The Light Dependence of Mesophyll Conductance and Relative Limitations on Photosynthesis in Evergreen Sclerophyllous Rhododendron Species

Abstract

Mesophyll conductance (g_m) limits CO₂ diffusion from sub-stomatal internal cavities to the sites of RuBP carboxylation. However, the response of g_m to light intensity remains controversial. Furthermore, little is known about the light response of relative mesophyll conductance limitation (l_m) and its effect on photosynthesis. In this study, we measured chlorophyll fluorescence and gas exchange in nine evergreen sclerophyllous Rhododendron species. g_m was maintained stable across light intensities from 300 to 1500 μmol photons m⁻² s⁻¹ in all these species, indicating that g_m did not respond to the change in illumination in them. With an increase in light intensity, l_m gradually increased, making g_m the major limiting factor for area-based photosynthesis (A_N) under saturating light. A strong negative relationship between l_m and A_N was found at 300 μmol photons m⁻² s⁻¹ but disappeared at 1500 μmol photons m⁻² s⁻¹, suggesting an important role for l_m in determining A_N at sub-saturating light. Furthermore, the light-dependent increase in l_m led to a decrease in chloroplast CO₂ concentration (C_c), inducing the gradual increase of photorespiration. A higher l_m under saturating light made A_N more limited by RuBP carboxylation. These results indicate that the light response of l_m plays significant roles in determining C_c, photorespiration, and the rate-limiting step of A_N.

1. Introduction

In southwest China, mountain forest ecosystems are characterized by the presence of evergreen sclerophyllous angiosperms. This vegetation type includes many species of the genus Rhododendron (Ericaceae) that exhibit low mesophyll conductance (g_m) [1]. Leaf CO₂ diffusion mainly includes diffusion from air to the intercellular cavity (stomatal conductance, g_s) and diffusion from the intercellular cavity to the site of RuBP carboxylation (g_m). Many previous studies indicated that sclerophyllous plants have relatively lower g_m values than herbaceous plants [1,2,3,4,5]. Such low levels of g_m in sclerophyllous plants limit A_N to a large extent when exposed to high light. Consequently, g_m is the major limiting factor for A_N under constant high light in sclerophyllous tree species, such as Mediterranean evergreens [4,6,7,8], the sclerophyllous genus Banksia [2], Eucalyptus camaldulensis [9,10], and sclerophyllous Rhododendron species [1]. Therefore, the response of g_m to the environment plays a critical role in controlling photosynthesis in sclerophyllous angiosperms.

Under natural field conditions, leaves experience rapid changes in light intensities on timescales of seconds or minutes [11,12]. g_s usually increases with increasing light in angiosperms, which raises the question whether g_m changes rapidly in response to the change in light intensity. Some studies observed that g_m increased with increasing irradiance in Eucalyptus species [13,14], Nicotiana tabacum [15], Camellia species [16], Triticum durum and Arbutus × ‘Marina’ [17], and rice (Oryza sativa) grown under high nitrogen concentrations [18]. Furthermore, a recent study found that with increasing irradiance, g_m gradually increased in all six studied angiosperms [19]. In contrast, other studies reported that light intensity did not affect g_m in Triticum aestivum [20], N. tabacum [21], and rice grown under low nitrogen concentrations [18]. Therefore, the response of g_m to increases in light intensity in angiosperms is species dependent and can be affected by nitrogen nutritional conditions. Previous studies mainly focused on the light response of g_m in herbaceous plants. However, the response of g_m to light intensity in sclerophyllous evergreen species is not well known.

Many previous studies have analyzed the quantitative relative stomatal, mesophyll, and biochemical limitations of A_N under saturating light conditions [4,22,23,24,25]. As we know, the value of A_N under saturating light largely determines the growth rate of plants [26,27,28]. However, some shade-tolerant plant species and leaves in lower parts of canopies may experience moderate light. As a result, the value of A_N at moderate light can significantly affect plant growth and crop productivity [29]. Therefore, understanding the relative limitations of A_N at moderate light may have broad applications in angiosperms and crops in particular. However, light response changes in the relative limitations of A_N are poorly understood.

