Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2.

Summary Two cDNAs encoding gibberellin 2‐oxidases were isolated from maturing pea seeds. The first, PsGA2ox1, was isolated by activity screening of a Lambda‐ZAP cDNA library excised into phagemid form and expressed in Escherichia coli. The second, PsGA2ox2, was obtained initially as a PCR product using degenerate primers designed according to conserved regions of plant 2‐oxoglutarate‐dependent dioxygenases. E. coli heterologous expression products of PsGA2ox1 and PsGA2ox2 converted GA1 to GA8, as shown by HPLC‐radiocounting, and gas chromatography‐MS. PsGA2ox1 converted GA20 to GA29, but GA20 was a poor substrate for the PsGA2ox2 expression product. Furthermore, PsGA2ox1 converted GA29 to GA29‐catabolite at a low level of efficiency while PsGA2ox2 did not catalyse this step. A cDNA of PsGA2ox1 isolated from plants of genotype sln contained a single base deletion which was predicted to produce a truncated protein and gibberellin 2‐oxidase activity could not be demonstrated from this cDNA. A 10 bp size difference between the introns of the SLN and sln PsGA2ox1 genes was used to show co‐segregation between the SLN and sln phenotypes and the size of the PCR products. PsGA2ox1 transcripts were more abundant in cotyledons than in shoots, while the reverse was the case for PsGA2ox2. The expression patterns of the genes, together with the effects of the sln mutation, indicate that PsGA2ox1 plays a major role in GA20 deactivation in both shoots and maturing seeds, while the PsGA2ox2 gene might be important for GA1 deactivation in the shoot.

The theory that bioactive gibberellins (GAs) act as inhibitors of inhibitors of plant growth was based originally on the slender pea (Pisum sativum) mutant (genotype la cry-s), but the molecular nature of this mutant has remained obscure. Here we show that the genes LA and CRY encode DELLA proteins, previously characterized in other species (Arabidopsis [Arabidopsis thaliana] and several grasses) as repressors of growth, which are destabilized by GAs. Mutations la and cry-s encode nonfunctional proteins, accounting for the fact that la cry-s plants are extremely elongated, or slender. We use the la and cry-s mutations to show that in roots, DELLA proteins effectively promote the expression of GA synthesis genes, as well as inhibit elongation. We show also that one of the DELLAregulated genes is a second member of the pea GA 3-oxidase family, and that this gene appears to play a major role in pea roots.It is well known that primary root growth is strongly influenced by the plant hormone GA (Davies, 2004). For example, the application of bioactive GA to roots treated with the growth inhibitor ancymidol completely restored growth to that of the untreated plants (Tanimoto, 1991). Yaxley et al. (2001) established the importance of GAs for root growth in peas (Pisum sativum) by using a variety of GA-deficient mutant plants. In the na-1 mutant, for example, root GA 1 levels, and root elongation, were significantly reduced compared with wild-type plants, and when the GA 1 content was restored to wild-type levels, so too was root elongation.The GAs act by destabilizing the growth inhibitory DELLA proteins (Peng et al., 1997;Harberd et al., 1998;Silverstone et al., 2001;Alvey and Harberd, 2005). In other words, GA acts as an ''inhibitor of an inhibitor '' (Harberd et al., 1998). Interestingly, there is also evidence that DELLA proteins promote the biosynthesis of active GAs. For example, in the Arabidopsis (Arabidopsis thaliana) DELLA mutant rga, the expression of the biosynthesis gene GA4 is reduced, indicating that high DELLA protein levels are associated with an up-regulation of GA synthesis genes (Silverstone et al., 2001 (Silverstone et al., 1998;Gubler et al., 2002). However, the available evidence indicates greater redundancy in dicots compared with monocots (Ikeda et al., 2001;Thomas and Hedden, 2006). There have been five DELLA genes isolated from Arabidopsis (GAI, RGA, RGL1, RGL2, and RGL3), yet only one in rice (SLR1), barley (SLN1), and maize (Zea mays; D8; Peng et al., 1997Peng et al., , 1999Silverstone et al., 1998;Ikeda et al., 2001;Chandler et al., 2002;Gubler et al., 2002), with the possibility of another DELLA gene in maize (D9; accession no. ABI84225). It should be noted, however, that DELLAs have been studied in fewer dicot model species than in monocot species. To date, DELLA-encoding genes from pea have not been reported, even though observations on the slender phenotype of pea triggered the early suggestion that GA acts an inhibitor of an inhibitor (Brian, 1957). The elongated slender phenotype, con...

We describe the isolation of the Le gene of pea, which controls internode elongation and originally was described by Mendel. Heterologous screening of a pea cDNA library yielded a partia1 clone that was 61 ?40 identical to coding regions of the putative Arabidopsis gibberellin 30-hydroxylase gene, GA4. DNA gel blot analysis with this cDNA revealed a Hindlll restriction fragment length polymorphism between pea isolines differing at Mendel's Le locus. Genomic clones of the GACrelated gene were isolated from the Le and /e isolines. Polymerase chain reaction combined with restriction fragment length polymorphism analysis were used to show that the gene mapped to the Le locus. A cDNA containing a complete open reading frame of the pea GACrelated gene was amplified by polymerase chain reaction from each isoline. Recombinant expression in Escbericbia co/i demonstrated that the product of the Le cDNA was a gibberellin 30-hydroxylase that is able t o convert GA, , to the bioactive GA,. Substantially reduced levels of gibberellin 3P-hydroxylase activity were measured, after expression of the /e cDNA, by using identical methods. This reduced activity was associated with an alanine-to-threonine substitution in the predicted amino acid sequence of the enzyme near its proposed active site.

