Auxin Regulates Sucrose Transport to Repress Petal Abscission in Rose (Rosa hybrida).

Developmental transitions in plants require adequate carbon resources, and organ abscission often occurs due to competition for carbohydrates/assimilates. Physiological studies have indicated that organ abscission may be activated by Suc deprivation; however, an underlying regulatory mechanism that links Suc transport to organ shedding has yet to be identified. Here, we report that transport of Suc and the phytohormone auxin to petals through the phloem of the abscission zone (AZ) decreases during petal abscission in rose (Rosa hybrida), and that auxin regulates Suc transport into the petals. Expression of the Suc transporter RhSUC2 decreased in the AZ during rose petal abscission. Similarly, silencing of RhSUC2 reduced the Suc content in the petals and promotes petal abscission. We established that the auxin signaling protein RhARF7 binds to the promoter of RhSUC2, and that silencing of RhARF7 reduces petal Suc contents and promotes petal abscission. Overexpression of RhSUC2 in the petal AZ restored accelerated petal abscission caused by RhARF7 silencing. Moreover, treatment of rose petals with auxin and Suc delayed ethylene-induced abscission, whereas silencing of RhARF7 and RhSUC2 accelerated ethylene-induced petal abscission. Our results demonstrate that auxin modulates Suc transport during petal abscission, and that this process is regulated by a RhARF7-RhSUC2 module in the AZ.


INTRODUCTION
Abscission, a common process in plants, involves the detachment of organs from the main body and is triggered by developmental and environmental cues (Bleecker and Patterson, 1997;Roberts et al., 2002;Lewis et al., 2006;Sawicki et al., 2015;Tucker and Kim, 2015). Organ abscission often occurs due to lack of nutrients and competition for carbohydrates (van Doorn, 2002). Carbohydrate transport is thus thought to play a critical role in regulating abscission.
Sucrose can serve as the principal long-distance transport form of carbohydrates and energy (Riesmeier et al., 1994). Sucrose distribution between source and sink organ depends on the sink strength, which in turn is determined by competition for nutrition between different organs (Marcelis et al., 2004;Sawicki et al., 2015;Yu et al., 2015). Plants sense sucrose distribution, and the associated signaling pathways regulate development. As an example in Arabidopsis (Arabidopsis thaliana), sucrose accelerates the transition from the juvenile to the adult stage by reducing levels of the micro-RNA miR156 (Yu et al., 2013). In pea (Pisum sativum), sucrose is considered as the initial regulator of apical dominance (Mason et al., 2014), and in rose (Rosa sp.), sucrose mediates the light-mediated control of bud burst (Henry et al., 2011). A major decline in sucrose levels in abscising organs has been observed in several plant species, including rose (Borochov et al., 1976), pepper (Capsicum annuum) (Aloni et al., 1997), citrus (Gomez-Cadenas et al., 2000), and apple (Malus domestica) (Zhu et al., 2011).
However, the significance of a reduced sucrose supply and the mechanism by which it is regulated have not been characterized.
Sucrose translocation is mainly mediated by two sucrose transporter families: Sucrose Carrier or Sucrose Transporter (SUC/SUT), and Sugar Will Eventually be Exported Transporter (SWEET). SUC proteins function as the principal mediators of long-distance sucrose transport, while SWEET proteins mainly play roles in sucrose loading and unloading (Chen et al., 2012;Eom et al., 2015). SUC proteins have 12 transmembrane domains (Lalonde et al., 2004) and are classified into 3 types, based on their structure and function Peng et al., 2014). Dicot-specific Type I and monocot-specific Type IIB members are associated with sucrose loading and transport (Sauer, 2007), while Type III proteins localize to the tonoplast and plasma membrane and are thought to facilitate sucrose release (Payyavula et al., 2011). In Arabidopsis, the Type I member SUC2 localizes to the phloem and plays a key role in sucrose loading and long-distance transport. In agreement, mutation of Arabidopsis SUC2 results in an impaired nutrition phenotype, as evidenced by stunted growth and the accumulation of carbohydrates in source organs (Gottwald et al., 2000). However, to date SUC proteins have not been shown to be involved in organ abscission.
The abscission process is tightly regulated by endogenous phytohormones, with auxin acting as a major inhibitor of abscission and ethylene as an accelerator (La Rue, 1936;Roberts et al., 2002;Ma et al., 2015). A continuous polar auxin flow passing through the abscission zone (AZ) inhibits abscission, and auxin depletion in the AZ results in abscission initiation as a consequence of enhancing the sensitivity of the AZ to ethylene (La Rue, 1936;Roberts et al., 2002;Ma et al., 2015). In addition, ethylene sensitivity can be significantly reduced by auxin during organ ripening and detachment (Sexton and Roberts, 1982;Olsson and Butenko, 2018;Shin et al., 2019). The regulatory genes involved in the auxin and ethylene signal transduction pathways have been shown to be involved in the regulation of abscission, and include AUXIN RESPONSE FACTOR (ARF)1, ARF2, ARF7 and ARF19 (Ellis et al., 2005;Lombardi et al., 2015), and the ethylene signaling gene ETHYLENE RESPONSE 1 (ETR1) and ETHYLENE INSENSITIVE 2 (EIN2) (Patterson and Bleecker, 2004). However, many aspects of the interaction between auxin and ethylene in organ abscission are not well understood.
Here, we investigate the regulatory mechanism of sucrose transport during rose petal abscission. We show that auxin regulates sucrose transport during petal abscission, and that a decrease in sucrose transport enhances ethylene sensitivity of the petal AZ. The molecular mechanism regulating this process is described.

