# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 2 Results
In fact, FPP, the primary substrate for squalene synthesis and the natural rubber initiator molecule, was only detected in leaf and not in stem or root tissue. Note that geranyl geranyl diphosphate (GGPP) was not detected in any tissues, and GPP was found mainly in root tissue (Table S1). In addition, mevalonate phosphate and pyrophosphate were at trace levels or not detected. In comparing results across genotypes, we note that in the quantification of plant metabolites, there were several cases where the VC concentrations deviated significantly from those for the WT control (Table S1). Differences were, at times, systematic across tissue types, i.e., pyruvate concentrations in VC plants were higher than the WT in all 3 tissues; MEP and HMB-PP levels for VC were lower than the WT, also for all three tissues. While it is possible that gene insertion may have produced unintended effects, the specific reason for these differences is not known. Our analysis, therefore, primarily considered comparisons between _SQSi_ and WT plants. Figure 3: Multivariate analysis visualization of precursor and intermediate compounds involved in natural rubber and squalene biosynthesis across all control and _SQSi_ lines and tissues. Individual samples with different genotype and tissue type appear on the vertical axis, and the pathway of each identified measured compound is represented in the horizontal axis. Relative abundance of targeted metabolites is represented by color; orange indicates high relative abundance, while blue indicates low abundance. [PERSON]’s correlation-based hierarchical cluster analysis indicates a strong separation across different tissue types (column-wise), while no clear clustering pattern was observed in terms of pathways (row-wise). The heatmap scale ranges from \(-\)1 to 5 (data pre-treated with autoscaling). Downregulation of _SQS_ impacted concentrations of isoprenoid pathway intermediates in a tissue-specific manner. In stem tissues, MVA pathway intermediates acetyl co-A and mevalonate showed significantly reduced concentrations in _SQSi_ lines compared with controls (Figure 5, Table S1). In contrast, for root tissues, the mevalonate concentration was higher in the _SQSi_ lines and both hydroxymethylglutaryl coenzyme A (HMG-CoA) and GPP were enriched vs. WT (but not VC). Mevalonate was also slightly enriched in leaf but not to statistical significance. Figure 4: Isoprenoid metabolism in WT guayule. Concentrations (\(\mu\)g/g dry weight) of metabolites in 5-month-old guayule plant tissues including: (1) the cytosolic mevalonic acid Pathway intermediates: acetyl coenzyme A (AC-CoA); acetoacetyl coenzyme A (Acac-CoA); hydroxymethylglutaryl coenzyme A (HMG-CoA); mevalonate (MVA); mevalonate-5-phosphate (MVA-P); and mevalonate diphosphate (MVA-PP). (2) The methylerititrol phosphate pathway (chloroplast) intermediates: pyruvate and glyceraldehyde-3-phosphate (G3P); 1-deoxyylulose-5-phosphate (DXP); methylerythitol-4-phosphate (MEP); 4-(cytidine-5\({}^{\prime}\)diphospho-methylerythrict (CDP-ME); methylerythnitol-2,4-cyclodiphosphate (CDP-MEP); 2-C-methyl-d-eryththrict 2,4-cyclodiphosphate (MEePP); and 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP). Also shown, common products of both pathways: isoprenenyl pyrophosphate (IPP)/dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), squalene, and natural rubber. Error bars represent \(+/-1\) standard error. Entering the MEP pathway, pyruvate was detected at the highest levels of all upstream isoprenoid pathway compounds in all tissues and all genotypes, as might be expected for a primary metabolite [9] (Table S1). pyruvate concentrations for _SQSi_ lines did not vary significantly from controls (except higher pyruvate in stem for SQSi-M). However, the important intermediate 2-C-methylethylmitol 4-phosphate (MEP) was higher in stem and root for _SQSi_ lines compared to WT (Figure 5, Table S1). In addition, HMBPP was present at higher concentrations in root tissue of _SQSi_ lines compared to controls.