CRISPR/Cas9-mediated mutagenesis of FT/TFL1 in petunia improves plant architecture and early flowering.

Petunias are renowned ornamental species widely cultivated as pot plants for their aesthetic appeal both indoors and outdoors. The preference for pot plants depends on their compact growth habit and abundant flowering. While genome editing has gained significant popularity in many crop plants in addressing growth and development and abiotic and biotic stress factors, relatively less emphasis has been placed on its application in ornamental plant species. Genome editing in ornamental plants opens up possibilities for enhancing their aesthetic qualities, offering innovative opportunities for manipulating plant architecture and visual appeal through precise genetic modifications. In this study, we aimed to optimize the procedure for an efficient genome editing system in petunia plants using the highly efficient multiplexed CRISPR/Cas9 system. Specifically, we targeted a total of six genes in Petunia which are associated with plant architecture traits, two paralogous of FLOWERING LOCUS T (PhFT) and four TERMINAL FLOWER-LIKE1 (PhTFL1) paralogous genes separately in two constructs. We successfully induced homogeneous and heterogeneous indels in the targeted genes through precise genome editing, resulting in significant phenotypic alterations in petunia. Notably, the plants harboring edited PhTFL1 and PhFT exhibited a conspicuously early flowering time in comparison to the wild-type counterparts. Furthermore, mutants with alterations in the PhTFL1 demonstrated shorter internodes than wild-type, likely by downregulating the gibberellic acid pathway genes PhGAI, creating a more compact and aesthetically appealing phenotype. This study represents the first successful endeavor to produce compact petunia plants with increased flower abundance through genome editing. Our approach holds immense promise to improve economically important potting plants like petunia and serve as a potential foundation for further improvements in similar ornamental plant species.

Plant-specific calreticulin is localized in the nuclei of highly specialized cells in the pistil-new observations for an old hypothesis.

One of the first cellular locations of the calreticulin (CRT) chaperone in eukaryotic cells, apart from its obvious localization in the endoplasmic reticulum (ER), was the cell nucleus (Opas et al. 1991). The presence of CRT has been detected inside the nucleus and in the nuclear envelope of animal and plant cells, and a putative nuclear localization signal (NLS) in the CRT amino acid sequence has been mapped in several animal and plant species. Over the last 30 years, other localization sites of this protein outside the ER and cell nucleus have also been discovered, suggesting that CRT is a multifunctional Ca2+-binding protein widely found in various cell types. In our previous studies focusing on plant developmental biology, we have demonstrated the presence of CRT inside and outside the ER in highly specialized plant cells, as well as the possibility of CRT localization in the cell nucleus. In this paper, we present a detailed analysis of immunocytochemical localization of CRT inside nuclei of the pistil transmission tract somatic cells before and after pollination. We show a similar pattern of the nuclear CRT localization in relation to exchangeable Ca2+ for two selected species of angiosperms, dicotyledonous Petunia and monocot Haemanthus, that differ in anatomical structure of the pistil and discuss the potential role of CRT in the cell nucleus.

Comprehensive Analyses of Four PhNF-YC Genes from Petunia hybrida and Impacts on Flowering Time.

Nuclear Factor Y (NF-Y) is a class of heterotrimeric transcription factors composed of three subunits: NF-A, NF-YB, and NF-YC. NF-YC family members play crucial roles in various developmental processes, particularly in the regulation of flowering time. However, their functions in petunia remain poorly understood. In this study, we isolated four PhNF-YC genes from petunia and confirmed their subcellular localization in both the nucleus and cytoplasm. We analyzed the transcript abundance of all four PhNF-YC genes and found that PhNF-YC2 and PhNF-YC4 were highly expressed in apical buds and leaves, with their transcript levels decreasing before flower bud differentiation. Silencing PhNF-YC2 using VIGS resulted in a delayed flowering time and reduced chlorophyll content, while PhNF-YC4-silenced plants only exhibited a delayed flowering time. Furthermore, we detected the transcript abundance of flowering-related genes involved in different signaling pathways and found that PhCO, PhGI, PhFBP21, PhGA20ox4, and PhSPL9b were regulated by both PhNF-YC2 and PhNF-YC4. Additionally, the transcript abundance of PhSPL2, PhSPL3, and PhSPL4 increased only in PhNF-YC2-silenced plants. Overall, these results provide evidence that PhNF-YC2 and PhNF-YC4 negatively regulate flowering time in petunia by modulating a series of flowering-related genes.

