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.

Arguments in the evo-devo debate: say it with flowers!

A key question in evolutionary developmental biology is how DNA sequence changes have directed the evolution of morphological diversity. The widely accepted view was that morphological changes resulted from differences in number and/or type of transcription factors, or even from small changes in the amino acid sequence of similar proteins. Research over the last two decades indicated that most of the developmental and genetic mechanisms that produce new structures involve proteins that are deeply conserved. These proteins are encoded by a type of genes known as 'toolkit' genes that control a plethora of processes essential for the correct development of the organism. Mutations in these toolkit genes produce deleterious pleiotropic effects. In contrast, alterations in regulatory regions affect their expression only at specific sites in the organism, facilitating morphological change at the tissue and organ levels. However, some examples from the animal and plant fields indicate that coding mutations also contributed to phenotypic evolution. Therefore, the main question at this point is to what extent these mechanisms have contributed to the evolution of morphological diversity. Today, an increasing amount of data, especially from the plant field, implies that changes in cis-regulatory sequences in fact played a major role in evolution.

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.

Leaf-Like Sepals Induced by Ectopic Expression of a SHORT VEGETATIVE PHASE (SVP)-Like MADS-Box Gene from the Basal Eudicot Epimedium sagittatum.

Epimedium L. (Berberidaceae, Ranales), a perennial traditional Chinese medicinal herb, has become a new popular landscape plant for ground cover and pot culture in many countries based on its excellent ornamental characteristics and, distinctive and diverse floral morphology. However, little is known about the molecular genetics of flower development in Epimedium sagittatum. Here, we describe the characterization of EsSVP that encodes a protein sharing 68, 54, and 35% similarity with SVP, AGAMOUS-like 24 (AGL24) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) in Arabidopsis, respectively. Quantitative RT-PCR (qRT-PCR) indicated that EsSVP transcripts were principally found in petiole and leaf tissues, with little expression in roots and flowers and no in fruits. The highest EsSVP expression was observed in leaves. The flowering time of 35S::EsSVP in most Arabidopsis thaliana and in all petunia plants was not affected in both photoperiod conditions, but 35S::EsSVP 5# and 35S::EsSVP 1# Arabidopsis lines induced late and early flowering under long day (LD, 14 h light/10 h dark) and short day (SD, 10 h light/14 h dark) conditions, respectively. The 35S::EsSVP Arabidopsis produced extra secondary inflorescence or floral meristems in the axils of the leaf-like sepals with excrescent trichomes, and leaf-like sepals not able to enclose the inner three whorls completely. Moreover, almost all transgenic Arabidopsis plants showed persistent sepals around the completely matured fruits. Upon ectopic expression of 35S::EsSVP in Petunia W115, sepals were enlarged, sometimes to the size of leaves; corollas were greenish and did not fully open. These results suggest that EsSVP is involved in inflorescence meristem identity and flowering time regulation in some conditions. Although, the SVP homologs might have suffered functional diversification among diverse species between core and basal eudicots, the protein functions are conserved between Arabidopsis/Petunia and Epimedium.

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.

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.

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.

Enhanced arsenate reduction by a CDC25-like tyrosine phosphatase explains increased phytochelatin accumulation in arsenate-tolerant Holcus lanatus.

Decreased arsenate [As(V)] uptake is the major mechanism of naturally selected As(V) hypertolerance in plants. However, As(V)-hypertolerant ecotypes also show enhanced rates of phytochelatin (PC) accumulation, suggesting that improved sequestration might additionally contribute to the hypertolerance phenotype. Here, we show that enhanced PC-based sequestration in As(V)-hypertolerant Holcus lanatus is not due to an enhanced capacity for PC synthesis as such, but to increased As(V) reductase activity. Vacuolar transport of arsenite-thiol complexes was equal in both ecotypes. Based on homology with the yeast As(V) reductase, Acr2p, we identified a Cdc25-like plant candidate, HlAsr, and confirmed the As(V) reductase activity of both HlAsr and the homologous protein from Arabidopsis thaliana. The gene appeared to be As(V)-inducible and its expression was enhanced in the As(V)-hypertolerant H. lanatus ecotype, compared with the non-tolerant ecotype. Homologous ectopic overexpression of the AtASR cDNA in A. thaliana produced a dual phenotype. It improved tolerance to mildly toxic levels of As(V) exposure, but caused hypersensitivity to more toxic levels. Arabidopsis asr T-DNA mutants showed increased As(V) sensitivity at low exposure levels and enhanced arsenic retention in the root. It is argued that, next to decreased uptake, enhanced expression of HlASR might act as an additional determinant of As(V) hypertolerance and As transport in H. lanatus.

FLOOZY of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture.

The mechanisms that determine the relative positions of floral organs, and thereby their numbers, is a poorly understood aspect of flower development. We isolated a petunia mutant, floozy (fzy), in which the formation of floral organ primordia in the outermost three floral whorls and one of the two bracts at the base of the flower is blocked at an early stage. In addition, fzy mutants fail to generate secondary veins in leaves and bracts and display a decreased apical dominance in the inflorescence. FZY encodes an enzyme with homology to flavin mono-oxygenases and appears to be the ortholog of YUCCA genes of Arabidopsis. FZY is expressed in young leafs and bracts and in developing flowers. In young floral meristems FZY is expressed in the center of the meristem dome and, later, expression becomes localized on the flanks of the initiating petal and stamen primordia and at several sites in maturing anthers and carpels. These findings indicate that FZY is involved in synthesizing a signaling compound that is required for floral organ initiation and specification of the vascularization pattern in leaves. Although fzy mutants contain normal auxin levels, ectopic expression of FZY results in excessive auxin accumulation, suggesting that the signaling compound is auxin.

Toward the analysis of the petunia MADS box gene family by reverse and forward transposon insertion mutagenesis approaches: B, C, and D floral organ identity functions require SEPALLATA-like MADS box genes in petunia.

We have initiated a systematic functional analysis of the MADS box, intervening region, K domain, C domain-type MADS box gene family in petunia. The starting point for this has been a reverse-genetics approach, aiming to select for transposon insertions into any MADS box gene. We have developed and applied a family signature insertion screening protocol that is highly suited for this purpose, resulting in the isolation of 32 insertion mutants in 20 different MADS box genes. In addition, we identified three more MADS box gene insertion mutants using a candidate-gene approach. The defined insertion lines provide a sound foundation for a systematic functional analysis of the MADS box gene family in petunia. Here, we focus on the analysis of Floral Binding Protein2 (FBP2) and FBP5 genes that encode the E-function, which in Arabidopsis has been shown to be required for B and C floral organ identity functions. fbp2 mutants display sepaloid petals and ectopic inflorescences originating from the third floral whorl, whereas fbp5 mutants appear as wild type. In fbp2 fbp5 double mutants, reversion of floral organs to leaf-like organs is increased further. Strikingly, ovules are replaced by leaf-like structures in the carpel, indicating that in addition to the B- and C-functions, the D-function, which specifies ovule development, requires E-function activity. Finally, we compare our data with results obtained using cosuppression approaches and conclude that the latter might be less suited for assigning functions to individual members of the MADS box gene family.