## 4 Discussion  
Sterols in the _E. rituximab_ latex, including euphol and \(\beta\)-sitosterol, are thought to be related to various medicinal properties of this plant in previous phytochemical studies [35]. Interestingly, in this study, a contrasting tissue-specificity was revealed for euphol and \(\beta\)-sitosterol in _E. rituximab_, as euphol was highly accumulated in the latex with the absence of \(\beta\)-sitosterol. This phytochemical data indicated that the expressions of genes involved in sterol biosynthesis were distinctly regulated in different _E. rituximab_ tissues. To decipher the mechanism underlying the tissue-specific biosynthesis of sterols in _E. rituximab_, a transcriptome database integrated from the root, stem, leaf and latex of the plant was constructed, and the genome-wide expression was extensively analyzed. With respect to the upstream pathway genes, most of the MVA pathway genes were up-regulated in latex compared to the other tissues, which was consistent with the high-level accumulation pattern of euphol in latex. On the other hand, the expression levels of most MEP pathway genes were significantly down-regulated in the latex compared to the other tissues. These gene expression data agreed with the well-accepted conclusion that the biosynthesis of triterpenoids or plant sterols is mainly derived from the MVA pathway rather than the MEP pathway [36]. In the pathway after IPP, the transcript abundance of most of the pathway genes, such as those encoding FPPS, SQS, and SE, was also significantly higher in latex than that in the other tissues, which further demonstrated that the tissue-specificity for euphol biosynthesis was likely due to the transcriptional regulation of the pathway genes. In fact, the biosynthesis of euphol and \(\beta\)-sitosterol shares the common intermediate 2,3-oxidosqualene, which is then channeled to \(\beta\)-sitosterol and euphol by CAS and euphol synthase, respectively [37]. Although lanosterol was recently reported to be a substrate for the biosynthesis of \(\beta\)-sitosterol [16], none of the unigenes encoding lanosterol synthase was identified in the _E. rituximab_ transcriptome, suggesting that the biosynthesis of \(\beta\)-sitosterol in _E. rituximab_ was mediated by CAS. Based on the phylogenetic tree and transcript level analysis, EtOSC2 is likely the putative gene encoding CAS, while EtOSC5 and EtOSC6 seem to be the candidates for euphol synthase. Thus, these three unigenes are possibly specific to steroid biosynthesis. Although the accumulation of \(\beta\)-sitosterol was not detected in latex, the expressions of a great number of unigenes in the pathway converting to \(\beta\)-sitosterol were identified in the latex (Table S6). It was confusing that the expression of _EtOSC2_ seemed to be inconsistent with the absence of \(\beta\)-sitosterol in the latex, since a notable expression of _EtOSC2_ was observed in the latex. One possible reason is that \(\beta\)-sitosterol could be synthesized in the latex and subsequently conveyed to other tissue cells. Alternately, in the latex, the metabolic flux of the intermediate 2,3-oxidosqualene was diametrically channeled to the pathway of euphol biosynthesis, whereas the carbon flux towards \(\beta\)-sitosterol was tightly attenuated, leading \(\beta\)-sitosterol being at a detectable level. This assumption is based on the observation of much higher transcript levels of _EtOSC5_ and _EtOSC6_ than _EtOSC2_ in the latex. In conclusion, a tissue specific distribution of plant sterols in _E. rituximab_ was investigated. The comprehensive transcriptome database derived from the root, stem, leaf and latex of _E. rituximab_ was integrated, which formed a strong basis for identifying the putative genes involved in the biosynthesis of sterols or triterpenoids in this plant, and especially for exploiting these OSC candidate genes responsible for euphol biosynthesis. Further functional characterizations of these OSCs are urgently required to confirm their roles in triterpenoid biosynthesis.