We used seeds of Persea americana Mill. cv. Hass, a cultivar bred in California from Mexican-Guatemalan ancestry and currently cultivated worldwide. The fruits (origin Aconcagua basin, Chile) were bought from a local fruit shop. The seeds were planted in 2 L clay pots and grown at the plant growth room of the Estonian University of Life Sciences under the following conditions: light intensity of 500-700 μmol m^-2 s^-1 with day/night temperatures of 28/24 °C for a 12 h light period and 60% relative humidity. The growth substrate was a commercial organic garden soil and a mixture of peat and sand (0–2 mm, Bauhof, Tartu, Estonia). The plants were watered every other day to soil field capacity and fertilized with 10 g slow-release fertilizer with microelements (N:P:K = 14:11:25, AS Baltic Agro, Tartu, Estonia) once per month. The seedlings were grown under these conditions until they were 50–70 cm tall and had utilized all seed reserves. Before the start of CO₂ treatments, the plants were transplanted into 5 L pots and the foliated parts of the plants were removed leaving ca. 5 cm long stems. Subsequently, the plants were transferred to the plant growth chamber (FITOCLIMA S600PLLH, Aralab, Lisbon, Portugal) to either ambient (400 μmol mol^-1) or elevated [CO₂] (800 μmol mol^-1). Chamber conditions were consistent with those in the plant growth room, except that light intensity was maintained at 1000 μmol m^-2 s^-1. The plants were watered every other day to the soil field capacity and fertilized in the beginning and in the middle of the growth period (see Abiola et al., 2025a for details of the growth conditions). At the time of the experiment, the plants had approximately 30–40 leaves and were 80–100 cm tall, after a growth period of four months. In this experiment, new leaves were formed continuously, and the plastochron (interval between the formations of successive leaves) was 11–12 days. Fully expanded young-mature leaves were ca. 60–70 days old, and old-mature non-senescent leaves were ca. 110–120 days old. We estimated that the available C and N stored in the root system could have been responsible for less than 10% of new growth, and thus, most of the above-ground biomass developed after removal of above-ground plant parts and transfer of plants to the new growth environment resulted from de novo carbon fixation and nutrient uptake from soil.

A custom-made open gas-exchange system was used to measure the foliage gas-exchange rate (Copolovici and Niinemets, 2010 for the full description of the system). The measurement system was specifically designed for simultaneous measurement of photosynthesis, transpiration, and volatile organic compound (VOC) emission rates, with all components constructed from glass, stainless steel, and Teflon^®. The 1.2 L measurement chamber was made with double glass walls. Water with preset temperature circulated between the chamber walls to regulate the chamber temperature (Copolovici and Niinemets, 2010). The CO₂ and H₂O vapor concentrations at the chamber inlets and outlets were measured with an infra-red dual-channel gas analyzer operated in differential mode (CIRAS III, PP-Systems, Amesbury, MA, USA).

After the measurement leaf was enclosed in the chamber, following standard conditions were established: light intensity at the leaf surface of 700 μmol m^-2 s^-1, chamber temperature of 24°C, CO₂ concentration of 390-410 μmol mol^-1 (ambient [CO₂] measurements), relative air humidity 60%, and leaf-to-air vapor pressure deficit of 1.7 kPa. After the steady-state gas exchange rates were achieved, typically between 25–30 minutes after leaf enclosure, differences in CO₂ and water vapor concentrations were recorded. Net assimilation rate (A), transpiration rate (E), stomatal conductance (g _s) and intercellular CO₂ concentration (C _i) were calculated according to von Caemmerer and Farquhar (1981). The apparent maximum Rubisco carboxylase activity (V _cmax) was computed according to Niinemets et al., 1999 as explained in detail in De Kauwe et al. (2016). In these calculations, average Rubisco kinetic characteristics for “warm” C₃ species from Galmés et al. (2016) were used. As C _i was used as the substitute for chloroplastic CO₂ concentration, the estimates of V _cmax provide an estimate of foliage photosynthetic activity without stomatal effects, but they do not consider possible variation in mesophyll conductance.

