# A single MYB transcription factor with multiple functions during flower development

Mathilde Chopy, Marta Binaghi, Gina Cannarozzi, Rayko Halitschke, Benoît Boachon, Roel Heutink, Dikki Pedenla Bomzan, Lea Jäggi, Geert van Geest, Julian Verdonk, et al.

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## To cite this version:

Mathilde Chopy, Marta Binaghi, Gina Cannarozzi, Rayko Halitschke, Benoît Boachon, et al. A single MYB transcription factor with multiple functions during flower development. New Phytologist, 2023, 239 (5), pp.2007-2025. 10.1111/nph.19096. hal-04704330

HAL Id: hal-04704330

https://hal.science/hal-04704330v1

Submitted on 20 Sep 2024

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1 A single MYB transcription factor with multiple functions during flower development
2
3 Mathilde Chopy¹, Marta Binaghi¹, Gina Cannarozzi¹, Rayko Halitschke², Benoît Boachon³, Roel
4 Heutink⁴, Dikki Pedenla Bomzan³, Lea Jäggi¹, Geert van Geest⁵, Julian C. Verdonk⁴ and Cris
5 Kuhlemeier¹

6
7 ¹Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern, Switzerland;
8 ²Department of Mass Spectrometry and Metabolomics, Max-Planck-Institute for Chemical
9 Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany; ³Université Jean Monnet Saint-Etienne,
10 CNRS, LBVpam UMR 5079, F-42023, Saint-Etienne, France ; ⁴Horticulture and Product
11 Physiology, Plant Science Group, Wageningen University and Research, 6708 Wageningen,
12 Netherlands; ⁵Interfaculty Bioinformatics Unit, University of Bern, Baltzerstrasse 6, 3012 Bern,
13 Switzerland
14
15 ORCID details for authors:
16

17 Mathilde Chopy
18 Email: mathilde.chopy@unibe.ch
19 ORCID: 0000-0003-4403-5769

20 Affiliation: Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern,
21 Switzerland

22 Twitter: @MathildeChopy
23

24 Marta Binaghi
25 Email: marta.binaghi@unibe.ch
26 ORCID: 0000-0001-6951-792X

27 Affiliation: Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern,
28 Switzerland

29 Twitter: @martabinaghi
30

31 Gina Cannarozzi
32 Email: cannarozzi@chem.ethz.ch
33 ORCID: 0000-0002-0704-663X

34 Affiliation: Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern,

35 Switzerland

36 Twitter: @GinaCannarozzi

37

38 **Rayko Halitschke**

39 Email: rhalitschke@ice.mpg.de

40 ORCID: 0000-0002-1109-8782

41 Affiliation: Department of Mass Spectrometry and Metabolomics, Max-Planck-Institute for

42 Chemical Ecology, Hans-Knöll-Str. 8, 07745 Jena, Germany

43

44 **Benoît Boachon**

45 Email: benoit.boachon@univ-st-etienne.fr

46 ORCID: 0000-0002-2362-2238

47 Affiliation: Université Jean Monnet Saint-Etienne, CNRS, LBVpam UMR 5079, F-42023, Saint-

48 Etienne, France

49 Twitter: @BB_VOCsPoP

50

51 **Roel Heutink**

52 Email: roel.heutink@wur.nl

53 ORCID: 0000-0002-6084-2395

54 Affiliation: Horticulture and Product Physiology, Plant Science Group, Wageningen University

55 and Research, 6708 Wageningen, Netherlands

56

57 **Dikki Pedenla Bomzan**

58 Email: dikki.pedenla.bomzan@univ-st-etienne.fr

59 ORCID: 0000-0003-0595-0569

60 Affiliation: Université Jean Monnet Saint-Etienne, CNRS, LBVpam UMR 5079, F-42023, Saint-

61 Etienne, France

62

63 **Lea Jäggi**

64 Email: lea.jaeggi@unibe.ch
65 ORCID: no ORCID number

66 Affiliation: Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern,

67 Switzerland

68

69 **Geert van Geest**

70 Email: geert.vangeest@unibe.ch

71 ORCID: 0000-0002-1561-078X

72 Affiliation: Interfaculty Bioinformatics Unit, University of Bern, Baltzerstrasse 6, 3012 Bern,

73 Switzerland

74 Twitter: @geertvangeest

75

76 **Julian Verdonk**

77 Email: julian.verdonk@wur.nl

78 ORCID: 0000-0002-1237-7951

79 Affiliation: Horticulture and Product Physiology, Plant Science Group, Wageningen University

80 and Research, 6708 Wageningen, Netherlands

81 Twitter: @JulianVerdonk

82

83 **Cris Kuhlemeier**

84 Email: cris.kuhlemeier@unibe.ch

85 ORCID: 0000-0003-2440-5937

86 Affiliation: Institute of Plant Sciences, University of Bern, Altenbergrain 21, 3013 Bern,

87 Switzerland

88

89 Author for correspondence:

90 *Cris Kuhlemeier*

91 Tel: 031 3510317 or 077 4359223

92 Email: cris.kuhlemeier@ips.unibe.ch

93

94
## 95 SUMMARY

96
97
Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines.

98
99
Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function.

102
103
To clarify the function of the SG19 transcription factors, we used a single system, *Petunia axillaris*, and targeted its two SG19 members (*EOB1* and *EOB2*) by CRISPR-Cas9.

104
105
106
107
108
109
110
Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The *eob2* knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism.

111
112
113
Here we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

114

## 115 KEY WORDS

116
117
Flower development, flower maturation, Petunia, R2R3-MYB transcription factors, senescence, starch metabolism, terpenoids/isoprenoids/carotenoids, volatile compounds
118 INTRODUCTION

119 Flower development can be divided into floral organ identity establishment, growth, maturation, 120 and senescence. Organ identity is established by the combinatorial action of the ABC class genes 121 (Coen & Meyerowitz, 1991), and how it varies between species has been studied in detail (Soltis 122 et al., 2007). Once established, the organs grow out and differentiate to carry out their specific 123 functions in pollinator attraction and reward. After pollination, petals and stamens senesce and 124 seeds are produced within the ovary. Members of subgroup 19 (SG19) of the R2R3-MYB 125 transcription factor family are key players in the later stages of floral organ development and 126 maturation. A number of studies, mainly in Solanaceae and Arabidopsis, have documented their 127 functions in flower opening (Colquhoun et al., 2011; Reeves et al., 2012; Liu & Thornburg, 128 2012; Niwa et al., 2018; Schubert et al., 2019), senescence (Colquhoun et al., 2011), nectary 129 development (Liu et al., 2009; Liu & Thornburg, 2012; Schmitt et al., 2018), stamen 130 development (Mandaokar et al., 2006; Cheng et al., 2009; Song et al., 2011; Reeves et al., 2012; 131 Qi et al., 2015; Huang et al., 2017; Battat et al., 2019; Huang et al., 2020), ovule fertility 132 (Schubert et al., 2019), pistil length (Reeves et al., 2012; Schubert et al., 2019; Yarahmadov et 133 al., 2020), as well as production of secondary metabolites such as scent compounds (Spitzer- 134 Rimon et al., 2010, 2012; Van Moerkercke et al., 2012; Medina-Puche et al., 2015; Ke et al., 135 2021), flavonols or terpenes (Battat et al., 2019; Shan et al., 2020; Yang et al., 2020; Zhang et 136 al., 2021b,a) (Fig. 1a). These later stages have been studied in a variety of species, often with a 137 variety of molecular tools. Consequently, it is often not clear whether contrasting mutant 138 phenotypes are species-specific, or are due to the methods used, to the focus of the study on a 139 particular organ, or to the multi-functionality of the SG19 MYB factors. For example, RNAi lines 140 targeting tobacco MYB305 led to nectary maturation defects in one study (Liu et al., 2009; Liu & 141 Thornburg, 2012), and to flower opening defects in another study (Colquhoun et al., 2011). 142 Different phenotypes were also observed in Petunia hybrida, where targeting EOB2 by VIGS 143 exclusively affected scent production (Spitzer-Rimon et al., 2010) whereas RNAi caused flower 144 opening defects (Colquhoun et al., 2011).

145
146 The R2R3-MYB SG19 transcription factors are characterized by two main functional domains:
147 an R2R3-MYB domain (RMD<sup>SG19</sup>) located at the N-terminus and a transcriptional activation
148 domain (TAD) located at the C-terminus (Liu *et al.*, 2009; Schubert *et al.*, 2019; Wu *et al.*,
149 2021b) (**Fig. 1b**). The RMD<sup>SG19</sup> and the TAD molecular functions have been individually
characterized using different experimental and computational techniques. In summary, the RMD<sup>SG19</sup> has at least three roles: (1) interact with different proteins (bHLHs, JAZs, DELLAs,...) (Song *et al.*, 2011; Qi *et al.*, 2015; Huang *et al.*, 2017; Liu *et al.*, 2017; Schubert *et al.*, 2019; Huang *et al.*, 2020; Yang *et al.*, 2020; Ke *et al.*, 2021; Zhang *et al.*, 2021b), (2) bind DNA through the recognition of particular cis-element targets ([G/A]TT[A/T]GG[T/C]) (Weirauch *et al.*, 2014; Medina-Puche *et al.*, 2015), and (3) reach the nucleus through the nuclear localization signal (NLS) (Liu *et al.*, 2009). On the other hand, the TAD recruits the transcriptional machinery and through synergic action, the TAD and RMD, regulate the expression of a wide range of targets (Sablowski *et al.*, 1994; Moyano *et al.*, 1996; Liu *et al.*, 2009; Spitzer-Rimon *et al.*, 2010; Liu & Thornburg, 2012; Moerkercke *et al.*, 2011; Medina-Puche *et al.*, 2015; Battat *et al.*, 2019; Kurilla *et al.*, 2019; Yang *et al.*, 2020; Bian *et al.*, 2021; Zhang *et al.*, 2021b; Wu *et al.*, 2021b; Ke *et al.*, 2021; Zhang *et al.*, 2021a; Wang *et al.*, 2022) (Tables S1, S2). R2R3-MYB SG19 protein sequences have the typical features of transcriptional activators. No characteristic repression motifs (EAR or TLLLRF) (Ma & Constabel, 2019) were found and all the targets identified in the literature (except one) were directly activated by R2R3-MYB SG19 members (Table S2). According to these results, SG19 members associate with a plethora of interacting proteins and promoters providing the potential to regulate many different processes of flower development.

The complex and conflicting data on the function of SG19s during flower development necessitates an in-depth structural and functional characterization of its members. To do so, we used a single system, *P. axillaris*, where two members of the SG19 clade have been identified: EMISSION OF BENZENOIDS I and II (EOB1 and EOB2). Those two genes have a flower-specific expression pattern. We generated mutations in *EOB1* and *EOB2* using CRISPR-Cas9, and analyzed their different functions. Domain-mutants also reveal the specific contributions of the two domains, RMDSG19 and TAD, in EOB2 functions. These discoveries made it clear that EOB2 is a complex multi-functional protein during floral organ development. Our study contributes to explain the complexity of the R2R2-MYB SG19 associated functions observed within and between species and in a broader perspective to study flower maturation.

## MATERIALS AND METHODS
182 **Plant material and growth conditions**

183 *Petunia axillaris* (Lam.) ssp. axillaris P (referred to as *P. ax* P or WT) originates from the
184 University of Bern Botanical Garden (Hoballah *et al.*, 2005). Plants were grown in a growth
185 chamber under a light : dark regime of 15 h : 9 h at 22°C : 17°C, in commercial soil (70%
186 Klasman substrate, 15% Seramis clay granules, 15% quartz sand), and fertilized once a week
187 with a nitrogen-phosphorous-potassium and iron fertilizer.

188
189 **Generation of phylogenetic tree**
190 Phylogenetic reconstruction focused on R2R3-MYB subgroup 19 from *Arabidopsis thaliana* (At),
191 *Solanum lycopersicum* (Sl), *Nicotiana tabacum* (Nt), *Petunia hybrida* (Ph) and *Petunia axillaris*
192 (Pax). Protein sequences alignment was performed with the plug-in version of MUSCLE for
193 UGENE, version 40.1, default parameters (Edgar, 2004; Okonechnikov *et al.*, 2012). Tree was
194 built with PhyML version 20120412 in UGENE, with LG substitution matrix, branch support
195 calculated with SH-like method (Guindon *et al.*, 2010).

196
197 **Vectors construction**

198 The CRISPR-Cas9 Vectors (pHSE401, dual gRNA), called VB191229-3063hgg and VB190510-1037erq targeting *EOB1* or *EOB2* respectively, were commercially synthesized by VectorBuilder (https://en vectorbuilder.com/design/retrieve.html). For each vector, two unique gRNAs were selected (gRNA sequences are listed in Table S3).

202
203 **Petunia axillaris P stable transformation with Agrobacterium**

204 Stable transformation procedure is based on a classical transformation protocol (Chopy et al., 2020) with some modifications. Briefly, leaves from 4-6 week old plants of *P. axillaris* P were surface sterilized and cut with a scalpel blade into one cm² small pieces. Sterilized leaf disks were put in a liquid *A. tumefaciens* (strain LBA4404) suspension for 30 minutes, dried between two layers of sterile filter papers and transferred to a co-culture plate medium for 5 days at 24°C (in the dark with a progressive increase of light). Then, once a week, leaf fragments were transferred to a fresh selective medium containing the appropriate selection agent and were kept at 24°C with moderate light, under long day conditions until the apparition of shoots. Each individual shoot was excised from the calli and transferred to a rooting medium. After rooting, plants were
transplanted to soil and grown in a growth chamber. The screening was then performed on these
plants after one week to allow them to acclimatize to the soil conditions.

**Overexpression constructs and transient expression**

The full-length cDNAs of *EOB1*, *EOB2*, *eob2-2* and *eob2-3* from *P. axillaris* wild-type, *eob2-2* or *eob2-3* were cloned (**Table S4**) in the pDONR221 vector (Invitrogen) and transferred to the pGWB402 vector (NAKAGAWA *et al.*, 2007). Transient transformation of *Petunia axillaris* P leaves using *A. tumefaciens* (strain GV3101) was performed as in (Moerkercke *et al.*, 2011) with some modifications. Petunia leaves from 4-6 week old plants were syringe-infiltrated. Three days later, four replicates per construct were harvested (one replicate consists of 3 leaf disks of 8 mm of diameter near to the agroinfiltrated position) and RT-qPCR were performed using *RAN1* as reference gene.

**DNA extraction and genotyping**

Genomic DNA was extracted from fresh leaf samples using a modified SDS extraction protocol (Edwards *et al.*, 1991). Primer pairs used for the genotyping are listed in **Table S4**.

**Color images**

Flower and branch images were recorded with a Canon EOS 60D camera and Canon 35 mm lens.
Floral organ pictures were photographed under a stereoscopic microscope (Nikon, SMZ1500)
equipped with a camera (Leica, DMC6200)

**Phenotypic measurements**

Before phenotyping, the *eob1* or *eob2* single homozygous mutants or heterozygous lines, cas9-negative lines were selected and we confirmed by sequencing that our gRNAs specifically targeted either *EOB1* or *EOB2*. Phenotypic measurements included tube length, opening angle, limb area, nectar volume and scent emission. All the flowers used for phenotyping were harvested one day post anthesis right before the onset of dark in the growth chamber.
Petal morphological traits (tube length, opening angle, limb area) were measured by photographing front and side view of 12 flowers per genotype. Images were analyzed using the ImageJ software. Nectar volumes were quantified according to the protocol described in (Brandenburg *et al.*, 2012). Methylbenzoate and benzaldehyde compounds were analyzed as
described previously by proton transfer reaction mass spectrometry (PTR-MS, Ionicon®) (Amrad et al., 2016). Five flowers per plant were analyzed as biological replicates.

### RNA extractions, cDNA preparation and quantitative RT-PCR

The different tissue samples used in this study were collected in three biological replicates and stored at -80°C until further processing. RNA extractions were performed using an innuPREP DNA/RNA Mini Kit (Analytik Jena; code 845-KS-20800250). cDNA was synthesized using the qScriber cDNA synthesis kit (HighQu; code RTK0104) and for RT-qPCR the ORA SEE qPCR Green ROX L mix (HighQu; code QPD0505) was used according to the manufacturer's recommendations. The amplification was performed using a QuantStudio 5 Real-Time PCR Instrument 346 (Applied Biosystems). The reference genes used to analyze the data were *RAN1* and *ACTIN11* (Mallona et al., 2010). Primer pairs are listed in **Table S4**.

### RNA sequencing

Total RNA was isolated from tissues listed in **Table S5** using an innuPREP DNA/RNA Mini Kit (Analytik Jena; code: 845-KS-20800250). The quantity and quality of the purified total RNA was assessed using a Thermo Fisher Scientific Qubit 4.0 fluorometer with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific, Q10211) and an Advanced Analytical Fragment Analyzer System using a Fragment Analyzer RNA Kit (Agilent, DNF-471), respectively. Sequencing libraries were made using an Illumina TruSeq Stranded mRNA Library Prep kit (Illumina, 20020595) in combination with TruSeq RNA UD Indexes (Illumina, 20022371) according to Illumina's guidelines. Pooled cDNA libraries were sequenced paired-end using a shared Illumina NovaSeq 6000 S1 Reagent Kit (200 cycles; Illumina, 20028318) on an Illumina NovaSeq 6000 instrument. The run produced a minimum of 36 million reads/sample. The quality of the sequencing run was assessed using Illumina Sequencing Analysis Viewer (Illumina version 2.4.7) and all base call files were demultiplexed and converted into FASTQ files using Illumina bcl2fastq conversion software v2.20. The quality control assessments, generation of libraries and sequencing were conducted by the Next Generation Sequencing Platform, University of Bern.

