ABSTRACT
Plant glutathione peroxidases (GPXs) consist of non-heme thiol peroxidases that are vital in maintaining hydrogen peroxide homeostasis and regulating plant environmental stress responses. A comparative genomic analysis of the GPX gene family in Ipomoea trifida and I. triloba using their respective genomes was performed. Six GPX genes were identified in each species, which were unevenly located in 4 of the 15 chromosomes of the closest ancestors of the sweet potato genomes (I. trifida and I. triloba). The presence of gene duplications and positive selection were highlighted, suggesting the evolutionary significance of the GPX genes in these species. Based on the phylogenetic analysis, the GPX genes of I. trifida, I. triloba, Arabidopsis thaliana and Oryza sativa can be classified into four groups (I, II, III and IV). The in silico expression analysis in different tissues and development stages revealed tissue-specific expression patterns, hinting at specialized roles for the GPX genes in different plant organs. Nonetheless, the ItfGPX5 and ItbGPX5 genes were highly expressed in most the studied tissues.
KEYWORDS:
Comparative genomics; Ipomoea species; gene expression; phylogenetic classification
RESUMO
As glutationas peroxidases vegetais (GPXs) consistem em peroxidases não-heme tiol vitais na manutenção da homeostase do peróxido de hidrogênio e na regulação das respostas ao estresse ambiental das plantas. Foi efetuada uma análise genômica comparativa da família de genes GPX em Ipomoea trifida e I. triloba, utilizando-se seus respectivos genomas. Foram identificados 6 genes GPX em cada espécie, que estavam localizados de forma desigual em 4 dos 15 cromossomos dos ancestrais mais próximos dos genomas da batata-doce (I. trifida e I. triloba). Apresença de duplicações gênicas e a seleção positiva foram destacadas, sugerindo o significado evolutivo dos genes GPX nessas espécies. Com base na análise filogenética, os genes GPX de I. trifida, I. triloba, Arabidopsis thaliana e Oryza sativa podem ser classificados em quatro grupos (I, II, III e IV). A análise da expressão in silico em diferentes tecidos e estágios de desenvolvimento mostraram padrões de expressão específicos de tecidos, sugerindo papéis especializados para os genes GPX em diferentes órgãos vegetais. No entanto, os genes ItfGPX5 e ItbGPX5 foram altamente expressos na maioria dos tecidos estudados.
PALAVRAS-CHAVE:
Genômica comparativa; espécies de Ipomoea; expressão gênica; classificação filogenética
Plants often experience various types of stress (Santos et al. 2022SANTOS, T. B.; RIBAS, A. F.; SOUZA, S. G. H. de; BUDZINSKI, I. G. F.; DOMINGUES, D. S. Physiological responses to drought, salinity, and heat stress in plants: a review. Stresses, v. 2, n. 1, p. 113-135, 2022.). These include both biotic and abiotic stresses, being the latter one of the main challenges that crops face (Bhat et al. 2020BHAT, J. A.; DESHMUKH, R.; ZHAO, T.; PATIL, G.; DEOKAR, A.; SHINDE, S.; CHAUDHARY, J. Harnessing high-throughput phenotyping and genotyping for enhanced drought tolerance in crop plants. Journal of Biotechnology, v. 324, n. 1, p. 248-260, 2020., Santos et al. 2022SANTOS, T. B.; RIBAS, A. F.; SOUZA, S. G. H. de; BUDZINSKI, I. G. F.; DOMINGUES, D. S. Physiological responses to drought, salinity, and heat stress in plants: a review. Stresses, v. 2, n. 1, p. 113-135, 2022.). Different types of abiotic stress can trigger the production of a large number of reactive oxygen species, which can damage essential molecules, membranes and cell organelles, potentially leading to plant cell death (Waszczak et al. 2018WASZCZAK, C.; CARMODY, M.; KANGASJÄRVI, J. Reactive oxygen species in plant signaling. Annual Review of Plant Biology, v. 69, n. 1, p. 209-236, 2018., Zafar et al. 2020ZAFAR, S. A.; HAMEED, A.; ASHRAF, M.; KHAN, A. S.; QAMAR, Z. U.; LI, X.; SIDDIQUE, K. H. M. Agronomic, physiological and molecular characterisation of rice mutants revealed the key role of reactive oxygen species and catalase in high-temperature stress tolerance. Functional Plant Biology, v. 47, n. 5, p. 440-453, 2020.). Enzymes of the antioxidant system include superoxide dismutases (EC 1.15.1.1), catalases (EC 1.11.1.6), glutathione peroxidases (GPXs) (EC 1.11.1.11) and peroxiredoxins (EC 1.11.1.15) (Meitha et al. 2020MEITHA, K.; PRAMESTI, Y.; SUHANDONO, S. Reactive oxygen species and antioxidants in postharvest vegetables and fruits. International Journal of Food Science, v. 2020, e8817778, 2020.).
