ADHERENCE OF AGROBACTERIA TO MOSS PROTONEMA AND GAMETOPHORES VIEWED BY SCANNING ELECTRON MICROSCOPY

The attachment of certain Agrobacterium species to moss protonema was examined by scanning electron microscopy. The bacteria cultured with the moss adhered to the protonemal filaments of Pylaisiella selwynii and Funaria hygrometrica throughout the fixation and washing procedure prior to examination. The number of bacteria adhering per unit length of filament did not correlate with the ability of four species (A. tumefaciens, A. rubi, A. rhizogenes, A. radiobacter) to induce gametophore formation in P. selwynii. Little difference was found between the number of A. tumefaciens on P. selwynii or on F. hygrometrica, and although this bacterial species induced gametophores on P. selwynii, it had no effect on F. hygrometrica. A. rhizogenes, which is capable of inducing rhizoid formation on both P. selwynii and F. hygrometrica without physical attachment, did associate with filaments of both mosses. Larger numbers of A. tumefaciens and A. rubi, which induce gametophores or callus, were associated with the spore and germ tube of P. selwynii than were A. rhizogenes or A. radiobacter which do not initiate these changes. These results suggest that the germ tube may constitute the site where effective attachment occurs. The number of A. tumefaciens adhering to P. selwynii was greatest at 2–6 h after addition and then decreased by 26 h, raising the possibility that some bacteria lyse following attachment. None of the bacteria adhered to the gametophore walls of either moss, indicating that a change in cell wall surface may occur. The four species of Agrobacterium differed in their orientation when adhering to the moss. These results suggest that the process of bacterial adherence to higher plant cells in Agrobacterium infection is similar to that which we observe in mosses and which leads to developmental changes.     

INFLUENCE OF CERTAIN PLANT GROWTH REGULATORS AND CROWN-GALL RELATED SUBSTANCES ON BUD FORMATION AND GAMETOPHORE DEVELOPMENT OF THE MOSS PYLAISIELLA SELWYNII

Bud formation and gametophore development were studied in the moss Pylaisiella selwynii (Kindb.) Steere and Anderson grown from spores in a liquid medium consisting of inorganic salts. Indoleacetic acid and ethrel increased bud formation within a narrow concentration range. Copious bud formation was obtained with the five cytokinins tested at concentrations varying from 5 X 10⁻⁶ to 5 X 10⁻¹⁴ M. Except for about 10 % of the buds obtained with 6-γ, γ-dimethylallylaminopurine at 5 times 10⁻¹⁴ m, the cytokinin-induced buds failed to develop into normal gametophores. Octopine, lysopine, and octopinic acid, substances obtained from crown-gall tumors, increased bud formation at 10⁻³ m. On lysopine-treated plants these buds developed into typical gametophores. Gemma-like structures were obtained with octopine but no gametophores. l -arginine and l -lysine, the amino acids which respectively occur in octopine and lysopine, failed to induce gametophore formation although buds were obtained with 10⁻³ m lysine. γ-Guanidinobutyric acid induced bud formation at 10⁻³ m, but these buds developed into highly abnormal gametophores. The failure of buds obtained with many of these treatments to develop into gametophores appeared to result from the formation of new cell walls in other than the normal geometrical relationship during initial divisions of the pro-bud. The relevance of the findings to the crown-gall problem is discussed.     

