Comparative Evaluation of Physiological Response and Drought Tolerance between Cunninghamia unica and C. lanceolata Seedlings under Drought Stress

Abstract

Cunninghamia plays an important role in China’s southern forest industry in the face of increasingly arid climate; thus it is urgent to identify and screen drought-tolerant varieties. In this study, 2-year-old seedlings of C. unica and C. lanceolata from four provenances were subjected to water-break tests, and their physiological responses to different drought conditions were observed. The results showed that with the aggravation of drought stress, C. unica had more stable changes in relative water content (RWC), water potential (Ψw) and intercellular CO₂ concentration (Ci) with more cumulative amounts of proline (PRO) than C. lanceolata, and its H₂O₂ maintained at a lower level, along with antioxidant enzyme activities decreasing later as compared with C. lanceolata. Moreover, comprehensive evaluation showed that C. unica had a higher drought tolerance than C. lanceolata as a whole, which could have been shaped by maintaining Ψw and opening stomata in its relative drought conditions. This work provides a theoretical basis for understanding the drought tolerance of C. unica and C. lanceolate individuals, so as to accelerate selective breeding in Chinese fir.

1. Introduction

Against the background of global climatic changes, drought is predicted to be increasingly more common in the future [1]. Drought can alter the physiological characteristics of plant leaves; for instance, lowering leaf photosynthetic and transpiration rates, enzyme activity, uptake of water and minerals and stomatal conductance [2]. Drought also affects tree morphology, such as decreasing leaf size, stem length, root length and disrupting phloem transport. These will threaten tree survival and lead to forest degradation and decline. For example, when the function of phloem is impacted by drought, it will cause a change in carbon allocation within the tree, a change in ecosystem carbon cycling and a change in forest mortality [3]. Currently, increasing drought events are inducing forest dieback [4].

With the decrease in natural forests and the increase in artificial forests, plantations are playing an increasingly important role in mitigating global climate change [5]. Cunninghamia lanceolata (Chinese fir), belonging to the genus Cunninghamia of Taxodiaceae, is mainly distributed in the warm and moist areas of southern China. Chinese fir, as one of the most important cultivated plants for wood production, has the largest planting area of 9.9 million ha in China [6,7]. Therefore, vigorously developing Chinese fir plantations can not only maximize economic benefits based on the original forestry, but also effectively improve the ecological environment. As a shallow-rooted tree species, traditional C. lanceolata is a drought-sensitive species, showing dependence on loose, moist and fertile soil and disfavoring strong radiation and strong winds [8,9]. Excessive drought causes soil moisture loss and even hardening, which limits the normal growth and development of C. lanceolata. In the face of the increasingly arid environment, screening drought-tolerant varieties seem to be the key to solving this problem.

Evidence shows that different populations of the same tree species show different adaptability to the environment, and differences in drought tolerance between populations are associated with both environmental and genetic factors [10,11]. A similar phenomenon is also observed in the genus Cunninghamia. Cunninghamia. unica is a new species of Cunninghamia identified in the 1970s, which is mainly distributed in arid-hot valley zones, such as Dechang County, Yanyuan County, Liangshan Prefecture and Miyi County, Panzhihua City [12,13,14]. The ecology and climate of these zones are mainly characterized by low rainfall, a high evapotranspiration rate and strong radiation [14], which is quite a contrast to the ecology and climate of the growing area of C. lanceolata. At present, there is a lack of knowledge regarding the difference in drought tolerance between C. unica and C. lanceolata, and the mechanism of its drought tolerance remains unclear.

Drought tolerance, as a complex mechanism, is related to adaptation, morphology, anatomy and the physiological and biochemical reactions of plants [15,16]. Usually, the physiological characteristics of leaves (water physiology, photosynthetic physiology, antioxidant enzymes, etc.) are taken as important observation indexes for drought tolerance evaluation [17]. Based on this, seedlings of C. unica and C. lanceolata from four different provenances were treated with water-break to simulate different drought conditions, indexes of water, photosynthetic and antioxidant physiology relating to drought tolerance were measured to comprehensively evaluate and elaborate: compared with C. lanceolata, does C. unica possess better drought tolerance and have the potential as a drought-resistant germplasm resource, and how is the drought tolerance mechanism of C. unica different?

