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Article

Stomatal Density Variation Within and Among Different Soybean Cultivars Across Various Growth Stages

by
Syada Nizer Sultana
1,2,
Hyun Jo
1,3,
Jong Tae Song
1,
Kihwan Kim
1,3 and
Jeong-Dong Lee
1,4,*
1
Department of Applied Biosciences, Kyungpook National University, Daegu 41566, Republic of Korea
2
College of Agricultural Sciences, IUBAT-International University of Business Agriculture and Technology, Dhaka 1230, Bangladesh
3
Upland Field Machinery Research Center, Kyungpook National University, Daegu 41566, Republic of Korea
4
Department of Integrative Biology, Kyungpook National University, Daegu 41566, Republic of Korea
*
Author to whom correspondence should be addressed.
Agriculture 2024, 14(11), 2028; https://doi.org/10.3390/agriculture14112028
Submission received: 6 September 2024 / Revised: 1 November 2024 / Accepted: 4 November 2024 / Published: 11 November 2024
(This article belongs to the Section Crop Production)
Browse Figures
Versions Notes

Abstract

:
Stomata regulate CO2 and water vapor exchange between leaves and the atmosphere, serving as a vital indicator of climate change resilience. Therefore, understanding the difference in stomatal numbers and patterns among different soybean cultivars across growth stages is essential to comprehending the complex mechanisms underlying soybean adaptation to climate change. The accurate measurements of stomatal density in soybean leaves are essential to understanding the complexity of stomatal density by environmental conditions. We demonstrated that the five epidermal sections and five microscopic images taken from both sides of each epidermal section at each leaf position (tip, middle, and bottom) were sufficient for stomatal measurements. Furthermore, we investigated variations in stomatal density among leaflet locations (left, right, and central) and leaf position across different growth stages. Notably, while there was no significant variation between the two leaves of the vegetative cotyledon (VC) stage and among the three leaflets of the V1 (first trifoliate) to V4 (fourth trifoliate) growth stages, leaves of the VC stage exhibited the lowest stomatal density, whereas those of the V4 stage exhibited the highest stomatal density. These findings could serve as a valuable tool for evaluating stomatal density, analyzing physiological differences under adverse climatic conditions, and phenotyping a large-scale population to identify the genetic factors responsible for stomatal density variations in soybean genotypes.

1. Introduction

Stomata, two small symmetric guard cells on the epidermis of higher plants, play a crucial role in regulating gas exchange between the leaf’s interior air space and the surrounding environment [1]. Stomata are separated by intervening cells, including epidermal cells and subsidiary cells, of various shapes, sizes, and numbers, arranged in specific patterns on the leaf surface [2]. While stomatal development is typically coordinated with the architecture and function of mesophyll [3] and xylem tissues [4,5], some plants deviate from these patterns and produce stomata arranged in clusters [6,7,8].
Stomatal density (SD), the number of stomata per unit leaf area, is influenced by cell division, differentiation, and expansion patterns during organ development [9]. Stomata are situated on both the adaxial (upper) and abaxial (lower) leaf surfaces in amphistomatous leaves, the most prevalent configuration. Alternatively, they may exclusively reside on the abaxial surface, in hypostomatous leaves, and less commonly, on the adaxial surface, in hyperstomatous leaves [10]. Abaxial and adaxial stomata differ from each other in various ways. Firstly, stomatal density is typically higher on the abaxial surface than on the adaxial surface of leaves [11]. Secondly, abaxial stomata play a major role in the gas exchange between a leaf or leaflet and the atmosphere and are more sensitive to environmental changes, whereas adaxial stomata contribute minimally to gas exchange activities [12,13]. Stomatal density is a reliable indicator of a plant’s adaptation to changing environmental conditions on broad regional scales because of its consistency [14]. Stomatal density directly impacts photosynthesis and transpiration [1]. The size and density of stomata, their positioning on the abaxial and adaxial sides of the leaf, and the thickness of the boundary layer collectively determine the limitations of gas exchange rates [15]. Significant variations in the stomatal density between species lead to differences in gas exchange rates. Stomatal density is regulated across different plant surfaces and in diverse environments, resulting in variable stomatal numbers and distribution patterns in mature organs [16,17]. This implies that numerous genes are involved in developmental pathways to produce various stomatal patterns and densities while ensuring their functionality [18]. The distribution of stomata on the leaf surface significantly impacts leaf function. Evidence indicates that the distribution of stomata between the two surfaces of a laminar leaf offers a fitness advantage under many conditions [19]. Researchers have long debated the impacts of changes in plant stomatal density on photosynthesis and growth [20,21]. Stomatal development adapts to environmental conditions throughout the plant growth cycle, frequently by altering the density and size of stomata on the plant epidermis [22]. Elevated light intensity, high humidity, or low atmospheric CO2 concentrations typically increase stomatal density [23]. Conversely, drought, reduced light, or elevated atmospheric CO2 concentrations decrease stomatal density [24,25,26,27]. When temperature increases, stomatal density decreases in Arabidopsis [28], whereas it increases in rice [22,29].
Interestingly, although the opening and closing of stomata serve as short-term responses to environmental changes [30], the form, distribution, and behavior of stomata fluctuate in response to short-term and long-term environmental changes [14]. Stomatal form and arrangement are mostly influenced by genetic characteristics and phenotypic plasticity, illustrating how plants adapt to their growing environments over time. Due to its importance in plant functioning and adaptability, as well as its role as an indicator of plant evolution, stomatal density has garnered substantial research attention [8]. The majority of our existing knowledge stems from the model plant Arabidopsis thaliana, and research on legume crops, especially soybean [Glycine max (L.) Merr], remains limited. Soybean is amphistomatic, with a greater stomatal density on the abaxial side than on the adaxial side [31]. Stomata are distributed randomly on the leaf surface of soybean [32]. Identifying the diversity in stomatal density among different soybean cultivars is crucial for elucidating the underlying reasons for their differential fitness under adverse conditions. Additionally, the identification of genetic factors associated with such variations is essential for conducting an efficient breeding program aimed at regulating stomatal density [33].
Several methods have been developed for determining the stomatal density of plants [34]. These methods typically involve microscopic imaging of the intact or detached leaf epidermis or a replica of the leaf surface to assess the number of stomata [33]. Recently, the deep learning-based method has been developed to measure the stomatal density of soybeans [35]. However, publications rarely mention the leaf sampling procedure, the number of epidermal sections, or microscopic images from each section. It is important to use specific sampling techniques to evaluate leaf stomatal characteristics. Within a single plant, fully expanded leaves exhibit ontogenetic gradients in stomatal spacing and uneven stomatal differentiation across the leaf surface, leading to variations in stomatal densities. Even when investigating leaves from the same species, evaluating differences in stomatal density across the leaf surface is crucial [36].
The present study aimed to determine the optimal number of epidermal sections to sample from a single leaf and to assess the variations in stomatal density within a leaf and between different leaves at different growth stages. Additionally, we aimed to evaluate variations in stomatal density among different soybean cultivars at different growth stages based on our deep learning for determining the stomatal density strategies.

