Water use dynamics of dryland wheat grown under elevated CO₂ with supplemental nitrogen

Plant materials

Wheat cvv. Yitpi and Wyalkatchem were selected for their similar grain yield potential but contrasting agronomic features. Yitpi is a free-tillering cultivar with long coleoptile and broad adaptation to most soil types and rainfall regions across Australia’s wheatbelt (Seednet 2005), whereas Wyalkatchem is characterised as having poor early vigour, short coleoptiles, high harvest index and adaption to low-rainfall regions (Intergrain 2001). Under low background soil N, cv. Wyalkatchem has been found to maintain higher grain protein than cv. Yitpi (Malik et al. 2016), most likely due to superior N acquisition and utilisation efficiencies as shown by its performance on a sandy, non-alkaline soil in Western Australia (Alhabbar et al. 2018).

Experimental design

This experiment was conducted in 2016 within the AGFACE facility in Horsham (36°44′57″S, 142°06′50″E; 127 m a.m.s.l.), Victoria, Australia. The study site has been described in detail by Mollah et al. (2009) and Fitzgerald et al. (2022), but the present experiment was conducted on an adjacent site with low background soil N (Walker et al. 2017). Soil type at the site is a Vertosol (Isbell 1996) clay soil (~35% clay at the surface and 60% at 1.4 m depth). The soil at this site has an alkaline pH throughout the profile with moderate sodicity (mainly >20 cm depth) and high levels of boron and salt (>40 cm depth) (Table 1). The site has a Mediterranean climate with cool, wet winters and dry summers (Hutchinson et al. 2005). Long-term (30-year, 1981–2010) average annual rainfall is 445 mm, with 314 mm typically falling during the wheat growing season (May–November) (Bureau of Meteorology 2017, Polkemmet Road #079023). For the same time period, average maximum and minimum temperatures were 17.8°C and 5.3°C, respectively. During the study year, total annual rainfall was 550 mm, of which 406 mm fell during May–November. Average daily maximum (17.6°C) and minimum (5.3°C) temperatures were similar to the long-term average. A summary of the environmental variables recorded by an on-site weather station (MEA Premium Weather Station 103; Measurement Engineering Australia, Magill, SA, Australia) during the studied growing season is given in Fig. 1.

Fig. 1.

Daily maximum and minimum temperatures, rainfall and reference evapotranspiration (ETₒ) recorded on-site at the Australian Grains FACE (AGFACE) facility in Horsham, Victoria, Australia in 2016. Arrows indicate stem-elongation (growth stage DC31; Zadoks et al. 1974), anthesis (DC65) and physiological maturity (DC90) of wheat cvv. Yitpi (—) and Wyalkatchem (....).

The experimental was a nested split-plot design with six replicate plots (octagonal ‘rings’) of 16 m diameter for a[CO₂] (~400 μmol mol⁻¹) and four rings for e[CO₂] (~550 μmol mol⁻¹) spaced at 60 m centre-to-centre. Normalised differences vegetation index (NDVI), profile probe, aboveground biomass and final harvest measurement were collected from the six a[CO₂] rings and the four e[CO₂] rings, whereas other periodic measurements (e.g. leaf gas exchange, green leaf area, N-Pen, SPAD and root growth) were acquired in four rings only for both a[CO₂] and e[CO₂] treatments. Within each ring, cvv. Yitpi and Wyalkatchem were randomly allocated to two subplots each, and each subplot was treated without (−N, 0 kg N ha⁻¹) or with (+N, 100 kg N ha⁻¹) supplemental N (see Supplementary Fig. S1) as granular urea lightly incorporated into the soil at sowing. Each subplot was 4 m long by 1.46 m wide and consisted of six rows with 0.244 m spacing. Plots were dry seeded (by drilling into 3–5 cm depth) in north–south orientation on 31 May 2016. At sowing, granular fertiliser (8.8% phosphorus (P), 11% sulfur (S)) was incorporated into the soil at rates of 8 kg P ha⁻¹ and 10 kg S ha⁻¹. Initial soil mineral N was 90.3 kg NO₃⁻-N ha⁻¹ (0–120 cm depth of soil profile).

