Molecular studies on the starch-granule-bound protein (R1 protein) and genetic approaches for its inhibition in the potato tubers

Abstract

The potato (Solanum tuberosum L.) is one of the most important food crops capable of nourishing the world’s vast population. Starch is the major component of the dry matter accounting for approximately 70% of the total solids. The other components include total sugars, proteins, fibres, lipids, riboflavin, niacin, vitamin A, vitamin C, minerals, ash and very small amount of glycoalkaloids. Potato plant is an important model crop both in terms of basic and applied research. Starch is synthesized in the amyloplasts of potato tubers. It comprises linear (amylose, approx. 25-30%) and branched (amylopectin, approx. 70-80%) glucose polymers arranged in to highly ordered, semicrystalline structures-the starch granule. Starch phosphorylation is an important aspect of starch metabolism. The level of starch phosphorylation usually varies between the starches from different plant species. Approximately 60-70% of the phosphate groups are linked to C-6 of the glucose residues, the rest to C-3 positions of glucosyl residues, and only 1% may be linked to C-2. In potato tuber starch, approx. 0.1-0.5% of the glucose moieties is found to be phosphorylated. It is commonly accepted that starch phosphorylation influences the overall secondary structures of the starch granule-a prerequisite for its degradation. There is a growing interest on the isolation and characterization of R1 protein i.e. -glucan/glucan-like, water dikinase (GWD) from different plant species. Since GWD is known to catalyze starch phosphorylation both in leaves and different plant storage organs. In the present study, a 4789-bp full-length cDNA (submitted to the NCBI data base under GenBank Acc No EU599037) encoding a GWD isoform was isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR approach using tuber RNA. The predicted protein consisted of 1463 amino acids (designated as KC-GWD) having N-terminal 77-amino acid transit peptide, and 1386-amino acid mature protein shorter by one amino acid as compared to the other mature GWDs from potato and tomato. The mature GWD showed 98% sequence identity with the GWD isolated earlier from the potato cv. Desiree. Variations were found at 25 locations representing mostly non-conservative substitutions. The KC-GWD represents a distinct isoform from potato, as revealed by sequence and phylogenetic analyses. Amino acid composition, segment-wise hydrophobic characters, predicted secondary structures were also analyzed and documented in this report. Broadly, the level of GWD expression as analyzed by semi-quantitative RT-PCR approach was found to be nearly uniform both in the mature tubers and leaves from most of the potato cultivars. By immunodetection technique, a band corresponding to ~155 kDa protein was detected only in the tuber protein extracts. The tuber starch-bound phosphorus content data showed minor variations between the potato cultivars. The storage of potato tubers at low temperatures results in high accumulation of reducing sugars such as glucose and fructose, a phenomenon known as cold-induced sweetening. The overall status of tuber starch phosphorylation and its degradability are implicated in the process of cold sweetening in potato tubers during storage at low temperatures. Therefore, manipulation of R1 (GWD) gene function became necessary in redesigning the potato crop through transgenics. In order to inhibit the R1 gene in potato, the following approaches were adopted in this study. One of the objectives of this work was to use the tuber-specific promoter in making some of the binary genetic constructs. Two partial GBSSI alleles, designated as GKC1 and GKC2 (submitted to the NCBI under the GenBank Acc Nos EU548081 and EU548082, respectively) were isolated from the potato cultivar Kufri Chandramukhi. The 767 bp promoter region of the GKC1 allele (refers to tuber-specific promoter) was used in making a GBSSI-GUS gene fusion, and the resulting binary vector was designated as pAN-GB01. Efforts were made in making a series of both antisense and sense genetic constructs under both constitutive (i.e. CaMV 35S) and tuber-specific (GBSSI) promoters using different regions of the KC-GWD cDNA (more specifically ~1.3 kb towards the 5′-terminus and ~1.2 kb towards the 3′-terminus). The orientation and intactness of the inserts in the individual genetic constructs were further checked through restriction digestion, and PCR using specific primers. Kufri Chipsona-1 (a commercially important potato cultivar) was used during Agrobacterium-mediated genetic transformation. The transgenic potato tubers from the independent transgenic lines corresponding to the individual genetic construct showed variations in the reducing sugar level during storage at low temperatures. It was observed that transgenic potato tubers corresponding to a number of transgenic lines generated by different antisense and sense genetic constructs appeared to be promising as they showed considerably lower accumulation of reducing sugars during storage at 40C. The overall reducing sugar levels of some of the transgenic tubers was found to be in the range of 1.5-2.5 mg g-1 FW which was less as compared to the control potato tubers (3.80-4.90 mg g-1 FW). Interestingly, the reducing sugar level was considerably reduced in a number of transgenic potato tubers corresponding to the different genetic constructs as used in this study, but tuber starch-bound phosphorus content remained more or less unaltered in these cases. However, tuber starch-bound phosphorus content was reduced significantly only in the transgenic lines generated by a few sense genetic constructs. All the data supporting the above observations are clearly and categorically described in the thesis work. Based on the results, at present it is difficult to find an exact correlation between the starch-bound phosphorus content and the extent of reducing sugar level in the cold-stored tubers. Since the earlier conceived notion was that more phosphate content in the starch leads to its more degradability. But the transgenic potato tubers showing lower levels of reducing sugars at low temperatures, also showed lower GWD level (as evident from protein gel blot analysis). Apart from starch phosphorylation, it may be presumed that the R1 protein possibly stimulate the activities of different starch hydrolytic enzymes involved in starch degradation through protein-protein interactions. In fact, more investigation is required for in-depth understanding of GWD functionality. The thesis work is quite relevant with regard to the basic aspects of starch research, and a step forward in terms of redesigning of a commercially important Indian potato cultivar.