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The contrasting N management of two oilseed rape genotypes reveals the mechanisms of proteolysis associated with leaf N remobilization and the respective contributions of leaves and stems to N storage and remobilization during seed filling.

Girondé A, Etienne P, Trouverie J, Bouchereau A, Le Cahérec F, Leport L, Orsel M, Niogret MF, Nesi N, Carole D, Soulay F, Masclaux-Daubresse C, Avice JC - BMC Plant Biol. (2015)

Bottom Line: Oilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds.Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling.The results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Oilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds. This weak N use efficiency is associated with a low foliar N remobilization, leading to a significant return of N to the soil and a risk of pollution. Contrary to what is observed during senescence in the vegetative stages, N remobilization from stems and leaves is considered efficient during monocarpic senescence. However, the contribution of stems towards N management and the cellular mechanisms involved in foliar remobilization remain largely unknown. To reach this goal, the N fluxes at the whole plant level from bolting to mature seeds and the processes involved in leaf N remobilization and proteolysis were investigated in two contrasting genotypes (Aviso and Oase) cultivated under ample or restricted nitrate supply.

Results: During seed filling in both N conditions, Oase efficiently allocated the N from uptake to seeds while Aviso favoured a better N remobilization from stems and leaves towards seeds. Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling. Under N limitation, Aviso had a better N remobilization from leaves to stems before the onset of seed filling. Afterwards, the higher N remobilization from stems and leaves of Aviso led to a higher final N amount in seeds. This high leaf N remobilization is associated with a better degradation/export of insoluble proteins, oligopeptides, nitrate and/or ammonia. By using an original method based on the determination of Rubisco degradation in the presence of inhibitors of proteases, efficient proteolysis associated with cysteine proteases and proteasome activities was identified as the mechanism of N remobilization.

Conclusion: The results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms. In addition, the stem may act as transient storage organs in the case of an asynchronism between leaf N remobilization and N demand for seed filling.

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Rubisco large subunit degradation in a source leaf with or without protease inhibitors (A) and the inhibition of the protease activities by protease inhibitors (B). The Rubisco large subunit (LSU) in the soluble protein extract (PE) of the source leaf (7 days after bolting) is visualized on stain free SDS-PAGE and quantified for the four biological repetitions by Image Lab software (Bio-Rad) at (t0) and after 1 h of incubation at 37°C (t1h) without inhibitors (PE, control conditions) or with specific protease inhibitors: iodoacetamide (PE + CPI; cystein protease inhibitor), aprotinin (PE + SPI; serine protease inhibitor), methanol (PE + Me), methanol and 1–10 phenanthroline (PE + Me + MI; metalloprotease inhibitor), methanol and pepstatin A (PE + Me + API; aspartic protease inhibitor), DMSO (PE + DMSO) or DMSO and MG132 (PE + DMSO + PI; proteasome inhibitor). The most representative biological repetition is shown in panel A and the percentage of degradation (mean value ± SE, n = 4 plants) are indicated below. Panel B presents the inhibition of the protease activities by the proteases inhibitors (expressed as % of LSU degradation observed in control conditions (PE)). In panel B, data are indicated as the mean value ± SE. An asterisks means that the LSU degradation is significantly different between N treatment and # means a significant differences between genotypes (n = 4 plants; * or #= p < 0.05).
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Fig9: Rubisco large subunit degradation in a source leaf with or without protease inhibitors (A) and the inhibition of the protease activities by protease inhibitors (B). The Rubisco large subunit (LSU) in the soluble protein extract (PE) of the source leaf (7 days after bolting) is visualized on stain free SDS-PAGE and quantified for the four biological repetitions by Image Lab software (Bio-Rad) at (t0) and after 1 h of incubation at 37°C (t1h) without inhibitors (PE, control conditions) or with specific protease inhibitors: iodoacetamide (PE + CPI; cystein protease inhibitor), aprotinin (PE + SPI; serine protease inhibitor), methanol (PE + Me), methanol and 1–10 phenanthroline (PE + Me + MI; metalloprotease inhibitor), methanol and pepstatin A (PE + Me + API; aspartic protease inhibitor), DMSO (PE + DMSO) or DMSO and MG132 (PE + DMSO + PI; proteasome inhibitor). The most representative biological repetition is shown in panel A and the percentage of degradation (mean value ± SE, n = 4 plants) are indicated below. Panel B presents the inhibition of the protease activities by the proteases inhibitors (expressed as % of LSU degradation observed in control conditions (PE)). In panel B, data are indicated as the mean value ± SE. An asterisks means that the LSU degradation is significantly different between N treatment and # means a significant differences between genotypes (n = 4 plants; * or #= p < 0.05).

