Overexpression of tocopherol biosynthesis genes in guayule (Parthenium argentatum) reduces rubber, resin and argentatins content in stem and leaf tissues.

Natural rubber produced in stems of the guayule plant (Parthenium argentatum) is susceptible to post-harvest degradation from microbial or thermo-oxidative processes, especially once stems are chipped. As a result, the time from harvest to extraction must be minimized to recover high quality rubber, especially in warm summer months. Tocopherols are natural antioxidants produced in plants through the shikimate and methyl-erythtiol-4-phosphate (MEP) pathways. We hypothesized that increased in vivo guayule tocopherol content might protect rubber from post-harvest degradation, and/or allow reduced use of chemical antioxidants during the extraction process. With the objective of enhancing tocopherol content in guayule, we overexpressed four Arabidopsis thaliana tocopherol pathway genes in AZ-2 guayule via Agrobacterium-mediated transformation. Tocopherol content was increased in leaf and stem tissues of most transgenic lines, and some improvement in thermo-oxidative stability was observed. Overexpression of the four tocopherol biosynthesis enzymes, however, altered other isoprenoid pathways resulting in reduced rubber, resin and argentatins content in guayule stems. The latter molecules are mainly synthesized from precursors derived from the mevalonate (MVA) pathway. Our results suggest the existence of crosstalk between the MEP and MVA pathways in guayule and the possibility that carbon metabolism through the MEP pathway impacts rubber biosynthesis.

Downregulation of Squalene Synthase Broadly Impacts Isoprenoid Biosynthesis in Guayule.

Production of natural rubber by Parthenium argentaum (guayule) requires increased yield for economic sustainability. An RNAi gene silencing strategy was used to engineer isoprenoid biosynthesis by downregulation of squalene synthase (SQS), such that the pool of farnesyl diphosphate (FPP) substrate might instead be available to initiate natural rubber synthesis. Downregulation of SQS resulted in significantly reduced squalene and slightly increased rubber, but not in the same tissues nor to the same extent, partially due to an apparent negative feedback regulatory mechanism that downregulated mevalonate pathway isoprenoid production, presumably associated with excess geranyl pyrophosphate levels. A detailed metabolomics analysis of isoprenoid production in guayule revealed significant differences in metabolism in different tissues, including in active mevalonate and methylerythritol phosphate pathways in stem tissue, where rubber and squalene accumulate. New insights and strategies for engineering isoprenoid production in guayule were identified.

RNASeq analysis of drought-stressed guayule reveals the role of gene transcription for modulating rubber, resin, and carbohydrate synthesis.

The drought-adapted shrub guayule (Parthenium argentatum) produces rubber, a natural product of major commercial importance, and two co-products with potential industrial use: terpene resin and the carbohydrate fructan. The rubber content of guayule plants subjected to water stress is higher compared to that of well-irrigated plants, a fact consistently reported in guayule field evaluations. To better understand how drought influences rubber biosynthesis at the molecular level, a comprehensive transcriptome database was built from drought-stressed guayule stem tissues using de novo RNA-seq and genome-guided assembly, followed by annotation and expression analysis. Despite having higher rubber content, most rubber biosynthesis related genes were down-regulated in drought-stressed guayule, compared to well-irrigated plants, suggesting post-transcriptional effects may regulate drought-induced rubber accumulation. On the other hand, terpene resin biosynthesis genes were unevenly affected by water stress, implying unique environmental influences over transcriptional control of different terpene compounds or classes. Finally, drought induced expression of fructan catabolism genes in guayule and significantly suppressed these fructan biosynthesis genes. It appears then, that in guayule cultivation, irrigation levels might be calibrated in such a regime to enable tunable accumulation of rubber, resin and fructan.

Genetic Engineering of Lesquerella with Increased Ricinoleic Acid Content in Seed Oil.

