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Iron (Fe) is an essential micronutrient for plants. Due to the low Fe bioavailability in cultivated soils, Fe deficiency is a widespread agricultural problem. In this study, we present the functional characterization of a petunia (Petunia hybrida) basic-helix-loop-helix transcription factor PhbHLH121 in response to Fe shortage. Real-time PCR revealed that the expression of PhbHLH121 in petunia roots and shoots was downregulated under Fe-limited conditions. CRISPR/Cas9-edited phbhlh121 mutant plants were generated to investigate the functions of PhbHLH121 in petunia. Loss-of-function of PhbHLH121 enhanced petunia tolerance to Fe deficiency. Further investigations revealed that the expression level of several structural genes involved in Fe uptake in petunia, such as IRT1 and FRO2, was higher in phbhlh121 mutants compared to that in wild-type under Fe-limited conditions, and the expression level of several genes involved in Fe storage and Fe transport, such as VTL2, FERs and ZIF1, was lower in phbhlh121 mutants compared to that in wild-type under Fe-deficient conditions. Yeast one-hybrid assays revealed that PhbHLH121 binds to the G-box element in the promoter of genes involved in Fe homeostasis. Yeast two-hybrid assays revealed that PhbHLH121 interacts with petunia bHLH IVc proteins. Taken together, PhbHLH121 plays an important role in the Fe deficiency response in petunia. [ABSTRACT FROM AUTHOR]

The ARF gene family plays a vital role in regulating multiple aspects of plant growth and development. However, detailed research on the role of the ARF family in regulating flower development in petunia and other plants remains limited. This study investigates the distinct roles of PhARF5 and PhARF19a in Petunia hybrida flower development. Phylogenetic analysis identified 29 PhARFs, which were grouped into four clades. VIGS-mediated silencing of PhARF5 and PhARF19a led to notable phenotypic changes, highlighting their non-redundant functions. PhARF5 silencing resulted in reduced petal number and limb abnormalities, while PhARF19a silencing disrupted corolla tube formation and orientation. Both genes showed high expression in the roots, leaves, and corollas, with nuclear localization. The transcriptomic analysis revealed significant overlaps in DEGs between PhARF5 and PhARF19a silencing, indicating shared pathways in hormone metabolism, signal transduction, and stress responses. Phytohormone analysis confirmed their broad impact on phytohormone biosynthesis, suggesting involvement in complex feedback mechanisms. Silencing PhARF5 and PhARF19a led to differential transcription of numerous genes related to hormone signaling pathways beyond auxin signaling, indicating their direct or indirect crosstalk with other phytohormones. However, significant differences in the regulation of these signaling pathways were observed between PhARF5 and PhARF19a. These findings reveal the roles of ARF genes in regulating petunia flower development, as well as the phylogenetic distribution of the PhARFs involved in this process. This study provides a valuable reference for molecular breeding aimed at improving floral traits in the petunia genus and related species. [ABSTRACT FROM AUTHOR]

Key message: An endoplasmic reticulum-localized Cu transporter, PhHMA5II1, interacts with copper chaperones and plays an important role in Cu detoxification in petunia. Copper (Cu) is an essential element for plant growth but toxic when present in excess. In this study we present the functional characterization of a petunia (Petunia hybrida) P1B-type heavy-metal ATPases (HMAs), PhHMA5II1. Heterologous expression of PhHMA5II1 in yeast (Saccharomyces cerevisiae) showed Cu transport activity. The expression of PhHMA5II1 in roots and shoots was unaffected by excess Cu. CRISPR/Cas9-edited mutant lines and PhHMA5II1 overexpressing transgenic plants were generated to investigate the functions of PhHMA5II1 in petunia. The PhHMA5II1 knockout mutant was hypersensitive to excess Cu and accumulated more Cu in roots compared to wild-type petunia. Overexpression of PhHMA5II1 enhanced Cu tolerance and reduced Cu accumulation in roots. Furthermore, PhHMA5II1 localized in endoplasmic reticulum, and the localization was unaffected by excess Cu. Yeast two-hybrid experiments and bimolecular fluorescence complementation assays demonstrate that PhHMA5II1 interact with petunia copper chaperons, PhATX1 and PhCCH. Finally, RNA-sequencing revealed that knockout PhHMA5II1 affected the expression of genes involved in cell-wall organization, copper ion homeostasis, and response to oxidative stress. Taken together, PhHMA5II1 plays an important role in Cu detoxification in petunia. [ABSTRACT FROM AUTHOR]

This experiment was carried out in the Plant Cells and Tissue Culture Laboratory of Horticulture and Landscaping Design Department / College of Agriculture / University of Kirkuk - IRAQ, to study the effect of auxin’s in different concentrations and MS media (full and ½) salt strength on rooting response of Petunia hybrida. The regenerated shoots explants from the multiplication stage were transferred to the rooting stage cultured on MS media ( full and ½ ) salts strength supplemented with different concentrations of IBA at ( 0.0, 0.25, 0.50, 0.75, and 1.0 ) mg.L-1, and NAA at ( 0.0, 0.25, 0.50, 0.75, and 1.0 ) mg.L-1. After 6 weeks of transforming, data showed that, the response percentage was ( 100% ) for MS media ( full and ½ ) salt strength supplemented with different concentrations of IBA and NAA beside control treatment. The best number of roots ( 29.70 root.part-1 ) and the length of the longest root ( 7.44 cm ) were recorded by MS ½ strength supplemented with IBA (1.0 mg.L-1) compared with other concentrations, and it was the highest results compared with MS full strength supplemented with different concentration of IBA. Moreover, results obtained that MS ½ strength supplemented with NAA ( 1.0 mg.L-1 ) recorded the best number of roots ( 18.70 root.part-1 ) and the length of the longest root ( 2.97 cm ) compared with the other concentrations, while it was the best result compared with MS full strength supplemented with different concentrations of NAA.

In this work, we assessed the effects of increasing ozone (O3) on four petunia varieties with different floral pigmentation (pink, red, rose-red, and white). Plants were exposed, in open-top chambers located in China, to three O3 concentrations, i.e., (i) ambient air (AA), (ii) AA + 60 ppb O3 (AA + 60), and (iii) AA + 120 ppb O3 (AA + 120), for 85 days (9 h day−1). Flower diameter and duration were assessed, together with leaf chlorophyll and flavonoid contents. White petunia showed a reduced flower diameter and longevity under AA + 60 (−7 and −6%, respectively, in comparison to AA), whereas pink and red petunias only showed this under AA + 120 (−8 and −7%, on average, respectively). Chlorophyll loss occurred in all varieties under AA + 60 (−30%, on average), and at AA + 120 in white and red petunias (−54%, on average). The total flavonoid content in the pink and white varieties increased only under AA + 120 (around +85%), while it grew at both AA + 60 and AA + 120 (+92% and two-fold higher, respectively) in the red variety. Increasing O3 concentrations did not affect particularly the red-rose variety. The white variety showed the strongest correlations among flower and leaf properties, confirming a variety-related O3 response, as well as demonstrating that it had the highest O3 sensitivity. [ABSTRACT FROM AUTHOR]

