2.1 Plant Materials and growth condition
In 2000, the plant seeds were purchased from a seedling company in Shandong Province, China, and were sown on the campus of Northwest Agriculture and Forestry University Yangling demonstration area, Shaanxi Province(108°5′18″E, 34°5′4″N) in the same period. After two Carex species have grown in the shade for years, they naturally formed a lawn with high ornamental value, thus playing a role in beautifying the campus environment (Fig. 1)(Table 1). The optimal growing season was selected for plants in summer (end of July) for this experiment。
Table 1
Index | CH | CB |
turfgrass height(cm) | 26.507 | 15.750 |
turfgrass density(plant/cm− 2) | 1.2 | 1 |
ground over age(%) | 98 | 95 |
length of leaves(cm) | 60.160 | 26.283 |
width of leaves(cm) | 0.3 | 0.2 |
leaf index | 238.630 | 68.259 |
The introduction site is characterized by a temperate continental monsoon climate. The annual precipitation is 635.1mm, Average evaporation 993.2 mm, the annual average temperature is 12.9°C, the annual average sunshine hours are 2163.8h, and the annual accumulated temperature ≥ 10°C is 4184°C. The distribution of precipitation in summer (June to August) is extremely uneven, often accompanied by summer drought and sub-drought. In early summer, it is often dry and less rainy, with more than 5 strong winds. Hot and high temperature, the average temperature is higher than 25 ℃, and the extreme maximum temperature can reach 42 ℃. Furthermore, the soil of the introduction land is poor, and the contents of organic matter, nitrogen and available phosphorus are low(0.06–0.1%)(Dou et al. 2015).
2.2 Diurnal variation of photosynthetic parameters
Photosynthetic Diurnal variation parameters were developed using a LI-6400XT portable photosynthesis measurement system, and the light source was natural sunlight. The middle position of the leaves was measured, and the following contents were examined every 2h from 8:00 to 18:00, including net photosynthetic rate (Pn), transpiration rate (Trmmol), stomatal conductance (Gs), air CO2 concentration (Ca), intercellular CO2 concentration (Ci), air temperature(Ta), relative humidity(RH) and photosynthetically active radiation (PAR). One plant was repeated four times. The following calculation was conducted after the measurement: light use efficiency (LUE) = P n/PAR, water use efficiency (WUE) = P n/Tr, Stomatal limitation(Ls)=(Ca-Ci)/Ca.
2.3 Photosynthetic light–Response Curves Measurements
LI-6400XT portable photosynthesis system (Li-Cor Inc., Lincoln, Nebraska, USA) with a red–blue LED light source (6400-02B), in 9:00–11:00, setting PAR of 1200, 1000, 800, 600, 500, 400, 300, 200, 150, 100, 50, 20, 0 µmol/m2/s, the CO2 injection system setting value was 400 µmol/mol, and the flow rate was 500 µmol/s.
The light–response curves were fitted using a modified Right-Angle Hyperbolic Model(Ye 2007; Ye & Yu 2008), The fitting model formula was as Eq. (1). After fitting and calculation, the light saturation point (LSP), light compensation point (LCP), apparent quantum efficiency (α), maximum net photosynthetic rate (Pnmax), and dark breathing rate (Rd) of the two types of plants can be obtained.
$$Pn(I)=\alpha \frac{1-\beta I}{1+\gamma I}I-Rd$$
1
Where I denotes the photosynthetically active radiation; β and γ represent coefficients
2.4 CO2–Response Curves Measurements
When the photosynthetic CO2 response was performed, the generated reaction substrate had a certain hysteresis. Thus, the CO2 concentration controlled by the instrument is first reduced from the atmospheric concentration (nearly 400 µmol/mol) to 50 µmol/mol during the observation. The CO2 concentration of the sample chamber is 400, 300, 200, 150, 100, 50, 400, 400, 600, 800, 1000, 1200, 1500 µmol/mol. Photosynthetically active radiation was set to 600 µmol/mol.
The light–response curves were fitted using a modified Right-Angle Hyperbolic Model(Ye & Yu 2009). The fitting model formula was as Eq. (2). After fitting and calculation, the CO2 saturation point (CSP), CO2 compensation point (CCP), initial carboxylation rate (η), photorespiration rate (Rp)and maximum net photosynthetic rate (Pnmax) can be calculated. The maximum carboxylation rate and maximum electron conductivity of the two plants were calculated using the fitaci function.
$$Pn(Ca)=\eta \frac{1-\beta Ca}{1+\gamma Ca}Ca-Rp$$
2
Where β and γ represent coefficients
2.5 Leaf functional traits measurements
Physiological characters: Using the third leaf at the top of each plant, the plants were determined for leaf area(LA), leaf thickness(LT), leaf saturated fresh weight(LSFW), Leaf fresh weight (LFW), and leaf dry weight(LDW) Collected 3 leaves of each plant species for 4 replicates, and calculate after measurement: Specific leaf area(SLA) = LA/LDW, Leaf dry matter content(LDMC) = LDW/LSFW, Leaf relative water content(LRWC)=(LFW-LDW)/(LSFW-LDW), Leaf tissue density(LTD) = LDW/(LT*LA).
Anatomical characters: The complete leaves of each plant growing in the sun were selected, and the leaf tissue near the middle or lower position was cut. First, the cut tissue blocks were quickly put into 4% glutaraldehyde at 4°C overnight for more than 12 h. Then rinsed and dehydrated with PBS buffer solution and different concentration gradients (30%, 50%, 70%, 80%, 90%, 100%) of ethanol. The samples were dried with a critical point dryer (EMCPD300), sprayed with gold by a sputter coater (Q150TS), and lastly magnified to 300 times with a field emission scanning electron microscope (Nano SEM-450) for observation and photography. Lastly, the high of siliceous papillosethe, the thickness of the stratum corneum(CUT), the thickness of the upper(UET) and lower epidermal cells (LET), thickness of the main vein(MVT), the vessel area (VA), main vessel vascular bundle structure area(VBA), vessel area/vascular bundle area(VA/VBA), the number of vesicle cells(BC) were calculated. 3–5 leaves of the respective plant species were collected for four replicates.
Coefficient of Variation(CV) = SD/mean*100%, Plasticity index(PI)=(MAX-MIN)/MAX.
2.6 Statistical analyses
Photosynthetic parameters were calculated in the Zipao Ye photosynthetic calculation model software 4.1.1. The data were analyzed using one-way analysis of variance using SPSS 26. The correlation was analyzed through Pearson and redundancy analysis. Before the RDA analysis, detrended correspondence analysis (DCA) was conducted on the leaf photosynthetic parameters. The results indicated that the maximum gradient length was 0.4, less than 3. Accordingly, the linear response model and Canoco 5.0 software were used for RDA analysis. Plotted in Origin 2019. Data in the graph are expressed as the mean ± SD.