Characterization and Expression Analysis of Four Cadmium-Tolerance-Associated Genes of Avicennia marina (Forsk.)

Simple Summary The present study initially identified four full-length genes (peroxidase1, AmPRX1; peroxidase2 AmPRX2; pectate lyase, AmPL; phytosulfokine, AmPSK) associated with metal tolerance in Avicennia marina, one of the pioneer mangrove species with adaptive features. These genes showed the characteristic features of the respective protein families, indicating their evolutionarily conserved relationship with other plant proteins (e.g., CrPRX in Catharanthus roseus, MtPRX in Medicago truncatula, and NnPSK in Nelumbo nucifera). Real-time quantitative PCR revealed that AmPRX1, AmPRX2, and AmPL were most abundantly expressed in the leaves, while AmPSK displayed similar transcript levels across leaves, stems, and roots. Additionally, increased expression levels of these genes were detected in A. marina leaves under cadmium stress. The study thus suggests the role of these four genes in the cadmium stress response and provides a basis for detailed functional studies. Abstract Mangroves colonize the intertidal area of estuaries (e.g., Pichavaram, Payardia, and Mai Po) with remarkable cadmium (Cd) pollution. A study on the mechanism of mangrove plant response to Cd pollution can help to understand the adaptive characteristics of plants under Cd stress. This study explored the roles of peroxidase (PRX), pectate lyase (PL), and phytosulfokine (PSK) genes in cadmium tolerance of mangrove Avicennia marina. Full-length sequences of four genes (i.e., AmPRX1, AmPRX2, AmPL, and AmPSK) associated with metal tolerance were identified with suppression subtractive hybridization and rapid amplification of cDNA ends. These genes showed the characteristic features of the respective protein family, indicating functions similar to other plant proteins. Real-time quantitative PCR analysis demonstrated that cadmium exposure resulted in differences in expression patterns among the tissues. Our findings emphasize the complex regulatory mechanism of these four genes in response to trace metal pollution and reveal their functions in metabolic signaling during the stress response.


Introduction
Mangroves are evergreen plant communities consisting of shrubs or trees commonly found on the coastline of tropical, subtropical, and temperate regions. They are well known for their various ecological services and socio-economic benefits [1,2]. The mangrove wetlands serve as a natural habitat for several species and act as an important resource for timber, fish, and medicinal herbs [3,4]. Mangroves also bind the soil particles together, reduce erosion, and protect the coastal regions. Mangrove sediments are known to effectively trap trace metals due to the large proportion of fine particles, high organic content, and low pH [5]. Consequently, mangroves possess an excellent tolerance for trace metal CuSO 4 , 0.7 µM ZnSO 4 , 9.1 µM MnCl 2 , 28 mM FeSO 4 , and 0.1 µM (NH 4 ) 6 Mo 7 O 24 ; pH adjusted to 6.0 with 1 M NaOH. Hoagland's solution was replaced every 3 days.
After 9 months, the hypocotyls were transferred onto a net and exposed to cadmium stress by submerging in half-strength Hoagland's solution containing 10‰ NaCl and 2 mg L −1 cadmium chloride (CdCl 2 ) for 1, 3, and 7 days. The Cd level was set to simulate the Cd stress in mangrove wetlands under pollution [38]. Plants submerged in halfstrength Hoagland's solution without CdCl 2 under the same conditions as treatment were maintained as the control group. Plants were harvested after 1, 3, and 7-day exposure and washed thoroughly with deionized water. Then plant tissues were stored at −80 • C before analysis.

Identification of the Full-Length cDNA Sequences
Total RNA was extracted from samples following the CTAB method [39]. RNase-free DNase I (TaKaRa, Tokyo, Japan) was added to the CTAB extract to remove genomic DNA. After removing the genomic DNA, the RNA purity and integrity were assessed based on the A260/A280 ratio using Nanodrop and agarose gel (1.0%) electrophoresis. Then, 1 µg of the total RNA was used to synthesize the single-stranded cDNA using SMARTer™ PCR cDNA Synthesis Kit (Clontech, Mountain View, CA, USA).
The 5 and 3 end cDNA sequences of AmPRX1, AmPRX2, AmPL, and AmPSK were obtained via rapid amplification of cDNA ends (RACE) performed using the total RNA extracted from leaves of A. marina with the SMARTer TM RACE Kit (Clontech, Mountain View, CA, USA). The first round of PCR was performed using the gene-specific primer (GSP) (Table S1) and the universal primer mix (UPM), and a second nested PCR was performed with the product of the first PCR using a nested gene-specific primer (NGSP) (Table S1) and the nested universal primer A (NUP). The final PCR products were analyzed on agarose gels, cloned into the pMD-18T vector (Takara, Tokyo, Japan), and sequenced. Finally, the 5 and 3 end sequences were assembled with an overlapping fragment to obtain the full-length cDNA sequences using Lasergene 7 (DNASTAR, WI, USA).

