3 Biotech

, 9:254 | Cite as

Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage

  • Gul Nawaz
  • Yue Han
  • Babar Usman
  • Fang Liu
  • Baoxiang Qin
  • Rongbai LiEmail author
Original Article


Proline-rich proteins (PRPs) play multiple physiological and biochemical roles in plant growth and stress response. In this study, we reported that the knockout of OsPRP1 induced cold sensitivity in rice. Mutant plants were generated by CRISPR/Cas9 technology to investigate the role of OsPRP1 in cold stress and 26 mutant plants were obtained in T0 generation with the mutation rate of 85% including 15% bi-allelic, 53.3% homozygous, and 16.7% heterozygous and 16 T-DNA-free lines in T1 generation. The conserved amino acid sequence was changed and the expression level of OsPRP1 was reduced in mutant plants. The OsPRP1 mutant plants displayed more sensitivity to cold stress and showed low survival rate with decreased root biomass than wild-type (WT) and homozygous mutant line with large fragment deletion was more sensitive to low temperature. Mutant lines accumulated less antioxidant enzyme activity and lower levels of proline, chlorophyll, abscisic acid (ABA), and ascorbic acid (AsA) content relative to WT under low-temperature stress. The changes of antioxidant enzymes were examined in the leaves and roots with exogenous salicylic acid (SA) treatment which resulted in increased activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) under cold stress, while enzyme antioxidant activity was lower in untreated seedlings which showed that exogenous SA pretreatment could alleviate the low-temperature stress in rice. Furthermore, the expression of three genes encoding antioxidant enzyme activities (SOD4, POX1, and OsCAT3) was significantly down-regulated in the mutant lines as compared to WT. These results suggested that OsPRP1 enhances cold tolerance by modulating antioxidants and maintaining cross talk through signaling pathways. Therefore, OsPRP1 gene could be exploited for improving cold tolerance in rice and CRISPR/Cas9 technology is helpful to study the function of a gene by analyzing the phenotypes of knockout mutants generated.


Cold Stress CRISPR/Cas9 Mutation Rice OsPRP1 Proline 



We would like to thank Prof. Liu Yaoguang from South China Agricultural University, who provided us pYCRISPR/Cas9 and gRNA vectors (pYL-U6a-b-gRNAs). We would also like to thank Mr. Mohsin Niaz and Mr. Zhao Neng for the helpful discussion and invaluable comments to make this research meaningful.

Author contributions

GN and YH conceived, designed, and performed the experiments. BU was responsible for vector construction and wrote the paper. GN and BU were responsible for final data analysis and wrote the final draft. BQ and FL participated in the experimental design, result analysis, and field trials. RL visualized the project, supervised the methodology, given feedback on data presentation, and reviewed the final draft. All the authors have read the manuscript and approved the submission.


This research was funded by the Key Technology Research and Development Program Guike, Guangxi (Guike AB16380066; Guike AB16380093).

Compliance with ethical standards

Conflict of interest

The authors declared no conflict of interest.

Accession numbers

Sequence data from this article can be found in the GenBank data library under accession numbers GenBank: KR029105 and KR029107 for the sgRNA intermediate plasmid and GenBank: KR029109 for the CRISPR/Cas9 binary vector.

Supplementary material

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Supplementary material 1 (DOCX 406 kb)
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Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  • Gul Nawaz
    • 1
  • Yue Han
    • 1
  • Babar Usman
    • 1
  • Fang Liu
    • 1
  • Baoxiang Qin
    • 1
  • Rongbai Li
    • 1
    Email author
  1. 1.College of Agriculture, State Key Laboratory for Conservation and Utilization of Subtropical Agro-BioresourcesGuangxi UniversityNanningChina

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