www.nature.com/scientificreports OPEN First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana Received: 12 January 2017 tabacum Accepted: 25 April 2017 Published: xx xx xxxx 1 1 1 2 2 3 1 Hangjun Sun , Xiaowei Liu , Fangfang Li , Wei Li , Jing Zhang , Zhixin Xiao , Lili Shen , 1 1 1 Ying Li , Fenglong Wang & Jinguang Yang 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. Post-translational modification (PTM) is a covalent modification process resulting from the proteolytic cleavage or addition of a functional group to one amino acid. Thus far, more than 200 PTMs have been characterized (http://www.uniprot.org/help/post-translational_modification). These processes modulate protein functions by altering their localization, activity state and interactions with other proteins. Among all PTMs, lysine acetyla- tion, originally identified in histones , is one of the most studied PTMs. Early studies on lysine acetylation have 2, 3 focused on nuclear proteins, such as histones and transcriptional factors . These studies suggested that lysine 4, 5 acetylation was restricted to the nucleus . The discovery of lysine acetylation on tubulin and mitochondrial proteins suggested an important role for lysine acetylation in cellular biology in addition to chromatin biol- 6–8 ogy . Using high-resolution mass spectrometry, the high abundance of lysine acetylation outside the nucleus has been identified. Lysine acetylation is abundant in most metabolic pathways, such as glycolysis, gluconeogenesis, 9–13 the tricarboxylic acid (TCA) cycle, and conserved in both eukaryotes and prokaryotes . In addition to lysine acetylation, some new types of PTMs, such as malonylation and lysine succinylation, were identified using mass spectrometry combined with the affinity purification of modified peptides using antibodies directed against these 14–20 modifications . Similar to lysine acetylation, lysine malonylation and succinylation are important in regulating 21–24 cellular metabolism, and both processes exist in eukaryotes and prokaryotes . Histone lysine crotonylation has recently been detected from yeast to humans and is primarily associated with active transcription . Similar to histone acetylation, crotonylation also occurs on the ε-amino group of lysine but distinguishes itself from acetylation by its four-carbon length and planar orientation. Lysine crotonylation, but Key Laboratory of Tobacco Pest Monitoring Controlling & Integrated Management, Tobacco Research Institute of Chinese Academy of Agricultural Sciences, Qingdao, 266101, China. Baoshan Branch, Yunnan tobacco company, Baoshan, 678000, China. Hongyunhonghe Tobacco (Group) Co., Ltd., Kunming, 650231, China. Correspondence and requests for materials should be addressed to F.W. (email: email@example.com) or J.Y. (email: yangjinguang@ caas.cn) Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 1 www.nature.com/scientificreports/ 26, 27 not acetylation, preferentially marks “escapee genes” during post-meiotic sex inactivation in mouse testes . Lysine crotonylation and acetylation sites overlap in histones and are catalysed through p300/CBP, a well-known histone acetyltransferase . Moreover, Sirtuin family members SIRT1-3, well-studied histone deacetylases, remove crotonylation in a site-specific manner. SIRT3 is present in both mitochondria and nuclei and is expressed in the kidneys and metabolically active tissues . These studies lead to a question that whether cytoplasmic proteins undergo lysine crotonylation, similar to acetylation, and play an important role in regulating cellular metabolism. Reflecting their sessile feature, plants rapidly change their endogenous status to adapt to adverse environmen- tal conditions. Compared with the regulation of transcription and translation, PTMs could trigger a much faster response, representing a major concern in plant science. However, studies of lysine acylation of the proteome in plant cells have primarily focused on acetylation and succinylation, confirmed in only a limited number of plant 10, 30, 31 11, 32 33 34 35 36 37 38 39 species, including Arabidopsis , rice , wheat , soybean , pea , grape , tomato , potato , strawberry , and Brachypodium distachyon L . Moreover, relatively few proteins have been modified through acetylation or succinylation. In these plants, both lysine acetylation and succinylation have been implicated in the regulation of diverse metabolic processes, such as carbon metabolism, glycolysis, pyruvate metabolism, the TCA cycle, and 33, 37, 40 photosynthesis . Common tobacco (Nicotiana tabacum) is a versatile model organism for fundamental biology research and biotechnology applications . It is the source of the BY-2 plant cell line, which is a key tool for plant molecular research. Moreover, tobacco is also one of the most widely cultivated non-food crops worldwide. In the present study, we investigated the global lysine crotonylation proteome of tobacco using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. In total, we identified 2044 lysine crotonylation sites in 637 proteins. The identified crotonylated proteins, primarily localized to the chloroplast, cytosol, nucleus, and mitochondria, were primarily involved in carbon metabolism, photosynthesis, protein biosynthesis, folding, degradation, and chromatin organization. To our knowledge, this study is the first to describe lysine crotonyla- tion in the global proteome, thereby expanding the current understanding of the effect of lysine crotonylation on nonhistone proteins. Results Detection of lysine-crotonylated proteins in tobacco leaves. To characterize the global crotonyl- ation proteome of tobacco, a proteomic method based on sensitive immune-affinity purification and high-res- olution LC-MS/MS was applied to identify crotonylated proteins and their modification sites in tobacco. An overview of the experimental procedures is shown in Fig. 1a. A total of 2044 lysine crotonylation sites distributed in 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom (Table 1). MS/MS information related to these crotonylated peptides were deposited to iProX database with accession number IPX0000889000 (http://www.iprox.org). Detailed information for all identified croton- ylated peptides and their corresponding proteins was shown in Supplementary Table S1, the scores for protein and peptide identification were shown in Supplementary Table S2. Among the 637 crotonylated proteins, 357 (56%) proteins contained one or two crotonylation sites, and 80 (13%) proteins had 7 or more crotonylation sites (Fig. 1b). Most peptides ranged from 7 to 28 amino acids in length, consistent with the properties of tryptic pep- tides (Fig. 1c). To confirm the validation of the MS data, the mass error of all identified peptides was assessed. The distribution of the mass error was near zero, and most of these proteins were less than 0.02 Da, suggesting that the mass accuracy of the MS data met the requirement (Fig. 1d). Motifs and secondary structures of lysine crotonylated peptides. To evaluate