Effects of Seasonal Changes on Chlorophyll Fluorescence and Physiological Characteristics in the Two Taxus Species

Taxus is a rare and endangered woody plant worldwide with important economic and ecological values. However, the weak environmental adaptability of Taxus species, in particular the unstable photosynthetic activity in different seasons, always affects its normal growth and development and limits its conservation and exploitation. To improve the survival of Taxus trees in cultivated areas, the seasonal dynamics of chlorophyll fluorescence (CF) and key physiological parameters were comprehensively investigated in T. media and T. mairei. The results demonstrated that the photosynthetic activity of both Taxus species was sensitive to local summer and winter environmental conditions, with the heterogeneity of fluorescence signatures intuitively presented on the needle surface by CF-Imaging detection, while images of maximum quantum efficiency of PSII photochemistry (Fv/Fm) demonstrated values below 0.7 in the blue–green sectors in winter. The distribution of light energy was regulated by the photosynthetic apparatus in both Taxus species to maintain a stable actual quantum yield of PSII photochemistry (φPSII), which was around 0.4–0.5. Based on a redundancy discriminant analysis, the interpretation rate of light intensity and air temperature ranked as the top two in both Taxus species, which were considered the main environmental factors affecting the photosynthetic performance of Taxus by disturbing the electron transport chain. In the winter, T. mairei exhibited weaker electron transport activity than T. media, thus caused lower photochemistry and more severe photosynthetic damages. Interestingly, both Taxus species demonstrated consistent response patterns, including diverse energy dissipation strategies and enhancement of osmoregulatory substances and antioxidative activities, thus maintaining stable photosynthetic functions in response to environmental changes.


Introduction
Taxus is a small coniferous tree or shrub, which belongs to the genus Taxus and family Taxaceae, with great medicinal, ornamental, and ecological values. The Taxus species is well-known for the important biological active compound taxol, an effective drug used in the treatment of various cancers such as lung, breast, and ovarian [1,2]. However, the content of taxol in Taxus species is extremely low; only 1 kg of taxol could be extracted from thousand-century-old Taxus trees [3]. As the main and reliable source for taxol and its precursors, the Taxus species has almost suffered extinction due to over-exploitation and illegal trade, while all the members of the genus Taxus have been listed as species for first-grade protection in the National Key Protected Wild Plants List in China [4]. Therefore, Botanical Garden Mem. Sun Yat-Sen for cultivation and targeted research. In such conditions, the comparison of the photosynthetic responses to seasonal changes in Taxus species from different geographical sources could be conducted smoothly. It was predicted that these two Taxus species could possess different photosynthetic response strategies and protection mechanisms for the photosynthetic apparatus in different seasons based on the geographical distribution pattern of their wild resources and many years of local phenological observations. In this study, these two representatives were selected for dynamic measurements of Chl fluorescence and key physiological parameters in different months. The aim was to investigate the seasonal differences in photosynthetic and physiological characteristics, explore the specificity among different Taxus species and confirm the main environmental limiting factors at different time scales, thus comprehensively elaborate the process and related mechanism of photosynthetic physiological responses for Taxus. The results of this study will provide a reference for the assessment of survival states and rational conservation and application of Taxus species.

Seasonal Dynamics of Chlorophyll Fluorescence Parameters and Images in T. media and T. mairei
As shown in Table 1, the Fv/Fm of T. media and T. mairei demonstrated a fluctuating upward trend from January to April, and remained at 0.77-0.83 from April to September, then gradually decreased. The lowest values in both Taxus species appeared in January, around 0.60-0.70. Significant differences of Fv/Fm values between the two Taxus species were observed in January-March and June. The Fv /Fm showed similar trends as Fv/Fm from January to April, then maintained relatively stable levels until December. The values of T. media in March, May, June, August, and November were significantly higher than that of T. mairei. The NPQ of T. mairei increased from January to April, and remained above 2 from April to November (except for June), but dropped to 1.35 in December; while that of T. media fluctuated during the measurements, and was below 2 after July. The lowest NPQ values of T. media and T. mairei were observed in January around 1.22 and 0.88, respectively. The trends of qP in Taxus species were opposite to those of Fv /Fm . The highest qP values of T. media and T. mairei appeared in January, around 0.86 and 0.96, respectively. The trends of ϕPSII were relatively stable throughout the annual observation. The ϕPSII values of T. media were higher than that of T. mairei after February, and significant differences were observed in March, July, and August. Figure 1 shows representative false color images of Fv/Fm, Fv /Fm , NPQ, qP, and ϕPSII by CF-Imaging, which visually represented the spatial differences over the leaf surface of T. media and T. mairei in different months. Among them, the images of Fv/Fm exposed significant heterogeneity. In most cases, the leaves of both Taxus species were an orange-red color, which turned blue-green in January, and yellow-orange in February and March, as well as green-yellow after October, along with decreasing Fv/Fm values in varying degrees. Moreover, the leaves of T. mairei were heterogeneous within orange sectors between June and August, which also affected the comprehensive values. The images of Fv /Fm and ϕPSII in the two Taxus species showed significant differences between the green and blue-cyan sectors. The leaves in most cases showed blue-cyan colors, which represented relatively high levels as compared with the green ones observed in the Fv /Fm of both Taxus species in January, as well as ϕPSII of T. mairei in February and that of T. media in August. This was confirmed by the numeric data. The qP images resembled that of Fv /Fm and ϕPSII, revealing a heterogeneity of this parameter within the blue-cyan area. Moreover, orange-red sectors were also observed in the leaves of Taxus species within several months, which showed higher levels than the blue-cyan parts. The NPQ in T. media leaves was lower than in T. mairei ones after July, and lower in the green parts than in the blue-cyan areas. This parameter was also low in the brown parts as compared with the green ones, which could be observed in January and December. Notably, the heterogeneity of Chl fluorescence occurred not only on the whole plant leaves, but also on part of the whole plant leaves or parts of the leaves, such as leaf margins and veins, which determined the degree of changes in comprehensive values of Chl fluorescence parameters. This was particularly evident for Fv/Fm and ϕPSII, which also reflected the complexity of the photosynthetic response.

