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. 2017 Nov 2;5(4):29.
doi: 10.3390/proteomes5040029.

Proteomic Investigations of Proteases Involved in Cotyledon Senescence: A Model to Explore the Genotypic Variability of Proteolysis Machinery Associated With Nitrogen Remobilization Efficiency During the Leaf Senescence of Oilseed Rape

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Proteomic Investigations of Proteases Involved in Cotyledon Senescence: A Model to Explore the Genotypic Variability of Proteolysis Machinery Associated With Nitrogen Remobilization Efficiency During the Leaf Senescence of Oilseed Rape

Marine Poret et al. Proteomes. .
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Abstract

Oilseed rape is characterized by a low nitrogen remobilization efficiency during leaf senescence, mainly due to a lack of proteolysis. Because cotyledons are subjected to senescence, it was hypothesized that contrasting protease activities between genotypes may be distinguishable early in the senescence of cotyledons. To verify this assumption, our goals were to (i) characterize protease activities in cotyledons between two genotypes with contrasting nitrogen remobilization efficiency (Ténor and Samouraï) under limiting or ample nitrate supply; and (ii) test the role of salicylic acid (SA) and abscisic acid (ABA) in proteolysis regulation. Protease activities were measured and identified by a proteomics approach combining activity-based protein profiling with LC-MS/MS. As in senescing leaves, chlorophyll and protein contents decrease in senescing cotyledons and are correlated with an increase in serine and cysteine protease activities. Two RD21-like and SAG-12 proteases previously associated with an efficient proteolysis in senescing leaves of Ténor are also detected in senescing cotyledons. The infiltration of ABA and SA provokes the induction of senescence and several cysteine and serine protease activities. The study of protease activities during the senescence of cotyledons seems to be a promising experimental model to investigate the regulation and genotypic variability of proteolysis associated with efficient N remobilization.