Dorsoventrally asymmetric expression of miR319/TCP generates dorsal-specific venation patterning in petunia corolla tube.

Vein-associated pigmentation (venation) is a type of floral coloration adopted by plants to attract pollinators. Several petunia (Petunia hybrida) lines generate dorsoventrally asymmetric venation patterning of the corolla tube, in which venation is only present in the dorsal tube. The molecular mechanism underlying this trait is unknown. Here, we demonstrate that miR319 is preferentially expressed in the dorsal corolla tube, leading to dorsoventrally asymmetric expression of its target genes. Transgenic lines overexpressing phy-miR319a generated uniform venation patterning of the corolla tube. Knockout of TCP genes targeted by miR319 promoted venation patterning in the ventral and dorsal tube, while overexpression of the miR319 target gene, PhTCP6, completely inhibited corolla tube venation patterning. In addition, miR319-targeted TCPs negatively regulated venation patterning, probably by repressing the regulator of venation patterning, AN4. Together, our data demonstrate that asymmetric expression of miR319 promotes venation patterning in the petunia dorsal tube alone by repressing the expression of its target TCP genes, which negatively regulate corolla tube venation patterning. These findings provide novel insights into how the dorsoventrally asymmetric distribution of venation patterning is established in zygomorphic flowers.

Enhancing cryopreservation survival in Petunia × Calibrachoa 'Light Yellow' callus: Insights into material characteristics and genetic integrity.

Petunia × Calibrachoa 'Light Yellow' (× Petchoa 'Light Yellow') is a kind of perennial herbaceous flower obtained through intergeneric hybridization of Petunia and Calibrachoa with high ornamental value and wide application, facing challenges in seed acquisition. Expanding propagation through tissue culture is an economically efficient means. Hence, establishing an effective procedure for the storage of callus is essential for × Petchoa 'Light Yellow'. Cryopreservation is an effective method for the in vitro propagation and long-term preservation of × Petchoa 'Light Yellow' germplasms. For formulating the optimization of the vitrification procedure, first, an orthogonal experimental design was employed to pinpoint critical steps in the vitrification protocol (pre-culture, osmoprotection, dehydration, and dilution) for Petunia × Calibrachoa callus tissues and then five additional factors (pre-culture, osmoprotection I and II, dehydration, and dilution) were optimized to further reduce the sample water content and enhance cell viability levels. The vitrification procedure was described as follows: callus tissues were precultured in MS solid medium with 0.3 M sucrose for 5 d, incubated with osmoprotection solution I and II for 15 min at 25 °C, respectively, cryoprotected with PVS2 for 30 min at 0 °C, and rapidly immersed in liquid nitrogen. Cryopreserved callus tissues were then diluted in MS liquid medium with 1.2 M sucrose for 20 min at 25 °C and recovered on MS solid medium with 0.5 mg/L 6-BA and 0.1 mg/L NAA, and sucrose. The cell viability measured by TTC staining was approximately 16 %-18 % after 72 h-recovery. Following 45 days, the relative survival of callus reached up to 49.48 %. Furthermore, EST-SSR analysis showed no significant difference in the genetic stability of cryopreserved callus compared to the control. Based on the cryopreservation of × Petchoa 'Light Yellow' callus, we further evaluated the response of callus water contents to the osmotic stress in the optimized and original protocols (CK) for a higher cryopreservation survival. A comparative analysis of water content demonstrated that the procedure of gradual and gentle dehydration significantly improved water content and cell survival. Ultrastructural changes between cryopreserved and non-cryopreserved callus were examined and high vacuolation emerged as a key determinant, indicating its substantial impact on the low survival of cryopreserved cells, which should help us to understand the effectiveness of osmotic protectants in dehydration.

