Project description:We report the application of sequencing-by-synthesis technology for high-throughput profiling of small RNAs involved in Chalcone synthase A (CHS-A) sense cosuppression in petunia. Sense cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a transgene overexpressing the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. Though sense cosuppression is recognized as an RNA silencing mechanism, little evidence has been yet provided demonstrating its association with the generation of individual small interfering RNAs (siRNAs) that are the assumed determinants. In this work, the deep sequencing of small RNAs in cosuppressed transgenic petunia and WT petunia respectively allowed for the identication of siRNAs that vastly predominate in the silenced flower and guide prominent cleavage events in CHS-A mRNA.
Project description:We report the application of sequencing-by-synthesis technology for high-throughput profiling of small RNAs involved in Chalcone synthase A (CHS-A) sense cosuppression in petunia. Sense cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a transgene overexpressing the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. Though sense cosuppression is recognized as an RNA silencing mechanism, little evidence has been yet provided demonstrating its association with the generation of individual small interfering RNAs (siRNAs) that are the assumed determinants. In this work, the deep sequencing of small RNAs in cosuppressed transgenic petunia and WT petunia respectively allowed for the identication of siRNAs that vastly predominate in the silenced flower and guide prominent cleavage events in CHS-A mRNA. Examination of 2 small RNA populations from WT and cosuppressed petunia petals respectively
Project description:Deoxyhypusine synthase (DHS) is encoded by a nuclear gene and is the key enzyme involved in the post-translational activation of the eukaryotic translation initiation factor eIF5A. DHS plays important roles in plant growth and development. To gain a better understanding of DHS, the petunia (Petunia hybrida) PhDHS gene was isolated, and the role of PhDHS in plant growth was analysed. PhDHS protein was localized to the nucleus and cytoplasm. Virus-mediated PhDHS silencing caused a sectored chlorotic leaf phenotype. The chlorophyll levels was reducted and the development of chloroplasts was abnormal in PhDHS-silenced leaves These features were not observed in DHS-suppressed Arabidopsis thaliana and Solanum lycopersicum. These results indicated that PhDHS is required for development in petunia. A proteome assay showed that 308 proteins are up-regulated and 266 proteins are down-regulated in PhDHS-silenced plants compared with control. Among them, 20 proteins in photosystem I and photosystem II and 11 thylakoid proteins were down-regulated in PhDHS-silenced leaves, further supporting the involvement of PhDHS in photosynthesis in petunia.
Project description:Acetyl-coenzyme A (acetyl-CoA) is a central metabolite and the acetyl source for protein acetylation. The formation of cytosolic acetyl-CoA is catalyzed by ATP-citrate lyase (ACL) from citrate. However, the effects of acetyl-CoA on protein acetylation were not well known. In this study, four genes, PhACLA1, PhACLA2, PhACLB1, PhACLB2, encoding ACLA and ACLB submits in petunia (Petunia hybrida) were identified. VIGS-mediated PhACLA1-A2 and PhACLB1-B2 silencing leads to abnormal leave and flower development and reduced total anthocyanins content, while silencing of any single PhACL gene did not result in visible phenotype change. We quantitatively investigated the petunia proteome, acetylome, and the association between them in petunia corollas treated with pTRV2-PhACLB1-B2 and pTRV2. In total, 6200 protein groups were identified from petunia, among which 5343 proteins were quantified. A total of 345 proteins were up-regulated and 182 proteins were down-regulated (with a threshold of 1.2-fold) in PhACLB1-B2-silenced plant compared with the control (P <0.05). A total of 2210 lysine acetylation sites in 1148 protein groups were identified, among which 1744 sites in 921 proteins were accurately quantified (Additional file 2: Table S7). We subsequently used the quantification results of the global proteome to normalize the acetylome quantification data. From these, 68 sites in 54 lysine acetylation proteins were quantified as up-regulated targets and 40 sites in 38 lysine acetylation proteins were quantified as down-regulated targets at a threshold of 1.2 (P < 0.05). The global proteome and acetylome were negatively correlated, implying that proteome expression levels were negatively regulated by acetylation in PhACLB1-B2-silenced plants compared with the control.
Project description:Petunia floral scent production and emission is highly regulated, with a major role for the transcription factor ODORANT1 (ODO1) in directing activation of volatile biosynthesis. Using ChIP-seq of tagged ODO1 protein from petunia flowers, and RNA-seq of wild-type and odo1i RNAi flowers, the ODORANT1-regulated gene network of petunia is described, which extends to branches involved in phenylpropanoid intermediate production and S-adenosyl-methionine biosynthesis to potentiate production and emission of volatiles. Analysis of direct targets of regulation has also enabled the identification of an ODO1 binding motif.
Project description:Protein acetylation and crotonylation are important post-translational modifications (PTMs) of lysine. In animal cells, histone lysines are crotonylated or acetylated depends on the relative intracellular concentrations of crotonyl-CoA and acetyl-CoA, and adding crotonate to cell cultures, or reducing the intracellular levels of acetyl-CoA, leads to an increase in histone crotonylation through the direct production of crotonyl-CoA. However, in plant the relationship of acetylation and crotonylation and the effect of the concentration of acetyl-CoA on protein crotonylation were not well known. Our previous study showed that PhACL silencing changed the content of acetyl CoA in petunia corollas. In this study, we performed a global crotonylation proteome analysis of petunia (Petunia hybrida) and found that protein crotonylation have close relationship with protein acetylation and the protein with more crotonylation sites often had more acetylation sites. Crotonylated proteins and acetylated proteins enriched in many common KEGG pathway. However, PhACL silencing resulted in different KEGG pathway enrichment of proteins with different level of crotonylated sites and acetylated sites. Cytoplasm non-histone lysine crotonylation may not depend on the concentrations of acetyl-CoA in petunia corollas. There was a positive correlation between crotonylome and acetylome expression levels in corollas of PhACL-silenced plants and control.
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:To obtain insights about the roles of VvMYB5a and VvMYB5b, here we perform complementation analyses using petunia regulatory mutants impaired in pigment accumulation in flower epidermis, proven to be a valid tool for gene functional studies. We created three transgenic petunia lines overexpressing VvMYB5a, VvMYB5b and VvMYBA1 and we compared petal transcriptomes of each overexpressors with the untransformed one.
Project description:An inter-regional cortical tract is one of the most fundamental architectural motifs that integrates neural circuits to orchestrate and generate complex functions of the human brain. To understand the mechanistic significance of inter-regional projections on development of neural circuits, we investigated an in vitro neural tissue model for inter-regional connections, in which two cerebral organoids are connected with a bundle of reciprocally extended axons. The connected organoids produced more complex and intense oscillatory activity than conventional or directly fused cerebral organoids, suggesting the inter-organoid axonal connections enhance and support the complex network activity. In addition, optogenetic stimulation of the inter-organoid axon bundles could entrain the activity of the organoids and induce robust plasticity of the macroscopic circuit. These results demonstrated that the projection axons could serve as a structural hub that boosts functionality of the organoid-circuits. This model could contribute to further investigation on development and functions of macroscopic neuronal circuits in vitro.
Project description:Six different Solanaceae species, Potato (Solanum tuberosum), Tomato (Lycopersicum esculentum), Pepper (Capsicum annuum), Tobacco (Nicotiana tabacum), Petunia and Nicotiana benthamiana were grown at 25C, 16h light and 8h darkness. Mature leaves were harvested after 4-6 weeks. RNA was isolated using Qiagen RNeasy. Tomato, pepper, petunia tobacco and N. benthamiana samples were hybridized against potato samples. Keywords: Direct comaprison