Plant GPXs are the main enzymes of the antioxidant defense system that sustain hydrogen peroxide homeostasis and regulate the response of plants to abiotic stress conditions. These enzymes are widely distributed among living organisms, including plants, animals and microorganisms. They mainly neutralize organic peroxides and other reactive oxygen species, maintaining the cellular redox balance (Passaia & Margis-Pinheiro 2015PASSAIA, G.; MARGIS-PINHEIRO, M. Glutathione peroxidases as redox sensor proteins in plant cells. Plant Science, v. 234, n. 1, p. 22-26, 2015.). In general, GPXs in plants contain cysteine at their functional sites, whereas GPXs in mammals contain selenocysteine residues as cysteine substitutes (Bela et al. 2015BELA, K.; HORVÁTH, E.; GALLÉ, Á.; SZABADOS, L.; TARI, I.; CSISZÁR, J. Plant glutathione peroxidases: emerging role of the antioxidant enzymes in plant development and stress responses. Journal of Plant Physiology, v. 176, n. 1, p. 192-201, 2015., Islam et al. 2015ISLAM, T.; MANNA, M.; KAUL, T.; PANDEY, S.; REDDY, C. S.; REDDY, M. K. Genome-wide dissection of Arabidopsis and rice for the identification and expression analysis of glutathione peroxidases reveals their stress-specific and overlapping response patterns. Plant Molecular Biology Reporter, v. 33, n. 5, p. 1413-1427, 2015.). Numerous studies have recently shown that increasing or regulating the GPX enzyme activity and expression, as well as regulating GPX genes, can help plants to cope with various environmental stresses (Zhou et al. 2018ZHOU, Y.; LI, J.; WANG, J.; YANG, W.; YANG, Y. Identification and characterization of the glutathione peroxidase (GPX) gene family in watermelon and its expression under various abiotic stresses. Agronomy, v. 8, n. 10, e206, 2018., Wang et al. 2021WANG, X.; LIU, X.; AN, Y.; ZHANG, H.; MENG, D.; JIN, Y.; HUO, H.; YU, L.; ZHANG, J. Identification of glutathione peroxidase gene family in Ricinus communis and functional characterization of RcGPX4 in cold tolerance. Frontiers in Plant Science, v. 12, e707127, 2021., Wang et al. 2022WANG, Y.; CAO, S.; SUI, X.; WANG, J.; GENG, Y.; GAO, F.; ZHOU, Y. Genome-wide characterization, evolution, and expression analysis of the ascorbate peroxidase and glutathione peroxidase gene families in response to cold and osmotic stress in Ammopiptanthus nanus. Journal of Plant Growth Regulation, v. 42, n. 8, p. 502-522, 2022.). GPXs can be located in different subcellular compartments in plants, including the nucleus, mitochondria, chloroplasts, plasma membrane, cytosol and apoplast (Herbette et al. 2007HERBETTE, S.; ROECKEL-DREVET, P.; DREVET, J. R. Seleno-independent glutathione peroxidases: more than simple antioxidant scavengers. FEBS Journal, v. 274, n. 9, p. 2163-2180, 2007., reviewed by Margis et al. 2008MARGIS, R.; DUNAND, C.; TEIXEIRA, F. K.; MARGIS-PINHEIRO, M. Glutathione peroxidase family: an evolutionary overview. FEBS Journal, v. 275, n. 15, p. 3959-3970, 2008.). Members of the GPX family have been identified and characterized in many plants, including Thellungiella salsuginea (Gao et al. 2014GAO, F.; CHEN, J.; MA, T.; LI, H.; WANG, N.; LI, Z.; ZHANG, Z.; ZHOU, Y. The glutathione peroxidase gene family in Thellungiella salsuginea: genome-wide identification, classification, and gene and protein expression analysis under stress conditions. International Journal of Molecular Sciences, v. 