Genome-wide analysis of respiratory burst oxidase homolog gene family in pea (Pisum sativum L.)

Plant respiratory burst oxidase homologs (RBOHs) are key enzymes regulating superoxide production, which is important for plant development and responses to biotic and abiotic stresses. This study aimed to characterize the RBOH gene family in pea (Pisum sativum L.). Seven PsRBOH genes were identified in the pea genome and were phylogenetically clustered into five groups. Collinearity analyses of the RBOHs identified four pairs of orthologs between pea and soybean. The gene structure analysis showed that the number of exons ranged from 6 to 16. Amino acid sequence alignment, conserved domain, and conserved motif analyses showed that all seven PsRBOHs had typical features of plant RBOHs. The expression patterns of PsRBOH genes in different tissues provided suggested their roles in plant growth and organ development. In addition, the expression levels of PsRBOH genes under different abiotic stresses were analyzed via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results demonstrated that PsRBOH genes exhibited unique stress-response characteristics, which allowed for functional diversity in response to different abiotic stresses. Furthermore, four PsRBOHs had a high probability of localization in the plasma membrane, and PsRBOH6 was localized to the plasma membrane and endoplasmic reticulum. The results of this study provide valuable information for further functional analysis of pea RBOH genes and their role in plant adaptation to climate-driven environmental constraints.


Introduction
Abiotic stress tolerance of major staple crops was significantly weakened or even lost during their domestication (Palmgren et al., 2015;Rawat et al., 2022).Major abiotic stresses such as drought, salinity, flooding, and extreme temperatures are increasing due to the current climate changes driven by global warming (Liu et al., 2020).Therefore, improving abiotic stress tolerance in crops is critical for future food security.
Despite different plants having different mechanisms for adapting to abiotic stresses, these stresses increase superoxide levels in all plants, and the superoxide is transformed into reactive oxygen species (ROS) via different pathways in different cellular compartments (Sandalio et al., 2021).ROS were initially considered toxic byproducts of aerobic metabolism and deemed harmful to plants; however, their central role in complex adaptive signaling networks has been recently reported (Mittler et al., 2011).While excessive ROS accumulation is toxic, causing DNA backbone damage, protein and lipid oxidation, and inducing apoptosis (Patel et al., 2018;Wang et al., 2020), transient stress-induced ROS spikes are considered central to systemic signaling and adaptation in plants (Bostock et al., 2014;Zhang et al., 2022).
Previous studies showed that many plants have multiple RBOH members, and different members exhibit different expression profiles with distinct functions.For example, there are ten RBOHs in Arabidopsis (AtRBOHA-J) (Sagi and Fluhr, 2006) and nine in rice (OsNox1-9) (Wang et al., 2013).Additional studies showed that AtRBOHD and AtRBOHF were expressed throughout the plant, while AtRBOHA-C, AtRBOHG and AtRBOHI were specifically expressed in the roots.Moreover, AtrbohH and AtrbohJ were specifically expressed in pollens and are thus required for normal pollen tube growth (Sagi and Fluhr, 2006;Kaya et al., 2014).OsNox1-2, OsNox5-6, and OsNox9 were ubiquitously expressed, while OsNox3, OsNox4, OsNox7, and OsNox8 showed tissue-specific expression in rice (Wang et al., 2013).Moreover, OsNox9 were associated with root development (Takeda et al., 2008;Yamauchi et al., 2017).
As a classic model plant and the second most important grain legume, pea (Pisum sativum L., 2n = 14) has been widely used in genetics and developmental biology studies since Mendel (Kreplak et al., 2019;Chen et al., 2022;Chen et al., 2023;Li et al., 2023).Pea has become an important legume crop and green vegetable favored by many people worldwide.Fresh pea is rich in soluble protein, starch, carotenoid, and flavonoids (Yang et al., 2022).However, pea is sensitive to most common abiotic stresses (e.g., drought, cold, high temperatures, and salinity), which cause massive yield losses.
Given the critical role of RBOH in plant adaptive responses to abiotic stresses, in this work we aimed to provide a comprehensive analysis of pea RBOH genes.This study analyzed the chromosomal distribution, developmental evolution, gene structure, conserved domains, subcellular location, and description of cis-acting elements of pea RBOH genes.We also studied the tissue specificity and expression patterns of pea RBOH genes under different abiotic stresses.The reported data provide valuable information for an in-depth understanding of the biological functions of the RBOH gene family in pea, paving the way for improving its abiotic stress tolerance.

