MOLECULAR PLANT

Please cite this article as: Liu Z., Boachon B., Lugan R., Tavares R., Erhardt M., Mutterer J., DemaisV., Pateyron S., Brunaud V., Ohnishi T., Pencik A., Achard P., Gong F., Hedden P., Werck-Reichhart D., and Renault H. (2015). A conserved cytochrome P450 evolved in seed plants regulatesflower maturation. Mol. Plant. doi: 10.1016/j.molp.2015.09.002.

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A conserved cytochrome P450 evolved in seed plants regulates flower maturation

Zhenhua Liu1,&, Benoît Boachon1, Raphaël Lugan1,§, Raquel Tavares2, Mathieu Erhardt1,

Jérôme Mutterer1, Valérie Demais3, Stéphanie Pateyron4, Véronique Brunaud5, Toshiyuki

Ohnishi6, Ales Pencik7, Patrick Achard1, Fan Gong8,#, Peter Hedden8, Danièle Werck-

Reichhart1,9,10,* and Hugues Renault1,9,10

1Institute of plant molecular biology, Centre national de la recherche scientifique (CNRS) - University

of Strasbourg, France 2Laboratoire de biométrie et biologie évolutive, Université Lyon 1 - CNRS, Villeurbanne, France 3Plateforme d’Imagerie in vitro, IFR 37 de Neurosciences, Strasbourg, France4Transcriptomic platform and 5Bioinformatics for predictive genomics, Unité de recherche en

génomique végétale (URGV), INRA - Université d'Evry Val d'Essonne - CNRS, France 6Graduate School of Agriculture, Shizuoka University, Japan 7Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre of the

Region Haná for Biotechnological and Agricultural Research, Faculty of Science, Palacký University &

Institute of Experimental Botany AS CR, Olomouc, Czech Republic 8Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK 9University of Strasbourg Institute for Advanced Study (USIAS), Strasbourg, France 10Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, Germany

& Current address: 158 Emerson Hall, Section of Plant Biology, School of Integrative Plant Science,

Cornell University, Ithaca, NY 14853, USA § Current address: Laboratoire Physiologie des Fruits et Légumes - EA 4279, Campus Agroparc,

Avignon, France # Current address: Home Office Science – Centre for Applied Science and Technology, Woodcock Hill,

Sandridge, St Albans, Herts AL4 9HQ UK

* Contact: Danièle Werck-Reichhart

tel: +33 3 68851854

fax: +33 3 54861921

e.mail: daniele.werck@ibmp-cnrs.unistra.fr

Running title: A P450-dependent signal regulates flower maturation

Short summary: This paper proposes a new phylogenomic approach to identify genes with

essential functions in plant signalling metabolism. CYP715, a candidate selected based on

its high conservation in plant genomes is shown to regulate flower maturation.

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Abstract Global inspection of plant genomes identifies genes maintained in low copies across

taxa and under strong purifying selection. Those are likely to have essential functions. Based

on this rational, we investigated the function of the low-duplicated CYP715 cytochrome P450

gene family that appeared early in seed plants and evolved under strong negative selection.

Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of

flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines revealed a

strong defect in petal development, and transient alteration of pollen intine deposition.

Comparative expression analysis pointed to downregulation of genes involved in pollen

development, cell wall biogenesis, hormone homeostasis and floral sesquiterpene

biosynthesis, in particular TPS21 and the key floral development regulators MYB21, MYB24

and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1

mutants. Flower hormone profiling in addition indicated a modification of gibberellins

homeostasis and a strong disturbance of the jasmonic acid derivatives turnover. Petal growth

was partially restored by the active gibberellin GA3 and the functional analog of jasmonoyl-

isoleucine, coronatine. CYP715 thus appears as a key regulator of flower maturation,

synchronizing petal expansion and volatile emission. It is thus expected to be an important

determinant for flower-insect interaction.

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Introduction The availability of the first plant genomes revealed extensive duplication in some

gene families and predicted an unsuspected complexity of plant metabolism and regulation

networks. Sequencing of a larger number of plant genomes brings a new outlook on the

global picture and for example highlights some genes that are under strong purifying

selection, with low duplication number in most plant genomes, and well conserved across

plant taxa and sometimes in other organisms (De Smet et al., 2013). Such genes are most

often involved in essential housekeeping functions such as DNA or RNA-related processes,

photosynthesis and plastid organisation, cofactor metabolic processes or embryonic

development (De Smet et al., 2013). Although less frequent, some single-copy genes can be

found also in large superfamilies encoding transcription factors or enzymes (Airoldi and

Davies, 2012; De Smet et al., 2013; Nelson and Werck-Reichhart, 2011). In the latter case, a

comparative genomics approach might thus support identification of genes with important

developmental functions. A survey of the largest family of genes coding for metabolic

enzymes, cytochromes P450 (P450s), on eight land plant genomes showed that most P450

families with essential housekeeping functions, for example involved in the biosynthesis of

lignin precursors or in hormone homeostasis are present in low, sometimes single, copy

number and broadly distributed across plant taxa (Nelson and Werck-Reichhart, 2011). It

also pointed to a few orphan P450 genes with similar characteristics.

CYP715A1 (At5g52400) is the sole member of its P450 family in Arabidopsis thaliana.

A single CYP715 family member is found also in larger dicot or monocot genomes such as

those of grapevine or rice (Nelson and Werck-Reichhart, 2011). The CYP715 family, in

addition, belongs to the CYP72 clan of P450 enzymes that encompasses several families

(CYP734, CYP735, CYP714) contributing to hormone homeostasis (Bak et al., 2011).

CYP734s are brassinolide 26-hydroxylases, involved in the catabolism of the brassinosteroid

hormones (Neff et al., 1999; Turk et al., 2003), while CYP735s catalyze hydroxylation of the

isoprenoid chain of cytokinin precursors for the biosynthesis of trans-zeatin (Takei et al.,

2004). Members of the CYP714 family were recently shown to function in gibberellin

deactivation and homeostasis in rice via 16α,17-epoxidation or 13-hydroxylation (Magome et

al., 2013; Zhu et al., 2006). Genetic evidence suggests that CYP714s play a similar role in

Arabidopsis (Nomura et al., 2013; Zhang et al., 2011). The function of the CYP715 proteins

has however not been reported. The strong selection pressure maintaining the single-copy

status of CYP715 genes and their membership of the CYP72 clan led us to postulate that

they play a role in plant hormone metabolism and development. We provide here evidence

that CYP715A1 in Arabidopsis regulates petal development, floral hormone homeostasis and

volatile terpenoid emission.

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Results CYP715 is a single or low-copy gene evolved with early seed plants

A systematic mining of genomic data available in Phytozome

(http://www.phytozome.org) and in the OnekP sequencing project database

(http://www.onekp.com) was first carried out. It indicated a broad distribution of the CYP715

family across seed plants (Figure 1). The CYP715 family is detected in all spermatophytes

(i.e. seed plants) including gymnosperms and angiosperms (Nelson and Werck-Reichhart,

2011). The CYP714 family, which is found exclusively in angiosperms, seems to have a

more recent origin. In most cases (21 out of 32), a single CYP715 member could be retrieved

for each taxon. In some of them however, usually plants that have undergone recent whole

genome duplications, a few gene duplicates can be found, for example in Fabaceae (i.e.

legumes) with up to six copies in the paleoploid soybean genome (Schmutz et al., 2010)

(Figure 1). Single copy genes usually also exhibit high sequence conservation (De Smet et

al., 2013). In order to investigate the selection regimes acting on the remarkably few CYP715

genes, we calculated the ratios of non-synonymous to synonymous substitutions (ω = dN/dS)

in the whole family using the one ratio model from PAML software (Yang, 2007). This model

assumes the same value for all the lineages. The calculated ω of 0.11781 indicates a

strong purifying selection for the CYP715 family in angiosperms (Table S1). Furthermore,

site models in PAML allowing the ratio to vary among sites were tested using the nearly

neutral model (M1a) and the selection model (M2a). Within both models, 85% of the sites

have an ω value of 0.09073 and nearly 15% of the sites have an ω value of 1. No sites under

positive selection were significantly identified on the CYP715 sequences (Table S1). The

CYP715 family thus seems to have evolved under high purifying selection. The reasons for

such high conservation and negative selection pressure were investigated by studying the

function of CYP715A1 in A. thaliana.

