Organellar Introns in Fungi, Algae, and Plants.

Introns are ubiquitous in eukaryotic genomes and have long been considered as 'junk RNA' but the huge energy expenditure in their transcription, removal, and degradation indicate that they may have functional significance and can offer evolutionary advantages. In fungi, plants and algae introns make a significant contribution to the size of the organellar genomes. Organellar introns are classified as catalytic self-splicing introns that can be categorized as either Group I or Group II introns. There are some biases, with Group I introns being more frequently encountered in fungal mitochondrial genomes, whereas among plants Group II introns dominate within the mitochondrial and chloroplast genomes. Organellar introns can encode a variety of proteins, such as maturases, homing endonucleases, reverse transcriptases, and, in some cases, ribosomal proteins, along with other novel open reading frames. Although organellar introns are viewed to be ribozymes, they do interact with various intron- or nuclear genome-encoded protein factors that assist in the intron RNA to fold into competent splicing structures, or facilitate the turn-over of intron RNAs to prevent reverse splicing. Organellar introns are also known to be involved in non-canonical splicing, such as backsplicing and trans-splicing which can result in novel splicing products or, in some instances, compensate for the fragmentation of genes by recombination events. In organellar genomes, Group I and II introns may exist in nested intronic arrangements, such as introns within introns, referred to as twintrons, where splicing of the external intron may be dependent on splicing of the internal intron. These nested or complex introns, with two or three-component intron modules, are being explored as platforms for alternative splicing and their possible function as molecular switches for modulating gene expression which could be potentially applied towards heterologous gene expression. This review explores recent findings on organellar Group I and II introns, focusing on splicing and mobility mechanisms aided by associated intron/nuclear encoded proteins and their potential roles in organellar gene expression and cross talk between nuclear and organellar genomes. Potential application for these types of elements in biotechnology are also discussed.

MiR8181 is involved in the cell growth regulation of Saccharina japonica.

Aureochrome, a blue-light receptor specifically found in photosynthetic stramenopiles, plays an important role in algal growth and development. It holds a reversed effector-sensor topology for the reception of blue light, acting as a candidate of optogenetic tool in transcriptional regulation. However, the inner regulatory mechanism of aureochrome is still unclear. In this study, we explored the potential regulatory relationship between microRNAs (miRNAs) and mRNAs by small RNA, transcriptome and degradome sequencing in Saccharina japonica. Through screening miRNA-mRNA interaction networks at the whole-genome level, we found that 18 miRNAs perfectly paired with aureochrome. Among these screened miRNAs, miR8181 was negatively correlated with aureochrome5 with high credibility, exhibiting tissue-specific expression in sporophyte of S. japonica. Degradome analysis further revealed the exact cleavage site of miR8181 on aureochrome5, confirming their targeting relationship. For the 54 target genes of miR8181, nine genes that exhibited similar expression to that of aureochrome5 competed for the same binding site, thus establishing a competing endogenous RNA network. Functional enrichment of the target genes revealed that miR8181 was involved in the regulation of cell differentiation and development in S. japonica. Moreover, overexpression of miR8181 resulted in significant decreases in the cell growth rates of Phaeodactylum tricornutum, suggesting negative roles of miR8181 in regulating cell growth. Our study revealed that miR8181, the targeting miRNA of aureochrome5, played negative roles in cell growth and development.

Weighted gene co-expression network analysis of the salt-responsive transcriptomes reveals novel hub genes in green halophytic microalgae Dunaliella salina.

Despite responses to salinity stress in Dunaliella salina, a unicellular halotolerant green alga, being subject to extensive study, but the underlying molecular mechanism remains unknown. Here, Empirical Bayes method was applied to identify the common differentially expressed genes (DEGs) between hypersaline and normal conditions. Then, using weighted gene co-expression network analysis (WGCNA), which takes advantage of a graph theoretical approach, highly correlated genes were clustered as a module. Subsequently, connectivity patterns of the identified modules in two conditions were surveyed to define preserved and non-preserved modules by combining the Zsummary and medianRank measures. Finally, common and specific hub genes in non-preserved modules were determined using Eigengene-based module connectivity or module membership (kME) measures and validation was performed by using leave-one-out cross-validation (LOOCV). In this study, the power of beta = 12 (scale-free R2 = 0.8) was selected as the soft-thresholding to ensure a scale-free network, which led to the identification of 15 co-expression modules. Results also indicate that green, blue, brown, and yellow modules are non-preserved in salinity stress conditions. Examples of enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in non-preserved modules are Sulfur metabolism, Oxidative phosphorylation, Porphyrin and chlorophyll metabolism, Vitamin B6 metabolism. Moreover, the systems biology approach was applied here, proposed some salinity specific hub genes, such as radical-induced cell death1 protein (RCD1), mitogen-activated protein kinase kinase kinase 13 (MAP3K13), long-chain acyl-CoA synthetase (ACSL), acetyl-CoA carboxylase, biotin carboxylase subunit (AccC), and fructose-bisphosphate aldolase (ALDO), for the development of metabolites accumulating strains in D. salina.

