Initial classifications of nsLTPs were based on molecular mass and are categorized into two main types: Type I nsLTPs, which are roughly 9 kDa, and Type II nsLTPs, at around 7 kDa (Kader, 1996). nsLTPs are defined by a conserved eight-cysteine motif (8CM), crucial for their lipid-binding function (Madni et al., 2020; Wang et al., 2012; José-Estanyol et al., 2004). This 8CM, typically arranged as C-Xn-C-XnC-C-Xn-C-Xn-C-Xn-C-C (where ‘C’ is cysteine and ‘Xn’ is a variable number of amino acids), forms a hydrophobic cavity lined by hydrophobic amino acid side chains.

Classifications were later expanded upon by several bioinformatics labs (Boutrot et al., 2008; Santos-Silva et al., 2023; Wang et al., 2012; Amador et al., 2021; Fleury et al., 2019). A side-by-side comparison of the key criteria used in these evolving classification systems, from Kader (1996) to Huang et al. (2023), is provided in Supplementary Table S1 . Boutrot et al. (2008) proposed nine types (I-IX) based on sequence similarity and cysteine spacing, with subsequent additions including groups X and XI (Liu et al., 2010). The major classification differences between Type I and Type II nsLTPs are shown in Table 1 .

nsLTPs have been found to accommodate a diverse group of lipids such as fatty acids, phospholipids, and sterols (Salminen et al., 2016; Fleury et al., 2019). nsLTPs have also been found to bind to other hydrophobic molecules. This ability to bind a variety of lipids in addition to other diverse ligands further emphasizes the versatility of nsLTPs and their involvement in a wide range of cellular processes as highlighted in Table 2 .

Type I nsLTPs are characterized by a conserved disulfide bond pattern, typically C1-C6, C2-C3, C4-C7, C5-C8 (Wang et al., 2012; Liu et al., 2015). This arrangement underpins a single, elongated, tunnel-like hydrophobic cavity that is highly conducive to binding and transporting a single lipid molecule. Proteindocking simulations indicate that ligands often lack a preferred orientation within these cavities, with hydrophobic interactions strongly dominating the protein-ligand interface (Fleury et al., 2019).

In contrast, Type II nsLTPs exhibit a different set of disulfide linkages, commonly C1-C5, C2-C3, C4-C7, C6-C8 (Liu et al., 2015). This altered bonding leads to a markedly different internal architecture, often featuring two adjacent hydrophobic cavities or a more triangular conformation (Liu et al., 2015). These variations suggest that Type II nsLTPs may accommodate different types or multiple ligands, or bind through distinct mechanisms. Molecular dynamics (MD) simulations have revealed the importance of specific amino acid residues for binding particular fatty acids, such as myristic acid and oleic acid in Ajwain [(Trachyspermum ammi)] nsLTP1 (Nazeer et al., 2019). Furthermore, subtle sequence differences, particularly in loop regions like H1–H2 and H1, influence ligand binding modes, as observed between barley HvLTP1.1 and maize ZmLTP1.6 (Salminen et al., 2016). The orientation of the ligand cavity entrance differs between the two folds. In Type I fold, it’s along an axis perpendicular to the C-terminal loop, while in Type II fold, it’s approximately parallel to it (Fleury et al., 2019).

The eight cysteines form four disulfide bridges, stabilizing the compact, α-helical rich fold and maintaining cavity integrity, facilitating lipid encapsulation and transfer. The N-terminal signal peptides often direct nsLTPs to the extracellular apoplast via the secretory pathway, or other compartments such as the endoplasmic reticulum, mitochondria, chloroplast, or vacuoles, depending on the type of nsLTP and associated specific signal peptide (Levesque-Tremblay et al., 2009; Nishimura et al., 2008; Li et al., 2024; Santos-Silva et al., 2023).

The nsLTP superfamily has been organized into at least eleven types based on a combination of features, including molecular mass, sequence homology, the spacing of cysteines within the 8CM domains, posttranslational modifications, and subcellular localization (Edstam et al., 2011; Boutrot et al., 2008; Santos-Silva et al., 2023) as shown in Supplementary Table S2 . These classifications align with clear structural distinctions described above. While all nsLTPs share a conserved right-handed superhelix fold, a fundamental structural dichotomy exists between the “Type I fold” and the “Type II fold.” This divergence is driven by differences in amino acid sequence and disulfide bond patterns, which result in significant variations in their 3D architecture, internal cavities, and ligand-binding characteristics (Fleury et al., 2019).

The classification and understanding of nsLTPs are being revolutionized by advanced computational tools. The European Bioinformatics Institute’s InterPro database (Blum et al., 2024) is an invaluable bioinformatics resource, unifying protein classification by integrating predictive signatures from various member databases like Pfam, SMART, PROSITE, CATH-Gene3D, and SUPERFAMILY. This approach mitigates redundancy and provides crucial insights into sequence conservation and predicted features. For instance, InterPro’s largest nsLTP family, “Bifunctional inhibitor/plant lipid transfer protein/seed storage helical domain” (IPR016140), comprises over 43,000 proteins, 362 domain architectures, and over 2,000 taxons. It also integrates data from 75 PDB solved structures and over 27,000 AlphaFold 3 predicted structures, partly based on PROSITE DOC (PDOC00516) and the PLANT LTP signature (PS00597): [LIVM]-[PA]-x(2)-C-x(1,2)-[LIVM]-x(1,2)-[LIVMST]-x-[LIVMFY]x(1,2)-[LIVMF]-[STRD]-x(3)-[DN]-C-x(2)-[LIVM].

Beyond broad databases, custom scripting, particularly in Python within the Anaconda environment (Anaconda, 2016), offers unparalleled flexibility for detailed sequence analysis. This enables researchers to precisely identify and categorize disulfide bond linkages (e.g., C1-C6, C2-C3, C4-C7, C5-C8 for Type I versus C1-C5, C2-C3, C4-C7, C6-C8 for Type II nsLTPs) in a flexible and scalable manner in addition to looking for novel patterns. For instance, by extending the 8CM spacing from n = 8–30 to n = 8–50, a new class of nsLTPs was identified in algae (Huang et al., 2023). Algal nsLTPs have divergent 8CM spacing and binding pocket residue property differences compared to land plants.

