{"paper_id":"2d07c8a8-ed6e-4b89-8284-6adab4b0fecc","body_text":"Chickpea root exudation unlocks legacy phosphorus for intercropped wheat in calcareous soils | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Chickpea root exudation unlocks legacy phosphorus for intercropped wheat in calcareous soils Sudeep Tawari, Avner Gross, Moshe Halpern This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8917717/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Aims Decades of phosphorus (P) fertilization created vast 'legacy P' reserves in agricultural soils, yet much remains locked in sparingly soluble forms inaccessible to crops. We tested whether wheat and chickpea differ in P acquisition from calcium-bound (Ca-P) versus iron-bound (Fe-P) sources, whether elevated CO₂ modifies these abilities, and whether intercropping enables wheat to access Ca-P through chickpea facilitation. Methods In two greenhouse experiments, we compared P uptake of monocropped wheat and chickpea on four P sources (hydroxyapatite Ca-P, FePO₄ Fe-P, KH₂PO₄, or no P) under ambient and elevated CO₂ (Experiment 1), and intercropping effects on P acquisition (Experiment 2). Growth parameters and tissue elemental composition were analysed. Results Chickpea and wheat displayed a crossover interaction: chickpea achieved 3.7-fold higher P uptake from Ca-P but not Fe-P, while wheat achieved 5.5-fold higher P uptake from Fe-P but could not access Ca-P (species × P-source interaction, p = 0.005). When intercropped with chickpea, wheat P uptake from Ca-P increased 7.0-fold (p = 0.008), while chickpea was unaffected. Land equivalent ratios for P uptake under Ca-P (4.03) confirmed facilitation beyond complementarity. Elevated CO₂ enhanced P uptake only when species accessed their specific P source. Leaf Mn, a proxy for carboxylate exudation, correlated positively with P in chickpea (r = + 0.53) but negatively in wheat (r = − 0.49), supporting carboxylate-mediated mobilization. Conclusions Chickpea and wheat occupy distinct 'P niches'. Intercropping enables wheat to access legacy Ca-P through rhizosphere modification by chickpea, a sustainable strategy for P-use efficiency in calcareous agricultural systems. Phosphorus acquisition Intercropping Legacy phosphorus Carboxylate exudation Rhizosphere facilitation Manganese proxy Elevated CO₂ Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Phosphorus (P) is essential for plant growth, yet global agriculture faces a P crisis. Economically viable phosphate rock reserves are concentrated in a few countries and may be substantially depleted within decades to a century at current consumption rates (Cordell et al. 2009 ; Lambers 2022 ), while inefficient P fertilization contributes to eutrophication of aquatic systems (Sharpley et al. 2013 ). Paradoxically, decades of intensive fertilization have created vast reserves of ‘legacy phosphorus’ in agricultural soils, fixed in sparingly soluble forms unavailable to most crop species. In calcareous and alkaline soils, P is often bound as calcium phosphates such as hydroxyapatite, while in acidic soils, P is adsorbed to iron and aluminum oxides (Shen et al. 2011 ). This ‘phosphorus tax’ (Roy et al. 2016 ) represents a vast but largely untapped nutrient reserve: recovering these legacy pools through the action of root exudates that solubilize fixed P could significantly offset demand for rock phosphate, improve P acquisition efficiency, and reduce dependence on mineral fertilizers (Zhu et al. 2018 ; Menezes-Blackburn et al. 2018 ). Although plants have evolved various strategies to mobilize sparingly soluble P, their effectiveness varies considerably among species (Wang and Lambers 2020 ), ranging from ‘P-scavenging’ strategies that expand root surface area to explore soil volume, to ‘P-mining’ strategies that utilize root exudates to chemically mobilize recalcitrant P pools (Wen et al. 2019 ). Legumes and cereals differ markedly in their P acquisition mechanisms, a divergence rooted in their distinct iron-acquisition strategies. Legumes such as chickpea ( Cicer arietinum ) employ Strategy I iron acquisition, a physiological suite characterized by rhizosphere acidification and the exudation of carboxylates, particularly malonate, citrate, and malate (Marschner and Römheld 1994 ). While this strategy evolved primarily for iron mobilization, it functions as a potent phosphorus-mining mechanism in calcareous soils; these organic anions solubilize calcium-bound P by chelating Ca 2+ and competing for sorption sites (Veneklaas et al. 2003 ; Pang et al. 2018 ). This carboxylate-releasing strategy is highly effective against calcium phosphates but less effective against iron-bound P. Mobilizing P from iron-bound complexes typically requires either reductive dissolution or strong chelation of ferric iron Fe 3+ rather than acidification alone (Hinsinger 2001 ). Cereals such as wheat ( Triticum aestivum ) primarily employ a scavenging strategy, utilizing extensive root systems to explore soil volume. Chemically, wheat utilizes Strategy II iron acquisition, releasing phytosiderophores that chelate Fe 3+ with high specificity to liberate iron-bound P (Marschner and Römheld 1994 ). This divergence in strategy reflects a fundamental evolutionary trade-off: species investing in metabolically expensive carboxylate exudation such as chickpea typically exhibit reduced root morphological plasticity compared to scavenging species such as wheat (Wen et al. 2019 ). These contrasting mechanisms suggest that legumes and cereals may differ in which soil P pools they can effectively access, a hypothesis tested in this study. Intercropping legumes and cereals has long been practiced to improve nitrogen use efficiency through biological N fixation (Fujita et al. 1992 ). More recently, attention has turned to potential P benefits. Hinsinger et al. ( 2011 ) distinguished two mechanisms by which intercropping can enhance P acquisition. Complementarity arises when intercropped species partition different P pools, reducing competition without altering P availability itself; for example, one species depleting organic P while the other takes up inorganic P. Facilitation, by contrast, occurs when one species converts P from an unavailable pool into an available form that the neighbouring species can access, effectively adding to the P available to its neighbour. Distinguishing these mechanisms experimentally is challenging (Loreau and Hector 2001 ), but a key prediction follows: if facilitation drives the intercropping benefit, the advantage should scale with the supply of the P substrate that the facilitating species can mobilize. A global meta-analysis of cereal-legume intercropping found mean LERP of 1.24 across 97 records and approximately 21% lower P fertilizer requirements for the same yield, with stronger effects where P-mobilizing legumes such as chickpea were involved (Tang et al. 2021 ). Wheat-legume intercropping systems have shown promise (Li et al. 2014 ; de Oliveira et al. 2022 ), but the specific P forms involved and the conditions under which facilitation occurs remain poorly characterized. Rising atmospheric CO₂ concentrations add another dimension to plant P nutrition. Elevated CO₂ generally stimulates photosynthesis and growth, but this response can be constrained by nutrient availability (Reich et al. 2006 ; Sardans et al. 2017 ). Plants grown under elevated CO₂ often exhibit reduced tissue nutrient concentrations, a phenomenon attributed to both dilution effects and impaired nutrient uptake (Loladze 2002 ). Crucially, elevated CO₂ may enhance P acquisition by increasing the carbon supply available for root exudation, which can stimulate the release of carboxylates that mobilize insoluble P pools (Pang et al. 2018 ). However, it remains poorly understood whether CO₂ fertilization effects depend specifically on P-source availability, meaning whether the growth response is contingent on the specific chemical forms of P that a plant is physiologically equipped to access. This study examines whether chickpea and wheat differ in their ability to acquire P from calcium-bound (hydroxyapatite) versus iron-bound (FePO₄) sources, and whether intercropping can leverage any such differences to improve P acquisition through facilitation. We also tested whether elevated CO₂ modifies these responses, and analysed plant elemental content to determine whether species-specific acquisition mechanisms leave distinct nutritional signatures. Two greenhouse experiments were conducted: the first compared P uptake by monocropped wheat and chickpea supplied with different P sources under ambient and elevated CO₂; the second tested whether intercropping wheat with chickpea enhances P acquisition from sparingly soluble P sources. Materials and methods Experimental site and growth conditions Two experiments were conducted in climate-controlled chambers interfaced with a CO₂ controller (custom-built, Emproco, Israel) equipped with an NDIR CO₂ sensor (Gascard NG, Edinburgh Instruments, Livingston, UK) at the Gilat Research Station, Israel. The first experiment was conducted from June 17 to August 18, 2021, and the second from December 20, 2022, to February 9, 2023. Temperature was maintained at 25°C during the day and 17°C at night, and plants received natural sunlight without supplemental lighting. Soil and plant material Plants were grown in Hamra soil collected from the Rehovot region of the Israeli coastal plain. The soil had a pH of 7.7, electrical conductivity of 0.28 dS m⁻¹ Olsen-extractable P < 6 mg kg⁻¹, and total P of 50 mg kg⁻¹ (0.005%), indicating a calcareous, severely P-limited system typical of Mediterranean agricultural soils. Pots were filled with 2.5 kg of soil and irrigated as needed to maintain optimal soil moisture. Nutrients other than phosphorus were supplied in non-limiting amounts via irrigation water, including 54 mg L⁻¹ N, 150 mg L⁻¹ K, 120 mg L⁻¹ Ca, and 24 mg L⁻¹ Mg (Gross et al. 2021 ). Wheat (Triticum aestivum L. cv. Gadera) and chickpea (Cicer arietinum L. cv. Zehavit) were used in both experiments. These cultivars are widely grown in Israeli agriculture and represent common modern varieties adapted to Mediterranean conditions. Both experiments were conducted with 4–6 replicates per treatment. Experiment 1: Elevated CO₂ and P uptake in monocrops Wheat and chickpea were grown in monocrop systems under two CO₂ conditions: ambient (400 µmol mol⁻¹) and elevated (850 µmol mol⁻¹). Plants were subjected to four P treatments: (1) Control: no additional P supplied; (2) KH₂PO₄: soluble P provided via irrigation water at 11.2 mg L⁻¹ P daily; (3) Hydroxyapatite (Ca-P; Sigma-Aldrich Israel, Rehovot, Israel): sparingly soluble calcium-bound P, 0.01 g P per pot; and (4) FePO₄ (Fe-P; Sigma-Aldrich Israel, Rehovot, Israel): sparingly soluble iron-bound P, 0.01 g P per pot. Each pot contained four wheat plants or two chickpea plants. The total number of pots was 55, with 2–6 replicates per treatment combination, unbalanced due to plant mortality. Due to this plant mortality, the KH₂PO₄ treatment under elevated CO₂ was excluded from analysis. Experiment 2: Intercropping effects on P uptake Wheat and chickpea were grown either in monocrop (four wheat plants or two chickpea plants per pot) or intercrop systems (two wheat plants and one chickpea plant per pot). Pots were subjected to three P treatments: (1) Control: no additional P supplied; (2) Hydroxyapatite (Ca-P): 0.01 g P per pot; and (3) FePO₄ (Fe-P): 0.01 g P per pot. In intercrop pots, wheat and chickpea were harvested and analysed separately, yielding two samples per pot (one per species) The total number of pots was 45, with 4–6 replicates per treatment combination. Plant growth and elemental analyses At the conclusion of each experiment, plants were harvested to measure aboveground biomass and nutrient uptake. Aboveground biomass was determined after drying plant material at 60°C to constant weight. For elemental analyses, dried plant material was finely ground, and subsamples were ashed at 500°C for 5 h in a muffle furnace. The ash was extracted with 1 M HCl. In Experiment 1, P concentration in the extract was determined colorimetrically using the molybdate blue method (Murphy and Riley 1962 ) on a Thermo Scientific Gallery Aqua Master discrete analyzer. In Experiment 2, elemental concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES), quantifying tissue