Physcomitrium patens (Hedw.) Mitt. growing in the dark defaults to an auxin- and cytokinin-independent developmental programme.

Auxin and cytokinin partially restore Physcomitrium (formerly Physcomitrella ) patens gametophores that have developed in the dark to a form more typical of those grown in light. Auxin synthesis and/or transport in gametophores decrease with time spent in the dark. Auxin synthesis resumes in the apices of dark-grown gametophores upon their return to the light. Red light and to a lesser extent blue light are sufficient for this. The mas and GH3 promoters are both auxin-inducible but respond differentially to spatial cues.

Sporogenesis in Physcomitrium patens: Intergenerational collaboration and the development of the spore wall and aperture.

Although the evolution of spores was critical to the diversification of plants on land, sporogenesis is incompletely characterized for model plants such as Physcomitrium patens. In this study, the complete process of P. patens sporogenesis is detailed from capsule expansion to mature spore formation, with emphasis on the construction of the complex spore wall and proximal aperture. Both diploid (sporophytic) and haploid (spores) cells contribute to the development and maturation of spores. During capsule expansion, the diploid cells of the capsule, including spore mother cells (SMCs), inner capsule wall layer (spore sac), and columella, contribute a locular fibrillar matrix that contains the machinery and nutrients for spore ontogeny. Nascent spores are enclosed in a second matrix that is surrounded by a thin SMC wall and suspended in the locular material. As they expand and separate, a band of exine is produced external to a thin foundation layer of tripartite lamellae. Dense globules assemble evenly throughout the locule, and these are incorporated progressively onto the spore surface to form the perine external to the exine. On the distal spore surface, the intine forms internally, while the spiny perine ornamentation is assembled. The exine is at least partially extrasporal in origin, while the perine is derived exclusively from outside the spore. Across the proximal surface of the polar spores, an aperture begins formation at the onset of spore development and consists of an expanded intine, an annulus, and a central pad with radiating fibers. This complex aperture is elastic and enables the proximal spore surface to cycle between being compressed (concave) and expanded (rounded). In addition to providing a site for water intake and germination, the elastic aperture is likely involved in desiccation tolerance. Based on the current phylogenies, the ancestral plant spore contained an aperture, exine, intine, and perine. The reductive evolution of liverwort and hornwort spores entailed the loss of perine in both groups and the aperture in liverworts. This research serves as the foundation for comparisons with other plant groups and for future studies of the developmental genetics and evolution of spores across plants.

Reactive oxygen species are required for spore wall formation in Physcomitrella patens.

A robust spore wall was a key requirement of terrestrialization by early plants. Sporopollenin in spore and pollen grain walls is thought to be polymerized and cross-linked to other macromolecular components partly through oxidative processes involving H2O2. Therefore, we investigated effects of scavengers of reactive oxygen species (ROS) on formation of spore walls in the moss, Physcomitrella patens. Exposure of sporophytes, containing spores in the process of forming walls, to ascorbate, dimethylthiourea or 4-hydroxy-TEMPO prevented normal wall development in a dose, chemical and stage-dependent manner. Mature spores, exposed while developing to a ROS scavenger, burst when mounted in water on a flat slide under a coverslip (a phenomenon we named "augmented osmolysis" since they did not burst in phosphate-buffered saline or in water on a depression slide). Additionally, walls of exposed spores were more susceptible to alkaline hydrolysis than those of control spores and some were characterized by discontinuities in the exine, anomalies in perine spine structure, abnormal intine and aperture and occasionally wall shedding. Our data support involvement of oxidative cross-linking in spore wall development, including sporopollenin polymerization or deposition, as well as a role for ROS in intine/aperture development.

PpORS, an ancient type III polyketide synthase, is required for integrity of leaf cuticle and resistance to dehydration in the moss, Physcomitrella patens.

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. 2'-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.

PpASCL, the Physcomitrella patens Anther-Specific Chalcone Synthase-Like Enzyme Implicated in Sporopollenin Biosynthesis, Is Needed for Integrity of the Moss Spore Wall and Spore Viability.

