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Background Development ornamental varieties with enhanced floral traits and distinctive characteristics is a central goal in floriculture. Anthocyanins, the principal flavonoid pigments in floral tissues, are synthesized via the well-characterized flavonoid biosynthesis pathway. Chalcone isomerase ( CHI ) catalyzes the first committed reaction in the flavonoid biosynthetic pathway. Method In this study, we investigated the effects of CHI suppression via RNA interference (RNAi) in three Petunia hybrida cultivars exhibiting distinct petal colors. Leaf discs from three Petunia hybrida lines exhibiting distinct petal colors (blue, pink, and purple) were inoculated with Agrobacterium tumefaciens harboring the recombinant plasmid pBI121 containing the RNAi construct targeting Phchi . Transgenic plants were regenerated on MS medium via microshoots emerging from wounded explant regions and verified by PCR using two primer pairs specific to the CHI silencing vector. Gene expression analysis was conducted for chi and associated pigment biosynthesis genes, including flavanone 3-hydroxylase (F3H) , flavonoid 3′-hydroxylase (F3′H) , flavonoid 3′,5′-hydroxylase (F3′5′H) , and dihydroflavonol 4-reductase (DFR) , comparing transgenic and wild-type plants. Chalcone and naringenin contents were quantified using a NanoDrop ELISA plate reader. Data analysis was performed using SPSS version 16, and mean comparisons were evaluated via T-test at a significance level of α = 0.05. Results Transformation efficiencies for CHI -silenced Petunia lines were 57.89%, 72.0% and 84.0% for the pink, blue, and purple phenotypes, respectively. Phenotypic evaluation revealed not only altered pigment distributions but also novel floral morphologies, such as tetramerous corollas and twisted tubular petal margins, in the CHI -suppressed lines. Quantitative metabolic profiling demonstrated a significant reduction in naringenin levels across all transgenic lines, whereas, chalcone accumulation was significantly elevated in a subset of lines. The expression of CHI gene was markedly reduced in all lines carrying the CHI -RNAi construct; however, transcript levels did not differ significantly between the aberrant floral phenotypes and the control. Transcript levels of key downstream flavonoid biosynthesis pathway genes, including F3H , F3'H , F3'5'H , and DFR were significantly attenuated in CHI -silenced lines compared with wild-type controls. This concerted down‐regulation indicates that suppression of the CHI ‐catalyzed step exerts a broader repressive effect on subsequent pathway components. Despite the overall repression of flavonoid-biosynthetic genes in CHI -silenced lines, certain novel phenotypes maintained or even increased expression of specific downstream enzymes. Notably, blue-flowered transformants exhibited F3'H transcript levels comparable to wild-type control, and pink-flowered lines retained F3'5'H expression. These exceptions highlight the nuanced, tissue-specific regulatory responses triggered by CHI suppression. Conclusion Collectively, our results demonstrate that RNAi- mediated silencing of CHI gene is an effective strategy for generating novel pigmentation patterns and floral morphology in Petunia hybrida . Moreover, this work provides critical insights into how targeted manipulation of a single enzymatic step can influence the broader transcriptional network governing anthocyanin biosynthesis.

Biosynthesis of benzoic acid from Phe requires shortening of the side chain by two carbons, which can occur via the β-oxidative or nonoxidative pathways. The first step in the β-oxidative pathway is cinnamoyl-CoA formation, likely catalyzed by a member of the 4-coumarate: CoA ligase (4CL) family that converts a range of frans-cinnamic acid derivatives into the corresponding CoA thioesters. Using a functional genomics approach, we identified two potential CoA-ligases from petunia [Petunia hybrids) petal-specific cDNA libraries. The cognate proteins share only 25% amino acid identity and are highly expressed in petunia corollas. Biochemical characterization of the recombinant proteins revealed that one of these proteins (Ph-4CL1) has broad substrate specificity and represents a bona fide 4CL, whereas the other is a cinnamateiCoA ligase (Ph-CNL). RNA interference suppression of Ph-4CL1 did not affect the petunia benzenoid scent profile, whereas downregulation of Ph-CNL resulted in a decrease in emission of benzylbenzoate, phenylethylbenzoate, and methylbenzoate. Green fluorescent protein localization studies revealed that the Ph-4CL1 protein is localized in the cytosol, whereas Ph-CNL is in peroxisomes. Our results indicate that subcellular compartmentalization of enzymes affects their involvement in the benzenoid network and provide evidence that cinnamoyl-CoA formation by Ph-CNL in the peroxisomes is the committed step in the β-oxidative pathway.