2. Materials and Methods

2.1. Determination of the Optimal Number of Epidermal Sections to Sample from a Single Leaf

The soybean cultivars Williams 82 [37] was used to determine the number of epidermal sections that should be sampled from a single leaf for counting stomatal density during microscopic observation. Soybean seeds were planted on March 15, 2021, at the greenhouse of Kyungpook National University (KNU) in Daegu, Korea (35°87′14″ N, 128°60′14″ E). Soybean seeds were sown in 50-hole plastic trays (530 mm × 270 mm × 110 mm) with horticultural soil (Hanareum, Shinsung Mineral, Seongnam-si, Korea). Four seeds were initially sown and later thinned to a final stand of one seedling. One fully expanded unifoliate leaf was sampled from a single plant 20 days after planting.
The steps from leaf collection to leaf preservation for microscopy were performed following the method described [36]. Subsequently, the leaf was divided into three positions (tip, middle, and bottom) using scissors, and each position was sectioned into several pieces (Figure 1). Thirty epidermal sections (approximately 3 × 3 mm) were taken from each position, resulting in a total of 90 epidermal sections from the entire leaf. Ten microscopic images were taken from both the abaxial and adaxial sides of each epidermal section. We randomized the number of epidermal sections from a leaf and the number of photos taken from individual epidermal sections to ascertain the minimal amount required for microscopic analysis.

2.2. Variation in Stomatal Density Within a Leaf and Between Different Leaves Across Various Growth Stages

To investigate the variation in stomatal density within a leaf and between different leaves across various growth stages, the soybean cultivar Williams 82 was cultivated in the greenhouse at KNU. Seeds were planted on 2 August 2021, in a 50-hole tray, with two seeds per hole and three replications. Following germination, seedlings were thinned to a single seedling per hole. Subsequently, they were transplanted into medium-sized pots containing actual soil. Three plants were placed in three separate pots, with each plant regarded as a replication. The plants were then grown until they reached the onset of the flowering stage, which began at the fourth trifoliate stage (V4) [38].
In this experiment, two unifoliate and trifoliate leaves were collected from each growth stage of a single plant. All leaves from the vegetative cotyledon (VC) to the V4 stage were sampled with three replications on the same day when they reached at V4 stage. Based on the results of Experiment 1, five epidermal sections were extracted from each of the three positions (tip, middle, and bottom) in a leaf. Subsequently, five microscopic photographs of each leaf section were captured. Sampling was conducted for both the abaxial and adaxial sides.

2.3. Evaluation of Stomatal Density Among Different Cultivars Across Various Growth Stages

To investigate the variation in stomatal density among different cultivars, we conducted an experiment in the greenhouse at KNU using five soybean cultivars, including Williams 82 [37], and four Korean cultivars: Pungsannamul [39], Seonpung [40], Hosim [41], and Daepung [42]. Sowing was performed on 28 March 2022, in a 50-hole tray containing horticultural soil (Hanareum, Shinsung Mineral, Seongnam-si, Korea). Six seeds were sown per cultivar, with one seed per hole. At the VC stage [38] seedlings were transplanted into actual soil-containing pots, with one plant per pot. The plants were then grown up to the V4, as in our previous experiment. Leaf samples were collected from each of the five soybean cultivars, with three replications, considering a plant as a replication.
To assess stomatal density, a unifoliate leaf and the central leaflet from each trifoliate were sampled on the same day for each growth stage. During microscopy, we sampled five epidermal sections from each leaf position, ultimately capturing five microscopic images from each side of a single epidermal section for a total of fifteen epidermal sections. Sampling was conducted for both the abaxial and adaxial sides.

