The<Emphasis Type="Italic"> Arabidopsis KAKTUS</Emphasis> gene encodes a HECT protein and controls the number of endoreduplication cycles

In animals and plants, many cell types switch from mitotic cycles to endoreduplication cycles during differentiation. Little is known about the way in which the number of endoreduplication cycles is controlled in such endopolyploid cells. In this study we have characterized at the molecular level three mutations in the Arabidopsis gene KAKTUS ( KAK), which were previously shown specifically to repress endoreduplication in trichomes. We show that KAK is also involved in the regulation of the number of endoreduplication cycles in various organs that are devoid of trichomes. KAK encodes a protein with sequence similarity to HECT domain proteins. As this class of proteins is known to be involved in ubiquitin-mediated protein degradation, our finding suggests that the number of endoreduplication cycles that occur in several cell types is controlled by this pathway. The KAK gene defines a monophylogenetic subgroup of HECT proteins that also contain Armadillo-like repeats.Communicated by G. Jürgens     

Phytochrome controls the number of endoreduplication cycles in the Arabidopsis thaliana hypocotyl

A majority of the cells in the Arabidopsis hypocotyl undergo endoreduplication. The number of endocycles in this organ is partially controlled by light. Up to two cycles occur in light-grown hypocotyls, whereas in the dark about 30% of the cells go through a third cycle. Is the inhibition of the third endocycle in the light an indirect result of the reduced cell size in the light-grown hypocotyl, or is it under independent light control? To address this question, the authors examined the temporal and spacial patterns of endoreduplication in light- or dark-grown plants and report here on the following observations: (i) during germination two endocycles take place prior to any significant cell expansion; (ii) in the dark the third cycle is completed very early during cell growth; and (iii) a mutation that dramatically reduces cell size does not interfere with the third endocycle. The authors then used mutants to study the way light controls the third endocycle and found that the third endocycle is completely suppressed in far red light through the action of phytochrome A and, to a lesser extent, in red light by phytochrome B. Furthermore, no 16C nuclei were observed in dark-grown constitutive photomorphogenic 1 seedlings. And, finally the hypocotyl of the cryptochrome mutant, hy4, grown in blue light was about three times longer than that of the wild-type without a significant difference in ploidy levels. Together, the results support the view that the inhibition of the third endocycle in light-grown hypocotyls is not the consequence of a simple feed-back mechanism coupling the number of cycles to the cell volume, but an integral part of the phytochrome-controlled photomorphogenic program.     

The Arabidopsis STICHEL gene is a regulator of trichome branch number and encodes a novel protein

Here, we analyze the STICHEL (STI) gene, which plays an important role in the regulation of branch number of the unicellular trichomes in Arabidopsis. We have isolated the STI locus by positional cloning and confirmed the identity by sequencing seven independent sti alleles. The STI gene encodes a protein of 1,218 amino acid residues containing a domain with sequence similarity to the ATP-binding eubacterial DNA-polymerase III gamma-subunits. Because endoreduplication was found to be normal in sti mutants the molecular function of STI in cell morphogenesis is not linked to DNA replication and, therefore, postulated to represent a novel pathway. Northern-blot analysis shows that STI is expressed in all organs suggesting that STI function is not trichome specific. The analysis of sti alleles and transgenic lines overexpressing STI suggests that STI regulates branching in a dosage-dependent manner.     

The Arabidopsis gene CAD1 controls programmed cell death in the plant immune system and encodes a protein containing a MACPF domain

To clarify the processes involved in plant immunity, we have isolated and characterized a single recessive Arabidopsis mutant, cad1 (constitutively activated cell death 1), which shows a phenotype that mimics the lesions seen in the hypersensitive response (HR). This mutant shows spontaneously activated expression of pathogenesis-related (PR) genes, and leading to a 32-fold increase in salicylic acid (SA). Inoculation of cad1 mutant plants with Pseudomonas syringae pv tomato DC3000 shows that the cad1 mutation results in the restriction of bacterial growth. Cloning of CAD1 reveals that this gene encodes a protein containing a domain with significant homology to the MACPF (membrane attack complex and perforin) domain of complement components and perforin proteins that are involved in innate immunity in animals. Furthermore, cell death is suppressed in transgenic cad1 plants expressing nahG, which encodes an SA-degrading enzyme. We therefore conclude that the CAD1 protein negatively controls the SA-mediated pathway of programmed cell death in plant immunity.     

