Neil W. Ashton

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. patensCHS 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.

Revelation of ancestral roles of KNOX genes by a functional analysis of Physcomitrella homologues

KNOX genes are indispensable elements of indeterminate apical growth programmes of vascular plant sporophytes. Since little is known about the roles of such genes in non-vascular plants, functional analysis of moss KNOX homologues (MKN genes) was undertaken using the genetically amenable model plant, Physcomitrella patens. Three MKN genes were inactivated by targeted gene knockout to produce single, double and triple mutants. MKN2 (a class 1 KNOX gene) mutants were characterised by premature sporogenesis, abnormal sporophyte ontogeny and irregular spore development. MKN4 (a second class 1 gene) mutants were phenotypically normal. MKN1-3 (a class 2 KNOX gene) mutants exhibited defects in spore coat morphology. Analysis of double and triple mutants revealed that the abnormal sporophytic phenotype of MKN2 mutants was accentuated by mutating MKN4 and to a lesser degree by mutating MKN1-3. The aberrant spore phenotype of MKN1-3 and MKN2 mutants was exacerbated by mutating MKN4. This study provides the first instance in which an abnormal phenotype has been associated with the disruption of a class 2 KNOX gene as well as the first demonstrated case of functional redundancy between a class 1 and a class 2 KNOX gene. We conclude that KNOX genes play significant roles in programming sporophytic development in moss and we provide evidence that ancestral function(s) of this gene family were instrumental in the successful transition of plants to a terrestrial environment.

Estimation of indole-3-acetic acid in gametophytes of the moss, Physcomitrella patens

By means of gas chromatography-selected ion monitoring-mass spectrometry using an isotope-dilution assay with 4,5,6,7-tetradeutero-indole-3-acetic acid as the internal standard, indole-3-acetic acid has been estimated to be present in aseptically cultured gametophytes of wild-type Physcomitrella patens (Hedw.) B.S.G. at a level of 0.075 μg g−1 dry weight or 2.1 ng g−1 fresh weight.

Genetic analysis by somatic hybridization of cytokinin overproducing developmental mutants of the moss,Physcomitrella patens

SummaryCytokinins are important regulators of growth and development in lower and higher eukaryotic plants. Genetic analysis by means of somatic hybridization, achieved through protoplast fusion, revealed that, of 15 independently isolated gametophore and cytokinin over-producing (OVE) mutants in the model system,Physcomitrella patens, 14 carry recessive mutations responsible for this abnormal phenotype. Seven of these strains have been assigned to three complementation groups:OVEA, OVEB andOVEC. A further three strains have been demonstrated not to belong to theOVEA group and another mutant does not fall into groupOVEB. Phenotypic segregation ratios among progeny obtained following self-fertilization of a number of different somatic hybrids showed that severalOVE mutations behave as recessive alleles of single Mendelian genes.

Clues about the ancestral roles of plant MADS-box genes from a functional analysis of moss homologues

Classic MIKC-type MADS-box genes (MIKCc genes) are indispensable elements in the genetic programming of pattern formation, including the segmental organisation of angiosperm flowers, in seed plants. Since little is known about the functions of MIKCc genes in non-seed plants, a functional analysis of moss MIKCc homologues was performed using the genetically amenable, simple model plant, Physcomitrella patens. Expression of moss homologues was knocked down using an antisense RNA approach or abolished by generating transformants with gene knockouts. The knocked down (“antisense”) transformants displayed a multifaceted mutant phenotype comprising delayed gametangia formation, diminished sporophyte yield and, in the most extremely affected cases, abnormal sporophyte development and altered leaf morphogenesis. Knocked out transformants were phenotypically normal. Analysis of in situMIKCc gene expression using transgenic strains containing MIKCc promoter–GUS fusions showed that these genes are generally expressed ubiquitously in vegetative and reproductive tissues. We conclude that MIKCc genes play significant roles in morphogenetic programming of the moss. Functional redundancy characterises some members of the gene group. Our findings provide clues concerning the ancestral roles of some MIKCc genes that may be represented in the genomes of diverse extant plant taxa.

Selaginella Genome Analysis - Entering the "Homoplasy Heaven" of the MADS World.