Identification of the PsRBOH genes in pea
The DNA sequences and annotation files of the pea genome were downloaded from the Pea Genome Database (https:// www.peagdb.com/).In addition, 17 soybean, 8 tomato, and 10 Arabidopsis RBOH protein sequences were downloaded from the phytozome (https://phytozome-next.jgi.doe.gov/),Sol Genomics N e t w o r k ( h t t p s : / / w w w .s g n .c o r n e l l .e d u / ) and TAIR (www.arabidopsis.org/)databases, respectively.Arabidopsis is a model plant widely used in various plant studies, including gene family identification studies.Tomato is a horticultural crop that has been widely studied and is one of the most widely eaten vegetables in the world.Its molecular biology research is relatively in-depth.Soybeans, like peas, are important leguminous plants with high protein levels.The domain sequences (HMM model file) of the NADPH oxidase (PF08414), NAD binding (PF08030), FAD binding (PF08022), and Ferric reductase domains (PF01794) were downloaded from the Pfam database (http://pfam.xfam.org/)and used as the seed files to search the RBOH proteins in the pea genome file via a hidden Markov model (HMM) search (e-value 0.01).The conserved domains were verified using CD-Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).
Analysis of the chromosomal location, phylogenetics, collinearity, gene structure, conserved motifs, and cis-elements The online software MG2C (http://mg2c.iask.in/mg2c_v2.0/)was used for the chromosome localization analysis.The phylogenetic tree was constructed using the IQ-Tree Wrapper program in TBtools software (Chen et al., 2020) with 1000 bootstrap replicates.The collinearity between pea and soybean sequences was determined using TBtools software (Chen et al., 2020).The Gene Structure Display Server (http://gsds.gao-lab.org)was used to generate the gene structure map, and the online software MEME (http://meme-suite.org/tools/meme)was used to search for conserved motifs with the maximum conserved motif search value set to 10.The 2000-bp sequences upstream of PsRBOH genes were obtained from the pea genome database and used as the promoter, and the cis-regulatory elements of these promoters were identified using PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Plant materials, stress treatments and tissue expression analysis P. sativum cultivar Zhongwan6 (ZW6) were obtained from the pea seed breeding center in China (Gu'an, Hebei).The seeds were germinated in a liquid culture using a quarter (1/4)-strength modified Hoagland nutrient solution (pH 5.5) in a growth chamber under 25 ± 2°C with a 16 h/8 h light/dark cycle.The four-leaf stage seedlings were treated with 1/4-strength modified Hoagland nutrient solution [Ca (NO 3 ) 2  O 123.25 mg/L, pH 5.5] supplemented with 100 mM NaCl for the salt-stress treatment, 10% (w/v) polyethylene glycol (PEG) for the drought stress treatment, and 6 mM CdCl 2 for the cadmium (Cd) treatment.For the heat and cold stress, the seedlings were treated with 1/4-strength modified Hoagland nutrient solution (pH 5.5) at 38°C and 4°C, respectively.For the low-boron stress treatment, seedlings were initially pretreated with the modified Hoagland nutrient solution containing 0.025 mM boric acid (H 3 BO 3 ) (B treatment) at the first stage of culture to diminish variations in B concentrations in seeds.Subsequently, the lowboron-treated plants were treated with the modified Hoagland nutrient solution containing 0.025 mM H 3 BO 3 , and the controls were treated with the modified Hoagland nutrient solution with 25 mM H 3 BO 3 at the four-leaf-stage seedlings (Li et al., 2018).The treatments were conducted in triplicates with five seedlings per replicate.The leaves of treated seedlings were sampled after 0.5, 3,6,12,24,36,48, and 72 h of stress treatment.Pea roots, leaves, stems, flowers, tendrils, one-week-old pea seeds, one-week-old pod, and two-week-old pea seeds were sampled for tissue expression analysis.All the harvested samples were frozen in liquid nitrogen and stored at −80°C until analysis.Each treatment had three independent biological replicates.

