Genome-Wide Identification and Bioinformatics Analyses of Host Defense Peptides Snakin/GASA in Mangrove Plants

Host defense peptides (HDPs) are components of plant defensive barriers that resist microbial infection. Members of the Snakin/GASA protein family in plants have functions of regulating plant growth, defense, and bacteriostasis. Most mangrove plants grow in coastal zones. In order to survive in harsh environments, mangrove plants have evolved complex adaptations against microbes. In this study, Snakin/GASA family members were identified and analyzed in the genomes of three mangrove species. Twenty-seven, thirteen, and nine candidate Snakin/GASA family members were found in Avicennia marina, Kandelia obovata, and Aegiceras corniculatum, respectively. These Snakin/GASA family members were identified and categorized into three subfamilies via phylogenetic analysis. The genes coding for the Snakin/GASA family members were unevenly distributed on chromosomes. Collinearity and conservative motif analyses showed that the Snakin/GASA family members in K. obovata and A. corniculatum underwent multiple gene duplication events. Snakin/GASA family member expression in normal leaves and leaves infected with pathogenic microorganisms of the three mangrove species was verified using real-time quantitative polymerase chain reaction. The expression of KoGASA3 and 4, AcGASA5 and 10, and AmGASA1, 4, 5, 15, 18, and 23 increased after microbial infection. This study provides a research basis for the verification of HDPs from mangrove plants and suggests directions for the development and utilization of marine biological antimicrobial peptides.


Introduction
Plants have evolved sophisticated defense mechanisms in the natural environment to protect themselves from bacteria, fungi, viruses, and protozoa [1]. For example, in the constitutive defense, waxy cuticles and trichomes form physical barriers against the infiltration and spread of pathogenic microorganisms [2]. Alternatively, in the induced defense, by triggering a cascade of reactions that activate many defense-related genes, a variety of proteins and secondary metabolites are released to inhibit the growth of pathogenic microorganisms [3,4]. Among the defense molecules in plants, host defense peptides (HDPs) are common defense barriers that plants have evolved to resist microbial stress [5]. HDPs are part of the innate immune system inherent in almost all life forms and are found in microbes, arthropods, amphibians, mammals, and plants. HDPs significantly contribute to host defense against pathogens [6][7][8][9]. In plants, HDPs are typically peptides with masses less than 9000 Daltons; they are thermally stable and positively charged, and are infected by pathogenic microorganisms in their natural habitats. This study provides a resource for future research on HDPs from mangrove species and suggests directions for developing and utilizing marine biological antimicrobial peptides.

Materials and Methods
2.1. Gene Identification of Snakin/GASA Family in K. obovata, A. corniculatum, and A. marina The genomic data of K. obovata were obtained from Genome Warehouse (GWH) with the accession code PRJCA002330/GWHACBH00000000 [25]. The A. corniculatum genome data were obtained from the China National GeneBank (CNGB), accession number CNA0017738 [26]. A. marina genomic data were obtained from NCBI (www.ncbi.nlm.nih. gov/, 3 April 2022), Genebank numbers GCA_019155195.1 (file 1, assembled to the chromosomal level but no transcripts) and PRJNA392013 (file 2, assembled to the scaffold level with transcripts [27]). The hidden Markov model (HMM) profile of the GASA domain (PF02704) from the Pfam database was used as a query, and the putative Snakin/GASA family members were identified by HMMER searching against the K. obovata, A. corniculatum, and A. marina genomes. The complete GASA domains of the protein sequences were examined using Batch CD-Search tools (https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml, 3 April 2022) and Pfam (http://pfam.xfam.org/, 3 April 2022). Finally, conserved domains of all candidate protein sequences were verified by manually removing incomplete domain sequences.

Physicochemical Characterization, Chromosomal Location, and Sequence Alignments
Snakin/GASA family members' physicochemical properties were predicted using ProtParam (https://web.expasy.org/protparam/, 3 April 2022). The chromosomal locations of Snakin/GASA family members were confirmed in the gene annotation files of K. obovata and A. corniculatum and mapped using MapChart software [28]. The coding sequences of Snakin/GASA family members were confirmed in the genome file 2 of A. marina. Then the chromosomal location information of Snakin/GASA family members was obtained in file 1 by blastn and visualized using MapChart software [28]. Snakin/GASA family members of the three mangrove species were aligned using Genedoc software [29] with Snakin-1, Snakin-2, and Snakin-3 sequences of S. tuberosum.