The use of the RapGreen tool developed by the de Lamotte research group was used to organize the nsLTPs superfamily into an interactive phylogenetic tree which and included phylogenetic, structural, and both Plant Ontology (PO) and Gene Ontology (GO) information (Fleury et al., 2019; Dufayard et al., 2021). The RapGreen phylogenetic analysis of 797 nsLTPs can be explored using the following website https:// phylogeny.southgreen.fr/treedisplay/index.php?data=msdmind . The seven broad nsLTP classes in this phylogenetic tree range include Type I (409 nsLTPs) and Type II (118 nsLTPs) and range to Type VIII nsLTP. This type of analysis can be applied to new nsLTPs which are identified in protein databases and with advanced deep and machine learning tools (e.g., UniProt, RCSB Protein Data Bank (RCSB PDB), and AlphaFold). Frequently Aligned Symbol Tree (FAST) and Structural Trace Analysis (STD), revealed additional information about amino acid residues that might confer functional specificity in defense but require additional study to connect with protein function (Fleury et al., 2019).

The advent of AlphaFold for highly accurate 3D protein structure prediction (Jumper et al., 2021) has provided an unparalleled dataset of predicted structures, complementing laborious experimental methods. Tools like the RCSB PDB’s Pairwise Structure Alignment tool (Bittrich et al., 2024), utilizing algorithms such as TM-align (Zhang and Skolnick, 2005), efficiently compare predicted and experimentally determined structures. This allows for robust identification of structural homologs, revealing subtle variations in folds, loop regions, and cavity architectures that refine existing classifications and can identify novel structural subgroups (Gonçalves et al., 2024a; Morales-Quintana et al., 2024). As an example of this, we downloaded the 12,880 proteins (in FASTA format) classified in the Plant non-specific lipid-transfer protein (IPR000528) group from InterPro and removed the 981 redundant sequences using FASTA file manipulation tool seqkit2 (Shen et al., 2024). A python script (McLaughlin, 2025) was used to identify 8CM motifs revealing 11,861 individual proteins with one 8CM domain, 35 proteins with two 8CM domains (UniProt format: A0A067JWZ8, A0A0D3HHF7, A0A251VQ94, A0A498ILS8, A0A498J7V5, A0A4D6NFG6, A0A4S8IBN1, A0A4Y1RRI6, A0A5B6W1L1, A0A6P5FSB9, A0A7J6HF70, A0A7J6HFY0, A0A7J7KVB4, A0A7J7MH43, A0A803MFK2, A0A834TCD7, A0A835KDA7, A0A835KME6, A0A8J5YXI7, A0A8J5ZEW1, A0A9D3VEA1, A0A9D5CJ42, A0A9E7F1U6, A0A9E7F2M4, A0A9J5YS56, A0A9Q0F1H8, A0AA38W3R9, A0AA88R1U2, A0AAD5D2I7, A0AAD6QXP8, A0AAD6WBZ6, A0AAP0LW68, A0AAQ3STU6, A0AAV7H7B0, and A0ABD3GH35), two proteins with three 8CM domains (A0AAD8S0M5, A0A6N2MXE4), and one protein with four 8CM domains (A0A0D3HQM8). Multi 8CM domain proteins have been detected before within the nsLTP family (Edstam et al., 2011). In that work, four proteins (MpLTPg2 from Marchantia polymorpha and PpLTPg1, PpLTPg5, PpLTPj5 from Physcomitrella patens) were found to have two 8CMs, while one protein (PpLTPg7 from P. patens) was detected with three 8CMs.

A bioinformatic analysis of the UniProt database using a custom Python script identified two novel multidomain nsLTPs in African wild rice (Oryza barthii). These proteins, A0A0D3HHF7 and A0A0D3HQM8, were found to possess two and four eight-cysteine motif (8CM) domains, respectively. AlphaFold3 structures are available for these novel proteins. For comparison, the AlphaFold3 structures for a single 8CM domain (A0A0D3GPP4) (A,E) and a GPI-anchored, single 8CM domain (A0A0D3FEW3) (B,F), a two 8CM domain (A0A0D3HHF7) (C,G), and a four 8CM domain protein (A0A0D3HQM8) (D,H), all from Oryza barthii, are shown in Figures 1A–D , which represent AlphaFold3 predicted structures with predicted Local Distance Difference Test (pLDDT) scores. The pLDDT score is a per-residue confidence metric that indicates how closely a predicted protein structure is expected to match its experimentally determined 3D structure. Figures 1E–H represent the same structures but highlighting a domain defined by The Encyclopedia of Domains (TED). The TED domain represents a systematically identified and classified protein domain within the AlphaFold Protein Structure Database (Lau et al., 2024). To confirm lipid binding activity of these novel proteins, an assay using recombinant versions of these proteins challenged against PIP (phosphoinositides) strips could be used to identify potential protein-lipid interactions (Shirey et al., 2017). The nsLTP classification systems are summarized in Table 3 .

Furthermore, the integration of advanced molecular docking programs and MD simulations (Paggi et al., 2024; Neubergerová and Pleskot, 2024) allows for atomic-level modeling of nsLTP-ligand interactions. These simulations quantify interaction energies, identify critical binding residues, and explore ligand dynamics (Fleury et al., 2019; Nazeer et al., 2019; Salminen et al., 2016). This capability is invaluable for correlating structural variations with specific ligand preferences, adding a functional dimension to structural classification. In essence, the synergistic application of comprehensive bioinformatics databases (e.g., InterPro), flexible Perl/Python scripting, AlphaFold’s structural predictions, advanced structural comparison tools (e.g., TM-align), and sophisticated molecular docking/MD simulations enables a more granular and functionally relevant classification of nsLTPs. Future nsLTP classifications can take advantage of these tools and the accumulated knowledge built up from sequence and structural information that is available in UniProt and AlphaFold databases. The PDB Pairwise Structure Alignment tool, for instance, enables the simultaneous alignment of up to 10 protein 3D structures (Bittrich et al., 2024). This provides an excellent method to compare to Type I, Type II, GPI-anchored nsLTPs, and other nsLTPs in a single tool providing alignment quality structure-based scores, such as Root-Mean-Square-Deviation (RMSD, measured in Angstroms)˚ and TM-score (ranging from 0.00 to 1.00), alongside sequence-based metrics like percent identity and the number of aligned residues. This multidisciplinary computational approach will accelerate the discovery and characterization of novel subfamilies, providing a more complete picture of their diverse roles in plant physiology and defense.

The most complete discussion of the relationship between nsLTP structure and plant defense is given by the de Lamotte laboratory (Fleury et al., 2019; Dufayard et al., 2021). They utilized a comprehensive approach combining phylogenetic and structural information to classify nsLTPs and investigate their involvement in defense mechanisms. To understand nsLTP function and variability across the superfamily, researchers analyzed a large dataset of 797 nsLTP protein sequences, which included both experimental 3D structures, including X-ray crystallography, NMR spectroscopy, cryo-electron microscopy data, and computer modeling. Type I nsLTPs formed a well-supported monophyletic group and predominated, making up over half of their dataset (417 out of 797 sequences). Type II nsLTPs were the second most abundant, with 126 sequences. Using the structural information available with these sequences, the study then classified the nsLTPs into two distinct structural categories: the Type I fold and the Type II fold. As expected, phylogenetic Type I nsLTPs were consistently found to have the Type I fold.

