The Kinetics of Mesophyll Conductance and Photorespiration During Light Induction

Abstract

Mesophyll conductance to CO₂ (g_m) act as a significant limiting factor influencing the CO₂ assimilation rate (A_N) during photosynthetic induction. However, the effect of vapor pressure deficit (VPD) on g_m kinetics during light induction is not well clarified. We combined gas exchange with chlorophyll fluorescence measurements to assess the induction kinetics of g_m during light induction under contrasting vapor pressure deficit (VPD) in two tree species with different stomatal conductance (g_s) behavior, Catalpa fargesii and Pterocarya stenoptera. Our results revealed three key findings: (1) the coordination of g_m and g_s kinetics during light induction occurred in C. fargesii but not in P. stenoptera, and the model of g_s kinetics largely determines whether the coordination of g_s and g_m exist in a given species; (2) a high VPD induced simultaneous changes in g_s and g_m kinetics in C. fargesii but had separated effects on g_s and g_m kinetics in P. stenoptera, indicating that the response of g_m kinetics during light induction to VPD differs between species; and (3) the relative contribution of photorespiration to total electron flow was flexible in response to the change in relative diffusional and biochemical limitations, pointing out that photorespiration has a significant role in the regulation of photosynthetic electron flow during light induction. These results provide new sight into the species-dependent kinetics of g_m and photorespiration during light induction.

1. Introduction

In natural environments, plants experience rapid fluctuations in light intensity on their leaves due to various factors such as shading by neighboring vegetation, cloud cover, and wind-induced changes in leaf orientation. These fluctuations can occur within seconds to minutes and significantly impact photosynthetic processes [1,2,3]. When plants transition from shade to direct sunlight, photosynthesis enters an induction phase, characterized by a gradual increase in the rate of photosynthesis until a new steady state is achieved [4,5,6]. The duration of this induction period, which can range from a few minutes to several tens of minutes, is influenced by plant species, prior light exposure, and environmental conditions [7]. This dynamic response in photosynthesis is crucial for carbon fixation and, ultimately, plant growth [8,9,10,11].

Many previous studies have demonstrated that in fluctuating light environments, the induction phase of photosynthesis can significantly reduce daily carbon assimilation in many crop species compared to an ideal scenario where photosynthetic rates achieve a steady state immediately following a change in light intensity [3,8,12,13]. This highlights the critical importance of understanding the physiological mechanisms underlying photosynthetic induction to potentially improve crop yield [14,15,16,17,18]. The induction process, particularly following a transition from low to high light, is primarily governed by three key physiological processes: the induction rate of photosynthetic electron transport in the thylakoid membrane, the activation of Calvin–Benson cycle enzymes (notably Rubisco), and CO₂ diffusion conductance.

Photosynthetic electron transport induction is a rapid process that significantly limits photosynthetic efficiency only within the first 1–2 min after the onset of light induction [19]. In contrast, Rubisco activity limits photosynthetic efficiency over a longer period, as it takes 5–20 min for Rubisco to reach its maximum activity level after illumination [20,21]. CO₂ diffusion conductance involves two sequential components along the CO₂ diffusion pathway: stomatal conductance (g_s) from the leaf surface to the intercellular spaces and mesophyll conductance (g_m), from the intercellular spaces to the carboxylation sites within the chloroplasts [22]. Over the past decade, research has focused on stomatal kinetics and their impact on photosynthetic induction, revealing significant variations in stomatal opening speeds among and within species [5,23,24,25]. Stomatal conductance typically takes much longer to reach a steady state (from tens of minutes to over an hour) compared to the activation of Rubisco [26,27,28,29]. The slow induction of g_s imposes a significant limitation on photosynthesis in some crops, such as African cassava germplasm and tomato species [17].

