Multi-omics Analysis For The Production Mechanism Of Secondary Metabolites Affecting The Heartwood Color In Chinese Fir

Chinese fir(Cunninghamia lanceolata(Lamb.)Hook)is an important evergreen coniferous tree species,widely distributed in many southern provinces of my country,and has important economic value.Red-heart Chinese fir with a red color heartwood,has the advantages of high density and attractive color,which is popular in the market as ornamental plants.However,there are still few reports on the formation of heartwood color,especially the production mechanism of secondary metabolites that affect heartwood color.The presence of a large number of dead cells and wood fibers makes it difficult to study xylem at the metabolic and molecular levels with existing sampling strategies.In this study,the xylem was divided into three effective sampling areas: sapwood,transition zone and heartwood and their secondary metabolites,cell survival status and differentially expressed genes were investigated.At the same time,based on the core population of Chinese fir consisting of 300 elite clones constructed in the early stage and the developed high-density SNP marker loci,the simpled genome-wide association study of important growth-related traits of Chinese fir and the content of secondary metabolites in heartwood was carried out.The SNP markers significantly correlated with the important traits of white-and red-heart Chinese fir were obtained.The main findings are as follows:1.A total of 673 secondary metabolites were detected in the xylem of red-heart and white-heart Chinese fir,of which flavonoids accounted for the highest proportion.It was found that the quantity and types of flavonoid secondary metabolites in various parts of the xylem of red cedar were generally more than those of white cedar.By analyzing the significant differential metabolites in the flavonoid biosynthesis pathway,it was found that the main differential metabolites are divided into several types,namely flavonoids(galangin,luteolin-7-O-glucuronide-5-O-rhamnoside and luteolin-7-O-glucuronide-5-O-rhamnoside),flavonols(kaempferol-3-O-robinoside-7-O-rhamnoside,kaempferol-7-O-glucoside and isorhamnetin-3-O-neohesperidoside),chalcone(naringenin chalcone,phloretin and phlorizin chalcone),dihydroflavonoids(pinocembrin,naringenin and naringenin-7-O-glucoside)and dihydroflavonol(pinobanksin).And according to the chemical structures of metabolites in the synthetic pathway,the existence of differential metabolites is mainly divided into three directions,among which the most likely to be related to the color of red cedar wood is the synthesis of luteolin and its glycosylation products,because of its fold difference.It reaches 1000 times and mainly exists in heartwood,while the glycosylation products of flavonoids are more stable and partially glycosylated flavonoids are colored.Finally,the absolute quantification of some flavonoid metabolites was carried out by LC-MS/MS,which proved that the secondary metabolites of flavonoids in red cedar were indeed generally higher than those in white cedar,and also proved the reliability of the metabolome.2.Cytological observation of the sapwood,transition zone and heartwood area of the xylem of Sequoia and White-hearted cedar all found ray parenchyma cells,among which the survival rate of ray parenchyma between the two in the sapwood was high,but the red heart The nuclei of parenchyma cells in fir are oval,different from the round nuclei in white-hearted fir.In the transition zone,the survival rate of ray parenchyma cells in red heart was significantly lower than that in white heart,and live ray parenchyma cells with intact nuclei were rarely observed in the transition zone of red heart.In the heartwood,both ray cells were dead,and no intact nuclei were observed,but after lysis of red heart ray parenchyma cells,orange vesicles were observed around the cells,which may be secondary metabolites in the After the cell dies,it begins to be released into the surrounding wood fibers.In conclusion,the difference between red heart fir and white heart fir began to appear in the transition zone,and secondary metabolites may be produced in the transition zone and released to the surroundings when they became heartwood,causing the color change of the heartwood.3.Most of the differential genes in transcriptome studies were enriched for metabolic pathways.Combined with the difference comparison results of secondary metabolites,it was found that the upregulated flavonoid synthesis-related regulatory genes in the red heart transition zone were mainly enriched in the whole phenylalanine pathway,the upstream of the flavonoid biosynthesis pathway and the downstream of the pathway.Glycosyltransferase genes for flavonoids and free sugars,and some MYB transcription factors are also involved in the expression.The analysis found that in the transition zone of red heart,the significantly expressed differential genes may regulate the synthesis of secondary metabolites that affect wood color by regulating the production of substrates for flavonoid synthesis and flavonoid glycosylation,which in turn affects red heart.Heartwood color.Fluorescence quantitative PCR analysis found that the expression patterns of 17 regulatory genes and transcription factors were consistent with the sequencing results,which provided a preliminary theoretical basis for the subsequent screening and verification of regulatory genes.4.There are certain phenotypic differences between the red-hearted fir and the white-hearted fir in the Chinese fir core population.It was found that there were pairwise positive correlations between growth traits except basic density and heartwood ratio.There was a significant negative correlation between basic density and these traits.There was no significant correlation between heartwood ratio and growth traits and secondary metabolites..In the metabolite content phenotype,there were extremely significant positive correlations between naringenin,luteolin,taxifolin and dihydrokaempferol.elements are correlated.This shows that although they are both secondary metabolites of flavonoids,their metabolic levels are not consistent due to differences in chemical structures or differences in regulatory genes.The genotype effect analysis of the significantly correlated SNP loci showed that the loci related to growth traits were basically independent,indicating that the genes regulated by different growth traits may not be the same.However,among the loci related to secondary metabolite content,there are multiple loci associated with multiple metabolite phenotypes at the same time,reflecting the pleiotropy of genes,and it is also an SNP marker associated with secondary metabolites in Chinese fir heartwood.The study of the locus laid the foundation.