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Molecular Plant Research Article

A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation Zhenhua Liu1,11, Beno^ıt Boachon1, Raphae¨l Lugan1,12, Raquel Tavares2, Mathieu Erhardt1, ^ me Mutterer1, Vale´rie Demais3, Ste´phanie Pateyron4, Ve´ronique Brunaud5, Je´ro Toshiyuki Ohnishi6, Ales Pencik7, Patrick Achard1, Fan Gong8,13, Peter Hedden8, Danie`le Werck-Reichhart1,9,10,* and Hugues Renault1,9,10 1

Institute of Plant Molecular Biology, Centre National de la Recherche Scientifique (CNRS), University of Strasbourg, 67084 Strasbourg, France

2

Laboratoire de Biome´trie et Biologie E´volutive, Universite´ Lyon 1, CNRS, 69622 Villeurbanne, France

3

Plateforme d’Imagerie In Vitro, IFR 37 de Neurosciences, 67084 Strasbourg, France

4

Transcriptomic Platform, Unite´ de Recherche en Ge´nomique Ve´ge´tale (URGV), INRA, Universite´ d’Evry Val d’Essonne, CNRS, 91057 Evry, France

5

Bioinformatics for Predictive Genomics, URGV, INRA, Universite´ d’Evry Val d’Essonne, CNRS, 91057 Evry, France

6

Graduate School of Agriculture, Shizuoka University, Shizuoka, 422-8529 Japan

7

Laboratory of Growth Regulators & Department of Chemical Biology and Genetics, Centre of the Region Hana´ for Biotechnological and Agricultural Research, Faculty of Science, Palacky´ University & Institute of Experimental Botany AS CR, 771 47 Olomouc, Czech Republic

8

Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK

9

University of Strasbourg Institute for Advanced Study (USIAS), 67084 Strasbourg, France

10

Freiburg Institute for Advanced Studies (FRIAS), University of Freiburg, 79104 Freiburg, Germany

11Present

address: 158 Emerson Hall, Section of Plant Biology, School of Integrative Plant Science, Cornell University, Ithaca, NY 14853, USA

12Present

address: Laboratoire Physiologie des Fruits et Le´gumes – EA 4279, Campus Agroparc, Avignon, France

13Present

address: Home Office Science – Centre for Applied Science and Technology, Woodcock Hill, Sandridge, St Albans, Herts AL4 9HQ, UK

*Correspondence: Danie`le Werck-Reichhart ([email protected]) http://dx.doi.org/10.1016/j.molp.2015.09.002

ABSTRACT Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, 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 showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower–insect interaction. Keywords: flower development, phylogenomics, negative selection, jasmonate, gibberellins, volatile compounds Liu Z., Boachon B., Lugan R., Tavares R., Erhardt M., Mutterer J., Demais V., 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 Regulates Flower Maturation. Mol. Plant. 8, 1751–1765.

INTRODUCTION

Sequencing of a larger number of plant genomes brings a new outlook on the global picture and, for example, highlights some

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.

Published by the Molecular Plant Shanghai Editorial Office in association with Cell Press, an imprint of Elsevier Inc., on behalf of CSPB and IPPE, SIBS, CAS.

Molecular Plant 8, 1751–1765, December 2015 ª The Author 2015. 1751

Molecular Plant 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 organization, cofactor metabolic processes, or embryonic development (De Smet et al., 2013). Although less frequent, some single-copy genes can also be found in large superfamilies encoding transcription factors or enzymes (Nelson and Werck-Reichhart, 2011; Airoldi and Davies, 2012; De Smet et al., 2013). 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, involved for example in the biosynthesis of lignin precursors or hormone homeostasis, are present in low-copy, sometimes single-copy number, and broadly distributed across plant taxa (Nelson and WerckReichhart, 2011). It also indicated 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 also found 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 (GA) deactivation and homeostasis in rice via 16a,17epoxidation or 13-hydroxylation (Zhu et al., 2006; Magome et al., 2013). Genetic evidence suggests that CYP714s play a similar role in Arabidopsis (Zhang et al., 2011; Nomura et al., 2013). The function of the CYP715 proteins, however, has 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. Here, we provide evidence that CYP715A1 in Arabidopsis regulates petal development, floral hormone homeostasis, and volatile terpenoid emission.

