Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide

The phytohormone gibberellin (GA) is an important regulator of plant morphology and reproduction, and the biosynthesis and distribution of GA in planta is agriculturally relevant to past and current breeding efforts. Tools like biosensors, extensive molecular genetic resources in reference plants and mathematical models have greatly contributed to current understanding of GA homeostasis; however, these tools are difficult to tune or repurpose for engineering crop plants. Previously, we showed that a GA-regulated Hormone Activated CAS9-based Repressor (GAHACR) functions in planta (Khakhar et al., 2018). Here, we use GAHACRs to modulate the strength of feedback on endemic GA regulated genes, and to directly test the importance of transcriptional feedback in GA signaling. We first adapted existing mathematical models to predict the impact of targeting a GAHACR to different nodes in the GA biosynthesis pathway, and then implemented a perturbation predicted by the model to lower GA levels. Specifically, we individually targeted either the biosynthetic gene GA20 oxidase (GA20ox) or the GA receptor GID1, and characterized primary root length, flowering time and the transcriptome of these transgenic lines. Using this approach, we identified a strong connection between GA signaling status and the circadian clock, which can be largely attenuated by elevated carbon dioxide levels. Our results identify a node in the GA signaling pathway that can be engineered to modulate plant size and flowering time. Our results also raise concerns that rising atmospheric CO2 concentration are likely to reverse many of the gains of Green Revolution crops.

Methods
RNA-Seq

For ambient carbon dioxide concentrations, T4 seedlings were cultured on plates for 4 days without drug selection, and then transplanted to LS0 plates without drug for 10 days in either ambient or carbon dioxide-supplemented Conviron (Pembina, North Dakota) growth chambers. RNA was extracted using the Illustra RNAspin Mini Kit from GE, and RNA-Seq was performed by Amaryllis Nucleics (Oakland CA). In brief, RNA was checked for quality and quantity on a Bioanalyzer, and poly(A) mRNA was purified from total RNA. The library was constructed using the Amaryllis proprietary construction kit, followed by QC by E-gel & Bioanalyzer. Libraries were pooled and run on an Illumina NextSeq 500 SR75, with a target of approximately 20 million reads per sample. Read preprocessing, mapping, and differential gene expression were performed by Amaryllis, in addition to independent differential gene expression analysis in-house using EdgeR and DESeq2. Network analysis was performed in cytoscape. GO terminology enrichment was calculated using g:profiler (biit.cs.ut.ee/gprofiler/gost) and plotted in R using ggplot2 (see code deposited at https://github.com/achillobator/GAHACR).