Biochar is the product of heating biomass in the absence of or with limited air to above 250°C in a process called charring or pyrolysis (Lehmann and Joseph, 2015). Biochar is often used as an additive to improve the quantity of the environment and amend the soil (Lehmann and Joseph, 2015), and such additions can reportedly enhance plant growth characteristics (Waqas et al., 2018). For example, biochar treatments increased both plant height and leaf size in tomato (Graber et al., 2010); treatment with attapulgite clay/yak dung (50/50) biochar resulted in the highest pasture yield and promoted the nutritional quality of grass (Rafiq et al., 2017); and biochar alone or in a co-application stimulated growth in halophyte plants, including their germination, root development, and biomass (Zheng et al., 2018).

Biochar can affect plant growth via several plausible mechanisms: (1) by improving soil and regulating the soil microbial environment, which indirectly or directly affects plant root growth and thus affects the whole plant (Lehmann and Joseph, 2015; Zheng et al., 2018); (2) by providing nutrients for plants to uptake (Wang et al., 2018); (3) by organic molecules on the surface of biochar that can promote or inhibit plant growth (Graber et al., 2010; Lievens et al., 2014; Gale et al., 2016; Yuan et al., 2017); and (4) by affecting endogenous plant hormones, which can impact plant development and physiology (Yang et al., 2015; French and Iyerpascuzzi, 2018; Waqas et al., 2018). Recently reported effects of biochar on plant hormones include changes to jasmonic acid levels in two rice varieties that altered their resistance to herbivory (Waqas et al., 2018) and evidence suggesting that biochar promotes growth, in part, via stimulation of the Gibberellic acid (GA) pathway (French and Iyerpascuzzi, 2018). The previous paper dealt with low temperature (cold), yet the relationship between biochar and abscisic acid (ABA)—which is closely related to low temperature—has not been reported on.

Low temperature may negatively impact agricultural crop productivity (Li et al., 2015). ABA is an essential phytohormone that not only regulates seed dormancy, germination, and seedling growth but also is involved in plant responses to environmental stresses, such as drought, high salinity, and chilling (He et al., 2014). In the presence of ABA, the ABA receptor pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of ABA receptor (RCAR) undergoes conformational changes and mediates interactions with the negative regulator type 2C protein phosphatase (PP2C), thus inhibiting their phosphatase activity, which then activates the positive regulator Class III SNF1-related protein kinase 2 (SnRK2s) to turn on downstream gene expression (Hubbard et al., 2010; Cao et al., 2013). The rice (Oryza sativa) ortholog of the ABA receptor in OsPYL/RCAR5 was recently identified as a positive regulator in seed germination and early seedling growth (Kim et al., 2012, 2014). He et al. (2014) determined the crystal structure of the ABA–OsPYL2–OsPP2C06 ternary complex, and the first structure of the ABA receptor in rice revealed a molecular mechanism of ABA sensitivity and phosphatase inhibition of OsPYLs (He et al., 2014).

Naturally occurring small molecules have long been foci for study due to their diverse biological activities (Bhuiya et al., 2017). One way to investigate these molecular and protein interactions is through molecular docking, which is now the most frequently used computational method for studying the interactions between organic molecules and biological macromolecules (Yang et al., 2015). In this context, docking is able to predict the preferred position of a ligand inside a receptor binding site (Ramirez and Caballero, 2018).

In previous work, we showed that biochar additions have a positive impact on cold stress resistance in rice plants (Yuan et al., 2017). Yet, it remains unclear whether the organic molecules of biochar can interact with the ABA signaling pathway associated with cold resistance. This study treated rice seedlings with different leacheates of biochar and combined the use of experiments and bioinformatics (molecular docking) to determine which of the organic molecules of biochar’s leacheates could influence the ABA signaling pathway. A mechanism was postulated: organic molecules of biochar’s leacheates can successfully connect with the ABA receptor protein, thereby affecting the ABA pathway of rice, which eventually fosters their resistance to cold.

