Genome-wide identification of castor bean class III peroxidase genes and analysis of expression patterns under abiotic stresses

Abstract

Background

The peroxidase (PRX) gene family plays important roles in plant growth and development, antioxidant defense, immune response, cell wall synthesis, and environmental stress response. However, the genome-wide identification and analysis of PRX in castor bean (Ricinus communis L.) have not been comprehensively analyzed.

Results

Based on the data, the PRX gene family in castor bean genome was identified genome-wide and analyzed by bioinformatics methods. Sixty-three members of the PRX gene family were identified in castor bean. These genes were unevenly distributed on 10 chromosomes. Phylogenetic analysis showed that the RcPRX family members were grouped into five clusters, most of which were closely related to Arabidopsis thaliana. Analysis of cis-acting elements in the promoters showed that RcPRX promoters contained the highest number of antioxidant responsive elements and abscisic acid responsive elements, and these genes may mediate oxidative and osmotic stress responses. In addition, transcriptome analysis showed that the high expression of RcPRX genes in castor bean roots may promote root growth and development and enhance plant adaptation to adverse stress. Meanwhile, qRT-PCR expression analysis revealed that most of the RcPRX genes were significantly up-regulated under salt stress, drought stress. A plausible explanation for the observed differential stress resilience among tissues with cotyledons exhibiting comparatively enhanced tolerance to salt and drought stress relative to roots and true leaves may reside in their distinct physiological and biochemical profiles.

Conclusions

These findings provide new insights into the composition, evolution, and function of the castor RcPRX gene family and provide a basis for subsequent exploration of gene function.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06945-5.

Background

Peroxidases (PRXs) are a class of isoenzymes that play an important regulatory role in plant growth and life cycle, and are able to catalyze the participation of hydrogen peroxide as an electron acceptor in redox reactions, which plays an important role in the scavenging of ROS [1, 2]. Based on their catalytic properties and sequence characteristics, hemoglobin PRXs can be further classified into three distinct classes: class I, class II, and class III peroxidases. class I PRXs are widely found in animals, fungi, bacteria, protozoa, and other organisms, class II PRXs are found in fungi, and class III PRXs are found only in plants [3, 4]. Class III peroxidases, a polygenic family, exist only in various plants [5] and is a typical plant secreted PRX, which may plays a key role in lignification, cell elongation and seed germination [6].

The PRX gene family in higher plants serves as a critical regulatory component in mediating biotic and abiotic stress responses through sophisticated molecular mechanisms [7, 8]. While salt and drought stress conditions induce excessive accumulation of reactive oxygen species (ROS), it should be emphasized that these oxidative molecules exhibit dual functionality in plant stress physiology, acting both as cytotoxic agents and essential secondary messengers in stress signaling pathways [9]. PRX proteins are integral to the maintenance of ROS homeostasis through their catalytic decomposition of hydrogen peroxide (H₂O₂), a biochemical process requiring coordinated interactions with antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX), rather than functioning as isolated catalytic entities [10, 11]. Current research indicates that the ROS-scavenging capacity of PRX is precisely regulated through multiple layers of control, encompassing subcellular compartmentalization, transcriptional regulation of expression levels, and post-translational modifications, with its protective efficacy demonstrating conditional dependence on environmental parameters.

The precise molecular mechanism through which PRX alleviates PSII photoinhibition remains incompletely characterized. Current hypotheses propose that PRX-mediated protection may involve indirect mitigation of photo-oxidative damage to D1 proteins through suppression of hydroxyl radical (-OH) generation via H₂O₂ detoxification. However, experimental evidence suggests this protective capacity exhibits stress intensity- and duration-dependent limitations, proving insufficient to fully preserve PSII functionality under severe stress conditions [9, 12]. The observed upregulation of TaWRKY46 expression in drought-stressed wheat cultivars provides additional validation of PRX genes’ pivotal role in plant stress adaptation. Physiological analyses reveal significant enhancement of peroxidase activity in cotyledons under osmotic stress, with PRX encoded enzymes demonstrating efficient catalytic decomposition of H₂O₂ to alleviate oxidative cellular damage [13, 14]. In Gossypium species, GrPRX has been implicated in both stress response pathways and reproductive development, particularly male gametophyte formation [15].

Furthermore, PRX exhibits indirect regulatory influence on cell wall biosynthesis processes. Through modulation of lignin biosynthesis and cell wall structural modifications, these enzymes contribute to enhanced mechanical strength and architectural stability of plant cell walls critical adaptations that improve plant resilience to osmotic stresses [12, 16]. Accumulating evidence from diverse plant systems demonstrates the evolutionary conservation of PRX mediated stress responses: Arabidopsis thaliana ATPRX3 functions as a positive regulator of drought and salt tolerance [17]; Solanum tuberosum StPRX41 and StPRX57 exhibit 4-fold transcriptional induction under water deficit [18]; Triticum aestivum displays genotype-specific TaPRX expression patterns during drought exposure [19]; and Raphanus sativus RsPRX1 knockdown lines show significant reductions in catalase and peroxidase activities, suggesting its regulatory role in antioxidant enzyme networks under multiple stress conditions [20].This multifunctional involvement of PRX proteins in ROS metabolism, cell wall fortification, and cross-talk with other antioxidant systems enables plants to maintain physiological homeostasis under adverse environmental conditions. The integration of these molecular mechanisms not only sustains essential growth processes during drought and salinity challenges but also enhances overall plant survival capacity through improved stress acclimatization. Collectively, these findings establish PRX as a central player in plant adaptive responses to osmotic stress conditions.