15, n. 2, p. 3319-3335, 2014.), Gossypium hirsutum (Chen et al. 2017CHEN, M.; LI, K.; LI, H.; SONG, C. P.; MIAO, Y. The glutathione peroxidase gene family in Gossypium hirsutum: genome-wide identification, classification, gene expression and functional analysis. Scientific Reports, v. 7, n. 1, e44743, 2017.), Phoenix dactylifera L. (Jana & Yaish 2020JANA, G. A.; YAISH, M. W. Genome-wide identification and functional characterization of glutathione peroxidase genes in date palm (Phoenix dactylifera L.) under stress conditions. Plant Gene, v. 23, e100237, 2020.), Brassica napus L. (Li et al. 2021LI, W.; HUAI, X.; LI, P.; RAZA, A.; MUBARIK, M. S.; HABIB, M.; KHAN, R. S. A. Genome-wide characterization of glutathione peroxidase (GPX) gene family in rapeseed (Brassica napus L.) revealed their role in multiple abiotic stress response and hormone signaling. Antioxidants, v. 10, n. 9, e1481, 2021.), Cyprinus carpio (Xue et al. 2022XUE, Y.; CHEN, L.; LI, B.; XIAO, J.; WANG, H.; DONG, C.; XU, P. Genome-wide mining of GPX gene family provides new insights into cadmium stress responses in common carp (Cyprinus carpio). Gene, v. 821, e146291, 2022.), Ammopiptanthus nanus (Wang et al. 2022WANG, Y.; CAO, S.; SUI, X.; WANG, J.; GENG, Y.; GAO, F.; ZHOU, Y. Genome-wide characterization, evolution, and expression analysis of the ascorbate peroxidase and glutathione peroxidase gene families in response to cold and osmotic stress in Ammopiptanthus nanus. Journal of Plant Growth Regulation, v. 42, n. 8, p. 502-522, 2022.), Capsicum annuum L. (Wang et al. 2023WANG, W.; CHENG, Y.; RUAN, M.; YE, Q.; YAO, Z.; WANG, R.; WAN, H. Comprehensive identification of glutathione peroxidase (GPX) gene family in response to abiotic stress in pepper (Capsicum annuum L.). Gene, v. 881, e147625, 2023.) and Cicer arietinum L. (Parveen et al. 2024PARVEEN, K.; SADDIQUE, M. A. B.; ALI, Z.; REHMAN, S. U.; KHAN, Z.; WAQAS, M.; MUNEER, M. A. Genome-wide analysis of glutathione peroxidase (GPX) gene family in chickpea (Cicer arietinum L.) under salinity stress. Gene, v. 898, e148088, 2024.).
Eight GPX genes in Arabidopsis thaliana have been described, and these genes are regulated by abiotic factors (Islam et al. 2015ISLAM, T.; MANNA, M.; KAUL, T.; PANDEY, S.; REDDY, C. S.; REDDY, M. K. Genome-wide dissection of Arabidopsis and rice for the identification and expression analysis of glutathione peroxidases reveals their stress-specific and overlapping response patterns. Plant Molecular Biology Reporter, v. 33, n. 5, p. 1413-1427, 2015., Bela et al. 2018BELA, K.; RIYAZUDDIN, R.; HORVÁTH, E.; HURTON, Á.; GALLÉ, Á.; TAKÁCS, Z.; CSISZÁR, J. Comprehensive analysis of antioxidant mechanisms in Arabidopsis glutathione peroxidase-like mutants under salt- and osmotic stress reveals organ-specific significance of the AtGPXL’s activities. Environmental and Experimental Botany, v. 150, n. 1, p. 127-140, 2018.). The same authors concluded that GPX genes may play an essential role in plants under stress. Five CaGPX genes were identified in Capsicum annuum L. using a bioinformatics method, further highlighting the function of these genes in response to abiotic stress (Wang et al. 2023WANG, W.; CHENG, Y.; RUAN, M.; YE, Q.; YAO, Z.; WANG, R.; WAN, H. Comprehensive identification of glutathione peroxidase (GPX) gene family in response to abiotic stress in pepper (Capsicum annuum L.). Gene, v. 881, e147625, 2023.).
