Discovery of ultrafast myosin, its amino acid sequence, and structural features.

Cytoplasmic streaming with extremely high velocity (∼70 µm s-1) occurs in cells of the characean algae (Chara). Because cytoplasmic streaming is caused by myosin XI, it has been suggested that a myosin XI with a velocity of 70 µm s-1, the fastest myosin measured so far, exists in Chara cells. However, the velocity of the previously cloned Chara corallina myosin XI (CcXI) was about 20 µm s-1, one-third of the cytoplasmic streaming velocity in Chara Recently, the genome sequence of Chara braunii has been published, revealing that this alga has four myosin XI genes. We cloned these four myosin XI (CbXI-1, 2, 3, and 4) and measured their velocities. While the velocities of CbXI-3 and CbXI-4 motor domains (MDs) were similar to that of CcXI MD, the velocities of CbXI-1 and CbXI-2 MDs were 3.2 times and 2.8 times faster than that of CcXI MD, respectively. The velocity of chimeric CbXI-1, a functional, full-length CbXI-1 construct, was 60 µm s-1 These results suggest that CbXI-1 and CbXI-2 would be the main contributors to cytoplasmic streaming in Chara cells and show that these myosins are ultrafast myosins with a velocity 10 times faster than fast skeletal muscle myosins in animals. We also report an atomic structure (2.8-Å resolution) of myosin XI using X-ray crystallography. Based on this crystal structure and the recently published cryo-electron microscopy structure of acto-myosin XI at low resolution (4.3-Å), it appears that the actin-binding region contributes to the fast movement of Chara myosin XI. Mutation experiments of actin-binding surface loops support this hypothesis.

Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina.

BACKGROUND: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta. RESULTS: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24. CONCLUSIONS: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3'-extended 5.8S pre-rRNA.

An Algal Greening of Land.

Photosynthetic eukaryotes arose ∼1.5 billion years ago by endosymbiosis with a cyanobacterium. Algae then evolved for a billion years before one lineage finally colonized land. Why the wait? The Chara braunii genome details a decisive step linking plant origins with Earth's history.

The Chara Genome: Secondary Complexity and Implications for Plant Terrestrialization.

Land plants evolved from charophytic algae, among which Charophyceae possess the most complex body plans. We present the genome of Chara braunii; comparison of the genome to those of land plants identified evolutionary novelties for plant terrestrialization and land plant heritage genes. C. braunii employs unique xylan synthases for cell wall biosynthesis, a phragmoplast (cell separation) mechanism similar to that of land plants, and many phytohormones. C. braunii plastids are controlled via land-plant-like retrograde signaling, and transcriptional regulation is more elaborate than in other algae. The morphological complexity of this organism may result from expanded gene families, with three cases of particular note: genes effecting tolerance to reactive oxygen species (ROS), LysM receptor-like kinases, and transcription factors (TFs). Transcriptomic analysis of sexual reproductive structures reveals intricate control by TFs, activity of the ROS gene network, and the ancestral use of plant-like storage and stress protection proteins in the zygote.

Seasonality of water chemistry, carbonate production, and biometric features of two species of Chara in a shallow clear water lake.

The objective of this study was to analyze the temporal variability of biometric features and the carbonate production of two charophytes: Chara polyacantha A. Braun and Chara rudis A. Braun against the background of the physical-chemical properties of water. The investigation was carried out in a small, mid-forest Lake Jasne (western Poland). It is a polymictic, mesotrophic, hardwater ecosystem dominated by charophyte vegetation. Each month, 10 individuals of each species were characterized in terms of morphometric features, fresh and dry weight, and the percentage of calcium carbonate. Additionally, physical-chemical parameters of the water were studied. The results of physical-chemical analyses indicated similar habitat conditions for both species. Despite smaller dry weight C. polyacantha was characterized by greater morphological variability and higher rates of growth and percentage share of calcium carbonate in dry mass than C. rudis. The percentage of calcium carbonates in dry mass did not differ significantly between the species and exceeded 60%, reaching the maximum (76% in C. polyacantha) in July and August. For both species, distinct correlations between the structure of biomass and morphological features were found. The obtained results show the great importance of charophyte vegetation in carbon cycling and functioning of lake ecosystems.

