Histone crotonylation is a new lysine acylation type of post-translational modification (PTM) enriched at active gene promoters and potential enhancers in yeast and mammalian cells. However, lysine crotonylation in nonhistone proteins and plant cells has not yet been studied. In the present study, we performed a global crotonylation proteome analysis of Nicotiana tabacum (tobacco) using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine modification sites distributed on 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. Similar to lysine acetylation and succinylation in plants, lysine crotonylation was related to multiple metabolism pathways, such as carbon metabolism, the citrate cycle, glycolysis, and the biosynthesis of amino acids. Importantly, 72 proteins participated in multiple processes of photosynthesis, and most of the enzymes involved in chlorophyll synthesis were modified through crotonylation. Numerous crotonylated proteins were implicated in the biosynthesis, folding, and degradation of proteins through the ubiquitin-proteasome system. Several crotonylated proteins related to chromatin organization are also discussed here. These data represent the first report of a global crotonylation proteome and provide a promising starting point for further functional research of crotonylation in nonhistone proteins.

There is increasing evidence that root exudates play important roles in plant disease resistance. Black shank, caused by Phytophthora nicotianae, is a destructive soil-borne disease in tobacco (Nicotiana tabacum L.). The aim of the present study was to investigate the activity and composition of the root exudates from resistant and susceptible tobacco cultivars. The root exudates of the resistant cultivar Gexin 3 showed inhibitory activity against P. nicotianae, while the exudates of susceptible cultivar Xiaohuangjin 1025 stimulated the colony growth but had no effect on spore germination. Metabolic profiling using liquid chromatography/electrospray ionization-quadrupole-time-of-flight mass spectrometry depicted differing metabolic patterns of root exudates between Gexin 3 and Xiaohuangjin 1025. The activity and composition of root exudates was altered by P. nicotianae inoculation. Multivariate analysis showed that root exudates (including organic acids, alkaloids, fatty acids, and esters) were different between the two varieties. The defense substances in root exudates, such as tartaric acid, ferulic acid, and lauric acid, may represent a pre-infection prevention strategy for tobacco. Phenylpropanoids as well as inducers of salicylic acid, fatty acids, 6-hydroxyhexanoic acid, and hydrojasmonate may be involved in tobacco defense responses. Compared to the susceptible cultivar, the roots of the resistant cultivar exhibited high enzyme activities of phenylalanine ammonia-lyase, cinnamate-4-hydroxylase and 4-coumarate-CoA ligase, which may prompt the synthesis and secretion of phenylpropanoids. Our results indicated that the root exudates not only provide a pre-infection prevention strategy by exuding antimicrobial substances, but also increase tobacco disease resistance by eliciting plant defense responses. In addition, some defense compounds as well as compounds that play a role in inducing plant defense responses, showed potential for disease control application. This study provides an insight into possible disease resistance mechanisms of root exudates, and attempts the beneficial utilization of these secondary metabolites of plants.