Before the heat treatment, steady-state values of A and g _s were recorded under standard conditions (24 °C). Heat stress was applied by immersing the sample leaves in distilled water at 48 °C for 10 min according to the procedure detailed in previous studies (Copolovici et al., 2012; Kask et al., 2016; Okereke et al., 2022). During immersion, the leaves were enclosed in a chemically inert polyester bag to avoid direct contact with water. The control treatment consisted of leaf immersion in distilled water at 25°C for 10 min. A controlled temperature water bath (VWR International, West Chester, Pennsylvania, USA) was used to maintain a highly stable temperature of the immersion medium. We choose this procedure as it does not depend on stomatal effects that can alter leaf temperature at given air temperature due to transpiratory cooling. We have demonstrated that the heat stress applied this way is highly repeatable and the severity of heat stress scales quantitatively with photosynthetic reduction, volatile emission and ion leakage (e.g., Okereke et al., 2022; Sulaiman et al., 2023).

In these experiments, four replicates of young-mature and old-mature leaves from different plants were used. Foliage gas-exchange measurements were conducted at 0.25, 1, 3, 24, and 48 h after the treatment under the standard conditions (chamber temperature of 24 °C).

Data of nitrogen (N _M), carbon (C _M), and phosphorus (P _M) content per dry mass used in this study are those from (Abiola et al., 2025a). Fresh leaves were scanned at 300 dpi and leaf area was measured using ImageJ 1.8.0 (NIH, Bethesda, Maryland, USA). Leaf dry mass was estimated after oven-drying at 70°C for 48 h, and leaf dry mass per unit area (M _A) was calculated.

The measurements of control and heat-treated plants were conducted in four replicate plants (n = 4), with one young-mature and one old-mature leaf measured per plant, using different plants grown under ambient and elevated [CO₂]. The data were presented as averages ± SE. The degree of heat stress recovery of gas exchange characteristics for each plant age groups was calculated as:

where R _C,i is the relative change of trait i and x _{i,before stress} and x _{i,after stress} the trait values assessed before and after stress application (Liu et al., 2022). Linear mixed models (LMM) with heat stress and [CO₂] levels as fixed effects and time as a random effect were used to test the effects of individual and interactive effects of treatments and [CO₂] on gas exchange characteristics through the stress recovery time. Tukey’s honestly significant difference (HSD) test following one-way ANOVA was used to compare averages at different heat stress recovery time points. Two-way analyses of variance with leaf age and [CO₂] as main effects and leaf age x [CO₂] interaction were used to estimate the global effects of growth [CO₂], leaf age and their interaction. All statistical analyses were conducted with R statistical software ver. 4.2.0 (2021) and visualized with OriginPro 2018 (OriginLab Corporation, Northampton, USA). The data used for ANOVA and LMM were tested for normality of distribution (Kolmogorov-Smirnov test), and when necessary, the data were log-transformed to improve the normality of data. All statistical effects were considered significant at P < 0.05. All datasets analyzed during this study are available in the EMU DSpace repository (Abiola et al. 2025b).

In Persea americana, the net assimilation rates ranged from 6.42 to 8.61 µmol m^-² s^-¹, with the lowest value observed in mature leaves under ambient [CO₂] and the highest in young leaves under elevated [CO₂] ( Figure 1 ). The net assimilation rate (A) in unstressed young-mature leaves was 14% greater in elevated [CO₂]-grown plants compared to ambient [CO₂]-grown plants, indicated by a significant [CO₂] effect on (P < 0.05), whereas A was similar between old-mature leaves, regardless of growth [CO₂] ( Table 1 ). A similar A measured suggest photosynthetic downregulation in high-[CO₂]-grown plants, and this was supported by lower leaf nitrogen content per dry mass (N _M) and leaf phosphorus content per dry mass (P _M) under elevated [CO₂] (Abiola et al., 2025a) for the effect of growth [CO₂] on elemental contents). The apparent (C _i-based) maximum carboxylase activity of Rubisco (V _cmax) was higher in young-mature leaves under elevated [CO₂] (mean ± SE = 51.95 ± 2.19) compared to young leaves under ambient [CO₂] (42.38 ± 1.8, P < 0.001, Table 1 ). However, V _cmax was similar in old-mature leaves under both [CO₂] conditions (P > 0.05, Table 1 ) despite reduced nutrient content (Abiola et al., 2025a) for the effect of growth [CO₂] on elemental contents). For intercellular CO₂ concentration (C _i), young-mature leaves under elevated CO₂ exhibited significant main effects of growth [CO₂] (mean ± SE = 175.02 ± 9.55) compared to ambient young-mature (205.15 ± 9.84, P < 0.001, Table 1 ). Again, old-mature leaves exhibited no C _i difference between [CO₂] treatments (P > 0.05; Table 1 ).

