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Overexpression of acdS in petunia negatively affects seed germination by suppression of ethylene biosynthesis and signaling genes and induction of abscisic acid biosynthesis genes in the seeds. The acdS gene, which encodes 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, has been overexpressed in horticultural crops to improve their tolerance to abiotic stress. However, the role of acdS in the germination of crop seeds has not been investigated, despite its suppression of ethylene production. In this study, acdS overexpression significantly reduced seed weight and germination rate in transgenic petunia cv. Merage Rose (T5, T7, and T12) relative to wild type via the suppression of ethylene biosynthesis and signaling genes and induction of abscisic acid (ABA) biosynthesis genes. The germination rate of T7 was significantly lower than those of T5 and T12, which was linked to higher expression of acdS in the former than the latter. The addition of exogenous ACC and gibberellic acid (GA

The C2H2 zinc finger proteins (ZFPs) play essential roles in regulating cold stress responses. Similarly, raffinose accumulation contributes to freezing stress tolerance. However, the relationship between C2H2 functions and raffinose synthesis in cold tolerance remains uncertain. Here, we report the characterization of the cold-induced C2H2-type zinc finger protein PhZFP1 in Petunia hybrida. PhZFP1 was found to be predominantly localized in the nucleus. Overexpression of PhZFP1 conferred enhanced cold tolerance in transgenic petunia lines. In contrast, RNAi mediated suppression of PhZFP1 led to increased cold susceptibility. PhZFP1 regulated the expression of a range of abiotic stress responsive-genes including genes encoding proteins involved in reactive oxygen species (ROS) scavenging and raffinose metabolism. The accumulation of galactinol and raffinose, and the levels of PhGolS1-1 transcripts, were significantly increased in PhZFP1-overexpressing plants and decreased in PhZFP1-RNAi plants under cold stress. Moreover, the galactinol synthase (GolS)-encoding gene PhGolS1-1 was identified as a direct target of PhZFP1. Taken together, these results demonstrate that PhZFP1 functions in cold stress tolerance by modulation of galactinol synthesis via regulation of PhGolS1-1. This study also provides new insights into the mechanisms underlying C2H2 zinc finger protein-mediated cold stress tolerance, and has identified a candidate gene for improving cold stress tolerance.

Calibrachoa Cerv., Fabiana Ruiz & Pav., and Petunia Juss. form a clade within tribe Petunieae (Solanaceae). Phylogenetic studies of Petunieae, either as part of a family-wide analysis or focusing on the genera Calibrachoa and Petunia, have either left Fabiana unsampled or included only a single species. These studies have found conflicting relationships among the three genera with all three possible topologies obtained. Petunia patagonica (Speg.) Millán, originally described in the genus Nierembergia Ruiz & Pav., is morphologically distinct within Petunia and geographically disjunct from other members of the genus. For the first time, in this study we include multiple species of Fabiana, Calibrachoa, and Petunia, including P. patagonica. Using three chloroplast DNA regions and the nuclear gene GBSSI, or “waxy,” our results provide strong support for a sister group relationship between Calibrachoa and Fabiana and for the placement of P. patagonica within Fabiana. Since there is already a species Fabiana patagonica Speg., we provide the new name Fabiana australis Alaria nom. nov. to replace Petunia patagonica.

To investigate whether low-level lighting is necessary and which narrow-band light spectra are effective in seed germination, seeds of 14 genotypes from begonia (Begonia × tuberosa), echinacea (Echinacea spp.), gerbera (Gerbera jamesonii), petunia (Petunia × hybrida), and vinca (Catharanthus roseus) were germinated under ultraviolet-B (UVB), blue (B), green (G), red (R), far-red (FR) light, or darkness. Light-emitting diode (LED) fixtures provided all spectrum treatments except for UVB, which was provided by a narrow-band fluorescent light. The photon flux density at seed level was ≈18 μmol·m−2·s−1 for B, G, R, and FR, and 0.4 μmol·m−2·s−1 for UVB. Based on daily germination observations, final germination percentage, germination onset time, germination time spread, and germination speed were compared among different spectrum treatments for each of the plant genotypes. There were no promotive effects on final germination percentage, germination onset time, and germination time spread under the narrow-band lights compared with darkness. For all plant genotypes, B had a similar effect as darkness on seed germination. FR inhibited seed germination relative to darkness by reducing final germination percentage by 31%–88%, delaying germination onset time by 30%–40%, and decreasing germination speed by 11%–48% in some genotypes. Under R, G, and UVB compared with darkness, germination speed was promoted for begonia ‘Apricot Shade’, a light-requiring genotype, and inhibited for vinca ‘Burgundy’, a light-inhibited genotype. Therefore, lighting at low levels used in our study is unnecessary for seed germination of the tested species except light-requiring genotypes, where R, G, and UVB are the most promotive among the tested narrow-band lights.

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 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.

An R2R3-MYB transcription factor, CmMYB9a, activates floral coloration in chrysanthemum by positively regulating CmCHS, CmDFR and CmFNS, but inhibiting the expression of CmFLS. Chrysanthemum is one of the most popular ornamental plants worldwide. Flavonoids, such as anthocyanins, flavones, and flavonols, are important secondary metabolites for coloration and are involved in many biological processes in plants, like petunia, snapdragon, Gerbera hybrida, as well as chrysanthemum. However, the metabolic regulation of flavonoids contributing to chrysanthemum floral coloration remains largely unexplored. Here, an R2R3-MYB transcription factor, CmMYB9a, was found to be involved in flavonoid biosynthesis. Phylogenetic analysis and amino acid sequence analysis suggested that CmMYB9a belonged to subgroup 7. Transient overexpression of CmMYB9a in flowers of chrysanthemum cultivar 'Anastasia Pink' upregulated the anthocyanin-related and flavone-related genes and downregulated CmFLS, which led to the accumulation of anthocyanins and flavones. We further demonstrated that CmMYB9a independently activates the expression of CmCHS, CmDFR and CmFNS, but inhibits the expression of CmFLS. Overexpression of CmMYB9a in tobacco resulted in increased anthocyanins and decreased flavonols in the petals by upregulating NtDFR and downregulating NtFLS. These results suggest that CmMYB9a facilitates metabolic flux into anthocyanin and flavone biosynthesis. Taken together, this study functionally characterizes the role of CmMYB9a in regulating the branched pathways of flavonoids in chrysanthemum flowers.