CYP715A1 expression is restricted to anther filaments and tapetal cells CYP715 mutants have not so far emerged from genetic screens with a major impact

on plant development or viability. It was therefore necessary to focus on particular

developmental stages and tissues for a functional analysis of CYP715 using mutants. A

survey of publicly available transcriptome data (http://www-ibmp.u-

strasbg.fr/~CYPedia/CYP715A1/CoExpr_CYP715A1_Organs.html) and a qRT-PCR analysis

(Figure 2A-C) pointed to flowers as the main site of CYP715A1 expression. CYP715A1 was

highly expressed at anthesis (Figure 2B) and in stamens of mature flowers (Figure 2C).

Plants transformed with a CYP715A1pro:GUS fusion construct further revealed restricted and

tissue-specific expression in the tapetal cells during pollen development (flower stage 5-9;

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Figure 2D-F) and in the anther filament upon flower maturation (flower stage 12-15; Figure

2D and 2G). Staining did not reveal any gene expression in other organ of the plant grown

under standard conditions.

CYP715A1 regulates petal development and intine deposition The two cyp715a1 T-DNA insertion mutants (cyp715a1-1 and cyp715a1-2) and a

CYP715A1 overexpressor line (OE-2) (see supplemental Figure S2 for molecular validation)

did not display any significant alteration of whole plant development and architecture. Focus

on flower development, however, revealed a striking inhibition of petal growth in both null

mutants, with reduced petal surface area (Figure 3A-D, I) associated with reduced cell size

(Figure 3E-G, J). Absence of curvature of the shortened petals and defective flower opening

(Figure 3B-D) was typical of mutations affecting petal growth. The petal growth phenotype

could be rescued when plants were grown under long-day conditions (i.e. 16h-light regime),

especially in early arising flowers of the inflorescence (Supplemental Figure S3), while it was

maintained under 12h light over several weeks. Meanwhile, the wild-type phenotype was

totally restored by complementation of the cyp715a1-1 line with the wild-type CYP715A1

genomic locus (Supplemental Figure S4). No alteration in the growth of other floral organs,

such as stamen or pistil, was observed (Figure 3H, K, L). Ectopic overexpression of

CYP715A1 under control of a CaMV-35S promoter did not result in a significant change in

flower development except for a minor increase of petal and petal cell growth as seen in

Figure 3I-J.

The potential impact of CYP715 expression was also investigated by transmission

electron microscopy (TEM) analysis of pollen development since the tapetum is well

documented to provide the precursors required for the formation of the pollen wall (Quilichini

et al., 2014). After the first mitosis, pollen grains from wild-type plants formed outer wall exine

and inner wall intine (Figure 4A). Simultaneously, vesicular material accumulated in the

pollen grains (Figure 4), possibly invaginated from the tonoplast (Yamamoto et al., 2003).

Whereas exine formation was normal throughout pollen grain development, at the bicellular

stage, the intine was observed to be strongly undulated in the two cyp715a1 lines, while the

accumulation of vesicular structures detected in the cytosol of the wild-type pollen grains was

much less evident in the mutant lines (Figure 4B-C). The intine is the innermost layer of the

pollen wall that is located adjacent to the pollen plasma membrane. It is composed of

cellulose, pectin, and various proteins, and is secreted by the microspore (i.e. has a

gametophytic origin) at the ring-vacuolated microspore stage (Owen and Makaroff, 1995;

Vizcay-Barrena and Wilson, 2006). The absence of small ring vesicles can thus be correlated

with the observed undulated intine formation. However, no significant modification of final

pollen structure, tectum formation or pollen coat deposition (Figure 4D-I) was detected.

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Overall, alteration in flower development did not translate into any reduced fertility for

Arabidopsis plants grown in controlled conditions.

Loss of CYP715A1 function causes extensive transcriptional changes in reproductive tissues

The loss of CYP715A1 function impacted development of tissues that were different

from its main expression sites, suggesting its direct or indirect involvement in the production

of a mobile signal. To determine the targets and mode of action of this putative signalling

compound, a comparative transcriptomic analysis of the cyp715a1-2 mutant versus wild-type

flower buds was carried out using the CATMA6.2 microarray (see Materials and Methods

sections for experimental procedures). This analysis identified a total of 370 genes

differentially expressed in the mutant compared to wild type (log2 0.8, p-value < 0.05); 188

were up-regulated and 182 were down-regulated (complete list in Supplemental Dataset S1).

Gene ontology (GO) analysis with the FatiGO tool (Babelomics 4.3,

babelomics.bioinfo.cipf.es) uncovered several significantly enriched GO terms related to

pollen development, cell wall and secretory pathways among the up-regulated genes list

(Supplemental Table S2), which is consistent with the above-described pollen phenotype of

cyp715a1 mutants. Pollen wall-related genes were also found overrepresented in the down-

regulated genes list (Supplemental Table S2). Interestingly, the analysis also revealed that

genes associated with the biosynthesis of indole derivatives and auxin, and with jasmonic

acid (JA) biosynthesis and response, were significantly enriched in the down-regulated

genes list (Table 1 and Supplemental Table S2), suggesting that loss of CYP715A1 function

interferes with hormone signalling and homeostasis.

These data were confirmed and further refined by targeted qRT-PCR analysis. The

expression of a subset of genes differentially expressed in the microarray analysis was

determined in both flower buds and open flowers of the two cyp715a1 mutants and the

CYP715A1-overexpressor line compared to wild type. A putative tryptophan synthase

(At5g28237), together with CYP79B3 and CYP79B2, two cytochrome P450 genes involved

in the biosynthesis of indole-3-acetaldoxime, indole glucosinolates and possibly auxin

(Tivendale et al., 2014), were consistently found down-regulated in mutant lines, thus

confirming microarray results (Figure 5). The two JA repressor genes, JA ZIM DOMAIN

(JAZ) JAZ5 and JAZ9, were also significantly down-regulated in open flowers of both

cyp715a1 mutants, and moderately in flower buds (Figure 5). Likewise, expression of the JA-

regulated transcription factors MYB21 and MYB24 was significantly repressed in the

mutants, mainly in flower buds (Figure 5). None of the investigated genes showed a

consistent differential expression in the CYP715A1-overexpressor line.

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CYP715A1 regulates floral hormone homeostasis To determine how CYP715A1 impacts flower hormone homeostasis and to obtain

information on the pathway potentially involving CYP715A1, hormone profiling of buds and

open flowers was then carried out. Petal development is thought to be mainly controlled by

gibberellins (GAs), auxin (indole-3-acetic acid, IAA), and JA (Brioudes et al., 2009; Chandler

et al., 2011; Reeves et al., 2012). GAs are described as upstream and light-dependent

regulators of flower and petal development (Plackett et al., 2012; Reeves et al., 2012).

CYP715s form a sister clade to CYP714s which are involved in GA metabolism (Magome et

al., 2013; Zhu et al., 2006). GA analysis of the wild type and the two cyp715a1 insertion lines

revealed a consistent, but minor decrease in all 13-deoxy GA biosynthetic intermediates, and

a 25% and 20% reduction in GA1 and GA4, respectively, in the cyp715a1 mutants. All GA 2-

oxidase products were slightly increased (Figure 6A). Accordingly, spraying GA3 could relax

the flower phenotype in the mutants (Figure 7). This rescue however did not restore normal

petal growth on the whole inflorescence, but permitted delayed opening of the early arising

flowers of each inflorescence.

Transcriptome analysis suggested a down-regulation of auxin and JA pathways in

cyp715a1 suppressed lines (Table 1, Supplemental Table S2 and Figure 5). A broader

hormone profiling did not reveal any significant modification in IAA and ABA contents, but

indicated a profound perturbation in the JA metabolism (Figure 6B and Supplemental Table

S3). More striking was the decrease in the end products of JA catabolism such as

carboxylated jasmonate-isoleucine (12COOH-JA-Ile) and glycosylated tuberonic acid (12OH-

JA-Glc) in young buds and open flowers of cyp715a1 lines, whereas only moderate

decreases in JA and JA-isoleucine were observed in young buds. Conversely, buds

accumulated free tuberonic acid (12OH-JA). Consistent with the transcription data, metabolic

profiles thus indicate a down-regulation of the JA pathway in CYP715A1 deficient plants, with

only a minor decrease in JA and JA-isoleucine, the decrease being most likely attenuated by

the well documented JA negative feedback loop (Wasternack and Hause, 2013). Also

consistent with a role for CYP715A1 in JA cascade signalling, coronatine a bacterial toxin

and structural and functional analog of the active phytohormone jasmonoyl-L-isoleucine

(Staswick and Tiryaki, 2004), partially rescued the floral phenotype of the null cyp715a1

mutants (Figure 7). As for GA3, this rescue did not restore normal petal growth on the whole

inflorescence, but permitted delayed opening of the early arising flowers of each

inflorescence. Coronatine was however less effective than GA3.

