BACKGROUND
over the last four decades, oilseed rape  has become the third most widely grown oleaginous crop worldwide with a  <dig> -fold increase in seed production between  <dig> and  <dig>  <cit> .
this crop represents a major renewable resource for human food , animal feed  and numerous non-food uses .
to increase the level of seed production with current genotypes, the use of n fertilizers has increased by 430% over the last forty years  <cit> .
despite the high capacity of mineral n absorption of oilseed rape  <cit> , only 50% of n from fertilizer is recovered in seeds at harvest  <cit> .
this low n use efficiency  is related to the loss of n by leaf drop , reflecting the weak n remobilization efficiency  of oilseed rape.
therefore, in a context of imposed limitations on n-fertilizer inputs, improving nue is becoming a priority in order to maintain/increase seed yield and decrease  the risk of water pollution by nitrate,  the emission of greenhouse gases contributing to global warming, and  the economic costs of oilseed rape crops.
nue can be represented as two main components: the n uptake efficiency  and the n utilization efficiency , itself subdivided into two other components, n assimilation efficiency  and n remobilization efficiency   <cit> .
even if n uptake of winter oilseed rape is considered to be efficient at vegetative stages  <cit> , an n uptake that remains significant until flowering is associated with genotypes having high seed yield  <cit> .
a mineral n input during seed filling increases seed yield of spring oilseed rape  and recent studies on winter oilseed rape  <cit>  have reported that a n uptake during reproductive stages appears to be a determinant trait for seed yield of winter oilseed rape, especially in restricted n supply.
nevertheless, in field conditions, the mineral n availability highly fluctuates during reproductive stages due to environmental factors, such as water deficit in the soil.
to obtain a high nue, the n coming from uptake has to be well managed by the plant.
consequently, an improvement of nute is also necessary to enhance nue.
concerning the nae, transgenic approaches have been targeted to enzymes involved in n metabolism such as nitrate reductase, nitrite reductase or alanine aminotransferase  in various species such as arabidopsis and tobacco, with more or less success .
a promising result was obtained with oilseed rape plants overexpressing an alat, which need 40% less n fertilizer to reach similar yield as the wild type  <cit> .
however, a study of  <dig> spring oilseed rape genotypes has suggested that nre is a major determinant of nute  <cit>  and an efficient assimilation needs to be correlated to an enhanced n remobilization to improve the seed n filling, which is essential to improve/maintain seed yield, in a context of n input reduction.
the n remobilization associated with leaf senescence is considered as crucial for oilseed rape yield  <cit> .
during the vegetative stages, n is remobilized from the older leaves to the younger leaves along the axis of the plants via the sequential senescence, but senescent leaves can fall with a high level of residual n  leading to a significant return of n to the soil  <cit> .
by developing a modelling approach as a mean to identify potential methods for improving the nue of oilseed rape, it was estimated that a 1% decrease in the residual n in fallen leaves , resulting from an enhanced foliar n remobilization, may increase seed yield by 5-10%  <cit> .
after bolting, and especially during monocarpic senescence when the n is remobilized from vegetative organs to seeds, the low soil n availability during spring and at the beginning of summer  <cit>  makes n remobilization crucial for seed yield and seed filling.
during this period of development the main source organs are leaves, stems and pod walls  <cit> .
during reproductive stages, the n amount in source leaves is highly remobilized   <cit> , leading to a low residual n in dead leaves  and resulting in the reduction of the risk of n pollution.
these results highlight an efficient n remobilization from source organs during monocarpic senescence, but also reveal variations for the residual n amount in stems compared with leaves of four winter oilseed rape genotypes  <cit> .
in addition, a genotypic and n-supply interaction for n content in stems was found for  <dig> genotypes of spring canola  <cit>  and the genotype with the best n remobilization was able to better remobilize n from stems and pod walls to seeds  <cit> .
these results suggest that stems can also be important organ for improving seed n-filling, but the role of stems in n management at the whole plant level for winter oilseed rape remains largely unknown.
although nre seems to be a major lever for improving the nue in oilseed rape, the cellular mechanisms associated with n remobilization from senescing leaves  remain largely an enigma.
during leaf senescence, the degradation of proteins  into amino acids or peptides is performed by different classes of proteases.
studies on arabidopsis reported the predominant involvement of cysteine and serine proteases , but also a role for the proteasome in the degradation of carbonylated proteins, which are accumulated during senescence  <cit> .
the ribulose- <dig> -biphosphate carboxylase/oxygenase  can represent up to 65% of the soluble proteins in c <dig> plants  <cit>  and 20–30% of total leaf n  <cit> .
consequently, rubisco is the major source of n for remobilization and its budget is very relevant for the plant.
in winter oilseed rape, cysteine, aspartic, and metallo-proteases as well as the 26s proteasome are supposed to play an important role in foliar n remobilization during senescence in the vegetative stages  <cit>  but there is no evidence concerning the proteolysis activities that are involved in leaf senescence and degradation of rubisco after the bolting stage.
among the enzymes involved in the conversion of amino acids into transportable forms, a recent study highlighted the importance of asparagine synthetase  in n remobilization during senescence in arabidopsis  <cit> .
in addition, the glutamine synthetase  catalyses the assimilation of ammonium into glutamate to synthesize glutamine.
glutamine was found at high levels in phloem sap of oilseed rape  and consequently, gss are supposed to be largely involved in n remobilization processes during senescence in winter wheat  <cit>  and maize  <cit> , especially the cytosolic form  in the case of oilseed rape  <cit> .
in parallel, glutamate dehydrogenase  may catalyse a glutamate deamination  <cit>  which provides ammonium for the gs activity in senescing leaves.
in addition, in case of high levels of ammonium, gdh can incorporate ammonium on α-ketoglutarate to produce glutamate  <cit> .
a significant quantity of glutamate has been found in phloem sap of oilseed rape  <cit>  and an increase of both gdh activities in sliced leaves of oilseed rape was associated with a decrease in soluble proteins and ammonium  <cit> , suggesting an important role for gdh in foliar n remobilization.
the phloem loading of the resulting amino acids is regarded as not limiting in oilseed rape at the vegetative stages  <cit> .
the efficient export of amino acids and the involvement of gs <dig> and gdh in n remobilization need to be confirmed in leaves as they senesce after bolting because they are proposed to have an efficient n remobilization.
the aim of this study was to identify the physiological traits involved in the high nue and n remobilization in restricted n supply conditions, at whole plant  and cellular levels .
to reach this goal, two genotypes , known to have different strategies following a nitrate limitation at the vegetative stage  <cit> , were selected.
long term pulse-chase 15 n-labelling was performed to precisely define the endogenous and exogenous n fluxes at the whole plant level between the bolting and mature seed stages, under restricted and ample nitrate supply.
a specific 15 n-labelling was also used in order to determine a relevant estimation of nue and its different components .
this study  highlights the role of leaves and stems in the remobilization of n towards seeds and  allows the identification of the physiological traits associated with a high nue in response to restricted n supply.
moreover, a study of foliar n remobilization in source leaves was performed through the analysis of foliar n compounds, the gs and gdh activities, and the development of a new method to study proteolytic activities, using endogenous rubisco as a substrate, in order to determine the involvement of different classes of proteases in the n remobilization which occurs during this growth stage.
RESULTS
impact of n limitation on growth, yield components, seed and oil composition
when plants were well supplied with nitrate , the total dm  was higher for oase  compared with aviso  during seed filling , mainly due to differences in the stem dm .
under our experimental conditions, the occurrence of the different stages of development was not significantly different between both genotypes or between both n treatments for a given genotype.
in low nitrate  conditions, no difference in the total dm was observed between genotypes .
as expected, compared with hn, dm was reduced in leaves from d <dig>  in stems at d <dig> and in pod walls at d <dig>  in addition, dm declined significantly in siliques at d <dig> and increased in stems at d <dig> for aviso while dm of siliques and stems declined at d <dig> for oase.
the root dm at d <dig> was lower in aviso under ln compared with hn.
under ln supply, a similar decrease in seed production was observed for both genotypes .figure  <dig>
kinetics of dry matter  of aviso and oase under ample or low n supply.
the plants were supplied with ample  or low  concentrations of nitrate.
dry matter is expressed in g per plant for aviso  and oase  at  <dig> ,  <dig> ,  <dig>  and  <dig>  days after the beginning of bolting .
seeds and pod walls were grouped and called siliques at d <dig> and they were separated from d <dig> onwards.
data are indicated as the mean value ± standard error .
different letters  indicate that the total dry matter is significantly different between two dates.
hashes represent significant differences between genotypes in hn or ln conditions and the asterisks represent significant differences between n treatments .
in hn plants, the yield components ; table 1) as well as the seed composition ; table 1) were similar for both genotypes, except for the percentage of omega- <dig>  and omega- <dig>  precursors, which were higher for aviso than oase.
however, the c18:2/c18: <dig> ratio was similar for both genotypes .