Editing of ACO genes involved in ethylene biosynthesis pathway reduces ethylene production in petunia seeds and inhibits seed germination. Ethylene production in the seeds of Petunia hybrida cv. ‘Mirage Rose’ was associated with expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) genes (PhACO1, PhACO3, and PhACO4). Suppression of their expression by ethylene inhibitor silver thiosulphate (STS) significantly reduced ethylene production and inhibited seed germination. When it was combined with ethylene precursor ACC, ethylene production was re-promoted via activation of the genes and higher seed germination was restored. This was confirmed using the mutants editing the genes and WT. In the present study, compared with wild type plants, three different mutants (phaco1, phaco3, and phaco4) showed significantly decreased germination percentages as well as delayed germination time and seedling growth. These reductions were associated with lighter seed weight, lower ACO transcript levels, and lower ethylene production in mutants. Inhibited seed germination owing to reduced ethylene production was further verified by the supplementation of exogenous ACC and gibberellic acid (GA3) to growth medium, which restored high seed germination activity in all mutants via enhanced ethylene production. In this study, we reported a key regulatory role of ethylene in seed germination mechanisms in petunia. Further, we highlighted on need to consider the negative effects of ethylene reduction in seed germination and plant growth when editing genes in the ethylene biosynthesis pathway for the maintenance of postharvest fruit, vegetable, and flower quality.

Golovinomyces longipes is a widespread powdery mildew on Solanaceae (Nicotiana, Petunia and Solanum spp.). In the past, it has been reported only once on Verbena (Verbenaceae), a non-solanaceaous host. Recently, this powdery mildew has been found on the composite Matricaria chamomilla. The identification of the powdery mildew species on this unusual host has been proved by morphological studies and DNA sequence analysis. Both datasets coincide with the characteristic data for G. longipes on Solanaceae. First inoculation experiments with further composites resulted in an infection of Brachyscome hybrid ‘Surdaisy’. To our knowledge, this is the first report of G. lonigpes on hosts belonging to the important family of Asteraceae and an additional proof of the broader host range of G. longipes beyond the Solanaceae.

Background CRISPR/Cas9 is a rapidly developing genome editing technology in various biological systems due to its efficiency, portability, simplicity and versatility. This editing technology has been successfully applied in in several important plants of Solanaceae such as tomato, tobacco, potato, petunia and groundcherry. Wolfberry ranked the sixth among solanaceous crops of outstanding importance in China following potato, tomato, eggplant, pepper and tobacco. To date, there has been no report on CRISPR/Cas9 technology to improve Lycium ruthenicum due to the unknown genome sequencing and the lack of efficient regeneration and genetic transformation systems. Results In this study, we have established an efficientregeneration and genetic transformation system of Lycium ruthenicum. We have used this system to validate target sites for fw2.2, a major fruit weight quantitative trait locus first identified from tomato and accounted for 30% of the variation in fruit size. In our experiments, the editing efficiency was very high, with 95.45% of the transgenic lines containing mutations in the fw2.2 target site. We obtained transgenic wolfberry plants containing four homozygous mutations and nine biallelic mutations in the fw2.2 gene. Conclusions These results suggest that CRISPR-based gene editing is effective for the improvement of black wolfberry traits, and we expect this approach to be routinely applied to this important economic fruit.

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.

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.

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.

In self-incompatible Solanaceous species, the pistil S-RNase acts as cytotoxin to inhibit self-pollination but is polyubiquitinated by the pollen-specific non-self S-locus F-box (SLF) proteins and subsequently degraded by the ubiquitin-proteasome system (UPS), allowing cross-pollination. However, it remains unclear how S-RNase is restricted by the UPS. Here, we first show that Petunia hybrida (Ph) S3-RNase is largely ubiquitinated by K48-linked polyubiquitin chains at three regions, R I, II and III. R I is ubiquitinated in unpollinated, self- and cross-pollinated pistils, indicating its occurrence prior to PhS3-RNase uptake into pollen tubes, whereas R II and III are exclusively ubiquitinated in cross-pollinated pistils. Second, removal of R II ubiquitination resulted in significantly reduced seed sets from cross-pollination and that of R I and III in less extents, indicating their increased cytotoxicity. In consistent, the mutated R II of PhS3-RNase resulted in marked reduction of its degradation, whereas that of R I and III in less reductions. Taken together, our results demonstrate that PhS3-RNase R II functions as a major ubiquitination region for its destruction and R I and III as minor ones, revealing that its cytotoxicity is primarily restricted by a stepwise UPS mechanism for cross-pollination in P. hybrida.ONE SENTENCE SUMMARYBiochemical and transgenic analyses reveal that Petunia hybrida S3-RNase cytotoxicity is largely restricted by a stepwise ubiquitination and degradation pathway during cross-pollination.

Red flower color has arisen multiple times and is generally associated with hummingbird pollination. The majority of evolutionary transitions to red color proceeded from purple lineages and tend to be genetically simple, almost always involving a few loss-of-function mutations of major phenotypic effect. Here we report on the complex evolution of a novel red floral color in the hummingbird-pollinated Petunia exserta (Solanaceae) from a colorless ancestor. The presence of a red color is remarkable because the genus cannot synthesize red anthocyanins and P. exserta retains a nonfunctional copy of the key MYB transcription factor AN2. We show that moderate upregulation and a shift in tissue specificity of an AN2 paralog, DEEP PURPLE, restores anthocyanin biosynthesis in P. exserta. An essential shift in anthocyanin hydroxylation occurred through rebalancing the expression of three hydroxylating genes. Furthermore, the downregulation of an acyltransferase promotes reddish hues in typically purple pigments by preventing acyl group decoration of anthocyanins. This study presents a rare case of a genetically complex evolutionary transition toward the gain of a novel red color.

miR156/157 plays multiple pivotal roles during plant growth and development. In this study, we identified 11 miR156- and 5 miR157-encoding loci from the genome of Petunia axillaris and Petunia inflata, designated as PaMIR0156/157s and PiMIR0156/157s, respectively. Real-time quantitative reverse transcription PCR (qRT-PCR) analysis indicated that PhmiR156/157 was expressed predominantly in cotyledons, germinating seeds, flower buds, young fruits and seedlings. PhmiR156/157 levels declined in shoot apical buds and leaves of petunia before flowering as the plant ages; moreover, the temporal expression patterns of most miR156/157-targeted PhSPLs were complementary to that of PhmiR156/157. Ectopic expression of PhMIR0157a in Arabidopsis and petunia resulted in delayed flowering, dwarf plant stature, increased branches and reduced organ size. However, PhMIR0156f-overexpressing Arabidopsis and petunia plants showed only delayed flowering. In addition, downregulation of PhmiR156/157 level by overexpressing STTM156/157 led to taller plants with less branches, longer internodes and precocious flowering. qRT-PCR analysis indicated that PhmiR156/157 modulates these traits mainly by downregulating their PhSPL targets and subsequently decreasing the expression of flowering regulatory genes. Our results demonstrate that the PhmiR156/157-PhSPL module has conserved but also divergent functions in growth and development, which will help us decipher the genetic basis for the improvement of flower transition, plant architecture and organ development in petunia.

