Speed of light-induced stomatal movement is not correlated to initial or final stomatal conductance in rice

Abstract

In nature, plants are often confronted with wide variations in light intensity, which may cause a massive carbon loss and water waste. Here, we investigated the response of photosynthetic rate and stomatal conductance to fluctuating light among ten rice genotypes and their influence on plant acclimation and intrinsic water-use efficiency (WUE_i). Significant differences were observed in photosynthetic induction and stomatal kinetics across rice genotypes. However, no significant correlation was observed between steady-state and non-steady-state gas exchange. Genotypes with a greater range of steady-state and faster response rate of the gas exchange showed stronger adaptability to fluctuating light. Higher stomatal conductance during the initial phase of induction had little effect on the photosynthetic rate but markedly decreased the plant WUE_i. Clarification of the mechanism influencing the dynamic gas exchange and synchronization between photosynthesis and stomatal conductance under fluctuating light may contribute to the improvement of photosynthesis and water-use efficiency in the future.

Highlights

• Faster photosynthetic induction contributes to a stronger adaptation to fluctuating light

• No significant correlation was observed between steady-state and non-steady-state gas exchange

• Higher stomatal conductance during the initial phase of light induction decreased plant WUE_i

Introduction

Canopy photosynthesis is considered a major target for improving crops because of its importance for supporting plant growth and grain yield formation (Long et al. 2006, Lawson et al. 2012, Wu et al. 2019). Over the last decades, the steady-state leaf photosynthesis (amount of CO₂ assimilated per leaf area per time under a given environmental condition) has been widely studied and significant knowledge gaps have been filled. However, canopy photosynthesis in natural conditions is not always stable, due to environmental fluctuations, such as light, temperature, humidity, and ambient CO₂ concentration (Lawson et al. 2012, Kaiser et al. 2015, 2016, 2017; Adachi et al. 2019). Among those environmental factors, the light might be the most dynamic one, as its signals influence the response of both photosynthetic rate and stomatal conductance. In nature, incident irradiance on plant leaves often fluctuates due to changes in sun angle and cloud cover in addition to shading from overlapping leaves and neighboring plants (Pearcy et al. 1990, Kaiser et al. 2015). The acclimation of plants to light has been studied extensively and plants that grow under constant environmental conditions tend to have different morphology and biomass compared with the fluctuating environment (Poorter et al. 2016, Vialet-Chabrand et al. 2017a). Also, many studies have investigated the short-term acclimation of leaf gas-exchange parameters to changes of light intensity, which dominate the leaf carbon assimilation and water-use efficiency under fluctuating light (Lawson and Blatt 2014, Vialet-Chabrand et al. 2017b).

Stomatal aperture is controlled by guard cell turgidity, which is sensitive to light intensity. Thus, the kinetics of stomata play an important role in balancing the mesophyll demands for CO₂ against the need to maintain leaf water content under fluctuating irradiance (Lawson et al. 2014). However, the underlying mechanism of light-induced stomatal movement is still not fully understood (Kübarsepp et al. 2020, Lawson and Matthews 2020). Moreover, there is controversy about physical attributes affecting stomatal response times following environmental perturbations, since opposite relationships between g_s kinetics and stomatal morphology have been reported (Lawson and Blatt 2014, Elliott-Kingston et al. 2016, Vialet-Chabrand et al. 2016, Durand et al. 2019, Eyland et al. 2021). On the other hand, leaves with a higher initial or final steady state of the stomatal aperture also show a faster response rate to light fluctuations (Drake et al. 2013, Zhang et al. 2019), which is also consistent with the hypothesis that pre-dawn stomatal opening contributes to the faster response of stomata at early daytime (Auchincloss et al. 2014). In contrast, Acevedo-Siaca et al. (2021) showed that there is no correlation between steady- and non-steady-state gas exchange. In addition, De Souza et al. (2020) and Soleh et al. (2016) also showed a lack of significant correlation between steady- and non-steady-state photosynthesis in cassava and soybean, respectively. Therefore, further evidence is still needed to elucidate the relationship between steady- and non-steady-state gas exchange.

