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Increasing pollution in the environment calls for the precise determination of metal toxicity in plants as they are at the base of the food chain. Mosses are often employed as biomonitors and provide good models for testing metal adsorption. However, species may react differently and many studies only look at one metal at a time, even though toxicity levels are affected by metal combinations. In this study, the effects of CuCl2, MnCl2, FeCl2, and Sb-acetate were examined individually and in combinations on the moss species Pohlia drummondii and Physcomitrium patens. In general, the two species reacted differently to the presence of trace metals; although, for both, the tolerance limit was at 100 µM. Overall, individual metals were less toxic than combinations, with some exceptions for Fe and Mn in P. patens. Additionally, we demonstrate that multiple combinations of metals are especially toxic if Cu is present. [ABSTRACT FROM AUTHOR]

SUMMARY: Plants exposed to light fluctuations are protected from photodamage by non‐photochemical quenching (NPQ), a reversible mechanism that enables dissipation of excess absorbed energy as heat, which is essential for plant fitness and crop productivity. In plants NPQ requires the presence of the membrane protein PsbS, which upon activation interacts with antenna proteins, inducing their dissipative conformation. Here, we exploited base editing (BE) in the moss Physcomitrium patens to introduce specific amino acid changes in vivo and assess their impact on PsbS activity, targeting transmembrane regions to investigate their role in essential protein–protein interactions. This approach enabled the recognition of residues essential for protein stability and the identification of a hydrophobic cluster of amino acids impacting PsbS activity. This work provides new information on the molecular mechanism of PsbS while also demonstrating the potential of BE approaches for in planta gene function analysis. [ABSTRACT FROM AUTHOR]

RAD51 is involved in finding and invading homologous DNA sequences for accurate homologous recombination (HR). Its paralogs have evolved to regulate and promote RAD51 functions. The efficient gene targeting and high HR rates are unique in plants only in the moss Physcomitrium patens (P. patens). In addition to two functionally equivalent RAD51 genes (RAD1-1 and RAD51-2), other RAD51 paralogues were also identified in P. patens. For elucidation of RAD51's involvement during DSB repair, two knockout lines were constructed, one mutated in both RAD51 genes (Pprad51-1-2) and the second with mutated RAD51B gene (Pprad51B). Both lines are equally hypersensitive to bleomycin, in contrast to their very different DSB repair efficiency. Whereas DSB repair in Pprad51-1-2 is even faster than in WT, in Pprad51B, it is slow, particularly during the second phase of repair kinetic. We interpret these results as PpRAD51-1 and -2 being true functional homologs of ancestral RAD51 involved in the homology search during HR. Absence of RAD51 redirects DSB repair to the fast NHEJ pathway and leads to a reduced 5S and 18S rDNA copy number. The exact role of the RAD51B paralog remains unclear, though it is important in damage recognition and orchestrating HR response. [ABSTRACT FROM AUTHOR]

The hemicelluloses comprise a group of matrix glycans that interact with cellulose microfibrils in plant cell walls and play important roles in establishing wall architecture. The structures of hemicelluloses are determined by carbohydrate‐active enzymes (CAZymes) that synthesize, integrate, and break down these polymers. Specifically, endo‐glucanase 16 (EG16) enzymes, which are related to the well‐known xyloglucan endotransglycosylase/hydrolase (XTH) gene products in Glycoside Hydrolase Family 16 (GH16), have been implicated in the degradation of the β(1,4)‐linked backbone of mixed‐linkage β(1,3);β(1,4)‐glucans (MLG) and xyloglucans. EG16 members are single‐copy genes found in most plant clades but are absent from many eudicots, including the model plant Arabidopsis thaliana. Until recently, EG16 members had only been characterized in vitro, establishing their substrate specificity, protein structure, and phylogenetic history, but their biological function was unknown. Here we used a hybrid polar, Populus alba × Populus grandidentata (P39), as a model to examine EG16 expression, subcellular localization, and pheno‐ and chemotypes of EG16‐downregulated P39 plants. PopulusEG16 expression is strong in young tissues, but RNAi‐mediated downregulation did not impact plant growth nor the fine structure of the hemicellulose xyloglucan, suggesting a restricted or currently unknown role in angiosperm physiology. [ABSTRACT FROM AUTHOR]

In flowering plants, the LEAFY (LFY) gene controls floral meristem activity. In early land plants such as mosses and ferns, it, however, has a minimum role in cell division and development of diploid sporophyte. Homology modeling, an accurate and efficient protein structure prediction method, was used to construct a 3D model of the LEAFY protein in nonflowering and flowering plants. The present study examines the following species: Charophyte green algae, Physcomitrella, Ceratopteris, Picea, and Arabidopsis, as they are the popularly used model organisms for developmental studies. LEAFY protein sequences from the model organisms were aligned by multiple sequence alignment. 3D models of the LEAFY protein from all the model organisms was constructed using the PHYRE2 program with 100% confidence, and the constructed models were evaluated using the MolProbity tool. On the basis of the conserved regions, Charophyte green algae shared 38–46% sequence similarity with Physcomitrella sp., 37–46% similarity with Ceratopteris sp., 33–41% similarity with Picea sp., and 32–38% similarity with Arabidopsis sp. The Motif Finder server identified the protein family domain FLO_LFY and LFY_SAM, whose function is floral meristem development. Secondary structure prediction analysis indicated that the LEAFY protein belongs to the alpha (α) protein class, which is stable against mutation and thus limits structural changes in the LEAFY protein. The study findings reveal two distinct clusters of the LFY gene from the common ancestor green algae. One cluster is present in nonflowering plants that include mosses, pteridophytes, and gymnosperms, and the other cluster is present in flowering plants that include orchids, monocots, dicots, and angiosperms.

Increasing pollution in the environment calls for the precise determination of metal toxicity in plants as they are at the base of the food chain. Mosses are often employed as biomonitors and provide good models for testing metal adsorption. However, species may react differently and many studies only look at one metal at a time, even though toxicity levels are affected by metal combinations. In this study, the effects of CuCl2, MnCl2, FeCl2, and Sb-acetate were examined individually and in combinations on the moss species Pohlia drummondii and Physcomitrium patens. In general, the two species reacted differently to the presence of trace metals; although, for both, the tolerance limit was at 100 µM. Overall, individual metals were less toxic than combinations, with some exceptions for Fe and Mn in P. patens. Additionally, we demonstrate that multiple combinations of metals are especially toxic if Cu is present.

