Secretome profiling of Pinus nigra embryogenic cultures reveals a freely secreted protein fraction dominated by cell wall- and redox-related proteins

Somatic embryogenesis is widely recognised as an efficient method for conifer micropropagation and conservation, and embryogenic cultures provide an experimentally accessible system for studying extracellular proteins under controlled conditions. While secreted proteins have been studied in several plant species, the freely secreted protein fraction of conifer embryogenic cultures has not yet been comprehensively characterised by proteomic approaches. Here, we characterised the secretome of Pinus nigra embryogenic suspension cultures and evaluated the identified proteins according to their predicted secretion features and functional categories. In total, 211 proteins were identified. Most were predicted to follow the classical secretory pathway, while a smaller subset represented candidates for unconventional secretion, with several membrane-associated and GPI-anchored proteins. The identified secretome was dominated by proteins acting on cell wall carbohydrates, followed by oxidoreductases, proteases, signalling-related proteins, and several defence-associated and nutrient-mobilising enzymes. This protein profile suggests that the extracellular environment of P. nigra embryogenic cultures contains proteins potentially involved in cell wall polysaccharide modification, redox-related wall processes, extracellular matrix organisation and cell-surface communication. Defence-associated proteins were also detected, indicating that these protein families form part of the extracellular protein repertoire of embryogenic cultures, although their specific roles remain to be established. Proteins secreted into suspension culture media were associated with matrix organisation, cell-surface communication, and protein and nutrient turnover.