In₂O₃-NPs were purchased from American elements¹. In₂O₃-NPs are highly pure (99.99%), yellow spherical nano-powder with a specific surface area of 50 m²g^–1 and a bulk density of 7.18 g/cm³. In₂O₃-NPs size was less than 50 nm. These morphological features were validated (scanning electron microscope, SEM, JEOL JSM-6510). To avoid coarse aggregation of In₂O₃ in aqueous solution, NPs were sonicated.

To better understand the effects of In₂O₃-NPs on plants, this study was performed with conditions that approach environmentally realistic conditions. The seed of Sorghum bicolor (L. cv. Giza 3) (C4) and Triticum aestivum L., cv. Giza 155 (C3) were obtained. They are a commercial high yielding hybrid variety grown in large areas of Egypt, moreover, this variety showed moderate tolerance to heavy metal stress, so they have been used as a model. Healthy seeds were sterilized in a solution of Na-hypochlorite (5% v/v, 25 min) and washed thoroughly with distilled water. The sterilized seeds were grown in 15 cm diameter, 30 cm height polyvinyl chloride (PVC) tubes containing sandy soil (90%) sand at an adjusted pH 7.6. The soil initially contained 1.46% carbon, 23.6 mg nitrate-nitrogen (N), 1.25 mg ammonium-N, and 15.8 mg phosphorus kg^–1 air. The soil was daily watered to adjust the water soil capacity at 68%. Plants were grown in sunlit CO₂ and temperature-CO₂-controlled chambers. The top of sunlit chambers consisted of a transparent polycarbonate plate to allow light. The temperature was adjusted at 26/20°C (day and night) and the Photosynthetic Active Radiation (PAR) was measured by a SDEC, type JYP1000 quantum sensor. The healthy seeds of C3 (wheat) and C4 (Sorghum) were grown under four growth conditions: (1) current CO₂ (aCO₂, 385 ppm); (2) aCO₂ + In₂O₃ (250 mg/kg soil); (3) future climate CO₂ (eCO₂, 665 ppm); and 4) eCO₂ + In₂O₃ (250 mg/Kg soil). After 7 weeks of growth, the rhizosphere soil as well as plant samples, i.e., [1, 2, 3, and 4th leaf (old tissues) and 5, 6, and 7th leaf (young tissue)] were collected and kept in −80°C for further analysis. The fresh and dry weight of roots and shoots were determined.

Photosynthetic rate (μmol CO₂ m^–2 s^–1) (Asat, the light-saturated leaf-level) was measured by LI-COR LI- 6400, LI-COR Inc., following the method described by Miner et al. (2017). The photosynthetic-related enzyme [e.g., ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCo)] was measured according to Sulpice et al. (2007).

Sugars were extracted in 50 mM TAE buffer pH 7.5 containing 0.02% Na-azide, 10 mM mannitol, 0.15% polyclar, 12 mM NaHSO₃, 1 mM mercapto-ethanol, and 2 mM PMSF. The extracts were centrifuged at 15,000 g and 4°C, for 10 min. Part of the extract was heated for 5 min at 90°C and, after cooling and centrifugation at 14,000 g (4°C and 5 min), the supernatants were transferred to mixed bed Dowex column of 300 μl Dowex H⁺, 300 μl Dowex Ac^–; both 100–200 mesh. After elution with ddH₂O, glucose, fructose, sucrose, and raffinose concentrations were measured (HPAEC-PAD) (Vergauwen et al., 2000). Non-heated supernatant was used for measuring the activity of the key sugar enzymes (Madany and Khalil, 2017). The activity of the invertase enzymes was measured In TAE buffer with 100 mM sucrose. Reaction mixtures were incubated at 30°C, and the reactions were stopped using an aliquot for 5 min in a water bath at 90°C. Glucose and fructose concentrations were measured as described above.

The activity of sucrose phosphate synthase (SPS) was measured in 1 ml of HEPES buffer at pH 8 contained UDP- glucose and mM fructose-6-phosphate at 37°C for 20 min and stopped by adding NaOH (30%). The reaction mixture containing citrate and glycogen initiated the starch synthase (Nishi et al., 2001). A 0.2%v starch solution containing 0.05% of I2/KI (w/v) in 0.05% HCl was used to detect amylase activity at 620 nm (Madany and Khalil, 2017).

