Genome-wide identification, phylogeny and expression profiling of class III peroxidases gene family in Brachypodium distachyon

Gene 700 (2019) 149–162

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Research paper

Genome-wide identification, phylogeny and expression profiling of class III peroxidases gene family in Brachypodium distachyon

T

Ting Zhua, Fang Xina, ShuWei Weia, Yue Liua, YuCui Hana, Jiao Xiea, Qin Dingb, , LingJian Maa, ⁎

a b

⁎

College of Agronomy, Northwest A&F University, Yangling, 712100, Shanxi, China College of Horticulture, Northwest A&F University, Yangling, 712100, Shanxi, China

ARTICLE INFO

ABSTRACT

Keywords: Brachypodium distachyon Class III peroxidases Phylogenetic analysis Gene duplication Gene ontology annotation Expression patterns analysis

Class III peroxidases are classical secretory plant peroxidases belonging to a large multi-gene family. Class III peroxidases are involved in various physical processes and the response to biotic and abiotic stress to protect plants from environmental adversities. In this study, 151 BdPrx genes were identified using HMM and Blastp program. According to their physical location, the 151 BdPrx genes were mapped on five chromosomes. The results of Gene Structure Display Serve and MEME revealed that BdPrxs in the same subgroup shared similar gene structure, and their protein sequences were highly conserved. Based on the analysis of evolutionary relationships and Ka/Ks, 151 BdPrx genes were divided into 15 subgroups, they have undergone purifying selection. In addition, the result of GO annotation showed that 100% of the BdPrxs participated in antioxidant. The protein-protein interaction network was constructed using the orthology-based method, found that 66 BdPrxs were involved in the regulatory network and 183 network branches were identified. Furthermore, analysis of the transcriptome data indicated that the BdPrx genes responded to low concentration of exogenous phytohormones and exhibited different levels of expression in the different tissues. Subsequently, 19 genes were selected for quantitative real-time PCR and found to be mainly expressed in the roots, might preferentially respond to hydrogen peroxide and gibberellin. Our results provide a foundation for further evolutionary and functional study of Prx genes in B. distachyon.

1. Introduction Peroxidases (Prxs) are a family of thiol-based enzymes that are widely distributed in organisms, ranging from archaea to mammals (Pulido et al., 2009). They play an important role in the interaction between ROS and nitric oxide (NO) signaling. They can reduce hydrogen peroxide (H2O2), peroxynitrite, and organic peroxides, which are produced in the cells, by utilizing thiol-containing proteins (Calderon et al., 2017; Camejo et al., 2013). According to their structures, researchers have divided them into two categories, namely heme peroxidases and non-heme containing peroxidases (Mathe et al., 2010). Heme peroxidases are composed of a single peptide chain combined with an iron porphyrin prosthetic group and belong to two families: animal and non-animal Prxs. Three types of Prxs exist in non-animal Prxs: class I, class II, and class III. Class I and class III are unique to plants (Cosio and Dunand, 2009). All three types contain one protoporphyrin and Fe(III), and the 3D

structures are very similar, whereas show low similarity in their amino acid sequence, and they have different functions and subcellular localizations (Hiraga et al., 2001; Luthje and Martinez-Cortes, 2018; Luthje et al., 2011). Class I peroxidases exist in bacteria and plants and include APX (ascorbate peroxidase) and GPX (glutathione peroxidase). Class II peroxidases only encoded by fungi. Class III peroxidases (Prxs, EC 1. 11. 1. 7), which are typical plant-secreted peroxidases containing many secreted peroxide enzymes found in plants, including in switchgrass (Moural et al., 2017), Chinese pear (Cao et al., 2016b), and Populus (Ren et al., 2014). In higher plants, Class III peroxidases contain lots of isozymes and enzymatic reaction types that allow Prxs to participate in various functions in plants. Generally, Class III peroxidases perform two possible catalytic cycles, and stabilize the balance of peroxyl radicals in plants. Moreover, class III Prx members play crucial roles in plant growth, development, and stress responses. Class III Prx members are involved in auxin metabolism, lignification and lignin formation, cell

Abbreviations: Prx III, class III peroxidases; ROS, excessive reactive oxygen species; PEG, polyethylene glycol; ABA, abscisic acid; GA, gibberellic acid; JA, jasmonic acid; BR, brassinosteroids; SA, salicylic acid; qRT-PCR, quantitative real-time PCR; Ks, number of synonymous substitutions per synonymous site; Ka, number of nonsynonymous substitutions per non-synonymous site; NJ, neighbor-joining; bp, base pair; aa, amino acids; MW, molecular weight; pI, isoelectric point; Da, Dalton ⁎ Corresponding authors. E-mail addresses: [email protected] (Q. Ding), [email protected] (L. Ma). https://doi.org/10.1016/j.gene.2019.02.103 Received 28 December 2018; Received in revised form 4 February 2019; Accepted 21 February 2019 Available online 21 March 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.

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wall cross-linking of compounds, phytoalexin metabolism, and aging, thereby regulating plant growth and development (Beltramo et al., 2012; Cosio et al., 2009; Mei et al., 2009). They also participate in the plant stress response, including in pathogen defense (Almagro et al., 2009), freezing (Llorente et al., 2002), to protect cells against damage. Brachypodium distachyon is a new model plant that is closely related to common crop plants, including barley and wheat. It has many beneficial biological traits, such as a small genome, a rapid growth cycle, and a simple growing environment (Bevan et al., 2010). At present, Prx genes in many plants have been identified, but they have not been reported in B. distachyon. In this study, we used genome-wide bioinformatics analysis to investigate the Prx genes in B. distachyon. The BdPrx genes were localized to the chromosomes, and the gene structures, ciselements, phylogenetic relationships, as well as gene duplications were analyzed. The expression patterns of these BdPrx genes in different tissues and under different treatments were investigated using published RNA sequencing data. Finally, we used quantitative real-time (qRT) PCR to investigate the expression level of 19 BdPrx genes. These results will establish a foundation for future research on the functions of BdPrx genes, and provide a basis for the excavation of Prx genes in other species.

2.4. Gene duplication and collinearity analysis of B. distachyon, Oryza sativa, Zea mays, and Sorghum bicolor In order to analyze the gene duplication of BdPrxs, and the collinear correlations between the Prxs in B. distachyon, O. sativa, Z. mays, and S. bicolor. The MCScanX (Wang et al., 2012) software was used to detect syntenic regions in the whole genome. We then used the Circos 0.67 tool (Krzywinski et al., 2009) to visualize the gene duplication of BdPrxs and the synteny relationships between the aforementioned species. To analyze the evolutionary selection of BdPrx genes, we compared the CDS sequences. The KaKs_Calculator software (Wang et al., 2010) was used to calculate the KaKs ratio. And the divergence time of the four species were calculated [T = Ks/2λ × 10−6 Mya (λ = 6.5 × 10 −9)] (Wang et al., 2015b). 2.5. Gene ontology (GO) annotation analysis and protein–protein interaction network between BdPrxs and other proteins in B. distachyon The GO annotation of the Prx proteins of B. distachyon obtained from the Plant Transcriptional Regulatory Map (Jin et al., 2017) and PLAZE (https://bioinformatics.psb.ugent.be/plaza/). We used the WEGO online tool (http://wego.genomics.org.cn/cgi-bin/wego/index. pl) (Ye et al., 2018) to visualize the result. The AraNet V2 tool (Lee et al., 2015) and Cytoscape (Shannon et al., 2003) were used to construct an interaction network between BdPrxs and other B. distachyon proteins. We verified by the trusted value of Arabidopsis in the string database (http://string-db.org/) to confirm the interaction network of the BdPrx proteins, the protein interaction scores, which were > 0.8 were selected.

