A role for BELLRINGER in cell wall development is supported by loss-of-function phenotypes.

BACKGROUND: Homeodomain transcription factors play critical roles in metazoan development. BELLRINGER (BLR), one such transcription factor, is involved in diverse developmental processes in Arabidopsis, acting in vascular differentiation, phyllotaxy, flower and fruit development. BLR also has a redundant role in meristem maintenance. Cell wall remodelling underpins many of these processes, and BLR has recently been shown to regulate expression of PECTIN METHYL-ESTERASE 5 (PME5), a cell wall modifying enzyme in control of phyllotaxy. We have further explored the role of BLR in plant development by analysing phenotypes and gene expression in a series of plants over-expressing BLR, and generating combinatorial mutants with blr, brevipedicellus (bp), a member of the KNOX1 family of transcription factors that has previously been shown to interact with blr, and the homeodomain transcription factor revoluta (rev), required for radial patterning of the stem. RESULTS: Plants over-expressing BLR exhibited a wide range of phenotypes. Some were defective in cell size and demonstrated misregulation of genes predominantly affecting cell wall development. Other lines with more extreme phenotypes failed to generate lateral organs, consistent with BLR repressing transcription in the shoot apex. Cell wall dynamics are also affected in blr mutant plants, and BLR has previously been shown to regulate vascular development in conjunction with BP. We found that when bp and blr were combined with rev, a set of defects was observed that were distinct from those of bp blr lines. In these triple mutants xylem development was most strikingly affected, resulting in an almost complete lack of vessels and xylem parenchyma with secondary thickening. CONCLUSIONS: Our data support a role for BLR in ordering the shoot apex and, in conjunction with BP and REV, playing a part in determining the composition and organisation of the vascular system. Microarray analysis strongly indicates that the striking vascular phenotypes of blr bp rev triple mutants and plants over-expressing BLR result from the misregulation of a suite of genes, targets of BLR in wild type plants, that determine cell size and structure in the developing vasculature.

EXS, a putative LRR receptor kinase, regulates male germline cell number and tapetal identity and promotes seed development in Arabidopsis.

BACKGROUND: Plant germlines arise late in development from archesporial initials in the L2 layer of the anther and ovule primordia. These cells generate a radially symmetrical array of tissues that, in the Arabidopsis anther, comprises a core of sporogenous cells (meiocytes) and the enveloping tapetum, middle cell, and endothecium layers. The putative transcription factor NZZ/SPL is required for the specification of archesporial cells, but nothing is known of how their number is regulated, or what controls cell fate in the lineages they generate. Here, we report detailed characterization of extra sporogenous cells (exs), a male sterile mutant that generates extra meiocytes but lacks tapetal and middle cell layers. RESULTS: We identified the EXS locus by map-based cloning and found it to encode a putative LRR receptor kinase. In the anther, an increased number of L2 layer cells assume an archesporial fate and divide to generate a larger number of sporogenous cells. In seeds, the exs mutation results in smaller embryonic cells, delayed embryo development, and smaller mature embryos. Consistent with the observed phenotype, EXS is expressed in the inflorescence meristem, floral apices, anthers, and in developing seeds. CONCLUSIONS: EXS regulates the number of cells that divide in the L2 layer of the anther, and thus the number of functional male archesporial initials. In the young seed, EXS affects cell size in the embryo and the rate at which it develops. The apparently contrasting roles of EXS in the anther and embryo suggest that signaling through the EXS receptor kinase is a feature of a number of regulatory pathways in Arabidopsis.

VAAMANA--a BEL1-like homeodomain protein, interacts with KNOX proteins BP and STM and regulates inflorescence stem growth in Arabidopsis.

Plant shoot growth depends on the activity of the shoot apical meristem (SAM), where organ primordia are initiated. In turn, the function of the SAM depends on the activities of homeodomain (HD) proteins of the knotted1-like homeobox (KNOX) class [Long et al., Nature 379 (1996) 66; Vollbrecht et al., Development 127 (2000) 3161]. In plants, KNOX proteins have been shown to interact specifically with the BEL1-like (BELL) class of homeodomain proteins [Bellaoui et al., Plant Cell 13 (2001) 2455; Muller et al., Plant 27 (2001) 13; Smith et al., Proc. Natl. Acad. Sci. USA 99 (2002) 9579], through a domain conserved between plants and animals. We have isolated a mutation in a BELL homeobox gene VAAMANA (VAN) that causes a dwarf phenotype. In addition, van inflorescence stems have clusters of cauline leaves; typically three are produced at each node. VAN interacts specifically with the class I KNOX proteins SHOOTMERISTEMLESS (STM), BREVIPEDICELLUS (BP), and KNAT6 (K6), and nuclear localisation of a VAN-GFP fusion depends on co-expression of STM or BP in tobacco leaves. This suggests that localisation of VAN, like that of the animal PBC homeodomain protein [Rieckhof et al., Cell 91 (1997) 171; Berthelsen et al., Genes Dev. 13 (1999) 946], is also regulated by interaction with a partner homeodomain protein.

Gene structure and molecular analysis of Arabidopsis thaliana ALWAYS EARLY homologs.