the nature of the crotonylated lysines in tobacco, the sequence motifs in all identified crotonylated peptides were investigated using the Motif-X programme. As shown in Supplementary Table S3, a total of nine conserved motifs were retrieved. Particularly, motifs KcrE, EKcr and KcrD (Kcr indicates the crotonylated lysine) were strikingly conserved (Fig. 2a, Supplementary Table S4). Importantly, the significantly conserved amino acids in these motifs, namely E and D, were both negatively charged, which were rarely identified in other PTMs. These motifs are likely to represent a feature of crotonylation in tobacco. Hierarchical cluster analysis was also performed to further analyse these motifs. As shown in the heat map (Fig. 2b), the enrichment of positively charged K residues was observed in the −10 to −5 and +10 to +5 positions, while negatively charged residues D and E were markedly enriched in the −4 to +4 position. Short aliphatic A residues were frequently observed in the −10 to +10 position, while the sulphur-containing C residue was not observed. To explore the relationship between lysine crotonylation and protein secondary structures, a structural analysis of all crotonylated proteins was performed using the algorithm NetSurfP. As shown in Fig. 2c, approxi- mately 47% of the crotonylated sites were located in α-helices, and 12% of the sites were located in β-strands. The remaining 42% of the crotonylated sites were located in disordered coils. However, considering the similarity of the distribution pattern between crotonylated lysines and all lysines, there was no tendency towards lysine croton- ylation in tobacco. The surface accessibility of the crotonylated lysine sites was also evaluated. The results showed that 91% of the crotonylated lysine sites were exposed to the protein surface, close to that of all lysine residues (Fig. 2d). Therefore, lysine crotonylation likely does not ae ff ct the surface properties of modified proteins. Functional annotation and subcellular localization of crotonylated proteins. To obtain an over- view of the crotonylated proteins in tobacco, the Gene Ontology (GO) functional classification of all crotony - lated proteins based on their biological processes, molecular functions and subcellular locations was investigated (Supplementary Table S5, Supplementary Table S6). Within the biological processes category, the majority of cro- tonylated proteins were related to metabolic processes, cellular processes, and single-organism processes, respec- tively accounting for 36, 27 and 24% of all the crotonylated proteins (Fig. 3a). For the molecular function category, 45 and 40% of the crotonylated proteins were associated with catalytic activity and binding functions, respectively Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 2 www.nature.com/scientificreports/ Figure 1. Proteome-wide identification of lysine crotonylation sites in Nicotiana tabacum . (a) Overview of experimental procedures used in the present study. Kcr indicates the crotonylated lysine. (b) Distribution of lysine crotonylation in one protein. (c) Distribution of lysine crotonylation peptides based on their length. (d) Mass error distribution of all crotonylated peptides. (Fig. 3b). Subcellular localization analysis revealed that most of the crotonylated proteins were localized to the chloroplast (37%), cytosol (30%), nucleus (12%), and mitochondria (5%) (Fig. 3c). Functional enrichment analysis. To better understand the biological function of these crotonylated proteins, we performed an enrichment analysis of the GO (Supplementary Table S7), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (Supplementary Table S8), and Pfam domain databases (Supplementary Table S9). The enrichment analysis of the cellular components revealed that the crotonylated proteins were sig - nificantly enriched in the proteasome complex, thylakoid membrane, and photosystem II oxygen evolving com- plex (Fig. 4a). Based on the enrichment results of the molecular function category, most crotonylated proteins were related to NAD binding, threonine-type peptidase activity, endopeptidase activity, and calcium ion bind- ing (Fig. 4a). In the biological processes category, most of the crotonylated proteins were implicated in oxoacid metabolic processes, protein catabolic processes, cellular amino acid metabolic processes, protein folding, ubiquitin-dependent protein catabolic processes, and photosynthesis (Fig. 4a). The KEGG pathway enrichment Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 3 www.nature.com/scientificreports/ No. of No. of acylated Acylation acylation sites proteins Plant References 91 74 Arabidopsis thaliana 10 699 389 rice 32 416 277 wheat 33 400 245 soybean 34 664 358 pea 35 Lysine acetylation 138 97 grape 36 35 31 potato 38 1392 684 strawberry 39 Brachypodium distachyon 636 353 40 665 261 rice 32 347 202 tomato 37 Lysine succinylation Brachypodium distachyon 605 262 40 Lysine crotonylation 2044 637 tobacco This study Table 1. Comparison of tobacco crotonylation proteome with other published acylation proteome in plants. Figure 2. Properties of the lysine crotonylation sites. (a) Sequence probability logos of significantly enriched crotonylation site motifs around the lysine crotonylation sites. (b) Heat map of the amino acid compositions around the lysine crotonylation sites showing the frequency of different types of amino acids around this residue. Red indicates enrichment and green indicates depletion. (c) Probabilities of lysine crotonylation in different protein secondary structures (alpha helix, beta-strand and disordered coil). (d) Predicted surface accessibility of crotonylation sites. Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 4 www.nature.com/scientificreports/ Figure 3. GO classification of the crotonylated proteins based on biology process (a) molecular functional (b) and subcellular localization (c), respectively. analysis showed that a majority of the crotonylated proteins were related to carbon metabolism, carbon fixation in photosynthetic organisms, pyruvate metabolism, proteasome, amino acid biosynthesis, the citrate cycle, glyc- olysis, porphyrin and chlorophyll metabolism, and photosynthesis (Fig. 4b). Consistent with these observations, Pfam domains, including the NAD(P)-binding domain, ATPase core domain, chlorophyll a/b binding protein domain, aldolase-type TIM barrel, and thioredoxin domain, were significantly enriched in crotonylated proteins (Fig. 4c), implying an important role for lysine crotonylation in these processes. Crotonylated proteins involved in photosynthesis. Notably, 72 crotonylated proteins were implicated in photosynthesis processes, such as light harvesting, the electron transport chain, ATP synthesis and carbon fix- ation (Table 2). Significantly, 73% (8/11) of the enzymes in the Calvin cycle , including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase, tri- ose phosphate isomerase, fructose-1,6-bisphosphate aldolase, fructose-1,6-bisphosphatase, transketolase, and