2. Materials and Methods

2.1. Material

Four provenances of C. lanceolata including Sichuan Hongya (CL-SC), Fujian Shunchang (CL-FJ), Hunan Huitong (CL-HN) and Guangdong Shaoguan (CL-GD) were selected and compared with C. unica from Sichuan Dechang (CU-DC) in this experiment (Figure 1). One-year-old C. unica and C. lanceolata seedlings were used as materials and cultivated with planting bags (25 × 28 cm) using the mountain yellow soils for one year under conventional water and fertilizer management. Finally, the 2-year-old C. unica and C. lanceolata seedlings were obtained by pot cultivation for this experiment.

2.2. Experiment Design

Five treatments with different water-break days of 1 d, 3 d, 7 d, 14 d and 24 d were designed to simulate different degrees of drought stress, in which seedlings of C. unica and C. lanceolata from four different provenances for each treatment were pre-cultivated. To guarantee consistent conditions of index determination for all seedlings in five treatments, the initial water-break times of five treatments were on different dates, according to different water-break days to make the water-break termination of five treatments on the same day. Finally, three seedlings (each seedling as a biological repeat) of each treatment with consistent growth were selected as sample materials for index determination. The mature leaves with good growth in the middle of the third round of branches from top to bottom were collected to determine the indexes. The experiment was conducted in the greenhouse from 5 June 2022 to 29 June 2022.

2.3. Determination of Drought Tolerance Indexes

2.3.1. Determination of Relative Water Content, Water Potential and Osmoregulators

The relative water content (RWC) of leaves was measured. The fresh leaves were weighed, and then soaked in de-ionized water for 8 h until full turgidity. After that, the leaf samples were weighed again after removing the surface moisture, and then immersed in water for 1h until the sample weight did not change, followed by weighing the fully turgid weight. Finally, they were transferred into a hot air oven at 105 °C for 30 min for fixation and dried at 80 °C for 24 h before weighing the dry weight [18]. The RWC of each sample was calculated according to the following the formula.

$$RWC(\%)=\left(Fw- Dw\right)/\left(Tw-Dw\right)\times 100$$

(1)

where, Fw = fresh weight (in gram); Dw = dry weight (in gram); Tw = turgid weight (in gram).

The water potential (Ψw) was measured by a WP4C dew point water potential meter (WPC4, METER Group, Inc., Pullman, WA, USA) [19]. Soluble protein (SP) was determined using a Coomassie brilliant blue method kit, and soluble sugar (SS) and proline (PRO) were measured using a colorimetry kit [20].

2.3.2. Determination of Chlorophyll Content, Net Photosynthetic Rate, Intercellular CO₂ Concentration and Stomatal Conductance

The three adjacent healthy mature leaves in the middle of branches were taken as the research objects, and their net photosynthetic rate (P_N), stomatal conductance (Gs) and intercellular CO₂ concentration (Ci) between 9:00 and 12:00 A.M. were measured using a Li-6400 portable photosynthetic meter (Li-6400, Li-Cor Inc., Lincoln, NE, USA). The light intensity was set to 800 μmol m⁻² s⁻¹, the temperature was 30 °C, the humidity was 60% and the CO₂ concentration was 400 ppm. Chlorophyll (Chl), chlorophyll a (Chla) and chlorophyll b (Chlb) were extracted and quantified using the alcohol method. The extract was respectively colorimetered at 663 nm and 645 nm by UV spectrophotometer, and 95% ethanol was used as the blank control. The OD value was measured and the measurement was repeatedly conducted three times [21]. Chla, Chlb and total Chl were calculated according to the following equations.

$$Chla=\left(12.72\times O.D_{663}-2.59\times O.D_{645}\right)\times \frac{n}{F_w}\times V_T\times 1000$$

(2)

$$Chlb=\left(22.88\times O.D_{645}-4.67\times O.D_{663}\right)\times \frac{n}{F_w}\times V_T\times 1000$$

(3)

$$Total Chl=Chla+Chl$$

(4)

where, Fw = weight of fresh weight (in gram), V_T is the total volume of extract (in milliliter) and n = dilution multiple.