2. Materials and methods 2.1. Identification of Prx genes in B. distachyon We obtained the whole genome data of B. distachyon from the Ensembl plant database (http://plants.ensem-bl.org/index.html). The Prx domain (PF00141) downloaded from the PFAM database (http:// pfam.xfam.org/). Then HMMER 3.0 program with the threshold of E < 1e −5 was used to find proteins containing a Prx domain in B. distachyon (Cui et al., 2016). We downloaded the Prx protein sequences of rice (Passardi et al., 2004) and Arabidopsis (Tognolli et al., 2002) from Ensembl plant database to search against the B. distachyon protein sequence using the BLASTP program, the threshold set at an e-value of 1e−5 and 50% identity. We preliminarily identified the B. distachyon Prx genes by analyzing the results from HMM and BLASTP. We then used the NCBI-CDD web server (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and PFAM database (http://pfam.xfam.org/) to confirm the predicted Prx genes of B. distachyon (Marchler-Bauer et al., 2015). The cello web server (http://cello.life.nctu.edu.tw/) (Cui et al., 2016) was used to predict the subcellular localization of these genes, and the ExPASy server (http://www.expasy.org/) (Artimo et al., 2012) was used to obtain the theoretical isoelectric point (PI) and molecular weight (MW) of the B. distachyon Prx proteins.

2.6. Promoter analysis We downloaded the upstream 1.5 kb DNA sequence of the B. distachyon Prx genes from the Ensembl plant database and submitted these sequences to the PLACE database (http://www.dna.affrc.go.jp/PLACE/ ) to predict the cis-regulatory elements in the promoter regions. 2.7. Gene expression of Prx in B. distachyon The transcriptome data of the BdPrx genes in nine plant tissues (Davidson et al., 2012), and under different phytohormone treatments (Kakei et al., 2015) obtained from SRA database (http://www.ncbi.nlm. nih.gov/sra). These data were downloaded for use as a resource to analyze the expression patterns of BdPrx genes and were displayed in heatmaps.

2.2. Chromosomal location, gene structure, and conserved motif analysis of the B. distachyon Prxs

2.8. qRT-PCR analysis The B. distachyon cultivated in a greenhouse under a 16/8-h daynight photoperiod at 23 °C and 55–60% relative humidity in 2018. The roots, leaves, stems, and inflorescences were collected during the heading stage in B. distachyon. And two-week-old seedlings were exposed to 200 mM NaCl, 20% polyethylene glycol (PEG)6000, 10 mM H2O2, 100 μM gibberellic acid (GA), and 100 μM abscisic acid (ABA) for 2 h. The B. distachyon seedlings were collected after these treatments. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for RNA extraction with three biological replicates. Total RNA extracted using RNAiso Reagent (TaKaRa, Beijing, China). The RT Master Mix Perfect Real-Time kit (TaKaRa, Beijing, China) was used to synthesize cDNA. The q7 system was used to carry out qRT-PCR. The volume of each reaction was 20 μL. QuantStudio™ Real-Time PCR Software was used to analyze the data. The reference gene actin was used to normalize the expression of the BdPrx genes and the 2(−ΔΔCt) (Livak and Schmittgen, 2001) analysis method was used to determine the relative expression level. The primers used were listed in Table S2.

The genome annotation file of B. distachyon downloaded from Ensembl plant database. We then mapped the Prx genes to the B. distachyon chromosomes based on their physical location. The genes and coding sequences (CDS) of B. distachyon Prx were used to analyze the gene structure, and then the Gene Structure Display Server (http://gsds. cbi.pku.edu.cn/) (Hu et al., 2015) was used to illustrate the structure. MEME v4.9.0 (http://meme-suite.org/tools/meme) (Wang et al., 2015a) was used to identify the conserved motif of B. distachyon Prx proteins, with the optimum motif set at ≥10 and ≤250, and the maximum number of motifs set at 20. 2.3. Phylogenetic analysis The ClustalX 2.0 (Larkin et al., 2007) with the default parameters was used to conduct the multiple sequence alignment. We then used MEGA 6.0 (Tamura et al., 2013) software with 1000 bootstrap replications to construct an un-rooted neighbor-joining (NJ) tree. 150

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3. Results m otif m otif1

3.1. Identification and chromosomal location of BdPrxs

m otif2 m otif3 m otif4 m otif5

In this study, a total of 151 Prx genes of B. distachyon were obtained. Compared to other species, the number of Prx genes in B. distachyon was greater than Arabidopsis (73), maize (119), rice (138), and Populus (93) (Tognolli et al., 2002; Wang et al., 2015b; Passardi et al., 2004; Ren et al., 2014). All BdPrx genes were located on five chromosomes in B. distachyon and were named BdPrx001 to BdPrx151 based on their chromosomal location (Table S1, Fig. S1). As shown in Fig. S1, most BdPrx genes were distributed on chromosomes 1 and 2, accounting for 33.11% and 29.08%. The other BdPrx genes were located on chromosome 3 (15.24%), chromosome 4 (9.27%), and chromosome 5 (12.58%). We detected BdPrx clusters at the top of chromosome 1 and 2, and at the bottom of chromosome 2 and 5. Based on the physicochemical properties of the BdPrx proteins (Table S1), we found that 93% of the BdPrxs were highly consistent in amino acid number and MW. The proteins of the BdPrxs ranged from 230 to 436 aa in length, and each BdPrx contained an average of 333 aa. The MW of the BdPrxs ranged from 25 to 50 KD, with an average of 35 KD. The PI of the BdPrx proteins ranged from 4.47 to 10.01. Based on the subcellular localization result (Table S1), about 82% of the BdPrxs were located at a single position, with 60% of the BdPrxs being located in extracellular, 13% in chloroplast, 5% in cytoplasmic, 2% in mitochondrial, 3% in nuclear, and 1% in plasma membrane. The remaining 60 BdPrxs existed in two positions, and only one BdPrx (BdPrx126) was located in extracellular, mitochondrial, or chloroplast. Based on our statistics, 106 genes appeared in extracellular, suggesting that the BdPrxs were mainly extracellular.

m otif6 m otif7 m otif8 m otif9 m otif10 m otif11 m otif12 m otif13 m otif14 m otif15 m otif16 m otif17 m otif18 m otif19 m otif20 gene_structure exon upstream / downstream

