# 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  
In the _Para_ rubber tree, NRB is known to begin with IPP synthesis in MVA pathway [38; 39], which is a cytosolic pathway for rubber biosynthesis [40]. In the early steps of the MVA pathway, ACAT is important for generating acetacotyl-CoA, then HMGS and HMGR activate the supply of mevalonate substrates [2; 41]. ACAT can catalyze a Claisen-type condensation of two acetyl-CoA units to form acetacotyl-CoA, which is the first step in the MVA pathway and is also important for providing the malonyl-CoA substrate for the biosynthesis of fatty acids [42]. This enzyme was found to mainly accumulate in the mature TKS roots (Table S1; Table S3), but it could not be detected in total latex of the _Hevea_ rubber tree [2]. Through a series of enzymatic reactions, acetyl-CoA can be catalyzed by ACAT to form IPP [43]. Four ACAT genes were determined in the _Hevea_ rubber tree [10] and eight ACAT genes were found in the TKS genome [16]. Overexpression of _AtACAT_ results in the increased accumulation of ACAT in the latex of _T. brevicorniculatum_, and ultimately increased the pentacyclic triterpene and sterol levels [44]. Our proteomic results demonstrated that seven (ACAT 2-8) out of the eight ACAT members can be identified by the shotgun proteomic method, and one member (ACAT 7, spots 157, 183 and 424) can also be detected on the 2-DE gel (Figure 2), indicating that ACAT 7 is high-abundant in TKS roots. In the following steps, HMGS and HMGR activate the supply of mevalonate substrates [2; 41]. HMGS, the second enzyme in the MVA pathway, catalyzes aldol-type condensation of acetacetyl-CoA and acetyl-CoA to produce HMG-CoA, and it is located as a cytosolic protein [45; 46]. In our proteomic data, one HMGS member (HMGS 1, evm.model.upg 270.25; spot 146) was identified from both high-through and 2-DE gel-based proteomics methods (Figure 7; Table S6). Furthermore, HMG-CoA can be converted to MVA by HMGR [47]. HMGR controls the carbon flow and metabolic reaction rate in MVA [42], and it is also a critical player for the regulation of triterpenoid metabolism [38; 48]. It was reported that ethylene cannot activate the enzyme activity of HMGR [49]. Five gene members have been characterized in the genome of the _Para_ rubber tree [10], but only HMGR1 gene was found to be induced by ethylene stimulation [49]. In the TKS genome, 12 HMGR members were determined, and only two out of them (_TkHMGR_1 and _TkHMGR_2) were predominantly expressed in roots, with the highest expression level in latex [16]. In this proteomic study, two members of HMGR (HMGR1 and HMGR12) were detected from TKS roots by the shotgun method (Table S3). Based on these genomic and proteomic results, we consider that HMGR1 might be the most important player for NRB in TKS roots. In the following steps, mevalonate kinase (MVK) converts mevalonate into isopentyl pyrophosphate and phosphomevalonate kinase (PMVK) catalyzes mevalonate-5-phosphoate to form mevalonate-5-pyrophosphate [50]. In _Hevea_, three MVK genes were determined [10]; and in TKS genome, 11 MVK genes were observed [16]. In our proteomic data, we identified eight MVK protein members from the predicted 11 MVK genes, and five PMVK members from the six PMVK genes (Table S6). Finally, MVD catalyzes mevalonate-5-pyrophosphate to form IPP [49]. Two MVD members were determined from both _Hevea_ and TKS genomes [16], and in this proteomic study, they were identified from the mature TKS roots (Table S6). These results revealed that enzymes involved in the conversion of mevalonate into isopentyl pyrophosphate and diphosphate to form IPP are important for NRB in the mature TKS roots. The MEP pathway is mainly occurred in the plastid and it is an alternative IPP synthesis pathway [51; 52]. Recently, the plastid MEP pathway has also been considered as a possible route for NRB [53; 54; 55]. The \({}^{13}\)C-labelling of _Hevea_ seedlings revealed that MEP pathway mainly contributes IPP for carotenoid biosynthesis [38].