Short-term exposure to elevated CO2 stimulates growth and metabolic responses that alleviate early-stage iron deficiency symptoms in soybean

ABSTRACT Elevated CO2 (eCO2) increase plant biomass and might lead to nutritional losses. The results showed that eCO2 under Fe-deficiency stimulates root dry weight from 0.37–0.80 g plant−1 and shoot dry weight from 0.82–2.10 g plant−1, without compromising root/shoot ratio. Besides, eCO2 improved the chlorophyl content of Fe-deficient plants. However, downregulation of photosynthesis, reduction in stomatal conductance and transpiration rate, and an increase in water-use efficiency was observed due to eCO2. Moreover, under Fe-deficiency, eCO2 decreased K and Mg in roots, and Mg in leaves, whereas increased P and Zn in roots and leaves. In Fe-sufficient plants, eCO2 increased K, P, Mn, Zn and Fe in leaves and did not change the concentration in roots. The Fe-deficiency-induced responses in roots, including the increase in FCR activity, and the expression of Fe-uptake genes were stimulated by eCO2, but were not sufficient to increase Fe concentration in Fe-deficient plants.


Introduction
The global atmospheric carbon dioxide concentration increased by 48% since the beginning of the industrial era from 280 ppm to the current level of about 420 ppm, (http://www.esrl.noaa.gov/gmd/ccgg/trends/) and is predicted to reach 700-1000 ppm by the end of this century (IPCC 2014). This steady increase in atmospheric CO 2 is responsible for global climate change affecting crops worldwide (Bencke-Malato et al. 2019). Therefore, one of the great questions for agriculture in the upcoming future is to improve biomass production and plant product quality, and atmospheric CO 2 concentration and regulation of mineral concentrations are major actors in this scenario (Briat et al. 2015). Soybean is a consistent source of nutrients as consumption of soybean-based products is on the rise due to its high content of proteins, fatty acids, natural antioxidants, vitamins, and minerals (Zheng et al. 2020). Previous findings have demonstrated that elevated CO 2 (eCO 2 ) promotes photosynthetic CO 2 assimilation rate in C3 plants, increasing biomass and yield (Haase et al. 2008;Leakey et al. 2009;Bishop et al. 2015a;Kimball 2016;Bencke-Malato et al. 2019). For example, Kimball (2016) showed that biomass and seed yield increased in C3 species under FACE conditions. Yields of C3 grain crops increased by an average of 19%. In another study, eCO 2 stimulated above-ground biomass by 22% and seed yield by 9% among 18 soybean genotypes grown under field conditions (Bishop et al. 2015b). Plants exposed to eCO 2 changed their root size and activity to increase nutrient uptake and translocation for a given nutrient (Pérez-López et al. 2014Guo et al. 2015. The results from Guo et al. (2015) in rice suggest that eCO 2 might support the translocation of calcium (Ca), magnesium (Mg), iron (Fe), zinc (Zn), and manganese (Mn) from soil to stem and panicle. A great deal of attention has been dedicated to the effects of eCO 2 on the nutrient quality of crop plants and expanding this knowledge to Fe status is of particular interest. Fe is a crucial element of biomass production and plant quality due to its essential role in the structure and function of the photosynthetic electron transfer chain and chlorophyl synthesis (Briat et al. 2015). Besides, Fe intake for a significant proportion of the worldwide population is delivered by plant consumption (Smith et al. 2017). Even though the Fe concentration in soils regularly surpasses plant requirements, its bioavailability is often limited, predominantly in calcareous soils representing 30% of cultivated soils (Jin et al. 2009). Furthermore, Fe is also the most commonly deficient micronutrient in the human diet, impacting roughly an estimated 2 billion people (Briat et al. 2015;Smith et al. 2017). Recent studies revealed that C3 plants, legumes, and maize have lower Fe concentrations ranging from 4-10% when grown at eCO 2 (550 ppm) under field conditions (Myers et al. 2014). Similar findings were obtained by Loladze (2014) in C3 plants, reflecting foliar and edible tissues, FACE, and non-FACE studies. Consequently, Fe metabolism in higher plants is likely to be affected by the atmospheric CO 2 concentration, and it is crucial to address the impact of eCO 2 on plant Fe nutrition. Jin et al. (2009) reported a relative increase in tomato biomass and the root/shoot ratio at eCO 2 under Fe-limited and Fe-sufficient conditions. Under eCO 2 , the Fe-deficiency-induced responses in roots, including ferric chelate reductase (FCR) activity, proton secretion, subapical root hair development, and the expression of FER, FRO1, and IRT genes, were higher than in plants grown at aCO 2 . Also, eCO 2 has a positive effect on biomass when Fe is limited in barley (Haase et al. 2008). Thus, understanding the Fe-uptake mechanisms and Fe-metabolism under eCO 2 conditions is crucial for selecting nutrient-rich and more tolerant genotypes to Fedeficiency (Morrissey and Guerinot 2009). Moreover, little is known about the interaction of eCO 2 and Fe-deficiency in soybean plants, and the underlying mechanisms responsible for such changes. In plants, the Fe-uptake mechanism is divided into two different approaches: strategy I occur in nongraminaceous plants, and Fe 3 + is reduced via a membrane-bound reductase to make it accessible for uptake by a Fe 2 + transporter, and strategy II occurs in grasses which secrete phytosiderophores that readily bind Fe 3 + , and these complexes are then transported back into the roots (Jin et al. 2009 Morrissey andGuerinot 2009). In the present study, soybean plants were grown in a hydroponic culture system to report the effects of the interaction of eCO 2 and Fe-deficiency in the morphological, physiological, and molecular responses under controlled conditions. It was previously demonstrated in tomato (Jin et al. 2009) and barley (Haase et al. 2008) that eCO 2 has a positive impact on plant biomass when Fe is limited. Here, we aimed to understand if eCO 2 may have a positive effect on soybean growth under Fe-deficiency without impairing mineral accumulation, since Fe homeostasis and eCO 2 could be linked to the interplay occurring among nutrient uptake and translocation.

