An ABCB transporter regulates anisotropic cell expansion via cuticle deposition in the moss Physcomitrium patens.

Anisotropic cell expansion is crucial for the morphogenesis of land plants, as cell migration is restricted by the rigid cell wall. The anisotropy of cell expansion is regulated by mechanisms acting on the deposition or modification of cell wall polysaccharides. Besides the polysaccharide components in the cell wall, a layer of hydrophobic cuticle covers the outer cell wall and is subjected to tensile stress that mechanically restricts cell expansion. However, the molecular machinery that deposits cuticle materials in the appropriate spatiotemporal manner to accommodate cell and tissue expansion remains elusive. Here, we report that PpABCB14, an ATP-binding cassette transporter in the moss Physcomitrium patens, regulates the anisotropy of cell expansion. PpABCB14 localized to expanding regions of leaf cells. Deletion of PpABCB14 resulted in impaired anisotropic cell expansion. Unexpectedly, the cuticle proper was reduced in the mutants, and the cuticular lipid components decreased. Moreover, induced PpABCB14 expression resulted in deformed leaf cells with increased cuticle lipid accumulation on the cell surface. Taken together, PpABCB14 regulates the anisotropy of cell expansion via cuticle deposition, revealing a regulatory mechanism for cell expansion in addition to the mechanisms acting on cell wall polysaccharides.

NAC103 mutation alleviates DNA damage in an Arabidopsis thaliana mutant sensitive to excess boron.

Excess boron (B) is toxic to plants and thereby causes DNA damage and cell death in root meristems. However, the underlying mechanisms which link boron and DNA damage remain unclear. It has been reported that the rpt5a-6 mutant of the 26S proteasome is sensitive to excess boron, resulting in more frequent cell death in root meristem and reduced root elongation. In this study, we showed that a reduction in root growth in the rpt5a mutant in the presence of high boron levels is repressed by a mutation in NAC domain containing transcription factor NAC103, a substrate of the proteasome, which functions in the unfolded protein response pathway. The mutation in NAC103 alleviated excess-B-induced DNA damage and cell death in root meristems of the rpt5a mutant. Superoxide ( O 2 - ) staining with nitroblue tetrazolium revealed that boron stress causes O 2 - accumulation in root tips, which was higher in the rpt5a-6 mutant, whereas the accumulation was lower in the rpt5a-6 nac103-3 double mutant. Our work demonstrates the overall involvement of NAC103 in maintaining healthy root meristem under excess boron conditions in the absence of RPT5A proteasome subunit.

PECT1, a rate-limiting enzyme in phosphatidylethanolamine biosynthesis, is involved in the regulation of stomatal movement in Arabidopsis.

An Arabidopsis mutant displaying impaired stomatal responses to CO2 , cdi4, was isolated by a leaf thermal imaging screening. The mutated gene PECT1 encodes CTP:phosphorylethanolamine cytidylyltransferase. The cdi4 exhibited a decrease in phosphatidylethanolamine levels and a defect in light-induced stomatal opening as well as low-CO2 -induced stomatal opening. We created RNAi lines in which PECT1 was specifically repressed in guard cells. These lines are impaired in their stomatal responses to low-CO2 concentrations or light. Fungal toxin fusicoccin (FC) promotes stomatal opening by activating plasma membrane H+ -ATPases in guard cells via phosphorylation. Arabidopsis H+ -ATPase1 (AHA1) has been reported to be highly expressed in guard cells, and its activation by FC induces stomatal opening. The cdi4 and PECT1 RNAi lines displayed a reduced stomatal opening response to FC. However, similar to in the wild-type, cdi4 maintained normal levels of phosphorylation and activation of the stomatal H+ -ATPases after FC treatment. Furthermore, the cdi4 displayed normal localization of GFP-AHA1 fusion protein and normal levels of AHA1 transcripts. Based on these results, we discuss how PECT1 could regulate CO2 - and light-induced stomatal movements in guard cells in a manner that is independent and downstream of the activation of H+ -ATPases. [Correction added on 15 May 2023, after first online publication: The third sentence is revised in this version.].

Novel inhibitors of microtubule organization and phragmoplast formation in diverse plant species.

Cell division is essential for development and involves spindle assembly, chromosome separation, and cytokinesis. In plants, the genetic tools for controlling the events in cell division at the desired time are limited and ineffective owing to high redundancy and lethality. Therefore, we screened cell division-affecting compounds in Arabidopsis thaliana zygotes, whose cell division is traceable without time-lapse observations. We then determined the target events of the identified compounds using live-cell imaging of tobacco BY-2 cells. Subsequently, we isolated two compounds, PD-180970 and PP2, neither of which caused lethal damage. PD-180970 disrupted microtubule (MT) organization and, thus, nuclear separation, and PP2 blocked phragmoplast formation and impaired cytokinesis. Phosphoproteomic analysis showed that these compounds reduced the phosphorylation of diverse proteins, including MT-associated proteins (MAP70) and class II Kinesin-12. Moreover, these compounds were effective in multiple plant species, such as cucumber (Cucumis sativus) and moss (Physcomitrium patens). These properties make PD-180970 and PP2 useful tools for transiently controlling plant cell division at key manipulation nodes conserved across diverse plant species.

