Search

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.

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

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

Details of the foraging patterns of solitary bees are much less well known than those of social species, and these patterns are often adjusted to exploit floral resources of one or only a few species. The specialized flower-visiting bees of Petunia are good models for investigating such foraging patterns. Here we analysed the floral biology and pollen presentation schedule of the endangered Petunia mantiqueirensis in mixed Araucaria forests of Serra da Mantiqueira, Brazil. Pollinators and their pollen foraging behaviour and food specialization were determined through analyses of scopa pollen loads. Flowers opened throughout the day and presented all their pollen resources within the first 30 min of anthesis, thus providing their pollen resources in an asynchronous fashion in one-flower packages throughout the day. Females of Pseudagapostemon fluminensis were the most frequent flower visitors, contacting stigmas in 96% of their visits, and were the unique effective pollinators of Petunia mantiqueirensis. These pollinators were responsible for the first three visits to 115 individually monitored flowers at any daylight hour, removing ~86% of a flower’s total pollen supply during the first visit. Although female bees harvest the majority of pollen resources of Petunia mantiqueirensis, analyses of scopa loads revealed that most of them also collect pollen from plants of other families while foraging for pollen in Petunia flowers.

Phytohormones of the strigolactone (SL) family have been characterized as negative regulators of lateral bud outgrowth and triggers of symbioses between plants and mycorrhizal fungi. SLs and their precursors are synthesized in root tips as well as along shoot and root vasculature; they either move shoot-wards and regulate plant architecture or are exuded from roots into the soil to establish mycorrhizal symbiosis. Owing to the difficulty in quantification of SL in shoot tissues because of low abundance, it is not yet clear how SL distribution in plants is regulated at short- and long-distances from SL biosynthetic and target tissues. To address this question, we grafted wild-type scions and rootstocks from different petunia mutants for SL biosynthesis/transport and investigated SL activity by quantifying lateral bud outgrowth in the main shoot. Based on these results, we show that (i) the previously reported petunia SL transporter PLEIOTROPIC DRUG RESISTANCE 1 (PDR1) directly accounts for short-distance SL transport and (ii) long-distance transport of SLs seems to be partially and not directly dependent on PDR1. These data suggest that the root-to-shoot transport of SLs occurs either via the vasculature bundle through transporters other than PDR1 or involves SL precursors that are not substrates of PDR1.

Competitive binding and complex formation of MBW proteins has a functional relevance for anthocyanidin production and trichome development across a range of different species, which can be explained by changes in one amino acid., A protein complex consisting of a MYB, basic Helix-Loop-Helix, and a WDR protein, the MBW complex, regulates five traits, namely the production of anthocyanidin, proanthocyanidin, and seed-coat mucilage, and the development of trichomes and root hairs. For complexes involved in trichome and root hair development it has been shown that the interaction of two MBW proteins can be counteracted by the respective third protein (called competitive complex formation). We examined competitive complex formation for selected MBW proteins from Arabidopsis thaliana, Arabis alpina, Gossypium hirsutum, Petunia hybrida, and Zea mays. Quantitative analyses of the competitive binding of MYBs and WDRs to bHLHs were done by pull-down assays using ProtA- and luciferase-tagged proteins expressed in human HEC cells. We found that some bHLHs show competitive complex formation whilst others do not. Competitive complex formation strongly correlated with a phylogenetic tree constructed with the bHLH proteins under investigation, suggesting a functional relevance. We demonstrate that this different behavior can be explained by changes in one amino acid and that this position is functionally relevant in trichome development but not in anthocyanidin regulation.

In petals of picotee petunia (Petunia hybrida) cultivars, margin-specific post-transcriptional gene silencing (PTGS) of chalcone synthase A (CHSA) inhibits anthocyanin biosynthesis, resulting in marginal white tissue formation. In this study, we found that a low molecular mass compound, fluacrypyrim, inhibits PTGS of CHSA, and we explored the site-specific PTGS mechanism of operation. Fluacrypyrim treatment abolished the picotee pattern and eliminated site-specific differences in the levels of anthocyanin-related compounds, CHSA expression, and CHSA small interfering RNA (siRNA). In addition, fluacrypyrim abolished the petunia star-type pattern, which is also caused by PTGS of CHSA. Fluacrypyrim treatment was effective only at the early floral developmental stage and predominantly eliminated siRNA derived from CHS genes; i.e. siRNA derived from other genes remained at a comparable level. Fluacrypyrim probably targets the induction of PTGS that specifically operates for CHS genes in petunia picotee flowers, rather than common PTGS maintenance mechanisms that degrade mRNAs and generate siRNA. Upon treatment, the proportion of colored tissue increased due to a shift of the border between white and colored sites toward the margin in a time- and dose-dependent manner. These findings imply that the fluacrypyrim-targeted PTGS induction is completed gradually and its strength is attenuated from the margins to the center of petunia picotee petals.

The cultivated petunia (Petunia ×hybrida) is derived from the progenitor species P. axillaris and P. integrifolia. The hybridization dates back only to the 1830s, though intensive breeding efforts have yielded cultivars exhibiting incredible diversity for many traits, including growth habit, flower color, and flower size. Until now, little is known about the genetic diversity and genomic background of modern cultivars. Here we selected a panel of 13 cultivars with contrasting growth habits and three wild species (the progenitors and P. exserta) to estimate the genomic contribution from the ancestral species and to study whether the variation of the genetic origin could be associated with different breeding programs or morphological variability. Transcriptome sequencing identified 1,164,566 SNPs representing 98.4% (32,451) of the transcripts that cover 99.2% (of 52,697,361 bp) of the P. axillaris transcriptome. Cultivars with an upright growth habit had more homozygous alleles and more P. axillaris-derived alleles than trailing cultivars, while mounded cultivars had intermediate heterozygosity. Unlike previous studies, we found the proportions of alleles derived from each progenitor species varied across cultivars but overall were not biased toward one progenitor species, suggesting diverse selection during cultivar development. For trailing cultivars, alleles potentially introgressed from other wild species (“out” alleles) were enriched. The “out” alleles were clustered in particular regions of chromosomes, suggesting that these regions may be hotspots of introgression. Transcripts in these regions were enriched with gene ontology terms associated with growth habit. This study provides novel insight into the contributions of progenitor species to the genomic background of modern petunia cultivars and identifies genome regions that may harbor genes conferring the trailing growth habit for further exploration.