Demystifying biotrophs: FISHing for mRNAs to decipher plant and algal pathogen-host interaction at the single cell level.

Plant-pathogen interactions follow spatial and temporal developmental dynamics where gene expression in pathogen and host undergo crucial changes. Therefore, it is of great interest to detect, quantify and localise where and when key genes are active to understand these processes. Many pathosystems are not accessible for genetic amendments or other spatially-resolved gene expression monitoring methods. Here, we adapt single molecule FISH techniques to demonstrate the presence and activity of mRNAs at the single-cell level using phytomyxids in their plant and algal host in lab and field material. This allowed us to monitor and quantify the expression of genes from the clubroot pathogen Plasmodiophora brassicae, several species of its Brassica hosts, and of several brown algae, including the genome model Ectocarpus siliculosus, infected with the phytomyxid Maullinia ectocarpii. We show that mRNAs are localised along a spatiotemporal gradient, thus providing a proof-of-concept of the usefulness of single-molecule FISH to increase knowledge about the interactions between plants, algae and phytomyxids. The methods used are easily applicable to any interaction between microbes and their algal or plant host, and have therefore the potential to rapidly increase our understanding of key, spatially- and temporally-resolved processes underpinning complex plant-microbe interactions.

Darkness-induced effects on gene expression in Cosmarium crenatum (Zygnematophyceae) from a polar habitat.

Light is a key environmental regulator in all photosynthetic organisms. Many studies focused on the physiologic response to changes in light availability of species from the Zygnematophyceae, but the impact of the absence of light and the molecular acclimation process on the other side have been poorly understood. Here we present transcriptomic analyses of Cosmarium crenatum from a polar habitat exposed to darkness. The algae were cultured in dark for one week; cell number and quantum yield of photosystem II (Fv/Fm) were monitored. Cell number was stable, but the Fv/Fm decreased in both groups, darkness-treated and control. Gene expression analysis revealed a strong repression of transcripts associated with photosynthesis, photorespiration and cell wall development. General carbohydrate and lipid metabolism were differentially regulated, but starch is shown to be the primary energy source in these conditions. Additionally, C. crenatum induced mRNA responsible for epigenetic modifications which may be a specific response to an adaption and acclimation to polar conditions. Our study sheds light on the molecular acclimation process to darkness and provides ecological implications for new perspectives in this specialized group of green algae.

Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization.

BACKGROUND: Biological mineral formation (biomineralization) proceeds in specialized compartments often bounded by a lipid bilayer membrane. Currently, the role of membranes in biomineralization is hardly understood. RESULTS: Investigating biomineralization of SiO2 (silica) in diatoms we identified Silicanin-1 (Sin1) as a conserved diatom membrane protein present in silica deposition vesicles (SDVs) of Thalassiosira pseudonana. Fluorescence microscopy of GFP-tagged Sin1 enabled, for the first time, to follow the intracellular locations of a biomineralization protein during silica biogenesis in vivo. The analysis revealed incorporation of the N-terminal domain of Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines. CONCLUSIONS: Sin1 is the first identified SDV transmembrane protein, and is highly conserved throughout the diatom realm, which suggests a fundamental role in the biomineralization of diatom silica. Through interaction with long-chain polyamines, Sin1 could serve as a molecular link by which the SDV membrane exerts control on the assembly of biosilica-forming organic matrices in the SDV lumen.

miRNAs in the alga Chlamydomonas reinhardtii are not phylogenetically conserved and play a limited role in responses to nutrient deprivation.