concentrations of P, Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, S, Sr, and Zn. Total P uptake was calculated as the product of biomass and P concentration. Statistical analysis In Experiment 1, species × P treatment effects on biomass, P concentration, and P uptake were analysed using two-way ANOVA (pooled across CO₂ levels). To assess CO₂ effects, a three-way ANOVA with species, P treatment, and CO₂ level was performed; planned interaction contrasts were computed using the pooled mean square error from the full model to test whether the CO₂ response differed across P treatments within each species. In Experiment 2, effects of P treatment and cropping system on biomass, P concentration, and P uptake were analyzed using two-way ANOVA. Dunnett's test was used for comparisons against the control treatment, and Tukey's HSD test was used for pairwise comparisons, at a significance level of p < 0.05. For comparisons between monocrop and intercrop systems within each P treatment, Mann-Whitney U tests were used. Replication varied across treatments (n = 2–6) owing to seedling establishment failures; non-parametric tests were therefore preferred where sample sizes were small, and results should be interpreted with this limitation in mind. For mineral profile analysis in Experiment 2, treatment effects on individual element concentrations were assessed using t-tests or Mann-Whitney U tests as appropriate. To examine relationships between P acquisition mechanisms and tissue mineral composition, Pearson correlations between P concentration and concentrations of other elements were calculated separately for each species. All statistical analyses were performed using JMP version 13 (SAS Institute Inc., Cary, NC, USA) and Python (scipy, pandas). Results Species-specific P-source utilization Wheat and chickpea displayed contrasting abilities to acquire P from different mineral sources, evidenced by significant species × P treatment interactions for biomass (F = 9.27, p < 0.001), tissue P concentration (F = 15.65, p < 0.001), and total P uptake (F = 11.38, p < 0.001). Chickpea effectively utilized calcium-bound P (Ca-P), showing 2.1-fold higher biomass (Fig. 1 A), 1.7-fold higher tissue P concentration (p < 0.001; Fig. 1 B), and 3.7-fold higher P uptake (p < 0.05; Fig. 1 C) compared to unfertilized controls. In contrast, chickpea showed no significant response to iron-bound P (Fe-P; 1.2-fold biomass, 1.2-fold P concentration, 1.7-fold P uptake; all p > 0.05; Fig. 1 A–C). Wheat displayed the opposite pattern. Under Fe-P treatment, wheat achieved 2.3-fold higher biomass (p < 0.001; Fig. 1 A), 2.5-fold higher tissue P concentration (p < 0.001; Fig. 1 B), and 5.5-fold higher P uptake (p < 0.001; Fig. 1 C) compared to controls. However, wheat failed to access Ca-P, with biomass (0.8-fold), P concentration (1.1-fold), and P uptake (0.9-fold) statistically indistinguishable from controls (all p > 0.05; Fig. 1 A–C). Both species responded strongly to the soluble P control (KH₂PO₄), with P uptake increasing 7.3-fold in chickpea and 13.6-fold in wheat (both p < 0.001; Fig. 1 C), confirming that growth was P-limited. Elevated CO₂ responses depend on P accessibility The effect of elevated CO₂ on P nutrition depended on P source accessibility, as indicated by a significant three-way Species × P treatment × CO₂ interaction for P uptake (F = 3.92, p = 0.032; Fig. 2 ). For wheat grown without supplemental P (Control), elevated CO₂ reduced tissue P concentration by 0.25 mg g⁻¹ and P uptake by 0.40 mg, consistent with growth-induced P dilution (Fig. 2 B, C). This negative response reversed when wheat was supplied with Fe-P, the P source wheat can access: elevated CO₂ increased P concentration by 0.44 mg g⁻¹ and P uptake by 1.22 mg (P × CO₂ interaction within wheat: F = 4.36, p = 0.032 for P concentration; F = 4.77, p = 0.025 for P uptake; Fig. 2 B, C). Under Ca-P, which wheat cannot efficiently access (Fig. 1 ), the CO₂ response remained negligible. Chickpea showed an opposite pattern: elevated CO₂ appeared to enhance growth specifically under Ca-P, the P source accessible to chickpea (+ 0.88 g biomass, + 1.17 mg P uptake), while showing no benefit or reduced performance under Fe-P (− 0.49 g biomass; Fig. 2 A, C). However, the P × CO₂ interaction did not reach significance for chickpea (p > 0.16 for all variables), so these trends remain suggestive and require confirmation with larger sample sizes. Intercropping facilitates wheat P acquisition from calcium-bound P In Experiment 2, we tested whether intercropping with chickpea could enable wheat to access Ca-P. Consistent with Experiment 1, wheat grown in monoculture with Ca-P performed poorly, with per-plant biomass (0.12 g) and P uptake (0.09 mg) only marginally above control levels (Fig. 3 A–C). However, when intercropped with chickpea under the same Ca-P treatment, wheat performance increased dramatically: per-plant biomass reached 0.49 g (3.9-fold increase, p = 0.008), tissue P concentration increased from 0.70 to 1.19 mg g⁻¹ (1.7-fold), and P uptake reached 0.61 mg per plant (7.0-fold increase, p = 0.008). This facilitation effect was specific to Ca-P. Under control (no P) conditions, intercropped wheat showed a modest but significant 2.1-fold increase in biomass and P uptake compared to monoculture (Fig. 3 A), likely reflecting baseline complementarity effects. Under Fe-P, a source wheat can access independently, intercropping provided only a 1.8-fold biomass increase, substantially less than the benefit observed under Ca-P. Chickpea experienced no cost from facilitating wheat P acquisition. Under Ca-P supply, chickpea per-plant biomass (1.31 vs. 1.20 g), P concentration (2.59 vs. 2.76 mg g⁻¹), and P uptake (3.46 vs. 3.20 mg) were statistically indistinguishable between intercrop and monoculture systems (all p > 0.5; Fig. 3 D–F). This asymmetric outcome, wheat benefits substantially while chickpea is unaffected, is consistent with facilitation through chickpea root exudates that mobilize Ca-P in the shared rhizosphere. Land equivalent ratios confirm facilitation beyond baseline complementarity Land equivalent ratios confirm this interpretation quantitatively. If facilitation drives the intercropping benefit, the advantage should scale with the supply of Ca-P substrate that chickpea can mobilize (Tang et al. 2021 ). Under Control conditions, the intercrop showed modest benefits (LER = 1.50, LERP = 1.38; Fig. 4 ), likely reflecting spatial complementarity and possibly small-scale facilitation from native soil Ca-P. Under Ca-P supply, both metrics increased substantially: LER reached 2.52 (p = 0.002 vs Control) and LERP reached 4.03 (p = 0.015). Under Fe-P, a source chickpea cannot mobilize, LER was not elevated above Control (LER = 1.30, LERP = 1.10; both ns). The intercropping advantage thus scales with the availability of chickpea’s specific substrate, consistent with facilitation rather than complementarity. Elemental content supports distinct P acquisition mechanisms Multivariate analysis of tissue elemental concentrations revealed strikingly different element co-accumulation patterns between species. Chickpea tissue P concentration was positively correlated with all measured elements, where 13 of 14 correlations were significantly positive (p < 0.05; Fig. 5 A). This pattern suggests non-specific nutrient mobilization, where the mechanism chickpea uses to acquire P simultaneously releases other soil-bound elements into solution. In contrast, wheat tissue P showed no consistent relationship with other elements. Only 3 of 14 correlations were significant, all negative (B, Mn, Na; Fig. 5 A). This decoupled pattern is consistent with a more targeted P acquisition mechanism that does not mobilize other elements as a side effect. The divergence was most pronounced for manganese. In chickpea, P and Mn were positively correlated (r = + 0.53, p = 0.002; Fig. 5 B), whereas in wheat they were negatively correlated (r = − 0.49, p = 0.007; Fig. 5 C). The negative P–Mn correlation in wheat likely reflects growth dilution: as plants acquire more P and increase biomass, a relatively fixed Mn pool becomes diluted across more tissue. The positive correlation in chickpea indicates that Mn accumulates alongside P, consistent with co-mobilization by the same rhizosphere process. These opposing patterns were consistent across both monoculture and intercrop systems. Discussion Distinct phosphorus niches in legumes and cereals Our results confirm a fundamental physiological trade-off in P acquisition between legumes and cereals, providing clear evidence for niche differentiation in soil nutrient use. The 'crossover' interaction observed in Experiment 1, where chickpea mobilized Ca-P but failed on Fe-P, while wheat exhibited the exact opposite pattern, demonstrates that these species occupy distinct 'phosphorus niches' defined by soil chemistry. Chickpea's capacity to access Ca-P is driven by a 'chemical mining' strategy. As observed in previous hydroponic and soil studies, chickpea roots actively acidify the rhizosphere and exude substantial amounts of carboxylates, particularly citrate and malate, under P-limiting conditions (Veneklaas et al. 2003 ; Alloush 2003 ). While acidification is a primary driver, the mobilization of phosphorus is likely enhanced by ligand exchange mechanisms. Barrow et al. ( 2018 ) recently demonstrated that citrate anions can displace phosphate from adsorption sites through competitive binding, a process that operates synergistically with proton extrusion to maximize P desorption in calcareous matrices. This mechanism is specifically effective against calcium phosphates; the exudation of protons lowers rhizosphere pH, while organic anions chelate Ca²⁺, shifting the chemical equilibrium to release orthophosphate into the soil solution (Hinsinger 2001 ). Our findings align with Pearse et al. ( 2006 ), who found that while legumes could mobilize Ca-P through acidification, they struggled to access Fe-P, likely because the solubilization of iron phosphates requires reduction or specific chelation rather than acidification alone. In contrast, wheat failed to access Ca-P, confirming that its extensive root system functions primarily as a 'scavenging' tool for soluble nutrients rather than a 'mining' tool for acid-soluble P pools (Lyu et al. 2016 ). However, wheat's superior performance on Fe-P suggests a reliance on Strategy II iron acquisition mechanisms. Graminaceous species release phytosiderophores to chelate ferric iron (Fe³⁺); by stripping iron from sparingly soluble Fe-P complexes, these chelators effectively release the associated phosphate into the soil solution as a collateral benefit (Shen et al. 2011 ). This mechanism is chemically distinct from the acidification strategy used by chickpea, explaining why wheat could access Fe-P but remained starved in the Ca-P treatment (Fig. 6 A). The accumulation of Mn in chickpea tissue, which serves as a physiological proxy for rhizosphere carboxylate exudation, is supporting evidence for the suggested P uptake mechanism. Broad surveys across 727 species at 66 sites have validated leaf Mn concentration as a practical indicator of carboxylate-mediated P-mining activity (Lambers et al. 2021 ), as the acidification and ligand exchange processes required to solubilize Ca-P also mobilize soil Mn (Yan et al. 2024 ; Wang et al. 2024 ). The positive P–Mn correlation we observed in chickpea, but not wheat, is thus consistent with carboxylate-mediated nutrient mobilization. These elemental profiles further support that chickpea and wheat employ fundamentally different P acquisition mechanisms. The striking contrast, where chickpea co-accumulates P with nearly all elements, while wheat does not, is consistent with the distinction between non-specific chemical mobilization and targeted nutrient scavenging. Carboxylate exudation, the mechanism proposed for chickpea's Ca-P acquisition, is inherently non-selective. Organic acids that solubilize calcium phosphates also mobilize other cations and metal-bound nutrients through similar ligand exchange and pH-mediated processes (Lambers et al. 2015 ). This predicts that plants relying on carboxylate exudation should show correlated accumulation of multiple elements, exactly the pattern observed in chickpea. Notably, intercropped wheat under Ca-P also showed significantly elevated K (+ 24%, p = 0.01), consistent