Overexpression of acdS in petunia reduces ethylene production and improves tolerance to heat stress.

Petunia hybrida, widely grown as a bedding plant, has reduced growth and flower quality at temperatures above 30 °C (heat stress), primarily due to heat stress-induced ethylene (ET) production. The gene acdS encodes the 1-aminocyclopropane-1-carboxylate (ACC) deaminase (ACCD) enzyme, which is known for its role in reducing ET production by breaking down the ET precursor, ACC, in plant tissues. This study investigated the impact of heat stress on both 'Mirage Rose' WT petunia and its acdS-overexpressing transgenic lines. Heat stress-induced growth inhibition was observed in WT plants but not in transgenic plants. The increased stress tolerance of transgenic plants over WT plants was associated with lower ET production, ROS accumulation, higher SPAD values, water content, and relative water content. Furthermore, higher sensitivity of the WT to heat stress than the transgenic plants was confirmed by analysing ET signalling genes, heat shock transcription factor genes, and antioxidant- and proline-related genes, more strongly induced in WT than in transgenic plants. Overall, this study suggests the potential application of acdS overexpression in other floriculture plants as a viable strategy for developing heat stress-tolerant varieties. This approach holds promise for advancing the floricultural industry by overcoming challenges related to heat-induced growth inhibition and loss of flower quality.

Incomplete lineage sorting and hybridization underlie tree discordance in Petunia and related genera (Petunieae, Solanaceae).

Despite the overarching history of species divergence, phylogenetic studies often reveal distinct topologies across regions of the genome. The sources of these gene tree discordances are variable, but incomplete lineage sorting (ILS) and hybridization are among those with the most biological importance. Petunia serves as a classic system for studying hybridization in the wild. While field studies suggest that hybridization is frequent, the extent of reticulation within Petunia and its closely related genera has never been examined from a phylogenetic perspective. In this study, we used transcriptomic data from 11 Petunia, 16 Calibrachoa, and 10 Fabiana species to illuminate the relationships between these species and investigate whether hybridization played a significant role in the diversification of the clade. We inferred that gene tree discordance within genera is linked to hybridization events along with high levels of ILS due to their rapid diversification. Moreover, network analyses estimated deeper hybridization events between Petunia and Calibrachoa, genera that have different chromosome numbers. Although these genera cannot hybridize at the present time, ancestral hybridization could have played a role in their parallel radiations, as they share the same habitat and life history.

CRISPR/Cas9-mediated mutagenesis of FT/TFL1 in petunia improves plant architecture and early flowering.

Petunias are renowned ornamental species widely cultivated as pot plants for their aesthetic appeal both indoors and outdoors. The preference for pot plants depends on their compact growth habit and abundant flowering. While genome editing has gained significant popularity in many crop plants in addressing growth and development and abiotic and biotic stress factors, relatively less emphasis has been placed on its application in ornamental plant species. Genome editing in ornamental plants opens up possibilities for enhancing their aesthetic qualities, offering innovative opportunities for manipulating plant architecture and visual appeal through precise genetic modifications. In this study, we aimed to optimize the procedure for an efficient genome editing system in petunia plants using the highly efficient multiplexed CRISPR/Cas9 system. Specifically, we targeted a total of six genes in Petunia which are associated with plant architecture traits, two paralogous of FLOWERING LOCUS T (PhFT) and four TERMINAL FLOWER-LIKE1 (PhTFL1) paralogous genes separately in two constructs. We successfully induced homogeneous and heterogeneous indels in the targeted genes through precise genome editing, resulting in significant phenotypic alterations in petunia. Notably, the plants harboring edited PhTFL1 and PhFT exhibited a conspicuously early flowering time in comparison to the wild-type counterparts. Furthermore, mutants with alterations in the PhTFL1 demonstrated shorter internodes than wild-type, likely by downregulating the gibberellic acid pathway genes PhGAI, creating a more compact and aesthetically appealing phenotype. This study represents the first successful endeavor to produce compact petunia plants with increased flower abundance through genome editing. Our approach holds immense promise to improve economically important potting plants like petunia and serve as a potential foundation for further improvements in similar ornamental plant species.

