Distinct non-photochemical quenching characteristics under constant and fluctuating light conditions: comparison between local and imported tobacco varieties

Plant Physiology and Biochemistry Distinct non-photochemical quenching characteristics under constant and fluctuating light conditions: comparison between local and imported tobacco varieties --Manuscript Draft-- Manuscript Number: Article Type: Research Paper Section/Category: Photosynthesis; plant-microbe interactions (biotic stress + beneficial) Keywords: non-photochemical quenching; local and imported varieties; fluctuating light; xanthophyll cycle; tobacco Corresponding Author: Yingchao Lin Guizhou Tobacco Science Research Institute Guiyang, CHINA First Author: Hui Lyu Order of Authors: Hui Lyu Zhaoqi Tang Weijie Xu Dejun Kong Zhihong Wang Zhixiao Yang Jishun Zhang Hongqi Wu Zili Wang Dusan Lazar Yingchao Lin Abstract: To explore the variation in adaptability to fluctuating light environments and optimize their photosynthesis and growth, the non-photochemical quenching (NPQ) characteristics of a local tobacco variety Xiaofeng 8 and a foreign introduced variety K326, under both constant and fluctuating light conditions were examined. Under high light intensities, Xiaofeng 8 exhibited a higher NPQ value compared to K326, indicating an enhanced capacity for dissipating excess light energy as thermal energy and mitigating the potential risk of photodamage. The induction changes of NPQ and photosystem II efficiency (ΦPSII) from dark to low and high light conditions were analyzed, revealing distinct adaptability to high light environments and associated photoprotection mechanisms in Xiaofeng 8. While Xiaofeng 8 induced the regulatory non-photochemical quenching in high light, K326 realized the protection by the constitutive non-photochemical quenching, which preserved also to low light conditions and seem to be the reason for a lower growth of K326 than of Xiaofeng 8 in natural light condition, i.e., under fluctuating light. Additionally, the role of xanthophyll cycle pigments in regulating NPQ and energy dissipation under high light intensities was examined using mutants defective in violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). The results showed that defects in these enzymes affected the NPQ response and reduced the growth rate of the mutants under fluctuating light conditions, suggesting a crucial role of the xanthophyll cycle in plant growth and adaptation to variable light environments. Collectively, these findings emphasize the importance of exploring genetic resources improving plant NPQ under fluctuating light conditions from both domestic and imported varieties, thereby providing promising targets for selecting and breeding new crop varieties with improved yield, quality, and resistance, ultimately enhancing breeding efficiency and crop adaptability. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation 1/25 Distinct non-photochemical quenching characteristics under constant and fluctuating light conditions: comparison between local and imported tobacco varieties Hui Lyu1,†, Zhaoqi Tang2,†, Weijie Xu2, Dejun Kong3, Zhihong Wang3, Zhixiao Yang3, Jishun Zhang3, Hongqi Wu4, Zili Wang3, Dusan Lazar5, Yingchao Lin3,* 1 School of Biological Science and Agriculture, Qiannan Normal University for Nationalities, Duyun, China 3 Guizhou Academy of Tobacco Science, Guiyang, China 2 Shanghai Tobacco Group Co., Ltd, Shanghai, China 4 College of Tobacco Science, Guizhou University,Guiyang, China 5 Department of Biophysics, Faculty of Science, Palacky University, Olomouc, Czech Republic * Correspondence: [email protected] † These authors contributed equally to this work. Two genotype NPQ by YL.docx Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2/25 Abstract: To explore the variation in adaptability to fluctuating light environments and optimize their photosynthesis and growth, the non-photochemical quenching (NPQ) characteristics of a local tobacco variety Xiaofeng 8 and a foreign introduced variety K326, under both constant and fluctuating light conditions were examined. Under high light intensities, Xiaofeng 8 exhibited a higher NPQ value compared to K326, indicating an enhanced capacity for dissipating excess light energy as thermal energy and mitigating the potential risk of photodamage. The induction changes of NPQ and photosystem II efficiency (ΦPSII) from dark to low and high light conditions were analyzed, revealing distinct adaptability to high light environments and associated photoprotection mechanisms in Xiaofeng 8. While Xiaofeng 8 induced the regulatory non-photochemical quenching in