Characterization of the heterotrimeric G-protein complex and its regulator from the green alga Chara braunii expands the evolutionary breadth of plant G-protein signaling.

The lack of heterotrimeric G-protein homologs in the sequenced genomes of green algae has led to the hypothesis that, in plants, this signaling mechanism coevolved with the embryophytic life cycle and the acquisition of terrestrial habitat. Given the large evolutionary gap that exists between the chlorophyte green algae and most basal land plants, the bryophytes, we evaluated the presence of this signaling complex in a charophyte green alga, Chara braunii, proposed to be the closest living relative of land plants. The C. braunii genome encodes for the entire G-protein complex, the Gα, Gß, and Gγ subunits, and the REGULATOR OF G-PROTEIN SIGNALING (RGS) protein. The biochemical properties of these proteins and their cross-species functionality show that they are functional homologs of canonical G-proteins. The subunit-specific interactions between CbGα and CbGß, CbGß and CbGγ, and CbGα and CbRGS are also conserved, establishing the existence of functional G-protein complex-based signaling mechanisms in green algae.

Molecular and biochemical analysis of the first ARA6 homologue, a RAB5 GTPase, from green algae.

RAB5 GTPases are important regulators of endosomal membrane traffic in yeast, plants, and animals. A specific subgroup of this family, the ARA6 group, has been described in land plants including bryophytes, lycophytes, and flowering plants. Here, we report on the isolation of an ARA6 homologue in a green alga. CaARA6 (CaRABF1) from Chara australis, a member of the Characeae that is a close relative of land plants, encodes a polypeptide of 237 aa with a calculated molecular mass of 25.4 kDa, which is highly similar to ARA6 members from Arabidopsis thaliana and other land plants and has GTPase activity. When expressed in Nicotiana benthamiana leaf epidermal cells, fluorescently tagged CaARA6 labelled organelles with diameters between 0.2 and 1.2 µm, which co-localized with fluorescently tagged AtARA6 known to be present on multivesicular endosomes. Mutations in the membrane-anchoring and GTP-binding sites altered the localization of CaARA6 comparable to that of A. thaliana ARA6 (RABF1). In characean internodal cells, confocal immunofluorescence and immunogold electron microscopy with antibodies against AtARA6 and CaARA6 revealed ARA6 epitopes not only at multivesicular endosomes but also at the plasma membrane, including convoluted domains (charasomes), and at the trans-Golgi network. Our findings demonstrate that ARA6-like proteins have a more ancient origin than previously thought. They indicate further that ARA6-like proteins could have different functions in spite of the high similarity between characean algae and flowering plants.

Two parthenogenetic populations of Chara canescens differ in their capacity to acclimate to irradiance and salinity.

The parthenogens of Chara canescens (Charophyceae) occupy broader geographical and ecological ranges than their sexual counterparts. Two possible hypotheses explain the ubiquity of parthenogens: the occurrence of one or several parthenogens with wide niches, or of many parthenogens that are restricted to narrow ecological niches. For the purposes of this study, C. canescens individuals from two neighbouring populations of the Baltic Sea (Bodstedter Bodden = BB; Salzhaff = SH), which differed significantly in water transparency and salinity, were investigated for significant differences in physiological capacity. Individuals of both habitats acclimated quickly to daily changes in irradiances in the field, but the photosynthetic efficiency of PS II showed a significant decrease with increasing daily irradiance in the habitat BB, which has lower levels of salinity and water transparency. In addition to the field study, individuals were reared under different levels of environmental factors in the laboratory: four irradiances (70-600 µmol m(-2) s(-1)) and five salinity levels (0-24 psu). The individuals of both habitats grew almost equally well at intermediate salinity levels. Growth under the artificial light supply was highest at levels corresponding to the in situ conditions for each population. Total chlorophyll was highest at intermediate salinities (BB), or hardly changed with salinity (SH). The physiological capacity for individuals from SH clearly depends upon changing growth irradiance, whereas the capacity for individuals from BB was relatively independent of salinity and irradiance. These findings indicate that both parthenogenetic C. canescens populations are locally adapted to light. However, to test adaptive potential of the parthenogens, more than two populations should be tested in future.