Petunia PHYTOCHROME INTERACTING FACTOR 4/5 transcriptionally activates key regulators of floral scent.

Floral scent emission of petunia flowers is regulated by light conditions, circadian rhythms, ambient temperature and the phytohormones GA and ethylene, but the mechanisms underlying sensitivity to these factors remain obscure. PHYTOCHROME INTERACTING FACTORs (PIFs) have been well studied as components of the regulatory machinery for numerous physiological processes. Acting redundantly, they serve as transmitters of light, circadian, metabolic, thermal and hormonal signals. Here we identified and characterized the phylogenetics of petunia PIF family members (PhPIFs). PhPIF4/5 was revealed as a positive regulator of floral scent: TRV-based transient suppression of PhPIF4/5 in petunia petals reduced emission of volatiles, whereas transient overexpression increased scent emission. The mechanism of PhPIF4/5-mediated regulation of volatile production includes activation of the expression of genes encoding biosynthetic enzymes and a key positive regulator of the pathway, EMISSION OF BENZENOIDS II (EOBII). The PIF-binding motif on the EOBII promoter (G-box) was shown to be needed for this activation. As PhPIF4/5 homologues are sensors of dawn and expression of EOBII also peaks at dawn, the prior is proposed to be part of the diurnal control of the volatile biosynthetic machinery. PhPIF4/5 was also found to transcriptionally activate PhDELLAs; a similar positive effect of PIFs on DELLA expression was further confirmed in Arabidopsis seedlings. The PhPIF4/5-PhDELLAs feedback is proposed to fine-tune GA signaling for regulation of floral scent production.

Dorsoventrally asymmetric expression of miR319/TCP generates dorsal-specific venation patterning in petunia corolla tube.

Vein-associated pigmentation (venation) is a type of floral coloration adopted by plants to attract pollinators. Several petunia (Petunia hybrida) lines generate dorsoventrally asymmetric venation patterning of the corolla tube, in which venation is only present in the dorsal tube. The molecular mechanism underlying this trait is unknown. Here, we demonstrate that miR319 is preferentially expressed in the dorsal corolla tube, leading to dorsoventrally asymmetric expression of its target genes. Transgenic lines overexpressing phy-miR319a generated uniform venation patterning of the corolla tube. Knockout of TCP genes targeted by miR319 promoted venation patterning in the ventral and dorsal tube, while overexpression of the miR319 target gene, PhTCP6, completely inhibited corolla tube venation patterning. In addition, miR319-targeted TCPs negatively regulated venation patterning, probably by repressing the regulator of venation patterning, AN4. Together, our data demonstrate that asymmetric expression of miR319 promotes venation patterning in the petunia dorsal tube alone by repressing the expression of its target TCP genes, which negatively regulate corolla tube venation patterning. These findings provide novel insights into how the dorsoventrally asymmetric distribution of venation patterning is established in zygomorphic flowers.

Cell layer-specific expression of the homeotic MADS-box transcription factor PhDEF contributes to modular petal morphogenesis in petunia.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia × hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer-specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

Transcriptomic analysis implicates ABA signaling and carbon supply in the differential outgrowth of petunia axillary buds.