High concentration of biochar enhanced the growth of a bean under saline condition, which may have contributed to the reduction of Na uptake and enhancement of K, Ca, and Mg contents (Farhanqi-Abriz and Torabian, 2018), and application is known to preserve rice pollen given high-temperature stress (Fahad et al., 2015). Our results also reveal high concentrations of biochar leacheates led to enhancement of rice plant growth under low-temperature stress (Figure 1A). The heights of the plants treated with 10, 20, and 30 mg/L exogenous ABA were less than those lacking ABA and those of 20 mg/L treatment were slightly better (Figure 1B). This phenotype (Figure 1B) may arise because although ABA apart from a gradual increase in a certain low concentration can affect cold resistance in plants, its primary role is to slow plant growth (Mega et al., 2015). In this way, when temperatures are low and the plant grows slowly, it can better protect itself from this temporary stress (Mega et al., 2015). Under low temperature, both biochar and ABA can influence the growth and development of rice. However, within a certain concentration range, the biochar does not have the inhibiting effect of ABA (Figures 1A,B). Biochar and ABA can play their roles better under adverse environmental conditions (Kim et al., 2014; Fahad et al., 2015). Thus, in our study, there were no significant changes found in plant phenotypes under different concentrations of biochar leacheates and exogenous ABA treatments in the normal temperature control (Figures 1C,D).

A number of basic leucine zipper (bZIP) transcription factors are known to function in stress signaling in plants, but to date, few have been functionally characterized in rice: the OsABF1 and OsABF2 genes do encode a bZIP transcription factor (Hossain et al., 2010a, b). Their expression in seedling shoots and roots is reportedly induced by various abiotic stress treatments, such as anoxia, salinity, drought, oxidative stress, cold, and ABA (Hossain et al., 2010a, b). However, under the same low temperature, the expression of OsABF1 and OsABF2 genes within a plant may differ, since they can each respond to ABA (Figures 2A,B).

PsbR is known as the 10-kDa Photosystem II polypeptide. Although this plant PsbR is thought to play important roles in photosynthesis, little is actually known about its contribution to abiotic stress resistance (Li et al., 2017). In a recent study, the OsPsbR1 gene was upregulated in response to cold stress, while the upregulation of OsPsbR3 gene was observed when plants were treated with ABA (Li et al., 2017). We found that the expression of OsPsbR1 in each biochar treatment was similar to that of the control, except under the 10% concentration treatment (Figure 2C), while that of OsPsbR3 increased with all leacheate concentrations tested (Figure 2D). When ABA changes, the expression level of OsPsbR3 gene also changes, indicating that the surface substances of biochar may affect the endogenous ABA in rice. The OsABA45 gene in rice seedlings can be induced by low temperature, dehydration, high salt, and ABA (Rabbani et al., 2003); LEA3 and RAB16A genes are associated with stress and can be induced by ABA (Zou et al., 2008; Hossain et al., 2010b). At the same low temperature, the expression of OsABA45, LEA3, and RAB16A genes differed in response to ABA (Figures 2E–G). Therefore, we speculate that substances in the biochar leacheates can affect the ABA pathway.

As a phytohormone, ABA is extensively involved in plant responses to abiotic stresses, such as drought, low temperature, and osmotic stress. In response to cold stress, plants usually accumulate an increased amount of ABA, and many stress-inducible genes are regulated by the endogenous ABA that accumulates during conditions of stress (Shinozaki et al., 2003). We found that a high concentration of biochar leacheates could improve the content of ABA and its precursors carotenoids in rice seedlings (Figure 4A), as well as the expression of ABA and the cold-related genes OsABF1, OsABF2, OsABA45, OsLEA3, RAB16A, OsPsbR1, and OsPsbR3 (Figure 2). This result indicates that adding more biochar leacheate can induce endogenous ABA biosynthesis in rice seedlings, and this increased ABA could influence corresponding biological functions to help plants resist cold stress. High concentrations of biochar could affect the ABA signaling pathway, and exogenous ABA could also affect the ABA signaling pathway, indicating that some ABA analogs may exist on the surface of biochar. These ABA analogs likely caused a series of physiological and biochemical processes related to ABA and cold resistance within a certain concentration range, which eventually promoted the cold resistance of rice plants.

