Fatima Cvrčková

Yeast G1 cyclins CLN1 and CLN2 and a GAP-like protein have a role in bud formation.

Cyclin‐dependent protein kinases have a central role in cell cycle regulation. In Saccharomyces cerevisiae, Cdc28 kinase and the G1 cyclins Cln1, 2 and 3 are required for DNA replication, duplication of the spindle pole body and bud emergence. These three independent processes occur simultaneously in late G1 when the cells reach a critical size, an event known as Start. At least one of the three Clns is necessary for Start. Cln3 is believed to activate Cln1 and Cln2, which can then stimulate their own accumulation by means of a positive feedback loop. They (or Cln3) also activate another pair of cyclins, Clb5 and 6, involved in initiating S phase. Little is known about the role of Clns in spindle pole body duplication and budding. We report here the isolation of a gene (CLA2/BUD2/ERC25) that codes for a homologue of mammalian Ras‐associated GTPase‐activating proteins (GAPs) and is necessary for budding only in cln1 cln2 cells. This suggests that Cln1 and Cln2 may have a direct role in bud formation.

The exocyst complex in plants

Plant cell morphogenesis requires precise regulation of localized cell expansion and cell division. Vectorial exocytosis is a major morphogenetic process in plant cells, intimately interwoven with the dynamics of the cytoskeleton. Small GTPases of the Ras superfamily act as molecular switches participating in the control of both vesicle trafficking (Rab GTPases) and cytoskeleton dynamics (Rho GTPases). In tip-growing plant cells, such as the pollen tubes, wall formation is confined to the tip. The cytoplasm is highly polarized and grows by exocytosis in the apex, which makes pollen tubes an excellent model to study a large variety of dynamic events: targeted vesicle transport and docking, cell wall formation, guidance and maintenance of polarity, organelle movement, membrane recycling. We have previously shown that Rab homologues (especially those involved in the delivery of secretory vesicles to the plasma membrane) are the most abundant small GTPases in tobacco pollen tubes (Zarsky and Cvrckova, 1997) and cloned a tobacco pollen Rop (Rho of plants) homologue (Cvrckova and Zarsky, 1999). We have also shown the importance of GTPases (possibly Rop) in polarized pollen tube growth by microinjection of GTP/GDP analogues into growing pollen tubes (Elias et al., 2001). Both Rab and Rho GTPases are potential interactors of the exocyst (Sec6/8) complex, a multisubunit protein assembly involved in exocytosis that has been characterized in Saccharomyces cerevisiae and mammalian cells (reviewed in Hsu et al., 1999). The exocyst is composed of eight distinct subunits, referred to in yeast as Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p and Exo84p. The mammalian exocyst contains homologues of all these proteins. The complex is critical for specification of the site of vesicle docking and fusion and probably acts as a tethering complex before the steps mediated by SNARE and associated proteins. The yeast exocyst is known to interact directly with both Rab (Sec4) and Rho GTPases. Importantly, the Rho family GTPases Rho1 and Cdc42 are involved in the regulation of intracellular localization of the exocyst (Guo et al., 2001). Since the exocyst seems to be well conserved among distinct lineages of eukaryotes, it is relevant to ask whether plants also contain a similar module involved in exocytosis and secretion. Attempts to clone plant genes homologous to SEC15 via complementation of a yeast mutant with an Arabidopsis cDNA library were unsuccessful (Matsuda and Nakano, 1998). However, our searches through public sequence databases did identify genes significantly similar to all exocyst subunits (Cvrckova et al., 2001, and our unpublished data). The genome of Arabidopsis thaliana seems to code for two highly similar genes homologous to SEC3, SEC5, one homologue of SEC6, SEC8 and SEC10 each, two paralogues related to SEC15 and three copies of a putative EXO84. Surprisingly, there are as many as 23 potential Arabidopsis genes related to EXO70, although Saccharomyces, Drosophila, Caenorhabditis and perhaps also mammals possess only one copy of the EXO70 gene. This suggests that the plant exocyst might have some special features compared to other eukaryotes (Fig. 1). We have started molecular characterization of the putative Arabidopsis exocyst subunits. First we isolated a cDNA clone of the AtSec15b gene using a PCR-based library screen, and obtained cDNA clones potentially containing complete coding sequence of other exocyst * Corresponding author. Tel.: +420-2-21953179; fax: +420-2-21953306. E-mail address: zarsky@ueb.cas.cz (V. Zarsky). Cell Biology International 27 (2003) 199–201 Cell Biology International