BACKGROUND: Shoot branching of flowering plants exhibits phenotypic plasticity and variability. This plasticity is determined by the activity of axillary meristems, which in turn is influenced by endogenous and exogenous cues such as nutrients and light. In many species, not all buds on the main shoot develop into branches despite favorable growing conditions. In petunia, basal axillary buds (buds 1-3) typically do not grow out to form branches, while more apical axillary buds (buds 6 and 7) are competent to grow. RESULTS: The genetic regulation of buds was explored using transcriptome analyses of petunia axillary buds at different positions on the main stem. To suppress or promote bud outgrowth, we grew the plants in media with differing phosphate (P) levels. Using RNA-seq, we found many (> 5000) differentially expressed genes between bud 6 or 7, and bud 2. In addition, more genes were differentially expressed when we transferred the plants from low P to high P medium, compared with shifting from high P to low P medium. Buds 6 and 7 had increased transcript abundance of cytokinin and auxin-related genes, whereas the basal non-growing buds (bud 2 and to a lesser extent bud 3) had higher expression of strigolactone, abscisic acid, and dormancy-related genes, suggesting the outgrowth of these basal buds was actively suppressed. Consistent with this, the expression of ABA associated genes decreased significantly in apical buds after stimulating growth by switching the medium from low P to high P. Furthermore, comparisons between our data and transcriptome data from other species suggest that the suppression of outgrowth of bud 2 was correlated with a limited supply of carbon to these axillary buds. Candidate genes that might repress bud outgrowth were identified by co-expression analysis. CONCLUSIONS: Plants need to balance growth of axillary buds into branches to fit with available resources while allowing some buds to remain dormant to grow after the loss of plant parts or in response to a change in environmental conditions. Here we demonstrate that different buds on the same plant with different developmental potentials have quite different transcriptome profiles.

Evolutionary walks through flower colour space driven by gene expression in Petunia and allies (Petunieae).

The structure and function of biochemical and developmental pathways determine the range of accessible phenotypes, which are the substrate for evolutionary change. Accordingly, we expect that observed phenotypic variation across species is strongly influenced by pathway structure, with different phenotypes arising due to changes in activity along pathway branches. Here, we use flower colour as a model to investigate how the structure of pigment pathways shapes the evolution of phenotypic diversity. We focus on the phenotypically diverse Petunieae clade in the nightshade family, which contains ca 180 species of Petunia and related genera, as a model to understand how flavonoid pathway gene expression maps onto pigment production. We use multivariate comparative methods to estimate co-expression relationships between pathway enzymes and transcriptional regulators, and then assess how expression of these genes relates to the major axes of variation in floral pigmentation. Our results indicate that coordinated shifts in gene expression predict transitions in both total anthocyanin levels and pigment type, which, in turn, incur trade-offs with the production of UV-absorbing flavonol compounds. These findings demonstrate that the intrinsic structure of the flavonoid pathway and its regulatory architecture underlies the accessibility of pigment phenotypes and shapes evolutionary outcomes for floral pigment production.

Hormonal Signaling during dPCD: Cytokinin as the Determinant of RNase-Based Self-Incompatibility in Solanaceae.

Research into molecular mechanisms of self-incompatibility (SI) in plants can be observed in representatives of various families, including Solanaceae. Earlier studies of the mechanisms of S-RNase-based SI in petunia (Petunia hybrida E. Vilm.) demonstrate that programmed cell death (PCD) is an SI factor. These studies suggest that the phytohormon cytokinin (CK) is putative activator of caspase-like proteases (CLPs). In this work, data confirming this hypothesis were obtained in two model objects-petunia and tomato (six Solanaceae representatives). The exogenous zeatin treatment of tomato and petunia stigmas before a compatible pollination activates CLPs in the pollen tubes in vivo, as shown via the intravital imaging of CLP activities. CK at any concentration slows down the germination and growth of petunia and tomato male gametophytes both in vitro and in vivo; shifts the pH of the cytoplasm (PHc) to the acid region, thereby creating the optimal conditions for CLP to function and inhibiting the F-actin formation and/or destructing the cytoskeleton in pollen tubes to point foci during SI-induced PCD; and accumulates in style tissues during SI response. The activity of the ISOPENTENYLTRANSFERASE 5 (IPT5) gene at this moment exceeds its activity in a cross-compatible pollination, and the levels of expression of the CKX1 and CKX2 genes (CK OXIDASE/DEHYDROGENASE) are significantly lower in self-incompatible pollination. All this suggests that CK plays a decisive role in the mechanism underlying SI-induced PCD.