Working with tomato, Graber et al. (2010) found that biochar treatments positively enhanced its plant height and leaf size without any effect on its flower and fruit yield (Graber et al., 2010). Yet these positive impacts of biochar on plant responses were not due to direct or indirect effects on plant nutrition per se, as there were no differences between control and treatments in their leaf nutrient contents (Graber et al., 2010). Therefore, those authors considered that the organic molecules in biochar were impacting the growth of crops. In the absence of interference from other factors, we also think that the organic molecules of biochar’s leacheates are crucial for altering the growth of rice seedlings under low-temperature stress. To explore the mechanism by which such organic molecules could influence rice seedlings cold tolerance, we recently identified them (Yuan et al., 2017). In researching the direct effects of biochar on plants, most studies have now identified organic molecules from biochar via GC/MS and then determined whether they can affect plants’ growth and by which possible mechanism (Graber et al., 2010; Gale et al., 2016).

We used biochar leacheates to eliminate other interferences, so we believe that the organic molecules contained in biochar are indeed an important factor influencing the growth of rice seedlings under low temperature. But admittedly, the underlying mechanism of these organic molecules is not yet known. In recent years, the interaction between molecules and proteins has become a hot research topic. In plants, the function of organic molecules can include hormone response, signal transduction, or ligand interaction with proteins to exercise a series of related biological functions (Antunes et al., 2011; Yang et al., 2015; Bhuiya et al., 2017; Haruta and Sussman, 2017). Therefore, we put forward an experimental hypothesis: organic molecules of biochar’s leacheates can enter cells of the plant and interact with corresponding proteins in them, thereby driving a series of physiological and biochemical reactions that enable plants to resist the cold. We suspected our identified organic molecules may have interacted mechanistically with stress or ABA-related proteins to generate the cold stress effects we observed.

The direct homologous receptor OsPYL/RCAR5 has been shown to positively impact the growth of rice seedlings (Kim et al., 2012, 2014). He et al. (2014) identified the structure of OsPYL2 in rice, and its 3D structure was found in the RCSB database (He et al., 2014). We concluded that the organic molecule Cyah in biochar could be successfully docked with OsPYL2 (Figure 5). The organic molecule Cyah has been recorded in the PMN database given its participation in a variety of chemical reactions (Table 5), but whether it interacts with the ABA receptor protein remains is unknown. Nevertheless, the interaction between the ABA ligand and ABA receptor protein can reduce abiotic stress (Cao et al., 2013). The ABA analogs and ABA ligands are the same as ABA receptor proteins, and the interaction between ABA analogs and ABA receptor proteins can prompt plant responses to abiotic stress, thus indicating that ABA analogs also function much like ABA ligands. Cyah and ABA are the same as the ABA receptor protein OsPYL2, in that they are combined with hydrogen bonds, so Cyah may also be analogs to ABA, with the same function as ABA in regulating OsPYL2 to produce a series of related effects. A plausible mechanism is that the organic molecule Cyah in biochar combines with OsPYL2 of rice, and the combination of PP2C and Cyah-OsPYL2 then inhibits the activity of PP2C itself; this activates SnRK2 and regulates the ion channel, second messenger, and the expression of related ABA genes, which together improves the cold resistance of plants.

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

JY designed and carried out the experiments, analyzed the results, and wrote the manuscript. JM, YE, XL, XY, and WC designed the experiments. All authors contributed to the article and approved the submitted version.

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