a of Cultivar‐dependent increases in mycorrhizal nutrient acquisition by barley in response to elevated CO2

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors, Plants, People, Planet © New Phytologist Foundation 1Department of Animal and Plant Sciences, University of Sheffield, Sheffield, UK 2School of Biology, Faculty of Biological Sciences, University of Leeds, Leeds, UK


Societal Impact Statement
Modern agriculture is under pressure to meet yield targets while reducing reliance on finite resources to improve sustainability. Climate change represents an additional challenge-elevated atmospheric CO 2 concentrations may increase plant growth and boost yield, but the nutritional value of crops grown at elevated CO 2 is often reduced.
Arbuscular mycorrhizal fungi (AMF) can improve plant nutrition, although how this symbiosis will be affected by climate change is unclear. Here, we demonstrate mycorrhizal contribution to nitrogen and phosphorus nutrition in barley under current and future CO 2 concentrations. In one cultivar, AMF substantially increased phosphorus uptake at elevated CO 2 and prevented phosphorus dilution, suggesting the symbiosis may become more important for crop nutrient uptake in the future.

Summary
• Globally important cereals such as barley (Hordeum vulgare L.) often engage in symbiosis with arbuscular mycorrhizal fungi (AMF). The impact of elevated atmospheric CO 2 on nutrient exchange between these symbionts remains unknown.
• In controlled environment experiments, we used isotope tracers ( 15 N, 33 P, 14 C) to quantify nutrient fluxes between two barley cultivars (Moonshine and Riviera) and their associated AMF at ambient (440 ppm) and elevated (800 ppm) CO 2 .
• Elevated CO 2 reduced shoot N concentration in Moonshine, and shoot N and P concentration in Riviera. Elevated CO 2 substantially increased mycorrhizal 33 P acquisition in Moonshine. Mycorrhizal contribution to P uptake in Moonshine may have prevented dilution of tissue P concentration at elevated CO 2 . In Riviera, AMF did not improve 33 P acquisition. Both cultivars received 15 N from their AMF symbionts, and this acquisition was not influenced by CO 2 concentration, although Moonshine received more 15 N than Riviera.
• Our results suggest that AMF may provide substantial contributions to barley nutrition at current and projected future CO 2 concentrations. This is especially noteworthy for barley, which is generally considered to have low mycorrhizal receptivity. AMF may help alleviate or avoid nutrient dilution normally observed at

| INTRODUCTION
One of the greatest challenges facing humankind is that of generating enough food for the global population. Food production must be increased by an estimated 25%-70% to meet the demands of a projected human population of 9.8 billion people in 2050 (Alexandratos & Bruinsma, 2012;Hunter et al., 2017;Tilman et al., 2011) against the background of climate change (Parry et al., 2004;Smith & Gregory, 2013). During the "Green Revolution" of the 1950s and 60s, crop yield increases were achieved through intensive application of agrochemicals, irrigation and advances in plant breeding.
These agricultural innovations improved nutrition globally, sparing the conversion of further natural ecosystems into agricultural land (Tilman et al., 2002). Phosphorus (P) fertilizer application increased 3.5-fold between 1960 and 1995 (Tilman et al., 2002), while nitrogen (N) addition increased ten-fold over the same period (Hinsinger et al., 2011) promoting crop growth and primary production (Čapek et al., 2018;Robertson & Vitousek, 2009). With increasing application, the efficiency with which crops acquire fertilizer has decreased (Hinsinger et al., 2011). At the same time, negative ecological effects due to excess fertilizer deposition, runoff and leaching into the wider environment have become a global phenomenon (Hautier et al., 2014). Serious environmental, economic and political issues associated with fertilizer production and usage mean that sustainable alternatives must now be sought (Carpenter, 2008;Cordell et al., 2009;Hinsinger et al., 2011).
Future food security faces additional challenges stemming from the impacts of global climate change. Increasing concentrations of greenhouse gases such as CO 2 in the atmosphere are linked to rising global temperatures (Stocker et al., 2013) and increasing frequency of extreme weather events (Wheeler & Von Braun, 2013).
The agricultural sector is a significant producer of greenhouse gases, contributing c.10% of the UK's total GHG emissions (DEFRA, 2015), a significant proportion of which comes from fertilizer production (Goucher et al., 2017). Atmospheric CO 2 concentrations are currently 410-415 ppm (ESRL-NOAA Global Monitoring Division, 2020). If current emission rates are maintained, global CO 2 concentrations could rise to between 750 and 1,300 ppm by 2100 (Edenhofer, 2015). Field-based experiments have shown that crop growth in an elevated [CO 2 ] atmosphere (eCO 2 ) may initially increase photosynthetic C fixation and plant biomass, although the effects can decline over time as a result of CO 2 acclimation (Ainsworth & Long, 2005;Long et al., 2006). Increased plant C fixation and growth at eCO 2 is likely to increase plant demand for P under eCO 2 (Jin et al., 2015). Increased demand, together with the rapid depletion of the finite raw resources for P-based fertilizer production (Cordell et al., 2009), presents further problems for future crop production.
Simply increasing agricultural productivity will not solve the problems associated with future food security if done in an unsustainable manner. Future increases in food production must be coupled with sustainable management practices geared towards minimizing or even removing carbon emissions from agriculture. In order to achieve this, alternative strategies which reduce agricultural reliance on synthetic fertilizer application must be sought.