Sporopollenin is the main constituent of the exine layer of spore and pollen walls. The anther-specific chalcone synthase-like (ASCL) enzyme of Physcomitrella patens, PpASCL, has previously been implicated in the biosynthesis of sporopollenin, the main constituent of exine and perine, the two outermost layers of the moss spore cell wall. We made targeted knockouts of the corresponding gene, PpASCL, and phenotypically characterized ascl sporophytes and spores at different developmental stages. Ascl plants developed normally until late in sporophytic development, when the spores produced were structurally aberrant and inviable. The development of the ascl spore cell wall appeared to be arrested early in microspore development, resulting in small, collapsed spores with altered surface morphology. The typical stratification of the spore cell wall was absent with only an abnormal perine recognisable above an amorphous layer possibly representing remnants of compromised intine and/or exine. Equivalent resistance of the spore walls of ascl mutants and the control strain to acetolysis suggests the presence of chemically inert, defective sporopollenin in the mutants. Anatomical abnormalities of late-stage ascl sporophytes include a persistent large columella and an air space incompletely filled with spores. Our results indicate that the evolutionarily conserved PpASCL gene is needed for proper construction of the spore wall and for normal maturation and viability of moss spores.

Ancestral and more recently acquired syntenic relationships of MADS-box genes uncovered by the Physcomitrella patens pseudochromosomal genome assembly.

KEY MESSAGE: The Physcomitrella pseudochromosomal genome assembly revealed previously invisible synteny enabling realisation of the full potential of shared synteny as a tool for probing evolution of this plant's MADS-box gene family. Assembly of the sequenced genome of Physcomitrella patens into 27 mega-scaffolds (pseudochromosomes) has confirmed the major predictions of our earlier model of expansion of the MADS-box gene family in the Physcomitrella lineage. Additionally, microsynteny has been conserved in the immediate vicinity of some recent duplicates of MADS-box genes. However, comparison of non-syntenic MIKC MADS-box genes and neighbouring genes indicates that chromosomal rearrangements and/or sequence degeneration have destroyed shared synteny over longer distances (macrosynteny) around MADS-box genes despite subsets comprising two or three MIKC genes having remained syntenic. In contrast, half of the type I MADS-box genes have been transposed creating new syntenic relations with MIKC genes. This implies that conservation of ancient ancestral synteny of MIKC genes and of more recently acquired synteny of type I and MIKC genes may be selectively advantageous. Our revised model predicts the birth rate of MIKC genes in Physcomitrella is higher than that of type I genes. However, this difference is attributable to an early tandem duplication and an early segmental duplication of MIKC genes prior to the two polyploidisations that account for most of the expansion of the MADS-box gene family in Physcomitrella. Furthermore, this early segmental duplication spawned two chromosomal lineages: one with a MIKC (C) gene, belonging to the PPM2 clade, in close proximity to one or a pair of MIKC* genes and another with a MIKC (C) gene, belonging to the PpMADS-S clade, characterised by greater separation from syntenic MIKC* genes. Our model has evolutionary implications for the Physcomitrella karyotype.

The Selaginella genome identifies genetic changes associated with the evolution of vascular plants.

Vascular plants appeared ~410 million years ago, then diverged into several lineages of which only two survive: the euphyllophytes (ferns and seed plants) and the lycophytes. We report here the genome sequence of the lycophyte Selaginella moellendorffii (Selaginella), the first nonseed vascular plant genome reported. By comparing gene content in evolutionarily diverse taxa, we found that the transition from a gametophyte- to a sporophyte-dominated life cycle required far fewer new genes than the transition from a nonseed vascular to a flowering plant, whereas secondary metabolic genes expanded extensively and in parallel in the lycophyte and angiosperm lineages. Selaginella differs in posttranscriptional gene regulation, including small RNA regulation of repetitive elements, an absence of the trans-acting small interfering RNA pathway, and extensive RNA editing of organellar genes.

