Cytokinin and ALOG proteins regulate pluripotent stem cell identity in the moss Physcomitrium patens.

The shoot apical meristem (SAM) contains pluripotent stem cells that produce all the aerial parts of the plant. Stem cells undergo asymmetric cell divisions to self-renew and to produce differentiating cells. Our research focused on unraveling the mechanisms governing the specification of these two distinct cell fates following the stem cell division. For this purpose, we used the model organism Physcomitrium patens, which features a singular pluripotent stem cell known as the gametophore apical cell. We show that the activity of cytokinins, critical stem cell regulators, is restricted to the gametophore apical cell due to the specific localization of PpLOG, the enzyme responsible for cytokinin activation. In turn, PpTAW, which promotes differentiating cell identity of the merophyte, is excluded from the gametophore apical cell by the action of cytokinins. We propose a cytokinin-based model for the establishment of asymmetry in the pluripotent stem cell division.

Using Spinning Disk Microscopy to Observe the Mitotic and Cytokinetic Apparatus in Physcomitrium patens.

Protonemata of the moss Physcomitrium patens are ideal structures in which to observe cytoskeletal organization and dynamics. Special care is needed to prepare P. patens cultures for high-resolution microscopy. Here, we describe methods for spinning disk microscopy of dividing P. patens cells expressing sGFP-tubulin and H2B-mCherry, including detailed methods for culturing P. patens.

GRAS transcription factors regulate cell division planes in moss overriding the default rule.

Plant cells are surrounded by a cell wall and do not migrate, which makes the regulation of cell division orientation crucial for development. Regulatory mechanisms controlling cell division orientation may have contributed to the evolution of body organization in land plants. The GRAS family of transcription factors was transferred horizontally from soil bacteria to an algal common ancestor of land plants. SHORTROOT (SHR) and SCARECROW (SCR) genes in this family regulate formative periclinal cell divisions in the roots of flowering plants, but their roles in nonflowering plants and their evolution have not been studied in relation to body organization. Here, we show that SHR cell autonomously inhibits formative periclinal cell divisions indispensable for leaf vein formation in the moss Physcomitrium patens, and SHR expression is positively and negatively regulated by SCR and the GRAS member LATERAL SUPPRESSOR, respectively. While precursor cells of a leaf vein lacking SHR usually follow the geometry rule of dividing along the division plane with the minimum surface area, SHR overrides this rule and forces cells to divide nonpericlinally. Together, these results imply that these bacterially derived GRAS transcription factors were involved in the establishment of the genetic regulatory networks modulating cell division orientation in the common ancestor of land plants and were later adapted to function in flowering plant and moss lineages for their specific body organizations.

Molecular and physiological responses to desiccation indicate the abscisic acid pathway is conserved in the peat moss, Sphagnum.

Mosses of the genus Sphagnum are the main components of peatlands, a major carbon-storing ecosystem. Changes in precipitation patterns are predicted to affect water relations in this ecosystem, but the effect of desiccation on the physiological and molecular processes in Sphagnum is still largely unexplored. Here we show that different Sphagnum species have differential physiological and molecular responses to desiccation but, surprisingly, this is not directly correlated with their position in relation to the water table. In addition, the expression of drought responsive genes is increased upon water withdrawal in all species. This increase in gene expression is accompanied by an increase in abscisic acid (ABA), supporting a role for ABA during desiccation responses in Sphagnum. Not only do ABA levels increase upon desiccation, but Sphagnum plants pre-treated with ABA display increased tolerance to desiccation, suggesting that ABA levels play a functional role in the response. In addition, many of the ABA signalling components are present in Sphagnum and we demonstrate, by complementation in Physcomitrium patens, that Sphagnum ABI3 is functionally conserved. The data presented here, therefore, support a conserved role for ABA in desiccation responses in Sphagnum.

A PSTAIRE-type cyclin-dependent kinase controls light responses in land plants.