Meanwhile, in both growth [CO₂], A decreased with leaf age (P < 0.05 in ambient [CO₂] and P = 0.001 for elevated [CO₂]), whereas the interactive effects of leaf age x [CO₂] were not significant (P > 0.05; Table 2 ). There was no significant difference in V _cmax and stomatal conductance (g _s) between leaf ages in both growth [CO₂]. However, leaf dry mass per unit area (M _A) increased with leaf age, and the interaction between leaf age x [CO₂] was significant (P < 0.05, Table 2 ).

Heat stress (48 °C) led to immediate decreases in foliage photosynthetic characteristics in all cases with full or partial recovery depending on growth [CO₂] and leaf age. The degree of recovery varied between young- and old-mature leaves and dependence on the growth [CO₂] as well ( Figures 1 , 2 ; Table 1 ). Heat shock resulted in decreases in light saturated photosynthesis (A) in both young and old-mature leaves grown under different [CO₂] ( Figures 1A, B , 2A, B , Table 1 ). The degree of reductions in A in all the heat-stressed plants was proportional to reductions in V _cmax ( Figures 1A, B, G, H , 2A, B, E, F , Supplementary Figure S1 , Table 1 ). The application of heat stress uncoupled the relationship between A and g _s ( Figures 1C, D , 2C, D , Table 1 ), and thus, the heat stress-dependent changes in net assimilation rate through recovery were primarily driven by changes in V _cmax (biochemical limitation) for different combinations of leaf age and growth [CO₂] ( Supplementary Figure S1 ).

Reductions of A due to heat stress were the greatest in young-mature leaves of plants grown under ambient [CO₂] ( Figures 1A , 2A , Table 1 ). At the end of the 48 h heat stress recovery period, A, g _s and V _cmax in all the stressed plants recovered fully, except for the young-mature leaves grown under ambient [CO₂] ( Figures 1A–D, G, H , 2A–F ). In heat-stressed young-mature leaves grown under elevated [CO₂], full recovery of A (P > 0.6 between control and at 48 h after heat shock) was accompanied by a greater recovery of g _s and V _cmax and a decrease in intercellular CO₂ concentration (C _i) than in young-mature leaves grown under ambient [CO₂] ( Figures 1A–F , 2A–F ).

Changes in foliage net assimilation rate (A) can occur due to alterations in stomatal conductance (g _s), mesophyll conductance (g _m) and maximum carboxylase activity of Rubisco (V _cmax) (Ethier et al., 2006; Niinemets, 2018). In our study we looked at changes in g _s and apparent V _cmax that does not consider possible differences in g _m. Generally, A decreases in non-senescent leaves with increasing leaf age, but the underlying physiological mechanisms can be species-specific (Ethier et al., 2006; Kositsup et al., 2010; Abiola et al., 2025a). In the current study, A was lower in old-mature leaves in comparison to young-mature leaves. Given the similar nutrient contents in the leaves with varying age in P. americana (Abiola et al., 2025a), the age-related reduction in photosynthetic capacity was not associated with limiting nutrient contents, but might indicate a decrease in mesophyll conductance due to thickening of cell walls (Niinemets et al., 2005, 2006; Onoda et al., 2017). This is plausible given the increase of leaf dry mass per unit area (M _A) with increasing leaf age ( Table 2 ).

Elevated [CO₂] is expected to increase A as the result of enhanced CO₂ availability for photosynthesis (Reich et al., 2018; Zheng et al., 2019; Jiang et al., 2020). However, previous studies have shown that A in plants under elevated [CO₂] can increase, reduce or even remain unaffected (Urban et al., 2012; Abiola et al., 2025a). Unaffected or reduced A is indicative of photosynthetic downregulation (see Introduction). Typically, plants grown under elevated [CO₂] have lower nutrient contents and higher M _A (Sun et al., 2013a; Fleischer and Terrer, 2022; Abiola et al., 2025a). In our study, leaf nitrogen content per dry mass (N _M) and leaf phosphorus content per dry mass (P _M) were higher in both young-mature and old-mature leaves grown under ambient [CO₂] compared to elevated [CO₂]-grown plants (Abiola et al., 2025a). Previous studies have shown that decreases in foliage nutrient contents such as N and P contribute to decreases in photosynthesis under elevated [CO₂]-grown plants (Jifon and Wolfe, 2002; Sanz-Sáez et al., 2013; Reich and Hobbie, 2013; Arrizabalaga-Arriazu et al., 2020). Lower N _M is typically associated with lower photosynthetic capacity per dry mass (A _mass), while the rate of photosynthesis per area (A _area) also depends on M _A, i.e., A _area = M _A x A _mass (Onoda et al., 2017; Onoda and Wright, 2018). Thus, the enhancement of M _A under elevated [CO₂] might compensate for reductions in A _mass. However, as discussed above, a higher M _A can be associated with greater investment in cell walls, which reduces photosynthetic efficiency by lowering N allocation to photosynthetic proteins and decreasing CO₂ diffusion due to low mesophyll conductance associated with thick cell walls (Tosens et al., 2016; Osnas et al., 2018). We argue that further studies should look at elevated [CO₂] effects on mesophyll conductance in different aged leaves.

