| LBD(Lateral Organ Boundaries Domain)protein is a class of plant-specific transcription factors that play important roles in plant growth,development,metabolism and defense.Previous studies have shown that LBD6 gene of Arabidopsis plays an important role in the formation of lateral organs,polarity establishment of organs and the normal development process of pollen;the overexpression of its homologous gene Os AS2 in rice can inhibit the differentiation of buds and promote cell division;its homologous gene RAMOSA2 in maize determines the fate of branching meristem cells;its homologous gene Vrs4 in barley controls the formation and arrangement of spikelets.Therefore,it is speculated that their homologous genes also play an important role in the growth and development of trees.TcLBD6 was cloned by our lab from the bark of Taxus chinensis,an important medicinal gymnosperms,but the function of TcLBD6 in tree was not known.In order to study the role of TcLBD6 transcription factor in tree growth and metabolism,an over-expression vector of TcLBD6 gene was constructed and transformed into poplar 84K(Populus alba×Populus glandulosa).In view of the most obvious phenotypic differences in leaves of TcLBD6 over-expression plant,The metabolic,transcriptional and proteomic analysis for leaves of the overexpression plants and control plants were carried out.The functions of TcLBD6 were studied and the preliminary mechanism was discussed mainly from the downstream metabolite level.In addition,25 genes related to anthocyanin synthesis were identified in Taxus chinensis,and promoter binding prediction was conducted,as that the metabonomics analysis showed that anthocyanin was significantly up-regulated in TcLBD6 over-expressed plants.These results laid a foundation for further study of the molecular mechanism of TcLBD6 involved in leaf development,secondary metabolism and development of multi-dimensional medicinal value of Taxus chinensis.The main results were as followings:(1)The over-expression vector of TcLBD6 was constructed and transformed into 84 K poplar.The results of phenotypic analysis showed that the growth of typical TcLBD6-oe plants was inhibited compared with WT-84 K.The phenotypic of leaves was the most different,which showed that the leaves became smaller or even contracted into needle-like,the leaf length and leaf width were 35% and 2% of the control 84 K poplar,respectively.The vascular tissue distribution was scattered,no obvious palisade tissue structure was found,and cells similar to palisade tissue appeared on the abaxial.These results indicated that TcLBD6 overexpression significantly inhibited leaf growth and development.(2)To compare the phenotypic differences between heterologous and homologous overexpression,Pag LBD6,Pag LBD25 and Pag LBD36,homologous genes of TcLBD6,were cloned from 84 K poplar and their over-expression vectors were constructed and transformed into 84 K poplar.The overe-xpression plant of Pag LBD6 showed suppressed growth,shrinking and upward curling leaves,similar to the leave phenotype of TcLBD6-oe,indicating that the phenotypic effect of TcLBD6 overexpression on leaves was due to gene overexpression,which was unconnected to TcLBD6 came from coniferous tree species.(3)In order to study the effect of over-expression of TcLBD6 on metabolite changes,widely targeted metabolomics approach was performed to detective metabolites in leaves of TcLBD6-oe and WT-84 K,as the phenotypic of leaves was the most different.It showed that the overexpression of TcLBD6 caused up-or down-regulation of many metabolites,among which metabolites with medicinal value including 3,4-Dihydroxy-L-phenylalanine(L-DOPA),3,4-Dihydroxyphenyl-2-methylalanine(methyldopa),isorhamnetin,sakuranetin,luteolin O-hexosyl-O-gluconic acid,6-Methoxy-7,8-Dihydroxy coumarin,cyanidin and peonidin were up-regulated significantly.The highest up-regulation metabolite was L-DOPA,which had the biological function of inhibiting the growth of weeds.The most abundant type that was up-regulated or down-regulated was flavonoid,and anthocyanins were significantly up-regulated.These results indicated that TcLBD6 overexpression significantly increased the content of many secondary metabolites of medicinal value in leaves.(4)The L-DOPA contents in leaves of TcLBD6-oe and WT-84 K were analyzed qualitatively and quantitatively by LC-MS.The results showed that the L-DOPA content of TcLBD6-oe was significantly higher than that of WT-84 K,which provided the effect of L-DOPA biosynthesis of TcLBD6 overexpression.L-DOPA(2m M)treatment of 84 K showed that L-DOPA treatment significantly inhibited the growth and development of 84 K seedlings,which was similar to the phenotype of TcLBD6-oe.(5)In order to explore the mechanism of TcLBD6 promoting the synthesis of L-DOPA and anthocyanin in 84 K,the transcriptome sequencing of TcLBD6-oe and WT-84 K leaves was carried out.The analysis revealed 10 329 differentially expressed genes(DEGs),of which 6771 were up-regulated and 3 558 down-regulated.KEGG analysis showed that several genes in the L-DOPA biosynthesis pathway were up-regulated,among which the polyphenol oxidase(PPO)gene up-regulated by 6.85 times,while those in the anthocyanin biosynthesis pathway were generally up-regulated.The JASPAR software predictions also revealed that there were multiple LBD binding sites in the promoter region of the PPO gene and chalcone synthase(CHS)and flavanone 3-hydroxylase(F3H)genes in the anthocyanin biosynthesis pathway of poplar 84 K,which laid a foundation for further research on the interaction of TcLBD6 and its target genes.(6)In order to explore the reason of L-DOPA inhibiting leaf development,this paper focused on the replacement of non protein amino acid L-DOPA,and used lable-free method to analyze the proteome of TcLBD6-oe and WT-84 K leaves.A total of 2 473 proteins were identified,of which 130 were differentially expressed.Among the differentially expressed proteins,77 were up-regulated and 53 were down-regulated.The amino acid residues substitution analysis of TcLBD6-oe and WT-84 K showed that many proteins in TcLBD6-oe had the substitution of L-DOPA residue.For endochitinase populus?84k053195.T1,tyrosine residue at position 60 was replaced by L-DOPA and phenylalanine residue at position 82 was replaced by L-DOPA,indicating that overexpression of TcLBD6 could result in the substitution of tyrosine residue or phenylalanine residue by L-DOPA residue for some proteins in transgenic plants,which was a new discovery.These results provided a new idea to study the mechanism of TcLBD6 inhibiting leaf development through L-DOPA,which was worth further study in the future.(7)As metabolomic analysis showed that anthocyanins were significantly upregulated in TcLBD6 over-expressed 84 K plants,total of 25 anthocyanin synthesis-related genes including Tc CHS,Tc CH1-Tc HI2,Tc F3H1-Tc F3H5,Tc F3'H1-Tc F3'H4,Tc F3'5'H,Tc ANS,Tc DFR1-Tc DFR8,Tc ANR and TcLAR1-TcLAR2 were also identified in Taxus chinensis,through analysis of sequence characteristics,conserved domains and phylogenetic correlations of amino acid sequences encoded by these genes.Expression patterns of these genes were researched.The JASPAR software predictions showed the presence of LBD binding sites in the promoter region of Tc CHI1,Tc F3H3,Tc F3'H4 and Tc ANS.These results laid a foundation for the future study on the relationship between TcLBD6 and the anthocyanin biosynthetic genes in Taxus chinensis. |
PDF Full Text Request