The Plant Journal (2014) 78, 578–590

doi: 10.1111/tpj.12494

RhHB1 mediates the antagonism of gibberellins to ABA and ethylene during rose (Rosa hybrida) petal senescence € 1, Changqing Zhang1, Jitao Liu1, Xiaowei Liu1, Guimei Jiang1, Xinqiang Jiang1, Muhammad Ali Khan1, Liangsheng Peitao Lu 2 Wang , Bo Hong1 and Junping Gao1,* 1 Department of Ornamental Horticulture, China Agricultural University, Beijing 100193, China, and 2 Beijing Botanical Garden, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China Received 26 October 2013; revised 20 January 2014; accepted 17 February 2014; published online 3 March 2014. *For correspondence (e-mail [email protected]).

SUMMARY Rose (Rosa hybrida) is one of the most important ornamental plants worldwide; however, senescence of its petals terminates the ornamental value of the flower, resulting in major economic loss. It is known that the hormones abscisic acid (ABA) and ethylene promote petal senescence, while gibberellins (GAs) delay the process. However, the molecular mechanisms underlying the antagonistic effects amongst plant hormones during petal senescence are still unclear. Here we isolated RhHB1, a homeodomain-leucine zipper I transcription factor gene, from rose flowers. Quantitative RT-PCR and GUS reporter analyses showed that RhHB1 was strongly expressed in senescing petals, and its expression was induced by ABA or ethylene in petals. ABA or ethylene treatment clearly accelerated rose petal senescence, while application of the gibberellin GA3 delayed the process. However, silencing of RhHB1 delayed the ABA- or ethylene-mediated senescence, and resulted in higher petal anthocyanin levels and lower expression of RhSAG12. Moreover, treatment with paclobutrazol, an inhibitor of GA biosynthesis, repressed these delays. In addition, silencing of RhHB1 blocked the ABA- or ethylene-induced reduction in expression of the GA20 oxidase encoded by RhGA20ox1, a gene in the GA biosynthetic pathway. Furthermore, RhHB1 directly binds to the RhGA20ox1 promoter, and silencing of RhGA20ox1 promoted petal senescence. Eight senescence-related genes showed substantial differences in expression in petals after treatment with GA3 or paclobutrazol. These results suggest that RhHB1 mediates the antagonistic effect of GAs on ABA and ethylene during rose petal senescence, and that the promotion of petal senescence by ABA or ethylene operates through an RhHB1–RhGA20ox1 regulatory checkpoint. Keywords: Rosa hybrida, flower senescence, RhHB1, gibberellins, abscisic acid, ethylene.

INTRODUCTION Senescence is a terminal process that is manifested at the cellular, tissue or whole-organ level. In an agronomic context, it may significantly decrease crop yield and biomass accumulation, and contributes substantially to postharvest loss in fruits, vegetables and ornamental plants during storage and transportation (Gan and Amasino, 1997). Senescence has been studied in various plant organs, including leaves (Buchanan-Wollaston et al., 2005; Zhang and Gan, 2012), fruits (Qin et al., 2009; Kou et al., 2012) and flowers (Xu et al., 2007; Chen et al., 2011), using physiological, biochemical and molecular approaches. Physiological processes such as wilting, color fading and discoloration are phenotypic indicators of senescence € rtensteiner, 2004; van Doorn and Woltering, 2008), and (Ho it has been shown that these are mediated by multiple 578

complex structural and regulatory pathways associated with programmed cell death and diverse catabolic activities, such as polymer degradation, as well as de novo biosynthesis of nucleic acid, proteins and lipids. Accordingly, senescence has been shown to involve thousands of up- or down-regulated genes (Xu et al., 2007; Guo and Gan, 2012), including those encoding various transcription factors (TFs) and components of signal transduction pathways (Guo et al., 2004). Plant senescence may be precociously triggered by an array of biotic and abiotic stresses, such as pathogen infection, drought stress, nutrient deficiency, darkness and extreme temperature (Gan and Amasino, 1997). It is also an age-dependent process, and is regulated by senescence-promoting hormones, such as ABA and ethylene, as © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd

RhHB1 regulates rose petal senescence 579 well as those that delay it, such as gibberellins (GAs) and cytokinins (Reid and Chen, 2007; Zhang and Zhou, 2013). These phytohormones collectively modulate senescence through an intricate network of interactions (van Doorn and Woltering, 2008; Zhang and Zhou, 2013), as exemplified by numerous studies of senescence in flowers. ABA is considered to be a natural promoter of flower senescence (Reid and Chen, 2007), and application of ABA has been shown to accelerate senescence-associated events, such as the loss of membrane permeability and lipid peroxidation in the day lily Hemerocallis fulva (Panavas et al., 1998). Similarly, exogenously applied ABA has been shown to lead to premature accumulation of senescence-associated transcripts in the tepals of daffodil (Narcissus pseudonarcissus) (Hunter et al., 2004a). The gaseous hormone ethylene is also an important regulator that accelerates flower senescence in many plant species (van Doorn, 2001). In ethylene-sensitive species, petal senescence is associated with a burst of endogenous ethylene production, and may be greatly accelerated by treating flowers with exogenous ethylene, or delayed by applying inhibitors of ethylene biosynthesis or action (Reid, 2002). GAs are also thought to act as senescence-delaying hormones (Kappers et al., 1998), and GA treatments increase the vase life of cut flowers such as rose, carnation (Dianthus caryophyllus), Sandersonia aurantiaca and daffodil (Saks and van Staden, 1993; Sultan and Farooq, 1999; Eason, 2002; Hunter et al., 2004a,b). At the physiological level, a GA3-induced delay in the senescence of rose flowers was reported to be associated with maintenance of membrane integrity, as reflected by reduced electrolyte leakage (Sabehat and Zieslin, 1994). In addition to studies describing the influence of individual plant hormones on flower senescence, there have also been reports describing the importance of hormonal interactions. One example is the regulation of senescence in ethylene-sensitive flower species, such as rose and carnation, by ABA, as exogenous ABA triggers an early increase in ethylene production, and enhances sensitivity to ethylene (Mayak and Halevy, 1972; Mayak and Dilley, 1976). In addition, application of silver thiosulfate, an ethylene action inhibitor, to carnation flowers prevents ABA-mediated stimulation of senescence (Onoue et al., 2000). As another example, treatment with cytokinins delayed petal senescence by reducing petal sensitivity to ethylene (Chang et al., 2003), and ethylene has also been reported to promote degradation of cytokinins during floral senescence in petunias (Petunia hybrida) (Taverner et al., 1999). Furthermore, it is known that GA acts as an antagonist to ABA or ethylene action, thereby delaying flower senescence (Saks and van Staden, 1993; Hunter et al., 2004a,b). There have been numerous such reports of complex hormonal interactions involved in the regulation of flower senescence; however, the molecular mechanisms underlying cross-talk between hormone signaling pathways and

