High temperature inhibits photosynthesis of chrysanthemum (Chrysanthemum morifolium Ramat.) seedlings more than relative humidity

High relative humidity (RH) and high temperature are expected more frequently due to climate change, and can severely affect the growth of chrysanthemums. In order to analyze the interactive effects of RH and high temperature on the photosynthetic performance of chrysanthemum, a completely randomized block experiment was conducted with three factors, namely temperature (Day/night temperature, 35°C/18°C, 38°C/18°C, 41°C/18°C), RH (Whole day RH, 50%, 70%, 90%), and treatment duration (3d, 6d, 9d). The control (CK) temperature was 28°C/18°C and RH was 50%. The results showed that with the increase of temperature, the apparent quantum efficiency (AQE), maximum net photosynthetic rate (Pn-max), net photosynthetic rate (Pn), transpiration rate (Tr), water use efficiency (WUE), maximal recorded fluorescence intensity (Fm), PSII maximal photochemical efficiency (Fv/Fm), absorption flux per cross section (ABS/CSm), trapped energy flux per cross section (TRo/CSm), electron transport flux per cross section (ETo/CSm) and photosynthetic pigment content of leaves significantly decreased, the minimal recorded fluorescence intensity (Fo), fluorescence intensity at point J of the OJIP curve (Fj) and non-photochemical quenching per cross section (DIo/CSm) significantly increased, the fluorescence difference kinetics of the OJ phase of chrysanthemum leaves showed K-bands. Pn, AQE, Fm, Fv, Fv/Fm, ABS/CSm, TRo/CSm, ETo/CSm and photosynthetic pigment content were higher at 70% RH than the other two RH conditions. The dominant factor causing the decrease of Pn in leaves was stomatal limitation at 35°C,38°C, three RH conditions, 3d and 6d, but non-stomatal limitation at 41°C and 9d. There was an interaction between temperature and RH, with a significant impact on Pn. The temperature had the greatest impact on Pn, followed by RH. This study confirms that heat stress severely affects the photosynthesis of chrysanthemum leaves, and when the temperature reaches or exceeds 35°C, adjusting the RH to 70% can effectively reduce the impact of heat stress on chrysanthemum photosynthesis.


Introduction
Chrysanthemum (Chrysanthemum morifolium Ramat.) is one of the four main cut flowers in the world (Fanourakis et al., 2022).It has been cultivated for more than 3,000 years, and over 3,000 varieties have been developed (Su et al., 2019).In China, it has a long history of use as a medicinal and edible plant (Ning et al., 2023).In Europe, the cultivation of chrysanthemum for cut flower production is a highly profitable industry (Castello et al., 2022).Chrysanthemum is one of the main flowers exported from China, with as many as 1.911 million chrysanthemums exported in 2019 alone (Dong, 2020).Hence, chrysanthemum is an economically important plant.
The optimal temperature for the growth of chrysanthemum is 25°C (Lee et al., 2013).Chrysanthemum growth is inhibited above 25°C and basically stops growing at 40°C (Li et al., 2021).Chrysanthemums 'Shenma' are typically short-day plants, and if the light duration is longer than 14.5 hours, its flowering will be delayed (Nakano et al., 2013).In order to meet the market supply demand and prompt chrysanthemums to bloom in the long sunshine season, black shading materials are often used for photoperiodic treatment, but the high temperature environment caused by shading in summer often causes chrysanthemums to wilt.Janka et al. (Janka et al., 2015) found that excessive irradiation and high temperatures above 28°C produced photoinhibition of chrysanthemum.The right relative humidity (RH) is equally important for the growth and development of chrysanthemum, and excessive RH can increase the incidence of white rust (Yoo and Roh, 2014).Influenced by the East Asian Monsoon, high temperature and high RH are main characteristics in greenhouse environment in southern China, which severely affect crops growth in glasshouses (Zhang et al., 2022).With global warming, extreme surface temperatures and duration in East Asia will increase more frequently (Chevuturi et al., 2018), and thus, the frequency of high temperature and high RH environments in greenhouses will be more frequent (Zheng et al., 2023).March to June is the main period for the growth of chrysanthemum seedlings in greenhouse in China and the impact of high temperature and high RH e n v i r o n m e n t o n t h e g r o w t h o f g r e e n h o u s e -g r o w n chrysanthemum seedlings should be noticed.In previous studies, yield increase through moisture control has been demonstrated in strawberries (Zucchi et al., 2016) and roses (Mortensen and Gislerød, 2000), but not in chrysanthemum cultivation.
High temperature and RH affect various metabolic and physiological processes in plants.Photosynthetic parameters derived from the light response curve are important indicators of the light energy utilization capacity of plants in the study of adversity stress (Xu et al., 2022).Weng et al. (Weng et al., 2021) found that net photosynthetic rate (P n ) of melon seedlings was inhibited at 42°C and 90% RH, compared to 30°C and 60% RH.In the study of stress, photosynthetic pigment content is an important index for assessing the extent of damage to photosynthetic organs, and chlorophylls are considered important components of stress biology in higher plants (Agathokleous et al., 2020).Yang et al. (Yang et al., 2022) found that lettuce chlorophyll a and chlorophyll b were reduced at 35°C and growth was inhibited, compared to 22°C . Chlorophyll fluorescence is inextricably linked to plant photosynthesis and can respond to changes in the photosynthetic system of a plant under adversity stress (Baker, 2008;Hazrati et al., 2016).Sun et al. (Sun et al., 2008) found that high temperatures reduced the PSII potential activity (F v /F o ) and maximum photochemical efficiency (F v /F m ) of chrysanthemum leaves, chrysanthemum protected reaction centers from damage by reducing the capture of light energy with the efficiency of electron transfer through PSII.
The two main reasons for limiting photosynthetic rate are stomatal and non-stomatal limitation.The exploration of stomatal and non-stomatal limitation under different adverse environments has been the focus of research, especially under combined stressors.Zubaidi et al. (Zubaidi et al., 2021) found that compared to 25°C, reduction in P n in wheat at 32°C was not only due to lower stomatal conductance, but also non-stomatal effects as mesophyll conductance and quantum yield were lower.In addition, Barker et al. (Barker, 1990) found that increasing the RH of greenhouse significantly increased the stomatal conductance (G s ), which improved the heat tolerance of tomato.While high temperatures have many detrimental impacts on plants, damage can be mitigated by regulating RH (Suzuki et al., 2015;Shamshiri et al., 2018).Zheng et al. (Zheng et al., 2020) found that increasing the RH to 70% at 35°C reduced gibberellin concentration (GA 3 ) and increased abscisic concentration (ABA) in tomato shoots, which was favorable to the growth of tomato plants, compared to 28°C and 50% RH.Similarly, Xu et al. (Xu et al., 2020) found that high RH was effective in alleviating the limitation of tomato growth by high temperature and improving the root to crown ratio, compared to 50% RH.In rice, increasing RH by mist spray under heat stress increased chlorophyll content, P n and yield (Jiang et al., 2020).When the effect of low, medium and high humidity on flowering and fruiting of tomato plants was studied (Peet et al., 2003), 50% RH was the optimum humidity at 35°C.
To our knowledge, there have been many studies on chrysanthemums under various factors of stress, but almost all of them are single-factor.The study of multifactorial stresses is more useful for chrysanthemum cultivation due to the complex environment in greenhouses.We hypothesized that high temperature could inhibit the photosynthesis of chrysanthemums, and changing RH could regulate it.The objective of this study is to analyze the interaction between RH and high temperature on the photosynthetic performance of chrysanthemums, analyze the dominant factors affecting chrysanthemum photosynthesis, and select the optimal RH for chrysanthemum photosynthesis in high temperature environments.

