Abstract

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 (~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.

中文翻译：

---

在拟南芥中产生和表征 Rubisco 小亚基家族的单基因和多基因突变体。

摘要

主要的 CO ₂固定酶 Rubisco 限制了植物的生产力。Rubisco (SSU) 的小亚基可以影响 Rubisco 的整体水平和催化效率，现在作为提高 Rubisco 性能的潜在工程目标越来越受到关注。然而，SSU 是由植物中的一个核rbcS基因家族编码的，这使得它们对工程和研究具有挑战性。在这里，我们使用 CRISPR/Cas9 [成簇的规则间隔回文重复序列 (CRISPR)/CRISPR 相关蛋白 9] 和 T-DNA 插入线为拟南芥中rbcS家族的四个成员生成了一套单基因和多基因敲除突变体，包括两个新的突变体2b3b和1a2b3b. 1a2b3b含有非常低水平的 Rubisco（相对于野生型约为 3%），是第一个具有由单个 SSU 同种型 (1B) 组成的同质 Rubisco 池的突变体示例。在近室外光照水平下的生长证明了几种 SSU 突变体的 Rubisco 限制生长表型以及 1A 和 3B 同种型的重要性。我们还将1a1b确定为可能的致命突变，这表明在早期发育过程中表达最少的 1B 亚型具有关键作用。此处成功使用 CRISPR/Cas 表明这是探索 SSU 异构体在植物中的功能作用的可行方法。