The unicellular alga Chlamydomonas reinhardtii contains many types of small RNAs (sRNAs) but the biological role(s) of bona fide microRNAs (miRNAs) remains unclear. To address their possible function(s) in responses to nutrient availability, we examined miRNA expression in cells cultured under different trophic conditions (mixotrophic in the presence of acetate or photoautotrophic in the presence or absence of nitrogen). We also reanalyzed miRNA expression data in Chlamydomonas subject to sulfur or phosphate deprivation. Several miRNAs were differentially expressed under the various trophic conditions. However, in transcriptome analyses, the majority of their predicted targets did not show expected changes in transcript abundance, suggesting that they are not subject to miRNA-mediated RNA degradation. Mutant strains, defective in sRNAs or in ARGONAUTE3 (a key component of sRNA-mediated gene silencing), did not display major phenotypic defects when grown under multiple nutritional regimes. Additionally, Chlamydomonas miRNAs were not conserved, even in algae of the closely related Volvocaceae family, and many showed features resembling those of recently evolved, species-specific miRNAs in the genus Arabidopsis. Our results suggest that, in C. reinhardtii, miRNAs might be subject to relatively fast evolution and have only a minor, largely modulatory role in gene regulation under diverse trophic states.

Identifying Aspects of the Post-Transcriptional Program Governing the Proteome of the Green Alga Micromonas pusilla.

Micromonas is a unicellular motile alga within the Prasinophyceae, a green algal group that is related to land plants. This picoeukaryote (<2 µm diameter) is widespread in the marine environment but is not well understood at the cellular level. Here, we examine shifts in mRNA and protein expression over the course of the day-night cycle using triplicated mid-exponential, nutrient replete cultures of Micromonas pusilla CCMP1545. Samples were collected at key transition points during the diel cycle for evaluation using high-throughput LC-MS proteomics. In conjunction, matched mRNA samples from the same time points were sequenced using pair-ended directional Illumina RNA-Seq to investigate the dynamics and relationship between the mRNA and protein expression programs of M. pusilla. Similar to a prior study of the marine cyanobacterium Prochlorococcus, we found significant divergence in the mRNA and proteomics expression dynamics in response to the light:dark cycle. Additionally, expressional responses of genes and the proteins they encoded could also be variable within the same metabolic pathway, such as we observed in the oxygenic photosynthesis pathway. A regression framework was used to predict protein levels from both mRNA expression and gene-specific sequence-based features. Several features in the genome sequence were found to influence protein abundance including codon usage as well as 3' UTR length and structure. Collectively, our studies provide insights into the regulation of the proteome over a diel cycle as well as the relationships between transcriptional and translational programs in the widespread marine green alga Micromonas.

Rhodenigma contortum, an obscure new genus and species of Rhodogorgonales (Rhodophyta) from Western Australia.

An unknown microscopic, branched filamentous red alga was isolated into culture from coral fragments collected in Coral Bay, Western Australia. It grew well unattached or attached to glass with no reproduction other than fragmentation of filaments. Cells of some branch tips became slightly contorted and digitated, possibly as a substrate-contact-response seen at filament tips of various algae. Attached multicellular compact disks on glass had a very different cellular configuration and size than the free filaments. In culture the filaments did not grow on or in coral fragments. Molecular phylogenies based on four markers (rbcL, cox1, 18S, 28S) clearly showed it belongs to the order Rhodogorgonales, as a sister clade of Renouxia. Based on these results, the alga is described as the new genus and species Rhodenigma contortum in the Rhodogorgonaceae. It had no morphological similarity to either of the other genera in Rhodogorgonaceae and illustrates the unknown diversity in cryptic habitats such as tropical coral rubble.

Phylogenetic relationships of corallinaceae (Corallinales, Rhodophyta): taxonomic implications for reef-building corallines.

A new, more complete, five-marker (SSU, LSU, psbA, COI, 23S) molecular phylogeny of the family Corallinaceae, order Corallinales, shows a paraphyletic grouping of seven well-supported monophyletic clades. The taxonomic implications included the amendment of two subfamilies, Neogoniolithoideae and Metagoniolithoideae, and the rejection of Porolithoideae as an independent subfamily. Metagoniolithoideae contained Harveylithon gen. nov., with H. rupestre comb. nov. as the generitype, and H. canariense stat. nov., H. munitum comb. nov., and H. samoënse comb. nov. Spongites and Pneophyllum belonged to separate clades. The subfamily Neogoniolithoideae included the generitype of Spongites, S. fruticulosus, for which an epitype was designated. Pneophyllum requires reassesment. The generitype of Hydrolithon, H. reinboldii, was a younger heterotypic synonym of H. boergesenii. The evolutionary novelty of the subfamilies Hydrolithoideae, Metagoniolithoideae, and Lithophylloideae was the development of tetra/bisporangial conceptacle roofs by filaments surrounding and interspersed among the sporangial initials.

