Soybean leaves are heterogeneous, and the adaxial surface is the major contributor to carbon gain because the adaxial surface palisade tissue is rich in chloroplasts and exposed to direct radiation (Evans, 1999). However, some plant leaves are inverted or wobbly due to cultivation conditions (e.g., water and fertilizer) and wind (Zhang et al., 2016; Paradiso et al., 2020). Due to the difference in anatomy between the abaxial and adaxial surfaces of soybeans, their response to environmental conditions can vary, especially light conditions (Hughes and Smith, 2007). Therefore, studying the response of inverted leaves to the environment will provide a theoretical basis for exploring ways to minimize damage to the photosynthetic apparatus.

Soybean is one of the most important oil crops in the world (Cai et al., 2020); due to human factors and frequent natural disasters, the global average temperature will continue to rise rapidly in the future; and unfavorable high temperatures will affect plant growth and development (IPCC, 2019), usually causing reversible/irreversible damage to different organs of the plant, this is because leaf photosynthesis is one of the most sensitive processes to elevated temperatures in plants (Yamori and Shikanai, 2016; Mihaljević et al., 2020). Under natural conditions, the elevated temperatures at noon in summer are usually accompanied by other environmental stresses, such as high light, and the dual stress of heat and high light seriously affects the growth and development of soybeans, especially during the seed-filling stage, resulting in reduced soybean yields (Cohen et al., 2021; Kimm et al., 2021). As an important organ in direct contact with the environment, leaves are more sensitive to light and temperature, because photosystem II (PSII) is sensitive to heat and high irradiation stress during the process of carbon dioxide assimilation (Dongsansuk et al., 2013; Jiang et al., 2021). Exposure to high light and elevated temperature in summer can damage the photosynthetic apparatus of the plant and cause photoinhibition, which is manifested in the metabolic processes: reduced transpiration accompanied by increased leaf temperature, reduced antioxidant and photosynthetic enzyme activities, damage to the cytoplasmic membrane, destruction of chloroplast structure and function, reduced electron transport and carbon metabolism, and increased reactive oxygen species (ROS) (Janka et al., 2013; Gu et al., 2017; Blackhall et al., 2020; Mihaljević et al., 2020). Studies have found that the synergistic effect of elevated temperature and high light caused significant degradation of D1 protein in plants, causing damage to both the donor and acceptor side of PSII (Krieger-Liszkay et al., 2008; Kalaji et al., 2016). Sunburn occurs on leaves under the long-term high light, and sunspots were also found on some fruits, which seriously affects fruit quality and crop yield (Chen et al., 2012; Blackhall et al., 2020). Under natural conditions, heat and high light stress often occur simultaneously and tend to damage the photosynthetic apparatus of inverted leaves; nevertheless, the state of the photosynthetic system of inverted leaves under elevated temperature and high irradiation needs further study.

The rapid chlorophyll a fluorescence technique is a nondestructive and effective tool for monitoring the effects of abiotic stress on the photochemical efficiency of PSII and the health of the plant, because it quickly, noninvasively analyzes and provides powerful data related to photosynthesis (Strasser and Srivastava, 1995; Oukarroum et al., 2018). The typical rise in chlorophyll a fluorescence transient kinetics over 1 s is multiphase (OJIP curve), and the shape of the OJIP curve changes with the physiological condition of the plant, reflecting valuable information on the structure and function of the photosynthetic apparatus (Kalaji et al., 2016). Strasser et al. (2004) developed a data processing method (JIP-test) for rapid chlorophyll a fluorescence induction curves based on the theory of energy fluxes in thylakoid membranes. The specific flux of each reaction center (RC) and the apparent flux of excited leaf cross-section (CS_O) provide rich information about the redox state of PSII (Strasser et al., 2010; Kalaji et al., 2016). The JIP-test has been widely used to analyze crop tolerance to single abiotic stresses and to screen for indicators of resistance identification, for example, elevated temperature stress (Janka et al., 2013; Mihaljević et al., 2020), nutrient deficiencies (Kalaji et al., 2014), drought stress (Marcińska et al., 2017), and high light stress (Hazrati et al., 2016).