3.2. Gene structure and conserved motif analysis of BdPrx genes Gene structural diversity plays an important role in the evolutionary relationships of gene families (Xu et al., 2012). We used the gene sequences and the CDS sequences of the BdPrxs to analyze the gene structure (Fig. 1). Most subfamilies contained 1–4 exons and 1–3 introns, the same subfamilies of these BdPrxs shared similar gene structures and positions. A total of 16.56% (25) of BdPrx gene structures were identified as typical structure (containing four exons and three introns), which was previously proposed by Tognolli et al. (2002) in A. thaliana. We suspect that these 25 genes may have a close relationship with A. thaliana. Thirteen (8.61%) of the 151 BdPrx genes did not possess introns, including BdPrx011, -012, and -128. However, there were still nine genes in our results that differed from the above and contained at least eight exons and seven introns. Conserved motifs play an important role in the basic functions of gene families and classification. Using MEME, 20 conserved motifs were analyzed (Fig. 1). As indicated in Fig. 1, most BdPrx proteins possessed highly conserved motifs, except for ten BdPrx proteins. Compared with the other BdPrx proteins, these ten BdPrxs contained few motifs. In addition, the same subfamilies possessed similar motifs. Motif 1, 2, 4, 5, and 7 encoded the BdPrx domain, and motif 1 was repeated twice in BdPrx094 and BdPrx108, suggesting that Prx proteins are functionally similar between different members of the same subgroup. Motif 18 was found in the subgroup I, Motif 19 existed in two subfamilies, and motif 20 appeared in subgroup I, II, IX, XI, and X, suggesting that they have special functions in these subfamilies.

BdPrx039 BdPrx040 BdPrx041 BdPrx128 BdPrx011 BdPrx012 BdPrx013 BdPrx016 BdPrx014 BdPrx015 BdPrx076 BdPrx059 BdPrx079 BdPrx077 BdPrx078 BdPrx068 BdPrx075 BdPrx066 BdPrx067 BdPrx063 BdPrx064 BdPrx065 BdPrx060 BdPrx061 BdPrx029 BdPrx062 BdPrx085 BdPrx080 BdPrx081 BdPrx082 BdPrx083 BdPrx084 BdPrx089 BdPrx091 BdPrx090 BdPrx088 BdPrx086 BdPrx087 BdPrx050 BdPrx109 BdPrx049 BdPrx108 BdPrx118 BdPrx105 BdPrx151 BdPrx069 BdPrx121 BdPrx058 BdPrx020 BdPrx096 BdPrx036 BdPrx071 BdPrx095 BdPrx116 BdPrx072 BdPrx074 BdPrx073 BdPrx141 BdPrx037 BdPrx038 BdPrx003 BdPrx030 BdPrx032 BdPrx033 BdPrx017 BdPrx110 BdPrx103 BdPrx104 BdPrx094 BdPrx051 BdPrx122 BdPrx129 BdPrx138 BdPrx001 BdPrx035 BdPrx002 BdPrx113 BdPrx021 BdPrx107 BdPrx022 BdPrx143 BdPrx093 BdPrx117 BdPrx137 BdPrx146 BdPrx147 BdPrx070 BdPrx145 BdPrx150 BdPrx148 BdPrx149 BdPrx144 BdPrx125 BdPrx053 BdPrx111 BdPrx023 BdPrx024 BdPrx025 BdPrx099 BdPrx052 BdPrx097 BdPrx098 BdPrx028 BdPrx101 BdPrx139 BdPrx100 BdPrx102 BdPrx026 BdPrx027 BdPrx031 BdPrx126 BdPrx127 BdPrx119 BdPrx120 BdPrx047 BdPrx019 BdPrx046 BdPrx018 BdPrx045 BdPrx055 BdPrx056 BdPrx057 BdPrx092 BdPrx133 BdPrx135 BdPrx054 BdPrx005 BdPrx043 BdPrx106 BdPrx124 BdPrx132 BdPrx131 BdPrx142 BdPrx034 BdPrx010 BdPrx042 BdPrx008 BdPrx009 BdPrx006 BdPrx007 BdPrx112 BdPrx115 BdPrx136 BdPrx130 BdPrx004 BdPrx044 BdPrx114 BdPrx134 BdPrx048 BdPrx123 BdPrx140

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Fig. 1. Phylogenetic relationship, gene structure and conserved motifs analysis of BdPrxs. Each subfamily uses a different color to distinguish. Grey boxes represent exons and black lines represent introns, green boxes stand for the upstream and downstream regions. Each motif is represented by a colored box, the length of genes and protein sequences is indicated by the scale at bottom. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

151

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Bd Pr x052 OsPrx025 BdPrx099 OsPrx087 BdPrx024 OsPr x088 BdPrx025 OsPrx086 Bd Prx023 OsPrx136 1 OsPrx13 57 OsPr x0 42 Bd Pr x1 x034 Bd Pr 041 OsPrx x042 Bd Pr 9 rx10 OsP 0 rx01 BdP 113 rx OsP 4 rx11 OsP 112 rx OsP 111 rx OsP 110 rx OsP 009 rx Bd P 008 Prx 07 Bd 0 Prx 06 Bd 0 Prx Bd rx040 P Os r x043 P 5 Bd r x11 P 5 Os r x00 9 P Bd r x05 4 P Os r x14 0 P 6 Bd r x0 4 P 6 Os x0 63 Pr Os Pr x0 62 Os r x0 61 P Os Pr x0 50 Os Pr x1 49 1 8 Bd r x P 14 Bd Pr x 145 Bd Pr x 146 Bd Pr x 147 0 Bd Pr x 07 2 x Bd Pr 01 Bd t Pr x A

O O sPr O sPr x02 s x 1 Bd Pr x 022 Os Pr x 020 Bd Pr x 125 Bd Pr x 023 1 Bd Pr x0 11 P 5 Bd r x 3 0 Bd Pr x0 91 Bd Pr x0 89 Bd Pr x0 90 P 8 Bd r x0 8 87 Pr x Bd P 08 Bd r x0 6 8 P Os r x08 4 P 3 Bd r x05 P 3 Bd r x08 2 P Bd rx081 Prx 080 Os P Os rx010 Prx OsP 009 rx OsP 067 rx0 6 Bd P 5 rx OsP 085 rx OsP 099 rx OsP 008 rx Bd P 011 rx06 2 OsP rx07 9 Bd Pr x0 OsPrx 29 06 OsPrx 8 101 OsPr x100 OsPr x0 07 Bd Pr x0 61 Bd Prx06 0 OsPrx035 OsPrx072 BdPrx075 OsPrx016 BdPr x068 OsPrx015 BdPrx067 BdPrx066 BdPrx065 OsPrx014 BdPrx064 OsPrx013 OsPrx012 BdPrx063 OsPrx070 Bd Prx078 BdPrx077

OsPr x002 Bd Pr x097 OsPrx027 BdPrx098 OsPrx026 BdPr x028 BdPrx100 OsPrx030 BdPrx102 OsPrx029 OsPrx02 8 Bd Pr x1 39 Bd Pr x1 01 Bd Pr x124 Bd Pr x131 Bd Pr x132 OsPrx 130 OsP rx13 5 AtPrx 052 AtP rx06 7 AtP rx AtP 068 rx AtP 004 rx Bd P 005 rx Bd P 026 rx0 Os 27 P Os rx081 Prx 082 Os P Os rx083 P Os rx08 4 P Bd r x085 P Bd r x12 P 6 Os r x12 At Pr x1 7 P 2 Bd r x01 2 P 7 Os r x0 3 At Pr x0 1 P Bd r x0 77 Os Pr x 11 1 At Pr x 06 1 P Bd r x0 18 O Pr x 40 Bd sPr x 018 O Pr x 126 s At Pr x 045 P At r x 03 At Pr x 053 8 Pr 05 x0 4 59

