Activity-Based Profiling of Papain-like Cysteine Proteases in Different Plant Organs During Barley Development

Papain-like cysteine proteases (PLCPs) are vital enzymes involved in plant development, acting as key regulators of processes such as seed germination, nutrient mobilization, senescence, and programmed cell death. In the present study, we analyzed active PLCPs in various barley organs, including roots, leaves, stems, and seeds at different stages of plant development. Protein extracts obtained from barley samples (4-day-old seedlings; plants at 2, 4, 7, and 11 weeks after sowing; developing seeds from 11-week-old plants; and mature dry seeds) were subjected to anion-exchange chromatography. Fractions containing active PLCPs were pooled, biotinylated using the DCG-04 probe, affinity-purified using streptavidin-agarose, and subsequently analyzed via SDS-PAGE. Bands corresponding to biotinylated PLCPs (detected using streptavidin-peroxidase and a chemiluminescent substrate) were excised from the gel and analyzed by tandem mass spectrometry, enabling the identification of up to 23 distinct PLCPs belonging to nine known PLCP subfamilies. Among the identified PLCPs, HvPap-6 from the L-like D subfamily proved to be the most abundant across all barley samples. In seedlings, B-like and L-like D proteases constituted the largest proportion of all PLCP classes, and their levels continued to increase as the plants developed. Although the relative abundance of L-like B and L-like C proteases was high in seedlings, their levels declined in the roots and leaves of developing plants, as three PLCPs from the L-like B subfamily were identified only during the seedling stage. These results suggest that L-like B and L-like C proteases play an important role in seed germination and seedling development. Organ-specific expression was also observed for certain PLCPs: HvPap-26 from the L-Like C subfamily was identified only in the shoots and roots of seedlings; four PLCPs of the L-like E subfamily were detected solely in the roots, whereas two other proteases from this subfamily were identified exclusively in the leaves and shoots under our experimental conditions. Thus, our results suggest that certain active PLCPs are organ-specific, and that the relative importance of identified PLCPs varies within these organs during plant development.