ATP hydrolysis determines cold tolerance in rice by regulating available energy for glutathione synthesis

Background Glutathione (GSH) is important for plants to resist against abiotic stress, and a large amount of energy is required in the process. However, it is not clear how the energy status affects the accumulation of GSH in plants under cold stress. Results Two rice genotypes, Zhongzao39 (ZZ39) and its recombinant inbred line 82 (RIL82) were subjected to cold stress for 48 h. Under cold stress, RIL82 suffered more damages than ZZ39 plants, in which the latter had higher increases in APX activity and GSH content than the former compared with their respective controls. This indicated that GSH was mainly responsible for the different cold tolerance between these two rice plants. Interestingly, under cold stress, greater increases in contents of carbohydrate, NAD(H), NADP(H) and ATP as well as the expression levels of GSH1 and GSH2 were showed in RIL82 than ZZ39 plants. In contrast, ATPase activity in RIL82 plants was adversely inhibited by cold stress while it significantly increased in ZZ39 plants. This indicated that cold stress reduced the accumulation of GSH in RIL82 plants mainly due to the inhibition on ATP hydrolysis rather than energy deficit. Conclusion We inferred that the energy status determined by ATP hydrolysis involved in regulating the cold tolerance of plants by controlling GSH synthesis.

1 mmol·L -1 GSH and 25 mmol·L -1 buthionine sulfoximine (BSO, a GSH synthetic inhibitor, Noctor et al. 2012) as well as a 1 mmol·L -1 Poly (ADP-ribose) polymerase (PARP) synthetic inhibitor 3aminobenzamide (3-ab, Keppler et al. 2018) containing 0.1% (v/v) Tween20 as a surfactant were sprayed onto rice leaves with 10 mL per pot about 30 mins before cold stress conducted. The first fully expanded leaf samples were collected to determine REC and MDA 48 h later. According to the above results, the synergistic effects of GSH and 3-ab on cold tolerance in rice plant were also investigated. About 30 mins before the cold stress, these two chemicals containing 0.1% (v/v) Tween20 as a surfactant were sprayed on rice leaves with 10 mL per pot together. 48 h later, the first fully expanded leaves were collected to determine the H 2 O 2 , MDA, GSH and ATP levels and the activities of PARP and ATPase.

Measurements of chlorophyll content and fluorescence quantum efficiency
The chlorophyll concentration was measured using an ethonal extraction procedure (Sartory and Grobbelaar 1984), in which 0.1 g leaf sample was sliced and immersed in 20 mL 95% ethanol for 48 h in the dark. Chlorophyll concentration was determined at 665 nm and 649 nm using a spectrophotometer (Lambda25; Perkin Elmer, Freemont, CA, USA).
After a 30-min dark adaptation period, Fv/Fm and Y(II) of the leaves were measured using a portable chlorophyll fluorescence spectrometer (PAM-2500 chlorophyll fluorescence system; Heinz Walz, Effeltrich, Germany) (Zhang et al. 2018a).

Relative electrical conductance measurement
Following the method of Xiong et al. (2012), 0.5 g of fresh leaves were collected at the end of the cold stress, cut into 25-mm 2 pieces, and immediately immersed into a test tube with 12 mL deionized water for 2 h at 25 °C. After incubation, a conductivity meter (DDA-11A; Shanghai Hongyi Instrument Co. Ltd., Shanghai, China) was used to measure the electrical conductivity of the solution (EC1). The electrical conductivity (EC2) was measured again after the samples were heated at 80 °C for 2 h in their effusates and cooled to 25 °C. The relative ion leakage was calculated as the ratio between EC1 and EC2. supernatant was collected for analysis at 450 nm.

Quantitative real-time polymerase chain reaction (PCR) analysis
Total RNA was extracted from 0.3 g leaves using TRIpure reagent (Aidlab Biotechnologies, Beijing, China). RNA was converted to first-strand cDNA using ReverTra Ace qPCR RT Master Mix (TOYOBO, Shanghai, China). The SYBR Green I (TOYOBO) was used as a fluorescent reporter, and the resultant cDNA was used as a template for quantitative PCR amplification in a Thermal Cycler Dice Real Time System II (TaKaRa Biotechnology, Dalian, China). Primers were designed using PRIMER5 software (Rozen and Skaletsky 2000). The primers for genes examined were listed in Supplementary Table 1. The PCR and detection were performed as described above (Feng et al. 2013). Relative transcript levels were analyzed using 2 −ΔΔCT method and the experiments were performed in triplicate.

