PLoS One 15(6): e0220598

PMC: PMC7313738

PMID: 32579545

Drought resistance of ten ground cover seedling species during roof greening

1 Introduction

Roof greening, which is regarded as the “fifth surface greening”, is one of the fundamental measures for sponge cities and represents an important national policy for improving the relationship between city development and nature protection to maintain the hydrological balance in China [1,2]. As an important supplement of urban landscaping, roof greening can help mitigate the urban heat island effect [3], improve air quality [4] and enrich the biodiversity of cities [5]; hence, this landscaping style has expanded throughout all of China. Green roofs can be categorized roughly into two types: those that consist of diverse types plants (shrubs, trees, grasses, and flowers), namely, intensive green roofs (IGRs), and those that consist of simple herbaceous plant species, namely, extensive green roofs (EGRs) [6]. To grow on roofs, plants face many challenges. Taking Beijing as an example, plants that compose green roofs suffer from restricted rainfall in winter, spring, and autumn and evaporation always increases with high summer temperatures. Drought is considered as one of the most common environmental stresses that currently affects plant growth [7,8]. When plants experience drought stress, reactive oxygen species (ROS) are produced [9] including singlet oxygen (^O₂), superoxide radical (O₂^{), hydroxyl free radical (•OH), and hydrogen peroxide (H}₂O₂) [10]. ROS can reduce crop productivity and plant viability because they can cause oxidative damage to proteins, DNA, and lipids [11]. Accordingly, drought stress can not only disrupt leaf membrane permeability (MP) [12] but also reduce the chlorophyll concentration (Chl) [13] and the activity of superoxide dismutase (SOD) [14], peroxidase (POD) [15] and ascorbate peroxidase (APX) [16]. These indicators are used for measuring the degree of plant drought stress, and they are usually analyzed as a whole due to their close associations, such as the ability of antioxidative enzymes SOD, POD and APX [17] to quench ROS and protect the cell from damage.

Biological membranes are crucial aspects of living systems that control the organization and distribution of different chemical components [18], and maintain sufficient water in plant tissue to protect the organism from dehydration and carboxylation and prevent enzymes from inactivating [19]. Liposomes are colloid vesicles composed of a lipid bilayer membrane and a watery internal compartment [20], and they serve as transport carriers for the efflux of secreted proteins. Low temperature [21], drought [22], salt [23] and heavy metal [24] stress break the stability of the plant cell membrane system and proteins, thereby increasing biofilm fluidity, altering the conformation of proteins, and then leading to physiological, biochemical and metabolic imbalance and abnormalities [25]. MP is determined by electrolyte leakage [26] and could be estimated by measuring electrolytes seeping from the plant cells under environmental stress. Generally, the greater the value of MP, the more cell damage there is. Under the same water condition, a lower MP value implies a stronger adaptation of the plant species to the environment.

Chlorophyll, a green pigment, is widely distributed in plant leaves and stems [27]. It helps convert absorbed solar radiation into stored chemical energy [28] and binds to proteins within chloroplasts and affects the light-harvesting capability and photosynthesis of plants[29, 30]. Upon drought stress, plant Chl is mainly affected by the physical destruction of chloroplasts and the inhibition of Chl a and Chl b functionality. Drought stress also causes the chloroplast matrix lamella to bend and swell [31], thereby impeding Chl synthesis and reducing its production [32]. In addition, reactive oxygen species (ROS, ^O₂, O₂^{and •OH) can directly or indirectly lead to lipid peroxidation and thus Chl damage [}33].

The antioxidant system in plants consists mainly of nonenzymatic antioxidants and antioxidant enzymes. The most important antioxidant system in plants is composed of the antioxidant enzymes in chloroplasts and the cytoplasm [34]. SOD is an important enzyme that is ubiquitously expressed in aerobic organisms and catalyzes the dismutation of superoxide anions to hydrogen and molecular oxygen, which constitutes the first line of defense against ROS at the cellular level [35, 36]. Based on the prosthetic metal at the active site, SODs are classified into three groups, namely, CuZn-SODs, Mn-SODs, or Fe-SODs [37], of which Mn-SODs are closely related to mitochondria [38] and CuZn-SODs are mainly located in the cytoplasm and chloroplasts of plant cells [39]. McCord and Fridovich [40] described the principle chemical reaction under the elimination of ROS by SODs as O₂^+O₂^+2H^→O₂+H₂O₂. Existing in peroxisomes, glyoxysomes, vacuoles, the nucleus, and the extracellular matrix, SODs play a critical role in drought tolerance [41]. The SOD activity reflects the ability of plant species to adapt to environmental stress. Higher SOD activity values represent a stronger adaptation ability [42].

The antioxidant enzyme POD can scavenge and breakdown ROS [43] via the reaction RH₂+H₂O₂→2H₂O+R, in which H₂O₂ is thoroughly converted into H₂O [44, 45]. Chen et al [46] and Wu et al [47] found that increased POD activity helped cucumber and Dendrobium moniliform alleviate oxidative damage under drought stress. POD can further scavenge peroxides induced by SOD, and the synergistic action between these enzymes constitutes the protective enzyme system of the organism. Changes in the activity of these enzymes under stress may reflect the plant resistance ability in adverse environments.

APX is a member of the class I heme peroxidases and an important enzyme in plant antioxidant defense systems, and APX with several isoenzymes has a strong ability to scavenge ROS [48–51]. APX has been found in most eukaryotes, including higher plants[52], where it plays a key role in the metabolism of H₂O₂. Stronger APX activity would more quickly remove H₂O₂, thus preventing oxidative damage [53]. Different kinds of POD isoenzymes have obvious tissue and organ specialization. Similarly, APX is distributed in chloroplasts and the cytoplasm. POD and APX differ in their composition, structure, substrate specialization affinity, and stability during the purification process [54].

The membership function method is widely used to assess plant stress resistance. For example, it is has been used to assess the drought tolerance of Malus [55], maize [56] and potato [57] and the salt tolerance of Sorghum bicolor [58], Lactuca sativa [59], sugar beet [60], etc. The membership function weighted average method (D value) not only eliminates the one-sidedness associated with individual indexes but is also a relatively reliable evaluation method because the D value is the pure number within the closed interval of [0,1], which makes the difference in drought resistance of each test material comparable [61].

According to “Beijing local standards, roof greening specification (DB11/T 281–2005)” [62], more than 20 species can be used for ground cover and roof greening. Studies have focused on most of these species for their drought resistance, although these studies were limited to one species or one family. Because of the lack of studies comparing the drought resistance between these species, this study aims to screen plant species with strong resistance under drought stress to provide government policymakers with scientific plant species choices to improve plant survival rates and save maintenance costs during roof greening.

2 Study area overview

Milu Park is located 2 km far away from the South 5^{Ring Road in Beijing and surrounded by Nan-Haizi Suburb Park. The drought stress experiment was conducted under a rain shed in the core-protection area for David’s Deer in Milu Park (39.78°N, 116.47°E). During the test period, the daily average temperature was approximately 18.7°C, the daily average humidity was approximately 55.2%, and the daily average illumination intensity (at 12:00) was approximately 2000 lx. The experiment was performed in the middle of April to the end of May 2015.}

3 Materials and methods

This research has been held in the Beijing Milu Ecological Research Center (also known as Milu Park), located in Daxing district, Beijing, China. Milu Park is a place dedicated to ecological science research, as well as offered popular science education for the public for free. The authors, as staffs of Milu Park, in charge of conducting scientific research including biological science and environmental science. No additional permission is required for the authors to carry out the experiments here. Also, the 10 plant species used for experiments were all market-purchased, common ground cover plants. These plants are not endangered rare plants, not be protected.

3.1 Seedlings

One-year-old seedlings of ten species, i.e., Paeonia lactiflora, Hemerocallis dumortieri, Meehania urticifolia, Iris lactea var. chinensis, Hylotelephium erythrostictum, Sedum lineare, Iris germanica, Cosmos bipinnata, Hosta plantaginea, and Dianthus barbatus, were provided by the Yu-Quanying flower market, a large and popular wholesale market in Fengtai District, Beijing that supplies most ornamental plants for Beijing City. The plant species present various propagation modes and other characteristics (Table 1).

Table 1

Plant species and their propagation modes together with other basic features.

Latin name	Propagation mode	Life cycle	Family	Species characteristics
Paeonia lactiflora	division of suckers	perennial	Ranunculaceae	Popular in gardening, and roots used as traditional Chinese medicine
Hemerocallis dumortieri	sowing of seeds	perennial	Liliaceae	Native to Northeast China, North Korea, Japan and Russia
Meehania urticifolia	sowing of seeds	annual or perennial	Lamiaceae	Adapted to dark and moist environments
Iris lactea var. chinensis	sowing of seeds	perennial	Iridaceae	Tolerant to saline-alkaline conditions and presents a well-developed root system
Hylotelephium erythrostictum	cuttings of seeds	perennial	Crassulaceae	Traditional Chinese medicine
Sedum lineare	sowing of seeds	perennial	Crassulaceae	Traditional Chinese medicine
Iris germanica	rhizome cuttings	perennial	Iridaceae	Native to Europe
Cosmos bipinnata	sowing seedling	annual or perennial	Asteraceae	Native to Mexico
Hosta plantaginea	division of suckers	perennial	Liliaceae	Traditional Chinese medicine
Dianthus barbatus	sowing of seeds	perennial	Caryophyllaceae	Native to Europe

3.2 Field soil collection and preparation

The field soil was collected from a wild wetland area in Milu Park, and it was then air-dried and ground to powder for the transplantation experiment. The soil was moderately saline (pH = 7.89) and presented available nitrogen, available phosphorus, and available potassium contents of 24.7 mg·kg^{, 18.9 mg·kg}^{, and 322 mg·kg}^{, respectively [}63].

3.3 Plant transplanting

The transplantation program was as follows: first, approximately 400 g powdered soil was placed in a plastic pot that was 20 cm tall and 13 cm in diameter and had 3 small holes at the bottom for drainage. Second, after removing the plastic wrap surrounding the roots, the seedlings were carefully planted at the pot's center. Third, another approximately 300 g of powdered soil was placed into the pot to cover the roots and then compressed tightly by hand. The seedlings were watered every 10 min, three times in total, to ensure that enough water was available to support plant growth. The transplantation was a success if new leaves and fresh stems were developed. The seedling survival rate reached 99% one week after replanting.

3.4 Drought stress treatment design

Three treatments were designed for each of the ten species: two drought stress level treatments, which included moderate drought stress (MDS or moderate; the water content in the soil varied from 40±2% ~ 45±2%) and severe drought stress (SDS or severe; the water content in the soil was less than 30±2%), and one control group (CG or well-watered), which was under sufficient soil water conditions (the water content in the soil was over 75±2%) [64, 65]. For each treatment, three replicates were performed. Drought stress was dependent on natural evaporation. During the drought stress period, a WET-2^{sensor made by Delta-T Devices, Ltd., Cambridge, UK, was applied to measure the water content. Once the relative water content (RWC) of the soil met the requirements of the experiment, the plant seedlings were maintained under those conditions for approximately two days to ensure that changes in plant physiology and biochemistry had occurred. For the well-watered treatment, the seedlings were watered every four days.}

3.5 Leaf sampling

All plants grew new leaves ten days after transplanting, which indicated the plants’ roots had developed by the time leaves could react to the plant's physiological status. Referring to the sampling method in VDI-Guideline 3975 Part 11 [66], at least 15 g of healthy leaves was collected for each replicate. The leaf samples were placed into sealed plastic bags under a portable ice-box at 0~4°C before being transferred to the lab for further physiological and chemical analysis.

