Protonemal Cells Research Articles

Bending of protonema cells in a plastid glycolate/glycerate transporter knockout line of Physcomitrella patens.

Arabidopsis LrgB (synonym PLGG1) is a plastid glycolate/glycerate transporter associated with recycling of 2-phosphoglycolate generated via the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). We isolated two homologous genes (PpLrgB1 and B2) from the moss Physcomitrella patens. Phylogenetic tree analysis showed that PpLrgB1 was monophyletic with LrgB proteins of land plants, whereas PpLrgB2 was divergent from the green plant lineage. Experiments with PpLrgB–GFP fusion proteins suggested that both PpLrgB1 and B2 proteins were located in chloroplasts. We generated PpLrgB single (∆B1 and ∆B2) and double (∆B1/∆B2)-knockout lines using gene targeting of P. patens. The ∆B1 plants showed decreases in growth and photosynthetic activity, and their protonema cells were bent and accumulated glycolate. However, because ∆B2 and ∆B1/∆B2 plants showed no obvious phenotypic change relative to the wild-type or ∆B1 plants, respectively, the function of PpLrgB2 remains unclear. Arabidopsis LrgB could complement the ∆B1 phenotype, suggesting that the function of PpLrgB1 is the same as that of AtLrgB. When ∆B1 was grown under high-CO2 conditions, all novel phenotypes were suppressed. Moreover, protonema cells of wild-type plants exhibited a bending phenotype when cultured on media containing glycolate or glycerate, suggesting that accumulation of photorespiratory metabolites caused P. patens cells to bend.

The nitric oxide production in the moss Physcomitrella patens is mediated by nitrate reductase.

During the last 20 years multiple roles of the nitric oxide gas (•NO) have been uncovered in plant growth, development and many physiological processes. In seed plants the enzymatic synthesis of •NO is mediated by a nitric oxide synthase (NOS)-like activity performed by a still unknown enzyme(s) and nitrate reductase (NR). In green algae the •NO production has been linked only to NR activity, although a NOS gene was reported for Ostreococcus tauri and O. lucimarinus, no other Viridiplantae species has such gene. As there is no information about •NO synthesis neither for non-vascular plants nor for non-seed vascular plants, the interesting question regarding the evolution of the enzymatic •NO production systems during land plant natural history remains open. To address this issue the endogenous •NO production by protonema was demonstrated using Electron Paramagnetic Resonance (EPR). The •NO signal was almost eliminated in plants treated with sodium tungstate, which also reduced the NR activity, demonstrating that in P. patens NR activity is the main source for •NO production. The analysis with confocal laser scanning microscopy (CLSM) confirmed endogenous NO production and showed that •NO signal is accumulated in the cytoplasm of protonema cells. The results presented here show for the first time the •NO production in a non-vascular plant and demonstrate that the NR-dependent enzymatic synthesis of •NO is common for embryophytes and green algae.

Fern spore germination in response to environmental factors

Fern spore germination gives rise to the rhizoid and protonemal cell through asymmetric cell division, and then develops into a gametophyte. Spore germination is also a representative single-cell model for the investigation of nuclear polar movement, asymmetrical cell division, polarity establishment and rhizoid tip-growth. These processes are affected by various environmental factors, such as light, gravity, phytohormones, metal ions, and temperature. Here, we present a catalog of spore germination in response to different environmental factors. They are as follows: (1) Representative modes of light affecting spore germination from different fern species include red light-stimulated and far red light-inhibited spore germination, far red light-uninhibited spore germination, blue light-inhibited spore germination, and spore germination in the dark. The optimal light intensity and illumination time for spore germination are different among various fern species. Light response upon spore germination is initiated from the cell mitosis that regulated by phytochromes (PHYs) and cryptochromes (CRYs). AcPHY2, AcCRY3 and/or AcCRY4 are hypothesized to be involved in spore germination; (2) Gravity and calcium are crucial to early nuclear movement and polarity establishment of spores; (3) Gibberellin and antheridiogen can initiate and promote spore germination in many species, but abscisic acid, jasmonic acid, and ethylene pose only minor effects; (4) Spores can obtain the maximal germination rate in their favorable growth medium. Moreover, metal ions, pH, and spore density in the culture medium also affect spore germination; (5) Most fern spores germinate at 25°C, and an optimal CO2 concentration is necessary for spore germination of certain fern plants. These provide valuable information for understanding fern spore germination in response to environmental factors.

