Epidermal control of floral organ identity by class B homeotic genes in Antirrhinum and Arabidopsis.

To assess the contribution of the epidermis to the control of petal and stamen organ identity, we have used transgenic Antirrhinum and Arabidopsis plants that expressed the Antirrhinum class B homeotic transcription factors DEFICIENS (DEF) and GLOBOSA (GLO) in the epidermis. Transgene expression was controlled by the ANTIRRHINUM FIDDLEHEAD (AFI) promoter, which directs gene expression to the L1 meristematic layer and, later, to the epidermis of differentiating organs. Transgenic epidermal DEF and GLO chimeras display similar phenotypes, suggesting similar epidermal contributions by the two class B genes in ANTIRRHINUM: Epidermal B function autonomously controls the differentiation of Antirrhinum petal epidermal cell types, but cannot fully control the pattern of cell divisions and the specification of sub-epidermal petal cell-identity by epidermal signalling. This non-autonomous control is enhanced if the endogenous class B genes can be activated from the epidermis. The developmental influence of epidermal B function in Antirrhinum stamen development is very limited. In contrast, epidermal B function in Arabidopsis can control most if not all epidermal and sub-epidermal differentiation events in petals and stamens, without any contribution from the endogenous class B genes. Possible reasons for differences in the efficacy of B-function-mediated cell communication between the two species are discussed. Interestingly, our experiments uncovered partial incompatibility between class B functional homologues. Although the DEFICIENS/PISTILLATA heterodimer is functional in transgenic Arabidopsis plants, the APETALA3/GLOBOSA heterodimer is not.

In situ analysis of RNA and protein expression in whole mounts facilitates detection of floral gene expression dynamics.

UNLABELLED: A three-dimensional whole-mount technique for detection of mRNA and protein expression patterns of floral regulatory genes in inflorescences from Antirrhinum majus is reported. This technique allows the observation of complex expression patterns in situ in developing flowers at different developmental stages initiated sequentially on the same inflorescence and labelled under the same conditions. Thereby, reconstruction from serial two-dimensional sections can be circumvented. The technique was used to study early changes in the expression of DEFICIENS (DEF), a class B floral homeotic transcription factor. Whole-mount analysis revealed that the order of appearance of DEF mRNA and protein expression in the floral primordium is opposite to the order of initiation of organ primordia. As a consequence, stamen primordia express the DEF gene prior to their initiation in whorl three, while petal primordia in the second whorl are morphologically distinct structures when second whorl DEF expression becomes established. This interesting feature was not readily detectable by previous analysis of serial sections. The particular usefulness of in situ analyses in whole mounts is further demonstrated in floral mutants with variable phenotypes and unpredictable sites of aberrant organ development. KEYWORDS: whole mount, in situ hybridization, immunolocalization, Antirrhinum majus, flower development.

CHORIPETALA and DESPENTEADO: general regulators during plant development and potential floral targets of FIMBRIATA-mediated degradation.

Two Antirrhinum majus mutants, choripetala (cho) and despenteado (desp), exhibit identical highly pleiotropic phenotypes including petaloid transformation of first whorl floral organs, narrowing of both vegetative and floral organs, reduction in carpel size and fertility and delayed germination. The petaloid first whorl results from ectopic expression of the class B genes DEFICIENS and GLOBOSA and is correlated with the ectopic expression of the proposed class B/C gene regulator FIMBRIATA (FIM). Ectopic class B gene expression is apparent from the earliest point at which class B gene transcription can be detected in the wild type, indicating that the pre-patterning of the class B domain has been disrupted in these mutants. Single and double mutant analyses indicate that CHO and DESP also play a role in regulation of the class C domain. Interestingly, the cho and desp mutations partially suppress the phenotype of fim null mutants, suggesting that the F-box protein FIM may target a member of the CHO/DESP pathway for degradation. We propose that CHO and DESP are members of a 'basal regulatory function' influencing many processes throughout plant development and in particular are directly or indirectly required for the repression of class B and C genes during early stages of flower development.

Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis.

