Reconstitution of the Jasmonate Signaling Pathway in Plant Protoplasts.

Ning Li,Christiane Gatz,Joachim F Uhrig,Corinna Thurow,Li-Jun Huang

doi:10.3390/cells8121532

Ning Li, Christiane Gatz + Show 3 more

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https://doi.org/10.3390/cells8121532

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Abstract

The phytohormone jasmonic acid (JA) plays an important role in various plant developmental processes and environmental adaptations. The JA signaling pathway has been well-elucidated in the reference plant Arabidopsis thaliana. It starts with the perception of the active JA derivative, jasmonoyl-isoleucine (JA-Ile), by the F-box protein COI1 which is part of the E3-ligase SCFCOI1. Binding of JA-Ile enables the interaction between COI1 and JAZ repressor proteins. Subsequent degradation of JAZ proteins leads to the activation of transcription factors like e.g., MYC2. Here we demonstrate that the pathway can be reconstituted in transiently transformed protoplasts. Analysis of the stability of a JAZ1-fLuc fusion protein as a function of COI1 transiently expressed in coi1 protoplasts allows structure function analysis of both JAZs and COI1. Using this system, we found that conserved cysteines in COI1 influence steady state COI1 protein levels. Using a luciferase reporter gene under the control of the JAZ1 promoter enable to address those features of JAZ1 that are required for MYC2 repression. Interestingly, the conserved TIFY-motif previously described to interact with NINJA to recruit the corepressor TOPLESS is not necessary for repression. This result is in favor of the alternative repression mode that proposes a direct competition between repressive JAZs and promotive MEDIATOR25 at MYC2. Finally, using protoplasts from the aos coi1 double mutant, which is deficient in JA synthesis and perception, we provide a system that has the potential to study the activity of different COI1 variants in the presence of different ligands.

Highlights

Plants are constantly exposed to a wide variety of environmental stresses that negatively affect their growth and yield
Having established thatprocess the degradation of JAZ1-Firefly luciferase (fLuc) fusion protein can be used to study CORONATINE INSENSITIVE1 (COI1) activity in coi1 protoplasts, we addressed the functional significance of a cysteine residue close to the jasmonic acid (JA)-Ile contacting amino acid R496 in the 17th leucine-rich repeats tertiary (LRRs)-motif that caught our attention due to its strong conservation from the lower land plant Marchantia polymorpha to all known sequences of higher plants
In our protoplast system, single or double mutations of these conserved cysteines had no obvious impact in mediating JAZ1 degradation (Figure 3B)

Summary

Introduction

Plants are constantly exposed to a wide variety of environmental stresses that negatively affect their growth and yield. Specific signaling networks serve to activate specific genes that serve to combat adverse conditions and to coordinate plant growth [1,2,3]. Understanding the molecular mechanisms underlying those signaling pathways is key to designing plants with an improved stress tolerance and growth performance. The fatty acid-derived plant hormone jasmonic acid (JA). Regulates a number of plant developmental and environmental responses [4,5,6,7]. JA is produced by the oxylipin biosynthesis pathway [8,9]. JA is conjugated to isoleucine through the activity of a jasmonoyl-L-amino acid synthetase to form the major bioactive JA derivative, jasmonoyl-isoleucine (JA-Ile) [11,12]

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