Abstract The hemicelluloses comprise a group of matrix glycans that interact with cellulose microfibrils in plant cell walls and play important roles in establishing wall architecture. The structures of hemicelluloses are determined by carbohydrate‐active enzymes (CAZymes) that synthesize, integrate, and break down these polymers. Specifically, endo‐glucanase 16 (EG16) enzymes, which are related to the well‐known xyloglucan endotransglycosylase/hydrolase (XTH) gene products in Glycoside Hydrolase Family 16 (GH16), have been implicated in the degradation of the β(1,4)‐linked backbone of mixed‐linkage β(1,3);β(1,4)‐glucans (MLG) and xyloglucans. EG16 members are single‐copy genes found in most plant clades but are absent from many eudicots, including the model plant Arabidopsis thaliana. Until recently, EG16 members had only been characterized in vitro, establishing their substrate specificity, protein structure, and phylogenetic history, but their biological function was unknown. Here we used a hybrid polar, Populus alba × Populus grandidentata (P39), as a model to examine EG16 expression, subcellular localization, and pheno‐ and chemotypes of EG16‐downregulated P39 plants. Populus EG16 expression is strong in young tissues, but RNAi‐mediated downregulation did not impact plant growth nor the fine structure of the hemicellulose xyloglucan, suggesting a restricted or currently unknown role in angiosperm physiology.

Two-pore channels (TPCs) are members of the superfamily of ligand-gated and voltage-sensitive ion channels in the membranes of intracellular organelles of eukaryotic cells. The evolution of ordinary plant TPC1 essentially followed a very conservative pattern, with no changes in the characteristic structural footprints of these channels, such as the cytosolic and luminal regions involved in Ca2+ sensing. In contrast, the genomes of mosses and liverworts encode also TPC1-like channels with larger variations at these sites (TPC1b channels). In the genome of the model plant Physcomitrium patens we identified nine non-redundant sequences belonging to the TPC1 channel family, two ordinary TPC1-type, and seven TPC1b-type channels. The latter show variations in critical amino acids in their EF-hands essential for Ca2+ sensing. To investigate the impact of these differences between TPC1 and TPC1b channels, we generated structural models of the EF-hands of PpTPC1 and PpTPC1b channels. These models were used in molecular dynamics simulations to determine the frequency with which calcium ions were present in a coordination site and also to estimate the average distance of the ions from the center of this site. Our analyses indicate that the EF-hand domains of PpTPC1b-type channels have a lower capacity to coordinate calcium ions compared with those of common TPC1-like channels. [ABSTRACT FROM AUTHOR]

Summary: The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, where it acts via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad‐scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL‐like (WOX) proteins in bryophytes. The relationship between CLAVATA, hormone homeostasis and the evolution of land plant stem cell functions is unknown.Here we show that in the moss, Physcomitrella (Physcomitrium patens), CLAVATA affects stem cell activity by modulating hormone homeostasis. CLAVATA pathway genes are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching, plant spread and auxin synthesis. The receptor‐like kinase PpRPK2 plays the major role, and Pprpk2 mutants have abnormal responses to cytokinin, auxin and auxin transport inhibition, and show reduced expression of PIN auxin transporters.We propose a model whereby PpRPK2 modulates auxin gradients in filaments to determine stem cell identity and overall plant form.Our data indicate that CLAVATA‐mediated auxin homeostasis is a fundamental property of plant stem cell function, probably exhibited by the last shared common ancestor of land plants. [ABSTRACT FROM AUTHOR]

In flowering plants, the LEAFY (LFY) gene controls floral meristem activity. In early land plants such as mosses and ferns, it, however, has a minimum role in cell division and development of diploid sporophyte. Homology modeling, an accurate and efficient protein structure prediction method, was used to construct a 3D model of the LEAFY protein in nonflowering and flowering plants. The present study examines the following species: Charophyte green algae, Physcomitrella, Ceratopteris, Picea, and Arabidopsis, as they are the popularly used model organisms for developmental studies. LEAFY protein sequences from the model organisms were aligned by multiple sequence alignment. 3D models of the LEAFY protein from all the model organisms was constructed using the PHYRE2 program with 100% confidence, and the constructed models were evaluated using the MolProbity tool. On the basis of the conserved regions, Charophyte green algae shared 38-46% sequence similarity with Physcomitrella sp., 37-46% similarity with Ceratopteris sp., 33-41% similarity with Picea sp., and 32-38% similarity with Arabidopsis sp. The Motif Finder server identified the protein family domain FLO_LFY and LFY_SAM, whose function is floral meristem development. Secondary structure prediction analysis indicated that the LEAFY protein belongs to the alpha (a) protein class, which is stable against mutation and thus limits structural changes in the LEAFY protein. The study findings reveal two distinct clusters of the LFY gene from the common ancestor green algae. One cluster is present in nonflowering plants that include mosses, pteridophytes, and gymnosperms, and the other cluster is present in flowering plants that include orchids, monocots, dicots, and angiosperms. [ABSTRACT FROM AUTHOR]

Mosses (Bryophyta) are a key group occupying an important phylogenetic position in land plant (embryophyte) evolution. The class Bryopsida represents the most diversified lineage, containing more than 95% of modern mosses, whereas other classes are species‐poor. Two branches with large numbers of gene duplications were elucidated by phylogenomic analyses, one in the ancestry of all mosses and another before the separation of the Bryopsida, Polytrichopsida, and Tetraphidopsida. The analysis of the phylogenetic progression of duplicated paralogs retained on genomic syntenic regions in the Physcomitrella patens genome confirmed that the whole‐genome duplication events WGD1 and WGD2 were re‐recognized as the ψ event and the Funarioideae duplication event, respectively. The ψ polyploidy event was tightly associated with the early diversification of Bryopsida, in the ancestor of Bryidae, Dicranidae, Timmiidae, and Funariidae. Together, four branches with large numbers of gene duplications were unveiled in the evolutionary past of P. patens. Gene retention patterns following the four large‐scale duplications in different moss lineages were analyzed and discussed. Recurrent significant retention of stress‐related genes may have contributed to their adaption to distinct ecological environments and the evolutionary success of this early‐diverging land plant lineage. [ABSTRACT FROM AUTHOR]

Key message: Kleisin NSE4 and circular form of SMC5/6 is indispensable for DSB repair and necessary for gene targeting but is not enough for recovery of cells from DNA damage in Physcomitrella. Structural maintenance of chromosomes (SMC) complexes are involved in cohesion, condensation and maintenance of genome stability. Based on the sensitivity of mutants to genotoxic stress the SMC5/6 complex is thought to play a prominent role in DNA stabilization during repair by tethering DNA at the site of lesion by a heteroduplex of SMC5 and SMC6 encircled with non-SMC components NSE1, NSE3 and kleisin NSE4. In this study, we tested how formation of the SMC5/6 circular structure affects mutant sensitivity to DNA damage, kinetics of DSB repair and gene targeting. In the moss Physcomitrella (Physcomitrium patens), SMC6 and NSE4 are essential single copy genes and this is why we used blocking of transcription to reveal their mutated phenotype. Even slight reduction of transcript levels by dCas9 binding was enough to obtain stable lines with severe DSB repair defects and specific bleomycin sensitivity. We show that survival after bleomycin or MMS treatment fully depends on active SMC6, whereas attenuation of NSE4 has little or negligible effect. We conclude that circularization of SMC5/6 provided by the kleisin NSE4 is indispensable for the DSB repair, nevertheless there are other functions associated with the SMC5/6 complex, which are critical to survive DNA damage. [ABSTRACT FROM AUTHOR]