high light, K326 realized the protection by the constitutive non-photochemical quenching, which preserved also to low light conditions and seem to be the reason for a lower growth of K326 than of Xiaofeng 8 in natural light condition, i.e., under fluctuating light. Additionally, the role of xanthophyll cycle pigments in regulating NPQ and energy dissipation under high light intensities was examined using mutants defective in violaxanthin de-epoxidase (VDE) and zeaxanthin epoxidase (ZEP). The results showed that defects in these enzymes affected the NPQ response and reduced the growth rate of the mutants under fluctuating light conditions, suggesting a crucial role of the xanthophyll cycle in plant growth and adaptation to variable light environments. Collectively, these findings emphasize the importance of exploring genetic resources improving plant NPQ under fluctuating light conditions from both domestic and imported varieties, thereby providing promising targets for selecting and breeding new crop varieties with improved yield, quality, and resistance, ultimately enhancing breeding efficiency and crop adaptability. Key words: non-photochemical quenching; local and imported varieties; fluctuating light; xanthophyll cycle; tobacco 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3/25 Introduction In natural environments, plants often face fluctuations in light intensity due to various factors such as cloud cover, leaf movement in the wind, plant shading and changes in solar angle (Long et al., 2006; Taylor and Long, 2017). This fluctuation in light intensity can lead to rapid changes in the intensity of light received by plant leaves, have a significant impact on the photosynthesis of plants. On the one hand, fluctuations in light intensity can affect the rate and efficiency of photosynthesis in plants, thereby affecting their growth, development, and yield (Slattery et al., 2018). On the other hand, with the rapid increase in light intensity, when plants absorb more light energy than they need for photosynthesis, the excess light energy may cause damage to plant cells (Shi et al., 2022; Shafiq et al., 2021). To cope with such challenges, plants have evolved various photoprotective mechanisms, among which the energy dependent non-photochemical quenching (qE) plays a crucial role (Bielczynski et al., 2017; Shi et al., 2022; Lu et al., 2022; Niu et al., 2023). The qE is a rapidly inducible process that enables plants to dissipate excess absorbed light energy as heat, thereby protecting their photosynthetic apparatus from photodamage (Nicol et al., 2019). The qE requires low pH of thylakoid lumen to protonate PSII subunit S (PsbS) (Brooks et al., 2014; Li et al., 2000) and to activate violaxanthin-deepoxidase (VDE), which converts violaxanthin (V) via antheraxanthin (A) to zeaxanthin (Z) (Demmig-Adams 1990; Yamamoto et al., 1962). Due to different genetic characteristics and photosynthetic mechanisms, there are differences in the qE mechanisms of plant varieties from different regions and genotypes (Tanaka et al., 2019). For example, overexpression of three transgenes (AtVDE, AtPsbS, and AtZEP) in soybean and tobacco, which are required to enhance setting and decay of qE under high light or rapidly fluctuating conditions as widely described previously, accelerated the relaxation of NPQ during sun-to-shade transitions, resulting in increased photosynthetic efficiency and crop productivity under fluctuating light (Kromdijk et al., 2016; De Souza et al., 2022). At the same time, it was found that some rice and corn varieties have higher NPQ ability, which can better dissipate excess light energy, protect the photosynthetic system from light damage, maintain high photosynthetic rate and yield, while other varieties cannot effectively cope with high light induced photodamage, resulting in a decrease in photosynthetic rate and yield (Li et al., 2020; Wang et al., 2017; Tsai et al., 2019). There are differences in the NPQ ability of plant varieties with different origins to high light, which are mainly due to the different genetic backgrounds, and evolutionary histories of their respective growth environments. Understanding these differences helps us 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4/25 to better understand the photosynthesis mechanism and photoprotection strategies of plants, providing scientific basis and technical support for agricultural production. While NPQ has been extensively studied under stable light conditions, it is important to note that plants often face fluctuations in light intensity in nature, as described earlier. Therefore, understanding