Light is a critical signal perceived by plants to adapt their growth rate and direction. Although many signaling components have been studied, how plants respond to constantly fluctuating light remains underexplored. Here, we showed that in the moss Physcomitrium (Physcomitrella) patens, the PSTAIRE-type cyclin-dependent kinase PpCDKA is dispensable for growth. Instead, PpCDKA and its homolog in Arabidopsis thaliana control light-induced tropisms and chloroplast movements by probably influencing the cytoskeleton organization independently of the cell cycle. In addition, lower PpCDKA kinase activity was required to elicit light responses relative to cell cycle regulation. Thus, our study suggests that plant CDKAs may have been co-opted to control multiple light responses, and owing to the bistable switch properties of PSTAIRE-type CDKs, the noncanonical functions are widely conserved for eukaryotic environmental adaptation.

How plants grow under gravity conditions besides 1 g: perspectives from hypergravity and space experiments that employ bryophytes as a model organism.

Plants have evolved and grown under the selection pressure of gravitational force at 1 g on Earth. In response to this selection pressure, plants have acquired gravitropism to sense gravity and change their growth direction. In addition, plants also adjust their morphogenesis in response to different gravitational forces in a phenomenon known as gravity resistance. However, the gravity resistance phenomenon in plants is poorly understood due to the prevalence of 1 g gravitational force on Earth: not only it is difficult to culture plants at gravity > 1 g(hypergravity) for a long period of time but it is also impossible to create a < 1 genvironment (µg, micro g) on Earth without specialized facilities. Despite these technical challenges, it is important to understand how plants grow in different gravity conditions in order to understand land plant adaptation to the 1 g environment or for outer space exploration. To address this, we have developed a centrifugal device for a prolonged duration of plant culture in hypergravity conditions, and a project to grow plants under the µg environment in the International Space Station is also underway. Our plant material of choice is Physcomitrium (Physcomitrella) patens, one of the pioneer plants on land and a model bryophyte often used in plant biology. In this review, we summarize our latest findings regarding P. patens growth response to hypergravity, with reference to our on-going "Space moss" project. In our ground-based hypergravity experiments, we analyzed the morphological and physiological changes and found unexpected increments of chloroplast size and photosynthesis rate, which might underlie the enhancement of growth and increase in the number of gametophores and rhizoids. We further discussed our approaches at the cellular level and compare the gravity resistance in mosses and that in angiosperms. Finally, we highlight the advantages and perspectives from the space experiments and conclude that research with bryophytes is beneficial to comprehensively and precisely understand gravitational responses in plants.

Establishment of a Live-Imaging Analysis for Polarized Growth of Conchocelis in the Multicellular Red Alga Neopyropia yezoensis.

A wide range of tip-growing cells in plants display polarized cell growth, which is an essential cellular process for the form and function of individual cells. Understanding of the regulatory mechanisms underlying tip growth in terrestrial plants has improved. Cellular processes involved in tip growth have also been investigated in some algae species that form filamentous cells, but their regulatory mechanisms remain unclear. In the macro red alga Neopyropia yezoensis, for which genome information has recently been released, the conchocelis apical cell exhibits tip growth and forms a filamentous structure. Here, we report a live-imaging technique using high-resolution microscopy to analyze the tip growth and cell division of N. yezoensis conchocelis. This imaging analysis addressed tip growth dynamics and cell division in conchocelis apical cells. The directionality and tip growth expansion were disrupted by the application of cytoskeletal drugs, suggesting the involvement of microtubules (MTs) and actin filaments (AFs) in these processes. A growing apical cell mostly contained a single chloroplast that moved toward the expanding part of the apical cell. Drug application also inhibited chloroplast movement, implying that the movement may be dependent on the cytoskeleton. The study determined that live-imaging analysis is a versatile approach for exploring the dynamics of tip growth and cell division in N. yezoensis conchocelis, which provides insights into the regulatory mechanisms underlying cellular growth in multicellular red algae.

Physcomitrella STEMIN transcription factor induces stem cell formation with epigenetic reprogramming.