with access to nutrients co-mobilized by chickpea’s rhizosphere acidification; similar multi-element co-accumulation in intercropped wheat has been reported in wheat–white lupin systems (de Oliveira et al. 2022 ). In contrast, the decoupling of P from other elements except for Fe in wheat is consistent with a more targeted acquisition mechanism. Phytosiderophore-mediated iron acquisition is highly specific for Fe³⁺ chelation and would not be expected to mobilize other soil nutrients. The negative P–Mn correlation in wheat most likely reflects simple growth dilution: as plants acquire more P and increase biomass, a relatively fixed Mn pool becomes distributed across more tissue, reducing concentration. This interpretation is corroborated by the intercropping comparison: wheat leaf Mn decreased by 20% (p = 0.03) under Ca-P despite a doubling of P concentration, consistent with the 3.9-fold biomass increase diluting a relatively fixed Mn pool. CO₂ fertilization is constrained by P accessibility The strict dependence on soil P speciation also constrains the crop response to future atmospheric conditions. While elevated CO₂ generally acts as a 'carbon fertilizer,' our results demonstrate that this benefit is capped by the 'Law of the Minimum': carbon fertilization fails to translate into improved P nutrition when the available P pool is chemically inaccessible (Jin et al. 2015 ; Jiang et al. 2020 ; Terrer et al. 2019 ; Sardans et al. 2017 ). In Experiment 1, elevated CO₂ enhanced wheat P concentration and P uptake only when wheat was supplied with Fe-P, a source it can physiologically access. Under Ca-P and Control treatments, the CO₂ response was negligible or negative (Fig. 2 ). These results, combined with our intercropping findings, suggest a path forward. In Experiment 1, wheat failed to respond to elevated CO₂ under Ca-P supply due to P limitation. In Experiment 2, intercropping with chickpea relieved this P limitation under ambient CO₂. Together, these findings suggest that intercropping may be a necessary precondition for realizing CO₂ fertilization benefits in P-limited calcareous systems, a hypothesis that merits direct experimental testing combining intercropping with elevated CO₂. These results have important implications for predicting agricultural responses to climate change. While elevated CO 2 can enhance crop yields through increased photosynthesis, these benefits may be diminished or eliminated in nutrient-limited systems (Terrer et al. 2019 ). Our data demonstrate that P source accessibility, not just total P availability, fundamentally constrains the CO 2 fertilization response. In calcareous soils where much of the soil P exists in calcium-bound forms that cereals cannot efficiently access, the CO 2 fertilization effect may be particularly limited unless facilitative interactions with legumes enable access to these P pools. Facilitation through rhizosphere modification The most striking finding of this study is the magnitude of facilitation observed when divergent P acquisition strategies are combined (Fig. 6 B). Experiment 1 established that wheat cannot access Ca-P in monoculture; yet in Experiment 2, when grown alongside chickpea, wheat biomass increased 3.9-fold and P uptake increased 7.0-fold. Since wheat lacks the physiological machinery to solubilize calcium phosphates, this P must have been mobilized by chickpea's rhizosphere activity and subsequently taken up by neighbouring wheat roots. Chickpea, meanwhile, was unaffected; its biomass, P concentration, and P uptake were indistinguishable between monoculture and intercrop (all p > 0.5). This one-directional benefit, where one species converts an unavailable P pool into a form the neighbour can access at no cost to itself, matches the definition of facilitation proposed by Hinsinger et al. ( 2011 ). The LERP of 4.03 far exceeds the standard threshold for complementarity (> 1.0), confirming a powerful facilitative interaction driven by rhizosphere modification. However, facilitation in wheat-chickpea systems may not be solely driven by acidification. Betencourt et al. ( 2012 ) demonstrated that intercropping can promote P availability through complex chemical interactions that may include localized pH changes and microbial stimulation, suggesting that chickpea's rhizosphere modification operates through multiple solubility pathways. While white lupin is often cited as the model species for carboxylate exudation, studies indicate that wheat-lupin intercrops achieve facilitation through similar spatial and chemical complementarity (Cu et al. 2005 ; de Oliveira et al. 2022 ). Our results suggest chickpea functions as a functional analogue to lupin in calcareous soils, where lupin is often poorly adapted due to sensitivity to high pH (White and Robson, 1989 ). This facilitation was P-source specific. The comparison of LER under Ca-P to LER under control conditions provides a rigorous test for true facilitation versus baseline complementarity effects. While all intercrop treatments showed LER > 1, indicating some degree of niche differentiation, only the Ca-P treatment showed LER significantly exceeding the control baseline. This demonstrates that the intercropping benefit under Ca-P reflects genuine facilitation of P acquisition, not merely reduced competition or spatial complementarity. The mechanism of facilitation likely involves chickpea's rhizosphere modification creating a zone of elevated P availability that wheat roots can exploit. In the intercropped pots, wheat roots intermingled with chickpea roots would have access to the P solubilized by chickpea's carboxylate exudation. This validates previous work by Li et al. ( 2004 ) and Betencourt et al. ( 2012 ), who reported similar facilitation between cereals and legumes, though the magnitude of benefit in our study is notably higher, likely due to the severe P limitation of our soil and the confined pot system maximizing root intermingling. Root exudates may also stimulate rhizosphere microbial communities that contribute to P solubilization, amplifying the direct chemical effects of carboxylate exudation (Richardson et al. 2011 ). While we did not measure microbial responses in this study, this represents an important avenue for future investigation. Chickpea experienced no measurable cost from facilitating wheat P acquisition. Per-plant biomass, P concentration, and P uptake were all maintained in intercrop compared to monoculture. This asymmetric outcome, where wheat benefits substantially while chickpea is unaffected, suggests that chickpea's P mobilization capacity exceeded its own uptake requirements, leaving surplus mobilized P available for wheat roots. Practical implications Unlike the well-documented benefits of cereal-legume intercropping for nitrogen acquisition, where legumes supply N via biological fixation, our results highlight a critical, often overlooked mechanism for phosphorus mobilization. Wheat-chickpea intercropping represents a viable biological strategy to 'unlock' legacy P in calcareous soils. By pairing a species that chemically mines P with a species that efficiently scavenges it, agricultural systems can access P pools that are otherwise biologically unavailable, improving P-use efficiency and potentially reducing fertilizer requirements. This P-facilitation appears strictly context-dependent. Facilitation was absent in the control (no P) treatment, despite severe P stress, because chickpea's acidification mechanism requires a calcium-phosphate substrate to act upon. Similarly, facilitation was minimal under Fe-P, where chickpea's mechanism is ineffective. Thus, the interaction is not a general stress response but a specific chemical synergy contingent on soil P speciation. These findings suggest that the benefits of wheat-legume intercropping for P nutrition will be greatest in calcareous and alkaline soils where calcium-bound P predominates. Field studies in semi-arid calcareous soils support these findings; Souid et al. ( 2024 ) reported that durum wheat-chickpea intercropping increased available rhizosphere P by 28% and microbial biomass P by 34% relative to bulk soil. Conclusions This study provides physiological evidence that cereal-legume intercropping can mobilize soil phosphorus pools that are biologically unavailable to monocultures. By characterizing the distinct 'phosphorus niches' of wheat and chickpea, we demonstrate that the benefits of intercropping extend beyond the well-known mechanisms of nitrogen fixation to include active phosphorus mining. The ability of chickpea to acidify the rhizosphere and solubilize calcium-bound phosphorus acts as a biological 'key,' unlocking nutrient reserves that wheat's scavenging root system cannot access on its own. The elemental profiles provide a novel line of evidence supporting mechanistic interpretations: chickpea's non-specific element co-accumulation pattern is consistent with carboxylate-mediated mobilization, while wheat's decoupled profile supports a more targeted acquisition strategy. The use of tissue Mn concentration as a proxy for carboxylate exudation offers a practical diagnostic tool for future studies of P acquisition mechanisms. Our findings have critical implications for agriculture under changing climate conditions. The potential yield benefits of elevated atmospheric CO₂ are strictly constrained by soil nutrient availability; without a strategy to mobilize recalcitrant phosphorus, the 'CO₂ fertilization effect' fails to materialize. Wheat-chickpea intercropping represents a sustainable strategy to recover 'legacy phosphorus', the vast reserves of fertilizer-derived P that have accumulated in calcareous soils over decades of intensive agriculture but remain fixed in insoluble forms. By integrating species with complementary root traits, farmers can improve system-level phosphorus use efficiency, reduce dependence on synthetic inputs, and stabilize yields in low-input environments. The benefits of interspecific facilitation may extend beyond the current growing season: rotational studies have shown that P mobilized by legume exudates can remain available for subsequent cereal crops, offering a long-term strategy for accessing legacy soil P (Nuruzzaman et al. 2005a , b ). Direct confirmation of the P transfer pathway using 32P-labelled calcium phosphate would further strengthen the mechanistic basis for mineral-form-specific facilitation. Declarations Competing interests The authors declare no competing interests. Funding This study was supported by the Israel Science Foundation (ISF) grant number 267/24. Funding that the ISF grant specified was received by AG. Author contributions M.H. conceived and designed the study, conducted the experiments, analysed the data, and wrote the manuscript. S.T. contributed to study design, experimentation, and manuscript revision. A.G. contributed to study design, data interpretation, and critical revision of the manuscript. Data availability The datasets generated and analysed during the current study are available in the Zenodo repository: https://doi.org/10.5281/zenodo.18698449 References Alloush GA (2003) Dissolution and effectiveness of phosphate rock in acidic soil amended with cattle manure. Plant Soil 251:37–46 Barrow NJ, Debnath A, Sen A (2018) Mechanisms by which citric acid increases phosphate availability. 