A Proteomic Profiling Approach to Reveal a Novel Role of Brassica napus Drought 22 kD/Water-Soluble Chlorophyll-Binding Protein in Young Leaves during Nitrogen Remobilization Induced by Stressful Conditions

摘要

Despite its water-soluble chlorophyll-binding protein (WSCP) function, the putative trypsin inhibitor (TI) activity of the Brassica napus drought 22 kD (BnD22) protein and its physiological function in young leaves during leaf nitrogen (N) remobilization promoted by stressful conditions remains an enigma. Therefore, our objectives were to determine (1) if BnD22 is related to the 19-kD TI previously detected in B. napus young leaves, and (2) if the levels of BnD22 transcripts, BnD22 protein, and TI activity in young leaves are associated with plant responses to stress conditions (N starvation and methyl jasmonate [MeJA] treatments) that are able to modulate leaf senescence. Compared to control, N starvation delayed initiation of senescence and induced 19-kD TI activity in the young leaves. After 3 d with MeJA, the 19-kD TI activity was 7-fold higher than the control. Using two-dimensional electrophoresis gel, TI activity, and electrospray ionization liquid chromatography tandem mass spectrometry analysis, it was demonstrated that two 19-kD proteins with isoelectric points 5.0 and 5.1 harboring TI activity correspond to BnD22 perfectly. BnD22 gene expression, TI activities, and BnD22 protein presented similar patterns. Using polyclonal anti-WSCP antibodies of Brassica oleracea, six polypeptides separated by two-dimensional electrophoresis were detected in young leaves treated with MeJA. Electrospray ionization liquid chromatography tandem mass spectrometry analysis of six polypeptides confirms their homologies with WSCP. Results suggest that BnD22 possesses dual functions (WSCP and TI) that lead to the protection of younger tissues from adverse conditions by maintaining metabolism (protein integrity and photosynthesis). By sustaining sink growth of stressed plants, BnD22 may contribute to a better utilization of recycling N from sources, a physiological trait that improves N-use efficiency.