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Article

Comparative Study on the Morpho-Physiological Responses of White Clover Cultivars with Different Leaf Types to Water Deficiency

by
Xueying Zhao
1,†,
Zhi Tian
1,†,
Lintao Cheng
1,†,
Jia Jiang
2,
Yujiao Liu
2,
Lizhi Liu
1,
Chunxu You
1,
Xun Liu
2,
Fuchun Xie
2,
Ligang Qin
2 and
Yajun Chen
1,*
1
College of Horticulture and Landscaping, Northeast Agricultural University, Harbin 150030, China
2
College of Animal Science and Technology, Northeast Agricultural University, Harbin 150030, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(7), 1859; https://doi.org/10.3390/agronomy13071859
Submission received: 24 June 2023 / Revised: 11 July 2023 / Accepted: 12 July 2023 / Published: 14 July 2023
(This article belongs to the Special Issue Advances in Stress Biology of Forage and Turfgrass)
Browse Figures
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Abstract

:
White clover (Trifolium repens L.) is one of the legume herbages with high feed quality, but it is sensitive to water deficiency. The objectives of this study were to compare morpho-physiological responses to drought stress and post-drought recovery in four-leafed white clover cultivars. Under well-watered conditions, drought stress (3 d, 6 d, 9 d and 12 d), and rehydration, the relative water content (RWC), membrane lipid permeability, osmoregulatory substances, photosynthetic characteristics and stomatal features of Chinese native Longping No.1 (LP, small-leafed) and three introduced cultivars, namely Pudi (PD, small-leafed), Rivendel (RD, medium-leafed) and Koala (KL, large-leafed), were evaluated. Results showed that small-leafed white clover maintained higher RWC and better membrane stability and osmotic regulation with increased drought intensities, compared to the medium-leafed and large-leafed cultivars. Following rewatering, small-leafed white clover recovered more rapidly with all of the parameters than the other cultivars. The increase of drought stress intensity led to the decline of net photosynthetic rate (Pn), transpiration rate (Tr) and stomatal conductance (Cs). However, the decreased range of Pn, Tr and Cs in the small-leafed white clover was significantly smaller than that in the medium-leafed and large-leafed white clovers. Meanwhile, the small-leafed white clover showed a smaller size and higher density of stoma either under normal or drought conditions than the intermediate- and large-leafed cultivars. Taken together, the results indicated that the adaptability of small-leafed white clover to drought stresses and post-drought recovery was better than that of medium-leafed and large-leafed white clovers. The study will provide better insights into the mechanism of drought response and recovery potential in different white clover cultivars.

