Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide
Data files
Oct 16, 2025 version files 2.30 MB
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counts_750ppm_GAHACR.txt
1.13 MB
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counts_ambient_GAHACR.txt
1.17 MB
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README.md
3.38 KB
Abstract
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.
https://doi.org/10.5061/dryad.547d7wmgr
Description of the data and file structure
Dataset overview:
Counts tables for differential gene expression performed in the analysis of 'reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide' are provided for RNA-Seq experiments performed on Arabidopsis seedlings. We performed these at both Ambient and elevated (750ppm) carbon dioxide levels, with and without the GAHACR genetic perturbation, using GAHACRs to modulate the strength of feedback on endemic GA regulated genes, and to directly test the importance of transcriptional feedback in GA signaling. In order to facilitate future researchers to most easily incorporate our RNA-seq results, we have shared the count tables generated from our experiments.
Files and variables
Description of the data and file structure
In general, all genotypes within these files will follow the same naming convention, where the parental GAHACR lacking any additional guide RNA is referenced as its plant number: 3943. This is used as the baseline for control in all experiments. The two lines carrying the four guide RNAs to retarget the dCAS9-TPLN repressor are labeled as 4578 and 4582. Individual biological replicates of the experiments are denoted with an underscore and a number, such as "_1", etc. Therefore, the three biological replicates for the parental are denoted as: "3943_1, 3943_2, 3943_3".
- counts_ambient_GAHACR.txt
This file contains the raw counts from the HTSeq (htseq-count) function mapped to the Arabidopsis TAIR10 genome annotation. These samples were grown at ambient carbon dioxide levels (400-450ppm depending on fluctuations). The file has the following columns: AGI, 3943_1, 3943_2, 3943_3, 4578_1, 4578_2, 4578_3, 4582_1, 4582_2, 4582_3. The AGI term refers to the TAIR10 gene name. The columns are as described above, with three biological replicates for each of the genotypes.
- counts_750ppm_GAHACR.txt
This file contains the raw counts from the HTSeq (htseq-count) function mapped to the Arabidopsis TAIR10 genome annotation. These samples were grown at elevated carbon dioxide levels (750ppm supplemented into the chamber). The file has the following columns: AGI, 3943_1, 3943_2, 3943_3, 4578_1, 4578_2, 4578_3, 4582_1, 4582_2, 4582_3. The AGI term refers to the TAIR10 gene name. The columns are as described above, with three biological replicates for each of the genotypes.
Code/software
No specific code or software is required; these data files can be used to perform differential gene expression analysis in your program of choice, such as DESeq2 or EdgeR-type differential expression callers.
Access information
Other publicly accessible locations of the data:
- All code utilized to analyze this data is made publicly available at GitHub: https://github.com/achillobator/GAHACR
Data was derived from the following sources:
- All sequencing was performed at Amaryllis Nucleics by Mike Covington, and count tables were generated by their in-house pipeline.
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).
- Leydon, Alexander R; Flores, Leonel; Khakhar, Arjun; Nemhauser, Jennifer L (2025). Reprogramming feedback strength in gibberellin biosynthesis highlights conditional regulation by the circadian clock and carbon dioxide [Preprint]. Cold Spring Harbor Laboratory. https://doi.org/10.1101/2025.03.18.644045