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.

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.

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.

Crotonylation versus acetylation in petunia corollas with reduced acetyl-CoA due to PaACL silencing.

Protein acetylation and crotonylation are important posttranslational modifications of lysine. In animal cells, the correlation of acetylation and crotonylation has been well characterized and the lysines of some proteins are acetylated or crotonylated depending on the relative concentrations of acetyl-CoA and crotonyl-CoA. However, in plants, the correlation of acetylation and crotonylation and the effects of the relative intracellular concentrations of crotonyl-CoA and acetyl-CoA on protein crotonylation and acetylation are not well known. In our previous study, PaACL silencing changed the content of acetyl-CoA in petunia (Petunia hybrida) corollas, and the effect of PaACL silencing on the global acetylation proteome in petunia was analyzed. In the present study, we found that PaACL silencing did not significantly alter the content of crotonyl-CoA. We performed a global crotonylation proteome analysis of the corollas of PaACL-silenced and control petunia plants; we found that protein crotonylation was closely related to protein acetylation and that proteins with more crotonylation sites often had more acetylation sites. Crotonylated proteins and acetylated proteins were enriched in many common Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. However, PaACL silencing resulted in different KEGG pathway enrichments of proteins with different levels of crotonylation sites and acetylation sites. PaACLB1-B2 silencing did not led to changes in the opposite direction in crotonylation and acetylation levels at the same lysine site in cytoplasmic proteins, which indicated that cytoplasmic lysine acetylation and crotonylation might not depend on the relative concentrations of acetyl-CoA and crotonyl-CoA. Moreover, the global crotonylome and acetylome were weakly positively correlated in the corollas of PaACL-silenced and control plants.

Ph5GT silencing alters flower color and flavonoids metabolome profile in petunia.

Anthocyanins are important pigments in plants and glycosylation plays an important role in the stability of anthocyanins. Anthocyanin 5-O-glucosyltransferase (5GT) can glycosylate anthocyanin at the 5-O-position. Till now, the enzymatic activity characteristics of 5GT had been studied in vitro in a variety of plants. However, the subcellular localization of 5GT protein still remained unclear, and little genetic evidence on the roles of 5GT in plants has been reported. The full-length Ph5GT gene from petunia (Petunia hybrida) was isolated in this study. Green fluorescent fusion protein assays revealed that Ph5GT protein was localized to the cytoplasm. Ph5GT was found to be highly expressed in flowers, with highest levels of expression occurring during the coloring stage of flower development. Furthermore, Ph5GT silencing led to the change in flower color from purple to light purple and a significant reduction in total anthocyanin content. The metabolome analysis revealed that the content of malvidins and petunidins modified by glycosylation at the 5-O-position was significantly reduced, while the content of their precursor without glycosylation was significantly increased, implying that Ph5GT could glycosylate malvidin and petunidin derivatives and that the substrate types of Ph5GT were expanded in comparison to previous studies.

PhRHMs play important roles in leaf and flower development and anthocyanin synthesis in petunia.

Anthocyanins, vital metabolites in plants, are formed by anthocyanidins combined with various monosaccharides, including glucose, rhamnose, and arabinose. Rhamnose contributes greatly to the glycosylation of anthocyanidins. There are two kinds of rhamnose synthase (RS): rhamnose biosynthesis (RHM), and nucleotide-RS/epimerase-reductase (UER1). Nevertheless, no RS isoform was reported to be involved in anthocyanin synthesis. Here, three homologous PhRHM genes, namely PhRHM1, PhRHM2, and PhRHM3, and one PhUER1 gene from petunia were cloned and characterized. Green fluorescent protein fusion protein assays revealed that PhRHMs and PhUER1 are localized in the cytoplasm. We obtained PhRHM1 or/and PhRHM2 or PhUER1 silenced petunia plants and did not attempt to obtain PhRHM3 silenced plants since PhRHM3 mRNA was not detected in petunia organs examined. PhRHM1 and PhRHM2 (PhRHM1-2) silencing induced abnormal plant growth and decreased the contents of l-rhamnose, photosynthetic pigments and total anthocyanins, while PhUER1 silencing did not cause any visible phenotypic changes. Flavonoid metabolome analysis further revealed that PhRHM1-2 silencing reduced the contents of anthocyanins with rhamnose residue. These results revealed that PhRHMs contribute to the biosynthesis of rhamnose and that PhRHMs participate in the anthocyanin rhamnosylation in petunia, while PhUER1 does not.