Statistical analysis
Data were processed using SPSS software 11.5 (IBM Corp., Armonk, NY, USA) to detect differences.
The mean values and standard errors in the figures represented data from three experimental replicates unless otherwise stated. The t-test was performed on the normalized data. An analysis of variance (ANOVA) with two factors (temperature and treatment) was used to compare the differences in LSD test with p (p ≤ 0.05).

Changes of leaf morphology, photosynthesis and REC under cold stress
Rice plants ZZ39 and RIL82 showed different responses to cold stress ( Fig. 1). Under control conditions, there was no difference in leaf morphology between the two rice plants. However, the leaves of RIL82 plants withered under cold stress, while the leaves of ZZ39 plants remained flat ( Fig.   1a, b). The chlorophyll content of the leaves of ZZ39 plants maintained constant under cold stress, but it increased significantly in RIL82 plants compared with control (Fig. 1c). Similarly, higher increase in REC of leaf was found in RIL82 than ZZ39 plants under cold stress (Fig. 1d). In contrast, both Fv/Fm and Y(II) values deceased significantly in response to cold stress, and RIL82 plants decreased more than ZZ39 plants (Fig. 1e, f).

H 2 O 2 and MDA contents
The H 2 O 2 content in the leaves of ZZ39 plants was not affected by cold stress as there was no significant difference between the control and cold stress group ( Fig. 2A, a). However, the H 2 O 2 content in RIL82 plants increased significantly in response to cold stress. The MDA content of both plants increased significantly under cold stress ( Fig. 2A, b). Compared with the control groups, a greater increase in MDA content was found in RIL82 than ZZ39 plants under cold stress.

Antioxidant enzyme activities
The activities of SOD, POD, CAT and APX were determined to investigate the effects of cold stress on the antioxidant capacity (Fig. 2B). No difference in SOD activity was found between the control and cold stress groups of ZZ39 plants, while a significant increase in SOD activity was observed in RIL82 plants under cold stress ( Fig. 2B, a). The activities of POD and CAT were not affected by cold stress as no differences were showed between the control and cold stress groups (Fig. 2B, b and c). However, the APX activity of ZZ39 plants increased significantly under cold stress, while no significant difference was showed between the control and cold stress groups of RIL82 plants (Fig. 2B, d).

Heat shock proteins
The genes associated with heat shock proteins were determined, such as HSP71.1 and HSP24.1 (Fig.   2C). The expression level of HSP71.1 was significantly induced by cold stress in both rice plants, where higher increase was found in ZZ39 than RIL82 plants (Fig. 2C, a). Compared with the control, about 26-fold increase in expression level of HSP24.1 was showed in ZZ39 plants under cold stress, while no difference was found in RIL82 plants between the control and cold stress groups (Fig. 2C, b).

GSH metabolism
According to the above results, APX was mainly responsible for reducing the H 2 O 2 and MDA levels caused by cold stress, which was presumably related to GSH. Therefore, the metabolism of GSH was determined under cold stress. Compared with the control, the contents of GSH+GSSG, GSH, and GSSG in the leaves of ZZ39 increased significantly under cold stress, while they decreased clearly in RIL82 plants except for the GSSG (Fig. 3a-c). Regarding the GSH/GSSG, it was significantly reduced by cold stress, but no obvious difference in decrease was showed between these two rice plants (Fig. the plants treated with H 2 O, a remarkable reduction in REC and MDA was found in the plants of ZZ39 treated with 3-ab under cold stress (Fig. 8B). However, such results were not found in RIL82 plants, as no significant difference was showed between the treatments of H 2 O and 3-ab under cold stress.