the associated antagonistic effects are not well understood. One component of the regulatory machinery comprises homeodomain-leucine zipper (HD-Zip) TFs, which are unique to plants and encode proteins with a homeodomain (HD) and a closely associated leucine zipper motif (Ruberti et al., 1991). This family of TFs has been divided into four sub-families (I–IV), according to their DNA-binding specificities, sequence similarity inside and outside the conserved domains, and biological functions (Ariel et al., 2007). Previous studies have shown that genes from the HD-Zip I sub-family are involved in abiotic stress responses (Ariel et al., 2010; Cabello and Chan, 2012), fruit ripening (Lin et al., 2008), and leaf and flower senescence (Manavella et al., 2006; Xu et al., 2007). For example, an HD-Zip I TF from tomato (Solanum lycopersicum), LeHB1, was shown to target a gene (LeACO1) encoding 1-Aminocyclopropane1-carboxylic acid oxidase, a key enzyme in the ethylene biosynthetic pathway, during fruit ripening, and inhibiting LeHB1 expression both greatly reduced LeACO1 mRNA levels and inhibited ripening (Lin et al., 2008). In contrast, another HD-Zip I TF from sunflower (Helianthus annuus), HaHB4, functions as an ethylene-mediated senescence repressor through reduction of ethylene synthesis and inhibition of its signal transduction (Manavella et al., 2006). In addition, in Mirabilis jalapa, the abundance of HD-Zip TFs was reported to increase substantially during senescence (Xu et al., 2007). However, despite these observations, the modes of action of HD-Zip I TFs in hormone-regulated flower senescence are still unclear. The commodity value of rose flowers (Rosa hybrida) largely depends on the quality of the open flowers, requiring fully extended petals and a long vase life. Accordingly, the physiological mechanisms of rose flower senescence have been an economically important area of research for several decades. In the case of flower senescence involving multi-layered petals, such as rose flowers, individual petals have been reported to behave similarly to whole flowers, providing a simpler experimental system (Mor and Reid, 1980; Le Page-Degivry et al., 1991). Senescence of rose flowers is characterized by wilting, color loss and abscission of petals (Tripathi and Tuteja, 2007; van Doorn and Woltering, 2008), and petal senescence may be affected by ABA, ethylene and GAs (Reid and Chen, 2007). Our study provides insight into the molecular mechanisms underlying the synergistic regulation of petal senescence by plant hormones. RESULTS GA3 delays ABA- and ethylene-induced senescence of rose petals As the senescence of rose flowers may be influenced by application of plant hormones such as ABA, ethylene and

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

€ et al. 580 Peitao Lu € ller et al., 1999; GA3 (Sabehat and Zieslin, 1994; Mu Chamani et al., 2005), we wished to systematically examine their potential antagonistic roles. To do so, 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 (Figure S1). Similar increases in ion leakage and RhSAG12 expression, 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. We next assessed the visible color changes of petal discs upon application of ABA, ethylene and/or GA3 based on dose experiments (Figure S2). As shown in Figure 1, the control petal discs at 5 days after treatment were essentially the same dark red color as before incubation, but, after 10 days, the color had faded to pink. In ABA- or ethylene-treated flowers, approximately half of the petal discs had turned yellow at 5 days after treatment, and almost all the petal discs showed a severe color loss after 10 days of incubation. Compared with untreated controls, GA3 treatment alone clearly delayed the color loss, and, as expected, the petal discs treated with a combination of GA3 + ABA or GA3 + ethylene showed better color retention than those treated with ABA or ethylene alone. Taken together, these results indicate that GA3 delays petal senescence, and plays an antagonistic role in ABA- and ethylene-induced petal senescence. Expression of RhHB1 increases during senescence and after ABA and ethylene treatments in rose flowers In a comparative analysis of two databases of rose ESTs derived from experiments involving dehydration or appli-

