Molecular Mechanism Of FaWRKY44 And FaWRKY46 In Regulating Strawberry Anthocyanin Metabolism

Anthocyanins are not only important nutrients for strawberry fruit but also the main substance that determines the fruit’s color.In China,strawberries are mainly cultivated in facilities during winter and spring,and the insufficient lighting in the facilities leads to poor coloring and low commerciality of strawberry fruit.Therefore,it is important to analyze the regulatory mechanism of anthocyanin metabolism during the development and ripening of strawberry fruit.In this study,two WRKY transcription factors-Fa WRKY44 and Fa WRKY46 were newly identified,both of which can affect the accumulation of anthocyanins in strawberry fruits,but the regulatory mechanisms involved are unclear.Therefore,this study used bioinformatics,qRT-PCR,overexpression or silencing,transcriptomics and metabolomics,DAP-seq,yeast one/two-hybrid,and other techniques to explore the expression characteristics of Fa WRKY44 and Fa WRKY46,and their mechanism of regulating strawberry anthocyanin metabolism.The main research content and results are as follows:1.The CDS sequences of Fa WRKY44 and Fa WRKY46,as well as their homologous genes,were amplified using PCR from two types of diploid wild strawberries’Ruegen’and’Fragaria pentaphylla’,as well as two types of octoploid cultivated strawberries’Benihoppe’and’Toyonaka’,respectively.The results showed that the CDS or amino acid sequences of WRKY44 and WRKY46 were relatively conserved among these four strawberry species.Subcellular localization of Fa WRKY46 or Fa WRKY44 in tobacco leaves showed that both proteins were localized in the nucleus.By connecting Fa WRKY46 or Fa WRKY44 to p ET-32a(+)or p GEX-4T-1 prokaryotic expression vectors,the soluble target proteins could be induced by E.coli Rosetta(DE3)strain.The promoters of Fa WRKY44 and Fa WRKY46 were amplified from’Benihoppe’by PCR method.Sequence analysis showed that the promoter sequence(2,131 bp)of Fa WRKY44 was closer to that of wild strawberries.Two types of promoter sequences existed in Fa WRKY46,of which promoter 1(2,419 bp)was closer to that in wild strawberries,and promoter 2 was missing a segment of about 700bp.2.The tissue specificity and expression characteristics of Fa WRKY44 and Fa WRKY46 under different treatment conditions(low potassium,drought,low phosphorus,ABA,high salt,low temperature,red or blue light)were analyzed by qRT-PCR.The results showed that Fa WRKY46 showed a strong constitutive expression trend with very high expression levels in roots,stems,runners,young leaves,functional leaves,flowers,and fruits at different developmental stages of the’Benihoppe’strawberry.Fa WRKY44 showed a weaker constitutive expression trend with relatively high expression levels in functional leaves and flowers,while the expression level was slightly lower in fully red fruits.Fa WRKY46 was relatively more sensitive to low phosphorus,low temperature,and high salt stress,while Fa WRKY44 was more sensitive to low potassium and low temperature.Neither Fa WRKY46 nor Fa WRKY44could respond quickly to red or blue light treatment.3.VIGS or transient overexpression methods were used to alter the expression levels of Fa WRKY46 or Fa WRKY44.The results showed that silencing of either Fa WRKY44 or Fa WRKY46 in’Benihoppe’strawberry fruit could inhibit the accumulation of anthocyanins in the flesh,while overexpression of Fa WRKY46 in’Xiaobai’strawberry restored the accumulation of anthocyanins in the flesh,but overexpression of Fa WRKY44 did not.Further analysis by qRT-PCR and RNA-seq revealed that the regulation of anthocyanin metabolism in strawberry fruit by Fa WRKY44 or Fa WRKY46 was more likely to be achieved by affecting the later stages of anthocyanin transport,storage,or degradation(stability),while the impact on anthocyanin synthesis and related structural genes in the early stage is limited.Overexpression of Fa WRKY44 in Arabidopsis was found to increase the accumulation of proanthocyanidins in seeds of wild type or three ttg2 mutants(CS2106724,SALK＿204777C,and SALK＿206852C).4.qRT-PCR,HPLC,transcriptomics,targeted/quasi-targeted metabolomics,and DAP-seq were used to analyze the key genes regulating the flesh of’Benihoppe’and’Xiaobai’strawberries.The results showed that Fa MYB10 or Fa TT19 was not a key factor causing pigment deficiency in’Xiaobai’flesh.The Fa C4H in the phenylpropanoid biosynthesis pathway was crucial for controlling the coloration of strawberry flesh.Its transcriptional level was inhibited,resulting in the inability to accumulate anthocyanins in the’Xiaobai’flesh,and this repression was not caused by promoter sequence methylation.Fa WRKY46 affected the accumulation of anthocyanins by regulating the transcription of Fa C4H and the downstream target genes UFGT7 and H~+-ATPase.5.The interaction proteins and upstream regulatory genes of Fa WRKY44 or Fa WRKY46 were screened using methods such as yeast one-hybrid,yeast two-hybrid,Bi FC,and bioinformatics prediction.The results showed that there was protein interaction between Fa WRKY44 and MBW complex members Fa TTG1,Fa EGL3,or Fa LWD1-like,but no interaction with Fa MYB1 or Fa MYB10.There were interactions between Fa WRKY46 and Fa LWD1,Fa LWD1 like,Fa EGL3,Fa MYB9,Fa MYB10 or Fa MYB11,but no interaction with Fa MYB1 or Fa MYB5.In addition,Fa WRKY44may interact with PR1A,U-box 19,GL2,TT1,MYB59 or ABC transporter proteins.Fa WRKY46 may have protein interactions with nuclear transport factor 2,Dp-1transcription factor,PP2C37,MAP3K1,PP2C23,PP2C4,ERF4,b HLH92,MYB39,or ABI4.The promoter of Fa WRKY46 could be bound by MYC2,b HLH13,NAC18,Zinc finger protein 1,WRKY3,and FK506-binding protein 4-like.6.The effects of MYBs,MAPKs,and ABA signaling pathway genes on the transcription levels of Fa WRKY44 or Fa WRKY46 were analyzed using qRT-PCR and transient overexpression methods.The results showed that the expression levels of Fa WRKY44 or Fa WRKY46 in strawberry flesh were more easily regulated by Fa MYB5or Fa MYB10 compared to the skin.The phosphate kinases Fa MAPK3,Fa MAPK4-2,or Fa MAPK16 were able to affect the expression levels of Fa WRKY44 or Fa WRKY46in strawberry flesh.The Fa NCED1,Fa Sn RK2.2,or Fa Sn RK2.6 in the ABA signaling pathway promoted the expression of Fa WRKY46 in strawberry flesh,but have no significant effect on Fa WRKY44.The effect of Fa BT2 protein on Fa WRKY44 or Fa WRKY46 protein levels was analyzed using yeast two-hybrid and a modified dual-luciferase method.The results showed that there was no interaction between Fa BT2and Fa WRKY46 or Fa WRKY44 proteins,but Fa BT2 was able to strongly induce the degradation of Fa WRKY46 protein.