Starch represents the most important carbohydrate used for food and feed purposes. With the aim of increasing starch content, we decided to modulate the adenylate pool by changing the activity of the plastidial adenylate kinase in transgenic potato plants. As a result, we observed a substantial increase in the level of adenylates and, most importantly, an increase in the level of starch to 60% above that found in wild-type plants. In addition, concentrations of several amino acids were increased by a factor of 2-4. These results are particularly striking because this genetic manipulation also results in an increased tuber yield. The modulation of the plastidial adenylate kinase activity in transgenic plants therefore represents a potentially very useful strategy for increasing formation of major storage compounds in heterotrophic tissues of higher plants.
Pyrimidine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. In this study, we investigate the influence of a decreased expression of UMP synthase (UMPS), a key enzyme in the pathway of de novo pyrimidine synthesis, on biosynthetic processes in growing potato (Solanum tuberosum) tubers. Transgenic plants were generated expressing UMPS in the antisense orientation under the control of the tuber-specific patatin promoter. Lines were selected with markedly decreased expression of UMPS in the tubers. Decreased expression of UMPS restricted the use of externally supplied orotate for de novo pyrimidine synthesis in tuber tissue, whereas the uridine-salvaging pathway was stimulated. This shift in the pathways of UMP synthesis was accompanied by increased levels of tuber uridine nucleotides, increased fluxes of [ 14 C]sucrose to starch and cell wall synthesis, and increased amounts of starch and cell wall components in the tubers, whereas there were no changes in uridine nucleotide levels in leaves. Decreased expression of UMPS in tubers led to an increase in transcript levels of carbamoylphosphate synthase, uridine kinase, and uracil phosphoribosyltransferase, the latter two encoding enzymes in the pyrimidine salvage pathways. Thus, the results show that antisense inhibition of the de novo pathway of pyrimidine synthesis leads to a compensatory stimulation of the less energyconsuming salvage pathways, probably via increased expression and activity of uridine kinase and uracil phosphoribosyltransferase. This results in increased uridine nucleotide pool levels in tubers and improved biosynthetic performance.
The dynamics of a proteome can only be addressed with large-scale, high-throughput methods. To cope with the inherent complexity, techniques based on targeted quantification using proteotypic peptides are arising. This is an essential systems biology approach; however, for the exploratory discovery of unexpected markers, nontargeted detection of proteins, and protein modifications is indispensable. We present a rapid label-free shotgun proteomics approach that extracts relevant phenotype-specific peptide product ion spectra in an automated workflow without prior identification. These product ion spectra are subsequently sequenced with database search and de novo prediction algorithms. We analyzed six potato tuber cultivars grown on three plots of two geographically separated fields in Germany. For data mining about 1.5 million spectra from 107 analyses were aligned and statistically examined in approximately 1 day. Several cultivar-specific protein markers were detected. Based on de novo-sequencing a dominant protein polymorphism not detectable in the available EST-databases was assigned exclusively to a specific potato cultivar. The approach is applicable to organisms with unsequenced or incomplete genomes and to the automated extraction of relevant mass spectra that potentially cannot be identified by genome/EST-based search algorithms.
Three cDNAs encoding purple acid phosphatase (PAP) were cloned from potato (Solanum tuberosum L. cv. Désirée) and expression of the corresponding genes was characterised. StPAP1 encodes a low-molecular weight PAP clustering with mammalian, cyanobacterial, and other plant PAPs. It was highly expressed in stem and root and its expression did not change in response to phosphorus (P) deprivation. StPAP2 and StPAP3 code for high-molecular weight PAPs typical for plants. Corresponding gene expression was shown to be responsive to the level of P supply, with transcripts of StPAP2 and StPAP3 being most abundant in P-deprived roots or both stem and roots, respectively. Root colonisation by arbuscular mycorrhizal fungi had no effect on the expression of any of the three PAP genes. StPAP1 mRNA is easily detectable along the root axis, including root hairs, but is barely detectable in root tips. In contrast, both StPAP2 and StPAP3 transcripts are abundant along the root axis, but absent in root hairs, and are most abundant in the root tip. All three PAPs described contain a predicted N-terminal secretion signal and could play a role in extracellular P scavenging, P mobilisation from the rhizosphere, or cell wall regeneration.
Systems Biology is an approach to biology and medicine that has the potential to lead to a better understanding of how biological properties emerge from the interaction of genes, proteins, molecules, cells and organisms. The approach aims at elucidating how these interactions govern biological function by employing experimental data, mathematical models and computational simulations. As Systems Biology is inherently multidisciplinary, education within this field meets numerous hurdles including departmental barriers, availability of all required expertise locally, appropriate teaching material and example curricula. As university education at the Bachelor’s level is traditionally built upon disciplinary degrees, we believe that the most effective way to implement education in Systems Biology would be at the Master’s level, as it offers a more flexible framework. Our team of experts and active performers of Systems Biology education suggest here (i) a definition of the skills that students should acquire within a Master’s programme in Systems Biology, (ii) a possible basic educational curriculum with flexibility to adjust to different application areas and local research strengths, (iii) a description of possible career paths for students who undergo such an education, (iv) conditions that should improve the recruitment of students to such programmes and (v) mechanisms for collaboration and excellence spreading among education professionals. With the growing interest of industry in applying Systems Biology approaches in their fields, a concerted action between academia and industry is needed to build this expertise. Here we present a reflection of the European situation and expertise, where most of the challenges we discuss are universal, anticipating that our suggestions will be useful internationally. We believe that one of the overriding goals of any Systems Biology education should be a student’s ability to phrase and communicate research questions in such a manner that they can be solved by the integration of experiments and modelling, as well as to communicate and collaborate productively across different experimental and theoretical disciplines in research and development.
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