Low temperatures are an important limitation for geographic distributions of warm-season crops like cucumber (Cucumis sativus L.). Tolerance to low temperatures varies among different plant species and genotypes when changes in normal environmental cues occur. To cope with low temperature, biochemical and biophysical events should be coordinated to form a physiological response. We examined how light intensity influences the effects of low temperature on photosynthesis machinery and some biochemical traits. We used chlorophyll fluorescence imaging and polyphasic fluorescence transient (OJIP) to analyze cold stress (4 ºC) damage to photosynthetic electron transport chain (ETC) under different Photosynthetic Photon Flux Densities (PPFDs; 0, 300 and 600 μmol m-2 s-1), in four accessions of cucumber. The results showed that, the negative effects of cold stress are PPFD-dependent. The adverse effect of cold stress on ETC was more pronounced in plants exposed to 600 μmol m-2 s-1 compared to the control and dark-exposed plants; indicated by disturbance in ETC and higher energy dissipation. Moreover, biochemical traits including H2O2 content, ascorbate peroxidase activity and electrolyte leakage, and water-soluble carbohydrate was increased under low temperature by increase in PPFD, while chlorophyll and carotenoid contents decreased under low temperature by PPFD elevation. Low temperature induced H2O2 accumulation via suppressing APX activity in a PPFD-dependent manner. In conclusion, high PPFDs exacerbate the adverse effects of low temperature on the cucumber seedlings.
Research Article
Light Intensity: the Role Player in Cucumber Response to Cold Stress
https://doi.org/10.21203/rs.3.rs-948547/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 14 Jan, 2022
Version 1
posted
You are reading this latest preprint version
Cucumber is a warm-season crop, therefore, its growing season and geographical distribution are restricted by ambient temperature (Allen and Ort 2001). To overcome multiple environmental stresses, plants have been evolved complicated mechanisms through evolution that enables them to survive ever-changing environments. Understanding the underlying stress-tolerance mechanisms is an essential step for the development of stress-tolerant cultivars. Although these processes are not fully clarified, it has been known that a series of integrated events involving anatomical, morphological, biochemical, and molecular factors result in stress tolerance (Ibáñez et al. 2010; Shomali and Aliniaeifard 2020).
Light influences plant growth and development. Plants respond to different aspects of light including intensity, duration, wavelength, and direction. In general, their response differs according to the presence of other environmental cues, genotype, season, cultivation practices, and many others (Kozai 2016). Light is the only source of energy for photosynthesis; however, it can simultaneously act as a stress agent. Under high light stress or when plants are exposed to other abiotic stresses (e.g. cold conditions), supply from electron transport chain (ETC) (including ATP and NADPH) are more than the demand of Calvin-Benson reactions (Gu et al. 2017, Miyake et al. 2009); thus, in such conditions, the exorbitant light energy directly defects electron transfer machinery, especially photosystem II (PSII) (Roach and Krieger-Liszkay 2014). PSII impairment induces the production of reactive oxygen species (ROS), thereby resulting in the impairment of D1 protein, and also prevention of its repair known as photoinhibition (Demarsy et al. 2018; Vranová et al. 2002).
In cold stress conditions, the Calvin-Benson cycle reactions of photosynthesis are halted due to disturbance in enzymatic activity and structural destruction, resulting in a dead-end for products of light reaction to reach their downstream target. Therefore, the reductive power leads to the production of ROS further disturbs ETC machinery (Demarsy et al. 2018; Heldt 1997; Takahashi and Murata 2008). Through this circumstance, abiotic stresses target photosynthesis machinery and cause a reduction in the efficiency of photosynthesis. Maximum quantum efficiency of PSII, FV/FM, has been introduced as a useful index for detecting low- and high-temperature damage to components of photosynthesis machinery and to identify tolerant genotypes of faba bean to temperature stress (Zhou et al. 2018).
Chlorophyll fluorescence has also been used as an easily-applicable method for assessing the adverse effects of environmental stresses on plants (Bączek-Kwinta et al. 2011; Baker and Eva 2004; Baker 2008; Calatayud et al. 2006; Hazrati et al. 2016; Husičková et al. 2019; Moosavi-Nezhad et al. 2021; Moradi et al. 2021; Seif et al. 2021; Shomali et al. 2021), as well as an index for screening of genotype for cold tolerance (Dolstra et al. 1994; Hetherington et al. 1983; Schapendonk et al. 1989). This may suggest that chlorophyll fluorescence, which reflect energy excitation in PSII, can be used as an index for studying stress tolerance in plants. The merits of this index are its stability under changing climates and its fast and easy application (Roden et al. 1999; Roháček 2002; Walker 1987). The parameters reflected by chlorophyll fluorescence are the indication of the overall photochemical status of leaf but not the short-term status (Roden et al. 1999).
The so-called 'OJIP' test is a quick and non-destructive method for analysis of polyphasic fast chlorophyll transient that has been used in many studies for assessment of photosynthtic aparture under different stress conditions (Bayat et al. 2018; Dai et al. 2019; Estaji et al. 2019; Habibi 2018; Hazrati et al. 2016; Kalaji et al. 2012; Kalaji et al. 2014; Kalhor et al. 2018; Panda et al. 2008; Stirbet et al. 2018; Zushi et al. 2012). OJIP test reflects details about energy flow through thylakoid membrane specifically in PSII components (Kalaji et al. 2017) and provides information about the physiological state of PSII (Antal and Rubin 2008).
Response of plant to cold is always concomitant with exposure to different photon flux densities (PPFDs), however, evaluation of cold sensitivity/tolerance when plants are exposed to different PPFDs is largely overlooked in the previous reports. In this research, it is hypothesized that biophysical and biochemical response of plants may alter in response to cold conditions and the alternations might vary under different PPFDs. In addition, investigating the interaction between cold temperature and PPFD can advance our knowledge about the integrated effects of light and temperature as two vital requisites for plant growth, which can be potential stressors in non-optimal ranges.
Plant materials and growth condition
Seeds of four cucumber accessions including, Nunhems gouhar (N), Super sultan (S), Tabriz (T) and T-141 were provided by the gene bank of Seed and Plant Improvement Institute of Iran. After surface sterilization, seeds were planted in pots containing a mixture of cocopeat and perlite (1:1 v/v). Seedlings were planted in the greenhouse under 27/18 ± 3 °C (day/night) temperature and 50 ± 5% RH. After the emergence of true leaves, the seedlings were irrigated with half strength Hoagland solution two times a week.