The genome of Lyophyllum shimeji provides insight into the initial evolution of ectomycorrhizal fungal genomes.

Mycorrhizae are one of the most fundamental symbioses between plants and fungi, with ectomycorrhizae being the most widespread in boreal forest ecosystems. Ectomycorrhizal fungi are hypothesized to have evolved convergently from saprotrophic ancestors in several fungal clades, especially members of the subdivision Agaricomycotina. Studies on fungal genomes have identified several typical characteristics of mycorrhizal fungi, such as genome size expansion and decreases in plant cell-wall degrading enzymes (PCWDEs). However, genomic changes concerning the evolutionary transition to the ectomycorrhizal lifestyle are largely unknown. In this study, we sequenced the genome of Lyophyllum shimeji, an ectomycorrhizal fungus that is phylogenetically related to saprotrophic species and retains some saprotroph-like traits. We found that the genome of Ly. shimeji strain AT787 lacks both incremental increases in genome size and reduced numbers of PCWDEs. Our findings suggest that the previously reported common genomic traits of mycorrhizal fungi are not essential for the ectomycorrhizal lifestyle, but are a result of abolishing saprotrophic activity. Since Ly. shimeji is commercially consumed as an edible mushroom, the newly available genomic information may also impact research designed to enhance the cultivation of this mushroom.

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.

Calcium-mediated rapid movements defend against herbivorous insects in Mimosa pudica.

Animals possess specialized systems, e.g., neuromuscular systems, to sense the environment and then move their bodies quickly in response. Mimosa pudica, the sensitive plant, moves its leaves within seconds in response to external stimuli; e.g., touch or wounding. However, neither the plant-wide signaling network that triggers these rapid movements nor the physiological roles of the movements themselves have been determined. Here by simultaneous recording of cytosolic Ca2+ and electrical signals, we show that rapid changes in Ca2+ coupled with action and variation potentials trigger rapid movements in wounded M. pudica. Furthermore, pharmacological manipulation of cytosolic Ca2+ dynamics and CRISPR-Cas9 genome editing technology revealed that an immotile M. pudica is more vulnerable to attacks by herbivorous insects. Our findings provide evidence that rapid movements based on propagating Ca2+ and electrical signals protect this plant from insect attacks.

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.

Low-invasive 5D visualization of mitotic progression by two-photon excitation spinning-disk confocal microscopy.

Non-linear microscopy, such as multi-photon excitation microscopy, offers spatial localities of excitations, thereby achieving 3D cross-sectional imaging with low phototoxicity even in thick biological specimens. We had developed a multi-point scanning two-photon excitation microscopy system using a spinning-disk confocal scanning unit. However, its severe color cross-talk has precluded multi-color simultaneous imaging. Therefore, in this study, we introduced a mechanical switching system to select either of two NIR laser light pulses and an image-splitting detection system for 3- or 4-color imaging. As a proof of concept, we performed multi-color fluorescent imaging of actively dividing human HeLa cells and tobacco BY-2 cells. We found that the proposed microscopy system enabled time-lapse multi-color 3D imaging of cell divisions while avoiding photodamage. Moreover, the application of a linear unmixing method to the 5D dataset enabled the precise separation of individual intracellular components in multi-color images. We thus demonstrated the versatility of our new microscopy system in capturing the dynamic processes of cellular components that could have multitudes of application.

Topoisomerase 1α is required for synchronous spermatogenesis in Physcomitrium patens.

DNA topoisomerase 1 (TOP1) plays general roles in DNA replication and transcription by regulating DNA topology in land plants and metazoans. TOP1 is also involved in specific developmental events; however, whether TOP1 plays a conserved developmental role among multicellular organisms is unknown. Here, we investigated the developmental roles of TOP1 in the moss Physcomitrium (Physcomitrella) patens with gene targeting, microscopy, 3D image segmentation and crossing experiments. We discovered that the disruption of TOP1α, but not its paralogue TOP1ß, leads to a defect in fertilisation and subsequent sporophyte formation in P. patens. In the top1α mutant, the egg cell was functional for fertilisation, while sperm cells were fewer and infertile with disordered structures. We observed that the nuclei volume of wild-type sperm cells synchronously decreases during antheridium development, indicating chromatin condensation towards the compact sperm head. By contrast, the top1α mutant exhibited attenuated cell divisions and asynchronous and defective contraction of the nuclei of sperm cells throughout spermatogenesis. These results indicate that TOP1α is involved in cell division and chromatin condensation during spermatogenesis in P. patens. Our results suggest that the regulation of DNA topology by TOP1 plays a key role in spermatogenesis in both land plants and metazoans.