1. Introduction

White clover (Trifolium repens L.) is a perennial herbaceous plant belonging to the family Fabaceae. This species is native to southeastern Europe and southern Asia [1]. Its high environmental adaptability has allowed it to spread to the Arctic and to sub-tropical regions such as Queensland (Australia) by means of animal, human or spontaneous distributions [2]. White clover is characterized by being prostrate with branchy stolons, maintaining uniform and long-lasting green color and having high nutritive value and excellent nitrogen-fixing capacity [3]. These traits make white clover a legume that is extensively used worldwide in soil restoration, fruit gardens, athletic fields, ornamental landscapes and farms [4,5,6].
Although white clover has many benefits, its preference for cool climate and its shallow root systems make it extremely sensitive to drought stress [7]. Some introduced cultivars often suffer from drought injury in mid-summer dormancy and episodic water deficiency in Northern China. Drought stress could negatively impact white clover canopy production, stolon branching, texture, persistence and aesthetics by changing plant biological and physiological metabolism as well as interrupting tissue structures [8], and inhibiting white clover growth and development [9,10,11]. An early study found that several white clover genotypes differing in mean leaf area, stolon diameter and petiole length showed an 80% decrease in stolon numbers and a 30% decrease in relative water content under water-shortage conditions [12]. Morpho-physiological parameters like shoot and root length, leaf water content, electrolyte leakage, water use efficiency, soluble proteins, antioxidant enzymes and photosynthetic rate in white clover were closely associated with water deficiency [9,13]. Therefore, it is crucial for the whole industry to breed new white clover cultivars resistant to drought stress.
The mechanisms of drought resistance have been studied in many plant species worldwide. Phenotype plasticity and multiple complex physiological processes are involved in plant responses to water deficiency [11,14]. Acting as channels for water evaporation and CO2 absorption in plants, stomata have been confirmed to be related to drought resistance due to their numbers and distributions on leaf surfaces [15]. Rapid stomatal closure can decrease transpiration rates and increase stomatal resistance, which should be beneficial to protect plants from becoming desiccated [16]. To prevent water loss from leaves during drought stress, plants have adopted strategies of osmotic adjustments by accumulating osmolytes including proline, soluble sugar and soluble proteins to decrease cell osmotic potential for water retention and the maintenance of turgor pressure [2]. Osmotic adjustments also protect proteins, enzymes, cellular organelles and membranes from oxidative damage. Therefore, these osmolytes enable tissues to maintain metabolic and physiological functions at lower water levels [17,18]. Among the physiological functions, photosynthesis is most important, and a report indicated that the net photosynthetic rate would decrease due to limiting or non-limiting stomata factors during drought stress [16].
Over the past decades, many white clover cultivars, commercial ecotypes and lines have been developed and exchanged in different parts of the world [19,20,21], due to their allotetraploid nature, self-incompatibility and highly complex genetic composition [22,23]. Great genetic variations in morph physiology and persistence under drought stress have been observed in white clovers [10,24,25]. More recent studies revealed the ability to accumulate osmoregulatory solutes among small-, medium- and large-leafed white clover genotypes at the age of 18 months, and results indicated that the small-leafed genotypes possessed greater abilities to accumulate osmoregulatory solutes when subjected to a drying cycle [26]. This once again confirmed that small-leafed white clovers were more drought tolerant than the large-leafed types based on the evaluation of water status and cellularly protecting enzymes as well as lignin metabolism [24], suggesting that smaller-leafed genotypes had greater abilities of osmotic adjustment, which would be useful to screen superior drought-resistance germplasm for white clover. A few studies showed photosynthetic abilities under drought stress in various genotypes of white clover [27,28]. However, they observed only the instantaneous photosynthetic capacity but not the photosynthetic properties under diurnal conditions. In addition, the stomatal characteristics in white clover related to drought are not clear, especially for native cultivars in Northern China.
In this research, morpho-physiological characteristics of the native Longping No.1 (LP, small-leafed) from Northern China and three introduced cultivars, namely Pudi (PD, small-leafed), Rivendel (RD, medium-leafed) and Koala (KL, large-leafed), were compared under drought conditions. The objectives of this study were: (i) to compare stomatal attributes and density and the diurnal photosynthesis performance under drought stress among four cultivars; and (ii) to reveal the ability of cellular osmolyte accumulation for evaluating the physiological differences in drought resistance in these tested accessions. The information could help select or improve white clover cultivars under drought stress through genetic modification in breeding as well as cost-effective management practices.

2. Materials and Methods

2.1. Experimental Materials and Test Design

The seeds of four white clover cultivars were provided by the grass research center of the Northeast Agricultural University (Harbin, China; 128°04′ E, 44°50′ N), including Long Ping No.1 (LP), Pudi (PD), Rivendel (RD) and Koala (KL). LP and PD are small-leafed types, RD is a medium-leafed (intermediate) type, and KL is a large-leafed type (Figure 1, Table 1). LP originated from Northern China, PD and KL were introduced from Australia, and RD is produced in Denmark.
The experiment was conducted on 5 March 2021, at the Horticultural Experimental Station of Northeast Agricultural University. The growth conditions in the greenhouse were: relative humidity of 65–75%, temperature of 20–28 °C and 12 h of illumination (700 mmol·m−2·s−1) per day. White clover seeds with full and uniform grains were selected, disinfected and washed three times with distilled water before being sown in 70 × 30 × 10 cm seedling boxes at a seeding density of 10 g/m2. After 15 d of emergence, 10 uniform white clover seedlings were selected from each box and transplanted into PVC pots with a diameter of 20 cm and a height of 15 cm, filled with a mixture of black soil and vermiculite (3:1 v/v). Pots with seedlings were randomly arranged and routinely managed for 60 d, and were then moved into a growth chamber (NP-PRX-600D). The chambers were set to 20/15 °C (day/night temperature), 75% relative humidity and 12 h of illumination (600 mmol·m−2·s−1).
After 14 d of acclimation to the growth chamber, plants were exposed to different treatments: (i) well-watered control treatment (CK, plants were watered normally to ensure vigorous plant growth); (ii) drought stress treatments (the cessation of water supply for 3 d, 6 d, 9 d and 12 d); and (iii) rewatering treatment (normal watering for 3 d). Treatments were arranged as a completely randomized design with four replicates of each cultivar.

2.2. Physiological Measurements

2.2.1. Leaf Relative Water Content

The relative water content (RWC) was measured using 2.0 g fresh and fully expanded leaf material from each cultivar. Fresh leaves were collected and immediately weighed to obtain the fresh weight (Wf). Then, leaves were soaked in distilled water for 24 h. They were reweighed after being dried gently with paper to obtain the turgid weight (Wt). The leaves were then dried in an oven at 80 °C for 8–12 h and weighed to obtain the dry weight (Wd). The relative water content was calculated according to the formula [29]:
RWC (%) = (Wf − Wd)/(Wt − Wd) × 100%

2.2.2. Relative Electrical Conductivity

Fresh white clover leaves (0.1 g) of different cultivars for all treatments were collected, rinsed with deionized water and dried with filter paper. The samples were placed in a test tube containing 10 mL of deionized water, and then the conductivity (E0) was measured. Next, samples were evacuated under vacuum until the leaves were immersed in the water, and the conductivity (E1) was measured. After heating and boiling, and then cooling to room temperature, conductivity (E2) was measured after adding water to a constant volume. The relative electrical conductivity (REC) (%) was calculated as [(E1 − E0)/(E2 − E0)] × 100%.