3.6 Determining the MP, Chl, SOD activity, POD activity, and APX activity

The MP (%) of the leaves was calculated as $M P = \frac{L_{t} - L_{C g}}{{1 - L}_{C g}} \times 100$ , where L_t is the relative electrical conductivity of the plant material in the drought stress treatments and L_Cg is the relative electrical conductivity of the material in the control group. The relative electrical conductivity $L = \frac{S_{1} - S_{0}}{S_{2} - S_{0}}$ , where S₁ is the original conductivity of the deionized water with fractured fresh leaves, S₂ is the conductivity of the boiled deionized water with fractured leaves, and S₀ is the conductivity of deionized water [67]. The leaf MP was determined using a Thermo Scientific^{Orion 3-star inductivity- measuring device. Before the test, all sample leaves were flushed with deionized water 3 times and residual water on the leaf surface was removed by absorbent paper.}

Chl was estimated according to the method described by Arnon [68] and Zhang et al [69] in detail. Three grams of fresh leaf material was crushed with a mortar and extracted with 10 mL of 80% acetone for 15 min. The extracted solution was then centrifuged at 2500 rpm (F = 34.9 g) for 3 min and measured at wavelengths of 643 nm, 645 nm, and 663 nm via a spectrophotometer (Metash^{UV-6100A). Calculations were performed via the formulas below.}

Chlorophyll a (Chla, mg \cdot L^{- 1}) = 12.7 A_{643} - 2.69 A_{645}

Chlorophyll b (Chl b, mg \cdot L^{- 1}) = 22.9 A_{645} - 4.68 A_{663}

The total chlorophyll of the solution (C_{T}, mg \cdot L^{- 1}) = Chl a + Chl b

Chlorophyll concentration (Chl, mg \cdot g^{- 1}) = C_{T} * V / W / 1000

where Chl a and Chl b refers to the concentration of chlorophyll a and chlorophyll b of the extracted solution; A₆₄₃, A₆₄₅ and A₆₆₃ refer to the absorbance of the measured solution at wavelengths of 643 nm, 645 nm and 663 nm, respectively; C_T (mg/L) is the total chlorophyll of the solution; V represents the total volume of the extracted solution (mL); and W is the weight of the extracted leaf (g). In the final result, Chl (mg·g^{) refers to the chlorophyll content contained within each gram of leaf sample.}

Crude enzyme extracts from the leaves were used to measure the SOD, POD, and APX activity. Approximately 0.5 g fresh leaves was added with a slight amount of CaCO₃, high-purity quartz sand and 5 mL of phosphate buffer (0.05 mol·L^{) and then crushed into a powder in a mortar under freezing conditions. The mixture was subsequently transferred to a 10 mL centrifuge tube and then diluted with deionized water to 10 mL. The samples were then centrifuged at high speed (F = 13000 g) for 20 min at 0~4°C [}67].

The SOD and POD reaction systems were established as described by Zhang et al [67], and the APX reaction system was described by Tang et al [70] as shown below:

Two copies of the reaction system solution for each leaf sample were configured following Table 2-SOD mentioned above, one of which was put into a test tube and illuminated with 4000 lx for approximately 20~30 min at room temperature, the other one was put into the check tube wrapped in aluminum foil to avoid illumination. Once the color of the solution transition started, the reaction was immediately stopped. The final solution absorbance value was determined with a Metash^{UV-6100A spectrophotometer at a wavelength of 560 nm.}

SOD activity (U \cdot {mg}^{- 1}) = \frac{(A_{0} - A_{S}) \times V_{T}}{A_{0} \times 0.5 W \times V_{1}} \times d i l u t i o n r a t i o

where A₀ is the absorbance of the check tube solution; As is the absorbance of the test tube solution; V_T (mL) is the total volume of the samples; V₁ (mL) is the volume of the reaction system; and W (g) is the weight of the fresh leaves.

Table 2

Solution for the SOD, POD, and APX reaction systems.

Protective enzyme	Solution	mL	Final concentration
SOD	Phosphate buffer (0.05 mol·L⁾	1.5	-
	Met solution (130 mmol·L⁾	0.3	13.0 mmol·L^-1
	Nitro blue tetrazolium solution (750 μmol·L⁾	0.3	75.0 μmol·L^-1
	EDTA-Na₂ solution (100 μmol·L⁾	0.3	10.0 μmol·L^-1
	Riboflavin solution (20 μmol·L⁾	0.3	2.0 μmol·L^-1
	Crude enzyme	0.1	Illumination check replaced with phosphate buffer
	Distilled water	0.5	-
	Total volume	3.3	-
POD	Phosphate buffer (0.05 mol·L⁾	2.9	-
	H₂O₂ (2%)	0.5	-
	2-Hydroxyanisole solution (2%)	0.1	-
	Crude enzyme	0.1	-
APX	Phosphate buffer (pH = 7.0, 0.05 mol·L⁾	1.8	-
	Ascorbic acid solution	0.1	-
	H₂O₂ (0.3 mmol·L⁾	1.0	-
	Crude enzyme	0.1	-

All the components were put into a test tube, and then the components of the POD reaction system were added (Table 2-POD). The solution’s light absorption value was recorded for each tube (the wavelength was maintained at 470 nm). The solution was read every 1 min, and each solution was recorded 5 times in 5 min.

POD activity (U \cdot g^{- 1} \cdot {min}^{- 1}) = \frac{Δ A_{470} \times V_{T}}{W \times V_{s} \times 0.01 \times t}

where ΔA₄₇₀ is the change in absorbance during the reaction period, W (g) is the weight of the sample, t (min) is the reaction time, Vs (mL) is the volume of the reaction system, and V_T (mL) is the total volume of the sample.

With respect to the APX reaction system (Table 2-APX), the mixture was put into a test tube, after which the light absorption values were recorded at 290 nm every minute; this step was repeated 5 times. The formula to calculate the APX activity was as follows:

APX activity (U \cdot {min}^{- 1} \cdot g^{- 1} FW) = \frac{Δ A_{290} \times V_{1}}{0.01 \times V_{2} \times t \times W}

where ΔA₂₉₀ is the change in absorbance during 5 min, V₁ (mL) is the volume of the crude enzyme, V₂ (mL) is the volume of the crude enzyme involved the reaction (0.1 mL in this test), t (min) is the reaction time (5 min in this test) and W (g) is the weight of the fresh leaves. FW is short for fresh weight.

3.7 Data analysis

SPSS 17.0 and Excel 2010 for Windows were used to calculate the mean, SD, etc. Multiple comparisons of the means by the least significant difference (Tukey’s honestly significant difference [HSD]) test were performed on the 5 parameters (MP, Chl, SOD activity, POD activity, and APX activity) under the two drought stress treatments and the control group. ANOVA was used to determine significant differences between 10 species and between three treatments (P<0.05).

Following a drought resistance assessment method [71] for plant species based on the membership function value in fuzzy mathematics was used, the MP, Chl, SOD activity, POD activity, and APX activity results can be integrated into a single value for each species. The membership function value was calculated as follows:

{\hat{X}}_{ij} = \frac{X_{ij} - X_{imin}}{X_{i max} - X_{i min}}

(1)

{\hat{X}}_{ij} = 1 - \frac{X_{ij} - X_{imin}}{X_{i max} - X_{i min}}

(2)

where the lowercase “i” and “j” represent the plant species and the parameter type, respectively; “ ${\hat{X}}_{i j}$ ” is the mean value of the parameter “j” of the species “i”; “X_imax” and “X_imin” represent the maximum and minimum of the parameter “j” of the “i” species; and “ ${\hat{X}}_{i j}$ ” is the membership function value and represents the drought resistance of the seedlings. The average of the membership function value was then applied to estimate the adaptive capability of the plants under drought stress. After calculation according to formula (1) or (2), only positive “ ${\hat{X}}_{i j}$ ” value was chosen as the result. The formula for the average was as follows (where “n” represents the number of parameters, and “ ${\bar{X}}_{i}$ ” represents the average “ ${\hat{X}}_{i j}$ ”):

{\bar{X}}_{i} = \sum {\hat{X}}_{i j} / n

3.1 Seedlings

Table 1

Plant species and their propagation modes together with other basic features.

Latin name	Propagation mode	Life cycle	Family	Species characteristics
Paeonia lactiflora	division of suckers	perennial	Ranunculaceae	Popular in gardening, and roots used as traditional Chinese medicine
Hemerocallis dumortieri	sowing of seeds	perennial	Liliaceae	Native to Northeast China, North Korea, Japan and Russia
Meehania urticifolia	sowing of seeds	annual or perennial	Lamiaceae	Adapted to dark and moist environments
Iris lactea var. chinensis	sowing of seeds	perennial	Iridaceae	Tolerant to saline-alkaline conditions and presents a well-developed root system
Hylotelephium erythrostictum	cuttings of seeds	perennial	Crassulaceae	Traditional Chinese medicine
Sedum lineare	sowing of seeds	perennial	Crassulaceae	Traditional Chinese medicine
Iris germanica	rhizome cuttings	perennial	Iridaceae	Native to Europe
Cosmos bipinnata	sowing seedling	annual or perennial	Asteraceae	Native to Mexico
Hosta plantaginea	division of suckers	perennial	Liliaceae	Traditional Chinese medicine
Dianthus barbatus	sowing of seeds	perennial	Caryophyllaceae	Native to Europe

3.2 Field soil collection and preparation

3.3 Plant transplanting

3.4 Drought stress treatment design

3.5 Leaf sampling

3.6 Determining the MP, Chl, SOD activity, POD activity, and APX activity

Chlorophyll a (Chla, mg \cdot L^{- 1}) = 12.7 A_{643} - 2.69 A_{645}

Chlorophyll b (Chl b, mg \cdot L^{- 1}) = 22.9 A_{645} - 4.68 A_{663}

The total chlorophyll of the solution (C_{T}, mg \cdot L^{- 1}) = Chl a + Chl b

Chlorophyll concentration (Chl, mg \cdot g^{- 1}) = C_{T} * V / W / 1000

The SOD and POD reaction systems were established as described by Zhang et al [67], and the APX reaction system was described by Tang et al [70] as shown below:

SOD activity (U \cdot {mg}^{- 1}) = \frac{(A_{0} - A_{S}) \times V_{T}}{A_{0} \times 0.5 W \times V_{1}} \times d i l u t i o n r a t i o

Table 2

Solution for the SOD, POD, and APX reaction systems.

Protective enzyme	Solution	mL	Final concentration
SOD	Phosphate buffer (0.05 mol·L⁾	1.5	-
	Met solution (130 mmol·L⁾	0.3	13.0 mmol·L^-1
	Nitro blue tetrazolium solution (750 μmol·L⁾	0.3	75.0 μmol·L^-1
	EDTA-Na₂ solution (100 μmol·L⁾	0.3	10.0 μmol·L^-1
	Riboflavin solution (20 μmol·L⁾	0.3	2.0 μmol·L^-1
	Crude enzyme	0.1	Illumination check replaced with phosphate buffer
	Distilled water	0.5	-
	Total volume	3.3	-
POD	Phosphate buffer (0.05 mol·L⁾	2.9	-
	H₂O₂ (2%)	0.5	-
	2-Hydroxyanisole solution (2%)	0.1	-
	Crude enzyme	0.1	-
APX	Phosphate buffer (pH = 7.0, 0.05 mol·L⁾	1.8	-
	Ascorbic acid solution	0.1	-
	H₂O₂ (0.3 mmol·L⁾	1.0	-
	Crude enzyme	0.1	-

POD activity (U \cdot g^{- 1} \cdot {min}^{- 1}) = \frac{Δ A_{470} \times V_{T}}{W \times V_{s} \times 0.01 \times t}

APX activity (U \cdot {min}^{- 1} \cdot g^{- 1} FW) = \frac{Δ A_{290} \times V_{1}}{0.01 \times V_{2} \times t \times W}

3.7 Data analysis

{\hat{X}}_{ij} = \frac{X_{ij} - X_{imin}}{X_{i max} - X_{i min}}

(1)

{\hat{X}}_{ij} = 1 - \frac{X_{ij} - X_{imin}}{X_{i max} - X_{i min}}

(2)

{\bar{X}}_{i} = \sum {\hat{X}}_{i j} / n

4 Results and discussion

4.1 Soil relative water content

The soil relative water content between the three drought stress levels are significantly different, with the average of 74.8~85.3% for the well-watered control group, 38.3~45.2% for the moderate drought level, and 15.4~24.3% for the severe drought level (Table 3). These findings were consistent with the designed levels.