sedoheptulose-1,7-bisphosphatase, were extensively crotonylated at multiple sites. Among these pro- teins, Rubisco and phosphoglycerate kinase were crotonylated at 15 and 16 lysine sites, respectively (Table 2, Supplementary Table S1). According to the annotation in UniProt, the 15 crotonylated lysines in Rubisco were distributed around substrate binding sites (Supplementary Fig. S1a). Strikingly, the catalytic sites K201 and key amino acid residues K201 and K334 were precisely crotonylated. The same phenomenon was also observed on phosphoglycerate kinase, whose substrate-binding site and ATP binding site were surrounded with crotonylated lysines (Supplementary Fig. S1b). These results indicated that lysine crotonylation might change enzyme activ- ity, thereby regulating photosynthesis. Moreover, most of the proteins that participated in the synthesis of chlo- rophyll, including glutamyl-tRNA reductase (HEMA), glutamate-1-semialdehyde 2,1-aminomutase (HEML), 5-aminolevulinate dehydratase (HEMB), uroporphyrinogen III decarboxylase (HEME), coproporphyrinogen III oxidase (HEMF), protoporphyrinogen oxidase (HEMY), magnesium chelatase, magnesium proto IX methyl- transferase (CHLM), Mg-protoporphyrin IX monomethylester cyclase (CRD1), 3,8-Divinyl protochlorophyl- lide a 8-vinyl reductase (DVR), and protochlorophyllide oxidoreductase, were also modified by crotonyl groups (Supplementary Table S1). Crotonylated proteins involved in protein biosynthesis, folding, ubiquitin-dependent degra- dation. A total of 47 crotonylated proteins were identified as ribosomal proteins, translation initiation fac- tors, elongation factors, EF-1-alpha-related GTP-binding proteins and aminoacyl-tRNA synthetases (Table 3, Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 5 www.nature.com/scientificreports/ Figure 4. Enrichment analysis of crotonylated proteins. (a) GO-based enrichment analysis of crotonylated proteins in terms of cellular component, molecular function, and biological process. (b) KEGG pathway-based enrichment analysis. (c) Protein domain enrichment analysis. The numbers in X axes represent the value of significant analysis. When the value is greater than 1.3, the p value is less than 0.05, which means the data is statistically significant. Supplementary Table S1), suggesting that lysine crotonylation may be involved in protein biosynthesis. Several lysine residues of HSP70 (HEAT SHOCK 70 PROTEIN), HSP90, ER-resident molecular chaperone BiP 4 (luminal-binding protein 4), and BiP 5, were moddifie by crotonyl groups (Table 3, Supplementary Table S1). es Th e proteins assist in protein folding to avoid abnormal folding and aggregation. Ubiquitin and related proteins, such as ubiquitin extension protein, ubiquitin-conjugating enzyme, and ubiquitin activating enzyme, were also crotonylated (Table 3). Moreover, 14 proteasome subunits, which participated in ubiquitin-dependent protein degradation, were modified through crotonylation (Table 3). Protein interaction network of the crotonylated proteins in tobacco. To further identify the cel- lular processes regulated through crotonylation in tobacco, the crotonylated protein interaction network was established using an algorithm in Cytoscape software. A total of 264 acetylated proteins were mapped to the protein interaction database (Supplementary Table S10), presenting a global view of the diverse cellular functions of crotonylated proteins in tobacco. As shown in Fig. 5, crotonylated protein involved in ribosome, proteasome, carbon metabolism, oxidative phosphorylation, and terpenoid backbone biosynthesis were retrieved, comprising a dense protein interaction network. The physiological interactions among these crotonylated protein complexes likely contribute to their cooperation and coordination in tobacco. Discussion Histone crotonylation is a new lysine acylation type of PTM enriched at active gene promoters and potential 25 28 enhancers in mammalian cells . Crotonylation is catalysed through histone acetyltransferase p300/CBP , ‘read’ Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 6 www.nature.com/scientificreports/ Protein Protein name Protein Protein name Q40481 Chlorophyll a-b binding protein P27493 Chlorophyll a-b binding protein 21 Q6RUN3 Chlorophyll a-b binding protein P27495 Chlorophyll a-b binding protein 40 Antenna proteins Q0PWS7 Chlorophyll a-b binding protein Q0PWS6 Chlorophyll a-b binding protein Q40512 Chlorophyll a-b binding protein Q84TM7 Chlorophyll a-b binding protein Q0PWS5 Chlorophyll a-b binding protein Q5DNZ6 Chlorophyll a-b binding protein A0A140G1Q8 Photosystem II CP43 reaction center protein P12133 NAD(P)H-quinone oxidoreductase subunit H Q04126 photosystem II oxygen-evolving complex Q40459 Oxygen-evolving enhancer protein 1 Q9SMB4 Photosystem II 22 kDa protein Q7DM39 Oxygen-evolving enhancer protein 2-1 Photosystems II P06411 Photosystem II CP47 reaction center protein P18212 Oxygen-evolving enhancer protein 2-2 complex P69686 Photosystem II D2 protein Q04127 Oxygen-evolving enhancer protein 2-3 Q40519 Photosystem II 10 kDa polypeptide Q5EFR4 oxygen-evolving protein 16 kDa subunit Q84QE8 Oxygen evolving complex Q53UI6 PsbQ P06449 Cytochrome f P06247 Cytochrome b6 Cytochrome b6f complex Q02585 Cytochrome b6-f complex iron-sulfur subunit 2 P06249 Cytochrome b6-f complex subunit 4 Q84QE7 Putative photosystem I subunit III P06405 Photosystem I P700 chlorophyll a apoprotein A1 Q84QE6 Photosystem I reaction center subunit X psaK P06407 Photosystem I P700 chlorophyll a apoprotein A2 Photosystems I complex P62094 Photosystem I iron-sulfur center Q9T2H8 19.3 kDa photosystem I PSAD protein D2K7Z2 Photosystem I reaction center subunit P35477 Plastocyanin B’/B” Ferredoxin–NADP O04397 Ferredoxin–NADP reductase O04977 Ferredoxin–NADP reductase reductase A0A140G1S2 ATP synthase subunit beta P06286 ATP synthase subunit c W8SRJ3 ATP synthase subunit beta P06290 ATP synthase subunit b ATP synthesis complex Q5M9V4 ATP synthase subunit alpha P29790 ATP synthase gamma chain P00823 ATP synthase subunit alpha P32980 ATP synthase delta chain P00834 ATP synthase epsilon chain P00876 Ribulose bisphosphate carboxylase large chain Q006P9 Malic enzyme A0A075M9F5 Ribulose bisphosphate carboxylase small chain A0A077DCL8 Phosphoenolpyruvate carboxykinase Q42961 Phosphoglycerate kinase A0A076KWG2 Malate dehydrogenase P09043 Glyceraldehyde-3-phosphate dehydrogenase A Q9XQP4 NAD-malate dehydrogenase P09044 Glyceraldehyde-3-phosphate dehydrogenase B P27154 Phosphoenolpyruvate carboxylase A0A068JFR6 Triosephosphate isomerase A0A068JCD2 Chloroplast fructose-1,6-bisphosphatase Carbon fixation A0A068JD04 Fructose-bisphosphate aldolase A0A075F1V0 Malate dehydrogenase A0A068JIB0 Fructose-bisphosphate aldolase Q006Q0 Malic enzyme F2VJ75 Fructose-bisphosphate aldolase Q9FSF0 Malate dehydrogenase A0A068JD95 Fructose-1,6-bisphosphatase A0A0K2GP10 Glyceraldehyde-3-phosphate dehydrogenase C3RXI5 Plastid transketolase P09094 Glyceraldehyde-3-phosphate dehydrogenase A0A076KWG9 Chloroplast sedoheptulose-1,7-bisphosphatase Q42962 Phosphoglycerate kinase A0A075EZS4 Glyoxisomal malate dehydrogenase Table 2. Crotonylated proteins involved in photosynthesis pathway. 