Plant PIEZO homologs modulate vacuole morphology during tip growth.

In animals, PIEZOs are plasma membrane-localized cation channels involved in diverse mechanosensory processes. We investigated PIEZO function in tip-growing cells in the moss Physcomitrium patens and the flowering plant Arabidopsis thaliana PpPIEZO1 and PpPIEZO2 redundantly contribute to the normal growth, size, and cytoplasmic calcium oscillations of caulonemal cells. Both PpPIEZO1 and PpPIEZO2 localized to vacuolar membranes. Loss-of-function, gain-of-function, and overexpression mutants revealed that moss PIEZO homologs promote increased complexity of vacuolar membranes through tubulation, internalization, and/or fission. Arabidopsis PIEZO1 also localized to the tonoplast and is required for vacuole tubulation in the tips of pollen tubes. We propose that in plant cells the tonoplast has more freedom of movement than the plasma membrane, making it a more effective location for mechanosensory proteins.

Involvement of Lhcb6 and Lhcb5 in Photosynthesis Regulation in Physcomitrella patens Response to Abiotic Stress.

There are a number of highly conserved photosystem II light-harvesting antenna proteins in moss whose functions are unclear. Here, we investigated the involvement of chlorophyll-binding proteins, Lhcb6 and Lhcb5, in light-harvesting and photosynthesis regulation in Physcomitrella patens. Lhcb6 or Lhcb5 knock-out resulted in a disordered thylakoid arrangement, a decrease in the number of grana membranes, and an increase in the number of starch granule. The absence of Lhcb6 or Lhcb5 did not noticeably alter the electron transport rates. However, the non-photochemical quenching activity in the lhcb5 mutant was dramatically reduced when compared to wild-type or lhcb6 plants under abiotic stress. Lhcb5 plants were more sensitive to photo-inhibition, while lhcb6 plants showed little difference compared to the wild-type plants under high-light stress. Moreover, both mutants showed a growth malformation phenotype with reduced chlorophyll content in the gametophyte. These results suggested that Lhcb6 or Lhcb5 played a unique role in plant development, thylakoid organization, and photoprotection of PSII in Physcomitrella, especially when exposed to high light or osmotic environments.

Automated Image Acquisition and Morphological Analysis of Cell Growth Mutants in Physcomitrella patens.

This protocol describes the automated imaging and a quantitative analysis of the morphology of small plants from the moss Physcomitrella patens. This method can be used for the analysis of growth phenotypes produced by transient RNA interference or for the analysis of stable mutant plants. Furthermore, we describe how to acquire higher resolution images via the acquisition of a collection of multiple overlapping tiles from the same image. Information is presented to guide the investigator in the choice of vectors and basic conditions to perform transient RNA interference in moss. Detailed directions and examples for fluorescence image acquisition of small regenerating moss plants are provided. Instructions for stitching image tiles and for using an ImageJ-based macro for the quantitative morphological analysis of moss plants are also provided.

Acyl-CoA synthetases from Physcomitrella, rice and Arabidopsis: different substrate preferences but common regulation by MS188 in sporopollenin synthesis.

MAIN CONCLUSION: ACOS5, OsACOS12 and PpACOS6 are all capable of fatty acyl-CoA synthetase activity but exhibit different substrate preferences. The transcriptional regulation of ACOS for sporopollenin synthesis appears to have been conserved in Physcomitrella, rice and Arabidopsis during evolution. Sporopollenin is the major constituent of spore and pollen exines. In Arabidopsis, acyl-CoA synthetase 5 (ACOS5) is an essential enzyme for sporopollenin synthesis, and its orthologues are PpACOS6 from the moss Physcomitrella and OsACOS12 from monocot rice. However, knowledge regarding the evolutionary conservation and divergence of the ACOS gene in sporopollenin synthesis remains limited. In this study, we analysed the function and regulation of PpACOS6 and OsACOS12. A complementation test showed that OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis, while PpACOS6 did not rescue the acos5 phenotype. ACOS5, PpACOS6 and OsACOS12 all complemented the acyl-CoA synthetase-deficient yeast strain (YB525) phenotype, although they exhibited different substrate preferences. To understand the conservation of sporopollenin synthesis regulation, we constructed two constructs with ACOS5 driven by the OsACOS12 or PpACOS6 promoter. Both constructs could restore the fertility of acos5 plants. The MYB transcription factor MS188 from Arabidopsis directly regulates ACOS5. We found that MS188 could also bind the promoters of OsACOS12 and PpACOS6 and activate the genes driven by the promoters, suggesting that the transcriptional regulation of these genes was similar to that of ACOS5. These results show that the ACOS gene promoter region from Physcomitrella, rice and Arabidopsis has been functionally conserved during evolution, while the chain lengths of fatty acid-derived monomers of sporopollenin vary in different plant species.