Castor bean, an annual or perennial herb of the genus castor bean in the family Euphorbiaceae, is a characteristic oilseed crop that has been planted for a long time in Tongliao area. As a pioneer plant, castor bean can improve soil structure and increase soil fertility [20]. Through molecular breeding techniques, genes related to abiotic stresses in castor bean can be excavated and characterized [21], providing theoretical basis and genetic resources for breeding of other crops for tolerance. In recent years, the class III peroxidase family has been extensively studied in many species, such as 73 members identified in Arabidopsis [5], 119 members in maize [22], 102 members in potato [18], 75 members in carrots [23], and 374 members in wheat [19], etc., but the members of the castor PRX gene family and their functions are still unknown. In this research, the PRX gene family members in the castor bean genome were comprehensively identified using bioinformatics methods, and their physicochemical properties, chromosomal localization, phylogenetic tree and promoter cis-acting elements were thoroughly analyzed. And the expression patterns of RcPRX genes under salt stress and drought stress conditions were analyzed. These findings laid the foundation for further exploration of the function of RcPRcX gene.

Materials and methods

Genomic characterization of castor bean RcPRX genes

Castor bean gene and annotation files were obtained from the Oil Plant Database (http://oilplants.iflora.cn), Arabidopsis AtPRXs sequences were obtained from TAIR (https://www.arabidopsis.org/), and rice PRX sequences were obtained from (https://www.uniprot.org/), and the PRX hmm (PF01805) cryptomerial structural domain model was downloaded from the Pfam (http://pfam-legacy.xfam.org/) website. Candidate sequences were analyzed using HMMER search [24] and BLAST [25] with a significance threshold of e-value ≤ e⁻⁵. The NCBI CDD tool (https://www.ncbi.nlm.nih.gov/cdd/) was used in conjunction with the EBI InterPro tool (http://www.ebi.ac.uk/interpro/) for structural domain detection of candidate sequences [26, 27], and Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) was used for multiple comparison [28] to remove redundant and non-conserved sequences, and candidate members were named PRX according to their chromosomal positions Protein.

RcPRX genes analysis and chromosome localization

The amino acid number, isoelectric point and molecular weight of RcPRXs were analyzed using the ProtParam tool (https://web.expasy.org/protparam/) [29]. The subcellular localization of RcPRXs was predicted using the WoLF PSORT tool (https://wolfpsort.hgc.jp/) [30]. Chromosomal data of RcPRX genes were extracted from castor bean genome annotation data, localized and visualized using TBtools v2.309 [31].

RcPRX genes covariance analysis

The BLAST protein database was created using Makeblastdb. Comparative analysis of RcPRXs sequences was performed using BLASTP. Gene covariance was analyzed using the MCScan X algorithm (https://github.com/wyp1125/MCScanx).

RcPRX genes phylogenetic analysis

For evolutionary analysis of the RcPRX genes, the protein sequences of Arabidopsis thaliana, rice and castor bean were aligned using MEGA 11, and a phylogenetic tree was constructed using the neighbor-joining (NJ) method with 1,000 bootstrap replicates [32], and then embellished and processed at the Evolview (https://www.evolgenius.info/evolview/#/) website.

RcPRX genes phylogenetic analysis

The MEME (https://meme-suite.org/meme/doc/meme.html) website was used to predict its structural domain composition [33]. All data were visualized using TBtools v2.309.

RcPRX genes promoter cis-acting element analysis

To study the cis-acting elements of the RcPRX genes promoter, the genomic sequence 2000 bp upstream of the start codon of the RcPRX genes was extracted from the genomic sequence. The cis-acting elements were analyzed using the PlantCARE database [34].

Protein-protein interaction network analysis

The String website (https://string-db.org/) and Cytoscape 3.8.0 software (https://cytoscape.org/download.html) were utilized to evaluate castor bean RcPRXs interaction data and predict possible connections between family members [35, 36].

Plant material and stress treatments

To understand how RcPRXs regulate plant growth and development and response to salt and drought stress, the expression profiles of RcPRX were examined based on Bing Han [37] RNA-seq data. According to the study, the 12 tissues were: germinating seed, endosperm, embryo, 3 week old seedling, young leaf, root, stem, inflorescence, pollen, ovule and pod.

The castor bean self-cross lines were obtained from the Key Laboratory of Castor bean Breeding in Inner Mongolia Autonomous Region, Inner Mongolia University for Nationalities. Strains “2129” was selected. Four-leaved castor bean seedlings were selected for salt stress and drought stress using 10% PEG-6000 [38] and 200 mM NaCl [39] solutions, and equal amounts of distilled water were used as controls. Samples were taken at 0 h, 6 h, 12 h and 24 h after the stress treatments, respectively. Roots, true leaves and cotyledons treated at different times were frozen in liquid nitrogen and stored in −80 °C refrigerator.