29, 43–46 by YEATS2 and AF9, ‘erased’ by Sirtuin family members SIRT1-3 in yeast and mammals . However, the lysine crotonylation of nonhistone proteins and in plant cells has not yet been studied. To determine whether lysine crotonylation also exists in plants and to study its function in cellular processes, a global crotonylation tobacco proteome was realized using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine crotonylation sites distributed in 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. These crotonylated proteins were asso- ciated with diverse biological processes, including multiple metabolic pathways, chromatin organization, protein biosynthesis, folding, and degradation. The protein interaction network analysis also suggested that a wide range of interactions involved in these biological processes was likely modulated through protein crotonylation. Carbon is one of the most important macroelements, providing the backbone for biological macromolecules. Lysine acetylation and succinylation in plants have been implicated in carbon metabolism, glycolysis, pyruvate 32, 33, 37, 40 metabolism, TCA cycle, pentose phosphate pathway, glyoxylate and dicarboxylate metabolism . The results of the present study showed that numerous enzymes in these metabolism pathways were also modified through crotonylation. In plants, one of the most important metabolic processes is photosynthesis. In the present study, there are 236 crotonylated proteins were localized to the chloroplast. Among these proteins, a total of 72 proteins were involved in photosynthesis processes. For example, 10, 14, 4, 8, 2, 9, and 25 proteins, identified as members of antenna proteins, photosystems II complex, cytochrome b6f complex, photosystems I complex, ferredoxin-NADP reductase, ATP synthesis complex, and the carbon fixation pathway, respectively. Significantly, 73% (8/11) enzymes in the Calvin cycle were extensively crotonylated at multiple sites, with an average of 10. Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 7 www.nature.com/scientificreports/ Protein Protein name Protein Protein name 30S ribosomal protein S16, P06379 50S ribosomal protein L2, chloroplastic P06374 chloroplastic 30S ribosomal protein S18, O80361 50S ribosomal protein L4, chloroplastic P69660 chloroplastic 30S ribosomal protein S18, O80362 50S ribosomal protein L10, chloroplastic P69660 chloroplastic P06382 50S ribosomal protein L14, chloroplastic P25998 60S ribosomal protein L8 P06386 50S ribosomal protein L20, chloroplastic A0A0D3QSL6 60S ribosomal protein L17 P06391 50S ribosomal protein L23, chloroplastic Q07761 60S ribosomal protein L23a P30956 50S ribosomal protein L28, chloroplastic Q285L8 40S ribosomal protein S3a 40S ribosomal protein S6 Ribosome subunits P30956 50S ribosomal protein L28, chloroplastic P29345 (Fragment) 40S ribosomal protein S17- P02376 30S ribosomal protein S19, chloroplastic A0A077D9P0 like protein P06355 30S ribosomal protein S2, chloroplastic Q6TKQ9 Ribosomal protein L3B P06357 30S ribosomal protein S3, chloroplastic Q6TKR0 Ribosomal protein L3A Ribosomal protein L11-like P06359 30S ribosomal protein S4, chloroplastic Q9FSF6 (Fragment) Cytoplasmic ribosomal P62732 30S ribosomal protein S7, chloroplastic A0A076L4N7 protein S13 P62129 30S ribosomal protein S12, chloroplastic A0A076L2E2 Ribosomal protein S25 P06373 30S ribosomal protein S15, chloroplastic Eukaryotic translation initiation factor Eukaryotic translation Q40554 A0A075EYQ6 3 subunit A initiation factor 5A Eukaryotic translation initiation factor Eukaryotic translation P56821 A0A077D849 3 subunit B initiation factor 5A Translation initiation factors Eukaryotic translation Q40471 Eukaryotic initiation factor 4A-9 A0A075QVP3 initiation factor NCBP-like protein Translation initiation factor A0A075QPA9 Eukaryotic initiation factor iso4E A0A075EYP9 IF1 P93769 Elongation factor 1-alpha Q9FEL2 Elongation factor 2 Q40581 EF-1-alpha-related GTP-binding protein Q9FEL3 Elongation factor 2 Elongation factors A0A077DCL2 Elongation factor 1-delta-like isoform 2 Elongation factor Tu, P68158 chloroplastic P93352 Elongation factor 2 A0A077D7Q3 Cytoplasmic asparagine-tRNA ligase 1 Glutamate–tRNA ligase, Aminoacyl-tRNA synthetases Q43794 chloroplastic/mitochondrial Q9FEL1 Lysyl-tRNA synthetase Q03684 Luminal-binding protein 4 I7GVS5 Heat shock protein 70 Q03685 Luminal-binding protein 5 Q67BD0 Heat shock protein 70-3 Molecular chaperones G9MD86 Heat shock protein 90 P36182 Heat shock protein 82 G9MD87 Heat shock protein 90 Q9ZT13 101 kDa heat shock protein Q14TB1 Heat shock protein 90 Ubiquitin-conjugating enzyme E2 36- Ubiquinol oxidase 2, A0A075F2H4 Q40578 like protein mitochondrial B6A8D0 Ubiquitin Q45FL8 Ubiquitin extension protein Ubiquitin NADH-ubiquinone B6V765 Ubiquitin specific protease 12 Q5M9U1 oxidoreductase chain 6 Ubiquitin activating enzyme O49905 Polyubiquitin Q75VJ8 26S proteasome ATPase regulatory Proteasome subunit alpha Proteasome subunits L7UU40 Q93X34 subunit 6 type Proteasome subunit alpha P93395 Proteasome subunit beta type-6 Q93X35 type Probable 26S proteasome non-ATPase Putative alpha5 proteasome P93768 Q93X37 regulatory subunit 3 subunit Putative alpha4 proteasome Q93X30 Proteasome subunit beta type Q93X38 subunit Proteasome subunits Putative alpha3 proteasome Q93X31 Putative beta5 proteasome subunit Q93X39 subunit Proteasome subunit alpha Q93X32 Putative beta4 proteasome subunit Q9XG77 type-6 Putative preprocysteine Q93X33 Putative beta 3 proteasome subunit Q9XGH8 proteinase Table 3. Crotonylated proteins involved in protein biosynthesis, folding, Ubiquitin-dependent degradation. Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 8 www.nature.com/scientificreports/ Figure 5. Interaction networks of the crotonylated proteins in tobacco. For example, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the key carbon fixation enzyme, was crotonylated at 15 amino acid sites. The key amino acid residues of Rubisco, K201 and K334 which were identified as acetylated resulting in the downregulation of Rubisco activity , also modified through crotonylation. This result suggested that crotonylation might change Rubisco activity in coordination with acetylation. Moreover, the two Rubisco activase isoforms , involved in the light activation of Rubisco, were also crotonylated at 24 sites. Moreover, 67% (10/15) of the enzymes involved in chlorophyll synthesis were also modified through croton- ylation. To our knowledge, until recently, there have been no reports of lysine acylation in chlorophyll metab- olism. These results suggested that lysine crotonylation might play a role in regulating carbon metabolism and photosynthesis. Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 9 www.nature.com/scientificreports/ Proteins are macromolecules that, in addition to carbohydrates, perform a vast array of functions within organisms. Proteins comprise amino acids and are synthesized through translation. In plants, proteins can be degraded in two ways - proteolysis in the vacuole or via the ubiquitin-proteasome system. The data in the present study revealed that lysine crotonylation was related to the synthesis and degradation of multiple amino acids, such as lysine, valine, leucine and isoleucine. The ribosome serves as the factory of protein synthesis. In the present study, we identified 47 crotonylated proteins associated with translation, including 29 ribosome subunits, 8 translation initiation factors, 7 elongation factors, and 3 aminoacyl-tRNA synthetases. After synthesis in the ribosome, the polypeptide chain rapidly folds into its characteristic and functional three-dimensional structure from a random coil. This process is accomplished through the assistance of chaperones, such as the ER-resident 50–55 molecular chaperone BiP, the HSP70 family, and the HSP90 family . The data in the present study showed that lysine residues in members of HSP70 and HSP90 were extensively crotonylated in tobacco. Moreover, Bip 4 and Bip 5 were also extensively modified through crotonylation, suggesting an important role for lysine cro- tonylation in protein folding. If several rounds of chaperone-assisted folding are futile, unfolded or misfolded 56, 57 proteins are recognized and targeted by ubiquitin and subsequently degraded by proteasomes . In the present study, we found ubiquitin, ubiquitin extension protein, ubiquitin-conjugating enzyme, and ubiquitin-activating enzyme, are all modified through crotonylation. Furthermore, 14 proteasome subunits were also crotonylated. es Th e results indicated the likely involvement of lysine crotonylation in regulating protein synthesis, folding, and ubiquitin-dependent degradation. The organization of the eukaryotic genome into nucleosomes dramatically impacts the regulation of gene expression. The structure of the nucleosome core is relatively invariant in eukaryotic organisms, and includes a 147-bp segment of DNA and two copies of each of the four core histone proteins . Histone chaperone nucle- osome assembly protein 1 (Nap1) has been implicated in nucleosome assembly by eliminating competing, nonnucleosomal histone-DNA interactions . The data presented here showed that tobacco histones H1, H2A, H2B, H3, and H4, and nucleosome assembly proteins Nap1;2, Nap1;3, and Nap1;4, were modified through crotonylation, indicating a potential role for lysine crotonylation in nucleosome assembly or disassembly. As complementary evidence, topoisomerase I, required for efficient nucleosome disassembly at gene promoter regions , was also crotonylated in the present study. Nucleosomes are folded through a series of higher-order structures to eventually form a chromosome. An important factor in higher-order organization is the nuclear matrix, which serves as a scaffold for loops of chromatin . Nuclear matrix has been proposed to play a role in regulating transcription, DNA replication, and RNA processing . Chromosomal DNA was anchored to nuclear matrix by its matrix-associated regions (MARs), bound by matrix attachment region-binding protein . Histone acetyltransferase (HAT) p300 and deacetylase SIRT1 interacts with matrix attachment region-binding 64, 65 protein SAF-A and SATB1, respectively, and thereby regulates gene expression . Surprisingly, in the present study, a matrix attachment region binding filament-like protein (MFP1) was identified as crotonylated at 20 amino acid sites, and even its homologue was also crotonylated at 8 amino acid sites. MFP1 is a conserved nuclear and chloroplast DNA-binding protein in plants; however, its physiological function is not under- 66–68 stood . Considering that p300 and SIRT1 possess crotonylation and decrotonylation activities, respectively, 25, 28, 29 in animals , it is an interesting assumption that the crotonylated or decrotonylated form of MFP1 was also associated with the regulation of gene expression. In addition to these crotonylated protein that might be associated with the assembly of nucleosome and chromatin, we identified a G-strand-specific single-stranded telomere-binding protein (GTBP), associated with maintaining telomere stability, also modified through croto- 69, 70 nyl groups . These results indicated the likely involvement of lysine crotonylation in chromatin organization and gene regulation at least in tobacco. In summary, the present study provided the first global lysine crotonylation proteome in tobacco. These data revealed lots of crotonylated proteins associated with diverse aspects of cellular process, particularly car- bon metabolism, photosynthesis, protein biosynthesis, folding, degradation, and chromatin organization. These finding raised some questions that if the crotonylation of these proteins are related to biological functions and that if crotonylation changes in different situations. All these questions should be addressed in the future work. Nevertheless, the results presented here may provide a promising starting point for further functional research of crotonylation in nonhistone proteins. Materials and Methods Plant materials and growth conditions. Tobacco were grown in a greenhouse at 25 °C and a photoper- iod of 16/8 h (light/dark). The leaves were excised from 4-week-old seedlings with three biological replicates and immediately used for protein extraction. Protein Extraction. e s Th amples were grinded to powder in liquid nitrogen, and subsequently mixed with extraction buffer (8 M urea, 2 mM EDTA, 3 μM TSA, 50 mM NAM, 10 mM DTT and 1% Protease Inhibitor Cocktail, Millipore). The remaining debris was removed through centrifugation at 20,000 g for 10 min at 4 °C. Finally, the proteins were precipitated using cold 15% TCA for 2 h at −20 °C. After centrifugation at 4 °C for 10 min, the supernatant was discarded. The remaining precipitate was washed three times with cold acetone. The protein was redissolved in buffer (8 M urea, 100 mM NH CO , pH 8.0) and the protein concentration was deter- 4 3 mined using the 2-D Quant kit (GE Healthcare) according to the manufacturer’s instructions. Trypsin Digestion. For digestion, the protein solution was reduced with 10 mM DTT for 1 h at 37 °C and alkylated with 20 mM IAA for 45 min at room temperature in darkness. For trypsin digestion, the protein sample was diluted aer addin ft g 100 mM NH CO to a urea concentration of less than 2 M. Finally, trypsin was added at 4 3 Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 10 www.nature.com/scientificreports/ 1:50 trypsin-to-protein mass ratio for the first digestion overnight and a 1:100 trypsin-to-protein mass ratio for a second 4-h digestion. HPLC Fractionation. The sample was subsequently fractionated through high pH reverse-phase HPLC using an Agilent 300 Extend C18 column (5 μm particles, 4.6 mm ID, 250 mm length). Briefly, the peptides were separated into 80 fractions using a gradient of 2% to 60% acetonitrile in 10 mM ammonium bicarbonate, pH 10, over 80 min. Subsequently, the peptides were