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Plant Soil 141:155–175 Hinsinger P (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical changes: a review. Plant Soil 237:173–195 Gross A, Tiwari S, Shtein I, Erel R (2021) Direct foliar uptake of phosphorus from desert dust. New Phytol 230:2213–2225 Hinsinger P, Betencourt E, Bernard L, Brauman A, Plassard C, Shen J, Tang X, Zhang F (2011) P for two, sharing a scarce resource: soil phosphorus acquisition in the rhizosphere of intercropped species. Plant Physiol 156:1078–1086 Jiang M, Caldararu S, Zhang H, Fleischer K, Crous KY, Yang J, De Kauwe MG, Ellsworth DS, Reich PB, Tissue DT, Zaehle S, Medlyn BE (2020) Low phosphorus supply constrains plant responses to elevated CO₂: a meta-analysis. Glob Change Biol 26:5856–5873 Jin J, Tang C, Sale P (2015) The impact of elevated carbon dioxide on the phosphorus nutrition of plants: a review. Ann Bot 116:987–999 Lambers H (2022) Phosphorus acquisition and utilization in plants. Annu Rev Plant Biol 73:17–42 Lambers H, Hayes PE, Laliberté E, Oliveira RS, Turner BL (2015) Leaf manganese accumulation and phosphorus-acquisition efficiency. Trends Plant Sci 20:83–90 Lambers H, Wright IJ, Guilherme Pereira C, Bellingham PJ, Bentley LP, Boonman A, Cernusak LA, Foulds W, Gleason SM, Gray EF, Hayes PE, Kooyman RM, Malhi Y, Richardson SJ, Shane MW, Staudinger C, Stock WD, Swarts ND, Turner BL, Turner J, Veneklaas EJ, Wasaki J, Westoby M, Xu Y (2021) Leaf manganese concentrations as a tool to assess belowground plant functioning in phosphorus-impoverished environments. Plant Soil 461:43–61 Li L, Tilman D, Lambers H, Zhang FS (2014) Plant diversity and overyielding: insights from belowground facilitation of intercropping in agriculture. New Phytol 203:63–69 Li SM, Li L, Zhang FS, Tang C (2004) Acid phosphatase role in chickpea/maize intercropping. Ann Bot 94:297–303 Loladze I (2002) Rising atmospheric CO₂ and human nutrition: toward globally imbalanced plant stoichiometry? Trends Ecol Evol 17:457–461 Loreau M, Hector A (2001) Partitioning selection and complementarity in biodiversity experiments. Nature 412:72–76 Lyu Y, Tang H, Li H, Zhang F, Rengel Z, Whalley WR, Shen J (2016) Major crop species show differential balance between root morphological and physiological responses to variable phosphorus supply. Front Plant Sci 7:1939 Marschner H, Römheld V (1994) Strategies of plants for acquisition of iron. Plant Soil 165:261–274 Menezes-Blackburn D, Giles C, Darch T, George TS, Blackwell M, Stutter M, Shand C, Lumsdon D, Cooper P, Wendler R, Brown L, Almeida DS, Wearing C, Zhang H, Haygarth PM (2018) Opportunities for mobilizing recalcitrant phosphorus from agricultural soils: a review. Plant Soil 427:5–16 Murphy J, Riley JP (1962) A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta 27:31–36 Nuruzzaman M, Lambers H, Bolland MDA, Veneklaas EJ (2005a) Phosphorus benefits of different legume crops to subsequent wheat grown in different soils of Western Australia. Plant Soil 271:175–187 Nuruzzaman M, Lambers H, Bolland MDA, Veneklaas EJ (2005b) Phosphorus uptake by grain legumes and subsequently grown wheat at different levels of residual phosphorus fertiliser. Aust J Agric Res 56:1041–1047 Pang J, Ryan MH, Lambers H, Siddique KH (2018) Phosphorus acquisition and utilisation in crop legumes under global change. Curr Opin Plant Biol 45:248–254 Pearse SJ, Veneklaas EJ, Cawthray GR, Bolland MDA, Lambers H (2006) Triticum aestivum shows a greater biomass response to a supply of aluminium phosphate than Lupinus albus, despite releasing fewer carboxylates into the rhizosphere. New Phytol 169:515–524 Reich PB, Hobbie SE, Lee T, Ellsworth DS, West JB, Tilman D, Knops JM, Naeem S, Trost J (2006) Nitrogen limitation constrains sustainability of ecosystem response to CO₂. Nature 440:922–925 Richardson AE, Lynch JP, Ryan PR, Delhaize E, Smith FA, Smith SE, Harvey PR, Ryan MH, Veneklaas EJ, Lambers H, Oberson A, Culvenor RA, Simpson RJ (2011) Plant and microbial strategies to improve the phosphorus efficiency of agriculture. Plant Soil 349:121–156 Roy ED, Richards PD, Martinelli LA, Coletta LD, Lins SRM, Vazquez FF, Willig E, Spera SA, VanWey LK, Porder S (2016) The phosphorus cost of agricultural intensification in the tropics. Nat Plants 2:16043 Sardans J, Grau O, Chen HY, Janssens IA, Ciais P, Piao S, Peñuelas J (2017) Changes in nutrient concentrations of leaves and roots in response to global change factors. Glob Change Biol 23:3849–3856 Sharpley A, Jarvie HP, Buda A, May L, Spears B, Kleinman P (2013) Phosphorus legacy: overcoming the effects of past management practices to mitigate future water quality impairment. J Environ Qual 42:1308–1326 Shen J, Yuan L, Zhang J, Li H, Bai Z, Chen X, Zhang W, Zhang F (2011) Phosphorus dynamics: from soil to plant. Plant Physiol 156:997–1005 Souid A, Hamdi W, L’taief B, Attallah A, Hamdi N, Alshaharni MO, Zagrarni MF (2024) The potential of durum wheat–chickpea intercropping to improve the soil available phosphorus status and biomass production in a subtropical climate. PLoS ONE 19(5):e0300573 Tang X, Zhang C, Yu Y, van der Shen J, Zhang F (2021) Intercropping legumes and cereals increases phosphorus use efficiency; a meta-analysis. Plant Soil 460:89–104 Terrer C, Jackson RB, Prentice IC, Keenan TF, Kaiser C, Vicca S, Fisher JB, Reich PB, Stocker BD, Hungate BA et al (2019) Nitrogen and phosphorus constrain the CO₂ fertilization of global plant biomass. Nat Clim Change 9:684–689 Veneklaas EJ, Stevens J, Cawthray GR, Turner S, Grigg AM, Lambers H (2003) Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake. Plant Soil 248:187–197 Wang Y, Lambers H (2020) Root-released organic anions in response to low phosphorus availability: recent progress, challenges and future perspectives. Plant Soil 447:135–156 Wang Y, Yang M, Yu F (2024) Determine leaf manganese concentration to estimate rhizosheath carboxylates of mycorrhizal plants in forest ecosystems. Plant Soil 508:1027–1033 Wen Z, Li H, Shen Q, Tang X, Xiong C, Li H, Pang J, Ryan MH, Lambers H, Shen J (2019) Trade-offs among root morphology, exudation and mycorrhizal symbioses for phosphorus-acquisition strategies of 16 crop species. New Phytol 223:882–895 White PF, Robson AD (1989) Effect of soil pH and texture on the growth and nodulation of lupins. Aust J Agric Res 40(1):63–73 Yan L, Tang D, Pang J, Lambers H (2024) Root carboxylate release is common in phosphorus-limited forest ecosystems in China: using leaf manganese concentration as a proxy. Plant Soil 508:143–158 Zhu J, Li M, Whelan M (2018) Phosphorus activators contribute to legacy phosphorus availability in agricultural soils: a review. Sci Total Environ 612:522–537 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {\"props\":{\"pageProps\":{\"initialData\":{\"identity\":\"rs-8917717\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":599390947,\"identity\":\"af057f20-4c46-4b3c-ab9e-7454a994afe1\",\"order_by\":0,\"name\":\"Sudeep Tawari\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Sudeep\",\"middleName\":\"\",\"lastName\":\"Tawari\",\"suffix\":\"\"},{\"id\":599390948,\"identity\":\"0a59f173-e634-4497-b8e7-e75a601ae8b5\",\"order_by\":1,\"name\":\"Avner Gross\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Avner\",\"middleName\":\"\",\"lastName\":\"Gross\",\"suffix\":\"\"},{\"id\":599390949,\"identity\":\"89c9c5fe-5df0-407a-afdb-2efef34a0f0e\",\"order_by\":2,\"name\":\"Moshe Halpern\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYBACAyBmZmCwYWBgh3EZGBvAUmzseLWkgUk0Lcx4tRyGakEBOLSYix0+/Lmg4nxiPzPzw88FBQzy8tGHGxi/VBxm4MOhxXJ2Wpr0jDO3E2c2sxlLzzBgMNx4LrGBWebMYdwOu51jxszbdtvY4DAPgzSPAQPjxh7GBmbJtjR8Wow/8/47B9LC/BuoxZ4YLQbSvA0H5IBa2EC2JM7nYWxg/Nhmg0cL0C88x5LlJJvZzKx5DCSSNwC1HGY4Y8ODW0vy4c88NXY8/OzNj2/z/LGxnd/D/vDhjwoJOfn2Bux60IAEg8EBYDTxMDDwEKUeDOSBZjP+IF79KBgFo2AUDH8AAF5JTRUcy/f4AAAAAElFTkSuQmCC\",\"orcid\":\"https://orcid.org/0000-0003-2137-6835\",\"institution\":\"Hebrew University of Jerusalem\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Moshe\",\"middleName\":\"\",\"lastName\":\"Halpern\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2026-02-19 13:12:11\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-8917717/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-8917717/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":104105160,\"identity\":\"78820093-2b32-4a33-a88a-707b871cf710\",\"added_by\":\"auto\",\"created_at\":\"2026-03-06 21:31:04\",\"extension\":\"png\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":74588,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eSpecies-specific phosphorus source utilization. Chickpea and wheat differ in their ability to acquire P from calcium-bound (Ca-P, hydroxyapatite) versus iron-bound (Fe-P, FePO₄) sources. A Shoot biomass, B tissue P concentration, and C total P uptake for plants grown with no added P (Control), or treated with Ca-P, Fe-P, or soluble P (KH₂PO₄). Bars show means ± SEM. * p \\u0026lt; 0.05, ** p \\u0026lt; 0.01, *** p \\u0026lt; 0.001 vs Control (Dunnett's test).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage1.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/15d1fbaee27634637fd40bc9.png\"},{\"id\":104403224,\"identity\":\"07b8e984-9364-4b50-82a6-c558ca000253\",\"added_by\":\"auto\",\"created_at\":\"2026-03-11 12:17:46\",\"extension\":\"png\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":84908,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eCO₂ response (elevated − ambient) for shoot biomass (A), tissue P concentration (B), and total P uptake (C) of chickpea and wheat grown with no supplemental P (Control), calcium-bound P (Ca-P), or iron-bound P (Fe-P). Values above the dashed line (zero) indicate a positive response to elevated CO₂; values below indicate a negative response. Error bars represent ±1 SE of the difference between means. Asterisks denote significant P × CO₂ interaction contrasts, comparing each P treatment's CO₂ response to the Control CO₂ response within the same species (* p \\u0026lt; 0.05,** p \\u0026lt; 0.01). Data pooled across replicates (n = 4–6 per treatment × CO₂ combination).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage2.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/c007e92d5972c235c4861ff8.png\"},{\"id\":104403741,\"identity\":\"939031f5-a103-40a2-8a50-7fb620a96564\",\"added_by\":\"auto\",\"created_at\":\"2026-03-11 12:18:57\",\"extension\":\"png\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":272193,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eIntercropping with chickpea facilitates wheat P acquisition from Ca-P. Per-plant performance of wheat (A–C) and chickpea (D–F) grown in monoculture (blue) or intercrop (red) under different P treatments. A, D Biomass, B, E tissue P concentration, C, F P uptake. Bars show means ± SEM.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage3.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/579ea52a37f90a14142eb231.png\"},{\"id\":104403955,\"identity\":\"b84cea2d-bc9b-4691-ba50-f357afa40ebe\",\"added_by\":\"auto\",\"created_at\":\"2026-03-11 12:19:27\",\"extension\":\"jpeg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":242666,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eLand equivalent ratios confirm P-source-specific facilitation. A Biomass LER and B LERP (land equivalent ratio for phosphorus uptake) for wheat-chickpea intercrops under different P treatments. Dashed line indicates LER = 1 (no intercropping advantage). Bars show means +/- SEM. Significance stars indicate LER significantly different from 1 (one-sample t-test: *p \\u0026lt; 0.05, **p \\u0026lt; 0.01).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage4.jpeg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/5b1d6f486f3b2b84cf364545.jpeg\"},{\"id\":104105164,\"identity\":\"79fdcfaf-5e27-42ee-a529-56289fa6445d\",\"added_by\":\"auto\",\"created_at\":\"2026-03-06 21:31:05\",\"extension\":\"png\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":482706,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003ePlant elemental profiles reveal distinct nutrient co-accumulation patterns. A Correlation coefficients (Pearson r) between plant P concentration and 14 other elements in chickpea (blue) and wheat (red). Asterisks indicate significant correlations (p \\u0026lt; 0.05). B, C P versus Mn concentration in chickpea (B) and wheat (C). Filled circles: monoculture; open squares: intercrop. Colors indicate P treatment (gray: Control; green: Ca-P; red: Fe-P). Dashed lines show linear regression fits with Pearson correlation coefficients.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage5.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/76d70410c30883b4e3878fe0.png\"},{\"id\":104105163,\"identity\":\"3f7c5137-d1a7-4d47-8a13-bbfbf7b5160a\",\"added_by\":\"auto\",\"created_at\":\"2026-03-06 21:31:04\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4015081,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eRepresentative plants from Experiment 2 grown under ambient CO₂. (A) Wheat monoculture supplied with hydroxyapatite (Ca-P), iron phosphate (Fe-P), or no P addition (Control). Wheat showed a pronounced growth response to Fe-P but not to Ca-P, consistent with species-specific P acquisition through a reductive mechanism. (B) Wheat–chickpea intercrop with Ca-P or no P addition. Enhanced growth under Ca-P supply demonstrates facilitative P acquisition through chickpea’s rhizosphere modification.