Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats (original) (raw)
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Nucleic Acids Research, 1986
Twenty independent, petal-specific chalcone synthase (CHS) cDNA clones have been isolated from Petunia hybrida variety Violet 30 (V30). Sequence analysis shows that the largest of these clones contains the entire coding sequence. Using this clone in Southern blot analysis reveals the presence of multiple CHS gene copies in the genome of Petunia hybrida V30. Hybridization and sequence analysis of the CHS cDNA clones shows that they are all copied from a single mRNA species. This indicates the presence of only one transcriptionally active CHS gene in petals. Finally we report the identification, cloning and partial characterization of this gene. Plarnt variety, plasmids, phages and bacterial strains are listed in
Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types
THE PLANT CELL ONLINE, 1990
Chalcone synthase (CHS) catalyzes the first step in the biosynthesis of flavonoids that function in flower pigmentation, protection against stress, and induction of nodulation. The petunia genome contains eight complete chs genes, of which four are differentially expressed in floral tissues and UV-light-induced seedlings. The 5'-flanking regions of these four chs genes were fused to the 8-glucuronidase (GUS) reporter gene and introduced into petunia plants by Agrobacterium-mediated transformation. We show that expression of each construct is identical to the expression of the authentic chs gene, implying that the differences in expression pattern between these chs genes are caused at least in part by their promoters. Histochemical analyses of GUS expression show that chs promoters are not only active in pigmented cell types (epidermal cells of the flower corolla and tube and [sub] epidermal cells of the flower stem) but also in a number of unpigmented cell types (mesophylic cells of the corolla, several cell types in the ovary and the seed coat). Comparison of chs-GUS expression and flavonoid accumulation patterns in anthers suggests that intercellular transport of flavonoids and enzymes occurs in this organ. Analysis of the flavonoids accumulated in tissues from mutant lines shows that only a subset of the genes that control flavonoid biosynthesis in the flower operates in the ovary and seed. This implies that (genetic) control of flavonoid biosynthesis is highly tissue specific.
Plant Cell, 1990
We attempted to overexpress chalcone synthase (CHS) in pigmented petunia petals by introducing a chimeric petunia CHS gene. Unexpectedly, the introduced gene created a block in anthocyanin biosynthesis. Forty-two percent of plants with the introduced CHS gene produced totally white flowers and/or patterned flowers with white or pale nonclonal sectors on a wild-type pigmented background; none of hundreds of transgenic control plants exhibited such phenotypes. Progeny testing of one plant demonstrated that the novel color phenotype co-segregated with the introduced CHS gene; progeny without this gene were phenotypically wild type. The somatic and germina1 stability of the novel color patterns was variable. RNase protection analysis of petal RNAs isolated from white flowers showed that, although the developmental timing of mRNA expression of the endogenous CHS gene was not altered, the leve1 of the mRNA produced by this gene was reduced 50-fold from wild-type levels. Somatic reversion of plants with white flowers to phenotypically parenta1 violet flowers was associated with a coordinate rise in the steady-state levels of the mRNAs produced by both the endogenous and the introduced CHS genes.
Plant Molecular Biology, 1989
Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.
Chalcone isomerase in flowers of mutants of Petunia hybrida
Planta, 1983
The effect of the gene Po on the activity of chalcone isomerase was investigated in Petunia hybrida. Furthermore, isomerase activities isolated from petals were compared with those extracted from anthers. No effect of Po on the pH-dependence of the isomerase and its kinetic properties was observed. With respect to these criteria, the enzyme extracted from anthers behaved in an identical
Plant Cell, 1990
To evaluate the effect of increased expression of genes involved in flower pigmentation, additional dihydroflavonol-4-reductase (DFR) or chalcone synthase (CHS) genes were transferred to petunia. In most transformants, the increased expression had no measurable effect on floral pigmentation. Surprisingly, however, in up to 25% of the transformants, a reduced floral pigmentation, accompanied by a dramatic reduction of DFR or CHS gene expression, respectively, was observed. This phenomenon was obtained with both chimeric gene constructs and intact CHS genomic clones. The reduction in gene expression was independent of the promoter driving transcription of the transgene and involved both the endogenous gene and the homologous transgene. The gene-specific collapse in expression was obtained even after introduction of only a single gene copy. The similarity between the sense transformants and regulatory CHS mutants suggests that this mechanism of gene silencing may operate in naturally occurring regulatory circuits.