At the perimeter of the e[CO₂] octagonal rings, horizontal stainless-steel tubes were positioned about 15 cm above the canopy at any developmental stage. From sunrise to sunset, enrichment of the [CO₂] of ambient air to the target of 550 μmol mol⁻¹ was achieved by injecting pure CO₂ into the air through 0.3-mm laser-drilled holes (20 holes m⁻¹) on the upwind-side tubes. CO₂ then quickly mixed with the air and was blown throughout the plot by prevailing wind. CO₂ concentrations were monitored and recorded every minute with an infrared gas analyser (SBA-4; PP Systems, Amesbury, MA, USA) positioned at the centre of each ring. The performance of the CO₂ delivery system is characterised in Mollah et al. (2009) and spatial variations of [CO₂] within e[CO₂] rings are described by Mollah et al. (2011).

Soil-water content and water use

Soil water was measured weekly with a PR2/6 Profile Probe (Delta-T Devices, Cambridge, UK) with six sensors positioned at 10, 20, 30, 40, 60 and 100 cm depth. A 100-cm-long, thin-walled fibreglass access tube (ATS1/ATL1; Delta-T Devices) was inserted into the soil in each plot and left in situ for the entire growing season (Fig. S1). To take a measurement, the PR2/6 capacitance probe was inserted into the access tube. It measures the dielectric constant (ε) via emission of an electromagnetic field 100 mm into the surrounding soil at each sensor depth. The output from each sensor is a DC voltage (in mV), which is converted to volumetric water content (m³ m⁻³) via a site-specific calibration curve (Whalley et al. 2004) developed for each soil depth of the AGFACE facilities in the earlier years. Soil-water content (mm) for each depth was calculated from the volumetric soil water multiplied by the corresponding depth increment and summed up to 100 cm depth. No irrigation was applied; therefore, water use between physiologically important growth stages was determined by the following equation:

where P is precipitation (mm) and SWD (mm) is the change in water content (mm) of the soil profile between the two measurements. The final measurement of soil-water content coincided with maturity of cv. Wyalkatchem, which occurred several days earlier than that of cv. Yitpi, possibly leading to an underestimate of Yitpi water use. Cumulative water use over the season was calculated by summing water use estimates at different growth stages.

Normalised difference vegetation index

Non-destructive measurements of canopy reflectance were done in all treatment subplots by using a handheld CropCircle ACS-210 with GeoSCOUT GLS-400 data logger (Holland Scientific, Lincoln, NE, USA), from which the NDVI was calculated. Measurements were taken from 51 to 189 days after sowing (DAS) at approximately weekly intervals. The sensor was held ~0.5 m above the crop canopy and oriented to measure a foot-print across the middle rows of the plot. The NDVI is calculated directly by the sensor, and mean values of transects from non-disturbed areas within the subplot were used as a surrogate for canopy cover (Perry et al. 2012).

Root length

Root length was measured at ~2-week intervals with a mini-rhizotron (CI-600; CID Bio-Science, Camas, WA, USA), which enables non-destructive monitoring of the same individual roots over the selected time intervals (Andersson and Majdi 2005). Detailed description of installing acrylic clear rhizo-tubes and acquiring the images of the root system for a similar experimental set-up in the AGFACE facility is given in Bahrami et al. (2017). In short, one 1.05-m-long rhizo-tube with internal diameter 7 cm was pre-installed at a 45° angle in each of the four subplots per ring (Fig. S1). For each rhizo-tube, four images were captured at four depths (1, 0–16 cm; 2, 17–32 cm; 3, 33–48 cm; 4, 49–64 cm) below the soil surface. The images were analysed for the captured root length per image using the software RootSnap! version 1.2.8.23 (CID Bio-Science). Total root length was calculated as the sum of root length across all four depths. The ratios of root length between the deeper (sum of Depths 3 and 4) and upper (sum of Depths 1 and 2) soil layers are reported.

Leaf gas exchange measurements

Stomatal conductance (g_s) and net photosynthetic assimilation rate (A_net) of fully expanded flag leaves (or uppermost leaf when flag leaves were not yet fully expanded) were measured weekly from 90 to 170 DAS. An open-path infrared gas analyser (LI-6400; LI-COR, Lincoln, NE, USA) with a standard leaf chamber (clear-top with a maximum measurement area of 2 cm by 3 cm) was used to measure instantaneous gas exchange of a single plant per plot. The cuvette air flow rate was set to 500 μmol s⁻¹ with [CO₂] of either 400 or 550 μmol mol⁻¹ depending on the [CO₂] growth conditions. Measurements were performed on sunny days from 09:00 to 11:30, and photosynthetically active radiation ranged between 900 and 1250 μmol m⁻² s⁻¹ during measurement periods (Houshmandfar et al. 2015). Values were recorded after first stabilisation (generally after 90 s), and three measurements were recorded at an interval of 5 s and averaged. This allowed conditions in the cuvette to reach a steady state but did not allow stomata to adjust to the cuvette conditions. Vapour pressure deficit was between 0.9 and 2.2 kPa depending on measurement dates and was not different among samples and treatments on each date (Houshmandfar et al. 2015). After completion of the gas exchange measurements, the leaf surface area was calculated from the length and width of the part of the leaf enclosed in the cuvette. Values for all gas exchange parameters were calculated based on the surface area of the leaf inside the cuvette. Intrinsic WUE (iWUE) was calculated as A_net divided by g_s.

