3.1.1. Physiological Modifications during Cotyledon Senescence
In order to characterize physiological changes associated with the senescence of cotyledons, 20 day old plants of two genotypes of oilseed rape (cv. Ténor and Samouraï) were subjected to ample (HN: 3.75 mM NO
3−) or low nitrogen supply (LN: 0.375 mM NO
3−) for five days. As expected, the results showed that chlorophyll content decreased during the senescence of cotyledons after five days, regardless of the genotype (
Figure 1A,B). In cotyledons subjected to nitrate limitation, the chlorophyll content strongly declined (on average −70%) compared to only −25% in cotyledons supplied with HN solution (
Figure 1B). During leaf senescence, it has been demonstrated that the decrease in chlorophyll content is greater in Ténor than in Samouraï [
8,
9]. In contrast to the leaves, during cotyledon senescence under N limitation, there was no difference in the decline of chlorophyll levels between Ténor and Samouraï (
Figure 1B).
The soluble protein contents decreased significantly during cotyledon senescence after five days, particularly under LN treatment (−80%) compared with HN treatment (−46%) (
Figure 1C). Nevertheless, this decrease was not significantly different between Ténor and Samouraï. These observations confirm those previously described in cotyledons [
41] or leaves [
8,
9,
42] in oilseed rape for total proteins during senescence. However, unlike these observations in cotyledons, it was recently reported that the rate of proteolysis during leaf senescence induced by N limitation was stronger for Ténor than Samouraï [
8,
9]. Based on these results, it was confirmed that the senescence of cotyledons was accelerated by an N limitation similar to leaf senescence in oilseed rape [
41,
42]. However, even though Ténor was characterized by a higher N remobilization efficiency than Samouraï during the sequential leaf senescence associated with N limitation [
8,
9], there was no physiological manifestation of this genotypic difference during cotyledon senescence under our experimental conditions (i.e., after five days of N treatment). This could be due to the fact that the senescence of cotyledons in our experiment was particularly rapid, especially in response to N limitation treatment (
Figure 1). This means that it is necessary to have several observations between Day 2 and Day 5 to see if the changes in the chlorophyll content and rates of proteolysis associated with the contrasted N remobilization efficiency between genotypes during leaf senescence were already present in Ténor compared with Samouraï during the senescence of cotyledons.
3.1.2. Modifications of Protease Activities during the Senescence of Cotyledons
Standard protease activity profiling of SHs (using FP-Rh, a specific fluorescent probe of serine hydrolases) and CPs (using MV201, a specific fluorescent probe of papain-like cysteine proteases, PLCPs) was performed on soluble protein extracts from cotyledons after 0 and five days of LN and HN treatments.
Using the labeling of SHs with FP-Rh, it was observed that the total specific activity of SHs was significantly increased between 0 and five days, particularly under the LN condition for both genotypes (2.5 fold for Ténor and 3.7 fold for Samouraï;
Figure 2B). Indeed, activities at ~70, ~40, ~35, ~30, and ~25 kDa increased during senescence between D0 and D5 under LN conditions in Ténor and Samouraï cotyledons (
Figure 2A). Activities of SHs were higher in Ténor than Samouraï cotyledons after five days of HN treatment. Finally, the increase in SH activities was significantly correlated with the decrease in soluble proteins shown in
Figure 1C (r = −0.899;
p-value < 0.0001). These results were consistent with previous reports showing that a gene encoding a serine carboxypeptidase was up-regulated gradually from the green to yellow cotyledons of cucumber [
43]. In addition, during the leaf senescence of oilseed rape, the global activity of serine proteases is increased, particularly in response to N limitation [
9].
To identify labeled SHs (
Figure 2), activity-dependent labeling with FP-biotin followed by a pull-down of biotinylated proteins was performed on Ténor cotyledons after five days of LN treatment (
Figure 3,
Table 1 and
Table S1). The labeling allowed the detection of 12 bands of active SHs between 50 and 20 kDa (
Figure 3) and the bands were identified by LC-MS/MS (
Table 1;
Table S1). We identified 14 serine proteases corresponding to 12 carboxypeptidases (S10), one subtilisin-like protease (S8), and one Deg-protease. Furthermore, other SHs were identified, corresponding to four lipases, four carboxyl-esterases, four methyl-esterases, and two thiol-esterases (
Table S1). Interestingly, among the 14 identified serine proteases, 10 were also identified as active proteases during the sequential leaf senescence of oilseed rape [
29]. As observed for leaf senescence, the activity of SHs was also increased during the senescence of cotyledons associated with LN treatment, regardless of the genotype. Moreover, many activities of serine proteases implicated in the leaf senescence of Ténor were also found during cotyledon senescence. Based on these results, this type of senescence could be a promising model to study proteases associated with leaf senescence. However, the genotypic differences observed between Ténor and Samouraï during leaf senescence were not observed during the senescence of cotyledons. That is why SP activities did not seem to be the best target for distinguishing between genotypes at the cotyledon stage.
