Chitosan-induced biotic stress tolerance and crosstalk with phytohormones, antioxidants, and other signalling molecules

Several polysaccharides augment plant growth and productivity and galvanise defence against pathogens. Such elicitors have ecological superiority over traditional growth regulators, considering their amplified biocompatibility, biodegradability, bioactivity, non-toxicity, ubiquity, and inexpensiveness. Chitosan is a chitin-derived polysaccharide that has recently been spotlighted among plant scientists. Chitosan supports plant growth and development and protects against microbial entities such as fungi, bacteria, viruses, nematodes, and insects. In this review, we discuss the current knowledge of chitosan’s antimicrobial and insecticidal potential with recent updates. These effects are further explored with the possibilities of chitosan’s active correspondence with phytohormones such as jasmonic acid (JA), salicylic acid (SA), indole acetic acid (IAA), abscisic acid (ABA), and gibberellic acid (GA). The stress-induced redox shift in cellular organelles could be substantiated by the intricate participation of chitosan with reactive oxygen species (ROS) and antioxidant metabolism, including hydrogen peroxide (H2O2), superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD). Furthermore, we propose how chitosan could be intertwined with cellular signalling through Ca2+, ROS, nitric oxide (NO), transcription factors (TFs), and defensive gene activation.


Introduction
Chitosan is a polysaccharide derived from the second most abundant natural biopolymer on Earth, i.e., chitin. It is a linear cationic polysaccharide that is made up of b-(1,4)-joined N-acetyl-D-glucosamine (GlcNAc) and D-glucosamine (GLcN) units. This biopolymer is obtained commercially by the N-deacetylation of chitin from the crustacean exoskeleton. Some mushrooms, green algae, and yeasts can also biosynthesise it. The chemical structure of chitosan can be determined through three characteristics: the degree of polymerisation (the length of the polymer), the degree of acetylation (the percentage of acetylated units), and the pattern of acetylation (the sequence of GLcN and GlcNAc units) (Shokri et al., 2021). The chemical properties of chitosan depend on the degree of deacetylation (DDA), i.e., the extent to which amine groups have substituted N-acetyl groups in chitin (Mukarram et al., 2022). The more deacetylated the product, the more positive the charges and the higher the solubility under acidic conditions. The less deacetylated the chitosan, the higher the solubility under neutral and alkaline conditions (Aranaz et al., 2021;Linhorst et al., 2021). Figure 1 depicts the chemical structure of chitosan and its common derivatives. Chitosan has several excellent chemical and physical properties that make it useful for many applications, e.g., biocompatibility, biodegradability, and antibacterial activity. These traits have led to using chitosan in various areas, including agriculture, food science, medicine, paper science and technologies, and environmental sciences (Morin-Crini et al., 2019). It also is used as a chelating agent because of its ability to bind with cholesterol, fats, proteins, and metal ions. In agriculture, chitosan has been used as a natural pesticide and plant growth agent due to its ability to improve plant growth and tolerance to environmental stresses. It effectively increases the yield and quality of crops such as vegetables, fruit, and ornamental plants. Chitosan has been used as a wound-healing promoter in medicine due to its ability to stimulate tissue regeneration and antibacterial properties (Croisier and Jeŕome, 2013;Wang et al., 2020). In the food industry, chitosan has been used as a food stabiliser due to its ability to prevent food spoilage and improve the texture and stability of food products. In general, chitosan's unique chemical structure and properties make it a versatile and valuable material with many applications (Ibrahim and El-Zairy, 2015;Rahman and Goswami, 2021;Shahrajabian et al., 2021;Gal et al., 2023).
Chitosan has been shown to act as a growth elicitor in plants, meaning that it can stimulate plant growth and improve crop yields. It could be due to the ability of chitosan to mimic the effects of plant growth regulators such as auxins and stimulate biosynthesis of other regulators such as ethylene (Pichyangkura and Chadchawan, 2015). In addition, chitosan has been shown to improve plant tolerance to environmental stresses such as drought, extreme temperatures, salinity, and heavy metal toxicity by enhancing the plant's antioxidant defence system (Hidangmayum et al., 2019). The effectiveness of chitosan as a plant growth elicitor is significantly correlated with its chemical structure. The degree of polymerisation, degree of acetylation, the pattern of acetylation, and monomer unit sequences are all essential structural factors (Hosseinnejad and Jafari, 2016;Fan et al., 2023). Applying chitosan to plant roots, leaves, or seeds can increase plant height, root length, biomass production, and the improvement in crop yields. Chitosan has been shown to improve crop quality, including increased fruit size, vitamin and mineral content, and shelf life (Chamnanmanoontham et al., 2015;Faqir et al., 2021). In addition, it has also been used in the bioremediation of contaminated soils, as it can help to decrease the levels of heavy metals and other toxic ingredients in the soil. In general, the ability of chitosan to act as a growth elicitor in plants makes it a valuable compound for improving crop yields and quality and promoting sustainable agricultural methods (Faqir et al., 2021;Ingle et al., 2022;Kugarajah et al., 2023).