RNA-seq analysis of sulfur-deprived Chlamydomonas cells reveals aspects of acclimation critical for cell survival.

The Chlamydomonas reinhardtii transcriptome was characterized from nutrient-replete and sulfur-depleted wild-type and snrk2.1 mutant cells. This mutant is null for the regulatory Ser-Thr kinase SNRK2.1, which is required for acclimation of the alga to sulfur deprivation. The transcriptome analyses used microarray hybridization and RNA-seq technology. Quantitative RT-PCR evaluation of the results obtained by these techniques showed that RNA-seq reports a larger dynamic range of expression levels than do microarray hybridizations. Transcripts responsive to sulfur deprivation included those encoding proteins involved in sulfur acquisition and assimilation, synthesis of sulfur-containing metabolites, Cys degradation, and sulfur recycling. Furthermore, we noted potential modifications of cellular structures during sulfur deprivation, including the cell wall and complexes associated with the photosynthetic apparatus. Moreover, the data suggest that sulfur-deprived cells accumulate proteins with fewer sulfur-containing amino acids. Most of the sulfur deprivation responses are controlled by the SNRK2.1 protein kinase. The snrk2.1 mutant exhibits a set of unique responses during both sulfur-replete and sulfur-depleted conditions that are not observed in wild-type cells; the inability of this mutant to acclimate to S deprivation probably leads to elevated levels of singlet oxygen and severe oxidative stress, which ultimately causes cell death. The transcriptome results for wild-type and mutant cells strongly suggest the occurrence of massive changes in cellular physiology and metabolism as cells become depleted for sulfur and reveal aspects of acclimation that are likely critical for cell survival.

An RNA symbiont enhances heat tolerance and secondary homothallism in the oomycete Phytophthora infestans.

Some strains of Phytophthora infestans, the potato late blight pathogen, harbour a small extrachromosomal RNA called PiERE1. A previous study reported that this RNA symbiont does not noticeably affect its host. Here it is revealed that PiERE1 exerts subtle effects on P. infestans, which result in greater thermotolerance during growth and an increase in secondary homothallism, i.e. oospore formation in the absence of the opposite mating type. The interaction can be considered mutualistic since these traits may increase the fitness of P. infestans in nature. Assays of biomarkers for cellular stress revealed that an Hsp70 chaperone was upregulated by PiERE1. A genome-wide search for more members of the Hsp70 family identified ten belonging to the DnaK subfamily, one in the Hsp110/SSE subfamily, and pseudogenes. Four DnaK subfamily genes encoding predicted cytoplasmic or endoplasmic reticulum proteins were upregulated in strains harbouring PiERE1. This may explain the greater thermotolerance conferred by the RNA element, and suggests that Hsp70 may be a useful biomarker for testing organisms for the cellular effects of symbiotic elements.

MRL1, a conserved Pentatricopeptide repeat protein, is required for stabilization of rbcL mRNA in Chlamydomonas and Arabidopsis.

We identify and functionally characterize MRL1, a conserved nuclear-encoded regulator of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. The nonphotosynthetic mrl1 mutant of Chlamydomonas reinhardtii lacks ribulose-1,5-bisphosphate carboxylase/oxygenase, and the resulting block in electron transfer is partially compensated by redirecting electrons toward molecular oxygen via the Mehler reaction. This allows continued electron flow and constitutive nonphotochemical quenching, enhancing cell survival during illumination in spite of photosystem II and photosystem I photoinhibition. The mrl1 mutant transcribes rbcL normally, but the mRNA is unstable. The molecular target of MRL1 is the 5 ' untranslated region of rbcL. MRL1 is located in the chloroplast stroma, in a high molecular mass complex. Treatment with RNase or deletion of the rbcL gene induces a shift of the complex toward lower molecular mass fractions. MRL1 is well conserved throughout the green lineage, much more so than the 10 other pentatricopeptide repeat proteins found in Chlamydomonas. Depending upon the organism, MRL1 contains 11 to 14 pentatricopeptide repeats followed by a novel MRL1-C domain. In Arabidopsis thaliana, MRL1 also acts on rbcL and is necessary for the production/stabilization of the processed transcript, presumably because it acts as a barrier to 5 ' >3 ' degradation. The Arabidopsis mrl1 mutant retains normal levels of the primary transcript and full photosynthetic capacity.