Previous studies have shown that the photochemical efficiency of leaves is reduced when the leaf is inverted (Paradiso et al., 2020). In this study, we aimed to investigate the daily response of inverted leaves under specific conditions of high temperature and high irradiation at the photosynthetic level. We used JIP-test and gas exchange parameters to evaluate the photochemical adaptation of inverted leaves under elevated temperature and high light at noon. We hypothesized that PSII function is weaker and photochemical efficiency is lower in inverted leaves under high temperature and high light compared to normal leaves.

Under natural conditions, a combination of high light and heat stress in summer is the main environmental stress that leads to a decrease in plant photosynthesis, usually starting with a reduction in the production of photosynthetic assimilates under mild abiotic stress when leaves avoid water dissipation by reducing stomatal aperture and thus limiting the mesophyll conductance to CO₂, at which point diffusive limitation is the main cause of the decrease in leaf photosynthetic capacity (Das et al., 2015; Bahamonde et al., 2018; Fanourakis et al., 2019; Mihaljević et al., 2020). As the stress level increased and time increased, the leaf photosynthetic apparatus was damaged and PSII photochemical activity was reduced in addition to diffusive limitation, and at this time, the nondiffusion limitation was better than diffusion limitation (Hazrati et al., 2016; Jiang et al., 2021). In this study, the P_n and gs of inverted leaves and normal leaves were significantly reduced at 4 p.m. during the treatment, and C_i of normal leaves was also significantly reduced at noon, but C_i in inverted leaves behaved differently in both years, with no significant difference between 9 a.m. and 4 p.m. C_i in inverted leaves in 2020, while the difference reached a significant level in 2021, which could be caused by climatic factors between years but could at least suggest that the reduction in P_n of normal leaves can be explained by a reduction in gs (Pandey et al., 2007) and that inverted leaves may be more susceptible to photooxidative damage and damage to the photosynthetic apparatus at elevated temperature and high light compared to normal leaves, which is supported by the decrease in F_v/F_m values and performance indexes (PI_ABS and PI_total) and the increase in DI_O/RC values in inverted leaves at noon. Between the 2 years, the T_r values of normal leaves were slightly elevated or flat in the noon compared to the morning measurement, but T_r values of inverted leaves were significantly lower in the noon, which may also be one of the reasons for the lower P_n of inverted leaves compared to normal leaves, as the lower transpiration rate leads to higher leaf temperature, and the photoinhibition of photosynthesis depends on Temperature, the increase of leaf temperature will strengthen the photoinhibition, thereby greatly reducing the photosynthetic capacity of inverted leaves (Avola et al., 2008; Das et al., 2015; Gago et al., 2015; Durand et al., 2020). After the recovery of inverted leaves, the C_i values of normal and inverted leaves did not decrease with the decrease of P_n and gs values under elevated temperature and high light, and the C_i values of inverted leaves were higher than those of normal leaves, indicating that the irreversible damage to the photosynthetic apparatus of inverted leaves occurred under heat and high light (Mihaljević et al., 2020; Cohen et al., 2021), but the factor of shorter recovery time of inverted leaves could not be ignored. To obtain the adaptation of inverted leaves to light under the combined stress of high light and elevated temperature, the response of P_n to several light intensities of PPFD was evaluated. In this study, the P_Nmax, AQY, R_D, LCP, and LSP values of inverted leaves were lower than those of normal leaves under treatment and recovery conditions, indicating that the photosynthetic potential of leaves under elevated temperature and high light was reduced and the ability to utilize strong light was weakened in inverted leaves, similar to the studies by Proietti and Palliotti (1997), Xu et al. (2013), and Paradiso et al. (2020). It is worth noting that the P_Nmax of normal leaves decreases significantly from treatment to recovery, and this may be due to the fact that soybeans are susceptible to high temperature stress during the filling stage, which induced and accelerated the senescence of inverted leaves in advance (i.e., PI_total of inverted leaves was significantly lower than that of normal leaves under elevated temperature and high light), while the photosynthetic products of normal leaves efficiently transport to the reproductive organs during the recovery phase, accelerating leaf senescence, resulting in that the P_Nmax of the normal leaves was significantly lower during the recovery.