2.3.3. Determination of Cell Membrane Permeability, Malondialdehyde Content and Antioxidant Enzymes Activity

Cell membrane permeability was expressed by relative electric conductivity (REC). Healthy and complete leaves of the same size were selected, washed with distilled water and dried. Then, 0.5 g of leaves were taken and placed in a test tube containing 30 mL de-ionized water. After that, the tube was covered and soaked indoors for 24 h before using a BEC540 conductivity tester (BEC540, HangZhou DiogTech Co., Ltd., Hangzhou, CHN) to measure the conductivity (R1) of the extract. Subsequently, the test tube was heated in a boiling water bath for 30 min and cooled down to room temperature, followed by measuring the conductivity (R2) of the extract again [22]. The REC was calculated according to the following equation

$$REC(\%)=R1∕R2\times 100$$

(5)

Hydrogen peroxide (H₂O₂) was measured by colorimetric kit, malondialdehyde (MDA) was measured by TBA kit, peroxidase (POD) was measured by colorimetric kit, catalase (CAT) was measured by visible light kit, and superoxide dismutase (SOD) was measured by WST-1 kit [23].

2.4. Statistical Analysis

Microsoft Excel 2010 software was used for data recording and calculation, SPSS statistics 20 software was used for one-way ANOVA, principal component analysis and cluster analysis and Origin 2021 software was used for mapping.

One-way ANOVA: Firstly, the original values of each index were used to analyze the seedlings from the same provenance between different treatments, as well as the seedlings from five provenances under the same treatment. To eliminate the original differences between C. unica and C. lanceolata, the relative values of those under the same treatment were analyzed.

$$Relative value=\left(index value after treatment\right)∕\left(average index value of 1 d treatment\right)$$

(6)

In a comprehensive evaluation of drought tolerance, first, the comprehensive index value (Z_i) and membership function value μ(Z_i) were calculated. Then, combining with the weight of each comprehensive index (W_i), the comprehensive evaluation value (D) of C. unica and C. lanceolata under each treatment was obtained based on which of their drought tolerance was evaluated. Finally, cluster analysis of C. unica and C. lanceolata was carried out according to the D value [24]. Z_i, μ(Z_i), W_i and D values were calculated by the following equations: where, a_i = eigenvector corresponding to the eigenvalue of a single index, X_i = relative value of the index, Z_imax = maximum value of the i-th comprehensive index of various provenance, Z_imin = minimum value of the i-th comprehensive index of various provenance, P_i = contribution of the i-th comprehensive index of various provenance.

$$Z_i={\displaystyle \sum }_{i=1}^n{a}_i{X}_i$$

(7)

$$\mu \left(Z_i\right)=\left(Z_i-Z_{imin}\right)∕\left(Z_{imax}-Z_{imin}\right)$$

(8)

$$W_i=P_i∕{\displaystyle \sum }_{i=1}^n{P}_i$$

(9)

$$D={\displaystyle \sum }_{i=1}^n\left[\mu \left(Z_i\right)\times W_i\right]$$

(10)

3. Results

3.1. Water Pysiology of C. unica and C. lanceolata Seedlings

RWC and Ψw of the leaves of C. unica and four C. lanceolata gradually decreased with the increase in water-break days (Figure 2a,b). In comparison to C. lanceolata, C. unica showed a relatively late decrease in RWC (24 d), and had lower RWC values in the short- and mid-term water-break (1 d, 3 d, and 7 d) and higher RWC values in the long-term water-break (14 d and 24 d) (Figure 2a). Generally, Ψw of C. unica was at a relatively lower level than C. lanceolata in the short-term water-break (1 d and 3 d), yet its decline was more stable with the aggravation of drought stress, so the Ψw values were at the middle levels in the mid- and long-term water-break (7 d, 14 d and 24 d) (Figure 2b). Osmoregulators were largely accumulated in C. unica and C. lanceolata in response to drought stress; however, their changes were quite different, showing significant differences among species/provenances under the same treatment and significant differences among treatments within the same species/provenance (Figure 2c–e, p < 0.05). The contents of SS and SP in C. unica leaves increased in fluctuation under different treatments, which were higher than those in C. lanceolata in the short-term (1 d and 3 d) and a 24-d water-break on the whole (Figure 2c,d). PRO contents of both C. unica and C. lanceolata showed continuous accumulation with the aggravation of drought stress, but the significant accumulations were observed in 7-d and 24-d water-breaks for C. unica and in 14-d and 24-d water-breaks for C. lanceolata in general (except CL-SC). Moreover, the PRO of C. unica was significantly higher than that of C. lanceolata in a 24-d water-break. (Figure 2e, p < 0.05).