Light treatments under cold condition
After the four-leave stage, cucumber seedlings were transferred to growth chambers and were exposed to either cold conditions (4 °C) or optimum temperature (24 °C) at 50% RH for 15 hours. In the cold conditions, seedlings were exposed to different PPFDs (0, 300, and 600 μmol m-2 s-1) supplied by white LED modules (Iran grow light, Tehran, Iran) under 4 °C. One set of plants were also exposed to 300 μmol m-2 s-1 under 24 °C as the control (Fig. 7A). After application of treatments, plants were immediately transferred to the laboratory for further investigations.
Chlorophyll fluorescence imaging and OJIP test measurements
A chlorophyll fluorescence imaging was performed in leaves detached from plants of each treatment. For this purpose, biophysical measurements were performed after plants put in darkness for 20 minutes. Detached young leaves were used for Fv/FM assessment using a fluorometer equipped with an imager (Handy Fluorcam FC 1000-H, Photon Systems Instruments, PSI, Czech Republic). The calculation of FV/FM was based on a custom-made protocol (Aliniaeifard et al. 2014a; Aliniaeifard et al. 2014b; Aliniaeifard and van Meeteren 2014; Genty et al. 1989).
FV/FM is the maximum photochemical efficiency of PSⅡ, also known as the maximum quantum yield of the primary photochemistry. FM denotes the maximal chlorophyll fluorescence intensity when all PSII reaction centers are closed, and F0 is the fluorescence intensity when all PSII reaction centers (RCs) are open. FV stands for the variable chlorophyll fluorescence (FM–F0) (Estaji et al. 2019; Habibi 2018; Kalaji et al. 2017).
A polyphasic chlorophyll fluorescence induction curve (OJIP transient) was also obtained in leaves attached to plants of treatment. Fluorpen FP 100-MAX (Photon Systems Instruments, Drasov, Czech Republic) was used to obtain the parameters of polyphasic chlorophyll a fluorescence (OJIP) transients. Different biophysical and phenomenological parameters related to PSII status (Strasser 1995) were investigated by the OJIP test according to the protocol described by (Kalhor et al. 2018). Parameters derived from the OJIP test provide information on the energy fluxes of light absorption (ABS) and trapping (TR0) of the excitation energy and electron transport (ET0) per reaction center (RC), which are described in Table 1.
Photosynthetic pigments
The light regimes as well as temperature effects on photosynthetic pigments (chlorophyll a, b, and carotenoids) content were assessed. For determination of chlorophyll a, chlorophyll b, and carotenoids content, the protocol described by Lichtenthaler and Wellburn (1983) were applied.
Their absorbance was recorded at 470 nm with a spectrophotometer (Lambda 25 UV/VIS) according to Lichtenthaler and Buschmann (2001). The entire process was conducted in low light conditions and placing the samples on ice, and the results expressed as mg g−1 on a fresh weight basis.
Ascorbate peroxidase (APX) activity
For determination of Ascorbate peroxidase (APX) activity, method described by Nakano and Asada (1981) was used with minor modifications. APX activity was determined by oxidation of ascorbic acid (AA) at 265 nm (ε = 13.7 mM−1cm−1). The reaction mixture contained the enzyme extract, 50 mM potassium phosphate buffer (pH 7.0), 5 mM AA, 0.5 mM H2O2. The reaction was started by adding H2O2. The rates were corrected for the non-enzymatic oxidation of AA by the inclusion of a reaction mixture without the enzyme extract (blind sample). The enzyme activity was expressed in AA μmol-1 min−1 per g of fresh weight (Nakano and Asada 1981).
Hydrogen peroxide (H2O2) content
For measurement of H2O2, spectrophotometric method described by Patterson et al. (1984) was applied (Patterson et al. 1984). Measurement started by reaction of sample with potassium iodide (KI). The reaction mixture contained 0.5 mL of Trichloroacetic acid (TCA) (0.1 %), leaf extract supernatant, 0.5 mL of K-phosphate buffer (100 mm) and 2 mL reagent (1 M KI (w/v) in fresh double-distilled H2O). Absorbance was measured at 390 nm after development of the reaction for 1 h in darkness. A standard curve prepared with known concentrations of H2O2 as described by Patterson et al. (1984) was used for calculation of H2O2 content in the supernatant (Patterson et al. 1984).
Electrolyte leakage (EL)
Cell membrane stability is estimated in plant tissue by measurement of EL. Ten leaf discs (0.5 cm diameter) from the young fully-developed leaves were taken using a cork borer. Leaf disc samples washed with deionized water to remove surface-adhered electrolytes, and immersed in 10 mL deionized water in closed vials at room temperature for 24 h. Then, the electrical conductivity (EC) of the samples was recorded as primary leakage (PL) and after one hour boiling of the samples in a bain-marie (100 °C) the secondary leakage (SL) was recorded. EL was calculated using the following equation (Nakano and Asada 1981; Shiferaw and Baker 1996).
Water-Soluble Carbohydrates (WSC)
Total WSC in cucumber leaves was determined by the anthrone colorimetric reagent as described by Yemm and Willi (1954). Five mL of ethanol (80%) was added to 0.5 g fresh leaves for three times, following which they were centrifuged for 15 min at 1500×g. Then, 100 μL of the extract was added to 3 mL of 0.2% anthrone reagent. The solution mixture was boiled at 95 °C for 10 min. Subsequently, the reaction was terminated quickly in an ice bath. The absorbance of the samples was recorded spectrophotometrically at 620 nm using glucose as the standard and expressed as mg soluble carbohydrates g−1 fresh weight (Yemm and Willis 1954).
Statistical analysis
The experiment was conducted in randomized block design with three levels of light (0, 300, and 600 μmol m-2 s-1), 4 accessions (N. S, T, T141), and two temperature conditions (24 and 4 °C) with 5replications. The statistical analysis was done by SAS® software (version 9.4).