Petunia hybrida is an economically important ornamental plant species. Branching in ornamental plants is closely associated with their ornamental traits, and branching is a significant agronomic trait in petunia, which shapes plant architecture and production cost. Although there are few studies regarding the involvement of the SPEAR genes in lateral branch development, they are known to regulate the development of plant organs. The PhSPEAR1 gene in petunia, an ortholog of Arabidopsis SPEAR1, was isolated for study. According to the results of real-time quantitative PCR (qRT-PCR), PhSPEAR1 was primarily expressed in the roots. The fluorescence signal indicated that PhSPEAR1 was localized in the nucleus and cytoplasm. An increase in PhSPEAR1 expression was induced by cytokinin or decapitation. Overexpression of PhSPEAR1 increased lateral branches in Arabidopsis. Based on our findings, PhSPEAR1 participates significantly in the regulation of branch number in petunia. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Kirkuk University for Agricultural Sciences is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Kirkuk University for Agricultural Sciences is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Iron (Fe) is an essential micronutrient for plants. Due to the low Fe bioavailability in cultivated soils, Fe deficiency is a widespread agricultural problem. In this study, we present the functional characterization of a petunia (Petunia hybrida) basic-helix-loop-helix transcription factor PhbHLH121 in response to Fe shortage. Real-time PCR revealed that the expression of PhbHLH121 in petunia roots and shoots was downregulated under Fe-limited conditions. CRISPR/Cas9-edited phbhlh121 mutant plants were generated to investigate the functions of PhbHLH121 in petunia. Loss-of-function of PhbHLH121 enhanced petunia tolerance to Fe deficiency. Further investigations revealed that the expression level of several structural genes involved in Fe uptake in petunia, such as IRT1 and FRO2, was higher in phbhlh121 mutants compared to that in wild-type under Fe-limited conditions, and the expression level of several genes involved in Fe storage and Fe transport, such as VTL2, FERs and ZIF1, was lower in phbhlh121 mutants compared to that in wild-type under Fe-deficient conditions. Yeast one-hybrid assays revealed that PhbHLH121 binds to the G-box element in the promoter of genes involved in Fe homeostasis. Yeast two-hybrid assays revealed that PhbHLH121 interacts with petunia bHLH IVc proteins. Taken together, PhbHLH121 plays an important role in the Fe deficiency response in petunia.

Petunia hybrida is an economically important ornamental plant species. Branching in ornamental plants is closely associated with their ornamental traits, and branching is a significant agronomic trait in petunia, which shapes plant architecture and production cost. Although there are few studies regarding the involvement of the SPEAR genes in lateral branch development, they are known to regulate the development of plant organs. The PhSPEAR1 gene in petunia, an ortholog of Arabidopsis SPEAR1, was isolated for study. According to the results of real-time quantitative PCR (qRT-PCR), PhSPEAR1 was primarily expressed in the roots. The fluorescence signal indicated that PhSPEAR1 was localized in the nucleus and cytoplasm. An increase in PhSPEAR1 expression was induced by cytokinin or decapitation. Overexpression of PhSPEAR1 increased lateral branches in Arabidopsis. Based on our findings, PhSPEAR1 participates significantly in the regulation of branch number in petunia.

Thrips are a common pest in most horticultural and ornamental crops. They are particularly common in greenhouses in western Romania. In the present study, we investigated thrips preference for different coloured traps as well as specific structure and abundance in Petunia hybrida varieties in greenhouses. Thrips attraction to colour was assessed using three commercially available coloured sticky traps: yellow, orange and blue. Taxonomically, the diversity of thrips is reflected in the relatively large number of species, identified in Petunia, 6 species: Frankliniella intonsa, Frankliniella occidentalis, Frankliniella schultzei, Scirtothrips dorsalis, Thrips palmi and Thrips tabaci. Of these species, 2 are the most common and abundant: Frankliniella occidentalis (n= 172) and Thrips tabaci (n= 163). In the Petunia varieties, significant differences were observed depending on the colour of the trap, these were found between yellow-blue (p = 0.000) and yellow-orange (p = 0.000) and it is observed that there are no significant differences between blue-orange (p = 0.434). Most of the thrips species were caught with the yellow sticky traps (F=39.398, p=0.000<0.05). [ABSTRACT FROM AUTHOR]

The art of pressed flowers is a method of artistic expression involving the pressing of flowers, leaves, and other plant organs for artistic creative purposes. However, the pressing process often results in color variation of the plant material, which significantly diminishes the quality of artistic works and must be solved using appropriate techniques. During this research, phenylalanine (10 mmol.L-1) was used to treat the petals of postharvest Petunia flowers to investigate the impact of phenylalanine on mitigating color variation, and the effect of phenylalanine on inhibiting the color variation of Petunia petals during the pressing process was evaluated by color measurement, physicochemical indices, and gene expression level analyses. Using the CIEL*a*b* color measurements, the samples from the test group had significantly higher brightness (L*) and red coloration (a*) at the final stage (S4) than the control group. In addition, phenylalanine had a significant inhibitory effect on malondialdehyde and superoxide anion accumulations in Petunia petals during pressing and reduced the enzymatic activities of superoxide dismutase, polyphenol oxidase, and catalase. The quantitative reverse-transcription polymerase chain reaction analysis showed that the transcript levels of CHS, DFR, F3050H, and UFGT genes in the petals of the treatment group continued to increase during the pressing process, and the transcript levels of key genes in the anthocyanin metabolic pathway of the treated samples were higher than those of the control group at the final stage (S4). These results indicated that phenylalanine can effectively diminish the color variation of Petunia petals in the pressing process, which could serve as a theoretical basis for the development of a comprehensive technology system aimed at preserving the color of pressed horticultural plants. [ABSTRACT FROM AUTHOR]

Petunia (Petunia × hybrida Hort. Vilm.-Andr.) is a well-suited plant for sustainable landscape issues in borderline areas with irrigation with saline water. Salicylic acid (SA) as a modulator performs an imperative function in modulating plant salt tolerance. However, there are a few reports on the effect of SA on petunia plants irrigated with saline water. During the 2022/2023 season, a factorial pot experiment in a randomized complete block design was carried out in Riyadh, Saudi Arabia, to assess the effect of SA concentration (0, 500, 1000, 2000 mgL−1) on petunia plant growth, flowering, ion content, chlorophyll level, and proline concentration under irrigation with salty water (230, 1500, 3000 mgL−1). Saline water up to 3000 mgL−1 dramatically reduced plant growth, chlorophyll, ions, and flowering attributes, while the contrary was observed in proline and sodium concentrations as compared to the control treatments (irrigation with tap water). Foliar spraying with 1000 mgL−1 SA considerably boosted plant growth and flowering as well as chlorophyll, proline, and ion content compared to untreated plants under such salinity levels. Alternatively, the application of 1000 mgL−1 to normal or salinized water significantly decreased the Na content in non-treated plants under such a salinity level. Accordingly, using 1000 mgL−1 of SA under salt stress conditions could be a useful technique to lessen the mutilation induced by the use of salinized water in the era of climate change. [ABSTRACT FROM AUTHOR]

