PhMYB4 fine-tunes the floral volatile signature of Petunia x hybrida through PhC4H

Abstract

In Petunia × hybrida cv 'Mitchell Diploid' (MD), floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis is controlled spatially, developmentally, and daily at molecular, metabolic, and biochemical levels. Multiple genes have been shown to encode proteins that either directly catalyse a biochemical reaction yielding FVBP compounds or are involved in metabolite flux prior to the formation of FVBP compounds. It was hypothesized that multiple transcription factors are involved in the precise regulation of all necessary genes, resulting in the specific volatile signature of MD flowers. After acquiring all available petunia transcript sequences with homology to Arabidopsis thaliana R2R3-MYB transcription factors, PhMYB4 (named for its close identity to AtMYB4) was identified, cloned, and characterized. PhMYB4 transcripts accumulate to relatively high levels in floral tissues at anthesis and throughout open flower stages, which coincides with the spatial and developmental distribution of FVBP production and emission. Upon RNAi suppression of PhMYB4 (ir-PhMYB4) both petunia cinnamate-4-hydroxylase (PhC4H1 and PhC4H2) gene transcript levels were significantly increased. In addition, ir-PhMYB4 plants emit higher levels of FVBP compounds derived from p-coumaric acid (isoeugenol and eugenol) compared with MD. Together, these results indicate that PhMYB4 functions in the repression of C4H transcription, indirectly controlling the balance of FVBP production in petunia floral tissue (i.e. fine-tunes).

Fig. 1.

Predicted amino acid sequence alignment of homologous R2R3-MYB proteins from various species. Sequences represented are from Arabidopsis thaliana [accession: NM_119665 (MYB32) and AY070100 (MYB4)], Eucalyptus gunnii (AJ576024), Gossypium hirsutum (AF336286), Populus trichocarpa (XM_002306144), Vitis vinifera (EF113078), Humulus lupulus (AB292244), Solenostemon scutellarioides (EF522161), Petunia×hybrida (1359067), and Solanum lycopersicum (X95296). Sequences were aligned using the AlignX program of the Vector NTI Advance™ 11 software. Conserved domains depicted above the sequences are the R2 domain (light blue), R3 domain (green), C1 motif (black), and the EAR domain (red). Residues highlighted in: blue represent consensus residues derived from a block of similar residues at a given position, green represent consensus residues derived from the occurrence of greater than 50% of a single residue at a given position, and yellow represent consensus residues derived from a completely conserved residue at a given position. Petunia sequences are highlighted in red to the left.

Fig. 2.

PhMYB4 transcript accumulation analysis (one-step sqRT-PCR). Spatial analysis used root, stem, stigma, anther, leaf, petal tube, petal limb, and sepal tissues of MD harvested at 16.00 h: shown is 24 cycles of amplification (A). Floral developmental analysis used MD and 44568 flowers from 11 sequential stages collected on one day at 16.00 h: 25 cycles (B). Ethylene treatment (2 μl l⁻¹) analysis used excised MD and 44568 whole flowers treated for 0, 1, 2, 4, and 8 h: 24 cycles (C). Rhythmic analysis used MD plants acclimated in a growth chamber with a long-day photoperiod and samples collected every 3 h for a total of 36 h: 24 cycles (D). Ph18S was used as a loading control (16–17 cycles), and 50 ng total RNA was used per reaction in all cases. Shown are representative pictures from multiple biological and technical replications.

Fig. 3.

PhC4H1 and PhC4H2 floral developmental transcript accumulation analysis (one-step qRT-PCR). Floral developmental analysis used MD flowers from 11 sequential stages collected on one day at 16.00 h. Gene-specific primers were designed and optimized as well as template concentration, which was identified as 50 ng total RNA per reaction. PhFBP1 and Ph18S were used as endogenous control transcripts. Shown are representative histograms from multiple biological replications using ΔΔCt method (mean ±se; n=3).

Fig. 4.

Comparative transcript accumulation analysis between MD and the homozygous ir-PhMYB4 lines (two-step qRT-PCR). All cDNA templates were 1/10 dilutions of cDNA stock samples generated from 2 μg total RNA isolated from stage 8 flowers at 16.00 h. Histograms are representative of multiple experiments and multiple biological replicates, and analysed by ΔΔCt method with PhFBP1 and Ph18S as the internal references. Individual petunia transcripts analysed are PhMYB4 (A, B); PhC4H1 and PhC4H2 (B); PhODO1, PhCM1, PhPAL1, and PhPAL2 (C).

Fig. 5.

Floral volatile emission analysis of representative plants from two independent T₂ ir-PhMYB4 lines (12-12 and 60-7). Developmentally staged flowers (stage 8) were used to collect FVBP emission at 18.00 h (mean ±se; n=3). Twelve major FVBP compounds were identified and quantified with all measurements ng g⁻¹ fresh weight h⁻¹.

Fig. 6.

A condensed, schematic representation of the FVBP pathway beginning at phenylalanine in petunia. FVBP production consists of three main branch-points; phenylalanine, t-cinnamic acid, and ferulic acid. Floral volatile compounds derived from each branch-point are highlighted in pink and proteins are in red. Multiple arrows indicate multiple biochemical steps.