Auxin Regulates Sucrose Transport during Petal Abscission
To investigate the roles of sucrose and auxin in rose petal abscission, we chose an abscission-prone rose cultivar (Rosa hybrida cv. Golden Shower) and divided the process of flower opening and abscission into 6 stages (Gao et al., 2016) (Supplemental Figure 1). As determined by scanning electron microscopy, petal AZ cells appeared to be more densely packed than adjacent cells from stage 1 to stage 3, with looser packing from stage 4 to stage 5 and clear intercellular separation at stage 6 (Supplemental Figure 1). Accordingly, we selected stage 3 and stage 5 as marker stages before and after the initiation of abscission, respectively.
To gain a better understanding of sucrose dynamics during petal abscission, we used the fluorescent coumarin glucoside esculin, which is recognized by sucrose transporters (Chandran et al., 2003;Gora et al., 2012;Knox and Oparka, 2018), to simulate the transport of sucrose through the petal AZ. Images of longitudinal sections showed that the strength of the esculin signal in the area from the AZ to the petal was lower at stage 5 compared to stage 3 ( Figure 1A).
Transverse sections of petal AZs also revealed a much weaker esculin signal in the phloem of the AZ at stage 5 relative to stage 3 ( Figure 1A). To investigate auxin distribution during petal abscission, we performed immunolocalization studies of longitudinal sections with an anti-indole-3-acetic acid (IAA) monoclonal antibody, and observed a lower immunofluorescence signal in the area from the AZ to petal at stage 5 when compared to stage 3 ( Figure 1B). Liquid chromatography-mass spectrometry (LC-MS)/MS analysis of endogenous sucrose and auxin contents in the AZ confirmed that their abundance significantly decreased in the AZ at stage 5 compared to stage 3 ( Figures 1C and 1D).
We then examined the effects of applying exogenous sucrose or auxin to rose petals. Following sucrose treatment, the time from fully opened flowers (stage 5) to complete petal abscission was 7.6 ± 0.5 d, and 5.0 ± 0.7 d for the mock control (Figures 1E and 1F). To test whether this repressive effect of sucrose was dependent on sucrose hydrolysis or osmosis, we treated flowers with glucose or fructose as metabolic controls, and with mannitol as an osmotic control sugar (Supplemental Figures 2A and 2B). Sucrose application caused the most substantial delay in petal abscission relative to the other treatments, although glucose also caused a delay due to an increased energy supply (Supplemental Figures 2A and 2B). Treatment with the synthetic auxin naphthalene acetic acid (NAA) at 10 μM or 100 μM caused petals to wilt, but they barely abscised by the end of the experiment (15 d) (Figures 1E and 1F;Supplemental Figure 3). By contrast, flowers treated with the auxin transport inhibitor naphthalene acetic acid (NPA) showed accelerated petal abscission, with only 3.0 ± 0.7 d from fully opened flowers to complete petal abscission ( Figures 1E and 1F). Notably, following a combined treatment with sucrose and NPA, the time from fully opened flowers to abscission of all petals was 5.0 ± 0.7 d, which was similar to the mock treatment ( Figures 1E and 1F). These results therefore strongly suggest that sucrose and auxin act in the same pathway in petal abscission. We also tested petal abscission in response to the auxin signaling inhibitor auxinole, which binds the auxin receptors TRANSPORT INHIBITOR RESPONSE1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) to inhibit auxin-responsive gene expression (Hayashi et al., 2012). The flowers treated with auxinole exhibited accelerated petal abscission (average time to abscission 4.6 ± 0.5 d), while a combined treatment with sucrose and auxinole restored the accelerated petal abscission by auxinole alone (average time to abscission 7.2 ± 1.3 d, Supplemental Figure 3).
To further investigate the interaction between auxin and sucrose during petal abscission, we determined the effects of NAA treatment on the fluorescence signal resulting from staining with esculin. Images of longitudinal and transverse sections showed a much higher esculin signal after NAA treatment compared to the signal in stage 5 flowers without NAA treatment ( Figure 1A). In addition, the IAA signal was similar to the mock control after sucrose treatment ( Figure 1B). These results suggest that auxin regulates sucrose transport during petal abscission, but that sucrose does not affect the distribution of auxin.