Feature Prediction of Deduced Proteins
The theoretical isoelectric point (pI) and the molecular weight (MW) of the deduced proteins (AmPRX1, AmPRX2, AmPL, and AmPSK) were analyzed in Lasergene 7 (DNAS-TAR, WI, USA). A homology search was conducted with NCBI BLAST (http://blast.ncbi. nlm.nih.gov/Blast.cgi, accessed on 1 December 2022). The three-dimensional model of each protein was built by the SWISS-MODEL server (http://swissmodel.expasy.org, accessed on 1 December 2022). The subcellular localization was predicted using PSORT (http://psort.hgc.jp/form.html, accessed on 1 December 2022). Multiple alignments of amino acid sequences homologous to AmPRX1, AmPRX2, AmPL, and AmPSK were performed using T-Coffee (https://tcoffee.org, accessed on 1 December 2022) [40] to compare the evolution of these genes. Finally, a phylogenetic tree based on amino acid sequences was constructed in Simple Phylogeny. Phylogenetic analysis of the PRX, PL, and PSK proteins was conducted based on the similarity of amino acid sequences. Four amino acid sequences in this study and isozyme sequences from GenBank were combined for phylogenetic analysis.

Quantitative Reverse Transcription Real-Time PCR (RT-qPCR)
Furthermore, RT-qPCR was performed to assess the mRNA expression levels of the genes associated with trace metal tolerance ( Figure S1). The cDNA was synthesized using a PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, Tokyo, Japan), and primers for the RT-qPCR were designed using Primer 5.0 (PREMIER Biosoft, San Francisco, CA, USA). Primers (Table S2) for RT-qPCR were 20-23 bp in length with melting temperatures of 55-58 • C. The PCR products were 100-120 bp in length. The A. marina 18S rRNA gene was used as an internal control. The RT-qPCR was carried out in a reaction mixture (18 µL) containing 12 µL of FastStart Universal SYBR Green Master (ROX; Roche Applied Science, Indianapolis, IN, USA, 2014), 0.25 µL (10 µM) each of sense and antisense primers, 2 µL of cDNA, and 3.5 µL of nuclease-free water. Reactions with cDNA replaced by nuclease-free water were run with each primer pair as a blank control. Three technical replicates were maintained per reaction, and PCR for each EST was performed in triplicate. The PCR was carried out on an ABI 7500 real-time PCR system (Applied Biosystems, Pleasanton, CA, USA) using the following program: 2 min at 50 • C, 10 min denaturation at 95 • C, followed by 40 cycles of 10 s at 95 • C and 1 min at 60 • C; finally, a melting curve analysis was performed for 30 min from 65 • C to 95 • C. The relative gene expression levels were quantified using the 2 − Ct method [41] and expressed as mean values of triplicates relative to the mean values of control at 0 days.

Statistical Analysis
Three replicates were involved in each treatment. Statistical analysis of data was conducted using Statistical Package for Social Science software (16.0, IBM, New York, NY, USA) and R (4.1.3). One-way analysis of variance (ANOVA) was applied to compare gene expression in different organs; two-way ANOVA was applied to the analysis of temporal expression patterns. Duncan's multiple range test was performed to measure the differences between pairs of means. Differences in values were considered statistically significant at p < 0.05 unless otherwise stated.