We describe the isolation of the Le gene of pea, which controls internode elongation and originally was described by Mendel. Heterologous screening of a pea cDNA library yielded a partial clone that was 61% identical to coding regions of the putative Arabidopsis gibberellin 3 beta-hydroxylase gene, GA4. DNA gel blot analysis with this cDNA revealed a HindIII restriction fragment length polymorphism between pea isolines differing at Mendel's Le locus. Genomic clones of the GA4-related gene were isolated from the Le and le isolines. Polymerase chain reaction combined with restriction fragment length polymorphism analysis were used to show that the gene mapped to the Le locus. A cDNA containing a complete open reading frame of the pea GA4-related gene was amplified by polymerase chain reaction from each isoline. Recombinant expression in Escherichia coli demonstrated that the product of the Le cDNA was a gibberellin 3 beta-hydroxylase that is able to convert GA20 to the bioactive GA1. Substantially reduced levels of gibberellin 3 beta-hydroxylase activity were measured, after expression of the le cDNA, by using identical methods. This reduced activity was associated with an alanine-to-threonine substitution in the predicted amino acid sequence of the enzyme near its proposed active site.

Two distinct partial cDNAs, PRFl and PRF3, similar in sequence to previously described polygalacturonases, were amplified from ripe peach (Prunus persica 1. Batsch cv Flavorcrest) fruit cDNA by the polymerase chain reaction. PRF1-related RNA was present in fruit from early ripening at levels not detected by northern analysis. PRF3-related RNA was readily detectable in ripe fruit by northern analysis. PRF3 was used to isolate a cDNA with a complete open reading frame, PRFS, from a XZAP li cDNA library prepared from poly(A)+ RNA of ripe peach fruit. PRF5 coded for a predicted protein of 393 amino acids with a molecular mass of 41,500 D. The derived amino acid sequence of PRFS included a putative leader sequence of 23 amino acids, followed by a sequence that matched the N terminus of endopolygalacturonase protein purified from ripe peach fruit. By northern analysis, PRF3-related RNA was undetectable in firm, unripe Flavorcrest fruit. It appeared at low levels as a 1.7-kb transcript in fruit that had begun to ripen and soften and was very abundant in ripe fruit that had undergone the "melting" stage of softening. The marked increase in PRF3-related RNA levels took place over a period of less than 2 d at 20°C and coincided with the climacteric peak in ethylene evolution. Levels of 1 -aminocyclopropane-1 -carboxylate oxidase-related RNA increased during ripening at a much earlier stage than levels of PRF3-related RNA. Lower levels of 1.7-kb RNA transcript were detected by PRF3 in ripe fruit of the melting cultivar Fragar, which are firmer than Flavorcrest fruit. In ripe fruit of the nonmelting cultivar Carolyn, PRF3 detected a 1.45-kb RNA transcript that was present at low levels. Transcripts of a peach polygalacturonase-related genomic sequence were not detected in ripening fruit.

Southern analysis of two ripening-related polygalacturonase (PG) genes of peach [Prunus persica (L.) Batsch] detected a restriction fragment length polymorphism (RFLP) in one that had been previously identified as encoding the endoPG enzyme of melting flesh fruit. This RFLP distinguished the melting flesh cultivars Flavorcrest and Flordaking from the nonmelting flesh cultivars Carolyn, Early Gold Queen, Fla. 86-28C, and Fla. 9-26C. Complete deletion of endoPG-related genomic sequences was demonstrated in the nonmelting flesh variety Fla. 9-20C. In a blind trial, segregation of the endoPG RFLP was followed in relation to the melting flesh trait in a population of 20 trees from `Fla. 86-28C' × `Springcrest' in which the trait was segregating 1:1. Cosegregation of the RFLP with the trait occurred for 17 out of 20 trees. Practical aspects of scoring the melting flesh trait in a genetically variable population may account for incomplete segregation. EndoPG protein was detected by western blotting in fruit of the melting flesh cultivars Flavorcrest and Fragar, but not in fruit of the nonmelting flesh cultivar Carolyn. Results from this study and earlier work are used to discuss the hypothesis that the endoPG gene corresponds to the melting flesh (M) locus of peach.

The gluten analysis of foods has long had limitations, which have precluded food standards authorities from issuing standards for gluten-free foods based on final gluten content. The Codex Alimentarius and the Food and Drug Administration have taken steps towards such standards in which they favour the R5-enzyme-linked immunosorbent assay for gluten analysis. If this method is to be widely employed, its limitations should be recognised. Above all, it should be noted the ability of R5-enzyme-linked immunosorbent assay, and other methods, to measure gluten's toxicity toward celiac disease patients is not validated clinically. Gluten is a complex mixture of proteins and its toxicity is not fully understood. Analytical methods are a valuable tool in the definition of gluten-free foods, but they should be employed with appropriate caveats in ensuring the safety of the foods.