Plants respond to water deficit through several mechanisms. Among which, plant hormones play an important role in induction to abiotic stress tolerance. To bring this issue closer, an experiment was conducted to examine whether salicylic acid (SA) and gibberellic acid (GA3) exogenous applications through foliar spray could alleviate detrimental effects of water deficit on Persian petunia. Treatments consisted of water stress in three levels: 100% field capacity (F.C.) as control, 50, and 25% F.C linked with different concentrations of GA3 and SA sprayed at four levels (0, 100, 200 and 300 mg L–1). Results showed that the dry and fresh biomass, leaf area, photosynthetic pigments levels, relative water content (RWC) and the total number of flowers were significantly (P≤0.01) reduced in response to water deficit. However, free proline concentration and root-to-shoot ratio significantly increased in stressed plants. Plants subjected to severe water stress (25% F.C.), exhibit about 66% stomatal conductance rate lower than the plants grown at 100% F.C. The results showed that SA-treated plants exhibited reduced negative effect of water stress on most of the qualitative and quantitative attributes compared to control, while GA3 application had little impact on studied traits. Importantly, stomatal conductance and RWC were improved in water-stressed plants treated with SA. Moreover, SA-treated plants produced more flowers at all water deficit levels compared to GA3 sprayed plants. It can be concluded that Persian petunia plants respond to water deficit through adaptive changes at biochemical and morphological levels and that adverse effects of water deficit could be alleviated by exogenous application of SA.

Under light-limiting conditions, many ornamental greenhouse-grown plants show undesired morphological characteristics, such as plant elongation (hypocotyl and epicotyl length) and low dry mass, which reduce plant quality. Research has shown that use of plant growth regulators (PGRs) and changes in both light intensity and spectral composition can reduce these undesired characteristics. However, little is known about the role of the combined effects of supplemental lighting and PGRs on the production of ornamental seedlings. The objective of this study was to characterize the combined and independent effects of light intensity, spectral composition, and PGR applications on the greenhouse production of ornamental transplants. Petunia (Petunia × hybrida), geranium (Pelargonium × hortorum), pansy (Viola × wittrockiana) and dianthus (Dianthus chinensis) were grown for 32–42 days under three supplemental light (SL) treatments: 1) high-pressure sodium (HPS), 2) light-emitting diodes (LEDs) with a 6 blue (B):5 green (G):89 red (R) (percent photon flux ratio), and 3) LEDs with 19B:81R (100 μmol m−2 s−1, 18 h photoperiod for all treatments). A control (No SL) was also included. In addition, a portion of plants were also sprayed with the paclobutrazol PGR (PBZ and No PBZ). The synergistic effects of the combination of PBZ and supplemental lighting resulted in the most compact plants, caused by a reduction in plant height by PBZ and an increase in dry mass by SL. However, PBZ reduced shoot dry mass of most plant species and light combinations. Plant compactness was greater under the 6B:5G:89R LED composition for petunia and when combined with PBZ for geranium than for plants under HPS lighting. Root dry mass of petunia, geranium, and pansy plants increased in response to SL compared with no SL by 2.4–5.7-fold. Results from the two LED spectra were unexpected; plants under 6B:5G:89R were more compact (petunia, geranium), had higher anthocyanin concentrations (petunia), were shorter (petunia, pansy, dianthus) and had less leaf area (petunia, pansy, dianthus) than plants in the SL treatment with a higher B and lower G PF (19B:81R). Supplemental lighting and PBZ can be used in conjunction or independently to improve plant morphology. The increased light from SL provided the most benefits by improving dry mass, compactness, and leaf number for most plant species. However, when PBZ was used in combination with SL, plant compactness increased for some species. The spectral composition of SL had an impact on plant growth and morphology, warranting additional research on plant responses to small changes in the spectral composition of SL.

MicroRNA319 (miR319) plays an important role in leaf development. Although leaf discs are frequently used for plant transformation, little is known about the function of miR319 in shoot regeneration from leaf explants. To investigate the role of miR319 in de novo shoot organogenesis from leaf explants, the MIR319 gene was overexpressed in petunia (Petunia × hybrida), under the control of the 35S promoter. Petunia leaves overexpressing miR319 (miR319-OX) showed several morphological abnormalities, such as rounded, uneven, and curled leaf blade, larger leaf size, and smaller and rounded abaxial surface cells, which were differences compared to wild-type (WT) leaves. Additionally, miR319-OX leaf explants demonstrated enhanced shoot organogenesis on a growth medium containing 6-benzylaminopurine (BA) at shoot-inducing concentrations, and cytokinin-independent shoot regeneration on hormone-free medium compared with WT explants. Quantitative real-time PCR (qRT-PCR) analysis indicated that the expression of petunia NO APICAL MERISTEM (PhNAM) and SHOOT MERISTEMLESS (PhSTM) genes was markedly increased in miR319-OX explants compared with WT explants under shoot induction conditions, whereas the expression of petunia cytokinin-responsive type-A response regulator genes, PhRR2 and PhRR3, showed no differences in expression between WT and miR319-OX leaf explants. These results suggest that miR319 regulates the formation of shoot meristems from leaf explants in petunia during in vitro culture.

Arbuscular mycorrhiza fungi (AMF) are beneficial soil fungi that can promote the growth of their host plants. Accurate quantification of AMF in plant roots is important because the level of colonization is often indicative of the activity of these fungi. Root colonization is traditionally measured with microscopy methods which visualize fungal structures inside roots. Microscopy methods are labor-intensive, and results depend on the observer. In this study, we present a relative qPCR method to quantify AMF in which we normalized the AMF qPCR signal relative to a plant gene. First, we validated the primer pair AMG1F and AM1 in silico, and we show that these primers cover most AMF species present in plant roots without amplifying host DNA. Next, we compared the relative qPCR method with traditional microscopy based on a greenhouse experiment with Petunia plants that ranged from very high to very low levels of AMF root colonization. Finally, by sequencing the qPCR amplicons with MiSeq, we experimentally confirmed that the primer pair excludes plant DNA while amplifying mostly AMF. Most importantly, our relative qPCR approach was capable of discriminating quantitative differences in AMF root colonization and it strongly correlated (Spearman Rho = 0.875) with quantifications by traditional microscopy. Finally, we provide a balanced discussion about the strengths and weaknesses of microscopy and qPCR methods. In conclusion, the tested approach of relative qPCR presents a reliable alternative method to quantify AMF root colonization that is less operator-dependent than traditional microscopy and offers scalability to high-throughput analyses.

