Proteomics: An Essential Tool to Study Plant-Specialized Metabolism

Abstract

Plants are a valuable source of specialized metabolites that provide a plethora of therapeutic applications. They are natural defenses that plants use to adapt and respond to their changing environment. Decoding their biosynthetic pathways and understanding how specialized plant metabolites (SPMs) respond to biotic or abiotic stress will provide vital knowledge for plant biology research and its application for the future sustainable production of many SPMs of interest. Here, we focus on the proteomic approaches and strategies that help with the study of plant-specialized metabolism, including the: (i) discovery of key enzymes and the clarification of their biosynthetic pathways; (ii) study of the interconnection of both primary (providers of carbon and energy for SPM production) and specialized (secondary) metabolism; (iii) study of plant responses to biotic and abiotic stress; (iv) study of the regulatory mechanisms that direct their biosynthetic pathways. Proteomics, as exemplified in this review by the many studies performed to date, is a powerful tool that forms part of omics-driven research. The proteomes analysis provides an additional unique level of information, which is absent from any other omics studies. Thus, an integrative analysis, considered versus a single omics analysis, moves us more closely toward a closer interpretation of real cellular processes. Finally, this work highlights advanced proteomic technologies with immediate applications in the field.

Keywords: biosynthesis; mass spectrometry; natural products or compounds; proteomics; secondary metabolism; specialized plant metabolite.

Figure 2

Proteomic approaches and strategies typically applied to analyze plant proteomes. Here, the top-down approach follows a comprehensive-centric definition that refers to protein separation by 2-DE prior to MS, while bottom-up applies to sample separation at the peptide level prior to the peptide analysis by MS. When dealing with complex samples, they can be extensively fractionated before the protein digestion step by either SDS-PAGE [113,114] or HPLC. Wider proteome coverage can be achieved by 2D protein separation, where orthogonal separation is applied in combination with the second peptide separation by the standard RP-HPLC, coupled online to MS. The additional separation step can consist of up-front chromatography based on high pH chromatography or strong cation exchange (SCX), which are among the most widely used techniques. Then, the digested proteins from gel slices or chromatographic fractions are analyzed by LC-MS/MS. On the one hand, if the goal is to identify new proteins of interest, a discovery or hypothesis-free approach is undertaken. In this case, MS data acquisition can be done by either DDA or IDA. Protein abundance can be determined simultaneously to protein identification by either label-free or label-based quantification methods, of which iTRAQ/TMT is the most widely used. On the other hand, having identified proteins of interest, a targeted or hypothesis-driven approach can be applied with different goals, to do the following: accurately quantify a protein by absolute quantification; validate protein abundances obtained from a discovery-based approach; selectively analyze a metabolic pathway under different conditions. In this case, MS data acquisition can be done by either an SRM or a PRM analysis.

Figure 1

Schematic overview of the specialized (secondary) metabolism. The plant-specialized metabolic pathways related to phenolic compounds (in green), terpenes (in yellow), fatty acid-derived (in light blue) and N-containing compounds, glucosinolates, alkaloids, cyanogenic glucosides and non-proteinogenic amino acids (in orange) are shown. Dark blue denotes precursors and primary biosynthetic pathways. The structure of the representative examples of related compounds is represented in black. The basic skeleton structures for some groups of compounds are depicted in green, and those related to flavonoids in blue. Abbreviations: anthocyanidin 3-O-glucosyltransferase (UFGT); anthocyanidin reductase (ANR); chalcone synthase (CHS); chalcone isomerase (CHI); cinnamate 4-hydroxylase (C4H); coumarate CoA ligase (4CL); p-coumaroyl-CoA 2-hydroxylase (C2’H); dihydroflavonol 4-reductase (DFR); flavanone 3-hydroxylase (F3H); flavone synthase (FNS); flavonol synthase (FLS); hydroxycinnamoyl transferase (HCT); isoflavone synthase (IFS); leucoanthocyanidin reductase (LAR); leucoanthocyanidin dioxygenase/anthocyanidin synthase (LDOX/ANS); norcoclaurine synthase (NCS); phenylalanine ammonia lyase (PAL); secologanin synthase (SLS); shikimate dehydrogenase (SDH); stilbene synthase (STS); strictosidine synthase (STR); terpene synthase (TS); tryptophan decarboxylase (TDC).

Figure 3

Current proteomic approaches applied for the study of plant-specialized metabolism. The plant material and the sample preparation procedure are critical for gaining access to target-specialized metabolic pathways and, therefore, the target proteome. So, different strategies can be followed to enrich the desired metabolic pathways regarding the selection of suitable plant material (tissue, organ, cell-type, subcellular compartments or development stage) and the conditions that enhance, annul or trigger the production of the SPM under study (mutant generation, elicitation or specific environmental conditions). The successfully applied proteomic approaches were comparative or differential proteomics, co-expression analysis, activity-guided proteomics, targeted proteomics and proteomics-guided mining approaches.