Detailed and standardized protocols for plant cultivation in environmentally controlled conditions are an essential prerequisite to conduct reproducible experiments with precisely defined treatments. Setting up appropriate and well defined experimental procedures is thus crucial for the generation of solid evidence and indispensable for successful plant research. Non-invasive and high throughput (HT) phenotyping technologies offer the opportunity to monitor and quantify performance dynamics of several hundreds of plants at a time. Compared to small scale plant cultivations, HT systems have much higher demands, from a conceptual and a logistic point of view, on experimental design, as well as the actual plant cultivation conditions, and the image analysis and statistical methods for data evaluation. Furthermore, cultivation conditions need to be designed that elicit plant performance characteristics corresponding to those under natural conditions. This manuscript describes critical steps in the optimization of procedures for HT plant phenotyping systems. Starting with the model plant Arabidopsis, HT-compatible methods were tested, and optimized with regard to growth substrate, soil coverage, watering regime, experimental design (considering environmental inhomogeneities) in automated plant cultivation and imaging systems. As revealed by metabolite profiling, plant movement did not affect the plants' physiological status. Based on these results, procedures for maize HT cultivation and monitoring were established. Variation of maize vegetative growth in the HT phenotyping system did match well with that observed in the field. The presented results outline important issues to be considered in the design of HT phenotyping experiments for model and crop plants. It thereby provides guidelines for the setup of HT experimental procedures, which are required for the generation of reliable and reproducible data of phenotypic variation for a broad range of applications.

SUMMARYThe metabolic function of the predicted Arabidopsis tyrosine aminotransferase (TAT) encoded by the At5g53970 gene was studied using two independent knock-out mutants. Gas chromatography-mass spectrometry based metabolic profiling revealed a specific increase in tyrosine levels, supporting the proposed function of At5g53970 as a tyrosine-specific aminotransferase not involved in tyrosine biosynthesis, but rather in utilization of tyrosine for other metabolic pathways. The TAT activity of the At5g53970-encoded protein was verified by complementation of the Escherichia coli tyrosine auxotrophic mutant DL39, and in vitro activity of recombinantly expressed and purified At5g53970 was found to be specific for tyrosine. To investigate the physiological role of At5g53970, the consequences of reduction in tyrosine utilization on metabolic pathways having tyrosine as a substrate were analysed. We found that tocopherols were substantially reduced in the mutants and we conclude that At5g53970 encodes a TAT important for the synthesis of tocopherols in Arabidopsis.

SUMMARYBarley endosperm cells differentiate into transfer cells (ETCs) opposite the nucellar projection. To comprehensively analyse ETC differentiation, laser microdissection-based transcript and metabolite profiles were obtained from laser microdissected tissues and cell morphology was analysed. Flange-like secondary-wall ingrowths appeared between 5 and 7 days after pollination within the three outermost cell layers. Gene expression analysis indicated that ethylene-signalling pathways initiate ETC morphology. This is accompanied by gene activity related to cell shape control and vesicle transport, with abundant mitochondria and endomembrane structures. Gene expression analyses indicate predominant formation of hemicelluloses, glucuronoxylans and arabinoxylans, and transient formation of callose, together with proline and 4-hydroxyproline biosynthesis. Activation of the methylation cycle is probably required for biosynthesis of phospholipids, pectins and ethylene. Membrane microdomains involving sterols/sphingolipids and remorins are potentially involved in ETC development. The transcriptional activity of assimilate and micronutrient transporters suggests ETCs as the main uptake organs of solutes into the endosperm. Accordingly, the endosperm grows maximally after ETCs are fully developed. Up-regulated gene expression related to amino acid catabolism, C:N balances, carbohydrate oxidation, mitochondrial activity and starch degradation meets high demands for respiratory energy and carbohydrates, required for cell proliferation and wall synthesis. At 10 days after pollination, ETCs undergo further differentiation, potentially initiated by abscisic acid, and metabolism is reprogrammed as shown by activated storage and stress-related processes. Overall, the data provide a comprehensive view of barley ETC differentiation and development, and identify candidate genes and associated pathways.

The metabolic-oxidative stress profile of the rice flag leaf during drought stress in the reproductive stage is highly predictive for grain yield loss sensitivity of 292 accessions at harvest time.

HighlightAutomated imaging-based plant phenotyping combined with GC-MS-based metabolite profiling of four lentil accessions differing in their drought and salt tolerance shows common and specific responses and yields characteristic stress markers