The Type I family is the predominant group for defense-related nsLTPs, encompassing 28 proteins that have been functionally classified as defense-related. In contrast, the Type II group contained only 3 classified defense-related nsLTPs, including the known protein AtDIR1 (Q8W453). This skewed distribution strongly suggests that Type I nsLTPs play a more significant role in plant defense (Fleury et al., 2019). Further experimental validation is required to refine the functional classification of Type I and Type II nsLTPs, many of which currently lack specific GO annotations.

Investigation of the evolutionary history of nsLTPs can provide valuable insights into their functional diversity and role in plant adaptation to different environments and pathogens. nsLTPs are found in all land plants and recently in green algae (Edstam et al., 2011; Edqvist et al., 2018; Huang et al., 2023). The diversity of nsLTP subfamilies is more limited in non-seed plants compared to seed plants, suggesting that new nsLTPs may have evolved during land plant evolution (Edstam et al., 2011). Phylogenetic analyses indicate that nsLTPs have undergone both tandem and segmental duplications, contributing to their functional diversity (Edstam et al., 2011; Wei et al., 2025). The adoption of novel nsLTP types likely assisted plants in adjusting to the harsh new environments and disease pressures.

Differences in nsLTPs between plant species and within families have been revealed via structural analysis and bioinformatic comparisons. For example, a survey of nsLTPs in rice and Arabidopsis thaliana revealed 52 rice nsLTPs and 49 Arabidopsis nsLTPs (Boutrot et al., 2008). The authors employed comparative genomics, using the identified rice nsLTPs as a basis, to identify 156 putative nsLTPs in wheat. The nsLTP gene family in maize (Zea mays) includes 65 genes, which can be divided into six types (1, 2, C, D, G, and a unique type X), each with distinct expression patterns and functions (Fang et al., 2023). Similarly, in Brassica rapa, 63 nsLTP genes were identified and grouped into nine types (I, II, III, IV, V, VI, VIII, IX, and XI), with specific roles in defense, reproduction, and stress responses (Li et al., 2014). In barley, 70 nsLTPs were classified, based on phylogeny, protein characteristics and gene structures, and placed in groups 1, 2, C, D, and G (Zhang et al., 2019). This diversity highlights the extensive expansion and functional specialization of the nsLTP family across the plant kingdom. Supplementary Table S2 provides a comprehensive overview of the number, types, and key characteristics of nsLTPs identified in representative species, from green algae and bryophytes to angiosperms like rice and A. thaliana. Research into barley nsLTPs has been extensive due to the connections with the brewing industry as these proteins significantly influence key beer quality attributes such as foam stability, head retention, haze formation, and flavor stability (antioxidant capacity) during storage (Cai et al., 2019; Stanislava, 2007; Wu et al., 2011; Douliez et al., 2001).

The adaptation and evolution of nsLTPs in response to different pathogens or environments are driven by selective pressures, including those from pathogens. For instance, nsLTPs involved in pathogen defense have evolved to recognize and respond to specific PAMPs, impacting the plant’s immune response. A good example of this was shown by the work of Situ et al. (2024). The oomycete Peronophythora litchii secretes a pectin acetylesterase (PlPAE5), which was found to destabilize the litchi (Litchi chinensis) lipid transport protein (LcLTP1), reducing salicylic acid (SA) production and promoting infection. This interaction indicates that pathogens target LTPs to disrupt key plant defense signaling pathways. The interaction between PlPAE5 and LcLTP1 exemplifies a counter-defense strategy, where the pathogen interferes with the plant’s defense mechanism.

Additionally, nsLTPs have adapted to various environmental stresses, such as drought and salinity, by modulating lipid composition and signaling pathways (Xiao et al., 2023; Fang et al., 2023; Santos-Silva et al., 2023; Riahi et al., 2021). With their varied structures and evolutionary adaptations, nsLTPs play critical roles in plant defense, development, and stress responses. Investigating their molecular mechanisms and evolutionary history reveal valuable insights into their functions and potential applications in agricultural biotechnology.

As detailed in the nsLTP structure section, the highly conserved 8CM, which forms four disulfide bridges, stabilizes nsLTPs. This stabilization is crucial for maintaining the integrity of the hydrophobic cavity, a feature that allows these proteins to bind and encapsulate lipid molecules (Wang et al., 2012). The lipid-binding process begins when lipid molecules interact with the hydrophobic cavity of nsLTPs. The internal cavity, lined with hydrophobic amino acid residues, creates an environment conducive to the stable binding of lipid molecules. Key amino acid residues involved in lipid binding include hydrophobic residues such as leucine, isoleucine, valine, and phenylalanine, which interact with the lipid tails (Madni et al., 2020). Mutational and modeling approaches have been used to define key residues for both protein stability and antimicrobial properties (Fleury et al., 2019; Gonçalves et al., 2024a).

The structure of the hydrophobic cavity significantly influences lipid specificity. The cavity’s size, shape, and flexibility determine which lipid molecules can be accommodated (Reinisch and Prinz, 2021; Missaoui et al., 2022; Sawano et al., 2008). For example, the hydrophobic cavity of nsLTPs can adjust its volume to accommodate various ligands, ranging from C10 to C18 fatty acids (Han et al., 2001; Liu et al., 2015). This structural plasticity allows nsLTPs to bind a wide array of lipid molecules, including multiple lipids simultaneously, contributing to their non-specific binding nature (Wang et al., 2012; Sodano et al., 1997; Charvolin et al., 1999; Wong et al., 2019; Douliez et al., 2001). For example, Figure 2 presents a Pymol rendering of Solanum melongena nsLTP determined by X-ray crystallography which demonstrates the presence of two lauric acid molecules sequestered within the protein’s hydrophobic cavity (Madni et al., 2023).