Drosophila always early (aly) is essential for spermatogenesis, and is related to the LIN-9 protein of Caenorhabditis elegans; lin-9 is a class B Synthetic Multivulva gene (synMuvB) required for gonadal sheath development. Aly/LIN-9 have two conserved regions, called domains 1 and 2, which have been identified in homologous proteins from several multicellular eukaryotes, including the model plant Arabidopsis thaliana. We cloned and sequenced cDNAs of three different A. thaliana ALWAYS EARLY homologs (AtALY1, AtALY2 and AtALY3), analysed the expression pattern of these three genes and show that AtALY1, like Aly, is nuclear localised. We also demonstrate that the plant homologs of aly/lin-9 contain an additional N-terminal myb domain not present in the animal Aly/LIN-9 proteins, and that part of the ALY/LIN-9 conserved domain 1 in the predicted plant proteins is related to the TUDOR domain.

PAH-domain-specific interactions of the Arabidopsis transcription coregulator SIN3-LIKE1 (SNL1) with telomere-binding protein 1 and ALWAYS EARLY2 Myb-DNA binding factors.

The eukaryotic SIN3 protein is the central component of the evolutionarily conserved multisubunit SIN3 complex that has roles in regulating gene expression and genome stability. Here we characterise the structure of the SIN3 protein in higher plants through the analysis of SNL1 (SIN3-LIKE1), SNL2, SNL3, SNL4, SNL5 and SNL6, a family of six SIN3 homologues in Arabidopsis thaliana. In an Arabidopsis-protoplast beta-glucuronidase reporter gene assay, as well as in a heterologous yeast repression assay, full-length SNL1 was shown to repress transcription in a histone-deacetylase-dependent manner, demonstrating the conserved nature of SIN3 function. Yeast two-hybrid screening identified a number of DNA binding proteins each containing a single Myb domain that included the Arabidopsis ALWAYS EARLY proteins AtALY2 and AtALY3, and two telomere binding proteins AtTBP1 and AtTRP2/TRFL1 as SNL1 partners, suggesting potential functions for SNL1 in development and telomere maintenance. The interaction with telomere-binding protein 1 was found to be mediated through the well-defined paired amphipathic helix domain PAH2. In contrast, the AtALY2 interaction was mediated through the PAH3 domain of SNL1, which is structurally distinct from PAH1 and PAH2, suggesting that evolution of this domain to a more novel structural motif has occurred. These findings support a diverse role of SNL1 in the regulation of transcription and genome stability.

Hawaiian skirt: an F-box gene that regulates organ fusion and growth in Arabidopsis.

A fast neutron-mutagenized population of Arabidopsis (Arabidopsis thaliana) Columbia-0 wild-type plants was screened for floral phenotypes and a novel mutant, termed hawaiian skirt (hws), was identified that failed to shed its reproductive organs. The mutation is the consequence of a 28 bp deletion that introduces a premature amber termination codon into the open reading frame of a putative F-box protein (At3g61590). The most striking anatomical characteristic of hws plants is seen in flowers where individual sepals are fused along the lower part of their margins. Crossing of the abscission marker, Pro(PGAZAT):beta-glucuronidase, into the mutant reveals that while floral organs are retained it is not the consequence of a failure of abscission zone cells to differentiate. Anatomical analysis indicates that the fusion of sepal margins precludes shedding even though abscission, albeit delayed, does occur. Spatial and temporal characterization, using Pro(HWS):beta-glucuronidase or Pro(HWS):green fluorescent protein fusions, has identified HWS expression to be restricted to the stele and lateral root cap, cotyledonary margins, tip of the stigma, pollen, abscission zones, and developing seeds. Comparative phenotypic analyses performed on the hws mutant, Columbia-0 wild type, and Pro(35S):HWS ectopically expressing lines has revealed that loss of HWS results in greater growth of both aerial and below-ground organs while overexpressing the gene brings about a converse effect. These observations are consistent with HWS playing an important role in regulating plant growth and development.

AtREC8 and AtSCC3 are essential to the monopolar orientation of the kinetochores during meiosis.

The success of the first meiotic division relies (among other factors) on the formation of bivalents between homologous chromosomes, the monopolar orientation of the sister kinetochores at metaphase I and the maintenance of centromeric cohesion until the onset of anaphase II. The meiotic cohesin subunit, Rec8 has been reported to be one of the key players in these processes, but its precise role in kinetochore orientation is still under debate. By contrast, much less is known about the other non-SMC cohesin subunit, Scc3. We report the identification and the characterisation of AtSCC3, the sole Arabidopsis homologue of Scc3. The detection of AtSCC3 in mitotic cells, the embryo lethality of a null allele Atscc3-2, and the mitotic defects of the weak allele Atscc3-1 suggest that AtSCC3 is required for mitosis. AtSCC3 was also detected in meiotic nuclei as early as interphase, and bound to the chromosome axis from early leptotene through to anaphase I. We show here that both AtREC8 and AtSCC3 are necessary not only to maintain centromere cohesion at anaphase I, but also for the monopolar orientation of the kinetochores during the first meiotic division. We also found that AtREC8 is involved in chromosome axis formation in an AtSPO11-1-independent manner. Finally, we provide evidence for a role of AtSPO11-1 in the stability of the cohesin complex.