Two novel types of hexokinases in the moss Physcomitrella patens

Background  

Hexokinase catalyzes the phosphorylation of glucose and fructose, but it is also involved in sugar sensing in both fungi and plants. We have previously described two types of hexokinases in the moss Physcomitrella. Type A, exemplified by PpHxk1, the major hexokinase in Physcomitrella, is a soluble protein that localizes to the chloroplast stroma. Type B, exemplified by PpHxk2, has an N-terminal membrane anchor. Both types are found also in vascular plants, and localize to the chloroplast stroma and mitochondrial membranes, respectively.     

Effect of the energy supply on filamentous growth and development in Physcomitrella patens

The filamentous gametophyte of the moss Physcomitrella patens consists of two filament types called chloronemata and caulonemata. Chloronemal cells are photosynthetically active with numerous chloroplasts, while caulonemata help to spread the colony by radial growth. The balance between the two filament types is affected by external factors such as light and plant hormones. In the present study, caulonema formation and chloronemal branching have been monitored during high and low light conditions and in the presence of glucose, auxin, or cytokinin. These experiments were performed both in a wild-type strain and in a hxk1 knockout mutant which lacks the major hexokinase of Physcomitrella. It was found that caulonema formation is induced by high energy conditions such as high light and external glucose, while chloronemal branching is stimulated by low energy conditions such as reduced light, and in the hxk1 mutant. The hxk1 mutation also causes buds to appear on chloronemal filaments, which is rarely seen in the wild type, and shows increased sensitivity to cytokinin and abscisic acid. Based on these findings a model is proposed in which the energy supply of the moss colony regulates the balance between chloronemal and caulonemal growth.     

Subcellular distribution and kinetic properties of cytosolic and non-cytosolic hexokinases in maize seedling roots: implications for hexose phosphorylation.

Hexose phosphorylation by hexokinases plays an important role in glycolysis, biosynthesis and control of sugar-modulated genes. Several cytosolic hexokinase and fructokinase isoforms have been characterized and organelle-bound hexokinases have also been detected in higher plants. In this study a hexokinase activity is described that is inhibited by ADP (K(i)=30 microM) and mannoheptulose (K(i) congruent with 300 microM) in non-cytosolic fractions (mitochondria, Golgi apparatus and microsomes) obtained from preparations of seedling roots of maize (Zea mays L.). The catalytic efficiency (Vmax/Km) for both ATP and glucose in all non-cytosolic hexokinase fractions is more than one order of magnitude higher than that of cytosolic hexokinase and fructokinases. Low (30%) or no ADP and mannoheptulose inhibition is observed with hexokinase and fructokinase activities derived from the cytosolic compartment obtained after ion exchange and affinity chromatography. The soluble fructokinase (FK) shows fructose cooperativity (Hill n>2). The Vmax/Km ratio is about 3-fold higher for ATP than for other NTPs and no difference for hexose phosphorylation efficiencies is found between cytosolic hexokinase and fructokinase isoforms (FK1, FK2) with ATP as substrate. The K(i) for fructose inhibition is 2 mM for FK1 and 25 mM for FK2. The data indicate that low energy-charge and glucose analogues preferentially inhibit the membrane-bound hexokinases possibly involved in sugar-sensing, but not the cytosolic hexokinases and fructokinases.     

Snf1-related protein kinase 1 is needed for growth in a normal day-night light cycle

The yeast Snf1 protein kinase and its animal homologue, the AMP-activated protein kinase, play important roles in metabolic regulation, by serving as energy gauges that turn off energy-consuming processes and mobilize energy reserves during low-energy conditions. The closest homologue of these kinases in plants is Snf1-related protein kinase 1 (SnRK1). We have cloned two SnRK1-encoding genes, PpSNF1a and PpSNF1b, in the moss Physcomitrella patens, where gene function can be studied directly by gene targeting in the haploid gametophyte. A snf1a snf1b double knockout mutant is viable, but lacks all Snf1-like protein kinase activity. The mutant has a complex phenotype that includes developmental abnormalities, premature senescence and altered sensitivities to plant hormones. Remarkably, the double knockout mutant also requires continuous light, and is unable to grow in a normal day-night light cycle. This suggests that SnRK1 is needed for metabolic changes that help the plant cope with the dark hours of the night.     

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.     

