# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 2 Results  
RNA interference was used to downregulate _SQS_ in guayule line G7-11/AZ-2. This approach is a sequence-specific gene downregulation mediated by introducing double-stranded RNA into the cell, resulting in the degradation of the specific mRNA and function loss for the target gene [31; 32]. To confirm positive _SQS_-transformed plants, the plasmid construct segment of the inserted bialaphos (BAR) gene sequence (Figure 1A) was amplified by polymerase chain reaction (PCR) from leaves of 8-week-old tissue cultured plants. Eight lines were deemed positively transformed (Figure 1B) and labeled SQSi. Quantitative reverse transcription Polymerase Chain Reaction (qPCR) analysis verified that the _SQS_ gene was suppressed in the SQSi lines but not in the wildtype (WT) and vector control (VC) plants (Figure 1C). Phenotype screening of tissue-culture-grown plants showed, on average, 52% higher natural rubber content (Figure 1D) and 10% lower resin (squalene-containing fraction) content (not shown) for SQSi plants compared to the control. Three biological SQSi lines, J, L, and M, were selected for greenhouse/growth chamber phenotype characterization based on consistent results in replicated experiments. When grown in soil, at 5 months, plant architectural features of _SQSi_ lines did not show remarkable differences compared to WT in, for example, biomass, size, or SPAD (chlorophyll) readings (Figure S2), in contrast to reports in apple [29] and rice [30]. Surprisingly, significant differences were found between the WT control and the VC plants for number of branches and number of flowering buds. Guayule sequesters carbon dioxide and converts it to metabolites, including natural rubber, squalene, and'resin', a mixture of terpen, fatty acids, low molecular weight rubber, and other chemicals co-extracted during rubber biorefining processes. In our investigation, squalene content varied by tissue type and genotype for plants grown in greenhouse/growth chamber conditions. Analysis of control plants by gas chromatography-mass spectrometry (GC-MS) found the highest level of squalene in stem tissues (up to 30+ \(\mu\)g per g dry weight tissue), followed by leaf, then root (Figure 2A). All \(SQSi\) lines analyzed had less squalene in stem tissues, -50-60% reduction compared with the WT (\(p<0.05\); Figure 2A). No significant differences were found in squalene content for leaf and roots tissues in \(SQSi\) lines compared with controls. The natural rubber concentration for growth chamber-grown plants, at less than 1% dry weight (Figure 2B), was significantly lower than would be expected in field plants. The highest rubber concentration was found in root tissue, as had been previously reported in young [33] and mature [34] guayule. In root tissue, natural rubber production was enriched by up to 65% in \(SQSi\) lines (significantly in \(SQSi\)-M) compared to the WT but not compared to the VC line (Figure 2B). Less rubber was found in stem and leaf tissues; small increases in NR production in the \(SQSi\) lines' stem tissue compared to the WT were not statistically significant and surprisingly lower than the VC. Resin concentration (up to 6% dw) was highest in leaf tissue, as expected (Figure 2C), and did not vary significantly in leaf or root tissues. Differences in resin content of stem tissue between \(SQSi\) lines and the WT control were noted but inconsistent (mixed higher and lower). Figure 1: **(A)** T-DNA region of transformation constructs. pND9, \(\beta\)-glucuronidase plus (\(GUSPlus\)) reporter gene driven by _Medicago truncatula_ promoter (MtHP-P) with ubiquitin 3 terminator (Ub3-T) and a shortened potato polyvinyl promoter (409-Ps) controlling the expression of the neomycin phosphotransferase II (_nptII_) gene for kanamycin resistance with ocropine synthase terminator (OcsT). pND9_SQSi, the \(GUSPlus\) gene in pND9, is replaced with a hairpin RNAi construct built with two inverted repeats of the partial _P. argentatum SQS_ gene (SQSR and SQSF); a bialaphos resistance (\(BAR\)) gene was inserted between them.