## 3 Discussion
### Ethylene Improved NRB by the Accumulation of Rubber Producing-Related Proteins in SRPs  
Ethylene stimulation can sharply induce natural rubber production, but the expression level of most key genes in NRB, such as FADS [3], CPT [26] and hydroxymethylglutaryl coenzyme A reductase (HMGR) [27], are not significantly up-regulated [11; 12]. At the protein level, new evidence supports the accelerative effect of ethylene on NRB [28; 29]. The accumulation of several proteins, including glutamine synthetase [30], phosphoenolpyruvate carboxylase [17], acetyl-CoA C-acetyltransferase [12; 17], sucrose transporter [31], and aquaporin [32], are activated upon ethylene application. At the proteomic level, ethylene induces the accumulation of many enzymes involved in carbohydrate metabolism, energy production, and NRB [12; 17]. Here, we determined 305 ethylene responsive proteins from DIGE (Table S1) and iTRAQ (Table S3) and found that half of them were induced by ethylene stimulation (Figure 6).  
NRB is a typical isoprenoid metabolic process [33; 34], beginning with isopentenyl pyrophosphate (IPP) synthesis in the menvalonate (MVA) pathway [7; 35]. In early steps, acetyl-CoA C-acetyltransferase (ACAT) is important for generating acetoacetyl-CoA, where it catalyzes acetyl-CoA to form acetoacetyl-CoA, which is the first step in the MVA pathway. Then, HMGS and HMGR activate the supply of mevalonate substrates [12; 33]. Our study showed that, ACOC, as an important enzyme for the biosynthesis of fatty acids, was mainly accumulated in SRPs (Table S3), but could not be detected in the total rubber latex [12]. Through a series of enzymatic reactions, acetyl-CoA can be catalyzed to form IPP [34]. Three ACAT genes were determined in the rubber tree [7]. Our previous proteomic results revealed that both the gene and protein accumulation of ACAT are depressed upon ethylene stimulation, but one protein isoform is induced [12]. In this study, one isoform (D12) was identified as ACAT from SRPs by DIGE and iTRAQ experiments. Its abundance was induced after E24 treatment butdepressed under E48 treatment (Table S1 and S3), which is consistent with our previously published latex proteomics results [12].  
HMGS is the second enzyme in the MVA pathway [29; 36]. Here, two HMGS members were found in SRPs. HMG-CoA is further converted to MVA by HMGR. However, we did not detect HMGR, probably due to its low abundance in SRPs. MEVK controls the first stage in the conversion of mevalonate into isopentyl pyrophosphate. It has a higher accumulation in rubber latex and may be coded by a single gene [7]. However, three genes were determined in a recently annotated genome of the rubber tree [8]. In this proteomics study, we also identified five MEVK members in SRPs (Table S3), indicating that more MEVK members are involved in NRB after the application of ethylene. Mevalonate disphosphate decarboxylase (MEVD) catalyzes MVAPP to form IPP [35]. It was reported to be coded by a single gene [7], but genomic analysis resulted in two MEVD genes [8], and in this study, we also obtained two MEVD members in the SRPs (Table S3).  
In the natural rubber elongation process, geranyl pyrophosphate synthase (GPS) catalyzes IPP and dimethylallyl diphosphate (DMAPP) to form geranyl diphosphate (GPP), and FADS catalyzes famesyl diphosphate (FPP) to form polyisoprene by adding IPP [2; 37]. GGPS and geranylgeranyl pyrophosphate synthase (GGPP) catalyze famesyl diphosphate to form geranylgeranyl diphosphate (GGPP) [18; 33]. We identified GPS, FADS, and GGPPS in SRPs and determined a FADS member (I48) as an ethylene responsive protein (Table S3).