Plant culture
Soybean (Glycine max cv 'Winsconsin Black') seeds were surface sterilized with 75% (v/v) ethanol, rinsed four times with ultra-pure water, and germinated at 25°C in the dark for 5 days. Then, plants were transplanted to black plastic pots (5 L) filled with an aerated, full-strength nutrient solution with the following composition: , and 20 μM Fe(III)-EDDHA. The solution was buffered with the addition of 1mM MES (pH 5.5) and changed every 3 days. Plants were grown in the controlled environment at 75% humidity with a daily cycle of 16 h at 25°C (day) and 8 h at 20°C (night). The daytime light intensity was 325 μmol s −1 m −2 of the photosynthetic photon flux density at the plant level. After 7 days of pre-treatment in the full-strength nutrient solution, plants were transferred to a nutrient solution with Fe(III)-EDDHA at 0.5 μM (Fe-deficiency) or 20 μM (Fe-sufficiency) for 12 days. The plants were grown at 400 ± 10 ppm (aCO 2 ) or 800 ± 10 ppm (eCO 2 ). The CO 2 concentration was continuously monitored and maintained by an automated CO 2 control system, which measured and adjusted the CO 2 concentration from soybean planting to the end of the experiment.

Physiological parameters
At the end of the experiment, the chlorophyl content was evaluated using the first expanded trifoliate leaf with a chlorophyl meter (SPAD-502; Minolta). Then, plants were measured for length and separated into shoots and roots, and the material was dried at 70°C until constant weight and stored for ICP-OES analysis. Each analysis was performed from 5 plants and mean values calculated.

