From perception to function, characterization of karrikin-like signaling in Lotus japonicus

Carbonnel, Samy (2019): From perception to function, characterization of karrikin-like signaling in Lotus japonicus. Dissertation, LMU München: Fakultät für Biologie

Abstract

Phytohormones are small molecules and key regulators for plant development. They translate and integrate perceived environmental cues into physiological responses. Recently, karrikins (KAR), smoke-derived compounds, were shown to trigger plant developmental responses by mimicking an unknown phytohormone called karrikin-like (KL). KAR and KL are perceived by the -hydrolase KAI2 which interacts with the F-box protein MAX2. Upon KL perception, a protein complex is formed with the repressor SMAX1, which is marked by ubiquitination for proteasomal degradation. At the beginning of this thesis, knowledge of KL function in plants was limited. Few reports in Arabidopsis showed its importance in seed germination and hypocotyl development. In rice, the discovery that the KL receptor complex is required for arbuscular mycorrhiza symbiosis (AMS) led to the question: Is KL signaling function in AMS conserved among other plant species, and particularly dicotyledons? Arabidopsis being unable to establish AMS, a new model plant was required. Thus, the goal of this thesis was to establish Lotus japonicus as a new model plant to study the role of KL signaling in plant development and AMS. To this end, L. japonicus homozygous mutant of each known KL signaling components, KAI2, MAX2, and SMAX1, were generated. In contrast to single-copy genes MAX2 and SMAX1, the KL receptor is duplicated in legumes. These two copies are functional as both rescued the elongated hypocotyl phenotype of the Arabidopsis thaliana kai2-2 mutant. However, genetic analysis of the KL perception mutants revealed that KL signaling is not required for inhibition of hypocotyl elongation in L. japonicus. However, transcriptional and developmental hypocotyl responses to the presence of KAR were dependent on only one LjKAI2 copy. Functional analysis in complemented A. thaliana kai2-2 and in-vitro binding assay demonstrated that the two LjKAI2 versions showed different affinities to ligands. Three amino-acids located in the ligand-binding cavities were shown to be determinant for ligand binding specificity. In conclusion, these results potentially indicate the presence of several KL molecules in planta to control different physiological responses through the divergent receptors. I also investigated the role of KL signaling in AMS using the L. japonicus KL receptor mutants. The level of colonization in the L. japonicus KL perception mutants was reduced to 50% compared to the wild-type level, where the two KL receptors have a redundant function. In rice, kai2 and max2 do not support colonization, whereas, in Pea, max2 mutant was less colonized than the wild-type. Recently in petunia, a kai2 mutant was shown to be impaired in AMS. Thus, the relative importance of KL signaling during AMS emerges as specific to phylogenetic-groups. Plant hormones can act in a local as well as in a systemic manner. Complementation by hairy-root transformation of max2 expressing the wild-type MAX2 showed that root colonization was only rescued only in transformed roots indicating that KL signaling is required locally for the optimum colonization. Due to the importance of KL signaling in roots for AMS, additional functions in L. japonicus root development were explored. Roots specifically responded, transcriptionally and developmentally, to KAR1 treatment in a KL perception component dependent manner. The root growth regulatory potential of KL signaling was confirmed by aberrant root phenotypes of two independent smax1 mutants. An RNAseq experiment of smax1 mutant roots revealed an increased transcript accumulation of ethylene biosynthesis genes. This increased ethylene production was shown to be causative for the root phenotypes in smax1 mutants. However, several differentially-expressed-genes were shown to be ethylene-signaling-independently-regulated and appeared as likely directly regulated by KL signaling. Thus, a member of the Ethylene Response Factor family was discovered as an early marker gene of KL signaling. Encoding a transcription factor, ERF could potentially act as a regulator for secondary KAR/KL responses. Altogether, our results illustrated that KL signaling influences root architecture development through SMAX1 removal, which acts as an inhibitor of ethylene biosynthesis. Collectively, results of this thesis open new frontiers of research on KL receptor evolution and the presence of multiple KL ligands, but also on the interaction of KL and ethylene signaling and the transcriptional cascade responding to KL/KAR. This work provides genetic tools and research axis for exciting future research using L. japonicus as a model plant to study KL signaling.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: Karrikin, Strigolactone, Ethylene, root development, Lotus japonicus
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften, Biologie
• Fakultäten: Fakultät für Biologie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 12. November 2019
• 1. Bericht­erstatter:in: Gutjahr, Caroline