RESUMO
Accumulating evidence suggests that peptidoglycan, consistent with a bacterial cell wall, is synthesized around the chloroplasts of many photosynthetic eukaryotes, from glaucophyte algae to early-diverging land plants including pteridophyte ferns, but the biosynthetic pathway has not been demonstrated. Here, we employed mass spectrometry and enzymology in a two-fold approach to characterize the synthesis of peptidoglycan in chloroplasts of the moss Physcomitrium (Physcomitrella) patens. To drive the accumulation of peptidoglycan pathway intermediates, P. patens was cultured with the antibiotics fosfomycin, D-cycloserine, and carbenicillin, which inhibit key peptidoglycan pathway proteins in bacteria. Mass spectrometry of the trichloroacetic acid-extracted moss metabolome revealed elevated levels of five of the predicted intermediates from uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) through the uridine diphosphate N-acetylmuramic acid (UDP-MurNAc)-D,L-diaminopimelate (DAP)-pentapeptide. Most Gram-negative bacteria, including cyanobacteria, incorporate meso-diaminopimelic acid (D,L-DAP) into the third residue of the stem peptide of peptidoglycan, as opposed to L-lysine, typical of most Gram-positive bacteria. To establish the specificity of D,L-DAP incorporation into the P. patens precursors, we analyzed the recombinant protein UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2,6-diaminopimelate ligase (MurE) from both P. patens and the cyanobacterium Anabaena sp. (Nostoc sp. strain PCC 7120). Both ligases incorporated D,L-DAP in almost complete preference to L-Lys, consistent with the mass spectrophotometric data, with catalytic efficiencies similar to previously documented Gram-negative bacterial MurE ligases. We discuss how these data accord with the conservation of active site residues common to DL-DAP-incorporating bacterial MurE ligases and of the probability of a horizontal gene transfer event within the plant peptidoglycan pathway.
Assuntos
Parede Celular , Peptidoglicano , Bactérias/metabolismo , Parede Celular/metabolismo , Cloroplastos/metabolismo , Bactérias Gram-Negativas/metabolismo , Ligases/metabolismo , Lisina/metabolismo , Peptidoglicano/química , Peptidoglicano/genética , Peptidoglicano/metabolismo , Difosfato de Uridina/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis , Briófitas , Bryopsida , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Briófitas/metabolismo , Bryopsida/genética , Bryopsida/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , Ácidos Indolacéticos/metabolismo , Células-Tronco/metabolismoRESUMO
Gravity is a critical environmental factor regulating directional growth and morphogenesis in plants, and gravitropism is the process by which plants perceive and respond to the gravity vector. The cytoskeleton is proposed to play important roles in gravitropism, but the underlying mechanisms are obscure. Here we use genetic screening in Physcomitrella patens, to identify a locus GTRC, that when mutated, reverses the direction of protonemal gravitropism. GTRC encodes a processive minus-end-directed KCHb kinesin, and its N-terminal, C-terminal and motor domains are all essential for transducing the gravity signal. Chimeric analysis between GTRC/KCHb and KCHa reveal a unique role for the N-terminus of GTRC in gravitropism. Further study shows that gravity-triggered normal asymmetric distribution of actin filaments in the tip of protonema is dependent on GTRC. Thus, our work identifies a microtubule-based cellular motor that determines the direction of plant gravitropism via mediating the asymmetric distribution of actin filaments.
Assuntos
Bryopsida/fisiologia , Gravitropismo/fisiologia , Cinesinas/fisiologia , Proteínas de Plantas/fisiologia , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiologia , Sequência de Bases , Bryopsida/genética , Mapeamento Cromossômico , Citoesqueleto/química , Citoesqueleto/fisiologia , DNA de Plantas/genética , Genes de Plantas , Gravitropismo/genética , Cinesinas/química , Cinesinas/genética , Microtúbulos/química , Microtúbulos/fisiologia , Mutagênese , Mutação , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Domínios ProteicosRESUMO
Hornworts comprise a bryophyte lineage that diverged from other extant land plants >400 million years ago and bears unique biological features, including a distinct sporophyte architecture, cyanobacterial symbiosis and a pyrenoid-based carbon-concentrating mechanism (CCM). Here, we provide three high-quality genomes of Anthoceros hornworts. Phylogenomic analyses place hornworts as a sister clade to liverworts plus mosses with high support. The Anthoceros genomes lack repeat-dense centromeres as well as whole-genome duplication, and contain a limited transcription factor repertoire. Several genes involved in angiosperm meristem and stomatal function are conserved in Anthoceros and upregulated during sporophyte development, suggesting possible homologies at the genetic level. We identified candidate genes involved in cyanobacterial symbiosis and found that LCIB, a Chlamydomonas CCM gene, is present in hornworts but absent in other plant lineages, implying a possible conserved role in CCM function. We anticipate that these hornwort genomes will serve as essential references for future hornwort research and comparative studies across land plants.