RESULTS CYP715 Is a Single or Low-Copy Gene that 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; Matasci et al., 2014) 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,

A P450-dependent Signal Regulates Flower Maturation 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 also usually exhibit high sequence conservation (De Smet et al., 2013). To investigate the selection regimes acting on the remarkably few CYP715 genes, we calculated the ratios of non-synonymous to synonymous substitutions (u = dN/dS) in the whole family using the one-ratio model from PAML software (Yang, 2007). This model assumes the same u value for all the lineages. The calculated u of 0.11781 indicates a strong purifying selection for the CYP715 family in angiosperms (Supplemental Table 1). Furthermore, site models in PAML allowing the u 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 u value of 0.09073 and nearly 15% of the sites have an u value of 1. No sites under positive selection were significantly identified on the CYP715 sequences (Supplemental Table 1). 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–2C) indicated 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 stages 5–9; Figure 2D–2F) and in the anther filament upon flower maturation (flower stages 12–15; Figure 2D and 2G). Staining did not reveal any gene expression in other organs 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 2 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–3D, 3I) associated with reduced cell size (Figure 3E–3G, and 3J). Absence of curvature of the shortened petals and defective flower opening (Figure 3B–3D) was typical of mutations affecting petal growth. The petal growth phenotype could be rescued when plants

1752 Molecular Plant 8, 1751–1765, December 2015 ª The Author 2015.

A P450-dependent Signal Regulates Flower Maturation

Molecular Plant

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 the S. verticillata sequence was retrieved from transcriptome data; it was thus not possible to ascertain whether it is present as a singlecopy gene. Members of other CYP72 clan families (i.e. CYP714, CYP735, CYP709, CYP721, CYP734, and CYP72) were added to the analysis. A. 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.

were grown under long-day conditions (i.e. 16-h light regime), especially in early arising flowers of the inflorescence (Supplemental Figure 3), while it was maintained under 12-h 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 4). No alteration in the growth of other floral organs, such as stamen or pistil, was observed (Figure 3H, 3K, and 3L). Ectopic overexpression of CYP715A1 under control of a CaMV35S 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 and 3J.

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., 2015). 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


Molecular Plant


A

B

C

D

E

F

G

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 three to four 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).

vesicular structures detected in the cytosol of the wild-type pollen grains was much less evident in the mutant lines (Figure 4B and 4C). 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–4I) was detected. Overall, alteration in flower development did not translate into any reduced fertility for Arabidopsis plants grown under controlled conditions.

Loss of CYP715A1 Function Causes Extensive Transcriptional Changes in Reproductive Tissues The loss of CYP715A1 function affected the 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 signaling compound, a comparative transcriptomic analysis of the cyp715a1-2 mutant versus wild-type flower buds was carried out using the CATMA6.2 microarray (see Methods for

experimental procedures). This analysis identified a total of 370 genes differentially expressed in the mutant compared with wild-type (log2 R 0.8, P < 0.05); 188 were upregulated and 182 were downregulated (for complete list see Supplemental Dataset 1). 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 list of upregulated genes (Supplemental Table 2), which is consistent with the pollen phenotype of cyp715a1 mutants described above. Pollen wall-related genes were also found to be overrepresented in the downregulated genes list (Supplemental Table 2). 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 downregulated genes list (Table 1 and Supplemental Table 2), suggesting that loss of CYP715A1 function interferes with hormone signaling 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 with 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 to be downregulated in mutant lines, thus confirming microarray results (Figure 5). The two JA repressor genes, JA ZIM DOMAIN (JAZ) JAZ5 and JAZ9, were also significantly downregulated 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.