Most land plants entertain a mutualistic symbiosis known as arbuscular mycorrhiza with fungi (Glomeromycota) that provide them with essential mineral nutrients, in particular phosphate (Pi), and protect them from biotic and abiotic stress. Arbuscular mycorrhizal (AM) symbiosis increases plant productivity and biodiversity and is therefore relevant for both natural plant communities and crop production. However, AM fungal populations suffer from intense farming practices in agricultural soils, in particular Pi fertilization. The dilemma between natural fertilization from AM symbiosis and chemical fertilization has raised major concern and emphasizes the need to better understand the mechanisms by which Pi suppresses AM symbiosis. Here, we test the hypothesis that Pi may interfere with AM symbiosis via the phytohormone gibberellic acid (GA) in the Solanaceous model systems Petunia hybrida and Nicotiana tabacum. Indeed, we find that GA is inhibitory to AM symbiosis and that Pi may cause GA levels to increase in mycorrhizal roots. Consistent with a role of endogenous GA as an inhibitor of AM development, GA-defective N. tabacum lines expressing a GA-metabolizing enzyme (GA methyltransferase—GAMT) are colonized more quickly by the AM fungus Rhizoglomus irregulare, and exogenous Pi is less effective in inhibiting AM colonization in these lines. Systematic gene expression analysis of GA-related genes reveals a complex picture, in which GA degradation by GA2 oxidase plays a prominent role. These findings reveal potential targets for crop breeding that could reduce Pi suppression of AM symbiosis, thereby reconciling the advantages of Pi fertilization with the diverse benefits of AM symbiosis.

The popular ornamental plant Petunia is also a valuable model plant in tissue culture. Cellular conversions during differentiation and regeneration have been investigated using various combinations of phytohormones; however, studies on genes for reprogramming toward desired tissue identities have been limited. In this study, we isolated Petunia protoplasts and cultured them in the callus, rooting, or shooting stages, which were used to establish the optimal protoplast culture conditions and to identify genes that epigenetically function as tissue identifiers. The optimal conditions for plasmolysis and enzyme digestion to obtain healthy protoplasts were compared, in which combinations of Viscozyme, Celluclast, and Pectinex (VCP) enzymes were more efficient in isolating protoplasts when followed by 21 to 25% sucrose purification and washing processes. The filtered and washed protoplasts started to divide at 1 day and developed into colonies after 3 weeks of culture, which showed higher efficiency in the Murashige and Skoog (MS) salt culture media compared to that in the Kao and Michayluk (KM) salt media. The pluripotent colonies formed calli on the solid medium supplemented with 3% sucrose after 4 weeks, and were destined to the same cell mass, rooting, or shooting on the regeneration medium. Three epigenetic controllers, ATXR2, ATX4A, and ATX4B, were highly expressed in calli, shoots, and organs of shoots and roots, respectively, confirming that dedifferentiation and regeneration of tissue identity is plastic.

Ferredoxin-thioredoxin reductase (FTR) is an iron-sulfur protein that supplies electrons from photochemically reduced ferredoxin (Fd) to thioredoxin (Trx) in the ferredoxin/thioredoxin system in chloroplasts. The FTR is a heterodimer with a variable subunit (FTRv) and a catalytic subunit (FTRc). The function of FTRv is not well known. In petunia (Petunia hybrida), FTRv is a single-copy gene, which is named PhFTRv. In this study, the spatio-temporal expression of PhFTRv in petunia was analyzed, and PhFTRv transcription was found to be high in leaves and stems. A tobacco rattle virus gene silencing was used in this study. Virus induced gene silencing-mediated PhFTRv silencing resulted in large yellow-green leaves, delayed flowering, and reduced cold tolerance in petunia plants.

Hybridization between closely related plant species is a widespread phenomenon with significant evolutionary consequences, so natural hybrid zones provide exciting opportunities to study the processes of genetic differentiation and species formation. In Petunia, genetics, ecology and evolution of pollination syndromes have been thoroughly studied and related to the development of prezygotic isolation barriers between species. However, to date, no studies have explored the importance of extrinsic post-zygotic barriers such as environmental selection as drivers of reproductive isolation. In this study, we applied a population genetics approach to understand the speciation of two partially sympatric taxa of Petunia Juss that hybridize in the wild despite having strong differences in pollination syndrome, mating system and environmental requirements. We evaluated 58 individuals from seven allopatric and two contact zones of Petunia axillaris and P. exserta with genomic data to study the population genetic structure and gene exchange between these species and scan for potential loci under natural selection related to the preservation of species barriers. We explicitly evaluated the time, intensity and direction of interspecific migration, testing for alternative demographic scenarios. The genetic structure showed well-differentiated species-level lineages, and poor differentiation among populations of P. axillaris; P. exserta populations were highly structured, with several well-differentiated intraspecific lineages. Our data provide evidence for admixture with low asymmetric gene flow from P. axillaris to P. exserta associated with recent secondary contact, suggesting that pollinator specificity is not the only trait responsible for species integrity in this system. Combining the results from FST outlier and genotype–phenotype association methods, we identified 54 non-synonymous candidate variants under natural selection. Those variants are found in 35 coding sequences from which we found genes related to light-response networks, supporting that extrinsic factors such as habitat preferences can also provide a mechanism of reproductive isolation between Petunia spp. We argue that a set of pre- and post-zygotic barriers in conjunction with demographic processes maintain the species integrity.

Previous studies have shown that high light intensity can induce anthocyanin synthesis (AS) in petunia plants. To identify which kind of light quality plays a role in inducing such metabolic process, and what transcripts participate in controlling it, we carried out whole-transcriptome sequencing and analysis of petunia petals treated with different light-quality conditions. Among the red and white light treatments, a total of 2 205 differentially expressed genes and 15, 22, and 20 differentially expressed circRNAs, miRNAs, and lncRNAs, were identified respectively. The AS-related genes, including the structural genes CHSj, F3′H, F3′5′H, DFR, and ANS, and the regulatory genes AN4, DPL, PHZ and MYBx were found to be downregulated under red light condition compared with their levels under white light condition. Furthermore, the light photoreceptor Cryptochrome 3 (CRY3) and a series of light-dependent genes, such as PIF, HY5, and BBXs, were also determined to respond to the light treatments. The anthocyanin contents in early petunia petals under red light were significantly lower than that under white and blue light. The results of qRT-PCR further confirmed the expression pattern of some AS-related and light-response genes in response to different light quality. Yeast two-hybrid results showed that the key elements in the light signal pathway, HY5 can interact with BBX19, BBX24 and BBX25. And PHZ, the important AS regulator can induce anthocyanin synthesis in response to blue light quality from transient expression analysis in petunia petals. These findings presented here not only deepen our understanding of how light quality controls anthocyanin synthesis, but also allow us to explore potential target genes for improving pigment production in petunia flower petals.

The long-term success of populations is dependent on individual reproductive success, and in general, increased population size and genetic diversity contribute to population maintenance, reducing the risk of local extinction. Interspecific hybridization has consequences that can vary according to the hybrids' fate, which can be strongly influenced by the reproductive capacity of hybrids and canonical individuals from the contact zone. We examined the reproductive biology and morphology of two closely related Petunia (Solanaceae) species and their interspecific hybrids from Serra do Sudeste, Southern Brazil, and we measured their reproductive success under controlled conditions based on seed production and germination from five pollination treatments. We found differences in self-compatibility degree among individuals, lineages, and pollination treatments based on high total seed production (> 204,000 seeds) and germination (630 seedlings evaluated). No correlation was observed between corolla colour and reproductive success or between floral morphological traits and compatibility. High self-compatibility and inter-lineage compatibility can explain the hybrid populations' maintenance and origin, favouring the two analysed species' introgression.

