CRISPR-based Assessment Of Gene Specialization In The Gibberellin Metabolic Pathway In Rice

Gibberellin functions as a natural regulator of critical growth and development processes in plants.However,in each step of the gibberellin metabolic pathway,different copy numbers can be found in different species,as is the case with the 13 genes across four enzymatic steps in the pathway in rice.A common view is that such gene duplication creates homologs that buffer the organism against loss of function(LOF)mutations.Therefore,knockouts of any single homolog might be expected to have little effect.To test this question,we created CRISPR/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 gibberellin pathway doesn't have a redundancy route at these enzymatic steps.In addition to lethality,we observed defects in plant height and infertility in many of these homologs,which is consistent with other known phenotypes of GA defective lines,suggesting that these duplicated members still retain functions related to GA synthesis or degradation.We identified both subfunctionalization(e.g.three recently diversified homologs of OsKO1,OsKO2 and OsKO5)and neofunctionalization(e.g.OsKO3 and OsKO4),indicating that the function of each step is conservative,whereas the evolution of duplicates in the same step is diversifying.Interestingly,the CRISPR/Cas9 lines at the SD1 locus were typically sterile,while natural sdl mutants that contributed to the "Green Revolution"in rice show normal setting rate.Collectively our results indicate candidates for future control of gibberellin and give insight into the evolution of these four critically important gene families in plants.