Light Intensity Dependence of CO₂ Assimilation Is More Related to Biochemical Capacity Rather than Diffusional Conductance

Abstract

The response of CO₂ assimilation rate (A_N) to incident light intensity reflects the efficiency of light utilization. The light intensity dependence of A_N varies widely among different plant species, yet the underlying mechanisms remain poorly understood. To elucidate this issue, we measured the light intensity dependence of gas exchange and chlorophyll fluorescence in twelve tree species. The results indicated that (1) with increasing light intensity, the variation in A_N was closely related to stomatal conductance (g_s), mesophyll conductance (g_m), the maximum velocity of Rubisco carboxylation (V_cmax), and electron transport rate (ETR); (2) compared with A_N at sub-saturating light, the increase in A_N at saturating light was more strongly associated with V_cmax and ETR than with g_s and g_m; and (3) the increase in V_cmax and A_N from 600 to 2000 μmol photons m⁻² s⁻¹ were positively correlated with the maximum capacity of V_cmax. These findings suggest that V_cmax is an energy-dependent process that significantly regulates the light intensity dependence of A_N in plants. This provides valuable insights for crop improvement through the manipulation of V_cmax.

1. Introduction

Plants utilize photosynthesis to convert light energy into chemical energy in the form of ATP and NADPH, which are used to assimilate CO₂ and synthesize organic matter. The maximum A_N largely varies among different plants due to changes in stomatal conductance (g_s), mesophyll conductance (g_m), and biochemical capacity [1,2,3,4,5]. Under field environments, natural light intensity usually fluctuates during the daytime due to changes in solar position and cloud cover [6,7]. Light intensity significantly influences the CO₂ assimilation rate (A_N) through effects on g_s, g_m, Rubisco activation state, and electron transport rate (ETR) [8,9,10]. The response of A_N to incident light intensity plays a significant role in determining plant growth and crop yield [11].

With the increase in light intensity, A_N usually increases gradually to reach the maximum value at saturating light. Many previous studies have measured the light response curves of A_N in plants [8,12,13], pointing out that the light saturation point (LSP) of A_N largely differs between species. For example, LSP is lower than 300 μmol photons m⁻² s⁻¹ in some low-photosynthesis plants, such as Paphiopedilum species and Pleione aurita [12,13]. By comparison, LSP is approximately 1500 μmol photons m⁻² s⁻¹ in some high-photosynthesis plants, such as tobacco and tomato [10,14]. Although such species-dependent LSP is related to the maximum A_N under saturating light, mechanisms underlying the variation in light intensity dependence of A_N have not been well clarified.

At a given incident light intensity, the value of A_N can be limited by diffusional CO₂ conductance and/or biochemical capacities [15]. Specifically, A_N under low light is mainly limited by the availability of ATP and NADPH produced through ETR [16]. With the increase in light intensity, the major limitation of A_N gradually shifts from ETR to RuBP carboxylation/regeneration [17]. According to the photosynthesis model, ETR = PPFD × Φ_PSII × s, where PPFD is the photosynthetic photon flux density, Φ_PSII is the effective quantum yield of photosystem II (PSII) photochemistry, and s is the proportion of light energy absorbed by leaves allocated to PSII, with a typical value of 0.45. At a moderate illumination of 600 μmol photons m⁻² s⁻¹, ETR can theoretically reach 189 μmol photons m⁻² s⁻¹ when Φ_PSII is assumed to be 0.7. The value of A_N can theoretically reach 23.6 μmol CO₂ m⁻² s⁻¹ based on the assumption that A_N = ETR/8. Since the saturating A_N of most species is lower than 23.6 μmol CO₂ m⁻² s⁻¹, their LSP of A_N is theoretically lower than 600 μmol photons m⁻² s⁻¹. However, the actual LSP is usually higher than 600 μmol photons m⁻² s⁻¹. This discrepancy between theoretical and actual LSP is caused by the actual Φ_PSII, which is related to the consumption rates of ATP and NADPH required for Rubisco carboxylation. As we know, the capacity of Rubisco carboxylation is determined by V_cmax and chloroplast CO₂ concentration, the latter of which is tightly related to CO₂ diffusional conductance (g_s and g_m) [18,19,20]. Therefore, the light intensity dependence of A_N is theoretically determined by the light responses of V_cmax, g_s, and/or g_m. In angiosperms, V_cmax and g_s usually increase with increasing illumination, but g_m is maintained stable in some species and gradually increases in others [8,9,21,22,23]. However, it remains unclear whether the light intensity dependence of A_N is mainly related to V_cmax or diffusional CO₂ conductance (g_s and g_m).

