Biochar supplementation altered the expression of antioxidant proteins in rice leaf chloroplasts under high-temperature stress

the most important cereals in the world and serves as a staple food for more than half of the world’s population [2]. Global rice demand is projected to increase by 30% by 2050 [19]. Meanwhile, increasing temperature due to climate change poses a threat to rice production. Global temperature is projected to increase by 2 °C by 2050 [1], while global rice yield is projected to be reduced by 3% with a global increase in temperature of 1 °C [22]. Therefore, it is necessary to develop proactive strategies to alleviate the negative effects of global warming on rice production.


Introduction
Rice is one of the most important cereals in the world and serves as a staple food for more than half of the world's population [2].Global rice demand is projected to increase by 30% by 2050 [19].Meanwhile, increasing temperature due to climate change poses a threat to rice production.Global temperature is projected to increase by 2 °C by 2050 [1], while global rice yield is projected to be reduced by 3% with a global increase in temperature of 1 °C [22].Therefore, it is necessary to develop proactive strategies to alleviate the negative effects of global warming on rice production.mitigated by antioxidant pathways [3,13,20].Antioxidant enzymes such as superoxide dismutase, peroxidase, and glutathione reductase are the most important components for defense against oxidative stress [15].The thioredoxin system, comprised of thioredoxin, nicotinamide adenine dinucleotide phosphate, and thioredoxin reductase, provides electrons to thiol-dependent antioxidant enzymes (e.g., peroxidase and peroxiredoxin) to remove ROS at high reaction rates and hence is crucial for alleviating oxidative stress-induced cell damage [11].
In the present study, we determined the effect of biochar supplementation on the expression levels of antioxidant proteins (antioxidant enzyme and thioredoxin system proteins) in the chloroplasts of rice leaves under hightemperature stress.Our objective was to identify the key antioxidant defense systems used to cope with high-temperature stress in rice leaf chloroplasts following biochar supplementation.

Materials and methods
The data were obtained from an experiment conducted in 2020.Briefly, a high-yielding hybrid rice variety, Longliangyouhuazhan, was grown in pots (length × width × height = 20 cm × 15 cm × 25 cm) without (C0) and with biochar supplementation (C40, 40 g biochar kg -1 soil).Six consecutive days (6-11 days after transplanting) of daily mean temperatures (35-36 °C) beyond the critical high temperature for rice tillering (33 °C; [6]) were monitored during the experimental period.The experimental details can be found in [5].
The uppermost fully-expanded leaves were sampled from three pots (replicates) for each treatment.The samples were placed in plastic bags and kept cool on ice during transport to the laboratory, after which they were hulled and stored at − 80 °C until proteins were extracted.Total proteins were extracted using the cold acetone method, and then measured via protein DC assay (Bio-Rad) and calculated according to a bovine serum albumin protein standard curve.A proteomic analysis was carried out using iTRAQ (isobaric tags for relative and absolute quantitation) coupled LC-MS/MS (liquid chromatography-mass spectrometry/mass spectrometry).The details of protein extraction and proteomic analysis are provided in Supplementary File S1.
The functions of the identified proteins were defined according to the Gene Ontology (GO) database (http:// www.geneontology.org).The differentially expressed proteins related to antioxidant defense (antioxidant enzyme and thioredoxin system proteins) in chloroplasts were selected for further evaluation.The differentially expressed proteins were defined as those that were significantly different (p < 0.05) in the expression level between C40 and C0.The details of bioinformatics analysis are provided in Supplementary File S1.

Results and discussion
A total of sixteen differentially expressed antioxidant proteins in chloroplasts were identified, including five antioxidant enzyme proteins and eleven thioredoxin system proteins (Table 1).These differentially expressed antioxidant proteins had molecular weights of 18.4-60.3kDa.The sequence coverage of these differentially expressed antioxidant proteins varied between 10.6 and 45.8% and the number of peptides was 2-19.
Among the five antioxidant enzyme proteins, expression levels of two glutathione reductase proteins (A2XAX5 and A2XCU8) were 25-72% lower under C40 conditions compared to C0, whereas those of another three proteins of peroxidase (B8AJE7), superoxide dismutase (B8B8G3), and peroxiredoxin (P0C5D4) were 62-123% higher in C40 leaves compared to C0 (Fig. 1A; Table 1).This result demonstrates that peroxidase, superoxide dismutase, and peroxiredoxin are important components of the chloroplastic antioxidant defense system against high-temperature stress in rice leaves upon biochar supplementation.
It has been well documented that nitric oxide (NO) can boost the antioxidant activity in plants under abiotic stress conditions [17].The synthesis of NO in plants includes two pathways: (1) the enzymatic pathway (nitrate reductase, nitric oxide-like synthase, nitrite-NO reductase, and xanthine oxidase); and (2) the non-enzymatic pathway (nitrification and de-nitrification) [14].As both these two pathways of NO synthesis are related to nitrogen (N) supply, several studies have shown that N application can increase leaf antioxidant enzyme activities and reduce yield losses in rice under high temperature conditions [10,21].In this study, we observed that biochar supplementation increased shoot N uptake and up-regulated leaf N assimilation and transport proteins in rice (see [5] for details), indicating that biochar supplementation may alter the synthesis of NO and in turn regulate the antioxidant defense system in the chloroplasts of rice leaves exposed to high temperature (Fig. 2).However, further investigations are required to confirm this possibility.
The findings of this study suggest that the combined expression of antioxidant enzymes (peroxidase, Table 1 Identified differentially expressed proteins related to antioxidant defense in chloroplasts of rice leaves exposed to high temperature between without and with biochar supplementation superoxide dismutase, and peroxiredoxin) and the thioredoxin system are central to the biochar-mediated protection of rice leaves exposed to high-temperature stress.
This study provides insight into the physiological mechanisms by which biochar supplementation mitigates the negative effect of high-temperature stress on rice plants.

1 Fig. 1
Fig.1Expression levels of antioxidant enzyme proteins (A) and thioredoxin system proteins (B) in chloroplasts of rice leaves exposed to high temperature without (C0) and with biochar supplementation (C40, 40 g biochar kg -1 soil).Columns and bars represent the means and standard deviations for three replicates, respectively.Protein descriptions are provided in Table1

Fig. 2 A
Fig. 2 A potential process underlying the effect of biochar supplementation on antioxidant defense systems in rice leaf chloroplasts exposed to high temperature