as expected, an n limitation led to a strong decrease in the n amount of seeds .
compared with hn conditions, nhi significantly increased in oase and tended to increase in aviso  in response to ln treatment .
seeds of oase ln plants had a higher c:n ratio associated with a higher oil percentage, which was to the detriment of proteins .
for oil composition, an increase of oleic acid  proportion was observed under n limitation for both genotypes .
while c18: <dig> and c18: <dig> percentages did not differ under both n conditions for aviso, an increase in c18: <dig>  and in the c18:2/c18: <dig> ratio  occurred under ln for oase.
the proportion of erucic acid also increased under ln conditions for aviso .table  <dig>
seed composition and nitrogen harvest index of aviso and oase under ample or low n supply
aviso
oase
hn
ln
hn
ln
yield components
seed composition
the plants were supplied with an ample  or low  nitrate concentration.
the n amount in seed is estimated in milligrams.
c: n ratio: ratio c: n in seeds; nhi: n harvesting index .
the seed composition  and the oil composition  was determined by nirs method.
asterisks represent significant differences between n treatments and hashes represent significant differences between the two genotypes .
effects of nitrate limitation on n amount and nue components
in hn plants, the total n amount did not differ between the genotypes .
however, the n amount in leaves and flowers at the final stage of development was significantly higher for oase than for aviso.
as expected, the total n amount was reduced for both genotypes in ln conditions , resulting from a decline in the n amount in nearly all organs from d <dig>  exceptions were flowers present at any time during the experiment, roots at d <dig> for oase, stems at d <dig> and leaves at d <dig> for aviso, which all showed a similar n amount in both n conditions.
in ln plants, the final n amount in seeds and pod walls of oase was lower than in aviso .figure  <dig>
kinetics of the n amount in aviso and oase under ample or low n supply.
the plants were supplied with ample  or low  concentrations of nitrate.
the n amount is expressed in mg per plant for aviso  and oase  at  <dig> ,  <dig> ,  <dig>  and  <dig>  days after the beginning of bolting .
seeds and pod walls were grouped and called siliques at d <dig> and they were separated from d <dig> onwards.
data are indicated as the mean value ± standard error .
different letters  indicate that the total n amount is significantly different between two dates.
hashes represent significant differences between genotypes in hn or ln conditions and the asterisks represent significant differences between n treatments .
the n use efficiency , n utilization efficiency , and n remobilization efficiency  were negatively correlated to n supply while the n uptake efficiency  was positively correlated to n supply .
the nue was higher for oase  than for aviso  in hn conditions while no differences were observed in the ln treatment .
the nute increased in response to ln supply but no differences were observed between the genotypes, whatever the n supply.
a strong genotype/treatment interaction effect was observed for nupe between d <dig> and d <dig>  in hn conditions, the nupe of oase was around 81%, suggesting that the n distributed to seeds during this period is mainly provided by the n that is newly taken up by the roots.
the nupe of oase is about 2-fold higher than aviso in both n conditions.
the global nre, i.e.
the nre calculated on the whole growing cycle , reached about 55-60% for both genotypes, indicating that a large part of the n stored in source organs at the bolting stage  is remobilized during the experiment.
when nre was calculated stepwise , a regular decrease occurred throughout the steps, whatever the treatment and the genotype.
in hn plants, nre was  <dig> -fold higher in oase than aviso between d <dig> and d <dig> while it was reduced by about 50% after d <dig> .
in response to ln supply, global nre increases for both genotypes, but a genotypic effect was highlighted between d <dig> and d <dig>  where the nre of aviso was  <dig> -fold higher than oase .
however, no genotype/treatment interaction was observed for nre.table  <dig>
nue, nute, nupe and nre of aviso and oase under ample or low n supply
genotypes
source of variation
aviso
oase
g
n
g x n
n treatment
hn
ln
hn
ln
f
g
f
n
r
f
gxn
nuea
nuteb
global
stepwise
the plants were supplied with an ample  or low  nitrate concentration.
d <dig> corresponds to the early bolting stage, d <dig> to pod formation, d <dig> to seed filling and d <dig> to mature seeds.
the main source of variation is deduced from an anova test where genotypes, n treatment, and genotype x n treatment interaction were tested.
the resulting f values are presented below.
the r values correspond to the correlation between n supply and nue, nute, nupe and nre, respectively.
a:nue  corresponds to the mg of n in seeds per mg of the n amount in the whole plant relative to the seed dm  produced per g of total dm .
b:nute  corresponds to the g of seed dm produced per mg of n in shoots .
c:nupe  is estimated as the percentage of n derived from uptake in the total n  distributed to seeds between d <dig> and d <dig> .
d:nre  is determined as the percentage of n stored in all source organs which is remobilized  between d <dig>  and final harvest  or between d <dig> and d <dig>  d <dig> and d <dig> and d <dig> and d <dig>
asterisks represent significant differences between n treatments and hashes represent significant differences between the two genotypes .
n fluxes at the whole plant level in hn conditions
the 15 n labelling method used in this study gave the opportunity to determine precisely the n fluxes at the whole plant level  between d0-d <dig>  d42-d <dig> and d70-d <dig>  due to the fact that genotype and n treatment effects were observed for nre solely between d <dig> and d <dig> in hn plants , only the n fluxes between these two growing stages are given in figure  <dig> .
in hn conditions, n remobilization was around 3-fold higher in aviso  than oase .
for both genotypes, the stem was the main source organ: 47%  and 59%  of the total n remobilized in aviso and oase, respectively.
for aviso, the other source organs were the leaves, pod walls, flowers and roots while for oase the source organs were leaves and roots.
for oase, the n amount remobilized from leaves was  <dig> -fold lower than for aviso.
the n remobilized from source organs was mainly distributed to the seeds with a significantly greater amount in aviso  than oase .
contrary to aviso, the flowers of oase are sink organs  for remobilized n. there was no significant difference in the amount of n uptake between genotypes .
however, the allocation of n towards seeds is more important for oase  than for aviso .
despite this higher allocation of n towards seeds in oase, the total n distributed to oase seeds was not the same as in aviso .figure  <dig>
n fluxes in aviso  and oase  in hn conditions between d <dig> and d <dig>  the plants were supplied with an ample concentration of nitrate .
d <dig> corresponds to the start of seed filling and d <dig> to the mature seed stage.
fluxes of n from remobilization or uptake in the different organs are expressed as mg of n remobilized or taken up, respectively.
a shaded box means that the organ was not present during these growing stages.
for fluxes of n remobilization, the n amount is indicated with a minus sign  when n is remobilized from a source organ, or it is indicated with a plus sign  when remobilized n is redistributed towards a sink organ.
data are indicated as the mean value ± standard error.
hashes represent significant differences between genotypes .
n fluxes at the whole plant level in response to nitrate limitation
between d <dig> and d <dig>  n remobilization increased for aviso in response to ln treatment  compared with hn , due to a larger n remobilization from source leaves .
this remobilized n was mainly redistributed towards siliques, allowing a similar amount of n to be redistributed as hn plants .
in contrast to aviso, oase showed a similar total amount of remobilized n in both n conditions .
however, compared with hn, the redistribution of this remobilized n to siliques was higher in oase ln plants  due to a lower loss via dead leaves and a lower redistribution to sink leaves .
the n remobilization in oase was lower than aviso  due to a lower contribution of roots and flowers.
as expected, in ln plants the n uptake strongly decreased for aviso  and was not detected for oase .
in aviso ln plants, the main sink organs for the n uptake were siliques, leaves and stems .
thanks to the n uptake in aviso, which supplemented the n remobilization, the total n amount distributed to siliques was similar in both genotypes .figure  <dig>
n fluxes in aviso  and oase  in ln conditions between d <dig> and d <dig>  the plants were supplied with a low concentration of nitrate .
d <dig> corresponds to early bolting and d <dig> to pod formation.
fluxes of n from remobilization or uptake in the different organs are expressed as mg of n remobilized or taken up, respectively.
for fluxes of n remobilization, the n amount is indicated with a minus sign  when n is remobilized from a source organ, or it is indicated with a plus sign  when remobilized n is redistributed towards a sink organ.
data are indicated as the mean value ± standard error.
asterisks represent significant differences between treatments and hashes represent significant differences between genotypes .
between d <dig> and d <dig>  no n uptake was detectable and the total remobilized n amount decreased for both genotypes under n limitation  contrary to hn plants .
this was mainly related to lower contributions of leaves and pod walls, leading to a lower n redistribution to seeds.
the n remobilization was  <dig> -fold higher for aviso than oase , mainly due to a 2-fold higher n remobilization from leaves.
nevertheless, the n redistribution to seeds was not significantly different between the genotypes .
these results can be explained by the fact that flowers and stems are sink organs for aviso contrary to oase, and a higher n loss by dead leaves occurs for aviso.figure  <dig>
n fluxes in aviso  and oase  in ln conditions between d <dig> and d <dig>  the plants were supplied with a low concentration of nitrate .
