Andrea Leisse

Starch content and yield increase as a result of altering adenylate pools in transgenic plants

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.

High-density kinetic analysis of the metabolomic and transcriptomic response of Arabidopsis to eight environmental conditions

The time-resolved response of Arabidopsis thaliana towards changing light and/or temperature at the transcriptome and metabolome level is presented. Plants grown at 21°C with a light intensity of 150 μE m⁻² sec⁻¹ were either kept at this condition or transferred into seven different environments (4°C, darkness; 21°C, darkness; 32°C, darkness; 4°C, 85 μE m⁻² sec⁻¹; 21 °C, 75 μE m⁻² sec⁻¹; 21°C, 300 μE m⁻² sec⁻¹ ; 32°C, 150 μE m⁻² sec⁻¹). Samples were taken before (0 min) and at 22 time points after transfer resulting in (8×) 22 time points covering both a linear and a logarithmic time series totaling 177 states. Hierarchical cluster analysis shows that individual conditions (defined by temperature and light) diverge into distinct trajectories at condition-dependent times and that the metabolome follows different kinetics from the transcriptome. The metabolic responses are initially relatively faster when compared with the transcriptional responses. Gene Ontology over-representation analysis identifies a common response for all changed conditions at the transcriptome level during the early response phase (5-60 min). Metabolic networks reconstructed via metabolite-metabolite correlations reveal extensive environment-specific rewiring. Detailed analysis identifies conditional connections between amino acids and intermediates of the tricarboxylic acid cycle. Parallel analysis of transcriptional changes strongly support a model where in the absence of photosynthesis at normal/high temperatures protein degradation occurs rapidly and subsequent amino acid catabolism serves as the main cellular energy supply. These results thus demonstrate the engagement of the electron transfer flavoprotein system under short-term environmental perturbations.

A Bypass of Sucrose Synthase Leads to Low Internal Oxygen and Impaired Metabolic Performance in Growing Potato Tubers

Plants possess two alternative biochemical pathways for sucrose (Suc) degradation. One involves hydrolysis by invertase followed by phosphorylation via hexokinase and fructokinase, and the other route—which is unique to plants—involves a UDP-dependent cleavage of Suc that is catalyzed by Suc synthase (SuSy). In the present work, we tested directly whether a bypass of the endogenous SuSy route by ectopic overexpression of invertase or Suc phosphorylase affects internal oxygen levels in growing tubers and whether this is responsible for their decreased starch content. (a) Oxygen tensions were lower within transgenic tubers than in wild-type tubers. Oxygen tensions decreased within the first 10 mm of tuber tissue, and this gradient was steeper in transgenic tubers. (b) Invertase-overexpressing tubers had higher activities of glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and alcohol dehydrogenase, and (c) higher levels of lactate. (d) Expression of a low-oxygen-sensitive Adh1-β-glucuronidase reporter gene construct was more strongly induced in the invertase-overexpressing background compared with wild-type background. (e) Intact transgenic tubers had lower ATP to ADP ratios than the wild type. ATP to ADP ratio was restored to wild type, when discs of transgenic tubers were incubated at 21% (v/v) oxygen. (f) Starch decreased from the periphery to the center of the tuber. This decrease was much steeper in the transgenic lines, leading to lower starch content especially near the center of the tuber. (g) Metabolic fluxes (based on redistribution of 14C-glucose) and ATP to ADP ratios were analyzed in more detail, comparing discs incubated at various external oxygen tensions (0%, 1%, 4%, 8%, 12%, and 21% [v/v]) with intact tubers. Discs of Suc phosphorylase-expressing lines had similar ATP to ADP ratios and made starch as fast as wild type in high oxygen but had lower ATP to ADP ratios and lower rates of starch synthesis than wild type at low-oxygen tensions typical to those found inside an intact tuber. (h) In discs of wild-type tubers, subambient oxygen concentrations led to a selective increase in the mRNA levels of specific SuSy genes, whereas the mRNA levels of genes encoding vacuolar and apoplastic invertases decreased. (i) These results imply that repression of invertase and mobilization of Suc via the energetically less costly route provided by SuSy is important in growing tubers because it conserves oxygen and allows higher internal oxygen tensions to be maintained than would otherwise be possible.