The hydrophobic cavity’s ability to shield the hydrophobic tails of lipid molecules during transfer is essential for preventing the exposure of these tails to the aqueous cellular environment, thereby facilitating efficient lipid transport (Avula et al., 2021). The binding sites of nsLTPs exhibit such flexibility that they have even been shown to accommodate volatiles in Petunia hybrida (Liao et al., 2023). This research provides mechanistic insights into how hydrophobic compounds, including volatiles, cross the hydrophilic cell wall. Recent advancements have unveiled the molecular details of lipid transport by yeast LTP VPS13, providing crucial insights into its function (Adlakha et al., 2022; Melia and Reinisch, 2022; Park et al., 2016; Neiman, 2024). This work revealed that VPS13 forms a bridge containing a hydrophobic channel at membrane contact sites to facilitate bulk glycerolipid transport between organelles. This bridge-conduit model is highly relevant for plants, where proteins like ATG2 likely employ a similar mechanism to mediate the large-scale lipid flux required for processes such as autophagy-related membrane biogenesis and constitutive inter-organelle lipid homeostasis (Osawa et al., 2019; Valverde et al., 2019; McEwan and Ryan, 2022). These lessons from model organisms such as yeast provide important clues into lipid transport mechanisms of actions in plants.

nsLTPs facilitate non-vesicular lipid transfer, which is distinct from the energy-dependent vesicular transport (Wong et al., 2017). The energetics of lipid transfer by nsLTPs involve both enthalpic and entropic contributions (Wong et al., 2019). The binding of lipid molecules to nsLTPs is primarily driven by hydrophobic interactions, which release water molecules from the hydrophobic cavity, resulting in a favorable entropic gain (Mouritsen, 2013). nsLTPs and LTPs function by reducing the energy barrier for lipids to leave bilayers, as the lipid is transferred into the hydrophobic cavity of the protein rather than into the aqueous phase (Reinisch and Prinz, 2021). The formation of disulfide bridges within the 8CM also contributes to the stability of the lipid-protein complex, providing an enthalpic contribution to the binding process (Maldonado et al., 2002). In general, metabolic energy is not required for LTPs to transfer lipids. LTPs facilitate lipid transfer by lowering the energy needed for a lipid to detach from a membrane. This occurs because the lipid enters the protein’s hydrophobic cavity rather than the less favorable aqueous environment (Reinisch and Prinz, 2021). Active lipid transport, particularly against a concentration gradient, necessitates energy. ATP-binding cassette (ABC) transporters are often involved in these processes, using ATP hydrolysis to power the movement of lipids (Christensen and Kolomiets, 2011; Roston et al., 2012; Sakuragi and Nagata, 2023; Norris et al., 2024).

Mechanistically, nsLTPs orchestrate plant immunity through a multifaceted strategy. nsLTPs have been shown to play a role in immune responses against fungi, bacteria, and viruses (Wang et al., 2004; Molina et al., 1993; Ji et al., 2024; Zhu et al., 2023; Zhao et al., 2020; Sun et al., 2008). This includes direct antimicrobial actions, such as disrupting pathogen membranes (Missaoui et al., 2022), a process that may involve permeabilization, pore formation (Regente et al., 2005), and interaction with specific lipids like PA (Madni et al., 2020). They also directly inhibit pathogen growth by interfering with crucial metabolic pathways (McLaughlin et al., 2021; Molina et al., 1993; Jiang et al., 2013; Schmitt et al., 2018), including inhibition of glycosidases (Resende et al., 2023). A nsLTP, Ca-LTP1 isolated from Capsicum annuum is able to inhibit α-amylase in vitro (Bessiatti Fava Oliveira et al., 2025; Kirubakaran et al., 2008), and a nsLTP isolated from Ginkgo biloba can function to inhibit the aspartic acid proteinase, pepsin and the cysteine proteinase papain (Sawano et al., 2008). Finally, nsLTPs integrate with other defense pathways, collaborating with ROS signaling and phytohormone signaling. Their antioxidant properties (Aldakhil et al., 2023; Balmant et al., 2021) are integral to maintaining redox homeostasis and preventing excessive ROS-mediated damage.

Lipids are essential for plant immunity, serving diverse roles beyond their structural function in membranes. They form protective barriers such as the cuticle, composed of cutin and waxes, which prevent pathogen entry and water loss, serving as a first line of defense against pathogens (Zhao et al., 2020; Jacq et al., 2017). Additionally, membrane lipids, including phospholipids and sphingolipids, maintain cellular integrity and are crucial for proper cellular function and immune responses (Dodds et al., 2024; Pretorius et al., 2021; Situ et al., 2024; Sarowar et al., 2009; Guo et al., 2022). Lipids also act as signaling molecules, with phosphatidic acid (PA) and diacylglycerol (DAG) being key examples. PA is a lipid second messenger involved in various stress responses, including the activation of defense-related genes and the production of ROS (Yao and Xue, 2018). DAG plays a role in protein kinase activation and downstream defense signaling pathways (Kalachova et al., 2022). The transport of these signaling lipids by LTPs is crucial for modulating defense responses and intercellular communication. Lipids also serve as precursors for antimicrobial compounds, such as oxylipins and phytoalexins, which combat pathogen infection (Cavaco et al., 2021; Kachroo and Kachroo, 2009). Membrane lipids, such as phospholipids and sphingolipids, are essential for maintaining membrane fluidity and integrity, which are critical for proper cellular function and immune responses. Additionally, certain lipids act as precursors for defense compounds, such as oxylipins and phytoalexins, which are antimicrobial compounds produced by plants to combat pathogen infection (Pretorius et al., 2021; Blée, 1998).

nsLTPs directly support these diverse roles, functioning as important components of plant defense against pathogens (Gao et al., 2022; Kader, 1996; Shah and Chaturvedi, 2009). A survey of the literature highlights the complexity of lipid-mediated plant immunity but also the opportunity to better understand plant-pathogen interactions. Advances in understanding pathogen recognition, signaling pathways, pathogenesis-related proteins, and lipid homeostasis, particularly in relation to nsLTPs, offer a framework for designing bioengineered crops with enhanced resistance to pathogens (Dodds et al., 2024).

