Oligomeric Proanthocyanidins Confer Cold Tolerance in Rice through Maintaining Energy Homeostasis

Oligomeric proanthocyanidins (OPCs) are abundant polyphenols found in foods and botanicals that benefit human health, but our understanding of the functions of OPCs in rice plants is limited, particularly under cold stress. Two rice genotypes, named Zhongzao39 (ZZ39) and its recombinant inbred line RIL82, were subjected to cold stress. More damage was caused to RIL82 by cold stress than to ZZ39 plants. Transcriptome analysis suggested that OPCs were involved in regulating cold tolerance in the two genotypes. A greater increase in OPCs content was detected in ZZ39 than in RIL82 plants under cold stress compared to their respective controls. Exogenous OPCs alleviated cold damage of rice plants by increasing antioxidant capacity. ATPase activity was higher and poly (ADP-ribose) polymerase (PARP) activity was lower under cold stress in ZZ39 than in RIL82 plants. Importantly, improvements in cold tolerance were observed in plants treated with the OPCs and 3-aminobenzamide (PARP inhibitor, 3ab) combination compared to the seedling plants treated with H2O, OPCs, or 3ab alone. Therefore, OPCs increased ATPase activity and inhibited PARP activity to provide sufficient energy for rice seedling plants to develop antioxidant capacity against cold stress.


Introduction
Rice is one of the most important crops in East and Southeast Asia [1]. As a subtropical or tropical crop, rice is susceptible to cold stress during the seedling and reproductive stages. Cold stress that occurs at the seedling stage leads to chlorosis of leaves, a reduction in the number of tillers, damage to the root system, and death [2][3][4]. This damage significantly decreases the yield and quality of the rice and restricts the growth of planting area of early rice in China [5][6][7][8].
Cold stress inhibits normal active oxygen metabolism in plant and causes changes to the cell membrane structure, enzyme functions, osmotic substances, and stomatal conductance [9][10][11][12]. These changes can damage or kill plants, which show symptoms such as wilting, slow growth, yellowing, and local tissue necrosis [13][14][15][16][17]. Notably, some plants grow normally under cold stress, which is related to cold tolerance and cold acclimation. The acquisition of cold acclimation and cold tolerance is a complex process, and a large number of genes, as well as changes in antioxidant capacity and membrane structure, are involved in this process [18][19][20][21]. Among them, oxidative stress affects plant growth and development; thus, enhancing antioxidant capacity by salicylic acid, quercetin, melatonin, and abscisic acid is important for rice seedlings to survive in abiotic stress including cold stress [22][23][24].
According to the method of Xiong et al. [55], 0.5 g fresh leaves were collected at the end of the cold stress, cut into about 25-mm 2 diameter discs with a puncher avoiding the veins, and immediately immersed in a test tube containing 10 mL of deionized water for 24 h at 25 • C. After the incubation, a conductivity meter (DDA-11A; Shanghai Hongyi Instrument Co., Ltd., Shanghai, China) was used to measure the electrical conductivity (EC1) of the solution. After the sample was placed in a water bath at 80 • C for 2 h, it was cooled to 25 • C, and the electrical conductivity (EC2) was measured again. Ion leakage was calculated as the ratio between EC1 and EC2.
After the seedlings completed a 30 min dark adaptation period, the Fv/Fm values of the leaves were measured using a portable chlorophyll fluorescence spectrometer (PAM-2500 chlorophyll fluorescence system; Heinz Walz, Effeltrich, Germany) [56].

RNA Sequencing (RNA-seq) and Bioinformatics Analysis
The first fully expanded leaves of seedlings plants grown under control and cold temperature conditions were harvested. Total RNA was extracted from rice leaves using Trizol reagent (Shanghai Thermo Fisher Scientific Co., Ltd., Shanghai, China). The Nanodrop ND-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and Agilent Bioanalyzer 4150 (Agilent Technologies, Santa Clara, CA, USA) were used to detect RNA quality and concentration, respectively. The mRNA was purified with oligo (dT) magnetic beads, and the mRNA was fragmented in ABclonal First Strand Synthesis Reaction Buffer. Random primers and reverse transcriptase were used to synthesize the first-strand cDNA. The synthesized cDNA was amplified by polymerase chain reaction (PCR) and sequenced using the Illumina Novaseq 6000/MGISEQ-T7 sequencing platform. Transcriptome sequencing and analytical services were completed by Shanghai Zhongke New Life Biotechnology Co., Ltd. (Shanghai, China).