Mentions: Different protease inhibitors (against cysteine, serine, aspartic or metallo- proteases and proteasome) were used in order to identify the class of proteases involved in the strong degradation of soluble proteins occurring in the source leaf at D7 (Figure 7C). Due to the fact that Rubisco represents a large proportion of the soluble proteins in leaves [26], the characterization of proteases was determined via the analysis of the degradation of the Rubisco large subunit (LSU, Figure 9A). In source leaf of HN plants (Figure 9B), the LSU proteolysis was strongly inhibited by iodoacetamide for Aviso and Oase (35.5 and 31.3% of inhibition, respectively) and by MG132 for Oase (46.22% of inhibition), suggesting that the proteolysis is mainly carried out by cysteine proteases for Aviso and by the proteasome and cysteine proteases for Oase. The contribution of cysteine and aspartic proteases to the LSU degradation in Aviso was the same in both N conditions, while a slight increase of serine proteases (from 6.1 to 12.1% of inhibition; p = 0.12) and metalloproteases (from 7.9 to 14.1% of inhibition; p = 0.17) and a significant increase of proteasome activity (60% of inhibition) were observed (Figure 9B) in response to the LN treatment. The contribution of proteasome, cysteine and serine proteases for Oase remained similar in both N conditions. Compared with HN plants, the contribution of aspartic proteases decreased (4% of inhibition) while the participation of metalloproteases increased (27% of inhibition) in Oase LN plants (Figure 9B).Figure 9


The contrasting N management of two oilseed rape genotypes reveals the mechanisms of proteolysis associated with leaf N remobilization and the respective contributions of leaves and stems to N storage and remobilization during seed filling.

Girondé A, Etienne P, Trouverie J, Bouchereau A, Le Cahérec F, Leport L, Orsel M, Niogret MF, Nesi N, Carole D, Soulay F, Masclaux-Daubresse C, Avice JC - BMC Plant Biol. (2015)

Rubisco large subunit degradation in a source leaf with or without protease inhibitors (A) and the inhibition of the protease activities by protease inhibitors (B). The Rubisco large subunit (LSU) in the soluble protein extract (PE) of the source leaf (7 days after bolting) is visualized on stain free SDS-PAGE and quantified for the four biological repetitions by Image Lab software (Bio-Rad) at (t0) and after 1 h of incubation at 37°C (t1h) without inhibitors (PE, control conditions) or with specific protease inhibitors: iodoacetamide (PE + CPI; cystein protease inhibitor), aprotinin (PE + SPI; serine protease inhibitor), methanol (PE + Me), methanol and 1–10 phenanthroline (PE + Me + MI; metalloprotease inhibitor), methanol and pepstatin A (PE + Me + API; aspartic protease inhibitor), DMSO (PE + DMSO) or DMSO and MG132 (PE + DMSO + PI; proteasome inhibitor). The most representative biological repetition is shown in panel A and the percentage of degradation (mean value ± SE, n = 4 plants) are indicated below. Panel B presents the inhibition of the protease activities by the proteases inhibitors (expressed as % of LSU degradation observed in control conditions (PE)). In panel B, data are indicated as the mean value ± SE. An asterisks means that the LSU degradation is significantly different between N treatment and # means a significant differences between genotypes (n = 4 plants; * or #= p < 0.05).
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Related In: Results  -  Collection