Assuntos
Anthocerotophyta/genética , Evolução Biológica , Embriófitas/fisiologia , Genoma de Planta , Características de História de VidaRESUMO
Land plants are considered monophyletic, descending from a single successful colonization of land by an aquatic algal ancestor. The ability to survive dehydration to the point of desiccation is a key adaptive trait enabling terrestrialization. In extant land plants, desiccation tolerance depends on the action of the hormone abscisic acid (ABA) that acts through a receptor-signal transduction pathway comprising a PYRABACTIN RESISTANCE 1-like (PYL)-PROTEIN PHOSPHATASE 2C (PP2C)-SNF1-RELATED PROTEIN KINASE 2 (SnRK2) module. Early-diverging aeroterrestrial algae mount a dehydration response that is similar to that of land plants, but that does not depend on ABA: Although ABA synthesis is widespread among algal species, ABA-dependent responses are not detected, and algae lack an ABA-binding PYL homolog. This raises the key question of how ABA signaling arose in the earliest land plants. Here, we systematically characterized ABA receptor-like proteins from major land plant lineages, including a protein found in the algal sister lineage of land plants. We found that the algal PYL-homolog encoded by Zygnema circumcarinatum has basal, ligand-independent activity of PP2C repression, suggesting this to be an ancestral function. Similarly, a liverwort receptor possesses basal activity, but it is further activated by ABA. We propose that co-option of ABA to control a preexisting PP2C-SnRK2-dependent desiccation-tolerance pathway enabled transition from an all-or-nothing survival strategy to a hormone-modulated, competitive strategy by enabling continued growth of anatomically diversifying vascular plants in dehydrative conditions, enabling them to exploit their new environment more efficiently.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Carofíceas/fisiologia , Embriófitas/fisiologia , Ligantes , Proteína Fosfatase 2C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/metabolismo , Evolução Biológica , Regulação da Expressão Gênica de Plantas , Hepatófitas/metabolismo , Proteína Fosfatase 2C/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologiaRESUMO
[This corrects the article DOI: 10.1038/s42003-019-0281-1.].
RESUMO
The SNF1-related protein kinase 2 (SnRK2) family includes key regulators of osmostress and abscisic acid (ABA) responses in angiosperms and can be classified into three subclasses. Subclass III SnRK2s act in the ABA response while ABA-nonresponsive subclass I SnRK2s are regulated through osmostress. Here we report that an ancient subclass III SnRK2-based signalling module including ABA and an upstream Raf-like kinase (ARK) exclusively protects the moss Physcomitrella patens from drought. Subclass III SnRK2s from both Arabidopsis and from the semiterrestrial alga Klebsormidium nitens, which contains all the components of ABA signalling except ABA receptors, complement Physcomitrella snrk2 - mutants, whereas Arabidopsis subclass I SnRK2 cannot. We propose that the earliest land plants developed the ABA/ARK/subclass III SnRK2 signalling module by recruiting ABA to regulate a pre-existing dehydration response and that subsequently a novel subclass I SnRK2 system evolved in vascular plants conferring osmostress protection independently from the ancient system.
