Remobilization of vegetative nitrogen to developing grain in wheat (Triticum aestivum L.)

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Review

Remobilization of vegetative nitrogen to developing grain in wheat (Triticum aestivum L.) Lingan Kong ∗ , Yan Xie, Ling Hu, Bo Feng, Shengdong Li Crop Research Institute, Shandong Academy of Agricultural Sciences, Jinan 250100, China

a r t i c l e

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Article history: Received 6 April 2016 Received in revised form 19 June 2016 Accepted 20 June 2016 Available online xxx Keywords: Grain protein content Nitrogen remobilization Sink/source relationship Senescence Wheat

a b s t r a c t A substantial percentage of the grain nitrogen (N) in cereal crops originates from the remobilization of N stored in vegetative tissues before anthesis. In wheat (Triticum aestivum L.), the percentage can reach 90%. All vegetative plant parts, including the leaves, stem, sheath, chaff and root, reserve N prior to anthesis and function as sources of nutrients during the grain filling phase. The developing grain per se accumulates nutrients as a sink. Nitrogen remobilization (NR) may be initiated immediately after anthesis, as indicated by decreases in the N or protein contents of the flag leaves. Senescence, a process that involves high macromolecular degradation for grain filling, begins approximately 8–16 days after anthesis. The resulting large amount of N compounds, mainly amino acids, will be remobilized to the grain. Precise programming of the timing and duration of NR and senescence is important. A large body of evidence has demonstrated that NR and senescence are under genetic control. Functional Gpc-1 genes, in combination with the NAC and WRKY transcription factors and their important targets, are involved in early senescence and enhance the grain protein content (GPC). NR and senescence are also regulated by a delicate balance between sink strength and source capacity, environmental factors and field managements. A better understanding of all these steps will reveal potential strategies to further increase GPC and processing quality and to minimize environmental contamination due to excess fertilizer run-off and associated expenses. © 2016 Elsevier B.V. All rights reserved.

Contents 1. 2. 3.

4.

5.

6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 N sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Regulation of NR by sink and source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.1. Initiation of NR and grain N demand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.2. Source-sink relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 3.3. Negative correlation with post-anthesis N uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Relationship between NR and senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.1. Timing of the onset of NR and senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 4.2. Regulation of NR by senescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Key points of physiological regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.1. Proteases and protein degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.2. Glutamine synthetase and asparagine synthetase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.3. Glutamate dehydrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 5.4. Autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Genetic control of NR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.1. Genes for the NAC-domain and WRKY

∗ Corresponding author at: Crop Research Institute, Shandong Academy of Agricultural Sciences, 202 Gongyebei Road, Jinan City 250100, China. E-mail address: [email protected] (L. Kong). http://dx.doi.org/10.1016/j.fcr.2016.06.015 0378-4290/© 2016 Elsevier B.V. All rights reserved.

Please cite this article in press as: Kong, L., et al., Remobilization of vegetative nitrogen to developing grain in wheat (Triticum aestivum L.). Field Crops Res. (2016), http://dx.doi.org/10.1016/j.fcr.2016.06.015

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transcription factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.2. Gpc-B1 and NAM-B1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.3. Genes for protein degradation and N transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 6.4. Other genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 7. Environment modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 7.1. N management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 7.2. Water availability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 7.3. Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 8. Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction Nitrogen (N) is one of the essential nutrients required for plant growth; for example, 20–50 g of N is required by (Triticum aestivum L.), rice (Oryza sativa L.) and maize (Zea mays L.) to produce 1 kg of grain (Robertson and Vitousek, 2009). However, crop plants are only able to use 30%–40% of supplied N for productivity; the remainder collects in the environment, leading to severe environmental pollution (Hirel et al., 2007). N use by cereal crops to produce grain protein involves multiple processes, including N uptake, assimilation, translocation, and remobilization. N remobilization efficiency (NRE; defined as N remobilization divided by total N at anthesis) is considered as an important attribute for increasing the grain protein content (GPC) and the baking quality of flour (MasclauxDaubresse et al., 2008; Criado et al., 2009). During the reproductive growth of cereals, root activity and nutrient uptake generally decrease, leading to the initiation of remobilization processes and the provision of nutrients for the development of newly emerged sinks. In cereals, a large amount of grain N is derived from these remobilized stores, a process that is strongly dependent on genotype and environment (Kichey et al., 2007; Maillard et al., 2015). Thus, the rate of nitrogen remobilization (NR) in the post-anthesis period is closely associated with the rate of grain N filling (Slimane et al., 2013) and depends on the amount of N stored in the vegetative parts at anthesis (Masclaux-Daubresse et al., 2008; Criado et al., 2009). A greenhouse experiment showed that higher N-utilization efficiency (NUtE; the ratio of grain weight to total N in the plant at maturity) in an Nefficient compared to an N-inefficient cultivar was attributed to higher NRE (Tian et al., 2015). Therefore, improving NR would be beneficial to increase nitrogen use efficiency (NUE; defined as the grain yield per unit of soil N plus fertilizer N) and N acquisition during grain filling. Wheat plants accumulate most of their N prior to anthesis, and it is remobilized to the ear during grain filling, where it is used for the synthesis of grain proteins (Triboi and Triboi-Blondel, 2002). The proportion of grain N originating from remobilization is estimated to be approximately 40–90% in wheat (Slafer et al., 1990; Kichey et al., 2007; Masoni et al., 2007; Waters et al., 2009; Bogard et al., 2010; Ercoli et al., 2008, 2010) and even up to 95% (Palta and Fillery, 1995), which is significantly higher than the maximum of ∼40% in maize (Maillard et al., 2015). Most of this variability can be attributed to differences in genotypes, climate, soil types and field management. In addition, the environmental conditions, such as the climate factors and soil water and nutrient availability, during the pre- and post-anthesis periods are likely to have different effects on N accumulation. The accumulation of N before anthesis is the major source of grain N. The greater the amount of N accumulated before anthesis, the higher the translocation rates of N to the grain and the lower the risk of net N losses as NH3 volatilized directly from plants during grain filling (Palta and Fillery, 1995). Therefore, one possi-

