Pyrenoids are suborganelles formed by the accumulation of the CO₂-fixing enzyme RubisCO in chloroplasts and play a central role in the efficient CO₂-fixation reaction in aquatic environments. Pyrenoid research has a long history and has been conducted mainly in the field of morphological taxonomy. Recently, however, epoch-making studies such as the discovery of the liquid-liquid phase separation of green algal pyrenoids and the discovery of molecules necessary for the phase separation have been carried out, and research on the molecular mechanism of pyrenoid formation has made significant progress. Against this background, a symposium entitled “Pyrenoids: Cutting Edge of Phase-separated Organelles in Plants” was held at the 86th Congress of the Botanical Society of Japan under the joint sponsorship of the Japanese Society of Plant Morphology. The symposium featured lectures by researchers leading pyrenoid research using various algae and hornworts, sharing the forefront of pyrenoid research and discussing its diversity, universality, and future research prospects.

Pyrenoids are suborganelles in chloroplasts of algae and hornworts and can be observed under a light microscope. Pyrenoids are formed by aggregating the CO₂-fixing enzyme RubisCO and play a central role in the efficient CO₂ fixation reaction, especially in aquatic conditions. The molecular mechanism of pyrenoid formation and its relationship to the CO₂- concentration mechanism have been dramatically improved using the model green alga Chlamydomonas reinhardtii. Pyrenoid in Chlamydomonas is formed by the liquid-liquid phase separation of RubisCO and multivalent RubisCO-binding protein with disordered regions. This concept has been argued to apply to pyrenoid formation in other algal species. In this review, I introduce recent studies on the Chlamydomonas pyrenoid structure of pyrenoid and the CO₂-concentrating mechanism. I provide a perspective that the functional components of the pyrenoid, (1) pyrenoid matrix, (2) starch sheaths and peripheral proteins, and (3) pyrenoid tubules structure, are universal structures acquired by algae through convergent evolution across lineages to achieve the purpose of CO₂ concentration around RubisCO. Understanding the principles behind the regulation, structure, and formation of pyrenoids will provide not only a basic understanding of a nonmembrane organelle of cell biology but also a molecular basis for the future introduction of pyrenoids into land plants to enhance photosynthetic performance and yield.

Pyrenoids found in chloroplasts of various algae and hornworts are suborganelles composed mainly of RubisCO. Pyrenoids contribute to the CO₂-concentrating mechanism that allows efficient photosynthesis; however, pyrenoids have been independently lost in various lineages of algae and in the origin of land plants. Recently, it was suggested that the hydrophobicity of two helices on the RubisCO small subunit (RBCS) surface is important for pyrenoid formation in the green alga, Chlamydomonas reinhardtii. Furthermore, several molecules were reported to be important for pyrenoid formation, such as EPYC1 which mediates RubisCO aggregates. However, details of molecular basis of formation and evolutionary loss of pyrenoids remain unclear. In this article, I introduce Chloromonas lineage, a potential model lineage for studying the evolution of pyrenoids. The Chloromonas lineage belonging to the order Volvocales with Chlamydomonas reinhardtii, consists of several genera/clades that differ in pyrenoid characteristics, such as the Reticulata group, which contains both pyrenoid-possessing and pyrenoid-lacking species, and the genus Gloeomonas, which, unlike Chlamydomonas reinhardtii, has several pyrenoids without starch sheaths. In addition, I also introduce our recent study which compared RBCS sequences of three pyrenoid-containing and two pyrenoid-lacking species within the Reticulata group.

Cyanobacteria and many eukaryotic algae have a carbon-concentrating mechanism (CCM) within each cell or individual chloroplast to enable efficient photosynthesis even in CO₂-deficient environments. The ‘pyrenoids’, a chloroplast internal compartment responsible for CCM in eukaryotic algae, are absent in chloroplasts of land plants, and CCM at the cellular level appears to have been lost during the evolution of land plants. However, only hornworts have pyrenoids in their chloroplasts and are believed to possess CCM. This review summarizes the current status of research on the characteristics and functions of pyrenoids in hornworts and their evolutionary origin, and discusses why hornworts are the only land plants that possess pyrenoids.