CYP715A1 Regulates Floral Hormone Homeostasis To determine how CYP715A1 affects flower hormone homeostasis and to obtain information on the pathway potentially involving CYP715A1, we next carried out hormone profiling of buds and open flowers. Petal development is thought to be mainly controlled by 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 (Zhu et al., 2006; Magome et al., 2013). 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 was able to 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.


Molecular Plant

A P450-dependent Signal Regulates Flower Maturation A

B

C

D

E

F

H

I

J

K

L

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 mm. (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. Statistical significance was calculated by two-tailed Student’s t-test: *P < 0.05; ***P < 0.001. (J) Scanning electron micrographs (e.g. panels E–G) were analyzed 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. (K and 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.

Transcriptome analysis suggested a downregulation of auxin and JA pathways in cyp715a1-suppressed lines (Table 1, Supplemental Table 2, and Figure 5). A broader hormone profiling did not reveal any significant modification in IAA and abscisic acid (ABA) contents, but indicated a profound perturbation in the JA metabolism (Figure 6B and Supplemental Table 3). 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-JAGlc) in young buds and open flowers of cyp715a1 lines, whereas only moderate decreases in JA and JA-Ile were observed in young buds. Conversely, buds accumulated free tuberonic acid (12OH-JA). Consistent with the transcription data, metabolic profiles thus indicate a downregulation of the JA pathway in CYP715A1-deficient plants, with only a minor decrease in JA and JA-Ile, 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 signaling, 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, however, was less effective than GA3.

CYP715A1 Regulates Floral Volatile Emission Our transcriptome analysis indicated a strong downregulation of two terpene synthases (TPS) genes, TPS21 and TPS14, in the cyp715a1-2 line (Supplemental Dataset 1). According to previous work (Hong et al., 2012; Reeves et al., 2012), flower maturation, including petal expansion and TPS21- and TPS11dependent volatile emission, involves the JA-activated transcription factors MYB21 and MYB24 that were also downregulated in cyp715a1 mutants (Supplemental Dataset 1; Figure 5). TPS21 is reported to produce the major floral volatile b-caryophyllene in Arabidopsis (Tholl et al., 2005; Hong et al., 2012). 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). This 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 downregulation 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


Molecular Plant


A

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Figure 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 and C) compared with wildtype (A). At the tricellular stage, pollen of mutants appears similar to wild-type (D–F). Scale bar, 1 mm. in, intine, v, vesicle. (G–I) Scanning electron micrographs of wild-type and mutant mature pollen reveal no significant difference. Scale bar, 20 mm.

benthamiana leaves. Confocal microscopy of leaf epidermal cells showed a typical ER localization of the fusion protein (Supplemental Figure 5). CYP715A1 is thus more likely to use a substrate present in the cytosol, although contact transfer of a plastid-derived precursor cannot be excluded (Mehrshahi et al., 2014).

I

floral volatiles from mature inflorescences of wild-type, cyp715a1 mutants, and a cyp715a1-2 complemented line (COMP), and analyzed them by gas chromatography–mass spectrometry (GC-MS). As shown in Figure 8B and 8C, CYP715A1 inactivation resulted in a large decrease in the emission of volatile sesquiterpenes, particularly all TPS21 products (b-caryophyllene, a-humulene, and a-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 and 8C). 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 signaling, most likely through 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; Watson et al., 2001; Tian et al., 2004; Kim and Della Penna, 2006). CYP715A1 has an 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

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 6). CYP715A1 was thus also expressed in insect cells with better yield (Supplemental Figure 6). 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 (Eriksson et al., 2010; Chandler et al., 2011), revealing severe developmental alterations. Subtle growth regulation, however, may escape investigations that rely on visual inspection. This work demonstrates that genes leading to finetuning 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, they are usually in species that underwent relatively recent wholegenome 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.,


Molecular Plant


Locus

cyp715a1/wild-type ratio (log2)