Chitosan against plant pathogens and pests
Chitosan has been used as an artificial plant defence elicitor for over two decades. It has been tested in various economically essential crops against various plant pathogens (Rabea et al., 2003). Many laboratory studies have investigated the correspondence of chitosan with pathogens and showed that it has a critical role in the triggering plant defence against microbes (fungi, oomycetes, bacteria, and viruses), nematodes, and insect herbivores (El Hadrami et al., 2010;Badawy and Rabea, 2011;Hadwiger, 2013). Chitosan treatment induces several protective reactions inhibiting pathogens spread and providing systemic acquired resistance to plants (Rabea et al., 2003;Chakraborty et al., 2020), including biosynthesis of phytoalexins (soybean) and lignification (antibiotic and antifeeding compounds in plants) (Horbowicz et al., 2009). As discussed, the antimicrobial activity of chitosan depends upon several factors such as degree of polymerisation, type of chitosan (native or modified), the host, pH, molecular weight (MW), degree of acetylation, and climatic conditions (Tsai and Su, 1999). Several studies suggest that chitosan's pentamers and heptamers show higher antifungal activity than larger ones (Rabea et al., 2003). In other forms, antipathogenic activity increases with MW (Kulikov et al., 2006).
Chitosan-induced plant protection can be broadly categorised into direct and indirect defence. Direct defence affects the performance of attacking pathogens, including reduced growth rate (El Hadrami et al., 2010), lower fecundity (larviposition) (Haas et al., 2018), and higher mortality of pests in plants treated with chitosan and its derivatives (Ibrahim et al., 2022), while indirect defence shows enhanced recruitment of natural enemies (Naree et al., 2021;El-Seedi et al., 2022). The chitosan-induced direct defence could be due to the production of antibiotic and defensive compounds, such as reactive oxygen species (ROS), phytoalexins, and phenolic acid (Yin et al., 2012;Waewthongrak et al., 2015;Singh et al., 2017), which affect the physiological status of plant pathogens and emit plant volatiles to repel herbivores (Walling, 2000;Zhang and Chen, 2009).
Moreover, chitosan-treated plants have been found to release a qualitatively and quantitatively different blend of volatile organic compounds (VOCs) compared to untreated plants (Zhang and Chen, 2009;Badiali et al., 2018). The change in quality and quantity of VOCs depends on the concentration and duration of the induced phase of chitosan application (Yin et al., 2012). For instance, chitosan treatment of crop plants altered VOC emission, enhancing indirect plant defence by repelling and/or recruiting the natural enemies of insect herbivores (Obara et al., 2002). In particular, rice leaves treated with chitosan release a high amount of linalool, methyl salicylate (MeSA), (Z)-3-hexen-1-ol, and bcaryophyllene (Cartwright et al., 1977;Bailey, 1982;Kodama et al., 1992;Obara et al., 2002). Exogenous application of chitosan elicits expression of defence-related genes (1-deoxy-D-xylulose 5phosphate synthase (DXS), PaDXS2B, and PaDXS2B) in seedlings of Picea glauca (Lapointe et al., 2001). Similarly, tomato plants treated with chitosan show higher levels of jasmonic acid (JA) than untreated plants (Doares et al., 1995). Encapsulation of geraniol in chitosan controls whitefly (De Oliveira et al., 2018). Chitosan induces resistance in Solanum tuberosum against late blight by accumulating salicylic acid (SA) caused by activation of benzoate-2hydroxylase and hydrolysis of SA conjugates in S. tuberosum (Ozeretskovskaya et al., 2006) (Table 1).

