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Title: Molecular Studies on Some Important Enzymes Involved in Tuberization of an Indian Potato (Solanum tuberosum L.) Cultivar Kufri Chipsona-1
Authors: Kaur, Yadveer
Supervisor: Das, Niranjan
Keywords: Potato (Solanum tuberosum L.) cultivar Kufri Chipsona-1;Field condition;Tuberization;Gibberellin 2-oxidases (GA2oxs);Lipoxygenases (LOXs);Ascorbate peroxidases (APXs);cDNA cloning;Sequence alignment;Phylogenetic analysis;In silico analysis;Protein motifs/domains;3-D structures;Gene expression;Enzyme assay;Protein-protein interactions
Issue Date: 10-Apr-2023
Abstract: Potato, a Solanaceae family member, is a nutritionally rich non-grain food crop cultivated all over the world. It is a promising crop capable to fight both hunger and malnutrition. Potato occupies a distinct position only after the major cereals in terms of fulfilling nutritional requirements/consumer acceptance. Tuberization, a complex developmental morpho-physiological process, is the most sensitive phase of the potato plant life cycle. Multiple genetic, physio-biochemical and environmental factors are known to influence several successive stages of tuberization: stolon initiation and growth, tuber induction and initiation, cell growth and tuber enlargement accompanied by the accumulation of storage biomolecules namely starch and proteins. As evident in the multiple research reports/reviews, the developmental stages of tuberization are associated with the expression of multiple constitutive and organ/cell type-specific genes. Multiple complex signalling and metabolic pathways are known to be involved in this process. Phytohormone signalling, lipoxygenase (LOX) cascade and reactive oxygen species (ROS) metabolism are some examples having significant influence on the tuberization process. Most of the potato cultivars are autotetraploids and shows high levels of heterozygosity. Usually, a particular gene has multiple forms (allelic variants) which could vary with regard to their spatio-temporal nature of expression patterns, and importantly functional attributes. Considerable progress has been made at genetic, molecular and biochemical levels with regard to the process of tuberization in potato. In-depth studies at molecular and biochemical levels are still required to clearly understand this complex process. The thesis work mainly focussed on some genes in an Indian potato cultivar, Kufri Chipsona-1 (KC-1) encoding the crucial enzymes namely Gibberellin 2-oxidases (GA2oxs), Lipoxygenases (LOXs) and Ascorbate peroxidase (APXs). Since these enzymes are associated with some of the crucial metabolic pathways that profoundly influence the process of tuberization in potato. The molecular, biochemical and in silico approaches were adopted in this study. GA2oxs inactivate the bioactive GAs during stolon swelling and early stages of tuberization. Based on RT-PCR approach, a 1105-bp cDNA encoding a 340-aa GA2ox1 form, designated St-GA2ox1, was isolated and characterized using total RNA from growing tubers. In plants, 9-LOXs and 13-LOXs catalyse the oxygenation of polyunsaturated fatty acids to produce fatty acid hydroperoxides which are associated with growth, development and stress responses. In potato, 9-LOX pathway-derived aldehydes and oxoacids are known to promote cell division and enlargement in growing tubers. Here, a 2723-bp cDNA encoding a distinct 861-aa 9-LOX form, designated StKCLX-1, was isolated and characterized from KC-1. In view with cellular defence, reactive oxygen species (ROS) metabolism is an important aspect during tuber development. Ascorbate peroxidases (APXs), a class of the major enzymatic antioxidants, reduce H2O2 into H2O utilizing ascorbate as a specific electron donor in the cellular compartments. We isolated and characterized a 942-bp cDNA clone encoding a cytosolic 250-aa APX isoform (designated StKCAPX1) using tuber total RNA from KC-1. The cDNA sequences were analysed and the crucial motifs/domains were predicted in the corresponding predicted proteins. 3-D structure of each protein was predicted through AlphaFold and iTASSER tools, validated through the predicted local-distance difference test (pLDDT) and Ramachandran Plot. Putative ligand binding sites were predicted by molecular docking. A close inspection of the potato genome database revealed the sizes of the individual gene families: presence of 11 GA2ox genes, 17 LOX genes and 8 APX genes distributed on six, seven and six different chromosomes, respectively. Multiple sequence alignments using several forms of each enzyme revealed the crucial motifs/domains mostly conserved between the Solanaceae family members. Phylogenetic trees were constructed to know the evolutionary relationships between the members of different plant species. Expression patterns at both transcription (semi-quantitative RT-PCR approach) and translation (enzyme assay) levels were examined by both molecular/biochemical and in silico approaches (exploring the Expression Atlas database). GA2ox1 gene was found to be expressed during entire tuberization process (early to mature stage). 9-LOX activity/expression was noticed at early stages of tuberization, and significantly increased in the freshly-harvested mature tubers. Significantly higher APX activity was noticed in tuberising stolon as compared to other stages of tuber development. Expression patterns clearly suggested that GA2ox1, LOX and APX activities are crucial in tuberization. STRING database revealed the prospective protein-protein interactions for each enzyme of the study. Apart from molecular studies on some cDNAs encoding the crucial enzymes namely GA2ox, LOX and APX from an Indian potato cultivar, this is a comprehensive study with regard to the corresponding multigene families in potato. Sequence analysis, prediction of motifs/domains and 3-D structures, molecular docking, expression patterns and enzyme assays are important aspects of this work as most of these attributes were not reported earlier in such a comprehensive manner. Therefore, developing stress-tolerant potato varieties remains one of the thrust areas of potato research. All the data as presented in this report would be useful in developing stress tolerant potato varieties with improved tuber quality and yield through modern and facile molecular techniques/biotechnological means. This work would be useful in gaining insights into the genes and gene products under study, and importantly their importance at various stages of tuber development in potato. Moreover, it would be useful in understanding/predicting structure-function relationships of the GA2oxs, LOXs and APXs in the Solanaceae family members, particularly in potato. Such efforts are required in future for identifying the tuber-expressed important forms of these enzymes-a prerequisite in genetic manipulations and genome editing technologies.
Description: PhD thesis
Appears in Collections:Doctoral Theses@DBT

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