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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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Changes in glutamate dehydrogenase activity, glutamine synthetase activity and their amounts (GS1, GS2) in a source leaf. Plants were supplied with ample (HN, 3.75 mM) or low (LN, 0.375 mM) nitrate concentrations. These data were obtained from a selected ‘source leaf’, determined as mature at D0 (early bolting) and becoming senescent during the experiment. The activity of glutamate dehydrogenase (GDH; A) was quantified as the synthesis of glutamate and is expressed as nmol of NADH used.h−1.μg−1 proteins. The activity of glutamine synthetase (GS; B) was determined by the nmol of glutamine produced.h−1.μg−1 proteins. The GS1 and GS2 amounts (C) were quantified after western blotting with specific antibodies and the percentage of GS1 among the total GS amount was estimated. Data were observed at 0, 7 (early bolting), 14 (flower buds raised above the youngest leaves), 21 (first petals visible, but flower buds still closed) and 28 (flowering) days after the beginning of bolting (D0). Only one biological replicate remained at D28, and its value is indicated by a cross (x). In panels A and B, data are indicated as the mean value ± SE (vertical bars). Letters a, b and c represent differences in kinetics, asterisks mean significant differences between treatment and hashes represent significant differences between genotypes (n = 4 plants; p < 0.05).
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Fig8: Changes in glutamate dehydrogenase activity, glutamine synthetase activity and their amounts (GS1, GS2) in a source leaf. Plants were supplied with ample (HN, 3.75 mM) or low (LN, 0.375 mM) nitrate concentrations. These data were obtained from a selected ‘source leaf’, determined as mature at D0 (early bolting) and becoming senescent during the experiment. The activity of glutamate dehydrogenase (GDH; A) was quantified as the synthesis of glutamate and is expressed as nmol of NADH used.h−1.μg−1 proteins. The activity of glutamine synthetase (GS; B) was determined by the nmol of glutamine produced.h−1.μg−1 proteins. The GS1 and GS2 amounts (C) were quantified after western blotting with specific antibodies and the percentage of GS1 among the total GS amount was estimated. Data were observed at 0, 7 (early bolting), 14 (flower buds raised above the youngest leaves), 21 (first petals visible, but flower buds still closed) and 28 (flowering) days after the beginning of bolting (D0). Only one biological replicate remained at D28, and its value is indicated by a cross (x). In panels A and B, data are indicated as the mean value ± SE (vertical bars). Letters a, b and c represent differences in kinetics, asterisks mean significant differences between treatment and hashes represent significant differences between genotypes (n = 4 plants; p < 0.05).

Mentions: The activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS), involved in the metabolism and remobilization of amino acids during senescence, were similar and constant in both N conditions for Aviso, except for a 50% decrease in GS activity at D28 in both N conditions and a putative 6-fold increase (n = 1) of GDH activity at D28 in LN conditions (Figure 8A and B). Concerning Oase, the GDH activity remained low until D21 and increased by 1.5-fold at D28 in both N conditions (Figure 8A). In HN plants, the GS activity remained constant until D21 and decreased by 43% at D28 (n = 1). In LN plants, the GS activity increased (1.5-fold) at D21 and putatively decreased at D28 (n = 1, Figure 8B). The immunoblots of cytosolic (GS1) and chloroplastic (GS2) glutamine synthetase (Figure 8C) revealed that the proportion of GS1 was not impacted by LN treatment in both genotypes, but it remained higher for Oase throughout the experiment (53% for Oase and 39% for Aviso on average).Figure 8


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)

Changes in glutamate dehydrogenase activity, glutamine synthetase activity and their amounts (GS1, GS2) in a source leaf. Plants were supplied with ample (HN, 3.75 mM) or low (LN, 0.375 mM) nitrate concentrations. These data were obtained from a selected ‘source leaf’, determined as mature at D0 (early bolting) and becoming senescent during the experiment. The activity of glutamate dehydrogenase (GDH; A) was quantified as the synthesis of glutamate and is expressed as nmol of NADH used.h−1.μg−1 proteins. The activity of glutamine synthetase (GS; B) was determined by the nmol of glutamine produced.h−1.μg−1 proteins. The GS1 and GS2 amounts (C) were quantified after western blotting with specific antibodies and the percentage of GS1 among the total GS amount was estimated. Data were observed at 0, 7 (early bolting), 14 (flower buds raised above the youngest leaves), 21 (first petals visible, but flower buds still closed) and 28 (flowering) days after the beginning of bolting (D0). Only one biological replicate remained at D28, and its value is indicated by a cross (x). In panels A and B, data are indicated as the mean value ± SE (vertical bars). Letters a, b and c represent differences in kinetics, asterisks mean significant differences between treatment and hashes represent significant differences between genotypes (n = 4 plants; p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig8: Changes in glutamate dehydrogenase activity, glutamine synthetase activity and their amounts (GS1, GS2) in a source leaf. Plants were supplied with ample (HN, 3.75 mM) or low (LN, 0.375 mM) nitrate concentrations. These data were obtained from a selected ‘source leaf’, determined as mature at D0 (early bolting) and becoming senescent during the experiment. The activity of glutamate dehydrogenase (GDH; A) was quantified as the synthesis of glutamate and is expressed as nmol of NADH used.h−1.μg−1 proteins. The activity of glutamine synthetase (GS; B) was determined by the nmol of glutamine produced.h−1.μg−1 proteins. The GS1 and GS2 amounts (C) were quantified after western blotting with specific antibodies and the percentage of GS1 among the total GS amount was estimated. Data were observed at 0, 7 (early bolting), 14 (flower buds raised above the youngest leaves), 21 (first petals visible, but flower buds still closed) and 28 (flowering) days after the beginning of bolting (D0). Only one biological replicate remained at D28, and its value is indicated by a cross (x). In panels A and B, data are indicated as the mean value ± SE (vertical bars). Letters a, b and c represent differences in kinetics, asterisks mean significant differences between treatment and hashes represent significant differences between genotypes (n = 4 plants; p < 0.05).
Mentions: The activities of glutamate dehydrogenase (GDH) and glutamine synthetase (GS), involved in the metabolism and remobilization of amino acids during senescence, were similar and constant in both N conditions for Aviso, except for a 50% decrease in GS activity at D28 in both N conditions and a putative 6-fold increase (n = 1) of GDH activity at D28 in LN conditions (Figure 8A and B). Concerning Oase, the GDH activity remained low until D21 and increased by 1.5-fold at D28 in both N conditions (Figure 8A). In HN plants, the GS activity remained constant until D21 and decreased by 43% at D28 (n = 1). In LN plants, the GS activity increased (1.5-fold) at D21 and putatively decreased at D28 (n = 1, Figure 8B). The immunoblots of cytosolic (GS1) and chloroplastic (GS2) glutamine synthetase (Figure 8C) revealed that the proportion of GS1 was not impacted by LN treatment in both genotypes, but it remained higher for Oase throughout the experiment (53% for Oase and 39% for Aviso on average).Figure 8

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