Phosphoproteome analysis reveals the involvement of protein dephosphorylation in ethylene-induced corolla senescence in petunia.

BACKGROUND: Senescence represents the last stage of flower development. Phosphorylation is the key posttranslational modification that regulates protein functions, and kinases may be more required than phosphatases during plant growth and development. However, little is known about global phosphorylation changes during flower senescence. RESULTS: In this work, we quantitatively investigated the petunia phosphoproteome following ethylene or air treatment. In total, 2170 phosphosites in 1184 protein groups were identified, among which 2059 sites in 1124 proteins were quantified. To our surprise, treatment with ethylene resulted in 697 downregulated and only 117 upregulated phosphosites using a 1.5-fold threshold (FDR < 0.05), which showed that ethylene negatively regulates global phosphorylation levels and that phosphorylation of many proteins was not necessary during flower senescence. Phosphoproteome analysis showed that ethylene regulates ethylene and ABA signalling transduction pathways via phosphorylation levels. One of the major targets of ethylene-induced dephosphorylation is the plant mRNA splicing machinery, and ethylene treatment increases the number of alternative splicing events of precursor RNAs in petunia corollas. CONCLUSIONS: Protein dephosphorylation could play an important role in ethylene-induced senescence, and ethylene treatment increased the number of AS precursor RNAs in petunia corollas.

Mitochondrial citrate synthase plays important roles in anthocyanin synthesis in petunia.

Anthocyanins are important flavonoid pigments in plants. Malonyl CoA is an important intermediate in anthocyanin synthesis, and citrate, formed by citrate synthase (CS) catalysing oxaloacetate, is the precursor for the formation of malonyl-CoA. CS is composed of two isoforms, mitochondrial citrate synthase (mCS), a key enzyme of the tricarboxylic acid (TCA) cycle, and citrate synthase (CSY) localizated in microbodies in plants. However, no CS isoform involvement in anthocyanin synthesis has been reported. In this study, we identified the entire CS family in petunia (Petunia hybrida): PhmCS, PhCSY1 and PhCSY2. We obtained petunia plants silenced for the three genes. PhmCS silencing resulted in abnormal development of leaves and flowers. The contents of citrate and anthocyanins were significantly reduced in flowers in PhmCS-silenced plants. However, silencing of PhCSY1 and/or PhCSY2 did not cause a visible phenotype change in petunia. These results showed that PhmCS is involved in anthocyanin synthesis and the development of leaves and flowers, and that the citrate involved in anthocyanin synthesis mainly derived from mitochondria rather than microbodies in petunia.

Suppression of chorismate synthase, which is localized in chloroplasts and peroxisomes, results in abnormal flower development and anthocyanin reduction in petunia.

In plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. Chorismate synthase (CS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a single-copy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fluorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. Petunia plants with reduced PhCS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal flower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were significantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia.

PaACL silencing accelerates flower senescence and changes the proteome to maintain metabolic homeostasis in Petunia hybrida.