In this study, we measured light response data of gas exchange and chlorophyll fluorescence in twelve tree species with the aim of systematically understanding the main factor influencing the light intensity dependence of A_N. The results indicated that the relative increase in A_N at saturating light compared with that at sub-saturating light was mainly determined by the increased kinetics of V_cmax rather than g_s and g_m. Therefore, optimizing the light intensity dependence of V_cmax has the potential to enhance crop yield.

2. Materials and Methods

2.1. Plant Materials and Growth Condition

In this study, we used the seedlings of twelve tree species with variation in the photosynthetic capacity: Chaenomeles cathayensis (Hemsl.) C. K. Schneid., Cercis glabra Pamp., Pterocarya stenoptera C. DC., Idesia polycarpa Maxim., Idesia polycarpa var. vestita Diels, Melia azedarach L., Pistacia chinensis Bunge, Fraxinus retusifoliolata Feng ex P. Y. Bai, Hypericum monogynum L., Styrax grandiflorus Griff., Sunhangia elegans (DC.) H. Ohashi and K. Ohashi, and Alnus nepalensis D. Don. All plants were cultivated in greenhouses in Kunming, Yunnan, China. The day/night temperature is 30/20 °C, relative humidity is about 60%, and the maximum light intensity to which leaves were exposed is approximately 1000 μmol photons m⁻² s⁻¹. To avoid any water and nutrient stress, plants were cultivated using a soilless substrate and drip irrigation techniques.

2.2. Gas Exchange and Chlorophyll Fluorescence Measurements

In the morning of clear days in summer, gas exchange and chlorophyll fluorescence were measured at 25 °C using the chlorophyll fluorescence probe of the LI-6400XT portable photosynthesis system (LI-COR Inc., Lincoln, NE, USA). Mature sun leaves were initially exposed to high light (1500 µmol photons m⁻² s⁻¹, composed of 90% red light and 10% blue light) for at least 30 min to complete photosynthetic induction. Subsequently, we measured the light intensity dependence of photosynthesis by gradually decreasing light intensity from 2000 to 100 µmol photons m⁻² s⁻¹. After exposure to each light intensity (2000, 1500, 1000, 600, 400, 200, and 100 µmol photons m⁻² s⁻¹) for 3 min, the data for gas exchange and chlorophyll fluorescence were then recorded.

In our study, we utilized the multi-phase flash (MPF) protocol following standard procedures to determine the parameters of chlorophyll fluorescence [24]. The light intensity for measurement was set at 1 µmol m⁻² s⁻¹, while the maximum flash intensity reached 8000 µmol m⁻² s⁻¹. During the second phase of the MPF, the flash intensity was reduced by 60%, with the three flash phases lasting 0.3 s, 0.7 s, and 0.4 s, respectively. Subsequently, we calculated the effective quantum yield of photochemistry for photosystem II (Φ_PSII) and the total electron transport rate through photosystem II (ETR) using the following equations [25,26]:

$${\Phi }_{\mathrm{P}\mathrm{S}\mathrm{I}\mathrm{I}}={\displaystyle \frac{\left({F_{\mathrm{m}}}^{\prime }-F_{\mathrm{s}}\right)}{{F_{\mathrm{m}}}^{\prime }}}$$

$$\mathrm{E}\mathrm{T}\mathrm{R}={\mathit{\Phi }}_{\mathrm{P}\mathrm{S}\mathrm{I}\mathrm{I}}\times \mathrm{P}\mathrm{P}\mathrm{F}\mathrm{D}\times 0.45$$

where PPFD represents the light intensity and 0.45 represents the proportion of light energy absorbed by leaves allocated to photosystem II.