We demonstrated that reductions of A in all heat-stressed P. americana were primarily due to non-stomatal factors, specifically, reductions in ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity ( Figures 1 , 2 , Supplementary Figure S1 ). Rapid reductions in Rubisco maximum activity upon heat stress might be attributed to thermal inhibition of Rubisco enzymatic activity (Hüve et al., 2011; Kask et al., 2016; Djanaguiraman et al., 2018). However, photosynthesis recovered in all the heat shock-treated P. americana leaves except in young-mature leaves of P. americana grown under ambient [CO₂] ( Figures 1A, E ), likely indicating direct thermal damage of photosynthetic components in these leaves (Hüve et al., 2019; Sulaiman et al., 2023).

Previous studies have associated severe heat stress (>47°C) with rapid reductions in A in several tropical species (Okereke et al., 2021; 2022). The current study showed that severe heat resulted in an immediate decrease in A by ca. 82% in old-mature leaves of P. americana ( Figures 1B , 2B ; Table 1 ), similar to the reductions observed previously in foliage of old-mature tropical herbaceous species including Amaranthus cruentus, A. hybridus, Solanum aethiopicum, Telfairia occidentalis and Vigna unguiculata (Okereke et al., 2021), but less than the reductions observed in the foliage of the tree species Carica papaya (Okereke et al., 2022). Also, unlike in C. papaya (Okereke et al., 2022), A in old-mature P. americana recovered completely after severe heat stress application ( Figures 1B , 2B ), suggesting greater heat stress tolerance of old-mature P. americana leaves. The recovery of old-mature leaves under ambient [CO₂] indicates enhancement of photosynthetic apparatus tolerance of heat-stressed plants. As suggested in the Introduction, such an enhancement in heat resistance with increasing leaf age might have multiple causes, including greater antioxidative capacity, enhanced repair capacity and enhanced formation of stress-protective compounds, partly as the result of greater physiological activity (Albert et al., 2018; Krause et al., 2014; Drake et al., 2018; Tarvainen et al., 2022). However, in young-mature leaves of P. americana grown under ambient [CO₂] ( Figures 1A , 2A ), photosynthesis did not recover (<78%) after severe heat stress application, suggesting the photosynthetic sensitivity of the young-mature leaves.

Both young and old-mature leaves grown under elevated [CO₂] exhibit greater heat resistance than those grown under ambient [CO₂], as evidenced by their enhanced recovery to control conditions following heat shock ( Figures 1A, B , 2A, B ). This demonstrate that increased leaf age and elevated [CO₂] independently and synergistically improved heat shock tolerance and recovery of photosynthetic activities in P. americana. Typically, heat protection of photosynthesis by elevated [CO₂] is associated with higher concentration of sugars in leaves under elevated [CO₂] (Taub et al., 2000; Zhang et al., 2019). This is because of greater photosynthesis rate under elevated [CO₂], and nutrient limitation of growth (lower sink activity) (Leakey et al., 2009; Habermann et al., 2019).

In conclusion, our study demonstrates that P. americana is a relatively heat tolerant species. Nevertheless, under transient heatwaves at current ambient [CO₂], photosynthesis of young leaves is expected to be strongly reduced. Increases in ambient [CO₂] are expected to improve the heat resistance of young leaves and thus, elevation in [CO₂] might improve the whole canopy photosynthesis in heat-exposed avocado. Future research should examine effects of heat stress on a wider range of tropical woody species, including impacts on canopy-level carbon gain, reproductive development, and yield under future climate scenarios.