Pyrenoid is a proteinaceous membraneless organelle, mainly composed of Rubisco. Many oxygenic photosynthetic organisms in aquatic environment possess the pyrenoids, which are responsible for the carbon concentrating mechanism. Pyrenoid is probably liquid-like structure, so that its morphology is simple and close to spherical. However, its accessory structure, a pyrenoid-penetrating thylakoid, a structure in pyrenoid periphery, and a membrane sac wrapping a pyrenoid, give rise to a variety of pyrenoid morphology, resulting in its usage as a taxonomic feature. The accessory structures seem to occur sporadically and evolve convergently, as well as pyrenoid itself. In this paper, the morphological aspect of pyrenoid in Heterokontophyta, which support primary production in the hydrosphere, has been reviewed to discuss the roles of pyrenoid accessory structures by summarizing the diversity and commonality of their morphological characteristics.

Pyrenoid is a protein body in the chloroplast that sequestrates ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), a CO₂ fixing enzyme. The pyrenoid occurs in many algae and a part of hornworts, but the structure does not have membrane and has not been considered as a decisive organelle, thus the detailed function has not been studied well to date. Recent studies demonstrated that green algal pyrenoid in Chlamydomonas is a liquid-liquid phase separation constituted by a natural disorder protein, EPYC1. On the contrary, marine photosynthesis that plays an enormous role as the global gas-exchange system is also sustained by the pyrenoid-based systems. The marine photoautotrophs, such as diatoms, primarily comprise of red-type secondary chloroplast, whose pyrenoid structure is dissimilar to that of green type. Even though these pyrenoids are the products of hetero-origin convergent evolution, recent studies however reveal structural/functional similarity amongst different pyrenoids. For instance, the pyrenoids in the CCM-operating algae have thylakoid-like membrane which penetrates/invaginates the pyrenoid. This may suggest that algae faced to the low CO₂ environment after the Carboniferous were forced to optimize the usage of light and CO₂ supply into some structural/functional frames with few options. This review will describe implications of the pyrenoid structures from some common aspects observed in the diatom pyrenoid.

Pyrenoids consist of Rubisco proteins and play a central role in CO₂ fixation in algae. It is suggested that pyrenoids are formed by liquid-liquid phase separation in plastid stroma. Recently, the pyrenoid formation and CO₂-concentrating mechanisms have been reported in the model green alga Chlamydomonas. However, there is limited information given the huge diversity of algae. How have pyrenoids evolved in a wide variety of photosynthetic organisms? Is the molecular mechanism of pyrenoids universal among them? To answer these questions, investigations in other than the model species are necessary. I have been conducting research using chlorarachniophyte algae that acquired plastids through secondary symbiosis. Here I’d like to introduce the morphological diversity and evolution of pyrenoids in chlorarachniophytes.

As suggested by classical studies, recent advances in research have demonstrated that mechanical forces play a key role in plant growth and development. We organized the Journal of Plant Research (JPR) international symposium to examine the universal relationship between mechanical forces and plant growth and development from the diverging viewpoints of plant morphology and plant molecular physiology. This symposium was co-organized by the Japanese Society of Plant Morphology.

When cells divide, DNA condenses into chromosomes, which are then equally distributed between the two daughter cells. At metaphase, a specific DNA region called the centromere is pulled by the spindle microtubules to the poles. At telophase, the chromosomes decondense and the cell nucleus is reconstructed. If the distribution of centromeres pulled to the two poles remains unchanged, the cell nucleus becomes Rabl structure, and if the centromeres are no longer unevenly distributed on the inner nuclear membrane, the cell nucleus becomes a centromere-dispersed cell nucleus (non-Rabl structure). For example, the spatial arrangement of centromeres in the nuclei of yeast, Drosophila, and wheat, is Rabl structure, while that in humans, Arabidopsis thaliana, and Caenorhabditis elegans is non-Rabl structure. Imaging and molecular cell biological studies using A. thaliana mutants with non-Rabl-structured nuclei have revealed that there are two molecular processes involved in the establishment of non-Rabl-structured nuclei. The process of dispersion of the centromeres after anaphase involves the CII-LINC complex, which is composed of the condensin II complex and the LINC (linker of nucleoskeleton and cytoskeleton) complex. Subsequently, the nuclear lamina, CRWN, acts to stabilize the dispersed centromeres near the inner nuclear membrane. Mutants of these factors have normal developmental and differentiation phenotypes without the significant alternation of gene transcription. On the other hand, when DNA damage stress is applied by adding a DNA double-strand break inducer, the frequency of DNA double-strand breaks in CII and CRWN mutants increases, resulting in abnormal organogenesis. Thus, the proper arrangement of centromeres may play a role in preventing DNA double-strand breaks in the nucleus.