cation of exogenous ethylene (Dai et al., 2012; Pei et al., 2013), we identified a transcript, JK617961, encoding a putative homeobox family TF, that was present in both databases. We further confirmed by quantitative RT–PCR that ABA and ethylene treatments both induce JK617961 mRNA expression in petals (Figure 2c), suggesting a possible role in senescence. To investigate the function of this putative TF, the full-length cDNA was cloned, and sequence analysis indicated that JK617961 encodes a protein of 263 amino acids with a conserved HD (amino acids 37–96) and leucine zipper domain (amino acids 97–139) (Figure S3). Sequence alignment analysis further suggested that JK617961 belongs to the HD–Zip I TF family, and that it has a high degree of sequence homology to MtHB1 from Medicago truncatula (Figure 2a): the gene was therefore named RhHB1. We evaluated the expression of RhHB1 at various flower opening stages and after exogenous plant hormone treatments using quantitative RT–PCR analysis. RhHB1 transcript levels increased substantially in petals in parallel with flower opening from a completely opened bud (opening stage 2) to a partially opened flower (opening stage 4), and remained high until the onset of petal wilting (opening stage 6), further suggesting that the gene function may be related to petal senescence (Figure 2b). In contrast to treatment with exogenous ABA and ethylene, which induced expression of RhHB1, applying the hormones or hormone analogs GA, naphthalene acetic acid and brassinosteroids did not alter expression (Figure 2c). It has been reported that the outer layer in the multi-layered petals of a carnation flower is older than the inner layer (Wulster et al., 1982). Here, we evaluated the expression levels of RhSAG12 in various whorls of the rose flower by RT–PCR. As expected, the expression of RhSAG12 was much greater in petals of the outer whorl than in petals of the inner whorl (Figure S4a). To understand the difference in RhHB1 expression between the various petal layers, we assessed the activity of the RhHB1 promoter. A 1650 bp region immediately upstream of the RhHB1 coding sequence was fused to the GUS reporter Figure 1. Effects of exogenous gibberellic acid (GA3), ABA and ethylene on the senescence of rose petal discs. After 24 h pre-treatment with combinations of 100 lM ABA, 10 ll l
1 ethylene and 50 lM GA3 (as indicated), the petal discs were kept in water and the photos were taken on days 5 and 10, respectively.

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

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Figure 2. Phylogenetic analysis and expression patterns of RhHB1. (a) Phylogenetic tree generated using various HD–Zip I protein sequences from Arabidopsis, HaHB4 from sunflower, MtHB1 from Medicago truncatula, LeHB1 from tomato, and RhHB1. The phylogenetic tree file was produced using MEGA 5.2 (http://www.megasoftware.net/). Bootstrap values indicate the divergence of each branch, and the scale indicates branch length. (b) Quantitative RT–PCR analysis of RhHB1 expression in rose petals at various opening stages. RhUBI1 was used as an internal control. (c) Quantitative RT–PCR analysis of RhHB1 expression in rose petals in response to exogenous hormones: GA, 100 lM gibberellic acid; NAA, 50 lM naphthalene acetic acid; BR, 5 lM brassinosteroid; ABA, 100 lM ABA; Eth, 10 ll l
1 ethylene; 1–MCP, 2 ll l
1 1–methylcyclopropene. Rose flowers at opening stage 2 were analyzed after 24 h of hormone treatment. Mock samples were treated with dimethylsulfoxide without phytohormones. RhUBI1 was used as an internal control. Error bars represent the standard deviations of three biological replicates.

gene, and this construct was introduced into rose petal tissues at the partially opened stage (opening stage 3), using Agrobacterium-mediated transformation. After a 3 day incubation period, GUS expression driven by the RhHB1 promoter region was strongly detected in the outer layer of petals, but almost no activity was detected in the inner layer (Figure S4b), thus providing further evidence of a correlation between RhHB1 expression and petal senescence. We also observed that the GUS activity was very strong in ABA- or ethylene-treated middle layer petal discs, but barely visible in untreated discs (Figure S4c). RhHB1 silencing delays ABA- and ethylene-induced senescence To identify the potential roles of RhHB1 in rose petal senescence in the context of ABA and ethylene action, we observed the phenotypes of RhHB1-silenced petal discs using a virus-induced gene silencing (VIGS) approach. We chose the RhHB1-specific 30 end region to construct a

tobacco rattle virus vector (TRV-RhHB1) to specifically silence RhHB1 in rose petal discs. The specificity of the VIGS silencing was confirmed by measuring the transcript abundance of RhHB1 homologous genes in RhHB1silenced petals (Figure S5). However, we cannot totally exclude the possibility of silencing of some very closely related genes. In the TRV control, color fading started 5 days after 24 h ABA or ethylene treatment, and the discs had almost turned pink at day 10. In contrast, the RhHB1-silenced discs clearly exhibited a delayed senescence phenotype, with only slight color fading at 10 days after treatment with ABA or ethylene for 24 h (Figure 3a,d). Moreover, the total anthocyanin content was significantly higher in the RhHB1silenced petal discs than in the TRV controls during ABAor ethylene-induced senescence (Figure 3b,e). Expression of RhSAG12 was also significantly lower in RhHB1-silenced discs than in the TRV control treated with either ABA or ethylene (Figure 3c,f).