Cytoplasmic nucleation and atypical branching nucleation generate endoplasmic microtubules in Physcomitrella patens.

The mechanism underlying microtubule (MT) generation in plants has been primarily studied using the cortical MT array, in which fixed-angled branching nucleation and katanin-dependent MT severing predominate. However, little is known about MT generation in the endoplasm. Here, we explored the mechanism of endoplasmic MT generation in protonemal cells of Physcomitrella patens. We developed an assay that utilizes flow cell and oblique illumination fluorescence microscopy, which allowed visualization and quantification of individual MT dynamics. MT severing was infrequently observed, and disruption of katanin did not severely affect MT generation. Branching nucleation was observed, but it showed markedly variable branch angles and was occasionally accompanied by the transport of nucleated MTs. Cytoplasmic nucleation at seemingly random locations was most frequently observed and predominated when depolymerized MTs were regrown. The MT nucleator γ-tubulin was detected at the majority of the nucleation sites, at which a single MT was generated in random directions. When γ-tubulin was knocked down, MT generation was significantly delayed in the regrowth assay. However, nucleation occurred at a normal frequency in steady state, suggesting the presence of a γ-tubulin-independent backup mechanism. Thus, endoplasmic MTs in this cell type are generated in a less ordered manner, showing a broader spectrum of nucleation mechanisms in plants.

RNAi screening identifies the armadillo repeat-containing kinesins responsible for microtubule-dependent nuclear positioning in Physcomitrella patens.

Proper positioning of the nucleus is critical for the functioning of various cells. Actin and myosin have been shown to be crucial for the localization of the nucleus in plant cells, whereas microtubule (MT)-based mechanisms are commonly utilized in animal and fungal cells. In this study, we combined live cell microscopy with RNA interference (RNAi) screening or drug treatment and showed that MTs and a plant-specific motor protein, armadillo repeat-containing kinesin (kinesin-ARK), are required for nuclear positioning in the moss Physcomitrella patens. In tip-growing protonemal apical cells, the nucleus was translocated to the center of the cell after cell division in an MT-dependent manner. When kinesin-ARKs were knocked down using RNAi, the initial movement of the nucleus towards the center took place normally; however, before reaching the center, the nucleus was moved back to the basal edge of the cell. In intact (control) cells, MT bundles that are associated with kinesin-ARKs were frequently observed around the moving nucleus. In contrast, such MT bundles were not identified after kinesin-ARK down-regulation. An in vitro MT gliding assay showed that kinesin-ARK is a plus-end-directed motor protein. These results indicate that MTs and the MT-based motor drive nuclear migration in the moss cells, thus showing a conservation of the mechanism underlying nuclear localization among plant, animal and fungal cells.

NACK kinesin is required for metaphase chromosome alignment and cytokinesis in the moss Physcomitrella patens.