Abscission
To begin to elucidate the molecular mechanism behind long-distance sucrose transport in the petal AZ, we identified three members of the rose SUC gene family (RhSUC2, RhSUC3 and RhSUC4), all of which have been previously reported to be expressed in rose flowers (Henry et al., 2011). We examined the expression patterns of these SUC genes in the petal AZ during flower opening and abscission by real time quantitative RT-qPCR. RhSUC2 expression peaked at stage 3, before significantly decreasing at stage 5 (Supplemental Figure 4A), while the expression of RhSUC3 and RhSUC4 remained constant during flower opening and abscission (Supplemental Figure 4A). An analysis of RhSUC2 expression in specific tissues further revealed a more than two-fold decrease in expression in the petal AZ at stage 5 compared to stage 3, whereas the adjacent petal and receptacle tissues experienced no such change ( Figure 2A). In addition, NAA treatment significantly induced the expression of RhSUC2 in the petal AZ ( Figure 2B). We therefore selected RhSUC2 for further analysis of its potential function in petal abscission.
We tested the transport function of RhSUC2 in a complementation assay, involving its heterologous expression in the sucrose uptake-deficient yeast strain SUSY7/ura3, which cannot grow efficiently on medium with sucrose as the sole carbon source (Riesmeier et al., 1994;Wieczorke et al., 1999). A drop test showed that transformants with pDR196-RhSUC2 or empty vector (pDR196) grew on medium with 2% glucose, but only pDR196-RhSUC2 transformants grew well on medium with 2% sucrose ( Figure 2C), suggesting that RhSUC2 is a functional sucrose transporter. Transmembrane domain prediction using TMHMM indicated that RhSUC2 contains 12 transmembrane domains ( Figure 2D). Subcellular localization assays in Nicotiana benthamiana leaves showed that the RhSUC2green fluorescent protein (GFP) signal overlapped with the signal derived from a plasma membrane marker protein tagged with mCherry (PM-marker, CD3-1007), indicating that RhSUC2 localizes to the plasma membrane ( Figure 2E).
We then tested the consequences of RhSUC2 silencing using virus-induced gene silencing (VIGS). To this end, we inserted 534 bp of the RhSUC2 3' untranslated region (UTR) into the pTRV2 VIGS vector. The RhSUC2-silencing construct reduced the expression of RhSUC2 in transformed petals compared to the TRV empty vector control ( Figure 3A), while it did not alter the expression of the related genes RhSUC3 or RhSUC4 (Supplemental Figure 4B), confirming the specificity of the gene silencing. We noticed that the flower diameter and petal size of fully opened flowers were significantly smaller in RhSUC2-silenced plants compared to the TRV control plants ( Figure 3C; Supplemental Figure 5A). In the RhSUC2-silenced plants, the time from fully opened flowers to abscission of all the petals was 3.8 ± 0.8 d, compared to 7.0 ± 0.7 d in the TRV control ( Figures 3B and   3C). In addition, LC-MS/MS analysis showed that sucrose levels in the petals of RhSUC2-silenced plants were lower than in the TRV control ( Figure 3D). To further examine whether the decrease of sucrose levels in the RhSUC2-silenced petals was caused by suppression of sucrose transport, we simulated the transport of sucrose using esculin. We observed a dramatic decrease in esculin transport into the petals of the silenced plants compared to the TRV control ( Figure 3E).
Collectively, these results indicated that sucrose transport is associated with petal development and abscission.
We next tested the effects of NAA and NPA treatments on petal abscission in We also tested whether RhSUC3 and RhSUC4 function in petal abscission by silencing each separately using VIGS. RT-qPCR analysis confirmed the specificity of RhSUC3 and RhSUC4 silencing in plants (Supplemental Figure 6A). We observed no differences in the time of petal abscission for the RhSUC3-or RhSUC4-silenced plants when compared to the TRV control (Supplemental Figures 6B and 6C).