Arbuscular mycorrhizal fungi (AMF), of the sub phylum
Glomeromycotina (Spatafora et al., 2016), are found almost ubiquitously in agricultural soils and form intimate, intracellular associations with plant roots (Smith & Read, 2008). In exchange for plant carbon, AMF supply their hosts with phosphate, nitrogen and other mineral nutrients (Cavagnaro, 2008;Hodge & Storer, 2015;Parniske, 2008;Watts-Williams et al., 2017). AMF produce substantial hyphal networks that reach far beyond the rhizosphere, effectively extending the foraging range of the root system (Jansa et al., 2003;Puschel et al., 2016), and permitting the host plant access to soil pores which might otherwise be inaccessible (Allen, 2007). Integrating and exploiting AMF in more sustainable agricultural practices may potentially provide numerous benefits ranging from reduced fertilizer inputs, improved soil quality and increased plant nutrient uptake Thirkell et al., 2017).
The effects of eCO 2 in the future are likely to influence the dynamics of crop-AMF symbioses (Dong et al., 2018;Thirkell, Pastok, et al., 2019). Atmospheric CO 2 enrichment has been shown to affect carbon-for-nutrient exchange between AMF and plants, both non-domesticated and domesticated (Elliott et al., 2020;Field et al., 2012). Barley cultivars show a varied response to eCO 2 , (Mitterbauer et al., 2017) but the extent to which this may be influenced or determined by AMF has not been investigated. In wheat, eCO 2 has been shown to have cultivar-specific effects on the function of associated AMF (Thirkell, Pastok, et al., 2019), suggesting that AMF receptivity, function and responsiveness to atmospheric [CO 2 ] could be key traits for future sustainable wheat breeding programmes. To date, research into the influence of abiotic factors (including eCO 2 ) on cereal-AMF symbiosis has largely focused on wheat (Cabral et al., 2016;Elliott et al., 2020;Mathur et al., 2019), although other crops species are recently being studied, including maize (Watts-Williams, Smith, et al., 2019). Barley is currently the world's 4th most commonly grown agricultural crop (FAO, 2018) and shows variable response to mycorrhizal colonization, from negative (Campos et al., 2018;Grace et al., 2009) with distilled water (d. H 2 O) and added to the sterile sand/soil mix immediately prior to planting. Each pot received 15 ml of inoculum such that each plant was inoculated with 15,000 ± 1,500 R. irregularis spores. Spore density was quantified at 1,000 ± 100 spores per ml using a 100 µl aliquot placed on a microscope slide, inspected under a compound microscope at 40× magnification. Visual inspection of inoculum at this stage showed no physical damage to spores as a result of the brief homogenization process.
Plants were maintained in controlled environment growth cabinets (Snijder Labs) with the following conditions: 15 hr day (20°C and 70% humidity, day-time PAR, supplied by LED lighting, 225 µmol m −2 s −1 at canopy level) and 9 hr night (at 15°C and 70% humidity). CO 2 concentrations were 440 ppm (ambient atmospheric [CO 2 ] treatment, hereafter referred to as aCO 2 ) and 800 ppm (elevated [CO 2 ] treatment, hereafter referred to as eCO 2 ). Atmospheric [CO 2 ] was monitored using Vaisala sensors (Vaisala), maintained throughout by the addition of gaseous CO 2 .
The experiment took place within two growth chambers, such that there were 12 plants in each chamber. To mitigate for any unintended effects of growth chamber usage, plants were transferred between growth cabinets every 4 weeks and CO 2 concentrations amended accordingly. Each week, plants within each growth chamber were moved to randomize any possible effect of unintended FIGURE 1 Diagram of experimental setup. Barley (Hordeum vulgare L. cv Moonshine, Riviera) were grown in a 3:1 mixture of topsoil and autoclaved silica sand in 1.5 L pots. Pots received a supplementary inoculum of the arbuscular mycorrhizal fungus Rhizophagus irregularis. Plants were grown in two CO 2 concentration treatments-ambient (440 ppm) and elevated (800 ppm). Isotope tracing was used to quantify barley N and P acquisition; a labeling solution ( 15 N and 33 P) was added via capillary tubing to a mesh-walled core in each pot, into which AMF hyphae could grow but roots could not. In control pots (no fungal access to isotope tracers), mesh-walled cores were rotated through 90˚ every 48 hr to sever AMF hyphae. Cores which did not receive labeling solution received autoclaved, distilled H 2 O. Eleven days after 15 N and 33 P addition, plant allocation of carbon to fungi was quantified using a 14 CO 2 pulse label. Plants were enclosed in polypropylene bags to create an airtight labeling chamber. A solution of NaH 14 CO 3 was added to a spectrophotometer cuvette before the labeling chamber was sealed. A 10% solution of lactic acid was then added through the polypropylene bag using a hypodermic syringe to evolve 14 CO 2 . Below-ground 14 CO 2 was sampled by use of a third, glass wool-filled core. After 20-22 hr of exposure, the remaining 14 CO 2 in the headspace was captured by addition of 2M KOH to another cuvette. Gas sampling and KOH addition was done through the polypropylene bags using a hypodermic syringe. Plants were destructively harvested 24 hr after 14 CO 2 liberation environmental gradients (e.g. light, heat, humidity) within the cabinets. Starting 4 weeks after the experiment was established, plants were given 25 ml of a low P (25% of the original P quantity) Long Ashton Solution (LAS) twice weekly (Table S1). Plants were watered with tap water as required.