Adjusted P value

Annotation

Function

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

AT3G44300

NIT2

Auxin biosynthesis

Auxin and Indoles

1.32

1.63E 11

+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


+1.13

1.73E 7

AT3G20220

SAUR47


+1.04

6.92E 6

AOC2

Jasmonate biosynthesis

0.93

5.54E 4

Jasmonate AT3G25770 AT1G44350

ILL6

Jasmonate catabolism

0.93

6.26E 4

AT3G27810

MYB21

Jasmonate stimulus response

1.13

1.51E 7

AT5G40350

MYB24


0.85

1.01E 2

AT3G01530

MYB57


+1.00

4.55E 5

AT1G15750

TOPLESS


1.06

3.78E 6

AT1G17380

JAZ5


1.14

9.55E 8

AT1G70700

JAZ9


1.04

7.22E 6

AT3G16470

JR1


1.01

2.48E 5

AT4G23600

CORI3


1.36

0.00E+0

Table 1. Genes Differentially Expressed in the cyp715a1-2 Mutant and Related to Auxin/Indoles and Jasmonate.

2010). Most duplicates might thus feature ongoing pseudogenization (De Smet et al., 2013). Some others (e.g. in Setaria italica) may reflect duplicative bursts giving rise to taxaspecific defense pathways (Xu et al., 2007; Nelson and WerckReichhart, 2011; Geisler et al., 2013). Common gene duplicates maintained in the Papilionoideae lineage may also reflect elaborate developmental processes required to give rise to a complex floral architecture or nodulation. CYP715A1 expression is exclusively detected in the anther tapetum during flower development and in 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 to most reported mutants in hormonal pathways, and in particular of the JA regulatory cascade (Wasternack and Hause, 2013; Stitz et al., 2014), CYP715A1 seems to selectively affect 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 is necessary to consider, however, 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 a 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 growthpromoting KLUH (CYP78A5) must also 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


Molecular Plant


Figure 5. Targeted Transcriptional Analysis of Genes Related to Jasmonate Signaling 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 three to four independent replicates. Statistical significance was calculated by two-tailed Student’s t-test: *P < 0.05; **P < 0.01.

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 with GA or JA signaling, the CYP715A1-derived signal thus appears to dissociate petal growth and volatile emission from anther and gynoecium development, and hence insect pollination from selffertilization. Given the strong perturbation in gene expression and JA homeostasis resulting from CYP715A1 downregulation, and considering the responses of petals and stigma, the CYP715-derived signal seems to be perceived in several flower tissues. Based on transcriptomic and metabolic data, the signaling cascade triggered by CYP715A1 involves modifications of GA and JA homeostasis, and regulates components of the JA signaling pathway such as the JA-activated MYB21 and MYB24 transcription factors, the JAZ5 and JAZ9 proteins, and the JA and GA signaling mediator MYC2, as well as auxin signaling 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; Song et al., 2011; Reeves et al., 2012). By contrast CYP715A1, seemingly using MYB21 and MYB24, exclusively affects the last steps, such as petal elongation, flower opening, and volatile emission. It thus seems to trigger a specific signaling cascade for targeted GA/JA-dependent gene activation at the late stages of flower development. Based on phylogenetic con-

siderations, 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 tissuespecific 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 overexpressor lines is consistent with a role of CYP715A1 in the upstream biosynthesis of a signaling 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 a very small impact on plant development (Fleet et al., 2003; Swain et al., 2005). However, it 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 overexpression of the CYP714 genes shown to participate in GA catabolism (Zhu et al., 2006; Magome et al., 2013; Nomura et al., 2013). 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 that orchestrates flower maturation. Altogether, our data also suggest that by generating a specific signal coordinating the final steps of flower maturation, including petal expansion and emission of flower volatiles, both signals advertising for pollinator visit during flower maturation, CYP715 might be a major determinant of both the interaction of flowers with insects and fertilization.