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis
Total RNA was extracted and purified with the TRIPure reagent (Aidlab Biotechnologies, Beijing, China). RNA was reverse transcribed into single-stranded cDNA using the ReverTra Ace qPCR RT Master Mix (TOYOBO, Shanghai, China). The cDNA was used as the template for PCR amplification. SYBR Green I (TOYOBO) was used as the fluorescent dye, and the Thermal Cycler Dice Real-Time System II (TaKaRa Biotechnology, Dalian, China) was used for real-time fluorescent qPCR analysis. The primers were designed using PRIMER5 software and are listed in Supplementary Table S1. QRT-PCR was performed according to the method of Feng et al. [57], and the relative expression levels of the genes were analyzed by the 2 −∆∆CT method.

OPC Contents Measurement
Based on the method of Mitsunaga et al. [58], with slight modifications, about 0.1 g of fresh leaves were placed in a 2 mL solution containing 60% ethanol and ground into a homogenate. The homogenate was shaken and extracted at 60 • C for 2 h and then centrifuged at 10,000× g for 10 min. Vanillin hydrochloride solution was added to the supernatant, mixed well, incubated in a water bath at 30 • C for 30 min, and absorbance was measured at 500 nm using a spectrophotometer (Lambda 25; Perkin Elmer, Freemont, CA, USA).

H 2 O 2 and Lipid Peroxidation Measurements
H 2 O 2 content was determined based on the method of Brennan and Frenkel, [59] with slight modifications. Four mL of 10 mM 3-amino-1,2,4-triazole (Bio Basic Inc, Toronto, Canada) and 0.2 g of fresh leaves were ground into a homogenate and centrifuged at 6000× g for 25 min. Two mL of the supernatant was added to 1 mL of 0.1% titanium tetrachloride (Shanghai Lingfeng Chemical Reagent Co., Ltd., Shanghai, China) solution containing 20% H 2 SO 4 , mixed, centrifuged, and the absorbance of the supernatant was measured with a spectrophotometer at 410 nm.
Two mL of 5% trichloroacetic acid was added to 0.1 g of fresh leaves to form a homogenate. The concentration of thiobarbituric acid (Sinopharm Group Chemical Reagent Co., Ltd., Shanghai, China) reactive substances was determined to estimate MDA content [60].

Antioxidant Enzyme Activity Measurements
Peroxidase (POD) activity was measured by the method of Maehly and Chance, [61]. Superoxide dismutase (SOD) activity was determined by the method of Giannopolitis and Ries, [62]. Catalase (CAT) activity was determined using the method of Zhang et al. [63].

Carbohydrate Measurements
Soluble sugar and starch contents were determined by the sulfuric acid-anthrone colorimetry method [64]. About 0.2 g of fresh leaves were immersed in 10 mL of absolute ethanol, heated at 80 • C for 30 min, extracted three times, the supernatant was removed to a constant volume, and activated carbon was added and shaken for 1 h to decolorize. The decolorizing solution was used to determine soluble sugar contents, and the filter residue was used to extract starch. The total non-structural carbohydrate (NSC) content was the sum of soluble sugar and starch contents.

ATP Content
ATP content was determined using an ATP analysis kit (Shanghai Enzyme-Linked Biotechnology Co., Ltd., Shanghai, China). About 0.1 g of fresh leaves were mixed with 1 mL of 0.1 M pH 7.4 PBS. The mixture was fully ground in an ice bath and centrifuged at 3000× g for 20 min and the supernatant was collected. The supernatant, standard and horseradish peroxidase (HRP)-labeled detection antibody were sequentially added to the microwells coated with the ATP capture antibody, incubated at 37 • C for 60 min, and washed thoroughly. The color is developed with the substrate 3,3 ,5,5 -tetramethylbenzidine (TMB).
TMB is converted into blue under the catalysis of HRP and into yellow under the action of acid. Absorbance was measured at a wavelength of 450 nm with a microplate reader (Shanghai Thermo Fisher Instrument Co., Ltd., Shanghai, China).

