Genome-Wide Identification and Expression Analysis of Catalase Gene Families in Triticeae

Abstract

Aerobic metabolism in plants results in the production of hydrogen peroxide (H₂O₂), a significant and comparatively stable non-radical reactive oxygen species (ROS). H₂O₂ is a signaling molecule that regulates particular physiological and biological processes (the cell cycle, photosynthesis, plant growth and development, and plant responses to environmental challenges) at low concentrations. Plants may experience oxidative stress and ultimately die from cell death if excess H₂O₂ builds up. Triticum dicoccoides, Triticum urartu, and Triticum spelta are different ancient wheat species that present different interesting characteristics, and their importance is becoming more and more clear. In fact, due to their interesting nutritive health, flavor, and nutritional values, as well as their resistance to different parasites, the cultivation of these species is increasingly important. Thus, it is important to understand the mechanisms of plant tolerance to different biotic and abiotic stresses by studying different stress-induced gene families such as catalases (CAT), which are important H₂O₂-metabolizing enzymes found in plants. Here, we identified seven CAT-encoding genes (TdCATs) in Triticum dicoccoides, four genes in Triticum urartu (TuCATs), and eight genes in Triticum spelta (TsCATs). The accuracy of the newly identified wheat CAT gene members in different wheat genomes is confirmed by the gene structures, phylogenetic relationships, protein domains, and subcellular location analyses discussed in this article. In fact, our analysis showed that the identified genes harbor the following two conserved domains: a catalase domain (pfam00199) and a catalase-related domain (pfam06628). Phylogenetic analyses showed that the identified wheat CAT proteins were present in an analogous form in durum wheat and bread wheat. Moreover, the identified CAT proteins were located essentially in the peroxisome, as revealed by in silico analyses. Interestingly, analyses of CAT promoters in those species revealed the presence of different cis elements related to plant development, maturation, and plant responses to different environmental stresses. According to RT-qPCR, Triticum CAT genes showed distinctive expression designs in the studied organs and in response to different treatments (salt, heat, cold, mannitol, and ABA). This study completed a thorough analysis of the CAT genes in Triticeae, which advances our knowledge of CAT genes and establishes a framework for further functional analyses of the wheat gene family.

Keywords: Triticeae; abiotic stress; antioxidant enzymes; catalase; gene structure; reactive oxygen species.

Figure 1

Analyses of CAT genes/proteins identified in T. dicoccoide, T. Urartu, and T. spelta: (A) phylogenetic analyses of CAT proteins in each species constructed by MEGA 11 and showing the phylogenetic relationship between the identified genes present in each species; (B) exon/intron structure of each identified gene; and (C) identification of conserved CAT domains (CAT-like superfamily and CAT-related superfamily) present in Tricacea proteins. The abscissae in (B,C) represent the lengths of the different genes/proteins. The small bows in (B) represent the CDS/UTR regions of the genes.

Figure 2

Distribution of conserved motifs in 19 identified CAT proteins in T. dicoccoides, T. urartu, and T. spelta recognized by the MEME search tool. Each motif is represented by a colored box. The order of the motifs corresponds to the positions of the motifs in the individual protein sequences.

Figure 3

Chromosomal localisation of TiCAT (A), TuCAT (B), and (C) TsCAT genes using MG2C (v2.1). The blue color represents TdcCAT genes in the T. dicoccoide genome; the red and pink colors represent TsCAT genes in the T. spelta genome; the green color represents TuCAT genes in the T. urartu genome.

Figure 4

Synteny relationships of CAT proteins among the different species T. dicocoides, T. urartu, T. spelta, T.durum, and T. aestivum. Ribbons were colored based on their scores for identity, with green ≤ 75%, orange ≤ 99.9999%, and red > 99.9999%, as constructed by the Circoletto webtool.