In addition to RuBP carboxylation, Rubisco catalyzes RuBP oxygenation under current atmospheric environmental conditions [30,31,32]. The photorespiratory pathway converts phosphoglycolate (2PG) to 3-phosphoglycerate (3PGA), allowing the Calvin–Benson cycle to operate in the presence of molecular oxygen [33,34]. Under low light, A_N is mainly limited by a lack of light energy, and the resulting high chloroplast CO₂ concentration (C_c) restricts the rate of RuBP oxygenation (V_o). Under high light, the increased rate of RuBP carboxylation (V_c) leads to a decrease in C_c [9,21], increasing the V_o/V_c ratio and thus enhancing photorespiration [35,36]. Therefore, photorespiration usually increases with increasing light intensity in C₃ plants [16,37,38,39,40]. Meanwhile, either an increase in g_s or g_m can partially compensate for the CO₂ consumption in CO₂ fixation. However, it is unclear whether the light response of photorespiration is mainly determined by g_s or g_m.

At saturating light, A_N can be limited by RuBP carboxylation and/or RuBP regeneration [41,42,43]. Once the operating C_c is lower than the chloroplast CO₂ concentration (C_trans) at which the transition from RuBP carboxylation limitation to RuBP regeneration limitation occurs, A_N is limited by RuBP carboxylation. When C_c is higher than C_trans, then A_N tends to be limited by RuBP regeneration. The major rate-limiting step of A_N is species dependent and can be affected by leaf nitrogen content and measurement temperature [21,41,43]. However, the effect of g_m on the rate-limiting step of A_N is poorly understood. The leaf g_m is positively correlated to leaf nitrogen content [18,38,43]. Furthermore, A_N tends to be limited by RuBP regeneration in plants grown under high nitrogen concentrations but is limited by RuBP carboxylation in plants grown under nitrogen-deficient concentrations [43]. Therefore, we hypothesize that the rate-limiting step of A_N under saturating light is largely determined by g_m.

In this study, we measured light responses of gas exchange and chlorophyll fluorescence in nine evergreen sclerophyllous Rhododendron species. The aims of this study were (1) to investigate the light response changes in g_m and the relative limitations of A_N; (2) to assess whether the increase in V_o/V_c ratio under high light is mainly determined by g_s or g_m; and (3) to examine the effects of g_m and l_m on the rate-limiting step of A_N.

2. Results

2.1. Light Intensity Dependence of Photosynthesis and Mesophyll Conductance

The A₈₀₀/A₁₅₀₀ ratios ranged from 0.86 to 1.02, suggesting that A_N were saturated or almost saturated at 800 μmol photons m⁻² s⁻¹. Under a high light of 1500 μmol photons m⁻² s⁻¹, A_N were saturated in all Rhododendron species. The light-saturated A_N ranged from 10.3 (R. ciliicalyx) to 17.8 μmol CO₂ m⁻² s⁻¹ (R. glanduliferum), a total variation of 60% between species (Figure 1). Under such saturating light, the PSII electron transport rate (J_PSII) ranged from 151.9 (R. decorum subsp. diaprepes) to 205.2 μmol electrons m⁻² s⁻¹ (R. delavayi), leading to a variation of 35% in J_PSII (Figure 1).

We calculated g_m under different light intensities according to the method of [44]. The value of g_m at 300 μmol photons m⁻² s⁻¹ varied between species, ranging from 0.040 (R. ciliicalyx) to 0.20 mol m⁻² s⁻¹ (R. decorum subsp. diaprepes) (Figure 2). At 1500 μmol photons m⁻² s⁻¹, g_m ranged from 0.049 (R. ciliicalyx) to 0.16 mol m⁻² s⁻¹ (R. decorum subsp. diaprepes) (Figure 2). All species showed no significant change in g_m between 300 and 1500 μmol photons m⁻² s⁻¹ and g_m was maintained stable over the light intensity change (Figure 2). The average light response values of g_m ranged from 0.048 (R. ciliicalyx) to 0.18 mol m⁻² s⁻¹ (R. decorum subsp. diaprepes) (Figure 2).

