As the plant material, we selected Veronica pusanensis which is an endemic species of South Korea and has high ornamental value. We selected plants around the shoot height of 3 cm and shoot width of 5 cm and applied them to the study.

The study was conducted for a total of 5 weeks from May 9 to June 14, 2022, at the experimental greenhouse (37°38′40″ N 127°06′25″ E) in the Department of Environmental Horticulture, Sahmyook University. Plants were planted in round plastic pots with a diameter and height of 11 × 10.5 cm, at the center of the pot. Here, we used horticultural substrates containing fertilizers (Hanareumsangto, Shinsung Mineral, South Korea). We designed six shading levels: no shade (i.e., direct sunlight) (0%), greenhouse glass (35%), greenhouse glass and 1 layer of clear polyethylene (PE) shading film (45%), greenhouse glass and 1 layer of white PE shading film (60%), greenhouse glass and 2 layers of white PE shading film (75%), and greenhouse glass and 1 layer of black PE shading film (99%). For the photosynthetic photon flux density (PPFD) of each shading level, we calculated the mean and standard deviation of the measurements taken at 1 p.m. once a week on sunny days using a portable spectral radiometer (SpectraPen mini, Photon Systems Instruments, Czech Republic) (Table 1).

The plants of all treatments including the control (0% shading level) were arranged on beds that are 1 m high, and the average temperature in the greenhouse during the study was 20.9 ± 2.1°C, and the average temperature of the outdoor area where the control was placed was 20.1 ± 2.0°C (Fig. 1A). Here, the relative humidity in the green-house was 58.5 ± 9.5%, and the relative humidity of the outdoor area was 60.0 ± 9.6% (Fig. 1B). The average cloud cover was 4.6 ± 2.2 okta, and this was evaluated by visual observation, which is the same as the measurement method of Korea Meteorological Administration, at 1 p.m. every day (Fig. 1C). The plants were watered 3 times a week. In this study, watering continued until gravitational water drainage occurred.

We measured shoot height, shoot width, root length, shoot fresh and dry weight, root fresh and dry weight, ground cover, leaf length, leaf width, and leaf color of CIELAB L^*, a^*, and b^* values. Here, we measured shoot height from the soil surface in the pot up to the point where the plant is the tallest and shoot width by measuring the broadest area of the plant seen from above. For root length, we measured the longest of the plant roots. Secondary analysis was conducted for ground cover by squaring the shoot width of the plant width. Fresh weight was measured after washing the plant and naturally drying it for 12 hours in an enclosed space, and dry weight was measured after hot air drying of the plant for 24 hours using a hot air dryer (HK-DO135F, HANKUK S&I, South Korea). CIELAB values (L^*, a^*, and b^*) were measured by setting the spectrophotometer (CM-2600d, Konica Minolta, Japan) to CIELAB D65/10° with reference to the leaf color measurement method Lee et al. (2022a), after which we collected data including specular components. For RHS values, we compared the results of each of L^*, a^*, and b^* with the Royal Horticultural Society (RHS) colour charts edition V and evaluated the leaf colors by selecting 2 approximate RHS values for each treatment. In addition, for leaf colors, we converted the means of CIELAB L^*, a^*, and b^* to converted colors using Converting Colors designed by Zettl (2023).

We analyzed the chlorophyll content and photosynthetic state of V. pusanensis affected by shading levels. Here, we measured the chlorophyll content using a portable chlorophyll meter (SPAD-502, Konica Minolta, Japan) and estimated the chlorophyll content relative to the same area for each treatment using SPAD units. We measured chlorophyll fluorescence responses using a portable chlorophyll fluorometer (FluorPen FP 110/D, Photon Systems Instruments, Czech Republic) by measuring the part where the leaf veins do not pass among the central part of the fully developed leaf. Here, before measuring the chlorophyll fluorescence, the plant was dark-adapted for approximately 15 minutes using dark-adaptation leaf clips according to the manufacturer’s guidelines (PSI, 2023), and we measured 5 times each in 3 replications on June 14, 2022, which is the end date of the study. We selected and applied five chlorophyll fluorescence parameters that can be used as plant stress indicators. We used the following equations to calculate F_v/F_m (1) representing the maximum quantum yield of photosystem II (PSII), Φ_Do (2) representing the probability that absorbed photons will be dissipated, ABS/RC (3) representing absorption flux per reaction center, DI_o/RC (4) representing dissipated energy flux per reaction center, and PI_ABS (5) that represents the performance index of absorption basis (PSI, 2023; Stirbet and Govindjee, 2011).

Analysis of variance (ANOVA) was conducted using SAS 9.4 (SAS Institute, USA) to analyze the results of the study. To compare the means, statistical analysis was conducted using Duncan’s multiple range test at p < .05. The study was in a completely randomized design, with 5 plants assigned to each treatment in 3 replications.

