The Regulation And Biosynthesis Of The Terpenoid Volatile Involved Into The Floral Scent And Stress Defence In5Landscape Plant Species

Terpenoids constitute the largest class of secondary metabolites made by plants. Part ofthe terpenoids exhibit the volatile property, including semiterpenes, monoterpenes,sesquiterpenes and part of the diterpenes. Terpenoids play as the role of the interaction withother organisms in the environment, which is significant to the reproduction and defence forthe plants. Taking Gomphrena globosa, Wisteria, Cyperus Iria, Populus trichocarpa andSelaginella moellendorffii as the model, the purpose of this study is to investigate thecomposition, factors of regulation, biological function and biosynthesis of the terpenoids inthe landscape plants with different evolution level in taxonomy to elucidate the origin andevolution of the terpenoids in plants. The main contents in this dessertation contain thecomposition and regulation of the volatile terpenoids in the floral scent of the herbalG.globosa and the woody Wisteria, the effects of the biotic and abiotic stress to the emissionand biosynthesis of the terpenoids in monocot C.Iria and dicot P.trichocarpa, the effects ofthe biotic and abiotic stress to the emission and biosynthesis of the terpenoids in lycopodS.moellendorffii.The main results are as follows:(1) Volatile chemicals emitted from the flowers of four G.globosa cultivars werecollected using a dynamic headspace technique and analyzed using gas chromatography–massspectrometry.5,2,1,2voaltile terpenoids were identified from 'Fireworks','Las VegasWhite','Las Vegas Pink' and 'Las Vegas Purple' respectively. The emssion rates ofterpenoid account for63.3%,25.8%,19.3%and22.4%in the total floral volatiles in thesefour cultivars respectively.'FW' showed the highest level of emission of terpenoids amongthe four cultivars, with the monoterpene β-ocimene as the dominant compound in the floralscent which accounts for52.2%of the total floral volatile compounds. The emission rate ofthe terpenoids in 'FW' showed a diurnal pattern with the maximum emission occurring at lateafternoon13:00pm-17:00pm and the lowest level of emission occurring at the night21:00pm-1:00am. Further analysis showed that this diurnal emission of G.globosa floral terpenoidsvolatiles is independent of light and regulated by circadian clock. The emission of floralvolatiles from 'FW' flowers that were treated with several chemicals was also analyzed. Thetreatment with silver thiosulphate (STS), an ethylene inhibitor, led to enhanced emission of total volatiles. In contrast, the treatments with salicylic acid (SA) and jasmonic acid (JA) ledto enhanced emission of total floral volatiles at4h but reduced emission at24h after thetreatment.(2) Volatile chemicals emitted from the flowers of chinese wisteria (W.sinenesis) andjapanese wisteria (W.floribunda) were collected using a dynamic headspace technique andidentified using gas chromatography–mass spectrometry.6terpenoids compounds weredetected from chinese wisteria flowers, including3monoterpenes,1sesquiterpene and2homoterpenes with the total emission rate as16.9μg·h^-1·g^-1, while10terpenoids compoundswere detected from japanese wisteria flowers, including3monoterpnes,6sesquiterpenes and1homoterpene with the total emission rate as16.1μg·h^-1·g^-1. Two monoterpenes, β-ocimeneand linalool were the most abundant compounds emitted from both species. Chinese wisteriawas selected as a model for further study of volatile emission from different parts of flowers,emission dynamics, and regulation of terpenoid production.5,3,4,4terpenoids wereidentified from the petals, sepals, stamens coupled with pistils, and pedicels. Although somevolatiles were detected from all parts of chinese wisteria flowers including monoterpeneβ-ocimene, linalool, and homoterpene TMTT, other terpenoids showed tissue specificity.Petals showed the highest level of total emission rate of terpenoid. The emission of floralterpenoids displayed a diurnal pattern with the maximal emissions occurring at9:00am-10:00am and the lowest level of emission occurring at1:00am-2:00am. This rhythmic pattern wasdetermined to be light-dependent. Regulation of floral terpenoids emission by exogenouschemicals, including STS, SA and JA, also was analyzed. Generally, jasmonic acid promotedthe emission of floral volatiles. In contrast, neither silver thiosulphate nor salicylic acidshowed a significant effect on floral terpenoids emission.(3) Composition of these terpenoids and their concentrations in leaves, flowers and rootsof C.Iria at different developmental stages through the whole lifetime, were determined.During the immature stage, the total concentration of sesquiterpenoids increased and reachedthe maximum level at70d and then decreased until the emergence of inflorescence.Monoterpenes started to accumulate from90d only in leaves. During the mature stage, thetotal concentration of sesquiterpenoids increased dramatically. It indicates that thesesquiterpenoids were mainly produced in the new-born and more dynamic tissues. Somesesquiterpenoids showed temporal and saptial specificity. The total amount of monoterpenes,mainly accumulated in flower, kept increaseing until130d, displaying temporal and saptialspecificity. The effect of several biotic or abiotic stress to the produce of terpenoids wasstudied. The concentration of sesquiterpenoids can be promoted significantly by methyljasmonate (MeJA) in leaves. In contrast, SA had no significant effect to accumulation of the total sesquiterpenoids in leaves. Polyethylene Glycol8000(PEG8000) used to imitate thestress of drought could promote the concentration of total sesquiterpenoids significantly inleave. Infestation of beet armyworm in leaves promoted the concentration of totalsesquiterpenoids significantly and could induce the producing of more variety ofmonoterpenes and sesquiterpenoids. However, no effect of neither the fall armyworminfestation nor physical wounding to the concentration of terpenoid in C. Iria leaves can bedectected. The volatile of C.iria leaves was found to possess the anti-fungal effect toFusarium graminearum.