Sweet potato [Ipomoea batatas (L.) Lam., 2n = 6x = 90] is an important plant belonging to the Convolvulaceae family, and this crop is grown in more than 100 countries worldwide (Liu 2017LIU, Q. Improvement for agronomically important traits by gene engineering in sweet potato. Breeding Science, v. 67, n. 1, p. 15-26, 2017.). Sweet potato is a vital global food rich in antioxidants and fiber (Alam 2021ALAM, M. K. A comprehensive review of sweet potato (Ipomoea batatas [L.] Lam): revisiting the associated health benefits. Trends in Food Science and Technology, v. 115, n. 3, p. 512-529, 2021.). However, pests, diseases and environmental stresses hinder its production (Wang et al. 2019WANG, W.; QIU, X.; YANG, Y.; KIM, H. S.; JIA, X.; YU, H.; KWAK, S. S. Sweetpotato bZIP transcription factor IbABF4 confers tolerance to multiple abiotic stresses. Frontiers in Plant Science, v. 10, e630, 2019.). An understanding of its adaptation mechanisms is essential for developing stress-tolerant varieties.
Genomes of the hexaploid sweet potato and two diploid species, namely I. trifida NCNSP0306 (2n = 2x = 30) and I. trilobaNCNSP0323 (2n = 2x = 30) (Wu et al. 2018WU, S.; LAU, K. H.; CAO, Q.; HAMILTON, J. P.; SUN, H.; ZHOU, C.; ESERMAN, L.; GEMENET, D. C.; OLUKOLU, B. A.; WANG, H. Y.; CRISOVAN, E.; GODDEN, G. T.; JIAO, J.; WANG, X.; MERCY, K.; MANRIQUE-CARPINTERO, N.; VAILLANCOURT, B.; WIEGERT-RININGER, K.; YANG, X. S.; BAO, K.; SCHAFF, J.; KREUZE, J.; GRUNEBERG, W.; KHAN, A.; GHISLAIN, M.; MA, D. F.; JIANG, J. M.; MWANGA, R.; LEEBENS-MACK, J.; COIN, L.; YENCHO, C.; ROBIN, B. R.; FEI, Z. Genome sequences of two diploid wild relatives of cultivated sweet potato reveal targets for genetic improvement. Nature Communications, v. 9, n. 1, e4580, 2018.), were recently made available, facilitating the identification and characterization of specific genes. The GPX gene family has not been documented in the sweet potato ancestors I. triloba and I. trifida. Therefore, the present study was performed to obtain more information about the GPX genes in I. triloba and I. trifida. In silico analyses were performed through physicochemical characterization, genetic structure analysis, conserved motif identification, phylogenetic relationship assessment and functional transcriptional profiling using the data obtained from each genome.
The study was conducted at the Universidade do Oeste Paulista, in Presidente Prudente, São Paulo state, Brazil, in 2023.
The protein sequence of A. thaliana (AT2G25080.1) was used as a query to identify putative sequences. All predicted amino acid, genomic and coding DNA sequences were recovered from GPX using the sweet potato database. Then, for confirmation, all sequences were selected and submitted to the database of the National Center for Biotechnology Information (Altschul et al. 1997ALTSCHUL, S. F.; MADDEN, T. L.; SCHAFFER, A. A.; ZHANG, J.; ZHANG, Z.; MILLER, W.; LIPMAN, D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, v. 25, n. 17, p. 3389-3402, 1997.), using the Basic Local Alignment Search Tool for proteins (BLASTP) tool. The identified GPX genes were named with the prefix of each species (Itb for I. triloba and Itf for I. trifida), followed by their chromosomal order. A physicochemical characterization using the Protein Parameters (PROTPARAM) tool of the Expert Protein Analysis System (ExPASy) was also performed, determining the molecular weight (kDa), isoelectric points and gene lengths of the ItlbGPX and ItfGPX proteins. The grand average of hydropathy (GRAVY) value of the protein sequences was determined using the GRAVY calculator. Further, the subcellular locations of the identified proteins were predicted using the Plant-mPLoc server (Chou & Shen 2010CHOU, K. C.; SHEN, H. B. Plant-mPLoc: a top-down strategy to augment the power for predicting plant protein subcellular localization. PLoS One, v. 5, n. 6, e11335, 2010.). All identified GPX genes were named according to their locations and orders on the chromosomes.
