Project description:Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been rarely applied because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of the transcriptome of each species, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in both species. In addition, microarray analysis was applied to explore the molecular basis of the seed coat defects in Petunia hybrida mutants, homozygous for a null allele of the AN11 gene, encoding a WDR transcription regulator. Among the transcripts differentially expressed in an11 seeds compared to wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida.
Project description:Petunia is an excellent model system, especially for genetic, physiological and molecular studies. Thus far, however, genome-wide expression analysis has been rarely applied because of the lack of sequence information. We applied next-generation sequencing to generate, through de novo read assembly, a large catalogue of transcripts for Petunia axillaris and Petunia inflata. On the basis of the transcriptome of each species, comprehensive microarray chips for gene expression analysis were established and used for the analysis of global- and organ-specific gene expression in both species. In addition, microarray analysis was applied to explore the molecular basis of the seed coat defects in Petunia hybrida mutants, homozygous for a null allele of the AN11 gene, encoding a WDR transcription regulator. Among the transcripts differentially expressed in an11 seeds compared to wild type, many expected targets of AN11 were found but also several interesting new candidates that might play a role in morphogenesis of the seed coat. Our results validate the combination of next-generation sequencing with microarray analyses strategies to identify the transcriptome of two petunia species without previous knowledge of their genome, and to develop comprehensive chips as useful tools for the analysis of gene expression in P. axillaris, P. inflata and P. hybrida. The manuscript describes the creation by next generation sequencing of a large catalogue of the transcriptome of the two Petunia species, that are considered to represent the natural material from which the breeders selected their varieties. This submission represents the transcriptome component of study. The high throughput sequencing data were submitted to SRA (accession numbers: SRA027293, SRP004866.1, SRX036999.2, SRX036998.2).
Project description:MicroRNAs (miRNAs) are short, endogenous RNAs that play key roles in the regulation of numerous biological processes, including development and metabolic pathways. Here, we present a catalog of conserved miRNAs identified in small RNA data sets from young flower buds of Petunia axillaris (PaxiN) and P. inflata (PinfS6) on the basis of identity to miRNAs known from Arabidopsis and Solanaceae, and confirmed by the presence of (a) corresponding MIR loc(i)(us) in the Petunia genome sequences. We identified 44 miRNAs, belonging to 30 families, and covering 140 MIR loci, representing 120 miRNA*s. An additional 13 miRNAs overlapped at loci, supposedly representing additional family members. Our results are consistent with those in tomato and potato that showed 34 miRNA families, corresponding to 96 and 120 MIR genes, respectively, and other plant species, including monocots. The results are highly comparable between P. axillaris and P. inflata, that showed all but four MIR loci present in both species, and one quarter of the MIRs having identical sequences, whereas the remainder showed minor (< 5 %) sequence variation only. miRNA frequencies were also highly similar between P. axillaris and P. inflata and in good agreement with those reported for tomato and potato buds. miRNA target genes included well-known candidates, such as miRNA156-Squamosa Promoter Binding-like protein (SPL), and hints to flower development and anthocyanin biosynthesis regulatory pathways. The presented MIR sequences allow for functional analysis by using mutants. The high conservation between P. axillaris and P. inflata indicates the usability of our miRNA catalog in the entire genus.
Project description:Establishing the global proteomes of Petunia inflata flower protoplasts at S3 and S5 developmental stages to assess the presence of anthocyanin biosynthetic enzymes and determine the suitability of flowers in different developmental stages as material for transient expression of TurboID-tagged bait proteins for proximity labeling.
Project description:Proximity-dependent labeling allows untargeted determination of both direct and indirect protein interactions in vivo, and therefore stands as an attractive alternative to targeted binary assays for determining global proteinprotein interaction networks. We used TurboID-based proximity labeling to study protein interaction networks of the core phenylpropanoid and anthocyanin pathways in petunia. To do so, we coupled the endoplasmic reticulum (ER) membrane anchored cytochrome P450 cinnamic acid 4-hydroxylase (C4H, CYP73A412) from Petunia inflata to TurboID and expressed it in protoplasts derived from anthocyanin-rich petunia petals. We identified multiple soluble enzymes from the late anthocyanin pathway among enriched proteins, along with other C4H isoforms, and other ER membrane anchored CYPs. Several of these interactions were subsequently confirmed by bimolecular fluorescence complementation (BiFC).