CYP715A1 regulates floral volatile emission

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Our transcriptome analysis indicated a strong down-regulation of two terpene

synthases (TPS), TPS21 and TPS14, in the cyp715a1-2 line (Supplemental Dataset S1).

According to previous work (Hong et al., 2012; Reeves et al., 2012), flower maturation,

including petal expansion and TPS21- and TPS11-dependent volatile emission, involves the

JA-activated transcription factors MYB21 and MYB24 that were also down-regulated in

cyp715a1 mutants as well (Supplemental Dataset S1, Figure 5). TPS21 is reported to

produce the major floral volatile β-caryophyllene in Arabidopsis (Hong et al., 2012; Tholl et

al., 2005). The expression of the TPS genes involved in the production of floral volatiles was

thus investigated by qRT-PCR, together with the expression of MYC2 recently described as

a direct regulator of TPS21 (Hong et al., 2012) (Figure 8A). It confirmed a large decrease in

TPS21 expression in young buds and open flowers of both cyp715a1 lines. TPS03

expression was also strongly decreased at both stages, whereas TPS14 and TPS10

expression was only partially suppressed in young buds. No statistically significant decrease

in TPS11 expression was detected. MYC2 down-regulation was significant only in open

flowers.

From the observed reduced expression of the TPS genes we inferred that the

emission of flower volatiles would be suppressed in the cyp715a1 mutants. To test this

inference, we collected floral volatiles from mature inflorescences of wild type, cyp715a1

mutants and a cyp715a1-2 complemented line (COMP), and analysed them by GC-MS. As

shown in Figure 8B-C, CYP715A1 inactivation resulted in a large decrease in the emission of

volatile sesquiterpenes, particularly of all TPS21 products (β-caryophyllene, α-humulene and

α-copaene). Monoterpene emission was also reduced, but to a lesser extent. A partial

restoration of the emission of these products was observed in the complemented line (Figure

8B-C). CYP715A1 thus appears to selectively control terpene volatile emission occurring

upon anthesis via regulation of TPS genes. TPS21, responsible for the major volatile emitted

in Arabidopsis, appears as a main target of CYP715A1 signalling, most likely though the

control of MYC2 expression.

What is the signal synthesized by CYP715A1? A few P450 enzymes using plastid-generated substrates were previously reported to

be localized within plastids or on the plastid envelope (Froehlich et al., 2001; Helliwell et al.,

2001; Kim and Della Penna, 2006; Tian et al., 2004; Watson et al., 2001). CYP715A1 has a

N-terminal membrane anchoring sequence unusually rich in hydrophilic residues, but is

nevertheless predicted to be targeted to the endoplasmic reticulum (ER) by TargetP and

Predotar (Emanuelsson et al., 2000; Small et al., 2004). This was confirmed by transient

expression of a CYP715A1::eGFP fusion construct via Agrobacterium tumefaciens-mediated

transfection of Nicotiana benthamiana leaves. Confocal microscopy of leaf epidermal cells

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showed a typical ER localization of the fusion protein (Supplemental Figure S5). CYP715A1

is thus more likely to use a substrate present in the cytosol, although contact transfer of a

pastid-derived precursor cannot be excluded (Mehrshahi et al., 2014).

In an attempt to determine enzyme activity, the CYP715A1 protein was first

expressed in yeast. Extremely low enzyme expression of the Arabidopsis protein and of its

Brachypodium distachyon ortholog was detected in yeast microsomes based on CO-bound

reduced/reduced differential spectroscopy (Supplemental Figure S6). CYP715A1 was thus

also expressed in insect cells with better yield (Supplemental Figure S6). Enzyme assays

performed with yeast and insect cell microsomes incubated with ABA, ent-kaurene, ent-

kaurenoic acid, GA12, GA1, GA4, 12OH-JA-Ile and tuberonic acid did not lead to any

detectable conversion of these compounds.

Discussion A number of genes involved in hormonal control of plant growth and floral organ

differentiation have emerged from genetic screens (Chandler et al., 2011; Eriksson et al.,

2010) revealing severe developmental alterations. Subtle growth regulation may however

escape investigations that rely on visual inspection. This work demonstrates that genes

leading to fine tuning functions important enough to require high gene conservation and

purifying selection across a broad range of plant taxa can now also be identified from

extended genome analyses. We show here that CYP715s constitute a family of duplication-

resistant P450 genes in seed plants, present as singletons in most plant genomes and

evolving under strong purifying selection. When duplications are found in some species,

those are usually in species which underwent relatively recent whole genome duplication

events, such as soybean (Schmutz et al., 2010), Medicago truncatula (Pfeil et al., 2005),

Brassica rapa (Wang et al., 2011), cotton (Wang et al., 2012), and eucalyptus (Myburg et al.,

2011), or species reported to show slower rates of evolution following relatively recent

genome duplications, such as poplar (Tuskan et al., 2006) and apple (Velasco et al., 2010).

Most duplicates might thus feature on-going pseudogenization (De Smet et al., 2013). Some

others (e.g. in Setaria italica) may reflect duplicative bursts giving rise to taxa-specific

defence pathways (Geisler et al., 2013; Nelson and Werck-Reichhart, 2011; Xu et al., 2007).

Common gene duplicates maintained in the Papillionoideae lineage may also reflect

elaborate developmental processes required to give rise to a complex floral architecture or to

nodulation.

CYP715A1 expression is exclusively detected in the anther tapetum during flower

development and filaments during flower maturation. It does not seem to be expressed in

other tissues in plants grown under controlled environment. Accordingly, a defect in

CYP715A1 selectively affects the development of flowers and no other plant organ. Contrary

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to most reported mutants in hormonal pathways and in particular of the JA regulatory

cascade (Stitz et al., 2014; Wasternack and Hause, 2013), CYP715A1 seems to selectively

impact petal growth without alteration in anther filament elongation or dehiscence, with the

phenotype attenuated under long day conditions. Consequently, this defect occurs without

significant impact on fertility in Arabidopsis. At first sight, this seems quite surprising

considering the high CYP715 conservation. It has however to be considered that Arabidopsis

is an autogamous plant, especially under laboratory growth conditions. A significant impact

on fertility might be expected for outcrossing plants under natural conditions if, as in

Arabidopsis, CYP715s in addition to petal development regulate floral volatile emission. This

trait has to be confirmed in other angiosperms, together with its impact on insect pollination.

It is potentially also critical in gymnosperms.

The expression of CYP715A1 is restricted to the tapetum in flower buds resulting in a

transient perturbation of the intine formation and microspore vesicular trafficking, and to the

anther filament upon flower maturation, but its main developmental effect was observed in

petals. In addition, the floral caryophyllene emission was almost completely suppressed in

cyp715a1 mutants. The terpene synthase TPS21, which is responsible for the emission of

caryophyllene, was shown to be expressed mainly in the stigma (Tholl et al., 2005).

Altogether, this would suggest that CYP715A1 has a non-cell-autonomous mode of action

and generates an stamen-derived mobile signal translocated to petals and stigma to regulate

petal growth and volatile emission. Alternatively, it may produce a compound that regulates

the biosynthesis or translocation of this mobile signal.

All hormones are reported to contribute to flower development, but only auxin, GAs,

and JAs are well documented for regulation of petal growth during flower maturation

(Chandler et al., 2011; Reeves et al., 2012). The still elusive product of the growth promoting

KLUH (CYP78A5) has also to be considered (Eriksson et al., 2010). The genes involved in

the production of all three or four hormones are described to have quite broad expression

patterns and impacts on flower architecture and overall development (Chandler et al., 2011;

Plackett et al., 2012; Wasternack et al., 2013). Conversely, the signal generated by

CYP715A1 seems to be produced in very specific tissues during flower development, and

also to selectively affect petal growth, and, transiently, intine formation, in addition to volatile

terpenoid emission (although we cannot exclude other subtle effects that we did not detect).

