Diversity of genome size and chromosome number in homothallic and heterothallic strains of the Closterium peracerosum-strigosum-littorale complex (Desmidiales, Zygnematophyceae, Streptophyta).

The evolutionary transitions of mating systems between outcrossing and self-fertilization are often suggested to associate with the cytological and genomic changes, but the empirical reports are limited in multicellular organisms. Here we used the unicellular zygnematophycean algae, the Closterium peracerosum-strigosum-littorale (C. psl.) complex, to address whether genomic properties such as genome sizes and chromosome numbers are associated with mating system transitions between homothallism (self-fertility) and heterothallism (self-sterility). Phylogenetic analysis revealed the polyphyly of homothallic strains, suggesting multiple transitions between homothallism and heterothallism in the C. psl. complex. Flow cytometry analysis identified a more than 2-fold genome size variation, ranging from 0.53 to 1.42 Gbp, which was positively correlated with chromosome number variation between strains. Although we did not find consistent trends in genome size change and mating system transitions, the mean chromosome sizes tend to be smaller in homothallic strains than in their relative heterothallic strains. This result suggests that homothallic strains possibly have more fragmented chromosomes, which is consistent with the argument that self-fertilizing populations may tolerate more chromosomal rearrangements.

Erratum: Expanded male sex-determining region conserved during the evolution of homothallism in the green alga Volvox.

[This corrects the article DOI: 10.1016/j.isci.2023.106893.].

Expanded male sex-determining region conserved during the evolution of homothallism in the green alga Volvox.

Male and female genotypes in heterothallic (self-incompatible) species of haploid organisms, such as algae and bryophytes, are generally determined by male and female sex-determining regions (SDRs) in the sex chromosomes. To resolve the molecular genetic basis for the evolution of homothallic (bisexual and self-compatible) species from a heterothallic ancestor, we compared whole-genome data from Thai and Japanese genotypes within the homothallic green alga Volvox africanus. The Thai and Japanese algae harbored expanded ancestral male and female SDRs of ∼1 Mbp each, representing a direct heterothallic ancestor. Therefore, the expanded male and female ancestral SDRs may originate from the ancient (∼75 mya) heterothallic ancestor, and either might have been conserved during the evolution of each homothallic genotype. An expanded SDR-like region seems essential for homothallic sexual reproduction in V. africanus, irrespective of male or female origin. Our study stimulates future studies to elucidate the biological significance of such expanded genomic regions.

Extensive Copy Number Variation Explains Genome Size Variation in the Unicellular Zygnematophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Genome sizes are known to vary within and among closely related species, but the knowledge about genomic factors contributing to the variation and their impacts on gene functions is limited to only a small number of species. This study identified a more than 2-fold heritable genome size variation among the unicellular Zygnematophycean alga, Closterium peracerosum-strigosum-littorale (C. psl.) complex, based on short-read sequencing analysis of 22 natural strains and F1 segregation analysis. Six de novo assembled genomes revealed that genome size variation is largely attributable to genome-wide copy number variation (CNV) among strains rather than mating type-linked genomic regions or specific repeat sequences such as rDNA. Notably, about 30% of genes showed CNV even between strains that can mate with each other. Transcriptome and gene ontology analysis demonstrated that CNV is distributed nonrandomly in terms of gene functions, such that CNV was more often observed in the gene set with stage-specific expression. Furthermore, in about 30% of these genes with CNV, the expression level does not increase proportionally with the gene copy number, suggesting presence of dosage compensation, which was overrepresented in genes involved in basic biological functions, such as translation. Nonrandom patterns in gene duplications and corresponding expression changes in terms of gene functions may contribute to maintaining the high level of CNV associated with extensive genome size variation in the C. psl. complex, despite its possible detrimental effects.

Predictive biomarkers of ossification progression and bone metabolism dynamics in patients with cervical ossification of the posterior longitudinal ligament.