Many studies have assessed the harmful effects of heat on photosynthesis, where elevated temperatures reduce CO₂ fixation by inhibiting photosynthetic system activity. The inactivation of PSII in plants under complex stress in summer leads to a decrease in photosynthetic capacity (Fanourakis et al., 2019; Jiang et al., 2021). Some researchers have noted that F_v/F_m values of normal leaves range from 0.75 to 0.83 and that a decrease in this parameter indicates that PSII has been damaged (Krause and Weis, 1991). According to a certain one, an increase in F_O is one of the signs of photoinhibition (Uhrmacher et al., 1995). It has also been suggested that an increase in DI_O/RC and a decrease in Ψ_EO can be prepared to identify photoinhibition rather than F_v/F_m (Jiang et al., 2008). In this study, the F_v/F_m values of normal and inverted leaves were significantly lower at 4 p.m. compared to morning measurements, and the decrease was higher in inverted leaves than that of normal leaves. The F_v/F_m values ranged from 0.62 to 0.71 at 4 p.m. during treatment for inverted leaves, while normal leaves ranged from 0.71 to 0.75; after recovery of inverted leaves, the F_v/F_m of the inverted leaves in 2021 decreased significantly at 4 p.m. (F_v/F_m = 0.55) and that of the normal leaves is 0.61, At the same time, compared with the morning measurement, the F_v/F_m and Ψ_EO values of the inverted leaves decreased at 4 p.m., and the increase in DI_O/RC and F_O values were higher than those of the normal leaves. The LSP of the inverted leaf is between 528.4 and 577.3 μmol(CO₂) m^–2 s^–1, while the normal leaf is between 629.6 and 664.7 mol(CO₂) m^–2 s^–1, and the temperature at 4 p.m. in summer is 38.2°C, and the light intensity is 1,512 μmol(CO₂) m^–2 s^–1, which is much higher than its light saturation point, which causes the light system to be overexcited. All the above mentioned results indicate that the combined stress of elevated temperature and high light in summer promotes PSII inhibition of inverted leaves and stronger energy dissipation. This is supported by the significant increase in DI_O/RC value at 4 p.m. and the PCA results. This is similar to the study by Mihaljević et al. (2020) on apples.

In recent years, there has been considerable interest in using chlorophyll a fluorescence and related parameters to assess the effects of abiotic stress on the photosynthetic structure, using chlorophyll a fluorescence kinetics to characterize plant tolerance to abiotic stresses at the PSII level (Hussain et al., 2019; Rastogi et al., 2020). In fact, elevated temperature and high light stress result in significant changes in the shape of the chlorophyll fluorescence induction curve (Mihaljević et al., 2020). In this study, the combined high light and elevated temperature stresses significantly affected PSII performance during treatment and recovery of leaves, and these effects were visible in the variable fluorescence curves and relative variable fluorescence curves during treatment and recovery (Figures 3, 4). The typical shape of the OJIP transient curve has 3 main phases, namely, O-J, J-I, and I-P (Strasser and Srivastava, 1995). The J-I phase reflects the reduction of electron carriers (plastoquinone and plastocyanin) between PSII and PSI (Tóth et al., 2007). In this study, the increase in J-I transients in normal and inverted leaves differed by year, and an increase in J-I transients was observed in inverted leaves compared to normal leaves, which supports the vulnerability of the electron carriers of the inverted leaves to elevated temperature and high light and the partial reduction of the PQ pools between PSII to PSI, which is consistent with the reduction in the PQ pools under elevated temperature reported by Mihaljević et al. (2020). The I-P phase is the slowest fluorescence rise and is associated with a decrease in electron transport proteins (Ceppi et al., 2012). Previous studies have confirmed that I-P is relatively sensitive to various abiotic stress (Schansker et al., 2005). In this study, inverted leaves showed a significant positive peak change during recovery, suggesting that damage to the PSI structure, loss of function in inverted leaves, and electron transport on the PSI receptor side may have been inhibited.