Compared to GA or JA signalling, the CYP715A1-derived signal thus appears to dissociate

petal growth and volatile emission from anther and gynoecium development, and hence

insect pollination from self-fertilization.

Given the strong perturbation in gene expression and JA homeostasis resulting from

CYP715A1 down-regulation, and considering the responses of petals and stigma, the

CYP715-derived signal seems to be perceived in several flower tissues. Based on

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transcriptomic and metabolic data, the signalling cascade triggered by CYP715A1 involves

modifications of GA and JA homeostasis, and regulates components of the JA signalling

pathway such as the JA-activated MYB21 and MYB24 transcription factors, the JAZ5 and

JAZ9 proteins, the JA and GA signalling mediator MYC2, as well as auxin signalling and

transport via ARF3 and GNOM (Figure 9). The MYB21 and MYB24 transcription factors have

been shown to have a strong impact on flower development and to affect petal and

gynoecium growth in addition to pollen germination, anther dehiscence and filament

elongation with resulting male sterility (Mandaokar et al., 2006; Reeves et al., 2012; Song et

al., 2011). Whereas seemingly using MYB21 and MYB24, CYP715A1 exclusively affects the

last steps, such as petal elongation, flower opening and volatile emission. It thus seems to

trigger a specific signalling cascade for targeted GA/JA-dependent gene activation at the late

stages of the flower development. Based on phylogenetic considerations, implying that

CYP715s share a common ancestor with CYP714s, the most plausible substrate of CYP715

would be a GA-type compound. In this work, GA quantification as well as quantification of

other hormones was performed on the whole inflorescence. This suggested a global

perturbation, but did not provide a precise enough indication of the tissue-specific changes in

hormone content resulting from CYP715 inactivation and most likely partially blurred both

transcriptome and metabolic data. In addition, no conversion of available GAs or precursors

could be detected. The absence of any strong developmental phenotype of the CYP715A1

over-expressor lines is consistent with a role of CYP715A1 in the upstream biosynthesis of a

signalling compound such as a GA since overexpression of genes encoding the most

upstream enzymes in GA biosynthesis such as copalyl diphosphate synthase, ent-kaurene

synthase and ent-kaurene oxidase (CYP701) have no or very small impact on plant

development (Fleet et al., 2003; Swain et al., 2005). It however excludes the possibility of the

involvement of CYP715 in GA catabolism which would be expected to result in major

developmental defects as illustrated by the severe phenotype from over-expression of the

CYP714 genes shown to participate in GA catabolism (Magome et al., 2013; Nomura et al.,

2013; Zhu et al., 2006). A careful dissection of the spatiotemporal transcriptome and

metabolic response to CYP715 in a suitable model is required to properly describe its mode

of action and to identify the signal orchestrating flower maturation.

Altogether, our data also suggest that by generating a specific signal coordinating the

final steps of flower maturation, including petal expansion and flower volatile emission, both

signals advertising for pollinator visit during flower maturation, CYP715 might be a major

determinant of flower’s interaction with insects and of fertilization.

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Experimental procedures

Plant materials and growth conditionsThe Col-0 accession was used as wild type in this study. T-DNA insertion lines

salk_076001 (cyp715a1-1) and salk_030920 (cyp715a1-2) were obtained from the

Arabidopsis Biological Resource Center (Ohio, USA). Unless otherwise stated, plants were

grown on soil in a growth chamber under 12h/12h day/night cycle (light intensity of 50

μmoles.m-2.s-1), 21°C/18°C day/night temperature cycle and 70% re lative humidity. Floral

material was always collected from Boyes’ stage 6.30 plants (Boyes et al., 2001).

Cloning procedures and transgenic lines production A DNA fragment corresponding to the CYP715A1 open reading frame was first PCR-

amplified from cDNA of Arabidopsis Col-0 open flowers and cloned into the pGEM-T Easy

vector (Promega) by TA cloning. The validated CYP715A1 coding sequence was then re-

amplified by PCR and transferred to the pCAMBIA230035Su vector to generate a

35S:CYP715A1 construct, and to the pCAMBIA230035Su-eGFP vector to generate a

35S:CYP715A1::eGFP fusion construct using the USERTM cloning technique (Nour-Eldin et

al., 2006). The full genomic CYP715A1 locus including a 2-kb promoter sequence was PCR-

amplified and inserted into the pCAMBIA3300u vector for complementation analysis. A 3-kb

promoter sequence upstream of the CYP715A1 START codon was first amplified and cloned

into the pGEM-T Easy vector by TA cloning. This vector was then used as template for

amplifying a gateway-compatible fragment subsequently cloned into pDONR207. The

destination vector pGWB633 was used as final GUS expression vector. Primers used for

cloning are listed in Supplemental Table S4. All constructs were checked by sequencing.

The Agrobacterium tumefaciens GV3101 strain carrying the expression vector was

used to transform Arabidopsis plants as previously described (Matsuno et al., 2009).

Agrobacterium tumefaciens LBA4404 carrying the expression vector was used to transform

N. benthamiana leaves for subcellular localization as previously described (Bassard et al.,

2012).

For CYP715A1 ectopic overexpression, a total of 21 independent transgenic T2 lines

(Col-0 background) were selected for qRT-PCR analysis. Three lines with highest expression

were considered for growth and development phenotyping. Line 2 (OE-2) was used for

molecular studies (Supplemental Figure S1). For complementation, more than 20

independent T2 lines showed flower complementation of both mutants.

Floral organs growth analysis

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Petals surface was measured according to a published method (Brioudes et al.,

2009). Cell surface was measured from SEM (Hitachi TM1000) pictures and analyzed using

the ImageJ software. The cell surface (μm2) was measured over a 4000μm2 area of the

adaxial distal region (conical cells) of petals in wild type, two mutants and an overexpressor

line. For measurement of stamen filament and pistil length, sepals, petals and short stamens

were discarded from stage 15 flowers. Filament and pistil length was determined using the

ImageJ software from pictures of at least 15 flowers collected from three different plants.

RNA isolation and qRT-PCR determination of tissue-specific gene expressionFlower material was harvested from Boyes’ 6.30 stage plants (Boyes et al., 2001) and

immediately snap-frozen in liquid nitrogen. Total RNA was isolated by the lithium chloride–

phenol method and treated with DNase I (Fermantas, Thermo Fisher Scientific, Courtaboeuf,

France) according to the manufacturer’s instructions. cDNA was synthesized with

SuperScript III Reverse Transcriptase (Invitrogen, Life Technologies, Saint Aubin, France)

using Oligo(dT)18 primers (Fermantas, Thermo Fisher Scientific, Courtaboeuf, France) and 2

μg of total RNA. Quantitative RT-PCR plates were prepared with a Biomek 3000 pipetting

system (Beckman Coulter, Villepinte, France) and run on a LightCycler 480 II device

(Roche). Each reaction comprised 2 μL of 10-fold diluted cDNA, 5 μL of LightCycler 480

SYBR Green I Master (Roche) and 250 nM of each primer in a total volume of 10 μL.

Amplification profile was as follows: 95 °C for 10 min and 40 cycles (95°C denaturation for 10

s, annealing at 60°C for 15 s, extension at 72°C fo r 15 s), followed by a melting curve

analysis from 55 to 95°C to check amplification spe cificity. Reactions were performed in

duplicate. Crossing points (Cp) were determined using the manufacturer software. Cp values

were corrected according to primer pair PCR efficiency computed with the LinReg PCR

method (Ruijter et al., 2009). Relative expression levels of genes was calculated using the 2-

Cp equation and PP2AA3 (At1g13320), SAND (At2g28390), EXP (At4g26410) and TIP41

(At4g34270) as reference genes. List of qPCR primers is available in Supplemental Table

S4.

