Activation of the <Emphasis Type="Italic">WUS</Emphasis> gene induces ectopic initiation of floral meristems on mature stem surface in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A gain-of-function Arabidopsis mutant was identified via activation tagging genetic screening. The mutant exhibited clustered ectopic floral buds on the surface of inflorescence stems. The mutant was designated as sef for stem ectopic flowers. Our detailed studies indicate that the ectopic flower meristems are initiated from the differentiated cortex cells. Inverse PCR and sequence analysis indicated that the enhancer-containing T-DNA from the activation tagging construct, SKI015, was inserted upstream of the previously cloned WUS gene encoding a homeodomain protein. Studies from RT-PCR, RNA in situ hybridization and transgenic plant analysis further confirmed that the phenotypes of sef are caused by the overexpression of WUS. Our results suggest that overexpression of WUS could trigger the cell pluripotence and reestablish a new meristem in cortex. The type of new meristems caused by WUS overexpression was dependent upon the developmental and physiological stages of a plant. With the help of some undefined factors in the reproductive organs the new meristems differentiated into floral buds. In a vegetative growth plant, however, only the new vegetative buds can be initiated upon the overexpression of WUS. These studies provide new insights of WUS on flower development.     

<Emphasis Type="Italic">MIR166</Emphasis>/<Emphasis Type="Italic">165</Emphasis> genes exhibit dynamic expression patterns in regulating shoot apical meristem and floral development in <Emphasis Type="Italic">Arabidopsis</Emphasis>

The miR166/165 group and its target genes regulate diverse aspects of plant development, including apical and lateral meristem formation, leaf polarity, and vascular development. We demonstrate here that MIR166/165 genes are dynamically controlled in regulating shoot apical meristem (SAM) and floral development in parallel to the WUSCHEL (WUS)-CLAVATA (CLV) pathway. Although miR166 and miR165 cleave same target mRNAs, individual MIR166/165 genes exhibit distinct expression domains in different plant tissues. The MIR166/165 expression is also temporarily regulated. Consistent with the dynamic expression patterns, an array of alterations in SAM activities and floral architectures was observed in the miR166/165-overproducing plants. In addition, when a MIR166a-overexpressing mutant was genetically crossed with mutants defective in the WUS-CLV pathway, the resultant crosses exhibited additive phenotypic effects, suggesting that the miR166/165-mediated signal exerts its role via a distinct signaling pathway.     

<Emphasis Type="Italic">ZWILLE</Emphasis> buffers meristem stability in<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis>

The shoot apical meristem of higher plants consists of a population of stem cells at the tip of the plant body that continuously gives rise to organs such as leaves and flowers. Cells that leave the meristem differentiate and must be replaced to maintain the integrity of the meristem. The balance between differentiation and maintenance is governed both by the environment and the developmental status of the plant. In order to respond to these different stimuli, the meristem has to be plastic thus ensuring the stereotypic shape of the plant body. Meristem plasticity requires the ZWILLE (ZLL) gene. In zll mutant embryos, the apical cells are misspecified causing a variability of the meristems size and function. Using specific antibodies against ZLL, we show that the zll phenotype is due to the complete absence of the ZLL protein. In immunohistochemical experiments we confirm the observation that ZLL is solely localized in vascular tissue. For a better understanding of the role of ZLL in meristem stability, we analysed the genetic interactions of ZLL with WUSCHEL (WUS) and the CLAVATA1, 2 and 3 (CLV) genes that are involved in size regulation of the meristem. In a zll loss-of-function background wus has a negative effect whereas clv mutations have a positive effect on meristem size. We propose that ZLL buffers meristem stability non-cell-autonomously by ensuring the critical number of apical cells required for proper meristem function.Edited by G. JürgensAn erratum to this article can be found at     

<Emphasis Type="Italic">WUS</Emphasis> and <Emphasis Type="Italic">STM</Emphasis>-based reporter genes for studying meristem development in poplar