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

€ et al. 582 Peitao Lu As GA3 delays ABA- and ethylene-induced senescence of rose petals, we further investigated the effect of paclobutrazol (PAC), an inhibitor of GA biosynthesis, on RhHB1-silenced petal senescence induced by exogenous ABA and ethylene. Compared to treatment with ABA or ethylene alone, treatments including PAC accelerated petal disc senescence, and differences in color fading were clearly observed at 5 days after treatment (Figures 3 and 4). This indicates that PAC reverses the senescence-retarding effects caused by RhHB1 silencing in the petal discs (Figure 4a,c), with a similar loss of anthocyanin content between silenced and non-silenced petal discs (Figure 4b, d). Taken together, 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 RhHB1-silenced petal discs. RhGA20ox1 silencing promotes senescence in rose petals GA20–oxidase (GA20ox), a 2–oxoglutarate-dependent dioxygenase, is a key regulatory enzyme in the GA biosynthetic pathway (Pimenta Lange and Lange, 2006). As GA3 treatment delays flower senescence, we speculated that altering expression of the rose GA20ox gene, RhGA20ox1, may have a similar effect. Indeed, we found that suppressing RhGA20ox1 expression using VIGS clearly promoted

senescence in petal discs compared with TRV-treated controls, as was seen with the PAC treatments (Figure 5a). The anthocyanin contents in the RhGA20ox1-silenced and PAC-treated discs were also significantly reduced compared to those of the TRV controls (Figure 5b). Additionally, RhSAG12 transcript levels were significantly up-regulated in RhGA20ox1-silenced and PAC-treated discs compared with the TRV-treated control (Figure 5c). We also measured endogenous GA levels in petal discs treated with the TRV control, the TRV-RhGA20ox1 vector or the TRV control plus PAC (TRV+PAC). The results show that the levels of the bioactive GAs GA1, GA3 and GA4 in discs treated with the TRV-RhGA20ox1 vectors were only 60, 32 and 67%, respectively, of those in TRV controls. The abundance of GA3 in TRV+PAC-treated discs was only 15% of that in the TRV controls, and GA1 and GA4 were not detected. Thus, both PAC treatment and silencing of the GA20–oxidase gene reduced the endogenous levels of bioactive GAs in rose petals (Figure 5d). Collectively, these results suggest that RhGA20ox1 plays a role in delaying rose petal senescence. To obtain further insights into the molecular processes associated with the delayed senescence of GA3-treated petals, we investigated the transcript levels of senescencerelated genes at various stages of flower opening (Figure S6), and also in GA3- or PAC-treated petal discs (Figure 6). Eight well-characterized senescence-related genes were

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Figure 3. RhHB1 silencing delays ABA- and ethylene-induced senescence in rose petal discs. Expression of RhHB1 was silenced in petal discs by virus-induced gene silencing (VIGS), and the discs were treated with 100 lM ABA or 10 ll l
1 ethylene for 24 h. (a, d) The phenotypes of the petal discs were recorded and photographed at various time points (days) after hormone treatment. (b, c, e, f) The anthocyanin contents (b, e) and relative expression of RhSAG12 by quantitative RT–PCR (c, f) were determined. Values are means  SD (n = 3). Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, Student’s t test).

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

RhHB1 regulates rose petal senescence 583

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Figure 4. Effects of paclobutrazol (PAC) on RhHB1-silenced petal senescence after ABA or ethylene treatment. The expression of RhHB1 in petal discs was silenced by virus-induced gene silencing (VIGS), and discs were treated with 10 lM PAC plus 100 lM ABA or 10 ll l
1 ethylene for 24 h. The phenotypes of the petal discs were recorded (a, c), and the anthocyanin contents were determined (b, d). Values are means  SD (n = 3).

selected, including two cysteine protease genes (JK617354 and JK620911), an Arabidopsis NAC-like, Activated by AP3/ PI (AtNAP)-like gene (JK619963), RhACO1 (AF441282), RhACS1 (AY061946), an ethylene responsive factor (ERF) domain homolog gene (JK622912), a b–carotene hydroxylase gene (JK623072) and a trehalose-6–phosphate synthase gene (JK621488). All eight genes were up-regulated during rose flower senescence, with very high expression levels at opening stage 6 (Figure S6). The eight genes were up-regulated in water- and PAC-treated discs during senescence, whereas their transcripts accumulated at lower levels in GA3-treated discs (Figure 6).

significantly higher following the same treatments in RhHB1-silenced petals (Figure 7) and compared to TRVtreated controls (Figure S7a). Furthermore, the expression profiles of RhHB1 and RhGA20ox1 showed opposite trends over the time course of senescence (Figure 2b and Figure S7b), increasing and decreasing, respectively. To establish whether RhHB1 affects the expression of RhGA20ox1 gene homologs from Arabidopsis, we evaluated the expression levels of five GA20ox genes in RhHB1-overexpressing Arabidopsis (RhHB1-ox) plants. Expression of all five AtGA20ox genes was suppressed in the RhHB1-ox plants compared with empty vector controls (Figure S7c).

ABA and ethylene treatments reduce RhGA20ox1 expression in an RhHB1-dependent manner in rose flowers

RhHB1 binds to the RhGA20ox1 promoter

In Arabidopsis, AtHB12 has been reported to be involved in regulating the growth of the inflorescence stem by influencing the action of AtGA20ox1, a GA biosynthetic gene (Son et al., 2010). We reasoned that the RhHB1-mediated petal senescence promoted by ABA or ethylene treatment may also involve repression of RhGA20ox1 expression. We therefore monitored RhGA20ox1 expression in ABA- or ethylene-treated rose petals. Compared with the control, RhGA20ox1 transcripts were present at significantly lower levels in ABA- or ethylene-treated petals, but were

Based on the results described above, we speculate that RhHB1 may directly repress RhGA20ox1 expression in rose petals. To test this hypothesis, we performed a gel-shift assay to determine whether the RhHB1 protein binds to the RhGA20ox1 promoter. A 1427 bp promoter region immediately upstream of the RhGA20ox1 coding sequence was amplified, and a 35 bp fragment spanning positions
324 to
290 of the RhGA20ox1 promoter was used as a probe (Figure 8a). The probe contains a predicted homeobox ciselement AATATTATT that is similar to the binding sequence of the HD–Zip I TFs (CAAT(A/T)ATTG) (Ariel et al., 2007). A

