Isolation and Characterization of an AGAMOUS-Like Gene from Magnolia wufengensis (Magnoliaceae)

The floral homeotic C function gene AGAMOUS (AG) plays crucial roles in Arabidopsis development by specifying stamen and carpel identity, repressing A-class genes, as well as regulating floral meristem determination. Although the function of AG homologs from other core eudicots appears highly conserved, the role of AG orthologs in the design of floral architecture in basal angiosperm remains unknown. We isolated and identified an AG ortholog from Magnolia wufengensis, a woody basal angiosperm belonging to the Magnoliaceae. Sequence and phylogenetic analyses revealed that it is a clade member of the euAG lineage, and hence, the gene is referred to as MAwuAG (M. wu fengensis AGAMOUS). Moreover, two highly conserved motifs specific to C proteins, AG motifs I and II, are found in the C-terminal regions of the MAwuAG protein, but the N-terminal extensions that usually appear in euAG lineage members from eudicots were not found in MAwuAG. The cDNA has the first in-frame ATG immediately preceding the MADS domain. A semi-quantitative PCR analysis showed that the expression of MAwuAG was restricted to reproductive organs of stamens and carpels. The transgenic Arabidopsis containing 35S::MAwuAG displayed extremely early flowering, bigger stamens and carpels, and homeotic conversion of petals into staminoid organs, but ectopic expression of MAwuAG in the first whorls failed to convert the sepals into carpeloid structures that are usually observed in the overexpression transgenic Arabidopsis of AG orthologs from other core eudicots. In addition, the phenotype of the transgenic 35S::MAwuAG Arabidopsis revealed that the abscission of the outer three floral whorls (sepals, petals, and stamens) was inhibited.     

<Emphasis Type="Italic">AVAG2</Emphasis> is a putative D-class gene from an ornamental asparagus

Members of the AGAMOUS (AG) family of MADS-box genes play important roles in regulating the development of reproductive organs in flowering plants. To elucidate the molecular mechanisms of floral development in Asparagus virgatus, we isolated and characterized an Asparagus AG-homologue, AVAG2. AVAG2 contains an open reading frame that encodes a deduced protein with 234 amino acid residues. Phylogenetic analysis indicated that AVAG2 belongs to the D-lineage of the AG gene family. AVAG2 mRNA was detected in the flower, but not in vegetative organs. Moreover, in in situ hybridization experiments, AVAG2 signals were observed in the stamens and carpels during early flower development, and appeared in the ovule only at later developmental stages. This suggests that the AVAG2 gene is involved in ovule formation. Thus, our expression data support the phylogenetic analysis indicating that AVAG2 belongs to the D-class gene family.     

Characterization of an AGAMOUS homologue from the conifer black spruce ( Picea mariana ) that produces floral homeotic conversions when expressed in Arabidopsis

Advances in elucidating the molecular processes controlling flower initiation and development have provided unique opportunities to investigate the developmental genetics of non-flowering plants. In addition to providing insights into the evolutionary aspects of seed plants, identification of genes regulating reproductive organ development in gymnosperms could help determine the level of homology with current models of flower induction and floral organ identity. Based upon this, we have searched for putative developmental regulators in conifers with amino acid sequence homology to MADS-box genes. PCR cloning using degenerate primers targeted to the MADS-box domain revealed the presence of over 27 MADS-box genes within black spruce (Picea mariana), including several with extensive homology to either AP1 or AGAMOUS, both known to regulate flower development in Arabidopsis. This indicates that like angiosperms, conifers contain a large and diverse MADS-box gene family that probably includes regulators of reproductive organ development. Confirmation of this was provided by the characterization of an AGAMOUS-like cDNA clone called SAG1, whose conservation of intron position and tissue-specific expression within reproductive organs indicate that it is a homologue of AGAMOUS. Functional homology with AGAMOUS was demonstrated by the ability of SAG1 to produce homeotic conversions of sepals to carpels and petals to stamens when ectopically expressed in transgenic Arabidopsis. This suggests that some of the genetic pathways controlling flower and cone development are homologous, and antedate the 300-million-year-old divergence of angiosperms and gymnosperms.     

Isolation and characterization of the C-class MADS-box gene from the distylous pseudo-cereal <Emphasis Type="Italic">Fagopyrum esculentum</Emphasis>

In the model species Arabidopsis thaliana, the floral homeotic C-class gene AGAMOUS (AG) specifies reproductive organ (stamen and carpels) identity and floral meristem determinacy. Gene function analyses in other core eudicots species reveal functional conservation, subfunctionalization and function switch of the C-lineage in this clade. To identify the possible roles of AG-like genes in regulating floral development in distylous species with dimorphic flowers (pin and thrum) and the C function evolution, we isolated and identified an AG ortholog from Fagopyrum esculentum (buckwheat, Family Polygonaceae), an early diverging species of core eudicots preceding the rosids-asterids split. Protein sequence alignment and phylogenetic analysis grouped FaesAG into the euAG lineage. Expression analysis suggested that FaesAG expressed exclusively in developing stamens and gynoecium of pin and thrum flowers. Moreover, FaesAG expression reached a high level in both pin and thrum flowers at the time when the stamens were undergoing rapidly increased in size and microspore mother cells were in meiosis. FaesAG was able to substitute for the endogenous AG gene in specifying stamen and carpel identity and in an Arabidopsis ag-1 mutant. Ectopic expression of FaesAG led to very early flowering, and produced a misshapen inflorescence and abnormal flowers in which sepals had converted into carpels and petals were converted to stamens. Our results confirmed establishment of the complete C-function of the AG orthologous gene preceding the rosids-asterids split, despite the distinct floral traits present in early- and late-diverging lineages of core eudicot angiosperms.     