Against fungi and oomycetes
In an increasing population, the control of fungal plant disease has become vital to meet food supply needs. Phytopathogenic fungi are the most common plant pathogens and cause many severe diseases. Over time, the widespread use of synthetic fungicides to combat fungal crop diseases has increased resistance to fungal pathogens (Bagheri et al., 2019). Most vegetable diseases are due to fungal pathogens (Koike et al., 2007). The role of chitosan as a biocontrol agent against fungal pathogens has been studied extensively (Abbey et al., 2019;dos Santos Gomes et al., 2021;Riseh et al., 2023). Chitosan inhibits mycelial growth, sporulation, spore viability, and germination of the fungal pathogens, probably due to chitosan's ability to bind DNA to inhibit RNA synthesis in the target organism (Roller and Covill, 1999). Chitosan imposes fatalities against other fungi and oomycetes, such as Phytophthora cinnamomi, Phytophthora palmivora, Gremmeniella abietina, Cryphonectria parasitica, and Heterobasidion annosum (Kuyyogsuy et al., 2018;Silva-Castro et al., 2018;Matei et al., 2020). Chitosan protected the S. tuberosum plant against late Chemical structure of chitin, chitosan, and its derivatives. Mukarram et al. 10.3389/fpls.2023.1217822 Frontiers in Plant Science frontiersin.org

Macrophomina phaseolina
Inhibition of mycelial growth and spore germination.

Jute (Corchorus olitorius)
Water-soluble (schitosan, crab shell) (>85% deacetylation) 12.5 g/L Hand spray (Chatterjee et al., 2014) Rhizoctonia solani Inhibition of mycelial growth and spore germination. Inhibit the systemic propagation of viral/viroid infection. Accumulation of the virus was less on treated leaves. The resistance may be due to callose content and ribonuclease induction.
A plastic container was used to perform the experiment Chitosan (degree of 90% deacetylation) 0.3% Added into suspension (Zheng et al., 2021a) blight disease by eliciting the induced systemic resistance, pattern recognition receptors, and several other defence-related genes, hormones, and enzymes (Zheng et al., 2021b). Furthermore, it can restrict mycelial growth and spore germination by regulating several genes of metabolism, cell membrane structure and function, and ribosome biogenesis . Chitosan and its derivatives attained a growth inhibition of up to 100% against Phytophthora cambivora and significantly reduced the mycelial growth in Phytophthora plurivora and Phytophthora × alni (Silva-Castro et al., 2018). It could result from enhanced activities of catalase (CAT), peroxidase (POD), polyphenol oxidase, and phenylalanine ammonia lyase (PAL) and the expression of HbPR1, HbGLU, HbASI, and HbCAT genes with chitosan application (Kuyyogsuy et al., 2018). The same study found a positive correlation between chitosan and callose and lignin depositions in Hevea brasiliensis leaves, which could provide additional fungal resistance. Furthermore, Guo et al. (2013) reported that 0.5 mg/mL of chitosan reduced growth in Fusarium oxysporum (15%) and Alternaria solani (57%). Similar effects were observed by Younes et al. (2014), where chitosan effectively suppressed the growth of Aspergillus niger, F. oxysporum, and A. solani.

Against bacteria
The antibacterial activity of chitosan encompasses both discouraging bacterial growth (bacteriostatic) and destroying them (bactericidal). Although the mode of chitosan activity against bacteria is still under discussion, the most accepted model is the electrostatic interaction model governing chitosan binding to the bacterial membrane. The functional NH 2 groups of GlcNAc subunits give chitosan a polycationic nature. The positive moiety binds electrostatically with the negatively charged components of bacterial cell membrane including extracellular polymeric substances and proteins . A few studies noted that chitosan can also bind to DNA and cause extensive nucleic acid degradation (Dananjaya et al., 2016;Wang et al., 2022). Chitosan binding induces permeabilisation of the bacterial cell surface and facilitates intracellular leakage. It degrades bacterial biofilm and causes cell death. However, it all depends on how well chitosan can perforate the plasma membrane, which directly depends on its MW and the bacterial type (Verlee et al., 2017). Several other factors can regulate chitosan's antibacterial potential, such as the ratio of its monomeric units (GlcNAc and GLcN), the DDA, the acetylation pattern, solubility, and environmental effects (Li and Zhuang, 2020). Nonetheless, no substantial relation was found between the source of chitosan to its antibacterial activity. It is observed in several gram (+) and gram (−) bacteria that the minimum inhibitory concentration (MIC) of chitosan (similar MW) decreases with increasing DDA (Verlee et al., 2017). Thus, a higher DDA would mean more effective chitosan against bacteria. Antibacterial activity of chitosan has been noted against several important gram (+) bacteria (such as Staphylococcus aureus, Bacillus cereus, Enterococcus faecalis, Micrococcus luteus, and Bacillus subtilis) and gram (−) bacteria (including Escherichia coli, Vibrio cholera, Shigella dysenteriae, Bacteroides fragilis, and Pseudomonas aeruginosa) (Benhabiles et al., 2012;Younes et al., 2014;Goy et al., 2016;Yadav et al., 2020).

