The alteration of proteins and metabolites in leaf apoplast and the related gene expression associated with the adaptation of Ammopiptanthus mongolicus to winter freezing stress.

Ammopiptanthus mongolicus, an evergreen broad-leaved plant, can tolerate severe freezing stress (temperatures as low as -20 °C in winter). The apoplast is the space outside the plasma membrane that plays an important role in plant responses to environmental stress. Here, we investigated, using a multi-omics approach, the dynamic alterations in the levels of proteins and metabolites in the apoplast and related gene expression changes involved in the adaptation of A. mongolicus to winter freezing stress. Of the 962 proteins identified in the apoplast, the abundance of several PR proteins, including PR3 and PR5, increased significantly in winter, which may contribute to winter freezing-stress tolerance by functioning as antifreeze proteins. The increased abundance of the cell-wall polysaccharides and cell wall-modifying proteins, including PMEI, XTH32, and EXLA1, may enhance the mechanical properties of the cell wall in A. mongolicus. Accumulation of flavonoids and free amino acids in the apoplast may be beneficial for ROS scavenging and the maintenance of osmotic homeostasis. Integrated analyses revealed gene expression changes associated with alterations in the levels of apoplast proteins and metabolites. Our study improved the current understanding of the roles of apoplast proteins and metabolites in plant adaptation to winter freezing stress.