| Higher plants biosynthesize thousands of functional terpenoids via differentmetabolite pathways. These varied terpenoids play important roles in plant respiration,photosynthesis, regulation of growth and development. Irregular monoterpenes arecommon and important in the Asteraceae family. There are two classical metabolicpathways in different subcellular compartments, i.e. cytosolic mevalonate (MVA)pathway and2-C-methyl-D-derythritol-4-phosphate (MEP) pathway.Pyrethrins are a natural insecticide biosynthesized by Tanacetum cinerariifolium (T.cinerariaefolium). Meanwhile, there are a set of six structurally close monoterpeneesters produced by two monoterpenic acids (pyrethric acid and chrysanthemic acid)with either of three ketone alcohols(cinerolone, jasmolone and pyrethrolone)esterification in pyrethrins. Acid moieties of pyrethrins are terpenoids synthesized witha cyclopropane ring via the MEP pathway (2-C-methyl-D-derythritol-4-phosphatepathway), while rethrolone moieties are synthesized from linolenic acid via the oxylipinpathway.Up to now, little is known about the irregular isoprenoids synthesis at themolecular level except for the sterol and carotenoid pathways. Rivera et al. stated thatchrysanthemyl pyrophosphate in T. cinerariaefolium involved in the first reaction inpyrethrins synthesis by condensing two molecules of DMAPP with CDS. Even thoughCDS was firstly identified ten years ago, the molecular functions of T.cinerariaefoliumCDS have not yet been clearly clarified during pyrethrins biosynthesis.In this study, we cloned a CDS_CCI2gene from T.cinerariaefolium cDNA, andinvestigated its subcellular localization, expression patterns in different tissues or undervarious stress treatments. Meanwhile, CDS_CCI2was overexpressed in tomato plantsand silenced by VIGS strategy in T.cinerariaefolium as an attempt to characterize itsbiological function. Furthermore, an efficient Agrobacterium tumefaciens-mediatedtransformation system in T.cinerariaefolium was established, which is fundamental totransgenic study on pyrethrin biosynthesis.The main results and conclusions are as follows:1. Chrysanthemyl diphosphate synthase (CDS) gene named CDS_CCI2(GenBankaccession No.HQ235057) was isolated from T.cinerariaefolium. The cDNA ofCDS_CCI2is in length with an1188bp open reading frame (ORF) encoding a polypeptide of395amino acids. CDS_CCI2was classed into CDS family byphylogenetic analysis of putative CDS_CCI2protein.2. The subcellular localization of the CDS_CCI2protein was investigated byfusion to GFP under the control of the CaMV35S promoter, and was transientlyexpressed in tobacco protoplasts. Fluorescence microscopy analysis associated withimage overlapping techniques showed that CDS_CCI2was able to fully direct thefusion protein to the plastid, indicating that CDS_CCI2may have a regulatory functionin pyrethrin biosynthesis.3. Quantitative RT-PCR was performed to determine the expression pattern ofCDS_CCI2gene in different tissues of T.cinerariaefolium. It revealed that CDS_CCI2transcripts were detected in all tested tissues (Roots, leaves, stems, flower buds, flowers)and the CDS_CCI2expression was utmost in flower buds as well.4. The expression of CDS_CCI2gene was detected in T.cinerariaefolium seedlingsexposed to various abiotic stresses. The results revealed that CDS_CCI2transcriptswere up-regulated by abscisic acid (ABA), methyl jasmonate (MeJA) and ethreltreatment.5. To investigate the physiological roles of CDS_CCI2in T.cinerariaefoliumdevelopment, the CDS_CCI2cDNA was overexpressed in tomato. Transgenic35S::CDS_CCI2lines displayed various morphological defects, such as dwarf growthhabit, reduced flower fertility, small fruit and parthenocarpy compared with wild-typeplants. The hypocotyls in35S::CDS_CCI2seedlings were shorter than those inwild-type seedlings. Overexpression of CDS_CCI2also altered the production ofendogenous secondary metabolites. Our data indicated that CDS_CCI2affected thebiosynthesis of GA and ABA.6. To characterize CDS_CCI2functionally, a loss of function approach wasimplemented using the tobacco rattle virus (TRV)-mediated gene silencing (VIGS)strategy. The present study uncovered the role of CDS_CCI2in affecting chlorophyll,carotenoids and pyrethrins biosynthesis.7. Establishing an efficient Agrobacterium tumefaciens-mediated transformationsystem for T.cinerariaefolium is fundamental to transgenic study on pyrethrinsbiosynthesis. The results revealed that cotyledons from tissue culture were excellentexplants for transformation. |
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