EFFECT OF HORMONES AND VITAMIN B12 ON GAMETOPHORE DEVELOPMENT IN THE MOSS PYLAISIELLA SELWYNII

Bud formation in the moss Pylaisiella selwynii is greatly enhanced by cytokinins at concentrations as low as 10⁻¹²m , yet these buds usually fail to develop into normal gametophores. Various ratios at different concentrations of the cytokinin N-6-γ,γ-dimethylallylaminopurine to indoleacetic acid failed to enhance bud initiation over that obtained with cytokinin alone or to permit normal gametophore development. Deletion of the cobaltous ions from the culture medium prevented the appearance of the few gametophores usually formed in the complete medium, but different amounts of cobaltous ion did not significantly enhance initiation of gametophore development. Bud initiation was enhanced 3- to 20-fold by vitamin B₁₂ at 10⁻⁵m or by B₁₂ coenzyme at 10⁻⁴m , and the time of appearance of these buds was advanced by 6–12 days compared to control plants. At these concentrations of the B₁₂ compounds the buds formed normal gametophores, but at 10⁻⁴m vitamin B₁₂ they grew into callus-like masses similar to those obtained with cytokinins. Although the effects of B₁₂ on bud initiation and development mimicked those of cytokinins, except in permitting normal development, no additive or synergistic effects were observed when they were tested together. It is suggested that B₁₂ may play a regulatory role in the control of gametophore initiation and development in mosses.     

Role of bacterial lipopolysaccharide in attachment of agrobacterium to moss

Gametophore induction in moss by Agrobacterium tumefaciens was inhibited by addition of lipopolysaccharide (LPS) from A. tumefaciens. The LPS did not affect bacterial viability or appear to bind to bacterial cells. LPS from nonbinding Agrobacterium radiobacter was not effective in reducing gametophore formation. A. tumefaciens LPS, if added 24 hours after addition of viable bacterial cells, had no effect in reducing gametophore formation. The polysaccharide portion of the LPS was identified as the binding component necessary for attachment of agrobacteria for induction of gametophores in moss and tumors in higher plants.     

THE REQUIREMENT OF PHYSICAL CONTACT FOR MOSS GAMETOPHORE INDUCTION BY AGROBACTERIUM TUMEFACIENS

Gametophore induction in the moss Pylaisiella selwynii by Agrobacteriuin tumefaciens strain B6 does not occur in parabiotic chambers when the moss and bacterium are separated by filters of 0.22 μm pore diameter. The combination of moss plus bacteria which gives increased gametophore formation also had no effect on sterile moss plants separated from the mixture by these filters. Heat-killed B6 cells inhibit gametophore induction by viable B6 cells when added prior to or with the viable bacteria but not when added 6 or more hr later. The concentration dependence of both gametophore induction by viable B6 cells and inhibition of this process by heat-killed B6 cells appears linear, suggesting that each response may require only one particle. Gametophore induction by Agrobacterium, therefore, requires physical contact comparable to the bacteria-site attachment step demonstrated essential for tumor induction in higher plants and shows similar concentration dependence. The size of the moss and the absence of a wound requirement for this response makes the Agrobacterium-moss system particularly advantageous for further resolution of the site attachment step in Agrobacterium tumorigenesis.     

Expression of the bacterial ipt gene in Physcomitrella rescues mutations in budding and in plastid division

Development of Physcomitrella patens (Hedw.) B.S.G. starts with a filamentous protonema growing by apical cell division. As a developmental switch, some subapical cells produce three-faced apical cells, the so-called buds, which grow to form leafy shoots, the gametophores. Application of cytokinins enhances bud formation but no subsequent gametophore development in several mosses. We used the ipt gene of Agrobacterium tumefaciens, encoding a protein which catalyzes the rate-limiting step in cytokinin biosynthesis, to transform two developmental Physcomitrella mutants. One mutant (P24) was defective in budding (bud) and thus did not produce three-faced cells, while the other one (PC22) was a double mutant, defective in plastid division (pdi), thus possessing at the most one giant chloroplast per cell, and in gametophore development (gad), resulting in malformed buds which could not differentiate into leafy gametophores. Expression of the ipt gene rescued the mutations in budding and in plastid division but not the one in gametophore development. By mutant rescue we provide evidence for a distinct physiological difference between externally applied and internally produced cytokinins. Levels of immunoreactive cytokinins and indole-3-acetic acid were determined in tissues and in culture media of the wild-type moss, both mutants and four of their stable ipt transformants. Isopentenyl-type cytokinins were the most abundant cytokinins in Physcomitrella, whereas zeatin-type cytokinins, the major native cytokinins of higher plants, were not detectable. Cytokinin as well as auxin levels were enhanced in ipt transgenics, demonstrating a cross-talk between both metabolic pathways. In all genotypes, most of the cytokinin and auxin was found extracellularly. These extracellular pools may be involved in hormone transport in the non-vascular mosses. We suggest that both mutants are defective in signal-transduction rather than in cytokinin metabolism. Received: 24 October 1997 / Accepted: 20 March 1998     