Previous studies have demonstrated a strong correlation between photosynthetic rate (P_N) and stomatal conductance (g_s) under a steady state (Farquhar and Sharkey 1982, Peguero-Pina et al. 2017, Xiong and Flexas 2020). However, plants are often confronted with a wide range of light intensity at the spatial and temporal level under field conditions. Upon a step increase in irradiation, photosynthesis and stomatal conductance exhibit a typically delayed response until reaching a new steady state (Scafaro et al. 2012). Stomatal conductance has a magnitude slower response than that of the photosynthetic rate to fluctuating light, which may be determined by initial and final g_s and the response rate of stomatal movement, causing a stomatal limitation to photosynthetic rate under fluctuating light (Lawson and Vialet-Chabrand 2019). Adachi et al. (2019) suggested that the higher stomatal conductance during photosynthetic induction is the primary factor for the rapid response of photosynthesis in rice under fluctuating light. Also, this nonsynchronization between P_N and g_s can cause a decrease in WUE_i (intrinsic water-use efficiency) towards the end of induction, when P_N has reached its steady state, whilst g_s continues to increase at the end of light induction (McAusland et al. 2016).

In the present study, ten rice genotypes were pot-grown in a natural environment with sufficient nutrition. The objectives of this study were to investigate: (1) the potential variations of dynamic P_N and g_s among rice genotypes, and their influence on leaf acclimation under fluctuating light, (2) the relationship between the steady and non-steady state of photosynthesis and stomatal conductance, and (3) the influence of nonsynchronization of P_N and g_s on plant water-use efficiency during light induction.

Materials and methods

Plant growth conditions

Ten genotypes of conventional and hybrid rice, including Huanghuazhan (HHZ), IDRA, ShanYou63 (SY63), YangLiangYou6 (YLY6), MingHui63 (MH63), YangDao6 (YD6), LiangYouPeiJiu (LYPJ), ChaoYou1000 (CY1000), ZhenShan97 (ZS97), and N22, were used in this study (Table 1S, supplement). Rice seeds were sown in plates with holes and filled with soil in a growth chamber with a 12-h light (28°C) and 12-h dark (23°C) cycle, and PAR of 400 μmol(photon) m^–2 s^–1 at the soil surface. Three fifteen-day-old seedlings were transplanted to 10-L pots filled with 10 kg crushed dry field paddy soil in March 2017. The nitrogen fertilizer application was 3 g(N) per pot and split-applied at a ratio of 4:3:3 at three phases including basal, tillering stage, and panicle initiation, which was applied in the form of urea. Respectively, 1.5 g of phosphorus (P) and potassium (K) were mixed into each pot as basal fertilizer and in the form of superphosphate and potassium chloride. For each genotype, three pots were prepared, and the pots were randomly rearranged weekly. Plants were grown outdoor (at the campus of Huazhong Agricultural University, Wuhan, China), and watered daily to avoid water deficit.

Leaf gas-exchange measurements

Photosynthetic rate (P_N) and stomatal conductance to water vapor (g_s) were measured on the youngest fully expanded leaves using a Li-6400XT portable photosynthesis system equipped with a 6400-40 leaf chamber (Li-Cor Inc., Lincoln, NE, USA). One day before the measurement, the pots were moved into a Conviron growth chamber (Controlled Environments Limited, Manitoba, Canada), and the air temperature, PPFD on the top canopy, and the relative humidity were set to 28°C, 400 μmol m^–2 s^–1, and 75%, respectively. To investigate the dynamics of photosynthesis, the leaves were first equilibrated at a PPFD of 100 μmol m^–2 s^–1 until P_N and g_s reached the ‘steady state’, which was defined as g_s at a < 1% change in rate during a 5-min period. Once the steady state was reached, PPFD was increased to 1,500 μmol m^–2 s^–1 for 700 s of light induction. During the measurement, the CO₂ concentration in the reference chamber, the leaf temperature, and the VPD were 400 μmol m^–2 s^–1, 28°C (± 1), and 1.3 ± 0.1 kPa, respectively. Gas-exchange parameters were recorded every 10 s. All measurements were conducted on the youngest fully expanded leaves at the tillering stage.

Photosynthetic induction

The response of photosynthetic induction was calculated with a previously reported method (Chazdon and Pearcy 1986, Kaiser et al. 2017) as follows: photosynthetic induction = (P_N – P_i)/(P_f – P_i) × 100, where P_N [μmol m^–2 s^–1] is the value at 60 s, P_f represents the final rate of induction (mean value of 50 s), and P_i is the initial value (mean value of 50 s).