The plant formin AtFH4 interacts with both actin and microtubules, and contains a newly identified microtubule-binding domain

The dynamic behaviour of the actin cytoskeleton in plants relies on the coordinated action of several classes of actin-binding proteins (ABPs). These ABPs include the plant-specific subfamilies of actin-nucleating formin proteins. The model plant species Arabidopsis thaliana has over 20 formin proteins, all of which contain plant-specific regions in place of the GTPase-binding domain, formin homology (FH)3 domain, and DAD and DID motifs found in many fungal and animal formins. We have identified for the first time a plant-specific region of the membrane-integrated formin AtFH4 that mediates an association with the microtubule cytoskeleton. In vitro analysis shows that this region (named the GOE domain) binds directly to microtubules. Overexpressed AtFH4 accumulates at the endoplasmic reticulum membrane and co-aligns the endoplasmic reticulum with microtubules. The FH1 and FH2 domains of formins are conserved in plants, and we show that these domains of AtFH4 nucleate F-actin. Together, these data suggest that the combination of plant-specific and conserved domains enables AtFH4 to function as an interface between membranes and both major cytoskeletal networks.

Molecular diversity of phospholipase D in angiosperms

BackgroundThe phospholipase D (PLD) family has been identified in plants by recent molecular studies, fostered by the emerging importance of plant PLDs in stress physiology and signal transduction. However, the presence of multiple isoforms limits the power of conventional biochemical and pharmacological approaches, and calls for a wider application of genetic methodology.ResultsTaking advantage of sequence data available in public databases, we attempted to provide a prerequisite for such an approach. We made a complete inventory of the Arabidopsis thaliana PLD family, which was found to comprise 12 distinct genes. The current nomenclature of Arabidopsis PLDs was refined and expanded to include five newly described genes. To assess the degree of plant PLD diversity beyond Arabidopsis we explored data from rice (including the genome draft by Monsanto) as well as cDNA and EST sequences from several other plants. Our analysis revealed two major PLD subfamilies in plants. The first, designated C2-PLD, is characterised by presence of the C2 domain and comprises previously known plant PLDs as well as new isoforms with possibly unusual features-catalytically inactive or independent on Ca2+. The second subfamily (denoted PXPH-PLD) is novel in plants but is related to animal and fungal enzymes possessing the PX and PH domains.ConclusionsThe evolutionary dynamics, and inter-specific diversity, of plant PLDs inferred from our phylogenetic analysis, call for more plant species to be employed in PLD research. This will enable us to obtain generally valid conclusions.

Formin homology 2 domains occur in multiple contexts in angiosperms

BackgroundInvolvement of conservative molecular modules and cellular mechanisms in the widely diversified processes of eukaryotic cell morphogenesis leads to the intriguing question: how do similar proteins contribute to dissimilar morphogenetic outputs. Formins (FH2 proteins) play a central part in the control of actin organization and dynamics, providing a good example of evolutionarily versatile use of a conserved protein domain in the context of a variety of lineage-specific structural and signalling interactions.ResultsIn order to identify possible plant-specific sequence features within the FH2 protein family, we performed a detailed analysis of angiosperm formin-related sequences available in public databases, with particular focus on the complete Arabidopsis genome and the nearly finished rice genome sequence. This has led to revision of the current annotation of half of the 22 Arabidopsis formin-related genes. Comparative analysis of the two plant genomes revealed a good conservation of the previously described two subfamilies of plant formins (Class I and Class II), as well as several subfamilies within them that appear to predate the separation of monocot and dicot plants. Moreover, a number of plant Class II formins share an additional conserved domain, related to the protein phosphatase/tensin/auxilin fold. However, considerable inter-species variability sets limits to generalization of any functional conclusions reached on a single species such as Arabidopsis.ConclusionsThe plant-specific domain context of the conserved FH2 domain, as well as plant-specific features of the domain itself, may reflect distinct functional requirements in plant cells. The variability of formin structures found in plants far exceeds that known from both fungi and metazoans, suggesting a possible contribution of FH2 proteins in the evolution of the plant type of multicellularity.