Shading levels had diverse effects on the plant sizes of Veronica pusanensis (Fig. 2). The results showed that shoot height of V. pusanensis was highest at 14.78 cm under the 35% shading level (Fig. 3A). Sedum zokuriense showed the highest shoot height under the 65% shading level (Lee et al., 2021b), whereas Sarcandra glabra showed the highest shoot height under the 50% shading level and Ardisia crenata under the 35% shading level (Bae et al., 2016), indicating that the preferred shading levels vary among species. Meanwhile, shoot width was widest at 29.21 and 28.84 cm under the 35 and 45% shading levels, indicating that shade culture is better than cultivation under direct sunlight in order to significantly increase the plant shoot sizes (Fig. 3B). Root length was longest at 21.87 cm under the 60% shading level (Fig. 3C). A previous study reported that root length of V. nakaiana and V. pyrethrina was in inverse proportion to the increase of shading levels (Kwon et al., 2020). On the contrary, in this study, root length increased along with shading levels from 0% to 60%, showing conflicting results from a previous study, but the results from 75% to 99% shading levels were similar to a previous study. Like the results of shoot width, ground cover was high at 868.1 and 861.8 cm² under the 35 and 45% shading levels (Fig. 3D). Leaf length was highest at 3.57 cm under the 35% shading level (Fig. 3E). Meanwhile, leaf width was highest at 3.46 cm under the 35% shading level, which was similar to the results of shoot height, shoot width, and leaf length (Fig. 3F). A previous study showed similar results as this study with Litsea japonica showing the highest leaf length and leaf width under the 35% shading level, but Osmanthus fragrans var. aurantiacus and Pittosporum tobira showed the highest leaf length and leaf width under the 50% shading level (Choi et al., 2012a), indicating that the responses to leaf growth according to shading levels vary among species. Phenotypic plasticity is the ability of a plant to change its morphological characteristics so that it can adapt to the changing growth environment (Bradshaw, 1965; DeWitt et al., 1998; Sultan, 1987). Among phenotypic plasticity, shade-induced phenotype induces the plant to show the morphological characteristics suitable to shade (Weijschede et al., 2006). In a shade-induced phenotype, we can expect to see an increase in the plant’s shoot height, shoot width or ground cover, and leaf sizes (Lee and Nam, 2022b), and this change is expected to help buffer the negative impact on growth due to the reduced amount of light received in shade.

Shoot fresh weight was highest at 18.51 g under the 35% shading level (Fig. 4A). Meanwhile, V. rotunda var. subintegra showed the highest shoot fresh weight when grown under the 55% shading level in the seedling process (Lee et al., 2020b). Root fresh weight was highest at 3.53 g under the 0% shading level (Fig. 4B). Shoot dry weight was highest at 2.58 g under the 35% shading level (Fig. 4C). Similarly, stem dry weight of Illicium anisatum was highest under the 35% shading level (Choi et al., 2012b). Root dry weight was highest at 0.87 g under the 0% shading level (Fig. 4D). Both root fresh weight and dry weight were highest under the 0% shading level, which was contrary to the result that root length was highest under the 60% shading level. This may be because factors such as carbohydrate content and thickness in the main root increased while the length of the side roots relatively decreased as there was a greater amount of light received. In addition, stimulation by water stress may have also caused an increase in the root fresh weight and dry weight. In previous studies, root fresh weight and dry weight of V. nakaiana and V. pyrethrina (Kwon et al., 2020) and V. rotunda var. subintegra were highest under the 0% shading level (Lee et al., 2020a), and root dry weight of Distylium racemosum, Neolitsea aciculata, and Stauntonia hexaphylla was also highest under the 0% shading level, showing consistent results with this study (Choi et al., 2012b). In summary of the results of this study and previous studies, shading was better than direct sunlight to significantly increase shoot biomass. On the other hand, to significantly increase root biomass, it was relatively more beneficial to grow under direct sunlight. In addition, in the plant sizes and biomass analysis, the control (0% shading level) was placed outside the greenhouse and shading film due to limitations in the study, and thus it could have been affected by additional abiotic stress due to air temperature and soil temperature rise, humidity change, and damage caused by rainfall. In particular, as shown in the results, there may have been a negative effect on shoot growth, which is something to refer to.