Total ATPase and PARP Content
ATPase content and PARP content were determined using an enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's instructions (Shanghai Enzyme Link Biotechnology Co., Ltd., Shanghai, China). The kit was used to quantitatively detect the content of total ATPase and PARP in plant tissue samples in vitro. About 0.1 g of fresh leaves were extracted with 0.1 M pH 7.4 PBS and then centrifuged at 3000× g for 20 min at 4 • C, and the supernatant was collected. The supernatant, standard, and HRP-labeled detection antibody were added to the microwells pre-coated with ATPase and PARP-captured antibody in sequence, incubated at 37 • C for 60 min, and washed. The color was developed with the substrate TMB. TMB was converted into blue under the catalysis of HRP and into yellow under the action of acid. The shade of color is positively correlated with the ATPase and PARP content in the sample. Absorbance was measured at a wavelength of 450 nm with a microplate reader.

Statistical Analysis
Data were processed using SPSS 11.5 software (IBM Corp., Armonk, NY, USA) to detect differences. The mean values and standard errors represent data from three independent experiments. The t-test and two-factor analysis of variance (temperature and treatment) were used to compare the differences with the LSD test A. p-value < 0.05 was considered significant.

The Responses of the Rice Seedlings to Cold Stress
The two rice genotypes presented different morphologies after cold stress and recovery ( Figure 1). No difference in leaf morphology was detected between the two genotypes under the control condition. Under cold stress, more damage was found on the leaves of RIL82 than on those of ZZ39 plants (Figure 1a-d). A significantly higher REC caused by cold stress was observed in RIL82 than in ZZ39 plants compared to their respective controls (Figure 1i,j). Cold stress decreased Fv/Fm in both genotypes, and notable reductions were found in RIL82 compared to those of ZZ39 plants regardless of the 24 h cold stress or 48 h recovery after the end of cold stress (Figure 1k-n). The mortality rate of RIL82 was significantly higher than that of ZZ39 plants after 96 h of recovery from the cold stress ( Figure 1o). The reference scales of (a-h), all represent 10 cm. Vertical bars denote standard deviations (n = 5). A t-test was adopted to compare the differences between the control and cold stressed within a cultivar. * denotes p < 0.05. The reference scales of (a-h), all represent 10 cm. Vertical bars denote standard deviations (n = 5). A t-test was adopted to compare the differences between the control and cold stressed within a cultivar. * denotes p < 0.05.

Transcriptome Analysis of the Mechanism Underlying the Difference in Cold Tolerance between the Two Genotypes
Transcriptome analysis was conducted to reveal the mechanism underlying the difference in cold tolerance between the two rice genotypes. Totals of 10,126 and 11,356 differentially expressed genes (DEGs) (fold-change > 2, p < 0.05) were found in ZZ39 and RIL82 plants, respectively ( Figure 2a); 1752 upregulated and 1835 downregulated DEGs were detected in RIL82, while 1229 upregulated and 1128 downregulated DEGs were presented in ZZ39 plants, indicating to a certain extent, the two genotypes had distinct transcriptional differences under cold stress (Figure 2b). Gene ontology (GO) analysis showed that a great number of genes involving in biological process and metabolic process were disturbed by cold stress in both genotypes based on the total DEGs. However, the biosynthetic processes were more enriched in ZZ39 than in RIL82 plants, while the metabolic processes, particularly carbohydrate metabolism, were more enriched in ZZ39 than in RIL82 plants ( Figure S1). In the case of cellular component enrichment analysis, many DEGs in ZZ39 were involved in cytoplasm and non-membrane-bounded organelle, while those of RIL82 were involved in membrane components ( Figure S1). The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the flavonoid biosynthetic pathway was enriched in RIL82, while such a result was not presented in ZZ39 based on all of the DEGs ( Figure S2). These results suggested that genes involved in biosynthetic process and metabolic process were differentially regulated by cold stress in the two rice genotypes. Interestingly, the flavonoid biosynthetic pathway was enriched in RIL82 only based on the downregulated DEGs (Figure 2c,d). This finding suggested that the flavonoid pathway played a key role in contributing to the difference in cold tolerance between the two genotypes. In addition, genes related to OPC biosynthesis in ZZ39 were upregulated under cold stress or no difference between the control and cold stress, while they were downregulated in RIL82 plants ( Figure 3A,B). Similarly, the relative expressions of PAL5, F3H and ANS were decreased by cold stress, in which a smaller decrease was presented in ZZ39 than RIL82 plants ( Figure 3C(a,c,e)). In contrast, notable increase in the relative expressions of CHS1, DFR and LAR genes were presented in ZZ39 plants, while no significant difference was found in RIL82 ( Figure 3C(b,d,f)). Accordingly, a notable increase in OPC contents was detected in ZZ39 plants under cold stress compared to the control, while in RIL82 no difference was found between the cold stressed and control treatments ( Figure 3D). Additionally, the genes related to the antioxidant capacity and energy metabolism were also involved in this process ( Figure S3). We selected five genes for qRT-PCR, and similar expression patterns were found between the qRT-PCR and RNA-seq analyses of both genotypes ( Figure S4). Therefore, we inferred that OPCs, antioxidant capacity and energy metabolism might be involved in contributing to the cold tolerance between two rice genotypes ( Figure 3E).