Structural Characterization of AmPRX1, AmPRX2, AmPL, and AmPSK cDNA Clones
The characteristics of the four genes associated with trace metal tolerance in A. marina are presented in Table 1. Phylogenetic analysis showed that the PRX proteins clustered in two major clades: One included AmPRX1 and the other included AmPRX2. Poor homology was detected in the first 20-30 amino acid residues of AmPRX1 and AmPRX2 ( Figure S2a  Furthermore, PSORT predicted that AmPRX1, AmPRX2, AmPL, and AmPSK are secretory proteins with signal peptides detected at their N-terminal. These genes showed the characteristic features of the respective protein family, indicating functions similar to other plant proteins ( Figure 2). Furthermore, PSORT predicted that AmPRX1, AmPRX2, AmPL, and AmPSK ar cretory proteins with signal peptides detected at their N-terminal. These genes sho the characteristic features of the respective protein family, indicating functions simila other plant proteins ( Figure 2).

Spatial Expression of AmPRX1, AmPRX2, AmPL, and AmPSK Genes in Avicennia marina
Furthermore, the organ-specific expression patterns of AmPRX1, AmPRX2, AmPL, and AmPSK genes in A. marina under normal conditions were analyzed via RT-qPCR. These four genes were detected in all organs (leaves, stems, and roots) but with differences in expression levels (

Expression Levels of AmPRX1, AmPRX2, AmPL, and AmPSK Genes in Avicennia marina under Cadmium Stress
The temporal expression profiles of AmPRX1, AmPRX2, AmPL, and AmPSK genes in the leaves, stems, and roots of A. marina during seven days of cadmium exposure were determined using RT-qPCR. Cadmium stress induced the AmPRX1 gene in the leaves on The gene expression levels were analyzed for 9−month−old hypocotyls under normal conditions. The 18S rRNA gene was used as the reference to normalize the gene e xpression levels. Red lines represent the expression level in stems as baseline.
The relative expression data were log1p transformed.

Spatial Expression of AmPRX1, AmPRX2, AmPL, and AmPSK Genes in Avicennia marina
Furthermore, the organ-specific expression patterns of AmPRX1, AmPRX2, AmPL, and AmPSK genes in A. marina under normal conditions were analyzed via RT-qPCR. These four genes were detected in all organs (leaves, stems, and roots) but with differences in expression levels (Figure 3, ANOVA, p < 0.05). AmPRX1 exhibited a preference for expression in the root (Figure 3a). The expression levels of AmPRX2 and AmPL were the highest in the leaves (Figure 3b,c), followed by stems and roots. Meanwhile, the AmPSK gene displayed comparable transcript levels among leaves, stems, and roots (Figure 3d).

Spatial Expression of AmPRX1, AmPRX2, AmPL, and AmPSK Genes in Avicennia marina
Furthermore, the organ-specific expression patterns of AmPRX1, AmPRX2, AmPL, and AmPSK genes in A. marina under normal conditions were analyzed via RT-qPCR. These four genes were detected in all organs (leaves, stems, and roots) but with differences in expression levels (Figure 3, ANOVA, p < 0.05). AmPRX1 exhibited a preference for expression in the root (Figure 3a). The expression levels of AmPRX2 and AmPL were the highest in the leaves (Figure 3b,c), followed by stems and roots. Meanwhile, the AmPSK gene displayed comparable transcript levels among leaves, stems, and roots (Figure 3d).

Expression Levels of AmPRX1, AmPRX2, AmPL, and AmPSK Genes in Avicennia marina under Cadmium Stress
The temporal expression profiles of AmPRX1, AmPRX2, AmPL, and AmPSK genes in the leaves, stems, and roots of A. marina during seven days of cadmium exposure were determined using RT-qPCR. Cadmium stress induced the AmPRX1 gene in the leaves on

(d) AmPSK
The gene expression levels were analyzed for 9−month−old hypocotyls under normal conditions. The 18S rRNA gene was used as the reference to normalize the gene e xpression levels. Red lines represent the expression level in stems as baseline.
The relative expression data were log1p transformed.