Mineral analysis by ICP-OES
About 200 mg of the roots and leaves dried samples were mixed with 5 mL of 65% HNO 3 (v/v) and 1 mL of H 2 O 2 30% (v/v) in a Teflon reaction vessel and heated in a Speedwave TMMWS-3+ (Berghof, Germany) microwave system. The digestion procedure was described in detail by Santos et al. (2015), and the resulting solutions were diluted to 50 ml with ultra-pure water for further analysis. Determination of mineral concentrations was performed using the ICP-OES Optima7000 DV (Perkin Elmer, USA) with a radial configuration from five independent digestions of each plant organ from all the treatments. The assays were performed from 5 samples and mean values calculated.

Leaf gas exchange parameters
Gas exchange parameters were performed in the last fully expanded leaves, ten days after the Fe-treatment, using a portable photosynthesis system (LI-6400XT; LICOR, Inc.). The CO 2 in the leaf chamber was set to match the CO 2 treatment with a PPFD of 500 μmol photon m −2 s −1 at 25°C. A/Ci curves were also measured, and data were analyzed using the PS-FIT software (http://www.life.illinois.edu/bernacchi/ links.html). This software uses the leaf model of photosynthesis (Farquhar et al. 1980) to calculate the maximum rates of electron transport (J max ) and maximum velocity of carboxylation by Rubisco (V c,max ). Moreover, the transpiration rate (T r ), stomatal conductance (g s ), and water use efficiency (WUE = A/T r ) were determined. The assays were performed from 3 plants and mean values calculated.

Organic acids and sugar analysis
The extraction protocol was described in detail by López-Millán et al. (2009). About 100 mg of plant material was grounded using liquid nitrogen and suspended in 5 mM H 2 SO 4 , vortexed for 30 s, and then boiled for 30 min. The samples were centrifuged at 2320 x g for 10 min, the supernatant filtered through a 0.45 mm PTFE filter, and the volume adjusted to 2 ml and stored at -80°C until further analysis. The HPLC system consisted of an ion exchange aminex HPX-87H Column (300×7.8 mm) (Bio-Rad, USA) maintained at an oven temperature of 40°C and two detectors in series (Refractive Index and UV 210 nm; K-2301 and K-2501, Knauer, Germany). The mobile phase was 5 mM H 2 SO 4 at a flow rate of 0.6 mL/min. The assays were performed from 5 samples, and mean values were calculated.

Statistical analysis
The data for each dependent variable was subjected to the two-way ANOVA at P < 0.05 in all cases. All statistical analyses were performed with SPSS software (SPSS version 26.0).

Results
3.1. Effects of eCO 2 and fe-deficiency on chlorophyl synthesis, plant growth, and fe-uptake After 12 days of plant growth at aCO 2 and under Fedeficiency (0.5 µM Fe-EDTA), the soybean leaves were chlorotic ( Figure 1) with an average SPAD reading of 12.7 ( Figure 2a). However, leaves of plants grown in the same growth conditions but at eCO 2 had SPAD readings of 19.9 ( Figure 2a), indicating that eCO 2 conditions (P < 0.05; Table 1) significantly improved the chlorophyl synthesis of plants grown in Fe-deficient conditions. In Fe-sufficient plants (20 µM Fe-EDTA) the chlorophyl content was also higher under eCO 2 in comparison to aCO 2 conditions (P < 0.05; Figure 2a). The effects of CO 2 and Fe-supply were statistically significant on SPAD readings (P < 0.001), with a significant CO 2 x Fe interaction (P < 0.05, Table 1). Moreover, in Fe-sufficient plants, the height was higher compared to Fe-deficient plants, and this effect was exacerbated in response to eCO 2 (Figure 2b). In hydroponic growth, root dry weight was increased at eCO 2 by 66.4% in Fe-sufficient (P < 0.01) and by 119.7% in Fe-deficient (P < 0.001) plants, as demonstrated in Figure 3a. Shoot dry weight (Figure 3b) was also increased under eCO 2 by 40.1% in Fe-sufficient (P < 0.001) and by 157.6% in Fe-deficient plants (P < 0.001). Furthermore, the root/shoot ratio (Figure 3c) was not affected in Fe-deficient plants (P > 0.05) but increased in Fe-sufficient plants (P < 0.05) by 20.1% under eCO 2 conditions. The effects of CO 2 levels and Fe-supply were statistically significant on FCR activity (P < 0.001), with a significant CO 2 x Fe interaction (P < 0.001, Table 1). Therefore, FCR activity was induced in plants grown in Fedeficiency, and this induction was higher when combined with aCO 2 (Figure 4). Contrastingly, FCR activity was higher under eCO 2 conditions in Fe-sufficient conditions.