Because plant cells are glued together via their cell walls, plant cell growth continuously generates mechanical stress. Plant stems are pressurized cylinders, where the epidermis is under tension and the remaining inner components under compression and prescribes maximal tensile stress direction along the circumference. In plant stems, the epidermis is the main tension-load bearing layer that drives organ growth by restricting radial cell growth via oriented cellulose deposition guided by cortical microtubules that respond to stress. Accumulating evidence has shown the importance of biomechanical vision to disentangle plant development. In parallel to mechanical forces that contribute to plant organ morphogenesis, organ integrity also relies on mechanical regulation, as hinted by the longitudinal cracks emerging in the inflorescence stem of Arabidopsis clavata3 de-etiolated3 mutant. Failure to tolerate high levels of mechanical stress, generated from uncoordinated cell growth of inner and outer cell layers, lead to tissue breakage. In this review, we summarize our recent findings on how organ mechanical integrity is maintained in Arabidopsis inflorescence stems. Then, in the second part we instead focus on the cracks found on fleshy fruits. The development of fleshy fruits in most species is characterized by shorter cell division phase versus continuous cell expansion until ripening, which is reminiscent of stem cracking, where increasing tensile stress is thought to exceed the limit of epidermal strength. Although cylindrical stems and spherical fruits display completely different morphologies, both represent ideal systems to decipher how mechanical integrity is sustained during plant growth.

Plant morphology (or anatomy) is reaching a new phase with serial sectioning methods followed by three-dimensional (3D) reconstruction methods. Although the recent studies of 3D analysis have tended to depend on high-performance machines (such as FE-SEM, FIB-SEM, SBF-SEM, and etc.), we can also perform 3D reconstruction using a microtome and an ordinary light microscope. Here, I introduce utilities of serial section light microscopy (ssLM) followed by 3D reconstruction with the case study applied to the analysis of intercellular airspace, mesophyll cells, and chloroplasts in leaf tissues of rice (Oryza sativa L.).

The evolution from unicellular to multicellular organisms has occurred many times in the history of life and many eukaryotic lineages have acquired their multicellular complexity, such as three-dimensional body plans or germ-soma differentiation, independently. Volvocine green algae are one of suitable model organisms for investigation into the evolution of multicellularity. Within this group, Volvocaceae and Astrephomene exhibit parallel evolution of spheroidal colonies from ancestral flattened colonies and germ-soma differentiation. However, little is known about these multicellular traits of Astrephomene in molecular and cellular levels, in contrast to Volvox or Volvocaceae. Therefore, we first established a new strain of Astrephomene and conducted developmental analyses. We found rotation of daughter protoplasts during successive cell divisions in Astrephomene, which is a tactic of spheroidal colony formation different from inversion in Volvocaceae. We also analyzed the embryogenesis of volvocine genera Gonium and Tetrabaena, which form ancestral flattened colonies, and revealed that the ancestor of Astrephomene and Volvocaceae might have newly acquired the cellular mechanisms for spheroidal colony formation respectively. In addition, we conducted de novo whole genome sequencing and cell-type RNA-seq analysis of Astrephomene. Though regA, which is a master regulator of somatic cell differentiation in Volvox, was absent in Astrephomene genome, we found another somatic-specific transcription factors in Astrephomene. On the other hand, differentiated gene expression patterns between somatic and reproductive cells in Astrephomene were similar with those in Volvox.