Molecular Plant


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Figure 6. Hormone Profiling of cyp715a1 Mutant 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). Metabolic route of JAs 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.

tion vector pGWB633 was used as final GUS expression vector. Primers used for cloning are listed in Supplemental Table 4. All constructs were checked by sequencing.

METHODS Plant Materials and Growth Conditions The 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 12-/12-h day/night cycle (light intensity of 50 mmol m 2 s 1), 21 C/18 C day/night temperature cycle, and 70% relative 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 reamplified 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 USER 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 gatewaycompatible fragment subsequently cloned into pDONR207. The destina-

The A. tumefaciens GV3101 strain carrying the expression vector was used to transform Arabidopsis plants as previously described (Matsuno et al., 2009). A. 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 1). For complementation, more than 20 independent T2 lines showed flower complementation of both insertion mutants.

Floral Organs Growth Analysis Petals surface was measured according to a published method (Brioudes et al., 2009). Cell surface was measured from scanning electron microscopy (SEM) (Hitachi TM1000) pictures and analyzed using ImageJ software. The cell surface (mm2) was measured over a 4000-mm2 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


Molecular Plant A

A P450-dependent Signal Regulates Flower Maturation by a melting-curve analysis from 55 to 95 C to check amplification specificity. Reactions were performed in duplicate. Crossing points (Cp) were determined using the manufacturer’s 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 were calculated using the 2 DCp equation and PP2AA3 (At1g13320), SAND (At2g28390), EXP (At4g26410), and TIP41 (At4g34270) as reference genes. List of qPCR primers is available in Supplemental Table 4.

Transcriptome Studies

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Figure 7. Relaxation of the cyp715 Mutant Flower Phenotype by GA3 or Coronatine Treatments. Immature inflorescences were dipped for 2 s into GA3 (50 mM) and coronatine (1 mM) solution containing 0.1% ethanol and 0.02% Tween. Treatment was performed every 2 days for 2 weeks. Mock treatments were performed using a 0.1% ethanol and 0.02% Tween solution. (A) Pictures of a 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.

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

RNA Isolation and qRT–PCR Determination of Tissue-Specific Gene Expression Flower material was harvested from Boyes stage 6.30 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) and 2 mg of total RNA. qRT–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 ml of 10fold diluted cDNA, 5 ml of LightCycler 480 SYBR Green I Master (Roche), and 250 nM of each primer in a total volume of 10 ml. 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 for 15 s), followed

Microarray analysis was carried out using the CATMAv6.2 array based on Roche-NimbleGen technology. A single high-density CATMAv6.2 microarray slide contains 12 chambers, each containing 219 684 primers representing all the A. thaliana genes: 37 309 probes corresponding to TAIRv8 annotation (including 476 probes of mitochondrial and chloroplast genes), and 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 several 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 12-h light conditions as described above. Total RNA was extracted using the Nucleospin Plant RNA kit (Macherey-Nagel). Twenty micrograms of RNA was treated with 5 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 labeled with Cy3-dUTP or Cy5-dUTP (PerkinElmer-NEN Life Science Products). Thirty picomoles of each labeled cRNA was hybridized to the 12 3 280K CATMA slides at +42 C for 16 h. Two-micrometer scanning was performed with an InnoScan900 scanner (Innopsys, Carbonne, France), and raw data were extracted using Mapix software (Innopsys).

Statistical Analysis of Microarray Data Experiments were designed by the statistics group of the Unite´ de Recherche en Ge´nomique Ve´ge´tale. 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 values of %0.05 were considered as being differentially expressed. Analysis was conducted using R software. The function SqueezeVar of the LIMMA library was 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 were 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: RS1208_cyp715A1) according to the ‘‘Minimum Information About a Microarray Experiment’’ standards.