2.4. Quantification of Stomatal Density

The stomatal observation was performed following the method described Sultana et al. (2021) [35]. Briefly, leaf samples were fixed in absolute ethanol for 1 h and then washed with cold tap water for dehydration. The dehydrated leaf samples were transferred into a clearing agent (95% ethanol and NaOCl [6–14%] [1:1, v/v]) for 1 h to enhance leaf transparency.
Stomata were observed using a bright-field optical microscope, Leica DM2500 (Leica Microsystems Limited, Balgach, Switzerland). The photographs were captured in the 3840 × 2880 format using a DFC 450C-744780815 camera (Leica Microsystems Ltd, Heerbrugg, Switzerland). Grayscale images with a high dynamic range were captured at 40× magnification using the Leica application suite (LAS v.4.6) in the 0.3118 × 0.233 mm format.
At each growth stages (VC, vegetative cotyledon; V1, first trifoliate; V2, second trifoliate; V3, third trifoliate; V4, fourth trifoliate), the stomatal number was recorded following the method described Sultana et al. (2021) [35]. The stomatal number for each side of the epidermal segment was determined as the mean value of the stomatal number obtained from ten images collected on that side. Subsequently, the stomatal number was converted into stomatal density using the formula described [43].

2.5. Data Analysis

In the first experiment, a randomization test followed by a one-way analysis of variance (ANOVA) with a least significant difference (LSD) test was conducted to investigate the difference in stomatal density within a single leaf and to determine how many epidermal sections should be sampled from each position.
In the second experiment, ANOVA was performed using R version 3.6.1 to compare the variations in stomatal density across the three different positions within and between different leaflets at various growth stages on both the abaxial and adaxial sides of leaves. LSD test was conducted to compare the differences in stomatal density among leaflets in different growth stages and the differences in stomatal density among different positions in different leaflets at each growth stage for both sides of the leaves.
In the third experiment, ANOVA was performed using R version 3.6.1 to investigate the main effects of the cultivars, stages, and their interactions on stomatal density. LSD test was conducted for both the abaxial and adaxial sides of the leaves to examine specific differences.

3. Results

3.1. Optimal Number of Epidermal Sections for Representing the Whole Leaf

In this experiment, a single unifoliate leaf was used to determine the optimal number of epidermal sections required to obtain a representative sample from a whole leaf for accurate stomatal density measurement. Thirty epidermal sections were taken from each position (Table 1). As stomatal density varies within leaf surfaces, we sought to determine the optimal number of epidermal sections required from each position to obtain consistent results. The stomatal density measurements from seven randomly selected groups of sections (5, 7, 10, 15, 20, 25, and 30) were compared to each other. The results indicated no significant difference between any of the seven groups of sections from the tip, middle, and bottom positions on both the abaxial and adaxial sides. Thus, using the average value of five epidermal sections adequately represents each position. Additionally, we investigated the optimal number of microscopic images required from a single epidermal section. Ten microscopic images were captured from each side of ten epidermal sections from the tip, middle, and bottom positions on both the abaxial and adaxial sides. The three randomly selected groups of microscopic images (5, 7, and 10) were employed to determine the ideal number of microscopic images. The stomatal density from 5, 7, and 10 microscopic images did not exhibit significant differences (Supplementary Table S1), indicating that obtaining five microscopic images from each side of a single epidermal section is sufficient for accurately measuring stomatal density. Under the proposed optimal stomatal density measurement condition, it was observed that the abaxial side of leaves exhibited a higher stomatal density compared to the adaxial side. Interestingly, the differences in stomatal density among the tip, middle, and bottom positions of a single leaf were observed. On the abaxial surface, the tip position exhibited the highest stomatal density followed by the middle position and the bottom position regardless of the number of sections per position. However, on the adaxial surface, no difference in stomatal density was observed among the tip, middle, and bottom positions (Table 1). These findings are consistent with those reported in many research [10,11,12,13], thereby corroborating the appropriateness of the proposed optimal stomatal density measurement condition for assessing stomatal density.

