Identification and functional classification of identified CWPs
Totally, 3618 ECWPs were identified in C. sinensis leaves by sequential salt extractions and UPLC-MS/MS. Among them, 501 ECWPs and 3079 ECWPs were considered to be CWPs and intracellular proteins, respectively, via multiple bioinformatics analysis. Notably, intracellular proteins represent 85.1% of ECWPs, indicating ECWPs were subjected to the contamination during ECWPs preparation. Similarity, high contamination of intracellular proteins was also detected d in sugarcane  and rice , accounting for 81.6% and 80.5%, respectively. However, the study of cell wall proteome was still rare in plant species and thus CWPs extraction need be improved. However, our study still led to an enlargement of CWPs and offer new knowledge in C. sinensis in spite of the high contamination of intracellular proteins.
At the same time, 262 N-glycoproteins were identified in the leaves of C. sinensis. As expected, most N-glycoproteins (195, 74.4%) were targeted into the cell wall/extracellular/plasma membrane and thus were assigned as CWPs. The result was in good accordance with that in tomato fruit  and Brachypodium distachyon leaf  which 65% and 60% of N-glycoproteins were reported to be located in the apoplast/cell wall/plasma membrane, respectively, demonstrating that N-glycoproteome is a feasible method to identify and characterize CWPs.
Taken together, 501 CWPs and 195 CWPs were identified by cell wall proteomic and N-glycoproteomic analysis, respectively, and 118 of which was in common. Excitingly, 25 new CWPs being absent in WallProtDB were assigned in this study (Additional file 3: Table S3). The result suggested cell wall proteome is more effective method than N-glycoproteome for CWPs identification. However, it should be noted that the use of N-glycoproteome enhanced CWP identification. As a result, the combined strategy of cell wall proteome and N-glycoproteome should be considered during CWP identification and characterization.
To obtain a global view of the biological processes in which the identified CWPs were involved, 578CWPs were divided into nine functional groups according to their functional domains in this study. Unsurprisingly, PACs (147, 25.4%), Ps (94, 16.3%) and ORs (62, 10.7%) represent top three functional groups. The function distribution of CWPs was in good concordance with that of A. thaliana rosettes and B. distachyon leaves (Additional file 5: Fig. S3). Notably, the proportion of PSs (9.7%) in C. sinensis was obviously higher than that of A. thaliana rosettes and B. distachyon leaves with 3.7% and 4.0%, respectively [12, 20], which maybe account for the long lifecycle of the evergreen leaf in C. sinensis.
Possible roles of identified CWPs
Glycoside hydrolases (GHs). PACs constitute the first largest functional class, with GHs as the major representatives (Table 1). 110 GHs were identified in this experiment corresponding to 74.8% of PACs group, which can be divided into 23 families including GH1, GH3, GH5, GH9, GH10, GH13, GH16, GH17, GH18, GH19, GH20, GH27, GH28, GH29, GH31, GH32, GH35, GH37, GH38, GH51, GH65, GH79 and GH127 according to CAZy nomenclature based on sequence homology (Fig. 3). As expected, the most representative families were GH3 and GH17, as previously documented [12, 20, 26]. Moreover, GH1, GH5, GH16, GH18, GH19, GH27, GH28, GH31, GH35 and GH38 were also well represented families, with at least five members of each (Fig. 3).
Possible substrates of most of GHs families were hemicelluloses (xyloglucan, xylans, glucomannans) and pectin (galactans, homogalacturonan). Out of GHs identified in this study, GH16, GH29, GH31 and GH65 potentially act on xyloglucans, GH10 and GH51 show possibly action on xylans, and GH28 and GH35 could hydrolyze homogalacturonan and galactans, respectively [46–48] (Additional file 6: Table S5). Moreover, GH1, GH3 and GH5 possess broad substrates range, their enzymes are reported to be involved in the modification and/or breakdown of cell wall hemicelluloses and pectins [49, 50], and also be implicated in lignification and secondary metabolism . Identification of these GHs families suggested that hemicelluloses and pectins might undergo important structural changes in the leaves of C. sinensis. Furthermore, GH127 (DUF1680 domain protein), being characterized recently as a novel beta -L-arabinofuranosidase, might be implicated in the degradation of cell wall polysaccharides and hydroxyproline-rich glycoproteins , and GH9 was known to catalyze the endohydrolysis of cellulose.