Plant cuticles are a mixture of crystalline and amorphous waxes that restrict the exchange of molecules between the plant and the atmosphere. The multicomponent nature of cuticular waxes complicates the study of the relationship between the physical and transport properties. Here, a model cuticle based on the epicuticular waxes of Petunia hybrida flower petals was formulated to test the effect of wax composition on diffusion of water and volatile organic compounds (VOCs). The model cuticle was composed of an n-tetracosane (C

We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar ‘Madness Midnight’ has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer’s field.

Cymbidium faberi Rolfe (Orchidaceae) is an herbaceous plant native to China, where it has a long history of cultivation owing to its beautiful flower pattern and floral fragrance. Previously, we conducted a transcriptome analysis of the flower and vegetative buds to elucidate the mechanisms of flower development in C. faberi. In the present study, we found nine secondary metabolic pathways through the KEGG pathway database that were related to the biosynthesis of methyl jasmonate (MeJA) and other volatile organic compounds. qRT-PCR was performed to analyze the expression levels of four key genes in the MeJA pathway. Among these, CfJMT (jasmonic acid carboxyl methyltransferase) had higher transcript levels in sepals, petals and labella than in other tissues. CfJMT was cloned from the petals of full-bloom flowers of C. faberi. The predicted CfJMT protein sequence contains conserved jasmonic acid methyl transferase-7 domains, indicating that it belongs to the SABATH protein family. The CfJMT coding sequence driven by the CaMV35S promoter was successfully transformed into Petunia hybrida through an Agrobacterium-mediated method. Although MeJA could not be detected in either wild-type or transgenic petunia plants, the leaves of the transgenic plants were smaller than those of wild-type plants and pollen development was abnormal. These results indicate that heterologous expression of CfJMT may change the levels of endogenous jasmonic acid and other hormones, but that the content of MeJA is not increased significantly by transformation with CfJMT alone. Thus, other related genes and regulation factors may play important roles in this process.

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., PaACL silencing reduced anthocyanin contents, accelerated flower senescence, and changed the proteome and acetylome to maintain metabolic homeostasis in petunia

Wastewater generated from industries contains numerous contaminants, among which heavy metals (HMs) are non-degradable. This research work highlights the use of commonly used ornamental plants, Nicotiana alata L. and Petunia hydrida L., with compost (C) and peat moss (M), and rhizospheric bacterial augmentation using Pseudomonas japonica, for the phytostabilization of HMs from synthetic wastewater. After plant–soil acclimatization, plants were exposed for 6 weeks to synthetic wastewater, containing cadmium, chromium, copper, lead, nickel, and zinc concentrations (based on the HMs level of wastewaters collected from textile and pharmaceutical industry). Physiological response, biochemical status, and enzymatic fluctuations of plants and the distribution of HMs in plant parts and soil, were quantified. With the combined use (5% each v/v conditioner/soil) of C and M, in bio-augmented soil, physiological response and enzymatic status of both plants improved, with decreased stress injury due to HMs. Further, the plant HMs uptake was reduced, with better stabilization of HM in soil. For better phytostabilization of HMs in wastewater, the use of compost, peat moss, and bacterial augmentation is recommended with Nicotiana alata L. and Petunia hydrida L.

Theobjective of this study was to investigate the effects of different growingmedia on plant growth and nutrient contents of petunia (Petunia hybrida). The experiment was conducted in a chamber roomunder controlled conditions at the laboratory. Seven different media (soil, 3:1soil: peat (P1), 2:1 soil: peat (P2), 3:1 soil: barnyard manure (BYM1), 2:1soil: barnyard manure (BYM2), 3:1 soil: sugar beet pulp (SBP1), 2:1 soil: sugarbeet pulp (SBP2)) were used as plant growing media. The experiment was endedafter three months following transplanting of seedlings. Plant growth andflower parameters and macro-micro nutrient contents were determined inharvested plants. The highest stem diameter, branch number, flower diameter,flower stalk diameter, plant fresh weight and plant dry weight were in 2:1ratio of soil: peat mixture as 7.00 mm, 6.33, 8.91 cm, 3.59 mm, 48.47 g and4.52 g while the highest plant lenght, lateral branch number and flower numbermeans were found as 27.43 cm, 24.67 and 24.67 in ratio of 3:1 soil: peatgrowing media respectively. The highest potassium, magnesium and zinc means ofplants were obtained as 6.6%, 2.2% and 32.50 mg kg-1 in soil:barnyard manure (2:1) growing media while the highest phosphorus and calciummeans of plants were found as 0.27% and 4.5% in soil and peat growing mediarespectively. The highest iron and copper means of plants were determined as231 mg kg-1 and 32.50 mg kg-1 in ratios of 2:1 and 3:1 ofsoil: sugarbeet pulp growing media respectively., The objective of this study was to investigate the effects of different growing media on plant growth and nutrient contents of petunia (Petunia hybrida). The experiment was conducted in a chamber room under controlled conditions at the laboratory. Seven different media (soil, 3:1 soil: peat (P1), 2:1 soil: peat (P2), 3:1 soil: barnyard manure (BYM1), 2:1 soil: barnyard manure (BYM2), 3:1 soil: sugar beet pulp (SBP1), 2:1 soil: sugar beet pulp (SBP2)) were used as plant growing media. The experiment was ended after three months following transplanting of seedlings. Plant growth and flower parameters and macro-micro nutrient contents were determined in harvested plants. The highest stem diameter, branch number, flower diameter, flower stalk diameter, plant fresh weight and plant dry weight were in 2:1 ratio of soil: peat mixture as 7.00 mm, 6.33, 8.91 cm, 3.59 mm, 48.47 g and 4.52 g while the highest plant lenght, lateral branch number and flower number means were found as 27.43 cm, 24.67 and 24.67 in ratio of 3:1 soil: peat growing media respectively. The highest potassium, magnesium and zinc means of plants were obtained as 6.6%, 2.2% and 32.50 mg kg-1 in soil: barnyard manure (2:1) growing media while the highest phosphorus and calcium means of plants were found as 0.27% and 4.5% in soil and peat growing media respectively. The highest iron and copper means of plants were determined as 231 mg kg-1 and 32.50 mg kg-1 in ratios of 2:1 and 3:1 of soil: sugarbeet pulp growing media respectively.