Exocyst subunit Sec6 is positioned by microtubule overlaps in the moss phragmoplast prior to cell plate membrane arrival.

During plant cytokinesis a radially expanding membrane-enclosed cell plate is formed from fusing vesicles that compartmentalizes the cell in two. How fusion is spatially restricted to the site of cell plate formation is unknown. Aggregation of cell-plate membrane starts near regions of microtubule overlap within the bipolar phragmoplast apparatus of the moss Physcomitrella patens Since vesicle fusion generally requires coordination of vesicle tethering and subsequent fusion activity, we analyzed the subcellular localization of several subunits of the exocyst, a tethering complex active during plant cytokinesis. We found that the exocyst complex subunit Sec6 but not the Sec3 or Sec5 subunits localized to microtubule overlap regions in advance of cell plate construction in moss. Moreover, Sec6 exhibited a conserved physical interaction with an ortholog of the Sec1/Munc18 protein KEULE, an important regulator for cell-plate membrane vesicle fusion in Arabidopsis Recruitment of the P. patens protein KEULE and vesicles to the early cell plate was delayed upon Sec6 gene silencing. Our findings, thus, suggest that vesicle-vesicle fusion is, in part, enabled by a pool of exocyst subunits at microtubule overlaps, which is recruited independently of vesicle delivery.

Subdiffraction-resolution live-cell imaging for visualizing thylakoid membranes.

The chloroplast is the chlorophyll-containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live-cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three-dimensional structured illumination microscopy (3D-SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D-SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild-type and mutant strains. Using 3D-SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D-SIM. This study demonstrates that 3D-SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.

Spores potentially dispersed to longer distances are more tolerant to ultraviolet radiation: A case study in the moss genus Orthotrichum.

PREMISE OF THE STUDY: Ultraviolet (UV) radiation influences the viability of algal spores and seed-plant pollen depending on the species, the dose, and the wavelength. In bryophytes, one of the dominant groups of plants in many habitats, UV radiation could determine their spore dispersal strategy, and such data are critical for reconstructing the ancestral state in plants and for determining the distribution range and persistence of bryophyte species. METHODS: Spores of four bryophyte species of the moss genus Orthotrichum that were either hygrochastic or xerochastic (spores dispersed under wet or dry conditions, respectively) were exposed to realistic doses of UV radiation under laboratory conditions. Spore viability was evaluated through germination experiments and, for the first time in bryophytes, ultrastructural observations. Given that the UV-B doses used were relatively higher than the UV-A doses, the UV effect was probably due more to UV-B than UV-A wavelengths. KEY RESULTS: All four species reduced their spore germination capacity in a UV dose-dependent manner, concomitantly increasing spore ultrastructural damage (cytoplasmic and plastid alterations). Most spores eventually died when exposed to the highest UV dose. Interestingly, spores of hygrochastic species were much more UV-sensitive than those of xerochastic species. CONCLUSIONS: UV tolerance determines moss spore viability, as indicated by germination capacity and ultrastructural damage, and differs between spores of species with different dispersal strategies. Specifically, the higher UV tolerance of xerochastic spores may enable them to be dispersed to longer distances than hygrochastic spores, thus extending more efficiently the distribution range of the corresponding species.

In-field and in-vitro study of the moss Leptodictyum riparium as bioindicator of toxic metal pollution in the aquatic environment: Ultrastructural damage, oxidative stress and HSP70 induction.

This study evaluates the effects of toxic metal pollution in the highly contaminated Sarno River (South Italy), by using the aquatic moss Leptodictyum riparium in bags at 3 representative sites of the river. Biological effects were assessed by metal bioaccumulation, ultrastructural changes, oxidative stress, as Reactive Oxygen Species (ROS) production and Glutathione S-transferase (GST) activity, as well as Heat Shock Proteins 70 (HSP70s) induction. The results showed that L. riparium is a valuable bioindicator for toxic metal pollution of water ecosystem, accumulating different amounts of toxic metals from the aquatic environment. Toxic metal pollution caused severe ultrastructural damage, as well as increased ROS production and induction of GST and HSP70s, in the samples exposed at the polluted sites. To assess the role and the effect of toxic metals on L. riparium, were also cultured in vitro with Cd, Cr, Cu, Fe, Ni, Pb, Zn at the same concentrations as measured at the 3 sites. Ultrastructure, ROS, GST, and HSP70s resulted severely affected by toxic metals. Based on our findings, we confirm L. riparium as a model organism in freshwater biomonitoring surveys, and GST and HSP70s as promising biomarkers of metal toxicity.