the NPQ response of plants under fluctuating light conditions is of great significance. By studying the response of NPQ to fluctuating light, we can better understand how plants adapt to these environmental changes, and how to improve plant stress resistance, photosynthetic efficiency, and yield through genetic engineering and other means, providing new solutions for ensuring global food security. Tobacco is well-known as a smoking material but it has always been considered as a medicinal plant and used as a traditional medicine for common illnesses (Charlton 2004, Berlowitz et al. 2020). In present knowledge, nicotine contained in tobacco is an agonist to acetylcholine, the later being natural chemical involved in ligand-gated nicotinic acetylcholine receptors, i.e. ligand-gated ion channels. These ion channels are indispensable for proper transmission of electric signals in synapses between neurons and neuron and muscle cells (Hogg et al. 2003) Tobacco plants can be also used as a natural living laboratory to produce human therapeutic reagents (Sanchez-Ramos 2020). These facts indicate that planting tobacco might be useful but it is important to know, which variety of tobacco best survives in the environment. Xiaofeng 8 is a common tobacco variety in Guizhou, China. It is characterized by a more robust growth rate in the field and a higher productivity of biomass. On the other hand, K326 tobacco variety was exported to China. This variety is resistant to the common root-knot nematodes and it is known for high quality and curability (see https://crosscreekseed.com/usa-varieties/usa-flue-cured-tobacco-seed/k-326/). In this work, we studied if the seemingly superior K326 variant is better in grown in the field, i.e., under fluctuating light conditions. Namely, we investigate the NPQ characteristics of two tobacco cultivars, Xiaofeng 8 and K326, under both constant and fluctuating light conditions. By comparing their NPQ responses, we aimed to gain insights into how plants adapt to fluctuating light environments and optimize their photosynthesis and growth. Furthermore, we also examined the role of xanthophyll cycle pigments in regulating NPQ and energy dissipation under high light intensities. Our findings provided valuable insights into the photoprotective strategies of plants and have implications for crop breeding and genetic engineering to improve photosynthetic efficiency and yield under fluctuating light conditions. Materials and methods 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5/25 Plant materials and growth conditions For the field experiment, the tobacco cultivars Xiaofeng 8, a local landrace, and K326 that introduced from USA, were selected as study materials on the basis of plant size and growth rate according to previous observations. The trial was conducted in Qingzhen (lat. 26°31′27″N, long. 107°21′16″E) in 2022, at an altitude of 1280 m, in Guizhou, China. Ten-day average temperature and 10-day sunshine hour records from March to October are presented in Fig. 1. Note: what is 10-day average temperature and ten-day sunshine hour? Specify it differently so that it is clear what you mean. The soil properties (0–20 cm) at the study site are presented in Lin et al. (2020). Seeds were sown in an open greenhouse on 25 Feb. 2022 on transplanting disks as described previously in detail (Lin et al., 2017). A series of agricultural operations, including turning up the soil, ridging, plastic mulching, and planting hole preparation, were carried out as described previously in detail (Lin et al., 2015, 2017). Seedlings were transplanted into the test plots (15×7 m) with three replicates for each genotype on 26 Apr. 2023. For detailed observations and measurements, fifteen plants were randomly selected from both cultivars. The vde (LOC104218706) and zep (LOC104218557) knockout mutant were generated from Biorun Biosciences (Wuhan, China) by CRISPR–Cas9, using a single-guide RNA. To investigate the effects of xanthophyll cycle on seedlings pigments, NPQ characteristic and growth under fluctuating light conditions, confirmed vde and zep knockout mutant seeds were sown as described previously in detail (Lin et al., 2017). Seedlings that were 45-day old were transplanted to plastic pots (diameter 7 cm; height 7 cm) filled with organic soil and maintained in a growth chamber with a 16/8 light/dark cycle. A constant light intensity of 1000 µmol photons m-2 s-1 was set for pigments and NPQ characteristic investigation while the light intensity for growth phenotype experiment was set to fluctuate every 3 min, randomly among 70, 400, 700, 900 µmol m−2 s−1 with an