Physiological Comparison of Wheat and Maize Seedlings Responses to Water Stresses

Abstract

The aim of the study was to investigate specific responses of spring wheat (C₃ photosynthesis) and maize (C₄ photosynthesis) to drought and flooding stress. Analyses of water content, gas exchange intensity, photosynthetic apparatus activity, chlorophyll content, plant height and biological membrane integrity were performed on the 10th day of drought and flooding in both species at the third leaf stage. A specific response of wheat under both drought and flooding conditions involved an increase in ET_o/RC ratio, describing electron transport flux converted into a single reaction center in PSII. Correlations between electrolyte leakage and the probability of electron transport beyond the plastoquinone Q_A, and the amount of energy used for the electron transport were also found. A specific response of maize during flooding was the increase of stomatal conductance. Additionally, a significant correlation between P_N/C_i and relative water content was exhibited. Furthermore, the parameters differentiating the studied species only under stressful conditions were rendered. The application of such parameters can be widely used, e.g., for studying the reaction of a potential cultivars to drought and flooding. Providing such information to potential farmers can help better select cultivars for their environmental conditions.

1. Introduction

Most forecasts on climate changes predict increasing water shortages in the areas of cereal cultivation as well as weather anomalies such as periodic flooding, larger floods, or whirlwinds [1,2]. This is why the demand for species and cultivars with enhanced tolerance to these environmental stresses is on the rise [3].

Soil drought is a serious risk to plant productivity. A decrease in the quantity and quality of yields is the final result of soil drought, as a consequence of many physiological disturbances, such as increasing the degree of cell membrane damage and chlorophyll degradation or reducing the photochemical activity of PSII by disordering the energy flow in PSII [4]. The occurrence of too heavy a rainfall after a long drought is also not favorable. Too much rain results in excess soil water, limited oxygen and nutrient availability and therefore reduced plant growth. Finally, agricultural lands can be subject to the inundation of water over-spilling from ditches, rivers, and streams during periods of intense rainfall [5,6]. Our previous research shows that plant aging induced by soil drought can be continued and even intensified by too rapid irrigation in the rehydration phase [7]. Water availability is critical for wheat production, and drought is the major cause of yield losses [8,9]. This is a serious problem, because wheat is one of the most important foods and is a major source of energy and nutrition for many people, for example, bread wheat is the staple food for about 35% of the world’s population [10,11,12]. Maize is also of great importance for human and animal nutrition, and has been reported to be very sensitive to drought stress. Although maize is perceived as preferring warm areas, such as the Mediterranean, which would suggest its low water requirements, in fact its cultivation in these areas often has to be supported by additional irrigation. There is a group of current studies that focuses on modeling the actual needs of maize to reduce unnecessary water losses [13,14]. This is another proof that plants are very often subjected to opposing effects, such as lack and excess of water.

Therefore, our research focused on two opposing stresses to investigate the specific responses of spring wheat (C₃ photosynthesis) and maize plants (C₄ photosynthesis) to drought and flooding stress at early stages of growth. As the species differ in their type of photosynthesis, we assumed they would also react in different ways to these two stress factors. To verify this hypothesis, we investigated and compared their gas exchange, photosynthetic apparatus activity, chlorophyll content, growth dynamics, water ratios, and damage to biological membranes.

The studied species differ not only in the type of photosynthesis, but also in height and development of the plant. For this reason, comparing their stress response, without specifying the control groups, is virtually impossible. Therefore, the aim of the study was also to select the parameters differentiating both species under stressful conditions and with insignificant differences under control conditions. The knowledge of such parameters may be useful for comparative studies of species in natural conditions. We suppose that the parameters of the chlorophyll fluorescence may be the most useful. As compared with measuring gas exchange, chlorophyll fluorescence measurement is quick, inexpensive and simple to perform. Its huge advantage is its high sensitivity that allows for detecting early stress-related changes prior to the visible symptoms.

The novelty of these studies will be the selection of parameters for the evaluation of the reactions of morphologically different species with a different type of photosynthesis, based on the same level of these parameters under optimal conditions and showing differences only under water stresses. Then, it will be possible to conclude that these differences do not result from differences between these species, but from their sensitivity to the stress tested.

Currently, the registration of new cultivars in Poland does not require providing information on a detailed response to drought and flooding. The changing climate becoming more unpredictable may suggest that the inclusion of such information in the characteristics of cultivars is highly justified. The potential selection of such quick and reliable indicators of plant conditions after stress would facilitate this process, especially in field conditions.

