Polyamine oxidases (PAOs) are flavin adenine dinucleotide-dependent enzymes that are involved in polyamine catabolism and play an essential role in growth and developmental processes as well as the response to abiotic stresses. Although the PAO gene families have been intensively studied in many plants, the soybean (Glycine max (L.) Merr.) PAO gene family has not been systematically identified. Here, we identified six PAO genes in the soybean genome and named them GmPAO1–GmPAO6. The phylogenetic analysis revealed that plant PAO proteins are divided into four classes. GmPAO1 and GmPAO4 belong to class I; GmPAO2, GmPAO5, and GmPAO6 belong to class IV. Similar to most dicotyledonous plants, soybeans do not contain class II. Interestingly, we identified an additional SWIRM-domain PAO gene GmPAO3, which exists between classes III and IV. GmPAO3 had a different gene structure and expression. To determine the individual roles of GmPAOs, we analyzed their expression levels in various tissues and under abiotic stress. Each GmPAO gene can respond in a specific tissue under specific abiotic stress. The data can help to clarify the role of GmPAOs in abiotic stress responses in soybean and provide a breeding basis for enhancing soybean tolerance to abiotic stresses.
Introduction
Soybean (Glycine max (L.) Merr.) is an important oilseed crop. However, soybean growth and yield are severely reduced by abiotic stresses, such as drought, salt, and cold (Kidokoro et al. 2015; Valliyodan et al. 2017; Cao et al. 2018). Therefore, it is necessary to understand the environmental adaptability of soybean and enhance its tolerance to abiotic stresses.
Polyamines (PAs) are low-molecular-weight organic cations that are widely present in prokaryotes and eukaryotes (Alcázar et al. 2010; Mattoo et al. 2010). PAs include diamine putrescine (Put), triamine spermidine (Spd), tetramines spermine (Spm), and thermospermine (T-Spm) (Takano et al. 2012; Sobieszczuk et al. 2017). It has been proposed that in plants, these PAs play important roles in various physiological processes, such as growth, development, and the responses to environmental stresses (Rakesh et al. 2014; Heba et al. 2017). Many studies have confirmed that the level of Spm will increase under drought stress in plants. Spm activates antioxidants and promotes reactive oxygen species (ROS) scavenging under drought stress, saving biomolecules and membranes from damage. (Hasan and Jahan 2021). Polyamine oxidase (PAO, EC 1.5.3.11) is an enzyme involved in the PA catabolic process (Tavladoraki et al. 2016; Wang et al. 2019). It is a flavin adenine dinucleotide-dependent enzyme (Wu et al. 2003). Studies on PAO genes in a range of plant species have revealed two pathways that catalyze the oxidation of PAs at the secondary amino group: the back conversion (BC) pathway and the terminal catabolism (TC) pathway (Kusano et al. 2008). In the TC pathway, PAOs convert Spm and Spd to N-(3-aminopropyl)-4-aminobutanal and 4-aminobutanal, respectively, together with 1,3-diaminopropane (Moschou et al. 2008; Wang and Liu 2016). In the BC pathway, PAOs can convert Spm (or T-Spm) to Spd and then Put; e.g., all five Arabidopsis PAO (AtPAO1–AtPAO5) genes can catalyze PAs in this way (Tavladoraki et al. 2006; Kamada et al. 2008; Moschou et al. 2008; Fincato et al. 2011). Moreover, both of these pathways can produce H2O2 (Fincato et al. 2012).
PAO proteins in plants are usually divided into four subfamilies (Ono et al. 2012; Sagor et al. 2015; Bagni et al. 2001; Yu et al. 2019). Members of subfamily I primarily participate in BC pathway and are located in the cytoplasm; members of subfamilies III and IV also play a role in the BC pathway, and the former is located in the cytoplasm and the latter is located in peroxisomes (Kim et al. 2014). The members of subfamily II are responsible for the final catabolism of PAs and are located in exosomes or vacuoles (Gholizadeh and Mirzagadheri 2020). PAO genes are involved in plant responses to abiotic stresses. In maize under high salinity stress, ROS are produced by PAO genes activity in the apoplast to sustain leaf growth (Rodríguez et al. 2009).
