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

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


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 CO 2 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.

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 CO 2 assimilation at saturating light in Rhododendron species, followed by biochemical capacity and stomatal conductance.

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 (lb) ( Figure  3), owing to the lack of ATP and NADPH. With an increase in light intensity, lb gradually decreased ( Figure 3). Meanwhile, mesophyll conductance limitation (lm) gradually increased and stomatal conductance limitation (ls) 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 lb to lm, except for the species with the highest gm: R. decorum subsp. diaprepes (Figure 3). Under the saturating light of 1500 μmol photons m −2 s −1 , lm ranged from 0.37 to 0.71, lb from 0.15 to 0.37, and ls from 0.12 to 0.26 ( Figure 3). Therefore, gm is the major limiting factor for CO2 assimilation at saturating light in Rhododendron species, followed by biochemical capacity and stomatal conductance.
The relationships between gm, lm, and AN at sub-saturating and saturating light intensities were also analyzed in these studied species ( Figure 4). As expected, the values of gm were significantly positively correlated to AN. Interestingly, a closer relationship between gm and AN 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 lm and AN 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 gm and lm play more important roles in determining AN at sub-saturating light than at saturating light.  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

Light Intensity Dependence of Chloroplast CO2 Concentration and Photorespiration
With an increase in light intensity, intercellular CO2 concentration (Ci) and chloroplast CO2 concentration (Cc) gradually decreased in all Rhododendron species ( Figures 5A,B). At 1500 μmol photons m −2 s −1 , Ci ranged from 253 (R. decorum subsp. diaprepes) to 317 μmol mol −1 (R. glanduliferum) ( Figure 5A), and Cc ranged from 76 (R. hancockii) to 144 μmol mol −1 (R. decorum subsp. diaprepes) ( Figure 5B). Further analysis found that the drop in Cc was tightly correlated with an increase in lm ( Figure 5C). Therefore, the light dependence change in Cc was mainly caused by an increase in lm. Cc is known to be a key factor affecting the affinity of Rubisco to CO2 and O2, and thus determines the value of Vo/Vc. With an increase in illumination, the Vo/Vc ratio gradually increased ( Figure 6A). Furthermore, the light dependence of Vo/Vc was positively correlated to lm ( Figure 6B). These results indicate that the increased Vo/Vc with increasing irradiance was mainly caused by the enhanced lm.
At 1500 μmol photons m −2 s −1 , Ctrans was calculated to analyze the rate-limiting step of AN. The operating Cc values are significantly lower than the Ctrans values in these studied species, except for R. decorum subsp. diaprepes (Figure 7). Therefore, the saturating AN at current atmospheric CO2 concentration was mainly limited by RuBP carboxylation in these Rhododendron species. In R. decorum subsp. diaprepes, AN was limited by RuBP carboxylation and regeneration.

Light Intensity Dependence of Chloroplast CO 2 Concentration and Photorespiration
With an increase in light intensity, intercellular CO 2 concentration (C i ) and chloroplast CO 2 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 CO 2 and O 2 , 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 CO 2 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.

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 CO 2 diffusion from air to sub-stomatal internal cavities and thus to favor photosynthetic CO 2 assimilation [45][46][47]. Subsequently, mesophyll conductance limits the diffusion of CO 2 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.

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 CO 2 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 Plants 2020, 9, 1536 9 of 14 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 CO 2 consumption by A N under high light, increasing the affinity of Rubisco to O 2 , and thus enhancing photorespiration. Therefore, the light response of photorespiration is more determined by l m rather than l s ( Figure 6B).

CO 2 Assimilation under High Light Tends to be Limited by RuBP Carboxylation in Rhododendron Species
In C 3 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 C 3 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 (Figures 2 and 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 CO 2 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.

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.

Gas Exchange and Chlorophyll Fluorescence Measurements
Gas exchange and chlorophyll fluorescence were recorded using the 2-cm 2 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 CO 2 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 CO 2 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 CO 2 concentrations. For each CO 2 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]: 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.

Estimation of Mesophyll Conductance and Chloroplast CO 2 Concentration
We calculated mesophyll conductance according to the following equation [44]: where A N represents the net rate of CO 2 assimilation; C i is the intercellular CO 2 concentration; and Γ* is the CO 2 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 CO 2 concentration (C c ) according to the following equation [49,53]: To identify the rate-limiting step of CO 2 assimilation, we subsequently estimated C trans (the chloroplast CO 2 concentration at which the transition from RuBP carboxylation to RuBP regeneration occurred) [21,43]: 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 CO 2 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 .

Quantitative Limitation Analysis of A N
Relative photosynthetic limitations were assessed as follows [22]: l s = g tot /g s × ∂A N /∂C c g tot + ∂A N /∂C c l m = g tot /g m × ∂A N /∂C c g tot + ∂A N /∂C c l b = g tot g tot + ∂A N /∂C 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 CO 2 between the leaf surface and sites of RuBP carboxylation (calculated as 1/g tot = 1/g s + 1/g m ).

Modeling of V c and V o
The rates of RuBP carboxylation (V c ) and oxygenation (V o ) were calculated as follows [36]: and: V o = A n + R d (C c /2Γ * ) − 0.5

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.

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.