RhARF7 Modulates Sucrose Transport by Regulating RhSUC2 Expression
To investigate the regulation of RhSUC2, we analyzed 1,773 bp of the RhSUC2 promoter region upstream from the transcription start site using PLACE software (https://www.dna.affrc.go.jp/PLACE/?action=newplace). This analysis revealed a number of cis-elements, including three auxin responsive cis-elements, AuxRE (TGTCTC), which are typical binding sites for auxin response factor (ARF) proteins ( Figure 5A). We also analyzed the promoter regions of SUC2 in other plant species, including Arabidopsis, strawberry (Fragaria vesca), and peach (Prunus persica), and found that they all contained AuxRE elements ( Figure 5A). We next identified We next conducted a dual-luciferase (LUC) reporter assay to assess the regulation of the RhSUC2 promoter by RhARF7 and RhARF8 in vivo. We determined that N. benthamiana leaf cells expressing RhARF7 dramatically activated the expression of the RhSUC2pro:LUC reporter when compared to those expressing other RhARF genes ( Figure 5D). In addition, expressing the RhARF7 effector resulted in a 9.3-fold increase in LUC activity of the RhSUC2pro:LUC reporter, whereas RhARF8 as effector failed to raise LUC activity over control levels ( Figure 5E). We then conducted a yeast one-hybrid assay to analyze the interaction of RhARF7 with the RhSUC2 promoter: RhARF7 bound to all three AuxRE elements, but not to a mutated AuxRE motif ( Figure 5F). We confirmed these results in an electrophoretic mobility shift assay (EMSA), where again RhARF7 bound to all three AuxRE biotin-labeled probes. Increasing concentrations of unlabeled probes gradually attenuated the extent of binding, indicative of efficient competition. As in the dual-luciferase assays, we observed no binding using a mutated labeled AuxRE probe in EMSA ( Figure 5G). Finally, a subcellular localization assay by co-infiltrating a RhARF7-GFP construct and the nuclear marker construct NF-YA4-mCherry in N. benthamiana leaves demonstrated that RhARF7 accumulates in the nucleus (Supplemental Figure 7B).
Taken together, these results are consistent with RhARF7 functioning as a direct regulator of RhSUC2 expression.
To investigate whether RhARF7 plays a role in regulating petal abscission, we first evaluated its expression and measured higher transcript levels in the AZ than in petals or the receptacle (Supplemental Figure 7C).  Figure 3D).

RhARF7 Silencing
To further dissect the genetic interaction between RhSUC2 and RhARF7, we In TRV-RhARF7+RhSUC2OX plants, the expression of RhSUC2 increased 3.2 times over that seen in RhARF7-silenced plants ( Figure 3A). The time to complete petal abscission was 6.6 ± 0.5 d in TRV-RhARF7+RhSUC2OX, which represented a significant delay compared to RhARF7-silenced plants. This time to complete petal shedding was not significantly different from that of the TRV control ( Figure   3B and 3C).

Silencing of RhSUC2 and RhARF7 Increases in Petal Sensitivity to Ethylene
Ethylene is the main accelerator of petal abscission. To examine the influence of sucrose and auxin on ethylene-induced rose petal abscission, we therefore treated flowers with sucrose and auxin in an air-tight chamber containing 10 ppm ethylene. This treatment delayed ethylene-induced petal abscission compared to the mock control, and a combination of sucrose and auxin treatments had a greater retardation effect compared to either treatment alone ( Figure 6A; Supplemental video 1). When we tested the effects of RhSUC2 and RhARF7 silencing on ethylene-induced petal abscission, we observed that the time of petal abscission following ethylene treatment in RhSUC2-and RhARF7-silenced plants was 10.3 ± 2.1 h and 7.6 ± 1.0 h, respectively, compared with 21.5 ± 2.5 h for the TRV control plants ( Figure 6B). In addition, RT-qPCR analysis showed that the expression of genes related to ethylene biosynthesis and signaling was up-regulated in RhSUC2 and RhARF7-silenced plants compared to TRV control plants (Supplemental Finally, RT-qPCR analyses indicated that the expression levels of RhSUC2 and RhARF7 in the petal AZ decreased in response to ethylene treatment ( Figure   6C). Esculin transport assays corroborated reduced sucrose transport in the petal AZ under ethylene treatment ( Figure 6D). However, immunolocalization of IAA indicated that auxin distribution in the area from AZ to petal did not change following the ethylene treatment ( Figure 6E).

