Functional Analysis Of A RhPR10.1 Gene In Ethylene-induced Petal Senescence In Rose(Rosa Hybrida)

Rose is one of the most important commercial flowers,accounting for 32%of total sales of cut flowers in the worldwide.As the largest rose planting country,the cut roses sale numbers and sales have remained stably growth tread in the last decade in china.The sale numbers and sale values increased from 2.67 billion to 5.64 billion and 0.97 billion yuan to 3.02 billion yuan over the last ten years,respectively.As origin and consumer market are not in same areas,the supply of cut rose mainly depends on the long-distance transportation,which often results in great postharvest loss to the quality of cut roses with unnormal opening and accelerated senescence,specially that losses is up to 30%in china.So the hot research topics is how to delay the cut roses postharvest senescence worldwide.It has been reported that phytohormone ethylene(C2H4)promotes flower senescence,while cytokinin delays this process.However,the molecular mechanisms underlying the antagonistic effect between these two plant hormones during flower senescence are still largely unknown.Here,we explore the function of RhPR10.1 in antagonism of ethylene-induced petal senescence in rose(Rosa hybrida ’Samantha’)through analysis of biochemical characteristics,biological functions and regulatory mechanisms.1)Total number of seven PR-10 family genes were found in ethylene-treated rose petals transcriptome database.The expression pattern of all seven PR-10 family genes were up-regulated in senescing rose petals,specially for RU06312.The expression level of RU06312 was also induced in other senescent floral organs and leaves.A conserved motif ’P-loop’ was found by aligment the amino acid of this protein with other PR-10 family proteins from other species.The most homologous protein of RU06312 was VvPR10.1,which may regulate somatic embryogenesis in Vitis vinifera cultivar"Chardonnay",so was named RhPR10.1.The expression of RhPR10.1 was also indued by cytokinin and ethylene treatments,but inhibited by ABA.Another aspect is that ’the role of RhPR10.1 may be involved in rose petal senescence’.2)To better understand the potential biology function of RhPR10.1 in rose petal senescence,we silenced RhPR10.1 in rose flowers and petals using a virus-induced gene silencing approach(VIGS).Flower opening phase of RhPR10.1-silenced plant was indistinguishable from TRV control.However,the RhPR10.1-silencing exhibited an acclerated senescence phenotype,with only lasting 3.7±0.4 days from stage 5 to 6,whereas TRV-control lasting 5.1 ±0.5 days.Moreover,the senescence was markerly accelerated in RhPR10.1-silenced rose petal,resulting much higher ion leakage rate and the expression of senescence marker gene RhSAG12.On the contray,constitutive expression of RhPR1O.1 in Arabidopsis significantly delayed leaf senescence.The transcripts of three cytokinin-induced type A cytokinin signaling pathway genes RhRR3,RhRR8 and RhRR9 were significantly down-regulated in RhPR10.1-silenced rose petals.The content of bioactive cytokinin iPA was significantly reduced from 558.53 ± 103.44 pg/g in TRV control to 103.98 ±22.48 pg/g in RhPR10.1-silenced rose flower.However,The phynotype of RhPR10.1-silenced rose petals was recovered by cytokinin treatment,and no significantly difference was found both ion leakage rate and expression of RhSAG12.Keeping both the points in mind,RhPR10.1 is involved in rose petal senescence through regulating cytokinin content and its signaling pathway.3)To better understand the molecular mechanism of RhPR10.1 in regulation of rose petal senescence,a ’1378’ bp promoter region upstream of RhPR10.1 coding sequence was amplified by TAIL-PCR and analyzed using PLACE database.Here,potential of ATHB6 binding site(CAATTATTA)spanning positions-706 to-715 was found.A transcript RU12591 annotating as ATHB6 homolog protein was found in our rose transcriptome database.The expression of RhHB6 was also signicantly up-regulated in senescing rose flower and ethylene treatment,but inhibited by ABA treatment.RhHB6 can directly bind to RhPR10.1 promter by EMSA and yeast one hybrid assays.4)Like as RhPR10.1-silenced in rose,flower opening phase of RhHB6-silenced plant was indistinguishable from TRV control.However,the RhHB6-silencing exhibited an acclerated senescence phenotype,with only lasting 3.6±0.3 days from stage 5 to 6,whereas TRV-control lasting 5.6±0.5 days.Taking together,RhPR10.1 functions in antagonism of ethylene-induced rose petal senescence through regulation of cytokinin content and its signaling pathway.Furthermore,the antagonistic mechanism between ethylene and cytokinin regulation in rose petal senescence partially through RhHB6-RhPR10.1 checkpoint.