Spatial distribution of the RABBIT EARS protein and effects of its ectopic expression in Arabidopsis thaliana flowers

In many flowering plants, flowers consist of two peripheral organs, sepals and petals, occurring in outer two whorls, and two inner reproductive organs, stamens and carpels. These organs are arranged in a concentric pattern in a floral meristem, and the organ identity is established by the combined action of floral homeotic genes expressed along the whorls. Floral organ primordia arise at fixed positions in the floral meristem within each whorl. The RABBIT EARS (RBE) gene is transcribed in the petal precursor cells and primordia, and regulates petal initiation and early growth in Arabidopsis thaliana. We investigated the spatial and temporal expression pattern of a RBE protein fused to the green fluorescent protein (GFP). Expression of the GFP:RBE fusion gene under the RBE cis-regulatory genomic fragment rescues the rbe petal defects, indicating that the fusion protein is functional. The GFP signal is located to the cells where RBE is transcribed, suggesting that RBE function is cell-autonomous. Ectopic expression of GFP:RBE under the APETALA1 promoter causes the homeotic conversion of floral organs, resulting in sterile flowers. In these plants, the class B homeotic genes APETALA3 and PISTILLATA are down-regulated, suggesting that the restriction of the RBE expression to the petal precursor cells is crucial for flower development.     

Type specification and spatial pattern formation of floral organs: A dynamic development model

A mathematical model simulating spatial pattern formation (positioning) of floral organs is proposed. Computer experiment with this model demonstrated the following sequence of spatial pattern formation in a typical cruciferous flower: medial sepals, carpels, lateral sepals, long stamens, petals, and short stamens. The positioning was acropetal for the perianth organs and basipetal for the stamens and carpels. Organ type specification and positioning proceed non-simultaneously in different floral parts and organ type specification goes ahead of organ spatial pattern formation. Computer simulation of flower development in several mutants demonstrated that the AG and AP2 genes determine both organ type specification and formation of the zones for future organ development. The function of the AG gene is to determine the basipetal patterning zones for the development of the reproductive organs, while the AP2 gene maintains proliferative activity of the meristem establishing the acropetal patterning zone for the development of the perianth organs.     

Phosphatidic acid is a major phospholipid class in reproductive organs of Arabidopsis thaliana

Phospholipids are the crucial components of biological membranes and signal transduction. Among different tissues, flower phospholipids are one of the least characterized features of plant lipidome. Here, we report that floral reproductive organs of Arabidopsis thaliana contain high levels of phosphatidic acid (PA), a known lipid second messenger. By using floral homeotic mutants enriched with specific floral organs, lipidomics study showed increased levels of PA species in ap3-3 mutant with enriched pistils. Accompanied gene expression study for 7 diacylglycerol kinases and 11 PA phosphatases revealed distinct floral organ specificity, suggesting an active phosphorylation/dephosphorylation between PA and diacylglycerol in flowers. Our results suggest that PA is a major phospholipid class in floral reproductive organs of A. thaliana.     

Gymnosperm Orthologues of Class B Floral Homeotic Genes and Their Impact on Understanding Flower Origin

Class B floral homeotic genes play a key role in specifying the identity of male reproductive organs (stamens) and petals during the development of flowers. Recently, close relatives (orthologues) of these genes have been found in diverse gymnosperms, the sister group of the flowering plants (angiosperms). The fact that such genes have not been found so far, despite considerable efforts, in mosses, ferns or algae, has been taken as evidence to suggest that B genes originated 300–400 million years ago in a lineage that led to extant seed plants. Gymnosperms do not develop petals, and their male reproductive organs deviate considerably from angiosperm stamens. So what is the function of gymnosperm B genes? Recent experiments revealed that B genes from diverse extant gymnosperms are exclusively expressed in male reproductive organs (microsporophylls). At least for some of these genes it has been shown that they can partially substitute for the Arabidopsis B genes AP3 and PI in ectopic expression experiments, or even partially substitute these genes in different class B floral organ identity gene mutants. This functional complementation, however, is restricted to male organ development. These findings strongly suggest that gymnosperm and angiosperm B genes have highly related interaction partners and equivalent functions in the male organs of their different host species. It seems likely that in extant gymnosperms B genes have a function in specifying male reproductive organs. This function was probably established already in the most recent common ancestor of extant gymnosperms and angiosperms (seed plants) 300 million years ago and thus represents the ancestral function of seed plant B genes, from which other functions (e.g., in specifying petal identity) might have been derived. This suggests that the B gene function is part of an ancestral sex determination system in which B gene expression specifies male reproductive organ development, while the absence of B gene expression leads to the formation of female reproductive organs. Such a simple switch mechanism suggests that B genes might have played a central role during the origin of flowers. In the out-of-male and out-of-female hypotheses changes in B gene expression led to the origin of hermaphroditic flower precursors out of male or female gymnosperm reproductive cones, respectively. We compare these hypotheses with other recent molecular hypotheses on the origin of flowers, in which C/D and FLORICAULA/LEAFY-like genes is given a more prominent role, and we suggest how these hypotheses might be tested in the future.     