2.3. Calculation of the Mesophyll Conductance

Mesophyll conductance (g_m) was calculated by combining the gas exchange data and chlorophyll fluorescence data with the following equation [27]:

$$g_{\mathrm{m}}={\displaystyle \frac{A_{\mathrm{N}}}{C_{\mathrm{i}}-{Г}^{\mathrm{*}}(\mathrm{E}\mathrm{T}\mathrm{R}+8\left(A_{\mathrm{N}}+R_{\mathrm{d}}\right))/(\mathrm{E}\mathrm{T}\mathrm{R}-4\left(A_{\mathrm{N}}+R_{\mathrm{d}}\right))}}$$

where A_N represents the net photosynthetic rate, C_i represents the intercellular CO₂ concentration, Г* represents the CO₂ compensation point in the absence of mitochondrial respiration, and a typical value of 40 μmol mol⁻¹ is used [9]; R_d represents the dark respiration rate measured at night.

The chloroplast CO₂ concentration (C_c) was calculated as follows [28]:

$$C_{\mathrm{c}}=C_{\mathrm{i}}-{\displaystyle \frac{A_{\mathrm{N}}}{g_{\mathrm{m}}}}$$

The maximum carboxylation rate of Rubisco (V_cmax) was calculated as follows [18,29]:

$$V_{\mathrm{c}\mathrm{m}\mathrm{a}\mathrm{x}}={\displaystyle \frac{(A_{\mathrm{N}}+R_{\mathrm{d}})(C_{\mathrm{c}}+K_{\mathrm{c}}(1+{O/K}_{\mathrm{o}}\left)\right)}{(C_{\mathrm{c}}-{Г}^{\mathrm{*}})}}$$

where K_c (404 μmol mol⁻¹) and K_o (278 mmol mol⁻¹) are the Rubisco Michaelis–Menten constants for CO₂ and oxygen, respectively; O (210 mmol mol⁻¹) is the oxygen concentration in the chloroplasts.

2.4. Quantitative Calculation of Photosynthetic Limitation

The quantitative calculation formula for the photosynthetic limiting factor is as follows [15]:

$$L_{\mathrm{s}}={\displaystyle \frac{g_{\mathrm{t}\mathrm{o}\mathrm{t}}/g_{\mathrm{s}}\times \mathsf{\partial }{A}_{\mathrm{N}}/\mathsf{\partial }{C}_{\mathrm{c}}}{g_{\mathrm{t}\mathrm{o}\mathrm{t}}+\mathsf{\partial }{A}_{\mathrm{N}}/\mathsf{\partial }{C}_{\mathrm{c}}}}$$

$$L_{\mathrm{m}}={\displaystyle \frac{g_{\mathrm{t}\mathrm{o}\mathrm{t}}/g_{\mathrm{m}}\times \mathsf{\partial }{A}_{\mathrm{N}}/\mathsf{\partial }{C}_{\mathrm{c}}}{g_{\mathrm{t}\mathrm{o}\mathrm{t}}+\mathsf{\partial }{A}_{\mathrm{N}}/\mathsf{\partial }{C}_{\mathrm{c}}}}$$

$$L_{\mathrm{b}}={\displaystyle \frac{g_{\mathrm{t}\mathrm{o}\mathrm{t}}}{g_{\mathrm{t}\mathrm{o}\mathrm{t}}+\mathsf{\partial }{A}_{\mathrm{N}}/\mathsf{\partial }{C}_{\mathrm{c}}}}$$

