The strawberry FaMYB1 transcription factor suppresses anthocyanin and flavonol accumulation in transgenic tobacco.

Fruit ripening is characterized by dramatic changes in gene expression, enzymatic activities and metabolism. Although the process of ripening has been studied extensively, we still lack valuable information on how the numerous metabolic pathways are regulated and co-ordinated. In this paper we describe the characterization of FaMYB1, a ripening regulated strawberry gene member of the MYB family of transcription factors. Flowers of transgenic tobacco lines overexpressing FaMYB1 showed a severe reduction in pigmentation. A reduction in the level of cyanidin 3-rutinoside (an anthocyanin) and of quercetin-glycosides (flavonols) was observed. Expression of late flavonoid biosynthesis genes and their enzyme activities were adversely affected by FaMYB1 overexpression. Two-hybrid assays in yeast showed that FaMYB1 could interact with other known anthocyanin regulators, but it does not act as a transcriptional activator. Interestingly, the C-terminus of FaMYB1 contains the motif pdLNL(D)/(E)Lxi(G)/S. This motif is contained in a region recently proposed to be involved in the repression of transcription by AtMYB4, an Arabidopsis MYB protein. Our results suggest that FaMYB1 may play a key role in regulating the biosynthesis of anthocyanins and flavonols in strawberry. It may act to repress transcription in order to balance the levels of anthocyanin pigments produced at the latter stages of strawberry fruit maturation, and/or to regulate metabolite levels in various branches of the flavonoid biosynthetic pathway.

Transcriptional and posttranscriptional gene silencing are mechanistically related.

Two distinct gene-silencing phenomena are observed in plants: transcriptional gene silencing (TGS), which involves decreased RNA synthesis because of promoter methylation, and posttranscriptional gene silencing (PTGS), which involves sequence-specific RNA degradation. PTGS is induced by deliberate [1-4] or fortuitous production (R.v.B., unpublished data) of double-stranded RNA (dsRNA). TGS could be the result of DNA pairing [5], but could also be the result of dsRNA, as was shown by the dsRNA-induced inactivation of a transgenic promoter [6]. Here, we show that when targeting flower pigmentation genes in Petunia, transgenes expressing dsRNA can induce PTGS when coding sequences are used and TGS when promoter sequences are taken. For both types of silencing, small RNA species are found, which are thought to be dsRNA decay products [7] and determine the sequence specificity of the silencing process [8, 9]. Furthermore, silencing is accompanied by the methylation of DNA sequences that are homologous to dsRNA. DNA methylation is assumed to be essential for regulating TGS and important for reinforcing PTGS [10]. Therefore, we conclude that TGS and PTGS are mechanistically related. In addition, we show that dsRNA-induced TGS provides an efficient tool to generate gene knockouts, because not only does the TGS of a PTGS-inducing transgene fully revert the PTGS phenotype, but also an endogenous gene can be transcriptionally silenced by dsRNA corresponding to its promoter.

Role of inverted DNA repeats in transcriptional and post-transcriptional gene silencing.

Transgenes and endogenous genes are sensitive to silencing, in particular when the genes are tandemly repeated. Their expression can be transcriptionally or post-transcriptionally repressed, or both. It is remarkable that very often, two or more genes or parts of the genes are arranged as inverted repeats (IR). Many of such IRs are dominant silencing loci. They can repress the expression of homologous genes elsewhere in the genome in trans which is usually associated with an increase in the level of DNA methylation. Trans-silencing has been explained by DNA-DNA pairing between a repetitive silencing locus and a homologous target locus. However, there is accumulating evidence that the trans effect might be mediated by dsRNA transcribed from the IR (trans)genes. Besides dsRNA-directed DNA methylation, dsRNA in plants as well as in other systems also induces the degradation of homologous RNAs and silence genes post-transcriptionally. These findings indicate that several features associated with gene silencing can be attributed to the activities of dsRNA, which would explain why inverted transgene repeats are such efficient silencing loci.

anthocyanin1 of petunia encodes a basic helix-loop-helix protein that directly activates transcription of structural anthocyanin genes.