Table 1. Abbreviations, formula, and definitions of the OJIP parameters assessed in the current study (Kalaji 2017)
|
Abbreviation |
Equation |
Definition |
||
|
FV /F0 |
ϕ P0 /(1 − ϕ P0 ) |
Maximum efficiency of the water diffusion reaction on the donor side of PSII |
|
|
|
FV /FM |
TR0 /ABS = [1 − (F0 /FM )] |
Relative maximal variable fluorescence |
|
|
|
FM/F0 |
|
Structural damage to PSII |
|
|
|
ABS/RC |
M0 (1/VJ )(1/ϕP0 ) |
Light absorbance flux for PSII antenna Chlorophylls per reaction center |
|
|
|
TR0/RC |
M0 (1/VJ ) |
Trapped energy flux per reaction center |
|
|
|
ET0/ RC |
M0 (1/VJ ) ψ0 |
Electron transport flux per reaction center |
|
|
|
DI0 /RC |
(ABS/RC) − (TR0 /RC) |
Energy flux not intercepted by an RC, dissipated in the form of heat, fluorescence, or transfer to other systems, at time t = 0. |
|
|
|
PIabs |
(RC/ ABS) × (ϕP0/(1 –ϕP0)) × (ψ0/(1 - ψ0)) |
Performance index per absorbed light |
|
|
Biophysical response of cucumber seedlings to low temperature varies among plants exposed to different PPFDs
The energy content of different PPFDs is absorbed by the chlorophyll pigments, which straightly affect the ETC of the photosynthetic apparatus (Fig. 7C, D). Therefore, to assess the impacts of different PPFDs on the photosynthetic functionality, polyphasic Chl a fluorescence (OJIP) on the dark-adapted leaves of four cucumber accessions’ seedlings was analyzed. Analysis of chlorophyll fluorescence were done following exposure of cucumber seedlings to low temperature under different PPFDs. Specified energy fluxes per QA– (reduced PSII reaction center) was significantly (P > 0.05) affected by PPFD and low temperature. Light absorbance flux by PSII antenna per reaction center (ABS/RC) decreased when plant exposed to 300 μmol m-2 s-1 in a low temperature condition compared to the control (300 μmol m-2 s-1 and optimum temperature). In contrast, exposing plant to 600 μmol m-2 s-1 led to an average of 22.5% increase in ABS/RC (i.e. 20.4% for N, 22.5% for S, 22.9% for T and 24.1% for T-141). The lowest ABS/RC, on the other hand, was noted for all accessions in dark conditions (Fig. 1A). Trapped energy per reaction center (TR0/RC) was decreased by increase in PPFD under low temperature. TR0/RC remained relatively unchanged under darkness and 300 μmol m-2 s-1 ,whereas, an average of 8% decrease was detected in accessions under low temperature and 600 μmol m-2 s-1 (9% for N, 8% for S, 9% for T and 6% for T-141) compared to the control (300 μmol m-2 s-1 and normal temperature) (Fig. 1.B)
Electron transport flux per reaction center (ET0/RC) reduced by exposure to low temperature in a PPFD-dependent manner. Plants exposed to low temperature showed a reduced ET0/RC under darkness and 300 μmol m-2 s-1, while the highest increase was noted under 600 μmol m-2 s-1. This means their ET0/RC increased by an average of 8% (8 % for N, 6% for S, 8% for T and 8% for T-141) compared to the control (300 μmol m-2 s-1 and normal temperature; Fig. 1C). Low temperature also increased the energy flux not intercepted by a RC but dissipated in the form of heat (DI0/RC). Thus under low temperature, the value of DI0/RC remained relatively unchanged under darkness and increased in 300 μmol m-2 s-1 .This increase was more profound under 600 μmol m-2 s-1 compared to those of the control (300 μmol m-2 s-1 and normal temperature). Under low temperature, the increase in DI0/RC markedly increased as PPFD increased. DI0/RC was increased by an average of 77.72% (75% for N, 67% for S, 83.4% for T and 85.4% for T-141) in all accessions when exposed to low temperature and 600 μmol m-2 s-1 compared to the plants exposed to the dark conditions (Fig.1D).
The value for FV/FM of all four accessions decreased by an increase in PPFD (Fig. 2, 3A). In comparison to control (300 μmol m-2 s-1 and normal temperature), FV/FM markedly decreased by an average of 55% in four accessions of cucumber (54% for N, 51% for S, 82% for T and 42% for T-141) when exposed to 600 μmol m-2 s-1 and low temperature (Fig. 3A; 7E).
Maximum efficiency of the water diffusion reaction on the donor side of PSII (FV/F0) was reduced drastically by low temperature and the reduction showed a PPFD dependent manner (Fig. 3A). Plants exposed to low temperature and 600 μmol m-2 s-1 portrayed an average of 31% (31% for N, 24% for S, 43.4% for T and 26% for T-141) reduction in FV/F0 in comparison with those under control conditions (300 μmol m-2 s-1 and normal temperature) (Fig. 3B).
The relative value of maximal and minimum chlorophyll fluorescence (FM/F0), which portrays the structural damage to PSII (Ayyaz et al. 2020), was profoundly affected by both low temperature and PPFD. The lowest value of FM/F0 was detected in those plants placed under low temperature and simultaneously exposed to 600 μmol m-2 s-1 (67% decrease) followed by those exposed to 300 μmol m-2 s-1 and darkness, respectively (Fig. 3C).
Performance index per absorbed light (PIabs) indicates the potential for reduction of intersystem electron acceptors by photons absorbed via PSII (Strasser et al. 2010). The PIabs of cold-exposed cucumber accessions was considerably lower in seedlings exposed to 300 and 600 μmol m-2 s-1 in comparison with the PIABS of control and dark-exposed seedlings (Fig. 3D). The more drastic decrease in PIabs (in average 76% compared to control) was recorded under low temperature and 600 μmol m-2 s-1 (Fig. 3D).
Low temperature induced H2O2 accumulation via suppressing APX activity in a PPFD-dependent manner
The levels of H2O2 showed a stepwise increase by rising in PPFD under low temperature. The highest H2O2 level was recorded under 600 μmol m-2 s-1, by an average of 2.5-fold increase in four accessions of cucumber (2.1 fold for N, 2.7 fold for S, 3.3 fold for T and 2.4 fold for T-141) compared to control (Fig. 4A; 7B).
APX activity decreased when plants exposed to low temperature yet it was negligible under dark and profound under 300 μmol m-2 s-1 and 600 μmol m-2 s-1 compared to the control. The most noticeable decrease in APX activity was occurred when plants exposed to low temperature under 600 μmol m-2 s-1 , which showed an average of 28% decrease in four successions of cucumber (N: 28.1%, S: 18%, T: 32% and T-141: 36%) compared to control (Fig.4B; 7B).
A negative correlation was observed between H2O2 and APX with an average of -77%, for those placed in control environment, together with -13.5%, -58.5% and -78% for those placed in low temperature treatments either under darkness, 300 or 600 μmol m-2 s-1, respectively (Fig. 4C; 7B). This negative correlation shows removal of H2O2 by high activity of APX as strongly detected in control treatment. Whereas, the negative correlation between APX activity and H2O2 content under low temperature and 600 μmol m-2 s-1 is indicative of H2O2 accumulation in expense of suppression of APX activity due to low temperature injury.
The level of EL was affected by low temperature and PPFD. Regardless of PPFD, the value for EL in all treatments was higher under low temperature than control, though with a negligible increase under darkness. Under low temperature, EL was increased stepwise by increase in PPFD. The highest EL was obtained under 600 μmol m-2 s-1 showed an average of 65% increase in four accessions of cucumber (N: 29% for N, 68% for S, 125% for T and 36% for T-141) compared to control (300 μmol m-2 s-1 and normal temperature) (Fig. 4D; 7B).
