Phenotype microarray‑based assessment of metabolic variability in plant protoplasts

Background Productivity and fitness of cultivated plants are influenced by genetic heritage and environmental interactions, shaping certain phenotypes. Phenomics is the -omics methodology providing applicative approaches for the analysis of multidimensional phenotypic information, essential to understand and foresee the genetic poten- tial of organisms relevant to agriculture. While plant phenotyping provides information at the whole organism level, cellular level phenotyping is crucial for identifying and dissecting the metabolic basis of different phenotypes and the effect of metabolic-related genetic modifications. Phenotype Microarray (PM) is a high-throughput technol- ogy developed by Biolog™ for metabolic characterization studies at cellular level, which is based on colorimetric reactions to monitor cellular respiration under different conditions. Nowadays, PM is widely used for bacteria, fungi, and mammalian cells, but a procedure for plant cells characterization has not yet been developed, due to difficulties linked in identifying a suitable reporter of cell activities. Results Here, we tested for the first time, PM technology on plant cells using protoplasts as a means of evaluating metabolic activity. Indeed, studying the metabolism of plant protoplasts can be a valuable method for predicting the inherent metabolic potential of an entire plant organism. Protoplasts are indeed valuable tools in plant research and biotechnology because they offer a simplified, isolated cellular system where researchers can focus on intracellu- lar processes without interference from the cell wall. As a proof-of-principle, we used protoplasts of Solanum tubero- sum L. as model system. Protoplasts were isolated from leaf tissue of in vitro-grown plants, purified and then diluted until desired concentration. Microplates were inoculated with protoplasts suspension and various markers of redox potential as indicators of cell activity were tested. After identifying the optimal conditions for PM testing, metabolic tests were extended to protoplasts from S. lycopersicum L., evaluating plant response to different NaCl concentrations and some of the toxic compounds present in pre-configured ­ Biolog™ microplates. Conclusions The standardized high-throughput system developed was effective for the metabolic characterization of plant protoplasts. This method lays the foundation for plant cell metabolic phenotype studies enabling compara- tive studies at cellular level among cultivars, species, wild-type organisms, and genome-edited plants.