We compared the photosynthetic characteristics of N. tabacum and Eucalyptus camaldulensis Dehnh. The latter is a fast-growing species native to Australia that has been widely introduced into China for forest plantations. For this study, Eucalyptus samples were collected from plants grown in an open field at an elevation of 700 m in Dongchuan County, Kunming City, Yunnan Province, China. The monthly air temperature, total radiation and precipitation were displayed in Figure 1 (data were collected from 1961 to 1980). Seedlings of N. tabacum cv. k326 were cultivated in plastic pots in a phytotron at Kunming Institute of Botany, Yunnan, China. Growing conditions were 24/18°C (day/night), 60% relative humidity, and an atmospheric CO₂ concentration maintained at 400 μmol mol⁻¹. The phytotron used sunlight as the source of illumination, and plants were exposed to approximately 95% of full sunlight (maximum at noon ≈ 1990 μmol photons m⁻² s⁻¹). Photosynthetic parameters were measured in June of 2014. Measurements were made using four mature leaves from four independent plants per species. Fully expanded mature leaves on 13-week-old plants of N. tabacum were used for photosynthetic measurements. For E. camaldulensis, mature leaves that flushed in the spring on 3-year-old plants were used for measurements.

Photosynthetic parameters for gas exchange and chlorophyll fluorescence were monitored with an open gas exchange system that incorporated infrared CO₂ and water vapor analyzers (Li-6400XT; Li-Cor Biosciences, Lincoln, NE, USA) and a 2-cm² measuring head (6400-40 Leaf Chamber Fluorometer; Li-Cor Biosciences). Data were recorded at 25°C and a relative air humidity of 60–70%. The atmospheric CO₂ concentration was maintained at 400 μmol mol⁻¹ by the Li-6400XT. Both g_s and the CO₂ assimilation rate peaked after plants were exposed to saturating light (2000 μmol photons m⁻² s⁻¹) for 20 min. Immediately, light response curves were evaluated at 2-min intervals at different light intensities. For N. tabacum, light response curves were measured at a photosynthetic photon flux density (PPFD) of 2000, 1600, 1200, 1000, 800, 600, 400, 300, 200, 150, 100, 50, or 0 μmol photons m⁻² s⁻¹. For E. camaldulensis, light response curves were measured at a photosynthetic photon flux density (PPFD) of 2000, 1600, 1200, 1000, 800, 500, 300, 150, 100, 50, or 0 μmol photons m⁻² s⁻¹.

The fluorescence parameters F_m′ and F_s were evaluated as previously described (Baker and Rosenqvist, 2004), with F_m′ representing the maximum fluorescence after light-adaption and F_s being the light-adapted steady-state fluorescence. The effective quantum yield of PSII was calculated as Φ_PSII = (F_m′ − F_s)/F_m′ (Genty et al., 1989). The maximum fluorescence after dark adaptation (F_m) was examined after 30 min of dark adaptation following measurement of the light response curve. Non-photochemical quenching was calculated as NPQ = (F_m − F_m′)/F_m′.

Using the data of chlorophyll fluorescence parameters, total photosynthetic electron flow through PSII is calculated as follows (Krall and Edwards, 1992): JT=ΦPSII×PPFD×Labs×0.5 where Φ_PSII is the effective quantum yield of PSII and L_abs represents leaf absorbance. We applied the constant of 0.5 based on the assumption that photons were equally distributed between PSI and PSII.

