Volume 8, Issue 12, Pages 1751-1765 (December 2015) A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation  Zhenhua Liu, Benoît Boachon, Raphaël Lugan, Raquel Tavares, Mathieu Erhardt, Jérôme Mutterer, Valérie Demais, Stéphanie Pateyron, Véronique Brunaud, Toshiyuki Ohnishi, Ales Pencik, Patrick Achard, Fan Gong, Peter Hedden, Danièle Werck-Reichhart, Hugues Renault  Molecular Plant  Volume 8, Issue 12, Pages 1751-1765 (December 2015) DOI: 10.1016/j.molp.2015.09.002 Copyright © 2015 The Author Terms and Conditions

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 single-copy 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. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

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). Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

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. 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. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

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 wild-type (A). At the tricellular stage, pollen of mutants appears similar to wild-type (D–F). Scale bar, 1 μm. in, intine, v, vesicle. (G–I) Scanning electron micrographs of wild-type and mutant mature pollen reveal no significant difference. Scale bar, 20 μm. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

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. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

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. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

Figure 7 Relaxation of the cyp715 Mutant Flower Phenotype by GA3 or Coronatine Treatments. Immature inflorescences were dipped for 2 s into GA3 (50 μM) and coronatine (1 μM) 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. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

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-β-caryophyllene, α-humulene and (−)-α-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: (−)-α-copaene; 2: (−)-E-β-caryophyllene; 3: (+)-thujopsene; 4: E-β-farnesene; 5: α-humulene; 6: β-acoradiene; 7: unidentified; 8: (+)-β-chamigrene; 9: α-farnesene; 10: (−)-β-bisabolene; 11: cuparene; 12: β-sesquiphellandrene; 13: unidentified. IS, internal standard. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions

Figure 9 Model of CYP715A1 Function. Terms in bold are supported by experimental data. Molecular Plant 2015 8, 1751-1765DOI: (10.1016/j.molp.2015.09.002) Copyright © 2015 The Author Terms and Conditions