Effect of OPCs on the Morphology, REC, H2O2, and Fv/Fm of Rice Leaves under Cold Stress
Based on the above results, we speculate that OPCs might be involved in affecting cold tolerance between the two rice genotypes. To confirm this hypothesis, different OPC concentrations were sprayed onto the two rice genotypes under cold stress, and the plant morphology, REC and Fv/Fm as well as contents of H2O2 and MDA were investigated. The results indicated that the OPCs could improve the plant morphology of the two rice genotypes under cold stress (Figure 4a,b). The REC, as well as the MDA and H2O2 contents, increased significantly under cold stress, whereas these enhancements were reduced by the OPCs (Figure 4c,d,g,i). The lowest REC, MDA, and H2O2 values under cold stress were observed in plants treated with 0.1% OPCs, which was significantly lower than those of plants treated with H2O. The Fv/Fm value decreased significantly in response to the cold stress in both genotypes; a remarkable increase in Fv/Fm was observed in rice plants treated with 0.1% OPCs compared to those plants treated with H2O under cold stress, particularly in the RIL82 (Figure 4e,f). According to these results, rice seedling plants treated with 0.1% OPCs could obviously increase the cold tolerance through reducing REC as well as contents of H2O2 and MDA.

Effect of OPCs on the Morphology, REC, H 2 O 2 , and Fv/Fm of Rice Leaves under Cold Stress
Based on the above results, we speculate that OPCs might be involved in affecting cold tolerance between the two rice genotypes. To confirm this hypothesis, different OPC concentrations were sprayed onto the two rice genotypes under cold stress, and the plant morphology, REC and Fv/Fm as well as contents of H 2 O 2 and MDA were investigated. The results indicated that the OPCs could improve the plant morphology of the two rice genotypes under cold stress (Figure 4a,b). The REC, as well as the MDA and H 2 O 2 contents, increased significantly under cold stress, whereas these enhancements were reduced by the OPCs (Figure 4c

Effects of OPCs on Antioxidant Enzyme Activities of Leaves under Cold Stress
The activities of SOD, POD, and CAT were determined to reveal the functions of OPCs in the antioxidant capacity in rice plants under cold stress. The SOD activity of ZZ39 increased significantly under cold stress; in this stress conditions a remarkable increase in SOD activity were observed in the plants treated with 0.1% OPCs compared with that of H 2 O treatment (Figure 5a). Such results were not found in the RIL82 plants under cold stress, in which no obvious difference in SOD activity were detected among all the treatments (Figure 5b). Cold stress significantly increased CAT activity in the leaves of ZZ39 but did not affect the leaves of RIL82 plants (Figure 5c,d). CAT activity was induced by OPCs in the two genotypes under cold stress, and the highest values were found in the 0.1% OPC treatment, which was significantly higher than that of the H 2 O treatment. No significant difference in POD activity was detected between the control and cold stressed ZZ39 plants, while a significant reduction was observed in RIL82 caused by cold stress compared to the control (Figure 5e,f). Similarly, POD activity increased in response to the OPCs under cold stress in both genotypes; the POD activities of the 0.1% OPC and 0.2% OPC treatments were significantly higher than those of the H 2 O treatments. In sum, the antioxidant capacity including SOD, POD, and CAT activity were enhanced by 0.1-0.2% OPCs in rice plants under cold stress.

