Increase rate of light-induced stomatal conductance is related to stomatal size in the genus Oryza.

The rapid response of stomatal conductance (gs) to fluctuating irradiance is of great importance to maximize carbon assimilation while minimizing water loss. Smaller stomata have been proven to have a faster response rate than larger ones, but most of these studies have been conducted with forest trees. In the present study, the effects of stomatal anatomy on the kinetics of gs and photosynthesis were investigated in 16 Oryza genotypes. Light-induced stomatal opening includes an initial time lag (λ) followed by an exponential increase. Smaller stomata had a larger maximum stomatal conductance increase rate (Slmax) during the exponential increase phase, but showed a longer time lag and a lower initial stomatal conductance (gs,initial) at low light. Stomatal size was, surprisingly, negatively correlated with the time required to reach 50% of maximum gs and photosynthesis (T50%gs and T50%A), which was shown to be positively correlated with λ and negatively correlated with gs,initial. With a lower gs,initial and a larger λ, small stomata showed a faster decrease of intercellular CO2 concentration (Ci) during the induction process, which may have led to a slower apparent Rubisco activation rate. Therefore, smaller stomata do not always benefit photosynthesis as reported before; the influence of stomatal size on dynamic photosynthesis is also correlated with λ and gs,initial.

Introduction

Light intensity in a plant canopy inherently varies temporarily in magnitude (1–2000 µmol m−2 s−1) and time (subseconds to minutes or longer) (Pearcy ; Barradas ; Leakey ; Smith and Berry, 2013). Leaves in the understory of a canopy obtain 10–90% of energy from these transient sunflecks (Pearcy ; Leakey ; Lawson ; Pearcy and Way, 2012), depending on forest type and plant age. The ability to utilize this energy varies significantly between species (Kaiser , 2018), and improving the energy utilization efficiency of sunflecks is an attractive approach to increase canopy photosynthesis and food production. Simulation analysis showed that leaf cumulative CO2 assimilation of wheat could potentially be improved by 21% by increasing the response rate of photosynthetic rate (A, see Table 1 for a list of abbreviations) to sunflecks (Taylor and Long, 2017). Plant biomass of transgenetic tobacco with an improved recovery rate of PSII under fluctuating light is 15% higher than that of the wild type (Kromdijk ).

Table 1.

List of symbols including units and values

Variable	Symbol	Unit	Value (25 °C)
Steady-state photosynthetic rate	A steady	µmol m−2 s−1
Steady-state stomatal conductance	g s,steady	mol m−2 s−1
Steady-state intercellular CO2 concentration	C i,steady	µmol−1 mol
Initial stomatal conductance	g s,initial	mol m−2 s−1
Initial photosynthetic rate	A initial	µmol m−2 s−1
Initial intercellular CO2 concentration	C i,initial	µmol−1 mol
Cuticular conductance	g cut	mol m−2 s−1
Guard cell length	L	µm
Guard cell width	W	µm
Stomatal density at abaxial leaf surface	D aba	mm−2
Stomatal density at adaxial leaf surface	D ada	mm−2
Stomatal size at abaxial leaf surface	S aba	µm2
Stomatal size at adaxial leaf surface	S ada	µm2
Maximum theoretical stomatal conductance	g s,max	mol m−2 s−1
Diffusivity of water vapor in air	d	m−2 s−1	24.9 ×10–6 m−2 s−1
Molar volume of air	v	m3 mol−1	22.4×10–3 m3 mol−1
Maximum area of the open stomatal pore	a max	µm2	π(p/2)2
Stomatal pore length	p	µm	L/2
Stomatal pore depth	l	µm	W/2
Rate of the Rubisco activation	1/τ	s−1
Initial time lag for stomatal response	λ	s
Time constant for stomatal response	K	s
Maximum stomatal conductance increase rate	Slmax	mmol m−2 s−2
Time to 50% of the steady-state photosynthetic rate after shifting to high light	T 50%A	s
Time to 50% of the steady-state stomatal conductance after shifting to high light	T 50%gs	s
Vapor pressure deficit	VPD	kPa
Air relative humidity	RH	%
Photosynthetic photon flux density	PPFD	µmol m−2 s−1

The response rate of A to a step increase of irradiance is predominantly determined by the stomatal opening rate and the activation rate of Calvin cycle enzymes, especially of Rubisco (Lawson ; Kaiser ; Tomimatsu and Tang, 2016; Meinzer ). Stomata control both CO2 uptake and H2O transpiration, and hence a rapid response of stomatal conductance (gs) to fluctuating light can both maximize CO2 assimilation and minimize unnecessary water loss. However, stomatal response to a step change of irradiance is an order of magnitude slower than A (Vico ; McAusland ; Sun ). When the initial stomatal conductance (gs,initial) is low, the delayed response of gs in comparison with A will inevitably limit CO2 assimilation but improve water use efficiency. The temporal response of gs to fluctuating light has recently received a great deal of attention, and plenty of these studies have been focused on the influence of stomatal morphology and anatomy.