Effects of eCO 2 and fe-deficiency on the photosynthetic parameters
Leaf photosynthetic assimilation rate (A) decreased in Fedeficient conditions (P < 0.05; Table 2) when compared to Fe-sufficient conditions. However, in Fe-sufficient and Fedeficient plants the response of A was not affected by exposure to eCO 2 (P > 0.05), with no significant CO 2 x Fe interaction (P > 0.05, Table 1). Also, eCO 2 reduced stomatal conductance (g s ) from 0.37-0.13 mol H 2 O m −2 s −1 in Fesufficient (P < 0.05) and from 0.30-0.12 mol H 2 O m −2 s −1 in Fe-deficient (P < 0.05) plants. The transpiration rate (T r ) also decreased by eCO 2 , in both Fe-supplies. The water-use efficiency (WUE) increased significantly from 2.23-4.76 mmol mol −1 (P < 0.01) in Fe-sufficient plants, and from 2.17-3.97 mmol mol −1 (P < 0.01) in Fe-limited plants due to exposure to eCO 2 . The A/Ci response curves showed a reduction in the V c,max and in J max under eCO 2 in Fesufficient and Fe-deficient plants (P < 0.05, Table 2). However, the response of V c,max and J max were not affected by Fe-supply (P > 0.05) but a significant CO 2 x Fe interaction was found (P < 0.05, Table 1).

Effects of eCO 2 and fe-deficiency on the sugar and organic acid concentrations
In hydroponic culture, elevation of atmospheric CO 2 did not affect sucrose concentration (P > 0.05) in roots and leaves of Fe-sufficient and Fe-deficient plants (Table 3). Moreover, eCO 2 increased glucose concentration in roots of Fe-deficient Figure 2. SPAD readings (a) and height (b) of soybean plants grown in the nutrient solution, depending on Fe-supply (0.5 and 20 μM Fe-EDDHA) and atmospheric CO 2 concentration (400 and 800 ppm). Data are mean ± SEM (n = 5). *, Significant differences (P < 0.05) between aCO 2 and eCO 2 treatments. Table 1. Two-factorial ANOVA table for the effect of eCO 2 and Fe-supply on leaf chlorophyl content (SPAD values), plant height (cm), plant biomass (root and shoot dry weight, g plant −1 ), root/shoot ratio, FCR activity (μmol Fe g −1 FW h -1 ), A (µmol m −2 s −1 ), g s (mol m −2 s −1 ), T r (mol m −2 s −1 ), WUE (µmol mol −1 ), V c,max (µmol m −2 s −1 ), and J max (µmol m −2 s −1 ).   Data are mean ± SEM (n = 5). *, Significant differences (P < 0.05) between aCO 2 and eCO 2 treatments. plants (P < 0.05) and in leaves of Fe-sufficient plants (P < 0.05). Besides, fructose concentration increased in Fe-deficient and Fe-sufficient plants (P < 0.05) in both tissues analyzed due to CO 2 enrichment. The effect of Fe-supply was not statistically significant on the carbohydrate responses (P > 0.05), as well as the CO 2 x Fe interaction (P > 0.05, Table 4). The citric acid concentration increased by the exposure to eCO 2 from 13.64-17.40, and from 8.07-12.55 µmol g FW −1 in roots of Fe-sufficient and Fe-deficient plants, respectively. In leaves, citric acid was not affected under eCO 2 (P > 0.05, Table 3).
At the end of the growth period, eCO 2 increased malic acid concentration in roots from 25.57-41.33 in Fe-sufficient and from 11.85-22.13 µmol g FW −1 in Fe-deficient plants. By comparison, leaves of soybean plants grown in the same nutrient conditions, eCO 2 only increased malic acid in Fe-sufficient plants from 40.11-60.54 µmol g FW −1 . Moreover, the effect of Fe-supply was significant on citric and malic acid concentrations (P < 0.05, Table 4).

