Day Length and Temperature Strongly Influence Sexual Reproduction and Expression of a Novel MADS-Box Gene in the Moss Physcomitrella patens

Abstract: The effect of temperature and light conditions on sexual reproduction (sporophyte formation) of in vitro cultures of the moss Physcomitrella patens was analysed. All parameters tested, i.e., temperature, light intensity and day length had a strong impact on the number of sporophytes formed. The highest number of sporophytes, 559 g fresh weight, developed at 15 °C, 8 h light/day with an intensity of 20 μmol/m²/s. In contrast, at 25 °C, as well as with a day length of 16 h per day, the number of sporophytes was drastically reduced. Vegetative growth, determined as fresh weight per petri dish, was impeded under conditions favouring sporophyte formation, probably due to nutrient transfer to the sporophytes. Microscopic documentation of the developing sporophytes revealed that, although archegonia were arranged in bundles at the gametophore apices, usually only one archegonium per gametophore apex developed into a mature sporophyte. From an EST database six novel MADS-box genes were identified which, in phylogenetic analyses, did not cluster with the known groups of higher plant MADS-box genes. One of these genes was represented only as a singleton in a cDNA library specifically derived from gametophore apices and developing sporophytes, and, therefore, designated PpMADS-S. RNA amounts of PpMADS-S were two to three times higher under conditions that stimulate sporophyte development (15 °C, 8 h light per day) when compared to conditions favouring vegetative growth (25 °C, 16 h light per day), indicating a possible function in sexual reproduction of this moss. Thus, an efficient experimental system was established to study sex organ formation, fertilization and embryo development in Physcomitrella.     

蝴蝶兰PhalPI基因的克隆及在花器官突变体中的表达分析

为深入研究兰科植物花器官发育的调控机理,从蝴蝶兰花瓣中克隆了一个B类MADS-box转录因子PhalPI（GenBank登录号为KY020416）。序列分析表明,该基因的cDNA全长为944 bp,含完整的开放阅读框,可编码210个氨基酸,属于BGLO/PI蛋白家族,与蝴蝶兰属的PhPI10和PeMADS6基因关系最近;表达模式分析表明,PhalPI基因在生殖器官中表达,在营养器官中不表达,在授粉后的子房中,该基因的表达水平降低。在5种花器官突变体中,PhalPI基因在萼片唇瓣化突变体的萼片和蕊柱中表达水平明显升高;在雄蕊花瓣化突变体的萼片和侧瓣中表达水平降低,在其唇瓣和蕊柱中显著升高;在侧瓣合柱化突变体的蕊柱中,PhalPI基因的表达也发生了显著升高;PhalPI基因表达的改变与花器官形态的突变相关;而在侧瓣唇瓣化和侧瓣花药化突变体中,PhalPI基因的表达水平没有变化。推测该基因在决定蝴蝶兰侧瓣和唇瓣的发育中起重要的调控作用。     

Identification of a MADS-box gene, FLOWERING LOCUS M, that represses flowering

The timing of flowering is important for the reproductive success of plants. Here we describe the identification and characterization of a new MADS-box gene, FLOWERING LOCUS M (FLM), which is involved in the transition from vegetative to reproductive development. FLM is similar in amino-acid sequence to FLC, another MADS-box gene involved in flowering-time control. flm mutants are early flowering in both inductive and non-inductive photoperiods, and flowering time is sensitive to FLM dosage. FLM overexpression produces late-flowering plants. Thus FLM acts as an inhibitor of flowering. FLM is expressed in areas of cell division such as root and shoot apical regions and leaf primordia.     

A short history of MADS-box genes in plants

Evolutionary developmental genetics (evodevotics) is a novel scientific endeavor which assumes that changes in developmental control genes are a major aspect of evolutionary changes in morphology. Understanding the phylogeny of developmental control genes may thus help us to understand the evolution of plant and animal form. The principles of evodevotics are exemplified by outlining the role of MADS-box genes in the evolution of plant reproductive structures. In extant eudicotyledonous flowering plants, MADS-box genes act as homeotic selector genes determining floral organ identity and as floral meristem identity genes. By reviewing current knowledge about MADS-box genes in ferns, gymnosperms and different types of angiosperms, we demonstrate that the phylogeny of MADS-box genes was strongly correlated with the origin and evolution of plant reproductive structures such as ovules and flowers. It seems likely, therefore, that changes in MADS-box gene structure, expression and function have been a major cause for innovations in reproductive development during land plant evolution, such as seed, flower and fruit formation.     

