Characterization of ancestral myosin XI from Marchantia polymorpha by heterologous expression in Arabidopsis thaliana.

Previous studies have revealed duplications and diversification of myosin XI genes between angiosperms and bryophytes; however, the functional differentiation and conservation of myosin XI between them remain unclear. Here, we identified a single myosin XI gene from the liverwort Marchantia polymorpha (Mp). The molecular properties of Mp myosin XI are similar to those of Arabidopsis myosin XIs responsible for cytoplasmic streaming, suggesting that the motor function of myosin XI is able to generate cytoplasmic streaming. In cultured Arabidopsis cells, transiently expressed green fluorescent protein (GFP)-fused Mp myosin XI was observed as some intracellular structures moving along the F-actin. These intracellular structures were co-localized with motile endoplasmic reticulum (ER) strands, suggesting that Mp myosin XI binds to the ER and generates intracellular transport in Arabidopsis cells. The tail domain of Mp myosin XI was co-localized with that of Arabidopsis myosin XI-2 and XI-K, suggesting that all these myosin XIs bind to common cargoes. Furthermore, expression of GFP-fused Mp myosin XI rescued the defects of growth, cytoplasmic streaming and actin organization in Arabidopsis multiple myosin XI knockout mutants. The heterologous expression experiments demonstrated the cellular and physiological competence of Mp myosin XI in Arabidopsis. However, the average velocity of organelle transport in Marchantia rhizoids was 0.04 ± 0.01 µm s-1 , which is approximately one-hundredth of that in Arabidopsis cells. Taken together, our results suggest that the molecular properties of myosin XI are conserved, but myosin XI-driven intracellular transport in vivo would be differentiated from bryophytes to angiosperms.

Day/Night Separation of Oxygenic Energy Metabolism and Nuclear DNA Replication in the Unicellular Red Alga Cyanidioschyzon merolae.

The transition from G1 to S phase and subsequent nuclear DNA replication in the cells of many species of eukaryotic algae occur predominantly during the evening and night in the absence of photosynthesis; however, little is known about how day/night changes in energy metabolism and cell cycle progression are coordinated and about the advantage conferred by the restriction of S phase to the night. Using a synchronous culture of the unicellular red alga Cyanidioschyzon merolae, we found that the levels of photosynthetic and respiratory activities peak during the morning and then decrease toward the evening and night, whereas the pathways for anaerobic consumption of pyruvate, produced by glycolysis, are upregulated during the evening and night as reported recently in the green alga Chlamydomonas reinhardtii Inhibition of photosynthesis by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) largely reduced respiratory activity and the amplitude of the day/night rhythm of respiration, suggesting that the respiratory rhythm depends largely on photosynthetic activity. Even when the timing of G1/S-phase transition was uncoupled from the day/night rhythm by depletion of retinoblastoma-related (RBR) protein, the same patterns of photosynthesis and respiration were observed, suggesting that cell cycle progression and energy metabolism are regulated independently. Progression of the S phase under conditions of photosynthesis elevated the frequency of nuclear DNA double-strand breaks (DSB). These results suggest that the temporal separation of oxygenic energy metabolism, which causes oxidative stress, from nuclear DNA replication reduces the risk of DSB during cell proliferation in C. merolaeIMPORTANCE Eukaryotes acquired chloroplasts through an endosymbiotic event in which a cyanobacterium or a unicellular eukaryotic alga was integrated into a previously nonphotosynthetic eukaryotic cell. Photosynthesis by chloroplasts enabled algae to expand their habitats and led to further evolution of land plants. However, photosynthesis causes greater oxidative stress than mitochondrion-based respiration. In seed plants, cell division is restricted to nonphotosynthetic meristematic tissues and populations of photosynthetic cells expand without cell division. Thus, seemingly, photosynthesis is spatially sequestrated from cell proliferation. In contrast, eukaryotic algae possess photosynthetic chloroplasts throughout their life cycle. Here we show that oxygenic energy conversion (daytime) and nuclear DNA replication (night time) are temporally sequestrated in C. merolae This sequestration enables "safe" proliferation of cells and allows coexistence of chloroplasts and the eukaryotic host cell, as shown in yeast, where mitochondrial respiration and nuclear DNA replication are temporally sequestrated to reduce the mutation rate.

RAB GTPases in the Basal Land Plant Marchantia polymorpha.