d <dig> corresponds to pod formation and d <dig> to the start of seed filling.
fluxes of n from remobilization or uptake in the different organs are expressed as mg of n remobilized or taken up, respectively.
a shaded box means that the organ was not present during these growing stages.
for fluxes of n remobilization, the n amount is indicated with a minus sign  when n is remobilized from a source organ, or it is indicated with a plus sign  when remobilized n is redistributed towards a sink organ.
data are indicated as the mean value ± standard error.
asterisks represent significant differences between treatments and hashes represent significant differences between genotypes .
between d <dig> and d <dig>  the n remobilization from all source organs was low for both genotypes , leading to a lower total n remobilization in ln compared with hn plants .
consequently, a lower n amount was redistributed to seeds .
under ln conditions, aviso had a higher amount of total remobilized n than oase  resulting in a higher redistribution of remobilized n to seeds .
this was related to a higher remobilization from stems  and source leaves  for aviso compared with oase.
the flowers were sink organs for remobilized n in oase ln plants  in contrast to aviso ln plants.
it is noteworthy that contrary to the hn treatment, sink leaves were present for both genotypes .
unlike the previous period , a significant n uptake occurred in both genotypes under ln conditions .
the n uptake and allocation of n taken up into seeds were respectively  <dig> - and  <dig> -fold higher for oase than for aviso.
nevertheless, the total n amount distributed to the seeds of oase ln plants  remained lower than the total n amount distributed to seeds of aviso ln plants .figure  <dig>
n fluxes in aviso  and oase  in ln conditions between d <dig> and d <dig>  the plants were supplied with a low concentration of nitrate .
d <dig> corresponds to the start of seed filling and d <dig> to mature seeds.
fluxes of n from remobilization or uptake in the different organs are expressed as mg n remobilized or taken up, respectively.
for fluxes of n remobilization, the n amount is indicated with a minus sign  when n is remobilized from a source organ, or it is indicated with a plus sign  when remobilized n is redistributed towards a sink organ.
data are indicated as the mean value ± standard error .
asterisks represent significant differences between treatments and hashes represent significant differences between genotypes .
impacts of nitrate limitation on chlorophyll levels, n compounds and amino acid metabolism during n remobilization in a selected source leaf
in order to study the remobilization at the foliar level in response to n limitation applied at the bolting stage , selected mature leaves undergoing senescence during the experiment  were analysed in detail over 28 days.
the leaf biomass , leaf area  and chlorophyll content  in these selected source leaves were not significantly different between aviso and oase, meaning that the leaf initial status was similar between both genotypes.
consequently, it was possible to compare the processes involved in the n remobilization of source leaves of both genotypes.
in hn conditions, the chlorophyll content decreased at d <dig> and remained constant for aviso until d <dig>  while it decreased all along the experiment for oase.
in response to ln treatment, a decrease in chlorophyll content was observed from d <dig> for both genotypes .
in hn conditions, the n amount in the source leaf was 2-fold higher for aviso than for oase and remained nearly constant during the 28 days for both genotypes .
in response to ln conditions, the leaf n amount decreased significantly from d <dig> in aviso alone, resulting in a decline of 83%  between d <dig> and d <dig> .figure  <dig>
changes in chlorophyll level, total n, n-soluble proteins, n-amino acids and other n compounds in a source leaf.
plants of aviso and oase were supplied with ample  or low  nitrate concentrations.
these data were obtained on a selected “source leaf”, determined as mature at d <dig>  and becoming senescent during the experiment.
the chlorophyll amount  is expressed in an arbitrary unit.
the amount of total n , soluble proteins , amino acids  and other n compounds  are expressed as mg of n per leaf for each fraction.
the fraction of other n compounds that mainly corresponded to insoluble proteins, oligopeptides and ammonia, was determined as follows: mg of total n amount - .
all these data were quantified at  <dig>   <dig> ,  <dig> ,  <dig>  and  <dig>  days after the beginning of bolting .
concerning the soluble proteins and the other n compounds, only one biological replicate remained at d <dig>  and its value is indicated by a cross .
data are indicated as the mean value ± standard error .
letters a, b and c represent differences in kinetics, asterisks indicate significant differences between treatments and hashes represent significant differences between genotypes .
considering the different n fractions, it is noteworthy that whatever the genotype, a similar pattern of soluble protein degradation  was observed for both n conditions.
however, the only available biological replicate at d <dig>  suggested a lower amount of soluble proteins at d <dig> in ln plants compared with hn for both genotypes .
even though the level of free amino acids was  <dig> -fold higher in aviso than in oase, it decreased strongly in both genotypes at d <dig> in hn conditions .
in ln conditions, a lower amount of n-amino acids was observed for both genotypes at d <dig> compared with the hn treatment.
surprisingly, the fraction corresponding to the other n compounds increased in aviso between d <dig> and d <dig> in both n conditions .
thereafter, the fraction of other n compounds in the source leaf of aviso remained almost stable in hn plants while it decreased strongly in ln plants .
concerning oase, the other n compounds remained low and constant during all the experiments, whatever the n supply.
the activities of glutamate dehydrogenase  and glutamine synthetase , 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 d <dig> in both n conditions and a putative 6-fold increase  of gdh activity at d <dig> in ln conditions .
concerning oase, the gdh activity remained low until d <dig> and increased by  <dig> -fold at d <dig> in both n conditions .
in hn plants, the gs activity remained constant until d <dig> and decreased by 43% at d <dig> .
in ln plants, the gs activity increased  at d <dig> and putatively decreased at d <dig> .
the immunoblots of cytosolic  and chloroplastic  glutamine synthetase  revealed that the proportion of gs <dig> was not impacted by ln treatment in both genotypes, but it remained higher for oase throughout the experiment .figure  <dig>
changes in glutamate dehydrogenase activity, glutamine synthetase activity and their amounts  in a source leaf.
plants were supplied with ample  or low  nitrate concentrations.
these data were obtained from a selected ‘source leaf’, determined as mature at d <dig>  and becoming senescent during the experiment.
the activity of glutamate dehydrogenase  was quantified as the synthesis of glutamate and is expressed as nmol of nadh used.h− <dig> μg− <dig> proteins.
the activity of glutamine synthetase  was determined by the nmol of glutamine produced.h− <dig> μg− <dig> proteins.
the gs <dig> and gs <dig> amounts  were quantified after western blotting with specific antibodies and the percentage of gs <dig> among the total gs amount was estimated.
data were observed at  <dig>   <dig> ,  <dig> ,  <dig>  and  <dig>  days after the beginning of bolting .
only one biological replicate remained at d <dig>  and its value is indicated by a cross .
in panels a and b, data are indicated as the mean value ± se .
letters a, b and c represent differences in kinetics, asterisks mean significant differences between treatment and hashes represent significant differences between genotypes .
proteolytic activities in the source leaves
different protease inhibitors  were used in order to identify the class of proteases involved in the strong degradation of soluble proteins occurring in the source leaf at d <dig> .
due to the fact that rubisco represents a large proportion of the soluble proteins in leaves  <cit> , the characterization of proteases was determined via the analysis of the degradation of the rubisco large subunit .
in source leaf of hn plants , the lsu proteolysis was strongly inhibited by iodoacetamide for aviso and oase  and by mg <dig> for oase , 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  and metalloproteases  and a significant increase of proteasome activity  were observed  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  while the participation of metalloproteases increased  in oase ln plants .figure  <dig>
rubisco large subunit degradation in a source leaf with or without protease inhibitors  and the inhibition of the protease activities by protease inhibitors .
the rubisco large subunit  in the soluble protein extract  of the source leaf  is visualized on stain free sds-page and quantified for the four biological repetitions by image lab software  at  and after 1 h of incubation at 37°c  without inhibitors  or with specific protease inhibitors: iodoacetamide , aprotinin , methanol , methanol and 1– <dig> phenanthroline , methanol and pepstatin a , dmso  or dmso and mg <dig> .
the most representative biological repetition is shown in panel a and the percentage of degradation  are indicated below.
panel b presents the inhibition of the protease activities by the proteases inhibitors ).
