This study aimed to investigate the effects of chitin and chitosan to promote overall defense responses of the C. sativa root system. The results demonstrate that chitosan has a much stronger effect than chitin to activate plant root defense responses. Chitosan can induce the production of defense enzymes in root tissue (Fig. 4) and the secretion of defense proteins into root exudate (Fig. 6-7). The gene transcript levels of defense proteins were also upregulated (Fig. 8), and cellular levels of ABA and CA were increased in root tissues (Fig. 3). However, the levels of defense hormones including SA and JA were not changed. In contrast, none of the defense responses induced by chitosan were observed in chitin treatments.

In general, chitin and chitosan may be considered relatively similar in structure. Both are natural β-1,4-linked polysaccharides, with the predominant subunit of N-acetyl-D-glucosamine and D-glucosamine for chitin and chitosan, respectively. The difference is the absence of the acetyl group in chitosan subunits. Chitin may be transformed to chitosan by natural chitin deacetylase enzymes (Zhao et al., 2010) or chemical deacetylation reactions (Vicente et al., 2021). However, the conversion may not be fully complete, and chitosan may be present in a partially acetylated form. The degree of deacetylation determines the number of acetyl groups removed from the chitosan structure. This value along with crystallinity, viscosity and polymer size affect the chemical and physical properties of chitosan (Triunfo et al., 2022).

In plants, the outer cell membrane has receptors which can specifically bind to chitin. Chitin-binding receptor is a protein complex, containing multiple lysin motif (LysM) domain proteins and receptor-like kinases (RLKs) or receptor-like proteins (RLPs) (Shinya et al., 2015). Chitin oligomers (or short-chain chitins) have high affinity to bind the receptor (Cao et al., 2014; Hayafune et al., 2014; Gubaeva et al., 2018). Interactions between chitin oligomers and chitin-binding receptors and downstream signaling processes may vary across different plant species but ultimately binding results in an activation of plant cellular defense response (García et al., 2021). The signaling process is rendered through mitogen-activated protein kinase (MAPK) cascades and in association with plant defense hormones i.e., salicylic acid, jasmonic acid and ethylene (Ramirez-Prado et al., 2018; Gong et al., 2020). On the other hand, plant receptors that can specifically bind to chitosan have not yet been reported (Yin et al., 2016), even though chitosan has been well described as a plant elicitor to induce plant defense (Pichyangkura and Chadchawan, 2015). The mechanism underlying chitosan-promoted plant defense is still unclear. It could be via chitin-binding receptors because partially deacetylated chitosan retains chitin-like identity and may be able to bind to the receptors. Chitosan with 60% deacetylation had higher eliciting activity than chitin oligomer (8 subunits) to enhance H₂O₂ production in Arabidopsis seedlings (Gubaeva et al., 2018). However, fully deacetylated chitosan had no effect, suggesting the acetyl group in chitin or partially deacetylated chitosan is required for chito-polymers to interact with plant receptors (Gubaeva et al., 2018). The chitosan used in our study was 75-85% deacetylated (Sigma-Aldrich). Hence, chitosan-induced defense responses as observed might be triggered via the binding of chitosan to chitin-binding receptors. Nevertheless, it is also feasible that the effects were induced via the binding of chitosan to its own specific receptors, which have not yet been discovered. Alternatively, chitosan may alter plant cell membrane permeability and directly signal cytoplasmic secondary messengers, such as hydrogen peroxide, nitric oxide, and phytohormones, triggering their downstream pathways (Pichyangkura and Chadchawan, 2015), or bind to chromatin in the nucleus to regulate plant defense responses (Hadwiger, 2013).

Our results also suggest that exogenous intact chitin poorly activates plant defense responses, likely due to inability to bind plant cell membrane receptors (Shinya et al., 2015). Based on scanning electron microscope, crude chitin aggregates into particles approximately 20 µm in diameter which is much larger than approximate size, 8 nm in diameter, of the extracellular domain of toll-like receptor, one of the classical receptors in plant and animal immune system (Fuchs et al., 2018). This large size mismatch and lack of available soluble chitin may impede direct binding between crude chitin and eukaryotic cell membrane receptors. Long-chain chitin nanofibers (chitin fibrils embedded in a protein matrix) can enhance cellular H₂O₂ production and PR gene expressions in Arabidopsis, but it is modified to be highly dispersed in water and loosely agglomerated, thereby can be easily degraded by chitinase enzymes (Egusa et al., 2015). Our results support the concept that natural interaction between fungal chitin and plant receptors requires digestion steps to break down polymeric cell wall chitin into soluble, recognizable chitin fragments (Shinya et al., 2015).

