Down regulation of p-coumarate 3-hydroxylase in petunia uniquely alters the profile of emitted floral volatiles

Petunia × hybrida cv ‘Mitchell Diploid’ floral volatile benzenoid/phenylpropanoid (FVBP) biosynthesis ultimately produces floral volatiles derived sequentially from phenylalanine, cinnamic acid, and p-coumaric acid. In an attempt to better understand biochemical steps after p-coumaric acid production, we cloned and characterized three petunia transcripts with high similarity to p-coumarate 3-hydroxylase (C3H), hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT), and caffeoyl shikimate esterase (CSE). Transcript accumulation of PhC3H and PhHCT was highest in flower limb tissue during open flower stages. PhCSE transcript accumulation was also highest in flower limb tissue, but it was detected earlier at initial flower opening with a bell-shaped distribution pattern. Down regulation of endogenous PhC3H transcript resulted in altered transcript accumulation of many other FVBP network transcripts, a reduction in floral volatiles, and the emission of a novel floral volatile. Down regulation of PhHCT transcript did not have as large of an effect on floral volatiles as was observed for PhC3H down regulation, but eugenol and isoeugenol emissions were significantly reduced on the downstream floral volatiles. Together these results indicate that PhC3H is involved in FVBP biosynthesis and the reduction of PhC3H transcript influences FVBP metabolism at the network level. Additional research is required to illustrate PhHCT and PhCSE functions of petunia.

Transcript accumulation of PhC3H, PhHCT, and PhCSE in MD. Since C3H, HCT, and CSE are involved in phenylpropanoid biosynthesis in other plant species, we hypothesized that relative transcript accumulation of PhC3H, PhHCT, and PhCSE would be highest in open flower and petal limb tissue, consistent with previously characterized FVBP genes 25,[47][48][49][50] . Transcript accumulation was assayed using the ∆ΔCt qRT-PCR method with total RNA extracted from a spatial series of MD tissues: root, stem, stigma, anther, leaf, petal tube, petal limb, and sepal; along with total RNA from a staged floral developmental series of tissues including 11 consecutive stages MD flowers 48 .
Spatial transcript accumulation was calculated based on that of root tissue where most transcripts of petunia volatile genes were lowest in previous studies 31,33,48,51 . The transcript accumulation of PhC3H, PhHCT, and PhCSE was highest in petal limb tissue while lowest in reproductive organs, leaf and sepal tissues (Fig. 3a,c,e). An approximate five-fold increase of PhC3H and PhHCT transcript was detected in petal limb tissue compared to root tissue; whereas, an approximate twenty-fold increase of PhCSE transcript was detected for the same tissue comparison. The transcript accumulation level of PhC3H and PhHCT in root tissue relatively elevated compared to stigma, anther, leaf, and sepal tissue. These results do not consistent with the profiles of known FVBP genes of petunia 31,33,48,51 .
Floral developmental analyses demonstrated high levels of transcript accumulation only in open flowers from stage 7 to stage 10 for both PhC3H and PhHCT (Fig. 3b,d), while PhCSE transcript accumulation occurred earlier during flower development and showed a bell-shape with a peak at the flower opening (stage 7) (Fig. 3f). Focusing on flower developmental stage 9 (fully open corolla and sexually receptive flower), PhC3H transcript was accumulated approximately 20-fold more compared to stage 1 (initial flower bud). Similar trends were observed in the accumulation of PhHCT and PhCSE with an approximate 100-fold and 40-fold increase respectively. However, PhCSE accumulation peaked earlier, at flower stage 7 rather than stage 9, with approximately a 70-fold increase compared to stage 1.
Volatile analyses of ir-PhC3H and ir-PhHCT flowers. Standard volatile collection and GC-MS analysis methods were used to compare floral volatile profiles of ir-PhC3H and ir-PhHCT lines to control MD plants. In general, ir-PhC3H lines showed reduced emission for five major petunia floral volatiles: eugenol, isoeugenol, www.nature.com/scientificreports www.nature.com/scientificreports/ benzyl benzoate, benzaldehyde, and phenylacetaldehyde. Compared to MD, ir-PhHCT lines were reduced in phenylpropene and benzyl benzoate emission and showed a trend toward increased emission of benzaldehyde and phenylacetaldehyde (Fig. 5, see Supplementary Fig. S3). The most abundant constituent of the MD floral volatile profile, methyl benzoate, was also reduced in ir-PhC3H-7 flowers, but to a lesser extent. Phenethyl alcohol was the only floral volatile not significantly different in all three ir-PhC3H lines compared to MD. The benzenoid p-cresol, which is a novel volatile molecule for the MD genetic background, was detected from flowers of ir-PhC3H lines (Fig. 5).
Floral volatile emission from ir-PhHCT lines were significantly reduced for the phenylpropene volatiles, eugenol and isoeugenol, and the two conjugated volatiles benzyl benzoate and phenethyl benzoate (see Supplementary  Fig. S3). The ir-PhHCT lines exhibited elevated emission of benzaldehyde, benzyl alcohol, and phenylacetaldehyde compared to MD. Methyl benzoate was also increased in ir-PhHCT-14 flowers, but to a lesser extent. P-cresol was not detected from any ir-PhHCT flowers (data not shown). www.nature.com/scientificreports www.nature.com/scientificreports/