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 .
discussion
evidence of two different n management strategies that lead to a similar seed yield under ample nitrate nutrition
the 15n-labelling method performed in this study allowed the distinction between endogenous and exogenous n fluxes and, therefore, the relevant determination of the involvement of n uptake and remobilization during seed filling.
despite no significant differences in seed yield , the two genotypes of winter oilseed rape investigated in the present work showed contrasting n management during seed filling under ample nitrate supply.
in fact, even if both genotypes used exogenous and endogenous n for seed filling , oase had the highest nupe  while aviso had the highest nre  during seed filling .
our data suggest that allocation of n taken up into seeds is more efficient in oase than aviso, while the processes of n remobilization from source organs towards seeds during the final reproductive phases is higher in aviso.
in fact, during seed filling, the total n remobilization was 3-fold higher for aviso than for oase, which was a consequence of a higher remobilization from leaves, stems and pod walls , leading to a higher amount of n redistributed towards seeds .
these results confirm that leaves, stems and pod walls are the main source organs for n remobilization after flowering  <cit> .
under nitrate limitation, n management in aviso is more efficient than oase for seed filling
despite an increase of nhi, a decrease of seed yield was observed for oase and aviso in response to nitrate limitation  applied at the early bolting stage .
the seed oil percentage increased in response to ln conditions for both genotypes  but the nutritional quality of oil of both genotypes was affected: increase of  c18:2/c18: <dig> ratio for oase and  proportion of erucic acid for aviso .
these results confirm the strong relationship between n supply and oil quality  <cit>  and suggest a genotypic variability about the oil quality in response to n limitation.
as expected, in response to ln treatment, the n uptake was lower throughout the experiment  and the total n amount in seeds  decreased for both genotypes , leading to a lower protein content in parallel to the increase in oil content  <cit> .
nevertheless, a significant n uptake occurred during seed filling despite the low n supply, especially in oase , supporting the fact that there is a significant n uptake capacity after flowering in oilseed rape  <cit> .
interestingly, growth of new leaves was observed during seed filling in response to ln conditions .
even if these new organs represent a sink for n, new leaves may help maintaining photosynthetic capacities during seed filling and raise the potential seed yield by increasing the allocation of c to seeds  <cit> .
in addition, photosynthetically active leaves could participate in the improvement of n uptake  <cit> , as previously suggested for winter oilseed rape  <cit> .
indeed, the n uptake is associated with the availability of photoassimilates in the roots  <cit> .
consequently, the higher n uptake of oase during seed filling  may be related to the significant higher c content observed under n limitation in oase roots at d <dig> and d <dig> .
as in hn conditions, n from uptake is mainly used for seed filling in oase, but due to the low n supply, a lower n amount is distributed to seeds compared with aviso .
in addition, even though oase had the highest n uptake during seed filling, aviso had the highest total n uptake from the bolting to mature seeds stages  in comparison with oase  .
this result agrees with the previous studies reporting that the genotypic variability of n uptake is mainly observed under restricted n supply  <cit> .
however, because both genotypes have the same seed yield under ln conditions , the positive correlation between n uptake and seed yield demonstrated in previous works on oilseed rape  <cit>  was not confirmed in our experimental conditions.
the higher n amount in seeds of aviso could be due to the fact that, as in hn treatment, endogenous n is mainly used for seed filling.
indeed, aviso had a higher nre during seed filling , leading to a higher redistribution of the n remobilized from leaves and stems toward seeds in comparison with oase .
in order to better understand the importance of n remobilization from leaves and stems in aviso with its superior nre, these organs were studied in detail.
impact of leaf n remobilization and its associated cellular processes in coping with n limitation
as previously observed for oilseed rape cv.
capitol  <cit> , leaves are the main source organs between bolting and early pod formation, whatever the genotype or the n supply .
in fact, the maintenance of the amount of n distributed to siliques in aviso under ln conditions is linked to an increase in n remobilization from source leaves  associated with an increase in the number of dead leaves .
the n remobilization in leaves of oase  and the number of dead leaves  were similar in both n conditions, suggesting a similar progression of senescence for this genotype whatever the n supply, which has never been highlighted previously.
the mechanisms involved in n remobilization were then investigated in a selected source leaf, becoming senescent between the bolting and the flowering stages.
proteins are the main form of n storage in source leaves, especially the soluble protein rubisco  <cit> , and for both genotypes a massive degradation of the soluble proteins occurs 7 days after bolting under both n conditions .
the aspartic and serine proteases seem to contribute at a low level to the proteolysis in both genotypes , contrary to previous results reported for arabidopsis  <cit> .
our study reveals a higher involvement of metalloproteases during n remobilization in the source leaves of oase in response to ln supply .
these results are consistent with previous proteomics studies which showed the induction of metalloproteases  during leaf senescence at the vegetative stages of winter oilseed rape  in response to limitation/privation in nitrate  <cit> .
because ftshs are involved in the proteolysis of d <dig> or lhcb <dig> proteins localized in the thylakoid membrane  <cit> , our results suggests that a nitrate limitation may lead to an improvement in the degradation of thylakoid-bound proteins.
except for aviso in hn conditions, the classes of proteases having the greatest involvement in the massive in vitro proteolysis observed 7 days after bolting are cysteine proteases and the proteasome system .
the importance of cysteine proteases in leaf proteolysis is in agreement with previous studies on winter oilseed rape demonstrating that the cysteine protease sag <dig> is highly induced at the transcriptomic  <cit>  and proteomic  <cit>  levels during leaf senescence.
the efficient n remobilization observed for oase in both n conditions and for aviso in ln treatment seems to be correlated to a high contribution of the proteasome in the proteolysis mechanisms , which confirm the previous studies on oilseed rape  <cit>  and arabidopsis  <cit> .
in addition, in our in vitro conditions, the proteasome seems to be able to degrade the lsu of rubisco.
however, in cell, the proteasome complex is involved in degradation of proteins previously tagged with ubiquitin and is mainly localized in the nucleus and cytosol .
some studies in arabidopsis shown that rubisco is degraded in acidic organelles such as lytic vesicles or vacuoles  and/or directly in the stroma .
taken together, these studies suggest that rubisco is probably not degraded in planta by the proteasome system.
however, a chloroplastic e <dig> enzyme, involved in the selectivity of the ubiquitin-proteasome system during plastid formation was recently found in the outer membrane of chloroplasts  <cit> .
in addition, the proteolysis by the proteasome can occur without the involvement of ubiquitin in animal cells  <cit> , suggesting that could also happen in plant cells.
consequently, the involvement of the proteasome pathway in the degradation of chloroplastic proteins seems to be possible and should be investigated further.
the efficient degradation of soluble proteins observed in the senescing leaf of aviso under both n conditions  was not correlated with a concomitant decrease in the total n amount .
in our study, there was no transient accumulation of n in the amino acid fraction , confirming that the export of amino acids was not limiting, as indicated in earlier results obtained for winter oilseed rape cv.
lirajet  <cit> .
consequently, despite the decline in n-soluble proteins, the stability of total n in senescing leaf was related to a transient accumulation of other n compounds , including transmembrane proteins, peptides, nitrate and/or ammonia.
in addition, the degradation and/or export of these other n compounds seem to be responsible of the higher n remobilization of the source leaf observed at d <dig> under ln conditions .
in aviso ln plants, the high decrease of other n compounds at d <dig> is followed by an increase of the amination activity related to gdh , which catalyses the incorporation of ammonium on α-ketogluratate to produce glutamate in case of accumulation of ammonium  <cit>  released by the catabolism of proteins and peptides  <cit> .
the better degradation of transmembrane proteins and peptides in ln conditions could lead to an ammonium accumulation and, therefore, may explain the increase of amination by the gdh in order to detoxify the cell and produce glutamate, which is found in phloem sap of oilseed rape  <cit> .
if this hypothesis is true, the lower level of other n compounds observed in source leaf of oase in both n conditions  could be due to an efficient degradation of transmembrane proteins and/or peptides into amino acids.
the amino acids are not accumulated in the source leaf , suggesting that they are well exported towards the phloem vessels.
this could be related to the fact that gs <dig>  the cytosolic isoform of gs which is known to be involved in n remobilization processes  <cit> , is present in higher proportions in the source leaf of oase than in aviso one .
these data suppose that an efficient degradation of transmembrane proteins and/or peptides and a rapid conversion of amino acids into their transportable forms  by enzymes like gdh or gs <dig> in order to enhance their exportation from the mesophyll cells towards the phloem is an important physiological trait for the optimization of nre.
these results have to be confirmed and other enzymes involved in the conversion of amino acids, such as asparagine synthetase in arabidopsis  <cit> , have to be investigated in oilseed rape leaves.
stems play a pivotal role in n management as a transient n storage organ
in ln supply, the highest n amount in seeds of aviso compared with oase is linked to a large n remobilization  from the stem to seeds during seed filling .
as previously observed  <cit> , the highest n remobilization is not related to a lower residual n in the stem at d <dig> , but to a significant higher distribution of n towards the stem before seed filling .
consequently, the increase of n storage capacity in stems before the onset of seed filling may significantly improve the n remobilization from stems towards seeds.
the same conclusion was observed about leaves during seed filling.
however, while leaves were crucial source organs before early silique formation, their participation remained low during seed filling .
a high n remobilization from leaves towards the stem was previously associated with a high nue in spring oilseed rape  <cit> .
taken together, results suggest that the stem is a crucial buffering organ for transient n storage in order to compensate the delay between the large n remobilization from leaves and the sink n demand for seed filling.
