Checcucci Alice, Decorosi Francesca, Alfreducci Giulia, Natale Roberto, Bellabarba Agnese, Biricolti Stefano, Paffetti Donatella, Mengoni Alessio, Viti Carlo
Department of Agricultural, Environmental, Food and Forestry Science and Technology (DAGRI), University of Florence, Florence, Italy.
Interdepartmental Service Centre for Agricultural Chemical and Industrial Biotechnologies (CIBIACI), University of Florence, Florence, Italy.
Plant Methods. 2025 May 7;21(1):58. doi: 10.1186/s13007-025-01378-5.
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 potential 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 technology 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.
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 intracellular processes without interference from the cell wall. As a proof-of-principle, we used protoplasts of Solanum tuberosum 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.
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 comparative studies at cellular level among cultivars, species, wild-type organisms, and genome-edited plants.
栽培植物的生产力和适应性受到遗传特性和环境相互作用的影响，从而形成特定的表型。表型组学是一种组学方法，为多维表型信息分析提供应用方法，对于理解和预测与农业相关生物的遗传潜力至关重要。虽然植物表型分析在整个生物体水平上提供信息，但细胞水平的表型分析对于识别和剖析不同表型的代谢基础以及代谢相关基因修饰的影响至关重要。表型芯片（PM）是Biolog公司开发的一种用于细胞水平代谢特征研究的高通量技术，它基于比色反应来监测不同条件下的细胞呼吸。如今，PM已广泛应用于细菌、真菌和哺乳动物细胞，但由于难以确定合适的细胞活性报告分子，尚未开发出用于植物细胞特征分析的方法。
在此，我们首次使用原生质体作为评估代谢活性的手段，对植物细胞进行了PM技术测试。事实上，研究植物原生质体的代谢可以成为预测整个植物生物体固有代谢潜力的有价值方法。原生质体确实是植物研究和生物技术中的宝贵工具，因为它们提供了一个简化的、孤立的细胞系统，研究人员可以在其中专注于细胞内过程而不受细胞壁的干扰。作为原理验证，我们使用马铃薯（Solanum tuberosum L.）的原生质体作为模型系统。从体外培养植物的叶片组织中分离原生质体，纯化后稀释至所需浓度。用原生质体悬液接种微孔板，并测试各种氧化还原电位标记物作为细胞活性指标。在确定PM测试的最佳条件后，将代谢测试扩展到番茄（S. lycopersicum L.）的原生质体，评估植物对不同NaCl浓度以及预配置的Biolog微孔板中存在的一些有毒化合物的反应。
所开发的标准化高通量系统对于植物原生质体的代谢特征分析是有效的。该方法为植物细胞代谢表型研究奠定了基础，能够在细胞水平上对不同品种、物种、野生型生物体和基因组编辑植物进行比较研究。