Total RNA of castor was extracted by Trizol method and detected by 1% agarose gel electrophoresis. The relative expression of RcPRX in roots, true leaves and cotyledons at different treatment times was detected by qRT-PCR. The experimental design included three biological replicates. The RcSKIP gene was used as an internal reference gene and analyzed by the 2^−ΔΔCt method [40].

Results

Identification and analysis of the RcPRX gene family

Based on the joint analysis of BLAST and HMM, a total of 63 castor bean PRX gene family members were identified and named RcPRX1 ~ RcPRX63 according to the order of their genes on the chromosome.The physicochemical properties of the castor bean PRX gene family members were analyzed, and the results showed that (Table 1), The castor bean PRX family comprises 63 members with marked physicochemical diversity. Structurally, amino acid residues vary 12-fold (102–1273), yielding molecular weights from 11622.13 Da (RcPRX48) to 136972.58 Da (RcPRX25). Isoelectric points ranged from pI 4.6 (RcPRX48) to pI 9.46 (RcPRX32). Hydrophobicity analysis identified 82.5% hydrophilic members versus 17.5% hydrophobic. Significantly, chloroplast localization predominates, suggesting PRX proteins likely regulate photosynthesis-related processes through spatial compartmentalization.

Table 1.

Physicochemical properties of castor bean PRX family members: an in-depth overview of amino acid number, molecular weight, isoelectric point, instability index, total average hydrophobicity and subcellular localization

