Nutrient sink limitation constrains growth in two barley species with contrasting growth strategies

Abstract Mineral nutrients exert important limitations on plant growth. Growth is limited by the nutrient source when it is constrained by nutrient availability and uptake, which may simultaneously limit investment in photosynthetic proteins, leading to carbon source limitation. However, growth may also be limited by nutrient utilization in sink tissue. The relative importance of these processes is contested, with crop and vegetation models typically assuming source limitations of carbon and mineral nutrients (especially nitrogen). This study compared the importance of source and sink limitation on growth in a slower‐growing wild perennial barley (Hordeum bulbosum) and a faster‐growing domesticated annual barley (Hordeum vulgare), by applying a mineral nutrient treatment and measuring nitrogen uptake, growth, allocation, and carbon partitioning. We found that nitrogen uptake, growth, tillering, shoot allocation, and nitrogen storage were restricted by low nutrient treatments. Multiple lines of evidence suggest that low nutrient levels do not limit growth via carbon acquisition: (a) Carbohydrate storage does not increase at high nutrient levels. (b) Ratio of free amino acids to sucrose increases at high nutrient levels. (c) Shoot allocation increases at high nutrient levels. These data indicate that barley productivity is limited by the capacity for nutrient use in growth. Models must explicitly account for sink processes in order to properly simulate this mineral nutrient limitation of growth.