In general, dumbbell-shaped stomata could respond to light faster than kidney-shaped stomata (Hetherington and Woodward, 2003; Franks and Farquhar, 2007; McAusland ), and smaller dumbbell-shaped stomata can open faster than large ones (Drake ; McAusland ; Kardiman and Ræbild, 2018). It is hypothesized that the faster response of smaller stomata may be caused by a greater guard cell membrane surface area to volume ratio, which enables more rapid changes in solutes and guard cell volume. But this hypothesis may not be valid in kidney-shaped stomata (McAusland ). Moreover, Elliott-Kingston and Xiong found no correlation between stomatal size and stomatal closing rate in response to darkness in a wide range of plant species. This suggests that the effects of stomatal size on gs response should be studied in closely related plant species (Drake ; Lawson and Vialet-Chabrand, 2018). The effects of stomatal anatomy on stomatal movement have been discussed in detail in Lawson and Vialet-Chabrand (2018).

Most of these studies on the effects of stomatal anatomy on stomatal movement have been conducted with forest trees. This kind of study on cereal crops such as rice is rare. There are more than 20 species in the genus Oryza. Oryza sativa L. is the most important rice species and is widely cultivated across the world. Large variations in stomatal anatomy (Xiong ) and stomatal response rate to changing irradiance (Qu ) have been documented in the Oryza genus. But neither of these studies has investigated the differences in stomatal anatomy between O. sativa and wild rice species and the correlation between stomatal anatomy and stomatal kinetics.

Light-induced stomatal opening includes three processes, with an initial time lag (λ) followed by an exponential increase and a stabilization at steady-state (gs,steady) (Naumburg and Ellsworth, 2000; Vialet-Chabrand ). Most previous studies have used the parameter of Slmax, which represents the stomatal conductance increase rate at the exponential increase phase, as a measure of the rapidity of stomatal response (Franks and Farquhar, 2007; Drake ; Meinzer ). However, it is important to consider the effects of both stomatal delay and gs,initial on CO2 assimilation when leaves are exposed to short-duration sunflecks, where stomata do not have enough time to enter the exponential phase. In fact, λ varies significantly from 0.11 to 6.12 min among different plant species (McAusland ), and the rapidity of the photosynthetic response to a step increase of irradiance is highly and positively correlated with gs,initial (Kirschbaum and Pearcy, 1988; Naumburg and Ellsworth, 2000; Leakey ; Way and Pearcy, 2012). However, there is little information for the determinants of λ and gs,initial and for the effects of stomatal anatomy on these parameters.

In low light, more than 50% of total Rubisco is deactivated (Pearcy, 1988; Lan ; Carmo-Silva and Salvucci, 2013), and it should be reactivated in high light before CO2 assimilation can occur. In addition to increasing Rubisco activase, elevated CO2 concentration can also improve Rubisco activation rates (Mott and Woodrow, 1993; Woodrow ; Carmo-Silva and Salvucci, 2011, 2013; Kaiser ,). Moreover, the apparent Rubisco activation rate (1/τ) is reported to be faster with a slower decrease in intercellular CO2 concentration (Ci) during the photosynthetic induction process (Kaiser ,b), and with a larger initial gs before exposure to high light (Valladares , Allen and Pearcy, 2000; Urban ). This suggests that the stomatal opening rate in response to light may affect the Rubisco activation rate through internal CO2 concentration. However, to the best of our knowledge, there is no study investigating the correlation between stomatal anatomy and Rubisco activation rate.

In the present study, a pot experiment was conducted with 16 Oryza genotypes. Stomatal anatomy and the kinetics of gs and photosynthesis were investigated. The objectives of this study were: (i) to investigate the differences in stomatal anatomy between O. sativa and wild genotypes; (ii) to investigate the influence of stomatal anatomy on the kinetics of gs, including Slmax, λ, and gs,initial, in Oryza genus; and (iii) to investigate the influence of stomatal anatomy on apparent Rubisco activation rate.