We describe the development of a reporter system for monitoring meristem initiation in poplar using promoters of poplar homologs to the meristem-active regulatory genes WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM). When ~3 kb of the 5′ flanking regions of close homologs were used to drive expression of the GUSPlus gene, 50–60% of the transgenic events showed expression in apical and axillary meristems. However, expression was also common in other organs, including in leaf veins (40 and 46% of WUS and STM transgenic events, respectively) and hydathodes (56% of WUS transgenic events). Histochemical GUS staining of explants during callogenesis and shoot regeneration using in vitro stems as explants showed that expression was detectable prior to visible shoot development, starting 3–15 days after explants were placed onto callus inducing medium. A minority of WUS and STM events also showed expression in the cambium, phloem, or xylem of regenerated, greenhouse grown plants undergoing secondary growth. Based on microarray gene expression data, a paralog of poplar WUS was detectably up-regulated during shoot initiation, but the other paralog was not. Both paralogs of poplar STM were down-regulated threefold to sixfold during early callus initiation. We identified 15–35 copies of cytokinin response regulator binding motifs (ARR1AT) and one copy of the auxin response element (AuxRE) in both promoters. Several of the events recovered may be useful for studying the process of primary and secondary meristem development, including treatments intended to stimulate meristem development to promote clonal propagation and genetic transformation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">FORMOSA</Emphasis> controls cell division and expansion during floral development in <Emphasis Type="Italic">Antirrhinum</Emphasis><Emphasis Type="Italic">majus</Emphasis>

Control of organ size is the product of coordinated cell division and expansion. In plants where one of these pathways is perturbed, organ size is often unaffected as compensation mechanisms are brought into play. The number of founder cells in organ primordia, dividing cells, and the period of cell proliferation determine cell number in lateral organs. We have identified the Antirrhinum FORMOSA (FO) gene as a specific regulator of floral size. Analysis of cell size and number in the fo mutant, which has increased flower size, indicates that FO is an organ-specific inhibitor of cell division and activator of cell expansion. Increased cell number in fo floral organs correlated with upregulation of genes involved in the cell cycle. In Arabidopsis the AINTEGUMENTA (ANT) gene promotes cell division. In the fo mutant increased cell number also correlates with upregulation of an Antirrhinum ANT-like gene (Am-ANT) in inflorescences that is very closely related to ANT and shares a similar expression pattern, suggesting that they may be functional equivalents. Increased cell proliferation is thought to be compensated for by reduced cell expansion to maintain organ size. In Arabidopsis petal cell expansion is inhibited by the BIGPETAL (BPE) gene, and in the fo mutant reduced cell size corresponded to upregulation of an Antirrhinum BPE-like gene (Am-BPE). Our data suggest that FO inhibits cell proliferation by negatively regulating Am-ANT, and acts upstream of Am-BPE to coordinate floral organ size. This demonstrates that organ size is modulated by the organ-specific control of both general and local gene networks. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mutations in the <Emphasis Type="Italic">TORNADO2</Emphasis> gene affect cellular decisions in the peripheral zone of the shoot apical meristem of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">thaliana</Emphasis>

Summary An EMS (ethyl methanesulfonate) mutagenesis effector screen performed with the STM:GUS marker line in Arabidopsis thaliana identified a loss-of-function allele of the TORNADO2 gene. The histological and genetic analyses described here implicate TRN2 in SAM function, where the peripheral zone in trn2 mutants is enlarged relative to the central stem cell zone. The trn2 mutant allele partially rescues the phenotype of shoot meristemless mutants but behaves additively to wuschel and clavata3 alleles during the vegetative phase and in the outer floral whorls. The development of carpels in trn2 wus-1 double mutant flowers indicates that pluripotent cells persist in floral meristems in the absence of TRN2 function and can be recruited for carpel anlagen. The data implicate a membrane-bound plant tetraspanin protein in cellular decisions in the peripheral zone of the SAM.     

Characterization of <Emphasis Type="Italic">SpAPETALA3</Emphasis> and <Emphasis Type="Italic">SpPISTILLATA</Emphasis>, B class floral identity genes in <Emphasis Type="Italic">Spinacia oleracea</Emphasis>, and their relationship to sexual dimorphism

Floral organ identity B class genes are generally recognized as being required for development of petals and stamens in angiosperm flowers. Spinach flowers are distinguished in their complete absence of petals in both sexes, and the absence of a developed stamen whorl in female flowers. As such, we hypothesized that differential expression of B class floral identity genes is integral to the sexual dimorphism in spinach flowers. We isolated two spinach orthologs of Arabidopsis B class genes by 3 and 5 RACE. Homology assignments were tested by comparisons of percent amino acid identities, searches for diagnostic consensus amino acid residues, conserved motifs, and phylogenetic groupings. In situ hybridization studies demonstrate that both spinach B class genes are expressed throughout the male floral meristem in early stages, and continue to be expressed in sepal primordia in reduced amounts at later stages of development. They are also highly expressed in the third whorl primordia when they arise and continue to be expressed in these tissues through the development of mature anthers. In contrast, neither gene can be detected in any stage in female flowers by in situ analyses, although northern blot experiments indicate low levels of SpAP3 within the inflorescence. The early, strong expressions of both B class floral identity genes in male floral primordia and their absence in female flowers demonstrate that B class gene expression precedes the origination of third whorl primordia (stamen) in males and is associated with the establishment of sexual floral dimorphism as it initiates in the first (sepal) whorl. These observations suggest that regulation of B class floral identity genes has a role in the development of sexual dimorphism and dioecy in spinach rather than being a secondary result of organ abortion.Electronic Supplementary Material Supplementary material is available for this article at Edited by G. Jürgens     