2.2.3. Malondialdehyde Content

Malondialdehyde (MDA) content was determined by the thiobarbituric acid (TBA) colorimetric method [30]. White clover leaves (0.1 g) of different cultivars under different drought treatments were added to 10 mL trichloroacetic acid (10%) and ground into a homogenate; the homogenate was then centrifuged at 4000 rpm for 10 min, and the supernatant was extracted from the sample. Next, 5 mL of supernatant was placed in a test tube, 5 mL of 0.6% TBA was added, and the mixture was mixed well and boiled for 15 min in a 100 °C water bath. The mixture was cooled quickly and then centrifuged. The absorbance of the supernatant was then determined at 450, 532 and 600 nm, respectively.
MDA concentration (µmol·g−1) was calculated by the formula C = 6.45 × (D532 − D600) − 0.56 × D450.
MDA content (µmol·g−1) = CNW−1, where N refers to the volume of extract and W refers to the fresh weight of plant tissue.

2.2.4. Free Proline Content

Free proline contents were determined by the sulfosalicylic acid method [31]. Leaves (0.2 g) of the four white clover cultivars under different drought treatments were added to 5 mL sulfosalicylic acid (3%). The plant samples were extracted in a boiling water bath for 10 min. After samples were cooled to room temperature, 2 mL of supernatant was removed, and 2 mL of glacial acetic acid and 3 mL of color-developing solution were added. Next, the mixture was heated in a boiling water bath for 40 min, followed by the addition of 5 mL of toluene for extraction. The absorbance value was measured at 520 nm by a spectrophotometer. The amount of Pro was checked from the standard curve [32].

2.2.5. Soluble Sugar Content

Referring to the method of the sulfuric acid–anthrone color, the soluble sugar (SS) contents were determined [33]. Leaves (0.5 g) of each cultivar under different drought treatments were added to ethanol. Samples were then placed in a water bath. Centrifugation and decolorization were performed to collect the sugar extract. Then, anthrone was added to the sugar extract, the mixture was boiled, and the absorbance was measured with a spectrophotometer at a wavelength of 620 nm, which was used to calculate the SS.

2.2.6. Soluble Protein Content

For the soluble protein (SP) content, approximately 0.5 g of leaves of each cultivar under different drought treatments were mixed with 5 mL of extraction buffer (0.05 M Tris-HCl, pH 8.0). The mixture was homogenized in an ice bath, and then centrifuged at 10,000 rpm at 4 °C for 10 min. A 1.0 mL aliquot of supernatant was put into a test tube, and 5 mL of Coomassie Brilliant Blue G-250 was added. The absorbance was read at 595 nm [34].

2.2.7. Photosynthetic Parameter Assay

Using the portable gas exchange system Li-6400XT (LI-COR Inc., Lincoln, NE, USA), the photosynthesis rate (Pn), transpiration rate (Tr) and stomatal conductance (Cs) of white clover leaves were measured from 06:00 to 16:00 h during the diurnal period (measured every 2 h, 5 values measured repeatedly each time). The atmospheric temperature and light intensities during the measurement period were 25 ± 5 °C and 1000 µmol m−2·s−1. The average atmospheric CO2 concentration was 400 µmol·mol−1.

2.3. Leaf Stomatal Scanning

After washing with distilled water, the white clover leaves were cut into 0.5 cm segments, immediately put in a vial containing glutaraldehyde and fixed with 2 mL of 0.1 mol L−1 phosphate buffer (pH 6.8) 3 times, for 10 min each time. Leaves were then dehydrated in a graded series of ethanol (50, 70, 80, 90 and 100%) for 10–15 min each. Leaves were transferred to a pure tert-butanol solution and let stand for 20 min and then washed with an equal volume of anhydrous ethanol and tert-butanol once and pure tert-butanol twice, with submergence for 15 min each time. After being left at room temperature for 30 min, leaves were dried with a critical point dryer, and the dried sample was sprayed with gold. Finally, the samples were placed on a scanning electron microscope (S-3400N, Hitachi, Tokyo, Japan), and changes in leaf stomatal size and density were observed.

