Leaf phenotype response to different intensities monochromatic blue and red light affects stomatal conductance and photosynthetic capacity

doi:10.21203/rs.3.rs-1595351/v1

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Research Article

Leaf phenotype response to different intensities monochromatic blue and red light affects stomatal conductance and photosynthetic capacity

https://doi.org/10.21203/rs.3.rs-1595351/v1

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Background

Stomatal conductance is a limiting factor of plant photosynthesis. Studies on the influencing factors of stomatal conductance amongst different species are abundant, but the effect of leaf phenotype on stomatal conductance is usually ignored in study. Here, the experiment set different intensities monochromatic blue and red light. We investigated the upper and lower epidermis stomatal traits, leaf structure, the maximum operational stomatal conductance and net photosynthetic rate of soybean leaves. In addition, the contribution of leaf structure and stomatal characteristics to stomatal conductance was quantified by a new software package.

Results

In this study, net photosynthetic rate was tightly coupled with operational stomatal conductance, inhibited by red light and enhanced by blue light. Regardless of light environment, the maximum operational stomatal conductance was mainly affected by leaf thickness according to the analysis of contribution rate, rather than stomatal density of upper and lower epidermis. A significant positive correlation was found between leaf structure and stomatal density, but the correlation between stomatal density and stomatal size was not found. Regardless of the amounts of light intensities, stomatal density (SD), palisade tissue and specific leaf weight (SLW) were mainly regulated by blue light, whereas red light was more important to stomatal size and spongy tissue.

Conclusions

Although leaf thickness is more critical in affecting stomatal conductance than stomatal density, blue light mainly promotes leaf thickness and stomatal density, which coordinated each other to facilitate stomatal conductance, thereby positively regulating photosynthetic capacity.

light quality

leaf structure

stomatal traits

stomatal conductance

net photosynthetic rate

Stomata play an essential role in regulating gas exchange for photosynthetic carbon assimilation. Stomata only account for 0.3–5% of the total leaf area due to the existence of cuticle, but 95% of gas exchange is completed through the stomata [1]. Specifically, gaseous CO₂ diffuses across layers of resistance, finally reaching the carboxylation site (rubisco enzyme in chloroplasts). Under the diffusion of CO₂ over long distances inside the blade, the concentrations of CO₂ in the intercellular substance (Ci) and the chloroplast (Cc) are relatively lower than that of the atmosphere, thereby affecting the supply of CO₂ to the site of carboxylation [2]. Soybean photosynthesis, as a C3 crop, is generally limited by the ability to absorb CO₂ from the atmosphere into chloroplasts, resulting in unsaturated Cc and thus reduced CO₂ substrates available for A[2, 3]. Moreover, studies show that maximising stomatal conductance can effectively increase Ci and internal concentration gradients, Cc [4, 5].

Assuming that all pores are completely open, the potential maximum stomatal conductance (g_smax) can be calculated by stomatal density (SD) and stomatal size (SZ). SD and SZ determine the theoretical capacity of gas exchange, and in principle, an infinite combination of either of these parameters achieves g_smax [2, 6, 7]. Although this principle is anatomically possible, the plants’ operational stomatal conductance (g_op) is not near g_smax. Instead, it is maintained at about 20% of g_smax because it allows guard cells to most effectively regulate stomatal enlargement [8, 9]. However, previous studies have shown that g_smax is positively correlated with g_op [8, 10, 11], and stomatal characteristics (i.e., SD and SZ) are significantly related to g_op in angiosperms; other unknown traits may be involved in regulating g_op [11, 12].

Meanwhile, SD and SZ are affected by regulatory factors during stomatal development; they are mutually coordinated with leaf tissue development to establish an efficient and robust network that facilitates gas exchange for photosynthesis [13]. Previous studies showed that changes in stomatal development of the epidermal layer led to coupled changes in the underlying mesophyll tissues, which tended to coordinate matching leaf photosynthetic potential (V_max) with g_smax [14]. In addition, related work by Tanaka et al. [15] reported that Arabidopsis EPF mutants with 75% more stomata than wild-type counterparts demonstrated significantly higher rates of A under high light intensities. The authors attributed this enhancement in A and stomatal conductance to improvements in the homogeneity of Ci throughout the leaf and thus lateral diffusion of CO₂. Thus, variations in stomatal development and leaf anatomical structure have become an essential adaptive strategy for plants to respond to changes in long-term environmental signals and are key to plant evolution and adaptation to new environmental conditions [16].

