Novel Approach for High-Throughput Metabolic Screening of Whole Plants by Stable Isotopes.

Here, we demonstrate whole-plant metabolic profiling by stable isotope labeling and combustion isotope-ratio mass spectrometry for precise quantification of assimilation, translocation, and molecular reallocation of (13)CO2 and (15)NH4NO3 The technology was applied to rice (Oryza sativa) plants at different growth stages. For adult plants, (13)CO2 labeling revealed enhanced carbon assimilation of the flag leaf from flowering to late grain-filling stage, linked to efficient translocation into the panicle. Simultaneous (13)CO2 and (15)NH4NO3 labeling with hydroponically grown seedlings was used to quantify the relative distribution of carbon and nitrogen. Two hours after labeling, assimilated carbon was mainly retained in the shoot (69%), whereas 7% entered the root and 24% was respired. Nitrogen, taken up via the root, was largely translocated into the shoot (85%). Salt-stressed seedlings showed decreased uptake and translocation of nitrogen (69%), whereas carbon metabolism was unaffected. Coupled to a gas chromatograph, labeling analysis provided enrichment of proteinogenic amino acids. This revealed significant protein synthesis in the panicle of adult plants, whereas protein biosynthesis in adult leaves was 8-fold lower than that in seedling shoots. Generally, amino acid enrichment was similar among biosynthetic families and allowed us to infer labeling dynamics of their precursors. On this basis, early and strong (13)C enrichment of Embden-Meyerhof-Parnas pathway and pentose phosphate pathway intermediates indicated high activity of these routes. Applied to mode-of-action analysis of herbicides, the approach showed severe disturbance in the synthesis of branched-chain amino acids upon treatment with imazapyr. The established technology displays a breakthrough for quantitative high-throughput plant metabolic phenotyping.

Large-scale phenotyping of transgenic tobacco plants (Nicotiana tabacum) to identify essential leaf functions.

Two of the major challenges in functional genomics are to identify genes that play a key role in biological processes, and to elucidate the biological role of the large numbers of genes whose function is poorly characterized or still completely unknown. In this study, a combination of large-scale expressed sequence tag sequencing, high-throughput gene silencing and visual phenotyping was used to identify genes in which partial inhibition of expression leads to marked phenotypic changes, mostly on leaves. Three normalized tobacco (Nicotiana tabacum) cDNA libraries were prepared directly in a binary vector using different tissues of tobacco as an RNA source, randomly sequenced and clustered. The Agrobacterium-tobacco leaf disc transformation system was used to generate sets of antisense or co-suppression transgenic tobacco plants for over 20 000 randomly chosen clones, each representing an independent cluster. After transfer to the glasshouse, transgenic plants were scored visually after 10-14 days for changes in growth, leaf form and chlorosis or necrosis. Putative hits were validated by repeating the transformation. This procedure is more stringent than the analysis of knockout mutants, because it requires that even a partial decrease in expression generates a phenotype. This procedure identified 88 validated gene/phenotype relations. These included several previously characterized gene/phenotype relationships, demonstrating the validity of the approach. For about one-third, a function could be inferred, but a loss-of-function phenotype had not been described previously. Strikingly, almost one-half of the validated genes were poorly annotated, or had no known function. For 77 of these tobacco sequences, a single or small number of potential orthologues were identified in Arabidopsis. The genes for which orthologues were identified in Arabidopsis included about one-half of the genes whose function was completely unknown. Comparison with published gene/phenotype relations for Arabidopsis knockout mutants revealed surprisingly little overlap with the present study. Our results indicate that partial gene silencing identifies novel gene/phenotype relationships, which are distinct from those uncovered by knockout screens. They also show that it is possible to perform these analyses in a crop species in which full genome sequence information is lacking, and subsequently to transfer the information to a reference species in which functional studies can be performed more effectively.

On the mechanism of action and selectivity of the corn herbicide topramezone: a new inhibitor of 4-hydroxyphenylpyruvate dioxygenase.

Topramezone is a new, highly selective herbicide of pyrazole structure for the post-emergence control of broadleaf and grass weeds in corn. The biokinetic properties and mode of action of topramezone were investigated in plants of Setaria faberi Herrm, Sorghum bicolor (L.) Moench, Solanum nigrum L. and the crop species corn (Zea mays L.). Within 2-5 days after treatment, topramezone caused strong photobleaching effects on the shoot, followed by plant death of sensitive weeds. The selectivity of topramezone between corn and the weed species has been quantified as above 1000-fold. By virtue of the plant symptoms and the reversal of the effects in Lemna paucicostata L. by adding homogentisate, it was hypothesized that topramezone blocks the formation of homogentisate, possibly through inhibition of 4-hydroxyphenylpyruvate dioxygenase (4-HPPD). Indeed, topramezone strongly inhibited 4-HPPD activity in vitro, with I(50) values of 15 and 23 nM for the enzyme isolated from S. faberi and recombinant enzyme of Arabidopsis thaliana L. respectively. The enzyme activity from corn was approximately 10 times less sensitive. After root and foliar application of [(14)C]topramezone, equivalent to field rates of 75 g ha(-1), the herbicide was rapidly absorbed and systemically translocated in the plant. Only marginal differences between leaf uptake and translocation of topramezone by the weeds and corn were found. Metabolism of foliar-applied [(14)C]topramezone was far more rapid in corn than in the weeds. A more rapid metabolism combined with a lower sensitivity of the 4-HPPD target enzyme contributes to the tolerance of corn to topramezone.

