Heavy Metal Stress-Associated Proteins in Rice and Arabidopsis: Genome-Wide Identification, Phylogenetics, Duplication, and Expression Profiles Analysis

Abstract

Heavy metal exposure is a serious environmental stress in plants. However, plants have evolved several strategies to improve their heavy metal tolerance. Heavy metal-associated proteins (HMPs) participate in heavy metal detoxification. Here, we identified 46 and 55 HMPs in rice and Arabidopsis, respectively, and named them OsHMP 1-46 and AtHMP 1-55 according to their chromosomal locations. The HMPs from both plants were divided into six clades based on the characteristics of their heavy metal-associated domains (HMA). The HMP gene structures and motifs varied greatly among the different classifications. The HMPs had high collinearity and were segmentally duplicated. A cis-element analysis revealed that the HMPs may be regulated by different transcription factors. An expression profile analysis disclosed that only eight OsHMPs were constitutive in rice tissues. Of these, the expression of OsHMP37 was far higher than that of the other seven genes while OsHMP28 was expressed exclusively in the roots. For Arabidopsis, nine AtHMPs presented with very high transcript levels in all organs. Most of the selected OsHMPs were differentially expressed in various tissues under different heavy metal stresses. Only OsHMP09, OsHMP18, and OsHMP22 showed higher expression levels in all tissues under different heavy metal stresses. In contrast, most of the selected AtHMPs had nearly constant expression levels in different tissues under various heavy metal stresses. The AtHMP20, AtHMP23, AtHMP25, AtHMP31, AtHMP35, AtHMP46 expression levels under different heavy metal stresses were higher in the leaves and roots. The foregoing discoveries elucidated HMP evolution in monocotyledonous and dicotyledonous plants and may helpful functionally characterize HMPs in the future.

Keywords: Arabidopsis; gene duplication; heavy-metal stress; phylogenetic analysis; rice.

Figure 1

Chromosomal locations of heavy-metal-associated genes in rice (A) and Arabidopsis (B). Black bars represent the chromosomes. Chromosome numbers are shown at the tops of the bar. Heavy-metal-associated genes are labeled at the right of the chromosomes. Scale bar on the left indicates the chromosome lengths (Mb).

Figure 2

Schematic representations of segmental duplications of rice (A) and Arabidopsis (B) heavy-metal-associated genes. Different color lines indicate all synteny blocks in rice or Arabidopsis genome between each chromosome. Thick red lines indicate duplicated heavy-metal-associated gene pair on different chromosome, and thick blue lines indicate duplicated gene pair on same chromosome. The chromosome number is indicated at the bottom of each chromosome. Scale bar marked on the chromosome indicating chromosome lengths (Mb).

Figure 3

Synteny analysis of heavy-metal-associated genes between rice and Brachypodium distachyon, Oryza brachyantha, Triticum aestivum, Setaria italica, and Zea mays. Gray lines in the background indicate the collinear blocks within rice and other plant genomes, while the orange lines highlight the syntenic OsHMP gene pairs. The species names with the prefixes “O. sativa,” “B. distachyon,” “O. brachyantha,” “T. aestivum,” “Setaria italica,” and “Z. mays” indicate Oryza sativa, Brachypodium distachyon, Oryza brachyantha, Triticum aestivum, Setaria italica, and Zea mays, respectively. Different color bars represent the chromosomes of different species. The chromosome number is labeled at the top or bottom of each chromosome.

Figure 4

Synteny analysis of heavy-metal-associated genes between Arabidopsis thaliana and Brassica rapa, Cucumis sativus, Glycine max, Gossypium raimondii, and Solanum tuberosum. Gray lines in the background indicate the collinear blocks within rice and other plant genomes, while the blue lines highlight the syntenic AtHMP gene pairs. The species names with the prefixes “A. thaliana,” “B. rapa,” “C. sativus,” “G. max,” “G. raimondii,” and “S. tuberosum” indicate Arabidopsis thaliana and Brassica rapa, Cucumis sativus, Glycine max, Gossypium raimondii, and Solanum tuberosum, respectively. Different color bars represent the chromosomes of different species. The chromosome number is labeled at the top or bottom of each chromosome.

Figure 5

Multiple Alignment of rice and Arabidopsis HMP and selected heavy-metal-associated domain amino acid sequences. “ATCCS,” “H1,” “H2,” “H3,” “H4,” and “P1B-ATPase” represent different HMP proteins classification.