Gene name	Gene ID	Number of Amino Aicd	Molecular Weight ( Da )	pI	Instabi-lity Index	Grand Average of Hydrop-athicity	Subcellular Location
RcPRX1	Rc01T000544.1	328	35215.13	9.03	35.04	−0.066	Chloroplast
RcPRX2	Rc01T001020.1	406	44652.08	8.17	40.1	−0.119	Chloroplast
RcPRX3	Rc01T001944.1	166	18195.11	8.96	42.13	0.170	Plasma membrance
RcPRX4	Rc01T001945.1	225	24543.22	8.39	51.81	0.060	Chloroplast
RcPRX5	Rc02T003384.1	607	65414.8	8.91	34.46	−0.208	Chloroplast
RcPRX6	Rc02T003833.1	694	75608.18	6.15	40.04	−0.161	Vacuole
RcPRX7	Rc02T003875.1	478	53193.32	7.9	48.21	−0.145	Plasma membrance
RcPRX8	Rc02T003876.1	356	39126.18	5.57	37.28	−0.160	Extracellular
RcPRX9	Rc02T003966.1	361	39613.35	8.82	37.27	−0.058	Chloroplast
RcPRX10	Rc02T004075.1	379	40843.15	7.69	49.33	−0.380	Chloroplast
RcPRX11	Rc03T005159.4	333	36162.92	6.95	39.76	−0.198	Mitochondrion
RcPRX12	Rc03T005263.1	358	39746.96	9.4	37.33	−0.216	Chloroplast
RcPRX13	Rc03T006087.1	345	37861.93	6	42.82	−0.058	Chloroplast
RcPRX14	Rc03T007047.1	329	36380.72	9.43	35.68	−0.188	Vacuole
RcPRX15	Rc04T007142.1	387	43297	5.62	42.81	−0.284	Chloroplast
RcPRX16	Rc04T007501.1	383	42458.56	9.12	43.97	−0.251	Chloroplast
RcPRX17	Rc04T007662.1	328	35266.81	5.26	35.83	0.265	Extracellular
RcPRX18	Rc04T008001.1	329	36525.18	8.79	33.61	−0.106	Extracellular
RcPRX19	Rc04T008334.2	935	104807.83	6.28	37.99	0.049	Plasma membrance
RcPRX20	Rc04T008913.1	538	61261	4.65	54.97	−1.012	Chloroplast
RcPRX21	Rc05T009355.1	332	37750.26	8.32	38.1	−0.355	Vacuole
RcPRX22	Rc05T009405.1	330	36053.6	9.14	26.62	0.008	Chloroplast
RcPRX23	Rc05T009573.1	1126	127023.37	8.82	46.39	−0.464	Mitochondrion
RcPRX24	Rc05T009727.1	332	37320.63	5.33	40.72	−0.181	Extracellular
RcPRX25	Rc05T010285.2	1273	136972.58	5.98	38.2	−0.155	Plasma membrance
RcPRX26	Rc05T010286.1	324	34983.4	4.79	34.8	−0.020	Extracellular
RcPRX27	Rc05T010287.1	324	34960.36	4.8	32.71	−0.032	Extracellular
RcPRX28	Rc05T010288.1	324	34983.4	4.79	34.8	−0.020	Extracellular
RcPRX29	Rc05T010491.1	336	36419.51	4.89	31.53	−0.024	Plasma membrance
RcPRX30	Rc05T010600.1	353	38823.63	9.36	46.95	−0.203	Chloroplast
RcPRX31	Rc05T011514.1	340	37400.56	6.59	42.62	−0.092	Chloroplast
RcPRX32	Rc05T011521.1	976	105679.72	9.46	42.57	−0.139	Plasma membrance
RcPRX33	Rc05T011534.2	332	35663.47	8.05	46.97	−0.066	Chloroplast
RcPRX34	Rc05T012575.1	284	31262.5	6.72	38.5	−0.377	Cytoplasmic vesicles and cytoskeleton
RcPRX35	Rc05T012606.1	364	39548.38	9.36	40.9	−0.208	Mitochondrion
RcPRX36	Rc05T012706.1	331	36139.07	8.7	44.89	−0.226	Extracellular
RcPRX37	Rc06T013029.1	326	35784.01	8.98	38.57	−0.165	Vacuole
RcPRX38	Rc06T013090.1	980	106712.83	7.72	36.54	−0.113	Plasma membrance
RcPRX39	Rc06T014647.1	697	75281.96	4.73	31	−0.049	Plasma membrance
RcPRX40	Rc06T014716.1	324	34886.74	8.03	39.31	0.007	Extracellular
RcPRX41	Rc07T014959.1	256	28043.24	6.93	29.6	0.174	Endoplasmic reticulum
RcPRX42	Rc07T015264.1	260	28514.54	5.73	32.57	−0.28	Cytoplasmic vesicles and cytoskeleton
RcPRX43	Rc07T015452.1	353	39430.51	9.18	35.75	−0.232	Chloroplast
RcPRX44	Rc07T015982.1	111	12800.64	8.76	45.78	−0.331	Nuclear
RcPRX45	Rc07T016248.1	179	19996.01	7.02	45.44	−0.158	Extracellular
RcPRX46	Rc07T016755.1	1142	126982.36	5.35	51.43	−0.489	Nuclear
RcPRX47	Rc07T016978.1	751	80973.04	6.8	35.35	−0.383	Nuclear
RcPRX48	Rc07T017256.1	102	11622.13	4.6	51.37	−0.298	Chloroplast
RcPRX49	Rc08T019492.1	318	34183.44	8.8	35.52	−0.127	Chloroplast
RcPRX50	Rc09T020659.1	370	40676.79	9.09	34.61	−0.018	Vacuole
RcPRX51	Rc09T020666.1	324	34814.44	6.59	43.56	0.002	Chloroplast
RcPRX52	Rc09T020667.1	349	38225.5	6	43.04	−0.073	Extracellular
RcPRX53	Rc09T020670.1	276	30434.04	7.59	48.66	0.111	Chloroplast
RcPRX54	Rc09T021362.1	329	36364.66	5.13	41.6	−0.092	Extracellular
RcPRX55	Rc09T021457.1	328	36060.48	8.82	35.22	−0.052	Chloroplast
RcPRX56	Rc09T021604.1	334	36263.31	9.05	39.66	−0.081	Chloroplast
RcPRX57	Rc09T021738.1	324	35433.11	5.59	32.62	−0.082	Extracellular
RcPRX58	Rc09T022029.1	317	35110.8	5.21	42.08	−0.201	Extracellular
RcPRX59	Rc10T022101.2	282	31478.02	5.8	36.67	−0.208	Cytoplasmic vesicles and cytoskeleton
RcPRX60	Rc10T022125.1	328	36819.85	5.79	40.83	−0.261	Chloroplast
RcPRX61	Rc10T022617.2	335	35080	4.83	38.11	0.001	Chloroplast
RcPRX62	Rc10T023710.1	966	103931.55	6.07	41	−0.095	Chloroplast
RcPRX63	Rc10T023957.1	324	35088.14	6.43	31.22	0.033	Vacuole

Chromosomal localization analysis of the RcPRX gene family

The distribution of genes on chromosomes was closely related to the degree of chromosome involvement in the expression process and their importance in plant growth and development. To investigate the replication pattern and evolutionary mechanism of RcPRX genes, chromosomal localization analysis was performed (Fig. 1), which showed that 63 RcPRX genes were unevenly distributed on 10 chromosomes, RcPRX49 was uniquely located on Chr 1, all others were anchored to 9 chromosomes.

Fig. 1.

Chromosome distribution of RcPRX genes. Chromosome numbers are shown in yellow font on the side. RcPRX genes are shown in red italic font on the side. Chromosome sizes are in millions of bases (Mb)

Analysis of the covariance of the RcPRX gene family

In order to investigate the replication patterns and evolutionary mechanisms of PRX gene family members in castor bean, the whole genome of castor bean was subjected to covariance analysis (Fig. 2). A total of 7 covariance events occurred in the 63 RcPRX genes, among which the highest number of covariance occurred in Chr 5, with a total of 4 pairs of genes harboring covariance, Chr 10 had fewer co-linkage events and only 1 inter-chromosomal duplication occurred.The results indicate that two tandem duplications exist (RcPRX49 and RcPRX62; RcPRX23 and RcPRX18), and the rest are segmental duplications.These findings suggested that tandem and segmental duplications played a significant role in the diversification and evolution of RcPRX genes.

Fig. 2.