Chlorophyll and N content

Surrogate measurements of chlorophyll and N content of flag leaves were taken non-destructively by chlorophyll meter (SPAD-502; Konica Minolta Sensing, Tokyo, Japan) and N analyser (N-Pen N 100; Photon Systems Instruments, Drásov, Czech Republic), at approximately weekly intervals. Both SPAD and N-Pen readings were taken on one side of the midrib and around the midpoint of the topmost leaf or flag leaf (Xiong et al. 2015). For each replicate subplot, SPAD and N-Pen values were averaged from the representative measurements of 10 and six leaves, respectively.

Growth, yield and morpho-physiological parameters

For determination of green leaf area, destructive samples were collected at approximately weekly to 2-week intervals from one week before stem elongation (DC31; Zadoks et al. 1974) until physiological maturity (DC90). Uniform individual plants were initially tagged at 40 DAS at the soil surface level to identify them clearly as single plants in later stages. Then at each sampling date, three predetermined and representative plants per plot were harvested (excluding edge rows). Green leaf area was measured with a leaf area meter (LI-3100C Area Meter; LI-COR).

For estimation of the area-based aboveground biomass, a predetermined subplot of 0.29, 0.49 and 0.73 m² including the middle four rows was harvested at DC31, DC65 and DC90, respectively. At DC31 and DC65, samples were oven-dried for 72 h at 70°C, and at DC90, samples were oven-dried for the same time but at 40°C. Dry weights were recorded at each stage. At DC90, plant height was also recorded. Spikes were threshed to separate the grains, and both grain yield and 1000-grain weight were recorded. Harvest Index (HI) was calculated as grain yield divided by the aboveground biomass. WUE of biomass (WUEb) and grain yield (WUEy) were calculated by dividing aboveground biomass and grain yield with cumulative water use.

Statistical analyses

The experiment was a split-plot design with two [CO₂] treatments as main-plots (rings) and two cultivars and two N rates in full factorial combination as subplots, with four e[CO₂] and six a[CO₂] replications. The effects of CO₂, cultivars and N rates were considered fixed effects and ring and plot as random effects in a linear mixed effects model analysed with R package ‘nlme’ (Pinheiro et al. 2022). For repeatedly measured parameters, DAS was used as an additional fixed effect. Homogeneity of variances was evaluated visually, and the model was adjusted when stepwise evaluation of the model indicated an improvement after correction. Statistical effects were regarded as significant where P < 0.1. Data analyses were performed in R version 3.4.3 (R Core Team 2022) and mixed effects model P-values are reported in Supplementary Tables S1, S2 and S3.

Plant density and phenological development

Plant density was significantly affected by cultivar (P = 0.056) and N rate (P = 0.040), where cv. Yitpi had 10% more seedlings established than cv. Wyalkatchem, and under supplemental N, 11% fewer seedlings were established than without supplemental N (Table 2). Wyalkatchem started flowering earlier and senesced faster, resulting in harvest 10 days earlier than Yitpi (Fig. 1); however, the other treatment variables had no significant effects on phenological development of either cultivar.

Gas exchange parameters

Net assimilation rate (A_net) of wheat grown under e[CO₂] was 12% greater than under a[CO₂] averaged across all measurements (Fig. 2a, Table S1). With supplemental N, cv. Yitpi maintained higher A_net than cv. Wyalkatchem later in the season, but without supplemental N, differences between cultivars diminished as the season progressed (DAS × N × Cv, P = 0.002). Stomatal conductance was 25% lower under e[CO₂] than a[CO₂] averaged across all measurements, but this difference diminished as the growing season progressed (Fig. 2b). Yitpi maintained greater g_s with supplemental N throughout the growing season, whereas this trend was reversed for Wyalkatchem so that, later in the season, g_s was greater for wheat grown without supplemental N (DAS × N × Cv, P = 0.012). Elevated [CO₂] increased iWUE, and differences between a[CO₂] and e[CO₂] increased later in the season with decreasing g_s (Fig. 2c; DAS × CO₂, P < 0.001). Early in the season, iWUE was similar for the two cultivars, but as the season progressed Yitpi had greater iWUE than Wyalkatchem (DAS × Cv, P = 0.005).

