Shade effects on growth, photosynthesis and chlorophyll fluorescence parameters of three Paeonia species

Plant material and growth conditions

Five accessions were arranged in a completely randomized design, and the interval of each accession made sure the leaves of different plants were not covered. First, the three Paeonia species were introduced as plants with as much of the root system and underground buds as possible to the National Engineering Research Center for Floriculture, Changping district, Beijing, in August 2016. We used P. anomala plants (n = 17) from the Altay city population, P. intermedia plants (n = 24) from the Yumin population, Xinjiang Province, and P. veitchii plants (n = 22) from the Lanzhou population, Gansu Province. Deep, fertile and well-drained soil was selected for the field plantings. Before the seedlings were transplanted, the soil was tilled, stones and weeds were removed, and decomposed organic fertilizer (0.25 kg/m² cake fertilizer) was applied. Seedlings were set apart 60 cm ×60 cm from the neighbor ones and were watered in accordance with the local weather conditions. Fertilizer was applied three times a year, that is, 1. 5 × 10⁻² kg/m² fertilizer NPK 30-10-10 in early spring after the soil thawed, 7. 5 ×10⁻³ kg/m² fertilizer NPK 20-20-20 two weeks after flowering, and 7.5 × 10⁻³ kg/m² fertilizer NPK 15-10-30 before the soil froze over after autumn. Weeding was performed throughout the growing season. After two years of cultivation in Beijing, more than 80% of these seedlings survived. In addition, P. lactiflora ‘Da Fugui’ (which is suitable for solar greenhouse cultivation; (Han et al., 2014)) and ‘Qiao Ling’ (which is not suitable for solar greenhouse cultivation), two common cultivars grown in China, were planted and managed as the wild Paeonia species.

A single-factor experiment with each species was carried out in March 2018. Herbaceous Paeonia species needs to renew buds underground to germinate and develop crowns and flowers every year. Before germination in 2018, a black nylon net was placed above the planting location of the three species and two cultivars as shade treatment; under this net, the natural light experienced by the plants was approximately 30% of the sunlight intensity. Full sun exposure was used as a control treatment. Plants of each treatment received the same fertilizer and amount of watering. The daylength during the experiment was 12.21–14.86 h, and the average was 13.69 ± 0.80 h. The actual light intensity, air temperature, CO₂ concentration and relative humidity above and below the shade net were recorded by a LI-6400 Portable Photosynthesis Measurement System (LI-COR, USA) with the measurement of the net photosynthesis rate (Pn). Concurrently, from 8:00 to 16:00 h, the light intensity under full sun exposure was greater than 1000 µmol m⁻² s⁻¹, while it was between 297.23–523.23 µmol m⁻² s⁻¹ at the same time under shade. The CO₂ concentration was between 392.64–423.21 µmol mol⁻¹ under full sun exposure and 385.52–426.53 µmol mol⁻¹ under shade, respectively. Besides, the CO₂ concentration under the shade net was significantly lower than that above the net from 12:00–14:00 h, and during that time, the temperature under the shade net was lower than outside it by approximately 2.22–2.86 ° C (Fig. 1).

Morphological and floral measurements

Morphological traits were measured at flowering (i.e., 30–37 days after shading). Crown width, branch length, stem diameter, width and length of the third or fourth leaf from the top, and flower diameter were measured by a flexible ruler or a Vernier caliper, and we performed every measurement three times in three different individuals of each accession. In addition, the leaves were fully spread out on graph paper, and images were taken. The leaves were then outlined, and the leaf areas were calculated by Autodesk Computer Aided Design (AutoCAD, Autodesk, USA). Floral parameters, including flowering rate, flower number per pot and single flowering period duration, were recorded. Flower color was measured by a portable multifunction colorimeter (3nh, China). A D65 standard light source with an eight mm window diameter was selected as the measuring light source, and the outer surface of the petal was measured. The lightness (L^∗), red/green coordinate (a^∗) and yellow/blue coordinate (b^∗) color values defined by the International Commission on Illumination (CIE) were recorded, and the measurements were repeated three times on different flowers.

Photosynthetic measurements

Photosynthetic parameters were measured 20 days after the flowering of each accession, which was variable. The short time interval from germination to flowering and the energy store for vegetative propagation of the following year were considered in the selection of measurement time. Three plants were randomly selected per accession under conditions of full sun exposure and under shade to measure the photosynthetic parameters (using a LI-6400 Portable Photosynthesis Measurement System, LI-COR, USA), and three leaves (the third or fourth leaf from the top of plants in different stems) from each plant were measured. To obtain diurnal variation in photosynthesis, the Pn was measured every hour from 7:00 h to 18:00 h using a transparent leaf chamber, with three to six measurements per accession. During this process, stomatal conductance (Gs), intercellular CO₂ concentration (Ci) and transpiration rate (Tr) were recorded simultaneously. To construct light response curves, the Pn under different levels of photosynthetic photon flux density (PPFD) (i.e., 2,000, 1,800, 1,600, 1,400, 1,200, 1,000, 800, 600, 400, 200, 150, 100, 50 and 0 µmol m⁻² s⁻¹) was measured from 8:30–11:30 h, with a CO₂ concentration of 400 µmol mol⁻¹. Three replicates were measured at each PPFD. Before the measurements, photosynthesis in the selected leaves was induced by 1,500 µmol m⁻² s⁻¹ PPFD for 20 min.