International audience; To attract insects, flowers produce nectar, an energy-rich substance secreted by specialized organs called nectaries. For Arabidopsis thaliana, a rosid species with stamen-associated nectaries, the floral B-, C-, and E- functions were proposed to redundantly regulate nectary development. Here we investigated the molecular basis of carpel-associated nectary development in the asterid species Petunia hybrida. We show that its euAGAMOUS (euAG) and PLENA (PLE) C-lineage MADS-box proteins are essential for nectary development, while their overexpression is sufficient to induce ectopic nectaries on sepals. Furthermore, we demonstrate that Arabidopsis nectary development also fully depends on euAG/PLE C-lineage genes. In turn, we show that petunia nectary development depends on two homologs of CRABS CLAW (CRC), a gene previously shown to be required for Arabidopsis nectary development, and demonstrate that CRC expression in both species depends on the members of both euAG/PLE C-sublineages. Therefore, petunia and Arabidopsis employ a similar molecular mechanism underlying nectary development, despite otherwise major differences in the evolutionary trajectory of their C-lineage genes, their distant phylogeny and different nectary positioning. However, unlike in Arabidopsis, petunia nectary development is position-independent within the flower. Finally, we show that the TARGET OF EAT (TOE)-type BLIND ENHANCER (BEN) and APETALA2 (AP2)-type REPRESSOR OF B-FUNCTION (ROB) genes act as major regulators of nectary size.

A major bottleneck in plant breeding has been the much limited genetic base and much reduced genetic diversity in domesticated, cultivated germplasm. Identification and utilization of favorable gene loci or alleles from wild or progenitor species can serve as an effective approach to increasing genetic diversity and breaking this bottleneck in plant breeding. This study was conducted to identify quantitative trait loci (QTL) in wild or progenitor petunia species that can be used to improve important horticultural traits in garden petunia. An F7 recombinant inbred population derived between Petunia axillaris and P. exserta was phenotyped for plant height, plant spread, plant size, flower counts, flower diameter, flower length, and days to anthesis in Florida in two consecutive years. Transgressive segregation was observed for all seven traits in both years. The broad-sense heritability estimates for the traits ranged from 0.20 (days to anthesis) to 0.62 (flower length). A genome-wide genetic linkage map consisting of 368 single nucleotide polymorphism bins and extending over 277 cM was searched to identify QTL for these traits. Nineteen QTL were identified and localized to five linkage groups. Eleven of the loci were identified consistently in both years; several loci explained up to 34.0% and 24.1% of the phenotypic variance for flower length and flower diameter, respectively. Multiple loci controlling different traits are co-localized in four intervals in four linkage groups. These intervals contain desirable alleles that can be introgressed into commercial petunia germplasm to expand the genetic base and improve plant performance and flower characteristics in petunia.

Tomato spotted wilt orthotospovirus (TSWV) is a major disease in peanut, Arachis hypogaea L., across peanut producing regions of the United States and elsewhere. Two thrips, Frankliniella fusca Hinds and Frankliniella occidentalis Pergande (Thysanoptera: Thripidae), are considered important vectors of TSWV in peanut in the Southeast. We compared the efficiency of acquisition (by larvae) and transmission (adults) of both thrips species for TSWV (Texas peanut-strain) to leaf disks of peanut (Florunner), as well as to Impatiens walleriana Hook. f. (Dwarf White Baby) and Petunia hybrida Juss. 'Fire Chief' using double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA). Both species were competent TSWV vectors in peanut and Impatiens, although F. fusca was the more efficient vector overall, i.e., virus acquisition and transmission rates for F. fusca averaged over several bioassays were 51.7 and 26.6%, respectively, compared with 20.0 and 15.3% for F. occidentalis. Neither species effectively transmitted this TSWV strain to Petunia (i.e., ≤3.6% transmission). We found statistically similar virus acquisition and transmission rates between both sexes for each species. We also detected no differences in TSWV-acquisition and transmission frequency between macropterous and brachypterous (short-wing) forms of F. fusca collected from a field population in south Texas. DAS-ELISA failed to detect low levels of TSWV in a few thrips that subsequently proved to be competent vectors.

Petunia inflata possesses a self-incompatibility (SI) mechanism, which involves S-RNase and multiple S-locus F-box (SLF) genes at the polymorphic S-locus. For a given S-haplotype, each SLF is thought to interact with some of its non-self S-RNases, but not with its self S-RNase. In this work, we studied an allelic pair of SLF1, S2-SLF1 and S3-SLF1, which differ in 44 amino acids and show differential interactions with S3-RNase. We first used an in vivo transgenic assay to determine whether four chimeric proteins of S2-SLF1 and S3-SLF1, each with one of the three functional domains swapped, interact with S3-RNase. The results narrowed the candidate amino acids for specific interaction of S2-SLF1 with S3-RNase to the 16 in domain FD3. We then examined seven additional chimeric proteins by dividing FD3 into two subdomains and four mini-domains (A, B, C and D). The results further narrowed the candidate amino acids to four in mini-domain A and four in mini-domain D. Molecular modeling of interactions between S3-RNase and S2-SLF1 revealed that three of these eight are at the interaction surface, and all three are conserved in S1-SLF1 and S6a-SLF1, both of which interact with S3-RNase based on the in vivo transgenic assay. Three of the chimeric proteins were used for the in vivo transgenic assay to determine whether FD3 alone contains the amino acids required for S2-SLF1 to interact with S7-RNase and S13-RNase. The results revealed the diversity and complexity of interactions between SLF proteins and S-RNases.

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

Petal senescence is a complex programmed process. It has been demonstrated previously that treatment with ethylene, a plant hormone involved in senescence, can extensively alter transcriptome and proteome profiles in plants. However, little is known regarding the impact of ethylene on posttranslational modification (PTM) or the association between PTM and the proteome. Protein degradation is one of the hallmarks of senescence, and ubiquitination, a major PTM in eukaryotes, plays important roles in protein degradation. In this study, we first obtained reference petunia (Petunia hybrida) transcriptome data via RNA sequencing. Next, we quantitatively investigated the petunia proteome and ubiquitylome and the association between them in petunia corollas following ethylene treatment. In total, 51,799 unigenes, 3,606 proteins, and 2,270 ubiquitination sites were quantified 16 h after ethylene treatment. Treatment with ethylene resulted in 14,448 down-regulated and 6,303 up-regulated unigenes (absolute log2 fold change > 1 and false discovery rate < 0.001), 284 down-regulated and 233 up-regulated proteins, and 320 up-regulated and 127 down-regulated ubiquitination sites using a 1.5-fold threshold (P < 0.05), indicating that global ubiquitination levels increase during ethylene-mediated corolla senescence in petunia. Several putative ubiquitin ligases were up-regulated at the protein and transcription levels. Our results showed that the global proteome and ubiquitylome were negatively correlated and that ubiquitination could be involved in the degradation of proteins during ethylene-mediated corolla senescence in petunia. Ethylene regulates hormone signaling transduction pathways at both the protein and ubiquitination levels in petunia corollas. In addition, our results revealed that ethylene increases the ubiquitination levels of proteins involved in endoplasmic reticulum-associated degradation.

