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

Abstract

<jats:title>Abstract</jats:title> <jats:p>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.</jats:p>

A procedure to introduce point mutations into the Rubisco large subunit gene in wild‐type plants

“…Understanding the role of the small subunit in plant Rubisco catalysis is an expanding area, and will be an important complement to engineering the LSu via chloroplast transformation (e.g. Ishikawa et al, 2011;Morita et al, 2014;Atkinson et al, 2017;Khumsupan et al, 2020), alongside newly developed tools for rbcL-rbcS co-engineering in the plastome (Martin-Avila et al, 2020). Reliable plastid transformation procedures for many agriculturally important crops are not yet available.…”

“…Understanding of the pervasive role, in some cases, of the small subunit (encoded by the nuclear Rbc S family) on catalysis is rapidly increasing, and despite the complexity of manipulating highly similar gene families, often involving a large number of homologs, advances are being made in this area (e.g. Khumsupan et al ., 2020; Martin‐Avila et al ., 2020). In this study, we introduced 10 restriction sites with 26 silent mutations in a modified tobacco rbc L gene ( Nt‐rbc L m ), which allowed PCR‐RFLP screening of transformants after the first round of selection.…”

CRISPR-Cas9-Mediated Mutagenesis of the Rubisco Small Subunit Family in Nicotiana tabacum

“…Genomic DNA was extracted as previously described (Khumsupan et al, 2020 ). Kanamycin-resistant plantlets were first screened by PCR using primers for spCas9 to confirm the presence of the transgene.…”

“…Eight T 0 plants with varying leaf phenotypes were transferred to soil and confirmed to contain the SpCas9 transgene by PCR using primers specific for the SpCas9 expression cassette. Four of these had visibly smaller and paler leaves than the non-transformed tissue culture control (i.e., wild-type), which is typical of Rubisco-deficient mutants (Khumsupan et al, 2020;Martin-Avila et al, 2020) (Figure 2A). The remaining four plants had a mixed pale and wild-type leaf phenotype indicative of chimeric mutations.…”

“…Although structurally distant from the active site, SSUs are known to affect the catalytic properties of Rubisco (Genkov and Spreitzer, 2009 ; Ishikawa et al, 2011 ; Esquivel et al, 2013 ; Fukayama et al, 2019 ; Orr et al, 2020 ). Arabidopsis mutants that lack up to three out of four rbcS homologs have proven useful models for the expression of non-native SSUs to examine the effect of divergent sequences on Rubisco catalysis (Izumi et al, 2012 ; Atkinson et al, 2017 ; Khumsupan et al, 2020 ). However, replacing the native SSU family remains difficult in polyploid species (i.e., most crops), which can have up to 22 rbcS homologs (e.g., wheat) and tend to produce near-identical mature SSU peptides.…”