Comprehensive Analysis of the Catalase (CAT) Gene Family and Expression Patterns in Rubber Tree (Hevea brasiliensis) under Various Abiotic Stresses and Multiple Hormone Treatments

Catalase (CAT) is one of the key enzymes involved in antioxidant defense systems and mainly scavenges H2O2 and plays a vital role in plant growth, development, and various adverse stresses. To date, a systematic study of the CAT gene family in rubber tree has not been reported. In this study, five HbCAT gene family members were identified from the rubber tree genome, and these were mainly clustered into two subfamilies. Gene structure and motif analysis showed that exon-intron and motif patterns were conserved across different plant species. Sequence analysis revealed that HbCAT proteins contain one active catalytic site, one heme-ligand signature sequence, three conserved amino acid residues (His, Tyr, and Asn), and one peroxisome-targeting signal 1 (PTS1) sequence. Fragment duplication is a selection pressure for the evolution of the HbCAT family based on Ka/Ks values. Analysis of cis-acting elements in the promoters indicated that HbCAT gene expression might be regulated by abscisic acid (ABA), salicylic acid (SA), and MYB transcription factors; furthermore, these genes might be involved in plant growth, development, and abiotic stress responses. A tissue-specific expression analysis showed that HbCATs gradually increased with leaf development and were highly expressed in mature leaves. Gene expression profiling exhibited the differential expression of the HbCATs under cold, heat, drought, and NaCl stresses. Our results provide comprehensive information about the HbCAT gene family, laying the foundation for further research on its function in rubber tree.


Introduction
In nature, plants face severe threats from various environmental stresses, including biotic and abiotic stresses [1].Environmental stresses alter the cellular redox stationary phase, leading to the production of large amounts of reactive oxygen species (ROS), such as superoxide anions (O 2− ), hydroxyl radicals (OH − ), per-hydroxyl radicals (HO 2− ), alkoxyl radicals, hydrogen peroxide (H 2 O 2 ), singlet oxygen ( 1 O 2 ), and other oxygen radicals [2][3][4].ROS plays an indispensable role as signaling molecules in a majority of biological processes, such as the regulation of plant growth, development, and response to biotic and abiotic stresses [2,3,5].However, the excessive production of ROS in plant cells leads to lipid peroxidation, as well as damages nucleic acid and protein structures, and impedes carbohydrate synthesis and metabolism, which, in turn, affects plant growth and development, ultimately leading to lower yields [6][7][8].
Various studies have suggested that CAT plays a vital role in plant growth, development, and defense against adverse environmental stresses [20][21][22].In Arabidopsis, AtCAT1 is involved in scavenging H 2 O 2 , which is produced under low temperatures, drought, oxidative stress (3-aminotriazol, 3-AT), methyl viologen (MV), and H 2 O 2 stresses [11].It has been shown that AtCAT2 is significantly induced by cold, drought, and bright-light stress treatments.Additionally, AtCAT3 was shown to be significantly upregulated under abscisic acid (ABA) and oxidative treatments and significantly induced during the senescence stage [11].Under normal conditions, the catalase activity in AtCAT2 mutant leaves was only 20% of that of the wild-type and more H 2 O 2 was accumulated compared to the wild-type; furthermore, the sensitivity of the AtCAT2 mutant to H 2 O 2 , NaCl, cold, and bright-light stresses increased [23].In another study, the results showed that the catalase activity in the AtCAT2 mutant leaves was only 10% of that of the wild-type, and AtCAT3 mutants exhibited an approximate 20% reduction in catalase activity, while AtCAT1 mutants showed similar catalase activity to the wild-type.Double AtCAT1 AtCAT2 and AtCAT2 AtCAT3 mutants showed a similar decrease in leaf catalase activity compared to AtCAT2 mutants [24].In rice, OsCATCs can be phosphorylated and activated by STRK1, which regulates H 2 O 2 homeostasis and enhances tolerance to salt and oxidation stresses [25].A recent study showed that the OsCAT3 prokaryotically expressed protein can significantly remove H 2 O 2 [12].Additionally, OsCAT3 knockout plants showed low viability and significantly reduced CAT, POD, and SOD enzyme activities [12].
Natural rubber (cis-1,4-polyisoprene) represents an important industrial raw and strategic material for people's welfare, defense security, and public safety [26].Due to its unique physical and chemical properties, including elasticity, abrasion resistance, impact, effective thermal dispersion, and ductility at low temperatures [27,28], it has the irreplaceable properties of synthetic rubber, especially in the military, deep sea, and aerospace industries.The rubber tree (Hevea brasiliensis) is a major typical tropical tree species that is capable of producing commercial sources of natural rubber [29], accounting for more than 98% of the world's natural rubber production [30].Currently, rubber trees are cultivated in more than 40 countries around the world [31], with more than 90% of the natural rubber production taking place in South and Southeast Asia [26][27][28][29].With the escalating demand for natural rubber, rubber tree cultivation plantations have progressively expanded to nontraditional regions, such as the central highlands of Vietnam (12 • N), northern India (29 • N), the southern highlands of Brazil (23 • S), and northeastern Thailand, as well as southwestern China (22 • N) [32].Consequently, commercial rubber tree plantations are frequently exposed to various environmental stresses (drought, cold, strong wind), overtapping, and overstimulation, which can affect the growth and development of rubber tree, ultimately leading to a decline in production [33][34][35].
To the best of our knowledge, the CAT gene family has not been characterized in rubber tree.Therefore, a comprehensive genome-wide analysis of the CAT gene family in rubber tree was performed, and the physicochemical properties, gene structures, protein motifs, chromosomal locations, phylogenetic tree, cis-elements, gene duplication, and homology analyses were discussed in detail.Furthermore, the expression patterns of HbCAT genes under various abiotic stresses and exogenous hormone treatments were comprehensively analyzed.