overall fractional contribution of 0.1, 0.3, 0.4, 0.2, respectively, which ensured the average light intensity was close to 600 µmol photons m-2 s-1. Chlorophyll a fluorescence measurements Chlorophyll a fluorescence (ChlF) was measured using a portable gas exchange system LI-6400XT (LiCor Inc., Lincoln, NE, USA). The kinetics were first recorded on attached leaves of 30 min dark-adapted plants using actinic red light 70 µmol photons m-2 s-1 (low light) or 1000 µmol photons m-2 s-1 (high light) for 6 min. For experiments in fluctuating light, attached leaves of 30-min dark-adapted plants were illuminated for four cycles of 3 min low light (70 μmol photons m−2 s−1) and 3 min high light (1000 μmol photons m−2 s−1), NPQ induction and relaxation were monitored at 20 s intervals. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6/25 The following parameters were determined from measured ChlF values (see Lazár 2015 for a review): effective quantum yield of PSII photochemistry in light-adapted state (ΦPSII) calculated as (Fm′–F)/Fm′, relative PSII electron transport rate (ETR) as ΦPSII×PAR×0.84×0.5, and non-photochemical quenching parameter (NPQ) as (Fm–Fm′)/Fm′, where Fm is maximum ChlF in dark-adapted state, Fm′ is maximum ChlF in light-adapted state, and F is ChlF in the actinic light. Assays of Plant growth Leaf area was measured using the fresh leaf by Area meter (AM 300, ADC Bio-scientific Ltd., UK) and replicated for three times with 5 seedlings per replicate. Number of leaves was surveyed at different time for leaf number determination. Plant height was measured by the distance from the first leaf base from bottom to the growing points of shoots (the apical meristems). The dry weight (DW) of the leaves was determined using the mass method after drying the plants at 105°C for 48 h. Leaf pigment analysis Pigments were analysed by reverse-phase-performance liquid chromatography. For pigments extraction, leaf discs were frozen in liquid nitrogen and suspended in KOH methanol solution (20%), keep at 55 °C for 30 minutes. Extraction solution (acetone: ethyl acetate=1:2, v/v) was added after cooling and ultrasonic extraction for 40 minutes. After centrifuge for 15 (4 ℃, 8000 g), The supernatant was evaporated to dryness. After diluted with acetone, extracted pigments were filtered through a membrane filter (pore size 0.2 μm), and used for high-performance liquid chromatography analysis. The contents of Leaf pigments were measured simultaneously using a Welch Ultimate C30 (5 μm, 4.6×250 mm) (Welch, Shanghai, China) with detection wavelength and column flow rate were 450nm and 1.0ml/min, respectively. The xanthophyll cycle de-epoxidation state (DEPS) has been shown to have a better linear relationship with plant heat dissipation capacity, and it was calculated as DEPS=(Z+0.5A0/(V+Z+A) (Johnson et al., 1993). The data analysis Data analysis software SPSS 17.0 was used for one-way analysis of variance (ANOVA), and the significant differences between genotype were indicated with different lowercase letters (P≤0.05). Statistical data were expressed as average value ± standard error (SE). Results Seedlings of Xiaofeng 8 grew faster in the field experiment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7/25 After being transplanted for 20 d, significant phenotypic differences were observed between seedlings of Xiaofeng 8 and K326 (Fig. 2A). In terms of plant height, K326 exhibits a significantly lower growth than Xiaofeng 8, which indicates a more vigorous vertical growth pattern in Xiaofeng 8 compared to K326 (Fig. 2D). Other growth parameters such as leaf number, maximum leaf area, and fresh weight of Xiaofeng 8 were significantly higher than those ones of K326 (Fig. 2B, C, E and F). Overall, Xiaofeng 8 shows a more robust growth pattern compared to K326. A stronger ability to dissipate excess light energy in Xiaofeng 8 under high light intensity To investigate the photosynthetic acclimation of the two tobacco varieties to different light conditions, induction of NPQ parameter and effective quantum yield of PSII photochemsitry, ΦPSII, during the dark to low and high constant light conditions were analyzed. During the first 40 seconds on a shift from dark to low light conditions (70 μmol photons m-2 s-1), value of NPQ parameter increased and then gradually decreased in K326 seedlings, while the NPQ parameter gradually decreased in Xiaofeng 8 seedlings (Fig. 3A). On the other hand, a rapid increase in NPQ were observed in both K326 and Xiaofeng 8 seedlings on shift from dark to high light condition (1000 μmol photons m-2 s-1), while the steady state value of NPQ in Xiaofeng 8 was