Light, as an important environmental signal, mainly affects leaf structure, stomatal conductance and photosynthetic performance through light quality and intensity. According to previous comparative studies, under low light conditions, plant leaves become smaller and thinner, and SD, photosynthetic rate, transpiration and stomatal conductance decrease [17–20]. Amongst the many light qualities, blue and red light are the two most efficient bands of photosynthesis in plants [21]. Blue and red light positively regulate stomatal development through photochromes and cryptochrome receptors, respectively [22]; they alter different leaf traits that affect photosynthesis including anatomical changes in palisade and spongy mesophyll [23]. Light quality is predicted to also play a key role in this coordination, considering the observed coordination amongst leaf structure, g_op, and photosynthetic capacity frequently, but few studies have been conducted.

Here, with light intensity changed, we surveyed stomatal characteristics, leaf structure, actual photosynthetic capacity and gas exchange capacity under monochromatic blue and red light to address the following questions: when light intensity changes, how do leaf structure and stomatal characteristics coordinated differently in response to monochromatic blue and red light? How will this coordination affect the actual gas exchange capacity and photosynthetic capacity? It is expected to provide theoretical basis for selecting appropriate breeding strategies and cultivation measures to promote gas exchange capacity of soybean for adapting the increasing CO₂ concentration of environment in the future [24, 25]. This information will also provide new insights into the potential for optimising production through light quality in subsequent agricultural production.

Leaf structure responded to blue and red light under different light intensities

The response of leaf structure to different light quality was different (Table 1). When soybean leaves were exposed to monochromatic blue light, the light intensity decreased, and then most parameters, such as SLW, LT, UET, PT and ST, significantly decreased. P/C and LL between 400 and 100 B showed no difference, but LC significantly increased by 9% under 100 B. Meanwhile, most indexes under monochromatic red light decreased significantly with the decrease in light intensity, but LC and SLW had no significant difference, and LL increased significantly by 21.3%.

Table 1

Leaf structure characteristics under monochrome blue and red light with 400 and 100 light intensities: 400 B, 100 B, 400 R and 100 R, denoting high-intensity monochromatic blue light, low-intensity monochromatic blue light and low-intensity monochrome red light.
Treatments	400B	100B	400R	100R
SLW (mg/cm²)	0.223 ± 0.04a	0.185 ± 0.0b	0.14 ± 0.01c	0.125 ± 0.01c
LT (µm)	115.678 ± 4.4a	63.252 ± 4.5c	80.334 ± 6.2b	55.401 ± 2.9d
UET (µm)	12.346 ± 1.7a	5.813 ± 0.9b	12.976 ± 2.1a	6.581 ± 1.1b
LET (µm)	7.161 ± 1.2a	4.081 ± 1b	6.979 ± 1.2a	4.453 ± 0.7b
PT (µm)	68.541 ± 4.4a	40.826 ± 3.3b	49.209 ± 5.3c	24.386 ± 2.2d
ST (µm)	33.485 ± 3.8a	19.014 ± 2.8b	17.873 ± 2.1b	14.923 ± 1.5c
LC (%)	0.593 ± 0.04b	0.649 ± 0.08a	0.614 ± 0.07ab	0.622 ± 0.04ab
LL (%)	0.289 ± 0.03ab	0.298 ± 0.04a	0.221 ± 0.02c	0.268 ± 0.03b
P/C (%)	2.07 ± 0.25c	2.19b ± 0.35bc	2.805 ± 0.3a	2.35 ± 0.25b
Note: SLW-Specific leaf weight; LT-Leaf thickness; UET-Upper epidermal cell thickness; LET-Lower epidermal cell thickness; PT-Palisade tissue thickness; ST-Spongy tissue thickness; LC-Leaf tissue compactness; LL-Leaf tissue looseness; P/C-Ratio of palisade tissue to the spongy tissue. Each value is expressed as the mean ± SE. Significant differences between treatments are indicated in different lower-case letters (P < 0.05). The same denotations are followed subsequently.

