Influence of metabolic stress on the inheritance of cell determination in the moss, Pottia intermedia.

Epigenetically-determined apogamy in aposporous regenerants of the moss Pottia intermedia persists during vegetative propagation, the capacity of apogamy being inherited by individual aposporous protonemal cells. To test Bauer-Lazarenko's proposal that stable apogamy in mosses may be due to some self-replicating cytoplasmic factor, the effects of different metabolic stress treatments on the expression of apogamy have been tested. Chronic metabolic stress caused by long-term growth of autotrophic aposporous protonema on mineral medium with 0.25% of casamino acids and on Murashiga-Skoog (MS) medium with sucrose and phytohormones, as well as by transitory action of high kinetin concentration, have a much stronger influence on the expression of apogamy, than short-term stress treatments with RNase and Pb(2+). Apogamy has been found to be lost stably, after prolonged growth on MS medium containing kinetin and ABA. The proposal that the capacity for apogamy is related to the release of aposporous protonemal cells from a putative factor for apogamy is discussed.

The generation and modification of cell polarity.

The generation of polarity in the cells of eukaryotes in most cases requires an external asymmetrical input. This signal may originate from a random event such as the point of penetration of an egg by a sperm or from asymmetries present in the maternal environment as, for example, in megasporogenesis in flowering plants, or from an asymmetry in the physical environment usually involving either light or gravity. The latter affords the best opportunities for experimental manipulation and single cell systems in algae, mosses and ferns allow both cell biological and genetical investigation of the mechanisms establishing polarity. These simple systems have already shown that the generation of polarity involves two distinguishable processes, axis alignment and axis orientation, but a detailed understanding of the mechanisms by which the environmental inputs are transduced to set up a polar axis is still lacking. Non-motile organisms must also be able to modify cell polarity in response to changed environmental inputs, and here too, lower plant systems afford the best prospect for understanding the mechanisms responsible.

Phytochrome-controlled phototropism of protonemata of the moss ceratodon purpureus: physiology of the wild type and class 2 ptr-mutants

Phototropism and polarotropism in protonemata of the moss Ceratodon purpureus are controlled by the photoreceptor phytochrome. One class of phototropism mutants is characterised by growing randomly when kept for a prolonged time (5 d or longer) in unilateral red light. It was found that a subclass of these mutants grows faster than the wild type, the rate of cell division and the length of the cells being increased. This difference is found for light-grown and dark-grown filaments. It is therefore suggested that the mutant phenotype neither results from a defect in phytochrome photoconversion nor from a defect in phytochrome-gradient formation. Instead, it is possible that a factor which is involved in both signal transduction of phototropism and regulation of cell size and cell division is deregulated. If dark-grown mutant filaments are phototropically stimulated for 24 h, they show a weak phototropic response. Phototropism and polarotropism fluence-rate effect curves for mutants were flattened and shifted to higher fluence rates compared with those for the wild type. With wild-type filaments, a previously unreported response was observed. At a low fluence rate, half of the filaments grew positively phototropically, while the other half grew negatively phototropically. It seems that under these conditions, a phytochrome gradient with two maxima for the far-red-absorbing form of phytochrome (Pfr) within the cross-section of the cell is displayed by the response of the filaments. At higher fluence rates, all filaments of the wild type grew towards the light. These data and results from microbeam irradiation experiments and from phototropism studies with filaments growing within agar, indicate that light refraction plays an important role in the formation of the Pfr gradient in phototropism of Ceratodon.

Sequence analysis of expressed sequence tags from an ABA-treated cDNA library identifies stress response genes in the moss Physcomitrella patens.

Partial cDNA sequencing was used to obtain 169 expressed sequence tags (ESTs) in the moss, Physcomitrella patens. The source of ESTs was a random cDNA library constructed from 7 day-old protonemata following treatment with 10(-4) M abscisic acid (ABA). Analysis of the ESTs identified 69% with homology to known sequences, 61% of which had significant homology to sequences of plant origin. More importantly, at least 11 ESTs had significant similarities to genes which are implicated in plant stress-responses, including responses which may involve ABA. These included a cDNA associated with desiccation tolerance, two heat shock protein genes, one cold acclimation protein cDNA and five others that may be involved in either oxidative or chemical stress or both, i.e., Zn/Cu-superoxide dismutase, NADPH protochlorophyllide oxidoreductase (PorB), selenium binding protein, glutathione peroxidase and glutathione S transferase. Analysis of codon usage between P. patens and seed plants indicated that although mosses and higher plants are to a large extent similar, minor variations also exists that may represent the distinctiveness of each group.

A specific member of the Cab multigene family can be efficiently targeted and disrupted in the moss Physcomitrella patens.