Developmental consequences of the <Emphasis Type="Italic">tumorous shoot development1</Emphasis> mutation,a novel allele of the cellulose-synthesizing <Emphasis Type="Italic">KORRIGAN1</Emphasis> gene

This work describes the further characterization of the tumorous shoot development1 (tsd1) mutant of Arabidopsis thaliana, which develops disorganized tumorous-like shoot tissue instead of organized leaves and stems. Map-based cloning revealed that tsd1 is a novel strong allele of the KOR1 gene, encoding a membrane-bound endo-1,4-β-d-glucanase involved in cellulose synthesis. To study developmental changes accompanying the aberrant growth of the tsd1 mutant, patterning in the meristems and the hormonal status were analysed by marker genes. Expression of key regulators of meristem maintenance, the CLV3 and STM genes, indicated the presence of numerous meristems in the tsd1 shoot callus. Expression of the LFY::GUS marker supported the ability of the tsd1 callus to form organ primordia, which however failed to develop further. An epidermal marker showed that the L1 layer was maintained only in distinct areas of the tsd1 callus, which could be a reason of disorganized shoot growth. In the tsd1 root meristem, quiescent center activity was lost early after germination, which caused differentiation of the root meristem. The spatial expression of genes reporting the auxin and cytokinin status was altered in the tsd1 mutant. Modifying the endogenous levels of these hormones partially rescued shoot and root development of the tsd1 mutant. Together, the work shows that TSD1/KOR1 is required for maintaining a correct meristematic pattern and organ growth as well as for a normal hormonal response.     

Over-expression of the <Emphasis Type="Italic">IGI1</Emphasis> leading to altered shoot-branching development related to MAX pathway in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Shoot branching and growth are controlled by phytohormones such as auxin and other components in Arabidopsis. We identified a mutant (igi1) showing decreased height and bunchy branching patterns. The phenotypes reverted to the wild type in response to RNA interference with the IGI1 gene. Histochemical analysis by GUS assay revealed tissue-specific gene expression in the anther and showed that the expression levels of the IGI1 gene in apical parts, including flowers, were higher than in other parts of the plants. The auxin biosynthesis component gene, CYP79B2, was up-regulated in igi1 mutants and the IGI1 gene was down-regulated by IAA treatment. These results indicated that there is an interplay regulation between IGI1 and phytohormone auxin. Moreover, the expression of the auxin-related shoot branching regulation genes, MAX3 and MAX4, was down-regulated in igi1 mutants. Taken together, these results indicate that the overexpression of the IGI1 influenced MAX pathway in the shoot branching regulation.     

Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.     

The <Emphasis Type="Italic">paleoAP3</Emphasis>-type gene <Emphasis Type="Italic">CpAP3</Emphasis>, an ancestral B-class gene from the basal angiosperm <Emphasis Type="Italic">Chimonanthus praecox</Emphasis>, can affect stamen and petal development in higher eudicots

Wintersweet (Chimonanthus praecox), a basal angiosperm endemic to China, has high ornamental value for developing beautiful flowers with strong fragrance. The molecular mechanism regulating flower development in wintersweet remains largely elusive. In this project, we seek to determine the molecular features and expression patterns of the C. praecox paleoAP3-type gene CpAP3 and examine its potential role in regulating floral development via ectopic expression in Arabidopsis thaliana and Petunia hybrida. The expression of CpAP3 is tissue-specific, with the highest level in the tepals, moderate level in carpels, and weak levels in stamen and vegetative stem tissues. Its dynamic expression during flowering is associated with flower-bud formation. Ectopic expression of CpAP3 partially rescued stamen development in ap3 mutant Arabidopsis. Although no phenotypic effect has been observed in wild-type Arabidopsis, CpAP3 overexpression in petunia brought rich morphological changes and homeotic conversions to flowers, mainly involving disruption of petal and stamen development. Expressed in a broader range than those canonical B-function regulators, the ancestral B-class gene CpAP3 can affect petal and stamen development in higher eudicots. This gene also holds some bioengineering potential in creating novel floral germplasms.