Petunia embryos carrying the no apical meristem (nam) mutation fail to develop a shoot apical meristem. Occasional shoots on nam- seedlings bear flowers that develop ten instead of five primordia in the second whorl. Double mutants with the homeotic gene green petals show that nam acts independently of organ identify in whorl 2 and now also affects primordium number in whorl 3. The nam gene was isolated by transposon tagging. The encoded protein shares a conserved N-terminal domain with several other proteins of unknown function and thus represents a novel class of proteins. Strikingly, nam mRNA accumulates in cells at the boundaries of meristems and primordia. These data indicate a role for nam in determining positions of meristems and primordia.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase ( SAAT ) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli . The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short-and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).

Glutathione S -transferases (GSTs) traditionally have been studied in plants and other organisms for their ability to detoxify chemically diverse herbicides and other toxic organic compounds. Anthocyanins are among the few endogenous substrates of plant GSTs that have been identified. The Bronze2 ( Bz2 ) gene encodes a type III GST and performs the last genetically defined step of the maize anthocyanin pigment pathway. This step is the conjugation of glutathione to cyanidin 3-glucoside (C3G). Glutathionated C3G is transported to the vacuole via a tonoplast Mg-ATP-requiring glutathione pump (GS-X pump). Genetically, the comparable step in the petunia anthocyanin pathway is controlled by the Anthocyanin9 ( An9 ) gene. An9 was cloned by transposon tagging and found to encode a type I plant GST. Bz2 and An9 have evolved independently from distinct types of GSTs, but each is regulated by the conserved transcriptional activators of the anthocyanin pathway. Here, a phylogenetic analysis is presented, with special consideration given to the origin of these genes and their relaxed substrate requirements. In particle bombardment tests, An9 and Bz2 functionally complement both mutants. Among several other GSTs tested, only soybean GmGST26A (previously called GmHsp26A and GH2 / 4 ) and maize GSTIII were found to confer vacuolar sequestration of anthocyanin. Previously, these genes had not been associated with the anthocyanin pathway. Requirements for An9 and Bz2 gene function were investigated by sequencing functional and nonfunctional germinal revertants of an9-T3529 , bz2::Ds , and bz2::Mu1. INTRODUCTIONGlutathione S -transferases (GSTs; EC 2.5.1.18) conjugate the glutathione tripeptide ( ␥ -Glu-Cys-Gly; GSH) to a broad variety of substrates. They are an ancient and ubiquitous gene family encoding ‫ف‬ 25-to 29-kD proteins that form both homodimers and heterodimers in vivo. GSTs are as plentiful as they are diverse. As a group, maize GSTs are among the most abundant nonphotosynthetic enzymes in plant cells, making up as much as 1% of the soluble leaf protein (SariGorla et al., 1993). At least 38 plant GSTs have been identified (Marrs, 1996), and a search of the Arabidopsis expressed sequence tag (EST) database identifies Ͼ 200 distinct ESTs with substantial homology to GSTs. It is striking that the GST family is so highly divergent; only a motif of ‫ف‬ 15 amino acids is moderately conserved in sequence and position among all plant GSTs (Marrs, 1996).Mammalian GST genes are divided into five groups ( ␣ , , , , and ) based on sequence similarities, immunological cross-reactivity, and substrate specificity. All identified plant GSTs fall into the most ancient class, that of theta. Droog et al. (1995) recently proposed three subdivisions of plant GSTs-type I, type II, and type III-based on a combination of sequence conservation, immunological cross-reactivity, and intron/exon structure of the gene. Type I GSTs have two introns, and many are induced, both transcriptionally and translationally, by environmental perturbations, ...

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.

Fruit flavor is a result of a complex mixture of numerous compounds. The formation of these compounds is closely correlated with the metabolic changes occurring during fruit maturation. Here, we describe the use of DNA microarrays and appropriate statistical analyses to dissect a complex developmental process. In doing so, we have identified a novel strawberry alcohol acyltransferase (SAAT) gene that plays a crucial role in flavor biogenesis in ripening fruit. Volatile esters are quantitatively and qualitatively the most important compounds providing fruity odors. Biochemical evidence for involvement of the SAAT gene in formation of fruity esters is provided by characterizing the recombinant protein expressed in Escherichia coli. The SAAT enzyme showed maximum activity with aliphatic medium-chain alcohols, whose corresponding esters are major components of strawberry volatiles. The enzyme was capable of utilizing short- and medium-chain, branched, and aromatic acyl-CoA molecules as cosubstrates. The results suggest that the formation of volatile esters in fruit is subject to the availability of acyl-CoA molecules and alcohol substrates and is dictated by the temporal expression pattern of the SAAT gene(s) and substrate specificity of the SAAT enzyme(s).