Response of stomatal size of two-sided soybean leaf to blue and red light under different intensities

Changes in stomatal size (SZ) in the upper and lower epidermis on soybean leaves were shown under different light environments (Figs. 1A, C). Decreasing light intensity under red light, stomatal size was significantly reduced in the upper and lower epidermis by 17.5% and 11.7%, respectively, whereas the leaves had no effects on stomatal size in the upper and lower epidermis under blue light (Figs. 1B, D).

Response of stomatal density of two-sided soybean leaf to blue and red light under different intensities

Black dots indicate that the stomata distributed on the upper and lower epidermis of soybean leaves (Fig. 2A). Under monochromatic blue light treatment, the decrease in light intensity significantly reduced the stomatal density of the upper (33.2%) and lower (29.5%) epidermis (Figs. 2B, C). Under red light background, the change in light intensity had no significant effects on the stomatal density of the upper and lower epidermis. Compared with red light, blue light significantly increased stomatal density in the upper and lower epidermis with high light intensity, whereas under low light intensity, blue light significantly aggrandised stomatal density in upper epidermis, but had no significant impact on stomatal density in lower epidermis.

Response curves of operational stomatal conductance to different light intensities

The g_op of soybean leaves under red light treatment increased with light intensity and tended to be stable after reaching the maximum value. Under blue light, the g_op initially increased, then decreased, and gradually increased again. The maximum operational stomatal conductance (g_opmax) of 0.394 and 0.208 mol m^− 2s^− 1 under 400 B and 100 B treatments appeared when the optical quantum flux density was 2300 µmol m^− 2s^− 1 (Fig. 3A). In the whole response process, the g_op under 400 B treatment is the largest, but it has no difference under 100 B and 400 R treatment; the most petite is under 100 R treatment (Fig. 3B). Moreover, the figure shows that the g_opmax of soybean leaves were significantly reduced under blue light (400 B and 100 B), whereas the change in light intensity had no significant effect on maximum operational stomatal conductance under red light (400 R and 100 R).

Relationship between net photosynthetic rate and operational stomatal conductance

With the increase in PPFD, A_N under 100 B, 400 R, and 100 R treatments showed an increasing trend and then stabilised and finally reached the saturation point, with the maximum values of 11.96, 11.68, and 7.22 µmol CO₂ m^− 2s^− 1, respectively. Under 400 B treatment, the net photosynthetic rate of soybean leaves increased with light quantum flux density and reached the maximum value of 20.97 µmol CO₂ m^− 2s^− 1 when the PPFD was 2300 µmol CO₂ m^− 2s^− 1 (Fig. 4A). The significance analysis of A_max under different treatments (Fig. 4B) indicated that the A_max of soybean significantly reduced with the weakened light intensity, and red light has a negative effect on the photosynthetic rate of the plant compared with blue light.

Figures 4C, D, E, F showed the dynamic correlation between g_op and net photosynthetic rate of soybean leaves in response to changes in light intensity. The g_op was positively correlated with the net photosynthetic rate under 400 B, 100 B, 400 R and 100 R treatments, and the correlation coefficients were as high as 0.841, 0.876, 0.992 and 0.983, respectively. The g_op was positively correlated with the net photosynthetic rate under different light conditions.

Contribution of leaf structure and stomatal traits to maximum operational stomatal conductance

In the current study, we have found that leaf structure and stomatal morphology greatly affected g_opmax of soybean leaves. As shown in Fig. 5A, SLW had the highest contribution to the maximum operational stomatal conductance (19.43%), followed by LT, PT and ST (15.65%, 13.39% and 12.01%, respectively). Overall, the influence degree of leaf structure on the g_opmax could reach 60.48% and become far more than 24.22% of stomatal morphology. Specifically, USD and LSD were 9.75% and 9.7%, respectively, whereas LSZ and USZ were only 3.21% and 1.56%, respectively. The correlation analysis also proved that SLW, LT, ST, PT, USD and LSD had a highly significant positive correlation with g_opmax. Leaf structure and stomatal characteristics also influenced each other (Fig. 5B).