Phytochrome evolution: Phytochrome genes in ferns and mosses

We have isolated phytochrome genes from the moss Physcomitrella , the fern Psilotum and PCR-generated phytochrome sequences from a few other ferns. The phytochrome gene of the moss Physcomitrella turned out not to contain the aberrant C-terminal third of the phytochrome from the moss Ceratodon , but the transmitter module-like sequences found in other phytochromes. A series of different phytochrome genes was detected in Psilotum . Differences between the amino acid sequences derived from them ranged from about 5 to more than 22%. Some of these genes are likely pseudogenes. Analysis by phylogenetic tree constructions revealed that higher and lower plant phytochromes evolved with different velocities. Lower plant phytochromes form a separate family characterized by a high degree of similarity. The amino acid differences between phytochrome types detected in a single species of higher plants are about two-fold higher than the differences between phytochromes of species of lower plants belonging to different divisions ( Physcomitrella and Selaginella ). Future studies on phytochrome sequences may eventually also throw light on the significance of Psilotum in the evolution of vascular plants.     

植物分子生物学研究极具前景的模式系统--小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA发生同源重组,这些特点使它成为研究基因功能的良好材料.一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实.小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础.关于小立碗藓的ESTs数据库已经建立,已有67 000条ESTs信息.     

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.     

Glucose repression in Saccharomyces cerevisiae is directly associated with hexose phosphorylation by hexokinases PI and PII

Genetic and biochemical analyses showed that hexokinase PII is mainly responsible for glucose repression in Saccharomyces cerevisiae, indicating a regulatory domain mediating glucose repression. Hexokinase PI/PII hybrids were constructed to identify the supposed regulatory domain and the repression behavior was observed in the respective transformants. The hybrid constructs allowed the identification of a domain (amino acid residues 102-246) associated with the fructose/glucose phosphorylation ratio. This ratio is characteristic of each isoenzyme, therefore this domain probably corresponds to the catalytic domain of hexokinases PI and PII. Glucose repression was associated with the C-terminal part of hexokinase PII, but only these constructs had high catalytic activity whereas opposite constructs were less active. Reduction of hexokinase PII activity by promoter deletion was inversely followed by a decrease in the glucose repression of invertase and maltase. These results did not support the hypothesis that a specific regulatory domain of hexokinase PII exists which is independent of the hexokinase PII catalytic domain. Gene disruptions of hexokinases further decreased repression when hexokinase PI was removed in addition to hexokinase PII. This proved that hexokinase PI also has some function in glucose repression. Stable hexokinase PI overproducers were nearly as effective for glucose repression as hexokinase PII. This showed that hexokinase PI is also capable of mediating glucose repression. All these results demonstrated that catalytically active hexokinases are indispensable for glucose repression. To rule out any further glycolytic reactions necessary for glucose repression, phosphoglucoisomerase activity was gradually reduced. Cells with residual phosphoglucoisomerase activities of less than 10% showed reduced growth on glucose. Even 1% residual activity was sufficient for normal glucose repression, which proved that additional glycolytic reactions are not necessary for glucose repression. To verify the role of hexokinases in glucose repression, the third glucose-phosphorylating enzyme, glucokinase, was stably overexpressed in a hexokinase PI/PII double-null mutant. No strong effect on glucose repression was observed, even in strains with 2.6 U/mg glucose-phosphorylating activity, which is threefold increased compared to wild-type cells. This result indicated that glucose repression is only associated with the activity of hexokinases PI and PII and not with that of glucokinase.     

MRE11 and RAD50, but not NBS1, are essential for gene targeting in the moss Physcomitrella patens

The moss Physcomitrella patens is unique among plant models for the high frequency with which targeted transgene insertion occurs via homologous recombination. Transgene integration is believed to utilize existing machinery for the detection and repair of DNA double-strand breaks (DSBs). We undertook targeted knockout of the Physcomitrella genes encoding components of the principal sensor of DNA DSBs, the MRN complex. Loss of function of PpMRE11 or PpRAD50 strongly and specifically inhibited gene targeting, whilst rates of untargeted transgene integration were relatively unaffected. In contrast, disruption of the PpNBS1 gene retained the wild-type capacity to integrate transforming DNA efficiently at homologous loci. Analysis of the kinetics of DNA-DSB repair in wild-type and mutant plants by single-nucleus agarose gel electrophoresis revealed that bleomycin-induced fragmentation of genomic DNA was repaired at approximately equal rates in each genotype, although both the Ppmre11 and Pprad50 mutants exhibited severely restricted growth and development and enhanced sensitivity to UV-B and bleomycin-induced DNA damage, compared with wild-type and Ppnbs1 plants. This implies that while extensive DNA repair can occur in the absence of a functional MRN complex; this is unsupervised in nature and results in the accumulation of deleterious mutations incompatible with normal growth and development.     