Changes of Chlorophyll a Fluorescence Transient and JIP-Test Parameters in T. media and T. mairei
Given the sensitivity of primary photochemical reactions to environmental changes in Taxus species, the Chl a fluorescence transient has been applied to monitor changes in the structure and states of the photosynthetic apparatus in representative months. As shown in Figure 2, the polyphasic OJIP rise was observed in leaves of Taxus species under different months. Notably, the J-I and I-P phases of both Taxus species increased significantly in July compared with that in April; while same phases of T. media were also higher in October than that in April. Moreover, the fluorescence intensity of OJIP rise in January decreased significantly and caused changes in the curve shapes. There was no obvious K-band appearing at 0.3 ms; however, the curves became flat as the J-, I-, and P-steps decreased sharply, which exhibited the same pattern in both Taxus species. Plants 2023, 11, x FOR PEER REVIEW 5 of 18

Changes of Chlorophyll a Fluorescence Transient and JIP-Test Parameters in T. media and T. mairei
Given the sensitivity of primary photochemical reactions to environmental changes in Taxus species, the Chl a fluorescence transient has been applied to monitor changes in the structure and states of the photosynthetic apparatus in representative months. As shown in Figure 2, the polyphasic OJIP rise was observed in leaves of Taxus species under different months. Notably, the J-I and I-P phases of both Taxus species increased significantly in July compared with that in April; while same phases of T. media were also higher in October than that in April. Moreover, the fluorescence intensity of OJIP rise in January decreased significantly and caused changes in the curve shapes. There was no obvious Kband appearing at 0.3 ms; however, the curves became flat as the J-, I-, and P-steps decreased sharply, which exhibited the same pattern in both Taxus species. To further evaluate the condition of the photosynthetic apparatus in Taxus species, selected parameters were calculated from JIP-test (Table S2) and were normalized for spider plots with values measured in April used as the respective control ( Figure 3). In this study, multiple parameters of both Taxus species exhibited significant changes in January and July as compared with that in April. Among them, Vj and Vi of T. mairei in January increased by 24.09% and 6.66%; meanwhile, Sm of T. media increased by 31.67%. Vi of both To further evaluate the condition of the photosynthetic apparatus in Taxus species, selected parameters were calculated from JIP-test (Table S2) and were normalized for spider plots with values measured in April used as the respective control ( Figure 3). In this study, multiple parameters of both Taxus species exhibited significant changes in January and July as compared with that in April. Among them, Vj and Vi of T. mairei in January increased by 24.09% and 6.66%; meanwhile, Sm of T. media increased by 31.67%. Vi of both Taxus species increased in July, which was opposite to their trends of Sm and N. The ϕEo of T. media and T. mairei only decreased by 31.09% and 46.84% in January; while their ϕR 0 decreased significantly in January and July, respectively. Notably, both Taxus species demonstrated increased ABS/RC and DI 0 /RC in January; however, their ABS/CSm decreased sharply in January, similar to the trends of TR 0 /CSm and ET 0 /CSm. In contrast, ABS/RC, TR 0 /RC, and ET 0 /RC of both Taxus species in July were higher than those in April, which were consistent with their corresponding parameters per CS. Moreover, DI 0 /CSm of T. media and T. mairei also increased by 25.93% and 45.74% in July. Apparently, both Taxus species demonstrated lower PI abs and PI total values in January and July than in April, with the former decreased more sharply. To further evaluate the condition of the photosynthetic apparatus in Taxus species, selected parameters were calculated from JIP-test (Table S2) and were normalized for spider plots with values measured in April used as the respective control ( Figure 3). In this study, multiple parameters of both Taxus species exhibited significant changes in January and July as compared with that in April. Among them, Vj and Vi of T. mairei in January increased by 24.09% and 6.66%; meanwhile, Sm of T. media increased by 31.67%. Vi of both Taxus species increased in July, which was opposite to their trends of Sm and N. The φEo of T. media and T. mairei only decreased by 31.09% and 46.84% in January; while their φR0 decreased significantly in January and July, respectively. Notably, both Taxus species demonstrated increased ABS/RC and DI0/RC in January; however, their ABS/CSm decreased sharply in January, similar to the trends of TR0/CSm and ET0/CSm. In contrast, ABS/RC, TR0/RC, and ET0/RC of both Taxus species in July were higher than those in April, which were consistent with their corresponding parameters per CS. Moreover, DI0/CSm of T. media and T. mairei also increased by 25.93% and 45.74% in July. Apparently, both Taxus species demonstrated lower PIabs and PItotal values in January and July than in April, with the former decreased more sharply.

