Nanofibrillation is an Effective Method to Produce Chitin Derivatives for Induction of Plant Responses in Soybean

Chitin, an N-acetylglucosamine polymer, is well-known to have unique biological functions, such as growth promotion and disease resistance induction in plants. Chitin has been expectedly used for improving crop yield using its functions; however, chitin derivatives, such as chitin oligosaccharide (CO) and chitosan, are widely used instead since chitin is difficult to handle because of its insolubility. Chitin nanofiber (CNF), produced from chitin through nanofibrillation, retains its polymeric structure and can be dispersed uniformly even in water. Here, the effects of CO and CNF on plant responses were directly compared in soybeans (Glycine max) to define the most effective method to produce chitin derivatives for plant response induction. The growth promotion of aerial parts was observed only in CNF-treated plants. The transcriptome analysis showed that the number of differentially expressed genes (DEGs) in CNF-treated soybeans was higher than in CO-treated soybeans. Notably, the expression patterns of DEGs were mostly similar but were strongly induced by CNF treatment as compared with the CO group. These results reveal that CNF can induce stronger plant response to chitin than CO in soybeans, suggesting nanofibrillation, rather than oligomerization, as a more effective method to produce chitin derivatives for plant response induction.


Introduction
Chitin is a natural polymer with an N-acetylglucosamine-repeating structure, which is a highly abundant polysaccharide occurring mainly in the exoskeletons of arthropods, including crustaceans and insects, and in the cell walls of yeast and fungi [1,2]. Chitin and its deacetylated derivative chitosan are well-known to have various kinds of unique functions that can contribute to the crop yield improvement. Chitin derivatives directly induce defense responses and affect plant growth, as well as stimulate beneficial microbe activity in soils, promoting plant growth and disease resistance [1]. These highly sophisticated functions that increase the crop yield have been expectedly used for chitin-derived materials widely as an ecofriendly promising agent instead of chemicals, such as fertilizers and pesticides, in agriculture. Original polymeric chitin generally produced from crab or shrimp shell wastes is difficult to be insoluble in most solvents mainly because of the highly extended hydrogen-bonded semi-crystalline structure [3]. Instead of original chitin, the water-or

Effects of Chitins on the Plant Growth in Soybeans
Soybean plants were grown in soil supplemented with CO and CNF but without any nutrients, because soybeans can be grown only using their own nutrients that are stored in its seeds from germination to the primary growth stage. Using this growth condition, the growth-promoting effects of both CO and CNF associated with nitrogen uptake efficiency, which was already reported previously [10], can be eliminated. The significant increase in shoot length was observed only in CNF-treated plants, whereas the root length in CO-treated plants was higher than in the other groups ( Figure 1a). No biomass increase was observed both in the examined shoots and roots, because of the absence of exogenous nutrients (Figure 1b). Soybean plants were grown in soil supplemented with CO and CNF but without any nutrients, because soybeans can be grown only using their own nutrients that are stored in its seeds from germination to the primary growth stage. Using this growth condition, the growth-promoting effects of both CO and CNF associated with nitrogen uptake efficiency, which was already reported previously [10], can be eliminated. The significant increase in shoot length was observed only in CNFtreated plants, whereas the root length in CO-treated plants was higher than in the other groups ( Figure 1a). No biomass increase was observed both in the examined shoots and roots, because of the absence of exogenous nutrients (Figure 1b). The effects of chitin supplementation to soils on soybean plant growth. The soybean plants were grown on soil mixed with equal volume of 0.01% (w/v) chitin oligosaccharide (CO) solution or chitin nanofiber (CNF) dispersion for four weeks and then harvested for measurement of length (a) and dry weight (b) of shoots and roots. Data are shown as mean and SE (n = 4). Different letter types (uppercase and lower case) denote significant differences (Tukey's HSD test, P < 0.05). Data are representative of three independent biological experiments, showing similar results.