Elevated temperatures suppress cell division in developing petunia buds leading to smaller flowers, mediated by ABA. Flower size is one of the most important showy traits in determining pollinator attraction, and a central factor determining the quality of floricultural products. Whereas the adverse effects of elevated temperatures on showy traits have been described in detail, its underlining mechanisms is poorly understood. Here, we investigated the physiological mechanism responsible for the reduction of flower size in petunia under elevated temperatures. We found that the early stages of flower-bud development were most sensitive to elevated temperatures, resulting in a drastic reduction of flower diameter that was almost independent of flower load. We demonstrated that the temperature-mediated flower size reduction occurred due to a shorter growth period, and a lower rate of corolla cell division. Consistently, local application of cytokinin, a phytohormone that promotes cell division, resulted in recovery of flower dimensions when grown under elevated temperatures. Hormone analysis of temperature-inhibited flower buds revealed no significant changes in levels of cytokinin, and a specific increase of abscisic acid (ABA) levels, known to inhibit cell division. Moreover, local application of ABA on flower buds caused a reduction of flower dimensions as a result of lower levels of cell division, suggesting that ABA mediates the reduction of flower size at elevated temperatures. Taken together, our results shed light on the mechanism by which elevated temperatures decrease petunia flower size, and show that temperature-mediated reduction of flower size can be alleviated by increasing the cytokinin/ABA ratio.

Nutrient deficiency is known to constrain plant growth in numerous ways, but how it impacts floral displays and pollination success remains unclear. Here we investigate how insufficient availability of sulphur – a vital plant nutrient that is a limiting factor in natural and agricultural regions throughout the world – influences the production of floral displays in Brassica rapa, Physalis philadelphica and three Petunia species with differently coloured flowers. Sulphur deficiency led to a drastic reduction in the number of open flowers, an aberrant flower morphology and smaller pollen with an altered mineral nutrient content. Intriguingly, sulphur deprivation also led to a clear reduction in pigmentation of yellow flowers, but not in flowers with white, purple and red colours. The pale yellow flower colour was due to decreased amounts of violaxanthin, lutein and other carotenoids, suggesting that the carotenoid synthesis pathway is particularly susceptible to sulphur deficiency. Additional experiments with nitrogen and phosphorus depletion confirmed that observed colour and morphological changes were not a general nutrient limitation response, but could be ascribed to sulphur depletion specifically. Taken together, our results showed that (mild) sulphur deficiency deteriorates a suite of floral traits, and that the effects may cascade to pollinators and so have the potential to undermine (agro-)ecosystem functioning.

Scent bouquets produced by the flowers of Petunia spp. (petunia) are composed of a complex mixture of floral volatile benzenoid and phenylpropanoid compounds (FVBPs), which are specialized metabolites derived from phenylalanine (Phe) through an interconnected network of enzymes. The biosynthesis and emission of high levels of these volatiles requires coordinated transcriptional activation of both primary and specialized metabolic networks. The petunia R2R3-MYB transcription factor ODORANT 1 (ODO1) was identified as a master regulator of FVBP production and emission; however, our knowledge of the direct regulatory targets of ODO1 has remained limited. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq) in petunia flowers, we identify genome-wide ODO1-bound genes that are enriched not only in genes involved in the biosynthesis of the Phe precursor, as previously reported, but also genes associated with the specialized metabolic pathways involved in generating phenylpropanoid intermediates for FVBPs. ODO1-bound genes are also involved in methionine and S-adenosylmethionine metabolism, which could modulate methyl group supplies for certain FVBPs. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and RNA-seq analysis in an ODO1 RNAi knockdown line revealed that ODO1-bound targets are expressed at lower levels when ODO1 is suppressed. A cis-regulatory motif, CACCAACCCC, was identified as a potential binding site for ODO1 in the promoters of genes that are both bound and activated by ODO1, which was validated by in planta promoter reporter assays with wild-type and mutated promoters. Overall, our work presents a mechanistic model for ODO1 controlling an extensive gene regulatory network that contributes to FVBP production to give rise to floral scent.

Although anthocyanins are widely distributed in higher plants, betalains have replaced anthocyanins in most species of the order Caryophyllales. The accumulation of flavonols in Caryophyllales plants implies that the late step of anthocyanin biosynthesis from dihydroflavonols to anthocyanins may be blocked in Caryophyllales. The isolation and characterization of functional dihydroflavonol 4-reductase (DFR) and anthocyanidin synthase (ANS) from Caryophyllales plants has indicated a lack of anthocyanins due to suppression of DFR and ANS. In this study, we demonstrated that overexpression of DFR and ANS from Spinacia oleracea (SoDFR and SoANS, respectively) with PhAN9, which encodes glutathione S-transferase (required for anthocyanin sequestration) from Petunia induces ectopic anthocyanin accumulation in yellow tepals of the cactus Astrophytum myriostigma. A promoter assay of SoANS showed that the Arabidopsis MYB transcription factor PRODUCTION OF ANTHOCYANIN PIGMENT1 (PAP1) activated the SoANS promoter in Arabidopsis leaves. The overexpression of Arabidopsis transcription factors with PhAN9 also induced ectopic anthocyanin accumulation in yellow cactus tepals. PAP homologs from betalain-producing Caryophyllales did not activate the promoter of ANS. In-depth characterization of Caryophyllales PAPs and site-directed mutagenesis in the R2R3–MYB domains identified the amino acid residues affecting transactivation of Caryophyllales PAPs. The substitution of amino acid residues recovered the transactivation ability of Caryophyllales PAPs. Therefore, loss of function in MYB transcription factors may suppress expression of genes involved in the late stage of anthocyanin synthesis, resulting in a lack of anthocyanin in betalain-producing Caryophyllales plants.

The production of greenhouse ornamentals relies on high fertilizer inputs to meet scheduling deadlines and quality standards, but overfertilization has negative environmental impacts. The goals of this study were to identify plant-growth-promoting rhizobacteria (PGPR) that can improve greenhouse ornamental crop performance with reduced fertilizer inputs, and to identify the best measurements of plant performance for assessing the beneficial impact of PGPR on ornamentals. A high-throughput greenhouse trial was used to identify 14 PGPR isolates that improved the flower/bud number and shoot dry weight of Petunia × hybrida ‘Picobella Blue’ grown under low fertility conditions in peat-based media. These 14 PGPR were then applied to petunias grown under low fertility conditions (25 mg L−1 N). PGPR-treated plants were compared to negative (untreated at 25 mg L−1 N) and positive (untreated at 50, 75, 100, and 150 mg L−1 N) controls. Multiple parameters were measured in the categories of flowering, vegetative growth, and vegetative quality to determine the best measurements to assess improvements in ornamental plant performance. Caballeronia zhejiangensis C7B12-treated plants performed better in almost all parameters and were comparable to untreated plants fertilized with 50 mg L−1 N. Genomic analysis identified genes that were potentially involved in plant growth promotion. Our study identified potential PGPR that can be used as biostimulants to produce high-quality greenhouse ornamentals with lower fertilizer inputs.