### Reads processing, differential expression analysis and GO

The quality of the RNA-seq reads was assessed using fastqc v0.11.9 (Andrews, 2022). Adapters were trimmed with Cutadapt v3.4.1 (Martin, 2011). The reads were aligned to the *Petunia*
277 *axillaris* N reference genome version 4.03 (Peax403; available on NCBI GenBank under the
278 accession GCA_026929995.1) using hisat2 v2.2.1 (Kim *et al.*, 2015). FeatureCounts v2.0.1 (Liao
279 *et al.*, 2014) was used to count the number of reads overlapping with each gene as specified in the
280 genome annotation corresponding with Peax403 (gff file can be downloaded from CoGe with the
281 id 62433). The Bioconductor package DESeq2 v1.36.0 (Love *et al.*, 2014) was used to test for
differential gene expression between the experimental groups. Gene GO-term mappings were
282 obtained from (Patrick *et al.*, 2021), and GO-term enrichment was performed with clusterProfiler
283 v4.4.4 (Wu *et al.*, 2021a).
284

285
286 **1-MCP treatment**
287 To inhibit ethylene perception, WT, *eob2-1*<sup>KO</sup>, *eob2-2*<sup>KO</sup> and *eob2-3*<sup>LofTAD</sup> plants were placed in a
288 growth chamber (Percival, model: E-36L2) and treated with an ethylene receptor antagonist, 1-
289 MCP (1-methylcyclopropene) volatile treatment. To treat the plants two EthylBloc<sup>TM</sup> Sachets of
290 2.5 grams (AgroFresh) were used and replaced once a day during at least two weeks.
291

292
293
**Ethylene measurements**
294

Ethylene measurements were performed using a laser-based photoacoustic ethylene sensor (ETD-293
294 300, SensorSense, Nijmegen, Netherlands). For WT and *eob2-1*<sup>KO</sup> ethylene measurements, full
295 flower buds, dissected petals or pistils at stage 5 were used (three flowers per plant were analyzed
296 as biological replicates, for WT n = 30 and for *eob2-1*<sup>KO</sup> n = 45). Mutant samples were collected
297 before the first signs of senescence, when the petal tips were still rigid. Flower buds or dissected
298 organs were enclosed in 4-mL vials and an accumulation time of 5 hours was used between
299 harvest time and measurements. Ethylene concentrations were analyzed with a flow rate of 2.5 L
300 h<sup>-1</sup> and quantified by signal integration after curve fitting. Ethylene concentrations were
301 calculated based on measurements of a 500-ppb ethylene gas standard analyzed as a reference
302 sample.
303

304
305
**Quantification of sesquiterpene accumulation in Petunia pistils and emission from tube**
306
Experiments were essentially performed as previously described (Boachon *et al.*, 2019). For the
307 quantification of sesquiterpenes accumulation in pistils, 10 pistils per plant line were harvested at
308 flower stage S9 and placed in 3 mL hexane. Camphor (3 nM) was added as internal standard (IS).
309
Tissues were crushed in hexane with a potter, vortexed for 20 s, sonicated for 10 min at 40°C and
309 centrifuged at 2000 x g for 5 min. Supernatant was recovered, concentrated under nitrogen flow to
310 c. 300 µL and analyzed by GC-MS (for more details see **Method S1**).

311 For the analysis of sesquiterpene emission from the inner surface of the *Petunia* tube, *Petunia*
312 flowers at stage S9 were detached from their receptacle, their reproductive organs were carefully
313 removed with forceps and flowers were placed in 5% sucrose solution. Emitted sesquiterpenes
314 were collected by Stir Bar Sorptive Extraction (SBSE) by placing a magnetic Twister® (Gerstel,
315 Germany) coated with Polydimethylsiloxane (PDMS) placed inside the upper part of the *Petunia*
316 tube. After 48 h of collection, Twisters were eluted with 200 µL of hexane containing 2 nmol of
317 IS, vortexed for 10 s and samples were analyzed by GC-MS (for more details see **Method S1**).

318
319
**Starch and Carotenoid measurements**
320
Tissues collected for the analysis are described in **Table S6**. The secreted nectar from WT
321 ovaries was removed as much as possible using a tissue. WT and *eob2-3*<sup>LoftAD</sup> ovaries were cut
322 just above the nectary glands. Limb and basal ovary samples were immediately flash-frozen in
323 liquid nitrogen and stored at -80°C. Frozen samples were lyophilized using a Freeze Dry system
324 (Lyoquest, from Swiss Vacuum technologies) for 4 days at -50°C, 10 mbar, in the dark.
325 Carotenoids were extracted from ~10 mg of freeze-dried nectaries and petals. Quantification of
326 carotenoids was carried out with an HPLC system (UltiMate 3000, Thermo Scientific) (for more
327 details see **Method S2**). Carbohydrates were extracted and quantified from a ~3 mg freeze-dried,
328 ball-mill-ground, tissues with 1 mL of 80% ethanol for 20 min at 80 °C according to (Geest *et al.*,
329 2016) with modifications (see **Method S3**).

330
331
**Detection of starch by Lugol staining**
332
Ovaries were placed in Lugol's solution (Carl Roth, N052) and a vacuum infiltration of 1 min was
333 applied followed by a 10 min incubation. Samples were briefly rinsed with water before being
334 photographed under a stereoscopic microscope (Nikon, SMZ1500) equipped with a camera (Leica,
335 DMC6200). Flowers (w/o sepals) were cleared in 70% ethanol at 80 °C for 15 min, progressively
336 rehydrated, stained with Lugol's solution (Carl Roth, N052) for 10 min and rinsed in water.
337 Pictures were taken before and after staining using a Canon EOS 60D camera and Canon 35 mm
338 lens. For cellular resolution, images of the epidermal peel of petal limbs were captured with an
339 optical microscope (Leica, DM2000 LED).

340
341 **Confocal microscopy, imaging of epidermal cells and cell size measurements**

342 Epidermal peels were taken from the outer rim area of the petal limb and placed on microscopy
343 slides for confocal microscopy imaging. Epidermal peels of three flowers per line were taken for
344 comparison. Images were taken with a laser scanning microscope 55 (Leica, SP5) using the
345 differential interference contrast (DIC).
346

347 **Protein structure prediction**
348

The protein structures of EOB2 and eob2-2<sup>KO</sup> were predicted by AlphaFold (Jumper *et al.*, 2021).
349 The structures were visualized and aligned by PyMOL (Version 2.5.3, Schrödinger LLC).
350

351 **Microsynteny analysis**
352

The microsynteny analysis was performed using GEvo tool from CoGe (Lyons *et al.*, 2008), with
353 Peax4.03 genome for *P. axillaris* and ITAG release 2.4 genome for *S. lycopersicum*.
354

355 **Conserved cis-elements screening**
356

356 The promoter sequences of the selected genes were extracted (2kb upstream of the ATG) and
357 scanned for EOB2 homolog-binding profile matrices (MA1408.1 and MA1037.1 defined in the
358 JASPAR database), using FIMO v5.1.0 (Grant *et al.*, 2011). Identified motifs (*P* < 1e-4) were
359 counted http://meme-suite.org/.

360
361 **RESULTS**

362
363 **EOB1 and EOB2 are very similar in sequence and expression**

364 To determine the relationships between members of the SG19 R2R3-MYB family, we performed
365 a phylogenetic analysis (Fig. 1a). SG19 members belonging to the Solanaceae were clearly
366 separated from Arabidopsis. Nevertheless, protein alignments showed that the two main
367 functional domains (RMD<sup>SG19</sup> and TAD) were highly conserved among the different species and
368 among the different gene copies within species (Figs 1b, S1). Furthermore, microsynteny
369 analysis established that *P. axillaris* *EOB1* and *EOB2* are the orthologs of *S. lycopersicum*
370 *MYB24* and *MYB21*, respectively (Fig. S2).
Next, we performed a detailed analysis of the spatial and temporal expression of *P. axillaris* *EOB1* and *EOB2*. Different tissues and developmental stages of *P. axillaris* (Figs 1c, S3 for definition of stages) were used to perform RT-qPCR of *EOB1* and *EOB2* (Fig. 1d). *EOB1* and *EOB2* expression was detected in the petal tube, petal limb, pistil, and a bit in the floral stem, but not in roots or leaves. Within the pistil, *EOB1* and *EOB2* were highly expressed in the basal ovary (BO) and the stigma, suggesting functions in nectary and stigma development (Fig. 1d).
RT-qPCR on floral buds (without sepals) from S1 to S8 and on S10 open limbs from anthesis (D0) to 4 days after anthesis (D4) revealed that *EOB1* and *EOB2* started to be expressed from stage S3/S4, their expression increased over flower development and decreased 4 days after anthesis. However, RNA-sequencing data performed on basal ovaries at S5 and S10 and on petal limbs at S5, S7 and S10 (Fig. 1e) showed that *EOB1* expression is overall lower compared to that of *EOB2*. Moreover, while *EOB1* and *EOB2* are co-expressed in the flower, *EOB2* expression starts slightly before *EOB1*. Unlike previously thought, EOB1 and EOB2 functions are most likely not limited to regulation of the scent pathway and flower opening in *P. axillaris* (Spitzer-Rimon et al., 2010; Colquhoun et al., 2011; Spitzer-Rimon et al., 2012).

### CRISPR-Cas9 as a tool to decipher R2R3-MYB SG19 complexity

In order to obtain insight in the structure of the proteins, the proteins were characterized based on the literature. *EOB1* and *EOB2* genes encode for transcription factors of 202 and 197 amino acids (aa) respectively, that consist of an RMD<sup>SG19</sup> at the N-terminal and a TAD at the C-terminal part of the proteins (refer to the introduction for more details, Fig. 2b). The RMD<sup>SG19</sup> was previously shown to contain a nuclear localization signal (NLS). An *in silico* NLS mapper predictor (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) (Kosugi et al., 2009) identified a putative bipartite NLS at the end of the RMD<sup>SG19</sup> (<sup>89</sup>RWSKIAKHLPGRTDNEIKNYWRTRIQKHIK<sup>118</sup>) in both EOB1 and EOB2 (Fig. 2b Fig. S1).

To elucidate the molecular and biological functions of EOB1 and EOB2 during *P. axillaris* flower development, targeted mutagenesis using CRISPR-Cas9 in *P. axillaris* P background was carried out (Fig. 2a). In total, one allele for *EOB1* and four alleles for *EOB2* were isolated by CRISPR-Cas9 (Fig. 2b and Table S7) and classified in different categories depending on their predicted protein conformations and putative molecular functions (Fig. 2c). The *eob1-1* allele showed a deletion of 31bp and is predicted to lead to nonfunctional product due to the truncated
RMD<sup>SG19</sup> and the absence of both the NLS and the TAD. The *eob2-1* mutant carried a deletion of 364bp, which is also expected to cause a complete loss-of-function (Figs 2b,c, S4a,b). The *eob2-2* allele presented a deletion of 7bp, this frameshift mutation resulted in an abnormal protein product. Alphafold2 was used to predict the 3D structure of EOB2 and *eob2-2* proteins (Fig. 2d). The RMD<sup>SG19</sup> domain was intact in *eob2-2*. In contrast, the C-terminal part of EOB2 was not conserved in *eob2-2*. This unrelated protein sequence may lead to destabilization of the *eob2-2* protein and disrupt RMD<sup>SG19</sup> DNA/protein binding capabilities. Even though the *eob2-1* and *eob2-2* encode quite different protein sequences, the two mutants displayed the same strong phenotype (see below). Therefore, most likely *eob2-1* and *eob2-2* both represent knockout alleles, referred as *eob2-1<sup>KO</sup>* and *eob2-2<sup>KO</sup>*. The *eob2-3* and *eob2-4* alleles contained deletions of 5bp or 2bp, respectively. These mutations removed the transcriptional activation domain (TAD) but retained an intact RMD<sup>SG19</sup> (Fig. 2b,c). The *eob2-3* and *eob2-4* truncated proteins retain the nuclear localization signal, bind DNA, and can interact with protein partners but lack their transactivation activity. Thus, *eob2-3* and *eob2-4* are referred to as loss-of-TAD alleles (*eob2-3<sup>LoFTAD</sup>* and *eob2-4<sup>LoFTAD</sup>*).

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## EOB2 is a regulator of EOB1

The knockout mutants in *EOB1* and *EOB2* caused very different phenotypes. No visible developmental phenotypes were observed in *eob1-1<sup>KO</sup>* (Figs 2e,f, 3a), but the emission of the major scent compounds was reduced compared to the wild type (Fig. 3b) as previously observed in *Petunia hybrida* EOB1-RNAi lines (Spitzer-Rimon et al., 2012). Moreover, *IGS* expression was induced in Petunia leaves after *EOB1* transient expression (Fig. 3e). We conclude that EOB1 is essential for floral volatile benzenoid/phenylpropanoid (FVBP) production, even in the presence of active EOB2.

The flowers of the *eob2* knockout alleles, *eob2-1<sup>KO</sup>* and *eob2-2<sup>KO</sup>*, failed to enter anthesis and prematurely senesced as closed buds. Flower initiation was not affected but flower bud growth was delayed after reaching ~2 cm (stage S4) and the buds prematurely entered senescence at ~3.5 cm (S5) (Figs 2e,f, 3a, S5). After reaching S5, the flower bud stopped growing and gradually senesced: the petal limb tip started to soften, the flower bud turned yellow/brown and the flower bud was completely brown/dead. At 16 weeks after germination, the difference between mutant plants and wild-type was remarkable: 100% of fully opened flowers in the wild-type against 0% in the mutant lines (Fig. 3a). As the heterozygotes are phenotypically wild type, we conclude that

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the *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> alleles are recessive (Fig. S6). The fact that expression of *EOB2* precedes *EOB1* expression (Fig. 1e) suggests that EOB2 may be an activator of EOB1. Indeed, *EOB2* expression is independent of EOB1 (Fig. 3c,e) while *EOB1* expression is activated by EOB2 (Fig. 3d,e), probably by binding to the defined "SG19 MYB-binding site" in the *EOB1* promoter (Figs 3f, S7a). Moreover, no additional effects were observed when comparing the *eob2-2*<sup>KO</sup> single mutant to the *eob1-1*<sup>KO</sup>/*eob2-2*<sup>KO</sup> double mutant flowers (Figs 3a, S9).
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Therefore, EOB1 is downstream of EOB2.
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443 **The loss-of-TAD mutants display floral organ maturation defects**

The *eob2* knockout alleles described so far displayed early senescence and did not reach anthesis, which makes it impossible to investigate EOB2 function(s) beyond stage 5. Based on its expression pattern (Fig. 1d), *EOB2* may also function in later floral developmental stages. The loss-of-TAD (LoTAD) mutants, *eob2-3LoTAD* and *eob2-4LoTAD*, allowed us to study EOB2 function at the later developmental stages.
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The *eob2-3LoTAD* and *eob2-4LoTAD* homozygous single mutants caused identical pleiotropic floral phenotypes with floral organ maturation defects and juvenile characteristics at late stages (Fig. 2f). The *eob2-3LoTAD* mutant was used for further analysis. Compared to the wild type, petal tubes were shorter, petals did not fully open, limbs were smaller and had greener petal veins (Fig. 4a,f).
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Both wild-type and LoTAD limbs were UV-absorbing (Fig. S8). Concerning the reproductive organs, no stamen/pollen phenotypes were observed. In contrast, we noticed phenotypes in style, stigma, and nectary. Styles were twisted, which can be interpreted as an indirect phenotype due to the mechanical constraints applied by the reduced petal growth (Fig. 4b).
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While droplets of exudate were present on wild-type stigmas, the mutant had dry stigma surfaces.
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A key function of the stigma exudate is pollen hydration, a prerequisite for pollen tube elongation and fertilization. Indeed, *eob2-3LoTAD* failed to hydrate pollen, impairing seed set. Seed yield was increased when the dry *eob2-3LoTAD* stigmas were treated with wild-type stigma exudate (Fig. 4c). Ovule number and development were not impacted (Fig. 4d). After pollination and fertilization, the style was detached from the fruit in the wild type but not in the mutants (Fig. 4c,e). Nectar secretion was disrupted (Fig. 4g) and carotene levels were reduced (Fig. 4h) indicating defective nectary maturation.
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Several SG19 members have been shown to play a role in the production of diverse floral volatiles in a range of species (see introduction). In the *eob2-3LoTAD* petal limbs, benzaldehyde
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and methylbenzoate were absent, in line with a decreased expression of *ODO1* and *EOB1* (Fig. 4i,j). The emission of terpene volatiles from the floral tube was reduced, as well as their accumulation in the stigma (Fig. 4k,l). In *P. hybrida*, TPS1 was shown to regulate terpene volatile synthesis and emission from the tube (Boachon *et al.*, 2019). In *eob2-3LoftAD* petal tube, TPS1 expression was also reduced (Fig. 4m).

Next, we analyzed the F1 progenies. The *eob2-3LoftAD/+* and *eob2-4LoftAD/+* heterozygous flowers displayed an intermediate phenotype (intermediate flower angle opening, nectar volume and methylbenzoate level) indicating that LofTAD alleles are semi-dominant (Fig. S6). This intermediate phenotype is compatible with a competition between the products of the wild-type and the LofTAD mutants for the same DNA-cis-elements.

### EOB2 prevents flower senescence during flower development by inhibiting ethylene synthesis

The contrasting phenotypes of *eob2* knockout vs LofTAD might be explained by differences at the transcriptional level. Therefore, a transcriptome analysis of S5 petal limb from WT, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LoftAD</sup> was carried out (Fig. 5a). EOB2 expression was significantly downregulated in the knockout line while no significant expression differences were observed in the wild-type vs LofTAD comparison (Fig. S4c). This reinforces the notion that *eob2-1*<sup>KO</sup> is a complete knockout, while *eob2-3*<sup>LoftAD</sup> retains partial activity. Whereas 23% of the genes were differentially regulated in the knockout (*eob2-1*<sup>KO</sup>), only 4.5% were differentially expressed in the LofTAD (*eob2-3*<sup>LoftAD</sup>). Again, this is in line with partial activity of *eob2-3*<sup>LoftAD</sup>. Most of the 4.5% were regulated in the same direction (either up- or down-regulated) in the two genotypes, supporting that both alleles partly act on the same pathways. Interestingly, in both mutant backgrounds, the majority of the DEGs was up-regulated. As structural data strongly indicate that EOB2 is a transcriptional activator (see introduction), the observed upregulation must be an indirect effect (indirect targets and/or mediated by interacting factors). Moreover, an *in silico* scanning promoter analysis revealed that the down-regulated gene promoters displayed a significant enrichment of the SG19 binding site matrices, while it was not the case for the up-regulated gene promoters (Table S8 and Fig. S11).

Within the large proportion of *eob2-1*<sup>KO</sup>-specific DEGs, GO terms associated with senescence were over-represented (Fig. 5b). Among them, several ACS and ACO genes encoding enzymes responsible for ethylene synthesis were up-regulated (Fig. 5c and Table S9) as well as many
ethylene response factors (ERF) and senescence-associated genes (SAG). Genes related to cellular processes maintenance (translation, cellular trafficking, mitotic cell cycle, etc.) were down-regulated, presumably as a consequence of senescence.