In soybean, NaCl stress can increase PAO genes expression (Fang et al. 2020). Exogenous Spd can reduce the harmful effects of NaCl stress and increase the biomass and GABA content. RNA-seq analysis related to NaCl stress also showed that most of the PAO genes were upregulated under NaCl stress (Liu et al. 2019).
The identification of members of the PAO gene family has been reported in maize (Zea mays L.), Arabidopsis (A. thaliana (L.) Heynh.), rice (Oryza sativa L.), barley (Hordeum vulgare L.), tomato (Solanum lycopersicum L.), tea (Camellia sinensis (L.) Kuntze), and other plants (Yoda et al. 2006; Fincato et al. 2011; Ono et al. 2012; Gholizadeh and Mirzagadheri 2020; Li et al. 2020). However, no systematic identification of the soybean PAO gene family has been carried out. In this study, we identified six PAO genes (GmPAO1–GmPAO6) from the soybean genome. We observed GmPAOs expression under various abiotic stresses.
Materials and methods
Plant materials and abiotic stress treatment
Glycine max, c.v “Williams 82” was used for all experiments and was grown in the public basic experiment center of Northeast Agricultural University, Harbin, Heilongjiang, China. The soybeans were planted in a mixture of vermiculite and soil (1:1, v/v), and the growth chamber was set to 24°C with a photoperiod of 16h/8h per day (light/dark). When the unifoliolate leaf was fully developed and the first trifoliolate leaf appeared (at the VC growth stage), the seedlings were transferred to water, a 0.9% (w/w) (∼150mmol/L) NaCl solution, 20% PEG6000, or 4°C water for abiotic stress treatment. Primary root, stem, and leaf samples were harvested at 0h, 2h, 4h, 8h, 12h, and 24h after treatment, immediately frozen in liquid nitrogen, and stored at −80°C. Three independent sets of samples were collected for each time point.
Identification of the PAO gene family in soybean and sequence alignment
The protein sequences of five AtPAOs in Arabidopsis were downloaded from the TAIR database ( https://www.arabidopsis.org/), using the Arabidopsis protein sequences to identify all candidate PAO genes in soybean. Systematic BLASTp ( https://phytozome-next.jgi.doe.gov/) searches were performed against the soybean reference genome ( https://www.soybase.org/) and Phytozome database ( https://phytozome-next.jgi.doe.gov/), based on the published sequences of PAO proteins of Arabidopsis and other plants as queries. The screening criteria were E<e−5 and protein length>200aa ( Supplementary TableS1 (cjps-2022-0019suppla.docx)). The candidate genes were confirmed online using Pfam ( http://pfam.xfam.org/) and the specific gene name and typical functional domains (Pfam: PF01593) of PAO proteins. Finally, the soybean PAO genes were identified according to the position on the chromosome; the setting situation names the PAO genes in soybeans in turn. The physical and chemical properties of the GmPAO protein were analyzed. Multiple sequence alignments of soybean GmPAO and corn ZmPAO1 proteins were conducted based on DNAMAN software using default parameter values. The resulting files were visualized through GeneDoc software (Fincato et al. 2012; Hao et al. 2018).
Gene and protein structure analyses
The coding sequence length, number of amino acids, and the chromosomal localization of soybean PAO proteins were obtained from the soybean sequence database. The physical and chemical properties of the putative GmPAO protein sequences, including the molecular formula, the number of amino acids, grand average of hydropathicity (GRAVY) index, instability index, isoelectric point, and molecular weight were obtained from the Expasy website ( http://web.expasy.org/cgi-bin/protparam/protparam). The schematic diagram of the exon–intron structure of the GmPAO gene was analyzed by GeneStructureDisplayServe2.0 (GSDS). ( http://gsds.cbi.pku.edu.cn/index.php). The ExPaSy Online website ( https://web.expasy.org/protparam/) was used to predict the secondary structure of the GmPAO protein. Phyre 2.0 tool ( http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) was used to predict the tertiary structure of the GmPAO protein. The subcellular localization of the GmPAO protein was predicted using WoLF PSORT ( http://www.genscript.com/psort/wolf_psort.html). The conserved domains of the GmPAO protein were searched using the Pfam and MEME database, and the resulting files were visualized in TBtools software.