Sample Collection
Plant samples obtained in the field can provide a true and visual picture of plant populations' health status, the causes of disease, and plant response characteristics. In November 2021, infected and normal leaves of three mangrove species ( Figure S1) were collected in Baguang Yinye Wetland Park (22 • 38 N, 114 • 24 E), Shenzhen. After washing the leaf surface with sterile water, the collected samples were photographed ( Figure S1), wrapped in tinfoil, and immediately frozen in liquid nitrogen and stored at −80 • C.
2.6. RNA Extraction, cDNA Synthesis, and qRT-PCR Analysis RNA was extracted using an RNA Pure Plant Plus Kit (Tiangen Biotechnology, Beijing, China). According to the manufacturer's instructions, the extracted RNA was reverse transcribed into cDNA using the Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (YEASEN, Shanghai, China). NanoDrop nucleic acid concentration tester (Thermo Fisher Scientific, MA, USA) was used to determine the concentration and purity of RNA. Only samples with OD 260/OD 280 ratios between 1.8 and 2.0 could proceed to the next step. RNA samples were randomly selected and electrophoresed on 0.8% agarose gels to check the integrity of the RNA. The loading volume was 1 µg total RNA, and the voltage was 6 V/cm. The specific primers for Snakin/GASA family members from three mangrove species were designed using Premier 6.0. The specificity of primers was determined by 0.8% agarose gel electrophoresis. Real-time PCR was run with the qTower3 instrument (Analytik Jena AG, Jena, Germany) to detect the chemical SYBR Green. The established reaction system was as follows: 5 µL 2 × SYBR Green Pro Taq HS Premix (Accurate Biotechnology, Hunan, China), 0.5 µL of each forward and reverse primer (10 µM), 1 µL of diluted cDNA template, and RNase-free ddH 2 O were added until the total volume was 10 µL. The reaction procedure was as follows: 95 • C for 15 min, 40 cycles of 10 s at 95 • C, 15 s at 59 • C, and 20 s at 72 • C. The melting curve analysis was performed immediately after the PCR reaction, i.e., fluorescence intensity was measured for each degree increase from 60 • C to 95 • C. The relative template abundance in each PCR expansion mixture was calculated by the 2 −∆∆CT method. Three biological replicates were used for gene expression analysis, and the expression of the control samples was set to 1 for normalization. Data manipulation and structural visualization were performed with GraphPad Prism 6, and an LSD test was used to calculate the p-value. All primers for internal reference genes and Snakin/GASA family members in the three mangrove species are shown in Tables S1 and S2. Internal reference genes refer to Dr. Peng Yalan's research [31][32][33].

Identification of Pathogenic Microorganism
The infected part was cut out, and the Ezuo Column Fungal Genomic DNA Extraction Kit DNA (Sangon Biotech, Shanghai, China) instructions were followed to obtain. PCR amplification was performed using the fungus ITS universal primer pair (ITS1F: CTTG-GTCATTTAGAGGAAGTAA; ITS4R: TCCTCCGCTTATTGATATGC) that amplifies the ITS1-5.8S-ITS2 region, and the bacteria 16S universal primer pair (27F: AGAGTTTGATCMTG-GCTCAG; 1492R: TACGGYTACCTTGTTACGACTT) that amplifies the V1-V5 variable regions. The nucleic acid was recovered from an agarose gel and sent for sequencing. The sequencing results were compared to the NCBI database to identify the pathogenic microbes.

Statistical Analysis
GraphPad Prism 7.0 (GraphPad, San Diego, CA, USA) was selected for statistical analysis. Statistical significance was determined via a one-way analysis of variance. All data are presented as mean ± SE and p-value < 0.05 was considered statistically significant.