Some identified GHs could participate in defense against pathogens and various stress. Chitin and beta − 1,3- or beta − 1,6-glucan are main components of cell walls of various fungi. GH17 acts as beta − 1,3-glucanase, together with chitinases (GH18 and GH19) and GH20 that function as key hydrolyzed enzyme of chitin, have shown to possess antifungal activity by degrading their cell walls and then participate in defense against pathogens [47, 53]. Intriguing, chitinases being in response to abiotic stress were also reported [54, 43]. GH37, a non-reducing sugar, was identified to be a new CWP in this work without being documented as CWPs in WallprotKB. GH37 acts as a universal stabiliser of protein conformation, might contribute to various stress defense .
Several identified GHs including GH13, GH27 and GH32 might be implicated in mobilization, allocation and partitioning of storage reserves. GH13 is was associated with the hydrolysis of starch and glycogen to yield glucose and maltose , GH27 is one of three hydrolyzed enzymes of galactomannans as a cell wall storage polysaccharide , and GH32 as invertases is involved in long distance nutrient allocation and carbohydrate partitioning [58, 59]. Additionally, a couple of GHs enzymes including GH3, GH18, GH19, GH35, GH38 and GH79 were known to be involved in post-translational modifications (PTMs) of glycoproteins [32, 47]. Here, GH3, GH35, GH38 and GH79 were verified as N-glycoproteins.
Collectively, identified GHs potentially give rise to complex cell wall carbohydrates remodeling, pathogen and stress response, mobilization and allocation of storage reserves as well as glycoproteins PTMs. The high number of GHs associated with cell wall metabolism and defense response were found in this work, which is consistent with published reports of sugarcane stems and leaves , B. distachyon grains , Saccharum officinarum cell suspension . The results might be attributed to sustainability remodeling during plant growth and development and terrestrial habit of plants.
Other CWPs acting on polysaccharides. Less represented CWPs acting on polysaccharides including carbohydrate esterase [11 pectinesterase, knows as pectin methylesterases (PMEs) and 3 pectinesterase inhibitor (PMEIs)], GTs (4), expansins (6), PNGase A (4), PAEs (3), PLs (2) as well as carbohydrate acylation (trichome birefringence-like proteins, 4) were also identified.
PMEs, PAEs and PLs are pectin modifying enzymes. PMEs that catalyse the demethyl-esterification of homogalacturonan domain of pectin . The degree of pectin methylation/demethylation impacts on cell wall stiffening and access to enzymes . Demethyl-esterificated pectin more favor the cleavage of the acidic polygalacturonic chains by GH28 and PLs. In parallel, PAEs can regulate pectin deacetyltation by cleaving the acetylester bond from pectin . Overall, these enzymes play a major role in controlling cell wall plasticity/rheology by affecting pectin metabolism .
Trichome birefringence-like proteins and PNGase A are also two modification enzyme families of cell wall. The former was characterized as xylan acetyltransferases, and was believed to be implicated in the mediation of xylan O-acetylation, which being required for secondary wall deposition and pathogen resistance . The latter is one of deglycosylation enzyme and has been considered to be involved in the release of N-glycans from glycopeptides generated by the proteolysis of denatured glycoproteins .
Regarding expansins, known as non-enzymatic and the most important structural proteins, are believed to play a central role in cell wall extension via their action on the cellulose-hemicellulose network, suggesting be essential for primary cell wall structure during plant growth and development related processes . Besides, 4 cell wall GT families are represented including GT2, GT31, GT48 and GT68, which is associated with the biosynthesis of cell wall polymers.