Table 3

Soil relative water content.

Plant species	Well-watered group (%)		Moderate stress group (%)		Severe stress group (%)
Plant species	27th, April 2015^*	29th, April 2015^**	2ed, May 2015^*	4th, May 2015^**	7th, May 2015^*	9th, May 2015^**
P. lactiflora	80.1±2%	75.8±1%	45.2±4%	42.1±2%	20.3±2%	18.2±2%
He. dumortieri	82.3±4%	76.8±2%	41.6±2%	40.7±1%	17.6±4%	15.5±3%
M. urticifolia	78.8±5%	75.2±1%	41.9±3%	40.5±2%	17.2±2%	15.4±2%
I. lactea	85.3±1%	80.2±3%	42.5±3%	41.3±3%	21.3±3%	19.7±1%
He. dumortieri	82.9±2%	79.3±2%	40.8±3%	49.8±4%	21.1±2%	18.6±2%
S. lineare	78.4±1%	76.1±2%	43.4±1%	40.2±3%	24.3±3%	22.4±3%
I. germanica	85.3±2%	78.5±2%	44.7±2%	41.4±3%	22.4±2%	20.2±3%
C. bipinnata	77.1±2%	74.8±3%	40.1±3%	39.2±2%	20.7±4%	17.8±4%
Ho. plantaginea	83.8±3%	78.6±2%	42.8±2%	38.3±2%	23.4±3%	22.3±5%
D. barbatus	82.3±4%	79.0±4%	40.3±1%	39.6±2%	24.3±1%	22.5±2%

The values represent the mean ± SD (n = 30).

* is the day when the water content of the soil achieved the designated level.

** is the sampling day.

4.2 Membrane permeability

Plant cells dehydrate when they suffer drought stress, which leads to mechanical damage to the membranes [72]. Greater MP values led to more cytosolic exosmosis and further damage to the plant cellular structure. However, it was hard to distinguish which species had stronger or weaker drought resistance when they were under the well-watered control because they had not been affected by drought yet. In this study, C. bipinnata and D. barbatus presented significantly higher MP values than the other species (Fig 1), followed by the MP value of M. urticifolia and Hy. Erythrostictum, for which the MP value was significantly different relative to the remaining species. On the contrary, Liliaceae and Iridaceae family species presented low MP values under the well-watered control. The change of MP values implies the different physiological characteristics of various plants.

An external file that holds a picture, illustration, etc.
Object name is pone.0220598.g001.jpg

Fig 1

Variance of the membrane permeability for the ten species under drought stress.

The histogram shows the mean values. Above the histogram, the lowercase letters before the commas indicate statistical significance among the different plant species, and those after the commas indicate statistical significance among the well-watered, moderate and severe drought stress treatments. The different lowercase letters indicate a significant difference at P<0.05.

With the drought stress treatment, the above four plant species with higher MP values expressed different tolerance features. C. bipinnata did not survive under severe drought stress, and M. urticifolia did not survive under moderate or severe drought stress. Both species are annual herbs, and their root growth is strongly inhibited by the lack of water [73,74]. Although leaf sampling occurred only ten days after transplanting, the plant roots were transplanted with the original moist rooting medium and the seedlings were shaded and fully watered, which was beneficial for root development. Previous studies identified a strong relationship between new leaf germination and plant survival rate [75]. In addition, leaf biomass was positively correlated with root biomass, implying that root length developed when the plants grew new leaves [76]. In this study, plants under the well-watered control have all grown new leaves and even buds at sampling, thus demonstrating that the root has developed. Leaf sampling could be conducted in 3 days, 5 days, 10 days, etc. once the soil RWC matched the designed drought levels [77–79]. Therefore, the withering of C. bipinnata and M. urticifolia was induced by drought stress instead of a short growth period. The MP values of D. barbatus did not change significantly.

Under drought stress, most species showed significantly increased MP values, including P. lactiflora, He. dumortieri, I. lactea, S. lineare, I. germanica, and Ho. plantaginea, among of which P. lactiflora showed significantly increased MP values only under severe drought stress (Fig 1). The MP value of I. germanica increased the most by 36% and 56% under moderate and severe conditions, respectively, and that of S. lineare increased by 31% and 55%, respectively. These findings indicated that these species' membranes were damaged under drought stress.

Among all the plant species, only two, Hy. erythrostictum and D. barbatus, did not show significantly changed MP values under the drought stress treatment. Hy. erythrostictum was considered a kind of xerophilous plant with fleshy leaves [80], and the MP values were reduced at the early stage of drought stress [81]. Although fewer investigations have been performed on the effects of drought in D. barbatus, especially on its MP, the permeation regulation synchronized with damage to the protoplast membrane in Dianthus plumarius, another Caryophyllaceae plant [82]. Although both S. lineare and Hy. erythrostictum belong to the Crassulaceae family, their MP value variation trend was the opposite. The MP values of S. lineare were also significantly greater than that of Hy. erythrostictum under drought stress [83].

Plant drought resistance is closely related to its cell membrane system stability [31]. Usually, the cell membrane is first affected by drought stress [84], and then the cell structure is damaged and MP increases, which leads to the extravasation of extracellular electrolytes, which is why MP values increase when plants are subjected to drought stress. The stability of the MP values of these two species indicated that their cell membrane was undamaged. Therefore, the osmotic adjustment ability of multicolored carnation leaves is strong enough to avoid damage to the protoplast membrane under drought stress treatment.

4.3 Chlorophyll concentration

In the well-watered control, the studied plant species presented Chl concentrations from 6.06 to 47.69 mg·g^{FW, and the fleshy Crassulaceae species}H. erythrosticum and S. lineare presented the lowest Chl concentrations (Fig 2). The plants' Chl concentrations were affected by light intensity and environmental temperature, which affect the opening and closing of stomata and photosynthetic rates of plant leaves and then affect the accumulation of carbohydrates, which is consistent with the Chl concentration of plants [85]. In the study, all plants grew in a stable light intensity environment because the experimental area had a roof, which can prevent the effects of strong sunshine or rain. As for the ambient temperature, no extreme temperatures were encountered during the experiment. The leaves of these plants were collected at the same time after drought stress under the same environmental temperature. Thus, the changes in Chl concentration should be caused by drought stress instead of temperature or sunlight.

An external file that holds a picture, illustration, etc.
Object name is pone.0220598.g002.jpg

Fig 2

Variance of the total chlorophyll concentration in the ten species during the drought stress tests.

The histogram shows the mean values. Above the histogram, the lowercase letters before the commas indicate statistical significance among the different plant species, and the lowercase letters after the commas indicate statistical significance among the well-watered, moderate and severe drought stress treatments. The different lowercase letters indicate a significant difference at P<0.05.

Upon drought stress, change trends of plant Chl concentration were various. Four species showed significantly (P<0.05) increased Chl concentrations under the different extents of drought stress, including P. lactiflora, Hy. erythrostictum, S. lineare and Ho. plantaginea (Fig 2). The increased Chl concentration under moderate or severe drought stress might be due to the increase of the stem cell mass and cell number of the leaves, thus forming a Chl condensation phenomenon, as in P. lactiflora [86]. Additionally, Liu et al [87] reported that the Chl a and b of Hy. erythrostictum would be increased during the day but decreased during the night under drought stress, which probably indicates more photosynthetic pigments were produced to promote photosynthesis of Hy. erythrostictum under drought stress. However, the photosynthetic pigment content was decreased at night for maintaining its normal physiological activities.

The species I. germanica and I. lacteal showed increased Chl concentrations under moderate stress, and then these values decreased under severe drought stress (Fig 2). Most Iridaceae plants are shade plants [88], some of which feature colorless leaves [89] and possess lower Chl concentrations than sun plants leaves [90]. Zhou [91] researched seven Iris species and also found that I. germanica had a higher Chl concentration in the early stage of drought stress than the control group.

D. barbatus did not change the Chl concentration under drought stress treatment (Fig 2), which indicated that the species had a strong self-repair and regulate ability during drought stress, and its leaves had a relatively good physiological and biochemical state, which could maintain normal photosynthesis and strong resistance during drought.

Only one species, He. Dumortieri, significantly decreased the Chl concentration with the increase of drought stress (Fig 2), which indicated that chlorophyll synthesis was interrupted and the chlorophyll decomposed under drought.

4.4 Superoxide dismutase activity

SOD activity is very sensitive to drought [92]. In this study, the SOD activity of six species, He. dumortieri, I. lactea, Hy. erythrostictum, I. germanica, Ho. plantaginea, and D. barbatus, initially significantly (P<0.05) decreased under moderate drought stress but increased under severe drought stress (Fig 3). The reduction of SOD activity under the moderate condition implied that a considerable amount of ROS was produced to damage plant cells and tissues, thus leading the plant cells to undergo oxidative damage. The activity of the enzyme SOD is influenced by the concentration of the O₂^{substrate. Stress raises the production of O}₂^{, thus increasing the SOD activity [}93]. A previous investigation indicated that the increased SOD activity under severe drought was caused by the drought exercise under moderate drought stress [87]. The drought exercise was applied to enhance the resistance of rice to high temperatures [94] and the resistance of wheat to drought stress [95].

An external file that holds a picture, illustration, etc.
Object name is pone.0220598.g003.jpg

Fig 3

Variance of superoxide dismutase activity in the ten species during the drought stress tests.

The change trends of SOD activity can be adopted to judge the species’ drought resistance. Those species with higher SOD activity under drought can be considered to have strong drought resistance [96]. The SOD activity of P. lactiflora and S. lineare increased under either the moderate or severe drought stress. The difference of SOD activity might be caused by the expression of various isozymes, which induced the accumulation of the antioxidant substance in plants leaves that started up the antioxidant protection system when plants were under moderate and severe drought stress [97]. The degrees of SOD increase of S. lineare were higher than that of P. lactiflora, which is consistent with a previous study [98] in which S. lineare had stronger SOD activity.

Overall, in this study, the SOD activity in most species increased after severe drought stress, which suggested that drought stress-induced SOD activity increases in these plant species to help them eliminate ROS. These plant species presenting increased SOD activity showed advantages in terms of their drought stress response, and proper drought exposure could significantly improve plant resistance to sustained drought stress [95].

4.5 Peroxidase activity

In the study, all plant species showed decreased POD activity under the moderate or severe drought stress, with most showing declining POD activity as the drought stress increased (Fig 4). Compared with the well-watered control, the POD activity of He. dumortieri, Hy. erythrostictum, S. lineare, I. germanica, Ho. plantaginea and D. barbatus was reduced significantly (P<0.05) during the moderate drought stress. The POD activity of these species was significantly (P<0.05) reduced under the severe drought stress (Fig 4). Such declining trends of POD activity with drought stress were contrary to the increased SOD activity trends.

An external file that holds a picture, illustration, etc.
Object name is pone.0220598.g004.jpg

Fig 4

Variance of peroxidase activity of the ten species under the drought stress tests.

