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CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis

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Induced mutations in the waxy locus in rice endosperm did not abolish GBSS activity completely. Compensatory mechanisms in endosperm and leaves caused a major reprogramming of the starch biosynthetic machinery.

Abstract

The mutation of genes in the starch biosynthesis pathway has a profound effect on starch quality and quantity and is an important target for plant breeders. Mutations in endosperm starch biosynthetic genes may impact starch metabolism in vegetative tissues such as leaves in unexpected ways due to the complex feedback mechanisms regulating the pathway. Surprisingly this aspect of global starch metabolism has received little attention. We used CRISPR/Cas9 to introduce mutations affecting the Waxy (Wx) locus encoding granule-bound starch synthase I (GBSSI) in rice endosperm. Our specific objective was to develop a mechanistic understanding of how the endogenous starch biosynthetic machinery might be affected at the transcriptional level following the targeted knock out of GBSSI in the endosperm. We found that the mutations reduced but did not abolish GBSS activity in seeds due to partial compensation caused by the upregulation of GBSSII. The GBSS activity in the mutants was 61–71% of wild-type levels, similarly to two irradiation mutants, but the amylose content declined to 8–12% in heterozygous seeds and to as low as 5% in homozygous seeds, accompanied by abnormal cellular organization in the aleurone layer and amorphous starch grain structures. Expression of many other starch biosynthetic genes was modulated in seeds and leaves. This modulation of gene expression resulted in changes in AGPase and sucrose synthase activity that explained the corresponding levels of starch and soluble sugars.

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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 rice codon-optimized cas9, and the empty pU3-gRNA vector. We thank Professor Kimiko Itoh, Graduate School of Science and Technology (Niigata University, Japan) for providing the KUR and Musa Wx lines. 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 contributed equally to this work.

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, Gemma Farré, Julia Juanos, Ludovic Bassie, Vicente Medina, Ignacio Romagosa, Pilar Muñoz, Changfu Zhu & Paul Christou

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

    Erika Soto & Gemma Villorbina

  3. Department of Biochemistry, Molecular and Cellular Plant Biology, Experimental Station of the Zaidín, Superior Council of Scientific Investigations (CSIC), Granada, Spain

    Antonio Jesús Serrato, Mariam Sahrawy & Jose Antonio Rojas

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

    Paul Christou

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  1. Lucía Pérez
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Pérez, L., Soto, E., Farré, G. et al. CRISPR/Cas9 mutations in the rice Waxy/GBSSI gene induce allele-specific and zygosity-dependent feedback effects on endosperm starch biosynthesis. Plant Cell Rep 38, 417–433 (2019). https://doi.org/10.1007/s00299-019-02388-z

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