RNA isolation and RT-qPCR analysis
Total RNA was isolated from the samples using FreeZol Reagent R711 (Nanjing Vazyme Biotech Co., Ltd.), according to the manufacturer's instructions.Briefly, 50 mg of the ground sample was added into 500 ml of FreeZol Reagent for lysis.The lysate was centrifuged, and the supernatant was collected.A dilution buffer was added to the supernatant, and the mixture was precipitated with isopropanol.After centrifugation, the supernatant was discarded, and the pellet was washed with 75% ethanol and dissolved in RNase-free double distilled water (ddH 2 O).The quality and concentrations of the isolated RNA samples were determined via 1% agarose gel electrophoresis and a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).Reverse transcription PCR was conducted on a QuantStudio ™ 6Flex Real-Time PCR System (Applied Biosystems ™ , Carlsbad, CA, USA) using HiScript ® III RT SuperMix for qPCR with (+gDNA wiper) (R323-01) (Vazyme, Nanjin) and ChamQTM Universal SYBR ® qPCR Master Mix (Q711) (Vazyme).Three technical replicates were set for each biological sample, and the reaction conditions were as follows: 95°C for 30 s, followed by 40 cycles at 95°C for 10 s and 65°C for 20 s.A melting curve was generated by cooling from 95°C to 65°C then ramping to 95°C, followed by the final cooling to 50°C for 30 s.
PsACT (Knopkiewicz and Wojtaszek, 2019) was used as the internal control.The expression level of each PsRBOH gene was calculated using the delta-delta Ct (2 −DDCT ) method (Livak and Schmittgen, 2001).All analyses were conducted in three biological replicates and three technical replicates.All primer sequences used in this study were designed by Primer Premier 6.0 and are listed in Supplementary Table S1.

Subcellular localization analysis
The coding sequence (CDS) of RBOH6 was amplified from the ZW6 and fused to the N terminus of the green fluorescent protein (GFP) in the pAN580 (GFP) vector.To determine the subcellular localization of RBOH6, the RBHO6-GFP vector was transiently coexpressed with endoplasmic reticulum (ER) marker mCherry-HDEL into Arabidopsis protoplasts (Ishikawa et al., 2018).And, the FM4-64 dye was used to marked plasma membrane.The fluorescent signals were observed using a confocal microscope (Leica SP8, Germany).Fluorescence signals were detected using the following excitation and emission wavelengths: GFP (488 nm/507 nm), FM4-64 (515 nm/640 nm) and mCherry (587 nm/610 nm).

Chromosomal localization analysis of the RBOH gene family in pea
Seven putative PsRBOH genes were retrieved from the pea genome after removing the redundant and repeat sequences.We named these PsRBOH1-7 (Figure 1 and Supplementary Table S2) based on their localization in the P. sativum genome.The distribution of these RBOH genes did not show a certain regularity.There was one RBOH gene on chromosomes chr1LG5 (RBOH1), chr5LG3 (RBOH4), and chr6LG2 (RBOH5) each, while two were on chromosomes chr3LG5 (RBOH2 and 3) and chr7LG2 (RBOH6 and 7) each.

Phylogenetic analysis of the PsRBOHs
To investigate the evolutionary relationships of RBOHs among pea, soybean, tomato, and Arabidopsis, we constructed a neighborjoining phylogenetic tree using the alignments of seven pea RBOH proteins, 17 soybean RBOH proteins, eight tomato RBOH proteins, and ten Arabidopsis RBOH proteins (Figure 2A).The results showed that 42 RBOHs from pea, soybean, tomato, and Arabidopsis were divided into five groups (I, II, III, IV, and V).Groups I, II, III, IV, and V contained 8, 6, 2, 6, and 20 RBOH proteins, respectively, and 1, 1, 0, 1 and 4 PsRBOHs were clustered in groups I, II, III, IV, and V, respectively.
Gene structure, conserved motif and conserved domain analysis of the PsRBOHs A gene structure map of PsRBOHs was constructed based on the pea genome sequence (Figure 3A).The PsRBOHs had varying structures with different numbers of untranslated regions (UTRs) and exons.All PsRBOHs but PsRBOH1 and PsRBOH4 had no UTRs, and the number of exons ranged from six (PsRBOH1) to 14 (PsRBOH6).Ten conserved motifs were identified via the MEME online tool and were used to gain a deeper understanding of various motif compositions of the PsRBOHs.As shown in Figure 3B and Supplementary Figure S1, PsRBOH proteins contained 10 motifs, except PsRBOH1 and PsRBOH4, which had 4 and 9 motifs, respectively.Moreover, all PsRBOH proteins, except PsRBOH1, had four typical conserved domains, including the NADPH-Ox, Ferri-reduction, FAD-binding, and NAD-binding domains (Figure 3C).PsRBOH1 lacked the FAD-binding and NADbinding domains, and all RBOH proteins contained the EFhand domain.