The NACK kinesins (NACK1, NACK2 in tobacco and AtNACK1/HINKEL, AtNACK2/STUD/TETRASPORE in Arabidopsis), members of a plant-specific kinesin-7 family, are required for cytokinesis. Previous studies using tobacco and Arabidopsis cells showed that NACK1 and AtNACK1 at the phragmoplast midzone activate the MAP kinase cascade during the late M phase, which is critical for the cell plate formation. However, the loss-of-function phenotype has not been investigated in details in living cells and the molecular activity of this kinesin remains to be determined. Here, we report the mitotic roles and activity of the NACK kinesins in the moss Physcomitrella patens. When we simultaneously knocked down three PpNACKs by RNA-interference (RNAi) in protonemal cells, we observed a cytokinesis failure following a defect in phragmoplast expansion. In addition, misaligned chromosomes were frequently detected in the pre-anaphase spindle and the anaphase onset was significantly delayed, indicating that PpNACK also plays a role in pre-anaphase. Consistent with the appearance of early and late mitotic phenotypes, endogenous PpNACK was localised to the interpolar microtubule (MT) overlap from prometaphase through telophase. In vitro MT gliding assay and single motor motility assay showed that PpNACK-b is a processive, plus-end-directed motor, suggesting that PpNACK is capable of transporting cargoes along the spindle/phragmoplast MT. Our study using Physcomitrella patens demonstrated that PpNACK is an active motor protein and identified unexpected and conserved roles of PpNACK during the mitosis of P. patens.

Fine structure of kinetin-treated protonema and kinetin-induced gametophore buds in Funaria hygrometrica

Besides occasional hypertrophy of grana and disintegration of stroma thylakoids occurring in some chloroplasts, no significant changes were found in ultrastructure of typical protonema cells treated for six days with kinetin. On the other hand, the fine structure of cells in kinetin--induced gametophore buds differed much from that of the protonema cells and showed characteristics of cells of with high metabolic activity and high division rates. The results indicate that cytokinins enhance development and differentiation in the protonema by activating only some of its cells, whereas the others remain unchanged or show symptoms of destruction and ageing. This is supported by the fact that in the presence of chloramphenicol, which prevents bud induction, kinetin acts synergistically with the inhibitor in producing degeneration and destruction of chloroplasts.

The effect of various cytokinins and other factors on the protonemal celi divisions and the induction of gametophores in Ceratodon purpureus

The cytokinins specifically induce formation of gametophore buds in the protonema of <i>Ceratodon purpureus</i>. The responsein this species is less sensitive than in <i>Funaria hygrometrica</i>, but is independent of light. The cytokinins also stimulate protonemal cell divisions, this response, however, is not specific and affected by many other factors.

P025 Auxin polar transport regulates the tip growth of protonema cells in the moss Physcomitrella patens

P025 Auxin polar transport regulates the tip growth of protonema cells in the moss Physcomitrella patens

Studies on the activity of kinetin in cultures of Funaria hygrometrica. II. Effect of kinetin on isolated protonema cells

Studies on the activity of kinetin in cultures of Funaria hygrometrica. II. Effect of kinetin on isolated protonema cells

Morphogenetic Studies on Two Mosses, Bryum dichotomum and Entodon macropodus Grown In Vitro

Axenic cultures of two moss taxa viz. Bryum dichotomum Hedw., an acrocarpous moss and Entodon macropodus (Hedw.) Mull. Hal., a pleurocarpous moss were established by inoculating their spores into agar media. Germination, growth, development and comparative morphogenetic studies of these two growth forms were done. In B. dichotomum, protonemal buds developed on caulonema, while in E. macropodus, buds were produced on chloronema. Sub culturing of B. dichotomum gave rise to new individuals through chloronemal and caulonemal stage, while E. macropodus it directly regenerated into new gametophytes. In old culture under low moisture conditions, asexual diaspores namely, protonemal bulbils B. dichotomum and protonemal brood cells E. macropodus were formed.

Behaviour of repetitive non-coding DNA in response to heavy metal stress in the protonemata of Funaria hygrometrica

Studies on the leafy shoots of Funaria hygrometrica have shown that the genome reacts to heavy-metal stress with the selective amplification of specific (G+C)-rich highly repetitive/non-coding DNA sequences. These studies have also shown that, when the metal-induced stress ceases, almost all the stress-induced repetitive DNA is eliminated from the genome. In this study, in order to investigate whether this reaction of genome could be associated with the developmental stage of the target organism, we analysed the behaviour of repetitive non-coding DNA in response to metal-stress in the protonemata of F. hygrometrica. That is the juvenile stage of the gametophyte phase and that precedes the leafy shoot stage. In particular, we analysed the genome of chloronemal cells in plants treated with a lead–zinc mixture similar to that found in mines in Sardinia, Italy. Our results show that, in the chloronemal cells of protonema, metal-stress induces an amplification of (G+C)-rich repetitive/non-coding DNA sequences similar to that observed in the leafy-shoot cells. However, unlike that which occurs in the leafy-shoot stage, in the protonemal stage almost all of the stress-induced repetitive DNA remains within the nucleus also after the stimulus is removed. Some hypotheses are discussed.