Commodity risk assessment of Petunia spp. and Calibrachoa spp. unrooted cuttings from Guatemala.

The European Commission requested the EFSA Panel on Plant Health to evaluate the probability of entry of pests (likelihood of pest freedom at entry), including both, regulated and non-regulated pests, associated with unrooted cuttings of the genera Petunia and Calibrachoa produced under physical isolation in Guatemala. The relevance of any pest for this opinion was based on evidence following defined criteria, based on the methodology used for high-risk plants adapted for the specificity of this assessment. Nineteen EU regulated pests (Bemisia tabaci, pepper golden mosaic virus, pepper huasteco yellow vein virus, tomato severe leaf curl virus, tomato yellow leaf curl virus, tomato spotted wilt virus, Liriomyza huidobrensis, Liriomyza sativae, Liriomyza trifolii, Bactericera cockerelli, Eotetranichus lewisi, Epitrix subcrinita, Epitrix cucumeris, Helicoverpa zea, Chloridea virescens, Spodoptera ornithogalli, Ralstonia solanacearum, Ralstonia pseudosolanacearum, Xanthomonas vesicatoria) and one EU non-regulated (Phenacoccus solenopsis) pest fulfilled all relevant criteria and were selected for further evaluation. For these pests, the risk mitigation measures proposed in the technical dossier from Guatemala were evaluated taking into account the possible limiting factors, and an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. The limited and partially conflicting information provided in the dossier contributes to the wide estimates of pest freedom. The estimated degree of pest freedom varies among the pests evaluated, with Ralstonia spp. (R. solanacearum and R. pseudosolanacearum) being the pest most frequently expected on the imported cuttings. The expert knowledge elicitation indicated, with 95% certainty, that between 9916 and 10,000 bags containing unrooted cuttings per 10,000 would be free of Ralstonia spp.

Rhythmic histone acetylation acts in concert with day-night oscillation of the floral volatile metabolic network.

The biosynthesis of specialized metabolites is strictly regulated by environmental inputs such as the day-night cycle, but the underlying mechanisms remain elusive. In Petunia hybrida cv. Mitchell flowers, the biosynthesis and emission of volatile compounds display a diurnal pattern with a peak in the evening to attract nocturnal pollinators. Using petunia flowers as a model system, we found that chromatin level regulation, especially histone acetylation, plays an essential role in mediating the day-night oscillation of the biosynthetic gene network of specialized metabolites. By performing time-course chromatin immunoprecipitation assays for histone modifications, we uncovered that a specific group of genes involved in the regulation, biosynthesis, and emission of floral volatile compounds, which displays the greatest magnitude in day-night oscillating gene expression, is associated with highly dynamic histone acetylation marks H3K9ac and H3K27ac. Specifically, the strongest oscillating genes featured a drastic removal of histone acetylation marks at night, potentially to shut down the biosynthesis of floral volatile compounds during the morning when they are not needed. Inhibiting daytime histone acetylation led to a compromised evening induction of these genes. Overall, our study suggested an active role of chromatin modification in the diurnal oscillation of specialized metabolic network.

Resistance to Seven Site-Specific Fungicides in Botrytis cinerea from Greenhouse-Grown Ornamentals.