Molecular mechanisms of reprogramming of differentiated cells into stem cells in the moss Physcomitrium patens.

Plant and animal stem cells can self-renew and give rise to differentiated cells to form tissues or organs. Unlike differentiated cells in animals, those in land plants can be readily reprogrammed into stem cells, reflecting the plasticity of plant cell identity. The moss Physcomitrium patens (synonym: Physcomitrella patens) is highly regenerable, and its leaf cells can be reprogrammed into stem cells in response to wounding or by transient DNA damage without wounding. Wounding and DNA damage induce STEM CELL-INDUCING FACTOR 1, an APETALA2/ETHYLENE RESPONSE FACTOR. Here, we summarize the genetic networks that regulate cellular reprogramming in P. patens and the roles of STEMIN1 and discuss the generality and divergence of the molecular mechanisms underlying cellular reprogramming in land plants and animals.

Overexpression of ATG8/LC3 enhances wound-induced somatic reprogramming in Physcomitrium patens.

Animal and plant somatic cells have the capacity to switch states or reprogram into stem cells to adapt during stress and injury. This ability to deal with stochastic changes or reprogramming of somatic cells also needs macroautophagy/autophagy. Here, we expand on this notion and provide a primary example of how overexpression of ATG8/LC3 in the moss Physcomitrium patens enhances the ability to reprogram somatic cells into stem cells when subjected to severe wounding. This observation suggests that autophagy is not only required for cells to dedifferentiate but also makes cells more competent to do so.Abbreviation: ATG: autophagy related; atg5: AUTOPHAGY 5; ATG8/LC3: AUTOPHAGY 8/microtubule associated protein 1 light chain 3; GFP: green fluorescent protein.

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Rapid movements in plants.

Plant movements are generally slow, but some plant species have evolved the ability to move very rapidly at speeds comparable to those of animals. Whereas movement in animals relies on the contraction machinery of muscles, many plant movements use turgor pressure as the primary driving force together with secondarily generated elastic forces. The movement of stomata is the best-characterized model system for studying turgor-driven movement, and many gene products responsible for this movement, especially those related to ion transport, have been identified. Similar gene products were recently shown to function in the daily sleep movements of pulvini, the motor organs for macroscopic leaf movements. However, it is difficult to explain the mechanisms behind rapid multicellular movements as a simple extension of the mechanisms used for unicellular or slow movements. For example, water transport through plant tissues imposes a limit on the speed of plant movements, which becomes more severe as the size of the moving part increases. Rapidly moving traps in carnivorous plants overcome this limitation with the aid of the mechanical behaviors of their three-dimensional structures. In addition to a mechanism for rapid deformation, rapid multicellular movements also require a molecular system for rapid cell-cell communication, along with a mechanosensing system that initiates the response. Electrical activities similar to animal action potentials are found in many plant species, representing promising candidates for the rapid cell-cell signaling behind rapid movements, but the molecular entities of these electrical signals remain obscure. Here we review the current understanding of rapid plant movements with the aim of encouraging further biological studies into this fascinating, challenging topic.

A discordance of seasonally covarying cues uncovers misregulated phenotypes in the heterophyllous pitcher plant Cephalotus follicularis.

Organisms withstand normal ranges of environmental fluctuations by producing a set of phenotypes genetically programmed as a reaction norm; however, extreme conditions can expose a misregulation of phenotypes called a hidden reaction norm. Although an environment consists of multiple factors, how combinations of these factors influence a reaction norm is not well understood. To elucidate the combinatorial effects of environmental factors, we studied the leaf shape plasticity of the carnivorous pitcher plant Cephalotus follicularis. Clonally propagated plants were subjected to 12-week-long growth experiments in different conditions controlled by growth chambers. Here, we show that the dimorphic response of forming a photosynthetic flat leaf or an insect-trapping pitcher leaf is regulated by two covarying environmental cues: temperature and photoperiod. Even within the normal ranges of temperature and photoperiod, unusual combinations of the two induced the production of malformed leaves that were rarely observed under the environmentally typical combinations. We identified such cases in combinations of a summer temperature with a short-to-neutral day length, whose average frequency in the natural Cephalotus habitats corresponded to a once-in-a-lifetime event for this perennial species. Our results suggest that even if individual cues are within the range of natural fluctuations, a hidden reaction norm can be exposed under their discordant combinations. We anticipate that climate change may challenge organismal responses through not only extreme cues but also through uncommon combinations of benign cues.

Calcium dynamics during trap closure visualized in transgenic Venus flytrap.