Compared to the well-studied stomatal limitation, the impact of g_m on photosynthetic induction is less understood. Studies measuring g_m at different light levels suggest that g_m generally increases in response to short-term irradiance increases in a range of plant species, with some exceptions [25,30,31,32,33,34]. However, the response of g_m to a light shift is not equivalent to its induction kinetics. Only a few studies have attempted to track the time course of g_m during light induction in model herbaceous species such as Arabidopsis [4,20], tobacco [4,20], and tomato [35,36,37]. These results indicated that the full induction of g_m requires approximately 7–20 min, which imposes a significant limitation on photosynthesis during light induction. In contrast, the nonsteady-state kinetics of g_m in response to light induction remains poorly understood in tree species.

A recent study indicated that in the “open stomata” mutant (ost1) of Arabidopsis, enhanced g_s during light induction was accompanied by a more rapid induction rate of g_m, leading to higher photosynthetic efficiency under fluctuating light [4]. Conversely, tomato plants with nitrogen deficiency exhibited simultaneous delays in the induction speeds of both g_s and g_m after transitioning from low to high light [37]. Under drought stress, the decrease in g_s is associated with a slower induction rate of g_m in tomato plants, thereby restricting photosynthetic efficiency during light induction [35,36]. These results suggest that during light induction, the kinetics of g_s significantly influence the induction speed of g_m. However, the hypothesis that g_s and g_m kinetics are coordinated during light induction requires further investigation.

Fluctuating light is often accompanied by variations in vapor pressure deficit (VPD) due to diurnal changes in air temperature and relative humidity. VPD is an important environmental factor that affects steady-state photosynthesis primarily by influencing g_s [38,39,40]. Additionally, high VPD conditions can restrict photosynthesis under fluctuating light by decreasing both the absolute value and the induction rate of g_s [40,41]. Some studies have reported that elevated VPD induces simultaneous decreases in g_s and g_m under fluctuating light in species such as tomato and rose [40,41]. However, the effects of changing VPD on g_m kinetics in tree species remain poorly understood. Moreover, it is unclear whether the coordination of g_s and g_m kinetics during light induction can be altered by changes in VPD. Future research should focus on elucidating these interactions to better understand how VPD influences photosynthetic efficiency under dynamic light conditions.

Within the first minutes after transitioning from low to high light, relatively low diffusional conductance results in a reduced chloroplast CO₂ concentration [20]. This, in turn, leads to a decreased rate of CO₂ assimilation, which induces a decline in the ADP/ATP ratio. The reduced ADP/ATP ratio can inactivate chloroplast ATP synthase [42]. Concurrently, the low chloroplast CO₂ concentration makes photorespiration a significant primary metabolic pathway. Photorespiration consumes a substantial fraction of ATP and NADPH, thereby preventing the feedback inhibition of chloroplast ATP synthase [43]. Therefore, photorespiration may play a crucial role in regulating the photosynthetic electron transport rate during light induction [44]. While the response of photorespiration to light intensity has been studied at a steady state, few investigations have examined the temporal dynamics of photorespiration following an immediate transition from low to high light [45,46].

In the present study, we combined measurements of gas exchange and chlorophyll fluorescence to estimate the kinetics of g_m during light induction in two tree species with different g_s behaviors. The main aims were to (1) examine whether the coordination of g_s and g_m during light induction is affected by g_s behavior; (2) characterize the effect of VPD on g_m kinetics during photosynthetic induction; and (3) quantify the relationship between photorespiration and g_m kinetics during light induction.

2. Results

2.1. The Response of The Induction Kinetics of CO₂ Assimilation, g_s and g_m to VPD

We first measured the g_s kinetics in fluctuating light at a moderate VPD of 1.3 kPa. Upon the transfer from high to low light, g_s gradually decreased in C. fargesii with an exponential decline model (Figure 1A). By comparison, in the other species P. stenoptera, g_s largely decreased in the first minute and then gradually decreased with a linear model (Figure 1B). After the transition from low to high light, g_s gradually increased in C. fargesii with a model of exponential rise to the maximum (Figure 1A), whereas g_s largely increased in the first minute and then gradually increased with a linear model (Figure 1B). Therefore, these two tree species showed different g_s kinetics model in fluctuating light.

