| The formation of heartwood in trees is a genetically and metabolically regulated developmental process,the result of adaptation to the environment over a long period of ecological evolution.The formation of heartwood not only helps the tree to resist external fungal attack,but also gives wood products durable and beautiful properties.Scientific and effective regulation of the quantity and quality of heartwood formation is an effective means of increasing the economic value of forest trees,however,key biological processes such as changes in the physiological function and the synthesis and regulation of secondary metabolites in parenchyma cells during heartwood formation are still unclear.In this study,the valuable tree species Dalbergia odorifera T.Chen was used as experimental material.The number of starch grains,the morphology of the nucleus in parenchyma cells were observed continuously on the xylem radially by histochemical staining,and the metabolic pathways involved in the differential metabolite composition and their involvement were analysed by metabolomics.The genes involved in the regulation of programmed death and secondary metabolic synthesis in parenchyma cells were screened using transcriptomic analysis,and the transcription factors associated with heartwood formation were identified using bioinformatics and perform a functionally analysis.The main results are as follows:(1)From sapwood to heartwood,the nutrients and cell structure change as the viability of the parenchyma cells decreases.The outer to inner layer of sapwood is rich in starch grains,but the length to diameter ratio of the nucleus is decreasing and the morphology gradually tends to round from a long fusiform.In the outer layer of the transition zone(radial width approximately 2mm),the number of starch grains decreases abruptly and the rate of nucleus reduction slows down.In the inner layer of the transition zone(radial width approx.1 mm),the color of the heartwood begins to develop,the starch grains largely disappear and the nuclei become less fluorescent.The transition zone is the area where the number of starch grains begins to decrease until the nuclei disappear completely.The content of water and flavonoids in the heartwood is higher than in the sapwood,and the change in content occurs after the viability of the parenchyma cells has been weakened(inner layer of the transition zone),both of which are not directly responsible for the programmed death of the parenchyma cells.(2)Metabolites are indicators of the physiological state of parenchyma cells.With the consumption of primary metabolites and the production of secondary metabolites,the parenchyma cells complete their programmed death and the sapwood transforms into heartwood.A total of 954 metabolites were identified in the xylem,dominated by flavonoids(172),prenol lipids(105)and isoflavonoids(103).Sucrose and glutamate are characteristic metabolites of sapwood and provide the energy and material basis for the vital activities of parenchyma cells in sapwood.Heartwood characteristic metabolites such as naringenin,formononetin and nerolidol are mainly enriched in the flavonoid synthesis pathway(ko00943),isoflavone synthesis pathway(ko00941)and terpene synthesis pathway(ko00909),which affect the formation of xylem color and aroma.The distribution of characteristic secondary metabolites was observed to be heterogeneous across xylem tissue parts by various in situ analysis techniques such as mass spectrometry imaging,with flavonoid metabolites appearing first in parenchyma cells,with insignificant differences in content between axial parenchyma cells and peritubular axial parenchyma cells,and higher than in wood fibre tissue.The heartwood composition was mainly deposited in the ectoplast of the cell wall at the pits,cell corners and cell middle lamella.(3)The biosynthesis of secondary metabolites and the programmed death of parenchyma cells are regulated by numerous structural genes.Key genes involved in the primary metabolic pathway,such as sucrose synthase gene(Dod SS1),hexose phosphate isomerase gene(Dod GPI)and 3-deoxy-Darabinoheptulose-7-phosphate synthase gene(Dod DHAPS),are significantly up-regulated in the transition zone,providing material and energy for the downstream secondary metabolic pathway.Differential genes in the flavonoid,isoflavonoid and sesquiterpene synthesis pathways are up-regulated in the transition region,with the phenylalanine deaminase gene(Dod PAL),the caffeic acid oxymethyltransferase gene(Dod COMT1),the isoflavone 2’-hydroxylase gene(Dod I2’H),the chalcone synthase gene(Dod CHS1),the hydroxymethylglutaryl Co A reductase gene(Dod HMGR)and the terpene synthase gene(Dod TPS4)being key genes in heartwood formation,regulating the production of sesquiterpenes and flavonoid secondary metabolites in heartwood.Differential genes associated with programmed death in parenchyma cells were also altered,with the genes encoding vesicle lytic enzymes(Dod VPE2)and desiccated protein-related enzymes(Dod DRP)significantly upregulated in the transition region and aquaporin gene(Dod AQP2)downregulated in the transition zone associated with physiological events such as clearance of cell contents and dehydration of cells.Cells in the transitional zone prepare for death.(4)The R2R3-MYB transcription factor is involved in the regulation of heartwood formation.126 Dod R2R3-MYB transcription factors were identified and screened from the D.odorifera genome and classified into 33 subfamilies by sequence alignment and phylogenetic evolutionary relationships,with transcription factor proteins related to the flavonoid biosynthesis pathway concentrated in subfamilies S3-7,10,12-13,21 and N4-8.Members of the D.odorifera R2R3-MYB gene family are clustered on 10 chromochromes and 58co-linear MYB gene pairs were identified,with fragment duplication playing a major role in the expansion of the R2R3-MYB gene family in D.odorifera.Three R2R3-MYB genes(Dod MYB32,Dod MYB55 and Dod MYB88)specifically expressed in the transition zone layer were associated with heartwood formation.Further experimental studies have shown that the Dod MYB89 transcription factor,localised in the nucleus,has transcriptional activation activity and is involved in regulating the biosynthesis of secondary metabolites of heartwood by activating the promoters of the structural genes Dod I2’H and Dod COMT in the flavonoid synthesis pathway.In summary,this study focused on the physiological metabolism of parenchyma cells in the xylem of D.odorifera,clarified the location of the transition zone,established a metabolic dataset of the xylem,analysed the metabolic pathways involved in differential metabolites,and mined candidate genes related to biological pathways such as programmed death of parenchyma cells and secondary metabolite production,providing basic data to support the multi-channel regulation of heartwood formation in D.odorifera. |
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