Functional Characterization Of TgWRKY75 And TgNAC29 During Petal Senescence In Tulipa Gesneriana

Petal senescence is an irreversible process and closely related to ornamental and economic values of ornamental plants.Tulip,which causes“Tulip Fever”in Netherland,is one of the most popular bulbous plants with high ornamental value.However,the molecular mechanisms of petal senescence in tulip remain largely elusive.Therefore,identification of key senescence-responsive genes and characterization of the petal senescence mechanisms are important for the improvement of cut flower preservation and prolongation of flowering period to promote tulip industry.Based on tulip Pac Biol transcriptome（SRA:PRJNA703083）and petal RNA-seq data（GEO:GSE136183）at different developmental stages,two senescence promoting transcription factors,TgWRKY75 and TgNAC29,were selected for further analysis.In the current work the function and molecular mechanisms of these two genes in petal senescence were characterized.Main results of this research are shown as below.1.Salicylic acid（SA）and abscisic acid（ABA）promote petal senescence.Hormonal quantification data during tulip petal senescence showed that endogenous SA and ABA contents significantly increased at the last two petal senescent stages.Exogenous treatment of tulip petal discs with SA and ABA significantly promoted petal senescence,while the most obvious effect was obtained after the combination treatment with SA and ABA.2.TgWRKY75 and TgNAC29 were induced by senescence and plant hormones.TgWRKY75 and TgNAC29 were selected based on the Tulipa gesneriana transcriptome data.The CDS of TgWRKY75 gene is 543 bp,encoding 180 amino acids with a conserved WRKY domain at the C-terminal,indicating that TgWRKY75is a typical WRKY transcription factor.Subcellular localization showed that TgWRKY75 is located in the nucleus.Transcriptional activation assay showed that N terminus of TgWRKY75 is essential for this transactivation.The CDS of TgNAC29gene is 765 bp,encoding 254 amino acids with a conserved NAM domain at the N-terminal,indicating that TgNAC29 is a typical NAC transcription factor.Subcellular localization showed that TgNAC29 is located in the nucleus.Transcriptional activation assay showed that C terminus of TgNAC29 is essential for this transactivation.In addition,the expression level of TgWRKY75 was induced by exogenous ABA and SA,and the transcript level of TgNAC29 was induced by exogenous SA.3.TgWRKY75 and TgNAC29 play positive roles in the regulation of petal senescence,respectively.TgWRKY75 and TgNAC29 were silenced in flowers and petals using VIGS method.Compared with TRV2 control,TgWRKY75 and TgNAC29VIGS plants and discs showed a delayed flower and petal senescence phenotype.In addition,significantly increased anthocyanin content and reduced Tg SAG6（marker gene of senescence in tulip）transcript levels were observed in TRV2-TgWRKY75 and TRV2-TgNAC29 discs when compared to TRV2 control.By contrast,transgenic Arabidopsis lines overexpressing TgWRKY75 and TgNAC29 were generated which displayed accelerated leaf senescence phenotype compared with WT.Consistently,lower chlorophyll content,increased AtSAG12 and AtSAG13 transcript levels were observed in TgWRKY75-OE and TgNAC29-OE transgenic plants in relative to WT.4.TgWRKY75 plays a positive role in tulip petal senescence by enhancing SA and ABA biosynthesis.Endogenous SA and ABA contents showed significant increases in TgWRKY75 transgenic plants when compared to wild type（WT）.The transcript levels of SA and ABA biosynthesis-related genes（At ICS1（At SID2）,TgNAL1,TgNBS3,AtEPS1,AtEDS5 and AtNCED1,AtNCED3,AtNCED5,AtABA1 and AtABA2）were obviously up-regulated in overexpressing lines when compared with WT.The contents of SA and ABA reduced and the expression levels of SA-and ABA-synthesis related genes TgPAL1,TgICS1 and TgNCED3 were significantly down-regulated in TgWRKY75-silenced discs when compared with TRV2 control.In the presence of AIP（an inhibitor of SA）,petal senescence was obviously retarded in both TRV2 and TRV2-TgWRKY75 petal discs.Silencing TgWRKY75 attenuated the promoting effect of ABA and SA on petal senescence.By contrast,TgWRKY75-OE transgenic plants showed the opposite trend.Furthermore,Y1H,EMSA and LUC assays indicated that TgWRKY75 is able to bind to the promoters of Tg ICS1,TgPAL1 and TgNCED3 to activate their expressions.These results indicate that TgWRKY75 plays a positive role in tulip petal senescence by enhancing SA and ABA biosynthesis.5.TgNAC29 functions as a positive regulator in tulip petal senescence by mediating SA biosynthesis and ROS accumulation.Higher SA and H₂O₂contents and lower POD activity were observed in TgNAC29-OE transgenic plants in relative to WT.The transcript levels of SA and ROS biosynthesis-related genes（At SID2,TgNAL1,AtEPS5,AtEPS1,AtRboh A,AtRboh D and AtRboh F）were significantly up-regulated in overexpressing lines when compared with WT,while ROS scavenging genes（TgNrx4,TgNrx33 and TgNrx72）were significantly down-regulated.By contrast,decreased SA and H₂O₂content and increased POD activity were observed in TRV2-TgNAC29 discs when compared to TRV2 control.The transcript levels of SA biosynthesis-related genes（TgICS1 and TgPAL1）were significantly lower,and ROS scavenging genes（TgPOD12 and TgPOD17）were significantly higher in TgNAC29-silenced discs than those in TRV2 control.AIP treatment significantly delayed petal senescence in both TRV2 and TRV2-TgNAC29 petal discs.Silencing TgNAC29 weakened the promoting effect of H₂O₂and SA on petal senescence.By contrast,TgNAC29-OE transgenic plants showed the opposite trend.Y1H,EMSA and LUC assays proved that TgNAC29 binds to the promoters of SA biosynthetic genes（Tg ICS1 and Tg PAL1）to activate their expressions,however repress ROS scavenging genes（Tg POD12 and Tg POD17）through binding to the promoter regions of these two genes.These results indicate that TgNAC29 functions as a positive regulator in tulip petal senescence by mediating SA biosynthesis and ROS accumulation.Taken together,these results in this study indicated enhanced feed-back regulations between TgWRKY75 and ABA/SA biosynthesis pathways,and between TgNAC29 and SA biosynthesis/ROS accumulation pathways,resulting in an irreversible senescent process in tulip petals.This study partially characterized the molecular mechanisms of tulip petal senescence and offer more insights for prolonging blooming period and postharvest preservation of fresh cut flowers.