Phosphorylation of H2AX histone as indirect evidence for double-stranded DNA breaks related to the exchange of nuclear proteins and chromatin remodeling in Chara vulgaris spermiogenesis.

Phosphorylation of H2AX histone results not only from DNA damage (caused by ionizing radiation, UV or chemical substances, e.g. hydroxyurea), but also regularly takes place during spermiogenesis, enabling correct chromatin remodeling. Immunocytochemical analysis using antibodies against H2AX histone phosphorylated at serine 139 indirectly revealed endogenous double-stranded DNA breaks in Chara vulgaris spermatids in mid-spermiogenesis (stages V, VI and VII), when protamine-type proteins appear in the nucleus. Fluorescent foci were not observed in early (stages I-IV) and late (VIII-X) spermiogenesis, after replacement of histones by protamine-type proteins was finished. A similar phenomenon exists in animals. Determination of the localization of fluorescent foci and the ultrastructure of nuclei led to the hypothesis that DNA breaks at stage V, when condensed chromatin adheres to the nuclear envelope. This is transformed into a net-like structure during stage VI, probably allowing chromosome repositioning to specific regions in the mature spermatozoid. However, at stages VI and VII, DNA breaks are necessary for transformation of the nucleosomal structure into a fibrillar and finally the extremely condensed status of sleeping genes at stage X.

Tracing plant Mitochondrial DNA evolution: rearrangements of the ancient mitochondrial gene cluster trnA-trnT-nad7 in liverwort phylogeny.

Whereas frequent recombination characterizes flowering plant mitochondrial genomes, some mitochondrial gene arrangements may, in contrast, be conserved between streptophyte algae and early land plant clades (bryophytes). Here we explore the evolutionary fate of the mitochondrial gene arrangement trnA-trnT-nad7, which is conserved among the alga Chara, the moss Physcomitrella, and the liverwort Marchantia, although trnT is inverted in orientation in the latter. Surprisingly, we now find that the Chara-type gene arrangement is generally conserved in mosses, but that trnT is lacking between trnA and nad7 in all simple-thalloid and leafy (jungermanniid) liverworts. The ancient gene continuity trnA-trnT-nad7 is, however, conserved in Blasia, representing the sister lineage to all other complex-thalloid (marchantiid) liverworts. The recombinogenic insertion of short sequence stretches, including nad5 and rps7 pseudogene fragments copied from elsewhere in the liverwort mtDNA, likely mediated a subsequent inversion of trnT and flanking sequences in a basal grade of marchantiid liverworts, which was then followed by an independent secondary loss of trnT in derived marchantiid taxa later in evolution. In contrast to the previously observed extreme degree of coding sequence conservation and the assumed absence of active recombination in Marchantia mtDNA, this now reveals a surprisingly dynamic evolution of marchantiid liverwort mitochondrial genomes.

The chloroplast genome sequence of Chara vulgaris sheds new light into the closest green algal relatives of land plants.