Molecular Plant


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Figure 8. Floral Emission of Volatile Sesquiterpenes Is Greatly Reduced in cyp715a1 Mutants. (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-b-caryophyllene, a-humulene and ( )-a-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 labeled in red, green, and yellow are products of TPS21, TPS11, and TPS03, respectively. 1: ( )-a-copaene; 2: ( )-E-b-caryophyllene; 3: (+)-thujopsene; 4: E-b-farnesene; 5: a-humulene; 6: b-acoradiene; 7: unidentified; 8: (+)-b-chamigrene; 9: a-farnesene; 10: ( )-b-bisabolene; 11: cuparene; 12: b-sesquiphellandrene; 13: unidentified. IS, internal standard.

GUS Histochemical Analysis Tissues from T2 Arabidopsis transgenic plants harboring 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 1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronide (X-Gluc), 100 mM sodium phosphate (pH 7.0), 10 mM EDTA, 0.5 mM potassium ferricyanide, 0.5 mM potassium ferrocyanide, and 0.1% (v/v) Triton X-100. Samples were incubated at 37 C in the dark overnight. Tissues were cleared three times in 75% ethanol before imaging with a Nikon (ECLIPSE, E800) microscope.

Scanning Electron Microscopy and Transmission Electron Microscopy 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 verticillata 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 MEGA5 software (Tamura et al., 2011). Phylogeny was inferred from the proteins alignment by maximum-likelihood analysis using the WAG model and 1000 bootstrap replications.


Molecular Plant


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 was performed by comparing twice the difference in log-likelihood values between M1a and M2a models using a c2 distribution (Anisimova et al., 2001).

Hormone Profiling Gibberellins Quantitative analysis of GAs was carried out using 0.2–0.5 g of freezedried whole inflorescences of wild-type and two cyp715a1 mutants, as described previously (Rieu et al., 2008). Each GA was quantified relative to its 17-2H2-labeled analog as internal standard, obtained from Professor L. Mander (Australian National University, Canberra ACT, Australia). Other Phytohormones ABA, IAA, and JA and its derivatives were extracted from approximately 200 mg of fresh material. Samples were extracted twice with 1 ml of ice-cold 80% methanol containing 5 mM dihydrojasmonic acid as internal standard. The two supernatants were recovered, pooled, and dried under vacuum overnight. Metabolites were resuspended in 200 ml of 80% MeOH prior to ultra-performance liquid chromatography–tandem mass spectrometry (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 3 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 temperature was set to 135 C. The capillary voltage was set to 3.2 kV. Data acquisition and analysis were performed with 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 for 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 ml of sample. Ionization was in positive or negative mode. Transitions in positive mode: IAA, 176 > 130 (cone voltage [CV] 20 V, collision energy [CE] 17 V); JA-Ile, 324 > 131 (CV 25 V, CE 20 V); in negative mode: 12OH-JA, 225 > 59 (CV 25 V, CE 25 V); 12OH-JA-Ile, 338 > 130 (CV 25 V, CE 20 V); 12COOH-JA-Ile, 352 > 130 (CV 25 V, CE 20 V); JA, 209 > 59 (CV 25 V, CE 23 V); ABA, 263 > 153 (CV 25 V, CE 12 V). 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 30 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 of 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 of Tenax TA (20/35; Grace Scientific) and 100 mg of activated charcoal 20–40 mesh (Sigma Aldrich). The volatiles emitted by the flowers were trapped at the outlet on a cartridge containing 150 mg of Tenax TA. Volatiles were sampled for 7 h. After volatile collection, flowers of each sample were swiftly cut from the inflorescence and weighed to normalize the emission of volatiles. Tenax cartridges were analyzed on a PerkinElmer Clarus 680 equipped with a PerkinElmer Clarus 600T quadrupole mass spectrometer and a TurboMatrix 100 thermal desorber (PerkinElmer). Two microliters of 400 mM nonyl acetate (Sigma) was added to each cartridge as an internal standard. Tenax cartridges were first dry-purged

