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High-throughput profiling of key enzyme activities of carbon, nitrogen, and antioxidant metabolism is emerging as a valuable approach to integrate cell physiological phenotyping into a holistic functional phenomics approach. However, the analyses of the large datasets generated by this method represent a bottleneck, often keeping researchers from exploiting the full potential of their studies. We address these limitations through the exemplary application of a set of data evaluation and visualization tools within a case study. This includes the introduction of multivariate statistical analyses that can easily be implemented in similar studies, allowing researchers to extract more valuable information to identify enzymatic biosignatures. Through a literature meta-analysis, we demonstrate how enzyme activity profiling has already provided functional information on the mechanisms regulating plant development and response mechanisms to abiotic stress and pathogen attack. The high robustness of the distinct enzymatic biosignatures observed during developmental processes and under stress conditions underpins the enormous potential of enzyme activity profiling for future applications in both basic and applied research. Enzyme activity profiling will complement molecular -omics approaches to contribute to the mechanistic understanding required to narrow the genotype-to-phenotype knowledge gap and to identify predictive biomarkers for plant breeding to develop climate-resilient crops. [ABSTRACT FROM AUTHOR]

Background: Reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anions significantly accumulate during biotic and abiotic stress and cause oxidative damage and eventually cell death. There is accumulating evidence that ROS are also involved in regulating beneficial plant–microbe interactions, signal transduction and plant growth and development. Due to the relevance of ROS throughout the life cycle and for interaction with the multifactorial environment, the physiological phenotyping of the mechanisms controlling ROS homeostasis is of general importance. Results: In this study, we have developed a robust and resource-efficient experimental platform that allows the determination of the activities of the nine key ROS scavenging enzymes from a single extraction that integrates posttranscriptional and posttranslational regulations. The assays were optimized and adapted for a semi-high throughput 96-well assay format. In a case study, we have analyzed tobacco leaves challenged by pathogen infection, drought and salt stress. The three stress factors resulted in distinct activity signatures with differential temporal dynamics. Conclusions: This experimental platform proved to be suitable to determine the antioxidant enzyme activity signature in different tissues of monocotyledonous and dicotyledonous model and crop plants. The universal enzymatic extraction procedure combined with the 96-well assay format demonstrated to be a simple, fast and semi-high throughput experimental platform for the precise and robust fingerprinting of nine key antioxidant enzymatic activities in plants. [ABSTRACT FROM AUTHOR]

Background: To improve our understanding about the physiological mechanism of grain yield reduction at anthesis, three spring wheat genotypes [L1 (advanced line), L2 (Vorobey) and L3 (Punjab-11)] having contrasting yield potential under drought in field were investigated under controlled greenhouse conditions, drought stress was imposed at anthesis stage by withholding irrigation until all plant available water was depleted, while well-watered control plants were kept at 95% pot water holding capacity. Results: Compared to genotype L1 and L2, pronounced decrease in grain number (NGS), grain yield (GY) and harvest index (HI) were found in genotype L3, mainly due to its greater kernel abortion (KA) under drought. A significant positive correlation of leaf monodehydroascorbate reductase (MDHAR) with both NGS and HI was observed. In contrast, significant negative correlations of glutathione S-transferase (GST) and vacuolar invertase (vacInv) both within source and sink were found with NGS and HI. Likewise, a significant negative correlation of leaf abscisic acid (ABA) with NGS was noticed. Moreover, leaf aldolase and cell wall peroxidase (cwPOX) activities were significantly and positively associated with thousand kernel weight (TKW). Conclusion: Distinct physiological markers correlating with yield traits and higher activity of leaf aldolase and cwPOX may be chosen as predictive biomarkers for higher TKW. Also, higher activity of MDHAR within the leaf can be selected as a predictive biomarker for higher NGS in wheat under drought. Whereas, lower activity of vacInv and GST both within leaf and spike can be selected as biomarkers for higher NGS and HI. The results highlighted the role of antioxidant and carbohydrate-metabolic enzymes in the modulation of source-sink balance in wheat crops, which could be used as bio-signatures for breeding and selection of drought-resilient wheat genotypes for a future drier climate. [ABSTRACT FROM AUTHOR]

Objective: Enzymatic fingerprinting of key enzymes of glucose metabolism is a valuable analysis tool in cell physiological phenotyping of plant samples. Yet, a similar approach for mammalian cell line samples is missing. In this study, we applied semi-high throughput enzyme activity assays that were originally designed for plant samples and tested their feasibility in extracts of six frequently used mammalian cell lines (Caco2, HaCaT, C2C12, HEK293, HepG2 and INS-1E). Results: Enzyme activities for aldolase, hexokinase, glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, phosphoglucomutase, phosphofructokinase could be detected in samples of one or more mammalian cell lines. We characterized effects of sample dilution, assay temperature and repeated freeze–thaw cycles causing potential biases. After careful selection of experimental parameters, the presented semi-high throughput methods could be established as useful tool for physiological phenotyping of cultured mammalian cells. [ABSTRACT FROM AUTHOR]