Experimental design
The experiment was carried out in March 2022 at the Agricultural Meteorological Experiment Station of Nanjing University of Information Science and Technology (118°42′E, 32°1 2′N).Seedlings of C. morifolium cv.'Shenma' were first raised in a greenhouse seedbed with substrate soil, vermiculite: peat mixture of 1:1 (v: v) in a Venlo-type glass greenhouse.When the seedlings had four true leaves, they were planted into pots of 17.5 cm (height) × 15.0 cm (diameter).The contents of organic carbon, available phosphorus, available potassium and nitrogen of the soil substrate were 10,450, 32.8, 89.4 and 1540 mg•kg -1 .Soil texture was medium loam.Soil pH was 6.7.One plant was planted in each pot.Two weeks after planting, the plants were moved into artificial climate chambers (TGP-1260, Australia) in a laboratory.
The experiment was conducted to simulate a summer greenhouse by setting the environmental parameters of artificial climate chambers.There were three temperature (day/night temperature) conditions set as 35°C/18°C, 38°C/18°C and 41°C/ 18°C, and three RH conditions, namely 50%, 70% and 90%, in combination with each temperature condition.Moreover, the temperature treatment was conducted for 3, 6 and 9 d to study the time dependency of response to temperature and RH.The experiment was a completely randomized group design with a total of 27 treatment combinations (Table 1).The control (CK) temperature and RH were 28°C/18°C and 50% respectively, which are the optimum temperature and normal RH for chrysanthemums cultivation in Nanjing.The environmental conditions and management measures in the artificial climate chambers remained the same during the experiment, except for different settings of temperature and relative air humidity.The photoperiod (day/night time) was set to 7:00 a.m.-17:00 p.m./ 18:00 p.m. -6:00 a.m.The light intensity in the artificial climate chamber was 800 mmol•m -2 •s -1 during daytime and 0 mmol•m -2 •s -1 during nighttime.

Measurement
The light response curve, gas exchange parameters, photosynthetic pigment content and chlorophyll fluorescence of chrysanthemum leaves were measured at 4, 7 and d after the start of the artificial control experiment.

Light response curves
With three LI-6400 portable gas exchange analyzers (LI-COR Biosciences Inc, USA), the light response curves of leaves were recorded from 9:00 a.m.-11:30 a.m. on each observation day.Inside the leaf chamber, the CO 2 level was maintained at 400 mmol•mol -1 .The levels of photosynthetically active radiation (PAR) (mmol•m -2 •s -1 ) inside the leaf chamber were 1200, 1000, 800, 600, 400, 300, 200, 100, and 0. The maximum wait time after each change of light intensity was set to 180s and the minimum wait time was 120s.Photosynthetic parameters like apparent quantum efficiency (AQE), light saturation point (LSP), maximum net photosynthetic rate (P n-max ), light compensation point (LCP) and dark respiration rate (R d ) were estimated using photosynthetic model simulations of Ye (Ye, 2010).The third leaf that was fully expanded before the experiment began was measured by the light response curve.One leaf per plant and three plants per treatment were measured.