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V XI

10 x0 19 0 Pr At Pr x 125 Bd Pr x 046 s O Pr x 039 Bd Pr x 047 Os Pr x 37 0 Bd Pr x 19 Os Pr x1 20 1 Bd Pr x 24 Bd Pr x1 2 2 Os r x0 3 P At r x02 2 P At r x03 3 AtP r x03 4 AtP r x03 AtP r x037 8 AtP r x03 AtP rx054 P Bd rx005 P 09 Os rx0 AtP rx055 P Bd rx056 Bd P 057 rx Bd P 003 rx OsP 092 rx Bd P 017 rx OsP 133 rx Bd P 5 rx13 BdP 4 rx05 OsP x014 At Pr 15 x0 At Pr 49 At Pr x0 36 At Pr x0 72 At Pr x0 20 At Pr x0

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AtPrx BdPrx OsPrx BdPrx140 BdPrx123 BdPrx004

OsPrx078

Bd Pr x033 Bd Pr x032

OsPr x116 AtPrx042 AtPrx029 OsPrx073 BdPr x003 Bd Pr x030

Bd O Pr x Bd sPr 050 x At Pr x 034 Os Pr x 049 0 P Bd r x 24 Bd Pr x 138 1 Os Pr x 21 0 Bd Pr x0 69 Os Pr x0 04 P Os r x1 58 0 P At r x0 4 4 P At r x05 9 P 6 At r x02 P 7 AtP r x00 2 AtP r x00 1 r AtP x039 r AtP x030 Os rx003 P Bd rx024 Prx 0 Os Prx 96 0 Bd Prx 89 OsP 020 rx Bd P 019 rx OsP 095 rx Bd P 075 rx07 1 OsP rx Bd P 096 rx03 6 OsP rx10 OsPrx 3 095 Bd Pr x116 OsPrx 056 Bd Pr x141 OsPr x0 94 Bd Pr x0 37 OsPrx07 4 Bd Prx073 BdPrx074 BdPrx072 OsPrx134 OsPr x097 BdPrx038 OsPrx080

OsPrx071 Bd Prx076 OsPrx069 079 Bd Prx 06 OsPr x0 59 Bd Pr x0 x012 Bd Pr x013 Bd Pr 08 x1 OsPr 6 rx10 OsP x105 OsPr 6 rx01 Bd P 015 rx Bd P 014 rx Bd P 128 rx Bd P 044 rx OsP 043 rx OsP 042 rx OsP 041 Prx Bd rx045 P 040 Os Prx Bd r x048 P Os r x047 P 46 Os r x0 39 P Os r x0 7 P Bd r x10 1 P Os r x01 66 P 0 Bd r x 64 P At r x0 32 P 0 At r x 18 P 1 Os Pr x 47 Bd Pr x0 151 x 17 At Pr 1 Bd Pr x 105 Os Pr x 127 Bd Pr x 108 Os Pr x 128 Bd sPr x 109 33 O Pr x 0 x Bd sPr O

37 x1 5 Pr x05 7 Bd sPr x01 O Pr 102 Bd Pr x 07 0 s O Pr x 138 At Pr x 18 0 Bd Pr x 46 At r x0 51 P 0 At Pr x 01 Bd Pr x0 5 2 Os r x0 2 P 6 At r x0 1 P At r x07 9 P At r x06 0 P At r x07 2 AtP r x12 P 2 Bd r x13 P 3 Os r x13 P 9 Os rx12 P Bd 137 Prx 3 Os 1 rx0 AtP 43 rx0 AtP 061 rx AtP 026 rx AtP 060 rx AtP 129 rx OsP 110 rx Bd P 1 rx12 OsP 120 rx OsP 4 rx10 BdP x103 Bd Pr 08 At Pr x0 44 At Pr x0 28 At Pr x0 57 At Pr x0 2 OsPrx05 Bd Prx094 OsPrx018 AtPrx048 AtPr x021

BdPrx044 BdPrx114 BdPr x134 BdPrx115 BdPrx136 BdPrx130 Bd Prx112 Bd Prx048 OsPrx03 6 Bd Pr x0 01 OsPr x0 51 OsPr x098 Bd Pr x035 At Pr x016 At Pr x0 AtPrx 45 035 AtPrx 073 AtP rx AtP 050 rx Bd P 051 rx OsP 002 rx Bd P 050 rx OsP 113 rx1 Os 19 P AtP rx123 rx0 Bd 5 Prx 5 Os 021 P Os rx091 P Os r x09 P Bd r x09 2 P Bd r x1 3 0 Os Pr x0 7 2 P Bd r x0 2 Os Pr x1 90 Bd Pr x0 43 P 5 r x0 8 At Bd Pr x0 93 Os Pr x 19 1 O Pr x 17 At sPr x 031 P At r x 076 At Pr x 006 At Pr x 041 At Pr x 065 Pr 0 x0 31 63

Fig. 2. Phylogenetic analysis of Prx in B. distachyon, A. thaliana and rice. Red triangles represent BdPrxs, the green circles are OsPrxs, blue stars for AtPrxs. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

3.3. Phylogenetic analysis of BdPrxs

contained at least two species, except for subfamily I. Only 10 BdPrxs existed in subfamily I, and no AtPrxs or OsPrxs. Consistent with the results of the gene structure and MEME analysis, we found that because the genetic structure and motif of these genes differed from other BdPrxs, this resulted in differences in amino acid sequences, and the number of motifs was less than the other BdPrxs. It explains the distant evolutionary relationship with the Prxs from rice and A. thaliana.

The phylogenetic tree is a recognized biological evolution model based on biomolecular sequences (amino acid or DNA) (Chor and Tuller, 2005). In this study, we performed multiple sequence alignment and discovered that BdPrxs were highly conserved in amino acid. An NJ phylogenetic tree was constructed using the full length Prx proteins from B. distachyon, A. thaliana, and O. sativa (Fig. 2), and we discovered that a strong evolutionary relationship existed between them. According to the criteria of the Prxs of rice grouping, 362 Prx proteins were divided into 15 subfamilies, and each subfamily contained BdPrxs. Subfamily VIII was the largest family, including 25 BdPrxs, while subfamily IV was the smallest family, containing four BdPrxs and accounting for 2.65% of the BdPrxs. Interestingly, each subfamily

3.4. Gene duplication, Ka/Ks ratio, and collinear correlations of Prxs between B. distachyon, rice, maize, and sorghum genomes Gene duplication events, including tandem duplication of individual genes, segmental duplication of multiple genes, and background duplications. However, in our study, we analyzed the tandem duplication 152

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Fig. 3. Gene duplications in the B. distachyon (A), and correlations between B. distachyon and rice (B), maize (C), and sorghum (D) genomes. The light background represents synteny blocks in the whole genome of four species and dark lines stand for collinear gene pairs of BdPrxs.

and segmental duplication of BdPrx gene family. 50% genes constituted duplicated genes. We detected 45 pairs of BdPrx genes were identified as tandem duplication genes (Fig. 3A). In contrast, 16 pairs of BdPrx genes were segmentally duplicated and were located on five chromosomes (Fig. 3B). Tandem duplication was thus more prominent than segmental duplication. We predict that tandem duplication is the main gene duplication event in the Prx gene families in B. distachyon. It is necessary to calculate the Ka (nonsynonymous)/Ks (synonymous), in order determine the duplication time of the duplicated genes (Fay and Wu, 2003; Zhang et al., 2006). We thus calculated the Ka/Ks ratio of the duplicated BdPrxs (Table S3). The result showed that the Ka/Ks of BdPrxs was < 1, except for Bdprx021/BdPrx022 (1.034), indicating that they were subjected to purifying selection during expansion. The duplication time of the tandem duplication was predicted to occur around 50.11 Mya, which was later than the segmental duplication of approximately 59.20 Mya.