Interaction with diurnal and circadian regulation results in dynamic metabolic and transcriptional changes during cold acclimation in Arabidopsis

In plants, there is a large overlap between cold and circadian regulated genes and in Arabidopsis, we have shown that cold (4°C) affects the expression of clock oscillator genes. However, a broader insight into the significance of diurnal and/or circadian regulation of cold responses, particularly for metabolic pathways, and their physiological relevance is lacking. Here, we performed an integrated analysis of transcripts and primary metabolites using microarrays and gas chromatography-mass spectrometry. As expected, expression of diurnally regulated genes was massively affected during cold acclimation. Our data indicate that disruption of clock function at the transcriptional level extends to metabolic regulation. About 80% of metabolites that showed diurnal cycles maintained these during cold treatment. In particular, maltose content showed a massive night-specific increase in the cold. However, under free-running conditions, maltose was the only metabolite that maintained any oscillations in the cold. Furthermore, although starch accumulates during cold acclimation we show it is still degraded at night, indicating significance beyond the previously demonstrated role of maltose and starch breakdown in the initial phase of cold acclimation. Levels of some conventional cold induced metabolites, such as γ-aminobutyric acid, galactinol, raffinose and putrescine, exhibited diurnal and circadian oscillations and transcripts encoding their biosynthetic enzymes often also cycled and preceded their cold-induction, in agreement with transcriptional regulation. However, the accumulation of other cold-responsive metabolites, for instance homoserine, methionine and maltose, did not have consistent transcriptional regulation, implying that metabolic reconfiguration involves complex transcriptional and post-transcriptional mechanisms. These data demonstrate the importance of understanding cold acclimation in the correct day-night context, and are further supported by our demonstration of impaired cold acclimation in a circadian mutant.

Systemic Analysis of Inducible Target of Rapamycin Mutants Reveal a General Metabolic Switch Controlling Growth in Arabidopsis thaliana

The target of rapamycin (TOR) pathway is a major regulator of growth in all eukaryotes, integrating energy, nutrient and stress signals into molecular decisions. By using large-scale MS-based metabolite profiling of primary, secondary and lipid compounds in combination with array-based transcript profiling, we show that the TOR protein not only regulates growth but also influences nutrient partitioning and central energy metabolism. The study was performed on plants exhibiting conditional down-regulation of AtTOR expression, revealing strong regulation of genes involved in pathways such as the cell cycle, cell-wall modifications and senescence, together with major changes in transcripts and metabolites of the primary and secondary metabolism. In agreement with these results, our morphological and metabolic analyses disclosed major metabolic changes leading to massive accumulations of storage lipids and starch. The implications of these data in the context of the general role of TOR in eukaryotic systems are discussed in parallel with the plant-specific aspects of TOR function. Finally, we propose a role for harnessing the plant TOR pathway by utilizing it as a potent metabolic switch, offering a possible route for biotechnological optimization of plant energy content and carbon partitioning for the production of bioenergy.

Inhibition of de Novo Pyrimidine Synthesis in Growing Potato Tubers Leads to a Compensatory Stimulation of the Pyrimidine Salvage Pathway and a Subsequent Increase in Biosynthetic Performance

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 [14C]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 energy-consuming 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.