2. Materials and Methods

2.1. Plants Material, Growth Conditions, Treatments

In the experiment, spring wheat (Triticum aestivum L.) cv. ‘Nimfa’ and maize (Zea mays L.) cv. ‘Kosmo230’ were used as plant material. The wheat cv. ‘Nimfa’ (Plant Breeding Strzelce Station) is a mid–early cultivar, with perfect baking quality and a large mass of a thousand grain, as compared with other Polish cultivars. The producer describe the Nimfa as a very good facultative spring wheat cultivar (for late autumn sowing). In addition to possible better winter hardiness, it may be combined with a better use of water from winter rainfall and a greater tolerance to flooding. A mid–early cv. ‘Kosmo 230’ (Małopolska Plant Breeding Station Ltd., Kraków, Poland) was intended for both silage and grain yield. Additionally, it is a high yield cultivar with a good yield structure. The producer describes cv. ‘Kosmo230’ as repetitively and stably yielding in various environments. At the same time, it was mentioned that it is a cultivar suitable for cultivation on weaker soils, but rich in water, which suggests its greater demand for water.

The plants were grown in a growth chamber in 4 L pots (8 pots per treatment, 12 plants per pot), filled with a mixture of commercial peat soil and sand (1:1, v/v). The base of the substrate was soil with highly milled peat deacidified with chalk and supplemented with a starting dose of 0.6 kg m⁻³ of NPK+Mg compound fertilizer. The pH of the substrate was 5.5–6.5, and the salinity was below 1.9 NaCl dm³. Vegetation was maintained at a 15 h photoperiod, an irradiance of 450 μmol (photon) m⁻² s⁻¹ (provided by high pressure sodium lamps, 400 W; Philips SON-T AGRO, Belgium), a temperature of 23/18 °C (day/night), and 40% air humidity. The plants were irrigated with full-strength Hoagland’s nutrient solution once a week. Soil moisture was controlled every day to maintain 70% of field water capacity (FWC) by weighing the pots and adding enough water to achieve weight corresponding to 70% FWC. The weights assigned to specific FWC levels were calculated from a proportion based on a previous determination of the maximum and minimum water capacity. Additionally, soil humidity was measured with a HydroSense Soil Water Content Measurement System (Campbell Scientific Australia, Inc., Garbutt, QLD, Australia) at different points of the pot to check the homogeneity of water content.

Water stress was initiated from the third leaf stage of wheat and maize. For plants exposed to drought stress, water level in the pots was maintained at 30–35% FWC for 10 days. The FWC level was maintained by weight by adding water, as necessary. The flooded plants and water level (pH 7.5, dGH = 273 mg/dm³) of 0.5 cm above the soil surface was maintained for 10 days. Analyses of water content, gas exchange intensity, photosynthetic apparatus activity, chlorophyll content, plant height, and biological membrane integrity were performed on the tenth day of the stress treatment. For all measurements, one replicate means one plant.

2.2. Relative Water Content (RWC)

The measurements involved one 6 cm middle fragment from the second fully developed leaf from the top. RWC was determined according to the Barrs and Weatherley [15] formula:

RWC = [(FW − DW)/(TW − DW)] × 100%

(1)

where FW represents fresh weight, DW dry weight and TW turgid weight. First, a leaf fragment was weighed to determine the fresh weight (FW). Then, this fragment was placed in darkness for 24 h in vials containing water to allow complete rehydration. After that time the TW was determined. Next, the leaf fragment was dried at 85 °C for 48 h and then DW was measured. The measurements were taken in 7 replicates.