3.2. The Impact of Leaflet Location and Leaf Positions Across Growth Stages on Stomatal Density

To investigate the effect of plant development (cell division, differentiation, and expansion patterns) on stomatal density, microscopic images of stomatal density on the abaxial side of leaves of Williams 82 were captured at different growth stages (Figure 2). Stomatal density of Williams 82 was quantified on both sides of the leaves across different growth stage (VC, V1, V2, V3, and V4), leaflet location (left, right, and central), leaf position (tip, middle, and bottom) by using a deep learning-based method (Table 2). Overall, the stomatal density tended to increase on the abaxial side in the order of the tip, the middle, and the bottom at the same growth stage regardless of the leaflet location. Interestingly, both sides of leaves tended to increase stomatal density across growth stages except in the V2 and V3 growth stages.
Specifically, in the VC stage, the abaxial side average of leaflets (unrolled unifoliate leave) exhibited the highest stomatal density at the tip position (194.82/mm2), followed by the middle (174.12/mm2) and the bottom (169.81/mm2) positions. The stomatal density tended to increase in the order of the tip, the middle, and the bottom on both the left and right leaflet locations. The statistical significance showed the between each position on both the left and right leaflet locations except between the middle and bottom positions. Similarly, on the adaxial side average of leaflets exhibited the highest stomatal density at the tip position (68.73/mm2), followed by the middle (67.99/mm2) and the bottom (61.94/mm2) position. The stomatal density at the tip and the bottom, as well as the middle and the bottom position, showed a significant difference except between the tip and the middle position. Interestingly, even though no statistically significant differences in stomatal density were observed among the left and right leaflets at each position, the statistical significance showed only between the tip and the bottom, as well as the middle and the bottom in the right leaflet.
In the V1 stage, the abaxial side average of leaflets (first trifoliate leaf) exhibited the highest stomatal density at the tip position (369.93/mm2), followed by the middle (328.11/mm2) and bottom (307.02/mm2) positions. The significant differences showed between each position. When investigating the leaflet location individually, statistically significant differences in stomatal density were observed only between the tip and bottom positions in the left leaflet. Conversely, significant differences in stomatal density were observed among all three positions in the right leaflet. In the case of the central leaflet, significant differences in stomatal density were observed between the tip and the middle positions as well as the tip and bottom positions, whereas the difference in stomatal density between the middle and the bottom positions was not significant. Similarly, on the adaxial side average of leaflets exhibited the highest stomatal density at the tip position (139.85/mm2), followed by the middle (128.44/mm2) and the bottom (120.73/mm2) positions. The significant differences showed only the between the tip and the bottom position. Although no statistically significant differences were observed in the left leaflet, the statistically significant differences in stomatal density were observed between the tip and the middle as well as the tip and the bottom except between the middle and the bottom in the right leaflet. Also, the results of the central leaflet were similar to those of the right leaflet. Surprisingly, no statistically significant differences in stomatal density were observed among the left, right, and central leaflets at each position on the abaxial side. However, on the adaxial side, the statistically significant differences in stomatal density at the middle position were observed between the left and the right as well as the left and central leaflet.
In the V2 stage, the abaxial side average of leaflets (second trifoliate leaf) exhibited the highest stomatal density at the tip position (315.95/mm2), followed by the middle (289.52/mm2) and bottom (284.96/mm2) positions. The statistical significance showed only the between each position except the between the middle and the bottom. Although statistically significant differences in stomatal density on the right leaflet were not observed, statistically significant differences were observed between the tip and middle as well as the tip and the bottom positions in the left leaflet, and between the tip and the bottom position in the central leaflet. Conversely, on the adaxial side average of leaflets exhibited the highest stomatal density at the middle position (86.96/mm2), followed by the bottom (86.26/mm2) and the tip (82.19/mm2) positions. However, there were no statistically significant difference between each position regardless of leaflet locations. The differences in stomatal density among the three leaflet locations in each position were not statistically significant for both the abaxial and adaxial sides.
In the V3 stage, the abaxial side average of leaflets (third trifoliate leaf) exhibited the highest stomatal density at the tip position (325.73/mm2), followed by the middle (299.15/mm2) and bottom (286.86/mm2) positions. The statistical significance showed the between each position except the one between the middle and the bottom. The statistically significant differences in stomatal density between the tip and the middle positions as well as the tip and the bottom positions were significant on both the left and right leaflet, whereas those between the middle and the bottom positions were not significant. In the central leaflet, the statistically significant differences between the tip and the middle positions were not significant, whereas those between the tip and the bottom positions as well as the middle and bottom positions were significant. The statistically significant differences in stomatal density at the middle position were observed between the left and the central leaflet. Similar to the V2 stage, the adaxial side average of leaflets exhibited the highest stomatal density at the middle position (69.86/mm2), followed by the bottom (69.31/mm2) and the tip (67.20/mm2) positions. Also, no statistically significant difference in stomatal density was observed among the tip, the middle, and the bottom position on the adaxial side. Similar to the abaxial side, the statistically significant differences in stomatal density at the middle position were observed between the left and the central leaflet.
In the V4 stage, the abaxial side average of leaflets (fourth trifoliate leaf) exhibited the highest stomatal density at the tip position (395.11/mm2), followed by the middle (368.77/mm2) and bottom (351.46/mm2) positions. Consistent with the results according to growth stages, the statistical significance showed the between each position except between the middle and the bottom. The statistically significant differences in stomatal density between the tip and the middle positions as well as the tip and the bottom positions were significant on both the left and right leaflet, whereas those between the middle and the bottom positions were not significant. In the central leaflet, the statistically significant differences between the tip and the middle positions were not significant, whereas those between the tip and the bottom positions as well as the middle and bottom positions were significant. Surprisingly, unlike other growth stages, there were various statistically significant differences in leaf position depending on the leaflet location. Similarly, the adaxial side average of leaflets exhibited the highest stomatal density at the tip position (132.99/mm2), followed by the middle (131.96/mm2) and the bottom (125.96/mm2) positions. Furthermore, the statistical significance showed no significant differences between each position. No statistically significant differences in stomatal density were observed among the three positions in each leaflet location.

3.3. Interaction Effects of Growth Stage, Leaflet Location and Leaf Position on Stomatal Density

In this experiment, the variation in stomatal density on the abaxial and adaxial sides of a single leaf and between leaflets from different growth stages was investigated (Table 3). ANOVA results revealed highly significant differences (p < 0.001) in stomatal density due to the effect of growth stage (S) and significant differences (p < 0.05) due to the effect of leaf position (P) on both the abaxial and adaxial sides. However, the stomatal density of leaflet location (L) did not exhibit significant differences.
The interaction between the growth stage (S) and leaflet location (L) significantly influenced the stomatal density on the abaxial side, whereas the interaction between the growth stage (S) and leaf position (P) significantly influenced the stomatal density on the adaxial side. The interaction between leaflet location (L) and leaf position (P), as well as The interaction between growth stage (S), leaflet location (P), and leaf position (P), showed no significant differences in stomatal densities on both the abaxial and adaxial sides.