Fig. 2.

(a) Net photosynthetic assimilation rate (A_net), (b) stomatal conductance (g_s), and (c) intrinsic water use efficiency (iWUE) measured weekly during the growing season of wheat cvv. Yitpi (Yit) and Wyalkatchem (Wyal) grown under ambient [CO₂] (a[CO₂], ~400 μmol mol⁻¹) or elevated [CO₂] (e[CO₂], ~550 μmol mol⁻¹) with (100 kg N ha⁻¹) or without (0 kg N ha⁻¹) supplemental N. Data are means and standard errors of n = 4 replicates.

Green leaf area, chlorophyll and N content

Averaged across all measurement dates and treatments, green leaf area under e[CO₂] was 10% greater than under a[CO₂] (Fig. 3a, Table S1). Cv. Yitpi had greater green leaf area than cv. Wyalkatchem, with the difference increasing as the season progressed (DAS × Cv, P < 0.001). Supplemental N increased green leaf area and maintained greater leaf area for longer than when wheat was grown without supplemental N (DAS × N, P = 0.004). Supplemental N stimulated both leaf N and chlorophyll content, but to a greater extent for Yitpi than for Wyalkatchem (Fig. 3b, c). As the season progressed, Yitpi grown with supplemental N maintained greater leaf N and chlorophyll content than Wyalkatchem; however, these differences disappeared where no supplemental N was applied (DAS × N × Cv, P = 0.004 and <0.001 for leaf N and chlorophyll content, respectively).

Fig. 3.

(a) Green leaf area, (b) N concentration (N-Pen), and (c) chlorophyll content (SPAD) measured weekly during the growing season of wheat cvv. Yitpi (Yit) and Wyalkatchem (Wyal) grown under ambient [CO₂] (a[CO₂], ~400 μmol mol⁻¹) or elevated [CO₂] (e[CO₂], ~550 μmol mol⁻¹) with (100 kg N ha⁻¹) or without (0 kg N ha⁻¹) supplemental N. Data are means and standard errors of n = 4 replicates; each replicate of leaf area is an average of three plants; for N concentration and chlorophyll content, each replicate is an average of 6 and 10 measurements, respectively.

Normalised difference vegetation index

Both early and later in the season, NDVI of cv. Yitpi was greater than for cv. Wyalkatchem; however, this difference was not evident during canopy closure (maximum NDVI; Fig. 4, Table S1; DAS × Cv, P < 0.001). Wheat grown with supplemental N had higher NDVI values than wheat grown without supplemental N, and these differences increased throughout the season until maturity (DAS × N, P < 0.001).

Fig. 4.

Normalised differences vegetation index (NDVI) measured at weekly to 2-week intervals during the growing season of wheat cvv. Yitpi and Wyalkatchem grown under ambient [CO₂] (a[CO₂], ~400 μmol mol⁻¹) or elevated [CO₂] (e[CO₂], ~550 μmol mol⁻¹) with (100 kg N ha⁻¹) or without (0 kg N ha⁻¹) supplemental N. Data are means and standard errors of n = 6 and 4 replicates for a[CO₂] and e[CO₂], respectively.

Root length

Effects of CO₂, N rate and cultivar on root length depended on rooting depth (Fig. 5; CO₂ × N × Cv × depth, P < 0.001). At Depth 1 (0–16 cm), root length of cv. Yitpi was smaller under e[CO₂] than a[CO₂] regardless of N supply (Fig. 5a, Table S2). Root length of cv. Wyalkatchem with supplemental N was greater under e[CO₂] than a[CO₂]; without supplemental N, [CO₂] had no effect. At this depth, root length of Yitpi was greater than that of Wyalkatchem. The difference was greatest when maximum root length was achieved but was diminished before and after this time.

Fig. 5.

Captured root length at four depths of the soil profile: (a) 1, 0–16 cm; (b) 2, 17–32 cm; (c) 3, 33–48 cm; and (d) 4, 49–64 cm. (e) Total root length (sum of all depths); (f) ratio of root length between Depths 1 + 2 and Depths 3 + 4. Measurements taken at ~2-week intervals during the growing season of wheat cvv. Yitpi and Wyalkatchem grown under ambient [CO₂] (a[CO₂], ~400 μmol mol⁻¹) or elevated [CO₂] (e[CO₂], ~550 μmol mol⁻¹) with (100 kg N ha⁻¹) or without (0 kg N ha⁻¹) supplemental N. Data are means and standard errors of n = 4 replicates.