Two-pore channels (TPCs) are members of the superfamily of ligand-gated and voltage-sensitive ion channels in the membranes of intracellular organelles of eukaryotic cells. The evolution of ordinary plant TPC1 essentially followed a very conservative pattern, with no changes in the characteristic structural footprints of these channels, such as the cytosolic and luminal regions involved in Ca2+ sensing. In contrast, the genomes of mosses and liverworts encode also TPC1-like channels with larger variations at these sites (TPC1b channels). In the genome of the model plant Physcomitrium patens we identified nine non-redundant sequences belonging to the TPC1 channel family, two ordinary TPC1-type, and seven TPC1b-type channels. The latter show variations in critical amino acids in their EF-hands essential for Ca2+ sensing. To investigate the impact of these differences between TPC1 and TPC1b channels, we generated structural models of the EF-hands of PpTPC1 and PpTPC1b channels. These models were used in molecular dynamics simulations to determine the frequency with which calcium ions were present in a coordination site and also to estimate the average distance of the ions from the center of this site. Our analyses indicate that the EF-hand domains of PpTPC1b-type channels have a lower capacity to coordinate calcium ions compared with those of common TPC1-like channels.

Summary: Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the d‐mannose/l‐galactose (d‐Man/l‐Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophytes, while the d‐galacturonate (d‐GalUA) pathway is found in prasinophytes and euglenoids. Based on the presence of the complete set of genes encoding enzymes involved in the d‐Man/l‐Gal pathway and an orthologous gene encoding aldonolactonase (ALase) – a key enzyme for the d‐GalUA pathway – Physcomitrium patens may possess both pathways. Here, we have characterized the moss ALase as a functional lactonase and evaluated the ascorbate biosynthesis capability of the two pathways using knockout mutants. Physcomitrium patens expresses two ALase paralogs, namely PpALase1 and PpALase2. Kinetic analyses with recombinant enzymes indicated that PpALase1 is a functional enzyme catalyzing the conversion of l‐galactonic acid to the final precursor l‐galactono‐1,4‐lactone and that it also reacts with dehydroascorbate as a substrate. Interestingly, mutants lacking PpALase1 (Δal1) showed 1.2‐fold higher total ascorbate content than the wild type, and their dehydroascorbate content was increased by 50% compared with that of the wild type. In contrast, the total ascorbate content of mutants lacking PpVTC2‐1 (Δvtc2‐1) or PpVTC2‐2 (Δvtc2‐2), which encode the rate‐limiting enzyme GDP‐l‐Gal phosphorylase in the d‐Man/l‐Gal pathway, was markedly decreased to 46 and 17%, respectively, compared with that of the wild type. Taken together, the dominant ascorbate biosynthetic pathway in P. patens is the d‐Man/l‐Gal pathway, not the d‐GalUA pathway, and PpALase1 may play a significant role in ascorbate metabolism by facilitating dehydroascorbate degradation rather than ascorbate biosynthesis. Significance Statement: The d‐mannose/l‐galactose (d‐Man/l‐Gal) pathway is the dominant l‐ascorbate (ASC) biosynthetic pathway in vascular land plants and some green algae; however, an alternative pathway utilizing d‐galacturonate (d‐GalUA) has also been proposed. Physcomitrium patens possesses orthologs of genes encoding aldonolactonase (ALase), a key enzyme in the d‐GalUA pathway, in addition to the complete set of genes encoding enzymes involved in the d‐Man/l‐Gal pathway. The current study shows that, in P. patens, the dominant pathway for ASC biosynthesis is the d‐Man/l‐Gal pathway, not the d‐GalUA pathway, and ALase play a pivotal role in ASC metabolism via oxidized ascorbate degradation rather than ASC biosynthesis. [ABSTRACT FROM AUTHOR]

Abstract Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella patens, 4‐hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long‐chain base (LCB). To assess the functional significance of t18:0, CRISPR‐Cas9 mutagenesis was used to generate mutant lines lacking the sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content in sbh protonemata was 2.4‐fold higher than in wild‐type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl‐chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl‐chain pairing in glucosylceramides, in which 4,8‐sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. The sbh lines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content of sbh lines decreased to wild‐type levels, and the mutants exhibited phenotypes more similar to wild‐type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyte P. patens and angiosperm A. thaliana.

Sphingolipids have roles as membrane structural components and as bioactive molecules in plants. In Physcomitrella patens, 4‐hydroxysphinganine (phytosphingosine, t18:0) is the predominant sphingolipid long‐chain base (LCB). To assess the functional significance of t18:0, CRISPR‐Cas9 mutagenesis was used to generate mutant lines lacking the sole SPHINGOID BASE HYDROXYLASE (SBH) gene encoding the hydroxylase responsible for converting sphinganine (d18:0) to t18:0. Total sphingolipid content in sbh protonemata was 2.4‐fold higher than in wild‐type. Modest changes in glycosyl inositolphosphorylceramide (GIPC) glycosylation patterns occurred. Sphingolipidomic analyses of mutants lacking t18:0 indicated modest alterations in acyl‐chain pairing with d18:0 in GIPCs and ceramides, but dramatic alterations in acyl‐chain pairing in glucosylceramides, in which 4,8‐sphingadienine (d18:2) was the principal LCB. A striking accumulation of free and phosphorylated LCBs accompanied loss of the hydroxylase. The sbh lines exhibited altered morphology, including smaller chloronemal cell size, irregular cell shape, reduced gametophore size, and increased pigmentation. In the presence of the synthetic trihydroxy LCB t17:0, the endogenous sphingolipid content of sbh lines decreased to wild‐type levels, and the mutants exhibited phenotypes more similar to wild‐type plants. These results demonstrate the importance of sphingolipid content and composition to Physcomitrella growth. They also illuminate similarities in regulating sphingolipid content but differences in regulating sphingolipid species composition between the bryophyte P. patens and angiosperm A. thaliana. [ABSTRACT FROM AUTHOR]

Summary: Phylogenetic trees have permeated biology. However, an understanding of how to interpret phylogenies has lagged behind, notably in publications outside of evolutionary biology. Here I argue that some language commonly used in plant systematics has contributed to the confusion by describing phylogenetic trees using intuitive but misleading terms reminiscent of Aristotle's Scala Naturae. These terms (perhaps inadvertently) misrepresent evolution, not as a process acting on all living species, but rather as a progression of successively diverging lineages leading to a group that represents a subjectively defined endpoint. My goal here is to show how thinking of the tree of life in terms of early‐diverging lineages and higher groups can distort evolutionary literacy, confound interdisciplinary communication, and potentially bias research agendas. I focus on the relationship between bryophytes and angiosperms as a case study, but the theme applies to all branches of the tree of life. Fortunately, evolutionary biologists have developed an easily understood alternative framework – tree thinking – which I highlight as a means to promote a clear understanding of phylogenies across sub‐disciplines of biology, and between practicing biologists and students, or members the public which funds much of our work. [ABSTRACT FROM AUTHOR]