where L_s, L_m, and L_b represent the degree of limitation of photosynthesis by g_s, g_m, and biochemical capacity, respectively, and g_tot represents the overall CO₂ diffusive conductance, with g_tot and ∂A_N/∂C_c being calculated as follows, respectively:

$$g_{\mathrm{t}\mathrm{o}\mathrm{t}}={\displaystyle \frac{g_{\mathrm{s}}\times g_{\mathrm{m}}}{g_{\mathrm{s}}+g_{\mathrm{m}}}}$$

$${\mathsf{\partial }A}_{\mathrm{N}}/{\mathsf{\partial }C}_{\mathrm{c}}=V_{\mathrm{c}\mathrm{m}\mathrm{a}\mathrm{x}}{\displaystyle \frac{{Г}^{\mathrm{*}}+K_{\mathrm{c}}(1+{O/K}_{\mathrm{o}})}{{(C_{\mathrm{c}}+K_{\mathrm{c}}(1+{O/K}_{\mathrm{o}}\left)\right)}^2}}$$

2.5. Calculation of Electron Flow for Photorespiration

The rate of electron flow for photorespiration (J_O) was calculated according to the following equation [30]:

$$J_{\mathrm{O}}=2/3\times (\mathrm{E}\mathrm{T}\mathrm{R}-4\times (A_{\mathrm{N}}+R_{\mathrm{d}}\left)\right)$$

2.6. Statistical Analysis

Six independent leaves from six different plants were used for each species. The software SigmaPlot 10.0 was used for graphing and fitting.

3. Results

3.1. The Steady-State Photosynthesis at Saturating Light

We first measured steady-state photosynthesis at a saturating light intensity of 2000 μmol photons m⁻² s⁻¹. As shown in

Table 1. Area-based photosynthetic characteristics for evergreen and deciduous angiosperm tree species. Average values ± SE (n ≥ 5) are shown for area-based maximum net photosynthetic rate (A_N), stomatal conductance (g_s), mesophyll conductance (g_m), maximum velocity of Rubisco carboxylation (V_cmax), and electron transport rate (ETR).

Species	Aarea (μmol m−2 s−1)	gs (mol m−2 s−1)	gm (mol m−2 s−1)	Vcmax (μmol m−2 s−1)	ETR (μmol m−2 s−1)
Ch. cathayensis	13.1 ± 0.43	0.406 ± 0.031	0.095 ± 0.01	91.5 ± 11	89.6 ± 2.8
Ce. glabra	21.1 ± 0.85	0.615 ± 0.043	0.16 ± 0.02	111 ± 11	137 ± 8.2
Pt. stenoptera	14.7 ± 0.49	0.303 ± 0.042	0.12 ± 0.005	118 ± 14	104 ± 6.0
Id. polycarpa	16.3 ± 0.77	0.314 ± 0.037	0.16 ± 0.01	101 ± 9.1	105 ± 5.4
Id. Polycarpa var. vestita	16.0 ± 0.78	0.446 ± 0.039	0.18 ± 0.04	139 ± 25	116±8.3
Me. azedarach	16.3 ± 0.76	0.302 ± 0.05	0.12 ± 0.01	155 ± 25	126 ± 6.9
Pi. chinensis	12.5 ± 0.97	0.177 ± 0.037	0.091 ± 0.01	144 ± 24	111 ± 6.4
Fr. retusifoliolata	15.4 ± 0.23	0.285 ± 0.02	0.15 ± 0.02	108 ± 12	105 ± 3.0
Hy. monogynum	21.3 ± 0.45	0.588 ± 0.06	0.17 ± 0.02	161± 22	156 ± 7.6
St. grandiflorus	10.9 ± 0.66	0.142 ± 0.01	0.10 ± 0.01	120 ± 9.9	88.9 ± 4.3
Su. elegans	19.2 ± 0.71	0.359± 0.07	0.24 ± 0.04	162 ± 30	152 ± 12
Al. nepalensis	19.4 ± 1.6	0.428 ± 0.09	0.13 ± 0.01	200 ±15	176 ± 4.6