Conserved function of Rho-related Rop/RAC GTPase signaling in regulation of cell polarity in Physcomitrella patens.

Cell polarity is fundamentally important to growth and development in higher plants, from pollen tubes to root hairs. Basal land plants (mosses and ferns) also have cell polarity, developing protonemal apical cells that show polar tip growth. Flowering plants have a distinct group of Rho GTPases that regulate polarity in polarized cell growth. Rop/RAC signaling module components have been identified in non-flowering plants, but their roles remain unclear. To understand the importance and evolution of Rop/RAC signaling in polarity regulation in land plants, we examined the functions of PpRop and PpRopGEF in protonemal apical cells of the moss Physcomitrella patens. Inducible overexpression of PpRop2 or PpRopGEF3 caused depolarized growth of tip-growing apical cells. PpRop2 overexpression also caused aberrant cross wall formation. Fluorescent protein-tagged PpRop2 localized to the plasma membrane, including the cross wall membrane, and fluorescent-tagged PpRopGEF3 showed polarized localization to the tip region in apical cells. Thus, our results suggest common functions of PpRop and PpRopGEF in the tip-growing apical cells and the importance of a conserved Rop/RAC signaling module in the control of cell polarity in land plants.

MICROTUBULE-ASSOCIATED PROTEIN65 Is Essential for Maintenance of Phragmoplast Bipolarity and Formation of the Cell Plate inPhyscomitrella patens

The phragmoplast, a plant-specific apparatus that mediates cytokinesis, mainly consists of microtubules (MTs) arranged in a bipolar fashion, such that their plus ends interdigitate at the equator. Membrane vesicles are thought to move along the MTs toward the equator and fuse to form the cell plate. Although several genes required for phragmoplast MT organization have been identified, the mechanisms that maintain the bipolarity of phragmoplasts remain poorly understood. Here, we show that engaging phragmoplast MTs in a bipolar fashion in protonemal cells of the moss Physcomitrella patens requires the conserved MT cross-linking protein MICROTUBULE-ASSOCIATED PROTEIN65 (MAP65). Simultaneous knockdown of the three MAP65s expressed in those cells severely compromised MT interdigitation at the phragmoplast equator after anaphase onset, resulting in the collapse of the phragmoplast in telophase. Cytokinetic vesicles initially localized to the anaphase midzone as normal but failed to further accumulate in the next several minutes, although the bipolarity of the MT array was preserved. Our data indicate that the presence of bipolar MT arrays is insufficient for vesicle accumulation at the equator and further suggest that MAP65-mediated MT interdigitation is a prerequisite for maintenance of bipolarity of the phragmoplast and accumulation and/or fusion of cell plate-destined vesicles at the equatorial plane.

AP2-type transcription factors determine stem cell identity in the moss Physcomitrella patens