P₉₀ of P_N and P₅₀ of P_N was the time taken for P_N to increase 90 and 50% of the difference between the initial and final values during induction within 700 s after shifting to high light. The relative rate of increase in g_s (P₉₀ of g_s, P₅₀ of g_s) during photosynthetic induction was also calculated. Intrinsic water-use efficiency (WUE_i) was calculated as P_N/g_s, and the integrated amount of CO₂ assimilation (carbon gain) was calculated as P_t × d_t, where P_t represents the photosynthetic rate across the measured period from the initial to the final phase of 700 s, and d_t represents the integrated amount of time during 700 s of light induction.

Induction limitation analysis

Transient stomatal (LS) and biochemical (LB) limitation during photosynthetic induction were calculated according to Woodrow and Mott (1989) and Urban et al. (2007):

$$P^{\ast }=\frac{\left(P_{\text{N}}+R_{\text{d}}\right)\left(C_{\text{if}}-{\Gamma }^{\ast }\right)}{C_{\text{i}}-{\Gamma }^{\ast }}-R\text{d}$$

where P^* represents the rate of CO₂ assimilation without stomatal limitation, C_if is the final C_i at the end of the induction period, Γ^* is the chloroplast CO₂-compensation point in the absence of photorespiration, and R_d is the dark respiration rate. In the present study, a Γ^* value of 40 μmol mol^–1 and R_d value of 1 μmol m^–2 s^–1 were used for rice leaves (Yamori et al. 2011, Xiong et al. 2015). Subsequently, LS and LB during the photosynthetic induction phase were calculated as: LS = (P^* – P_N)/(P_f + R_d), LB = (P_f – P^*)/(P_f + R_d), where P_f is the final photosynthetic rate of light induction.

Statistical analysis

One-way analysis of variance (ANOVA) and the least-significant difference (LSD) test were used to assess the measured parameters among different genotypes using SPSS 21.0 (SPSS for Windows, Chicago, Illinois, USA). Linear regression was analyzed to test the correlation among measured parameters using SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA).

Results

Photosynthetic induction under fluctuating light

After a step increase in light intensity, P_N increased and rapidly reached the maximum value. However, the stomatal opening was rather slow and the g_s did not reach the maximum after 700 s of high light exposure (Fig. 1). The P₉₀ of P_N varied from 224 to 307 s and that of g_s varied from 134 to 434 s (Fig. 2C). The photosynthetic induction and stomatal opening were independent of their initial and/or final values (Fig. 3A,B). The carbon gain during photosynthetic induction differed significantly between genotypes (Fig. 2F). The values of both P_f – P_i and g_sf – g_si positively correlated with carbon gain during the light induction (Fig. 4A,B), but there was a lack of a link between gas-exchange induction (represented by P₅₀ or P₉₀) and carbon gain. Limitation analysis showed that during the initial phase, biochemical limitation accounted for approximately 80%, but declined rapidly at high light level (Fig. 5). Conversely, the stomatal limitation was low at the initial phase and increased gradually after exposure to high light. P_f and P₃₀₀ were positively correlated with g_sf and g_s,300, but no positive correlation was observed between P_i and g_si, indicating the nonsynchronization of P_N and g_s in the initial phase of induction (Fig. 6).

Fig. 1. Response of gas exchange to a step increase of light intensity among ten rice cultivars. (A) Photosynthetic rate (P_N), (B) stomatal conductance (g_s). Low light (shade area) and high light (open area) were 100 and 1,500 μmol m^–2 s^–1, respectively. Each point represents the mean of three replications.

Fig. 2. Calculations of gas-exchange parameters after a step increase in light intensity across ten rice genotypes. (A,B) Variations of range from minimum values to maximum values of photosynthesis and stomatal conductance, (C,D) the time taken for P_N and g_s to increase 90% of the difference between the first and final values (P₉₀ of P_N, P₉₀ of g_s), (E) the rate of photosynthetic induction at 60 s (IS₆₀), and (F) carbon assimilation during 700 s of photosynthetic induction. Each bar represents the mean (+ SD) of three replications across two pairs of diploid and tetraploid rice. Different letters indicate statistically significant differences (P<0.05) between rice genotypes.