Petunia is a very important flower in the global floriculture industry and has played a critical role as a model in plant genetic studies. Owing to limited genetic variability in commercial germplasm, development of novel petunia phenotypes and new varieties has become increasingly difficult. To enrich petunia germplasm and facilitate genetic improvement, it is important to explore genetic variation in progenitor species that may contain highly valuable genes/alleles. In this study, an interspecific recombinant inbred population (168 recombinant inbreds) derived from Petunia integrifolia × P. axillaris were phenotyped for days to anthesis (DTA), flower count (Flower_C), flower diameter (Flower_D), flower length (Flower_L), plant height (Plant_H), plant spread (Plant_S), and plant size (Plant_Z) in 2014 and 2015. Transgressive segregation was observed for all traits in both years. The broad-sense heritability on a 2-year basis varied from 0.38 (Flower_C) to 0.82 (Flower_L). Ten QTL were consistently identified in both years and by two mapping strategies [multiple QTL mapping (MQM) in MapQTL and inclusive composite interval mapping (ICIM) in IciMapping]. Major QTL explained up to 30.2, 35.5, and 47.1% of the total phenotypic variation for Plant_S, Flower_L, and Flower_D, respectively. These findings should be of significant values for introgression of desirable genes from wild petunias into commercial varieties and future genetic improvement of this important flower. Uncovering the genetics behind desirable traits in petunia plants could help improve commercial cultivars. While petunia plants are a key feature of the global horticultural industry, the limited genetic variability in commercial plants makes it difficult to improve desired plant traits. Zhanao Deng at the University of Florida, US, and co-workers conducted a genome-wide QTL identification study using a crossbred population combining two petunia species, P. integrifolia and P. axillaris. The team phenotyped the petunias in an open-air sub-tropical field rather than in an artificial environment like previous studies. They identified and characterized 17 genetic loci for seven important aesthetic traits, ranging from flower count to plant size. The petunia species had very different genetic backgrounds, likely stemming from their different geographic origins. The two species each can contribute novel genes for enhancing cultivated petunia cultivars.

Self-incompatibility (SI) in flowering plants is a genetic reproductive barrier to distinguish self- and non-self pollen to promote outbreeding. In Solanaceae, self-pollen is rejected by the ribonucleases expressed in the styles (S-RNases), via its cytotoxic function. On the other side, the male-determinant is the S-locus F-box proteins (SLFs) expressed in pollen. Multiple SLFs collaboratively detoxify non-self S-RNases, therefore, non-self recognition is the mode of self-/non-self discrimination in Solanaceae. It is considered that SLFs function as a substrate-recognition module of the Skp1-Cullin1-F-box (SCF) complex that inactivates non-self S-RNases via their polyubiquitination, which leads to degradation by 26S proteasome. In fact, PhSSK1 (Petunia hybrida SLF-interacting Skp1-like1) was identified as a specific component of SCFSLF and was shown to be essential for detoxification of S-RNase in Petunia However, different molecules are proposed as the candidate Cullin1, another component of SCFSLF, and there is as yet no definite conclusion. Here, we identified five Cullin1s from the expressed sequence tags (ESTs) derived from the male reproductive organ in Petunia Among them, only PhCUL1-P was co-immunoprecipitated with S7-SLF2. In vitro protein-binding assay suggested that PhSSK1 specifically forms a complex with PhCUL1-P in an SLF-dependent manner. Knockdown of PhCUL1-P suppressed fertility of transgenic pollen in cross-compatible pollination in the functional S-RNase-dependent manner. These results suggested that SCFSLF selectively uses PhCUL1-P. Phylogeny of Cullin1s indicates that CUL1-P is recruited into the SI machinery during the evolution of Solanaceae, suggesting that the SI components have evolved differently among species in Solanaceae and Rosaceae, despite both families sharing the S-RNase-based SI.

Highlight PhERF2, an ethylene-responsive element binding factor, acts as a positive transcriptional regulator in antiviral RNA silencing and is essential for efficient silencing of genes in plants., Virus-induced RNA silencing is involved in plant antiviral defense and requires key enzyme components, including RNA-dependent RNA polymerases (RDRs), Dicer-like RNase III enzymes (DCLs), and Argonaute proteins (AGOs). However, the transcriptional regulation of these critical components is largely unknown. In petunia (Petunia hybrida), an ethylene-responsive element binding factor, PhERF2, is induced by Tobacco rattle virus (TRV) infection. Inclusion of a PhERF2 fragment in a TRV silencing construct containing reporter fragments of phytoene desaturase (PDS) or chalcone synthase (CHS) substantially impaired silencing efficiency of both the PDS and CHS reporters. Silencing was also impaired in PhERF2- RNAi lines, where TRV-PhPDS infection did not show the expected silencing phenotype (photobleaching). In contrast, photobleaching in response to infiltration with the TRV-PhPDS construct was enhanced in plants overexpressing PhERF2. Transcript abundance of the RNA silencing-related genes RDR2, RDR6, DCL2, and AGO2 was lower in PhERF2-silenced plants but higher in PhERF2-overexpressing plants. Moreover, PhERF2-silenced lines showed higher susceptibility to Cucumber mosaic virus (CMV) than wild-type (WT) plants, while plants overexpressing PhERF2 exhibited increased resistance. Interestingly, growth and development of PhERF2-RNAi lines were substantially slower, whereas the overexpressing lines were more vigorous than the controls. Taken together, our results indicate that PhERF2 functions as a positive regulator in antiviral RNA silencing.

Cytosolic acetyl-CoA is an intermediate of the synthesis of most secondary metabolites and the source of acetyl for protein acetylation. The formation of cytosolic acetyl-CoA from citrate is catalysed by ATP-citrate lyase (ACL). However, the function of ACL in global metabolite synthesis and global protein acetylation is not well known. Here, four genes, PaACLA1, PaACLA2, PaACLB1, and PaACLB2, which encode the ACLA and ACLB subunits of ACL in Petunia axillaris, were identified as the same sequences in Petunia hybrida 'Ultra'. Silencing of PaACLA1-A2 and PaACLB1-B2 led to abnormal leaf and flower development, reduced total anthocyanin content, and accelerated flower senescence in petunia 'Ultra'. Metabolome and acetylome analysis revealed that PaACLB1-B2 silencing increased the content of many downstream metabolites of acetyl-CoA metabolism and the levels of acetylation of many proteins in petunia corollas. Mechanistically, the metabolic stress induced by reduction of acetyl-CoA in PaACL-silenced petunia corollas caused global and specific changes in the transcriptome, the proteome, and the acetylome, with the effect of maintaining metabolic homeostasis. In addition, the global proteome and acetylome were negatively correlated under acetyl-CoA deficiency. Together, our results suggest that ACL acts as an important metabolic regulator that maintains metabolic homeostasis by promoting changes in the transcriptome, proteome. and acetylome.