Colinearity analysis can be used as a reference to studying the evolutionary relationship between different species. The size of the collinear fragment is closely related to the divergence time (McCouch, 2001). In order to analyze the relationships of Prx genes among B. distachyon, rice, maize, and sorghum, we searched for homologous genes and analyzed their collinearity. A total of 99 B. distachyon Prx gene pairs had homologous genes in rice (Fig. 3B), 156 in maize (Fig. 3C), and 231 in sorghum (Fig. 3D). For example, BdPrx004 on chromosome 1 is homologous to genes in rice (Os03G0285700) chromosome 3, maize (GRMZM2G140667) chromosome 2, and sorghum (Sobic.002G431100) chromosome 2. The Ka/Ks between B. distachyon and rice, maize, sorghum was about 0.45, 0.56, and 0.64. The divergence time between rice and B. distachyon was around 45.90 Mya, which was later than sorghum at 63.71 Mya and maize at 59.48 Mya (Table S4–S6). These results imply that these genes experienced strong purifying selection, and the Prx genes in B. distachyon were highly

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intracinetracellular llular p art externa endomem e b nensvelope l encap sulatira ng struystem ctu plasmcaell periphere membra ry intrace ne llula cell a rt organreorganelle p p ll a e rt m in em membra tracellu ne-bou lar orgabnrane nded o e ll anelle extrace organrg e llular re elle part gion pa rt photos whole meapoplast yntheti mbrane c m membrane extraceembrane pa llular m rt atrix plasm intrinsic compo cell-cell ojudesma nent of n c ti n peroxid membrao oxidore ase ac ne ti ity ductas e activ vity ion bin organic c d g o c fa y c c to heteroc lic comp r bindin yclic co ound bin ing mpoun ding d re ding responresponse tosponse tobin s tr se to e a b ss ndogeniotic stimuelu o respon response us stimulus se to e to chem s x rnal respon ic se te timuluasl cellula develo biotic s r respto s pmenta ti m o u n lus se l pro e ss invo immunetorestimulus an tocm lved in sponse ical stru anatoam re p cture d roduction ical str e uc re cellula m rpvheolopment r devtu elopmo genesis develo ental pro multice developm pmental cess llula ntal ma growth rganisem cellular tura post-reo proces multice bryonic developmtion s involv llular m organis development ed in re m en multice producti se reproductiont o llular o rganisnmin multiceelld germina al repro ular orga tion nis ductive re cellular procesm compporoductive p nent org rocess anizati s cellular n metabocell growoth regulati cell com lic process m on of c positive ellularunication cell waregulationsoignal transpdrocess u ll organ f cellula ization r rocctieon or biop ss c e g ll organic respons ular detox enesis substa e to othe ification nce me r organ tabolic is procesm secon biosynth s tic proc positive regulatiodnary metabe ss regulati of meta olic proce on of m bolic pro ess etaboli ess c proc c positive oxidati cess on-readtabolic proc regula u e ti c o ti regulati n of biolo on process s gical p on of b ro ess iologic s al proc cess

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Fig. 4. Gene Ontology (GO) annotation analysis of BdPrxs, The GO annotation analysis result is divided into three main categories: cellular component, molecular function and biological process. The y-axis on the left represents the percentage of genes in each category, and the number of genes on the right.

homologous with sorghum, followed by maize and then rice.

developmental process (4%), anatomical structure morphogenesis (5%). As we all know that most plant physiological processes are accomplished by the interaction of proteins, rather than by single proteins. To better understand the molecular mechanism of BdPrxs, we constructed an interaction network between the BdPrx proteins and other B. distachyon proteins. A total of 183 interacting protein pairs were identified (Fig. 5). Sixty-six BdPrx proteins possessed interacting proteins. One third of the BdPrx proteins possessed at least four other proteins that interacted with them, which were marked in yellow in Fig. 5. Among them, most other B. distachyon proteins interacted with BdPrx076 proteins, accounting for 13.67% of the total, and thus might have an important role in the regulation of protein interaction networks. It was reported that Prxs were involved in lignification and participate in the metabolism of ROS, reactive nitrogen species (RNS), and plant hormones. In this study, we detected that BRADI1G2092 and BRADI3G08860 were dirigent proteins and participated in lignification. These two proteins interacted with the BdPrx proteins. Sottomayor

3.5. Gene ontology (GO) annotation analysis and protein–protein interaction network between BdPrxs and other proteins in B. distachyon In order to enhance our understanding of the biological functions of BdPrx proteins, we performed GO annotation analysis on 151 proteins (Fig. 4). Fifty-six GO terms were designated, and the different GO terms may have similar functionality. The GO annotation analysis included three functions, including cellular component, molecular function, and biological process. In cellular component, about 50% of the BdPrxs were involved in cell composition. < 10% of the BdPrxs were involved in cell junction and endomembrane composition. We know that Prxs can protect cells from ROS. Accordingly, we found that 100% of the BdPrxs participated in antioxidant, and response to stress. We thus predict the main function of BdPrxs to include the scavenging of ROS and response to stress. 100% participated in cellular detoxification. Furthermore, the biological process also included other terms related to

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Fig. 5. The protein–protein interaction network between BdPrxs and other B. distachyon proteins. Proteins with more interactions are marked. The BdPrxs are in yellow blocks, and the other B. distachyon proteins are in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

(Sottomayor et al., 2008) suggested that CroPrx01 interacted with arabinogalactan proteins (AGPs) to executive function, and accordingly, BRADI2G31980 (belonging to AGP) interacted with seven BdPrx proteins. In addition, the BdPrx proteins also interacted with proteins from other families, including V-ATPase_G, ATP-synt_D, and Ion_trans, to executive their functions in plants.