Target-based discovery of novel herbicides.

In the past 10 years, strategies for the first steps of herbicide discovery have switched from the testing of chemicals for efficacy on whole plants towards the use of in-vitro assays against molecular targets. Many different approaches have been developed to identify bona fide targets for in-vitro screening. Developments in functional genomics and in pharmaceutical research could aid the development of assay systems for the evaluation of chemicals for their suitability as lead structures in herbicide discovery.

In silico metabolic network analysis of Arabidopsis leaves.

BACKGROUND: During the last decades, we face an increasing interest in superior plants to supply growing demands for human and animal nutrition and for the developing bio-based economy. Presently, our limited understanding of their metabolism and its regulation hampers the targeted development of desired plant phenotypes. In this regard, systems biology, in particular the integration of metabolic and regulatory networks, is promising to broaden our knowledge and to further explore the biotechnological potential of plants. RESULTS: The thale cress Arabidopsis thaliana provides an ideal model to understand plant primary metabolism. To obtain insight into its functional properties, we constructed a large-scale metabolic network of the leaf of A. thaliana. It represented 511 reactions with spatial separation into compartments. Systematic analysis of this network, utilizing elementary flux modes, investigates metabolic capabilities of the plant and predicts relevant properties on the systems level: optimum pathway use for maximum growth and flux re-arrangement in response to environmental perturbation. Our computational model indicates that the A. thaliana leaf operates near its theoretical optimum flux state in the light, however, only in a narrow range of photon usage. The simulations further demonstrate that the natural day-night shift requires substantial re-arrangement of pathway flux between compartments: 89 reactions, involving redox and energy metabolism, substantially change the extent of flux, whereas 19 reactions even invert flux direction. The optimum set of anabolic pathways differs between day and night and is partly shifted between compartments. The integration with experimental transcriptome data pinpoints selected transcriptional changes that mediate the diurnal adaptation of the plant and superimpose the flux response. CONCLUSIONS: The successful application of predictive modelling in Arabidopsis thaliana can bring systems-biological interpretation of plant systems forward. Using the gained knowledge, metabolic engineering strategies to engage plants as biotechnological factories can be developed.

Physionomics and metabolomics-two key approaches in herbicidal mode of action discovery.

BACKGROUND: For novel herbicides identified in greenhouse screens, efficient research is important to discover and chemically optimise new leads with new modes of action (MoAs). RESULTS: The metabolic and physiological response pattern to a herbicide can be viewed as the result of changes elicited in the molecular and biochemical process chain. These response patterns are diagnostic of a herbicide's MoA. At the starting point of MoA characterisation, an array of bioassays is used for comprehensive physiological profiling of herbicide effects. This physionomics approach enables discrimination between known, novel or multiple MoAs of a compound and provides a first clue to a new MoA. Metabolic profiling is performed with the use of treated Lemna paucicostata plants. After plant extraction and chromatography and mass spectrometry, changes in levels of approximately 200 identified and 300 unknown analytes are quantified. Check for known MoA assignment is performed by multivariate statistical data analyses. Distinct metabolite changes, which can direct to an affected enzymatic step, are visualised in a biochemical pathway view. Subsequent target identification includes metabolite feeding and molecular, biochemical and microscopic methods. CONCLUSION: The value of this cascade strategy is exemplified by new herbicides with MoAs in plastoquinone, auxin or very-long-chain fatty acid synthesis.

On the mode of action of the herbicides cinmethylin and 5-benzyloxymethyl-1, 2-isoxazolines: putative inhibitors of plant tyrosine aminotransferase.

BACKGROUND: The mode of action of the grass herbicides cinmethylin and 5-benzyloxymethyl-1,2-isoxazolines substituted with methylthiophene (methiozolin) or pyridine (ISO1, ISO2) was investigated. RESULTS: Physiological profiling using a series of biotests and metabolic profiling in treated duckweed (Lemna paucicostata L.) suggested a common mode of action for the herbicides. Symptoms of growth inhibition and photobleaching of new fronds in Lemna were accompanied with metabolite changes indicating an upregulation of shikimate and tyrosine metabolism, paralleled by decreased plastoquinone and carotenoid synthesis. Supplying Lemna with 10 µM of 4-hydroxyphenylpyruvate (4-HPP) reversed phytotoxic effects of cinmethylin and isoxazolines to a great extent, whereas the addition of L-tyrosine was ineffective. It was hypothesised that the herbicides block the conversion of tyrosine to 4-HPP, catalysed by tyrosine aminotransferase (TAT), in the prenylquinone pathway which provides plastoquinone, a cofactor of phytoene desaturase in carotenoid synthesis. Accordingly, enhanced resistance to ISO1 treatment was observed in Arabidopsis thaliana L. mutants, which overexpress the yeast prephenate dehydrogenase in plastids as a TAT bypass. In addition, the herbicides were able to inhibit TAT7 activity in vitro for the recombinant enzyme of A. thaliana. CONCLUSION: The results suggest that TAT7 or another TAT isoenzyme is the putative target of the herbicides.