At Depth 2 (17–32 cm), root length was smaller under e[CO₂] than a[CO₂] in the absence of supplemental N, but with supplemental N, root length was greater under e[CO₂] than a[CO₂] (Fig. 5b, Table S2). At this depth, stimulation of root length by supplemental N was most pronounced during the period of maximum root length but the difference was less both before and after this time. Three-way interactions CO₂ × N × Cv were significant (P < 0.001) for root length of wheat at both Depth 3 (33–48 cm) and Depth 4 (49–64 cm; Fig. 5c, d, Table S2). At Depth 3, root length of cv. Yitpi grown without supplemental N was 25% smaller under e[CO₂] than a[CO₂], but when N was applied, it was 62% greater under e[CO₂] than a[CO₂]. However, root length of cv. Wyalkatchem when grown without supplemental N was 32% greater under e[CO₂] than a[CO₂] but was similar for both CO₂ treatments when grown with supplemental N. The trend of root length at Depth 4 was similar to that of Depth 3.

Supplementation with N stimulated total root length, with greater effect later in the season than at the early measurement dates (DAS × N, P = 0.033; Fig. 5e, Table S2). A four-way interaction DAS × CO₂ × N × Cv was significant (P < 0.001) for the ratio of root length in the deeper (Depth 3 + 4) versus the upper (Depth 1 + 2) soil layers (Fig. 5f, Table S2). Elevated [CO₂] increased this ratio for cv. Wyalkatchem when grown without supplemental N, but with supplemental N the ratio was greater under a[CO₂] than e[CO₂]. Yitpi showed an opposite trend with negligible difference between a[CO₂] and e[CO₂] treatments when grown without supplemental N, but with supplemental N, the ratio was greater under e[CO₂] than a[CO₂]. All those differences were more prominent later in the growing season.

Aboveground biomass at different growth stages

At DC31, e[CO₂] stimulated aboveground biomass by 28% when wheat was grown with supplemental N, whereas this effect was negligible (<5%) when N was not applied (CO₂ × N, P = 0.073; Fig. 6a). At the same growth stage, supplemental N stimulated the aboveground biomass of cv. Yitpi to a greater extent than cv. Wyalkatchem (58% vs 32%, N × Cv, P = 0.096). At DC65, aboveground biomass showed a significant three-way interaction (CO₂ × N × Cv, P = 0.030; Fig. 6b). Elevated [CO₂] stimulated the aboveground biomass of Yitpi by 28% when grown with supplemental N but decreased it by 9% when N was not applied. At the same growth stage, e[CO₂] stimulated the aboveground biomass of Wyalkatchem to a similar extent for both N rates. At DC90, e[CO₂] and supplemental N stimulated aboveground biomass by 11% (P = 0.03) and 15% (P = 0.002), respectively (Table 2, Fig. 6c). Yitpi had 12% greater (P = 0.01) aboveground biomass than Wyalkatchem.

Fig. 6.

Aboveground biomass at (a) DC31, (b) DC65, and (c) DC90 of wheat cvv. Yitpi and Wyalkatchem grown under ambient [CO₂] (a[CO₂], ~400 μmol mol⁻¹) or elevated [CO₂] (e[CO₂], ~550 μmol mol⁻¹) with (100 kg N ha⁻¹) or without (0 kg N ha⁻¹) supplemental N. Data are means and standard errors of n = 6 and 4 replicates for a[CO₂] and e[CO₂], respectively. Only significant (P < 0.1) P-values of the mixed effects model with main effects CO₂, N rate and cultivar (Cv) as well as their interactions are presented.

Grain yield, 1000-grain weight and harvest index

At harvest (DC90), e[CO₂] increased the plant height of cv. Wyalkatchem by 4%, whereas it reduced that of cv. ‘Yitpi’ by 3% (CO₂ × Cv, P = 0.011; Table 2). Supplemental N stimulated the grain yield of Yitpi by 14% while decreasing that of Wyalkatchem by 7% (N × Cv, P = 0.063; Table 2). Supplemental N decreased the HI of Wyalkatchem by 19% but did not affect that of Yitpi (N × Cv, P = 0.002). Elevated [CO₂] stimulated 1000-grain weight of Yitpi grown with supplemental N (9%) but had no effect when N was not applied. For Wyalkatchem, e[CO₂] decreased 1000-grain weight by 5% when grown with supplemental N and increased it by 3% when grown without supplemental N (CO₂ × N × Cv, P = 0.098; Table 2).