Amino acids were extracted in 2 ml of 80% (v/v) aqueous ethanol, spiked with norvaline as internal standard, and the homogenate was centrifuged at 14,500 rpm for 18 min. The supernatant was vacuum evaporated, and the pellet resuspended in 1 ml chloroform. Simultaneously, residue was re-extracted with 1 ml HPLC-grade water and, after centrifugation at 14,000 rpm for 20 min, the supernatant was mixed with the pellet suspended in chloroform. The combined extract was centrifuged for 10 min at 14,000 rpm and the aqueous phase was filtered through Millipore micro filters (0.2 μM pore size) before assaying amino acids levels. All amino acid individuals were determined by using a Waters Acquity UPLC-tqd system equipped with a BEH amide 2.1 × 50 column (Sinha et al., 2013).

The activities of key enzymes involved in proline metabolism, i.e., pyrroline-5- carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), pyrroline-5-carboxylate dehydrogenase (P5CDH), proline dehydrogenase (ProDH), ornithine amino transferase (OAT), and arginase (Arg), were extracted [Tris-HCl buffer (50 mM, pH 7.4)] (Shabbaj et al., 2021b). The activities of OAT and ARG were assessed in KPO4 buffer (50 mM KPO4 buffer pH 7.0). The activities of all enzymes (P5CS, P5CDH, PRODH, OAT, GS, and ARG) were measured by monitoring the reduction of NADH at A340 (Shabbaj et al., 2021b), the P5C-dependent NADH oxidation at A340 (Lutts et al., 1999), production γ-glutamyl hydroxamate at A535 (Temple et al., 1996), the reduction of 2,6-dichloroindophenol at A600 (Chen et al., 2004), the production by using the method of diacetyl monoxime described by Chen et al. (2004), and the glutamine-dependent NADH oxidation at A340 (Robinson et al., 1991). By using BSA as a standard, the protein concentrations were measured as assessed by Lowry et al. (1951).

Fatty acids were quantified using GC mass (Madany et al., 2020), where a weight of 0.5 g of LN-fine powdered plant leaves were extracted in aqueous methanol at 27°C. The GC/MS analysis was performed (Hewlett Packard 6890, MSD 5975 mass spectrometer, Hewlett Packard, Palo Alto, CA, United States) with HP-5 MS column of 30 m × 0.25 mm × 0.25 mm. The fatty acids were calculated from the standard curve using the analyte/internal standard ion yield ratios.

Plant samples were extracted in cold perchloric acid (Bregoli et al., 2002). After centrifugation for 30 min at 14,000 g, we applied dansyl chloride to derivatize the samples supernatants and standards in order to detect free polyamines. After hydrolyzation at 110°C overnight, in 6 N HCl was added for the formation of conjugated polyamines. The reverse phase HPLC (Shimadzu SIL10-ADvp; C18 column) was used to measure the concentrations of dansylated polyamine derivatives. For measuring the activities of polyamine related enzymes, plant shoot and roots were homogenized in 100 mM KPO4, pH 7.5, and they were centrifuged for 20 min at 25,000 RPM. Ornithine decarboxylase enzyme (ODC) and arginine decarboxylase (ADC) enzyme activity were measured by measuring the labeled CO₂ (Birecka et al., 1985), which was liberated from L-[l-14C] Ar (55 mCi mmol^–1) and L-[l-14C] Orn (55 mCi mmol^–1). The trapped radioactivity was measured in a liquid scintillation counter. The activity of spermidine synthase (SpdS) was assayed in a reaction mixture of pH 8.0 Tris-HCl (0.1 M) (Yoon et al., 2000). The production of 5′-deoxy-5′-methylthioadenosyne was quantified by using a fluorescence detection method (a reverse phase HPLC equipped with a fluorescence detector). Spermine synthase (SpmS) activity was measured by the production of methylthioadenosine (Cason et al., 2003).

All results were expressed as the mean of five biological replicates (n = 5). Statistical analysis was performed using one-way ANOVA in the SPSS 22 (Tukey test, P ≤ 0.05). Data normality was checked by using Levene’s test. Hierarchical cluster analysis Euclidean distance was performed using the R stat software package (version 4.5.0, the R).