Against viruses
Plant viruses negatively impact plants and cause a wide range of symptoms, including discolouration, distortion of plant parts, and loss of vigour, affecting yield. Chitosan application has been found significantly effective against several viruses, including potato virus X, tobacco mosaic and necrosis viruses, alfalfa mosaic virus, peanut stunt virus, cucumber mosaic virus, potato virus X (PVX), and beans mild mosaic virus (Phaseolus vulgaris) (Pospieszny et al., 1991;Pospieszny, 1997;Chirkov et al., 2001;Faoro et al., 2001). Since chitosan's antiviral activity depends on its MW, low MW chitosan shows higher antiviral activities than high MW. Chitosan application inhibits systemic propagation of viroid (Pospieszny et al., 1991) and induces plant resistance by callose formation and enhanced ribonuclease content. Treated leaves show less accumulation of viruses than the control (Chirkov et al., 2001).

How does chitosan work?
There are several theories regarding the antimicrobial mechanism of chitosan. Goy et al. (2009) proposed three faces of the antibacterial mechanism of chitosan: degradation of the cell wall by ionic surface interaction, inhibition of protein and mRNA synthesis through permeation of chitosan into nuclei of microorganisms, and limitation of nutrient availability for microorganism by the formation of external covering over the plant surface. Another researcher stated that the mechanism of chitosan action is based on the destruction of the cell membrane due to a burst of extracellular components, which has been observed in disrupting the growth of fungi (Xing et al., 2015). Recent studies suggested that chitosan is responsible for the hydrolysis of cell wall components (peptidoglycans), disrupting electrolytic balance and increasing pathogen mortality. The exogenous spray of chitosan induces resistance against insect herbivores. Chitosan has been extensively exploited to improve inducible plant defences against insect herbivores (Gatehouse, 2002). Chitosan treatment enhances plant response locally (around the infection sites) and systemically to alert healthy plant parts. These responses include signal transduction, synthesis of resistance-related compounds such as phytoalexins, pathogenesis-related protein (PR-protein) callose formation, lignification, and synthesis of proteinase inhibitors (Katiyar et al., 2015;Chun and Chandrasekaran, 2019). Evidence shows that chitosan also increases the endogenous 2-oxophytodeinoic and JA levels in many crops, including Oryza sativa (Rakwal et al., 2002). Furthermore, chitosan has been found responsible for activating the octadecanoic acid pathway that enhances the activity of chitinase, glucanase, and lipoxygenase and accumulates phytoalexins (El Hadrami et al., 1997;El Hadrami et al., 2010). Figure 2 proposes a modus operandi for chitosan-induced changes in the soil and plant and how they influence the cellular physiology of stressed plants. Chitosan seems to have intricate crosstalk with several other signalling pathways to confer biotic tolerance. These pathways can include phytohormones (SA, JA, and ET), ROS and antioxidant metabolisms, and other cell signals.