3.2. Photosynthetic Physiological Responses of C. unica and C. lanceolata Seedlings

P_N of C. unica and four C. lanceolata seedlings gradually declined with the aggravation of drought stress, yet P_N of C. unica declined slower than that of C. lanceolata during the short- to mid-term water-break (1 d–7 d), especially during the short-term water-break (1 d–3 d). Under severe stress (14 d and 24 d), P_N of C. unica and four C. lanceolata seedlings were negative (Figure 3a). The variation in Gs and Ci of C. unica and four C. lanceolata seedlings were quite different with the aggravation of drought stress (Figure 3b,c). Gs of C. unica stably changed during the short-term water-break (1 d–3 d), followed by an evident jump in the mid-term water-break (7 d) and then gradually declined during the long-term water-break (14 d–24 d), while C. lanceolata showed a continuous downward trend, except CL-GD, which is similar to C. unica. As a whole, Gs of C. unica was higher than that of C. lanceolate, except for the treatment of a 1-d water-break. The change in Ci of C. unica was quite stable in comparison to C. lanceolata, and the change in Ci was quite different among seedlings from different provenances. Three chlorophyll contents (Chla, Chlb and Chl) showed roughly the same trend with the aggravation of drought stress for C. unica and all C. lanceolata seedlings, but quite different trends among C. unica and four C. lanceolata seedlings (Figure 3d–f). Generally, the chlorophyll contents of C. unica were lower than those of C. lanceolata in 1-d, 3-d, 7-d and 14-d water-breaks, but higher than those of C. lanceolata in a 24-d water-break, except for a few cases.

3.3. Antioxidant Physiological Responses of C. lanceolata and C. lanceolata Seedlings

REC, MDA and H₂O₂ of C. unica and four C. lanceolata seedlings all showed wavelike rises with the aggravation of drought stress, but only exhibited significant increases in few cases of C. lanceolata, such as REC of CL-SC and CL-GD, MDA of CL-SC and H₂O₂ of CL-FJ, and three indexes of C. unica had no significant differences compared to most C. lanceolata seedlings in most treatments (Figure 4a–c, p < 0.05). Three antioxidant enzymes (POD, SOD and CAT) of C. unica and four C. lanceolata seedlings presented roughly similar trends of increasing first and then decreasing, yet the downward trend of C. unica would appear during the long-term water-break (14 d–24 d), which was later than that of C. lanceolata in general (Figure 4d–f).

3.4. Comprehensive Evaluation of Drought Tolerance of C. unica and C. lanceolata Seedlings

To comprehensively evaluate the drought tolerance of C. unica and four C. lanceolata seedlings, membership function and principal component analysis was conducted by taking a total of 17 personality indexes as variables (

Table 1. Comprehensive index values of Cunninghamia. unica and C. lanceolata.

Day	Provenance	Comprehensive Index Value
		Z1	Z2	Z3	Z4	Z5	Z6
3	CU-DC	9.52	3.29	1.91	3.63	3.49	4.10
	CL-SC	3.27	−0.33	−0.63	3.01	2.29	2.84
	CL-HN	1.72	2.84	0.08	1.96	2.02	2.29
	CL-FJ	1.76	1.96	1.33	2.54	1.96	2.10
	CL-GD	−0.03	1.44	0.68	2.98	2.16	2.28
7	CU-DC	13.19	3.23	−2.30	−0.36	—	—
	CL-SC	4.31	0.20	−1.74	0.34	—	—
	CL-HN	0.79	−0.32	3.70	2.76	—	—
	CL-FJ	0.15	4.17	1.23	2.71	—	—
	CL-GD	0.98	3.67	2.75	0.57	—	—
14	CU-DC	12.26	1.98	5.80	1.96	0.72	—
	CL-SC	13.25	−4.35	7.23	2.01	1.06	—
	CL-HN	18.94	−7.35	12.06	2.82	1.34	—
	CL-FJ	28.05	−4.11	12.36	3.04	0.59	—
	CL-GD	11.55	−2.20	7.53	2.24	0.88	—
24	CU-DC	1.56	−8.42	21.98	−27.16	21.10	—
	CL-SC	−1.44	−6.90	15.31	−15.02	11.93	—
	CL-HN	−1.08	−2.78	12.46	−16.48	14.41	—
	CL-FJ	−7.82	−4.92	20.10	−22.36	18.63	—
	CL-GD	1.23	−2.79	12.36	−11.18	10.05	—

). The drought tolerance of these seedlings was evaluated, where the D values of C. unica ranked first in 3 d and 7 d, third in 14 d, and second in 24 d (

Table 2. Weight of comprehensive index value (W_i), membership function value, comprehensive evaluation value (D) and drought tolerance ranking of C. unica and C. lanceolata.

