Journal of Experimental Botany Advance Access published April 30, 2014 Journal of Experimental Botany doi:10.1093/jxb/eru177

Review paper

Leaf senescence and nitrogen remobilization efficiency in oilseed rape (Brassica napus L.) Jean-Christophe Avice1,2,3,* and Philippe Etienne1,2,3 1 

Normandie University, F-14032 Caen, France UCBN, UMR INRA–UCBN 950 Ecophysiologie Végétale, Agronomie & nutritions N.C.S., F-14032 Caen, France 3  INRA, UMR INRA–UCBN 950 Ecophysiologie Végétale, Agronomie & nutritions N.C.S., F-14032 Caen, France 2 

*  To whom correspondence should be addressed. E-mail: [email protected]

Abstract Despite its worldwide economic importance for food (oil, meal) and non-food (green energy and chemistry) uses, oilseed rape has a low nitrogen (N) use efficiency (NUE), mainly due to the low N remobilization efficiency (NRE) observed during the vegetative phase when sequential leaf senescence occurs. Assuming that improvement of NRE is the main lever for NUE optimization, unravelling the cellular mechanisms responsible for the recycling of proteins (the main N source in leaf) during sequential senescence is a prerequisite for identifying the physiological and molecular determinants that are associated with high NRE. The development of a relevant molecular indicator (SAG12/Cab) of leaf senescence progression in combination with a 15N-labelling method were used to decipher the N remobilization associated with sequential senescence and to determine modulation of this process by abiotic factors especially N deficiency. Interestingly, in young leaves, N starvation delayed senescence and induced BnD22, a water-soluble chlorophyll-binding protein that acts against oxidative alterations of chlorophylls and exhibits a protease inhibitor activity. Through its dual function, BnD22 may help to sustain sink growth of stressed plants and contribute to a better utilization of N recycled from senescent leaves, a physiological trait that could improve NUE. Proteomics approaches have revealed that proteolysis involves chloroplastic FtsH protease in the early stages of senescence, aspartic protease during the course of leaf senescence, and the proteasome β1 subunit, mitochondria processing protease and SAG12 (cysteine protease) during the later senescence phases. Overall, the results constitute interesting pathways for screening genotypes with high NRE and NUE. Key words:  Brassica napus, leaf senescence, N remobilization efficiency, N transport, N use efficiency, proteolysis.

Introduction In order to satisfy the increased demand for food, feed, and biofuels that is resulting from human population growth, and to achieve this in a context of sustainable agriculture, the optimization of plant nutrient-use efficiency is now required more than ever. Because the energy cost of nitrogen (N) fertilizers is one of the highest budget items for farmers, research into genotypes having high N-use efficiency (NUE), in combination with optimization of N fertilization, is becoming

a priority for many crops, especially oilseed rape (Brassica napus L.). As the world’s second and third main crops for feed and food, respectively, oilseed rape represents a major renewable resource for human and animal food uses and for numerous non-food uses (biofuel, lubricants, cleansers, etc.). Over the last three decades, the world oilseed rape production has increased 5-fold (from less than 12 Mt in 1980 to 61.5 Mt in 2012) with a 10-fold increase in Europe (from 2 Mt in

Abbreviations: β-subunit A1, 20S proteasome β-subunit A1; GS, glutamine synthetase; LHC, light-harvesting complex; N, nitrogen; NAE, nitrogen-assimilation efficiency; NRE; nitrogen-remobilization efficiency; NUE, nitrogen use efficiency; NUpE, nitrogen uptake efficiency; NUtE, nitrogen utilization efficiency; MPP, mitochondrial processing peptidase; PGPH, peptidyl glutamyl peptide hydrolase; RCB, Rubisco-containing body, ROS, reactive oxygen species; Rubisco, ribulose 1,5 bisphosphate carboxylase/oxygenase; SAG, senescence-associated gene; SAG12, senescence-specific cysteine protease; SAV, senescence-associated vesicle; SDG, senescence-downregulated gene. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

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Received 14 January 2014; Revised 24 February 2014; Accepted 17 March 2014

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Fig. 1.  Schematic representation of N fluxes and associated components of NUE at the whole-plant level in B. napus. NUE can be subdivided into two main components: NUpE (yellow bubble) and NUtE (orange bubbles) that includes NAE and NRE. Flux of N taken up and of endogenous N (remobilization) are indicated in red and blue, respectively. Modified from Salon et al. (2014). 34S and 15N labelling to model S and N flux in plants and determine the different components of N and S use efficiency. Methods in Molecular Biology 1090, 335–346. “With kind permission from Springer Science and Business Media”.