Seasonal Dynamics of Chlorophyll and Carotenoid Contents in T. media and T. mairei
As shown in Figure 4, the Chl and carotenoid contents in T. media and T. mairei fluctuated in different months. For Chl a, the lower levels in both Taxus species were observed between January and March; while the highest was observed in October, around 1.41 and 1.64 mg g −1 , respectively. The Chl a content of T. media was higher than that of T. mairei in different months, and significant differences were observed in January and March-October ( Figure 4A). The Chl b content in T. media demonstrated a higher accumulation in May and September, with values around 0.47 and 0.44 mg g −1 , respectively; meanwhile, the peak in T. mairei was 0.36 mg g −1 in July. The lowest level of Chl b in both Taxus species was observed in January, which was 0.21 and 0.30 mg g −1 , respectively. Significant differences for Chl b values between T. media and T. mairei were demonstrated in January-April and October ( Figure 4B). The carotenoid content in T. media varied gently in different months, with a maximum of 0.39 mg g −1 in December; meanwhile, that in T. mairei demonstrated the lowest in May at 0.22 mg g −1 , and reached the highest at 0.35 mg g −1 in November with a strong fluctuating rise. Significant differences between T. media and T. mairei were in January-June and September ( Figure 4C). The trend of total Chl content in Taxus species was similar with that of Chl a content. The lowest content in T. media and T. mairei was observed in February and March, respectively, with values around 1.47 and 1.05 mg g −1 ; meanwhile, the highest was obtained in October, which was 1.77 and 2.04 mg g −1 . The total Chl content of T. media was significantly higher than that of T. mairei in January and March-September ( Figure 4D).
were in January-June and September ( Figure 4C). The trend of total Chl content in Taxus species was similar with that of Chl a content. The lowest content in T. media and T. mairei was observed in February and March, respectively, with values around 1.47 and 1.05 mg g −1 ; meanwhile, the highest was obtained in October, which was 1.77 and 2.04 mg g −1 . The total Chl content of T. media was significantly higher than that of T. mairei in January and March-September ( Figure 4D).  , carotenoid (C) and total chlorophyll (D) content in T. media and T. mairei. Data are shown as mean ± SE (n = 3). Asterisk indicates significant difference between two Taxus species in the same month according to Student's t-test (p < 0.05).

Seasonal Dynamics of Soluble Protein and Soluble Sugar Content in T. media and T. mairei
The soluble protein content in T. media and T. mairei demonstrated a fluctuating trend from January to May with a similar accumulation (except for January); after June, that of both increased strongly at first and then decreased rapidly, but the responses of T. mairei to environmental changes were earlier than that of T. media. The lowest value in both Taxus species was observed in February, around 0.65 and 1.88 mg g −1 , respectively; while the highest was 31.95 mg g −1 in September for T. mairei and was 27.93 mg g −1 in November for T. media ( Figure 5A). The soluble sugar content in two Taxus species demonstrated similar trends as their soluble protein from January to April, with values in T. mairei being significantly higher than that in T. media (except for January). Then, both Taxus species demonstrated fluctuating trends, but with insignificant difference of values within. The lowest value in T. mairei was at 1.12% in July; while that in T. media was at 0.70% in June. The highest value in both Taxus species appeared in December, which was at 7.77% and 7.60%, respectively ( Figure 5B). trends as their soluble protein from January to April, with values in T. mairei being significantly higher than that in T. media (except for January). Then, both Taxus species demonstrated fluctuating trends, but with insignificant difference of values within. The lowest value in T. mairei was at 1.12% in July; while that in T. media was at 0.70% in June. The highest value in both Taxus species appeared in December, which was at 7.77% and 7.60%, respectively ( Figure 5B).