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

€ et al. 584 Peitao Lu

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Figure 5. RhGA20ox1 silencing promotes senescence in petal discs. The expression of RhGA20ox1 in petal discs was silenced by virus-induced gene silencing (VIGS). Petal discs were also treated with the TRV control or with the TRV control plus 10 lM paclobutrazol (PAC) for 24 h. (a) The phenotypes of the petal discs were imaged. (b, c) The anthocyanin contents (b) and relative expression of RhSAG12 by quantitative RT–PCR (c) were determined. Values are means  SD (n = 3). Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, Student’s t test). (d) Levels of endogenous bioactive gibberellins in TRV control, TRV-RhGA20ox1 and TRV control + PAC-treated petal discs. The GA quantification analysis was performed using three biological replicates. Values are means (ng g
1 fresh weight)  SD. Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, Student’s t test).

recombinant form of the RhHB1 protein with a glutathione S–transferase tag (GST–RhHB1) was purified from Escherichia coli, then co-incubated and electrophoresed with biotin-labeled and/or non-labeled probe. As shown in Figure 8(b), a DNA-binding band was detected on addition of GST–RhHB1 and labeled DNA probes, but no band was detected in the GST control. When the concentrations of unlabeled DNA probe were gradually increased in the reaction mixture, the DNA-binding signal gradually weakened. To test the interaction of RhHB1 with the RhGA20ox1 promoter in vivo, we performed a yeast one-hybrid assay (Figure 8c). The RhGA20ox1 cis-element promoter fragment and its corresponding mutant, AcTcgcAcT, were used to drive expression of the LacZ reporter gene (Figure 8a). The RhHB1 open reading frame was fused to the yeast GAL4 activation domain (GAD) to create the effector construct (GAD–RhHB1). The results of the yeast one-hybrid

experiment confirmed that RhHB1 does indeed bind to RhGA20ox1 promoter baits, but not to the GAD promoter or the RhGA20ox1 mutant fragment. Finally, we tested the repression effects of RhHB1 on RhGA20ox1 expression by site-directed mutagenesis of the cis-element in the RhGA20ox1 promoter in Arabidopsis protoplasts (Figure S8). When the Super1300:RhHB1 effector construct and the PRhHB1:GUS reporter construct were introduced into Arabidopsis protoplasts, GUS activity was significantly decreased compared to the controls. In contrast, site-directed mutagenesis of the RhGA20ox1 promoter prevented RhHB1 repression, as a similar GUS activity to the vector control was observed (Figure S8). These results indicate that RhHB1 is capable of directly repressing RhGA20ox1 expression by binding to a 9 bp pseudopalindromic sequence, AATATTATT, in the RhGA20ox1 promoter.

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

RhHB1 regulates rose petal senescence 585

Figure 6. Expression analysis of senescence-related genes in GA3- or PAC-treated petal discs by quantitative RT–PCR. Petal discs were treated with 50 lM GA3 or 10 lM PAC for 24 h, and then kept in water for observation. The petal discs were sampled for gene expression analysis on days 5 and 10 after treatment. RhUBI1 was used as an internal control, and relative expression levels were normalized to the control. Values are means  SD (n = 3). Asterisks indicate statistically significant differences (*P < 0.05, **P < 0.01, Student’s t test).

DISCUSSION Flower senescence is a complex metabolic process that is influenced by both internal and external cues (Reid and Chen, 2007). Plant hormones play pivotal roles as internal cues, usually through combinatorial interactions (Beaudoin et al., 2000; Zhang and Zhou, 2013). In this study, we showed that ABA and ethylene cooperatively regulate rose petal senescence in part by inducing expression of RhHB1, a HD–Zip I TF. RhHB1 is capable of binding to the promoter of a GA biosynthetic gene, RhGA20ox1, thereby repressing expression of RhGA20ox1. This regulatory point mediates the interaction between plant hormone networks during petal senescence (Figure S9). RhHB1 is involved in petal senescence through repression of RhGA20ox1 expression in rose HD–Zip I TFs are unique to plants, and several reports have described their involvement in various plant development responses, including fruit ripening (Lin et al., 2008), and leaf and flower senescence (Manavella et al., 2006; Xu et al., 2007). Here we showed that RhHB1, a rose HD–Zip I TF, was also up-regulated in senescent petals (Figure 2b and Figure S4b) and was induced by ABA and ethylene (Figure 2c and Figure S4c). ABA- and ethylene-induced senescence was delayed in RhHB1-silenced petal discs (Figure 3), suggesting that RhHB1 plays a pivotal role in the regulation of flower senescence. The regulatory action of RhHB1/RhGA20ox1 was further suggested by the observation that RhHB1 bound directly to a 9 bp AATATTATT segment of the RhGA20ox1 promoter, as revealed using an electrophoretic mobility shift assay (EMSA) and yeast

one-hybrid analysis (Figure 8b,c). The Arabidopsis protein AtHB12, a homolog of RhHB1, was also reported to regulate the expression of AtGA20ox1, but did not directly bind to DNA fragments of the AtGA20ox1 promoter in vitro (Son et al., 2010). Furthermore, the 9 bp AATATTATT ciselement is distinct from some sequences that are bound by HD–Zip I family TFs, which have previously been reported to recognize the sequence CAAT(A/T)ATTG (Ariel et al., 2007). In addition, tomato LeHB1 was shown by EMSA to interact with the same 9 bp sequence of the LeACO1 promoter (Lin et al., 2008). Unlike the delayed senescence phenotype in RhHB1-silenced petal discs (Figure 3), silencing RhGA20ox1 accelerated senescence in rose petal discs, consistent with data from PAC-treated petal discs (Figure 4). These data suggest that RhHB1 directly binds to the promoter of RhGA20ox1, and represses the functions of RhGA20ox1, which is essential for delaying petal senescence. RhHB1 mediates the antagonistic effect between ABA, ethylene and GAs during rose petal senescence Plant developmental processes are generally regulated by multiple coordinated hormone signaling pathways, which interact in a synergistic or antagonistic manner (Beaudoin et al., 2000; Wang and Irving, 2011). For example, an antagonistic relationship has been determined for GA and ABA at various developmental stages of plant growth, including seed germination in Arabidopsis (Debeaujon and Koornneef, 2000), programmed cell death in barley (Hordeum vulgare L.) aleurone layer cells (Fath et al., 2000), and anthocyanin biosynthesis in Petunia hybrida

