Study On Mechanism Of Temperature And Light Regulating Growth And Podophyllotoxin Biosynthesis In Sinopodophyllum Hexandrum

Sinopodophyllum hexandrum Royle(family Berberidaceae)is a perennial rhizomatous plant,and its rhizomes are the major source of podophyllotoxin(PPT),as well as its derivatives that are used as anti-cancer,anti-viral and anti-oxidant.Currently,S.hexandrum is an endangered species in large part because of its unique pharmacological effects to meet the ever-increasing pharmaceutical demand.Therefore,there is an urgent need to accelerate the cultivation of S.hexandrum and cell culture to increase PPT yield.Our laboratory and other scholars have confirmed that high altitude can significantly promote PPT biosynthesis,and low temperature and UV-B are the main ecological factors in high altitude environment.In this study,base on transcriptome sequencing and tissue culture seedlings of S.hexandrum,cell/subcellular structure,growth characteristics,bioactive accumulation and key gene expression are investigated under low temperature and UV-B by the methods of scanning and transmission electron microscope,HPLC and qRT-PCR to further reveal the molecular mechanism of PPT biosynthesis promoted by high altitude.The results are as follows:1)Studying the effect of temperature on the induction of sterile seedlings,plant growth characteristics and microstructure,the results showed that seedling inducing rate(61.91%)at 15°C was 1.2-and 1.5-fold increase compared to 22 and 30°C;the biomass,chlorophyll content,leaf area,aerial parts height,diameter of stem and root,number of bulbs and roots as well as root length were significantly increased at 15°C compared to 22°C;stomatal density,shape,chloroplasts,mitochondria,starch granules,osmisophilic granule and thylakoid grana were significantly improved by observeing leaf micro-and ultra-structure.2)Studying the effect of temperature on metabolite accumulation and expression level of related genes,the results showed that PPT content was increased 2.3-fold on a per plant basis,and a 1.8-,1.4-and 1.3-fold increase of flavonoids and phenolics contents,antioxidant capacity by stimulating genes over-expression related to photosynthesis(e.g.ShMENG,ShmatK and ShndhF,etc.),PPT biosynthesis(ShPAL~Sh2-ODD),flavonoids biosynthesis(ShCHS1 and ShDTX41)as well as stress response(e.g.ShAPX3,ShEX2 and ShMYB,etc.).3)Studying the effects of temperature and light on plant growth,metabolite accumulation and expression level of related genes,the results showed that the biomass,root length and numbers of root were promoted under UV-B+15°C compared to 15°C,while other growth characteristics(chlorophyll content,leaf area,aerial parts height and number of bulbs,etc.)was adversed.Although soluble sugar,flavonoids and phenolics contents as well as antioxidant capacity were significantly enhanced by stimulating genes over-expression related to glyco-metabolism,flavonoids biosynthesis and stress response under UV-B+15°C,0.9-fold decrease of PPT content was observed compared to 15°C by stimulating downstream genes down-regulated with 0.26-(ShCCoAMT)~0.78-fold(ShCYP71CU1).In conclusion,low temperature(15°C)is more conducive to plant growth,accumulation of metabolites and expression level of genes.Although UV-B promote plant biomass,flavonoids and phenolics contents,antioxidant capacity as well as related genes expression at 15°C,PPT accumulation and expression level of genes related to biosynthesis were inhibited.These findings will provide a direct theoretical basis for guiding S.hexandrum cultivation and improving PPT production.