Response of leaf structure and stomatal traits to blue and red light

Light quality can affect the anatomical structure of plant leaves, including the arrangement of palisade and sponge cells, thereby affecting the photosynthetic efficiency of plants [23, 26]. However, leaf response to different light quality is different (Table 1). In many plant species, blue light (B) and blue and red light (RB) affect the growth of palisade parenchyma compared with red light and white light [23]. With decreasing light intensity, this study also showed that the leaf tissue compactness (LC) and SLW was unchanged under red light, suggesting that blue light may respond to the decrease in light intensity by increasing the proportion of palisade tissue in leaves and mainly regulate palisade tissue compared with red light. In addition, this finding is basically consistent with the results of previous studies, where SLW was shown to be much less affected than Amax in the spectrum containing relatively little blue [21, 27]. The result suggests that SLW is mainly regulated by the blue spectrum. Palisade tissue and specific leaf weight are important indicators of leaf thickness, and the above results also indicate that blue light plays a key role in regulating leaf thickness. In addition, when light intensity decreased, the leaf looseness (LL) of soybean leaves under blue light did not change significantly, indicating that red light adapted to the environment by adjusting the proportion of spongy tissue in the leaves.

For stomatal traits, a significantly negative correlation has been widely observed in different species between stomatal density and size, both of which determine the g_smax [28, 29]. Nevertheless, almost no connection between SZ and SD was found in our research (Fig. 5B); this finding is the same as that in the previous research, where stomatal density and length showed no coincident trend during the breeding process of japonica rice varieties [30]. Interestingly, in present the experiment, no significant difference is found in stomatal size under monochromatic blue light regardless of high or low intensity (Fig. 1). This finding suggests that stomatal size is mainly controlled by the red spectrum; but this result is contrary to that of Seif et al. [31]. Simultaneously, the light intensity was significantly reduced, but the stomatal density under monochromatic red light was unaffected and reduced observably under blue light, which also proved that blue light was more momentous in regulating SD (Fig. 2). Similar effects were shown, that is, increasing blue light under red light can increase stomatal density [32, 33]. Although numerous studies have indicated that different light quality could activate the corresponding light photoreceptors to trigger a cascade of biochemical reactions downstream for regulation of stomata development, the response of stomatal density and stomatal size to light quality is biased according to our data, and the underlying mechanism needs further study [34, 35].

Correlation between leaf structure and stomatal traits

A high correlation between leaf structural characteristics and stomatal traits (Fig. 5B) indicated that they could influence and coordinate with each other. This result is consistent with that of a previous study [13, 14], which found that stomatal development of stomata-speciﬁc regulators could alter leaf structural changes to obtain high g_smax. In the present study, the USD and LSD are more than 90% correlated with leaf anatomical structure, such as ST, PT and LT, amongst which the USD and LSD are most closely correlated with leaf thickness. On the contrary, no correlation was found between SZ and leaf anatomical characters regardless of upper and lower epidermis. These results suggest that specific regulators of SD are more pivotal in inducing leaf development, especially for leaf tissue development, than SZ for the high gas exchange capacity obtained. Specifically, the epidermal pattern factor (EPF) induces the density of palisade mesophyll cells below the paraxial epidermis [14, 36, 37]. Moreover, manipulating the SPCH activity can change the degree of stomatal lineage proliferation, thereby increasing leaf area and leaf thickness. However, the response speed to the environment under natural light remarkably affected SZ. The presence of small stomata usually accelerates stomatal response because the membrane surface area is larger relative to the volume ratio of guard cells. Thus, increasing the rate of ion flux is conducive to rapid response to fluctuating light environment, thereby improving the photosynthetic rate [28, 38, 39]. However, the relationship between stomatal size and stomatal response speed varies across a wide range of species [40, 41], and the molecular genetic mechanisms that control stomatal size remain unclear, requiring further research.