Crosstalk with phytohormones
It is suggested that chitosan induces plant defence through an intricate correspondence with several phytohormones such as JA, abscisic acid (ABA), indole acetic acid (IAA), SA, and gibberellic acid (GA) (Jogaiah et al., 2020;Ji et al., 2022). These phytohormones are critical to inducing defence against several plant pathogens and pests.
Chitosan promotes JA concentration in plant tissues under attack by activating the octadecanoic pathway (Rakwal et al., 2002;Iriti et al., 2009). The pathway is responsible for JA biosynthesis by oxidising linolenic acid and increasing phytoalexin content. Chitosan-induced JA signalling promoted the accumulation of several other secondary metabolites such as glucosinolates and anthocyanins glucosides (Iula et al., 2022). These compounds regulate stress-induced oxidative damage given their ROS scavenging nature. Furthermore, JA can trigger signal transduction and activates defence-related genes against pathogen invasion. Peian et al. (2021) reported that chitosan activated multiple JA-biosynthesis-related genes such as VvLOX, VvAOC, VvAOS, and VvCOI1 to induce production of methyl jasmonate, a vital defence hormone, in Vitis vinifera L. under fungal stress.
ABA-mediated signal transduction is critical for plants to respond to biotic and abiotic stresses Lee and Luan, 2012). Chitosan is reported to stimulate ABA biosynthesis to protect the plant against P. palmivora in H. brasiliensis (Kuyyogsuy et al., 2018). In Fragaria × ananassa ' Fugilia' , TOPLESS-related 3 (TPR3) and HISTONE DEACETYLASE 19 (HDA19) are overexpressed during pathogen attack. These proteins interact with the cell wall and reduce its stability. Nonetheless, both TPR3 and HDA19 were restricted by chitosan integration into such plants while restoring cell wall stability and fruit quality (Peian et al., 2021). Chitosan and ABA treatments induced the expression of HbPR1, HbGLU, HbASI, and HbCAST and the deposition of lignin and callose (Kuyyogsuy et al., 2018). ABA also regulates callose deposition intensity and speed (Flors et al., 2005). Furthermore, the same study (Kuyyogsuy et al., 2018) links chitosan with upregulated NCED activity, a crucial enzyme for ABA biosynthesis. Thus, chitosan and ABA seem to work together to defend plants against stressful scenarios. However, their precise crosstalk is still unclear. Chitosan can activate the pathogen-associated molecular pattern (PAMP) and promote H + and Ca 2+ entry in the cytosol; this activates mitogen-activated protein kinases (MAPKs) and the production of JA, ABA, and phytoalexins (El Hadrami et al., 2010;Iula et al., 2022).
In Helianthus annuus L., chitosan magnified IAA and phenol content during stress (Li et al., 2019;Bakhoum et al., 2020). Similarly, chitosan treatment upregulated IAA and SA content in Arabidopsis thaliana by altering their gene expression patterns in the roots. It resulted in restricted expression of WOX5 in the apical root meristem and arrested root development . Chitosan-induced IAA amassing could be triggered by upregulated genes of the tryptophan-dependent biosynthesis pathway (ami1, aao1, and yuc2) and reduced expression of IAA translocation gene (pin1) (Lopez-Moya et al., 2019).
Similar implications of chitosan treatment were observed in GA biosynthesis. Seed priming with chitosan improved germination rate, lipase activity, and seedling growth through increased GA levels in peanut plants (Zhou et al., 2002). Chitosan was also linked with the enhanced impact of GA on plant physiology in another study with P. vulgaris L. (Pereira et al., 2017). Nonetheless, the understanding of chitosan-GA crosstalk is still preliminary and needs more attention. Proposed modus operandi for chitosan-induced biotic stress tolerance in higher plants. These effects can be identified as indirect (A, B) and direct (C). Indirect effects include chitosan action on soil properties that improve water retention. Improved soil-water content promotes soil microbiota. Such microbiota include several chitinolytic organisms that can dissolve insect exoskeletons by chitinase enzymes (see panel A). Other beneficial soil organisms can promote source-sink potential. A higher nutrient status combined with increased soil-water retention promotes plant innate defence, e.g., antioxidant capacity (see panel B). In contrast, the direct effects of chitosan include cytotoxic action on pathogen/pest cells. Chitosan can cause hydrolysis of the cell wall components. This, along with metal chelation and cation efflux from the cytoplasm, disrupts ROS-antioxidant metabolism in pathogen/pest cells. This could result in cell degradation or death of plant invaders. It is suggested that these direct actions can rely on chitosan's intricate crosstalk with certain phytohormones such as JA, SA, ET, ABA, and GA. A few studies suggested a genomic approach for such adjustments such as activation of defensive genes and TFs (see panel C). Apart from this, other signalling pathways could be facilitating chitosan-induced biotic tolerance in plants. Nevertheless, their understanding is still in preliminary phase and demands more investigation. PSII, photosystem II; CA, carbonic anhydrase; NR, nitrate reductase; ROS, reactive oxygen species; SAR, systemic induced resistance; PAMP, pathogen-associated molecular pattern; NPR1, natriuretic peptide receptor A/guanylate cyclase A; SA, salicylic acid; JA, jasmonic acid; ET, ethylene; GA, gibberellic acid; ABA, abscisic acid; TFs, transcription factors.

