ThePhyscomitrium (Physcomitrella) patensPpKAI2L receptors for strigolactones and related compounds highlight MAX2 dependent and independent pathways

Abstract

In flowering plants, the α/β hydrolase DWARF14 (D14) perceives strigolactone (SL) hormones and interacts with the F-box protein MORE AXILLARY GROWTH2 (MAX2) to regulate developmental processes. The key SL biosynthetic enzyme, CAROTENOID CLEAVAGE DEOXYGENASE8 (CCD8), is present in the moss Physcomitrium (Physcomitrella) patens, and PpCCD8-derived compounds regulate plant extension. However, perception of these still unknown compounds does not require the PpMAX2 homolog. Putative candidate receptors are among the 13 PpKAI2LIKE-A to -L genes, homologous to the ancestral D14 paralog KARRIKIN INSENSITIVE2 (KAI2). In Arabidopsis, AtKAI2 is the receptor for karrikins and a still elusive endogenous KAI2-Ligand (KL). Based on germination assays using seeds of the parasitic plant Phelipanche ramosa, we propose that PpCCD8-derived compounds are non-canonical SLs. We show that all tested PpKAI2L proteins can bind and cleave SL analogs, some with similar affinities to AtKAI2. The PpKAI2L-H protein shows a strong hydrolytic activity not found for other PpKAI2L. Moss mutants for all PpKAI2L gene subclades were obtained and tested for their response to SL analogs. We show that PpKAI2L-A to -E genes encode redundant proteins that are not involved in PpCCD8-derived compound perception, but rather act in a PpMAX2-dependant pathway. In contrast, mutations in PpKAI2L-G, and -J genes abolish the response to the SL analog (+)-GR24, suggesting that both these encoded proteins are receptors for PpCCD8-derived molecules.

Crystal structure of Arabidopsis DWARF14‐LIKE2 (DLK2) reveals a distinct substrate binding pocket architecture

“…In a previous study, conformational differences of helix αD1 have been observed in S. hermonthica KAI2iB structures, suggesting that this helix could act as a gatekeeper for ligand entry and exit (Xu et al, 2016). In addition, a recent study has suggested that the conserved Phe28 residue in D14/KAI2 proteins may act as a gatekeeper and could be a possible explanation for the single turnover kinetics observed with some SL analogs (Lopez‐Obando et al, 2021). The recent interest of the scientific community in AlphaFold has prompted us to compare the experimental AtDLK2 structure with the in silico structure generated by the AlphaFold prediction tool.…”

Karrikin perception and signalling

“…Until very recently, it was assumed that substrate recognition and hydrolysis by KAI2 were essentially the same as for D14, but direct experimental evidence was lacking. We now know that Arabidopsis KAI2 and several homologues from P. patens form covalent adducts with a 96‐Da mass when incubated with the 2′ S ‐configured compounds GR24 ent ‐5DS and ent ‐5‐deoxystrigol (Bürger et al ., 2019; Lopez‐Obando et al ., 2021); in the latter case, these adducts were mapped to the active site histidines, as seen with D14. Even more compelling evidence has come from X‐ray crystallography studies of PsKAI2B from pea, which revealed the probable presence of 5‐hydroxy‐3‐methylbutenolide attached to the catalytic serine (Guercio et al ., 2022).…”

“…The DDK clade is much more divergent on a sequence level. It contains not only D14 and D14-LIKE2 (DLK2) homologues from angiosperms, but also one or more 'KAI2-like' sequences from mosses such as Physcomitrium patens (PpKAI2L-FK, PpKAI2L-HIL and PpKAI2L-GJM) (Lopez-Obando et al, 2021), liverworts such as Marchantia polymorpha (MpKAI2b) (Mizuno et al, 2021) and Selaginella moellendorffii (SmKAI2b) (Waters et al, 2015b) that had not resolved clearly with core KAI2 or D14 sequences in previous analyses (Waters et al, 2012;Lopez-Obando et al, 2016). In reality, many of these 'KAI2-like' DDK members are no more similar to eu-KAI2 than they are to eu-D14 (Bythell-Douglas et al, 2017).…”

“…The KAR/KL signalling pathway is found in lycophytes and bryophytes as well as angiosperms (Lopez‐Obando et al ., 2021; Mizuno et al ., 2021; Bonhomme & Guillory, 2022; Kodama et al ., 2022). In one case, a KAI2 gene from the lycophyte S. moellendorffii has been shown to partially rescue several Arabidopsis kai2 phenotypes, although it does not confer responses to KARs or other known KAI2 agonists.…”

Structural and Functional Analyses Explain Pea KAI2 Receptor Diversity and Reveal Stereoselective Catalysis During Signal Perception

“…Lines were resistant to hygromycin. The AtKAI2 native promoter (0.7 kb) was cloned into the pDONR-P4P1R vector, using Gateway recombination (Invitrogen) as described in (62).The 6xHA with linker and mCitrine tags were cloned into pDONR-P2RP3 (Invitrogen) as described in de Saint Germain et al (48). PsKAI2A.1, PsKAI2A.2 and PsKAI2B CDS were PCR amplified from Pisum cv.…”