CONCLUSIONS
as previously observed under restricted n supply, the higher n uptake from bolting to flowering is associated with the most efficient genotype for seed n filling .
nevertheless, under field conditions, the mineral n availability strongly fluctuates and is usually low during seed filling, meaning that a higher n uptake could not be sufficient to significantly improve the nue.
the investigation of the contrasting n management in aviso and oase genotypes confirms that remobilization during monocarpic senescence is efficient for leaves and stems.
consequently, an efficient remobilization and utilization of n taken up and n stored before the onset of seed filling is probably one of the main physiological trait for attaining a high nue in an agronomic context of mineral n restriction.
up to the early phase of seed filling, n is mainly stored in leaves, and therefore an efficient foliar n remobilization is important to limit n loss from dead leaves and improving seed n filling together with a limitation of pollution risk.
our findings suggest that an efficient foliar nre is mainly associated to  a high proteolysis by the cysteine proteases and proteasome system and  a strong coordination between proteolysis, the export/degradation of oligopeptides and the export of amino-acids .
future developments are needed to confirm the relevance of these classes of proteases before their potential use in a “candidate protein-strategy” to identify qtls related to high nre.
our study also highlights that the improvement of nue and seed n filling under low mineral n availability is highly related to an efficient storage of the n remobilized from leaves into the stem.
thus, it is likely that the stem may act as a transient storage organ in case of asynchronism between the massive remobilization of n coming from source leaves and its further utilization by seeds.
such physiological traits  may be particularly relevant to breeding programs aimed at identifying brassica napus genotypes having high nue in response to the fluctuation of nitrate availability and low n fertilizer inputs.

 over the last four decades, oilseed rape has become the third most widely grown oleaginous crop worldwide with a -fold increase in seed production between and. this crop represents a major renewable resource for human food, animal feed and numerous non-food uses. to increase the level of seed production with current genotypes, the use of n fertilizers has increased by 430% over the last forty years. despite the high capacity of mineral n absorption of oilseed rape, only 50% of n from fertilizer is recovered in seeds at harvest. this low n use efficiency is related to the loss of n by leaf drop, reflecting the weak n remobilization efficiency of oilseed rape. therefore, in a context of imposed limitations on n-fertilizer inputs, improving nue is becoming a priority in order to maintain/increase seed yield and decrease the risk of water pollution by nitrate, the emission of greenhouse gases contributing to global warming, and the economic costs of oilseed rape crops. nue can be represented as two main components: the n uptake efficiency and the n utilization efficiency, itself subdivided into two other components, n assimilation efficiency and n remobilization efficiency. even if n uptake of winter oilseed rape is considered to be efficient at vegetative stages, an n uptake that remains significant until flowering is associated with genotypes having high seed yield. a mineral n input during seed filling increases seed yield of spring oilseed rape and recent studies on winter oilseed rape have reported that a n uptake during reproductive stages appears to be a determinant trait for seed yield of winter oilseed rape, especially in restricted n supply. nevertheless, in field conditions, the mineral n availability highly fluctuates during reproductive stages due to environmental factors, such as water deficit in the soil. to obtain a high nue, the n coming from uptake has to be well managed by the plant. consequently, an improvement of nute is also necessary to enhance nue. concerning the nae, transgenic approaches have been targeted to enzymes involved in n metabolism such as nitrate reductase, nitrite reductase or alanine aminotransferase in various species such as arabidopsis and tobacco, with more or less success. a promising result was obtained with oilseed rape plants overexpressing an alat, which need 40% less n fertilizer to reach similar yield as the wild type. however, a study of spring oilseed rape genotypes has suggested that nre is a major determinant of nute and an efficient assimilation needs to be correlated to an enhanced n remobilization to improve the seed n filling, which is essential to improve/maintain seed yield, in a context of n input reduction. the n remobilization associated with leaf senescence is considered as crucial for oilseed rape yield. during the vegetative stages, n is remobilized from the older leaves to the younger leaves along the axis of the plants via the sequential senescence, but senescent leaves can fall with a high level of residual n leading to a significant return of n to the soil. by developing a modelling approach as a mean to identify potential methods for improving the nue of oilseed rape, it was estimated that a 1% decrease in the residual n in fallen leaves, resulting from an enhanced foliar n remobilization, may increase seed yield by 5-10%. after bolting, and especially during monocarpic senescence when the n is remobilized from vegetative organs to seeds, the low soil n availability during spring and at the beginning of summer makes n remobilization crucial for seed yield and seed filling. during this period of development the main source organs are leaves, stems and pod walls. during reproductive stages, the n amount in source leaves is highly remobilized, leading to a low residual n in dead leaves and resulting in the reduction of the risk of n pollution. these results highlight an efficient n remobilization from source organs during monocarpic senescence, but also reveal variations for the residual n amount in stems compared with leaves of four winter oilseed rape genotypes. in addition, a genotypic and n-supply interaction for n content in stems was found for genotypes of spring canola and the genotype with the best n remobilization was able to better remobilize n from stems and pod walls to seeds. these results suggest that stems can also be important organ for improving seed n-filling, but the role of stems in n management at the whole plant level for winter oilseed rape remains largely unknown. although nre seems to be a major lever for improving the nue in oilseed rape, the cellular mechanisms associated with n remobilization from senescing leaves remain largely an enigma. during leaf senescence, the degradation of proteins into amino acids or peptides is performed by different classes of proteases. studies on arabidopsis reported the predominant involvement of cysteine and serine proteases, but also a role for the proteasome in the degradation of carbonylated proteins, which are accumulated during senescence. the ribulose- -biphosphate carboxylase/oxygenase can represent up to 65% of the soluble proteins in c plants and 20–30% of total leaf n. consequently, rubisco is the major source of n for remobilization and its budget is very relevant for the plant. in winter oilseed rape, cysteine, aspartic, and metallo-proteases as well as the 26s proteasome are supposed to play an important role in foliar n remobilization during senescence in the vegetative stages but there is no evidence concerning the proteolysis activities that are involved in leaf senescence and degradation of rubisco after the bolting stage. among the enzymes involved in the conversion of amino acids into transportable forms, a recent study highlighted the importance of asparagine synthetase in n remobilization during senescence in arabidopsis. in addition, the glutamine synthetase catalyses the assimilation of ammonium into glutamate to synthesize glutamine. glutamine was found at high levels in phloem sap of oilseed rape and consequently, gss are supposed to be largely involved in n remobilization processes during senescence in winter wheat and maize, especially the cytosolic form in the case of oilseed rape. in parallel, glutamate dehydrogenase may catalyse a glutamate deamination which provides ammonium for the gs activity in senescing leaves. in addition, in case of high levels of ammonium, gdh can incorporate ammonium on α-ketoglutarate to produce glutamate. a significant quantity of glutamate has been found in phloem sap of oilseed rape and an increase of both gdh activities in sliced leaves of oilseed rape was associated with a decrease in soluble proteins and ammonium, suggesting an important role for gdh in foliar n remobilization. the phloem loading of the resulting amino acids is regarded as not limiting in oilseed rape at the vegetative stages. the efficient export of amino acids and the involvement of gs and gdh in n remobilization need to be confirmed in leaves as they senesce after bolting because they are proposed to have an efficient n remobilization. the aim of this study was to identify the physiological traits involved in the high nue and n remobilization in restricted n supply conditions, at whole plant and cellular levels. to reach this goal, two genotypes, known to have different strategies following a nitrate limitation at the vegetative stage, were selected. long term pulse-chase 15 n-labelling was performed to precisely define the endogenous and exogenous n fluxes at the whole plant level between the bolting and mature seed stages, under restricted and ample nitrate supply. a specific 15 n-labelling was also used in order to determine a relevant estimation of nue and its different components. this study highlights the role of leaves and stems in the remobilization of n towards seeds and allows the identification of the physiological traits associated with a high nue in response to restricted n supply. moreover, a study of foliar n remobilization in source leaves was performed through the analysis of foliar n compounds, the gs and gdh activities, and the development of a new method to study proteolytic activities, using endogenous rubisco as a substrate, in order to determine the involvement of different classes of proteases in the n remobilization which occurs during this growth stage.  