Gas exchange parameters
Gas exchange parameters of chrysanthemum leaves were measured using the transparent leaf chamber that comes with the LI-6400 portable gas exchange analyzer (LI-COR Biosciences Inc, USA).Measurement time, leaf position was the same as described in section 2.2.1.The net photosynthetic rate (P n ), stomatal conductance (G s ), intercellular CO 2 concentration (C i ), atmospheric CO 2 concentration (C a ) and transpiration rate (T r ) were measured.Vapor pressure deficit (VPD) was calculated as formula 2 (Howell and Dusek, 1995;Kadam et al., 2015).The stomatal restriction values (L s ) were calculated as formula 3 (Berry, 1982).The water use efficiency (WUE) was calculated as formula 4 (R A Fischer and Turner, 1978). (2)

Chlorophyll fluorescence parameters
Chlorophyll fluorescence induction curves can provide insight into know the response of the plant to changes in environmental factors.Therefore, here we measured the chlorophyll fluorescence of each treatment.Chlorophyll fluorescence parameters were measured by a plant efficiency analyzer (Pocket PEA, Hansatech, UK).Measurement time and blade position were the same as the photosynthetic parameters.Leaves were dark-adapted with leaf clips for 30 minutes before measurement.Following a 5000 mmol•m -2 •s -1 light pulse, the fast chlorophyll fluorescence-induced kinetic curve (OJIP) and its related fluorescence parameters of chrysanthemum leaves were measured with a duration of 1s.The minimal recorded fluorescence intensity (F o ), fluorescence intensity at point J of the OJIP curve (F j ), maximal recorded fluorescence intensity (F m ), PSII maximal photochemical efficiency (F v /F m ) were measured.The JIP-test indices and terminology mentioned in this paper are presented in Table 2 (Strasser et al., 2010).The variable fluorescence intensities were O-J normalized according to formula 10-11 (Yusuf et al., 2010).
Where, W oj means fluorescence difference kinetics.F t means instantaneous fluorescence at any moment.F o means minimal recorded fluorescence intensity.F j means fluorescence intensity at point J of the OJIP curve.

Terms and Formulas Illustrations
ABS/CSm ≈ F m absorption flux per cross section

Data processing
All values were the means of three replicates per treatment.In order to make comparisons across treatments with different temperature, RH and treatment duration, this study used the calculation of mean values of relevant treatment indicators for comparison.The values of T 35°C , T 38°C and T 41°C were the means of treatments which day/night temperature was set as 35°C/18°C, 38°C/18°C and 41°C/18°C, respectively.The values of RH 50% , RH 70% and RH 90% were the means of treatments which RH condition was set as 50%, 70% and 90%, respectively.The values of L 3d , L 6d and L 9d were the means of treatments which duration was set as 3d, 6d and 9d, respectively.Duncan's multiple range test at the 0.05 level of significance was used to detect differences between all treatments using SPSS 26.0 software (SPSS Inc., Chicago, IL).Figures were drawn using Origin Pro 2023b (Origin Lab Corporation, Northampton, MA, USA).

Interactive effects of relative humidity and high temperature on light response curves
Table 3 shows the parameters of light response curves of chrysanthemum leaves under different treatments.As to temperature, the values of LSP, AQE and P n-max in CK treatment were significantly higher than that of all temperature treatments.AQE and P n-max values decreased with increasing temperature.The values of LSP, AQE, and P n-max in T 41°C were the lowest among all temperature treatments, which were 36.67%,47.79% and 57.61% lower than that of CK treatment.The values of LCP and R d in three temperature treatments were higher than that of CK treatment.
Among RH treatments, the values of AQE in RH 70% were highest and significantly larger than that of other RH treatments.Although statistically non-significant, RH 70% had the highest values of LSP and P n-max , followed by the RH 50% and RH 90% .The value of R d in RH 70% was lower than that of RH 90% , indicating that the respiratory consumption of leaves was low and photosynthetic activity was high at 70% RH, while the respiratory consumption of leaves was high at 90% RH.
Regarding the treatment duration, although statistically nonsignificant, the values of LSP, AQE, and P n-max decreased over time.The values of LSP, AQE, and P n-max in L 9d were the lowest among L 3d , L 6d and L 9d , which were 34.82%, 39.82% and 49.11% lower than that of CK treatment.
The results of ANOVA are also shown in Table 3. Temperature and RH had a significant (P< 0.01) impact on light response curve parameters.Treatment duration had a significant (P< 0.01) impact on LCP, LSP and R d .The interaction between temperature and RH was significant for P n-max (P< 0.01) and LSP (P< 0.05).Besides, the  interaction between temperature and treatment duration had a significant impact on P n-max (P< 0.01), LSP and AQE (P< 0.05).Moreover, the interaction between RH and treatment duration had a significant impact on AQE and P n-max (P< 0.01) and LCP (P< 0.05).Further, the interaction of all three factors (temperature, RH and treatment duration) was significant for AQE and P n-max (P< 0.05).Contributions of temperature, RH and treatment duration are shown in Figure 1.Temperature had the most impact, since it contributed 57.14%, 36.70%,45.89%, 63.01% and 46.42% of the variation in LSP, AQE, P n-max , LCP and R d , followed by RH, and treatment duration was the least.
The interaction between temperature and RH was significant for P n-max (P< 0.01).Interactions affecting P n-max from looking at temperature effects at a given RH and RH effects at a given temperature were shown in Figure 2. At a given temperature, the values of P n-max at 70% RH were highest, followed by 50% RH and 90% RH, which indicated that 70% RH mitigated the negative effect of high temperature on P n-max , while high RH aggravated.At a given RH, the values of P n-max decreased with increasing temperature, which indicated that increasing temperature aggravated the effect of RH on P n-max .In addition, the values of P n-max decreased with increasing treatment duration.The value of P n-max was lowest at 41°C and 90% RH.