3.4. Variation in Stomatal Density Among Different Cultivars Across Various Growth Stages

Stomatal density on the abaxial and adaxial surfaces varied among soybean cultivars and with the advancement of growth stages (Table 4). The selected cultivars Pungsannamul, Hosim, Seonpung, and Daepung are utilized as representative Korean cultivars. Pungsannamul is a cultivar optimized for processing antioxidant compounds, including polyphenols and flavonoids [44], while Hosim is characterized by a high oleic acid content [41]. These cultivars were utilized to investigate the correlation between genetic traits associated with soybean quality and stomatal density. Seonpung [40] and Daepung [42] are high-yielding cultivars with large seed sizes, making them suitable as comparative cultivars for evaluating the impact of stomatal density on productivity. Consistent with the results according to growth stages, the VC stage exhibited the lowest stomatal density, and the highest stomatal density on the abaxial side was observed at the V4 stage across five soybean cultivars, except for Seonpung, which exhibited the highest stomatal density at the V1 stage. Overall, five soybean cultivars tended to increase the stomatal density on the both abaxial and adaxial sides in order of tip, middle, and bottom except V2 and V3 stages. Furthermore, five soybean cultivars tended to show lower stomatal density on the abaxial side across the growth stages compared to Williams 82 as used reference control.
Specifically, in the VC stage, the statistically significant differences in stomatal density on the abaxial surface were observed among five soybean cultivars, except between Hosim and Pungsannamul. Daepung (150.00/mm2) exhibited the lowest stomatal density, whereas Williams 82 (184.81/mm2) exhibited the highest stomatal density. On the adaxial side, Hosim (59.58/mm2) exhibited the lowest stomatal density, whereas Seonpung (71.14/mm2) exhibited the highest stomatal density. The Seonpung exhibited statistically significant differences from other cultivars including cultivar Williams 82.
In the V1 stage, Seonpung (343.62/mm2) exhibited the highest stomatal density on the abaxial side, whereas Daepung (236.81/mm2) exhibited the lowest stomatal density. Statistically significant differences were observed between each soybean cultivar except between Hosim and Pungsannamul as well as Hosim and Williams 82. On the adaxial side, Hosim (148.11/mm2) exhibited the highest stomatal density, whereas Daepung (114.09/mm2) exhibited the lowest stomatal density. Notably, on the adaxial side, no variations in stomatal density were observed between Daepung and Pungsannamul, Pungsannamul and Williams 82, Seonpung and Williams 82, and Daepung and Williams 82.
In the V2 stage, Williams 82 (286.91/mm2) exhibited the highest stomatal density on the abaxial side, whereas Daepung (218.88/mm2) exhibited the lowest stomatal density. Notably, the statistically significant differences between Daepung and Pungsannamul as well as Seonpung and Williams 82 were not significant. On the adaxial side, Williams 82 (90.60/mm2) exhibited the highest stomatal density, whereas Daepung (71.69/mm2) exhibited the lowest stomatal density. While the stomatal density of Daepung differed significantly from that of other cultivars, the differences between the stomatal density of other cultivars were not significant at this stage.
In the V3 stage, the statistically significant differences in stomatal density on the abaxial side were observed among all cultivars. On the abaxial side, Williams 82 (337.69/mm2) exhibited the highest stomatal density, whereas Pungsannamul (246.54/mm2) exhibited the lowest stomatal density. All cultivars exhibited significant differences in stomatal density, except between Hosim and Seonpung. On the adaxial side, Hosim (104.48/mm2), exhibited the highest stomatal density, whereas Pungsannamul (87.84/mm2) exhibited the lowest stomatal density. No statistically significant differences in stomatal density were observed between Daepung and Pungsannamul, Seonpung and Williams 82, and Hosim and Seonpung.
In the V4 stage, the statistically significant differences in stomatal density on the abaxial side were observed among all cultivars. The highest and lowest stomatal densities were observed in leaves of Williams 82 (482.74/mm2) and Pungsannamul (298.84/mm2), respectively. Similarly, significant variations in stomatal density on the adaxial side were observed among all cultivars, except between Daepung and Hosim. Williams 82 (159.85/mm2) exhibited the highest stomatal density, whereas Pungsannamul (86.07/mm2) exhibited the lowest stomatal density.
Stomatal density exhibited significant variation (p < 0.001) on both sides of the leaves due to both cultivars and growth stages. Additionally, a significant difference (p < 0.001) was observed in the interaction between cultivars (C) and growth stages (S) (Table 5). The (C × S) interaction between the cultivar (C) and growth stage (S) significantly influenced the stomatal density on the both abaxial and adaxial sides.