Introduction Flower color is one of the most significant characteristics in ornamental plant breeding. New varieties of various plants in relation to their flower color have been obtained by monitoring the expression levels of genes involved or regulating the flavonoid and anthocyanin biosynthesis pathway. Flavonoids possess significant and diverse biological functions. They are the major pigments for flowers, fruits, seeds, and leaves. They are natural products that contain a C6-C3-C6 carbon framework and are synthesized by a branched pathway that yields both colored and colorless compounds. The gene encoding chalcone isomerase (CHI) is among the genes and enzymes identified in the flavonoid pathway. This enzyme catalyzes the isomerization of naringenin chalcone into the corresponding flavanone. CHI enzyme belongs to the family of isomerases, specifically the class of intramolecular lyases. Chalcone isomerase has a core 2-layer alpha/beta structure and has attracted much attention recently due to its role in stress response and pigment production. One of the most effective methods of genetic engineering is the reduction of flower pigments by suppression of required enzymes for their biosynthesis. RNA interference (RNAi) has provided the tool for the investigation of genes involved in the production of flower color. Silencing of any gene in the anthocyanin biosynthetic pathway can result in reduced or inhibited anthocyanin production. RNAi technology is an effective gene silencing method and a powerful tool for studying gene function and development of new traits by transformation of viral RNA or hairpin RNA (hpRNA) constructs into plants. The processing of dsRNA into 21-23-nt small interfering RNAs (siRNAs), and the mediators of RNAi, triggers cognate mRNA degradation. The hpRNAi methodology simply requires a transgene construct containing an inversely-repeated sequence of the target gene flanked with a promoter and terminator which effectively function in plants. Material and Methods In this research, with the design and construction of chiRNAi, the transformation of the RNAi construct was carried out of Petunia plants. Potted plants of P. hybrida were grown under standard greenhouse conditions (16-17°C night temperature and 21-24°C day temperature and photoperiod 16/8 (light/dark)). The RNAi construct including the 530 bp cds of the chalcone isomerase (chi) gene and 741 bp of pdk gene as intron between chi sense and antisense were used for transient RNAi-induced silencing. The pBI121-chi530 plasmids were introduced into A. tumefaciens strain LBA4404 by electroporation method. Colonies of A. tumefaciens carrying the desired plasmid were screened by PCR with specific primers for chi gene. RNAi construct co-cultured with petunia’s leave. Samples was kept in dark condition for 3 days and then transferred to branch induction media. Samples were investigated for phenotypical changes and chi gene expression by qRT-PCR. Results and Discussion Transgenic lines showed a reduced number of pigments and a faded flower color. So that, in purple petunia, was shown 5 phenotypical groups. These groups was indicated different levels of chi gene silencing. In pink petunia was seen two groups of phenotypical changes. In these plants, chi-RNAi construct was reduced pigment production and so, these plants had faded colors in petals. Also, the chi gene expression was reduced in all transgenic lines. Generally, the results of this research showed that RNAi can be used as an efficient method for gene silencing. The application of gene silencing can indicate the gene’s function in biosynthesis pathways of various components such as anthocyanins. In addition, the chalcone isomerase gene was identified as one of the effective genes in anthocyanin biosynthesis pathway in Petunia plants that could be involved in the production of color in these plants; hence, chi gene silencing resulted in clear phenotypic alterations in this plant. Conclusion In general the concentration of the target mRNA in a particular tissue could be a factor that influences silencing efficiency. At very low levels of gene expression, small amounts of the silencing target, mRNA, could be completely degraded by the RNA-induced silencing complex (RISC), whereas the presence of higher amounts of the target mRNA may result in incomplete silencing, allowing some residual functional mRNA to be translated into the corresponding protein. This research demonstrated the hpRNA construct has been successfully established for floral tissues of P. hybrida. The hpRNA construct was developed for chi-RNAi silencing of one of the key genes in the anthocyanin biosynthetic pathway in Petunia flowers. The silencing of the chi gene is a prototype for the modification of the anthocyanin biosynthetic pathway in Petunia through gene suppression. This strategy could also be useful for rapid functional analysis of other genes involved in flower development.

The branch number of plants is an important agronomic trait that directly influences the ornamental characters and production costs of ornamental plants. Shoot branching has always been a hot topic for Petunia hybrida. During our research, we isolated the homologous gene of narrow-leaf 1 (NAL1), denoted as PhNAL1. The expression level of PhNAL1 was higher in leaves and axils than in roots, stems, and flowers. Pertinent to shoot apex removal and 6-benzyladenine treatments, both interventions demonstrated a suppressive effect on the expression of PhNAL1. Through subcellular localization analysis, we found that PhNAL1 predominantly localized in the nucleus. By using RNA interference targeting PhNAL1, we induced a noticeable increase in branch number while concurrently reducing plant height of petunia. These findings demonstrate that PhNAL1 is involved in regulating branch development within petunia. This study provides genetic resources for the subsequent cultivation of new cultivars of petunia endowed with distinct branching characteristics.

Mildew Locus O (MLO), which encodes a seven-transmembrane domain protein, is responsible for the development of powdery mildew disease symptoms in many plant species. Hence, we knocked out PhMLO1 from Petunia hybrida cv. Mirage Rose at the genomic level using the Clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) system and investigated involvement of the gene in powdery mildew disease development in the petunia. The MLO1 transcript levels observed in all mlo1 mutants were significantly lower than that in the wild type (WT). Specifically, the transcript level was the lowest in the mutants (line no. 1, 25, 33, and 85), followed by the mutants (line no. 14, 17, and 81), and line no. 6. Disease development was positively linked to the higher transcript levels of PhMLO1, because the disease severity observed in WT was greater than that observed in the mutants (line no. 6,14, 17, and 81), and disease symptoms were almost not observed in the mutants (line no. 1, 25, 33, and 85). In addition, the transmission of the edited alleles from the T0 to T1 generation was also confirmed, and the disease severity results were identical to those observed in the T0 mutant lines. Overall, the editing of PhMLO1 completely or partially prevented the development of this disease in this petunia, depending on MLO1 transcript level of the mutants. This study highlights the role of PhMLO1 in the development of powdery mildew disease in petunia and points to the need to edit the MLO genes in other ornamental plants to improve ornamental quality against powdery mildew disease. [ABSTRACT FROM AUTHOR]