The petunia loci anthocyanin1 (an1), an2, an4, and an11 are required for the transcription of anthocyanin biosynthetic genes in floral organs. The an2 and an11 loci were recently cloned and shown to encode a MYB-domain transcriptional activator and a cytosolic WD40 protein, respectively. Here, we report the isolation of an1 by transposon tagging. an1 encodes a new member of the basic helix-loop-helix family of transcription factors that is functionally and evolutionarily distinct from JAF13, the apparent petunia ortholog of maize RED1 and snapdragon DELILA. We provide genetic evidence that the transcription factors encoded by an1, an2, and an4 operate in an unexpectedly complex regulatory hierarchy. In leaves, ectopic expression of AN2 induces an1 expression, whereas in anthers, an1 expression depends on an4, encoding (or controlling) a MYB protein that is paralogous to AN2. Experiments with transgenic plants expressing a post-translationally controlled AN1-GLUCOCORTICOID RECEPTOR fusion protein indicated that independent of protein synthesis, AN1 directly activates the expression of the dfrA gene encoding the enzyme dihydroflavonol 4-reductase and of Pmyb27 encoding a MYB-domain protein of unknown function.

Distinct features of post-transcriptional gene silencing by antisense transgenes in single copy and inverted T-DNA repeat loci.

The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post-transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not sufficient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.

Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.

Posttranscriptional silencing of chalcone synthase (Chs) genes in petunia transformants occurs by introducing T-DNAs that contain a promoter-driven or promoterless Chs transgene. With the constructs we used, silencing occurs only by T-DNA loci which are composed of two or more T-DNA copies that are arranged as inverted repeats (IRs). Since we are interested in the mechanism by which these IR loci induce silencing, we have analyzed different IR loci and nonsilencing single-copy (S) T-DNA loci with respect to the expression and methylation of the transgenes residing in these loci. We show that in an IR locus, the transgenes located proximal to the IR center are much more highly methylated than are the distal genes. A strong silencing locus composed of three inverted T-DNAs bearing promoterless Chs transgenes was methylated across the entire locus. The host Chs genes in untransformed plants were moderately methylated, and no change in methylation was detected when the genes were silenced. Run-on transcription assays showed that promoter-driven transgenes located proximal to the center of a particular IR are transcriptionally more repressed than are the distal genes of the same IR locus. Transcription of the promoterless Chs transgenes could not be detected. In the primary transformant, some of the IR loci were detected together with an unlinked S locus. We observed that the methylation and expression characteristics of the transgenes of these S loci were comparable to those of the partner IR loci, suggesting that there has been cross talk between the two types of loci. Despite the similar features, S loci are unable to induce silencing, indicating that the palindromic arrangement of the Chs transgenes in the IR loci is critical for silencing. Since transcriptionally silenced transgenes in IRs can trigger posttranscriptional silencing of the host genes, our data are most consistent with a model of silencing in which the transgenes physically interact with the homologous host gene(s). The interaction may alter epigenetic features other than methylation, thereby impairing the regular production of mRNA.

Analysis of bHLH and MYB domain proteins: species-specific regulatory differences are caused by divergent evolution of target anthocyanin genes.

The regulatory anthocyanin loci, an1, an2, an4 and an11 of Petunia hybrida, and r and c1 from Zea mays, control transcription of different sets of target genes. Both an2 and c1 encode a MYB-type protein. This study reports the isolation of a P. hybrida gene, jaf13, encoding a basic helix-loop-helix protein that, on the basis of sequence homology and intron/exon structure, represents the P. hybrida orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 is sufficient for activation of the dihydroflavonol 4-reductase-A (dfrA) promoter and enhanced pigment accumulation in P. hybrida. This indicates that an2 and jaf13 play a key role in determining the tissue-specific expression pattern of structural genes. However, because chalcone synthase (chs) and flavanone-3-hydroxylase (f3h) are not activated, the pattern of pigmentation is not fundamentally altered. Expression of an2 in Z. mays complements a mutation in pl, a c1 paralogue, indicating that an2 activates a wider set of target genes in this host. Transient expression assays in Z. mays and P. hybrida tissues showed that C1 and R or AN2 and JAF13 can activate the promoter of the c2 gene, encoding Z. mays CHS, but not the chsA promoter from P. hybrida. These results indicate that regulatory anthocyanin genes are conserved between species and that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks.

