Functional Analysis Of A HD-Zip I Family Member RhHB1during Rose Petal Senescence

Rose is one of the most important ornamental plants and cut flowers in the global market. The economic value of rose is largely depended on the flower opening quality, which is decided by petal expansion and senescence. The supply of cut rose mainly depends on the long-distance transportation. However, inappropriate postharvest handling and long-distance transportation often results in great postharvest loss to the quality of cut roses with unnormal opening and accelerated senescence. It is clear that both abscisic acid (ABA) and ethylene can accelerate the process of flower senescence and shortened vase life. On the other hand, gibberellins (GAs) are able to delay flower senescence and improve the opening quality. To date, however, it is less known concerning how ABA, ethyene and GAs integratedly regulate the flower senescence. Previously, HD-Zip I transcription factors were reported to be involved in mediating phytohormone cross-talk. Here, we further analyzed the HD-Zip I candidate gene’s biochemical characteristics, biological functions and the regulatory mechanisms in rose (Rosa hybrida’Samantha’) petal senescence. The results are shown as belows.1) we isolated8HD-Zip I subfamily genes by searching two transcriptome databases of rose ESTs derived from experiments involving dehydration or application of exogenous ethylene. We conducted RT-PCR and identified a HD-Zip I subfamily member and named RhHBl, which is likely contribute petal senescence. RhHB1shared the typical basic structure chracteristics of HD-Zip I family proteins and contained conserved homeodomain and leucine zipper dimians. It has a high degree of sequence homology to AtHB7and AtHB12from Arabidopsis. We examined subcellular localization of RhHB1in onion epidermal cells. The GFP fluorescence of RhHBl-GFP was exclusively located in the nuclei of the cells. Yeast one-hybrid assay indicated that the full-length and C-terminal region of RhHBl have transcription activity in yeast. Expression of RhHBl increases during senescence and after ABA and ethylene treatments in rose flowers, suggesting that the gene function may be related to petal senescence.2) we investigated whether it was possible to use petal discs as an easily manipulated and replicated experimental system to study senescence. We therefore compared the senescence process between petal discs and whole petals, and assessed changes of soluble protein content, ion leakage, anthocyanin content and RhSAG12(the rose homolog of SAG12) expression as markers for senescence progression. Similar increases in ion leakage and RhSAG12expression, and similar decreases in soluble protein concentration and anthocyanin content, were observed during senescence of petal discs and whole petals, although senescence was more rapid in the discs. We therefore concluded that the overall senescence process was comparable, and that the petal discs provide an appropriate experimental system.3) Expression of RhHB1was silenced in petal discs by virus-induced gene silencing (VIGS), and the discs were treated with ABA or ethylene. Compared with TRV control, Silencing of RhHB1delayed the ABA-or ethylene-mediated senescence. However, treatment with paclobutrazol, an inhibitor of GA biosynthesis, repressed these delays. These results suggest that ABA and ethylene promote petal senescence in rose at least partially through induction of RhHB1, and that the delayed senescence phenotype is probably attributed to higher GAs in RhHBl-silenced petal discs.Based on the above results, we found that a key regulatory enzyme in the GA biosynthetic pathway, rose GA20ox gene RhGA20oxl, was significantly induced in RhHB1-silenced petals. Suppressing RhGA20oxl expression using VIGS clearly promoted senescence in petal discs compared with TRV-treated controls, as was seen with the PAC treatments. We further measured endogenous GA levels in petal discs treated with the TRV control, the TRV-RhGA20ox1vector or the TRV control plus PAC (TRV+PAC). The results show that the levels of the bioactive GAs in discs treated with the TRV-RhGA20oxl vectors or TRV+PAC-treated discs were significantly reduced as compared with TRV controls. Moreover, eight well-characterized senescence-related genes were up-regulated in water-and PAC-treated discs during senescence, whereas their transcripts accumulated at lower levels in GA3-treated discs. These results suggest that RhGA20oxl plays a role in delaying rose petal senescence, and RhHBl-mediated petal senescence may involve repression of RhGA20oxl expression and reduce endogenous bioactive GAs.RhGA20oxl transcripts were present at significantly lower levels in ABA-or ethylene-treated petals, but were significantly higher following the same treatments in RhHB1-silenced petals and compared to TRV treated controls. These results suggest that ABA and ethylene treatments reduce RhGA20oxl expression in an RhHBl-dependent manner in rose petals. EMS A and yeast one-hybrid assay further confirmed that RhHBl is capable of directly repressing RhGA20oxl expression by binding to a9bp pseudopalindromic sequence, AATATTATT, in the RhGA20oxl promoter.4) Taken together, ABA and ethylene cooperatively regulate rose petal senescence in part by inducing expression of RhHBl, a HD-Zip I TF. RhHBl is capable of binding to the promoter of a GA biosynthetic gene, RhGA20oxl, thereby repressing expression of RhGA20ox1. The RhHB1-RhGA20ox1regulatory checkpoint mediates the interaction between plant hormone networks during petal senescence.