# Proteomic Landscape of the Mature Roots in a Rubber-Producing Grass _Taraxacum Kok-saghyz_
## 3 Discussion
### Almost All NRB-Related Proteins Can Be Identified From the Mature TKS Roots
Genomic analysis demonstrated that all genes involved in the MEP pathway can be detected in both _Hevea_ and TKS, but most of the MEP-related genes have a medium or low expression level in the latex of the _Hevea_ rubber tree [10] and TKS [16]. However, our proteomics results demonstrated that 13 unique proteins involved in the MEP pathway were determined in the TKS roots; these proteins are DXS, DXR, CMS, CMK, HDS and HDR (Table S6). These results indicated that the MEP pathway may also contribute IPP for NRB in the mature TKS roots. In the initiator synthesis process, GPS and IPI catalyze IPP and DMAPP to form GPP, and FPS to produce farnesyl diphosphate by adding IPP onto GPP to form polyisoprene [40; 56], then GGPS and GPS catalyze farnesyl diphosphate to form geranylgeranyl diphosphate [31; 42]. Our proteomics data showed that four crucial enzymes, named IPI, GGPS, GPS and FPS, can be identified from the TKS roots. Then, in the natural rubber elongation process, cis-isoprene transferase (CPT), also named _Hevea_ rubber transferase, catalyzes multiple isoprene units (C5H8) and polymerizes into IPP long chain molecules and determine the size of the rubber molecule [46; 57]. It is closely associated with the rubber particle membrane and is widely known to be a key enzyme in rubber biosynthesis [28; 31]. Eleven CPT members were determined in the _Para_ rubber tree genome [10], but only CPT2 shows rubber transferase activity [40], and a rubber transferase activator (RTA) or CPT-like protein (CPTL) can interact with CPTs to activate NRB in the _Hevea_ rubber latex [57]. In the TKS genome, eight CPT members and two CPT-like proteins (CPTL1 and CPTL2) have been characterized, and _TKCPT1_, _TKCPT2_ and _TKCPT1_ genes play a critical role for the elongation of rubber polymers in the latex and roots of TKS [16]. However, our proteomics data only identified CPT1 in the TKS roots (Table S6), indicated CPT1 may be the crucial player for NRB in TKS roots. In the final elongation process, three rubber particle membrane binding proteins, named REF, SRPP and a Nogo-B receptor or HRT1-REF bridging protein (HRBP), are widely known to play crucial roles [35], and they were positively identified from the TKS roots by our proteomics study (Figure 7). Among them, REF is anchored inside rubber particle membrane by its auto-assembly ability [42], whereas SRPP largely covers rubber particle surface in an oriented anisotropic manner [58]. REF is also suggested to be a conservative regulator of lipid droplets and it is related to form an intracellular structure [59; 60]. However, proteomics of the detergent-washed rubber particles identified none lipid droplet proteins [28; 31], indicated that the maturation of rubber particles might be different from that in lipid droplets. Histochemical localization indicated that rubber biosynthesis capability in _Hevea_ laticifer is mostly concentrated in SRPs, and SRPP, a rubber biosynthesis-related protein that accumulated mainly in SRPs, is more important than REF for rubber biosynthesis in the _Hevea_ rubber tree [48]. Five SRPP genes (_TbSRPP 1-5_) were found in TBR [7]. Among them, except for _TbSRPP 2_[61], four members can enhance NRB. But the average molecular weight of rubber hydrocarbons has not been significantly affected by these genes [62]. Overexpression of _TbSRPP3_ only resulted in a slight change in the content of rubber in TBR root [63]. In this proteomics of TKS roots, eight out of 10 SRPP members were identified (Table S6). Different with that in _Hevea_ genome, which contains eight REF members, only two REF members have been characterized in the TKS genome [16]. Our proteomics data also identified the two REF members in the mature TKS roots (Table S6). These published results, as well as our proteomics of the 6M TKS roots, revealed that SRPP might be more important than REF for rubber biosynthesis in the mature roots of TKS, and more attention should be paid on determination of the detail roles of SRPP in TKS roots in future.