UDP-glycosyltransferase (UGT) plays an essential role in regulating the synthesis of hormones and secondary metabolites in plants. In this study, 129 members of the Petunia UGT family were identified and classified into 16 groups (A–P) based on phylogenetic analysis. The same subgroups have conserved motif compositions and intron/exon arrangement. In the promoters of the Petunia UGT genes, several cis-elements associated with plant hormones, growth and development, and abiotic stress have been discovered. Their expression profiles in five tissues were revealed by tissue expression based on RNA-seq data. Subcellular localization analysis showed that PhUGT51 was located in the nucleus and cell membrane. Salt stress caused an increase in the expression level of PhUGT51, but the expression level remained stable with the growth over time. In addition, the overexpression of PhUGT51 caused a significant increase in salt resistance. Our study systematically analyses the UGT gene family in Petunia for the first time and provides some valuable clues for the further functional studies of UGT genes.

Sustainable plant production practices have been implemented to reduce the use of synthetic fertilizers and other agrochemicals. One way to reduce fertilizer use without negatively impacting plant nutrition is to enhance crop uptake of nutrients with biostimulants. As the effectiveness of a biostimulant can depend on the origin, species, dose, and application method, the aim of this research was to evaluate the effect of a commercial animal-based protein hydrolysate (PH) biostimulant on the visual quality, biomass, macronutrient content, root morphology, and leaf gas exchange of a petunia (Petunia × hybrida Hort. “red”) under preharvest conditions. Two treatments were compared: (a) three doses of an animal-based PH biostimulant: 0 (D0 = control), 0.1 (D0.1 = normal), and 0.2 g L–1 (D0.2 = high); (b) two biostimulant application methods: foliar spray and root drenching. The dose × method interaction effect of PH biostimulant on the plants was significant in terms of quality grade and fresh and dry biomass. The high dose applied as foliar spray produced petunias with extra-grade visual quality (number of flowers per plant 161, number of leaves per plant 450, and leaf area per plant 1,487 cm2) and a total aboveground dry weight of 35 g, shoots (+91%), flowers (+230%), and leaf fresh weight (+71%). P and K contents were higher than in untreated petunias, when plants were grown with D0.2 and foliar spray. With foliar spray at the two doses, SPAD showed a linear increase (+21.6 and +41.0%) with respect to untreated plants. The dose × method interaction effect of biostimulant application was significant for root length, projected and total root surface area, and number of root tips, forks, and crossings. Concerning leaf gas exchange parameters, applying the biostimulant at both doses as foliar spray resulted in a significant improvement in net photosynthesis (D0.1: 22.9 μmol CO2 m–2 s–1 and D0.2: 22.4 μmol CO2 m–2 s–1) and stomatal conductance (D0.1: 0.42 mmol H2O m–2 s–1 and D0.2: 0.39 mmol H2O m–2 s–1) compared to control. These results indicate that application of PH biostimulant at 0.2 g L–1 as foliar spray helped to achieve extra-grade plants and that this practice can be exploited in sustainable greenhouse conditions for commercial production of petunia.

Out of the three aromatic amino acids, the highest flux in plants is directed towards phenylalanine, which is utilized to synthesize proteins and thousands of phenolic metabolites contributing to plant fitness. Phenylalanine is produced predominantly in plastids via the shikimate pathway and subsequent arogenate pathway, both of which are subject to complex transcriptional and post-transcriptional regulation. Previously, it was shown that allosteric feedback inhibition of arogenate dehydratase (ADT), which catalyzes the final step of the arogenate pathway, restricts flux through phenylalanine biosynthesis. Here, we show that in petunia (Petunia hybrida) flowers, which typically produce high phenylalanine levels, ADT regulation is relaxed, but not eliminated. Moderate expression of a feedback-insensitive ADT increased flux towards phenylalanine, while high overexpression paradoxically reduced phenylalanine formation. This reduction could be partially, but not fully, recovered by bypassing other known metabolic flux control points in the aromatic amino acid network. Using comparative transcriptomics, reverse genetics, and metabolic flux analysis, we discovered that transcriptional regulation of the d-ribulose-5-phosphate 3-epimerase gene in the pentose phosphate pathway controls flux into the shikimate pathway. Taken together, our findings reveal that regulation within and upstream of the shikimate pathway shares control over phenylalanine biosynthesis in the plant cell.

Biosynthesis of secondary metabolites relies on primary metabolic pathways to provide precursors, energy, and cofactors, thus requiring coordinated regulation of primary and secondary metabolic networks. However, to date, it remains largely unknown how this coordination is achieved. Using Petunia hybrida flowers, which emit high levels of phenylpropanoid/benzenoid volatile organic compounds (VOCs), we uncovered genome-wide dynamic deposition of histone H3 lysine 9 acetylation (H3K9ac) during anthesis as an underlying mechanism to coordinate primary and secondary metabolic networks. The observed epigenome reprogramming is accompanied by transcriptional activation at gene loci involved in primary metabolic pathways that provide precursor phenylalanine, as well as secondary metabolic pathways to produce volatile compounds. We also observed transcriptional repression among genes involved in alternative phenylpropanoid branches that compete for metabolic precursors. We show that GNAT family histone acetyltransferase(s) (HATs) are required for the expression of genes involved in VOC biosynthesis and emission, by using chemical inhibitors of HATs, and by knocking down a specific HAT gene, ELP3, through transient RNAi. Together, our study supports that regulatory mechanisms at chromatin level may play an essential role in activating primary and secondary metabolic pathways to regulate VOC synthesis in petunia flowers., Our study shows that post-translational modification of histones is essential for regulating the biosynthesis and emission of floral scent compounds, thus providing insights into the regulation of secondary metabolism at chromatin level.

Summary Foliar uptake of peroxyacetyl nitrate (PAN) was measured by the gas exchange method on nine herbaceous species. Susceptibility to PAN was also examined in the tested species in order to seek correlations with the uptake rate of PAN. PAN was synthesized by the nitration of peracetic acid in n-tridecane. Uptake rate of PAN by the leaves was kept at a steady level during the 90 min light period, while in the dark it declined rapidly and reached almost zero within 30–45 min. This suggests that adsorption of PAN on to the surface of the leaves is very small and most of it enters the leaf through open stomata. Among the species tested, sunflower showed the highest rate of PAN uptake, followed by radish, tomato and spinach. Maize and soyabean exhibited the lowest rate. A highly significant correlation was observed between the rate ol PAN uptake and stomatal conductance among the tested species. Petunia was most susceptible to PAN, followed by radish and tobacco, but maize and peanut were tolerant. There was no significant correlation between the uptake rate of PAN and susceptibility to it among the species. A higher susceptibility of petunia to PAN was not characterized by a higher rate of pollutant uptake. These results show that the main factor determining the differences between species in susceptibility to PAN is not the amount that gains access to the tissues, but some internal metabolic processes.

