Erik Souer

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.

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.

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.

Patterning of Inflorescences and Flowers by the F-Box Protein DOUBLE TOP and the LEAFY Homolog ABERRANT LEAF AND FLOWER of Petunia

Angiosperms display a wide variety of inflorescence architectures differing in the positions where flowers or branches arise. The expression of floral meristem identity (FMI) genes determines when and where flowers are formed. In Arabidopsis thaliana, this is regulated via transcription of LEAFY (LFY), which encodes a transcription factor that promotes FMI. We found that this is regulated in petunia (Petunia hybrida) via transcription of a distinct gene, DOUBLE TOP (DOT), a homolog of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis. Mutation of DOT or its tomato (Solanum lycopersicum) homolog ANANTHA abolishes FMI. Ubiquitous expression of DOT or UFO in petunia causes very early flowering and transforms the inflorescence into a solitary flower and leaves into petals. Ectopic expression of DOT or UFO together with LFY or its homolog ABERRANT LEAF AND FLOWER (ALF) in petunia seedlings activates genes required for identity or outgrowth of organ primordia. DOT interacts physically with ALF, suggesting that it activates ALF by a posttranslational mechanism. Our findings suggest a wider role than previously thought for DOT and UFO in the patterning of flowers and indicate that the different roles of LFY and UFO homologs in the spatiotemporal control of floral identity in distinct species result from their divergent expression patterns.

Role of EVERGREEN in the Development of the Cymose Petunia Inflorescence

Plants species diverge with regard to the time and place where they make flowers. Flowers can develop from apical meristems, lateral meristems, or both, resulting in three major inflorescence types known as racemes, cymes, and panicles, respectively. The mechanisms that determine a racemose architecture have been uncovered in Arabidopsis and Antirrhinum. To understand how cymes are specified, we studied mutations that alter the petunia inflorescence. Here we show that EVERGREEN (EVG) encodes a WOX homeodomain protein, which is exclusively expressed in incipient lateral inflorescence meristems (IMs), promoting their separation from the apical floral meristem (FM). This is essential for activation of DOUBLE TOP and specification of floral identity. Mutations that change the cymose petunia inflorescence into a solitary flower fully suppress the evg phenotype. Our data suggest a key role for EVG in the diversification of inflorescence architectures and reveal an unanticipated link between the proliferation and identity of meristems.

Epigenetic interactions among three dTph1 transposons in two homologous chromosomes activate a new excision-repair mechanism in petunia.

Unstable anthocyanin3 (an3) alleles of petunia with insertions of the Activator/Dissociation–like transposon dTph1 fall into two classes that differ in their genetic behavior. Excision of the (single) dTph1 insertion from class 1 an3 alleles results in the formation of a footprint, similar to the “classical” mechanism observed for excisions of maize and snapdragon transposons. By contrast, dTph1 excision and gap repair in class 2 an3 alleles occurs via a newly discovered mechanism that does not generate a footprint at the empty donor site. This novel mechanism depends on the presence of two additional dTph1 elements: one located in cis, 30 bp upstream of the an3 translation start in the same an3 allele, and a homologous copy, which is located in trans in the homologous an3 allele. Absence of the latter dTph1 element causes a heritable suppression of dTph1 excision–repair from the homologous an3 allele by the novel mechanism, which to some extent resembles paramutation. Thus, an epigenetic interaction among three dTph1 copies activates a novel recombination mechanism that eliminates a transposon insertion.

ABF transcription factors of Thellungiella salsuginea: structure, expression profiles and interaction with 14-3-3 regulatory proteins

ABF transcription factors are the key regulators of ABA signaling. Using RACE-PCR, we identified and sequenced the coding regions of four genes that encode ABF transcription factors in the extremophile plant Thellungiella salsuginea, a close relative of Arabidopsis thaliana that possesses high tolerance to abiotic stresses. An analysis of the deduced amino acid sequences revealed that the similarity between Thellungiella and Arabidopsis ABFs ranged from 71% to 88%. Similar to their Arabidopsis counterparts, Thellungiella ABFs share a bZIP domain and four conservative domains, including a highly conservative motif at the C-terminal tail, which was reported to be a canonical site for binding by 14-3-3 regulatory proteins. Gene expression analysis by real-time PCR revealed a rapid transcript induction of three of the ABF genes in response to salt stress. To check whether Thellungiella ABF transcription factors can interact with abundant 14-3-3 proteins, multiple constructs were designed, and yeast two-hybrid experiments were conducted. Six of the eight tested Ts14-3-3 proteins were able to bind the TsABFs in an isoform-specific manner. A serine-to-alanine substitution in the putative 14-3-3 binding motif resulted in the complete loss of interaction between the 14-3-3 proteins and the ABFs. The role of 14-3-3 interaction with ABFs in the salt and ABA signaling pathways is discussed in the context of Thellungiella survivability.