, the net CO₂ assimilation rate (A_N) ranged from 10.9 ± 0.66 to 21.3 ± 0.45 μmol m⁻² s⁻¹, stomatal conductance (g_s) ranged from 0.14 ± 0.01 to 0.62 ± 0.04 mol m⁻² s⁻¹, mesophyll conductance (g_m) ranged from 0.09 ± 0.01 to 0.24 ± 0.04 mol m⁻² s⁻¹, electron transport rate (ETR) ranged from 89.6 ± 2.8 to 175 ± 4.6 μmol m⁻² s⁻¹, and the maximum velocity of Rubisco carboxylation (V_cmax) ranged from 91.5 ± 11 to 199 ± 15 μmol m⁻² s⁻¹. These results indicated that photosynthetic capacity varied substantially among the studied tree species.

3.2. Light Intensity Dependence of Photosynthetic Parameters

We next measured gas exchange and chlorophyll fluorescence under light conditions ranging from 100 to 2000 μmol photons m⁻² s⁻¹. As shown in Figure 1A, A_N increased rapidly with increasing light intensity from 100 to 400 μmol photons m⁻² s⁻¹, but increased by an average of 18% when light intensity increased from 600 to 2000 μmol photons m⁻² s⁻¹. At a high light intensity of 1500 μmol photons m⁻² s⁻¹, A_N was almost saturated in all twelve tree species. Concomitantly, g_s displayed a slight increase throughout the entire light response curve in all studied species (Figure 1B), while g_m increased by an average of 32% with increasing light intensity from 600 to 2000 μmol photons m⁻² s⁻¹ (Figure 1C). For all studied species, ETR increased rapidly as light intensity increased from 100 to 600 μmol photons m⁻² s⁻¹. During the light course from 600 to 1000 μmol photons m m⁻² s⁻¹, ETR increased by an average of 12% (Figure 2A). In the twelve studied tree species, ETR was approximately saturated at a high light intensity of 1000 μmol photons m⁻² s⁻¹ (Figure 2A). Similar to the light response trend of ETR, V_cmax gradually increased with light intensity increasing from 400 to 1000 μmol photons m⁻² s⁻¹, and was almost saturated at 1000 μmol photons m⁻² s⁻¹ in these studied species (Figure 2B). Across the light intensity range from 400 to 2000 μmol photons m⁻² s⁻¹, the variation in A_N was tightly correlated with the variations in g_s (R² = 0.66, p < 0.0001, Figure 3A), g_m (R² = 0.47, p < 0.0001, Figure 3B), V_cmax (R² = 0.26, p < 0.0001, Figure 3C), and ETR (R² = 0.76, p < 0.0001, Figure 3D). These results suggest that variation in A_N among these tree species is related to variations in g_s, g_m, V_cmax, and ETR.

3.3. Correlation Between Physiological Parameters and CO₂ Assimilation Rate

Compared with A_N at 400 μmol photons m⁻² s⁻¹ (A_N-400), A_N at 2000 μmol photons m⁻² s⁻¹ (A_N-2000) reached 120–144% in the studied species, with g_s reaching 114–142%, g_m reaching 85–178%, V_cmax reaching 115–213%, and ETR reaching 116–158% (Figure 4). The variation in A_N-2000/A_N-400 was not significantly correlated with g_s-2000/g_s-400 (Figure 4A) or g_m-2000/g_m-400 (Figure 4B), but was significantly correlated with V_cmax-2000/V_cmax-400 (R² = 0.57, p = 0.005, Figure 4C) and ETR₂₀₀₀/ETR₄₀₀ (R² = 0.68, p = 0.001, Figure 4D). Similarly, the variation in A_N-2000/A_N-600 was not significantly correlated with g_s-2000/g_s-600 (Figure 5A) or g_m-2000/g_m-600 (Figure 5B), but was significantly correlated with V_cmax-2000/V_cmax-600 (R² = 0.64, p = 0.002, Figure 5C) and ETR₂₀₀₀/ETR₆₀₀ (R² = 0.66, p = 0.001, Figure 5D). Therefore, the relative increase in A_N at 2000 μmol photons m⁻² s⁻¹ compared with that at 400 and 600 μmol photons m⁻² s⁻¹ is mainly determined by the light intensity dependence of V_cmax and ETR rather than g_s and g_m.

