Cas9‐mediated mutagenesis of potato starch‐branching enzymes generates a range of tuber starch phenotypes

Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐me...

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Bibliographic Details
Published in:Plant biotechnology journal 2019-12, Vol.17 (12), p.2259-2271
Main Authors: Tuncel, Aytug, Corbin, Kendall R., Ahn‐Jarvis, Jennifer, Harris, Suzanne, Hawkins, Erica, Smedley, Mark A., Harwood, Wendy, Warren, Frederick J., Patron, Nicola J., Smith, Alison M.
Format: Article
Language:English
Subjects:
1,4-alpha-Glucan Branching Enzyme - genetics
Abnormalities
Amylopectin
Cas9‐mediated mutagenesis
Chain branching
Chains
CRISPR
CRISPR-Associated Protein 9
CRISPR-Cas Systems
Deoxyribonucleic acid
DNA
Enzymes
Genes
Genetic aspects
Genetic transformation
Genomes
Genomics
Genotypes
Granular materials
Granule cells
High-performance liquid chromatography
Isoforms
Liquid chromatography
Microscopy
Mutagenesis
Mutation
NMR
Nuclear magnetic resonance
Phenotype
Phenotypes
Plant Proteins - genetics
potato tuber
Potatoes
Proteins
Protoplasts
Reduction
Solanum tuberosum - enzymology
Solanum tuberosum - genetics
Starch
starch granule
starch structure
Starch-branching enzymes
Starches
starch‐branching enzyme
Synthetic biology
Tubers
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Summary:Summary We investigated whether Cas9‐mediated mutagenesis of starch‐branching enzymes (SBEs) in tetraploid potatoes could generate tuber starches with a range of distinct properties. Constructs containing the Cas9 gene and sgRNAs targeting SBE1, SBE2 or both genes were introduced by Agrobacterium‐mediated transformation or by PEG‐mediated delivery into protoplasts. Outcomes included lines with mutations in all or only some of the homoeoalleles of SBE genes and lines in which homoeoalleles carried several different mutations. DNA delivery into protoplasts resulted in mutants with no detectable Cas9 gene, suggesting the absence of foreign DNA. Selected mutants with starch granule abnormalities had reductions in tuber SBE1 and/or SBE2 protein that were broadly in line with expectations from genotype analysis. Strong reduction in both SBE isoforms created an extreme starch phenotype, as reported previously for low‐SBE potato tubers. HPLC‐SEC and 1H NMR revealed a decrease in short amylopectin chains, an increase in long chains and a large reduction in branching frequency relative to wild‐type starch. Mutants with strong reductions in SBE2 protein alone had near‐normal amylopectin chain‐length distributions and only small reductions in branching frequency. However, starch granule initiation was enormously increased: cells contained many granules of
ISSN:1467-7644
1467-7652
1467-7652
DOI:10.1111/pbi.13137