Ⅰ. INTRODUCTION
Eco-friendly agriculture has currently expanded to replace application of chemical fertilizers using various organic substances (Brunelle et al., 2024). Particularly, grasslands are a complexed eco-system composed of various living organisms, including animals, plants, microorganisms, and human activities, having crosstalk with the surrounding environments. Forage crops production is largely depends on the environmental changes, and current global warming causes adverse effects to their productivity due to the secondary abiotic stresses, including unexpected regional sudden climate changes (Hart et al., 2022). To cope the negative environmental effects, biofertilizers produced from manure, microbiome, and food by-products have adopted for increasing soil fertility and crop productivity (Mahmud et al., 2021;Mandal et al., 2024).
Humic substances (HS) are mixtures of supramolecular complex having irregular polymeric structure and divided into humic acid (HA), fulvic acid, and humin depending on their solubility (Cunha-Santino and Bianchini-Júnior, 2004;Jeon et al., 2018). Among these, HA are the major organic substances in grassland existing as amounts of two-third (Stevenson, 1982). HA positively affects not only to promote plant growth, but also to improve soil fertility (Trevisan et al., 2010). Due to wide range of HA’s biological activities, its significance is increasing in current agriculture industry. However, the HA quality is inconsistent depending on the extraction sources and accumulated regions (Stevenson, 1982;Metzer, 2010). To overcome this challenge, we have investigated to produce artificial HA-like fertilizers through top-down or bottom-up approaches. Then, we have reported that bottom-up approachdriven polymeric products by oxidative polymerization of catechol and vanillic acid displays HA-like biological activities in various plants including Arabidopsis, alfalfa, and Italian ryegrass (Cha et al., 2017; Khaleda et al., 2018a: Khaleda et al., 2018b).
Lignins are largely existed in soils produced by decomposition of plant biomass with the interaction of soil microbes. In addition, lignins share a similar structure with aromatic portions of HA (Shevchenko and Bailey, 1996). Thus, fungal-induced decomposition of lignin-related biomass can help humification (Chefetz et al., 1998). In particular, technical lignins are largely produced by cellulose-based fermentation from woody biomass for pulp industry (Calvo-Flores and Dobado, 2010). The technical lignins, such as Kraft and sulfite lignins, are produced as a byproduct in pulp industries. Kraft lignin is produced by the kraft process, which treat with aqueous sodium hydroxide and sodium hydrosulphide to woody biomass (Bilal et al., 2021). Sulfite lignin is produced by treating wood chips with sulfite and bisulfite ion solution, resulting in pure cellulose fibers (Pérez et al., 2023). Among both lignin pulping process, sulfite pulping process is high efficiency and less environmentally cost compared to Kraft process (Bajpai, 2021). We recently reported that Kraft lignins display HA-like biological activities promoting seed germination and salt stress tolerance in Arabidopsis (Jeong et al., 2018). However, the applicability of sulfite lignins has not yet been identified.
In this study, we investigated whether sulfite lignin applications could promote plant growth and stress tolerance, mimicking biological activities of HA. To evaluate the potential of sulfite lignin as a biostimulant, we conducted seed germination and salt tolerance assays using Arabidopsis plants, with the aim of expanding its applications to grasslands for sustainable agriculture.
Ⅱ. MATERIALS AND METHODS
1. Plant materials and growth conditions
Arabidopsis thaliana (ecotype Columbia) wild-type plants were used in this study. Plants were sown on MS agar medium (pH 5.8) plates with 2% (w/v) sucrose and grown in the controlled growth chamber with a 16-h-light/8-h-dark cycle condition.
2. Germination assay on HA, kraft lignin and sulfite lignin-containing medium
HA, kraft lignin and sulfite lignin were purchased from Sigma-Aldrich, and prepared in distilled water as described previously (Cha et al., 2017). HA, kraft lignin and sulfite lignin were mixed to be varying concentrations including 0, 8.6, 86, and 860 mg L-1 with MS medium. And, Arabidopsis seeds were sown onto the prepared medium and germinated. Germination rates were measured by counting the emergence of radicles and cotyledons at two and five days after treatments, respectively.
3. Salt stress tolerance assay
Arabidopsis seeds were germinated and grown in normal MS media for 7 days. Then, seedlings were transferred onto the MS media containing 860 mg L-1 HA, kraft lignin or sulfite lignin with or without 250 mM NaCl for another 7 days. Seedings exposed to HA or lignins were harvested and measured chlorophyll contents for determining salt stress tolerance.
4. Total chlorophyll content
Harvested plants were measured fresh weight and soaked in 80% (v/v) acetone with agitation (120 rpm) for 2 days under dark conditions. Total chlorophyll contents were measuring the absorbance at 645 and 663 nm using a Beckman UV/Vis spectrophotometer and quantitatively calculated as described previously (Cha et al., 2017).
5. Statistical analysis
All physiological experiments, including seed germination and chlorophyll content assays, were performed with three independent biological replicates. Statistical analyses were conducted using a two-tailed Student’s t-test in Microsoft Excel 2016.