The Arabidopsis CLASP gene encodes a microtubule-associated protein involved in cell expansion and division

Controlling microtubule dynamics and spatial organization is a fundamental requirement of eukaryotic cell function. Members of the ORBIT/MAST/CLASP family of microtubule-associated proteins associate with the plus ends of microtubules, where they promote the addition of tubulin subunits into attached kinetochore fibers during mitosis and stabilize microtubules in the vicinity of the plasma membrane during interphase. To date, nothing is known about their function in plants. Here, we show that the Arabidopsis thaliana CLASP protein is a microtubule-associated protein that is involved in both cell division and cell expansion. Green fluorescent protein-CLASP localizes along the full length of microtubules and shows enrichment at growing plus ends. Our analysis suggests that CLASP promotes microtubule stability. clasp-1 T-DNA insertion mutants are hypersensitive to microtubule-destabilizing drugs and exhibit more sparsely populated, yet well ordered, root cortical microtubule arrays. Overexpression of CLASP promotes microtubule bundles that are resistant to depolymerization with oryzalin. Furthermore, clasp-1 mutants have aberrant microtubule preprophase bands, mitotic spindles, and phragmoplasts, indicating a role for At CLASP in stabilizing mitotic arrays. clasp-1 plants are dwarf, have significantly reduced cell numbers in the root division zone, and have defects in directional cell expansion. We discuss possible mechanisms of CLASP function in higher plants.     

The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily

Specific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1. Previous work has shown that RPS5 encodes a member of the nucleotide binding site-leucine rich repeat class of plant disease resistance genes. Here we report that PBS1 encodes a putative serine-threonine kinase. Southern blot analysis revealed that the pbs1-1 allele contained a deletion of the 3' end of the PBS1 open reading frame. DNA sequence analysis of the pbs1-2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases. The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants. Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function. The Pto kinase of tomato, which is required for specific resistance to P. syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain. These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins. We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition.     

The cell morphogenesis gene SPIRRIG in Arabidopsis encodes a WD/BEACH domain protein

WD40/BEACH domain proteins have been implicated in membrane trafficking and membrane composition events in Dictyostelium and Drosophila . In this paper, we show that the Arabidopsis SPIRRIG ( SPI ) gene encodes a WD40/BEACH domain protein. The cellular analysis revealed fragmented vacuoles in root hairs similar to those found in the corresponding Dictyostelium mutants, suggesting a related cellular function. The phenotypic analysis revealed that spi mutants share all phenotypic aspects of mutants in the actin polymerization-regulating ARP2/3 pathway, including distorted trichomes, less lobing of epidermal pavement cells, disconnected epidermal cells on various organs, and shorter root hairs. This complete phenotypic overlap suggests that this WD40/BEACH domain protein and the actin-regulating ARP2/3 pathway are involved in similar growth processes.     

The Arabidopsis AtPNG1 gene encodes a peptide: N-glycanase

Deglycosylation of misfolded proteins by the endoplasmic reticulum-associated degradation (ERAD) pathway is catalyzed by peptide:N-glycanases (PNGases) that are highly conserved among mammals and yeast. The catalytic mechanism of PNGases employs a catalytic triad consisting of Cys, His and Asp residues, which is shared by other enzyme families such as cysteine proteases and protein cross-linking transglutaminases (TGases). In contrast to the yeast and mammalian systems, very little is known about ERAD in plants and the enzymes responsible for proper clearance of misfolded plant proteins. We have used a computer-based modeling approach to identify an Arabidopsis thaliana PNGase (AtPNG1). AtPNG1 is encoded by a single-copy gene and displays high structural homology with known PNGases. Importantly, heterologous expression of AtPNG1 restored N-glycanase activity in a PNGase-deficient Saccharomyces cerevisiae mutant. The AtPNG1 gene is uniformly and constitutively expressed at low levels throughout all developmental stages of the plant, and its expression does not appear to be subject to substantial regulation by external stimuli. Recently, recombinant AtPNG1 produced in Escherichia coli was reported to display TGase activity (Della Mea et al., Plant Physiol. 135, 2046-54, 2004). However, inactivation of the AtPNG1 gene did not result in decreased TGase activity in the mutant plant, and recombinant AtPNG1 produced in S. cerevisiae exhibited only residual TGase activity. We propose that the AtPNG1 gene encodes a bona fide peptide:N-glycanase that contributes to ERAD-related protein quality control in plants.     