ble avenue for increasing the grain N yield is improving the NRE. However, the mechanisms that regulate the rate of NR from the vegetative organs to the grain are far from clear in wheat (Barneix, 2007; Bancal, 2009). A better understanding of the regulation of NR at genetic and physiological levels under field conditions is required so that the NUE, grain yield, grain quality and air and underground water quality can be improved. Therefore, we carried out an extensive literature review to summarize the factors that regulate the processes of NR. In this review, NR is defined as total plant N at anthesis minus straw N at maturity; NRE is defined as NR divided by total N at anthesis according to Bogard et al. (2010). In these definitions, we did not take into account the post-anthesis plant N uptake and net N losses.

2. N sources In cereal crops, the leaves, stem, glumes and roots are considered as the N sources for the development of grains (Barbottin et al., 2005; Masoni et al., 2007). In barley, NR to fill the grain occurs predominantly from the leaves and stem (Egle et al., 2015). In wheat, the leaf and leaf sheath are important stores of N (Gaju et al., 2011; Pask et al., 2012; Barraclough et al., 2014) because they are generally more efficient than other organs with respect to NR (Xu et al., 2006; Bahrani et al., 2011) (Table 1). In particular, the amount of N contributed by the flag leaf was approximately 18%, and NR from the flag leaf was positively correlated with the N yield per spike and per area (Wang et al., 2008). Under N stress, the leaf NR was found to be a determinant of genetic variation in GPC but not of grain yield (Gaju et al., 2014). Stem N was also considered a major N pool because a large of amount of soluble N compounds was accumulated in transit in the vascular system at anthesis (Barraclough et al., 2014). Peduncle N was positively correlated, and quantitative trait loci (QTLs) were colocalized with grain N and flag leaf N assimilatory traits (Habash et al., 2001), suggesting that stem N can be indicative of the grain N status in wheat. Other organs such as ears have the capacity to accumulate large amounts of N during the pre-anthesis period. Notably, the glumes act as both a temporary sink and source of N that will soon be remobilized to the grains at early grain-filling stages (Lopes et al., 2006). In the glumes of domesticated wheat, Zou et al. (2015) reported that the amino acid permease (AAP) gene was up-regulated compared to the wild progenitor. Similarly, in barley, the regulation of HvAAP gene expression in glume development was closely associated with the remobilization and accumulation of N in grains (Kohl et al., 2015). However, root N appears to be a relatively less important source of remobilized N (Sun et al., 1996; Soon et al., 2008) (Table 1). Interestingly, the amount of N remobilized from organs other than leaves may have an important implication for grain filling as a mechanism of buffering leaf senescence in the post-anthesis period. For example, glumes were observed to lose twice as much of their total N content as that lost by the flag leaf between the milk and

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Table 1 Variations in N remobilization of total vegetative tissues or different organs. Source

NRE (%) (reference)

Determined by

Total vegetative tissues

40–90 (Slafer et al., 1990; Guitman et al., 1991; Barbottin et al., 2005; Tahir and Nakata, 2005; Kichey et al., 2007; Masoni et al., 2007; Gaju et al., 2014; Bogard et al., 2010) 75–76 (Sun et al., 1996; Kichey et al., 2007; Pask et al., 2012) 61 (Pask et al., 2012) 43 (Sun et al., 1996); 48 (Pask et al., 2012); 73 (Kichey et al., 2007) 47 (Sun et al., 1996) 56 (Pask et al., 2012); 73 (Kichey et al., 2007)