2.4. Data Analysis

Data were analyzed using one-way analyses of variance with SPSS v10.0 software (SPSS Inc., Chicago, IL, USA). The mean values were compared via the least significant difference test at the 0.05 probability level. GraphPad Prism v8 (GraphPad company, La Jolla, CA, USA) was used for plotting.

3. Results

3.1. Comparison of Leaf Relative Water Content and Membrane Lipid Permeability

3.1.1. Relative Water Content (RWC)

As drought levels increased, there were notable differences in RWC of leaves among the four cultivars. Throughout the whole drought period, the RWC of LP was the highest among the four cultivars, and RWC of KL leaves was significantly lower than the others (p < 0.05) (Figure 2A). Rewatering promoted the recovery of white clover that suffered from the drought. Compared with the 12 d drought stress, the leaf RWC increased by 56.11% in LP, 61.66% in PD, 57.92% in RD and 57.46% in KL after rewatering. In addition, the RWC in LP was restored to normal levels after rewatering, reaching 97.83%, while the RWC of PD, RD and KL was 92.34%, 82.33% and 78.92%, respectively (Figure 2A).

3.1.2. Relative Electrical Conductivity Rate (REC)

The plasma membrane permeabilities represented by REC in the four cultivars under normal conditions showed no significant differences. However, as drought stress prolonged, the REC increased for all cultivars, as shown in Figure 2B. The REC value of four cultivars sharply increased from the 6th to the 12th day of drought stress, with the highest REC in KL and the lowest in LP. Notably, there was no significant difference in REC between RD and PD on the 12th day. After rewatering, the REC of each cultivar decreased, with the order of REC among the cultivars being KL > PD ≈ RD > LP (Figure 2B).

3.1.3. Malondialdehyde (MDA)

Under well-watered conditions, the leaf MDA content among the four white clover cultivars was not apparently different. Throughout the entire drought stress process, the MDA content among the cultivars increased, as shown in Figure 2C. Furthermore, KL had significantly higher MDA content than the other three cultivars on the 3rd, 6th and 12th day of drought stress (p < 0.05), while MDA in RD was higher than in the other cultivars on the 9th day. On the 12th day of water shortage, the MDA content was similar between PD and RD, while the MDA content showed a significant difference between LP and KL (p < 0.05). LP had consistently lower MDA content, while KL was the opposite. After rewatering, the MDA content of each cultivar was close to the level on the 3rd day of drought stress; KL had significantly higher MDA content than the others (p < 0.05), and LP had the lowest MDA content (Figure 2C).

3.2. Changes in Osmoregulatory Substances

3.2.1. Free Proline (Pro)

From the beginning of drought stress (3rd day), each cultivar actively adapted to changes in water availability, and the content of Pro began to increase. On the 6th day of drought stress, the difference in the accumulation of Pro was significant among cultivars (p < 0.05). The Pro content in LP was significantly higher than that in the other three cultivars (Figure 3A). By contrast, KL had the lowest accumulation of Pro content, with the smallest increase. On the 12th day of drought stress, the accumulation of Pro in each cultivar reached its maximum value, which was significantly higher than that in the control group (Figure 3A). After rewatering, the Pro content in LP and PD remained high, followed by RD, and KL had the lowest content (Figure 3A).

3.2.2. Soluble Sugar (SS)

The SS content in white clover cultivars was similar except PD, which had a lower SS content than the other cultivars under well-watered conditions (Figure 3B). On the 3rd day of drought stress, there was little difference in SS content among the different cultivars. However, the SS content of LP was significantly higher than that of the other three cultivars during days 6 to 12, and the differences among the cultivars became extremely significant with increasing drought stress (Figure 3B). After rewatering, the SS content of each cultivar decreased to varying degrees. LP had the lowest SS content, while KL had the highest SS content.

3.2.3. Soluble Protein (SP)

Under normal conditions, there were slight differences in SP content among the different cultivars; LP had slightly higher SP content than PD, RD, and KL (Figure 3C). During the initial stage of drought stress (3rd day), the SP content of each cultivar increased, but the increase was relatively small (Figure 3C). As the duration of drought stress increased, the SP content of each cultivar began to decrease. On the 6th day of drought stress, the SP content of LP was significantly higher than that of the other three cultivars (p < 0.05). After rewatering, the SP content of LP increased the most, with an increase of 81.83% compared to the SP content on the 12th day of drought stress. Furthermore, PD, RD and KL increased by 81.19%, 78.98% and 77.66%, respectively (Figure 3C). LP showed a significantly higher recovery rate than the other three cultivars.