these genes are more sensitive low-stringency phytohormones. 3.8. Expression patterns of BdPrx genes in four tissues and under stress treatment by quantitative RT-PCR analysis Nineteen BdPrx genes from 15 subfamilies were randomly selected to investigate the expression patterns in four tissues and under stress treatment by qRT-PCR. As shown in Fig. 7A, all genes exhibited different profiles in the four tissues. Ten genes expressed low in leaves and inflorescence. Most genes were more highly expressed in the root than in the other three tissues, which corroborates previous findings in Arabidopsis and rice (Hiraga et al., 2001; Tognolli et al., 2002), indicating that genes have different functions in different tissues and that most BdPrx genes are essential for the roots during B. distachyon growth and development. In addition, BdPrx069 and BdPrx076 were detected to be specifically expressed in the inflorescences, suggesting that these two genes play an important role in flower development. BdPrx005, -23, -44 showed high expression level in the stems, and the BdPrx023 was only expressed in the stem, implying that they have an important function in the stem. Especially the BdPrx023. BdPrx005, -009, -44, -076, -085, -109 were expressed in four tissues, suggesting these genes are important to B. distachyon growth and development. Additionally, the expression patterns of 19 genes under different stress treatments were also investigated (Fig. 7B). Compared with the control, 19 BdPrx genes were significantly up- or down-regulated by the five different treatments. Six of the 19 genes were up-regulated by the different treatments. For instance, the BdPrx074, -0103, and -0109, were upregulated by the five treatments, while BdPrx085 was down-regulated by NaCl and PEG, and up-regulated by H2O2, GA, and ABA. BdPrx054 was up-regulated by the four treatments, but not by ABA. As shown in Fig. 7B, the highest expression level of the nine BdPrx genes were detected under the H2O2 treatment, and of these, BdPrx076 and BdPrx103 were strongly induced by H2O2. Among the five treatments, the BdPrx genes of the seedlings demonstrated the most obvious response to H2O2 and GA. However, the BdPrx064 did not respond to H2O2 treatment. Eight genes were highly expressed under the GA treatments. For the ABA and PEG treatments, BdPrx051 showed preferential expression under ABA, and BdPrx064 was high in the PEG treatment. These results reveal that when the seedlings of B. distachyon are treated with the five different stresses, most of the genes would response to five treatments, and they are preferentially expressed under the H2O2 and GA treatments.

3.6. Promoter analysis Promoters control the transcription and expression of genes by combining with specific transcription factors. We thus analyzed the ciselements in the BdPrx gene promoter regions (Table S9–S10). We discovered that the 151 BdPrxs contained a variety of cis-elements, which were divided into two types. One was related to response to stress and phytohormones. The other was related to plant growth and development, including light-responsive elements and morphologically-constructed elements. As shown in Table S9, we detected that the ABRE, CGTCA-motif, TGACG-motif, ARE, Box 4, G-box, and CAT-box elements were present in over 60% of BdPrx genes, and thus we suspect that these cis-acting elements have a great impact on the expression of BdPrx genes. 76.82% of BdPrx genes contained ABRE elements, which are involved in the ABA response. ABRE repeated nine times in BdPrx111 and BdPrx147 and seven times in four genes, suggesting that these genes can be induced by ABA. 3.7. Expression profiles of BdPrx genes The tissue-specific expression patterns of 94 BdPrx genes were investigated based on the transcription data of nine tissues. As shown in Fig. 6A, most BdPrx genes displayed different tissue-specific expression patterns. For example, nine genes, namely BdPrx006,-007,-009,-042,112,-114,-115,-116, and -140, were mainly expressed in the leaves, and six genes were found to be specifically expressed in the anthers. BdPrx110,-145, and -147 were highly expressed in the B. distachyon embryo tissue. Some genes exhibited higher expression levels only in individual tissues, such as BdPrx081, -083, -087, -090, -103, and -107, which were expressed in six tissues, excluding early inflorescence, emerging inflorescence, and pistil. These results indicate that BdPRrx genes have different functions in the tissues; some genes focus on the development of flowers, some genes play a role in the leaves, and some genes control pistil development. BdPRrx genes majorly influence the leaves, embryos, anthers, and seeds. In order to better understand the induced response of BdPrxs to phytohormones, we analyzed the expression patterns of 151 BdPrxs under eight different phytohormone treatments under high-stringency or low-stringency conditions (Fig. 6B). The result showed that 151 BdPrx genes were up- or down-regulated by the different phytohormones. Compared with the high-stringency treatments, the BdPrx genes exhibited greater differential expression under low-stringency conditions. For example, at low stringency, BdPrx058, -125, -128 were up-regulated by auxin; BdPrx065 was highly expressed under cytokinin treatment; BdPrx056,-082 were strongly induced by low salicylic acid (SA); and BdPrx077, -102 were strongly up-regulated by brassinosteroids (BR). BdPrx006, -116 were down-regulated by ABA; The expression of BdPrx028, -053 were decreased under jasmonic acid (JA); and BdPrx008, -018 were lowly expressed under GA treatment. However, no significant changes were observed at a high stringency, suggesting that

4. Discussion Class III peroxidases are components of multi-gene family proteins and belong to PR proteins, which are widely found in plants. They play a vital role in plant growth, development, and stress. In previous studies, the genome-wide identification and analysis of the Prx superfamily in A. thaliana, O. sativa, maize, Populus trichocarpa and Pyrus bretschneideri were reported (Tognolli et al., 2002; Passardi et al., 2004; Wang et al., 2015b; Ren et al., 2014; Cao et al., 2016b). However, the Prx superfamily in B. distachyon has not been reported. As a new generation model crop, the complete genome sequence of Bd21 was already completed in 2010 (‘Genome sequencing and analysis of the model grass Brachypodium distachyon’, 2010). This provided a basis for the identification of peroxidases in B. distachyon. Therefore, in this

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BdPrx009 BdPrx042 BdPrx115 BdPrx140 BdPrx112 BdPrx114 BdPrx116 BdPrx006 BdPrx007 BdPrx071 BdPrx040 BdPrx144 BdPrx003 BdPrx049 BdPrx064 BdPrx111 BdPrx120 BdPrx147 BdPrx110 BdPrx145 BdPrx124 BdPrx146 BdPrx119 BdPrx135 BdPrx033 BdPrx105 BdPrx128 BdPrx019 BdPrx138 BdPrx053 BdPrx055 BdPrx058 BdPrx137 BdPrx044 BdPrx136 BdPrx130 BdPrx023 BdPrx125 BdPrx002 BdPrx078 BdPrx005 BdPrx100 BdPrx004 BdPrx142 BdPrx133 BdPrx123 BdPrx107 BdPrx104 BdPrx103 BdPrx102 BdPrx094 BdPrx091 BdPrx090 BdPrx089 BdPrx087 BdPrx086 BdPrx084 BdPrx083 BdPrx081 BdPrx060 BdPrx041 BdPrx039 BdPrx036 BdPrx032 BdPrx027 BdPrx026 BdPrx025 BdPrx017 BdPrx013 BdPrx015 BdPrx148 BdPrx050 BdPrx122 BdPrx077 BdPrx126 BdPrx046 BdPrx020 BdPrx073 BdPrx072 BdPrx099 BdPrx056 BdPrx092 BdPrx063 BdPrx093 BdPrx131 BdPrx118 BdPrx001 BdPrx143 BdPrx057 BdPrx127 BdPrx069 BdPrx129 BdPrx150 BdPrx051 BdPrx076 BdPrx113 BdPrx141 BdPrx054 BdPrx149 BdPrx038 BdPrx062 BdPrx079 BdPrx030 BdPrx018 BdPrx048 BdPrx117 BdPrx021 BdPrx101 BdPrx031 BdPrx085 BdPrx121 BdPrx132 BdPrx074 BdPrx109 BdPrx108 BdPrx012 BdPrx134 BdPrx070 BdPrx097 BdPrx008 BdPrx106

