Differences in PItotal of Quercus liaotungensis seedlings between provenance

The performance index of overall photochemistry (PItotal) is widely used in photosynthesis research, but the PItotal interspecies differences are unclear. To this end, seeds of Quercus liaotungensis from 10 geographical provenances were planted in two different climate types. Two years later, leaf relative chlorophyll content (SPAD) and chlorophyll a fluorescence transient of seedlings were measured. Meanwhile, the environmental factors of provenance location, including temperature, precipitation, solar radiation, wind speed, transpiration pressure, and soil properties, were retrieved to analyze the trends of PItotal among geographic provenance. The results showed that, in each climate type, there was no significant difference in SPAD and electron transfer status between PSII and PSI, but PItotal was significantly different among geographic provenances. The major internal causes of PItotal interspecies differences were the efficiency of electronic transfer to final PSI acceptor and the number of active reaction centers per leaf cross-section. The main external causes of PItotal interspecies differences were precipitation of the warmest quarter, solar radiation intensity in July, and annual precipitation of provenance location. PItotal had the highest correlation with precipitation of the warmest quarter of origin and could be fitted by the Sine function. The peak location and fluctuating trend of precipitation—PItotal fitted curve were different in two climate types, largely due to the difference of precipitation and upper soil conductivity in the two test sites. Utilizing the interspecific variation and trends of PItotal might be a good strategy to screen high and stable photosynthetic efficiency of Q. liaotungensis provenance.

www.nature.com/scientificreports/ performance index of overall photochemistry (PI total ) 26 . PI total calculates by PI ABS and δ Ro (the efficiency of the electron from PQH 2 is transferred to final PSI acceptors), which can fully describe the photochemical activity of the linear photosynthetic electron transfer chain 25 . Data from numerous studies indicated that PI total decreased significantly in response to high PAR dose, high ambient temperature, low soil water content, K + -deficiency stress, Mg-deficiency stress, shade stress, and heat stress [27][28][29][30][31] . Simultaneously, PI total increased significantly during the light-induced plasticity of plant growth 32 and was considered the most salinity-sensitive parameter 33,34 . Widespread plant species often show extensive variation in morphological and growth characteristics as well as the substantial difference in stress resistance due to different individual selection pressures 35 . Existing studies have shown significant differences in the PSII photochemical activity from different provenances 36,37 . However, the mechanism and the trend of change for the interspecies differences of PSII photochemical activity are still unclear. Investigating PSII interspecies differences can help implement the OJIP-test to provenance trials, and obtain excellent germplasm resources with high and stable photosynthetic inorganic carbon assimilation ability. In this paper, 2-year-old Q. liaotungensis seedlings coming from 10 different provenances were used as test materials. The difference in PI total between provenances from internal factors (φ Po , ψ Eo , δ Ro , and RC/ABS) and external factors (temperature, precipitation, solar radiation, wind speed, transpiration pressure, and soil properties) in provenances was analyzed. Several studies have investigated that dry climate threatened Q. liaotungensis forest growth 38,39 . In order to verify the experimental results, two separate experiments were conducted in semi-arid and sub-humid distribution zones of Q. liaotungensis. We tested three different hypotheses: (1) There are differences among the Q. liaotungensis provenances of PI total . (2) These changes can be explained by the parameters used to calculate PI total and the environmental factors of provenance. (3) The difference trend may be predicted. Our expected results were to 1) evaluate the degree of PSII interspecific difference of Q. liaotungensis.
(2) Understand the patterns of variation observed.