The PSII and OEC are one of the main stress-sensitive sites in the photosynthetic apparatus (Adamski et al., 2011). Previous studies have shown that high light and elevated temperature reduce PSII activity and electron transfer efficiency (Boguszewska-Mańkowska et al., 2018). In this study, the active RC of normal and inverted leaves was significantly reduced at 4 p.m. (supported by the increase in ABS/RC). The increase in ABS/RC may be due to the increase in antenna size or partial PSII RC inactivation, which can be confirmed by a decrease in active RC per excitation cross-section (RC/CS). The inactivation of RC is considered to be a photoprotective mechanism, because part of the RC is transformed into a so-called “heat sink” by Stefanov et al. (2011), to dissipate excess excitation energy to prevent excessive excitation of PSII. The significant increase in dissipated energy (DI_O/RC) supports the change of RC function. Compared with the morning measurement, the TR_O/RC and DI_O/RC of the inverted leaves increased by 31.5% and 135.1%, respectively, at 4 p.m., while the normal leaves increased by 21.4% and 39.1%, respectively. These findings suggest that inverted leaves dissipate excitation energy in the form of more heat and fluorescence and more severe photoinhibition, which is consistent with the research results of Mihaljević et al. (2020). ET_O/RC describes the electron transport flux of each RC, which reflects the activity of active RCs. The ET_O/RC values decreased at 4 p.m., but some researchers have shown that ET_O/RC remained constant at heat and high light or that the ET_O/RC values increased at elevated temperatures (Faria-Silva et al., 2019; Mihaljević et al., 2020). In this study, the ET_O/RC values of inverted leaves decreased significantly at 4 p.m. compared with 9 a.m., while normal leaves remained essentially unchanged, which further supports the hypothesis that some of the RC is converted into a “heat sink” and also indicates that the activity of active RC in inverted leaves is reduced under high light and elevated temperature, which is consistent with the study by Mihaljević et al. (2020). Elevated temperature and high light also affect both the donor and acceptor sides of PSII (Buchner et al., 2015): on the donor side, OEC inactivated, as shown in this study by a significant increase in the positive K-band at 300 ms and V_K/V_J, which may be due to the loss of manganese cluster function in PSII at elevated temperature, resulting in an imbalance electron transport between the OEC and the PSII RC; while at the acceptor site, the electron transport between Q_A^– and Q_B^– is inhibited (Gu et al., 2017), and the disruption of the electron transport chain is due to the dissociation of the LHCII from PSII, which helps to avoid excessive reduction of PQ and protect PSII from damage (Galić et al., 2020). These findings can be supported by the significant reduction in the Area value of inverted leaves at 4 p.m. (because Area refers to the free PQ pool). A positive L-band indicates a lower energy connection (Kalaji et al., 2014; Jiang et al., 2021). Both inverted and normal leaves exhibited significant positive L- and K-bands at elevated temperature and high light, but the positive L- and K-bands were more pronounced in inverted leaves than in normal leaves, indicating more severe OEC damage. Elevated temperature and high light not only affect the function of PSII but also adversely affect the electron flow on the PSI acceptor side (φ_RO, R_EO/RC, and δ_RO) (Kalaji et al., 2014). A large number of studies have shown that δ_RO values increase in plants after heat stress (Oukarroum et al., 2009; Mihaljević et al., 2020). The same, but inverted leaves exhibit higher R_EO/RC and δ_RO than normal leaves, indicating that the reduction in electron transport efficiency from the intersystem electron carrier to the PSI receptor side of the inverted leaves is even greater. High light and elevated temperature did not appear to have an effect on φ_RO values, as ANOVA showed no significant differences between treatments; however, inverted leaves had significantly lower φ_RO compared with normal leaves, which may be due to a reduction in PSI content in inverted leaves (Yan et al., 2013). In this study, F_V/F_O was significantly lower in inverted and normal leaves under high light and elevated temperature, but the decrease was higher in inverted leaves than in normal leaves, indicating that electron transport was impaired during photosynthesis in inverted leaves compared to normal leaves, which is similar to the results of Janka et al. (2020) who treated microalgae (Tetradesmus wisconsinensis) with bicarbonate.