Generally using 15 N labeling

Leaf lamina Leaf sheath True stems Roots Chaff (spike)

early dough stages (Lopes et al., 2006). It was also hypothesized that under N deficiency, an increase in stem NR may delay senescence by buffering lamina NR to the grain (Foulkes et al., 2009). Stem and/or sheaths probably regulate grain filling by mobilizing N, although half of the mobilized N originated from the blades (Slimane et al., 2013). Similar results were observed by Gaju et al. (2014). 3. Regulation of NR by sink and source 3.1. Initiation of NR and grain N demand Investigation of the effects of the source-sink ratio during grain filling on grain growth may enhance better understanding of nutrient remobilization. Generally, NR is considered to be triggered by the N demand of developing grains (the main sink) and then regulated by the source-sink relationship (Zhang et al., 2012) (Fig. 2). Jenner et al. (1991) surmised that the N demands of developing grains induced leaf senescence. In contrast, stay-green phenotypes, characterized by their capacity to retain chlorophyll in the photosynthetic apparatus during senescence (Thomas and Howarth, 2000), result from a low sink N demand because the source vegetative N concentration at anthesis was not higher in the staygreen phenotype compared to the wild type (Derkx et al., 2012). Therefore, NR may be associated with sink demand and canopy senescence. Indeed, investigations involving source/sink manipulations have clearly shown that higher sink strength (for example, higher grain number per area) might accelerate both NR and leaf senescence during grain filling. By contrast, sink reduction (through spikelet removal) significantly delayed the onset of senescence in wheat and other cereal species (Mi et al., 2000; Bogard et al., 2011), whereas source reduction (through defoliation and shading the spike) accelerated senescence (Bogard et al., 2011). A cultivar with small spikes (low sink demand) showed a low capacity to take up more N after anthesis and exhibited rapid senescence (Mi et al., 2000). However, the opposite results have also been reported, i.e., where both NR and leaf senescence were delayed by defoliation, probably because the plant reacts to changes in the source-to-sink ratio by increasing the efficiency of the remaining source organs (Guitman et al., 1991). 3.2. Source-sink relationship Currently, controversy exists regarding how NR is regulated during grain filling. Martre et al. (2003) suggested that the rate of NR is not driven by grain N demand but rather by the source supply from the vegetative tissues. The final GPC has been correlated with the free amino acid concentrations in the flag leaf during grain filling (Caputo et al., 2001). The ditelosomic line of Chinese Spring wheat CSDT7BL, which is deficient in the export of several amino acids to the phloem, also shows a decreased GPC compared to the normal line (Caputo et al., 2001). Bancal (2009) reported that both NR and grain number were strongly correlated with the stem N content at anthesis. These physiological results with respect to

Dumas analysis using 15 N labeling Dumas analysis Dumas analysis using 15 N labeling Using 15 N labeling Dumas analysis using 15 N labeling

source-regulation of GPC have been confirmed through the use of simulation models under wide ranges of field managements and environments (Jamieson and Semenov, 2000; Martre et al., 2003). Furthermore, using the coupled model SiriusQuality1 to simulate the dynamics of total grain N and of the different grain protein fractions confirms that accumulation of total grain N is sourceregulated rather than sink-regulated, at least under non-luxury N conditions (Martre et al., 2006). Recently, source manipulation, by cutting blades, immediately reduced grain N filling, whereas wrapping blades did not change grain N filling, and it has been proven that grains apparently did not regulate their N-filling rate themselves (Slimane et al., 2013), strongly suggesting that NR may be largely source regulated. However, Fischer (2008) argues that the source limitation mainly occurs under N-limiting conditions, whereas under nonN-limiting conditions, grain growth is mainly sink limited. Similar results have been observed by Serrago et al. (2013) in wheat and barley. Interestingly, temporal changes in grain N concentration suggest that limitation of protein deposition in the grain shifts from sink to source in barley (Dreccer et al., 1997). These results indicate that N storage during the vegetative phase does not often limit the final GPC and grain yield. Using biotechnologies to increase the sink capacity with balanced source reserves is necessary to increase NRE.

3.3. Negative correlation with post-anthesis N uptake Post-anthesis N uptake during grain filling strongly affects NR and leaf senescence (Fig. 2). Ample availability of soil N during grain filling favors post-anthesis N uptake and reduces the remobilization of pre-anthesis N (Papakosta and Gagianas, 1991). The proportion of remobilized N in the grain is inversely proportional to available soil N (Barbottin et al., 2005; Suprayogi et al., 2011). An experiment conducted across 12 sites also demonstrated that NR is significantly negatively correlated with post-anthesis N uptake, with correlation coefficients ranging from −0.51 to −0.87 (Bogard et al., 2010). Negative correlations were observed between post-anthesis N uptake and lamina NR, i.e., r = −0.61 at low N and r = −0.79 at high N; the correlations between post-anthesis N uptake and stem NR were r = −0.57 at low N and r = −0.80 at high N. Among cultivars under low N conditions, post-anthesis N uptake was negatively correlated with lamina NR (r = −0.75) and stem NR (r = −0.63) or a weak negative correlation was observed in some cases (Gaju et al., 2014). In maize, regardless of the level of N fertilization, it appears that there is strong opposition between NR and post-anthesis N uptake (r = −0.80) (Gallais and Hirel, 2004). These results may be due to post-anthesis N uptake being preferentially transported to the spike to meet the N demand of developmental grains (Egle et al., 2015) and may be related to the biosynthesis and/or degradation of hormones (Fig. 2).