Comparison and Analysis of Differentially Expressed Genes (DEGs) in Chitin-treated Soybeans
Only CNF promoted the growth of aerial parts in soybeans under no exogenous nutrient supplement, which suggests that exclusively chitin-treated plants may be a better option to examine the differences in the effects of CO and CNF. Transcriptome analysis through RNA-seq of soybeans exclusively chitin-treated plants was conducted. The summary of sequencing, including the number (ca. 5.2-9.5M) and mapping efficiency (ca. 85%-90%), to reference the genome sequence of raw reads for three biological replicates of each treatment is shown in Table S1. After the expression profile comparison between control and chitin-treated soybeans, 10 and 40 genes showed significantly different expression levels in CO-and CNF-treated plants, respectively (false discovery rate [FDR] cutoff < 0.05) ( Table 1). Of these, four genes with annotations of syringolide-induced protein 14-1-1 (GLYMA_04G020700), glutamate decarboxylase (GLYMA_05G136100), naringenin-chalcone synthase 1 (GLYMA_08G109400), and C2H2-type domain-containing protein (GLYMA_10G295200) and two genes without annotations were determined as common DEGs in both chitin treatments ( Figure 2a, Table 1). Next, 44 genes determined as DEGs in CO-and/or CNF-treated plants were clustered based on fold change values, as compared with control ( Figure 2b). The clustered heatmap clearly showed that the expression patterns (upregulation or downregulation) of all DEGs were mostly similar in both chitin treatments, but DEGs expression observed in CNF-treated plants was strongly induced by CNF treatment, as compared with CO. Only DEGs in CNF-treated plants were further analyzed since the DEGs level in the CO-treated plants was not enough for functional analysis.
All DEGs in CNF-treated plants were subjected to gene ontology (GO) analysis to obtain deep

Comparison and Analysis of Differentially Expressed Genes (DEGs) in Chitin-treated Soybeans
Only CNF promoted the growth of aerial parts in soybeans under no exogenous nutrient supplement, which suggests that exclusively chitin-treated plants may be a better option to examine the differences in the effects of CO and CNF. Transcriptome analysis through RNA-seq of soybeans exclusively chitin-treated plants was conducted. The summary of sequencing, including the number (ca. 5.2-9.5M) and mapping efficiency (ca. 85-90%), to reference the genome sequence of raw reads for three biological replicates of each treatment is shown in Table S1. After the expression profile comparison between control and chitin-treated soybeans, 10 and 40 genes showed significantly different expression levels in CO-and CNF-treated plants, respectively (false discovery rate [FDR] cutoff < 0.05) ( Table 1). Of these, four genes with annotations of syringolide-induced protein 14-1-1 (GLYMA_04G020700), glutamate decarboxylase (GLYMA_05G136100), naringenin-chalcone synthase 1 (GLYMA_08G109400), and C2H2-type domain-containing protein (GLYMA_10G295200) and two genes without annotations were determined as common DEGs in both chitin treatments ( Figure 2a, Table 1). Next, 44 genes determined as DEGs in CO-and/or CNF-treated plants were clustered based on fold change values, as compared with control ( Figure 2b). The clustered heatmap clearly showed that the expression patterns (upregulation or downregulation) of all DEGs were mostly similar in both chitin treatments, but DEGs expression observed in CNF-treated plants was strongly induced by CNF Plants 2020, 9, 810 4 of 11 treatment, as compared with CO. Only DEGs in CNF-treated plants were further analyzed since the DEGs level in the CO-treated plants was not enough for functional analysis.
All DEGs in CNF-treated plants were subjected to gene ontology (GO) analysis to obtain deep functional characterization. The number of DEGs categorized by GO terms is shown in Table 2. The dominant number of GO terms were the following: "cell periphery" (GO:0071944) and "plasma membrane" (GO:0005886) in the cellular component category; "drug binding" (GO:0008144), "transporter activity" (GO:0005215), "localization" (GO:0051179), "establishment of localization" (GO:0051234), and "transmembrane transporter activity" (GO:0022857) in the molecular function category; and "response to stimulus" (GO:0050896) in the biological process category.