Co-linearity analysis of castor bean PRX family members. Co-linear relationships are indicated by red lines, and gene locations in the outer ring are indicated by black italicized letters.The 13 RcPRX genes are located on a ring formed by the 10 chromosomes (Chr 1-Chr 10) of castor bean

Evolutionary analysis of members of the RcPRX gene family

In order to study the evolutionary relationship of RcPRX genes, the protein sequences of 63 PRX family members of castor bean, 73 PRX family members of Arabidopsis and 138 PRX family members of rice were analyzed by phylogenetic tree analysis, and the results showed (Fig. 3) that the RcPRXs were clustered into five clusters with the members of AtPRXs and OsPRXs, in which cluster IV had the highest number of PRX family members, possessing 23 RcPRXs members; Cluster V has the lowest number of PRX family members, and the number of PRX family members possesses only 5. In summary, RcPRXs members were unevenly distributed in each cluster, indicating that castor bean was closely related to Arabidopsis.

Fig. 3.

Relationships among castor bean, Arabidopsis, and rice PRX were explored by phylogenetic analysis. Neighbor Joining (NJ) method was used to draw the phylogenetic tree using 1000 bootstrap replicates. Different groups are indicated by different colored branches. Red circles indicate castor bean PRX proteins; blue triangles indicate Arabidopsis PRX proteins; and green squares indicate rice PRX proteins.The five groups of members of the RcPRX gene family are I, II, III, IV, and V. Five different colors were used to point out the genes in each group

Analysis of conserved structural domains of RcPRX gene family members

In order to deeply explore the characteristics of the RcPRX genes family, 63 members were analyzed for conserved structural domains. The results of the analysis showed (Fig. 4) that all RcPRX members contain the structural domain of peroxidase, a core feature of the family, indicating that they had retained this key functional domain during evolution. In addition, some RcPRX gene family members contain other structural domains, such as the ascorbate peroxidase structural domain, and this diversity of structural domains may confer additional functions to the RcPRXs or regulate their activities, thus playing a broader role in the physiological processes of plants.

Fig. 4.

Conserved structural domains of the RcPRX protein

Cis-acting element analysis of members of the RcPRX gene family

In order to deeply investigate the expression regulation mechanism of RcPRX genes, the cis-acting elements of RcPRX genes were analyzed in detail (Fig. 5 and Fig S1). The results showed that 61 cis-acting elements were screened from 63 RcPRX genes, covering four major categories: growth and development, hormone response, light response and stress response.

Fig. 5.

Analysis of cis-acting elements in the promoter region of the RcPRX genes. The DNA sequence of 2000 bp upstream of the RcPRX genes was analyzed using Plant CARE software. Bubbles indicate the number of elements. The change in color represents the increase in the number of cis-acting elements

Among them, the highest number of elements related to light response indicated that RcPRX genes might play an important role in light signaling. This was followed by hormone response elements and growth and development-related elements, while the number of elements related to stress response was relatively small. Specifically, no hormone response-like elements were found in RcPRX40, RcPRX45 and RcPRX46, while stress response elements were lacking in RcPRX14, RcPRX44 and RcPRX63. In addition, no growth and developmental-like elements were detected in 12 genes including RcPRX4, RcPRX18, RcPRX24, RcPRX28 and RcPRX29.

In the hormone response class of elements, a total of 301 were identified, including abscisic acid (ABRE), growth hormone (AuxRR-core, TGA-element), methyl jasmonate (CGTCA-motif, TGACG-motif), gibberellin (GARE-motif, P-box, TATC-box), and salicylic acid (SARE, TCA-element). Among them, the highest number of elements related to abscisic acid was 102, and the RcPRX genes might be related to ABA-mediated environmental stress response. There were a total of 243 stress response elements, including antioxidant response elements (ARE, GC-motif), defense and stress response elements (TC-rich repeats), drought-induced elements (MBS, CCAAT-box), and low-temperature response elements (LTR), among which the number of antioxidant response elements was the highest at 107. In a word, the RcPRX genes, through the cis-acting elements in its promoter region, might be involved in physiological processes such as growth and development, hormone regulation, adversity stress and light regulation in castor bean.

Protein-protein interaction analysis

Protein interactions play a crucial role in organisms. Proteins do this by forming complexes and building multi-protein networks, and they regulate a wide range of functions in organisms. The results showed (Fig. 6) that there were 28 nodes in the whole interaction network. RcPRX34 played a central role and had extensive interactions with 12 proteins including RcPRX12, RcPRX10, and RcPRX28. In addition, RcPRX12, RcPRX18, RcPRX49, RcPRX27 and RcPRX28 interact with RcPRX59. Particularly, RcPRX3 interacted with RcPRX4 and RcPRX23 with RcPRX11, while none of the other PRX gene family members had any interactions with each other.

Fig. 6.