A nonlinear regression analysis was carried out according to the formula of the nonrectangular hyperbolic model, and a light response curve was generated. Linear regression of the Pn and PPFD in the range of 0–200 µmol m⁻² s⁻¹ was performed, and the apparent quantum yield (AQY), dark respiration rate (Rd), light-saturated photosynthesis rate (LSPn), light compensation point (LCP) and light saturation point (LSP) were calculated (Walker, 1989).

Chlorophyll content and fluorescence measurements

During the flowering period, the third or fourth newest leaf under the flowers was randomly collected, and we used three leaves from three individuals per accession. After cleaning, 0.2 g of fresh leaves were cut into pieces, soaked in 25 ml of 95% ethanol and kept under dark conditions at room temperature. After 48 h, the absorbance of the solutions was measured at 665 nm and 649 nm by a Biomate 3S UV-visible spectrophotometer (Thermo Fisher Scientific, USA). The chlorophyll a and b contents were subsequently calculated by previously described equations (Alsaadawi, Al-Hadithy & Arif, 1986).

Chlorophyll fluorescence parameters were measured by a PAM-2500 portable amplitude modulation fluorometer (Walz, Germany) on the third or fourth leaf from each selected individual per accession. The minimal fluorescence with all photosystem II [PSII] reaction centers open (Fo), maximal fluorescence in the absence of NPQ in the dark-adapted state (Fm), minimal and maximal fluorescence in the presence of NPQ during illumination (Fo’ and Fm’) and steady-state fluorescence after onset of illumination (Fs) were recorded after 20 min of dark adaptation. To obtain Fo, a light pulse of 3 µmol m⁻² s⁻¹ was applied, and the modulation frequency was 20 kHz. To obtain the Fm, a saturating light pulse at an intensity of 8000 µmol m⁻² s⁻¹ was applied for 0.8 s. The light intensity during the measurement of Fo’ and Fm’ was determined according to the default program of the PAM-2500 portable amplitude modulation fluorometer.

Fv is calculated by the difference of Fm and Fo, and it reflects the reduction of electron acceptors of PSII(QA). The maximal PSII efficiency of dark-adapted leaves (Fv/Fm), maximum primary photochemical yield (Fv/Fo) of PSII, nonphotochemical fluorescence quenching (NPQ), quenching coefficient of photochemical quenching (q_p) and relative PSII electron transport rate (ETR) were calculated according to various formulas (Demmig-Adams et al., 1996; Hu, Sun & Wang, 2007; Li et al., 2006). Similarly, the quantum yield of constitutive thermal energy dissipation (Φ_D), quantum yield of PSII photochemistry (Φ_PSII) and quantum yield of ΔpH- and xanthophyll-regulated thermal energy dissipation (Φ_NPQ) were calculated according to the methods reported in previous studies (Hendrickson, Furbank & Chow, 2004; Zivcak et al., 2014).

Evaluation of shade tolerance

The shade tolerance of plants is the result of many factors, and it cannot be judged from only a single index. The membership function method was used in conjunction with nine indexes of photosynthetic and chlorophyll fluorescence parameters to comprehensively evaluate the shade tolerance of the three wild Paeonia species and two cultivars. Fv/Fm is an acceptable parameter for evaluating whether a leaf is experiencing photoinhibition and its degree (Baker, 2008; Peng et al., 2017). Thus, we considered Fv/Fm a basic indicator for shade tolerance and calculated its correlation with five of the measured photosynthetic parameters (i.e., the AQY, LCP, LSP, Rd and change rate of the LSPn under shade) and three chlorophyll fluorescence parameters (i.e., Fv/Fo, ETR and Φ_PSII change under shade) (Table S1).

The membership function method was used to evaluate the shade tolerance of plants according to methods of previous studies (Liu et al., 2019; Wang et al., 2014). Formula (1) was used if the index was positively related to Fv/Fm, and formula (2) was used if the index was negatively related to Fv/Fm.

(1)${\displaystyle {\mathrm{Z}}_{\mathrm{ij}}=\left({\mathrm{X}}_{\mathrm{ij}}-{\mathrm{X}}_{\mathrm{i}min}\right)∕\left({\mathrm{X}}_{\mathrm{i}max}-{\mathrm{X}}_{\mathrm{i}min}\right)}$ (2)${\displaystyle {\mathrm{Z}}_{\mathrm{ij}}=\left({\mathrm{X}}_{\mathrm{i}max}-{\mathrm{X}}_{\mathrm{ij}}\right)∕\left({\mathrm{X}}_{\mathrm{i}max}-{\mathrm{X}}_{\mathrm{i}min}\right)}$

Z_ij is the shade tolerance value of the i index for the j plant accession according to the membership function, and X_ij is the measured value of the i index for the j plant accession. X_i min and X_i max are the minimum and maximum values of each index, respectively. The membership function values of each index were averaged per accession. The higher the average value, the greater the shade tolerance of the plant.