Materials and methods

Plant material and growth conditions

A pot experiment was conducted outdoors with 16 rice genotypes, including eight O. sativa genotypes and eight wild genotypes, at Huazhong Agricultural University (114.37°E, 30.48°N), Wuhan, China. After germination on a moist filter paper on 12 May 2018, seeds were transferred to nursery plates. When the seedlings had developed an average of 2.5 leaves, they were transplanted to 11.0-litre pots with a density of three seedlings per pot. Each pot was filled with 10.0 kg of soil. Nitrogen (N), phosphorus (P), and potassium (K) were applied at the rates of 2.0, 1.5, and 1.5 g pot−1, respectively. Fertilizers were applied by mixing them into the soil. Rice plants were watered daily, and a minimum layer of 2 cm of water was maintained to avoid drought stress. Pests were intensively controlled using chemical pesticides. Measurements were performed between 40 and 60 d after emergence.

Gas exchange measurements

To avoid environmental fluctuation and midday depression of photosynthesis, the seedlings were transferred to a controlled-environment growth room (Model GR48; Conviron, Controlled Environments Limited, Winnipeg, MB, Canada) on the night before the measuring date. Relative humidity and CO2 concentration in the growth room were controlled at 60% and 400 μmol mol–1, respectively. The photosynthetic photon flux density (PPFD) below the canopies is very low, and irradiance greater than the background irradiance of 10 μmol m−2 s−1 is usually regarded as sunflecks (Kaiser ). To simulate the low-irradiance environment, the PPFD inside the growth room was kept at room irradiance, which was about 10 μmol m–2 s–1 at the leaf level. Gas exchange measurements were conducted on the youngest fully expanded leaves, using a portable photosynthesis system (LI-6400XT; LI-COR Inc., Lincoln, NE, USA) with a 6400-02B light source.

Leaf temperature, relative humidity, and CO2 concentration inside the leaf chamber of the LI-6400XT were controlled at 28 °C, 60%, and 400 μmol mol−1, respectively. The PPFD inside the leaf chamber was set to 10 μmol m−2 s−1. Measurements were left to stabilize for ~2 min, after which data were recorded. The recorded A, gs and Ci were represented as Ainitial, gs,initial, and Ci,initial, respectively. Then, the PPFD was increased to 1200 μmol m−2 s−1, and the data were automatically recorded every 5 s until A and gs stabilized (usually within 50 min). The stabilized A and gs were represented as Asteady, and gs,steady, respectively. Times to 50% of Asteady and gs,steady (T50%gs and T50%A) were used to represent the response rates of A and gs to the step-change of irradiance.

Cuticular conductance measurements

Cuticular conductance (gcut) was measured according to Sack and Scoffoni (2011). Briefly, the youngest fully expanded leaves were excised and immediately photographed for leaf area measurement. Then, the leaves were dried in air at a room temperature of 25 °C and a light intensity <10 μmol m−2 s−1. Leaf weight was measured every 5 min over 4 h using a digital balance (Sartorius BP 2215, Gottingen, Germany). The slope of water loss versus time generally becomes less steep within the first hour of leaf drying, which suggests progressive stomatal closure. About 1 h later, leaf weight will show a linear decline with time, suggesting closed stomata (Sack ). The linear part was used to calculate the minimum transpiration rate, which is generally considered to be the cuticular transpiration rate, although water vapor may also diffuse though any leaky stomata (Sack and Scoffoni, 2011). Two temperature and humidity sensors (HOBs, H21-002; Onset Computer Corporation, Bourne, MA, USA) were placed next to the leaves to record air temperature and humidity. The value of gcut was calculated as the cuticular transpiration rate divided by the vapor pressure deficit (VPD) of air, with the assumptions that leaf internal air is fully water-saturated (Pearcy ; Buckley ) and leaf temperature is close to ambient air temperature due to low transpiration rate at low light (Cavender-Bares ).

where T is air temperature and RH is air relative humidity.