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"floatimage6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/9c26b5e1b0dac115eaed1415.png\"},{\"id\":106965957,\"identity\":\"3b57963f-ed6d-4587-9f53-a2ce332112d9\",\"added_by\":\"auto\",\"created_at\":\"2026-04-15 09:57:56\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":7060334,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-8917717/v1/ef29928c-6129-499e-8904-d760711378ec.pdf\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Chickpea root exudation unlocks legacy phosphorus for intercropped wheat in calcareous soils\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003ePhosphorus (P) is essential for plant growth, yet global agriculture faces a P crisis. Economically viable phosphate rock reserves are concentrated in a few countries and may be substantially depleted within decades to a century at current consumption rates (Cordell et al. \\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e2009\\u003c/span\\u003e; Lambers \\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), while inefficient P fertilization contributes to eutrophication of aquatic systems (Sharpley et al. \\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e2013\\u003c/span\\u003e). Paradoxically, decades of intensive fertilization have created vast reserves of \\u0026lsquo;legacy phosphorus\\u0026rsquo; in agricultural soils, fixed in sparingly soluble forms unavailable to most crop species. In calcareous and alkaline soils, P is often bound as calcium phosphates such as hydroxyapatite, while in acidic soils, P is adsorbed to iron and aluminum oxides (Shen et al. \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). This \\u0026lsquo;phosphorus tax\\u0026rsquo; (Roy et al. \\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e) represents a vast but largely untapped nutrient reserve: recovering these legacy pools through the action of root exudates that solubilize fixed P could significantly offset demand for rock phosphate, improve P acquisition efficiency, and reduce dependence on mineral fertilizers (Zhu et al. \\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e; Menezes-Blackburn et al. \\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). Although plants have evolved various strategies to mobilize sparingly soluble P, their effectiveness varies considerably among species (Wang and Lambers \\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e), ranging from \\u0026lsquo;P-scavenging\\u0026rsquo; strategies that expand root surface area to explore soil volume, to \\u0026lsquo;P-mining\\u0026rsquo; strategies that utilize root exudates to chemically mobilize recalcitrant P pools (Wen et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eLegumes and cereals differ markedly in their P acquisition mechanisms, a divergence rooted in their distinct iron-acquisition strategies. Legumes such as chickpea (\\u003cem\\u003eCicer arietinum\\u003c/em\\u003e) employ Strategy I iron acquisition, a physiological suite characterized by rhizosphere acidification and the exudation of carboxylates, particularly malonate, citrate, and malate (Marschner and R\\u0026ouml;mheld \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e). While this strategy evolved primarily for iron mobilization, it functions as a potent phosphorus-mining mechanism in calcareous soils; these organic anions solubilize calcium-bound P by chelating Ca\\u003csup\\u003e2+\\u003c/sup\\u003e and competing for sorption sites (Veneklaas et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Pang et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). This carboxylate-releasing strategy is highly effective against calcium phosphates but less effective against iron-bound P. Mobilizing P from iron-bound complexes typically requires either reductive dissolution or strong chelation of ferric iron Fe\\u003csup\\u003e3+\\u003c/sup\\u003e rather than acidification alone (Hinsinger \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eCereals such as wheat (\\u003cem\\u003eTriticum aestivum\\u003c/em\\u003e) primarily employ a scavenging strategy, utilizing extensive root systems to explore soil volume. Chemically, wheat utilizes Strategy II iron acquisition, releasing phytosiderophores that chelate Fe\\u003csup\\u003e3+\\u003c/sup\\u003e with high specificity to liberate iron-bound P (Marschner and R\\u0026ouml;mheld \\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e1994\\u003c/span\\u003e). This divergence in strategy reflects a fundamental evolutionary trade-off: species investing in metabolically expensive carboxylate exudation such as chickpea typically exhibit reduced root morphological plasticity compared to scavenging species such as wheat (Wen et al. \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). These contrasting mechanisms suggest that legumes and cereals may differ in which soil P pools they can effectively access, a hypothesis tested in this study.\\u003c/p\\u003e \\u003cp\\u003eIntercropping legumes and cereals has long been practiced to improve nitrogen use efficiency through biological N fixation (Fujita et al. \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e1992\\u003c/span\\u003e). More recently, attention has turned to potential P benefits. Hinsinger et al. (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e) distinguished two mechanisms by which intercropping can enhance P acquisition. Complementarity arises when intercropped species partition different P pools, reducing competition without altering P availability itself; for example, one species depleting organic P while the other takes up inorganic P. Facilitation, by contrast, occurs when one species converts P from an unavailable pool into an available form that the neighbouring species can access, effectively adding to the P available to its neighbour. Distinguishing these mechanisms experimentally is challenging (Loreau and Hector \\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e), but a key prediction follows: if facilitation drives the intercropping benefit, the advantage should scale with the supply of the P substrate that the facilitating species can mobilize. A global meta-analysis of cereal-legume intercropping found mean LERP of 1.24 across 97 records and approximately 21% lower P fertilizer requirements for the same yield, with stronger effects where P-mobilizing legumes such as chickpea were involved (Tang et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Wheat-legume intercropping systems have shown promise (Li et al. \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e2014\\u003c/span\\u003e; de Oliveira et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e), but the specific P forms involved and the conditions under which facilitation occurs remain poorly characterized.\\u003c/p\\u003e \\u003cp\\u003eRising atmospheric CO₂ concentrations add another dimension to plant P nutrition. Elevated CO₂ generally stimulates photosynthesis and growth, but this response can be constrained by nutrient availability (Reich et al. \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e; Sardans et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). Plants grown under elevated CO₂ often exhibit reduced tissue nutrient concentrations, a phenomenon attributed to both dilution effects and impaired nutrient uptake (Loladze \\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e2002\\u003c/span\\u003e). Crucially, elevated CO₂ may enhance P acquisition by increasing the carbon supply available for root exudation, which can stimulate the release of carboxylates that mobilize insoluble P pools (Pang et al. \\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e). However, it remains poorly understood whether CO₂ fertilization effects depend specifically on P-source availability, meaning whether the growth response is contingent on the specific chemical forms of P that a plant is physiologically equipped to access.\\u003c/p\\u003e \\u003cp\\u003eThis study examines whether chickpea and wheat differ in their ability to acquire P from calcium-bound (hydroxyapatite) versus iron-bound (FePO₄) sources, and whether intercropping can leverage any such differences to improve P acquisition through facilitation. We also tested whether elevated CO₂ modifies these responses, and analysed plant elemental content to determine whether species-specific acquisition mechanisms leave distinct nutritional signatures. Two greenhouse experiments were conducted: the first compared P uptake by monocropped wheat and chickpea supplied with different P sources under ambient and elevated CO₂; the second tested whether intercropping wheat with chickpea enhances P acquisition from sparingly soluble P sources.\\u003c/p\\u003e\"},{\"header\":\"Materials and methods\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eExperimental site and growth conditions\\u003c/h2\\u003e \\u003cp\\u003eTwo experiments were conducted in climate-controlled chambers interfaced with a CO₂ controller (custom-built, Emproco, Israel) equipped with an NDIR CO₂ sensor (Gascard NG, Edinburgh Instruments, Livingston, UK) at the Gilat Research Station, Israel. The first experiment was conducted from June 17 to August 18, 2021, and the second from December 20, 2022, to February 9, 2023. Temperature was maintained at 25\\u0026deg;C during the day and 17\\u0026deg;C at night, and plants received natural sunlight without supplemental lighting.\\u003c/p\\u003e \\u003c/div\\u003e\\n\\u003ch3\\u003eSoil and plant material\\u003c/h3\\u003e\\n\\u003cp\\u003ePlants were grown in Hamra soil collected from the Rehovot region of the Israeli coastal plain. The soil had a pH of 7.7, electrical conductivity of 0.28 dS m⁻\\u0026sup1; Olsen-extractable P\\u0026thinsp;\\u0026lt;\\u0026thinsp;6 mg kg⁻\\u0026sup1;, and total P of 50 mg kg⁻\\u0026sup1; (0.005%), indicating a calcareous, severely P-limited system typical of Mediterranean agricultural soils. Pots were filled with 2.5 kg of soil and irrigated as needed to maintain optimal soil moisture. Nutrients other than phosphorus were supplied in non-limiting amounts via irrigation water, including 54 mg L⁻\\u0026sup1; N, 150 mg L⁻\\u0026sup1; K, 120 mg L⁻\\u0026sup1; Ca, and 24 mg L⁻\\u0026sup1; Mg (Gross et al. \\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eWheat (Triticum aestivum L. cv. Gadera) and chickpea (Cicer arietinum L. cv. Zehavit) were used in both experiments. These cultivars are widely grown in Israeli agriculture and represent common modern varieties adapted to Mediterranean conditions. Both experiments were conducted with 4\\u0026ndash;6 replicates per treatment.\\u003c/p\\u003e\\n\\u003ch3\\u003eExperiment 1: Elevated CO₂ and P uptake in monocrops\\u003c/h3\\u003e\\n\\u003cp\\u003eWheat and chickpea were grown in monocrop systems under two CO₂ conditions: ambient (400 \\u0026micro;mol mol⁻\\u0026sup1;) and elevated (850 \\u0026micro;mol mol⁻\\u0026sup1;). Plants were subjected to four P treatments: (1) Control: no additional P supplied; (2) KH₂PO₄: soluble P provided via irrigation water at 11.2 mg L⁻\\u0026sup1; P daily; (3) Hydroxyapatite (Ca-P; Sigma-Aldrich Israel, Rehovot, Israel): sparingly soluble calcium-bound P, 0.01 g P per pot; and (4) FePO₄ (Fe-P; Sigma-Aldrich Israel, Rehovot, Israel): sparingly soluble iron-bound P, 0.01 g P per pot. Each pot contained four wheat plants or two chickpea plants. The total number of pots was 55, with 2\\u0026ndash;6 replicates per treatment combination, unbalanced due to plant mortality. Due to this plant mortality, the KH₂PO₄ treatment under elevated CO₂ was excluded from analysis.\\u003c/p\\u003e\\n\\u003ch3\\u003eExperiment 2: Intercropping effects on P uptake\\u003c/h3\\u003e\\n\\u003cp\\u003eWheat and chickpea were grown either in monocrop (four wheat plants or two chickpea plants per pot) or intercrop systems (two wheat plants and one chickpea plant per pot). Pots were subjected to three P treatments: (1) Control: no additional P supplied; (2) Hydroxyapatite (Ca-P): 0.01 g P per pot; and (3) FePO₄ (Fe-P): 0.01 g P per pot. In intercrop pots, wheat and chickpea were harvested and analysed separately, yielding two samples per pot (one per species) The total number of pots was 45, with 4\\u0026ndash;6 replicates per treatment combination.