Effects of OPCs on NSC and Energy Metabolism of Rice under Cold Stress
Sugars and energy are important factors involved in OPCs affecting antioxidant capacity in rice seedlings under cold stress, and thus the contents of NSC, ATP, ATPase and PARP were determined. The NSC contents in the rice plants increased in response to the cold stress, but no significant difference was observed between the genotypes (Figure 6a,b). Under cold stress, the 0.2% and 0.4% OPC treatments had greater effects on the NSC content of ZZ39, both of which were significantly higher than the H 2 O treatment. However, no significant difference was observed among all of the cold stress treatments in RIL82 plants.
The effect of cold stress on ATP content differed between the genotypes (Figure 6c,d). A notable reduction in ATP content was found in ZZ39 plants under cold stress compared to the control, while ATP increased significantly in RIL82 under cold stress. The ATP content of ZZ39 plants under cold stress treated with 0.1, 0.2, or 0.4% OPCs was significantly lower than that of the H 2 O treatment, in which the lowest value was found in plants treated with 0.1% OPCs. In contrast, the ATP contents of the 0.2 and 0.4% OPC treatments were significantly higher than that of the H 2 O treatment in RIL82 plants; no difference in ATP content was detected between the 0.1% OPC and H 2 O treatments. Cold stress had little effect on ATPase activity in ZZ39 compared to the control, whereas a significant decrease in ATPase activity was found in RIL82 plants (Figure 6e,f). The ATPase activity of the 0.1% OPC treatment was significantly higher in ZZ39 plants under cold stress than in the other treatments, and no differences were observed among the other treatments. OPCs alleviated the inhibited ATPase activity caused by cold stress in RIL82 plants; the ATPase activity of the OPC treatments was significantly higher than that of the H 2 O treatments under cold stress, in which the highest value was in the 0.1% OPC treatment. PARP can be activated by cold stress, and a greater increase in PARP activity was found in RIL82 than in ZZ39 plants under cold stress compared to their respective controls (Figure 6g,h). The PARP activity of the 0.1% and 0.2% OPC treatments in ZZ39 was lower than that of the H 2 O treatment, and significant decreases in PARP activity were found in the 0.1, 0.2, and 0.4% OPC treatments compared to the H 2 O treatments in RIL82 plants. It was considered that, 0.1% OPCs could improve the energy status in rice seedling plants mainly ascribing to its function in enhancing the ATPase content but inhibiting the PARP content. Antioxidants 2023, 12, x FOR PEER REVIEW 12 of 24 Different letters indicate significant differences among the control and cold stress treatments within a genotype by two-way analysis of variance (temperature and treatment) (p < 0.05). Different letters indicate significant differences among the control and cold stress treatments within a genotype by two-way analysis of variance (temperature and treatment) (p < 0.05).

Effect of the OPCs + 3ab Combination on Fv/Fm, H 2 O 2 , and Energy Metabolism under Cold Stress
Our results indicate that OPCs might enhance cold tolerance in rice seedling plants by improving antioxidant capacity and energy status. To confirm this hypothesis, the OPCs and 3ab alone or combination were sprayed onto rice seedling plants under cold stress. The 3ab, a PARP inhibitor, can suppress the PARP activity to reduce the consumption of NAD + , that increases the energy production efficiency and improve energy status in plants. Thus, the Fv/Fm, and contents of H 2 O 2 , ATP, ATPase, and PARP were determined to investigate the energy status in seedling plants under cold stress. OPCs, 3ab, and OPCs + 3ab alleviated the cold damage compared to the plants treated with H 2 O in the two genotypes, particularly the OPCs + 3ab treatment (Figure 7a,b). No difference in Fv/Fm was observed among the OPCs, 3-ab, or OPCs + 3ab treatments in ZZ39 plants under cold stress, whereas they were significantly higher than the H 2 O treatment. The highest Fv/Fm value in RIL82 plants was observed in the OPCs and OPCs + 3ab treatments, which were significantly higher than those of the H 2 O and 3ab treatments; no difference in Fv/Fm was showed between the H 2 O and 3ab treatments in RIL82 plants under cold stress (Figure 7c,d). H 2 O 2 contents increased significantly in the two genotypes under cold stress compared to the control (Figure 7e,f). Among these treatments, the lowest values were found in the OPCs + 3ab treatment, followed by the 3ab treatment in ZZ39 plants, which was significantly lower than the H 2 O treatment under cold stress. In RIL82 plants, the lowest H 2 O 2 content was found in the OPCs + 3ab treatment, followed by the OPC treatments, both of which were significantly lower than the H 2 O treatment under cold stress. All these indicated that synergistic effect was existed in OPCs and 3ab, since a notable increase in cold tolerance was presented in OPCs + 3ab treatment compared with the other treatments.
Regarding the energy status, the highest ATP content under cold stress was detected in the 3ab treatment, followed by the H 2 O treatment, while the lowest values were observed in the OPCs + 3ab treatments of the two genotypes (Figure 8a,b). In contrast, the highest ATPase activity was observed in plants treated with OPCs + 3ab, followed by the OPCs treatments, both of which were significantly higher than that of the H 2 O treatments in the two genotypes under cold stress (Figure 8c,d). PARP activity decreased significantly compared to the H 2 O treatment when plants were sprayed with the OPCs, 3-ab, and OPCs + 3ab treatments under cold stress in the two genotypes; the lowest values were found in the OPCs + 3ab treatment (Figure 8e,f). Thus, these results suggested that OPCs could synergy with 3ab to increase cold tolerance in rice seedling plants by improving energy status. Regarding the energy status, the highest ATP content under cold stress was detected in the 3ab treatment, followed by the H2O treatment, while the lowest values were observed in the OPCs + 3ab treatments of the two genotypes (Figure 8a,b). In contrast, the highest ATPase activity was observed in plants treated with OPCs + 3ab, followed by the OPCs treatments, both of which were significantly higher than that of the H2O treatments in the two genotypes under cold stress (Figure 8c,d). PARP activity decreased significantly compared to the H2O treatment when plants were sprayed with the OPCs, 3-ab, and OPCs + 3ab treatments under cold stress in the two genotypes; the lowest values were found in the OPCs + 3ab treatment (Figure 8e,f). Thus, these results suggested that OPCs could synergy with 3ab to increase cold tolerance in rice seedling plants by improving energy status.