Seasonal Dynamics of SOD and POD Activity in T. media and T. mairei
The SOD activity of both Taxus species maintained relatively high levels with an insignificant difference from January to April as well as from October to December. In May, the SOD activity of T. mairei was 11.27% higher than that of T. media with a significant difference; from July to September, that of both demonstrated a trend of decreasing first then increasing, and the values in T. media were significantly higher than that in T. mairei. The lowest value in both Taxus species appeared in August, which was 391.20 and 536.11 U/g, respectively ( Figure 6A). The POD activity of T. mairei demonstrated an obvious fluctuating trend during the measurements with two extremely high peaks occurred in March and July, respectively. The trend of POD activity in T. media was similar with that in T. mairei, but the first peak appeared in April, whose responses to environmental changes were relatively late; while the second peak was less pronounced. Notably, the values of T. mairei in most months were significantly higher than that of T. media ( Figure 6B). media. Data are shown as mean ± SE (n = 3). Asterisk indicates significant difference between two Taxus species in the same month according to Student's t-test (p < 0.05).

Seasonal Dynamics of SOD and POD Activity in T. media and T. mairei
The SOD activity of both Taxus species maintained relatively high levels with an insignificant difference from January to April as well as from October to December. In May, the SOD activity of T. mairei was 11.27% higher than that of T. media with a significant difference; from July to September, that of both demonstrated a trend of decreasing first then increasing, and the values in T. media were significantly higher than that in T. mairei. The lowest value in both Taxus species appeared in August, which was 391.20 and 536.11 U/g, respectively ( Figure 6A). The POD activity of T. mairei demonstrated an obvious fluctuating trend during the measurements with two extremely high peaks occurred in March and July, respectively. The trend of POD activity in T. media was similar with that in T. mairei, but the first peak appeared in April, whose responses to environmental changes were relatively late; while the second peak was less pronounced. Notably, the values of T. mairei in most months were significantly higher than that of T. media ( Figure 6B).

Pearson Correlation Analysis of Chl Fluorescence and Physiological Indicators in T. media and T. mairei
The Pearson correlation analysis was performed to evaluate the relationships among the Chl fluorescence and physiological indicators in the two Taxus species (Figure 7). In T. media, the Fv/Fm was highly positively correlated with Fv′/Fm′ and NPQ, and highly negatively correlated with qP (p < 0.01), as was the correlation between Fv′/Fm′ and qP (p < 0.01). Moreover, the Fv′/Fm′ was positively correlated with Chl a, Chl b, and the total Chl

Pearson Correlation Analysis of Chl Fluorescence and Physiological Indicators in T. media and T. mairei
The Pearson correlation analysis was performed to evaluate the relationships among the Chl fluorescence and physiological indicators in the two Taxus species (Figure 7). In T. media, the Fv/Fm was highly positively correlated with Fv /Fm and NPQ, and highly negatively correlated with qP (p < 0.01), as was the correlation between Fv /Fm and qP (p < 0.01). Moreover, the Fv /Fm was positively correlated with Chl a, Chl b, and the total Chl content, respectively (p < 0.05); meanwhile, the qP was highly negatively correlated with Chl b (p < 0.01), and negatively correlated with Chl a and the total Chl content (p < 0.05). There was also a highly positive correlation in the Chl a, Chl b, and total Chl content, and a highly negative correlation between ϕPSII and POD activity (p < 0.01). Among other indicators, the SOD activity was negatively correlated with Fv/Fm and Fv /Fm (p < 0.05); the carotenoid content was negatively correlated with Fv/Fm and NPQ, and positively correlated with soluble sugar content (p < 0.05). The correlation trend of Chl fluorescence parameters in T. mairei was basically the same as that in T. media; meanwhile, the NPQ in T. mairei was additionally highly positively correlated with Fv /Fm (p < 0.01) and negatively correlated with qP (p < 0.05). Interestingly, T. mairei demonstrated highly positive correlations in photosynthetic pigment content (p < 0.01), except between Chl b and carotenoid content. Moreover, there were positive correlations between Fv /Fm and Chl b or the total Chl content as well as the NPQ and soluble protein content in T. mairei (p < 0.05).