PURPOSE: This study aimed to establish biomarkers to predict the progression of ossification by examining ossification volume and bone metabolism dynamics in patients with ossification of the posterior longitudinal ligament (OPLL). METHODS: We assessed OPLL progression using computed tomography-based three-dimensional (3D) image analysis and examined bone metabolism dynamics in 107 patients with OPLL (men, 72; women, 35; mean age, 63.6 years). The volume of OPLL was calculated twice during the follow-up period, and OPLL progression was evaluated by the annual rate of ossification increase. Bone metabolism dynamics were assessed by routine blood tests and analysis of various serum biomarkers (including 25-hydroxyvitamin D, intact parathyroid hormone, fibroblast growth factor 23, intact N-terminal propeptide of type 1, tartrate-resistant acid phosphatase isoform 5b, sclerostin, and Dickkopf-1) and bone mineral density (BMD). Patients were classified into the progression (P) or non-progression (NP) group according to the annual rate of increase in previous 3D image analyses, and associated factors between these groups were compared. RESULTS: The P and NP groups consisted of 29 patients (23 men and 6 women) and 78 patients (49 men and 29 women), respectively. Univariate analysis revealed significant differences in terms of age, body mass index, serum phosphorus, serum sclerostin, and BMD. In multivariate analysis, age, serum phosphorus, and serum sclerostin were identified as independent factors associated with OPLL progression. CONCLUSION: Younger age, hypophosphatemia, and high serum sclerostin are risk factors for OPLL progression. Serum phosphorus and sclerostin could serve as important biomarkers for predicting ossification progression.

Evaluating flexibility and predicting curve correction using fulcrum-bending radiographs in Lenke type 2 adolescent idiopathic scoliosis.

BACKGROUND: Fulcrum-bending (FB) correction is considered to provide the best estimation of main thoracic (MT) curve flexibility and postoperative correction in surgical treatment for adolescent idiopathic scoliosis (AIS). However, few studies evaluated the usefulness of FB radiographs for proximal thoracic (PT) curve. We aimed to perform flexibility assessments using both active side-bending (SB) and FB radiographs and evaluate surgical outcomes after posterior spinal fusion (PSF) for Lenke type 2 AIS. METHODS: This study included 38 consecutive patients with Lenke type 2 AIS who underwent PSF using a pedicle screw construct with a minimum 2-year follow-up. Radiographic parameters, including correction rate, SB and FB flexibility, and FB correction index (FBCI: [correction rate/FB flexibility] × 100), were evaluated preoperatively, immediately after surgery, and at the 2-year follow-up. The clinical outcomes were preoperatively evaluated using the Scoliosis Research Outcomes Instrument-22 and at the follow-up. RESULTS: All scoliosis curves significantly improved and shoulder balance shifted toward left shoulder elevation (all comparisons, p < 0.0001). There were significant differences between the SB and FB corrections in the PT and MT curves (p < 0.0001). The magnitudes of the discrepancies between the SB and FB corrections in the PT and MT curves were 11.2° ± 5.2° and 11.6° ± 7.2°, respectively. FB correction did not differ from postoperative Cobb angles correction immediately after surgery or at the 2-year follow-up; the mean FBCIs in the PT and MT curves were 98.8% and 105.5%, respectively. The self-image domain SRS-22 scores had significantly increased at the 2-year follow-up (p < 0.0001). CONCLUSIONS: There were significant differences between the SB and FB corrections, and FB correction tended to approximate the postoperative curve correction (FBCI = 100%) for PT and MT curves in patients with Lenke type 2 AIS. FB flexibility is more reliable than SB flexibility in evaluating actual curve flexibility even for the PT curve.

Zygnematophycean algae: Possible models for cellular and evolutionary biology.

Plant terrestrialization was a critical event for our planet. For the study of plant evolution, charophytes have received a great deal of attention because of their phylogenetic position. Among charophytes, the class Zygnematophyceae is the closest lineage to land plants. During sexual reproduction, they show isogamous conjugation by immotile gametes, which is characteristic of zygnematophycean algae. Here, we introduce the genera Mougeotia, Penium, and Closterium, which are representative model organisms of Zygnematophyceae in terms of chloroplast photorelocation movement, the cell wall, and sexual reproduction, respectively.

