A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation.

Our goal was to identify the leaf proteomic changes which appeared during N remobilisation that were associated or not associated with senescence of oilseed rape in response to contrasting nitrate availability. Remobilisation of N and leaf senescence status were followed using (15)N tracing, patterns of chlorophyll level, total protein content and a molecular indicator based on expression of senescence-associated gene 12/Cab genes. Three phases associated with N remobilisation were distinguished. Proteomics revealed that 55 proteins involved in metabolism, energy, detoxification, stress response, proteolysis and protein folding, were significantly induced during N remobilisation. Four proteases were specifically identified. FtsH, a chloroplastic protease, was induced transiently during the early stages of N remobilisation. Considering the dynamics of N remobilisation, chlorophyll and protein content, the pattern of FtsH expression indicated that this protease could be involved in the degradation of chloroplastic proteins. Aspartic protease increased at the beginning of senescence and was maintained at a high level, implicating this protease in proteolysis during the course of leaf senescence. Two proteases, proteasome beta subunit A1 and senescence-associated gene 12, were induced and continued to increase during the later phase of senescence, suggesting that these proteases are more specifically involved in the proteolysis processes occurring at the final stages of leaf senescence.

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.

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.

N-protein mobilisation associated with the leaf senescence process in oilseed rape is concomitant with the disappearance of trypsin inhibitor activity.

Brassica napus L. (oilseed rape) is an important crop plant characterised by low nitrogen (N) use efficiency. This is mainly due to a weak N recycling from leaves that is related to incomplete protein degradation. Assuming that protease inhibitors are involved throughout protein mobilisation, the goal of this study was to determine their role in the control of N mobilisation associated with leaf senescence. Results showed that a 19-kDa polypeptide exhibiting trypsin inhibitor (TI) activity presented an increased gradient from the older to the younger leaves. According to the SAG12/Cab gene expression profile, which is an indicator of leaf senescence, mature leaves of nitrate-deprived plants presented an earlier initiation of senescence and a decrease in protein concentration when compared with nitrate-replete plants. This coincided with disappearance of both TI activity and a reduction in the transcript level of the BnD22 gene (encoding a protein sharing homology with Künitz protease inhibitor). In young leaves of N-deprived plants, initiation of senescence was delayed; soluble protein concentration was maintained while both TI activity and BnD22 transcripts were high. This indicates that in oilseed rape growing under nitrate deprivation, the more efficient N recycling from mature leaves contributes to the maintenance of growth in young leaves. The data suggest a significant role for protease inhibitors in the regulation of proteolytic processes associated with N mobilisation during leaf senescence.