Ⅲ. RESULTS AND DISCUSSION
1. Sulfite lignin promotes seed germination
HA exhibts various biostimulant activities such as promoting plant growth and stress tolerance (Trevisan et al., 2010). In particular, seed germination is one of representative physiological parameters driven by HA-mediated bioactivities, which are interest of agronomists. Thus, to determine the HA-like bioactivities of sulfite lignin, we prepared Arabidopsis wild-type seeds on MS media containing varying concentrations of HA, kraft lignin or sulfite lignin. While seeds grown in MS control after 2 days incubation were slightly germinated, treatments of HA, sulfite lignin or kraft lignin showed enhanced germination of Arabidopsis seeds in a dose-dependent manner. Moreover, seeds grown in maximum concentrations (860 mg L-1) of HA and lignins were removed seed coat, indicating entirely germinated compared to MS control. Interestingly, seeds germinated in sulfite lignin at the concentration of 860 mg L-1 exhibited promoted root hair formation compared to HA or kraft lignin-treated seeds (Fig. 1). These observation clearly showed that sulfite lignin treatments to plant seeds promoted seed germination.
To quantitatively analyze whether sulfite lignin treatments to seeds enhance germination, we measured germination rates using radicle and cotyledon emergences under HA and lignins. First, we checked the radicle emergence, which is a primary indicator for seed germination. In the treatment of HA as a positive control, it displayed that gradual increase of HA treatment promoted the radicle emergence in a dose-dependent manner (Fig. 2A), consistent with our previous reports (Cha et al., 2017). Interestingly, sulfite lignin-treated seeds showed highest radicle emergence rates at 8.6 mg L-1, which is not significantly different with 86 and 860 mg L-1 treatements of sulfite lignin treatments as similar effect with kraft lignin treatments (Jeong et al., 2018). In addition, sulfite and kraft lignins showed significantly higher radicle emergence than HA at 8.6 mg L-1 treatment (Fig. 2A). Next, we determined the cotyledon emergence as another indicator for seed germination. Sulfite lignin treatment gradually increased cotyledon emergence in a dose-dependent manner as similar as HA and kraft lignin. Interestingly, sulfite lignin treatment at 8.6 mg L-1 showed significantly higher cotyledon emergence than HA and kraft lignin (Fig. 2B). Both germination assays indicates that sulfite lignin promotes seed germinations with higher biological activity at low concentrations, 8.6 mg L-1 than HA and kraft lignin. It suggests that sulfite lignin has agricultural scalability that can promote plant growth even with a small application dose, and can further increase the utilization of pulp industry by-products.
2. Sulfite lignin enhances salt stress tolerance
HA exhibits priming effects for stress-indced transcriptomes, resulting in various stress tolerance including heat and salt stresses (Cha et al., 2020;Cha et al., 2021). In addition, kraft lignin treatment to Arabidopsis wild-type plants enhanced salt stress tolerance (Jeong et al., 2018). Thus, we also examined whether sulfite lignin can enhance stress tolerance to excessive salt concentrations. Seven-day old wild-type seedlings were exposed to 250 mM NaCl in the absence or presence of HA, sulfite lignin, and kraft lignin for another 7 days. While plants grown in 250 mM NaCl showed leaf chlorosis caused by chlorophyll breakdown, the plants grown in co-treatments of sulfite lignin and NaCl had healthy green leaves as similar as HA and kraft lignin (Fig. 3A). To validate quantitatively the salt stress tolerance, chlorophyll contents were measured. In the absence of salt stress, plants grown in HA, sulfite lignin, or kraft lignin were significantly higher chlorophyll contents than MS control, suggesting that HA and both lignins promotes photosynthesis, contributing the production of chemical energy source for plant growth (Simkin et al., 2019). Under salt stress conditions, sulfite lignin treatment had significantly higher chlorophyll contents than HA, similar to HA and kraft lignin (Fig. 3B). Thus, these results indicate that sulfite lignin displays salt stress tolerance in Arabidopsis, and mimics HA-like biological activities.
We have previously reported that kraft lignin varients resulted from one-pot Fenton oxidations dramatically elevates biological activites such as seed germination and salt stress tolerance, compared to non-manipulated kraft lignin (Jeong et al., 2018). Futhermore, we need to more focus on the modification of HA-like substances. Humic-Fe complexes are widely existing in the soil environments (Pinton et al., 1997), and in fact, one of major coal sources of HA are lignite, which contain significant amounts of Fe ions (Kermer et al., 2017). Given that Fe ions are essential micronutrients for plant growth (Li et al., 2023), Fenton oxidation process for lignin compounds may help to increase the biological functionalities of sulfite lignin to plants. Thus, it raises the necessity of further research whether the Fenton reactions also elevate the HA-like bioactivities of sulfite lignin. In addition, our findings having sulfite lignin-induced bioactivities need to be elusive in molecular levels, such as transcriptome and proteome analyses, to reveal the precise molecular mechanisms.
Ⅳ. CONCLUSION
This study highlights the potential of sulfite lignin as an eco-friendly biostimulant for promoting seed germination and enhancing salt stress tolerance in Arabidopsis. The findings demonstrate that sulfite lignin mimics HA-like biological activities, particularly at low concentrations, and outperforms HA and kraft lignin in promoting seed germination. Furthermore, sulfite lignin enhances stress tolerance, which is comparable with HA and kraft lignin. These results underscore the feasibility of utilizing sulfite lignin, a byproduct of pulp industries, as a cost-effective and sustainable biofertilizer in agriculture, particularly in grassland and forage fields. From a practical perspective, sulfite lignin’s ability to function as a biostimulant and stress tolerance enhancer provides an innovative approach to addressing challenges posed by global warming and its associated abiotic stresses on crop productivity. Its promising performance at low concentrations highlights its economic and environmental benefits in comparison to traditional chemical fertilizers.