Low temperature decreased the leaf photosynthesis pigment contents in a PPFD-dependent manner
Cold stress can damage photosynthesis pigments in plants. In the present study, both low temperature and PPFD affected chlorophyll (a+b) of cucumber accessions. Regardless of PPFD, chlorophyll (a+b) content reduced when plants were exposed to low temperature and the level of reduction was more drastic under 600 μmol m-2 s-1 compared to the dark, 300 μmol m-2 s-1 , and control. Furthermore, an average of 49% (30% for N, 36% for S, 77% for T and 53% for T-141) decrease in chlorophyll (a+b) content was observed under 600 μmol m-2 s-1 and low temperature compared to the control condition (Fig. 5A).
Carotenoid was also affected by both PPFD and low temperature. A stepwise decrease in carotenoid content was observed by increase in PPFD under low temperature. Under low temperature and 600 μmol m-2 s-1, the decrease in carotenoid was more drastic than 300 μmol m-2 s-1, dark, and control; showed an average of 64% (48% for N, 57% for S, 84% for T and 69% for T-141) decrease in carotenoid content compared to control (Fig. 5B). To better understand the association between photosynthesis pigments and photosynthesis efficiency, the correlation between chlorophyll (a+b) and carotenoid with PIabs was analyzed. So, both chlorophyll (a+b) and carotenoid were negatively correlated with PIabs and the correlation was weakened by low temperature and PPFD. The correlation between PIabs and chlorophyll (a+b) reduced from an average of 74% (72% for N, 63% for S, 88% for T and 73% for T-141) in control to an average of 29% (29% for N, 38% for S, 21 for T% and 30% for T-141) for plants exposed to 600 μmol m-2 s-1 under low temperature (Fig. 5C). The correlation between carotenoid and PIabs showed a stepwise decrease by increase in PPFD under low temperature compared to control. The highest correlation between PIabs and carotenoid was observed in control (94% for N, 90% for S,95% for T and 99% for T-141) and the lowest correlation was obtained in plants placed in low temperature condition and 600 μmol m-2 s-1 (59% for N, 4% for S,10% for T and 15% for T-141) (Fig. 5D).
Water soluble carbohydrate (WSC) accumulates in plants under low temperature
Both PPFD and low temperature affected WSC in cucumber accessions. The effect of low temperature was negligible on WSC of plants exposed to darkness, while increase in PPFD led to a considerable increase in WSC. More accumulation of WSC was observed in seedlings placed under low temperature condition and 600 μmol m-2 s-1 than those placed under 300 μmol m-2 s-1 and control, which showed an average of 4.5-fold increase in four accessions of cucumber (6 fold for N, 3 fold for S, 2 fold for T and 5 fold for T-141) (Fig. 6A). By increase in PPFD under low temperature, correlation between WSC and PIabs increased compared to the control (300 μmol m-2 s-1 and normal temperature). In control condition, WSC and PIabs showed an average of 34% correlation (23% for N, 43% for S, 33% for T and 38% for T-141) while under low temperature and 600 μmol m-2 s-1 the correlation increased to an average of 60% in four accessions of cucumber (53% for N, 66% for S, 59% for T and 62% for T-141) (Fig. 6B).
The physiological impacts of cold stress on plants depends on the ability of acclimatization, treatment, plants developmental stage and PPFD (Raju et al. 2018). Light intensity is one of the most important factors that affect the magnitude of perceived stress by plants. By imposing oxidative stress, light influences both molecular and whole plant response to imposed stresses (Raju et al. 2018). In the present study, seedlings of four cucumber accessions were exposed to either low temperature (4 °C) under simultaneous different light intensities (i.e. 0, 300, 600 μmol m-2 s-1) or optimum temperature (24 °C) under 300 μmol m-2 s-1. Photosynthetic performance of the cucumber plants were decreased by exposure to low temperature and simultaneous higher PPFDs. For instance, FV/FM decreased under low temperature, obtaining the lowest value under low temperature and 600 μmol m-2 s-1. However, the value of FV/FM in both darkness-exposed and control treatments was close to 0.8. In accordance, it has been shown that low temperature and light stresses reduced FV/FM and quantum yield of PSII in Zea maize and Pinus leucodermis (Colom et al. 2003; Fracheboud et al. 1999). Decrease in FV/FM due to low temperature stress has been also reported in nine Stevia rebaudiana cultivars (Hajihashemi et al. 2018b). These results indicate that low temperature-light stress caused photoinhibition particularly when PPFD increased from 0 to 600 μmol m-2 s-1. In line with the findings of the present study, the occurrence of photoinhibition resulted from higher rate of photo damage than repair on PSII under low temperature-light stress have been reported in a study on three Paphiopedilum species (Yang et al. 2017). Low temperature consequences in depression of chemical reactions including inhibition of the Calvin–Benson Cycle, thereby blocking the photosynthetic electron transport passway from PSII to NADP+ (Allakhverdiev and Murata 2008). The results obtained from this study showed that the maximum efficiency of the water-diffusion reaction on the donor side of PSII dropped drastically under 600 μmol m-2 s-1, indicated by the lower FV/F0 when compared to that of other treatments. The maximum FV/F0 was related to dark-exposed and control seedlings and its minimum value was observed in seedlings under low temperature and 600 μmol m-2 s-1. This is a common phenomenon in stress conditions, specifically light stress (Baker and Eva 2004; Fu et al. 2012; Hazrati et al. 2016; Maxwell and Johnson 2000). Oxygen-evolving complex (OEC) is the most sensitive link in ETC and is responsible for the transfer of electrons from water to ETC (Heldt 1997; Kalaji et al. 2017). Therefore, our results suggests that the disruption of OEC that associates with structural damage to PSII (Ayyaz et al. 2020) exacerbated by increase in PPFD under low temperature, which is in line with the results of Fracheboud et al. (1999).
In the current study, more reduction in ET0/RC was associated with higher PPFDs, while only a slight reduction was observed in ET0/RC of darkness-exposed and control seedlings. It should be noted that ET0/RC depends on both the energy flux produced through the transfer of a high-energy electron from QA- to the ETC acceptors (Stirbet et al. 2018) and the plant's demand for the photosynthetic products. This is defined by plant’s physiological conditions, which is controlled by environmental factors, in particular, abiotic stresses. Therefore, any disturbance in light reactions of photosynthesis causes reduction in the rate of ET0/RC (Kalaji et al. 2017).