| 33 P, 15 N and 14 C isotope tracing
Assimilation of N and P via AMF was quantified using 33 P and 15 N tracers when plants were 11 weeks old and were at anthesis. Based on the methods of Thirkell, Pastok, et al. (2019), three cores constructed from PVC tubing (length 80 mm, diameter 18 mm), with windows (50 mm × 12 mm) cut in the lower two-thirds of each side were inserted into each of the plant pots ( Figure 1). These windows and the bottom of each core were covered in a 20 µm nylon mesh which prevented penetration by plant roots but allowed AMF hyphal ingrowth. Mesh windows represented c. 70% of the area of the lower two-thirds of the cores. Plastic capillary tubing, 140 mm in length and 1.02 mm in diameter, perforated along the entire length with holes (c. 0.5 mm diameter) was positioned and glued using AquaMate silicone sealant (Everbuild Building Products) to the base inside the cores. Two of the cores were filled with the same soil and sand substrate as the bulk soil, plus 3 g/L crushed basalt (particle size <1 mm), to act as AMF "bait" (Quirk et al., 2012). Each pot also contained a third mesh-windowed core, loosely packed with glass wool (Acros Organics) and then the top was sealed with an airtight septum (SubaSeal ® Perkin Elmer) through which gas sampling can be conducted with a hypodermic syringe, in order to measure belowground respiration throughout the course of the experiment.
To ensure only AMF-mediated 33 P and 15 N tracer movement was measured, one of the mesh-windowed soil cores in each pot was rotated by 90° immediately prior to isotope tracer additions. This rotation severed the fungal connections between the plant and the core contents, preventing direct transfer of the isotope tracers to the host plants via extraradical mycorrhizal fungal mycelium. In half of pots (n = 6 per cultivar), 15 N and 33 P labeling solution was added to the static core, and in the remaining microcosms (n = 6 per cultivar), to the rotated core. Core rotation was repeated every 48 hr between isotope tracer addition and harvest to prevent hyphal re-entry to cores. The second core in each pot remained static, thereby preserving the hyphal connections between the core contents and the host plant. After 10 weeks of growth, 150 µl labeling solution, con- Geiger monitor (Series 900 mini monitor -ThermoFisher Scientific).
As each pot contained one static and one rotated core, the levels of disturbance to the bulk soil and rhizosphere were consistent across all treatments. It is possible that core rotation may have influenced root uptake of isotope labels, but an experimental testing of the method suggests that such effects are likely be minimal (Leifheit et al., 2014).