Interactive effects of relative humidity and high temperature on photosynthetic pigment content
Figure 3 shows the photosynthetic pigment contents of chrysanthemum leaves under different treatments.The values of chlorophyll a, chlorophyll b, carotenoids, and chlorophyll (a+b) generally decreased with increasing temperature.The values of chlorophyll a, chlorophyll b, carotenoids, and chlorophyll (a+b) in T 41°C were the lowest among three temperature treatments, which were 44.86%, 40.63%, 47.62% and 43.78% lower than that of CK treatment.
The value of carotenoids in RH 70% was highest and significantly higher than that of other RH treatments.Although without statistically significant, the values of chlorophyll a, chlorophyll b, and chlorophyll (a+b) decreased in RH 50% and RH 90% , compared to RH 70% .There was no significant effect of treatment duration on photosynthetic pigment content.
Temperature had the most contribution to the variation in the values of chlorophyll a, chlorophyll b, carotenoids and chlorophyll (a+b), which was 66.34%, 83.731%, 70.95% and 72.07%, followed by RH, and treatment duration was the least.
The interaction between temperature and RH was significant for carotenoids (P< 0.01).Interactions affecting carotenoids from looking at temperature effects at a given RH and RH effects at a given temperature were shown in Figure 4.At a given temperature, the values of carotenoids at 70% RH were highest, which indicated that 70% RH mitigated the negative effect of high temperature on carotenoids.The values of carotenoids at 50% RH and 90% RH were significantly lower than that of CK, which indicated that 50% RH and 90% RH aggravated the negative effect of high temperature on carotenoids.At a given RH, the values of carotenoids decreased with increasing temperature, which indicated that increasing temperature aggravated the effect of RH on carotenoids.In addition, the values of carotenoids decreased with increasing treatment duration.Table 4 shows the gas exchange parameters of chrysanthemum leaves under different treatments.The value of P n in CK treatment was significantly higher than that of all treatments.With increasing temperature, the value of P n significantly decreased and to reach the lowest in T 41°C , which was 57.61% lower than that of CK.Among RH treatments, although statistically non-significant, the value of P n was highest in RH 70% , followed by RH 50% , and RH 90% was the lowest.There was no significant effect of treatment duration on P n .
Compared to CK treatment, the values of G s and C i in all treatments significantly decreased.The lowest values of G s and C i were recorded in T 38°C , which were 52.00% and 42.40% lower than that of CK treatment.Among RH treatments, the value of G s was highest in RH 70% , followed by RH 50% , and RH 90% was the lowest.The value of C i in RH 70% was higher than that of RH 90% .The values of G s and C i in L 6d were the lowest among L 3d , L 6d and L 9d , which were 48.00% and 38.66% lower than that of CK treatment.
The value of L s in CK treatment was significantly lower than that of all treatments.Among temperature treatments, the value of L s was highest in T 38°C , which was 79.17% higher than that of CK treatment, followed by T 41°C , and T 35°C was the lowest.The value of  Effect of interaction between temperature and RH on maximum net photosynthetic rate of chrysanthemum leaves.
L s in RH 70% was lower than that of RH 50% and RH 90% .There was no significant effect of treatment duration on L s .
In order to determine whether the decrease in P n is caused by obstruction of CO 2 diffusion or decrease in enzyme activity, we conducted stomatal and non-stomatal limitation analyses of photosynthesis.The evaluation of stomatal and non-stomatal limitation mainly depends on the change direction of C i and L s (Xu, 1997).The increase of L s and the decrease of C i indicate that the main reason for P n decrease is stomatal limitation, while the decrease of L s and the increase of C i indicate the reason is nonstomatal.Compared to CK, L s increased and C i decreased in T 35°C , indicating that stomatal limitation was the reason for P n decrease.Effect of interaction between temperature and RH on carotenoids of chrysanthemum leaves.
In addition, the reason for T 38°C was also stomatal limitation because L s increased and C i decreased, compared to T 35°C .In T 41°C , L s decreased while C i increased, compared to T 38°C , indicating that the non-stomatal limitation was the reason for P n decrease.Under three RH conditions, compared to CK, L s increased and C i decreased, indicating that RH affects P n through stomatal factors.L s increased and C i decreased under L 3d and L 6d treatments, indicating that the major factor for P n decrease from 0d to 6d was stomatal limitation.Compared to L 6d , L s decreased and C i increased in L 9d treatment, indicating that the decrease in P n from 6d to 9d was non-stomatal limitation.
For the water regimes of chrysanthemums, the values of T r and WUE in CK treatment were significantly higher than that of all treatments.Among temperature treatments, the value of T r was highest in T 35°C , followed by T 41°C , and T 38°C was the lowest, which was 12.07% lower than that of CK treatment.With increasing temperature, the value of WUE significantly decreased and reached the lowest in T 41°C , which was 54.98% lower than that of CK treatment.The value of T r in RH 50% and RH 70% was significantly higher than that of RH 90% .Although statistically non-significant, the value of WUE was highest in RH 70% , followed by RH 50% , and RH 90% was the lowest.The value of T r was highest in L 3d , followed by L 9d , and L 6d was the lowest, which was 12.07% lower than that of CK treatment.There was no significant effect of treatment duration on WUE.
The results of ANOVA are also shown in Table 4. Temperature and RH had significant impacts (P<0.01) on P n , G s , C i and L s .Temperature and treatment duration had significant impacts (P<0.01) on T r .The interaction of temperature and RH was significant for P n and WUE (P< 0.01).Furthermore, the interaction of temperature and treatment duration was significant for G s and P n (P< 0.05).Temperature had the greatest impacts on P n , G s , C i , L s and T r with contribution rates of 67.76%, 51.45%, 44.59%, 44.95% and 50.67%, followed by RH.The interaction of temperature and RH had the greatest impact on WUE with contribution rates of 52.09%.
The interaction between temperature and RH was significant for P n (P< 0.01).Interactions affecting P n from looking at temperature effects at a given RH and RH effects at a given temperature were shown in Figure 5.At a given RH, the values of P n decreased with increasing temperature, which indicated that increasing temperature aggravated the effect of RH on P n .At a given temperature, the values of P n at 70% RH were highest, followed by 50% RH and 90% RH, which indicated that 70% RH mitigated the negative effect of high temperature on P n , while high RH aggravated.In addition, the values of P n decreased with increasing treatment duration.The value of P n was lowest at 41°C and 90% RH.