Functional identification of PhMATE1 in flower color formation in petunia.

Multidrug and toxic compound extrusion (MATE) transporter proteins are a class of secondary transporter proteins that can transport flavonoids. Anthocyanins, a kind of flavonoid, are important secondary metabolites widely found in higher plants; they determine the flower color of most angiosperms. TT12 in Arabidopsis was the first MATE protein identified to be involved in flavonoid transport. Petunia (Petunia hybrida) is an important ornamental plant and is one of the ideal plants for studying plant flower color. However, there are few reports on anthocyanin transport in petunia. In this study, we characterized a homolog of Arabidopsis TT12 in the petunia genome, PhMATE1, that shares the highest amino acid sequence identity with Arabidopsis TT12. PhMATE1 protein contained 11 transmembrane helices. PhMATE1 showed a high transcription level in corollas. The silencing of PhMATE1 mediated by both virus-induced gene silence and RNA interference changed flower color and reduced anthocyanin content in petunia, suggesting that PhMATE1 is involved in anthocyanin transport in petunia. Furthermore, PhMATE1 silencing downregulated the expression of the structural genes of the anthocyanin synthesis pathway. The results of this study supported the hypothesis that MATEs are involved in the sequestration of anthocyanins during flower color formation.

Altered profile of floral volatiles and lignin content by down-regulation of Caffeoyl Shikimate Esterase in Petunia.

BACKGROUND: The floral volatile profile of Petunia x hybrida 'Mitchell diploid' (MD) is dominated by phenylpropanoids, many of which are derived from p-coumaric acid. However, the downstream processes involved in the production of caffeoyl-CoA and feruloyl-CoA from p-coumaric acid are complex, as the genes and biosynthesis steps are associated with flavonoids and lignin synthesis as well as floral volatiles benzenoid/phenylpropanoid (FVBP). Caffeoyl shikimate esterase (CSE) converts caffeoyl shikimate to caffeic acid and is considered one of the essential regulators in lignin production. Moreover, CSE in involved in phenylpropanoid production. To investigate the roles of CSE in FVBP biosynthesis, we used RNAi-mediated CSE down-regulated (ir-PhCSE) petunias. RESULTS: Lowered CSE transcript accumulation in ir-PhCSE plants resulted in reduced lignin layers in the stems and stunted growth, suggesting a positive correlation between lignin layers and lignin content. The altered CSE level influenced the expression of many FVBP genes, including elevated transcripts of p-coumarate-3-hydroxylase (C3H), hydroxycinnamoyl transferase (HCT), and 4-coumaric acid: CoA ligase (4CL). In particular, the expression of C4H in ir-PhCSE plants was more than twice the expression in MD plants. Moreover, the production of volatile compounds was alterend in ir-PhCSE plants. Most floral volatiles decreased, and the amounts of phenylalanine and caffeic acid were significantly lower. CONCLUSIONS: Reduced lignin layers in the stems and stunted growth in ir-PhCSE plants suggest that PhCSE is essential for lignin production and plant growth in petunia. The decreased CSE level influenced the expression of many FVBP genes, and interference of shikimate derivates altered volatile compound production. Significantly decreased caffeic acid, but not ferulic acid, in ir-PhCSE plants suggest that CSE is primarily involved in the reaction of caffeoyl shikimate. Higher C3H and C4H transcripts seem to alleviate accumulated p-coumaric acid resulting from altered CSE. Finally, alteration in C3H, HCT, and 4CL in CSE down-regulated plants suggests an interaction of the FVBP genes, leading to the regulation of floral volatiles of petunia.

Genetic architecture of a pollinator shift and its fate in secondary hybrid zones of two Petunia species.

