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Oryza sativa heat-induced RING finger protein 1 (OsHIRP1) positively regulates plant response to heat stress

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OsHIRP1 is an E3 ligase that acts as a positive regulator in the plant response to heat stress, thus providing important information relating to adaptation and regulation under heat stress in plant.

Abstract

Extreme temperature adversely affects plant growth, development, and productivity. Here, we report the molecular functions of Oryza sativa heat-induced RING finger protein 1 (OsHIRP1), which might play an important role in the response to heat. Transcription of the OsHIRP1 was upregulated in response to heat and drought treatment. We found that the OsHIRP1-EYFP fusion protein was localized to the nucleus after heat treatment (45 °C). Two interacting partners, OsARK4 and OsHRK1, were identified via yeast-two-hybrid screening, which were mainly targeted to the nucleus (OsARK4) and cytosol (OsHRK1), and their interactions with OsHIRP1 were confirmed by biomolecular fluorescence complementation (BiFC). An in vitro ubiquitination assay showed that OsHIRP1 E3 ligase directly ubiquitinates its interacting proteins, OsAKR4 and OsHRK1, as substrates. Using an in vitro cell-free degradation assay, we observed a clear reduction in the levels of the two proteins under high temperature (45 °C), but not under low temperature conditions (4 °C and 30 °C). Seeds of OsHIRP1-overexpressing plants exhibited high germination rates compared with the control under heat stress. The OsHIRP1-overexpressing plants presented high survival rates of approximately 62–68%, whereas control plants displayed a low recovery rate of 34% under condition of acquired thermo-tolerance. Some heat stress-inducible genes (HsfA3, HSP17.3, HSP18.2 and HSP20) were up-regulated in OsHIRP1-overexpressing Arabidopsis than control plants under heat stress conditions. Collectively, these results suggest that OsHIRP1, an E3 ligase, positively regulates plant response to heat stress.

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Acknowledgements

This work was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science, and Technology (Grant No. 2016R1A2B4015626) and Cooperative research program for agriculture science and technology development (Grant No. PJ013429012018).

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  1. Plant Genomics Laboratory, Department of Bio-resources Sciences, Kangwon National University, Chuncheon, 200-713, South Korea

    Ju Hee Kim, Sung Don Lim & Cheol Seong Jang

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  1. Ju Hee Kim
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Ju Hee Kim, Sung Don Lim and Cheol Seong Jang have contributed to this work.

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Correspondence to Cheol Seong Jang.

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Supplementary material 2 Fig. S1 Expression of the specific expression marker genes in response to abiotic stress. qRT-PCR analysis was performed under different types of abiotic stress [salt (200 mM NaCl), drought, heat (45°C) and ABA (0.1 mM)] in rice roots. OsNAC10 for NaCl, OsSalt for drought and ABA and, OsHSP90-1 for heat (heat 45°C). qRT-PCR was performed with three biological replicates per each time point (n = 9). Relative expression of the specific expression markers was determined by qRT-PCR, with OsACTINII levels used as an internal control. Asterisks represent a statistically significant difference according to two-tailed Student’s t-test; *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively. Fig. S2 The multiple amino acid sequence alignments of the OsHIRP1 and orthologous gene, from Aegilops tauschii (F775_02460), Arabidopsis (AT3G05670), Brachypodium distachyon (BRADI1G64740), Triticum aestivum (Traes_4DL_6E50CBE1B) (A). The obtained sequences were aligned using ClustalW2 (ftp://ebi.ac.uk/pub/software/clustalw2/). Comparison of amino acid sequence similarity of the OsHIRP1 with its homologs (B). Fig. S3 Subcellular localization of the OsHIRP1 protein under heat stresses (38°C and 45°C for 15 min). The 35S:EYFP-fused OsHIRP1 protein incubated at room temperature (22°C) overnight after PEG-mediated transformation, and observed in non-treated or heat-treated (38°C and 45°C for 15 min) rice protoplasts. Fig. S4 Positive interaction protein with the OsHIRP1 from yeast two-hybrid screening. Fig. S5 Subcellular localization of the OsHRK1 with various organelle markers (ER; ER-rk CD3-959, Golgi: G-rk CD3-967, mitochondria; mt-rk CD3-991, plastid; pt-rk CD3-999, and peroxisome; px-rk CD3-983). Fig. S6 Subcellular localization of the OsHIRP1/OsAKR4 complex. The 35S:OsMeCP-DsRed2 was used as a marker of nuclear localization in rice protoplasts. Fig. S7 Subcellular localization of the 35S:EYFP-fused OsARK4 and OsHRK1 under heat stresses (38°C and 45°C for 15 min) in rice protoplasts. Fig. S8In vitro degradation assays of substrate proteins regulated by OsHIRP1 E3 ligase. The extracted OsAKR4- and OsHRK1-His-Trx were mixed with E.V (empty vector), and mixtures were incubated at different temperatures (4°C, 30°C, and 45°C) for 2 h (A, B). Anti-Nus and anti-Trx antibodies were used for immunoblotting analyses. MG132 was used to inhibit the 26S proteasome pathway. Ponceau S staining of the Rubisco proteins was used as a loading control. Fig. S9 Germination rate of the OsHIRP1-overexpressing plants (35S:OsHIRP1-EYFP) and control (35S:EYFP) under salt and PEG. (A) Seeds were germinated on half MS agar medium with 0, 100, and 150 mM NaCl or -0.25, -0.5, and -0.75 MPa PEG. Data represent means ± standard deviation (SD) (n = 25) from three biological replicates. Fig. S10 Protein interactions of two Arabidopsis homologous protein. The multiple amino acid sequence alignments in between OsHIRP1 partner proteins and its homologous proteins (A; AtARK4 and OsAKR4, B; AtHRK1 and OsHRK1). (C) Comparison of amino acid sequence similarity of OsHIRP1 partner proteins with its homologs. Yeast two-hybrid (Y2H) assay for the in vivo interaction of OsHIRP1 with two Arabidopsis homologous proteins (C). The full-length OsHIRP1 was cloned into pGBKT7-BD (DNA-binding domain), and homologous partner genes (AtAKR4 and AtHRK1) were cloned into pGADT7-AD (activating domain). Each of the constructed vectors was co-transformed into the Y2H Gold yeast strain in DDO/A (left) and QDO/X/A (right) medium. Co-transformation of BD-53/AD-T served as a positive control. Fig. S11 The subcellular localizations of the OsHIRP1-EYFP in Arabidopsis protoplasts. The 35S:EYFP and 35S:EYFP-fused OsHIRP1 were observed in Arabidopsis protoplasts under normal conditions (22°C) and under heat-treatments at 38°C and 45°C (A, B). Each of the constructed vectors was transiently expressed in Arabidopsis protoplasts for 16 h. 35S:EYFP was used as a negative control. (PPTX 17421 KB)

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Kim, J.H., Lim, S.D. & Jang, C.S. Oryza sativa heat-induced RING finger protein 1 (OsHIRP1) positively regulates plant response to heat stress. Plant Mol Biol 99, 545–559 (2019). https://doi.org/10.1007/s11103-019-00835-9

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