Our showed that, TR0/RC decreased under low temperature synergistically with increasing in PPFD. When TR0/RC decreases, not only oxidation of QA- to QA slows down to a cessation (Strasser et al. 2000), but also it causes a reduction in the rate of ET0/RC. Indeed, when TR0 is diminished, ETC is fed by the lower level of absorbed energy (Sarkar and Ray 2016). High result in QA- over-reduction due to inhibition of ET0 to the downstream electron receptors located in ETC (Foyer et al. 2012).
ABS/RC increased with the raise in light intensity under low temperature. Presumably, with the increase in light PPFD, absorption flux cannot proceed through electron acceptors in RC due to the reduced status of QA-. As a result, energy flux dissipates as heat (Strasser et al. 2000). In accordance, our results revealed that dissipated energy in the form of DI0/RC increased with the increase in light intensity. This shows the route where absorbed light energy is directed under light stress condition. Under stress condition, the energy flux that is not intercepted by RC consequences in reduced electron transport through ETC and as a result the energy dissipates in the form of heat or fluorescence (Kalaji et al. 2017). Increase in DI0/RC is another indicator of stress (Bayat et al. 2018; Rodríguez et al. 2015; Shomali et al. 2021). In the current research, the maximum DI0/RC obtained from plants exposed to 600 and 300 μmol m-2 s-1, respectively; whereas plants under control condition showed the lowest DI0/RC. Results obtained from this research allowed us to claim that in normal conditions the share of energy that participates in photochemical work outweighs that of dissipated in other routes indicated by lower DI0/RC in control plants compared to that of plants under other treatments. Low temperature shifted the direction of energy to the route of dissipation. Besides, increase in light intensity under low temperature, as a consequence increased the share of energy passed through the rout of dissipation instead of photochemical activity.
In this research, the PIabs of leaves in all four accessions decreased under low temperature stress and the decline following exposure to 300 and 600 μmol m-2 s-1when compared to its value in darkness-exposed and control seedlings. A very slight reduction in PIabs was observed in low temperature- and darkness-exposed seedlings compared to PIabs of control seedlings, which confirms the previous reports regarding the significant effect of light stress on PIabs (Gericke 2018). Reduction in PIabs underlies increase in absorbed (ABS/RC) and dissipated (DI0/RC) energy together with the reduction in ET0/RC (Bayat et al. 2018). PIabs is the most applicable performance index (PI) (Stirbet et al. 2018) that states the potential for reduction of intersystem electron acceptors by photons absorbed via PSII (Strasser et al. 2010), more specifically, energy flux at the onset of absorption process up to the reduction of plastoquinone (PQ) (Strasser et al. 2000). PI is also an index for the overall vitality of PSII and known as the most sensitive indicator in this regard (Kalaji et al. 2017; Van Heerden et al. 2007).
In this study, chlorophyll a, chlorophyll b, and carotenoid content were measured to characterize the effect of light intensity and low temperature stress on cucumber accessions. Chlorophylls are the most abundant pigments in plants, which capture light energy during photosynthesis. When plants face to biotic or abiotic stresses, impaired chlorophyll biosynthesis or accelerated chlorophyll degradation cause reduction in chlorophyll content (Perveen et al. 2010). Therefore, investigation of photosynthetic pigments provides more evidence for photosynthesis downregulation under low temperature. Both chlorophylls and carotenoids decreased by seedlings exposure to low temperature and the highest decline was observed in plants exposed to 600 μmol m-2 s-1 compared with those exposed to 300 μmol m-2 s-1, darkness and control. Such effect of low temperature stress on photosynthetic pigments has also been observed in rice cultivars (Han et al. 2017). The correlation between PIabs with other parameters has been used to understand the way different stressors affect plants’ photosynthesis (Shomali et al. 2021). In our study, a strong correlation was detected between chlorophylls and carotenoids with PIabs in control, while the correlation weakened by increase in PPFD under low temperature. This suggest that, not only chlorophylls and carotenoids degraded under low temperature, but also existing chlorophylls couldn’t efficiently contribute to improving photosynthetic performance of cucumber accessions under low temperature.
In our study, H2O2 content of leaves was detected following plant exposure to low temperature stress. The production of ROS in response to various environmental stresses has been considered as a hallmark of stress (Steffens 2014). The impairment of electron flow through ETC induces ROS production that further scavenge through enzymatic and non-enzymatic mechanisms (Ahmad et al. 2010; Pospíšil 2016). ROS production occurs when abiotic stresses inhibit the PSII complex from being coupled with electron transport, leading to the complete reduction of the PQ pool and blockage of the electron flow (Pospíšil 2016). In this circumstance, H2O2 produces when an electron from QA- is transported to an O2 molecule, which decreases the reduction of the PQ pool (Caverzan et al. 2012). In this study, the increase in H2O2 was more drastic when low temperature stress was concomitant with 600 μmol m-2 s-1, which is in agreement with the result of previous studies that have shown that H2O2 content increased in watermelon seedlings (Rivero et al. 2002) and rice in response to low temperature stress.
The activity of APX reduced after exposing plants to low temperature, surging the level of this enzyme by increasing in the level of light intensity from 0 to 600 μmol m-2 s-1. APX is an antioxidant enzyme that plays an important role in removing ROS in plants via cleavage of H2O2 into H2O and O2 (Asada 1999; Scandalios 2002). When APX activity decreases, the accumulation of H2O2 occurs and adversely affect electron transport in stressed plant. In agreement with the result of this study, the reduction in APX activity in response to low temperature stress in rice (Xu et al. 2014; Zhang et al. 2013) and cassava (Xu et al. 2014) as well as under light stress in rose (Bayat et al. 2018; Xu et al. 2014) has been previously reported.
A negative correlation observed between H2O2 and APX, the strength of which increased in a PPFD-dependent manner under low temperature. A strong negative correlation was observed in both control plants and plants exposed to simultaneous low temperature and 600 μmol m-2 s-1; however, the reasoning for strong negative correlation differs. Considering the level of H2O2 and APX as well as the correlation between them in different treatments demonstrates that the strong correlation between those traits in control plants raised from the high activity of APX coupled with minute content of H2O2. However, the strong correlation between APX and H2O2 under low temperature parallel with 600 μmol m-2 s-1 suggests the increase in H2O2 in expense of suppressed APX activity due to low temperature injury.
The results of the present study revealed that low temperature impacts on plants was trivial under darkness while was profound under 600 μmol m-2 s-1. Production of ROS has a close relationship with the impairment of electron transport (Pospíšil 2016) and the fact that light is the driving force for electron production and transport, allows us to conclude that light plays a pivotal role on occurrence and the magnitude of low temperature stress.
