HY5 Participates In Leaves Phototropism In Grape(vitis Vinifera L.)

Light,which can be sensed and transmitted by photoreceptors of plants,is an informational signal and undoubtly one of the most important environmental factors.Light serves as a signal to modulate many aspects of plant development throughout its life cycle,such as phototropism.Phototropism is of great importance in orienting their photosynthetic organs towards the light so as to optimize the light capture capacity of the aerial parts in seedlings of higher plants.However,the genetic regulatory mechanisms that govern phototropism in grape(Vitis vinifera L.)leaves remained elusive.Approximately 20-day-old grape leaves were the fastest in response to phototropism.Leave blades and petioles were sampled from shoots of V.vinifera cv.‘Italian Riesling'trees that were artificially fixed on the wire with strings under the condition of the blade abaxial side face-upward in the field(fixation of shoots did not interfere with leaves rotation),respectively.Leaves growing in the natural state(not artificially fixed on the wire)as control.The treated blades rotate from the abaxial side to the adaxial side after fixation for 2 h,and the phototropic response was completed after 6-10 hours.After irradiating the abaxial side of treated blades for 2 h,leaves were sampled for RNA sequencing analysis.Compared with the previous 3 h transcriptome data,the key candidate transcription factors related to phototropic response of grape leaves were screened.HY5 is a member of the basic leucine zipper protein(b ZIP)transcription factor(TF)family.It plays an important role in the photomorphogenesis of Arabidopsis thaliana,and it can regulate the expression of several hormones related genes.Although many functions of HY5 in Arabidopsis thaliana have been widely studied,few studies have been done in grape.In Arabidopsis,GA is considered to be a factor that inhibits phototropism,but its function in grape leaf phototropism has not been determined.Phytochrome interacting factors(PIFs)belong to basic helix loop helix(b HLH)proteins.Their functions in Arabidopsis light signal have been widely studied,but few in grape.In this study,‘Ialian Riesling'was used as the experiment materials,Exogenous GA₃ was sprayed to determine the role of GA₃ in the phototropism response of grape leaves,VvHY5 and VvPIF3 transcription factors were cloned,and gene function verification was carried out.RNA was extracted from the leaves and petioles of grape leaves during the grape leaf phototropic response,a yeast two hybrid c DNA library was prepared and p GBKT7-VvHY5 vector was constructed.The interaction proteins of VvHY5 were screened by mating method.The main results are as follows:1.Transcriptome sequencing results showed that hormone signaling pathway was involved in the phototropic response of grape leaves;GA may negatively regulate the phototropism of grape leaves,and VvPIF3,the negative regulator of light signaling pathway in GA,plays an important role in the negative regulation of phototropism of grape leaves.In addition,The positive regulator of the light signaling pathway,HY5,was significantly up-regulated in treated leaves and petioles at both time points,suggesting that VVHY5 plays an important role in positively regulating the phototropic response of grape leaves.Therefore,this study completed the functional verification from two aspects of positive regulation and negative regulation.2.The transcription factor VvHY5 was successfully cloned by RT-PCR,and the sequencing results showed that VvHY5 contained a complete open reading frame of510 bp,encoding 169 amino acids.Real-time fluorescence quantification PCR(q RT-PCR)analysis showed that VvHY5 was expressed in both leaves and petioles,but there were some differences in different tissues,and the expression level in leaves was higher than that in petioles.The expression of VvHY5 in leaves and petioles increased at first and then decreased during the phototropic response.Overexpression of VvHY5in Arabidopsis promoted phototropism of hypocotyl,and the bending angle of hypocotyl was significantly larger than that of wild type.The bending angle of the transgenic plant was 39.38 degrees,and that of the wild type was 8.7 degrees.In transgenic Arabidopsis and grapevine calli overexpressed with VvHY5,the expression of IAA19 was increased and the expression of TIR1 was decreased,indicating that VvHY5 can regulate phototropism by regulating the expression of phototropism related genes.3.RNA was extracted from the leaves and petioles of grape leaves during the grape leaf phototropic response,and a yeast two hybrid c DNA library was prepared and p GBKT7-VvHY5 vector was constructed.The interaction proteins of VvHY5 were screened by mating method,including two light signal related genes(VvHY5-like)VvHYH and VvBBX24.The heterodimer of HYH and HY5 could be used as a coactivator to improve its phototropism in Arabidopsis etiolated seedlings.The results of yeast two hybrid point-to-point verification showed that the combination of VvHY5 and VvHYH,VvHY5 and VvBBX24 into Y2H yeast competent cells could grow on SD/-Trp-Leu-His-Ade medium and make X-?-gal blue.Yeast one hybrid showed that VvHY5 could bind to the promoter of VvABI5.4.The results of exogenous GA₃ treatment showed that GA₃ significantly inhibited the phototropic response of grape leaves.Moreover,transcriptome sequencing was conducted on exogenous GA₃-treated grape leaves and petioles and water-sprayed grape leaves and petioles.The results showed that the transcription factor VvPIF3 of GA pathway was up-regulated in the leaves and petioles treated with GA₃,which indicated that VvPIF3 negatively regulated the phototropism of grape leaves Furthermore,VvPIF3 plays a key role in GA negatively regulated phototropism of grape leaves.5.The transcription factor VvPIF3 was successfully cloned by RT-PCR,and the sequencing results showed that VvPIF3 contained a complete open reading frame of603 bp,encoding 200 amino acids.q RT-PCR analysis showed that the expression of VvPIF3 in leaves and petioles decreased during the whole phototropism response.The expression of VvPIF3 in Lck was lower than that in PCK at different time points,but the expression of VvPIF3 in L was higher than that in P at different time points.Overexpression of VvPIF3 in Arabidopsis inhibited the phototropism of hypocotyls,and the phototropic bending angle of hypocotyl was significantly smaller than that of wild type.The bending angle of the transgenic plant was 14.13 degrees,and the bending angle of the wild type was between 4.38 and 4.87 degrees.Moreover,the GA content of VvPIF3 overexpressed Arabidopsis and grapevine callus was significantly higher than that of wild-type Arabidopsis and grape callus,while the IAA content was significantly lower than that of wild-type Arabidopsis and grape callus.