In plants, flowering is triggered by endogenous and environmental signals. CONSTANS (CO) promotes flowering of Arabidopsis in response to day length. Four early target genes of CO were identified using a steroid-inducible version of the protein. Two of these genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1) and FLOWERING LOCUS T (FT), are required for CO to promote flowering; the others are involved in proline or ethylene biosynthesis. The SOC1 and FT genes are also regulated by a second flowering-time pathway that acts independently of CO. Thus, early target genes of CO define common components of distinct flowering-time pathways.

PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development.

We report the discovery of an Antirrhinum MADS-box gene, FARINELLI (FAR), and the isolation of far mutants by a reverse genetic screen. Despite striking similarities between FAR and the class C MADS-box gene PLENA (PLE), the phenotypes of their respective mutants are dramatically different. Unlike ple mutants, which show homeotic conversion of reproductive organs to perianth organs and a loss of floral determinacy, far mutants have normal flowers which are partially male-sterile. Expression studies of PLE and FAR, in wild-type and mutant backgrounds, show complex interactions between the two genes. Double mutant analysis reveals an unexpected, redundant negative control over the B-function MADS-box genes. This feature of the two Antirrhinum C-function-like genes is markedly different from the control of the inner boundary of the B-function expression domain in Arabidopsis, and we propose and discuss a model to account for these differences. The difference in phenotypes of mutants in two highly related genes illustrates the importance of the position within the regulatory network in determining gene function.

Molecular analysis of a second functional A1 gene (dihydroflavonol 4-reductase) in Zea mays.

Some genes involved in anthocyanin biosynthesis in Zea mays are duplicated and differentially expressed. From the analysis of the A1 gene (dihydroflavonol 4-reductase), which is involved in this pathway, no molecular evidence for gene duplication was known to date. Isolation and analysis of A1 homologous genomic clones revealed the presence of a second A1 gene in maize and also two copies of the gene in Teosinte guerrero. The duplicated genes are structurally very similar and, at least in maize, the second gene is expressed.

STYLOSA and FISTULATA: regulatory components of the homeotic control of Antirrhinum floral organogenesis.

The identity and developmental pattern of the four organ types constituting the flower is governed by three developmental functions, A, B and C, which are defined by homeotic genes and established in two adjacent whorls. In this report we morphologically and genetically characterise mutants of two genes, STYLOSA (STY) and FISTULATA (FIS) which control floral homeotic meristem- and organ-identity genes and developmental events in all floral whorls. The morphology of the reproductive organs in the first and second whorls of sty fis double mutant flowers indicate that the two genes are part of the mechanism to prevent ectopic expression of the C-function in the perianth of wild-type flowers. This is verified by the detection of the expansion of the expression domain of the class C gene PLENA (PLE) towards the perianth. Interestingly, in the second whorl of sty and fis mutants, spatial differences in stamenoid features and in the pattern of ectopic expression of the PLE gene were observed. This suggests that, with respect to the negative control of PLE, petals are composed of two regions, a lateral and a central one. Mutation in ple is epistatic to most of the sty/fis-related homeotic defects. PLE, however, is not the primary target of STY/FIS control, because dramatic reduction of expression of FIMBRIATA, meristem identity genes (FLORICAULA and SQUAMOSA) and of class B organ identity genes (GLOBOSA) occur before changes in the PLE expression pattern. We propose that STY/FIS are hierarchically high-ranking genes that control cadastral component(s) of the A-function. SQUAMOSA as a potential target of this control is discussed. Retarded growth of second whorl organs, subdivision of third whorl primordia and the failure to initiate them in sty/fis mutants may be mediated by the FIMBRIATA gene.

Pollen-specific expression of DEFH125, a MADS-box transcription factor in Antirrhinum with unusual features.

MADS-box genes encode transcription factors that regulate different processes of early and late floral development. A novel type of MADS-box gene, DEFH125, was isolated from a stamen specific cDNA library from Antirrhinum majus. The DEFH125 protein shows extensive similarity over the entire length to AGL17, a root-specific MADS-box protein of Arabidopsis. By sharing amino acid deviations from the consensus MADS-box sequence not found in other MADS-box families, these two proteins constitute a novel MADS-box subfamily. However, in contrast to members of other subfamilies the overall structural similarity between the DEFH125 and AGL17 proteins does not coincide with a similarity of expression patterns and functions. The DEFH125 gene is expressed at detectable levels only in the third whorl when the meiotic division of the pollen mother cell is already accomplished. The DEFH125 protein has been located in the cytoplasm of the vegetative cell within the maturing pollen. Surprisingly, after pollination, the DEFH125 protein is also found in nuclei of cells within the transmitting tract of the carpel. The intriguing role of DEFH125, the first MADS-box transcription factor of this type, in aspects of fertilization, such as pollen maturation, pollen tube formation or pollen tube guidance in the carpel, is discussed.