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Fig9: Rubisco large subunit degradation in a source leaf with or without protease inhibitors (A) and the inhibition of the protease activities by protease inhibitors (B). The Rubisco large subunit (LSU) in the soluble protein extract (PE) of the source leaf (7 days after bolting) is visualized on stain free SDS-PAGE and quantified for the four biological repetitions by Image Lab software (Bio-Rad) at (t0) and after 1 h of incubation at 37°C (t1h) without inhibitors (PE, control conditions) or with specific protease inhibitors: iodoacetamide (PE + CPI; cystein protease inhibitor), aprotinin (PE + SPI; serine protease inhibitor), methanol (PE + Me), methanol and 1–10 phenanthroline (PE + Me + MI; metalloprotease inhibitor), methanol and pepstatin A (PE + Me + API; aspartic protease inhibitor), DMSO (PE + DMSO) or DMSO and MG132 (PE + DMSO + PI; proteasome inhibitor). The most representative biological repetition is shown in panel A and the percentage of degradation (mean value ± SE, n = 4 plants) are indicated below. Panel B presents the inhibition of the protease activities by the proteases inhibitors (expressed as % of LSU degradation observed in control conditions (PE)). In panel B, data are indicated as the mean value ± SE. An asterisks means that the LSU degradation is significantly different between N treatment and # means a significant differences between genotypes (n = 4 plants; * or #= p < 0.05).
Mentions: Different protease inhibitors (against cysteine, serine, aspartic or metallo- proteases and proteasome) were used in order to identify the class of proteases involved in the strong degradation of soluble proteins occurring in the source leaf at D7 (Figure 7C). Due to the fact that Rubisco represents a large proportion of the soluble proteins in leaves [26], the characterization of proteases was determined via the analysis of the degradation of the Rubisco large subunit (LSU, Figure 9A). In source leaf of HN plants (Figure 9B), the LSU proteolysis was strongly inhibited by iodoacetamide for Aviso and Oase (35.5 and 31.3% of inhibition, respectively) and by MG132 for Oase (46.22% of inhibition), suggesting that the proteolysis is mainly carried out by cysteine proteases for Aviso and by the proteasome and cysteine proteases for Oase. The contribution of cysteine and aspartic proteases to the LSU degradation in Aviso was the same in both N conditions, while a slight increase of serine proteases (from 6.1 to 12.1% of inhibition; p = 0.12) and metalloproteases (from 7.9 to 14.1% of inhibition; p = 0.17) and a significant increase of proteasome activity (60% of inhibition) were observed (Figure 9B) in response to the LN treatment. The contribution of proteasome, cysteine and serine proteases for Oase remained similar in both N conditions. Compared with HN plants, the contribution of aspartic proteases decreased (4% of inhibition) while the participation of metalloproteases increased (27% of inhibition) in Oase LN plants (Figure 9B).Figure 9

Bottom Line: Oilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds.Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling.The results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Oilseed rape is the third largest oleaginous crop in the world but requires high levels of N fertilizer of which only 50% is recovered in seeds. This weak N use efficiency is associated with a low foliar N remobilization, leading to a significant return of N to the soil and a risk of pollution. Contrary to what is observed during senescence in the vegetative stages, N remobilization from stems and leaves is considered efficient during monocarpic senescence. However, the contribution of stems towards N management and the cellular mechanisms involved in foliar remobilization remain largely unknown. To reach this goal, the N fluxes at the whole plant level from bolting to mature seeds and the processes involved in leaf N remobilization and proteolysis were investigated in two contrasting genotypes (Aviso and Oase) cultivated under ample or restricted nitrate supply.

Results: During seed filling in both N conditions, Oase efficiently allocated the N from uptake to seeds while Aviso favoured a better N remobilization from stems and leaves towards seeds. Nitrate restriction decreased seed yield and oil quality for both genotypes but Aviso had the best seed N filling. Under N limitation, Aviso had a better N remobilization from leaves to stems before the onset of seed filling. Afterwards, the higher N remobilization from stems and leaves of Aviso led to a higher final N amount in seeds. This high leaf N remobilization is associated with a better degradation/export of insoluble proteins, oligopeptides, nitrate and/or ammonia. By using an original method based on the determination of Rubisco degradation in the presence of inhibitors of proteases, efficient proteolysis associated with cysteine proteases and proteasome activities was identified as the mechanism of N remobilization.

Conclusion: The results confirm the importance of foliar N remobilization after bolting to satisfy seed filling and highlight that an efficient proteolysis is mainly associated with (i) cysteine proteases and proteasome activities and (ii) a fine coordination between proteolysis and export mechanisms. In addition, the stem may act as transient storage organs in the case of an asynchronism between leaf N remobilization and N demand for seed filling.

Show MeSH
Related in: MedlinePlus