Previously, it was proved that indium adversely affects the growth and development of higher plants (Syu et al., 2017). The treatment of C3 plants with In₂O₃-NPs reduced biomass accumulation (FW, DW), photosynthesis, and RuBisco activity in young leaves by about 30, 14, 50, and 65%, respectively, in comparison to control plants (Figures 1A–D). Like C3 plants, old leaves of C3 plants treated with In₂O₃-NPs reduced biomass accumulation (FW and DW), RuBisco activity (by about 20%), and photosynthesis (by 40%) as compared with control plants. Additionally, the young leaves of C3 plants suffered from a remarkable reduction in their FW, DW, photosynthesis, and RuBisco (60, 50, 60, and 80% reduction) in response to treatment with In₂O₃-NPs. On the other hand, extreme levels of CO₂ were reported to alleviate the adverse effects of different environmental conditions (Shabbaj et al., 2021a). In this context, the exposure of C4 plants to eCO₂ significantly augmented the biomass (FW and DW) as well as the photosynthetic related parameters (photosynthesis and RuBisco). A further enhancement in the biomass was observed in the old leaves of C4 plants in which the increment reached 40 and 100% in FW and DW, respectively (Figure 1). Similarly, a noticeable improvement in the FW and DW as well as the photosynthesis and RuBisco activity was observed upon treatment with eCO₂ either alone or in combination with In₂O₃-NPs as compared with their counter control (Figure 1). Moreover, elevated levels of CO₂ caused a slight increase in the biomass and photosynthesis of young leaves C3 plants (∼10–20% increase) as compared to their respective controls. Consistently, CO₂ enrichment was found to augment the FW, DW, photosynthesis, and RuBisco activity of C4 plants (at the young leaves), that reached up to 40% when compared to their counter control plants.

In₂O₃-NPs significantly retarded the growth of both young and old leaves of C3 and C4 plants. The observed decreases in wheat and sorghum growth may be ascribed to the negative impact of In₂O₃-NPs on the vital biological activities such as cell division, nutrient uptake, photosynthesis, and respiration (Rastogi et al., 2017). This harmful effect could be due to the potency of In₂O₃-NPs to penetrate the cell and elicit molecular and cellular events in the cell (Buzea et al., 2007) including an increase in the cell wall rigidity, diminishing the cell wall growth, and finally cell rupturing (Kopittke et al., 2009). In line with our findings, the photosynthesis of wheat and maize showed a remarkable retardation after treatment with NiO and HgO nanoparticles (Saleh et al., 2019; AbdElgawad et al., 2020). Also, in nanoparticles strikingly less nutrient uptake in rice plants was observed due their adverse effect on the nutrient translocation to the shoots (Syu et al., 2017).

Besides being a prospective tool that enhances plant growth (Shabbaj et al., 2021a), eCO₂ reduced the hazardous impact of In₂O₃-NPs. This alleviative impact of eCO₂ was demonstrated in the recovery of plant growth and photosynthesis (Figure 1). Generally, C4 plants respond better to eCO₂ at growth level. Consistently, photosynthetic efficiency was highly augmented in In₂O₃-contaminted old and young leaves In line with our findings, eCO₂, within the physiological range, caused a remarkable restoration for the crops’ growth by enhancing their photosynthetic carbon assimilation and, accordingly, carbohydrate partitioning (Shabbaj et al., 2021a). To improve photosynthesis machinery, eCO₂ is known to upregulate the RuBisco activity in different plant species under NPs stress conditions (AbdElgawad et al., 2020; Selim et al., 2021a). Increased CO₂ substrate availability for RuBisco instantaneously inhibits photorespiration (Dusenge et al., 2019).