Measurements of leaf morphological and anatomical features

Three small leaf discs (approximately 5×5 mm) from the middle of each leaf were collected with the fixative 2.5% glutaric aldehyde in 0.1 mol l−1 phosphate buffer (pH 7.6), and then infiltrated in a vacuum chamber (DZF-6050; Shanghai Hasuc Co. Ltd). The leaf samples were stored at 4 °C until analysis. For each genotype, three leaves from different plants were selected. Four images of both abaxial and adaxial sides were taken using a scanning electron microscope (JSM-6390LV, Tokyo, Japan) under vacuum condition. Stomatal density (D), guard cell length (L), and guard cell width (W) on each leaf side were measured using ImageJ (Wayne Rasband/NIH, Bethesda, MD, USA). In this study, the stomatal size (S) was calculated based on the assumption that stomata are elliptical in shape with their major axis equal to L and their minor axis equal to W (Ouyang ; Xiong ):

Maximum theoretical stomatal diffusive conductance (gs,max), which was determined by stomatal anatomy, was estimated for each genotype according to Franks and Beerling (2009):

where d is the diffusivity of water vapor in air, v is the molar volume of air, amax is the maximum area of the open stomatal pore, and l is the stomatal pore depth for fully open stomata. The gs,max for each leaf was calculated as the sum of gs,max on the abaxial and adaxial sides.

Calculations

The value of 1/τ was calculated following Woodrow and Mott (1989) and Soleh . It is derived from the plot of the logarithmic difference between instantaneous A and Asteady against the time of induction (see Supplementary Fig. S1 at JXB online). The value of 1/τ was determined using linear regression in the range of 2–5 min after induction. We would like to clarify that 1/τ represents the rapidity of photosynthesis increase, which may be correlated with the amount of Rubisco activase (Hammond ) and/or interior CO2 concentration (Kaiser ,b). To avoid the effects of stomatal opening on 1/τ, A was normalized according to the following equation:

An analytical model was used to describe the temporal response of gs to a step-change in PPFD (Supplementary Fig. S2; McAusland ), using four parameters: a time constant (K), an initial time lag (λ), gs,initial, and gs,steady:

where t represents the time after the step-increase of PPFD. The parameters of K and λ were calculated using the sigmodal model with a spreadsheet provided by Vialet-Chabrand . According to McAusland , Slmax during the exponential increase phase was computed as:

Statistical analysis

One-way analysis of variance (ANOVA) and the least-significant difference (LSD) test were used to assess the measured traits in tables among different genotypes using Statistix 9 software (Analytical software, Tallahassee, FL, USA). The correlations were analysed using SigmaPlot 12.5 (Systat Software Inc., San Jose, CA, USA), and the regressions that are best fit for the data are shown in the figures.

Results

Genotypic variation in stomatal anatomy

Stomatal features varied greatly between the selected Oryza species. In general, stomatal size was smaller for abaxial side (lower surface) than adaxial side (upper surface), inversely, stomatal density was higher for abaxial side than adaxial side (Table 2). Saba and Sada ranged from 96 to 210 µm2 and from 98 to 230 µm2, respectively, among genotypes. With respect to stomatal density, the ranges were 174–915 mm−2 and 208–848 mm−2, respectively, for Daba and Dada. On average, stomatal size was 28% smaller in O. sativa than wild genotypes, while stomatal density was 80% larger in O. sativa. This resulted in a significantly larger gs,max in O. sativa, although gs,steady measured at 1200 μmol m−2 s−1 was similar between O. sativa and wild genotypes (Table 3). Stomatal size and density were negatively correlated for each side (see Supplementary Fig. S3).

Table 2.

Intraspecific variations in stomatal size and density in rice plants

Species	Genotype	Stomatal size (μm2)		Stomatal density (mm−2)
		S aba	S ada	D aba	D ada
Oryza sativa L.	Yangliangyou 6	142±23	127±17	811±101	684±49
Oryza sativa L.	Liangyoupei 9	96±15	98±17	912±64	848±35
Oryza sativa L.	Yongyou 12	142±21	147±15	610±84	500±157
Oryza sativa L.	Ase bolong ka	101±11	135±13	896±24	617±27
Oryza sativa L.	Huanghuazhan	127±24	130±15	915±100	686±128
Oryza sativa L.	Trembese	146±14	134±25	444±42	433±60
Oryza sativa L.	Yangdao 6	143±18	135±15	690±60	767±57
Oryza sativa L.	Buluh bawu	126±12	150±14	864±17	538±39
Average		128±20 B	132±16 B	768±171 A	634±140 A
Oryza rufipogon L.	S7705	152±11	159±22	350±58	514±51
Oryza latifolia L.	E9-23	150±37	194±36	381±80	246±32
Oryza alta L.	E1-6	188±33	205±20	174±36	208±49
Oryza australiansis L.	Aus	210±23	230±26	358±34	312±39
Oryza punctata L.	E16-21	145±20	159±27	454±32	368±42
Oryza minuta L.	E13-10	185±12	191±20	533±34	296±41
Oryza officinalis L.	2203	176±23	194±29	539±19	438±56
Oryza minuta L.	ABC	181±22	185±36	522±38	549±73
Average		173±23 A	190±23 A	414±125 B	366±124 B
ANOVA
Species		***	***	***	**