Using the basic equation of leaf carbon dioxide gas exchange and Rubisco specificity for carboxylation relative to oxygenation (von Caemmerer and Farquhar, 1981; Sharkey, 1988; Walker et al., 2016), the rate of Rubisco carboxylation (V_c) and that of Rubisco oxygenation (V_o) are calculated according to. Vc=An+ Rd1−(Γ*/Cc)Vo=An+ Rd(Cc/2Γ*)−0.5 where A_n represented the net rate of CO₂ assimilation, R_d was the rate of mitochondrial respiration as measured after 30 min of dark adaptation, Γ^* was the CO₂ compensation point in the absence of daytime respiration (Farquhar et al., 1980; Brooks and Farquhar, 1985), and C_c was the chloroplast CO₂ concentration. The electron flow for photorespiratory carbon oxidation can be expressed as: Je(PCO)=4×Vo The NADPH demands from CO₂ assimilation and photorespiration were calculated according to the models of Farquhar et al. (1980). Using the data from gas exchange measurements, we determined the rate of electron transport for NADPH required by carboxylation and oxygenation of RuBP (J_g) as follows (Zivcak et al., 2013; Walker et al., 2014) Jg=4×(An+ Rd)×(Ci+2Γ*)/(Ci-Γ*) where C_i was the intercellular CO₂ concentration. The alternative electron flow was calculated as follows (Makino et al., 2002; Zivcak et al., 2013; Huang et al., 2016):  Ja=JT-Jg

Values for mesophyll conductance (g_m) were estimated through a combination analysis of gas exchange and chlorophyll fluorescence, and according to the following equation (Harley et al., 1992; Loreto et al., 1992; Warren and Dreyer, 2006; Yamori et al., 2010, 2011): gm=AnCi-Γ*(JT+8(An+Rd))/(JT-4(An+Rd)) Using the estimated g_m, we calculated the chloroplast CO₂ concentration (C_c) according to the following equation (Long and Bernacchi, 2003; Warren and Dreyer, 2006; Yamori et al., 2010, 2011): Cc=Ci- Angm The response of net CO₂ assimilation rate to CO₂ concentration was examined at 2000 μmol photons m⁻² s⁻¹ and 25°C. Before A/C_i measurement, leaves were light adapted at 2000 μmol photons m⁻² s⁻¹ and 400 μmol mol⁻¹ CO₂ concentration for at least 20 min to obtain the maximum values of g_s and A_n. Afterwards, the CO₂ concentrations were set to 50 μmol mol⁻¹ and increased stepwise. For E. camaldulensis, CO₂ concentrations were set to 0, 50, 100, 150, 200, 300, 400, 600, 800, 1000, and 1200 μmol mol⁻¹. The CO₂ concentrations in A/C_i measurement in N. tabacum were set to 0, 50, 100, 150, 200, 300, 400, 600, 800, 1000, 1200, 1600, 2000 μmol mol⁻¹. Each stepwise measurement was completed within 2–3 min. Using A/C_i curves, we calculated the maximum rates of RuBP regeneration (J_max) and RuBP carboxylation (V_cmax) according to the method of Long and Bernacchi (2003). To identify the limiting step of CO₂ assimilation, we determined the chloroplast CO₂ concentration at which the transition from RuBP carboxylation to RuBP regeneration occurred (C_trans) as follows (Yamori et al., 2010, 2011): Ctrans= Kc(1+O/Ko)Jmax/4Vcmax-2Γ*1-Jmax/4Vcmax where K_c (μmol mol⁻¹) and K_o (mmol mol⁻¹) were the Michaelis constants for CO₂ and O₂, respectively (Farquhar et al., 1980), and were assumed to be 406.8 μmol mol⁻¹ and 277 mmol mol⁻¹, respectively, at 25°C (Long and Bernacchi, 2003); O was the partial pressure of O₂ and was assumed to be 210 (Farquhar et al., 1980); J_max was the maximum rate of RuBP regeneration; and V_cmax was the maximum rate of RuBP carboxylation. The rate-limiting step for CO₂ assimilation was then determined by comparing the values of C_c and C_trans.