Kinetin-induced Formation of Gametophores in Dark Cultures of Ceratodon purpureus

The effects of various concentrations of kinetin on the developmentof gametophytes were studied in light and dark cultures of Ceratodonpurpureus in liquid media. There was no bud formation in eitherlight or dark control cultures. Buds were produced under theinfluence of kinetin in light as well as in dark cultures, theeffect of a given concentration of kinetin being a little strongerin the light. Kinetin has been thus found to be an essential factor of budformation in a moss which hardly produces gametophores evenin the light. This is believed to be the first report of gametophore formationin the dark since Keil's short communication in 1949. It alsorepresents a new example replacing a morphogenetic light stimulusby kinetin.     

A CELLULOSE SYNTHASE (CESA) gene essential for gametophore morphogenesis in the moss Physcomitrella patens

In seed plants, different groups of orthologous genes encode the CELLULOSE SYNTHASE (CESA) proteins that are responsible for cellulose biosynthesis in primary and secondary cell walls. The seven CESA sequences of the moss Physcomitrella patens (Hedw.) B. S. G. form a monophyletic sister group to seed plant CESAs, consistent with independent CESA diversification and specialization in moss and seed plant lines. The role of PpCESA5 in the development of P. patens was investigated by targeted mutagenesis. The cesa5 knockout lines were tested for cellulose deficiency using carbohydrate-binding module affinity cytochemistry and the morphology of the leafy gametophores was analyzed by 3D reconstruction of confocal images. No defects were identified in the development of the filamentous protonema or in production of bud initials that normally give rise to the leafy gametophores. However, the gametophore buds were cellulose deficient and defects in subsequent cell expansion, cytokinesis, and leaf initiation resulted in the formation of irregular cell clumps instead of leafy shoots. Analysis of the cesa5 knockout phenotype indicates that a biophysical model of organogenesis can be extended to the moss gametophore shoot apical meristem.     

Acclimation and endogenous abscisic acid in the moss Physcomitrella patens during acquisition of desiccation tolerance

The moss Physcomitrella patens has been used as a model organism to study the induction of desiccation tolerance (DT), but links between dehydration rate, the accumulation of endogenous abscisic acid (ABA) and DT remain unclear. In this study, we show that prolonged acclimation of P. patens at 89% relative humidity (RH) [?16 MPa] can induce tolerance of desiccation at 33% RH (?153 MPa) in both protonema and gametophore stages. During acclimation, significant endogenous ABA accumulation occurred after 1 day in gametophores and after 2 days in protonemata. Physcomitrella patens expressing the ABA‐inducible EARLY METHIONINE promoter fused to a cyan fluorescent protein (CFP) reporter gene revealed a mostly uniform distribution of the CFP increasing throughout the tissues during acclimation. DT was measured by day 6 of acclimation in gametophores, but not until 9 days of acclimation for protonemata. These results suggest that endogenous ABA accumulating when moss cells experience moderate water loss requires sufficient time to induce the changes that permit cells to survive more severe desiccation. These results provide insight for ongoing studies of how acclimation induces metabolic changes to enable DT in P. patens.     

Development of novel methods for the initiation of in vitro bryophyte cultures for conservation

The value of ex situ plant collections for conservation is increasingly recognised. The Royal Botanic Gardens, Kew (RGB, Kew) has been pioneering methods for the development of such collections with the establishment of the Millennium Seed Bank and a project for the ex situ conservation of threatened UK bryophytes (mosses, liverworts, hornworts). A broad range of bryophytes are currently held in a tissue culture collection at RBG, Kew on sucrose-free ¼ or ½ Murashige &; Skoog or Knops minimal medium. The efficacy of the novel sterilising agent Sodium dichloroisocyanurate (NaDCC) was tested on a range of taxa, utilising a variety of bryophytic initiation material. Concentrations of 1% (w/v) for 3 min and 0.5% (w/v) for 2 min, without the addition of detergent, were found to be successful in initiating cultures from sporophytes and leafy gametophores respectively. Initiation of cultures from wild-collected sporophytes was more successful than from wild-collected gametophores. However, pre-culturing of gametophore material was found to enhance success rate of procedures. Transferring visibly clean material away from contamination post-initiation was also determined to increase protocol success rate. The ability of the biocide Plant Preservative Mixture^TM (PPM^TM) to control fungal and bacterial contamination in bryophyte protonemal cultures was also tested, but not found to be suitable. However, results obtained indicated that contamination may confer resistance on the moss against the phytotoxic effects of the biocide. Methods developed have wider applicability for the establishment of in vitro collections of other threatened plants.     