nsLTPs are also recognized to play critical roles in the symbiotic relationships between plants and microorganisms, a prime example being the role of nsLTPs in legume–rhizobia symbioses (Zhou et al., 2019; Gao et al., 2022; Gasser et al., 2023; Wei et al., 2022; Chen et al., 2023). In Medicago truncatula (Barrel Medic), the nsLTP MtN5 displays a dual role by acting as an antimicrobial agent for plant defense while also being essential for promoting symbiotic root nodulation with Sinorhizobium meliloti (Pii et al., 2009). The expression level of the MtN5 gene directly dictates the quantity of root nodules, as silencing the gene reduced nodule formation by 50% while overexpressing it resulted in a threefold increase in nodule formation. A nodule-specific nsLTP in Chinese Milk Vetch (Astragalus sinicus), AsE246, was found to play an important role in the symbiosis with nitrogen-fixing bacterium Mesorhizobium huakuii (Lei et al., 2014). Similar to the findings for MtN5 in M. truncatula, the expression level of AsE246 was shown to be a key determinant of symbiotic efficacy. Overexpression of AsE246 promoted an increase in nodule formation while silencing the gene resulted in a suite of symbiotic defects, including reduced nodulation, lower lipid content within nodules, impaired nitrogen fixation, and aberrant symbiosome development. Later, AsE246, was found to directly bind the high temperature protein G (HtpG) from M. huakuii (Zhou et al., 2019), impacting the lipid profile of the root nodules. Gasser et al. (2023) provides a recent review of nsLTPs in nitrogen-fixing symbiosis. Interestingly, the role of nsLTPs in common bean (Phaseolus vulgaris) and soybean (Glycine max) nodule development is not clear. RT-qPCR expression analysis of roots inoculated with rhizobia has shown the upregulation of PvLTPs (PvLTPd.4, PvLTPd.6, PvLTPd.10, and PvLTPg.11) and GmLTPs GmLTPd.1 and GmLTP1.1 but functional studies are needed (Fonseca-García et al., 2021).

The lipid biology underlying plant defense mechanisms is complex and dynamic. During pathogen challenge, plants rapidly remodel their membrane composition, produce signaling lipids, and mobilize antimicrobial lipid compounds (Pretorius et al., 2021). nsLTPs orchestrate many of these responses by ensuring the appropriate trafficking of lipid molecules to their required locations for membrane repair, signal transmission, or direct pathogen confrontation (Santos-Silva et al., 2023; Boutrot et al., 2008; Blein et al., 2002; Buhot et al., 2001; Cheng et al., 2004; de Oliveira Carvalho and Gomes, 2007; Edqvist et al., 2018).

Some nsLTPs exhibit direct antimicrobial activity, functioning as antimicrobial peptides (AMPs) that target and disrupt pathogen membranes (Gao et al., 2022; Santos-Silva et al., 2023; Amador et al., 2021). Molina et al. (1993) provided an early study on the antifungal nature of nsLTPs. nsLTPs purified from barley (Hordeum vulgare) (Cw18 and Cw21) and maize (Zea mays) leaves (Cw41) showed activity against phytopathogenic bacteria and fungi. In vitro assays using a recombinant nsLTP Ltp 3F1 cloned from wheat (Sumai 3), was shown to have broad antifungal properties, including Aspergillus species, Candida species, Fusarium species, Rhizoctonia solani, Pyricularia oryzae, Alternaria species, Botrytis cinerea, Pythium debaryanum, Phytophthora infestans, and Magnaporthe poae (Kirubakaran et al., 2008).

The rice LTP110 protein was shown to directly inhibit fungal spores of P. oryzae (Ge et al., 2002). Using site-directed mutagenesis, the authors showed that the Cys50–Cys89 disulfide bridge was not essential for antimicrobial activity (Ge et al., 2003). Enhanced resistance to Alternaria solani and B. cinerea was observed in transgenic Arabidopsis plants that expressed the TdLTP4 gene (Safi et al., 2015). Further research found that recombinant TdLTP4 protein had a broad antimicrobial spectrum and was effective against bacteria and fungi, including S. aureus, L. monocytogenes, F. oxysporum, F. g. (Hsouna et al., 2021).

McLaughlin et al. (2021) showed that recombinant AtLTP4.4 expressed in Pichia pastoris was able to inhibit F. g. in zone of inhibition assays, possibly by disrupting membranes based on its ability to bind and transfer lipids, leading to alterations in membrane permeability and integrity. Antifungal activity was also noted for the recombinant Brassica rapa BrLTP2.1 protein when tested against F. oxysporum and P. syringae pv. tomato. Site-directed mutagenesis of select cysteine residues, for example, Cys69 of BrLTP2.1 impacted the antifungal nature of the protein (Schmitt et al., 2018). Recently, a potato (S. tuberosum nsLTP, SpLTPa was shown to bind to and disrupt the plasma membrane (PM) of the oomycete pathogen P. infestans but not the PM of the potato cells (Chen et al., 2024). The ability of plant LTPs, including Coffea canephora Cc-LTP1 and Cc-LTP2, to permeabilize the membranes of Candida albicans, a fungal pathogen associated with nosocomial infections, was also observed (Zottich et al., 2011; Bard et al., 2016).

Do nsLTPs specifically interact with fungal membranes? There are multiple lines of evidence that there are direct interactions. The Ha-AP10 nsLTP from common sunflower (Helianthus annuus) was the first nsLTP shown to permeabilize fungal cell membranes, specifically inhibiting F. solani spore germination (Regente and De La Canal, 2000). Ha-AP10 was subsequently shown, using fluorescent probes and a liposome leakage assay, to interact directly with phospholipids and this produces the fungicidal effect (Regente et al., 2005). Using chitin affinity chromatography, chitin-binding nsLTPs were isolated from Capsicum chinense were identified and characterized (Gonçalves et al., 2024b). These proteins were found to have antifungal activity against Candida and Fusarium (Gonçalves et al., 2024a, c). Chen et al. (2024) found that PI(3,5)P2 and PI(3)P phosphoinositides competitively inhibit the binding of StLTPa to fungal plasma membranes, diminishing the inhibitory effect of StLTPa. Madni et al. (2020) also demonstrated that a nsLTP isolated from eggplant (Solanum melongena) can disrupt fungal membranes. They used the SYTOX Green uptake assay, which measures membrane integrity, to show this disruption (characterized as “bleaching” or increased porosity) via the dye entering the fungal cells upon exposure to the nsLTP.

The plant cuticle is composed of a complex mixture of lipids, including cutin and waxes, which create a hydrophobic layer that prevents water loss and protects against pathogen entry. The lipids on the plant surface consist of cutin and waxes (very-long-chain fatty acids (VLCFAs) and their derivatives) (Serrano et al., 2014). Cutin consists of a polymeric network formed by C16 and C18 fatty acids, cross-linked through ester bonds. nsLTPs transport cutin monomers and wax to the plant surface for cuticle assembly and deposition (Jacq et al., 2017; Zhao et al., 2020; Missaoui et al., 2022; Nakamura et al., 2016). nsLTPs facilitate the transport and deposition of lipid molecules essential for cuticle formation by binding and transferring various lipids, such as fatty acids, phospholipids, and glycolipids, from their synthesis sites in the endoplasmic reticulum to epidermal cells. This process ensures a continuous supply of lipids necessary for cuticle development and maintenance. Some evidence of this has come from overexpression studies in plants. For instance, Arabidopsis plants engineered to overexpress an nsLTP from saltwater cress (Thellungiella salsuginea), TsnsLTP4, showed an increase in epicuticular wax deposition (Sun et al., 2015). Likewise, a nsLTP in tomato, SlLTPG3, was found to play a crucial role in transporting cuticular wax and cutin to the tomato fruit surface, contributing to enhanced cuticle thickness and reduced permeability. Overexpression of SlLTPG3 resulted in increased cuticular wax and cutin accumulation, leading to delayed fruit softening and an extended shelf life in tomatoes compared to wild-type plants (Wang et al., 2022).