Cytosolic acetyl-CoA is an intermediate of the synthesis of most secondary metabolites and the source of acetyl for protein acetylation. The formation of cytosolic acetyl-CoA from citrate is catalysed by ATP-citrate lyase (ACL). However, the function of ACL in global metabolite synthesis and global protein acetylation is not well known. Here, four genes, PaACLA1, PaACLA2, PaACLB1, and PaACLB2, which encode the ACLA and ACLB subunits of ACL in Petunia axillaris, were identified as the same sequences in Petunia hybrida 'Ultra'. Silencing of PaACLA1-A2 and PaACLB1-B2 led to abnormal leaf and flower development, reduced total anthocyanin content, and accelerated flower senescence in petunia 'Ultra'. Metabolome and acetylome analysis revealed that PaACLB1-B2 silencing increased the content of many downstream metabolites of acetyl-CoA metabolism and the levels of acetylation of many proteins in petunia corollas. Mechanistically, the metabolic stress induced by reduction of acetyl-CoA in PaACL-silenced petunia corollas caused global and specific changes in the transcriptome, the proteome, and the acetylome, with the effect of maintaining metabolic homeostasis. In addition, the global proteome and acetylome were negatively correlated under acetyl-CoA deficiency. Together, our results suggest that ACL acts as an important metabolic regulator that maintains metabolic homeostasis by promoting changes in the transcriptome, proteome. and acetylome.

The N1-Methyladenosine Methylome of Petunia mRNA.

N1-methyladenosine is a unique type of base methylation in that it blocks Watson-Crick base pairing and introduces a positive charge. m1A is prevalent in yeast and mammalian mRNA and plays a functional role. However, little is known about the abundance, dynamics, and topology of this modification in plant mRNA. Dot blotting and liquid chromatography tandem mass spectrometry analyses revealed a dynamic pattern of m1A mRNA modification in various tissues and at different developmental stages in petunia (Petunia hybrida), a model system for plant growth and development. We performed transcriptome-wide profiling of m1A in petunia mRNA by m1A mRNA immunoprecipitation followed by a deep-sequencing approach (m1A-seq, using an m1A-specific antibody). m1A-seq analysis identified 4,993 m1A peaks in 3,231 genes expressed in petunia corollas; there were 251 m1A peaks in which A residues were partly replaced by thymine and/or reverse transcription stopped at an adenine site. m1A was enriched in coding sequences, with single peaks located immediately after start codons. Ethylene treatment upregulated 400 m1A peaks in 375 mRNAs and downregulated 603 m1A peaks in 530 mRNAs in petunia corollas; 975 m1A peaks in mRNA were only detected in corollas treated with air and 430 were only detected in corollas treated with ethylene. Silencing of petunia tRNA-specific methyltransferase 61A (PhTRMT61A) reduced the m1A level in mRNA in vivo and in vitro. In addition, PhTRMT61A silencing caused abnormal leaf development, and the PhTRMT61A protein was localized to the nucleus. Thus, m1A in mRNA is an important epitranscriptome marker and plays a role in plant growth and development.

PhDHS Is Involved in Chloroplast Development in Petunia.

Deoxyhypusine synthase (DHS) is encoded by a nuclear gene and is the key enzyme involved in the post-translational activation of the eukaryotic translation initiation factor eIF5A. DHS plays important roles in plant growth and development. To gain a better understanding of DHS, the petunia (Petunia hybrida) PhDHS gene was isolated, and the role of PhDHS in plant growth was analyzed. PhDHS protein was localized to the nucleus and cytoplasm. Virus-mediated PhDHS silencing caused a sectored chlorotic leaf phenotype. Chlorophyll levels and photosystem II activity were reduced, and chloroplast development was abnormal in PhDHS-silenced leaves. In addition, PhDHS silencing resulted in extended leaf longevity and thick leaves. A proteome assay revealed that 308 proteins are upregulated and 266 proteins are downregulated in PhDHS-silenced plants compared with control, among the latter, 21 proteins of photosystem I and photosystem II and 12 thylakoid (thylakoid lumen and thylakoid membrane) proteins. In addition, the mRNA level of PheIF5A-1 significantly decreased in PhDHS-silenced plants, while that of another three PheIF5As were not significantly affected in PhDHS-silenced plants. Thus, silencing of PhDHS affects photosynthesis presumably as an indirect effect due to reduced expression of PheIF5A-1 in petunia. Significance: PhDHS-silenced plants develop yellow leaves and exhibit a reduced level of photosynthetic pigment in mesophyll cells. In addition, arrested development of chloroplasts is observed in the yellow leaves.

PhERF6, interacting with EOBI, negatively regulates fragrance biosynthesis in petunia flowers.

