AUTHOR=Poulet Axel , Zhao Min , Peng Yongyan , Tham FangFei , Jaudal Mauren , Zhang Lulu , van Wolfswinkel Josien C. , Putterill Joanna 

TITLE=Gene-edited Mtsoc1 triple mutant Medicago plants do not flower

JOURNAL=Frontiers in Plant Science

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/plant-science/articles/10.3389/fpls.2024.1357924

DOI=10.3389/fpls.2024.1357924

ISSN=1664-462X

ABSTRACT=<p>Optimized flowering time is an important trait that ensures successful plant adaptation and crop productivity. <italic>SOC1-like</italic> genes encode MADS transcription factors, which are known to play important roles in flowering control in many plants. This includes the best-characterized eudicot model <italic>Arabidopsis thaliana</italic> (Arabidopsis), where <italic>SOC1</italic> promotes flowering and functions as a floral integrator gene integrating signals from different flowering-time regulatory pathways. <italic>Medicago truncatula</italic> (Medicago) is a temperate reference legume with strong genomic and genetic resources used to study flowering pathways in legumes. Interestingly, despite responding to similar floral-inductive cues of extended cold (vernalization) followed by warm long days (VLD), such as in winter annual Arabidopsis, Medicago lacks FLC and CO which are key regulators of flowering in Arabidopsis. Unlike Arabidopsis with one <italic>SOC1</italic> gene, multiple gene duplication events have given rise to three <italic>MtSOC1</italic> paralogs within the <italic>Medicago</italic> genus in legumes: one Fabaceae group A <italic>SOC1</italic> gene, <italic>MtSOC1a</italic>, and two tandemly repeated Fabaceae group B <italic>SOC1</italic> genes, <italic>MtSOC1b</italic> and <italic>MtSOC1c</italic>. Previously, we showed that <italic>MtSOC1a</italic> has unique functions in floral promotion in Medicago. The <italic>Mtsoc1a Tnt1</italic> retroelement insertion single mutant showed moderately delayed flowering in long- and short-day photoperiods, with and without prior vernalization, compared to the wild-type. In contrast, <italic>Mtsoc1b Tnt1</italic> single mutants did not have altered flowering time or flower development, indicating that it was redundant in an otherwise wild-type background. Here, we describe the generation of <italic>Mtsoc1a Mtsoc1b Mtsoc1c</italic> triple mutant lines using CRISPR-Cas9 gene editing. We studied two independent triple mutant lines that segregated plants that did not flower and were bushy under floral inductive VLD. Genotyping indicated that these non-flowering plants were homozygous for the predicted strong mutant alleles of the three <italic>MtSOC1</italic> genes. Gene expression analyses using RNA-seq and RT-qPCR indicated that these plants remained vegetative. Overall, the non-flowering triple mutants were dramatically different from the single <italic>Mtsoc1a</italic> mutant and the Arabidopsis <italic>soc1</italic> mutant; implicating multiple <italic>MtSOC1</italic> genes in critical overlapping roles in the transition to flowering in Medicago.</p>