A pollen grain lands on a stigma, swells with water, and sprouts a pollen tube, which races through the style to deliver sperm to the ovule below. When the pollen arises from the flower itself, this process often stops in its tracks. Self-incompatibility (SI) between pollen and pistil promotes

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

Anthocyanins are a chemically diverse class of secondary metabolites that color most flowers and fruits. They consist of three aromatic rings that can be substituted with hydroxyl, sugar, acyl, and methyl groups in a variety of patterns depending on the plant species. To understand how such chemical diversity evolved, we isolated and characterized METHYLATION AT THREE2 (MT2) and the two METHYLATION AT FIVE (MF) loci from Petunia spp., which direct anthocyanin methylation in petals. The proteins encoded by MT2 and the duplicated MF1 and MF2 genes and a putative grape (Vitis vinifera) homolog Anthocyanin O-Methyltransferase1 (VvAOMT1) are highly similar to and apparently evolved from caffeoyl-Coenzyme A O-methyltransferases by relatively small alterations in the active site. Transgenic experiments showed that the Petunia spp. and grape enzymes have remarkably different substrate specificities, which explains part of the structural anthocyanin diversity in both species. Most strikingly, VvAOMT1 expression resulted in the accumulation of novel anthocyanins that are normally not found in Petunia spp., revealing how alterations in the last reaction can reshuffle the pathway and affect (normally) preceding decoration steps in an unanticipated way. Our data show how variations in gene expression patterns, loss-of-function mutations, and alterations in substrate specificities all contributed to the anthocyanins’ structural diversity.

Plants require sophisticated regulatory mechanisms to ensure the degree of anthocyanin pigmentation is appropriate to myriad developmental and environmental signals. Central to this process are the activity of MYB-bHLH-WD repeat (MBW) complexes that regulate the transcription of anthocyanin genes. In this study, the gene regulatory network that regulates anthocyanin synthesis in petunia (Petunia hybrida) has been characterized. Genetic and molecular evidence show that the R2R3-MYB, MYB27, is an anthocyanin repressor that functions as part of the MBW complex and represses transcription through its C-terminal EAR motif. MYB27 targets both the anthocyanin pathway genes and basic-helix-loop-helix (bHLH) ANTHOCYANIN1 (AN1), itself an essential component of the MBW activation complex for pigmentation. Other features of the regulatory network identified include inhibition of AN1 activity by the competitive R3-MYB repressor MYBx and the activation of AN1, MYB27, and MYBx by the MBW activation complex, providing for both reinforcement and feedback regulation. We also demonstrate the intercellular movement of the WDR protein (AN11) and R3-repressor (MYBx), which may facilitate anthocyanin pigment pattern formation. The fundamental features of this regulatory network in the Asterid model of petunia are similar to those in the Rosid model of Arabidopsis thaliana and are thus likely to be widespread in the Eudicots.

The highly polymorphic S (for self-incompatibility) locus regulates self-incompatibility in Petunia inflata; the S-RNase regulates pistil specificity, and multiple S-locus F-box (SLF) genes regulate pollen specificity. The collaborative non-self recognition model predicts that, for any S-haplotype, an unknown number of SLFs collectively recognize all non-self S-RNases to mediate their ubiquitination and degradation. Using a gain-of-function assay, we examined the relationships between S2-SLF1 (for S2-allelic product of Type-1 SLF) and four S-RNases. The results suggest that S2-SLF1 interacts with S7- and S13-RNases, and the previously identified S1- and S3-RNases, but not with S5- or S11-RNase. An artificial microRNA expressed by the S2-SLF1 promoter, but not by the vegetative cell-specific promoter, Late Anther Tomato 52, suppressed expression of S2-SLF1 in S2 pollen, suggesting that SLF1 is specific to the generative cell. The S2 pollen with S2-SLF1 suppressed was compatible with S3-, S5-, S7-, S11-, and S13-carrying pistils, confirming that other SLF proteins are responsible for detoxifying S5- and S11-RNases and suggesting that S2-SLF1 is not the only SLF in S2 pollen that interacts with S3-, S7-, and S13-RNases. Petunia may have evolved at least two types of SLF proteins to detoxify any non-self S-RNase to minimize the deleterious effects of mutation in any SLF.

Autophagy is one of the main mechanisms of degradation and remobilization of macromolecules, and it appears to play an important role in petal senescence. However, little is known about the regulatory mechanisms of autophagy in petal senescence. Autophagic processes were observed by electron microscopy and monodansylcadaverine staining of senescing petals of petunia (Petunia hybrida); autophagy-related gene 8 (ATG8) homologues were isolated from petunia and the regulation of expression was analysed. Nutrient remobilization was also examined during pollination-induced petal senescence. Active autophagic processes were observed in the mesophyll cells of senescing petunia petals. Pollination induced the expression of PhATG8 homologues and was accompanied by an increase in ethylene production. Ethylene inhibitor treatment in pollinated flowers delayed the induction of PhATG8 homologues, and ethylene treatment rapidly upregulated PhATG8 homologues in petunia petals. Dry weight and nitrogen content were decreased in the petals and increased in the ovaries after pollination in detached flowers. These results indicated that pollination induces autophagy and that ethylene is a key regulator of autophagy in petal senescence of petunia. The data also demonstrated the translocation of nutrients from the petals to the ovaries during pollination-induced petal senescence.

Flower scent is a highly dynamic trait, under developmental, spatial, and diurnal regulation. The mechanism governing scent production is only beginning to be unraveled. In petunia (Petunia hybrida), EMISSION OF BENZENOIDS II (EOBII) controls transcription of both the shikimate pathway-regulating MYB factor ODORANT1 (ODO1) and phenylpropanoid scent-related structural genes. A promoter-activation screen identified an R2R3-MYB–like regulatory factor of phenylpropanoid volatile biosynthesis acting downstream of EOBII, designated EOBI. EOBI silencing led to downregulation of ODO1 and numerous structural scent-related genes from both the shikimate and phenylpropanoid pathways. The ability of EOBI to directly activate ODO1, as revealed by electrophoretic mobility shift assay and yeast one-hybrid analysis, place EOBI upstream of ODO1 in regulating substrate availability for volatile biosynthesis. Interestingly, ODO1-silenced transgenic petunia flowers accumulated higher EOBI transcript levels than controls, suggesting a complex feedback loop between these regulatory factors. The accumulation pattern of EOBI transcript relative to EOBII and ODO1, and the effect of up/downregulation of EOBII on transcript levels of EOBI and ODO1, further support these factors' hierarchical relationships. The dependence of scent production on EOBI expression and its direct interaction with both regulatory and structural genes provide evidence for EOBI's wide-ranging involvement in the production of floral volatiles.