(4) The threatment of MeJA, SA and alamethicin (Ala) showed signifiacant effect ofpromotion on the emission of terpenoids volatile from the leaves of P.trichocarpa in the longterm (24h). Moreover, the production of monoterpene (β-Ocimene), sesquiterpenes(α-Bergamotene, Nerolidol Z and E), homoterpenes (DMNT and TMTT) can be inducedspecificly by the treatment of Ala after24h. The infestation of the willow leaf beetle (WLB)larve could promote the emission rate of terpenoids from the leaves of poplar and couldinduce the producing of monoterpne (β-Ocimene), sesquiterpenes (β-Caryophyllene,Germacrene D, α-Humulene, Nerolidol Z and E) and homoterpenes (DMNT and TMTT)specificly. Four putative sesquiterpene synthase genes, PtTPS1-PtTPS4, were identified andcharacterized by the alignment and phylogenetic analysis of terpene synthase (TPS) candidategenes in P.trichocarpa. The semi-quantitatively expression analysis showed that PtTPS1andPtTPS2could express in all the tisssues with the highest expression in the stem; PtTPS3could only express in the young leaves; PtTPS4could express in all the tisssues with thehighest expression in the young leaves. PtTPS1only expressed in the leaves with SAtreatment after both2h and24h; PtTPS2expressed in the leaves of all the treatment exceptthe control after24h; PtTPS3only expressed in the leaves infested by WLB; PtTPS4couldexpress in the leaves after all the treatments.The full length of PtTPS1-4were cloned. Theresults of sequencing found that PtTPS1is a pseudogene, while other3genes are functionalbased on their protein sequence. PtTPS2-PtTPS4were expressed in E.coli and theirbiochemical activity assays were conducted. It displayed that all these three expressedrecombinant protein could catalyze to produce the sesquiterpene with FPP as the substrate.The main products of PtTPS2were β-Farnesene and Elemol; The main products of PtTPS3were β-Caryophyllene and α-Humulene; The main products of PtTPS4were Elemol,β-Eudesmol and α-Eudesmol.The products of sesquiterpene of PtTPS2, PtTPS3and PtTPS4included almost all the sesquiterpene products emitted from the leaves infested by WLB,suggesting they are the sesquiterpene synthase in P.trichocarpa involved in the indirectdefence after the infestation of WLB. (5) After being treated with Ala, a fungal peptide antibiotic, S.moellendorffii plants areinduced to emit a triterpene—squalene as the only volatile terpenoid. The emission ofsqualene from S.moellendorffii also can be induced by a number of stress factors, includingheat, physical wounding, abscisic acid (ABA), SA and MeJA. To understand the biosynthesisof stress-induced squalene, a single squalene synthase (SQS) gene was identified from theS.moellendorffii genome. The encoded protein, SmSQS, shows high level of sequencesimilarity to SQSs from higher plants. Gene expression analysis verified that SmSQStranscript accumulation induced by various stress factors correlates well with squaleneemission under the same treatments. The stress induced SmSQS gene expression and a basallevel expression of SmSQS in untreated control plants suggests that SmSQS functions in bothprimary metabolism and specialized metabolism. The full-length cDNA of SmSQS wascloned and expressed in E. coli. Recombinant SmSQS was shown to convert FPP to squalene.Expression of green fluorescent protein (GFP)-SmSQS in onion epidermal cells demonstratedthat SmSQS is targeted to the cytoplasm rather than endoplasmic reticulum (ER) membranedue to the absence of the C-terminal hydrophobic transmembrane domain.The analysis ofSmSQS promoter regions revealed the presence of elements that are related to plant responseto stresses. The transgenic Arabidopsis expressed with the fusion of promoter-SmSQSshowed higher emission of squalene and improved resistance to the stress of Cd.(6) The composition of the volatile terpenoids from five species studied in thisdessertation were compared systematically. The non-seed, S.moellendorffii, produces the leastvariety of volatile terpenoids, with the triterpene squalene as the unique isoprene compoundsafter stresses. The profile of the volatile terpenoids from the leaves of C.iria, P.trichocarpaand flowers of Wisteria showed similarity to some extent, sharing6terpnoids (1monoterpeneand5sesquiterpenes). There are diverse types of biosynthesis and accumulation of theterpenoid compounds in these five species. Some terpenoids were present in both the leavesand flowers, including1,8-Cineole,(E)-β-ocimene, β-Caryophyllene, α-Humulene,β-Farnesene and Elemol. Part of the volatile terpenoids showed tissue specifity, includingGeranyl acetone, Geranyl propionate and Geranyl isovalerate only in flowers, γ-eudesmol,β-Eudesmol and α-Eudesmol only in leaves. Others can only be emitted by the induction ofbiotic and abiotic stresses, including Germacrene D, Nerolidol,(-)-Aristolene and squalene.