Materials and methods
Test sites. The test was carried out simultaneously in Yangqu and Sanjiao test sites. The Yangqu test site is located in Yangqu County, Shanxi, China (38.0981° N, 112.7346° E, and 961 m above sea level), is a warm temperate semi-arid continental monsoon climate. The annual mean temperature is 8.5 °C, the annual rainfall is 430.4 mm, the annual average frost-free period is 164 days. The Sanjiao test site is located in Fushan County, Shanxi, China (35.9649° N, 112.0766° E, and 1197 m above sea level), is a warm temperate semi-humid continental monsoon climate. The annual mean temperature is 9.1 °C, the annual rainfall is 569.6 mm, the annual average frost-free period is 191 days. The soil type of the two test sites is loam.
Test materials. In the autumn of 2017, the seeds of Q. liaotungensis were collected from 10 provenances in the species' natural distribution range. The names, geographical locations, climate conditions, and soil types of all provenances were summarized in Fig. 1 and Table 1. For each provenance, seeds were collected from plus trees with plant spacing was greater than 50 m in the middle-aged forests. The fully mixed seeds were used as the provenance seed. Malathion was used to kill insects in seeds. The seeds were sown in the field in the autumn of the same year. The experiment had a randomized block design with three replications. At least 8000 seeds per replication were sown, and each seed was sowed at a distance of about 20 cm. Field management followed the normal agricultural practices in the two test sites. Two years later, vigorous seedlings from germinated seeds were evaluated for relative chlorophyll content (SPAD) and chlorophyll a fluorescence. All parameters were measured in August 2019.
Parameter measurement methods. The mature fully expanded and unshaded leaves of 30 vigorous seedlings from each provenance were used to monitor the chlorophyll a fluorescence transient and SPAD. SPAD and chlorophyll a fluorescence transient were sequentially measured on the same leaf. One measurement per seedling was taken, resulting in 30 measurements per provenance.
The SPAD value of leaf was measured with a chlorophyll meter (SPAD-502Plus, KONICA MINOLTA, Japan) and chlorophyll a fluorescence transient was measured with a PAM-fluorometer (FluorPen FP110, Photon Systems Instruments, Czech Republic). The chlorophyll a fluorescence transient measurement was made from leaves that were dark adapted for 20 min using leaf clips. The mesophyll was illuminated with saturated blue light (2 100 μmol m −2 s −1 ) for 1 s, and the fluorescence signals at intervals of 10 μs (before 600 μs), 100 μs (between 600 μs and 15 ms), 1 ms (between 15 and 100 ms), and 10 ms (after 100 ms) were recorded. The OJIP-derived parameters ( Table 2) were calculated with reference to Stirbet et al. 25 and Holland et al. 40 .
Environmental factors (11 temperature factors, 8 precipitation factors, 12 monthly solar radiation intensity factors, 12 monthly average wind speed factors, and 12 monthly transpiration pressure factors) were retrieved from the WorldClim database (https:// www. world clim. org/ data/ world clim21. html) with a resolution of 10 arcminutes based on the geographical location of the provenance. 32 soil factors were retrieved from the Harmonized World Soil Database (HWSD v1.2) provided by the National Tibetan Plateau Data Center. Eliminating collinearity was executed when the Pearson correlation coefficient between environmental factors was greater than 0.9. 22 factors were finally retained (Table 3). Data processing. ArcGIS  www.nature.com/scientificreports/ test at 0.05 and 0.01. The coefficient of variation (CV = (Standard deviation/mean) × 100%) was calculated in order to investigate the degree of dispersion of φ Po , ψ Eo , δ Ro , and RC/ABS. The relationship between PI total (the mean of the provenances) and provenance's environmental factor was examined using the Maximal Information Coefficient correlation analysis (MIC). MIC was calculated using the "Minerva" of R package. The relationship between PI total and the warmest quarter precipitation of provenance was determined using regression analyses    It indicated that the material basis for light energy absorption of the tested leaves were similar in the same test site (Fig. 2).