Effects of eCO 2 and fe-deficiency on the mineral concentrations
The mineral-based responses under different Fe-supplies in soybean plants were also influenced by eCO 2 , as demonstrated in Figures 5 and 6. Elevation of atmospheric CO 2 concentration did not affect Ca concentration in root and leaf tissues, but it was significantly reduced in Fe-deficient plants (P < 0.05, Table 4). eCO 2 increased K concentration (P < 0.05) from 24.6-41.4 μg g −1 in leaves of Fe-sufficient plants, and decreased K concentration (P < 0.05) from 75.7-52.3 μg g −1 in roots of Fe-deficiency plants. The concentration of P was generally higher under eCO 2 conditions (P < 0.05), except in roots of Fe-sufficient plants. Furthermore, eCO 2 did not affect Mg concentration (P > 0.05) in Fe-sufficient plants, but a significant decrease was achieved in Fe-deficient plants (P < 0.05). Regarding to micronutrients, Mn concentration was not changed by CO 2 treatment A, Photosynthetic carbon assimilation rate (µmol m −2 s −1 ); g s , stomatal conductance (mol m −2 s −1 ); T r , transpiration rate (mol m −2 s −1 ); WUE, water-use efficiency (µmol mol −1 ); V c,max , maximum velocity of carboxylation by Rubisco (µmol m −2 s −1 ); J max , maximum rate of electron transport (µmol m −2 s −1 ). Data are mean ± SEM (n = 3). *, Significant differences (P < 0.05) between aCO 2 and eCO 2 treatments. 62,26 ± 8,38 Suc, sucrose; Gluc, glucose; Frut, fructose; CA, citric acid; MA, malic acid. Data are mean ± SEM (n = 5). *, Significant differences (P < 0.05) between aCO 2 and eCO 2 treatments. Results from the analysis of variance with degrees of freedom (d.f.), F ratios and probabilities (P) for some parameters. Significant effects are shown in boldface.
in Fe-deficient and Fe-sufficient plants (P > 0.05), apart from the increase in Mn concentration occurring in leaves of Fesufficient plants (P < 0.05) from 56.7-155.8 μg g −1 . CO 2 enrichment led to a significant increase in Zn concentration, except in roots of Fe-sufficient plants (P > 0.05). The concentration of Fe was significantly higher in Fe-sufficient plants as expected and was not changed by the CO 2 treatment, excluding the leaves of Fe-sufficient plants.

Effects of eCO 2 and fe-deficiency on the expression of genes involved in fe-uptake mechanisms
The effects of eCO 2 and Fe-deficiency on genes related to the Fe-uptake mechanisms -DMT1, FER, FRO2, IRT1, and ferritinwere analyzed in soybean roots using qPCR. Overall, higher gene expression levels were associated with Fedeficiency responses as demonstrated in Figure 7. In the case of the FRO2 gene, the expression differed between treatments, with higher expression levels observed in Fedeficiency plants, and this change was higher under eCO 2 conditions. Similar findings were obtained in DMT1 and FER gene expression levels. Moreover, IRT1 gene expression was higher in Fe-deficient plants, but eCO 2 did not affect the expression levels. Contrastingly, the ferritin gene expression was detected only in Fe-sufficient plants, and this change was higher under eCO 2 conditions.