combined into 6 fractions and dried using vacuum centrifugation. Affinity Enrichment. To enrich Kcro peptides, tryptic peptides dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, and 0.5% NP-40, pH 8.0) were incubated with pre-washed antibody beads (PTM Biolabs) at 4 °C overnight with gentle shaking. The beads were washed four times with NETN buffer and twice with ddH O. The bound peptides were eluted from the beads using 0.1% TFA. The eluted fractions were com- bined and vacuum-dried. The resulting peptides were cleaned with C18 ZipTips (Millipore) according to the manufacturer’s instructions, followed by LC-MS/MS analysis. Quantitative Proteomic Analysis by LC-MS/MS. e p Th eptides were dissolved in 0.1% FA and directly loaded onto a reversed-phase pre-column (Acclaim PepMap 100, Thermo Scientific). Peptide separation was performed using a reversed-phase analytical column (Acclaim PepMap RSLC, Thermo Scientific). The gradient comprised an increase from 6% to 22% solvent B (0.1% FA in 98% ACN) for 24 min, 22% to 40% for 8 min and climbing to 80% in 5 min, subsequently holding at 80% for the last 3 min, all at a constant flow rate of 300 nl/min TM on an EASY-nLC 1000 UPLC system, the resulting peptides were analysed using the Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (ThermoFisher Scientific). The peptides were subjected to NSI source TM followed by tandem mass spectrometry (MS/MS) in Q Exactive plus (Thermo) coupled online to the UPLC. Intact peptides were detected in the Orbitrap at a resolution of 70,000. The peptides were selected for MS/MS using NCE setting as 30; ion fragments were detected using Orbitrap at a resolution of 17,500. A data-dependent procedure that alternated between one MS scan followed by 20 MS/MS scans was applied for the top 20 precursor ions above a threshold ion count of 5E3 in the MS survey scan with 15.0 s dynamic exclusion. The electrospray voltage applied was 2.0 kV. Automatic gain control (AGC) was used to prevent overfilling of the Orbitrap; 5E4 ions were accumulated for generation of MS/MS spectra. For MS scans, the m/z scan range was 350 to 1800. Fixed first mass was set as 100 m/z. Database Search. The resulting MS/MS data was processed using MaxQuant with integrated Andromeda search engine (v.188.8.131.52). Tandem mass spectra were searched against UniProt tobacco database concatenated with reverse decoy database. Trypsin/P was specified as cleavage enzyme allowing up to 4 missing cleavages, 5 modifications per peptide and 5 charges. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification and oxidation on Met, cro - tonylation on Lys and crotonylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for protein, peptide and modification sites were specified at 1%. Minimum peptide length was set at 7. All the other parameters in MaxQuant were set to default values. The site localiza- tion probability was set as >0.75. Bioinformatics Methods. Motif-X software (http://motif-x.med.harvard.edu/) was used to analyse the model of sequences constituted with amino acids in specific positions of acetyl-21-mers (10 amino acids upstream and downstream of the site) in all protein sequences . For further hierarchical clustering based on categories, all the acetylation substance categories obtained after enrichment were first collated along with their p -values, and subsequently filtered for those categories at least enriched in one of the clusters with a p -value < 0.05. This filtered p-value matrix was transformed by the function x = −log (p-value), and the x values for each category were z-transformed. These z scores were subsequently clustered using one-way hierarchical clustering (Euclidean distance, average linkage clustering) in the Genesis programme. The cluster membership was visualized using a heat map through the “heatmap.2” function in the “gplot2” R-package. Secondary structures were predicted using NetSurfP. Gene Ontology (GO) annotation proteome was derived from the UniProt-GOA database (http:// www.ebi.ac.uk/GOA/). The proteins were classified using Gene Ontology annotation based on three categories: biological process, cellular component and molecular function. e Th protein subcellular localization was analysed using Wolfpsort (http://www.genscript.com/wolf-psort.html). The KEGG was used to annotate protein pathways. GO term, protein domain, and KEGG pathway enrichment were performed using the DAVID bioinformatics resources 6.7. Fisher’s exact test was used to examine the enrichment or depletion (two-tailed test) of specific annotation terms among members of resulting protein clusters. Correction for multiple hypothesis testing was performed using standard false discovery rate control methods. Any terms with adjusted p-values below 0.05 in any of the clusters were treated as significant. The Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database (http://string-db.org/) was used for PPI analysis. Cytoscape (version 3.0) software was used to display the network . References 1. Allfrey, V. G., Faulkner, R. & Mirsky, A. E. Acetylation and methylation of histones and their possible role in the regulation of rna synthesis. Proceedings of the National Academy of Sciences of the United States of America 51, 786–794 (1964). 2. Gu, W. & Roeder, R. G. Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595–606 (1997). 3. Roth, S. Y., Denu, J. M. & Allis, C. D. Histone acetyltransferases. Annual review of biochemistry 70, 81–120, doi:10.1146/annurev. biochem.70.1.81 (2001). Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 11 www.nature.com/scientificreports/ 4. Lee, K. K. & Workman, J. L. Histone acetyltransferase complexes: one size doesn’t fit all. Nature reviews. Molecular cell biology 8, 284–295, doi:10.1038/nrm2145 (2007). 5. Shahbazian, M. D. & Grunstein, M. Functions of site-specific histone acetylation and deacetylation. Annual review of biochemistry 76, 75–100, doi:10.1146/annurev.biochem.76.052705.162114 (2007). 6. Hubbert, C. et al. HDAC6 is a microtubule-associated deacetylase. Nature 417, 455–458, doi:10.1038/417455a (2002). 7. Onyango, P., Celic, I., McCaffery, J. M., Boeke, J. D. & Feinberg, A. P. SIRT3, a human SIR2 homologue, is an NAD-dependent deacetylase localized to mitochondria. Proceedings of the National Academy of Sciences of the United States of America 99, 13653–13658, doi:10.1073/pnas.222538099 (2002). 8. Schwer, B., North, B. J., Frye, R. A., Ott, M. & Verdin, E. The human silent information regulator (Sir)2 homologue hSIRT3 is a mitochondrial nicotinamide adenine dinucleotide-dependent deacetylase. The Journal of cell biology 158, 647–657, doi:10.1083/ jcb.200205057 (2002). 9. Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science (New York, N.Y.) 325, 834–840, doi:10.1126/science.1175371 (2009). 10. Finkemeier, I., Laxa, M., Miguet, L., Howden, A. J. & Sweetlove, L. J. Proteins of diverse function and subcellular location are lysine acetylated in Arabidopsis. Plant Physiol 155, 1779–1790, doi:10.1104/pp.110.171595 (2011). 