Reporter Gene-Facilitated Detection of Compounds in Arabidopsis Leaf Extracts that Activate the Karrikin Signaling Pathway.

Yueming K Sun,Steven M Smith,Gavin R Flematti,Mark T Waters

doi:10.3389/fpls.2016.01799

Abstract

Karrikins are potent germination stimulants generated by the combustion of plant matter. Treatment of Arabidopsis with karrikins triggers a signaling process that is dependent upon a putative receptor protein KARRIKIN INSENSITIVE 2 (KAI2). KAI2 is a homolog of DWARF 14 (D14), the receptor for endogenous strigolactone hormones. Genetic analyses suggest that KAI2 also perceives endogenous signal(s) that are not strigolactones. Activation of KAI2 by addition of karrikins to Arabidopsis plants induces expression of transcripts including D14-LIKE 2 (DLK2). We constructed the synthetic reporter gene DLK2:LUC in Arabidopsis, which comprises the firefly luciferase gene (LUC) driven by the DLK2 promoter. Here we describe a luminescence-based reporter assay with Arabidopsis seeds to detect chemical signals that can activate the KAI2 signaling pathway. We demonstrate that the DLK2:LUC assay can selectively and sensitively detect karrikins and a functionally similar synthetic strigolactone analog. Crucially we show that crude extracts from Arabidopsis leaves can also activate DLK2:LUC in a KAI2-dependent manner. Our work provides the first direct evidence for the existence of endogenous chemical signals that can activate the KAI2-mediated signaling pathway in Arabidopsis. This sensitive reporter system can now be used for the bioassay-guided purification and identification of putative endogenous KAI2 ligands or their precursors, and endogenous compounds that might modulate the KAI2 signaling pathway.

Highlights

Karrikins (KAR) are potent compounds in wildfire smoke that stimulate germination of many plant species (Flematti et al, 2004), including Arabidopsis thaliana (Nelson et al, 2009)
We generated transgenic Arabidopsis expressing the reporter construct in line was then crossed with kai2-2 (Ler) ecotype, and crossed a suitably responding transgenic line with kai2-2
We found that both D14-LIKE 2 (DLK2) and luciferase gene (LUC) transcripts were induced by KAR2 treatments (Figure 1B)

Summary

Introduction

Karrikins (KAR) are potent compounds in wildfire smoke that stimulate germination of many plant species (Flematti et al, 2004), including Arabidopsis thaliana (Nelson et al, 2009). Evidence for Endogenous KAI2 Activator(s) mutations in SUPPRESSOR OF MAX2 1 (SMAX1) and its paralog SMAX1-LIKE2 (SMXL2) induce constitutive KAR responses (Stanga et al, 2013, 2016) These mutant phenotypes suggest that the karrikin signaling pathway defined by KAI2, MAX2, and SMAX1/SMXL2 has endogenous functions in plant development that extend beyond mediating responses to KAR. Several reports have demonstrated interaction of KAR with Arabidopsis KAI2 and its homologs (Guo et al, 2013; Kagiyama et al, 2013; Xu et al, 2016), but no covalent interaction has been reported Additional butenolides such as synthetic SL isomer GR24ent−5DS activate KAI2 signaling, while mutation of the catalytic triad abolishes hydrolysis and signaling (Scaffidi et al, 2014; Waters et al, 2015b). We examine the KL hypothesis directly by asking whether the signaling pathway defined by KAI2-MAX2-SMAX1 can be activated by metabolites in plant extracts

Methods

Results

Conclusion