2.3. Photochemical Efficiency

Measurements involved the central part of the first fully expanded leaf and were performed with a Handy PEA (Hansatech Ltd., King’s Lynn, UK). They were taken after 30 min of leaf adaptation to darkness. The excitation irradiance was 3000 μmol (quantum) m⁻² s⁻¹ (peak at 650 nm). Changes in fluorescence were registered automatically by the fluorimeter during irradiation between 10 μs and 1 s. During the initial 2 ms, data were collected every 10 μs with a 12-bit resolution. After this period, the frequency of the automatic data set was dropped. The collected data were analyzed with a JIP test, based on the theory of energy flow in PSII [16,17,18,19]. The following parameters were calculated: absorbed energy flux (ABS), trapped energy flux (TR_o), electron transport flux (ET_o), and dissipated energy flux (DI_o), as well as the area of the photosynthetic sample (RC/CS_m). The parameters are classified as specific, i.e., expressed as calculated per a reactive center (RC), and phenomenological, i.e., expressed in terms of the excited photosynthetic surface (cross section, CS) of the sample. Finally, we calculated the following yield ratios: the probability (at t = 0) of electron transport beyond the plastoquinone Q_A (ψ_o), the quantum yield of electron transport (at t = 0) (φ_Eo), the maximum quantum yield of PSII photochemistry (at t = 0) (F_v/F_m). PI (overall performance index of PSII photochemistry) was also determined. Measurements involved 16 replicates.

2.4. Gas Exchange

Gas exchange was measured using an infrared gas analyzer LCpro-SD (ADC BioScientific Ltd., Hoddesdon, UK) automatically controlling the measurement conditions. The following parameters were measured: photosynthetic rate (P_N), transpiration (E), stomatal conductance (g_s), as well as the intercellular concentration of CO₂ (C_i). The instantaneous water use efficiency index (WUE) was determined based on the quotient of photosynthetic rate and transpiration (P_N/E). Apparent carboxylation efficiency (P_N/C_i) was also calculated. The conditions in the measurement chamber were as follows: carbon dioxide concentration 360 µmol mol⁻¹ air, air humidity equal to ambient humidity, temperature 23 °C, PAR intensity 600 µmol photons m⁻² s⁻¹. The measurements were carried out between 10:00 a.m. and 12:30 p.m. and involved the first fully expanded leaf in 7 replicates.

2.5. Chlorophyll Content

Measurements based on the leaf greenness index were performed in the first fully developed leaf from the top with a hand-held chlorophyll meter, SPAD-502 (Konica-Minolta, Inc., Tokyo, Japan). Light absorption at 650 nm (absorbed by Chl) and 940 nm (absorbed by the other structures) was determined. The readings were converted using a microprocessor and presented in contractual units, called SPAD readings (with maximum at 60 units). SPAD readings are directly proportional to Chl content. The measurements were taken in 18 replicates.

2.6. Plant Height Analysis

Plant height was determined by measuring the entire aboveground part (from the ground level to the end of the longest leaf).

2.7. Electrolyte Leakage (EL)

For each cultivar, three 3 cm middle leaf fragments were excised from the first fully developed leaf from the top. The samples were washed twice in deionized water and immersed in 25 cm³ of deionized water. After 24 h (t₁) of shaking at room temperature, the samples were frozen at −35 °C for 24 h, then heated and shaken again (24 h, room temperature, t₂). EL was calculated as follows:

EL = (EL₁/EL₂) × 100%,

(2)

where EL₁ and EL₂ represent the specific electrical conductance at t₁ and t₂, respectively. Measurements of electrical conductance were performed with a microcomputer conductivity meter CC-317 (Elmetron, Zabrze, Poland). They were taken with 6 replicates.

2.8. Statistical Analysis

A two-way analysis (stress/control and species) of variance using the Duncan’s test at p < 0.05 was performed in order to determine the significance of differences within treatment (drought, waterlogging), measured separately for each parameter. All data were checked for normality before analysis using the Shapiro–Wilk test. All data were analyzed using Statistica 13.1 software (Statsoft, Inc., Tulsa, OK, USA).

3. Results and Discussion

3.1. Plant Height, Relative Water Content (RWC) and Electrolyte Leakage (EL)

Table 1. Effects of 10-day drought and root flooding on plant height [cm] (n = 20), relative water content (RWC) [%] (n = 7) and electrolyte leakage (EL) [%] (n = 6) of wheat and maize leaves. Means indicated with the same letters within drought or flooding treatment for each parameter are not significantly different at p < 0.05. The grey areas point to differences between species within each parameter under stress conditions with no differences under control conditions.

