Response of photosynthesis, the xanthophyll cycle, and wax in Japanese yew (Taxus cuspidata L.) seedlings and saplings under high light conditions

Plant material

This experiment was performed at a nursery at the Northeast Forestry University (45°72′N, 126°63′E) on July 2020. Japanese yews were planted in the nursery for the purposes of this study. We designed a comparative experiment between two groups: (1) the Japanese yew seedling group and (2) the Japanese yew sapling group. Each group included 10 samples. Yew seedlings and saplings were classified by plant height according to the definitions provided in Devaney’s study (Devaney, Whelan & Jansen, 2015): yew seedlings, approximately 0–0.5 m; yew saplings, 0.51–2 m. Three-year-old yew seedlings and 11-year-old yew saplings were selected for the study. The average height, crown width, and base diameter of the seedings were 0.25 m, 27 cm, and 1.26 cm, respectively; the average height, crown width, and base diameter of the saplings were 1.55 m, 96 cm, and 4.16 cm, respectively. Seedlings were planted in plastic pots that were 25 cm in diameter and 20 cm in height (25 × 20 cm), and saplings were planted in plastic pots that were 60 cm in diameter and 55 cm in height (60 × 55 cm). All seedlings and saplings were grown from seed and cultivated under shade cloths situated 2 m above the ground. Shaded plants were grown under a maximum photon flux density (PFD) of about 700 μmol·m⁻²·s⁻¹ (40% full sun). Light intensity was measured using a quantum radiometer (LI-189; LI-COR, Lincoln, NE, USA). Plants were watered every 5 days. In the spring, five grams of compound fertilizer was applied to each seeding and ten grams of compound fertilizer was applied to each sapling. Current-year shoots of both seedling and sapling plants were chosen from the mid-crown of each plant to measure pigments, photosynthetic rate, chlorophyll fluorescence, and wax content.

Measurement of pigments

The chlorophyll (Chl) content and total carotenoids (Car) in the leaves were extracted using 80% acetone. Calcium carbonate was added to prevent the pheophytinization of Chl a. The extracts were filtered through filter paper and analyzed with a UV-2800 system (Hitachi, Shinagawa-ku, Japan). A spectrophotometer was used to estimate chlorophyll content and total carotenoids using extinction coefficients and equations previously described by Lichtenthaler (1987). Xanthophyll cycle components, including violaxanthin (V), antheraxanthin (A) and zeaxanthin (Z), were measured according to Magney et al. (2017). Plants were illuminated at 1,200 μmol·m⁻²·s⁻¹ (PFD) for 2 h with a blue-red (8:1) light-emitting diode (LED) after a night of dark-adaptation. Then, the leaf samples were rapidly frozen in liquid nitrogen and extracted with 85% acetone. The quantification of pigments was determined by high-performance liquid chromatography (HPLC) with a Waters 600 solvent pump, a Waters 2,996 diode array detector, a Waters 717 autosampler (Waters Spa, Milford, MA, USA) set to record at 445 nm, and a YMC Carotenoid C30 reverse phase column (5 μm particle size, 250 × 4.6 mm I.D.; YMC America, Inc., Allentown, PA, USA).

Measurement of the photosynthetic rate

The net photosynthetic rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) were measured using a LI-6400 portable photosynthesis system (LI-COR, Lincoln, Nebraska, USA). The photosynthetic rate (Pn) response curves to PFD were determined according to outdoor temperature and CO₂ atmospheric concentration using a coniferous leaf chamber at 30 °C and 380 μmol·mol⁻¹ CO₂ to obtain the light-saturation point (LSP) and light-compensation point (LCP). PFDs were fixed in a sequence of: 1,800; 1,500; 1,200; 1,000; 800; 400; 200; 100; 50; and 0 μmol·m⁻²·s⁻¹.

Photorespiration (Pr) was calculated with the following formula:

$\mathrm{P}\mathrm{r}=\mathrm{P}{\mathrm{n}}_{\mathrm{O}2}^1-\mathrm{P}{\mathrm{n}}_{\mathrm{O}2}^2$where Pn_O2¹ is the photosynthetic rate measured at 21% O₂ +350 μmol·mol⁻¹CO₂, and Pn_O2² is the photosynthetic rate measured at 2% O₂ +350 μmol·mol⁻¹CO₂.

Measurement of chlorophyll fluorescence parameters

To examine the response of PSII in seedlings and saplings to high light conditions, chlorophyll (Chl) fluorescence was measured in the laboratory with an FMS-2 pulse-modulated fluorometer (Hansatech, Norfolk, UK). Seedling and sapling plants were exposed to 1,200 μmol·m⁻²·s⁻¹ PFD irradiance for 2 h using a plant growth lamp (ECO-014, Guangzhou, China). During the 2 h of high light exposure, the maximum quantum yield of photosystem II (Fv/Fm), actual PSII efficiency ( $\mathrm{\Phi }$PSII), non-photochemical quenching (NPQ), and high-energy fluorescence quenching (qE) were measured one time, at 30 min intervals. Fv/Fm recovery in darkness for 1.5 h followed the high light exposure. Before measuring Fv/Fm, the sample leaf was dark-adapted for 35 min. Fv/Fm, $\mathrm{\Phi }$PSII, NPQ, and qE measurements were taken following the methods described by Johnson et al. (1993).

