## 3 Discussion
### Different Protoforms in SRPs May Play Different Roles in NRB  
Genomic analysis demonstrated that most proteins involved in MVA pathways are encoded by multiple genes, meeting the formation of different isoprenoids in the Para rubber tree [8; 38; 39]. HRT or CPT is a key enzyme in natural rubber biosynthesis, and it uses pyrophosphates to initiate rubber molecules [36; 40]. It is a rubber particle membrane protein [18]. A total of eleven HRT members have been determined in the recently published rubber tree genome [8], but only HRT2 shows HRTase activity [2]. Its activity is higher in SRPs than in LRPs [21]. At the gene level, the expression of CPT1 and CPT2 are both depressed by ethylene [11; 12]. However, our previous results revealed ethylene significantly improved HRT2 abundance in the rubber latex [12]. Here, we further proved HRT2 abundance (I116) was significantly induced by ethylene in SRPs (Figure S5, Table S3), indicating HRT2 plays a more important role in ethylene-induced latex production in SRPs. During the elongation process of rubber biosynthesis, REF, SRPP, and a Nogo-B receptor HRBP are known as RP membrane binding proteins, playing a role as a natural rubber biosynthetic machinery [18; 41]. We identified HRBP (I69) in SRPs and found that it could be significantly induced by ethylene (Table S3). REF and SRPP are closely combined with rubber particles [42]. A recent report indicates that SRPP can recruit CPT to the endoplasmic reticulum to interact with HRBP [22]. In _Heeva_, 18 REF/SRPP family members are observed, and their genes exhibit distinct expression patterns in different tissues; most genes are located in Scaffold1222 in a single 205-kb genome site [8; 15]. In 2-DE gels, we determined 28 protein isoforms from five REF/SRPP members, and multiple protein isoforms were identified [15]. Generally, the gene expression and protein accumulation for these REF/SRPP members is decreased or not changed after ethylene treatment. However, individual REF/SRPP isoforms display differently changed patterns upon ethylene stimulation in different rubber tree clones [8; 15]. In this SRPs proteomics study, 23 REF/SRPP isoforms were identified as DAPs upon ethylene application, and they were induced by ethylene treatment for 24 h (Table S1). Among them, many amino acids were phosphorylated upon ethylene stimulation (Table S2). It is widely known that both phosphorylation and dephosphorylation are predominant intracellular posttranslational modification mechanisms, playing important roles in different cell-signaling pathways [43]. Two key classes of enzymes that are essential in controlling the opposite action of these posttranslational modifications are kinases and phosphatases (responsible for phosphorylation and dephosphorylation, respectively), which correspondingly initiate and terminate cellular signals. Our phosphoproteomicsanalysis of the rubber latex has revealed that many phosphorylated proteins are substantially more apparent after ethylene treatment, and a large amount of ethylene-responsive proteins with phosphatase/phosphotransferase activity and kinase activity are enriched [15]. We also found that the abundance of some latex proteins in the rubber tree were significantly reduced after ethylene stimulation [15]. Recently, [PERSON] et al. collected proteins from the C-serum, lutoids, and rubber particle layers of the rubber latex, determining 73 post-translationally modified peptides in the 47 identified rubber latex proteins, including the main PTMs, such as phosphorylation, lysine acetylation, and ubiquitination [43]. In this study, we noticed that four isoforms of the identified 23 REF/SRPP members were decreased after ethylene stimulation for 48 h (Figure 2I, Table S1), and the phosphorylated amino acids were decreased (Figure 3B; Table S2), indicating that the dephosphorylation of proteins might also be important in NRB.