Stomatal responses at different vegetative stages of selected maize varieties of Bangladesh under water deficit condition

Drought stress causes stomatal behavior change in most plants. Water deficit condition caused by drought is one of the most significant abiotic factors reducing plant growth, development, reproductive efficiency, and photosynthesis, resulting in yield loss. Maize (Zea mays L.) holds a superior position among all the cereals due to its versatile use in the food, feed, and alcohol industries. A common demonstrative feature of a complex network of signaling pathways led by predominantly abscisic acid under drought conditions is stomatal aperture reduction or stomatal closure, which allows the plant to reduce water loss through the stomatal pore and to sustain a long time on water deficit condition. This study analyses the stomatal density, stomatal closure percentages, and guard cell aperture reduction using a microscopy-based rapid & simple method to compare guard cell response & morphological variations of three hybrid maize varieties viz. BHM (BARI hybrid maize)-7, BHM-9, and BHM-13 developed by Bangladesh Agricultural Research Institute (BARI). A drought treatment was applied to all varieties at two different vegetative stages, vegetative stage 3 (V3) and V5, until they reach V4 and V6, respectively. After drought exposure at the V4 stage, the percentage of closed stomata of BHM-7, BHM-9, and BHM-13 was 21%, 23%, and 33%, respectively. The reduction in the guard cell aperture ratio of BHM-7, BHM-9, and BHM-13 was 14.83%, 10.92%, and 33.85%, respectively. At the V6 stage, for the second set of plants, the closed stomata of BHM-7, BHM-9, and BHM-13 were 18%, 21%, and 34%, respectively. The rate of reduction in guard cell aperture ratio of BHM-7, BHM-9, and BHM-13 was 5.52%, 2.48%, and 38.75%, respectively. Therefore, BHM-13 showed maximum drought adaptation capacity compared to BHM-7 and BHM-9 due to the highest percentage of closed stomata and the highest percentage of reduction in aperture ratio.


Introduction
Globally, drought is the most harmful environmental phenomenon that comes with financial 48 hardship among farmers in developed countries, malnutrition, and even famine in third-world 49 countries (1). It adversely affects almost every physiological process in the plant, such as 50 membrane fluidity and function, decreases photosynthesis, causes injury, aberrant physiology, 51 limitation of growth, and increases susceptibility to insects and disease-causing pests (2). Plants    leaf of the plant. Sharp forceps were used to take off a small portion of the leaf epidermal peel 137 (<0.5 cm) as stomata are located in this area. The leaf epidermis was placed in a glass slide and a 138 drop of water was added to the epidermis. The glass slide was then covered by a coverslip.

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Epidermal peel was collected at V3 and V5 stage just before drought exposure, at V4 and V6 stage 140 just after drought exposure, and at V5 and V7 stage after re-watering of both control and treated 141 plants.  The guard cell aperture ratio was counted by the width/length method described by Russell will have 0.18 to less than 0.29, and fully closed stomata will have 0. Specific numbers of stomata 159 were taken randomly per mm2 to measure the guard cell aperture ratio in the second leaf of plants 160 in each variety at different vegetative stages.

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After re-watering at the V7 stage, the mean aperture ratio of BHM-7 was 0.078±0.0079 (Mean ± 428 SE) and 0.065±0.007 (Mean ± SE) for the control and treated groups, respectively (Fig. 6B). The 429 mean aperture ratio of BHM-9 was 0.0612±0.006 (Mean ± SE) and 0.058±0.006 (Mean ± SE) for 430 the control and treated groups, respectively (Fig. 6B). The mean aperture ratio of BHM-13 was 431 0.0561±0.0058 (Mean ± SE) and 0.0550±0.0078 (Mean ± SE) for the control and treated groups, 432 respectively (Fig. 6B). After re-watering, the differences between aperture ratio of BHM-7 control 433 and treated, BHM-9 control and treated, BHM-13 control and treated were 16.50%, 27.74%, and  The stomatal density of BHM-7, BHM-9 and BHM-13 varies each other with the age of plants and 466 treatment condition (Fig. 1). Stomatal density was increased in all plants in V4 and V5 stage (Fig.   467 1C and Fig. 1E) compared to V3 (Fig. 1A). However, stomatal density was decreased in BHM-7 468 at V7 stage (Fig. 1F) compared to V5 (Fig. 1B) and V6 stage (Fig. 1D). Drought has a strong 469 influence in stomatal density, guard cell size and aperture ratio. It has been found that severe  to BHM-7 and BHM-9 both at the V4 stage (Fig. 2C) and at the V6 stage (Fig. 3C). By synthesizing 480 abscisic acid, it effectively reduces water loss in drought conditions than the other two varieties 481 regarding stomata closing phenomena (20). After re-watering for 7 days, no stomata were closed 482 in BHM-13 (Fig. 4C), however, BHM-7 and BHM-9 had 7% and 15% closed stomata, respectively 483 at V5 stage ( Fig. 4A and Fig. 4B). This was due to a physiologically younger form of leaves of 484 stressed plants following turgor regaining (21). During endosmosis or the entry of water, stomata became turgid, which resulted in stomatal opening. When the turgor develops within the two guard 486 cells, the thin outer walls bulge outward and force the inner walls into a crescent shape to open the 487 stomata. This is the state during which the exchange of oxygen, carbon dioxide, and water vapor 488 loss occurred through pores (3, 22).

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At the V3 stage and V5 stage, before the drought, BHM-13 had the lowest aperture ratio (Fig. 5A   490 and 5B). After drought exposure at V4 and V6 stage, a highest reduction of the aperture ratio was 491 found in BHM-13 treated plants compared to control plants ( Fig. 5C and 5D). This is probably due 492 to the production of high abscisic acid in the treated plants under water deficit condition, that 493 subsequently leads to a reduction of the aperture between guard cells (20). A similar aperture ration 494 was found in control and treated plants, both for BHM-7 and BHM-9 ( Fig. 5C and 5D). After 495 drought exposure followed by re-watering, the aperture ratio of the treated group was very close 496 to the control group for BHM-13 ( Fig. 6A and 6B). However, the aperture ratio is varied for the 497 control and treated plants, both for the BHM-7 and BHM-9 ( Fig. 6A and 6B). Plants' abiotic stress 498 adaptation mechanisms are very complex; however, abscisic acid is the crucial regulator of 499 adaptation mechanisms (23). 500 Therefore, BHM-13 is the most drought-resistant variety amongst the three tested varieties (BHM-501 7, BHM-9, and BHM-13). It has been found that BHM-13 is morphologically short and capable 502 of developing side branching from its lower nodes under heat stress condition (24). Since drought-503 resistant plants should combine a better root system, stomatal regulation, water-use efficiency, and 504 hormonal balance, further morphological, biochemical & yield analyses are recommended to find 505 out the drought-resistant variety more precisely.

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Acknowledgments 509 We thank Biotechnology and Genetic Engineering Discipline and Agrotechnology Discipline, 510 Khulna University for their institutional and technical support.