Expression Levels of AmPRX1, AmPRX2, AmPL, and AmPSK Genes in Avicennia marina under Cadmium Stress
The temporal expression profiles of AmPRX1, AmPRX2, AmPL, and AmPSK genes in the leaves, stems, and roots of A. marina during seven days of cadmium exposure were determined using RT-qPCR. Cadmium stress induced the AmPRX1 gene in the leaves on the first day ( Figure 4a). Meanwhile, AmPRX2 exhibited fluctuating expression in the leaves after cadmium exposure (Figure 4b). The expression level of AmPL was detectable on the first day and gradually increased until the end of the experiment (Figure 4c). The maximum expression level was detected on the seventh day, 13.5 times as many as the initial level. The expression levels of AmPL in the stems and roots were significantly lower than the ones in the leaves and even undetectable. PEER REVIEW 7 of 13 the first day ( Figure 4a). Meanwhile, AmPRX2 exhibited fluctuating expression in the leaves after cadmium exposure (Figure 4b). The expression level of AmPL was detectable on the first day and gradually increased until the end of the experiment (Figure 4c). The maximum expression level was detected on the seventh day, 13.5 times as many as the initial level. The expression levels of AmPL in the stems and roots were significantly lower than the ones in the leaves and even undetectable.

Discussion
The present study initially identified four full-length genes associated with metal tolerance (peroxidase, AmPRX1 and AmPRX2; pectate lyase, AmPL; phytosulfokine, AmPSK) in A. marina, one of the pioneer dominant mangrove species, which might have adaptive features to metal stress. These genes showed the characteristic features of the respective protein family, indicating functions similar to other plant proteins (Figures 1 and 2). RT-qPCR was used to reveal the regulation pattern of these genes in A. marina leaves under cadmium stress.
Peroxidase plays a crucial role in many physiological and developmental processes in plants [28,29]. The AmPRX1 and AmPRX2 proteins analyzed in this study showed a high similarity to the peroxidases of other plants. The AmPRX2 protein has a 3D structure highly similar to PePRX (Figure 2). The highly conserved structure suggests that the AmPRX2 protein might play important roles similar to other plant peroxidases. In general, the prosthetic group of peroxidase consists of protein-bound heme, which usually requires a histidine residue as a proximal ligand [44]. However, differences were detected in the N-terminal and C-terminal regions between the AmPRX1 and AtPRX2 proteins, which may be related to the adaptability of different organs of A. marina in response to the specific stresses in the intertidal wetlands.
The study identified AmPRX1 and AtPRX2 as secretory peroxidases with signal peptides detected at their N-terminal. These genes belong to class III of the plant heme-dependent peroxidase superfamily, which contains several conserved features, including four disulfide bridges and two calcium-binding sites [45]. Members of this class are implicated in hydrogen peroxide regulation during cell protection and signaling under abi-

(d) AmPSK
The 18S rRNA gene was used as the reference to normalize the gene e xpression levels. Red lines represent the expression level in initial stems (0 d) as baseline.
The relative expression data were log1p transformed.