In present study, we investigated the effect of light intensity and low temperature stress by detecting EL that reflects damage to cell membrane. EL has been introduced as a direct stress marker for indication of cell membrane damage from low-temperature stress (Cui et al. 2019). In this study, the EL increased following the exposure of plants to low temperature and the increase in PPFD led to the higher EL level compared with that of plants exposed to control and darkness while in absence of light only negligible differences observed in EL of plants compared to control. It can be proposed that, increase in PPFD amplifies the cellular damage caused by low temperature. The low temperature-induced EL has been reported in previous studies on tomato (Kim et al. 2002) and wheat (Shahryar and Maali-Amiri 2016). However, the involvement of light on the low temperature damage has not been addressed, yet.
Despite the decline in PIabs and FV/FM, WSC increased under low temperature stress. The increase in WSC under low temperature stress have been explained as a defense mechanism to prevent cellular freezing (Améglio et al. 2001). In line with our findings, the increase in WSC in response to low temperature have been reported in different plant species (Hajihashemi et al. 2018a; Wang et al. 2013; Yuanyuan et al. 2009). Moreover, when light reactions of photosynthesis is impaired, a feedback inhibition signaling bring about to prevent photosynthesis and inhibit the foliar transport of carbohydrates (Adams et al. 2013), which was highlighted by increase in correlation of PIabs and WSC by raise in PPFD under low temperature.
Light and temperature fluctuations are among the factors always present in the scene of plants life. However, fluctuation in PPFD or drop in ambient temperature negatively influences the plant performance. To study the mechanisms underlay plant tolerance to low temperature-light stress, cucumber plants were exposed to low temperature (4 °C) and different intensities of PPFD (including 0, 300, and 600 μmol m-2 s-1). Analyzing of plant biophysical responses through chlorophyll a fluorescence imaging and OJIP transient was used as a tool to characterize and evaluate the response of four low temperature-exposed cucumber accessions to light intensities. OJIP parameters altered in response to low temperature-light stress in an accession- and PPFD-specific manner, in such a way that, TR0/RC, and ET0/RC and the downstream signals were retarded. In addition, the share of energy dissipation outweighs that of trapped to be utilized for chemical work. All of the observed alternations in OJIP parameters were more pronounced when seedlings were exposed to low temperature-light stress rather than low temperature stress alone. Moreover, beside photosynthesis pigment degradation, the activity of antioxidant enzymes such as APX suppressed in a PPFD-dependent manner under low temperature, which led to increase in H2O2 and the subsequent photoinhibition that were detected by OJIP. The resulting photoinhibition caused a feedback inhibition for Calvin cycle, further resulted in WSC accumulation. In conclusion, low temperature injures plants by adversely affecting the photosynthesis machinery and the magnitude of low temperature stress damage is closely depends on PPFD.
Author contribution statement
S.A., and J.A-K. took part in designing and planning the experiments, preparing material for the experiment. T.A. carried out the experiment. SA and Sh.A. made substantial contributions to conception and design. A.Sh. performed statistical analysis, and wrote the manuscript. M.M-N. and SA critically revised the final manuscript. M.M-N draw the schematic figures.
Compliance with ethical standards (e.g. Conflict of interest)
Authors declare no conflict of interest.
Funding statement
We would like to thank Iran National Science Foundation (INSF) (grant number 96006991) and University of Tehran for their supports.
Data availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author.
Additional information
The experimental research and field studies complied with relevant institutional, national, and international guidelines and legislation
Adams WW, Muller O, Cohu CM, Demmig-Adams B (2013) May photoinhibition be a consequence, rather than a cause, of limited plant productivity? Photosynthesis research 117: 31-44.
Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S (2010) Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Critical reviews in biotechnology 30: 161-175.
Aliniaeifard S, Malcolm Matamoros P, van Meeteren U (2014a) Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling? Physiol Plant 152: 688-699.
Aliniaeifard S, Malcolm Matamoros P, van Meeteren UJPP (2014b) Stomatal malfunctioning under low VPD conditions: induced by alterations in stomatal morphology and leaf anatomy or in the ABA signaling? Journal of Experimental Botany 152: 688-699.
Aliniaeifard S, van Meeteren U (2014) Natural variation in stomatal response to closing stimuli among Arabidopsis thaliana accessions after exposure to low VPD as a tool to recognize the mechanism of disturbed stomatal functioning. J Exp Bot 65: 6529-6542.
Allakhverdiev SI, Murata N (2008) Salt stress inhibits photosystems II and I in cyanobacteria. Photosynthesis research 98: 529-539.
Allen DJ, Ort DR (2001) Impacts of chilling temperatures on photosynthesis in warm-climate plants. Trends Plant Sci 6: 36-42.
Améglio T, Cochard H, Ewers FW (2001) Stem diameter variations and cold hardiness in walnut trees. Journal of Experimental Botany 52: 2135-2142.
Antal T, Rubin A (2008) In vivo analysis of chlorophyll a fluorescence induction. Photosynthesis res 96: 217-226.
Asada K (1999) The water-water cycle in chloroplasts: scavenging of active oxygens and dissipation of excess photons. Annual review of plant biology 50: 601-639.
Ayyaz A, Amir M, Umer S, Iqbal M, Bano H, Gul HS, Noor Y, Javed M, Athar HR, Zafar ZU (2020) Melatonin induced changes in photosynthetic efficiency as probed by OJIP associated with improved chromium stress tolerance in canola (Brassica napus L.). Heliyon 6: e04364.
Bączek-Kwinta R, Kozieł A, Seidler-Łożykowska K (2011) Are the fluorescence parameters of German chamomile leaves the first indicators of the anthodia yield in drought conditions? Photosynthetica 49: 87-97.
Baker N, Eva R (2004) Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. J Exp Bot 55: 1607-1621.
Baker NR (2008) Chlorophyll fluorescence: a probe of photosynthesis in vivo. Ann rev plant biol 59: 89-113.
Bayat L, Arab M, Aliniaeifard S, Seif M, Lastochkina O, Li T (2018) Effects of growth under different light spectra on the subsequent high light tolerance in rose plants. AoB Plants 10: ply052.
Calatayud A, Roca D, Martínez P (2006) Spatial-temporal variations in rose leaves under water stress conditions studied by chlorophyll fluorescence imaging. Plant Physiol Bioch 44: 564-573.
Caverzan A, Passaia G, Rosa SB, Ribeiro CW, Lazzarotto F, Margis-Pinheiro M (2012) Plant responses to stresses: role of ascorbate peroxidase in the antioxidant protection. Genetics and molecular biology 35: 1011-1019.
Colom M, Prato EP, Giannini R (2003) Chlorophyll fluorescence and photosynthetic response to light in 1-year-old needles during spring and early summer in Pinus leucodermis. Trees 17: 207-210.
Cui G, Chai H, Yin H, Yang M, Hu G, Guo M, Yi R, Zhang P (2019) Full-length transcriptome sequencing reveals the low-temperature-tolerance mechanism of Medicago falcata roots. BMC plant biology 19: 1-16.