DISCUSSION
The shedding of superfluous organs is necessary for normal plant development and survival (Patterson, 2001), and the abscission process often occurs due to deprivation of, or competition for, carbohydrates/assimilates (Addicott and Lynch, 1955). Here, we report the role and regulatory mechanism of sucrose transport during petal abscission in rose flowers.
The exogenous application of sucrose is well-known to suppress organ abscission or senescence in plants. In Dendrobium and pepper, sucrose feeding was reported to inhibit flower abscission (Aloni et al., 1997;Pattaravayo et al., 2013), and in citrus, branch girdling (that is, the removal of the bark and phloem) can lead to an increase in sugar content and a reduction in fruit abscission rates (Iglesias et al., 2006), while defoliation reduces carbohydrate levels and increases fruit abscission (Mehouachi et al., 1995). In this study, we determined the dynamics of sucrose transport during petal abscission in vivo, and showed that sucrose import into petals was impaired during petal abscission (Figures 1A and 1C). Sugar transport includes symplastic and apoplastic pathways, with sucrose transporters mainly functioning in the apoplastic pathway. SUC proteins with high-affinity sucrose activity serve in phloem loading, long-distance transport and unloading (Sauer, 2007;Kuhn and Grof, 2010). The Arabidopsis SUC2 transporter belongs to the high sucrose-affinity type and plays an essential role in sucrose transport and distribution between source and sink tissues (Durand et al., 2018). In the context of rose bud growth, RhSUC2, RhSUC3 and RhSUC4 have been reported to be expressed in buds. However, only RhSUC2 transcript levels displayed an upregulation in response to light in the buds of beheaded plants, hinting at the special role of RhSUC2 in light-induced bud break (Henry et al., 2011). Here, we also observed the down-regulation of RhSUC2 expression during petal abscission, whereas the expression of RhSUC3 and RhSUC4 did not change (Supplemental Figure 4A). Notably, RhSUC2 expression exhibited a down-regulation in the AZ but not in the receptacle or petals (Figure 2A). Moreover, RhSUC2 silencing led to decreased sucrose import to the petals and promoted petal abscission (Figure 3).
Taken together, these results demonstrate that RhSUC2 plays a role in the regulation of petal abscission, in addition to bud outgrowth.
Auxin is a well-known inhibitor of abscission and its depletion is a prerequisite for abscission (Lombardi et al., 2015). In Arabidopsis, overexpression of an auxin synthesis gene in the AZ was reported to effectively reduce petal abscission (Basu et al., 2013), and in tomato (Solanum lycopersicum), depletion of auxin by the removal of flowers resulted in promoting pedicel abscission (Meir et al., 2010). In our study, auxin distribution decreased from the receptacle to the petal at stage 5 compared to stage 3 in rose ( Figures 1B and 1D), and auxin application inhibited petal abscission (Figures 1E and 1F).
The influence of sucrose on auxin metabolism and signaling has been described in previous studies. In Arabidopsis, sucrose application led to elevated auxin levels and increased polar auxin transport in seedlings (Lilley et al., 2012).