Identification of Snakin/GASA Genes in Three Mangrove Plants and Chromosomal Location
Chromosome-scale assemblies of the genomes of K. obovata and A. corniculatum were reported in 2020 [25] and 2021 [26]. A reference-grade genome of A. marina was pub-lished in 2021 [27]. We first used the conserved GASA domain (PF02704) to search for Snakin/GASA family members in the three mangrove species using Hmmersearch. Thirteen candidate Snakin/GASA family members were found in A. corniculatum; nine candidate Snakin/GASA family members were found in K. obovate; and twenty-seven candidate Snakin/GASA family members were found in A. marina. The Snakin/GASA family members of the three mangrove species were named according to their locations on the chromosomes ( Figure 1). As shown in Figure 1, the A. corniculatum genome contained 13 Snakin/GASA family members spread across six chromosomes. Eight Snakin/GASA family members of A. corniculatum (AcGASA1-AcGASA8) were located on chromosome 1. Nine Snakin/GASA family members were spread across six chromosomes in the K. obovata genome. On chromosomes 4 and 2, there were three and two Snakin/GASA family members, respectively. AmGASA27 was not placed on a scaffold and could not be located on a chromosome. The remaining 26 Snakin/GASA family members were distributed on 15 chromosomes in the A. marina genome. Furthermore, three Snakin/GASA family members were respectively identified on A. marina's chromosomes 2, 20, 22, and 25.

Physicochemical Properties Prediction
As shown in Table 1, after ProtParam prediction, among the Snakin/GASA family members in A. marina, AmGASA22 had 88 amino acids and was the smallest. AmGASA19 had 307 amino acids. Among the Snakin/GASA family members of K. obovata, KoGASA9 was the largest with 186 amino acids. There were 421 amino acids in AcGASA6 of A. corniculatum, the Snakin/GASA family member with the most significant number of amino acids among the three mangrove species. AcGASA7 and AcGASA8 had 346 and 387 amino acids, respectively. KoGASA1, 4, 5, and 8 in K. obovata, AcGASA5, 9, 10, 12, and 13 in A. corniculatum, and AmGASA2, 5,11,16,17,21,22, and 23 in A. marina had fewer than 100 amino acids.  Table 1 displays the predicted properties of Snakin/GASA family members in three different mangrove species. The majority of these proteins were classified as basic proteins with a theoretical isoelectric point (pI) value above 8. The proteins AmGASA4 and AmGASA19 had the highest and lowest pI values, with 9.83 and 5.39, respectively.

Subcellular Localization Prediction
This study predicted the subcellular localization of Snakin/GASA family members in three mangrove species. For A. corniculatum, AcGASA2, 5, 10, and 12 were predicted to be localized in the extracellular matrix, AcGASA6 in the nucleus, and AcGASA3, 4, 8, and 9 in the chloroplast (Table S3). AcGASA1, 11, and 13 were predicting localized in the chloroplast and extracellular matrix. In K. obvoata, the predicted localization of KoGASA2, 4, 5, 6, 7, and 8 was in the extracellular matrix. KoGASA1 was distributed in the extracellular and Golgi apparatus. KoGASA9 was localized in the mitochondrion and chloroplast. In A. marina, AmGASA3 and 12 localized in the cytoplasm. The predicted localization of AmGASA1, 4-6, 8-11, 14-18, 20, 21, 23, and 27 was in the extracellular matrix.

Phylogeny, and Protein Sequence Analysis of Snakin/GASA Family Members in Three Mangrove Species
To illustrate the evolutionary relationships of the Snakin/GASA family members of the three mangrove species, a phylogenetic tree of conserved GASA domains was constructed ( Figure 2). The GASA domains of all species were divided into three groups. Group III had the most members. There were 11 Snakin/GASA family members of A. corniculatum belonging to Group I. There were nine Snakin/GASA family members of K. obovata distributed among the three groups. There were 8, 6, and 13 Snakin/GASA family members of A. marina in groups I, II, and III, respectively. The three mangrove plants were more phylogenetically related to V. vinifera L. and P. trichocarpa. The Snakin-1, Snakin-2, and Snakin-3 of potato were aligned with the Snakin/GASA family members of the three mangrove species. The Snakin/GASA family members of A. corniculatum and K. obovata were divided into three groups ( Figure 3A,B). Snakin/GASA family members of A. marina were divided into four groups. Sequence analysis of the predicted proteins of A. marina species showed that the family has four distinct structural domain features, while the fourth family members are missing some of the conserved domains. AmGASA3, 12, and 15 were dissimilar from the three Snakins of potato ( Figure 3C). KoGASA4 and AmGASA11 were adjacent to StGASA15 (Snakin-3); AmGASA7 and Am-GASA26 were adjacent to StGASA2 (Snakin-2).