Water use at different growth stages and water use efficiency

During early growth stages (i.e. sowing until DC31), e[CO₂] resulted in 45% higher water use than a[CO₂] (P = 0.009; Fig. 7a). However, during this stage, e[CO₂]-induced stimulation of water use was greater for cv. Yitpi than cv. Wyalkatchem (74% vs 21%, CO₂ × Cv; P = 0.073). From DC65 to DC90, Yitpi used 34% more water than Wyalkatchem (P = 0.029; Fig. 7c). Cumulative water use of Yitpi was 6% higher than that of Wyalkatchem (P = 0.079; Fig. 7d). Wheat grown with supplemental N had 5% higher cumulative water use than wheat grown without supplemental N (P = 0.099). Supplemental N increased the WUEb by 10% (P = 0.059), and Wyalkatchem had 17% lower WUEy than Yitpi (P = 0.008; Table 2).

Fig. 7.

Water use (a) from 21 DAS to DC31, (b) from DC31 to DC65, (c) from DC65 to DC90, and (d) cumulative through all growth stages of wheat cvv. Yitpi and Wyalkatchem grown under ambient [CO₂] (a[CO₂], ~400 μmol mol⁻¹) or elevated [CO₂] (e[CO₂], ~550 μmol mol⁻¹) with (100 kg N ha⁻¹) or without (0 kg N ha⁻¹) supplemental N. Data are means and standard errors of n = 6 and 4 replicates for a[CO₂] and e[CO₂], respectively. Only significant (P < 0.1) P-values of the mixed effects model with main effects CO₂, N rate and cultivar (Cv) as well as their interactions are presented.

Increased early biomass and leaf area under e[CO₂] led to increased water use, offsetting leaf level intrinsic water use efficiency

Plants grown under e[CO₂] conditions commonly exhibit a reduction in g_s (Ainsworth and Long 2005). Our study quantified a 25% decrease in g_s under e[CO₂], which, in combination with a 12% increase in A_net, resulted in a 40% enhancement in iWUE compared with a[CO₂] conditions. Although this indicates an improvement in leaf-level WUE, it is crucial to consider the implications at larger scales such as the plant, field or regional level. The well-known CO₂ fertilisation effect stimulates plant biomass, including leaf area, which can counteract the water savings achieved at the leaf level (Ainsworth and Long 2005; Bunce 2016). Previous studies (Wu et al. 2004; Ukkola et al. 2016) support this notion. Our investigation, specifically during the early growth stage (from sowing until DC31), revealed that the increased biomass and leaf area offset the gains in leaf-level WUE, resulting in higher water use under e[CO₂] than a[CO₂]. Consequently, greater canopy transpiration likely contributed to soil water depletion, aligning with recent studies conducted in both controlled environment (Uddin et al. 2018d) and field (Gray et al. 2016) conditions. The elevated water use during the early growth stages under e[CO₂] may subsequently lead to reduced water availability during critical stages (i.e. flowering and grain filling). This, combined with terminal drought events during low-rainfall years, can result in the phenomenon of haying-off, significantly impacting grain yield (van Herwaarden et al. 1998; Nuttall et al. 2012). To ensure optimal soil-water availability under e[CO₂] for grain filling (and to capitalise on the CO₂ fertilisation effect), multiple adaptations are likely necessary. These adaptations may involve advancements in genetics for enhanced transpiration efficiency (Christy et al. 2018), early-season vigour to facilitate rapid soil coverage (Bourgault et al. 2013), and strategic soil-water management practices to minimise soil evaporation (Lascano and Baumhardt 1996).

Differences in water use between a[CO₂] and e[CO₂] conditions during the early growth stages were no longer evident in the later growth stages, resulting in similar cumulative water use. The study area is characterised by low and unpredictable rainfall during the grain-filling period, which coupled with high evaporative demand (Fig. 1) driven by elevated temperatures and strong winds can mean that all available soil water is transpired (Sadras and Rodriguez 2007; Uddin et al. 2018a). The greater canopy cover under e[CO₂] during anthesis may have mitigated the impact of evaporative water loss, leading to lower water use (although not statistically significant, P = 0.138). Despite the study period experiencing above-average rainfall primarily driven by a single large event (DAS 100–105), there were only a few small rainfall events (<5 mm) accompanied by a steady increase in reference evapotranspiration from anthesis onward, which likely masked any minor differences in water use between a[CO₂] and e[CO₂].