Cis-element analysis of the putative PsRBOH promoters
To further explore the function of PsRBOH genes in peas, we predicted the cis-elements of the promoters of these using the PlantCARE database (Figure 3D).The results showed that the lightresponsive elements (LREs) were the most abundant (84) among the seven PsRBOH promoter sequences and widely distributed in all promoter sequences, followed by ethylene-responsive elements (EREs) (20), which was also distributed in all promoter sequences Genomic distribution of the RBOH genes in pea chromosomes.Liu et al. 10.3389/fpls.2023.1321952Frontiers in Plant Science frontiersin.org(Figure 3D).We also found that the promoter sequences of PsRBOH

Tissue-specific expression of PsRBOHs in pea
To determine the expression patterns of the PsRBOH genes in different tissues, we studied the tissue-specific transcriptional activity of seven genes in the root, stem, tendril, leaf, flower, pod, The gene structures, conserved motifs, conserved domain, and cis-elements analysis of putative promoters of PsRBOHs.one-week-old pea seeds (Pea1W), and two-weeks-old pea seeds (Pea2W) of pea (Figure 4).The results showed that the expressions of the PsRBOH genes had significant differences in different tissues.
PsRBOH5 was highly expressed in the roots, while PsRBOH4, PsRBOH6, and PsRBOH7 were highly expressed in the stem.
PsRBOH2 was highly expressed in all tissues.PsRBOH6 was highly expressed in the leaves, but PsRBOH3 was not detected in the leaves.Moreover, PsRBOH1 and PsRBOH3 were highly expressed in tendrils, and all seven PsRBOH genes had relatively lower expression levels in the pod and Pea1W.PsRBOH5 and PsRBOH7 were highly expressed in the roots and Pea2W, respectively, but PsRBOH1 was not expressed in flowers and pods.

Expression of PsRBOHs under different abiotic stresses
To further elucidate the expression patterns of pea RBOH genes under different abiotic stresses, we evaluated the expression profiles of PsRBOHs in the leaves and roots under heat stress, cold stress, salt stress, Cd stress, PEG-induced drought stress (PEG), and lowboron (LB) stress at various time points after the treatment.
The abiotic stresses induced or inhibited the expression of genes in a highly specific manner, with the most significant changes occurring at the early stages of stress treatment (Figure 5).The expression levels of all genes increased in the leaves in all six treatments (Figures 5A-F), except for PsRBOH4 under salt stress (Figure 5C) and PsRBOH5 under Cd stress (Figure 5D).All PsRBOH genes were up-regulated at 6 h under all treatments (Figures 5A-F) except for PsRBOH4 under cold stress (Figure 5B), PsRBOH5 under Cd stress (Figure 5D) and PsRBOH4-6 under salt stress (Figure 5C).PsRBOH1 was up-regulated at 3 h under all six treatments but downregulated at 24 h under heat (Figure 5A), cold (Figure 5B) and Cd stresses (Figure 5D).PsRBOH3 was not expressed in the leaves under all treatments (Figures 5A-F).Furthermore, PsRBOH4 was downregulated from 0.5 to 48 h but up-regulated at 72 h under salt stress (Figure 5C).PsRBOH5 was induced at 0.5 h under heat stress (Figure 5A) but down-regulated at 12 h under heat (Figure 5B), salt (Figure 5C), Cd (Figure 5D), and PEG (Figure 5E) stresses.In addition, PsRBOH6 was induced at 6 h under LB stress (Figure 5F) and down-regulated at all time points except 3 h and 6 h under salt stress (Figure 5C).
The expression patterns of PsRBOHs had similarities and differences in the roots and leaves.In the roots, PsRBOH5 was induced at 0.5 h under heat (Figure 6A), salt (Figure 6C), PEG (Figure 6E), and LB (Figure 6F) stresses and maintained the high expression until 72 h under salt (Figure 6C), PEG (Figure 6E) and LB (Figure 6F) stresses.PsRBOH1 was up-regulated at 3 h and maintained a high expression level until 72 h under cold (Figure 6B) and salt stresses (Figure 6C) except at 12 h under cold stress.However, PsRBOH1 was down-regulated at all time points under LB treatment (Figure 6F).PsRBOH6 induced at 0.5 h under salt (Figure 6C) and Cd (Figure 6D) treatments but down-regulated from 6 to 72 h under Cd (Figure 6D) and LB (Figure 6F) treatments.Additionally, PsRBOH6 had a similar expression pattern with PsRBOH1 under LB stress, except at the 3 h time-point (Figure 6F).