On the origin of floral morphological novelties

Floral morphological novelties, like homeotic changes of whorl 1 organs, can easily arise by modifying existing regulatory networks. Ectopic expression of B-function MADS-box genes in whorl 1 leads to a replacement of sepals by petals, as is found in the Liliaceae. In cases where leaf-like sepals or even inflated calyces develop, which ultimately envelop the mature fruit as in Physalis, ectopic expression of a vegetative MADS-box gene seems to be responsible. Current knowledge concerning the origin of such morphological novelties is reviewed.     

Transforming petals into sepaloid organs in<Emphasis Type="Italic"> Arabidopsis</Emphasis> and oilseed rape: implementation of the hairpin RNA-mediated gene silencing technology in an organ-specific manner

Oilseed rape (Brassica napus L.) genotypes with no or small petals are thought to have advantages in photosynthetic activity. The flowers of field-grown oilseed rape form a bright-yellow canopy that reflects and absorbs nearly 60% of the photosynthetically active radiation (PAR), causing a severe yield penalty. Reducing the size of the petals and/or removing the reflecting colour will improve the transmission of PAR to the leaves and is expected to increase the crop productivity. In this study the hairpin RNA-mediated (hpRNA) gene silencing technology was implemented in Arabidopsis thaliana (L.) Heynh. and B. napus to silence B-type MADS-box floral organ identity genes in a second-whorl-specific manner. In Arabidopsis, silencing of B-type MADS-box genes was obtained by expressing B. napus APETALA3 (BAP3) or PISTILLATA (BPI) homologous self-complementary hpRNA constructs under control of the Arabidopsis A-type MADS-box gene APETALA1 (AP1) promoter. In B. napus, silencing of the BPI gene family was achieved by expressing a similar hpRNA construct as used in Arabidopsis under the control of a chimeric promoter consisting of a modified petal-specific Arabidopsis AP3 promoter fragment fused to the AP1 promoter. In this way, transgenic plants were generated producing male fertile flowers in which the petals were converted into sepals (Arabidopsis) or into sepaloid petals (B. napus). These novel flower phenotypes were stable and heritable in both species.Abbreviations PAR photosynthetically active radiation - ST-LS1 potato light-inducible tissue-specific ST-LS1 gene - GUS -glucuronidase     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

A new role of the Arabidopsis SEPALLATA3 gene revealed by its constitutive expression

During Arabidopsis flower development a set of homeotic genes plays a central role in specifying the distinct floral organs of the four whorls, sepals in the outermost whorl, and petals, stamens, and carpels in the sequentially inner whorls. The current model for the identity of the floral organs includes the SEPALLATA genes that act in combination with the A, B and C genes for the specification of sepals, petals, stamens and carpels. According to this new model, the floral organ identity proteins would form different complexes of proteins for the activation of the downstream genes. We show that the presence of SEPALLATA proteins is needed to activate the AG downstream gene SHATTERPROOF2, and that SEPALLATA4 alone does not provide with enough SEPALLATA activity for the complex to be functional. Our results suggest that CAULIFLOWER may be part of the protein complex responsible for petal development and that it is fully required in the absence of APETALA1 in 35S::SEP3 plants. In addition, genetic and molecular experiments using plants constitutively expressing SEPALLATA3 revealed a new role of SEPALLATA3 in activating other B and C function genes. We molecularly prove that the ectopic expression of SEPALLATA3 is sufficient to ectopically activate APETALA3 and AGAMOUS. Remarkably, plants that constitutively express both SEPALLATA3 and LEAFY developed ectopic petals, carpels and ovules outside of the floral context.