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4. Relationship between NR and senescence 4.1. Timing of the onset of NR and senescence A continuous decrease in N or the protein concentration in vegetative organs after anthesis in wheat (Guitman et al., 1991; Feng et al., 2009; Wang et al., 2013) and barley (Dreccer et al., 1997), which likely constitutes the development of various spike tissues and peduncle elongation, ovary growth, seed set and grain filling, indicates that NR occurs immediately after anthesis. In some cases, the initiation of NR from vegetative to reproductive organs is even observed after heading, e.g., Soon et al. (2008). Senescence, the programmed degradation of cell constituents, makes nutrients available for remobilization to developing seeds (Uauy et al., 2006a, 2006b). A series of metabolic events, including chloroplast degeneration, hydrolysis and remobilization of macromolecules and the disintegration of cellular organelles, occurs during this process. Ultrastructural observation using transmission electron microscopy (TEM) showed that at 16 days after anthesis (DAA) in comparison to 8 DAA, there was obvious chloroplast degradation and chromatin condensation in the cells of vegetative tissues as well as a marked increase in the number of plastoglobuli in wheat (Kong et al., 2015a, 2015b) (Fig. 1). This suggests that programmed senescence might be initiated between 8 and 16 DAA. This postulation is strongly supported by the constant values of chlorophyll fluorescence (Kong et al., 2015a) and net photosynthesis (Wang et al., 2013; Xu et al., 2016) and a similar or slight increase in the chlorophyll content (an indicator of a senescence biomarker) of the flag leaf at the early stages of grain filling (Guitman et al., 1991; Kichey et al., 2005; Derkx et al., 2012; Guttieri et al., 2013; Zhao et al., 2015). Similar results were reported by Pearce et al. (2014). These authors observed that during the first 12 DAA, the SPAD values increased compared with those measured at anthesis and no visible signs of senescence appeared; during this period, some mechanisms were required to actively prepare the plant for the upcoming senescence. Kohl et al. (2015) reported that some NAC (NAM, ATAF 1.2 and CUC2) transcription factors were upregulated in the glumes at 14 DAA and were obviously associated with developmental senescence. In addition, it is well known that decreasing the sink capacity would decrease the N demand and the rate of NR and would delay plant senescence, as observed in sourcesink manipulation experiments (Sinclair and de Wit, 1975; Martre et al., 2006). In this scenario, we may conclude that NR occurs before the onset of canopy senescence, but the N remobilized before senescence might originate from storage proteins as opposed to those with metabolic functions, at least up to an amount that does not affect essential physiological processes such as photosynthesis. Indeed, the patterns of NR before the initiation of senescence are different from those after the initiation because the transition between these phases is accompanied by changed expression of specific N transporters (Kohl et al., 2015). Furthermore, considering that senescence occurs at least 8 DAA while NR occurs immediately after anthesis, source N deficiency induces earlier leaf senescence, and post-anthesis N uptake can meet the sink N demand and delay leaf senescence. Consequently, it is hypothesized that presenescence NR from the leaves to the spike seems to be involved in the onset of leaf senescence (Gregersen et al., 2008; Bogard et al., 2011). 4.2. Regulation of NR by senescence After the initiation of canopy senescence, the rate of NR from the vegetative organs to the grain is considered to be governed by programmed senescence (Gregersen et al., 2008; Bogard et al., 2011). Accelerated senescence favors high NR, leading to a high

