Knockout of OsPRP1, a gene encoding proline-rich protein, confers enhanced cold sensitivity in rice (Oryza sativa L.) at the seedling stage
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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.
KeywordsCold 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.
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.
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.
- Aebi H (1984) Catalase in vitro. In: Methods in enzymology, vol 105. Academic Press, pp 121–126Google Scholar
- Forcat S, Bennett MH, Mansfield JW, Grant MR (2008) A rapid and robust method for simultaneously measuring changes in the phytohormones ABA, JA and SA in plants following biotic and abiotic stress. Plant Methods 4(1):16. https://doi.org/10.1186/1746-4811-4-16 CrossRefPubMedPubMedCentralGoogle Scholar
- Fowler TJ, Bernhardt C, Tierney ML (1999) Characterization and expression of four proline-rich cell wall protein genes in Arabidopsis encoding two distinct subsets of multiple domain proteins. Plant Physiol 121(4):1081–1091. https://doi.org/10.1104/pp.121.4.1081 CrossRefPubMedPubMedCentralGoogle Scholar
- Gupta N, Susa K, Yoda Y, Bonventre JV, Valerius MT, Morizane R (2018) CRISPR/Cas9-based targeted genome editing for the development of monogenic diseases models with human pluripotent stem cells. Curr Protoc Stem Cell Biol 45(1):e50. https://doi.org/10.1002/cpsc.50 CrossRefPubMedPubMedCentralGoogle Scholar
- Han Y, Luo D, Usman B, Nawaz G, Zhao N, Liu F, Li R (2018) Development of high yielding glutinous cytoplasmic male sterile rice (Oryza sativa L.) lines through CRISPR/Cas9 based mutagenesis of Wx and TGW6 and proteomic analysis of anther. Agronomy 8:290. https://doi.org/10.3390/agronomy8120290 CrossRefGoogle Scholar
- Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6(2):271–282. https://doi.org/10.1046/j.1365-313x.1994.6020271.x CrossRefPubMedGoogle Scholar
- Huang J, Sun SJ, Xu DQ, Yang X, Bao YM, Wang ZF, Tang HJ, Zhang H (2009) Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245. Biochem Biophys Res Commun 389(3):556–561. https://doi.org/10.1016/j.bbrc.2009.09.032 CrossRefPubMedGoogle Scholar
- Kanneganti V, Gupta AK (2008) Overexpression of OsiSAP8, a member of stress associated protein (SAP) gene family of rice confers tolerance to salt, drought and cold stress in transgenic tobacco and rice. Plant Mol Biol 66(5):445–462. https://doi.org/10.1007/s11103-007-9284-2 CrossRefPubMedGoogle Scholar
- Poire R, Wiese-Klinkenberg A, Parent B, Mielewczik M, Schurr U, Tardieu F, Walter A (2010) Diel time-courses of leaf growth in monocot and dicot species: endogenous rhythms and temperature effects. J Exp Bot 61(6):1751–1759. https://doi.org/10.1093/jxb/erq049 CrossRefPubMedPubMedCentralGoogle Scholar
- Quarrie SA, Whitford PN, Appleford NE, Wang TL, Cook SK, Henson IE, Loveys BR (1988) A monoclonal antibody to (S)-abscisic acid: its characterisation and use in a radioimmunoassay for measuring abscisic acid in crude extracts of cereal and lupin leaves. Planta 173(3):330–339. https://doi.org/10.1007/bf00401020 CrossRefPubMedGoogle Scholar
- Venekamp JH (1989) Regulation of cytosol acidity in plants under conditions of drought. Physiol Plant 76(1):112–117. https://doi.org/10.1111/j.1399-3054.1989.tb05461.x CrossRefGoogle Scholar
- Xu RF, Li H, Qin RY, Li J, Qiu CH, Yang YC, Ma H, Li L, Wei PC, Yang JB (2015) Generation of inheritable and “transgene clean” targeted genome-modified rice in later generations using the CRISPR/Cas9 system. Sci Rep 5:11491. https://doi.org/10.1038/srep11491 CrossRefPubMedPubMedCentralGoogle Scholar