In the model plant Arabidopsis thaliana (Arabidopsis), where the gibberellin (GA) signaling pathway has been best studied, GAs promote flowering. However, in many woody perennial plants such as apple (Malus x domestica Borkh.), GAs generally have a repressive role in flowering. The genetic and molecular mechanisms of GA-mediated flowering repression in apple and other plants remain unknown. In this study, we carried out transcriptional profiling of apple shoot apices during the anticipated period of floral induction and examined how gene regulation is influenced by exogenous GAs. GA treatment resulted in downregulation of a diversity of genes participating in GA biosynthesis, transport and reception, and strong upregulation of the GA catabolic GA2-ox (GA2-oxidase) genes, suggesting GA homeostasis is mediated at multiple levels in these tissues. We also observed rapid and strong upregulation of both of two copies of a gene previously observed to inhibit flowering in apple, MdTFL1 (TERMINAL FLOWER 1), offering a potential explanation for the flowering-inhibitory effects of GA in apple. These results provide a context for investigating factors that may transduce the GA signal in apple and contribute to a preliminary genetic framework for the repression of flowering by GAs in a woody perennial plant. GA2ox expression and the enzyme activity determines concentrations of specific GAs in the right structure at the right time and contributes to enabling GAs to execute their function precisely. In return, their expression is under tight regulation by GA. Taking advantage of this tight link between GA2ox and GA activity, we aimed to develop regulation of GA2ox as a model to evaluate GA activity and function in both apple and Arabidopsis with a focus on floral induction. Through analysis of high-quality apple genomes and our transcriptional datasets, we identified 16 canonical GA2ox-like genes in the apple genome, representing eight pairs of homoeologous genes. By analyzing their spatial and developmental expression, we found that at least half of the genes were expressed in all the structures studied. In addition, individual members of homoeologous pairs displayed both overlapping and distinct expression patterns, suggesting function redundance and divergency. Finally, we found that two pairs of homoeologous genes (MdGA2ox1A, -1B, -2A and -2B) may be responsible for regulating GA activity in the shoot apex during floral induction. These findings provide biological and evolutionary insights into GA biology in apple, particularly in floral induction. In Arabidopsis, where GA2ox has been relatively well studied, we developed a genetic tool kit comprising both CRISPR/Cas-induced, transgene-free single knockout mutants and transcriptional and translational GUS fusion lines for the seven canonical Arabidopsis GA2ox genes (AtGA2ox1-AtGA2ox8). Mutations in the loss-of-function mutants have presumably disrupted function of both two functional domains of the GA2ox proteins. By comparing GUS activity in the GUS fusion lines, we found that the transcribed region is required for appropriate expression for several genes. This tool kit can be used for a wide spectrum of GA biology.
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