Dai Y, Yuan L, Zhang S, Wang J, Xie S, Zhao M, Chen G, Sun R, Wang C (2019) Comprehensive Evaluation for Cold Tolerance in Wucai (Brassica campestris L.) by the Performance Index on an Absorption Basis (PIabs). Agronomy 9: 61.
Demarsy E, Goldschmidt-Clermont M, Ulm R (2018) Coping with ‘dark sides of the sun’through photoreceptor signaling. Trends Plant Sci 23: 260-271.
Dolstra O, Haalstra S, Van der Putten P, Schapendonk A (1994) Genetic variation for resistance to low-temperature photoinhibition of photosynthesis in maize (Zea mays L.). Euphytica 80: 85-93.
Estaji A, Kalaji H, Karimi H, Roosta H, Moosavi-Nezhad S (2019) How glycine betaine induces tolerance of cucumber plants to salinity stress. Photosynthetica 57: 753-761.
Foyer CH, Neukermans J, Queval G, Noctor G, Harbinson J (2012) Photosynthetic control of electron transport and the regulation of gene expression. J Exp Bot 63: 1637-1661.
Fracheboud Y, Haldimann P, Leipner J, Stamp P (1999) Chlorophyll fluorescence as a selection tool for cold tolerance of photosynthesis in maize (Zea mays L.). Journal of experimental botany 50: 1533-1540.
Fu W, Li P, Wu Y (2012) Effects of different light intensities on chlorophyll fluorescence characteristics and yield in lettuce. Sci Hort 135: 45-51.
Genty B, Briantais J-M, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990: 87-92.
Gericke WA (2018). Effects of light manipulation through different photo selected net colours and LEDs (Lactuca Sativa L.) and cabbage as estimated by chlorophyl fluoressence parameters, macro and micro element content and physical measurements (Stellenbosch: Stellenbosch University).
Habibi G (2018) Antioxidant metabolism and performance index of photosystem II in maize seedlings exposed to high concentration of salicylic acid. J Plant Physiol 23: 360-368.
Hajihashemi S, Noedoost F, Geuns J, Djalovic I, Siddique KH (2018a) Effect of cold stress on photosynthetic traits, carbohydrates, morphology, and anatomy in nine cultivars of Stevia rebaudiana. Frontiers in plant science 9: 1430.
Hajihashemi S, Noedoost F, Geuns JM, Djalovic I, Siddique KH (2018b) Effect of Cold Stress on Photosynthetic Traits, Carbohydrates, Morphology, and Anatomy in Nine Cultivars of Stevia rebaudiana. Front plant sci 9.
Han Q-H, Huang B, Ding C-B, Zhang Z-W, Chen Y-E, Hu C, Zhou L-J, Huang Y, Liao J-Q, Yuan S (2017) Effects of melatonin on anti-oxidative systems and photosystem II in cold-stressed rice seedlings. Frontiers in Plant Science 8: 785.
Hazrati S, Tahmasebi-Sarvestani Z, Modarres-Sanavy SAM, Mokhtassi-Bidgoli A, Nicola S (2016) Effects of water stress and light intensity on chlorophyll fluorescence parameters and pigments of Aloe vera L. Plant physiology and biochemistry 106: 141-148.
Heldt H-W (1997). Plant biochemistry and molecular biology (Oxford University Press).
Hetherington S, Smillie R, Hardacre A, Eagles H (1983) Using chlorophyll fluorescence in vivo to measure the chilling tolerances of different populations of maize. Function Plant Biol 10: 247-256.
Husičková A, Humplík JF, Hýbl M, Spíchal L, Lazár D (2019) Analysis of Cold-Developed vs. Cold-Acclimated Leaves Reveals Various Strategies of Cold Acclimation of Field Pea Cultivars. Remote Sensing 11: 2964.
Ibáñez H, Ballester A, Muñoz R, Quiles MJ (2010) Chlororespiration and tolerance to drought, heat and high illumination. Journal of plant physiology 167: 732-738.
Kalaji HM, Carpentier R, Allakhverdiev SI, Bosa K (2012) Fluorescence parameters as early indicators of light stress in barley. Photochem Photobiol B: Biology 112: 1-6.
Kalaji HM, Oukarroum A, Alexandrov V, Kouzmanova M, Brestic M, Zivcak M, Samborska IA, Cetner MD, Allakhverdiev SI, Goltsev V (2014) Identification of nutrient deficiency in maize and tomato plants by in vivo chlorophyll a fluorescence measurements. Plant physiol biochem 81: 16-25.
Kalaji MH, Goltsev VN, Żuk-Gołaszewska K, Zivcak M, Brestic M (2017). Chlorophyll fluorescence: understanding crop performance—basics and applications (CRC Press).
Kalhor MS, Aliniaeifard S, Seif M, Asayesh EJ, Bernard F, Hassani B, Li T (2018) Enhanced salt tolerance and photosynthetic performance: Implication of ɤ-amino butyric acid application in salt-exposed lettuce (Lactuca sativa L.) plants. Plant physiol biochem 130: 157-172.
Kim TE, Kim SK, Han TJ, Lee JS, Chang SC (2002) ABA and polyamines act independently in primary leaves of cold‐stressed tomato (Lycopersicon esculentum). Physiologia Plantarum 115: 370-376.
Maxwell K, Johnson GN (2000) Chlorophyll fluorescence—a practical guide. J Exp Bot 51: 659-668.
Moosavi-Nezhad M, Salehi R, Aliniaeifard S, Tsaniklidis G, Woltering EJ, Fanourakis D, Żuk-Gołaszewska K, Kalaji HM (2021) Blue light improves photosynthetic performance during healing and acclimatization of grafted watermelon seedlings. International journal of molecular sciences 22: 8043.
Moradi S, Kafi M, Aliniaeifard S, Salami SA, Shokrpour M, Pedersen C, Moosavi-Nezhad M, Wróbel J, Kalaji HM (2021) Blue Light Improves Photosynthetic Performance and Biomass Partitioning toward Harvestable Organs in Saffron (Crocus sativus L.). Cells 10: 1994.
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and cell physiology 22: 867-880.
Panda D, Sharma S, Sarkar R (2008) Fast chlorophyll fluorescence transients as selection tools for submergence tolerance in rice (Oryza sativa). Indian J Agri Sci 78: 933-939.
Patterson BD, MacRae EA, Ferguson IB (1984) Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analytical biochemistry 139: 487-492.
Perveen S, Shahbaz M, Ashraf M (2010) Regulation in gas exchange and quantum yield of photosystem II (PSII) in salt-stressed and non-stressed wheat plants raised from seed treated with triacontanol. Pak J Bot 42: 3073-3081.