The fungal pathogen Botrytis cinerea is a notorious problem on many floriculture greenhouse hosts including petunia, geranium, and poinsettia; these key crops contribute to the $6.43 billion U.S. ornamental industry. While growers use cultural strategies to reduce relative humidity and free moisture to limit Botrytis blight, fungicides remain a primary component of control programs. Isolates (n = 386) of B. cinerea sampled from symptomatic petunia, geranium, and poinsettia in Michigan greenhouses from 2018 to 2021 were screened for resistance to eight fungicides belonging to seven Fungicide Resistance Action Committee (FRAC) groups. Single-spored isolates were subjected to a germination-based assay using previously defined discriminatory doses of each fungicide. Resistance was detected to thiophanate-methyl (FRAC 1; 94%), pyraclostrobin (FRAC 11; 80%), boscalid (FRAC 7; 67%), iprodione (FRAC 2; 65%), fenhexamid (FRAC 17; 38%), cyprodinil (FRAC 9; 38%), fludioxonil (FRAC 12; 21%), and fluopyram (FRAC 7; 13%). Most isolates (63.5%) were resistant to at least four FRAC groups, with 8.7% of all isolates demonstrating resistance to all seven FRAC groups tested. Resistance frequencies for each fungicide were similar among crops, production regions, and growing cycles but varied significantly for each greenhouse. Phenotypic diversity was high, as indicated by the 48 different fungicide resistance profiles observed. High frequencies of resistance to multiple fungicides in B. cinerea populations from floriculture hosts highlight the importance of sustainable and alternative disease management practices for greenhouse growers.

Petunia as a model for MYB transcription factor action under salt stress.

Salinity is a current and growing problem, affecting crops worldwide by reducing yields and product quality. Plants have different mechanisms to adapt to salinity; some crops are highly studied, and their salinity tolerance mechanisms are widely known. However, there are other crops with commercial importance that still need characterization of their molecular mechanisms. Usually, transcription factors are in charge of the regulation of complex processes such as the response to salinity. MYB-TFs are a family of transcription factors that regulate various processes in plant development, and both central and specialized metabolism. MYB-TFs have been studied extensively as mediators of specialized metabolism, and some are master regulators. The influence of MYB-TFs on highly orchestrated mechanisms, such as salinity tolerance, is an attractive research target. The versatility of petunia as a model species has allowed for advances to be made in multiple fields: metabolomic pathways, quality traits, stress resistance, and signal transduction. It has the potential to be the link between horticultural crops and lab models, making it useful in translating discoveries related to the MYB-TF pathways into other crops. We present a phylogenetic tree made with Petunia axillaris and Petunia inflata R2R3-MYB subfamily sequences, which could be used to find functional conservation between different species. This work could set the foundations to improve salinity resistance in other commercial crops in later studies.

Induced production of specialized steroids by transcriptional reprogramming in Petunia hybrida.

Plants produce specialized metabolites with defensive properties that are often synthesized through the coordinated regulation of metabolic genes by transcription factors in various biological contexts. In this study, we investigated the regulatory function of the transcription factor PhERF1 from petunia (Petunia hybrida), which belongs to a small group of ETHYLENE RESPONSE FACTOR (ERF) family members that regulate the biosynthesis of bioactive alkaloids and terpenoids in various plant lineages. We examined the effects of transiently overexpressing PhERF1 in petunia leaves on the transcriptome and metabolome, demonstrating the production of a class of specialized steroids, petuniolides, and petuniasterones in these leaves. We also observed the activation of many metabolic genes, including those involved in sterol biosynthesis, as well as clustered genes that encode new metabolic enzymes, such as cytochrome P450 oxidoreductases, 2-oxoglutarate-dependent dioxygenases, and BAHD acyltransferases. Furthermore, we determined that PhERF1 transcriptionally induces downstream metabolic genes by recognizing specific cis-regulatory elements in their promoters. This study highlights the potential of evolutionarily conserved transcriptional regulators to induce the production of specialized products through transcriptional reprogramming.

The nature and organization of satellite DNAs in Petunia hybrida, related, and ancestral genomes.