Studies investigating C3 and C4 species responses to climate conditions have so far mostly addressed changes in biomass, photosynthesis, and nutrient relations (Drake, 2014). Comparatively, much less is known about the effects of In₂O₃-NPs and/or eCO₂ and associated heavy metal NPs stress in C3 and C4 at the carbon and nitrogen metabolism level. Like other heavy metal nanoparticles, In₂O₃-NPs threaten plant growth by retarding vital metabolic processes such as carbon and nitrogen partitioning (Rahoui et al., 2015). Our results revealed a remarkable alternation in carbon and nitrogen assimilation in both sorghum and wheat, particularly in young leaves. For instance, a reduction in starch level and an elevation of sucrose metabolizing enzymes was observed. It is well known that soluble sugars such as glucose and sucrose are the most pivotal structural and energetic organic compounds in higher plants (Shabbaj et al., 2021b). Besides being osmoregulators, sugars possess obvious ROS scavenging properties (Van den Ende, 2013).

Photosynthesis products and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism (Ainsworth and Rogers, 2007). For instance, sugars are involved in the organic acids’ biosynthesis. Here, growing of C3 and C4 plants under In₂O₃-NPs accumulated organic acids, particularly in young leaves of C4, indicated their role in reducing uptake by plant roots and restricting their translocation to leaves (Panfili et al., 2009). Organic acids also act as stored pools of fixed carbon (Igamberdiev and Eprintsev, 2016), and as a bridge to connect C and N metabolism together such as N assimilation and amino acid biosynthesis (Raven and Farquhar, 1990). In addition to organic acid intermediates, amino acids biosynthesis also needs energy derived from sugar catabolism during dark respiration (Bolton, 2009). In turn, the accumulated amino acids, e.g., proline, improved plant tolerance to environmental challenges (Shabbaj et al., 2021b). Several amino acids can also act as substrate to produce polyamines, for instance the diamine Put is synthesized directly from ornithine by ODC or from arginine by ADC (Pal Bais and Ravishankar, 2002). Polyamines retrieve the different environmental abiotic stresses, including metal and metalloid stresses in plants (Hasanuzzaman et al., 2019). Besides being essential for plant growth and development, polyamines are crucial in maintaining the integrity of nucleic acids and ensuring their translocation (Cason et al., 2003). Sugars also provide substrates for de novo fatty acid biosynthesis (Zhai et al., 2021). Under environmental stress conditions, fatty acids mainly contribute to maintaining structural integrity and supplying energy for several plant metabolic activities. This is consistent with the elevation in the levels of tocopherols that scavenge ROS to preserve polyunsaturated fatty acids from lipid peroxidation (Krieger-Liszkay and Trebst, 2006). Contamination with In₂O₃-NPs caused a noticeable reduction in the levels of fatty acids, especially in young leaves of both C3 plants.

On the other hand, maintaining the photosynthetic machinery in stressed plants under eCO₂ is very essential to maintain a continuous supply with non-structural carbohydrates that play a crucial role in enhancing plant tolerance with the continuous supply of amino, organic, and fatty acids (Thompson et al., 2017). eCO₂ in combination with In₂O₃-NPs significantly reduced the sucrose levels with a concomitant increase in starch levels. In parallel with this finding, a noticeable reduction in the activities of sucrose-P-synthase in both C3 and C4 plants, particularly the young leaves of C4 plants, was observed. The accumulation of soluble sugars rather than storage sugars could proclaim the retrieval of the osmotic imbalance triggered by In₂O₃-NPs. In line with our results, the recovery of tomato leaves from salt stress was accompanied by low levels of sucrose and hexose with concomitant elevation in starch levels (Khelil et al., 2007).

To reduce In₂O₃-NPs uptake by plant, soluble sugars may be consumed under eCO₂ to produce organic acids through respiration. Here, succinate and fumarate levels were induced, particularly in the old leaves of C3 plants, when treated with eCO₂ under In₂O₃-NPs. Increased levels of succinate and fumarate can be explained by the ability of eCO₂ to boost succinate dehydrogenase activity (Igamberdiev and Eprintsev, 2016). eCO₂-induced organic acids’ accumulation contributes to manipulating the energy and redox status in the cell, therefore supplying the cell with the required precursors for the amino acids’ biosynthesis. Besides being essential for protein biosynthesis, amino acids are also involved in a wide array of osmolytes to maintain cellular turgor pressure (Suprasanna et al., 2016). Notably, eCO₂ provoked the levels of glutamate and proline in both C3 and C4 crops, particularly young leaves of C3 plants, under In₂O₃-NPs contamination.