Symplasmic and apoplasmic transport inside feather moss stems of Pleurozium schreberi and Hylocomium splendens.

Background and Aims: The ubiquitous feather mosses Pleurozium schreberi and Hylocomium splendens form a thick, continuous boundary layer between the soil and the atmosphere, and play important roles in hydrology and nutrient cycling in tundra and boreal ecosystems. The water fluxes among these mosses and environmental factors controlling them are poorly understood. The aim of this study was to investigate whether feather mosses are capable of internal transport and to provide a better understanding of species-specific morphological traits underlying this function. The impacts of environmental conditions on their internal transport rates were also investigated. Methods: Cells involved in water and food conduction in P. schreberi and H. splendens were identified by transmission electron microscopy. Symplasmic and apoplasmic fluorescent tracers were applied to the moss stems to determine the routes of internal short- and long-distance transport and the impact of air humidity on the transport rates. Key Results: Symplasmic transport over short distances occurs via food-conducting cells in both mosses. Pleurozium schreberi is also capable of apoplasmic internal long-distance transport via a central strand of hydroids. These are absent in H. splendens. Reduced air humidity significantly increased the internal transport of both species, and the increase was significantly faster for P. schreberi than for H. splendens. Conclusions: Pleurozium schreberi and Hylocomium splendens are capable of internal transport but the pathway and conductivity differ due to differences in stem anatomy. These results help explain their varying desiccation tolerance and possibly their differing physiology and autecology and, ultimately, their impact on ecosystem functioning.

Unique Motifs and Length of Hairpin in Oleosin Target the Cytosolic Side of Endoplasmic Reticulum and Budding Lipid Droplet.

Plant cytosolic lipid droplets (LDs) are covered with a layer of phospholipids and oleosin and were extensively studied before those in mammals and yeast. Oleosin has short amphipathic N- and C-terminal peptides flanking a conserved 72-residue hydrophobic hairpin, which penetrates and stabilizes the LD Oleosin is synthesized on endoplasmic reticulum (ER) and extracts ER-budding LDs to cytosol. To delineate the mechanism of oleosin targeting ER-LD, we have expressed modified-oleosin genes in Physcomitrella patens for transient expression and tobacco (Nicotiana tabacum) BY2 cells for stable transformation. The results have identified oleosin motifs for targeting ER-LD and oleosin as the sole molecule responsible for budding-LD entering cytosol. Both the N-terminal and C-terminal peptides are not required for the targeting. The hairpin, including its entire length, initial N-portion residues, and hairpin-loop of three Pro and one Ser residues, as well as the absence of an N-terminal ER-targeting peptide, are necessary for oleosin targeting ER and moving onto budding LDs and extracting them to cytosol. In a reverse approach, eliminations of these necessities allow the modified oleosin to enter the ER lumen and extract budding LDs to the ER lumen. Modified oleosin with an added vacuole signal peptide transports the ER-luminal LDs to vacuoles. The overall findings define the mechanism of oleosin targeting ER-LDs and extracting budding LDs to the cytosol as well as reveal potential applications.

The Physcomitrella patens exocyst subunit EXO70.3d has distinct roles in growth and development, and is essential for completion of the moss life cycle.

The exocyst, an evolutionarily conserved secretory vesicle-tethering complex, spatially controls exocytosis and membrane turnover in fungi, metazoans and plants. The exocyst subunit EXO70 exists in multiple paralogs in land plants, forming three conserved clades with assumed distinct roles. Here we report functional analysis of the first moss exocyst subunit to be studied, Physcomitrella patens PpEXO70.3d (Pp1s97_91V6), from the, as yet, poorly characterized EXO70.3 clade. Following phylogenetic analysis to confirm the presence of three ancestral land plant EXO70 clades outside angiosperms, we prepared and phenotypically characterized loss-of-function Ppexo70.3d mutants and localized PpEXO70.3d in vivo using green fluorescent protein-tagged protein expression. Disruption of PpEXO70.3d caused pleiotropic cell elongation and differentiation defects in protonemata, altered response towards exogenous auxin, increased endogenous IAA concentrations, along with defects in bud and gametophore development. During mid-archegonia development, an abnormal egg cell is formed and subsequently collapses, resulting in mutant sterility. Mutants exhibited altered cell wall and cuticle deposition, as well as compromised cytokinesis, consistent with the protein localization to the cell plate. Despite some functional redundancy allowing survival of moss lacking PpEXO70.3d, this subunit has an essential role in the moss life cycle, indicating sub-functionalization within the moss EXO70 family.