Chitosan crosstalk with ROS and antioxidant metabolism
Chitosan triggers signal transduction for phytoalexins production, secondary metabolites, and enzymatic and nonenzymatic antioxidants in defence responses to (a)biotic stresses (Pongprayoon et al., 2022). Chitosan triggered signalling pathways in strawberry fruits during oxidative stresses. It induces chloroplastrelated genes. Peroxiredoxin-ROS scavenger genes have related to the cellular levels of ROS in the signalling networks of the chloroplast (Awad et al., 2015). Chitosan application in arbuscular mycorrhizal (AM) tomato (Solanum lycopersicum L.) promoted the growth of plants. Among the possibilities to evaluate the influence of chitosan on tomato growth and flowering may be its anti-transpirant properties to activate ROS scavenging to increase stomatal conductance and xylem vessel growth. In addition, applying chitosan to leaves increased plants' photosynthesis rate and consequently improved plant growth and development (El Amerany et al., 2020). Plants treated with chitosan developed an increased capacity to produce enzymatic antioxidants such as CAT, POD, superoxide dismutase (SOD), and glutathione reductase (GR) to mitigate the effects of oxidative stress in salinity stress (Alkahtani et al., 2020). Chitosan's involvement with vide supra specialised molecules has been reported in several plant species under various stresses. Chitosan stimulates the activity of several defence-related enzymes such as POD in Prunus persica L. fruits or Phoenix dactylifera L. roots (Abdellatef et al., 2022). Chitosan treatment can amplify PAL activity as well in many crops like Triticum aestivum, V. vinifera, and O. sativa, resulting in increased levels of phenolic and flavonoid compounds through phenylpropanoid pathway . In V. vinifera, chitosan elevated PAL activity and enhanced the antioxidant defence mechanisms against Botrytis cinerea by upregulated CAT, POD, and SOD activities (Peian et al., 2021).
The foliar application of chitosan encouraged O 2 − scavenging and restricted H 2 O 2 generation and lipid peroxidation to manage stress in sweet peppers (Alkahtani et al., 2020). In white clover, chitosan in dehydration-responsive element-binding protein (DREB) responsive pathway upregulated DREB2, DREB4, and DREB5 genes (Ling et al., 2022). It activated Y2K and Y2SK genes, which encode dehydrins (DHNs) that produce water-stress tolerance. Notably, these genes are critical to stress tolerance and antioxidant defence.