Protein interaction network of castor bean PRX family members by using the STRING database to construct the interaction network. Circles represent castor bean PRX proteins. Color changes represent the level of importance. Solid lines indicate the presence of interaction between two proteins

Analysis of tissue expression patterns of RcPRX genes

Based on the analysis of RNA-seq data, the expression patterns of RcPRX genes in 12 different tissue sites were investigated. The results of tissue expression profiling showed that the RcPRX gene family exhibited significant tissue specificity (Fig. 7). Designate log₂(FPKM) > 1 as a highly expressed gene. The results showed that RcPRX32 and RcPRX33 exhibited high expression in developing seeds; whereas RcPRX1, RcPRX7, RcPRX43, and RcPRX48 were significantly highly expressed in germinating seeds. In addition, RcPRX25 showed high expression in stem tissues; RcPRX10 and RcPRX56 were highly expressed in inflorescences; and RcPRX57 and RcPRX62 were highly expressed in pollen. These genes showed significant expression differences in different tissues, suggesting that they may have different functions or be subject to different regulatory mechanisms in different tissues. Especially, RcPRX5, RcPRX35, RcPRX40, RcPRX44, RcPRX49, RcPRX50, RcPRX51, RcPRX52, RcPRX59, and RcPRX61 were expressed at similar levels in multiple tissues, suggesting that these genes may play relatively stable functions in these tissues. The diversity of tissue expression patterns suggests that the RcPRX gene family might play diverse roles in regulating different tissues and developmental stages of castor bean. In addition, the most RcPRX genes were expressed at high levels in roots, suggesting that members of the castor bean PRX family may play important biological functions in castor bean roots.

Fig. 7.

Heatmap of RcPRX genes expression levels in different tissues of castor bean(Seed1 ~ Seed5 are seeds at 10, 20, 35, 45, and 55 days after pollination, respectively, and G_seed is seeds germinated for 2 days). The transcript levels of each specific RcPRXs in different tissues were normalized using the fragment per exon per million fragment mapped exons (FPKM) value as a standard reference. Changes in the color scale indicate differences in the expression level of each gene

Using Origin to plot and analyze the correlation coefficient map of RcPRX genes (Fig. 8), a significant positive correlation was shown between RcPRX1 and RcPRX2, RcPRX3, RcPRX4 and other genes. This result suggests that these genes may exhibit consistent change trends under similar environments or conditions, implying that they may be subject to common regulatory mechanisms or play synergistic roles in similar biological processes. On the contrary, a negative correlation was shown between RcPRX1 and RcPRX57, suggesting that possibly an increase in RcPRX1 may be correlated with a decrease in RcPRX57 under certain conditions. The strong positive correlation between RcPRX33 and RcPRX34, RcPRX35 and other genes further revealed a possible close connection between them. This strong correlation may imply that these genes were influenced by the same factors or that they play a synergistic role in the same physiological or metabolic pathways.

Fig. 8.

Correlation coefficients for the RcPRX genes. Color shades are the magnitude of Pearson correlation coefficients

Expression patterns of RcPRX genes under salt and drought stresses

To better understand the expression pattern of RcPRX genes under salt stress and drought stress conditions. After salt stress treatment of castor seedlings at the four-leaf stage for different times (Fig. 9), it was found that the expression of RcPRX genes in different tissues differed significantly at different times of salt stress treatment. In roots, RcPRX6 and RcPRX50 were up-regulated, while RcPRX7, RcPRX15 and RcPRX53 were down-regulated. In true leaves, RcPRX7 was significantly up-regulated with the extension of treatment time, while RcPRX6 and RcPRX15 were up-regulated after 24 h of treatment. In cotyledons, RcPRX15 was significantly up-regulated with increasing treatment time, while RcPRX7 and RcPRX50 were up-regulated after 6 h and 24 h of treatment, respectively. RcPRX7 was strongly induced in true leaves, RcPRX6 in roots and RcPRX15 in cotyledons after salt stress treatment.

Fig. 9.

Differential analysis of RcPRX genes in roots (A), true leaves (B) and cotyledons (C) of castor bean in salt stress treatments. All data are expressed as the mean ± standard error (SE) of three independent replicates. x-axis indicates the different treatment times, and y-axis indicates the relative expression levels. T-test (*P < 0.05, **P < 0.01, ***P < 0.001) was used to determine the significance of differences. Different colors represent different genes of castor

Meanwhile, castor seedlings at the four-leaf stage were subjected to drought stress treatments of different durations (Fig. 10). In roots, RcPRX7 was up-regulated at 6 h of treatment, and RcPRX15 and RcPRX50 were up-regulated at 12 h of treatment; in true leaves, RcPRX7 was significantly up-regulated, and the remaining four RcPRX genes were down-regulated; in cotyledons, RcPRX7 and RcPRX15 were up-regulated, and RcPRX50 was up-regulated after 12 h of treatment. With the prolongation of drought stress treatment, RcPRX50 was found to be highly induced in roots and reached its maximum value at 12 h. RcPRX7 was strongly induced in true leaves and RcPRX15 in cotyledons.

Fig. 10.

