CARMIL is a bona fide capping protein interactant

CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the alpha- and beta-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is approximately 0.4 microm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, approximately 200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer <--> dimer equilibrium with an association constant of 1.0 x 10(6) m(-1). Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins ( approximately 1 and 2 microm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.     

The C. elegans SYS-1 protein is a bona fide beta-catenin

C. elegans SYS-1 has key functional characteristics of a canonical beta-catenin, but no significant sequence similarity. Here, we report the SYS-1 crystal structure, both on its own and in a complex with POP-1, the C. elegans TCF homolog. The two structures possess signature features of canonical beta-catenin and the beta-catenin/TCF complex that could not be predicted by sequence. Most importantly, SYS-1 bears 12 armadillo repeats and the SYS-1/POP-1 interface is anchored by a conserved salt-bridge, the "charged button." We also modeled structures for three other C. elegans beta-catenins to predict the molecular basis of their distinct binding properties. Finally, we generated a phylogenetic tree, using the region of highest structural similarity between SYS-1 and beta-catenin, and found that SYS-1 clusters robustly within the beta-catenin clade. We conclude that the SYS-1 protein belongs to the beta-catenin family and suggest that additional divergent beta-catenins await discovery.     

Ribosomal protein L27a is required for growth and patterning in Arabidopsis thaliana

Ribosomal proteins are integral to ribosome biogenesis, and function in protein synthesis. In higher eukaryotes, loss of cytoplasmic ribosomal proteins results in a reduced growth rate as well as developmental defects. To what extent and how ribosomal proteins affect development is currently not known. Here we describe a semi-dominant mutation in the cytoplasmic ribosomal protein gene RPL27aC that affects multiple aspects of plant shoot development, including leaf patterning, inflorescence and floral meristem function, and seed set. In the embryo, RPL27aC is required to maintain the growth rate and for the transition from radial to bilateral symmetry associated with initiation of cotyledons. rpl27ac-1d embryos undergo stereotypical patterning to establish a globular embryo. However, a temporal delay in initiation and outgrowth of cotyledon primordia leads to development of an enlarged globular embryo prior to apical domain patterning. Defects in embryo development are coincident with tissue-specific ectopic expression of the shoot meristem genes SHOOT MERISTEMLESS (STM) and CUP-SHAPED COTYLEDON2 (CUC2), in addition to delayed expression of the abaxial gene FILAMENTOUS FLOWER (FIL) and mis-regulation of the auxin efflux effector PIN-FORMED1 (PIN1). Genetic interactions with other ribosomal protein mutants indicate that RPL27aC is a component of the ribosome. We propose that RPL27aC regulates discrete developmental events by controlling spatial and temporal expression of developmental patterning genes via an as yet undefined process involving the ribosome.     

Arabidopsis thaliana NOP10 is required for gametophyte formation

The female gametophyte is crucial for sexual reproduction of higher plants, yet little is known about the molecular mechanisms underlying its development. Here,we report that Arabidopsis thaliana NOP10(AtNOP10) is required for female gametophyte formation. AtNOP10 was expressed predominantly in the seedling and reproductive tissues, including anthers, pollen grains, and ovules.Mutations in AtNOP10 interrupted mitosis of the functional megaspore during early development and prevented polar nuclear fusion in the embryo sacs. AtNOP10 shares a high level of amino acid sequence similarity with Saccharomyces cerevisiae(yeast) NOP10(ScNOP10), an important component of the H/ACA small nucleolar ribonucleoprotein particles(H/ACA sno RNPs) implicated in 18 S r RNA synthesis and r RNA pseudouridylation. Heterologous expression of ScNOP10 complemented the mutant phenotype of Atnop10. Thus, AtNOP10 influences functional megaspore mitosis and polar nuclear fusion during gametophyte formation in Arabidopsis.     

DRB2 is required for microRNA biogenesis in Arabidopsis thaliana

Background

The Arabidopsis thaliana (Arabidopsis) DOUBLE-STRANDED RNA BINDING (DRB) protein family consists of five members, DRB1 to DRB5. The biogenesis of two developmentally important small RNA (sRNA) species, the microRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) by DICER-LIKE (DCL) endonucleases requires the assistance of DRB1 and DRB4 respectively. The importance of miRNA-directed target gene expression in plant development is exemplified by the phenotypic consequence of loss of DRB1 activity (drb1 plants).

Principal Findings

Here we report that the developmental phenotype of the drb235 triple mutant plant is the result of deregulated miRNA biogenesis in the shoot apical meristem (SAM) region. The expression of DRB2, DRB3 and DRB5 in wild-type seedlings is restricted to the SAM region. Small RNA sequencing of the corresponding tissue of drb235 plants revealed altered miRNA accumulation. Approximately half of the miRNAs detected remained at levels equivalent to those of wild-type plants. However, the accumulation of the remaining miRNAs was either elevated or reduced in the triple mutant. Examination of different single and multiple drb mutants revealed a clear association between the loss of DRB2 activity and altered accumulation for both the elevated and reduced miRNA classes. Furthermore, we show that the constitutive over-expression of DRB2 outside of its wild-type expression domain can compensate for the loss of DRB1 activity in drb1 plants.