Fig. 3. Relationship between steady-state and dynamic response rate of stomatal conductance and photosynthesis. (A–D) Relationship between dynamic response rate of gas exchange and initial values, (E,F) relationship between dynamic response rate of gas exchange and final values. Each point represents the mean (+ SD) of three replications.

Fig. 4. Relationship between carbon gain during light induction and gas exchange. (A,B) Relationship between carbon gain and variations from the initial phase to the final phase of stomatal conductance (g_s), (C,D) relationship between carbon gain and variations from the initial phase to the final phase of photosynthetic rate (P_N). Each point represents the mean (+ SD) of three replications.

Fig. 5. Transient stomatal (LS) and biochemical limitation (LB) during photosynthetic induction of ten rice cultivars. The gray points represent the stomatal limitation, and the orange points are biochemical limitation to photosynthesis after a step increase in light intensity. Each curve represents the mean of three replications.

Fig. 6. Relationship between photosynthesis and stomatal conductance under different light intensity. (A,C) Relationship of stomatal conductance and photosynthesis under low light level and high light level, (B) relationship of stomatal conductance and photosynthesis after 300 s of induction, and (D) relationship between variations of stomatal conductance and photosynthetic rate from the initial phase to the final phase. Each point represents the mean (+ SD) of three replications.

Variation of initial and final gas exchange across rice genotypes

The steady-state gas-exchange parameters varied significantly among rice genotypes. The g_si ranged from 0.09 to 0.28 mol m^–2 s^–1 and g_sf ranged from 0.46 to 0.82 mol m^–2 s^–1, respectively (Table 1). Consistently, across the investigated genotypes, the P_f ranged from 24.7 to 34.0 μmol m^–2 s^–1, and P_i from 4.36 to 7.88 μmol m^–2 s^–1, respectively. The difference between initial and final gas-exchange parameters (P_f – P_i, g_sf – g_si) was calculated. Substantial variations in the value of P_f – P_i (18.8–27.4 μmol m^–2 s^–1) and g_sf – g_si (0.29–0.55 mol m^–2 s^–1) were observed across rice genotypes (Fig. 2A,B; Table 1). The genotypes with higher g_sf – g_si, including Huanghuazhan, IDRA, Yangdao6, Yangliangyou6, Shanyou63, tended to have higher P_f – P_i values. The significant difference was observed in WUE_i among ten rice genotypes under different light conditions, particularly under low light (W_i) (Table 1). Moreover, W_i and W_f were strongly correlated with g_si and g_sf, respectively, but not with P_f (Fig. 7).

Table 1. Gas-exchange parameters of initial photosynthetic rate (P_i), final photosynthetic rate (P_f), initial stomatal conductance (g_si), final stomatal conductance (g_sf), initial water-use efficiency (W_i), and final water-use efficiency (W_f) during the initial and final phases of light induction. All data are shown as mean ± SD of three replications. The data with different lowercase letters in each column were significantly different at P<0.05 level.

Genotypes	Pi [μmol m–2 s–1]	Pf [μmol m–2 s–1]	gsi [mol m–2 s–1]	gsf [mol m–2 s–1]	Wi [μmol mol–1]	Wf [μmol mol–1]
HHZ	4.49 ± 1.39c	31.9 ± 5.0abc	0.28 ± 0.08a	0.82 ±0.14a	16.1 ± 3.1d	38.7 ± 0.4cd
IDRA	6.93 ± 1.48ab	34.0 ± 1.1a	0.17 ± 0.04bc	0.60 ± 0.04bc	43.0 ± 16.4bcd	56.7 ± 1.7ab
YD6	6.23 ± 0.16abc	32.8 ± 0.7a	0.23 ± 0.05ab	0.78 ± 0.08bc	28.5 ± 6.3bcd	42.5 ± 3.9bcd
YLY6	7.88 ± 1.42a	32.6 ± 1.4ab	0.14 ± 0.04bc	0.63 ± 0.20abc	61.2 ± 25.0ab	55.4 ± 17.5ab
SY63	7.26 ± 0.35ab	31.9 ± 2.4abc	0.14 ± 0.01bc	0.66 ± 0.14abc	53.3 ± 7.2abc	49.3 ± 7.2abcd
CY1000	4.36 ± 1.51c	27.1 ± 4.2cd	0.23 ± 0.08ab	0.77 ± 0.11ab	22.6 ± 17.0cd	36.4 ± 11.4d
MH63	6.23 ± 0.99abc	27.5 ± 3.9bcd	0.09 ± 0.04c	0.46 ± 0.07c	83.2 ± 37.5a	59.7 ± 0.8a
ZS97	6.27 ± 0.47abc	26.0 ± 0.8d	0.19 ± 0.06ab	0.54 ± 0.10c	47.7 ± 12.8bcd	49.1 ± 10.8abcd
LYPJ	5.95 ± 0.12abc	25.5 ± 1.2d	0.19 ± 0.05ab	0.48 ± 0.07c	32.6 ± 9.3bcd	54.1 ± 10.9abc
N22	5.87 ± 0.54bc	24.7 ± 2.7d	0.22 ± 0.04ab	0.62 ± 0.09abc	27.7 ± 7.6cd	40.1 ± 1.9bcd