Chalcone synthase as a reporter in virus-induced gene silencing studies of flower senescence
Plant Molecular Biology, 2004
Agrobacterium-mediated infection of petunia (Petunia hybrida) plants with tobacco rattle virus (TRV) bearing fragments of Petunia genes resulted in systemic infection and virus-induced gene silencing (VIGS) of the homologous host genes. Infection with TRV containing a phytoene desaturase (PDS) fragment resulted in reduced abundance of PDS transcripts and typical photobleaching of photosynthetic tissues. Infection with TRV containing a chalcone synthase (CHS) fragment resulted in silencing of anthocyanin production in infected flowers. The silencing phenotype ranged from scattered white spots on the normal purple background to entirely white flowers. Symptoms in the V26 cultivar were a diffuse mosaic, but infection of some purple-flowered commercial cultivars resulted in large white sectors and even entirely white flowers. Abundance of CHS transcripts in the white flowers was less than 4% of that in purple flowers on the same plant. Infection with TRV containing a tandem construct of PDS and CHS resulted in leaf photobleaching and white patterns on the flowers. Transcripts of CHS and PDS were reduced both in leaves and in flowers confirming simultaneous silencing of both genes by the tandem construct. We tested the effects of infection with TRV containing CHS and a fragment of a petunia gene encoding for 1aminocyclopropane-1-carboxylate oxidase (ACO4) Abundance of transcripts encoding ACO4 and ACO1 were reduced (by 5% and 20%, respectively) in infected flowers. Whether the flowers were treated with ACC or pollinated, the white (silenced) flowers or flower sectors produced less ethylene and senesced later than purple (non-silenced) tissues. These results indicate the value of VIGS with tandem constructs containing CHS as reporter and a target gene as a tool for examining the function of floral-associated genes.
Chalcone Synthase and Flavonol Accumulation in Stigmas and Anthers of Petunia hybrida
1993
Flavonol aglycones are required for pollen germination in pe- tunia (Petunia hybrida 1.). Mutant plants lacking chalcone synthase (CHS), which catalyzes the first committed step in flavonoid syn- thesis, do not accumulate flavonols and are self-sterile. lhe mutant pollen can be induced to germinate by supplementing it with kaempferol, a flavonol aglycone, either at the time of pollination or by
Plant Journal, 1998
A single-copy sense Chalcone synthase (Chs) transgene driven by a strong promoter and producing a fully translatable transcript was converted to an allelic antisense Chs transgene by Cre-lox-mediated DNA recombination in petunia. The sense Chs allele suppressed flower pigmentation in a simple pattern determined by cells at the junctions between adjacent petals, as is typical of single-copy sense Chs transgenes of this type, whereas the antisense Chs allele produced a different pattern of Chs suppression with white petal edges and reduced pigmentation throughout the petal limbs, as is typical of antisense Chs transgenes. In plants carrying a lox-flanked Chs transgene, the presence of Cre protein can cause both sense-specific and antisense-specific patterns to be superimposed in the same flower, suggesting that sense and antisense suppression by single-copy transgenes are mediated by different mechanisms or occur in different cellular or developmental compartments. The presence of Cre also causes the production of numerous, non-clonal white spots, suggesting that the turnover state is not cell-autonomous.
Plant Cell Reports
The first enzyme in the flavonoid pathway, chalcone synthase, is encoded by a gene (CHS) whose expression is normally under developmental control. In our previous studies, an 896-bp promoter region of a flower-specific CHS gene was isolated from Lilium orential ‘Sorbonne’, and designated as PLoCHS. Here, the PLoCHS promoter was fused to the β-glucuronidase (GUS) gene to characterize its spatial and temporal expression in Petunia hybrida ‘Dreams Midnight’ using an Agrobacterium-mediated leaf disc transformation method. Our results demonstrated that GUS expression was present in flowers, but reduced or absent in the other tissues (leaf and stem) examined. In petals, GUS activity reached its peak at flower developmental stage 4, and decreased at later stages. Deletion analysis indicated that even a 307-bp fragment of the PLoCHS promoter could still direct flower-specific expression. Further deletion of the region from −261 to −72 bp resulted in weak expression in different organs, including flowers, leaves and stems. This evidence combined with prediction of cis-acting elements in the PLoCHS promoter suggests that the TACPyAT box located in this promoter plays a key role in the regulation of organ-specific expression.