Subcellular localization analysis
The subcellular localization of pea RBOH proteins was predicted using WoLFPSORT (Table 1).The results indicated that four PsRBOH proteins were highly likely to be located in the plasma membrane.PsRBOH4 and PsRBOH5 were presumably located in the nucleus, and PsRBOH4 in the Evolutionary analysis showed that PsRBOH6 was homologous to AtRBOHF and GmRBOHF1/2.It has been shown that multiple calcineurin B-like (CBL) interacting protein kinases (CIPKs) target Arabidopsis RBOHF by directly binding Ca 2+ to its EF-hands to fine-tune superoxide production in response to different stimuli (Drerup et al., 2013;Han et al., 2019).Since AtRBOHF localizes in the plasma membrane (Drerup et al., 2013), PsRBOH6 was selected for a transient expression assay using Arabidopsis mesophyll protoplasts (Figure 7).The PsRBOH6-GFP plasmid was transiently co-expressed with the plasma membrane and endoplasmic reticulum markers, FM4-64 (Figure 7A) and mCherry-HDEL (Figure 7B), in Arabidopsis leaf protoplasts, respectively.As shown in Figure 7, GFP-PsRBOH6 fluorescent signals were extensively co-localized with FM4-64 and mCherry-HDEL, suggesting that PsRBOH6 localizes in the plasma membrane and endoplasmic reticulum.

