## 2 Results
### Integrative Analyses of DAPs in Different SRPs  
We further performed western blotting to determine the general accumulation patterns of 9 typical proteins in SRPs (Figure 5B) and calculated their relatively changed ratios (Figure 5C). Among them, 8 proteins could be clearly detected in D24, where only the accumulation of the ethylene-inducible protein (EIP) was relatively low. In E24, six proteins, namely, SRPP, _Hewa_ rubber cis-prenyltransferase (HRT), EIP, plasma membrane intrinsic protein (PMIP), heat shock protein 70 (HSP70), and acetyl-CoA carboxylase (ACOC) were significantly induced, and three proteins, named REF, ascorbate peroxidase (APX) and glutathione reductase (GLR), were not changed significantly. In D48, the abundance of almost all proteins (except for REF) was significantly up-regulated, but half of them were decreased under E48 treatment (Figure 5C). SRPP and REF, as the two most abundant proteins, showed differently changed patterns upon ethylene stimulation in SRPs. The E24 and D48 treatments could significantly increase the accumulation of SRPP, but decreased the abundance of REF. Western blotting confirmed that ethylene stimulation at both 24 and 48 h could sharply induce the accumulation of HRT. EIP was  
Figure 5: Accumulation patterns of the typical ethylene-responsive proteins in SRPs. The 4 columns represent the 46 sharply changed proteins under the E24/D24, D48/D24, E48/D48, and E48/E24 treatments (marked as E24, D-D, E48, and E-E). The up- or down-accumulated proteins are indicated in red or green color, respectively. The protein number from iTRAQ (I-) is indicated on left side (**A**). Western blotting showed the general protein accumulation patterns (**B**). The dotted line represents the changed ratio value of 1.0 (**C**). * \(p\), 0.05, **_p_, 0.01, and *** \(p\), 0.001.  
significantly up-regulated by the ethylene and D48 treatments, and PMIP was mainly accumulated under the E24 and D48 treatments. HSP70 could be induced by all of the three treatments. APX and GLR showed similarly changed patterns, and they were rich in E48, but had a low abundance in E24. ACOC was also significantly enhanced by ethylene stimulation in SRPs (Figure 5C, Table S3).  
Given the results from this SRPs proteomics study and the recent published literature, we summarized the main functions of the identified proteins and their potential locations in rubber latex, proposing a possible schematic representation of the intrinsically proteome-based mechanism for the possible regulation details of proteins in SRPs (Figure 6). These results reveal that the ethylene stimulation of rubber latex production performs on the protein accumulation level in rubber particles, especially in SRPs. The post-translational modification of some key proteins and many metabolic enzymes might play crucial roles in the control of NRB process in SRPs.  
Figure 6: Schematic diagram of SRP proteins involved in natural rubber biosynthesis (NRB). A schematic diagram of enzyme localization and the regulation of ethylene-induced biochemical pathways on the protein level in SRPs are both highlighted. In this schematic diagram, the identified proteins involved in NRB from SRPs are marked with different colors (green, unchanged protein; red, changed protein), and the changed patterns determined by 2-D DIGE (D-) and iTRAQ (I-) for these DAPs are provided in the parentheses. The up arrow stands for upregulation, the down arrow stands for downregulation, and minus sign the protein is not significantly changed after ethylene application.