Sucrose has also been shown to promote auxin signaling to regulate the irondeficiency response in Arabidopsis (Lin et al., 2016). In maize (Zea mays), sugar levels regulate auxin biosynthesis in developing seeds (Le et al., 2010), and in rose, sucrose regulates bud outgrowth as an early modulator of relative phytohormone content during bud development (Barbier et al., 2015). In particular, the expression of genes encoding auxin biosynthesis and efflux carriers was induced during bud outgrowth (Barbier et al., 2015). However, we observed that sucrose had no effect on the distribution of auxin during petal abscission ( Figure   1B), whereas auxin promoted sucrose transport during abscission ( Figure 1A), indicating that auxin regulates sucrose distribution during petal abscission. The expression of RhSUC2 was reported to be reduced in response to auxin in rose buds (Henry et al., 2011), but we saw an up-regulation in RhSUC2 expression by auxin in the petal AZ ( Figure 2B). These differences may reflect distinct auxinsucrose regulatory relationships during bud outgrowth and petal abscission.
Moreover, we observed that flower diameter and petal size of fully opened flowers were smaller in both RhSUC2-and RhARF7-silenced plants compared to TRV control (Supplemental Figure 5). Previous studies suggested that defects in sucrose transport or auxin signaling resulted in smaller size of flower. In Arabidopsis, the sizes of petals were significantly reduced in the arf6 arf8 double mutant (Nagpal et al., 2005). In cucumber (Cucumis sativus), silencing of the sucrose transporter CsSUT1 resulted in smaller male flowers (Sun et al., 2019).
It has been reported that ARF1, ARF2, ARF7 and ARF19 contribute to petal abscission in Arabidopsis (Ellis et al., Okushima et al., 2005), while we found that RhARF7 is involved in the regulation of petal abscission in rose (Figure 3), and directly regulates the expression of RhSUC2 (Figures 5D-5G). Sequence analysis identified auxin responsive cis-elements (AuxRE) in the SUC2 promoter that are conserved in the promoters of homologs from Arabidopsis, strawberry and peach ( Figure 5A). This suggests that the regulation of sucrose transport in the AZ by auxin may be a common phenomenon in plants.
The balance of auxin and ethylene constitutes a major organ abscission regulatory module in plants (Patterson, 2001), with auxin inhibiting ethylene sensitivity (Taylor and Whitelaw, 2001 (Zhou et al., 1998;Shi et al., 2003). Our results showed that sucrose treatment can suppress ethylene-induced petal abscission in rose ( Figure 6A; Supplemental video 1), and that the strongest suppression of ethylene-induced petal abscission resulted from a co-treatment with auxin and sucrose ( Figure 6A; Supplemental video 1). These results indicate that sucrose acts as a mediator of the interaction between auxin and ethylene in organ abscission. Intriguingly, we observed that ethylene-induced abscission can be accelerated by silencing of RhSUC2 or RhARF7 ( Figure 6B). Our results suggest that auxin and sucrose may have other functions in petal abscission, bypassing ethylene sensitivity during abscission.
Ethylene was reported to affect sucrose distribution and accumulation as a feedback regulation. In carnation, ethylene promotes sucrose mobilization from petals to other organs in the flower (Nichols and Ho, 1975), while in rice (Oryza sativa), ethylene inhibitors enhance sucrose biosynthesis during grain filling (Naik and Mohapatra, 2000). Moreover, ETHYLENE RESPONSIVE FACTOR 72 (ERF72) suppresses sucrose biosynthesis in cassava (Manihot esculenta Crantz) (Liu et al., 2018). Here, we showed that an ethylene treatment reduced sucrose transport in the petal AZ ( Figure 6D), but did not affect auxin distribution ( Figure   6E), suggesting that ethylene reduces sucrose transport as part of a feedback mechanism during petal abscission.
In conclusion, our results demonstrate that auxin induces sucrose transport to repress petal abscission in the early stages of flower development. The reduction of auxin levels in the petal AZ leads to an attenuation of sucrose transport and promotes the initiation of petal abscission. In this process, the RhARF7-RhSUC2 module mediates the regulation by auxin of sucrose transport (Figure 7).