BACKGROUND: Theory suggests that the genetic architecture of traits under divergent natural selection influences how easily reproductive barriers evolve and are maintained between species. Divergently selected traits with a simple genetic architecture (few loci with major phenotypic effects) should facilitate the establishment and maintenance of reproductive isolation between species that are still connected by some gene flow. While empirical support for this idea appears to be mixed, most studies test the influence of trait architectures on reproductive isolation only indirectly. Petunia plant species are, in part, reproductively isolated by their different pollinators. To investigate the genetic causes and consequences of this ecological isolation, we deciphered the genetic architecture of three floral pollination syndrome traits in naturally occurring hybrids between the widespread Petunia axillaris and the highly endemic and endangered P. exserta. RESULTS: Using population genetics, Bayesian linear mixed modelling and genome-wide association studies, we found that the three pollination syndrome traits vary in genetic architecture. Few genome regions explain a majority of the variation in flavonol content (defining UV floral colour) and strongly predict the trait value in hybrids irrespective of interspecific admixture in the rest of their genomes. In contrast, variation in pistil exsertion and anthocyanin content (defining visible floral colour) is controlled by many genome-wide loci. Opposite to flavonol content, the genome-wide proportion of admixture between the two species predicts trait values in their hybrids. Finally, the genome regions strongly associated with the traits do not show extreme divergence between individuals representing the two species, suggesting that divergent selection on these genome regions is relatively weak within their contact zones. CONCLUSIONS: Among the traits analysed, those with a more complex genetic architecture are best maintained in association with the species upon their secondary contact. We propose that this maintained genotype-phenotype association is a coincidental consequence of the complex genetic architectures of these traits: some of their many underlying small-effect loci are likely to be coincidentally linked with the actual barrier loci keeping these species partially isolated upon secondary contact. Hence, the genetic architecture of a trait seems to matter for the outcome of hybridization not only then when the trait itself is under selection.

SCARECROW-like GRAS protein PES positively regulates petunia floral scent production.

Emission of scent volatiles by flowers is important for successful pollination and consequently, reproduction. Petunia (Petunia hybrida) floral scent is formed mainly by volatile products of the phenylpropanoid pathway. We identified and characterized a regulator of petunia scent production: the GRAS protein PHENYLPROPANOID EMISSION-REGULATING SCARECROW-LIKE (PES). Its expression increased in petals during bud development and was highest in open flowers. Overexpression of PES increased the production of floral volatiles, while its suppression resulted in scent reduction. We showed that PES upregulates the expression of genes encoding enzymes of the phenylpropanoid and shikimate pathways in petals, and of the core regulator of volatile biosynthesis ODORANT1 by activating its promoter. PES is an ortholog of Arabidopsis (Arabidopsis thaliana) PHYTOCHROME A SIGNAL TRANSDUCTION 1, involved in physiological responses to far-red (FR) light. Analyses of the effect of nonphotosynthetic irradiation (low-intensity FR light) on petunia floral volatiles revealed FR light as a scent-activating factor. While PHYTOCHROME A regulated scent-related gene expression and floral scent production under FR light, the influence of PES on volatile production was not limited by FR light conditions.

Physiological changes besides the enhancement of pigmentation in Petunia hybrida caused by overexpression of PhAN2, an R2R3-MYB transcription factor.

KEY MESSAGE: Ectopic expression of PhAN2 in vegetative tissue can improve regeneration and adventitious rooting but inhibit axillary bud outgrowth of petunia, while overexpression specifically in flowers could shorten longevity. Anthocyanin 2 has been only treated as a critical positive regulation factor of anthocyanin biosynthesis in petunia flowers. To determine if this gene had other functions in plant growth, we overexpressed this gene in an an2 mutant petunia cultivar driven by promoters with different strengths or tissue specificity. Various physiological processes of transformants in different growth stages and environments were analyzed. Besides the expected pigmentation improvement in different tissues, the results also showed that ectopic expression of AN2 could improve the regeneration skill but inhibit the axillary bud germination of in vitro plants. Moreover, the rooting ability of shoot tips of transformants was significantly improved, while some transgenic lines' flower longevity was shortened. Gene expression analysis showed that the transcripts level of AN2, partner genes anthocyanin 1 (AN1), anthocyanin 11 (AN11), and target gene dihydroflavonol 4-reductase (DFR) was altered in the different transgenic lines. In addition, ethylene biosynthesis-related genes 1-aminocyclopropane-1-carboxylic acid synthase (ACS1) and ACC oxidase (ACO1) were upregulated in rooting and flower senescence processes but at different time points. Overall, our data demonstrate that the critical role of this AN2 gene in plant growth physiology may extend beyond that of a single activator of anthocyanin biosynthesis.