Under drought stress, stronger POD activity might be attributed to the plant defense mechanisms against free radical formation resulting from water deficit [99]. According to Fig 4, the POD activity of I. lacteal was reduced under both the moderate and severe drought stress and was significantly (P<0.05) higher than that of the species that survived under severe drought stress except for He. dumortieri and Ho. plantaginea. This finding may indicate the species that have stronger resistance to drought.

Under severe drought stress, P. lactiflora, S. lineare, I. germanica and D. barbatu showed significantly lower POD activity values compared with the other species and other water conditions (Fig 4), which may be related to the POD enzyme reaching its tolerance limit and decreasing rapidly.

In general, the POD activity of all the species tended to decrease when the seedlings experienced drought stress. As a special enzyme to eliminate H₂O₂, the reduced POD activity in this study might have been caused by ROS elimination because different protective enzymes work together as a whole, with the elimination of O₂^{SOD increasing H}₂O₂ production. However, a very high concentration of H₂O₂ was beyond the reach of POD activity [100], which indicates that although SOD and POD are both antioxidases and can cooperate in scavenging ROS, drought stress might lead to a different enzyme system to resist adverse drought environments.

4.6 Ascorbate peroxidase activity

Under well-watered conditions, P. lactiflora presented the highest APX activity value at 0.98 U·min^·g^{FW, although the other species had APX activity from 0.09 to 0.26 U}·min^·g^{FW. Seven species showed increased APX activity, with}P. lactiflora, He. dumortieri, Hy. erythrostictum, I. germanica, Ho. plantaginea, and D. barbatus presenting significantly higher APX values under moderate drought than under well-watered and severe drought stress conditions (P<0.05). However, the APX activity of S. lineare decreased significantly (P<0.05) under moderate and severe drought stress. At severe stress, most plant species reduced their APX value no more than 0.50 U·min^·g^{FW, with the lowest at 0.07 U·min}^·g^{FW for}Ho. plantaginea (Fig 5).

An external file that holds a picture, illustration, etc.
Object name is pone.0220598.g005.jpg

Fig 5

Variance of ascorbate peroxidase activity of the ten species under the drought stress tests.

An interesting phenomenon in this study was that the SOD and APX activity of some plants seemed to complement each other. The SOD activity of He. dumortieri, I. lactea, Hy. erythrostictum, I. germanica, Ho. plantaginea, and D. barbatus decreased under moderate drought stress and increased under severe stress (Fig 3), whereas the APX activity displayed the opposite pattern. Moreover, the highest values of APX activity in these species were recorded in the moderate drought stress treatment, which may suggest that APX activity was first activated by the early or moderate drought stress to scavenge ROS. When the SOD activity increased under severe drought stress, the APX activity decreased at the same time, which suggests that SOD plays a dominant role in ROS scavenging during severe drought stress. The weakening of APX activity under severe drought stress indicates that its antioxidant capability is temporary and limited [101].

4.7 Assessment of plant drought resistance

We determined the drought resistance of ten ground cover species through six physiological indicators: MP, Chl, SOD activity, POD activity, and APX activity. However, it is difficult to judge which plant species has better drought resistance only based on individual parameters. Therefore, it is reasonable to use the membership function method that applies fuzzy mathematics to weigh these indicators and ultimately assess the drought resistance of the ten species. The calculated results following the formula of the membership function are shown in Table 4. The findings indicate that He. dumortieri was the most drought resistant species while C. bipinnata and M. urticifolia were not suitable for moderate or severe drought stress due to withering. In addition, P. lactiflora survived the weakest drought resistance. The order of plant resistance to drought stress was as follows: He. dumortieri > I. germanica > I. lactea > D. barbatus > Hy. erythrostictum > S. lineare > Ho. plantaginea > P. lactiflora > M. urticifolia > C. bipinnata (Table 4).

Table 4

Drought resistance under the drought stress test.

Plant species	MP	Chl	SOD	POD	APX	${\bar{X}}_{i}$	Drought resistance rank
P. lactiflora	0.34	0.45	0.39	0.46	0.37	0.4021	8
He. dumortieri	0.60	0.60	0.66	0.37	0.34	0.5140	1
M. urticifolia	-	-	-	-	-	Non available	9
I. lactea	0.46	0.44	0.62	0.35	0.52	0.4772	3
Hy. erythrostictum	0.42	0.50	0.60	0.36	0.42	0.4608	5
S. lineare	0.52	0.46	0.42	0.35	0.52	0.4542	6
I. germanica	0.55	0.47	0.60	0.42	0.36	0.4795	2
C. bipinnata	-	-	-	-	-	Non available	10
Ho. plantaginea	0.53	0.36	0.65	0.34	0.35	0.4460	7
D. barbatus	0.33	0.49	0.63	0.52	0.35	0.4640	4

${\bar{X}}_{i}$ refers to the Membership function value.

Higher ${\bar{X}}_{i}$ value is, stronger plant drought resistance is.

4.1 Soil relative water content

Table 3

Soil relative water content.

Plant species	Well-watered group (%)		Moderate stress group (%)		Severe stress group (%)
Plant species	27th, April 2015^*	29th, April 2015^**	2ed, May 2015^*	4th, May 2015^**	7th, May 2015^*	9th, May 2015^**
P. lactiflora	80.1±2%	75.8±1%	45.2±4%	42.1±2%	20.3±2%	18.2±2%
He. dumortieri	82.3±4%	76.8±2%	41.6±2%	40.7±1%	17.6±4%	15.5±3%
M. urticifolia	78.8±5%	75.2±1%	41.9±3%	40.5±2%	17.2±2%	15.4±2%
I. lactea	85.3±1%	80.2±3%	42.5±3%	41.3±3%	21.3±3%	19.7±1%
He. dumortieri	82.9±2%	79.3±2%	40.8±3%	49.8±4%	21.1±2%	18.6±2%
S. lineare	78.4±1%	76.1±2%	43.4±1%	40.2±3%	24.3±3%	22.4±3%
I. germanica	85.3±2%	78.5±2%	44.7±2%	41.4±3%	22.4±2%	20.2±3%
C. bipinnata	77.1±2%	74.8±3%	40.1±3%	39.2±2%	20.7±4%	17.8±4%
Ho. plantaginea	83.8±3%	78.6±2%	42.8±2%	38.3±2%	23.4±3%	22.3±5%
D. barbatus	82.3±4%	79.0±4%	40.3±1%	39.6±2%	24.3±1%	22.5±2%

The values represent the mean ± SD (n = 30).

* is the day when the water content of the soil achieved the designated level.

** is the sampling day.

4.2 Membrane permeability

Fig 1

Variance of the membrane permeability for the ten species under drought stress.

4.3 Chlorophyll concentration

Fig 2

Variance of the total chlorophyll concentration in the ten species during the drought stress tests.

4.4 Superoxide dismutase activity

Fig 3

Variance of superoxide dismutase activity in the ten species during the drought stress tests.

4.5 Peroxidase activity

Fig 4

Variance of peroxidase activity of the ten species under the drought stress tests.

4.6 Ascorbate peroxidase activity

Fig 5

Variance of ascorbate peroxidase activity of the ten species under the drought stress tests.

4.7 Assessment of plant drought resistance

Table 4

Drought resistance under the drought stress test.

Plant species	MP	Chl	SOD	POD	APX	${\bar{X}}_{i}$	Drought resistance rank
P. lactiflora	0.34	0.45	0.39	0.46	0.37	0.4021	8
He. dumortieri	0.60	0.60	0.66	0.37	0.34	0.5140	1
M. urticifolia	-	-	-	-	-	Non available	9
I. lactea	0.46	0.44	0.62	0.35	0.52	0.4772	3
Hy. erythrostictum	0.42	0.50	0.60	0.36	0.42	0.4608	5
S. lineare	0.52	0.46	0.42	0.35	0.52	0.4542	6
I. germanica	0.55	0.47	0.60	0.42	0.36	0.4795	2
C. bipinnata	-	-	-	-	-	Non available	10
Ho. plantaginea	0.53	0.36	0.65	0.34	0.35	0.4460	7
D. barbatus	0.33	0.49	0.63	0.52	0.35	0.4640	4

${\bar{X}}_{i}$ refers to the Membership function value.

Higher ${\bar{X}}_{i}$ value is, stronger plant drought resistance is.

5 Conclusions

This study investigated how ten common plant species were tolerant to levels of drought stress and showed that drought stress disrupted plant growth because the same conditions were not observed under the well-watered treatment. Five parameters (MP, Chl, SOD, POD, and APX activity) changed under moderate and severe drought stress. The main results are as follows.

First, C. bipinnata and M. urticifolia failed to survive the drought stress and were not suitable for both moderate and severe drought stress.

Second, each plant species had quite different physiological and biochemical parameters. He. dumortieri, I. lactea, and Ho. plantaginea maintained a stable MP value after experiencing drought stress. Most species (except P. lactiflora and S. lineare) showed reduced SOD activity under moderate drought stress but increased activity under severe drought stress. However, the plant species showed decreased POD activity and APX activity when the drought stresses increased.

Third, complementary relationships might occur among SOD, POD and APX activity, and SOD may play a dominant role in scavenging ROS under severe drought stress while APX and POD are responsible under moderate drought stress.

Finally, C. bipinnata and M. urticifolia were very sensitive to drought stress and thus are unfit for roof greening, especially in arid regions. However, He. dumortieri, I. germanica, I. lactea, D. barbatus, Hy. erythrostictum, S. lineare, Ho. plantaginea, and P. lactiflora could be applied as roof greening in Beijing and other northern Chinese cities.

Supporting information

S1 Data

(XLS)

Click here for additional data file.^{(35K, xls)}

^{Beijing Biodiversity Conservation Research Center, Beijing, China}

^{Beijing Gardening and Greening Bureau, Beijing, China}

^{Beijing Center for Physical and Chemical Analysis, Beijing, China}

Department of Agronomy, University of Agriculture, Faisalabad, PAKISTAN

^{Contributed equally.}

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: moc.liamtoh@ujnayuil

Received 2019 Jul 31; Accepted 2020 Jun 2.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Abstract

Roof greening is an important national policy for maintaining the hydrological balance in China; however, plant growth is limited by drought stress. This study aims to identify strong drought resistant plant species for roof greening from ten common species: Paeonia lactiflora, Hemerocallis dumortieri, Meehania urticifolia, Iris lactea var. chinensis, Hylotelephium erythrostictum, Sedum lineare, Iris germanica, Cosmos bipinnata, Hosta plantaginea, and Dianthus barbatus. By controlling the soil relative water content (RWC), we designed three treatments: moderate drought stress (40±2% < RWC < 45±2%), severe drought stress (RWC < 30±2%) and well-watered control (RWC > 75±2%). After the seedlings were provided different levels of water, their membrane permeability (MP), chlorophyll concentration (Chl), and superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) activity were measured. Finally, the membership function method was used to assess the drought resistance of these species. The results showed that C. bipinnata and M. urticifolia were not suitable for moderate or severe drought stress and did not survive. The other species presented variations in physiological and biochemical parameters. The MP of He. dumortieri, I. lactea and Ho. plantaginea showed minor changes between the well-watered control and drought stress. Most of the species showed reduced SOD activity under moderate drought stress but increased activity under severe stress. All of the plant species showed decreases in the protective enzymes POD and APX with increasing drought stress. The membership function method was applied to calculate the plant species' drought resistance, and the following order of priority of the roof-greening plant species was suggested: He. dumortieri > I. germanica > I. lactea > D. barbatus > Hy. erythrostictum > S. lineare > Ho. plantaginea > P. lactiflora.