In addition, we found a positive correlation between V_cmax-2000/V_cmax-400 and ETR₂₀₀₀/ETR₄₀₀ (R² = 0.95, p < 0.0001, Figure 6A), a negative correlation between V_cmax-2000/V_cmax-400 and C_c-2000/C_c-400 (R² = 0.75, p = 0.0003, Figure 6B), a negative correlation between V_cmax-2000/V_cmax-400 and L_b-2000/L_b-400 (R² = 0.67, p = 0.001, Figure 6C), and a positive correlation between V_cmax-2000/V_cmax-400 and J_O-2000/J_O-400 (R² = 0.95, p < 0.0001, Figure 6D). Similarly, we observed a positive correlation between V_cmax-2000/V_cmax-600 and ETR₂₀₀₀/ETR₆₀₀ (R² = 0.94, p < 0.0001, Figure 7A), a negative correlation between V_cmax-2000/V_cmax-600 and C_c-2000/C_c-600 (R² = 0.81, p < 0.0001, Figure 7B), a negative correlation between V_cmax-2000/V_cmax-600 and L_b-2000/L_b-600 (R² = 0.81, p < 0.0001, Figure 7C), and a positive correlation between V_cmax-2000/V_cmax-600 and J_O-2000/J_O-600 (R² = 0.92, p < 0.0001, Figure 7D). These results indicate that the coordinated changes in V_cmax and ETR compensate for the decrease in C_c and the increase in J_O and diminish the biochemical limitation of A_N.

4. Discussion

The light intensity dependence of photosynthesis varies widely among different plants and is tightly related to plant growth and crop yield [11]. According to the C₃ photosynthesis model, the CO₂ assimilation rate (A_N) at a given incident light intensity can be limited by light energy (as indicated by ETR), diffusional CO₂ conductance (g_s and g_m), and V_cmax [18]. With increasing illumination from sub-saturating to saturating light, angiosperms typically display gradual increases in ETR and g_s, and g_m remains stable in some species but gradually increases in others [8,9,21,22]. However, the major factor affecting the light intensity dependence of A_N remains unclear.

In this study, we found that light intensity dependence of A_N, V_cmax, and ETR varied among the twelve studied tree species (Figure 1 and Figure 2). Furthermore, the increase in A_N from sub-saturating to saturating light among the twelve studied tree species was tightly related to changes in V_cmax and ETR, with no significant correlation to changes in g_s and g_m (Figure 4 and Figure 5). Therefore, variation in the light intensity dependence of A_N among species is primarily determined by biochemical capacity rather than diffusional conductance. Compared with sub-saturating light, the increase in V_cmax under saturating light suggests an elevation in the Rubisco activation state [31], allowing for increases in Rubisco carboxylation and oxygenation. The ETR-dependent regeneration rates of ATP and NADPH are mainly determined by their consumption rates, which in turn are influenced by primary metabolism, including CO₂ assimilation and photorespiration [32,33,34]. When CO₂ assimilation is restricted, the inactivation of chloroplast ATP synthase results in an increase in proton gradient (ΔpH) across the thylakoid membranes, which slows down the electron flow at cytochrome b₆/f complex [32,35]. Therefore, the coordination between V_cmax and ETR tightly determines the light intensity dependence of A_N.

