Integrated Metabolome And Transcriptome Reveals The Metabolic Regulation Of Anoectochilus Roxburghii In Response To Light Quality

Anoectochilus roxburghii(Orchidaceae)is a perennial herb of orchidaceous family,which is used for medicine,food and ornamental.Light plays essential roles in regulating the plant growth,morphogenesis and physiological metabolism.And light has been considered an effective factor for improving plant growth and the biosynthesis of metabolites in A.roxburghii.In this study,the tissue culture seedlings of A.roxburghii were used as the experimental materials under six light qualities,including blue light(B),red : blue light = 1:2(R1B2),red : blue light = 1:1(R1B1),red : blue light = 2:1(R2B1),red light(R)and white light(W).The effects were studied for different light qualities on the growth and metabolism.Firstly,the biomass accumulation and physicochemical parameters of A.roxburghii were evaluated.Then,to obtain comprehensive metabolite information,five metabolomics technologies were used to comprehensively analyzed primary and secondary metabolic changes of A.roxburghii under different light qualities,including gas chromatography-mass spectrometry(GC-MS),ultra performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry(UPLCQTOF-MS),high performance liquid chromatography(HPLC),neutral desorption electrospray extraction ionization mass spectrometry(ND-EESI-MS)and nanoelectrospray ionization mass spectrometry(Nano-ESI-MS),as well as the metabolic response of polyamines.Meanwhile,the differentially expressed genes(DEGs)and related metabolic pathways were analyzed by transcriptomics.The results showed that R2B1 significantly increased the biomass,soluble sugar and soluble protein accumulation of A.roxburghii.And R2B1 significantly promoted the accumulation of secondary metabolites of phenolic acids and flavonoids,including caffeic acid,ferulic acid,p-coumaric acid,chlorogenic acid,kaempferol,rutin and kinsenoside,and enhanced amino acid metabolism,sugar metabolism,phenylpropane metabolism,flavonoid biosynthesis and kinsenoside biosynthesis in A.roxburghii.Similarly,transcriptomics results further confirmed that R2B1 promoted the accumulation of main metabolites in A.roxburghii.In addition,in this study,polyamine metabolites were also deduced to be negatively correlated with phenolic acids and flavonoids.Based on the above results,we proposed the metabolic regulation mechanism of A.roxburghii in response to light qualities.On the one hand,the red-blue combined light,especially R2B1,directly enhanced photosynthesis and promoted the accumulation of primary metabolites through photosynthetic pigments on the chloroplast.On the other hand,red-blue combined light acted on the photoreceptors on the plasma membrane,activating the light signalling pathway,regulating downstream gene expression and the synthesis of secondary metabolites through second messengers.In this mechanism,under R2B1,polyamines,as second messengers,possibly played an inhibitory role in the biosynthesis of phenolic acids and flavonoids.R2B1 reduced the metabolic response of polyamines,thus activating the pathway of phenylpropane metabolism,flavonoid biosynthesis and kinsenoside biosynthesis inhibited by polyamines at the transcriptional level,and finally promoting the accumulation of total phenols,total flavonoids and some other bioactive substances in A.roxburghii.This study revealed the metabolic regulation mechanism of A.roxburghii in response to light qualities,which provided a reference for the photobiology studies of other plants.At the same time,this study confirmed that R2B1 could be used as the optimal light quality for A.roxburghii in vitro,providing a scientific basis for the production and cultivation of A.roxburghii.