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Universal stress protein HRU1 mediates ROS homeostasis under anoxia

Nature Plants volume 1, Article number: 15151 (2015) Cite this article

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Abstract

Plant survival is greatly impaired when oxygen levels are limiting, such as during flooding or when anatomical constraints limit oxygen diffusion. Oxygen sensing in Arabidopsis thaliana is mediated by Ethylene Responsive Factor (ERF)-VII transcription factors, which control a core set of hypoxia- and anoxia-responsive genes responsible for metabolic acclimation to low-oxygen conditions. Anoxic conditions also induce genes related to reactive oxygen species (ROS). Whether the oxygen-sensing machinery coordinates ROS production under anoxia has remained unclear. Here we show that a low-oxygen-responsive universal stress protein (USP), Hypoxia Responsive Universal Stress Protein 1 (HRU1), is induced by RAP2.12 (Related to Apetala 2.12), an ERF-VII protein, and modulates ROS production in Arabidopsis. We found that HRU1 is strongly induced by submergence, but that this induction is abolished in plants lacking RAP2.12. Mutation of HRU1 through transfer DNA (T-DNA) insertion alters hydrogen peroxide production, and reduces tolerance to submergence and anoxia. Yeast two-hybrid and bimolecular fluorescence complementation (BiFC) analyses reveal that HRU1 interacts with proteins that induce ROS production, the GTPase ROP2 and the NADPH oxidase RbohD, pointing to the existence of a low-oxygen-specific mechanism for the modulation of ROS levels. We propose that HRU1 coordinates oxygen sensing with ROS signalling under anoxic conditions.

At the molecular level, the response to both hypoxia and anoxia is characterized by the induction of a set of hypoxia-responsive genes, which are required to adjust the metabolism of the plant to low oxygen1–5. A relatively large number of genes induced by hypoxia/anoxia are of an unknown function6. The molecular mechanism regulating the induction of hypoxia-responsive genes has been described for Arabidopsis thaliana7–9. Under aerobic conditions, members of group VII ERF transcription factors are constitutively transcribed but their relative proteins are degraded through an oxygen-dependent branch of the N-end rule pathway for protein proteolysis (NERP)8,9. Under anaerobiosis, the ERF-VII proteins are stabilized and translocated to the nucleus, where they activate transcription of the hypoxia/anoxia-responsive genes9. Another consequence of anoxia is the inhibition of the terminal step of the mitochondrial electron transport chain (mETC), which stimulates mitochondria-associated ROS production10. In addition, the activation of a RHO (Ras homologous)-like small G protein of plants (ROP2) under anoxia induces hydrogen peroxide (H2O2) production mediated by a NADPH oxidase (in plants also known as ‘respiratory burst oxidase homologue’ (Rboh))11. Production of H2O2 under anoxia was abolished in a mutant expressing a dominant-negative version of ROP2, linking ROS production by Rbohs under anoxia to the regulative role of small GTPases11. As a consequence of ROS production, a specific set of genes is induced, including typical markers of oxidative stress, such as ZAT10 and ZAT12, as well as those largely overlapping with heat-regulated genes12.

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Figure 1: HRU1 is a hypoxia-inducible universal stress protein.
Figure 2: Phenotypic characterization of the hru1-1 mutant.
Figure 3: HRU1 interacts with the small GTPase ROP2, RbohD and TRXh.
Figure 4: FRAP analysis of protein localization, dynamics and interactions.
Figure 5: Altered HRU1 expression affects hydrogen peroxide accumulation, gene expression and tolerance to low oxygen.

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Acknowledgements

We thank J. Bailey-Serres for reviewing an early version of this manuscript, A. Galli for helpful suggestions about the Y2H assay and C. Kiferle for helpful assistance in data analysis. We acknowledge Z. Yang (University of California, Riverside) for providing us with the plasmid carrying the coding sequence for the RIC1-Maltose Binding Protein (MBP) fusion protein.

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Author notes

  1. Silvia Gonzali and Elena Loreti: These authors contributed equally to this work.

Authors and Affiliations

  1. PlantLab, Institute of Life Sciences, Scuola Superiore Sant'Anna, Via Mariscoglio 34, 56124, Pisa, Italy

    Silvia Gonzali, Giacomo Novi, Sandro Parlanti, Chiara Pucciariello, Laura Bassolino, Valeria Banti, Francesco Licausi & Pierdomenico Perata

  2. nanoPlant Center @NEST, Institute of Life Sciences, Scuola Superiore Sant'Anna, Piazza San Silvestro 12, 56127, Pisa, Italy

    Silvia Gonzali, Chiara Pucciariello, Francesco Licausi & Pierdomenico Perata

  3. Institute of Agricultural Biology and Biotechnology, National Research Council, Via Moruzzi 1, 56100, Pisa, Italy

    Elena Loreti

  4. Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Piazza San Silvestro 12, 56127, Pisa, Italy

    Francesco Cardarelli

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  1. Silvia Gonzali
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Contributions

S.G., E.L., L.B., S.P., C.P. and F.L. performed the molecular biology experiments, Y2H, BiFC and split-luciferase assays. G.N. and S.P. obtained the mutants and performed the phenotypic characterization of the mutants and transgenic lines. G.N. performed the submergence and hypoxia tolerance assays. E.L. and P.P. performed the transient expression analysis in Nicotiana. V.B. and C.P. performed the ROP pull-down assays. F.C. designed, performed and analysed the FRAP experiments. P.P., S.G. and E.L. designed the experiments. P.P. and E.L. wrote the manuscript. All authors discussed and commented on the content of the paper.

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Correspondence to Pierdomenico Perata.

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Gonzali, S., Loreti, E., Cardarelli, F. et al. Universal stress protein HRU1 mediates ROS homeostasis under anoxia. Nature Plants 1, 15151 (2015). https://doi.org/10.1038/nplants.2015.151

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