Effects of GSH and 3-ab combination on rice plants under cold stress
The above results indicated that exogenous GSH enhanced cold tolerance in these two rice genotypes, while such result was only found in ZZ39 plants when treated with PARP inhibitor (3-ab).
Thus, we wonder whether there is a synergistic effect between GSH and 3-ab in enhancing cold tolerance in plants. According to the photos, the leaves of ZZ39 treated with H 2 O or 3-ab wilted slightly under cold stress, while the plants treated with GSH or GSH+3-ab maintained flat (Fig. 9a, b).
In contrast, the leaves of RIL82 plants treated with H 2 O and 3-ab severely wilted under cold stress, whereas these effects were reversed by GSH or GSH+3-ab, especially for the old leaves ( Fig. S2).
Under cold stress, similar changing patterns of MDA and H 2 O 2 were found in plants treated with GSH or 3-ab alone ( Fig. 9c-f). Additionally, the lowest MDA and H 2 O 2 levels were showed in the plants treated with GSH+3-ab under cold stress. Compared with 3-ab treatment, slight decreases in MDA and H 2 O 2 levels were showed in ZZ39 plants treated with GSH+3-ab, while a remarkable reduction was observed in RIL82 plants. Regarding the GSH content, it clearly increased in plants treated with GSH or GSH+3-ab treatments compared with H 2 O treatment in both rice genotypes in response to cold stress (Fig. 9g, h). Indeed, higher GSH content was found in both plants treated with 3-ab than those plants treated with H 2 O, but significant difference was only found in ZZ39 plants.
Rice plants treated with GSH, 3-ab or GSH+3-ab attained lower PARP activity than H 2 O treatment in both rice genotypes under cold stress (Fig. 9i, j). However, significant difference was only found in the treatments of 3-ab or GSH+3-ab compared with H 2 O under cold stress. As to the ATP, the highest levels were showed in the plants treated with 3-ab in both plants under cold stress, which was significantly higher than other treatments (Fig. 9k, l). Interestingly, the lowest value was observed in GSH treatment, but the difference was not significant compared with H 2 O treatment. Under cold stress, the highest activities of ATPase were showed in the treatments of GSH and GSH+3ab in both rice genotypes, while the lowest activities were found in plants treated with H 2 O and 3-ab (Fig. 9m, n). In ZZ39 plants, lower ATPase activity was found in H 2 O treatment than 3-ab treatment under cold stress, while no significant differences between these two treatments were showed in RIL82 plants.

Discussion
The function of GSH in conferring cold tolerance in rice plants The present results indicated that cold stress caused more damages to RIL82 than ZZ39 plants ( Fig. 1), since excess MDA and H 2 O 2 were showed in the former than latter ( Fig. 2A) In this study, remarkable increases were found in contents of GSH + GSSG, GSH and GSSG of ZZ39 compared with RIL82 under cold stress, while such effects were not found in GSH/GSSG and GR (Fig. 3). This suggested that GSH might be the main factor resulting in different cold tolerance between these two rice plants.
Importantly, exogenous GSH significantly enhanced cold tolerance in both rice plants, whereas this was impaired by its synthetic inhibitor BSO (Fig. 8A).

The role of ATP hydrolysis in GSH synthesis in rice plants under cold stress
The GSH accumulation is determined by GSH-S and GR in plants, and the former are responsible for the GSH synthesis using the γ-EC and Gly while the latter reduces the GSSG to GSH (Rao and Reddy 2008;Noctor et al. 2012). According to the present results, the GSH-S rather than GR is responsible for GSH accumulation in plants under cold stress (Fig. 3). It was reported that GSH1 and GSH2 mainly responsible for the synthesis of GSH in plants (Cairns et al. 2006;Pasternak et al. 2008). However, higher increases in expression levels of GSH1 and GSH2 were showed in RIL82 than ZZ39 plants under cold stress (Fig. 3). These paradoxical results might be mainly ascribed to the energy status in plants under cold stress, since these two pathways is ATP dependent (Buwalda et al. 1990;Noctor et al. 1997;Ogawa et al. 2004). ). However, it was the energy ultilization ability rather than energy shortage that mainly contributed to the different cold tolerance between these two rice plants (Figs. 4 and 5). The ATPase activity and its expression level significantly increased under cold stress in ZZ39 plants, while a large decrease was found in RIL82 plants (Fig. 4). This suggested that ATP hydrolysis in RIL82 plants were adversely inhibited by cold stress, and thus the lower GSH synthesis because of the higher unavailable ATP (Puhakainen et al. 1999;Mendoza et al. 2000;Deng et al. 2015;Muzi et al. 2016). This hypothesis was confirmed by the present results that the PARP inhibitor (3-ab) only enhanced the cold tolerance and GSH content in ZZ39 plants under cold stress ( Fig. 8B and 9), though the ATP content increased significantly in both rice plants (Fig. 9k and l).
It is puzzling that the ATP hydrolysis increased in RIL82 plants under cold stress in the present of exogenous GSH (Fig. 9n). This indicated that exogenous GSH could activate ATPase activity to provide energy for the GSH synthesis under cold stress. Similar results have been not documented previously that how GSH activates ATPase in plants under cold stress remains unclear.