Data are shown as means ±SD of 5–10 replicates. **P<0.01; ***P<0.001. The data with different uppercase letters in each column were significant at P<0.05 level.

Table 3.

Intraspecific variations in steady-state photosynthesis, CO2 diffusion conductance and dynamic photosynthetic parameters in rice plants

Species	Genotype	A steady	g s,max	g s,steady	g s,intial	g cut	T 50%A	T 50%gs	λ	Slmax	1/τ
		(μmol m−2 s−1)	(mol m−2 s−1)	(mol m−2 s−1)	(mol m−2 s−1)	(mol m−2 s−1)	(s)	(s)	(s)	(mmol m−2 s−2)	×10–5 (s−1)
Oryza sativa L.	Yangliangyou 6	20.6±1.6	5.7±0.2	0.43±0.04	0.026±0.004	0.009±0.001	639±133	749±142	487±113	0.96±0.01	238±197
Oryza sativa L.	Liangyoupei 9	19.1±1.3	5.9±0.2	0.43±0.08	0.026±0.008	0.007±0.001	615±29	718±46	402±109	0.76±0.25	325±80
Oryza sativa L.	Yongyou 12	22.3±1.2	5.8±0.3	0.58±0.06	0.028±0.001	0.010±0.001	610±68	700±128	449±90	1.05±0.08	243±118
Oryza sativa L.	Ase bolong ka	22.8±1.8	5.0±0.1	0.58±0.17	0.028±0.007	0.009±0.001	460±68	560±61	337±31	1.46±0.42	547±221
Oryza sativa L.	Huanghuazhan	19.3±1.9	5.8±0.2	0.39±0.07	0.030±0.003	0.012±0.001	580±73	568±54	428±50	1.90±0.57	61±31
Oryza sativa L.	Trembese	17.0±1.3	3.5±0.1	0.40±0.06	0.033±0.011	0.013±0.001	559±108	651±154	350±92	1.31±0.58	174±111
Oryza sativa L.	Yangdao 6	17.7±1.4	5.5±0.5	0.38±0.07	0.034±0.005	0.014±0.001	447±112	495±135	225±51	0.61±0.33	757±530
Oryza sativa L.	Buluh bawu	25.7±0.9	5.0±0.2	0.57±0.07	0.034±0.011	0.011±0.001	476±73	556±60	368±56	1.52±0.21	422±219
Average		20.5±2.9 A	5.3±0.8 A	0.47±0.09 A	0.030±0.003 B	0.011±0.002 B	548±76 A	625±92 A	381±81 A	1.20±0.43 A	346±223 B
Oryza rufipogon L.	S7705	15.5±2.6	3.6±0.1	0.33±0.03	0.042±0.016	0.024±0.001	431±36	431±38	232±43	1.08±0.33	473±133
Oryza latifolia L.	E9-23	13.9±1.5	3.2±0.1	0.31±0.04	0.050±0.009	0.011±0.001	196±76	383±16	42±30	0.45±0.23	1259±241
Oryza alta L.	E1-6	16.9±1.5	1.9±0.0	0.51±0.03	0.072±0.015	0.022±0.001	215±62	353±94	67±31	0.83±0.19	2023±746
Oryza australiansis L.	Aus	15.5±2.5	3.4±0.2	0.26±0.06	0.089±0.018	0.026±0.005	137±12	124±13	23±24	0.30±0.04	972±196
Oryza punctata L.	E16-21	15.7±1.9	3.5±0.1	0.51±0.07	0.090±0.006	0.012±0.001	125±28	160±55	30±9	0.72±0.28	1933±638
Oryza minuta L.	E13--10	15.3±1.5	3.6±0.1	0.49±0.07	0.110±0.026	0.014±0.001	140±44	188±74	38±17	0.58±0.18	1823±346
Oryza officinalis L.	2203	17.3±1.4	4.0±0.2	0.54±0.05	0.138±0.049	0.018±0.001	143±18	124±45	57±22	0.43±0.06	1452±358
Oryza minuta L.	ABC	17.0±2.5	5.0±0.1	0.67±0.12	0.139±0.043	0.018±0.001	160±70	376±26	78±31	0.91±0.23	2238±668
Average		15.9±1.1 B	3.5±0.9 B	0.45±0.14 A	0.091±0.037 A	0.018±0.006 A	193±101 B	267±130 B	71±68 B	0.66±0.27 B	1521±598 A
ANOVA
Species		***	***	ns	***	**	***	***	***	**	***