have reported that the low NUE observed for oilseed rape is mainly related to a weak NRE. Indeed, the recycling of foliar N at vegetative stages of growth, which occurs during ‘sequential’ leaf senescence (i.e. a senescence progression with an acropetal gradient along the axis of the plant that affects leaves as they reach maturity) is not optimal and may result in a high level of residual N in fallen leaves. This type of senescence is characterized by the redistribution of nutrients (carbohydrates, lipids and proteins) degraded in the oldest leaves (source organ) in favour of growing tissues (i.e. the young sink leaves). Thus, during vegetative stages, the return of N to the soil from dead leaves can reach up to 140 kg N ha–1 year–1, directly affecting the economic and environmental assessments of this crop production. In addition, it is currently assumed that this lack of mobilization of foliar N during sequential senescence also affects the agronomic performance. For instance, the removal of 50% of rosette leaves at the beginning of the bolting stage resulted in a significant reduction (–30%) in the growth of reproductive organs of oilseed rape compared with plants that retained all rosette leaves (Noquet et  al., 2004). By using an 15N labelling technique and developing a modelling approach as a tool for identifying potential methods for improving the N harvest index of oilseed rape, Malagoli et  al. (2005b) showed by simulation that N content or seed yield could be improved by approximately 15% through optimizing leaf N remobilization during the vegetative stages or by reducing the residual percentage of N in dead leaves. During the last decade, a few studies have shown possibilities for improving NUE in oilseed rape via optimization of NUpE-related root traits, as reported by Schulte auf’m Erley et al. (2007). Transgenic approaches have also demonstrated that enhancement of NAE through overexpression of enzymes involved in the metabolism of N assimilation processes (alanine aminotransferase, asparagine transferase) may result in improvements to the NUE or N harvest index of oilseed rape (reviewed by Good and Beatty, 2011). Nevertheless, in a context of reduced fertilizer N inputs, the optimization of leaf NRE is probably one of the main levers for significantly improving the NUE in oilseed rape. Indeed, increasing N remobilization from senescent organs or damaged tissues represents an important adaptive trait allowing (i) better recycling in plants of N resources from the vegetative parts, (ii) limits to N loss in the dry remains, (iii) reductions in N fertilizer inputs, and (iv) finally increases in the agro-environmental balance of crop plants (MasclauxDaubresse et al., 2010; Gregersen et al., 2013). In leaves of oilseed rape, proteins and especially chloroplastic proteins represent the main source of N that might be remobilized during sequential senescence. During the vegetative stages, it seems that the weak NRE associated with sequential senescence might not be due to a limitation of the amino acid transport systems from mesophyll cells to phloem but is primarily related to an incomplete hydrolysis of foliar proteins (Noiraud et al., 2003; Tilsner et al., 2005). Therefore, unravelling the cellular mechanisms responsible for efficient remobilization of leaf proteins during sequential leaf senescence is a prerequisite for attaining improvements in the NUE in oilseed rape via NRE optimization. As reviewed by Roberts

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1980 to 19.2 Mt in 2012; Food and Agriculture Organization, http://www.fao.org). Oilseed production has a fundamental dependence on inorganic N with a current requirement for N fertilization between 140 and 200 kg N ha–1 year–1. Despite its high capacity for mineral N absorption, oilseed rape is characterized by a low NUE, with only 50% of the N absorbed by the plant being present at harvest in the seeds (Schjoerring et  al., 1995). Consequently, this can contribute to a serious concern regarding N loss in the field, giving rise to soil and water pollution from nitrate leaching, as well as air pollution from greenhouse gas emissions. In order to reconcile the environmental considerations and the agronomic goal of high seed yield and quality, an adjustment of N fertilization in combination with the use of genotypes having a high NUE has become a major objective for many crops and is a priority challenge for oilseed rape. NUE, for which the definitions and equations for estimation have been given in detail in previous reviews (Good et  al., 2004; Masclaux-Daubresse et  al., 2010; Good and Beatty, 2011; Hawkesford, 2011; Hirel and Lea, 2011; Kant et al., 2011; Xu et al., 2012), can be subdivided into two main components: N uptake efficiency (NUpE) and N utilization efficiency (NUtE) including assimilation/storage [N assimilation efficiency (NAE)], and remobilization ([N remobilization efficiency (NRE)] within/from plant organs (Fig.  1). Several studies in controlled or field conditions (Malagoli et  al., 2005a, b; Tilsner et  al., 2005; Gombert et  al., 2006)

Leaf senescence and NUE in oilseed rape  |  Page 3 of 12

Sequential leaf senescence and regulation Molecular indicators of leaf senescence progression in B. napus Leaf senescence is a genetically programmed developmental process whose timing is difficult to establish. The progression of leaf senescence is usually evaluated by different physiological indicators such as leaf yellowing or changes in protein and/or chlorophyll contents (Levey and Wingler, 2005). However, as these physiological traits are associated with steps that occur earlier or later during the senescence progression, comparisons of results from different studies are often tricky. As a consequence, it is now almost impossible to have a clear vision of the timing of all cellular and molecular events associated with leaf senescence. However, elucidation of the mechanisms involved in establishment and regulation of senescence is essential for both scientific knowledge and practical applications (Wu et al., 2012; Xu et al., 2012). As with other types of programmed cell death, plant senescence is accompanied by a decrease in protein synthesis (e.g. ribulose 1,5 bisphosphate carboxylase/oxygenase (Rubisco)], expression of senescence-downregulated genes (SDGs) often related to photosynthesis (e.g. Cab gene encoding a chlorophyll a/b-binding protein) and up-regulation