CIELAB is the color space made in 1976 by the International Commission on Illumination (Commission Internationale de l’Eclairage; CIE) in France (Lee et al., 2022c). CIELAB was developed in 1976 to make up for the deficiencies of Hunter Lab developed in 1958, and unlike Hunter Lab indicated in L, a, and b, it is indicated in L^*, a^*, and b^* that include asterisk (Lee and Nam, 2022a). In this study, the leaf color analysis of V. pusanensis using CIELAB showed different results by shading level (Table 2). CIELAB L^*, which is the color space coordinate representing lightness, was highest at 46.90 under the 0% shading level. Meanwhile, CIELAB a^* that represents green-red opponent colors showed the same significance level under the 35–60% shading levels. CIELAB b^* that represents blue-yellow opponent colors was highest at 24.64 under the 0% shading level. b^* tended to be relatively high in the treatment where growth parameters such as shoot height, shoot width, fresh weight, and dry weight were low. The results of previous studies showed some similarities with this study, proving that the growth rates of Orostachys japonica and O. boehmeri (Lee et al., 2022c), and Delosperma cooperi (Lee et al., 2022d) had a negative correlation with b^*. Meanwhile, L^* and b^* showed a positive correlation with each other (Kim et al., 2022; Lee et al., 2022c; 2022d). Similarly, this study also showed that L^* and b^* increased together under the 0% shading level, implying that it would be possible to use L^* and b^* as stress indicators for the leaf color of some plants including V. pusanensis. Royal Horticultural Society (RHS) values were 146B and 147B under the 0% shading level, showing that the color was relatively closer to yellow. On the other hand, the values were 137A and N137D under the 35–60% shading levels, showing that the leaf color was relatively green.

As a result, assuming that increased plant sizes and dark green leaf color contribute positively to the purchasing power of consumers when supplying V. pusanensis as an ornamental flower crop, it is recommended to grow V. pusanensis under the 35% shading level in order to significantly increase plant sizes and maintain dark green leaf color.

Chlorophyll content (SPAD units) was highest in the order of 35 to 45% shading levels at 50.94 and 50.55, respectively, showing the same results as shoot width and ground cover (Fig. 5A). This is consistent with the results of previous studies that, when growing Hylotelephium telephium (Nam et al., 2022), Hoya carnosa and Spathiphyllum wallisii (Lee et al., 2021a), and S. zokuriense (Lee et al., 2021b) grown under conditions of optimal shading levels and light intensity, an increase in chlorophyll content was observed concomitant with high shoot width. Meanwhile, V. rotunda var. subintegra showed relatively higher chlorophyll content under the 55–75% shading levels compared to the 0% shading level (Kwon et al., 2020). In summary of the results above, some plants including V. pusanensis seem to have homeostasis to maintain a constant total amount of carbon dioxide assimilation by maximizing plant sizes and chlorophyll density under low light intensity. F_v/F_m, which represents maximum quantum yield of photosystem II (PSII), was highest at 0.85 under the 35% shading level, and lowest at 0.81 under the 99% shading level (Fig. 5B). F_v/F_m, which is a parameter that represents a maximum quantum yield of PSII, is known to range from 0.78 to 0.84 in higher plants species that are not stressed (Björkman and Demmig, 1987; Butler and Kitajima, 1975; Genty et al., 1989; Govindjee, 1995; Govindjee, 2004; Paillotin, 1976; Yoo et al., 2012). F_v/F_m was slightly higher than the normal range under the 35% shading level, whereas it was within the normal range in other treatments. In particular, the maximum quantum yield was significantly low under the 99% shading level where the light intensity was extremely low, but it did not have a negative effect so much as to significantly deteriorate the function of the reaction center of PSII. As a result, plant sizes or biomass of V. pusanensis was relatively low under the 99% shading level, but shade tolerance was excellent, and growth rates are expected to be quickly recovered by moving the plant to an environment with high light intensity. Φ_Do that represents the probability that the absorbed photon will be dissipated was highest at 0.18 under the 99% shading level (Fig. 5C). ABS/RC that represents absorption flux per reaction center was highest at 1.99 under the 99% shading level (Fig. 5D). ABS/RC, one of chlorophyll fluorescence parameters, represents the state of the reaction center that is inactivated as the number of reaction centers in reduction state increases in PSII (Spoustová et al., 2013). Meanwhile, DI_o/RC that represents dissipated energy flux per reaction center was highest at 0.38 under the 99% shading level, indicating that the energy use efficiency has decreased (Fig. 5E). PI_ABS is a performance index with an absorption basis (Srivastava et al., 1999). This index represents the degree of photosynthetic activity and is a set of light energy absorbing capacity, electron transfer efficiency, and electron fixation efficiency of PSII (Thach et al., 2007). PI_ABS represents the overall vitality of the photosynthetic apparatus (Strasser et al., 2000; Živčák et al., 2008), and it is used as an index that represents the soundness of plants (Oukarroum et al., 2007). PI_ABS was highest at 8.13 under the 35% shading level (Fig. 5F). This result was consistent with the results of chlorophyll content, shoot fresh weight, and dry weight, indicating that higher PI_ABS leads to higher chlorophyll density and more carbon dioxide assimilation.

In conclusion, as a result of comprehensively evaluating the plant sizes, biomass, leaf color, and photosynthetic responses analysis of V. pusanensis affected by shading levels, we discovered that shading levels significantly increase the size of V. pusanensis and maintain dark green leaf color. It is recommended to cultivate the plant under the 35% shading level to maintain the activity of PSII in the optimal state.