Functional Research Of CmCADs In Lignin Biosynthesis In Oriental Melon(Cucumis Melo L.)under Abiotic Stresses

Oriental melon(Cucumis melo var.makuwa Makino)is a kind of economic plant in Cucurbitaceae,which is widely planted in China and some south-east Asia countries.However,the weak ability of resistance is the main reason leading to severe loss in protected field production.Thus it is important to improve the resistant ability of oriental melon.So far,cinnamyl alcohol dehydrogenase(CAD)is a key enzyme in lignin monomer synthesis,which has been identified from kinds of species and usually present as a family containing different numbers of gene members.Different members may fulfill different functions but most members can be regulated by stresses and some signaling substances.In addition,lignin is taken as an important substance in resisting or responding to stresses,and thus it is meaningful to investigate the relationship between CAD and stresses for enhancing the ability of stress resistance of oriental melon.However,little is known about the relation between CAD and stress since five CmCAD genes were identified from melon genome database and named CmCAD1?5,respectively.To investigate the relationship between CmCAD genes and stresses and their functions fulfilled in lignin synthesis,we use 'Caihong 7' oriental melon cultivar and Arabidopsis cadc cadd double mutant as experimental materials.The effects of the three abiotic stresses,drought(8%and 15%PEG6000),wound(leaf wound,LW;stem wound,SW)and salt(50 and 100 mmol/L NaCl),on the expressions of CmCADs and deposition of lignin were investigated,and then we further investigate the relationship between CmCADs as well as lignin synthesis and abscisic acid(ABA),hydrogen peroxide(H2O2),jasmonic acid(JA)under drought stress.According to tissue expression and phylogenetic analysis,we further investigate the functions of CmCAD1,CmCAD2 and CmCAD3 in lignin synthesis and drought tolerance through overexpressed each member in Arabidopsis cadc cadd double mutant and VIGS silencing in oriental melon seedlings.Results are listed as followings:1.The lignin synthesis in oriental melon seedlings is promoted under drought,wound and salt stresses.The three abiotic stresses promoted the accumulation of lignin in stems and roots,and had no effects on lignin content in leaves.The activities of phenylalanine ammonia-lyase(PAL),CAD and peroxidase(POD)in melon seedlings were generally induced significantly,while the activity of LAC was only induced by 8%PEG treatment.Under drought stress,all five CmCAD genes in stems were generally induced significantly or extreme significantly.Under wound stress,all the five CmCAD genes increased fast within 3 h and then declined gradually.Among the members,CmCAD3 was the most highly induced gene.CmCAD1,CmCAD3 and CmCAD4 exhibited systemic responses.Under salt stress,the five CmCAD genes showed differential and tissue specific responses.2.Under drought stress simulated by 8%PEG treatment,lignin synthesis and CmCAD genes in oriental melon seedlings were regulated by ABA,H2O2 and JA.ABA,H2O2 and JA were all induced by 8%PEG treatment and ABA and H2O2 responded faster.The three signals may play roles in regulating lignin synthesis under drought stress.The three signals positively regulate the activities of CAD and LAC,while diversely regulate the activities of PAL and POD.Gene expression analysis revealed that CmCAD1,CmCAD2 and CmCAD3 can be positively regulated by ABA and H2O2,CmCAD4 only can be regulated by H2O2 and CmCAD5 is regulated by H2O2 and JA.ABA and H2O2 could generally regulate the expressions of the lignifying genes,while JA could only regulate CmPAL1-Like?CmPOD2-like and CmLAC17-Like genes,indicating ABA and H2O2 seems to be the main signals in regulating lignin synthesis under drought condition.In addition,inhibited either ABA or H2O2 could strongly inhibited the other one from increasing under drought stress,and exogenous applied ABA or H2O2 had limited promotion to endogenous ABA or H2O2 when pretreated with inhibitor,and lignin synthesis was still inhibited,suggesting the cooperation promotion