The leaves of the carnivorous plant Venus flytrap, Dionaea muscipula (Dionaea) close rapidly to capture insect prey. The closure response usually requires two successive mechanical stimuli to sensory hairs on the leaf blade within approximately 30 s (refs. 1-4). An unknown biological system in Dionaea is thought to memorize the first stimulus and transduce the signal from the sensory hair to the leaf blade2. Here, we link signal memory to calcium dynamics using transgenic Dionaea expressing a Ca2+ sensor. Stimulation of a sensory hair caused an increase in cytosolic Ca2+ concentration ([Ca2+]cyt) starting in the sensory hair and spreading to the leaf blade. A second stimulus increased [Ca2+]cyt to an even higher level, meeting a threshold that is correlated to the leaf blade closure. Because [Ca2+]cyt gradually decreased after the first stimulus, the [Ca2+]cyt increase induced by the second stimulus was insufficient to meet the putative threshold for movement after about 30 s. The Ca2+ wave triggered by mechanical stimulation moved an order of magnitude faster than that induced by wounding in petioles of Arabidopsis thaliana5 and Dionaea. The capacity for rapid movement has evolved repeatedly in flowering plants. This study opens a path to investigate the role of Ca2+ in plant movement mechanisms and their evolution.

Corrigendum: Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori.

[This corrects the article DOI: 10.3389/fmicb.2020.01628.].

Ethylene signaling mediates host invasion by parasitic plants.

Parasitic plants form a specialized organ, a haustorium, to invade host tissues and acquire water and nutrients. To understand the molecular mechanism of haustorium development, we performed a forward genetics screening to isolate mutants exhibiting haustorial defects in the model parasitic plant Phtheirospermum japonicum. We isolated two mutants that show prolonged and sometimes aberrant meristematic activity in the haustorium apex, resulting in severe defects on host invasion. Whole-genome sequencing revealed that the two mutants respectively have point mutations in homologs of ETHYLENE RESPONSE 1 (ETR1) and ETHYLENE INSENSITIVE 2 (EIN2), signaling components in response to the gaseous phytohormone ethylene. Application of the ethylene signaling inhibitors also caused similar haustorial defects, indicating that ethylene signaling regulates cell proliferation and differentiation of parasite cells. Genetic disruption of host ethylene production also perturbs parasite invasion. We propose that parasitic plants use ethylene as a signal to invade host roots.

Networking and Specificity-Changing DNA Methyltransferases in Helicobacter pylori.

Epigenetic DNA base methylation plays important roles in gene expression regulation. We here describe a gene expression regulation network consisting of many DNA methyltransferases each frequently changing its target sequence-specificity. Our object Helicobacter pylori, a bacterium responsible for most incidence of stomach cancer, carries a large and variable repertoire of sequence-specific DNA methyltransferases. By creating a dozen of single-gene knockout strains for the methyltransferases, we revealed that they form a network controlling methylome, transcriptome and adaptive phenotype sets. The methyltransferases interact with each other in a hierarchical way, sometimes regulated positively by one methyltransferase but negatively with another. Motility, oxidative stress tolerance and DNA damage repair are likewise regulated by multiple methyltransferases. Their regulation sometimes involves translation start and stop codons suggesting coupling of methylation, transcription and translation. The methyltransferases frequently change their sequence-specificity through gene conversion of their target recognition domain and switch their target sets to remodel the network. The emerging picture of a metamorphosing gene regulation network, or firework, consisting of epigenetic systems ever-changing their specificity in search for adaptation, provides a new paradigm in understanding global gene regulation and adaptive evolution.

DNA damage triggers reprogramming of differentiated cells into stem cells in Physcomitrella.

DNA damage can result from intrinsic cellular processes and from exposure to stressful environments. Such DNA damage generally threatens genome integrity and cell viability1. However, here we report that the transient induction of DNA strand breaks (single-strand breaks, double-strand breaks or both) in the moss Physcomitrella patens can trigger the reprogramming of differentiated leaf cells into stem cells without cell death. After intact leafy shoots (gametophores) were exposed to zeocin, an inducer of DNA strand breaks, the STEM CELL-INDUCING FACTOR 1 (STEMIN1)2 promoter was activated in some leaf cells. These cells subsequently initiated tip growth and underwent asymmetric cell divisions to form chloronema apical stem cells, which are in an earlier phase of the life cycle than leaf cells and have the ability to form new gametophores. This DNA-strand-break-induced reprogramming required the DNA damage sensor ATR kinase, but not ATM kinase, together with STEMIN1 and closely related proteins. ATR was also indispensable for the induction of STEMIN1 by DNA strand breaks. Our findings indicate that DNA strand breaks, which are usually considered to pose a severe threat to cells, trigger cellular reprogramming towards stem cells via the activity of ATR and STEMINs.