Genetic control of dihydroflavonol 4-reductase gene expression in Petunia hybrida.

The functions of four loci (An1, An2, An4, and An6) which control pigmentation in flowers of Petunia hybrida have been characterized. Linkage-analysis and molecular complementation experiments showed that the An6 locus contains the structural dfrA gene, encoding the enzyme dihydroflavonol 4-reductase (DFR). Analysis of gus gene expression driven by the dfrA promoter in transgenic plants showed that the dfrA promoter is highly active in the flower corolla, the anthers and seeds and, at a lower level, in ovules and the flower stem. These data are discussed in relation to the expression of other pigmentation genes and the accumulation pattern of anthocyanins. The expression of the drfA-gus transgene was dependent on the genes an1 (in every tissue), an2 (in the flower limb only) and an4(in anthers), demonstrating that these genes encode regulatory factors that control drfA promoter activity.

Conditional inhibition of beta-glucuronidase expression by antisense gene fragments in petunia protoplasts.

Antisense RNA-mediated inhibition of gene expression is a valuable tool to induce mutant phenotypes. We are interested in the application of antisense gene fragments with the aim to improve the efficiency of inhibition and to be able to selectively suppress gene family members in plants. Protoplasts may provide a rapid system to screen the efficiency of antisense gene segments. As a first step, we set up a transient expression system for leaf protoplasts of Petunia hybrida and used as a model system the inhibition of beta-glucuronidase (uidA) expression by uidA antisense gene segments. Both GUS enzyme activities and uidA RNA levels were measured. Co-introducing equal amounts of a full-length uidA antisense gene and a uidA sense gene reduced GUS activity by 60-70%. Various uidA antisense fragments also inhibited expression although with different efficiencies and we show that strong antisense fragments can be retrieved from weak antisense gene fragments. A promoter-less antisense gene did not reduce uidA expression indicating that the inhibition is mediated by antisense transcripts. Using quantitative PCR on first-strand cDNA we show that expression of functional antisense genes lead to reduced levels of uidA mRNA. This suggests that the mechanism of antisense RNA inhibition in protoplasts is similar to that in transgenic plants and that the protoplast system in combination with PCR can be used to preselect antisense fragments of any gene.

The petunia homologue of the Antirrhinum majus candi and Zea mays A2 flavonoid genes; homology to flavanone 3-hydroxylase and ethylene-forming enzyme.

The synthesis of anthocyanins in higher plants involves many enzymatic steps. Here we describe the isolation and characterization of a cDNA, ant17, which encodes a protein that has 73% amino acid sequence identity with the candi gene product of Antirrhinum majus and 48% with that of the maize a2 gene. This protein may therefore be involved in the synthesis of anthocyanins in the steps after the action of dihydroflavonol 4-reductase. This is consistent with the absence of ant17 expression in the regulatory anthocyanin mutants of petunia an1, an2 and an11. Furthermore, ant17 is predominantly expressed in corollas and anthers and is induced by gibberellic acid.

Establishment of hairy root cultures of Linum flavum producing the lignan 5-methoxypodophyllotoxin.

Hairy root cultures were induced from leaf explants of Linum flavum by infection with Agrobacterium rhizogenes. The transformed nature of tissue was confirmed by the production of opines. The cultures produced 1.5 to 3.5% of the lignan 5-methoxypodophyllotoxin (5-MPT) on a dry weight basis, which was 2 to 5 times higher than the 5-MPT content in untransformed root cultures and 5 to 12 times higher than in L. flavum cell suspensions. The 5-MPT production as a function of time was up to four times higher than that in cell suspensions.

Flavonols stimulate development, germination, and tube growth of tobacco pollen.

The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 mum), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.

Differential expression of two MADS box genes in wild-type and mutant petunia flowers.