GPC, but may have a detrimental effect on grain yield due to a reduction in the duration of C assimilation (Triboi and TriboiBlondel, 2002), probably because most C in the grain originates from post-anthesis photosynthesis and the amount of canopy N remobilized post-anthesis was negatively associated with the duration of post-anthesis senescence. However, the onset of senescence was negatively correlated with NRE among wheat genotypes under low N supply (Gaju et al., 2011). In contrast, a delay in vegetative senescence (stay-green trait) increased the duration of leaf photosynthesis during grain filling but resulted in a delay in NR and a reduction in NRE and had a negative impact on protein deposition in the grain (Van Oosterom et al., 2010a,b). The delayed senescence of TaNAM-RNAi plants decreased GPC and only provided a yield advantage under optimal temperatures as opposed to heat or water stress (Guttieri et al., 2013). In this scenario, unfavorably delayed leaf senescence is a concern with respect to low NR and grain yield in wheat due to large amounts of nutrients retained in vegetative organs. Consequently, the patterns of senescence should correspond to NR. A locus associated with early leaf senescence and increased GPC was identified on chromosome 6B in wild emmer wheat (Triticum turgidum subsp. dicoccoides). Recombinant wheat with this Gpc-B1 locus displayed early leaf senescence and improved remobilization of amino acids from the flag leaf, thereby increasing GPC without reducing grain yield (Kade et al., 2005; Uauy et al., 2006a, 2006b). 5. Key points of physiological regulation Members of the amino acid transporter family and the nitrate/peptide transporter family are key components in NR (Kohl et al., 2015). Approximately 80% of the N in wheat grain is supplied by amino acids that are disassembled from proteins and recycled and remobilized from vegetative tissues during grain filling (Barneix, 2007; Masclaux-Daubresse et al., 2008; Waters et al., 2009). A large number of enzymes are involved in these processes. 5.1. Proteases and protein degradation The rate of N uptake after anthesis is low due to decreasing root activity and hence the plant N demand cannot be fully met. This situation reduces the biosynthesis of plant hormones such as cytokinin, leading to protein degradation of vegetative plant parts (Fig. 2). During senescence, proteases are rapidly activated to degrade leaf proteins into amino acids (Guitman et al., 1991). Plastidial proteins are hydrolyzed to amino acids by proteolytic enzymes and then exported to the phloem and eventually transported to the developing grains (Hirel and Gallais, 2006; Okumoto and Pilot, 2011). Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC: 4.1.1.39) is mostly known as a key enzyme involved in CO2 assimilation during the Calvin cycle; however, comparatively little is known about its role as a pool of N storage in green tissues (Aranjuelo et al., 2015). In diploid wheat, Rubisco degradation was mediated by high endoproteolytic activities, which led to a higher rate of NR and a higher senescence rate compared to the tetraploids and hexaploids (Srivalli and Khannachopra, 2004). The induction of two subtilisin-like proteases (P1 and P2) was temporally associated with the degradation of the Rubisco small and large subunits in the flag leaf as well as with GPC, leading to the conclusion that these proteases might participate in NR to developing grains. Interestingly, these enzymes might be regulated by a cytokinin-mediated mechanism because during dark-induced senescence of the detached leaves, the induction of P1 was completely prevented and the induction of P2 was reduced (Roberts et al., 2011). Serine proteases are the most important family of proteases participating in NR during grain filling, acting as

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Fig. 1. TEM micrographs showing the ultrastructure of the chloroplast at different stages of the flag leaves (A–E) and glumes (F–J). At the end of anthesis and 8 DAA, chloroplasts are well differentiated in cells of both organs, containing fully developed grana with numerous layers and well-developed stroma lamellae (A, B, F and G). At 16 DAA, the thylakoid membranes are slightly dilated, the thylakoid stacks are irregularly arranged, and the number of plastoglobuli markedly increased, indicating the onset of senescence (C and H). At 24 DAA, the shape of the chloroplasts changed from lens-like to round and the ultrastructure of the chloroplasts is characterized by a disintegrating envelope, irregularly shaped thylakoids and irregularly arranged thylakoid grana with fewer stacks (D and I). At 32 DAA, thylakoid membranes are disintegrated and the entire structure of the chloroplasts is ruptured (E and J). Abbreviations: Ch, chloroplast; G, granum; P, plastoglobule; S, starch grain. Scale bars: 500 nM.

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Fig. 2. Diagram summarizing a model for the regulation of NR to the grain in wheat. NR is under genetic control and is regulated by the interactions between sink strength and source capacity, between ABA and other hormones and between the environment and physiological processes. The blue arrows indicate the promoting effects. The red lines indicate the inhibitory effects. The dashed line indicates the amino acid export to the phloem and the filling grain. CKs, cytokinins; NR, nitrogen remobilization; TF, transcription factor; ZR, zeatin-riboside. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

major regulators and executors in wheat and barley (Hollmann et al., 2014). However, the mechanism of Rubisco degradation by the senescence-associated proteases remains unknown; the way in which proteolysis and the export rate of amino acids are regulated is far from clear (Barneix, 2007; Gregersen et al., 2008). 5.2. Glutamine synthetase and asparagine synthetase Glutamine and asparagine are considered as the main amino acids involved in NR in the phloem sap of many plant species (Masclaux-Daubresse et al., 2008). Using cultivars exhibiting contrasting NUE (Kichey et al., 2007) and a quantitative genetic approach (Habash et al., 2001), GS activity has been shown to be closely associated with the extent of NR in wheat. The localization of cytosolic GS in the vascular tissues and the developmental regulation of cytosolic GS suggest that GS is important for NR (Habash et al., 2001; Bernard et al., 2008; for a recent review, see Bernard and Habash, 2009) (Fig. 2). QTLs for GS activity were invariably co-localized with those for grain N, with increased activity associated with higher grain N (but with no or negative correlations with grain yield components). One reason may be that wheat plants over-expressing GS in green tissues produce more biomass and accumulate more N during vegetative growth, clearly contributing to higher NR during grain filling (Habash et al., 2001). The other reason may be that during senescence, higher GS with the concerted action of glutamate synthase produces a larger amount of glutamine and glutamate that are then transported from the leaves to the developing grains. However, Peeters and Vanlaere (1994) reported that as senescence progresses, total GS and especially ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) activities declined continuously, but the cytosolic GS (GS1) was shown to be very persistent. A large amount of GS1 was detected in the connections between the mestome sheath cells and the vascular cells of mature