Changes in cis-regulatory elements of a key floral regulator are associated with divergence of inflorescence architectures.

Higher plant species diverged extensively with regard to the moment (flowering time) and position (inflorescence architecture) at which flowers are formed. This seems largely caused by variation in the expression patterns of conserved genes that specify floral meristem identity (FMI), rather than changes in the encoded proteins. Here, we report a functional comparison of the promoters of homologous FMI genes from Arabidopsis, petunia, tomato and Antirrhinum. Analysis of promoter-reporter constructs in petunia and Arabidopsis, as well as complementation experiments, showed that the divergent expression of LEAFY (LFY) and the petunia homolog ABERRANT LEAF AND FLOWER (ALF) results from alterations in the upstream regulatory network rather than cis-regulatory changes. The divergent expression of UNUSUAL FLORAL ORGANS (UFO) from Arabidopsis, and the petunia homolog DOUBLE TOP (DOT), however, is caused by the loss or gain of cis-regulatory promoter elements, which respond to trans-acting factors that are expressed in similar patterns in both species. Introduction of pUFO:UFO causes no obvious defects in Arabidopsis, but in petunia it causes the precocious and ectopic formation of flowers. This provides an example of how a change in a cis-regulatory region can account for a change in the plant body plan. Summary: Alterations of floral meristem identity gene expression patterns during evolution involve changes in both cis-regulatory elements and upstream trans-acting factors.

Brassinosteroid biosynthesis and signalling in Petunia hybrida

Brassinosteroids (BRs) are steroidal plant hormones that play an important role in the growth and development of plants. The biosynthesis of sterols and BRs as well as the signalling cascade they induce in plants have been elucidated largely through metabolic studies and the analysis of mutants in Arabidopsis and rice. Only fragmentary details about BR signalling in other plant species are known. Here a forward genetics strategy was used in Petunia hybrida, by which 19 families with phenotypic alterations typical for BR deficiency mutants were identified. In all mutants, the endogenous BR levels were severely reduced. In seven families, the tagged genes were revealed as the petunia BR biosynthesis genes CYP90A1 and CYP85A1 and the BR receptor gene BRI1. In addition, several homologues of key regulators of the BR signalling pathway were cloned from petunia based on homology with their Arabidopsis counterparts, including the BRI1 receptor, a member of the BES1/BZR1 transcription factor family (PhBEH2), and two GSK3-like kinases (PSK8 and PSK9). PhBEH2 was shown to interact with PSK8 and 14-3-3 proteins in yeast, revealing similar interactions to those during BR signalling in Arabidopsis. Interestingly, PhBEH2 also interacted with proteins implicated in other signalling pathways. This suggests that PhBEH2 might function as an important hub in the cross-talk between diverse signalling pathways.

Isolated Thellungiella shoots do not require roots to survive NaCl and Na2SO4 salt stresses

Shoots of Thellungiella derived by micropropagation were used to estimate the plants’ salt tolerance and ability to regulate Na+ uptake. Two species with differing salt tolerances were studied: Thellungiella salsuginea (halophilla), which is less tolerant, and Thellungiella botschantzevii, which is more tolerant. Although the shoots of neither ecotype survived at 700 mM NaCl or 200 mM Na2SO4, micropropagated shoots of T. botschantzevii were more tolerant to Na2SO4 (10 – 100 mM) and NaCl (100 – 300 mM). In the absence of roots, Na2SO4 salinity reduced shoot growth more dramatically than NaCl salinity. Plantlets of both species were able to adapt to salt stress even when they did not form roots. First, there was no significant correlation between Na+ accumulation in shoots and Na+ concentration in the growth media. Second, K+ concentrations in the shoots exposed to different salt concentrations were maintained at equivalent levels to control plants grown in medium without NaCl or Na2SO4. These results suggest that isolated shoots of Thellungiella possess their own mechanisms for enabling salt tolerance, which contribute to salt tolerance in intact plants.