Down-Stream molecular Responses to Volatile Signaling in Roots

Abstract

Plants, fungi, and prokaryotic organisms emit volatiles for inter-kingdom communication. Some volatiles are emitted in large quantities into the environment and act as climate relevant gases, others are released for pollination or are involved direct or indirect in herbivore defense. Most studies focused on volatiles produced above ground by plants. Fungi also have complex volatiles patterns, which can impact growth and development of plants effects or mediate communication between plants, bacteria, and fungi. For example, volatiles released by the ectomycorrhizal fungus L. bicolor induce lateral root (LR) formation and root hair growth in the potential host poplar and in the non-host Arabidopsis thaliana. However, until today it remained unclear, which receptors and signaling pathways in plants are involved in the L. bicolor volatile-induced LR formation. In this study, we investigated down-stream molecular responses to L. bicolor volatile signaling in A. thaliana and Populus x canescens. To gain insights in the volatile-induced responses, we employed forward and reverse genetic approaches using Arabidopsis mutant lines with roles in hormone signaling and response to volatiles. We exposed mutant lines with potential roles in LR formation associated with ROS homeostasis (per45 and per59), strigolactone biosynthesis and signaling (max2 and max4) and germination (germin-like 3, GLP3) to L. bicolor volatiles and tested the influence on LR density compared to wildtype plants. None of the tested mutant lines showed a different behavior compared to wildtype plants upon volatile exposure. Since cysteine rich receptor like kinases (CRKs) play roles in stress, growth, and development, we screened a crk knock-out library containing 42 crk lines. We found evidence that CRK4, CRK42 and CRK46 may be involved in volatile perception. However, further analyses of those lines are necessary to strengthen this result. Furthermore, we investigated the response dose and exposure time to L. bicolor volatiles. By removing the fungus from co-culture with the plant and co-culture the plant with different numbers of fungal plugs we found that co-cultivation with one fungal plug was sufficient to induce enhanced LR formation. With 2, 3, and 4 fungal plugs, the Arabidopsis plants showed similar enhancement of LR densities as those exposed to one plug. This result suggests a repletion of volatile receptors or signaling. Furthermore, we showed that a pulse of volatiles exposure of 2 days is sufficient to induced enhanced LR formation. To identify volatile-induced candidate genes, time-course experiments with increasing duration of volatile exposure were performed and the transcriptomes and metabolomes analyzed. In these studies, we identified a consistently down-regulated gene of unknown function (At4g31330). We further investigated the LR density of At4g31330 T DNA insertion upon volatile exposure and transformed A. thaliana plants with CRIPSR/Cas9 to generate At4g31330 loss-of function mutant lines. Furthermore, we generated At4g31330 overexpression lines and investigated their LR density upon volatile exposure. Compared to wildtype, the At4g31330 CRISPR/lines showed rather low LR density, independent of volatile exposure. The At4g31330 overexpression lines behaved like wildtype upon volatile exposure. We also detected plant hormone related genes with involvements in stress or LR formation in the transcriptome data. The genes detected by this approach were predominantly related to abscisic acid (ABA). Thus, we exposed ABA-mutant lines to L. bicolor volatiles and found reduced LR response in aao3-4 and pyl8-1 and enhanced response in aba2-1 compared to wildtype, indicating that ABA might be involved in volatile perception or signaling in plants. Taken together, we showed that LR formation in response to L. bicolor volatiles in planta is a complex process, involving yet unknown proteins, CRKs, and ABA signaling. Since LRs are important for plant water and nutrient uptake, further studies how CRKs and ABA are influenced by fungal volatiles to eventually reprogram root development are needed.