flag leaves (Kichey et al., 2005). Similarly, in rice, GS1 was only found to be localized in the vascular tissue in mature or senescing leaves (Tobin and Yamaya, 2001). These findings indicate that an active transfer of organic N molecules might occur within the vascular system and that GS1 plays a special role in the NR process, probably being solely responsible for the synthesis of glutamine that is exported to the grains for storage protein synthesis. Although an important physiological role of GS1 in the modulation of amino acids export in wheat has been demonstrated, contrary results were also reported by Caputo et al. (2009): N depletion and/or 6benzylaminopurine application induced a higher expression of GS1 but a decrease in the exudation of amino acids to the phloem. Asparagine and arginine are two of the main amino acids involved in N recycling and remobilization. Consequently, asparagine synthetase (AS) is proposed to have an important function in NR in maize (Li et al., 2016), and arginase encoded by OsARG, a key enzyme in arginine catabolism, plays a critical role during panicle development, especially under conditions of insufficient exogenous N in rice (Ma et al., 2013). However, their role in the regulation of NR during the grain filling of wheat is not well defined.

5.3. Glutamate dehydrogenases Glutamate dehydrogenases (GDH) seem to play an important role in NR (Bernard et al., 2008; Bernard and Habash, 2009). GDH aminating activity was strongly induced in the mitochondria and the cytosol of phloem companion cells of senescing flag leaves, suggesting that the shift in GDH cellular compartmentation is important during leaf NR (Kichey et al., 2005). However, TercéLaforgue et al. (2004) concluded that GDH does not play a direct role during the process of NR but rather is induced following a build up of ammonium. Therefore, the metabolic or sensing role of this enzyme remains to be elucidated (Kichey et al., 2005).

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5.4. Autophagy

6.2. Gpc-B1 and NAM-B1

Autophagy is a primary route for nutrient recycling in plants by which superfluous or damaged cytoplasmic material and organelles are encapsulated and delivered to the vacuole for breakdown (Li et al., 2015). This process has already been shown to play a critical role in the remobilization of N to growing seeds through its role in supporting the dismantling of the chloroplast as an essential aspect of the nutrient remobilization process (Guiboileau et al., 2012) (Fig. 2). It is well known that chloroplasts in green leaves represent a major pool of reduced N. Newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplastic proteins in rice exposed to darkening (Izumi et al., 2015). In barley, the autophagy factors APG7 and ATG18F are strongly upregulated during flag leaf senescence (Hollmann et al., 2014). In atg plants, NR is impaired, resulting in significant decreases in the N harvest index and grain yield of Arabidopsis and maize. Hollmann et al. (2014) suggest autophagy might be important for NR through its negative impact on cell-death processes. An autophagy-related gene TdAtg8 was cloned from wild emmer wheat and was shown to play a role in the responses to N deficiency and osmotic stress; for example, TdAtg8 yeast transformants grew better than the control under N starvation (Kuzuoglu-Ozturk et al., 2012). These results suggest that the route of autophagy might exist in common wheat and may be involved in N redistribution. However, its role in NR during grain filling remains to be characterized.

Gpc-B1 is functional in ancestral wild wheat, whereas it is deleted or nonfunctional in modern wheat because the allele has a 1-bp frameshift mutation in the 5 end (Uauy et al., 2006a, 2006b). This gene, which encodes an NAC transcription factor (NAM-B1) that is responsible for the Gpc-B1 QTL on chromosome 6BS, has been shown to accelerate canopy senescence during grain filling and to be responsible for a higher NR and a better partitioning of N to the grain without reducing grain yield (Uauy et al., 2006a, 2006b; Waters et al., 2009; Distelfeld et al., 2012; Bogard et al., 2011) (Fig. 2). Knockout of all Gpc-1 homoeologs resulted in a reduction in the rate of total N accumulation in the grains, suggesting that the Gpc-1 mutants have a lower efficiency to disassemble leaf proteins and transport them to the grains (Avni et al., 2014). RNAi knockdown of NAM-B1 resulted in delayed leaf senescence, which reduced NR from the leaves and consequently resulted in a lower GPC (Uauy et al., 2006a, 2006b; Waters et al., 2009; Guttieri et al., 2013). Transcriptome analysis identified 691 differentially regulated genes during senescence between the wild type and TaNAM-RNAi (Cantu et al., 2011). It is unlikely that the NAM proteins directly regulate all of these genes, and some of them are probably part of secondary networks. More interestingly, the expression of NAM genes in the flag leaf also controlled the senescence of the peduncle and spike, which remained green in TaNAM-RNAi plants (Guttieri et al., 2013).