Ethylene is a key hormone promoting flower senescence and blocking ethylene sensitivity partially restored the RNAi-*EOB2* flower phenotypes in *P. hybrida* and *N. attenuata* (Colquhoun *et al.*, 2011). To obtain direct proof that ethylene production is activated prematurely in *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> compared to wild-type flowers, we quantified ethylene emission from S5 flower buds before the first signs of senescence. Ethylene levels in *eob2-1*<sup>KO</sup> flower buds at S5 were higher compared to wild-type (Fig. 5d). Since *EOB2* is expressed in both petal and pistil (Fig. 1c), ethylene measurements were performed on dissected petal and pistil from S5 flower buds.

We noticed a strong contribution from both floral organs in releasing ethylene. In parallel, a 1-MCP treatment (ethylene action inhibitor) was continuously applied on wild-type, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LofTAD</sup> plants. 1-MCP treatment caused a partial rescue of the *eob2-1*<sup>KO</sup> flower senescence phenotype but negligible floral developmental effects on wild-type and *eob2-3*<sup>LofTAD</sup> (Figs 5e, S9, S10). Similar results were obtained with the other allele *eob2-2*<sup>KO</sup>. Though *eob2-1*<sup>KO</sup> or *eob2-2*<sup>KO</sup> flowers reached anthesis after 1-MCP treatment, they did not fully open, were scentless and morphologically similar to *eob2-3*<sup>LofTAD</sup> or *eob2-4*<sup>LofTAD</sup> flowers.

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## EOB2 activates pathways of secondary metabolism

We took advantage of the powerful *eob2-3*<sup>LofTAD</sup> mutant to (1) identify EOB2 transcriptional targets and (2) unravel to what extent nectary and petal limb maturation are regulated by the same genetic pathway. To do so, we compared the transcriptomes of wild-type and *eob2-3*<sup>LofTAD</sup> petal limb and nectary, at S5 and S10 (Fig. 6a). The analysis of the differentially expressed genes (DEGs) revealed a higher proportion of up-regulated genes in each condition and the number of DEGs was higher at S10 than S5 in both floral organs (Fig. 6a). Since EOB2 is thought to be a transcriptional activator (see introduction), the down-regulated genes are potential direct targets. Among the down-regulated genes, a significant enrichment of promoters presented SG19 binding sites, which was not the case for the up-regulated genes (Figs 6b, S11 and Table S8). Sixty genes were commonly down-regulated in the four different conditions (Fig. 6c). Among them, we found seven genes encoding enzymes of the shikimate/phenylpropanoid and four carotenoid biosynthetic genes (Fig. 6d). These genes are likely to be direct targets of EOB2 and to support this finding some homologs were also identified as direct targets in other species (Table S2).

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Indeed, SG19 binding sites were identified at least once in each of the 11 promoters tested, while only five promoters presented the exact motif characteristic for SG19 (Fig. 6d). This data provides evidence that EOB2 is a direct activator candidate of genes involved in shikimate, phenylpropanoid and carotenoid maturation pathways in both limb and nectary tissues (Fig. S12).

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## 536 Carbohydrate metabolism is involved in floral organ maturation

Nectary and limb are mostly sink tissues, dependent on the surrounding photosynthetic tissues to provide carbohydrates for their active growth. Starch that is stored at early stages is mobilized later to support respiration, growth and maturation (Streb & Zeeman, 2012). A GO-term enrichment analysis was performed on the down-regulated genes from the four conditions. Only two GO-terms were significantly co-enriched in the four conditions: ‘TCA cycle’ and ‘glycolytic process’, meaning that high energy carbohydrate breakdown was impaired in both *eob2-3*<sup>LoTAD</sup> nectary and limb (Fig. 7a). We also evaluated whether other components of carbohydrate metabolism were impacted. One gene encoding a beta-amylase (*BAM*) was strongly down-regulated in both *eob2-3*<sup>LoTAD</sup> floral organs compared to wild-type (Fig. 7b), suggesting that starch degradation was also impaired (Fig. 7c).

We next evaluated starch levels in wild-type vs *eob2-3*<sup>LoTAD</sup> in both petals and nectaries using IKI staining. During wild-type flower development, we observed an accumulation of large amounts of starch at stage S5 and decreased levels at stage 10, while starch levels remained high in *eob2-3*<sup>LoTAD</sup> (Fig. 7d,e). In nectary at S10, starch was only detected within the wild-type stomata, whereas *eob2-3*<sup>LoTAD</sup> nectary stained heavily for starch, comparable to S5 immature nectary (Fig. 7d). Full petal and petal limb epidermal peels, also revealed that *eob2-3*<sup>LoTAD</sup> retained more starch granules compared to wild-type (Fig. 7e,f). In addition, *eob2-3*<sup>LoTAD</sup> petal limb epidermal cells were smaller. The Lugol results were confirmed by starch content quantification (Fig. 7g). These results indicated that the carbohydrate breakdown was reduced in both nectary and limb of the *eob2-3*<sup>LoTAD</sup> mutant (Fig. 7c). This is consistent with the juvenile character of the *eob2-3*<sup>LoTAD</sup> mutant.

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## 559 DISCUSSION

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Different promoter sequences and subtle differences in protein coding sequences drive the functional divergence of *EOB1* and *EOB2*

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The *EOB1* and *EOB2* genes encode closely related members of subgroup 19 R2R3-MYB transcription factors. Nevertheless, *eob1* and *eob2* mutants display radically different phenotypes. Loss-of-function of EOB1 causes a decrease in scent emission, whereas EOB2 is extremely pleiotropic. Thus, there must be subtle differences in protein sequence and/or expression that are responsible for these highly distinct phenotypes. *EOB2* is expressed slightly earlier than *EOB1*, EOB2 induces *EOB1* expression and a potential EOB2-binding site is present in the *EOB1* promoter, potentially explaining their extremely similar expression patterns. Phylogenetic and microsynteny analysis reveal that species with a single SG19 member, the gene is closer to the petunia *EOB2* than to *EOB1*. It might be that *EOB2* ancestor evolved as a driver of flower maturation, while petunia *EOB1* appeared later during evolution from *EOB2* duplication and gained a specific function in scent production (scent booster), notably due to its divergent promoter (Fig. S7) and the associated delayed expression pattern compared to *EOB2*, with a peak at anthesis.

As *EOB1* expression was strongly reduced in an *eob2*<sup>KO</sup> background, while *eob1*<sup>KO</sup> mutants had normal *EOB2* expression, EOB2 must be a major activator of *EOB1*. In parallel, a previous study revealed that PhERF6 interacts with EOB1 to regulate scent biosynthesis, but not with EOB2 (Liu *et al.*, 2017). Promoter differences as well as subtle differences in protein-protein interactions underlie the loss of scent phenotype in the *eob1*<sup>KO</sup> mutant.

## Nectary and limb are sink tissues, that rely on common pathways for their maturation

At later developmental stages, the LofTAD mutant flowers retained starch, did not produce benzenoid/phenylpropanoid volatiles, produced less terpenoids and presented a smaller, greener, not fully opened corolla (Figs 4,7). In line with this, transcriptomic data revealed that genes encoding enzymes of both primary and secondary metabolisms were decreased in immature tissues compared to those of the WT. We consider following options: it could be that both primary and secondary metabolisms are dependent on EOB2 or that EOB2 directly regulates only one of these two processes and that the other process is reduced as a consequence. The presence of EOB2-binding sites in promoters of genes in the shikimate/phenylpropanoid and carotenoid pathways would support the second option. In that case, the decrease of energy-demanding secondary metabolites production could reduce carbohydrate flux through feedback inhibition.

## Comparisons with the R2R3-MYB SG19 functions previously reported in the literature
In the introduction, we asked the question whether the conflicting data on the functions of SG19 R2R3-MYBs reported within and between different species were due to species specificity, to the methods used, to the focus of the study on a particular organ or to the multi-functionality of the SG19 R2R3-MYBs. Here, we used well-defined mutants and revealed in a single system the complexity of the SG19 R2R3-MYB functions.

Comparable to *eob2* KO, premature flower senescence has been described for strong RNAi lines targeting *EOB2* homologs in *P. hybrida* and *N. attenuata* (Colquhoun *et al.*, 2011). By contrast and similar to *eob2* LofTAD, immature floral organ phenotypes were reported in other tobacco RNAi lines targeting *MYB305* (Liu *et al.*, 2009; Liu & Thornburg, 2012). We can speculate that those RNAi lines had an increased proportion of alternative splicing variants with an intact R2R3-MYB and no TAD (RNAi targeted the C-ter) or that the silencing was incomplete making it possible to block the senescence but not enough to induce transcription. The VIGS performed in *P. hybrida* targeting *EOB2* only revealed scent related phenotypes, so it could be that the silencing was incomplete or that *EOB1* instead of *EOB2* was targeted in this study.

The expression pattern of tobacco *MYB305* (Liu *et al.*, 2009) was similar to *Petunia* *EOB2*, while expression of Arabidopsis *AtMYB21* and *AtMYB24* was also detected in stamens and these proteins play a major role in Arabidopsis anther maturation (Qi *et al.*, 2015). Out of those different expression patterns and functions, we concluded that in Arabidopsis SG19 R2R3-MYB members were recruited for stamen maturation while other factors were selected in pollinator-dependent species such as *Petunia* or *Nicotiana*. Except for the stamens, Arabidopsis and *Petunia* SG19 genes have similar spatial expression patterns and share functions (nectary maturation, flower opening), however other functions are specific for *Petunia* (senescence, FVBP) or for Arabidopsis (flavonols) (Zhang *et al.*, 2021b). In addition to the differences in floral organ expression, it appears that during evolution, different partners and target genes were recruited by the SG19 R2R3-MYBs (**Tables S1, S2**).

## One gene, two mutations, contrasting phenotypes

The *eob2*<sup>KO</sup> mutations display premature flower senescence, whereas the semi-dominant *eob2*<sup>LofTAD</sup> alleles retain the RMD<sup>SG19</sup> functions specifically and *eob2*<sup>LofTAD</sup> mutants and show multiple flower maturation defects resulting in juvenile flowers (**Fig. 8**). The conclusions are the following: (1) *EOB2* is a multifunctional protein, (2) *EOB2* transcriptional activity drives flower maturation and (3) the R2R3-MYB domain is, in addition to its DNA-binding role in
transcription, involved in the regulation of indirect senescence-related targets through an
unknown mechanism. Cases of nonfunctional versus partially functional gene product showing
different phenotypes are rare in the literature, an example is the tomato MADS-box gene *RIN* (for
*ripening inhibitor*) where the *rin-knockout* (Ito *et al.*, 2017) and semi-dominant *rinG2* (Ito *et al.*,
2021) alleles were leading to different tomato fruit phenotypes. Proper conclusions about the
function(s) of the protein require a careful analysis of the protein structure before targeting a TF
gene by mutagenesis.

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635 Complexity of SG19 MYB factors protein functions
636 The genetic processes that underlie the initiation and determination of the floral organs have been
637 well studied in Arabidopsis, snapdragon and *Petunia* (Coen & Meyerowitz, 1991;
638 Vandenbussche *et al.*, 2004; Morel *et al.*, 2018), and are quite well conserved between species
639 (Soltis *et al.*, 2007). Floral diversity mainly arises during subsequent maturation and serves to
640 adapt flowers to specific guilds of pollinators (Schiestl & Johnson, 2013). This aspect may be
641 negligible in a selfing species such as *Arabidopsis thaliana* but is essential in animal-pollinated
642 species. The versatility of SG19 MYB factors and their many interacting regulatory factors
643 appear to be well suited to adapt flowers for optimal pollination. SG19 MYB factors affect
644 multiple aspects of flower maturation, with potential impact on the functions of the flower. These
645 include floral morphology (Yarahmadov *et al.*, 2020), scent (Spitzer-Rimon *et al.*, 2010), nectar
646 (Liu *et al.*, 2009, p. 305; Liu & Thornburg, 2012) and color (Wang *et al.*, 2022), but also plant
647 defense against microorganisms mediated by the antimicrobial activity of terpenes (Boachon *et
648 al.*, 2019) or compounds present in the stigma exudate/nectar (Kurilla *et al.*, 2019).

649 While part of the confusion about the roles of SG19 MYB factors in flower development may be
650 due to the use of different techniques, it is also clear that data on EOB2 and its homologs cannot
651 be extrapolated from one species to the next, possibly reflecting the diversity of plant-pollinator
652 interactions.
653

654
655 ACKNOWLEDGEMENTS
656 This work was supported by grants from the Swiss National Science Foundation
657 (31003A_182340) and the European Union (ERC-ADG 741354). We would like to thank the
658 Next Generation Sequencing Platform of the University of Bern for performing the high-
throughput sequencing experiments and the Interfaculty Bioinformatics Unit of the University of Bern for providing computational infrastructure and support with bioinformatic analyses. We thank Christopher Ball, Jasmin Sekulovski, and Sarah Dolder for taking care of our *Petunia* plants. We are grateful to Patrick Ryan and Ying Li for providing their GO annotations, Diane Bonnet for advice on microscopy, Aya Assamene for technical support, and Chaobin Li for bioinformatics supports as well as helpful discussion and proof-reading.

AUTHOR CONTRIBUTIONS
M.C. and C.K. conceived and designed the experiments. M.C. generated the CRISPR-Cas9 lines, performed the phenotyping/pictures, the 1-MCP treatments, the starch staining using Lugol, the RT-qPCR with the technical support of L.J.. G.C. performed the protein structures prediction with AlphaFold. R.Halitschke performed the ethylene measurements. B.B. and D.P.B. performed the terpene measurements. R.Heutink and J.V. performed the starch and carotenoid measurements. M.C., M.B. and G.G. performed the RNA-seq analysis. M.C. and C.K. wrote the article.

DATA AVAILABILITY
Raw reads are available on NCBI Sequence Read Archive (SRA), under the BioProject accession PRJNA797226 (reviewer link: https://dataview.ncbi.nlm.nih.gov/object/PRJNA797226?reviewer=pa7i8vmprfql6vvu67ui6tqviu). Plasmids and seeds produced in this study are available from C.K. on request.

COMPETING INTERESTS

None declared.

REFERENCES

Amrad A, Moser M, Mandel T, de Vries M, Schuurink RC, Freitas L, Kuhlemeier C. 2016. Gain and Loss of Floral Scent Production through Changes in Structural Genes during Pollinator-Mediated Speciation. *Current biology: CB 26*: 3303–3312.

Andrews S. 2022. Babraham Bioinformatics - FastQC A Quality Control tool for High Throughput Sequence Data.
691 Battat M, Eitan A, Rogachev I, Hanhineva K, Fernie A, Tohge T, Beekwilder J, Aharoni A.

692 2019. A MYB Triad Controls Primary and Phenylpropanoid Metabolites for Pollen Coat

693 Patterning. *Plant Physiology* **180**: 87–108.

694 Bian S, Sui X, Wang J, Tian T, Wang C, Zhao X, Liu X, Fang N, Zhang Y, Liu Y, et al.

695 2021. NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1a promoter to

696 regulate nicotine biosynthesis. *Plant Physiology*.

697 Boachon B, Lynch J, Ray S, Yuan J, Caldo K, Junker R, Kessler S, Morgan J, Dudareva N.

698 2019. Natural fumigation as a mechanism for volatile transport between flower organs. *Nature*

699 *Chemical Biology* **15**: 583–588.

700 Brandenburg A, Kuhlemeier C, Bshary R. 2012. Hawkmoth Pollinators Decrease Seed Set of

701 a Low-Nectar Petunia axillaris Line through Reduced Probing Time. *Current Biology* **22**: 1635–

702 1639.

703 Cheng H, Song S, Xiao L, Soo HM, Cheng Z, Xie D, Peng J. 2009. Gibberellin Acts through

704 Jasmonate to Control the Expression of MYB21, MYB24, and MYB57 to Promote Stamen

705 Filament Growth in Arabidopsis. *PLOS Genetics* **5**: e1000440.

706 Chopy M, Morel P, Rodrigues Bento S, Vandenbussche M. 2020. Genome editing by

707 CRISPR-Cas9 technology in Petunia hybrida. *Acta Horticulturae*: 209–218.

708 Coen ES, Meyerowitz EM. 1991. The war of the whorls: genetic interactions controlling flower

709 development. *Nature* **353**: 31–37.

710 Colquhoun TA, Schwieterman ML, Wedde AE, Schimmel BCJ, Marciniak DM, Verdonk

711 JC, Kim JY, Oh Y, Gális I, Baldwin IT, et al. 2011. EOBII Controls Flower Opening by

712 Functioning as a General Transcriptomic Switch. *Plant Physiology* **156**: 974–984.

713 Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high

714 throughput. *Nucleic Acids Research* **32**: 1792–1797.

715 Edwards K, Johnstone C, Thompson C. 1991. A simple and rapid method for the preparation

716 of plant genomic DNA for PCR analysis. *Nucleic Acids Research* **19**: 1349.

717 Geest G van, Choi YH, Arens P, Post A, Liu Y, Meeteren U van. 2016. Genotypic differences

718 in metabolomic changes during storage induced-degreening of chrysanthemum disk florets.

719 *Postharvest Biology and Technology* **115**: 48–59.

720 Grant CE, Bailey TL, Noble WS. 2011. FIMO: scanning for occurrences of a given motif.

721 *Bioinformatics* **27**: 1017–1018.

722 Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O. 2010. New

723 Algorithms and Methods to Estimate Maximum-Likelihood Phylogenies: Assessing the

724 Performance of PhyML 3.0. *Systematic Biology* **59**: 307–321.
725 Hoballah ME, Stuurman J, Turlings TCJ, Guerin PM, Connétable S, Kuhlemeier C. 2005.
726 The composition and timing of flower odour emission by wild Petunia axillaris coincide with the
727 antennal perception and nocturnal activity of the pollinator Manduca sexta. *Planta* **222**: 141–150.

728 Huang H, Gao H, Liu B, Qi T, Tong J, Xiao L, Xie D, Song S. 2017. Arabidopsis MYB24
729 Regulates Jasmonate-Mediated Stamen Development. *Frontiers in Plant Science* **8**.

730 Huang H, Gong Y, Liu B, Wu D, Zhang M, Xie D, Song S. 2020. The DELLA proteins
731 interact with MYB21 and MYB24 to regulate filament elongation in Arabidopsis. *BMC Plant
732 Biology* **20**: 64.