Relative importance of impact factor to actual gas exchange capacity

The intense influence role of gas exchange capacity on photosynthesis has been widely proven by previous studies [7, 42-44]. Here, we further demonstrated that net photosynthetic rate evolved and was tightly associated with shifts in g_s under blue and red light (Fig. 4C, D, E and F). Leaf thickness affects the length of water and CO₂ transport path; it is a factor affecting g_s in the previous studies [29, 45, 46]. In particular, Carriqui et al. [47] discovered that the structural differences between the leaves of angiosperms and ferns partly explain the decline in gas exchange capacity in ferns according to the comparison of the leaf anatomical characteristics between ferns and angiosperms. In addition, the stomatal conductance is related to stomatal density and size [28, 48, 49]. Furthermore, a tendency to increase g_op within species was realised by decreasing stomatal size and increasing stomatal density [28, 38]. However, this trend may not apply to all species; thus, some differences exist. We determine the relative importance of individual structural variables in g_opmax by rdacca. hp [50] indicated that under different light quality conditions, the gas exchange capacity of soybean leaves was mainly affected by leaf thickness (SLW, LT, PT and ST) and SD rather than SZ (Fig. 5). The result is similar to that of the previous study, that is, no significant correlation was observed between stomatal size and g_opmax, but g_opmax increased with stomatal density [11]. In addition, leaf and leaf tissue thickness have a more significant ability to regulate g_opmax than stomatal density by experimental consequence. Especially in a monochromatic red-light environment, the changes between SD and SLW were concordant with g_opmax, and they were unaffected by decreasing light intensity. However, A_max decreased significantly. The reason may be that the effect of light intensity on significant reduction exceeds its self-regulation ability on plant growth and development in this experiment because SLW harmonizing with stomatal density to compensate plant growth is limited. These results indicate that leaf thickness and stomatal density of the upper and lower epidermis are mainly regulated by blue light, and they are in harmony with each other to significantly improve gas exchange capacity and photosynthetic capacity; but leaf thickness seems to be more critical in influencing stomatal conductance. Therefore, some studies found that the amount of the stomatal density in plants is reduced by about half, and no adverse effects on plant growth and development are observed [6, 51, 52]. However, broader studies are needed to elucidate the specific mechanisms, providing new insights for future breeding and cultivation measures.

Summary

When light intensity decreased, palisade tissue thickness, leaf thickness and upper and lower epidermal cell thickness were significantly reduced, resulting in significantly reduced photosynthetic rate. However, the leaf structure and stomatal traits of soybean responded to monochromatic blue and red light conditions differently. Red light was more critical to stomatal size and spongy tissue development, whereas palisade tissue, specific leaf weight and stomatal density of upper and lower epidermis were mainly regulated by blue light, which coordinate each other to greatly promote operational stomatal conductance of soybean leaves. Stomatal conductance was positively correlated with net photosynthetic rate regardless of any light treatments. Thus, blue light can promote leaf thickness and stomatal density, which coordinate each other, and can increase stomatal conductance, improving photosynthetic capacity on soybean leaves (Fig.6). Due to the gradual increase of CO₂ concentration in the environment, this is conducive to select suitable breeding strategies and cultivation measures to promote the absorption of CO₂, so as to improve the gas exchange capacity and photosynthetic potential of soybean.

Plant material

Soybean ‘Rongxian Dongdou’ (light sensitive soybean variety) was selected for the response to growth conditions under a different light environment. Experiment was conducted in a greenhouse in the College of Agronomy, Sichuan Agricultural University, Chengdu, Sichuan province, China (30°71′20″N, 103°86′87″E). Prior to the experiment, soybean seeds were cleaned by 75% ethanol and deionised water for 5 min and germinated on a wet sterile gauze for 48 h in the dark at 25 ℃. After germination, two seeds were sown in 400 mL pots filled with a mixed matrix of PINDSTRUP organic soil (Pindstrup Mosebrug A/S, Ryomgaard, Denmark) and vermiculite (v:v, 3:1) in a light chamber with 25 ℃ day and night temperature, 60% relative humidity, 400 µmol mol^− 1 CO₂ and12 h/12 h photoperiod. The soybean seedlings were watered every two days with 0.2% Hoagland’s solution (water–culture method for growing plants without soil, 1950). When the soybean seedlings have grown to VC stage [53], the pots were transferred to different light treatments with the same environment conditions as mentioned above. During the period of the V3, the fully unfolded penultimate compound leaves were sampled to measure their morphological and physiological parameters.

