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Figure S1.PhCM1 and PhCM2 CDS alignment using the Align X program in Vector NTI advance 10.3.0 software package (Invitrogen; Carlsbad, CA, USA). PhCM1 and PhCM2 share 46.1% identity at the nucleotide level.

Figure S2. qRT-PCR transcript accumulation analysis of PhCM1 and PhCM2 in petunia. Spatial analysis used root, stem, stigma, anther, leaf, petal tube, petal limb, and sepal tissues of MD harvested at 16:00 h (a). The spatial experiment consisted of one biological replicate used for sqRT-PCR and one separate biological replicate with two technical replicates per biological replicate. Floral developmental analysis used MD flowers from 11 sequential stages at 16:00 h (b). The developmental analysis consisted of two biological replicates separate from the biological replicates used for the sqRT-PCR with three technical replicates. Ethylene treatment (2 μl L-1) analysis used excised MD and 44568 whole flowers treated for 0, 1, 2, 4, and 8 h (c). The ethylene treated series consisted of one biological replicate used in the sqRT-PCR with two technical replicates per biological replicate. PhFBP1 and Ph18S were used as references throughout these experiments.

Figure S3. Schematic representation and nucleotide comparison of RNAi region used for the production of petunia PhCM1 RNAi transgenic lines. 213 bases at the 3′ end of the coding sequence of PhCM1 were chosen for the RNAi construct. This region shared 58.2% identity with the corresponding nucleotide region from PhCM2.

Figure S4. sqRT-PCR transcript accumulation analysis in floral tissues of three independent T1ir-PhCM1 lines. MD, 2-4, 24-9, 33-9 were used with primers specific for floral volatile benzenoid/phenylpropanoid, shikimate, and phenylpropanoid transcripts. The number of cycles used for amplification of each transcript is shown on the right. Ph18S was used as a loading control in all cases.

Figure S5. sqRT-PCR transcript accumulation analysis in floral tissues of two independent, homozygous T2ir-PhCM1 lines. Individuals and biological replicates from MD, 24-9, 33-8 were used with primers specific for PhCM1. The number of cycles used for amplification of each transcript is shown on the right. Ph18S was used as a loading control.

Figure S6. Physiological comparison between MD and representative independent T2ir-PhCM1 homozygous lines 24-9 and 33-8 in 9 week old petunia seedlings (mean ± SE; n = 5).

Figure S7. Stem cross-sections (between 7–8 node from apical meristem) from 9 week old petunias stained with Phlorogucinol. Shown are MD and representative individuals from two independent ir-PhCM1 homozygous T2 lines, 24-9 and 33-8. Pictures are from light microscopy at 4 × on a Leica MZ 16F and are representative of three biological replicates.

Table S1. Functional complementation of CM-deficient E. coli KA12/pKIMP-UAUC. M9c minimal media was used for all experiments and supplemented with 20 μg ml−1 of L-phenylalanine and L-tyrosine where stated. Antibiotics used were chloramphenicol [Ch] (30 μg ml−1) for selection of the pKIMP plasmid and carbenicillin [Ca] (100 μg ml−1) for selection of the pET-32 plasmid. KA12/pKIMP-UAUC is not a λDE3 lysogenic E. coli, so bacteriophage CE6 (Novagen, cat# 69390) infection was used to induce transcription from the pET-32 T7 promoter where stated and no CE6 administered (NA) where stated. Transformants were incubated at 37°C for two days, and growth was scored as a plus (+) or minus (−). A sample of positive colonies was picked and colony PCR was performed for confirmation of pET-32-CM1 or pET-32-CM2 plasmids.

Table S2. Gene specific primers used for the transcript accumulation analyses throughout this study.

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.