Mild Reductions in Mitochondrial Citrate Synthase Activity Result in a Compromised Nitrate Assimilation and Reduced Leaf Pigmentation But Have No Effect on Photosynthetic Performance or Growth

Transgenic tomato (Solanum lycopersicum) plants, expressing a fragment of the mitochondrial citrate synthase gene in the antisense orientation and exhibiting mild reductions in the total cellular activity of this enzyme, displayed essentially no visible phenotypic alteration from the wild type. A more detailed physiological characterization, however, revealed that although these plants were characterized by relatively few changes in photosynthetic parameters they displayed a decreased relative flux through the tricarboxylic acid cycle and an increased rate of respiration. Furthermore, biochemical analyses revealed that the transformants exhibited considerably altered metabolism, being characterized by slight decreases in the levels of organic acids of the tricarboxylic acid cycle, photosynthetic pigments, and in a single line in protein content but increases in the levels of nitrate, several amino acids, and starch. We additionally determined the maximal catalytic activities of a wide range of enzymes of primary metabolism, performed targeted quantitative PCR analysis on all three isoforms of citrate synthase, and conducted a broader transcript profiling using the TOM1 microarray. Results from these studies confirmed that if the lines were somewhat impaired in nitrate assimilation, they were not severely affected by this, suggesting the presence of strategies by which metabolism is reprogrammed to compensate for this deficiency. The results are discussed in the context of carbon-nitrogen interaction and interorganellar coordination of metabolism.

Mild Reductions in Mitochondrial NAD-Dependent Isocitrate Dehydrogenase Activity Result in Altered Nitrate Assimilation and Pigmentation But Do Not Impact Growth

Transgenic tomato (Solanum lycopersicum) plants were generated expressing a fragment of the mitochondrial NAD-dependent isocitrate dehydrogenase gene (SlIDH1) in the antisense orientation. The transgenic plants displayed a mild reduction in the activity of the target enzyme in the leaves but essentially no visible alteration in growth from the wild-type. Fruit size and yield were, however, reduced. These plants were characterized by relatively few changes in photosynthetic parameters, but they displayed a minor decrease in maximum photosynthetic efficiency (Fv/Fm). Furthermore, a clear reduction in flux through the tricarboxylic acid (TCA) cycle was observed in the transformants. Additionally, biochemical analyses revealed that the transgenic lines exhibited considerably altered metabolism, being characterized by slight decreases in the levels of amino acids, intermediates of the TCA cycle, photosynthetic pigments, starch, and NAD(P)H levels, but increased levels of nitrate and protein. Results from these studies show that even small changes in mitochondrial NAD-dependent isocitrate dehydrogenase activity lead to noticeable alterations in nitrate assimilation and suggest the presence of different strategies by which metabolism is reprogrammed to compensate for this deficiency.

Global mRNA changes in microarray experiments.