\\u003c/p\\u003e\\n\\u003ch3\\u003ePlant growth and elemental analyses\\u003c/h3\\u003e\\n\\u003cp\\u003eAt the conclusion of each experiment, plants were harvested to measure aboveground biomass and nutrient uptake. Aboveground biomass was determined after drying plant material at 60\\u0026deg;C to constant weight. For elemental analyses, dried plant material was finely ground, and subsamples were ashed at 500\\u0026deg;C for 5 h in a muffle furnace. The ash was extracted with 1 M HCl.\\u003c/p\\u003e \\u003cp\\u003eIn Experiment 1, P concentration in the extract was determined colorimetrically using the molybdate blue method (Murphy and Riley \\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e1962\\u003c/span\\u003e) on a Thermo Scientific Gallery Aqua Master discrete analyzer. In Experiment 2, elemental concentrations were determined by inductively coupled plasma optical emission spectrometry (ICP-OES), quantifying tissue concentrations of P, Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, S, Sr, and Zn. Total P uptake was calculated as the product of biomass and P concentration.\\u003c/p\\u003e \\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eStatistical analysis\\u003c/h2\\u003e \\u003cp\\u003eIn Experiment 1, species \\u0026times; P treatment effects on biomass, P concentration, and P uptake were analysed using two-way ANOVA (pooled across CO₂ levels). To assess CO₂ effects, a three-way ANOVA with species, P treatment, and CO₂ level was performed; planned interaction contrasts were computed using the pooled mean square error from the full model to test whether the CO₂ response differed across P treatments within each species. In Experiment 2, effects of P treatment and cropping system on biomass, P concentration, and P uptake were analyzed using two-way ANOVA. Dunnett's test was used for comparisons against the control treatment, and Tukey's HSD test was used for pairwise comparisons, at a significance level of p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05. For comparisons between monocrop and intercrop systems within each P treatment, Mann-Whitney U tests were used. Replication varied across treatments (n\\u0026thinsp;=\\u0026thinsp;2\\u0026ndash;6) owing to seedling establishment failures; non-parametric tests were therefore preferred where sample sizes were small, and results should be interpreted with this limitation in mind.\\u003c/p\\u003e \\u003cp\\u003eFor mineral profile analysis in Experiment 2, treatment effects on individual element concentrations were assessed using t-tests or Mann-Whitney U tests as appropriate. To examine relationships between P acquisition mechanisms and tissue mineral composition, Pearson correlations between P concentration and concentrations of other elements were calculated separately for each species. All statistical analyses were performed using JMP version 13 (SAS Institute Inc., Cary, NC, USA) and Python (scipy, pandas).\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec10\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eSpecies-specific P-source utilization\\u003c/h2\\u003e \\u003cp\\u003eWheat and chickpea displayed contrasting abilities to acquire P from different mineral sources, evidenced by significant species \\u0026times; P treatment interactions for biomass (F\\u0026thinsp;=\\u0026thinsp;9.27, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), tissue P concentration (F\\u0026thinsp;=\\u0026thinsp;15.65, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001), and total P uptake (F\\u0026thinsp;=\\u0026thinsp;11.38, p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001). Chickpea effectively utilized calcium-bound P (Ca-P), showing 2.1-fold higher biomass (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA), 1.7-fold higher tissue P concentration (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), and 3.7-fold higher P uptake (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC) compared to unfertilized controls. In contrast, chickpea showed no significant response to iron-bound P (Fe-P; 1.2-fold biomass, 1.2-fold P concentration, 1.7-fold P uptake; all p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA\\u0026ndash;C). Wheat displayed the opposite pattern. Under Fe-P treatment, wheat achieved 2.3-fold higher biomass (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA), 2.5-fold higher tissue P concentration (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB), and 5.5-fold higher P uptake (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC) compared to controls. However, wheat failed to access Ca-P, with biomass (0.8-fold), P concentration (1.1-fold), and P uptake (0.9-fold) statistically indistinguishable from controls (all p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.05; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA\\u0026ndash;C). Both species responded strongly to the soluble P control (KH₂PO₄), with P uptake increasing 7.3-fold in chickpea and 13.6-fold in wheat (both p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.001; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC), confirming that growth was P-limited.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eElevated CO₂ responses depend on P accessibility\\u003c/h2\\u003e \\u003cp\\u003eThe effect of elevated CO₂ on P nutrition depended on P source accessibility, as indicated by a significant three-way Species \\u0026times; P treatment \\u0026times; CO₂ interaction for P uptake (F\\u0026thinsp;=\\u0026thinsp;3.92, p\\u0026thinsp;=\\u0026thinsp;0.032; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). For wheat grown without supplemental P (Control), elevated CO₂ reduced tissue P concentration by 0.25 mg g⁻\\u0026sup1; and P uptake by 0.40 mg, consistent with growth-induced P dilution (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, C). This negative response reversed when wheat was supplied with Fe-P, the P source wheat can access: elevated CO₂ increased P concentration by 0.44 mg g⁻\\u0026sup1; and P uptake by 1.22 mg (P \\u0026times; CO₂ interaction within wheat: F\\u0026thinsp;=\\u0026thinsp;4.36, p\\u0026thinsp;=\\u0026thinsp;0.032 for P concentration; F\\u0026thinsp;=\\u0026thinsp;4.77, p\\u0026thinsp;=\\u0026thinsp;0.025 for P uptake; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB, C). Under Ca-P, which wheat cannot efficiently access (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e), the CO₂ response remained negligible. Chickpea showed an opposite pattern: elevated CO₂ appeared to enhance growth specifically under Ca-P, the P source accessible to chickpea (+\\u0026thinsp;0.88 g biomass, +\\u0026thinsp;1.17 mg P uptake), while showing no benefit or reduced performance under Fe-P (\\u0026minus;\\u0026thinsp;0.49 g biomass; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA, C). However, the P \\u0026times; CO₂ interaction did not reach significance for chickpea (p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.16 for all variables), so these trends remain suggestive and require confirmation with larger sample sizes.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eIntercropping facilitates wheat P acquisition from calcium-bound P\\u003c/h2\\u003e \\u003cp\\u003eIn Experiment 2, we tested whether intercropping with chickpea could enable wheat to access Ca-P. Consistent with Experiment 1, wheat grown in monoculture with Ca-P performed poorly, with per-plant biomass (0.12 g) and P uptake (0.09 mg) only marginally above control levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA\\u0026ndash;C). However, when intercropped with chickpea under the same Ca-P treatment, wheat performance increased dramatically: per-plant biomass reached 0.49 g (3.9-fold increase, p\\u0026thinsp;=\\u0026thinsp;0.008), tissue P concentration increased from 0.70 to 1.19 mg g⁻\\u0026sup1; (1.7-fold), and P uptake reached 0.61 mg per plant (7.0-fold increase, p\\u0026thinsp;=\\u0026thinsp;0.008).\\u003c/p\\u003e \\u003cp\\u003eThis facilitation effect was specific to Ca-P. Under control (no P) conditions, intercropped wheat showed a modest but significant 2.1-fold increase in biomass and P uptake compared to monoculture (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA), likely reflecting baseline complementarity effects. Under Fe-P, a source wheat can access independently, intercropping provided only a 1.8-fold biomass increase, substantially less than the benefit observed under Ca-P.\\u003c/p\\u003e \\u003cp\\u003eChickpea experienced no cost from facilitating wheat P acquisition. Under Ca-P supply, chickpea per-plant biomass (1.31 vs. 1.20 g), P concentration (2.59 vs. 2.76 mg g⁻\\u0026sup1;), and P uptake (3.46 vs. 3.20 mg) were statistically indistinguishable between intercrop and monoculture systems (all p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.5; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD\\u0026ndash;F). This asymmetric outcome, wheat benefits substantially while chickpea is unaffected, is consistent with facilitation through chickpea root exudates that mobilize Ca-P in the shared rhizosphere.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eLand equivalent ratios confirm facilitation beyond baseline complementarity\\u003c/h2\\u003e \\u003cp\\u003eLand equivalent ratios confirm this interpretation quantitatively. If facilitation drives the intercropping benefit, the advantage should scale with the supply of Ca-P substrate that chickpea can mobilize (Tang et al. \\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e). Under Control conditions, the intercrop showed modest benefits (LER\\u0026thinsp;=\\u0026thinsp;1.50, LERP\\u0026thinsp;=\\u0026thinsp;1.38; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), likely reflecting spatial complementarity and possibly small-scale facilitation from native soil Ca-P. Under Ca-P supply, both metrics increased substantially: LER reached 2.52 (p\\u0026thinsp;=\\u0026thinsp;0.002 vs Control) and LERP reached 4.03 (p\\u0026thinsp;=\\u0026thinsp;0.015). Under Fe-P, a source chickpea cannot mobilize, LER was not elevated above Control (LER\\u0026thinsp;=\\u0026thinsp;1.30, LERP\\u0026thinsp;=\\u0026thinsp;1.10; both ns). The intercropping advantage thus scales with the availability of chickpea\\u0026rsquo;s specific substrate, consistent with facilitation rather than complementarity.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec14\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eElemental content supports distinct P acquisition mechanisms\\u003c/h2\\u003e \\u003cp\\u003eMultivariate analysis of tissue elemental concentrations revealed strikingly different element co-accumulation patterns between species. Chickpea tissue P concentration was positively correlated with all measured elements, where 13 of 14 correlations were significantly positive (p\\u0026thinsp;\\u0026lt;\\u0026thinsp;0.05; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). This pattern suggests non-specific nutrient mobilization, where the mechanism chickpea uses to acquire P simultaneously releases other soil-bound elements into solution.\\u003c/p\\u003e \\u003cp\\u003eIn contrast, wheat tissue P showed no consistent relationship with other elements. Only 3 of 14 correlations were significant, all negative (B, Mn, Na; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA). This decoupled pattern is consistent with a more targeted P acquisition mechanism that does not mobilize other elements as a side effect.\\u003c/p\\u003e \\u003cp\\u003eThe divergence was most pronounced for manganese. In chickpea, P and Mn were positively correlated (r\\u0026thinsp;=\\u0026thinsp;+\\u0026thinsp;0.53, p\\u0026thinsp;=\\u0026thinsp;0.002; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB), whereas in wheat they were negatively correlated (r\\u0026thinsp;=\\u0026thinsp;\\u0026minus;\\u0026thinsp;0.49, p\\u0026thinsp;=\\u0026thinsp;0.007; Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig5\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eC). The negative P\\u0026ndash;Mn correlation in wheat likely reflects growth dilution: as plants acquire more P and increase biomass, a relatively fixed Mn pool becomes diluted across more tissue. The positive correlation in chickpea indicates that Mn accumulates alongside P, consistent with co-mobilization by the same rhizosphere process. These opposing patterns were consistent across both monoculture and intercrop systems.