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CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the corresponding small subunit gene APS2b in rice leaves

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Abstract

The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.

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Acknowledgements

We thank Dr. Caixia Gao (Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China) for providing pJIT163-2NLSCas9 containing the cas9 gene codon-optimized for rice, and the empty pU3-gRNA vector for the introduction of sgRNAs into rice. This work was supported by funding from the Spanish Ministry of Economy and Competitiveness (MINECO) (BIO2014-54426), and a Juan de la Cierva fellowship to GF (IJCI- 2014-19528). LP is the recipient of MINECO fellowship. ES is the recipient of a PhD fellowship from the University of Lleida (BIO2014-54441-P).

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  1. Lucía Pérez and Erika Soto have contributed equally to the study.

Authors and Affiliations

  1. Department of Plant Production and Forestry Science, School of Agrifood and Forestry Science and Engineering (ETSEA), University of Lleida-Agrotecnio Center, Av. Alcalde Rovira Roure 191, 25198, Lleida, Spain

    Lucía Pérez, Ludovic Bassie, Vicente Medina, Pilar Muñoz, Teresa Capell, Changfu Zhu, Paul Christou & Gemma Farré

  2. Department of Chemistry, University of Lleida-Agrotecnio Center, Lleida, Spain

    Erika Soto & Gemma Villorbina

  3. Catalan Institute for Research and Advanced Studies (ICREA), Barcelona, Spain

    Paul Christou

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  1. Lucía Pérez
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  3. Gemma Villorbina
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Correspondence to Paul Christou or Gemma Farré.

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Pérez, L., Soto, E., Villorbina, G. et al. CRISPR/Cas9-induced monoallelic mutations in the cytosolic AGPase large subunit gene APL2 induce the ectopic expression of APL2 and the corresponding small subunit gene APS2b in rice leaves. Transgenic Res 27, 423–439 (2018). https://doi.org/10.1007/s11248-018-0089-7

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