A U-box E3 ubiquitin ligase OsPUB67 is positively involved in drought tolerance in rice

  • Qiao Qin
  • Yinxiao Wang
  • Liyu Huang
  • Fengping Du
  • Xiuqin Zhao
  • Zhikang Li
  • Wensheng WangEmail author
  • Binying FuEmail author

Key message

OsPUB67, a U-box E3 ubiquitin ligase, may interact with two drought tolerance negative regulators (OsRZFP34 and OsDIS1) and improve drought tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure.


E3 ubiquitin ligases are major components of the ubiquitination cascade and contribute to the biotic and abiotic stress response in plants. In the present study, we show that a rice drought responsive gene, OsPUB67, encoding the U-box E3 ubiquitin ligase was significantly induced by drought, salt, cold, JA, and ABA, and was expressed in nuclei, cytoplasm, and membrane systems. This distribution of expression suggests a significant role for OsPUB67 in a wide range of biological processes and abiotic stress response. Over-expression of OsPUB67 improved drought stress tolerance by enhancing the reactive oxygen scavenging ability and stomatal closure. Bimolecular fluorescence complementation assays revealed that a few E2s interacted with OsPUB67 with unique functional implications in different cell components. Further evidence showed that several E3 ubiquitin ligases interacted with OsPUB67, especially OsRZFP34 and OsDIS1, which are negative regulators of drought tolerance. This interaction on the stomata implied OsPUB67 might function as a heterodimeric ubiquitination complex in response to drought stress. Comprehensive transcriptome analysis revealed OsPUB67 participated in regulating genes involved in the abiotic stress response and transcriptional regulation in an ABA-dependent manner. Our findings revealed OsPUB67 mediated a multilayered complex drought stress tolerance mechanism.


Rice Drought tolerance U-box ubiquitin ligase ABA Stomata 



This work was supported by grants from the National Natural Science Foundation of China (Nos. 31571631 and 31501291), the CAAS Innovative Team Award to WSW and BYF, and National High-level personnel of special support program to WSW.

Author contributions

QQ performed the physiological analysis, subcellular localization assay and helped revised manuscript; YXW and LYH performed the vector construction and genetic transformation; FPD and XQZ performed RNA-seq analysis; ZKL discussed and revised this manuscript; WSW and BYF designed the experiments, wrote and revised this manuscript. All authors have participated in this research and approved the final manuscript.

Compliance with ethical standards

Conflict of interest

The authors have no conflicts of interest to declare.

Supplementary material

11103_2019_933_MOESM1_ESM.pptx (396 kb)
Supplementary material 1—Fig. S1. Phenotypes of the OsPUB67 over-expressing (A) and CRISPR/Cas9 mutant (B) transgenic lines under drought conditions at the seedling stage. A: 7 days after drought stress; B: 5 days after drought stress (PPTX 395 kb)
11103_2019_933_MOESM2_ESM.pptx (627 kb)
Supplementary material 2—Fig. S2. Basic information of the OsPUB67 over-expressing and CRISPR/Cas9 mutant transgenic plants in response to drought stress. A: The relative expression of the OsPUB67 over-expression lines and WT. B: Generation of the Ospub67-M1 mutant by CRISPR/Cas9. CRISPR/Cas9-mediated double-nucleotide deletion in the OsPUB67 target sequence led to a truncated OsPUB67 protein at the U-box domain. C and D: Phenotypes of OsPUB67 OE3, WT, and M1 plants under control and drought stress conditions. E: The survival rates of the OsPUB67 OE3, WT, and M1 plants (PPTX 626 kb)
11103_2019_933_MOESM3_ESM.xlsx (22 kb)
Supplementary material 3—Supplementary Table 1 (XLSX 21 kb)
11103_2019_933_MOESM4_ESM.xlsx (23 kb)
Supplementary material 4—Supplementary Table 2 (XLSX 22 kb)
11103_2019_933_MOESM5_ESM.xlsx (15 kb)
Supplementary material 5—Supplementary Table 3 (XLSX 15 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Qiao Qin
    • 1
  • Yinxiao Wang
    • 1
  • Liyu Huang
    • 1
    • 2
  • Fengping Du
    • 1
  • Xiuqin Zhao
    • 1
  • Zhikang Li
    • 1
    • 3
  • Wensheng Wang
    • 1
    • 3
    Email author
  • Binying Fu
    • 1
    Email author
  1. 1.Institute of Crop Sciences/National Key Facility for Crop Gene Resources and Genetic ImprovementChinese Academy of Agricultural SciencesBeijingChina
  2. 2.School of AgricultureYunnan UniversityYunnanChina
  3. 3.College of Agronomy, Anhui Agricultural UniversityHefeiChina

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