Although the increase in water use due to supplemental N was marginal during each growth stage, it led to greater cumulative water use. This observation can be primarily attributed to the significant increase in various growth parameters, including green leaf area, biomass and canopy cover, resulting from N supplementation. Of the two cultivars tested, Yitpi consistently outperformed Wyalkatchem in these growth parameters, ultimately exhibiting higher cumulative water use. Notably, during the grain-filling period, Yitpi exhibited higher water use than Wyalkatchem (Fig. 6c). Water use during this critical stage has a higher conversion efficiency into grain production (Kirkegaard et al. 2007; Wasson et al. 2012), which likely contributed to the grain yield and WUEy advantage of Yitpi over Wyalkatchem.

Stimulation of root length under e[CO₂] increased crop water use through improved soil-water acquisition

The stimulation of root length observed under e[CO₂] conditions in our study is consistent with earlier findings from an adjacent site with high background soil N (Bahrami et al. 2017; Uddin et al. 2018a). This consistency is further supported by reports from chamber studies (Chaudhuri et al. 1990; Benlloch-Gonzalez et al. 2014a) and other FACE experiments (Pacholski et al. 2015; Gray et al. 2016). Averaged across all measurement dates and treatments, the stimulation of root length under e[CO₂] was greater (22%) than the stimulation of aboveground biomass (11%) and grain yield, which aligns with trends reported in previous studies investigating CO₂ enrichment effects (Kimball et al. 2002; Benlloch-Gonzalez et al. 2014b).

The response of root growth to e[CO₂] can be influenced by various factors including cultivar differences (Benlloch-Gonzalez et al. 2014a; Uddin et al. 2018a), growing conditions such as N and water availability (Stulen and den Hertog 1993; Wechsung et al. 1999; Burkart et al. 2004), and crop growth stage (Wechsung et al. 1999; Weigel and Manderscheid 2012). In the present study, the effect of supplemental N on the response of root length to e[CO₂] was dependent on the cultivar and growth stage. The pattern observed in root length (at soil Depths 3 and 4) of both cultivars in response to either supplemental N or e[CO₂] aligned with their respective biomass and grain yield responses. The root length in Depths 3 and 4, total root length, and ratio of root length in the deeper (Depths 3 and 4) versus the upper layers (Depths 1 and 2) were affected by complex interactions among all treatment variables. Yitpi exhibited greater stimulation of root growth with supplemental N, whereas Wyalkatchem showed the opposite trend, with greater e[CO₂]-induced stimulation of root growth, particularly in the lower soil profile, when grown without supplemental N. However, there was no interaction between N rate and [CO₂] in terms of grain yield, although supplemental N had a greater impact on increasing the grain yield of Yitpi than of Wyalkatchem. The stimulation of root growth by e[CO₂] and/or supplemental N may be attributed to indirect effects related to increased total plant biomass and adaptive mechanisms for improved water and nutrient acquisition (Pacholski et al. 2015). Furthermore, the stimulation of root growth under e[CO₂] may be necessary to sustain the increase in biomass and grain yield (Contador et al. 2015).

Elevated [CO₂] stimulated the growth of roots at all measured soil depths, indicating a potential impact on the spatial pattern of soil-water and nutrient uptake within the soil profile (Fig. S2). The greater stimulation of root growth near the soil surface can be advantageous for dryland crops, because it enables them to utilise surface water that becomes temporarily available following small rainfall events (Sadras and Rodriguez 2007). Simultaneously, the stimulation of root length under e[CO₂] may allow extraction of water from deeper soil layers during the critical grain-filling periods, which is often associated with terminal drought in dryland Mediterranean agroecosystems (Passioura 1983). The uptake of subsoil water during grain filling has the potential to make a substantial contribution to the grain yield of dryland crops (Angus and van Herwaarden 2001; Kirkegaard et al. 2007).

A study conducted in the AGFACE facility, utilising similar CO₂ exposure treatments to those used in our study but with contrasting irrigation regimes on a soil with high background N levels, reported a positive correlation between seasonal water use and root length in deeper soil layers (Uddin et al. 2018a). Stimulation of root growth in the deeper soil layers plays a significant role in maintaining plant physiological processes (Gallardo et al. 1994; Uddin et al. 2018b) because these roots are younger and more efficient in extracting and supplying water than roots in the upper soil layers (Gregory et al. 1978). The higher water use under e[CO₂] during the early growth stages suggests that root growth stimulation improves water acquisition. However, these early-stage responses were subsequently overshadowed by the prevailing dryland condition at the study site as the growing season progressed (Uddin et al. 2018a). Although early depletion of soil water can have negative effects on grain yield, particularly during seasons with dry finishes, the small variation in cumulative water use observed in this study would have a negligible impact on stored soil moisture for the following season.

