| Torreya grandis cv.merrillii is mainly distributed in the subtropical hilly areas of China.It is an economic tree species widely cultivated for the purpose of nut production.It integrates a variety of uses such as seed,oil,medicinal,wood,greening,and ornamental.T.grandis cv.merrillii seed kernel is rich in a variety of nutrients and health care ingredients.In recent years,the health care value of other organs besides seed kernel has also attracted widespread attention.In this study,the leaves of T.grandis cv.merrillii were used as the research object,and the changes of essential oil composition and contents under different site and management measures(goose-raising model,stand age,altitude,variety)were determined by GC-MS.Based on the results of the dominant ecological factors resulting in the changes of essential oils,an experiment of the nutrient(N,P)addition on non-volatile metabolites in T.grandis cv.merrillii leaves was conducted.LC-MS method was used to determine the widely targeted metabolites,and Illumina performed transcriptome sequencing and comprehensively analysing the regulation mechanism of nutrient addition on the metabolites of T.grandis cv.merrillii.It has important guiding significance for revealing the essential oil formation mechanism,precise nutrient regulation and quality improvement of the leaves of T.grandis.The main results are as follows.(1)There were monoterpene,sesquiterpene,monoterpene oxygen-containing derivatives,esters and sesquiterpene oxygen-containing derivatives in the foliar essential oil of T.grandis cv.merrillii by GC-MS.A total of 31 metabolites were detected in the foliar essential oil under different management models(goose-raising and non-goose-raising).D-limonene,a-pinene and isoledene were the main components,and the variation rate of differential metabolites was 16.13%.There existed 33 kinds of metabolites in the foliar essential oil of T.grandis cv.merrillii with different ages(including 6-,8-,10-and 12-year-old).D-limonene,?-pinene and isoledene were the main components,and the variation rate of the differential metabolites was 21.21-25%.Among the two altitudes of 350 m and 600 m,a total of 29 kinds of metabolites was detected in the foliar essential oil.D-limonene,?-pinene and ?-cadinene were the main components and the variation rate was 3.44-3.57%.26 kinds of compounds were detected in the two varieties of T.grandis cv.merrillii,and D-limonene,?-pinene and isoledene were the main components.The variation rate was 22.73-23.81%.The variability of essential oil for different ages and varieties of T.grandis cv.merrillii was significantly greater than that for different management models and altitude gradient.Those results indicated that the endogenous regulation(forest age,variety)of essential oils in T.grandis cv.merrillii leaves was stronger than ethe xternal factors(goose-raising,altitude).(2)The content of P in T.grandis cv.merrillii leaves was significantly increased and the content of Ca was significantly reduced in geese-raising model.Soil NO3--N,P,Ca,and Mg were significantly increased in geese-raising model,but MBN was significantly decreased.The dominant ecological factors were plant TC,TP,Ca and soil TP,Ca,Mg,NO3--N.In the age-sequence of T.grandis cv.merrillii stands,foliar TN and Ca were significantly reduced,several soil parameters appeared inflection point in 10-and 12-year-old stands.The ecological factors were foliar TN,K and Ca and soil K.The effect of different altitudes on plant and soil nutrients were not significant,and the ecological factors resulting in the variation were the foliar K and Ca and soil pH and NH4+-N.The contents of foliar TC and TP of two varieties of T.grandis cv.merrillii were significantly different,and the dominant ecological factors were TC,TP,TN and Ca.Through comprehensive analysis,it was shown that the C,N,P,K,and Ca were the dominant factors controlling the expression of differential metabolites in the foliar essential oils of T.grandis cv.merrillii.(3)Using LC-MS for wide target metabolism detection,there were 201 metabolites in the leaves of T.grandis cv.merrillii,among which the primary metabolites were small peptides,the secondary metabolites were flavonoids and organic acids.The enriched differential metabolites were mainly concentrated in the alanine,aspartate and glutamate metabolism,purine metabolism,tryptophan metabolism,galactose metabolism and glutathione metabolism pathways.The results showed that after the addition of N and P,the differential metabolites of T.grandis cv.merrillii changed,mainly affecting the pathways related to carbon and nitrogen metabolism.(4)Using Illumina sequencing and Trinity software for assembly,a total of 100644 gene sequences were obtained,which were compared to four databases NR,Swissprot,KEGG and COG/KOG,and 16558 gene sequences reached common annotation.There are 20457 transcripts annotated to KOG,and the top 10 transcription factors are ERF,bHLH,C2H2,MYB_related,Trihelix,MYB,WRKY,GRAS,C3H and NAC families.2663 transcripts were R genes,among which N,RLP and TNL families were the most.Go functional annotation of differential genes mainly concentrates on protein processing and protein maturation in biological process,peptidase activity,hydrolase activity and nucleotide phosphate binding of molecular function.According to GO annotations,the 20 most functionally differentiated genes were screened out,of which 7 were transporters involved in N metabolism.The main metabolic pathway of KEGG enrichment are phenylpropanoid biosynthesis,metabolism pathway,biosynthesis of secondary meabolites,amino sugar and nucleotide sugar metabolism.(5)Correlation analysis of differential metabolites and differential genes,raffinose,melezitose and related genes clustered together,other compounds(mostly small peptides)clustered with related genes.The abundance of raffinose was significantly decreased after the addition of N and P.The annotation on the galactose metabolism pathway was related to the changes in gene expression of galactinol synthase,raffinose synthase and?-galactosyltransferase.From the above results,it is known that the functional components of the essential oil of T.grandis cv.merrillii leaves have a certain development value,and the variation characteristics of its content are affected by various ecological factors.The precise N and P regulation not only significantly affects the C and N cycles of plants,but also significantly affects the raffinose content related to plant growth and development,and stress.The enzyme genes involved in the regulation of galactose metabolism pathway have great potential for application in the food and feed industry,pulp and paper,sugar,and pharmaceutical industries. |
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