733 Ito Y, Nakamura N, Kotake-Nara E. 2021. Semi-dominant effects of a novel ripening inhibitor
734 (rin) locus allele on tomato fruit ripening. *PLOS ONE* **16**: e0249575.

735 Ito Y, Nishizawa-Yokoi A, Endo M, Mikami M, Shima Y, Nakamura N, Kotake-Nara E,
736 Kawasaki S, Toki S. 2017. Re-evaluation of the rin mutation and the role of RIN in the
737 induction of tomato ripening. *Nature Plants* **3**: 866–874.

738 Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, Tunyasuvunakool K,
739 Bates R, Žídek A, Potapenko A, et al. 2021. Highly accurate protein structure prediction with
740 AlphaFold. *Nature* **596**: 583–589.

741 Ke Y, Abbas F, Zhou Y, Yu R, Fan Y. 2021. Auxin-Responsive R2R3-MYB Transcription
742 Factors HcMYB1 and HcMYB2 Activate Volatile Biosynthesis in Hedychium coronarium
743 Flowers. *Frontiers in Plant Science* 0.

744 Kim D, Langmead B, Salzberg SL. 2015. HISAT: a fast spliced aligner with low memory
745 requirements. *Nature Methods* **12**: 357–360.

746 Kosugi S, Hasebe M, Tomita M, Yanagawa H. 2009. Systematic identification of cell cycle-
747 dependent yeast nucleocytoplasmic shuttling proteins by prediction of composite motifs.
748 *Proceedings of the National Academy of Sciences of the United States of America* **106**: 10171–
749 10176.

750 Kurilla A, Toth T, Dorgai L, Darula Z, Lakatos T, Silhavy D, Kerenyi Z, Dallmann G.
751 2019. Nectar- and stigma exudate-specific expression of an acidic chitinase could partially
752 protect certain apple cultivars against fire blight disease. *Planta* **251**: 20.

753 Liao Y, Smyth GK, Shi W. 2014. featureCounts: an efficient general purpose program for
754 assigning sequence reads to genomic features. *Bioinformatics* **30**: 923–930.

755 Liu G, Ren G, Guirgis A, Thornburg RW. 2009. The MYB305 Transcription Factor Regulates
756 Expression of Nectarin Genes in the Ornamental Tobacco Floral Nectary. *The Plant Cell* **21**:
757 2672–2687.

758 Liu G, Thornburg RW. 2012. Knockdown of MYB305 disrupts nectary starch metabolism and
759 floral nectar production. *The Plant Journal* **70**: 377–388.
760 **Liu F, Xiao Z, Yang L, Chen Q, Shao L, Liu J, Yu Y. 2017.** PhERF6, interacting with EOBI, negatively regulates fragrance biosynthesis in petunia flowers. *New Phytologist* **215**: 1490–1502.

762 **Love MI, Huber W, Anders S. 2014.** Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. *Genome Biology* **15**: 550.

764 **Lyons E, Pedersen B, Kane J, Alam M, Ming R, Tang H, Wang X, Bowers J, Paterson A, Lisch D, et al. 2008.** Finding and Comparing Syntenic Regions among Arabidopsis and the Outgroups Papaya, Poplar, and Grape: CoGe with Rosids. *Plant Physiology* **148**: 1772–1781.

767 **Ma D, Constabel CP. 2019.** MYB Repressors as Regulators of Phenylpropanoid Metabolism in Plants. *Trends in Plant Science* **24**: 275–289.

769 **Mallona I, Lischewski S, Weiss J, Hause B, Egea-Cortines M. 2010.** Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. *BMC Plant Biology* **10**: 4.

772 **Mandaokar A, Thines B, Shin B, Lange BM, Choi G, Koo YJ, Yoo YJ, Choi YD, Choi G, Browse J. 2006.** Transcriptional regulators of stamen development in Arabidopsis identified by transcriptional profiling. *The Plant Journal* **46**: 984–1008.

775 **Martin M. 2011.** Cutadapt removes adapter sequences from high-throughput sequencing reads. *EMBnet.journal* **17**: 10–12.

777 **Medina-Puche L, Molina-Hidalgo FJ, Boersma M, Schuurink RC, López-Vidriero I, Solano R, Franco-Zorrilla J-M, Caballero JL, Blanco-Portales R, Muñoz-Blanco J. 2015.** An R2R3-MYB Transcription Factor Regulates Eugenol Production in Ripe Strawberry Fruit Receptacles. *Plant Physiology* **168**: 598–614.

781 **Moerkercke AV, Haring MA, Schuurink RC. 2011.** The transcription factor EMISSION OF BENZENOIDS II activates the MYB ODORANT1 promoter at a MYB binding site specific for fragrant petunias. *The Plant Journal* **67**: 917–928.

784 **Morel P, Heijmans K, Ament K, Chopy M, Trehin C, Chambrier P, Bento SR, Bimbo A, Vandenbussche M. 2018.** The Floral C-Lineage Genes Trigger Nectary Development in Petunia and Arabidopsis. *The Plant Cell* **30**: 2020–2037.

787 **Moyano E, Martínez-Garcia JF, Martin C. 1996.** Apparent redundancy in myb gene function provides gearing for the control of flavonoid biosynthesis in antirrhinum flowers. *The Plant Cell* **8**: 1519–1532.

790 **NAKAGAWA T, SUZUKI T, MURATA S, NAKAMURA S, HINO T, MAEO K, TABATA R, KAWAI T, TANAKA K, NIWA Y, et al. 2007.** Improved Gateway Binary Vectors: High-Performance Vectors for Creation of Fusion Constructs in Transgenic Analysis of Plants. *Bioscience, Biotechnology, and Biochemistry* **71**: 2095–2100.

794 **Niwa T, Suzuki T, Takebayashi Y, Ishiguro R, Higashiyama T, Sakakibara H, Ishiguro S. 2018.** Jasmonic acid facilitates flower opening and floral organ development through the
796 upregulated expression of SlMYB21 transcription factor in tomato. *Bioscience, Biotechnology, and Biochemistry* **82**: 292–303.
797

798 **Okonechnikov K, Golosova O, Fursov M, the UGENE team. 2012.** Unipro UGENE: a unified
799 bioinformatics toolkit. *Bioinformatics* **28**: 1166–1167.

800 **Patrick RM, Huang X-Q, Dudareva N, Li Y. 2021.** Dynamic histone acetylation in floral
801 volatile synthesis and emission in petunia flowers. *Journal of Experimental Botany* **72**: 3704–
802 3722.

803 **Qi T, Huang H, Song S, Xie D. 2015.** Regulation of Jasmonate-Mediated Stamen Development
804 and Seed Production by a bHLH-MYB Complex in Arabidopsis. *The Plant Cell* **27**: 1620–1633.

805 **Reeves PH, Ellis CM, Ploense SE, Wu M-F, Yadav V, Tholl D, Chételat A, Haupt I,**
806 Kennerley BJ, Hodgens C, et al. 2012. A Regulatory Network for Coordinated Flower
807 Maturation. *PLOS Genetics* **8**: e1002506.

808 **Sablowski R w., Moyano E, Culianez-Macia F a., Schuch W, Martin C, Bevan M. 1994.** A
809 flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. *The
810 EMBO Journal* **13**: 128–137.

811 **Schiestl FP, Johnson SD. 2013.** Pollinator-mediated evolution of floral signals. *Trends in
812 Ecology & Evolution* **28**: 307–315.

813 **Schmitt AJ, Roy R, Klinkenberg PM, Jia M, Carter CJ. 2018.** The Octadecanoid Pathway,
814 but Not COI1, Is Required for Nectar Secretion in Arabidopsis thaliana. *Frontiers in Plant
815 Science* **9**: 1060.

816 **Schubert R, Dobritzsch S, Gruber C, Hause G, Athmer B, Schreiber T, Marillonnet S,**
817 Okabe Y, Ezura H, Acosta IF, et al. 2019. Tomato MYB21 Acts in Ovules to Mediate
818 Jasmonate-Regulated Fertility. *The Plant Cell* **31**: 1043–1062.

819 **Shan X, Li Y, Yang S, Yang Z, Qiu M, Gao R, Han T, Meng X, Xu Z, Wang L, et al. 2020.**
820 The spatio-temporal biosynthesis of floral flavonols is controlled by differential phylogenetic
821 MYB regulators in Freesia hybrida. *New Phytologist* **228**: 1864–1879.

822 **Soltis DE, Chanderbali AS, Kim S, Buzgo M, Soltis PS. 2007.** The ABC Model and its
823 Applicability to Basal Angiosperms. *Annals of Botany* **100**: 155–163.

824 **Song S, Qi T, Huang H, Ren Q, Wu D, Chang C, Peng W, Liu Y, Peng J, Xie D. 2011.** The
825 Jasmonate-ZIM Domain Proteins Interact with the R2R3-MYB Transcription Factors MYB21
826 and MYB24 to Affect Jasmonate-Regulated Stamen Development in Arabidopsis. *The Plant Cell*
827 **23**: 1000–1013.

828 **Spitzer-Rimon B, Farhi M, Albo B, Cna'ani A, Zvi MMB, Masci T, Edelbaum O, Yu Y,**
829 Shklarman E, Ovadis M, et al. 2012. The R2R3-MYB-Like Regulatory Factor EOBI, Acting
830 Downstream of EOBII, Regulates Scent Production by Activating ODO1 and Structural Scent-
831 Related Genes in Petunia. *The Plant Cell* **24**: 5089–5105.
832 Spitzer-Rimon B, Marhevka E, Barkai O, Marton I, Edelbaum O, Masci T, Prathapani N-K, Shklarman E, Ovadis M, Vainstein A. 2010. EOBII, a Gene Encoding a Flower-Specific Regulator of Phenylpropanoid Volatiles' Biosynthesis in Petunia. *The Plant Cell* **22**: 1961–1976.

835 Streb S, Zeeman SC. 2012. Starch Metabolism in Arabidopsis. *The Arabidopsis Book* / *American Society of Plant Biologists* **10**: e0160.

837 Van Moerkercke A, Galván-Ampudia CS, Verdonk JC, Haring MA, Schuurink RC. 2012. Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals. *Journal of Experimental Botany* **63**: 3157–3171.

840 Vandenbussche M, Zethof J, Royaert S, Weterings K, Gerats T. 2004. The duplicated B-class heterodimer model: whorl-specific effects and complex genetic interactions in Petunia hybrida flower development. *The Plant Cell* **16**: 741–754.

843 Wang Y, Zhou L-J, Wang Y, Geng Z, Ding B, Jiang J, Chen S, Chen F. 2022. An R2R3-MYB transcription factor CmMYB21 represses anthocyanin biosynthesis in color fading petals of chrysanthemum. *Scientia Horticulturae* **293**: 110674.

846 Weirauch MT, Yang A, Albu M, Cote AG, Montenegro-Montero A, Drewe P, Najafabadi HS, Lambert SA, Mann I, Cook K, et al. 2014. Determination and Inference of Eukaryotic Transcription Factor Sequence Specificity. *Cell* **158**: 1431–1443.

849 Wu T, Hu E, Xu S, Chen M, Guo P, Dai Z, Feng T, Zhou L, Tang W, Zhan L, et al. 2021a. clusterProfiler 4.0: A universal enrichment tool for interpreting omics data. *The Innovation* **2**: 100141.

852 Wu Z, Li T, Liu X, Yuan G, Hou H, Teng N. 2021b. A novel R2R3-MYB transcription factor L1MYB305 from Lilium longiflorum plays a positive role in thermotolerance via activating heat-protective genes. *Environmental and Experimental Botany* **184**: 104399.

855 Yang Z, Li Y, Gao F, Jin W, Li S, Kimani S, Yang S, Bao T, Gao X, Wang L. 2020. MYB21 interacts with MYC2 to control the expression of terpene synthase genes in flowers of Freesia hybrida and Arabidopsis thaliana. *Journal of Experimental Botany* **71**: 4140–4158.

858 Yarahmadov T, Robinson S, Hanemian M, Pulver V, Kuhlemeier C. 2020. Identification of transcription factors controlling floral morphology in wild Petunia species with contrasting pollination syndromes. *The Plant Journal* **104**: 289–301.

861 Zhang C, Dai Z, Ferrier T, Matus JT. 2021a. The grape MYB24 mediates the coordination of light-induced terpene and flavonol accumulation in response to berry anthocyanin sunscreen depletion. *Preprint bioRxiv*.

864 Zhang X, He Y, Li L, Liu H, Hong G. 2021b. Involvement of the R2R3-MYB transcription factor MYB21 and its homologs in regulating flavonol accumulation in Arabidopsis stamen. *Journal of Experimental Botany* **72**: 4319–4332.

867
868 SUPPORTING INFORMATION

869 The following Supporting information is available for this article.

870 **Fig. S1** Multiple sequence alignment of SG19 R2R3-MYB proteins.

871 **Fig. S2** Microsynteny analysis comparing genomic regions surrounding *EOB1* or *EOB2* in *P. axillaris* and *S. lycopersicum*.

873 **Fig. S3** Description of the different *Petunia axillaris* P floral developmental stages used in this study.

875 **Fig. S4** Construction of *eob1* and *eob2* mutant alleles by CRISPR-Cas9.

876 **Fig. S5** Observation of the internal floral organs of *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> flower buds.

877 **Fig. S6** The *eob2-3LofTAD* and *eob2-4LofTAD* are semi-dominant alleles.

878 **Fig. S7** *EOB1* and *EOB2* promoter sequences are highly divergent.

879 **Fig. S8** Additional phenotyping of *eob2-3*<sup>LofTAD</sup>.

880 **Fig. S9** Branches of WT, *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> before and after 1-MCP treatment.

881 **Fig. S10** *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> flowers after 1-MCP treatment, looked similar to *eob2-3*<sup>LofTAD</sup> and *eob2-4*<sup>LofTAD</sup>.

883 **Fig. S11** Percentage of genes with a 2kb promoter containing at least one putative R2R3-MYB SG19 binding site.

885 **Fig. S12** Model showing the direct activation of secondary metabolite related genes by EOB2.

886
887 **Table S1** Summary of protein-protein interactions involving members of the R2R3-MYB SG19 reported in the literature.

889 **Table S2** Summary of the direct target genes of members of the R2R3-MYB SG19 reported in the literature.

891 **Table S3** gRNA sequences used to target EOB1 and EOB2.

892 **Table S4** Sequence description of primers used for this work.

893 **Table S5** Description of the samples collected for the RNA sequencing experiment.

894 **Table S6** Description of the samples collected for the starch and carotenoid measurements.

895 **Table S7** From the T0 transgenic lines obtained by CRISPR-Cas9 to the final homozygous mutant lines used in this study.

897 **Table S8** Fraction of genes with a 2kb promoter containing at least one putative R2R3-MYB SG19 binding site.
**Table S9** Gene ID of genes used in this study.

**Method S1** Quantification of sesquiterpene accumulation in Petunia pistils and emission from tube by GC-MS.

**Method S2** Carotenoid extraction and quantification with an HPLC system.

**Method S3** Starch extraction and quantification with an HPLC system.

## FIGURE LEGENDS

**Fig. 1** Analysis of the R2R3-MYB Subgroup 19 (SG19) family complexity and focus on *P. axillaris* EOB1 and EOB2 members.

(a) Phylogenetic analysis of members of the SG19 R2R3-MYB in *Arabidopsis thaliana* (At), *Solanum lycopersicum* (Sl), *Nicotiana tabacum* (Nt), *Petunia hybrida* (Ph) and *Petunia axillaris* (Pax). Numbers at nodes represent bootstrap values and scale bar indicates the number of amino acid substitutions per site. Gene functions reported in the literature are indicated by black squares in the associated table (flower opening, flower senescence, stamen development, nectary development, ovule development, pistil length, flavonol, terpene and scent production). (b) Diagram describing the SG19 R2R3-MYB protein structure with domain organization. The R2R3-MYB domain (RMD<sup>SG19</sup>) is represented in red and the C-terminal motif

NyWS<sup>V</sup>/<sub>M</sub><sup>E</sup>/<sub>D</sub><sup>D</sup>/<sub>L</sub>W<sup>P</sup>/<sub>S</sub> which is a transcriptional activation domain (TAD) in blue. The RMD<sup>SG19</sup> is involved in DNA binding of specific target genes, protein-protein interactions (with repressor proteins such as DELLAs, JAZs or bHLHs) and contains a nuclear localization signal (NLS) (See Tables S1 and S2 for more details). The dashed line below the RMD<sup>SG19</sup> represents the position of the putative bipartite NLS. The R2R3-MYB domain binds DNA and the TAD activates the target genes. (c) Floral developmental stages, floral parts and pistil parts of *P. axillaris* used in this study. Scale bar = 1 cm. S1 to S10 = Stage 1 to Stage 10, more details about stages are available on Fig. S3. (d) RT-qPCR analysis of *EOB1* and *EOB2* expression in *P. axillaris* P. The different floral tissues have been dissected from open flower (S10). Stem = floral stem, BO = Basal ovary, AO = Apical ovary; D0-D4 = Number of days post anthesis. Relative expression values are means of three biological replicates with standard deviation, normalized against *ACT11* and *RAN1*. (e) RNA sequencing analysis of *EOB1* and *EOB2* expression in petal limb and basal ovaries (BO) at different stages (details are available in Table S5).
**Fig. 2 Generation of EOB2 domain-variants allowed to dissect and characterize EOB2 domain functions during P. axillaris flower development.**

(a) DNA sequences of wild-type *EOB1* and *EOB2*. Exons are shown as grey boxes, solid lines represent introns. Position of the double gRNAs used to target *EOB1* (gRNA1 and gRNA2) and *EOB2* (gRNA3 and gRNA4) are indicated. (b) Diagrams of EOB1 and EOB2 wild-type (WT) proteins illustrating the R2R3-MYB domain (RMD<sup>SG19</sup>) and the transcriptional activation domain (TAD) with their respective amino acid regions, aligned with predicted proteins obtained after CRISPR-Cas9 mutagenesis. Boxes with dashed lines represent unrelated amino acid sequences compared to the wild type. Left: allele names. Right: The DNA column describes the deletions obtained in base pairs (bp) and the protein column predicts the associated protein length in amino acids (aa) followed by the predicted protein activities according to the domain composition (*eob1-1*, *eob2-1*, *eob2-3*, *eob2-4*) or protein conformation (*eob2-2*). (c) Diagrams of protein conformation and domains composition of EOB1 and EOB2 wild-type and mutant alleles sorted by knockout (KO) and loss-of-TAD (LoFTAD) categories. (d) EOB2 and *eob2-2* protein structures prediction with AlphaFold, figure was drawn with PyMOL. (e) Pictures of WT, *eob1-1*<sup>KO</sup>, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LoF/TAD</sup> flower development over time. S2 flower buds were used at day 0. No visible phenotype between WT and *eob1-1*<sup>KO</sup>. Premature senescence observed in *eob2-1*<sup>KO</sup> (same phenotype for *eob2-2*<sup>KO</sup>) and juvenile flowers in *eob2-3*<sup>LoF/TAD</sup> (as well as *eob2-4*<sup>LoF/TAD</sup>). (f) Pictures of flower, ovary with nectary glands (NG) and stigma surface of WT, *eob1-1*<sup>KO</sup>, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LoF/TAD</sup> at stage 5 (S5) and stage 10 (S10). Before S5, no differences observed between the different genotypes. After reaching S5, *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> stop growing and rapidly enter senescence. At S10, *eob2-3*<sup>LoF/TAD</sup> petals did not fully open, stigma surface and nectary were dry and immature compared to the WT or *eob1-1*. (e) and (f) scale bars are 1 cm for flowers and 1 mm for ovaries and stigmas.