Identification of the CAT Gene Family in Rubber Tree
In the present study, five CAT genes were identified in the complete genome of the rubber tree via HMMER search and BLASTP methods.Detailed information on the five HbCAT genes is presented in Table 1.These genes are named HbCAT1-HbCAT5 according to their order of localization on the chromosomes.Further analysis indicated that the coding sequence (CDS) lengths of these HbCAT genes ranged from 1236 bp (HbCAT5) to 1704 bp (HbCAT2), and the lengths of the encoded protein sequences ranged from 411 aa (HbCAT5) to 567 aa (HbCAT2).The molecular weights ranged from 47.15 kDa (HbCAT4) to 65.13 kDa (HbCAT2), and the isoelectric points ranged from 6.45 (HbCAT4) to 7.60 (HbCAT1).

Phylogenetic Analysis of CAT Genes
To investigate the phylogenetic relationships of HbCAT genes between other plant species, we obtained three AtCAT genes from a dicotyledonous model plant the Arabidopsis (Arabidopsis thaliana) database.Additionally, we identified OsCAT, PtCATs, and MeCATs from monocotyledonous model plant rice (Oryza sativa), woody model plant populus (Populus trichocarpa) [36][37][38], and euphorbiaceae family cassava (Manihot esculenta) genomes using the same methods (HMMER and BLASTP).Three CAT genes were identified in rice, four in populus, and six in cassava (Table S2).A phylogenetic tree was constructed using the neighbor-joining (NJ) method based on the alignment of 21 CAT protein sequences, which were classified into four groups (Groups I-IV) (Figure 1).As shown in Figure 1, five CAT members (AtCAT1, AtCAT3, OsCAT1, MeCAT3, and MeCAT6) were clustered in group I; five CAT members (HbCAT2, HbCAT4, HbCAT5, MeCAT4, and MeCAT5) were clustered in group II; four CAT members (OsCAT2, OsCAT3, PtCAT1, and PtCAT3) were clustered in group III; and seven CAT members (AtCAT2, MeCAT1, MeCAT2, HbCAT1, HbCAT3, PtCAT2, and PtCAT4) were clustered in group IV.Furthermore, HbCAT genes were mainly clustered into two evolutionary branches (groups II and IV), and group II represented the unique branch of the euphorbiaceous family (Figure 1).
HbCAT1, HbCAT3, PtCAT2, and PtCAT4) were clustered in group IV.Furthermore, HbCAT genes were mainly clustered into two evolutionary branches (groups II and IV), and group II represented the unique branch of the euphorbiaceous family (Figure 1).

Gene Structures and Conserved Motifs of CATs
To better understand the HbCAT gene family, we extracted the information on the CAT gene structures from the genome annotation file and visualized them using TBtools software (v1.0987663) (Figure 2B).We observed that all HbCAT genes exhibited a consistent pattern of eight exons and seven introns, except for HbCAT2, which had nine exons and eight introns.Furthermore, we found that the intron-exon structural patterns of CAT genes are highly conserved across species.For example, 12 (57.14%)out of the 21 CAT genes contained eight exons and seven introns.To further explore the functional diversity of CAT genes, conserved motifs of CAT proteins were analyzed using the MEME online website, and eight motifs were identified (Figure 2C).Detailed information on the eight motifs is presented in supplementary Table S3 and Figure S1.Almost all CAT genes from different species contained eight conserved motifs, except MeCAT5 (7), HbCAT5 (7), MeCAT1 (6), and PtCAT4 (4).In conclusion, CAT genes from different species have highly similar conserved gene structures and motif patterns, and the ones on the same evolutionary branch have similar gene structures and conserved motif patterns.These results strongly support evolutionary taxonomic identity and suggest that CAT genes are conserved during evolution (Figure 2A).
A multiple protein sequence alignment analysis indicated that except for the HbCAT5 gene, which has no heme-ligand signature sequence (RIFSYSDTQ) and tyrosine (Tyr), all HbCAT genes contained one active catalytic site (FDRERIPERVVHARGASA), one heme ligand signature sequence (RIFSYSDTQ), and three conserved amino acid residues, such

Gene Structures and Conserved Motifs of CATs
To better understand the HbCAT gene family, we extracted the information on the CAT gene structures from the genome annotation file and visualized them using TBtools software (v1.0987663) (Figure 2B).We observed that all HbCAT genes exhibited a consistent pattern of eight exons and seven introns, except for HbCAT2, which had nine exons and eight introns.Furthermore, we found that the intron-exon structural patterns of CAT genes are highly conserved across species.For example, 12 (57.14%)out of the 21 CAT genes contained eight exons and seven introns.To further explore the functional diversity of CAT genes, conserved motifs of CAT proteins were analyzed using the MEME online website, and eight motifs were identified (Figure 2C).Detailed information on the eight motifs is presented in supplementary Table S3 and Figure S1.Almost all CAT genes from different species contained eight conserved motifs, except MeCAT5 (7), HbCAT5 (7), MeCAT1 (6), and PtCAT4 (4).In conclusion, CAT genes from different species have highly similar conserved gene structures and motif patterns, and the ones on the same evolutionary branch have similar gene structures and conserved motif patterns.These results strongly support evolutionary taxonomic identity and suggest that CAT genes are conserved during evolution (Figure 2A).
A multiple protein sequence alignment analysis indicated that except for the HbCAT5 gene, which has no heme-ligand signature sequence (RIFSYSDTQ) and tyrosine (Tyr), all HbCAT genes contained one active catalytic site (FDRERIPERVVHARGASA), one heme ligand signature sequence (RIFSYSDTQ), and three conserved amino acid residues, such as histidine (His), tyrosine (Tyr), and aspartic acid (Asn).In addition, all proteins contained one peroxisome targeting signal type 1 (PTS1) sequence (QKL) (Figure 3).