The RAB GTPase is an evolutionarily conserved machinery component of membrane trafficking, which is the fundamental system for cell viability and higher order biological functions. The composition of RAB GTPases in each organism is closely related to the complexity and organization of the membrane trafficking pathway, which has been developed uniquely to realize the organism-specific membrane trafficking system. Comparative genomics has suggested that terrestrialization and/or multicellularization were associated with the expansion of membrane trafficking pathways in green plants, which has yet to be validated in basal land plant lineages. To obtain insight into the diversification of membrane trafficking systems in green plants, we analyzed RAB GTPases encoded in the genome of the liverwort Marchantia polymorpha in a comprehensive manner. We isolated all genes for RAB GTPases in Marchantia and analyzed their expression patterns and subcellular localizations in thallus cells. While a majority of MpRAB GTPases exhibited a ubiquitous expression pattern, specific exceptions were also observed; MpRAB2b, which contains a sequence similar to an intraflagellar transport protein at the C-terminal region; and MpRAB23, which has been secondarily lost in angiosperms, were specifically expressed in the male reproductive organ. MpRAB21, which is another RAB GTPase whose homolog is absent in Arabidopsis, exhibited endosomal localization with RAB5 members in Marchantia. These results suggest that Marchantia possesses unique membrane trafficking pathways involving a unique repertoire of RAB GTPases.

Acidophilic green algal genome provides insights into adaptation to an acidic environment.

Some microalgae are adapted to extremely acidic environments in which toxic metals are present at high levels. However, little is known about how acidophilic algae evolved from their respective neutrophilic ancestors by adapting to particular acidic environments. To gain insights into this issue, we determined the draft genome sequence of the acidophilic green alga Chlamydomonas eustigma and performed comparative genome and transcriptome analyses between Ceustigma and its neutrophilic relative Chlamydomonas reinhardtii The results revealed the following features in Ceustigma that probably contributed to the adaptation to an acidic environment. Genes encoding heat-shock proteins and plasma membrane H+-ATPase are highly expressed in Ceustigma This species has also lost fermentation pathways that acidify the cytosol and has acquired an energy shuttle and buffering system and arsenic detoxification genes through horizontal gene transfer. Moreover, the arsenic detoxification genes have been multiplied in the genome. These features have also been found in other acidophilic green and red algae, suggesting the existence of common mechanisms in the adaptation to acidic environments.

Chloroplast division checkpoint in eukaryotic algae.

Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase-specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.

SNARE Molecules in Marchantia polymorpha: Unique and Conserved Features of the Membrane Fusion Machinery.

The membrane trafficking pathway has been diversified in a specific way for each eukaryotic lineage, probably to fulfill specific functions in the organisms. In green plants, comparative genomics has supported the possibility that terrestrialization and/or multicellularization could be associated with the elaboration and diversification of membrane trafficking pathways, which have been accomplished by an expansion of the numbers of genes required for machinery components of membrane trafficking, including soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. However, information regarding membrane trafficking pathways in basal land plant lineages remains limited. In the present study, we conducted extensive analyses of SNARE molecules, which mediate membrane fusion between target membranes and transport vesicles or donor organelles, in the liverwort, Marchantia polymorpha. The M. polymorpha genome contained at least 34 genes for 36 SNARE proteins, comprising fundamental sets of SNARE proteins that are shared among land plant lineages with low degrees of redundancy. We examined the subcellular distribution of a major portion of these SNARE proteins by expressing Citrine-tagged SNARE proteins in M. polymorpha, and the results showed that some of the SNARE proteins were targeted to different compartments from their orthologous products in Arabidopsis thaliana. For example, MpSYP12B was localized to the surface of the oil body, which is a unique organelle in liverworts. Furthermore, we identified three VAMP72 members with distinctive structural characteristics, whose N-terminal extensions contain consensus sequences for N-myristoylation. These results suggest that M. polymorpha has acquired unique membrane trafficking pathways associated with newly acquired machinery components during evolution.

A nitrogen source-dependent inducible and repressible gene expression system in the red alga Cyanidioschyzon merolae.