Conclusions/Significance

Our results suggest that in the SAM region, DRB2 is both antagonistic and synergistic to the role of DRB1 in miRNA biogenesis, adding an additional layer of gene regulatory complexity in this developmentally important tissue.     

Nucleostemin-like 1 is required for embryogenesis and leaf development in Arabidopsis

Arabidopsis NSN1 encodes a nucleolar GTP-binding protein and is required for flower development. Defective flowers were formed in heterozygous nsn1/+?plants. Homozygous nsn1 plants were dwarf and exhibited severe defects in reproduction. Arrests in embryo development in nsn1 could occur at any stage of embryogenesis. Cotyledon initiation and development during embryogenesis were distorted in nsn1 plants. At the seedling stage, cotyledons and leaves of nsn1 formed upward curls. The curled leaves developed meristem-like outgrowths or hyperplasia tissues in the adaxial epidermis. Long and enlarged pavement cells, characteristic of the abaxial epidermis of wild type plants, were found in the adaxial epidermis in nsn1 leaves, suggesting a disoriented leaf polarity in the mutant. The important role of NSN1 in embryo development and leaf differentiation was consistent with the high level expression of the NSN1 gene in the developing embryos and the primordia of cotyledons and leaves. The CLAVATA 3 (CLV3) gene, a stem cell marker in the Arabidopsis shoot apical meristem (SAM), was expressed in expanded regions surrounding the SAM of nsn1 plants, and induced ectopically in the meristem-like outgrowths in cotyledons and leaves. The nsn1 mutation up-regulated the expression levels of several genes implicated in the meristem identity and the abaxial cell fate, and repressed the expression of other genes related to the specification of cotyledon boundary and abaxial identity. These results demonstrate that NSN1 represents a novel GTPase required for embryogenesis, leaf development and leaf polarity establishment in Arabidopsis.     

PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana

Phosphatidylserine (PS) has many important biological roles, but little is known about its role in plants, partly because of its low abundance. We show here that PS is enriched in Arabidopsis floral tissues and that genetic disruption of PS biosynthesis decreased heterozygote fertility due to inhibition of pollen maturation. At1g15110, designated PSS1, encodes a base-exchange-type PS synthase. Escherichia coli cells expressing PSS1 accumulated PS in the presence of l-serine at 23°C. Promoter-GUS assays showed PSS1 expression in developing anther pollen and tapetum. A few seeds with pss1-1 and pss1-2 knockout alleles escaped embryonic lethality but developed into sterile dwarf mutant plants. These plants contained no PS, verifying that PSS1 is essential for PS biosynthesis. Reciprocal crossing revealed reduced pss1 transmission via male gametophytes, predicting a rate of 61.6%pss1-1 pollen defects in PSS1/pss1-1 plants. Alexander's staining of inseparable qrt1-1 PSS1/pss1-1 quartets revealed a rate of 42% having three or four dead pollen grains, suggesting sporophytic pss1-1 cell death effects. Analysis with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI) showed that all tetrads from PSS1/pss1-1 anthers retain their nuclei, whereas unicellular microspores were sometimes anucleate. Transgenic Arabidopsis expressing a GFP-LactC2 construct that binds PS revealed vesicular staining in tetrads and bicellular microspores and nuclear membrane staining in unicellular microspores. Hence, distribution and/or transport of PS across membranes were dynamically regulated in pollen microspores. However, among unicellular microspores from PSS1/pss1-2 GFP-LactC2 plants, all anucleate microspores showed little GFP-LactC2 fluorescence, suggesting that pss1-2 microspores are more sensitive to sporophytic defects or show partial gametophytic defects.     

Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana

Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and γGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of γ-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.     

A nucleosome interaction module is required for normal function of Arabidopsis thaliana BRAHMA

The BRAHMA (BRM) gene encodes the SNF2-type ATPase of the putative Arabidopsis thaliana SWI/SNF chromatin remodelling complex. This family of ATPases is characterized by the presence of a conserved catalytic domain and an arrangement of auxiliary domains, whose functions in the remodelling activity remains unclear. Here, we characterize, at the molecular and functional level, the carboxy-terminal part of Arabidopsis BRM. We have found three DNA-binding regions that bind various free DNA and nucleosomal probes with different specificity. One of these regions contains an AT-hook motif. The carboxy terminus also contains a bromodomain able to bind histones H3 and H4. We propose that this array of domains constitute a nucleosome interaction module that helps BRM to interact with its substrate. We also characterize an Arabidopsis mutant that expresses a BRM protein lacking the last 454 amino acid residues (BRM-DeltaC), encompassing the bromodomain and two of the three DNA-binding activities identified. This mutant displays an intermediate phenotype between those of the wild-type and a null allele mutant, suggesting that the nucleosome interaction module is required for the normal function of BRM but it is not essential for the remodelling activity of BRM-containing SWI/SNF complexes.     