6. Genetic control of NR New cultivars with increasing yield tend to have higher N accumulation in leaves and higher NR to the grain compared with old cultivars (Wang et al., 2008). A −0.52% year−1 straw N concentration at physiological maturity was found by Cormier et al. (2013) due to higher NRE in modern cultivars, revealing the potential of genetic improvement in NR. An increasing body of evidence has demonstrated that the genetic control of NR is linked to the regulation of leaf senescence in wheat (Masclaux et al., 2001; Uauy et al., 2006a, 2006b), maize (Masclaux et al., 2001) and sorghum (Sorghum bicolor (L.) Moench) (Van Oosterom et al., 2010a,b). 6.1. Genes for the NAC-domain and WRKY transcription factors A number of regulatory genes were up-regulated in the leaf during early grain filling or in the developing grain of wheat, notably NAC-domain and WRKY transcription factors (Gregersen and Holm, 2007; Guo et al., 2012) (Fig. 2). These factors have previously been identified as being associated with senescence in many species (Gregersen et al., 2013). In barley glumes, the specific NAC and WRKY transcription factors, in combination with hormones (ABA and jasmonic acid), have been shown to be involved in the regulation of transition between early grain filling and developmental senescence (Kohl et al., 2015). The transcription factor genes HvNAC026, HvNAC001, HvNAC005, HvNAC013, HvWRKY12 and MYB were up-regulated in flag leaves during general senescence processes (Hollmann et al., 2014). TaNAC-S, a novel NAC1-type transcription factor was identified in wheat, with gene expression located primarily in the leaf/sheath tissues. Transgenic wheat plants overexpressing TaNAC-S resulted in delayed leaf senescence, leading not only to increased GPC but also to increased grain yields; this result further confirmed the improved NR from vegetative organs to growing grain in transgenic lines (Zhao et al., 2015). OsNAC5, a novel senescence-associated ABA-dependent NAC transcription factor, could be involved in the ABA-induced senescence process and amino acid remobilization from flag leaves to developing grains (Sperotto et al., 2009).

6.3. Genes for protein degradation and N transport Protein degradation and translocation routes are pivotal processes of NR that must be under genetic control during grain filling (Fig. 2). In barley, predominant genes, including genes encoding the transcription factor serine type protease SCPL51, genes encoding the papain-like cysteine peptidases HvPAP14 and HvPAP20 and a subtilase gene, were more strongly up-regulated during flag leaf senescence under standard N conditions (Hollmann et al., 2014). The elevated expression of these genes in senescing leaves of plants with standard N supply indicates important roles of the corresponding proteins in NR. In addition to the genes of autophagy factors, over-expression of an alanine aminotransferase (AlaAT) gene significantly increased the yield of rice (Beatty et al., 2009) and contributed to a high NRE in wheat (Tian et al., 2015). Therefore, AlaAT may serve as a potential target to enhance NUtE in crops. These results indicate that the transport of N compounds might be an important process affecting NR. However, in wheat, their functions need to be further confirmed and elucidated.

6.4. Other genes Previous studies found that genes responsible for N metabolism were involved in the NR. The over-expression of GS genes could increase the yield of some cereal crops, such as maize, rice and wheat (Habash et al., 2001; Martin et al., 2006; Brauer et al., 2011; Li et al., 2016). More recently, it was reported that the expression of the TaGS1c gene increased significantly and contributed to the high NRE in wheat cultivars with high NUtE (Tian et al., 2015). Furthermore, the specific roles of ZmGln1-3 and ZmAS3 were highlighted in contributing to higher NRE in maize under post-silking drought (Li et al., 2016). Over-expression of the PPDK (cytosolic pyruvate orthophosphate dikinase) gene was reported to be involved in NR during leaf senescence, increasing the seed weight of transgenic Arabidopsis (Taylor et al., 2010) and contributing to the high NRE (Tian et al., 2015).

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7. Environment modification 7.1. N management Vegetative senescence and NR are controlled by many internal and external factors and can be accelerated or delayed by the alteration of these signals (Fig. 2). Because the amount of N remobilized to the grain is highly dependent on N stored at anthesis, an adequate N supply can simultaneously increase the accumulation of protein in the grain as well as grain yield by delaying the onset of senescence and increasing the amount of reserve N (Barbottin et al., 2005; Masoni et al., 2007; Zhang et al., 2012). Post-anthesis NRE increased from 67% to 71% with increasing N application (P < 0.001) (Gaju et al., 2011), largely due to the variation in the total duration of senescence under different N supply levels (Gaju et al., 2014). Alternatively, splitting the same N dose in three amendments improved NRE and thus increased GPC, although the N reserves prior to anthesis did not increase (Fuertes-Mendizábal et al., 2012). However, abundant experimental evidence shows that under heavy N fertilizer regimes, NR was inhibited, the GPC reached a maximum and then remained stable, whereas the straw N concentration continued to increase (Triboi and Triboi-Blondel, 2002; Barbottin et al., 2005; Barneix, 2007; Bahrani et al., 2011; Pask et al., 2012). 7.2. Water availability Ample availability of soil water favored post-anthesis root uptake and grain requirement of N, which delayed NR from the vegetative parts and thereby delayed the onset of senescence in maize (Ciampitti and Vyn, 2011). By contrast, a moderate soil water deficit enhanced the remobilization of pre-stored N to the grains by weakening the availability of soil N (Xu et al., 2006), resulting in 13% higher NRE compared to the control (Bahrani et al., 2011). In maize, post-silking drought resulted in a higher leaf NR, which may be attributed to the up-regulation of the GS and AS genes (Li et al., 2016). However, severe drought stress enhanced senescence in wheat by accelerating the loss of leaf N and leaf chlorophyll and increasing lipid peroxidation, which would therefore reduce NRE and NUE (Ercoli et al., 2008) even under high N level conditions (Giuliani et al., 2011). Considering that water stress accelerates senescence and the grain filling rate as well as increases ABA but reduces cytokinin concentrations in the vegetative plant parts of wheat (Yang et al., 2003), we speculate that these hormones might be involved in water stress-induced plant senescence and NR (Fig. 2). 7.3. Temperature Generally, daily cumulative temperature and the diurnal temperature difference during grain filling are positively associated with the GPC, but daily average temperature higher than 22 ◦ C is negatively associated with the GPC (see review Kong et al., 2013). The rate of NR was accelerated by temperature increase, whereas the N concentration in the stem and leaves decreased after flowering (Corbellini et al., 1997; Dreccer et al., 1997). The effects of temperature on NR and GPC may be explained as follows: moderate heat stress accelerates leaf senescence and the degradation of chlorophyll proteins, and NR from the leaves thus increases to meet the N demand of the actively growing grains. 8. Perspectives Desired crop yields and optimal quality attributes can be achieved by the finely controlled remobilization of canopy N to the developing grain (Hawkesford, 2014). Because genetic diversity exists amongst modern cultivars, selection for post-anthesis