Pospíšil P (2016) Production of reactive oxygen species by photosystem II as a response to light and temperature stress. Frontiers in plant science 7: 1950.
Raju SKK, Barnes AC, Schnable JC, Roston RL (2018) Low-temperature tolerance in land plants: Are transcript and membrane responses conserved? Plant science 276: 73-86.
Rivero RM, Ruiz JM, García PC, López-Lefebre LR, Sánchez E, Romero L (2002) Response of oxidative metabolism in watermelon plants subjected to cold stress. Functional plant biology 29: 643-648.
Roach T, Krieger-Liszkay A (2014) Regulation of photosynthetic electron transport and photoinhibition. Curr Protein Pept Sci 15: 351-362.
Roden JS, Egerton JJ, Ball MC (1999) Effect of elevated [CO2] on photosynthesis and growth of snow gum (Eucalyptus pauciflora) seedlings during winter and spring. Func Plant Biol 26: 37-46.
Rodríguez VM, Soengas P, Alonso-Villaverde V, Sotelo T, Cartea ME, Velasco P (2015) Effect of temperature stress on the early vegetative development of Brassica oleracea L. BMC plant biol 15: 145.
Roháček K (2002) Chlorophyll fluorescence parameters: the definitions, photosynthetic meaning, and mutual relationships. Photosynthetica 40: 13-29.
Sarkar R, Ray A (2016) Submergence-tolerant rice withstands complete submergence even in saline water: probing through chlorophyll a fluorescence induction OJIP transients. Photosynthetica 54: 275-287.
Scandalios JG (2002) The rise of ROS. Trends in biochemical sciences 27: 483-486.
Schapendonk A, Dolstra O, Van Kooten O (1989) The use of chlorophyll fluorescence as a screening method for cold tolerance in maize. Photosynthesis res 20: 235-247.
Seif M, Aliniaeifard S, Arab M, Mehrjerdi MZ, Shomali A, Fanourakis D, Li T, Woltering E (2021) Monochromatic red light during plant growth decreases the size and improves the functionality of stomata in chrysanthemum. Functional Plant Biology 48: 515-528.
Shahryar N, Maali-Amiri R (2016) Metabolic acclimation of tetraploid and hexaploid wheats by cold stress-induced carbohydrate accumulation. Journal of plant physiology 204: 44-53.
Shiferaw B, Baker DA (1996) An evaluation of drought screening techniques for Eragrostis tef. Tropical Science (United Kingdom).
Shomali A, Aliniaeifard S (2020). Overview of Signal Transduction in Plants Under Salt and Drought Stresses. In Salt and Drought Stress Tolerance in Plants (Springer), pp. 231-258.
Shomali A, Aliniaeifard S, Didaran F, Lotfi M, Mohammadian M, Seif M, Strobel WR, Sierka E, Kalaji HM (2021) Synergistic Effects of Melatonin and Gamma-Aminobutyric Acid on Protection of Photosynthesis System in Response to Multiple Abiotic Stressors. Cells 10: 1631.
Steffens B (2014) The role of ethylene and ROS in salinity, heavy metal, and flooding responses in rice. Frontiers in plant science 5: 685.
Stirbet A, Lazár D, Kromdijk J (2018) Chlorophyll a fluorescence induction: can just a one-second measurement be used to quantify abiotic stress responses? Photosynthetica 56: 86-104.
Strasser B (1995) Measuring fast fluorescence transients to address environmental questionsL; the JIP-test. Photosynthesis: from light to biosphere: 977-980.
Strasser RJ, Srivastava A, Tsimilli-Michael M (2000) The fluorescence transient as a tool to characterize and screen photosynthetic samples. Probing photosynthesis: mechanisms, regulation and adaptation: 445-483.
Strasser RJ, Tsimilli-Michael M, Qiang S, Goltsev V (2010) Simultaneous in vivo recording of prompt and delayed fluorescence and 820-nm reflection changes during drying and after rehydration of the resurrection plant Haberlea rhodopensis. BBA 1797: 1313-1326.
Takahashi S, Murata N (2008) How do environmental stresses accelerate photoinhibition? Trend plant sci 13: 178-182.
Van Heerden P, Swanepoel J, Krüger G (2007) Modulation of photosynthesis by drought in two desert scrub species exhibiting C3-mode CO2 assimilation. Environ Exp Bot 61: 124-136.
Vranová E, Inzé D, Van Breusegem F (2002) Signal transduction during oxidative stress. J Exp Bot 53: 1227-1236.
Walker D (1987) The use of the oxygen electrode and fluorescence probes in simple measurements of photosynthesis. Sheffield: Research Institute for Photosynthesis, University of Sheffield: 212.
Wang K, Shao X, Gong Y, Zhu Y, Wang H, Zhang X, Yu D, Yu F, Qiu Z, Lu H (2013) The metabolism of soluble carbohydrates related to chilling injury in peach fruit exposed to cold stress. Postharvest Biology and Technology 86: 53-61.
Xu J, Yang J, Duan X, Jiang Y, Zhang P (2014) Increased expression of native cytosolic Cu/Zn superoxide dismutase and ascorbate peroxidase improves tolerance to oxidative and chilling stresses in cassava (Manihot esculenta Crantz). BMC Plant Biology 14: 1-14.
Yang Y-J, Chang W, Huang W, Zhang S-B, Hu H (2017) The effects of chilling-light stress on photosystems I and II in three Paphiopedilum species. Botanical studies 58: 1-12.
Yemm E, Willis A (1954) The estimation of carbohydrates in plant extracts by anthrone. Biochemical journal 57: 508-514.
Yuanyuan M, Yali Z, Jiang L, Hongbo S (2009) Roles of plant soluble sugars and their responses to plant cold stress. African Journal of Biotechnology 8.
Zhang Z, Zhang Q, Wu J, Zheng X, Zheng S, Sun X, Qiu Q, Lu T (2013) Gene knockout study reveals that cytosolic ascorbate peroxidase 2 (OsAPX2) plays a critical role in growth and reproduction in rice under drought, salt and cold stresses. PloS one 8: e57472.
Zhou R, Hyldgaard B, Yu X, Rosenqvist E, Ugarte RM, Yu S, Wu Z, Ottosen C-O, Zhao T (2018) Phenotyping of faba beans (Vicia faba L.) under cold and heat stresses using chlorophyll fluorescence. Euphytica 214: 68.
Zushi K, Kajiwara S, Matsuzoe N (2012) Chlorophyll a fluorescence OJIP transient as a tool to characterize and evaluate response to heat and chilling stress in tomato leaf and fruit. Sci Hort 148: 39-46.
No competing interests reported.