Transcriptome StudiesMicroarray analysis was carried out using the CATMAv6.2 array based on Roche-

NimbleGen technology. A single high density CATMAv6.2 microarray slide contains twelve

chambers, each containing 219 684 primers representing all the Arabidopsis thaliana genes:

37 309 probes corresponding to TAIRv8 annotation (including 476 probes of mitochondrial

and chloroplast genes), 1796 probes corresponding to EUGENE software predictions.

Moreover, it included 5328 probes corresponding to repeat elements, 1322 probes for

miRNA/MIR, 329 probes for other RNAs (rRNA,tRNA, snRNA, soRNA) and finally several

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controls. Each long primer was in technical triplicate and in both strands (Forward and

Reverse sense) in each chamber for robust analysis. Three independent biological replicates

were produced, each replicate consisting of pooled RNA from eight plants. Flower buds

were collected on plants at developmental growth stage 6.30 (Boyes et al., 2001), cultivated

in 12h-light conditions as described above. Total RNA was extracted using the Nucleospin

Plant RNA kit (Macherey-Nagel). Twenty micrograms of RNA were treated with five units of

RQ1 DNase I (Promega) and subsequently purified using the Nucleospin RNA Clean-up kit

(Macherey-Nagel). For each comparison, one technical replicate with fluorochrome reversal

was performed for each biological replicate (i.e. four hybridizations per comparison). The

cRNAs were labelled with Cy3-dUTP or Cy5-dUTP (Perkin-Elmer-NEN Life Science

Products). Thirty pmol of each labelled cRNA were hybridized to the 12x280K CATMA slides

at +42°C for 16 hours. Two micron scanning was perf ormed with InnoScan900 scanner

(InnopsysR, Carbonne, France) and raw data were extracted using MapixR software

(InnopsysR, Carbonne, France).

Statistical Analysis of Microarray Data Experiments were designed by the statistics group of the Unité de Recherche en

Génomique Végétale. For each array, the raw data comprised the logarithm of median

feature pixel intensity at wavelengths 635 nm (red) and 532 nm (green). For each array, a

global intensity-dependent normalization using the LOESS procedure (Yang et al., 2002) was

performed to correct the dye bias. The differential analysis is based on the log ratios

averaging over the duplicate probes and over the technical replicates. Hence the numbers of

available data for each gene equal the number of biological replicates and are used to

calculate the moderated t-test (Smyth, 2004).Under the null hypothesis, no evidence that the

specific variances vary between probes is highlighted by the LIMMA library and consequently

the moderated t-statistic is assumed to follow a standard normal distribution. To control the

false discovery rate, adjusted p-values are calculated using the optimized FDR approach

(Storey and Tibshirani, 2003). Adjusted p-value 0.05 were considered as being

differentially expressed. Analysis was done with the R software. The function SqueezeVar of

the LIMMA library has been used to smooth the specific variances by computing empirical

Bayes posterior means. Kerfdr was used to calculate the adjusted p-values.

Microarray data from this article was deposited at Gene Expression Omnibus

(http://www.ncbi.nlm.nih.gov/geo/, accession No.GSE52269) and at CATdb

(http://urgv.evry.inra.fr/CATdb/; Project: RS12-08_cyp715A1) according to the “Minimum

Information About a Microarray Experiment” standards.

GUS histochemical analysis

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Tissues from T2 Arabidopsis transgenic plants harbouring the CYP715A1pro:GUS

construct were incubated in 90% acetone solution for 20 min on ice, rinsed with water, and

transferred to a GUS solution containing 1mM 5-bromo-4-chloro-3-indolyl- -D-glucuronide

(X-Gluc), 100mM sodium phosphate (pH 7.0), 10mM EDTA, 0.5mM potassium ferricyanide,

0.5mM potassium ferrocyanide and 0.1% (v/v) Triton X-100. Samples were incubated at

37°C in the dark overnight. Tissues were cleared th ree times in 75% ethanol before imaging

with a Nikon (ECLIPSE, E800) microscope.

Scanning Electron Microscopy (SEM) and Transmission electron microscopy (TEM)Open flowers were infiltrated with a solution of glutaraldehyde (2.5%) and

paraformaldehyde (2%) in phosphate buffer (0.1 M, pH 7.2), dehydrated in a graded series of

ethanol, transferred to a mixture of ethanol and hexamethyldisilazane at increasing

concentrations of 25%, 50%, 75%, and 100%, and dried overnight. Dried anthers were

mounted on specimen stubs using double-sided copper tape. The samples were coated with

gold: palladium in a sputter coater (Balzers SCD 030, Leica, Vienna, Austria) and imaged

using SEM (S800; Hitachi, Tokyo, Japan). Petals were directly observed with a Hitachi TM-

1000 table-top SEM. Single flowers from young buds were selected for TEM analysis as

described previously (Cheminant et al., 2011).

Bioinformatics CYP715 coding sequences from Angiosperms were retrieved from Phytozome

(http://www.phytozome.net). CYP715 from the gymnosperm species Picea abies and

Sciadopitys sverticillata were retrieved from ConGenIE (http://congenie.org/) and the OnekP

project database (http://www.onekp.com), respectively. Other out-group P450s were

retrieved from CYPedia (http://www-ibmp.u-strasbg.fr/~CYPedia/). Protein sequences were

aligned with MUSCLE (Edgar, 2004) using the MEGA5 software (Tamura et al., 2011).

Phylogeny was inferred from the proteins alignment by Maximum Likelihood analysis using

the WAG model and 1000 bootstraps replications.

The calculation of ratios of non-synonymous to synonymous substitutions was carried

out by codeML analysis from PAML version 4.6 (Yang, 2007). A likelihood ratio test (LRT)

was performed by comparing twice the difference in log likelihood values between M1a and

M2a models using a 2 distribution (Anisimova et al., 2001).

Hormone profiling Gibberellins (GAs)

Quantitative analysis of GAs was carried out using 0.2-0.5 g of freeze-dried whole

inflorescences of wild type and two cyp715a1 mutants as described previously (Rieu et al.,

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2008). Each GA was quantified relative to its 17-2H2-labeled analogue as internal standard,

obtained from Prof. L. Mander (Australian National University, Canberra ACT, Australia).

Other phytothormones

ABA, IAA, JA and its derivatives, were extracted from approximately 200 mg of fresh

material. Samples were extracted twice with 1mL of ice-cold 80% methanol containing 5μM

dihydrojasmonic acid as internal standard. The two supernatants were recovered, pooled

and dried under vacuum overnight. Metabolites were resuspended in 200 μL of 80% MeOH

prior to UPLC-MS/MS analysis using a Waters Quattro Premier XE (Waters, Milford, MA)

equipped with an electrospray ionization source coupled to an Acquity UPLC system

(Waters) fitted with an Acquity UPLC BEH C18 column (100 x 2.1 mm, 1.7 m; Waters) and

pre-column. Nitrogen was used as the drying and nebulizing gas in-source. The nebulizer

gas flow was set to 50 L/h, and the desolvation gas flow was set to 900 L/h. The interface

temperature was set to 400 °C, and the source tempe rature was set to 135 °C. The capillary

voltage was set to 3.2 kV. Data acquisition and analysis were performed with the MassLynx

software and relative quantitation was performed by peak integration without smoothing. The

mobile phase consisted of water (A) and methanol (B), both containing 0.1% formic acid. The

run started by 2 min of 95% A, then a linear gradient was applied to reach 100% B at 12 min

followed by isocratic run using B during 2 min. Return to initial conditions was achieved in 3

min, with a total run time of 17 min. The column was maintained at 35 °C with a flow rate of

0.35 ml/min, injecting 5 μl of sample. Ionization was in positive or negative mode. Transitions

in positive mode: IAA, 176 > 130 (CV 20V, CE 17V); JA-Ile, 324 > 131 (CV 25V, CE 20V); in

negative mode: 12OH-JA, 225 > 59 (CV 25V, CE 25V); 12OH-JA-Ile, 338 > 130 (CV 25V, CE

20V); 12COOH-JA-Ile, 352 > 130 (CV 25V, CE 20V); JA, 209 > 59 (CV 25V, CE 23 V); ABA,

263 > 153 (CV 25V, CE 12V). Methods for intermediates of the IAA cascade quantification

have been reported previously (Novak et al., 2012).