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Fig. 6. The expression profiles of 94 BdPrx genes in nine tissues (A); The expression profiles of 151 BdPrxs under high and low-stringency of 8 phytohormones (B); H stands for high-stringency, L represents low-stringency. The higher expression levels are shown in red. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

study, we identified 151 Prx genes from the whole genome of B. distachyon. The evolutionary analysis divided the 151 Prx genes of B. distachyon into 15 subfamilies, with 61 pairs of genes involved in gene duplication events. In previous studies, segmental duplication is the main duplication event in multiple gene families in B. distachyon, such as AP2/ERF, bHLH, ARF, PP2C, and TIFY (Cui et al., 2016; Niu et al., 2017; Zhou et al., 2018; Cao et al., 2016a; Zhang et al., 2015). In contrast, in this study, tandem duplication contributes majorly to the expansion of BdPrx genes, which is similar to that observed in poplar (Ren et al., 2014). The Ka/Ks ratios of most duplicated pairs detected are < 1, which indicate that the Prx genes of B. distachyon expand on purifying selection, and the time of the segmental duplications is much earlier than the tandem duplication. The result of the phylogenetic analysis is consistent with the gene duplication analysis, and the result of MEME, implying that these genes, which are in the same subfamily, may perform the same or similar functions during plant growth or the stress response. However, BdPrx064 and BdPrx076 belong to the same subfamily, but they respond differently to H2O2, suggesting that members of the same subgroup may function differently. Gene structure analysis indicates that only a small part of the gene structures constitute typical gene structure. The 151 BdPrx genes contain different numbers of introns/exons, indicating that they are rich in gene function. A previous study showed that introns have a profound impact on biological evolution, which evolution can be reflected in the changing trend of introns content (Mount and Henikoff, 1993). We thus speculate that the evolution of introns leads to changes in the number of introns in the gene, resulting in a diverse genetic structure. Based on the evolutionary relationships between rice and Arabidopsis, the 362 Prxs were divided into 15 subfamilies. The result showed that a strong evolutionary relationship existed between them. All families except subfamily I contained two species, and only 10 BdPrxs existed in subfamily I. We thus predict that these 10 genes are unique to B. distachyon. Collinear analysis indicated close relationships between B. distachyon and rice, maize, and sorghum. BdPrx genes with higher homology to the three species could have similar functions. Previous studies have reported on the functional diversity of Prx genes in rice and A. thaliana. For example, Kidwai et al. (Kidwai et al., 2019) reported that the overexpression of OsPRX38 (homologous to Bdprx045) in A. thaliana not only enhanced arsenic tolerance, but also affected plant growth. OsPRX2 was induced by potassium deficiency in rice, and its overexpression improved potassium deficiency tolerance in rice (Mao et al., 2018). This gene had a strong evolutionary relationship with the BdPrxs to subfamily 14 and 12. AtPrx12 (Klok et al., 2002) was close to the BdPrxs of subfamily 10, it participated in the low oxygen response. AtPrx57, which shared a close relationship to the BdPrxs of subfamily 4, responded to arsenic stress (Abercrombie et al., 2008). The transgenic plants AtPrx33 and AtPrx34 (Bindschedler et al., 2006) were involved in promoting cell elongation in roots. These two genes shared high homology with the BdPrxs of subfamily 14. According to results, the functions of the BdPrx genes of subfamilies 4, 10, 12 and 14 can be predicted. The functional diversity of BdPrx was also reflected in the GO annotation results. BdPrxs are involved in plant growth, development, and stress in different ways, and are thus vital to plants. Our results provide a new foundation for studying the functions of BdPrx genes. The study of gene expression patterns provides a means of investigating the function of BdPrx genes. Available transcriptome data of B. distachyon were downloaded for use as a resource to study the gene

expression patterns. Based on this data, we discovered many genes that contribute to the leaves, embryos, anthers, and seeds, providing a reference for the study of reproductive growth in B. distachyon. However, the expression of BdPrx genes in the root was not reported. Some studies have indicated the presence of many Prxs in plant roots, such as maize (Mika et al., 2008; Hadzi-Taskovic Sukalovic et al., 2015) and A. thaliana (Klok et al., 2002; Kumari et al., 2008; Passardi et al., 2006). According to the analysis of qRT-PCR, we studied the tissue-specific expression of 19 genes in the root, stem, leaf, and inflorescence, 19 BdPrx genes with different expression levels in the four tissues, indicating that BdPrx genes play a role in specific developmental stages in B. distachyon. Compared with the stem, leaf, and inflorescence, BdPrx genes play a vital role in the root of B. distachyon, which is also true for maize (Mika et al., 2010; Mika et al., 2008). Additionally, researchers have reported that numerous Prx genes help plants to adjust to environmental stresses. For instance, the overexpression of CpPrx01 changed the growth pattern of plants and reduced the endogenous indole-3-acetic acid (IAA) levels (Cosio et al., 2009). CaPrx02 participated in regulating H2O2 levels (Choi et al., 2007), and in tomato, LePrx06 could be induced by H2O2 (Coego et al., 2005). We therefore analyzed the expression patterns of 19 genes under five different stress treatments. Compared to CK, 19 BdPrxs were significantly up or down regulated. Among the five treatments, the BdPrx genes of the seedlings demonstrated the most obvious response to H2O2 and GA. However, the BdPrx064 did not respond to H2O2 treatment, the reason may be that the function of BdPrx064 is redundant after tandem duplication event. From the results of qRT-PCR, we consider that the BdPrx genes have a strong response to H2O2 and GA. Under PEG treatment, BdPrx064 was highly expressed, and BdPrx051 was strongly induced by ABA, suggesting that BdPrx051 and BdPrx064 have important functions in the drought regulatory network. These findings corroborate the promoter analysis. For instance, BdPrx051 and BdPrx064 were highly expressed in the seedlings following ABA and PEG treatment, which are similar to the finding that the promoter of BdPrx051 contains two ABRE cis-elements, and that BdPrx064 contains five ABRE cis-elements, all of which were involved in drought responsiveness. BdPrx069 was up-regulated by GA and contained one P-box (gibberellin-responsive element) and one GARE-motif (gibberellin-responsive element). These results provide new information for the discovery of resistance genes in B. distachyon. Based on all the results, we consider that the BdPrx gene family is sensitive to induction of hydrogen peroxide. We speculate that this gene family balances the concentration into reactive oxygen species in plants and reduces the toxicity caused by the accumulation of reactive oxygen species. 5. Conclusion Class III Prx is superfamily with a large number of members that is widely distributed in different organisms. This gene family plays essential roles in the plants growth and development. It is necessary to study the plant Prx gene family. However, the Prx gene family has not been reported in B. distachyon. In this study, we for the first time identified the 151 BdPrx genes, which could defined as candidates for studying of functions. The 151 BdPrx genes were subjected to chromosomal location, phylogenetic construction, conserved motif and expression pattern analysis, functional prediction. And the qRT-PCR results of 19 BdbPrx genes in four tissues and under five treatments were