Phylogenetic tree construction
The gene accession number reported in the literature was used to download the PAO proteins' sequence of multiple plants from NCBI ( https://www.ncbi.nlm.nih.gov/) and Phytozome ( https://phytozome-next.jgi.doe.gov/) ( TableS1 (cjps-2022-0019suppla.docx)). The amino acid sequences of these PAOs were determined using MEGA6 software, and a phylogenetic tree was constructed via the MEGA 6.0 software using the neighbor-joining algorithm with a bootstrap support value of 1000 replicates (Tamura et al. 2011). The online site iTOL ( http://itol.embl.de/) was used to display the data.
Tissue-specific expression of six GmPAOs
The Phytozome database ( https://phytozome-next.jgi.doe.gov/) was used to analyze GmPAO genes' expression. The GmPAO genes' expression data (RPKM values) of various tissues and organs of soybean were downloaded, and the heat map was generated from log10(RPKM) transformed values. HEML1.0 software was used for mapping.
RNA extraction and qrt-PCR analysis
Total RNA was extracted using Trizol reagent, and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (TOYOBO, Osaka, Japan). In this study, all primers were obtained from the qPrimerDB database (shown in Table1) (Lu et al. 2018). Real-time PCR (rt-PCR) was performed using an ABI7500 instrument and TOYOBO SYBR Green Real-time PCR Master Mix. Quantitative real-time PCR (qrt-PCR) was carried out by initial denaturation at 95°C for 3min, 40 cycles at 95°C for 15s, 60°C for 30s, 72°C for 30s, and final extension at 72°C for 5min. GmActin4 (Glyma.12G063400) served as an internal control (Zhang et al. 2022). The qRT-PCR results were evaluated using the cycle threshold (Ct) values and were calculated using the 2−ΔΔCt method (Czechowski et al. 2005).
Table 1.
Primers used for quantitative real-time PCR.
Statistical analysis
All of the experiments were performed with at least three biological replicates. To assess the impact of abiotic stress on the expression of GmPAO genes, the data were statistically analyzed with a one-way Analysis of variance (ANOVA). Differences were considered statistically significant at P<0.05, and differences were considered highly significant at P<0.01. All analyses were performed using GraphPad Prism 8.0.2.
Results
Characterization of polyamine oxidase gene families in soybean
Based on Arabidopsis AtPAO proteins, six PAO genes were identified in the soybean genome, which were named according to their chromosomal positions, i.e., GmPAO1–GmPAO6. Additionally, the physical and chemical properties of the GmPAO genes and GmPAO proteins were analyzed using the online website ExPaSy. The encoded proteins of the PAOs ranged from 385 to 734 amino acids, and the predicted molecular weight ranged from 43.3 to 80.6KDa. The isoelectric point was between 5.18 and 6.09, indicating weak acidity (Table2). Additional details of these genes are shown in Table3, including secondary protein structure and subcellular localization predictions. In addition, MEGA6 software was used to multiply the GmPAO and ZmPAO1 alignment of the sequences. The results showed that GmPAO members and ZmPAO1 had different sequence homologies, and the GmPAO members also showed differences. The analysis also predicted the presence of peroxisome-targeting sequences in GmPAO6 (Fig.1).
Fig. 1.
Alignment of the amino acid sequences of six soybean polyamine oxidase (GmPAOs) and one maize polyamine oxidase (ZmPAO1) proteins. The signal peptide of ZmPAO1 is underlined. Green boxes indicate the residues of ZmPAO1 protein that are involved in catalytic activity and conserved in the different GmPAO proteins. Conserved residues within GmPAOs are marked with yellow boxes. Non-conserved residues in GmPAOs that are different from those of the PAO catalytic site in ZmPAO1 are marked with orange boxes. In the C-termini of GmPAOs sequences, the putative type-I peroxisomal-targeting signals (PTS1) are indicated with a blue background.