The analysis of phenotypic change resulting from gene disruption following homologous recombination provides a powerful technique for the study of gene function. This technique has so far been difficult to apply to plants because the frequency of gene disruption following transformation with constructs containing DNA homologous to genomic sequences is low (0.01 to 0.1%). It has recently been shown that high rates of gene disruption (up to 90%) can be achieved in the moss Physcomitrella patens using genomic sequences of unknown function. We have used this system to examine the specificity of gene disruption in Physcomitrella using a member of the Cab multigene family. We have employed the previously characterised Cab gene ZLAB1 and have isolated segments of 13 other closely related members of the Cab gene family. In the 199-bp stretch sequenced, the 13 new members of the Cab family show an average of 8.5% divergence from the DNA sequence of ZLAB1. We observed 304 silent substitutions and 16 substitutions that lead to a change in the amino acid sequence of the protein. We cloned 1029 bp of the coding region of ZLAB1 (including 177 of the 199 bp with high homology to the 13 new Cab genes) into a vector containing a selectable hygromycin resistance marker, and used this construct to transform P. patens. In three of nine stable transformants tested, the construct had inserted in, and disrupted, the ZLAB1 gene. There was no discernible phenotype associated with the disruption. We have therefore shown that gene disruption is reproducible in P. patens and that the requirement for sequence homology appears to be stringent, therefore allowing the role of individual members of a gene family to be analysed in land plants for the first time.

A positively gravitropic mutant mirrors the wild-type protonemal response in the moss Ceratodon purpureus.

Wild-type Ceratodon purpureus (Hedw.) Brid. protonemata grow up in the dark by negative gravitropism. When upright wild-type protonemata are reoriented 90 degrees, they temporarily grow down soon after reorientation ("initial reversal") and also prior to cytokinesis ("mitotic reversal"). A positively gravitropic mutant designated wrong- way response (wwr-1) has been isolated by screening ultraviolet light-mutagenized Ceratodon protonemata. Protonemata of wwr-l reoriented from the vertical to the horizontal grow down with kinetics comparable to those of the wild-type. Protonemata of wwr-1 also show initial and mitotic reversals where they temporarily grow up. Thus, the direction of gravitropism, initial reversal, and mitotic reversal are coordinated though each are opposite in wwr-1 compared to the wild-type. Normal plastid zonation is still maintained in dark-grown wwr-1 apical cells, but the plastids are more numerous and plastid sedimentation is more pronounced. In addition, wwr-1 apical cells are wider and the tips greener than in the wild-type. These data suggest that a functional WWR gene product is not necessary for the establishment of some gravitropic polarity, for gravitropism, or for the coordination of the reversals. Thus, the WWR protein may normally transduce information about cell orientation.

Phytochrome control of phototropism and chlorophyll accumulation in the apical cells of protonemal filaments of wildtype and an aphototropic mutant of the moss Ceratodon purpureus.

The aphototropic mutant line ptr116 of the moss Ceratodon purpureus shows characteristics of a deficiency in the phytochrome chromophore. Photoreversibility measurements indicate an approximately 20 time lower concentration of spectrally active phytochrome compared to wild-type, whereas normal phytochrome apoprotein levels are found on immunoblots. Feeding with the tetrapyrroles biliverdin, the proposed precursor of the phytochrome chromophore, or phycocyanobilin, which may replace the phytochrome chromophore, resulted in the rescue of ptr116 phototropism. The ptr116 mutant and the phenotypically-related mutant ptr1 contain lower chlorophyll levels than the wild-type. Chlorophyll content of wildtype and mutant tissue grown under different light conditions was estimated using conventional spectrophotometry of extracts and fluorimetrically, on single apical cells. Dark-grown tissue contained about 100 times less chlorophyll than tissue grown under standard white light conditions. Red light given for 24 h to dark adapted filaments induced an increase in the chlorophyll content in the wildtype, but not in ptr116. Blue light induced an increase in chlorophyll both in wildtype and in ptr116. The red light effect on the wildtype was partially reversible with far-red. If ptr116 was grown on phycocyanobilin, an increase in chlorophyll was also found when cells were irradiated with red light. The results indicate that phytochrome as well as a blue light photoreceptor regulate chlorophyll accumulation in C. purpureus protonemata. It can be assumed that in ptr116, the synthesis of the phytochrome chromophore is blocked specifically beyond the synthesis common to chlorophyll and the phytochrome chromophore and affects an enzymatic step between protoporphyrin and biliverdin.

Growth and development of fern gametophytes in an airlift fermenter.