Collinearity Analysis among Snakin/GASA Family Members
There are two versions of the A. marina genome. The annotation of the genome assembled to the scaffold level was relatively complete, while the annotation of the genome assembled to the chromosome level was lacking. Analyzing the intergenic collinearity of Snakin/GASA family members in the A. marina genome is temporarily impossible. To illustrate the family expansion pattern of Snakin/GASA family members in the three mangrove species, we analyzed gene duplication events in their genomes by McsanX. In the K. obovata genome, two repetitive events occurred in the evolution of Snakin/GASA family members ( Figure 4A). No gene tandem duplication events were found in K. obovata. In A. corniculatum, nonsynonymous substitution rate (Ka) and synonymous substitution rate (Ks) and their ratio were estimated for Snakin/GASA family members. The gene duplication event of Snakin/GASA family members occurred on chromosome 1 ( Table 2). The negative Ka/Ks values of two duplicated gene pairs (AcGASA4 and AcGASA3, AcGASA5 and AcGASA9, Ka/Ks values < 1) suggested negative or purifying selection pressure during evolution. T = Ks/2r, where r is the expected clock sample rate of synonymous substitution in dicotyledons, and r = 1.5 × 10 8 substitutions/synonymous sites/year is the formula used to determine divergence time. The range of divergence times of two gene pairs calculated using Ks values was 32.28 to 33.13 million years ago (MYA) ( Table 2). AcGASA5 and AcGASA6 had signatures of neutral evolution, as suggested by Ka/Ks values equal to 1, while the Ka/Ks values of other gene pairs were greater than one, suggesting positive selection.  Figure 4B and Table 3 display the collinearity analysis of Snakin/GASA family members among different species. Four collinear gene pairs were identified between K. obovata and A. thaliana. Between A. corniculatum and A. thaliana, two collinear gene pairs of Snakin/GASA family members were found. Between K. obovata and P. trichocarpa and K. obovata and V. vinifera, there were nine and seven collinear gene pairs of Snakin/GASA family members, respectively. Two and three collinear gene pairs of Snakin/GASA family members existed between A. corniculatum and V. vinifera, respectively. Finally, K. obovata and V. vinifera shared three gene pairs with Snakin/GASA family traits.

Gene Structure and Motif Identification among Snakin/GASA Family Members
As shown in Figure

Protein Structure Prediction
One representative of each Snakin/GASA family member in each mangrove species was selected for three-dimensional (3D) protein structure prediction. The predicted structures of these Snakin/GASA family members were similar, with all of them having random coils, extended strands, and two long α-helices (Figure 7).

Responses to Pathogenic Microbial Threats
To investigate the role of the GASA family in response to environmental stress, infected leaves were collected from the Dapeng mangrove forest (22 • 38 N, 114 • 24 E) in Shenzhen, China ( Figure S1).
Microbiological testing of black spots on collected environmental leaf samples revealed that fungal infections caused black spots on the leaves, while no bacterial infections were detected (Table S4). It was found that Tropicoporus texanus caused marginal and vein scorching in A. corniculatum. K. obovata became ulcerated as a result of Jattaea spp. infection. It should be Berkeleyomyces basicola that caused the infection of A. marina (Supplementary  Tables S4 and S5). The expression levels of Snakin/GASA family members in the infected leaves were measured to explore their role in responding to fungal infections.
After collecting infected and normal leaves of three mangrove plants in the field, qRT-PCR analysis was performed to examine the expression of Snakin/GASA family members ( Figure 8A-C). In the infected leaves of A. marina, the expression of all selected Snakin/GASA family members was significantly up-regulated, with AmGASA18 showing the most significant difference. Compared with the normal leaves, the expression of AmGASA18 in the leaves infected by pathogenic microbial was increased by more than fivefold. In A. corniculatum, AcGASA1, 2, 8, and 12 were significantly down-regulated in the infected leaves. Compared with normal leaves, the expression levels of AcGASA5, AcGASA6, AcGASA9, and AcGASA10 in infected leaves were significantly increased by 2.820, 2.123, 1.563, and 1.819-fold, respectively. AcGASA5 showed the most intense response to infection. In K. obovata, KoGASA1, 2, 5, 6, 7, and 8 were significantly down-regulated after being infected by pathogenic microorganisms. KoGASA3 and KoGASA4 were significantly up-regulated by 2.453 and 2.261-fold, respectively, in infected leaves.