Day	Provenance	Membership Function Value						D Value	Rank
		μ(Z1)	μ(Z2)	μ(Z3)	μ(Z4)	μ(Z5)	μ(Z6)
3	CU-DC	1.00	1.00	1.00	1.00	1.00	1.00	1.00	1
	CL-SC	0.35	0.00	0.00	0.63	0.22	0.37	0.24	5
	CL-HN	0.18	0.88	0.28	0.00	0.04	0.09	0.30	3
	CL-FJ	0.19	0.63	0.77	0.35	0.00	0.00	0.38	2
	CL-GD	0.00	0.49	0.52	0.61	0.13	0.09	0.29	4
	Wi	0.34	0.20	0.18	0.13	0.08	0.07	—	—
7	CU-DC	1.00	0.79	0.00	0.00	—	—	0.61	1
	CL-SC	0.32	0.12	0.09	0.22	—	—	0.20	5
	CL-HN	0.05	0.00	1.00	1.00	—	—	0.35	4
	CL-FJ	0.00	1.00	0.59	0.98	—	—	0.55	2
	CL-GD	0.06	0.89	0.84	0.30	—	—	0.50	3
	Wi	0.36	0.31	0.20	0.13	—	—	—	—
14	CU-DC	0.04	1.00	0.00	0.00	0.18	—	0.24	3
	CL-SC	0.10	0.32	0.22	0.05	0.63	—	0.20	5
	CL-HN	0.45	0.00	0.95	1.08	1.00	—	0.53	2
	CL-FJ	1.00	0.35	1.00	1.00	0.00	—	0.79	1
	CL-GD	0.00	0.55	0.26	0.25	0.39	—	0.20	4
	Wi	0.48	0.20	0.14	0.10	0.07	—	—	—
24	CU-DC	1.00	0.00	1.00	0.00	1.00	—	0.66	2
	CL-SC	0.68	0.27	0.31	0.76	0.17	—	0.49	4
	CL-HN	0.72	1.00	0.01	0.67	0.39	—	0.61	3
	CL-FJ	0.00	0.62	0.80	0.30	0.78	—	0.38	5
	CL-GD	0.96	1.00	0.00	1.00	0.00	—	0.72	1
	Wi	0.39	0.21	0.19	0.13	0.08	—	—	—

).

3.5. Cluster Analysis

The drought tolerance of C. unica and four C. lanceolata seedlings could be categorized by cluster analysis of the D values. It was found that C. unica (CU-DC) was the most drought-tolerant type compared with C. lanceolata, and the four C. lanceolata seedlings, including Hunan Huitong (CL-HN), Guangdong Shaoguan (CL-GD) and Sichuan Hongya (CL-SC), were the most non-drought-tolerant type, while Fujian Shunchang (CL-FJ) ranked in the intermediate position (Figure 5).

4. Discussion

With the increase in water-break days, RWC and Ψw of both C. unica and C. lanceolata seedlings decreased in varying degrees and responded to the drought by accumulating osmotic-regulating substances. For plants, normal water content is of great significance for maintaining the basic functions of cells and their normal physiological activities [25]. The RWC of leaves is an important index by which to measure the water status of plant tissues and the dynamic water absorption-loss balance [26]. To a certain extent, it can reflect the tolerance of plants to drought stress [27,28]. If the decline rate of the RWC of plants under drought stress is slow and the range is small, it means the drought tolerance is strong, and vice versa [29]. During the whole water-break duration, the RWC of C. unica significantly and slowly decreased late (p < 0.05), indicating that it has stronger drought tolerance. The decline in Ψw of plant leaf is an important indicator of plant water shortage. Moreover, the value and decline range of Ψw can be used as an important index by which to identify plant drought tolerance [30]. Under the same soil water content, the value and decline range of leaf Ψw are negatively correlated with drought tolerance; that is, the lower the Ψw, the smaller the decline range, the stronger the water absorption capacity, and the stronger the drought tolerance. Compared with C. lanceolata, C. unica had a lower Ψw value and a slower decline rate during the whole water-break period (p < 0.05), indicating that C. unica can better maintain its Ψw and has stronger drought tolerance. Sapes et al. observed the same results in their drought stress experiments on Pinus ponderosa populations, wherein RWC and Ψw decreased with the increase in drought stress, and they pointed out that RWC and Ψw were accurate predictors of drought risk [28].