The total amount of ATP demand from Rubisco carboxylation and oxygenation was obtained with the following formula (Walker et al., 2014): vATP=(An + Rd) (3Ci + 3.5Γ*α)(Ci-Γ*) Assuming that the stoichiometry of ATP/NADPH produced by LEF (electron transport from PSII to NADP⁺) is 1.29 (Sacksteder et al., 2000; Seelert et al., 2000; Walker et al., 2014), the amount of ATP produced by LEF was calculated as:  vATP(LEF)=1/2×Jg×1.29 Rates of ATP supply from other flexible mechanisms were determined by subtracting the amount of ATP produced by LEF from v_ATP according to:  vATP(Flex)=vATP-vATP(LEF)

Photosynthetic limitations in E. camaldulensis and N. tabacum were assessed according to the method of Grassi and Magnani (2005) and Carriqui et al. (2015). The values for stomatal (l_s), mesophyll conductance (l_mc), and biochemical (l_b) limitations represented measures of the relative importance of stomatal diffusion, mesophyll diffusion, and photosynthetic biochemistry in setting the observed value of A_n. Relative photosynthetic limitations were calculated as follows (Grassi and Magnani, 2005; Carriqui et al., 2015): ls=gtot/gs×∂An/∂Ccgtot+∂An/∂Cclmc=gtot/gm×∂An/∂Ccgtot+∂An/∂Cclb=gtotgtot+∂An/∂Cc where g_tot was total conductance to CO₂ between the leaf surface and carboxylation sites (calculated as 1/g_tot = 1/ g_s + 1/ g_m).

All results were displayed as mean values of four independent measurements. We used one-way ANOVA and SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) to examine differences between the two species. Those differences were considered significant at P < 0.05.

The A/C_i curves indicated that the maximum rate of photosynthesis was higher in N. tabacum than in E. camaldulensis (Figure 2A). Values for the maximum rate of RuBP regeneration (J_max) and RuBP carboxylation (V_cmax) were significantly higher in N. tabacum (Figure 2B). Both species showed similar ratios of J_max/V_cmax (Figure 1B) as well as the same value for A₄₀₀ at 400 μmol mol⁻¹ CO₂. Because the CO₂ compensation point in the absence of daytime respiration (Γ^*) has an important impact on g_m, C_c, C_trans, and limitations on the stomata (l_s), mesophyll conductance (l_mc), and biochemical functions (l_b), we conducted a sensitivity analysis and examined the rate-limiting step for CO₂ assimilation to Γ^* (range from 30 to 40 μmol mol⁻¹). For E. camaldulensis, l_mc was the most important constraining factor for photosynthesis, followed by l_s and l_b (Figure 2C). By contrast, biochemical limitations were the most significant in N. tabacum, followed by l_mc and l_s (Figure 2C). Furthermore, irrespective of Γ^*, the value of C_c at 400 μmol mol⁻¹ CO₂ was significantly lower than C_trans in E. camaldulensis (Table 1), suggesting that the rate-limiting step of A₄₀₀ tended to be RuBP carboxylation. By comparison, in N. tabacum the value of C_c at 400 μmol mol⁻¹ CO₂ was significantly higher than C_trans (Table 1), indicating A₄₀₀ tended to be limited by RuBP regeneration.

The light response curves demonstrated that the response of A_n to incident light was similar between E. camaldulensis and N. tabacum (Figure 3A). However, the maximum value of g_s was much higher in E. camaldulensis (Figure 3B), while C_i was slightly higher in that species (Figure 3C). When Γ^* was assumed to be 40 μmol mol⁻¹, then C_c under saturating light was much lower in E. camaldulensis (Figure 3D). At 2000 μmol photons m⁻² s⁻¹ in light response curves, the value of C_c was 124 μmol mol⁻¹ in E. camaldulensis and 226 μmol mol⁻¹ in N. tabacum (Figure 3D). This large difference of C_c between E. camaldulensis and N. tabacum was principally caused by the contrast in g_m.