Adventitious root formation (ARF) in the model plant Petunia hybrida cv. Mitchell has been analysed in terms of anatomy, gene expression, enzymatic activities and levels of metabolites. This study focuses on the involvement of wound response and primary metabolism. Microscopic techniques were complemented with targeted transcript, enzyme and metabolite profiling using real time polymerase chain reaction (PCR), Northern blot, enzymatic assays, chromatography and mass spectrometry. Three days after severance from the stock plants, first meristematic cells appeared which further developed into root primordia and finally adventitious roots. Excision of cuttings led to a fast and transient increase in the wound-hormone jasmonic acid, followed by the expression of jasmonate-regulated genes such as cell wall invertase. Analysis of soluble and insoluble carbohydrates showed a continuous accumulation during ARF. A broad metabolite profiling revealed a strong increase in organic acids and resynthesis of essential amino acids. Substantial changes in enzyme activities and metabolite levels indicate that specific enzymes and metabolites might play a crucial role during ARF. Three metabolic phases could be defined: (i) sink establishment phase characterized by apoplastic unloading of sucrose and being probably mediated by jasmonates; (ii) recovery phase; and (iii) maintenance phase, in which a symplastic unloading occurs.

Petunia possesses self-incompatibility, by which pistils reject self-pollen but accept non-self-pollen for fertilization. Self-/nonself-recognition between pollen and pistil is regulated by the pistil-specific S-RNase gene and by multiple pollen-specific S-locus F-box (SLF) genes. To date, 10 SLF genes have been identified by various methods, and seven have been shown to be involved in pollen specificity. For a given S-haplotype, each SLF interacts with a subset of its non-self S-RNases, and an as yet unknown number of SLFs are thought to collectively mediate ubiquitination and degradation of all non-self S-RNases to allow crosscompatible pollination. To identify a complete suite of SLF genes of P. inflata, we used a de novo RNA-seq approach to analyze the pollen transcriptomes of S₂-haplotype and S₃-haplotype, as well as the leaf transcriptome of the S₃S₃ genotype. We searched for genes that fit several criteria established from the properties of the known SLF genes and identified the same seven new SLF genes in S₂-haplotype and S₃-haplotype, suggesting that a total of 17 SLF genes constitute pollen specificity in each S-haplotype. This finding lays the foundation for understanding how multiple SLF genes evolved and the biochemical basis for differential interactions between SLF proteins and S-RNases.

Carotenoids and carotenoid cleavage products play an important and integral role in plant development. The Decreased apical dominance1 (Dad1)/PhCCD8 gene of petunia (Petunia hybrida) encodes a hypothetical carotenoid cleavage dioxygenase (CCD) and ortholog of the MORE AXILLARY GROWTH4 (MAX4)/AtCCD8 gene. The dad1-1 mutant allele was inactivated by insertion of an unusual transposon (Dad-one transposon), and the dad1-3 allele is a revertant allele of dad1-1. Consistent with its role in producing a graft-transmissible compound that can alter branching, the Dad1/PhCCD8 gene is expressed in root and shoot tissue. This expression is upregulated in the stems of the dad1-1, dad2, and dad3 increased branching mutants, indicating feedback regulation of the gene in this tissue. However, this feedback regulation does not affect the root expression of Dad1/PhCCD8. Overexpression of Dad1/PhCCD8 in the dad1-1 mutant complemented the mutant phenotype, and RNA interference in the wild type resulted in an increased branching phenotype. Other differences in phenotype associated with the loss of Dad1/PhCCD8 function included altered timing of axillary meristem development, delayed leaf senescence, smaller flowers, reduced internode length, and reduced root growth. These data indicate that the substrate(s) and/or product(s) of the Dad1/PhCCD8 enzyme are mobile signal molecules with diverse roles in plant development.