Low water potential dehydration is one of the important ways for plants to adapt to drought stress, which is beneficial for reducing cell Ψw, maintaining cell turgor, and increasing plant water absorption by increasing the cell content of osmoregulators [31,32]. Therefore, osmoregulators are one of the important indexes of plant water physiology, which is of great significance for plant osmotic regulation. SS, SP and PRO are key substances of osmotic regulation, and the significant accumulation of these substances improves the drought tolerance of plants [33,34,35]. Under mild drought stress (short-term water-break), the contents of SS and SP of C. unica were at a higher level than C. lanceolata, which may be because C. unica were in a relatively arid environment for a long time and accumulated more osmoregulators to deal with drought. Compared with SS and SP, PRO significantly changed, indicating that PRO is the main osmoregulator of C. unica and C. lanceolata. Interestingly, the main osmoregulators of different tree species are not the same. Wang et al. found that, under drought stress, Populus euphratica Oliv accumulated a large amount of SS to participate in osmotic regulation, while Populus pruinosa Schrenk accumulated a large amount of PRO [36], which accounts for the difference in drought tolerance strategies among different tree species. PRO has a very strong hydration ability, which can prevent protein denaturation from dehydration under osmotic stress conditions and can be used as a cytoplastic protective agent for enzymes and cell structure [37]. Therefore, one of the drought tolerance strategies of C. unica and C. lanceolata is to enhance anti dehydration in tissues and prevent SP decomposition to maintain high water content and low osmotic potential by accumulating a large amount of PRO. C. unica had a better performance of this strategy than C. lanceolata, which explains why it can better maintain REC and Ψw under the conditions of increased drought stress.

Under drought stress, the P_N value of both C. unica and C. lanceolata decreased with the increase in water-break days. Generally, the factors affecting plant photosynthesis can be divided into stomatal factors and non-stomatal factors [38,39]. The stomatal and non-stomatal limiting factors causing the decrease in P_N can be judged according to the changes in Ci and stomatal limiting value, in which Ci is a more important factor [40]. Some studies have indicated that a decrease in photosynthesis is usually caused by stomatal limitation under mild to moderate drought conditions when both Gs and Ci decline [41,42], while the more reliable criterion of non-stomatal limitation is the increase in Ci or the decrease in Gs [43]. In the present study, P_N, Gs and Ci of C. unica were maintained at a relatively stable level, while P_N and Gs decreased in different degrees and Ci increased in different degrees in C. lanceolata during the short-term water-break (1 d–3 d), indicating that C. unica can better maintain stomatal opening and ensure normal P_N under mild drought (short-term water-break), while the decrease in P_N of C. lanceolata is mainly affected by non-stomatal limitation. The reason for the different photosynthetic physiological responses of C. unica and C. lanceolata under mild drought may be that C. unica has lived at high altitude for a long time and has been subjected to longer sunshine duration and stronger light intensity, so it can avoid photo-inhibition, allowing the excess light energy to be excited in time, thus having a decreased carbon assimilation rate, producing divergent adaptation to maintain stomatal opening under mild drought, and demonstrating stronger drought tolerance than C. lanceolata under mild drought stress. In the study of the interaction between continuous planting and drought stress on two kinds of C. lanceolata, Bian et al. found that, under normal water conditions to moderate drought conditions [44], the two kinds of C. lanceolata showed the same trend as C. lanceolata in our study under different soil conditions. In summary, we speculate that the maintenance of stomatal openings in C. unica under mild drought may be a unique mechanism in the genus Cunninghamia. Under moderate (mid-term water-break) to severe drought stress (long-term water-break), the P_N of C. unica and C. lanceolata further decreased from a near-zero value to a negative value, indicating that the photosynthetic mechanism of seedlings was seriously damaged, and the main factor affecting the decline in P_N was the non-stomatal limitation for both C. unica and C. lanceolata [45]. Severe drought stress mainly damages the photosystemII (PSII) of the photosynthetic organs of plants [46], which is a non-stomatal limiting factor for the decline in P_N. PS II can respond to the reduction of CO₂ assimilation capacity by adjusting the electron transfer rate and photochemical efficiency to avoid or reduce the damage caused by excess light energy in the form of heat dissipation [47]. Chl is a pivotal pigment affecting plant photochemical efficiency, which helps to convert absorbed solar radiation into stored chemical energy and binds to proteins within chloroplasts, affecting the light-harvesting capability and photosynthesis of plants [48,49,50]. Plants that can maintain higher Chl content under water stress conditions are considered to have higher use efficiency of light energy, and therefore, are thought to have higher drought tolerance [32]. Under severe drought stress (long-term water-break), only the contents of Chl (Chla, Chlb and Chl) of C. unica showed an upward trend and were higher than those of C. lanceolata during a 24-d water-break. In contrast, the contents of Chl (Chla, Chlb and Chl) in C. lanceolata decreased to varying degrees. Higher content of Chl is conducive to capturing a higher amount of light, meaning the damage of PS II system may be reduced and P_N may be higher because light energy has changed into the chemical energy [51], indicating that C. unica have stronger drought tolerance under severe drought stress. However, most previous studies have shown that Chl content significantly declines with the increase in drought stress [52,53,54], and some studies have revealed that Chl content increased first and then decreased with the aggravation of drought stress [55]. Furthermore, it is puzzling that the Chl content of C. unica decreased first and then increased with the increase in drought stress. For this phenomenon, we cannot give a reasonable explanation at present, but only speculate that it may be related to the high content of PRO as an enzyme protector in C. unica.