Parameter	Treatment	Drought		Flooding
		Wheat	Maize	Wheat	Maize
Plant height	Control	46.9 ± 0.90 bc	64.1 ± 1.63 a	46.9 ±0.90 c	64.1 ± 1.63 a
	Stress	43.6 ± 0.85 c	49.3 ± 1.66 b	44.5 ± 0.85 c	59.0 ± 1.78 b
RWC	Control	96.0 ± 1.11 a	97.0 ± 1.09 a	96.0 ± 1.11 a	97.0 ± 1.09 a
	Stress	84.3 ± 2.09 b	88.1 ± 1.41 b	90.7 ± 2.08 b	90.9 ± 1.15 b
EL	Control	6.2 ± 0.28 c	8.7 ± 0.45 bc	6.2 ± 0.28 c	8.7 ± 0.45 b
	Stress	9.0 ± 0.38 b	11.6 ± 1.68 a	6.6 ± 0.54 bc	35.4 ± 1.46 a

shows the results on plant height, relative water content (RWC) and electrolyte leakage (EL). Only in maize did both soil drought and flooding significantly decrease plant height (Table 1). Plant growth inhibition is a visible effect of environmental stresses, including drought and flooding [20,21,22]. A significant positive correlation between net photosynthesis of winter wheat and its height difference was noted by the authors, indicating that the height was affected by photosynthesis. However, the impact of drought on both these parameters depends on the development phase of wheat. Bread wheat research has shown that plant height also has a significant positive correlation with grain yield [23,24]. However, in maize, both stressors resulted in a significant drop in leaf water content (RWC) (Table 1), and we found it more likely that plant growth under both stresses was reduced as a result of changes in photosynthesis and photosynthetic apparatus activity. It has been shown that photosynthesis and photosynthetic apparatus activity are negatively affected by changes in RWC [25].

The results on relative water content (RWC) indicated a similar level of leaf hydration in both species experiencing drought and flooding (Table 1). According to Hsiao [26], this level of RWC drop corresponds to mild or moderate water stress. Soil drought-induced decrease in water content in wheat and maize was also reported by other authors [27,28]. Azizi, et al. [29] explained a significantly lower leaf RWC in flooded Populus euphratica by limited water uptake by the plant root system. A similar decline in leaf water content during flooding stress was seen in sorghum and soybean [30,31].

Drought stress was also associated with increased damage to the biological membranes (higher EL) observed in both investigated species (Table 1). The same response of wheat and maize to drought was witnessed in other studies [4,32].

The integrity of biological membranes was particularly sensitive to flooding in maize, as manifested by the highest value of EL (35.4 %) (Table 1). Similar disturbances to the biological membrane integrity in flooded maize plants were also observed by other authors [33,34,35]. Yordanova and Popova [36] proved that the increase in electrolyte leakage under flooding stress was linked to root oxygen deficiency that induced photooxidative damage in maize leaves. Additionally, ROS accumulation induced by flooding was another destructive factor for cell membranes [37].

Significant correlations between electrolyte leakage (EL) and the probability (at t = 0) of electron transport beyond the plastoquinone Q_A (ψ_o) (Figure 1A), and the amount of energy used for the electron transport (ET_o/CS_m) were found (Figure 1B). The results point to the fact that flooding conditions disturb electron transport between PSII and PSI in maize.

3.2. Leaf Gas Exchange and Water Use Efficiency (WUE)

During drought, both wheat and maize experienced a significant decrease in net photosynthesis (P_N), transpiration intensity (E), and stomatal conductance (gs) (

Table 2. Effects of 10-day drought and flooding of roots on leaf gas exchange (n = 7) in wheat and maize. P_N—net photosynthesis intensity (µmol(CO₂) m⁻² s⁻¹), E—transpiration intensity (mmol(H₂O) m⁻² s⁻¹), g_s—stomatal conductance [mol(H₂O) m⁻² s⁻¹], C_i—intracellular concentration of CO₂—(µmol(CO₂) mol(air)⁻¹), WUE—photosynthetic ratio of water use (µmol(CO₂)mmol⁻¹(H₂O)), P_N/C_i—apparent carboxylation efficiency (mol(air) m⁻² s⁻¹). Means indicated with the same letters within drought or flooding treatment, for each parameter are not significantly different at p < 0.05. The grey areas point to differences between species within each parameter under stress conditions with no differences under control conditions.