Anthocyanins are responsible for the color spectrum of both ornamental and natural flowers. However, not all plant species produce all colors. For example, roses are not blue because they do not naturally possess a hydroxylase that opens the pathway for delphinidin and its derivatives. It is more intriguing why some plants do not carry orange or scarlet red flowers with anthocyanins based on pelargonidin, because the precursor for these anthocyanins should be available if anthocyanins are made at all. The key to this is the substrate specificity of dihydroflavonol 4-reductase (DFR), an enzyme located at the branch point between flavonols and anthocyanins. The most common example is petunia, which does not bear orange flowers unless the enzyme is complemented by biotechnology. We changed a few amino acids in the active site of the enzyme and showed that the mutated petunia DFR started to favor dihydrokaempferol, the precursor to orange pelargonidin, in vitro. When transferred to petunia, it produced an orange hue and dramatically more pelargonidin-based anthocyanins in the flowers. [ABSTRACT FROM AUTHOR]

Summary: Pathogenic fungi use secreted effector proteins to suppress immunity and support their infection, but effectors have also been reported from fungi that engage in nutritional symbioses with plants. Sequence‐based effector comparisons between pathogens and symbiotic arbuscular mycorrhizal (AM) fungi are hampered by the huge diversity of effector sequences even within closely related microbes.To find sequence‐divergent but structurally similar effectors shared between symbiotic and pathogenic fungi, we compared secreted protein structure models of the AM fungus Rhizophagus irregularis to known pathogen effectors. We identified proteins with structural similarity to known Fusarium oxysporum f. sp. lycopersici dual domain (FOLD) effectors, which occur in low numbers in several fungal pathogens. Contrastingly, FOLD genes from AM fungi (MycFOLDs) are found in enlarged and diversified gene families with higher levels of positive selection in their C‐terminal domains.Our structure model comparison suggests that MycFOLDs are similar to carbohydrate‐binding motifs. Different MycFOLD genes are expressed during colonisation of different hosts and MycFOLD‐17 transcripts accumulate in plant intracellular arbuscules.The exclusive presence of MycFOLDs across unrelated plant‐colonising fungi, their inducible expression, lineage‐specific sequence diversification and transcripts in arbuscules suggest that FOLD proteins act as effectors during plant colonisation of symbiotic and pathogenic fungi. [ABSTRACT FROM AUTHOR]

The present study tested the effect of aerated (AM), non-aerated (NAM), a 1:1 combination of both compost teas (A-NAM), and bioslurry (B) as bio-fertilizers in two ornamental plants growing in different substrates. The present study aimed to evaluate the products as biostimulants in ornamental plants. Compost teas were brewed from a composted mixture of residues, while bioslurry was prepared, with fresh material and no aeration. Two ornamental plant species were used: Petunia hybrida and Impatiens walleriana. Experimental presented a completely randomized factorial design, with two factors: Biostimulants with 6 levels: AM, NAM, A-NAM, B, F (traditional fertilizer), and H2O (control); and Substrate, with 3 levels: sand, commercial, and compost. Bio-inputs presented variable properties and effects on plant growth, with significant interaction between factors. In sand, no effect of bioproducts on plant biomass, SPAD index and pigments content were observed. In compost, B significantly increased I. walleriana and P. hybrida dry biomass (1.75 ± 0.42 g and 1.54 ± 0.28 g, respect to 1.33 ± 0.18 g for H2O), while in the commercial substrate, only in P. hybrida (1.35 ± 0.44 g, regarding 0.42 ± 0.11 g for H2O). All bio-inputs increased pigments content of both plant species in all substrates. To our knowledge, this is the first record that B was tested, which increased plant biomass at similar values to traditional fertilizer. We demonstrated that the biostimulants brewed from organic residues might present the same performance as commercial fertilizers. [ABSTRACT FROM AUTHOR]

Aquilegia buergeriana is a native plant in Korea with blue flowers. Flavonoid 3′,5′ hydroxylase (F3′5′H) is a key gene involved in the synthesis of delphinidin pigment responsible for the flower's blue color. We isolated the F3′5′H from the petals of A. buergeriana (AbF3′5′H) and introduced the AbF3′5′H gene into Petunia hybrida using Agrobacterium-mediated transformation. Forty-five plants were acquired from a kanamycin-supplemented medium. Fifteen of these were identified as transgenic plants using polymerase chain reaction (PCR). Quantitative real-time PCR (qRT-PCR) analysis revealed that the AbF3′5′H was expressed in the petal, corolla tube, and stigma of P. hybrida. AbF3′5′H-transgenic plant (T0) flower color was darker than that of non-transgenic plants (NTs). Particularly, the stigma color was dramatically changed, from light yellow green (145C) to purple (N77C or N79D). The segregation ratio of the three transgenic (T1) lines was identified as 3:1 by PCR analysis of AbF3′5′H and neomycin phosphotransferase-II. The flower color change of the transgenic lines (T1) was similar to that of T0. qRT-PCR analysis showed that AbF3′5′H-transgenic T1 lines had a higher AbF3′5′H expression than NT in all floral organs. Moreover, delphinidin was confirmed to be accumulated in both corolla tube and stigma and was enhanced in the petals of AbF3′5′H-transgenic T1 lines through UPLC analysis. Our findings indicate the role of AbF3′5′H in flower color change. These results also indicate the functionality of AbF3′5′H in bluish flower modifications. [ABSTRACT FROM AUTHOR]

Scientific relevance. The garden petunia, Petunia hybrida, is a popular and wide spread ornamental crop from the family Solanaceae. It is a promising model plant for molecular and genetic research. In vitro micropropagation plays an important role in the distribution of the garden petunia because the survivability and quality of seed material decreases significantly in every subsequent generation. Besides, micropropagation reduces the cost of production substantially. Considering that very few researchers addressed this question in the Russian Federation, this direction of research is still worthy of attention.Materials and methods. The experiments were conducted by the Laboratory of Breeding and Genetic Research on Field Crops at FSBSI “Federal Scientific Center of Agricultural Biotechnology of the Far East named after A.K. Chaiki”. Seeds of Petunia hybrida (double-flowered) were used as primary explants. Liquid bleacher ACE diluted with distilled water in the proportion 1:9 was used as a sterilizing agent (the working solution contained 0.50% NaOCl). The total time of exposure was 15 minutes. The primary explants were subcultured onto a hormone-free Murashige and Skoog basal medium containing 20 g/L sucrose and 6 g/L agar. Isolated in vitro objects were cultured in test tubes with cotton-gauze plugs at an illuminance of 4000 lx, a temperature of 22–25 °C, and a 16h photoperiod in a culture room. The duration of one passage was 60 days. Micropropagation was carried out using 7- 10 mm cuttings with one or two nodes. The pot culture of the regenerants was established under controlled conditions in a light room (photoperiod was 16 hours, temperature was 23°С).Results. The optimal method for introducing Petunia hybrida into cell culture is the use of seeds treated with the solution of bleacher ACE that was diluted with distilled water in the proportion 1:9. The optimal time of exposure is 15 minutes. Petunia hybrida demonstrated a high regeneration rate on the hormone-free MS medium – it had a fast growth and development rate, and good rhizogenesis; the reproductive rate was 8.77. For the micropropagation of the garden petunia, it is advisable to use cuttings of test tube plants, which should be placed onto a hormone-free MS medium. The test tube plants of Petunia hybrida acclimatized successfully on a soil substrate. This shows the high plasticity of the culture.