Discussion
RBOH genes are involved in plant signal transmission, morphogenesis and development, and responses to biotic and abiotic stresses.However, the RBOH gene family has been identified in various plants but not in pea.These studies will be useful for understanding the role of RBOH genes in plant responses to abiotic stresses.
Determining the phylogenetic relationships among species is fundamental for many biological studies (Kapli et al., 2020).Phylogenetic analysis indicated that members of the RBOH gene families in pea, soybean, tomato, and Arabidopsis could be divided into five groups, indicating that RBOHs are conserved in various species (Chang et al., 2020;Chen and Yang, 2020;Gui et al., 2022;Du et al., 2023).
In soybeans, the ancestral Papilionoideae whole-genome duplication event and Glycine-specific duplication event resulted in nearly 75% of the genes present in multiple copies (Schmutz et al., 2010).While there has no a recent whole-genome duplication but reflects the ancestral Papilionoideae whole-genome duplication event in pea (Kreplak et al., 2019).This also explains why the amount of RBOHs in pea is less than that in soybean and the collinear gene pairs of four PsRBOHs (PsRBOH2, PsRBOH4, PsRBOH6, and PsRBOH7) in soybean.
In Arabidopsis, all ten RBOHs had four typical conserved domains: NADPH-Ox, Ferri-reduction, FAD-binding, and NADbinding domains (Sagi and Fluhr, 2006).However, in wheat, 4 out of 36 NADPH oxidases had the NADPH_Ox domain but lacked one or two other conserved domains (Hu et al., 2018).In eggplant, 5 out of 8 SmRBOHs lacked the FAD-binding domain, which was substituted by the NOX_Duox_like_FAD_NADP domain (Du et al., 2023).In this study, all PsRBOHs, except PsRBOH1, had the four typical conserved domains.PsRBOH1 lacked the FADbinding and NAD-binding domains which are crucial for electron transfer and ROS production (Wang et al., 2022).Therefore, the possible function of RBOH1 in the production of superoxide needs further verification.
The tissue-specific expression of genes is crucial for plant growth and development and provides important insights into understanding gene function.The tissue-specific expression patterns of the RBOHs have been reported in many plants; however, the expression patterns are distinct in different plants.Two out of ten, four out of seven, and all seven RBOH genes were expressed throughout Arabidopsis (Sagi and Fluhr, 2006), strawberry (Zhang et al., 2018) and grape (Cheng et al., 2013), respectively.However, only four out of ten AtRBOH genes were specifically expressed in the roots and elongation zone (Sagi and , 2006).In this study, five RBOHs (PsRBOH2 and PsRBOH4-7) were expressed in all tissues, while PsRBOH1 was not detected in flowers and pods, and PsRBOH3 was not expressed in the leaves.In addition, the expression of PsRBOH2 was higher in all tissues, especially in the leaves.In soybeans, GmRBOHB1, which was orthologous with PsRBOH2 and clustered in one cluster, was highly expressed in the leaves (Liu et al., 2019).These results indicated the tissue specificity of the PsRBOH genes, suggesting that these genes may have different functions during plant development.
Cis-acting elements regulate the expression of target genes by binding the trans-acting factors (Yamaguchi-Shinozaki and Shinozaki, 2005).It has been reported that RBOH family members in various plants are induced by different abiotic stress stimuli, such as drought (Duan et al., 2009), salt (Xie et al., 2011), heat (Li et al., 2014;Xia et al., 2014), wounding (Sagi et al., 2004), and cold stress (Zhang et al., 2018).Moreover, RBOHs respond to environmental stimuli through hormonal signaling networks involving abscisic, salicylic, jasmonic acid, and ethylene (Overmyer et al., 2003).In the present study, many hormoneresponsive elements (such as ARE, ERE, MeJARE, and SARE) and abiotic response elements (such as DRE and LTR) were found in the promoter of PsRBOHs, indicating their potential roles in pea response to phytohormones and stresses.
The involvement of the RBOH gene family members in abiotic stress responses has been reported in many plants.In grapes, VvRBOHs were significantly increased under salt, drought, powdery mildew, salicylic acid, and abscisic acid treatments (Cheng et al., 2013).It was reported that the expression level of AtRBOHD was significantly increased in Arabidopsis at an early stage in response to hypoxia (Sagi and Fluhr, 2006).Soybean RBOH genes were significantly induced by salt, PEG, cold, and Cu stresses, and GmRBOHD2 was significantly induced by salt, polyethylene glycol, low temperature, and Cu toxicity in the roots (Liu et al., 2019).In the present study, all PsRBOH genes responded to all six stresses.Moreover, PsRBOH3, which had a close evolutionary relationship with GmRBOHD2, was expressed in the roots and not in leaves, and its expression occurred earlier (0.5 -6 h) after heat, cold, and LB stress treatments.The expression of PsRBOH1 was up-regulated in the leaves 3 h after the abiotic stress treatments and lasted for up to 24 h, after which it was down-regulated under heat, cold, salt and Cd treatments.However, PsRBOH1 was upregulated in the roots by heat, cold and NaCl treatments but downregulated by LB treatment.A similar phenomenon was also observed in cotton (Wang et al., 2020).These results indicate that the PsRBOH gene family could be involved in the abiotic stress response, but the regulatory mechanisms are still unclear.
Superoxide can be synthesized in different cellular compartments, such as chloroplasts (Foyer and Hanke, 2022), mitochondria (Postiglione and Muday, 2022), peroxisome (Sandalio et al., 2021), and the plasma membrane oxidoreductase system (Lherminier et al., 2009;Jimeńez-Quesada et al., 2022;Miller and Mittler, 2023).In leaves from pea plants grown with 50 µm CdCl 2 , the accumulation of H 2 O 2 was observed mainly in the plasma membrane of transfer, mesophyll and epidermal cells, as well as in the tonoplast of bundle sheath cells (Romero-Puertas et al., 2004).RBOH is present in the plasma membrane systems of almost all animals and plants (Chen and Yang, 2020).Subcellular location is a key characteristic that determines the function of many proteins, indicating that proteins in different subcellular locations have different functions (Koroleva et al., 2005).All the RBOHs in Arabidopsis (Sagi and Fluhr, 2006), rice (Groom et al., 1996) and wheat (Hu et al., 2018) were predicted to localize to the plasma membrane.Some RBOHs were shown to localize to the chloroplast thylakoid membrane of grapes (Cheng et al., 2013) and strawberry (Zhang et al., 2018).Moreover, in Gossypium barbadense, 71 out of 87 RBOHs were located in the cytoplasm (Chang et al., 2020).Tobacco RBOHD was localized in the plasma membrane and Golgi cisternae (Noirot et al., 2014).In P. vulgaris, PvRBOHA was localized in the plasma membrane of root hair (Arthikala et al., 2017), whereas PvRBOHB was localized in the central apical dome (Montiel et al., 2012).These different subcellular distributions of RBOHs in root hairs resulted in two models that explain root hair development (Arthikala et al., 2017).In pea, four out of the seven RBOHs had a high probability of being localized in the plasma membrane, indicating similar functions.Two of PsRBOHs were presumably located in the nucleus, while PsRBOH6 was localized in the cell membrane and endoplasmic reticulum, suggesting that they may regulate ROS production at different subcellular locations (Lee et al., 2023).