2.2. Light Intensity Dependence of Relative Limitations of Photosynthesis

The relative limitations of photosynthesis were significantly affected by the light intensity. Under low light, the rate of photosynthesis was largely limited by biochemical capacity (l_b) (Figure 3), owing to the lack of ATP and NADPH. With an increase in light intensity, l_b gradually decreased (Figure 3). Meanwhile, mesophyll conductance limitation (l_m) gradually increased and stomatal conductance limitation (l_s) changed slightly (Figure 3). At a moderate light intensity of 500 μmol photons m⁻² s⁻¹, the major limiting factor for photosynthesis shifted from l_b to l_m, except for the species with the highest g_m: R. decorum subsp. diaprepes (Figure 3). Under the saturating light of 1500 μmol photons m⁻² s⁻¹, l_m ranged from 0.37 to 0.71, l_b from 0.15 to 0.37, and l_s from 0.12 to 0.26 (Figure 3). Therefore, g_m is the major limiting factor for CO₂ assimilation at saturating light in Rhododendron species, followed by biochemical capacity and stomatal conductance.

The relationships between g_m, l_m, and A_N at sub-saturating and saturating light intensities were also analyzed in these studied species (Figure 4). As expected, the values of g_m were significantly positively correlated to A_N. Interestingly, a closer relationship between g_m and A_N was found at 300 μmol photons m⁻² s⁻¹ than that at 1500 μmol photons m⁻² s⁻¹ (Figure 4A). Furthermore, a close negative correlation was found between l_m and A_N at 300 μmol photons m⁻² s⁻¹ (Figure 4B). However, this significant relationship disappeared at 1500 μmol photons m⁻² s⁻¹ (Figure 4B). These results indicate that g_m and l_m play more important roles in determining A_N at sub-saturating light than at saturating light.

2.3. Light Intensity Dependence of Chloroplast CO₂ Concentration and Photorespiration

With an increase in light intensity, intercellular CO₂ concentration (C_i) and chloroplast CO₂ concentration (C_c) gradually decreased in all Rhododendron species (Figure 5A,B). At 1500 μmol photons m⁻² s⁻¹, C_i ranged from 253 (R. decorum subsp. diaprepes) to 317 μmol mol⁻¹ (R. glanduliferum) (Figure 5A), and C_c ranged from 76 (R. hancockii) to 144 μmol mol⁻¹ (R. decorum subsp. diaprepes) (Figure 5B). Further analysis found that the drop in C_c was tightly correlated with an increase in l_m (Figure 5C). Therefore, the light dependence change in C_c was mainly caused by an increase in l_m. C_c is known to be a key factor affecting the affinity of Rubisco to CO₂ and O₂, and thus determines the value of V_o/V_c. With an increase in illumination, the V_o/V_c ratio gradually increased (Figure 6A). Furthermore, the light dependence of V_o/V_c was positively correlated to l_m (Figure 6B). These results indicate that the increased V_o/V_c with increasing irradiance was mainly caused by the enhanced l_m.

At 1500 μmol photons m⁻² s⁻¹, C_trans was calculated to analyze the rate-limiting step of A_N. The operating C_c values are significantly lower than the C_trans values in these studied species, except for R. decorum subsp. diaprepes (Figure 7). Therefore, the saturating A_N at current atmospheric CO₂ concentration was mainly limited by RuBP carboxylation in these Rhododendron species. In R. decorum subsp. diaprepes, A_N was limited by RuBP carboxylation and regeneration.

3. Discussion

3.1. Rapid Response of g_m to Changes in Light Intensity is Consistent among Rhododendron Species

With an increase in light intensity, stomatal conductance gradually increased to enhance the CO₂ diffusion from air to sub-stomatal internal cavities and thus to favor photosynthetic CO₂ assimilation [45,46,47]. Subsequently, mesophyll conductance limits the diffusion of CO₂ from intercellular cavities to the sites of carboxylation, and may respond rapidly to changes in light intensity. Some previous studies observed that g_m rapidly increased with increasing irradiance in N. tabacum [15], Triticum durum, and Arbutus × ‘Marina’ [17], and three Eucalyptus species E. globules, E. saligna, and E. sieberi [13,14]. A recent study reported that g_m rapidly responded to changes in light intensity in six studied angiosperms [19]. By contrast, some authors found that g_m was not responsive to irradiance in Triticum aestivum [20] and N. tabacum [21]. Therefore, the response of g_m to light intensity highly differs between species, presenting an important photosynthetic response to environmental change. However, the light response of g_m in evergreen sclerophyllous angiosperms is poorly understood.