impact of n limitation on growth, yield components, seed and oil composition when plants were well supplied with nitrate, the total dm was higher for oase compared with aviso during seed filling, mainly due to differences in the stem dm. under our experimental conditions, the occurrence of the different stages of development was not significantly different between both genotypes or between both n treatments for a given genotype. in low nitrate conditions, no difference in the total dm was observed between genotypes. as expected, compared with hn, dm was reduced in leaves from d in stems at d and in pod walls at d in addition, dm declined significantly in siliques at d and increased in stems at d for aviso while dm of siliques and stems declined at d for oase. the root dm at d was lower in aviso under ln compared with hn. under ln supply, a similar decrease in seed production was observed for both genotypes.figure kinetics of dry matter of aviso and oase under ample or low n supply. the plants were supplied with ample or low concentrations of nitrate. dry matter is expressed in g per plant for aviso and oase at,, and days after the beginning of bolting. seeds and pod walls were grouped and called siliques at d and they were separated from d onwards. data are indicated as the mean value ± standard error. different letters indicate that the total dry matter is significantly different between two dates. hashes represent significant differences between genotypes in hn or ln conditions and the asterisks represent significant differences between n treatments. in hn plants, the yield components; table 1) as well as the seed composition; table 1) were similar for both genotypes, except for the percentage of omega- and omega- precursors, which were higher for aviso than oase. however, the c18:2/c18: ratio was similar for both genotypes. as expected, an n limitation led to a strong decrease in the n amount of seeds. compared with hn conditions, nhi significantly increased in oase and tended to increase in aviso in response to ln treatment. seeds of oase ln plants had a higher c:n ratio associated with a higher oil percentage, which was to the detriment of proteins. for oil composition, an increase of oleic acid proportion was observed under n limitation for both genotypes. while c18: and c18: percentages did not differ under both n conditions for aviso, an increase in c18: and in the c18:2/c18: ratio occurred under ln for oase. the proportion of erucic acid also increased under ln conditions for aviso.table seed composition and nitrogen harvest index of aviso and oase under ample or low n supply aviso oase hn ln hn ln yield components seed composition the plants were supplied with an ample or low nitrate concentration. the n amount in seed is estimated in milligrams. c: n ratio: ratio c: n in seeds; nhi: n harvesting index. the seed composition and the oil composition was determined by nirs method. asterisks represent significant differences between n treatments and hashes represent significant differences between the two genotypes. effects of nitrate limitation on n amount and nue components in hn plants, the total n amount did not differ between the genotypes. however, the n amount in leaves and flowers at the final stage of development was significantly higher for oase than for aviso. as expected, the total n amount was reduced for both genotypes in ln conditions, resulting from a decline in the n amount in nearly all organs from d exceptions were flowers present at any time during the experiment, roots at d for oase, stems at d and leaves at d for aviso, which all showed a similar n amount in both n conditions. in ln plants, the final n amount in seeds and pod walls of oase was lower than in aviso.figure kinetics of the n amount in aviso and oase under ample or low n supply. the plants were supplied with ample or low concentrations of nitrate. the n amount is expressed in mg per plant for aviso and oase at,, and days after the beginning of bolting. seeds and pod walls were grouped and called siliques at d and they were separated from d onwards. data are indicated as the mean value ± standard error. different letters indicate that the total n amount is significantly different between two dates. hashes represent significant differences between genotypes in hn or ln conditions and the asterisks represent significant differences between n treatments. the n use efficiency, n utilization efficiency, and n remobilization efficiency were negatively correlated to n supply while the n uptake efficiency was positively correlated to n supply. the nue was higher for oase than for aviso in hn conditions while no differences were observed in the ln treatment. the nute increased in response to ln supply but no differences were observed between the genotypes, whatever the n supply. a strong genotype/treatment interaction effect was observed for nupe between d and d in hn conditions, the nupe of oase was around 81%, suggesting that the n distributed to seeds during this period is mainly provided by the n that is newly taken up by the roots. the nupe of oase is about 2-fold higher than aviso in both n conditions. the global nre, i.e. the nre calculated on the whole growing cycle, reached about 55-60% for both genotypes, indicating that a large part of the n stored in source organs at the bolting stage is remobilized during the experiment. when nre was calculated stepwise, a regular decrease occurred throughout the steps, whatever the treatment and the genotype. in hn plants, nre was -fold higher in oase than aviso between d and d while it was reduced by about 50% after d. in response to ln supply, global nre increases for both genotypes, but a genotypic effect was highlighted between d and d where the nre of aviso was -fold higher than oase. however, no genotype/treatment interaction was observed for nre.table nue, nute, nupe and nre of aviso and oase under ample or low n supply genotypes source of variation aviso oase g n g x n n treatment hn ln hn ln f g f n r f gxn nuea nuteb global stepwise the plants were supplied with an ample or low nitrate concentration. d corresponds to the early bolting stage, d to pod formation, d to seed filling and d to mature seeds. the main source of variation is deduced from an anova test where genotypes, n treatment, and genotype x n treatment interaction were tested. the resulting f values are presented below. the r values correspond to the correlation between n supply and nue, nute, nupe and nre, respectively. a:nue corresponds to the mg of n in seeds per mg of the n amount in the whole plant relative to the seed dm produced per g of total dm. b:nute corresponds to the g of seed dm produced per mg of n in shoots. c:nupe is estimated as the percentage of n derived from uptake in the total n distributed to seeds between d and d. d:nre is determined as the percentage of n stored in all source organs which is remobilized between d and final harvest or between d and d d and d and d and d asterisks represent significant differences between n treatments and hashes represent significant differences between the two genotypes. n fluxes at the whole plant level in hn conditions the 15 n labelling method used in this study gave the opportunity to determine precisely the n fluxes at the whole plant level between d0-d d42-d and d70-d due to the fact that genotype and n treatment effects were observed for nre solely between d and d in hn plants, only the n fluxes between these two growing stages are given in figure . in hn conditions, n remobilization was around 3-fold higher in aviso than oase. for both genotypes, the stem was the main source organ: 47% and 59% of the total n remobilized in aviso and oase, respectively. for aviso, the other source organs were the leaves, pod walls, flowers and roots while for oase the source organs were leaves and roots. for oase, the n amount remobilized from leaves was -fold lower than for aviso. the n remobilized from source organs was mainly distributed to the seeds with a significantly greater amount in aviso than oase. contrary to aviso, the flowers of oase are sink organs for remobilized n. there was no significant difference in the amount of n uptake between genotypes. however, the allocation of n towards seeds is more important for oase than for aviso. despite this higher allocation of n towards seeds in oase, the total n distributed to oase seeds was not the same as in aviso.figure n fluxes in aviso and oase in hn conditions between d and d the plants were supplied with an ample concentration of nitrate. d corresponds to the start of seed filling and d to the mature seed stage. fluxes of n from remobilization or uptake in the different organs are expressed as mg of n remobilized or taken up, respectively. a shaded box means that the organ was not present during these growing stages. for fluxes of n remobilization, the n amount is indicated with a minus sign when n is remobilized from a source organ, or it is indicated with a plus sign when remobilized n is redistributed towards a sink organ. data are indicated as the mean value ± standard error. hashes represent significant differences between genotypes. n fluxes at the whole plant level in response to nitrate limitation between d and d n remobilization increased for aviso in response to ln treatment compared with hn, due to a larger n remobilization from source leaves. this remobilized n was mainly redistributed towards siliques, allowing a similar amount of n to be redistributed as hn plants. in contrast to aviso, oase showed a similar total amount of remobilized n in both n conditions. however, compared with hn, the redistribution of this remobilized n to siliques was higher in oase ln plants due to a lower loss via dead leaves and a lower redistribution to sink leaves. the n remobilization in oase was lower than aviso due to a lower contribution of roots and flowers. as expected, in ln plants the n uptake strongly decreased for aviso and was not detected for oase. in aviso ln plants, the main sink organs for the n uptake were siliques, leaves and stems. thanks to the n uptake in aviso, which supplemented the n remobilization, the total n amount distributed to siliques was similar in both genotypes.figure n fluxes in aviso and oase in ln conditions between d and d the plants were supplied with a low concentration of nitrate. d corresponds to early bolting and d to pod formation. fluxes of n from remobilization or uptake in the different organs are expressed as mg of n remobilized or taken up, respectively. for fluxes of n remobilization, the n amount is indicated with a minus sign when n is remobilized from a source organ, or it is indicated with a plus sign when remobilized n is redistributed towards a sink organ. data are indicated as the mean value ± standard error. asterisks represent significant differences between treatments and hashes represent significant differences between genotypes. between d and d no n uptake was detectable and the total remobilized n amount decreased for both genotypes under n limitation contrary to hn plants. this was mainly related to lower contributions of leaves and pod walls, leading to a lower n redistribution to seeds. the n remobilization was -fold higher for aviso than oase, mainly due to a 2-fold higher n remobilization from leaves. nevertheless, the n redistribution to seeds was not significantly different between the genotypes. these results can be explained by the fact that flowers and stems are sink organs for aviso contrary to oase, and a higher n loss by dead leaves occurs for aviso.figure n fluxes in aviso and oase in ln conditions between d and d the plants were supplied with a low concentration of nitrate. d corresponds to pod formation and d to the start of seed filling. fluxes of n from remobilization or uptake in the different organs are expressed as mg of n remobilized or taken up, respectively. a shaded box means that the organ was not present during these growing stages. for fluxes of n remobilization, the n amount is indicated with a minus sign when n is remobilized from a source organ, or it is indicated with a plus sign when remobilized n is redistributed towards a sink organ. data are indicated as the mean value ± standard error. asterisks represent significant differences between treatments and hashes represent significant differences between genotypes. between d and d the n remobilization from all source organs was low for both genotypes, leading to a lower total n remobilization in ln compared with hn plants. consequently, a lower n amount was redistributed to seeds. under ln conditions, aviso had a higher amount of total remobilized n than oase resulting in a higher redistribution of remobilized n to seeds. this was related to a higher remobilization from stems and source leaves for aviso compared with oase. the flowers were sink organs for remobilized n in oase ln plants in contrast to aviso ln plants. it is