In petunia flowers, the loci anl, an2, and anll control the pigmentation of the flower by stimulating the transcription of anthocyanin biosynthetic genes. The anl and an2 locus were recently cloned and encode a basic helix-loop-helix (bHLH) and MYB-domain transcriptional activator, respectively. Here, we report the isolation of the anll locus by transposon tagging. RNA gel blot experiments show that anll is expressed independently from anl and an2 throughout plant development, as well as in tissues that do not express the anthocyanin pathway. It encodes a novel WD-repeat protein that is highly conserved even in species that do not produce anthocyanins such as yeast, nematodes, and mammals. The observation that the human anll homolog partially complements the anll petunia mutant in transient assays shows that sequence similarity reflects functional conservation. Overexpression of an2 in anll-petals restored the activity of a structural anthocyanin gene in transient assays, indicating that AN11 acts upstream of AN2. Cell fractionation experiments show that the bulk of the ANll protein is localized in the cytoplasm. Taken together, this indicates that ANll is a cytoplasmic component of a conserved signal transduction cascade that modulates AN2 function in petunia, thereby linking cellular signals with transcriptional activation.

The shape and color of flowers are important for plant reproduction because they attract pollinators such as insects and birds. Therefore, it is thought that alterations in these traits may result in the attraction of different pollinators, genetic isolation, and ultimately, (sympatric) speciation. Petunia integrifolia and P. axillaris bear flowers with different shapes and colors that appear to be visited by different insects. The anthocyanin2 ( an2 ) locus, a regulator of the anthocyanin biosynthetic pathway, is the main determinant of color differences. Here, we report an analysis of molecular events at the an2 locus that occur during Petunia spp evolution. We isolated an2 by transposon tagging and found that it encodes a MYB domain protein, indicating that it is a transcription factor. Analysis of P. axillaris subspecies with white flowers showed that they contain an2 Ϫ alleles with two alternative frameshifts at one site, apparently caused by the insertion and subsequent excision of a transposon. A third an2 ؊ allele has a nonsense mutation elsewhere, indicating that it arose independently. The distribution of polymorphisms in an2 ؊ alleles suggests that the loss of an2 function and the consequent changes in floral color were not the primary cause for genetic separation of P. integrifolia and P. axillaris. Rather, they were events that occurred late in the speciation process, possibly to reinforce genetic isolation and complete speciation. INTRODUCTIONFlowers are the structures containing the male and female sex organs of angiosperms. Flowers of diverse species display a wide range of different morphologies and pollination strategies. For instance, flowers of wind-pollinated species usually possess small and inconspicuous petals or no petals at all, whereas flowers of insect-pollinated plants usually possess large, brightly colored, and patterned petals that serve as visual signals and a landing site for visiting insects.Recent experiments suggest that the wide variety of plant and flower morphologies may have depended on the evolution of a relatively small number of genes. First, mutations at single loci, usually isolated by breeders or researchers, are sufficient to cause fundamental alterations in inflorescence architecture (Doebley et al., 1997;Souer et al., 1998). Similarly, the different shapes, colors, and color patterns of naturally occurring Mimulus (monkeyflower) spp are due to alterations at only a few (major) loci (Bradshaw et al., 1995).Second, even very different inflorescence and flower architectures appear to be determined by genes that often encode conserved proteins but that differ in their expression patterns (reviewed in Doebley and Lukens, 1998).The isolation of key regulatory loci and analysis of the molecular alterations that have taken place in them will provide new insights into the evolution and diversification of flower morphology. The biosynthesis of anthocyanin flower pigments is particularly suited for such studies, because it is a well-defined biochemical pathway that is being...

The shape and color of flowers are important for plant reproduction because they attract pollinators such as insects and birds. Therefore, it is thought that alterations in these traits may result in the attraction of different pollinators, genetic isolation, and ultimately, (sympatric) speciation. Petunia integrifolia and P. axillaris bear flowers with different shapes and colors that appear to be visited by different insects. The anthocyanin2 ( an2 ) locus, a regulator of the anthocyanin biosynthetic pathway, is the main determinant of color differences. Here, we report an analysis of molecular events at the an2 locus that occur during Petunia spp evolution. We isolated an2 by transposon tagging and found that it encodes a MYB domain protein, indicating that it is a transcription factor. Analysis of P. axillaris subspecies with white flowers showed that they contain an2 Ϫ alleles with two alternative frameshifts at one site, apparently caused by the insertion and subsequent excision of a transposon. A third an2 ؊ allele has a nonsense mutation elsewhere, indicating that it arose independently. The distribution of polymorphisms in an2 ؊ alleles suggests that the loss of an2 function and the consequent changes in floral color were not the primary cause for genetic separation of P. integrifolia and P. axillaris. Rather, they were events that occurred late in the speciation process, possibly to reinforce genetic isolation and complete speciation. INTRODUCTIONFlowers are the structures containing the male and female sex organs of angiosperms. Flowers of diverse species display a wide range of different morphologies and pollination strategies. For instance, flowers of wind-pollinated species usually possess small and inconspicuous petals or no petals at all, whereas flowers of insect-pollinated plants usually possess large, brightly colored, and patterned petals that serve as visual signals and a landing site for visiting insects.Recent experiments suggest that the wide variety of plant and flower morphologies may have depended on the evolution of a relatively small number of genes. First, mutations at single loci, usually isolated by breeders or researchers, are sufficient to cause fundamental alterations in inflorescence architecture (Doebley et al., 1997;Souer et al., 1998). Similarly, the different shapes, colors, and color patterns of naturally occurring Mimulus (monkeyflower) spp are due to alterations at only a few (major) loci (Bradshaw et al., 1995).Second, even very different inflorescence and flower architectures appear to be determined by genes that often encode conserved proteins but that differ in their expression patterns (reviewed in Doebley and Lukens, 1998).The isolation of key regulatory loci and analysis of the molecular alterations that have taken place in them will provide new insights into the evolution and diversification of flower morphology. The biosynthesis of anthocyanin flower pigments is particularly suited for such studies, because it is a well-defined biochemical pathway that is being...