Seeds of castor (Ricinus communis) are enriched in oil with high levels of the industrially valuable fatty acid ricinoleic acid (18:1OH), but production of this plant is limited because of the cooccurrence of the ricin toxin in its seeds. Lesquerella (Physaria fendleri) is being developed as an alternative industrial oilseed because its seeds accumulate lesquerolic acid (20:1OH), an elongated form of 18:1OH in seed oil which lacks toxins. Synthesis of 20:1OH is through elongation of 18:1OH by a lesquerella elongase, PfKCS18. Oleic acid (18:1) is the substrate for 18:1OH synthesis, but it is also used by fatty acid desaturase 2 (FAD2) and FAD3 to sequentially produce linoleic and linolenic acids. To develop lesquerella that produces 18:1OH-rich seed oils such as castor, RNA interference sequences targeting KCS18, FAD2 and FAD3 were introduced to lesquerella to suppress the elongation and desaturation steps. Seeds from transgenic lines had increased 18:1OH to 1.1-26.6% compared with that of 0.4-0.6% in wild-type (WT) seeds. Multiple lines had reduced 18:1OH levels in the T2 generation, including a top line with 18:1OH reduced from 26.7% to 19%. Transgenic lines also accumulated more 18:1 than that of WT, indicating that 18:1 is not efficiently used for 18:1OH synthesis and accumulation. Factors limiting 18:1OH accumulation and new targets for further increasing 18:1OH production are discussed. Our results provide insights into complex mechanisms of oil biosynthesis in lesquerella and show the biotechnological potential to tailor lesquerella seeds to produce castor-like industrial oil functionality.

Transcriptomic and evolutionary analysis of the mechanisms by which P. argentatum, a rubber producing perennial, responds to drought.

BACKGROUND: Guayule (Parthenium argentatum Gray) is a drought tolerant, rubber producing perennial shrub native to northern Mexico and the US Southwest. Hevea brasiliensis, currently the world's only source of natural rubber, is grown as a monoculture, leaving it vulnerable to both biotic and abiotic stressors. Isolation of rubber from guayule occurs by mechanical harvesting of the entire plant. It has been reported that environmental conditions leading up to harvest have a profound impact on rubber yield. The link between rubber biosynthesis and drought, a common environmental condition in guayule's native habitat, is currently unclear. RESULTS: We took a transcriptomic and comparative genomic approach to determine how drought impacts rubber biosynthesis in guayule. We compared transcriptional profiles of stem tissue, the location of guayule rubber biosynthesis, collected from field-grown plants subjected to water-deficit (drought) and well-watered (control) conditions. Plants subjected to the imposed drought conditions displayed an increase in production of transcripts associated with defense responses and water homeostasis, and a decrease in transcripts associated with rubber biosynthesis. An evolutionary and comparative analysis of stress-response transcripts suggests that more anciently duplicated transcripts shared among the Asteraceae, rather than recently derived duplicates, are contributing to the drought response observed in guayule. In addition, we identified several deeply conserved long non-coding RNAs (lncRNAs) containing microRNA binding motifs. One lncRNA in particular, with origins at the base of Asteraceae, may be regulating the vegetative to reproductive transition observed in water-stressed guayule by acting as a miRNA sponge for miR166. CONCLUSIONS: These data represent the first genomic analyses of how guayule responds to drought like conditions in agricultural production settings. We identified an inverse relationship between stress-responsive transcripts and those associated with precursor pathways to rubber biosynthesis suggesting a physiological trade-off between maintaining homeostasis and plant productivity. We also identify a number of regulators of abiotic responses, including transcription factors and lncRNAs, that are strong candidates for future projects aimed at modulating rubber biosynthesis under water-limiting conditions common to guayules' native production environment.

Downregulation of a CYP74 Rubber Particle Protein Increases Natural Rubber Production in Parthenium argentatum.