Phyllotaxis, the geometry of leaf arrangement around stems, determines plant architecture. Molecular interactions coordinating the formation of phyllotactic patterns have mainly been studied in multicellular shoot apical meristems of flowering plants. Phyllotaxis evolved independently in the major land plant lineages. In mosses, it arises from a single apical cell, raising the question of how asymmetric divisions of a single-celled meristem create phyllotactic patterns and whether associated genetic processes are shared across lineages. We present an overview of the mechanisms governing shoot apical cell specification and activity in the model moss, Physcomitrium patens , and argue that similar molecular regulatory modules have been deployed repeatedly across evolution to operate at different scales and drive apical function in convergent shoot forms. Convergent phyllotactic patterns shape architectural diversity in land plants. Phyllotaxis is determined by rotating divisions of a single apical cell in mosses. 4D patterns of apical cell division plane orientation rely on geometrical, physical, and biochemical cues. Shared molecular players act at different scales to drive leafy shoot morphogenesis and pattern phyllotaxis in distantly related plant lineages. [ABSTRACT FROM AUTHOR]

DNMT2 is a DNA/tRNA cytosine methyltransferase that is highly conserved in structure and function in eukaryotes. In plants however, limited information is available on the function of this methyltransferase. We have previously reported that in the moss Physcomitrella patens, DNMT2 plays a crucial role in stress recovery and tRNAAsp transcription/stability under salt stress. To further investigate the role of PpDNMT2 at genome level, in this study we have performed RNA sequencing of ppdnmt2. Transcriptome analysis reveals a number of genes and pathways to function differentially and suggests a close link between PpDNMT2 function and osmotic and ionic stress tolerance. We propose PpDNMT2 to play a pivotal role in regulating salt tolerance by affecting molecular networks involved in stress perception and signal transduction that underlie maintenance of ion homeostasis in cells. We also examined interactome of PpDNMT2 using affinity purification (AP) coupled to mass spectrometry (AP-MS). Quantitative proteomic analysis reveals several chloroplast proteins involved in light reactions and carbon assimilation and proteins involved in stress response and some not implicated in stress to co-immunoprecipitate with PpDNMT2. Comparison between transcriptome and interactome datasets has revealed novel association between PpDNMT2 activity and the antioxidant enzyme Superoxide dismutase (SOD), protein turnover mediated by the Ubiquitin-proteasome system and epigenetic gene regulation. PpDNMT2 possibly exists in complex with CuZn-SODs in vivo and the two proteins also directly interact in the yeast nucleus as observed by yeast two-hybrid assay. Taken together, the work presented in this study sheds light on diverse roles of PpDNMT2 in maintaining molecular and physiological homeostasis in P. patens. This is a first report describing transcriptome and interactome of DNMT2 in any land plant.

COR413 genes belong to a poorly characterized group of plant-specific cold-regulated genes initially identified as part of the transcriptional activation machinery of plants during cold acclimation. They encode multispanning transmembrane proteins predicted to target the plasma membrane or the chloroplast inner membrane. Despite being ubiquitous throughout the plant kingdom, little is known about their biological function. In this study, we used reverse genetics to investigate the relevance of a predicted chloroplast localized COR413 protein (PpCOR413im) from the moss Physcomitrella patens in developmental and abiotic stress responses. Expression of PpCOR413im was strongly induced by abscisic acid (ABA) and by various environmental stimuli, including low temperature, hyperosmosis, salinity and high light. In vivo subcellular localization of PpCOR413im-GFP fusion protein revealed that this protein is localized in chloroplasts, confirming the in silico predictions. Loss-of-function mutants of PpCOR413im exhibited growth and developmental alterations such as growth retardation, reduced caulonema formation and hypersensitivity to ABA. Mutants also displayed altered photochemistry under various abiotic stresses, including dehydration and low temperature, and exhibited a dramatic growth inhibition upon exposure to high light. Disruption of PpCOR413im also caused altered chloroplast ultrastructure, increased ROS accumulation, and enhanced starch and sucrose levels under high light or after ABA treatment. In addition, loss of PpCOR413im affected both nuclear and chloroplast gene expression in response to ABA and high light, suggesting a role for this gene downstream of ABA in the regulation of growth and environmental stress responses. Developmental alterations exhibited by PpCOR413im knockout mutants had remarkable similarities to those exhibited by hxk1, a mutant lacking a major chloroplastic hexokinase, an enzyme involved in energy homeostasis. Based on these findings, we propose that PpCOR413im is involved in coordinating energy metabolism with ABA-mediated growth and developmental responses.

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana, moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release.

Alternative electron pathways contribute to regulation of photosynthetic light reactions to adjust to metabolic demands in dynamic environments. The chloroplast NADH dehydrogenase-like (NDH) complex mediates the cyclic electron transport pathway around PSI in different cyanobacteria, algae, and plant species, but it is not fully conserved in all photosynthetic organisms. In order to assess how the physiological role of this complex changed during plant evolution, we isolated Physcomitrella patens lines knocked out for the NDHM gene that encodes a subunit fundamental for the activity of the complex. ndhm knockout mosses indicated high PSI acceptor side limitation upon abrupt changes in illumination. In P. patens , pseudo-cyclic electron transport mediated by flavodiiron proteins (FLVs) was also shown to prevent PSI over-reduction in plants exposed to light fluctuations. flva ndhm double knockout mosses had altered photosynthetic performance and growth defects under fluctuating light compared with the wild type and single knockout mutants. The results showed that while the contribution of NDH to electron transport is minor compared with FLV, NDH still participates in modulating photosynthetic activity, and it is critical to avoid PSI photoinhibition, especially when FLVs are inactive. The functional overlap between NDH- and FLV-dependent electron transport supports PSI activity and prevents its photoinhibition under light variations. [ABSTRACT FROM AUTHOR]

DNMT2 is a DNA/tRNA cytosine methyltransferase that is highly conserved in structure and function in eukaryotes. In plants however, limited information is available on the function of this methyltransferase. We have previously reported that in the moss Physcomitrella patens , DNMT2 plays a crucial role in stress recovery and tRNAAsp transcription/stability under salt stress. To further investigate the role of PpDNMT2 at genome level, in this study we have performed RNA sequencing of ppdnmt2. Transcriptome analysis reveals a number of genes and pathways to function differentially and suggests a close link between PpDNMT2 function and osmotic and ionic stress tolerance. We propose PpDNMT2 to play a pivotal role in regulating salt tolerance by affecting molecular networks involved in stress perception and signal transduction that underlie maintenance of ion homeostasis in cells. We also examined interactome of PpDNMT2 using affinity purification (AP) coupled to mass spectrometry (AP-MS). Quantitative proteomic analysis reveals several chloroplast proteins involved in light reactions and carbon assimilation and proteins involved in stress response and some not implicated in stress to co-immunoprecipitate with PpDNMT2. Comparison between transcriptome and interactome datasets has revealed novel association between PpDNMT2 activity and the antioxidant enzyme Superoxide dismutase (SOD), protein turnover mediated by the Ubiquitin-proteasome system and epigenetic gene regulation. PpDNMT2 possibly exists in complex with CuZn-SODs in vivo and the two proteins also directly interact in the yeast nucleus as observed by yeast two-hybrid assay. Taken together, the work presented in this study sheds light on diverse roles of PpDNMT2 in maintaining molecular and physiological homeostasis in P. patens. This is a first report describing transcriptome and interactome of DNMT2 in any land plant. [ABSTRACT FROM AUTHOR]