We isolated and characterized two flower-specific genes from petunia. The protein products of these genes, designated floral binding protein 1 (FBP1) and 2 (FBP2), are putative transcription factors with the MADS box DNA binding domain. RNA gel blot analysis showed that the fbp1 gene is exclusively expressed in petals and stamen of petunia flowers. In contrast, the FBP1 protein was only detectable in petals and not in stamens, suggesting post-transcriptional regulation of the fbp1 gene in these tissues. The fbp2 gene is expressed in petals, stamen, carpels, and at a very low level in sepals but not in vegetative tissues. We analyzed the spatial expression of these fbp genes in floral organs of two homeotic flower mutants. In the blind mutant, whose flower limbs are transformed into antheroid structures on top of normal tubes, identical expression levels of both genes were observed in the antheroid structures as in normal anthers. In the homeotic mutant green petals, the petals are replaced by sepaloid organs in which the expression of fbp1 is strongly reduced but not completely abolished. Our results suggest a regulation of the fbp1 gene expression by the green petals (gp) gene. Expression of the fbp2 gene was not affected in the green petals mutant. In contrast to the proposed models describing floral morphogenesis, our data indicated that homeotic genes can be functional in one whorl only.

The TACPyAT repeats in the chalcone synthase promoter of Petunia hybrida act as a dominant negative cis-acting module in the control of organ-specific expression.

Analysis of the expression of the GUS reporter gene driven by various regions of the Petunia hybrida chalcone synthase (chsA) promoter revealed that the developmental and organ-specific expression of the chsA gene is conferred by a TATA proximal module located between -67 and -53, previously designated as the TACPyAT repeats. Histochemical analysis of GUS reporter gene expression revealed that the organ-specific 67 bp promoter fragment directs the same cell-type specificity as a 530 bp promoter, whereas additional enhancer sequences are present within the more TATA distal region. Moreover, the region between -800 and -530 is also involved in extending the cell-type specificity to the trichomes of flower organs and of young seedlings. The mechanism by which the TACPyAT repeats modulate expression during plant development was studied by analysing the expression of the GUS gene driven by chimeric promoters consisting of the CaMV 35S enhancer (domain B, -750 to -90) fused to various chsA 5' upstream sequences. Detailed enzymatic and histochemical analysis revealed that in the presence of the TACPyAT module the CaMV 35S region only enhances GUS activity in those organs in which the chsA promoter is normally active. Furthermore, this analysis shows that enhancement in the presence of the CaMV 35S domain B is accomplished by increasing the number of cell types expressing the GUS gene within the organ, rather than enhancement of the chsA cell-type-specific expression within these organs. Deletion of the TACPyAT sequences in the chimeric promoter construct completely restores the well-documented CaMV 35S domain B cell-type specificity, showing that the TACPyAT module acts as a dominant negative cis-acting element which controls both organ and developmental regulation of the chsA promoter activity.

Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility.

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.

Transactivation of Ds by Ac-transposase gene fusions in tobacco.

To study regulation of the (Ds) transposition process in heterologous plant species, the transposase gene of Ac was fused to several promoters that are active late during plant development. These promoters are the flower-specific chalcone synthase A promoter (CHS A), the anther-specific chalcone isomerase B promoter CHI B and the pollen-specific chalcone isomerase A2 promoter CHI A2. The modified transposase genes were introduced into a tobacco tester plant. This plant contains Ds stably inserted within the leader sequence of the hygromycin resistance (HPT II) gene. As confirmed with positive control elements, excision of Ds leads to the restoration of a functional HPT II gene and to a hygromycin resistant phenotype. No hygromycin resistance was observed in negative control experiments with Ac derivatives lacking 5' regulatory sequences. Although transactivation of Ds was observed after the introduction of transposase gene fusions in calli, excision in regenerated plants was observed only for the CHS A- or CHI B-transposase gene fusions. With these modified transposase genes, somatic excision frequencies were increased (68%) and decreased (22%), respectively, compared to the situation with the Ac element itself (38%). The shifts in transactivation frequencies were not associated with significant differences in the frequencies of germinally transmitted excision events (approximately 5%). The relative somatic stability of Ds insertions bearing the CHI B-transposase gene fusion suggests the usefulness of this activator element for transposon tagging experiments.