Interactive effects of relative humidity and high temperature on chlorophyll fluorescence
Chlorophyll fluorescence kinetic curves respond to the photosynthetic efficiency and potential of chrysanthemum leaves.As seen in Figure 6A, the fluorescence values of curve O-P in CK treatment, T 35°C , T 38°C and T 41°C were 318~1595, 336~1547, 367~1470 and 393~1264, indicating that the fluorescence values of the leaves decreased with increasing temperature.As seen in Figure 6B, the fluorescence values of curve O-P in RH 50% , RH 70% and RH 90% were 356~1475, 322~1604 and 363~1441.It showed that the fluorescence value of curve O-P in RH 70% was highest, followed by RH 50% , and RH 90% was the lowest.As seen in Figure 6C, the fluorescence values of curve O-P in L 3d , L 6d and L 9d were 332~1570, 355~1385 and 389~1282, indicating that the fluorescence values of chrysanthemum leaves decreased with increasing treatment duration.
As seen in Table 5, F o and F j were lower in CK treatment than that of all temperature treatments.With temperature increasing, F o and F j increased and reached the highest in T 41°C , which were 23.53% and 13.93% higher than that of CK treatment.F o and F j in Effect of interaction between temperature and RH on net photosynthetic rate of chrysanthemum leaves.Zhou et al. 10.3389/fpls.2023.1272013Frontiers in Plant Science frontiersin.orgRH 70% were close to that of CK treatment, while lower than that of other RH treatments.With treatment duration increasing, F o and F j increased and reached the highest in L 9d , which were 22.29% and 15.59% higher than that of CK treatment.F m and F v /F m were higher in CK than that of all temperature treatments.With temperature increasing, F m and F v /F m decreased and reached the lowest in T 41°C , which were 20.69% and 13.75% lower than that of CK treatment.F m and F v /F m in RH 70% were close to that of CK treatment, while  higher than that of other RH treatments.With treatment duration increasing, F m and F v /F m decreased and reached the lowest in L 9d , which were 19.56% and 13.75% lower than that of CK treatment.As seen in Figure 7A, ABS/CSm, TRo/CSm and ETo/CSm were higher in CK treatment than that of all treatments, while DIo/CSm was lowest.With temperature increasing, ABS/CSm, TRo/CSm and ETo/CSm decreased and reached the lowest in T 41°C , which were 20.69%, 31.87% and 49.37% lower than that of CK treatment.While DIo/CSm increased with temperature increasing, the highest DIo/ CSm was observed in T 41°C , which was 23.53% higher than that of CK treatment.As seen in Figure 7B, ABS/CSm, TRo/CSm, ETo/ CSm, and DIo/CSm in RH 70% were close to that of CK treatment.ABS/CSm, TRo/CSm and ETo/CSm in RH 50% and RH 50% treatments were significantly lower than that of CK treatment, while DIo/CSm was higher.As seen in Figure 7C, With treatment duration increasing, ABS/CSm, TRo/CSm and ETo/CSm decreased and reached the lowest in L 9d , which were 19.56%, 30.15% and 48.69% lower than that of CK treatment.While DIo/CSm increased with treatment duration increasing, the highest DIo/CSm was observed in L 9d , which was 22.29% higher than that of CK treatment.
The fluorescence difference kinetics DW OJ of the different treated OJ phases reveal their respective K-bands.As seen in Figure 8A, the DW OJ curves in T 38°C and T 41°C followed the same trend and both showed a K-band, while DW OJ curves in T 35°C did not show a clear K-band.As seen in Figure 8B, the DW OJ curves in RH 50% and RH 90% followed the same trend and both showed a Kband, while DW OJ curves in RH 70% did not show a clear K-band.As seen in Figure 8C, the DW OJ curves in L 6d and L 9d followed the same trend and both showed a K-band, while DW OJ curves in L 3d did not show a clear K-band.