Keywords: Brassica napus L.; cotyledon; genotypic variability; nitrogen remobilization efficiency; phytohormones; protease activity; senescence.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Chlorophyll (A,B) and soluble protein (C) contents during cotyledon senescence in two genotypes of oilseed rape supplied with high (HN) or low (LN) nitrate for five days. Cotyledons (15 days old) of two different genotypes (Ténor and Samouraï) were subjected to ample (HN: 3.75 mM NO3) or low nitrogen supply (LN: 0.375 mM NO3) for five days. Vertical bars indicate ± SD of the mean (n = 3). Different letters indicate statistical differences (p < 0.05, ANOVA, Newman-Keuls test).
Figure 2
Figure 2
Serine hydrolase (SH) activity at pH 7.5 during cotyledon senescence in two genotypes of oilseed rape supplied with high (HN) or low (LN) nitrate for five days. Soluble proteins of cotyledons were extracted from Ténor or Samouraï plants after 0 and five days of HN (3.75 mM NO3) or LN (0.375 mM NO3) treatment. Labeling of protease activity with FP-Rh (specific fluorescent probe of SHs) (pH 7.5; 1 h labeling) was performed. After SDS-PAGE, the fluorescence was detected with a scanner (A) Ténor; (B) Samouraï. As a control, Mix refers to a mix of 0 and five day extracts (HN and LN) in the presence of FP-Rh, NPC (no probe control) refers to the same mix in the absence of FP-Rh, and DFP refers to the same mix in the presence of FP-Rh and DFP (specific inhibitor of serine proteases). Positions of active proteases are represented by black arrowheads. SH global activity characterized by the fluorescence intensity was calculated with the Image J® (C) software. The presented gel is representative of three biological replicates. Gel stained with Coomassie brilliant blue (cbb) shows the RuBisCO large sub-unit (RBCL) and the decline of this major protein indicated that proteolysis and N remobilization are induced. Vertical bars indicate ± SD of the mean (n = 3). Different letters indicate statistical differences (p < 0.05, ANOVA, Newman-Keuls test).
Figure 3
Figure 3
Detection of SHs (A) and PLCPs (B) labeled with FP-biotin and DCG04 in senescent cotyledons of oilseed rape (cv. Ténor) after five days of LN treatment. To characterize SHs and PLCPs observed in Figure 2 and Figure 4, a pull-down of biotinylated proteins was performed after labeling with the biotin-tagged probes FP-biotin (A) and DCG04 (B). After SDS-PAGE, proteins were detected by staining with silver nitrate (for details see “Materials and Methods”). NPC refers to the no probe controls. Black arrowheads indicate the excised zones.
Figure 4
Figure 4
PLCP activities at pH 5.5 during cotyledon senescence in two genotypes of oilseed rape supplied with high (HN) or low (LN) nitrate for five days. Soluble proteins were extracted from Ténor or Samouraï cotyledons after 0 and five days of HN (3.75 mM NO3) or LN (0.375 mM NO3) treatment. Labeling of protease activity was carried out with MV201 (specific fluorescent probe for PLCPs, pH 5.5, 4 h labeling). After SDS-PAGE, the fluorescence was detected with a scanner (A) Ténor; (B) Samouraï. As a control, Mix refers to a mix of 0 and five day extracts (HN and LN) in the presence of MV201, NPC (no probe control) refers to the same mix in the absence of MV201, and E-64 refers to the same mix in the presence of MV201 and E-64 (specific inhibitor of PLCPs). The total amount of input proteins after incubation is represented by protein gels stained with Coomassie brilliant blue (cbb). Positions of active proteases are represented by black arrowheads. The white square and arrowhead indicate new activity during senescence in Ténor but not in Samouraï. PLCP global activity as characterized by fluorescence intensity was calculated with Image J® (C) software. The gels are representative of three biological replicates. Gel stained with Coomassie brilliant blue (cbb) shows the RuBisCO large sub-unit (RBCL) and the decline of this major protein indicated that proteolysis and N remobilization are induced. Vertical bars indicate ± SD of the mean (n = 3). Different letters indicate statistical differences (p < 0.05, ANOVA, Newman-Keuls test).
Figure 5
Figure 5
Chlorophyll (A) and soluble protein (B) contents of cotyledons in oilseed rape (cv. Ténor) at 0 or three days after water, SA, or ABA infiltrations. Plants (15 days old) were infiltrated with 1 mL of water, salicylic acid (SA, 500 μM), or abscisic acid (ABA, 50 μM). The infiltration was performed on the abaxial surface of the cotyledons with a needleless syringe by simple pressure. Vertical bars indicate ± SD of the mean (n = 3). Different letters indicate statistical differences (p < 0.05, ANOVA, Newman-Keuls test).
Figure 6
Figure 6
PLCP and SH activities of cotyledons of oilseed rape (cv. Ténor) at 0 or three days after water, SA, or ABA infiltrations. Plants (15 days old) were infiltrated with 1 mL of water, salicylic acid (SA, 500 μM), or abscisic acid (ABA, 50 μM). Soluble proteins of cotyledons were extracted from Ténor plants after 0 and three days of infiltration treatments. Labeling of protease activity was carried out with MV201 or FP-Rh (specific fluorescent probes of PLCPs and SHs respectively, pH 5.5, 4 h labeling for MV201 and pH 7.5; 1 h labeling for FP-Rh). After SDS-PAGE, the fluorescence was detected with a scanner ((A) PLCPs; (C) SHs). As a control, Mix refers to a mix of 0 and five day extracts (HN and LN) in the presence of MV201 or FP-Rh. NPC (no probe control) refers to the same mix in the absence of MV201 or FP-Rh. In addition, competition experimentation was performed with the same mix in the presence of E-64 or DFP, corresponding to specific inhibitors of PLCPs or SHs, respectively. Positions of active proteases are represented by black arrowheads. PLCP or SH global activity as characterized by the fluorescence intensity was calculated with Image J® software (B) PLCPs; (D) SHs). The presented gels are representative of three biological replicates. Gel stained with Coomassie brilliant blue (cbb) shows the RuBisCO large sub-unit (RBCL) and the decline of this major protein indicated that proteolysis and N remobilization are induced. Vertical bars indicate ± SD of the mean (n = 3). Different letters indicate statistical differences (p < 0.05, ANOVA, Newman-Keuls test).

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References

    1. Carré P., Pouzet A. Rapeseed market, worldwide and in Europe. Oilseeds Fats Crop. Lipids. 2014;21:1–12. doi: 10.1051/ocl/2013054. - DOI
    1. Rathke G.W., Christen O., Diepenbrock W. Effects of nitrogen source and rate on productivity and quality of winter oilseed rape (Brassica napus L.) grown in different crop rotations. Field Crops Res. 2005;94:103–113. doi: 10.1016/j.fcr.2004.11.010. - DOI
    1. Schjoerring J.K., Bock J.G.H., Gammelvind L., Jensen C.R., Mogensen V.O. Nitrogen incorporation and remobilization in different shoot components of field-grown winter oilseed rape (Brassica napus L.) as affected by rate of nitrogen application and irrigation. Plant Soil. 1995;177:255–264. doi: 10.1007/BF00010132. - DOI
    1. Malagoli P., Laîné P., Rossato L., Ourry A. Dynamics of nitrogen uptake and mobilization in field-grown winter oilseed rape (Brassica napus) from stem extension to harvest II. An 15N-labelling-based simulation model of N partitioning between vegetative and reproductive tissues. Ann. Bot. 2005;95:1187–1198. doi: 10.1093/aob/mci131. - DOI - PMC - PubMed
    1. Malagoli P., Laîné P., Rossato L., Ourry A. Dynamics of nitrogen uptake and mobilization in field-grown winter oilseed rape (Brassica napus) from stem extension to harvest: I. Global N flows between vegetative and reproductive tissues in relation to leaf fall and their residual N. Ann. Bot. 2005;95:853–861. doi: 10.1093/aob/mci091. - DOI - PMC - PubMed
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