Another stress related amino acid, proline, is a crucial osmoregulator that enhances cell integrity and maintains cellular structure and enzymatic activity (Madany et al., 2020). A rate limiting enzyme denoted as P5CS can modulate proline biosynthesis from either ornithine or glutamate pathways. eCO₂ clearly triggered both glutamine and ornithine pathways in old leaves and to a higher extent in young leaves of wheat In₂O₃-NPs stressed plants, however, ornithine pathway seems to be more abundant in both old and young leaves of C4 plants equally. This enhancement in the activities of proline biosynthetic enzymes was concomitant with a remarkable reduction in the activities of biodegrading enzymes (PRODH), particularly in young leaves of C4 plants.

One of the mechanisms involved in the stress mitigation impact of eCO₂ is the biosynthesis of polyamines. Of note, the aforementioned polyamines have been proven to scavenge free radicles in vitro (Sengupta et al., 2015). Under stress conditions, polyamines can also act as signaling molecules that can modulate the ion homeostasis and consequently organize the ion transportation by interacting with the ion channels (Podlešáková et al., 2019). They also play a pivotal role in diminishing ROS accumulation and preventing lipid peroxidation, hence stabilizing the membrane (Liu et al., 2007; Spormann et al., 2021). In this context, Quartacci et al. (2001) found that the oxidative damage caused by Cd²⁺ or Cu²⁺ was ameliorated by the exogenous application of Spm upon wheat leaves by maintaining the membrane integrity via stabilizing lipids and avoiding solute leakage.

The co-application of future climate eCO₂ with In₂O₃-NPs strikingly enhanced the accumulation of fatty acids, particularly unsaturated fatty acids (18:1, 18:2, and 18:3), in young leaves of both C3 and C4 crops. The increased levels of oleic acid (18:1) could contribute to restoring the plant defense signaling (Nandi et al., 2003). In addition, the elevation in polyunsaturated fatty acids and tocopherols levels in response to eCO₂ is a good sign of membrane stability under In₂O₃-NPs pollution. On the other side, fatty acids provide several plant metabolic processes with energy and structural integrity, and so play a crucial role in regulating signal transduction under different environmental challenges.

To get a better view on the differential responses to the effect of In₂O₃-NPs stress and eCO₂, we applied principal component analysis (PCA) using all measured nitrogen-related metabolites (Figure 6A) and carbon-related metabolites (Figure 6B). At nitrogen metabolites level, principal component 1 (PC1) and principal component 2 (PC2) explained 57 and 18% of the total variance, respectively. However, at carbon metabolites level, principal component 1 (PC1) and principal component 2 (PC2) explained 39 and 21% of the total variance, respectively. The PCA shows a clear separation in Cr treatment with PC1. In general, the PCA analysis revealed a clear separation of the two treatments of eCO₂ (either alone or in combination with In₂O₃-NPs) on development in the two plant species in either old or young leaves (Figure 6A). At nitrogen metabolism level, eCO₂ clearly induced more levels of amino acids and polyamines under control or stress conditions, which explain 57% of the variation (Figure 6A, PC1). Moreover, the old leaves of both C3 and C4 plants showed more specific responses, particular under the control conditions. Similar, the second PCA (Figure 6B) showed that the effect of ambient CO₂ on carbon metabolism was lower compared to elevated CO₂ levels. The main drivers for PC1 (39%) were unsaturated fatty acid and soluble sugars.

For species specific responses, we found the C4 data were clustered together and separated from C3 data across PC2 (21%, Figure 6B). The pattern was more diffuse for leaf types. For instance, our PCA analysis declared that the increment in sucrose was species dependent as its level was increased in both old and young leaves of C4 plants in response to eCO₂ under In₂O₃-NPs treatment. At the growth level, fewer reductions were observed for C4, indicating that C4 crops are more tolerant to heavy metal stress than C3 crops (Srivastava et al., 2012). This enhanced tolerance of C4 plants could be attributed to the efficient photosynthetic activity and so enhance growth rates as compared to C3 crops (AbdElgawad et al., 2020). Together this suggests that the primary processes affected by elevated CO₂ under abiotic stress conditions may not be identical in young and old leaves of both C3 and C4 species groups.