Epigenetic modifications, including histone modifications, stabilize cell-specific gene expression programmes to maintain cell identities in both metazoans and land plants1-3. Notwithstanding the existence of these stable cell states, in land plants, stem cells are formed from differentiated cells during post-embryonic development and regeneration4-6, indicating that land plants have an intrinsic ability to regulate epigenetic memory to initiate a new gene regulatory network. However, it is less well understood how epigenetic modifications are locally regulated to influence the specific genes necessary for cellular changes without affecting other genes in a genome. In this study, we found that ectopic induction of the AP2/ERF transcription factor STEMIN1 in leaf cells of the moss Physcomitrella patens decreases a repressive chromatin mark, histone H3 lysine 27 trimethylation (H3K27me3), on its direct target genes before cell division, resulting in the conversion of leaf cells to chloronema apical stem cells. STEMIN1 and its homologues positively regulate the formation of secondary chloronema apical stem cells from chloronema cells during development. Our results suggest that STEMIN1 functions within an intrinsic mechanism underlying local H3K27me3 reprogramming to initiate stem cell formation.

Physcomitrella MADS-box genes regulate water supply and sperm movement for fertilization.

MIKC classic (MIKCC)-type MADS-box genes encode transcription factors that function in various developmental processes, including angiosperm floral organ identity. Phylogenetic analyses of the MIKCC-type MADS-box family, including genes from non-flowering plants, suggest that the increased numbers of these genes in flowering plants is related to their functional divergence; however, their precise functions in non-flowering plants and their evolution throughout land plant diversification are unknown. Here, we show that MIKCC-type MADS-box genes in the moss Physcomitrella patens function in two ways to enable fertilization. Analyses of protein localization, deletion mutants and overexpression lines of all six genes indicate that three MIKCC-type MADS-box genes redundantly regulate cell division and growth in the stems for appropriate external water conduction, as well as the formation of sperm with motile flagella. The former function appears to be maintained in the flowering plant lineage, while the latter was lost in accordance with the loss of sperm.

The Mitotic Function of Augmin Is Dependent on Its Microtubule-Associated Protein Subunit EDE1 in Arabidopsis thaliana.

The augmin complex plays an essential role in microtubule (MT)-dependent MT nucleation by recruiting the γ-tubulin complex to MT walls to generate new MTs [1]. The complex contains eight subunits (designated AUG) including AUG8, which is an MT-associated protein (MAP). When this complex is isolated from etiolated seedlings consisting of primarily interphase cells in Arabidopsis thaliana, AUG8 is an integral component [2]. EDE1 (Endosperm DEfective 1) is homologous to AUG8 [3]. Here, we demonstrate that EDE1, but not AUG8, is associated with acentrosomal spindle and phragmoplast MT arrays in patterns indistinguishable from those of the AUG1-7 subunits and the γ-tubulin complex proteins (GCPs) that exhibit biased localization toward MT minus ends. Consistent with this colocalization, EDE1 directly interacts with AUG6 in vivo. Moreover, a partial loss-of-function mutation, ede1-1, compromises the localization of augmin and γ-tubulin on the spindle and phragmoplast MT arrays and leads to serious distortions in spindle MT remodeling during mitosis. However, mitosis continues even when kinetochore fibers are not obviously discernable, and cytokinesis takes place following the formation of elongated bipolar phragmoplast MT arrays in the mutant. Hence, we conclude that the mitotic function of augmin is dependent on its MAP subunit EDE1, which cannot be replaced by AUG8, and that the cell-cycle-dependent function of augmin can be differentially regulated by employing distinct MAP subunits. Our results also illustrate that plant cells can respond flexibly to serious challenges of compromised MT-dependent MT nucleation to complete mitosis and cytokinesis.

Genome of the pitcher plant Cephalotus reveals genetic changes associated with carnivory.

Carnivorous plants exploit animals as a nutritional source and have inspired long-standing questions about the origin and evolution of carnivory-related traits. To investigate the molecular bases of carnivory, we sequenced the genome of the heterophyllous pitcher plant Cephalotus follicularis, in which we succeeded in regulating the developmental switch between carnivorous and non-carnivorous leaves. Transcriptome comparison of the two leaf types and gene repertoire analysis identified genetic changes associated with prey attraction, capture, digestion and nutrient absorption. Analysis of digestive fluid proteins from C. follicularis and three other carnivorous plants with independent carnivorous origins revealed repeated co-options of stress-responsive protein lineages coupled with convergent amino acid substitutions to acquire digestive physiology. These results imply constraints on the available routes to evolve plant carnivory.