Anthocyanins are responsible for the color spectrum of both ornamental and natural flowers. However, not all plant species produce all colors. For example, roses are not blue because they do not naturally possess a hydroxylase that opens the pathway for delphinidin and its derivatives. It is more intriguing why some plants do not carry orange or scarlet red flowers with anthocyanins based on pelargonidin, because the precursor for these anthocyanins should be available if anthocyanins are made at all. The key to this is the substrate specificity of dihydroflavonol 4-reductase (DFR), an enzyme located at the branch point between flavonols and anthocyanins. The most common example is petunia, which does not bear orange flowers unless the enzyme is complemented by biotechnology. We changed a few amino acids in the active site of the enzyme and showed that the mutated petunia DFR started to favor dihydrokaempferol, the precursor to orange pelargonidin, in vitro. When transferred to petunia, it produced an orange hue and dramatically more pelargonidin-based anthocyanins in the flowers.

Effect of growing media and Jeevamrit on potted petunia (Petunia hybrida Vilm) was studied in a field experiment laid out in completely randomized design (factorial). Petunias were grown in pots consisting of three growing media viz soil + sand + FYM (1:1:1, v/v), soil + cocopeat + FYM (1:1:1 v/v) and soil + cocopeat + vermicompost (1:1:1, v/v). Jeevamrit (10%) was applied to pots in the form of foliar spray and drench at an interval of 15 and 30 days. Maximum plant height (23.32 cm), earliest peak flowering (49.32 days), larger diameter flowers (8.59 cm) and maximum pot presentability score (94.01) were recorded in growing medium comprising soil + cocopeat + FYM (1:1:1, v/v). Among different Jeevamrit treatments, its drench application at fortnightly and monthly interval produced plants with more height of 23.44 cm and 23.24 cm, took minimum days of 39.45 and 39.64 to first flower opening, took minimum period for peak flowering of 48.75 and 49.27 days and recorded highest pot presentability score of 96.93 and 95.04 respectively. Maximum plant spread (36.29 cm) and flowering duration (65.28 days) were recorded in treatment combination soil + cocopeat + FYM (1:1:1, v/v) and Jeevamrit drenching at fortnightly interval. It was concluded that for obtaining quality potted petunias, these should be grown on medium comprising soil + cocopeat + FYM (1:1:1, v/v) which should be applied with Jeevamrit (10%) as drench at fortnightly interval. [ABSTRACT FROM AUTHOR]

Saline-alkaline stress not only leads to reduced crop yields but also diminishes the ornamental value of flowers. While petunia exhibits tolerance to saline-alkaline stress, research on the mechanisms underlying this tolerance remains unreported. We investigated the physiological and molecular mechanisms underlying saline-alkaline stress tolerance using two petunia genotypes (Haishishenlou and Mitchell Diploid) with differing tolerance levels. Haishishenlou exhibited less inhibition of growth under saline-alkaline stress compared to Mitchell Diploid, as indicated by higher biomass. Higher endogenous concentration of abscisic acid (ABA) and greater expression levels of ABA biosynthetic genes and lower expression levels of ABA catabolic genes in Haishishenlou than in Mitchell Diploid were observed when challenged by saline-alkaline stress, suggesting that a higher concentration of ABA may underpin the greater tolerance of Haishishenlou to saline-alkaline stress than that of Mitchell Diploid. Under saline-alkaline conditions, Haishishenlou displayed higher chlorophyll content, photosynthetic rates, Pro and soluble sugars content, as well as higher activities of catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD), and a lower Na+/K+ ratio. Exogenous application of ABA alleviated the growth inhibition induced by saline-alkaline stress, promoted the accumulation of proline and soluble sugar, reduced reactive oxygen species (ROS) content and Na+/K+ ratio, and improved antioxidative capacity. These results indicated that a higher endogenous concentration of ABA may underpin the greater tolerance of petunia seedlings to saline-alkaline stress, and exogenous ABA improves the saline-alkaline tolerance of petunia seedlings. • Growth of Haishishenlou was less inhibited by saline-alkaline compared to Mitchell due to higher endogenous abscisic acid. • Exogenous application of abscisic acid alleviates saline-alkaline-induced inhibition by reducing the Na+/K+ ratio. • Exogenous application of abscisic acid alleviates saline-alkaline-induced inhibition by enhancing osmotic regulation. • Exogenous application of abscisic acid alleviates saline-alkaline-induced inhibition by improving antioxidative capacity. [ABSTRACT FROM AUTHOR]

Petunia (Petunia × hybrida Hort. Vilm.-Andr.) is a well-suited plant for sustainable landscape issues in borderline areas with irrigation with saline water. Salicylic acid (SA) as a modulator performs an imperative function in modulating plant salt tolerance. However, there are a few reports on the effect of SA on petunia plants irrigated with saline water. During the 2022/2023 season, a factorial pot experiment in a randomized complete block design was carried out in Riyadh, Saudi Arabia, to assess the effect of SA concentration (0, 500, 1000, 2000 mgL−1) on petunia plant growth, flowering, ion content, chlorophyll level, and proline concentration under irrigation with salty water (230, 1500, 3000 mgL−1). Saline water up to 3000 mgL−1 dramatically reduced plant growth, chlorophyll, ions, and flowering attributes, while the contrary was observed in proline and sodium concentrations as compared to the control treatments (irrigation with tap water). Foliar spraying with 1000 mgL−1 SA considerably boosted plant growth and flowering as well as chlorophyll, proline, and ion content compared to untreated plants under such salinity levels. Alternatively, the application of 1000 mgL−1 to normal or salinized water significantly decreased the Na content in non-treated plants under such a salinity level. Accordingly, using 1000 mgL−1 of SA under salt stress conditions could be a useful technique to lessen the mutilation induced by the use of salinized water in the era of climate change.

Low-temperature stress limits plant growth, and reduces aesthetics of many ornamental plants. Plants have developed different adaptive mechanisms to cope with low-temperature stress, in which NAC transcription factor family members playing an important role in low-temperature tolerance. However, their roles in petunia in response to low temperature are still largely unknown. Here, we found that a NAC transcription factor, namely, PhNH10, negatively regulates low-temperature response in petunia. PhNH10-silenced and -CRISPR/Cas9 mutant plants displayed higher survival rate, anthocyanin content and abscisic acid concentration than PhNH10-overexpression and wild-type plants under low-temperature condition. PhNH10 can directly bind to the PhABA8ox promoter to active its expression, which further promotes the abscisic acid catabolism, while silencing of PhABA8ox increased the ABA concentration and low-temperature tolerance. In addition, PhNH10 interact with a low-temperature-related E2 ubiquitin-conjugating enzyme, PhUBC2-1, which in turn inhibited the binding capacity of PhNH10 on PhABA8ox promoter. Our research has elucidated an extensive mechanistic network underlying the PhNH10-mediated regulation of low-temperature response in petunia. This finding not only presents a new viewpoint in understanding the low-temperature tolerance mechanisms but also delineates a promising pathway for transgenic petunia with improved low-temperature resistance., (© 2024 John Wiley & Sons Ltd.)