A divergent RWP-RK transcription factor determines mating type in heterothallic Closterium.

The Closterium peracerosum-strigosum-littorale complex (Closterium, Zygnematophyceae) has an isogamous mating system. Members of the Zygnematophyceae are the closest relatives to extant land plants and are distantly related to chlorophytic models, for which a genetic basis of mating type (MT) determination has been reported. We thus investigated MT determination in Closterium. We sequenced genomes representing the two MTs, mt+ and mt-, in Closterium and identified CpMinus1, a gene linked to the mt- phenotype. We analyzed its function using reverse genetics methods. CpMinus1 encodes a divergent RWP-RK domain-containing-like transcription factor and is specifically expressed during gamete differentiation. Introduction of CpMinus1 into an mt+ strain was sufficient to convert it to a phenotypically mt- strain, while CpMinus1-knockout mt- strains were phenotypically mt+. We propose that CpMinus1 is the major MT determinant that acts by evoking the mt- phenotype and suppressing the mt+ phenotype in heterothallic Closterium. CpMinus1 likely evolved independently in the Zygnematophyceae lineage, which lost an egg-sperm anisogamous mating system. mt- specific regions possibly constitute an MT locus flanked by common sequences that undergo some recombination.

Highly efficient transformation of the model zygnematophycean alga Closterium peracerosum-strigosum-littorale complex by square-pulse electroporation.

The zygnematophycean algae occupy an important phylogenetic position as the closest living relatives of land plants. Reverse genetics is quite useful for dissecting the functions of genes. However, this strategy requires genetic transformation, and there are only a few reports of successful transformation in zygnematophycean algae. Here, we established a simple and highly efficient transformation technique for the unicellular zygnematophycean alga Closterium peracerosum-strigosum-littorale complex using a square electric pulse-generating electroporator without the need for cell wall removal. Using this method, the transformation efficiency increased > 100-fold compared with our previous study using particle bombardment. We also succeeded in performing CRISPR/Cas9-based gene knockout using this new method. Our method requires only small amounts of labor, time and incubator space. Moreover, our technique could also be utilized to transform other charophycean algae with available genome information by optimizing the electric pulse conditions.

Three sex phenotypes in a haploid algal species give insights into the evolutionary transition to a self-compatible mating system.

Mating systems of haploid species such as fungi, algae, and bryophytes are either heterothallic (self-incompatible) with two sex phenotypes (male and female, or mating type minus and plus in isogamous species) or homothallic (self-compatible) with only a bisexual phenotype producing zygotes within a clone. The anisogamous volvocine green alga Pleodorina starrii is a haploid species previously reported to have a heterothallic mating system. Here, we found that two additional culture strains originating from the same water system of P. starrii were taxonomically identified as P. starrii and produced male and female gametes and zygotes within a clone (bisexual). Sequences of rapidly evolving plastid genome regions were identical between the bisexual and unisexual (male or female) P. starrii strains. Intercrossings between the bisexual and unisexual strains demonstrated normal thick-walled zygotes and high survivability of F1 strains. Thus, these strains belong to the same biological species. Pleodorina starrii has a new haploid mating system that is unique in having three sex phenotypes, namely, male, female, and bisexual. Genetic analyses suggested the existence of autosomal "bisexual factor" locus independent of volvocine male and female determining regions. The present findings increase our understanding of the initial evolutionary step of transition from heterothallism to homothallism.

Expression of Genes from Paternal Alleles in Rice Zygotes and Involvement of OsASGR-BBML1 in Initiation of Zygotic Development.