Cells reprogramming to stem cells inhibit the reprogramming of adjacent cells in the moss Physcomitrella patens.

Under certain circumstances differentiated cells can be reprogrammed to form stem cells in land plants, but only a portion of the cells reprograms successfully. A long-distance inhibitory signal from reprogrammed cells to surrounding cells has been reported in some ferns. Here we show the existence of anisotropic inhibitory signal to regulate stem cell formation in the moss Physcomitrella patens. When single cells were isolated from a gametophore leaf, over 90% of them were reprogrammed to stem cells with characteristic nuclear expansion. By contrast, when two adjacent cells were isolated, the nuclei of both cells expanded, but successful reprogramming of both cells occurred only in approximately one fifth of the pairs. When three aligned cells were isolated, the reprogramming rate of both edge cells decreased with a living middle cell but did not with a dead middle cell. Furthermore, unequal conversion into stem cells was more prominent in cell pairs aligned parallel to the proximal-distal leaf axis than in those perpendicular to the axis. This study gives an insight into the role of the inhibitory signal in development and evolution as well as the efficient stem cell induction from differentiated cells.

A Lin28 homologue reprograms differentiated cells to stem cells in the moss Physcomitrella patens.

Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3'-untranslated region (3'-UTR). Removal of the 3'-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28.

WOX13-like genes are required for reprogramming of leaf and protoplast cells into stem cells in the moss Physcomitrella patens.

Many differentiated plant cells can dedifferentiate into stem cells, reflecting the remarkable developmental plasticity of plants. In the moss Physcomitrella patens, cells at the wound margin of detached leaves become reprogrammed into stem cells. Here, we report that two paralogous P. patens WUSCHEL-related homeobox 13-like (PpWOX13L) genes, homologs of stem cell regulators in flowering plants, are transiently upregulated and required for the initiation of cell growth during stem cell formation. Concordantly, Δppwox13l deletion mutants fail to upregulate genes encoding homologs of cell wall loosening factors during this process. During the moss life cycle, most of the Δppwox13l mutant zygotes fail to expand and initiate an apical stem cell to form the embryo. Our data show that PpWOX13L genes are required for the initiation of cell growth specifically during stem cell formation, in analogy to WOX stem cell functions in seed plants, but using a different cellular mechanism.

Contribution of NAC transcription factors to plant adaptation to land.

The development of cells specialized for water conduction or support is a striking innovation of plants that has enabled them to colonize land. The NAC transcription factors regulate the differentiation of these cells in vascular plants. However, the path by which plants with these cells have evolved from their nonvascular ancestors is unclear. We investigated genes of the moss Physcomitrella patens that encode NAC proteins. Loss-of-function mutants formed abnormal water-conducting and supporting cells, as well as malformed sporophyte cells, and overexpression induced ectopic differentiation of water-conducting-like cells. Our results show conservation of transcriptional regulation and cellular function between moss and Arabidopsis thaliana water-conducting cells. The conserved genetic basis suggests roles for NAC proteins in the adaptation of plants to land.

Kinesins have a dual function in organizing microtubules during both tip growth and cytokinesis in Physcomitrella patens.

Microtubules (MTs) play a crucial role in the anisotropic deposition of cell wall material, thereby affecting the direction of growth. A wide range of tip-growing cells display highly polarized cell growth, and MTs have been implicated in regulating directionality and expansion. However, the molecular machinery underlying MT dynamics in tip-growing plant cells remains unclear. Here, we show that highly dynamic MT bundles form cyclically in the polarized expansion zone of the moss Physcomitrella patens caulonemal cells through the coalescence of growing MT plus ends. Furthermore, the plant-specific kinesins (KINID1) that are is essential for the proper MT organization at cytokinesis also regulate the turnover of the tip MT bundles as well as the directionality and rate of cell growth. The plus ends of MTs grow toward the expansion zone, and KINID1 is necessary for the stability of a single coherent focus of MTs in the center of the zone, whose formation coincides with the accumulation of KINID1. We propose that KINID-dependent MT bundling is essential for the correct directionality of growth as well as for promoting growth per se. Our findings indicate that two localized cell wall deposition processes, tip growth and cytokinesis, previously believed to be functionally and evolutionarily distinct, share common and plant-specific MT regulatory components.