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BdPrx030 BdPrx003 BdPrx102 BdPrx056 BdPrx135 BdPrx016 BdPrx088 BdPrx072 BdPrx104 BdPrx059 BdPrx039 BdPrx089 BdPrx086 BdPrx090 BdPrx048 BdPrx103 BdPrx012 BdPrx014 BdPrx066 BdPrx078 BdPrx007 BdPrx075 BdPrx149 BdPrx081 BdPrx145 BdPrx097 BdPrx011 BdPrx100 BdPrx083 BdPrx113 BdPrx055 BdPrx141 BdPrx064 BdPrx073 BdPrx110 BdPrx108 BdPrx087 BdPrx015 BdPrx017 BdPrx036 BdPrx074 BdPrx101 BdPrx025 BdPrx010 BdPrx033 BdPrx021 BdPrx032 BdPrx041 BdPrx013 BdPrx024 BdPrx002 BdPrx065 BdPrx060 BdPrx062 BdPrx111 BdPrx118 BdPrx092 BdPrx150 BdPrx132 BdPrx040 BdPrx084 BdPrx045 BdPrx139 BdPrx027 BdPrx051 BdPrx085 BdPrx119 BdPrx143 BdPrx001 BdPrx144 BdPrx129 BdPrx151 BdPrx029 BdPrx070 BdPrx037 BdPrx148 BdPrx068 BdPrx142 BdPrx008 BdPrx077 BdPrx147 BdPrx061 BdPrx018 BdPrx146 BdPrx094 BdPrx035 BdPrx123 BdPrx028 BdPrx031 BdPrx093 BdPrx038 BdPrx112 BdPrx019 BdPrx043 BdPrx022 BdPrx098 BdPrx006 BdPrx054 BdPrx053 BdPrx116 BdPrx020 BdPrx057 BdPrx133 BdPrx117 BdPrx107 BdPrx106 BdPrx091 BdPrx026 BdPrx080 BdPrx109 BdPrx137 BdPrx105 BdPrx082 BdPrx131 BdPrx095 BdPrx050 BdPrx071 BdPrx120 BdPrx009 BdPrx046 BdPrx067 BdPrx134 BdPrx136 BdPrx044 BdPrx114 BdPrx140 BdPrx004 BdPrx130 BdPrx121 BdPrx126 BdPrx069 BdPrx076 BdPrx138 BdPrx096 BdPrx052 BdPrx115 BdPrx124 BdPrx063 BdPrx058 BdPrx079 BdPrx005 BdPrx023 BdPrx049 BdPrx125 BdPrx128 BdPrx127 BdPrx042 BdPrx122 BdPrx034 BdPrx047 BdPrx099

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Fig. 7. Quantitative RT-PCR analysis of 19 randomly selected BdPrx genes. The relative expression level of the 19 genes in 4 tissues including root, stem, leaf, inflorescence (A); The relative expression level of 19 genes with different treatments including 10 mM H2O2, 100 μM ABA, 100 μM GA, 20% PEG6000, 200 mM NaCl (B).

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analyzed. These results provide a foundation for the functional verification and evolution of BdPrx genes, and give a reference to the discovery of homologous genes in major cereal crops.

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Declarations of interest None Author contributions Ting Zhu conceived the study, performed the bioinformatics analysis and the experimental work, and wrote the manuscript. Fang Xin and ShuWei Wei took part in the experiments and construct the figures and tables. Yue Liu carried out the software analyses. YuCui Han and Xiejiao helped in revamping the manuscript. Qin Ding and LingJian Ma conceived and guided the experiments, and revised the manuscript, and contributed with valuable discussion. All authors read and approved the final manuscript. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.02.103. Funding This study was supported by the National “13th Five-Year” Key R&D Project (Grant No. 2016YFD0101602) and Shaanxi Science & Technology Co-ordination & Innovation Project of China (Grant No. 2016KTCQ02-02). Conflicts of interest The authors declare that they have no competing interests. Acknowledgements We thank for the Instrument sharing platform of Northwest Agricultural and Forestry University and lab members for their assistance in this study. We thank Letpub (http://www.letpub.com.cn/) for editing the English text of a draft of this manuscript. References Abercrombie, J.M., Halfhill, M.D., Ranjan, P., Rao, M.R., Saxton, A.M., Yuan, J.S., Stewart Jr., C.N., 2008. Transcriptional responses of Arabidopsis thaliana plants to As (V) stress. BMC Plant Biol. 8, 87. Almagro, L., Gomez Ros, L.V., Belchi-Navarro, S., Bru, R., Ros Barcelo, A., Pedreno, M.A., 2009. Class III peroxidases in plant defence reactions. J. Exp. Bot. 60, 377–390. Artimo, P., Jonnalagedda, M., Arnold, K., Baratin, D., Csardi, G., de Castro, E., Duvaud, S., Flegel, V., Fortier, A., Gasteiger, E., Grosdidier, A., Hernandez, C., Ioannidis, V., Kuznetsov, D., Liechti, R., Moretti, S., Mostaguir, K., Redaschi, N., Rossier, G., Xenarios, I., Stockinger, H., 2012. ExPASy: SIB bioinformatics resource portal. Nucleic Acids Res. 40, W597–W603. Beltramo, C., Torello Marinoni, D., Perrone, I., Botta, R., 2012. Isolation of a gene encoding for a class III peroxidase in female flower of Corylus avellana L. Mol. Biol. Rep. 39, 4997–5008. Bevan, M.W., Garvin, D.F., Vogel, J.P., 2010. Brachypodium distachyon genomics for sustainable food and fuel production. Curr. Opin. Biotechnol. 21, 211–217. Bindschedler, L.V., Dewdney, J., Blee, K.A., Stone, J.M., Asai, T., Plotnikov, J., Denoux, C., Hayes, T., Gerrish, C., Davies, D.R., Ausubel, F.M., Bolwell, G.P., 2006. Peroxidase-dependent apoplastic oxidative burst in Arabidopsis required for pathogen resistance. Plant J. 47, 851–863. Calderon, A., Lazaro-Payo, A., Iglesias-Baena, I., Camejo, D., Lazaro, J.J., Sevilla, F., Jimenez, A., 2017. Glutathionylation of pea chloroplast 2-Cys Prx and mitochondrial Prx IIF affects their structure and peroxidase activity and sulfiredoxin deglutathionylates only the 2-Cys Prx. Front. Plant Sci. 8, 118. Camejo, D., Romero-Puertas Mdel, C., Rodriguez-Serrano, M., Sandalio, L.M., Lazaro, J.J., Jimenez, A., Sevilla, F., 2013. Salinity-induced changes in S-nitrosylation of pea mitochondrial proteins. J. Proteome 79, 87–99. Cao, J., Jiang, M., Li, P., Chu, Z., 2016a. Genome-wide identification and evolutionary analyses of the PP2C gene family with their expression profiling in response to multiple stresses in Brachypodium distachyon. BMC Genomics 17, 175. Cao, Y., Han, Y., Meng, D., Li, D., Jin, Q., Lin, Y., Cai, Y., 2016b. Structural, evolutionary, and functional analysis of the class III peroxidase gene family in Chinese pear (Pyrus bretschneideri). Front. Plant Sci. 7, 1874.

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