Identified CWPs involved in proteases. Proteases (Ps; 94) were the second largest class, representing 16.8% of total CWPs, with Asp proteases (28), Ser carboxypeptidases (28), Ser proteases (19) and Cys proteases (14) as main families. Although the biological roles of proteases are remarkable diverse, proteases certainly play crucial roles in the plant developmental and in response to environmental stresses through turnover and maturation of CWPs, the generation of active peptides in the cell wall . Overall, the importance of this class was expected because proteases are responsible for the degradation or the maturation of cell wall modifying enzymes.
Identified CWPs involved in redox. The third abundant functional class found in tea leaves was ORs (62, 10.7%), mainly comprises class III peroxidase (PODs, 29), multicopper oxidases (13), BBE (berberine bridge enzyme) (S)-reticulin (6) and laccases (5). Class III PODs, a large multigene families, corresponded to one half of the OR functional class.
Class III PODs are known to be involved in lignin metabolism by catalyzing the oxidative polymerization of monolignols , stress responses and signaling via consuming hydrogen peroxide and generating reactive oxygen species . Class III PODs also could mediate cross-linking of cell wall compounds such as structural proteins, monolignols as well as of aromatic amino acids with polysaccharides [70–72]. Laccases, like Class III PODs, are candidates for polymerizing monolignol unit into lignin, suggesting be required for cell wall lignification [73, 74].
BBE-like proteins, act as monolignol oxidoreductases, may participate in the mobilization and oxidation of monolignols required for polymerization processes . All in all, three high represented enzyme families in the class were considered to be involved in ligin production and subsequence the reinforcement of cell walls strength and rigidity, which favoring plant defense against adverse environmental factors.
Other CWPs related to redox processes including monocopper oxidase-like proteins (SKU5 and SKS1), blue copper proteins and ascorbate oxidases were identified, which probably play a role in both cell wall loosening, expansion and reticulation processes[24, 76].
Identified CWPs involved in signaling. Identified CWPs from the class mainly contain fasciclin-like arabinogalactan proteins (FLAs, 9) and receptor-like protein kinases (RLKs) superfamily proteins (38). FLAs, heavily O-glycosylated CWPs, have been found to be correlated with cell wall formation , cell-to-cell adhesion and communication  and abiotic stress response . In the present study, RLKs comprise 21 LRR-RLKs, 6 cysteine-rich receptor-like protein kinases, 3 S-locus receptor kinase subfamily proteins, 2 wall-associated receptor kinases and 6 lectin receptor kinase (LRK) subfamily proteins. RLKs, primary cell wall “sensors”, are responsible for the control of diverse signaling events , has been found to possess important functions in a wide variety of developmental and defense-related processes by recognition of an extracellular ligand which leads to activation of the intracellular kinase domain and subsequent transduction of downstream signaling pathways .
Identified CWPs related to lipid metabolism. The class of CWPs is that of proteins predicted to be related to lipid metabolism, mainly consist of lipid-transfer proteins (LTPs, 10) and GDSL esterase/lipases (GDSLs, 16). Besides, other CWPs related to PLMs like glycerophosphodiester phosphodiesterases (3), phosphoesterases (2), embryo-specific protein ATS3B-like (4) as well as neutral ceramidase (1) were identified.
LTPs have been shown to be required for lipid export to the cell surface and be closely associated with cutin and wax formation . A LTP was also suggested to be involved in cell wall extension by interacting with the cellulose/xyloglucan network . GDSLs, a newly discovered subclass of lipolytic enzymes, possess multifunctional properties which are assumed to play important roles not only in the formation of surface cutin and epi-cuticular wax , but also function in tolerance to biotic and abiotic stresses [85, 86]. In summary, numerous LTPs and GDSLs might play important roles in cuticle assemble during the growth and development of C. sinensis leaf. The identification of CWPs related to PLMs is easy to understand for leathery leaf of C. sinensis.
Identified CWPs related to other function. Identified MPs mainly encompasses purple acid phosphatases (PAPs, 17), blue copper binding proteins (BCPs, 9), dirigent proteins (DIRs, 8), germin-like proteins (GLPs, 5), Thaumatins (7) and proteins having a cupin domain (5).