In petunia, the production of volatile benzenoids/phenylpropanoids determines floral aroma, highly regulated by development, rhythm and ethylene. Previous studies identified several R2R3-type MYB trans-factors as positive regulators of scent biosynthesis in petunia flowers. Ethylene response factors (ERFs) have been shown to take part in the signal transduction of hormones, and regulation of metabolism and development processes in various plant species. Using virus-induced gene silencing technology, a negative regulator of volatile benzenoid biosynthesis, PhERF6, was identified by a screen for regulators of the expression of genes related to scent production. PhERF6 expression was temporally and spatially connected with scent production and was upregulated by exogenous ethylene. Up-/downregulation of the mRNA level of PhERF6 affected the expression of ODO1 and several floral scent-related genes. PhERF6 silencing led to a significant increase in the concentrations of volatiles emitted by flowers. Yeast two-hybrid, bimolecular fluorescence complementation and co-immunoprecipitation assays indicated that PhERF6 interacted with the N-terminus of EOBI, which includes two DNA binding domains. Our results show that PhERF6 negatively regulates volatile production in petunia flowers by competing for the binding of the c-myb domains of the EOBI protein with the promoters of genes related to floral scent.

PhCESA3 silencing inhibits elongation and stimulates radial expansion in petunia.

Cellulose synthase catalytic subunits (CESAs) play important roles in plant growth, development and disease resistance. Previous studies have shown an essential role of Arabidopsis thaliana CESA3 in plant growth. However, little is known about the role of CESA3 in species other than A. thaliana. To gain a better understanding of CESA3, the petunia (Petunia hybrida) PhCESA3 gene was isolated, and the role of PhCESA3 in plant growth was analyzed in a wide range of plants. PhCESA3 mRNA was present at varying levels in tissues examined. VIGS-mediated PhCESA3 silencing resulted in dwarfing of plant height, which was consistent with the phenotype of the A. thaliana rsw1 mutant (a temperature-sensitive allele of AtCESA1), the A. thaliana cev1 mutant (the AtCESA3 mild mutant), and the antisense AtCESA3 line. However, PhCESA3 silencing led to swollen stems, pedicels, filaments, styles and epidermal hairs as well as thickened leaves and corollas, which were not observed in the A. thaliana cev1 mutant, the rsw1 mutant and the antisense AtCESA3 line. Further micrographs showed that PhCESA3 silencing reduced the length and increased the width of cells, suggesting that PhCESA3 silencing inhibits elongation and stimulates radial expansion in petunia.

The acyl-activating enzyme PhAAE13 is an alternative enzymatic source of precursors for anthocyanin biosynthesis in petunia flowers.

Anthocyanins, a class of flavonoids, are responsible for the orange to blue coloration of flowers and act as visual attractors to aid pollination and seed dispersal. Malonyl-CoA is the precursor for the formation of flavonoids and anthocyanins. Previous studies have suggested that malonyl-CoA is formed almost exclusively by acetyl-CoA carboxylase, which catalyzes the ATP-dependent formation of malonyl-CoA from acetyl-CoA and bicarbonate. In the present study, the full-length cDNA of Petunia hybrida acyl-activating enzyme 13 (PhAAE13), a member of clade VII of the AAE superfamily that encodes malonyl-CoA synthetase, was isolated. The expression of PhAAE13 was highest in corollas and was down-regulated by ethylene. Virus-induced gene silencing of petunia PhAAE13 significantly reduced anthocyanin accumulation, fatty acid content, and cuticular wax components content, and increased malonic acid content in flowers. The silencing of PhAAE3 and PhAAE14, the other two genes in clade VII of the AAE superfamily, did not change the anthocyanin content in petunia flowers. This study provides strong evidence indicating that PhAAE13, among clade VII of the AAE superfamily, is specifically involved in anthocyanin biosynthesis in petunia flowers.

Proteomes and Ubiquitylomes Analysis Reveals the Involvement of Ubiquitination in Protein Degradation in Petunias.