Disrupted tRNA gene diversity and possible evolutionary scenarios.

The following unusual tRNAs have recently been discovered in the genomes of Archaea and primitive Eukaryota: multiple-intron-containing tRNAs, which have more than one intron; split tRNAs, which are produced from two pieces of RNA transcribed from separate genes; tri-split tRNAs, which are produced from three separate genes; and permuted tRNA, in which the 5' and 3' halves are encoded with permuted orientations within a single gene. All these disrupted tRNA genes can form mature contiguous tRNA, which is aminoacylated after processing by cis or trans splicing. The discovery of such tRNA disruptions has raised the question of when and why these complex tRNA processing pathways emerged during the evolution of life. Many previous reports have noted that tRNA genes contain a single intron in the anticodon loop region, a feature common throughout all three domains of life, suggesting an ancient trait of the last universal common ancestor. In this context, these unique tRNA disruptions recently found only in Archaea and primitive Eukaryota provide new insight into the origin and evolution of tRNA genes, encouraging further research in this field. In this paper, we summarize the phylogeny, structure, and processing machinery of all known types of disrupted tRNAs and discuss possible evolutionary scenarios for these tRNA genes.

Cysteine modification of a specific repressor protein controls the translational status of nucleus-encoded LHCII mRNAs in Chlamydomonas.

The cytosolic RNA-binding protein NAB1 represses translation of LHCII (light-harvesting complex of photosystem II) encoding mRNAs by sequestration into translationally silent mRNP complexes in the green alga Chlamydomonas reinhardtii. NAB1 contains 2 cysteine residues, Cys-181 and Cys-226, within its C-terminal RRM motif. Modification of these cysteines either by oxidation or by alkylation in vitro was accompanied by a decrease in RNA-binding affinity for the target mRNA sequence. To confirm the relevance of reversible NAB1 cysteine oxidation for the regulation of its activity in vivo, we replaced both cysteines with serines. All examined cysteine single and double mutants exhibited a reduced antenna at PSII caused by a perturbed NAB1 deactivation mechanism, with double mutations and Cys-226 single mutations causing a stronger and more distinctive phenotype compared with the Cys-181 mutation. Our data indicated that the responsible redox control mechanism is mediated by modification of single cysteines. Polysome analyses and RNA co-immunoprecipitation experiments demonstrated the interconnection of the NAB1 thiol state and its activity as a translation repressor in vivo. NAB1 is fully active in its dithiol state and is reversibly deactivated by modification of its cysteines. In summary, this work is an example that cytosolic translation of nucleus encoded photosynthetic genes is regulated via a reversible cysteine-based redox switch in a RNA-binding translation repressor protein.

Rapid and effective method of RNA isolation from green microalga Ankistrodesmus convolutus.

The rapid and effective method for the isolation of RNA from green microalga Ankistrodesmus convolutus based on homogenization in a simple CTAB buffer and selective precipitation of RNA with lithium chloride is developed. This procedure avoids the use of toxic chaotropic agents and phenol while high concentration of dithiothreitol is used to inhibit RNase activity and prevent oxidative cross-linking of nucleic acids by phenolics. The extraction procedure was able to produce high quality and intact RNA from A. convolutus. The yield of total RNA was 0.69-0.73 mg/g of fresh weight, with A(260)/A(280) ratio of 1.79-1.86. The obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR and cDNA library construction. The procedure may also have wider applicability for total RNA isolation from other green microalgae species.

Chlorovirus-mediated membrane depolarization of Chlorella alters secondary active transport of solutes.