Chromosomal Localization and Synteny Analysis of CAT Genes
Chromosomal location analysis indicated that five HbCAT genes were unevenly anchored to only three of the eighteen chromosomes of the rubber tree.HbCAT1 and HbCAT2 were located on Chr2 and Chr5, respectively, and HbCAT3, HbCAT4, and HbCAT5 were located on Chr14 (Figure 4A).and the heme-ligand signature sequence (RLFSYNDTQ) are highlighted in blue, and the peroxisometargeting signal 1 (PTS1) sequence is highlighted in green.

Chromosomal Localization and Synteny Analysis of CAT Genes
Chromosomal location analysis indicated that five HbCAT genes were unevenly anchored to only three of the eighteen chromosomes of the rubber tree.HbCAT1 and HbCAT2 were located on Chr2 and Chr5, respectively, and HbCAT3, HbCAT4, and HbCAT5 were located on Chr14 (Figure 4A).To better understand the evolution of HbCAT genes, replication events of HbCAT genes were analyzed using the TBtools software (v1.0987663) [39].As shown in Figure 4B, there were eight homologous HbCAT gene pairs in the rubber tree, originating from fragment replication, which included HbCAT1/HbCAT2, HbCAT1/HbCAT3, HbCAT1/HbCAT4, HbCAT2/HbCAT4, HbCAT2/HbCAT3, HbCAT2/HbCAT5, HbCAT3/HbCAT4, and HbCAT4/HbCAT5 (Figure 4B, Table 2).Although the HbCAT3, HbCAT4, and HbCAT5 genes were located on the same chromosome (Chr14), they were separated by at least one megabase (Table 2), suggesting that these genes may have been formed by fragmentary replication.In conclusion, these results suggest that the amplification of the HbCAT gene family was primarily derived from fragment replication.The Ka, Ks, and Ka/Ks values of HbCAT homologous gene pairs were evaluated, and the results showed that the Ka/Ks values of all homologous gene pairs were less than 1 (Table 2), indicating that purifying selection is the selective pressure for HbCAT gene evolution.
To further investigate the evolutionary origins and orthologous relationships of CAT genes among different plant species, a synteny analysis of four dicotyledonous plants (H.brasiliensis, P. trichocarpa, A. thaliana, and M. esculenta) and one monocotyledonous plant (O.sativa) was performed.As shown in Figure 5, there are four orthologous CAT gene To better understand the evolution of HbCAT genes, replication events of HbCAT genes were analyzed using the TBtools software (v1.0987663) [39].As shown in Figure 4B, there were eight homologous HbCAT gene pairs in the rubber tree, originating from fragment replication, which included HbCAT1/HbCAT2, HbCAT1/HbCAT3, HbCAT1/HbCAT4, Hb-CAT2/HbCAT4, HbCAT2/HbCAT3, HbCAT2/HbCAT5, HbCAT3/HbCAT4, and HbCAT4/HbCAT5 (Figure 4B, Table 2).Although the HbCAT3, HbCAT4, and HbCAT5 genes were located on the same chromosome (Chr14), they were separated by at least one megabase (Table 2), suggesting that these genes may have been formed by fragmentary replication.In conclusion, these results suggest that the amplification of the HbCAT gene family was primarily derived from fragment replication.The Ka, Ks, and Ka/Ks values of HbCAT homologous gene pairs were evaluated, and the results showed that the Ka/Ks values of all homologous gene pairs were less than 1 (Table 2), indicating that purifying selection is the selective pressure for HbCAT gene evolution.
To further investigate the evolutionary origins and orthologous relationships of CAT genes among different plant species, a synteny analysis of four dicotyledonous plants (H.brasiliensis, P. trichocarpa, A. thaliana, and M. esculenta) and one monocotyledonous plant (O.sativa) was performed.As shown in Figure 5, there are four orthologous CAT gene pairs between H. brasiliensis and A. thaliana, nineteen pairs between H. brasiliensis and P. trichocarpa, thirteen pairs between H. brasiliensis and M. esculenta, and seven pairs between H. brasiliensis and O. sativa.Four HbCAT genes (HbCAT1, HbCAT2, HbCAT3, and HbCAT4) had homologous gene pairs for O. sativa, A. thaliana, P. trichocarpa, and M. esculenta, indicating that these genes possibly evolved from genes that existed before the differentiation of monocotyledons and dicotyledons.Homologous gene pairs of the HbCAT5 gene were present in woody plants, such as P. trichocarpa (HbCAT5/PtCAT1/2/3) and M. esculenta (HbCAT5/MeCAT5), but not in the herbaceous plants A. thaliana and O. sativa (Table S4), suggesting that HbCAT5 may have formed after the differentiation of herbaceous and woody plants.

Analysis of Cis-Elements in the Promoters of HbCAT Genes
To further understand the potential regulatory mechanisms of the HbCAT genes under abiotic stresses, cis-acting elements in the promoter regions of each HbCAT gene were scanned using the plantCARE.A total of 25 cis-acting elements were detected in the promoter regions of the HbCAT genes, which were mainly categorized into four categories:

Analysis of Cis-Elements in the Promoters of HbCAT Genes
To further understand the potential regulatory mechanisms of the HbCAT genes under abiotic stresses, cis-acting elements in the promoter regions of each HbCAT gene were scanned using the plantCARE.A total of 25 cis-acting elements were detected in the promoter regions of the HbCAT genes, which were mainly categorized into four categories: hormone-responsive, environmental stress-related, organizational development, and lightresponsive.Detailed information on these cis-acting elements is presented in Table S5.Hormone-responsive elements were identified in several HbCAT genes (Figure 6A,B), e.g., the abscisic acid (ABA) response element ABRE was detected in the HbCAT1/3/5 genes, and the salicylic acid (SA) response element TCA was identified in the HbCAT1/3 genes, suggesting that the expression of these genes may be regulated by the ABA and SA hormone signaling pathways.In addition, at least one abiotic stress-related responsive element was identified in almost all HbCAT genes.For example, HbCAT1/2/3/5 contained defense and stress-responsive elements (TC-rich repeats), HbCAT1/3/4/5 contained LTR elements involved in low-temperature responsiveness, and HbCAT1/2/4/5 contained woundresponsive elements (WUN motifs), suggesting that these genes may be involved in the response to various abiotic stresses.HbCAT2/4/5 contained a cis-acting regulatory element related to meristem expression, implying that these genes may be involved in meristem development.HbCAT2 and HbCAT3 contained MYB transcription factor-binding sites (Figure 6B), suggesting that the HbCAT2 and HbCAT3 genes may be regulated by MYB transcription factors.Moreover, almost all HbCAT genes contained various light-responsive elements (TCT-motif, LAMP-element, chs-CMA1a, Box 4, I-box, chs-CMA2a, AT1-motif, ATC-motif, GT1-motif, MRE, G-Box, AE-box, GATA-motif, TCCC-motif, and ACE), and Box4 and G-box elements were abundantly enriched (Figure 6A,B; Table S5), suggesting that HbCAT genes may play vital roles in light stress response.

Functional Gene Ontology (GO) Annotation of HbCAT Genes
To further understand the potential functions of the HbCAT genes, we performed a gene ontology (GO) annotation and enrichment analysis.The GO_BP enrichment analysis showed that these genes were primarily involved in responding to abiotic and oxidative stresses, e.g., response to reactive oxygen species (GO:0000302), stress (GO:0006950), hy-

Expression Patterns of HbCAT Genes in Different Tissues
The expression patterns of the HbCAT genes in seven different tissues were detected using the RT-qPCR technique to elucidate their transcript levels in various tissues.As shown in Figure 8, HbCAT genes were constitutively expressed in the bark, root, latex, budburst leaf, copper-brown leaf, light-green leaf, and mature leaf of the rubber tree cultivar GT1, albeit at different levels.For instance, HbCAT1 and HbCAT2 were highly expressed in all tissues.All HbCAT genes showed high expression levels in the root and latex, except for HbCAT4, which exhibited a low level in latex.The expression levels of all HbCAT genes gradually increased with leaf development and remained on a higher level in mature leaves compared with the other stages, suggesting that these genes may play vital roles in the growth and development of rubber tree leaves.

Expression Patterns of HbCAT Genes in Different Tissues
The expression patterns of the HbCAT genes in seven different tissues were detected using the RT-qPCR technique to elucidate their transcript levels in various tissues.As shown in Figure 8, HbCAT genes were constitutively expressed in the bark, root, latex, budburst leaf, copper-brown leaf, light-green leaf, and mature leaf of the rubber tree cultivar GT1, albeit at different levels.For instance, HbCAT1 and HbCAT2 were highly expressed in all tissues.All HbCAT genes showed high expression levels in the root and latex, except for HbCAT4, which exhibited a low level in latex.The expression levels of all HbCAT genes gradually increased with leaf development and remained on a higher level in mature leaves compared with the other stages, suggesting that these genes may play vital roles in the growth and development of rubber tree leaves.

Expression Patterns of HbCAT Genes under Different Abiotic Stresses and Exogenous Hormone Treatments
To confirm whether the HbCAT genes responded to different abiotic stresses and exogenous hormone treatments, the expression patterns of the HbCAT genes under low temperature (4 °C), high temperature (42 °C), drought, and high salt (300 mM NaCl) stresses were analyzed using the RT-qPCR technique.The results indicated that the expression levels of most of the HbCAT genes were significantly induced by most of the treatments (Figure 9).For instance, the expression level of HbCAT3 increased gradually with the duration of cold stress and peaked after 24 h, which was 133.06-fold that of the control (Figure 9A).HbCAT3 was also strongly upregulated under high temperature, drought, and NaCl treatments and peaked after 48 h (Figure 9B,D).The expression levels of HbCAT1/2 initially increased and then decreased under cold, drought, and salt stresses.In contrast, HbCAT4/5 genes were not significantly altered at early treatment stages (1 h, 3 h, 6 h, and 12 h); however, they were significantly upregulated at the late treatment stages (24 h and 48 h) under cold and drought stresses (Figure 9A,B,D).Under heat stress, the expression levels of HbCAT2/4 were significantly downregulated at all time points during the treatment (Figure 9C).The expression level of HbCAT1 was strongly induced by ABA and MeJA and significantly upregulated at all time points during the treatments.In contrast, HbCAT3 was strongly downregulated under ABA and MeJA treatments (Figure 9E,F).

Expression Patterns of HbCAT Genes under Different Abiotic Stresses and Exogenous Hormone Treatments
To confirm whether the HbCAT genes responded to different abiotic stresses and exogenous hormone treatments, the expression patterns of the HbCAT genes under low temperature (4 • C), high temperature (42 • C), drought, and high salt (300 mM NaCl) stresses were analyzed using the RT-qPCR technique.The results indicated that the expression levels of most of the HbCAT genes were significantly induced by most of the treatments (Figure 9).For instance, the expression level of HbCAT3 increased gradually with the duration of cold stress and peaked after 24 h, which was 133.06-fold that of the control (Figure 9A).HbCAT3 was also strongly upregulated under high temperature, drought, and NaCl treatments and peaked after 48 h (Figure 9B,D).The expression levels of HbCAT1/2 initially increased and then decreased under cold, drought, and salt stresses.In contrast, HbCAT4/5 genes were not significantly altered at early treatment stages (1 h, 3 h, 6 h, and 12 h); however, they were significantly upregulated at the late treatment stages (24 h and 48 h) under cold and drought stresses (Figure 9A,B,D).Under heat stress, the expression levels of HbCAT2/4 were significantly downregulated at all time points during the treatment (Figure 9C).The expression level of HbCAT1 was strongly induced by ABA and MeJA and significantly upregulated at all time points during the treatments.In contrast, HbCAT3 was strongly downregulated under ABA and MeJA treatments (Figure 9E,F).