of ABA and H2O2 to lignin synthesis under drought stress.3.It is speculated that CmCAD1,CmCAD2 and CmCAD3 are the main genes in lignin synthesis in melon.Tissue expression analysis revealed that all the five CmCAD genes expressed higher in stem,CmCAD1,CmCAD2 and CmCAD5 showed higher and similar expression patterns in stem,while CmCAD3 is the most highly expressed gene among the family members and strongly responded to drought and wound,which suggests that CmCAD3 may be a bona fide CAD gene for lignin synthesis in oriental melon.Phylogenetic analysis of CmCADs revealed that CmCAD1 and CmCAD2 belong to the bona fide CAD group,CmCAD3 and CmCAD5 belong to the group in which members functions remain to be explored.While CmCAD4 is placed in the fake CAD group.4.CmCAD1,CmCAD2 and CmCAD3 can differentially recover the lignin composition in Arabidopsis cadc cadd double mutant.CmCAD1,CmCAD2 and CmCAD3 is overexpressed in Arabidopsis cadc cadd double mutant respectively and there shows no obvious difference in phenotype between transgenic plants and WT and mutant plants,however lignin content and CAD activity are significantly improved.Lignin staining and cell wall ultra-structure observations of floral stem sections revealed that transferred CmCAD1 into mutant recover lignin composition and secondary cell wall structure to some extent,while transferred CmCAD2 or CmCAD3 can recover lignin composition and secondary cell wall structure very well.Combined with the shift of staining color,it indicates that CmCAD1 can catalyze sinapyl aldehyde,CmCAD2 and CmCAD3 can efficiently catalyze coniferal-and sinapyl-aldehyde to coniferal-and sinapyl-alcohol and then participate the cell wall construction.5.VIGS transient silencing CmCAD1,CmCAD2 and CmCAD3 respectively or collectively can differentially inhibit lignin synthesis and thus delay or inhibit melon seedling growth.The phenotype of CmCAD1 silencing plants is quite similar to that of control,while CmCAD2 silencing,CmCAD3 silencing or co-silencing plants exhibit significantly reduced leaf area,plant height,internode length and internode diameter However,all silencing plants show reduced CAD activity and lignin content,and CmCAD2-,CmCAD3-and co-silencing plants show severely decreased phloroglucinol-HCl staining of xylem and size of vascular,as well as abnormal phenotype of secondary cell wall.However,Maule staining observed yellow xylem in control and all silencing plants,suggesting lignin in oriental melon is mainly composed by G unit.At the meanwhile,significant increases of cell numbers in a certain area of stem pith are observed in CmCAD3 silencing and co-silencing plants,indicating the important role of CmCAD3 in cell expansion.What's more,delayed or inhibited in Casparian strip formation are observed in all silencing plants,indicating Casparian strip is mainly composed by lignin and CmCADl,CmCAD2 and CmCAD3 are responsible for Casparian strip formation.6.The ability of drought tolerance of Arabidopsis double mutant is enhanced after transferred CmCAD genes into it and of oriental melon seedlings is weakened after silencing the genes.Transgenic Arabidopsis plants were treated with drought stress and found that CmCAD2 and CmCAD3 transgenic lines can recover the drought tolerance ability of mutant to a certain and water potential in floral stems significantly.What's more,CmCAD2 and CmCAD3 transgenic lines show similar phenotypes and root length,as well as leakage ratio,with WT under drought stress simulated by mannitol,indicating recover the composition of lignin and deposition,which is helpful for water status recovery and thus improve the ability of drought tolerance.The drought treatment to VIGS silencing plants revealed disrupted drought resistance of CmCAD2-,CmCAD3-and co-silencing plants,which may due to the abnormal vascular development and thus affect water transportation.Though lignin accumulations in silencing plants are still promoted under drought stress,the lignin contents in stems of CmCAD2-,CmCAD3-and co-silencing plants are still significantly lower than control,thus can't rescue the ability of drought resistance.