The phylum Streptophyta comprises all land plants and six monophyletic groups of charophycean green algae (Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales, and Charales). Phylogenetic analyses of four genes encoded in three cellular compartments suggest that the Charales are sister to land plants and that charophycean green algae evolved progressively toward an increasing cellular complexity. To validate this phylogenetic hypothesis and to understand how and when the highly conservative pattern displayed by land plant chloroplast DNAs (cpDNAs) originated in the Streptophyta, we have determined the complete chloroplast genome sequence (184,933 bp) of a representative of the Charales, Chara vulgaris, and compared this genome to those of Mesostigma (Mesostigmatales), Chlorokybus (Chlorokybales), Staurastrum and Zygnema (Zygnematales), Chaetosphaeridium (Coleochaetales), and selected land plants. The phylogenies we inferred from 76 cpDNA-encoded proteins and genes using various methods favor the hypothesis that the Charales diverged before the Coleochaetales and Zygnematales. The Zygnematales were identified as sister to land plants in the best tree topology (T1), whereas Chaetosphaeridium (T2) or a clade uniting the Zygnematales and Chaetosphaeridium (T3) occupied this position in alternative topologies. Chara remained at the same basal position in trees including more land plant taxa and inferred from 56 proteins/genes. Phylogenetic inference from gene order data yielded two most parsimonious trees displaying the T1 and T3 topologies. Analyses of additional structural cpDNA features (gene order, gene content, intron content, and indels in coding regions) provided better support for T1 than for the topology of the above-mentioned four-gene tree. Our structural analyses also revealed that many of the features conserved in land plant cpDNAs were inherited from their green algal ancestors. The intron content data predicted that at least 15 of the 21 land plant group II introns were gained early during the evolution of streptophytes and that a single intron was acquired during the transition from charophycean green algae to land plants. Analyses of genome rearrangements based on inversions predicted no alteration in gene order during the transition from charophycean green algae to land plants.

Characterization of MADS-box genes in charophycean green algae and its implication for the evolution of MADS-box genes.

The MADS-box genes of land plants are extensively diverged to form a superfamily and are important in various aspects of development including the specification of floral organs as homeotic selector genes. The closest relatives of land plants are the freshwater green algae charophyceans. To study the origin and evolution of land plant MADS-box genes, we characterized these genes in three charophycean green algae: the stonewort Chara globularis, the coleochaete Coleochaete scutata, and the desmid Closterium peracerosum-strigosum-littorale complex. Phylogenetic analyses suggested that MADS-box genes diverged extensively in the land plant lineage after the separation of charophyceans from land plants. The stonewort C. globularis mRNA was specifically detected in the oogonium and antheridium together with the egg and spermatozoid during their differentiation. The expression of the C. peracerosum-strigosum-littorale-complex gene increased when vegetative cells began to differentiate into gametangial cells and decreased after fertilization. These expression patterns suggest that the precursors of land plant MADS-box genes originally functioned in haploid reproductive cell differentiation and that the haploid MADS-box genes were recruited into a diploid generation during the evolution of land plants.

Importance of the converter region for the motility of myosin as revealed by the studies on chimeric Chara myosins.

A long alpha-helix in myosin head constitutes a lever arm together with light chains. It is known from X-ray crystallographic studies that the first three turns of this lever arm alpha-helix are inserted into the converter region of myosin. We previously showed that chimeric Chara myosin in which the motor domain of Chara myosin was connected to the lever arm alpha-helix of Dictyostelium myosin had motility far less than that expected for the motor domain of Chara myosin. Here, we replaced the inserted three turns of alpha-helix of Dictyostelium myosin with that of the Chara myosin and found that the replacement enhanced the motility 2.6-fold without changing the ATPase activity so much. The result clearly showed the importance of interaction between the converter region and the lever arm alpha-helix for the efficient motility of myosin.

Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity.

The mechanism and structural features that are responsible for the fast motility of Chara corallina myosin (CCM) have not been elucidated, so far. The low yields of native CCM that can be purified to homogeneity were the major reason for this. Here, we describe the expression of recombinant CCM motor domains, which support the fast movement of actin filaments in an in vitro motility assay. A CCM motor domain without light chain binding site moved actin filaments at a velocity of 8.8 microm/s at 30 degrees C and a CCM motor domain with an artificial lever arm consisting of two alpha-actinin repeats moved actin filaments at 16.2 microm/s. Both constructs displayed high actin-activated ATPase activities ( approximately 500 Pi/s/head), which is indicative of a very fast hydrolysis step. Our results provide an excellent system to dissect the specific structural and functional features that distinguish the myosin responsible for fast cytoplasmic streaming.