Plant Materials and Growth Conditions
We used the abscission-prone rose cultivar R. hybrida cv. Golden Shower as material for the experiments in this study, except for the VIGS assays. We   Figure 3). We therefore used Rosa hybrida cv. Samantha for VIGS assays. R. hybrida cv. Samantha plants were propagated by tissue culture as previously described (Wu et al., 2017). We used The distal and proximal sides of the petal AZ are connected to the petal and receptacle, respectively. We collected petal AZ samples by excising both sides at the base of the petals (< 2 mm in length) and the area of the receptacles adjacent to the petals (< 2 mm in length) (Gao et al., 2016).

Quantification of Endogenous Sucrose and Auxin Levels
We measured sucrose levels as previously described . Briefly, we ground freeze-dried petals or AZs using a mixer mill with zirconia beads for 1.5 min at 30 Hz. We then extracted 100 mg powder overnight at 4ºC with 1 mL 70% (v/v) methanol. Following centrifugation at 10,000g for 10 min, we filtered the supernatants through a 0.22 μm membrane filter (SCAA-104, 0.22 μm pore size, ANPEL) for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis.
For auxin extraction, we collected 120 mg fresh rose AZ tissue, followed by flash-freezing and grinding in liquid nitrogen, before extraction with 1.2 mL methanol/water (8/2, v/v) at 4ºC overnight. We centrifuged the extract at 12,000g at 4ºC for 15 min. We collected the supernatant and allowed it to evaporate to dryness under a nitrogen gas stream. We dissolved the final pellet in 100 μL

Esculin Feeding and Confocal Microscopy
We diluted esculin hydrate (Sigma-Aldrich) to 10 mM in deionized water. We performed vacuum infiltration by immersing leaves adjacent to the rose flower in esculin solution under vacuum at 0.7 MPa. We sectioned petal AZ by hand with a razor blade 10 h after esculin treatment and immediately immersed the sections in 80% (v/v) glycerol before mounting on glass slides. We recorded fluorescence using a laser scanning confocal microscope (Olympus FV1000, Japan), with an excitation wavelength of 405 nm and emission wavelength of 454 nm.

Immunolocalization of IAA
We examined IAA distribution as previously described (Sakata et al., 2010).

RNA extraction and RT-qPCR
We extracted total RNA using the hot borate method as previously described (Gao et al., 2016). We synthesized first-strand cDNAs using 1 μg of total RNA with oligo d(T) and random primers in a final volume of 20 μL. For RT-qPCR, we used 1 μL cDNA as the template. We performed RT-qPCR (40 cycles of denaturation for 5 sec at 95ºC and annealing for 30 sec at 60ºC) using the Step One Plus TM realtime PCR system (Applied Biosystems) with KAPATM SYBR® FAST quantitative PCR kits (Kapa Biosystems). We used RhUBI2 as the reference gene (Meng et al., 2013). Each experiment was performed independently three times. The primers used in this study are listed in Supplemental Data Set 1.

Subcellular Localization of RhSUC2 and RhARF7
We determined the subcellular localization of RhSUC2 and RhARF7 in N.
benthamiana leaf epidermal cells. We PCR-amplified the open reading frames (ORFs) for RhSUC2 and RhARF7, and inserted the PCR products at the SalI/SpeI restriction sites in the pSuper1300 vector to form SUPERpro:RhSUC2-GFP and SUPERpro:RhARF7-GFP, respectively. We introduced the resulting vectors into Agrobacterium (Agrobacterium tumefaciens) strain GV3101. We grew (SUPERpro:NF-YA4-mCherry) were used as plasma membrane and nuclear markers, respectively (Nelson et al., 2007;Zhou et al., 2015). The GFP fluorescence signals were observed at an excitation wavelength of 488 nm and emission wavelength of 506-538 nm, and mCherry signals were detected using excitation with a 587 nm laser and emission with a 575-675 nm band pass filter.
The primers used in this study are listed in Supplemental Data Set 1.

Virus Induced Gene Silencing (VIGS)
We performed VIGS-mediated gene silencing as previously described (Wu et al., 2017). Briefly, we PCR-amplified 534 bp and 414 bp fragments specific to RhSUC2 and RhARF7 3' UTR regions, respectively, and inserted them separately into the pTRV2 vector, which we then introduced into Agrobacterium strain GV3101.

Sucrose Uptake Assay
We cloned the ORF for RhSUC2 into the yeast expression vector pDR196 and transformed the resulting construct into the sucrose uptake-deficient yeast strain SUSY7/ura3 by the lithium acetate method. The pDR196 empty vector was used as the control. We spotted transformed pDR196-RhSUC2 and pDR196 colonies as serial dilutions and cultured them on synthetic dropout medium (SD -Ura)_ with 2% glucose or 2% sucrose as the sole carbon source at 30ºC for 3 d. We recorded the growth of the various yeast strains by taking photographs.