Spores of the fernsPteridium aquilinum andAnemia phyllitidis were grown in an airlift fermenter and subsequent growth and development of gametophytes was monitored. Both species produced greater biomass than that generated in any other solid- or liquid-based culture system tested.Pteridium generated more tissue thanAnemia in every system. The morphology of airlift-grown gametophytes was similar to that of soil-grown plants; fewer gametophytes with perturbed development were observed in airlift cultures than in the other liquid-based systems. No attempt was made to optimise airlift conditions for the species and tissue employed, so it is concluded that airlift cultivation is a promising system for the bulk production of fern gametophytic tissue.

The moss, Physcomitrella patens, transformed with apoaequorin cDNA responds to cold shock, mechanical perturbation and pH with transient increases in cytoplasmic calcium.

The gene for apoaequorin has been used previously to indicate cytosolic calcium changes in higher plants. Here we report the transformation of the moss Physcomitrella patens with the cDNA for apoaequorin. Stable transformants were obtained in the wild type which reconstitute the calcium-sensitive luminescent protein aequorin in vivo after incubation in coelenterazine, and continue to grow normally. The wild type responds to cold-shock (0-10 degrees C) with increases in cytosolic calcium. Mechanical perturbation, in the form of touch, also induces transient increases in cytosolic calcium. A smaller response to pH, distinct from the touch response and exhibiting different kinetics, can also be detected.

Cushing's syndrome and hypothalamic-pituitary adrenal axis suppression induced by medroxyprogesterone acetate.

The referral of a patient with features of Cushing's syndrome but with suppressed plasma cortisol and adrenocorticotrophic hormone concentrations prompted us to study the effect of medroxyprogesterone acetate (MPA) therapy on the adrenal axis. 11 women (aged 54-82 years) who were receiving 200-400 mg/day MPA were studied. Of these, four had subnormal plasma cortisol responses to a short synacthen test, and two more had borderline responses (30 min post-synacthen plasma cortisol results of 411 and 511 nmol/L). We conclude that suppression of the adrenal axis occurs relatively frequently in patients on MPA and that such patients should be checked for evidence of suppression before MPA therapy is withdrawn.

Genetic analysis of the effects of re-transformation of transgenic lines of the moss Physcomitrella patens.

Genetic analysis of the progeny of crosses involving strains of the moss Physcomitrella patens obtained by re-transforming a stable transgenic line, indicates that the plasmid used for re-transformation inserts at or near the chromosomal location of the related plasmid used to obtain the original transgenic line. The resulting structure may be subject to gene silencing.

The alignment of the axis of asymmetry in regenerating protoplasts of the moss, Ceratodon purpureus, is determined independently of axis polarity.

Ceratodon protoplasts regenerate by polar outgrowth to form cell filaments. The kinetics of regeneration show that some cellular event has to be completed before regeneration can be initiated. The development of the regeneration axis is strongly influenced by light, with axis alignment and axis polarity being fixed independently. We define axis alignment as the relationship of the regeneration axis to the incident light, independent of polarity. Thus protoplasts regenerating directly towards, or directly away from the light source are defined as being similarly aligned but with opposite polarity. Protoplasts that regenerate in unidirectional red light form axes that are aligned parallel to the light direction, with about 70% being polarised towards the light and about 30% away. In unidirectional blue or white light, almost all protoplasts regenerate towards the light but axis alignment is determined less stringently. Re-orientation of protoplasts regenerating in unidirectional light shows that axis alignment is fixed between 8 and 9 hours before protoplasts regenerate and that axis polarity is fixed later. When protoplasts are removed from directional light to either non-directional light or to darkness, regeneration axes continue to be aligned by the earlier directional stimulus for at least 24 hours. Thus although axis alignment is fixed only about 8 hours before regeneration, in the absence of contradictory information about directionality in the light environment, protoplasts retain a memory of light direction for much longer. However, both reorientation and removal from a directional light field have profound effects on axis polarity; the pattern observed in undisturbed protoplasts being lost. To account for these observations, we propose that separate gradients are established independently to determine the alignment and polarity of the regeneration axis respectively. The alignment gradient is established rapidly and is steeper in red than in blue or white light, the polarity gradient is established slowly and is steeper in white or blue light than in red. These studies will now allow a genetic dissection of these processes in moss.

Molecular Responses to Abscisic Acid and Stress Are Conserved between Moss and Cereals.

Promoter elements from the wheat Em gene have been characterized. These elements are inducible by abscisic acid (ABA) and by osmotic stress. In this study, we demonstrated that the same promoter elements function in a distantly related plant species, the moss Physcomitrella patens. Transient and stable expression of the [beta]-glucuronidase reporter gene was used to determine that the heterologous wheat promoter also responds to osmotic stress and ABA in moss. Mutational analysis of the promoter indicated that the mechanism of gene regulation is conserved in both species. Gel retardation and DNase I footprint analyses were conducted to characterize further the interaction of moss transcription factors with the Em promoter. In addition, the synthesis of stress-related polypeptides in moss was observed. The evolutionary significance of these data and the potential for studying the entire ABA perception-response pathway in moss are discussed.