We report functional genomics studies of a CYP74 rubber particle protein from Parthenium argentatum, commonly called guayule. Previously identified as an allene oxide synthase (AOS), this CYP74 constitutes the most abundant protein found in guayule rubber particles. Transgenic guayule lines with AOS gene expression down-regulated by RNAi (AOSi) exhibited strong phenotypes that included agricultural traits conducive to enhancing rubber yield. AOSi lines had higher leaf and stem biomass, thicker stembark tissues, increased stem branching and improved net photosynthetic rate. Importantly, the rubber content was significantly increased in AOSi lines compared to the wild-type (WT), vector control and AOS overexpressing (AOSoe) lines, when grown in controlled environments both in tissue-culture media and in greenhouse/growth chambers. Rubber particles from AOSi plants consistently had less AOS particle-associated protein, and lower activity (for conversion of 13-HPOT to allene oxide). Yet plants with downregulated AOS showed higher rubber transferase enzyme activity. The increase in biomass in AOSi lines was associated with not only increases in the rate of photosynthesis and non-photochemical quenching (NPQ), in the cold, but also in the content of the phytohormone SA, along with a decrease in JA, GAs, and ABA. The increase in biosynthetic activity and rubber content could further result from the negative regulation of AOS expression by high levels of salicylic acid in AOSi lines and when introduced exogenously. It is apparent that AOS in guayule plays a pivotal role in rubber production and plant growth.

Modular assembly of transposable element arrays by microsatellite targeting in the guayule and rice genomes.

BACKGROUND: Guayule (Parthenium argentatum A. Gray) is a rubber-producing desert shrub native to Mexico and the United States. Guayule represents an alternative to Hevea brasiliensis as a source for commercial natural rubber. The efficient application of modern molecular/genetic tools to guayule improvement requires characterization of its genome. RESULTS: The 1.6 Gb guayule genome was sequenced, assembled and annotated. The final 1.5 Gb assembly, while fragmented (N50 = 22 kb), maps > 95% of the shotgun reads and is essentially complete. Approximately 40,000 transcribed, protein encoding genes were annotated on the assembly. Further characterization of this genome revealed 15 families of small, microsatellite-associated, transposable elements (TEs) with unexpected chromosomal distribution profiles. These SaTar (Satellite Targeted) elements, which are non-autonomous Mu-like elements (MULEs), were frequently observed in multimeric linear arrays of unrelated individual elements within which no individual element is interrupted by another. This uniformly non-nested TE multimer architecture has not been previously described in either eukaryotic or prokaryotic genomes. Five families of similarly distributed non-autonomous MULEs (microsatellite associated, modularly assembled) were characterized in the rice genome. Families of TEs with similar structures and distribution profiles were identified in sorghum and citrus. CONCLUSION: The sequencing and assembly of the guayule genome provides a foundation for application of current crop improvement technologies to this plant. In addition, characterization of this genome revealed SaTar elements with distribution profiles unique among TEs. Satar targeting appears based on an alternative MULE recombination mechanism with the potential to impact gene evolution.

Transcriptome and gene expression analysis in cold-acclimated guayule (Parthenium argentatum) rubber-producing tissue.

Natural rubber biosynthesis in guayule (Parthenium argentatum Gray) is associated with moderately cold night temperatures. To begin to dissect the molecular events triggered by cold temperatures that govern rubber synthesis induction in guayule, the transcriptome of bark tissue, where rubber is produced, was investigated. A total of 11,748 quality expressed sequence tags (ESTs) were obtained. The vast majority of ESTs encoded proteins that are similar to stress-related proteins, whereas those encoding rubber biosynthesis-related proteins comprised just over one percent of the ESTs. Sequence information derived from the ESTs was used to design primers for quantitative analysis of the expression of genes that encode selected enzymes and proteins with potential impact on rubber biosynthesis in field-grown guayule plants, including 3-hydroxy-3-methylglutaryl-CoA synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, farnesyl pyrophosphate synthase, squalene synthase, small rubber particle protein, allene oxide synthase, and cis-prenyl transferase. Gene expression was studied for field-grown plants during the normal course of seasonal variation in temperature (monthly average maximum 41.7 °C to minimum 0 °C, from November 2005 through March 2007) and rubber transferase enzymatic activity was also evaluated. Levels of gene expression did not correlate with air temperatures nor with rubber transferase activity. Interestingly, a sudden increase in night temperature 10 days before harvest took place in advance of the highest CPT gene expression level.