COR413 genes belong to a poorly characterized group of plant-specific cold-regulated genes initially identified as part of the transcriptional activation machinery of plants during cold acclimation. They encode multispanning transmembrane proteins predicted to target the plasma membrane or the chloroplast inner membrane. Despite being ubiquitous throughout the plant kingdom, little is known about their biological function. In this study, we used reverse genetics to investigate the relevance of a predicted chloroplast localized COR413 protein (PpCOR413im) from the moss Physcomitrella patens in developmental and abiotic stress responses. Expression of PpCOR413im was strongly induced by abscisic acid (ABA) and by various environmental stimuli, including low temperature, hyperosmosis, salinity and high light. In vivo subcellular localization of PpCOR413im-GFP fusion protein revealed that this protein is localized in chloroplasts, confirming the in silico predictions. Loss-of-function mutants of PpCOR413im exhibited growth and developmental alterations such as growth retardation, reduced caulonema formation and hypersensitivity to ABA. Mutants also displayed altered photochemistry under various abiotic stresses, including dehydration and low temperature, and exhibited a dramatic growth inhibition upon exposure to high light. Disruption of PpCOR413im also caused altered chloroplast ultrastructure, increased ROS accumulation, and enhanced starch and sucrose levels under high light or after ABA treatment. In addition, loss of PpCOR413im affected both nuclear and chloroplast gene expression in response to ABA and high light, suggesting a role for this gene downstream of ABA in the regulation of growth and environmental stress responses. Developmental alterations exhibited by PpCOR413im knockout mutants had remarkable similarities to those exhibited by hxk1 , a mutant lacking a major chloroplastic hexokinase, an enzyme involved in energy homeostasis. Based on these findings, we propose that PpCOR413im is involved in coordinating energy metabolism with ABA-mediated growth and developmental responses. [ABSTRACT FROM AUTHOR]

Mosses are an ancient land plant lineage and are therefore important in studying the evolution of plant developmental processes. Here, we describe stomatal development in the model moss species Physcomitrium patens (previously known as Physcomitrella patens) over the duration of sporophyte development. We dissect the molecular mechanisms guiding cell division and fate and highlight how stomatal function might vary under different environmental conditions. In contrast to the asymmetric entry divisions described in Arabidopsis thaliana , moss protodermal cells can enter the stomatal lineage directly by expanding into an oval shaped guard mother cell (GMC). We observed that when two early stage P. patens GMCs form adjacently, a spacing division can occur, leading to separation of the GMCs by an intervening epidermal spacer cell. We investigated whether orthologs of Arabidopsis stomatal development regulators are required for this spacing division. Our results indicated that bHLH transcription factors PpSMF1 and PpSCRM1 are required for GMC formation. Moreover, the ligand and receptor components PpEPF1 and PpTMM are also required for orientating cell divisions and preventing single or clustered early GMCs from developing adjacent to one another. The identification of GMC spacing divisions in P. patens raises the possibility that the ability to space stomatal lineage cells could have evolved before mosses diverged from the ancestral lineage. This would have enabled plants to integrate stomatal development with sporophyte growth and could underpin the adoption of multiple bHLH transcription factors and EPF ligands to more precisely control stomatal patterning in later diverging plant lineages. We also observed that when P. patens sporophyte capsules mature in wet conditions, stomata are typically plugged whereas under drier conditions this is not the case; instead, mucilage drying leads to hollow sub-stomatal cavities. This appears to aid capsule drying and provides further evidence for early land plant stomata contributing to capsule rupture and spore release. [ABSTRACT FROM AUTHOR]

Physcomitrium (Physcomitrella) patens is a bryophyte highly tolerant to different stresses, allowing survival when water supply is a limiting factor. This moss lacks a true vascular system, but it has evolved a primitive water-conducting system that contains lignin-like polyphenols. By means of a three-step protocol, including ammonium sulfate precipitation, adsorption chromatography on phenyl Sepharose and cationic exchange chromatography on SP Sepharose, we were able to purify and further characterize a novel class III peroxidase, PpaPrx19, upregulated upon salt and H2O2 treatments. This peroxidase, of a strongly basic nature, shows surprising homology to angiosperm peroxidases related to lignification, despite the lack of true lignins in P. patens cell walls. Moreover, PpaPrx19 shows catalytic and kinetic properties typical of angiosperm peroxidases involved in oxidation of monolignols, being able to efficiently use hydroxycinnamyl alcohols as substrates. Our results pinpoint the presence in P. patens of peroxidases that fulfill the requirements to be involved in the last step of lignin biosynthesis, predating the appearance of true lignin.

Summary: The Arabidopsis COP1/SPA complex is a key repressor of photomorphogenesis that suppresses light signaling in the dark. Both COP1 and SPA proteins are essential components of this complex. Although COP1 also exists in humans, SPA genes are specific to the green lineage.To elucidate the evolution of SPA genes we analyzed SPA functions in the moss Physcomitrella patens by characterizing knockout mutants in the two Physcomitrella SPA genes PpSPAa and PpSPAb.Light‐grown PpspaAB double mutants exhibit smaller gametophores than the wild‐type. In the dark, PpspaAB mutant gametophores show enhanced continuation of growth but etiolate normally. Gravitropism in the dark is reduced in PpspaAB mutant protonemata. The expression of light‐regulated genes is mostly not constitutive in PpspaAB mutants. PpSPA and PpCOP1 interact; PpCOP1 also interacts with the transcription factor PpHY5 and, indeed, PpHY5 is destabilized in dark‐grown Physcomitrella. Degradation of PpHY5 in darkness, however, does not require PpSPAa and PpSPAb.The data suggest that COP1/SPA‐mediated light signaling is only partially conserved between Arabidopsis and Physcomitrella. Whereas COP1/SPA interaction and HY5 degradation in darkness is conserved, the role of SPA proteins appears to have diverged. PpSPA genes, unlike their Arabidopsis counterparts, are only required to suppress a subset of light responses in darkness. See also the Commentary on this article by Hiltbrunner, 224: 1412–1414. [ABSTRACT FROM AUTHOR]

Introduction.Aphanorrhegma (Physcomitrella) patens (Hedw.) Lindb. is an attractive model system for comparative analyses of leaf development because it evolved leaves (phyllids) independently to flowering plants, yet its genome contains homologues of many gene families that regulate angiosperm leaf development. In addition, A. patens phyllids are primarily a single cell layer thick, making it simple to identify the cellular basis of defects that perturb shape. Identification of gene functions in shape determination depends on like-for-like comparison of mutant versus wild-type plants. Key results. Here we show that, if heteroblasty is not perturbed, such comparisons should use phyllid L13 or above in the heteroblastic series, and fully expanded phyllids above P7 in the developmental series. Using a quantitative approach, we show that heteroblastic size variation reflects differences in cell proliferation rather than cell size and shape. A comparison of control to pinA pinB mutant phyllid development verifies that PIN proteins promote cell proliferation and suppress expansion to determine phyllid shape. Conclusion. The results and approach that we have generated will be applicable to any study of A. patens phyllid development to reveal the cellular basis of phyllid size and shape variations. [ABSTRACT FROM AUTHOR]