4. Discussion

Stomata are a pivotal focus for plant biologists due to their role in regulating plant growth and environmental responses [45]. Stomatal features vary considerably within and between leaves [36], across species, and even among individuals within a species [46], depending on variables such as genotype [47], growth stage [48], and environment [19]. However, studies exploring the stomatal variation in soybean using precise testing methods are limited. In this study, we aimed to elucidate the variation in stomatal density in soybean using a newly developed method [35].
Studies on stomatal variation rarely specify the leaf sampling techniques employed. The ideal approach for sampling leaves has not yet been established, leaving uncertainties regarding the optimal number of specimens or microscopic images required from a single leaf. Various sampling techniques have been employed to examine stomatal distribution in soybean. For example, Amaliah et al. (2019) [32] obtained a stomatal section from both sides of the soybean leaf and observed stomata from three different fields of view. Sakoda et al. (2019) [33] determined stomatal density by sampling the central leaflets on the first trifoliate in their greenhouse experiment. They prepared replicas of the abaxial side of the tip, middle, and bottom positions of each leaflet, and acquired three microscopic images for each replica [33]. Additionally, they sampled fully expanded and central leaflets on the upper-most trifoliate in their field experiment [33]. They prepared one replica from the middle position of the central leaflet and captured 6–9 microscopic images for each accession [33]. However, these sampling procedures lack logical justification. Evaluating stomatal characteristics necessitates a well-defined sampling technique. Given the variations in stomatal density within a leaf, epidermal sectioning should be performed separately from the tip, middle, and bottom positions. Our findings indicated that at least five epidermal sections from each position and five microscopic images from each side of a single epidermal section should be obtained for any stomatal measurement (Table 1). In this study, we assessed the variation in stomatal density within a single leaf. Our results revealed significant differences in stomatal density between the abaxial and adaxial surfaces of soybean leaves, with the abaxial surface containing more stomatal density than the adaxial surface (Table 1). This result is consistent with the findings reported previously in soybean. Additionally, we found that stomatal density was higher in the tip position than in the middle and bottom positions on the abaxial surface of the leaf (Table 1), aligning with the findings reported previously [33]. Only a few studies have investigated how stomatal density varies across different positions on a single soybean leaf. Poole et al. (1996) [36] revealed that stomatal density was higher at the margins and tips of both monocotyledonous and dicotyledonous herbs, compared to other positions in a leaf.
We also observed differences in stomatal density between unifoliate leaves and among trifoliate leaves in soybean. The results indicated that any of the leaflets can be sampled for measuring stomatal density because no discernible variations were observed between the two unifoliate leaves and among the three leaflets of a trifoliate leaf (Table 2). Additionally, we found that stomatal density on the abaxial surface changed significantly in response to growth stages, exhibiting an increasing pattern except at the V2 stage. However, on the adaxial surface, stomatal density changed in a randomly oriented pattern. Furthermore, mature leaves in the VC stage exhibited the lowest stomatal density, whereas developing leaves in the V4 stage exhibited the highest stomatal density on both leaf surfaces. Several studies have highlighted the differences in stomatal density among different stages in various crops. It was revealed that terminal leaves exhibited higher stomatal density than basal leaves in alfalfa (Medicago sativa L.) [49]. It found differences in stomatal frequencies across different plant positions and concluded that leaflets on the seventh node exhibited a higher number of stomata than leaflets on the first node in faba bean (Vicia faba L.) [50]. A previous study reported that stomatal density was higher in the upper leaves than in the lower leaves of the common bean (Phaseolus vulgaris L.) [51]. Another study revealed that rice leaves exhibit higher stomatal density at later stages of growth [52]. Conversely, some studies have shown that younger leaves exhibit larger stomatal densities. It discovered that the flag leaf in rice exhibits larger stomatal density at the heading stage than at the mid-growth stage [53,54]. Besides genotype and environmental conditions, mature leaves may play an integral role in the stomatal development of new leaves through the transmission of systemic signals because stomatal development in developing leaves is not completely autonomous [55]. In dicots, epidermal cell expansion after proliferative and formative divisions during leaf growth significantly impacts the stomatal density exhibited during the final stages of leaf growth [48].
Stomatal frequency is widely assumed to be species-specific [47]. Under identical environmental conditions, we observed differences in stomatal density across cultivars at different growth stages (Table 4). Previous studies have demonstrated diversity in stomatal density among various soybean germplasm at different stages. Stomatal density ranged from 242 to 345 mm−2 across 43 soybean accessions in the third trifoliate stage, from 192 to 334 mm−2 across 77 soybean accessions in the seed-filling stage [31], and from 93 ± 3 to 166 ± 4 mm−2 across 90 soybean accessions in the first trifoliate stage. Tanaka et al. (2010) [31] discovered a considerable difference in stomatal density between U.S. cultivars and Japanese soybean cultivars. In another study, Sultana et al. (2021) [35] revealed that stomatal density at the VC stage ranged from 54.79 to 328.8 mm−2 across 386 accessions, with a mean of 134.0 mm−2. Surprisingly, each of the five soybean cultivars had unique stomatal density characteristics across the plant development. Based on the water-use efficiency depending on stomatal density, these findings provide a perspective on the breeding approach for each cultivar by growth stages.

5. Conclusions

In this study, we quantified the variation in stomatal density within and between soybean leaves at different growth stages. This study is the first to report a non-significant variation between two unifoliate leaves and among the three leaflets of a trifoliate leaf in each stage. Stomatal density also varied between different stages among different cultivars. These results suggest that any unifoliate leaf or any leaflet from a trifoliate can be considered for sampling throughout the measurement process.
Based on the results of the present study, we propose the following method as an improved way of sampling: (1) collect a unifoliate or a leaflet from a trifoliate at any growth stage, (2) take five epidermal sections from each leaf position (tip, middle, and bottom) for microscopy, and (3) capture five microscopic images from both sides of a single epidermal section. This approach will provide sufficient data for measuring stomatal density in soybean. While there are few reports on soybean stomatal density, the effects of genetic differences on stomatal density in soybean remain largely unexplored. The phenotypic differences observed in the assessed traits predominantly stem from genetic factors, indicating potential implications for future plant breeding goals and the selection of promising plant materials. From this perspective, the variables discussed in this research hold considerable significance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14112028/s1, Table S1: Quantitative assessment of the stomatal density on the both abaxial and adaxial sides across sequential imaging sections.