RESUMO
Plant peroxisomes are important components of cellular antioxidant networks, dealing with ROS generated by multiple metabolic pathways. Peroxisomes respond to environmental and cellular conditions by changing their size, number, and proteomic content. To investigate the role of peroxisomes in response to drought, dehydration and ABA treatment we took an evolutionary and comparative genomics approach. Colonisation of land required evolution of dehydration tolerance in the absence of subsequent anatomical adaptations. Therefore, the model bryophyte Physcomitrella patens, the model dicot Arabidopsis thaliana and wheat (Tricitcum aestivum), a globally important cereal crop were compared. Three sets of genes namely 'PTS1 genes' (a proxy for genes encoding peroxisome targeted proteins), PEX genes (involved in peroxisome biogenesis) and genes involved in plant antioxidant networks were identified in all 3 species and their expression compared under drought (dehydration) and ABA treatment. Genes encoding enzymes of ß-oxidation and gluconeogenesis, antioxidant enzymes including catalase and glutathione reductase and PEX3 and PEX11 isoforms showed conserved up-regulation, and peroxisome proliferation was induced by ABA in moss. Interestingly, expression of some of these genes differed between drought sensitive and resistant genotypes of wheat in line with measured photosynthetic and biochemical differences. These results point to an underappreciated role for peroxisomes in drought response.
Assuntos
Arabidopsis/genética , Bryopsida/genética , Regulação da Expressão Gênica de Plantas/genética , Peroxissomos/metabolismo , Transcrição Gênica , Triticum/genética , Ácido Abscísico/metabolismo , Dessecação , Secas , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
How genes shape diverse plant and animal body forms is a key question in biology. Unlike animal cells, plant cells are confined by rigid cell walls, and cell division plane orientation and growth rather than cell movement determine overall body form. The emergence of plants on land coincided with a new capacity to rotate stem cell divisions through multiple planes, and this enabled three-dimensional (3D) forms to arise from ancestral forms constrained to 2D growth. The genes involved in this evolutionary innovation are largely unknown. The evolution of 3D growth is recapitulated during the development of modern mosses when leafy shoots arise from a filamentous (2D) precursor tissue. Here, we show that a conserved, CLAVATA peptide and receptor-like kinase pathway originated with land plants and orients stem cell division planes during the transition from 2D to 3D growth in a moss, Physcomitrella. We find that this newly identified role for CLAVATA in regulating cell division plane orientation is shared between Physcomitrella and Arabidopsis. We report that roles for CLAVATA in regulating cell proliferation and cell fate are also shared and that CLAVATA-like peptides act via conserved receptor components in Physcomitrella. Our results suggest that CLAVATA was a genetic novelty enabling the morphological innovation of 3D growth in land plants.
Assuntos
Bryopsida/genética , Proliferação de Células/genética , Evolução Molecular , Proteínas de Plantas/genética , Evolução Biológica , Bryopsida/crescimento & desenvolvimento , Bryopsida/metabolismo , Proteínas de Plantas/metabolismoRESUMO
The development of 'designer' organelles could be a key strategy to enable foreign pathways to be efficiently controlled within eukaryotic biotechnology. A fundamental component of any such system will be the implementation of a bespoke protein import pathway that can selectively deliver constituent proteins to the new compartment in the presence of existing endogenous trafficking systems. Here we show that the protein-protein interactions that control the peroxisomal protein import pathway can be manipulated to create a pair of interacting partners that still support protein import in moss cells, but are orthogonal to the naturally occurring pathways. In addition to providing a valuable experimental tool to give new insights into peroxisomal protein import, the variant receptor-signal sequence pair forms the basis of a system in which normal peroxisomal function is downregulated and replaced with an alternative pathway, an essential first step in the creation of a designer organelle.Designer organelles could allow the isolation of synthetic biological pathways from endogenous components of the host cell. Here the authors engineer a peroxisomal protein import pathway orthogonal to the naturally occurring system.
Assuntos
Proteínas de Arabidopsis/metabolismo , Peroxissomos/metabolismo , Arabidopsis/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Transporte ProteicoRESUMO
Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth's continents by permitting efficient shoot-atmosphere gas exchange and plant hydration1. However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood2-4 and their function has remained a matter of debate for a century5. Here, we show that genes encoding the two basic helix-loop-helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata. Protein-protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss-angiosperm gene complementations6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana7. Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes.
