Functional Analysis Of Transcription Factors Related To Petunia Flower And Apple Fruit Senescence

Flower senescence leads to petal wilty and death of the whole flower,which seriously affects its ornamental value;while fruit senescence causes loss of edibleness,in short,senescence irreversibly reduces the commercial value of horticultural products.Flower senescence is a process stimulated by endogenous signals and environmental cues,while postharvest fruit senescence mainly affected by environmental condition.Both flower and fruit senescence are regulated by hormones,and require gene transcription,posttranscriptional and post-translational regulation.Gene transcription is regulated by transcription factors.So far,there is only several transcription factors reported to be related with flower senescence,but how they regulated senescence process is less studied.The purpose of this study is to select several transcription factors that regulate petunia flower senescence process,illustrate how they regulate flower senescence by genetic engineering methods.In this study,the virus-induced gene silencing(VIGS)method was used to identify the function of transcription factors up-regulated during petunia flower senescence.To further demonstrate the function of senescence-related transcription factors,apple fruit was used to check the expression pattern of related transcription factors during fruit senescence process.The results are as following:1.30 transcription factors were selected from routine screening of flower senescenceassociated up-regulated genes using customer-designed Nimblegen microarray.The 30 transcription factors were grouped to 11 different families:Zinc-finger(9),ERF(3),HD-Zip(2),bHLH(3),Nam-like(3),GRAS(1),leucine zipper(1),LIM(1),MYB(1),ARF(1)and MADS-box(1),and there were four DNA-binding proteins not belong to any family.2.According to the sequences of the 30 transcription factors in DFCI database,gene specific primers for each transcription factor gene were designed and RT-PCR was used to analyze their expressions during petunia flower senescence.The results showed that:during flower senescence,the expression levels of TF4(PhEREBF10)and TF8(Zinc-finger)decreased at the first two days after flower opening,then increased at the late stage of senescence with the same level as the day opening;TF13(Leucine zipper)and TF26(Nucleoid DNA binding protein)with no significant changes;the expression levels of TF12(Zinc-finger),TF16(MYB)and TF22(Nucleoid DNA binding protein)went down;the expressions of these transcription factors were not correspondence with the results in microarray database;the other transcription factors were all up-regulated during petunia flower senescence in accordance with microarray data.3.VIGS method was used to identify the functions of the 30 transcription factors on petunia flower senescence.Flower longevity of wild type,vector control and gene silenced petunia plants were recorded after gene silencing of each transcription factor.The results were:compared with wild type and vector control,flower longevity of TF4(PhEREBF10)and TF14(bHLH)silenced plants were extended by two days;TF5(Zinc-finger)and TF7(HDZip)silenced plants were extended by one day;TF2(GRAS)silenced plants were extended by half day;however,TF17(ARF)silenced plants accelerated flower senescence by one day;silence of the three Nam-like protein genes,the other family genes and the four unknown family DNA-binding protein genes did not change petunia flower longevity.4.Flower longevity of TF14(bHLH)silenced plants were extended by two days by VIGS method.Detailed studies on TF14(bHLH)were conducted to further identify how it regulated flower senescence.According to sequence alignment analysis,this gene showed high identity with bHLH transcription factors of several species,so we named it Ph bHLH in this study.Ph_bHLH expressed in stem,leaf and flower organs of petunia,and with high expression level in senescent leaf and flower;Ph_bHLH expression was induced by ethylene and inhibited by 1-MCP;expression levels of ethylene biosynthesis genes(ACO1,ACO4 and ACS)and senescence-associated genes(SAG 12 and SAG29)were down-regulated in Ph_bHLH silenced flowers.The results suggested that transcription factor Ph_bHLH affects flower senescence by regulating expression of senescence-related genes.5.Flower longevity of TF7(HD-Zip)silenced plants were extended by one day by VIGS method.Detailed studies on TF7(HD-Zip)were conducted to further identify how it regulated flower senescence.According to sequence alignment analysis,this gene showed high identity with pepper(CaHD-Zip),tobacco(NaHD20)and Arabidopsis(ATHB 7 and ATHB12)HDZip transcription factors,so we named it PhHD-Zip in this study.Bioinformatics analysis showed that predicted subcellular location of PhHD-Zip may be nucleus or microbody(peroxisome),and its predicted function was growth factor and transcription regulation;PhHD-Zip expressed in stem,leaf and flower organs of petunia,and with highest expression level in flower;PhHD-Zip transcript level was induced by hormones(ethylene,ABA)and abiotic stress conditions(dehydration,NaCl and cold).6.Natural and pollinated flower longevities were both extended in PhHD-Zip silenced flowers by VIGS methods.Silence of PhHD-Zip reduced ethylene production and transcript abundance of ethylene biosynthesis-related genes(ACO1,ACO4 and ACS),ABA biosynthesis-related gene(NCED)and senescence-related genes(SAG12 and SAG29);On the other hand,over-expression of this gene(35S::PhHD-Zip)in petunia caused earlier flower senescence.The results suggest that transcription factor PhHD-Zip plays an important role in regulating petunia flower senescence,mediating through the ethylene and ABA biosynthesis pathways and may play a role in crosstalk between the two hormones.7.According to the results from petunia,HD-Zip,bHLH,ERF,Zinc-finger and ARF transcription factors related to flower senescence,so we suppose these transcription factors may play a role during apple fruit senescence.Apple homologs with the higest similarity to the five petunia transcription factor genes were selected based on DFCI database,and there expression patterns during postharvest apple fruit storage process were analyzed.Results showed that expression levels of apple MdHD-Zip,MdbHLH and MdERF genes increased during fruit senescence;which suggests these transcription factors may play a role in fruit senescence process.