Discussion
Abiotic stress is a vital factor affecting rubber tree growth, development, latex yield, and latex quality [40][41][42], especially in China, which is located in a nontraditional rubber plantation belt.Catalase (CAT) has been confirmed to play a vital role in plant abiotic stresses [43][44][45].However, the characterization of the HbCAT gene family of rubber tree and its biological function under abiotic stress has not been reported.In our study, a total of five HbCAT genes were identified in rubber tree based on a genome-wide search using catalase (PF00199) and catalase-related (PF06628) Hidden Markov Model (HMM) files as references and comparative analyses with AtCAT protein sequences, which were localized on chromosomes Chr2 (HbCAT1), Chr5 (HbCAT2), and Chr14 (HbCAT3/4/5) (Figure 4A).We identified four PtCAT gene family members in P. trichocarpa and six MeCAT gene family members in M. esculenta, using the same methods (HMMER and BLASTP).Previous

Discussion
Abiotic stress is a vital factor affecting rubber tree growth, development, latex yield, and latex quality [40][41][42], especially in China, which is located in a nontraditional rubber plantation belt.Catalase (CAT) has been confirmed to play a vital role in plant abiotic stresses [43][44][45].However, the characterization of the HbCAT gene family of rubber tree and its biological function under abiotic stress has not been reported.In our study, a total of five HbCAT genes were identified in rubber tree based on a genome-wide search using catalase (PF00199) and catalase-related (PF06628) Hidden Markov Model (HMM) files as references and comparative analyses with AtCAT protein sequences, which were localized on chromosomes Chr2 (HbCAT1), Chr5 (HbCAT2), and Chr14 (HbCAT3/4/5) (Figure 4A).We identified four PtCAT gene family members in P. trichocarpa and six MeCAT gene family members in M. esculenta, using the same methods (HMMER and BLASTP).Previous studies have also shown that the number of CAT gene families is small in plants, e.g., there are three CAT genes in A. thaliana [11], rice [10,12], and small radish [13], four in cucumber and soybeans [14,15], seven in cotton [16], ten in wheat [17], fourteen in rapeseed [18], and sixteen in sugarcane [19].Typical CAT protein is a tetrameric enzyme with four identical subunits, and each subunit contains a heme prosthetic group in the catalytic center [46].In this study, we found that the HbCAT (HbCAT1-HbCAT4) genes had one active catalytic site (FDRERIPERVVHARGASA), one heme ligand signature motif (RIFSYSDTQ), and three conserved amino acid residues (HIS, Tyr, and Asn) (Figure 3).In addition, all HbCAT (HbCAT1-HbCAT5) genes included one peroxisome targeting signal type 1 (PTS1) sequence (QKL).These results are consistent with previous research on cucumber and sugarcane [14,19].Peroxisomal matrix proteins primarily interact with the cytoplasmic receptor PEX5 via the C-terminal tripeptide of PTS1, thereby causing efficient transport to the peroxisome [47,48].In pumpkin (Cucumis sativus), CAT1 contains a C-terminal PTS1 (peroxisome-targeting signal 1) sequence QKL, which deviates from the conventional SKL motif of typical PTS1 signals [49].In Arabidopsis, the import of CAT1 into peroxisomes relies on its dependence on the cytoplasmic PTS receptor PEX 5p, which is similar to typical PTS1 import; however, unlike typical PTS1, CAT1 specifically interacts with the N-terminal structural domain of PEX 5 rather than its C-terminal structural domain [50].Although the precise mechanism of plant CAT entry into the peroxisome remains unclear, it is noteworthy that the C-terminal PST1 sequence plays a pivotal role in this process.These results indicated that almost all members of the HbCAT gene family have conserved CAT structural domains and exert functions related to CAT activity.
According to the phylogenetic tree analysis, 21 CATs were clustered into four groups, which is consistent with previous research on CAT evolutionary relationships between the BnCAT, BoCAT, BraCAT, and AtCAT genes [18].HbCATs were primarily clustered into groups II and IV along with AtCATs and MeCATs (Figure 1), suggesting that the evolutionary pedigrees of rubber tree, Arabidopsis, and cassava are comparable.Notably, group II contained only HbCAT and MeCAT genes, implying that these genes may be unique to euphorbiaceous plants and have distinct biological functions.The study of gene intron-exon structural patterns can contribute to our understanding of the phylogenetic history of the CAT gene family.The gene exon-intron structure represents a vital feature of gene evolution.In general, closely related genes have similar exon-intron patterns and protein structures [51,52].In our study, all HaCAT genes contained seven introns and eight exons, except HbCAT2, which had eight introns and nine exons (Figure 2B), suggesting that the number of introns and exons of HbCAT genes has not changed significantly during the evolutionary process.CAT genes from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, and M. esculenta showed highly conserved structures.For example, 12 (57.14%) of the 21 CAT genes contained seven introns and eight exons.A previous study reported that the ancestral copy of the CAT gene included seven introns, and these positions are conserved [53].In addition, all HbCAT proteins contained catalase (PF00199) and catalase-related (PF06628) domains, and together these results confirmed that CAT genes are highly conserved during plant evolution, with a few exceptions.Similar findings were obtained in several previous studies [17,18].