Conclusions
This study comprehensively analyzed the RBOH gene family in the pea genome.Seven PsRBOH genes were identified and were divided into five groups, which were distributed on five chromosomes.In addition, we analyzed collinearity, gene structure, conserved domains, conserved motifs, cis-elements, and subcellular distribution of the PsRBOH genes.The PsRBOHs exhibited tissue specificity and functional diversity during plant growth and response to different abiotic stresses.Overall, these results provide valuable information which could be used for further functional analysis of pea RBOH genes in response to climate-driven environmental constraints.
(A) Gene structure of PsRBOHs.The green block represents the coding sequence (CDS), the yellow block shows the 5' or 3' untranslated regions (UTRs), and the black line indicates the intron.The scale bar indicates the length of the DNA sequences.(B) The conserved motifs in PsRBOHs were identified by MEME tools.Each motif is indicated by a rectangular box of a different color and the motifs are numbered from 1 to 10.The black line scale represents the length of amino acids.(C) Conserved domain compositions of PsRBOHs.Only major domains are presented in the figure based on our searches in the Pfam database.The black line scale represents the length of amino acids.(D) Cis-elements analysis of putative promoters of PsRBOHs.The black line scale represents the length of promoter amino acids.The cis-elements are as follows: MeJA-responsive elements (MeJARE), light-responsive elements (LREs), ethylene-responsive elements (EREs), drought-inducible elements (DRE), abscisic acid-responsive element (ARE), salicylic acidresponsive elements (SARE), and low temperature-responsive element (LTR).

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FIGURE 2Phylogenetic and collinearity analyses of the PsRBOHs.(A) Phylogenetic analysis of RBOHs of pea, soybean, tomato, and Arabidopsis.(B) Collinearity analysis of RBOHs between pea and soybean.The colored lines represent RBOHs collinearity between species, and the gray lines are collinearity of all gene members between species.Yellow and green boxes represent pea and soybean chromosomes, respectively.

FIGURE 4
FIGURE 4Expression patterns of PsRBOH genes in different tissues.Pea1W: One-week-old pea seeds; Pea2W: Two-week-old pea seeds.Error bars represent standard deviations for three biological replicates.Different letters indicating significant differences among tissues (p<0.05,Duncan's test).n.d.:Not detected.
FIGURE 6Root expression of PsRBOHs under heat stress (A), cold stress (B), salt stress (C), cadmium (Cd) stress (D), polyethylene glycol (PEG)-induced drought stress (PEG) (E), and low boron (LB) stress (F).The log-transformed relative expression levels were used to generate the heatmaps.The color scale is shown on the right.

FIGURE 7
FIGURE 7 Subcellular localization of PsRBOH6 protein.(A) The plasma membrane was stained by FM4-64.(B) Transient co-expression of PsRBOH6-GFP with endoplasmic reticulum marker mCherry-HDEL.Empty vector was as control.Scale bar = 10 mm.Single optical sections obtained by CLSM. 2