On the other hand, enhanced defense responses as observed from chitosan treatments have confirmed its eliciting properties (Chandra et al., 2017a; Lopez-Moya et al., 2021; López-Velázquez et al., 2021). As summarized in Fig. 9, our analyses using various techniques (including enzymatic assays, exudate proteomics and qPCR method) were well correlated, showing increased peroxidase and chitinase activities, transcript levels of defense genes in the root tissues and increased secretion of defense proteins in the exudates. Correlations between expression levels of defense genes in the tissues and their protein intensities in the exudates were also positive (Supplementary Fig. S5), implying that increased production of defense genes and proteins in tissue is correlated to secretion of the proteins into exudate. To the best of our knowledge, this is the first report showing increased secretion of defense proteins into exudate in response to chitosan. The effect appeared to be dose dependent as it was strongest in the 0.5% chitosan treatment and gradually lower in the 0.2% and 0.1% chitosan treatments. The majority of significantly increased proteins in exudate are classified as PR proteins which are usually increasingly expressed once a plant is attacked by pathogens. PR protein 1 has long been used as a genetic marker for plant systemic acquired resistance (Breen et al., 2017). Chitinases are key enzymes that protect plants against fungal pathogens (Vaghefi et al., 2013; Kaur et al., 2022). Thaumatin-like protein has been shown to play a role in wheat resistance against leaf rust fungus, Puccinia triticina (Zhang et al., 2018). Mulatexin protein has a strong toxicity against silkworms, helping protect mulberry tree from pest attack (Wasano et al., 2009). Additionally, a previous study on tomato root demonstrated that chitosan could depolarize root cell membrane and induced secretions of phytohormones, lipid signaling and phenolic compounds in root exudate (Suarez-Fernandez et al., 2020). Combining this information with our root exudate proteome data, it could be concluded that the impact of chitosan is not limited to plant tissues but can be extended to root metabolite and protein secretions.

The molecular mechanism underlying chitosan promotion of plant defense is still unclear. We hypothesized that chitosan could somehow mediate biosynthesis of phytohormones, leading to a regulation of defense genes in the nucleus. In our study, phytohormone measurement showed only slight increases of ABA hormone and CA compound in the root tissue of 0.5% chitosan treatment. The levels of known defense hormones, SA and JA, were not changed in any conditions (Fig. 3). In our experiment, the sampling timepoint could be a key factor as tissue samples were collected one week after the treatment, but upregulations of genes or transcription factors related to phytohormone biosynthesis could happen within 24 hours of chitosan treatment (Colman et al., 2019). Higher levels of ABA detected upon chitosan treatment suggests that ABA might contribute to the increased levels of defense genes and proteins. ABA has long been proposed to associate with abiotic stress, but recently its role in relation to biotic stress has been increasingly reported (Shigenaga and Argueso, 2016). ABA signaling pathway cross talks with SA and JA defense hormone pathways, implying ABA is also involved in regulating plant disease resistance (Anderson et al., 2004; Berens et al., 2017). This is evidenced by several studies, for example, spraying 100 µM of exogenous ABA induced expression levels of PR4 and PR10 genes in lentil seedlings (Ford et al., 2017). Additionally, ABA hormone can modulate rice MAPK5 gene and protein biosynthesis, influencing downstream signaling processes of both disease resistance and abiotic stress tolerance (Xiong and Yang, 2003). Therefore, ABA could be one of the players, triggering defense responses in C. sativa root system as observed.

Moreover, we found that root growth was largely interrupted by chitosan, which is consistent with previous observations in Arabidopsis root (Lopez-Moya et al., 2017; Iglesias et al., 2019). The inhibition is due to upregulation of transcription factors involved in the auxin biosynthesis pathway, resulting in auxin accumulation at the root tips (Lopez-Moya et al., 2019). However, in our study auxin levels, IAA and Me-IAA, were comparable to control. In turn, higher levels of ABA and CA were detected. It is possible that auxin accumulation may take place at the early stage after chitosan exposure. The effect may ease off through time as expression level of the YUC2 gene, encoding one of the key enzymes in the IAA biosynthesis pathway, leveled off within three days after the treatment (Lopez-Moya et al., 2017). Increases in cellular CA level may be another factor affecting root growth since treating plants with exogenous CA were evidenced to decrease root length, alter activity of IAA oxidase, an IAA degradation enzyme and inhibit auxin efflux (Salvador et al., 2013; Steenackers et al., 2017). Ultimately, the stalling of root growth suggests that the plant may transform energy from expanding existing roots or generating new roots to consolidate their defense system in response to chitosan. This plant adaptation process is known as growth-defense tradeoffs, which can be triggered by any abiotic and biotic factors (Huot et al., 2014; He et al., 2022). Our findings demonstrate that chitosan appears to activate this process in C. sativa root system. Further exploration is required to identify the underlying biological mechanisms impacting root growth and causing upregulation of defense responses. The knowledge gained would benefit crop improvement to maximize crop yield with a balance of disease resistance (Silva et al., 2019). After all, chitosan could be a potential elicitor for agricultural application, especially in hydroponic setup to counteract fungal pathogen attacks under safe and practical conditions.