. Appropriate fertiliser application will reduce the financial, environmental, and ecological costs of intensive farming, yet the crop requirement for nutrients must be met in order to maintain and increase yields. Nutrient use efficiency is therefore a major target for crop improvement, with nitrogen being the key element of interest (Perchlik & Tegeder, 2018;Tegeder & Masclaux-Daubresse, 2018). The timing of fertiliser application has been extensively researched from an agronomic perspective, and much is known at the molecular level about nitrate transport and signalling mechanisms (Kiba, Kudo, Kojima, & Sakakibara, 2011;Miller, Fan, Orsel, Smith, & Wells, 2007;Sakakibara et al., 2006). However, the whole-plant physiological behaviour linking the ecology of crops on one hand, and molecular physiology on the other, remains poorly characterised.
The relationships between sources and sinks are vital determinants of growth (Chang & Zhu, 2017;White, Rogers, Rees, & Osborne, 2016). Source strength may be defined as the capacity of the plant to take up a resource from the external environment-in this case, mineral nutrients from the soil-whilst sink strength is the internal capacity of the plant to utilize that resource in storage or growth. In each case, strength is the product of the size and activity of the source or sink tissue (Geiger & Shieh, 1993;White et al., 2016). For the primary mineral nutrient nitrogen, the source:sink ratio is regulated by interacting molecular mechanisms, yet our understanding of how, and the extent to which, nitrogen source and sink strengths limit growth at different times during ontogeny remains incomplete (White et al., 2016). Broadly speaking, plants are thought to transit from carbon sink to source limitation during development (Arp, 1991;Marschner, 1995), but the generality of this principle is unclear because differences in carbon source and sink limitation have been observed for a variety of crops, particularly at grain filling (Acreche & Slafer, 2009;Álvaro, Royo, García del Moral, & Villegas, 2008;Jaikumar, Snapp, Flore, & Loescher, 2014;Peterhansel & Offermann, 2012;Slewinski, 2012). Many studies focus on reproductive growth in order to examine source-sink relations with respect to yield, such that understanding of source-sink relations is especially deficient for the vegetative stage (Burnett, Rogers, Rees, & Osborne, 2016). The majority of studies focus on sources and sinks for carbon which are of great importance (Paul, Oszvald, Jesus, Rajulu, & Griffiths, 2017) yet nutrient sources and sinks are critical factors that interact with carbon sources and sinks (Ruiz-Vera, De Souza, Long, & Ort, 2017) and underpin plant growth in their own right (Sonnewald & Fernie, 2018;White et al., 2016).
Growth is controlled by a network of processes, the strengths of which vary with nutrient supply (White et al., 2016). Therefore, understanding how nutrient uptake and utilization limits growth is important for improving crop yield and sustainability (Burnett et al., 2016;White et al., 2016). In particular, the processes responsible for nutrient limitation of growth are contested. For example, low nitrogen uptake by the root could primarily constrain growth by one of two mechanisms: limiting the synthesis of photosynthetic proteins and thereby causing carbon source limitation; or directly limiting the synthesis of proteins and other compounds required for sink tissue expansion (Fatichi, Leuzinger, & Korner, 2014;Körner, 2015;Poorter, Anten, & Marcelis, 2013;Stitt & Schulze, 1994). Process-based vegetation and crop models often represent nitrogen limitation by focusing solely on root nitrogen uptake and nitrogen use in photosynthesis (Bao, Hoogenboom, McClendon, & Vellidis, 2017;Oleson et al., 2010;Wärlind, Smith, Hickler, & Arneth, 2014), without considering the direct limiting effects of nitrogen availability on tissue growth. However, there are several exceptions (Zaehle et al., 2014).
The natural diversity of plant growth rates can be used to investigate the factors that limit growth, and ecological research has advanced our understanding of how growth and nutrient use are adapted to different soil conditions. Growth rate is considered an important adaptation to variation in soil fertility, such that nutrientpoor environments are dominated by slower-growing plants, and nutrient-rich environments are dominated by faster-growing plants (Aerts & Chapin, 2000). This relationship is hypothesised to be a significant factor underlying global trait variation (Díaz et al., 2016;Wright et al., 2004). Life history strategy (i.e. annual or perennial) is also an important axis of growth rate variation among wild species (Garnier, 1992;Grime & Hunt, 1975), and the growth rate differences between annuals and perennials are well documented (Houghton, Thompson, & Rees, 2013). Annuals grow quickly in order to make a high investment in reproduction during their single year of life, whereas perennials grow more slowly to conserve resources for future years (Bennett, Roberts, & Wagstaff, 2012;Garnier, 1992;Iwasa, 2000). Therefore, annuals generally have a higher relative growth rate (RGR) than perennials, which is especially clear when congeneric species are compared (Garnier, 1992). Despite the dominance of slow growers in nutrient-poor environments (Aerts & Chapin, 2000), fast-growing species still grow faster than slow-growing ones in infertile soil (Campbell & Grime, 1992). Fast-growers have a high nutrient uptake capacity and a greater flexibility to alter their uptake capacity in response to nutrient availability, that is, a greater physiological plasticity (Aerts & Chapin, 2000;Garnier, Koch, Roy, & Mooney, 1989).
This study compares the responses of two barley species to a nutrient gradient, in order to investigate how nutrient availability and uptake limit vegetative growth. It aims to elucidate the relative importance of indirect limitation via carbon source strength, by measuring carbohydrate content and amino acid:sucrose ratio, and direct limitation of sink tissue expansion growth, by measuring relative growth rate (RGR) and tissue composition. The work compares an elite fast-growing domesticated annual spring malting barley (Hordeum vulgare cv. NFC Tipple) with a slower-growing wild perennial relative (Hordeum bulbosum). By working with species with different life history strategies, this approach uses pre-existing variation in growth rate to probe the nature of the annual crop system. Previous work on these species during the vegetative growth stage shows significant carbon sink limitation of growth in the annual, as evidenced by a lack of plasticity of photosynthesis and allocation, and a weak growth response to elevated CO 2 concentration (Burnett et al., 2016). In contrast, the perennial exhibits carbon source limitation, and is able to increase sink development and utilize the additional carbon available from photosynthesis under elevated CO 2 (Burnett et al., 2016). This study investigates the role of nutrient limitation in this system, testing the alternative hypotheses that: (a) nutrients (primarily nitrogen) limit photosynthesis, thereby causing carbon limitation of growth or (b) that nutrients directly limit expansion growth. It also tests the hypothesis (c) that vegetative growth in the fast-growing annual is more limited by its ability to take up soil nutrients (source limitation), while the slow-growing perennial is more limited by its ability to utilize these nutrients (sink limitation).