3.3. Comparison of Photosynthetic Characteristics and Stomatal Features

3.3.1. Photosynthetic Rate (Pn)

The diurnal variation in Pn of the four white clover cultivars followed a bimodal curve under both normal conditions and drought stress (Figure 4). The Pn of all cultivars showed an increasing trend with increasing light intensity, and reached maximum values around 10:00 h (Figure 4). A midday depression in photosynthesis occurred around 12:00 h. Then, Pn gradually increased in the afternoon and reached the second peak around 14:00 h (Figure 4). Furthermore, the morning peak was generally higher than the afternoon peak. With the increase in drought degree and duration, the Pn of all cultivars decreased gradually at 10:00 h. On the 12th day of water deficiency, the Pn of PD was 16.03% lower than that under normal conditions (CK) at 10:00 h, while LP, RD and KL decreased by 22.01%, 30.71% and 31.40%, respectively (Figure 4A–D). The degree of KL under drought stress changed the most. The Pn of LP, PD, RD and KL was 8.86, 8.66, 8.63 and 8.50 μmol·m−2s−1 under normal conditions, respectively, and there was no significant difference (Figure 4E). On the 6th day of water deficiency, the Pn of LP was 10.81%, 9.95% and 6.64% higher than that of KL, RD and PD at 10:00 h, respectively (Figure 4F).

3.3.2. Transpiration Rate (Tr)

Similar to the Pn, the diurnal variation in Tr of the four white clover cultivars under normal conditions and drought stress also showed a bimodal curve (Figure 5). The Tr of LP, PD, RD and KL under normal conditions (CK) at 10:00 h was 5.75, 5.52, 5.26 and 5.24 mmol·m−2s−1, respectively, and there were no significant differences. Compared with the CK, the Tr of LP decreased the least (22.26%) at 10:00 h on the 12th day of water shortage, and the PD, RD and KL decreased by 24.63%, 27.57% and 34.22% respectively (Figure 5A–D). Both under normal conditions and on the 6th day of drought stress, the Tr of LP was higher than that of the other three cultivars at 10:00 h, and KL had the lowest Tr value at 10:00 h among the four cultivars (Figure 5E,F).

3.3.3. Stomatal Conductance (Cs)

The diurnal variation in Cs showed a similar trend to that of Pn and Tr. Under normal conditions (CK), there were no significant differences in Cs (10:00 h) among different white clover cultivars. With the increase in drought intensity, the Cs value of the four white clover cultivars decreased gradually at 10:00 h (Figure 6A–D). Compared with the CK, the Tr of KL decreased the most, by 60.13%, on the 12th day of water deficiency, and the RD, PD and LP decreased by 55.19%, 49.26% and 45.40%, respectively (Figure 6A–D). The Cs of KL, RD, PD and LP on the 6th day of drought stress at 10:00 h was 35.91%, 32.46%, 23.81% and 22.98% lower than that under normal conditions, respectively (Figure 6E,F).

3.3.4. The Characteristics of Leaf Epidermal Stomata

The stomatal lengths, widths and densities of the four white clover cultivars have their own characteristics under well-watered condition, and KL has the largest stomata size. By contrast, LP has the smallest stomata length, and PD has the smallest stomata width. LP has the highest density of stomata compared to the others under normal conditions (Table 2, Figure 7). After drought stress, the lengths and widths of stomata decreased, while the densities increased, with varying degrees of change among cultivars. The lengths and widths of stomata decreased less in LP, followed by PD and RD, and KL showed the most decreased level after drought stress. Compared to CK, the stomatal lengths of LP, PD, RD and KL under drought stress decreased by 3.40 µm (13.78%), 3.42 µm (13.95%), 4.29 µm (16.38%) and 4.76 µm (17.23%), respectively, and the stomatal widths decreased by 4.48 µm (25.25%) in LP, by 6.11 µm (36.52%) in PD, by 5.45 µm (29.85%) in RD, and by 8.39 µm (42.29%) in KL (Table 2). In addition, it was observed that the variation in stomatal widths of the four white clover cultivars after drought stress was greater than that of stomatal lengths. After drought stress, the stomatal densities on the leaf surface increased by 16.30/mm2 (8.69%) in LP, by 22.90/mm2 (13.60%) in PD, by 20.90/mm2 (13.47%) in RD, and by 26.20/mm2 (20.37%) in KL (Table 2). The change in stomatal densities of LP was relatively less than in the other cultivars (Figure 7).