Chlorophyll a fluorescence transient analysis. Both normalized chlorophyll a fluorescence transient
showed the typical OJIP shape in two test sites. Differences in each step's relative fluorescence intensity between provenances were not evident, indicating that the electron transfer status between PSII and PSI of all provenances were similar (Fig. 3).
Chlorophyll a fluorescence parameter analysis. PI total reflected the photochemical activity of the electron transfer chain from the PSII oxygen-evolving complex to the final electron acceptors of PSI. Figure 4 showed that there were significant differences in PI total between provenances in each test site. In Yangqu test site, SJ provenance had the highest PI total , with an average value of 4.07, reaching 2.21-fold higher (p < 0.01) than that of the lowest PI total (KC provenance) in the same test site. In Sanjiao test site, the average PI total value of LK provenance was 5.21, reaching 1.83 -fold higher (p < 0.05) than that of the lowest PI total (ZW provenance) in the same test site. PI total is based on four independent parameters: φ Po , ψ Eo , δ Ro , and RC/ABS. φ Po , ψ Eo , and δ Ro represent the probabilities that electron is transferred to the Q A , PQ, and final PSI acceptor side. RC/ABS represents the density of PSII reaction centers. Table 4 showed the analysis of φ Po , ψ Eo , δ Ro , and RC/ABS of Q. liaotungensis seedlings between provenances. The variation analysis (using CV) showed the data-sparse in descending order were δ Ro ,  www.nature.com/scientificreports/ ψ Eo , and φ Po between provenances. The differences of δ Ro and ψ Eo between provenances were highly significant (p < 0.01) in both test sites. The CV of the PSII reaction center was 7.53 (in Yangqu test site) and 7.61 (in Sanjiao test site), implying the data-sparse of RC/ABS between provenances were prominent in both test sites. These results suggest that the difference in probability of electron transfer from PSII to PSI and the number of active reaction centers in PSII were gradually increased between provenances, causing a significant difference in PI total between provenances in the same test site.

Environmental factor analysis. MIC correlation analysis is used to find linear and nonlinear correlations
between variables. The MIC value ranges from 0 to 1. The closer the MIC value to 1.0, the higher the correlation of the variable is. As shown in Fig. 5, the warmest quarter precipitation (B18) had the highest MIC value in both test sites. The results showed that the warmest quarter precipitation (B18) in provenance was closely related to the PSII photochemical activity of Q. liaotungensis seedlings. Additionally, the solar radiation intensity in July (R7) and the annual precipitation (B12) also significantly affected the PSII photochemical activity in the Sanjiao test site. The regression analysis was used to predict the precipitation of the warmest quarter (B18) of provenance and PI total in the two test sites. The results showed that the precipitation of the warmest quarter (B18) and PI total could be fitted by the Sine function in the two test sites (R 2 was 0.90 in Yangqu test site and was 0.77 in Sanjiao test site). However, the peak location of the fitted curves differed between the two test sites. The fitting curve of Yangqu test site had a peak of around 310 mm, while in Sanjiao test site, the peak was around 350 mm. Compared with the fitted curve of Yangqu, the peak location of the Sanjiao's fit curve was shifted to the right. Interestingly, Fig. 6 showed that the precipitation of Sanjiao was shifted to the right compared with Yangqu. Figure 4. PI total analysis of Quercus liaotungensis seedlings between provenances. P-value indicates the differential significance of PI total between provenances in the same test site. YQ represents the Yangqu test site, and SJ represents the Sanjiao test site. Different lowercase letters indicate a significant difference at the 0.05 level, and uppercase letters indicate a significant difference at the 0.01 level. Table 4. φ Po , ψ Eo , δ Ro and RC/ABS analysis of Quercus liaotungensis seedlings between provenances. Mean and SD represent the average value and standard deviation of the parameter, respectively. CV and P-values represent the coefficient of variation and difference significance between provenances, respectively.  Table 5 showed the influence of provenance and test site on SPAD and chlorophyll a fluorescence parameter. Test site had significant effect on parameters and test site:provenance had significant combined effect on φ Po and RC/ABS, while provenance had no effect. For PI total , only test site had a significant influence (P = 0.04).
The average values of SPAD, ψ Eo , and δ Ro of all provenances were significantly higher, and the average values of φ Po and RC/ABS were lower in Sanjiao test site compared with Yangqu test site (Fig. 7). These results indicated that SPAD, ψ Eo , and δ Ro were the key parameters which caused the difference in PI total between the two test sites.
In Sanjiao test site, the annual precipitation (B12) and the coldest quarter precipitation (B19) were 5.6 and 3.7 times higher than those in Yangqu test site, respectively. Additionally, the topsoil salinity (S7) and the topsoil sand fraction (S2) in Yangqu test site were 3.2 times higher than those in Sanjiao test site (Fig. 8). The result demonstrated precipitation, topsoil salinity, and sand fraction were the main environmental factors that cause differences in PI total between the two test sites.  Table 3 for the meaning of environmental factor code. Black bars indicate the same environmental factor with the highest MIC between test sites. www.nature.com/scientificreports/