Another notable example is Arabidopsis LTPG (Type G nsLTP), which is a glycosylphosphatidylinositol-anchored lipid transfer protein identified as crucial for exporting lipids to the plant surface. It plays a significant role in transporting cutin and wax precursors, thereby contributing to the proper formation and integrity of the cuticle. Research by DeBono et al. (2009) highlighted the importance of Arabidopsis LTPG in maintaining cuticle integrity and function. nsLTPs contribute to cuticle development through their hydrophobic cavity, which allows for stable binding and transport of lipid molecules, as well as targeted delivery facilitated by signal peptide sequences that guide these proteins to specific subcellular locations. Disrupting the nsLTP GPI-anchored 1 (LTPG1) gene in Arabidopsis altered the plant’s cuticular lipid composition and ultrastructure, negatively impacting its immunity as shown by increased susceptibility to fungal infection (Alternaria brassicicola) (Lee et al., 2009).

Additionally, nsLTPs play a role in the plant’s defense mechanisms against abiotic stresses such as drought and salinity by reducing water loss and protecting against desiccation (Nakamura et al., 2016). Their antimicrobial properties further reinforce the cuticle’s barrier function against pathogen attack (Kuźniak and Gajewska, 2024). In summary, nsLTPs, including Arabidopsis LTPG, are integral to the development and maintenance of the plant cuticle, enhancing the plant’s ability to withstand environmental stresses and pathogen invasion through the continuous transport of lipid molecules required for cuticle formation.

The broad classes of nsLTP1 and nsLTP2 are known to participate in the transport of phospholipids and glycolipids, contributing to the maintenance of membrane integrity and the formation of lipid-based defense barriers (Aldakhil et al., 2023; Gangadhar et al., 2016; Gasser et al., 2023; Jacq et al., 2017; Schmitt et al., 2018). The integrity of the cuticle is directly linked to plant resistance as a compromised cuticle significantly increases susceptibility to both pathogen invasion and dehydration. Studies have revealed that an nsLTP from Arabidopsis plays a structural role in maintaining the adhesion between the hydrophobic cuticle and the hydrophilic cell wall (Jacq et al., 2017; Hairat et al., 2018). During fungal infection, fungal cutinases release cutin which can activate defense responses (Serrano et al., 2014; Arya et al., 2021). In rice, the application of cutin monomer 16-hydroxypalmitic acid (HPA) induces OsLTP5 expression along with other defense genes (Kim et al., 2008), indicating that plants respond to cuticle damage.

While nsLTPs are traditionally associated with their ability to bind and transport lipids, emerging evidence underscores their versatile role in protein-protein interactions. This protein-binding capability extends beyond simple lipid binding and exchange, offering a direct mechanism for nsLTPs to modulate cellular processes and interfere with pathogen virulence.

A prominent example of nsLTP protein binding involves their interaction with alpha-amylases. Studies have demonstrated that nsLTPs can bind to and inhibit the enzymatic activity of these hydrolytic enzymes (da Silva et al., 2018b; Bessiatti Fava Oliveira et al., 2025; Zottich et al., 2011). This suggests a resistance mechanism against insect alpha-amylases, potentially disrupting their function and thereby inhibiting carbohydrate digestion, which would serve as a potent plant defense mechanism against herbivory (da Silva et al., 2018b; Saxena et al., 2023). Subsequent research using C. canephora Cc-LTP1 and a recombinant Vigna unguiculata Vu-LTP1 confirmed that nsLTPs cause potent inhibition of insect intestinal alpha-amylases, leading to reduced larval development in the cowpea weevil, Callosobruchus maculatus and supporting the role of these nsLTPs as an active defense mechanism against insect attack by disrupting their digestive processes (da Silva et al., 2018a).

The ability of nsLTPs to modulate carbohydrate metabolism could also play a crucial role in plant stress responses to other threats, such as fungal or bacterial challenge, by influencing the availability of energy and building blocks needed for defense or recovery. A recent study found a potential link between nsLTPs, rice germination rates, and carbohydrate levels in seeds. CRISPR knockouts of OsLTPL23 negatively impacted rice germination rates and resulted in significantly lower starch levels and higher soluble sugar levels in the edited seeds, the authors hypothesizing that OsLTPL23 may have alpha-amylase inhibitor activity (Li et al., 2023). Further research is needed to understand the exact role of alpha-amylase inhibition by nsLTPs in plants and if there is a role in disease resistance, specifically how this might affect the availability of sugars crucial for both plant defense and pathogen growth.

More recently, the ability of nsLTPs to directly interact with and modulate pathogen-derived proteins has garnered significant attention. Although a mechanism was not identified, antiviral and antiproliferative activities have been demonstrated for a nsLTP derived from bunch-flowered daffodil (Narcissus tazetta) (Ooi et al., 2008). However, more recent work has shown how nsLTPs exert antiviral action. For instance, the cowpea Vigna unguiculata LTP1 has been shown to bind to and inhibit the proteolytic activity of the cysteine protease encoded by the CPMV (Ji et al., 2024). This interaction directly interferes with a key viral protein necessary for replication and spread, showcasing a novel antiviral defense mechanism mediated by nsLTPs. The implications of this protein-binding capacity extends beyond viral defense. It is plausible that nsLTPs can interact with other pathogen-derived proteins, such as bacterial or fungal effectors, thereby disrupting their virulence functions (Guo et al., 2022). Exploring the diversity of pathogen proteins targeted by nsLTPs is crucial for understanding the full scope of their contribution to plant immunity. Cataloging the contents of nsLTPs in plants using proteomic and lipomics would help better understand the scope of nsLTP involvement in plant disease response.