Discussion
The light response curves parameters are important indicators that reflect the light energy utilization capacity and efficiency of plants under different environmental conditions.High temperatures affect the photosynthetic properties of plants (Li and Li, 2009).This study showed that values of AQE and P n-max decreased significantly with increasing temperature, while the value of R d increased.This indicated that the respiratory consumption of chrysanthemum leaves was high and photosynthetic activity was low under high temperature environments, which is in accordance with the study of Su et al. (Su and Liu, 2005) RH also affects plant photosynthesis.For example, 60% RH is the optimal level for  Zhou et al. 10.3389/fpls.2023.1272013Frontiers in Plant Science frontiersin.orgcabbage to grow under high temperature environments, while at 90% RH under high temperature environments, cabbage was damaged and P n was reduced (Han et al., 2019).In this study, AQE at 70% RH were higher than that at 50% and 90% RH.This suggested that adjusting the RH to 70% could alleviate the inhibition of high temperature stress on photosynthesis.The values of LSP, AQE, and P n-max were lowest at 90% RH, suggesting that the inhibition of photosynthesis was greater under high RH environments.Similar findings were reported in different studies too (Shin et al., 2007).The prolongation of the stress time increases the damage to plant photosynthesis (Han et al., 2018).The values of LSP, AQE, and P n-max were decreased with increasing treatment duration.This indicated that the degree of photosynthetic inhibition increased with prolonged stress.Chlorophyll content is an important indicator to assess stress in photosynthetic organs and usually reflects chloroplast development and photosynthetic performance (Lu et al., 2019).Chlorophyll synthesis is a series of enzymatic reactions, and high temperature stress causes protein denaturation and lipid peroxidation of cell membranes, reducing the rate of chlorophyll synthesis (Liao et al., 2004).This study showed that the values of chlorophyll a, chlorophyll b, and chlorophyll (a+b) decreased with increasing temperature.It suggested that chloroplasts were damaged or the rate of chlorophyll synthesis was reduced under high temperature environments, contributing to the decrease in P n , which is in line with the study of Yang et al. (Yang et al., 2019).
High temperature leads to the decrease in chlorophyll content of plant leaves, firstly, because high temperature reduces the rate of chlorophyll synthesis (Li et al., 2020), and secondly, because the accumulation of reactive oxygen species at high temperature accelerates the degradation of chlorophyll (Yuan et al., 2017).Carotenoids are both photosynthetic pigments and endocytic source of antioxidants, which can absorb excess energy in chloroplast, quench reactive oxygen species and prevent membrane lipid peroxidation (Zhou et al., 2016).This study showed that the content of carotenoid decreased with increasing temperature, indicating that carotenoids were damaged by high temperature stress and their functions were damaged, which can lead to reactive oxygen accumulation.This is consistent with the findings of Lokesha (Lokesha et al., 2019).This also indicated that under high temperature environments, the presence of nonstomatal limiting factors for photosynthesis inhibition.Moreover, this study found that the value of carotenoids at 70% RH was highest under high temperatures.It indicated that 70% RH was able to maintain the carotenoid content of chrysanthemum leaves under high temperatures, thus absorbing excess energy from chloroplasts, quenching reactive oxygen species, and increasing P n .In this study, it was found that the values of chlorophyll a, chlorophyll b, and chlorophyll (a+b) decreased at 50% and 90% RH, compared to 70% RH.Hence, 70% RH could alleviate the damage of chloroplasts in chrysanthemum leaves by high temperature.The difference of photosynthetic pigment content between RH 50% and RH 90% was statistically non-significant.A recent study pointed out that at 46°C, lower RH amplifies the inhibition of the photosystem by high temperature (Lysenko et al., 2023).For high or low RH, further experiments are needed as to which has a greater effect on chlorophyll content.Stomata are channels for the exchange of carbon and water between chloroplasts and the atmosphere and have an impact on plant physiology (Du et al., 2018).This study showed that the dominant factor in the decrease of P n at 35°C and 38°C was stomatal limitation, which is concordance with the work of Fan et al. (Fan et al., 2010).In this environment, high temperature led to a large number of stomatal closures, reduced G s and blocked CO 2 diffusion, which contributed to the reduction of P n .Wu et al. (Wu et al., 2001) found that stomatal limitation caused inhibition of photosynthesis when plants were under mild heat stress, but inhibition of photosynthesis under extreme heat stress was caused by non-stomatal limitation.In this study, when the temperature increased to 41°C, the dominant factor was non-stomatal limitation.As can be seen from the discussion below, this dominant factor was high temperature led to disruption of the internal structure of PSII and inhibition of its activity.Stomatal morphological characteristics are related to their function and also influenced by VPD (Sinclair et al., 2007).Alineaeifard et al. (Aliniaeifard and van Meeteren, 2016) found that chrysanthemums exposed to low VPD (0.23 kPa) had larger stomatal sizes, wider pore diameters, and greater stomatal densities, resulting in higher G s compared to chrysanthemums grown in a 1.05 kPa VPD environment.This study showed that the value of G s was higher in a 2.00 kPa VPD environment (RH 70% ) than that in a 3.34kPa VPD environment (RH 50% ).Among three RH conditions, the value of P n at 70% RH was the maximum, while R d was the minimum, suggesting that 70% RH mitigated the inhibitory effect of high temperature on P n .High and low RH in high temperature environments can exacerbate the effects (Bunce, 2002).The study showed that the value of P n decreased at 90% and 50% RH, indicating that high and low RH inhibit photosynthesis in high temperature environments.The dominant factor causing the decrease in P n from 0d to 6d was stomatal limitation, but non-stomatal limitation from 6 d to 9 d.As can be seen from the discussion below, the dominant factor for the decrease in P n from 6d to 9d was that prolonged stress led to disruption of the internal structure of PSII and inhibition of its activity.