Transcript accumulation of FVBP related genes in ir-PhC3H and ir-PhHCT flowers. C3H is
reported as a key protein concerning regulation of metabolically related protein aggregates 12 . As PhC3H transcript levels were low in petunia flowers, most of the volatiles were reduced (Fig. 5). Transcript accumulation of the metabolically related genes, PhC4Hs, PhPALs, and PhHCT showed decreased levels in ir-PhC3H flowers compared to MD. Many other FVBP genes including PhBSMT, PhCCR2, PhCFAT, PhCSE, PhIGS1, PhMYBA, PhMYB4, PhODO1, PhPAAS, and PhPAR demonstrated reduced transcript accumulation in ir-PhC3H flowers (Fig. 6). However, down regulation of PhHCT did not affect transcript accumulation of PhCSE as much as that of PhC3H (see Supplementary Fig. S4).

Discussion
The transcript accumulation profile, enzyme function, protein localization, and protein-protein interactions of coumarate 3-hydroxylase (C3H) have been investigated in model plant systems like Arabidopsis 12,35,42,43,55,56 and poplar 24,57 . C3H is an integral protein feature of the cellular machinery leading to produce monolignols which serve as precursors to lignin and lignin production in plants 58 . Coniferyl alcohol, a common monolignol, can serve as a precursor to volatile phenylpropene biosynthesis in petunia floral tissue where the requirement for lignin is relatively low 59 . Around 7:00 PM, a petunia flower can emit over 10 ug*gFW −1 *h −1 of a volatile phenylpropene, isoeugenol 48,50 , and the rate of phenylpropene and benzenoid emission is least influenced by a limiting pool of phenylalanine, the initial substrate for the phenylpropanoid pathway, indicating a strong regulation of www.nature.com/scientificreports www.nature.com/scientificreports/ carbon flux toward monolignol production 52,60 . When arogenate dehydratase, which converts arogenate to phenylalanine, was down regulated in petunia, the emission of many petunia volatiles was reduced 60 .
Throughout this work, three transcripts were cloned and sequenced from Petunia × hybrida cv 'MD' flower tissue. Single and unique transcripts with homology to C3H, HCT, and CSE were identified using publicly available genomic and transcriptomic databases, although the possibilities for the multiple copies of each gene family member could not be excluded (Fig. 2, see Supplementary Figs S1 and S2). All three petunia transcripts accumulated to their relatively highest levels in flower limb tissue compared to other parts of the plant (Fig. 3), which is in line with FVBP network transcripts 48 . PhC3H and PhHCT exhibited typical accumulation patterns of genes involved in FVBP biosynthesis during floral development 20,25,29,31,33,48,49,[51][52][53]60 . The transcript levels were relatively low in young or developing floral buds, highest in open flowers, and then showed a dramatic reduction at senescence (Fig. 3) 48 . Compared to PhC3H, PhHCT and other known genes in the FVBP biosynthesis pathway, PhCSE transcript accumulated earlier during development and displayed an almost normal distribution rather than a developmentally-delayed distribution (Fig. 3). Interestingly, PhCSE followed a developmental bell-shaped accumulation profile similar to that of the petunia R2R3-MYB transcription factor, PhEOBII, which appears to have a positive regulatory effect on flower opening and FVBP biosynthesis 61,62 .