Expression of myb-related genes in the moss, Physcomitrella patens.

Three cDNA clones encoding proteins containing a myb-related DNA binding domain have been isolated from a cDNA library prepared from protonemal tissue of the moss, Physcomitrella patens. The three cDNA clones between them encode two different classes of myb-like proteins, termed Pp1 and Pp2, that, outside of the myb domain, show no regions of significant homology. Acidic domains, capable of forming alpha-helical structures, are present in the carboxy-termini of the derived amino acid sequences from Pp1 and Pp2cDNAs suggesting that, like other myb genes, these proteins probably function as transcriptional activators. In contrast to other plants, where extensive myb-related gene families are present in the genome, a relatively small family is present in P. patens. Analyses of transcript levels during development of P. patens showed that maximum levels of transcription of the two genes occurred in young wild-type protonemal tissue that correlated with the time of maximum mitotic index. A decline in the expression of both genes occurs with increasing age of the wild-type tissue. Aberrant levels of expression of the two genes were observed in developmental mutants of P. patens which, as well as carrying specific morphological mutations, have greatly retarded protonemal growth rates. Transformation of wild-type P. patens with antisense constructs derived from Pp1 and Pp2 cDNA clones led to a dramatically reduced frequency of transformants when the expression of the reporter gene within the constructs was selected. Taken together, the data strongly suggest that expression of Pp1 and Pp2 is essential for cell growth during normal gametophytic development of P. patens.

Gene transfer strategies in plants.

In recent years, a large number of gene transfer methods have been developed. However, the results of these studies have often been published in such a way that it has been extremely difficult for researchers to assess the reliability and efficiency of the method, and to judge whether or not integrative transformation has occurred. Thus although an abundance of knowledge exists within the area of gene transfer, its documentation remains disjointed. This report summarises the recent progress which has been made in the field of gene transfer systems in plants and discusses the associated advantages, disadvantages and limitations in an attempt to clarify this issue.

Genetic analysis of a mutant class of Physcomitrella patens in which the polarity of gravitropism is reversed.

In the moss Physcomitrella patens, single-cell protonemata and multicellular gametophores respond to reorientation relative to the gravity vector by growing negatively gravitropically. A mutant class in which the protonemata, but not the gametophores, respond by growing towards gravity has been identified. In this paper, we describe the isolation of additional mutants of this class. Complementation and segregation ratio analyses were carried out on these mutants, which indicate that a single gene may mutate to switch the polarity of gravitropism.

Stable transformation of the moss Physcomitrella patens.

We report the stable transformation of Physcomitrella patens to either G418 or hygromycin B resistance following polyethylene glycol-mediated direct DNA uptake by protoplasts. The method described in this paper was used successfully in independent experiments carried out in our two laboratories. Transformation was assessed by the following criteria: selection of antibiotic-resistant plants, mitotic and meiotic stability of phenotypes after removal of selective pressure and stable transmission of the character to the offspring; Southern hybridisation analysis of genomic DNA to show integration of the plasmid DNA; segregation of the resistance gene following crosses with antibiotic-sensitive strains; and finally Southern hybridisation analysis of both resistant and sensitive progeny. In addition to stable transformants, a heterogeneous class of unstable transformants was obtained.

Developmental genetic studies of the moss, Physcomitrella patens.

The development of the haploid gametophyte stage of Physcomitrella patens presents excellent opportunities for the detailed study of plant morphogenesis at the cellular level. The filamentous protonema undergoes a number of developmental transitions that can be observed directly in living material using time-lapse video microscopy and that can be manipulated both by treatment with phytohormones and by environmental stimuli. Mutants affecting these processes can be isolated and can be analysed using conventional as well as parasexual methods. Molecular biological techniques are now being established since these will be essential if the mechanisms that bring about morphogenetic processes are to be understood at the molecular level. A technique for genetic transformation has been devised and is being exploited to attempt to tag developmentally-relevant genes using maize transposons. Changes in gene activity associated with developmental transitions and in response to treatment with phytohormones and environmental stimuli are being studied using cDNA library subtraction techniques. Heterologous genes involved in the control of transcription and of the cell cycle are being used to probe the P. patens genome to identify possible homologues involved in developmental regulation.

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

Cytokinins 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 and OVEC. A further three strains have been demonstrated not to belong to the OVEA group and another mutant does not fall into group OVEB. Phenotypic segregation ratios among progeny obtained following self-fertilization of a number of different somatic hybrids showed that several OVE mutations behave as recessive alleles of single Mendelian genes.