| Plant harvest and sample preparation
Plants were destructively harvested 24 hr after 14 CO 2 labeling, 88 days after planting. Each microcosm was separated into shoots, roots, bulk soil, static core soil, and rotated core soil before being freeze-dried for 48 hr and weighed. A small sub-sample of roots was separated out before freeze-drying and stored for assessment of colonization by AMF. After weighing, plant materials were homogenized and stored in airtight desiccators prior to analysis.

| Assessment of mycorrhizal colonization
Plant roots were stained using the "ink and vinegar" staining method described by Vierheilig et al. (1998) and 12 C) transferred to the mycelial network from each plant. It was assumed that 13 C enrichment did not differ between treatments, and 13 C was not included in calculations owing to its negligible contribution to total CO 2 in atmospheric air. Total fixed carbon was calculated as a function of total CO 2 in each labeling system and proportion of 14 CO 2 which had been fixed by each plant. Total 14 C and 12 C assimilated by plants and transferred to AMF was calculated using the following formulae (Cameron et al., 2006): where T fp is the total C transferred from plant to AMF (g), A is sample radioactivity (Bq), A sp is the specific activity of the source (Bqmol −1 ), m a is the atomic mass of 14 C (14), P r is the proportion of the total supplied 14 C present in plant tissue and m c is the mass of C in the CO 2 present within the labeling system (g) (using the ideal gas law, below).
where m cd is the mass of CO 2 (g), M cd is the molecular mass of CO 2 (44.01 g/mol), P is pressure (kPa), V cd is volume of CO 2 in the system (0.003 m 3 ), m c is mass of unlabeled C ( 12 C) in the labeling system (g), M is the molar mass of C (12.011 g), R is the universal gas constant (JK −1 / mol), T is the absolute temperature (K), m c is the mass of C in the CO 2 present in the labeling system (g), where 0.27292 is proportion of C in CO 2 (27.292%; Cameron et al., 2008).

| Plant nutrient content
Total phosphorus content within plant and soil material (i.e., nontracer P) was quantified following the colorimetric determination of phosphorus methods adapted from Murphy and Riley (1962

| Statistics
Statistical analyses were carried out using the "RStudio" interface of R statistical software, version 3.4.3. (R Core Team, 2020;RStudio Team, 2015). For tissue nutrient content, biomass and mycorrhizal colonization, data were tested by two-way ANOVA (using base R functions), where cultivar and [CO 2 ] were used as predictor variables. For 15 N, 33 P and 14 C enrichment, data were analyzed separately by cultivar so that CO 2 concentration and core rotation treatment were predictor variables. Where ANOVA gave p < .05 for interaction or main effects, Tukey's honestly significant difference tests were used to identify statistical differences between groups and performed using the emmeans package in R (Lenth, 2020). Prior to running analyses, data were tested for normality using Shapiro-Wilk test, by visual inspection of residual plots and model fit was compared using Akaike information criterion (AIC) testing. Where relevant assumptions were not met, data were log 10 transformed.
Data were plotted using the packages ggplot2 (Wickham, 2016) and multcompView (Graves et al., 2019). The data that support the findings of this study are available from the corresponding author upon reasonable request.