Table 2.
Basic information of the six soybean polyamine oxidase (GmPAO) gene.
Table 3.
Protein secondary structure analysis and subcellular predication of six soybean polyamine oxidase (GmPAO) gene.
Phylogenetic analysis of GmPAO genes
To study the phylogenetic relationship of PAO proteins in different species, a phylogenetic tree was constructed using the neighbor-joining method based on the seven different species (Fig.2). The full-length amino acid sequences of PAO proteins from maize, Arabidopsis, Brachypodium distachyon (L.) P.Beauv., rice, barley, and soybean were obtained from the Phytozome. The results showed that all PAO proteins were grouped into four (I–IV) subfamilies in the phylogenetic tree. Among them, class I included two soybean PAO proteins (GmPAO1 and GmPAO4). No GmPAO protein was classified as class II and class III. Class IV included three soybean PAO proteins (GmPAO2, GmPAO5, and GmPAO6). It is worth noting that the phylogenetic analysis placed GmPAO3 protein between class III and IV.
Motif, domain, promoter, and structure analysis of GmPAO genes
Phylogenetic analysis of the soybean PAO genes alone (Fig.3A) and corresponding genetic structure analyses using the GSDS website were utilized to reveal the exon and intron structures of GmPAOs. The GmPAO2, GmPAO4, GmPAO5, and GmPAO6 genes had 10 exons and 9 introns, and GmPAO1 had 9 exons and 8 introns, but only two exons (Fig.3B).
Fig. 3.
Bioinformatic analysis of the soybean polyamine oxidase (GmPAO) gene family. (A) Phylogenetic tree was generated based on the protein sequences of GmPAOs. (B) Exon–intron structures of GmPAO genes, exons, and introns are indicated by red boxes and black lines, and the untranslated regions (UTRs) are indicated by blue boxes. (C) Conserved domains of GmPAOs proteins. (D) Conserved motifs of GmPAOs proteins; each motif is indicated by a colored box. (E) Prediction of cis-acting elements within GmPAO genes. (F) The number and types of cis-acting elements in the promoter regions of GmPAO genes.
Structural analysis of protein domain for GmPAO1–GmPAO6 demonstrated that all members contained typical amino-oxidase catalytic domain. GmPAO3 protein has an additional SWIRM domain and NAD binding domain. The GmPAO2, GmPAO5, and GmPAO6 proteins had all six motifs, while GmPAO1 and GmPAO4 had motifs 1, 3, and 6. However, the GmPAO3 protein lacked three motifs. Motif 1 and motif 3 were present in all GmPAO proteins (Fig.3D).
A large number of light-responsive elements, hormone-responsive elements, and abiotic forced-response elements are distributed in the promoter of GmPAO genes (Fig.3E), suggesting that these genes might play essential functions in light-responsive, hormone-responsive, growth and development, and abiotic and biotic stresses. Furthermore, we also count the number of cis-regulatory elements (Fig.3F). These differences indicate that GmPAO was relatively conservative in the evolutionary process, and different classes evolved similar gene structures and structural domain arrangements. GmPAO3 may have different expression and regulation modes.
These six genes were located on chr 2, chr 7, chr 9, chr 14, and chr 18. A physical map of the location of the GmPAO genes on the chromosomes is illustrated in Fig.4.
Expression profiles of soybean polyamine oxidase genes in diverse tissues
The Phytozome database was used to analyze the expression patterns of GmPAOs in different tissues. After transforming the RPKM values using log10, we constructed a heat map for further analysis. As demonstrated in Fig.5, six GmPAO genes were expressed in all tissues, and the expression of GmPAO genes varied among the different tissues. However, GmPAO3 had the most stable expression across the different tissues, while other members were most prominently expressed in flowers relative to other tissues. GmPAO6 was ubiquitously and highly expressed in all tissues relative to the other GmPAO genes, and the expression level of GmPAO1 in all tissues was relatively low except in the flowers.