**Fig. 3 EOB2 is a regulator of EOB1.**

(a) Flower growth measurements of WT and crispr mutants from around 1 cm buds until senescence. Ten flowers per genotype were used for the analysis: WT, *eob1-1*<sup>KO</sup>, *eob2-1*<sup>KO</sup>, *eob2-2*<sup>KO</sup>, and *eob1-1*<sup>KO</sup> *eob2-2*<sup>KO</sup> double mutant. The black arrows indicate the first signs of senescence. The right panel represents the percentage of open flowers 16 weeks after germination (n = 100). (b) PTR-MS measurements for the major scent compounds, benzaldehyde and methylbenzoate, performed just before dark on 1 DPA flowers from WT and *eob1-1*<sup>KO</sup> plants (n =
15; error bar = SD; significant differences *, P<0.05; Student's t-test). (c) RT-qPCR analysis of *ODO1* and *EOB2* expression in WT and *eob1-1*<sup>KO</sup> limb at S10, 1DPA, just before dark (n = 3; error bar = SD. (d) RNA sequencing analysis of *EOB1* expression in WT and *eob2-1*<sup>KO</sup> S5 petal limb (n = 3; error bar = SD; significant differences *, P<0.05; Student's t-test). (e) Schematic representation of a Petunia plant showing the extraction of three leaf-disks in the infiltration area. RT-qPCR analysis of *EOB1*, *EOB2* and *IGS* normalized expression in *Petunia axillaris* P leaves, 3 days after agroinfiltration with transient expression constructs (n = 4; error bar = SD; significant differences *, P<0.05; Student's t-test). *P35S::GFP* represents the negative control. (f) Illustration showing a possible interaction between *EOB2* and the promoter of *EOB1*. *EOB1* and *EOB2* promoter sequence analysis (1000bp before the ATG) revealed the presence of a putative SG19 MYB-binding site 450bp before the START codon of *EOB1*.

Fig. 4 Different organs impacted in their maturation in *eob2-3*<sup>LoftAD</sup> mutants.

(a) Pictures of WT (top row) and *eob2-3*<sup>LoftAD</sup> (bottom row) flowers at stage 10 (S10). Side view, top view, bottom view, stigma surface, stigma surface 24h after pollination of freshly opened flowers and close up of the nectary gland. Scale bar is 1 cm. (b) Pistil pictures of WT and *eob2-3*<sup>LoftAD</sup>, 5 days after S3. Left: no petal removal (S10). Right: with petal removal performed at around S3 (w/o). Twisted styles in *eob2-3*<sup>LoftAD</sup> are due to petal mechanical constraints. (c) Capsules one month after pollination. The mean number of seeds per capsule is indicated (n = 6). No seeds in the *eob2-3*<sup>LoftAD</sup> capsule without stigma exudate. (d) Side view of ovaries with carpel wall removed, comparable number of ovules was observed. (e) Two weeks after pollination the style detached from the fruit/capsule of WT while persisting in *eob2-3*<sup>LoftAD</sup>. (f) to (m) WT is represented in white and *eob2-3*<sup>LoftAD</sup> in dark grey (error bar = SD, except (k) and (l) where error bar = SE; significant differences *, P<0.05; Student's t-test). (f) Petal morphological measurements at S10: petal tube length, petal opening angle and petal limb area (n = 12). (g) Nectar volume measurements at S10 (n = 20). (h) Carotenoid content from nectary at S10. (i) PTR-MS measurements of the two main floral volatiles benzenoid/phenylpropanoid (FVBP) (n = 15). (j) RNA-seq of *ODO1* and *EOB1* expression in WT and *eob2-3*<sup>LoftAD</sup> limb at S10. (k) Terpene volatiles (*TPS1* products) emitted from WT and *eob2-3*<sup>LoftAD</sup> petal tubes (n = 5). (l) Terpene volatiles accumulated on stigmas during anthesis (S9) (n = 4). (m) RT-qPCR of *TPS1* from petal tube at S10.
<caption>**Fig. 5** Premature production of ethylene by flower buds is responsible for the knockout flower senescence phenotype.</caption>

(a) RNA-seq experiment summary. Right: White triangles show the petal limb tissue part from S5 buds collected for the RNA-seq experiment (just before the first signs of senescence for *eob2-1*<sup>KO</sup>). Middle: pie charts showing the proportion of DEGs in the *P. axillaris* transcriptome (total of 32768 genes). Significant DEGs = log2FC >1 or <-1 and p-adjusted value < 0.01. Left: Comparison of S5 limb total DEGs from *eob2-1*<sup>KO</sup> vs WT and *eob2-3*<sup>LoftAD</sup> vs WT. (b) Gene ontology (GO) term enrichment analysis of specific up-regulated genes in *eob2-1*<sup>KO</sup>. (c) RNA-seq results of genes belonging to ACS and ACO multi-gene families encoding for enzymes of the ethylene biosynthetic pathway. Only genes from the family expressed in either WT or *eob2-1* are shown (ACS 8/22 and ACO 6/13) (see **Table S9** for gene ID). Error bar = SD. (d) Detection of ethylene released from WT and *eob2-1*<sup>KO</sup> S5 full flower buds, S5 dissected petal or S5 dissected pistil, quantified using a laser-based photoacoustic ethylene sensor (n = 30 for WT and n = 45 for *eob2-1*<sup>KO</sup>; error bar = SE; significant differences *, P<0.05; Student's t-test). Mutant samples were collected before the first signs of senescence, when the petal tips were still rigid. (e) Side view of flowers of WT, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LoftAD</sup> with or without 1-MCP treatment. Partial rescue of the *eob2-1*<sup>KO</sup> phenotype was observed. Scale bars = 1 cm.

<caption>**Fig. 6** EOB2 activates the expression of common targets in two different floral organs.</caption>

(a) RNA-seq experiment summary. Top: limb and basal ovary tissues collected just before dark at S5 and S10 for the RNA-seq experiment. Bottom: Number of DEGs between *eob2-3*<sup>LoftAD</sup> and WT. (b) The graph corresponds to the difference in fraction of genes (up vs all / down vs all) with a 2kb promoter containing at least one putative R2R3-MYB SG19 binding site (matrix MA1408.1 (*FaEOBII*)). Similar results were obtained with MA1037.1 (*AtMYB24*) (**Table S8**). (c) Summary of overlaps between down-regulated genes (*eob2-3*<sup>LoftAD</sup><sub>WT</sub>) in limb and nectary at stages S5 and S10. (d) Table showing the expression data in limb and nectary at S10 of a selection of 11 genes over a total of 60 (from **Fig. 6b**). n.r.c., normalized read counts, L2FC, Log2 fold change. The presence of a R2R3-MYB SG19 binding site in 2kb promoter was predicted using FIMO. Motifs with grey nucleotides should also be recognized by SG19 proteins. Emission Of Benzenoids 1 (EOB1), Chorismate Mutase 1 (CM1), Benzoic acid/Salicylic acid
carboxyl MethylTransferase (BSMT), Eugenol Synthase (EGS), Isoeugenol Synthase (IGS).
Carotenoid related: ζ-carotene isomerase (Z-ISO), ζ-carotene desaturase (ZDS), Lycopene Beta
Cyclase (LCY B), Carotenoid cleavage dioxygenase (CCD). See **Table S9** for gene ID.

**Fig. 7 Carbohydrate metabolism is disrupted in *eob2-3<sup>LofTAD</sup>* nectary and limb.**

(a) GO terms from down-regulated genes co-enriched in *eob2-3<sup>LofTAD</sup>* limb and nectary at S5 and S10 (pvalue<0.005).

(b) RNA-seq result of *BETA-AMYLASE (BAM)* in nectary and limb at S10.

See **Table S9** for gene ID.

(c) Simplified representation of the carbohydrate metabolism pathway and the derived primary and secondary metabolites (blue). PYR: pyruvate, PEP: phosphoenolpyruvate, E4P: erythrose-4-phosphate, Rubisco: ribulose-1,5-bisphosphate carboxylase/oxygenase and TCA cycle: tricarboxylic acid cycle, also known as the Krebs cycle.

(d) and (e) Visualization of starch in nectaries and cleared petal (S5 and S10) by staining with Lugol's solution.

(f) Adaxial epidermal cell peeling from petal limb at S10. Left: Lugol's staining. Right: differential interference contrast (DIC) observation. Arrows indicate the starch granules inside the limb epidermal cells. Scale bar is 30 µm. Basal epidermal cell area (µm²) of petal limb at S10 (n = 90 cells; error bar = SD; significant differences *, P<0.05; Student's t-test).

(g) Starch content in WT and *eob2-3<sup>LofTAD</sup>* nectary and limb tissues at different stages. The black arrow indicates the tendency towards an accumulation of starch in *eob2-3<sup>LofTAD</sup>* limb S10 compared to the WT (n = 3; error bar = SD; significant differences *, P<0.05; ns, non-significance; Student's t-test).

**Fig. 8 One gene, two mutations, contrasting phenotypes.**

Model to illustrate EOB2 multi-functionality. The knockout (KO) lines and LofTAD lines do not present the same floral phenotype, while both lines are unable to activate target genes. This is the proof that EOB2 is involved in other functions. Direct targets are most likely related to maturation-related genes like the secondary metabolism related genes, while indirect targets are probably senescence-related proteins/genes.
<figure><img src="image_3.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_4.png" /></figure>

Petunia axillaris P

<figure><img src="image_5.png" /></figure>

Floral parts

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>(e)</figcaption></figure>

<figure><img src="image_14.png" /></figure>
**Fig. 1 Analysis of the R2R3-MYB Subgroup 19 (SG19) family complexity and focus on *P. axillaris* EOB1 and EOB2 members.**

(a) Phylogenetic analysis of members of the SG19 R2R3-MYB in *Arabidopsis thaliana* (At), *Solanum lycopersicum* (S1), *Nicotiana tabacum* (Nt), *Petunia hybrida* (Ph) and *Petunia axillaris* (Pax). Numbers at nodes represent bootstrap values and scale bar indicates the number of amino acid substitutions per site. Gene functions reported in the literature are indicated by black squares in the associated table (flower opening, flower senescence, stamen development, nectary development, ovule development, pistil length, flavonol, terpene and scent production).

(b) Diagram describing the SG19 R2R3-MYB protein structure with domain organization. The R2R3-MYB domain (RMD<sup>SG19</sup>) is represented in red and the C-terminal motif NyWSV<sub>M</sub><sup>E</sup>/<sub>D</sub>D<sup>I</sup>/<sub>L</sub>W<sup>P</sup>/<sub>S</sub> which is a transcriptional activation domain (TAD) in blue. The RMD<sup>SG19</sup> is involved in DNA binding of specific target genes, protein-protein interactions (with repressor proteins such as DELLAs, JAZs or bHLHs) and contains a nuclear localization signal (NLS) (See Tables S1 and S2 for more details). The dashed line below the RMD<sup>SG19</sup> represents the position of the putative bipartite NLS. The R2R3-MYB domain binds DNA and the TAD activates the target genes. (c) Floral developmental stages, floral parts and pistil parts of *P. axillaris* used in this study. Scale bar = 1 cm. S1 to S10 = Stage 1 to Stage 10, more details about stages are available on Fig. S3. (d) RT-qPCR analysis of *EOB1* and *EOB2* expression in *P. axillaris* P. The different floral tissues have been dissected from open flower (S10). Stem = floral stem, BO = Basal ovary, AO = Apical ovary; D0-D4 = Number of days post anthesis. Relative expression values are means of three biological replicates with standard deviation, normalized against *ACT11* and *RAN1*. (e) RNA sequencing analysis of *EOB1* and *EOB2* expression in petal limb and basal ovaries (BO) at different stages (details are available in **Table S5**).
<figure><img src="image_15.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>(c)</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>(d)</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>(e)</figcaption></figure>

<figure><img src="image_19.png" /><figcaption>(eob2-1KO)
(or eob2-2KO)</figcaption></figure>

<caption>(eob1-1KO)</caption>

<caption>WT</caption>
Fig. 2 Generation of EOB2 domain-variants allowed to dissect and characterize EOB2 domain functions during *P. axillaris* flower development.

(a) DNA sequences of wild-type *EOB1* and *EOB2*. Exons are shown as grey boxes, solid lines represent introns. Position of the double gRNAs used to target *EOB1* (gRNA1 and gRNA2) and *EOB2* (gRNA3 and gRNA4) are indicated. (b) Diagrams of EOB1 and EOB2 wild-type (WT) proteins illustrating the R2R3-MYB domain (RMD<sup>SG19</sup>) and the transcriptional activation domain (TAD) with their respective amino acid regions, aligned with predicted proteins obtained after CRISPR-Cas9 mutagenesis. Boxes with dashed lines represent unrelated amino acid sequences compared to the wild type. Left: allele names. Right: The DNA column describes the deletions obtained in base pairs (bp) and the protein column predicts the associated protein length in amino acids (aa) followed by the predicted protein activities according to the domain composition (*eob1-1*, *eob2-1*, *eob2-3*, *eob2-4*) or protein conformation (*eob2-2*). (c) Diagrams of protein conformation and domains composition of EOB1 and EOB2 wild-type and mutant alleles sorted by knockout (KO) and loss-of-TAD (LofTAD) categories. (d) EOB2 and *eob2-2* protein structures prediction with AlphaFold, figure was drawn with PyMOL. (e) Pictures of WT, *eob1-1*<sup>KO</sup>, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LofTAD</sup> flower development over time. S2 flower buds were used at day 0. No visible phenotype between WT and *eob1-1*<sup>KO</sup>. Premature senescence observed in *eob2-1*<sup>KO</sup> (same phenotype for *eob2-2*<sup>KO</sup>) and juvenile flowers in *eob2-3*<sup>LofTAD</sup> (as well as *eob2-4*<sup>LofTAD</sup>). (f) Pictures of flower, ovary with nectary glands (NG) and stigma surface of WT, *eob1-1*<sup>KO</sup>, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LofTAD</sup> at stage 5 (S5) and stage 10 (S10). Before S5, no differences observed between the different genotypes. After reaching S5, *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> stop growing and rapidly enter senescence. At S10, *eob2-3*<sup>LofTAD</sup> petals did not fully open, stigma surface and nectary were dry and immature compared to the WT or *eob1-1*. (e) and (f) scale bars are 1 cm for flowers and 1 mm for ovaries and stigmas.
<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /></figure>

<figure><img src="image_25.png" /></figure>

<figure><img src="image_26.png" /></figure>

<figure><img src="image_27.png" /></figure>

<figure><img src="image_28.png" /><figcaption>Fig. 3 EOB2 is a regulator of EOB1.</figcaption></figure>

(a) Flower growth measurements of WT and crispr mutants from around 1 cm buds until senescence. Ten flowers per genotype were used for the analysis: WT, *eob1-1*<sup>KO</sup>, *eob2-1*<sup>KO</sup>, *eob2-2*<sup>KO</sup>, and *eob1-1*<sup>KO</sup> *eob2-2*<sup>KO</sup> double mutant. The black arrows indicate the first signs of senescence. The right panel represents the percentage of open flowers 16 weeks after germination (n = 100). (b) PTR-MS measurements for the major scent compounds, benzaldehyde and methylbenzoate, performed just before dark on 1 DPA flowers from WT and *eob1-1*<sup>KO</sup> plants (n = 15; error bar = SD; significant differences *, P<0.05; Student's t-test). (c) RT-qPCR analysis of *ODO1* and *EOB2* expression in WT and *eob1-1*<sup>KO</sup> limb at S10, 1DPA, just before dark (n = 3; error bar = SD. (d) RNA sequencing analysis of *EOB1* expression in WT and *eob2-1*<sup>KO</sup> S5 petal limb (n = 3; error bar = SD; significant differences *, P<0.05; Student's t-test). (e) Schematic representation of a Petunia plant showing the extraction of three leaf-disks in the infiltration area. RT-qPCR analysis of *EOB1*, *EOB2* and *IGS* normalized expression in *Petunia axillaris* P leaves, 3 days after agroinfiltration with transient expression constructs (n = 4; error bar = SD; significant differences *, P<0.05; Student's t-test). *P35S::GFP* represents the negative control. (f) Illustration showing a possible interaction between EOB2 and the promoter of *EOB1*, *EOB1* and *EOB2* promoter sequence analysis (1000bp before the ATG) revealed the presence of a putative SG19 MYB-binding site 450bp before the START codon of *EOB1*.
<figure><img src="image_29.png" /><figcaption>(a)</figcaption></figure>

WT

eob2-3 LoFTAD

<figure><img src="image_30.png" /><figcaption>(b)</figcaption></figure>

<figure><img src="image_31.png" /></figure>

WT

eob2-3

LoFTAD

<figure><img src="image_32.png" /></figure>

WT
eob2-3
LoFTAD

<figure><img src="image_33.png" /><figcaption>(c)</figcaption></figure>

~570
seeds

no
seeds

~238
seeds

LoFTAD

<figure><img src="image_34.png" /><figcaption>(d)</figcaption></figure>

WT

eob2-3

LoFTAD

<figure><img src="image_35.png" /><figcaption>(e)</figcaption></figure>

WT

eob2-3

LoFTAD

<figure><img src="image_36.png" /><figcaption>(f)</figcaption></figure>

w/o petal

<figure><img src="image_37.png" /><figcaption>(h)</figcaption></figure>

<figure><img src="image_38.png" /></figure>

<figure><img src="image_39.png" /></figure>

FVBP

<figure><img src="image_40.png" /></figure>

Benzaldehyde
Methylbenzoate

<figure><img src="image_41.png" /></figure>

ODO1 EOB1

petal
limb S10

<figure><img src="image_42.png" /></figure>

Germacrene D
Bicyclogermacrene
β-cadinene

Germacrene D-4-ol

<figure><img src="image_43.png" /></figure>

Terpene volatiles

stigma

<figure><img src="image_44.png" /></figure>

TPS1
petal
tube S10
Fig. 4 Different organs impacted in their maturation in *eob2-3*<sup>LofTAD</sup> mutants.