\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec16\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eDistinct phosphorus niches in legumes and cereals\\u003c/h2\\u003e \\u003cp\\u003eOur results confirm a fundamental physiological trade-off in P acquisition between legumes and cereals, providing clear evidence for niche differentiation in soil nutrient use. The 'crossover' interaction observed in Experiment 1, where chickpea mobilized Ca-P but failed on Fe-P, while wheat exhibited the exact opposite pattern, demonstrates that these species occupy distinct 'phosphorus niches' defined by soil chemistry.\\u003c/p\\u003e \\u003cp\\u003eChickpea's capacity to access Ca-P is driven by a 'chemical mining' strategy. As observed in previous hydroponic and soil studies, chickpea roots actively acidify the rhizosphere and exude substantial amounts of carboxylates, particularly citrate and malate, under P-limiting conditions (Veneklaas et al. \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e; Alloush \\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e2003\\u003c/span\\u003e). While acidification is a primary driver, the mobilization of phosphorus is likely enhanced by ligand exchange mechanisms. Barrow et al. (\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2018\\u003c/span\\u003e) recently demonstrated that citrate anions can displace phosphate from adsorption sites through competitive binding, a process that operates synergistically with proton extrusion to maximize P desorption in calcareous matrices. This mechanism is specifically effective against calcium phosphates; the exudation of protons lowers rhizosphere pH, while organic anions chelate Ca\\u0026sup2;⁺, shifting the chemical equilibrium to release orthophosphate into the soil solution (Hinsinger \\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e2001\\u003c/span\\u003e). Our findings align with Pearse et al. (\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e2006\\u003c/span\\u003e), who found that while legumes could mobilize Ca-P through acidification, they struggled to access Fe-P, likely because the solubilization of iron phosphates requires reduction or specific chelation rather than acidification alone.\\u003c/p\\u003e \\u003cp\\u003eIn contrast, wheat failed to access Ca-P, confirming that its extensive root system functions primarily as a 'scavenging' tool for soluble nutrients rather than a 'mining' tool for acid-soluble P pools (Lyu et al. \\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e2016\\u003c/span\\u003e). However, wheat's superior performance on Fe-P suggests a reliance on Strategy II iron acquisition mechanisms. Graminaceous species release phytosiderophores to chelate ferric iron (Fe\\u0026sup3;⁺); by stripping iron from sparingly soluble Fe-P complexes, these chelators effectively release the associated phosphate into the soil solution as a collateral benefit (Shen et al. \\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). This mechanism is chemically distinct from the acidification strategy used by chickpea, explaining why wheat could access Fe-P but remained starved in the Ca-P treatment (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eA).\\u003c/p\\u003e \\u003cp\\u003e \\u003c/p\\u003e \\u003cp\\u003eThe accumulation of Mn in chickpea tissue, which serves as a physiological proxy for rhizosphere carboxylate exudation, is supporting evidence for the suggested P uptake mechanism. Broad surveys across 727 species at 66 sites have validated leaf Mn concentration as a practical indicator of carboxylate-mediated P-mining activity (Lambers et al. \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e2021\\u003c/span\\u003e), as the acidification and ligand exchange processes required to solubilize Ca-P also mobilize soil Mn (Yan et al. \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e; Wang et al. \\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e). The positive P\\u0026ndash;Mn correlation we observed in chickpea, but not wheat, is thus consistent with carboxylate-mediated nutrient mobilization.\\u003c/p\\u003e \\u003cp\\u003eThese elemental profiles further support that chickpea and wheat employ fundamentally different P acquisition mechanisms. The striking contrast, where chickpea co-accumulates P with nearly all elements, while wheat does not, is consistent with the distinction between non-specific chemical mobilization and targeted nutrient scavenging.\\u003c/p\\u003e \\u003cp\\u003eCarboxylate exudation, the mechanism proposed for chickpea's Ca-P acquisition, is inherently non-selective. Organic acids that solubilize calcium phosphates also mobilize other cations and metal-bound nutrients through similar ligand exchange and pH-mediated processes (Lambers et al. \\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e). This predicts that plants relying on carboxylate exudation should show correlated accumulation of multiple elements, exactly the pattern observed in chickpea. Notably, intercropped wheat under Ca-P also showed significantly elevated K (+\\u0026thinsp;24%, p\\u0026thinsp;=\\u0026thinsp;0.01), consistent with access to nutrients co-mobilized by chickpea\\u0026rsquo;s rhizosphere acidification; similar multi-element co-accumulation in intercropped wheat has been reported in wheat\\u0026ndash;white lupin systems (de Oliveira et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). In contrast, the decoupling of P from other elements except for Fe in wheat is consistent with a more targeted acquisition mechanism. Phytosiderophore-mediated iron acquisition is highly specific for Fe\\u0026sup3;⁺ chelation and would not be expected to mobilize other soil nutrients. The negative P\\u0026ndash;Mn correlation in wheat most likely reflects simple growth dilution: as plants acquire more P and increase biomass, a relatively fixed Mn pool becomes distributed across more tissue, reducing concentration. This interpretation is corroborated by the intercropping comparison: wheat leaf Mn decreased by 20% (p\\u0026thinsp;=\\u0026thinsp;0.03) under Ca-P despite a doubling of P concentration, consistent with the 3.9-fold biomass increase diluting a relatively fixed Mn pool.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec17\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eCO₂ fertilization is constrained by P accessibility\\u003c/h2\\u003e \\u003cp\\u003eThe strict dependence on soil P speciation also constrains the crop response to future atmospheric conditions. While elevated CO₂ generally acts as a 'carbon fertilizer,' our results demonstrate that this benefit is capped by the 'Law of the Minimum': carbon fertilization fails to translate into improved P nutrition when the available P pool is chemically inaccessible (Jin et al. \\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e2015\\u003c/span\\u003e; Jiang et al. \\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e2020\\u003c/span\\u003e; Terrer et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e; Sardans et al. \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e2017\\u003c/span\\u003e). In Experiment 1, elevated CO₂ enhanced wheat P concentration and P uptake only when wheat was supplied with Fe-P, a source it can physiologically access. Under Ca-P and Control treatments, the CO₂ response was negligible or negative (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThese results, combined with our intercropping findings, suggest a path forward. In Experiment 1, wheat failed to respond to elevated CO₂ under Ca-P supply due to P limitation. In Experiment 2, intercropping with chickpea relieved this P limitation under ambient CO₂. Together, these findings suggest that intercropping may be a necessary precondition for realizing CO₂ fertilization benefits in P-limited calcareous systems, a hypothesis that merits direct experimental testing combining intercropping with elevated CO₂.\\u003c/p\\u003e \\u003cp\\u003eThese results have important implications for predicting agricultural responses to climate change. While elevated CO\\u003csub\\u003e2\\u003c/sub\\u003e can enhance crop yields through increased photosynthesis, these benefits may be diminished or eliminated in nutrient-limited systems (Terrer et al. \\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e2019\\u003c/span\\u003e). Our data demonstrate that P source accessibility, not just total P availability, fundamentally constrains the CO\\u003csub\\u003e2\\u003c/sub\\u003e fertilization response. In calcareous soils where much of the soil P exists in calcium-bound forms that cereals cannot efficiently access, the CO\\u003csub\\u003e2\\u003c/sub\\u003e fertilization effect may be particularly limited unless facilitative interactions with legumes enable access to these P pools.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec18\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003eFacilitation through rhizosphere modification\\u003c/h2\\u003e \\u003cp\\u003eThe most striking finding of this study is the magnitude of facilitation observed when divergent P acquisition strategies are combined (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig6\\\" class=\\\"InternalRef\\\"\\u003e6\\u003c/span\\u003eB). Experiment 1 established that wheat cannot access Ca-P in monoculture; yet in Experiment 2, when grown alongside chickpea, wheat biomass increased 3.9-fold and P uptake increased 7.0-fold. Since wheat lacks the physiological machinery to solubilize calcium phosphates, this P must have been mobilized by chickpea's rhizosphere activity and subsequently taken up by neighbouring wheat roots. Chickpea, meanwhile, was unaffected; its biomass, P concentration, and P uptake were indistinguishable between monoculture and intercrop (all p\\u0026thinsp;\\u0026gt;\\u0026thinsp;0.5). This one-directional benefit, where one species converts an unavailable P pool into a form the neighbour can access at no cost to itself, matches the definition of facilitation proposed by Hinsinger et al. (\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). The LERP of 4.03 far exceeds the standard threshold for complementarity (\\u0026gt;\\u0026thinsp;1.0), confirming a powerful facilitative interaction driven by rhizosphere modification.\\u003c/p\\u003e \\u003cp\\u003eHowever, facilitation in wheat-chickpea systems may not be solely driven by acidification. Betencourt et al. (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e) demonstrated that intercropping can promote P availability through complex chemical interactions that may include localized pH changes and microbial stimulation, suggesting that chickpea's rhizosphere modification operates through multiple solubility pathways. While white lupin is often cited as the model species for carboxylate exudation, studies indicate that wheat-lupin intercrops achieve facilitation through similar spatial and chemical complementarity (Cu et al. \\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e2005\\u003c/span\\u003e; de Oliveira et al. \\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e2022\\u003c/span\\u003e). Our results suggest chickpea functions as a functional analogue to lupin in calcareous soils, where lupin is often poorly adapted due to sensitivity to high pH (White and Robson, \\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e1989\\u003c/span\\u003e).\\u003c/p\\u003e \\u003cp\\u003eThis facilitation was P-source specific. The comparison of LER under Ca-P to LER under control conditions provides a rigorous test for true facilitation versus baseline complementarity effects. While all intercrop treatments showed LER\\u0026thinsp;\\u0026gt;\\u0026thinsp;1, indicating some degree of niche differentiation, only the Ca-P treatment showed LER significantly exceeding the control baseline. This demonstrates that the intercropping benefit under Ca-P reflects genuine facilitation of P acquisition, not merely reduced competition or spatial complementarity.\\u003c/p\\u003e \\u003cp\\u003eThe mechanism of facilitation likely involves chickpea's rhizosphere modification creating a zone of elevated P availability that wheat roots can exploit. In the intercropped pots, wheat roots intermingled with chickpea roots would have access to the P solubilized by chickpea's carboxylate exudation. This validates previous work by Li et al. (\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e2004\\u003c/span\\u003e) and Betencourt et al. (\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e2012\\u003c/span\\u003e), who reported similar facilitation between cereals and legumes, though the magnitude of benefit in our study is notably higher, likely due to the severe P limitation of our soil and the confined pot system maximizing root intermingling.