Upon fertilization in angiosperms, one sperm cell fuses with the egg cell to produce a zygote, and, via karyogamy, the parental genetic information is combined to form the diploid zygotic genome. Recently, analyses with parentally imbalanced rice zygotes indicated that parental genomes are utilized synergistically in zygotes with different functions, and that genes transcribed from the paternal or maternal allele might play important roles in zygotic development. Herein, we first conducted single nucleotide polymorphism-based mRNA-sequencing using intersubspecific rice zygotes. Twenty-three genes, with paternal allele-specific expression in zygotes, were identified, and, surprisingly, their allele dependencies in the globular-like embryo tended to be biallelic. This suggests that the paternal-dependent expression of these genes is temporary, occurring during the early stages of zygote development. Of the 23 genes, we focused on Oryza sativa Apospory-specific Genome Region (ASGR)-BABY-BOOM LIKE (BBML) 1 (OsASGR-BBML1), presumed to encode an AP2-transcription factor, due to its reported role in zygotic development. Interestingly, ectopic expression of OsASGR-BBML1 in egg cells induced nuclear and cell divisions, indicating that exogenously expressed OsASGR-BBML1 converts the proliferation status of the egg cell from quiescent to active. In addition, the suppression of the function of OsASGR-BBML1 and its homologs in zygotes resulted in the developmental arrest, suggesting that OsASGR-BBML1 possesses an important role in initiating zygotic development. Monoallelic or preferential gene expression from the paternal genome in the zygote might be a safety mechanism allowing egg cells to suppress the gene expression cascade toward early embryogenesis that is normally triggered by fusion with a sperm cell.

The genus Closterium, a new model organism to study sexual reproduction in streptophytes.

Contents Summary 99 I. Introduction 99 II. Life cycle of Closterium 100 III. Sexual reproductive processes in the heterothallic Closterium peracerosum-strigosum-littorale complex 101 IV. Homothallism in the C. psl. complex 102 V. Sexual reproduction and inheritance of mating types in Closterium ehrenbergii 102 VI. mt-determining gene of the C. psl. complex 103 VII. Future perspectives 103 Acknowledgements 103 References 103 SUMMARY: Closterium occupies a key phylogenetic position as an ancestor of land plants and is the best-characterized Charophycean alga in terms of the process of sexual reproduction. Zygospores form as a result of sexual reproduction between genetically determined mating type plus (mt+ ) and mating type minus (mt- ) cells in heterothallic strains, or between clonal cells in homothallic strains. Here we review knowledge on the intercellular communication and mating type determination for successful sexual reproduction in Closterium. Using genomic information and transgenic techniques, the genus could be a model organism to study the mechanisms and evolution of sexual reproduction in streptophytes.

Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants.

Evolutionary mechanisms underlying innovation of cell types have remained largely unclear. In multicellular eukaryotes, the evolutionary molecular origin of sperm differentiation is unknown in most lineages. Here, we report that in algal ancestors of land plants, changes in the DNA-binding domain of the ancestor of the MYB transcription factor DUO1 enabled the recognition of a new cis-regulatory element. This event led to the differentiation of motile sperm. After neo-functionalization, DUO1 acquired sperm lineage-specific expression in the common ancestor of land plants. Subsequently the downstream network of DUO1 was rewired leading to sperm with distinct morphologies. Conjugating green algae, a sister group of land plants, accumulated mutations in the DNA-binding domain of DUO1 and lost sperm differentiation. Our findings suggest that the emergence of DUO1 was the defining event in the evolution of sperm differentiation and the varied modes of sexual reproduction in the land plant lineage.

Identification of a new mating group and reproductive isolation in the Closterium peracerosum-strigosum-littorale complex.

