Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum        

Abstract

Certain Taraxacum species, such as Taraxacum koksaghyz and Taraxacum brevicorniculatum, produce large amounts of high-quality natural rubber in their latex, the milky cytoplasm of specialized cells known as laticifers. This high-molecular mass biopolymer consists mainly of poly(cis-1,4-isoprene) and is deposited in rubber particles by particle-bound enzymes that carry out the stereospecific condensation of isopentenyl diphosphate units. The polymer configuration suggests that the chain-elongating enzyme (rubber transferase; EC 2.5.1.20) is a cis-prenyltransferase (CPT). Here, we present a comprehensive analysis of transgenic T. brevicorniculatum plants in which the expression of three recently isolated CPTs known to be associated with rubber particles (TbCPT1 to -3) was heavily depleted by laticifer-specific RNA interference (RNAi). Analysis of the CPT-RNAi plants by nuclear magnetic resonance, size-exclusion chromatography, and gas chromatography-mass spectrometry indicated a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and the main storage carbohydrate, inulin. Transmission electron microscopy revealed that the laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. We also observed lower activity of hydroxymethylglutaryl-coenzyme A reductase, the key enzyme in the mevalonate pathway, reflecting homeostatic control of the isopentenyl diphosphate pool. To our knowledge, this is the first in planta demonstration of latex-specific CPT activity in rubber biosynthesis.

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

“…The transformation of T. brevicorniculatum and T. koksaghyz plants was carried out as previously described with slight modifications (Post et al ., 2012). T. koksaghyz leaf discs were incubated on callus induction medium containing 400 mg/L amoxicillin, and shoot induction medium was supplemented with 1 mg/L kinetin, 100 μg/L indole acetic acid and 200 mg/L amoxicillin.…”

“…This is supported by the observation that wild‐grown T. koksaghyz can accumulate NR during dormancy, when the inulin level decreases (Ulmann, 1951). The opposite process was shown in T. brevicorniculatum plants that accumulate more inulin due to the inhibition of CPT activity by RNA interference, which resulted in lower levels of NR (Post et al ., 2012). The excess IPP was first used for the formation of other isoprenoids such as sterols and pentacyclic triterpenes, and the eventual saturation of this relief pathway led to the accumulation of MVA pathway precursors, affecting upstream flux and redirecting carbon to the storage product inulin (Post et al ., 2012).…”

Manoyl Oxide (13R), the Biosynthetic Precursor of Forskolin, Is Synthesized in Specialized Root Cork Cells in Coleus forskohlii

“…Plant anatomical features and cellular structures typically associated with the biosynthesis and storage of large amounts of terpenoids are well studied and include glandular trichomes (Gershenzon et al, 2000;Iijima et al, 2004;Siebert, 2004;Schilmiller et al, 2008;Xie et al, 2008;Chatzopoulou et al, 2010;Lane et al, 2010), laticifer cells (Mahlberg, 1993;Post et al, 2012), resin cells, resin blisters, or resin ducts (Martin et al, 2002;Zulak and Bohlmann, 2010), and glandular cavities lined by epithelial cells (Heskes et al, 2012;Voo et al, 2012). Here, we showed accumulation of the partly lipophilic diterpenoid forskolin and the presence of its precursor, (13R) manoyl oxide, together with a series of other labdane-and abietane-type diterpenoids, in root cork cells of C. forskohlii.…”

A Lettuce (Lactuca sativa) Homolog of Human Nogo-B Receptor Interacts with cis-Prenyltransferase and Is Necessary for Natural Rubber Biosynthesis

“…During the course of this work, RNAi-mediated silencing of three highly homologous CPTs (TbCPT1-3; Ͼ96% amino acid identity to each other) in dandelion (Taraxacum brevicorniculatum) showed the reduction of NR in this plant (20). TbCPTs display very low homology to CPTL2 (17% amino acid identity), and importantly they possess all five conserved motifs required for CPT activity.…”

“…Recently, three dandelion CPTs displaying Ͼ96% amino acid identity were silenced by RNAi (20). The resulting transgenic dandelions showed elimination of NR in the latex, thereby proving the necessity of CPT in NR biosynthesis, although purified recombinant CPT activity was not reported.…”