The energy allocation for rice plants to survive in cold stress
It has been reported that the ATP synthetic rates are adversely inhibited in abiotic stress conditions (Gibbs and Greenway 2003), where higher rates of glycolysis and activities of fermentative enzymes were observed in plants ( Gibbs et al. 2000;Saika et al. 2006). In this case, the complementary responses could be used by the plants with low energy status to stabilize energy charge, including that ATP-regenerating pathways such as glycolysis become derepressed to maximize energy production and retard ATP-utilizing pathways to conserve ATP (Gibbs et al. 2000). In this study, more energy consumption was found in the ZZ39 plants than RIL82 under cold stress (Figs. 4 and 5). This strategy was not beneficial for plants to resist against cold stress. However, there is a hierarchical down-regulation of ATP consumption during periods of ATP shortage (Atwell et al. 1982;Greenway and Gibbs 2003), in which the protein consumed the largest proportion of ATP synthesis (Edwards et al. 2012). This explained the remarkable decrease in ATP in ZZ39 plants, but higher increases in content of GSH and expression levels of heat shock proteins than RIL82 plants under cold stress.
Thus, we inferred that the ZZ39 plants consumed more energy for the synthesis of GSH and heat shock proteins to resist against cold stress, rather than the plant growth and development. Without exception, notably higher increases in expression levels of SnRK1A and SnRK1B were found in ZZ39 than RIL82 plants under cold stress (Fig. 6b, c), which was consistent with the previous results (Valledor et al. 2013;Lin et al. 2014;Yu et al. 2018). However, a large decrease in expression level of TOR was showed in RIL82, rather than ZZ39 plants under cold stress (Fig. 6d). Clearly, this changing pattern between SnRK1 and TOR don't follow the "yin-Yang" model (Rodriguez et al. 2019). It has been reported that the TOR can be activated to induce the synthesis of GSH and heat shock proteins and confer cold and drought tolerance in plants (Dobrenel et al. 2013;Xiong and Sheen, 2015;Bakshi et al. 2017;Speiser et al. 2018;Rodriguez et al. 2019). This suggests that the antagonism between the SnRK1 and TOR may be ambiguous and the kinases may act in a different way under certain physiological circumstances (Rodriguez et al. 2019). The target genes of TOR and SnRK1 kinases only partially, and not always antagonistically overlay under energy deficiency (Wu et al. 2019).
Additionally, the TOR was reported to be activated by ATPase (Zoncu et al. 2011), which could explain the lower expression level of TOR showed in RIL82 than ZZ39 under cold stress. Therefore, we inferred that the ATPase might function in the process of SnRK1 and TOR acting together to regulate the energy homeostasis in plants under cold stress.

Conclusion
Cold stress caused more damages to RIL82 than ZZ39 plants, since higher increases in REC, MDA and H 2 O 2 were found in the former than the latter. Among the antioxidants including SOD, POD, CAT, APX, GSH, and GR, there were only APX and GSH involved in regulating cold tolerance between the two rice plants. The APX activity and GSH content increased significantly in ZZ39 plants under cold stress, while in RIL82 plants no obvious differences were showed between the control and cold stress.
However, significantly higher increases in expression levels of GSH1 and GSH2 as well as contents of carbohydrates, NAD(H), NADP(H) and ATP were found in RIL82 under cold stress, rather than the ZZ39 plants. These findings indicated that lower GSH accumulation in RIL82 plants was not due to the energy deficit caused by cold stress. It's worth noting that, the ATPase activity and its expression level increased obviously in ZZ39 plants under cold stress, while a remarkable decrease was found in RIL82 plants. This suggested that the ATP hydrolysis by ATPase play a key role in GSH accumulation.
Therefore, we inferred that the ATPase was the main factor responsible for determining cold tolerance between these two rice plants via regulating the GSH accumulation.   (  Expression levels of heat shock proteins. Vertical bars denote standard deviations (n=3). A t-test was conducted to compare the difference between control and cold stress within a cultivar. * denotes P < 0.05.

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