Under all light intensities, E. camaldulensis had significantly higher effective quantum yield of PSII (Φ_PSII) compared to N. tabacum, especially under high light (Figure 4A). Concomitantly, NPQ values were lower in E. camaldulensis than N. tabacum (Figure 4B). According to the data of Φ_PSII, E. camaldulensis had significantly higher values of total electron flow through PSII (J_T) than N. tabacum (Figure 5A). Meanwhile, the ratios of the rate of Rubisco carboxylation (V_c) to that of Rubisco oxygenation (V_o) under saturating light intensities were much higher in E. camaldulensis (Figure 5B). On assumptions of Γ^* being 40 μmol mol⁻¹ and leaf absorbance being 0.85 in E. camaldulensis and N. tabacum, at 2000 μmol photons m⁻² s⁻¹, comparative values for J_T and the V_o/V_c ratio in E. camaldulensis vs. N. tabacum were 280 vs. 178 and 0.63 vs. 0.38, respectively (Figures 5A,B). Furthermore, under light intensities above 300 μmol photons m⁻² s⁻¹, electron flux for photorespiratory carbon oxidation [Je(PCO)] were significantly higher in E. camaldulensis (Figure 5C). Values for Je(PCO) at 2000 μmol photons m⁻² s⁻¹ were 100 and 48 μmol electrons m⁻² s⁻¹ in E. camaldulensis and N. tabacum, respectively. Plotting the V_o/V_c ratio and C_c indicated that the difference in the V_o/V_c ratio between E. camaldulensis and N. tabacum was mainly determined by the difference in C_c (Figure 6), which in turn caused by the change in g_m.

Under high light, E. camaldulensis had significantly increased alternative electron flow, as indicated by the higher values of J_a and the J_a/J_g ratio (Figure 7). To examine the relationship between the alternative electron flow and photorespiration, we evaluated possible associations between J_a and Je(PCO) under light intensities higher than 300 μmol photons m⁻² s⁻¹. Interestingly, J_a was positively and linearly correlated with Je(PCO) (Figure 7A, P < 0.0001). Similarly, the J_a/J_g ratio was positively and linearly correlated with V_o/V_c ratio (Figure 7B, P < 0.0001). According to photosynthesis model, an increase in the V_o/V_c ratio requires a higher ATP/NADPH energy demand from other flexible mechanisms such as cyclic electron flow and alternative electron flow. Increased alternative electron flow promotes the formation of a proton gradient across the thylakoid membrane (ΔpH), which can be used for activating NPQ and ATP synthesis. We found that under high light the rate of ATP supplied from alternative electron sinks [v_ATP(Flex)] were much higher in E. camaldulensis than in N. tabacum (Figure 8A), and the rate of alternative electron flow was positively correlated to v_ATP(Flex) (Figure 8B, P < 0.0001). Furthermore, NPQ values under high light were significantly lower in E. camaldulensis than in N. tabacum (Figure 4B), suggesting that the main role for alternative electron flow in E. camaldulensis is not to activate NPQ but to provide extra ATP. The greater capacity of the photorespiratory pathway in E. camaldulensis plants is sustained by the enhanced alternative electron flow.

In N. tabacum leaves, l_b was the most important constraining factor for photosynthesis, followed by l_mc and l_s, being consistent with high levels of stomatal and mesophyll conductance. By comparison, in E. camaldulensis, l_mc had the greatest influence while l_s was the least limiting factor. This was consistent with the higher g_s and lower g_m measured for that species. These results confirmed that, the major limiting factor for photosynthesis differs intrinsically between high-photosynthesis herbaceous and sclerophyllous plants. Diffusional limitations of CO₂ are the main reason for lower photosynthetic rates in ferns than in angiosperms (Gago et al., 2013; Carriqui et al., 2015). Mesophyll conductance is the most constraining factor for photosynthesis in ferns (Carriqui et al., 2015). These findings suggested that, not only in low-photosynthesis species (e.g., ferns) but also in some high-photosynthesis sclerophyllous species such as E. camaldulensis, mesophyll conductance is the primary limiting factor for photosynthesis. The difference of g_m between E. camaldulensis and N. tabacum is probably determined by their leaf anatomy (Terashima et al., 2006). The thicker cell walls lead to a lower g_m in E. camaldulensis when compared with N. tabacum.