The qrt-PCR analysis of the expression of GmPAO genes under abiotic stresses
To investigate the expression level of GmPAO genes under three abiotic stresses (drought, salt, and low temperature), qRT-PCR was used to measure the expression of the GmPAO genes in the roots, stems, and leaves (Figs.6–8). The qRT-PCR results showed that GmPAO1, GmPAO4, GmPAO5, and GmPAO6 changed significantly under NaCl stress. In roots, GmPAO4 was upregulated 7-fold under NaCl stress for 8h. In leaves, GmPAO4 was also upregulated 12-fold under NaCl stress for 8h.
Fig. 6.
Quantitative real-time PCR analysis of soybean polyamine oxidase (GmPAO) genes in response to salt stress. Three independent biological replicates were carried out. Asterisks above bars denote a statistically significant difference by Student's t test (*P<0.05 and **P<0.01).
Fig. 7.
Quantitative real-time PCR analysis of soybean polyamine oxidase (GmPAO) genes in response to low temperature stress. Three independent biological replicates were carried out. Asterisks above bars denote a statistically significant difference by Student's t test (*P<0.05 and **P<0.01).
Fig. 8.
Quantitative real-time PCR analysis of soybean polyamine oxidase (GmPAO) genes in response to low drought stress. Three independent biological replicates were carried out. Asterisks above bars denote a statistically significant difference by Student's t test (*P<0.05 and **P<0.01).
Under low-temperature stress (4°C), almost all GmPAOs, including GmPAO1, GmPAO3, GmPAO4, GmPAO5, and GmPAO6, were upregulated in roots treated for 8 or 12h, and GmPAO2 was upregulated most obviously in leaves. However, GmPAO3 was upregulated 8-fold in the stems.
We used 20% PEG6000 to simulate drought stress. Drought stress had a great impact on soybean roots. GmPAO1 was upregulated 9-fold in roots under drought stress for 4h, while GmPAO4 was upregulated 11-fold in roots for 8h. GmPAO5 was upregulated 15-fold in roots for 8h. GmPAO1 and GmPAO6 were mainly upregulated in stems, and GmPAO6 was upregulated up to 5-fold at 8h under drought stress. In leaves, all the GmPAOs changed significantly, and GmPAO6 was upregulated 34-fold at 12h under drought stress. The response of GmPAO genes to abiotic stress in the stems was not as significant in roots and leaves. GmPAO3 was upregulated in the stems under low-temperature stress, which was the most obvious at 12h, i.e., an 8-fold change.
Discussion
Abiotic stresses, including drought, salinity, and cold, can negatively affect soybean growth and yield (Heba et al. 2017; Zhu et al. 2017; Jabeen et al. 2021). Therefore, the identification of tolerant genes to abiotic stress is important for the breeding of abiotic stress-tolerant soybean cultivars. PAO genes play a vital role in plant protection against abiotic stresses (Hasan and Jahan 2021). In recent years, the identification of members of the PAO family has been reported in multiple species ( Yoda et al. 2006; Fincato et al. 2011; Ono et al. 2012; Gholizadeh and Mirzagadheri 2020; Li et al. 2020). However, systematic analysis of the identification, characteristics, and functions of the soybean PAO family has not been reported. In this study, six GmPAO genes were identified in soybean, and then we used a phylogenetic tree to classify them. Contrary to the previous studies, the six soybean members identified in this study were only included in class I and class IV, while GmPAO3 protein exists between class III and IV, and phylogeny showed that it was more closely related to class III, but it falls outside of class III and IV.