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€ et al. 586 Peitao Lu

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Figure 7. RhGA20ox1 expression in RhHB1-silenced petal discs after ABA or ethylene treatments. Expression of RhHB1 in petals was silenced by virus-induced gene silencing (VIGS), and discs were then treated with 100 lM ABA (TRV-RhHB1+ABA) or 10 ll l
1 ethylene (TRV-RhHB1+ethylene) for 24 h, before evaluating RhGA20ox1 expression by quantitative RT–PCR analysis. Petals that were not subjected to hormone treatments were used as a negative control. RhUBI1 was used as an internal control. Values are means  SD (n = 3). Asterisks indicate statistically significant differences (*P < 0.05, Student’s t test). (a) RhGA20ox1 expression after ABA treatment. ABA, petals treated with 100 lM ABA for 24 h. (b) RhGA20ox1 expression after ethylene treatment. Eth, petals treated with 10 ll l
1 ethylene for 24 h.

flowers (Weiss et al., 1995). In daffodil, a delay in flower senescence results from the neutralizing effect of GA on ABA action (Hunter et al., 2004a). Furthermore, GA has been reported to antagonize ethylene-mediated senescence in several species of cut flowers (Reid and Chen, 2007), and exogenous application of GA delays the senescence of cut carnation flowers by reducing ethylene production (Saks and van Staden, 1993). Similar hormonal antagonism was also observed in the present study, as both ABA and ethylene clearly promoted rose petal senescence, but GA3 delayed it, even when petal senescence had already been induced by ABA or ethylene (Figure 1). Studies have shown that senescence is controlled by complex regulatory networks involving several plant

Figure 8. RhHB1 binding to cis-elements in the promoter of RhGA20ox1. (a) Wild-type and mutant probes derived from the RhGA20ox1 promoter. The wild-type cis-element and its nucleotide substitutions in the mutants are underlined. (b) Interaction between GST–RhHB1 and the biotin-labeled probe on a native PAGE gel. Purified protein (3 lg) was incubated with 25 pM of the biotin-labeled wild-type probe. Non-labeled probe at various concentrations (10–100-fold) was added for the competition test. (c) Transactivation activity of the RhGA20ox1 promoter by RhHB1 in yeast. GAD–RhHB1, but not GAD itself, activates expression of the LacZ reporter gene driven by the wild-type 35 bp fragment of the RhGA20ox1 promoter. The mutated fragment does not activate LacZ reporter gene expression.

hormones (Zhang and Zhou, 2013). As an example, the TF WRKY53 is a component of the signaling pathway crosstalk between senescence and biotic stress responses, which is most likely modulated by the equilibrium between jasmonic and salicylic acid (Miao and Zentgraf, 2007). In addition, AtSARK, a senescence-associated receptor-like kinase, regulates leaf senescence via the synergistic actions of auxin and ethylene (Xu et al., 2011), and over-expression of the TF CBF2 in Arabidopsis was reported to suppress hormone-induced leaf senescence (Sharabi-Schwager et al., 2010). There are also examples of HD–Zip I TFs functioning in the integration of plant hormones in growth and development, such as HaHB4, which was shown to integrate signals from the jasmonic acid and ethylene pathways, which are considered to be abiotic stress-related and biotic stress-related, respectively (Manavella et al., 2008). The corresponding TF encoded by AtHB4 has been shown to modulate shade avoidance syndrome responses by controlling responsiveness to several hormones (Sorin et al., 2009), and, in Medicago truncatula, an ABA-responsive TF, MtHB1, represses the auxin-regulated gene LBD1 in order

© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 78, 578–590

RhHB1 regulates rose petal senescence 587 to control lateral root emergence under adverse environmental conditions (Ariel et al., 2010). In the present study, ABA and ethylene accelerated senescence, but this pattern was delayed in GA3-treated and RhHB1-silenced rose petal discs (Figures 1 and 3), and PAC treatment repressed these delays (Figure 4). Additionally, ABA and ethylene treatments reduced the expression of RhGA20ox1, a GA biosynthesis rate-limiting enzyme, in an RhHB1-dependent manner (Figures 7 and 8b,c). These results suggest that RhHB1 mediates an antagonistic relationship of GAs with ABA and ethylene during rose petal senescence. EXPERIMENTAL PROCEDURES Plant material and growth conditions Rose flowers (Rosa hybrida ‘Samantha’ and ‘Honey’) were harvested at opening stage 2 (Ma et al., 2005) from a local commercial greenhouse, placed immediately in tap water, and delivered to the laboratory within 1 h. Stems were then re-cut underwater to approximately 25 cm, and uniform flowers without any defects were selected and kept in deionized water until further processing. Arabidopsis seeds were sterilized and placed on MS medium, then stratified at 4°C for 3 days to allow germination. Seven-dayold seedlings were transplanted into pots containing a 1:1 mixture of vermiculite and peat moss under the following conditions: 22  2°C, 50% relative humidity and a 16 h light per 8 h dark photoperiod. Appropriate concentrations of plant growth regulators were determined based on dose–response experiments (Figure S2). Rose stems (approximately 25 cm, opening stage 2) were placed in a vase containing 100 lM gibberellic acid (GA3), 50 lM naphthalene acetic acid, 5 lM brassinosteroid or 100 lM ABA for 24 h. Mock samples were treated with 0.1% dimethylsulfoxide without any phytohormones. For ethylene or 1–methylcyclopropene treatment, the rose flowers (opening stage 2) were exposed to 10 ll l
1 ethylene or 2 ll l
1 1–methylcyclopropene in an airtight chamber for 24 h. NaOH solution (1 M) was also present in the chamber to prevent accumulation of CO2 (Ma et al., 2006).