Plant responses to drought stress vary depending on the severity of stress and the stage of drought progression. To improve the understanding of such responses, the leaf physiology, abscisic acid (ABA) concentration, and expression of genes associated with ABA metabolism and signalling were investigated in Petunia × hybrida. Plants were exposed to different specific substrate water contents (θ = 0.10, 0.20, 0.30, or 0.40 m(3)·m(-3)) to induce varying levels of drought stress. Plant responses were investigated both during the drying period (θ decreased to the θ thresholds) and while those threshold θ were maintained. Stomatal conductance (g(s)) and net photosynthesis (A) decreased with decreasing midday leaf water potential (Ψ(leaf)). Leaf ABA concentration increased with decreasing midday Ψ(leaf) and was negatively correlated with g(s) (r = -0.92). Despite the increase in leaf ABA concentration under drought, no significant effects on the expression of ABA biosynthesis genes were observed. However, the ABA catabolism-related gene CYP707A2 was downregulated, primarily in plants under severe drought (θ = 0.10 m(3)•m(-3)), suggesting a decrease in ABA catabolism under severe drought. Expression of phospholipase Dα (PLDα), involved in regulating stomatal responses to ABA, was enhanced under drought during the drying phase, but there was no relationship between PLDα expression and midday Ψ(leaf) after the θ thresholds had been reached. The results show that drought response of plants depends on the severity of drought stress and the phase of drought progression.

The molecular bases of the gametophytic self-incompatibility (GSI) system of species of the subtribe Pyrinae (Rosaceae), such as apple and pear, have been widely studied in the last two decades. The characterization of S-locus genes and of the mechanisms underlying pollen acceptance or rejection have been topics of major interest. Besides the single pistil-side S determinant, the S-RNase, multiple related S-locus F-box genes seem to be involved in the determination of pollen S specificity. Here, we collect and review the state of the art of GSI in the Pyrinae. We emphasize recent genomic data that have contributed to unveiling the S-locus structure of the Pyrinae, and discuss their consistency with the models of self-recognition that have been proposed for Prunus and the Solanaceae. Experimental data suggest that the mechanism controlling pollen-pistil recognition specificity of the Pyrinae might fit well with the collaborative 'non-self' recognition system proposed for Petunia (Solanaceae), whereas it presents relevant differences with the mechanism exhibited by the species of the closely related genus Prunus, which uses a single evolutionarily divergent F-box gene as the pollen S determinant. The possible involvement of multiple pollen S genes in the GSI system of Pyrinae, still awaiting experimental confirmation, opens up new perspectives to our understanding of the evolution of S haplotypes, and of the evolution of S-RNase-based GSI within the Rosaceae family. Whereas S-locus genes encode the players determining self-recognition, pollen rejection in the Pyrinae seems to involve a complex cascade of downstream cellular events with significant similarities to programmed cell death.

Many species in Rosaceae, Solanaceae, and Plantaginaceae exhibit S-RNase-based self-incompatibility (SI). In this system, the pistil and pollen specificities are determined by S-RNase and the S locus F-box protein, respectively. The pollen S determinant F-box protein in Prunus (Rosaceae) is referred to by two different terms, SFB (for S-haplotype-specific F-box protein) and SLF (for S locus F box), whereas it is called SLF in Solanaceae and Plantaginaceae. Prunus SFB is thought to be a molecule indispensable for its cognate S-RNase to exert cytotoxicity and to arrest pollen tube growth in incompatible reactions. Although recent studies have demonstrated the molecular function of SCFSLF in the SI reaction of Solanaceae and Plantaginaceae, how SFB participates in the Prunus SI mechanism remains to be elucidated. Here we report the identification of sweet cherry (Prunus avium) SFB (PavSFB)-interacting Skp1-like1 (PavSSK1) using a yeast (Saccharomyces cerevisiae) two-hybrid screening against the pollen cDNA library. Phylogenetic analysis showed that PavSSK1 belongs to the same clade as Antirrhinum hispanicum SLF-interacting Skp1-like1 and Petunia hybrida SLF-interacting Skp1-like1 (PhSSK1). In yeast, PavSSK1 interacted not only with PavSFBs from different S haplotypes and Cullin1-likes (PavCul1s), but also with S-locus F-box-likes. A pull-down assay confirmed the interactions between PavSSK1 and PavSFB and between PavSSK1 and PavCul1s. These results collectively indicate that PavSSK1 could be a functional component of the SCF complex and that PavSFB may function as a component of the SCF complex. We discuss the molecular function of PavSFB in self-/nonself-recognition in the gametophytic SI of Prunus.

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

For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity.This review focuses on the work from the authors' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil.The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism.

Maintenance of indeterminacy is fundamental to the generation of plant architecture and a central component of the plant life strategy. Indeterminacy in plants is a characteristic of shoot and root meristems, which must balance maintenance of indeterminacy with organogenesis. The Petunia hybrida HAIRY MERISTEM (HAM) gene, a member of the GRAS family of transcriptional regulators, promotes shoot indeterminacy by an undefined non-cell-autonomous signaling mechanism(s). Here, we report that Arabidopsis (Arabidopsis thaliana) mutants triply homozygous for knockout alleles in three Arabidopsis HAM orthologs (Atham1,2,3 mutants) exhibit loss of indeterminacy in both the shoot and root. In the shoot, the degree of penetrance of the loss-of-indeterminacy phenotype of Atham1,2,3 mutants varies among shoot systems, with arrest of the primary vegetative shoot meristem occurring rarely or never, secondary shoot meristems typically arresting prior to initiating organogenesis, and inflorescence and flower meristems exhibiting a phenotypic range extending from wild type (flowers) to meristem arrest preempting organogenesis (flowers and inflorescence). Atham1,2,3 mutants also exhibit aberrant shoot phyllotaxis, lateral organ abnormalities, and altered meristem morphology in functioning meristems of both rosette and inflorescence. Root meristems of Atham1,2,3 mutants are significantly smaller than in the wild type in both longitudinal and radial axes, a consequence of reduced rates of meristem cell division that culminate in root meristem arrest. Atham1,2,3 phenotypes are unlikely to reflect complete loss of HAM function, as a fourth, more distantly related Arabidopsis HAM homolog, AtHAM4, exhibits overlapping function with AtHAM1 and AtHAM2 in promoting shoot indeterminacy.

