AtPAP2 Overexpression Induces The Accumulation Of Flavonoids And Its Effect On The Growth Of Tomato Plants

Anthocyanins are naturally occurring flavonoid-derived compounds,belonging to an important end product of the branch of phenylpropanoid metabolism.They are widely distributed in plants responsible for the coloration of many flowers and fruits.Anthocyanins not only play an important role in the physiology and biochemistry of plants,but also have an effect in fighting against certain cancers,cardiovascular diseases and diabetes due to their antioxidant activity and ability to modulate signaling pathways in animal cells.Accumulation of anthocyanins is the result of the coordinated expression of structural genes encoding anthocyanin biosynthesis.Through the study of different plant species,this synergistic expression is through the interaction of the R2R3-MYB transcription factor,the bHLH(basic helix-loop-helix)transcription factor,and the WD40 repeat protein to form a transcription complex,which is then regulated at the transcriptional level.Arabidopsis MYB90/PAP2(production of anthocyanin pigment 2)gene encodes a R2R3-MYB transcription factor.Its constitutive expression in Arabidopsis and tobacco induces the production of purple plants,Therefore,this study intends to overexpress PAP2(AtPAP2)gene in tomato to obtain tomato with increased anthocyanin content,and then study the effect of accumulation of flavonoids(anthocyanin)on plant growth.The results showed that the transgenic lines had different degrees of anthocyanin accumulation in different organs,and the transgenic plants showed phenotypes such as reduced plant height and reduced lateral root growth.We further analyzed the possible causes of phenotypic changes in transgenic plants by transcriptome sequencing,real-time PCR,and exogenous auxin treatment.The main results of this study are as follows:1.In this experiment,the MYB90/PAP2 gene in Arabidopsis thaliana was introduced into tomato through genetic transformation,and three homozygous positive lines L1,L2,and L15 were obtained after screening the progeny.The transgenic lines had different degrees of anthocyanin accumulation in different organs,and the purple color was intensely pigmented in the stamen.In fruits,anthocyanins are dotted in the form of purple dots on the peel and in the flesh.Meanwhile,structural genes associated with anthocyanin synthesis and a bHLH transcription factor gene SIAN1 were significantly up-regulated in transgenic plants.Therefore,AtPAP2 can induce the accumulation of anthocyanins in transgenic plants by activating expression of anthocyanin biosynthesis-associated structural and regulatory genes.In addition,MYB protein S1AN2 associated with the biosynthesis of anthocyanins in tomato can interact with two endogenous bHLH proteins of S1AN1 and S1JAF13,while AtPAP2 only interacts with S1JAF13 but not with S1AN1.This may be a contributing factor to the limited accumulation of anthocyanin in AtPAP2 transgenic tomato fruit.2.The growth of transgenic tomato shoots was inhibited.The plant height,stem diameter,and number of nodes in L1 5 were reduced to approximately 80%of the wild type.Similarly,AtPAP2 transgenic tobacco was also significantly dwarfed,and the stem thickness and number of stem sections were also significantly reduced.The results showed that the expression of genes related to lignin biosynthesis,another important end product on the branch of phenylpropanoid metabolism,has also been affected in tomato stems,including cinnamate 4-hydroxylase(C4H),hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase(HCT)and p-coumarate 3'-hydroxlase(C3'H)all declined markedly in transgenic lines.Therefore,AtPAP2 can negatively regulate the biosynthesis of lignin.The dwarf phenotype of transgenic plants may be partly due to the decrease of lignin content,resulting in impaired xylem function and affecting the transport of water and the assimilation of nutrients.3.The number of lateral roots and lateral root primordia in transgenic tomato was significantly reduced.After 6 days of seed germination,the average number of lateral roots in wild-type tomato was 9 and the average number of lateral roots in transgenic line L1 was 5.6,while almost no lateral roots appeared in transgenic line L15.After 11 days of germination,the average number of lateral roots of L15 and L1 was 7.6 and 28.9,respectively,while the number of lateral roots of wild-type tomato reached 40 in the meantime.In addition,L15 still had no lateral root primordium formation 4 days after seed germination,while the average number of lateral root primordium per main root of WT has reached 23.2.Therefore,the heterologous expression of AtPAP2 inhibited the formation of lateral roots,which was probably due to the destruction of the initiation of the lateral root.4.Using RNA-Seq analysis,the expression differences of wild-type and transgenic tomato roots at the whole genome level were analyzed,and 19 differentially expressed genes related to lateral root formation were screened out.Among them,three LBD/ASL genes SlLBD16,SlLBD 16-like and SlLBD6,in the auxin signaling pathway,and two ATP-binding cassette(ABC)transporter subfamily ABCB/P-glycoprotein(ABCB/PGP)genes SIABCB21-like and SlABCBll-like related to auxin transport were down-regulated in transgenic plants.In addition,the SIPIN5 gene encoding auxin transport protein was up-regulated about 5 folds in transgenic tomato roots.S1PIN5 transports active auxin in the cytoplasm to the endoplasmic reticulum for passivation,thereby reducing the concentration of active auxin in the cell.Therefore,AtPAP2 may through regulating the concentration of active auxin in the cells and reliance on the LBD family genes and ABCB subfamily genes to regulate auxin signal transduction and auxin transport processes,and then,affecting lateral roots formation.5.Exogenous application of high concentrations of auxin to the roots of transgenic tomatoes could partly restore lateral root initiation,Meanwhile,the lateral root primordia and lateral roots of the transgenic plants were relatively slow to develop compared to the wild type,which also applied auxin in the roots.In addition,by using the lateral root induction system,we detected the expression of related genes,and found the expression level of SIPIN5 was significantly up-regulated in transgenic plants compared with wild-type plants.Therefore,we speculate that the change of auxin concentration and transport rate caused by the differential expression of SlPIN5 in transgenic plants may be an important reason for the slower formation of lateral root primordia in transgenic tomatoes treated with high concentrations of auxin.In summary,in this study,we overexpressed the MYB transcription factor gene AtPAP2 in tomato to obtain transgenic plants with higher content of anthocyanins,in order to study the effect of accumulation of flavonoids(anthocyanins)on the growth of tomato plants.Some genes that may be involved in regulating the initiation or elongation of lateral roots of tomato were excavated through transcriptome sequencing and other methods,providing a reference for further research on the molecular mechanism of tomato root development.