Cloning, plasmid construction and plant transformation The open reading frames and promoter sequences of RhHB1 and RhGA20ox1 were amplified using the RACE method and nested PCR, according to the manufacturer’s instructions (Clontech, http:// www.clontech.com/). All PCR products were sub-cloned into the pGEM–T Easy vector (Promega, http://www.promega.com/), and then transformed into Escherichia coli DH5a cells and sequenced. All primer sequences used in this study are listed in Table S1. For construction of the RhHB1 VIGS vector, a 347 bp fragment at the 30 end of RhHB1 was PCR-amplified using primers RhHB1F1 (containing a BamHI site at the 50 end) and RhHB1R1 (containing an XhoI site at the 30 end) to generate the pTRV2-RhHB1 construct. For construction of the RhGA20ox1 VIGS vector, a 345 bp fragment from the 30 end of the gene was used. In order to express the RhHB1 recombinant protein in E. coli for EMSA, the RhHB1 open reading frame was amplified using primers RhHB1F2 and RhHB1R2. The PCR products were digested with BamHI and EcoRI, and ligated into the corresponding sites of the pGEX–2T (Lin et al., 2007), allowing production of the GST–RhHB1 fusion protein. The GST tag in pGEX–2T was used to facilitate purification of the fusion protein.

For the yeast one-hybrid assay, the RhHB1 open reading frame was cloned into the EcoRI and XhoI sites of the pJG4–5 vector (Clontech) to produce the GAD–RhHB1 construct. To generate a construct expressing the LacZ reporter gene driven by the RhGA20ox1 promoter with a wild-type or mutant motif, 35 bp oligonucleotides were synthesized using the complementary primers RhGApYF and RhGApYR for the wild-type, and RhGApmYF and RhGApmYR for the mutant. The annealed oligonucleotides were ligated into the EcoRI and XhoI sites of pLacZi2l (Lin et al., 2007), resulting in PRhGA20ox1:LacZ and PRhGA20ox1 m:LacZ, respectively.

Silencing of RhHB1 and RhGA20ox1 in rose petals by VIGS Silencing of RhHB1 and RhGA20ox1 by VIGS was performed as described by Dai et al. (2012), with some minor modifications. The pTRV1, pTRV2, pTRV2-RhHB1 and pTRV2-RhGA20ox1 vectors were transformed individually into Agrobacterium tumefaciens GV3101, and the transformed A. tumefaciens lines were cultured for 24 h in Luria–Bertani medium supplemented with 10 mM MES, 20 mM acetosyringone, 50 lg ml
1 kanamycin and 50 lg ml
1 gentamycin sulfate. The cultures were harvested, and suspended in infiltration buffer (10 mM MgCl2, 200 mM acetosyringone, 10 mM MES, pH 5.6) to a final OD600 of approximately 1.8. Mixtures of cultures containing an equal ratio (v/v) of pTRV1 and pTRV2, pTRV1 and pTRV2-RhHB1 or pTRV1 and pTRV2RhGA20ox1, were used for the TRV control, TRV-RhHB1 and TRV2-RhGA20ox1 experiments, respectively. The mixtures were placed at room temperature in the dark for 4 h before vacuum infiltration. Rose petals from the middle whorl at opening stage 2 were collected, and 1 cm diameter discs were excised from the center of the petals using a hole punch. Vacuum infiltration was performed by immersing rose petals or discs in the bacterial suspension solution and infiltrating under a vacuum at 0.7 MPa. After release of the vacuum, petals and discs were washed in deionized water and kept in deionized water for 3 days at 8°C, followed by an equilibrium step at 23°C for 1 day. The samples were treated with 100 lM ABA or 10 ll l
1 ethylene for 24 h. For RNA isolation, petals were kept in deionized water at 23°C until sampling. The phenotypes of petal discs were observed daily until necrosis was observed.

Extraction and quantification of anthocyanins Anthocyanins were extracted as described by Wang et al. (2001), with some minor modifications. Petal disc material was extracted using 5 ml methanol containing 2% formic acid, and the extract was filtered through a 0.22 lm reinforced nylon membrane filter (ANPEL, http://www.anpel.com.cn/) for HPLC-Diode Array Detection and HPLC-MS analyses. Replicates of each sample were analyzed in triplicate. The chromatographic separation was performed on a Dionex (http://www.dionex.com/) system equipped with an UltiMate3000 HPLC pump, an UltiMate 3000 autosampler, a TCC–100 thermostated column compartment and a PDA100 photodiode array detector. A 20 ll aliquot of solution was injected, and analyzed on an ODS–80Ts QA C18 column (250 mm length 9 4.6 mm inner diameter, Tosoh, http://www.tosoh.com/), protected with a C18 guard cartridge (ANPEL). Eluent A consisted of a 1% formic acid aqueous solution, and eluent B was 15% methanol in acetonitrile. A gradient elution with the following composition was used: 5% B at 0 min, 35% B at 25 min, 5% B at 30 min. The flow rate was 0.8 ml min
1, and chromatograms were acquired at 525 nm for anthocyanins, with DAD data being recorded from 200–800 nm. The total content of anthocyanins in each sample was measured semi-quantitatively by linear regression of the commercially

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€ et al. 588 Peitao Lu available standard cyanidin 3–glucoside (Sigma, http://www. sigmaaldrich.com/).