(a) Pictures of WT (top row) and *eob2-3*<sup>LofTAD</sup> (bottom row) flowers at stage 10 (S10). Side view, top view, bottom view, stigma surface, stigma surface 24h after pollination of freshly opened flowers and close up of the nectary gland. Scale bar is 1 cm. (b) Pistil pictures of WT and *eob2-3*<sup>LofTAD</sup>, 5 days after S3. Left: no petal removal (S10). Right: with petal removal performed at around S3 (w/o). Twisted styles in *eob2-3*<sup>LofTAD</sup> are due to petal mechanical constraints. (c) Capsules one month after pollination. The mean number of seeds per capsule is indicated (n = 6). No seeds in the *eob2-3*<sup>LofTAD</sup> capsule without stigma exudate. (d) Side view of ovaries with carpel wall removed, comparable number of ovules was observed. (e) Two weeks after pollination the style detached from the fruit/capsule of WT while persisting in *eob2-3*<sup>LofTAD</sup>. (f) to (m) WT is represented in white and *eob2-3*<sup>LofTAD</sup> in dark grey (error bar = SD, except (k) and (l) where error bar = SE; significant differences *, P<0.05; Student's t-test).

(f) Petal morphological measurements at S10: petal tube length, petal opening angle and petal limb area (n = 12). (g) Nectar volume measurements at S10 (n = 20). (h) Carotenoid content from nectary at S10. (i) PTR-MS measurements of the two main floral volatiles benzenoid/phenylpropanoid (FVBP) (n = 15). (j) RNA-seq of *ODO1* and *EOB1* expression in WT and *eob2-3*<sup>LofTAD</sup> limb at S10. (k) Terpene volatiles (*TPS1* products) emitted from WT and *eob2-3*<sup>LofTAD</sup> petal tubes (n = 5). (l) Terpene volatiles accumulated on stigmas during anthesis (S9) (n = 4). (m) RT-qPCR of *TPS1* from petal tube at S10.
<figure><img src="image_45.png" /></figure>
**Fig. 5** Premature production of ethylene by flower buds is responsible for the knockout flower senescence phenotype.

(a) RNA-seq experiment summary. Right: White triangles show the petal limb tissue part from S5 buds collected for the RNA-seq experiment (just before the first signs of senescence for *eob2-1*<sup>KO</sup>). Middle: pie charts showing the proportion of DEGs in the *P. axillaris* transcriptome (total of 32768 genes). Significant DEGs = log2FC >1 or < -1 and p-adjusted value < 0.01. Left: Comparison of S5 limb total DEGs from *eob2-1*<sup>KO</sup> vs WT and *eob2-3*<sup>LofTAD</sup> vs WT. (b) Gene ontology (GO) term enrichment analysis of specific up-regulated genes in *eob2-1*<sup>KO</sup>. (c) RNA-seq results of genes belonging to ACS and ACO multi-gene families encoding for enzymes of the ethylene biosynthetic pathway. Only genes from the family expressed in either WT or *eob2-1* are shown (ACS 8/22 and ACO 6/13) (see **Table S9** for gene ID). Error bar = SD. (d) Detection of ethylene released from WT and *eob2-1*<sup>KO</sup> S5 full flower buds, S5 dissected petal or S5 dissected pistil, quantified using a laser-based photoacoustic ethylene sensor (n = 30 for WT and n = 45 for *eob2-1*<sup>KO</sup>; error bar = SE; significant differences *, P<0.05; Student's t-test). Mutant samples were collected before the first signs of senescence, when the petal tips were still rigid. (e) Side view of flowers of WT, *eob2-1*<sup>KO</sup> and *eob2-3*<sup>LofTAD</sup> with or without 1-MCP treatment. Partial rescue of the *eob2-1*<sup>KO</sup> phenotype was observed. Scale bars = 1 cm.
<figure><img src="image_46.png" /><figcaption>**Fig. 6 EOB2 activates the expression of common targets in two different floral organs.**</figcaption></figure>

(a) RNA-seq experiment summary. Top: limb and basal ovary tissues collected just before dark at S5 and S10 for the RNA-seq experiment. Bottom: Number of DEGs between *eob2-3*<sup>LoTAD</sup> and WT. (b) The graph corresponds to the difference in fraction of genes (up vs all / down vs all) with a 2kb promoter containing at least one putative R2R3-MYB SG19 binding site (matrix MA1408.1 (*FaEOBII*)). Similar results were obtained with MA1037.1 (*AtMYB24*) (**Table S8**).

(c) Summary of overlaps between down-regulated genes (*eob2-3*<sup>LoTAD</sup><*WT*) in limb and nectary at stages S5 and S10. (d) Table showing the expression data in limb and nectary at S10 of a selection of 11 genes over a total of 60 (from **Fig. 6b**). n.r.c., normalized read counts, L2FC, Log2 fold change. The presence of a R2R3-MYB SG19 binding site in 2kb promoter was predicted using FIMO. Motifs with grey nucleotides should also be recognized by SG19 proteins. Emission Of Benzenoids 1 (EOB1), Chorismate Mutase 1 (CM1), Benzoic acid/Salicylic acid carboxyl MethylTransferase (BSMT), Eugenol Synthase (EGS), Isoeugenol Synthase (IGS). Carotenoid related: ζ-carotene isomerase (Z-ISO), ζ-carotene desaturase (ZDS), Lycopene Beta Cyclase (LCY B), Carotenoid cleavage dioxygenase (CCD). See **Table S9** for gene ID.
<figure><img src="image_47.png" /><figcaption>Fig. 7 Carbohydrate metabolism is disrupted in *eob2-3*<sup>LoFTAD</sup> nectary and limb.</figcaption></figure>

(a) GO terms from down-regulated genes co-enriched in *eob2-3*<sup>LoFTAD</sup> limb and nectary at S5 and S10 (pvalue<0.005). (b) RNA-seq result of *BETA-AMYLASE* (*BAM*) in nectary and limb at S10. See **Table S9** for gene ID. (c) Simplified representation of the carbohydrate metabolism pathway and the derived primary and secondary metabolites (blue). PYR: pyruvate, PEP: phosphoenolpyruvate, E4P: erythrose-4-phosphate, Rubisco: ribulose-1,5-bisphosphate carboxylase/oxygenase and TCA cycle: tricarboxylic acid cycle, also known as the Krebs cycle. (d) and (e) Visualization of starch in nectaries and cleared petal (S5 and S10) by staining with Lugol's solution. (f) Adaxial epidermal cell peeling from petal limb at S10. Left: Lugol's staining. Right: differential interference contrast (DIC) observation. Arrows indicate the starch granules inside the limb epidermal cells. Scale bar is 30 µm. Basal epidermal cell area (µm²) of petal limb at S10 (n = 90 cells; error bar = SD; significant differences *, P<0.05; Student's t-test). (g) Starch content in WT and *eob2-3*<sup>LoFTAD</sup> nectary and limb tissues at different stages. The black arrow indicates the tendency towards an accumulation of starch in *eob2-3*<sup>LoFTAD</sup> limb S10 compared to the WT (n = 3; error bar = SD; significant differences *, P<0.05; ns, non-significance; Student's t-test).
<figure><img src="image_48.png" /><figcaption>**Fig. 8** One gene, two mutations, contrasting phenotypes.</figcaption></figure>

Model to illustrate EOB2 multi-functionality. The knockout (KO) lines and LofTAD lines do not present the same floral phenotype, while both lines are unable to activate target genes. This is the proof that EOB2 is involved in other functions. Direct targets are most likely related to maturation-related genes like the secondary metabolism related genes, while indirect targets are probably senescence-related proteins/genes.
New Phytologist Supporting Information

**Article title:** A single MYB transcription factor with multiple functions during flower development

**Authors:** Mathilde Chopy, Marta Binaghi, Gina Cannarozzi, Rayko Halitschke, Benoît Boachon, Roel Heutink, Dikki Pedenla Bomzan, Lea Jäggi, Geert van Geest, Julian C. Verdonk and Cris Kuhlemeier

**Article acceptance date:** 22-May-2023
<figure><img src="image_49.png" /><figcaption>Fig. S1 Multiple sequence alignment of SG19 R2R3-MYB proteins.</figcaption></figure>

Sequence analysis of SG19 R2R3-MYB transcription factors from *Arabidopsis thaliana* (At) and different Solanaceae species: *Petunia axillaris* (Pa), *Petunia hybrida* (Ph), *Solanum lycopersicum* (Sl) and *Nicotiana tabacum* (Nt). The conserved R2R3 MYB-DNA-binding domain is highlighted in red (R2 in light red, R3 in dark red) and the C-terminal motif NyWSV/M E/D D/L WP/S which is a transcriptional activation domain (TAD) is highlighted in blue. The amino acid (AA) differences observed in the TAD most likely have no impact on the proteins transcriptional activation due to conservative substitutions (M/Y/V/I/L are hydrophobics, E/D are negatively charged) (Figure 5 from Liu *et al.*, 2009). The putative bHLH binding motif and bipartite NLS are framed by an orange or purple rectangle respectively.
<figure><img src="image_50.png" /><figcaption>Fig. S2 Microsynteny analysis comparing genomic regions surrounding *EOB1* or *EOB2* in *P. axillaris* and *S. lycopersicum*.</figcaption></figure>

(a) The microsynteny analysis was performed using GEvo from CoGe. The SG19 MYB's genomic locations are indicated in the left part. Rectangles indicate SG19 MYB genes, circles indicate the surrounding genes and lines indicate gene-poor regions.

Syntenic genes are linked by dashed lines. Left: Genomic region surrounding *PaEOB1* (Peaxi162Scf00129g01231.1) compared with *SIMYB24* (Solyc02g086690.2). Right: Genomic region surrounding *PaEOB2* (Peaxi162Scf00080g00064.1) compared with *SIMYB21* (Solyc02g067760.2). No clear synteny was observed comparing *P. axillaris* and *A. thaliana*.

Concerning *N. tabacum*, the genome assembly quality did not allow to perform the analysis. (b) Gene ID and coordinates of the genes used in the microsynteny analysis.
<figure><img src="image_51.png" /><figcaption>Fig. S3 Description of the different *Petunia axillaris* P floral developmental stages used in this study.</figcaption></figure>

(a) Table describing the different floral developmental stages used in this study over time, S1 to S10 = Stage 1 to Stage 10. (b) Pictures of the floral developmental stages of *P. axillaris* P. Scale bar = 1 cm. (c) Overview of the stigma surface and the nectary gland over the different developmental stages. The maturation phase of the stigma and nectary gland start at S5. The stigma secretes exudate after S5 and from S8 the stigma surface is covered by exudate droplets resulting in a wet stigma surface. The nectary gland starts to turn orange after S5 due to an accumulation of carotenoids and this process is accompanied by progressive nectar secretion, visible from S6 in the picture series. A metabolic switch may happen in nectary glands and stigma after stage 5.
<figure><img src="image_52.png" /><figcaption>Fig. S4 Construction of *eob1* and *eob2* mutant alleles by CRISPR-Cas9.</figcaption></figure>

(a) *eob1-1* allele generated by CRISPR-Cas9. (b) *eob2-1* to 4 alleles generated by CRISPR-Cas9. Allele *eob2-1* consists of a 364bp deletion, leading to the removal of the last intron-exon junction affecting the normal splicing process. The primer pairs used for the genotyping (*EOB1-pp1* and *EOB2-pp1*) and RT-qPCR (*EOB1-pp2* and *EOB2-pp2*) are indicated. (c) Left: RNA sequencing analysis of *EOB2* expression in WT, *eob2-1* and *eob2-3* S5 petal limb (error bar = SD; significant differences *, P<0.05; ns, non-significance; Student's t-test). Right: IGV software was used to construct Sashimi plots depicting splice junctions for the *EOB2* gene, from aligned RNA-seq data derived from S5 limb tissue. The library sizes (13 – 15 M for *eob2-1* and 25 – 60 M for WT) can explain the difference between the number of counts before and after normalization. But overall *EOB2* is lower compared to the WT.
<figure><img src="image_53.png" /><figcaption>**Fig. S5** Observation of the internal floral organs of *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> flower buds.
The dissected flower buds revealed that styles are twisted, nectary are not mature (yellow) and stamens can released fertile
pollen several days after senescence.</figcaption></figure>
<figure><img src="image_54.png" /></figure>

**Fig. S6** The *eob2-3*<sup>LoTTAD</sup> and *eob2-4*<sup>LoTTAD</sup> are semi-dominant alleles.

(a) Phenotype of the heterozygous mutant flowers. *eob2-3*<sup>LoTTAD/+</sup> or *eob2-4*<sup>LoTTAD/+</sup> displayed an intermediate phenotype between the WT and homozygous mutants. *eob2-1*<sup>KO/+</sup> or *eob2-2*<sup>KO/+</sup> resembled the WT. (b) Nectar measurements (n = 20; error bar = SD). (c) Methylbenzoate measurements using a PTR-MS (n = 15; error bar = SD).
<figure><img src="image_55.png" /><figcaption>Fig. S7 EOB1 and EOB2 promoter sequences are highly divergent.</figcaption></figure>

(a) *Petunia axillaris* EOB1 and EOB2 promoter sequences (2kb). The defined "SG19 MYB-binding site" in the EOB1 promoter is underlined. (b) percent identity matrix created by Clustal2.1.
<figure><img src="image_56.png" /><figcaption>**Fig. S8** Additional phenotyping of *eob2-3*<sup>LOFTAD</sup>. Visible and UV pictures of WT and *eob2-3*<sup>LOFTAD</sup>. All flowers contain UV-absorbing pigments.</figcaption></figure>
<figure><img src="image_57.png" /><figcaption>**Fig. S9** Branches of WT, *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> before and after 1-MCP treatment. (a) Phenotypes of untreated WT, *eob2-1*<sup>KO</sup>, *eob2-2*<sup>KO</sup>, and the double mutant *eob1-1*<sup>KO</sup> *eob2-2*<sup>KO</sup> branches. (b) and (c) Partial rescue of the *eob2-1*<sup>KO</sup> (b) and *eob2-2*<sup>KO</sup> (c) phenotypes on 3 independent plant branches after 1-MCP treatment. Scale bars = 2cm.</figcaption></figure>
<figure><img src="image_58.png" /><figcaption>**Fig. S10** *eob2-1*<sup>KO</sup> and *eob2-2*<sup>KO</sup> flowers after 1-MCP treatment, looked similar to *eob2-3*<sup>LoftAD</sup> and *eob2-4*<sup>LoftAD</sup>, **(a)** Phenotype of untreated WT, *eob2-3*<sup>LoftAD</sup> and *eob2-4*<sup>LoftAD</sup> flowers. **(b)** and **(c)** Partial rescue of the *eob2-1*<sup>KO</sup> (**b**) and *eob2-2*<sup>KO</sup> (**c**) phenotypes on 4 independent flowers after 1-MCP treatment. Scale bars = 1cm.</figcaption></figure>
(a) MA1037.1 (AtMYB24)

<table><thead><tr><th></th><th>WT vs eob2-3 limb S5</th><th>WT vs eob2-3 nec S5</th><th>WT vs eob2-3 limb S10</th><th>WT vs eob2-3 nec S10</th><th>WT vs eob2-3 common to the 4 conditions</th><th>WT vs eob2-1 limb S5</th></tr></thead><tbody><tr><td>UP-regulated eob2-3<sup>LoITAD</sup>>WT</td><td>19.6%<br>212 over 1084</td><td>18.1%<br>179 over 989</td><td>21.1%<br>686 over 3255</td><td>19.2%<br>485 over 2523</td><td>18.3%<br>22 over 120 ▲</td><td>21%<br>944 over 4485 +</td></tr><tr><td>DOWN-regulated eob2-3<sup>LoITAD</sup><<WT</td><td>39.3%<br>153 over 389</td><td>35%<br>292 over 834</td><td>30.5%<br>501 over 1645</td><td>28.5%<br>529 over 1854</td><td>48.3%<br>29 over 60 ▲</td><td>27.4%<br>867 over 3162 +</td></tr><tr><td>common not DEGs<br>padj>0.01; -1&lt;log2FC&lt;1</td><td></td><td></td><td>22.9%<br>1267 over 5522</td><td></td><td>not DEGs<br>padj>0.01; -1&lt;log2FC&lt;1 ▲</td><td>22.9%<br>1393 over 6085 +</td></tr><tr><td>total in the Pax genome</td><td></td><td></td><td>23%<br>7517 over 32768 ▲</td><td></td><td>total in the Pax genome</td><td>23%<br>7517 over 32768 +</td></tr></tbody></table>

(b) MA1408.1 (FaEOBII)

<table><thead><tr><th></th><th>WT vs eob2-3 limb S5</th><th>WT vs eob2-3 nec S5</th><th>WT vs eob2-3 limb S10</th><th>WT vs eob2-3 nec S10</th><th>WT vs eob2-3 common to the 4 conditions</th><th>WT vs eob2-1 limb S5</th></tr></thead><tbody><tr><td>UP-regulated eob2-3<sup>LoITAD</sup>>WT</td><td>28.5%<br>309 over 1084</td><td>26.3%<br>260 over 989</td><td>30.9%<br>1005 over 3255</td><td>26.8%<br>677 over 2523</td><td>26.7%<br>32 over 120 ○</td><td>30.9%<br>1387 over 4485 ◇</td></tr><tr><td>DOWN-regulated eob2-3<sup>LoITAD</sup><<WT</td><td>40.6%<br>158 over 389</td><td>44.6%<br>372 over 834</td><td>39.4%<br>648 over 1645</td><td>39.6%<br>734 over 1854</td><td>53.3%<br>32 over 60 ○</td><td>36.7%<br>1162 over 3162 ◇</td></tr><tr><td>common not DEGs<br>padj>0.01; -1&lt;log2FC&lt;1</td><td></td><td></td><td>32%<br>1767 over 5522</td><td></td><td>not DEGs<br>padj>0.01; -1&lt;log2FC&lt;1 ○</td><td>32.3%<br>1963 over 6085 ◇</td></tr><tr><td>total in the Pax genome</td><td></td><td></td><td>31.9%<br>10439 over 32768 ○</td><td></td><td>total in the Pax genome</td><td>31.9%<br>10439 over 32768 ◇</td></tr></tbody></table>