of senescence-associated genes (SAGs), as well as de novo synthesis of proteins (Buchanan-Wollaston et  al., 2003). Therefore, in addition to the range of physiological and biochemical markers conventionally used to quantify symptoms of leaf senescence, SDGs and SAGs constitute a pool of target genes available for acquiring the most suitable molecular indicators of senescence progression (Lim et  al., 2007; Balazadeh et al., 2008). Thus, in oilseed rape, Gombert et al. (2006) developed an accurate molecular indicator to monitor the spatio-temporal progression of sequential leaf senescence by quantifying the expression levels of two genes (Fig.  2): SAG12 (SAG encoding a cysteine protease up-regulated during senescence) and Cab LHCII type I (SDG encoding a chlorophyll a-binding protein associated with the light-harvesting complex II, which is down-regulated during senescence). For instance, for a given leaf rank of oilseed rape, the theoretical date of the onset of senescence can be determined by following kinetic expression of the SAG12 and Cab genes through semi-quantitative reverse transcription-PCR analysis. The intersection point corresponding to the concomitant up- and down-regulation of the SAG12 and Cab genes indicates the onset of foliar senescence (Fig. 2A). Moreover, this molecular indicator is also reliable for determining the last theoretical senescing leaf rank (i.e. the youngest leaf rank that has started to senesce) on the axis of whole plant at a given step of development in winter oilseed rape (Gombert et al., 2006). Indeed, the kinetic determination of the last theoretical senescing leaf rank allows the rate of leaf senescence progression during vegetative development to be obtained (Fig. 2B; Etienne et al., 2007). This is especially suitable for appraising the impact of biotic or abiotic stresses on the progression of leaf senescence and/or to compare oilseed rape genotypes for their ability to mobilize foliar N.

Abiotic stresses modulate sequential senescence progression in B. napus As for all plants, B.  napus must endure many abiotic stress conditions such as salinity, extreme temperatures, drought and/or low soil nutrient availability during its developmental cycle. In many instances, these harsh conditions induce morphological, physiological, and molecular modifications conferring tolerance to abiotic stresses. These changes are often associated with an acceleration of leaf senescence (called abiotic stress-induced senescence) in order to improve remobilization of nutrients from old leaves to young leaves, and finally to ensure the survival of the species. It is generally accepted that abiotic stress-induced senescence results in a significant reduction in crop yield (Gepstein and Glick, 2013). However, in oilseed rape, some studies have reported that unfavourable environmental conditions are not necessarily associated with an acceleration of leaf senescence. For instance, in response to limited S nutrition during the vegetative stages, it has been demonstrated via the SAG12/Cab molecular indicator that plants with high initial endogenous sulfate have a delay in leaf senescence (Fig. 2B2) and maintain shoot growth compared with control plants that are well supplied with S (Dubousset et  al., 2009). Thus, in these conditions of S limitation, S

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et al. (2012), the current knowledge on senescence-associated proteases has been achieved mainly in Arabidopsis using large-scale gene expression analysis or through the identification and characterization of enzymes harbouring proteolytic activity during senescence. Despite recent reports in cereals (Liu et al., 2008 for rice; Ruuska et al., 2008 for barley; Roberts et al., 2011 for wheat) and oleaginous crops (Otegui et  al., 2005 for soybean; Tilsner et  al., 2005; Desclos et  al., 2009 for oilseed rape), our knowledge concerning identification of the proteolytic systems involved in efficient remobilization of proteins during leaf senescence remains incomplete or vaguely understood in the case of plants with agronomic interests. In this review, the main features concerning the physiological and molecular processes involved in NRE will be described, highlighting the importance of remobilization associated with sequential leaf senescence in oilseed rape. Based on the development of a relevant and original molecular indicator of leaf senescence progression (SAG12/Cab), the first part will depict the phenomenon of sequential leaf senescence and how this process is mediated by abiotic factors such as soil N, sulfur (S) restriction, and/or light limitation in B.  napus. In the second part, the main changes in the plant physiological mechanisms and the main molecular actors that are specifically involved in leaf senescence will be described, with a special emphasis on N remobilization processes (proteolysis, amino acid and oligopeptide transport, etc.). In conclusion, we discuss the opportunity to use these physiological and molecular determinants as key markers to screen oilseed rape genotypes with improved NUE, especially in the context of low-nitrate fertilization.

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remobilization in favour of growing leaves involves the induction of sulfate transporters that is independent of the leaf senescence process (Dubousset et  al., 2009). In contrast, if plants with low initial S reserves (i.e. severe S deficiency) are exposed to S limitation, the mobilization of sulfate by specific transporters is not sufficient to maintain shoot growth (Abdallah et al., 2011). In this case, oilseed rape plants induce leaf senescence to degrade endogenous organic S compounds such as soluble proteins in order to improve S (and N) mobilization for emerging leaves. This work underlines that the

initial endogenous S reserves are a determining factor controlling the plant’s response to S deficiency (Abdallah et al., 2011). The impact of light restriction was also recently studied on biomass production and the progression of senescence in leaves of B.  napus (Brunel-Muguet et  al., 2013). Thus, a shading treatment corresponding to around a 43% reduction in the photosynthetically active radiations (i) induced the development of leaves on the main stem and ramifications, and (ii) provoked a delay in senescence in mature leaves (in