All gene structures and positions, including introns and exons, were analyzed using the Gene Structure Display Server (Hu et al. 2015HU, B.; JIN, J.; GUO, A. Y.; ZHANG, H.; LUO, J.; GAO, G. GSDS 2.0: an upgraded gene feature visualization server. Bioinformatics, v. 31, n. 8, p. 1296-1297, 2015.). This tool uses the genomic and coding sequence of each gene to generate the corresponding figure. Sequence motifs were identified and analyzed using the Multiple Em for Motif Elicitation (MEME) web server (Bailey et al. 2009BAILEY, T. L.; BODEN, M.; BUSKE, F. A.; FRITH, M.; GRANT, C. E.; CLEMENTI, L.; REN, J.; LI, W. W.; NOBLE, W. S. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research, v. 37, suppl. 2, p. W202-W208, 2009.). The number of motifs was set to 10, while all other parameters were set to default values.
The physical locations of the GPX genes were obtained from the Sweet Potato Database, and the chromosome location map was constructed using the Mapchart software (Voorrips 2002VOORRIPS, R. MapChart: software for the graphical presentation of linkage maps and QTLs. Journal of Heredity, v. 93, n. 1, p. 77-78, 2002.). The synonymous (Ks) and non-synonymous (Ka) substitution rates of the paralogous genes were investigated using the Ka_Ks Calculator 2.0 (Zhang et al. 2006ZHANG, Z.; LI, J.; ZHAO, X. Q.; WANG, J.; WONG, G. K.; YU, J. KaKs_Calculator: calculating Ka and Ks through model selection and model averaging. Genomics, Proteomics & Bioinfomatics, v. 4, n. 4, p. 259-263, 2006.). Amino acid sequences of all GPX proteins were aligned using the ClustalW software (Thompson et al. 1994THOMPSON, J. D.; HIGGINS, D. G.; GIBSON, T. J. CLUSTAL, W. Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, v. 22, n. 22, p. 4673-4680, 1994.) with the default parameters. A phylogenetic tree was then constructed using the Molecular Evolutionary Genetics Analysis 7.0 (MEGA7.0) software (Kumar et al. 2016KUMAR, S.; STECHER, G.; TAMURA, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, v. 33, n. 7, p. 1870-1874, 2016.). Additionally, protein sequences from model species such as A. thaliana and Oryza sativa were included for comparative analysis. The phylogenetic tree was constructed using the neighbor-joining method, with bootstrapping of 1,000 replicates and a cut-off value of 50 % (Kumar et al. 2016KUMAR, S.; STECHER, G.; TAMURA, K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, v. 33, n. 7, p. 1870-1874, 2016.).
In addition, bioinformatics tools were used to analyze the expression patterns of these genes in the following tissues: flower, flower bud, leaf, root1, root2 and stem in the I. triloba genome and flower_callus, stem_callus, flower, flower bud, leaf, root1, root2 and stem in the I. trifida genome. For both species, information was obtained from the RNA sequencing data available in the database of these species. All GPX gene expression levels were quantified using fragments per kilobase of exon per million fragments mapped (FPKM) values. The GPX gene heatmaps were drafted using the CIMMiner algorithm.