Figure 5

Phylogenetic analysis of CATs in bread wheat (T. aestivum L. Ta), durum wheat (T. turgidum ssp durum L. Td), Arabidopsis (Arabidopsis thaliana L. At), oat (Avena sativa L. Av), rice (Oryza sativa L. Os), tobacco (Nicotiana plumbaginifolia L. Np), spelta (T. spelta L. Ts), T. dicoccoides (Tdc), and T. urartu (Tu). The tree was generated with the full-length CAT protein sequences for bread wheat (pink triangle), durum wheat (Cyan rectabgle), Arabidopsis (cyan circle), oat (orange star), rice (blue circle), tobacco (yellow rhombus), spelta (blue six-pointed star), T. dicoccoides (Tdc), and T. urartu (purple rhombus). Six elementary phylogenetic groupings were displayed in the phylogenetic tree, and each group was denoted by a different background color (Scale: 0.1). The phylogenetic tree was constructed using test maximum likelihood with 1000 bootstraps using MEGA 11 software and then visualized using the iTOL web tool. The accession numbers for the CAT proteins used in this figure are: A. sativa L. (AVESA.00001b.r3.1Dg0003456.1; AVESA.00001b.r3.4Ag0002488.4; AVESA.00001b.r3.4Cg0001036.2; AVESA.00001b.r3.7Dg0000025.2; AVESA.00001b.r3.7Dg0002783.2; AVESA.00001b.r3.7Dg0002783.1; AVESA.00001b.r3.6Cg0000037.1; AVESA.00001b.r3.2Dg0000518.1; AVESA.00001b.r3.1Ag0002627.3; AVESA.00001b.r3.6Cg0001322.3); T. turgidum ssp durum (TdCAT1 WDD45561.1; TdCAT2 VAI41949.1; TdCAT3 VAI53367.1; TdCAT4 VAI53366.1; TdCAT5 VAI10245.1; TdCAT6 VAI53365.1); O. sativa ssp japonica (OsCATD XP_015636098.1; OsCATA: XP_015625395; OsCATC: Q10S82.1; OsCATB: XP_015643077); A. thaliana (AtCAT2: AAL66998.1; AtCAT1: AAQ56816.1; AtCAT3: NP_564120.1); N. plumbaginifolia (NpCAT1: P49315.1; NpCAT2: P49316.1; NpCAT3: P49317.1); T. aestivum (TaCAT1-D: TraesCS4D02G322700; TaCAT1-B: TraesCS5A02G498000; TaCAT2-A: TraesCS6A02G04170; TaCAT3-A2: TraesCS7A02G549900; TaCAT2-B: TraesCS6B02G056800; TaCAT1-A: TraesCS4B02G325800; TaCAT3-A1: TraesCS7A02G549800; TaCAT3-B: TraesCS7B02G473400; TaCAT2-D: TraesCS6D02G048300; TaCAT3-U: TraesCSU02G105300); T. urartu: (TuG1812G0700005868.01.T01, TuG1812G0700005870.01.T01, TuG1812G0700005318.01.T01, TuG1812G0600000378.01.T01); T. spelta: (TraesTSP4B01G347300.1, TraesTSP4D01G343400.1, TraesTSP5A01G526000.1, TraesTSP6A01G043000.1, TraesTSP6B01G059900.1, TraesTSP7A01G595800.1, TraesTSP7B01G508100.1, TraesTSP7D01G591500.1); and T. Dicoccoides (TRIDC4BG054740.1, TRIDC7AG076360.6, TRIDC7BG073240.1, TRIDC6BG007200.1, TRIDC6BG007200.2, TRIDC6AG004940.1, TRIDC6AG004940.2).

Figure 6

The 3D structures of CAT proteins in (A) T. dicocoide, (B) T. spelta, and (C) T. urartu built by SWISS-MODEL.

Figure 7

Prediction of subcellular localization of CAT proteins in T. dicocoide, T. spelta, and T. urartu using the Wolf PSORT online server and visualization via Tbtools software. Grey colors suggest “no prediction” of the protein in this cellular compartment.

Figure 8

Gene ontology predictions for the TdcCAT, TsCAT, and TuCAT proteins using PANNZER and generated by SRplot webtool. The reliability of the prediction results is visualized via the intensity of the blue color and the sizes of the circles.

Figure 9

Molecular functions of TdcCAT, TsCAT, and TuCAT proteins as predicted by PANNZER and generated by the SRplot webtool. The reliability of the prediction results is visualized via the intensity of the orange color and the sizes of the circles.

Figure 10

Putative cis element numbers for TdcCAT, TsCAT, and TuCAT gene promotors using Plantcare and visualized using Tbtools v1.123. Grey colors mean no prediction.

Figure 11

Relative expression levels of (A) TdcCAT1, TuCAT1, and TsCAT1 and (B) TdcCAT4, TuCAT4, and TsCAT4 in roots, stems, and leaves in normal conditions. Error bars represent standard deviation.

Figure 12

TdcCAT1 gene expression analysis under stressful conditions. Error bars represent standard deviation (n = 15 plants). Letters indicate significant differences (two-way ANOVA test with Tukey’s pairwise comparison).

Figure 13

TdcCAT4 gene expression analysis under stressful conditions. The error bars represent standard deviation (n = 15 plants). Letters indicate significant differences (two-way ANOVA test with Tukey’s pairwise comparison).