Induction of budding on chloronemata and caulonemata of the moss,Physcomitrella patens,using isopentenyladenine

The bud-inducing effect of the cytokinin N⁶-(²-isopentenyl)-adenine (i⁶-Ade) was examined in the moss Physcomitrella patens growing in liquid culture. Under these conditions, buds could be induced on chloronemata as well as on caulonemata. By application of i⁶-Ade, bud-formation was accelerated in both types of tissue. The number of buds, their size and their site of development were dependent on the concentration of the cytokinin in the range of 10^-7 M to 10^-5 M. Moreover, the percentage of caulonema cells increased with a cytokinin concentration of 10^-5 M. These results indicate that chloronema cells may also function as target cells for exogenous cytokinins. The composition of proteins from caulonemata and chloronemata of two different species (P. patens and Funaria hygrometrica), grown on solid medium were compared. No differences could be detected between the protein patterns of caulonemata and chloronemata of the same species while between the two species the differences were obvious.Abbreviations i⁶-Ade N⁶-(²-isopentenyladenine) - Da dalton - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Light requirements for regeneration of protoplasts of the moss Physcomitrella patens

Protoplasts prepared by enzymic treatment of protonemata of the moss Physcomitrella patens regenerate rapidly in white light (15 W m^?2). The great majority of protoplasts follow a simple regenerative sequence, namely: cell wall synthesis; formation of an asymmetric cell; division of the asymmetric cell, and further extension and division to produce a new chloronemal filament. Only cell wall formation occurs independently of light. The production of an asymmetric cell requires relatively high photon fluence rates of blue or red light and ceases upon transfer to darkness. The subsequent stages of regeneration require much lower photon fluence rates, and red light is considerably more effective than blue or far-red light in permitting cell division. This system is of interest in the study of the induction of cell polarity in plants.     

Unique Tissue-Specific Cell Cycle in Physcomitrella

Abstract: The moss Physcomitrella patens (Hedw.) B.S.G. is a novel tool in plant functional genomics as it has an inimitable high gene targeting efficiency facilitating the establishment of gene/function relationships.
Here we report, based on flow cytrometric (FCM) data, that the basic nuclear DNA content per cell of Physcomitrella is 0.53 pg, equating to a genome size of 1 C = 511 Mbp. Furthermore, we describe a unique tissue-specific cell cycle change in this plant. Young plants consisting of only one cell type (chloronema) displayed one single peak of fluorescence in FCM analyses. As soon as the second cell type (caulonema) developed from chloronema, a second peak of fluorescence at half the intensity of the previous one became detectable, indicating that caulonema cells were predominantly at the G1/S transition, whereas chloronema cells were mainly accumulating at the G2/M transition. This conclusion was validated by further evidence: i) The addition of ammonium tartrate arrested Physcomitrella in the chloronema state and in G2/M. ii) Two different developmental mutants, known to be arrested in the chloronema/caulonema transition, remained in G2/M, regardless of age and treatment. iii) The addition of auxin or cytokinin induced the formation of caulonema, as well as decreasing the amount of cells in G2/M phase. Additionally, plant growth regulators promoted endopolyploidisation.
Thus, cell cycle and cell differentiation are closely linked in Physcomitrella and effects of plant hormones and environmental factors on both processes can be analysed in a straight forward way. We speculate that this unique tissue-specific cell cycle arrest may be the reason for the uniquely high rate of homologous recombination found in the Physcomitrella nuclear DNA.     

A novel type of chloroplast stromal hexokinase is the major glucose-phosphorylating enzyme in the moss Physcomitrella patens

Hexokinase catalyzes the first step in the metabolism of glucose but has also been proposed to be involved in sugar sensing and signaling both in yeast and in plants. We have cloned a hexokinase gene, PpHXK1, in the moss Physcomitrella patens where gene function can be studied directly by gene targeting. PpHxk1 is a novel type of chloroplast stromal hexokinase that differs from previously studied membrane-bound plant hexokinases. Enzyme assays on a knock-out mutant revealed that PpHxk1 is the major glucose-phosphorylating enzyme in Physcomitrella, accounting for 80% of the total activity in protonemal tissue. The mutant is deficient in the response to glucose, which in wild type moss induces the formation of caulonemal filaments that protrude from the edge of the colony. Growth on glucose in the dark is strongly reduced in the mutant. Sequence data suggest that most plants including Physcomitrella and Arabidopsis have both chloroplast-imported hexokinases similar to PpHxk1 and traditional membrane-bound hexokinases. We propose that the two types of plant hexokinases have distinct physiological roles.     

Cryptochrome light signals control development to suppress auxin sensitivity in the moss Physcomitrella patens

The blue light receptors termed cryptochromes mediate photomorphological responses in seed plants. However, the mechanisms by which cryptochrome signals regulate plant development remain obscure. In this study, cryptochrome functions were analyzed using the moss Physcomitrella patens. This moss has recently become known as the only plant species in which gene replacement occurs at a high frequency by homologous recombination. Two cryptochrome genes were identified in Physcomitrella, and single and double disruptants of these genes were generated. Using these disruptants, it was revealed that cryptochrome signals regulate many steps in moss development, including induction of side branching on protonema and gametophore induction and development. In addition, the disruption of cryptochromes altered auxin responses, including the expression of auxin-inducible genes. Cryptochrome disruptants were more sensitive to external auxin than wild type in a blue light-specific manner, suggesting that cryptochrome light signals repress auxin signals to control plant development.     