Introduction: The garden petunia, Petunia hybrida (Solanaceae) is a fertile, diploid, annual hybrid species (2n=14) originating from P. axillaris and P. inflata 200 years ago. To understand the recent evolution of the P. hybrida genome, we examined tandemly repeated or satellite sequences using bioinformatic and molecular cytogenetic analysis. Methods: Raw reads from available genomic assemblies and survey sequences of P. axillaris N (PaxiN), P. inflata S6, (PinfS6), P. hybrida (PhybR27) and the here sequenced P. parodii S7 (PparS7) were used for graph and k-mer based cluster analysis of TAREAN and RepeatExplorer. Analysis of repeat specific monomer lengths and sequence heterogeneity of the major tandem repeat families with more than 0.01% genome proportion were complemented by fluorescent in situ hybridization (FISH) using consensus sequences as probes to chromosomes of all four species. Results: Seven repeat families, PSAT1, PSAT3, PSAT4, PSAT5 PSAT6, PSAT7 and PSAT8, shared high consensus sequence similarity and organisation between the four genomes. Additionally, many degenerate copies were present. FISH in P. hybrida and in the three wild petunias confirmed the bioinformatics data and gave corresponding signals on all or some chromosomes. PSAT1 is located at the ends of all chromosomes except the 45S rDNA bearing short arms of chromosomes II and III, and we classify it as a telomere associated sequence (TAS). It is the most abundant satellite repeat with over 300,000 copies, 0.2% of the genomes. PSAT3 and the variant PSAT7 are located adjacent to the centromere or mid-arm of one to three chromosome pairs. PSAT5 has a strong signal at the end of the short arm of chromosome III in P. axillaris and P.inflata, while in P. hybrida additional interstitial sites were present. PSAT6 is located at the centromeres of chromosomes II and III. PSAT4 and PSAT8 were found with only short arrays. Discussion: These results demonstrate that (i) repeat families occupy distinct niches within chromosomes, (ii) they differ in the copy number, cluster organization and homogenization events, and that (iii) the recent genome hybridization in breeding P. hybrida preserved the chromosomal position of repeats but affected the copy number of repetitive DNA.

Varying the expression pattern of the strigolactone receptor gene DAD2 results in phenotypes distinct from both wild type and knockout mutants.

The action of the petunia strigolactone (SL) hormone receptor DAD2 is dependent not only on its interaction with the PhMAX2A and PhD53A proteins, but also on its expression patterns within the plant. Previously, in a yeast-2-hybrid system, we showed that a series of a single and double amino acid mutants of DAD2 had altered interactions with these binding partners. In this study, we tested the mutants in two plant systems, Arabidopsis and petunia. Testing in Arabidopsis was enabled by creating a CRISPR-Cas9 knockout mutant of the Arabidopsis strigolactone receptor (AtD14). We produced SL receptor activity in both systems using wild type and mutant genes; however, the mutants had functions largely indistinguishable from those of the wild type. The expression of the wild type DAD2 from the CaMV 35S promoter in dad2 petunia produced plants neither quite like the dad2 mutant nor the V26 wild type. These plants had greater height and leaf size although branch number and the plant shape remained more like those of the mutant. These traits may be valuable in the context of a restricted area growing system such as controlled environment agriculture.

Petunia dihydroflavonol 4-reductase is only a few amino acids away from producing orange pelargonidin-based anthocyanins.

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.

Flower Development in the Solanaceae.

Flower development is the process leading from a reproductive meristem to a mature flower with fully developed floral organs. This multi-step process is complex and involves thousands of genes in intertwined regulatory pathways; navigating through the FLOR-ID website will give an impression of this complexity and of the astonishing amount of work that has been carried on the topic (Bouché et al., Nucleic Acids Res 44:D1167-D1171, 2016). Our understanding of flower development mostly comes from the model species Arabidopsis thaliana, but numerous other studies outside of Brassicaceae have helped apprehend the conservation of these mechanisms in a large evolutionary context (Moyroud and Glover, Curr Biol 27:R941-R951, 2017; Smyth, New Phytol 220:70-86, 2018; Soltis et al., Ann Bot 100:155-163, 2007). Integrating additional species and families to the research on this topic can only advance our understanding of flower development and its evolution.In this chapter, we review the contribution that the Solanaceae family has made to the comprehension of flower development. While many of the general features of flower development (i.e., the key molecular players involved in flower meristem identity, inflorescence architecture or floral organ development) are similar to Arabidopsis, our main objective in this chapter is to highlight the points of divergence and emphasize specificities of the Solanaceae. We will not discuss the large topics of flowering time regulation, inflorescence architecture and fruit development, and we will restrict ourselves to the mechanisms included in a time window after the floral transition and before the fertilization. Moreover, this review will not be exhaustive of the large amount of work carried on the topic, and the choices that we made to describe in large details some stories from the literature are based on the soundness of the functional work performed, and surely as well on our own preferences and expertise.First, we will give a brief overview of the Solanaceae family and some of its specificities. Then, our focus will be on the molecular mechanisms controlling floral organ identity, for which extended functional work in petunia led to substantial revisions to the famous ABC model. Finally, after reviewing some studies on floral organ initiation and growth, we will discuss floral organ maturation, using the examples of the inflated calyx of the Chinese lantern Physalis and petunia petal pigmentation.