Replication events are the basis for the divergence of homologous gene functions and the primary driver of gene family membership expansion [54], which is the most assessed method in gene family expansion analyses.In the present study, gene duplication event analysis suggested that fragment duplication was the primary driver of HbCAT gene evolution.Numerous studies have reported that segmental duplication was the primary driving force of expansion for CAT gene family members [15,19].Evaluation of the Ka, Ks, and Ka/Ks values for HbCAT homologous gene pairs showed that the purification selection is a selection pressure for HbCAT gene evolution (Table 2).Meanwhile, to investigate the evolutionary relationship of HbCATs, we performed a synteny analysis of HbCAT genes between AtCATs, OsCATs, PtCATs, and MeCATs.The results showed that the highest to lowest homologous pairs were populus (19), cassava (13), rice (7), and Arabidopsis (4) (Table S4).Interestingly, HbCAT5 had no homologous pairs with rice and Arabidopsis; however, it had three and one homologous gene pairs with populus and cassava, respectively, which may be unique to woody plants, and this gene may have an irreplaceable function in plants.
Cis-acting elements play a significant regulatory role in gene transcription and can bind sites for transcription factors with core promoters and regulate the transcription of target genes [55,56].Previous studies have suggested that CAT promoters contain numerous light-, hormone-, stress-responsive cis-acting elements, meristem expression relevant elements, and MYB binding sites.Furthermore, CAT genes can be induced by different abiotic stresses, such as cold [17][18][19], drought [11,14,15,18,19], heat [57,58], and salt [14,17,18] stresses.In our study, promoter analyses revealed that all five HbCAT genes contained the core components, such as the "TATA box", and a series of stress-related elements (TC-rich repeats, LTR elements, and WUN-motif), light-responsive elements (TCT-motif, LAMP-element, chs-CMA1a, Box 4, I-box, chs-CMA2a, AT1-motif, ATC-motif, GT1-motif, MRE, G-Box, AE-box, GATA-motif, TCCC-motif, and ACE), hormone-related cis-acting elements (ABRE, TCA, and ARE) (Figure 6A,B), and several genes (HbCAT2, and HbCAT3) contained MYB binding sites.Moreover, these cis-acting elements were irregularly dispersed in the promoter region of the HbCAT genes (Figure 6).Cis-acting elements distributed in different locations facilitate the regulation of protein functions under various environmental stimuli.These results suggested that HbCAT genes may be involved in the regulation of various stresses and biological processes.
Numerous studies have suggested that CAT genes play a vital role in plant growth, development, and defense against adverse environmental stresses.In Arabidopsis, the expression of AtCAT1 was low under normal conditions; however, it was significantly upregulated by cold, drought, and oxidative stresses (H 2 O 2 , 3-aminotriazol, and methyl viologen) and by treatments with the plant hormones SA and ABA; AtCAT2 was significantly upregulated under drought and H 2 O 2 stresses and AtCAT3 was significantly increased under oxidative stress and ABA treatments [11].Further studies found that AtCAT2 mutants exhibited enhanced sensitivity to H 2 O 2 , NaCl, cold, and bright light stresses [23].In sugarcane, the expression of ScCAT1 was significantly upregulated under S. scitamineum stress.Furthermore, overexpression of the ScCAT1 gene in tobacco could enhance resistance to pathogen infection by scavenging excessively toxic ROS [19].In durum wheat, the expression of TaCAT2 and TaCAT3 was significantly upregulated after salt, mannitol, cold, heat, and ABA treatments [59].In soybean, the expression of the GmaxCAT17.1 gene under drought stress exhibited a continuous upregulation at all treatment points (4 h, 8 h, and 12 h), and GmaCAT6.1,GmaCAT4, and GmaCAT17.1 were significantly upregulated under combined drought and heat stresses [15].In rapeseed, Raza et al. [18] reported that BnCAT1, BnCAT2, BnCAT3, BnCAT12, and BnCAT13 were primarily induced by ABA, gibberellic acid (GA), IAA, cold, and salinity treatments.In our study, the expression of the HbCAT3 gene was strongly upregulated by low temperature (4 • C), high temperature (42 • C), drought, and high salt (300 mM NaCl) stresses, and by ABA (100 µM) and MeJA (100 µM) treatments (Figure 9).The expression levels of the HbCAT1/2 initially increased and then decreased under cold, drought, and salt stresses.In contrast, the expression levels of the HbCAT4/5 genes were not significantly changed at early treatment stages (1 h, 3 h, 6 h, and 12 h); however, they were significantly upregulated at the late treatment stages (24 h and 48 h) under cold and drought stresses (Figure 9A,B,D).The expression level of HbCAT1 was significantly upregulated at all time points under ABA and MeJA treatments.In contrast, the expression level of HbCAT3 was strongly downregulated (Figure 9E,F), indicating a close correlation between the expression of these HbCAT genes and the ABA and MeJA signaling pathways in rubber tree.These findings suggest that HbCAT genes play crucial roles in responding to cold, heat, drought, and salt stress.CATs are key enzymes responsible for regulating cellular H 2 O 2 levels to protect normal physiological processes from damage under abiotic stresses [9].Wu et al. [19] found that the expression of the sugarcane ScCAT1 gene exhibited a "rising and then declining" pattern in response to S. scitamineum stress in both YT93-159 (smut resistant) and ROC22 (smut susceptible) sugarcane cultivars.However, the CAT enzyme activity exhibited a sustained increase, suggesting that each member of the CAT gene family may make distinct contributions to the activity of catalase.Consequently, the relationship between HbCAT gene expression and CAT activity under abiotic stresses requires further investigation.In summary, these results lay the foundation for further exploration of the functions of stress-related CAT genes and further understanding of the roles of these genes in stress-tolerance mechanisms.