植物分子生物学研究极具前景的模式系统——小立碗藓

小立碗藓作为植物分子生物学研究极具前景的模式系统已日益受到人们的重视,它的生活史周期短,易于培养,转基因植株易于分析,核基因组容易和外源DNA 发生同源重组,这些特点使它成为研究基因功能的良好材料。一些成功的基因敲除和基因破坏已经在小立碗藓中实现,这些基因的功能也通过小立碗藓转化植株的特点得以证实。小立碗藓标签突变文库已经建立,其应用为小立碗藓基因的进一步研究打下了基础。关于小立碗藓的ESTs 数据库已经建立,已有67 000 条ESTs 信息。     

Efficient gene targeting in the moss Physcomitrella patens

The moss Physcomitrella patens is used as a genetic model system to study plant development, taking advantage of the fact that the haploid gametophyte dominates in its life cycle. Transformation experiments designed to target three single-copy genomic loci were performed to determine the efficiency of gene targeting in this plant. Mean transformation rates were 10-fold higher with the targeting vectors and molecular evidence for the integration of exogenous DNA into each targeted locus by homologous recombination is provided. The efficiency of gene targeting determined in these experiments is above 90%, which is in the range of that observed in yeast and several orders of magnitude higher than previous reports of gene targeting in plants. Thus, gene knock-out and allele replacement approaches are directly accessible to study plant development in the moss Physcomitrella patens . Moreover, efficient gene targeting has so far only been observed in lower eukaryotes such as protozoa, yeasts and filamentous fungi, and, as shown here the first example from the plant kingdom is a haplobiontic moss. This suggests a possible correlation between efficient gene targeting and haplo-phase in eukaryotes.     

Cyclin D-knockout uncouples developmental progression from sugar availability

Multicellular organisms need to modulate proliferation and differentiation in response to external conditions. An important role in these processes plays the mitogen-stimulated induction of cyclin D (cycD) gene expression. D-type cyclins have been identified as the crucial intracellular sensors for cell-cycle regulation in all eukaryotes. However, cycD deletions have been found to cause specific phenotypic alterations in animals but not yet in plants. An insertional mutation of a so far uncharacterized Arabidopsis cycD gene did not alter the plant phenotype. To gain new insights into CycD function of land plants, we generated targeted cycD gene knockouts in the moss Physcomitrella patens and observed a surprisingly limited disruption phenotype. While wild-type plants reacted to exogenous glucose sources with prolonged growth of juvenile stages and retarded differentiation, cycD knockouts exhibited developmental progression independent of sugar supply. On the other hand, growth rate, cell sizes or plant size were not affected. Thus, we conclude that Physcomitrella CycD might not be essential for cell-cycle regulation but is important for coupling the developmental progression to nutrient availability.     

Involvement of mitochondrial-targeted RecA in the repair of mitochondrial DNA in the moss, Physcomitrella patens

Homologous recombination is a universal process that contributes to genetic diversity and genomic integrity. Bacterial-type RecA generally exists in all bacteria and plays a crucial role in homologous recombination. Although RecA homologues also exist in plant mitochondria, there have been few reports about the in vivo functions of these homologues. We identified a recA gene orthologue (named PprecA1) in a cDNA library of the moss, Physcomitrella patens. N-terminal fusion of the putative organellar targeting sequence of PpRecA1 to GFP caused a targeting of PpRecA1 to mitochondria. PprecA1 partially complemented the effects of a DNA damaging agent in an Escherichia coli recA deficient strain. Additionally, the expression of PprecA1 was induced by treating the plants with DNA damaging agents. Disruption of PprecA1 by targeted replacement resulted lower rate of the recovery of the mitochondrial DNA from methyl methan sulfonate damage. This is the first report about the characteristics of a null mutant of bacterial-type recA gene in plant. The data suggest that PprecA1 participates in the repair of mitochondrial DNA in P. patens.     

Comparison of gene targeting efficiencies in two mosses suggests that it is a conserved feature of Bryophyte transformation

The moss, Physcomitrella patens, is a novel tool in plant functional genomics due to its exceptionally high gene targeting efficiency that is so far unique for plants. To determine if this high gene targeting efficiency is exclusive to P. patens or if it is a common feature to mosses, we estimated gene-targeting efficiency in another moss, Ceratodon purpureus. We transformed both mosses with replacement vectors corresponding to the adenine phosphoribosyl transferase (APT) reporter gene. We achieved a gene targeting efficiency of 20.8% for P. patens and 1.05% for C. purpureus. Our findings support the hypothesis that efficient gene targeting could be a general mechanism of Bryophyte transformation.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.