Ethylene-responsive element-binding factor (ERF) genes constitute one of the largest transcription factor gene families in plants. In Arabidopsis and rice, only a few ERF genes have been characterized so far. Flower senescence is associated with increased ethylene production in many flowers. However, the characterization of ERF genes in flower senescence has not been reported. In this study, 13 ERF cDNAs were cloned from petunia. Based on the sequence characterization, these PhERFs could be classified into four of the 12 known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Expression analyses of PhERF mRNAs were performed in corollas and gynoecia of petunia flower. The 13 PhERF genes displayed differential expression patterns and levels during natural flower senescence. Exogenous ethylene accelerates the transcription of the various PhERF genes, and silver thiosulphate (STS) decreased the transcription of several PhERF genes in corollas and gynoecia. PhERF genes of group VII showed a strong association with the rise in ethylene production in both petals and gynoecia, and might be associated particularly with flower senescence in petunia. The effect of sugar, methyl jasmonate, and the plant hormones abscisic acid, salicylic acid, and 6-benzyladenine in regulating the different PhERF transcripts was investigated. Functional nuclear localization signal analyses of two PhERF proteins (PhERF2 and PhERF3) were carried out using fluorescence microscopy. These results supported a role for petunia PhERF genes in transcriptional regulation of petunia flower senescence processes.

Studies of flower development have lots of information about petals, carpels, and stamens, but let’s not forget the birds and the bees—and the flies and moths—and what draws pollinators to insect-pollinated flowers, including flower color, shape, and rewards that provide energy. For example

Zinc finger nucleases (ZFNs) are a powerful tool for genome editing in eukaryotic cells. ZFNs have been used for targeted mutagenesis in model and crop species. In animal and human cells, transient ZFN expression is often achieved by direct gene transfer into the target cells. Stable transformation, however, is the preferred method for gene expression in plant species, and ZFN-expressing transgenic plants have been used for recovery of mutants that are likely to be classified as transgenic due to the use of direct gene-transfer methods into the target cells. Here we present an alternative, nontransgenic approach for ZFN delivery and production of mutant plants using a novel Tobacco rattle virus (TRV)-based expression system for indirect transient delivery of ZFNs into a variety of tissues and cells of intact plants. TRV systemically infected its hosts and virus ZFN-mediated targeted mutagenesis could be clearly observed in newly developed infected tissues as measured by activation of a mutated reporter transgene in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida) plants. The ability of TRV to move to developing buds and regenerating tissues enabled recovery of mutated tobacco and petunia plants. Sequence analysis and transmission of the mutations to the next generation confirmed the stability of the ZFN-induced genetic changes. Because TRV is an RNA virus that can infect a wide range of plant species, it provides a viable alternative to the production of ZFN-mediated mutants while avoiding the use of direct plant-transformation methods.

Floral scent, which is determined by a complex mixture of low molecular weight volatile molecules, plays a major role in the plant's life cycle. Phenylpropanoid volatiles are the main determinants of floral scent in petunia (Petunia hybrida). A screen using virus-induced gene silencing for regulators of scent production in petunia flowers yielded a novel R2R3-MYB–like regulatory factor of phenylpropanoid volatile biosynthesis, EMISSION OF BENZENOIDS II (EOBII). This factor was localized to the nucleus and its expression was found to be flower specific and temporally and spatially associated with scent production/emission. Suppression of EOBII expression led to significant reduction in the levels of volatiles accumulating in and emitted by flowers, such as benzaldehyde, phenylethyl alcohol, benzylbenzoate, and isoeugenol. Up/downregulation of EOBII affected transcript levels of several biosynthetic floral scent-related genes encoding enzymes from the phenylpropanoid pathway that are directly involved in the production of these volatiles and enzymes from the shikimate pathway that determine substrate availability. Due to its coordinated wide-ranging effect on the production of floral volatiles, and its lack of effect on anthocyanin production, a central regulatory role is proposed for EOBII in the biosynthesis of phenylpropanoid volatiles.

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

A gene encoding a preprohydroxyproline-rich systemin, SnpreproHypSys, was identified from the leaves of black nightshade (Solanum nigrum), which is a member of a small gene family of at least three genes that have orthologs in tobacco (Nicotiana tabacum; NtpreproHypSys), tomato (Solanum lycopersicum; SlpreproHypSys), petunia (Petunia hybrida; PhpreproHypSys), potato (Solanum tuberosum; PhpreproHypSys), and sweet potato (Ipomoea batatas; IbpreproHypSys). SnpreproHypSys was induced by wounding and by treatment with methyl jasmonate. The encoded precursor protein contained a signal sequence and was posttranslationally modified to produce three hydroxyproline-rich glycopeptide signals (HypSys peptides). The three HypSys peptides isolated from nightshade leaf extracts were called SnHypSys I (19 amino acids with six pentoses), SnHypSys II (20 amino acids with six pentoses), and SnHypSys III (20 amino acids with either six or nine pentoses) by their sequential appearance in SnpreproHypSys. The three SnHypSys peptides were synthesized and tested for their abilities to alkalinize suspension culture medium, with synthetic SnHypSys I demonstrating the highest activity. Synthetic SnHypSys I was capable of inducing alkalinization in other Solanaceae cell types (or species), indicating that structural conformations within the peptides are recognized by the different cells/species to initiate signal transduction pathways, apparently through recognition by homologous receptor(s). To further demonstrate the biological relevance of the SnHypSys peptides, the early defense gene lipoxygenase D was shown to be induced by all three synthetic peptides when supplied to excised nightshade plants.

The programmed degradation of macromolecules during petal senescence allows the plant to remobilize nutrients from dying to developing tissues. Ethylene is involved in regulating the timing of nucleic acid degradation in petunia, but it is not clear if ethylene has a role in the remobilization of phosphorus during petal senescence. To investigate ethylene's role in nutrient remobilization, the P content of petals (collectively called the corolla) during early development and senescence was compared in ethylene-sensitive wild type Petunia x hybrida 'Mitchell Diploid' (MD) and transgenic petunias with reduced sensitivity to ethylene (35S::etr1-1). When compared to the total P content of corollas on the day of flower opening (the early non-senescing stage), P in MD corollas had decreased 74% by the late stage of senescence (advanced wilting). By contrast, P levels were only reduced by an average of 32% during etr1-1 corolla (lines 44568 and Z00-35-10) senescence. A high-affinity phosphate transporter, PhPT1 (PhPht1;1), was cloned from senescing petunia corollas by RT-PCR. PhPT1 expression was up-regulated during MD corolla senescence and a much smaller increase was detected during the senescence of etr1-1 petunia corollas. PhPT1 mRNA levels showed a rapid increase in detached corollas (treated at 1 d after flower opening) following treatment with low levels of ethylene (0.1 microl l(-1)). Transcripts accumulated in the presence of the protein synthesis inhibitor, cycloheximide, indicating that PhPT1 is a primary ethylene response gene. PhPT1 is a putative phosphate transporter that may function in Pi translocation during senescence.