Physcomitrella patens auxin-resistant mutants affect conserved elements of an auxin-signaling pathway.

Auxin regulates most aspects of flowering-plant growth and development, including key developmental innovations that evolved within the vascular plant lineage after diverging from a bryophyte-like ancestor nearly 500 million years ago. Recent studies in Arabidopsis indicate that auxin acts by directly binding the TIR1 subunit of the SCF(TIR1) ubiquitin ligase; this binding results in degradation of the Aux/IAA transcriptional repressors and de-repression of auxin-responsive genes. Little is known, however, about the mechanism of auxin action in other plants. To characterize auxin signaling in a nonflowering plant, we utilized the genetically tractable moss Physcomitrella patens. We used a candidate-gene approach to show that previously identified auxin-resistant mutants of P. patens harbor mutations in Aux/IAA genes. Furthermore, we show that the moss Aux/IAA proteins interact with Arabidopsis TIR1 moss homologs called PpAFB and that a reduction in PpAFB levels results in a phenotype similar to that of the auxin-resistant mutants. Our results indicate that the molecular mechanism of auxin perception is conserved in land plants despite vast differences in the role auxin plays in different plant lineages.

Genome-wide analysis of the chalcone synthase superfamily genes of Physcomitrella patens.

Enzymes of the chalcone synthase (CHS) superfamily catalyze the production of a variety of secondary metabolites in bacteria, fungi and plants. Some of these metabolites have played important roles during the early evolution of land plants by providing protection from various environmental assaults including UV irradiation. The genome of the moss, Physcomitrella patens, contains at least 17 putative CHS superfamily genes. Three of these genes (PpCHS2b, PpCHS3 and PpCHS5) exist in multiple copies and all have corresponding ESTs. PpCHS11 and probably also PpCHS9 encode non-CHS enzymes, while PpCHS10 appears to be an ortholog of plant genes encoding anther-specific CHS-like enzymes. It was inferred from the genomic locations of genes comprising it that the moss CHS superfamily expanded through tandem and segmental duplication events. Inferred exon-intron architectures and results from phylogenetic analysis of representative CHS superfamily genes of P. patens and other plants showed that intron gain and loss occurred several times during evolution of this gene superfamily. A high proportion of P. patens CHS genes (7 of 14 genes for which the full sequence is known and probably 3 additional genes) are intronless, prompting speculation that CHS gene duplication via retrotransposition has occurred at least twice in the moss lineage. Analyses of sequence similarities, catalytic motifs and EST data indicated that a surprisingly large number (as many as 13) of the moss CHS superfamily genes probably encode active CHS. EST distribution data and different light responsiveness observed with selected genes provide evidence for their differential regulation. Observed diversity within the moss CHS superfamily and amenability to gene manipulation make Physcomitrella a highly suitable model system for studying expansion and functional diversification of the plant CHS superfamily of genes.

MADS about MOSS.

Classic MIKC-type MADS-box genes (MIKC(c)) play diverse and crucial roles in angiosperm development, the most studied and best understood of which is the specification of floral organ identities. To shed light on how the flower evolved, phylogenetic and functional analyses of genes involved in its ontogeny, such as the MIKC(c) genes, must be undertaken in as broad a selection as possible of plants with disparate ancestries. Since little is known about the functions of these genes in non-seed plants, we investigated the developmental roles of a subset of the MIKC(c) genes present in the moss, Physcomitrella patens, which is positioned informatively near the base of the land plant evolutionary tree. We observed that transgenic lines possessing an antisense copy of a MIKC(c) gene characteristically displayed knocked-down expression of the corresponding native MIKC(c) gene as well as multiple diverse phenotypic alterations to the haploid gametophytic and diploid sporophytic generations of the life cycle. In this addendum, we re-examine our findings in the light of recent pertinent literature and provide additional data concerning the effects of simultaneously knocking out multiple MIKC(c) genes in this moss.