Redox homeostasis is tightly regulated for proper cellular activities. Specific protein-protein interactions between redox active molecules such as thioredoxin (Trx) and target proteins constitute the basis for redox-regulated biological processes. The use of cysTMT quantitative proteomics for studying Trx reactions enabled identification of potential Trx targets that provide important insights into the redox regulation in guard cells, a specialized plant cell type responsible for sensing of environmental signals, gas exchange and plant productivity.
Arabidopsis MAP
kinase 4 (MPK4) has been proposed
to be a negative player in plant immunity, and it is also activated
by pathogen-associated molecular patterns (PAMPs), such as flg22.
The molecular mechanisms by which MPK4 is activated and regulates
plant defense remain elusive. In this study, we investigated Arabidopsis defense against a bacterial pathogen Pseudomonas syringae pv tomato (Pst) DC3000
when Brassica napus MPK4 (BnMPK4) is overexpressed. We showed an increase in pathogen resistance
and suppression of jasmonic acid (JA) signaling in the BnMPK4 overexpressing (OE) plants. We also showed that the OE plants have
increased sensitivity to flg22-triggered reactive oxygen species (ROS)
burst in guard cells, which resulted in enhanced stomatal closure
compared to wild-type (WT). During flg22 activation, dynamic phosphorylation
events within and outside of the conserved TEY activation loop were
observed. To elucidate how BnMPK4 functions during
the defense response, we used immunoprecipitation coupled with mass
spectrometry (IP-MS) to identify BnMPK4 interacting
proteins in the absence and presence of flg22. Quantitative proteomic
analysis revealed a shift in the MPK4-associated protein network,
providing insight into the molecular functions of MPK4 at the systems
level.
Activation of mitogen-activated protein kinases (MAPKs) under diverse biotic and abiotic factors and identification of an array of downstream MAPK target proteins are hot topics in plant signal transduction. Through interactions with a plethora of substrate proteins, MAPK cascades regulate many physiological processes in the course of plant growth, development, and response to environmental factors. Identification and quantification of potential MAPK substrates are essential, but have been technically challenging. With the recent advancement in phosphoproteomics, here we describe a method that couples metal dioxide for phosphopeptide enrichment with tandem mass tags (TMT) mass spectrometry (MS) for large-scale MAPK substrate identification and quantification. We have applied this method to a transient expression system carrying a wild type (WT) and a constitutive active (CA) version of a MAPK. This combination of genetically engineered MAPKs and phosphoproteomics provides a high-throughput, unbiased analysis of MAPK-triggered phosphorylation changes on the proteome scale. Therefore, it is a robust method for identifying potential MAPK substrates and should be applicable in the study of other kinase cascades in plants as well as in other organisms.
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