Volatile collection and quantification by GC-MS Flower volatile collection was carried out as previously described (Ginglinger et al.,

2013). About thirty plants per genotype were cultivated until flowering and pooled in three

replicates for headspace collection. Approximately 60 inflorescences per line and per

replicate were gathered as a bunch and inserted in a small glass vial filled with 4 mL water.

Bunches were then placed in sealed 1-L glass jars equipped with inlet and outlet connectors

adapted for metal cartridges (140 mm length, 4 mm diameter) containing sorbent. A vacuum

pump was used to draw air through the glass jar at 100 mL/min. The incoming air was

purified through a cartridge containing 100 mg Tenax TA (20/35; Grace Scientific) and 100

mg activated charcoal 20-40 mesh (Sigma Aldrich). The volatiles emitted by the flowers were

trapped at the outlet on a cartridge containing 150 mg Tenax TA. Volatiles were sampled for

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7 h. After volatile collection, flowers of each sample were swiftly cut from the inflorescence

and weighed in order to normalize the emission of volatiles. Tenax cartridges were analyzed

on a PerkinElmer Clarus 680 equipped with a Perkin Elmer Clarus 600T quadrupole mass

spectrometer and a TurboMatrix 100 thermal desorber (TDS) (PerkinElmer). 2μL of 400 μM

nonyl acetate (Sigma) was added to each cartridge as an internal standard. Tenax cartridges

were first dry-purged with nitrogen at 50mL/min for 3 min at ambient temperature. Volatiles

were released from Tenax cartridges by heating at 250°C for 5 min, with a nitrogen flow of 50

mL/min. Desorbed volatiles were focused on a Tenax cold trap electronically cooled at -30°C.

Volatiles were then injected on the Perkin Elmer GC-MS device described above in 1/10 split

mode under a 15 psi constant He pressure used as the vector gas into the analytical column

by heating the cold trap to 280°C for 5 min. Compou nds were separated on a HP5-MS

column (30 m, 0.25 mm, 0.25 μm thickness; Agilent Technologies) with a temperature

program of 1 min at 50 °C, 20°C/min to 320 °C, and 5 min at 320 °C and analyzed using

electron-impact MS spectra (70eV, m/z 50–300, scan time 0.3 s). Volatiles were identified on

the basis of their retention time and mass specta and compared to authentic standards when

available (Sigma Aldrich). Quantification of terpene emission was carried out by integration of

the specific 93 and 126 m/z ion peaks at the retention time of each terpenes and nonyl

acetate respectively, and using standard curves established with concentration ranges of

authentic standards when available or extrapolated when not available. Emission was

calculated as the mass of terpenes emitted per mass of fresh weight of flowers per hour.

Production of recombinant proteins Expression in yeast

The USERTM cloning technique (Nour-Eldin et al., 2006) was used to insert the

CYP715A1 coding sequence into the USER compatible yeast expression vector pYeDP60u2

(Höfer et al., 2013) using the primers listed in Supplemental Table S4. The CYP715

sequence from B. distachyon was generated by gene synthesis and optimized for codon

usage in yeast (Genecust) (Supplemental Fig. S7). The recoded fragment was first cloned

into the pUC57 vector flanked with BamHI and KpnI restriction sites, before transfer to the

pYeDP60 yeast expression vector. WAT11 and WAT21 yeast strain transformation,

cultivation and preparation of microsomal membranes were carried out as described by

Gavira et al. (Gavira et al., 2013). The Saccharomyces cerevisiae WAT11 and WAT21

strains, expressing the ATR1 and ATR2 cytochrome P450 reductase from Arabidopsis

thaliana, respectively, under the galactose-inducible promoter GAL10-CYC1 were gifts from

D. Pompon (Urban et al., 1997). Expression of functional cytochrome P450 was determined

by differential spectrophotometry according to former report (Omura and Sato, 1964).

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Expression in insect cells

Recombinant CYP715A1 protein was prepared by expressing the full lengths of the

Arabidopsis CYP715A1 cDNA in a baculovirus expression vector system using the Bac-to-

Bacbaculovirus expression system (Life Technologies) and Spodoptera furugiperda cells

(Sf9; Life Technologies) (Ohnishi et al., 2006). Briefly, The ORF of CYP715A1 cDNA was

amplified by PCR using primers TO-427 and TO-428 (Supplemental Table S4). The PCR

amplified product was cloned via the GATEWAY entry vector pENTR/D-TOPO (Life

Technologies) into pDEST8 to generate a baculovirus–insect cell expression clone.

The pDEST8 construct was then used for the preparation of a recombinant bacmid

DNA by transformation of Escherichia coli strain DH10Bac (Life Technologies), and

transfection of the insect cells was done according to the manufacturer's instructions (Life

Technologies). For large-scale expression, Sf9 cells were propagated as suspension cultures

in Grace's insect medium containing 0.1% (w/v) Pluronic F-68 (Life Technologies), and

incubated in a rotary shaker at 27°C and 150 rpm. F or expression of recombinant CYP715A1

protein, Sf9 cells were cultured in the above Grace's insect medium supplemented with

100 m 5-aminolevulinic acid and 100 m ferrous citrate to compensate for the low heme

synthetic capacity of the insect cells. Microsomal fraction of the insect cells expressing

recombinant CYP715A1 was obtained from the infected cells (500 mL of suspension-cultured

cells). Infected cells were washed with PBS buffer and suspended in buffer A consisting of

20 mM potassium phosphate (pH 7.25), 20% (v/v) glycerol, 1 mM EDTA, and 1 mM DTT.

The cells were sonicated and cell debris was removed by centrifugation at 10,000g for 15

min. The supernatant was further centrifuged at 100,000g for 1 h and the pellet was

homogenized with buffer A to provide the microsomal fractions. The microsomal fractions

were stored at –80oC before the enzyme assay.

Enzyme assays When carried out with yeast microsomes, enzyme assays contained in a final volume

of a 100 μL, 10 μL of microsomes, 20 mM potassium phosphate buffer (pH 7.5), 100 μM

substrate and 500 μM NADPH. For insect cell microsomes, the reaction mixture consisted of

10 μL microsomes, 0.1 unit of Arabidopsis NADPH-cytochrome P450 reductase 2 (ATR2),

100 mM potassium phosphate (pH 7.25), 100 μM substrate, and 1 mM NADPH. The reaction

was initiated by addition of NADPH, then incubated at 28°C for 30 min and terminated with

10 μL of 50% acetic acid. The mix was vortexed, supplemented with 40 μL of acetonitrile and

centrifuged prior to liquid chromatography analysis.

Samples were analyzed by either UPLC-MS/MS as described above or reverse-

phase HPLC (Alliance 2695, Waters) with photo-diode array detection at 265nm for ABA

(Photodiode 2996, Waters). For this purpose, 50 μL of sample were injected onto a NOVA-

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PAK C18 4.6 x 250 mm column heated at 37°C. Separat ion was carried out using 0.2%

acetic acid in water (A) and 0.2% acetic acid in acetonitrile (B) at a 1 mL.min-1 flow rate.

Elution program was as follows: 5% to 100% B in 15 minutes (curve 8), 100% B for 2 min.

Assays with 14C-labeled GAs and precursors were analysed by HPLC with on-line

radiomonitoring as described previously (Ward et al., 2010).

Accession Numbers Sequence data from this article can be found in the Arabidopsis Genome Initiative or the

Rice Genome Annotation Project databases under the following accession numbers:

CYP715A1 (AT5G52400), CYP714A1 (AT5G24910), CYP714A2 (AT5G24900), Os-

CYP714C2 (LOC_Os12g02640), CYP735A1 (AT5G38450), CYP735A2 (AT1G67110),

CYP709B1 (AT2G46960), CYP721A1 (AT1G75130), CYP734A1 (AT2G26710), CYP72C1

(AT1G17060), CYP98A3 (AT2G40890), Tryptophan synthase (AT5G28237), CYP79B2

(AT4G39950), CYP79B3 (AT2G22330), JAZ5 (AT1G17380), JAZ9 (AT1G70700), MYB21

(AT3G27810), MYB24 (AT5G40350), MYC2 (AT1G32640), TPS03 (AT4G16740), TPS10

(AT2G24210), TPS11 (AT5G44630), TPS14 (AT1G61680), TPS21 (AT5G23960). PP2AA3

(AT1G13320), SAND (AT2G28390), EXP (AT4G26410) AND TIP41 (AT4G34270).