Using the labeling of CPs with MV201, it was observed that the total specific PLCP activity was significantly increased between 0 and five days, especially under the LN condition for both genotypes (2.6 fold for Ténor and 2.4 fold for Samouraï;
Figure 4B). Indeed, activities at ~40 and ~30 kDa increased during senescence between D0 and D5, while a new activity appeared at ~25 kDa under LN conditions in Ténor and Samouraï cotyledons (
Figure 4A). Under LN supply, Ténor was characterized by the appearance of a new proteolytic activity at ~35 kDa not present in Samouraï (
Figure 4A). The increase in PLCP activities was significantly correlated with the decrease in soluble proteins observed in
Figure 1C (r = −0.827;
p-value < 0.0001). It was already shown that several CPs were expressed in cotyledons. Indeed, genes encoding CPs were up-regulated during the development of cotyledons of soybean and common bean [
44,
45]. Further, the expression of γ and αVPEs (vacuolar processing enzymes) has been shown 17 days after germination in
Arabidopsis thaliana cotyledons [
46], while in upland cotton, cysteine proteases accumulate to high levels only in the yellowing cotyledons [
47]. In leaves of oilseed rape, many PLCP activities increased during senescence, especially in response to nitrate limitation [
29]. Additionally, a recent study showed that Ténor was characterized by higher total PLCP activity than Samouraï, and this was related to the appearance of new cysteine protease activities (RD21-like, RD19-like, SAG12-like, cathepsin-B, XBCP3-like, and aleurain-like proteases) [
35]. Interestingly, as in senescent leaves, senescing cotyledons of Ténor presented additional CP activity compared with Samouraï.
In order to identify labeled CPs (
Figure 4), an activity-dependent labeling with DCG04 followed by a pull-down of biotinylated proteins was performed on cotyledons of Ténor after five days of LN treatment (
Figure 3,
Table 1 and
Table S2). The DCG04-labeling allowed the detection of four bands of active PLCPs between 37 and 25 kDa (
Figure 3). Five active PLCPs were identified by LC-MS/MS (
Table 1 and
Table S2) and corresponded to four RD21-like proteases and one SAG12-like protease according to the classification of Richau et al. (2012) [
37] (
Table 1 and
Table S2). All of these active proteases were also identified during leaf senescence under LN treatment in oilseed rape [
29].
Moreover, activities at ~35 kDa in Ténor compared with Samouraï were associated with three different RD21-like proteases (BnaA06g36920D [
Brassica napus]/A0A078G7A3; BnaA10g05390D [
Brassica napus]/A0A078EXH0; BnaA08g04080D [
Brassica napus]/A0A078FVG4). Interestingly, two of these (BnaA10g05390D [
Brassica napus]/A0A078EXH0; BnaA08g04080D [
Brassica napus]/A0A078FVG4) were also identified during the sequential leaf senescence of oilseed rape induced with N limitation in Ténor but not in Samouraï [
35]. However, the induction of specific CP activity was observed in senescing cotyledons of Ténor in contrast to Samouraï. Interestingly, the same induced-active proteases were characteristic of Ténor during both cotyledon and leaf senescence and were associated with the higher N remobilization efficiency of Ténor than Samouraï during sequential leaf senescence.
These results showed that, even if there is no physiological difference between genotypes during the senescence of cotyledons, the PLCPs associated with the high leaf N remobilization efficiency in Ténor are found at an early stage of development. Unlike SPs, the study of CP activities at the cotyledon stage seems to be a promising way to distinguish between genotypes with contrasting proteolysis machinery and N remobilization efficiency. Furthermore, to confirm these results, it will be necessary to undertake a large-scale screening of CP activities during cotyledon senescence in many genotypes characterized by contrasting N remobilization efficiency at the leaf level.