Mosses are one of the earliest land plants that diverged from fresh-water green algae. They are considered to have acquired a higher capacity for thermal energy dissipation to cope with dynamically changing solar irradiance by utilizing both the "algal-type" light-harvesting complex stress-related (LHCSR)-dependent and the "plant-type" PsbS-dependent mechanisms. It is hypothesized that the formation of photosystem (PS) I and II megacomplex is another mechanism to protect photosynthetic machinery from strong irradiance. Herein, we describe the analysis of the PSI–PSII megacomplex from the model moss, Physcomitrella patens, which was resolved using large-pore clear-native polyacrylamide gel electrophoresis (lpCN-PAGE). The similarity in the migration distance of the Physcomitrella PSI–PSII megacomplex to the Arabidopsis megacomplex shown during lpCN-PAGE suggested that the Physcomitrella PSI–PSII and Arabidopsis megacomplexes have similar structures. Time-resolved chlorophyll fluorescence measurements show that excitation energy was rapidly and efficiently transferred from PSII to PSI, providing evidence of an ordered association of the two photosystems. We also found that LHCSR and PsbS co-migrated with the Physcomitrella PSI–PSII megacomplex. The megacomplex showed pH-dependent chlorophyll fluorescence quenching, which may have been induced by LHCSR and/or PsbS proteins with the collaboration of zeaxanthin. We discuss the mechanism that regulates the energy distribution balance between two photosystems in Physcomitrella. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a devastating slowly progressive neurodegenerative disorder with no cure. While there are many hypotheses, the exact mechanism causing this pathology is still unknown. Among many other features, AD is characterized by brain hypometabolism and decreased sugar availability, to which neurons eventually succumb. In light of this aspect of the disease, we hypothesized that boosting fuel supply to neurons may help them survive or at least alleviate some of the symptoms. Here we demonstrate that live moss Physcomitrella patens cells can be safely co-cultured with human fibroblasts in vitro and thus have a potential for providing human cells with energy and other vital biomolecules. These data may form the foundation for the development of novel approaches to metabolic bioengineering and treatment of diseased cells based on live plants. In addition, by providing alternative energy sources to human tissues, the biotechnological potential of this interkingdom setup could also serve as a springboard to foster innovative dietary processes addressing current challenges of mankind such as famine or supporting long-haul space flight. [ABSTRACT FROM AUTHOR]

Summary: Double‐stranded breaks can be repaired by different mechanisms such as homologous recombination (HR), classical nonhomologous end joining (C‐NHEJ) and alternative end joining (Alt‐EJ). Polymerase Q (POLQ) has been proposed to be the main factor involved in Alt‐EJ‐mediated DNA repair.Here we describe the role of POLQ in DNA repair and gene targeting in Physcomitrella patens. The disruption of the POLQ gene does not influence the genetic stability of P. patens nor its development.The polq mutant shows the same sensitivity as wild‐type towards most of the genotoxic agents tested (ultraviolet (UV), methyl methanesulfonate (MMS) and cisplatin) with the notable exception of bleomycin for which it shows less sensitivity than the wild‐type. Furthermore, we show that POLQ is involved in the repair of CRISPR‐Cas9‐induced double‐stranded breaks in P. patens. We also demonstrate that POLQ is a potential competitor and/or inhibitor of the HR repair pathway.This finding has a consequence in terms of genetic engineering, as in the absence of POLQ the frequency of gene targeting is significantly increased and the number of clean two‐sided HR‐mediated insertions is enhanced. Therefore, the control of POLQ activity in plants could be a useful strategy to optimize the tools of genome engineering for plant breeding. [ABSTRACT FROM AUTHOR]

Moss‐aGalactosidase A (moss‐aGal) is a moss‐derived version of human α‐galactosidase developed for enzyme replacement therapy in patients with Fabry disease. It exhibits a homogenous N‐glycosylation profile with >90% mannose‐terminated glycans. In contrast to mammalian cell produced α‐galactosidase, moss‐aGal does not rely on mannose‐6‐phosphate receptor mediated endocytosis but targets the mannose receptor for tissue uptake. We conducted a phase 1 clinical trial with moss‐aGal in six patients with confirmed diagnosis of Fabry disease during a 28‐day schedule. All patients received a single dose of 0.2 mg/kg moss‐aGal by i.v.‐infusion. Primary endpoints of the trial were safety and pharmacokinetics; secondary endpoints were pharmacodynamics by analyzing urine and plasma Gb3 and lyso‐Gb3 concentrations. In all patients, the administered single dose was well tolerated. No safety issues were observed. Pharmacokinetic data revealed a stable nonlinear profile with a short plasma half‐life of moss‐aGal of 14 minutes. After one single dose of moss‐aGal, urinary Gb3 concentrations decreased up to 23% 7 days and up to 60% 28 days post‐dose. Plasma concentrations of lyso‐Gb3 decreased by 3.8% and of Gb3 by 11% 28 days post‐dose. These data reveal that a single dose of moss‐aGal was safe, well tolerated, and led to a prolonged reduction of Gb3 excretion. As previously shown, moss‐aGal is taken up via the mannose receptor, which is expressed on macrophages but also on endothelial and kidney cells. Thus, these data indicate that moss‐aGal may target kidney cells. After these promising results, phase 2/3 clinical trials are in preparation. [ABSTRACT FROM AUTHOR]

Highlights • PnJAZ1 is a stress-induced gene from Antarctic moss and its protein levels are reduced by OPDA treatment. • PnJAZ1 interacts with the AtMYC2 transcription factor and mediates the conserved jasmonate signalling pathway. • The overexpression of PnJAZ1 increases the tolerance of transgenic plants to salt and osmotic stresses and ABA treatment. • PnJAZ1 regulates interactions between the jasmonate and ABA signalling pathways. Abstract Abscisic acid (ABA) and jasmonates (JAs) are the primary plant hormones involved in mediating salt tolerance. In addition, these two plant hormones exert a synergistic effect to inhibit seed germination. However, the molecular mechanism of the interaction between ABA signalling and JA signalling is still not well documented. Here, a moss jasmonate ZIM-domain gene (PnJAZ1), which encodes a nucleus-localized protein with conserved ZIM and Jas domains, was cloned from Pohlia nutans. PnJAZ1 expression was rapidly induced by various abiotic stresses. The PnJAZ1 protein physically interacted with MYC2 and was degraded by exogenous 12-oxo-phytodienoic acid (OPDA) treatment, implying that the JAZ protein-mediated signalling pathway is conserved in plants. Transgenic Arabidopsis and Physcomitrella plants overexpressing PnJAZ1 showed increased tolerance to salt stress and decreased ABA sensitivity during seed germination and early development. The overexpression of PnJAZ1 inhibited the expression of ABA pathway genes related to seed germination and seedling growth. Moreover, the transgenic Arabidopsis lines exhibited enhanced tolerance to auxin (IAA) and glucose, mimicking the phenotypes of abi4 or abi5 mutants. These results suggest that PnJAZ1 acts as a repressor, mediates JA-ABA synergistic crosstalk and enhances plant growth under salt stress. [ABSTRACT FROM AUTHOR]