Data are shown as means±SD of 5–10 replicates. **P<0.01; ***P<0.001; ns, not significant at P=0.05 level. The data with different uppercase letters in each column were significant at P<0.05 level. 1/τ, rate of Rubisco activation; λ, initial lag time for the gs response; Asteady, steady-state photosynthetic rate; gcut, cuticular conductance; gs,initial, initial stomatal conductance; gs,max, maximum theoretical stomatal conductance; gs,steady, steady-state stomatal conductance; Slmax, maximum stomatal conductance increase rate; T50%A, times to 50% of the steady-state photosynthetic rate after shifting to high light; T50%gs, times to 50% of the steady-state stomatal conductance after shifting to high light.

Correlation between stomatal anatomy and stomatal response rate to a step increase of PPFD

T 50%gs is determined by Slmax and λ, both of which showed a large variation among genotypes (Table 3). Slmax ranged from 0.30 to 1.90 mmol m−2 s−2, and λ ranged from 23 to 487 s. Slmax was negatively correlated with both Saba (P<0.05) and Sada (P<0.05), and similarly, λ was also negatively correlated with both Saba (P<0.01) and Sada (P<0.0001) (Supplementary Table S1; Fig. 1). This suggested that smaller stomata in the Oryza genus open faster than large ones at the exponential increase phase, but smaller stomata showed a longer lag time before reaching the exponential increase phase.

Fig. 1.

Effects of stomatal size at the abaxial (Saba) (A, C) and adaxial sides (Sada) (B, D) on maximum stomatal conductance increase rate (Slmax) and initial time lag (λ) for stomatal response across Oryza genotypes. Data are means ±SD of 5–10 replicates. The lines represent the regressions that are best fit for the data.

T 50%gs was positively correlated with both Slmax and λ (Figs 2A, B). The negative correlations between stomatal size and Slmax and λ resulted in negative correlations between T50%gs and stomatal size (Figs 2C, D). This suggested that with the presence of a large variation in time lag, time lag is more important than stomatal opening rate at the exponential phase in determining the time required for stomatal opening. The time required for stomatal opening was shorter in larger stomata, although the stomatal opening rate at the exponential increase phase was slower than with smaller stomata.

Fig. 2.

The correlations of T50%gs with Slmax (A), λ (B), Saba (C) and Sada (D) across Oryza genotypes. λ, initial time lag for stomatal response; Saba, stomatal size at abaxial side; Sada, stomatal size at adaxial side; Slmax, maximum stomatal conductance increase rate. Data are means ±SD of 5–10 replicates. The lines represent the regressions that are best fit for the data.

g s,initial was positively correlated with both Saba (P<0.01) and Sada (P<0.01) (Supplementary Table S1; Fig. 3), and gs,initial was negatively correlated with λ (Supplementary Table S1; Fig. 4). It should be noted that the contribution of gcut to the apparent gs will increase with a low gs, for example at night or under drought conditions. gcut varied from 0.0073 to 0.0259 mol m−2 s−1, with an average of 0.014 mol m−2 s−1, which accounted for 24% of gs,initial (Table 3). gs,initial was highly correlated with gs,initial–gcut, and to a lesser extent with gcut (see Supplementary Table S1). This suggested that stomatal opening is still the major determinant for gs,initial, although gcut contributes strongly to it.

Fig. 3.

Effects of stomatal size at the abaxial (Saba) (A) and adaxial sides (Sada) (B) on initial stomatal conductance (gs,initial) across Oryza genotypes. Data are means ±SD of 5–10 replicates. The lines represent the regressions that are best fit for the data.

Fig. 4.

Relationship between initial stomatal conductance (gs,initial) and initial time lag for the gs response (λ) across Oryza genotypes. Data are means ±SD of 5–10 replicates. The lines represent the regressions that are best fit for the data.