GmPAO1 and GmPAO4 belong to class I, based on subcellular localization prediction, while the GmPAO1 protein is located in the vacuole, similar to CsPAO6 protein, suggesting that it may play a role in the regulation of osmotic pressure (Yoda et al. 2006), whereas the GmPAO4 protein is located in the endoplasmic reticulum. These results are consistent with the results of most species for the class PAO gene families that catalyze PAs using the BC pathway. Classes II and III PAO proteins have not been identified in soybeans. Three soybean PAO proteins, GmPAO2, GmPAO5, and GmPAO6, belong to class IV. Moreover, based on subcellular localization prediction, GmPAO2 and GmPAO6 proteins are located in the peroxisome, and GmPAO6 has peroxisomal targeting sequences. GmPAO5 is located in the endoplasmic reticulum, and class IV members are involved in the BC pathway (Fincato et al. 2012). Of course, to verify the specific catalytic form of each soybean PAO gene family member, biochemical and enzymatic characterizations are still needed.
Tissue expression profile analysis revealed that flower organs were relatively richly expressed, and the expression of class IV was greater than that of class I in soybean, which was similar to that of tomato (Yoda et al. 2006). Except for GmPAO3, the other members have similar gene structures. Genes with GmPAO3-like structures have also been identified in rice and Arabidopsis (Moschou et al. 2012; Ono et al. 2012). The main function of this domain is to catalyze the demethylation of the H3K4 histone lysine, which belongs to the clade of histone lysine-specific demethylases (Moschou et al. 2012).However, due to the apparent lack of class III GmPAO proteins and the unique structure of the GmPAO3 gene, we hypothesize that the GmPAO3 gene may act as a coded class III GmPAO protein under undetermined circumstances. Of course, the function of the GmPAO3 gene needs further study.
In Arabidopsis, the AtPAO1 protein belongs to class I; AtPAO1 gene is mainly expressed during the elongation zone of roots (Fincato et al. 2012). The PAO1 single mutant and other single mutants were sensitive to salt and drought stress, but the PAO1 PAO5 double mutant was tolerant to salt and drought stresses, although the exact cause of this phenomenon is unclear (Sagor et al. 2016). GmPAO1 and GmPAO4 proteins belong to class I. Similar to AtPAO1 protein, they are the most sensitive to salt and drought stresses in roots and leaves (Figs.6 and 8). In tomato, SiPAO2, SiPAO3, SiPAO4, and SiPAO5 belong to Class IV; all the Class IV members were responsive to cold stress (Hao et al. 2018). GmPAO2, GmPAO5, and GmPAO6 proteins belong to Class IV, and similar to SiPAO2-SiPAO5 proteins, GmPAO2, GmPAO5, and GmPAO6 genes also were responsive to cold stress in the roots and leaves.
In conclusion, GmPAO4 and GmPAO1 genes had the greatest response in roots and leaves under salt stress. Under low-temperature cold stress, the GmPAO2 gene was responsive in leaves, and GmPAO3 was responsive in stems. The most obvious changes in the GmPAO genes occurred under drought stress. Additionally, except for GmPAO3, other GmPAO genes responded to drought stress in roots, and the response of GmPAO6 was strongest. To understand the exact mechanism of PAOs, further genetic and biochemical experiments are needed. Our research results provide a reference for further research on the function of GmPAO proteins.
Funding
This study was financially supported by the Chinese National Natural Science Foundation (31871650, 31971967, 32001570); the Heilongjiang Provincial Project (GX17B002, C2018016, GJ2018GJ0098, 2019ZX16B01); the National Key R&D Project (2017YFD0101306, 2017YFD0101302); the National Project (2014BAD22B01, 2016ZX08004001-007); the Youth Leading Talent Project of the Ministry of Science and Technology in China (2015RA228); the National Ten-thousand Talents Program; the Postdoctoral Fund in Heilongjiang Province (LBH-Z15017, LBH-Q17015); the national project (CARS-04-PS06); and the “Academic Backbone” Project of Northeast Agricultural University (17XG22). The funding bodies had no role in the study design, data collection, analysis, decision to publish, or preparation of the manuscript.
Author contributions
XZ and YPH conceived and designed the experiments; KWY and CN performed the experiments; KWY and XCZ analyzed the data; KWY, KZZ, YHZ, and NX contributed materials/analysis tools; and KWY prepared the manuscript.
Supplementary material
Supplementary data are available with the article at https://doi.org/10.1139/cjps-2022-0019.