This article is a Commentary on Storti et al. (2020), 228: 1316–1326. [ABSTRACT FROM AUTHOR]

Summary: In flowering plants, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1)/TERMINAL FLOWER 2 (TFL2) is known to interact with polycomb group (PcG) and non‐PcG proteins and control developmental programs. LHP1/TFL2 is an ancient protein and has been characterized in the early‐divergent plant Physcomitrella patens. However, interacting partners of PpLHP1 other than the chromomethylase PpCMT have not been identified to date. Also, while functional polycomb repressive complex 2 (PRC2) is known to exist in P. patens, there is no experimental evidence to support the existence of PRC1‐like complexes in these mosses. In this study, using protein−protein interaction methods, transient expression assays and targeted gene knockout strategy, we report the conserved properties of LHP1/TFL2 using the Physcomitrella system. We show that a PRC1‐like core complex comprising of PpLHP1 and the putative PRC1 Really Interesting New Gene (RING)‐finger proteins can form in vivo. Also, the interaction between PpRING and the PRC2 subunit PpCLF further sheds light on the possible existence of combinatorial interactions between the Polycomb Repressive Complex (PRC) in early land plants. Based on the interaction between PpLHP1 and putative hnRNP PpLIF2‐like in planta, we propose that the link between PpLHP1 regulation and RNA metabolic processes was established early in plants. The conserved subnuclear distribution pattern of PpLHP1 in moss protonema further provides insight into the manner in which LHP1/TFL2 are sequestered in the nucleoplasm in discrete foci. The PpLHP1 loss‐of‐function plants generated in this study share some of the pleiotropic defects with multiple aberrations reported in lhp1/tfl2. Taken together, this work documents an active role for PpLHP1 in epigenetic regulatory network in P. patens. Significance Statement: We provide evidence for the existence of a PRC1‐like complex in P. patens and conservation of LHP1 function in mosses and Arabidopsis. Interaction of PpLHP1 with PRC1 Really Interesting New Gene (RING)‐finger proteins that further interact with PRC2 PpCLF advocates the existence of a transcriptional repression mechanism in these early plants. Association of PpLHP1 with hnRNP PpLIF2‐like 1 suggests the conservation of a link between PpLHP1 regulation and RNA processes in land plants. pplhp1 plants share morphological aberrations with Arabidopsis lhp1/tfl2. [ABSTRACT FROM AUTHOR]

Mosses, liverworts, hornworts and lycophytes represent transition stages between the aquatic to terrestrial/land plants. Several morphological and adaptive novelties driven by genomic components including emergence and expansion of new or existing gene families have played a critical role during and after the transition, and contributed towards successful colonization of terrestrial ecosystems. It is crucial to decipher the evolutionary transitions and natural selection on the gene structure and function to understand the emergence of phenotypic and adaptive diversity. Plants at the “transition zone”, between aquatic and terrestrial ecosystem, are also the most vulnerable because of climate change and may contain clues for successful mitigation of the challenges of climate change. Identification and comparative analyses of such genetic elements and gene families are few in mosses, liverworts, hornworts and lycophytes. Ovate family proteins (OFPs) are plant-specific transcriptional repressors and are acknowledged for their roles in important growth and developmental processes in land plants, and information about the functional aspects of OFPs in early land plants is fragmentary. As a first step towards addressing this gap, a comprehensive in silico analysis was carried out utilizing publicly available genome sequences of Marchantia polymorpha (Mp), Physcomitrella patens (Pp), Selaginella moellendorffii (Sm) and Sphagnum fallax (Sf). Our analysis led to the identification of 4 MpOFPs, 19 PpOFPs, 6 SmOFPs and 3 SfOFPs. Cross-genera analysis revealed a drastic change in the structure and physiochemical properties in OFPs suggesting functional diversification and genomic plasticity during the evolutionary course. Knowledge gained from this comparative analysis will form the framework towards deciphering and dissection of their developmental and adaptive role/s in early land plants and could provide insights into evolutionary strategies adapted by land plants. [ABSTRACT FROM AUTHOR]

Two-component systems (TCSs) are signal transduction mechanisms for responding to various environmental stimuli. In angiosperms, TCSs involved in phytohormone signaling have been intensively studied, whereas there are only a few reports on TCSs in basal land plants. The moss Physcomitrella patens possesses several histidine kinases (HKs) that are lacking in seed plant genomes. Here, we studied two of these unique HKs, PAS-histidine kinase 1 (PHK1) and its paralog PHK2, both of which have PAS (Per–Arnt–Sim) domains, which are known to show versatile functions such as sensing light or molecular oxygen. We found homologs of PHK1 and PHK2 only in early diverged clades such as bryophytes and lycophytes, but not in seed plants. The PAS sequences of PHK1 and PHK2 are more similar to a subset of bacterial PAS sequences than to any angiosperm PAS sequences. Gene disruption lines that lack either PHK1 or PHK2 or both formed gametophores earlier than the wild-type, and consistently, more caulonema side branches were induced in response to light in the disruption lines. Therefore, PHK1 and PHK2 delay the timing of gametophore development, probably by suppressing light-induced caulonema branching. This study provides new insights into the evolution of TCSs in plants. [ABSTRACT FROM AUTHOR]

Summary: The plant hormone auxin regulates many aspects of plant growth and development. Auxin signaling involves hormone perception by the TRANSPORT INHIBITOR RESPONSE/AUXIN F‐BOX (TIR1/AFB)–Aux/IAA co‐receptor system, and the subsequent degradation of the Aux/IAA transcriptional repressors by the ubiquitin proteasome pathway. This leads to the activation of downstream gene expression and diverse physiological responses. Here, we investigate how the structural elements in the Aux/IAAs determine their function in Auxin perception and transcriptional repression. We took advantage of the facile genetics of the moss Physcomitrella patens to determine the activity of wild‐type and mutant PpIAA1a proteins in a Δaux/iaa null background. In this way, Aux/IAA function was characterized at the molecular and physiological levels without the interference of genetic redundancy. We identified and characterized degron variants in Aux/IAAs that affect their stability and Auxin response. We also demonstrated that neither the Aux/IAA EAR motif nor Aux/IAA oligomerization is essential for the repressive function of Aux/IAA. Our study demonstrates how key elements within the Aux/IAA proteins fine tune stability and repressor activity, as well as the long‐term developmental outcome. [ABSTRACT FROM AUTHOR]