Redundancy Discriminant Analysis between Environmental Factors and Chlorophyll Fluorescence and Physiological Indicators in T. media and T. mairei
The effects of different environmental factors on Chl fluorescence and physiological characteristics in the two Taxus species were analyzed by RDA (Figure 8). The representative environmental factors were clearly distributed on the left side of the RDA1 axis in both Taxus species. The interpretation rate in T. media was ranked as In > Ta > RH > P; while that in T. mairei was ranked as Ta > In > RH > P. Among them, In, Ta, and RH in both Taxus species significantly affected Chl fluorescence and physiological parameters (p < 0.1). For T. media and T. mairei, the environmental factors were closely related to Chl fluorescence indicators and ROS scavenging enzymes. According to the principle of RDA, the included angle between environmental factors and Chl fluorescence and physiological indicators is acute, indicating that they are positively correlated, while obtuse angles indicate negative correlations. Therefore, the SOD activity, qP, and φPSII were negatively correlated with environmental factors, while other indicators were positively correlated with environmental factors.

Redundancy Discriminant Analysis between Environmental Factors and Chlorophyll Fluorescence and Physiological Indicators in T. media and T. mairei
The effects of different environmental factors on Chl fluorescence and physiological characteristics in the two Taxus species were analyzed by RDA (Figure 8). The representative environmental factors were clearly distributed on the left side of the RDA1 axis in both Taxus species. The interpretation rate in T. media was ranked as In > Ta > RH > P; while that in T. mairei was ranked as Ta > In > RH > P. Among them, In, Ta, and RH in both Taxus species significantly affected Chl fluorescence and physiological parameters (p < 0.1). For T. media and T. mairei, the environmental factors were closely related to Chl fluorescence indicators and ROS scavenging enzymes. According to the principle of RDA, the included angle between environmental factors and Chl fluorescence and physiological indicators is acute, indicating that they are positively correlated, while obtuse angles indicate negative correlations. Therefore, the SOD activity, qP, and ϕPSII were negatively correlated with environmental factors, while other indicators were positively correlated with environmental factors. characteristics in the two Taxus species were analyzed by RDA (Figure 8). The representative environmental factors were clearly distributed on the left side of the RDA1 axis in both Taxus species. The interpretation rate in T. media was ranked as In > Ta > RH > P; while that in T. mairei was ranked as Ta > In > RH > P. Among them, In, Ta, and RH in both Taxus species significantly affected Chl fluorescence and physiological parameters (p < 0.1). For T. media and T. mairei, the environmental factors were closely related to Chl fluorescence indicators and ROS scavenging enzymes. According to the principle of RDA, the included angle between environmental factors and Chl fluorescence and physiological indicators is acute, indicating that they are positively correlated, while obtuse angles indicate negative correlations. Therefore, the SOD activity, qP, and φPSII were negatively correlated with environmental factors, while other indicators were positively correlated with environmental factors.