Discussion
Cold stress damaged the rice plants in both genotypes. The mortality rates of the ZZ39 and RIL82 plants under cold stress were 40% and 100% respectively, suggesting that the ZZ39 had higher cold tolerance than that of the RIL82 plants ( Figure 1). This finding was consistent with previous results reported by Yu et al. [39], who reported that glutathione (GSH) was the main factor contributing to the difference in cold tolerance between these two genotypes. According to the transcriptome analysis, genes related to GSH were induced and were involved in regulating the cold response in ZZ39 and RIL82 plants (Figure 2c,d). However, the genes related to flavonol in RIL82 plants decreased in response to the cold stress, whereas no significant difference was observed between the control and cold stressed in ZZ39 plants ( Figure 3A,B), suggesting that flavonols also notably affected cold response in these two genotypes [65][66][67].
As the final product of the flavonol pathway in plants, OPCs are abundant but complex class of polyphenols found in foods and botanicals, which has been largely utilized in animals and humans to treat disease because of their strong antioxidant properties [30]. Several researches have reported that the OPCs could affect the resistance to abiotic stress [68][69][70][71]. However, the role of OPCs in rice plants under abiotic stress has rarely been documented [72], particularly under cold stress. The present results revealed a significant increase in OPCs contents in ZZ39 plants under cold stress compared to the control, while no difference in OPCs contents was detected in RIL82 plants between the control and cold stressed ( Figure 3D). Importantly, exogenous OPCs alleviate cold damage in rice plants, particularly in the cold susceptible cultivar RIL82 (Figure 4), indicating that OPCs also conferred cold tolerance to the rice seedlings, which was consistent with the result conducted with apple [34].
As reported, many genes and transcription factors are involved in the OPCs synthesis and stress response in plants [31,[73][74][75][76]. Similarly, such results were also found in rice seedling plants according to the transcriptome analysis (Figures 2 and 3). Several genes including PAL5, F3H, ANH, CHS1, DFR and LAR played important roles in the difference accumulation of OPCs and cold tolerance between these two rice genotype ( Figure 3C). As well-known, many PAs are oligomers of the catechin and epicatechin flavonoid compounds, and key steps in the synthesis of these two building blocks are catalyzed by leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR), respectively. Therefore, among these genes, the ANR and LAR are two key genes responsible for the synthesis of OPCs [74], both of which confer abiotic or biotic resistance to plants. However, the ANR were largely documented in affecting abiotic or biotic stress compared with LAR [31,77], in particular under cold stress; The R2R3-MYB transcription factor MdMYB23 could interact with the promoter of ANR to enhance the cold tolerance and proanthocyanidins accumulation in apple [34]. Up to now, the effect of LAR on rice plants under cold stress has not been reported. However, the expression levels of LAR were increased in plants under cold stress compared with those of controls in two rice genotypes, while large decreases were presented in ANS (Figure 3). This suggested that LAR could also enhance cold tolerance in rice seedling plants by accumulating OPCs, whereas more researches are required to reveal the underlying mechanism.
Large increases in the activities of SOD, POD, and CAT, as well as large decreases in MDA and H 2 O 2 contents, were found in the plants treated with the OPCs under cold stress compared to those plants treated with H 2 O (Figures 4 and 5). Thus, the anti-stress function of OPCs in plants may be because of their strong antioxidant capacity [31,78,79]. Antioxidant capacity in plants is very energy costly; thus, stress tolerance may become impaired under low energy conditions [37]. This could explain why plants with higher stress tolerance always compromise biomass, yield, or quality, as most of the energy is allocated to maintain respiration, rather than growth [38,80]. Interestingly, the ATP content in the rice plants treated with OPCs was significantly lower than that of plants treated with H 2 O under cold stress in ZZ39 seedling plants (Figure 6c,d). This result suggested that OPCs enhanced cold tolerance by increasing energy utilization efficiency, which was determined by ATPase activity. It has been reported that the ATP hydrolysis blocked by the lower ATPase activity has been considered as the main factor resulting in more damage to RIL82 plants under cold stress [39]. Furthermore, OPCs could pose significant effects on the fermentation via glucose transport, the energy and redox homeostasis as well as the activities of rate-limiting enzymes in glycolysis [81]. Thus, OPCs increased energy use efficiency by regulating ATPase activity, which is responsible for the strong antioxidant capacity in plants under cold stress. This finding was confirmed by the result that cold tolerance was enhanced in rice plants treated with OPCs + 3ab compared to the other treatments in the two genotypes, but no difference in cold tolerance was found between the H 2 O and 3ab treatments of RIL82 plants; a significantly higher ATP content was found in the 3ab than the H 2 O treatment under cold stress (Figures 7 and 8).