The unicellular red alga Cyanidioschyzon merolae is a model organism for studying the basic biology of photosynthetic organisms. The C. merolae cell is composed of an extremely simple set of organelles. The genome is completely sequenced. Gene targeting and a heat-shock inducible gene expression system has been recently established. However, a conditional gene knockdown system has not been established, which is required for the examination of function of genes that are essential to cell viability and primary mutant defects. In the current study, we first evaluated the expression of a transgene from two chromosomal neutral loci located in the intergenic region between CMD184C and CMD185C, and a region upstream of the URA5.3 gene. There was no significant difference in expression between them and this result suggests that both may be used as neutral loci. We then designed an inducible and repressible gene expression by using promoters of nitrate-assimilation genes. The expression of nitrate-assimilation genes such as NR (nitrate reductase), NIR (nitrite reductase), and NRT (the nitrate/nitrite transporter) are reversibly regulated by their dependence on nitrogen sources. We constructed stable strains in which a cassette containing the NR, NIR, or NRT promoter and sfGFP gene was inserted in a region upstream of URA5.3 and examined the efficacy of the promoters. The NR, NIR, and NRT promoters were constitutively activated in the nitrate medium, whereas their activities were extremely low in presence of ammonium. The activation of each promoter was immediately inhibited within a period of 1 h by the addition of ammonium. Thus, a conditional knockdown system in C. merolae was successfully established. The activity varies among the promoters, and each is selectable according to the expression level of a target gene estimated by RNA-sequencing. This method is applicable to defects in genes of interest in photosynthetic organism.

Target of rapamycin (TOR) plays a critical role in triacylglycerol accumulation in microalgae.

Most microalgae produce triacylglycerol (TAG) under stress conditions such as nitrogen depletion, but the underlying molecular mechanism remains unclear. In this study, we focused on the role of target of rapamycin (TOR) in TAG accumulation. TOR is a serine/threonine protein kinase that is highly conserved and plays pivotal roles in nitrogen and other signaling pathways in eukaryotes. We previously constructed a rapamycin-susceptible Cyanidioschyzon merolae, a unicellular red alga, by expressing yeast FKBP12 protein to evaluate the results of TOR inhibition (Imamura et al. in Biochem Biophys Res Commun 439:264-269, 2013). By using this strain, we here report that rapamycin-induced TOR inhibition results in accumulation of cytoplasmic lipid droplets containing TAG. Transcripts for TAG synthesis-related genes, such as glycerol-3-phosphate acyltransferase and acyl-CoA:diacylglycerol acyltransferase (DGAT), were increased by rapamycin treatment. We also found that fatty acid synthase-dependent de novo fatty acid synthesis was required for the accumulation of lipid droplets. Induction of TAG and up-regulation of DGAT gene expression by rapamycin were similarly observed in the unicellular green alga, Chlamydomonas reinhardtii. These results suggest the general involvement of TOR signaling in TAG accumulation in divergent microalgae.

Expression of Cyanobacterial Acyl-ACP Reductase Elevates the Triacylglycerol Level in the Red Alga Cyanidioschyzon merolae.

Nitrogen starvation is known to induce the accumulation of triacylglycerol (TAG) in many microalgae, and potential use of microalgae as a source of biofuel has been explored. However, nitrogen starvation also stops cellular growth. The expression of cyanobacterial acyl-acyl carrier protein (ACP) reductase in the unicellular red alga Cyanidioschyzon merolae chloroplasts resulted in an accumulation of TAG, which led to an increase in the number and size of lipid droplets while maintaining cellular growth. Transcriptome and metabolome analyses showed that the expression of acyl-ACP reductase altered the activities of several metabolic pathways. The activities of enzymes involved in fatty acid synthesis in chloroplasts, such as acetyl-CoA carboxylase and pyruvate dehydrogenase, were up-regulated, while pyruvate decarboxylation in mitochondria and the subsequent consumption of acetyl-CoA by the tricarboxylic acid (TCA) cycle were down-regulated. Aldehyde dehydrogenase, which oxidizes fatty aldehydes to fatty acids, was also up-regulated in the acyl-ACP reductase expresser. This activation was required for the lipid droplet accumulation and metabolic changes observed in the acyl-ACP reductase expresser. Nitrogen starvation also resulted in lipid droplet accumulation in C. merolae, while cell growth ceased as in the case of other algal species. The metabolic changes that occur upon the expression of acyl-ACP reductase are quite different from those caused by nitrogen starvation. Therefore, there should be a method for further increasing the storage lipid level while still maintaining cell growth that is different from the metabolic response to nitrogen starvation.

The nuclear-encoded sigma factor SIG4 directly activates transcription of chloroplast psbA and ycf17 genes in the unicellular red alga Cyanidioschyzon merolae.

The plant organelle chloroplast originated from the endosymbiosis of a cyanobacterial-like photosynthetic bacterium, and still retains its own genome derived from this ancestor. We have been focusing on a unicellular red alga, Cyanidioschyzon merolae, as a model photosynthetic eukaryote. In this study, we analyzed the transcriptional specificity of SIG4, which is one of four nuclear-encoded chloroplast RNA polymerase sigma factors in this alga. Accumulation of the SIG4 protein was observed in response to nitrogen depletion or high light conditions. By comparing the chloroplast transcriptomes under nitrogen depletion and SIG4-overexpressing conditions, we identified several candidate genes as SIG4 targets. Together with the results of chromatin immunoprecipitation analysis, the promoters of the psbA (encoding the D1 protein of the photosystem II reaction center) and ycf17 (encoding a protein of the early light-inducible protein family) genes were shown to be direct activation targets. The phycobilisome (PBS) CpcB protein was decreased by SIG4 overexpression, which suggests the negative involvement of SIG4 in PBS accumulation.