The biological significance of V_cmax in the light response of A_N aligns with the general understanding that V_cmax is an energy-dependent process. For example, V_cmax gradually increases upon transition from low to high light, imposing a significant on A_N [22,36]. With increasing maximum V_cmax, the time required to reach 95% of the maximum V_cmax during light induction also gradually increases [37]. In Arabidopsis thaliana, the value of V_cmax at 1200 μmol photons m⁻² s⁻¹ is 2-fold higher than that at 100 μmol photons m⁻² s⁻¹ [22]. Consistently, we found that V_cmax at 600 μmol photons m⁻² s⁻¹ was substantially higher than that at 400 μmol photons m⁻² s⁻¹ in the twelve tree species (Figure 2). Furthermore, as V_cmax-2000/V_cmax-400 and V_cmax-2000/V_cmax-600 increased, ETR₂₀₀₀/ETR₄₀₀ and ETR₂₀₀₀/ETR₆₀₀ also increased simultaneously (Figure 6 and Figure 7), suggesting that ETR provides the essential energy for the increase in V_cmax through Rubisco activation.

Accompanying the increase in V_cmax under saturating light, the value of C_c decreased (Figure 6 and Figure 7), resulting in an inevitable increase in the rate of Rubisco oxygenation (photorespiration). Photorespiration begins with the oxygenation of RuBP catalyzed by Rubisco, producing 2-phosphoglycolate (2-PG). Given that 2-PG inhibits central enzymes in the Calvin–Benson cycle and glycolysis, it must be converted into glycerate-3-P through the photorespiratory pathway. This process requires additional ATP and NADPH produced by ETR. Therefore, J_O-2000/J_O-400 and J_O-2000/J_O-600 positively increased with the increases in V_cmax-2000/V_cmax-400 and V_cmax-2000/V_cmax-600 (Figure 6 and Figure 7). Under conditions of high V_cmax, the rate of RuBP oxygenation was inevitably elevated in normal air. Such enhancement of photorespiration accelerates the consumption rate of ATP and NADPH. This process not only prevents substrate limitation of chloroplast ATP synthase but also favors electron transfer from PSI to NADP⁺ [38,39]. Therefore, photorespiration participates in the light intensity dependence of V_cmax and ETR.

Considering the strong effect of V_cmax on the light intensity dependence of A_N, it is surprising that the light intensity dependence of V_cmax varies among different species (Figure 2). We found that the values of V_cmax-2000/V_cmax-600 and A_N-2000/A_N-600 were positively correlated with the steady-state V_cmax at 2000 μmol photons m⁻² s⁻¹ (Figure 8). As a result, the light intensity dependence of V_cmax and A_N is largely determined by the maximum capacity of V_cmax. In plants with low V_cmax capacity, such as Chaenomeles cathayensis, V_cmax can reach its maximum value at 600 μmol photons m⁻² s⁻¹ (Figure 2). By comparison, in plants with high V_cmax capacity, such as Alnus nepalensis, V_cmax saturates at 1500 μmol photons m⁻² s⁻¹ (Figure 2). In wild-type plants, the maximum value of leaf V_cmax is tightly related to leaf Rubisco content or leaf nitrogen content [5,40,41]. Therefore, variation in the light intensity dependence of V_cmax and A_N among tree species may be determined by leaf Rubisco content or leaf nitrogen content.

5. Conclusions

We document that the light intensity dependence of A_N is more related to the light response of V_cmax rather than the light response of g_s and g_m. Furthermore, the light response of V_cmax is in turn influenced by its maximum capacity. Therefore, the maximum V_cmax plays a significant role in determining the light intensity dependence of A_N. We propose that increasing the maximum V_cmax or enhancing the nitrogen distribution into Rubisco has the potential to improve A_N under sub-saturating and saturating light conditions. This strategy provides insight into crop improvement.