Truncation of LEAFY COTYLEDON1 protein is required for asexual reproduction in Kalanchoë daigremontiana.

Kalanchoë daigremontiana reproduces asexually by generating numerous plantlets on its leaf margins. The formation of plantlets requires the somatic initiation of organogenic and embryogenic developmental programs in the leaves. However, unlike normal embryogenesis in seeds, leaf somatic embryogenesis bypasses seed dormancy to form viable plantlets. In Arabidopsis (Arabidopsis thaliana), seed dormancy and embryogenesis are initiated by the transcription factor LEAFY COTYLEDON1 (LEC1). The K. daigremontiana ortholog of LEC1 is expressed during leaf somatic embryo development. However, KdLEC1 encodes for a LEC1-type protein that has a unique B domain, with 11 unique amino acids and a premature stop codon. Moreover, the truncated KdLEC1 protein is not functional in Arabidopsis. Here, we show that K. daigremontiana transgenic plants expressing a functional, chimeric KdLEC1 gene under the control of Arabidopsis LEC1 promoter caused several developmental defects to leaf somatic embryos, including seed dormancy characteristics. The dormant plantlets also behaved as typical dormant seeds. Transgenic plantlets accumulated oil bodies and responded to the abscisic acid biosynthesis inhibitor fluridone, which broke somatic-embryo dormancy and promoted their normal development. Our results indicate that having a mutated form of LEC1 gene in K. daigremontiana is essential to bypass dormancy in the leaf embryos and generate viable plantlets, suggesting that the loss of a functional LEC1 promotes viviparous leaf somatic embryos and thus enhances vegetative propagation in K. daigremontiana. Mutations resulting in truncated LEC1 proteins may have been of a selective advantage in creating somatic propagules, because such mutations occurred independently in several Kalanchoë species, which form plantlets constitutively.

Evolution of asexual reproduction in leaves of the genus Kalanchoë.

Plant somatic cells have the remarkable ability to regenerate an entire organism. Many species in the genus Kalanchoë, known as "mother of thousands," develop plantlets on the leaf margins. Using key regulators of organogenesis (STM) and embryogenesis (LEC1 and FUS3) processes, we analyzed asexual reproduction in Kalanchoë leaves. Suppression of STM abolished the ability to make plantlets. Here, we report that constitutive plantlet-forming species, like Kalanchoë daigremontiana, form plantlets by coopting both organogenesis and embryogenesis programs into leaves. These species have a defective LEC1 gene and produce nonviable seed, whereas species that produce plantlets only upon stress induction have an intact LEC1 gene and produce viable seed. The latter species are basal in the genus, suggesting that induced-plantlet formation and seed viability are ancestral traits. We provide evidence that asexual reproduction likely initiated as a process of organogenesis and then recruited an embryogenesis program into the leaves in response to loss of sexual reproduction within this genus.