Acyl sugars containing branched-chain fatty acids (BCFAs) are exuded by glandular trichomes of many species in Solanaceae, having an important defensive role against insects. From isotope-feeding studies, two modes of BCFA elongation have been proposed: (1) fatty acid synthase-mediated two-carbon elongation in the high acyl sugar-producing tomato species Solanum pennellii and Datura metel; and (2) α-keto acid elongation-mediated one-carbon increments in several tobacco (Nicotiana) species and a Petunia species. To investigate the molecular mechanisms underlying BCFAs and acyl sugar production in trichomes, we have taken a comparative genomic approach to identify critical enzymatic steps followed by gene silencing and metabolite analysis in S. pennellii and Nicotiana benthamiana. Our study verified the existence of distinct mechanisms of acyl sugar synthesis in Solanaceae. From microarray analyses, genes associated with α-keto acid elongation were found to be among the most strongly expressed in N. benthamiana trichomes only, supporting this model in tobacco species. Genes encoding components of the branched-chain keto-acid dehydrogenase complex were expressed at particularly high levels in trichomes of both species, and we show using virus-induced gene silencing that they are required for BCFA production in both cases and for acyl sugar synthesis in N. benthamiana. Functional analysis by down-regulation of specific KAS I genes and cerulenin inhibition indicated the involvement of the fatty acid synthase complex in BCFA production in S. pennellii. In summary, our study highlights both conserved and divergent mechanisms in the production of important defense compounds in Solanaceae and defines potential targets for engineering acyl sugar production in plants for improved pest tolerance.

MADS-domain proteins are important transcription factors involved in many biological processes of plants. Interactions between MADS-domain proteins are essential for their functions. In tomato (Solanum lycopersicum), the number of MIKC(c)-type MADS-domain proteins identified has totalled 36, but a large-scale interaction assay is lacking. In this study, 22 tomato MADS-domain proteins were selected from six functionally important subfamilies of the MADS-box gene family, to create the first large-scale tomato protein interaction network. Compared with Arabidopsis and petunia (Petunia hybrida), protein interaction patterns in tomato displayed both conservation and divergence. The majority of proteins that can be identified as putative orthologues exhibited conserved interaction patterns, and modifications were mostly found in genes underlining traits unique to tomato. JOINTLESS and RIN, characterized for their roles in abscission zone development and fruit ripening, respectively, showed enlarged interaction networks in comparison with their Arabidopsis and petunia counterparts. Novel interactions were also found for members of the expanded subfamilies, such as those represented by AP1/FUL and AP3/PI MADS-domain proteins. In search for higher order complexes, TM5 was found to be the preferred bridge among the five SEP-like proteins. Additionally, 16 proteins with the MADS-domain removed were used to assess the role of the MADS-domain in protein-protein interactions. The current work provides important knowledge for further functional and evolutionary study of the MADS-box genes in tomato.

Ethylene is a gaseous plant hormone involved in many physiological processes including senescence, fruit ripening, and defence. Here the effects of pollination and wound-induced ethylene signals on transcript accumulation of benzoyl CoA:benzyl alcohol/phenylethanol benzoyltransferase (PhBPBT) are shown in Petuniaxhybrida cv. Mitchell 'Diploid' (MD). In petunia, PhBPBT is responsible for the biosynthesis of both benzyl benzoate and phenylethyl benzoate from benzyl alcohol and phenylethanol, respectively. RNAi-silenced lines, with reduced PhBPBT transcript, displayed reduced benzyl benzoate emission, and increased benzyl alcohol levels. Detailed expression analysis showed that PhBPBT is regulated by both light and an endogenous circadian rhythm, while it is also differentially regulated in response to ethylene in a tissue-specific manner. Twenty-four hours following pollination of MD flowers, expression of PhBPBT decreases in the corolla, while it increases in the ovary after 48 h. This is caused by ethylene that is emitted from the flower coinciding with fertilization as this is not observed in transgenic ethylene-insensitive plants (CaMV35S::etr1-1; 44568). Ethylene is also emitted from vegetative tissue of petunia following mechanical wounding, resulting in an increase in PhBPBT expression in the leaves where expression is normally below detection levels. Indicative of this pattern of expression, we hypothesize that PhBPBT and subsequent benzyl benzoate production is involved in defence-related processes in the corolla prior to pollination, in the ovary immediately following fertilization, and in vegetative tissue in response to wounding.

The floral scent emission and endogenous level of its components in Petunia axillaris under different conditions (20, 25, 30, and 35 degrees C) were investigated under the hypothesis that floral scent emission would be regulated by both metabolic and vaporization processes. The total endogenous amount of scent components decreased as the temperature increased, the total emission showing a peak at 30 degrees C. This decrease in endogenous amount was compensated for by increased vaporization, resulting in an increase of floral scent emission from 20 degrees C to 30 degrees C. The ambient temperature differently and independently influenced the metabolism and vaporization of the scent compounds, and differences in vapor pressure among the scent compounds were reduced as the temperature increased. These characteristics suggest the operation of an unknown regulator to change the vaporization of floral scent.

Petunia inflata possesses S-RNase–based self-incompatibility (SI), which prevents inbreeding and promotes outcrossing. Two polymorphic genes at the S-locus, S-RNase and P. inflata S-locus F-box (Pi SLF), determine the pistil and pollen specificity, respectively. To understand how the interactions between Pi SLF and S-RNase result in SI responses, we identified four Pi SLF–like (Pi SLFL) genes and used them, along with two previously identified Pi SLFLs, for comparative studies with Pi SLF2. We examined the in vivo functions of three of these Pi SLFLs and found that none functions in SI. These three Pi SLFLs and two other Pi SLFs either failed to interact with S3-RNase (a non-self S-RNase for all of them) or interacted much more weakly than did Pi SLF2 in vitro. We divided Pi SLF2 into FD1 (for Functional Domain1), FD2, and FD3, each containing one of the Pi SLF–specific regions, and used truncated Pi SLF2, chimeric proteins between Pi SLF2 and one of the Pi SLFLs that did not interact with S3-RNase, and chimeric proteins between Pi SLF1 and Pi SLF2 to address the biochemical roles of these three domains. The results suggest that FD2, conserved among three allelic variants of Pi SLF, plays a major role in the strong interaction with S-RNase; additionally, FD1 and FD3 (each containing one of the two variable regions of Pi SLF) together negatively modulate this interaction, with a greater effect on interactions with self S-RNase than with non-self S-RNases. A model for how an allelic product of Pi SLF determines the fate of its self and non-self S-RNases in the pollen tube is presented.