Fig. 7. Relationship between water-use efficiency and gas exchange. (A,B) Relationship between W_i and g_si, as well as W_i and P_i under low light level, (C,D) relationship between W_f and g_sf, as well as W_f and P_f under high light level. Each point represents the mean (+ SD) of three replications.

Discussion

The steady-state gas exchange varies greatly among rice genotypes

In nature, plants usually experience a wide range of spatial and temporal variations in light intensity, which leads to simultaneous fluctuations in leaf carbon assimilation and water loss (Pearcy et al. 1990, Lawson and Blatt 2014). When a shaded leaf is suddenly exposed to irradiation, the photosynthesis will slowly increase to reach a new stable steady state. This process is called photosynthetic induction, which takes seconds to hours and depends on stomatal and biochemical limitations (Kaiser et al. 2017, Zhang et al. 2018). Significant differences were observed between rice genotypes in their response rate of photosynthesis to light fluctuations, especially in the early phase of induction (Acevedo-Siaca et al. 2020). Moreover, no correlation was found between different growth stages in steady and dynamic gas-exchange parameters in rice (Acevedo-Siaca et al. 2021). Similarly, we observed significant differences in photosynthetic induction (IS₆₀) and response rate (P₅₀ of P_N, P₉₀ of P_N) across ten rice genotypes under a stepwise increase in irradiance (Fig. 2). However, the significant differences were more likely to be found during the whole process, rather than only in the initial phase (Fig. 1). Consistently, significant differences were also observed in the response rate of stomatal conductance to fluctuating light (P₉₀ of g_s) (Fig. 3D). Generally, stomatal response to changing conditions is an order of magnitude slower than the photosynthetic response in some plant species, which possibly causes a 10–15% stomatal limitation on photosynthesis (McAusland et al. 2016, Lawson and Vialet-Chabrand 2019).

In this study, the rate of steady-state leaf photosynthesis varied widely among rice cultivars (Table 1), which is consistent with previous results (Kanemura et al. 2007). However, little research has noticed the scope of photosynthetic rate and stomatal conductance ranges from low light to high light conditions. Significantly, we observed great variations in P_f – P_i under fluctuating light (Fig. 2A,B). Interestingly, the genotypes with higher P_f – P_i values (HHZ, IDRA, YLY6, YD6, SY63) also exhibited faster photosynthetic responses to light fluctuations, especially for P₉₀ of P_N and P₉₀ of g_s, which would result in higher carbon assimilation (Fig. 2F). Furthermore, great variations were also observed in the g_sf – g_si values. The rice genotypes with higher g_sf – g_si values, including HHZ, YD6, YLY6, and SY63, exhibited a faster response rate of stomatal opening to light fluctuations (Fig. 2B,D), which is significant for breeding research, as these genotypes may have stronger adaptability to fluctuating light (Fig. 2A,C), as well as higher carbon assimilation and WUE_i in the field. A higher photosynthetic rate has always been a major target for improving crop performance (Yamori et al. 2016). A faster response rate can help maintain higher photosynthetic efficiency under increasing irradiation and therefore contribute to higher biomass in a natural environment.

