Long non-coding RNA-mediated competing endogenous RNA regulatory network during flower development and color formation in Melastoma candidum.

M. candidum, an evergreen shrubby flower known for its superior adaptation ability in South China, has gained increased attention in garden applications. However, scant attention has been paid to its flower development and color formation process at the non-coding RNA level. To fill this gap, we conducted a comprehensive analysis based on long non-coding RNA sequencing (lncRNA-seq), RNA-seq, small RNA sequencing (sRNA-seq), and widely targeted metabolome detection of three different flower developmental stages of M. candidum. After differentially expressed lncRNAs (DElncRNAs), differentially expressed mRNAs (DEmRNAs), differentially expressed microRNAs (DEmiRNAs), and differentially synthesized metabolites (DSmets) analyses between the different flower developmental stages, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were conducted to identify some key genes and metabolites in flavonoid, flavone, anthocyanin, carotenoid, and alkaloid-related GO terms and biosynthetic pathways. Three direct-acting models, including antisense-acting, cis-acting, and trans-acting between lncRNAs and mRNAs, were detected to illustrate the direct function of lncRNAs on target genes during flower development and color formation. Based on the competitive endogenous RNA (ceRNA) regulatory theory, we constructed a lncRNA-mediated regulatory network composed of DElncRNAs, DEmiRNAs, DEmRNAs, and DSmets to elucidate the indirect role of lncRNAs in the flower development and color formation of M. candidum. By utilizing correlation analyses between DERNAs and DSmets within the ceRNA regulatory network, alongside verification trials of the ceRNA regulatory mechanism, the study successfully illustrated the significance of lncRNAs in flower development and color formation process. This research provides a foundation for improving and regulating flower color at the lncRNA level in M. candidum, and sheds light on the potential applications of non-coding RNA in studies of flower development.

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.

Genome-Wide Identification and Characterization of TCP Gene Family Members in Melastoma candidum.

It has been confirmed that the plant-specific Teosinte-branched 1/Cycloidea/Proliferating (TCP) gene family plays a pivotal role during plant growth and development. M. candidum is a native ornamental species and has a wide range of pharmacodynamic effects. However, there is still a lack of research on TCP's role in controlling M. candidum's development, abiotic stress responses and hormone metabolism. A comprehensive description of the TCP gene family in M. candidum is urgently needed. In this study, we used the HMMER search method in conjunction with the BLASTp method to identify the members of the TCP gene family, and a total of 35 TCP genes were identified. A domain analysis further confirmed that all 35 TCPs contained a TCP superfamily, a characteristic involved in dimerization and DNA binding that can be found in most genes from this gene family, suggesting that our identification was effective. As a result of the domain conservation analysis, the 35 TCP genes could be classified into two classes, TCP-P and TCP-C, based on the conservative regions of 55 and 59 amino acids, respectively. Gene-duplication analysis revealed that most TCP genes were present in duplication events that eventually led to TCP gene expansion in M. candidum. All the detected gene pairs had a Ka/Ks value of less than one, suggesting that purification selection is the most important factor that influences the evolution of TCP genes. Phylogenetic analysis of three species displayed the evolutionary relationship of TCP genes across different species and further confirmed our results. The real-time quantitative PCR (qRT-PCR) results showed that McTCP2a, McTCP7a, McTCP10, McTCP11, McTCP12a, McTCP13, McTCP16, McTCP17, McTCP18, McTCP20 and McTCP21 may be involved in leaf development; McTCP4a, McTCP1, McTCP14, McTCP17, McTCP18, McTCP20, McTCP22 and McTCP24 may be involved in flower development; and McTCP2a, McTCP3, McTCP5a, McTCP6, McTCP7a, McTCP9, McTCP11, McTCP14 and McTCP16 may be involved in seed development. Our results dissect the TCP gene family across the genome of M. candidum and provide valuable information for exploring TCP genes to promote molecular breeding and property improvement of M. candidum in the future.

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.

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.