Differential analysis of RcPRX genes in roots (A), true leaves (B) and cotyledons (C) of castor bean in drought stress treatments. All data are expressed as the mean ± standard error (SE) of three independent replicates. x-axis indicates the different treatment times, and y-axis indicates the relative expression levels. T-test (*P < 0.05, **P < 0.01, ***P < 0.001) was used to determine the significance of differences. Different colors represent different genes of castor

The results showed that RcPRX7 in true leaves and RcPRX15 in cotyledons exhibited up-regulation after salt and drought stress treatments. The RcPRX genes in true leaves and cotyledons were more tolerant to salt stress than those in roots, and the RcPRX genes in roots and cotyledons were more tolerant to drought stress than those in true leaves.

Discussion

Peroxiredoxins have important regulatory roles in plant growth and development and abiotic adversity response. Currently, the peroxidase family has been demonstrated to be biologically important in several species, including Arabidopsis [17], rice [2], and maize [22]. Here, the castor bean PRX gene family was systematically analyzed by bioinformatics analysis, and a total of 63 castor bean PRX gene family members were identified and categorized into five subfamilies based on the similarity of their gene structure and conserved motif composition, which is basically consistent with the studies of cereal [15], nodular kale [41], and Chinese cabbage [42], etc. The members of the RcPRX gene family were unevenly distributed across the chromosomes. The results of evolutionary analyses showed that the RcPRX gene family was conserved during plant evolution, as well as functional differentiation and specificity.

The results of subcellular localization of gene family members showed that most of the RcPRX genes were localized in chloroplasts. Chloroplasts produce ROS during photosynthesis, and excessive ROS can cause oxidative damage to cells. The peroxidase encoded by the RcPRX genes may be involved in the scavenging of ROS in chloroplasts to protect chloroplasts from oxidative damage. Maintaining the normal function of chloroplasts [43]. And the RcPRX genes, which is localized in the extracellular or plasma membrane, can directly scavenge ROS in the extracellular or near the plasma membrane and reduce the direct attack of ROS on the cell membrane [44]. This study is in agreement with the studies of GE Wen-dong [42] and ZHANG Hui-Hui [9]. So it is hypothesized that the PRX family members of castor bean may play a role in oxidative stress and other processes.

Further analysis of gene replication types revealed that the 63 RcPRX genes underwent seven covariation events and exhibited segmental and tandem replication. These events likely increased the number of RcPRX genes and laid the foundation for their functional diversification [45]. In addition, protein interaction network analysis identified 28 nodes in the network, with RcPRX34 potentially being a key protein in executing the functions of PRX family members in the cell.

PRX genes affect plant responses to abiotic stresses through different mechanisms. For example, in nodular kale (Brassica oleracea L.) [41], PRX gene family members had the highest expression in chloroplasts and were induced to up-regulate their expression under salt and drought stress, suggesting that these genes play a role in adversity stress response. In cereals [15], the expression of class III PRX gene family members changed significantly under drought stress, implying their importance in drought stress response in cereals. ThPRX1 and ThPRX2 in Willow montana [46], 2-CysPrx in Arabidopsis [47] and ScPOD02 in sugarcane [47] are involved in the scavenging of reactive oxygen species, which help plants to cope with abiotic stresses. In wheat transgenics overexpressing TaPRX-2 A, which was highly expressed in roots, tolerance to abiotic stress was enhanced by enhanced oxidative stress tolerance [48]. In soybean GsPRX9 was strongly induced by salt stress and enhanced salt tolerance by enhancing antioxidant enzyme activity and reducing ROS accumulation [49]. 61 cis-acting elements were identified in the RcPRX family, including four categories: growth and development, hormone response, light response and stress response. The presence of these cis-acting elements suggests that RcPRX genes may be involved in growth and development, hormone regulation, adversity stress and light regulation in castor bean. Especially, RcPRX contained the highest number of antioxidant response elements and elements responsive to abscisic acid, a feature that suggests that the relevant genes may have important roles in antioxidant defense mechanisms. ARE, which can respond to oxidative stress signals and regulate the expression of antioxidant genes by binding to transcription factors. In plants, activation of ARE induces the expression of several antioxidant enzymes, such as SOD, POD, and CAT, which enhances the antioxidant defense of plants [50, 51]. Under osmotic stress, plants produce a large amount of ROS, which cause oxidative damage to cells. ABA can reduce the accumulation of ROS by regulating the activity of antioxidant enzymes, thus alleviating the damage caused by oxidative stress to plants [52]. So it is hypothesized that RcPRX may play a driving role in oxidative stress and osmotic stress.

Class III peroxidase enhances drought tolerance in plants by scavenging reactive oxygen species and mitigating oxidative stress damage to plant cells in response to oxidative stress and drought stress [12]. RcPRX genes contain a high number of ARE and MBS cis-acting elements, and MBS can be recognized and bound by MYB transcription factors, thereby regulating gene expression [53]. ARE and MBS cis-acting elements enhance plant tolerance to oxidative and abiotic stresses by regulating the expression of related genes. The results showed that RcPRX genes also exhibited tolerance to drought stress under drought stress treatment. Taken together, RcPRX genes may induce castor bean hormone signaling in response to abiotic stress.