| Barley growth stimulated by elevated eCO 2
Barley shoot biomass was significantly greater when plants were grown in eCO 2 compared to aCO 2 (Figure 2a; T ables S2 and S3;  Table S3).

| Mycorrhizal colonization in barley was unaffected by eCO 2 or variety
All plants of both cultivars were colonized by AMF (Figure 3a-c), and the extent of fungal proliferation in roots was not affected by [CO 2 ] ( Figure 3a; F 2,44 = 0.977, p > .05) or variety (Figure 3a, F 2,44 = 2.016, p > .05). Mean root length colonization ranged from just over 10% in Riviera at aCO 2 to 18% in Moonshine at aCO 2 (Table S2). Overall, arbuscule frequency was low; ranging from around 2% in Riviera at aCO 2 to around 4% in Riviera at eCO 2 and was not significantly influenced by CO 2 or cultivar (Figure 3b). Similarly, vesicle frequency was low across treatment groups, ranging from 0.18% in Riviera at aCO 2 to 0.68% in Moonshine at eCO 2 , although the majority of plants sampled had no vesicles recorded (Table S2).

| Elevated CO 2 dilutes mineral nutrition of barley
Aboveground phosphorus content was significantly greater in cv.
Mycorrhizal P uptake in Moonshine was strongly dependent upon CO 2 concentration-there was a significant interaction between  Table S3; F 2,20 = 16.90, p < .001). While there was no difference between static and rotated treatments for aCO 2 (Tukey p > .05), under eCO 2 , there was significantly more 33 P in Moonshine shoots of the static treatment than the rotated treatment (Tukey p < .001), indicating substantial contribution of AMF to barley P nutrition (Figure 4c). By contrast, there was no clear evidence of mycorrhizal P uptake in cv. Riviera, as the 33 P content of static core treatment was not different from the rotated core treatment (Figure 4d; Figure S1).
Riviera had significantly higher N content (Figure 4a) and concentration ( Figure 5b) than Moonshine, and eCO 2 caused significantly reduced N concentration (F 2,43 = 21.48, p < .001), a trend which was stronger in Riviera (Tukey p = .002) than it was in Moonshine (Tukey p = .069). 15 N uptake was significantly enhanced by AMF in both Moonshine (Figure 5c; Figure S1) and Riviera (Figure 5d; Figure S1), demonstrated by higher 15 N content in static core plants compared to rotated core plants. There was reduced 15 N uptake in eCO 2 compared to aCO 2 , a trend seen across all treatments (Figure 5c,d). Core rotation did not affect shoot N or P concentration ( Figure S2a-d).

| Carbon transfer from plants to fungi
All treatments showed similar amounts of C transfer from plants to fungi in both varieties, quantified as plant-fixed C detected in static and rotated cores (Figure 6a,b; Tables S2 and S3). Root length colonization data (Figure 3a-c) corroborate the pattern seen in carbon allocation data, that neither [CO 2 ] nor cultivar significantly affect carbon allocation to AMF.

| DISCUSSION
As future atmospheric CO 2 c o n c e n t r a t i o n s a r e p r o j e c t e d t o continue rising (Le Quéré et al., 2015), crop growth is also expected to increase, due to enhanced photosynthetic C assimilation (Ainsworth & Long, 2005;Dong et al., 2018;Mitterbauer et al., 2017;Terrer et al., 2016). Our data support this trend, as both cultivars had greater shoot biomass at eCO 2 compared to ambient [CO 2 ], although root biomass appeared to be less affected ( Figure 2). While the biomass response to eCO 2 was similar in both cultivars examined here, it is important to note that significant variation in barley growth responses to eCO 2 has been demonstrated elsewhere (Mitterbauer et al., 2017). The mechanisms responsible for this variation are not entirely clear; crop genetic diversity will prove critical when adapting agriculturally important species to climate change factors such as drought and eCO 2 . Crop cultivars have demonstrated differing susceptibility to photosynthetic acclimation to eCO 2 , where predicted increases in photosynthesis are not observed (Tausz-Posch et al., 2020). The extent to which this occurs in cereals remains to be resolved, although it is worth noting that photosynthetic acclimation to eCO 2 may depend on nitrogen availability and water use efficiency-two factors which themselves may be influenced by AMF.
Concerns have been raised that any "CO 2 fertilisation" effect on crop growth may exacerbate problems of malnutrition; despite potential increases in yield, the nutritional quality of the grains is often decreased at eCO 2 (Myers et al., 2014). This is largely because carbohydrate assimilation accounts for the majority of the yield increases observed at eCO 2 , thus the relative concentrations of mineral nutrients and protein become "diluted" (Cotrufo et al., 1998;Manderscheid et al., 1995).