4. Discussion

White clover is an important forage legume with excellent feed quality that is widely cultivated in the world [35]. However, it is severely affected by drought stress owing to its shallow root system and inefficient transpiration control [36]. Therefore, better understanding of its physiological mechanisms of stress tolerance would benefit white clover breeding and management programs [37]. In this study, we compared the drought tolerance and post-drought recovery of four white clover cultivars with different leaf types associated with membrane lipid permeability, osmoregulatory substances, photosynthetic characteristics and stomatal features. Our research will help further understand the mechanisms of small-leafed and large-leafed white clover tolerance to drought stress and select more tolerant white clover cultivars.
RWC is one of the main indicators for defining plant water status, and reflects the balance between water supply and the transpiration rate in plants [38]. In this research, LP and PD (small-leafed cultivars) maintained higher RWC than the other cultivars throughout the drought stress and rewatering (Figure 2A). This result was also consistent with a previous study which demonstrated that small-leafed white clover (PI 288084) was able to maintain higher RWC level than a large-leafed cultivar (“Chuanyin Ladino”) under the same duration of drought treatment [24]. The membrane permeability is a parameter negatively affected by water deficiency. The increase in relative electrical conductivity rate may indicate a loss of cell membrane ability to regulate ion transportation under water deficiency [39]. MDA is a key indicator for peroxidative deterioration of membrane lipids under adverse environments, and its content can indicate the degree of membrane damage [40,41]. In the present study, the REC and MDA contents of the four cultivars increased with prolonged periods of drought, but the increases in REC and MDA contents in the small-leafed cultivars were significantly lower than those in the medium- and large-leafed cultivars under drought (Figure 2B,C). The results of our research indicate that the small-leafed clovers maintained better membrane stability under drought stress. We speculated that this could contribute to greater stress tolerance in LP. After rewatering, the REC and MDA (Figure 2B,C) decreased rapidly in the four cultivars. Particularly, the two parameters REC and MDA were significantly lower in LP (small-leafed) than in the other three cultivars, indicating that small-leafed white clover has better membrane-repairing ability. A previous study showed that the recovery of REC and MDA in a small-leafed white clover that suffered from drought was faster than in large-leafed clover after rewatering, which was close to the well-watered level [24].
The preservation or improvement of the cell osmotic regulation ability under drought stress is a crucial factor for plant drought resistance [42]. Pro, SS and SP are the three primary osmotic adjustment compounds, and their relationships with plant drought resistance have been extensively studied [43,44]. Our findings showed that the Pro and SS contents of the four white clover cultivars increased during drought stress (Figure 3A,B). After rewatering, the SS content of LP (small-leafed) was close to that of the control (Figure 3B). The degree of drought stress directly affected the change in SP. With the increase in stress intensity, the SP content of the four white clover cultivars showed a trend of rising first and falling subsequently (Figure 3C). The SP content of LP (small-leafed) increased the greatest after rewatering, followed by PD (small-leafed). It was evident that white clovers with small leaves had stronger recovery capacity. Furthermore, when plants were facing drought stress, many osmotic adjustment substances responded to change, causing swelling pressure such as the opening and closing of stomata, cell growth and photosynthesis [45].
The leaf is the main organ for plant photosynthesis. It is also the most sensitive and plastic part to environmental changes [46]. Stomata in the leaf epidermis are channels for transpiration and absorption of CO2 [47]. Stomatal densities and sizes as well as the state of opening or closing control the photosynthetic and transpiration functions of plants [48,49]. Stomatal activities are very sensitive to water deficiency. A slight water shortage can cause a decrease in stomatal conductance, reduce CO2 concentration in the channels and slow down the output of photosynthetic products accumulated in the leaves, thus producing inhibition feedback on photosynthesis [50]. Water is a major participant in photosynthesis which excites chlorophyll molecules in the photosynthetic system to produce high-energy electrons and is ultimately used to produce photosynthates for plant growth and development [51]. Meanwhile, water molecules play a lubricative role in the stomatal opening and closing processes and assist the stomatal movement [52,53]. Therefore, water shortage will directly cause a decline in plant photosynthesis and stomatal functions. In the current study, four cultivars of white clover varied in their stomatal densities and sizes. Under drought conditions, stoma length and width decreased, but stomatal density increased concurrently with decreased leaf water content and diurnal photosynthetic characteristics. However, these changes were still different among the four cultivars. Small-leafed white clover showed smaller sizes and higher densities of stomata both under normal conditions and under drought stress compared to the large-leafed type (Table 2, Figure 7). A previous study revealed that small-leafed white clover had a higher instantaneous net photosynthetic rate, stomatal conductance and physiological water use efficiency during drought and water recovery [24], indicating that the small-leafed cultivar had stronger cellular water retention and drought resistance than large-leafed whiter clover under drought stress. In our study, the small-leafed cultivars LP and PD showed similar photosynthetic characteristics to the study by Li et al. [24]. Although these indices in the current study were dealing with diurnal processes, they suggested that higher stomatal density can facilitate CO2 and water exchange in cellular metabolism, thus enhancing the ability of photosynthesis. According to research by Caradus et al. [19], the leaf size of white clover decreases with increased altitude and latitude. Therefore, it could be inferred that the ancestors of the cultivars LP and PD in this study came from high-latitude and high-altitude regions with harsh environmental factors which shaped them with smaller leaf areas and stronger drought resistance.