Parameters	Treatments	Drought		Flooding
		Wheat	Maize	Wheat	Maize
PN	Control	14.25 ± 2.09 ab	17.85 ± 1.58 a	14.25 ± 2.09 b	17.85 ± 1.58 a
	Stress	10.47 ± 1.79 c	12.64 ± 1.20 b	13.46 ± 1.91 b	16.59 ± 1.85 a
E	Control	4.95 ± 0.75 b	5.67 ± 0.83 a	4.95 ± 0.75 ab	5.67 ± 0.83 a
	Stress	3.17 ± 0.43 c	3.12 ± 0.54 c	4.34 ± 0.28 b	4.93 ± 0.40 ab
gs	Control	0.046 ± 0.0036 b	0.064 ± 0.0042 a	0.046 ± 0.0036 b	0.064 ± 0.0042 ab
	Stress	0.025 ± 0.0045 c	0.036 ± 0.0021 b	0.039 ± 0.0021c	0.075 ± 0.0062 a
Ci	Control	371.3 ± 23.12 bc	468.3 ± 31.43 a	371.3 ± 23.12 c	468.3 ± 31.43 a
	Stress	369.2 ± 19.51 c	406.3 ± 25.18 ab	382.2 ± 31.65 c	411.2 ± 27.78 b
WUE	Control	2.88 ± 0.47 b	3.15 ± 0.27 ab	2.88 ± 0.47 b	3.15 ± 0.27 ab
	Stress	3.30 ± 0.39 ab	4.05 ± 0.62 a	3.10 ± 0.31 ab	3.37 ± 0.19a
PN/Ci	Control	0.038 ± 0.0051 a	0.039 ± 0.0047 a	0.038 ± 0.0051 a	0.039 ± 0.0047 a
	Stress	0.029 ± 0.0039 b	0.032 ± 0.0035 ab	0.036 ± 0.0029 a	0.041 ± 0.0040 a

). No major changes were found in the intercellular concentration of CO₂ (C_i) or water use efficiency (WUE). Drought stress induced alterations in apparent carboxylation efficiency (P_N/C_i) only in wheat (Table 2). However, a significant correlation between P_N/C_i and RWC for both species was found (Figure 2).

A drought-induced drop in the photosynthetic activity of wheat and maize leaves was also reported in other studies [32,38]. In both species exposed to drought and experiencing reduced photosynthetic activity, the water use efficiency was maintained at the control level, which may be associated with the partial closure of stomata and limited transpiration. It has been shown that WUE may differ depending not only between species, but also on the intensity of the drought [39].

Flooding caused a significant reduction in stomatal conductivity (gs) in wheat and a significant decrease in the intercellular concentration of carbon dioxide (Ci) in maize. The other parameters of gas exchange and WUE were similar in both species (Table 2). Stomatal closure under flooding stress has been already explained by negative hydraulic message and a negative ABA message from oxygen-deficient roots. Both effects result from limited water conductance into the roots and an inhibition of the oxygen-depending ABA biosynthetic pathway. The consequence of the negative hydraulic message was a loss of leaf turgor that induced stomata to close [40,41].

3.3. Photosynthetic Apparatus Activity and Chlorophyll Content

Changes in chlorophyll fluorescence parameters indicated a clear reduction in the activity of the photosynthetic apparatus in both species exposed to soil drought (

Table 3. Effects of 10-day drought and flooding of wheat and maize roots on chlorophyll fluorescence parameters (n = 16) and chlorophyll content (n = 18). Means indicated with the same letters within drought or flooding treatment for each parameter are not significantly different at p < 0.05. The grey areas point to differences between species within each parameter under stress conditions and with no differences under control conditions.