PAPs, might associated with the degradation of xyloglucan and oligosaccharides via dephosphorylating CWPs like alpha xylosidase and beta glucosidase . DIRs, are linked to lignin polymerization [88, 89] and play important roles in various stress responses and controlling cell wall modification/reinforcement during cell wall integrity maintenance . Regarding BCPs, GLPs, cupins and Thaumatins were previously reported to be associated with stress responses in plants [91–94].
Five structure proteins were identified in present study including three leucine-rich repeat extensin-like protein (LRR-EXTs), non-classical arabinogalactan protein 31-like (AGP) and hydroxyproline-rich glycoprotein. LRR-EXTs have known to influence mechanical properties of cell wall by their ability to form insolubilized, covalently crosslink to cell wall components , as well as function as perceive extracellular signals and indirectly relay into the cytoplasm to regulate plant growth and salt tolerance, thereby suggesting they are important for cell wall development, plant growth and stress tolerance . Non-classical AGPs have both a proline-rich domain and a non-proline-rich domain, may be function in metal ion-binding, defense response and interact with pectin [97, 98]. As for hydroxyproline-rich glycoprotein, which is an important structural components of plant cell walls and are thought to be implicated to structural integrity, cell-cell interaction and intercellular communication .
Several enzymes of CWPs inhibitor were also detected in this study. PMEIs that inhibited partly the activity of PMEs, adjust the degree of pectin methyl-esterification. PGIPs (polygalacturonase inhibitor-like) specifically bind with polygalacturonases (GH28), thereby they can inhibit the hydrolyzation of pectin and then regulate pectin degradation, which can trigger defense against microbes and insects . In summary, two couple of PMEIs and PME, PGIPs and PG occurred coincidentally and modulate precisely pectin metabolism. As for Cys proteinase inhibitor possess inhibitory activities against specific Cys proteases, probably play a role in insect predation .
Identified CWPs emphasizing on plant cell wall formation and defense response
Under dynamically changing environmental conditions, plant grow and develop continuously, and always encounter variable stresses and deleterious attack of insects and microbes. To acclimate, plant cell walls that acting as the first barrier change constantly, whereas CWPs play central roles in altering cell wall properties.
Doubtlessly, to meet normal growth and development, a large amount of CWPs could be triggered to adjust vigorously cell wall structure. Here, identified numerous CWPs related to PACs, mainly including GH1, GH3, GH5, GH9, GH10, GH16, GH28, GH29, GH31, GH35, GH51 and GH65, might contribute to the rearrangement of cell wall structure. In contrast, expansins probably lead to cell wall extension. Certainly, several CWPs associated with the formation and metabolism of secondary cell wall, like Class III PODs, BBEs, laccases, LTPs, GDSLs and DIRs, maybe favor to the reinforcement/modification of cell wall (Fig. 4).
Facing to adverse environment, C. sinensis, a terrestrial plant, have no ability to escape. Therefore, they have evolved in the context of altering cell wall properties for improved defense responses. Today, ample identified CWPs were potentially involved in various defense. GH17, GH18, GH19 and GH20 were reported to be involved mainly in against pathogens as well as abiotic stress by hydrolyzing chitin. Class III PODs, monocopper oxidase-like proteins, blue copper proteins and ascorbate oxidases were known to be implicated in respond to various biotic and abiotic stresses by redox reaction. LTPs, GDSLs and DIRs were also associated with defense response through the regulation of secondary cell wall. PGIPs and Cys proteinase inhibitor might function in improving protection against insects and pathogens  via inhibiting the activity of degradation enzymes of invaders. Likewise, BCPs, GLPs, cupins and Thaumatins also serve functions in defense response (Fig. 4).
To sense changed environment and the status of complex cell wall structures, plants have developed cell wall integrity-sensing pathway to transduce signals into cytoplasm. A number of sensors at the plasma membrane including RLKs and FLAs were identified in present study, which enable C. sinensis to coordinate the processes of the cell wall and the cytoplasm (Fig. 4).
In summary, a work model of identified CWPs were proposed (Fig. 4), which emphasizing on plant cell wall formation and defense response, and further making a bit explanation for plant internal activities during normal growth under natural environment.