Pandey, Ashutosh; Upadhyay, Santosh Kumar
Boron (B) is an essential micronutrient of plants. Plants grapple with a narrow range of B between its toxicity and deficiency. B homeostasis mechanism is required to rescue plants from such a quagmire. B transporters are specialized proteins involved in the homeostasis of B. In the present study, a total of 29 BOR genes were identified in five major cereals, including three BORs in each Brachypodium distachyon and Sorghum bicolor, four in Oryza sativa, six in Zea mays, and 13 in Triticum aestivum. Multiple sequence alignments, domain structure analyses, and phylogenetic analysis indicated the conserved nature of the BOR protein family. Duplication events and Ka/Ks analysis of TaBORs showed the role of segmental duplication events and purifying selection in the expansion of the BOR family in T. aestivum. Furthermore, in silico expression and co-expression analyses under biotic and abiotic stress conditions depicted their involvement in combating such conditions. Moreover, qRT-PCR of TaBORs in B treatment suggested the roles of BOR genes in B stress management. The present study hints at the conserved nature of BOR proteins and their different aspects. The study will lay down a way for several crop improvement programs.
TaCCS1-B expression modulates copper, enzymatic antioxidants and polyphenols contents and provides abiotic stress tolerance in transgenic Arabidopsis
(John Wiley & Sons, 2024) Tyagi, Shivi; Shumayla; Singh, Samar; Pandey, Ashutosh; Upadhyay, Santosh Kumar
Abiotic stress, including osmotic and salinity stress, significantly affects plant growth and productivity. Copper chaperone for superoxide dismutase (CCS) is essential for copper homeostasis and oxidative stress management. In this study, we investigated the role of the TaCCS1-B gene of bread wheat in enhancing stress tolerance in yeast and transgenic Arabidopsis. Expression of TaCCS1-B increased abiotic stress tolerance in recombinant yeast cells. Phenotypic analysis of Arabidopsis TaCCS1-B expressing lines demonstrated that they exhibited significantly higher germination rates, increased root length and better growth under osmotic and salinity stress than wild type. Additionally, the transgenic lines exhibited higher copper accumulation and enhanced photosynthetic pigments and proline level, coupled with reduced hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents. They also showed higher enzymatic antioxidants' activities, indicating reduced oxidative stress in transgenic lines, resulting in reduced flavonoid content. Gene expression analysis indicated modulated expression of stress-responsive genes in the transgenic lines under stress conditions. These findings suggested the role of TaCCS1-B in enhancing stress tolerance by improving copper homeostasis and regulating key stress-responsive genes. This study highlights the potential of TaCCS1-B for the development of better stress resilience crops, which is critical for sustaining agricultural productivity for food security under adverse environmental conditions.
TaGPX1-D overexpression provides salinity and osmotic stress tolerance in Arabidopsis
(Elsevier B.V., 2023) Tyagi, Shivi; Shumayla; Sharma, Yashraaj; Madhu; Sharma, Alok; Pandey, Ashutosh; Singh, Kashmir; Upadhyay, Santosh Kumar
Glutathione peroxidases (GPXs) are known to play an essential role in guarding cells against oxidative stress by catalyzing the reduction of hydrogen peroxide and organic hydroperoxides. The current study aims functional characterization of the TaGPX1-D gene of bread wheat (Triticum aestivum) for salinity and osmotic stress tolerance. To achieve this, we initially performed the spot assays of TaGPX1-D expressing yeast cells. The growth of recombinant TaGPX1-D expressing yeast cells was notably higher than the control cells under stress conditions. Later, we generated transgenic Arabidopsis plants expressing the TaGPX1-D gene and investigated their tolerance to various stress conditions. The transgenic plants exhibited improved tolerance to both salinity and osmotic stresses compared to the wild-type plants. The higher germination rates, increased antioxidant enzymes activities, improved chlorophyll, carotenoid, proline and relative water contents, and reduced hydrogen peroxide and MDA levels in the transgenic lines supported the stress tolerance mechanism. Overall, this study demonstrated the role of TaGPX1-D in abiotic stress tolerance, and it can be used for improving the tolerance of crops to environmental stressors, such as salinity and osmotic stress in future research.