Tuber development in potato (Solanum tuberosum L.): molecular cloning, sequence analyses and expression patterns of some crucial genes

Abstract

Potato (Solanum tuberosum L.) is a major non-grain food crop grown all over the world and belongs to the Solanaceae family. Currently, the major objectives of global potato research include optimization of production by producing resistant varieties towards various challenging biotic and abiotic stresses to ensure both high yield and improvement of the nutritional values. In potato life cycle, tuberization is developmentally-regulated and depends on the activities of several interdependent complex processes. Considerable progress has been made towards understanding the multiple signaling pathways that lead to activation of many proteins/enzymes along with expression of many genes which are crucial at the different stages of tuber development. Tuberization is associated with sucrose, fructose and starch metabolism, calcium (Ca2+) signaling and reactive oxygen species (ROS). Tuberization in potato involves many constitutive and developmentally-regulated genes with multiple forms which still remain unknown. Likewise, an enzyme or protein could exist in different forms in a plant; therefore, we need to understand the corresponding genes/allelic variants and their expression patterns/regulations particularly at different stages of tuber development. Keeping in view, the focus was on molecular and biochemical studies on a few crucial enzymes namely Sucrose synthase (SuSy), Fructokinase (FRK), Calciumdependent protein kinases (CDPK) and Catalase (CAT) in an Indian potato cultivar. In silico approaches were also adopted for sequence analyses. Sucrose synthase (SuSy, EC 2.4.1.13) refers to a glycosyltranferase (GT) that plays a crucial role in sugar metabolism mainly in the sink tissues of plants. Here, we report isolation and characterization of a 2,668-bp cDNA encoding a distinct full-length SuSy4 form, corresponding to SUS I group gene family, from a commercially important Indian potato (Solanum tuberosum L.) cultivar, Kufri Chipsona-1 by RT-PCR using tuber total RNA. The predicted protein, designated as KC-SuSy, consisted of 805 amino acids (protein_id QWW18611). Fructokinase (FRK) (ATP: D-fructose 6-phosphotransferase; EC 2.7.1.4) is a key enzyme in fructose metabolism in the sink organs of plants. In developing potato (Solanum tuberosum L.) tubers, fructose released by sucrose synthase (SuSy)-mediated sucrolytic pathway is converted to fructose-6-phosphate by FRKs. We report isolation of a 1110-bp cDNA clone encoding a 319-aa FRK2 isoform (designated St-FRK2, QIS79145) from tuber total RNA along with two more cDNAs encoding a 256-aa FRK2 variant (QIV66775) and a 266-aa FRK-like protein (designated StFLN, QIV66777) using total RNA from tuber and leaf, respectively. Calcium-dependent protein kinases (CDPK) act as sensor-transducers in decoding the calcium (Ca2+) ions associated with multiple signal transduction pathways. In this study, a 1560-bp cDNA clone encoding 515-aa CDPK2 isoform (designated StCDPK2, QIS79146) was isolated and characterized using tuber total RNA. Catalase (CAT; EC 1.11.1.6) is regarded as one of the major enzymatic antioxidants in plants and plays a crucial role in regulation of growth and stress metabolism. Through RT-PCR using tuber RNA, a 1600-bp cDNA clone was isolated which encoded a novel 492-aa CAT1 isoform designated KC-CAT1 (QWW18612). Apart from the cDNA cloning studies, more members corresponding to the above genes were retrieved from the published reports and other genome databases. Genome wide characterization of the gene families, prediction of the crucial catalytic motifs, chromosomal localization of the genes and phylogenetic analyses were carried out. This thesis work constitutes a comprehensive report providing an insight into the gene families and their classification in potato. Multiple sequence alignments with the catalytic motifs, crucial amino acids and secondary structures were presented; 3-D structures were predicted. Gene-specific expression patterns were shown. Significantly higher level of SuSy, FRK and CAT activities were noticed during the early stages of tuber development implicating their crucial roles in the process of tuberization i,e., from initial swelling of underground stolon to the mature tuber. Protein-protein interaction studies could provide clues of the cross talk between these signalling pathways. Significantly higher expression levels of Susy4, FRK2, CDPK2 and CAT1 genes in the developing tubers indicated that Suc, Fru and starch metabolism, Ca2+ signaling and ROS metabolism are important in the process of tuberization. These results would be quite useful in improving not only the potato crops but the other members of the Solanaceae family as well.