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Fig. 2.  Expression of SAG12/Cab genes, an accurate molecular indicator to monitor spatio-temporal progression of leaf senescence in B. napus (cv. Capitol). (A) Accumulation of transcripts of SAG12/Cab genes reveals the temporal progression of leaf senescence in B. napus. The different stages of leaf development correspond to: young leaf (YL), mature leaf (ML), senescing leaf (SL), and dead leaf (DL). (A1) Kinetic accumulation of SAG12 and Cab transcripts during leaf development of B. napus 25S rRNA was used as a cDNA synthesis and amplification control. (A2) Quantification of steady-state transcript levels of SAG12 and Cab genes during leaf development [expressed as a percentage of the maximum level (100%) observed in DL and in YL for SAG12 and Cab, respectively]. For a given leaf rank, the theoretical date of the onset of senescence is determined by the intersection point resulting from the concomitant up- and down-regulation of the SAG12 and Cab genes, respectively. (A3) Total N amount (in mg per leaf) in B. napus leaves at different stages of development. The theoretical date of leaf senescence matches the drastic decrease in the total amount of leaf N corresponding to the N-mobilization period. (Adapted from Gombert et al., 2006. The expression patterns of SAG12/Cab genes reveal the spatial and temporal progression of leaf senescence in Brassica napus L. with sensitivity to the environment. Journal of Experimental Botany 57, 1949–1956, by permission of Oxford University Press). (B) Accumulation of SAG12/Cab transcripts reveals the spatial progression of leaf senescence in B. napus in response to the variation in N or S nutrition. (B1) Schematic representation of the N status of B. napus leaves according to their nodal position along the axis and to the accumulation of SAG12/Cab transcripts. The last theoretical senescence leaf rank (*) subjected to the sink/source transition for N is determined by the intersection point corresponding to concomitant up- and downregulation of the SAG12 and Cab genes, respectively (Adapted from Etienne et al., 2007. N-protein mobilisation associated with the leaf senescence process of oilseed rape is concomitant with the disappearance of a trypsin inhibitor activity. Functional Plant Biology 34, 895–906. CSIRO Publishing). (B2) Schematic representation of kinetics progression of leaf senescence along the axis of B. napus in control (+N, +S), N-deprived (–N, +S), or S-deprived (+N, –S) plants. At each time of treatment, the last theoretical senescent leaf rank corresponding to the sink/source transition for N (*) is characterized by a concomitant up- and down-regulation of the SAG12 and Cab genes, respectively, as described in (B1). The slope (a, a′ and a′′) of each straight line provides information about the rate of progression of leaf senescence for the control (+N, +S), N-deprived (–N, +S), and S-deprived (+N, –S) plants. The coordinate at the origin (b) indicates the last theoretical senescing leaf at the beginning of the experiment, i.e. before treatment applications (Adapted from Dubousset et al., 2009. Remobilization of leaf S compounds and senescence in response to restricted sulphate supply during the vegetative stage of oilseed rape are affected by mineral N availability. Journal of Experimental Botany 60(11), 3239–3253, by permission of Oxford University Press; and from Abdallah et al., 2011. Do initial S reserves and mineral S availability alter leaf S-N mobilization and leaf senescence in oilseed rape? Plant Science 180, 511–520, with permission from Elsevier).

Leaf senescence and NUE in oilseed rape  |  Page 5 of 12 in young leaves showing a significant delay in senescence. These authors pointed out that the senescence delay in young leaves was concomitant with the induction of BnD22 (Fig. 3A, B), a 22 kDa B. napus serine protease inhibitor of the Künitz type (Reviron et al., 1992), which presents a basipetal gradient of accumulation that is opposite to the acropetal gradient of sequential leaf senescence (Fig. 3C). Interestingly, BnD22 is also known to be a water-soluble chlorophyllbinding protein (WSCP) of class II (specific class including Brassicaceae; Nishio and Satoh, 1997; Satoh et  al., 1998; Schmidt et  al., 2003; Reinbothe et  al., 2004; Desclos et  al., 2008). Interestingly, Horigome et  al. (2007) reported that, during leaf senescence, BnD22/WSCP forms a tetrameric BnD22–chlorophyll complex to protect chlorophyll against photodegradation. Moreover, in Brassica oleracea, Takahashi et al. (2012) proposed that WSCP/BnD22 might act as a scavenger of Chl when chloroplasts are injured during stressful conditions in order to avoid oxidative cell damage caused by radical species derived from chlorophylls. In B. napus, several studies have demonstrated that BnD22 and other WSCPs are accumulated in young leaves in response to various abiotic

Fig. 3.  Putative roles of a 22 kDa BnD22/WSCP in young leaves during N remobilization associated with leaf senescence in B. napus (cv. Capitol). L, ladder of molecular weight markers (kDa; Precision Plus Protein Dual Color Standards, Bio-Rad). (A) SDS-PAGE of soluble proteins extracted from a young leaf of control (+N) or N-deprived (–N) B. napus plants after 14, 21, and 28 d of treatment. Boxes indicate BnD22 (19 kDa) differentially accumulated in control (black box) or N-deprived (red box) young leaves. (B) Putative dual function of BnD22 protein in B. napus leaves. (B1) Partial two-dimensional gel electrophoresis immunoblot from young leaves performed with polyclonal anti-cauliflower WSCP of B. oleracea (Nishio and Satoh, 1997). Polypeptides recognized by the antibody were excised, digested, and identified by electrospray ionization/liquid chromatography/random mass spectrometry and matched with the BnD22 amino acid sequence (GenBank Protein accession no. X65637). (B2) BnD22 19 kDa tryspin inhibitor activity of the Künitz type in young leaves from B. napus supplied with (+N) or without (–N) nitrate over 21 d. Details of the method of revelation of trypsin inhibitor activity are available in Etienne et al. (2007). (C) Schematic representation of the acropetal senescence progression and basipetal gradient of BnD22 accumulation and trypsin inhibitor activity along the axis of B. napus plants supplied with (+N, black frame) or without (–N, red frame) nitrate. N deprivation induced an acceleration of senescence in basal old leaves associated with an earlier disappearance of BnD22. In contrast, BnD22 was highly accumulated in young leaves, which showed a slowdown in senescence progression. Physiological events in upper young leaves and basal old leaves associated with BnD22 and senescence modifications in response to N deprivation are indicated in the green and yellow shaded boxes, respectively (Adapted from Etienne et al., 2007. N-protein mobilisation associated with the leaf senescence process of oilseed rape is concomitant with the disappearance of a trypsin inhibitor activity. Functional Plant Biology 34, 895–906, CSIRO Publishing; and from Desclos et al., 2008. A proteomic profiling approach to reveal a novel role of BnD22 (Brassica napus drought 22 kD)/Water Soluble Chlorophyll binding protein in young leaves during nitrogen remobilization induced by stressful conditions. Plant Physiology 147, 1830–1844, Copyright 2008 American Society of Plant Biologists).