FtsZ-less prokaryotic cell division as well as FtsZ- and dynamin-less chloroplast and non-photosynthetic plastid division.

The chloroplast division machinery is a mixture of a stromal FtsZ-based complex descended from a cyanobacterial ancestor of chloroplasts and a cytosolic dynamin-related protein (DRP) 5B-based complex derived from the eukaryotic host. Molecular genetic studies have shown that each component of the division machinery is normally essential for normal chloroplast division. However, several exceptions have been found. In the absence of the FtsZ ring, non-photosynthetic plastids are able to proliferate, likely by elongation and budding. Depletion of DRP5B impairs, but does not stop chloroplast division. Chloroplasts in glaucophytes, which possesses a peptidoglycan (PG) layer, divide without DRP5B. Certain parasitic eukaryotes possess non-photosynthetic plastids of secondary endosymbiotic origin, but neither FtsZ nor DRP5B is encoded in their genomes. Elucidation of the FtsZ- and/or DRP5B-less chloroplast division mechanism will lead to a better understanding of the function and evolution of the chloroplast division machinery and the finding of the as-yet-unknown mechanism that is likely involved in chloroplast division. Recent studies have shown that FtsZ was lost from a variety of prokaryotes, many of which lost PG by regressive evolution. In addition, even some of the FtsZ-bearing bacteria are able to divide when FtsZ and PG are depleted experimentally. In some cases, alternative mechanisms for cell division, such as budding by an increase of the cell surface-to-volume ratio, are proposed. Although PG is believed to have been lost from chloroplasts other than in glaucophytes, there is some indirect evidence for the existence of PG in chloroplasts. Such information is also useful for understanding how non-photosynthetic plastids are able to divide in FtsZ-depleted cells and the reason for the retention of FtsZ in chloroplast division. Here we summarize information to facilitate analyses of FtsZ- and/or DRP5B-less chloroplast and non-photosynthetic plastid division.

Microtubule stability affects the unique motility of F-actin in Marchantia polymorpha.

Actin microfilaments play crucial roles in diverse plant functions. Some specific cellular processes require interaction between F-actin and microtubules, and it is believed that there are direct or indirect connections between F-actin and microtubules. We previously reported that actin microfilaments exhibit unique dynamic motility in cells of the liverwort, Marchantia polymorpha; the relevance of this activity to microtubules has not been explored. To examine whether the dynamics of F-actin in M. polymorpha were somehow regulated by microtubules, we investigated the effects of stabilization or destabilization of microtubules on dynamics of actin bundles, which were visualized by Lifeact-Venus. To our surprise, both stabilization and destabilization of microtubules exerted similar effects on F-actin motility; apparent sliding movement of F-actin in M. polymorpha cells was accelerated by both oryzalin and paclitaxel, with the effect of paclitaxel more evident than that of oryzalin. Immunofluorescence staining revealed that some F-actin bundles were arrayed along with microtubules in M. polymorpha thallus cells. These results suggest that microtubules play regulatory roles in the unique F-actin dynamics in M. polymorpha.

Application of Lifeact reveals F-actin dynamics in Arabidopsis thaliana and the liverwort, Marchantia polymorpha.

Actin plays fundamental roles in a wide array of plant functions, including cell division, cytoplasmic streaming, cell morphogenesis and organelle motility. Imaging the actin cytoskeleton in living cells is a powerful methodology for studying these important phenomena. Several useful probes for live imaging of filamentous actin (F-actin) have been developed, but new versatile probes are still needed. Here, we report the application of a new probe called Lifeact for visualizing F-actin in plant cells. Lifeact is a short peptide comprising 17 amino acids that was derived from yeast Abp140p. We used a Lifeact-Venus fusion protein for staining F-actin in Arabidopsis thaliana and were able to observe dynamic rearrangements of the actin meshwork in root hair cells. We also used Lifeact-Venus to visualize the actin cytoskeleton in the liverwort Marchantia polymorpha; this revealed unique and dynamic F-actin motility in liverwort cells. Our results suggest that Lifeact could be a useful tool for studying the actin cytoskeleton in a wide range of plant lineages.