Six GPX genes were identified in each species (I. triloba and I. trifida) (Table 1). In I. triloba, the ItbGPX gene length ranged from 510 bp (ItbGPX5) to 741 bp (ItbGPX6), and the number of amino acids ranged from 167 (ItbGPX4) to 246 (ItbGPX6). The molecular weight in this species ranged from 18.70 kDa (ItbGPX5) to 27.06 kDa (ItbGPX6). In I. trifida, the ItfGPXgene length ranged from 507 bp (ItfGPX4) to 741 bp (ItfGPX6), and the number of amino acids from 168 (ItfGPX4) to 246 (ItfGPX6). The molecular weight in this species ranged from 18.67 kDa (ItfGPX5) to 26.98 kDa (ItfGPX6). The theoretical isoelectric point in I. triloba ranged from 5.70 (ItbGPX4) to 9.55 (ItbGPX6), and that in I. trifida from 6.12 (ItfGPX4) to 9.48 (ItfGPX6) (Table 1). The GRAVY value of all proteins was above −0.099, indicating that GPX is hydrophilic with high solubility in water (Table 1). The basic physicochemical properties of GPX and the results of the other analyses are shown in Table 1. The subcellular location of most genes was predicted to be in mitochondria and chloroplasts. The same was observed in C. annuum L. (Wang et al. 2023WANG, W.; CHENG, Y.; RUAN, M.; YE, Q.; YAO, Z.; WANG, R.; WAN, H. Comprehensive identification of glutathione peroxidase (GPX) gene family in response to abiotic stress in pepper (Capsicum annuum L.). Gene, v. 881, e147625, 2023.). GPX members may be involved in the response mechanism to abiotic stresses in these studied species.
By investigating the exon/intron structures of the GPX genes, it is possible to understand the organization of these genes in terms of coding and non-coding sequences in I. triloba and I. trifida (Figures 1A and 2A). This information regarding the genomic structure and expression of these genes is valuable. The sequence motifs were analyzed in two ancestral sweet potatoes. The preserved motif analysis results showed that the motifs 1, 2 and 3 were observed among all members studied in I. triloba and I. trifida (Figures 1B and 2B). All sequences were submitted to the database (Paysan-Lafosse et al. 2023PAYSAN-LAFOSSE, T.; BLUM, M.; CHUGURANSKY, S.; GREGO, T.; PINTO, B. L.; SALAZAR, G. A.; BATEMAN, A. InterPro in 2022. Nucleic Acids Research, v. 51, n. D1, p. D418-D427, 2023.), and it was observed that they presented the specific domain for GPX (IPR000889).
Exon/intron structure analysis of GPX and conserved motifs in Ipomoea triloba. A) the green boxes, gray lines and black boxes represent exons, introns and untranslated regions, respectively; B) conserved motifs in predicted GPX proteins identified by the MEME tool.
Exon/intron structure analysis of GPX and conserved motifs in Ipomoea trifida. A) the green boxes, gray lines and black boxes represent exons, introns and untranslated regions, respectively; B) conserved motifs in predicted GPX proteins identified by the MEME tool.
The chromosomal distribution of GPX genes showed the same pattern in both I. trifida and I. triloba. Chromosomal location analysis showed that the GPX genes were randomly distributed on four chromosomes in I. trifida and I. triloba (Figures 3A and 3B). In both I. trifida and I. triloba, two GPX genes were located on the chromosome 4, two on the chromosome 8, one on the chromosome 7 and one on the chromosome 9. Based on this chromosomal distribution, the expansion of the GPX genes was examined in the I. triloba and I. trifida genomes, being found four duplication pairs in I. trifida with a high rate of sequence similarity (Figure 3; Table 2). No tandem duplication was found between the GPX genes in the two studied species; only segmental duplications were found (four and three for I. trifida and I. triloba, respectively). The findings of the present study suggest that GPX genes may have arisen through gene duplication, with segmental duplication emerging as a predominant driving force for expansion within the investigated species. Similar studies have focused on different gene families in sweet potato (Liu et al. 2023LIU, E.; LI, Z.; LUO, Z.; XU, L.; JIN, P.; JI, S.; ZHOU, G.; WANG, Z.; ZHOU, Z.; ZHANG, H. Genome-wide identification of DUF668 gene family and expression analysis under drought and salt stresses in sweet potato [Ipomoea batatas (L.) Lam]. Genes, v. 14, n. 1, e217, 2023., Zhang et al. 2023ZHANG, J. Z.; HE, P. W.; XU, X. M.; LÜ, Z. F.; CUI, P.; GEORGE, M. S.; LU, G. Q. Genome-wide identification and expression analysis of the xyloglucan endotransglucosylase/hydrolase gene family in sweet potato [Ipomoea batatas (L.) Lam]. International Journal of Molecular Sciences, v. 24, n. 1, e775, 2023.). The duplication process is implicated in augmenting functional divergence, a pivotal factor facilitating adaptation to dynamic climatic changes (Conant & Wolfe 2008CONANT, G. C.; WOLFE, K. H. Probabilistic cross-species inference of orthologous genomic regions created by whole-genome duplication in yeast. Genetics, v. 179, n. 3, p. 1681-1692, 2008.).