A hypergravity environment increases chloroplast size, photosynthesis, and plant growth in the moss Physcomitrella patens.

The physiological and anatomical responses of bryophytes to altered gravity conditions will provide crucial information for estimating how plant physiological traits have evolved to adapt to significant increases in the effects of gravity in land plant history. We quantified changes in plant growth and photosynthesis in the model plant of mosses, Physcomitrella patens, grown under a hypergravity environment for 25 days or 8 weeks using a custom-built centrifuge equipped with a lighting system. This is the first study to examine the response of bryophytes to hypergravity conditions. Canopy-based plant growth was significantly increased at 10×g, and was strongly affected by increases in plant numbers. Rhizoid lengths for individual gametophores were significantly increased at 10×g. Chloroplast diameters (major axis) and thicknesses (minor axis) in the leaves of P. patens were also increased at 10×g. The area-based photosynthesis rate of P. patens was also enhanced at 10×g. Increases in shoot numbers and chloroplast sizes may elevate the area-based photosynthesis rate under hypergravity conditions. We observed a decrease in leaf cell wall thickness under hypergravity conditions, which is in contrast to previous findings obtained using angiosperms. Since mosses including P. patens live in dense populations, an increase in canopy-based plant numbers may be effective to enhance the toughness of the population, and, thus, represents an effective adaptation strategy to a hypergravity environment for P. patens.

Approaches to Characterize Organelle, Compartment, or Structure Purity.

The function of subcellular structures is defined by their specific sets of proteins, making subcellular protein localization one of the most important topics in organelle research. To date, many organelle proteomics workflows involve the (partial) purification of the desired subcellular structure and the subsequent analysis of the proteome using tandem mass spectrometry (MS/MS). This chapter gives an overview of the methods that have been used to assay the purity and enrichment of subcellular structures, with an emphasis on quantitative proteomics using differently enriched subcellular fractions. We introduce large-scale-based criteria for assignment of proteins to subcellular structures and describe in detail the use of 15N metabolic labeling in moss to characterize plastid and mitochondrial proteomes.

The phenotype of the CRINKLY4 deletion mutant of Physcomitrella patens suggests a broad role in developmental regulation in early land plants.

MAIN CONCLUSIONS: Deletion of the ancestral gene of the land plant multigene family of receptor like kinase CR4 in Physcomitrella patens demonstrates involvement in developmental control of gametophytic and sporophytic organs. The CRINKLY4 (CR4) family of receptor kinases in angiosperms consists of three clades, one including CR4, the CR4-related CCR1 and CCR2, a second including CCR3 and CCR4 family members, and a third and more distant clade. In addition to crinkly leaves in maize, which gave rise to the mutant gene name, CR4 is implicated in ovule, embryo, flower and root development in Arabidopsis thaliana. In root tips of the same species the module including a CLAVATA3/ESR-related protein, an Arabidopsis CR4, a CLAVATA1 and a WUSCHEL-related homeobox 5 (CLE40-ACR4-CLV1-WOX5) is implicated in meristem cell regulation. In embryos and shoots, CR4 acts together with A. thaliana MERISTEM LAYER 1 and PROTODERMAL FACTOR 2 to promote A. thaliana epidermis differentiation. Phylogenetic analysis has demonstrated that early land plants, e.g. mosses carry a single ancestral CR4 gene, together with genes encoding the other members of the CLE40-ACR4-CLV1-WOX5 signaling module. Here we show that CR4 serves as a broad regulator of morphogenesis both in gametophyte phyllids, archegonia and in sporophyte epidermis of the moss Physcomitrella patens. The phenotype of the CR4 deletion mutant in moss provides insight into the role of the ancestral CR4 gene as a regulator of development in early land plants.

Spatio-temporal patterning of arginyl-tRNA protein transferase (ATE) contributes to gametophytic development in a moss.

The importance of the arginyl-tRNA protein transferase (ATE), the enzyme mediating post-translation arginylation of proteins in the N-end rule degradation (NERD) pathway of protein stability, was analysed in Physcomitrella patens and compared to its known functions in other eukaryotes. We characterize ATE:GUS reporter lines as well as ATE mutants in P. patens to study the impact and function of arginylation on moss development and physiology. ATE protein abundance is spatially and temporally regulated in P. patens by hormones and light and is highly abundant in meristematic cells. Further, the amount of ATE transcript is regulated during abscisic acid signalling and downstream of auxin signalling. Loss-of-function mutants exhibit defects at various levels, most severely in developing gametophores, in chloroplast starch accumulation and senescence. Thus, arginylation is necessary for moss gametophyte development, in contrast to the situation in flowering plants. Our analysis further substantiates the conservation of the N-end rule pathway components in land plants and highlights lineage-specific features. We introduce moss as a model system to characterize the role of the NERD pathway as an additional layer of complexity in eukaryotic development.