Stem cells are formed at particular times and positions during the development of multicellular organisms. Whereas flowering plants form stem cells only in the sporophyte generation, non-seed plants form stem cells in both the sporophyte and gametophyte generations. Although the molecular mechanisms underlying stem cell formation in the sporophyte generation have been extensively studied, only a few transcription factors involved in the regulation of gametophyte stem cell formation have been reported. The moss Physcomitrella patens forms a hypha-like body (protonema) and a shoot-like body (gametophore) from a protonema apical cell and a gametophore apical cell, respectively. These apical cells have stem cell characteristics and are formed as side branches of differentiated protonema cells. Here, we show that four AP2-type transcription factors orthologous to Arabidopsis thaliana AINTEGUMENTA, PLETHORA and BABY BOOM (APB) are indispensable for the formation of gametophore apical cells from protonema cells. Quadruple disruption of all APB genes blocked gametophore formation, even in the presence of cytokinin, which enhances gametophore apical cell formation in the wild type. All APB genes were expressed in emerging gametophore apical cells, but not in protonema apical cells. Heat-shock induction of an APB4 transgene driven by a heat-shock promoter increased the number of gametophores. Expression of all APB genes was induced by auxin but not by cytokinin. Thus, the APB genes function synergistically with cytokinin signaling to determine the identity of the two types of stem cells.

Quantitative analysis of organelle distribution and dynamics in Physcomitrella patens protonemal cells.

BackgroundIn the last decade, the moss Physcomitrella patens has emerged as a powerful plant model system, amenable for genetic manipulations not possible in any other plant. This moss is particularly well suited for plant polarized cell growth studies, as in its protonemal phase, expansion is restricted to the tip of its cells. Based on pollen tube and root hair studies, it is well known that tip growth requires active secretion and high polarization of the cellular components. However, such information is still missing in Physcomitrella patens. To gain insight into the mechanisms underlying the participation of organelle organization in tip growth, it is essential to determine the distribution and the dynamics of the organelles in moss cells.ResultsWe used fluorescent protein fusions to visualize and track Golgi dictyosomes, mitochondria, and peroxisomes in live protonemal cells. We also visualized and tracked chloroplasts based on chlorophyll auto-fluorescence. We showed that in protonemata all four organelles are distributed in a gradient from the tip of the apical cell to the base of the sub-apical cell. For example, the density of Golgi dictyosomes is 4.7 and 3.4 times higher at the tip than at the base in caulonemata and chloronemata respectively. While Golgi stacks are concentrated at the extreme tip of the caulonemata, chloroplasts and peroxisomes are totally excluded. Interestingly, caulonemata, which grow faster than chloronemata, also contain significantly more Golgi dictyosomes and fewer chloroplasts than chloronemata. Moreover, the motility analysis revealed that organelles in protonemata move with low persistency and average instantaneous speeds ranging from 29 to 75 nm/s, which are at least three orders of magnitude slower than those of pollen tube or root hair organelles.ConclusionsTo our knowledge, this study reports the first quantitative analysis of organelles in Physcomitrella patens and will make possible comparisons of the distribution and dynamics of organelles from different tip growing plant cells, thus enhancing our understanding of the mechanisms of plant polarized cell growth.

Coarse-Grained Model of Cooperative Chloroplast Transport in Moss

Organelle motility is essential for the functioning of the eukaryotic cell. Actively modifying intracellular structures allows cells to change and adapt to different conditions. One of these cellular structures is the microtubule cytoskeleton, which is comprised of polarized filaments that function as tracks to transport cargo via molecular motors. Recent studies have revealed the importance of cooperative transport in living cells. In the moss Physcomitrella patens, reorganization of the chloroplasts is critical to adapt to changes in light quality and intensity. In this work, we performed detailed analysis of the transport of chloroplasts in protonemal moss cells, and showed that their transport is facilitated by molecular motors and the microtubule cytoskeleton. Our findings were recapitulated using coarse-grained modeling of this cargo transport over different microtubule network topologies. Our simulations include a detailed description of motor and microtubule dynamics, in the presence of thermal fluctuations. Our modeling sheds light into the molecular mechanisms at play in the photo-relocation response of chloroplasts in moss.