| Plant material, growth conditions, and nutrient treatment
Seeds of Hordeum vulgare cv. NFC Tipple from the UK HGCA (2014) recommended list and Hordeum bulbosum (Accessions GRA1031 and GRA947) from Turkey (von Bothmer, 1996) were obtained from Syngenta and IPK Gatersleben, respectively. Data were collected until 42 days after germination, in order to focus measurements on the period of maximal vegetative growth, since previous work had found that maximum RGR occurs approximately 28 days after germination during the vegetative growth stage in these species (Burnett et al., 2016). Seeds were first germinated on wet filter paper, then transplanted to 4-L pots filled with a 1:10 sand:vermiculite mix topped with an additional layer of sand to aid seedling root development.
This mix was designed to provide a very low-nutrient, nitrogen-free substrate to which varying levels of nutrient solution could be added.
Plants were grown at the University of Sheffield in controlled environment plant growth chambers (BDR 16, Conviron, Isleham, UK), modified to scrub CO 2 using soda lime to achieve the current (2015) ambient atmospheric level of 400 μmol/mol CO 2 . Plants were randomised between three chambers, with the following growth conditions: 12-hr photoperiod with day/night temperatures of 20/ 18°C, 65% relative humidity, 400 μmol/mol CO 2 , and daytime light levels of 600 μmol photons m −2 s −1 to provide a daily light integral of 25.9 mol m −2 day −1 .
For the first week, plants were watered daily with Reverse Osmosis water. Thereafter, plants were watered three times per week with 250 ml Long Ashton nutrient solution, applied at different concentrations (nutrient treatments): "low nutrients" (1% of Long Ashton stock solution), "medium nutrients" (20% of stock), and "high nutrients" (100% of stock to the requirements of the plant, so the treatment altered the application of all mineral nutrients in accordance with these proportions. Plants did not display visible signs of mineral deficiency or toxicity (Supporting Information Figure S1). and adjusted for percentage labelled atom fed, and values from control samples which did not receive 15 N were subtracted from these data in order to give 15 N enrichment relative to the baseline level for each organ type and nutrient treatment (baseline 15 N of control samples did not differ between the two species).

| Growth and allocation
Shoot area was obtained by photographing plants twice per week starting 8 days after germination, using the method described by Burnett et al. (2016)

| Metabolites
Metabolite harvests were carried out on plants from the main study.

| Statistical methods
All data were analysed in R (R Core Team, 2015) using Type II ANOVA. Natural logarithmic transformations were performed prior to analysis to satisfy the normality assumptions of ANOVA.

| RESULTS
Net nitrogen uptake ( Figure 1) decreases significantly as nutrient treatment is lowered (F 2,10 = 54.6, p < 0.001), but there is no significant species effect (either main effect or interaction, p > 0.5 in each case) on net nitrogen uptake ( Figure 1). Relative growth rate (RGR; Figure 2) is consistently higher in annual than in perennial barley, suggesting that the annual invests more resources into growth than the perennial, even at low nutrient levels (significant effect of species: F 1,11 = 9.77, p < 0.01). This shows that the nutrient use efficiency of growth is higher in the annual. RGR decreases significantly in annual and perennial barley when nutrient treatment is lowered Allocation to roots increases at lower nutrient supplies (p < 0.001) and allocation to leaves and sheaths decreases at lower nutrient supplies (p < 0.001). These data are obtained from the subset of 29 additional plants harvested for biomass calibration described in the Materials and Methods section. For root:shoot ratio, root mass is divided by the sum of leaf and sheath mass for each individual and the results averaged. For leaf, sheath, and root mass ratios, the ratio is the dry mass of that organ divided by the dry mass of the whole plant. 18 annuals and 11 perennials were harvested at five timepoints. Data show mean ± SE (at low, medium, high nutrients, annual n = 6, 6, 5; perennial n = 4, 2, 5). For brevity, age effects have not been included in this summary, but they are discussed elsewhere in the manuscript.
F I G U R E 1 Net nitrogen uptake rate of annual (filled circles, solid line) and perennial barley (hollow circles, dashed line) decreases as nutrient treatment level is lowered (p < 0.001). Uptake was measured over 24 hr before harvesting plants 42 days after germination. Data show mean ± SE (n = 3) (F 2,11 = 7.98, p < 0.01), and this effect is especially strong when nutrients are decreased from the medium to low treatment level.
Biomass partitioning is also affected by nutrient treatment. In accordance with the change in RGR, tillering decreases substantially at low nutrient levels ( Figure 3); this effect is greater in older plants (significant nutrient × age interaction for tillering: F 4,24 = 13.1, p < 0.001). However, whilst the nutrient effect is strong, there is neither a difference between species nor a nutrient × species interaction (p > 0.6 in each case). Allocation to roots (Table 1)   TNC in the leaf is significantly higher in the annual (F 1,24 = 14.8, p < 0.001), as would be expected if the annual is more carbon sink limited than the perennial. Leaf TNC is also affected by nutrient treatment (F 2,24 = 6.1, p < 0.01): leaf TNC increases when nutrient treatment is lowered from medium to low in younger plants (Figure 5a), yet decreases at low nutrient levels in older plants (Figure 5b,c). However, contrary to expectations that carbon source limitation would increase at low nutrient levels, leaf TNC increases when nutrient treatment is lowered from high to medium, that is, leaf TNC accumulates more at medium nutrient treatment levels than at high levels. This indicates that carbon source limitation does not increase when nutrient treatment is decreased. Rather, carbon source limitation is greater at high nutrient treatment levels. Root TNC shows a significant nutrient treatment × age interaction (F 4,25 = 9.3, p < 0.001): the increase in TNC at lower nutrient treatment is most pronounced 14 days after germination (Figure 5f).
Elemental N content (Figure 6a-c) decreases at lower nutrient treatment level in leaf (nutrient treatment × age interaction: F 4,24 = 6.5, p < 0.01), sheath (nutrient treatment effect: F 1,11 = 50.8, p < 0.001), and root (nutrient treatment × age interaction:  Figure 7), indicating greater carbon source limitation in perennial than annual barley. There was no significant effect of age on this ratio. Contrary to hypothesis (1), root amino acid:sucrose ratio increases at higher nutrient treatment levels (F 2,25 = 64.9, p < 0.001), especially between low and medium nutrient levels (Figure 7b), suggesting that carbon source limitation is increased rather than decreased at high nutrient levels, rather than carbon source limitation increasing at low nutrient levels.

