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
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.
KeywordsCRISPR/Cas9 AGPase large subunit gene Rice Ectopic expression Monoallelic mutations
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).
- Farré G, Sudhakar D, Naqvi S, Sandmann G, Christou P, Capell T, Zhu C (2012) Transgenic rice grains expressing a heterologous ρ-hydroxyphenylpyruvate dioxygenase shift tocopherol synthesis from the γ to the α isoform without increasing absolute tocopherol levels. Transgenic Res 21:1093–1097CrossRefPubMedGoogle Scholar
- Johnson PE, Patron NJ, Bottrill AR et al (2003) A low-starch barley mutant, risø 16, lacking the cytosolic small subunit of ADP-glucose pyrophosphorylase, reveals the importance of the cytosolic isoform and the identity of the plastidial small subunit. Plant Physiol 131:684–696CrossRefPubMedPubMedCentralGoogle Scholar
- Preiss J (1994) Regulation of the C3 reductive cycle and carbohydrate synthesis. In: Tolbert NE, Preiss J (eds) Regulation of atmospheric CO2 and O2 by photosynthetic carbon metabolism, 1st edn. Oxford University Press, New York, pp 93–102Google Scholar
- Rychter AM, Rao IM (2005) Role of phosphorus in photosynthetic carbon metabolism. In: Pessarakli M (ed) Handbook of photosynthesis, 2nd edn. Taylor y Francis group, Tucson, pp 123–148Google Scholar
- Steup M (1990) Starch degrading enzymes. In: Dey PM, Harborne JB (eds) Methods in plant biochemistry. Academic Press, London, pp 103–128Google Scholar
- Valdez M, Cabrera-Ponce JL, Sudhakar D, Herrera-Estrella L, Christou P (1998) Transgenic central american, west african and asian elite rice varieties resulting from particle bombardment of foreign DNA into mature seed-derived explants utilizing three different bombardment devices. Annu Bot 82:795–801CrossRefGoogle Scholar
- Yoshida S, Forno DA, Cock JH, Gomez KA (1976) Determination of sugar and starch in plant tissue, 3erd edition. Laboratory manual for physiological studies of rice. The international rice research institute, Laguna Philippines, pp 46–49Google Scholar