Discussion
In the two test sites, there were significant differences in PI total between the 10 provenances of Q. liaotungensis when they were grown in the same environment. This finding was similar to the results of the research on interspecies differences in PI ABS of European beech (Fagus sylvatica) by Kurjak, et al. 36 , reflecting the difference in PSII photochemistry activity between provenances. PI ABS is related to only the process of electron transport to the PQ pool 25 . In this study, PI total was used to evaluate the state of the linear photosynthetic electron transfer chain www.nature.com/scientificreports/ between PSII and PSI. Therefore, the results of this study could be interpreted as the total PSII photochemical activity of Q. liaotungensis of different geographic provenance. From the perspective of electron transfer from PSII to PSI, the CV of δ Ro was the highest, and the P-value of δ Ro was 0.001 (Table 4). This phenomenon indicated that electron transfer between PQ and PSI electron acceptors of Q. liaotungensis appeared to be most sensitive to environmental changes. On the other hand, this phenomenon might indicate that the PSI acceptor side of Q. liaotungensis's structure was relatively unstable and had higher diversity between different provenances. The data-sparse of RC/ABS between provenances of Q. liaotungensis was also rather large. Several reports have confirmed that environmental stress can cause decreased PSII active reaction centers [41][42][43] . In this study, the normalized OJIP curves of provenances did not show obvious signs of stress. Hence, a possible explanation for the data-sparse of RC/ABS is that some provenances encountered mild stress in the test site due to ecological distance (between the original field site and the common garden).
The study results showed that the precipitation of the warmest quarter of provenance location was closely related to the PSII photochemical activity of Q. liaotungensis in both test sites. Yangqu and Sanjiao test sites were both characterized by a temperate continental monsoon climate, with high temperatures and plentiful rainfall in the summer. Related studies have found that the net photosynthetic rate of Q. liaotungensis reached maximum in mid-July 44 . These results suggest that the PSII photochemistry activity of different Q. liaotungensis provenances was closely related to the water supply during the growing season. This speculation is similar to the conclusion of Wu et al. that sufficient water supply during the growing season can significantly increase the carbon assimilation rate of plant 45 .
The precipitation-PI total fitted curves first raised and then fell in the two test sites. Studies have confirmed that drought stress 46,47 and flooding stress 48,49 will significantly reduce the photochemical activity of PSII. Ecological distance might lead to excess or lack of water for some provenance. As a result, the curves first raised and then fell. Furthermore, the peak location of the Sanjiao's fitted curve was shifted to the right compared with the Yangqu's curve. GLMM analysis revealed a significant effect of the test site on PI total (Table 4) and the Yangqu test site with lower precipitation in the warmest quarter than the Sanjiao test site (Fig. 8). Based on this hypothesis, precipitation can be considered as the main cause of this phenomenon, and more provenances may suffer from mild drought stress in the Yangqu test site.
PI total and the peak location of the precipitation-PI total fitted curves were varied between the two test sites. As shown in Fig. 7, the internal factors causing this phenomenon were the difference in leaf chlorophyll content and the probability of electron transfer to PQ and final PSI acceptors. Because photosynthetic pigment play an essential role in absorption and transfer of light energy 50 , and φ Po , ψ Eo , δ Ro , and RC/ABS were key indicators of the total PSII photochemical activity. However, other internal factors such as morphological and physiological traits may also affect the PSII photochemical activity between provenances 37 . Precipitation and soil conductivity have significant effects on PSII photochemical activity of plant leaves 51 . Figure 8 also showed that the external factors resulting in this phenomenon are the different precipitation and upper soil conductivity in the two test sites.

Conclusion
We have shown that the differences in PI total of Q. liaotungensis seedlings between provenances do exist, and the different trends of PI total can be fitted by the Sine function in the two test sites. These results helped to screen the provenance of Q. liaotungensis with high and stable photosynthetic efficiency by using PI total . In the future, it is necessary to eliminate genetic differences within provenance, increase the number of provenances, and incorporate more environmental factors to improve the accuracy of the OJIP-test.