Non-cell-autonomous function of the Antirrhinum floral homeotic proteins DEFICIENS and GLOBOSA is exerted by their polar cell-to-cell trafficking.

In Antirrhinum majus, petal and stamen organ identity is controlled by two MADS-box transcription factors, DEFICIENS and GLOBOSA. Mutations in either of these genes result in the replacement of petals by sepaloid organs and stamens by carpelloid organs. Somatically stable def and glo periclinal chimeras, generated by transposon excision events, were used to study the non-cell-autonomous functions of these two MADS-box proteins. Two morphologically distinct types of chimeras were analysed using genetic, morphological and molecular techniques. Restoration of DEF expression in the L1 cell layer results in the reestablishment of DEF and GLO functions in L1-derived cells only; inner layer cells retain their mutant sepaloid features. Nevertheless, this activity is sufficient to allow the expansion of petal lobes, highlighting the role of DEF in the stimulation of cell proliferation and/or cell shape and elongation when expressed in the L1 layer. Establishment of DEF or GLO expression in L2 and L3 cell layers is accompanied by the recovery of petaloid identity of the epidermal cells but it is insufficient to allow petal lobe expansion. We show by in situ immunolocalisation that the non-cell-autonomy is due to direct trafficking of DEF and GLO proteins from the inner layer to the epidermal cells. At least for DEF, this movement appears to be polar since DEF acts cell-autonomously when expressed in the L1 cell layer. Furthermore, the petaloid revertant sectors observed on second whorl mutant organs and the mutant margins of petals of L2L3 chimeras suggest that DEF and GLO intradermal movement is limited. This restriction may reflect the difference in the regulation of primary plasmodesmata connecting cells from the same layer and secondary plasmodesmata connecting cells from different layers. We propose that control of intradermal trafficking of DEF and GLO could play a role in maintaining of the boundaries of their expression domains.

Functional analysis of the Antirrhinum floral homeotic DEFICIENS gene in vivo and in vitro by using a temperature-sensitive mutant.

Flowers of the temperature-sensitive DEFICIENS (DEF) mutant, def-101, display sepaloid petals and carpelloid stamens when grown at 26 degrees C, the non-permissive temperature. In contrast, when cultivated under permissive conditions at 15 degrees C, the morphology of def-101 flowers resembles that of the wild type. Temperature shift experiments during early and late phases of flower development revealed that second and third whorl organ development is differentially sensitive to changes in DEF expression. In addition, early DEF expression seems to control the spatially correct initiation of fourth whorl organ development. Reduction of the def-101 gene dosage differentially affects organogenesis in adjacent whorls: at the lower temperature development of petals in the second whorl and initiation of carpels in the centre of the flower is not affected while third whorl organogenesis follows the mutant (carpelloid) pattern. The possible contribution of accessory factors to organ-specific DEF functions is discussed. In situ analyses of mRNA and protein expression patterns during def-101 flower development at 15 degrees C and at 26 degrees C support previously proposed combinatorial regulatory interactions between the MADS-box proteins DEF and GLOBOSA (GLO), and provide evidence that the autoregulatory control of DEF and GLO expression by the DEF/GLO heterodimer starts after initiation of all organ primordia. Immunolocalisation revealed that both proteins are located in the nucleus. Interestingly, higher growth temperature affects the stability of both the DEF-101 and GLO proteins in vivo. In vitro DNA binding studies suggest that the temperature sensitivity of the def-101 mutant is due to an altered heterodimerisation/DNA-binding capability of the DEF-101 protein, conditioned by the deletion of one amino acid within the K-box, a protein region thought to be involved in protein-protein interaction. In addition, we introduce a mutant allele of GLO, glo-confusa, where insertion of one amino acid impairs the hydrophobic carboxy-terminal region of the MADS-box, but which confers no strong phenotypic changes to the flower. The strong mutant phenotype of flowers of def-101/glo-conf double mutants when grown in the cold represents genetic evidence for heterodimerisation between DEF and GLO in vivo. The potential to dissect structural and functional domains of MADS-box transcription factors is discussed.