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate < 0.001), 284 down-regulated and 233 up-regulated proteins, and 320 up-regulated and 127 down-regulated ubiquitination sites using a 1.5-fold threshold (P < 0.05), indicating that global ubiquitination levels increase during ethylene-mediated corolla senescence in petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation.

Molecular Characterization and Functional Analysis of Two Petunia PhEILs.

Ethylene plays an important role in flower senescence of many plants. Arabidopsis ETHYLENE INSENSITIVE3 (EIN3) and its homolog EIL1 are the downstream component of ethylene signaling transduction. However, the function of EILs during flower senescence remains unknown. Here, a petunia EIL gene, PhEIL2, was isolated. Phylogenetic tree showed that PhEIL1, whose coding gene is previously isolated, and PhEIL2 are the homologs of Arabidopsis AtEIL3 and AtEIL1, respectively. The expression of both PhEIL1 and PhEIL2 is the highest in corollas and increased during corolla senescence. Ethylene treatment increased the mRNA level of PhEIL1 but reduced that of PhEIL2. VIGS-mediated both PhEIL1 and PhEIL2 silencing delayed flower senescence, and significantly reduced ethylene production and the expression of PhERF3 and PhCP2, two senescence-associated genes in petunia flowers. The PhEIL2 protein activating transcription domain is identified in the 353-612-amino acids at C-terminal of PhEIL2 and yeast two-hybrid and bimolecular fluorescence complementation assays show that PhEIL2 interacts with PhEIL1, suggesting that PhEIL1 and PhEIL2 might form heterodimers to recognize their targets. These molecular characterizations of PhEIL1 and PhEIL2 in petunia are different with those of in Vigna radiata and Arabidopsis.

Expression and functional analysis of PhEOL1 and PhEOL2 during flower senescence in petunia.

The ethylene biosynthesis pathway controls flower senescence. Previous studies have shown that Arabidopsis ETHYLENE-OVERPRODUCER1 (ETO1) interacts specifically with and negatively regulates type 2 1-aminocyclopropane-1-carboxylate synthases (ACSs), the rate-limiting enzymes of ethylene biosynthesis. The ethylene biosynthesis pathway controls flower senescence in petunias (Petunia hybrida Juss.). However, the role of ETO1-like genes (EOLs) during flower senescence has not been investigated. Here, two full-length petunia EOL cDNAs, PhEOL1 and PhEOL2, were isolated. RT-PCR assays indicated that the expression of PhEOL1 and PhEOL2 increased after exogenous ethylene treatment. The VIGS-mediated silencing of PhEOL1 accelerated flower senescence and produced more ethylene than the control condition, whereas the silencing of PhEOL2 did not. Notably, the effects caused by PhEOL1 suppression were not enhanced by PhEOL2 suppression in corollas. In addition, the expression of two petunia type 2 PhACS genes increased during flower senescence and after ethylene treatment. A yeast two-hybrid assay showed that PhEOL1 interacts with both PhACS2 and PhACS3. It is possible that PhEOL1 is involved in flower senescence by interacting with type 2 PhACSs in petunias.

Functional characterization of PhGR and PhGRL1 during flower senescence in the petunia.

KEY MESSAGE: Petunia PhGRL1 suppression accelerated flower senescence and increased the expression of the genes downstream of ethylene signaling, whereas PhGR suppression did not. Ethylene plays an important role in flowers senescence. Homologous proteins Green-Ripe and Reversion to Ethylene sensitivity1 are positive regulators of ethylene responses in tomato and Arabidopsis, respectively. The petunia flower has served as a model for the study of ethylene response during senescence. In this study, petunia PhGR and PhGRL1 expression was analyzed in different organs, throughout floral senescence, and after exogenous ethylene treatment; and the roles of PhGR and PhGRL1 during petunia flower senescence were investigated. PhGRL1 suppression mediated by virus-induced gene silencing accelerated flower senescence and increased ethylene production; however, the suppression of PhGR did not. Taken together, these data suggest that PhGRL1 is involved in negative regulation of flower senescence, possibly via ethylene production inhibition and consequently reduced ethylene signaling activation.