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. 2023 Jun 9:14:1180647.
doi: 10.3389/fpls.2023.1180647. eCollection 2023.

Bioinformatic analysis of short-chain dehydrogenase/reductase proteins in plant peroxisomes

Yuchan Zhang  1   2 , Xiaowen Wang  1 , Xinyu Wang  1 , Yukang Wang  1 , Jun Liu  1 , Saisai Wang  1 , Weiran Li  1 , Yijun Jin  1 , Delara Akhter  1   3 , Jiarong Chen  1 , Jianping Hu  4 , Ronghui Pan  1   2
Affiliations

Bioinformatic analysis of short-chain dehydrogenase/reductase proteins in plant peroxisomes

Yuchan Zhang et al. Front Plant Sci. .

Abstract

Peroxisomes are ubiquitous eukaryotic organelles housing not only many important oxidative metabolic reactions, but also some reductive reactions that are less known. Members of the short-chain dehydrogenase/reductase (SDR) superfamily, which are NAD(P)(H)-dependent oxidoreductases, play important roles in plant peroxisomes, including the conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid (IAA), auxiliary β-oxidation of fatty acids, and benzaldehyde production. To further explore the function of this family of proteins in the plant peroxisome, we performed an in silico search for peroxisomal SDR proteins from Arabidopsis based on the presence of peroxisome targeting signal peptides. A total of 11 proteins were discovered, among which four were experimentally confirmed to be peroxisomal in this study. Phylogenetic analyses showed the presence of peroxisomal SDR proteins in diverse plant species, indicating the functional conservation of this protein family in peroxisomal metabolism. Knowledge about the known peroxisomal SDRs from other species also allowed us to predict the function of plant SDR proteins within the same subgroup. Furthermore, in silico gene expression profiling revealed strong expression of most SDR genes in floral tissues and during seed germination, suggesting their involvement in reproduction and seed development. Finally, we explored the function of SDRj, a member of a novel subgroup of peroxisomal SDR proteins, by generating and analyzing CRISPR/Cas mutant lines. This work provides a foundation for future research on the biological activities of peroxisomal SDRs to fully understand the redox control of peroxisome functions.

Keywords: benzaldehyde biosynthesis; fatty acid β-oxidation; peroxisomal targeting signal type 1 (PTS1); peroxisome; short-chain dehydrogenase/reductase (SDR).