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lower positions in the canopy) allowing an extension of their lifespan. As a consequence, these mature leaves from shaded plants had a more efficient remobilization of their N compounds and they fell with lower residual N contents. All these studies suggest that oilseed rape is able to respond to abiotic stresses by induction of architectural changes and increasing the lifespan of mature leaves through delayed leaf senescence so as to optimize light capture and N remobilization from source organs (mature and old leaves) towards sink organs, i.e. young leaves and reproductive organs (Brunel-Muguet et al., 2013). In contrast, other abiotic stresses such as N deficiency are known to systematically accelerate sequential leaf senescence of oilseed rape. Thus, some studies carried out under greenhouse (Gombert et al., 2006; Etienne et al., 2007) or field conditions (Gombert et al., 2010) shown that, compared with N-supplied plants, B.  napus N-deprived plants present an earlier senescence of older leaves (Fig. 2B2) associated with a better mobilization of endogenous N compounds. Etienne et al. (2007) also demonstrated that, in addition to the induction of senescence in the older leaves, N deficiency also induces some physiological and molecular modifications

Page 6 of 12  |  Avice and Etienne stress conditions such as N limitation (Etienne et  al., 2007; Desclos et  al., 2009), S deficiency (D’Hooghe et  al., 2013), drought, or salinity (Reviron et al., 1992). All these data suggest that, in response to stressful conditions, accumulation of BnD22 (with a dual function: protease inhibitor and WCSP) and other WSCPs could contribute towards limiting protein and chlorophyll degradation in order to preserve metabolic activities (such as photosynthesis). This may result in improvement to the NUE through an increase in leaf lifespan and maintenance of the sink strength of these young leaves (Satoh et al., 2001; Desclos et al., 2008) until plants recover under more favourable conditions. From these studies, it could be suggested that protease inhibitors and WSCPs are determinant factors capable of finely regulating leaf senescence progression. Thus, it is likely that oilseed rape genotypes with contrasting accumulation levels of BnD22 and/or WSCPs in their leaves could show contrasting NRE and be more or less resistant to stressful conditions.

Based on recent results using a pulse–chase 15N-labelling method (which is particularly reliable for the determination of endogenous N fluxes; for details, see Salon et  al., 2014) and SAG12/Cab as a molecular indicator of the progression of leaf senescence (see above), it has been possible to study concomitantly the flux of N remobilization and the main metabolic changes associated with protein degradation from the initiation/early stages of senescence until the abscission of source leaves in oilseed rape (cv. Capitol) (Fig. 4). These integrative studies have allowed the establishment of the putative chronology of the physiological, biochemical, and molecular events associated with the processes of N remobilization/ recycling in source leaves of oilseed rape undergoing sequential senescence (Fig. 5).

What are the processes involved in N remobilization and proteolysis between leaf maturity and the early stages of leaf senescence in oilseed rape? As reported by several studies focused on the fluxes of N remobilization during the life span of oilseed rape leaves (Malagoli et al., 2005a; Etienne et al., 2007), it appears that N remobilization occurs precociously when leaf maturity has been attained and before the real commencement of senescence processes (Fig. 4). Thus, compared with the initial level observed at maturity, the N amount declines by 40% by the theoretical date of the onset of leaf senescence (Fig. 4C). This is concomitant with a 50% reduction in initial protein content (Fig. 4D) and a decrease of 35% in the initial amino acid content during the same period (Desclos, 2008). The analysis of protease activities by zymograms of gelatine–polyacrylamide gels reveals that the decline in proteins during this first phase of N remobilization is mainly ensured by constitutive

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Physiological processes and molecular actors involved in protein degradation and N remobilization during sequential leaf senescence in B. napus