Abstract

Click here for additional data file.^{(35K, xls)}

Acknowledgments

The authors thank the anonymous reviewers and editor Saddam Hussain for valuable comments, and Fen Qin (a business development manager for the China office from Power Systems Research) for helping with the manuscript preparation.

Acknowledgments

Funding Statement

This work was supported by the Young Core Plan of the Beijing Academy of Science and Technology (BJAST) (No. 201528), the Beijing Natural Science Foundation (No. 8142017) and the National Natural Science Foundation of China (No. 41475133).

Funding Statement

Data Availability

All relevant data are within the paper and its supporting information files.

Data Availability

20 Aug 2019

PONE-D-19-20236

Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening

PLOS ONE

Dear Dr. Liu,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

PLOS ONE requires that all submitted research be written in clear, correct, unambiguous English (https://journals.plos.org/plosone/s/criteria-for-publication#loc-5). In this case, we feel that your manuscript does not meet this criterion. At this time, we would therefore ask that you have your manuscript thoroughly copyedited for spelling and grammar.

We would appreciate receiving your revised manuscript by Oct 04 2019 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out.

We look forward to receiving your revised manuscript.

Kind regards,

Dr Jamie Males

Associate Editor

PLOS ONE

on behalf of

Dorin Gupta, Ph.D.

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments (if provided):

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files to be viewed.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email us at gro.solp@serugif. Please note that Supporting Information files do not need this step.

18 Sep 2019

Dear, editor,

I appreciate your attention to the manuscript.

We have edited the manuscript very carefully in this version:

Firstly, we revised the grammar and spelling of the manuscript completely that included tense, voice, syntactical structure, and some spelling mistakes.

Secondly, we deleted some words that had nothing to do with the manuscript itself.

Kind regards,

Dr. Yanju-Liu

Attachment

Submitted filename: 4.Response to Reviewers.docx

Click here for additional data file.^{(15K, docx)}

7 Jan 2020

PONE-D-19-20236R1

Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening

PLOS ONE

Dear Dr. Liu,

We would appreciate receiving your revised manuscript by Feb 21 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Saddam Hussain

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

After careful consideration of myself and based on the evaluation of external reviewers, I feel that the manuscript is interesting, but is not suitable for publication as it currently stands. A "Major Revision" is required as the comments of reviewers. I am in the view of reviewer 1, that the authors provided a large set of data, but failed to provide any justification or explanation of results, or why they found inconsistent patterns in the data. Results and Discussion section should be thoroughly improved. Present your key findings in the conclusion section. Change the description of treatments as well-watered’, ‘moderate’ and ‘severe’ drought. Title should also be improved. Replace number (10) with text (ten). Authors should also carefully improve the language of the draft, to omit the minor grammatical and typo mistakes.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: This paper assesses the suitability of 10 species for use on green roofs according to their drought tolerance. Drought tolerance was quantified using a number of enzymes extracted from leaves after exposure to three treatments modifying water availability. A composite drought tolerance index was then used to rank species according to their suitability for use on green roofs.

Overall, the paper is well-written, and the methods are largely sound. I find, however, that the results and discussion do not do a good job of putting the results in the context of the literature. What I mean is, for each response variable measured (i.e., for each subsection of the results/discussion), a paragraph is first given which describes how to effectively interpret the response variable and what conditions affect the variable in question. Take Section 4.2 for example. The authors explain what a high/low value of MP means and what can affect it --- in very general terms. Then, the remaining paragraphs are equivalent to just ‘results’ --- that is, the next three paragraphs describe the trends and statistical differences among species in relation to watering treatment, but do not attempt to explain or discuss what they have observed in the context of literature. Further, the implications of the findings are not expressed, and no conclusions are presented. All we have is a description of the results, but no further explanation. For example --- why have certain species been less affected by the drought with regard to membrane permeability? What do we know about these species, their adaptations, where they come from --- that is, are the results you found logical? As the reader, we cannot assess whether your findings are sensible, nor can we walk away from this paper with anything except species rankings, without an explanation of the results. The MP results are clear, compared with SOD. Looking at Figure 3, we can see for most species SOD is high for Cg, low in T1, then high again in T2 --- why is this? As it stands, the species are ranked only within treatment (i.e., all species exposed to Cg are ranked, then ranked within T1, then ranked within T2 --- so three different rankings are produced only within each treatment). What we need to see is your explanation as to why we did not see a clear increase or decrease in SOD in relation to declining water ability --- i.e., the comparison among treatments, within each species. I cannot accept the rankings as they are, because there is no clear trend in SOD in relation to treatment and more importantly, no attempt is made to explain why we see the results as presented in Figure 3. Section 4.6 has great potential to present a synthesis of why the variables contributing to ‘Xi’ respond to watering treatment in the way do --- that is, once the trends in each response variable have been explained in isolation, a more sophisticated discussion of the combined response of all variables is required --- such that we can understand the overall strategy of each species and therefore present a justified species ranking --- that is, a ranking supported by what we know and understand about these species, such that the ranking as presented according to the synthesised variable Xi can be properly assessed. As it is now, we must take your word for it that this ranking is sensible. In my view, this is the major weakness of the paper, as all sections in the results/discussion fail to properly interrogate and explain the trends in the data.

Some criticism of the length of time plants were exposed to their treatments prior to measurement is likely. Plants were only exposed to their treatment soil water content for two days, which is quite short. However, differences in some response variables were observed, however, they were inconsistent at times which may be due to the way drought was imposed. Further justification of the suitability of this type of drought exposure would help. I can see the average soil moisture content values in Table 3, which I presume is the content for the two days of exposure. However, the treatments began when the watering regime was imposed, so I suggest a figure is required to show the decline in soil moisture content over time, to get an understanding of the severity of drought treatments over time, not just in the two days prior to sampling. I am not familiar with how long it takes for the five measured parameters to change in response to drought, so discussion of this is required.

I also suggest that the use of ‘Cg’, ‘T1’ ‘T2’ and are not intuitive labels when interpreting the results and would suggest re-labelling them as something like: ‘well-watered’, ‘moderate’ and ‘severe’ drought treatments which makes it easier for the reader. Similarly, codes for species should be used on the x-axis, as numbering species is not intuitive and makes the reader work hard to find out which species is what. This should be done in the text of the results/discussion, as well as on all tables and figures. Further, log scales on figures such as Figures 4 and 5 may improve their readability, rather than using broken y-axes.

Reviewer #2: Title: Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening from Zhang et al. which was submitted to PloS One has meaningful results and well conducted experiment. The use of different plant species i.e. Paeonia lactiflora, Hemerocallis dumortieri, Physostegia virginiana, Iris lacteal, Hylotelephium erythrostictum, Sedum lineare, Iris germanica, Cosmos bipinnata, Hosta plantaginea and Dianthus barbatus for determining the drought tolerant and drought sensitive species is a novel research. I recommend this article should be publish in PloS One before changing some minor mistakes which are as follow:

On the abstract: You have mentioned Cosmos bipinnata and Physostegia virginiana died while other plant species survived. I recommend you please change the terminology like died is not suitable for the plant you can write in a better way.

On the introduction: The information regarding ROS or oxidative stress is too limited. You should add at least 2-3 sentences about their mechanism, effect and actions of antioxidants in response to oxidative stress. https://doi.org/10.1016/j.ecoenv.2019.109915. doi:10.3390/plants8120545. https://doi.org/10.1007/s11356-019-07264-7. Please see these latest articles which just published recently and find some relevant information about your topic and cites these in introduction.

On Material and Method: You have mentioned “The seedlings were provided by the Yu-quanying flower market, a large and popular market in Beijing” can you provide more details about it (if possible).

You have mentioned “mg•kg-1” Please see above mention articles and see how we can correctly write this unit and write correctly throughout your manuscript.

You have mentioned “Leaves were placed into sealed plastic bags and kept in a portable ice box at 0 ~ 4℃” Scientifically when you are collecting your samples please took in liquid nitrogen. You can taken in the ice box. It’s ok but more convenient to take in liquid nitrogen.

On Results and discussions: Better to write chl a like this: use first time full abbreviation.

“Drought stress causes changes in chlorophyll content in plants” Please modify this sentence.

You have find that “The chlorophyll content of Physostegia virginiana was the highest, at 49.07 mg/g•FW” This is the maximum chlorophyll contents and determined by Arnon’s (1949) method. Are you sure that you determined too much chlorophyll in this plant species. For the best of our knowledge we did not saw too much high chlorophyll contents. Please verify your formula or your method and make sure that chlorophyll is very high? Please see it carefully.

“The chlorophyll content of Hosta plantaginea was the highest, at 59.11 mg/g•FW” Is it?

Please verifily the units of antioxidants you have determined “U•min-1 •g -1 FW” or “U•mg-1” Please make sure that you are going through with right units.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Attachment

Submitted filename: Reviewers Comments.docx

Click here for additional data file.^{(13K, docx)}

25 Feb 2020

Response to Reviewers

First, we are very grateful to the reviewers for their careful work and useful advice, which are truly beneficial for improving the quality of the article. We have accepted the comments of the reviewers and editor and have tried our best to revise the article as needed.

� Response to Reviewer #1’s main comments:

Summary comment: The section of results and discussion required a “Major Revision”.

Summary response: Yes. Thanks for review’s nice suggestions. We have revised the manuscript according to each of the comments as follows.

Comments 1: The explain of MP, Chl, SOD, POD, and AsAPOD(APX) means required greater depth.

Response 1: We looked up a large number of papers and cited 31 of them to elaborate further on the importance of the MP, Chl, SOD activity, POD activity, and APX activity indicators. Not only was the first paragraph of each section (section 4.2 to 4.6) supplemented with explanations of the indicators but also some scientific discussions were interspersed throughout other paragraphs.

Comments 2: The describes of statistical differences among species is not enough, it needs further explain why species have such change under the watering condition.

Response 2: We have strengthened the discussion of each indicator. On the basis of the objective elaboration of the statistical data, an interpretation of the changes in the data has been added.

For instance, in section 4.2, we further explained the cause of death of Cosmos bipinnata and M. urticifolia under drought stress, explained the reason why C. bipinnata presented increased MP values and further explained the why the stability of the various membrane systems of the cells means strong resistance to drought stress.

In section 4.3, we first summarized the general trends of plant Chl before the detailed discussion. We added a paragraph to discuss the changes in different species from the family perspective, which made the discussion are more reasonable.

Similarly, we have made in-depth revisions to section 4.4. We summarized three suggestions: (1) drought stress induced an increase in SOD activity, and by increasing their SOD activity, the abovementioned plant species eliminated ROS; (2) the plant species presenting increased SOD activity showed advantages in terms of their drought stress response; and (3) proper drought exposure could significantly improve plant resistance to sustained drought stress.

We also strengthened the link among the POD, APX, and SOD protective enzymes in the following two sections. We found that there may be a complementary relationship among POD, APX, SOD. On the basis of the change trends of the activity of these three protective enzymes, SOD may play a dominant role in scavenging ROS during severe drought stress (or during the late period of drought stress), and APX and POD may play a dominant role in scavenging ROS during moderate drought stress (or during the early period of drought stress).

In accordance with the reviewer's suggestion, we added details to Table 3 (the moisture content of the soil and the increases in soil RWC on the sampling day).

Comments 3: Figures 4 and 5 may improve their readability, rather than using broken y-axes.

Response 3: We improved the figures in the paper. At first, “Cg”, “T1” and “T2” were used instead of “well-watered”, “moderate” and “severe”, respectively. Secondly, Figures 4 and 5 have been changed into log-scale figures.