Wax determination and removal

To quantify the total weight of the wax on the adaxial and abaxial Japanese yew leaf surfaces, 2 g of leaves were submerged in 30 mL of hexane for 30 s and then rinsed with 2 mL of hexane. Wax extracts (hexane) were then evaporated using N2 gas. The total weight of the wax was expressed based on leaf area, which was determined using a photo image system (LA-S, Hangzhou, China). To identify the impact of epicuticular wax on photosynthetic protection, the wax from the adaxial surface of Japanese yew leaves were removed by gently pressing Blu-Tack (Bostik, Stafford, UK) against the leaf surface a number of times, without causing damage to the leaf surface, according to the methods outlined by Mohammadian, Watling & Hill (2007).

Statistical analysis

Excel 2022 was used for data input and storage, and the mean values in the table and the figures represent 3–5 replications. All statistical analyses were performed using SPSS statistical software (IBM SPSS Statistics 19.0; Chicago, IL, USA). Student’s t-test was used to analyze the differences between the seedlings and saplings. The experimental data were also subjected to a homogeneity of variance test and a normality test. All charts in this article were plotted in the Origin 9.0 software.

Effect of sunlight on appearance of seedling and sapling leaves

When Japanese yew seedlings were exposed to high light conditions, their foliage turned a bronze color (the left side of Fig. 1A); however, Japanese yew saplings live in open environments (Fig. 1B). Photodamage occurred in seedling leaves exposed to high light conditions, indicating there may be some differences in photoprotection between Japanese yew seedlings and saplings.

Pigment content

Seedlings and saplings grown under the same light conditions did not show significant differences in Chl b and Chl (a+b; Table 1, P > 0.05). Chl a, Car, the Chl a/b ratio, and the Car/Chl ratio in sapling leaves were significantly higher than in seedling leaves (Table 1, P < 0.05).

Photosynthesis and photorespiration

The Pn-PFD curves are shown in Fig. 2. The light saturation points (LSP) in sapling and seedling leaves were approximately 1,000 μmol·m⁻²·s⁻¹ and 800 μmol·m⁻²·s⁻¹ PFD, respectively. The maximum photosynthetic rates (Pn-max) under LSP were approximately 12.1 μmol·m⁻²·s⁻¹ in saplings and 8.2 μmol·m⁻²·s⁻¹ in seedlings. The sapling leaves had a significantly higher CO₂ assimilation capacity beyond LSP than the seedling leaves (Fig. 2, P < 0.01). As shown in Fig. 2, 1,200 μmol·m⁻²·s⁻¹ PFD is a strong light condition for the Japanese yew.

Compared with sapling leaves, photorespiration (Pr), gross photosynthetic rate (Pg), and the Pr/Pg ratio were all lower in seedling leaves, but only the difference in Pg between sapling and seedling leaves reached statistical significance (Fig. 3, P > 0.05).

The maximum quantum yield of photosystem II

After a period of dark adaptation, the maximum quantum yields of photosystem II (Fv/Fm) in seedling and sapling leaves were 0.83 and 0.84, respectively (Fig. 4, P > 0.05). Under high light conditions, the Fv/Fm in both seedling and sapling leaves decreased. However, a more significant decrease in Fv/Fm was observed in seedling leaves at 120 min (Fig. 4, P < 0.01), indicating that seedling leaves were more susceptible to high light conditions than sapling leaves. When the plants were returned to dark conditions, Fv/Fm recovery in seedling leaves was significantly slower than in sapling leaves (Fig. 4, P < 0.05). After 90 m of darkness, the Fv/Fm was still not completely restored in seedling leaves.

Chlorophyll fluorescence changes under high light conditions

When seedling and sapling leaves were exposed to high light conditions, there was a pronounced decline of $\mathrm{\Phi }$PSII observed over time (Fig. 5A). However, $\mathrm{\Phi }$PSII in sapling leaves was higher than in seedling leaves (P < 0.05). NPQ and qE in sapling leaves also remained higher than in seedling leaves (Figs. 5B and 5C, P < 0.05).

Change in xanthophyll cycle pigments

Energy dissipation is closely related to xanthophyll cycle pigments. Compared within seedling leaves, the size of the xanthophyll cycle pool was higher in sapling leaves (Figs. 6A and 6B, P < 0.05). There were no significant differences in de-epoxidation levels in the components of xanthophyll cycle pigments in the seedling or sapling leaves exposed to 1,200 μmol·m⁻²·s⁻¹ PFD for 2 h (Fig. 6C, P < 0.05).

The effect of epicuticular wax on gas-exchange

When treated with 500 PFD to 2,000 PFD, the photosynthetic rate in sapling leaves without wax was significantly higher than in leaves with wax (Fig. 7B, P < 0.05). However, there was no significant difference in the photosynthetic rate in the seedling leaves with or without wax (Fig. 7A, P > 0.05). The sapling leaves without wax also had higher Gs and Tr values compared to the sapling leaves with wax (Figs. 7D and 7F, P < 0.05). However, there was no significant difference in Gs and Tr values in the seedling leaves with or without wax (Figs. 7C and 7E, P > 0.05).

The total wax weight was significantly higher on sapling leaves than on seedling leaves (Fig. 8, P < 0.01).

Chlorophyll fluorescence with or without wax

Leaves with and without wax showed a reduction in Fv/Fm when grown under full sun for 2 h (Fig. 9). Fv/Fm values were restored to pre-exposure values after returning to low light conditions for 1.5 h. The Fv/Fm in sapling leaves with wax was significantly higher than in sapling leaves without wax after exposure to full sun for 2 h (Fig. 9B, P < 0.05). However, there was no significant difference in Fv/Fm in the seedling leaves with or without wax (Fig. 9A, P > 0.05).