Author Contributions

Writing—original draft, S.N.S.; visualization, S.N.S.; methodology, S.N.S.; investigation, S.N.S.; formal analysis, S.N.S.; data curation, S.N.S.; writing—review and editing, H.J., J.T.S., K.K. and J.-D.L.; conceptualization, J.-D.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01416803)” Rural Development Administration, Jeonju, Korea.

Institutional Review Board Statement

Not applicable.

Data Availability Statement

The data presented in this study are available on request from the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Agriculture 14 02028 g001
Figure 1. Single leaf showing three different positions (tip, middle, and bottom). Each position was cut into thirty epidermal sections and then microscopic images were collected from each section. The size of a single epidermal section is approximately 3 × 3 mm.
Figure 1. Single leaf showing three different positions (tip, middle, and bottom). Each position was cut into thirty epidermal sections and then microscopic images were collected from each section. The size of a single epidermal section is approximately 3 × 3 mm.
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Figure 2. Single-layer microscopic images of stomatal arrangement on the abaxial side of leaves at different growth stages in Williams 82.
Figure 2. Single-layer microscopic images of stomatal arrangement on the abaxial side of leaves at different growth stages in Williams 82.
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Table 1. The difference of stomatal density in different leaf positions on the abaxial (lower) and adaxial (upper) sides of a single leaf.
Table 1. The difference of stomatal density in different leaf positions on the abaxial (lower) and adaxial (upper) sides of a single leaf.
No.
Sections per Position		Number of Stomata/mm2
AbaxialAdaxial
TipMiddleBottomMeanLSD (x)TipMiddleBottomMeanLSD (x)
5143.15± 3.39134.11 ± 3.80129.61 ± 3.98135.634.5853.45 ± 5.2255.83 ± 2.9455.47 ± 4.3054.925.26
7143.84 ± 2.27133.25 ± 3.50129.65 ± 3.75135.583.9853.05 ± 4.8156.28 ± 3.9855.64 ± 2.3855.004.74
10143.47 ± 2.56133.40 ± 2.04129.31 ± 1.78135.402.6453.7 ± 2.9255.28 ± 3.1654.64 ± 2.5854.553.56
15144.14 ± 2.10133.85 ± 1.41129.12 ± 1.41135.712.0553.04 ± 1.6754.75 ± 2.9154.87 ± 2.2054.222.85
20143.66 ± 1.40134.13 ± 1.40129.17 ± 0.89135.661.5453.62 ± 1.2654.76 ± 1.4254.97 ± 1.4054.451.67
25143.41 ± 0.70133.82 ± 0.96129.14 ± 1.01135.461.1153.53 ± 0.8154.65 ± 0.6255.03 ± 1.2754.411.15
30143.65 ± 0.00133.56 ± 0.00129.70 ± 0.00135.641.4154.04 ± 0.0054.92 ± 0.0055.05 ± 0.0054.681.05
LSD (y)2.432.652.691.71 3.542.982.782.31
(x) LSD (0.05) for comparison values for each row. (y) LSD (0.05) for comparison value for each column.
Table 2. The differences in stomatal density in different leaf positions among leaflet locations across growth stages.
Table 2. The differences in stomatal density in different leaf positions among leaflet locations across growth stages.
Growth StagesLeafletAbaxial (Number of Stomata/mm2)Adaxial (Number of Stomata/mm2)
TipMiddleBottomLSD (y)TipMiddleBottomLSD (y)
VCLeft193.99175.45173.1613.7967.6366.7162.408.24
Right195.64172.79166.4612.7569.8369.2861.486.97
LSD (x)14.0911.9914.39 6.518.058.60
V1Left368.34331.50319.7139.58134.62138.57125.1722.83
Right366.69327.88300.8120.11144.89126.36120.3915.71
Central374.77324.94300.5324.67140.03120.39116.6315.57
LSD (x)35.2529.5124.16 17.5617.6216.57
V2Left314.11284.19285.3926.6877.5483.5184.3313.92
Right322.73296.31294.9328.6087.7385.7190.2012.57
Central310.99288.05274.5630.2781.3091.6786.3411.79
LSD (x)29.6525.8430.00 11.1412.0314.89
V3Left332.00288.78289.5234.7766.2562.3169.289.46
Right314.20291.26283.3720.6765.4368.9265.3411.32
Central330.99317.41287.6823.2469.9278.3773.3210.92
LSD (x)31.9527.0623.89 8.4211.0512.00
V4Left406.97377.06357.5222.09119.84132.88120.7620.11
Right397.43367.24366.3319.91141.32134.62129.9423.56
Central380.92362.01330.5425.82137.83128.38127.1922.98
LSD (x)24.7322.2221.11 23.08-22.1521.55
Mean LSD (w) LSD (w)
VC194.82174.12169.8110.1968.7367.9961.945.08
V1369.93328.11307.0220.83139.85128.44120.7313.29
V2315.95289.52284.9626.2182.1986.9686.269.53
V3325.73299.15286.8623.4067.2069.8669.317.34
V4395.11368.77351.4619.62132.99131.96125.9619.61
LSD (z)59.2244.4053.72 31.5329.8830.77