Extraction and quantification of endogenous gibberellins Endogenous GAs were extracted and quantified as described by Pan et al. (2010). Petal disc material (approximately 100 mg) was frozen in liquid nitrogen, ground to a fine powder, and extracted using extraction solvent (2:1:0.002 v/v/v 2–propanol/H2O/concentrated HCl; ratio of sample to solvent 1:10) on a rotary shaker (100 rpm) at 4°C for 30 min. Dichloromethane (1 ml) was added to each sample, which was then shaken for 30 min at 4°C. After centrifugation (13 000 g, 4°C, 5 min), two phases were present, and the lower phase (approximately 1.5 ml) was collected. The solvent mixture was concentrated to near dryness using a concentrator (Concentrator plus, Eppendorf, http://www.eppendorf.com/), and then re-dissolved in 0.1 ml methanol. The sample solution was detected by HPLC electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS).

QUANTITATIVE RT-PCR For quantitative RT-PCR analysis, 1 lg DNase-treated total RNA was used to synthesize cDNA using a reverse transcription system (Promega) according to the manufacturer’s instructions, with a 20 ll reaction volume. A 2 ll aliquot of cDNA was used as the template in a 20 ll quantitative RT-PCR reaction using the StepOnePlusTM real-time PCR system (Applied Biosystems, http:// www.appliedbiosystems.com/) with KAPATM SYBRâ FAST quantitative PCR kits (Kapa Biosystems, http://www.kapabiosystems. com/). Eight senescence-associated genes were analyzed: RhSAG12 (GenBank accession number JK620911), a cysteine protease gene (JK617354), an AtNAP-like gene (JK619963), RhACO1 (AF441282), RhACS1 (AY061946), an ERF domain homolog gene (JK622912), a b-carotene hydroxylase gene (JK623072) and a trehalose-6-phosphate synthase gene (JK621488). The rose Ubiquitin1 gene (RhUBI1, JK622648) was used as the internal control. All reactions were performed in triplicate.

Purification of recombinant protein and EMSA The EMSA was performed as previously described (Dai et al., 2012). Briefly, expression of the GST–RhHB1 fusion protein was induced in 100 ml cultures of transformed E. coli BL21 cells (see above) by adding isopropyl-b–D–thiogalactopyranoside to a final concentration of 0.2 mM, and the cultures were incubated at 28°C for 6 h. The recombinant protein was purified using glutathione Sepharose 4B beads (GE Healthcare, http://www.gehealthcare. com/) according to the manufacturer’s instructions. EMSA was performed using a LightShift chemiluminescent EMSA kit (Pierce, http://www.piercenet.com/), according to the manufacturer’s instructions. Briefly, biotin-labeled DNA fragments (50 -GGTTACTGTAATATTATTGAAAGGTATT-30 ) were synthesized, annealed and used as probes, with unlabeled DNA of the same sequence being used as a competitor.

Yeast one-hybrid assay Yeast one-hybrid assays were performed as described by Lin et al. (2007). Briefly, plasmid expressing the GAD–RhHB1 fusion was cotransformed with various LacZ reporter gene constructs into yeast strain EGY48 as described in the Yeast Protocols Handbook (Clontech). Transformants were grown on synthetic dextrose plates lacking uracil and tryptophan but containing X–gal (5-bromo-4– chloro-3–indolyl-b–D–galactopyranoside) to observe the color development of yeast colonies.

ADDITIONAL EXPERIMENTAL PROCEDURES Additional experimental procedures are described in Methods S1.

ACKNOWLEDGEMENTS We thank Zhizhong Gong (China Agricultural University, Beijing, China) for providing the Super1300 and pGEX–2T vectors, and Rongcheng Lin (Chinese Academy of Sciences, Beijing, China) for providing the pJG4–5 and pLacZi2l plasmids and yeast strain EGY48. We thank PlantScribe (www.plantscribe.com) for editing our manuscript. This work was supported by the National Natural Science Foundation of China (grant numbers 31130048, 31171992 and 31071827).

SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Senescence parameters were measured for rose petals discs and isolated petals. Figure S2. Dose–response experiments for exogenous GA3, PAC, brassinosteroid, naphthalene acetic acid, ABA and ethylene for rose flower and petal discs. Figure S3. Comparison of RhHB1 amino acid sequence with HD– Zip I family members from other plant species. Figure S4. Transient GUS expression assay in rose petals driven by the RhHB1 promoter. Figure S5. Quantitative RT–PCR of four RhHB1 and one RhGA 20ox1 homologous genes in RhHB1- and RhGA20ox1-silenced petals. Figure S6. Semi-quantitative RT–PCR analysis of eight candidate genes associated with senescence at various flower opening stages. Figure S7. Expression of RhGA20ox1 is negatively correlated with that of RhHB1. Figure S8. Regulation of RhGA20ox1 promoter activity by RhHB1 in Arabidopsis mesophyll protoplasts. Figure S9. Proposed model for RhHB1 mediation of the antagonistic effect of GAs on ABA and ethylene during rose petal senescence. Table S1. List of primers used in this study. Methods S1. Additional experimental procedures.

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