Light-Harvesting Complex Stress-Related Proteins Catalyze Excess Energy Dissipation in Both Photosystems of Physcomitrella patens.

Two LHC-like proteins, Photosystem II Subunit S (PSBS) and Light-Harvesting Complex Stress-Related (LHCSR), are essential for triggering excess energy dissipation in chloroplasts of vascular plants and green algae, respectively. The mechanism of quenching was studied in Physcomitrella patens, an early divergent streptophyta (including green algae and land plants) in which both proteins are active. PSBS was localized in grana together with photosystem II (PSII), but LHCSR was located mainly in stroma-exposed membranes together with photosystem I (PSI), and its distribution did not change upon high-light treatment. The quenched conformation can be preserved by rapidly freezing the high-light-treated tissues in liquid nitrogen. When using green fluorescent protein as an internal standard, 77K fluorescence emission spectra on isolated chloroplasts allowed for independent assessment of PSI and PSII fluorescence yield. Results showed that both photosystems underwent quenching upon high-light treatment in the wild type in contrast to mutants depleted of LHCSR, which lacked PSI quenching. Due to the contribution of LHCII, P. patens had a PSI antenna size twice as large with respect to higher plants. Thus, LHCII, which is highly abundant in stroma membranes, appears to be the target of quenching by LHCSR.

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.

Conservation of Male Sterility 2 function during spore and pollen wall development supports an evolutionarily early recruitment of a core component in the sporopollenin biosynthetic pathway.

The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.

Developmental changes in guard cell wall structure and pectin composition in the moss Funaria: implications for function and evolution of stomata.

BACKGROUND AND AIMS: In seed plants, the ability of guard cell walls to move is imparted by pectins. Arabinan rhamnogalacturonan I (RG1) pectins confer flexibility while unesterified homogalacturonan (HG) pectins impart rigidity. Recognized as the first extant plants with stomata, mosses are key to understanding guard cell function and evolution. Moss stomata open and close for only a short period during capsule expansion. This study examines the ultrastructure and pectin composition of guard cell walls during development in Funaria hygrometrica and relates these features to the limited movement of stomata. METHODS: Developing stomata were examined and immunogold-labelled in transmission electron microscopy using monoclonal antibodies to five pectin epitopes: LM19 (unesterified HG), LM20 (esterified HG), LM5 (galactan RG1), LM6 (arabinan RG1) and LM13 (linear arabinan RG1). Labels for pectin type were quantitated and compared across walls and stages on replicated, independent samples. KEY RESULTS: Walls were four times thinner before pore formation than in mature stomata. When stomata opened and closed, guard cell walls were thin and pectinaceous before the striated internal and thickest layer was deposited. Unesterified HG localized strongly in early layers but weakly in the thick internal layer. Labelling was weak for esterified HG, absent for galactan RG1 and strong for arabinan RG1. Linear arabinan RG1 is the only pectin that exclusively labelled guard cell walls. Pectin content decreased but the proportion of HG to arabinans changed only slightly. CONCLUSIONS: This is the first study to demonstrate changes in pectin composition during stomatal development in any plant. Movement of Funaria stomata coincides with capsule expansion before layering of guard cell walls is complete. Changes in wall architecture coupled with a decrease in total pectin may be responsible for the inability of mature stomata to move. Specialization of guard cells in mosses involves the addition of linear arabinans.

Visualizing structural dynamics of thylakoid membranes.

To optimize photosynthesis, light-harvesting antenna proteins regulate light energy dissipation and redistribution in chloroplast thylakoid membranes, which involve dynamic protein reorganization of photosystems I and II. However, direct evidence for such protein reorganization has not been visualized in live cells. Here we demonstrate structural dynamics of thylakoid membranes by live cell imaging in combination with deconvolution. We observed chlorophyll fluorescence in the antibiotics-induced macrochloroplast in the moss Physcomitrella patens. The three-dimensional reconstruction uncovered the fine thylakoid membrane structure in live cells. The time-lapse imaging shows that the entire thylakoid membrane network is structurally stable, but the individual thylakoid membrane structure is flexible in vivo. Our observation indicates that grana serve as a framework to maintain structural integrity of the entire thylakoid membrane network. Both the structural stability and flexibility of thylakoid membranes would be essential for dynamic protein reorganization under fluctuating light environments.