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The Characterization of Arabidopsis Photorespiration D-glycerate 3-Kinase Mutants Generated by CRISPR/Cas9 and Identification of Its Interacting Proteins

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Abstract

D-Glycerate 3-kinase (GLYK, EC2.7.1.31) catalyzes the phosphorylation of glycerate to form 3-phosphoglycerate. However, knowledge about GLYK regulation is limited. In this study, the Arabidopsis Ler background glyk mutants were generated using CRISPR/Cas9 technology to elucidate the genetic regulation mechanisms of GLYK in future. Specifically, two sgRNAs targeting the Arabidopsis GLYK genomic sequence (At1g80380) were constructed into egg cell-specific expression CRISPR/Cas9 vector, and Ler plants were transformed by the floral dip method. A high-throughput screening and sequencing combined PCR (SSC-PCR) was developed to identify mutants generated by CRISPR/Cas9 technology using the DNA of T1 positive transgenic lines. By using this method, the edited GLYK lines were efficiently screened out from T1-positive transgenic lines. Thereafter, two independent homozygous lines, glyk-1 and glyk-3, were selected and further confirmed their genetic stability and absence of off-targets. The glyk-1 and glyk-3 showed typical photorespiration phenotype, and the chlorophyll content and Fv/Fm ratio of glyk-1 and glyk-3 gradually decreased when they were moved from 3000 ppm CO2 to 400 ppm CO2. Finally, these two mutants became lethal after nine-day exposure to 400 ppm CO2. Furthermore, six proteins have been screened out for their potential interactions with AtGLYK using yeast two-hybrid system in a previous study, while in this study, only two proteins, At1g78380 (AtGSTU19) and At4g37980 (AtCAD7), were further confirmed their interactions with AtGLYK using BiFC and LCI approaches, respectively. In summary, the developed SSC-PCR method here can be applied for the quick screening and identification of CRISPR/Cas9-edited mutants in plants; the glyk mutants and the GLYK-interacting proteins reported in this study will facilitate future understanding of the genetic regulation mechanisms of AtGLYK.

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Data Availability

We declared that all the materials and data reported in this article would be available upon request.

Code Availability

Not applicable.

Abbreviations

BiFC:

Bimolecular fluorescence complementation

Cas9:

CRISPR-associated protein 9

ORF:

Open reading frame

CoIP:

Co-immunoprecipitation

CRISPR:

Clustered regularly interspaced short palindromic repeats

GLYK:

D-Glycerate 3-kinase

LCI:

Luciferase complementation imaging

NHEJ:

Non-homologous end-joining

PAM:

Protospacer adjacent motif

2-PG:

2-Phosphoglycolate

3-PGA:

3-Phosphoglycerate

ppm:

Parts per million

RuBP:

Ribulose-1, 5-disphosphate

sgRNA:

Single guide RNA

SSC-PCR:

Screening and sequencing combined PCR

TALENs:

Transcription activator-like effector nucleases

ZFNs:

Zinc finger nucleases

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Acknowledgements

We thank Dr Qijun Chen (China Agricultural University, Beijing) for kindly providing the CRISPR/Cas9 vector system.

Funding

This research was sponsored by the Science and Technology Funding of Guangzhou (Grant No. 201707010032).

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Authors and Affiliations

  1. Center for Photosynthesis and Plant Stress Biology, College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China

    Yingjie Li, Zhengshiyu Lai, Qi Zhong, Feng Wu, Shuhan Yang, Biying Xie, Yawen Li, Wenting Sun, Xinxiang Peng & Xuewen Hou

  2. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, College of Life Sciences, South China Agricultural University, Guangzhou, 510642, China

    Xinxiang Peng & Xuewen Hou

  3. Departments of Animal Biosciences, University of Guelph, Guelph, ON, N1G2W1, Canada

    Weijun Wang

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  1. Yingjie Li
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  2. Zhengshiyu Lai
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  4. Qi Zhong
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Contributions

XH and XP: designed and supervised the research. YL, ZL, QZ, FW, SY, BX, YL, and WS: conducted the experiments. YL and ZL: analyzed the data. XH: wrote the manuscript. XH and WW: finalized the manuscript. All authors read and approved the final version of the manuscript.

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Correspondence to Xuewen Hou.

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Li, Y., Lai, Z., Wang, W. et al. The Characterization of Arabidopsis Photorespiration D-glycerate 3-Kinase Mutants Generated by CRISPR/Cas9 and Identification of Its Interacting Proteins. J Plant Growth Regul 42, 2458–2473 (2023). https://doi.org/10.1007/s00344-022-10716-z

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