Lipids are dynamic participants in plant immunity, functioning beyond structural roles to generate potent signaling molecules during plant-pathogen interactions (Li et al., 2024). The lipid landscape itself is a dynamic battleground where plants modify lipids for defense, while pathogens exploit them for survival. Upon perception of microbial threats, plants rapidly remodel their lipidome, producing signaling lipids such as phosphatidic acid (PA), lysophosphatidic acid (LPA), oxylipins like jasmonic acid (JA) and 12oxo-phytodienoic acid (OPDA), and sphingolipids such as ceramides (Kuźniak and Gajewska, 2024; Pretorius et al., 2021). These lipid mediators activate diverse pathways, including ROS production, calcium signaling via phosphoinositides, and programmed cell death, collectively shaping robust defense responses. Sphingolipids, like ceramides, induce programmed cell death (PCD), and glycosyl inositolphosphoryl ceramides (GIPCs) contribute to membrane structure and are known to be bound to nsLTPs (Berkey et al., 2012; Gonzalez-Klein et al., 2021). Phosphoinositides (PIs), such as PI4P and PI(4,5)P2, serve as precursors for second messengers like IP3, regulating calcium signaling (Boss and Im, 2012). Additionally, fatty acid derived signals like N-acylethanolamines (NAEs) modulate stress responses (Chapman, 2004; Blancaflor et al., 2014; Hu et al., 2023). These lipid mediators initiate complex signaling cascades, ultimately leading to defense gene expression.

nsLTPs emerge as central regulators of these lipid-mediated defenses, contributing to antimicrobial lipid delivery, membrane reinforcement, lipid signal transduction (e.g., PA, LPC), and long-distance signaling (e.g., DIR1 in SAR) (Santos-Silva et al., 2023; Amador et al., 2021; Chen et al., 2017; Salminen et al., 2016). They facilitate the targeted movement of signaling lipids, reinforce membrane barriers, and orchestrate long-distance immune communication, notably through SAR (Chen et al., 2017; Finkina et al., 2016; Li et al., 2024). The Arabidopsis DIR1 protein exemplifies this function, mediating the movement of lipid-based systemic signals (Carella et al., 2017; Champigny et al., 2013; Lascombe et al., 2008). Beyond mere transport, nsLTPs actively amplify signaling cascades by mobilizing lipid messengers, including PA and lysophosphatidylcholine (LPC). By controlling the spatial and temporal distribution of these lipids, nsLTPs integrate localized and systemic immune responses.

nsLTPs operate within a broader lipid signaling network through coordinated interactions with lipid kinases, phosphatases, and membrane transporters. By delivering lipid substrates to diacylglycerol kinases and phosphoinositide kinases, nsLTPs potentiate lipid signal amplification, while their collaboration with lipid phosphatases ensures signal resolution (Lev, 2010). In parallel, they complement energy-dependent export mediated by ATP-binding cassette (ABC) transporters, particularly during defense-induced membrane remodeling (Roston et al., 2012). At membrane contact sites (MCS), LTPs contribute to lipid exchange between organelles, linking lipid homeostasis with stress responses (Lahiri et al., 2015; Michaud and Jouhet, 2019; Prinz et al., 2020; Zhang et al., 2022). Thus, nsLTPs and LTPs serve not merely as passive shuttles but as dynamic integrators of lipid signaling, tightly coordinating immune activation and cellular resilience.

nsLTPs play diverse roles in plant defense, as illustrated by the following specific examples. A. thaliana DIR1, a nsLTP, is crucial for SAR by facilitating long-distance transport of lipid-based signals (Champigny et al., 2013). Medicago sativa MsLTP1 exhibits broad-spectrum antimicrobial activity, potentially via a pore-forming mechanism in pathogen membranes (Barashkova et al., 2023). AtLTP4.4 enhances resistance to Fusarium head blight through antifungal and antioxidant activities (McLaughlin et al., 2021). Nicotiana benthamiana NbLTP1 boosts immunity against tobacco mosaic virus (TMV) by upregulating SA biosynthesis and downstream signaling components like NPR1 (Zhu et al., 2023).

nsLTPs interact with pattern recognition receptors (PRRs), triggering pattern-triggered immunity (PTI). They are also involved in activating mitogen-activated protein kinase (MAPK) pathways, crucial for defense signaling, partly by transporting lipid-based secondary messengers like phosphatidic acid (PA). Furthermore, nsLTPs integrate with hormonal signaling (SA, JA, ET), enhancing SA biosynthesis and signaling and modulating JA and ET pathways.

The expression of nsLTP genes is regulated by biotic and abiotic stress factors. PAMP recognition by PRRs initiates signaling cascades that activate defense-related genes, including nsLTPs. Key transcription factors (WRKY, MYB, NAC) bind to specific cis-regulatory elements (e.g., W-box, MYB-box) in nsLTP gene promoters. Post-translational modifications, such as glycosylation and phosphorylation, are also important. Glycosylation can enhance nsLTP stability, activity, and localization, while phosphorylation can alter conformation, affecting interactions and activity.

The nsLTPs often contain N-terminal signal peptides and are directed to specific cellular locations like the extracellular apoplast (via the secretory pathway), endoplasmic reticulum, the chloroplast, or vacuoles (Boutrot et al., 2008; Liu et al., 2010; Wang et al., 2012; Nishimura et al., 2008; Chiu et al., 2020). The presence of numerous nsLTP genes raises questions about functional redundancy. While some nsLTPs show overlapping functions in lipid binding and transport, others have specialized roles in cuticle formation, pathogen defense, or reproduction, potentially providing robust defense. Some nsLTPs exhibit direct antimicrobial activity, while others are crucial for SAR by facilitating long-distance transport of lipid signals or maintaining cuticle integrity. The redox-sensitive nsLTP, LTP-II, was shown to be important for guard cell closure in response to the bacterial protein flg22 (Balmant et al., 2021).

SAR is a long-lasting, broad-spectrum immune response triggered by an initial localized pathogen attack. Following local infection, mobile signals are generated and transported to distal tissues, priming them for enhanced defense upon subsequent pathogen exposure. Lipids, lipid-derived molecules, and lipid-associated proteins, particularly nsLTPs, are key mediators of this systemic immune communication.

Among nsLTPs, Arabidopsis DIR1 is a well-characterized example essential for SAR establishment (Champigny et al., 2013; David et al., 2021; Lascombe et al., 2008; Carella et al., 2017). DIR1 facilitates the movement of lipid-based signals through the plant vascular system. Loss of DIR1 function impairs systemic defenses despite normal local responses. Structural and biochemical studies suggest DIR1 carries lipid molecules, likely glycerolipids or phospholipid-derived messengers, crucial for priming distal tissues. While the exact cargo is still under investigation, candidates include lipid derivatives like azelaic acid (AzA) and dehydroabietinal (DA) (Champigny et al., 2013; Lascombe et al., 2008; Carella et al., 2017; David et al., 2021).