Phylogenetic Tree, Gene Structures, and Conserved Motifs
To further clarify the evolutionary relationships between the HbCATs and CATs of other plant species, we downloaded the genome annotation and protein sequence files of the rice (O.sativa Japonica Group), Poplar (P.trichocarpa) [36][37][38], and cassava (M.esculenta) from the Ensembl Plant genome database (https://plants.ensembl.org/index.html,accessed on 5 March 2023) [70].Similarly, HMMER searches based on catalase-conserved domains (PF00199 and PF06628) and BLASTP searches with AtCAT protein sequences as references were performed to obtain CAT gene families from different species.A neighbor-joining (NJ) phylogenetic tree was constructed using MEGA X [71] with 1000 independent replications, and the phylogenetic tree was further beautified using the Evolview online website (https: //www.evolgenius.info/,accessed on 3 June 2023) [72].Protein-conserved motifs were analyzed using the MEME website (http://meme-suite.org/tools/meme,accessed on 13 March 2023) [73], and the number of motifs was set to 8 and the sequence length to being between 6 bpm and 100 bp.Intron-exon information for CAT genes was extracted from the genome annotation file and visualized using TBtools software (v1.0987663) [39].

Chromosomal Localization, Gene Duplication, and Synteny Analysis
The chromosome location information of the HbCAT genes was obtained from the gene annotation file of the rubber tree genome, and each HbCAT gene was mapped to a chromosome using TBtools.The genome-wide replication events and replication relationships of CATs were analyzed using the MCScanX module of the TBtools software (v1.0987663) [39] with default values.Additionally, with the use of TBtools, the collinear relationships within the HbCAT gene family and between other species were visualized, and the nonsynonymous (ka)/synonymous (ks) substitutions of CAT gene pairs were identified.

Cis-Acting Elements Analysis
To further investigate the potential functions of HbCAT genes, we extracted 2000 bp DNA sequences upstream of the HbCAT gene start codon (ATG) as the promoter sequence of the HbCAT genes using TBtools software (v1.0987663) [39] and submitted these sequences to the PlantCARE online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 3 April 2023) [74] to predict potential cis-acting elements for each promoter region.Subsequently, the prediction results were visualized using TBtools.

GO Enrichment Analysis of HbCAT Genes
Gene functional annotation analysis was performed using the online eggNOG database (http://eggnog-mapper.embl.de/,accessed on 20 June 2023) [75].The GO annotation-based package hierarchy file (go-basic.obo)was downloaded, followed by a GO functional enrichment analysis using the "GO enrichment" module of TBtools software (v1.0987663) [39].

Plant Materials and Stress Treatments
The 12-year-old rubber tree cultivar GT1 clone was used to determine the tissuespecific expression grown on the Experimental Farm of Yunnan Institute of Tropical Crop (100.788E, 22.036 N).The trees were regularly tapped for latex collection using a half-spiral pattern every four days (S/2, d/4) without ethylene (ET) stimulation.Samples of leaves at different developmental stages (budburst, copper-brown, light-green, and mature stages), bark, root, and latex were collected from ten healthy tapping trees, with three biological replicates.
The expression patterns of the HbCAT gene family under various abiotic stresses and exogenous hormone (ABA, MeJA) treatments were analyzed using asexual GT1 bagged seedlings that had developed one extension unit, and the leaves were completely mature.Plantlets were transferred into a growth chamber under a 16 h light/8 h dark cycle at 28 • C with 80% relative humidity.Two days later, plantlets were treated in a growth chamber at 4 • C and 42 • C for low/high-temperature stress treatments.For the drought stress treatment group, plantlets were removed from all culture bags and soil, and bare-root cultivated in a growth chamber at 28 • C with an 80% relative humidity [63,76].For the salt stress treatment, plantlets were irrigated with 300 mM NaCl solution [63].For the hormone treatments, plantlets were sprayed with 100 µM abscisic acid (ABA) and 100 µM methyl jasmonate (MeJA) solution, and the control group was treated with 0.02% Tween-20.Leaf samples were collected at various time points (0, 1, 3, 6, 12, 24, and 48 h) during treatments and immediately frozen in liquid nitrogen and stored at −80 • C for further analysis.There were three independent biological replicates in each sample from five plantlets (n = 5).

RNA Extraction and RT-qPCR Analysis
Total RNA was extracted from each sample using an RNAprep Pure Plus kit (Tiangen, Beijing, China).First-strand cDNA was synthesized using a RevertAid TM First Strand cDNA Synthesis kit (Thermo Scientific, Vilnius, Lithuania).Specific primers were designed using the NCBI database (https://www.ncbi.nlm.nih.gov/,accessed on 29 April 2022) and synthesized by Sangon Biotech (Shanghai) Co., Ltd.(Shanghai, China).Detailed primer sequences are listed in Table S1.RT-qPCR analysis was performed using TB Green ® Premix Ex Taq™ (Takara, Beijing, China) from the TaKaRa Company and a qTOWER3G real-time fluorescence quantification system (Shanghai, China).The HbActin7a gene was used as an internal reference.Three technical replicates were performed for RT-qPCR.Raw data were normalized as described previously [63,76].

Figure 1 .
Figure 1.Phylogenetic analysis of CAT proteins from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, and M. esculenta.The sequences of five HbCAT proteins (red circle), three AtCAT proteins (green rectangles), three OsCAT proteins (dark-violet rectangles), four PtCAT proteins (yellow triangles), and six MeCAT proteins (blue stars) were multiply aligned using Clustal W. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replications.Four subgroups (Groups I-IV) were classified with different background colors.

Figure 1 .
Figure 1.Phylogenetic analysis of CAT proteins from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, and M. esculenta.The sequences of five HbCAT proteins (red circle), three AtCAT proteins (green rectangles), three OsCAT proteins (dark-violet rectangles), four PtCAT proteins (yellow triangles), and six MeCAT proteins (blue stars) were multiply aligned using Clustal W. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1000 bootstrap replications.Four subgroups (Groups I-IV) were classified with different background colors.

Figure 2 .
Figure 2. Phylogenetic tree, gene structures, and conserved motifs of the CAT genes from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, and M. esculenta.(A) A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replications.(B) Gene structure of CAT genes.Upstream/downstream sequences are represented by light green boxes, and exons and introns are represented by yellow boxes and black horizontal lines, respectively.(C) Conserved motifs of CAT proteins.Different colored boxes show various motifs.