Drought increases cellular levels of reactive oxygen species (ROS), such as superoxide radicals (O₂·⁻), hydroxyl radicals (·OH) and H₂O₂, leading to the lipid peroxidation of membranes and increases in the REC and the content of MDA [21,56,57,58]. Therefore, changes in REC and MDA can indirectly reflect the injury degree of the plant cell membrane. REC and MDA of C. unica and C. lanceolata showed an upward trend as a whole, indicating that the cell membrane of seedlings would be damaged to varying degrees under drought stress. The REC and MDA of C. unica were at a high level during the whole water-break duration, indicating that the plasma membrane was seriously damaged. To protect cells against the deleterious effects of excessive ROS, plants have evolved a series of sophisticated enzymatic antioxidant defense mechanisms to maintain the homeostasis of the intracellular redox state; for instance, plants produce antioxidants, flavonoids and secondary metabolites that play the role of protecting the plant for detoxifying ROS and protect the plant against stress conditions by stabilizing the protein and amino acid. [32]. SOD, POD and CAT are important antioxidant enzymes for scavenging ROS in plants, wherein SOD first converts O₂·⁻ into H₂O₂ in plants, and then scavenges H₂O₂ by enzymes such as POD and CAT [59,60]. On the whole, the activities of SOD, POD and CAT increased first and then decreased with the increase in drought degree, indicating that drought stress could induce an increase in enzyme activity in the antioxidant system at the beginning of drought stress. With the aggravation of drought stress, it eventually exceeded the tolerance range of seedlings and the production of ROS exceeded the scavenging capacity of cells; then, the antioxidant enzyme system was damaged and the activities of three antioxidant enzymes decreased in varying degrees. Therefore, the point in time when antioxidant enzyme activity decreased can be regarded as the limited degree of tolerance of C. unica and C. lanceolata. Under moderate (mid-term water-break) to severe drought (long-term water-break) stress, the antioxidant enzyme activities of CAT and POD in C. unica decreased later than C. lanceolata, indicating that C. unica had a stronger tolerance to drought. Drought stress increased the activity of CAT the most, followed by SOD and POD, indicating that CAT is more sensitive to drought stress and it is the main protective enzyme of drought stress in C. unica, which accounts for the low content of H₂O₂ in the stress process and the reduction of the damage of H₂O₂ to plant cells.

The drought tolerance of plants is the result of many factors, which cannot be accurately and comprehensively reflected by a certain index [32]. Consequently, the combination of principal component analysis and membership function was adopted in this work to make the evaluation results more accurate [61]. The D value of C. unica was the largest in 3 d and 7 d of water-breaks, indicating that it had the strongest drought tolerance under mild and moderate drought stress. In a 24-d water-break, C. unica ranked second, illustrating that it had strong drought tolerance under the most serious drought stress.

5. Conclusions

This study verified that C. unica had better drought tolerance than C. lanceolata based on physiological measurement. The drought environments under which C. unica grows have likely shaped its drought-tolerance by maintaining Ψw and open stomata to suit drought conditions. The result of this study indicates that C. unica provides potential materials for the selection of drought-tolerant Chinese fir germplasm resources, and for the further study of drought tolerance of the genus of Cunninghamia at the genetic and molecular levels.