Laser capture of tomato pericarp tissues for microscale carotenoid analysis by supercritical fluid chromatography

Authors: Ramsey, John - John; Fish, Tara; Thannhauser, Theodore - Ted; Giovannoni, James

Submitted to: Methods in Enzymology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/9/2022
Publication Date: 7/21/2022
Citation: Ramsey, J.S., Fish, T., Thannhauser, T.W., Giovannoni, J.J. 2022. Laser capture of tomato pericarp tissues for microscale carotenoid analysis by supercritical fluid chromatography. Methods in Enzymology. 670:213-233. https://doi.org/10.1016/bs.mie.2022.01.014.
DOI: https://doi.org/10.1016/bs.mie.2022.01.014

Interpretive Summary: Laser microdissection enables the collection of specific tissues and cell types for extraction of biological molecules of interest. Separate collection and sample preparation of unique cell types enable high-resolution analysis of biological function that is specific to a given tissue but remains difficult to achieve. We are especially interested in fruit ripening and the compounds that result in appealing flavors, colors and nutrient value. We implemented a number of novel collection and extraction procedures to enable efficient recovery and analysis of nutritional metabolite of tomato, specifically carotenoids. Carotenoids are responsible for tomato color and the colors of many fruits, vegetables and flowers. They provide antioxidant activity and some forms are converted in the body to the essential nutrient vitamin A. Successful development of these extraction and analysis procedures allows for precise determination of which cell and tissue types of the fruit are responsible for production and accumulation of these compounds. This knowledge in turn will assist breeders and geneticists in development of more flavorful, attractive and nutritional tomatoes. These approaches are likely applicable to additional fruit and vegetables.

Technical Abstract: Plant organs and tissues are comprised of an array of cell types often superimposed on a gradient of developmental stages. As a result, the ability to analyze and understand the synthesis, metabolism, and accumulation of plant biomolecules requires improved methods for cell- and tissue-specific analysis. Tomato (Solanum lycopersicum) is the world's most valuable fruit crop and is an important source of health-promoting dietary compounds, including carotenoids. Furthermore, tomato possesses unique genetic activities at the cell and tissue levels, making it an ideal system for tissue- and cell-type analysis of important biochemicals. A sample preparation workflow was developed for cell-type-specific carotenoid analysis in tomato fruit samples. Protocols for hyperspectral imaging of tomato fruit samples, cryoembedding and sectioning of pericarp tissue, laser microdissection of specific cell types, metabolite extraction using cell wall digestion enzymes and pressure cycling, and carotenoid quantification by supercritical fluid chromatography were optimized and integrated into a working protocol. The workflow was applied to quantify carotenoids in the cuticle and noncuticle component of the tomato pericarp during fruit development from the initial ripening to full ripe stages. Carotenoids were extracted and quantified from cell volumes less than 10 nL. This workflow for cell-type-specific metabolite extraction and quantification can be adapted for the analysis of diverse metabolites, cell types, and organisms.