Paramecium bursaria chlorella virus 1 (PBCV-1) is the prototype of a family of large, double-stranded DNA, plaque-forming viruses that infect certain eukaryotic chlorella-like green algae from the genus Chlorovirus. PBCV-1 infection results in rapid host membrane depolarization and potassium ion release. One interesting feature of certain chloroviruses is that they code for functional potassium ion-selective channel proteins (Kcv) that are considered responsible for the host membrane depolarization and, as a consequence, the efflux of potassium ions. This report examines the relationship between cellular depolarization and solute uptake. Annotation of the virus host Chlorella strain NC64A genome revealed 482 putative transporter-encoding genes; 224 are secondary active transporters. Solute uptake experiments using seven radioactive compounds revealed that virus infection alters the transport of all the solutes. However, the degree of inhibition varied depending on the solute. Experiments with nystatin, a drug known to depolarize cell membranes, produced changes in solute uptake that are similar but not identical to those that occurred during virus infection. Therefore, these studies indicate that chlorovirus infection causes a rapid and sustained depolarization of the host plasma membrane and that this depolarization leads to the inhibition of secondary active transporters that changes solute uptake.

Unstable RNAi effects through epigenetic silencing of an inverted repeat transgene in Chlamydomonas reinhardtii.

RNA interferences in the unicellular green alga, Chlamydomonas reinhardtii, can be silenced. We have used the silencing of a transgene (aadA) that confers resistance to spectinomycin to investigate the mechanisms responsible for silencing by an artificial inverted repeat (IR) of the aadA gene. The IR construct provided strong silencing, but the RNAi efficiency varied among subclones of a single RNAi-transformed strain with successive cell divisions. Northern blot analyses revealed an inverse correlation between the copy number of the hairpin RNA and the spectinomycin resistance of the subclones. There is an inverse correlation between the efficiency of RNAi and the frequency of methylated CpG (*CpG) in the silenced region. No significant methylated cytosine was observed in the target aadA gene, which suggests the absence of RNA-directed DNA methylation in trans. Several experiments suggest the existence of an intrinsic IR sequence-dependent but a transcription-independent DNA methylation system in C. reinhardtii. The correlation between the *CpG levels and the IR transcript implies the existence of IR DNA-dependent DNA methylation. Treatment of RNAi-induced cells with a histone deacetylase inhibitor, Trichostatin A, rapidly increased the amount of the hairpin RNA and suggests that transcription of the silencer construct was repressed by *CpG-related silencing mechanisms.

Stress induces the assembly of RNA granules in the chloroplast of Chlamydomonas reinhardtii.

Eukaryotic cells under stress repress translation and localize these messenger RNAs (mRNAs) to cytoplasmic RNA granules. We show that specific stress stimuli induce the assembly of RNA granules in an organelle with bacterial ancestry, the chloroplast of Chlamydomonas reinhardtii. These chloroplast stress granules (cpSGs) form during oxidative stress and disassemble during recovery from stress. Like mammalian stress granules, cpSGs contain poly(A)-binding protein and the small, but not the large, ribosomal subunit. In addition, mRNAs are in continuous flux between polysomes and cpSGs during stress. Localization of cpSGs within the pyrenoid reveals that this chloroplast compartment functions in this stress response. The large subunit of ribulosebisphosphate carboxylase/oxygenase also assembles into cpSGs and is known to bind mRNAs during oxidative stress, raising the possibility that it plays a role in cpSG assembly. This discovery within such an organelle suggests that mRNA localization to granules during stress is a more general phenomenon than currently realized.

Structural features and gene-expression profiles of actin homologs in Porphyra yezoensis (Rhodophyta).

The marine red alga Porphyra yezoensis contains an actin gene family consisting of at least four isoforms (PyACT1, 2, 3 and 4). The amino acid identity between isoforms exceeds 83%, and each contains a putative nuclear export signal (NES). We scanned the sequences for amino acids in regions homologous to the intermonomeric interface of actin filaments. Few residues expected to engage in cross-linking were conserved between the four isoforms. The results of the sequence analyses suggest that PyACT2 probably functions in the nucleus as a monomer (G-actin) or in other unconventional forms. In addition, the distribution and position of the introns were different from those in florideophycean actin genes. The expression level of PyACT3 in matured gametophytes was significantly higher than in those in a vegetative state, although the mRNA was detected at similar levels in both apical and basal parts of thalli. The expression levels of PyACT2 and 4, on the other hand, did not change significantly between the matured and vegetative gametophytes. The PyACT3 may serve as a molecular marker for monitoring thallus maturation in this species.