Petunia RNAi (ir) lines for PhC3H and PhHCT were generated to test the effects of reduced transcript levels of each gene on FVBP pathway. Multiple lines of ir-PhC3H showed a reduction of endogenous PhC3H transcript by 75.6-98.2% in floral tissue, and ir-PhHCT lines were reduced in endogenous transcript by 48.7-82.3% (Fig. 4). It is unclear whether a reduction of PhHCT transcript beyond 82.3% is lethal, but no obvious growth phenotypes were observed any of the ir-PhHCT or ir-PhC3H lines. Floral volatile analysis of the ir-PhC3H lines demonstrated a clear but unexpected volatile phenotype compared to the control petunia volatile emission phenotype (Fig. 5). The reduction of endogenous PhC3H resulted in very low levels of emitted phenylpropenes, isoeugenol and eugenol, as expected. Additionally, most of the emitted benzenoid volatiles were also reduced, along with phenylacetaldehyde which is generated directly from the initial substrate of the phenylpropanoid pathway, phenylalanine. A novel floral volatile molecule, p-cresol was detected from ir-PhC3H lines at considerable concentrations (~200-800 ng*gFW -1 *h −1 ). It was an unexpected product because p-cresol has not been reported in FVBP biosynthesis of MD before but detected in other floral volatile profiles such as Petunia × hybrida (V26) having purple flowers, Satyrium pumilum (African orchid), and Jasminum polyanthum (Pink Jasmin) [63][64][65] . P-cresol is known to be converted from 4-hydroxyphenylacetic acid which is derived from tyrosine 65,66 . www.nature.com/scientificreports www.nature.com/scientificreports/ The volatile phenotype of the ir-PhC3H lines suggested that a disruption of C3H activity cause a downregulation of the majority of the phenylpropanoid pathway in petal tissue of petunia flowers. Based on the transcript accumulation assay, 16 of the 18 FVBP related genes demonstrated clear reductions in the ir-PhC3H lines compared to controls (Figs 1, 6). Known positive and negative regulating transcription factors 20,61,66 , core phenylpropanoid pathway enzymes 20,25 , enzymes that produce specific FVBP compounds 33,67 , and even enzymes responsible for biochemical steps after C3H 29,31 were all significantly reduced in transcript accumulation. For www.nature.com/scientificreports www.nature.com/scientificreports/ example, the small family of phenylalanine ammonia-lyase (PAL1, 2, 3) transcripts was reduced 52.3-66.8%, which would severely limit phenylpropanoid metabolism in petal tissue, especially at the elevated rates normally found in petunia flower petal tissue.
It is unclear at this point what mechanisms are involved in the downregulation of the general phenylpropanoid pathway. In Arabidopsis and poplar, C3H appears to be a major driver of protein-protein interaction at the ER, where C4H and C3H form homodimer and heterodimer protein complexes with elevated enzymatic activity 12,24,37,43 . The P450 protein complexes can associate with soluble phenylpropanoid pathway enzymes like PALs, 4CLs, and HCT to form supramolecular structures. These large, ER tethered protein aggregates are thought to concentrate required enzymes to accommodate for a high metabolic demand of the phenylpropanoid pathway in specific conditions or tissues 12 . We demonstrated that the down regulation of PhC3H in petunia resulted in the change of flower volatiles and expression of related FVBP genes, but further studies are required to elucidate the role of PhC3H in the stability of a large phenylpropanoid related protein complex in petunia flower limb tissue. www.nature.com/scientificreports www.nature.com/scientificreports/