Discussions
The advantage of CF-Imaging technology was permitting the study of photosynthetic activity over the entire leaf surface. Through the false color palettes, where different colors encode for different values of Chl fluorescence parameters, the spatial and temporal heterogeneity of Chl fluorescence signatures could be represented pixel by pixel in the images [25,30]. In this study, CF-Imaging provides intuitive and accurate information on the seasonal responses of photosynthetic performance in Taxus leaves. Among them, images of Fv/Fm in Taxus demonstrated healthy orange-red colors throughout the entire leaf area in spring and autumn; meanwhile, the heterogeneity was mainly represented as green-yellow and blue-green sectors in summer and winter. The Fv/Fm is a reliable indicator to detect some kinds of early stresses in plants, such as high irradiance and low temperature, which persists in the normal range of 0.8 or so under suitable environmental conditions, and always decreases due to the changes in the use of light energy and photoinactivation of PSII RCs [37,38]. However, Epron et al. [39] also reported that Fv/Fm could be unaffected under drought stress until the complete cessation of CO 2 assimilation. The changes of Fv/Fm in this study indicated that the photosynthetic apparatus of Taxus differed in sensitivity to different seasons and was partially or fully susceptible in summer and winter environments. This also implied that water deficit was not the main factor affecting the photosynthetic performance of Taxus during seasonal changes. Moreover, images of other Chl fluorescence parameters also demonstrated a distribution of heterogeneity with different visualization effects during the measurements; but their accuracy or sensitivity is less than that of Fv/Fm. According to the results of the evaluation of all Chl fluorescence parameters, the Fv/Fmbased CF-Imaging has the potential to be developed as a simple and effective tool for assessing the physiological stress states of Taxus.
Literatures have reported that the Chl and carotenoid contents were the basis for the interpretation of Chl fluorescence parameters and the origin of multicolor fluorescence imaging [40][41][42]. Therefore, the Chl and carotenoid contents of the two Taxus species in different months could provide a reasonable explanation for their photochemistry and image heterogeneity. Overall, the Chl contents of Taxus demonstrated significant positive correlations with Fv /Fm , and negative correlations with qP. This suggested that light energy utilization for photochemistry in Taxus leaves could be improved by controlling the number of functioning PSII RCs under light conditions [43]. Interestingly, qP of both Taxus species increased to varying degrees in winter, which was the opposite of NPQ. Combined with the analysis of other Chl fluorescence parameters, it suggested a low level of PSII, presumably as a result of energy migration occurring between PSII and PSI, based on the contribution of Chl and carotenoids to form aggregates of photosystem complexes [44]. Carotenoids involved in photosynthesis are bound to the light-harvesting pigment-protein complexes of photosystems [45]. The significant correlations between carotenoid and Chls as well as between carotenoid and Chl fluorescence parameters were respectively observed in T. mairei and T. media, which further emphasized the crucial roles of carotenoids on the light harvesting, energy transfer and photoprotection in these two Taxus species [46]. All the efforts were aimed at maintaining a relatively consistent ϕPSII, which also benefits from the feedback regulation of photosynthetic carbon assimilation [22]. This could be an important mechanism for photochemical responses of Taxus to different environmental conditions, and should be considered for the detection of injury degree.
The Chl a fluorescence transient is characterized by a polyphasic fluorescence increase, which reflects the information on the O-P phases of Chl fluorescence kinetics [47]. The JIPtest analysis, based on the so-called "Theory of Energy Fluxes in Biomembranes", involves translating the fluorescence transients into several phenomenological and biophysical expressions [48,49]. In this study, a series of basic fluorescence parameters involved in specific energy fluxes, phenomenological energy fluxes, quantum efficiencies, and a performance index were selected to further study the photosynthetic responses of Taxus to seasonal changes. For both Taxus species, the specific energy fluxes (ABS/RC, TR 0 /RC, and ET 0 /RC) increased significantly in July compared with that in April; while the corresponding phenomenological energy fluxes (ABS/CSm, TR 0 /CSm, and ET 0 /CSm) also demonstrated a consistent trend. This suggested that the absorption and conversion of light energy in Taxus were enhanced in the summer environment. However, the decrease in Sm and N indicated a decreased pool size of electron carriers, which potentially affected the reduction events of Q A [50]. Moreover, the increased Vi further implied diminished electrons transport to Q B , which ultimately affected the reduction at the PSI acceptor side (ϕR 0 ). This situation was basically consistent with the theory term as "energy trapping" [22], which was considered a specific mechanism to protect the photosynthetic apparatus from potential environmental stresses. However, it simultaneously sacrificed the photosynthetic performance, which was reflected by a significant decrease in the performance indexes (PI total and PI abs ). Moreover, the ϕE 0 and ϕR 0 of both Taxus species were lower in January than that in April, indicating a weak activity of the electron transport chain; while the increased ABS/RC and DI 0 /RC represented a strong absorption and dissipation of light energy at the level of the antenna Chl. Interestingly, the phenomenological energy fluxes decreased sharply, which could be the result of a decrease in the opening ratio of active RCs. These combined results further decreased the performance indexes compared with that in July; meanwhile, the difference of the parameters above in the two Taxus species could reasonably explain their different photochemistry. Fortunately, this situation demonstrated a recovery in April, which further implied that the Taxus has the potential of a reversible deactivation for RCs in winter. It appears that the continuous and diverse energy dissipation mechanism is the key to the survival of evergreen conifers, such as Taxus [51].
The RDA is a two-table method in which the gradient found in the Chl fluorescence and physiological parameters could be directly related to the external environmental factors [52]. The results of RDA demonstrated that In and Ta were the main factors influencing the photochemistry of T. media and T. mairei, and their sensitivities to these two environmental factors were different. Therefore, the protection of Taxus from potential environmental stresses induced by seasonal changes need to comprehensively consider the tree species and priority of main influencing factors. The seasonal changes disturbed the photosynthetic physiology of Taxus; simultaneously, a series of physiological chain reactions occurred to maintain the normal photosynthetic process. As important osmoregulatory substances, the soluble sugar and soluble protein demonstrated almost the same response patterns in the two Taxus species throughout the measurements. They increased sharply in spring and midsummer; while the soluble sugar content in both species was also higher in early winter. Previous studies have reported that tree species with higher levels of soluble sugars accumulation could maintain cell turgor and water potential to ensure stomatal opening and stability of photosynthesis [53]. Therefore, the Taxus that accumulate more soluble sugars and consume them at a lower rate are presumed to be more tolerant to environmental stress. The soluble proteins are mostly enzymes for various biological processes, which increased in content at the beginning of environmental stresses, and gradually degraded as stresses continued or seasons changed. Interestingly, T. media always maintained higher levels of soluble proteins than T. mairei in winter. This could be an important clue for the stronger tolerance of T. media to the winter environment. Moreover, changes in the photosynthetic physiological states are also due to activity of the protective enzyme system, the increase in which could further increase the Chl fluorescence yield [54,55]. In this study, both Taxus species demonstrated similar trends in SOD activity, with no changes in winter but a significant decrease in summer. This suggested that the SOD activity for reactive oxygen scavenging are less important as a photoprotective mechanism for environmental acclimation, which was consistent with the results reported by Verhoeven et al. [7]. However, the POD activity demonstrated a seasonal increase with a different sensitivity in different Taxus species, indicating that the POD activity appeared to be the main protective enzyme involved in the protection of the photosynthetic apparatus in Taxus. The results of the Pearson correlation analysis and RDA further confirmed the impact of the protective enzyme system on the photochemistry of Taxus induced by environmental factors and the potential interspecific differences. Since the Chl fluorescence dynamics and physiological metabolisms of Taxus are continuous processes, combined with the complex and varied environmental factors in the field, which makes the results in this study still need the support of large-scale observations for many years, this further demonstrates the diversity of CF-imaging. The subsequent research should focus on the optimization of the combination and analysis mode for the measured data and environmental factors, which is crucial for the surveillance and conservation management of Taxus.