11. Nallamilli, B. R. et al. Global analysis of lysine acetylation suggests the involvement of protein acetylation in diverse biological processes in rice (Oryza sativa). PLoS One 9, e89283, doi:10.1371/journal.pone.0089283 (2014). 12. Kim, S. C. et al. Substrate and functional diversity of lysine acetylation revealed by a proteomics survey. Mol Cell 23, 607–618, doi:10.1016/j.molcel.2006.06.026 (2006). 13. Zhao, S. et al. Regulation of cellular metabolism by protein lysine acetylation. Science (New York, N.Y.) 327, 1000–1004, doi:10.1126/ science.1179689 (2010). 14. Peng, C. et al. The first identification of lysine malonylation substrates and its regulatory enzyme. Molecular & cellular proteomics: MCP 10, M111.012658, doi:10.1074/mcp.M111.012658 (2011). 15. Zhang, Z. et al. Identification of lysine succinylation as a new post-translational modification. Nature chemical biology 7, 58–63, doi:10.1038/nchembio.495 (2011). 16. Xie, Z. et al. Lysine succinylation and lysine malonylation in histones. Molecular & cellular proteomics: MCP 11, 100–107, doi:10.1074/mcp.M111.015875 (2012). 17. Park, J. et al. SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways. Mol Cell 50, 919–930, doi:10.1016/j. molcel.2013.06.001 (2013). 18. Weinert, B. T. et al. Lysine succinylation is a frequently occurring modification in prokaryotes and eukaryotes and extensively overlaps with acetylation. Cell reports 4, 842–851, doi:10.1016/j.celrep.2013.07.024 (2013). 19. Xie, L. et al. First succinyl-proteome profiling of extensively drug-resistant Mycobacterium tuberculosis revealed involvement of succinylation in cellular physiology. Journal of proteome research 14, 107–119, doi:10.1021/pr500859a (2015). 20. Pan, J., Chen, R., Li, C., Li, W. & Ye, Z. Global Analysis of Protein Lysine Succinylation Profiles and Their Overlap with Lysine Acetylation in the Marine Bacterium Vibrio parahemolyticus. Journal of proteome research 14, 4309–4318, doi:10.1021/acs. jproteome.5b00485 (2015). 21. Hirschey, M. D. & Zhao, Y. Metabolic Regulation by Lysine Malonylation, Succinylation, and Glutarylation. Molecular & cellular proteomics: MCP 14, 2308–2315, doi:10.1074/mcp.R114.046664 (2015). 22. Colak, G. et al. Proteomic and Biochemical Studies of Lysine Malonylation Suggest Its Malonic Aciduria-associated Regulatory Role in Mitochondrial Function and Fatty Acid Oxidation. Molecular & cellular proteomics: MCP 14, 3056–3071, doi:10.1074/mcp. M115.048850 (2015). 23. Qian, L. et al. Global Profiling of Protein Lysine Malonylation in Escherichia coli Reveals Its Role in Energy Metabolism. Journal of proteome research 15, 2060–2071, doi:10.1021/acs.jproteome.6b00264 (2016). 24. Heiling, S. et al. Using the knowns to discover the unknowns: MS-based dereplication uncovers structural diversity in 17-hydroxygeranyllinalool diterpene glycoside production in the Solanaceae. e P Th lant journal: for cell and molecular biology 85, 561–577, doi:10.1111/tpj.13119 (2016). 25. Tan, M. et al. Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification. Cell 146, 1016–1028, doi:10.1016/j.cell.2011.08.008 (2011). 26. Montellier, E., Rousseaux, S., Zhao, Y. & Khochbin, S. Histone crotonylation specifically marks the haploid male germ cell gene expression program: post-meiotic male-specific gene expression. BioEssays: news and reviews in molecular, cellular and developmental biology 34, 187–193, doi:10.1002/bies.201100141 (2012). 27. Baumann, K. Post-translational modifications: Crotonylation versus acetylation. Nature reviews. Molecular cell biology 16, 265, doi:10.1038/nrm3992 (2015). 28. Sabari, B. R. et al. Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation. Mol Cell 58, 203–215, doi:10.1016/j.molcel.2015.02.029 (2015). 29. Bao, X. et al. Identification of ‘erasers’ for lysine crotonylated histone marks using a chemical proteomics approach. eLife 3, doi:10.7554/eLife.02999 (2014). 30. Wu, X. et al. Lysine acetylation is a widespread protein modification for diverse proteins in Arabidopsis. Plant Physiol 155, 1769–1778, doi:10.1104/pp.110.165852 (2011). 31. Konig, A. C., Hartl, M., Boersema, P. J., Mann, M. & Finkemeier, I. The mitochondrial lysine acetylome of Arabidopsis . Mitochondrion 19(Pt B), 252–260, doi:10.1016/j.mito.2014.03.004 (2014). 32. He, D. et al. Global Proteome Analyses of Lysine Acetylation and Succinylation Reveal the Widespread Involvement of both Modification in Metabolism in the Embryo of Germinating Rice Seed. Journal of proteome research 15, 879–890, doi:10.1021/acs. jproteome.5b00805 (2016). 33. Zhang, Y. et al. Comprehensive profiling of lysine acetylproteome analysis reveals diverse functions of lysine acetylation in common wheat. Scientific reports 6, 21069, doi:10.1038/srep21069 (2016). 34. Smith-Hammond, C. L., Swatek, K. N., Johnston, M. L., Thelen, J. J. & Miernyk, J. A. Initial description of the developing soybean seed protein Lys-N(epsilon)-acetylome. Journal of proteomics 96, 56–66, doi:10.1016/j.jprot.2013.10.038 (2014). 35. Smith-Hammond, C. L., Hoyos, E. & Miernyk, J. A. The pea seedling mitochondrial Nepsilon-lysine acetylome. Mitochondrion 19(Pt B), 154–165, doi:10.1016/j.mito.2014.04.012 (2014). 36. Melo-Braga, M. N. et al. Modulation of protein phosphorylation, N-glycosylation and Lys-acetylation in grape (Vitis vinifera) mesocarp and exocarp owing to Lobesia botrana infection. Molecular & cellular proteomics: MCP 11, 945–956, doi:10.1074/mcp. M112.020214 (2012). 37. Jin, W. & Wu, F. Proteome-Wide Identification of Lysine Succinylation in the Proteins of Tomato (Solanum lycopersicum ). PLoS One 11, e0147586, doi:10.1371/journal.pone.0147586 (2016). 38. Salvato, F. et al. The potato tuber mitochondrial proteome. Plant Physiol 164, 637–653, doi:10.1104/pp.113.229054 (2014). 39. Fang, X. et al. Global analysis of lysine acetylation in strawberry leaves. Front Plant Sci 6, 739, doi:10.3389/fpls.2015.00739 (2015). 40. Zhen, S. et al. First Comprehensive Proteome Analyses of Lysine Acetylation and Succinylation in Seedling Leaves of Brachypodium distachyon L. Scientific reports 6, 31576, doi:10.1038/srep31576 (2016). 41. Sierro, N. et al. The tobacco genome sequence and its comparison with those of tomato and potato. Nature communications 5, 3833, doi:10.1038/ncomms4833 (2014). Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 12 www.nature.com/scientificreports/ 42. Michelet, L. et al. Redox regulation of the Calvin-Benson cycle: something old, something new. Front Plant Sci 4, 470, doi:10.3389/ fpls.2013.00470 (2013). 43. Zhang, Q. et al. Structural Insights into Histone Crotonyl-Lysine Recognition by the AF9 YEATS Domain. Structure (London, England: 1993) 24, 1606–1612, doi:10.1016/j.str.2016.05.023 (2016). 44. Li, Y. et al. Molecular Coupling of Histone Crotonylation and Active Transcription by AF9 YEATS Domain. Mol Cell 62, 181–193, doi:10.1016/j.molcel.2016.03.028 (2016). 45. Zhao, D. et al. YEATS2 is a selective histone crotonylation reader. Cell research 26, 629–632, doi:10.1038/cr.2016.49 (2016). 