Chalcone Synthase Promoters in Petunia Are Active in Pigmented and Unpigmented Cell Types.

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[prime]-flanking regions of these four chs genes were fused to the [beta]-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.

Overexpression of petunia chalcone isomerase in tomato results in fruit containing increased levels of flavonols.

Tomatoes are an excellent source of the carotenoid lycopene, a compound that is thought to be protective against prostate cancer. They also contain small amounts of flavonoids in their peel ( approximately 5-10 mg/kg fresh weight), mainly naringenin chalcone and the flavonol rutin, a quercetin glycoside. Flavonols are very potent antioxidants, and an increasing body of epidemiological data suggests that high flavonoid intake is correlated with a decreased risk for cardiovascular disease. We have upregulated flavonol biosynthesis in the tomato in order to generate fruit with increased antioxidant capacity and a wider range of potential health benefit properties. This involved transformation of tomato with the Petunia chi-a gene encoding chalcone isomerase. Resulting transgenic tomato lines produced an increase of up to 78 fold in fruit peel flavonols, mainly due to an accumulation of rutin. No gross phenotypical differences were observed between high-flavonol transgenic and control lines. The phenotype segregated with the transgene and demonstrated a stable inheritance pattern over four subsequent generations tested thus far. Whole-fruit flavonol levels in the best of these lines are similar to those found in onions, a crop with naturally high levels of flavonol compounds. Processing of high-flavonol tomatoes demonstrated that 65% of flavonols present in the fresh fruit were retained in the processed paste, supporting their potential as raw materials for tomato-based functional food products.

High level fructan accumulation in a transgenic sugar beet.

We have transformed sugar beet into a crop that produces fructans. The gene encoding 1-sucrose:sucrose fructosyl transferase (1-SST), which was isolated from Helianthus tuberosus, was introduced into sugar beet. In H. tuberosus, 1-SST mediates the first steps in fructan synthesis through the conversion of sucrose (GF) into low molecular weight fructans GF2, GF3, and GF4. In the taproot of sugar beet transformed with the 1-sst gene, the stored sucrose is almost totally converted into low molecular weight fructans. In contrast, 1-sst expression in the leaves resulted in only low levels of fructans. Despite the storage carbohydrate having been altered, the expression of the 1-sst gene did not have any visible effect on phenotype and did not affect the growth rate of the taproot as observed under greenhouse conditions.

Regulation of plant gene expression by antisense RNA.

Regulation of gene expression by antisense RNA was first discovered as a naturally-occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down-regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that 'sense' genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA-RNA interaction.

The application of DNA microarrays in gene expression analysis.

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Promoter activity of a putative pollen monosaccharide transporter in Petunia hybrida and characterisation of a transposon insertion mutant.

For the growth of the male reproductive cells of plants, the pollen, the presence of sufficient sucrose or monosaccharides is of vital importance. From Petunia hybrida a pollen-specific putative monosaccharide transporter designated PMT1 (for petunia monosaccharide transporter) has been identified previously. The present work provides an in-depth analysis and characterisation of PMT1 in the context of pollen development with the GUS reporter gene and an insertion mutant. The promoter of the pollen-specific putative PMT1 gene has been isolated by inverse PCR and sequenced. Analysis of plants transformed with the promoter-GUS fusion confirmed the specificity of this gene, belonging to the late pollen-specific expressed genes. GUS activity was detected even after 24 h of in vitro pollen germination, at the pollen tube tip. To elucidate the importance of PMT1 for gametophyte development and fertilisation, we isolated a mutant plant containing a transposon insertion in the PMT1 gene by the dTph1 transposon-tagging PCR-based assay. The PMT1 mutant contained a dTph1 insertion in position 1474 bp of the transcribing part of the gene, before the last two transmembrane-spanning domains. Analysis of the progeny of the heterozygous mutant after selfing revealed no alterations in pollen viability and fertility. Mature pollen grains of a plant homozygous for the transposon insertion were able to germinate in vitro in a medium containing sucrose, glucose, or fructose, which indicates that PMT1 is not essential for pollen survival. Several explanations for these results are discussed in the present work.

A novel purification procedure for chalcone flavanone isomerase from Petunia hybrida and the use of its antibodies to characterize the Po mutation.

We have purified chalcone flavanone isomerase (CHI) from flowerbuds of Petunia hybrida to high purity. We made use of an affinity matrix consisting of Sepharose-bound Dextran Blue that is known to bind proteins containing the dinucleotide fold [S. T. Thompson, K. H. Cass, and E. Stellwagen (1975) Proc. Natl. Acad. Sci. USA 72, 669-672]. The final step, consisting of preparative elution from a denaturing acrylamide gel, yielded an approximately 2000-fold purified CHI protein. The enzyme is a single polypeptide with Mr = 29,000, and highly specific antiserum was raised against it. Using this antiserum it was shown that corolla and anther tissues express different forms of the enzyme as judged by pI. Furthermore, the absence of immunoreactive CHI was demonstrated in a mutant of P. hybrida (genotype popo) which accumulates 2',4,4',6'-tetrahydroxy-chalcone in anthers as a consequence of lack of enzyme activity.