5. Conclusions

In this research, four white clover cultivars with different leaf sizes were investigated by assessing their morpho-physiological features under drought and post-drought recovery. In response to drought, the small-leafed cultivars (LP and PD) maintained higher RWC, Pro, SS and SP contents, lower REC and MDA contents and stronger photosynthetic rate than medium-leafed (RD) and large-leafed (KL) cultivars. Meanwhile, small-leafed white clovers showed smaller sizes and higher densities of stomata than the medium- and large-leafed types under both well-watered and drought conditions. These results demonstrated that small-leafed white clovers had stronger drought tolerance and post-drought recovery owing to greater abilities to accumulate more osmoregulatory substances for retaining cellular water content and cell membrane stability as well as greater photosynthetic capacity. This information will help us to better understand the mechanisms of drought resistance in white clovers with different leaf types, and assist in further breeding new white clover cultivars resistant to drought.

Author Contributions

X.Z., Z.T. and L.C. performed the experiments and wrote the paper; J.J., Y.L. and L.L. analyzed the data; C.Y. and X.L. discussed the results; F.X. supervised the experiment; L.Q. and Y.C. designed the experiments and revised the paper. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the China Postdoctoral Science Foundation (2022MD723773), Heilongjiang Postdoctoral Fundation (LBH-Z21009), Academic Backbone Project of Northeast Agricultural University (21XG25), and College Student Innovation and Entrepreneurship Training Program at Northeast Agriculture University.

Data Availability Statement

Not applicable.