A single MYB transcription factor with multiple functions during flower development.

Members of the R2R3-MYB transcription factor subgroup 19 (SG19) have been extensively studied in multiple plant species using different silenced or mutated lines. Some studies have proposed a function in flower opening, others in floral organ development/maturation, or specialized metabolism production. While SG19 members are clearly key players during flower development and maturation, the resulting picture is complex, confusing our understanding in how SG19 genes function. To clarify the function of the SG19 transcription factors, we used a single system, Petunia axillaris, and targeted its two SG19 members (EOB1 and EOB2) by CRISPR-Cas9. Although EOB1 and EOB2 are highly similar, they display radically different mutant phenotypes. EOB1 has a specific role in scent emission while EOB2 has pleiotropic functions during flower development. The eob2 knockout mutants reveal that EOB2 is a repressor of flower bud senescence by inhibiting ethylene production. Moreover, partial loss-of-function mutants (transcriptional activation domain missing) show that EOB2 is also involved in both petal and pistil maturation through regulation of primary and secondary metabolism. Here, we provide new insights into the genetic regulation of flower maturation and senescence. It also emphasizes the function of EOB2 in the adaptation of plants to specific guilds of pollinators.

PhCHS5 and PhF3'5'H Genes Over-Expression in Petunia (Petunia hybrida) and Phalaenopsis (Phalaenopsis aphrodite) Regulate Flower Color and Branch Number.

Flower breeders are continually refining their methods for producing high-quality flowers. Phalaenopsis species are considered the most important commercially grown orchids. Advances in genetic engineering technology have provided researchers with new tools that can be used along with traditional breeding methods to enhance floral traits and quality. However, the application of molecular techniques for the breeding of new Phalaenopsis species has been relatively rare. In this study, we constructed recombinant plasmids carrying flower color-related genes, Phalaenopsis Chalcone synthase (PhCHS5) and/or Flavonoid 3',5'-hydroxylase (PhF3'5'H). These genes were transformed into both Petunia and Phalaenopsis plants using a gene gun or an Agrobacterium tumefaciens-based method. Compared with WT, 35S::PhCHS5 and 35S::PhF3'5'H both had deeper color and higher anthocyanin content in Petunia plants. Additionally, a phenotypic comparison with wild-type controls indicated the PhCHS5 or PhF3'5'H-transgenic Phalaenopsis produced more branches, petals, and labial petals. Moreover, PhCHS5 or PhF3'5'H-transgenic Phalaenopsis both showed deepened lip color, compared with the control. However, the intensity of the coloration of the Phalaenopsis lips decreased when protocorms were co-transformed with both PhCHS5 and PhF3'5'H. The results of this study confirm that PhCHS5 and PhF3'5'H affect flower color in Phalaenopsis and may be relevant for the breeding of new orchid varieties with desirable flowering traits.

Developmental and temporal changes in petunia petal transcriptome reveal scent-repressing plant-specific RING-kinase-WD40 protein.

