# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 2 Results
LB, left border, RB, right border. **(B)** Confirmation of transgenic plants. Guayule transgenic plants were confirmed with PCR using genomic DNA as a template. Primers for the BAR gene were used to confirm the guayule plants harboring pND9_SQS_ T-DNA. The expected product size is 551 bp in the downregulated lines. **(C)** Quantitative-RT-PCR (qPCR) of stemback tissues. Expression level of 170 bp PCR product in the \(SQS\) coding sequence compared with WT. The \(18S\) gene (195 bp) was used as an internal control. The asterisks (*) indicate a significant difference in comparison with WT at \(p\leq 0.05\). **(D)** Rubber content in tissue-cultured plants, as determined by ASE microassay. WT = G7-11 wildtype; VC = vector control pND9; SQSi-A, -E, -F, -H, -J, -K, -L, and -M represent independent gene downregulation events. So, quite interestingly, downregulation of _SQS_ resulted in significantly reduced squalene in all lines and enhanced rubber concentrations in some but not in the same tissues nor to the same extent. To gain insight into the pathway flux, concentrations of 13 additional precursor and intermediate compounds involved in natural rubber and squalene biosynthesis were quantified by liquid chromatography-mass spectrometry (LC-MS) (Table S1). Multivariate analysis of the LC-MS results across both control and transgenic lines (Figure 3) illustrates distribution patterns in stem, leaf, and root tissue. The resulting heat map reveals the primary level of separation is by tissue type, dominating over genotype. Moreover, in stem tissue, WT and VC lines are grouped perfectly away from transgenic lines, suggesting the _SQSi_ genetic modification has a more profound effect on metabolism in young stem tissue, where squalene is most concentrated in guayule. Levels of metabolites were dramatically different, especially between leaf tissue and stem tissue, as shown by the high abundance of orange blocks in the left upper corner and right lower corner of Figure 3. However, these blocks could not be assigned to specific pathways since MEP (chloroplastic) and MVA (cytosolic) pathways did not show patterns of clustering (as shown by the left axis pathways' color block distribution), suggesting a mixed and more  
Figure 2: Secondary metabolites in guayule. Concentration of targeted metabolites in 5-month-old guayule plant tissues: WT = wildtype, VC = vector control, squalene synthase downregulated lines _SQSi_-J, -L, -M represent 3 independent transformation events. (**A**) Squalene (ug/g dw), determined from purified acetone extracted tissue samples using gas chromatography-mass spectrometry (GC-MS). (**B**) Natural rubber (% dw), and (**C**) resin (% dw), quantified by accelerated solvent extraction. Average value of 3 biological replicates; error bars are \(+/-\) standard error; symbol \({}^{*}\) denotes significantly different from WT at \(p<0.05\); **\({}^{**}\) at \(p<0.005\). complex underlying regulation between these two metabolic pathways. Further, correlation analysis (Figure S3) revealed strong and significant positive correlations between rubber and GPP (r = 0.69, \(p<\) 0.05), and resin and FPP (r = 0.77, \(p<\) 0.05). Squalene was positively correlated with both acetyl CoA and mevalonate-5-phosphate (MVAP). Next, a more detailed analysis of the LC-MS data, the first of its kind for guayule, provided new insight into the isoprenoid pathway flux in guayule. The results for WT plants (Figure 4) clearly illustrate higher concentrations of mevalonate pathway metabolites, especially acetyl co-A, HMG-CoA, and mevalonate, in WT stem tissues compared with leaf and root tissues. MEP pathway intermediates, including the precursor pyruvate, were also more abundant in stem tissue (except 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP)). We note that for guayule, stem tissue, where rubber biosynthesis occurs, includes soft bark parenchyma tissue rich in chloroplasts. Leaf tissue presented the highest concentrations of HMBPP, isopenetyl pyrophosphate (IPP)/di-methylallyl diphosphate (DMAPP), and FPP.