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AtA6PR1 and AtA6PR2 encode putative aldose 6-phosphate reductases that are cytosolically localized and respond differentially to cold and salt stress in Arabidopsis thaliana

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Abstract

Sorbitol is synthesized in Rosaceae species, especially in source organs, in a pathway involving aldose 6-phosphate reductase (A6PR, EC 1.1.1.200). As compatible solutes, sorbitol and other sugar alcohols assist in the ability of the plant to withstand abiotic stress conditions. Here, we identify two tandemly-duplicated genes in a non-Rosaceae species (Arabidopsis thaliana L.), and show that the proteins encoded by At2g21250 (AtA6PR1) and At2g21260 (AtA6PR2) possess the molecular characteristics of A6PRs. Consistent with bioinformatic predictions, we determined that green fluorescent protein-tagged versions of AtA6PR1 and AtA6PR2 are cytosolically localized, a finding supported by immunoblotting using a specific anti-AtA6PR1 antisera after subcellular fractionation of Arabidopsis leaves. We also show that under standard growth conditions, both genes are widely-expressed, whilst AtA6PR1 protein accumulates in both source and sink organs. Under short term (24 h) cold (4 °C) or saline (150 mM NaCl) stress, both genes respond differentially and reciprocally in leaf and root tissues, suggesting a sub-functionalization of their roles. The identification of homozygous T-DNA insertional ata6pr1-mutants will facilitate the functional characterization of this gene, and help to determine its role under abiotic stress conditions.

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Fig. 1
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Abbreviations

A6PR:

Aldose 6-phosphate reductase

GFP:

Green fluorescent protein

NADP(H):

Nicotinamide adenine dinucleotide phosphate (reduced)

SDH:

Sorbitol dehydrogenase

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Acknowledgements

This work was financed by Fondecyt 1140527 and 1181198 (M.H.). We thank Dr. M. R. Bono and Dr. L. Vargas for producing the anti-AtA6PR1 antisera, NASC Nottingham Arabidopsis Stock Centre (UK) for supplying the ata6pr1-1 and ata6pr1-2 seeds, and all past and present members of the Centro de Biología Molecular Vegetal for valuable discussions, especially Karina Olivos.

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Authors and Affiliations

  1. Centro de Biología Molecular Vegetal (CBMV), Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Santiago, Chile

    Bárbara Rojas, Joel Wurman, María Sofía Zamudio, Amanda Donoso, Pamela Cabedo, Francisca Díaz, Claudia Stange & Michael Handford

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  1. Bárbara Rojas
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  2. Joel Wurman
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  3. María Sofía Zamudio
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  4. Amanda Donoso
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Correspondence to Michael Handford.

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Supplementary material 1 (DOCX 21 kb)

Supplementary Fig. S1

Sequence analysis of AtA6PRs of Arabidopsis. Alignment of the protein sequences of AtA6PR1 (At2g21250), AtA6PR2 (At2g21260) and MdA6PR (from apple, Genbank NP_001280957; Kanayama et al. 1992; Figueroa and Iglesias 2010) using ESPript 3.0. Black shading and boxed lettering indicate identical residues and conservative substitutions, respectively. The 3 aldo/keto reductase signature family regions are underlined, and asterisks highlight the conserved IPKS/T motif (TIFF 7567 kb)

Supplementary Fig. S2

Specificity of anti-AtA6PR1 antisera. Total soluble proteins was isolated from 100 mg of 10 d-old seedlings of Arabidopsis grown on MS plates (Murashige and Skoog 1962). Samples (25 µg) were resolved by SDS-PAGE. WT, wild-type plants; ata6pr1-1 and ata6pr1-2, SALK T-DNA insertional mutants (SALK_021705 and SALK_028553, respectively). Upper panel: Immunoblot analysis using mouse polyclonal anti-AtA6PR1 primary antisera (1:4000) and an anti-mouse alkaline phosphatase-conjugated secondary antibody (Sigma, goat, 1:25,000). Lower panel: Coomassie-stained gel (TIFF 2543 kb)

Supplementary Fig. S3

Response of positive control genes in Arabidopsis to cold and salt stress. Transcript levels were analyzed by real-time quantitative RT-PCR, using total RNA isolated at different time points from one-month old plants subjected to a cold stress (4 °C, AtCOR27) or b saline stress (150 mM NaCl, AtWRKY33), respectively. The data were normalized using At1g13320 (PP2A) levels as a control (Czechowski et al. 2005), and transcript levels calibrated to 1.0 at the start of the experiment (0 h) for each gene in each organ. Different letters indicate significant differences as determined using one-way ANOVA and a Tukey post-test (p < 0.05); standard error bars are displayed (n = 3) (TIFF 1592 kb)

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Rojas, B., Wurman, J., Zamudio, M.S. et al. AtA6PR1 and AtA6PR2 encode putative aldose 6-phosphate reductases that are cytosolically localized and respond differentially to cold and salt stress in Arabidopsis thaliana. J. Plant Biochem. Biotechnol. 28, 114–119 (2019). https://doi.org/10.1007/s13562-018-0459-5

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