Molecular cloning and characterization of constans-like cDNA clones of the fuji apple

Two cDNA clones,MdCOL1 andMdCOL2, encoding CONSTANS (CO)-like proteins were isolated from an apple (Malus domestica cv. Fuji) fruit cDNA library. Both proteins contain two zinc finger motifs at the amino terminal end and a putative nuclear localization domain at the carboxyl terminal end. Genomic DNA blot analysis suggests that theCO-like genes are members of a small multigene family. RNA blot and RT-PCR analyses revealed that these genes are expressed in every organ that was examined. However, the expression levels were higher in floral buds and fruits at their early developmental stages compared to late reproductive stages or vegetative organs. Such expression patterns are quite different from those of theCO-like genes fromArabidopsis, which show strong organ specificity in either roots, cauline leaves, or flowers. These results indicate that the appleCO-like genes are significantly different from theArabidopsis genes and that they appear to play important roles in reproductive organ development.     

Histone Acetylation Accompanied with Promoter Sequences Displaying Differential Expression Profiles of B-Class MADS-Box Genes for Phalaenopsis Floral Morphogenesis

Five B-class MADS-box genes, including four APETALA3 (AP3)-like PeMADS2∼5 and one PISTILLATA (PI)-like PeMADS6, specify the spectacular flower morphology in orchids. The PI-like PeMADS6 ubiquitously expresses in all floral organs. The four AP3-like genes, resulted from two duplication events, express ubiquitously at floral primordia and early floral organ stages, but show distinct expression profiles at late floral organ primordia and floral bud stages. Here, we isolated the upstream sequences of PeMADS2∼6 and studied the regulatory mechanism for their distinct gene expression. Phylogenetic footprinting analysis of the 1.3-kb upstream sequences of AP3-like PeMADS2∼5 showed that their promoter regions have sufficiently diverged and contributed to their subfunctionalization. The amplified promoter sequences of PeMADS2∼6 could drive beta-glucuronidase (GUS) gene expression in all floral organs, similar to their expression at the floral primordia stage. The promoter sequence of PeMADS4, exclusively expressed in lip and column, showed a 1.6∼3-fold higher expression in lip/column than in sepal/petal. Furthermore, we noted a 4.9-fold increase in histone acetylation (H3K9K14ac) in the translation start region of PeMADS4 in lip as compared in petal. All these results suggest that the regulation via the upstream sequences and increased H3K9K14ac level may act synergistically to display distinct expression profiles of the AP3-like genes at late floral organ primordia stage for Phalaenopsis floral morphogenesis.     

MOSAIC FLORAL ORGANS1, an AGL6-Like MADS Box Gene,Regulates Floral Organ Identity and Meristem Fate in Rice

Floral organ identity and meristem determinacy in plants are controlled by combinations of activities mediated by MADS box genes. AGAMOUS-LIKE6 (AGL6)-like genes are MADS box genes expressed in floral tissues, but their biological functions are mostly unknown. Here, we describe an AGL6-like gene in rice (Oryza sativa), MOSAIC FLORAL ORGANS1 (MFO1/MADS6), that regulates floral organ identity and floral meristem determinacy. In the flower of mfo1 mutants, the identities of palea and lodicule are disturbed, and mosaic organs were observed. Furthermore, the determinacy of the floral meristem was lost, and extra carpels or spikelets developed in mfo1 florets. The expression patterns of floral MADS box genes were disturbed in the mutant florets. Suppression of another rice AGL6-like gene, MADS17, caused no morphological abnormalities in the wild-type background, but it enhanced the phenotype in the mfo1 background, indicating that MADS17 has a minor but redundant function with that of MFO1. Whereas single mutants in either MFO1 or the SEPALLATA-like gene LHS1 showed moderate phenotypes, the mfo1 lhs1 double mutant showed a severe phenotype, including the loss of spikelet meristem determinacy. We propose that rice AGL6-like genes help to control floral organ identity and the establishment and determinacy of the floral meristem redundantly with LHS1.     