Chitosan crosstalk with signalling molecules
Chitosan activates signalling pathways in cells by binding to specific cellular receptors, activating important secondary messengers such as Ca 2+ , nitric oxide (NO), ROS, and transcription factors (TFs) (vide supra section 4.2 for ROS). These molecules play a critical role in triggering several biochemical responses. It is worth noting that chitosan, along with Ca 2+ treatment, increases Ca 2+ influx into the cytosol. This elevated cytosolic Ca 2+ level is associated with enhanced callose formation. Notably, chitosan without the Ca 2+ application did not form callose, suggesting that chitosan-induced callose synthesis depends on the presence of Ca 2+ (Koühle et al., 1985).
Similarly, NO plays a critical role in diverse vital physiological phenomena and provides defence against stress scenarios (Corpas et al., 2001;Kolbert et al., 2019). Chitosan promotes the generation of NO and phosphatidic acid. Nonetheless, it inhibits the phospholipase-mediated signalling pathway in the presence of an NO scavenger. Thus, it seems plausible that chitosan elicits defence responses in a NO-dependent pathway (Tocci et al., 2011). Moreover, chitosan can regulate photosynthesis and stomatal movement (Mukarram et al., 2023) in NO-dependent signalling, considering NO has critical roles in stomatal movement in stressed plants (Garcııá-Mata and Lamattina, 2001;Neill et al., 2008).
Many chitosan-modulated genes, including defence-related genes and TFs related to signalling pathways, are involved in biotic stress responses. Povero et al. (2011) demonstrated that several WRKY genes of A. thaliana respond to chitosan treatment. WRKY gene family is attributed to defending against pathogens or pathogen-mimicking stimuli. Among these chitosanelicited WRKY TFs, At5g13080 (WRKY75), At3g01970 (WRKY45), At2g46400 (WRKY46), and At4g31800 (WRKY18) are specifically involved in pathogen responses. Chitosan also overexpressed other MYB TFs such as MYB31 (At1g74650) and MYB15 (At3g23250). The modulation of these TFs by chitosan highlights its impact on regulating biotic stress responses and emphasises its potential as a valuable tool in enhancing plant defence mechanisms.
Chitosan microparticles (CS-MPs) or nanoparticles (CS-NPs) are even smaller chitosan derivatives than COSs. CS-MPs are produced from the aqueous solution of chitosan mixed with dilute acid. The resulting suspension loses its aqueous phase at low pressure and forms microparticles. CS-MPs formed from low chitosan concentration can stabilise Pickering emulsion (oil-in-water) (Mwangi et al., 2016). This opens new perspectives for optimising stimulus-responsive emulsion and their stable storage. Several studies with CS-MPs or CS-NPs concluded their enhanced beneficial role in agriculture, food storage, and biomedical sectors over chitosan polymers (Mwangi et al., 2016;Iglesias et al., 2019;Malerba and Cerana, 2020). Further, CS-MPs and CS-NPs have special relevance in vaccine delivery, inflammatory diseases, and cancer treatment (see Prabaharan and Mano, 2004;Naskar et al., 2019). These bioactivities could also be true against microbial and pest communities. CS-NPs exhibit cytotoxic effects against many gram (+) and gram (−) bacteria, e.g., E. coli, Staphylococcus choleraesuis, Staphylococcus typhimurium, and S. aureus (Qi et al., 2004). Ahmed and Aljaeid (2016) suggested the crucial roles of CS-MPs and CS-NPs with the influenza vaccine, cholera toxin, hepatitis B surface protein, and antigen protein against several other fungi and viruses. Integrating CS-NPs with certain metals, phenolics, and essential oils produces enhanced antioxidants and scavenging activities against microbes (Hasheminejad et al., 2019;Fahimirad et al., 2021;Rashki et al., 2021). This makes chitosan particles an ideal candidate for encapsulating agents in the food packaging and preservation industry as well as a delivery vehicle for bioactive compounds such as nutrients, essential oils, vitamins, and antioxidants (Hasheminejad et al., 2019;Maleki et al., 2022).

Conclusion and perspectives
The past few decades have witnessed exponential growth in chitosan studies in several aspects of crop defence to biomedical applications. It is established now that chitosan boosts plant development and yield during optimal and stressful environments. Nonetheless, there is a vast gap in the molecular understanding of chitosan and its derivatives. In particular, a comprehensive cognition of chitosan's crosstalk with phytohormones, antioxidants, and signalling molecules is lacking. It is pertinent for future studies to explore the signalling potential of chitosan itself. Although our knowledge of chitosan's antimicrobial potential has widened over the past two decades, multiple inconsistencies and a well-defined mechanism must be solved. Therefore, another aspect worth exploring is the modus operandi of chitosan against microbial entities such as fungi, bacteria, and viruses. Recent advances in structural modification in chitosan conferred superior results over chitosan. It could be interesting to know whether these modified chitosan oligomers or nanoparticles adopt different pathways or signalling molecules for enhanced bioactivities.

Author contributions
All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.

Funding
This work was supported by funding from the Slovak scientific grant agency VEGA (1/0117/22 and 1/0108/23).

Acknowledgments
Figures 1 and 2 were created with BioRender.com.

Conflict of interest
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.

Publisher's note
All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Aranaz, I., Alcańtara, A. R., Civera, M. C., Arias, C., Elorza, B., Heras Caballero, A., et al. (2021). Chitosan: An overview of its properties and applications. Polymers 13, 3256. doi: 10.3390/polym13193256 Awad, J., Stotz, H. U., Fekete, A., Krischke, M., Engert, C., Havaux, M., et al. (2015). 2-cysteine peroxiredoxins and thylakoid ascorbate peroxidase create a water-water cycle that is essential to protect the photosynthetic apparatus under high light stress conditions. Plant Physiol. 167, 1592Physiol. 167, -1603Physiol. 167, . doi: 10.1104 Badawy, M. E. I., and Rabea, E. I. (2011). A biopolymer chitosan and its derivatives as promising antimicrobial agents against plant pathogens and their applications in crop protection. Int. J. Carbohydr. Chem. 2011, 1-29. doi: 10.1155/2011