FIL2, an extracellular Leucine-Rich Repeat protein, is specifically expressed in Antirrhinum flowers.

The expression of the Antirrhinum gene FIL2 is affected in mutants of the homeotic transcription factor DEFICIENS. Northern and Western blot analyses showed that FIL2 in wild-type Antirrhinum flowers is expressed weakly in the petals and more abundantly in the reproductive organs; the gene is active in the filaments and anthers of stamens, and in the stigma and transmitting tissue of the carpels. The FIL2 protein is glycosylated with high mannose type glycan chains and is located in the middle lamella of the extracellular matrix. The amino acid sequence contains 10 tandem repeats, the composition of which is similar to the Leucine-Rich Repeat (LRR) motif found in mammals, Drosophila and yeast. The possibility that FIL2 might be a component of a cellular signalling mechanism, involving LRR-mediated protein-protein interactions is discussed.

Floral development and expression of floral homeotic genes are influenced by cytokinins.

Tobacco plants that are somatic mosaics for the expression of a cytokinin-synthesizing gene have viviparous leaves. Epiphyllous buds can be either vegetative or floral. Floral adventitious buds can be either normal or abnormal. Abnormalities of floral development correlate with: (i) a local activation of the cytokinin-synthesizing gene, (ii) a drastic increase in floral cytokinin content, and (iii) a decrease in the steady-state levels of mRNA homologous of the homeotic genes DEFA, GLO and PLENA of Antirrhinum majus. Thus, these data show in planta that cytokinins, a class of phytohormones, are able to alter the development of floral organs and to decrease the expression of three homeotic floral genes.

GLOBOSA: a homeotic gene which interacts with DEFICIENS in the control of Antirrhinum floral organogenesis.

GLOBOSA (GLO) is a homeotic gene whose mutants show sepaloid petals and carpelloid stamens. The similarity of Glo mutants to those of the DEFICIENS (DEFA) gene suggests that the two genes have comparable functions in floral morphogenesis. The GLO cDNA has been cloned by virtue of its homology to the MADS-box, a conserved DNA-binding domain also contained in the DEFA gene. We have determined the structure of the wild type GLO gene as well as of several glo mutant alleles which contain transposable element insertions responsible for somatic and germinal instability of Glo mutants. Analyses of the temporal and spatial expression patterns of the DEFA and GLO genes during development of wild type flowers and in flowers of various stable and unstable defA and glo alleles indicate independent induction of DEFA and GLO transcription. In contrast, organ-specific up-regulation of the two genes in petals and stamens depends on expression of both DEFA and GLO. In vitro DNA-binding studies were used to demonstrate that the DEFA and GLO proteins specifically bind, as a heterodimer, to motifs in the promoters of both genes. A model is presented which proposes both combinatorial and cross-regulatory interactions between the DEFA and GLO genes during petal and stamen organogenesis in the second and third whorls of the flower. The function of the two genes controlling determinate growth of the floral meristem is also discussed.

Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: evidence for DNA binding and autoregulation of its persistent expression throughout flower development.

We have determined the structure of the floral homeotic deficiens (defA) gene whose mutants display sepaloid petals and carpelloid stamens, and have analysed its spatial and temporal expression pattern. In addition, several mutant alleles (morphoalleles) were studied. The results of these analyses define three functional domains of the DEF A protein and identify in the deficiens promoter a possible cis-acting binding site for a transcription factor which specifically upregulates expression of deficiens in petals and stamens. In vitro DNA binding studies show that DEF A binds to specific DNA motifs as a heterodimer, together with the protein product of the floral homeotic globosa gene, thus demonstrating that the protein encoded by deficiens is a DNA binding protein. Furthermore, Northern analysis of a temperature sensitive allele at permissive and non-permissive temperatures provides evidence for autoregulation of the persistent expression of deficiens throughout flower development. A possible mechanism of autoregulation is discussed.

The MADS box gene family in tomato: temporal expression during floral development, conserved secondary structures and homology with homeotic genes from Antirrhinum and Arabidopsis.