In possession of smaller stomata, O. sativa showed a larger Slmax but a longer λ than wild genotypes (Table 3). This resulted in a longer time required for stomatal opening (T50%gs) in O. sativa than in wild genotypes. Moreover, gs,initial was lower in O. sativa than in wild genotypes.

Genotypic variation in 1/τ and the response of photosynthesis to a step increase of PPFD

There was significant genotypic variation for 1/τ among the selected genotypes (Table 3); 1/τ varied from 6.1×10–4 to 2.2×10–2 s−1. Interestingly, 1/τ was negatively correlated with both T50%gs and the relative rate of Ci depletion (Fig. 5). This suggested that the response rate of stomatal conductance may affect apparent Rubisco activation rate by regulating leaf interior CO2 concentration. Apparent Rubisco activation rate in O. sativa was significantly slower than in wild genotypes (Table 3).

Fig. 5.

The correlations of 1/τ with λ (A) and the rate of Ci depletion () (B) during the first 5 min of induction across Oryza genotypes. 1/τ, the rate of Rubisco activation; λ, initial time lag for the gs response. Data are means ±SD of 5–10 replicates. The lines represent the regressions that are best fit for the data.

T 50%A was positively correlated with T50%gs and negatively correlated with 1/τ (see Supplementary Table S1). This suggested that both the time required for stomatal opening and apparent Rubisco activation rate determine the rapidity of the photosynthetic response to step changes of PPFD. T50%A in O. sativa was significantly larger than in wild genotypes (Table 3).

Genotypic variation in Asteady

A steady varied significantly among the selected genotypes (Table 3), with a range of 13.9–25.7 μmol m−2 s−1. Asteady in O. sativa was significantly larger than in wild genotypes.

Discussion

Stomata control gas exchange between the leaf interior and the atmosphere. A large number of small stomata are frequently suggested to be more efficient than fewer, larger stomata for both photosynthesis and its response to environmental perturbations. Interestingly, intraspecific variation in stomatal anatomy among Oryza species also had a significant effect on stomatal kinetics (Fig. 1; Supplementary Table S1).

Stomatal size in O. sativa, the widely grown rice species, was smaller than in wild genotypes (Table 2). This is in agreement with previous studies (Giuliani ; Kondamudi ). Stomatal number and size are suggested to be an adaptive mechanism in plants to environmental stresses and to affect photosynthesis capacity (Kondamudi ). It is speculated that the possession of larger, fewer stomata in wild species may of benefit for their survival in unfavorable environments. In contrast, smaller, more numerous stomata can potentially improve the maximum stomatal conductance and photosynthesis (Table 3; Franks and Beerling, 2009; Kondamudi ). Thus, the smaller, more numerous stomata in cultivated rice might be the result of the selection processes for higher yields (Kondamudi ). Yangliangyou 6, Liangyoupei 9 and Yongyou 12 are three high-yielding ‘super’ hybrid rice cultivars that can yield 10–12 t ha−1 under favorable conditions (Wei , 2017). However, compared with other O. sativa cultivars, they showed no consistent trend in stomatal size and density (Table 2). This suggests that the ability of rice cultivars to acclimate to environmental perturbations has not been improved in recent high-yielding rice cultivars.

Stomatal response to a step change in irradiance

Stomatal response is mainly derived from gas exchange measurements, instead of direct measurement of stomatal aperture. However, it should be noted that gs and stomatal aperture are not linearly correlated (Vialet-Chabrand ). In fact, a hyperbolic relationship was found in previous studies (Kaiser and Kappen, 1997; Kaiser and Kappen, 2000), where the influence of stomatal aperture on gs decreases rapidly with the magnitude of stomatal opening. l in Eq. 3 can also represent the distance that gas molecules have to diffuse through the stomatal pore, which is suggested to be positively correlated with stomatal size (Franks and Beerling, 2009). Therefore, when aperture size on a leaf area basis increases to the same extent in response to a step change in irradiance, with a low value of l, a large number of small stomata can thus possess a higher gs than fewer large ones (Franks and Beerling, 2009). This would explain, together with a larger guard cell membrane surface area to volume ratio, the faster stomatal response rate at the exponential phase in smaller stomata (Fig. 1; Table 3).