The overall photosynthetic performance of both normal and inverted leaves was reduced under elevated temperature and high light complex stress conditions, which can be explained by a decrease in the performance indexes (PI_ABS and PI_total). The expression of performance index PI_ABS is the product of three independent characteristics: the density of active RC per PSII antenna chlorophyll (RC/ABS), the maximum quantum efficiency of PSII (F_v/F_m), and the electron transport beyond Q_A (Ψ_EO) (Strasser et al., 2000; Kalaji et al., 2016). In this study, the decrease in PI_ABS appeared to be associated with a decrease in RC/ABS and Ψ_EO values, as the decrease in their values is greatest at elevated temperatures and high light. The PI_ABS values of normal and inverted leaves decreased by 63.2%–66% and 68%–90% at 4 p.m., respectively, compared to 9 a.m. Compared with PI_ABS, the PI_total values not only reflect PSII photosynthetic electron transfer activity but also relate to changes in PSI-related processes (Kalaji et al., 2014; Jiang et al., 2021). Therefore, some researchers suggest that PI_total is more sensitive to abiotic stresses than PI_ABS. Some researchers also concluded that the sensitivity of PI_ABS and PI_total to abiotic stress varied depending on environmental factors (Mallick and Mohn, 2003; Mihaljević et al., 2020). In this study, the PI_total values of normal and inverted leaves decreased by 24.1%–25.9% and 5.9%–66.7%, respectively, at 4 p.m. compared to 9 a.m. This is consistent with the trend in P_N values. The decrease in PI_total may be related to the loss of PSII activity, leading to a reduction in the electron transport chain and disruption of PSI function (Kalaji et al., 2014; Oukarroum et al., 2018). In this study, the PI_ABS values of inverted leaves appeared to be more sensitive to elevated temperature and high light compared with PI_total. The fact that PI_ABS and PI_total were still not restored to the levels of normal leaves after the recovery of inverted leaves may be due to the higher degree of damage suffered by inverted leaves under elevated temperature and high light, and the effective repair capacity of the photosynthetic apparatus (synthesis of new proteins to replace damaged core proteins) of inverted leaves remained lower than the photooxidative damage capacity after recovery.

Stress resistance is a very important factor for the successful production of soybean in the increasingly demanding agroecological conditions. Our data indicate that a more severe photooxidative damage occurs in inverted leaves under high light and elevated temperature conditions compared to normal leaves, as evidenced by OEC inactivation, inhibition of electron transport, and inactivation of some PSII RCs. High light and elevated temperature significantly reduced the performance indexes (PI_ABS and PI_total) of inverted leaves. The PI_ABS values of inverted leaves were more sensitive to elevated temperature and high light. The inhibition of electron transport and the inactivation of PSII RCs in inverted leaves under combined high light and elevated temperature stresses were responsible for the significant reduction in P_n. Due to climate change, in the future, it will be required a better understanding of interactions between soybean and their environment to achieve better adaptability and thus the productivity of future cultivars.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

CW and JZ designed the research. CW, JR, QG, LZ, and CL performed the research. CW, CL, and JZ analyzed the data. CW, LZ, and JZ wrote the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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