Study On The Mechanism Of Wound-induced Defensive Responses Of Dracaena Spp. To Produce Resin

Dragon’s blood is an extract from resinous wood of Dracaena.spp..It is a traditional Chinese medicine that has been applied over thousands of years.Dragon’s blood could activate blood,dissolve stasis,kill pain and regenerate tissues to heal wounds.In China,the source plants of dragon’s blood are D.cochinchinensis and D.cambodiana.In nature conditions,the resin formation process would be timeconsuming.The traditional method of manufacturing severely destroys the plant resources.As the demand of dragon’s blood keeps growing in recent years,the world plant resources become more and more deficient.As little is known about the resin formation mechanism of Dracaena spp.,no efficient artificial induction technique is available,which restrict the sustainable utilization of dragon,s bloodThe present study works on the D.cochinchinensis and D.cambodiana.First of all,histochemical stain method and imaging mass spectrometry technique were used to confirm the structure characteristics and material basis of wound-induced resin formation process.Then the chemical reagent chromogenic method and liquid chromatography-mass spectrometry were used to detect the content change of mutiple signal molecules within stems of D.cambodiana.Finally,muti-omics analysis was used to comprehensively reveal the molecular mechanism of would-induced resin formation process of Dracaena spp.from multiple aspects,including the genetic basis,gene expression patterns,wounding signal regulation and miRNA regulation.The findings are as followed:1.Structure characteristics and materials basis of wound-induced resin formation processCortex in healthy stems of D.cambodiana have abundant of calcium oxalate raphide that play roles in physical defenses.Vascular bundles are well connected with surrounding parenchymal cells through plenty of pits in their cell walls.When the damage only reaches cortex,the stem would not produce resin.The injured cortex would be suberized to form new bark.When the damage reaches the ground tissue,the injured ground tissue would be obviously layered:decomposed layer,resin layer and healthy stem.After being damaged,resin initially occures in phloem,then fills the xylem within vascular bundles and finally spreads to the surrounding parenchymal cells.As time goes after wounded,the resin,the flavonoid compounds and saccharides show similar variation trend.After being wounded,the content of phenylalanine within vascular bundle phloem decreased and two flavones(3,4-hydroxyflavone and galangin)are generated in the same spots.Vascular bundles are the resin formation location of dragon trees and the saccharides is speculated to be the material basis of the woundinduced resin formation process.2.Concentration change of wounding signals during the wound-induced resin formation process.Among mutiple plant signal molecules,the content of H2O2 and jasmonate dramatically increased after the stem were wounded.Exogenous H2O2 and jasmonate could significantly improve the flavonoid content within stems of D.cambodiana.3.Genome characteristics of D.cochinchinensis.Using next Generation Sequencing,Biopac sequencing and Hi-C sequencing,the high-quality genome of D.cochinchinensis at chromosomal level was obtained 1.26G.DNA damage repair related genes and flavonoid biosynthesis related genes were positively selected,which could be the genetic basis of the wound-induced defensive responses to produce resin.4.After damaged,adjusts mutiple metabolism pathways(centered on flavonoid biosynthesis)to produce resin thus form the physical and chemical defense.Both the gene expression pattern and metabolites accumulation pattern showed characteristics of stages after the stems of D.cochinchinensis were wounded.In the short term after damage,genes involved in vairous of pathways were up-regulated including TCA cycle,glycolysis,starch and surcrose metabolism,etc.and promote the flux from primary metabolism to secondary metabolism.During this stage,different types of metabolites were synthesized;In the middle and late stages,many genes annotated in the flavonoid biosynthesis including CHS,CHI,DFR,LAR,OPP were significantly up-regulated and the flavonoid compounds is the main enriched metabolite type.MYBs an bHLH might be the main transcriptome factor regulating the flavonoid biosynthesis in Dracaena spp..Besides,ROS generation and scavenging related genes were differentially expressed after wounded.Biosynthesis and receptor genes of multiple hormones including jasmonate,gibberellin and abscisic acid were upregulated.The exogenous H2O2 and JA could enhance the expression of CHS,OMTand PPO.It is known that the pathways and signals mentioned above is closely related to plant wounding responses.The flavonoid compounds and flavone glycoside form the physical and chemical defenses.5.miRNAs are widely involved in the wound-induced defense responses and resin formation process through multiple pathways.MiRNA profile significantly changed after the D.cambodiana stem were damaed.A novel miRNA was discovered to target a CHS gene.The expression of this miRNA were decreased while that of the CHS gene were increased after wounding.The present study initially applies imaging mass spectrometry to show flavonoids were synthezied and accumulated in vascular bundles.The study first reports the chromosomal level genome of D.cochinchinensis,which could provide data foundations for the biological characteristics research on spp..The study initially proposes that dragon trees showed stage characteristics in the metabolic process from short term to long term after wounding.Flavonoid biosyhthesis is the main process in the defensive responses of dragon trees.H2O2 and JA are important signals in the defensice responses.The study first reports the miRNA profile of D.cambodiana and analyzes the role of miRNAs in the defensive responses.The present study demonstrates the relationship between wound-stress and resin formation,confirms the resin formation spot and excavates the potent genes related to the quality of dragon’s blood,which could provide theoretical basis for the development of high efficient induction technique.