# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 3 Discussion  
Plants synthesize and accumulate an immense variety of isoprenoid metabolites under complex developmental and environmental controls. Strategically, metabolic engineering of a key enzyme in a crucial biosynthetic pathway can be a powerful approach to increasing a valuable end product. Thus, the hypothesis was posited that by diverting the carbon metabolic flux away from squalene synthesis, the production of NR would be increased in guayule. [PERSON], [PERSON] et al. [35] reported a single copy _SQS_ gene in guayule, moderately expressed in stem tissue of field plants, which was the target of our RNAi construct (Figure 1A). Constitutive downregulation of _SQS_ using this construct was successful based on gene expression results (Figure 1C) and the confirmation of lower squalene concentrations in stem tissue compared with controls (Figure 2A). We showed that squalene was more concentrated in stem tissues (Figure 2A), where, frequently, most rubber biosynthesis occurs, supporting the notion that NR initiation and squalene synthesis compete for the same substrate (FPP) in the same tissue. The extent of reduction of squalene (\(\sim\)60%) was quite similar to that reported in other plants, _A. annua_[27] and _W. somifera_[28]. Despite lowered squalene levels, in greenhouse/growth chamber _SQSi_ guayule plants, pools of substrate, i.e., FPP, were not detected. Since FPP is a precursor to dozens of downstream products, it may be too  
Figure 6: (**A**) Relative expression of metabolic and putative NR biosynthesis and structural genes in _SQSi_ lines’ background. Plant phenotypes for controls and SQS lines, RT-PCR for the isoprenoid pathway, and natural rubber biosynthesis-associated genes. (**B**) Quantitative expression of isoprenoid biosynthesis genes. Expression levels in stem tissue were compared to WT and normalized to the 18S reference gene. Values are averages of three biological replicates +/ – SD; (*) indicates a significant difference in comparison to _WT_ at _p_\(>\) 0.05. _HMGR_ = 3-hydroxy-3 methylglutaryl-coenzyme A reductase; _FPPS_ = famesyl pyrophosphate synthase; _SQS_ = squalene synthase; _CPT_ = _cis_-phenyltransferase; _SRPP_ = small rubber particle protein; _CBP_ = _CPT_ binding protein. quickly metabolized for detection. However, when SQS was downregulated, GPP levels in root tissue were enhanced, perhaps due to the downregulation of _FPPS_. Results then suggest available FPP and/or GPP (as suggested by the positive correlation between GPP and rubber (\(p<0.05\), Figure S3)) contributed to increased natural rubber production in root tissue. In _Taraxacum kok-saghyz_, another rubber-producing plant, squalene content in rubber-producing root tissues was not affected by the downregulation of _SQS_[36]. However, downregulation of _SQE_, which catalyzes the next step in the metabolic pathway, increased squalene content by 27-fold, with no effect on isoprenoid biosynthesis (pentacyclic triterpenenes and sterols; NR not reported). Isoprenoid pathway regulation is known to be complex. Silencing of _SQS_ in _W. somifera_ resulted in the upregulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (_HMGR_) and other MVA biosynthetic genes [28], attributed to a feedback regulation mechanism. In tobacco (_Nicotiana tabacum_), inhibition of _SQS_ likewise upregulated _HMGR_[37]. In contrast, we found that downregulation of _SQS_ in guayule lowered the expression of _HMGR_, _FPPS_, and _MVK_ (Figure 6). High levels of FPP are known to downregulate the expression of _HMGR_ in humans [38; 39] and insects [40], and prenyl phosphates similarly inhibit _MVK_ in animals [41; 42]. In addition, it is well recognized that excess prenyl phosphates are inhibitory in the microbial production of terpenes [43; 44]. Less is known about MVA feedback regulation by prenyl phosphates in plants. However, [PERSON] and [PERSON] [45] identified HMGR activity as rate-controlling in Hevea rubber biosynthesis, and its mevalonate kinase activity was inhibited by GPP, FPP, and GGPP [46].