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The substitution pattern of anthocyanin pigments is a main determinant of f lower color. Flavonoid 3,5-hydroxylase (F35H) is a cytochrome P450 enzyme (Cyt P450) that catalyzes the 3,5-hydroxylation of dihydrof lavonols, the precursors of purple anthocyanins. Species such as rose and carnation lack F35H activity and are, therefore, unable to generate purple or blue f lowers. Petunia, on the other hand, contains two loci, termed hf1 and hf2, that encode a Cyt P450 with F35H activity. Here we report the identification of an additional petunia gene that is required for 3,5 substitution of anthocyanins and purple f lower colors. It encodes a cytochrome b 5 and is expressed exclusively in the f lower. Inactivation of the gene by targeted transposon mutagenesis reduced F35H enzyme activity and the accumulation of 5-substituted anthocyanins, resulting in an altered f lower color. However, no phenotypic effect on the activity of other Cyt P450s, involved in the synthesis of hormones or general phenylpropanoids, was observed. These data provide in vivo evidence for the regulation of the activity of specific Cyt P450s by a cytochrome b 5 .

Cassava is a poor man's crop which is mainly grown as a subsistence crop in many developing countries. Its commercial use was first as animal feed (also known as tapioca), but has shifted since the late sixties to a source of native starch. The availability of native starches, which on the one hand do not require substantial chemical derivatisation and on the other hand have improved properties, would make cassava also for small farmers a potentially attractive cash crop. Since breeding is difficult in this polyploid, vegetatively propagated, crop a transgenic approach would be ideal to improve certain characteristics. We have created a cassava genotype producing amylose-free starch by genetic modification. The absence of amylose increased the clarity and stability of gels made with the transgenic starch, without requiring treatment with environment-unfriendly chemicals such as epoxides (propylene oxide, ethylene oxide) and acetic anhydride, which are normally used to improve stability. The amylose-free starch showed no changes in particle size distribution, chain length distribution or phosphorous content when compared to amylose-containing starch, but the granule melting temperature was increased by almost 2°C. Furthermore, the amylose-free cassava starch shows enhanced clarity and stability properties. These improved functionalities are desired in technical applications in paper and textile manufacturing, but also in the food industry for the production of sauces, dairy products and noodles.

The development and testing in the field of genetically modified -so called- orphan crops like cassava in tropical countries is still in its infancy, despite the fact that cassava is not only used for food and feed but is also an important industrial crop. As traditional breeding of cassava is difficult (allodiploid, vegetatively propagated, outbreeding species) it is an ideal crop for improvement through genetic modification. We here report on the results of production and field testing of genetically modified low-amylose transformants of commercial cassava variety Adira4 in Indonesia. Twenty four transformants were produced and selected in the Netherlands based on phenotypic and molecular analyses. Nodal cuttings of these plants were sent to Indonesia where they were grown under biosafety conditions. After two screenhouse tests 15 transformants remained for a field trial. The tuberous root yield of 10 transformants was not significantly different from the control. Starch from transformants in which amylose was very low or absent showed all physical and rheological properties as expected from amylose-free cassava starch. The improved functionality of the starch was shown for an adipate acetate starch which was made into a tomato sauce. This is the first account of a field trial with transgenic cassava which shows that by using genetic modification it is possible to obtain low-amylose cassava plants with commercial potential with good root yield and starch quality.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-011-9507-9) contains supplementary material, which is available to authorized users.

SummaryThe Petunia hybrida line W138 contains more than 200 copies of the transposable element dTphl. In W138 progeny these elements give rise to new unstable mutations at high frequency. With the aim of isolating these mutated genes a method was developed to isolate dTphl flanking sequences unique for mutant plants. This method is based on differential screening of cloned inverse polymerese chain reaction (IPCR) products originating from the mutated plant. It directly yields e probe for the mutated gene which can be used to screen pre-existing cDNA and genomic libraries. This method may be generally applicable to isolate genes tagged by other high copy number transposable elements, like Mutator (Mu) or Dissociation (Ds) in Zea mays.