Chromosomal distribution and duplication events of GPX genes in sweet potato for Ipomoea triloba (A) and I. trifida (B). The black lines represent duplicated genes (see Table 2). The number of chromosomes and their size in Mb are indicated at the top of each bar. The vertical scale represents the size of the chromosome.
Duplication data of paralogous gene pairs among Ipomoea trifida and I. triloba GPX genes. Ka is the non-synonymous substitution number per non-synonymous site, Ks the number of the synonymous substitution site and Ka/Ks the ratio of non-synonymous to synonymous substitutions.
The direction and magnitude of pressure selection can be inferred based on the Ka/Ks ratio, where Ka/Ks > 1 indicates a positive selection, Kα/Ks = 1 a neutral evolution and Ka/Ks < 1 a purifying selection (Ali et al. 2017ALI, H.; LIU, Y.; AZAM, S. M.; PRIYADARSHANI, S. V. G. N.; LI, W.; HUANG, X.; QIN, Y. Genomic survey, characterization, and expression profile analysis of the SBP genes in pineapple (Ananas comosus L.). International Journal of Genomics, v. 2017, e1032846, 2017.). To detect the selection pressure acting on GPX genes, the Kα, Ks and Ka/Ks values were analyzed in the two Ipomea species (Table 2). In I. triloba, only one pair of one gene (ItlbGPXl/ItlbGPX3) had a Ka/Ks ratio of < 1, indicating that these genes evolved through purifying selection. The Ka/Ks ratios of all four GPX gene pairs in I. tripod and two gene pairs in I. triloba were > 1. Thus, a positive selection (Darwinian selection) may have resulted in the accumulation of progressive mutations and spread them throughout the population (Si et al. 2022SI, Z.; WANG, L.; QIAO, Y.; ROYCHOWDHURY, R.; JI, Z.; ZHANG, K.; HAN, J. Genome-wide comparative analysis of the nucleotide-binding site-encoding genes in four Ipomoea species. Frontiers in Plant Science, v. 13, e960723, 2022.).
To elucidate the evolutionary relationship of GPX proteins in I. triloba and I. trifida, a phylogenetic tree was constructed using four species (I. triloba, I. trifida, A. thaliana and O. sativa) (Figure 4). All GPX proteins from I. triloba and I. trifida were unequally distributed across the branches of the phylogenetic tree (Figure 4). According to these results, the clades were named Group I, II, III and IV, respectively (Figure 4). The findings indicated that the number and type of GPX protein in each sweet potato group may differ from those in its two diploid relatives. The data regarding the number of groups corroborate those reported by Wang et al. (2023)WANG, W.; CHENG, Y.; RUAN, M.; YE, Q.; YAO, Z.; WANG, R.; WAN, H. Comprehensive identification of glutathione peroxidase (GPX) gene family in response to abiotic stress in pepper (Capsicum annuum L.). Gene, v. 881, e147625, 2023..
Phylogenetic tree of GPX protein sequences from Ipomoea triloba, I. trifida, Arabidopsis thaliana and Oryza sativa. A neighbor-joining phylogenetic tree was constructed using the MEGA7.0 software with 1,000 bootstrap replicates. Groups I, II, III and IV are indicated by square brackets.