Assuntos
Bryopsida/crescimento & desenvolvimento , Bryopsida/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/fisiologia , Proteínas de Plantas/genética , Estômatos de Plantas/crescimento & desenvolvimento , Proteínas de Plantas/metabolismo , Estômatos de Plantas/genéticaAssuntos
Briófitas , Bryopsida/genética , Retroelementos , Nicotiana/genética , Transformação GenéticaRESUMO
The model bryophyte Physcomitrella patens is unique among plants in supporting the generation of mutant alleles by facile homologous recombination-mediated gene targeting (GT). Reasoning that targeted transgene integration occurs through the capture of transforming DNA by the homology-dependent pathway for DNA double-strand break (DNA-DSB) repair, we analysed the genome-wide transcriptomic response to bleomycin-induced DNA damage and generated mutants in candidate DNA repair genes. Massively parallel (Illumina) cDNA sequencing identified potential participants in gene targeting. Transcripts encoding DNA repair proteins active in multiple repair pathways were significantly up-regulated. These included Rad51, CtIP, DNA ligase 1, Replication protein A and ATR in homology-dependent repair, Xrcc4, DNA ligase 4, Ku70 and Ku80 in non-homologous end-joining and Rad1, Tebichi/polymerase theta, PARP in microhomology-mediated end-joining. Differentially regulated cell-cycle components included up-regulated Rad9 and Hus1 DNA-damage-related checkpoint proteins and down-regulated D-type cyclins and B-type CDKs, commensurate with the imposition of a checkpoint at G2 of the cell cycle characteristic of homology-dependent DNA-DSB repair. Candidate genes, including ATP-dependent chromatin remodelling helicases associated with repair and recombination, were knocked out and analysed for growth defects, hypersensitivity to DNA damage and reduced GT efficiency. Targeted knockout of PpCtIP, a cell-cycle activated mediator of homology-dependent DSB resection, resulted in bleomycin-hypersensitivity and greatly reduced GT efficiency.
Assuntos
Bryopsida/genética , Quebras de DNA de Cadeia Dupla , Transcrição Gênica , Bleomicina/farmacologia , Bryopsida/fisiologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Transcrição Gênica/fisiologiaRESUMO
The patterning of stomata plays a vital role in plant development and has emerged as a paradigm for the role of peptide signals in the spatial control of cellular differentiation. Research in Arabidopsis has identified a series of epidermal patterning factors (EPFs), which interact with an array of membrane-localised receptors and associated proteins (encoded by ERECTA and TMM genes) to control stomatal density and distribution. However, although it is well-established that stomata arose very early in the evolution of land plants, until now it has been unclear whether the established angiosperm stomatal patterning system represented by the EPF/TMM/ERECTA module reflects a conserved, universal mechanism in the plant kingdom. Here, we use molecular genetics to show that the moss Physcomitrella patens has conserved homologues of angiosperm EPF, TMM and at least one ERECTA gene that function together to permit the correct patterning of stomata and that, moreover, elements of the module retain function when transferred to Arabidopsis Our data characterise the stomatal patterning system in an evolutionarily distinct branch of plants and support the hypothesis that the EPF/TMM/ERECTA module represents an ancient patterning system.
Assuntos
Bryopsida/metabolismo , Estômatos de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Epiderme Vegetal/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
The anatomically simple plants that first colonized land must have acquired molecular and biochemical adaptations to drought stress. Abscisic acid (ABA) coordinates responses leading to desiccation tolerance in all land plants. We identified ABA nonresponsive mutants in the model bryophyte Physcomitrella patens and genotyped a segregating population to map and identify the ABA NON-RESPONSIVE (ANR) gene encoding a modular protein kinase comprising an N-terminal PAS domain, a central EDR domain, and a C-terminal MAPKKK-like domain. anr mutants fail to accumulate dehydration tolerance-associated gene products in response to drought, ABA, or osmotic stress and do not acquire ABA-dependent desiccation tolerance. The crystal structure of the PAS domain, determined to 1.7-Å resolution, shows a conserved PAS-fold that dimerizes through a weak dimerization interface. Targeted mutagenesis of a conserved tryptophan residue within the PAS domain generates plants with ABA nonresponsive growth and strongly attenuated ABA-responsive gene expression, whereas deleting this domain retains a fully ABA-responsive phenotype. ANR orthologs are found in early-diverging land plant lineages and aquatic algae but are absent from more recently diverged vascular plants. We propose that ANR genes represent an ancestral adaptation that enabled drought stress survival of the first terrestrial colonizers but were lost during land plant evolution.