WUE is the amount of CO 2 assimilated per unit mass of water lost by leaf transpiration (Hatfield and Dold, 2019).Given that stomata control water balance, stomatal behavior has a significant effect on WUE (Li and Liu, 2022).This study showed that the value of T r decreased at 35°C and 38°C.This was due to that stomatal limitation at 35°C and 38°C was a major factor in reducing P n , and the massive closure of stomata led to the decrease of T r .While the value of T r increased at 41°C, because that non-stomatal limitation at 41°C was a major factor in reducing P n , and the increase of G s led to the increase of T r .The value of WUE decreased with increasing temperature.This may be related to chrysanthemum leaf senescence under high temperature environments.Bunce et al. (Bunce, 2002) found that under excessively high RH conditions, increased VPD was caused by higher T r , which lead to the increase of G s .This study showed that VPD and the value of T r were lower at 90% RH than that of other RH conditions.This mean that 90% RH was not favorable for transpiration of chrysanthemum leaves.Numerically, the value of WUE was higher at 70% RH than 50% RH, suggesting that 70% RH may be better for water utilization by chrysanthemum leaves than 50% RH.The value of T r decreased at 3d and 6d, because that stomatal limitation at 3d and 6d was a major factor in reducing P n , and the massive closure of stomata led to the decrease of T r .While the value of T r increased at 9d, because that non-stomatal limitation at 9d was a major factor in reducing P n , and the increase of G s led to the increase of T r .
By analyzing chlorophyll fluorescence, it is possible to know the response of the plant to changes in environmental factors (Maxwell and Johnson, 2000).PS II is one of the most sensitive parts of the photosynthetic system to temperature stress and is closely linked to chlorophyll fluorescence (Murata et al., 2007).This study found that with increasing temperature, F o and F j increased.Photoinactivation usually leads to oxidative damage and inactivation of PSII reaction centers, which further leads to an increase in F o (Liu et al., 2022).It indicated that high temperature resulted in inactivation of PSII reaction centers.Changes in cystoid membrane structure and organization may result in changes in F m during many stress treatments (Baker, 2008).This study found that with increasing temperature, F m decreased, suggesting that high temperature resulted in the changes in cystoid membrane structure and organization.F v /F m is used to measure the maximum efficiency of PSII (Liu et al., 2020).A decrease in F v /F m is often observed when plants are in a stress state, which represents impaired PSII function (Guidi et al., 2019).This study found that with increasing temperature, F v /F m decreased, suggesting that high temperature resulted in impaired PSII function.In addition, this study found that ABS/CSm, TRo/CSm and ETo/CSm decreased, and DIo/CSm increased in chrysanthemum leaves under high temperature environments.This suggested that high temperatures may affect the structure of the PSII functional antenna, which reduces the ability to capture light (ABS/CSm), leading to a decrease in the excitation energy of the reduced Q A (TRo/CSm) and its ability to be used for electron transfer (ETo/CSm), and an increase in heat dissipation (DIo/CSm).To further analyze the changes of PSII in chrysanthemum leaves, experiments were conducted to investigate the kinetics of fluorescence differences in the OJ phases of different treatments.DW OJ can reflect the PSII functional antenna size and the activity of the manganese complex-dominated exocytosis complex.It was shown that K-bands appeared and DW OJ > 0 at 38 °C and 41°C, indicating that the oxygen release complex of PSII in chrysanthemum leaves was inactivated under high temperature environments (Wakjera et al., 2013), the efficiency of plastoquinone Q A in transferring electrons was decreased (Hermans et al., 2005), and PSII functional antenna size changed (Li et al., 2009).It also suggested that the main factor for the decrease in P n at 41°C was that high temperature led to disruption of the internal structure of PSII and inhibition of its activity.For the different RH conditions, F o , F j , F m and F v /F m were close to those of CK at 70% RH, indicating that 70% RH mitigated the effect of high temperature on PSII activity.While F o , F j , and DIo/CSm increased, F m , F v /F m , ABS/CSm, TRo/CSm and ETo/CSm decreased at 50% and 90% RH.It was also shown that K-bands appeared and DW OJ > 0 at 50% and 90% RH, indicating that both 50% and 90% RH inactivated the oxygen release complex of PSII, reduced the electron transfer efficiency of Q A and PSII functional antenna size changed.With treatment duration increasing, F o , F j , and DIo/CSm increased, while F m , F v / F m , ABS/CSm, TRo/CSm and ETo/CSm decreased.It was also shown that K-bands appeared and DW OJ > 0 at 6d and 9d.Kband at 9d was higher than that at 6d, suggesting that the inhibition of PSII activity was exacerbated with prolonged stress.It also suggested that the main factor for the decrease in P n from 6d to 9d was that prolonged stress led to disruption of the internal structure of PSII and inhibition of its activity.
Under high temperature environment, photosynthesis is often suppressed before other cellular functions are compromised.And RH can influence photosynthesis differently according to environmental changes (Rodrigues et al., 2016).The results of this study showed that high temperature had a greater effect on photosynthesis in chrysanthemum seedlings than RH.This indicates that high temperature dominates the effect of photosynthesis under high temperature and high RH.Meanwhile, the study also showed that there was an interactive effect of high temperature and RH on photosynthesis of chrysanthemum leaves, and the interaction of the two had a significant effect on P n (P<0.01).This indicates that changes in RH under high temperature conditions can significantly affect the photosynthetic rate.Excessive RH aggravated the inhibitory effect of high temperature on photosynthetic rate.This was mainly due to the fact that high RH led to low VPD, the closure of stomata, the reduction of T r , and the reduction of CO 2 exchange between inside and outside the leaves (Carvalho et al., 2015).In addition, high RH produces leaf thermal overload.Elevated leaf temperatures exacerbate damage to leaf photosynthetic functions.Lieten et al. (Lieten, 2002) found that strawberry leaves under high RH conditions showed leaf tip burn.In this study, we found that the inhibition of photosynthesis rate at 90% RH was also significantly higher at 50% RH than at 70% RH under high temperature conditions.This was because stomatal closure affected leaf CO 2 exchange and heat dissipation under low RH conditions (Ferrante and Mariani, 2018).At 70% RH, G s was significantly greater than that at 50% and 90% RH, which was favorable to alleviate the inhibition of photosynthesis by high temperature.