The influences of initial stomatal opening state on light-induced stomatal kinetics

Previous studies have suggested that light-induced stomatal kinetics is related to stomatal morphology including stomatal size, density, and shape (Franks and Beerling 2009, Drake et al. 2013, Raven 2014, Lawson and Blatt 2014, McAusland et al. 2016). It has also been demonstrated that plant species with a higher density of small stomata tend to have a faster stomatal response rate to environmental fluctuations (Franks and Beerling 2009, Drake et al. 2013, Vialet-Chabrand et al. 2016). However, Elliott-Kingston et al. (2016) suggested that darkness-induced stomatal closing rate was not correlated with stomatal size but related to atmospheric CO₂ concentration at the time of taxa diversification (Elliott-Kingston et al. 2016). In addition, plant species with dumbbell-shaped guard cells have much faster stomatal kinetics under fluctuating light than those species with elliptical-shaped guard cells (McAusland et al. 2016), since dumbbell-shaped guard cells require lower energy to change the stomatal aperture than elliptical-shaped guard cells (Hetherington and Woodward 2003, Franks and Farquhar 2007, Raven 2014). Recently, several studies have noticed that stomatal kinetics may be related to minimum and maximum stomatal conductance during light induction (Zhang et al. 2019). One hypothesis concerning nocturnal transpiration is that ‘pre-opening’ at dawn may help the stomata reach the maximum aperture more rapidly, and reduce the diffusional limitation of CO₂ uptake in the early daytime (Dawson et al. 2007, Drake et al. 2013). In a previous study, one-hour low-humidity treatments to reduce predawn nocturnal stomatal aperture do affect the response rate of stomatal conductance and photosynthesis at the first several minutes after dawn (Auchincloss et al. 2014). However, in the present study, no correlation was found between g_si and the response rate of stomatal conductance (P₅₀ of g_s, P₉₀ of g_s), as well as P_i and the response rate of photosynthesis (P₅₀ of P_N, P₉₀ of P_N) (Fig. 3A,B,D,E). The disconnection between initial and response rate suggested that more research attention should be paid to the specific mechanisms of these dynamic processes, which largely determine the carbon assimilation of plants in the natural environment.

Stomatal size and density are potential determinants of leaf diffusive conductance to CO₂ and water vapor (Franks et al. 2009). There is usually a negative relationship between stomatal size and density (Xiong et al. 2018). Smaller stomata are generally coupled with a higher maximum stomatal conductance and higher photosynthetic capacity (Franks and Beerling 2009), enhance plant fitness in a broader range of environments, and are capable of achieving a faster response rate (Hetherington and Woodward 2003, Raven 2014, Lawson and Vialet-Chabrand 2019). However, Acevedo-Siaca et al. (2020, 2021) recently suggested that there is still a lack of further evidence for the correlation between a steady-state and dynamic gas exchange since little correlation was found between the maximum value and the response rate. This is consistent with the present study (Fig. 3C). One possible explanation may be the distribution of resources for photosynthetic proteins, including the content of Rubisco and Rubisco activase, which may dominate the steady-state and dynamic process of photosynthesis (Acevedo-Siaca et al. 2021). Similarly, no correlation was found between the maximum value and response rate of stomatal conductance under fluctuating light in this study. This might be partly attributed to the mechanism underlying light-induced stomatal movement, in which red light induction is believed to connect stomatal kinetics and mesophyll CO₂ assimilation (Matthews et al. 2020), though the exact ‘mesophyll signals’, which are transferred from mesophyll or chloroplast to guard cells and trigger the guard cell function, have not been fully elucidated (Lawson et al. 2014). Besides, the supply of osmoticum and energy by guard cell photosynthesis may also contribute to the stomatal movement under fluctuating light (Santelia and Lawson 2016). Overall, the light-induced stomatal behavior was not correlated with steady-state values and might be associated with the inside ‘signals’ stimulated by a fluctuation of environments outside.