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. [ABSTRACT FROM AUTHOR]

Several families of transcription factors (TFs) control the progression of senescence. Many key TFs belonging to the WRKY family have been described to play crucial roles in the regulation of leaf senescence, mainly in Arabidopsis thaliana. However, little is known about senescence-associated WRKY members in floricultural species. Delay of senescence in leaves and petals of Petunia hybrida, a worldwide ornamental crop are highly appreciated traits. In this work, starting from 28 differentially expressed WRKY genes of A. thaliana during the progression of leaf senescence, we identified the orthologous in P. hybrida and explored the expression profiles of 20 PhWRKY genes during the progression of natural (age-related) leaf and corolla senescence as well as in the corollas of flowers undergoing pollination-induced senescence. Simultaneous visualization showed consistent and similar expression profiles of PhWRKYs during natural leaf and corolla senescence, although weak expression changes were observed during pollination-induced senescence. Comparable expression trends between PhWRKYs and the corresponding genes of A. thaliana were observed during leaf senescence, although more divergence was found in petals of pollinated petunia flowers. Integration of expression data with phylogenetics, conserved motif and cis-regulatory element analyses were used to establish a list of candidates that could regulate more than one senescence process. Our results suggest that several members of the WRKY family of TFs are tightly linked to the regulation of senescence in P. hybrida. [ABSTRACT FROM AUTHOR]

Deficiency or excess of iron (Fe) and improper medium pH will inhibit the growth and development of plants, reduce the transfer and utilization of energy from the root to the leaf, and affect the utilization efficiency of inorganic nutrients. The most common symptom of Fe deficiency in plants is chlorosis of the young leaves. In this study, the effects of the iron source, in combination with the medium pH, on plant growth and development, plant pigment synthesis, and nutrient uptake in a model plant Petunia hybrida cultured in vitro were investigated. Iron sulfate (FeSO4·7H2O) or iron chelated with ethylenediaminetetraacetic acid (Fe-EDTA) were supplemented to the MNS (a multipurpose nutrient solution) medium at a concentration of 2.78 mg·L−1 Fe, and the treatment without any Fe was used as the control. The pH of the agar-solidified medium was adjusted to either 4.70, 5.70, or 6.70 before autoclaving. The experiment was carried out in an environmentally controlled culture room with a temperature of 24 °C with 100 µmol·m−2·s−1 photosynthetic photon flux density (PPFD) supplied by white light emitting diodes (LEDs) during a photoperiod of 16 h a day, 18 °C for 8 h a day in the dark, and 70% relative humidity. Regardless of the Fe source including the control, the greatest number of leaves was observed at pH 4.70. However, the greatest lengths of the leaf and root were observed in the treatment with Fe-EDTA combined with pH 5.70. The contents of the chlorophyll, carotenoid, and anthocyanin decreased with increasing medium pH, and contents of these plant pigments were positively correlated with the leaf color. The highest soluble protein content and activities of APX and CAT were observed in the Fe-EDTA under pH 5.70. However, the GPX activity was the highest in the control under pH 4.70. In addition, the highest contents of ammonium (NH4+) and nitrate (NO3−) were measured in the FeSO4-4.7 and EDTA-5.7, respectively. More than that, the treatment of Fe-EDTA combined with pH 5.70 (EDTA-5.7) enhanced nutrient absorption, as proven by the highest tissue contents of P, K, Ca, Mg, Fe, and Mn. The genes' ferric reduction oxidase 1 and 8 (PhFRO1 and PhFRO8), iron-regulated transporter 1 (PhIRT1), nitrate transporter 2.5 (PhNRT2.5), and deoxyhypusine synthase (PhDHS) were expressed at the highest levels in this treatment as well. In the treatment of EDTA-5.7, the reduction and transport of chelated iron in P. hybrida leaves were enhanced, which also affected the transport of nitrate and catalyzed chlorophyll level in leaves. In conclusion, when the medium pH was adjusted to 5.70, supplementation of chelated Fe-EDTA was more conducive to promoting the growth and development of, and absorption of mineral nutrients by, the plant and the expression of related genes in the leaves. [ABSTRACT FROM AUTHOR]

Emissions of volatile organic compounds (VOCs) from plants are critical for processes such as pollination and seed dispersal. They also serve as a defense mechanism against parasites, pathogens, and herbivores and contribute to the pleasant smells of fruits, vegetables, flowers, and leaves. These compounds are primarily made up of phenylpropanoids/benzenoids, terpenoids, fatty acid derivatives, and amino acid derivatives. There has been considerable advancement in understanding the biosynthesis of VOCs over the past decades, but the specifics of how these compounds exit the cells and enter the environment remain less clear. Historically, it was believed that VOCs diffused passively out of cells. However, recent research involving petunia flowers has shed light on an active biological process involving an ABC transporter that aids in the emission of volatiles. In our study utilizing Petunia hybrida, which emits a high volume of volatiles derived from phenylalanine (Phe), we aimed to elucidate the mechanism of VOC release and the potential involvement of the cuticle and non-specific lipid transfer proteins in this process. Employing reverse-genetic and chemical methodologies, we identified a cell-wall localized non-specific lipid transfer protein (nsLTP), PhnsLTP3, that plays a part in transporting VOCs through the cell wall to the cuticle. Inhibiting PhnsLTP3 led to a decrease in VOC emissions and altered the distribution of VOCs, resulting in fewer of them reaching the cuticle, without affecting the total VOC content. Our findings also reveal that while the cuticle significantly hinders the movement of VOCs, it acts as a pivotal sink and concentrator for these compounds. Intracellular VOC accumulation can trigger a feedback mechanism that reduces their biosynthesis by downregulating the supply of the Phe precursor, a protective measure against the toxicity of these hydrophobic substances. This research offers new avenues for the metabolic engineering of VOCs to enhance desired fragrances or diminish the release of unwanted odors from plants. [ABSTRACT FROM AUTHOR]