Reproductive isolation is essential for the process of speciation. In order to understand speciation, it is necessary to compare one mating group with other phylogenetically related but reproductively isolated groups. The Closterium peracerosum-strigosum-littorale (C. psl.) complex is a unicellular isogamous zygnematophycean alga, which is believed to share a close phylogenetic relationship with the land plants. In this study, we identified a new mating group, named group G, of C. psl. complex and compared its physiological and biochemical characteristics with the mating group I-E, which was closely related to the mating group G. Zygospores are typically formed as a result of conjugation between mating-type plus (mt+) and mating-type minus (mt-) cells in the same mating group during sexual reproduction. Crossing experiments revealed mating groups G and I-E were reproductively isolated from each other, but the release of lone protoplasts from mt- cells of mating group G was induced in the presence of mt+ cells of mating group I-E. In fact, the sex pheromone, protoplast-release-inducing protein of mating group I-E induced the release of protoplasts from mt- cells of mating group G. When mt+ and mt- cells of both mating groups I-E and G were co-cultured (multiple-choice matings), the zygospore formation of mating group G, but not that of mating group I-E, was inhibited. Based on these results, we propose a possible mechanism of reproductive isolation between the two mating groups and suggest the presence of sexual interference between mating group G and mating group I-E.

CRISPR/Cas9-based knockouts reveal that CpRLP1 is a negative regulator of the sex pheromone PR-IP in the Closterium peracerosum-strigosum-littorale complex.

Heterothallic strains of the Closterium peracerosum-strigosum-littorale (C. psl.) complex have two sexes, mating-type plus (mt+) and mating-type minus (mt-). Conjugation between these two sexes is regulated by two sex pheromones, protoplast-release-inducing protein (PR-IP) and PR-IP Inducer, which are produced by mt+ and mt- cells, respectively. PR-IP mediates the release of protoplasts from mt- cells during mating. In this study, we examined the mechanism of action of CpRLP1 (receptor-like protein 1), which was previously identified in a cDNA microarray analysis as one of the PR-IP-inducible genes. Using CRISPR/Cas9 technology, we generated CpRLP1 knockout mutants in mt- cells of the C. psl. complex. When the knockout mt- cells were mixed with wild-type mt+ cells, conjugation was severely reduced. Many cells released protoplasts without pairing, suggesting a loss of synchronization between the two mating partners. Furthermore, the knockout mutants were hypersensitive to PR-IP. We conclude that CpRLP1 is a negative regulator of PR-IP that regulates the timing of protoplast release in conjugating C. psl. cells. As the first report of successful gene knockout in the class Charophyceae, this study provides a basis for research aimed at understanding the ancestral roles of genes that are indispensable for the development of land plants.

Molecular evolutionary analysis of a gender-limited MID ortholog from the homothallic species Volvox africanus with male and monoecious spheroids.

Volvox is a very interesting oogamous organism that exhibits various types of sexuality and/or sexual spheroids depending upon species or strains. However, molecular bases of such sexual reproduction characteristics have not been studied in this genus. In the model species V. carteri, an ortholog of the minus mating type-determining or minus dominance gene (MID) of isogamous Chlamydomonas reinhardtii is male-specific and determines the sperm formation. Male and female genders are genetically determined (heterothallism) in V. carteri, whereas in several other species of Volvox both male and female gametes (sperm and eggs) are formed within the same clonal culture (homothallism). To resolve the molecular basis of the evolution of Volvox species with monoecious spheroids, we here describe a MID ortholog in the homothallic species V. africanus that produces both monoecious and male spheroids within a single clonal culture. Comparison of synonymous and nonsynonymous nucleotide substitutions in MID genes between V. africanus and heterothallic volvocacean species suggests that the MID gene of V. africanus evolved under the same degree of functional constraint as those of the heterothallic species. Based on semi quantitative reverse transcription polymerase chain reaction analyses using the asexual, male and monoecious spheroids isolated from a sexually induced V. africanus culture, the MID mRNA level was significantly upregulated in the male spheroids, but suppressed in the monoecious spheroids. These results suggest that the monoecious spheroid-specific down regulation of gene expression of the MID homolog correlates with the formation of both eggs and sperm in the same spheroid in V. africanus.

Sexual reproduction and sex determination in green algae.