(x) LSD (0.05) for comparison mean in each position of different leaflets. (y) LSD (0.05) for comparison mean among different positions in each leaflet. (w) LSD (0.05) for comparing mean among position in each growth stages. (z) LSD (0.05) for comparison mean of each position in different growth stages.
Table 3. Analysis of variance for stomatal density (number of stomatal/mm2) on the abaxial and adaxial side of soybean leaf across grow stage (S), leaflet location (L), and leaf position (P).
Table 3. Analysis of variance for stomatal density (number of stomatal/mm2) on the abaxial and adaxial side of soybean leaf across grow stage (S), leaflet location (L), and leaf position (P).
Abaxial SideAdaxial Side
Source of VariationDfF-ValuePr (>F)F-ValuePr (>F)
Growth stage (S)4432.14<2.2 × 10−16 ***286.17<2.2 × 10−16 ***
Leaflet location (L)21.380.251.520.22
Leaf position (P)276.15<2.2 × 10−16 ***3.610.03 *
S × L72.500.02 *1.670.11
S × P81.810.072.280.02 *
L × P41.160.331.020.39
S × L × P140.430.960.80.67
Residuals588
Total629
Asterisks indicated statistically significant differences by ANOVA (* p < 0.05, *** p < 0.001)
Table 4. The differences in stomatal density on abaxial and adaxial sides in different cultivars among across growth stages.
Table 4. The differences in stomatal density on abaxial and adaxial sides in different cultivars among across growth stages.
Abaxial Side (Number of Stomata/mm2)
CultivarsVCIndexV1Index (a)V2Index (a)V3Index (a)V4Index (a)LSD (y)
Daepung150.0081.16236.8176.07218.8876.29260.0076.99456.894.6316.49
Hosim158.9986.03293.2894.21249.5386.97301.1189.17422.4287.5017.79
Pungsannamul159.4886.29274.1988.07230.4580.32246.5473.01298.8461.908.94
Seonpung174.8494.61343.62110.38280.2597.68297.3788.06329.1368.1813.63
Williams 82184.81100.00311.32100.00286.91100.00337.69100.00482.74100.0013.39
LSD (x)6.46 20.16 12.73 11.66 16.95
Adaxial Side (Number of Stomata/mm2)
CultivarsVCIndexV1Index (a)V2Index (a)V3Index (a)V4Index (a)LSD (y)
Daepung60.8795.77114.0993.1171.6979.1391.0393.53146.3991.5811.78
Hosim59.5893.74148.11120.8787.8496.95104.48107.35146.7091.7710.77
Pungsannamul61.4296.63114.1693.1686.8795.8887.8490.2586.0753.847.08
Seonpung71.14111.93129.45105.6495.00104.8698.98101.70116.0572.608.46
Williams 8263.56100.00122.54100.0090.60100.0097.33100.00159.85100.009.21
LSD (x)5.54 12.86 8.46 6.96 12.15
(x) LSD (0.05) for comparison the mean value among cultivars in specific growth stage. (y) LSD (0.05) for comparison mean value among growth stages within individual cultivars. (a) Index of all cultivars in different stages were computed relative to Williams 82.
Table 5. Analysis of variance of stomatal density (number of stomata/mm2) on the abaxial and adaxial side of soybean leaf among the different cultivars across different growth stages.
Table 5. Analysis of variance of stomatal density (number of stomata/mm2) on the abaxial and adaxial side of soybean leaf among the different cultivars across different growth stages.
Abaxial SideAdaxial Side
Source of VariationDfF-ValuePr (>F)F-ValuePr (>F)
Cultivar (C)4158.33<2.2 × 10−16 ***32.19<2.2 × 10−16 ***
Stage (S)41301.64<2.2 × 10−16 ***330.17<2.2 × 10−16 ***
C × S1657.81<2.2 × 10−16 ***17.61<2.2 × 10−16 ***
Residuals1100
Total1124
Asterisks indicated statistically significant differences by ANOVA (*** p < 0.001)
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Sultana, S.N.; Jo, H.; Song, J.T.; Kim, K.; Lee, J.-D. Stomatal Density Variation Within and Among Different Soybean Cultivars Across Various Growth Stages. Agriculture 2024, 14, 2028. https://doi.org/10.3390/agriculture14112028

AMA Style

Sultana SN, Jo H, Song JT, Kim K, Lee J-D. Stomatal Density Variation Within and Among Different Soybean Cultivars Across Various Growth Stages. Agriculture. 2024; 14(11):2028. https://doi.org/10.3390/agriculture14112028

Chicago/Turabian Style

Sultana, Syada Nizer, Hyun Jo, Jong Tae Song, Kihwan Kim, and Jeong-Dong Lee. 2024. "Stomatal Density Variation Within and Among Different Soybean Cultivars Across Various Growth Stages" Agriculture 14, no. 11: 2028. https://doi.org/10.3390/agriculture14112028

APA Style

Sultana, S. N., Jo, H., Song, J. T., Kim, K., & Lee, J. -D. (2024). Stomatal Density Variation Within and Among Different Soybean Cultivars Across Various Growth Stages. Agriculture, 14(11), 2028. https://doi.org/10.3390/agriculture14112028

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