The ATPases, including the phosphorylated intermediate-type (P-type) and vacuolartype (V-type) H + -ATPases, are important ATP-driven proton pumps that generate membrane potential and provide proton motive force for secondary active transport [71]. The expression and activity of both P-and V-type H + -ATPases are highly regulated by hormones and environmental cues, which are involved in plant growth and stress adaptation [71]. Indeed, the H + -ATPase is required for proanthocyanidins synthesis in the Arabidopsis thaliana seeds or Gossypium hirsutum [28,49,50,82]. Interestingly, some of the flavonoids, including Myricetin, quercetin (C) and gossypin were found to be inhibitors of K (+) -ATPase, which was competitive with respect to ATP [83][84][85][86]. However, the OPCs could increase the activity of ATPase and fermentation efficiency [81]. Moreover, OPCs significantly reduced the concentration of free Ca 2+ and elevated Ca 2+ -ATPase activity in sciatic nerves of rats [87]. This finding was consistent with the present results that higher ATPase activity were found in plants treated with OPCs than those plants treated with H 2 O (Figure 6e,f). This novel function of OPCs may be related to their ability to reduce excess ROS (Figure 4i,j), which always inhibits ATPase in plants under abiotic stress [88][89][90][91].
Indeed, the ATPase including Na + /K + , Ca 2+ , and H + pumping P-type ATPases, and V-ATPase are subject to redox regulation in mammals, yeast and plants [88]. Oxidative inhibition of the ATPase is ascribed to disulfide-bond formation between conserved cysteine residues at the catalytic site of subunit A, which can be induced by the reactive oxygen species [89]. The Cys-327 functions as a protective residue in the plasma membrane H + -ATPase, and other P-type ATPases [91]. It has been reported that the oligomeric proanthocyanidin-L-cysteine complexes presented higher bioavailability and antioxidant capacity and enhanced survival time in the animal test groups [92]. Thus, OPCs was inferred to be directly combined with cysteine residues to stabilize ATPase caused by cold stress in a ROS independent pathway, which has not been documented previously ( Figure 9). However, increased H + -ATPase could improve oxidative stress in Candida glabrata, Tamarix hispida Willd and tea plants [93][94][95].
The function of OPCs in conferring strong antioxidant capacity in rice plants to maintain ROS homeostasis under cold stress could reduce energy consumption ( Figure 9). Knowingly, PARP uses NAD + to produce the post-translationally modified PAR that attaches to PARP itself or other target proteins [96]. This process costs a large amount of energy. PARP is activated by abiotic stressors, such as hot and cold [37,39,97,98]. A significant increase in PARP activity was detected in plants under cold stress, in which the greater increase was observed in RIL82 than in ZZ39 plants (Figure 6g,h). Interestingly, OPCs inactivated PARP, and conferred cold tolerance in rice plants, particularly in the cold-susceptible RIL82 cultivar. This finding suggests that OPCs could also reduce energy consumption in plants under cold stress (Figure 9). This function of OPCs may be related to their ability to scavenge excess ROS, which activates PARP under cold stress [39,45]. , OPCs combine with Cys residues to form a complex at the plasma membrane to scavenge intracellular ROS to stabilize ATPase activity under cold stress [92]. Taken together, OPCs could improve energy status to enhance cold tolerance in rice plants. OPCs, oligomeric proanthocyanidins; PARP, poly (ADP-ribose) polymerase; T-AOC, total antioxidant capacity; ROS, reactive oxygen species; Cys residue, cysteine residue.
The function of OPCs in conferring strong antioxidant capacity in rice plants to maintain ROS homeostasis under cold stress could reduce energy consumption ( Figure 9). Knowingly, PARP uses NAD + to produce the post-translationally modified PAR that attaches to PARP itself or other target proteins [96]. This process costs a large amount of energy. PARP is activated by abiotic stressors, such as hot and cold [37,39,97,98]. A significant increase in PARP activity was detected in plants under cold stress, in which the greater increase was observed in RIL82 than in ZZ39 plants (Figure 6g,h). Interestingly, OPCs inactivated PARP, and conferred cold tolerance in rice plants, particularly in the cold-susceptible RIL82 cultivar. This finding suggests that OPCs could also reduce energy consumption in plants under cold stress ( Figure 9). This function of OPCs may be related to their ability to scavenge excess ROS, which activates PARP under cold stress [39,45].