Parameters	Treatments	Drought		Flooding
		Wheat	Maize	Wheat	Maize
ABS/CSm	Control	2409 ± 61.3 a	1945 ± 52.0 b	2409 ± 61.3 a	1945 ± 52.0 b
	Stress	2187 ± 116.9 a	1481 ± 71.3 c	2248 ± 68.5 a	1426 ± 69.9 c
TRo/CSm	Control	1914 ± 52.9 a	1553 ± 44.8 b	1914 ± 52.9 a	1553 ± 44.8 b
	Stress	1453 ± 85.4 b	959 ± 72.7 c	1606 ± 56.9 b	940 ± 67.7 c
ETo/CSm	Control	857 ± 38.6 a	731 ± 28.5 b	857 ± 38.6 a	731 ± 28.5 b
	Stress	619 ± 46.6 c	304 ± 41.2 d	729 ± 29.8 b	310 ± 34.2 c
DIo/CSm	Control	496 ± 15.1 b	393 ± 17.9 c	496 ± 15.1 b	393 ± 17.9 c
	Stress	734 ± 49.9 a	522 ± 14.1 b	642 ± 26.6 a	487 ± 30.1 b
RC/CSm	Control	687 ± 27.0 a	650 ± 17.4 a	687 ± 27.0 a	650± 17.4 a
	Stress	425 ± 29.9 b	383 ± 23.9 b	514 ± 25.4 b	368 ± 27.7 c
ABS/RC	Control	3.54 ± 0.076 c	3.00 ± 0.044 d	3.54 ± 0.076 c	3.00 ± 0.044 d
	Stress	5.30 ± 0.230 a	3.92 ± 0.083 b	4.45 ± 0.141 a	3.98 ± 0.128 b
TRo/RC	Control	2.81 ± 0.047 b	2.39 ± 0.035 c	2.81 ± 0.047 b	2.39 ± 0.035 d
	Stress	3.47 ± 0.076 a	2.48 ± 0.052 c	3.16 ± 0.059 a	2.56 ± 0.055 c
ETo/RC	Control	1.24 ± 0.020 b	1.12 ± 0.022 b	1.24 ± 0.020 b	1.12 ± 0.022 b
	Stress	1.47 ± 0.083 a	0.79 ± 0.049 c	1.45 ± 0.087 a	0.78 ± 0.057 c
DIo/RC	Control	0.73 ± 0.030 c	0.61 ± 0.011 c	0.73 ± 0.030 b	0.61 ± 0.011 b
	Stress	1.83 ± 0.158 a	1.44 ± 0.105 b	1.29 ± 0.088 a	1.41 ± 0.143 a
Fv/Fm	Control	0.79 ± 0.005 a	0.80 ± 0.002 a	0.79 ± 0.005 a	0.80 ± 0.002 a
	Stress	0.66 ± 0.016 b	0.64 ± 0.020 b	0.71 ± 0.010 b	0.63 ± 0.024 c
ψo	Control	0.45 ± 0.012 ab	0.47 ± 0.011 a	0.45 ± 0.012 ab	0.47 ± 0.011 a
	Stress	0.42 ± 0.019 b	0.30 ± 0.018 c	0.46 ± 0.021 a	0.31 ± 0.025 b
φEo	Control	0.35 ± 0.010 a	0.38 ± 0.009 a	0.35 ± 0.010 ab	0.38 ± 0.019 a
	Stress	0.28 ± 0.013 b	0.20 ± 0.015 c	0.32 ± 0.012 b	0.20 ± 0.020 c
PI	Control	0.92 ± 0.083 b	1.19 ± 0.062 a	0.92 ± 0.083 b	1.19 ± 0.062 a
	Stress	0.31 ± 0.040 c	0.24 ± 0.039 c	0.50 ± 0.039 c	0.27 ± 0.055 d
Chl	Control	32.1 ± 0.37 bc	38.0 ± 0.79 a	32.1 ± 0.37 c	38.0 ± 0.79 a
	Stress	31.1 ± 0.79 c	34.2 ± 0.98 b	30.3 ± 0.95 c	35.4 ± 0.32 b

). Out of 13 chlorophyll fluorescence parameters, 11 changed significantly in response to wheat leaf dehydration (except for ABS/CS_m and ψ_o). In maize, the changes were significant for 12 parameters, except for the quantum yield of electron transport φ_Eo (Table 3). Flooding resulted in significant alterations in all chlorophyll fluorescence parameters in maize (Table 3), and 10 parameters in wheat (except for ABS/CS_m, ψ_o and φ_Eo). The parameter that differentiated the species under both stresses was ET_o/RC, as it significantly increased in stressed wheat but decreased in drought- and flooding-exposed maize.

Chlorophyll fluorescence measurements revealed substantial disturbances in the performance of the photosynthetic apparatus in stressed wheat and maize plants. The parameter is currently commonly used to compare intraspecific responses of the photosynthetic apparatus to environmental stresses and it serves as a quick and reliable tool for the selection of plants with improved tolerance to abiotic stresses [4,42,43].