Acknowledgments

We thank Jun Liu from Bayer Company (Canada) for his great help to improve the manuscript and English. We are very grateful to the editor and reviewers for their comments that helped to greatly improve the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Leaf phenotypic features of the four white clover cultivars (T. repens L.). (A) LP; (B) PD; (C) RD; (D) KL.
Figure 1. Leaf phenotypic features of the four white clover cultivars (T. repens L.). (A) LP; (B) PD; (C) RD; (D) KL.
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Figure 2. Analysis of the leaf relative water content and membrane lipid permeability of four white clover (T. repens L.) cultivars under different treatments. (A) Leaf relative water content (%); (B) relative electrical conductivity rate (%); (C) malondialdehyde content. The bars indicate the standard errors of the means (±SE). The different lowercase letters indicate significant differences among the four cultivars (p < 0.05).
Figure 2. Analysis of the leaf relative water content and membrane lipid permeability of four white clover (T. repens L.) cultivars under different treatments. (A) Leaf relative water content (%); (B) relative electrical conductivity rate (%); (C) malondialdehyde content. The bars indicate the standard errors of the means (±SE). The different lowercase letters indicate significant differences among the four cultivars (p < 0.05).
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Figure 3. Analysis of osmoregulatory substances of four white clover (T. repens L.) cultivars under different treatments. (A) Free proline content; (B) soluble sugar content; (C) soluble protein content. The bars indicate the standard errors of the means (± SE). The different lowercase letters indicate significant differences among the four cultivars (p < 0.05).
Figure 3. Analysis of osmoregulatory substances of four white clover (T. repens L.) cultivars under different treatments. (A) Free proline content; (B) soluble sugar content; (C) soluble protein content. The bars indicate the standard errors of the means (± SE). The different lowercase letters indicate significant differences among the four cultivars (p < 0.05).
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Figure 4. Diurnal changes in the photosynthesis rate (Pn) of four white clover (T. repens L.) cultivars: (A) LP; (B) PD; (C) RD; (D) KL under drought stress. (E) Diurnal changes in the Pn of four white clover cultivars under normal conditions. (F) Diurnal changes in the Pn of four white clover cultivars on the 6th day of drought stress. Vertical bars indicate LSD (p < 0.05).
Figure 4. Diurnal changes in the photosynthesis rate (Pn) of four white clover (T. repens L.) cultivars: (A) LP; (B) PD; (C) RD; (D) KL under drought stress. (E) Diurnal changes in the Pn of four white clover cultivars under normal conditions. (F) Diurnal changes in the Pn of four white clover cultivars on the 6th day of drought stress. Vertical bars indicate LSD (p < 0.05).
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Figure 5. Diurnal changes in the transpiration rate (Tr) of four white clover (T. repens L.) cultivars: (A) LP; (B) PD; (C) RD; (D) KL under drought stress. (E) Diurnal changes in the Tr of four white clover cultivars under normal conditions. (F) Diurnal changes in the Tr of four white clover cultivars on the 6th day of drought stress. Vertical bars indicate LSD (p < 0.05).
Figure 5. Diurnal changes in the transpiration rate (Tr) of four white clover (T. repens L.) cultivars: (A) LP; (B) PD; (C) RD; (D) KL under drought stress. (E) Diurnal changes in the Tr of four white clover cultivars under normal conditions. (F) Diurnal changes in the Tr of four white clover cultivars on the 6th day of drought stress. Vertical bars indicate LSD (p < 0.05).
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Figure 6. Diurnal changes in the stomatal conductance (Cs) of four white clover (T. repens L.) cultivars: (A) LP; (B) PD; (C) RD; (D) KL under drought stress. (E) Diurnal changes in the Cs of four white clover cultivars under normal conditions. (F) Diurnal changes in the Cs of four white clover cultivars on the 3rd day of drought stress. Vertical bars indicate LSD (p < 0.05).
Figure 6. Diurnal changes in the stomatal conductance (Cs) of four white clover (T. repens L.) cultivars: (A) LP; (B) PD; (C) RD; (D) KL under drought stress. (E) Diurnal changes in the Cs of four white clover cultivars under normal conditions. (F) Diurnal changes in the Cs of four white clover cultivars on the 3rd day of drought stress. Vertical bars indicate LSD (p < 0.05).
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Figure 7. Stomatal apparatus characteristics of white clover (T. repens L.) captured under normal and drought conditions using scanning electron microscopy (×500). (A–D) Stoma status of LP, PD, RD and KL under normal conditions; (A1–D1) Stoma status of LP, PD, RD and KL under drought stress. White arrows indicate stomatal status.
Figure 7. Stomatal apparatus characteristics of white clover (T. repens L.) captured under normal and drought conditions using scanning electron microscopy (×500). (A–D) Stoma status of LP, PD, RD and KL under normal conditions; (A1–D1) Stoma status of LP, PD, RD and KL under drought stress. White arrows indicate stomatal status.
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Table
Table 1. Phenotypic indicators of the four white clover cultivars (T. repens L.).
Table 1. Phenotypic indicators of the four white clover cultivars (T. repens L.).
IndicatorsLPPDRDKL
Plant height/cm8.57 ± 1.43 c8.83 ± 1.07 c10.23 ± 1.07 b15.52 ± 1.31 a
Leaflet area/cm21.02 ± 0.29 c1.18 ± 0.37 c1.97 ± 0.21 b3.12 ± 0.42 a
Branch number7.6 ± 0.28 a5.3 ± 0.49 c5.7 ± 0.57 b4.9 ± 0.31 d
The lowercase letters represent significant differences (p < 0.05) among four cultivars under the same indicator.
Table
Table 2. The characteristics of leaf epidermal stomata in white clover (T. repens L.).
Table 2. The characteristics of leaf epidermal stomata in white clover (T. repens L.).
TreatmentsCultivarsStomata Length (µm)Stomata Width (µm)Stomata Density (Number/mm2)
CKLP24.67 ± 1.63 d17.78 ± 2.12 c187.5 ± 6.34 a
PD25.24 ± 1.93 c16.73 ± 2.47 d168.4 ± 8.46 b
RD26.18 ± 2.78 b18.26 ± 1.76 b155.2 ± 13.71 c
KL27.62 ± 1.55 a19.84 ± 3.39 a128.6 ± 9.73 d
DroughtLP21.27 ± 2.26 c13.29 ± 1.44 a203.8 ± 10.52 a
PD21.82 ± 3.31 b10.62 ± 1.85 d191.3 ± 11.67 b
RD21.89 ± 1.49 b12.81 ± 2.26 b176.1 ± 7.48 c
KL22.86 ± 2.74 a11.45 ± 2.63 c154.8 ± 5.36 d
Note: CK—well-watered condition. Drought—the 12th day of drought stress. The lowercase letters represent significant differences (p < 0.05) among four cultivars under the same conditions.
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Zhao, X.; Tian, Z.; Cheng, L.; Jiang, J.; Liu, Y.; Liu, L.; You, C.; Liu, X.; Xie, F.; Qin, L.; et al. Comparative Study on the Morpho-Physiological Responses of White Clover Cultivars with Different Leaf Types to Water Deficiency. Agronomy 2023, 13, 1859. https://doi.org/10.3390/agronomy13071859

AMA Style

Zhao X, Tian Z, Cheng L, Jiang J, Liu Y, Liu L, You C, Liu X, Xie F, Qin L, et al. Comparative Study on the Morpho-Physiological Responses of White Clover Cultivars with Different Leaf Types to Water Deficiency. Agronomy. 2023; 13(7):1859. https://doi.org/10.3390/agronomy13071859

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Zhao, Xueying, Zhi Tian, Lintao Cheng, Jia Jiang, Yujiao Liu, Lizhi Liu, Chunxu You, Xun Liu, Fuchun Xie, Ligang Qin, and et al. 2023. "Comparative Study on the Morpho-Physiological Responses of White Clover Cultivars with Different Leaf Types to Water Deficiency" Agronomy 13, no. 7: 1859. https://doi.org/10.3390/agronomy13071859

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