| AMF may mediate barley P assimilation response to eCO 2
Substantial 33 P enrichment in Moonshine shoots at eCO 2 (Figure 4c,d) indicates that increased transfer of P at eCO 2 by AMF helped maintain tissue P concentrations across [CO 2 ] treatments for this cultivar.
In contrast, cv. Riviera received no more 33 P from AMF symbionts at eCO 2 than at ambient [CO 2 ], and P concentration became diluted as biomass increased. Although little is known about the mechanisms underpinning the effects of eCO 2 on mycorrhizal cereal crop nutrient acquisition, a variety of responses are evident in the literature.
In general, eCO 2 tends to enhance P uptake in mycorrhizal plants Low N availability in the substrate may partly explain the decreased N concentrations at eCO 2 , as both cultivars here were grown in nutrient-limited conditions. N and P dilution was probably more pronounced in cv. Riviera than Moonshine at eCO 2 because Riviera achieved a greater biomass, and as such had a higher nutrient demand. Previously, N availability has been identified as the most significant limitation for eCO 2 fertilization in AM plants (Terrer et al., 2016). In both cultivars, allowing AMF access to the labeled core resulted in greater 15 N label assimilation in barley shoots, suggesting AMF contributed to N uptake. Mycorrhizal acquisition of N has been demonstrated in barley (Wilkinson et al., 2019), and wheat (Miransari et al., 2009;Thirkell, Pastok, et al., 2019;Zhu et al., 2016), where it has been shown to increase under eCO 2 (Zhu et al., 2018).
By contrast, there was no effect of eCO 2 (Figure 5c,d) on mycorrhizal 15 N uptake here. Notably, mycorrhizal acquisition of 15 N in cv. Moonshine was around double that in cv. Riviera (Figure 5c,d).
Variation in mycorrhizal functioning among cultivars of crops is wellknown from the literature (Hetrick et al., 1992;Sawers et al., 2017;Watts-Williams, Emmett, et al., 2019;Zhang et al., 2019). Indeed, cultivar specificity in mycorrhizal 15 N uptake has been shown previously in a barley field study (Thirkell, Cameron, et al., 2019). It is clear from our data that cv. Riviera receives less nutritional contribution from its mycorrhizal symbionts than Moonshine does (Figures 4c,d and 5c,d), although it is not clear why this is the case. A recent metaanalysis of AMF influence over grain yields suggests that older varieties typically benefit more from AMF than do modern varieties (Zhang et al., 2019). Similarly, an experimental comparison of five barley cultivars showed that modern cultivars generally responded more negatively to AMF inoculation than older ones (Al Mutairi et al., 2020). cv. Riviera is indeed a newer cultivar than Moonshine, however both cultivars were developed relatively recently ( vs. 1994( , SASA, 2020. As such, cultivar age is unlikely to be a contributory factor here.