Discussion
Most members of the Snakin/GASA family are small peptides with multiple functions. They are regulated by various hormones involved in plant development, stress response, and antibacterial activities [10,14]. The biological processes, physicochemical properties, and gene structures of Snakin/GASA family members in various plants (including G. max, V. vinifera, and Populus spp.) have been reported [18,19,21].
Peptide lengths vary greatly among Snakin/GASA family members in the same plant species. For instance, the smallest Snakin/GASA protein in A. thaliana has 87 amino acids, while the largest has 275 amino acids [34]. The smallest Snakin/GASA family member in wheat has 261 amino acids, while the largest has 1099 amino acids [35]. The peptide lengths of the Snakin/GASA family members of the three mangroves varied significantly. HDPs are typically proteins with less than 100 amino acids [36]. However, many proteins with more than 100 amino acids have good antibacterial effects and may have other biological functions [37]. KoGASA1, 4, 5, and 8 in K. obovata, AcGASA5, 9, 10, 12, and 13 in A. corniculatum, and AmGASA2, 5,11,16,17,21,22, and 23 in A. marina had fewer than 100 amino acids. Their amino acid numbers met the criteria for HDPs, but their antibacterial functions remain elusive.
In cotton and potato, longer chromosomes did not necessarily contain more Snakin/GASA family members. This suggests that the number of Snakin/GASA family members on each chromosome of the three mangrove species was unrelated to chromosome length. Additionally, the pI values (from 4.11 to 10.14) also vary widely among different plant species and individual members [10]. The apple MdGASA23 is currently the known Snakin/GASA family member with the lowest pI value (pI value = 4.11) [38]. The non-alkaline Snakin/GASA family members in the three mangrove species may have different functions compared to the basic Snakin/GASA family members. Typically, HDPs usually contain more cationic amino acid residues and are basic [36,37]. Therefore, electrically neutral AmGASA19 and AcGASA6 may not have normal HDP functions.
The GASA domain of Snakins is typically situated at the C-terminal and consists of approximately 60 residues, including 12 conserved cysteines [10,39]. The GASA domains of KoGASA9, AcGASA7, 8, and AmGASA19 are not located at their C-terminal. Based on their sequence features, they may not be considered part of the Snakin/GASA family. Subcellular localization provides insights into the functions of proteins [40][41][42]. Existing studies have shown that Snakin/GASA family members are distributed in various locations within plants, including nuclei, cell walls, cytoplasm, and extracellular spaces [10,17,21,43]. For example, AtGASA5 of A. thaliana is present in the cell wall and extracellular matrix [10,44], and HbGASA5 and HbGASA9 proteins of Hevea brasiliensis are present in the nucleus and the cytoplasm [45]. The transition between the cell periphery and the nucleus may be important concerning their antimicrobial function [10]. Based on the predictions, several members of the Snakin/GASA family in the three mangrove species, including AmGASA1, 4-6, 8-11, 14-18, 20, 21, 23, 27, KoGASA2, 4-8, AcGASA2, 5, 10, and 12, may have antimicrobial function and are predicted to be located in the extracellular space. KoGASA9 and AcGASA6-8 did not have signal peptides, which may be the reason why they were predicted to be localized in the intracellular space. The subcellular localization of proteins can be influenced by various factors, including post-translational modifications, electrostatic interactions, and covalent bonds with membrane lipids [10,13]. As a result, the predictions of the two methods were somewhat inconsistent. At present, it is not possible to conclusively determine whether Snakin/GASA family members have antibacterial function only based on subcellular localization.
The adjacent Snakin/GASA family members in the evolutionary tree had similar sequences and thus may have similar functions. Although many plant Snakin/GASA family members have been identified, few have been functionally validated [16,19,44,46]. The tandem and segmental duplication of genes play important roles in functional regulation, domestication, evolution, and response to biotic and abiotic stresses [42,47,48]. Segmentally duplicated genes also show similar functions and stable expression [18,49]. The motif and exon-intron analyses and Ka/Ks analysis showed that Snakin/GASA family members of A. corniculatum may have evolved leading to variation in motifs and introns in some groups. Some members of the Snakin/GASA family can be strongly induced to respond to temperature variation [10]. The Oligocene (33 million to 23 million years ago) was an important turning point in the rapid transformation of the Earth's climate from "greenhouse" to "ice chamber" [50], which may be the reason for negative or purifying selection pressure during the evolution of GASA/Snakin family members in A. corniculatum. AtGASA4, 6, and 14 in A. thaliana are critical in promoting plant development [16,34,51]. Inhibition of both AtGASA4 and 6 has been shown to cause delayed flowering [16]. KoGASA6 and AmGASA13 are adjacent to AtGASA4 and 6 ( Figure 2), and thus they may have the same function in regulating flower development. AtGASA4 and AtGASA14 can interact with the cell membrane-localized receptor-like kinase protein VH1/BRL2, which participates in leaf vein development [34,51]. KoGASA4 and AmGASA11 are adjacent to AtGASA5, which are negative regulators of GA-induced flowering and stem growth [44]. KoGASA6 is adjacent to VvGASA7, which regulates seed development, and they are collinear genes (Figure 2 and Table 3). Therefore, KoGASA6 may regulate seed development [19]. VvGASA5 can regulate ovule abortion. AcGASA11 and VvGASA5 are collinear genes with the same function [19]. VvGASA2 is thought to play a role in seed development [19]. Collinear relationships were observed between VvGASA2 and KoGASA1, KoGASA5 and AcGASA10, and KoGASA8 and AcGASA12. KoGASA1 and KoGASA5, KoGASA1 and KoGASA8, are intra-genome collinear gene pairs that belonged to the same group in the evolutionary tree; their amino acid sequences consisted of motifs 2, 3, and 5, and their patterns of response to pathogenic microorganisms were consistent. This suggests that their functions may be similar. Therefore, KoGASA1, 5, 8, and AcGASA10 and 12 may regulate seed development. Snakin-1, Snakin-2, and Snakin-3 had good antibacterial effects. KoGASA4 and AmGASA11 were adjacent to StGASA15 (Snakin-3); AmGASA7 and AmGASA26 were adjacent to StGASA2 (Snakin-2). Therefore, KoGASA4, AmGASA7, 11, and 26 may have the same function as Snakin-2 and Snakin-3.