Discussion
In this study, we showed that CNF supplementation to soils, rather than CO, promoted the growth of aerial parts and induced a stronger plant response to chitin. This is the first report that directly and comprehensively compared the effects of high-and low-molecular-weight chitins on plants by transcriptome analysis using RNA-seq. In a previous study [10], both CO and CNF showed growth-promoting action on the aerial parts of tomato by improving nitrogen uptake efficiency, which is inconsistent with the results of promoting effects on soybean growth found in this study. The transcriptome analysis of CNF-treated tomato indicated that the expression levels of genes related to nitrogen acquisition and assimilation and nutrient allocation were changed [10]. As previously described in this study, the effects of chitin treatments on soybean growth without exogenous nutrient application were examined, which eliminates the influence of nitrogen acquisition. Therefore, the inconsistency on the growth-promoting action by both chitins may be caused by the CNF-specific function involved in the regulation of nitrogen assimilation and nutrient allocation. However, the transcriptome analysis in this study demonstrated that all the DEGs' expression patterns were mostly similar in both chitin treatments, but DEGs expression was strongly induced by CNF treatment, as compared with CO treatment. This indicates that both CO and CNF show a mode of action that is common in plants, but the degree of plant response to chitin by CNF is stronger than by CO. This is also supported by the evidence that CNF induced more ROS production than CO in Arabidopsis, but ROS production was completely impaired in chitin receptor CERK1 mutant cerk1-2 [5,13]. We, therefore, concluded that the growth-promoting effects on the aerial parts of soybeans by CNF would not be Plants 2020, 9, 810 8 of 11 caused by CNF-specific function and may be attributed to the different levels of plant response to chitin caused by CO and CNF.
In plants, chitin is recognized by the ectodomains of LysM (lysin motif) receptors, such as CEBiP and CERK1, localized on plasma membranes, and then chitin recognition information is transduced by LysM receptor phosphorylation to activate downstream signaling [4,13,14]. Arabidopsis CERK1 binds to polymeric chitin and plays an essential role in chitin signaling [15,16], and CNF is also recognized by CERK1 in Arabidopsis similar to CO, because of the lack of ROS production by CNF in cerk1-2 [5], suggesting that the primary site of action by CNF is in the apoplast, similar to CO. The molecular basis that explains the reason why CNF can induce stronger plant response to chitin than CO is still unclear because it has not been clarified whether CNF directly binds to CERK1. The rapid and massive CO production by chitinase presented or secreted from plant cells in the apoplast may cause CNF's stronger induction since CNF can be immediately degraded to CO by chitinase [5].
The GO analysis of DEGs in CNF-treated plants showed that most of the genes differentially expressed by CNF treatment in soybeans may be involved in the function in the apoplast and the communication between inner and outer plasma membranes according to the categorized GO terms like "cell periphery," "plasma membrane," "transporter activity," and "response to stimulus," which is consistent with the aforementioned possible primary site of CNF action. The GO enrichment analysis and KEGG pathway analysis of DEGs in CNF-treated plants demonstrated the potential biological functions of chitins because the mode of action by CO and CNF in plants is common. The overrepresented GO terms "carbohydrate transport" and "glutamate catabolic process" may be associated with the growth-promoting effects of chitins. This is consistent with previous findings of increased carbon content by both CO and CNF treatments associated with its growth-promoting action, and the genes involved in glutamate catabolic process, such as glutamate synthase and glutamate dehydrogenase, were upregulated in CNF-treated tomato [10]. The genes involved in the "butanoate metabolism" pathway are also overrepresented. In this metabolic pathway, a non-protein amino acid, gamma-aminobutyric acid (GABA), is primarily biosynthesized from glutamate by glutamate decarboxylase in plants, which modulates plant growth [17,18]. The gene encoding glutamate decarboxylase (GLYMA_05G136100) was upregulated by both CO and CNF treatments, which suggests that the function of GABA is required for growth promotion in chitin-treated plants. Moreover, the overrepresented GO terms "calcium ion binding," "peroxidase activity," and KEGG-related "MAPK signaling pathway" seem to be associated with chitin-induced defense response, because MAMPs, including chitin, quickly induce Ca 2+ influx, expression of peroxidase, ROS production, and MAPK signaling activation in plants [19,20]. Chitin rapidly upregulated genes encoding NADPH oxidases, which are involved in ROS production on plasma membrane, rbohD and RbohF in Arabidopsis [21]. Likewise, the expression level of gene (GLYMA_20G236200) encoding unknown protein homologous to NADPH oxidase was increased by both CO and CNF treatments in soybeans.
In summary, we demonstrated that nanofibrillation, rather than generally oligomerization, is an effective method to produce chitin derivatives to induce the plant response in soybeans. To develop ecofriendly chitin derivatives that are agriculturally usable, several studies on applying chitin and chitosan nanoparticles have recently been reported [22,23]. However, the production cost of these nanoparticles may be more than CNF, which only demands machines, such as a grinder, for physical treatment because these nanoparticles still demand the usage of chemicals [2,24]. Considering not only the advantage of biological function but also its unique physical features and chitin production without hazardous chemicals, nanofibrillation is the best choice to develop practical and promising ecofriendly chitin derivatives, which can be agriculturally applied to improve crop yield. Chemicals are not required for CNF production from original chitin, but hazardous chemicals are necessary to produce original chitins from raw materials, such as crab and shrimp shells. The nanofibrillation technique of raw materials can be used, so that using chemicals during the whole process can be avoided, since CNF composites prepared directly from crab shells showed similar effects on disease