When the vapor pressure deficit (VPD) increased from 1.3 to 2.7 kPa, the g_s kinetics during light induction were altered in both species. In C. fargesii, g_s first increased to a peak in 10 min and then gradually decreased to a lower steady-state value. By comparison, in P. stenoptera the absolute value of g_s was depressed over time. The kinetics of the net CO₂ assimilation rate (A_N) showed similar trends to g_s in both species (Figure 1C,D). In C. fargesii, A_N gradually increased to the steady-state value at 1.3 kPa VPD but first increased to a peak and then gradually decreased at 2.7 kPa VPD, leading to a lower steady-state A_N at 2.7 kPa than at 1.3 kPa. In P. stenoptera, A_N gradually increased to the steady-state value during light induction, and the absolute value was substantially depressed by the high VPD condition. However, the increased VPD condition had no significant effect on photosynthetic electron transport rate (ETR) during light induction (Figure 1E,F).

During light induction, the kinetics of g_m was altered by the high vapor pressure deficit in both species. In C. fargesii, g_m gradually increased in the light induction period (20 min) at 1.3 kPa VPD but first increased to a peak in 15 min and then slightly decreased at 2.7 kPa VPD (Figure 2A). In P. stenoptera, g_m gradually increased to its peak in approximately 6 and 13 min at 1.3 and 2.7 kPa VPD conditions, respectively, and then remained stable (Figure 2B). Compared with 1.3 kPa VPD, the steady-state value of g_m at 2.7 kPa VPD was depressed in C. fargesii but remained stable in P. stenoptera (Figure 2A,B). The kinetics of chloroplast CO₂ concentration (C_c) in light induction showed a similar response to VPD as well as g_m (Figure 2C,D). By comparison, the rise in VPD hardly affected the kinetics of the Rubisco carboxylation rate (V_cmax) in both species (Figure 2E,F). Owing to the different effects of VPD on CO₂ diffusion and V_cmax, the ratio between diffusional and biochemical photosynthesis limitations (L_d:L_b) at 2.7 kPa VPD substantially increased at a steady state in C. fargesii (Figure 2G) and increased in P. stenoptera during light induction (Figure 2H).

2.2. Correlation Between g_s and g_m During Light Induction

The relationships between g_s, g_m and A_N during the induction period are examined in Figure 3. In C. fargesii, A_N was positively correlated to g_s and g_m (Figure 3A,C), and a coordination between g_s and g_m was observed (Figure 3E). In P. stenoptera, the relationship between A_N and g_s differed between 1.3 and 2.7 kPa VPD conditions and the same A_N was accompanied by a much lower g_s at 2.7 kPa VPD (Figure 3B). Similarly to C. fargesii, A_N was tightly related to g_m in P. stenoptera, especially at 2.7 kPa VPD (Figure 3D). The coordination between g_s and g_m in P. stenoptera was observed at 2.7 kPa VPD but disappeared at 1.3 kPa VPD, suggesting that the induction of g_m was independent of g_s in P. stenoptera.

2.3. The Time-Integrated Limitations of A_N During Light Induction

At 60% relative humidity, the time-integrated limitations of g_s (σ_stom), g_m (σ_mesophyll), and biochemistry (σ_biochem) during light induction were 45%, 42%, and 13%, respectively, in C. fargesii (Figure 4A). When the atmospheric VPD increased from 1.3 to 2.7 kPa, σ_stom, σ_mesophyll, and σ_biochem changed to 30%, 58%, and 12%, respectively (Figure 4A). Therefore, CO₂ diffusional conductance imposed a major limitation on A_N during light induction in C. fargesii. In P. stenoptera, the values for σ_stom, σ_mesophyll, and σ_biochem at 1.3 kPa VPD were 17%, 28%, and 56%, respectively; and they changed to 42%, 37%, and 21% at 2.7 kPa VPD, respectively. This result suggests that a high VPD shifted the major limitation of A_N from biochemistry to CO₂ diffusional conductance in P. stenoptera.