Discussion
The beneficial effects of eCO 2 on stimulating plant growth and biomass accumulation of several crops have been widely conditions. Data are mean ± SEM (n = 5). *, Significant differences (P < 0.05) between aCO 2 and eCO 2 treatments.
studied (Ainsworth et al. 2002;Long et al. 2004;Ziska and Bunce 2007;Bunce 2008;Vasconcelos et al. 2014Bunce 2015Santos et al. 2015;Bishop et al. 2015b;Bunce 2016;Kimball 2016). However, there is a lack of information about the influence of eCO 2 on the changing demands for Fe-supply and is crucial to consider how eCO 2 affects and might alleviate the Fe-deficiency responses. In this sudy, we treated soybean plants with limited (0.5 μM) and sufficient (20 μM) Fe-supply under eCO 2 conditions. Supplying soybean plants with 0.5 µM Fe is sufficiently low to induce FCR activity. Furthermore, soybean plants revealed clear Fe chlorosis symptoms in young leaves at aCO 2 but not at eCO 2 conditions, which affected biomass accumulation and growth (Figure 1). An increase from 0.5-20 μM Fe in hydroponic solution alleviated the chlorosis symptoms in soybean plants. As reported for other species (Wu et al. 2004;Bourgault et al. 2016;Bunce 2016;Bourgault et al. 2017), soybean growth increased at eCO 2 conditions, as well as the requirement for nutrients and water, to sustain such CO 2 -based stimulation. Consequently, more nutrients will need to be applied, or plants will have to become more effective at obtaining those nutrients from the soil (Jin et al. 2009). In this study, eCO 2 led to an increase in biomass, even at reduced Fe-supply, similar to plants grown at Fesufficient conditions (Figure 3). Therefore, in Fe-sufficient plants, root and shoot dry weight increased by 66.4% and 40.1%, respectively, whereas in Fe-deficient plants increased by 119.7% and 157.4% due to eCO 2 conditions (Figure 3).  Similar findings were reported in tomato (Jin et al. 2009) and barley (Haase et al. 2008) grown at eCO 2 with or without Fesupply. The root/shoot ratio increased in Fe-sufficient plants due to eCO 2 and was not affected in Fe-deficient plants, suggesting that the biomass accumulation changed with Fe-supply. The reason proposed might be that under eCO 2 and Fe-supply, upregulation of the root over the shoot growth was observed appearing in a higher root/shoot ratio. Also, eCO 2 alleviated the Fe-deficiency chlorosis symptoms, as demonstrated by the increase in chlorophyl concentration (Figure 1). The driving force for increased soybean growth at eCO 2 is a higher A (Ainsworth et al. 2002). We noticed that the A was not changed due to CO 2 enrichment and could not predict the magnitude of biomass increase. However, the photosynthetic capacity was affected by Fesupply (P < 0.001, Table 1), with a concomitant decrease in Fe-deficient plants of about 20% (Table 2). Therefore, under eCO 2 photosynthetic acclimation was observed and attributed to a decrease in V c,max , and J max . Nevertheless, despite acclimation of photosynthetic capacity, biomass production is greater in plants exposed to eCO 2 . It is generally accepted that Fe-deficiency decreased photosynthesis in higher plants (Pestana et al. 2001Jiang et al. 2007), since the Fe-status is a crucial parameter that affects metabolic changes, photosynthesis, and consequently plant growth at eCO 2 (Briat et al. 2015). eCO 2 reduces the transpiration-driven mass flow due to the stomatal closure (Leakey et al. 2009;Prior et al. 2010). In our study, eCO 2 decreased the g s by 60 −64.9% and T r by 44.7−52.9% compared to aCO 2 conditions ( Table 2). The decrease in g s at eCO 2 was associated with higher WUE and consistent with previous studies (Prior et al. 2010;Zheng et al. 2020). Our results suggested that soybean plants may have increased drought tolerance under eCO 2 conditions. The decrease in the T r might be a modification to maximize the carbon fixation under eCO 2 conditions (Jauregui et al. 2015). The carbohydrate accumulation, mainly glucose and fructose, which are assimilates from photosynthesis, exhibited significant variation with eCO 2 in leaf and root tissues resulting in differential carbon allocation, as demonstrated in Table 3. Therefore, eCO 2 increased carbohydrate accumulation independently of Fe-supply, while an increase of photosynthesis capacity was not confirmed. A possible reason for this phenomenon, at eCO 2 , is that plants exceed what they are capable of using or distributing to sinks, increasing the carbohydrates levels, and leading to feedback inhibition of photosynthesis (Thompson et al. 2017). Furthermore, the results showed a differential variation of sugars in plant tissues, but this variation was independent of Fe-supply (P > 0.05, Table 4). Therefore, variation in nutrient uptake mechanisms should be less or partially influenced by sugars under Fe-deficiency. However, there are new insights on sugar sensing and signaling pathways of how plants are influenced by the higher sugars content produced under eCO 2 conditions (Thompson et al. 2017). Sugars are recognized to crosstalk with hormones (e.g. auxin, nitric oxide and ethylene) and act on gene regulation and therefore modify nutrient uptake and transport, among other functions (Thompson et al. 2017). Hindt and Guerinot (2012) proposed a