This article investigated the rapid response of g_m to light intensity in nine sclerophyllous Rhododendron species. We found that the values of g_m did not change between 300 and 1500 μmol photons m⁻² s⁻¹ in either of these species (Figure 2). Therefore, these nine Rhododendron species showed the same response model of g_m to changes in light intensity. Consistently, the three Eucalyptus species E. globules, E. saligna, and E. sieberi also showed the same trend in the light response of g_m [13,14]. Therefore, we propose that the rapid response of g_m to the change in light intensity may be a conservative photosynthetic trait in a given genus. This conclusion is applicable only under normal growth conditions because water and/or nutrition stresses can alter the rapid response of g_m to irradiance.

3.2. Light Response Changes in Relative Limitations of Photosynthesis

Many studies have documented that plant growth and crop productivity are largely linked to A_N under high light [26,27,28]. However, global rice productivity in agricultural fields was not determined by the maximum A_N under saturating light but was linked to the A_N under low light [29]. Therefore, in order to optimize the cultivation strategy, we should take into consideration the relative limitations of photosynthesis under different light intensities. While the relative limitations of A_N under high light are widely studied, information on the light response of relative limitations is very limited. In this article, we analyzed the relative limitations of A_N under different light intensities in nine Rhododendron species. The stomatal conductance limitation was the most insignificant factor for A_N in these species, irrespective of light intensity (Figure 3). Under low light, the light-dependent production of ATP and NADPH was restricted by limiting light energy, and A_N was mainly limited by biochemical factors (Figure 3). With an increase in light intensity, ETRII rapidly increased (Figure 1), leading to the increased production rates of ATP and NADPH. In contrast, the value of C_c gradually decreased (Figure 5B). Therefore, the change in C_c was out-of-step with the change in energy supply, generating an imbalance between CO₂ supply and production of ATP and NADPH. When exposed to a moderate light of 500 μmol photons m⁻² s⁻¹, the most limiting factor for A_N shifted from biochemical capacity to g_m (Figure 3). Therefore, g_m was the major limiting factor for A_N in these species when exposed to high light. Furthermore, the values of A_N at 300 μmol photons m⁻² s⁻¹ were largely correlated to the values of g_m and l_m (Figure 4), indicating that increasing g_m has a significant potential to increase A_N under sub-saturating light. Taking into consideration that different plants have different light saturating points, measuring the light dependence changes in relative limitations of A_N may have broad applications in tree breeding and crop improvement.

With an increase in light intensity, electron transport for photorespiration gradually increased [16,37,38,39]. However, it is unclear whether the light response of photorespiration was caused by the change of l_m or l_s. In this study, we found that l_s showed only small responses to changes in light intensity (Figure 3). By comparison, l_m gradually increased with light intensity (Figure 3). Meanwhile, C_c gradually decreased (Figure 5B) and the ratio of V_o/V_c gradually increased (Figure 6A). Furthermore, with an increase in light intensity, the decrease in C_c was tightly correlated to an increased l_m (Figure 5C). Thus, the stable g_m was insufficient to compensate for CO₂ consumption by A_N under high light, increasing the affinity of Rubisco to O₂, and thus enhancing photorespiration. Therefore, the light response of photorespiration is more determined by l_m rather than l_s (Figure 6B).

3.3. CO₂ Assimilation under High Light Tends to be Limited by RuBP Carboxylation in Rhododendron Species

In C₃ plants, A_N under saturating light can be limited by RuBP carboxylation and/or RuBP regeneration [21,42,43]. Once the value of C_c is lower than the value of C_trans, A_N tends to be limited by RuBP carboxylation [43]. When C_c is higher than C_trans, A_N is then limited by RuBP regeneration. However, the rate-limiting step of A_N in sclerophyllous species is poorly understood. In this study, we found that the values of operating C_c under saturating light were significantly lower than the values of C_trans in these species, apart from in R. decorum subsp. disprepes (Figure 7). Therefore, A_N under saturating light in these Rhododendron species was mainly limited by RuBP carboxylation.