| DISCUSSION
In this study, annual and perennial barley were grown along a nutrient gradient to examine the processes through which mineral nutrients limit growth (hypotheses 1, 2) and elucidate the relative contributions of nutrient source and sink strengths to growth in each species (3). The work focused on measurements of nitrogen uptake and concentration, since nitrogen is the primary mineral nutrient.
There are multiple lines of evidence consistent with the hypothesis that nutrients directly limit expansion growth (2)  Predicted biomass increases with age especially in annual barley, and especially at higher nutrient treatment levels (p < 0.001 for each of these interactions). These data are for plants in the main study. Biomass (g) is predicted from leaf area data, using the correlation obtained for the subset of additional plants harvested for biomass calibration. Predicted biomass is derived from photographs preceding destructive harvests (carried out at 14, 28, and 42 days after germination). Data show mean ± SE, with n decreasing over time due to destructive harvests (at 12 days, at low, medium, high nutrients, annual n = 17, 18, 18; perennial n = 7, 13, 15; at 22 days, at low, medium, high nutrients, annual n = 10, 10, 10; perennial n = 6, 6, 9; at 40 days, at low, medium, high nutrients, annual n = 5, 5, 5; perennial n = 3, 3, 4). nutrient level. However, this is not always the case ( Figure 5), even though leaf protein shows a small increase in the higher nutrient treatment levels (Figure 4), indicating that carbon source limitation is not alleviated by nutrients. In addition, amino acid:sucrose ratio, another indicator of carbon source limitation, increases in the root at higher nutrient treatment levels, further indicating that carbon source limitation increases at high nutrient levels rather than decreasing (Figure 7b). Consistent with this hypothesis, carbon becomes an increasingly limiting resource at higher nutrient treatment levels, and the leaf and sheath mass ratios in both species increase to compensate for this effect (Table 1). Taken together, this evidence shows that the nutrient limitation on growth is mediated by a direct constraint on expansion growth (hypothesis (2)) rather than acting via carbon limitation (hypothesis (1)).
Regarding hypothesis (3), the investment of nitrogen into growth and storage shows large decreases when nutrient treatment is decreased from a medium to a low supply, but this response is smaller when nutrients are decreased from a high to a medium nutrient supply, especially in annual barley. These results indicate a significant nutrient source limitation at low nutrient levels, but a nutrient sink limitation at high nutrient levels (Figures 2, 4 and 6). Annual barley grows faster overall ( Figure 2) and is larger than the perennial (Table 3) indicating higher nutrient use efficiency of growth. RGR is more strongly limited by nutrient supply in the perennial, suggesting that the lower nutrient efficiency of growth in the perennial leads to stronger nutrient limitation when nutrient supply is restricted. The perennial shows a relatively greater growth response to nutrient level than the annual (Figure 2) and has a higher leaf nitrogen concentration (Figure 6a). This suggests that, under nitrogen-limited conditions (i.e. the low nutrient treatment), the perennial preferentially allocates nitrogen to storage rather than growth, consistent with the hypothesis that the perennial will conserve mineral nutrients (Campbell & Grime, 1992). However, contrary to the hypothesis that growth in the perennial would be more Contrary to expectations, the perennial has a higher SLA (Table 2) and higher leaf nitrogen concentration ( Figure 6) than the annualtraits that are generally associated with fast-growing species in the ecological literature (Reich et al., 2003;Wright et al., 2004). This is consistent with previous work on this species (Burnett et al., 2016), which showed carbon source limitation of growth in the perennial, since it is investing in carbon acquisition by the leaves in order to match carbon and nitrogen supply. Indeed, the higher SLA and leaf nitrogen concentration observed here for the perennial indicate that it may have the potential to be a rather fast-growing species despite its perennial life history strategy. Potential RGR has previously been correlated with nitrogen uptake capacity (Garnier et al., 1989) and here the net nitrogen uptake rates are very similar for annual and perennial barley, although the perennial never matches the RGR of the annual.
RGR (Figure 2) is higher in the annual, but LAR (the product of SLA and LMR) does not differ between species (Table 2). Whilst SLA tends to be the major contributor to LAR and thus RGR in herbaceous species (Poorter & van der Werf, 1998), some studies have found that RGR correlates with LMR rather than SLA (references within Garnier, 1992), which is consistent with these data. Furthermore, since RGR is the product of net assimilation rate (NAR) and leaf area ratio (LAR; Poorter & Remkes, 1990), the higher RGR of annual compared to perennial barley seen here is likely due to higher photosynthetic assimilation, as seen in previous work with this species (Burnett et al., 2016). Two further points of contention are: whether RGR regulates resource uptake or whether resource uptake is regulated by RGR (Garnier et al., 1989;Rodgers & Barneix, 1988); and the extent to which uptake is regulated by demand (Taulemesse, Le Gouis, Gouache, Gibon, & Allard, 2015). Indeed, nitrate itself is an important regulator of nitrogen uptake (Masclaux-Daubresse et al., 2010). In addition to elucidating the relative contribution of nutrient source and sink to growth, a deeper understanding of the molecular drivers that underpin regulation of nutrient uptake will be an important component of improving crop nutrient use efficiency. 28, 42 days after germination) due to lack of significant age effects ± SE (at low, medium, high nutrients, annual n = 9, 9, 9 in leaf and root and 1, 6, 6 for sheath; perennial n = 4, 7, 9 for leaf, 0, 5, 5 for sheath, and 5, 7, 9 for root). There are more samples for CHN analysis than for metabolite analysis due to the small size of some samples. Points are offset with respect to x-axis position, to increase readability