\\u003c/p\\u003e \\u003cp\\u003eRoot exudates may also stimulate rhizosphere microbial communities that contribute to P solubilization, amplifying the direct chemical effects of carboxylate exudation (Richardson et al. \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e2011\\u003c/span\\u003e). While we did not measure microbial responses in this study, this represents an important avenue for future investigation.\\u003c/p\\u003e \\u003cp\\u003eChickpea experienced no measurable cost from facilitating wheat P acquisition. Per-plant biomass, P concentration, and P uptake were all maintained in intercrop compared to monoculture. This asymmetric outcome, where wheat benefits substantially while chickpea is unaffected, suggests that chickpea's P mobilization capacity exceeded its own uptake requirements, leaving surplus mobilized P available for wheat roots.\\u003c/p\\u003e \\u003c/div\\u003e \\u003cdiv id=\\\"Sec19\\\" class=\\\"Section2\\\"\\u003e \\u003ch2\\u003ePractical implications\\u003c/h2\\u003e \\u003cp\\u003eUnlike the well-documented benefits of cereal-legume intercropping for nitrogen acquisition, where legumes supply N via biological fixation, our results highlight a critical, often overlooked mechanism for phosphorus mobilization. Wheat-chickpea intercropping represents a viable biological strategy to 'unlock' legacy P in calcareous soils. By pairing a species that chemically mines P with a species that efficiently scavenges it, agricultural systems can access P pools that are otherwise biologically unavailable, improving P-use efficiency and potentially reducing fertilizer requirements.\\u003c/p\\u003e \\u003cp\\u003eThis P-facilitation appears strictly context-dependent. Facilitation was absent in the control (no P) treatment, despite severe P stress, because chickpea's acidification mechanism requires a calcium-phosphate substrate to act upon. Similarly, facilitation was minimal under Fe-P, where chickpea's mechanism is ineffective. Thus, the interaction is not a general stress response but a specific chemical synergy contingent on soil P speciation. These findings suggest that the benefits of wheat-legume intercropping for P nutrition will be greatest in calcareous and alkaline soils where calcium-bound P predominates. Field studies in semi-arid calcareous soils support these findings; Souid et al. (\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e2024\\u003c/span\\u003e) reported that durum wheat-chickpea intercropping increased available rhizosphere P by 28% and microbial biomass P by 34% relative to bulk soil.\\u003c/p\\u003e \\u003c/div\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eThis study provides physiological evidence that cereal-legume intercropping can mobilize soil phosphorus pools that are biologically unavailable to monocultures. By characterizing the distinct 'phosphorus niches' of wheat and chickpea, we demonstrate that the benefits of intercropping extend beyond the well-known mechanisms of nitrogen fixation to include active phosphorus mining. The ability of chickpea to acidify the rhizosphere and solubilize calcium-bound phosphorus acts as a biological 'key,' unlocking nutrient reserves that wheat's scavenging root system cannot access on its own.\\u003c/p\\u003e \\u003cp\\u003eThe elemental profiles provide a novel line of evidence supporting mechanistic interpretations: chickpea's non-specific element co-accumulation pattern is consistent with carboxylate-mediated mobilization, while wheat's decoupled profile supports a more targeted acquisition strategy. The use of tissue Mn concentration as a proxy for carboxylate exudation offers a practical diagnostic tool for future studies of P acquisition mechanisms.\\u003c/p\\u003e \\u003cp\\u003eOur findings have critical implications for agriculture under changing climate conditions. The potential yield benefits of elevated atmospheric CO₂ are strictly constrained by soil nutrient availability; without a strategy to mobilize recalcitrant phosphorus, the 'CO₂ fertilization effect' fails to materialize. Wheat-chickpea intercropping represents a sustainable strategy to recover 'legacy phosphorus', the vast reserves of fertilizer-derived P that have accumulated in calcareous soils over decades of intensive agriculture but remain fixed in insoluble forms. By integrating species with complementary root traits, farmers can improve system-level phosphorus use efficiency, reduce dependence on synthetic inputs, and stabilize yields in low-input environments. The benefits of interspecific facilitation may extend beyond the current growing season: rotational studies have shown that P mobilized by legume exudates can remain available for subsequent cereal crops, offering a long-term strategy for accessing legacy soil P (Nuruzzaman et al. \\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e2005a\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003eb\\u003c/span\\u003e). Direct confirmation of the P transfer pathway using 32P-labelled calcium phosphate would further strengthen the mechanistic basis for mineral-form-specific facilitation.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e \\u003ch2\\u003eCompeting interests\\u003c/h2\\u003e \\u003cp\\u003eThe authors declare no competing interests.\\u003c/p\\u003e \\u003c/p\\u003e\\u003ch2\\u003eFunding\\u003c/h2\\u003e \\u003cp\\u003eThis study was supported by the Israel Science Foundation (ISF) grant number 267/24. Funding that the ISF grant specified was received by AG.\\u003c/p\\u003e\\u003ch2\\u003eAuthor contributions\\u003c/h2\\u003e \\u003cp\\u003eM.H. conceived and designed the study, conducted the experiments, analysed the data, and wrote the manuscript. S.T. contributed to study design, experimentation, and manuscript revision. A.G. contributed to study design, data interpretation, and critical revision of the manuscript.\\u003c/p\\u003e\\u003ch2\\u003eData availability\\u003c/h2\\u003e \\u003cp\\u003eThe datasets generated and analysed during the current study are available in the Zenodo repository: \\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://doi.org/10.5281/zenodo.18698449\\u003c/span\\u003e\\u003cspan address=\\\"10.5281/zenodo.18698449\\\" targettype=\\\"DOI\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\u003cli\\u003e\\u003cspan\\u003eAlloush GA (2003) Dissolution and effectiveness of phosphate rock in acidic soil amended with cattle manure. Plant Soil 251:37\\u0026ndash;46\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eBarrow NJ, Debnath A, Sen A (2018) Mechanisms by which citric acid increases phosphate availability. 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PLoS ONE 19(5):e0300573\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTang X, Zhang C, Yu Y, van der Shen J, Zhang F (2021) Intercropping legumes and cereals increases phosphorus use efficiency; a meta-analysis. Plant Soil 460:89\\u0026ndash;104\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eTerrer C, Jackson RB, Prentice IC, Keenan TF, Kaiser C, Vicca S, Fisher JB, Reich PB, Stocker BD, Hungate BA et al (2019) Nitrogen and phosphorus constrain the CO₂ fertilization of global plant biomass. Nat Clim Change 9:684\\u0026ndash;689\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eVeneklaas EJ, Stevens J, Cawthray GR, Turner S, Grigg AM, Lambers H (2003) Chickpea and white lupin rhizosphere carboxylates vary with soil properties and enhance phosphorus uptake. Plant Soil 248:187\\u0026ndash;197\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang Y, Lambers H (2020) Root-released organic anions in response to low phosphorus availability: recent progress, challenges and future perspectives. Plant Soil 447:135\\u0026ndash;156\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWang Y, Yang M, Yu F (2024) Determine leaf manganese concentration to estimate rhizosheath carboxylates of mycorrhizal plants in forest ecosystems. Plant Soil 508:1027\\u0026ndash;1033\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWen Z, Li H, Shen Q, Tang X, Xiong C, Li H, Pang J, Ryan MH, Lambers H, Shen J (2019) Trade-offs among root morphology, exudation and mycorrhizal symbioses for phosphorus-acquisition strategies of 16 crop species. New Phytol 223:882\\u0026ndash;895\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eWhite PF, Robson AD (1989) Effect of soil pH and texture on the growth and nodulation of lupins. Aust J Agric Res 40(1):63\\u0026ndash;73\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eYan L, Tang D, Pang J, Lambers H (2024) Root carboxylate release is common in phosphorus-limited forest ecosystems in China: using leaf manganese concentration as a proxy. Plant Soil 508:143\\u0026ndash;158\\u003c/span\\u003e\\u003c/li\\u003e \\u003cli\\u003e\\u003cspan\\u003eZhu J, Li M, Whelan M (2018) Phosphorus activators contribute to legacy phosphorus availability in agricultural soils: a review. Sci Total Environ 612:522\\u0026ndash;537\\u003c/span\\u003e\\u003c/li\\u003e\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Phosphorus acquisition, Intercropping, Legacy phosphorus, Carboxylate exudation, Rhizosphere facilitation, Manganese proxy, Elevated CO₂\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-8917717/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-8917717/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003ch2\\u003eAims\\u003c/h2\\u003e \\u003cp\\u003eDecades of phosphorus (P) fertilization created vast 'legacy P' reserves in agricultural soils, yet much remains locked in sparingly soluble forms inaccessible to crops. We tested whether wheat and chickpea differ in P acquisition from calcium-bound (Ca-P) versus iron-bound (Fe-P) sources, whether elevated CO₂ modifies these abilities, and whether intercropping enables wheat to access Ca-P through chickpea facilitation.\\u003c/p\\u003e\\u003ch2\\u003eMethods\\u003c/h2\\u003e \\u003cp\\u003eIn two greenhouse experiments, we compared P uptake of monocropped wheat and chickpea on four P sources (hydroxyapatite Ca-P, FePO₄ Fe-P, KH₂PO₄, or no P) under ambient and elevated CO₂ (Experiment 1), and intercropping effects on P acquisition (Experiment 2). Growth parameters and tissue elemental composition were analysed.\\u003c/p\\u003e\\u003ch2\\u003eResults\\u003c/h2\\u003e \\u003cp\\u003eChickpea and wheat displayed a crossover interaction: chickpea achieved 3.7-fold higher P uptake from Ca-P but not Fe-P, while wheat achieved 5.5-fold higher P uptake from Fe-P but could not access Ca-P (species \\u0026times; P-source interaction, p\\u0026thinsp;=\\u0026thinsp;0.005). When intercropped with chickpea, wheat P uptake from Ca-P increased 7.0-fold (p\\u0026thinsp;=\\u0026thinsp;0.008), while chickpea was unaffected. Land equivalent ratios for P uptake under Ca-P (4.03) confirmed facilitation beyond complementarity. Elevated CO₂ enhanced P uptake only when species accessed their specific P source. Leaf Mn, a proxy for carboxylate exudation, correlated positively with P in chickpea (r\\u0026thinsp;=\\u0026thinsp;+\\u0026thinsp;0.53) but negatively in wheat (r\\u0026thinsp;=\\u0026thinsp;\\u0026minus;\\u0026thinsp;0.49), supporting carboxylate-mediated mobilization.\\u003c/p\\u003e\\u003ch2\\u003eConclusions\\u003c/h2\\u003e \\u003cp\\u003eChickpea and wheat occupy distinct 'P niches'. Intercropping enables wheat to access legacy Ca-P through rhizosphere modification by chickpea, a sustainable strategy for P-use efficiency in calcareous agricultural systems.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Chickpea root exudation unlocks legacy phosphorus for intercropped wheat in calcareous soils\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2026-03-06 21:30:55\",\"doi\":\"10.21203/rs.3.rs-8917717/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"ec7c121a-74fa-4631-bbc5-9854c6ec2228\",\"owner\":[],\"postedDate\":\"March 6th, 2026\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2026-04-15T09:19:32+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2026-03-06 21:30:55\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-8917717\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-8917717\",\"identity\":\"rs-8917717\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}