Authors’ contributions Conceptualization, Z.H., H.R. and D.W-R.; Investigation, Z.H., H.R., B.B., R.L., J.M., M.E.,

V.D., S.P., T.O., A.P., F.G.; Formal Analysis, R.T., V.B. and R.L.; Writing – Original Draft,

Z.H., H.R., R.T. and D.W-R.; Writing – Review & Editing, Z.H., H.R., R.T., P.A., P.H. and

D.W-R.; Visualization, H.R.; Supervision, H.R., P.A., P.H. and D.W-R.; Project

administration, D.W-R.; Funding Acquisition, Z.L. and D.W-R.

Acknowledgements ZL is grateful to the China Scholarship Council and the Région Alsace for co-funding a PhD

scholarship. HR and DWR acknowledge the support of the Agence Nationale pour la

Recherche via the PHENOWALL ANR-10-BLAN-1528 project, and of the Freiburg Institute

for Advanced Studies (FRIAS) and the University of Strasbourg Institute for Advanced Study

(USIAS) for the METABEVO grant. The European Fund for Regional Development in the

programme INTERREG IVA Broad Region EU invests in your future supported the study on

flower volatiles. AP acknowledges support of the Ministry of Education, Youth and Sport of

the Czech Republic (grant LO1204 from the National Program of Sustainability I). We are

very grateful to Pr. D. Nelson (University of Tennessee, Memphis) for making available to us

his extended annotation of the CYP715 family in higher plants.

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Table 1. Genes differentially expressed in the cyp715a1-2 mutant and related to auxin/indoles and jasmonate.

Locus Annotation Function cyp715a1/WT ratio (log2)

Adjustedp-value

Auxin and Indoles

AT5G28237 Tryptophan synthase Tryptophan biosynthesis -2.47 0.00E+0

AT4G39950 CYP79B2 Indole-3-acetaldoxime biosynthesis -0.82 2.96E-2

AT2G22330 CYP79B3 Indole-3-acetaldoxime biosynthesis -1.32 1.63E-11

AT3G44300 NIT2 Auxin biosynthesis +1.23 2.13E-9

AT1G13980 GNOM Auxin transport -0.95 2.36E-4

AT2G36910 ABCB1 Auxin transport -0.85 1.13E-2

AT2G33860 ARF3 Auxin stimulus response -0.82 2.35E-2

AT1G54070 Auxin/dormancy

associated family protein Auxin stimulus response +1.13 1.73E-7

AT3G20220 SAUR47 Auxin stimulus response +1.04 6.92E-6

Jasmonate

AT3G25770 AOC2 Jasmonate biosynthesis -0.93 5.54E-4

AT1G44350 ILL6 Jasmonate catabolism -0.93 6.26E-4

AT3G27810 MYB21 Jasmonate stimulus response -1.13 1.51E-7

AT5G40350 MYB24 Jasmonate stimulus response -0.85 1.01E-2

AT3G01530 MYB57 Jasmonate stimulus response +1.00 4.55E-5

AT1G15750 TOPLESS Jasmonate stimulus response -1.06 3.78E-6

AT1G17380 JAZ5 Jasmonate stimulus response -1.14 9.55E-8

AT1G70700 JAZ9 Jasmonate stimulus response -1.04 7.22E-6

AT3G16470 JR1 Jasmonate stimulus response -1.01 2.48E-5

AT4G23600 CORI3 Jasmonate stimulus response -1.36 0.00E+0

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Figures legends

Figure 1. Phylogeny of the CYP715 family. Maximum likelihood tree illustrating phylogenetic relationships of CYP715 proteins. Species containing

CYP715 as a single copy gene are highlighted in red. Note that S. verticillata sequence was retrieved

from transcriptome data: it was thus not possible to ascertain if it is present as a single copy gene.

Members of other CYP72 clan families (i.e. CYP714, CYP735, CYP709, CYP721, CYP734 and

CYP72) were added to the analysis. Arabidopsis thaliana CYP98A3 (AtCYP98A3) was used to root

the tree. Phylogeny consistency was tested with 1000 bootstrap iterations; only values above 50% are

displayed on branches. At, Arabidopsis thaliana; Os, Oryza sativa.

Figure 2. CYP715A1 is expressed in the tapetum during pollen development and anther filaments during flower maturation. (A-C) qRT-PCR monitoring of CYP715A1

expression in various plant organs (A), flower stages (B) and organs of mature flowers (C) using total

RNA isolated from Boyes’ stage 6.30 plants. Error bars represent the SE of 3-4 independent

replicates. (D-G) Typical GUS staining pattern in flowers: whole inflorescence (D), close-up on flower

buds (E), flower buds anther cross section (F) and close-up on an open flower (G).

Figure 3. The cyp715a1 null mutation prevents petal elongation but not stamen or pistil development. (A-I) Defect in petal and petal cell growth in the cyp715a1 mutants. (A-C) Flowers of cyp715a1

mutants failed to open. (D) Typical petal phenotypes in mature flowers. (E-G) Typical scanning

electron micrographs of petal surface open flowers, scale bar, 20 μm. (H) Stamen and pistil in mature

flowers. Short stamens were discarded before imaging. (I) Average petal area of stage 15 flowers.

Error bars represent the SE of 46-50 independent measurements. (J) Scanning electron micrographs

(e.g. panels E-G) were analysed to determine average size of petal cells from open flowers. Error bars

represent the SE of five independent measurements made on five different flowers. Statistical

significance was calculated by two-tailed Student’s t-test: *P< 0.05; ***P< 0.001. (J-L) CYP715

transiently affects intine formation and pollen development. (K) Average long stamen filament length of

stage 15 flowers. Error bars represent the SE of 47-58 independent measurements. (L) Average pistil

length of stage 15 flowers. Error bars represent the SE of 13-18 independent measurements.

Statistical significance was calculated by two-tailed Student’s t-test: *P< 0.05; ***P< 0.001.

Fig. 4. Transient defect in intine formation in the cyp715a1 mutants. (A-F) Typical transmission electron micrographs of pollen sections from wild type and insertion

mutants. Bicellular pollen of cyp715a1 mutants shows a wavy intine layer and an absence of vesicular

structures (B-C) compared to wild type (A). At the tricellular stage, pollen of mutants appears similar to

wild type (D-F). Scale bar, 1 μm. in, intine, v, vesicule. (G-H) Scanning electron micrographs of wild-

type and mutants mature pollen reveal no significant difference. Scale bar, 20 μm.

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Figure 5. Targeted transcriptional analysis of genes related to jasmonate signalling and auxin/indole biosynthesis. Expression of genes found differentially expressed in transcriptome analysis was assessed in flower

buds and open flowers of the two cyp715a1 mutants, the CYP715A1-overexpressor line (OE-2) and

the wild type (Col-0). Error bars represent the SE of 3-4 independent replicates. Statistical significance

was calculated by two-tailed Student’s t-test: *P< 0.05; **P< 0.01.

Figure 6. Hormones profiling of cyp715a1 mutants flowers. (A) Gibberellins (GAs) profiling. Absolute quantification of GAs was carried out in whole inflorescence

of wild type (Col-0) and cyp715a1 mutants. Error bars represent the SD of two independent replicates.

The values are displayed according to biosynthetic sequences with the GA2-oxidase (GA2ox) products

shown separately. (B) IAA, ABA and JAs profiling. Relative concentration was evaluated by UPLC-

MS/MS in flower buds and mature flowers of the two cyp715a1 lines, the CYP715A1 overexpressor

(OE-2) and wild type (Col-0). JAs metabolic route is indicated with black arrows. Error bars represent

the SE of three independent replicates. Statistical significance was calculated by two-tailed Student’s

t-test: *P< 0.05; **P< 0.01. ABA, abscisic acid; IAA, indole-3-acetic acid; JA, jasmonic acid; JA-Ile;

jasmonate-isoleucine conjugate; 12OH-JA-Glc, 12-hydroxyjasmonate-glucose conjugate.

