The non-specific lipid transfer proteins (nsLTPs) are a plant-specific superfamily of cysteine-rich AMPs (antimicrobial peptides). They received this name due to their ability to bind to several hydrophobic molecules, such as phospholipids and fatty acids, among others. nsLTPs are characterized by their reduced size (6.5–10.5 kDa) and the presence of eight cysteine residues (8CM domain), which form four disulfide bonds [1]. They are associated with various plant biological processes, such as growth and development, abiotic stress responses, besides plant defense ^{[2][3][4][5][6][7][8]}[2,3,4,5,6,7,8]. The mentioned plant-originated AMPs have demonstrated remarkable efficacy against bacterial and fungal pathogens [9]. The discovery of cysteine-rich AMPs such as nsLTPs has paved the way for exploring plants as potential biofactories for synthesizing antimicrobial compounds, which holds significant promise for their application as biotherapeutic agents in the field of antimicrobial drug development [9]. It has been proposed that such antimicrobial activity is due to the nsLTPs’ ability to disrupt the permeability and integrity of the pathogens’ outer membranes, similar to other plant AMPs ^[7][10][7,10]. However, further studies are necessary to understand all their biological roles.

Previous works indicate that nsLTPs are encoded by a large gene family, presenting more than 50 loci in many angiosperm genomes and up to 50 loci in bryophytes, ferns, and gymnosperms [1]. Some classification systems are available for nsLTPs. These, however, are heterogeneous in terms of subgroups’ numbers and nomenclature. The classification initially proposed for nsLTPs—division into ‘nsLTP1’ and ‘nsLTP2’ subfamilies—was based on their molecular mass, sequence identity (<30% of similarity between nsLTP1s and nsLTP2s), and lipid transfer efficiency ^[11][12][11,12]. Disulfide bond patterns in nsLTPs may also differ. ‘nsLTP1s’ displays ‘Cys1-Cys6, Cys2-Cys3, Cys4-Cys7, and Cys5-Cys8’ pattern [13], whereas ‘nsLTP2s’ exhibit that of ‘Cys1-Cys5, Cys2-Cys3, Cys4-Cys7, and Cys6-Cys8’ [14]. Such differences in disulfide bond configuration play a crucial role in the stability of these proteins, constraining their conformational dynamics [1]. Another notable difference regards the hydrophobic cavity. The subgroup nsLTP1s may have a long tunnel-like cavity ^[15][16][15,16], while nsLTP2s may have two adjacent hydrophobic cavities [14]. However, it is worth mentioning that there is no specific rule in this regard since the hydrophobic cavities can vary according to the sequence of amino acid residues, the number of disulfide bonds present in the structure, and the lipid binding specificity. Boutrot et al. [17] proposed a different classification approach. These authors introduced phylogenetic grouping as a key classification criterion. The proposition includes early diverging nsLTP homologs found in mosses and liver plants. The mentioned classification established nine groups (types ‘1’ to ‘9’) stratified with their respective consensus cysteine motifs (8CM domain)—Cys-Xn-Cys-Xn-CysCys-Xn-CysXCys-Xn-Cys-Xn-Cys (X: different amino acids; n: variable number of amino acids)—and the inter-cysteine amino acid residues diversity. Later, another proposition, by Edstam et al. [1], sought to break the limitations inherent in the sequence conservation, considering post-translational modifications in the glycosylphosphatidylinositol (GPI) anchoring sites, intron positions, and spacing in the regions between cysteine residues, besides sequence similarity. This classification system suggests five major groups (‘LTP1’, ‘LTP2’, ‘LTPc’, ‘LTPd’, and ‘LTPg’) and four minor groups (‘LTPe’, ‘LTPf’, ‘LTPh’, ‘LTPj’, and ‘LTPk’). Despite the efforts made on the new nsLTP classification systems, the conventional classification of ‘LTP1’ and ‘LTP2’ is still widely used due to a lack of consensus among different studies. Since the nsLTP gene family is so complex and diversified, no established classification guidelines are final [18].