In general, the rate-limiting step of A_N can be influenced by temperature and leaf nitrogen content [41,43]. For example, at normal growth temperatures, A_N is limited by RuBP carboxylation under nitrogen deficiency but tends to be limited by RuBP regeneration at high nitrogen conditions in C₃ crop species [43]. In this article, we found that the rate-limiting step of A_N by RuBP carboxylation in Rhododendron species was mainly caused by a relatively lower g_m (Figure 2 and Figure 7). In rice (Oryza sativa), wheat (Triticum aestivum), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), values for g_m at saturating light were higher than 0.2 mol m⁻² s⁻¹ [43,48]. By comparison, the studied Rhododendron species displayed g_m values ranging from 0.048 to 0.18 mol m⁻² s⁻¹ (Figure 2). Such low g_m in these Rhododendron species limited the diffusion of CO₂ from intercellular cavities to chloroplasts, resulting in C_c being lower than C_trans. Therefore, g_m has the potential to alter the rate-limiting step of A_N under high light.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

Nine evergreen sclerophyllous Rhododendron species from China were studied: R. decorum, R. glanduliferum, R. hancockii, R. decorum subsp. disprepes, R. ciliicalyx, R. delavayi, R. davidii, R. fortunei, and R. pachypodum. All plants of these species are cultivated in a common garden at the Kunming Botanical Garden, Yunnan, China (102°44′31″ E, 25°08′24″ N, 1950 m of elevation). We chose fully expanded but not senescent sun leaves for photosynthetic measurements. For each species, at least four leaves from different individual plants were measured.

4.2. Gas Exchange and Chlorophyll Fluorescence Measurements

Gas exchange and chlorophyll fluorescence were recorded using the 2-cm² measuring head of LI-6400XT (Li-Cor Biosciences, Lincoln, NE, USA). All measurements were conducted at approximately 25 °C and relative air humidity near 60%. After photosynthetic induction at 1500 μmol photons m⁻² s⁻¹ for 20 min, light response curves were recorded at a 400 μmol mol⁻¹ CO₂ concentration, and photosynthetic parameters were monitored after exposure to each light intensity for 2 min. After light adaptation at 1500 μmol photons m⁻² s⁻¹ and 400 μmol mol⁻¹ CO₂ concentration for 20 min, A/C_i measurements were made at 50, 100, 150, 200, 300, 400, 600, 800, 1000, and 1200 μmol mol⁻¹ CO₂ concentrations. For each CO₂ concentration, photosynthetic measurement was completed in 2 to 3 min. Using the A/C_i curves, the maximum rates of RuBP regeneration (J_max) and carboxylation (V_cmax) were calculated [49].

The quantum yield of photosystem II (PSII) photochemistry was calculated as Φ_PSII = (F_m’ − F_s)/F_m’ [50], where F_m’ and F_s represent the maximum and steady-state fluorescence after light adaption, respectively [51]. The total electron transport rate through PSII (J_PSII) was calculated as follows [52]:

$$J_{\mathrm{PSII}}={\mathsf{\Phi }}_{\mathrm{PSII}}\times \mathrm{PPFD}\times L_{\mathrm{abs}}\times 0.5,$$

where PPFD is the photosynthetic photon flux density and leaf absorbance (L_abs) is assumed to be 0.84. We applied the constant of 0.5 based on the assumption that photons were equally distributed between PSI and PSII.

4.3. Estimation of Mesophyll Conductance and Chloroplast CO₂ Concentration

We calculated mesophyll conductance according to the following equation [44]:

$$g_{\mathrm{m}}=\frac{A_{\mathrm{N}}}{C_{\mathrm{i}}-{\Gamma }^{\ast }\left(J_{\mathrm{PSII}}+8\left(A_{\mathrm{N}}+R_{\mathrm{d}}\right)\right)/\left(J_{\mathrm{PSII}}-4\left(A_{\mathrm{N}}+R_{\mathrm{d}}\right)\right)},$$

where A_N represents the net rate of CO₂ assimilation; C_i is the intercellular CO₂ concentration; and Γ* is the CO₂ compensation point in the absence of daytime respiration [41,48], and we used a typical value of 40 umol mol⁻¹ in our current study [19]. Respiration rate in the dark (R_d) was considered to be the half of the dark-adapted mitochondrial respiration rate as measured after 10 min of dark adaptation [23].