2.4. Induction Kinetics of Photorespiration

The kinetics of photorespiration is shown in Figure 5. Photosynthetic electron flow to Rubisco oxygenation (J_O) gradually increased during light induction in both species, and the changing VPD did not significantly influence the kinetics of J_O in them (Figure 5A,B). Similarly, the changing VPD slightly affected the kinetics of photosynthetic electron flow to Rubisco carboxylation (J_C). In C. fargesii, J_C gradually increased during the induction period (20 min) at 1.3 kPa VPD but reached the maximum in 12 min at 2.7 kPa VPD (Figure 5C). In P. stenoptera, the changing VPD condition did not alter the increase trend in J_C but decreased the absolute value (Figure 5D). Owing to the different effects of changing VPD on J_O and J_C in C. fargesii, the J_O/J_C ratio gradually decreased at 1.3 kPa VPD but first decreased and then gradually increased at 2.7 kPa VPD (Figure 5E). As a result, after light induction for 20 min, the J_O/J_C ratio at 2.7 kPa VPD was substantially higher than that at 1.3 kPa VPD (Figure 5E). By comparison, in P. stenoptera, the J_O/J_C ratio remained stable at 0.5 and 0.6 during light induction under 1.3 and 2.7 kPa VPD conditions, respectively (Figure 5F). Plotting the data of J_O/J_C ratio and L_d:L_b indicated that a non-linear positive relationship was found (Figure 6). When L_d:L_b was extremely high within the first seconds after the transition to high light, photorespiration acted as a major electron sink to favor the operation of photosynthetic electron flow. When L_d:L_b was lowered at the later phase of light induction, the contribution of photorespiration to the total electron flow was diminished. Therefore, electron flow to photorespiration is flexible according to the change in L_d:L_b.

3. Discussion

3.1. Relationship Between g_s and g_m During Light Induction Is Species-Dependent

During light induction, g_s gradually increases and imposes significant limitations on photosynthesis in model species such as Arabidopsis thaliana, tobacco, tomato and Aferican cassava [4,17,20,35,36]. A few studies have investigated the dynamic kinetics of g_m during light induction; the results of these studies proposed a hypothesis that g_s and g_m coordinated to optimize photosynthesis during light induction [4,20]. We found that the tree species C. fargesii exhibited a fine coordination between g_s and g_m kinetics during light induction (Figure 3E). However, such a coordination between g_s and g_m disappeared in the other tree species P. stenoptera (Figure 3F), suggesting that the induction kinetics of g_s is independent of that of g_m in P. stenoptera. Therefore, coordination between g_s and g_m kinetics during light induction is not universal in angiosperms.

After the transition from low to high light, all studied angiosperms showed a similar trend of g_m kinetics with a model of exponential increase to the maximum, as shown in Arabidopsis thaliana [4,20], tobacco [4,20], tomato [35], and the studied tree species C. fargesii and P. stenoptera (Figure 2A,B). However, the model of g_s kinetics during light induction varied between species. Generally, there are three different models of g_s kinetics during light induction among angiosperms: (1) an exponential increase to the maximum, as shown in Arabidopsis thaliana [4,20], tobacco [20], and the studied tree species C. fargesii (Figure 1C), (2) a sigmoidal increase to the maximum, as shown in tomato [35,37], and (3) a rapid increase in the first minute and then a gradual increase with a linear model, as shown in the studied tree species P. stenoptera (Figure 1D). Apparently, in P. stenoptera, the different induction models of g_s and g_m led to the discoordination between gs and gm during light induction. Therefore, the model of g_s kinetics largely determines whether the coordination of g_s and g_m exist in a given species.

3.2. Differential Effects of VPD on g_m Kinetics During Light Induction

A high VPD can delay the induction rate of photosynthetic CO₂ assimilation. One explanation is that a high VPD slows the induction rate of g_s [5,40,41], but the effect of VPD on g_m kinetics during light induction is not well understood [41]. Here, we found that the response of g_m kinetics to VPD was similar to that of g_s in C. fargesii (Figure 1C and Figure 2A). In detail, under the high VPD condition, g_m first gradually increased to its peak and then gradually decreased to the steady state, with a significantly lower steady-state value than that under the low VPD condition (Figure 2A). By comparison, a high VPD had different effects on the induction kinetics of g_s and g_m in P. stenoptera (Figure 1D and Figure 2B). Specifically, a high VPD just delayed the induction rate of g_m but did not significantly affect the steady-state value (Figure 2B). Therefore, the response of g_m kinetics during light induction to VPD differs between species.