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

with nitrogen at 50 ml/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 PerkinElmer 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. Compounds were separated on an HP5-MS column (30 m, 0.25 mm, 0.25 mm 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 (70 eV, m/z 50–300, scan time 0.3 s). Volatiles were identified on the basis of their retention time and mass spectra, and compared with 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 terpene 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 USER cloning technique (Nour-Eldin et al., 2006) was used to insert the CYP715A1 coding sequence into the USER-compatible yeast expression vector pYeDP60u2 (Ho¨fer et al., 2013) using the primers listed in Supplemental Table 4. The CYP715 sequence from B. distachyon was generated by gene synthesis and optimized for codon usage in yeast (Genecust) (Supplemental Figure 7). 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. (2013). The Saccharomyces cerevisiae WAT11 and WAT21 strains, expressing the ATR1 and ATR2 cytochrome P450 reductase from A. thaliana, respectively, under the galactoseinducible promoter GAL10-CYC1, were gifts from D. Pompon (Urban et al., 1997). Expression of functional cytochrome P450 was determined by differential spectrophotometry according to a former report (Omura and Sato, 1964). 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


Molecular Plant

A P450-dependent Signal Regulates Flower Maturation Technologies) and Spodoptera furugiperda cells (Sf9; Life Technologies) (Ohnishi et al., 2006). In brief, The open reading frame of CYP715A1 cDNA was amplified by PCR using primers TO-427 and TO-428 (Supplemental Table 4). 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. For expression of recombinant CYP715A1 protein, Sf9 cells were cultured in Grace’s insect medium supplemented with 100 mm of 5-aminolevulinic acid and 100 mm of 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 10 000 g for 15 min. The supernatant was further centrifuged at 100 000 g for 1 h, and the pellet was homogenized with buffer A to provide the microsomal fractions. The microsomal fractions were stored at 80 C before the enzyme assay.

SUPPLEMENTAL INFORMATION Supplemental Information is available at Molecular Plant Online.

FUNDING Z.L. is grateful to the China Scholarship Council and the Re´gion Alsace for co-funding a PhD scholarship. H.R. and D.W.-R. 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 program INTERREG IVA Broad Region EU Invests in Your Future supported the study on flower volatiles. A.P. acknowledges support of the Ministry of Education, Youth and Sport of the Czech Republic (grant LO1204 from the National Program of Sustainability I).

AUTHOR 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., and 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 and 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.

ACKNOWLEDGMENTS We are very grateful to Prof. D. Nelson (University of Tennessee, Memphis) for making available to us his extended annotation of the CYP715 family in higher plants. No conflict of interest declared.

Enzyme Assays When carried out with yeast microsomes, enzyme assays contained, in a final volume of 100 ml, 10 ml of microsomes, 20 mM potassium phosphate buffer (pH 7.5), 100 mM substrate, and 500 mM NADPH. For insect cell microsomes, the reaction mixture consisted of 10 ml of microsomes, 0.1 unit of Arabidopsis NADPH-cytochrome P450 reductase 2 (ATR2), 100 mM potassium phosphate (pH 7.25), 100 mM 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 ml of 50% acetic acid. The mix was vortexed, supplemented with 40 ml of acetonitrile, and centrifuged prior to liquid chromatography analysis. Samples were analyzed by either UPLC–MS/MS as described above or reverse-phase high-performance liquid chromatography (HPLC) (Alliance 2695, Waters) with photodiode array detection at 265 nm for ABA (Photodiode 2996, Waters). For this purpose, 50 ml of sample was injected onto a NOVA-PAK C18 4.6 3 250-mm column heated at 37 C. Separation was carried out using 0.2% acetic acid in water (A) and 0.2% acetic acid in acetonitrile (B) at a flow rate of 1 ml min 1. Elution program was as follows: 5%–100% B in 15 min (curve 8), 100% B for 2 min. Assays with 14C-labeled GAs and precursors were analyzed by HPLC with online 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).

Received: March 18, 2015 Revised: August 31, 2015 Accepted: September 5, 2015 Published: September 17, 2015

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Molecular Plant


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