Abstract
Main conclusion
Induced mutations in the SC-uORF of the tomato transcription factor gene SlbZIP1 by the CRISPR/Cas9 system led to the high accumulation of sugar and amino acid contents in tomato fruits.
Abstract
Tomato (Solanum lycopersicum) is one of the most popular and consumed vegetable crops in the world. Among important traits for tomato improvement such as yield, biotic and abiotic resistances, appearance, post-harvest shelf life and fruit quality, the last one seems to face more challenges because of its genetic and biochemical complexities. In this study, a dual-gRNAs CRISPR/Cas9 system was developed to induce targeted mutations in uORF regions of the SlbZIP1, a gene involved in the sucrose-induced repression of translation (SIRT) mechanism. Different induced mutations in the SlbZIP1-uORF region were identified at the T0 generation, stably transferred to the offspring, and no mutation was found at potential off-target sites. The induced mutations in the SlbZIP1-uORF region affected the transcription of SlbZIP1 and related genes in sugar and amino acid biosynthesis. Fruit component analysis showed significant increases in soluble solid, sugar and total amino acid contents in all SlbZIP1-uORF mutant lines. The accumulation of sour-tasting amino acids, including aspartic and glutamic acids, raised from 77 to 144%, while the accumulation of sweet-tasting amino acids such as alanine, glycine, proline, serine, and threonine increased from 14 to 107% in the mutant plants. Importantly, the potential SlbZIP1-uORF mutant lines with desirable fruit traits and no impaired effect on plant phenotype, growth and development were identified under the growth chamber condition. Our result indicates the potential utility of the CRISPR/Cas9 system for fruit quality improvement in tomato and other important crops.
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Abbreviations
- bZIP :
-
Basic leucine zipper
- CRISPR:
-
Clustered regularly interspaced short palindromic repeats
- gRNA:
-
Guide RNA
- sgRNA:
-
Single-guide RNA
- MS:
-
Murashige and Skoog medium
- uORF:
-
Upstream open reading frame
- SC-ORF:
-
Sucrose control open reading frame
- SC-uORF:
-
Sucrose-controlled upstream open reading frame
- SIRT:
-
Sucrose-induced repression of translation
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Acknowledgements
The authors thank Dr. Van Tuong Nguyen for great suggestion and proofreading on the manuscript.
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This work was fund by the Vietnam Academy of Science and Technology (ĐLTE00.10/20-21).
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Supplementary Information
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425_2023_4089_MOESM1_ESM.jpg
Supplementary file1 The multiple alignment in SlbZIP1-uORF regions. WT, wild-type plants; 9.1a, 14.1, and 15.2, T1 generation of mutant lines. Red letters indicate the PAM sites, blue underlined letters indicate the single guide RNA (sgRNA) sequences, blue rectangle border indicates the uORF1, the yellow highlight indicates the uORF2, purple rectangle border indicates the uORF3, and dark orange rectangle border indicates the uORF4 (JPEG 1022 KB)
425_2023_4089_MOESM2_ESM.jpg
Supplementary file2 The procedure of Agrobacterium mediated transformation in tomato using cultivar PT18. a Infected cotyledon segments on the co-cultivation medium. b Shoot induction on selection medium containing 50 mg/L kanamycin. c Shoot development on regeneration medium with 50 mg/L kanamycin. d Regenerated plants on rooting medium added 50 mg/L kanamycin. e Transgenic plants under the growth chamber (JPEG 637 KB)
425_2023_4089_MOESM3_ESM.jpg
Supplementary file3 Potential off-target analysis. a The SlbZIP2 gene map. b Sequencing of the off-target site from tested tomato mutant lines. WT, wild-type plants; 9.1a, 14.1, and 15.2, T1 generation of mutant lines. Red letters indicate the potential off-target sequences, bold underlined letters indicate the PAM site, and the yellow highline indicates the start codon of SlbZIP2-Morf (JPEG 257 KB)
425_2023_4089_MOESM4_ESM.jpg
Supplementary file4 Tomato ripe fruit characterization. The whole (above) and cross sections (below) of the ripe fruits (JPEG 189 KB)
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Nguyen, N.H., Bui, T.P., Le, N.T. et al. Disrupting Sc-uORFs of a transcription factor bZIP1 using CRISPR/Cas9 enhances sugar and amino acid contents in tomato (Solanum lycopersicum). Planta 257, 57 (2023). https://doi.org/10.1007/s00425-023-04089-0
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DOI: https://doi.org/10.1007/s00425-023-04089-0