model of the Fe-deficiency response, where auxin, ethylene, and nitric oxide were involved as positive regulators of the Fe acquisition genes FIT, FRO2, and IRT1. A recent study of Lin et al. (2016) proposed sucrose as the upstream signaling molecule of auxins, causing an increase in auxin and a subsequent increase in nitric oxide, ultimately causing FIT-mediated transcriptional regulation of FRO2 and IRT1 genes and inducing Fe-uptake. In this study, soybean plants allocate greater amounts of sugars (mainly glucose and frutose) to root and leaf tissues during exposure to eCO 2 conditions, which might cause an imbalance in carbon and nitrogen metabolism. Therefore, Fe-deficiency signals with sugar variation induced by eCO 2 conditions might act in concert and affect Fe-uptake mechnisms. Besides, if these genes are regulated by the increase of sugars, then it stands to reason that an increase in sugar content in roots mediated by exposure to eCO 2 might also induce Fedeficiency induced responses. The levels of organic acids increased at eCO 2 in roots, and remained almost unchanged in leaves. The increased levels of carbohydrates and organic acids in plants exposed to eCO 2 indicates a higher energy condition capable of maintaining the greater carbon allocation (Jauregui et al. 2015). Moreover, Pavlovic et al. (2013) demonstrated that increased concentration of citrate and malate in the root tissues of cucumber was also important in alleviating Fe deficient responses. Since eCO 2 and sugars increase plant root growth, it was expected greater uptake of nutrients, thus alleviating nutrient deficiencies, however, there are other mechanisms that are affected by eCO 2 that lead to nutrient deficiencies (Thompson et al. 2017). The influence of eCO 2 and Fe-deficiency, on plant mineral concentrations, were also addressed. The roots and leaves were differentially affected by eCO 2 and Fe-supply, as described in Figures 5 and 6. Overall, eCO 2 increased K, P, Mn, Zn, and Fe concentrations in leaves of Fe-sufficient plants, and P and Zn in Fe-deficient plants but did not affect the other minerals. In roots of Fe-sufficient plants, the mineral concentrations were not affected by eCO 2 , whereas in Fe-deficient plants, eCO 2 increased P and Zn, and Ca, Mn, and Fe remained unchanged. Consequently, our results suggested that eCO 2 can induce the accumulation of minerals particularly in soybean leaves and at Fe-sufficient conditions probably indicating that the mineral uptake and upward transport by soybean roots was not restricted. As also reported by others (Haase et al. 2008;Jin et al. 2009;Guo et al. 2015) CO 2 enrichment could considerably increase the mineral uptake by the roots promoting the transport from soil to leaves. Also, it is recognized that increase in biomass at eCO 2 changes the demands for nutrients (Seneweera 2011). In this study, with a consistent supply of nutrients under flooded conditions, the bioavailability was relatively higher to maintain that nutrient supply under eCO 2 . Thus, the increase in the root/shoot ratio, mainly in Fe-sufficient plants, under eCO 2 can promote nutrient uptake and possibly lead to higher nutrient concentrations under eCO 2 conditions. Also, we found a decrease in K (roots) and Mg (roots and leaves) in Fe-deficient plants. This reduction could be attributed to the unbalanced mineral translocation from the roots to the leaves or by differential mineral uptake.
The adaptation of soybean plants to Fe-deficiency under eCO 2 conditions was also demonstrated by evaluating the expression of genes involved in Fe-uptake and the activity of root FCR. It was reported in tomato and Arabidopsis, that the FER gene is upregulated by Fe-deficiency and encodes a root-specific bHLH transcription factor regulating the expression of some Fe-responsive genes, such as the ferric chelate reductase FRO2 and the transporter IRT1 (Connolly et al. 2003Jin et al. 2009Paolacci et al. 2014). In the present study, we found typical Fe-deficiency responses in soybean plants grown with different Fe-supplies at eCO 2 under controlled conditions. In agreement with previous findings (Jin et al. 2009), we found that FCR activity increased in response to Fe-deficiency. Moreover, DMT1, FER, FRO2, and IRT1 gene expression levels were also upregulated by Fe-deficiency, and this phenomenon usually increased by exposure to eCO 2 (Figure 7). Graziano and Lamattina (2007) argued that these are crucial Fe-deficiency responses, but they are not sufficient to significantly increase Fe concentration in Fe-deficient plants ( Figure 6). Furthermore, expression of the ferritin protein gene increased in Fe-sufficient plants, particularly at eCO 2 conditions. Iron homeostasis is strongly dependent on ferritin expression levels produced in response to Fe as previously demonstrated (Wei and Theil 2000;Arnaud et al. 2006;Briat et al. 2010). Fe homeostasis has to be precisely controlled, to prevent starvation that impairs the metabolism, and to avoid excess that may lead to cell death (Arnaud et al. 2006). Therefore, at eCO 2 conditions Fe-sufficient plants have more Fe content in their roots ( Figure 6), and this could be a strategy (i.e. increasing ferritin expression) that plants use to control the Fe excess.

