Hiroshi Teramura

DELLAs Function as Coactivators of GAI-ASSOCIATED FACTOR1 in Regulation of Gibberellin Homeostasis and Signaling in Arabidopsis

This work reports the discovery of the DELLA-binding transcription factor GAF1 and shows that DELLAs and TPR act as coactivators and a corepressor with GAF1, respectively. GA converts the GAF1 complex from transcriptional activator to repressor via degradation of DELLAs. Accordingly, DELLAs turn on or off two sets of GA-regulated genes by dual functions, namely titration and coactivation. Gibberellins (GAs) are essential regulators of plant development, and DELLAs are negative regulators of GA signaling. The mechanism of GA-dependent transcription has been explained by DELLA-mediated titration of transcriptional activators and their release through the degradation of DELLAs in response to GA. However, the effect of GA on genome-wide expression is predominantly repression, suggesting the existence of unknown mechanisms of GA function. In this study, we identified an Arabidopsis thaliana DELLA binding transcription factor, GAI-ASSOCIATED FACTOR1 (GAF1). GAF1 shows high homology to INDETERMINATE DOMAIN1 (IDD1)/ENHYDROUS. GA responsiveness was decreased in the double mutant gaf1 idd1, whereas it was enhanced in a GAF1 overexpressor. GAF1 binds to genes that are subject to GA feedback regulation. Furthermore, we found that GAF1 interacts with the corepressor TOPLESS RELATED (TPR) and that DELLAs and TPR act as coactivators and a corepressor of GAF1, respectively. GA converts the GAF1 complex from transcriptional activator to repressor via the degradation of DELLAs. These results indicate that DELLAs turn on or off two sets of GA-regulated genes via dual functions, namely titration and coactivation, providing a mechanism for the integrative regulation of plant growth and GA homeostasis.

Establishment of a modified CRISPR/Cas9 system with increased mutagenesis frequency using the translational enhancer dMac3 and multiple guide RNAs in potato

CRISPR/Cas9 is a programmable nuclease composed of the Cas9 protein and a guide RNA (gRNA) molecule. To create a mutant potato, a powerful genome-editing system was required because potato has a tetraploid genome. The translational enhancer dMac3, consisting of a portion of the OsMac3 mRNA 5′-untranslated region, greatly enhanced the production of the protein encoded in the downstream ORF. To enrich the amount of Cas9, we applied the dMac3 translational enhancer to the Cas9 expression system with multiple gRNA genes. CRISPR/Cas9 systems targeting the potato granule-bound starch synthase I (GBSSI) gene examined the frequency of mutant alleles in transgenic potato plants. The efficiency of the targeted mutagenesis strongly increased when the dMac3-installed Cas9 was used. In this case, the ratio of transformants containing four mutant alleles reached approximately 25% when estimated by CAPS analysis. The mutants that exhibited targeted mutagenesis in the GBSSI gene showed characteristics of low amylose starch in their tubers. This result suggests that our system may facilitate genome-editing events in polyploid plants.