� Response to Reviewer #2’s comments:

Comments 1: On the abstract: You have mentioned Cosmos bipinnata and Physostegia virginiana died while other plant species survived. I recommend you please change the terminology like died is not suitable for the plant you can write in a better way.

Response 1: We used the word “is not suitable for” instead of “died”.

Comments 2: On the introduction: The information regarding ROS or oxidative stress is too limited. You should add at least 2-3 sentences about their mechanism, effect and actions of antioxidants in response to oxidative stress.

Response 2: Following reviewer’s suggestion, we not only supplemented some descriptions of ROS but also added many descriptions to other parts of the discussion.

Comments 3: On Material and Method: You have mentioned “The seedlings were provided by the Yu-quanying flower market, a large and popular market in Beijing” can you provide more details about it (if possible).

Response 3: We have added some a specific description of the market.

Comments 4: You have mentioned “mg•kg-1” Please see above mention articles and see how we can correctly write this unit and write correctly throughout your manuscript.

Response 4: We checked the reference of “ZHU Minghao, et al. The impact of Elaphurus davidianus in different habitats on soil physical and chemical properties [J]. Environmental Chemistry, 2016, 35(1): 208-217. DOI:10.7524/j.issn.0254-6108.2016.01.2015070803”. Again, units of “mg•kg-1” were correct.

Comments 5: Better to write chl a like this: use first time full abbreviation.

Response 5: We have replaced “Chl a” by “Chlorophyll a” when it appeared first time.

Attachment

Submitted filename: Response to Reviewers.doc

Click here for additional data file.^{(43K, doc)}

24 Mar 2020

PONE-D-19-20236R2

Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening

PLOS ONE

Dear Dr. Liu,

==============================

ACADEMIC EDITOR: Although, the authors tried to improve the manuscript compared with first draft, but failed to respond/deal with the comments raised by me (editor) and reviewer 1. A major revision and detailed point by point response is required prior to publication. Also respond to the editor's comments raised during revision 1.

Authors should give proper explanation of results, or why they found inconsistent patterns in the data. Results and Discussion section should be thoroughly improved. Present your key findings in the conclusion section. Change the description of treatments as well-watered’, ‘moderate’ and ‘severe’ drought. Title should also be improved. Replace number (10) with text (ten). Authors should also carefully improve the language of the draft, to omit the minor grammatical and typo mistakes.

==============================

We would appreciate receiving your revised manuscript by May 08 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Saddam Hussain

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Although, the authors tried to improve the manuscript compared with first draft, but failed to respond/deal with the comments raised by me (editor) and reviewer 1. A major revision and detailed point by point response is required prior to publication. Also respond to the editor's comments raised during revision 1.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: No

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #1: General

I think that perhaps my comments were not clear on the first revision. The structure of the results/discussion needs to be significantly improved as it does not attempt to explain your results in depth or detail. The first paragraph of each section in the results/discussion is generic and not related to explaining/interpreting your results. Therefore, this information belongs in the introduction where it is required to justify your approach. So, for each section of the discussion, re-locate the first paragraph to the introduction. But please ensure that the introduction is not a ‘cut and paste’ disjointed section as a result, it needs to flow and be logical. The remaining paragraphs in each section of the results/discussion have largely not changed since my first review. For the majority, it seems pointless to rank species within treatment --- these paragraphs are largely unreferenced and the reader is confused by you continually ranking species. Save the ranking for your strongest indicator of tolerance which is the change in each response variable from well-watered to severe drought conditions. That should be the focus and first paragraph of each section. Indeed, there really only needs to be one decent paragraph in each section. Species showing the most dramatic change from well-watered to droughted conditions are clearly the most sensitive, therefore this is the only ranking you require. Do this for each variable, then for the combined variable at the end. Comment then on the relative importance of each variable in section 4.6 --- for instance, does the ranking produced by the membership function relate well to any individual measure, and why? I hope this is clear. I have provided detailed comments for section 4.2 below to help, but please apply this logic throughout your paper. With regard to other general comments, it is very, very important that in a prominent position early in your results and discussion that you comment on the short length of exposure to drought AND (as you state in the first sentence in Section 3.5) that the sampling was done only 10 days after transplant. It is highly likely these two factors affected the outcome of your experiment and people will be highly critical of the short duration of exposure to experimental conditions unless you address it early. For example, sampling only 10 days after planting, it is highly likely that roots did not develop and therefore manipulation of soil moisture is largely irrelevant. The results you observed may simply be transplant shock with such a short timeframe. Comment must be made on these issues if people are to have confidence in the results.

Section 4.1

There is inconsistency between how soil water content is discussed --- e.g. in the methods, it is referred to as relative water content (RWC), then as soil moisture content in the subsection heading for Section 4.1, then as soil water content in the first sentence of that section. Please be consistent with terminology throughout the paper.

Section 4.2

Paragraph 1: is not very good because it does not talk about your data/your study first, which is what the results/discussion is for --- the focus needs to be on your data, using the literature to support. The background about why measuring membrane permeability is useful for quantifying drought tolerance is all very useful information, but it belongs in the introduction --- not the results/discussion. My suggestion is that, given the introduction is so short, and that the results/discussion is so long, these ‘general information’ paragraphs in each section of the results/discussion should be moved to the introduction of the paper. Still on the example of section 4.2, your first paragraph would be the one currently under Figure 1 (the current paragraph 2).

Paragraph 2: In the second paragraph, we are told that MP values under well-watered conditions reflect normal/healthy status. Why then do you rank species by well-watered MP in the next sentence? This is not logical to me --- if you have a healthy, well-watered treatment group, then the MP value is “ideal” --- therefore the only relevant thing to talk about here is how MP changes when you impose drought stress (i.e., the last paragraph in this section). For instance, are well-watered D.barbatus plants less drought tolerant because they have very high MP under well-watered conditions? Not if you consider a well-watered D.barbatus plant to be healthy…What I am saying here is your focus should be on looking at the change within species, from well-watered to droughted conditions to define drought tolerance. There should be only one species ranking based on MP and I would do this by starting with the least tolerant (the species which died), then rank the remainder based on the magnitude of the change in MP between well-watered and droughted. The remaining discussion on who changed from moderate to severe is not required.

Paragraph 3: Similarly, you focus the third paragraph on C. bipinnata --- you suggest that the high MP for well-watered C. bipinnata means that it is not tolerant to adversity. Firstly, “adversity” is not specific, you conducted a drought experiment, so be specific and refer to drought --- but secondly, your opening sentence in paragraph two (which is unreferenced, by the way…and needs to be!) states clearly that MP values under well-watered conditions reflect “healthy”…therefore, you cannot say that the high MP value for C. bipinnata implies a lack of tolerance because this value is taken from the well-watered treatment. On the other hand, the fact that this species died in response to both moderate and severe drought is good evidence of a higher sensitivity. You refer to the study by Wu et al (2018) who found the same as you ---- I thought you said in the introduction that the drought tolerance of these species has not been quantified before, therefore that statement is also incorrect and needs to be changed. BUT, more to the point, WHAT did Wu et al (2018) say about this species, did they come up with an explanation for high MP? And more to the point, what is your explanation for it? For each result, I am looking for an explanation --- e.g. the last sentence in the last paragraph of section 4.2 --- here, you make a good link between your two sensitive species and findings from the literature with regard to root growth --- but your treatment was very short, therefore it likely did not cause poor root development, but instead may indicate these plants have inherently superficial/poor root systems….this is good discussion and this is what I need to see for each result you present --- not only who was bigger/smaller etc., but why you think that might be the case. Every main result needs to be explained.

Paragraph 4: Contains no references and is largely unnecessary --- ranking species here is pointless.

Paragraph 5: Again, the ranking within treatment is largely unnecessary, the CHANGE from well-watered to droughted is relevant. The reference is generic about MP and doesn’t explain your results.

Paragraph 6: The reference is again generic and the first two sentences should have been used earlier in the discussion. This paragraph should be the main focus of section 4.2 and requires expansion to discuss not only what happened (species ranking), but further, more in-depth assessment of why. I know that your motivation for this paper is to rank species by drought tolerance, but we need a good explanation of the observed trends in the data, using the literature, before we can utilise/have confidence in that ranking. Considering this, have you thought about making section 4.6 the first section in your results/discussion? This is the ultimate ranking of species drought tolerance, so why not just make a ranking here, then you can explore the detail of the constituent components of the contributors to the integrated variable, each of which is ‘less interesting’ than the overall aim of your paper. This is using the logic of the newspaper article, where the most important information comes first, and the least important/interesting comes last.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

19 May 2020

Response to Reviewers

We authors appreciate Reviewer #1 and editor for your detailed guide to further revise the manuscript. We have renewed the content following your suggestions.

Summary of Reviewer #1’s viewpoints:

Reviewer #1 took the section 4.2 as an example and showed us how to organize the article structure.

According to the suggestions, we made the following major changes in Results and Discussion:

1. Move the first paragraphs in "Result and Discussion" to the “Introduction" section.

2. For each parameter, statistically significant changes were described instead of detailed species data lists.

3. We focued on the important phenomenon and their scientific explanation.

4. We summarised the major finding points in the last paragraph for MP, SOD and POD.

In addition, we corrected the spelling mistakes , grammatical and other errors.

Attachment

Submitted filename: Response to reviewers.docx

Click here for additional data file.^{(16K, docx)}

2 Jun 2020

Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening

PONE-D-19-20236R3

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at gro.solp@gnillibrohtua.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact gro.solp@sserpeno.

Kind regards,

Saddam Hussain

Academic Editor

PLOS ONE

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: Dear Authors,

Thanks for your efforts in addressing my comments. I am happy with the revisions made now, the paper is much improved.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

10 Jun 2020

PONE-D-19-20236R3

Drought Resistance of 10 Ground Cover Seedling Species during Roof Greening

Dear Dr. Liu:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact gro.solp@sserpeno.

If we can help with anything else, please email us at gro.solp@enosolp.

Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Saddam Hussain

Academic Editor

PLOS ONE

References

1. Wang PY, Zhang Y. Discussion on the design of water storage and drainage sheaf on the green roof plate. Chinese Landscape Architecture. 2015, 31(11): 13–17. (In Chinese with English abstract) [PubMed]
2. Yu KJ, Li DH, Yuan H, Fu W, Qiao Q, Wang SS. “Sponge city”: theory and practice. Planning Studies. 2015,39(6): 26–36. (In Chinese with English abstract) [PubMed]
3. Ondimu SN, Murase H. Combining Galerkin methods and neural network analysis to inversely determine thermal conductivity of living green roof materials. Biosystems Engineering. 2007, 96(4): 541–550. [PubMed]
4. Baik JJ, Kwak KH, Park SB, Ryu YH. Effects of building roof greening on air quality in street canyons. Atmospheric Environment. 2012, 61(12): 48–55. [PubMed]
5. Köhler M, Clements AM. Green Roofs, Ecological Functions. Springer; New York: 2013. [PubMed]
6. Peng LLH, Jim CY. Economic evaluation of green-roof environmental benefits in the context of climate change: The case of Hong Kong. Urban Forestry & Urban Greening. 2015, 14: 556–561. [PubMed]
7. Nahar K, Hasanuzzaman M, Alam MM, Fujita M. Glutathione-induced drought stress tolerance in mung bean: coordinated roles of the antioxidant defence and methylglyoxal detoxification systems. AoB Plants. 2015, July 1, 7:plv069. 10.1093/aobpla/plv069 ] [
8. Romain M, Erwin D, Marc V, Francis MD, Didier D, Jean-Michel P, et al. Impact of drought on productivity and water use efficiency in 29 genotypes of Populus deltoides×Populus nigra. New Phytologist. 2006, 169(4): 765–777. 10.1111/j.1469-8137.2005.01630.x [] [[PubMed]
9. Sharma P, Dubey RS. Drought induces oxidative stress and enhances the activities of antioxidant enzymes in growing rice seedlings. Plant Growth Regulation. 2005, 46(3): 209–221. [PubMed]
10. Smirnoff N. The role of active oxygen in the response of plants to water deficit and desiccation. New Phytologist. 1993, 125: 27–58. [PubMed]
11. Apel K, Hirt H. 2004. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annual Review of Plant Biology, 55(1): 373–399. [[PubMed]
12. Bai LP, Sui FG, Ge TD, Sun ZH, Lu YY, Zhou GS. Effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of Maizet. Pedosphere. 2006, 16(3): 326–332. [PubMed]
13. Rulcová J, Pospíšilová J. Effect of Benzylaminopurine on rehydration of bean plants after water stress. Biologia Plantarum. 2001, 44(1): 75–81. [PubMed]
14. Bedard K, Krause K. The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology. Physiological Reviews. 2007, 87(1):245–313. 10.1152/physrev.00044.2005 [] [[PubMed]
15. Bahari AA, Sokhtesaraei R, Chaghazardi HR, Masoudi F, Nazarli H. Effect of water deficit stress and foliar application of salicylic acid on antioxidants enzymes activity in leaves of Thymus daenensis subsp. Lancifolius. Cercetari Agronomice in Moldova. 2015, 48(1): 57–67. [PubMed]
16. Ghozlene I, Mohammed-Reda D, Rachid R, Houria B. ROS and antioxidant system of triticum durum after water stress. Annual Research & Review in Biology. 2014, 4(8): 1241–1249. [PubMed]
17. Chang EM, Shi SQ, Liu JF, Yang WJ, Jiang ZP. ROS production and its elimination in old platycladus orientalis's leaves. Journal of Northeast Forestry University. 2011, 39(11): 8–11. (In Chinese with English abstract) [PubMed]
18. Russell JP, Veronica V. Phenylalanine increases membrane permeability. Journal of the American Chemical Society. 2017, 10.1021/jacs.7b09219 [] [[PubMed]
19. Sanchez-Rodriguez E, Rubio WM, Cervilla LM, Blasco B, Rios JJ, Rosales M, et al. Genotypic differences in some physiological parameters symptomatic for oxidative stress under moderate drought in tomato plants. Plant Sci. 2010, 178: 30–40. [PubMed]
20. Jovanović A, Balanč B, Đorđević V, Šavikin K, Nedović V, Bugarski B, et al. Fluorescence analysis of liposomal membranes permeability. Tehnika. 2019, 74: 493–498. [PubMed]
21. Shen M. Preliminary study on the relations between membrane permeability, endogenous hormones and cold resistance of Ivy. Acta Horticulturae Sinica. 2005, 32(1): 141–144. (In Chinese with English abstract) [PubMed]
22. Dai GX, Peng KQ, Xiao LT, Deng GF. Effect of Drought Stress Simulated by PEG on Malonaldehyde, Proline Contents and Superoxide Dismutase Activity in Low Potassium Tolerant Rice Seedlings. Chinese Journal of Rice Science. 2006, 20: 557–559. (In Chinese with English abstract) [PubMed]
23. Du RF, Hao WF, Wang LF. Dynamic response on anti-oxidative defense system and lipid peroxidation of Lespedeza davurica to drought stress and re-watering. Acta Prataculuturae Sinica. 2012, 21(2): 51–61. (In Chinese with English abstract) [PubMed]
24. De Vos CHRD, Schat H, Vooijs R, Ernst Wilfried HO. Copper-induced Damage to the Permeability Barrier in Roots of Silene cucubalus. Journal of Plant Physiology. 1989, 135(2): 164–169. [PubMed]
25. Zhang GL, Chen LY, Zhang ST, Xiao YH, He ZZ, Lei DY. Effect of high temperature stress on protective enzyme activities and membrane permeability of flag leaf in rice. Acta Agronomica Sinica. 2006, 32(9): 1306–1310. (In Chinese with English abstract) [PubMed]
26. Ruchika P, Brendan RC, Richard E. Growth and Physiological Responses of Temperate Pasture Species to Consecutive Heat and Drought Stresses. Plants. 2019, 10.3390/plants8070227 ] [
27. Yao LN, Zhou QY, Yang HX. Discussion on the molecular structure of chlorin. Education In Chemistry. 2005, 5: 58–59, 41. (In Chinese) [PubMed]
28. Croft H, Chen JM, Wang R, Mo G, Luo S, Luo X, et al. The global distribution of leaf chlorophyll content. Remote Sensing of Environment. 2020, 10.1016/j.rse.2019.111479 [[PubMed]
29. Paulsen H, Schmid VHR. Analysis and Reconstitution of Chlorophyll-Proteins. Heme, Chlorophyll, and Bilins. 2008, 10.1385/1-59259-243-0:235 [[PubMed]
30. Mork‑Jansson AE, Eichacker LA. A strategy to characterize chlorophyll protein interaction in LIL3. Plant Methods. 2019, 10.1186/s13007-018-0385-5 ] [
31. Jian LC, Wang H. Plant stress biology, Science Press, Beijing, China: 2009, pp 123, 174. (In Chinese) [PubMed]
32. Zhang MS, Xie B, Tan F, Zhang QT. Relationship among Soluble Protein, Chlorophyll and ATP in Sweet potato under water stress with drought resistance. Scientia Agriculture Sinica. 2003, 36(1): 13–16. (In Chinese with English abstract) [PubMed]
33. Zhang MS, Tan F. Relationship between ratio of Chlorophyll a and b under water stress and drought resistance of different Sweet Potato varieties. Seed. 2001, 4: 23–25. (In Chinese with English abstract) [PubMed]
34. Qin P, Liu FH, Liang XN. Superoxide Dismutase and Plant Resistance to the Environmental Stress. Heilongjiang Agricultural Sciences. 2002, (1): 31–34. (In Chinese with English abstract) [PubMed]
35. Noctor G, Foyer CH. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Biology, Annual Review of Plant Biology. 1998, 49: 249–279. [[PubMed]
36. Alscher RG, Erturk N, Heath LS. Role of superoxide dismutases (SODs) in controlling oxidative stress in plants. Journal of experimental botany. 2002, 53: 1331–1341. [[PubMed]
37. Fridovich ISuperoxide dismutases In: Meister A, ed. Advances in Enzymology and Related Areas of Molecular Biology. New York: John Wiley & Sons; 1986, 58: 61–97. 10.1002/9780470123041.ch2 [] [[PubMed][Google Scholar]
38. White JA, Scandalios JG. In vitro synthesis, importation and processing of Mn-superoxide dismutase (SOD-3) into maize mitochondria. Biochimica et Biophysica Acta. 1987, 926: 16–25. 10.1016/0304-4165(87)90178-4 [] [[PubMed]
39. Baum JA, Scandalios JG. Developmental expression and intracellular localization of superoxide dismutases in maize. Differentiation. 1979, 13: 133–140. [PubMed]
40. Mccord JM, Fridovich I. Superoxide Dismutase an enzymic function for erythrocuprein (Hemocuprein). Journal of Biological Chemistry. 1969, 244(22): 6049–6055. [[PubMed]
41. Molina-Rueda JJ, Tsai CJ, Kirby EG. The Populus Superoxide Dismutase Gene Family and Its Responses to Drought Stress in Transgenic Poplar Overexpressing a Pine Cytosolic Glutamine Synthetase (GS1a). PloS One. 2013, 8(2): e56421 10.1371/journal.pone.0056421 ] [
42. Xu XY, Zhang FY, Zeng QW. Effect of NaCl and Na₂SO₄ stress on seed germination of Cosmos bipinnatus. Journal of Northeast Agricultural University. 2014, 45(4): 55–59. (In Chinese with English abstract) [PubMed]
43. Mohammadi MHS, Etemadi N, Arab MM, et al. Molecular and physiological responses of Iranian Perennial ryegrass as affected by Trinexapac ethyl, Paclobutrazol and Abscisic acid under drought stress. Plant Physiology and Biochemistry. 2016, 111: 129–143. 10.1016/j.plaphy.2016.11.014 [] [[PubMed]
44. Asada K. Ascorbate peroxidase- a hydrogen peroxide- scavenging enzyme in plants. Physiologia Plantarum. 1992, 85(2): 235–241. [PubMed]
45. Qin P, Liu YJ, Liu FH. Effects of drought stress on SOD and POD activities in tobacco leaves. Chinese tobacco science. 2005, (2):28–30. (In Chinese with English abstract) [PubMed]
46. Chen Y, Wang M, Hu LL, Liao WB, Dawuda MM, Li CL. Carbon Monoxide Is Involved in Hydrogen Gas-Induced Adventitious Root Development in Cucumber under Simulated Drought Stress. Frontiers in Plant Science. 2017, 8(230). 10.3389/fpls.2017.00128 ] [
47. Wu XL, Yuan J, Luo AX, Chen Y, Fan YJ. Drought stress and re-watering increase secondary metabolites and enzyme activity in dendrobium moniliforme. Industrial Crops & Products. 2016, 94: 385–393. [PubMed]
48. Jiang MY, Guo SC. Oxidative stress and antioxidation induced by water deficiency in plants. Plant Physiology Community. 1996, 32(2): l44–l50. (In Chinese with English abstract) [PubMed]
49. Shigeoka S, Ishikawa T, Tamoi M, Miyagawa Y, Takeda T, Yabuta Y, et al. Regulation and function of ascorbate peroxidase isoenzymes. Journal of Experimental Botany. 2002, 53(372):1305–1319. [[PubMed]
50. Li ZQ, Li JX, Zhang GF. Expression regulation of plant ascorbate peroxidase and its tolerance to abiotic stresses. HEREDITAS (Beijing). 2013, 35(1): 45–54. (In Chinese with English abstract) [[PubMed]
51. Sukweenadhi J, Kim YJ, Rahimi S, Silva J, Myagmarjav D, Kwon WS, et al. Overexpression of a cytosolic ascorbate peroxidase from Panax ginseng enhanced salt tolerance in Arabidopsis thaliana. Plant Cell Tissue and Organ Culture. 2017, 1–14. [PubMed]
52. Anjum NA, Sharma P, Gill SS, Hasanuzzaman M, Khan EA, Kachhap K, et al. Catalase and ascorbate peroxidase-representative H₂O₂-detoxifying haeme enzymes in plants. Environmental Science and Pollution Research. 2016, 10.1007/s11356-016-7309-6 [] [[PubMed]
53. Wu QS, Xia RX. Effects of arbuscular mycorrhizal fungi on leaf solutes and root absorption areas of trifoliate orange seedlings under water stress conditions. Frontiers of Forestry in China. 2006, 1: 312–317. [PubMed]
54. Tian GZ, Li HF, Qiu WF. Advances on research of plant peroxidases. Journal of Wuhan Botanical Research. 2001, 19(4): 332–344. (In Chinese with English abstract) [PubMed]