The sexual reproductive processes of some representative freshwater green algae are reviewed. Chlamydomonas reinhardtii is a unicellular volvocine alga having two mating types: mating type plus (mt+) and mating type minus (mt-), which are controlled by a single, complex mating-type locus. Sexual adhesion between the gametes is mediated by sex-specific agglutinin molecules on their flagellar membranes. Cell fusion is initiated by an adhesive interaction between the mt+ and mt- mating structures, followed by localized membrane fusion. The loci of sex-limited genes and the conformation of sex-determining regions have been rearranged during the evolution of volvocine algae; however, the essential function of the sex-determining genes of the isogamous unicellular Chlamydomonas reinhardtii is conserved in the multicellular oogamous Volvox carteri. The sexual reproduction of the unicellular charophycean alga, Closterium peracerosum-strigosum-littorale complex, is also focused on here. The sexual reproductive processes of heterothallic strains are controlled by two multifunctional sex pheromones, PR-IP and PR-IP Inducer, which independently promote multiple steps in conjugation at the appropriate times through different induction mechanisms. The molecules involved in sexual reproduction and sex determination have also been characterized.

Concanavalin A Disrupts the Release of Fibrous Material Necessary for Zygote Formation of a Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

The Closterium peracerosum-strigosum-littorale (C. psl.) complex is the best characterized charophycean alga with respect to the processes of sexual reproduction. We examined the effect of concanavalin A (Con A) on physiological and ultrastructural changes during the conjugation of the C. psl. complex. Two heterothallic gametangial cells formed a sexual pair as usual; however, the release of gametes was completely blocked by the addition of Con A. Fluorescein isothiocyanate-labeled Con A bound to the outermost layer of the conjugation papillae of paired cells. In the absence of Con A, the disruption of outer cell walls on the conjugation papillae and the secretion of fibrous materials from the conjugation papillae were observed using a transmission electron microscope, but Con A-treated cells did not show these changes. Instead, a highly electron-dense layer was observed in the outermost papillae, and the excess fibrous materials remained at the inside of the layer. These results suggest that an unknown molecule(s) recognized by Con A is essential for the diffusion of fibrous materials at the conjugation papillae, which is an indispensable step for gamete release during conjugation of the C. psl. complex.

Algal ancestor of land plants was preadapted for symbiosis.

Colonization of land by plants was a major transition on Earth, but the developmental and genetic innovations required for this transition remain unknown. Physiological studies and the fossil record strongly suggest that the ability of the first land plants to form symbiotic associations with beneficial fungi was one of these critical innovations. In angiosperms, genes required for the perception and transduction of diffusible fungal signals for root colonization and for nutrient exchange have been characterized. However, the origin of these genes and their potential correlation with land colonization remain elusive. A comprehensive phylogenetic analysis of 259 transcriptomes and 10 green algal and basal land plant genomes, coupled with the characterization of the evolutionary path leading to the appearance of a key regulator, a calcium- and calmodulin-dependent protein kinase, showed that the symbiotic signaling pathway predated the first land plants. In contrast, downstream genes required for root colonization and their specific expression pattern probably appeared subsequent to the colonization of land. We conclude that the most recent common ancestor of extant land plants and green algae was preadapted for symbiotic associations. Subsequent improvement of this precursor stage in early land plants through rounds of gene duplication led to the acquisition of additional pathways and the ability to form a fully functional arbuscular mycorrhizal symbiosis.

A Receptor-Like Kinase, Related to Cell Wall Sensor of Higher Plants, is Required for Sexual Reproduction in the Unicellular Charophycean Alga, Closterium peracerosum-strigosum-littorale Complex.

Here, we cloned the CpRLK1 gene, which encodes a receptor-like protein kinase expressed during sexual reproduction, from the heterothallic Closterium peracerosum-strigosum-littorale complex, one of the closest unicellular alga to land plants. Mating-type plus (mt(+)) cells with knockdown of CpRLK1 showed reduced competence for sexual reproduction and formed an abnormally enlarged conjugation papilla after pairing with mt(-) cells. The knockdown cells were unable to release a naked gamete, which is indispensable for zygote formation. We suggest that the CpRLK1 protein is an ancient cell wall sensor that now functions to regulate osmotic pressure in the cell to allow proper gamete release.