PhAAT1, encoding an anthocyanin acyltransferase, is transcriptionally regulated by PhAN2 in petunia.

Anthocyanins widely exist in plants and they are important pigments for color of petals and fruits. They are produced through a multi-step pathway controlled by transcription factor complexes. The anthocyanin skeleton modification is the last reaction in the anthocyanin synthesis pathway, which improves the stability of anthocyanins. Acylation modification is an important modification of anthocyanins. However, the identification and function of anthocyanin acyltransferase genes and their expression regulation are rarely reported. In this study, we identified the petunia anthocyanin acyltransferase gene, PhAAT1. PhAAT1 is located in the cytoplasm and PhAAT1 silencing changed flower color and reduced the stability of anthocyanin. Metabolomics analysis showed that PhAAT1 silencing led to the reduction of p-coumaroylated and caffeoylated anthocyanins. In addition, PhAAT1 was positively regulated by the MYB transcription factor, PhAN2, which directly interacts with the promoter of PhAAT1.

Protoplast isolation and transient gene expression in different petunia cultivars.

The protocol optimized for Petunia hybrida cv. Mirage Rose produced high protoplast yields in 3 out of other 11 cultivars (Damask White, Dreams White, and Opera Supreme White). Factors optimized in the protoplast transfection process showed that the best transfection efficiency (80%) was obtained using 2.5 × 105 protoplast density, 40% polyethylene glycol (PEG) concentration, 10 µg plasmid DNA, and 15 min of transfection time. Assessing the usability of the protocol for other cultivars (Damask White, Dreams White, and Opera Supreme White), a reasonable protoplast transfection efficiency (⁓50%) was observed in the cultivars Dreams White and Opera Supreme White, with lower efficiency (⁓50%) observed in the cv. Damask White. The transient expression of enhanced green fluorescent protein (eGFP) in the nucleus of the transfected protoplasts of all cultivars was confirmed using PCR. This system could be valuable for genome editing of unwanted genes in petunias using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology. Furthermore, it could contribute to other studies on protein subcellular localization, protein-protein interactions, and functional gene expression in the petunias.

Shikimate Kinase Plays Important Roles in Anthocyanin Synthesis in Petunia.

In plants, the shikimate pathway is responsible for the production of aromatic amino acids L-tryptophan, L-phenylalanine, and L-tyrosine. L-Phenylalanine is the upstream substrate of flavonoid and anthocyanin synthesis. Shikimate kinase (SK) catalyzes the phosphorylation of the C3 hydroxyl group of shikimate to produce 3-phosphate shikimate (S3P), the fifth step of the shikimate pathway. However, whether SK participates in flavonoid and anthocyanin synthesis is unknown. This study characterized the single-copy PhSK gene in the petunia (Petunia hybrida) genome. PhSK was localized in chloroplasts. PhSK showed a high transcription level in corollas, especially in the coloring stage of flower buds. Suppression of PhSK changed flower color and shape, reduced the content of anthocyanins, and changed the flavonoid metabolome profile in petunia. Surprisingly, PhSK silencing caused a reduction in the shikimate, a substrate of PhSK. Further qPCR analysis showed that PhSK silencing resulted in a reduction in the mRNA level of PhDHQ/SDH, which encodes the protein catalyzing the third and fourth steps of the shikimate pathway, showing a feedback regulation mechanism of gene expression in the shikimate pathway.