Planta

, Volume 241, Issue 5, pp 1119–1129 | Cite as

Rice RING E3 ligase may negatively regulate gamma-ray response to mediate the degradation of photosynthesis-related proteins

  • Yong Chan Park
  • Jung Ju Kim
  • Dong Sub Kim
  • Cheol Seong Jang
Original Article
First Online:
Received:
Accepted:

Abstract

Main conclusion

In this study, our findings regarding the regulation of GA irradiation-induced OsGIRP1 in relation to the levels of photosynthesis-related proteins such as OsrbcL1 and OsrbcS1 and hypersensitive responses of overexpressing plants to GR irradiation provide insight into the molecular functions of OsGIRP1 as a negative regulator in response to the stress of radiation.

Abstract

The RING (Really Interesting New Gene) finger proteins are known to play crucial roles in various abiotic stresses in plants. Here, we report on RING finger E3 ligase, Oryza sativa gamma rays-induced RING finger protein1 gene (OsGIRP1), which is highly induced by gamma rays (GR) irradiation. In vitro ubiquitination assay demonstrated that a single amino acid substitution (OsGIRP1C196A) of the RING domain showed no E3 ligase activity, supporting the notion that the activity of most E3s is specified by a RING domain. We isolated at least 6 substrate proteins of OsGIRP1, including 2 Rubisco subunits, OsrbcL1 and OsrbcSl, via yeast two-hybridization and bimolecular fluorescence complementation assays. OsGIRP1 and its partner proteins were targeted to the cytosol and the cytosol or chloroplasts, respectively; however, florescence signals of the complexes with OsGIPR1 were observed in the cytosol. Protein degradation in cell extracts showed that OsGIRP1 mediates proteolysis of 2 substrates, OsrbcS1 and OsrbcL1, via the 26S proteasome degradation pathway. The Arabidopsis plants overexpressing OsGIRP1 clearly exhibited increased sensitivity to GR irradiation. These results might suggest that OsGIRP1 acts as a negative regulator of GR response to mediate the degradation of photosynthesis-related proteins.

Keywords

26S proteasome Gamma rays RING E3 ligase Rubisco subunits Ubiquitination 

Abbreviations

RING

Really interesting new gene

GR

Gamma rays

BiFC

Bimolecular fluorescence complementation

Rubisco

Ribulose biphosphate carboxylase/oxygenase

DDO/X/A

Synthetic defined medium lacking Leu and Trp supplemented with 40 μg ml−1 X-α-Gal and 70 ng ml−1 aureobasidin A (AbA)

QDO/X/A

Synthetic defined medium lacking Ade, His, Leu, and Trp with 40 μg ml−1 X-α-Gal and 70 ng ml−1 AbA

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Notes

Acknowledgments

This work was supported by the National Research Foundation of Korea Funded by the Ministry of Education, Science, and Technology (NRF-2013R1A1A4A01011064) and by grants from the Nuclear R&D Program of the Ministry of Science, ICT, and Future Planting (MSIP), Republic of Korea.

Supplementary material

425_2015_2242_MOESM1_ESM.pdf (269 kb)
Supplementary material 1 (PDF 269 kb) Fig. S1 Subcellular localization and BiFC assay of 4 OsGIRP1-interacting proteins. Four full-length genes were cloned into 35S:EYFP vector (for subcellular localization) and SPYNE(R) vector (for BiFC assay), respectively. Each construct vector was transfected into rice protoplasts. OsPPT1 (a), OsDNLZ1 (b), OsMSR1 (c), and Os4NPP1 (d) were separately transfected for subcellular localization, and transfected with OsGIRP1-SPYCE(M) for BiFC assay into rice protoplastsFig. S2 Semi-quantitative RT-PCR of OsGIRP1-overexpressing Arabidopsis and control plants. Two-week-old seedlings of each of T3 transgenic plants were used for RT-PCR. AtUBC was used for the internal controlFig. S3 The transcript levels of OsrbcS1 (a) and OsrbcL1 (b) genes in irradiated rice. Irradiated rice samples were used for quantitative RT-PCR. The cDNA was mixed with a with TOPreal™ qPCR2XPreMix with SYBR green (Enzynomics, South Korea) and 10 pmol of each of the primers. PCR was performed using the CFX96 real-Time PCR Detection System (BioRad). The transcript level was standardized on the basis of cDNA amplification with Actinll as an internal control
425_2015_2242_MOESM2_ESM.pdf (115 kb)
Supplementary material 2 (PDF 114 kb) Table S1 Primer list for cloning and semi-quantitative RT-PCR used in this study

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Yong Chan Park
    • 1
  • Jung Ju Kim
    • 1
  • Dong Sub Kim
    • 2
  • Cheol Seong Jang
    • 1
  1. 1.Plant Genomics Lab, Department of Applied Plant SciencesKangwon National UniversityChuncheonRepublic of Korea
  2. 2.Advanced Radiation Technology Institute, Korea Atomic Energy Research InstituteJeongeupSouth Korea

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