Rosa rugosa is an important natural perfume plant in China. Rose essential oil is known as ‘liquid gold’ and has high economic and health values. Monoterpenes are the main fragrant components of R. rugosa flower and essential oil. In this study, a member of the hydrolase gene family RrNUDX1 was cloned from Chinese traditional R. rugosa ‘Tang Hong’. Combined analysis of RrNUDX1 gene expression and the aroma components in different development stages and different parts of flower organ, we found that the main aroma component content was consistent with the gene expression pattern. The RrNUDX1 overexpressed Petunia hybrida was acquired via Agrobacterium-mediated genetic transformation systems. The blades of the transgenic petunias became wider and its growth vigor became strong with stronger fragrance. Gas chromatography with mass spectrometry analysis showed that the contents of the main aroma components of the transgenic petunias including methyl benzoate significantly increased. These findings indicate that the RrNUDX1 gene plays a role in enhancing the fragrance of petunia flowers, and they could lay an important foundation for the homeotic transformation of RrNUDX1 in R. rugosa for cultivating new R. rugosa varieties of high-yield and -quality essential oil.

Summary Cucumber mosaic virus (CMV) is a highly prevalent viral pathogen causing substantial damage to the bulb and cut‐flower production of Lilium spp. Here, we performed an Illumina RNA sequencing (RNA‐Seq) study on the leaf tissues of a virus‐resistant species Lilium regale inoculated with mock control and CMV. A total of 1346 differentially expressed genes (DEGs) were identified in the leaves of L. regale upon CMV inoculation, which contained 34 up‐regulated and 40 down‐regulated DEGs that encode putative transcription factors (TFs). One up‐regulated TF, LrNAC35, belonging to the NAM/ATAF/CUC (NAC) superfamily, was selected for further functional characterization. Aside from CMV, lily mottle virus and lily symptomless virus infections provoked a striking increase in LrNAC35 transcripts in both resistant and susceptible Lilium species. The treatments with low temperature and several stress‐related hormones activated LrNAC35 expression, contrary to its reduced expression under salt stress. Ectopic overexpression of LrNAC35 in petunia (Petunia hybrida) resulted in reduced susceptibility to CMV and Tobacco mosaic virus infections, and enhanced accumulation of lignin in the cell walls. Four lignin biosynthetic genes, including PhC4H, Ph4CL, PhHCT and PhCCR, were found to be up‐regulated in CMV‐infected petunia lines overexpressing LrNAC35. In vivo promoter‐binding tests showed that LrNAC35 specifically regulated the expression of Ph4CL. Taken together, our results suggest a positive role of transcriptome‐derived LrNAC35 in transcriptional modulation of host defence against viral attack.

Plant-specific NAM, ATAF, and CUC (NAC) transcription factors (TFs) play clear roles in plant development and abiotic stress responses. Chinese dwarf cherry (Cerasus humilis) is an economically important shrub, that has strong resistance to drought. In this study, we isolated and functionally characterized a novel NAC TF from C. humilis. The ChNAC1 ORF contained 894 nucleotides, encoding 297 amino acid residues. ChNAC1 amino acid sequences had the highest similarity with homologous petunia (Petunia hybrida) and tomato (Solanum lycopersicum) NAM proteins. The ChNAC1 transcripts were most abundant in the leaves of seedlings and significantly up-regulated by drought stress. The nuclear localization and transcriptional activity of the C-terminal domain further confirmed that ChNAC1 functions as a TF. Yeast two-hybrid results showed that ChNAC1 can homodimerize in yeast cells. Next, we transformed ChNAC1 into wild-type Arabidopsis thaliana and found that the ectopic expression of ChNAC1 increased chlorophyll, water, proline, and protein contents as well as higher peroxidase (POD) and superoxide dismutase (SOD) activities while decreasing electrolyte conductivity and reactive oxygen species (ROS) contents compared to wild-type and mutant lines. Further, overexpression of ChNAC1 positively regulated ABA-responsive genes under drought stress and increased ABA sensitivity during root growth. Collectively, these results demonstrate the key role of ChNAC1 in drought stress tolerance.

A study was carried out to investigate the effects of growing media on physiology, flowering behaviour and longevity of potted petunia plants (Petunia grandiflora Juss.) under green house condition. A total of 30 potted petunia plants were used in this experiment and five different growing media namely top soil (control), vermicompost, biochar, cocopeat and peatmoss were tested. Leaf area, lateral branching, stomatal conductance and net photosynthetic rate significantly increased when the plant was grown in peatmoss and cocopeat. The highest length of lateral branch was recorded in the control treatment. In addition, peatmoss and cocopeat medium increased the number of flower, flower diameter, weight of individual flower and petal thickness significantly. Furthermore, chlorophyll a & b contents in leaves, carotenoids and anthocyanin content in flowers were also increased significantly with peatmoss and cocopeat applications. Ethylene production was reduced significantly and flower longevity of potted petunia plants increased in different growing media. There was a positive correlation between petal thickness and flower senescence, and between anthocyanin content with flower senescence in potted petunia plants. It can be concluded that peatmoss and cocopeat growing media improved the growth, quality and longevity of potted petunia flowers under green house condition.

We describe and illustrate a new species of Petunia, P. correntina, from southwestern Corrientes, Argentina, in southern South America. The species has funnel-form corolla with whitish-green tube and purple lobes, apex of longer filaments nearly straight, apex of medium filaments curved laterally and opposite each other, connivent anthers, bluish pollen, stigma located between anthers of the large and medium stamens and inflexed pedicels in fruiting stage. This suit of characters is unique into the genus. Few populations are known, inhabiting sandy soils of the Paraná basin. A key to the Argentinian species of Petunia with purple corolla is given.

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.

Petunia × hybrida Vilm. is a fast-growing ornamental plant that was cultured under varying storage conditions to address seasonal fluctuations in microcutting demand. The effects of storage period (16 to 32 wk), temperature (15 to 23°C), low and high light intensity, sucrose (1 to 5% w/v), and mannitol (0 to 4% w/v) in factorial arrangements were analyzed. Stored and non-stored shoots were compared for microcutting production, harvested twice at 3-wk intervals, and were subsequently transferred to the greenhouse for 17 d. Nearly all plants from 16-wk storage survived well at all treatment conditions and the quality and quantity of microcuttings were enhanced from shoots stored at 15°C and 3% (w/v) sucrose, without mannitol for all storage periods. Another experiment tested 11- to 25-wk storage period with Petunia hybrida ‘Ragtime’ and ‘Suncatcher’ at 12°C and low light intensity. Repeated cycles of microcutting at 2-wk intervals were extended with ex vitro rooting in the greenhouse for 15 d. More and better quality microcuttings were obtained from the second and third cutting cycles than from the first or fourth cycles. By reducing temperature and light intensity, Petunia hybrida was successfully stored for 32 wk (without mannitol). A seasonal schedule with a short production window, followed by cutting large numbers of high-quality shoot-tips, could be affected and efficiently managed through storage. The value of stored shoots was enhanced by extending the number of times a shoot could be cut over repeated cutting cycles with a gain in microcutting quality.