Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Abstract

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.

Proteome and metabolome profiling of cytokinin action in Arabidopsis identifying both distinct and similar responses to cytokinin down- and up-regulation

“…RuBisCO-depleted samples were pooled and extracted by acetone/trichloroacetric acid (TCA) extraction (Damerval et al , 1986). Dried protein was solubilized and separated, essentially as previously described (Lochmanová et al , 2008; Hradilová et al , 2010; Černý et al , 2011 a , b ). Briefly, portions of extracts containing 150 µg of protein were applied to 7cm IPG strips with a non-linear pH gradient (3–10; Bio-Rad, ), isoelectrically focused, then treated with buffers containing dithiothreitol (DTT) and iodoacetamide (Sigma-Aldrich) to reduce and alkylate the proteins, which were subsequently separated by SDS–PAGE.…”

The impact of heat stress targeting on the hormonal and transcriptomic response in Arabidopsis

“…Total protein was extracted and separated as previously described [16]. Briefly, 250-300 mg of frozen material was homogenized in liquid nitrogen and extracted by standard acetone/TCA extraction [17].…”

Proteome Analysis in Arabidopsis Reveals Shoot- and Root-Specific Targets of Cytokinin Action and Differential Regulation of Hormonal Homeostasis

“…CKs and CK primary response genes have been shown to regulate the stability of transporters of another plant hormone, auxin (Pernisová et al, 2009;R u zi cka et al, 2009;Marhavý et al, 2011;Zhang et al, 2011). CKs mediate the feedback regulation of CK signaling via CK-mediated proteolysis of B-type ARABIDOPSIS RESPONSE REGULATOR (ARR), ARR2 (Kim et al, 2012), and changes in the proteome and phosphoproteome have been shown to be part of the fast CK response in Arabidopsis ( Cerný et al, 2011). These findings suggest that posttranscriptional regulatory activity may indeed be a mechanism involved in the contrasting behavior and response of the different plant tissues.…”