Growth conditions

The experiment sets four light treatments of two types of light quality (monochromatic blue and monochromatic red) and different light intensities (400 µmol m^− 2s^− 1 and 100 µmol m^− 2s^− 1) by light chambers. The specific light quality (Fig. 7A) and intensity (Fig. 7B) was treated as follows: high intensity monochromatic blue light (400 B; PPFD: 400 µmol m^− 2s^− 1; blue wavelength: 400, 450 and 480 nm); low intensity monochromatic blue light (100 B; PPFD: 100 µmol m^− 2s^− 1; wavelength: 400, 450 and 480 nm), high intensity monochromatic red light (400 R: PPFD: 400 µmol m^− 2s^− 1; wavelength: 625, 660 and 690 nm) and low intensity monochromatic red light (100 R; PPFD: 100 µmol m^− 2s^− 1; wavelength: 625, 660 and 690 nm). The pots were moved daily to avoid boundary effects, and the same light intensity was maintained during growth under one treatment. Spectral irradiance and light intensity were measured by HR-450 (Hipoint Inc., Gaoxiong, Taiwan, China).

Measurement of morphological characteristics

Penultimate compound leaves (fully expanded) of four soybean seedlings were selected. Samples were over-dried at 105 ℃ for 0.5 h to destroy the tissues and then dried at 80 ℃ for 72 h to constant weight. The specific leaf weight (SLW, mg/cm²) was calculated using the proportion of penultimate compound leaf and the quality of dry matter in the leaf area. The average value is considered the final result.

Leaf anatomical feature measurements

Leaf piece (4 mm²); about four replications for each treatment without midribs were fixed in formalin–acetic acid–alcohol solution (FAA; 90% ethanol; 5% formaldehyde; 5% glacial acetic acid, v/v/v) at 4 ℃ for three days. The leaf samples were dehydrated in a graded series of ethanol solutions (95%, 75%, 50%, 25% and 10% of ethanol for each treatment for 30 min, three times) and embedded in paraffin; the tissue sections were cut to 4 µm thickness with a rotary microtome (RM2235, Leica Microsystems Ltd., Germany). The tissue sections were stained with toluidine blue, observed with a light microscope (ECLIPSE Ts2, Nikon Instruments Inc., Japan) and captured by a digital camera (Digital Sight DS-U3, Nikon Instruments Inc., Japan). The thickness of the whole leaf (LT), spongy (ST), palisade tissue (PT) and epidermis cells on the upper (UET) and lower (LET) sections was measured by ImageJ. Furthermore, other parameters were obtained using the following formula [54]:

$$\text{Leaf tissue compactness(LC)=}\frac{\text{The thickness of palisade tissue}}{\text{ leaf thickness}}\text{×100\%}$$

$$\text{Leaf tissue looseness}\text{ (LL)=}\frac{\text{The thickness of spongy tissu}\text{e}}{\text{ leaf thickness}}\text{×100\%}$$

$$\text{R}\text{atio of palisade tissue to spongy tissue (P/C)=}\frac{\text{thickness of palisade tissue}}{\text{ spongy tissue thickness}}\text{×100\%}$$

Determination of stomatal structure characteristics

A small piece of 1 cm×1 cm size was cut from the base 1/3 of the fully unfolded penultimate compound leaf near the central vein. After being removed, it was immediately placed into FAA fixative solution and fixed in a refrigerator at 4 ℃ for more than 48 h. Step by step dehydration with ethanol was performed, as follows: 50% ethanol soaked for 1 h, 70% ethanol soaked for 1 h, 85% ethanol soaked for 1 h, 95% ethanol soaked for 40 min twice, 100% 40 min ethanol soaked for 40 min twice, to complete dehydration. The dehydrated samples were soaked in 5% sodium hydroxide solution for 42 h or more until the leaves turned white. The samples were transferred to deionised water for cleaning two to three times, and then transferred to saturated trichloroacetaldehyde solution. After the samples were completely transparent, the trichloroacetaldehyde was utterly cleaned with deionised water. Finally, it was stained with 1% toluidine blue aqueous solution and observed with an optical microscope (Nikon Eclipse 50I, Japan), and images were captured by a digital camera (Nikon Digital Sight DS-U3, Japan)[55]. Stomatal size (stomatal long axis length) and stomatal density (mm^− 2) in the upper and lower epidermis were measured by ImageJ in five visual fields for four replicates of each treatment.

