Ronald Koes

The No Apical Meristem Gene of Petunia Is Required for Pattern Formation in Embryos and Flowers and Is Expressed at Meristem and Primordia Boundaries

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.

How genes paint flowers and seeds

Abstract Mutant analyses have given insight into the various parameters that contribute to flower colour and pattern, which is so important for pollination. One important factor is the accumulation of orange, red and purple anthocyanin pigments in the cell vacuole—patterns arise by cell-specific expression of combinations of regulatory proteins. The overall colour perceived is also influenced by vacuolar pH, co-pigmentation and the shape of the petal cells. Although understanding of the biochemistry and genetics of anthocyanin and flavonol biosynthesis is well developed, this is not the case for pH and cell-shape control.

Molecular Analysis of the anthocyanin2 Gene of Petunia and Its Role in the Evolution of Flower Color

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- 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.

Regulatory Genes Controlling Anthocyanin Pigmentation Are Functionally Conserved among Plant Species and Have Distinct Sets of Target Genes.

In this study, we demonstrate that in petunia at least four regulatory genes (anthocyanin-1 [an1], an2, an4, and an11) control transcription of a subset of structural genes from the anthocyanin pathway by using a combination of RNA gel blot analysis, transcription run-on assays, and transient expression assays. an2- and an11- mutants could be transiently complemented by the maize regulatory genes Leaf color (Lc) or Colorless-1 (C1), respectively, whereas an1- mutants only by Lc and C1 together. In addition, the combination of Lc and C1 induces pigment accumulation in young leaves. This indicates that Lc and C1 are both necessary and sufficient to produce pigmentation in leaf cells. Regulatory pigmentation genes in maize and petunia control different sets of structural genes. The maize Lc and C1 genes expressed in petunia differentially activate the promoters of the chalcone synthase genes chsA and chsJ in the same way that the homologous petunia genes do. This suggests that the regulatory proteins in both species are functionally similar and that the choice of target genes is determined by their promoter sequences. We present an evolutionary model that explains the differences in regulation of pigmentation pathways of maize, petunia, and snapdragon.

Functional Complementation of Anthocyanin Sequestration in the Vacuole by Widely Divergent Glutathione S-Transferases

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.

anthocyanin1 of Petunia Encodes a Basic Helix-Loop-Helix Protein That Directly Activates Transcription of Structural Anthocyanin Genes

The petunia loci anthocyanin1 (an1), an2, an4, and an11 are required for the transcription of anthocyanin biosynthetic genes in floral organs. The an2 and an11 loci were recently cloned and shown to encode a MYB-domain transcriptional activator and a cytosolic WD40 protein, respectively. Here, we report the isolation of an1 by transposon tagging. an1 encodes a new member of the basic helix-loop-helix family of transcription factors that is functionally and evolutionarily distinct from JAF13, the apparent petunia ortholog of maize RED1 and snapdragon DELILA. We provide genetic evidence that the transcription factors encoded by an1, an2, and an4 operate in an unexpectedly complex regulatory hierarchy. In leaves, ectopic expression of AN2 induces an1 expression, whereas in anthers, an1 expression depends on an4, encoding (or controlling) a MYB protein that is paralogous to AN2. Experiments with transgenic plants expressing a post-translationally controlled AN1–GLUCOCORTICOID RECEPTOR fusion protein indicated that independent of protein synthesis, AN1 directly activates the expression of the dfrA gene encoding the enzyme dihydroflavonol 4-reductase and of Pmyb27 encoding a MYB-domain protein of unknown function.

Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes.

In this paper we report the isolation of cDNA clones encoding the flavonoid‐biosynthetic enzyme chalcone flavanone isomerase (CHI) from Petunia hybrida. A nearly full size cDNA clone, isolated from a corolla‐specific expression library, was characterized by sequence analysis. Using this CHI cDNA and the previously cloned flavonoid‐specific chalcone synthase (CHS) cDNA we show that CHI and CHS genes are coordinately and tissue‐specifically expressed in a developmental and light‐regulated manner. Furthermore, coordinate induction of both mRNAs is observed after continuous irradiation of Petunia plantlets with UV light, probably as part of the plants UV defence mechanism. The two CHI genes, denoted A and B, were isolated from a genomic library of the Petunia inbred line V30. Both genes are transcriptionally active: gene A is transcribed in corolla, tube and UV‐irradiated plantlets (1.0 kb mRNA), whereas gene B is only transcribed in immature anthers (1.0 kb mRNA). In combination with Southern blot analysis these data implicate the presence of two distinct non‐allelic CHI genes in the genome of the P. hybrida line V30. Unexpectedly, mature anthers accumulate a 0.3 kb larger CHI RNA. This RNA is transcribed from CHI gene A and has a 0.3 kb 5′ extension relative to the gene A transcript found in corolla tissue. Furthermore it is neither coordinately expressed with CHS mRNA nor UV inducible. Its biological function is still obscure, since no active CHI enzyme could be demonstrated in the same tissue.

The chalcone synthase multigene family of Petunia hybrida (V30): differential, light-regulated expression during flower development and UV light induction

We have analysed the expression of the 8–10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.

PH4 of Petunia Is an R2R3 MYB Protein That Activates Vacuolar Acidification through Interactions with Basic-Helix-Loop-Helix Transcription Factors of the Anthocyanin Pathway

The Petunia hybrida genes ANTHOCYANIN1 (AN1) and AN2 encode transcription factors with a basic-helix-loop-helix (BHLH) and a MYB domain, respectively, that are required for anthocyanin synthesis and acidification of the vacuole in petal cells. Mutation of PH4 results in a bluer flower color, increased pH of petal extracts, and, in certain genetic backgrounds, the disappearance of anthocyanins and fading of the flower color. PH4 encodes a MYB domain protein that is expressed in the petal epidermis and that can interact, like AN2, with AN1 and the related BHLH protein JAF13 in yeast two-hybrid assays. Mutation of PH4 has little or no effect on the expression of structural anthocyanin genes but strongly downregulates the expression of CAC16.5, encoding a protease-like protein of unknown biological function. Constitutive expression of PH4 and AN1 in transgenic plants is sufficient to activate CAC16.5 ectopically. Together with the previous finding that AN1 domains required for anthocyanin synthesis and vacuolar acidification can be partially separated, this suggests that AN1 activates different pathways through interactions with distinct MYB proteins.

ANTHOCYANIN1 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.