The rate of CO₂ assimilation can be limited by RuBP carboxylation and/or RuBP regeneration in C₃ plants (Farquhar et al., 1980; Yamori et al., 2010, 2011). The specific rate-limiting step of CO₂ assimilation is determined by the relative values of C_c and C_trans. When the value of C_c is higher than C_trans, the CO₂ assimilation rate is mainly limited by RuBP regeneration. Otherwise, the CO₂ assimilation rate is limited by RuBP carboxylation when C_c is lower than C_trans. In N. tabacum leaves, the value of C_c under saturating light was higher than C_trans, and thus the rate-limiting step of CO₂ assimilation tended to be RuBP regeneration (Table 1). On the contrary, the value of C_c under saturating light was lower than C_trans in E. camaldulensis, and thus the rate of CO₂ assimilation was limited by RuBP carboxylation (Table 1). According to the calculation of C_c = C_i − A_n/g_m, the value of C_c can be affected by three parameters C_i, A_n, and g_m. Here, because A_n and C_i values under saturating light were similar in E. camaldulensis and N. tabacum (Figures 3A,C), Because the value of C_c can be largely determined by g_m, the difference in the main rate-limiting step of CO₂ assimilation between N. tabacum and E. camaldulensis was mainly caused by the contrasts in their mesophyll conductance.

In the herbaceous N. tabacum, which has thin, flexible leaves, g_m can reach 0.5 mol m⁻² s⁻¹ when plants are exposed to high nitrogen concentrations (Yamori et al., 2011). By comparison, sclerophyllous plants have relatively lower g_m values that range between 0.09 and 0.25 mol m⁻² s⁻¹ (Lloyd et al., 1992; Hassiotou et al., 2009). Conductance in the mesophyll can be influenced by leaf anatomical traits such as the cell surface area and the chloroplast surface area that is exposed to intercellular air spaces (Evans et al., 1994; Oguchi et al., 2005; Terashima et al., 2011), chloroplast rearrangements (Tholen et al., 2008), cell wall thickness (Terashima et al., 2006, 2011; Flexas et al., 2012), and LMA (Flexas et al., 2008; Hassiotou et al., 2009). The value of LMA for in E. camaldulensis is approximately 169.5 g m⁻² (Suganuma et al., 2006), which is much higher than N. tabacum (approximately 25 g m⁻²) (Yamori et al., 2010). The large differences of leaf anatomical traits between E. camaldulensis and N. tabacum might result in the diversity of g_m.

We found that the capacity of the photorespiratory pathway was greater for E. camaldulensis than for N. tabacum (Figures 5B,C). Rubisco is a dual functional enzyme that catalyzes the carboxylation of RuBP, but also oxygenates RuBP in photorespiration. A lower C_c in E. camaldulensis would increase the rate of RuBP oxygenation by Rubisco. Photorespiration begins with RuBP oxygenation that generates glycolate-2-phosphate and glycerate-3-phosphateare. To maintain a steady-state high rate of photosynthesis in E. camaldulensis, the RuBP pool must remain stable. In chloroplast, the steady-state of RuBP pool depends on two different RuBP regeneration pathways: the Calvin-Benson cycle and the photorespiratory pathway. Impairment of photorespiratory pathway induced a gradient decrease in photosynthetic rate at atmospheric CO₂ concentration (Takahashi et al., 2007). Importantly, during steady-state phases, the rates of RuBP oxygenation and RuBP regeneration through the photorespiratory pathway must be balanced. Under high light, E. camaldulensis accelerate the photorespiratory pathway to favor the regeneration of RuBP via glycerate-3-phosphate, thereby preventing the RuBP pool from shrinking.