Three classes of proteinase inhibitor gene have distinct but overlapping patterns of expression in Pisum sativum plants

Genes representative of three gene classes encoding proteinase inhibitor proteins, with distinct spatial expression patterns, were isolated and characterized from Pisum.Under standard plant growth conditions, one class is expressed exclusively in seeds, whereas the other two make minor contributions to seed inhibitor proteins but are also expressed in other organs, predominantly in root endodermal and floral reproductive tissues. Two of the gene classes contain few genes and are genetically linked at the Tri locus, whereas the third class displays complex hybridization patterns to genomic DNA and maps to diverse genetic loci. Expression analysis of this last class suggests that only a small number of these genes are expressed. The quantitative effect of the Tri locus on root and floral inhibitor gene expression was examined in near-isogenic lines of pea. The proteins encoded by the three classes are all members of the same family (Bowman-Birk) of enzyme inhibitors but are distinct in terms of overall sequence, active site sequences and inhibitor function.     

Expression of floral MADS-box genes in Sinofranchetia chinensis (Lardizabalaceae): implications for the nature of the nectar leaves

Background and Aims

The perianths of the Lardizabalaceae are diverse. The second-whorl floral organs of Sinofranchetia chinensis (Lardizabalaceae) are nectar leaves. The aim of this study was to explore the nature of this type of floral organ, and to determine its relationship to nectar leaves in other Ranunculales species, and to other floral organs in Sinofranchetia chinensis.

Methods

Approaches of evolutionary developmental biology were used, including 3′ RACE (rapid amplification of cDNA ends) for isolating floral MADS-box genes, phylogenetic analysis for reconstructing gene evolutionary history, in situ hybridization and tissue-specific RT-PCR for identifying gene expression patterns and SEM (scanning electron microscopy) for observing the epidermal cell morphology of floral organs.

Key Results

Fourteen new floral MADS-box genes were isolated from Sinofranchetia chinensis and from two other species of Lardizabalaceae, Holboellia grandiflora and Decaisnea insignis. The phylogenetic analysis of AP3-like genes in Ranunculales showed that three AP3 paralogues from Sinofranchetia chinensis belong to the AP3-I, -II and -III lineages. In situ hybridization results showed that SIchAP3-3 is significantly expressed only in nectar leaves at the late stages of floral development, and SIchAG, a C-class MADS-box gene, is expressed not only in stamens and carpels, but also in nectar leaves. SEM observation revealed that the adaxial surface of nectar leaves is covered with conical epidermal cells, a hallmark of petaloidy.

Conclusions

The gene expression data imply that the nectar leaves in S. chinensis might share a similar genetic regulatory code with other nectar leaves in Ranunculales species. Based on gene expression and morphological evidence, it is considered that the nectar leaves in S. chinensis could be referred to as petals. Furthermore, the study supports the hypothesis that the nectar leaves in some Ranunculales species might be derived from stamens.     

Dwarf and deformed flower 1, encoding an F‐box protein,is critical for vegetative and floral development in rice (Oryza sativa L.)

Recent studies have shown that F‐box proteins constitute a large family in eukaryotes, and play pivotal roles in regulating various developmental processes in plants. However, their functions in monocots are still obscure. In this study, we characterized a recessive mutant dwarf and deformed flower 1‐1 (ddf1‐1) in Oryza sativa (rice). The mutant is abnormal in both vegetative and reproductive development, with significant size reduction in all organs except the spikelet. DDF1 controls organ size by regulating both cell division and cell expansion. In the ddf1‐1 spikelet, the specification of floral organs in whorls 2 and 3 is altered, with most lodicules and stamens being transformed into glume‐like organs and pistil‐like organs, respectively, but the specification of lemma/palea and pistil in whorls 1 and 4 is not affected. DDF1 encodes an F‐box protein anchored in the nucleolus, and is expressed in almost all vegetative and reproductive tissues. Consistent with the mutant floral phenotype, DDF1 positively regulates B‐class genes OsMADS4 and OsMADS16, and negatively regulates pistil specification gene DL. In addition, DDF1 also negatively regulates the Arabidopsis LFY ortholog APO2, implying a functional connection between DDF1 and APO2. Collectively, these results revealed that DDF1, as a newly identified F‐box gene, is a crucial genetic factor with pleiotropic functions for both vegetative growth and floral organ specification in rice. These findings provide additional insights into the molecular mechanism controlling monocot vegetative and reproductive development.