Anthocyanini of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms

ANTHOCYANIN1 (AN1) of petunia is a transcription factor of the basic helix-loop-helix (bHLH) family that is required for the synthesis of anthocyanin pigments. Here, we show that AN1 controls additional aspects of cell differentiation: the acidification of vacuoles in petal cells, and the size and morphology of cells in the seed coat epidermis. We identified an1 alleles, formerly known as ph6, that sustain anthocyanin synthesis but not vacuolar acidification and seed coat morphogenesis. These alleles express truncated proteins lacking the C-terminal half of AN1, including the bHLH domain, at an ∼30-fold higher level than wild-type AN1. An allelic series in which one, two, or three amino acids were inserted into the bHLH domain indicated that this domain is required for both anthocyanin synthesis and vacuolar acidification. These findings show that AN1 controls more aspects of epidermal cell differentiation than previously thought through partially separable domains

Transgenes and protein localization: myths and legends

Fluorescent protein (FP) fusions are frequently used to localize and follow the movement of proteins in living cells. However, a consensus is missing about the experimental design and controls that guarantee the reliability of the results. Here, we discuss possible artifacts and try to navigate through the many methods, preferences, and assumptions that surround protein localization in plants that make it difficult to design a universal approach to achieve reliable results. © 2013 Elsevier Ltd

New Challenges for the Design of High Value Plant Products:Stabilization of Anthocyanins in Plant Vacuoles

In the last decade plant biotechnologists and breeders have made several attempt to improve the antioxidant content of plant-derived food. Most efforts concentrated on increasing the synthesis of antioxidants, in particular anthocyanins, by inducing the transcription of genes encoding the synthesizing enzymes. We present here an overview of economically interesting plant species, both food crops and ornamentals, in which anthocyanin content was improved by traditional breeding or transgenesis. Old genetic studies in petunia and more recent biochemical work in brunfelsia, have shown that after synthesis and compartmentalization in the vacuole, anthocyanins need to be stabilized to preserve the color of the plant tissue over time. The final yield of antioxidant molecules is the result of the balance between synthesis and degradation. Therefore the understanding of the mechanism that determine molecule stabilization in the vacuolar lumen is the next step that needs to be taken to further improve the anthocyanin content in food. In several species a phenomenon known as fading is responsible for the disappearance of pigmentation which in some case can be nearly complete. We discuss the present knowledge about the genetic and biochemical factors involved in pigment preservation/destabilization in plant cells. The improvement of our understanding of the fading process will supply new tools for both biotechnological approaches and marker-assisted breeding

Analysis of bHLH and MYB-domain proteins: species-specific regulatory differences caused by divergent evolution of target anthocyanin genes

The regulatory anthocyanin loci, an1, an2, an4 and an11 of Petunia hybrida, and r and c1 from Zea mays, control transcription of different sets of target genes. Both an2 and c1 encode a MYB-type protein. This study reports the isolation of a P. hybrida gene, jaf13, encoding a basic helix-loop-helix protein that, on the basis of sequence homology and intron/exon structure, represents the P. hybrida orthologue of the Z. mays r genes. Ectopic expression of an2 and jaf13 is sufficient for activation of the dihydroflavonol 4-reductase-A (dfrA) promoter and enhanced pigment accumulation in P. hybrida. This indicates that an2 and jaf13 play a key role in determining the tissue-specific expression pattern of structural genes. However, because chalcone synthase (chs) and flavanone-3-hydroxylase (f3h) are not activated, the pattern of pigmentation is not fundamentally altered. Expression of an2 in Z. mays complements a mutation in pl, a c1 paralogue, indicating that an2 activates a wider set of target genes in this host. Transient expression assays in Z. mays and P. hybrida tissues showed that C1 and R or AN2 and JAF13 can activate the promoter of the c2 gene, encoding Z. mays CHS, but not the chsA promoter from P. hybrida. These results indicate that regulatory anthocyanin genes are conserved between species and that divergent evolution of the target gene promoters is responsible for the species-specific differences in regulatory networks