As mentioned earlier, stomata present a significant time lag before the exponential increase of gs. After the step increase of irradiance, the guard cells begin to swell for some time before the stomatal pore widens (Kaiser and Kappen, 2001; Franks and Farquhar, 2007). This leads to the time lag, which can last up to 1 h (Kaiser and Kappen, 2001). However, gs can be significantly larger than zero without the presence of a visible stomatal pore (Kaiser and Kappen, 2000, 2001). This may be due to a small change of aperture, below the resolution of the microscope, which can contribute to a large gs. gs at this ‘spannungsphase’ is significantly related to guard cell complex area between the dorsal cell walls of the guard cells (SCA) (Kaiser and Kappen, 2001). This may explain the lower gs,initial with smaller stomata (Fig. 3; Supplementary Table S1), where SCA can potentially be smaller.

The reason λ is larger in smaller stomata with a lower gs,initial (Fig. 1C, D; Table 3) is not known. However, we hypothesized that smaller stomata may be less swollen under low light condition, or the counterpressure of surrounding mesophyll cells is initially harder to overcome than for larger guard cells. In fact, gs,initial has been frequently found to be negatively correlated with the time required for increase in A (Valladares ; Naumburg ; Kaiser ; Taylor and Long, 2017), which is consistent with the negative correlations between gs,initial and T50%gs and T50%A in the present study (see Supplementary Table S1).

Responses of 1/τ and photosynthesis to the step change of irradiance

To the best of our knowledge, this is the first study to demonstrate the negative correlations between stomatal size and the response rates of 1/τ and photosynthesis to a step change of irradiance (see Supplementary Table S1). Rubisco is a bifunctional enzyme, and sufficient CO2 supply can increase the Rubisco carboxylation rate by inhibiting the oxygenation of ribulose-1,5-bisphosphate (Woodrow ; Kaiser ). This may account for the faster apparent Rubisco activation rate at elevated CO2 concentrations and with a larger gs,initial (Mott and Woodrow, 1993; Woodrow ; Urban ; Carmo-Silva and Salvucci, 2011, 2013; Kaiser ,b). This would also explain the significant and negative relationship between 1/τ and the rate of Ci depletion as observed in this study (Fig. 5). The intraspecific variation in the rate of Ci depletion should be related to different gs,initial (Supplementary Fig. S4), because a larger gs,initial can not only supply more CO2 to Rubisco but also improve the gs response rate by decreasing λ (Fig. 4). With a lower gs,initial, small stomata showed a lower Rubsico activation rate and thus a longer T50%A (Supplementary Table S1).

Implications

The present study demonstrated that smaller stomata can not only improve stomatal response rate, but also result in a longer time lag and a lower initial stomatal conductance. Whether smaller stomata are beneficial for decreasing the times required for gs and A to increase, in response to a step increase of irradiance, is dependent on the magnitude of gs,initial.

The background irradiance received by the upper leaves of cereal crops, which are more important than understory leaves for food production, should be largely higher than the background low light of 10 μmol m−2 s−1 used in the present study. However, the background irradiance received by the understory species in forests and by the lower leaves of cereal crops can be sufficiently low to get a low gs,initial (Pearcy ; Barradas ; Nishimura ; Kaiser ), although the movement of leaves in the presence of wind can result in greater penetration of light to lower leaves (Burgess ).

Under high background irradiance, gs,initial should be much higher and λ would be dramatically decreased (Kaiser ). Then, the times required for gs and A to increase should be determined more by Slmax, and should be lower in leaves with smaller stomata. Under low background irradiance, however, the possession of large stomata may be beneficial for utilizing sunflecks (Xiong ), which might be the reason for the understory species in forests, such as ferns, having large stomata. Moreover, gs, including gs,initial, can be significantly decreased under abiotic stresses, such as drought and salt stress (Sun ; Zhang ). Whether large stomata can decrease the times required for gs and A to increase under these abiotic stresses needs to be further investigated.

Supplementary data

Supplementary data are available at JXB online.

Fig. S1. Illustration of the semilogarithmic time course of photosynthetic rate.

Fig. S2. Temporal response of stomatal conductance (gs) to a step change in PPFD from 0 to 1200 μm m−2 s−1.

Fig. S3. Correlations between the stomatal density and stomatal size at both abaxial and adaxial side across Oryza genotypes.

Fig. S4. Relationships between the rate of Ci depletion () during the first 5 min of induction and the initial stomatal conductance (gs,initial).

Table S1. Correlation matrix between studied traits.

Click here for additional data file.