The effect of supplemental N on grain yield was cultivar-specific

Elevated CO₂ consistently stimulated aboveground biomass throughout the growing season, and significant interactions were observed among [CO₂], N rate and cultivar treatments during the early growth stages, with positive interactions observed between e[CO₂] and supplemental N. At maturity, e[CO₂] resulted in similar stimulation of both aboveground biomass and grain yield, leading to an insignificant HI response to [CO₂]. The lack of significance regarding the effect of [CO₂] on grain yield (P = 0.108) may be attributed to the smaller sample size and larger variability of the yield data in this study. Previous FACE studies, including those conducted over multiple seasons and with larger sample sizes, have reported e[CO₂]-induced stimulation of biomass and grain yield within the AGFACE facility (Fitzgerald et al. 2016; Tausz et al. 2017a; Fitzgerald et al. 2022), as well as in other locations worldwide (Weigel and Manderscheid 2012; Ziska et al. 2012; Kimball 2016; Ainsworth and Long 2021). Similarly, no significant interaction was observed between [CO₂] and N rate or cultivar for grain yield, which aligns with findings from earlier FACE studies (Weigel and Manderscheid 2012; Bourgault et al. 2016; Fitzgerald et al. 2016; Tausz et al. 2017a, 2017b).

Previous research examining the interactive effect of e[CO₂] and N rates has primarily focused on high-yielding systems, such as those with high rainfall or continuous irrigation, where grain yields exceed 5 t ha⁻¹ and N rates >250 kg ha⁻¹ stimulate the CO₂ fertilisation effect (Kimball et al. 2001; Manderscheid et al. 2009). In a low-yielding, water-limited environment with high background soil N (~280 kg NO₃⁻-N ha⁻¹), the addition of supplemental N (50–60 kg N ha⁻¹) has been found to have no significant impact on biomass and grain yield, and it does not alter the CO₂ fertilisation effect (Tausz et al. 2017b). By contrast, in a similar environment with low background soil N, a significant positive interaction between supplemental N and e[CO₂] was observed affecting aboveground biomass and grain yield in wheat (Walker et al. 2017). In the present study, the addition of supplemental N influenced grain yield, with variations depending on the cultivar studied, but not CO₂ concentration.

Despite the initial expectation that cv. Wyalkatchem would outperform cv. Yitpi owing to its reputed greater N use efficiency (Alhabbar et al. 2018), the results of this study showed the opposite trend. Yitpi performed better than Wyalkatchem across various growth parameters and in grain yield, particularly when supplemental N was applied. One of the mechanisms underpinning the stimulation of grain yield by supplemental N in soil environments with low background N is the maintenance of higher A_net for an extended period (Ziska et al. 1996). During grain filling, wheat plants supplemented with N retained greater leaf N and chlorophyll contents, indicating greater photosynthetic capacity (Cannella et al. 2016). In this study, supplemental N maintained greater post-anthesis leaf N and chlorophyll content for cv. Yitpi under a low soil-N system. The combination of greater canopy cover (or green leaf area) and higher A_net in cv. Yitpi likely improved the supply of assimilates to developing grains. Late-season photosynthesis plays a crucial role in increasing grain yield under water-limited conditions (Liang et al. 2018), because most vegetative growth is completed and new photosynthates are primarily allocated to the growth and development of larger grains. This ultimately resulted in a greater yield benefit for cv. Yitpi in our study.

By contrast, application of supplemental N accelerated the post-anthesis senescence of cv. Wyalkatchem, resulting in a shorter critical grain-filling period. In dryland conditions, the remobilisation of stem water-soluble carbohydrate plays a significant role in wheat grain yield (Davidson and Chevalier 1992; van Herwaarden et al. 2003). In our study, Wyalkatchem showed early flowering and fast senescence compared with Yitpi, which is consistent with the findings of Nguyen et al. (2016). This early senescence adversely affects both the assimilate supply and complete remobilisation of stem water-soluble carbohydrate to the developing grains, leading to a substantial decrease in grain size and yield, and ultimately resulting in lower HI. The lack of a grain yield response to supplemental N in a cultivar with reputed high N use efficiency suggests that N use efficiency and responsiveness may need to be considered as separate factors (Ulas et al. 2013; Mahjourimajd et al. 2016; Ivić et al. 2021; Pujarula et al. 2021).