Background: Salinity stress is one of the most important rising problems worldwide. It significantly reduces plant growth and development, mainly by provoking excessive uptake of ions such as Na+. The Salt Overly Sensitive (SOS) machinery is a well-known signaling pathway that help plants to maintain ion homeostasis by reducing Na+ accumulation in plant cells. Overexpression of key components of this pathway has been reported to increase salinity stress tolerance in some plant species. Methods and results: In this study, SOS3 cDNA isolated from Arabidopsis seedlings was transferred into the Petunia genome by two common plant transformation methods, Agrobacterium and biolistic gun. Transgene integration and expression in putative lines were evaluated by PCR and RT-PCR techniques. In vitro and greenhouse evaluation of transgenic plants for salt tolerance showed that, compared to the wild type, transgenic plants overexpressing AtSOS3 gene exhibit enhanced salt tolerance in response to high NaCl concentrations. Conclusions: These results not only demonstrate the potential of SOS pathway components to improve salt tolerance in Petunia, but also provide more evidence for functional conservation of the SOS salt tolerance signaling pathway among different plant families. [ABSTRACT FROM AUTHOR]

The WRKY transcription factors (TFs) participate in various physiological, growth and developmental processes of plants. In our study, a total of 79 WRKY family members were identified and classified into three groups (Group I, Group IIa–e, and Group III) based on phylogenetic and conservative domain analyses. Conserved motif analysis showed that seven WRKYGQK domains changed. The promoter sequence analysis suggested that there were multiple stress- and hormone-related cis-regulatory elements in the promoter regions of PhWRKY genes. Expression patterns of PhWRKYs based on RNA-seq data revealed their diverse expression profiles in five tissues and under different treatments. Subcellular localization analysis showed that PhWRKY71 was located in the nucleus. In addition, overexpression of PhWRKY71 caused a significant increase in branch number. This indicated that PhWRKY71 played a critical role in regulating the shoot branching of Petuniahybrida. The above results lay the foundation for further revealing the functions of PhWRKY genes. [ABSTRACT FROM AUTHOR]

Pollen tube growth depends on several complex processes, including exo/endocytosis, cell wall biogenesis, intracellular transport, and cell signaling. Our previous results provided evidence that calreticulin (CRT)—a prominent calcium (Ca2+)-buffering molecular chaperone in the endoplasmic reticulum (ER) lumen—is involved in pollen tube formation and function. We previously cloned and characterized the CRT gene belonging to the CRT1/2 subgroup from Petunia hybrida (PhCRT1/2), and found that post-transcriptional silencing of PhCRT1/2 expression strongly impaired pollen tube growth in vitro. Here, we report cloning of a new PhCRT3a homolog; we identified the full-length cDNA sequence and described its molecular characteristics and phylogenetic relationships to other plant CRT3 genes. Using an RNA interference (RNAi) strategy, we found that knockdown of PhCRT3a gene expression caused numerous defects in the morphology and ultrastructure of cultivated pollen tubes, including disorganization of the actin cytoskeleton and loss of cytoplasmic zonation. Elongation of siPhCRT3a pollen tubes was disrupted, and some of them ruptured. Our present data provide the first evidence that PhCRT3a expression is required for normal pollen tube growth. Thus, we discuss relationships between diverse CRT isoforms in several interdependent processes driving the apical growth of the pollen tube, including actomyosin-dependent cytoplasmic streaming, organelle positioning, vesicle trafficking, and cell wall biogenesis. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: Petunia floral scent production and emission is highly regulated, with a major role for the transcription factor ODORANT 1 (ODO1). This paper describes the ODO1‐regulated gene network of petunia, which extends to branches involved in the production of phenylpropanoid intermediates and S‐adenosylmethionine biosynthesis, along with the identification of an ODO1‐binding motif. [ABSTRACT FROM AUTHOR]

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.

β-Ionone, sustainably derived from Petunia hybrida as a natural bioresource, was identified as a lead compound for integrated aphid management. A series of β-ionone derivatives containing ester groups were designed and synthesized for the purpose of discovering renewable botanical-based products. The odorant-binding protein (OBP) binding test indicated that β-ionone and its derivatives displayed binding affinities with Acyrthosiphon pisum OBP9 (ApisOBP9) and Harmonia axyridis OBP15 (HaxyOBP15). Bioactivity assays revealed that most β-ionone derivatives exhibited a higher repellent activity than that of β-ionone. β-Ionone and derivatives 4g and 4l displayed attractiveness to H. axyridis . Specifically, 4g was a highly promising derivative, possessing good repellent activity against A. pisum and attractiveness to H. axyridis . Molecular dynamics simulations revealed that integrating the hydrophobic ester group into the β-ionone framework strengthened the van der Waals interactions of 4g with ApisOBP9/HaxyOBP15, improving the binding affinity with OBPs and producing higher push-pull activity than β-ionone; 4g also had low toxicity toward nontarget organisms. Thus, 4g is a potential ecofriendly, botanical-based option for aphid management.

Background: Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca2+). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca2+-binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca2+ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development. Results: Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca2+ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca2+ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca2+ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. Conclusion: Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca2+ homeostasis during pollen grains formation. This Ca2+-buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca2+ homeostasis are strictly required during the multi-step process of pollen development. [ABSTRACT FROM AUTHOR]

SUMMARY: 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. Significance Statement: An increase in phenylalanine levels is a frequent target of metabolic engineering strategies for efficient production of phenylalanine‐derived metabolites. Phenylalanine is produced predominantly in plastids via the shikimate pathway and is subject to complex transcriptional and post‐transcriptional regulation, which occurs not only within the pathway but also upstream in the pentose phosphate pathway. [ABSTRACT FROM AUTHOR]

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.

An efficient plant regeneration protocol was developed from in vitro germinated seeds of Petunia hybrida an ornamentally important plant in the family Solanaceae. Shoot tip and node explants of Petunia hybrida were cultured on MS basal medium supplemented with different concentrations and combinations of Benzyl amino purine (BAP), 1-Naphthaleneacetic acid (NAA), Indole-3-butyric acid (IBA) and Gibberellic acid (GA3). The highest shoot length was obtained from MS medium supplemented with 1 mg/l BAP + 1 mg/l NAA. The highest shoot number (3 shoots/explant) were obtained from MS medium supplemented with 0.6 mg/l BAP + 0.5 mg/l IBA. The isolated shoots were transferred to MS basal medium supplemented with different concentrations of GA3 ranging from 0.05, 0.2, 0.5 and 1 mg/l for shoot elongation. The highest shoot length (5.75 cm) was recorded from the MS medium supplemented with 0.2 mg/l GA3 +0.2 mg/l BAP. Rooting of regenerated shoots were achieved on MS medium supplemented with 0.1-1 mg/1 IBA and NAA. The regenerated shoots with well developed roots were successfully acclimatized and established in pots containing sterilized peat moss and grown under laboratory conditions with 70% survival rates.

