Synthesis of flavonoid pigments in flowers requires the co‐ordinated expression of genes encoding enzymes in th phenylpropanoid biosynthetic pathway. Some cis‐elements involved in the transcriptional control of these genes have been defined. We report binding of petal‐specific activities from tobacco and Antirrhinum majus (snapdragon) to an element conserved in promoters of phenylpropanoid biosynthetic genes and implicated in expression in flowers. These binding activities were inhibited by antibodies raised against Myb305, a flower‐specific Myb protein previously cloned from Antirrhinum by sequence homology. Myb305 bound to the same element and formed a DNA‐protein complex with the same mobility as the Antirrhinum petal protein in electrophoretic mobility shift experiments. Myb305 activated expression from its binding site in yeast and in tobacco protoplasts. In protoplasts, activation also required a G‐box‐like element, suggesting co‐operation with other elements and factors. The results strongly suggest a role for Myb305‐related proteins in the activation of phenylpropanoid biosynthetic genes in flowers. This is consistent with the genetically demonstrated role of plant Myb proteins in the regulation of genes involved in flavonoid synthesis.

The flavor of strawberry (Fragaria 3 ananassa) fruit is dominated by an uncommon group of aroma compounds with a 2,5-dimethyl-3(H)-furanone structure. We report the characterization of an enzyme involved in the biosynthesis of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol), the key flavor compound in strawberries. Protein extracts were partially purified, and the observed distribution of enzymatic activity correlated with the presence of a single polypeptide of ;37 kD. Sequence analysis of two peptide fragments showed total identity with the protein sequence of a strongly ripeninginduced, auxin-dependent putative quinone oxidoreductase, Fragaria 3 ananassa quinone oxidoreductase (FaQR). The open reading frame of the FaQR cDNA consists of 969 bp encoding a 322-amino acid protein with a calculated molecular mass of 34.3 kD. Laser capture microdissection followed by RNA extraction and amplification demonstrated the presence of FaQR mRNA in parenchyma tissue of the strawberry fruit. The FaQR protein was functionally expressed in Escherichia coli, and the monomer catalyzed the formation of HDMF. After chemical synthesis and liquid chromatography-tandem mass spectrometry analysis, 4-hydroxy-5-methyl-2-methylene-3(2H)-furanone was confirmed as a substrate of FaQR and the natural precursor of HDMF. This study demonstrates the function of the FaQR enzyme in the biosynthesis of HDMF as enone oxidoreductase and provides a foundation for the improvement of strawberry flavor and the biotechnological production of HDMF.

Two Myb-related transcription factors, Myb305 and Myb340, are expressed specifically in flowers of Antirrhinum. The proteins are structurally very similar throughout their DNA binding domains, implying that they bind to common target motifs. This binding has been demonstrated experimentally. Myb305 has been shown to activate the gene encoding the first enzyme of phenylpropanoid metabolism, phenylalanine ammonia-lyase. We show that Myb340 can also activate transcription from the phenylalanine ammonia-lyase gene promoter and that both transcription factors can activate two other genes involved in flavonoid metabolism, thereby linking early and later steps in plant secondary metabolism. Myb340 is a stronger activator than Myb305, but relatively more Myb305 than Myb340 protein is able to bind to target promoters when both proteins are synthesized in yeast or Escherichia coli, probably as a result of inhibition of Myb340 binding by phosphorylation. This means that Myb305 can compete with Myb340 to reduce its effective transcriptional activation when both transcription factors are expressed in the same cell. This competitive interaction has been demonstrated in plant cells. Expression patterns determined by in situ hybridization showed that the two transcription factors are expressed within the same cells of the flower and imply that the detailed specializations in function of these two apparently redundant transcription factors may be used to provide gears that adjust the rate of induction of secondary metabolism to floral development.

Two genomic clones corresponding to putative pectate lyase genes (plA and plB) were isolated and characterized in strawberry (Fragaria x ananassa cv. Chandler). The corresponding ORFs for the plA and plB genes revealed deduced proteins of 451 and 439 amino acids, respectively, that differ from that of the previously isolated strawberry plC gene. Southern blot analysis has shown that while the plB gene is a single copy gene, the plA gene is probably encoded by a small multigene family. By using specific probes corresponding to the untranslated 3' terminal region of the pl genes, and QRT-PCR methodology, the spatio-temporal expression pattern of both strawberry pl genes have been compared with that of the plC gene. The three transcripts were specifically expressed only in fruit and mainly during the ripening stages. Moreover, the expression of the plA and plB genes was induced in green de-achened fruit, but this increase was reduced by the external application of auxins as was the expression of plC. The expression of both pl genes was also strongly reduced in harvested fruit kept in controlled atmosphere (CA) containing high CO(2) levels. Immunolocalization studies using antibodies raised against the strawberry PL proteins placed the proteins in the cell wall of parenchymatic cells of the fruit receptacle. The role of pl genes in cell-wall disassembly and fruit ripening softening is discussed.

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.