SABRE is required for stabilization of root hair patterning in Arabidopsis thaliana

Patterned differentiation of distinct cell types is essential for the development of multicellular organisms. The root epidermis of Arabidopsis thaliana is composed of alternating files of root hair and non‐hair cells and represents a model system for studying the control of cell‐fate acquisition. Epidermal cell fate is regulated by a network of genes that translate positional information from the underlying cortical cell layer into a specific pattern of differentiated cells. While much is known about the genes of this network, new players continue to be discovered. Here we show that the SABRE (SAB) gene, known to mediate microtubule organization, anisotropic cell growth and planar polarity, has an effect on root epidermal hair cell patterning. Loss of SAB function results in ectopic root hair formation and destabilizes the expression of cell fate and differentiation markers in the root epidermis, including expression of the WEREWOLF (WER) and GLABRA2 (GL2) genes. Double mutant analysis reveal that wer and caprice (cpc) mutants, defective in core components of the epidermal patterning pathway, genetically interact with sab. This suggests that SAB may act on epidermal patterning upstream of WER and CPC. Hence, we provide evidence for a role of SAB in root epidermal patterning by affecting cell‐fate stabilization. Our work opens the door for future studies addressing SAB‐dependent functions of the cytoskeleton during root epidermal patterning.     

Disabled is a bona fide component of the Abl signaling network

Abl is an essential regulator of cell migration and morphogenesis in both vertebrates and invertebrates. It has long been speculated that the adaptor protein Disabled (Dab), which is a key regulator of neuronal migration in the vertebrate brain, might be a component of this signaling pathway, but this idea has been controversial. We now demonstrate that null mutations of Drosophila Dab result in phenotypes that mimic Abl mutant phenotypes, both in axon guidance and epithelial morphogenesis. The Dab mutant interacts genetically with mutations in Abl, and with mutations in the Abl accessory factors trio and enabled (ena). Genetic epistasis tests show that Dab functions upstream of Abl and ena, and, consistent with this, we show that Dab is required for the subcellular localization of these two proteins. We therefore infer that Dab is a bona fide component of the core Abl signaling pathway in Drosophila.     

The PsbP protein is required for photosystem II complex assembly/stability and photoautotrophy in Arabidopsis thaliana

Interfering RNA was used to suppress the expression of the genes At1g06680 and At2g30790 in Arabidopsis thaliana, which encode the PsbP-1 and PsbP-2 proteins, respectively, of photosystem II (PS II). A phenotypic series of transgenic plants was recovered that expressed intermediate and low amounts of PsbP. Chlorophyll fluorescence induction and Q(A)(-) decay kinetics analyses were performed. Decreasing amounts of expressed PsbP protein led to the progressive loss of variable fluorescence and a marked decrease in the fluorescence quantum yield (F(V)/F(M)). This was primarily due to the loss of the J to I transition. Analysis of the fast fluorescence rise kinetics indicated no significant change in the number of PS II(beta) centers present in the mutants. Analysis of Q(A)(-) decay kinetics in the absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea indicated a defect in electron transfer from Q(A)(-) to Q(B), whereas experiments performed in the presence of this herbicide indicated that charge recombination between Q(A)(-) and the oxygen-evolving complex was seriously retarded in the plants that expressed low amounts of the PsbP protein. These results demonstrate that the amount of functional PS II reaction centers is compromised in the plants that exhibited intermediate and low amounts of the PsbP protein. Plants that lacked detectable PsbP were unable to survive in the absence of sucrose, indicating that the PsbP protein is required for photoautotrophy. Immunological analysis of the PS II protein complement indicated that significant losses of the CP47 and D2 proteins, and intermediate losses of the CP43 and D1 proteins, occurred in the absence of the PsbP protein. This demonstrates that the extrinsic protein PsbP is required for PS II core assembly/stability.     

A toolkit for studying cellular reorganization during early embryogenesis in Arabidopsis thaliana

Considerable progress has been made in understanding the influence of physical and genetic factors on the patterns of cell division in various model systems. However, how each of these factors directs changes in subcellular structures has remained unclear. Generic machineries for the execution of cell expansion and division have been characterized, but how these are influenced by genetic regulators and physical cell properties remains an open question. To a large degree, the complexity of growing post‐embryonic tissues and a lack of precise predictability have prevented the extraction of rigid correlations between subcellular structures and future orientation of cell division. The Arabidopsis embryo offers an exquisitely predictable and simple model for studying such correlations, but so far the tools and methodology for studying subcellular structures in the early embryo have been lacking. Here, we describe a set of markers to visualize a range of subcellular structures in the early Arabidopsis embryo. We have designed a series of fluorescent cellular reporters optimized for embryos, and demonstrate the effectiveness of using these ‘ACE’ reporters with simple three‐dimensional imaging procedures that preserve delicate cellular structures. We describe the ontogeny of subcellular structures in the early embryo and find that central/peripheral cell polarity is established much earlier than suspected. In addition, we show that the actin and microtubule cytoskeleton has distinct topologies in the embryo. These tools and methods will allow detailed analysis of the events of cellular reorganization that underlie morphogenesis in the Arabidopsis embryo.