Conclusion
This experiment investigated the effects of high temperature, RH and treatment duration on photosynthesis of "Shenma" chrysanthemum leaves.The results showed that heat stress above 35°C affects chlorophyll fluorescence parameters of chrysanthemum leaves, significantly reduced the content of photosynthetic pigments, and severely inhibited photosynthesis.Under high temperature environment, the decrease of P n and photosynthetic pigment content at 70% RH was lower than the other two RH conditions and it reduced the damage of high temperatures to photosynthesis system.The dominant factor causing the decrease of P n in leaves was stomatal limitation at 35°C,38°C, three RH conditions, 3d and 6d, but nonstomatal limitation at 41°C and 9d.There was an interaction between temperature and RH, with a significant impact on P n (P<0.01).Under high temperature and RH environments, temperature is the main factor affecting photosynthesis, followed by RH.When the temperature reached or exceeded 35°C, adjusting the RH to 70% could effectively reduce the damage of high temperature stress on chrysanthemum leaves.

FIGURE 1
FIGURE 1Contributions of temperature, RH and treatment duration.Contribution rate (%) =SS F ×100/(SS T -SS E -SS B ). SS F means sum of squares away from the mean difference.SS T , SS E and SS B mean sum of squares for total, error and block, respectively.T, RH and L represent temperature, relative humidity and treatment duration, respectively.

FIGURE 3
FIGURE 3Effect of different temperature, RH and treatment duration on photosynthetic pigment content of chrysanthemum leaves.

FIGURE 4
FIGURE 4 FIGURE 6 Effect of different temperature (A), RH (B) and treatment duration (C) on rapid fluorescence kinetic curve of chrysanthemum leaves.T, RH and L represent temperature, relative humidity and treatment duration, respectively.
FIGURE 7 Effect of different temperature (A), RH (B) and treatment duration (C) on light energy absorption, capture, and transferenergy of chrysanthemum leaves.ABS/CSm means absorption flux per cross section.TRo/CSm means trapped energy flux per cross section.ETo/CSm means electron transport flux per cross section.DIo/CSm means non-photochemical quenching per cross section.T, RH and L represent temperature, relative humidity and treatment duration, respectively.
FIGURE 8 Effect of different temperature (A), RH (B) and treatment duration (C) on the fluorescence differential kinetics of OJ phase of chrysanthemum leaves.T, RH and L represent temperature, relative humidity and treatment duration, respectively.

TABLE 1
Experimental scheme of artificial climate chambers.

TABLE 2
JIP-test indices and terminology used in the study.

TABLE 3
Parameters of light response curve of chrysanthemum leaves.

TABLE 4
Gas exchange parameters of chrysanthemum leaves.

TABLE 5
Chlorophyll fluorescence parameters of chrysanthemum leaves.
Values are the means of three replicates per treatment.T, RH and L represent temperature, relative humidity and treatment duration, respectively.± indicates standard deviation.Means are not significantly different between different treatments when followed by the same lowercase letter, means are significantly different between different treatments (P < 0.05) when followed by different lowercase letters.LSP means light saturation point.F o means minimal recorded fluorescence intensity.F j means fluorescence intensity at point J of the OJIP curve.F m means maximal recorded fluorescence intensity.F v /F m means PSII maximal photochemical efficiency.