Five genes with homology to the floral homeotic genes deficiens of Antirrhinum and agamous of Arabidopsis were isolated from tomato. Each of the five genes is unique in the genome and could be localized to a different chromosome by RFLP mapping. Four of the tomato genes (hereafter TM) are flower-specific with distinguishable temporal expression. TM4 and TM8 are 'early', while TM5 and TM6 are 'late' genes. TM4 is homologous to squamous and TM6 is similar to deficiens, which are, respectively, 'early' and 'late' bona fide homeotic genes in Antirrhinum. The proteins encoded by the five tomato genes, like several known homeotic genes from other plants, contain within their N-terminus a highly conserved DNA-binding domain, the MADS box. All known plant MADS box genes also share, however, other properties. They all contain a central, moderately conserved, and rather basic domain, and a highly divergent or even missing C-terminal domain. Furthermore, molecular modelling predicts the presence of a conserved amphipatic alpha helix, at a constant distance from the MADS box in each of these proteins. The common properties of eight MADS box proteins from three plant families indicate that all their domains were coded for by the same ancestor gene. The sequence homology between pairs of MADS genes from different species indicates that the MADS ancestor gene multiplied and diverged in an ancestor plant common to several dicotyledon families.

Genetic Control of Flower Development by Homeotic Genes in Antirrhinum majus.

Homeotic mutants have been useful for the study of animal development. Such mutants are also known in plants. The isolation and molecular analysis of several homeotic genes in Antirrhinum majus provide insights into the underlying molecular regulatory mechanisms of flower development. A model is presented of how the characteristic sequential pattern of developing organs, comprising the flower, is established in the process of morphogenesis.

Deficiens, a homeotic gene involved in the control of flower morphogenesis in Antirrhinum majus: the protein shows homology to transcription factors.

Deficiens (defA+) is a homeotic gene involved in the genetic control of Antirrhinum majus flower development. Mutation of this gene (defA-1) causes homeotic transformation of petals into sepals and of stamina into carpels in flowers displaying the 'globifera' phenotype, as shown by cross sections and scanning electronmicroscopy of developing flowers. A cDNA derived from the wild type defA+ gene has been cloned by differential screening of a subtracted 'flower specific' cDNA library. The identity of this cDNA with the defA+ gene product has been confirmed by utilizing the somatic and germinal instability of defA-1 mutants. According to Northern blot analyses the defA+ gene is expressed in flowers but not in leaves, and its expression is nearly constant during all stages of flower development. The 1.1 kb long mRNA has a 681 bp long open reading frame that can code for a putative protein of 227 amino acids (mol. wt 26.2 kd). At its N-terminus the DEF A protein reveals homology to a conserved domain of the regulatory proteins SRF (activating c-fos) in mammals and GRM/PRTF (regulating mating type) in yeast. We discuss the structure and the possible function of the DEF A protein in the control of floral organogenesis.

Cin4, an insert altering the structure of the A1 gene in Zea mays, exhibits properties of nonviral retrotransposons.

A wild-type allele of the A1 gene of Zea mays contains a 1.1-kb-long insert termed Cin4-1, which alters the structure of the transcription unit compared to other A1 alleles. The Cin4-1 element is a member of a family of elements occurring in 50-100 copies in the maize genome. Genomic cloning and sequence analysis of several family members and their flanking regions allowed classification of Cin4 as a nonviral retrotransposon. Individual Cin4 elements terminate in an oligo(A) track of variable size (6-11 residues) at their 3'-end. The 5'-ends of family members are heterogeneously truncated with respect to the longest Cin4 element. Cin4 elements are flanked by small direct duplications, the size of which varies between 3 and 16 bp. On the basis of a comparison of the target sequence and the sequence of Cin4 we suggest and discuss a model of the mechanism of Cin4 integration via in situ cDNA synthesis on an RNA template. The longest Cin4 element analysed so far has two non-overlapping open reading frames (ORFs) comprising 2793 nucleotides (ORF1) and 3489 nucleotides (ORF2). The putative 1163 amino acid long Cin4 protein derived from the sequence of ORF2 has the capacity to encode a reverse transcriptase-like protein and a DNA-binding domain. The conservation pattern of these two domains and the overall organisation of Cin4 is similar to that detected in nonviral retrotransposons in animals. The origin and function of Cin4 are discussed.