Figure 7. Relaxation of the cyp715 mutants flower phenotype by GA3 or coronatine treatments. Immature inflorescences were dipped for two seconds into GA3 (50μM) and coronatine (1μM)

solution containing 0.1% ethanol and 0.02% tween. Treatment was performed every two

days for two weeks. Mock treatments were performed using a 0.1% ethanol and 0.02%

tween solution. (A) Pictures of bunch of five inflorescences each. (B) The number of fully

open flowers per inflorescence was determined. Data are the mean ± SE of measurements

made on 19-24 inflorescences derived from four different plants. Statistical significance

(mock versus treatment) was calculated by two-tailed Student’s t-test: **P < 0.01, ***P <

0.001.

Figure 7. Relaxation of the cyp715 mutants flower phenotype by GA3 or coronatine treatments. Immature inflorescences were dipped for two seconds into GA3 (50μM) and coronatine (1μM)

solution containing 0.1% ethanol and 0.02% tween. Treatment was performed every two

days for two weeks. Mock treatments were performed using a 0.1% ethanol and 0.02%

tween solution. (A) Pictures of bunch of five inflorescences each. (B) The number of fully

open flowers per inflorescence was determined. Data are the mean ± SE of measurements

made on 19-24 inflorescences derived from four different plants. Statistical significance

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(mock versus treatment) was calculated by two-tailed Student’s t-test: **P < 0.01, ***P <

0.001.

Figure 8. Floral emission of volatile sesquiterpenes is greatly reduced in cyp715a1mutants. (A) Relative expression of MYC2 and floral terpene synthases (TPS) was evaluated by qRT-PCR in

flower buds and mature flowers of the two cyp715a1 mutants, the CYP715A1 overexpressor (OE-2)

and wild type (Col-0). Error bars represent the SE of four independent replicates. Statistical

significance was calculated by two-tailed Student’s t-test: *P< 0.05; **P< 0.01. (B) Quantitative

determination of floral volatile sesquiterpenes emitted from wild type (Col-0), the two cyp715a1

mutants and the cyp715a1-2 complemented with the wild-type CYP715A1 locus (COMP). Only results

for TPS21-derived volatiles [i.e. (–)-E- -caryophyllene, -humulene and (–)- -copaene] are shown.

Error bars represent the SE of three independent volatiles collections. Statistical significance was

calculated by two-tailed Student’s t-test: *P< 0.05; **P< 0.01. (C) Typical GC-MS chromatograms

focused on floral volatile sesquiterpenes elution window. Peaks labelled in red, green and yellow are

products of TPS21, TPS11 and TPS03, respectively. 1: (–)- -copaene; 2: (–)-E- -caryophyllene; 3:

(+)-thujopsene; 4: E- -farnesene; 5: -humulene; 6: -acoradiene; 7: unidentified; 8: (+)- -

chamigrene; 9: -farnesene; 10: (–)- -bisabolene; 11: cuparene; 12: -sesquiphellandrene; 13:

unidentified. IS, internal standard.

Figure 9. Model of CYP715A1 function. Terms in bold are supported by experimental data.

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Medicago truncatula 1Phaseolus vulgaris 2

Phaseolus vulgaris 3Glycine max 3

Glycine max 2Glycine max 6

Glycine max 5Glycine max 1Glycine max 4

Phaseolus vulgaris 1

Prunus persica 2Prunus persica 1

Cucumis sativusAquilegia coerulea

Manihot esculenta 1Manihot esculenta 2

Ricinus communisLinum usitatissimum

Populus trichocarpa 1Populus trichocarpa 2

Carica papayaVitis vinifera

Solanum lycopersicumSolanum tuberosum 2Solanum tuberosum 1

Nicotiana attenuataEucalyptus grandis 1

Eucalyptus grandis 2Eucalyptus grandis 3

Eucalyptus grandis 4Citrus sinensis

Theobroma cacaoGossypium raimondii 1

Gossypium raimondii 2Brassica rapa 2

Eutrema salsugineumBrassica rapa 1

Capsella rubellaAtCYP715A1

Arabidopsis lyrataSorghum bicolor

Zea maysOryza sativa

Brachypodium distachyonSetaria italica 1

Setaria italica 2Setaria italica 3

Amborella trichopodaSciadopitys verticillata

Picea abiesAmborella trichopoda CYP714

OsCYP714C2AtCYP714A1

AtCYP714A2AtCYP735A1

AtCYP735A2AtCYP709B1

AtCYP721A1AtCYP734A1

AtCYP72C1AtCYP98A3

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CYP715 family

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Extra CYP72 clan families

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0

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6

9

12

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

0.0

0.5

1.5

2.0

2.5

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Rel

ativ

e m

RN

A le

vel

Tryptophansynthase

** **** **

JAZ5 JAZ9

*

* *

*

** **

Flower buds

Open flowers

MYB21 MYB24

* ** *

CYP79B2 CYP79B3

*

* *

** **** **

0.0

1.0

2.0

3.0

4.0

5.0R

elat

ive

mR

NA

leve

l

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.00

0.05

0.10

0.15

0.20

1.0

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Flower buds Open flowers

0

3

6

9

12

15

Rel

ativ

e un

its

ABA

0

20

40

60

80 IAA

0

3

6

9

12

15 JA

0

20

40

60

80

100 JA-Ile

0

10

20

30

40

50

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Rel

ativ

e un

its

12OH-JA-Ile

0

5

10

15

20

25

30

35

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

12OH-JA

0

40

80

120

160

200 12COOH-JA-Ile

0

5

10

15

20

25

30

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

12OH-JA-Glc

*

*

***

*

** **

*** ** **

** **

* *

Col-0 cyp715a1-1 cyp715a1-2

GA1 pathway(13-ox pathway)

GA

2ox

prod

.

0 2 4 6 8 10 12

GA12

GA15

GA24

GA9

GA4

GA51

GA34

GA53

GA44

GA19

GA20

GA1

GA29

GA8

Gibberellin level (ng.g -1 DW)

GA

2ox

prod

. bi

osyn

thes

is

Rel

ativ

e un

its

bios

ynth

esis

GA4 pathway(13-deox pathway)

A B

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

0

1

2

3

Col-0

cyp7

15a1

-1

cyp7

15a1

-2

cyp7

15a1

-1

cyp7

15a1

-2

cyp7

15a1

-1

cyp7

15a1

-2

Num

ber o

f ful

ly o

pen

flow

ers

pe

r inf

lore

scen

ce **

***

**

Mock GA3 Coronatine

Col-0

cyp715a1-1

cyp715a1-2

cyp715a1-1

cyp715a1-2

cyp715a1-1

cyp715a1-2

Mock GA3 CoronatineA

B

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPT

Rel

ativ

e ab

unda

nce

of th

e io

ns c

urre

nt m

/z 9

3 an

d m

/z 1

26 (%

) 0

100

50

Col-0

cyp715a1-1

cyp715a1-2

COMP

IS

1

2

3 4

5

6 78 9

10 11 12

10 1312

13 Time (min)

11

C

0

100

50

0

100

50

0

100

50

A

B

0

20

40

60

80

Col-0

cyp7

15a1

-1

cyp7

15a1

-2

COMP

ng e

mitt

ed.h

-1.g

-1 fl

ower

s

0

2

4

6

8

10

12

Col-0

cyp7

15a1

-1

cyp7

15a1

-2

COMP 0

2

4

6

8

Col-0

cyp7

15a1

-1

cyp7

15a1

-2

COMP

**

*

** ** **

**

**

(–)- -copaene-humulene(–)-E- -caryophyllene

0.0

1.0

2.0

3.0

4.0

5.0R

elat

ive

mR

NA

leve

l

0.0

0.5

1.0

1.5

2.0

2.5

3.0TPS21

Flower buds Open flowers

* * ** *** *

TPS14

0

3

6

9

12

15

0.0

0.2

0.4

0.6

0.8

1.0

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Rel

ativ

e m

RN

A le

vel

** **

* *

TPS03

0.0

2.0

4.0

6.0

8.0

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

TPS10

0.0

1.0

2.0

3.0

4.0

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

Col-0

cyp7

15a1

-1

cyp7

15a1

-2OE-2

TPS11

MYC2

* *

MANUSCRIP

T

ACCEPTED

ACCEPTED MANUSCRIPTCYP715A1

JASMONATES GIBBERELLINS AUXINTransport & signaling

GNOM/ARF3

JAZ5/JAZ9MYB21/MYB24

MYC2DELLA

Volatiles emission

Intinedeposition

Petalgrowth

Flower maturation