proteases and peptidase activities (Desclos, 2008). Among these constitutive proteolytic enzymes, there are probably some chloroplastic and vacuolar proteases as well as the 26S proteasome complex in the cytosol (Fig. 5A). Following this first phase, the large N remobilization observed during the early stages of senescence is characterized by a rapid degradation of chlorophylls (Fig. 4C) and proteins (from 50 to 10% of initial total protein content, Fig. 4D), which is concomitant with the onset of dismantling of chloroplasts (Fig. 4A). As the main source of nutrients, chloroplasts are the first organelles to deteriorate during senescence and the initial steps of chlorophyll and chloroplastic protein degradation are likely to take place within the plastid itself (reviewed by Martínez et al., 2008). Thus, it is likely that N remobilization during the earlier stages of leaf senescence would mainly be associated with the degradation of stromal proteins (especially Rubisco) accumulated in the chloroplasts. Additionally, proteomics analysis using two-dimensional gel electrophoresis (Fig.  4E1) reveals that aspartic protease and chloroplastic FtsH are induced during the initiation of senescence and remain highly expressed during the early phases of senescence (Fig. 4E2). FtsH proteases (family M41) are membrane-bound ATP-dependent metalloproteases with a zinc-binding domain (reviewed by Wagner et  al., 2012). Among the 12 FtsH proteases, nine FtsH proteases have been localized in chloroplasts and are well known for contributing to plastid differentiation, degrading the core protein D1 in the photosystem II repair cycle (Nixon et al., 2010) and breaking down proteins of light-harvesting complexes (LHCII or Lhcb3) in Arabidopsis (Żelisko and Jackowski, 2004). Therefore, the specific induction of chloroplastic FtsH suggests that proteins associated with thylakoid membranes (proteins involved in the light-harvesting complexes of photosystems and in the electron transfer chain) are also degraded at early stages of leaf senescence in oilseed rape. The proteolysis of thylakoidal membrane proteins involved in the electron transfer chain may cause a rapid oxidative stress leading to the subsequent overproduction of reactive oxygen species (ROS) (Zimmermann and Zentgraf, 2005) and the acceleration of senescence. Accordingly, Bieker et al. (2012) recently demonstrated that an earlier increase of H2O2 levels coincided with an acceleration of leaf senescence in oilseed rape (cv. Mozart). ROS overproduction may lead to deleterious effects and to the fragmentation of numerous chloroplastic proteins that are then more suitable for complete proteolysis (Ishida et al., 1997). Interestingly, while the thylakoidal membranes were clearly altered from the early stages of senescence, the chloroplastic envelope remains intact for longer, as observed in transmission electronic microscopy (Fig. 4A). This suggests that protease pathways inside and outside the chloroplasts may cooperate to achieve the complete degradation of chloroplastic proteins (as reported in other plants including Arabidopsis) where the later steps of Rubisco proteolysis may take place within the central vacuole (reviewed by Martínez et  al., 2008). The translocation of plastid proteins to the central vacuole (lytic vacuole rich in serine, aspartic, and cysteine proteases) could be done via trafficking of senescence-associated vesicles (SAVs) or Rubisco-containing

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Fig. 4.  Changes in ultrastructure of mesophyll cells (A1, A2), and modifications of chlorophyll levels (B), leaf N amount (C), leaf protein amounts (D), and proteome profiles of the main identified proteases (E1, E2) during the progression of leaf senescence in B. napus (cv. Capitol). When the leaf became mature, N remobilization was observed precociously between the date corresponding to the maximum level of leaf protein amount (initial protein level=100%) and the theoretical date of the onset of senescence (C). This first phase of N remobilization also corresponded approximately to a 50% reduction in total initial protein content (D). The large N remobilization during the early stages of senescence was characterized by rapid degradation of chlorophylls (B) and proteins (from 50 to 10% of initial total protein content, D), which was concomitant with the onset of the dismantling of chloroplasts (A2) and the induction of proteases such as chloroplastic FtsH and aspartic protease (E2) (Desclos et al. 2009). The N remobilization associated with the later stages of senescence were characterized by a decline in protein amount (D), which was concomitant with the onset of the dismantling of many organelles in mesophyll cells (A2) and the induction of proteases such as aspartic protease, the 20S proteasome β subunit A1 (β1) and a senescence specific cysteine protease (SAG12) (E2). The theoretical date of the onset of leaf senescence was determined using the SAG12/Cab indicator (see Fig. 2 for details). (A1) and (A2) show transmission electron microscopy images of mesophyll cells before [Soil Plant Analysis Development, chlorophyll meter (SPAD): 50–60] and after (SPAD: 30–40) the theoretical date of the onset of senescence (a magnification of a chloroplast is inserted in each picture). In (C), N remobilization is expressed as the percentage of initial amount of N and determined using an 15N-labelling pulse–chase method. In (E2), profiles were defined on the basis of analyses of changes in protein abundance after two-dimensional gel electrophoresis determined every 7 d from leaf maturity to the final leaf senescence stages (Adapted from Desclos et al. 2009. A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomic events associated with N remobilization during leaf senescence induced by nitrate limitation or starvation. Proteomics 9, 3580–3608, Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).

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Fig. 5.  Schematic representation of the putative cellular events associated with N remobilization during the early (A) and later (B) stages of sequential leaf senescence in mesophyll cells of B. napus (cv. Capitol). (A) During the earlier stages of leaf senescence, N remobilization from proteins is associated mainly with the degradation of stromal proteins (especially Rubisco) accumulated in the chloroplasts. The specific induction of chloroplastic FtsH suggests that proteins associated with thylakoid membranes are also degraded at early stages of leaf senescence. The degradation of the proteins involved in the electron transfer chain associated with the photosystems may cause a rapid oxidative stress. The subsequent overproduction of reactive oxygen species (ROS) could lead to deleterious effects on proteins and to the fragmentation of numerous stromal proteins that are then more

Leaf senescence and NUE in oilseed rape  |  Page 9 of 12 protein involved in the transport of oligopeptides (Karim et  al., 2005) during N remobilization in senescent leaves of oilseed rape (Fig.  5 and unpublished data). Although these results need to be confirmed (especially for other amino acid and oligopeptide transporters), it seems that the phloem loading of amino acids and peptides is not the limiting factor in N remobilization in leaves of oilseed rape.