(c)

<figure><img src="image_59.png" /></figure>

(d)

<figure><img src="image_60.png" /><figcaption>**Fig. S11** Percentage of genes with a 2kb promoter containing at least one putative R2R3-MYB SG19 binding site. The presence of a R2R3-MYB SG19 binding site in 2kb promoter was predicted using FIMO and two independent SG19 predicted motifs (a) MA1037.1 (AtMYB24) and (b) MA1408.1 (FaEOBII) (P < 1e-4). The symbols indicate the percentage values used in (c) WT vs eob2-3<sup>LoITAD</sup> and (d) WT vs eob2-1<sup>KO</sup>.</figcaption></figure>
<figure><img src="image_61.png" /><figcaption>**Fig. S12** Model showing the direct activation of secondary metabolite related genes by EOB2.</figcaption></figure>

The promoter of secondary metabolite related genes is represented in white. The reduced expression of these genes in *eob2-3*<sup>LOFTAD</sup> limb and nectary tissues is most likely due to the missing TAD. Both EOB2 and *eob2-3*<sup>LOFTAD</sup> protein can bind the MYB-DNA-binding site ([G/A]TT[A/T]GG[T/C]) but only EOB2 can activate the transcription.
<table><thead><tr><th>Organism</th><th>Protein-protein interactions</th><th>Methods</th><th>N- or C-terminus part interaction</th><th>Transcriptional function influence</th><th>Publication</th></tr></thead><tbody><tr><td rowspan="6">Arabidopsis thaliana</td><td>JAZs* - AtMYB21<br>JAZs* - AtMYB24<br>AtMYB21 - AtMYB21<br>AtMYB24 - AtMYB24<br>AtMYB21 - AtMYB24</td><td>Y2H, LCI,<br>BiFC</td><td>N-terminus</td><td>inhibition</td><td>Song et al. (2011)</td></tr><tr><td>AtIIle bHLH - AtMYB21<br>AtIIle bHLH - AtMYB24</td><td>Y2H, Co-IP,<br>BiFC</td><td>N-terminus</td><td>bHLH-MYB<br>transcription<br>complex</td><td>Qi et al. (2015)</td></tr><tr><td>JAZs** - AtMYB21<br>JAZs** - AtMYB24<br>AtMYB24 - AtMYB21<br>AtMYB24 - AtMYB24</td><td>Y2H, LCI</td><td>N-terminus N-<br>and C-<br>terminus</td><td>inhibition</td><td>Huang et al. (2017)</td></tr><tr><td>AtMYC2 - AtMYB21</td><td>Y2H, BiFC, in<br>vitro pull-down</td><td>not tested</td><td>inhibition</td><td>Yang et al. (2020)</td></tr><tr><td>DELLAs - AtMYB21<br>DELLAs - AtMYB24<br>AtJAZ1 - AtMYB21</td><td>Y2H, in vitro<br>pull-down</td><td>N-terminus</td><td>inhibition</td><td>Huang et al. (2020)</td></tr><tr><td>AtSnRK2.4 - AtMYB21</td><td>Y2H, in vitro<br>pull-down,<br>BiFC</td><td>N- and C-<br>terminus</td><td>act together to<br>mediate salt<br>stress responses</td><td>Zhang et al. (2021)</td></tr><tr><td>Petunia hybrida</td><td>PhERF6 - EOB1</td><td>Y2H, BiFC,<br>Co-IP</td><td>N-terminus</td><td>inhibition</td><td>Liu et al. (2017)</td></tr><tr><td>Solanum lycopersicum</td><td>SIJAZ9 - SIMYB21</td><td>Y2H, BiFC,<br>splitTALE</td><td>not tested</td><td>not tested</td><td>Schubert et al. (2019)</td></tr><tr><td>Freesia hybrida</td><td>FhMYC2 - FhMYB21L2</td><td>Y2H, BiFC,</td><td>not tested</td><td>inhibition</td><td>Yang et al. (2020)</td></tr><tr><td>Hedychium coronarium</td><td>HcMYB1 - HclAA4</td><td>Y2H, BiFC</td><td>not tested</td><td>inhibition</td><td>Ke et al. (2021)</td></tr></tbody></table>

**Table S1** Summary of protein-protein interactions involving members of the R2R3-MYB SG19 reported in the literature. List of abbreviations: Bimolecular Fluorescence Complementation (BiFC), Yeast-two-Hybrid (Y2H), Co-Immunoprecipitation (Co-IP), Luciferase Complementation Imaging (LCI), Jasmonate zim-domain (JAZ), Ethylene Response Factor (ERF), repressors of gibberellin (DELLAs), Sucrose non-fermenting-1-related protein kinase 2 (salt tolerance, SnRK2). JAZs* = JAZ1, JAZ8, JAZ11, JAZs** = JAZ1, JAZ2, JAZ3, JAZ4, JAZ5, JAZ6, JAZ8, JAZ10, JAZ11, JAZ12, DELLAs = AtRGA, AtGAI, AtRGL1, AtRGL2, AtRGL3 and IIle bHLH = bHLH TFs of the IIle clade such as MYC2, MYC3, MYC4, MYC5.
<table><caption>**Table S2** Summary of the direct target genes of members of the R2R3-MYB SG19 reported in the literature.</caption><thead><tr><th>Organism</th><th>Direct target gene</th><th>Methods</th><th>Publication</th></tr></thead><tbody><tr><td rowspan="5">Arabidopsis thaliana</td><td>AtMYB21 → PAL1, PAL2<br>AtMYB21 → UGT73B2<br>AtMYB21 → UGT79B6<br>AtMYB21 → AtMYB24<br>AtMYB24 → AtMYB21</td><td>DL</td><td>Battat et al. (2019)</td></tr><tr><td>AtMYB21 → AtTPS14<br>AtMYB21 → AtTPS21</td><td>transient protoplast, EMSA</td><td>Yang et al. (2020)</td></tr><tr><td>AtMYB21 → AtFLS1</td><td>Y1H, DL, ChIP, EMSA</td><td>Zhang et al. (2021)</td></tr><tr><td>Nicotiana tabacum<br>Antirrhinum majus</td><td>MYB305 → PAL2<br>MYB305 → CHS</td><td>Transactivation, EMSA</td><td>Sablowski et al. (1994)<br>Moyano et al. (1996)</td></tr><tr><td>Nicotiana tabacum</td><td>MYB305 → NEC1<br>MYB305 → NEC5</td><td>EMSA</td><td>Liu et al. (2009)</td></tr><tr><td rowspan="3">Petunia hybrida</td><td>MYB305 → AGP</td><td>indirect</td><td>Liu et al. (2012)</td></tr><tr><td>NtMYB305a → NtPMT1a</td><td>Y1H, ChIP, EMSA</td><td>Bian et al. (2021)</td></tr><tr><td>PhEOB2 → PhPAL<br>PhEOB2 → PhIGS</td><td>Transient protoplast<br>transfection</td><td>Spitzer-Rimon et al. (2010)</td></tr><tr><td rowspan="4">Freesia vesca</td><td>PhEOB2 → PhODO1</td><td>Transactivation, EMSA</td><td>Moerkercke et al. (2011)</td></tr><tr><td>PhEOB2 → PhEOB1<br>PhEOB1 → PhPAL<br>PhEOB1 → PhIGS<br>PhEOB1 → PhODO1</td><td>Transient protoplast<br>transfection, EMSA, Y1H,</td><td>Spitzer-Rimon et al. (2012)</td></tr><tr><td>FaEOB2 → FvCAD1<br>FaEOB2 → PhODO1</td><td>Transactivation</td><td>Medina-Puche et al. (2015)</td></tr><tr><td>FhMYB21L2 → FhTPS1</td><td>Transient protoplast<br>transfection, ChIP, EMSA</td><td>Yang et al. (2020)</td></tr><tr><td>Malus domestica</td><td>MYB305 → Machi3-1</td><td>Expression correlation<br>analysis</td><td>Kurilla et al. (2019)</td></tr><tr><td>Lilium longiflorum</td><td>LIMYB305 → LIHSC70</td><td>Y1H, DL</td><td>Wu et al. (2021)</td></tr><tr><td rowspan="2">Hedychium coronarium</td><td>HcMYB2 → HcBSMT2<br>HcMYB1 → HcBSMT2<br>HcMYB1 → HcTPS5</td><td>Y1H, DL</td><td>Ke et al. (2021)</td></tr><tr><td>VvMYB24 → 20 terpene related genes<br>(VvTPS35)<br>VvMYB24 → 6 carotenoid related genes<br>(GGPS1, CRTISO2, LCYE)<br>VvMYB24 → 30 photosynthesis and<br>light-response genes VvHYH</td><td>DAP-seq, DL</td><td>Zhang et al. (2021)<br>(PREPRINT)</td></tr><tr><td>Chrysanthemum morifolium</td><td>CmMYB21 → CmDFR</td><td>Y1H, DL</td><td>Wang et al. (2022)</td></tr></tbody></table>

R2R3-MYB SG19 are prominently activators, with one exception identified in *Chrysanthenum morifolium*. List of abbreviations: Chromatin ImmunoPrecipitation (ChIP), Electrophoretic Mobility Shift Assay (EMSA), Yeast one-Hybrid (Y1H), Dual-Luciferase (DL), DNA Affinity Purification sequencing (DAP-seq). Shikimate/Phenylpropanoids/Benzaldehyde: Emission Of Benzenoids (EOB), Phenylalanine Ammonia-Lyase (PAL), Chalcone Synthase (CHS), Isoeugenol Synthase (IGS), Cinnamyl Alcohol Dehydrogenase (CAD), Odorant 1 (ODO1). Nectary related: ADP-glucose pyrophosphorylase (small subunit, AGPs), Chitinase (Machi). Terpene related: Terpene synthase (TPS). Flavonoid related: Dihydroflavonol 4-Reductase (DFR), UDP-Glucuronosyltransferase (UGT), Flavonol Synthase (FLS). Carotenoid related: Geranylgeranyl Diphosphate Synthase 1 (GGPS1), Carotenoid Isomerase (CRTISO2), Lycopene Epsilon Cyclase (LCYE). Photosynthesis and light-response genes: HY5 Homolog (HYH). Nicotine related: Putrescine N-Methyltransferase (PMT). Others: Heat Shock protein (HSC).
<table><caption>Table S3 gRNA sequences used to target EOB1 and EOB2.</caption><thead><tr><th>Target gene</th><th>gRNA#</th><th>gRNA sequence (from 5'-3')</th></tr></thead><tbody><tr><td>EOB1_ Peaxi162Scf00129g01231.1</td><td>gRNA1</td><td>GAAAAAGTTGTCGACTTCGG</td></tr><tr><td>EOB1_ Peaxi162Scf00129g01231.1</td><td>gRNA2</td><td>CTCCGGCCAGATGTCCGGAG</td></tr><tr><td>EOB2_ Peaxi162Scf00080g00064.1</td><td>gRNA3</td><td>TACTCTCCCACTTCATACTC</td></tr><tr><td>EOB2_ Peaxi162Scf00080g00064.1</td><td>gRNA4</td><td>CACTTCATACTCTGGAAATG</td></tr></tbody></table>