Delay of flower senescence and abscission in Arabidopsis transformed with an FOREVER YOUNG FLOWER homolog from Oncidium orchid

The ectopic expression of FOREVER YOUNG FLOWER (FYF), a MADS box gene in Arabidopsis, caused significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. It was proposed that the function of the FYF gene was related to the regulation of senescence and abscission. This hypothesis was further supported by one line of evidence reported in this study. The evidence is the similar delay of flower senescence and abscission observed in transgenic Arabidopsis ectopically expressing OnFYF, an FYF homolog from the Oncidium orchid, a monocot. This data suggested that the function of FYF homologs in regulating flower senescence and abscission was highly conserved in both dicot and monocot plants.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.     

Regulation of flower development in Arabidopsis by SCF complexes

SCF complexes are the largest and best studied family of E3 ubiquitin protein ligases that facilitate the ubiquitylation of proteins targeted for degradation. The SCF core components Skp1, Cul1, and Rbx1 serve in multiple SCF complexes involving different substrate-specific F-box proteins that are involved in diverse processes including cell cycle and development. In Arabidopsis, mutations in the F-box gene UNUSUAL FLORAL ORGANS (UFO) result in a number of defects in flower development. However, functions of the core components Cul1 and Rbx1 in flower development are poorly understood. In this study we analyzed floral phenotypes caused by altering function of Cul1 or Rbx1, as well as the effects of mutations in ASK1 and ASK2. Plants homozygous for a point mutation in the AtCUL1 gene showed reduced floral organ number and several defects in each of the four whorls. Similarly, plants with reduced AtRbx1 expression due to RNA interference also exhibited floral morphological defects. In addition, compared to the ask1 mutant, plants homozygous for ask1 and heterozygous for ask2 displayed enhanced reduction of B function, as well as other novel defects of flower development, including carpelloid sepals and an inhibition of petal development. Genetic analyses demonstrate that AGAMOUS (AG) is required for the novel phenotypes observed in the first and second whorls. Furthermore, the genetic interaction between UFO and AtCUL1 supports the idea that UFO regulates multiple aspects of flower development as a part of SCF complexes. These results suggest that SCF complexes regulate several aspects of floral development in Arabidopsis.     

Separation of AG function in floral meristem determinacy from that in reproductive organ identity by expressing antisense AG RNA

The Arabidopsis floral homeotic gene AGAMOUS (AG) is a regulator of early flower development. The ag mutant phenotypes suggest that AG has two functions in flower development: (1) specifying the identity of stamens and carpels, and (2) controlling floral meristem determinacy. To dissect these two AG functions, we have generated transgenic Arabidopsis plants carrying an antisense AG construct. We found that all of the transgenic plants produced abnormal flowers, which can be classified into three types. Type I transgenic flowers are phenocopies of the ag-1 mutant flowers, with both floral meristem indeterminacy and floral organ conversion; type II flowers are indeterminate and have partial conversion of the reproductive organs; and type III flowers have normal stamens and carpels, but still have an indeterminate floral meristem inside the fourth whorl of fused carpels. The existence of type III flowers indicates that AG function can be perturbed to affect only floral meristem determinacy, but not floral organ identity. Furthermore, the fact that floral meristem determinacy is affected in all transformants, but floral organ identity only in a subset of them, suggests that the former may required a higher level of AG activity than the latter. This hypothesis is supported by the levels of AG'mRNA detected in different transformants with different frequencies of distinct types of abnormal antisense AG transgenic flowers. Finally, since AG inhibits the expression of another floral regulatory gene AP1, we examined AP1 expression in antisense AG flowers, and found that AP1 is expressed at a relatively high level in the center of type II flowers, but very little or below detectable levels in the inner whorls of type III flowers. These results provide further insights into the interaction of AG and AP1 and how such an interaction may control both organ identity and floral meristem determinacy.     

Cucumber SUPERMAN Has Conserved Function in Stamen and Fruit Development and a Distinct Role in Floral Patterning

The Arabidopsis SUPERMAN (SUP) gene encodes a C2H2 type zinc finger protein that is required for maintaining the boundaries between stamens and carpels, and for regulating development of ovule outer integument. Orthologs of SUP have been characterized in bisexual flowers as well as dioecious species, but it remains elusive in monoecious plants with unisexual flowers on the same individual. Here we isolate the SUP ortholog in Cucumis sativus L (CsSUP), a monoecious vegetable. CsSUP is predominantly expressed in female specific organs: the female flower buds and ovules. Ectopic expression of CsSUP in Arabidopsis can partially complement the fruit development in sup-5 mutant, and its over-expression in wide-type leads to reduced silique length, suppressed stamen development and distorted petal patterning. Our data suggest that CsSUP plays conserved as well as distinct roles during flower and fruit development, and it may function in the boundaries and ovules to balance petal patterning, stamen and ovule development in Arabidopsis.