In moth-pollinated petunias, production of floral volatiles initiates when the flower opens and occurs rhythmically during the day, for optimal flower-pollinator interaction. To characterize the developmental transcriptomic response to time of day, we generated RNA-Seq databases for corollas of floral buds and mature flowers in the morning and in the evening. Around 70% of transcripts accumulating in petals demonstrated significant changes in expression levels in response to the flowers' transition from a 4.5-cm bud to a flower 1 day postanthesis (1DPA). Overall, 44% of the petal transcripts were differentially expressed in the morning vs. evening. Morning/evening changes were affected by flower developmental stage, with a 2.5-fold larger transcriptomic response to daytime in 1DPA flowers compared to buds. Analyzed genes known to encode enzymes in volatile organic compound biosynthesis were upregulated in 1DPA flowers vs. buds-in parallel with the activation of scent production. Based on analysis of global changes in the petal transcriptome, PhWD2 was identified as a putative scent-related factor. PhWD2 is a protein that is uniquely present in plants and has a three-domain structure: RING-kinase-WD40. Suppression of PhWD2 (termed UPPER - Unique Plant PhEnylpropanoid Regulator) resulted in a significant increase in the levels of volatiles emitted from and accumulated in internal pools, suggesting that it is a negative regulator of petunia floral scent production.

A pathogen effector FOLD diversified in symbiotic fungi.

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.

The Amsterdam petunia germplasm collection: A tool in plant science.

Petunia hybrida is a plant model system used by many researchers to investigate a broad range of biological questions. One of the reasons for the success of this organism as a lab model is the existence of numerous mutants, involved in a wide range of processes, and the ever-increasing size of this collection owing to a highly active and efficient transposon system. We report here on the origin of petunia-based research and describe the collection of petunia lines housed in the University of Amsterdam, where many of the existing genotypes are maintained.

Altered profile of floral volatiles and lignin content by down-regulation of Caffeoyl Shikimate Esterase in Petunia.

BACKGROUND: The floral volatile profile of Petunia x hybrida 'Mitchell diploid' (MD) is dominated by phenylpropanoids, many of which are derived from p-coumaric acid. However, the downstream processes involved in the production of caffeoyl-CoA and feruloyl-CoA from p-coumaric acid are complex, as the genes and biosynthesis steps are associated with flavonoids and lignin synthesis as well as floral volatiles benzenoid/phenylpropanoid (FVBP). Caffeoyl shikimate esterase (CSE) converts caffeoyl shikimate to caffeic acid and is considered one of the essential regulators in lignin production. Moreover, CSE in involved in phenylpropanoid production. To investigate the roles of CSE in FVBP biosynthesis, we used RNAi-mediated CSE down-regulated (ir-PhCSE) petunias. RESULTS: Lowered CSE transcript accumulation in ir-PhCSE plants resulted in reduced lignin layers in the stems and stunted growth, suggesting a positive correlation between lignin layers and lignin content. The altered CSE level influenced the expression of many FVBP genes, including elevated transcripts of p-coumarate-3-hydroxylase (C3H), hydroxycinnamoyl transferase (HCT), and 4-coumaric acid: CoA ligase (4CL). In particular, the expression of C4H in ir-PhCSE plants was more than twice the expression in MD plants. Moreover, the production of volatile compounds was alterend in ir-PhCSE plants. Most floral volatiles decreased, and the amounts of phenylalanine and caffeic acid were significantly lower. CONCLUSIONS: Reduced lignin layers in the stems and stunted growth in ir-PhCSE plants suggest that PhCSE is essential for lignin production and plant growth in petunia. The decreased CSE level influenced the expression of many FVBP genes, and interference of shikimate derivates altered volatile compound production. Significantly decreased caffeic acid, but not ferulic acid, in ir-PhCSE plants suggest that CSE is primarily involved in the reaction of caffeoyl shikimate. Higher C3H and C4H transcripts seem to alleviate accumulated p-coumaric acid resulting from altered CSE. Finally, alteration in C3H, HCT, and 4CL in CSE down-regulated plants suggests an interaction of the FVBP genes, leading to the regulation of floral volatiles of petunia.