$$\text{Stomatal density (}\text{SD}\text{)}\text{=}\frac{\text{number of stomata under a yield}}{\text{ the area of yield}}$$

Photosynthesis measurements

The fully unfolded penultimate compound leaves of five plants under each treatment were used to analyse the photosynthetic responses to light intensity and quality changes at around 10:00 AM. Leaf gas exchange analysis was evaluated in a 6 cm² leaf chamber with a CO₂ concentration of 400 µmol mol^− 1 using a portable infrared gas analyser (Li-6400, LI-COR Inc., Lincoln NE, United States). Fourteen light intensity levels (0, 20, 50, 100, 150, 200, 400, 600, 800, 1000, 1200, 1500, 2000 and 2300 µmol m^− 2s^− 1 at a time step of 2 min) were imposed. The net photosynthetic and stomatal conductance response curve to light under different treatments were fitted with a smooth curve.

Statistical analysis

In this experiment, SPSS statistics 25 was used to compare the differences between treatments via a one-way analysis of variance. For all statistical analyses, mean ± standard error (SE) values are presented, and at least three biological replicates were analysed for significant differences at the 5% level in all parameters. Microsoft Excel 2016 was used for data calculation, and the figures were drawn by Origin 2021b.

Ci: Intercellular carbon dioxide concentration; Cc: Chloroplast carbon dioxide concentration; g_s: stomatal conductance; g_smax: potential maximum stomatal conductance; SD: stomatal density; SZ: stomatal size; SLW: Specific leaf weight; LT: Leaf thickness; PT: Palisade tissue thickness; ST: Spongy tissue thickness; UET: Upper epidermal cell thickness; LET: Lower epidermal cell thickness; LC: Leaf tissue compactness; LL: Leaf tissue looseness; P/C: Ratio of palisade tissue to the spongy tissue; USD: Stomatal density of upper epidermis ; LSD: Stomatal density of lower epidermis; USZ: Stomatal size of upper epidermis; LSZ: Stomatal size of lower epidermis; g_op: Operational stomatal conductance; g_opmax: Maximum operational stomatal conductance; A_N: Net photosynthetic rate; A_max: Maximum net photosynthetic rate

Ethics approval and consent to participate

Experimental research on the plants complies with relevant institutional, national, and international guidelines and legislation.

Consent for publication

Not applicable.

Availability of data and materials

All data are fully available without restriction.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by National Natural Science Foundation of China (32071963), the International S & T Cooperation Projects of Sichuan Province (2020YFH0126), and the Program on Industrial Technology System of National Soybean (CARS-04-PS19).

Authors' contributions

FY and WY carried out the design of this research work; JC, JG, SL, YF, PC, QW, TT performed the experiment; JC analyzed the data and wrote the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Authors' information

¹College of Agronomy, Sichuan Agricultural University, Huimin Road 211, Wenjiang District, Chengdu 611130, People’s Republic of China.

²Sichuan Engineering Research Center for Crop Strip Intercropping System, Chengdu 611130, People’s Republic of China.

³Key Laboratory of Crop Ecophysiology and Farming System in Southwest, Ministry of Agriculture, Chengdu 611130, People’s Republic of China.

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No competing interests reported.

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Leaf phenotype response to different intensities monochromatic blue and red light affects stomatal conductance and photosynthetic capacity

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Abstract

Background

Results

Conclusions

Figures

Background

Results

Leaf structure responded to blue and red light under different light intensities

Response curves of operational stomatal conductance to different light intensities

Relationship between net photosynthetic rate and operational stomatal conductance

Contribution of leaf structure and stomatal traits to maximum operational stomatal conductance

Discussion

Methods

Plant material

Growth conditions

Measurement of morphological characteristics

Leaf anatomical feature measurements

Determination of stomatal structure characteristics

Photosynthesis measurements

Statistical analysis

Abbreviations

Declarations

References

Additional Declarations

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