Figure 6

Phylogenetic relationships among 156 heavy-metal-associated proteins in rice, Arabidopsis, soybean, maize, potato, wheat, sorghum, wild rice, Brachypodium distachyon, and Populus trichocarpa. The maximum likelihood tree was created using MEGA v. 7.0 (bootstrap value = 1,000) and the bootstrap value of each branch is displayed. The black solid circles, hollow circles, and yellow solid circles represent heavy-metal-associated proteins from rice, Arabidopsis, and other species, respectively.

Figure 7

Phylogenetic analysis, gene structure and motif analysis of heavy-metal-associated genes in rice and Arabidopsis. (A) Phylogenetic tree of HMP proteins between rice and Arabidopsis. The maximum likelihood tree was created using MEGA v. 7.0. P1B-ATPase, ATCCS, H1, H2, H3, and H4 are marked with different colors. (B) Gene structure of HMP genes in rice and Arabidopsis. A schematic diagram was constructed by the Gene Structure Display Server 2.0. Exons, introns, and untranslated regions are marked by green double-sided wedge, black lines, and blue round-corner rectangles, respectively. The scale bar at the bottom estimates the lengths of the exons, introns, and untranslated regions. (C) Motif composition of HMP proteins in rice and Arabidopsis. Motif analysis was performed using the MEME program. Boxes of different colors represent the various motifs. Their location in each sequence is marked. Motif sequence logo is shown in Figure S4. The scale bar at the bottom indicates the lengths of the HMP protein sequences.

Figure 8

Predicted cis-elements in the promoter regions of the rice heavy-metal-associated genes. All promoter sequences (−1,500 bp upstream genomic sequence) were analyzed. The heavy-metal-associated genes are shown on the left side of the figure. The scale bar at the bottom indicates the length of promoter sequence. Different cis-elements were labeled by rectangle of different color.

Figure 9

Predicted cis-elements in the promoter regions of the Arabidopsis heavy-metal-associated genes. All promoter sequences (−1,500 bp upstream genomic sequence) were analyzed. The heavy-metal-associated genes are shown on the left side of the figure. The scale bar at the bottom indicates the length of promoter sequence. Different cis-elements were labeled by rectangle of different color.

Figure 10

Expression pattern of the rice heavy-metal-associated gene family in various tissues or stages. (A) Numbers of expressed genes in each organ. Expression data of the rice HMP genes were downloaded from the Expression Atlas database. Extremely high: TPM > 100, high: 100 ≥TPM >50, medium: 50 ≥ TPM > 5, low: 5 ≥ TPM > 0; (B) Expression patterns of the rice HMP genes in various tissues. Heatmaps were generated using HemI from the normalized value by row for the signatures in transcripts per million (TPM). Transcript levels are depicted by different colors on the scale. Green and red represent low and high expression levels, respectively.

Figure 11

Expression pattern of the Arabidopsis heavy-metal-associated gene family in various tissues or stages. (A) Numbers of expressed genes in each organ. Expression data of the Arabidopsis HMP genes were downloaded from the Expression Atlas database. Extremely high: TPM > 100, high: 100 ≥ TPM > 50, medium: 50 ≥ TPM > 5, low: 5 ≥ TPM > 0; (B) Expression patterns of the Arabidopsis HMP genes in various tissues. Heatmaps were generated using HemI from the normalized value by row for the signatures in transcripts per million (TPM). Transcript levels are depicted by different colors on the scale. Green and red represent low and high expression levels, respectively.

Figure 12

Expression profiles of 12 selected OsHMPs in response to Cu²⁺(A), Cd²⁺(B), Zn²⁺(C), and Pb²⁺(D) stresses in 2-weeks old rice seedlings after treatment for 1, 3, 12, and 24 h. Data represent means (±SD) of three biological replicates. Vertical bars indicate standard deviations. Asterisks indicate corresponding genes significantly upregulated or downregulated between the treatment and control (n = 12, *p < 0.05; **p < 0.01; Student's t-test).

Figure 13

Expression profiles of 9 selected AtHMPs in response to Cu²⁺(A), Cd²⁺(B), Zn²⁺(C), and Pb²⁺(D) stresses in 2-weeks old Arabidopsis seedlings after treatment for 1, 3, 12, and 24 h. Data represent means (±SD) of three biological replicates. Vertical bars indicate standard deviations. Asterisks indicate corresponding genes significantly upregulated or downregulated between the treatment and control (n = 12, *p < 0.05; **p < 0.01; Student's t-test).