Novel approaches to functional studies of stress-related proteins in wheat and barley: Genetic engineering, proteoforms, organellar proteomics, interactomics and proxiomics

• Protein homeology and functional redundancy cause impediments to protein functional studies in Triticeae. • Gene editing, namely multiplex CRISPR/Cas, represents a promising way to protein functional studies. • Studies on proteoforms and protein subcellular localizations represent tedious tasks due to the demands on plant material and need of protein enrichment. • Interactomics studies use classical approaches such as Y2H or BiFC but often rely on bioinformatics data gained in Arabidopsis. Barley and common wheat are important crops from the Triticeae tribe which are characterized by large genomes and a high number of homoeologous genes. Environmental stresses induce plant responses resulting in altered biomolecular profiles, proteoforms, subcellular localization, and protein-protein interactions. Gene homoeology limits the potential of classical mutational approaches in protein functional studies and mostly only transient knockdown approaches based on RNA interference could be used to suppress the protein expression and gain a new phenotype. The advent of genome editing approaches based on programmable site-directed nucleases, most notably CRISPR/Cas systems, has substantially expanded the experimental toolbox in wheat and barley by enabling precise, heritable manipulation of coding and regulatory sequences. Multiplex targeting of homoeologous genes overcomes functional redundancy and allows the generation of allelic, dosage-dependent and regulatory variants, providing finer resolution for dissecting protein function and trait variation. In this review, the main focus is given on experimental tools used for protein functional studies in Triticeae crops. We summarize the use of genome editing alongside established approaches such as overexpression and RNA interference for genomic studies in Triticeae crops. We further discuss how gene homoeology contributes to protein functional diversification and review current progress in proteoforms research. In addition, advances in organellar proteomics, namely nuclear, chloroplast, and mitochondrial proteomics are highlighted. Finally, modern approaches routinely used in Arabidopsis thaliana to study in vivo protein cell localization (spatial proteomics based on advanced fluorescence microscopy) as well as protein-protein interactions (fluorescent microscopy, FLIM-FRET, and enzyme-based proximity labeling) are briefly summarised in the context of their applicability to Triticeae species. All approaches presented in the text can significantly contribute to elucidation of protein biological functions in Triticeae crops´ responses to environmental stresses.