Cytokinin induces the developmentally restricted synthesis of an extracellular protein in Physcomitrella patens

Early development of the moss Physcomitrella patens follows a simple course leading to the formation of a filamentous protonema containing only two cell-types, chloronema and caulonema. The addition of the hormone cytokinin leads to the induction of multicellular buds from such protonema. The spectrum of extracellular proteins (ECPs) synthesized by P. patens has been investigated at defined stages of development and under defined hormone treatments. It is found that in contrast to the limited changes in intracellular protein synthesis detectable, in the extracellular environment major and specific changes in the patterns of proteins synthesized occur. For example, the presence of caulonema cells is characterized by the synthesis of a 25 kDa ECP whereas early chloronema differentiation is distinguished by the presence of a 38 kDa ECP. The analysis of the pattern of ECPs synthesized by developmental mutants altered in bud formation, and in response to cytokinin in tunicamycin treated protonema (in which bud induction is blocked) indicate that the synthesis of a 14 kDa ECP is specifically induced by cytokinin. This protein represents a novel cytokinin-induced ECP. These data show that the differentiation of particular cell types in plants is associated with the synthesis of particular ECPs, and suggest that hormones which induce specific morphogenic events may do so via the synthesis of specific ECPs.     

Gravitropic responses of wild-type and mutant strains of the moss Physcomitrella patens

The gravitropic responses of dark-grown caulonemata and gametophores of wild-type and mutant strains of the moss Physcomitrella patens have been investigated. In the wild-type both caulonemata and gametophores show negative orthogravitropism. No gravitropic response is observed when plants are rotated slowly on a clinostat and the inductive effect of gravity can be replaced by centrifugal force. The gravitropic response of caulonemanta is biphasic, consisting of an initial phase producing a bend of about 20 degrees within 12 h of 90 degrees reorientation and a subsequent slower phase leading to completion of the 90 degrees curvature. No obvious sedimentation of statoliths accompanies this response. Several mutants have been isolated that are either partially or completely impaired in caulonemal gravitropism and one mutant shows a positive gravitropic response. Complementation analysis using somatic hybrids obtained following protoplast fusion indicates that at least three genes can mutate to give an altered gravitropic phenotype. None of these mutants is altered in gametophore gravitropism, suggesting that the gravitropic response of caulonemal filaments may require at least some gene products that are not required for the response of the multicellular gametophores. One class of mutant with impaired caulonemal gravitropism shows a pleiotropic alteration in leaf shape.     

Moss biology and phytohormones--cytokinins in Physcomitrella

Mosses present several advantages for the analysis of phytohormone physiology. Their enormous regeneration capacity, the possibility of controlling their whole life cycle under in vitro culture conditions, as well as the small number of cell types facilitate studies of hormone homeostasis. This review focuses on the metabolism and biosynthesis of cytokinins, mostly summarising data obtained using the moss Physcomitrella patens (Hedw.) B.S.G. which has served as a model system for cytokinin research for many years. A comparison of metabolic differences with respect to seed plants is presented, pointing out an important role of adenosine kinase for the formation of nucleotides during cytokinin interconversion in Physcomitrella. Results on cytokinin biosynthesis in Physcomitrella are summarised with respect to the OVE mutants, which can be considered unique in the plant kingdom due to their strong overproduction of cytokinins. The OVE phenotype is correlated with both increased activity in early stages of cytokinin biosynthesis as well as increased conversion of cytokinin riboside to the base. Cytokinin interconverting reactions can contribute to the increased levels of cytokinins in OVE mutants. Further studies on hormone physiology in moss will help to complete our understanding of hormonal homeostasis by elucidating the situation in an evolutionary early embryophyte.     

Physiological properties of Saccharomyces cerevisiae from which hexokinase II has been deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H(+)-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.     

Strigolactones regulate protonema branching and act as a quorum sensing-like signal in the moss Physcomitrella patens

Strigolactones are a novel class of plant hormones controlling shoot branching in seed plants. They also signal host root proximity during symbiotic and parasitic interactions. To gain a better understanding of the origin of strigolactone functions, we characterised a moss mutant strongly affected in strigolactone biosynthesis following deletion of the CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) gene. Here, we show that wild-type Physcomitrella patens produces and releases strigolactones into the medium where they control branching of protonemal filaments and colony extension. We further show that Ppccd8 mutant colonies fail to sense the proximity of neighbouring colonies, which in wild-type plants causes the arrest of colony extension. The mutant phenotype is rescued when grown in the proximity of wild-type colonies, by exogenous supply of synthetic strigolactones or by ectopic expression of seed plant CCD8. Thus, our data demonstrate for the first time that Bryophytes (P. patens) produce strigolactones that act as signalling factors controlling developmental and potentially ecophysiological processes. We propose that in P. patens, strigolactones are reminiscent of quorum-sensing molecules used by bacteria to communicate with one another.     

