期刊
JOURNAL OF EXPERIMENTAL BOTANY
卷 71, 期 19, 页码 5963-5975出版社
OXFORD UNIV PRESS
DOI: 10.1093/jxb/eraa316
关键词
Arabidopsis thaliana; chloroplast; CRISPR/Cas9; photosynthesis; protoplasts; Rubisco; SpCas9; T-DNA
资金
- UK Biotechnology and Biological Sciences Research Council [BB/I024488/1, BB/M006468/1, BB/S015531/1]
- Leverhulme Trust [RPG-2017-402]
- Darwin Trust of Edinburgh
- BBSRC [BBS/E/T/000PR9819, BB/S015531/1, BB/S015337/1, BB/I024488/1, BB/M006468/1] Funding Source: UKRI
The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (similar to 3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.
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