To the editor: Microarray mRNA expression profiling is of wide utility in all areas of the life sciences1,2. Almost all normalization algorithms used to interpret microarray experiments calculate changes in transcript abundance based on the assumption that no changes occur in the total amount of mRNA. This assumption is, however, violated under many experimental conditions, often resulting in the false classification of thousands of genes as differentially expressed3. The potential for environmental and developmental effects on global mRNA have been demonstrated for heat shock and serum deprivation in mammalian cells and yeast stationary phase, respectively3. In addition, two papers4,5 published in a previous issue from the MicroArray Quality Control (MAQC) project highlighted the potential for tissue-specific changes, reporting global mRNA differences between human universal reference and brain RNA samples. Here, we present a simple method to generate external controls for Affymetrix (Santa Clara, CA, USA) GeneChip oligonucleotide microarrays to allow these platforms to measure absolute mRNA expression at the global level. The principal approach for correcting for global mRNA changes is the addition of defined amounts of external RNAs obtained by in vitro transcription to each sample. These are usually added to the total RNA, allowing them to be treated in parallel to the endogenous mRNAs during the labeling and hybridization processes6. The expression estimates of endogenous RNAs can then be normalized using those of the external RNAs. As mRNA accounts for a small proportion (~2%) of total RNA, total RNA is considered a more stable quantity for normalization; furthermore, total RNA quantification allows normalization on a per cell basis3. A clear prerequisite for any robust application of external controls is that they are of sufficient number, cover the array’s dynamic concentration range and are representative for the studied genome6. At present, these requirements are obviously not fulfilled in the platform most widely used for expression analysis—Affymetrix GeneChips—which currently offer just four external mRNA controls derived from Bacillus subtilis (see Supplementary Fig.1 online). Probes not detecting endogenous mRNAs in a particular RNA sample have been successfully used as spike-in controls for validation of different array platforms7,8. Even so, searching for genes absent in a single RNA sample and using cDNA clones (that is, expressed genes) to generate controls is not useful for developing external controls of general use across diverse sample types. The principle of the approach we present below relies on the observation that, when analyzing expression data across the thousands of publicly available samples, some probes never seem to record positive hybridization signals. To stringently identify such probe sets, we primarily used the Affymetrix MAS5 present/absent P-values from ~1,000 arrays, but also considered probe set signal (see Supplementary Methods online). When applied to the most recent Affymetrix expression GeneChips for rat, mouse, Arabidopsis and human, our selection identified a large number of candidate probes in all cases (Table 1). We then used BLAST analyses, both to ensure that the sequence spanning the probe set could be amplified from genomic DNA—eliminating the need for cDNA clones—and to select sequences with low potential for cross-hybridization. With additional criteria, between 313 and 3,458 external RNA controls were selected for each array (Table 1). To validate our approach, and to generate controls for our own experiments, we used 94 of the controls selected for Arabidopsis. Of these, after PCR amplification, in vitro transcription and quality control, 75 yielded high-quality synthetic RNAs to use as external controls. These controls were subsequently used to prepare a dilution series covering three orders of magnitude (see Table 1 in Supplementary Notes online), including the upper and lower detection limits of the ATH1 array (Fig. 1a and Supplementary Fig. 2 online). Initially, to simulate global mRNA differences, we performed a validation study in which we spiked the same RNA sample with different amounts of the external controls. This demonstrated that relative changes between the amount of mRNA and the external controls could be detected and normalized against (Fig. 1b and Supplementary Results and Discussion online). Importantly, and as observed by others7, the measured change in mRNA amount (that is, the control ratios) is dependent on intensity—with technical influences of detection and saturation at the extremes (Supplementary Results and Discussion). Without many representative controls at multiple concentrations, the estimated shift in global mRNA will be less accurate and the dynamic limits are unknown, thus normalization of global mRNA changes will result in wrongly called differential expression. Previous uses of external controls either neglect this9,10 or require custom arrays that are not widely available3. Our approach accurately detects the global shift and helps reveal dynamic limits and thus also substantially improves inference. As a biological test case for our method, we performed a cold acclimation experiment, investigating the response of Arabidopsis to low temperature. We and others have previously shown this response involves massive expression changes11; however, whether or not cold acclimation

Heterologous expression of a ketohexokinase in potato plants leads to inhibited rates of photosynthesis, severe growth retardation and abnormal leaf development

In the present paper we investigated the effect of heterologous expression of a rat liver ketohexokinase in potato (Solanum tuberosum L.) plants with the aim of investigating the role of fructose 1-phosphate in plant metabolism. Plants were generated that contained appreciable activity of ketohexokinase but did not accumulate fructose 1-phosphate. They were, however, characterised by a severe growth retardation and abnormal leaf development. Studies of 14CO2 assimilation and metabolism, and of the levels of photosynthetic pigments, revealed that these lines exhibited restricted photosynthesis. Despite this fact, the levels of starch and soluble sugars remained relatively constant. Analysis of intermediates of starch and sucrose biosynthesis revealed large increases in the triose phosphate and fructose 1,6-bisphosphate pools but relatively unaltered levels of inorganic phosphate and 3-phosphoglycerate, and these lines were also characterised by an accumulation of glyceraldehyde. The transformants neither displayed consistent changes in the activities of Calvin cycle enzymes nor in enzymes of sucrose synthesis but displayed a metabolic profile partially reminiscent of that brought about by end-product limitation, but most likely caused by an inhibition of photosynthesis brought about by the accumulation of glyceraldehyde. Analysis of the metabolite contents in lamina and vein fractions of the leaf, and of the enzymes of carbohydrate oxidation indicate that the phloem-enriched veins of ketohexokinase-expressing leaves tend toward hypoxia and indicate a problem of phloem transport.