Conclusions
Cold stress caused more damage to RIL82 than ZZ39 plants. Transcriptome analysis indicated that OPCs were involved in affecting the cold tolerance between the two genotypes. This hypothesis was confirmed by that data that 0.1% OPCs significantly enhanced cold tolerance in plants by improving antioxidant capacity and reducing excess ROS. ATP content was lower in plants under cold stress treated with OPCs than that in plants treated with H2O, while higher ATPase activity was found in plants treated with OPCs than plants treated with H2O. This result indicates that OPCs enhanced energy use efficiency to provide sufficient energy for antioxidant capacity under cold stress. Further, OPCs inactivated PARP to reduce energy consumption in plants under cold stress. Taken together, OPCs enhanced ATPase activity and inhibited PARP activity to improve energy status and confer cold tolerance in plants by maintaining the oxidant balance.
Supplementary Materials: The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, Figure S1: Gene Ontology (GO) analysis (TOP30); Figure S2: Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis (TOP20); Figure S3: Heat map of genes associated with antioxidant capacity and energy metabolism; Figure S4: Histogram of five selected genes with , OPCs combine with Cys residues to form a complex at the plasma membrane to scavenge intracellular ROS to stabilize ATPase activity under cold stress [92]. Taken together, OPCs could improve energy status to enhance cold tolerance in rice plants. OPCs, oligomeric proanthocyanidins; PARP, poly (ADP-ribose) polymerase; T-AOC, total antioxidant capacity; ROS, reactive oxygen species; Cys residue, cysteine residue.

Conclusions
Cold stress caused more damage to RIL82 than ZZ39 plants. Transcriptome analysis indicated that OPCs were involved in affecting the cold tolerance between the two genotypes. This hypothesis was confirmed by that data that 0.1% OPCs significantly enhanced cold tolerance in plants by improving antioxidant capacity and reducing excess ROS. ATP content was lower in plants under cold stress treated with OPCs than that in plants treated with H 2 O, while higher ATPase activity was found in plants treated with OPCs than plants treated with H 2 O. This result indicates that OPCs enhanced energy use efficiency to provide sufficient energy for antioxidant capacity under cold stress. Further, OPCs inactivated PARP to reduce energy consumption in plants under cold stress. Taken together, OPCs enhanced ATPase activity and inhibited PARP activity to improve energy status and confer cold tolerance in plants by maintaining the oxidant balance.