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Subcellular localization of peroxisomal SDR proteins newly identified in this study. (A) Arabidopsis peroxisomal SDR proteins, with the 5 newly identified in bold. (B) Confocal microscopy images of the peroxisome localization of YFP-SDRf, YFP-SDRg, YFP-SDRh, YFP-SDRi, and YFP-SDRj in tobacco leaf epidermal cells. Each YFP fusion was co-expressed with the peroxisome marker CFP-PTS1. Scale bars = 10 mm. (C) Confocal images of several partial proteins of SDRf and SDRh in tobacco leaf epidermal cells. 15aa indicates the last 15 amino acids of the protein. Δ3aa indicates the protein without the last 3 amino acids. Each mVenus fusion was co-expressed with the peroxisome marker moxCerulean3-PTS1. Scale bars = 10 mm.
Figure 2
Figure 2
Phylogenetic analysis of SDR proteins in Arabidopsis thaliana (At) (red words) and their homologs (blue words) in Physcomitrella patens (Pp), Ginkgo biloba (Gb) and Oryza sativa (Os). Besides of the Arabidopsis peroxisomal SDRs involved in this study, previously reported peroxisomal SDR proteins in human and plants (*) were included for functional reference. Several non-peroxisomal but functionally established SDR proteins (●) showing high sequence similarity with SDRj were also included in the phylogenetic tree for functional reference of SDRj. The phylogenetic tree was constructed using MEGAX (Kumar et al., 2018) by Maximum Likelihood method and the Jones-Taylor-Thornton (JTT) model. Ca, Capsicum annuum; Cm: Cucumis melo; Hs: Homo sapiens; Ph: Petunia hybrida; Pm: Persicaria minor; Ps: Papaver somniferum; St: Solanum tuberosum.
Figure 3
Figure 3
Analysis of the phylogenetic relationship (A), gene structure (B) and conserved motifs (C) of peroxisomal SDR genes in Arabidopsis and their orthologs from rice. The maximum likelihood (ML) tree under the Jones-Taylor-Thornton (JTT) model was constructed using MEGAX and full-length protein sequences. The exon-intron structures of these genes were graphically displayed by Tbtools using the CDS and genome sequence of the SDRs. The MEME Suite web server (http://meme.nbcr.net/meme/intro.html) was used to predict conserved motifs in the proteins.
Figure 4
Figure 4
Gene expression profile analysis of Arabidopsis peroxisomal SDR genes in various tissues. The expression data of Arabidopsis SDRs were collected from the Arabidopsis eFP Browser (https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi). (A) Relative expression levels of each gene in different types of tissue. Relative expression data of each gene within each column was shown as z-score. (B) Relative expression levels of different SDR genes in each type of tissue. Relative expression data of different genes the same tissue type within each row was shown as z-score. z-score was calculated as described in Materials and Methods. Red or yellow represents relatively high expression, blue represents relatively low expression, and white represents the average expression level.
Figure 5
Figure 5
Generation and characterization of the sdrj mutants. (A) SDRj gene structure. Open box, UTR; black box, exon; solid line, intron. CRISPR target site is indicated. (B) Mutation sites in the sdrj mutants. A 1-bp insertion was found in the second exon in both mutants. (C) Appearance of 3-week-old plants. Bar = 1 cm. (D) Sucrose dependence assays. For each mutant, all data were normalized to the data in sucrose-containing media. Error bars indicate standard deviations (n=4, the average length of more than 8 seedlings was used as one biological replicate). **** means P < 0.0001 from WT1 or WT2, and ns means no significant difference from WT1 or WT2, as determined by one-way ANOVA. (E–H) 2,4-DB, IBA, propionate and isobutyrate response assays. Root lengths on medium containing different concentration of 2,4-DB, IBA, propionate and isobutyrate are shown. Error bars indicate standard deviations (n=4, the average length of more than 12 seedlings was used as one biological replicate). **** means P < 0.0001, *** means P < 0.001, ** means P < 0.01, from 0 concentration, and ns means no significant difference from 0 concentration, as determined by one-way ANOVA.
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References

    1. Armenteros J. J. A., Salvatore M., Emanuelsson O., Winther O., Von Heijne G., Elofsson A., et al. . (2019). Detecting sequence signals in targeting peptides using deep learning. Life Sci. Alliance 2, 1–14. doi: 10.26508/lsa.201900429 - DOI - PMC - PubMed
    1. Babiychuk E., Kushnir S., Belles-Boix E., Van Montagu M., Inze D. (1995). Arabidopsis thaliana NADPH oxidoreductase homologs confer tolerance of yeasts toward the thiol-oxidizing drug diamide. J. Biol. Chem. 270, 26224–26231. doi: 10.1074/jbc.270.44.26224 - DOI - PubMed
    1. Bachem C. W. B., Horvath B., Trindade L., Claassens M., Davelaar E., Jordi W., et al. . (2001). A potato tuber-expressed mRNA with homology to steroid dehydrogenases affects gibberellin levels and plant development. Plant J. 25, 595–604. doi: 10.1046/j.1365-313X.2001.00993.x - DOI - PubMed
    1. Bailey T. L., Johnson J., Grant C. E., Noble W. S. (2015). The MEME suite. Nucleic Acids Res. 43, W39–W49. doi: 10.1093/nar/gkv416 - DOI - PMC - PubMed
    1. Baker A., Hogg T. L., Warriner S. L. (2016). Peroxisome protein import: a complex journey. Biochem. Soc Trans. 44, 783–789. doi: 10.1042/BST20160036 - DOI - PMC - PubMed