Abstract
Nitric oxide (NO) plays an important role in stomata closure induced by environmental stimuli including pathogens. During pathogen challenge, nitric oxide (NO) acts as a second messenger in guard cell signaling networks to activate downstream responses leading to stomata closure. One means by which NO’s action is achieved is through the posttranslational modification of cysteine residue(s) of target proteins. Although the roles of NO have been well studied in plant tissues and seedlings, far less is known about NO signaling and, more specifically, protein S-nitrosylation (SNO) in stomatal guard cells. In this study, using iodoTMTRAQ quantitative proteomics technology, we analyzed changes in protein SNO modification in guard cells of reference plant Arabidopsis thaliana in response to flg22, an elicitor-active peptide derived from bacterial flagellin. A total of 41 SNO-modified peptides corresponding to 35 proteins were identified. The proteins cover a wide range of functions, including energy metabolism, transport, stress response, photosynthesis, and cell–cell communication. This study creates the first inventory of previously unknown NO responsive proteins in guard cell immune responses and establishes a foundation for future research toward understanding the molecular mechanisms and regulatory roles of SNO in stomata immunity against bacterial pathogens.
Highlights
Crop stress due to bacterial pathogens results in sizeable losses in economic revenue annually and threatens global food security [1,2]
Using a double-labeling strategy termed iodoTMTRAQ, in which redox changes such as SNO and protein level changes can be monitored in one experiment, we identified a total of 41 SNO-modified peptides, which corresponded to 35 SNO-responsive proteins, and they were significantly changed in response to flg22 treatment
Gshuoawrd tcheallts 1w0erμeMabfllegt2o2ptrroedatumceenRtOiSndauncdeNd OROwSith(Ftihgeurheig3h)esatnldevNelOs apt r1o5dmucintiuotnes(Faingdur3e0 m4)ininuttehse, reenspriecchteivdeslyto, amftaetralthgeutarredatcmelelsn.tGs.uTahredsecerlelssuwltesrseuagbgleesttothparot dRuOcSe, RNOOS, aanndd/NorOovweirtahlltghueahridghceelsltrleedvoexls at 15 minutes and 30 minutes, respectively, after the treatments. These results suggest that Reactie Oxygen Species (ROS), Nitric oxide (NO), and/or overall guard cell redox state play significant roles in guard cell signaling that leads to stomatal closure
Summary
Crop stress due to bacterial pathogens results in sizeable losses in economic revenue annually and threatens global food security [1,2]. Several of the predicted cysteine(s) were the same as those identified as having the SNO modification Biological processes of these potential redox-regulated proteins include lipid metabolic process, photosynthesis, and nitrogen compound metabolic process (Figure 5). NIT1 and SAHH2, previously reported to undergo protein nitrosylation in Arabidopsis seedlings [31], were identified in guard cells and were oxidized at both early and late stages of stomatal closure. None of these proteins showed significant protein level changes. Analysis of GO terms revealed that the biological processes of the 21 potentially nitrosylated proteins include defense response, transcription, carbohydrate metabolic process, response to ABA, proteolysis, oxidation–reduction process, response to hypoxia, lipid metabolic process, and electron transport, amino acid biosynthetic process (Figure 5)
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