What are the processes involved in N remobilization and proteolysis during the later stages of leaf senescence in oilseed rape? During the later stages of leaf senescence, the chloroplasts are transformed into gerontoplasts and show numerous plastoglobules (Fig.  4A2), while the other organelles are also subjected to degradation (Fig. 5B). This general dismantling of organelles in mesophyll cells is associated with the induction of proteases such as the 20S proteasome β-subunit A1 (β1, proteolytic subunit of the 26S proteasome harbouring peptidylglutamyl peptide hydrolase activity, PGPH), the mitochondrial processing peptidase (MPP), and SAG12, a senescence-specific cysteine protease localized in vacuoles and especially in SAVs (Fig. 4E). In the 20S core particle of the 26S proteasome, β-subunit A1 is responsible for PGPH activity and cleaves misfolded, damaged, and/or partially degraded proteins previously polyubiquitinated by ubiquitin-conjugating machinery (Ciechanover, 1998). Moreover, during plant defence reactions, the β1-subunits of proteasomes are also known to act as negative regulators of NADPH oxidase (Lequeu et  al., 2005). The later stages of leaf senescence are also characterized by the occurrence of an oxidative burst resulting to toxic ROS effects on proteins released into the cytosol. These damaged proteins could be rapidly degraded by the 26S proteasome. Thus, because the 20S proteasome β-subunit A1 is induced during senescence in leaf of oilseed rape (Desclos et al., 2009), it can be proposed that 26S proteasome may play an important role in N recycling and in the modulation of ROS production (Fig. 5B). The induction of MPP also suggests an important role of this peptidase in N remobilization. MPP is an integral component of the respiratory cytochrome bc1 complex and cleaves some cytosolic protein precursors targeted to the mitochondrial matrix or inner membrane (Gakh et  al., 2002).

suitable for complete proteolysis. The translocation of plastid proteins to the central vacuole (lytic vacuole rich in aspartic protease) could be done via the trafficking of senescence-associated vesicles (SAVs) or Rubisco-containing bodies (RCBs). A RCB is an autophagosome obtained from a stromule (tubular extension of the chloroplastidial membranes). (B) During the later stages of leaf senescence, the chloroplasts are transformed into gerontoplasts and show numerous plastoglobules, while the other organelles are also subjected to degradation. This is associated with the induction of proteases such as the 20S proteasome β-subunit A1 (β1, catalytic particle of the 26S proteasome harbouring a peptidylglutamyl peptide hydrolase activity), the mitochondrial processing peptidase (MPP) and SAG12, a senescence-specific cysteine protease localized in vacuoles and especially in SAVs. Autophagy activities may be involved in the recycling of peroxisomal proteins (pexophagy) and mitochondrial proteins (mitophagy). The later stages of leaf senescence are also characterized by the occurrence of an oxidative burst resulting in toxic ROS effects on proteins released into the cytosol that might facilitate their degradation by the 26S proteasome. Moreover, the 26S proteasome may be involved in the negative regulation of ROS production as described previously by Lequeu et al. (2005). The translocation of oligopeptides and amino acids from the vacuole to the cytosol and from the mesophyll cells towards the companion cells prior to phloem loading appeared to not be limiting in B. napus (Tilsner et al., 2005). These observations suggest that the amino acid and the oligopeptide transporters as well as the conversion of amino acids into glutamine using the cytosolic isoform of glutamine synthetase 1 (GS1) are very efficient.

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bodies (Fig.  5A). SAVs are small vacuolar compartments that contain chloroplastic proteins such as Rubisco and glutamine synthetase 2 and are characterized by their high protease activities, especially SAG12 (Otegui et  al., 2005; Martínez et  al., 2008). This vacuolar transfer and processing of chloroplastic proteins has been observed at an early stage of natural leaf senescence in Arabidopsis (Ono et  al., 2013). Rubisco-containing bodies are autophagosomes obtained from stromules (tubular extensions of the chloroplastic envelope) (Chiba et al., 2003; Ishida and Yoshimoto, 2008). Moreover, recent studies have reported that autophagy machinery is induced during leaf ageing, in response to N starvation, and may control N remobilization in Arabidopsis under both limiting and ample nitrate conditions (Guiboileau et al., 2012). Aspartic proteases are localized principally in vacuoles and plastids and are the second-largest protease class  in plants after serine proteases (van der Hoorn, 2008). Interestingly, it was demonstrated in tobacco leaves that a chloroplastic aspartic protease (CND41) was involved in the in vitro degradation of Rubisco (Kato et al., 2004). In leaves of Arabidopsis plants subjected to N mineral limitation, the induction of a putative CND41 protease was accompanied by a decrease in Rubisco and correlated with the progress of senescence in recombinant inbred lines exhibiting different leaf senescence rates (Diaz et al., 2008). All these data are consistent with the hypothesis that aspartic proteases may play an important role in the degradation of chloroplastic proteins in the chloroplast itself and/or the lytic vacuole during the early steps of senescence in oilseed rape (Fig. 5A). Protein degradation generates oligopeptides and amino acids, thereafter being transported towards companion cells and exported to the sink organs via phloem loading (Fig.  5A). Results obtained from senescing leaves of oilseed rape (cv. Capitol) under greenhouse (Desclos, 2008) or field (Noiraud et al., 2003) conditions have shown that the amounts of amino acids decreased during the N recycling associated with senescence. This decrease is concomitant with a strong accumulation of transcripts encoding amino acid type BnAAP1 carriers (Noiraud et  al., 2003; Desclos, 2008). These results are similar to those obtained by Tilsner et al. (2005) in senescent leaves of oilseed rape. Additionally, recent transcriptomic approaches have revealed the induction of a major facilitator superfamily