Materials and Methods
Plant materials and Cloning. Petunia × hybrida cv 'MD' was used as a control and genetic background for all experiments. Plants were grown in glass greenhouses as previously described by Dexter et al. 33 .
Multiple data sources including the National Center for Biotechnology Information (NCBI -http://www.ncbi. nlm.nih.gov), the Sol Genomics Network (SGN -https://solgenomics.net), and the 454 petunia database (http:// biosrv.cab.unina.it/454petuniadb/protocol.php) were employed to search for petunia nucleotide sequences with similarity to Arabidopsis thaliana p-coumarate 3-hydroxylase (AtC3H, AT2G40890), Nicotiana tabacum hydroxycinnamoyl transferase (NtHCT, AJ507825), and Arabidopsis thaliana caffeoyl shikimate esterase (AtCSE, AT1G52760). The target petunia sequences were collected and assembled into contigs using a software package (Vector NTI Advance ™ 11.3) and a SMARTer ™ RACE cDNA Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA) according to the manufacturer's protocol. This approach resulted in three in silico candidate sequences for PhC3H, PhHCT, and PhCSE. The full sequences were amplified using PfuTurbo DNA Polymerase (Agilent Technologies, Santa Clara, CA) (primers on Table S1) and cloned into a pGEM-T easy vector (Promega, Madison, WI) using similar methods as Colquhoun et al. 20 . Nucleotide sequencing with multiple clones from multiple amplifications was performed at an on campus Sanger sequencing core (Interdisciplinary Center for Biotechnology Research, University of Florida, FL) using Big Dye V1-2. The resulted high-quality sequence was then used as a query to search the petunia genome database at SGN (https://solgenomics.net), which supported that each sequence most likely originating from a single locus.

bp sequence of
PhC3H and a 335 bp sequence of PhHCT were amplified for RNAi vector construction (primers on Table S1). The RNAi gene driven by a flower specific constitutive promoter, pFMV in pHK vector was introduced into MD leaf discs using Agrobacterium-mediated transformation methods 68 . Detailed methods for this procedure have been described by Dexter et al. 33 and Underwood et al. 50 . All T 0 plant tissues were collected for floral volatile analyses and transcript accumulation analyses, and then the flowers were self-pollinated.

Analyses of transcript accumulation.
To observe transcript accumulation based on spatial and flower development, petunia MD tissues were collected following the method of Colquhoun et al. 48 . The spatial series consisted of root, stem, stigma, anther, leaf, petal tube, petal limb, and sepal. The developmental series included 11 stages of flowers, bud < 0.5 cm (stage 1); bud 0.5 to 1.5 cm (stage 2); bud 1. Volatile collection. Petunia flowers were harvested at 16.00 h and volatiles were collected for 1 hour in glass tubes using a push-pull dynamic headspace collection system as previously described 33,51,70 . Volatiles collected from at least three biological replicate flowers on glass columns containing approximately 50 mg HaySep Q 80-100 porous polymer adsorbent (Hayes Separations Inc., Bandera, TX) were eluted with methylene chloride. Quantification of volatiles from the elution matrix was performed on an Agilent 7890A Series gas chromatograph (GC) equipped with an Agilent 5977A single quadrupole mass spectrum detector (MSD). Parameters of the GC were used as follows: Helium carrier gas fixed at 11.5 psi, split injector at 20:1 split, inlet temperature 220 °C, injection volume 2 uL, and the syringe wash solvents were acetone and hexane. Sample analytes were separated using an equipped DB-5 column (Agilent Technologies, Santa Clara, CA, USA). Oven temperatures were programmed as follows: the initial oven temperature of 40 °C was held for 0.5 minutes then ramped 5 °C*minute −1 to 250 °C and held for 4 minutes. The MSD was equipped with an extractor ion source and tuned for sensitivity and mass accuracy just prior to sample analysis. Parameters for the MSD were maintained as follows: www.nature.com/scientificreports www.nature.com/scientificreports/ MassHunter Quantitative Analysis program. Compound identity was verified by extracting and comparing the mass spectral data of each compound peak to the 2011 NIST mass spectral library and comparing retention time and mass spectral profiles to authentic standards run under identical machine parameters. Volatile mass emission rates (ng*gFW −1 *hr −1 ) were calculated based on each compound individual peak area relative to the peak area of an elution standard, nonyl acetate, within each sample and standardized for each sample corresponding biological mass. Dilutions for volatile standards were run on the GC-MS in duplicate to obtain a response factor for each compound that was used in the calculation of volatile emission mass. Mean separation and comparison of ir-PhC3H and ir-PhHCT floral volatiles to MD controls were performed using Duncan's multiple range test (one-way ANOVA, P < 0.05) with the JMP Pro v.12 statistical software package (SAS Institute Inc., Cary, NC).

Data Availability
All submitted manuscripts including figures and tables are available on Scientific report.