Conclusion
We have demonstrated that changes in CO 2 concentration and Fe-supply can induce a set of morphological, physiological, and molecular responses in soybean plants. CO 2 enrichment has a decisive influence on the adaptation to Fedeficiency in soybean plants. According to our results, biomass accumulation was significantly increased by eCO 2 irrespective of Fe-supply. Therefore, our results suggest that eCO 2 alleviated symptoms of Fe-deficiency and might support biomass increase in plants grown in calcareous soils, at least during plant's earlier developmental stages, which is usually the most sensitive to Fe-deficiency. However, it would be interesting to replicate the responses under eCO 2 of soybean plants grown on alkaline and calcareous soils which can induce Fe-deficiency chlorosis. Improved acquisition of several minerals under sufficient Fe-supply may suggest that eCO 2 can support the unbalanced translocation of minerals to the leaves. eCO 2 also mitigates the Fedeficiency-induced chlorosis and enhances Fe-acquisition mechanisms but they are not sufficient to increase the Fe-status of plants grown in Fe-deficient conditions. It will be meaningful to understand if these observed effects will be sustained under a longer-term exposure and what will be the reported influence of eCO 2 on nutritional value of soybean grains.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Data availability statement
The data that support the findings of this study are available from the corresponding author, José C. Soares, upon reasonable request.

Funding
This work was supported by National Funds from FCT -Fundação para a Ciência e Tecnologia through project UIDB/50016/2020 and by FCT project PTDC/AGR-PRO/3972/2014 The authors gratefully acknowledge the International PhD Programme in Biotechnology, Portugal for providing  to José Carvalho Soares.