Snakin/GASA family members are regulated by gibberellin (GA), abscisic acid (ABA), and other plant hormones [10,19]. The predictions via the Plant CARE website suggested that the promoters of the three mangrove GASA family members had significant differences in the number of cis-acting elements in response to abscisic acid, gibberellin, methyl jasmonate, low temperature, drought, and pathogenic microorganisms. MeJA, as a damagerelated plant hormone and signaling molecule, can stimulate the expression of plant defense genes and induce plant chemical defenses [52,53]. In this study, AmGASA1-7, 10,12,13,15,18,21,23, and 25 had MeJA and defense regulatory elements upstream, consistent with their up-regulated expression after pathogenic microorganism infection. Not all Snakin/GASA family members of the three mangrove species with MeJA and defense regulatory elements were up-regulated after infection by pathogenic microorganisms. Salicylic acid (SA) can act as a signal to promote the expression of downstream defense genes and limit the growth of pathogenic microorganisms [54,55]. Four SA regulatory elements were upstream of KoGASA4, and its expression was up-regulated after pathogenic microorganism infection. KoGASA4 is a secreted protein with a signal peptide and was predicted to localize in the extracellular milieu. Therefore, KoGASA4 has some characteristics of plant HDPs, and its function is worthy of further verification. Although AcGASA6 had two SA upstream regulatory elements and its expression could be induced by pathogenic microorganisms, it did not have a signal peptide and was predicted to localize in the nucleus. Therefore, after pathogenic microorganisms have invaded the plants, the increased level of SA induces the expression of AcGASA6. A recent report has shown that the type one protein phosphatases (TOPP)-SnRK2 module of ABA signaling in A. thaliana was disturbed by the pathogenic effector AvrE, resulting in up-regulation of ABA signaling and stomatal closure that promoted the generation of interstitial waterlogging and pathogenic infection [56]. Four ABA regulatory elements upstream of AcGASA10 may account for its up-regulation after infection by pathogenic microorganisms. The regulatory relationship between the Snakin/GASA family members of the three mangrove species and hormones should be further investigated.
Based on prediction, the structure of Snakin/GASA family members is highly conserved [39]. The structures of these Snakin/GASA family members were similar to previous studies [39,42]. These results confirmed the structural conservation of Snakin/GASA family members. Furthermore, the 3D structures of Snakin/GASA family members in the three mangrove species were similar to those of potato Snakins [13].
Microorganisms often cause diseases in plants. For instance, T. texanus can cause browning of the leaf edges and veins, Jattaea spp. will cause yellowing and ulceration of plant leaves; and Fusarium spp. will cause the plant to wilt [57][58][59]. Genes involved in plant pathogen defense are often up-regulated after microbial infection. The synthesis of most HDPs occurs precisely under conditions of infection or other stress factors. Multiple studies have shown that Snakin/GASA family members are involved in pathogen defense. For example, overexpression of the Snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants [60]. Expression of Snakin-2 is up-regulated after infection of potato tubers with the compatible fungus B. cinerea [61]. The new CaSn gene from the Snakin family induces resistance against root-knot nematode infection in Capsicum annuum [62]. Snakin-3 was induced 24 h after infection by Pseudomonas syringae pv. Tabaci [13]. PnSN1 in P. notoginseng roots was induced 24 h after infection by F. solani [17]. TcGASA12 and TcGASA13 of Theobroma cacao were up-regulated after infection by Phytophthora megakarya [42]. Snakin/GASA family members whose expression was not induced after being infected by pathogenic microorganisms did not necessarily lack antibacterial activity. The Snakin/GASA family members whose expression is induced by pathogens are more likely to have functions involved in plant defense and antibacterial effects. According to the RT-qPCR results of the infected leaves, the expression levels of KoGASA3, 4, AcGASA5, 6, 9, 10, AmGASA1-5, 7, 12, 13, 15, 18, 23, and 25 were significantly higher than those of normal leaves. They may be candidates for HDP and deserve further verification. The prediction of HDPs relies on multiple criteria, including sequence features such as the presence of conserved motifs, charge, hydrophobicity, and secondary structure. Additionally, subcellular localization and phylogenetic analysis provide valuable information on the potential function of the protein. Based on these criteria, it is suggested that AcGASA6 may not be an HDP, as it lacks a signal peptide and is predicted to be localized in the nucleus. Regarding AcGASA9, its GASA domain is incomplete, which may affect its functionality as an HDP. AmGASA12 was predicted to be located in chloroplasts, and its GASA domain is incomplete. According to sequence characteristics, amino acid number, and subcellular localization prediction, they may not be HDPs. AmGASA1-5, 7, 13, 15, 18, 23, and 25 can be regulated by ABA or MeJA, while KoGASA3, 4, and AcGASA5, 10 are regulated by ABA. These genes may be induced by pathogenic microorganisms, resulting in increased expression. Thus, they may play a role in the plant's defense against pathogenic microorganisms. Further verification is needed to confirm their functions. That is very worthy of study; there has been much discussion and in-depth excavation, and it is also one of the directions for future work.