Assuntos
Ácido Abscísico/farmacologia , Bryopsida/efeitos dos fármacos , Bryopsida/metabolismo , Proteínas de Plantas/metabolismo , Bryopsida/genética , Cristalografia por Raios X , Dessecação , Secas , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/genética , Mutação , Pressão Osmótica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Estrutura Secundária de ProteínaRESUMO
Peroxisomal biogenesis factor 11 (PEX11) proteins are found in yeasts, mammals and plants, and play a role in peroxisome morphology and regulation of peroxisome division. The moss Physcomitrella patens has six PEX11 isoforms which fall into two subfamilies, similar to those found in monocots and dicots. We carried out targeted gene disruption of the Phypa_PEX11-1 gene and compared the morphological and cellular phenotypes of the wild-type and mutant strains. The mutant grew more slowly and the development of gametophores was retarded. Mutant chloronemal filaments contained large cellular structures which excluded all other cellular organelles. Expression of fluorescent reporter proteins revealed that the mutant strain had greatly enlarged peroxisomes up to 10 µm in diameter. Expression of a vacuolar membrane marker confirmed that the enlarged structures were not vacuoles, or peroxisomes sequestered within vacuoles as a result of pexophagy. Phypa_PEX11 targeted to peroxisome membranes could rescue the knock out phenotype and interacted with Fission1 on the peroxisome membrane. Moss PEX11 functions in peroxisome division similar to PEX11 in other organisms but the mutant phenotype is more extreme and environmentally determined, making P. patens a powerful system in which to address mechanisms of peroxisome proliferation and division.
Assuntos
Bryopsida/citologia , Bryopsida/genética , Peroxissomos/genética , Proteínas de Plantas/genética , Bryopsida/crescimento & desenvolvimento , Técnicas de Inativação de Genes , Membranas Intracelulares/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Família Multigênica , Mutação , Peroxinas , Peroxissomos/metabolismo , Peroxissomos/patologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The bryophytes are a morphologically and ecologically diverse group of plants that have recently emerged as major model systems for a variety of biological processes. In particular, the genome sequence of the moss, Physcomitrella patens, has significantly enhanced our understanding of the evolution of developmental processes in land plants. However, to fully explore the diversity within bryophytes, we need additional genomic resources. Here, we describe analyses of the transcriptomes of a male and a female isolate of the moss, Ceratodon purpureus, generated using the 454 FLX technology. Comparative analyses between C. purpureus and P. patens indicated that this strategy generated nearly complete coverage of the protonemal transcriptome. An analysis of the overlap in gene sets between C. purpureus and P. patens provides new insights into the evolution of gene family composition across the land plants. In spite of the overall transcriptomic similarity between the two species, Ka /Ks analysis of P. patens and C. purpureus suggests considerable physiological and developmental divergence. Additionally, while the codon usage was very similar between these two mosses, C. purpureus genes showed a slightly greater codon usage bias than P. patens genes potentially because of the contrasting mating system of the two species. Finally, we found evidence of a genome doubling ~65-76 MYA that likely coincided with the contemporaneous polyploidy event inferred for P. patens but postdates the divergence of P. patens and C. purpureus. The powerful laboratory tools now available for C. purpureus will enable the research community to fully exploit these genomic resources.
Assuntos
Bryopsida/genética , Perfilação da Expressão Gênica , Evolução Molecular , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
The early evolution of plants required the acquisition of a number of key adaptations to overcome physiological difficulties associated with survival on land. One of these was a tough sporopollenin wall that enclosed reproductive propagules and provided protection from desiccation and UV-B radiation. All land plants possess such walled spores (or their derived homologue, pollen). We took a reverse genetics approach, consisting of knock-out and complementation experiments to test the functional conservation of the sporopollenin-associated gene MALE STERILTY 2 (which is essential for pollen wall development in Arabidopsis thaliana) in the bryophyte Physcomitrella patens. Knock-outs of a putative moss homologue of the A. thaliana MS2 gene, which is highly expressed in the moss sporophyte, led to spores with highly defective walls comparable to that observed in the A. thaliana ms2 mutant, and extremely compromised germination. Conversely, the moss MS2 gene could not rescue the A. thaliana ms2 phenotype. The results presented here suggest that a core component of the biochemical and developmental pathway required for angiosperm pollen wall development was recruited early in land plant evolution but the continued increase in pollen wall complexity observed in angiosperms has been accompanied by divergence in MS2 gene function.