## 3 Discussion
### Comprehensive Proteomics Analysis Revealed SRP Might Be an Efficient Machine for NRB  
In the rubber tree, cis-1,4-polyisoprene is generated through the MVA pathway, involving initiation, elongation, and termination [7; 33; 35]. The enzymes for polyisoprene initiation include ACAT, HMGS, HMGR, MEVK, and phosphomevalonate kinase (PMVK). During the elongation process, REF, SRPP, CPT (HRT), GPS, GGPPS, FADS and HRBP are key proteins, and they were all identified from SRPs (Table S3), and most of them were induced after ethylene application (Figure 6). These proteins and cofactors may formulate a natural living carbocation polymerization (NLCP) mechanism for carotenoid biosynthesis [33]. The proteomics study of rubber particles and interaction network analyses revealed a protein complex consisting of HRT, REF, and HRBP, which function as rubber biosynthetic machinery in the rubber particles [18]. Our results herein demonstrated that SRP with enzymes should be a more active NLCP machine for NRB. In a recent proteomics study, we have noticed that ethylene can improve SRPs generation and prolongs the latex flowing time [12]. Here, we observed that, the general abundance of pro-hevein was not significantly changed after ethylene treatment (Table S3), but six isoforms of pro-hevein were determined as ethylene-responsive proteins on DIGE gels (Figure 2). Among them, five isoforms were significantly depressed by E24 treatment, but four isoforms were induced under E48 treatment (Figure 6). Hevein and its precursor pro-hevein are traditionally considered as major proteins in lutoids, they can inhibit rubber particle aggregation and latex coagulation [3; 14; 45]. The decreased accumulation of hevein in latex maintains the flow of latex and finally improves latex yield after ethylene application [12]. Here, many SRP proteins were directly or indirectly involved in NRB. We identified 24 members of peptidyl-prolyl cis-trans isomerase (PP isomerase) from SRPs, but only one member (I176) was induced by ethylene stimulation (Table S3), indicating that this isoform may be more important than others in SRPs. In the SRPs, we also identified an albumin-2 like protein (I12), and it was sharply induced by ethylene stimulation (Table S3). This protein may contribute to osmotic regulation in laticiferous cells upon ethylene stimulation. A universal stress protein was also previously observed in total rubber particles (TRPs) [20]. In SRPs, its two members were sharply accumulated after ethylene treatment for 24 h but decreased in the E48 treatment (Figure 6). Methyltransferase can transfer a methyl group from S-adenosylmethionine to their substrates. This has been identified from the rubber latex [12; 17; 19] and C-serum [46]. Its abundance was significantly up-regulated upon the application of ethylene to the total rubber latex [12] and SRPs (Table S3). Vacuolar protein sorting-associated protein (VPSP) has been detected from rubber latex [47]. Here, we found VPSP4, as well as REF175, elongation factor 1 and ribosomal protein L18-2, were sharply up-regulated after ethylene treatment for 24 h in SRPs (Figure 6). Given our in-depth proteomics data in SRPs and the results from the recent literature, we proposed an intrinsically subcellular-based mechanism associated with both the localizations and responses of major SRP proteins after ethylene stimulation. We suggested a general scheme of the ethylene-induced biochemical pathways in SRP at the protein level (Figure 6). This new schematic model considers SRP as a natural rubber biosynthetic machine in response of ethylene stimulation. The newly obtained proteomics data revealed that SRPs contain almost all proteins involved in NRB, and many proteoforms are induced by ethylene. The changed patterns of some proteoforms are different from the observations of that in the gene expression level [11; 12] and general protein accumulation patterns [12; 17].