Yeast One-Hybrid Assay
We inserted RhSUC2 promoter fragments into the pAbAi vector (Clontech) and introduced the resulting constructs into the yeast one-hybrid Y1H gold strain. The transformed cells were grown on synthetic dropout medium (SD -Ura) plus 100 ng mL -1 aureobasidin A (AbA). We then introduced the recombinant vector pGADT7-RhARF7 into a yeast strain harboring the pRhSUC2 (AuxRE)-AbAi and pRhSUC2 (mAuxRE)-AbAi. The transformed cells were grown on synthetic dropout medium (SD -Ura)_plus 200 ng mL -1 AbA for 3 d. The primers used in this study are listed in Supplemental Data Set 1.

Transactivation and dual Luciferase Reporter Assays
We analyzed the transactivation of the RhSUC2 promoter by ARFs as previously described (Kong et al., 2015;Wang et al., 2018). We inserted the ORFs for the ARF genes into the pGreenII0029 62-SK vector (Hellens et al., 2005) to generate effector constructs under control of the 35S promoter; we also cloned the RhSUC2 promoter into the pGreenII0800-LUC vector (Hellens et al., 2005) to drive expression of the luciferase reporter. We used empty vectors as negative controls.
We introduced all constructs into Agrobacterium strain GV3101 harboring the pSoup plasmid, and grew bacterial cultures overnight as described above. We collected cells by centrifugation at 3214g for 5 min at room temperature and resuspended the pellets in infiltration buffer (10 mM MgCl2, 200 μM acetosyringone, 10 mM MES, pH 5.6) to a final OD600 of 0.8 before mixing cell suspensions in a 1:1 (v/v) ratio. We then infiltrated into young N. benthamiana leaves. After 3 d, we harvested the infiltrated leaves and sprayed them with 50 mg L -1 D-luciferin (Promega). We captured images of LUC signals with a CDD camera (CHEMIPROHT 1300B/LND, 16 bits, Roper Scientific) at -110℃.
For dual luciferase reporter assays, we co-infiltrated different bacterial mixtures described above with Agrobacterium cells harboring a 35Spro:REN construct (where the Renilla luciferase gene (REN) is under the control of the 35S promoter) into N. benthamiana leaves as previously described (Wei et al., 2017).
After 3 d, we measured LUC and REN activities with the dual-luciferase reporter assay reagents (Promega) and a GloMax 20/20 luminometer (Promega). The primers used in this study are listed in Supplemental Data Set 1.

Electro Mobility Shift Assay (EMSA)
We performed EMSA assays according to the instructions of the Light Shift chemiluminescent EMSA kit (Thermo Scientific). We cloned the RhARF7 ORF into the GST vector pGEX-4T-2 (GE Healthcare) and transformed the resulting construct into E. coli Rosetta cells. We induced the production of the GST-RhARF7 fusion protein by the addition of 0.6 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) in 100 mL LB medium at 16ºC for 12 h. We extracted and purified the fusion protein with glutathione Sepharose 4B beads (GE Healthcare) following the manufacturer's instructions. The biotin-labeled probes were designed as described in Supplemental Data Set 1.

Sequence Analysis
We aligned amino acid sequences with default parameters using ClustalW (https://www.genome.jp/tools-bin/clustalw). We constructed the phylogenetic tree based on the alignment result using the neighbor-joining method with MEGA version 5.05 (Tamura et al., 2011) with the following parameters: 1,000 bootstrap replicates, Poisson correction, partial deletion and uniform rates. The protein transmembrane prediction was performed on the TMHMM server (http://www.cbs.dtu.dk/services/TMHMM-2.0/) (Moller et al., 2001).

Accession Numbers
Rose gene sequences from this article can be found in GenBank    (B), based on RT-qPCR analysis. RhUBI2 was used as the internal control. Letters indicate significant differences according to Tukey-Kramer test (P<0.05). Asterisks indicate statistically significant differences between NAA treatment and mock determined by two-tailed Student's t-test (**P<0.01). (C) RhSUC2 functions as a sucrose transporter, and can rescue the growth of the sucrose uptake-deficient yeast strain SUSY7/ura3 on 2% sucrose medium. The empty vector pDR196 was used as control, and 2% glucose served as the medium control. (D) Transmembrane domains of RhSUC2, as predicted by TMHMM. (E) Subcellular localization of RhSUC2 in Nicotiana benthamiana leaves. Green fluorescent protein (GFP) was fused to the C terminus of RhSUC2. A mCherry-labelled plasma membrane marker (PM-marker, CD3-1007) was co-infitlrated with RhSUC2-GFP to indicate membrane localization. Scale bars, 100 μm.  This information is current as of August 26, 2020