NR may have value in breeding cultivars with optimized NR and senescence duration as well as higher NUE, GPC and grain yield (Gaju et al., 2014). Utilizing land races and ancestral germplasm to widely exploit the current germplasm pool can be used to screen the NRassociated genes and then substantially improve the genetics of NR (Hawkesford, 2014). Considering that the timing of onset of senescence explained a high amount of the variation in NR and NUtE (Martre et al., 2003; Gaju et al., 2011; Barraclough et al., 2014), it seems reasonable to select cultivars with remobilization of stored N in preference to photosynthetic N from vegetative tissues to help delay senescence while meeting the needs for high GPC. Because the over-expression of some genes results in delayed senescence and higher GPC, manipulation of these genes, such as TaNAC-S, PPDK, may be a direction for future wheat improvement strategies (Taylor et al., 2010; Zhao et al., 2015). Previous findings strongly suggest an important role of GS1 in improving NR, and maize plants over-expressing different isoforms of GS1 achieved higher NR and grain yield (Martin et al., 2006). Therefore, the specific manipulation of GS activity in leaves seems to be a useful method for improving NR and increasing the grain number and size in wheat. There is a link between N remobilization and Rbcs expression, and Rbcs has already been shown to colocalize with a QTL for GPC (Laperche et al., 2006); thus, Rbcs and similar genes have to be considered as potential targets for crop improvement. Hormones have differential effects on NR during plant senescence, probably through activities of GS and glutamate pyruvate transaminase (Xie et al., 2004). For example, ABA, as opposed to ZR, GA and IAA, facilitates the post-anthesis senescence course and NR from vegetative organs to the grain (Xie et al., 2003; Xie et al., 2004). Further investigations are needed to explore the possible regulation mechanisms of endogenous hormones in NR, which will help temporally manipulate plant senescence, NR and GPC. Integrating analysis using systems biology platforms (e.g., genomics, transcriptomics, proteomics and metabolomics) can link the NR-related genetic basis to physiological functions and help characterize the regulatory, signaling and execution processes that are involved in NR and natural leaf senescence. Interpretation of these data can accelerate identification of potential candidate genes. Transcription factors such as the NAM genes can potentially regulate a large number of direct targets and downstream genes (Guttieri et al., 2013); thus, increased identification of direct NAM targets and their downstream networks could enable targeting of specific components that may allow us to select for the beneficial effects of delayed senescence while avoiding the detrimental effects on NR and GPC. The N content of leaves during the grain-filling period plays a key role in determining the efficiency of NR during grain filling as well as a high photosynthetic capacity. A nondestructive method based on near-infrared spectroscopy may be valuable to monitor changes in leaves and grain N concentrations (Bogard et al., 2010, 2011; Vilmus et al., 2014) and thus to assess the dynamics of NR. Nutrient remobilization requires mostly phloem transport. Phloem loading is crucial for efficient NR because the amino acids that are released due to protein degradation are exported to the grains via the phloem. The pathways of N to the grain are not fully defined, and specific sites of phloem loading for transport to the grain are not known (Simpson et al., 1983; Guttieri et al., 2013). Therefore, a better understanding of the export process would provide valuable target genes for manipulating and improving the yield and quality of proteins in grains. Studies of these putative transporter and regulator proteins may help in understanding the elusive molecular mechanisms of amino acid export in plants (Okumoto and Pilot, 2011).

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Acknowledgements This work was supported by the Shandong Modern Agricultural Technology & Industry System (SDAIT-01-06), National Earmarked Fund for Modern Agro-industry Technology Research System (CARS-3-1-21), the Special Fund for Agroscientific Research on Public Causes MOA of China (201303109-7) and the Program of Major Independently Innovative Key Technology of Shandong Province (2014GJJS0201).

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