The Arabidopsis DIMINUTO/DWARF1 gene encodes a protein involved in steroid synthesis.

We have identified the function of the Arabidopsis DIMINUTO/DWARF1 (DIM/DWF1) gene by analyzing the dim mutant, a severe dwarf with greatly reduced fertility. Both the mutant phenotype and gene expression could be rescued by the addition of exogenous brassinolide. Analysis of endogenous sterols demonstrated that dim accumulates 24-methylenecholesterol but is deficient in campesterol, an early precursor of brassinolide. In addition, we show that dim is deficient in brassinosteroids as well. Feeding experiments using deuterium-labeled 24-methylenecholesterol and 24-methyldesmosterol confirmed that DIM/DWF1 is involved in both the isomerization and reduction of the Delta24(28) bond. This conversion is not required in cholesterol biosynthesis in animals but is a key step in the biosynthesis of plant sterols. Transient expression of a green fluorescent protein-DIM/DWF1 fusion protein and biochemical experiments showed that DIM/DWF1 is an integral membrane protein that most probably is associated with the endoplasmic reticulum.     

The Arabidopsis AMP1 gene encodes a putative glutamate carboxypeptidase

Arabidopsis amp1 mutants show pleiotropic phenotypes, including altered shoot apical meristems, increased cell proliferation, polycotyly, constitutive photomorphogenesis, early flowering time, increased levels of endogenous cytokinin, and increased cyclin cycD3 expression. We have isolated the AMP1 gene by map-based cloning. The AMP1 cDNA encodes a 706;-amino acid polypeptide with significant similarity to glutamate carboxypeptidases. The AMP1 mRNA was expressed in all tissues examined, with higher expression in roots, stems, inflorescences, and siliques. Microarray analysis identified four mRNA species with altered expression in two alleles of amp1, including upregulation of CYP78A5, which has been shown to mark the shoot apical meristem boundary. The similarity of the AMP1 protein to glutamate carboxypeptidases, and in particular to N-acetyl alpha-linked acidic dipeptidases, suggests that the AMP1 gene product modulates the level of a small signaling molecule that acts to regulate a number of aspects of plant development, in particular the size of the apical meristem.     

The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats.

Arabidopsis Landsberg erecta is one of the most popular ecotypes and is used widely for both molecular and genetic studies. It harbors the erecta (er) mutation, which confers a compact inflorescence, blunt fruits, and short petioles. We have identified five er mutant alleles from ecotypes Columbia and Wassilewskija. Phenotypic characterization of the mutant alleles suggests a role for the ER gene in regulating the shape of organs originating from the shoot apical meristem. We cloned the ER gene, and here, we report that it encodes a putative receptor protein kinases. The deduced ER protein contains a cytoplasmic protein kinase catalytic domain, a transmembrane region, and an extracellular domain consisting of leucine-rich repeats, which are thought to interact with other macromolecules. Our results suggest that cell-cell communication mediated by a receptor kinase has an important role in plant morphogenesis.     

The TORMOZ gene encodes a nucleolar protein required for regulated division planes and embryo development in Arabidopsis

Embryogenesis in Arabidopsis thaliana is marked by a predictable sequence of oriented cell divisions, which precede cell fate determination. We show that mutation of the TORMOZ (TOZ) gene yields embryos with aberrant cell division planes and arrested embryos that appear not to have established normal patterning. The defects in toz mutants differ from previously described mutations that affect embryonic cell division patterns. Longitudinal division planes of the proembryo are frequently replaced by transverse divisions and less frequently by oblique divisions, while divisions of the suspensor cells, which divide only transversely, appear generally unaffected. Expression patterns of selected embryo patterning genes are altered in the mutant embryos, implying that the positional cues required for their proper expression are perturbed by the misoriented divisions. The TOZ gene encodes a nucleolar protein containing WD repeats. Putative TOZ orthologs exist in other eukaryotes including Saccharomyces cerevisiae, where the protein is predicted to function in 18S rRNA biogenesis. We find that disruption of the Sp TOZ gene results in cell division defects in Schizosaccharomyces pombe. Previous studies in yeast and animal cells have identified nucleolar proteins that regulate the exit from M phase and cytokinesis, including factors involved in pre-rRNA processing. Our study suggests that in plant cells, nucleolar functions might interact with the processes of regulated cell divisions and influence the selection of longitudinal division planes during embryogenesis.