A reduction in C_c also accelerates the production of photorespiratory intermediates, e.g., glycine and glycerate, which inhibit the Calvin-Benson cycle (Chastain and Ogren, 1989; Eisenhut et al., 2007; Timm et al., 2012, 2015). In Arabidopsis thaliana plants with elevated glycine decarboxylase activity, the rapid acceleration of the photorespiratory pathway lowers the accumulation of photorespiratory metabolites including those which impair Rubisco activation and possibly the activity of other enzymes of the Calvin-Benson cycle, increasing the performance of the Calvin-Benson cycle (Timm et al., 2012, 2015). Furthermore, N. tabacum plants grown under high light and high nitrogen concentration up-regulate photorespiratory pathway to maintain high rates of photosynthesis (Huang et al., 2014, 2016). To overcome those detrimental effects, the photorespiratory pathway should be enhanced in E. camaldulensis. In addition, although CO₂ is released in the mitochondria in the photorespiratory pathway, C₃ plants can trap photorespired CO₂ within individual mesophyll cell. This causes chloroplast CO₂ concentrations to rise and, ultimately, improves the rate of photosynthesis in C₃ plants (Busch et al., 2013). Taking together, the enhanced photorespiratory capacity strongly contributes to the high rate of CO₂ assimilation in E. camaldulensis.

We were surprised to learn that, under saturating illumination, alternative electron flow was enhanced in E. camaldulensis when compared with N. tabacum. The water-water cycle, nitrate reduction and malate shunt are potential candidates responsible for this increase in alternative electron flow (Yi et al., 2014). When plants are illuminated at atmospheric CO₂ and O₂, most of this alternative flow accounts for the electron flux to oxygen and oxidized ascorbic acid (Miyake and Yokota, 2000; Makino et al., 2002). Thus, we concluded that the WWC activity was greater in E. camaldulensis. During the early phase of photosynthetic induction in rice, the WWC first generates a ΔpH across the thylakoid membranes to form NPQ and supply ATP for carbon assimilation (Neubauer and Yamamoto, 1992; Miyake and Yokota, 2000; Makino et al., 2002). However, when photosynthesis reaches a steady-state rate, the WWC no longer maintains a high NPQ but instead provides additional ATP for primary metabolism in rice leaves (Makino et al., 2002). Our light response curves indicated that NPQ values under high light were significantly lower in E. camaldulensis (Figure 4B). Because the NPQ activation relies on the acidification of thylakoid lumen, and which also depresses electron transport through Cyt b₆/f complex via “photosynthetic control,” this result suggested the higher levels of lumen acidification in leaves of N. tabacum when illuminated at high light. Therefore, the steady-state rate of WWC under intense illumination in E. camaldulensis mainly contributed to additional ATP synthesis rather than lumen acidification, which is consistent with previous studies on the role of the WWC (Makino et al., 2002; Huang et al., 2016).

In electron flow from PSII to NADP⁺, the stoichiometry of the ATP/NADPH ratio is thought to be 1.29 (Sacksteder et al., 2000; Seelert et al., 2000). By comparison, each Rubisco oxygenation consumes 3.5 ATP and 2 NADH equivalents in total. Therefore, the occurrence of photorespiratory pathway needs a higher ATP/NADPH ratio than 1.29 to maintain primary metabolism (Edwards and Walker, 1983; Walker et al., 2014). Under high light and ambient CO₂, the ATP/NADPH ratio required by CO₂ assimilation, photorespiration, and nitrite assimilation is approximately 1.6 (Walker et al., 2014). Furthermore, the ATP/NADPH energy demand for primary metabolism will rise as photorespiration increases. As a result, the rate of ATP supplied from other flexible pathways must be higher in E. camaldulensis due to its higher rate of photorespiratory pathway. Cyclic electron flow and the WWC are the main flexible pathways that contribute to extra ATP synthesis under high light and atmospheric CO₂ concentrations (Makino et al., 2002; Walker et al., 2014; Huang et al., 2015, 2016). Here, we found that the greater rate of photorespiration was accompanied by higher alternative electron flow (Figure 7), and the rate of ATP supplied from other flexible mechanisms was positively correlated to the rate of electron flow to alternative sinks (Figure 8B). Therefore, it appears that E. camaldulensis enhances the alternative electron flow to balance the ATP/NADPH energy demand for high rates of photorespiration.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.