Development of an Agrobacterium-mediated stable transformation method for the sensitive plant Mimosa pudica.

The sensitive plant Mimosa pudica has long attracted the interest of researchers due to its spectacular leaf movements in response to touch or other external stimuli. Although various aspects of this seismonastic movement have been elucidated by histological, physiological, biochemical, and behavioral approaches, the lack of reverse genetic tools has hampered the investigation of molecular mechanisms involved in these processes. To overcome this obstacle, we developed an efficient genetic transformation method for M. pudica mediated by Agrobacterium tumefaciens (Agrobacterium). We found that the cotyledonary node explant is suitable for Agrobacterium-mediated transformation because of its high frequency of shoot formation, which was most efficiently induced on medium containing 0.5 µg/ml of a synthetic cytokinin, 6-benzylaminopurine (BAP). Transformation efficiency of cotyledonary node cells was improved from almost 0 to 30.8 positive signals arising from the intron-sGFP reporter gene by using Agrobacterium carrying a super-binary vector pSB111 and stabilizing the pH of the co-cultivation medium with 2-(N-morpholino)ethanesulfonic acid (MES) buffer. Furthermore, treatment of the explants with the detergent Silwet L-77 prior to co-cultivation led to a two-fold increase in the number of transformed shoot buds. Rooting of the regenerated shoots was efficiently induced by cultivation on irrigated vermiculite. The entire procedure for generating transgenic plants achieved a transformation frequency of 18.8%, which is comparable to frequencies obtained for other recalcitrant legumes, such as soybean (Glycine max) and pea (Pisum sativum). The transgene was stably integrated into the host genome and was inherited across generations, without affecting the seismonastic or nyctinastic movements of the plants. This transformation method thus provides an effective genetic tool for studying genes involved in M. pudica movements.

System for stable ß-estradiol-inducible gene expression in the moss Physcomitrella patens.

Inducible transgene expression provides a useful tool to analyze gene function. The moss Physcomitrellapatens is a model basal land plant with well-developed research tools, including a high efficiency of gene targeting and substantial genomics resources. However, current systems for controlled transgene expression remain limited. Here we report the development of an estrogen receptor mediated inducible gene expression system, based on the system used in flowering plants. After identifying the appropriate promoters to drive the chimeric transducer, we succeeded in inducing transcription over 1,000-fold after 24 h incubation with ß-estradiol. The P. patens system was also effective for high-level long-term induction of gene expression; transcript levels of the activated gene were maintained for at least seven days on medium containing ß-estradiol. We also established two potentially neutral targeting sites and a set of vectors for reproducible expression of two transgenes. This ß-estradiol-dependent system will be useful to test genes individually or in combination, allowing stable, inducible transgenic expression in P. patens.

KNOX2 genes regulate the haploid-to-diploid morphological transition in land plants.

Unlike animals, land plants undergo an alternation of generations, producing multicellular bodies in both haploid (1n: gametophyte) and diploid (2n: sporophyte) generations. Plant body plans in each generation are regulated by distinct developmental programs initiated at either meiosis or fertilization, respectively. In mosses, the haploid gametophyte generation is dominant, whereas in vascular plants-including ferns, gymnosperms, and angiosperms-the diploid sporophyte generation is dominant. Deletion of the class 2 KNOTTED1-LIKE HOMEOBOX (KNOX2) transcription factors in the moss Physcomitrella patens results in the development of gametophyte bodies from diploid embryos without meiosis. Thus, KNOX2 acts to prevent the haploid-specific body plan from developing in the diploid plant body, indicating a critical role for the evolution of KNOX2 in establishing an alternation of generations in land plants.

The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.