higher than those of K326 (Fig. 3B). Quantum yield of PSII photochemistry in light-adapted state (ΦPSII) of both K326 and Xiaofeng 8 initially decreased on shift from dark to both low and high light, followed by an increase with increasing time (Fig. 3C and D). While the initial decrease was more pronounced upon transition to high light than to low light, the following increase was higher upon transition to low light than to high light. Nevertheless, no obvious differences were detected in changes of ΦPSII between the two varieties under same light intensity (Fig. 3C and D). More detailed exploration of dependence of the steady-state values of the NPQ parameters, ΦPSII, and of the relative PSII electron transport rate (ETR) on intensities of illumination by constant photosynthetic active radiation (PAR) is presented in Fig. 4. There is no significant difference in ETR and ΦPSII between K326 and Xiaofeng 8 varieties during exposure to an increasing amount of constant PAR (Fig. 4A and 4B) and the same is true for the NPQ parameter at PAR levels < 600 μmol photons m2 s-1 (Fig. 4C). However, a higher steady state NPQ values were detected at constant PAR > 400 μmol photons m-2 s-1 in Xiaofeng 8 than those of K326 (Fig. 4C). Characteristics of NPQ and ΦPSII in fluctuating light in K326 and Xiaofeng 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8/25 To investigate the response of NPQ parameter to continuously fluctuating light, seedling leaves of both two tobacco varieties were exposed to alternating cycles of 180 s at high light and 180 s low light. As for the dark-to-low light transition, Xiaofeng 8 displayed insignificantly lower NPQ parameter value compared with those of K326. After transitions from low to high light of every alternating cycle, value of NPQ parameter of Xiaofeng 8 increased rapidly and reached a higher level than those of K326, and this pattern was also detected during the rest high light period throughout the entire light cycle. After transitions from high to low light, the value of NPQ parameter decreased rapidly and no obvious change were detected between K326 and Xiaofeng 8 (Fig. 5A). Interestingly, no obvious change were detected in ΦPSII between the two plants throughout the entire low-high light cycle (Fig. 5B). During low light periods, the value of ΦPSII is relatively high. However, when the light intensity increases to high light, the ΦPSII value rapidly decreases. Responses of xanthophyll cycle pigments in K326 and Xiaofeng 8 to high light In order to underlie the mechanisms regulating their ability to dissipate excess light energy under high light, response pattern of xanthophyll cycle pigments in K326 and Xiaofeng 8 seedlings were analyzed after exposure to high light (1000 μmol photons m-2 s-1). The results showed that, no significant difference were detected in the three pigments, and the DEPS state between the two varieties in dark. After exposure to high light, the content of V in Xiaofeng 8 decreased rapidly within the first 10 min and reduced to half at most after 10 min. On the other hand, the contents of A, and Z were increased after transition from dark to high light (Fig. 6). The pattern of changes of xanthophyll cycle pigments in K326 is similar to that of Xiaofeng 8, except that the content of V is lower in Xiaofeng 8 than in K326 in high light and content of A, Z, and DEPS is higher in Xiaofeng 8 than in K326 in high light (Fig 6). Altered xanthophyll cycle pigments affected seedlings NPQ under high light intensity To further investigate the role of xanthophyll cycle pigments in regulating plants ability to dissipate excess energy under rapidly fluctuating light conditions, mutants with malfunctional VDE or zeaxanthinepoxidase (ZEP), enzymes catalyzing the de-epoxidation of V via A to Z and the epoxidation of Z via A to V, respectively, were analyzed under various light conditions and compared with the wild type plant (K326) as control. Lack of the two enzymes has significantly altered xanthophyll cycle pool size. 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Biophys. 97, 168-73. https://doi.org/10.1016/0003-9861(62)900607. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 17/25 Distinct non-photochemical quenching characteristics under constant and fluctuating light conditions: comparison between local and imported tobacco varieties Hui Lyu1，†, Zhaoqi Tang2，†, Weijie Xu2, Dejun Kong3, Zhihong Wang3, Zhixiao Yang3, Jishun Zhang3, Hongqi Wu4, Zili Wang3, Dusan Lazar5, Yingchao Lin3,* 1 School of Biological Science and Agriculture, Qiannan Normal University for Nationalities, Duyun, China 2 Shanghai Tobacco Group Co., Ltd, Shanghai, China 3 Guizhou Academy of Tobacco Science, Guiyang, China 4 College of Tobacco Science, Guizhou University,Guiyang, China 5 Department of Biophysics, Faculty of Science, Palacky University, Olomouc, Czech Republic * Correspondence: [email protected] † These authors contributed equally to this work. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 18/25 Figure legends Figure 1. Ten-day average sunshine hour and 10-d average temperature from April to October at Qingzhen in 2023. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 19/25 Figure 2. Comparison of growth of K326 and Xiaofeng 8 seedlings grown in the field after transplanting for 20 d. (A) Phenotype of K326 (right ridge) and Xiaofeng 8 (left ridge) in the field. (B to F) Leaf number (B), maximum leaf area (C), plant height (D), plant fresh weight (E) and dry weight (F) of seedlings grown in the field after transplanting for 20 d in Qingzhen, 2022. Data are presented as means ± S.E.. (n > 10 biological replicates) and the different letters indicate significant differences (P < 0.05). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 20/25 Figure 3. Kinetics of NPQ (A and B) and ΦPSII (C and D) on transition from dark to constant low light (70 µmol photons m-2 s-1, A and C) and constant high light (1000 µmol photons m-2 s-1, B and D). Measurements were made after 30 min dark adaptation in the seedlings grown in the field after transplanting for 20 d in Qingzhen, 2022. Data are the means ±S.E.. Where not visible, error bars are smaller than the symbols. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 21/25 Figure 4. Light dependence of steady-state chlorophyll fluorescence parameters of the relative PSII electron transport rate (ETR, A), quantum yield of PSII photochemistry in light-adapted state (ΦPSII, B), and the non-photochemical quenching parameter (NPQ, C) in seedlings grown in the field after transplanting for 20 d in Qingzhen, 2022. Each point represents the mean of 5 measurements and error bars indicate the S.E. and the asterisk in (C) indicate significant differences (P < 0.05). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 22/25 Figure 5. Dynamics of NPQ parameter (A) and ΦPSII (B) to fluctuating light conditions simulated by four cycles of 3 min low light (70 μmol photons m−2 s−1, gray background) and 3 min high light (1000 μmol photons m−2 s−1, white background) as indicated. Measurements were made after 30 min dark adaptation in the seedlings grown in the field after transplanting for 20 d in Qingzhen, 2022. Data are the means ± S.E. Where not visible, error bars are smaller than the symbols. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 23/25 Figure 6. Changes in the content of violaxanthin (A), antheraxanthin (B), zeaxanthin (C), and the xanthophyll cycle de-epoxidation state (DEPS, D) during illumination with 1000 µmol photons m−2 s−1 in the youngest mature leaves of K326 and Xiaofeng 8 seedlings grown in the field after transplanting for 20 d in Qingzhen, 2022. Fully expanded leaves of seedlings that prior dark adapted for 30 min were collected after 0, 1 min, 5 min, 10 min, 20 min and 30 min of illumination at 1000 µmol photons m-2 s-1. Data represent means ± S.E. of n=5 individual plants. If not shown, S.E. is smaller than symbol. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 24/25 Figure 7. (A) and (B), Generation of mutations in VDE (LOC104218706, A) and ZEP (LOC104218557, B) by CRISPR–Cas9, using a single-guide RNA. Sequences of VDE and ZEP in wild-type (WT) and vde and zep mutant plants are shown. The deletion is indicated by a dashed line. (C-F), Contents of violaxanthin (C), antheraxanthin (D), zeaxanthin (E), and the xanthophyll cycle de-epoxidation state (DEPS, F) in 60-day-old wild type (K326), vde and zep knockout mutant plants. (G) and (H), Dynamics of NPQ parameter (G) and ΦPSII (H) to fluctuating light conditions simulated by four cycles of 3 min low light (70 μmol photons m−2 s−1, gray bars) and 3 min high light (1000 μmol photons m−2 s−1, white bars) as indicated in K326, vde and zep knockout mutant plants grown under constant light (1000 μmol photons m−2 s−1). Seedlings that were 45-day-old were moved to growth chamber with a 16/8 light/dark cycle and a constant light intensity of 1000 µmol photons m-2s-1. For (H) and (I), measurements were made after 30 min dark adaptation in more than five fully individual plans. Data are the means ±S.E.. Where not visible, error bars are smaller than the symbols, the different letters in (C-F) indicate significant differences (P < 0.05). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65