46. Andrews, F. H. et al. e Th Taf14 YEATS domain is a reader of histone crotonylation. Nature chemical biology 12, 396–398, doi:10.1038/ nchembio.2065 (2016). 47. Gao, X. et al. Downregulation of Rubisco Activity by Non-enzymatic Acetylation of RbcL. Molecular plant 9, 1018–1027 (2016). 48. Henderson, J. N., Hazra, S., Dunkle, A. M., Salvucci, M. E. & Wachter, R. M. Biophysical characterization of higher plant Rubisco activase. Biochimica et biophysica acta 1834, 87–97, doi:10.1016/j.bbapap.2012.09.006 (2013). 49. Nagata, N., Tanaka, R., Satoh, S. & Tanaka, A. Identification of a vinyl reductase gene for chlorophyll synthesis in Arabidopsis thaliana and implications for the evolution of Prochlorococcus species. Plant Cell 17, 233–240, doi:10.1105/tpc.104.027276 (2005). 50. Margaritopoulou, T. et al. HSP90 canonical content organizes a molecular scaffold mechanism to progress flowering. The Plant journal: for cell and molecular biology 87, 174–187, doi:10.1111/tpj.13191 (2016). 51. Tillmann, B. et al. Hsp90 Is Involved in the Regulation of Cytosolic Precursor Protein Abundance in Tomato. Molecular plant 8, 1128, doi:10.1016/j.molp.2015.05.011 (2015). 52. Xu, Z. S. et al. Heat shock protein 90 in plants: molecular mechanisms and roles in stress responses. International journal of molecular sciences 13, 15706–15723, doi:10.3390/ijms131215706 (2012). 53. Yu, A. et al. Roles of Hsp70s in Stress Responses of Microorganisms, Plants, and Animals. Biomed Res Int 2015, 510319, doi:10.1155/2015/510319 (2015). 54. Valente, M. A. et al. The ER luminal binding protein (BiP) mediates an increase in drought tolerance in soybean and delays drought- induced leaf senescence in soybean and tobacco. Journal of experimental botany 60, 533–546, doi:10.1093/jxb/ern296 (2009). 55. Liu, J. X. & Howell, S. H. Managing the protein folding demands in the endoplasmic reticulum of plants. e Th New phytologist 211, 418–428, doi:10.1111/nph.13915 (2016). 56. Liu, Y. & Li, J. Endoplasmic reticulum-mediated protein quality control in Arabidopsis. Front Plant Sci 5, 162, doi:10.3389/ fpls.2014.00162 (2014). 57. Sadanandom, A., Bailey, M., Ewan, R., Lee, J. & Nelis, S. e Th ubiquitin-proteasome system: central modifier of plant signalling. The New phytologist 196, 13–28, doi:10.1111/j.1469-8137.2012.04266.x (2012). 58. Cutter, A. R. & Hayes, J. J. A brief review of nucleosome structure. FEBS letters 589, 2914–2922, doi:10.1016/j.febslet.2015.05.016 (2015). 59. Andrews, A. J., Chen, X., Zevin, A., Stargell, L. A. & Luger, K. The histone chaperone Nap1 promotes nucleosome assembly by eliminating nonnucleosomal histone DNA interactions. Mol Cell 37, 834–842, doi:10.1016/j.molcel.2010.01.037 (2010). 60. Durand-Dubief, M., Persson, J., Norman, U., Hartsuiker, E. & Ekwall, K. Topoisomerase I regulates open chromatin and controls gene expression in vivo. e EMB Th O journal 29, 2126–2134, doi:10.1038/emboj.2010.109 (2010). 61. Bode, J., Benham, C., Knopp, A. & Mielke, C. Transcriptional augmentation: modulation of gene expression by scao ff ld/matrix- attached regions (S/MAR elements). Critical reviews in eukaryotic gene expression 10, 73–90 (2000). 62. Nickerson, J. A., Blencowe, B. J. & Penman, S. The architectural organization of nuclear metabolism. International review of cytology 162a, 67–123 (1995). 63. Cockerill, P. N. & Garrard, W. T. Chromosomal loop anchorage of the kappa immunoglobulin gene occurs next to the enhancer in a region containing topoisomerase II sites. Cell 44, 273–282 (1986). 64. Martens, J. H., Verlaan, M., Kalkhoven, E., Dorsman, J. C. & Zantema, A. Scao ff ld/matrix attachment region elements interact with a p300-scao ff ld attachment factor A complex and are bound by acetylated nucleosomes. Mol Cell Biol 22, 2598–2606 (2002). 65. Xue, Z. et al. SIRT1 deacetylates SATB1 to facilitate MAR HS2-MAR epsilon interaction and promote epsilon-globin expression. Nucleic acids research 40, 4804–4815, doi:10.1093/nar/gks064 (2012). 66. Meier, I., Phelan, T., Gruissem, W., Spiker, S. & Schneider, D. MFP1, a novel plant filament-like protein with affinity for matrix attachment region DNA. Plant Cell 8, 2105–2115, doi:10.1105/tpc.8.11.2105 (1996). 67. Samaniego, R., Jeong, S. Y., Meier, I. & de la Espina, S. M. Dual location of MAR-binding, filament-like protein 1 in Arabidopsis , tobacco, and tomato. Planta 223, 1201–1206, doi:10.1007/s00425-005-0168-x (2006). 68. Harder, P. A., Silverstein, R. A. & Meier, I. Conservation of matrix attachment region-binding filament-like protein 1 among higher plants. Plant Physiol 122, 225–234 (2000). 69. Lee, Y. W. & Kim, W. T. Tobacco GTBP1, a homolog of human heterogeneous nuclear ribonucleoprotein, protects telomeres from aberrant homologous recombination. Plant Cell 22, 2781–2795, doi:10.1105/tpc.110.076778 (2010). 70. Lee, Y. W. & Kim, W. T. Roles of NtGTBP1 in telomere stability. Plant Signal Behav 6, 523–525 (2011). 71. Schwartz, D. & Gygi, S. P. An iterative statistical approach to the identification of protein phosphorylation motifs from large-scale data sets. Nat Biotechnol 23, 1391–1398, doi:10.1038/nbt1146 (2005). 72. Cline, M. S. et al. Integration of biological networks and gene expression data using Cytoscape. Nature protocols 2, 2366–2382, doi:10.1038/nprot.2007.324 (2007). Acknowledgements This work was supported by Shandong Provincial Natural Science Foundation (ZR2015YL065, ZR2014CQ025), State Tobacco Monopoly Bureau (110201601024(LS-04)), Hongyunhonghe Tobacco (Group) Co., Ltd. (HYHH2016YL02), and Yunnan Tobacco Company of China National Tobacco Corporation (2016YL02). Author Contributions J.Y. and F.W. designed research; H.S., X.L., F.L., W.L., J.Z. and Z.X. performed search; J.Y., H.S., L.S. and Y.L. analyzed data; H.S. wrote the paper. Additional Information Supplementary information accompanies this paper at doi:10.1038/s41598-017-03369-6 Competing Interests: The authors declare that they have no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 13 www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2017 Scientific Repo R ts | 7: 3013 | DOI:10.1038/s41598-017-03369-6 14
Scientific Reports – Springer Journals
Published: Jun 7, 2017
It’s your single place to instantly
discover and read the research
that matters to you.
Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.
All for just $49/month
Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly
Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.
Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.
Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.
All the latest content is available, no embargo periods.
“Hi guys, I cannot tell you how much I love this resource. Incredible. I really believe you've hit the nail on the head with this site in regards to solving the research-purchase issue.”Daniel C.
“Whoa! It’s like Spotify but for academic articles.”@Phil_Robichaud
“I must say, @deepdyve is a fabulous solution to the independent researcher's problem of #access to #information.”@deepthiw
“My last article couldn't be possible without the platform @deepdyve that makes journal papers cheaper.”@JoseServera