# Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule
## 1 Introduction  
Guayule (_Paruthenium argentatum_ Gray), a perennial shrub indigenous to the Chihuahuan desert of Mexico and southwestern Texas, is one of 2000+ natural rubber-producing plant species [1, 2] and is under development as a drought-tolerant industrial crop for the western United States. Natural rubber, a plant secondary metabolite, is a strategic and irreplaceable material vital to modern transportation, medicine, and defense (Critical Agricultural Materials Act 7, Public Law 107-293, U.S.C 178. 2019). In guayule, natural rubber is biosynthesized in stemblast tissues [3], especially in response to abiotic and biotic stresses [4] such as cold, non-freezing temperatures [5, 6, 7]. Natural rubber biosynthesis requires the 15-carbon initiator, farnesyl pyrophosphate (FPP), and the 5-carbon monomer, isopentenyl pyrophosphate (IPP), both derived from the isoprenoid pathway. Plants are major sources of isoprenoids and produce a myriad of them through the cytosolic mevalonic acid (MVA) and chloroplastic methyleryththird phosphate (MEIP) pathways [8, 9, 10].  
Attempts to increase rubber yield in plants have included overexpression of farnesyl pyrophosphate synthase (_FPPS_) [11] and other genes for isoprenoid pathway enzymes [12, 13, 14]. Remarkably, overexpression of isoprenoid production genes in both rubber-producing [13] and non-rubber producing [15] plants led to very high production of the C-30 isoprenoid squalene, which accumulated in subcellular microbodies. Squalene is produced by the condensation of two molecules of FPP in a reaction catalyzed by squalene synthase (SQS, EC 2.5.1.21); consequently, biosynthesis of natural rubber and squalene sharea common isoprenoid pathway substrate at the branching point between triterpene/steroid and polyisoprene biosynthesis [8].  
Squalene synthase (SQS) is a structurally conserved enzyme found ubiquitously in nature [16; 17]; it synthesizes precursors for critical isoprenoids [16]. SQS is considered an essential control point for the management of carbon flux in, for example, forming cholesterol in mammals and sterols and triterpens in plants, depending on the cellular environment [8; 9]. In plants, phytosterols are vital in plant growth and development [18; 19], and triterpene compounds play an essential role in biotic responses [20]; thus, genetic manipulation of _SQS_ has been studied in plants with considerable interest.  
Examples of _SQS_ engineering in plants include the manipulation of triterpene [20] and phytosterol production [19; 21]. Overexpression of _SQS_ from _Panax ginseng_ (_PgSS1_) [22; 23], _Bupleurum falcatum_ (_BfSS1_) [24], _Arabidopsis thaliana_ (_AtSSI_) [25], and _Glycine max_ (_Gm-SQS1_) [26] increased phytosterol and triterpene biosynthesis, establishing _SQS_'s role in metabolic regulation. Suppression of _SQS_ in _Artemisia annua_ upregulated artemisinin biosynthetic genes, increasing the content of the antimalarial compound artemisinin [27]. Silencing SQS in _Withnoldes somifera_, an important medicinal plant in India, led to reduced squalene and withanolide content [28].  
Physiologically and phenotypically, SQS-silenced plants showed decreased sterol content, white leaf pigmentation, and missshaped leaves in apple (_Malus_ x _domestic_) [29]. In rice (_Oryza sativa_), RNAi-mediated transformation of maize squalene synthase impacted root morphology and reduced stomatal conductance, resulting in improved drought tolerance [30].  
While considerable private and public investment over the last decade has verified guayule's potential as an important industrial crop, increased natural rubber (NR) yield remains the primary goal of guayule improvement. In this report, we genetically downregulated squalene synthase expression in guayule by RNAi technology, such that the pool of FPP might be available to enhance natural rubber synthesis (Figure S1).