Based on the estimated g_m, we then calculated the chloroplast CO₂ concentration (C_c) according to the following equation [49,53]:

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

To identify the rate-limiting step of CO₂ assimilation, we subsequently estimated C_trans (the chloroplast CO₂ concentration at which the transition from RuBP carboxylation to RuBP regeneration occurred) [21,43]:

$$C_{\mathrm{trans}}=\frac{K_{\mathrm{c}}\left(1+O/K_{\mathrm{o}}\right)J_{\max }/4V_{\mathrm{cmax}}-2{\Gamma }^{\ast }}{1-J_{\max }/4V_{\mathrm{cmax}}}$$

where K_c (μmol mol⁻¹) and K_o (mmol mol⁻¹) are assumed to be 407 μmol mol⁻¹ and 277 mmol mol⁻¹ at 25 °C, respectively (Long and Bernacchi 2003); O was assumed to be 210 mmol mol⁻¹ (Farquhar et al., 1980). The rate-limiting step for CO₂ assimilation was analyzed by comparing the values of C_c and C_trans. A_N tends to be limited by RuBP carboxylation when C_c is lower than C_trans and tends to be limited by RuBP regeneration when C_c is higher than C_trans.

4.4. Quantitative Limitation Analysis of A_N

Relative photosynthetic limitations were assessed as follows [22]:

$$l_s=\frac{g_{\mathrm{tot}}/g_{\mathrm{s}}\times \partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}{g_{\mathrm{tot}}+\partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}$$

$$l_m=\frac{g_{\mathrm{tot}}/g_{\mathrm{m}}\times \partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}{g_{\mathrm{tot}}+\partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}$$

$$l_b=\frac{g_{\mathrm{tot}}}{g_{\mathrm{tot}}+\partial A_{\mathrm{N}}/\partial C_{\mathrm{c}}}$$

where l_s, l_m, and l_b represent the relative limitations of stomatal conductance, mesophyll conductance, and biochemical capacity, respectively, in setting the observed value of A_N. g_tot is the total conductance of CO₂ between the leaf surface and sites of RuBP carboxylation (calculated as 1/g_tot = 1/g_s + 1/g_m).

4.5. Modeling of V_c and V_o

The rates of RuBP carboxylation (V_c) and oxygenation (V_o) were calculated as follows [36]:

$$V_{\mathrm{c}}=\frac{A_{\mathrm{n}}+R_{\mathrm{d}}}{1-\left({\Gamma }^{\ast }/C_{\mathrm{c}}\right)}$$

and:

$$V_{\mathrm{o}}=\frac{A_{\mathrm{n}}+R_{\mathrm{d}}}{\left(C_{\mathrm{c}}/2{\Gamma }^{\ast }\right)-0.5}$$

4.6. Statistical Analysis

Data were displayed as means ± SE (n = 4–5). After testing for normality and homogeneity of variances, one-Way ANOVA tests were used at α = 0.05 significance level to determine whether significant differences existed between different averages.

5. Conclusions

In this study, we examined the light response of g_m and its effect on photosynthesis in nine evergreen sclerophyllous Rhododendron species. The results indicated that all species showed no significant response of g_m to variations in illumination. Therefore, the response of g_m to rapid changes in irradiance may be a conservative photosynthetic trait in a given genus, at least in the genus Rhododendron. Furthermore, we found that the light response of photorespiration was mainly determined by g_m limitation rather than g_s limitation. At saturating light, g_m limitation significantly affected the differentials between C_trans and C_c, thus altering the rate-limiting step of A_N. We propose that examining the light dependence changes in relative limitations of A_N can provide some valuable means for tree breeding and crop improvement.