noteworthy that contrary to the hn treatment, sink leaves were present for both genotypes. unlike the previous period, a significant n uptake occurred in both genotypes under ln conditions. the n uptake and allocation of n taken up into seeds were respectively - and -fold higher for oase than for aviso. nevertheless, the total n amount distributed to the seeds of oase ln plants remained lower than the total n amount distributed to seeds of aviso ln plants.figure n fluxes in aviso and oase in ln conditions between d and d the plants were supplied with a low concentration of nitrate. d corresponds to the start of seed filling and d to mature seeds. fluxes of n from remobilization or uptake in the different organs are expressed as mg n remobilized or taken up, respectively. for fluxes of n remobilization, the n amount is indicated with a minus sign when n is remobilized from a source organ, or it is indicated with a plus sign when remobilized n is redistributed towards a sink organ. data are indicated as the mean value ± standard error. asterisks represent significant differences between treatments and hashes represent significant differences between genotypes. impacts of nitrate limitation on chlorophyll levels, n compounds and amino acid metabolism during n remobilization in a selected source leaf in order to study the remobilization at the foliar level in response to n limitation applied at the bolting stage, selected mature leaves undergoing senescence during the experiment were analysed in detail over 28 days. the leaf biomass, leaf area and chlorophyll content in these selected source leaves were not significantly different between aviso and oase, meaning that the leaf initial status was similar between both genotypes. consequently, it was possible to compare the processes involved in the n remobilization of source leaves of both genotypes. in hn conditions, the chlorophyll content decreased at d and remained constant for aviso until d while it decreased all along the experiment for oase. in response to ln treatment, a decrease in chlorophyll content was observed from d for both genotypes. in hn conditions, the n amount in the source leaf was 2-fold higher for aviso than for oase and remained nearly constant during the 28 days for both genotypes. in response to ln conditions, the leaf n amount decreased significantly from d in aviso alone, resulting in a decline of 83% between d and d.figure changes in chlorophyll level, total n, n-soluble proteins, n-amino acids and other n compounds in a source leaf. plants of aviso and oase were supplied with ample or low nitrate concentrations. these data were obtained on a selected “source leaf”, determined as mature at d and becoming senescent during the experiment. the chlorophyll amount is expressed in an arbitrary unit. the amount of total n, soluble proteins, amino acids and other n compounds are expressed as mg of n per leaf for each fraction. the fraction of other n compounds that mainly corresponded to insoluble proteins, oligopeptides and ammonia, was determined as follows: mg of total n amount -. all these data were quantified at,, and days after the beginning of bolting. concerning the soluble proteins and the other n compounds, only one biological replicate remained at d and its value is indicated by a cross. data are indicated as the mean value ± standard error. letters a, b and c represent differences in kinetics, asterisks indicate significant differences between treatments and hashes represent significant differences between genotypes. considering the different n fractions, it is noteworthy that whatever the genotype, a similar pattern of soluble protein degradation was observed for both n conditions. however, the only available biological replicate at d suggested a lower amount of soluble proteins at d in ln plants compared with hn for both genotypes. even though the level of free amino acids was -fold higher in aviso than in oase, it decreased strongly in both genotypes at d in hn conditions. in ln conditions, a lower amount of n-amino acids was observed for both genotypes at d compared with the hn treatment. surprisingly, the fraction corresponding to the other n compounds increased in aviso between d and d in both n conditions. thereafter, the fraction of other n compounds in the source leaf of aviso remained almost stable in hn plants while it decreased strongly in ln plants. concerning oase, the other n compounds remained low and constant during all the experiments, whatever the n supply. the activities of glutamate dehydrogenase and glutamine synthetase, 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 d in both n conditions and a putative 6-fold increase of gdh activity at d in ln conditions. concerning oase, the gdh activity remained low until d and increased by -fold at d in both n conditions. in hn plants, the gs activity remained constant until d and decreased by 43% at d. in ln plants, the gs activity increased at d and putatively decreased at d. the immunoblots of cytosolic and chloroplastic glutamine synthetase revealed that the proportion of gs was not impacted by ln treatment in both genotypes, but it remained higher for oase throughout the experiment.figure changes in glutamate dehydrogenase activity, glutamine synthetase activity and their amounts in a source leaf. plants were supplied with ample or low nitrate concentrations. these data were obtained from a selected ‘source leaf’, determined as mature at d and becoming senescent during the experiment. the activity of glutamate dehydrogenase was quantified as the synthesis of glutamate and is expressed as nmol of nadh used.h− μg− proteins. the activity of glutamine synthetase was determined by the nmol of glutamine produced.h− μg− proteins. the gs and gs amounts were quantified after western blotting with specific antibodies and the percentage of gs among the total gs amount was estimated. data were observed at,, and days after the beginning of bolting. only one biological replicate remained at d and its value is indicated by a cross. in panels a and b, data are indicated as the mean value ± se. letters a, b and c represent differences in kinetics, asterisks mean significant differences between treatment and hashes represent significant differences between genotypes. proteolytic activities in the source leaves different protease inhibitors were used in order to identify the class of proteases involved in the strong degradation of soluble proteins occurring in the source leaf at d. due to the fact that rubisco represents a large proportion of the soluble proteins in leaves, the characterization of proteases was determined via the analysis of the degradation of the rubisco large subunit. in source leaf of hn plants, the lsu proteolysis was strongly inhibited by iodoacetamide for aviso and oase and by mg for oase, 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 and metalloproteases and a significant increase of proteasome activity were observed 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 while the participation of metalloproteases increased in oase ln plants.figure rubisco large subunit degradation in a source leaf with or without protease inhibitors and the inhibition of the protease activities by protease inhibitors. the rubisco large subunit in the soluble protein extract of the source leaf is visualized on stain free sds-page and quantified for the four biological repetitions by image lab software at and after 1 h of incubation at 37°c without inhibitors or with specific protease inhibitors: iodoacetamide, aprotinin, methanol, methanol and 1– phenanthroline, methanol and pepstatin a, dmso or dmso and mg. the most representative biological repetition is shown in panel a and the percentage of degradation are indicated below. panel b presents the inhibition of the protease activities by the proteases inhibitors ). 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. discussion evidence of two different n management strategies that lead to a similar seed yield under ample nitrate nutrition the 15n-labelling method performed in this study allowed the distinction between endogenous and exogenous n fluxes and, therefore, the relevant determination of the involvement of n uptake and remobilization during seed filling. despite no significant differences in seed yield, the two genotypes of winter oilseed rape investigated in the present work showed contrasting n management during seed filling under ample nitrate supply. in fact, even if both genotypes used exogenous and endogenous n for seed filling, oase had the highest nupe while aviso had the highest nre during seed filling. our data suggest that allocation of n taken up into seeds is more efficient in oase than aviso, while the processes of n remobilization from source organs towards seeds during the final reproductive phases is higher in aviso. in fact, during seed filling, the total n remobilization was 3-fold higher for aviso than for oase, which was a consequence of a higher remobilization from leaves, stems and pod walls, leading to a higher amount of n redistributed towards seeds. these results confirm that leaves, stems and pod walls are the main source organs for n remobilization after flowering. under nitrate limitation, n management in aviso is more efficient than oase for seed filling despite an increase of nhi, a decrease of seed yield was observed for oase and aviso in response to nitrate limitation applied at the early bolting stage. the seed oil percentage increased in response to ln conditions for both genotypes but the nutritional quality of oil of both genotypes was affected: increase of c18:2/c18: ratio for oase and proportion of erucic acid for aviso. these results confirm the strong relationship between n supply and oil quality and suggest a genotypic variability about the oil quality in response to n limitation. as expected, in response to ln treatment, the n uptake was lower throughout the experiment and the total n amount in seeds decreased for both genotypes, leading to a lower protein content in parallel to the increase in oil content. nevertheless, a significant n uptake occurred during seed filling despite the low n supply, especially in oase, supporting the fact that there is a significant n uptake capacity after flowering in oilseed rape. interestingly, growth of new leaves was observed during seed filling in response to ln conditions. even if these new organs represent a sink for n, new leaves may help maintaining photosynthetic capacities during seed filling and raise the potential seed yield by increasing the allocation of c to seeds. in addition, photosynthetically active leaves could participate in the improvement of n uptake, as previously suggested for winter oilseed rape. indeed, the n uptake is associated with the availability of photoassimilates in the roots. consequently, the higher n uptake of oase during seed filling may be related to the significant higher c content observed under n limitation in oase roots at d and d. as in hn conditions, n from uptake is mainly used for seed filling in oase, but due to the low n supply, a lower n amount is distributed to seeds compared with aviso. in addition, even though oase had the highest n uptake during seed filling, aviso had the highest total n uptake from the bolting to mature seeds stages in comparison with oase. this result agrees with the previous studies reporting that the genotypic variability of n uptake is mainly observed under restricted n supply. however, because both genotypes have the same seed yield under ln conditions, the positive correlation between n uptake and seed yield demonstrated in previous works on oilseed rape was not confirmed in our