Fungal endophytes can improve the growth of plants and are considered as an effective tool for metal remediation. Trametes hirsuta was tested for Cd and Pb tolerance, further Petunia hybrida Vilm. was infected at seedling stage by T. hirsuta and was tested to see the impact of Cd and Pb toxicity. On potato dextrose agar media plates T. hirsuta showed growth till 500 mg/L of Cd and 1000 mg/L of Pb, respectively. P. hybrida inoculated with fungus showed increased fresh biomass of 33.1, 20.1 and 51.8% in roots, stems and leaves, respectively, for Cd exposed plant, along with 53.5% higher chlorophyll content as compared to their respective non-inoculated plants. In case of Pb, plants inoculated with fungus showed 39% higher stem and 42% root fresh biomass along with 64% higher chlorophyll content. Significantly higher Cd and Pb accumulation in the roots and stems was observed in case of T. hirsuta infected plants as compared to their respective non-infected ones. While antioxidant production reduced significantly in case of T. hirsuta infected plants. Our results strongly suggest that T. hirsuta is capable to promote P. hybrida growth and also improves the extraction of Cd and Pb. Therefore, endophytic fungus assisted phytoremediation can have great potential to restore contaminated soils. [ABSTRACT FROM AUTHOR]

[Display omitted] An efficient protocol for in-vitro propagation of an important ornamental crop, Petunia hybrida Vilm. Cv. "Bravo" was developed. The explants that were used to carry out the experiment were Leaf segments, nodal segments and shoot tips. Nodal segments recorded highest per cent asepsis followed by shoot tips and leaf segments. Asepsis was found to be highest when the explants were sterilized with Fungicide (Carbendazim) 0.02% for the duration of 30 min followed by 0.1% HgCl 2 for duration of 10 min and then ethanol 70% for 10 s. Longer duration of the sterilant treatment showed more necrotic effects on the explants, thus mercuric chloride treatment when given for 5 min proved to be more effective in terms of survival of the explants. Maximum establishment per cent was recorded in Murashige and Skoog (MS) media fortified with BAP (1.5 mg L−1) and IBA (0.5 mg L−1) in shoot tips and nodal segments, i.e. 97.90 and 95.74% respectively. Callus was efficiently induced and developed when PGR amalgamation of BAP (0.1 mg L−1) and 2,4-D (1.5mg L−1) was used. Kinetin at the concentration of 2.0 mg L−1 along with IBA at 0.5mg L−1 recorded highest callus regeneration in both leaf and internodal segment derived callus. Maximum proliferation percent of shoots (97.90%), highest number of shoots (20.50 explant−1) and maximum length of shoot (2.70 cm) was recorded in PGR combination of IBA and BAP both at 0.5 mg L−1 concentration level. Rhizogenesis was recorded to be highest in the MS media containing IBA 1.00 mg L−1. Best hardening media which recorded maximum survival per cent 92.50% was noticed on the media formulation comprised of equal ratio of perlite and vermiculite mix, under poly house conditions. [ABSTRACT FROM AUTHOR]

Venation is a common anthocyanin pattern displayed in flowers that confers important ornamental traits to plants. An anthocyanin-related R2R3-MYB transcription factor, DPL, has been proposed to regulate corolla tube venation in petunia plants. Here, however, we provide evidence redefining the role of DPL in petunia. A CRISPR/Cas9-mediated mutation of DPL resulted in the absence of the vein-associated anthocyanin pattern above the abaxial surface of the flower bud, but not corolla tube venation, thus indicating that DPL did not regulate the formation of corolla tube venation. Alternately, quantitative real-time PCR analysis demonstrated that the spatiotemporal expression pattern of another R2R3-MYB gene, AN4, coincided with the formation of corolla tube venation in petunia. Furthermore, overexpression of AN4 promoted anthocyanin accumulation by increasing the expression of anthocyanin biosynthesis genes. CRISPR/Cas9-mediated mutation of AN4 led to an absence of corolla tube venation, suggesting that this gene in fact determines this key plant trait. Taken together, the results presented here redefine the prime regulator of corolla tube venation, paving the way for further studies on the molecular mechanisms underlying the various venation patterns in petunia. [ABSTRACT FROM AUTHOR]

Summary: The intimate association of host and fungus in arbuscular mycorrhizal (AM) symbiosis can potentially trigger induction of host defence mechanisms against the fungus, implying that successful symbiosis requires suppression of defence.We addressed this phenomenon by using AM‐defective vapyrin (vpy) mutants in Petunia hybrida, including a new allele (vpy‐3) with a transposon insertion close to the ATG start codon. We explore whether abortion of fungal infection in vpy mutants is associated with the induction of defence markers, such as cell wall alterations, accumulation of reactive oxygen species (ROS), defence hormones and induction of pathogenesis‐related (PR) genes.We show that vpy mutants exhibit a strong resistance against intracellular colonization, which is associated with the generation of cell wall appositions (papillae) with lignin impregnation at fungal entry sites, while no accumulation of defence hormones, ROS or callose was observed. Systematic analysis of PR gene expression revealed that several PR genes are induced in mycorrhizal roots of the wild‐type, and even more in vpy plants. Some PR genes are induced exclusively in vpy mutants.Our results suggest that VPY is involved in avoiding or suppressing the induction of a cellular defence syndrome that involves localized lignin deposition and PR gene induction. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Main conclusion: Sodium butyrate applied to Petunia hybrida seeds under a long-day photoperiod has a negative impact (reduced seedling length, decreased production of photosynthetic pigments, and accumulation of DNA damage) on early seedling development, whereas its administration under dark/light conditions (complete dark conditions for 5 days followed by exposure to long-day photoperiod for 5 days) bypasses some of the adverse effects. Genotoxic stress impairs plant development. To circumvent DNA damage, plants activate DNA repair pathways in concert with chromatin dynamics. These are essential during seed germination and seedling establishment, and may be influenced by photoperiod variations. To assess this interplay, an experimental design was developed in Petunia hybrida, a relevant horticultural crop and model species. Seeds were treated with different doses of sodium butyrate (NaB, 1 mM and 5 mM) as a stress agent applied under different light/dark conditions throughout a time period of 10 days. Phenotypic (germination percentage and speed, seedling length, and photosynthetic pigments) and molecular (DNA damage and gene expression profiles) analyses were performed to monitor the response to the imposed conditions. Seed germination was not affected by the treatments. Seedling development was hampered by increasing NaB concentrations applied under a long-day photoperiod (L) as reflected by the decreased seedling length accompanied by increased DNA damage. When seedlings were grown under dark conditions for 5 days and then exposed to long-day photoperiod for the remaining 5 days (D/L), the damaging effects of NaB were circumvented. NaB exposure under L conditions resulted in enhanced expression of HAT/HDAC (HISTONE ACETYLTRANSFERASES/HISTONE DEACTEYLASES) genes along with repression of genes involved in DNA repair. Differently, under D/L conditions, the expression of DNA repair genes was increased by NaB treatment and this was associated with lower levels of DNA damage. The observed DNA damage and gene expression profiles suggest the involvement of chromatin modification- and DNA repair-associated pathways in response to NaB and dark/light exposure during seedling development. [ABSTRACT FROM AUTHOR]