55. Yang F, Liu C, Jiang LJ, Guan QM. Comprehensive evaluation on drought tolerance in Malus. Journal of Northwest A & F University. 2020, 48(8): 119–128. (In Chinese with English abstract) [PubMed]
56. Yang XY, Xu YY, Zhao MA, Zeng JQ, Han J, Pei YH, et al. Evaluation of Drought Resistance of Different Maize Varieties under Drought Stress Simulation at Germination Stage. Journal of Maize Sciences. 2019, 27(6): 25–30. (In Chinese with English abstract) [PubMed]
57. Du PB, Zhang YF, Bai XD, Fan XB, Yang C, Qi HY, et al. 2019 Evaluation of Drought Resistance of Potato Varieties by Principal Component Analysis and Membership Function Method. Seed, 38(8): 120–126. (In Chinese with English abstract) [PubMed][Google Scholar]
58. Gao X, Zhu L, Su T. 2018. Comprehensive evaluation on salt tolerance of Sorghum bicolor at booting stage by membership function method. Journal of Southern Agriculture, 49(9): 1736–1744. (In Chinese with English abstract) [PubMed]
59. Wang JF, Zhang LH, Zhao RF, Xie ZK. Responses of plant growth of different life-forms to precipitation changes in desert grassland. Chinese Journal of Applied Ecology. 2020. [[PubMed]
60. Zang ZQ, Bai C, Zhang HZ, Li XD, Fu ZJ, Zhao SM, et al. Salt Tolerance Analysis of Sugar Beet Germplasm Resources by Membership Function Method. Sugar Crops of China. 2019, 41(4): 54–57. (In Chinese with English abstract) [PubMed]
61. He XY, Wen RL, Wu CR, et al. Analysis of maize drought resistance at seeding stage by fuzzy subordination method. Southwest China Journal of Agricultural Sciences. 2008, 21(1): 52–56. (In Chinese with English abstract) [PubMed]
62. Beijing Municipal Administration of Quality and Technology Supervision. 2005. The local standard of roof greening in Beijing (DB11/T 281–2005).
63. Zhu MH, Liu YJ, Zhang TT, Cheng ZB, Yang Z. The impact of Elaphurus davidianus in different habitats on soil physical and chemical properties, Environmental Chemistry. 2016, 35(1): 208–217. (In Chinese with English abstract) [PubMed]
64. Bai JJ, Wu JW, Li JY, He Q, Qiu Q, Pan X. Effects of drought stress on chlorophyll fluorescence parameters of two fast-growing tree species. Journal of South China Agricultural University. 2015, 36(1): 85–90. (In Chinese with English abstract) [PubMed]
65. Bu LD, Zhang RH, Chang Y, Xue JQ, Han MM. Response of photosynthetic characteristics to water stress of maize leaf in seeding. Acta Ecologica Sinica. 2010, 30(5):1184–1191. (In Chinese with English abstract) [PubMed]
66. Verein Deutscher Ingenieure, Düsseldorf. Biological Measuring Techniques for the Determination and Evaluation of Effects of Air Pollution on Plants. VDI 3957. 2007: Part 11, pp. 6–12.
67. Zhang SQ, Li Y. Experimental Technique of Phytophysiology. Beijing: Science Press Ltd; 2011: 191–193, 203–204. (In Chinese with English abstract) [PubMed]
68. Arnon DI. Copper enzymes in isolated chloroplasts polyphenol oxidase in Beta vulgaris. Plant Physiol. 1949, 24(1): 1–15. 10.1104/pp.24.1.1 ] [
69. Zhang PQ, Liu YJ, Chen X, Yang Z, Zhu MH, Li YP. Pollution resistance assessment of existing landscape plants on Beijing streets based on air pollution tolerance index method. Ecotoxicology and Environmental Safety. 2016, 132: 212–223. 10.1016/j.ecoenv.2016.06.003 [] [[PubMed]
70. Tang SH, Luo C. Experimental Technique of Phytophysiology. Chongqing: Southwest China Normal University Press; 2012, 213–214. (In Chinese) [PubMed]
71. Chen RM, Yang XJ, Liang FS, Lu SY, Zhang RZ, Zhao HR. Comprehensive evaluation on wheat drought-resistance traits by membership function values analysis. Journal of Agricultural University of Hebei. 2002, 25(2):7–9. (In Chinese with English abstract) [PubMed]
72. Oparka KJ. Plasmolysis: new insights into an old process, New Phytol. 1994, 126: 571–591. [PubMed]
73. Wang F, Wang B, Yu JH, Jie JM, Feng Z, Liao WB, et al. Comprehensive Evaluation on Substrates for Lettuce Soilless Culture by Membership Function Method. Acta Agriculture Boreali-occidentalis Sinica. 2020, 29(1): 117–126. (In Chinese with English abstract) [PubMed]
74. Xu K, Li FL, Gou SY, Bao WK. Root functional traits and trade-offs in one-year-old plants of 25 species from the arid valley of Minjiang River. Acta Ecologica Sinica. 2012, 32(1): 215–225. (In Chinese with English abstract) [PubMed]
75. Chen ZH, Xia CH, Wu Z. Studies on tissue plants nursing on moist tray of Gerbera jamesonii. Journal of Yunnan Agricultural University(Natural Science). 2014, 29(02): 223–228. (In Chinese with English abstract) [PubMed]
76. Cai JH, Li JF, Wei XY, Zhang L. Dynamic change in biomass, root vigor and replacement rate during the green leaf period of Lycoris radiat. Journal of Nanjing Forestry University (Natural Science Edition). 2018, 42(01): 55–59. (In Chinese with English abstract) [PubMed]
77. Niu W. Studies on photosynthetic characteristics and drought Resistance of four Caraganas species. Lanzhou, Gansu Province, China: Gansu Agriculture University; 2018: 32. (In Chinese with English abstract) [PubMed]
78. Yang B. Response of Robinia pseudoacacia L. Seedlings to Soil Water Stress: Based on Growth, Physiology and the Allocation and Dynamics of Non-structural Carbohydrates. Yangling, Shanxi Province, China: Northwest A & F University; 2019: 19. (In Chinese with English abstract) [PubMed]
79. Wu JC, Tian SQ, Xue RR, Xu Z, Zhang XX. Physiological and biochemical response of three species of Lina seedlings to natural drought stress. Guizhou Agriculture Science. 2017, 45(12): 25–29. (In Chinese with English abstract) [PubMed]
80. Liu ZJ, Yuan R, Wang N, Li BY, Jia SC, Zong YZ, et al. Effects of drought Stress on leaf physiology of Hylotelephium erythrostictum. Journal of Shanxi Agricultural Sciences. 2018, 46(04), 548–553. (In Chinese with English abstract) [PubMed]
81. Fu BC, Bo W, Qin GJ, Wang S. Physiological response of drought stress in Sedum species and evaluation of drought resistance. Molecular Plant Breeding. 2017, 15(03): 1096–1103. (In Chinese with English abstract) [PubMed]
82. Sun X. Preliminary study on drought-resistance characteristics of Dianthus plunarius. Haerbin, Heilongjiang province, China: Northeast Forestry University; 2006: 20. (In Chinese with English abstract) [PubMed]
83. He YC, Zu WH, Wang Q, Li QB, Ma FF, Hu HR. Comparison of drought tolerance in six succulents. Acta Agriculturae Boreali-occidentalis Sinica. 2010, 19(3): 127–130. (In Chinese with English abstract) [PubMed]
84. Yang PH, Li GQ, Guo L, Wu SJ. Effect of drought stress on plasma mambrane permeality of soybean varieties during flowering-poding stage. Agricultural research in the arid areas. 2003, 21(3): 127–130. (In Chinese with English abstract) [PubMed]
85. Chen JY, Feng LY, Gao J, Shi JW, Zhou YC, Tu FT, et al. Influence of light intensity on stoma and photosynthetic characteristics of Soybean leaves. Scientia Agricultura Sinica. 2019, 52(21): 3773–3781. (In Chinese with English abstract) [PubMed]
86. Chang QS, Zhang LX, Wang JZ, Wang Z, Xu SJ, Kang L, et al. Effects of drought stress and rewatering on physiological indexes of four Paeonia lactiflora cultivars and evaluation of their drought resistance. Journal of Nanjing Forestry University(Natural Sciences Edition). 2018, 42(06): 44–50. (In Chinese with English abstract) [PubMed]
87. Liu YL, Guo SX, Ma MS. Effect of drought stress and rewatering at seedlings stage on light energy utilization and antioxidant enzymes activities of spring maize leaves. Journal of Soil and Water Conservation. 2018, 32(1): 339–343. (In Chinese with English abstract) [PubMed]
88. Tian X. The studies on the introduction and adaptability of Crassulaceae plants and their propagation techniques. Hefei City, Anhui Province, China: Anhui Agriculture University; 2009, 1. (In Chinese with English abstract) [PubMed]
89. Cui J. Effect of the stress on the three kinds of Crassulaceae Echeveria succulent leaves coloring. Shenyang City, Liaoning Province, China: Shenyang Agriculture University; 2016: 1. (In Chinese with English abstract) [PubMed]
90. Huang QC, Wei YH. Comparative Analysis of Chlorophyll Content on Several Sun Plants and Shade Plants. Hubei Agricultural Sciences. 2009, 48(8): 1923–1924, 1929. (In Chinese with English abstract) [PubMed]
91. Zhou Y. Studies on the drought resistance of 7 Iris species. Urumqi, Xinjiang Uygur Autonomous Region, China: Xinjiang Agriculture University; 2006: 14. (In Chinese with English abstract) [PubMed]
92. Ding YF, Sun JP, Shang XY, Li XJ, Sun H, Zhu JW, et al. Effects of drought on cell membrane damage and activities of protective enzyme of different flue-cured tobacco leaves. Southwest China Journal of Agricultural Sciences. 2015, 28(6): 2746–2749. (In Chinese with English abstract) [PubMed]
93. Yin YQ, Hu JB, Deng MJ. Latest development of antioxidant system and responses to stress in plant leaves. Chinese Agricultural Science Bulletin. 2007, 23(1): 105–110. (In Chinese with English abstract) [PubMed]
94. Shang RX, Yu X, You CC, He HB, Li J, Wu LQ. Adaptation physiological mechanism of rice under dual stress of drought stress and high temperature in booting stage. Journal of Gansu Agricultural University. 2019, 12(54): 39–46. (In Chinese with English abstract) [PubMed]
95. Li TH, Wang X, Cai J, Zhou Q, Dai TB, Jiang D. Comprehensive evaluation of drought priming on plant tolerance in different wheat cultivars. Journal of Triticeae Crops. 2018, 38(1): 65–73. (In Chinese with English abstract) [PubMed]
96. Zuo YM, Yang WZ, Yang TM, Yang MQ, Xu ZL, Yang SB, et al. Comparison of resistant physiological index among four species in the Genus Panax under water stress. Crop. 2016, (03): 84–88. (In Chinese with English abstract) [PubMed]
97. Dong QQ, Ai X, Zhang YZ, Zhang KC, Zhou DY, Wang XG, et al. Effect of drought stress on physiological characteristics and yield in different tolerant peanut. Journal of Shenyang Agricultural University. 2020, 51(01): 18–26. (In Chinese with English abstract) [PubMed]
98. Chen L, Shen XR, Zhao HY, Liu K. Optimization of treatment conditions for extracting SOD in Sedum lineare. Grassland and Turf. 2009,1(132): 1–6. (In Chinese with English abstract) [PubMed]
99. Ruppenthal V, Zoz T, Steiner F, Lana MDC, Castagnara DD. Silicon does not alleviate the adverse effects of drought stress in soybean plants. Semina: Ciências Agrárias, Londrina. 2016, 37(6): 3941–3954. [PubMed]
100. Sun GR, Peng YZ, Yan XF, Zhang R, Jiang LF. Effect of drought stress on activity of cell defense enzymes and lipid peroxidation in leaves of Betula Platyphylla seedlings. Scientia silvae sinicae. 2003, 39(1): 165–167. [PubMed]
101. Li Y, Deng XP, Sang-Soo K, Kiyoshi T. Drought tolerance of transgenic sweet potato expressing both Cu/Zn Superoxide Dismutase and Ascorbate Peroxidase. Journal of Plant Physiology and Molecular Biology. 2006, 32(4): 451–457. (In Chinese with English abstract) [[PubMed]