Conclusions
In this study, the number of Snakin/GASA family members varied among the three mangrove species. Specifically, K. obovata, A. corniculatum, and A. marina had 9, 13, and 27 candidate Snakin/GASA family members, respectively. Sequence alignment showed that the cysteine residues in the GASA domains of Snakin/GASA family members were relatively conserved within the three mangrove species. Additionally, the peptide lengths of the Snakin/GASA family members in the three mangrove species were different. Through evolutionary analysis, prediction of physicochemical properties, and qRT-PCR results, we have identified several potential candidates for HDPs. Taken together, these findings suggest that AmGASA1-5, 7, 13, 15, 18, 23, 25, KoGASA3, 4, AcGASA5, and 10 are most likely involved in plant defense. These candidates warrant further investigation to determine their involvement in plant defenses. This study lays the foundation for further investigation and exploration of the HDPs present in mangrove plants.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes14040923/s1, Figure S1: Normal leaves and leaves infected with pathogenic microorganisms of A. corniculatum, K. obovata and A. marina; Table S1: Primers used for qRT-PCR; Table S2: Reference genes were used in qRT-PCR; Table S3: Subcellular localization prediction of Snakin/GASA family members in A. marina, K. obovata and A. corniculatum; Table S4: Microbial detection of infected leaves. Table S5: Identification of pathogenic microorganism.

Data Availability Statement:
The authors confirm that the data supporting the results of this study can be found within the article or its Supplementary Materials.