Beyond DIR1, other lipid-associated proteins contribute to SAR. AZI1 (Azelaic Acid Induced 1), an LTP-like protein from the hybrid proline-rich protein (HyPRP) family, is implicated in amplifying SAR signaling, potentially by facilitating azelaic acid mobilization (Priya Reddy and Oelmüller, 2024; Gao et al., 2022). AZI1 functions in concert with DIR1, suggesting cooperative action of multiple lipid carriers for robust signal fidelity. The transport of lipid signals by nsLTPs likely occurs through the apoplast and phloem, enabling rapid, energy-efficient dissemination of immune signals.

At the molecular level, SAR involves transcriptional reprogramming, including systemic upregulation of PR genes and increased SA biosynthesis (Vidhyasekaran, 2015). nsLTPs likely interface with these pathways by delivering lipid signals that trigger SA accumulation and by reinforcing membrane and cell wall integrity, enhancing overall stress resilience. Thus, through lipid transport and signal integration, nsLTPs act as key orchestrators of SAR, linking local pathogen recognition to global plant-wide immune readiness.

ROS are highly reactive molecules produced as byproducts of normal cellular metabolism. While ROS are essential signaling molecules in various physiological processes, including defense, excessive accumulation of ROS leads to oxidative stress, causing damage to lipids, proteins, and DNA (Mittler, 2017; Shetty et al., 2008; Wang et al., 2024). nsLTPs mitigate oxidative stress during pathogen attack through direct ROS scavenging or by indirectly upregulating antioxidant genes (McLaughlin et al., 2021, 2015; Yang et al., 2023; Gangadhar et al., 2016; Xu et al., 2018; Wang et al., 2014; Balmant et al., 2021; Hsouna et al., 2021; Safi et al., 2015). In Nicotiana benthamiana, overexpression of the type-I nsLTP, NbLTP1, activated genes related to ROS scavenging and enhanced resistance to TMV (Zhu et al., 2023). Under thermal stress, tobacco plants overexpressing NtLTPI.38 exhibited a significant upregulation of genes encoding antioxidant enzymes and thermal stress-related proteins (Song et al., 2023). Similarly, in NtLTP4 overexpression lines, several important ROS-scavenging enzyme encoding genes, such as SOD, APX, CAT, and GST, dramatically increased (Xu et al., 2018). Overexpression of NtLTP25 significantly increased the enzyme activities of CAT, GST, APX, and SOD, as well as the transcription levels of their encoding gene (Li et al., 2024).

The ROS scavenging activity of nsLTPs has profound implications for plant immunity. By neutralizing ROS, nsLTPs mitigate oxidative damage, protecting cellular components and ensuring the proper function of essential cellular processes. Furthermore, because ROS also act as signaling molecules, nsLTPs can fine-tune ROS levels, influencing the activation of defense pathways and preventing excessive, damaging oxidative stress. Ultimately, by scavenging ROS, nsLTPs enhance the overall stress tolerance of plants, allowing them to better cope with adverse environmental conditions, including pathogen attack. Understanding the mechanisms of their ROS scavenging activity provides valuable insights for improving plant resistance to both biotic and abiotic stresses.

nsLTPs contribute significantly to the plant’s antioxidant defense system. Several mechanisms underlie their ROS scavenging activity. First, the cysteine-rich nature of nsLTPs provides abundant free thiol groups (Wu et al., 2011). These thiol groups can, theoretically, directly interact with and neutralize ROS, such as H₂O₂ and superoxide radicals . Second, nsLTPs impact the degree of lipid peroxidation, a chain reaction initiated by ROS that severely damages cellular membranes (Priya Reddy and Oelmüller, 2024; Levesque-Tremblay et al., 2009). For instance, overexpression of NtLTP25 in tobacco significantly reduced the degree of lipid peroxidation, as indicated by a reduction in malondialdehyde (MDA) levels in the leaves (Li et al., 2024). By stabilizing lipid membranes, nsLTPs may prevent the propagation of lipid peroxidation and protect cellular integrity (Song et al., 2023).

Several examples illustrate the ROS-scavenging capabilities of nsLTPs. Barley LTP1 exhibits strong antioxidant activity, effectively scavenging ROS and protecting cells from oxidative damage (Cai et al., 2019; Stanislava, 2007; Wu et al., 2011). CmnsLTP6.9, from Chinese chestnut, regulates ROS scavenging and remodels lipid profiles, contributing to stress tolerance (Xiao et al., 2023). Similarly, tobacco NtLTPI.38 displays antioxidant capacity, suggesting its involvement in ROS detoxification (Yang et al., 2023). To better understand how the sugar beet responses to abiotic stress, quantitative redox proteomics (iodoTMTRAQ) was used to identify and quantify redox posttranslational modifications (PTMs). Several proteins were identified to be chemically reduced during salt stress, including a nsLTP (A0A0K9RNM7), a novel discovery (Li et al., 2021a). Another example is the LTP-II, which was found to be redox-responsive in response to flg22 during stomatal closure and played a role in plant resistance to Pseudomonas syringe pv. tomato DC3000 (Balmant et al., 2021).

How might the cysteines in nsLTPs function to scavenge or otherwise impact ROS accumulation and thereby provide some measure of resistance against oxidative stress? Molecular details from the study of the serine protease inhibitor (serpin) superfamily protein maspin in mouse mammary cells may provide useful information (Mahajan et al., 2013). Maspin is rich in redox-sensitive cysteine residues, containing a total of eight, similar to that found in nsLTPs. It was shown that structurally exposed cysteine thiols were oxidized in the presence of sulfenic acid and that the protein binds to glutathione S-transferase (GST). Another research group, following up on that work, showed that oxidized maspin increases GST, which may lead to the inhibition of oxidative stress-induced ROS generation (Yin et al., 2005). These compelling studies provide a powerful framework for investigating nsLTPs in plants and their potential to modulate ROS. A critical question emerges: does oxidized nsLTP, analogous to maspin, bind to GST in plants, thereby directly influencing its activity and the plant’s capacity to manage oxidative stress? If so, that might help explain why glutathione levels were found to be higher in transgenic Arabidopsis overexpressing AtLTP4.4 and why ROS levels were significantly lower upon exposure to oxidizing agents such as trichothecenes and H₂O₂ (McLaughlin et al., 2015). Other nsLTPs may contribute indirectly to ROS accumulation by regulating the expression of antioxidant enzymes or by transporting lipids that are involved in ROS detoxification (Song et al., 2023; Xu et al., 2018; Zhu et al., 2023; Li et al., 2024; Hsouna et al., 2021).