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      Chickpea Biofortification for Cytokinin Dehydrogenase via Genome Editing to Enhance Abiotic-Biotic Stress Tolerance and Food Security

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          Abstract

          Globally more than two billion people suffer from micronutrient malnutrition (also known as “hidden hunger”). Further, the pregnant women and children in developing nations are mainly affected by micronutrient deficiencies. One of the most important factors is food insecurity which can be mitigated by improving the nutritional values through biofortification using selective breeding and genetic enhancement techniques. Chickpea is the second most important legume with numerous economic and nutraceutical properties. Therefore, chickpea production needs to be increased from the current level. However, various kind of biotic and abiotic stresses hamper global chickpea production. The emerging popular targets for biofortification in agronomic crops include targeting cytokinin dehydrogenase ( CKX). The CKXs play essential roles in both physiological and developmental processes and directly impact several agronomic parameters i.e., growth, development, and yield. Manipulation of CKX genes using genome editing tools in several crop plants reveal that CKXs are involved in regulation yield, shoot and root growth, and minerals nutrition. Therefore, CKXs have become popular targets for yield improvement, their overexpression and mutants can be directly correlated with the increased yield and tolerance to various stresses. Here, we provide detailed information on the different roles of CKX genes in chickpea. In the end, we discuss the utilization of genome editing tool clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) to engineer CKX genes that can facilitate trait improvement. Overall, recent advancements in CKX and their role in plant growth, stresses and nutrient accumulation are highlighted, which could be used for chickpea improvement.

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          Most cited references121

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          The Medicago Genome Provides Insight into the Evolution of Rhizobial Symbioses

          Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation 1 . Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Mya). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species 2 . Medicago truncatula (Mt) is a long-established model for the study of legume biology. Here we describe the draft sequence of the Mt euchromatin based on a recently completed BAC-assembly supplemented with Illumina-shotgun sequence, together capturing ~94% of all Mt genes. A whole-genome duplication (WGD) approximately 58 Mya played a major role in shaping the Mt genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the Mt genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max (Gm) and Lotus japonicus (Lj). Mt is a close relative of alfalfa (M. sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the Mt genome sequence provides significant opportunities to expand alfalfa’s genomic toolbox.
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            Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity.

            Cytokinins are hormones that regulate cell division and development. As a result of a lack of specific mutants and biochemical tools, it has not been possible to study the consequences of cytokinin deficiency. Cytokinin-deficient plants are expected to yield information about processes in which cytokinins are limiting and that, therefore, they might regulate. We have engineered transgenic Arabidopsis plants that overexpress individually six different members of the cytokinin oxidase/dehydrogenase (AtCKX) gene family and have undertaken a detailed phenotypic analysis. Transgenic plants had increased cytokinin breakdown (30 to 45% of wild-type cytokinin content) and reduced expression of the cytokinin reporter gene ARR5:GUS (beta-glucuronidase). Cytokinin deficiency resulted in diminished activity of the vegetative and floral shoot apical meristems and leaf primordia, indicating an absolute requirement for the hormone. By contrast, cytokinins are negative regulators of root growth and lateral root formation. We show that the increased growth of the primary root is linked to an enhanced meristematic cell number, suggesting that cytokinins control the exit of cells from the root meristem. Different AtCKX-green fluorescent protein fusion proteins were localized to the vacuoles or the endoplasmic reticulum and possibly to the extracellular space, indicating that subcellular compartmentation plays an important role in cytokinin biology. Analyses of promoter:GUS fusion genes showed differential expression of AtCKX genes during plant development, the activity being confined predominantly to zones of active growth. Our results are consistent with the hypothesis that cytokinins have central, but opposite, regulatory functions in root and shoot meristems and indicate that a fine-tuned control of catabolism plays an important role in ensuring the proper regulation of cytokinin functions.
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              Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew.

              Sequence-specific nucleases have been applied to engineer targeted modifications in polyploid genomes, but simultaneous modification of multiple homoeoalleles has not been reported. Here we use transcription activator-like effector nuclease (TALEN) and clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (refs. 4,5) technologies in hexaploid bread wheat to introduce targeted mutations in the three homoeoalleles that encode MILDEW-RESISTANCE LOCUS (MLO) proteins. Genetic redundancy has prevented evaluation of whether mutation of all three MLO alleles in bread wheat might confer resistance to powdery mildew, a trait not found in natural populations. We show that TALEN-induced mutation of all three TaMLO homoeologs in the same plant confers heritable broad-spectrum resistance to powdery mildew. We further use CRISPR-Cas9 technology to generate transgenic wheat plants that carry mutations in the TaMLO-A1 allele. We also demonstrate the feasibility of engineering targeted DNA insertion in bread wheat through nonhomologous end joining of the double-strand breaks caused by TALENs. Our findings provide a methodological framework to improve polyploid crops.
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                Author and article information

                Contributors
                Journal
                Front Genet
                Front Genet
                Front. Genet.
                Frontiers in Genetics
                Frontiers Media S.A.
                1664-8021
                20 May 2022
                2022
                : 13
                : 900324
                Affiliations
                [1] 1 Indian Agricultural Research Institute (ICAR) , New Delhi, India
                [2] 2 Department of Genetics and Plant Breeding , UAS , Bangalore, India
                [3] 3 University School of Biotechnology , Guru Gobind Singh Indraprastha University , New Delhi, India
                [4] 4 NCoE-SAM , Department of Pediatrics , KSCH , Lady Hardinge Medical College , New Delhi, India
                [5] 5 Institute of Himalayan Bioresource Technology (CSIR) , Palampur, India
                [6] 6 Boyce Thompson Institute , Cornell University , Ithaca, NY, United States
                [7] 7 Centre of Food Technology , University of Allahabad , Prayagraj, India
                Author notes

                Edited by: Santosh Kumar Gupta, National Institute of Plant Genome Research (NIPGR), India

                Reviewed by: Surendra Pratap Singh, Chhatrapati Shahu Ji Maharaj University, India

                Satya Prakash, University of Warwick, United Kingdom

                Abhijit Dey, Presidency University, India

                Humira Sonah, Laval University, Canada

                Pranav Pankaj Sahu, Global Change Research Centre (ASCR), Czechia

                *Correspondence: Rajendra Kumar, rajendrak64@ 123456yahoo.co.in

                This article was submitted to Plant Genomics, a section of the journal Frontiers in Genetics

                Article
                900324
                10.3389/fgene.2022.900324
                9164125
                35669196
                61603b8c-7089-4903-84c3-11221d6e183c
                Copyright © 2022 Mahto, Ambika, Singh, Chandana, Singh, Verma, Gahlaut, Manohar, Yadav and Kumar.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 20 March 2022
                : 22 April 2022
                Categories
                Genetics
                Systematic Review

                Genetics
                biofortification,chickpea,cytokinindehydrogenase (ckx),genome-editing,stress
                Genetics
                biofortification, chickpea, cytokinindehydrogenase (ckx), genome-editing, stress

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