A New Set of ESTs from Chickpea (Cicer arietinum L.) Embryo Reveals Two Novel F-Box Genes, CarF-box_PP2 and CarF-box_LysM, with Potential Roles in Seed Development

Considering the economic importance of chickpea (C. arietinum L.) seeds, it is important to understand the mechanisms underlying seed development for which a cDNA library was constructed from 6 day old chickpea embryos. A total of 8,186 ESTs were obtained from which 4,048 high quality ESTs were assembled into 1,480 unigenes that majorly encoded genes involved in various metabolic and regulatory pathways. Of these, 95 ESTs were found to be involved in ubiquitination related protein degradation pathways and 12 ESTs coded specifically for putative F-box proteins. Differential transcript accumulation of these putative F-box genes was observed in chickpea tissues as evidenced by quantitative real-time PCR. Further, to explore the role of F-box proteins in chickpea seed development, two F-box genes were selected for molecular characterization. These were named as CarF-box_PP2 and CarF-box_LysM depending on their C-terminal domains, PP2 and LysM, respectively. Their highly conserved structures led us to predict their target substrates. Subcellular localization experiment revealed that CarF-box_PP2 was localized in the cytoplasm and CarF-box_LysM was localized in the nucleus. We demonstrated their physical interactions with SKP1 protein, which validated that they function as F-box proteins in the formation of SCF complexes. Sequence analysis of their promoter regions revealed certain seed specific cis-acting elements that may be regulating their preferential transcript accumulation in the seed. Overall, the study helped in expanding the EST database of chickpea, which was further used to identify two novel F-box genes having a potential role in seed development.