Background Anthocyanins are a class of brightly colored, glycosylated flavonoid pigments that imbue their flower and fruit host tissues with hues of predominantly red, orange, purple, and blue. Although all anthocyanins exhibit pH-responsive photochemical changes, distinct structural decorations on the core anthocyanin skeleton also cause dramatic color shifts, in addition to improved stabilities and unique pharmacological properties. In this work, we report for the first time the extension of the reconstituted plant anthocyanin pathway from (+)-catechin to O -methylated anthocyanins in a microbial production system, an effort which requires simultaneous co-option of the endogenous metabolites UDP-glucose and S -adenosyl- l -methionine (SAM or AdoMet). Results Anthocyanin O -methyltransferase (AOMT) orthologs from various plant sources were co-expressed in Escherichia coli with Petunia hybrida anthocyanidin synthase ( Ph ANS) and Arabidopsis thaliana anthocyanidin 3- O -glucosyltransferase ( At 3GT). Vitis vinifera AOMT ( Vv AOMT1) and fragrant cyclamen ‘Kaori-no-mai’ AOMT ( Ckm OMT2) were found to be the most effective AOMTs for production of the 3′- O -methylated product peonidin 3- O -glucoside (P3G), attaining the highest titers at 2.4 and 2.7 mg/L, respectively. Following modulation of plasmid copy number and optimization of Vv AOMT1 and Ckm OMT2 expression conditions, production was further improved to 23 mg/L using Vv AOMT1. Finally, CRISPRi was utilized to silence the transcriptional repressor MetJ in order to deregulate the methionine biosynthetic pathway and improve SAM availability for O -methylation of cyanidin 3- O -glucoside (C3G), the biosynthetic precursor to P3G. MetJ repression led to a final titer of 51 mg/L (56 mg/L upon scale-up to shake flask), representing a twofold improvement over the non-targeting CRISPRi control strain and 21-fold improvement overall. Conclusions An E. coli strain was engineered for production of the specialty anthocyanin P3G using the abundant and comparatively inexpensive flavonol precursor, (+)-catechin. Furthermore, dCas9-mediated transcriptional repression of metJ alleviated a limiting SAM pool size, enhancing titers of the methylated anthocyanin product. While microbial production of P3G and other O -methylated anthocyanin pigments will likely be valuable to the food industry as natural food and beverage colorants, we expect that the strain constructed here will also prove useful to the ornamental plant industry as a platform for evaluating putative anthocyanin O -methyltransferases in pursuit of bespoke flower pigment compositions.

One of the key factors that defines plant form is the regulation of when and where branches develop. The diversity of form observed in nature results, in part, from variation in the regulation of branching between species. Two CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes, CCD7 and CCD8, are required for the production of a branch-suppressing plant hormone. Here, we report that the decreased apical dominance3 (dad3) mutant of petunia (Petunia hybrida) results from the mutation of the PhCCD7 gene and has a less severe branching phenotype than mutation of PhCCD8 (dad1). An analysis of the expression of this gene in wild-type, mutant, and grafted petunia suggests that in petunia, CCD7 and CCD8 are coordinately regulated. In contrast to observations in Arabidopsis (Arabidopsis thaliana), ccd7ccd8 double mutants in petunia show an additive phenotype. An analysis using dad3 or dad1 mutant scions grafted to wild-type rootstocks showed that when these plants produce adventitious mutant roots, branching is increased above that seen in plants where the mutant roots are removed. The results presented here indicate that mutation of either CCD7 or CCD8 in petunia results in both the loss of an inhibitor of branching and an increase in a promoter of branching.