Experimental Site and Plant Material
The experimental site is in the germplasm nursery for Taxus species located in Nanjing botanical garden Mem. Sun Yat-Sen, Nanjing, China (32 • 3 N, 118 • 49 E), characterized by a typical subtropical humid monsoon climate with an annual average temperature of 16.2 • C, and an annual average precipitation of 1013 mm. The soil type at the experimental site is yellow-brown earth with a pH of 6.68 and moderate fertility. The twelve-year-old trees of T. media and T. mairei that were used in this study were planted in the germplasm nursery according to the planting density of 1 m × 1.5 m under the same conditions, such as sunshine and water. Ten individuals with uniform and well-grown conditions were prepared as per Taxus species for the subsequent measurements. All the field measurements were carried out on clear, cloudless days between the 10th and 15th of each month from January to December, 2021. Mature and healthy leaves from the third to sixth branch (from top to bottom) per plant were marked for the measurement of Chl a fluorescence transient. Then, the leaves were collected for CF-Imaging and measurements of other physiological parameters. The monthly climatic data including the net radiation intensity (In), relative humidity (RH), precipitation amount (P), and mean temperature (Ta) were assessed on the basis of climatic service by Wheat A software (version 1.4.9a) (accessed on 20 August 2022 http://www.wheata.cn) (Table S1).

Measurement of Chlorophyll a Fluorescence Transient
The Chl a fluorescence transient was measured with a Handy PEA (Hansatech, Instruments Ltd., Norfolk, UK) on the marked leaves of two Taxus species. Before the measurements, all the leaves were dark-adapted for 30 min with leaf clips. Then, the OJIP kinetics of transients were induced with a pulse of saturating red light of 3000 µmol (photon) m -2 s -1 . The fluorescence intensity was recorded from 10 µs to 2 s, and the data were analyzed using the JIP-test [21]. For each Taxus species, measurements were repeated four times per month. The introduced basic fluorescence parameters were listed below: Vj, relative variable fluorescence at the J-step; Vi, relative variable fluorescence at the I-step; Sm, standardized area above the fluorescence curve between Fo and Fm is proportional to the pool size of the electron acceptors Q A on the reducing side of PSII; N, the times Q A is reduced while fluorescence reaches its maximal value (number of Q A redox turnovers until Fm is reached); ϕE 0 , the quantum yield for electron transport from Q A − to plastoquinone; ϕR 0 , the quantum yield for reduction in the end electron acceptors at the PSI acceptor side; ABS/RC, average absorbed photon flux per PSII reaction center (RC); TR 0 /RC, trapping flux leading to Q A reduction per RC; ET 0 /RC, electron transport flux per RC at t = 0; DI 0 /RC, dissipated energy flux per RC at t = 0; ABS/CSm, absorbed photon flux per excited cross section (CS) at t = t Fm ; TR 0 /CSm, maximum trapped exciton flux per CS at t = t Fm ; ET 0 /CSm, electron transport flux per CS at t = t Fm ; DI 0 /CSm, dissipated energy flux per CS at t = t Fm ; PI abs , performance index for energy conservation from photons absorbed by PSII antenna to the reduction of Q B ; PI total , performance index for energy conservation from photons absorbed by PSII antenna to the reduction of PSI acceptors.

Measurement of Chlorophyll Fluorescence-Imaging
Fluorescence images of the leaf surface were obtained using a CF-Imaging system (CF Imager, Technologica, Essex, UK) following the manufacturer's instructions.