Although chlorophyll fluorescence is often used to assess the condition of plants under stress, there are no studies using this interspecies comparison tool that were used in our research. Despite the many changes in the response of both species, we chose ET_o/RC, ψ_o, φ_Eo as the ones that differentiate the response of wheat and maize to both water stresses. In addition, under the stress of drought, the differentiating parameter was DI_o/RC and under flooding conditions they were F_v/F_m and RC/CS_m. We discarded all those parameters that differed under stress conditions, and also those that differed under control conditions, because they are due to interspecies differences. The ET_o/RC parameter turned out to be the reason why it was possible to observe the specific response of the studied species in the stress condition (increase in wheat and decrease in maize). Environmental stresses, including drought and flooding, limit the photosynthetic metabolism of carbon and reduce the utilization of light-phase products (NADPH₂, ATP), which is why some of the absorbed light energy may damage PSII [44,45]. Most changes in chlorophyll fluorescence parameters, especially those expressed per excited surface (CS) of the photosynthetic sample, corroborated those reported in other studies [3,4,46,47].

A specific response involving a significant spike in ET_o/RC was observed in drought- and flooding-exposed wheat, while maize responded to the stresses with a drop in this parameter (Table 3). In drought-stressed leaves, the increase of ET_o/RC may indicate that other sinks for electrons from PSII, such as photorespiration and/or Mehler peroxidase reactions, were sustaining the electron flow [48,49]. Another, more probable explanation for the ET_o/RC spike under soil drought may be not so much increased energy supply to the reaction centers as the reduced number of the active reaction centers (RC/CS_m) [50]. The drop in energy flow through thylakoid membranes accompanied by a reduction in RC/CS_m observed in drought- and flooding-exposed maize was also reported by Golebiowska-Pikania, et al. [51]. PSII reaction centers are inactivated following the degradation of D1 protein [52]. This inhibits the electron transport per active reaction center (ET_o/RC) and limits the probability of electron transport beyond QA (ψ_o). Overall, these effects disturb the transfer of the excitation energy, a parameter that is connected with the increase in non-photochemical dissipation energy (DI_o/RC) and the reduction in the quantum yield of PSII photochemistry (F_v/F_m) (Table 3). Similar changes in the quantum yield of PSII photochemistry and energy dissipation under stress conditions were described in other studies [53,54,55]. Still, Antonkiewicz and Rapacz [56] claimed that F_v/F_m should not be treated as an indicator of damage to a photosynthetic apparatus but rather as a symptom of its adaptive changes under stress conditions.

A significant drop in Chl content was found only in flooding-exposed maize (Table 3). A decrease in this pigment levels during drought and flooding was probably another factor limiting the intensity of photosynthesis (Table 2) and the activity of the photosynthetic apparatus (Table 3). Apart from non-photochemical dissipation of energy (DI_o/RC), lowering chlorophyll content may be a defense response that allows for limiting light absorption (lower values of ABS/CS_m in drought- and flooding-exposed maize) and diminishing photoinhibition damage to the photosynthetic apparatus [46,57,58]. Plant responses in the form of chlorophyll content drop under drought and flooding was reported in other studies [3,33,47]. However, it has been shown that plants tolerant to flooding do not exhibit a decrease in chlorophyll content or that the decrease is smaller than in susceptible plants [29]. Despite the widespread use of chlorophyll content to assess plant responses to stress, our research indicates that it should not be used for interspecies comparisons, as these differences may be due to factors other than stress factors.

4. Conclusions

Our experimental system involving a 10-day long exposure of wheat and maize plants at three-leaf stage to drought or flooding allowed us to observe some specific responses to these stress factors. A specific response of wheat, both under drought and flooding, was the increase in ET_o/RC reflecting electron transport flux per single reaction center in PSII. Additionally, the parameters differentiating the studied species only under stressful conditions were rendered. During a drought, these were EL, P_N, ET_o/RC, DI_o/RC, ψ_o, φ_Eo and during flooding conditions, these were g_s, RC/CS_m, ET_o/RC, F_v/F_m, ψ_o, φ_Eo. For most of these parameters, the direction of these changes between the control and stress plants is the same in both species, and the differences are due to the level of these differences. A specific response of maize during flooding was the increase in the level of stomatal conductance which was not observed in the second species.

Further studies with more advanced methodologies and measurements are necessary to prove the potential role of these findings. However, the experimental design presented here and the specific responses of wheat and maize to drought and flooding seem to be promising tools for assessing a large pool of plants for the selection of genotypes with an improved tolerance to both stress factors at early stages of growth. To this end, follow-up large-scale experiments with high number of genotypes, lines, or strains are needed.

Selected indicators of plant responses to drought and flooding in the future may help in a more complete characterization of the registered cultivars.