Arabidopsis exocyst subunits SEC8 and EXO70A1 and exocyst interactor ROH1 are involved in the localized deposition of seed coat pectin

• Polarized deposition of cell wall pectins is a key process in Arabidopsis thaliana myxospermous seed coat development. The exocyst, an octameric secretory vesicle tethering complex, has recently been shown to be involved in the regulation of cell polarity in plants. Here, we used the Arabidopsis seed coat to study the participation of the exocyst complex in polarized pectin delivery. • We characterized the amount of pectinaceous mucilage and seed coat structure in sec8 and exo70A1 exocyst mutants. Using a yeast two-hybrid screen, we identified a new interactor of the exocyst subunit Exo70A1, termed Roh1, a member of the DUF793 protein family. • T-DNA insertions in SEC8, EXO70A1 caused considerable deviations from normal seed coat development, in particular reduced pectin deposition and defects in the formation of the central columella of seed epidermal cells. A gain-of-function mutation of ROH1 also caused reduced pectin deposition. Interestingly, we observed a systematic difference in seed coat development between primary and secondary inflorescences in wild-type plants: siliques from secondary branches produced seeds with thicker seed coats. • The participation of exocyst subunits in mucilage deposition provides direct evidence for the role of the exocyst in polarized cell wall morphogenesis.

Evolution of the Land Plant Exocyst Complexes

Exocyst is an evolutionarily conserved vesicle tethering complex functioning especially in the last stage of exocytosis. Homologs of its eight canonical subunits – Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84 – were found also in higher plants and confirmed to form complexes in vivo, and to participate in cell growth including polarized expansion of pollen tubes and root hairs. Here we present results of a phylogenetic study of land plant exocyst subunits encoded by a selection of completely sequenced genomes representing a variety of plant, mostly angiosperm, lineages. According to their evolution histories, plant exocyst subunits can be divided into several groups. The core subunits Sec6, Sec8, and Sec10, together with Sec3 and Sec5, underwent few, if any fixed duplications in the tracheophytes (though they did amplify in the moss Physcomitrella patens), while others form larger families, with the number of paralogs ranging typically from two to eight per genome (Sec15, Exo84) to several dozens per genome (Exo70). Most of the diversity, which can be in some cases traced down to the origins of land plants, can be attributed to the peripheral subunits Exo84 and, in particular, Exo70. As predicted previously, early land plants (including possibly also the Rhyniophytes) encoded three ancestral Exo70 paralogs which further diversified in the course of land plant evolution. Our results imply that plants do not have a single “Exocyst complex” – instead, they appear to possess a diversity of exocyst variants unparalleled among other organisms studied so far. This feature might perhaps be directly related to the demands of building and maintenance of the complicated and spatially diverse structures of the endomembranes and cell surfaces in multicellular land plants.

Are plant formins integral membrane proteins

BackgroundThe formin family of proteins has been implicated in signaling pathways of cellular morphogenesis in both animals and fungi; in the latter case, at least, they participate in communication between the actin cytoskeleton and the cell surface. Nevertheless, they appear to be cytoplasmic or nuclear proteins, and it is not clear whether they communicate with the plasma membrane, and if so, how. Because nothing is known about formin function in plants, I performed a systematic search for putative Arabidopsis thaliana formin homologs.ResultsI found eight putative formin-coding genes in the publicly available part of the Arabidopsis genome sequence and analyzed their predicted protein sequences. Surprisingly, some of them lack parts of the conserved formin-homology 2 (FH2) domain and the majority of them seem to have signal sequences and putative transmembrane segments that are not found in yeast or animals formins.ConclusionsPlant formins define a distinct subfamily. The presence in most Arabidopsis formins of sequence motifs typical or transmembrane proteins suggests a mechanism of membrane attachment that may be specific to plant formins, and indicates an unexpected evolutionary flexibility of the conserved formin domain.