Flavonols are not essential for fertilization in Arabidopsis thaliana.

Flavonols are plant metabolites suggested to serve a vital role in fertilization of higher plants. Petunia and maize plants mutated in their flavonol biosynthesis are not able to set seed after self-pollination. We have investigated the role of these compounds in Arabidopsis thaliana. Like in all other plant species, high levels of flavonols could be detected in pollen of wild-type A. thaliana. No flavonols were detected in reproductive organs of the A. thaliana tt4 mutant in which the chs gene is mutated. Surprisingly, this mutant did set seed after self-fertilization and no pollen tube growth aberrations were observed in vivo. The role of flavonols during fertilization of Arabidopsis is discussed.

Petal and stamen formation in petunia is regulated by the homeotic gene fbp1.

For Arabidopsis and Antirrhinum, the so-called ABC model has been developed, which postulates that the determination of floral organ primordia is controlled by the action of three classes of homeotic genes. A number of these ABC genes encode putative transcription factors with the MADS box DNA binding motif. This paper reports on the functional analysis of the petunia MADS box gene fbp1. The temporal and spatial expression of fbp1 has been investigated in detail in transgenic plants containing the beta-glucuronidase (GUS) reporter gene fused to an fbp1 promoter fragment. fbp1-driven GUS activity was specifically detected in emerging petal and stamen primordia, suggesting a function of fbp1 in the control of second and third floral whorl identity. To test this hypothesis, transgenic petunia plants were generated in which fbp1 expression was inhibited by a co-suppression approach. The flowers of such plants exhibited homeotic conversions of petals towards sepals and stamens towards carpels. Occasionally, the third whorl carpels are fused forming a pentalocular gynoecium. This dominant fbp1 mutation acted as a single Mendelian trait in genetic crosses. These results strongly indicate that fbp1 is a petunia class B homeotic gene which is required for the correct initiation and determination of petals and stamens.

Regulation of chalcone flavanone isomerase (CHI) gene expression inPetunia hybrida: the use of alternative promoters in corolla, anthers and pollen.

In this paper we report on the organization and expression of the two chalcone flavanone isomerase (CHI) genes A and B from thePetunia hybrida inbred line V30. From a combination of sequence data, primer extension and RNAse protection experiments we infer the presence of two promoters PA1 and PA2 upstream of the CHI gene A coding region. It is shown that both promoters are used differentially in various flower tissues: the PA1 promoter is active in corolla and tube tissue whereas the PA2 promoter, which gives rise to a 437 bp longer transcript, is only active in late stages of anther development and more specifically in pollen grains. The CHI-B gene, on the other hand, has only one promoter (PB) which is active only in immature anther tissue. Thus, in addition to the use of two alternative promoters in front of the same CHI coding region (CHI-A), the promoters in front of the two distinct CHI gene copies are also used differentially as a mechanism to regulate their expression. Comparison of PB with other flavonoid gene promoters active in immature anther tissue revealed a highly conserved region which was designated as 'anther box'. We hypothesize that it plays a regulatory role in anther-specific gene expression. Finally, a model describing the evolutionary relationship between both CHI genes is presented.

Co-suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem.

The function of the petunia MADS box gene fbp2 in the control of floral development has been investigated. Inhibition of fbp2 expression in transgenic plants by a co-suppression approach resulted in the development of highly aberrant flowers with modified whorl two, three and four organs. This mutant flower phenotype inherited as a single Mendelian trait. The flowers possess a green corolla which is reduced in size. Furthermore, the stamens are replaced by green petaloid structures and the inner gynoecial whorl is dramatically reduced. No ovules or placenta are formed and instead two new inflorescences developed in the axils of the carpels. These homeotic transformations are accompanied by a complete down-regulation of the petunia MADS box gene fbp6 which is highly homologous to the Arabidopsis and Antirrhinum genes agamous (ag) and plena (ple). In contrast to this, two other petunia MADS box genes, exclusively expressed in whorls two and three, are still transcribed. Our results indicate that the fbp2 gene belongs to a new class of morphogenesis genes involved in the determination of the central part of the generative meristem.

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.

Functional interaction between the homeotic genes fbp1 and pMADS1 during petunia floral organogenesis.