Statistical analysis

We compared every parameter under shade and sun exposure via the least significant difference method (LSD) after one-way ANOVA was performed (SPSS 18.0). Microsoft Excel 2016 and R 3.5.1 (R Core Team, 2019) were used to plot the results.

Morphological and floral characteristics

The single flowering period of P. intermedia and P. veitchii was prolonged by shade, while their flowering rate and flower diameter were not affected (Table 1). P. anomala could not flower under any light condition in Beijing (Fig. 2). Moreover, the flower color of P. veitchii faded under shade, and it presented significantly higher L^∗ and b^∗ color values and lower a^∗ values, showing an increase in lightness and a decrease in red and blue (Fig. 2). P. anomala and P. intermedia had larger leaf areas under shade than under full sun. No differences were observed in crown width, branch length or stem diameter for any of the three species under any light condition (Table 1).

Photosynthetic characteristics

The photosynthesis diurnal variation of the three species was bimodally distributed under sun exposure, peaking at approximately 10:00 h and 15:00 h (Fig. 3). Under shade, single-peak photosynthesis curves were detected for the three species, and at those moments, Pn was significantly higher under shade than under sun. For the two cultivars, both the sun and shade groups presented single-peak curves, while peaking at 10:00 h under sun and at 11:00h or 12:00h, under shade, respectively. No significant differences were detected in ‘Da Fugui’ at midday between the two light conditions, and the Pn of ‘Qiao Ling’ under shade at midday was significantly lower than that under full sun. For all five accessions, the Pn in the morning (7:00–10:00 h) and afternoon (14:00–18:00 h) in the sun was often higher than that under shade.

The Pn increased linearly within the PPFD range of 0–200 µmol m⁻² s⁻¹, continuously increased at a lower rate in the PPFD range of 200–1,000 µmol m⁻² s⁻¹, and then remained unchanged or only slightly changed under higher PPFD (Figs. 4A–4E). Significant differences in the Pn between the two light conditions occurred under only a few light intensities. When the PPFD was 50–150 and 800–1.000 µmol m⁻² s⁻¹, the Pn of P. anomala under shade was significantly higher than that under sun exposure (Fig. 4A). For ‘Da Fugui’, differences between the sun and shade groups occurred only at 0 and 50 µmol m⁻² s⁻¹ (Fig. 4D), and when the PPFD was 20–200 µmol m⁻² s⁻¹, the Pn of ‘Qiao Ling’ was significantly higher than that under shade (Fig. 4E); in both cases, Pn increased.

The AQY significantly increased in P. intermedia and ‘Qiao Ling’ but decreased in ‘Da Fugui’ under shade (Fig. 4F). The LSPn, LSP and LCP decreased to different extents, with the exception of those of ‘Da Fugui’, which remained unchanged (Figs. 4G–4I). Among them, with respect to P. anomala, shade significantly decreased only the LCP (Fig. 4I); for P. intermedia, the LSP was significantly decreased in response to shade (Fig. 4H); and for P. veitchii, both the LSPn and LSP decreased significantly in response to shade (Figs. 4G–4H). All three parameters decreased in ‘Qiao Ling’ under shade (Figs. 4G–4I).

The chlorophyll content tended to increase in response to shade, while the changes in chlorophyll a and b in P. veitchii and chlorophyll b in P. anomala were not significant. The Chl a/b significantly increased in response to shade in P. anomala and P. intermedia, whereas it significantly decreased in ‘Da Fugui’ (Fig. 5).

Chlorophyll fluorescence characteristics

The Fv/Fm and Fv/Fo of P. anomala, P. intermedia, P. veitchii and ‘Da Fugui’ increased significantly in response to shade (Figs. 6A–6B). The NPQ of P. anomala, P. intermedia and P. veitchii increased, and the q_p of the last two accessions decreased significantly (Fig. 6C). With respect to ‘Qiao Ling’, only q_p decreased significantly in response to shade (Fig. 6C), and the Fv/Fm, Fv/Fo and NPQ remained unchanged (Figs. 6A–6B, 6D). Moreover, only the ETR of P. anomala increased with shade treatment (Fig. 6E).

Regarding the distribution of the absorbed light energy, both Φ_D and Φ_PSII of P. anomala decreased significantly in response to shade, and no significant differences were observed in these two parameters for the other four accessions (Figs. 6F–6G). The Φ_NPQ tended to increase under shade in all the samples, although the differences were significant only for P. anomala, P. intermedia and ‘Qiao Ling’ (Fig. 6H).

Evaluation of shade tolerance of five accessions

The average scores of the three wild species were similar and significantly higher than those of ‘Da Fugui’ and ‘Qiao Ling’, indicating that the shade tolerance of P. anomala, P. intermedia and P. veitchii was greater than that of the common cultivars grown in China. In addition, the average score of ‘Da Fugui’ was slightly higher than that of ‘Qiao Ling’, but this difference was not significant (Table 2).