Quantitative Phosphoproteomics Analysis of Nitric Oxide–Responsive Phosphoproteins in Cotton Leaf

Abstract

Knowledge of phosphorylation events and their regulation is crucial to understanding the functional biology of plant proteins, but very little is currently known about nitric oxide–responsive phosphorylation in plants. Here, we report the first large-scale, quantitative phosphoproteome analysis of cotton (Gossypium hirsutum) treated with sodium nitroprusside (nitric oxide donor) by utilizing the isobaric tag for relative and absolute quantitation (iTRAQ) method. A total of 1315 unique phosphopeptides, spanning 1528 non-redundant phosphorylation sites, were detected from 1020 cotton phosphoproteins. Among them, 183 phosphopeptides corresponding to 167 phosphoproteins were found to be differentially phosphorylated in response to sodium nitroprusside. Several of the phosphorylation sites that we identified, including RQxS, DSxE, TxxxxSP and SPxT, have not, to our knowledge, been reported to be protein kinase sites in other species. The phosphoproteins identified are involved in a wide range of cellular processes, including signal transduction, RNA metabolism, intracellular transport and so on. This study reveals unique features of the cotton phosphoproteome and provides new insight into the biochemical pathways that are regulated by nitric oxide.

NO to drought-multifunctional role of nitric oxide in plant drought: Do we have all the answers?

“…Detection and functional analysis of S-nitrosylated proteins is demanding under drought because of its instability and reversibility. A number of S-nitrosylated proteins were identified in A. thaliana, Brassica juncea, Citrus aurantium and Z. mays after stress treatment [85,86]. GSNO detected notably under biotic and abiotic stress conditions is also an important example of S-nitrosylation.…”

“…The K + channel at the guard cell plasma membrane or a closely associated regulatory protein is modified through S-nitrosylation facilitating stomatal closure during drought [56]. NO fumigation of A. toxicaria prevented the inactivation of the antioxidant enzymes by S-nitrosylation, and thus reduced H 2 O 2 levels, thereby increasing desiccation tolerance of seeds [86,87]. It is evident that NO-mediated transcriptomic, …”

Two-Dimensional Gel Electrophoresis-Based Proteomic Analysis Reveals N-terminal Truncation of the Hsc70 Protein in Cotton Fibers In Vivo

“…However, it is limited due to its low sensitivity (usually no more than 2,000 protein spots can be detected) and its low identification rate of less abundant protein spots. In recent years, gel-free high-throughput MS technologies have been widely used instead of 2-DE gels, benefitting from their high sensitivity and high throughput for identifying proteins78910111213. However, 2-DE gel technology can still provide advantages that are not available for gel-free approaches, such as visualization maps of protein profiles, information about the MW and pI of individual protein spots and, most importantly, reliable evidence for existing protein isoforms14.…”

“…These findings supplement transcriptional-level research on cotton fiber development, especially on post-translational modification mechanisms78. Gel-free high-throughput mass spectrometry (MS) approaches have been applied in recent years to identify proteins on a larger scale with higher sensitivity and reveal new aspects of the protein-level regulatory mechanism of cotton fiber development910111213. However, 2-DE technology is irreplaceable because it yields visualization maps of protein profiles, which provide information on the abundance of proteins and reliable evidence for existing protein isoforms14.…”

Recent advances and challenges in plant phosphoproteomics

“…Quantitation is achieved in MS/MS where the tags are released, and the differences among the tag intensities reflect the original quantities of peptides and proteins in different samples . In phosphoproteomics research, these technologies have been extensively used (Table ). However, isobaric tag labeled peptides showed reduced identification efficiency as much as 50% in multistage activation (MSA) MS/MS due to the high charge state induced by the tag in ESI.…”