Staining of Protonemal Cells of Ceratodon Purpureus (Hedw.) Brid. for Better Highlighting Micronuclei in Bryophytes

The aim of this study is to develop a technique of visualization and staining of nuclei of protonemal cells of Ceratodon purpureus (Hedw.) Brid. in order to establish a test of genotoxicity (micronucleus assay). This technique is part of the development of methodology for environmental risk assessment. Bryophytes are often used as biological models in biomonitoring and ecotoxicology. For ecotoxicology studies, there are bioassays, including micronucleus assay. The genotoxicity assay is often practiced in higher plants; we wanted to expand its use to lower plants, like mosses and especially Ceratodon purpureus. To meet the criteria of micronucleus assay, the cells of protonemata of Ceratodon purpureus were selected as a model. The liquid culture of protonemata provides the quantity of biomass needed to achieve a bioassay. First, the development of a reproducible method for visualization and staining of the nuclei of protonemal cells was a necessary and prior step to the realization of micronucleus assay. So for this staining stage, the coloring agent used was the acetic orcein. It is the reference coloring agent for the micronucleus test on Vicia faba (AFNOR, 2004). With our assays of staining, we could develop a reproducible technique of staining of protonemal nuclei. Then, to attempt to visualize micronuclei, we used our method of staining after an exposure of protonemata to cadmium. The first test of staining of micronuclei following exposure of protonemata to Cd failed to demonstrate micronuclei in the conditions of the experiment. Further research must be conducted using other clastogenic agents to validate our results. Assays on micronuclei of cells of protonemata of Ceratodon purpureus are a first in scientific research. Its innovative aspect encourages further research.

Morphological Adaptations to Drought and Reproductive Strategy of the Moss Syntrichia caninervis in the Gurbantunggut Desert, China

The biological soil crusts (BSCs) in the Gurbantunggut Desert, the largest fixed and semi-fixed desert in China, feature moss-dominated BSCs, which play an indispensable role in sand fixation. Syntrichia caninervis Mitt. (S. caninervis) serves as one of the most common species in BSCs in the desert. In this study we examined the morphological structure of S. caninervis from leafy gametophyte to protonema using light and scanning electron microscopy (SEM). We also examined the relationships between the morphological structure of S. caninervis and environmental factors. We found that: (1) this moss species is commonly tufted on the sand surface, and its leaves are folded upwards and twisted around the stem under dry conditions; (2) the cells on both upper and lower leaf surfaces have C-shaped dark papillae, which may reflect sunlight to reduce the damage from high temperature; (3) the leaf costa is excurrent, forming an awn with forked teeth; and (4) the protonema cells are small and thickset with thick cell walls and the cytoplasm is highly concentrated with a small vacuole. In addition, we also found that the protonema cells always form pouches on the tip of the mother cells during the process of cell polarization. Our results suggest that S. caninervis has, through its life cycle, several morphological and structural characteristics to adapt to dry environmental conditions. These morphological features of S. caninervis may also be found in other deserts in the world due to the world-wide distribution of the species.

Spores before sporophytes: hypothesizing the origin of sporogenesis at the algal–plant transition

Fossil spores from mid-Ordovician deposits (475 million yr old) are the first indication of plants on land and predate megafossils of plants by 30-50 million yr. Sporopollenin-walled spores distinguish land plants from algae, which typically have heavy-walled zygotes that germinate via meiosis into motile or protonemal cells. All land plants are embryophytes with spores produced by the sporophyte generation. It is generally assumed that retention of the zygote and delay in meiosis led to matrotrophic embryo development and intercalation of the diploid sporophyte before spore production. However, new data on the cell biology of sporogenesis in extant bryophytes suggest that spores were produced directly from zygotes in protoembryophytes. The mechanism of wall transfer from zygote to meiospores was a three-phase heterochrony involving precocious initiation of cytokinesis, acceleration of meiosis, and concomitant delay in wall deposition. In bryophyte sporogenesis, cytokinesis is typically initiated in advance of meiosis, and quadrilobing of the cytoplasm is followed by development of a bizarre quadripolar spindle that assures coordination of nuclear distribution with predetermined spore domains. This concept of the innovation of sporogenesis at the onset of terrestrialization provides a new perspective for interpreting fossil evidence and understanding the evolution of land plants.