Gibberellic Acid Regulates Chalcone Synthase Gene Transcription in the Corolla of Petunia hybrida.

The pigmentation of Petunia hybrida corollas is regulated by gibberellic acid (GA(3)). It controls the increase of flavonoid enzyme levels and their corresponding mRNAs. We have used an in vitro culture system for corollas to study the regulatory role of GA(3) in the expression of flavonoid genes. By determining steady-state mRNA levels, we show that the accumulation of chalcone synthase (chs) mRNA in young corollas is dependent on the presence of both sucrose and GA(3) in the culture medium. Whereas sucrose had a general metabolic effect on gene expression, the stimulatory role of GA(3) was specific. Analysis of nascent transcripts in isolated corolla nuclei showed that changes in steady-state chs mRNA levels correlated very well with changes in the transcription rate. We therefore conclude that GA(3) controls the expression of chs at the transcriptional level. Preculturing the corollas in sucrose medium without GA(3) resulted in a lower chs mRNA level. The expression could be reinduced by the addition of GA(3). The hormone is thus required for the induction but also for the maintenance of chs transcription. The delayed reinduction of chs expression, the lag time in the kinetics of chs mRNA accumulation, and the inhibitory effect of cycloheximide on the action of GA(3) suggest that GA(3) controls chs transcription in an indirect manner. Our data support a model in which GA(3) induces the production of a regulatory protein such as a receptor or a trans-acting factor that is directly involved in chs transcription.

Regulation and manipulation of flavonoid gene expression in anthers of petunia: the molecular basis of the Po mutation.

Molecular mechanisms governing development of the male reproductive organs of flowers, the anthers, are largely unknown. In this article, we report on the investigation of the molecular basis of a mutation involving the expression of a gene encoding the flavonoid biosynthesis enzyme chalcone flavanone isomerase (CHI) in anthers of petunia. In petunia, the gene Po regulates the expression of CHI in anthers: PoPo petunia lines contain CHI enzyme activity in petals and anthers, whereas popo lines contain the CHI enzyme only in petals but not in anthers. As a result of the Po mutation, the substrate of CHI accumulates and therefore the pollen of a popo line are yellow or greenish. The genome of petunia contains two chi genes, chiA and chiB. In a restriction fragment length polymorphism analysis, a 100% linkage was observed between Po and chiA. This result suggested that Po is identical to chiA and that Po is not a regulatory gene of chiA. Introduction of a chiA gene isolated from a PoPo line into a popo line resulted in a complementation of the mutation that was directly visible because the pollen color shifted from yellow to white. This proved that chiA and Po are identical. Because chiA encodes a functional CHI enzyme in flower petals of a popo line, we propose that the Po mutation is a mutation in the regulatory region of chiA abolishing chiA promoter activity in anthers but not in corollas. This change in anther color is a fine illustration of how floral pigmentation can be manipulated in a predictable way and suggests the use of CHI as a visible marker.

Stamens and Gibberellic Acid in the Regulation of Flavonoid Gene Expression in the Corolla of Petunia hybrida.

Stamen removal at an early stage of flower development inhibits anthocyanin synthesis and chalcone flavanon isomerase (CHI) enzyme activity in corollas of Petunia hybrida. The inhibition can be overcome by gibberellic acid (GA(3)) application. Gibberellin also induces anthocyanin synthesis in detached, young green corollas, grown in vitro in a sucrose medium and promotes CHI enzyme activity. Western blot analysis indicates an increase in chalcone synthase (CHS) and CHI protein levels following GA(3) treatment in both the in vivo and the in vitro systems. Northern blot analysis shows a higher level of steady-state mRNAs for CHS and CHI 24 hours after GA(3) application. In corollas from a transgenic plant containing a beta-glucuronidase gene driven by a CHI promoter, a sixfold increase of beta-glucuronidase activity was measured following GA(3) application. The mode of action of stamens and GA(3) control over flavonoid gene expression is discussed.