Effects of Water and Alkaline Solution on Durability of Carbon-Glass Hybrid Fiber Reinforced Polymer Bars.

The glass fiber reacts with the hydroxyl owing to the concrete pore solution. A thin coat of carbon fiber wraps around the internal GFRP bars to improve the durability of internal GFRP bars in harsh environments. This paper investigates the effect of a thin carbon fiber coat on the durability of the carbon-glass hybrid fiber reinforced polymer bars (HFRP bars) in water, and compares the performance of FRP bars in alkaline solution. To this end, the water absorption behavior, interlaminar shear strength of both the GFRP bars and the HFRP bars was characterized in water and alkaline solution. The results indicate that the diffusivity coefficient of the carbon fiber coat is higher than that of internal GFRP in water. Compared to the GFRP bars in water, the HFRP bars have a higher diffusivity coefficient and saturation water absorption. It caused that the interlaminar shear strength of the HFRP bars aged in water at a temperature of 60 °C for 140 days decreases more markedly than that of the GFRP bars aged under similar conditions. Finally, it was proved that the thin carbon fiber coat does not slow the deterioration of the GFRP bars in water, while the carbon fiber coat significantly improves the retention of the interlaminar shear strength of the HFRP bars in the alkaline solution owing to the prevention of internal glass fiber reactivated by alkali ions.

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.

Genomic identification and expression analysis of the BBX transcription factor gene family in Petunia hybrida.

The B-box proteins (BBXs) are a class of zinc finger transcription factors containing one or two B-BOX domains that play important roles in plant growth, development and stress response. The petunia (Petunia hybrida) is a model ornamental plant, and its draft genome has been published. However, no systematic study of the BBX gene family in Petunia has been reported. In this study, a total of 28 BBX members from the Petunia genome were identified. We performed analyses of their phylogenetic relationships, structures, conserved motifs, promoter regions, and expression patterns. Based on the phylogenetic relationship, the PhBBXs were divided into six groups. Analysis of the gene structures and conserved motifs further confirmed the closer relationships in each group. Based on the RNA-seq data, the transcript abundance of PhBBXs in different tissues were divided into two major groups. The analysis of cis-elements showed that many stress responsive elements appeared in the promoter region of most PhBBX genes. The stress response patterns of PhBBXs were detected under drought, salinity, cold and heat treatments. Based on the RNA-seq data, we found that 3 genes responded to drought, 8 genes responded to salt, 18 genes responded to cold, and 15 genes responded to heat. In conclusion, this study may facilitate further functional studies of BBXs 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.

Genome-Wide Identification and Expression Profile Analysis of the NF-Y Transcription Factor Gene Family in Petunia hybrida.

Nuclear Factor Ys (NF-Ys) are a class of heterotrimeric transcription factors that play key roles in many biological processes, such as abiotic stress responses, flowering time, and root development. The petunia (Petunia hybrida) is a model ornamental plant, and its draft genome has been published. However, no details regarding the NF-Y gene family in petunias are available. Here, 27 NF-Y members from the petunia genome were identified, including 10 PhNF-YAs, 13 PhNF-YBs, and 4 PhNF-YCs. Multiple alignments showed that all PhNF-Y proteins had clear conserved core regions flanked by non-conserved sequences. Phylogenetic analyses identified five pairs of orthologues NF-YB proteins from Petunia and Arabidopsis, and six pairs of paralogues NF-Y proteins in Petunia. Analysis of the gene structure and conserved motifs further confirmed the closer relationship in each subfamily. Bioinformatics analysis revealed that 16 PhNF-Ys could be targeted by 18 miRNA families. RNA-seq results showed that expression patterns of PhNF-Ys among four major organs (leaf, stem, flower, and root) were clustered into six major groups. The stress response pattern of PhNF-Ys was identified under cold, heat, drought, and salinity treatments. Based on the RNA-seq data, we found that 3 genes responded to drought, 4 genes responded to salt, 10 genes responded to cold, and 9 genes responded to hot. In conclusion, this study provides useful information for further studying the functions of NF-Ys in stress response.

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.