To determine the function of cytosolic phosphorylase (Pho2; EC 2.4.1.1), transgenic potato plants were created in which the expression of the enzyme was inhibited by introducing a chimeric gene containing part of the coding region for cytosolic phosphorylase linked in antisense orientation to the 35S CaMV promotor. As revealed by Northern blot analysis and native polyacrylamide gel electrophoresis, the expression of cytosolic phosphorylase was strongly inhibited in both leaves and tubers of the transgenic plants. The transgenic plants propagated from stem cuttings were morphologically indiscernible from the wild-type. However, sprouting of the transgenic potato tubers was significantly altered: compared with the wild-type, transgenic tubers produced 2.4 to 8.1 times more sprouts. When cultivated in the greenhouse, transgenic seed tubers produced two to three times more shoots than the wild-type. Inflorescences appeared earlier in the resulting plants. Many of the transgenic plants flowered two or three times successively. Transgenic plants derived from seed tubers formed 1.6 to 2.4 times as many tubers per plant as untransformed controls. The size and dry matter content of the individual tubers was not noticeably altered. Tuber yield was significantly higher in the transgenic plants. As revealed by carbohydrate determination of freshly harvested and stored tubers, starch and sucrose pools were not noticeably affected by the antisense inhibition of cytosolic phosphorylase; however, glucose and fructose levels were markedly reduced after prolonged storage. These results favour the view that cytosolic phosphorylase does not participate in starch degradation. The possible links between the reduced levels of cytosolic phosphorylase and the observed changes with respect to sprouting and flowering are discussed.

BackgroundThe moss Physcomitrella patens is an attractive model system for plant biology and functional genome analysis. It shares many biological features with higher plants but has the unique advantage of an efficient homologous recombination system for its nuclear DNA. This allows precise genetic manipulations and targeted knockouts to study gene function, an approach that due to the very low frequency of targeted recombination events is not routinely possible in any higher plant.ResultsAs an important prerequisite for a large-scale gene/function correlation study in this plant, we are establishing a collection of Physcomitrella patens transformants with insertion mutations in most expressed genes. A low-redundancy moss cDNA library was mutagenised in E. coli using a derivative of the transposon Tn1000. The resulting gene-disruption library was then used to transform Physcomitrella. Homologous recombination of the mutagenised cDNA with genomic coding sequences is expected to target insertion events preferentially to expressed genes. An immediate phenotypic analysis of transformants is made possible by the predominance of the haploid gametophytic state in the life cycle of the moss. Among the first 16,203 transformants analysed so far, we observed 2636 plants ( = 16.2%) that differed from the wild-type in a variety of developmental, morphological and physiological characteristics.ConclusionsThe high proportion of phenotypic deviations and the wide range of abnormalities observed among the transformants suggests that mutagenesis by gene-disruption library transformation is a useful strategy to establish a highly diverse population of Physcomitrella patens mutants for functional genome analysis.

A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes. Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low anity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in¯owers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds. This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds. Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation. The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates. Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined. A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species. In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates. These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.

Based on the relative ease of performing targeted nuclear gene knockout, the moss Physcomitrella patens has recently been developed as a model system for plant functional genomics. To address the need for new promoters that could drive expression of transgenes in this moss, we tested two fragments of the promoter region of the gene for the sugar beet (Beta vulgaris) V-type H + -ATPase subunit isoform c. Four gene knockout constructs were tested in which the neomycin phosphotransferase II selection marker gene was put under the control of two distinct V-type H + -ATPase promoter fragments, the NOS promoter, or the CaMV 35S promoter. In each case the selection cassettes were flanked by moss FtsZ1 cDNA sequences to facilitate chromosomal targeting. From a total of more than 440 transformed plants, the number of plants generated per construct was monitored and found to be in the range of 5 to 11 stable transgenics per transformation. Both V-type H + -ATPase promoter fragments lead to NPTII expression levels that were sufficiently high to generate large numbers of stable transgenic plants. The numbers of plants obtained with the two V-type H + -ATPase promoter fragments were comparable to those with constructs containing the standard NOS and 35S promoters. We propose that the higher plant V-type H + -ATPase promoter can be used for the expression of transgenes in the bryophyte P. patens.

A full-length cDNA encoding plastidic phosphorylase (Pho1, EC 2.4.1.1) from spinach (Spinacia oleracea L.) has been isolated. Analysis of the deduced protein sequence revealed considerable homologies with the corresponding proteins from other plants, animals and prokaryotes. Escherichia coli cells carrying the entire cDNA for Pho1 expressed an active phosphorylase, which resembled the properties of the plastidic isozyme of spinach with respect to its low affinity to glycogen. Expression of Pho1 was studied in spinach at the level of both mRNA and enzyme activity. Plastidic phosphorylase was transcribed in flowers and leaves, but the highest Pho1 transcript levels were found in mature fruits/seeds. This is in agreement with the enzyme activity levels, as Pho1 activity was detected in all tissues tested, but the highest activity was also present in mature fruits/seeds. Since developing seeds are strong sink organs, which import sucrose and accumulate starch, this observation may indicate that plastidic phosphorylase plays a role in starch formation. The assumption has been tested further by a series of induction experiments in which leaf discs from spinach and potato plants were incubated with various carbohydrates. Following incubation, phosphorylase steady-state transcript levels as well as levels of neutral sugars and starch were determined. A similar induction behaviour was found for Pho1 from spinach and Pho1a from potato, indicating the presence of related sugar signal transduction pathways in these two species. In addition, the expression of Pho1a and Agp4 (the large submit of ADPglucose synthase) from potato seems to be partly coordinately regulated by carbohydrates. These data may suggest that the regulation of Pho1 expression is linked to the carbohydrate status of the respective tissue.