The moss genes <Emphasis Type="Italic">PpSKI1</Emphasis> and <Emphasis Type="Italic">PpSKI2</Emphasis> encode nuclear SnRK1 interacting proteins with homologues in vascular plants

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant’s ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.     

Changes in water potential and its components during shoot formation in tobacco callus

Addition of plant growth regulators (5 nM NAA and 5μM BAP) to a defined basal medium stimulated adventitious bud formation of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco) cotyledon explants in culture. Cytoplasmic soluble proteins synthesized during early stages of adventitious bud formation were analyzed by electrophoresis of ³H- and ¹⁴C-leucine labeled proteins on SDS polyacrylamide gels. Increased synthesis of low molecular weight proteins (16,000 to 20,000 daltons) was detected after 2 days in culture and reached a maximal level at day 4. When cotyledon explants cultured on bud medium for 2 days were transferred to callus medium (which suppressed adventitious bud formation), suppression of the synthesis of low molecular weight proteins was also observed, suggesting that these proteins may be associated with early stages of adventitious bud formation.     

Encapsulation of moss buds: an efficient method for the in vitro conservation and regeneration of the endangered moss <Emphasis Type="Italic">Splachnum ampullaceum</Emphasis>

In vitro culture techniques are usually employed for ex situ conservation of endangered plant species. However, encapsulation to preserve threatened bryophytes is scarcely used, and only as a pretreatment prior to cryopreservation. In our study, two different methods of germplasm conservation, involving calcium-alginate encapsulation of moss material, were assessed. The plant material used was gametophyte buds (gametophores) of Splachnum ampullaceum Hedw., a rare species of moss. Moss regeneration was evaluated at different periods of time to examine the efficacy of the technique for moss germplasm conservation. The effects of encapsulation and cold storage on developmental parameters such as protonematal colony diameter, bud length, and number of buds were also studied. The results suggest that moss encapsulation with no prior treatment may be a suitable method for germplasm conservation during long periods of time. With our method survival rates as high as 50% could be reached after 2.5 years of cold storage versus 0% of 24-h cryopreserved beads. This technique together with cold storage, avoiding freezing, may be especially important in desiccation intolerant mosses.     

Promotion of Pylaisiella selwynii growth and gametophore formation by octopine and cytokinin

Growth, branching and gametophore formation by protonema developing from spores of the moss Pylaisiella selwynii were promoted by octopine, an unusual amino acid found in crown gall tumors. Cytokinin (0.01 μ M ) in combination with octopine (0.1 μ M to 1 m M ) increased the number of gametophores formed and decreased the time required for their development. The combined effect of these two compounds was similar to that obtained with virulent agrobacteria. At higher cytokinin concentrations, protonemal buds formed abnormal masses of cells rather than normal gametophores. Common amino acids and auxins, alone or in combination with cytokinin, had relatively little effect on the development of the moss.     

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio‐lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1‐1 and 1‐2). Both PpAN1 genes complemented the A. thaliana an‐1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1‐promoter– uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1‐1 and PpAN1‐2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip‐growing protonemata. These results indicate that both PpAN1 genes in P. patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P. patens, transformed lines expressing P. patens α‐tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1‐1/1‐2 double‐knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.     