Figure 3 .
Figure 3. Multiple protein sequence alignment of the HbCAT gene family.Three conserved amino acids, His (a), Asn (b), and Tyr (c), are highlighted in red.The active catalytic site (FDRERIPERVVHARGASA)

Figure 2 .
Figure 2. Phylogenetic tree, gene structures, and conserved motifs of the CAT genes from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, and M. esculenta.(A) A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replications.(B) Gene structure of CAT genes.Upstream/downstream sequences are represented by light green boxes, and exons and introns are represented by yellow boxes and black horizontal lines, respectively.(C) Conserved motifs of CAT proteins.Different colored boxes show various motifs.

Figure 2 .
Figure 2. Phylogenetic tree, gene structures, and conserved motifs of the CAT genes from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, and M. esculenta.(A) A phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replications.(B) Gene structure of CAT genes.Upstream/downstream sequences are represented by light green boxes, and exons and introns are represented by yellow boxes and black horizontal lines, respectively.(C) Conserved motifs of CAT proteins.Different colored boxes show various motifs.

Figure 3 .Figure 3 .
Figure 3. Multiple protein sequence alignment of the HbCAT gene family.Three conserved amino acids, His (a), Asn (b), and Tyr (c), are highlighted in red.The active catalytic site (FDRERIPERVVHARGASA) Figure 3. Multiple protein sequence alignment of the HbCAT gene family.Three conserved amino acids, His (a), Asn (b), and Tyr (c), are highlighted in red.The active catalytic site (FDRERIPERVVHARGASA) and the heme-ligand signature sequence (RLFSYNDTQ) are highlighted in blue, and the peroxisome-targeting signal 1 (PTS1) sequence is highlighted in green.

Figure 4 .
Figure 4. Chromosomal localization and replication events of HbCAT genes.(A) Chromosomal distribution of the five HbCAT genes.The scale on the left side of the chromosome refers to the location of the genes in H. brasiliensis.Chromosome numbers (Chr2, Chr5, and Chr14) are shown on the left of the chromosomes.(B) Circle map of duplicated gene pairs for HbCAT genes.The heat map and rectangular histogram with blue lines indicate gene density on the chromosomes.The orange indicates segment duplicated HbCAT gene pairs, while the gray line presents all the syntenic blocks in the H. brasiliensis genome.

Figure 4 .
Figure 4. Chromosomal localization and replication events of HbCAT genes.(A) Chromosomal distribution of the five HbCAT genes.The scale on the left side of the chromosome refers to the location of the genes in H. brasiliensis.Chromosome numbers (Chr2, Chr5, and Chr14) are shown on the left of the chromosomes.(B) Circle map of duplicated gene pairs for HbCAT genes.The heat map and rectangular histogram with blue lines indicate gene density on the chromosomes.The orange indicates segment duplicated HbCAT gene pairs, while the gray line presents all the syntenic blocks in the H. brasiliensis genome.

Figure 5 .
Figure 5. Synteny analysis of HbCAT genes between AtCATs, OsCATs, PtCATs, and MeCATs.Gray lines in the background represent all collinearity blocks in the genomes of different species, and red lines represent syntenic CAT gene pairs.

Figure 5 .
Figure 5. Synteny analysis of HbCAT genes between AtCATs, OsCATs, PtCATs, and MeCATs.Gray lines in the background represent all collinearity blocks in the genomes of different species, and red lines represent syntenic CAT gene pairs.

20 Figure 6 .
Figure 6.Cis-acting elements in the promoter region of HbCAT genes.(A) Classification and statistical analysis of cis-regulatory elements.The number in the colored rectangles indicates the number of cis-acting elements.(B) The distribution of cis-acting elements in the HbCAT promoter region (−2000 bp).Different colored rectangles indicates different cis-elements.

Figure 6 .
Figure 6.Cis-acting elements in the promoter region of HbCAT genes.(A) Classification and statistical analysis of cis-regulatory elements.The number in the colored rectangles indicates the number of cisacting elements.(B) The distribution of cis-acting elements in the HbCAT promoter region (−2000 bp).Different colored rectangles indicates different cis-elements.

Figure 7 .
Figure 7. GO enrichment analysis of HbCAT in rubber tree.

Figure 7 .
Figure 7. GO enrichment analysis of HbCAT in rubber tree.

20 Figure 8 .
Figure 8. Expression profiles of HbCAT genes in different tissues (bark, root, latex, budburst leaf, copper brown leaf, light green leaf, and mature leaf).The color bar shows the relative expression levels of the genes calculated based on the bark.

Figure 8 .
Figure 8. Expression profiles of HbCAT genes in different tissues (bark, root, latex, budburst leaf, copper brown leaf, light green leaf, and mature leaf).The color bar shows the relative expression levels of the genes calculated based on the bark.

Figure 9 .
Figure 9. Expression patterns of the HbCAT genes under different abiotic stresses and exogenous ABA and MeJA treatments (A-F).Color bars represent different expression levels ranging from low levels (blue) to high levels (red).Numbers represent the relative expression levels of HbCAT genes computed based on the expression level at 0 h and the mean of three replicates.

Figure 9 .
Figure 9. Expression patterns of the HbCAT genes under different abiotic stresses and exogenous ABA and MeJA treatments (A-F).Color bars represent different expression levels ranging from low levels (blue) to high levels (red).Numbers represent the relative expression levels of HbCAT genes computed based on the expression level at 0 h and the mean of three replicates.

Table 1 .
Detailed information on HbCAT genes identified in rubber tree.

Table 2 .
The Ka and Ks values of HbCAT gene pairs in rubber tree.
Note: Groups A and B represent collinear gene pairs of HbCAT genes in H. brasiliensis.Int.J. Mol.Sci.2024, 25, x FOR PEER REVIEW 8 of 20