Functional Analysis Of RhFerritin1 Involved In Dehydration Tolerance And Senescence Of Cut Rose Flowers

Rose(Rosa hybrid)is one of the most important cut flowers in the world.Cut rose flowers are harvested at the bud stage,experience typical reversible dehydration stresses during the postharvest handling from production areas to consumers.However,the regulatory mechanisms involved in rose flower dehydration tolerance are not known.Ferritin is a type of multi-subunit protein,which is commonly found in plants.It has been reported ferritin is involved in response to many biotic and abiotic stress responses.Previous studies have found that JK616449,a putative ferritin gene,was significatly induced by dehydration in rose petals.But the function of JK616449 in response to dehydration stress was still unclear.In this study,we investigated the basic biochemical characteristics,biological functions,and transcriptional regulation patterns of Ferritin by proceeding yeast one hybrid,qRT-PCR,VIGS etc approach.Those results are shown as follows:1)We cloned the full-length JK616449 gene,and obtained 1518 bp cDNA with an 807-bp open reading frame(ORF),encoding a polypeptide of 268 amino acids.This putative protein has a plant-specific extended polypeptide(EP)and a conserved Fe storage and transfer sequence.Phylogenetic analysis indicated that the rose ferritin protein was highly homologous to A.thaliana AtFer1,and so the rose gene was renamed RhFerl.We observed that RhFerl expression increased after dehydration and exogenous ABA treatment in rose petals.2)The relative Fe concentration was increased significantly after dehydration in rose petals,but there is no concomitant increase in free Fe2+.qRT-PCR analysis showed that the expression of Fe transport genes were repressed in dehydrated rose petals.This result indicated that the maintenance of the Fe homeostasis did not depend on Fe transporter genes.And we investigated subcellular Fe localization in rose petals and found that Fe accumulated in the vascular bundles and cell cytoplasm,but not in the vacuole.Furthermore,the levels of H2O2 and the activities of ROS-scavenging enzymes also showed no obvious induction.These results indicate that the maintaining of Fe levels was mainly depended on RhFerl during rose petals dehydration.3)Silencing RhFerl reduced dehydration tolerance in rose petals.Fe concentrations were similar between RhFerl-silenced rose petals and those of the TRV control,whereas in vivo staining of ferrous Fe(Fe2+)revealed more free Fe2+ in RhFerl-silenced petals compared with the TRV controls.Furthemore,the levels of H2O2 and the activities of ROS-scavenging enzymes were much higher in RhFerl-silenced petals than in those of the controls.This result indicated that there was a more severe oxidation stress in Rher1-silenced rose petals.4)The expression of RhFerl was in a RhABF2-dependent manner in dehydrated rose petals.Silencing RhABF2 down-regulated the expression of RhFerl.RhABF2 could bind to the RhFerl promoter probe,which containing the ABRE element,by processed EMSA approach in vitro.To test for a possible interaction between RhABF2 and the RhFer1 promoter in vivo,we performed a yeast one-hybrid and transiently transformed tobacco assay.The results showed that RhABF2 was capable of directly regulating RhFer1 expression by binding to the promoter of RhFer1.5)Silencing RhABF2 reduced dehydration tolerance in rose petals.Silencing RhABF2 resulted in a higher concentration of free Fe2+,breakdown in Fe homeostasis and accumulating H2O2,which caused a more severe oxidative stress in rose petals.6)ABA production was induced at the flower senescence stages,and the expression levels of both RhABF2 and RhFer1 were considerably higher at stages 4-6 than at stage 2.In addition,expression of the four Fe transporter genes,RhYSL3,RhOPT3,RhIRT3 and RhNRamp3,but not RhVIT1,increased sharply during flower senescence.Silencing-RhABF2 or-RhFer1 accelerateed senescence in rose petals.Those results indicated that the regulatory module RhABF2/RhFer1 plays synergistic roles with transporter genes during flower senescence.In conclusion,the regulatory module RhABF2/RhFer1 contributes to the maintenance of Fe levels and enhances dehydration tolerance through the action of RhFer1 locally sequestering free Fe2+ under dehydration conditions,and plays synergistic roles with transporter genes during flower senescence.