Both chloronemal and caulonemal cells expand by tip growth in the moss Physcomitrella patens

Tip growth is a mode of cell expansion in which all growth is restricted to a small area that forms a tip in an elongating cell. In green plants, tip growth has been shown to occur in root hairs, pollen tubes, rhizoids, and caulonema. Each of these cell types has a longitudinally elongated shape, longitudinally oriented microtubules and actin microfilaments, and a characteristic cytoplasmic organization at the growing tip which is required for growth. Chloronema are elongated cylindrical shaped cells that form during the development of the moss protonema. Since there are no published reports on the precise mode of chloronema elongation and conflicting interpretations of its cytology, the mechanism of cell growth has remained unclear. To determine if chloronema elongate by tip or diffuse growth, time-lapse light microscopy was employed to follow the movement of fluorescent microspheres attached to the surface of growing cells. It is shown here that chloronemal cells elongate by a form of tip growth. However, the slower growth of chloronema compared with caulonema is probably the result of differences in cytological organization of the growing tip.     

Blue-light irradiation up-regulates the ent-kaurene synthase gene and affects the avoidance response of protonemal growth in Physcomitrella patens

Main conclusion

We report a novel physiological response to blue light in the moss Physcomitrella patens . Blue light regulates ent -kaurene biosynthesis and avoidance response to protonemal growth.

Abstract

Gibberellins (GAs) are a group of diterpene-type plant hormones biosynthesized from ent-kaurenoic acid via ent-kaurene. While the moss Physcomitrella patens has part of the GA biosynthetic pathway, from geranylgeranyl diphosphate to ent-kaurenoic acid, no GA is found in this species. Caulonemal differentiation in a P. patens mutant with a disrupted bifunctional ent-copalyl diphosphate synthase/ent-kaurene synthase (PpCPS/KS) gene is suppressed under red light, and is recovered by application of ent-kaurene and ent-kaurenoic acid. This indicates that derivatives of ent-kaurenoic acid, not GAs, might act as endogenous developmental regulators. Here, we found unique responses in the protonemal growth of P. patens under unilateral blue light, and these regulators were involved in the responses. When protonemata of the wild type were incubated under blue light, the chloronemal filaments grew in the opposite direction to the light source. Although this avoidance was not observed in the ent-kaurene deficient mutant, chloronemal growth toward a blue-light source in the mutant was suppressed by application of ent-kaurenoic acid, and the growth was rescued to that in the wild type. Expression analysis of the PpCPS/KS gene showed that the mRNA level under blue light was rapidly increased and was five times higher than under red light. These results suggest that regulators derived from ent-kaurenoic acid are strongly involved not only in the growth regulation of caulonemal differentiation under red light, but also in the light avoidance response of chloronemal growth under blue light. In particular, growth under blue light is regulated via the PpCPS/KS gene.     

Enhancing the growth of Physcomitrella patens by combination of monochromatic red and blue light - a kinetic study

In the current work we demonstrate the relevance of monochromatic light conditions in moss plant cell culture. Light intensity and illumination wavelength are important cultivation parameters due to their impact on growth and chlorophyll formation kinetics of the moss Physcomitrella patens. This moss was chosen as a model organism due to its capability to produce complex recombinant pharmaceutical proteins. Filamentous moss cells were cultivated in mineral medium in shaking flasks. The flasks were illuminated by light emitting diodes (LED) providing nearly monochromatic red and blue light as well as white light as a reference. A maximum growth rate of 0.78 day((1) was achieved under additional CO(2) aeration and no growth inhibition was observed under high light illumination. The application of dual red and blue light is the most effective way to reach high growth and chlorophyll formation rates while minimizing energy consumption of the LEDs. These observations are discussed as effects of photo sensory pigments in the moss. The combination of monochromatic red and blue light should be considered when a large scale process is set up.     

Role of PSBS and LHCSR in Physcomitrella patens acclimation to high light and low temperature

Photosynthetic organisms respond to strong illumination by activating several photoprotection mechanisms. One of them, non-photochemical quenching (NPQ), consists in the thermal dissipation of energy absorbed in excess. In vascular plants NPQ relies on the activity of PSBS, whereas in the green algae Chlamydomonas reinhardtii it requires a different protein, LHCSR. The moss Physcomitrella patens is the only known organism in which both proteins are present and active in triggering NPQ, making this organism particularly interesting for the characterization of this protection mechanism. We analysed the acclimation of Physcomitrella to high light and low temperature, finding that these conditions induce an increase in NPQ correlated to overexpression of both PSBS and LHCSR. Mutants depleted of PSBS and/or LHCSR showed that modulation of their accumulation indeed determines NPQ amplitude. All mutants with impaired NPQ also showed enhanced photosensitivity when exposed to high light or low temperature, indicating that in this moss the fast-responding NPQ mechanism is also involved in long-term acclimation.     

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.     

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. The hxk2 mutant had a higher mitochondrial H⁺-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2 mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.