The petunia MADS box floral binding protein (fbp) gene 1 represents a class B homeotic gene determining the identity of second and third floral whorl organs. Suppression of fbp1, which is highly homologous to the Antirrhinum gene globosa and Arabidopsis gene pistillata, results in the conversion of petals to sepals and stamens to carpels. In contrast to fbp1, the petunia homeotic gene pMADS1, encoding a protein homologous to the Antirrhinum protein DEFICIENS, has been shown to be involved in the formation of petals only. We demonstrated that the induction of fbp1 is established independent of pMADS1, whereas at later developmental stages, fbp1 is up-regulated by pMADS1 in petals. On the other hand, the induction and maintenance of pMADS1 expression are not affected by fbp1. To obtain information about the functional interaction between fbp1 and pMADS1, an fbp1 cosuppression mutant with mild phenotypic alterations was crossed with a green petals mutant in which pMADS1 expression was abolished. Progeny plants, heterozygous for the pMADS1 gene, had flowers with a more pronounced reversion from petals into sepals than was observed for the parent fbp1 mutant. The morphology of the third whorl organs was not changed. These observations, together with expression levels of pMADS1 and fbp1 in mutant flowers, provide evidence for functional control of fbp1 by PMADS1 in vivo.

Hexose transport in growing petunia pollen tubes and characterization of a pollen-specific, putative monosaccharide transporter.

We investigated the molecular and physiological processes of sugar uptake and metabolism during pollen tube growth and plant fertilization. In vitro germination assays showed that petunia (Petunia hybrida) pollen can germinate and grow not only in medium containing sucrose (Suc) as a carbon source, but also in medium containing the monosaccharides glucose (Glc) or fructose (Fru). Furthermore, high-performance liquid chromatography analysis demonstrated a rapid and complete conversion of Suc into equimolar amounts of Glc and Fru when pollen was cultured in a medium containing 2% Suc. This indicates the presence of wall-bound invertase activity and uptake of sugars in the form of monosaccharides by the growing pollen tube. A cDNA designated pmt1 (petunia monosaccharide transporter 1), which is highly homologous to plant monosaccharide transporters, was isolated from petunia. Pmt1 belongs to a small gene family and is expressed specifically in the male gametophyte, but not in any other vegetative or floral tissues. Pmt1 is activated after the first pollen mitosis, and high levels of mRNA accumulate in mature and germinating pollen. A model describing the transport of sugars to the style, the conversion of Suc into Glc and Fru, and the active uptake by a monosaccharide transporter into the pollen tube is presented.

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.

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.

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.

Bovine alpha 1----3-galactosyltransferase: isolation and characterization of a cDNA clone. Identification of homologous sequences in human genomic DNA.

We have isolated, by immunological screening of a lambda gt11 expression library, a cDNA clone that represents the complete coding sequence for bovine alpha 1----3-galactosyltransferase. The coding sequence predicts a membrane-bound protein with three distinct structural features: a large, potentially glycosylated COOH-terminal domain (346 amino acids), a single transmembrane domain (16 amino acids), and a short NH2-terminal domain (6 amino acids). Thus, the domain structure for this transferase is similar to that deduced for beta 1----4-galactosyltransferase (Shaper, N. L., Hollis, G. F., Douglas, J. G., Kirsch, I. R., and Shaper, J. H. (1988) J. Biol. Chem. 263, 10420-10428) and alpha 2----6-sialyltransferase (Weinstein, J., Lee, E. V., McEntee, K., Lai, P.-H., and Paulson, J. C. (1987) J. Biol. Chem. 262, 17735-17743). S1 analysis demonstrates that two sets of mRNAs, which are heterogeneous at their 5' ends, are transcribed. Because both sets initiate upstream of the translational start site, only one protein is encoded by this gene. alpha 1----3-Galactosyltransferase is widely expressed in different mammalian species, with the notable exception of man and Old World monkeys (Galili, U., Shohet, S. B., Kobrin, E., Stults, C.L.M., and Macher, B. A. (1988) J. Biol. Chem. 263, 17755-17762). By Northern blot analysis we were indeed unable to detect transcripts for this enzyme in various human and Old World monkey cell lines; transcripts were readily detected in other mammalian species. However, by Southern blot analysis, homologous sequences for alpha 1----3-galactosyltransferase were identified in human genomic DNA. This suggests that the gene, although present in the human genome, is normally not expressed. These observations have potential medical implications. Because many humans have high levels of circulating antibodies directed against the enzymatic product of alpha 1----3-galactosyltransferase (Gal alpha 1----3Gal beta 1----4GlcN Ac) (Galili, U., Clark, M. R., Shohet, S. B., Buehler, J., and Macher, B. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 1369-1373), it has been suggested that activation of this normally silent gene may play a role in autoimmune disease in man (Etienne-Decerf, J., Malaise, M., Mahieu, P., and Winand, R. (1987) Acta Endocrinol. 115, 67-74).

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.