TY - JOUR

T1 - Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis

AU - Khumsupan, Panupon

AU - Kozlowska, Marta

AU - Orr, Douglas

AU - Andreou, Andreas

AU - Nakayama, Naomi

AU - Patron, Nicola

AU - Carmo-Silva, Elizabete

AU - McCormick, Alistair

N1 - This is a pre-copy-editing, author-produced PDF of an article accepted for publication in Journal of Experimental Botany following peer review. The definitive publisher-authenticated version Panupon Khumsupan, Marta A Kozlowska, Douglas J Orr, Andreas I Andreou, Naomi Nakayama, Nicola Patron, Elizabete Carmo-Silva, Alistair J McCormick, Generating and characterising single- and multi-gene mutants of the Rubisco small subunit family in Arabidopsis, Journal of Experimental Botany, 71, (19), https://doi.org/10.1093/jxb/eraa316 is available online at: https://academic.oup.com/jxb/article/71/19/5963/5868035

PY - 2020/10/1

Y1 - 2020/10/1

N2 - 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 for by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 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 (ca. 3% relative to WT) 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 this is a viable approach for exploring the functional roles of SSU isoforms in plants.

AB - 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 for by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 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 (ca. 3% relative to WT) 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 this is a viable approach for exploring the functional roles of SSU isoforms in plants.

KW - Arabidopsis thaliana

KW - chloroplast

KW - CRISPR/Cas9

KW - photosynthesis

KW - protoplasts

KW - Rubisco

KW - SpCas9

KW - T-DNA

U2 - 10.1093/jxb/eraa316/5868035

DO - 10.1093/jxb/eraa316/5868035

M3 - Journal article

VL - 71

SP - 5963

EP - 5975

JO - Journal of Experimental Botany

JF - Journal of Experimental Botany

SN - 0022-0957

IS - 19

ER -