2. Omics Studies for nsLTPs

2.1. Understanding nsLTPs from Previous Studies

Previous studies (Table 1) involving nsLTP gene search and plant transcriptional expression analyses were scrutinized. Based on this search, different views on nsLTP abundance, types (Table 1), and functions were found. The seminal work by Edstam et al. [1] proposes a greater nsLTP abundance in terrestrial plants and their absence in green algae (chlorophytes and charophytes) (Table 1), suggesting that nsLTP genes evolved soon after the terrestrial environment conquest. In favor of the mentioned proposition, a limited number of representatives and types of nsLTPs are observed when comparing lower plants (as bryophytes and lichens) to spermatophytes, which indicates the emergence of new types of nsLTPs in higher plants ^[1][19][20][1,19,20]. Table 1). Chromosome distribution and collinearity analyses suggested that the expansion of the StnsLTP gene family was enhanced by tandem duplications. In turn, Ka/Ks analysis showed that 47 pairs of duplicated genes have gone through purifying selection during evolution. StnsLTP genes were expressed mainly in younger tissues. Furthermore, StnsLTPs contained a large number of stress-responsive, cis-acting elements in their promoter regions. These results indicated that StnsLTPs might play significant and functionally varied roles in potato plants. In Arachis duranensis, Song et al. [31] discovered 64 AdnsLTPs (Arachis duranensis nsLTPs) genes, which were divided into six groups (‘1’, ‘2’, ‘C’, ‘D’, ‘E’, and ‘G’; Table 1), anchored over nine chromosomes. Considering the AdnsLTPs’ expansion mechanisms, the study revealed some gene clustering by tandem duplication, while other family members showed segmental duplication in several chromosomes. Following treatments with high salt (NaCl, 250 mM), PEG, low temperature (4 °C), and abscisic acid, the AdnsLTPs’ expression levels were altered. Three AdnsLTPs were linked to nematode infection resistance. The DOF and WRI1 transcription factors were suggested as potential controllers of the AdnsLTP response to nematode infection. Fang et al. [30] found 330 TansLTPs (Triticum aestivum nsLTPs) genes in wheat (T. aestivum) (Table 1). Such a quantitative result can be considered an update of the 461 nsLTP loci found by Kouidri et al. [26] for the same species. To date, T. aestivum is the plant with the highest number of nsLTPs. The TansLTPs clustered into five groups (‘1’, ‘2’, ‘C’, ‘D’, and ‘G’) by phenetic analysis (Table 1). Gene structure and MEME pattern analyses showed that different groups of nsLTPs had similar structural compositions. Chromosome anchoring revealed that all five groups were distributed on 21 chromosomes. Furthermore, 31 gene clusters were identified as tandem duplications, and 208 gene pairs were identified as segmental duplications. Data mining of RNA-seq libraries, covering multiple stress conditions, showed that the transcript levels of some of the nsLTP genes could be strongly up-regulated by drought and high salt (NaCl, 250 mM) stresses. In another context, Liang et al. [35] scrutinized the Brassica napus pangenome for BnnsLTPs (B. napus nsLTPs). These authors identified 246 BnnsLTP genes, divided into five groups (‘1’, ‘2’, ‘C’, ‘D’, and ‘G’; Table 1). Different BnnLTP genes were identified among the eight studied B. napus varieties (ZS11, Gangan, Zheyou7, Shengli, Tapidor, Quinta, Westar, and No2127). BnnsLTPs showed different duplication patterns in different varieties. Cis-regulatory elements that respond to biotic and abiotic stresses were anchored at all BnnsLTP genes. Finally, RNA-Seq analysis showed that the BnnsLTP genes were involved in responses to the fungus Sclerotinia sclerotiorum infection. Vangelisti et al. [36], studying sunflower (Helianthus annuus) HansLTPs (Helianthus annuus nsLTPs), observed the existence of four (‘1’, ‘2’, ‘3’, and ‘4’) groups (Table 1). The authors did not explicitly classify the observed groups according to the available classification systems. The HansLTPs (101 in total) were further examined by looking into potential gene duplication sources, which revealed a high prevalence of tandem- in addition to whole-genome duplication (WGD) events. This finding is consistent with polyploidization events that occurred during the evolution of the sunflower genome. Three (‘1’, ‘3’, and ‘4’) of the four HansLTP groups responded uniquely to environmental cues, including auxin, abscisic acid, and the saline environment. Interestingly, sunflower seeds were the only source of expression for HansLTP group ‘2’ genes. In line with the reports mentioned above and other works in Table 1, it is observed that the nsLTP genes act multifunctionally and show genetic variability even within accessions of the same species. nsLTPs are present in a wide range of plants, showing gene expression in different tissues, developmental stages, and stressful conditions.

2.2. Filling the Gap: Discovering and Classifying nsLTPs in New Plant Genomes

To provide genomic information for nsLTPs in plants not yet studied in the previous topic, and to update nsLTP data for some species with improved genome versions that have been made available, the following plant genomes were scrutinized (Table 2): (1) Marchantia polymorpha; (2) Ceratopteris richardii; (3) Selaginella moellendorffii; (4) Thuja plicata; (5) Gossypium hirsutum; (6) Lactuca sativa; (7) Manihot esculenta; (8) Mimulus guttatus; (9) Populus trichocarpa; (10) Sinapis alba; (11) Solanum tuberosum; and (12) Spinacea oleracea. The mentioned species were chosen to diversify the number of analyzed clades.