Floral fragrance is responsible for attracting pollinators as well as repelling pathogens and pests. As such, it is of immense biological importance. Molecular dissection of the mechanisms underlying scent production would benefit from the use of model plant systems with big floral organs that generate an array of volatiles and that are amenable to methods of forward and reverse genetics. One candidate is petunia (Petunia hybrida), which has emerged as a convenient model system, and both RNAi and overexpression approaches using transgenes have been harnessed for the study of floral volatiles. Virus-induced gene silencing (VIGS) is characterized by a simple inoculation procedure and rapid results relative to transgenesis. Here, we demonstrate the applicability of the tobacco rattle virus-based VIGS system to studies of floral scent. Suppression of the anthocyanin pathway via chalcone synthase silencing was used as a reporter, allowing easy visual identification of anthocyaninless silenced flowers/tissues with no effect on the level of volatile emissions. Use of tobacco rattle virus constructs containing target genes involved in phenylpropanoid volatile production, fused to the chalcone synthase reporter, allowed simple identification of flowers with suppressed activity of the target genes. The applicability of VIGS was exemplified with genes encoding S-adenosyl-l-methionine:benzoic acid/salicylic acid carboxyl methyltransferase, phenylacetaldehyde synthase, and the myb transcription factor ODORANT1. Because this high-throughput reverse-genetics approach was applicable to both structural and regulatory genes responsible for volatile production, it is expected to be highly instrumental for large-scale scanning and functional characterization of novel scent genes.

Petunia plants that exhibit a white-flowering phenotype as a consequence of chalcone synthase transgene-induced silencing occasionally give rise to revertant branches that produce flowers with wild-type pigmentation. Transcription run-on assays confirmed that the production of white flowers is caused by post-transcriptional gene silencing (PTGS), and indicated that transgene transcription is repressed in the revertant plants, providing evidence that induction of PTGS depends on the transcription rate. Transcriptional repression of the transgene was associated with cytosine methylation at CpG, CpNpG and CpNpN sites, and the expression was restored by treatment with either 5-azacytidine or trichostatin A. These results demonstrate that epigenetic changes occurred in the PTGS line, and these changes interfere with the initiation of transgene transcription, leading to a reversion of the PTGS phenotype.

Aromatic scent-related compounds in flowers were comprehensively analyzed by high-performance liquid chromatography (HPLC) based on their absorption spectra to understand regulation of metabolism leading to floral scent diversity in Petunia axillaris lines. An unknown compound occurring at similar levels to scent compounds in some plant lines was identified to be dihydroconiferyl acetate. Based on the structure, dihydroconiferyl acetate is likely to be a biosynthetically closely related compound to aromatic scent compounds, especially iso-eugenol. Similar time-course changes of the concentrations suggest that the metabolism of dihydroconiferyl acetate is underlaid by the similar regulation to aromatic scent compounds. Dihydroconiferyl acetate and iso-eugenol occurred almost exclusively, implying that metabolism of the common precursors to each compound is selectively regulated in these plants. The branching of the biosynthetic pathway into dihydroconiferyl acetate and iso-eugenol is probably one of regulatory steps leading to scent diversity in P. axillaris lines.

The response of plants to drought stress includes reduced transpiration as stomates close in response to increased abscisic acid (ABA) concentrations. Constitutive overexpression of 9-cis-epoxycarotenoid dioxygenase (NCED), a key enzyme in ABA biosynthesis, increases drought resistance, but causes negative pleiotropic effects on plant growth and development. We overexpressed the tomato NCED (LeNCED1) in petunia plants under the control of a stress-inducible promoter, rd29A. Under water stress, the transgenic plants had increased transcripts of NCED mRNA, elevated leaf ABA concentrations, increased concentrations of proline, and a significant increase in drought resistance. The transgenic plants also displayed the expected decreases in stomatal conductance, transpiration, and photosynthesis. After 14 days without water, the control plants were dead, but the transgenic plants, though wilted, recovered fully when re-watered. Well-watered transgenic plants grew like non-transformed control plants and there was no effect of the transgene on seed dormancy.

Control of branch development is a major determinant of architecture in plants. Branching in petunia (Petunia hybrida) is controlled by the DECREASED APICAL DOMINANCE (DAD) genes. Gene functions were investigated by plant grafting, morphology studies, double-mutant characterization, and gene expression analysis. Both dad1-1 and dad3 increased branching mutants can be reverted to a near-wild-type phenotype by grafting to a wild-type or a dad2 mutant root stock, indicating that both genes affect the production of a graft-transmissible substance that controls branching. Expression of the DAD1 gene in the stems of grafted plants, detected by quantitative reverse transcription-polymerase chain reaction correlates with the branching phenotype of the plants. The dad2-1 mutant cannot be reverted by grafting, indicating that this gene acts predominantly in the shoot of the plant. Double-mutant analysis indicates that the DAD2 gene acts in the same pathway as the DAD1 and DAD3 genes because the dad1-1dad2-1 and dad2-1dad3 double mutants are indistinguishable from the dad2-1 mutant. However, the dad1-1dad3 double mutant has an additive phenotype, with decreased height of the plants, delayed flowering, and reduced germination rates compared to the single mutants. This result, together with the observation that the dad1-1 and dad3 mutants cannot be reverted by grafting to each other, suggests that the DAD1 and DAD3 genes act in the same pathway, but not in a simple stepwise fashion.

Cytokinins (CKs) are involved in the regulation of plant development including plastid differentiation and function. Partial location of CK biosynthetic pathways in plastids suggests the importance of CKs for chloroplast development. The impact of genetically modified CK metabolism on endogenous CK, indole-3-acetic acid, and abscisic acid contents in leaves and isolated intact chloroplasts of Nicotiana tabacum was determined by liquid chromatography/mass spectrometry and two-dimensional high-performance liquid chromatography, and alterations in chloroplast ultrastructure by electron microscopy. Ectopic expression of Sho, a gene encoding a Petunia hybrida isopentenyltransferase, was employed to raise CK levels. The increase in CK levels was lower in chloroplasts than in leaves. CK levels were reduced in leaves of tobacco harbouring a CK oxidase/dehydrogenase gene, AtCKX3. The total CK content also decreased in chloroplasts, but CK phosphate levels were higher than in the wild type. In a transformant overexpressing a maize beta-glucosidase gene, Zm-p60.1, naturally targeted to plastids, a decrease of CK-O-glucosides in chloroplasts was found. In leaves, the changes were not significant. CK-O-glucosides accumulated to very high levels in leaves, but not in chloroplasts, of plants overexpressing a ZOG1 gene, encoding trans-zeatin-O-glucosyltransferase from Phaseolus lunatus. Manipulation of the CK content affected levels of indole-3-acetic and abscisic acid. Chloroplasts of plants constitutively overexpressing Sho displayed ultrastructural alterations including the occasional occurrence of crystalloids and an increased number of plastoglobuli. The other transformants did not exhibit any major differences in chloroplast ultrastructure. The results suggest that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of CK metabolism.