| Mycorrhizal nutrient acquisition patterns are uncoupled in barley
Our data suggest that mycorrhizal acquisition of N and P are not intrinsically linked, i.e., plants which receive N from their AMF symbionts do not necessarily also receive P (Figures 4c,d and 5c,d). As N and P transfer from fungi to a plant host occurs via transporters specific for ammonium (Guether et al., 2009;Kobae et al., 2010;Perez-Tienda et al., 2011) and phosphate (Harrison et al., 2002(Harrison et al., , 2010, this is not surprising. Uncoupled fungal transfer of N and P may reflect plant demand, as both cultivars appeared more N-limited than P-limited FIGURE 3 Arbuscular mycorrhizal colonization-total fungal biomass (a) arbuscule frequency (b) and vesicle frequency (c) of spring barley (Hordeum vulgare L. cv. Moonshine and Riviera) grown in ambient (440 ppm, white boxes) and elevated (800 ppm, gray boxes) atmospheric CO 2 , n = 12. ANOVA p-values are included for main effects and interaction between main effects. Data were log10 transformed where assumptions for statistical tests were not satisfied in our experiments (Figures 4b and 5b), given the typical demand from cereals for these macronutrients (Maathuis & Diatloff, 2013;Marschner, 2011). It is unclear from the literature how mycorrhizal benefit would be affected by higher nutrient availabilities; some evidence suggests that limitation in either N or P is sufficient to stimulate plant hosts to rely on mycorrhizal nutrient uptake (Nouri et al., 2014).  Further evidence suggests the contrary, that limited P and sufficient (or luxury) N supply should promote the greatest transfer of P and N from fungi to hosts (Johnson et al., 2015). Without experimental testing, it may not be possible to determine how to maximize the contribution of the AMF to P and N nutrition of these cultivars, given the substantial functional diversity in mycorrhizal functioning arising from plant genotype (Baon et al., 1993;Hetrick et al., 1992;Sawers et al., 2017;Watts-Williams, Emmett, et al., 2019).

| Mycorrhizal C acquisition largely unresponsive to cultivar or CO 2 concentration
Using 14 C tracing, we found no evidence that plant-to-fungus car- meta-analyses (Alberton et al., 2005;Treseder, 2004). However, our results are consistent with data previously reported in wheat in similar experimental systems (Thirkell, Pastok, et al., 2019). Our root length colonization data suggest that allocation to intraradical fungal structures was likewise unaffected by eCO 2 (Figure 3ac). The amounts of C transferred to fungi here were perhaps too low to detect by the 14 C labeling, a technique which can create noisy data ( Figure 6;
Moreover, how rising atmospheric [CO 2 ] will affect AM symbioses is also uncertain (Cotton, 2018). As the combined pressures of climate change, population growth and environmental accountability mount, and demand for sustainable food production increases through the 21st century, innovative agricultural solutions must be found. Exploiting the soil microbial community has been suggested as one potential tool which could be used to achieve sustainable intensification in agriculture (Rillig et al., 2016;Thirkell et al., 2017).
As we used an unsterilized farm soil in our growth media, it is possible that the differences we observed in carbon-for-nutrient exchange in our experiments were a result of changes in AMF fungal community structure and composition as a result of the [CO 2 ] treatments in our experiments (Cotton et al., 2015;Panneerselvam et al., 2020). Little is currently known about how or why these changes may occur (Cotton, 2018) but it is clear that different AMF isolates and species show strongly contrasting symbiotic phenotypes (Mensah et al., 2015;Munkvold et al., 2004).
As such, [CO 2 ]-induced changes in AMF community structure and composition may affect C-for-nutrient exchange with host plants.
Unfortunately, we did not investigate changes in AMF community composition between [CO 2 ] treatments in our experiments, but this is certainly worth future investigation, particularly within the context of future climate change.
Our results, together with those in previous research (Thirkell, Cameron, et al., 2019;Thirkell, Pastok, et al., 2019) suggest that cultivar identity is an important factor in regulating the response of mycorrhizal cereal nutrient acquisition in barley to eCO 2 . Our finding that AMF might limit, or even prevent, [CO 2 ]-induced dilution of P in barley shoots is intriguing, and must be validated in further trials, including those which grow plants to yield, before ultimately being tested in the field (Lekberg & Helgason, 2018). With a greater understanding of the factors regulating carbon-for-nutrient exchange between mycorrhizal symbionts, it should be possible using existing breeding techniques to maximize the benefit of cereal mycorrhizas.