Large-scale analysis of 73 329 physcomitrella plants transformed with different gene disruption libraries: production parameters and mutant phenotypes

Gene targeting in the moss Physcomitrella patens has created a new platform for plant functional genomics. We produced a mutant collection of 73 329 Physcomitrella plants and evaluated the phenotype of each transformant in comparison to wild type Physcomitrella. Production parameters and morphological changes in 16 categories, such as plant structure, colour, coverage with gametophores, cell shape, etc., were listed and all data were compiled in a database (mossDB). Our mutant collection consists of at least 1804 auxotrophic mutants which showed growth defects on minimal Knop medium but were rescued on supplemented medium. 8129 haploid and 11 068 polyploid transformants had morphological alterations. 9 % of the haploid transformants had deviations in the leaf shape, 7 % developed less gametophores or had a different leaf cell shape. Other morphological deviations in plant structure, colour, and uniformity of leaves on a moss colony were less frequently observed. Preculture conditions of the plant material and the cDNA library (representing genes from either protonema, gametophore or sporophyte tissue) used to transform Physcomitrella had an effect on the number of transformants per transformation. We found correlations between ploidy level and plant morphology and growth rate on Knop medium. In haploid transformants correlations between the percentage of plants with specific phenotypes and the cDNA library used for transformation were detected. The number of different cDNAs present during transformation had no effect on the number of transformants per transformation, but it had an effect on the overall percentage of plants with phenotypic deviations. We conclude that by linking incoming molecular, proteome, and metabolome data of the transformants in the future, the database mossDB will be a valuable biological resource for systems biology.     

Day Length and Temperature Strongly Influence Sexual Reproduction and Expression of a Novel MADS-Box Gene in the Moss Physcomitrella patens

Abstract: The effect of temperature and light conditions on sexual reproduction (sporophyte formation) of in vitro cultures of the moss Physcomitrella patens was analysed. All parameters tested, i.e., temperature, light intensity and day length had a strong impact on the number of sporophytes formed. The highest number of sporophytes, 559 g fresh weight, developed at 15 °C, 8 h light/day with an intensity of 20 μmol/m²/s. In contrast, at 25 °C, as well as with a day length of 16 h per day, the number of sporophytes was drastically reduced. Vegetative growth, determined as fresh weight per petri dish, was impeded under conditions favouring sporophyte formation, probably due to nutrient transfer to the sporophytes. Microscopic documentation of the developing sporophytes revealed that, although archegonia were arranged in bundles at the gametophore apices, usually only one archegonium per gametophore apex developed into a mature sporophyte. From an EST database six novel MADS-box genes were identified which, in phylogenetic analyses, did not cluster with the known groups of higher plant MADS-box genes. One of these genes was represented only as a singleton in a cDNA library specifically derived from gametophore apices and developing sporophytes, and, therefore, designated PpMADS-S. RNA amounts of PpMADS-S were two to three times higher under conditions that stimulate sporophyte development (15 °C, 8 h light per day) when compared to conditions favouring vegetative growth (25 °C, 16 h light per day), indicating a possible function in sexual reproduction of this moss. Thus, an efficient experimental system was established to study sex organ formation, fertilization and embryo development in Physcomitrella.     

THE QUANTITATIVE DETERMINATION OF THE INFECTIVITY OF AGROBACTERIUM TUMEFACIENS

A bioassay relating number of Agrobacterium tumefaciens cells in the inoculum quantitatively to the number of crown-gall tumors initiated on primary pinto bean leaves is described. Variability in estimation of infectious titers by this assay is similar to that observed in comparable plant virus assays, most determinations showing standard errors of 20% of the mean tumor per leaf value. The assay has the advantages of speed and practicality. The efficiency of the system is low, typically requiring between 10⁵ and 10⁶ bacteria for each tumor initiated. Infectivity titers of 10³-10⁴, however, are readily obtained from stationary phase cultures. Statistical analysis of the infectivity titration curve indicates that a single bacterium is the usual infectious unit. The assay is specific within the family Rhizobiaceae to the species Agrobacterium tumefaciens and Agrobacterium rubi. A. tumefaciens strains IIBNV6 and ATCC # 11095 were non-infectious, while strain B6 was the most infectious of the strains tested. The infectivity of the latter strain is shown to decrease about 4-fold between early log and stationary phases of growth. Changes in the growth medium or in the dilution-inoculation medium failed to alter the infectivity of the bacterium.