Measurement of Photosynthetic Pigment Content
The Chl a, Chl b, total Chl and carotenoid contents in the leaves of T. media and T. mairei were measured with an 80% acetone extraction according to the method by Wang et al. [20]. The samples (0.1 g) were homogenized in 10 mL of 80% acetone, and homogenates were centrifuged at 4 • C for 15 min (6000× g). The supernatants were analyzed by a UV-2102PC/PCS ultraviolet spectrophotometer (UNICO, Shanghai, China). The absorbance was determined at 665, 649, and 470 nm spectrophotometrically. Contents of these pigments were calculated following Lichtenthaler and Buschmann [56].

Measurement of Soluble Protein and Soluble Sugar
Soluble protein content was measured using the Coomassie Brilliant Blue G-250 method described by Bradford [57]. Fresh needles of Taxus species (0.5 g) were manually ground and extracted with pre-cooled phosphate buffer (pH 7.0). After centrifugation, the supernatant was mixed with Coomassie Brilliant Blue G-250, and the absorbance of the blue color was read at 595 nm by a spectrophotometer. The amount of soluble protein was calculated with bovine serum albumin as a standard using the following equation: y = 0.007x + 0.0488.
Soluble sugar content was measured with the anthrone-sulfuric acid method [58]. Distilled water was added to the samples (0.1 g) and warmed at 100 • C for 30 min. The samples were extracted two times, each on 10 mL, and the extract was diluted to 25 mL by distilled water. Then, the extract was mixed with anthrone ethyl acetate and 98% sulfuric acid. After heating in a boiling water bath for 1 min, the absorbance of the mixture was read at 625 nm. The amount of soluble sugar was calculated with glucose as a standard using the following equation: y = 0.0037x + 0.0091.

Measurement of SOD and POD Activity
The SOD and POD activity was measured using the method reported by Zu et al. [14]. Fresh needles of Taxus species were ground and extracted with a pre-cooled phosphate buffer (pH 7.0). After centrifugation, the supernatant was collected and used for enzyme activities. SOD was assayed by a reaction of the extract mixed with 50 mM phosphate buffer (pH 7.8), 0.1 mM EDTA, 13 mM methionine, 0.02 mM riboflavin, and 0.75 mM nitro blue tetrazolium, which was carried out under illumination at 25 • C for 20 min. Then, the absorbance was measured at 560 nm to determine the SOD activity. The POD was determined by a reaction of the extract mixed with 50 mM phosphate buffer (pH 7.0) and 0.5 mM ascorbic acid. This reaction was started by adding 0.1 mM H 2 O 2 , and POD activity was determined by the rate of guaiacol oxidation at 470 nm.

Statistical Analysis
In this study, the experiments were replicated twice independently. The results were expressed as mean ± SE of at least three biological replicates. The student's t-test was performed using SPSS 19.0 for statistical analysis. Data normality and variance homogeneity were tested with Shapiro and Levene's tests in SPSS 19.0 prior to the statistical analysis. Pearson correlation analysis was performed to reveal a correlated degree of Chl fluorescence and physiological indicators by using an online tool ChiPlot (accessed on 23 August 2022 https://www.chiplot.online/). A redundancy discriminant analysis (RDA) was performed to reveal the interactions between the environmental factors and measured indicators by using Canoco 5.0 (Microcomputer Power, Ithaca, NY, USA). ChiPlot and GraphPad Prism 8.0 were used to draw the plots.

Conclusions
In summary, the photosynthetic physiology of T. media and T. mairei was mainly influenced by In and Ta in the experimental site with seasonal changes. The Fv/Fm-based CF-Imaging technology provided information on the photosynthetic activity and visualized the heterogeneity over the leaf phenotypes, which was expected to be an effective tool to evaluate physiological stress states of Taxus. Notably, both Taxus species could maintain a stable ϕPSII under various environmental conditions. This was mainly due to the regulation on the absorption and conversion of light energy by their photosynthetic apparatus, which should be fully considered when determining the damage degree to which Taxus was exposed. According to the results of Chl a transient, the activity of the electron transport chain in the two Taxus species differed to varying degrees in the representative months, which could explain the different values of photochemical parameters for both species growing in similar environmental conditions. Moreover, the diverse energy dissipation approaches were crucial for environmental acclimation. During this period, the osmotic regulation and increase in antioxidant activity were also observed, which contributed to a stable photosynthetic structure and function.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/plants12142636/s1, Table S1: Monthly climatic data of Nanjing city in 2021; Table S2: Selected JIP-test parameters of T. media and T. mairei in January, April, July, and October.

Data Availability Statement:
The data supporting the findings of this study are available within the article (and its supplementary information files).