Discussion
The present study initially identified four full-length genes associated with metal tolerance (peroxidase, AmPRX1 and AmPRX2; pectate lyase, AmPL; phytosulfokine, AmPSK) in A. marina, one of the pioneer dominant mangrove species, which might have adaptive features to metal stress. These genes showed the characteristic features of the respective protein family, indicating functions similar to other plant proteins (Figures 1 and 2). RT-qPCR was used to reveal the regulation pattern of these genes in A. marina leaves under cadmium stress.
Peroxidase plays a crucial role in many physiological and developmental processes in plants [28,29]. The AmPRX1 and AmPRX2 proteins analyzed in this study showed a high similarity to the peroxidases of other plants. The AmPRX2 protein has a 3D structure highly similar to PePRX (Figure 2). The highly conserved structure suggests that the AmPRX2 protein might play important roles similar to other plant peroxidases. In general, the prosthetic group of peroxidase consists of protein-bound heme, which usually requires a histidine residue as a proximal ligand [44]. However, differences were detected in the N-terminal and C-terminal regions between the AmPRX1 and AtPRX2 proteins, which may be related to the adaptability of different organs of A. marina in response to the specific stresses in the intertidal wetlands.
The study identified AmPRX1 and AtPRX2 as secretory peroxidases with signal peptides detected at their N-terminal. These genes belong to class III of the plant hemedependent peroxidase superfamily, which contains several conserved features, including four disulfide bridges and two calcium-binding sites [45]. Members of this class are implicated in hydrogen peroxide regulation during cell protection and signaling under abiotic stress [46]. Mangroves have an efficient antioxidant defense system. Evidence showing mangrove tissue differences in gene expression will be helpful to reveal the maintenance of cellular homeostasis in plants.
Furthermore, the RT-qPCR revealed differences in the expression levels of AmPRX1 and AmPRX2 among the various organs (leaves, stems, and roots) under normal conditions. AmPRX1 and AmPRX2 were highly expressed in the leaves of A. marina. For instance, AmPRX2 showed the highest expression level among these genes, 9.1 times higher than that in the roots. These differences in expression patterns among the organs may be associated with their various physiological functions. Several researchers have reported tissue specificity of peroxidase paralogous [47], suggesting that differences in tissue expression and regulation are key parts of species evolution that need to be further explored. Specifically, a high peroxidase expression in the leaves can effectively prevent ROS-induced chloroplast damage, ensuring efficient energy acquisition and photosynthesis to encounter stress.
By regulating the hydrolysis of pectic polysaccharides, which are prominent components of plant cell walls, pectate lyase not only plays essential roles in plant growth and development but is also closely involved in signaling and defense responses [48][49][50][51]. The expression of AmPL was much higher in leaves than in stems and roots ( Figure 3). Meanwhile, the expression of AmPL remained continuously up-regulated during 7 days of Cd exposure ( Figure 4). This evidence suggests the specificity of AmPL in leaf resistance to Cd stress. Pectate lyases are depolymerizing plant enzymes involved in cell wall degradation. They catalyze the cleavage of pectate, the de-esterified product of pectin, which is the major component that maintains the structural integrity of cell walls in higher plants [52]. In Arabidopsis, PL-like proteins control stomatal development; they are known to cleave pectins and regulate stomatal dynamics, likely playing essential roles during metal toxicity.
Phytosulfokine, a sulfated pentapeptide growth factor, is an important part of the stimulation of plant proliferation [53] and integrates the growth and defense of plants to balance the competing metabolic costs [54]. Unlike the other three genes in this study, the expression of AmPSK was slightly higher in stems than in leaves and roots but did not show significant differences overall. Furthermore, the expression of AmPSK differed among tissues during Cd exposure. In leaves, AmPSK expression peaked on days 1-3, while in stems and roots, days 3-7 were the periods when AmPSK was primarily upregulated. We speculate that this is related to the overall regulation of AmPSK as a growth factor. PSK is a plant peptide growth factor with hormone-like mitogenic activity; it promotes plant growth via cellular growth and expansion in plants [55].
It must be noted that AmPRX2 was upregulated in the leaves under cadmium stress, while AmPRX1 and AmPRX2 were downregulated in the stems and roots, suggesting a potential, complex role of PRX genes under cadmium stress. Considering the correlation between trace metal tolerance and the upregulation of PRX genes and the downregulation in the stems and roots, it can be assumed that multiple mechanisms are involved in trace metal tolerance in A. marina. In the present study, these four genes were identified in the leaves. Clearly, there are some differential Cd adaptation mechanisms in the roots and stems [38,56,57]. This also represents a wide range of adaptive features of organisms in adaptation to stress, and more refined regulatory mechanisms need to be shown in future studies with the help of spatial transcriptome tools [58].

Conclusions
This study reveals the expression patterns of four genes associated with trace metal tolerance in A. marina. For instance, a significant upregulation (13.5 times) of AmPL was detected in leaves after 7 days of cadmium exposure; however, no significant differences in AmPL expression were detected. Additionally, the AmPSK gene of A. marina exhibited upregulation in the leaves, stems, and roots after cadmium exposure. These observations collectively reveal the precise regulation of the metal tolerance-associated genes at the organ level. However, detailed studies are necessary to explore their roles in providing tolerance, elucidate the complex network of molecular mechanisms, and analyze the synergistic effect of multiple genes in mangrove plants under trace metal stress. Further research is necessary to understand the specific functions and regulatory mechanisms of A. marina, especially in the response mechanisms of stems and roots.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/biology12020216/s1, Figure S1. Diagram for the RT-qPCR protocol; Figure S2. Multiple sequence alignment of PRX (a,b), PL (c), and PSK (d) proteins; Table S1. Primers used for RACE in this study; Table S2. Primers used for qRT-PCR analysis in this study.  Acknowledgments: We are grateful to Chenjuan Zheng of Xiamen University for their help in the process of experimentation The authors are also grateful to the anonymous referees for their constructive comments.

Conflicts of Interest:
The authors declare no conflict of interest.