Stomatal kinetics and the implications for carbon and water economics under light fluctuation

Stomata are micropores composed of pairs of guard cells, which control nearly all CO₂ absorption and water loss of plant leaves (Caird et al. 2007). The stomatal movement under fluctuating light plays a key role in leaf carbon assimilation and WUE_i (Ooba and Takahashi 2003, Vico et al. 2011, McAusland et al. 2016). Delay in the increase or decrease in g_s response after a step change in irradiance has been reported in many experiments, which may result in a nonsynchronous stomatal conductance and photosynthetic rate (Lawson et al. 2010, Vico et al. 2011, Lawson and Blatt 2014). The g_s is significantly correlated with P_N between species in a natural environment, as a higher CO₂ assimilation rate may require a larger pore aperture (Peguero-Pina et al. 2017). This is consistent with our result under high light level, as final stomatal conductance (g_sf) was positively correlated with the final photosynthetic rate (P_f), P₃₀₀, and g_s,300 as well (Fig. 6B,C). Differently, no positive correlation was observed between the initial stomatal conductance (g_si) and initial photosynthetic rate (P_i), which might indicate that nonsynchronous stomatal conductance and photosynthesis existed at the beginning of photosynthetic induction (Fig. 6A) and this nonsynchronicity after a step change in light intensity is consistent with previous results (Lawson and Blatt 2014).

C_i decreased rapidly at first and then reached a steady state gradually with a step increase in irradiance. Compared with the initial phase, C_i was lower at the steady state (Fig. 1S, supplement), which, to some extent, suggested g_si was higher than needed for carboxylation. The stomatal limitation was lower approximately less than 10% during photosynthetic induction across ten rice genotypes, especially at the beginning of induction (Fig. 5), again indicating that g_si was exorbitant. This is consistent with Acevedo-Siaca et al. (2020) and photosynthetic induction was strongly limited by nonstomatal limitations, and stomatal limitation only increased gradually from 2% to 10–15% over the first 300 s. Furthermore, W_i was lower during the initial phase and mainly dominated by stomatal conductance (Fig. 7A,B; Fig. 2S, supplement), which might indicate that higher stomatal conductance during the initial phase decreased leaf W_i and had little influence on photosynthetic induction. Modeled synchrony behavior in stomatal conductance and photosynthesis has been shown to theoretically increase WUE_i by 20% in a bean leaf exposed to dynamic light (Lawson and Blatt 2014). Improving synchronous photosynthesis and stomatal conductance at the beginning of induction will, to some extent, benefit the improvement of plant WUE_i under natural conditions. As it has been shown above, leaf W_i and W_f were mainly determined by stomatal conductance at low light and high light levels (Fig. 7). The results suggested that decreasing stomatal conductance during the initial phase of induction might benefit the balance between carbon assimilation and water loss under fluctuating light.

Conclusion

This study demonstrates significant differences between ten rice genotypes in steady-state and dynamic photosynthesis and stomatal conductance. No significant correlation was observed between steady-state and non-steady-state gas exchange. The genotypes with greater variations in steady-state gas exchange and faster response rate of dynamic gas exchange could have higher carbon assimilation and may have stronger adaptability to the natural environment than other genotypes. Higher stomatal conductance during the initial phase of induction has little influence on photosynthetic rate but reduces plant WUE_i. The findings of the present study might contribute to the exploration of the deeper mechanism of dynamic photosynthetic rate and stomatal movement under fluctuating light.

Abbreviations

C _i

intercellular CO₂ concentration

C _if

final intercellular CO₂ concentration

g _s

stomatal conductance

g_s,300

stomatal conductance at 300 s of induction

g _sf

final stomatal conductance

g _si

initial stomatal conductance

LB

transient biochemical limitation

LS

transient stomatal limitation

P _f

final photosynthetic rate

P _i

initial photosynthetic rate

P _N

photosynthetic rate

P₅₀ of g_s

the time taken for g_s to increase 50% of the difference between the first and final values

P₉₀ of g_s

the time taken for g_s to increase 90% of the difference between the first and final values

P₅₀ of P_N

the time taken for P_N to increase 50% of the difference between the first and final values

P₉₀ of P_N

the time taken for P_N to increase 90% of the difference between the first and final values

P ₃₀₀

photosynthetic rate at 300 s of induction

R _d

dark respiration rate

W_f

final intrinsic water-use efficiency

W_i

initial intrinsic water-use efficiency

WUE_i

intrinsic water-use efficiency

Γ^*

CO₂-compensation point in the absence of photorespiration

Conflict of interest

The authors declare that they have no conflict of interest.