In the tree species P. stenoptera, g_s imposed a minor limitation on A_N during light induction at a low VPD but acted as a major limitation at a high VPD (Figure 4B). By comparison, the limitation of g_m imposed on A_N just increased slightly under the high VPD condition (Figure 4B). Furthermore, the same value of A_N during light induction was accompanied with a lower g_s when illuminated at a high VPD (Figure 3B). As a result, the intrinsic water use efficiency (WUEi) was enhanced at a high VPD compared to a low VPD. Concomitantly, the maximum velocity of Rubisco carboxylation (V_cmax) was not altered by the increase in VPD (Figure 2F). Therefore, such an increase in dynamic WUEi under the high VPD condition could not be explained by the change in V_cmax. Alternatively, the dynamic g_m was slightly affected by the increase in VPD, which compensated for the large decrease in g_s. Consequently, the extent of the decrease in chloroplast CO₂ concentration (C_c) under the high VPD condition was much lower than that of g_s (Figure 1D and Figure 2D), which was accompanied by a slight decrease in A_N (Figure 1B). Therefore, the insusceptibility of g_m kinetics to VPD has the potential to increase WUEi under high VPD conditions.

3.3. Modulation of Photorespiration in Response to Photosynthetic Limitation

Here, we found that within the first few minutes after the transition to high light, photorespiration was a major alternative electron sink in the C. fargesii, with the J_O/J_C ratio being approximately 1.0 (Figure 5E). By comparison, the J_O/J_C ratio was substantially lower in P. stenoptera than in C. fargesii (Figure 5E). Therefore, the contribution of photorespiration to total electron flow during light induction significantly differed between C. fargesii and P. stenoptera. Photorespiration is determined by Rubisco activity and chloroplast CO₂ concentration (C_c). As shown in Figure 2, the value of V_cmax was higher in C. fargesii than in P. stenoptera, while the value of C_c was lower in C. fargesii than in P. stenoptera. Therefore, the higher J_O/J_C in C. fargesii than in P. stenoptera was mainly caused by their differences in Rubisco activity and chloroplast CO₂ concentration.

In addition, we found a non-linear positive relationship between the J_O/J_C ratio and L_d:L_b in these two studied species (Figure 6), indicating that the relative contribution of photorespiration to total electron flow was flexible in response to the change in relative diffusional and biochemical limitations. When CO₂ assimilation was limited by the high L_d:L_b within the first few minutes after the transition from low to high light, the restriction of CO₂ assimilation increased the ATP/ADP and NADPH/NADP⁺ ratios, rendering chloroplast ATP synthase activity and electron transport from photosystem I to NADP⁺ limited [42]. Photorespiration consumes a significant fraction of ATP and NADPH to maintain the regeneration of RuBP [47] and consequently prevents the feedback inhibition of chloroplast ATP synthase activity and facilitates the operation of the electron downstream of photosystem I [43]. Consistently, photorespiration acted as the major electron sink pathway when L_d:L_b was extreme high. Alternatively, when CO₂ assimilation operated efficiently under low L_d:L_b conditions, the contribution of photorespiration to total electron flow was diminished. Therefore, photorespiration has a significant role in the regulation of photosynthetic electron flow during light induction.

4. Materials and Methods

4.1. Plant Materials and Growing Conditions

In this study, we used the seedlings of two tree species with different stomatal conductance (g_s) behavior, Catalpa fargesii and Pterocarya stenoptera. They are deciduous, arboreal trees native to the subtropical regions of southwestern China. The two tree species are light-demanding and typically thrive under full sunlight. Therefore, they have similar light requirements. All plants were cultivated in greenhouses in Kunming, Yunnan, China. The day/night temperature is 30/20 °C, the relative humidity is about 60%, and the maximum light intensity to which leaves are 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.