experimental conditions. the higher n amount in seeds of aviso could be due to the fact that, as in hn treatment, endogenous n is mainly used for seed filling. indeed, aviso had a higher nre during seed filling, leading to a higher redistribution of the n remobilized from leaves and stems toward seeds in comparison with oase. in order to better understand the importance of n remobilization from leaves and stems in aviso with its superior nre, these organs were studied in detail. impact of leaf n remobilization and its associated cellular processes in coping with n limitation as previously observed for oilseed rape cv. capitol, leaves are the main source organs between bolting and early pod formation, whatever the genotype or the n supply. in fact, the maintenance of the amount of n distributed to siliques in aviso under ln conditions is linked to an increase in n remobilization from source leaves associated with an increase in the number of dead leaves. the n remobilization in leaves of oase and the number of dead leaves were similar in both n conditions, suggesting a similar progression of senescence for this genotype whatever the n supply, which has never been highlighted previously. the mechanisms involved in n remobilization were then investigated in a selected source leaf, becoming senescent between the bolting and the flowering stages. proteins are the main form of n storage in source leaves, especially the soluble protein rubisco, and for both genotypes a massive degradation of the soluble proteins occurs 7 days after bolting under both n conditions. the aspartic and serine proteases seem to contribute at a low level to the proteolysis in both genotypes, contrary to previous results reported for arabidopsis. our study reveals a higher involvement of metalloproteases during n remobilization in the source leaves of oase in response to ln supply. these results are consistent with previous proteomics studies which showed the induction of metalloproteases during leaf senescence at the vegetative stages of winter oilseed rape in response to limitation/privation in nitrate. because ftshs are involved in the proteolysis of d or lhcb proteins localized in the thylakoid membrane, our results suggests that a nitrate limitation may lead to an improvement in the degradation of thylakoid-bound proteins. except for aviso in hn conditions, the classes of proteases having the greatest involvement in the massive in vitro proteolysis observed 7 days after bolting are cysteine proteases and the proteasome system. the importance of cysteine proteases in leaf proteolysis is in agreement with previous studies on winter oilseed rape demonstrating that the cysteine protease sag is highly induced at the transcriptomic and proteomic levels during leaf senescence. the efficient n remobilization observed for oase in both n conditions and for aviso in ln treatment seems to be correlated to a high contribution of the proteasome in the proteolysis mechanisms, which confirm the previous studies on oilseed rape and arabidopsis. in addition, in our in vitro conditions, the proteasome seems to be able to degrade the lsu of rubisco. however, in cell, the proteasome complex is involved in degradation of proteins previously tagged with ubiquitin and is mainly localized in the nucleus and cytosol. some studies in arabidopsis shown that rubisco is degraded in acidic organelles such as lytic vesicles or vacuoles and/or directly in the stroma. taken together, these studies suggest that rubisco is probably not degraded in planta by the proteasome system. however, a chloroplastic e enzyme, involved in the selectivity of the ubiquitin-proteasome system during plastid formation was recently found in the outer membrane of chloroplasts. in addition, the proteolysis by the proteasome can occur without the involvement of ubiquitin in animal cells, suggesting that could also happen in plant cells. consequently, the involvement of the proteasome pathway in the degradation of chloroplastic proteins seems to be possible and should be investigated further. the efficient degradation of soluble proteins observed in the senescing leaf of aviso under both n conditions was not correlated with a concomitant decrease in the total n amount. in our study, there was no transient accumulation of n in the amino acid fraction, confirming that the export of amino acids was not limiting, as indicated in earlier results obtained for winter oilseed rape cv. lirajet. consequently, despite the decline in n-soluble proteins, the stability of total n in senescing leaf was related to a transient accumulation of other n compounds, including transmembrane proteins, peptides, nitrate and/or ammonia. in addition, the degradation and/or export of these other n compounds seem to be responsible of the higher n remobilization of the source leaf observed at d under ln conditions. in aviso ln plants, the high decrease of other n compounds at d is followed by an increase of the amination activity related to gdh, which catalyses the incorporation of ammonium on α-ketogluratate to produce glutamate in case of accumulation of ammonium released by the catabolism of proteins and peptides. the better degradation of transmembrane proteins and peptides in ln conditions could lead to an ammonium accumulation and, therefore, may explain the increase of amination by the gdh in order to detoxify the cell and produce glutamate, which is found in phloem sap of oilseed rape. if this hypothesis is true, the lower level of other n compounds observed in source leaf of oase in both n conditions could be due to an efficient degradation of transmembrane proteins and/or peptides into amino acids. the amino acids are not accumulated in the source leaf, suggesting that they are well exported towards the phloem vessels. this could be related to the fact that gs the cytosolic isoform of gs which is known to be involved in n remobilization processes, is present in higher proportions in the source leaf of oase than in aviso one. these data suppose that an efficient degradation of transmembrane proteins and/or peptides and a rapid conversion of amino acids into their transportable forms by enzymes like gdh or gs in order to enhance their exportation from the mesophyll cells towards the phloem is an important physiological trait for the optimization of nre. these results have to be confirmed and other enzymes involved in the conversion of amino acids, such as asparagine synthetase in arabidopsis, have to be investigated in oilseed rape leaves. stems play a pivotal role in n management as a transient n storage organ in ln supply, the highest n amount in seeds of aviso compared with oase is linked to a large n remobilization from the stem to seeds during seed filling. as previously observed, the highest n remobilization is not related to a lower residual n in the stem at d, but to a significant higher distribution of n towards the stem before seed filling. consequently, the increase of n storage capacity in stems before the onset of seed filling may significantly improve the n remobilization from stems towards seeds. the same conclusion was observed about leaves during seed filling. however, while leaves were crucial source organs before early silique formation, their participation remained low during seed filling. a high n remobilization from leaves towards the stem was previously associated with a high nue in spring oilseed rape. taken together, results suggest that the stem is a crucial buffering organ for transient n storage in order to compensate the delay between the large n remobilization from leaves and the sink n demand for seed filling.  as previously observed under restricted n supply, the higher n uptake from bolting to flowering is associated with the most efficient genotype for seed n filling. nevertheless, under field conditions, the mineral n availability strongly fluctuates and is usually low during seed filling, meaning that a higher n uptake could not be sufficient to significantly improve the nue. the investigation of the contrasting n management in aviso and oase genotypes confirms that remobilization during monocarpic senescence is efficient for leaves and stems. consequently, an efficient remobilization and utilization of n taken up and n stored before the onset of seed filling is probably one of the main physiological trait for attaining a high nue in an agronomic context of mineral n restriction. up to the early phase of seed filling, n is mainly stored in leaves, and therefore an efficient foliar n remobilization is important to limit n loss from dead leaves and improving seed n filling together with a limitation of pollution risk. our findings suggest that an efficient foliar nre is mainly associated to a high proteolysis by the cysteine proteases and proteasome system and a strong coordination between proteolysis, the export/degradation of oligopeptides and the export of amino-acids. future developments are needed to confirm the relevance of these classes of proteases before their potential use in a “candidate protein-strategy” to identify qtls related to high nre. our study also highlights that the improvement of nue and seed n filling under low mineral n availability is highly related to an efficient storage of the n remobilized from leaves into the stem. thus, it is likely that the stem may act as a transient storage organ in case of asynchronism between the massive remobilization of n coming from source leaves and its further utilization by seeds. such physiological traits may be particularly relevant to breeding programs aimed at identifying brassica napus genotypes having high nue in response to the fluctuation of nitrate availability and low n fertilizer inputs. 
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. 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. 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. however, the contribution of stems towards n management and the cellular mechanisms involved in foliar remobilization remain largely unknown. 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 cysteine proteases and proteasome activities and a fine coordination between proteolysis and export mechanisms. nitrate restriction decreased seed yield and oil quality for both genotypes but aviso had the best seed n filling. contrary to what is observed during senescence in the vegetative stages, n remobilization from stems and leaves is considered efficient during monocarpic senescence. this high leaf n remobilization is associated with a better degradation/export of insoluble proteins, oligopeptides, nitrate and/or ammonia. 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 cultivated under ample or restricted nitrate supply. 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. under n limitation, aviso had a better n remobilization from leaves to stems before the onset of seed filling. 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. afterwards, the higher n remobilization from stems and leaves of aviso led to a higher final n amount in seeds. brassica napusleaf senescencen remobilization efficiencyn use efficiencyproteolysisproteasomeissue-copyright-statement© the author 2015
 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 cultivated under ample or restricted nitrate supply.  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.  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 cysteine proteases and proteasome activities and 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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords brassica napusleaf senescencen remobilization efficiencyn use efficiencyproteolysisproteasomeissue-copyright-statement© the author 2015 