Nuclear phosphoproteome of developing chickpea seedlings (Cicer arietinum L.) and protein-kinase interaction network

• Intact nuclei were isolated using two independent methods: HSB and PDG.
• Putative phosphoproteins were enriched by IMAC and resolved by 1-DE.
• Mass spectrometry led to the identification of 107 phosphoproteins.
• This study represents the first generation nuclear phosphoproteome profile of a non-model plant.

Nucleus, the control centre of eukaryotic cell, houses most of the genetic machineries required for gene expression and their regulation. Post translational modifications of proteins, particularly phosphorylation control a wide variety of cellular processes but its functional connectivity, in plants, is still elusive. This study profiled the nuclear phosphoproteome of a grain legume, chickpea, to gain better understanding of such event. Intact nuclei were isolated from 3-week-old seedlings using two independent methods, and nuclear proteins were resolved by 2-DE. In a separate set of experiments, phosphoproteins were enriched using IMAC method and resolved by 1-DE. The separated proteins were stained with phosphospecific Pro-Q Diamond stain. Proteomic analyses led to the identification of 107 putative phosphoproteins, of which 86 were non-redundant. Multiple sites of phosphorylation were predicted on several key elements, which included both regulatory and functional proteins. The analysis revealed an array of phosphoproteins, presumably involved in a variety of cellular functions, viz., protein folding (24%), signalling and gene regulation (22%), DNA replication, repair and modification (16%), and metabolism (13%), among others. These results represent the first nucleus-specific phosphoproteome map of a non-model legume, which would provide insights into the possible function of protein phosphorylation in plants.Biological significanceChickpea is grown over 10 million hectares of land worldwide, and global production hovers around 8.5 million metric tons annually. Despite its nutritional merits, it is often referred to as ‘orphan’ legume and has remained outside the realm of large-scale functional genomics studies. While current chickpea genome initiative has primarily focused on sequence information and functional annotation, proteomics analyses are limited. It is thus important to study the proteome of the cell organelle particularly the nucleus, which harbors most of the genetic information and gene expression machinery. Phosphorylation-dependent modulation of gene expression plays a vital role but the complex networks of phosphorylation are poorly understood. This inventory of nuclear phosphoproteins would provide valuable insights into the dynamic regulation of cellular phenotype through phosphorylation.This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Figure optionsDownload high-quality image (245 K)Download as PowerPoint slide