4.2. Gas Exchange and Chlorophyll Fluorescence Measurements

Leaves were exposed to high light (1500 µmol photons m⁻² s⁻¹, composed of 90% red light and 10% blue light) using the chlorophyll fluorescence probe of the LI-6400XT portable photosynthesis system for at least 30 min on a sunny summer morning. Steady-state data for gas exchange and chlorophyll fluorescence were then recorded. Subsequently, the light intensity was reduced to 100 µmol photons m⁻² s⁻¹ to simulate a sun-to-shade transition, lasting for 20 min. The light intensity was then returned to 1500 µmol photons m⁻² s⁻¹ to simulate a shade-to-sun transition, also lasting for 20 min. During both sun-to-shade and shade-to-sun transitions, data were recorded at 1-minute intervals. The air temperature was maintained at 25 °C, and the vapor pressure deficit (VPD) was controlled at 1.3 kPa (relative humidity of 60%) and 2.7 kPa (relative humidity of 15%) for different experimental conditions.

In our study, we utilized the multi-phase flash (MPF) protocol following standard procedures to determine the parameters of chlorophyll fluorescence [48]. The light intensity for the 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 [49]:

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

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

where PPFD represents the light intensity and 0.45 represents the proportion of light energy absorbed by leaves allocated to photosystem II [50].

4.3. 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 [51]:

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

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

The chloroplast CO₂ concentration (C_c) was calculated as follows [52,53]:

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

The maximum carboxylation rate of Rubisco (V_cmax) was calculated as follows [54]:

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

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.

4.4. Quantitative Calculation of Photosynthetic Limitation

The quantitative calculation formula for the photosynthetic limiting factor is as follows [55]:

$$L_{\mathrm{s}}={\displaystyle \frac{g_{\mathrm{t}\mathrm{o}\mathrm{t}}/g_{\mathrm{s}}\times \partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}{g_{\mathrm{t}\mathrm{o}\mathrm{t}}+\partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}}$$

$$L_{\mathrm{m}}={\displaystyle \frac{g_{\mathrm{t}\mathrm{o}\mathrm{t}}/g_{\mathrm{m}}\times \partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}{g_{\mathrm{t}\mathrm{o}\mathrm{t}}+\partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}}$$

$$L_{\mathrm{b}}={\displaystyle \frac{g_{\mathrm{t}\mathrm{o}\mathrm{t}}}{g_{\mathrm{t}\mathrm{o}\mathrm{t}}+\partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}}$$

where L_s, L_m, and L_b represent the degree of the 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 calculated as follows, respectively:

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

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

The total CO₂ diffusional limitation (L_d) was calculated as follows:

$$L_{\mathrm{d}}=L_{\mathrm{s}}+L_{\mathrm{m}}$$

The time-integrated relative limitations imposed by V_cmax (σ_biochem), g_m (σ_mesophyll) and g_s (σ_stom) during the 20 min light induction were calculated according to the detailed method described in [4].

4.5. Calculation of Electron Flow for Photorespiration

The rate of electron flow for photorespiration (J_O) and Rubisco carboxylation (J_C) were calculated according to the following equations [56]:

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

$$J_{\mathrm{C}}=1/3\times (\mathrm{E}\mathrm{T}\mathrm{R}+8\times (A_{\mathrm{N}}+R_{\mathrm{d}}\left)\right)$$

4.6. Statistical Analysis

Six independent leaves from six different plants were used for each measurement. One-way ANOVA (the Tukey comparison test) was used to determine whether significant differences (α = 0.05) existed between the low and high VPD treatments. The software SigmaPlot 10.0 was used for graphing and fitting.

5. Conclusions

Our findings indicate that the coordination of g_s and g_m during light induction is determined by g_s kinetics. The effect of VPD on g_m kinetics during light induction is species-dependent. Concomitantly, the flexibility of photorespiration plays an important role in the regulation of photosynthetic electron flow in response to the kinetics of g_m. Therefore, we call for more studies to investigate the role of photorespiration during light induction with the change in g_m kinetics under environmental stresses.