There is tissue specificity in the expression of PRXs gene in different plants. These tissue-specific expression of PRXs gene may play important roles in plant growth and development, response to adversity stress and immune regulation. Based on the analysis of RNA-seq data, significant differences in the expression patterns of RcPRX genes at different developmental periods were found. Most of the RcPRX genes were highly expressed in roots, and a few RcPRX genes were differentially expressed at other stages.Most of the RcPRX genes were highly expressed in roots, and RcPRX6 was strongly induced in roots under salt stress treatments in qRT-PCR results, which was hypothesized to possibly play an important role in root growth and development. This diversity of expression patterns suggests that the RcPRX genes family may play diverse roles in regulating different tissues and developmental stages of castor bean. Roots are the primary site where plants feel oxidative stress, and WU Tian-Tian showed that hydrogen sulfide can enhance the antioxidant capacity of root cells and mitigate the cellular damage caused by oxidative stress by regulating the Nrf2-ARE pathway [54]. Root cells contain a variety of antioxidant enzymes capable of scavenging ROS and attenuating cellular damage from oxidative stress. This study is consistent with the studies of Luo W [55], Xue Li [56], Jiang Lili [57], Marzol E [58], which confirmed the authenticity and accuracy of this study. It is hypothesized that RcPRX plays a facilitating role in root differentiation and may indirectly affect the overall structure and function of the root system. PRX may affect the lignin content, which decreases under stress, and PRX may have an effect on it, leading to thickening of the cell wall and changes in water absorption [58]. In addition to osmotic stress, salt stress produces ROS and oxidative stress in castor bean. In summary, the high expression of RcPRX genes in castor bean roots may play an important role in promoting root growth and development, improving plant adaptation to adversity stress, participating in plant immune regulation, and influencing the structure and function of the root system. These speculations provide a theoretical basis for the subsequent further study of the function of RcPRX genes in castor bean.

Under salt and drought stress treatments, RcPRX genes showed different responses in different tissues at different treatment times. The results of qRT-PCR studies showed that cotyledons had higher gene expression under salt and drought stress than roots and true leaves. The high expression of some adversity-responsive genes in cotyledons helped them to maintain normal physiological functions under stress conditions [59]. The high expression of these adversity-responsive genes helps cotyledons to maintain higher metabolic activity under stress conditions, thereby increasing their tolerance. Cotyledons are the main photosynthetic organs during the early stages of plant seedling development and can provide a large amount of photosynthetically assimilated substances for seedling growth. The photosynthetic capacity enables the cotyledons to maintain a higher energy supply under adverse conditions, thus enhancing their tolerance [60]. The high expression of these adversity-responsive genes helps cotyledons maintain higher metabolic activity under stress conditions, thereby enhancing their tolerance. This enhanced tolerance can be attributed to the unique physiological and biochemical properties of cotyledons. As a key organ for nutrient storage and development in the early stages of castor bean seedlings, cotyledons may have evolved specific mechanisms to cope with unfavorable environmental conditions. Cotyledons are the main photosynthetic organs during the early stages of seedling development and are capable of providing a large amount of photosynthetically assimilated substances for seedling growth. The photosynthetic capacity enables cotyledons to maintain a high energy supply under adverse conditions, thereby increasing their tolerance [61]. Plants are better adapted to environmental stress by regulating the expression of photosynthesis-related genes under adverse conditions to increase photosynthetic efficiency [24]. Therefore, the RcPRX genes may be involved in a variety of physiological processes in plants under salt and drought stress, including ROS scavenging and gene expression regulation. However, the exact characterization of this pathway in castor bean remains unclear.

Conclusions

The present study reveals 63 members of the RcPRX gene family were identified in castor bean and subjected to several analyses, including physicochemical properties and subcellular localization analysis, chromosomal localization, analysis of covariance, phylogenetic analysis, cis-acting element analysis, protein interaction network analysis, and tissue expression pattern analysis. This study found that the RcPRX gene family has gene duplication events during evolution and is closely related to rice. Cis-acting element analysis showed that the promoter region of the RcPRX genes had a variety of elements related to growth and development, hormone response, light response, and stress response, such as ABRE and MBS. The RcPRX genes showed different response patterns under salt stress and drought stress treatments, suggesting that these genes play an important role in regulating plant responses to abiotic stresses. RcPRX7 and RcPRX15 provided the basis for subsequent functional analysis. The results of this study provide a basis for further investigation of the function of the RcPRX gene family under abiotic stress in castor bean.

SupplementaryInformation

Supplementary Material 1. ^{(188.8KB, zip)}

Authors’ contributions

Jixing Zhang conceived and designed the experiments. Jiayu Li performed the experiments. Jiayu Li, Mubo Fan, Shuqi Yang, Hongyan Huo, Weiquan Fan, Shiyou Lü, and Jixing Zhang analyzed the data. Jiayu Li and Mubo Fan wrote the manuscript. All authors reviewed and edited the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (31760399) and projects of Inner Mongolia Collaborative Innovation Center for Castor Industry (MDK2023083, MDK20230779, MDK2023077).

Data availability

All data generated or analysed during this study are included in this article and can be found in Supplementary Tables S1 and Figure S1.

Declarations

Ethics approval and consent to participate

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Consent for publication

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Competing interests

The authors declare no competing interests.