# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 3 Discussion
In our study, the MVA pathway was clearly impacted; when _HMGR_, _MVK_, and _FPPS_ were downregulated in _SQSi_ lines, higher concentrations of their respective substrates (HMG-coA, mevalonate, and GPP) accumulated, mainly in root tissues (Figure 5; Table S1). Taken together, the results suggest the intriguing possibility that GPP may be the preferred rubber biosynthesis initiator [47] in guayule root tissue and/or in the absence of FPP. Further evidence indicates that elevated GPP was responsible for the observed negative feedback regulation in the MVA pathway in guayule or that the concentration of FPP needed to suppress gene transcription was below detection limits (0.01 \(\mathrm{\SIUnitSymbolMicro g}\)/g dw tissue). Interestingly, in stem tissue, expression of the gene encoding rubber biosynthesis-associated _cis_-prenyl transferase 3 (CPT3) significantly increased in _SQSi_ lines compared to WT controls, while levels of the gene encoding CPT3-binding protein (CBP) were unaffected (Figure 6), indicating these genes are regulated differently. CPT3 and CBP proteins are hypothesized to form an enzymatic complex (along with other yet-to-be-identified factors) necessary for rubber biosynthesis [48; 49]. Despite the fact that _CPT3_ transcription is stimulated by MVA pathway disruptions, no increase in rubber was detected in stem tissue, possibly due to the unchanged levels of _CBP_ expression and concomitant translation to form the necessary active enzymatic complex. Expression levels of the gene encoding SRPP, a protein known to associate with the rubber particle, were also unaffected in stem tissue of _SQSi_ lines compared to controls. SRPP has an indirect rubber biosynthesis function [50; 51], and it appears to be insensitive to MVA pathway changes. Characterization of isoprenoid pathway intermediates in this study revealed new insights beyond phenotypes for engineered guayule. In WT plants, for example, higher concentrations of HMBPP, IPP/DMAPP, and FPP found in the leaf are consistent with reported high terpene resin content in guayule leaves [52; 53; 54]. Considering the MEV pathway as a whole, tissue-specific accumulation patterns of mevalonate were, intriguingly, the inverse of the accumulation of rubber (Figure 4). Root tissues showed less accumulation of mevalonate compared to stems and lower IPP/DMAPP compared to leaves, suggesting higher flux through the MVA pathway, which may explain why more rubber was concentrated in root tissues for these greenhouse plants. Interestingly, the accumulation of MEcPP in the stem and root, as well as HMBPP in the leaf, suggests that further optimization of the MEP pathway (e.g., overexpressing _ispG_ and _ispH_) could result in greater production of rubber in these tissues. While the MVA pathway is considered the preferential route for C\({}_{30}\) isoprenoid formation in plants [55], downregulation of _SQS_ in guayule impacted the metabolite pools in both the MVA pathway in the cytosol and the MEP pathway in chloroplasts. Evidence for active exchange between intermediates in these pathways has been established [56; 57; 58] but poorly understood in guayule. In our investigation, up to five-fold higher mevalonate concentration and up to double MEP concentrations were found in plant tissues of _SQSi_ guayule. While the feedback mechanisms controlling both the MVA and MEP pathways are numerous and complex [55], our results provide evidence of crosstalk in the feedback regulation of both isoprenoid pathways in guayule. The metabolite data also suggests that rerouting carbon from squalene, especially in the stem, might increase rubber production, consistent with our strategy. In our work, downregulation of squalene synthase reduced squalene content by more than half in guayule stems, potentially creating a pool of excess FPP. If half of the excess FPP had been used to initiate new rubber molecules, about 33,000 \(\upmu\)g/g dw rubber would have resulted, an enormous increase in rubber content of +3% dw, which was not observed.