Evolutionary Dynamics Of Genome Size And GC Content In Genus Nicotiana/Genetic Modulation Of Ent-Kaurenoic Acid Oxidase Gene In Nicotiana Tabaccum

Hybridization and Polyploidization is one of the most common phenomena observed in plants especially in the genus Nicotiana leading to the duplication of genome. Our study used cytogenomic approach to unfold the extent of genome size changes and GC contents variation over evolutionary time scale in genus Nicotiana. The genome sizes of three species derived from complete sequence data (17mer) were found15%smaller than our FCM genome size estimates. The tetraploid species in the genus Nicotiana reveals both up and downsizing in their genome sizes. The largest section Suaveolentes reveals genome downsizing of25%to50%from the very small genome of N.simulans (2C=3.28pg) to the large genome of N.suaveolentes (2C=11.88pg) respectively. Whereas the genome sizes of three homoploid hybrids were found in close proximity to their progenitor species (5.30-7.11pg) with the exception of N.noctiflora(9.53pg). Large amount of genome sequence loss appeared in the evolutionary more aged species (>10Myr) as compared to the recently evolved species (<0.2Myr). The GC contents were found homogenous with a difference of only1.31%from ancestral diploid (39.97%) to tetraploids (41.28%) whereas the coefficient of variation between average GC and GC3was also found in the same range. The net results of our study have been that genome size change appeared in either direction in the genus Nicotiana. The GC contents were found more homogenous among the Genus Nicotiana and the slight difference observed from ancestral diploid to tetraploid descendants might possibly arisen from mutational bias. Gibberellin is an important class of growth hormone that plays a pivotal role in plant growth and development. A wide range of mutational, overexpression and gene silencing studies have been carried out to elucidate the metabolic pathway of GA hormone. Most of the genes involved in this pathway have been identified, functionally characterized and their regulatory role has been studied in detail in various plant species. The regulatory and rate limiting role of the middle step of GA biosynthetic pathway involving the three step oxidation of ent-Kaurenoic acid oxidase has not been well understood. The aim of our studies was to clone and functionally characterized KAO gene and to elucidate there regulatory and rate limiting role in GA pathway in Nicotiana tabaccum. To understand this regulation, we modulated the expression of KAO gene of gibberellin pathway in tobacco plants. To evaluate how up-and down regulation of the middle step affects the biosynthetic pathway, we have produced transgenic tobacco plants that either overexpress or silence KAO gene. The overexpression construct carried KAO gene driven by35s promoter whereas silencing was done by knock out suppression using TALEN. Moreover, we studied its effects on the morphological phenotype, downstream gibberellin levels and the expression pattern of endogenous gibberellin metabolism genes. Our results demonstrate the regulatory and rate limiting role of ent-kaurenoic acid oxidase gene in GA biosynthetic pathway.