Roots of angiosperm formins: The evolutionary history of plant FH2 domain-containing proteins

BackgroundShuffling of modular protein domains is an important source of evolutionary innovation. Formins are a family of actin-organizing proteins that share a conserved FH2 domain but their overall domain architecture differs dramatically between opisthokonts (metazoans and fungi) and plants. We performed a phylogenomic analysis of formins in most eukaryotic kingdoms, aiming to reconstruct an evolutionary scenario that may have produced the current diversity of domain combinations with focus on the origin of the angiosperm formin architectures.ResultsThe Rho GTPase-binding domain (GBD/FH3) reported from opisthokont and Dictyostelium formins was found in all lineages except plants, suggesting its ancestral character. Instead, mosses and vascular plants possess the two formin classes known from angiosperms: membrane-anchored Class I formins and Class II formins carrying a PTEN-like domain. PTEN-related domains were found also in stramenopile formins, where they have been probably acquired independently rather than by horizontal transfer, following a burst of domain rearrangements in the chromalveolate lineage. A novel RhoGAP-related domain was identified in some algal, moss and lycophyte (but not angiosperm) formins that define a specific branch (Class III) of the formin family.ConclusionWe propose a scenario where formins underwent multiple domain rearrangements in several eukaryotic lineages, especially plants and chromalveolates. In plants this replaced GBD/FH3 by a probably inactive RhoGAP-like domain, preserving a formin-mediated association between (membrane-anchored) Rho GTPases and the actin cytoskeleton. Subsequent amplification of formin genes, possibly coincident with the expansion of plants to dry land, was followed by acquisition of alternative membrane attachment mechanisms present in extant Class I and Class II formins, allowing later loss of the RhoGAP-like domain-containing formins in angiosperms.

Analysis of the hybrid proline-rich protein families from seven plant species suggests rapid diversification of their sequences and expression patterns.

BackgroundPlant hybrid proline-rich proteins (HyPRPs) are putative cell wall proteins consisting, usually, of a repetitive proline-rich (PR) N-terminal domain and a conserved eight-cysteine motif (8 CM) C-terminal domain. Understanding the evolutionary dynamics of HyPRPs might provide not only insight into their so far elusive function, but also a model for other large protein families in plants.ResultsWe have performed a phylogenetic analysis of HyPRPs from seven plant species, including representatives of gymnosperms and both monocot and dicot angiosperms. Every species studied possesses a large family of 14–52 HyPRPs. Angiosperm HyPRPs exhibit signs of recent major diversification involving, at least in Arabidopsis and rice, several independent tandem gene multiplications. A distinct subfamily of relatively well-conserved C-type HyPRPs, often with long hydrophobic PR domains, has been identified. In most of gymnosperm (pine) HyPRPs, diversity appears within the C-type group while angiosperms have only a few of well-conserved C-type representatives. Atypical (glycine-rich or extremely short) N-terminal domains apparently evolved independently in multiple lineages of the HyPRP family, possibly via inversion or loss of sequences encoding proline-rich domains. Expression profiles of potato and Arabidopsis HyPRP genes exhibit instances of both overlapping and complementary organ distribution. The diversified non-C-type HyPRP genes from recently amplified chromosomal clusters in Arabidopsis often share their specialized expression profiles. C-type genes have broader expression patterns in both species (potato and Arabidopsis), although orthologous genes exhibit some differences.ConclusionHyPRPs represent a dynamically evolving protein family apparently unique to seed plants. We suggest that ancestral HyPRPs with long proline-rich domains produced the current diversity through ongoing gene duplications accompanied by shortening, modification or loss of the proline-rich domains. Most of the diversity in gymnosperms and angiosperms originates from different branches of the HyPRP family. Rapid sequence diversification is consistent with only limited requirements for structure conservation and, together with high variability of gene expression patterns, limits the interpretation of any functional study focused on a single HyPRP gene or a couple of HYPRP genes in single plant species.