Summary: Forward genetics is now straightforward in the moss Physcomitrella patens, and large mutant populations can be screened relatively easily. However, perturbation of development before the formation of gametes currently leaves no route to gene discovery. Somatic hybridization has previously been used to rescue sterile mutants and to assign P. patens mutations to complementation groups, but the cellular basis of the fusion process could not be monitored, and there was no tractable way to identify causative mutations. Here we use fluorescently tagged lines to generate somatic hybrids between Gransden (Gd) and Villersexel (Vx) strains of P. patens, and show that hybridization produces fertile diploid gametophytes that form phenotypically normal tetraploid sporophytes. Quantification of genetic variation between the two parental strains reveals single nucleotide polymorphisms at a frequency of 1/286 bp. Given that the genetic distinction between Gd and Vx strains exceeds that found between pairs of strains that are commonly used for genetic mapping in other plant species, the spore populations derived from hybrid sporophytes provide suitable material for bulk segregant analysis and gene identification by genome sequencing. [ABSTRACT FROM AUTHOR]

Cryo-scanning electron microscopy shows that nascent intercellular spaces (ICSs) in bryophytes are liquid-filled, whereas these are gas-filled from the outset in tracheophytes except in the gametophytes of Lycopodiales. ICSs are absent in moss gametophytes and remain liquid-filled in hornwort gametophytes and in both generations in liverworts. Liquid is replaced by gas following stomatal opening in hornworts and is ubiquitous in moss sporophytes even in astomate taxa. New data on moss water relations and sporophyteweights indicate that the latter are homiohydric while X-ray microanalysis reveals an absence of potassium pumps in the stomatal apparatus. The distribution of ICSs in bryophytes is strongly indicative of very ancient multiple origins. Inherent in this scenario is either the dual or triple evolution of stomata. The absence, in mosses, of any relationship between increases in sporophyte biomass and stomata numbers and absences, suggests that CO2 entry through the stomata, possible only after fluid replacement by gas in the ICSs, makes but a minor contribution to sporophyte nutrition. Save for a single claim of active regulation of aperture dimensions in mosses, all other functional and structural data point to the sporophyte desiccation, leading to spore discharge, as the primeval role of the stomatal apparatus. [ABSTRACT FROM AUTHOR]

Stem cells self-renew and produce precursor cells that differentiate to become specialized cell types. Land plants generate several types of stem cells that give rise to most organs of the plant body and whose characters determine the body organization. The moss Physcomitrella patens forms eight types of stem cells throughout its life cycle. Under gametangiuminducing conditions, multiple antheridium apical stem cells are formed at the tip of the gametophore and each antheridium apical stem cell divides to form an antheridium. We found that the gametophore apical stem cell, which typically forms leaf and stem tissues, changes to become a new type of stem cell, which we term the antheridium initial stem cell. This antheridium initial stem cell produces multiple antheridium apical stem cells, resulting in a cluster of antheridia at the tip of gametophore. This is the first report of a land plant stem cell directly producing another type of stem cell during normal development. Notably, the antheridium apical stem cells are distally produced from the antheridium initial stem cell, similar to the root cap stem cells of vascular plants, suggesting the use of similar molecular mechanisms and a possible evolutionary relationship. [ABSTRACT FROM AUTHOR]

Main conclusion : PpORS knockout mutants produced abnormal leaves with increased dye permeability and were more susceptible to dehydration, consistent with PpORS products being constituents of a cuticular structure in the moss. Type III polyketide synthases (PKSs) have co-evolved with terrestrial plants such that each taxon can generate a characteristic collection of polyketides, fine-tuned to its needs. 2ʹ-Oxoalkylresorcinol synthase from Physcomitrella patens (PpORS) is basal to all plant type III PKSs in phylogenetic trees and may closely resemble their most recent common ancestor. To gain insight into the roles that ancestral plant type III PKSs might have played during early land plant evolution, we constructed and phenotypically characterized targeted knockouts of PpORS. Ors gametophores, unless submerged in water while they were developing, displayed various leaf malformations that included grossly misshapen leaves, missing or abnormal midribs, multicellular protuberances and localized necrosis. Ors leaves, particularly abnormal ones, showed increased permeability to the hydrophilic dye, toluidine blue. Ors gametophores lost water faster and were more susceptible to dehydration than those of the control strain. Our findings are consistent with ors leaves possessing a partially defective cuticle and implicate PpORS in synthesis of the intact cuticle. PpORS orthologs are present in a few moss species but have not been found in other plants. However, conceivably an ancestral ORS in early land plants may have contributed to their protection from dehydration. [ABSTRACT FROM AUTHOR]

Autophagy is the process responsible for degrading cytoplasmic components in lysosomes and vacuoles. Autophagy-deficient mutants are generally more sensitive to environmental stress than their wild-type (WT) plants. In this study, however, we found that autophagy-deficient (atg) mutants of Physcomitrium , in which an autophagy-related gene, either ATG5 or ATG7 , is disrupted, are more tolerant to extreme desiccation stress than WT plants. The colonies were treated with the phytohormone abscisic acid, desiccated using silica gel, and hydrated on a nutrient-sufficient medium. atg mutant colonies survived this stress program and produced green colonies whereas the WT colonies did not. Evans Blue staining revealed obvious cell death in both colonies, but the rate was higher in WT than in atg5 colonies. atg cells had a higher protein content, accumulated more sucrose, and had greater water-holding capacity than WT cells. These factors likely reduce the rate of cell death with less damage during the stress program. On the other hand, inhibition of autophagy during the hydration step with the inhibitor 3-methyladenine increased survival, suggesting that autophagy occurring during the hydration step is involved in cell death. We conclude that Physcomitrium autophagy-deficient cells are more tolerant to extreme desiccation stress than WT cells due to higher water-holding capacity and the promotion of cell death by autophagy through an unknown mechanism. These findings challenge the conventional understanding of the role of autophagy in cell survival against stress and highlight the importance of further research in this area. • Physcomitrium autophagy-deficient mutants have higher desiccation and freezing tolerance. • Autophagy appears to promote cell death following desiccation stress. • Constitutive autophagy reduces protein content and water-holding capacity, leading to more cell damage and death during desiccation and hydration. • Autophagy occurring during hydration promotes cell damage and death by an as yet unknown autophagic cell death mechanism. [ABSTRACT FROM AUTHOR]

We analyzed tissue-specific transcriptomes of Arabidopsis thaliana and identified 66 gene families with a high frequency of “gradient genes” – genes showing a significant expression gradient between tissues. Gradient gene families include many with roles in hormone and peptide signaling, cell wall synthesis and remodeling, secondary metabolism, transcriptional regulation, and transport between cells. We compared the size of the gradient gene families among the genomes of four plant species with radically different body plans – a single-celled algae, a moss, a eudicot, and a monocot – and found that most of the gradient gene families (58/66) expanded in parallel with the evolution of morphological complexity. A novel measure of tissue diversity was used to show that members of any one gradient gene family tend not to be clustered in a single tissue, but are rather apportioned evenly across the tissues studied. Considered together, our results suggest that the diversification of these gene families supported the diversification of tissue types and the evolution of body plan complexity in plants.