Study On The Mechanism Of The Color Formation Of The Heartwood Of Catalpa Bungei

Catalpa bungei belongs to the Catalpa genus of the Bignoniaceae family,and it is a precious timber and garden ornamental tree species unique to China.It is widely distributed and rich in germplasm resources.Catalpa bungei var.is a geographical variety of C.bungei.Its heartwood is golden yellow and has good mechanical properties,and it is a high-quality wood with many uses.In this study,the sapwood and heartwood of 21-year-old C.bungei were subjected to widely targeted metabolomics analysis.A total of 633 metabolites were detected,among which 341metabolites were significantly different in content.Thirteen quinone metabolites and three flavonoid metabolites were obtained by screening the metabolites related to heartwood color from the differential metabolites.Through the analysis of the content and color of metabolites,it was determined that the naphthoquinone metabolites（α-lapachone and its derivatives）were the main substances in forming the color of the heartwood of C.bungei.The main differential metabolite of sapwood and heartwood of C.bungei was isolated by HPLC and normal chromatography,and it was identified as 4,9-dihydroxy-α-lapachone by NMR.According to the results of metabolite detection and literature reports,the biosynthetic pathway ofα-lapaquinone and its derivatives was speculated as follows:OSB→1,4-dihydroxy-2-naphthoic acid→deoxylapachol→lapachol→α-lapachone→9-hydroxy-α-lapachone→4,9-dihydroxy-α-lapachone.The color of lapachol,α-lapachone,9-hydroxy-α-lapachone and 4,9-dihydroxy-α-lapachone solution are light yellow,bright yellow to rose red,which are directly related to the color of the hearthwood.The sapwood was divided into three parts（GS1,GS2,GS3）according to a single growth ring,and the heartwood was divided into four parts（GH1,GH2,GH3,GH4）according to 4-5 growth rings.The content of each metabolite in the biosynthetic pathway was detected by LC-MS,and the other six metabolites were detected except 1,4-dihydroxy-2-naphthoic acid.The distribution of main metabolites in the pathway was as follows:the content of metabolites in GS1 was significantly higher than that in GS2 and GS3,and there was no difference between GS2 and GS3;the content of metabolites gradually decreased from GH4 to GH1.It was inferred that the target metabolites were synthesized only in sapwood and reached the peak when sapwood transformed to heartwood（GS1）.The synthesis stopped and the metabolites degraded gradually in heartwood.The biosynthetic pathway ofα-lapaquinone and its derivatives was modified as follows:OSB→OSB-Co A→DHNA-Co A→DHNA→deoxylapachol→lapachol→α-lapachone→9-hydroxy-α-lapachone→4,9-dihydroxy-α-lapachone.In addition,two components were isolated from the MS signal peak at5.94 min.The molecular formula of the principal component was C₁₅H₁₆O₃,and the structure was analyzed as 2,2-dihydro-2H-benzo[g]chromene-5,10-diol.The molecular formula of the other component was C₁₅H₁₄O₄,which was speculated to be 4-hydroxy-α-lapachone.They may also participate in the color formation of heartwood.RNA was not extracted from the heartwood of C.bungei,but the concentration of RNA extracted from the sapwood showed a very obvious trend of GS1<GS2<GS3.A total of 28657genes were identified by RNA-seq,of which 26465 were aligned with the reference genome of C.bungei,and 2192 were new genes.1502 common differentially expressed genes were obtained from GS1 vs GS2 and GS1 vs GS3.Eight profiles were obtained from expression trend analysis,and the gene expression trend in Profile 1 was consistent with the distribution of target metabolites.Eight modules were obtained by weighted gene co-expression network analysis,and the red module was positively correlated with target metabolites.The GO enrichment analysis of the above results showed that the GO term with the highest significance was GO:0016705,and the functional annotations were consistent with the chemical properties of hydroxylation reactions in the biosynthetic pathway.Thirteen genes annotated as CYP450 were selected from the cross set of three gene sets as the primary gene set,and five target CYP450 genes were further selected from the primary gene set,which were Cbu CYP71D15、Cbu CYP82A3A、Cbu CYP82A3B、Cbu CYP81E8 and Cbu CYP81E7.Five Cbu CYP450 genes were successfully cloned,and the target function of Cbu CYP82A3（Cbu CYP82A3A and Cbu CYP82A3B）and Cbu CYP71D15 was verified by transient transformation in Nicotiana benthamiana.Cbu CYP82A3 catalyzes the hydroxylation reaction ofα-lapachone→9-hydroxy-α-lapachone,and Cbu CYP71D15 catalyzes the hydroxylation reaction of 9-hydroxy-α-lapachone→4,9-dihydroxy-α-lapachone.They are hydroxylases that catalyze the last two reactions in the biosynthetic pathway ofα-lapachone and its derivatives.