In flowering plants, arguably the most significant transcription factors regulating development are MADS-domain proteins, encoded by Type I and Type II MADS-box genes. Type II genes are divided into the MIKCC and MIKC* groups. In angiosperms, these types and groups play distinct roles in the development of female gametophytes, embryos, and seeds (Type I); vegetative and floral tissues in sporophytes (MIKCC); and male gametophytes (MIKC*), but their functions in other plants are largely unknown. The complete set of MADS-box genes has been described for several angiosperms and a moss, Physcomitrella patens. Our examination of the complete genome sequence of a lycophyte, Selaginella moellendorffii, revealed 19 putative MADS-box genes (13 Type I, 3 MIKCC, and 3 MIKC*). Our results suggest that the most recent common ancestor of vascular plants possessed at least two Type I and two Type II genes. None of the S. moellendorffii MIKCC genes were identified as orthologs of any floral organ identity genes. This strongly corroborates the view that the clades of floral organ identity genes originated in a common ancestor of seed plants after the lineage that led to lycophytes had branched off, and that expansion of MIKCC genes in the lineage leading to seed plants facilitated the evolution of their unique reproductive organs. The number of MIKC* genes and the ratio of MIKC* to MIKCC genes is lower in S. moellendorffii and angiosperms than in P. patens, correlated with reduction of the gametophyte in vascular plants. Our data indicate that Type I genes duplicated and diversified independently within lycophytes and seed plants. Our observations on MADS-box gene evolution echo morphological evolution since the two lineages of vascular plants appear to have arrived independently at similar body plans. Our annotation of MADS-box genes in S. moellendorffii provides the basis for functional studies to reveal the roles of this crucial gene family in basal vascular plants.

A parsimonious model of lineage-specific expansion of MADS-box genes in Physcomitrella patens

Key messageThe MADS-box gene family expanded in the lineage leading to the moss,Physcomitrella patens, mainly as a result of polyploidisations and/or large-scale segmental duplication events and to a lesser extent by tandem duplications.AbstractPlant MADS-box genes comprise a large family best known for the roles of type II MIKCC genes in floral organogenesis, but also including type II MIKC* genes, some of which have been implicated in male gametophytic development, and type I genes, a few of which are involved in ontogeny of female gametophytes, seeds and embryos. Genome-wide analyses of the MADS-box family in angiosperms have revealed numeric predominance of type I and MIKCC genes and cross-species phylogenetic clustering of the Mα, Mβ and Mγ subtypes of type I genes and of 12 major subgroups of MIKCC genes. The genome sequence of Physcomitrella patens has facilitated investigation of its full complement of 26 MADS-box genes, including 6 MIKCC genes, 11 MIKC* genes, seven type I genes and two pseudogenes. A much higher degree of similarity in sequence and architecture within the MIKCC and MIKC* gene subtypes exists in Physcomitrella than in Arabidopsis. Furthermore, MADS-box and K-box sequence is highly conserved between the MIKCC and MIKC* subgroups in Physcomitrella. Nine MIKC* genes and two MIKCC genes are located in pairs or triplets on individual DNA scaffolds. Phylogenetic gene clustering, gene architectures and gene linkages (directly determined from examination of the genome sequence) underpin a parsimonious model of two tandem duplications and three segmental duplication events, which can account for lineage-specific expansion of the MADS-box gene family in Physcomitrella from 4 members to 26. Two of these segmental duplication events may be indicative of polyploidisations, one of which has been postulated previously.

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.

Heteroblasty in the moss, Aphanoregma patens (Physcomitrella patens), results from progressive modulation of a single fundamental leaf developmental programme

Abstract Leaves at the apex of a mature Aphanoregma patens (Hedw.) Lindb. (Physcomitrella patens (Hedw.) Bruch Schimp. in B.S.G.) gametophore differ markedly in size and form from those at its base. To determine how these differences are produced during development, we first examined qualitative and quantitative differences between successive leaves along the stem and among leaves at different developmental stages. Differences between successive leaves were slight and cumulative. Local changes in cell number and size combined to produce a regularly shaped and approximately bilaterally symmetrical leaf suggesting that cell division and cell expansion are regionally regulated and coordinated at the organ level. The midrib and marginal teeth are discrete characters, which were prefigured by changes in cell shape in leaves that lacked these characters. In leaf primordia, cell proliferation was responsible for most of the changes in leaf form and size early in development and may have continued as cell expansion took over as the primary contributor to leaf growth and morphogenesis. Thus, leaf heteroblasty in Physcomitrella probably results from modulation of a single developmental programme by external and/or internal forces, which alter progressively in intensity as a gametophore grows. We applied exogenous cytokinin and auxin separately to growing cultures to explore their effects on leaf growth. Cytokinin and auxin stimulated leaf cell division and leaf cell elongation, respectively. Also, young upper leaves of gametophores exposed to exogenous auxin closely resembled basal leaves of untreated plants. Therefore, endogenous cytokinins and auxins may be among the modulating internal forces involved in leaf morphogenesis and the establishment of leaf heteroblasty.