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Conclusion and future developments The knowledge summarized in this review underlines the importance of developing a relevant leaf senescence indicator (SAG12/Cab) in order to determine the fine detail of sequential leaf senescence for a given leaf rank or at the whole-plant level in oilseed rape. Future developments will benefit from use of this molecular indicator as a means of verifying if there is a correlation between the duration of N

remobilization and the residual N contents of fallen leaves. Indeed, as the theoretical date of the onset of leaf senescence (determined by the SAG12/Cab indicator) is the starting point of the N remobilization period, and the fall of the leaf corresponds to the end of the N recycling phase, it will be easy to deduce the duration of N recycling to determine the level of correlation of this parameter with the amount of residual N in dead leaves. If such a correlation is established, a screening of genotypic variability could be performed based on use of the SAG12/Cab indicator in order to characterize genotypes with contrasting NRE. An integrative approach using the SAG12/Cab indicator, which includes a specific 15N-labelling method for determining the N fluxes of remobilization and analysis of the leaf proteome, has also highlighted the main proteins implicated in N remobilization during early and later stages of leaf senescence. Thus, the major proteases that are putatively involved in recycling of leaf proteins during sequential senescence have been identified. On the other hand, some studies suggest that other proteins (such as BnD22 with its dual function WSCP and protease inhibitor), may help to maintain the sink growth of stressed plants and contribute to a better utilization of recycled N from senescent leaves, a physiological trait that could improve NUE in combination with a high NRE in senescent leaves. To determine the importance of these proteins in the N remobilization associated with sequential senescence and the maintenance of NUE in young sink organs, mutagenesis/transgenic approaches could be achieved in future investigations. Another strategy would be to explore the genotypic variability of NRE to verify if these proteins are associated with a better N remobilization during senescence and a higher NUE (Masclaux-Daubresse and Chardon, 2011). In a recent comparative study of genotypes presenting contrasted NUE (unpublished data), our work revealed that genotypes with the highest NUE under nitrate limitation are mainly characterized by a better NRE in senescent leaves. Moreover, the young growing leaves of these genotypes are also characterized by a better utilization of N originating from both the senescent leaves and nutrient solution. Otherwise, genotypes having high NRE are characterized by an efficient proteolysis of insoluble and soluble proteins in senescent leaves while insoluble protein proteolysis is less efficient for genotypes with low NRE. The first analysis of leaf proteome and protease activities in these genotypes suggests a significant role for FtsH proteases that are specifically involved in the degradation of insoluble proteins associated with thylakoids (unpublished data). Additionally, a proteomics characterization of proteins specifically involved in efficient N remobilization in senescent leaves and maintenance of NUtE in young leaves could lead to a ‘candidate proteins’ strategy to identify quantitative trait loci related to NUE. Such candidates may be particularly relevant to breeding programmes aimed at identifying B.  napus genotypes with efficient nutrient management (especially N) in response to fluctuating nutritional availabilities and low fertilizer inputs.

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These results suggest that MPP may have a function in leaf senescence by facilitating importation of cytosolic proteins into mitochondria to maintain mitochondrial functions for energy production for as long as possible. Among the mechanisms that could contribute to the remobilization of N compounds originating from organelles such as mitochondria, it is also important to mention that specific autophagy activities may be involved in the recycling of mitochondrial proteins (mitophagy) or peroxisomal proteins (pexophagy) (Fig. 5B). The cysteine protease SAG12 (BnSAG12-1) was induced 17-fold during the last stages of leaf senescence (Fig. 4E). Using transgenic Arabidopsis plants, it was reported that SAG12 localizes to SAVs that were present in the peripheral cytoplasm of mesophyll and guard cells and was characterized by an intense proteolytic activity (Otegui et al., 2005; Prins et al., 2008). These data suggest that SAG12, the 26S proteasome, and also aspartic protease, which remain abundant (Fig.  4E), are proteases more specifically involved in proteolysis occurring at the final stages of leaf senescence in oilseed rape. As observed during the early stages of senescence, the translocation of oligopeptides and amino acids from the vacuole to the cytosol and from the mesophyll cells towards the companion cells prior to phloem loading will not be limiting in the last phases of senescence in oilseed rape (Fig. 5B) (Tilsner et al., 2005; Desclos, 2008). These observations suggest that amino acid and oligopeptide transporters as well as the conversion of amino acids into glutamine using the cytosolic isoform of glutamine synthetase 1 (GS1) are efficient as far as possible. In accordance with this hypothesis, proteomics approaches have revealed the induction of glutamate ammonia ligase (BnGSR2), i.e. GS1 in B. napus, during this phase of leaf senescence (Desclos et al., 2009). GS1 is a key enzyme responsible for ammonia incorporation into glutamate leading to the formation of glutamine, one of the main amino acids alongside asparagine, glutamate, and aspartate that is remobilized from senescing leaves to growing parts of plants through the phloem (Ochs et al., 1999; Masclaux et al., 2000; Diaz et  al., 2008). The upregulation of BnGSR2 observed during leaf senescence (Desclos et al., 2009) suggests a crucial role for this enzyme in the recovery of N from senescing leaves. In addition to GS1, asparagine synthetase, an enzyme that transfers the glutamine-amide group to aspartate forming asparagine and glutamate in companion cells, could also be essential for N distribution and remobilization via the phloem during the vegetative stage, as reported recently in Arabidopsis thaliana by Gaufichon et al. (2013).

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Acknowledgements The authors thank Dr Marie Desclos and Dr Lucie Dubousset for their invaluable contributions to this work, and Maxence James and Dr Didier Goux (CMABio, Centre de Microscopie Appliquée à la Biologie, Université de Caen Basse-Normandie) for their assistance in the achievement of observations by confocal and transmission electron microscopy. The authors also wish to acknowledge Dr Laurence Cantrill for proofreading and English correction.

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