Acylsugars are synthesized in the glandular trichomes of the Solanaceae family and are implicated in protection against abiotic and biotic stress. Acylsugars are composed of either sucrose or glucose esterified with varying numbers of acyl chains of differing length. In tomato (Solanum lycopersicum), acylsugar assembly requires four acylsugar acyltransferases (ASATs) of the BAHD superfamily. Tomato ASATs catalyze the sequential esterification of acyl-coenzyme A thioesters to the R4, R3, R3ʹ, and R2 positions of sucrose, yielding a tetra-acylsucrose. Petunia spp. synthesize acylsugars that are structurally distinct from those of tomato. To explore the mechanisms underlying this chemical diversity, a Petunia axillaris transcriptome was mined for trichome preferentially expressed BAHDs. A combination of phylogenetic analyses, gene silencing, and biochemical analyses coupled with structural elucidation of metabolites revealed that acylsugar assembly is not conserved between tomato and petunia. In P. axillaris, tetra-acylsucrose assembly occurs through the action of four ASATs, which catalyze sequential addition of acyl groups to the R2, R4, R3, and R6 positions. Notably, in P. axillaris, PaxASAT1 and PaxASAT4 catalyze the acylation of the R2 and R6 positions of sucrose, respectively, and no clear orthologs exist in tomato. Similarly, petunia acylsugars lack an acyl group at the R3ʹ position, and congruently, an ortholog of SlASAT3, which catalyzes acylation at the R3ʹ position in tomato, is absent in P. axillaris. Furthermore, where putative orthologous relationships of ASATs are predicted between tomato and petunia, these are not supported by biochemical assays. Overall, these data demonstrate the considerable evolutionary plasticity of acylsugar biosynthesis.

The majority of carotenoids in petals are xanthophylls and most of these xanthophylls are esterified with fatty acids. Although petunia ( Petunia x hybrida ) is an important ornamental plant, it cannot accumulate enough carotenoids to have deep-yellow flowers. Our previous study suggested that low esterification activity causes low carotenoid accumulation in petunia corollas. Here, we introduced xanthophyll esterase ( XES ) from the petals of Ipomoea obscura , tomato ( Solanum lycopersicum ), and marigold ( Tagetes erecta ) into a pale-yellow-flowered cultivar of petunia to see whether these affect carotenoid accumulation in petunia corollas. Carotenoid contents and the proportions of esterified xanthophylls were elevated in the corollas of XES -overexpressing ( XES -OX) transformants. Expression analysis showed that the transcript levels of endogenous carotenoid biosynthetic genes, which included geranylgeranyl diphosphate synthase 2, ζ-carotene desaturase , and lycopene β-ring cyclase in corolla tubes were upregulated in XES -OX plants. In addition, we discovered a difference in the composition of esterified xanthophylls among XES -OX plants, which may be caused by differences in the substrate specificity of their respective XESs. We conclude that esterification is an important process for carotenoid accumulation and XES is a useful tool for the quantitative and qualitative control of carotenoid accumulation in petals.

Flavonoids are known to be involved in pollen fertility in petunia (P. hybrida) and maize (Zea mays). As a first step toward elucidating the role of flavonoids in pollen, we have identified a glycosyltransferase that is responsible for the terminal modification of petunia pollen-specific flavonoids. An in silico search of the petunia transcriptome database revealed four candidate UDP-glycosyltransferase (UGT) genes. UGT79B31 was selected for further analyses based on a correlation between the accumulation pattern of flavonol glycosides in various tissues and organs and the expression profiles of the candidate genes. Arabidopsis ugt79b6 mutants that lacked kaempferol/quercetin 3-O-glucosyl(1 → 2)glucosides, were complemented by transformation with UGT79B31 cDNA under the control of Arabidopsis UGT79B6 promoter, showing that UGT79B31 functions as a flavonol 3-O-glucoside: 2\"-O-glucosyltransferase in planta. Recombinant UGT79B31 protein can convert kaempferol 3-O-galactoside/glucoside to kaempferol 3-O-glucosyl(1 › 2)galactoside/glucoside. UGT79B31 prefers flavonol 3-O-galactosides to the 3-O-glucosides and rarely accepted the 3-O-diglycosides as sugar acceptors. UDP-glucose was the preferred sugar donor for UGT79B31. These results indicated that UGT79B31 encodes a flavonoid 3-O-glycoside: 2\"-O-glucosyltransferase. Transient expression of UGT79B31 fused to green fluorescent protein (GFP) in Nicotiana benthamiana showed that UGT79B31 protein was localized in the cytosol.