| Annual and perennial barley do not store excess nitrogen as protein
Nitrate is a labile store and therefore shows a particularly strong response to nutrient treatment ( Figure 4); this metabolite also shows a strong response to nitrogen treatment in wheat (Devienne, Mary, & Lamaze, 1994) and Arabidopsis (Tschoep et al., 2009) and, like protein, constitutes a key store for nitrogen in herbaceous plants (Millard, 1988 (Figure 4): rather than regulating leaf protein concentration as leaves get older, barley plants create more leaf tissue and maintain the same protein concentrations; this contradicts the way in which many earth system models deal with nutrient limitation and is therefore an important avenue for further investigation.

| High nutrient levels increase carbon source limitation
Storage of nitrogen as nitrate and amino acids suggests that plants are carbon source limited as protein synthesis requires additional carbon. Both species show a decrease in N concentration and an increase in C:N ratio as the nutrient treatment level is lowered (Figure 6). This corresponds with a shift from excess nitrogen to excess carbon in the plants (Stitt & Krapp, 1999). However, this effect is observed when nutrient treatment is decreased from medium to low, but not between high and medium nutrient levels ( Figure 6) despite a high nitrate availability and uptake rate, suggesting that plants are reaching their maximum capacity for nitrogen storage at high nutrient treatment levels, and are carbon source limited. Leaf TNC data ( Figure 5) also show carbon source limitation at high nutrient levels.
The higher LMR and SMR in both species at higher nutrient treatment levels (Table 1) enables greater acquisition of carbon, which becomes an increasingly limiting resource at higher nutrient treatment levels; conversely, more biomass is allocated to roots in low nutrient environments (Aerts & Chapin, 2000). Not only does LMR increase with nutrient treatment in both species, but LAR also increases (Tables 1 and 2), as observed by Garnier et al. (1989), enabling greater photosynthetic carbon acquisition since there is a greater, thinner leaf area for light harvesting. This suggests an increase in carbon source limitation at high nutrient levels. Both species show an increase in tillering as nutrient treatment is increased (Figure 3), as observed in wheat by Taulemesse et al. (2015), and especially when plants are older, facilitating a rapid increase in allocation to shoots and thus enabling greater carbon acquisition.
In addition to these structural changes, and the differences in elemental carbon and nitrogen concentrations, root amino acid:sucrose ratio increases at higher nutrient treatment levels ( Figure 7b).
This indicates that carbon source limitation is increasing, since the available amino acids outsupply the corresponding supply of available carbon necessary to fuel growth (Isopp, Frehner, Long, & Nösberger, 2000;Paul & Driscoll, 1997;Stitt & Krapp, 1999). The increase is particularly pronounced between low and medium F I G U R E 7 Amino acid:sucrose ratio is higher in perennial (hollow circles, dashed line) than in annual (filled circles, solid line) barley in both (a) leaf and (b) root (p < 0.001). In the root, amino acid:sucrose ratio increases with increasing nutrient treatment level (p < 0.001). Data show mean across all ages (14, 28, 42 days after germination) ± SE (in leaf at low, medium, high nutrients, annual n = 6, 8, 9, perennial n = 9, 8, 9; in root at low, medium, high nutrients, annual n = 2, 7, 8, perennial n = 3, 6, 7). Insufficient data were available for sheath nutrient treatment levels. The higher amino acid:sucrose ratio in the perennial, and lower TNC concentration indicates that the perennial is more carbon source limited than the annual (Figures 5-7); this corroborates the evidence from previous work on these species (Burnett et al., 2016).

