CRISPR-Based Assessment of Gene Specialization in the Gibberellin Metabolic Pathway in Rice

Gibberellin (GA) functions as an essential natural regulator of growth and development in plants. For each step of the GA metabolic pathway, different copy numbers can be found in different species, as is the case with the 13 genes across four enzymatic steps in rice (Oryza sativa). A common view is that such gene duplication creates homologs that buffer organisms against loss-of-function (LOF) mutations. Therefore, knockouts of any single homolog might be expected to have little effect. To test this question, we generated clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) knockouts for these homologs and measured effects on growth and reproduction. Surprisingly, we report here that there is consistently one or more essential gene at each enzymatic step, for which LOF mutation induces death or sterility-suggesting that the GA pathway does not have a redundancy route and that each gene family is essential for GA metabolism. In most of these genes from the same gene family, we observed defects in plant height and infertility, suggesting that the duplicated members retain functions related to GA synthesis or degradation. We identified both subfunctionalization of the three recently diversified homologs OsKO1, OsKO2, and OsKO5 and neofunctionalization in OsKO3 and OsKO4 Thus, although the function of each step is conserved, the evolution of duplicates in that step is diversified. Interestingly, the CRISPR/Cas9 lines at the SD1 locus were typically sterile, whereas the natural sd1 mutants, related to the "Green Revolution" in rice, show normal setting rates. Collectively, our results identify candidates for control of GA production and provide insight into the evolution of four critical gene families in plants.