<table><caption>Table S4 Sequence description of primers used for this work.
Boldface: recombination site for cloning</caption><thead><tr><th>Target gene</th><th>Application</th><th>Primer pair</th><th>Forward primer (from 5'-3')</th><th>Reverse primer (from 5'-3')</th></tr></thead><tbody><tr><td>EOB1_ Peaxi162Scf00129g01231.1</td><td>Genotyping</td><td>EOB1-pp1</td><td>CGACAACTTATTTGAGATTGAGACG</td><td>CACTTAGCATGCAGTTCCATAATC</td></tr><tr><td>EOB2_ Peaxi162Scf00080g00064.1</td><td>Genotyping</td><td>EOB2-pp1</td><td>CAAATACATGGTGTACAGGGC</td><td>TAACCATAGGCACCTCCATG</td></tr><tr><td>EOB1_ Peaxi162Scf00129g01231.1</td><td>RT-qPCR</td><td>EOB1-pp2</td><td>CAGCTCTTGATTATGGAACTGC</td><td>GTGCTTCTGTATCCTAGTCCTC</td></tr><tr><td>EOB2_ Peaxi162Scf00080g00064.1</td><td>RT-qPCR</td><td>EOB2-pp2</td><td>GAGGAAAGGACCTTGGACTATG</td><td>ACCGAAGCCGACAACTTT</td></tr><tr><td>ODO1_ Peaxi162Scf00002g00037.1</td><td>RT-qPCR</td><td>ODO1-pp1</td><td>TGCTTCAACCATGTCGAATTG</td><td>TCCGTGCCTGTTCTCTACGTT</td></tr><tr><td>TPS1_ Peaxi162Scf00074g00143.1</td><td>RT-qPCR</td><td>TPS1-pp1</td><td>GCAACTGAAGCGCCTATGTT</td><td>TGTGTATCCATCCGCCTCTT</td></tr><tr><td>RAN1_ Peaxi162Scf01372g00049.1</td><td>RT-qPCR</td><td>RAN1-pp1</td><td>AAGCTCCCACCTGTCTGGAAA</td><td>AACAGATTGCCGGAAGCCA</td></tr><tr><td>ACTIN11_ Peaxi162Scf00258g00618.1</td><td>RT-qPCR</td><td>ACT11-pp1</td><td>TGCACTCCCACATGCTATCCT</td><td>TCAGCCGAAGTGGTGAAAGAG</td></tr><tr><td>EOB1_ Peaxi162Scf00129g01231.1</td><td>RT-qPCR for transient</td><td>EOB1-pp3</td><td>TGTGAGCACAATGATCAACAAAG</td><td>TCCAGTGTATGAAGATGGAGAATAG</td></tr><tr><td>EOB2_ Peaxi162Scf00080g00064.1</td><td>RT-qPCR for transient</td><td>EOB2-pp3</td><td>GCCAAATGTCTAATGGTCCAAAT</td><td>TTAGGGCCTGCTTGGAAAG</td></tr><tr><td>CAS9_ pHSE401 vector</td><td>Genotyping</td><td>CAS9-pp1</td><td>CTGCAGAATGAGAAGCTCTAC</td><td>GACGATATTCACTTGTGGCATG</td></tr><tr><td>EOB1_ Peaxi162Scf00129g01231.1</td><td>Cloning</td><td>EOB1-pp4</td><td>GGGGACAAGTTTGTACAAAAAAGCAGGCTT</td><td>GGGGACCACTTTGTACAAGAAAGCTGGGTC</td></tr><tr><td>EOB2_ Peaxi162Scf00080g00064.1</td><td>Cloning</td><td>EOB2-pp4</td><td>GGGGACAAGTTTGTACAAAAAAGCAGGCTT</td><td>GGGGACCACTTTGTACAAGAAAGCTGGGTC</td></tr><tr><td>eob2-2_ Peaxi162Scf00080g00064.1</td><td>Cloning</td><td>eob2-2-pp1</td><td>GGGGACAAGTTTGTACAAAAAAGCAGGCTT</td><td>GGGGACCACTTTGTACAAGAAAGCTGGGTC</td></tr><tr><td>eob2-3_ Peaxi162Scf00080g00064.1</td><td>Cloning</td><td>eob2-3-pp1</td><td>GGGGACAAGTTTGTACAAAAAAGCAGGCTT</td><td>GGGGACCACTTTGTACAAGAAAGCTGGGTC</td></tr></tbody></table>
<table><caption>**Table S5** Description of the samples collected for the RNA sequencing experiment. Day post anthesis (DPA)</caption><thead><tr><td>Samples</td><td>Genotype</td><td>Stage</td><td>Tissue</td></tr></thead><tbody><tr><td>Sample 1</td><td>eob2-1</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 2</td><td>eob2-1</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 3</td><td>eob2-1</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 4</td><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 5</td><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 6</td><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 7</td><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 8</td><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 9</td><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>3 limbs</td></tr><tr><td>Sample 10</td><td>eob2-3</td><td>S7 (flower bud of ~5.5 cm, just before dark)</td><td>2 limbs</td></tr><tr><td>Sample 11</td><td>eob2-3</td><td>S7 (flower bud of ~5.5 cm, just before dark)</td><td>2 limbs</td></tr><tr><td>Sample 12</td><td>eob2-3</td><td>S7 (flower bud of ~5.5 cm, just before dark)</td><td>2 limbs</td></tr><tr><td>Sample 13</td><td>PaxP WT</td><td>S7 (flower bud of ~5.5 cm, just before dark)</td><td>2 limbs</td></tr><tr><td>Sample 14</td><td>PaxP WT</td><td>S7 (flower bud of ~5.5 cm, just before dark)</td><td>2 limbs</td></tr><tr><td>Sample 15</td><td>PaxP WT</td><td>S7 (flower bud of ~5.5 cm, just before dark)</td><td>2 limbs</td></tr><tr><td>Sample 16</td><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>1 limb</td></tr><tr><td>Sample 17</td><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>1 limb</td></tr><tr><td>Sample 18</td><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>1 limb</td></tr><tr><td>Sample 19</td><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>1 limb</td></tr><tr><td>Sample 20</td><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>1 limb</td></tr><tr><td>Sample 21</td><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>1 limb</td></tr><tr><td>Sample 25</td><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 26</td><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 27</td><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 28</td><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 29</td><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 30</td><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 31</td><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 32</td><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 33</td><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 34</td><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 35</td><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>5 basal ovaries</td></tr><tr><td>Sample 36</td><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>5 basal ovaries</td></tr></tbody></table>
<table><caption>**Table S6** Description of the samples collected for the starch and carotenoid measurements. Day post anthesis (DPA). yes and no indicate which compounds were measured.</caption><thead><tr><th>Genotype</th><th>Stage</th><th>Tissues</th><th>Starch</th><th>Carotenoids</th></tr></thead><tbody><tr><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 limbs</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 limbs</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 limbs</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>3 limbs</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>3 limbs</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>3 limbs</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 limbs</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 limbs</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>5 limbs</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>3 limbs</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>3 limbs</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>3 limbs</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>~67 basal ovaries</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>~67 basal ovaries</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>~67 basal ovaries</td><td>yes</td><td>no</td></tr><tr><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>~55 basal ovaries</td><td>yes</td><td>yes</td></tr><tr><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>~55 basal ovaries</td><td>yes</td><td>yes</td></tr><tr><td>eob2-3</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>~55 basal ovaries</td><td>yes</td><td>yes</td></tr><tr><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>~40 basal ovaries</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>~40 basal ovaries</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S5 (flower bud of ~3.5 cm, just before dark)</td><td>~40 basal ovaries</td><td>yes</td><td>no</td></tr><tr><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>~45 basal ovaries</td><td>yes</td><td>yes</td></tr><tr><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>~45 basal ovaries</td><td>yes</td><td>yes</td></tr><tr><td>PaxP WT</td><td>S10 (open flower; 1 DPA, just before dark)</td><td>~45 basal ovaries</td><td>yes</td><td>yes</td></tr></tbody></table>
<table><caption>Table S7 From the T0 transgenic lines obtained by CRISPR-Cas9 to the final homozygous mutant lines used in this study.</caption><thead><tr><th>Alleles</th><th>T0 plants</th><th>x</th><th>T1 plant selected</th><th>x</th><th>Final homozygous mutant lines</th><th></th></tr></thead><tbody><tr><td>eob1-1 (-31bp)</td><td>PaxP background eob1-1/EOB1 CAS9 positive</td><td>x</td><td>PaxP background eob1-1/EOB1 CAS9 negative</td><td>x</td><td>PaxP background eob1-1/eob1-1 CAS9 negative</td><td></td></tr><tr><td>eob2-1 (-364bp)</td><td>PaxP background eob2-1/EOB2 CAS9 positive</td><td>x</td><td>PaxP background eob2-1/EOB2 CAS9 negative</td><td>x</td><td>PaxP background eob2-1/eob2-1 CAS9 negative</td><td>maintained by cuttings</td></tr><tr><td>eob2-2 (-7bp)</td><td>PaxP background eob2-2/eob2-4 CAS9 positive</td><td>x</td><td>PaxP background eob2-2/EOB2 CAS9 negative</td><td>x</td><td>PaxP background eob2-2/eob2-2 CAS9 negative</td><td>maintained by cuttings</td></tr><tr><td>eob2-3 (-5bp)</td><td>PaxP background eob2-3/EOB2 CAS9 positive</td><td>x</td><td>PaxP background eob2-3/EOB2 CAS9 negative</td><td>x</td><td>PaxP background eob2-3/eob2-3 CAS9 negative</td><td></td></tr><tr><td>eob2-4 (-2bp)</td><td>PaxP background eob2-2/eob2-4 CAS9 positive</td><td>x</td><td>PaxP background eob2-4/EOB2 CAS9 negative</td><td>x</td><td>PaxP background eob2-4/eob2-4 CAS9 negative</td><td></td></tr><tr><td>eob1-1 (-31bp) eob2-2 (-7bp)</td><td>PaxP background eob1-1/EOB1 CAS9 positive</td><td>x</td><td>PaxP background eob2-2/eob2-4 CAS9 positive</td><td>x</td><td>PaxP background eob1-1/eob1-1 eob2-2/eob2-2 CAS9 negative</td><td>maintained by cuttings</td></tr></tbody></table>
<table><caption>**Table S8** Fraction of genes with a 2kb promoter containing at least one putative R2R3-MYB SG19 binding site.</caption><thead><tr><th>comparison</th><th>motif</th><th>regulation</th><th>with_<br>regulated</th><th>without_<br>regulated</th><th>with</th><th>without</th><th>fraction</th><th>expected_<br>fraction</th><th>p_value</th><th>pAdjusted</th><th>log10_<br>padj</th><th>fraction_diff</th></tr></thead><tbody><tr><td>WT vs eob2-3 limb S5</td><td>MA1408.1 up</td><td></td><td>309</td><td>775</td><td>10439</td><td>22329</td><td>0.285055351</td><td>0.318572998</td><td>0.016958118</td><td>0.020349742</td><td>1.69</td><td>-0.033517647</td></tr><tr><td>WT vs eob2-3 nec S5</td><td>MA1408.1 up</td><td></td><td>260</td><td>729</td><td>10439</td><td>22329</td><td>0.26289181</td><td>0.318572998</td><td>0.000117851</td><td>0.000217572</td><td>3.66</td><td>-0.055681188</td></tr><tr><td>WT vs eob2-3 limb S10</td><td>MA1408.1 up</td><td></td><td>1005</td><td>2250</td><td>10439</td><td>22329</td><td>0.30875576</td><td>0.318572998</td><td>0.211787372</td><td>0.23104077</td><td>0.64</td><td>-0.009817238</td></tr><tr><td>WT vs eob2-3 nec S10</td><td>MA1408.1 up</td><td></td><td>677</td><td>1846</td><td>10439</td><td>22329</td><td>0.268331352</td><td>0.318572998</td><td>1.20842E-08</td><td>2.90021E-08</td><td>7.54</td><td>-0.050241646</td></tr><tr><td>common to the 4 conditions</td><td>MA1408.1 up</td><td></td><td>32</td><td>88</td><td>10439</td><td>22329</td><td>0.266666667</td><td>0.318572998</td><td>0.239775753</td><td>0.250200785</td><td>0.6</td><td>-0.051906331</td></tr><tr><td>WT vs eob2-1 limb S5</td><td>MA1408.1 up</td><td></td><td>1387</td><td>3098</td><td>10439</td><td>22329</td><td>0.309253066</td><td>0.318572998</td><td>0.15226853</td><td>0.174021177</td><td>0.76</td><td>-0.009319932</td></tr><tr><td>WT vs eob2-3 limb S5</td><td>MA1408.1 down</td><td></td><td>158</td><td>231</td><td>10439</td><td>22329</td><td>0.406169666</td><td>0.318572998</td><td>0.000290813</td><td>0.0004653</td><td>3.33</td><td>0.087596668</td></tr><tr><td>WT vs eob2-3 nec S5</td><td>MA1408.1 down</td><td></td><td>372</td><td>462</td><td>10439</td><td>22329</td><td>0.446043165</td><td>0.318572998</td><td>8.07268E-15</td><td>9.68722E-14</td><td>13.01</td><td>0.127470167</td></tr><tr><td>WT vs eob2-3 limb S10</td><td>MA1408.1 down</td><td></td><td>648</td><td>997</td><td>10439</td><td>22329</td><td>0.393920973</td><td>0.318572998</td><td>3.80814E-11</td><td>1.52325E-10</td><td>9.82</td><td>0.075347975</td></tr><tr><td>WT vs eob2-3 nec S10</td><td>MA1408.1 down</td><td></td><td>734</td><td>1120</td><td>10439</td><td>22329</td><td>0.395900755</td><td>0.318572998</td><td>4.95218E-13</td><td>2.64087E-12</td><td>11.58</td><td>0.077327757</td></tr><tr><td>common to the 4 conditions</td><td>MA1408.1 down</td><td></td><td>32</td><td>28</td><td>10439</td><td>22329</td><td>0.533333333</td><td>0.318572998</td><td>0.000711522</td><td>0.001067283</td><td>2.97</td><td>0.214760335</td></tr><tr><td>WT vs eob2-1 limb S5</td><td>MA1408.1 down</td><td></td><td>1162</td><td>2000</td><td>10439</td><td>22329</td><td>0.367488931</td><td>0.318572998</td><td>8.82443E-10</td><td>2.64733E-09</td><td>8.58</td><td>0.048915933</td></tr></tbody></table><table><thead><tr><th>comparison</th><th>motif</th><th>regulation</th><th>with_<br>regulated</th><th>without_<br>regulated</th><th>with</th><th>without</th><th>fraction</th><th>expected_<br>fraction</th><th>p_value</th><th>pAdjusted</th><th>log10_<br>padj</th><th>fraction_diff</th></tr></thead><tbody><tr><td>WT vs eob2-3 limb S5</td><td>MA1037.1 up</td><td></td><td>212</td><td>872</td><td>7517</td><td>25251</td><td>0.195571956</td><td>0.229400635</td><td>0.006554535</td><td>0.00873938</td><td>2.06</td><td>-0.033828679</td></tr><tr><td>WT vs eob2-3 nec S5</td><td>MA1037.1 up</td><td></td><td>179</td><td>810</td><td>7517</td><td>25251</td><td>0.1809909</td><td>0.229400635</td><td>0.00019159</td><td>0.000328441</td><td>3.48</td><td>-0.048409735</td></tr><tr><td>WT vs eob2-3 limb S10</td><td>MA1037.1 up</td><td></td><td>686</td><td>2569</td><td>7517</td><td>25251</td><td>0.210752688</td><td>0.229400635</td><td>0.007373347</td><td>0.009313701</td><td>2.03</td><td>-0.018647947</td></tr><tr><td>WT vs eob2-3 nec S10</td><td>MA1037.1 up</td><td></td><td>485</td><td>2038</td><td>7517</td><td>25251</td><td>0.19223147</td><td>0.229400635</td><td>2.78724E-06</td><td>6.08124E-06</td><td>5.22</td><td>-0.037169164</td></tr><tr><td>common to the 4 conditions</td><td>MA1037.1 up</td><td></td><td>22</td><td>98</td><td>7517</td><td>25251</td><td>0.183333333</td><td>0.229400635</td><td>0.276273132</td><td>0.276273132</td><td>0.56</td><td>-0.046067301</td></tr><tr><td>WT vs eob2-1 limb S5</td><td>MA1037.1 up</td><td></td><td>944</td><td>3541</td><td>7517</td><td>25251</td><td>0.210479376</td><td>0.229400635</td><td>0.001154161</td><td>0.001629404</td><td>2.79</td><td>-0.018921259</td></tr><tr><td>WT vs eob2-3 limb S5</td><td>MA1037.1 down</td><td></td><td>153</td><td>236</td><td>7517</td><td>25251</td><td>0.393316195</td><td>0.229400635</td><td>3.77189E-13</td><td>2.64087E-12</td><td>11.58</td><td>0.163915561</td></tr><tr><td>WT vs eob2-3 nec S5</td><td>MA1037.1 down</td><td></td><td>292</td><td>542</td><td>7517</td><td>25251</td><td>0.350119904</td><td>0.229400635</td><td>1.34272E-15</td><td>3.22252E-14</td><td>13.49</td><td>0.120719269</td></tr><tr><td>WT vs eob2-3 limb S10</td><td>MA1037.1 down</td><td></td><td>501</td><td>1144</td><td>7517</td><td>25251</td><td>0.304559271</td><td>0.229400635</td><td>5.50182E-13</td><td>2.64087E-12</td><td>11.58</td><td>0.075158636</td></tr><tr><td>WT vs eob2-3 nec S10</td><td>MA1037.1 down</td><td></td><td>529</td><td>1325</td><td>7517</td><td>25251</td><td>0.285329018</td><td>0.229400635</td><td>8.71986E-09</td><td>2.32529E-08</td><td>7.63</td><td>0.055928384</td></tr><tr><td>common to the 4 conditions</td><td>MA1037.1 down</td><td></td><td>29</td><td>31</td><td>7517</td><td>25251</td><td>0.483333333</td><td>0.229400635</td><td>1.72634E-05</td><td>3.45268E-05</td><td>4.46</td><td>0.253932699</td></tr><tr><td>WT vs eob2-1 limb S5</td><td>MA1037.1 down</td><td></td><td>867</td><td>2295</td><td>7517</td><td>25251</td><td>0.274193548</td><td>0.229400635</td><td>5.96085E-10</td><td>2.04372E-09</td><td>8.69</td><td>0.044792914</td></tr></tbody></table>

The presence of a R2R3-MYB SG19 binding site in 2kb promoter was predicted using FIMO and two independent SG19 predicted motifs MA1408.1 (*FaEOBII*) and MA1037.1 (*AtMYB24*) (P < 1e-4). "with-regulated" is for up or down regulated genes with a motif in the promoter and "with" is for the total number of genes identified in Petunia (32,768) with a motif in the promoter.
<table><caption>**Table S9** Gene ID of genes used in this study.</caption><thead><tr><th></th><th>Gene ID _ Figure 5C</th></tr></thead><tbody><tr><td>ACS #1</td><td>Peaxi162Scf00074g01725.1</td></tr><tr><td>ACS #2</td><td>Peaxi162Scf00620g00121.1</td></tr><tr><td>ACS #3</td><td>Peaxi162Scf00102g01634.1</td></tr><tr><td>ACS #4</td><td>Peaxi162Scf00381g00219.1</td></tr><tr><td>ACS #5</td><td>Peaxi162Scf00192g00920.1</td></tr><tr><td>ACS #6</td><td>Peaxi162Scf00102g01343.1</td></tr><tr><td>ACS #7</td><td>Peaxi162Scf00096g01846.1</td></tr><tr><td>ACS #8</td><td>Peaxi162Scf00822g00212.1</td></tr><tr><td></td><td>Gene ID _ Figure 5C</td></tr><tr><td>ACO #1</td><td>Peaxi162Scf00521g00613.1</td></tr><tr><td>ACO #2</td><td>Peaxi162Scf00294g00812.1</td></tr><tr><td>ACO #3</td><td>Peaxi162Scf01096g00025.1</td></tr><tr><td>ACO #4</td><td>Peaxi162Scf00047g01927.1</td></tr><tr><td>ACO #5</td><td>Peaxi162Scf01333g00015.1</td></tr><tr><td>ACO #6</td><td>Peaxi162Scf01333g00016.1</td></tr><tr><td></td><td>Gene ID _ Figure 6C</td></tr><tr><td>EOB1</td><td>Peaxi162Scf00129g01231.1</td></tr><tr><td>CM1</td><td>Peaxi162Scf00166g00931.1</td></tr><tr><td>BSMT1</td><td>Peaxi162Scf00047g01123.1</td></tr><tr><td>BSMT2</td><td>Peaxi162Scf00047g00116.1</td></tr><tr><td>EGS</td><td>Peaxi162Scf00020g01714.1</td></tr><tr><td>IGS1</td><td>Peaxi162Scf00889g00229.1</td></tr><tr><td>IGS3</td><td>Peaxi162Scf00185g01622.1</td></tr><tr><td>Z-ISO</td><td>Peaxi162Scf00378g00631.1</td></tr><tr><td>ZDS</td><td>Peaxi162Scf00404g00021.1</td></tr><tr><td>LCY B</td><td>Peaxi162Scf00091g00064.1</td></tr><tr><td>CCD</td><td>Peaxi162Scf00953g00316.1</td></tr><tr><td></td><td>Gene ID _ Figure 7B</td></tr><tr><td>BAM</td><td>Peaxi162Scf00715g00216.1</td></tr></tbody></table>

Gene IDs of the eight 1-Aminocyclopropane-1-Carboxylic Acid Synthase (ACS) genes and six 1-Aminocyclopropane-1-Carboxylic Acid Oxidase (ACO) genes used in Figure 5C, eleven genes used in Figure 6C and one BETA-AMYLASE (BAM) gene used in Figure 7B.
**Method S1** Quantification of sesquiterpene accumulation in Petunia pistils and emission from tube by GC-MS.

Samples were analyzed on an Agilent 9000 Intuvo gas chromatograph system (Agilent Technologies), connected to an Agilent 5977B mass detector (Agilent Technologies). 2 µL of samples were injected in a split/splitless injector set at 250°C with a 2-fold split. Temperature of the guard chip was set to 75°C at the beginning of the run before following the temperature of the oven in track oven mode. Bus temperature was set at 280°C. Samples were analyzed on a HP-5MS-UI column (30 m x 0.25 mm x 0.25 µm) (Agilent Technologies) using a program consisting of 1 min at 50°C, followed by 20°C.min-1 to 310°C, then 4 min at 310°C, with helium as carrier gas set at 1.2 mL.min-1. Detector was set at 250 °C, ionization energy was set at 70 eV and analysis was realized in scan mode for analysis of sesquiterpene accumulation in pistils (Mass spectra scanned from 30 to 300 amu) and in single ion monitoring mode for analysis of sesquiterpene emission from tubes using m/z 161 to quantify sesquiterpenes and m/z 95 to quantify IS. Products were identified based on their retention times and electron ionization mass spectra compared to those of authentic standards (Germacrene D) or those present in the NIST2017 and WILEY libraries. Quantification of compounds was performed using the Mass Hunter quantitative software (Agilent Technologies) using response factors of authentic germacrene D relative to the IS and normalized to the weight of tissues.

**Method S2** Carotenoid extraction and quantification with an HPLC system.

Tissues were ground using a ball mill (Retch MM400, Retch). Extraction was performed for 15 min in microcentrifuge tubes containing 400 ul 80% acetone in darkness under slight agitation (50 rpm). After this, samples were centrifuged in a microcentrifuge for 7 min at max speed. 100 ul of the supernatant was transferred to an HPLC tube with insert. Pigments were separated with a YMC carotenoid S-5 µm column (250 x 2.1 mm; YMC Europe GMBH). Pigments were eluted using 100% solvent A (Methanol: Methyl tert-butyl ether: H2O, 90:6:4) for the first 12 min followed by an 84 min linear gradient to 100% solvent B (MeOH/MTBE/H20 (25:71:4)). The column temperature was 35 °C, the flow rate 0.2 ml/min. The pigments were detected by their absorbance at 480 nm. Standards were used to identify carotenoids.

**Method S3** Starch extraction and quantification with an HPLC system.

After extraction, tubes were centrifuged at 25k x g for 5 min. For starch quantitation, the pellet was washed three times with 80% ethanol. The pellet was vacuum dried, and starch was catalyzed to glucose by a 2-step enzymatic reaction. For the first step, the pellet was resuspended in 0.8 mL α-amylase solution (1 mg/ml in water, Rohalase® A3 from *Bacillus subtilis*, 44 U/mg, Serva) and incubated for 30 min at 90 °C while agitating. Subsequently, 0.4 ml amyl-glucosidase solution (0.5 mg/ml in 50 mM citrate buffer, pH 4.6, from *Aspergillus Niger*, 70U/mg, Sigma) was added, shortly mixed by vortex, and incubated for 10 min at 60 °C under agitation. Finally, samples were centrifuged, the supernatant was transferred to a new tube and diluted 20-fold. Glucose levels were analyzed with the HPLC, as described in **Method S2**, this time eluted with 100 mM NaOH + 25 mM sodium acetate instead of only 100 mM NaOH.