| Annual barley is more nutrient sink limited than perennial barley at moderate nutrient supply
In addition to the differences in the nutrient responses of each species, there are broad similarities revealed by the nutrient treatment.
The treatment conditions are sufficiently strong that, at low nutrient treatment, growth in both annual and perennial barley is strongly nutrient source limited, as shown by high C:N ratios (Figure 6d-f), low nutrient uptake rates ( Figure 1) and low growth rates compared to medium nutrient levels (Figure 2). At high nutrient levels, growth in the annual is more nutrient sink limited than the perennial, shown by its lower relative ability to increase growth ( Figure 2). Growth at the medium nutrient supply appears to be more nutrient sink limited in annual than perennial barley, since increasing the nutrient level further has a much greater effect in the perennial.
In general, the higher RGR of annual plants arises from their large investment in leaf area and photosynthetic capacity: specific leaf area (SLA, mm 2 leaf per gram leaf), nitrogen content, and partitioning of that nitrogen to the photosynthetic machinery are higher in the leaves of annuals, enabling greater carbon acquisition due to a larger light-harvesting area per unit leaf mass and a greater nitrogen density associated with the photosynthetic machinery, thus facilitating high rates of carbon assimilation and faster growth (Garnier & Laurent, 1994;Grime et al., 1997;Pierce, Brusa, Vagge, & Cerabolini, 2013;Poorter & van der Werf, 1998). In contrast, perennials tend to have a lower SLA and nitrogen concentration, and invest more resources in the construction of robust, long-lived leaves. Plants with lower SLA may also invest proportionately less leaf nitrogen in the photosynthetic machinery (Hikosaka, Hanba, Hirose, & Terashima, 1998).
The nutrient sink limitation uncovered here for annual barley at medium nutrient treatment levels implies that barley crops are unable to invest excess nutrients into growth and storage during the vegetative stage. Although nitrogen is taken up and stored as nitrate, the subsequent reduction of nitrate to organic forms of nitrogen is lacking, coupled with a lack of proportional increases in expansion growth to create a nutrient sink. Both the efficiency of nutrient acquisition and the efficiency of nutrient utilisation are important for breeders (Santa-Maria, Moriconi, & Oliferuk, 2015), such that sink development in addition to source strength is vital for realising improved crop productivity (Burnett et al., 2016;White et al., 2016).
Regarding the key mineral nutrient nitrogen, nitrogen transporters have been identified as a key target for improving the nitrogen source:sink balance (Tegeder & Masclaux-Daubresse, 2018), whilst nitrogen allocation patterns are important for nitrogen use efficiency and yield (Perchlik & Tegeder, 2018). Additional nutrient storage, in order to build up nutrient reserves for subsequent grain filling, would require larger nutrient sinks-such as increased capacity for expansion growth-to develop during the vegetative growth stage. These could allow farmers to reduce the dosage level of fertiliser application later in development and still increase crop yield (by increasing grain size and number rather than by increasing grain nitrogen concentration), which is of interest for breeders and farmers working with malting barley (Syngenta Crop Protection, 2011). The source: sink balance of the primary mineral nutrient nitrogen is important for crop improvement (Sonnewald & Fernie, 2018), and this element is of global ecological importance (Taylor & Menge, 2018).