The presence of a homolog of each GPX protein from I. triloba and I. trifida in the A. thaliana groups suggests a possible evolutionary conservation of these genes between the two studied species. The similarity between the GPX genes of these sweet potato ancestors and A. thaliana, for example, may indicate similar functions of these proteins, in terms of the antioxidant response and protection against oxidative damage within the cell. Furthermore, the recent divergence between the genomes of I. triloba and I. trifida may be responsible for the high sequence identity between the GPX genes of these two species. According to Wu et al. (2018)WU, S.; LAU, K. H.; CAO, Q.; HAMILTON, J. P.; SUN, H.; ZHOU, C.; ESERMAN, L.; GEMENET, D. C.; OLUKOLU, B. A.; WANG, H. Y.; CRISOVAN, E.; GODDEN, G. T.; JIAO, J.; WANG, X.; MERCY, K.; MANRIQUE-CARPINTERO, N.; VAILLANCOURT, B.; WIEGERT-RININGER, K.; YANG, X. S.; BAO, K.; SCHAFF, J.; KREUZE, J.; GRUNEBERG, W.; KHAN, A.; GHISLAIN, M.; MA, D. F.; JIANG, J. M.; MWANGA, R.; LEEBENS-MACK, J.; COIN, L.; YENCHO, C.; ROBIN, B. R.; FEI, Z. Genome sequences of two diploid wild relatives of cultivated sweet potato reveal targets for genetic improvement. Nature Communications, v. 9, n. 1, e4580, 2018., a whole-genome triplication event occurred in an ancient Ipomoea lineage ancestor approximately 46.1 million years ago (Mya). This event occurred before both the approximately 3.6 Mya divergence of I. nil from the lineage that includes I. trifida and I. triloba and the approximately 2.2 Mya divergence between I. trifida and I. triloba (Wu et al. 2018WU, S.; LAU, K. H.; CAO, Q.; HAMILTON, J. P.; SUN, H.; ZHOU, C.; ESERMAN, L.; GEMENET, D. C.; OLUKOLU, B. A.; WANG, H. Y.; CRISOVAN, E.; GODDEN, G. T.; JIAO, J.; WANG, X.; MERCY, K.; MANRIQUE-CARPINTERO, N.; VAILLANCOURT, B.; WIEGERT-RININGER, K.; YANG, X. S.; BAO, K.; SCHAFF, J.; KREUZE, J.; GRUNEBERG, W.; KHAN, A.; GHISLAIN, M.; MA, D. F.; JIANG, J. M.; MWANGA, R.; LEEBENS-MACK, J.; COIN, L.; YENCHO, C.; ROBIN, B. R.; FEI, Z. Genome sequences of two diploid wild relatives of cultivated sweet potato reveal targets for genetic improvement. Nature Communications, v. 9, n. 1, e4580, 2018.).
To better understand the behavior of these genes, RNA sequencing data (FPKM values) were obtained from the I. trifida and I. triloba genomes from the database. As shown in Figure 5, the ItfGPX5 gene in I. trifida had a higher expression profile than the other genes (Figure 5A). Other genes were also expressed (Figure 5A). The ItbGPX5 gene was highly expressed in all studied tissues. The ItbGPX3 gene was expressed in the flower bud, leaf and stem tissues. However, mild expression levels were also observed in other tissues (Figure 5B). With respect to the biological mechanism, it is believed that genes can be expressed according to the type of stress and specific tissue.
In silico expression profiles for Ipomoea trifida (A) and I. triloba (B). The color bar represents the fragments per kilobase of exon per million fragments mapped (FPKM) value obtained in the genome.
These results represent a preliminary exploration of GPX genes and facilitate future investigation on the biological functions of GPX proteins in sweet potato.
Six ItfGPX (Ipomoea trifida) and six ItbGPX (I. triloba) genes were identified and characterized in this study.
The phylogenetic analysis showed that the GPX genes of I. trifida, I. triloba, A. thaliana and O. sativa are classified into four groups;
The GPX genes in I. trifida and I. triloba were highly conserved, when compared with those in A. thaliana and O. sativa;
The expression patterns in various tissues and development stages may involve different plant growth and development processes.
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Publication Dates
-
Publication in this collection
16 Aug 2024 -
Date of issue
2024
History
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Received
05 Mar 2024 -
Accepted
11 June 2024 -
Published
05 July 2024