Application of dimethyl labeling-based quantitative interactomics in the study of ethylene signaling components in Arabidopsis thaliana

Abstract

In higher plants, Lectin receptor-like kinases (LecRKs) are abundant in prevalent. They are responsible for plant immunity, plant defense mechanism, protection of plants from fungi, pathogens and insects. In general, the association of pattern recognition receptors (PRRs) with LecRKs can result in the formation of cell-surface multiprotein immune complexes for combating microbe-and host-derived molecular patterns. Nevertheless. their physiological roles in plant hormone signaling are less reported.

In this study, we focused on identifying protein interactors of EIN2, a central ethylene signaling regulator in Arabidopsis thaliana. In total, the results from quantitative dimethyl labelling-based affinity purification mass spectrometry (AP-MS) approach demonstrated altered significant expression of 31 EIN2 protein partners that are involved in ethylene signaling. The Arabidopsis L-type lectin receptor kinase IV.1 (LecRK-IV.1) was identified through the quantitative interactome analysis. Loss-of-function mutation leads to slight ethylene insensitivity in etiolated seedlings and adult plants. Two members of LecRK-IV subfamily have been isolated by conserved motif analysis and mutations of LecRK-IV.1/2/4 conferred enhanced ethylene insensitivity. Molecular genetic analysis of etr1/2/lecRK-IV.1/2/4 and ctr1/lecRK-IV.1/2/4 CRISPR/Cas9-mediated knockout mutants revealed that LecRK-IV.1, -IV.2 and -IV.4 act downstream of at least two ethylene receptors, ETR1 and ETR2, as well as CTR1 kinase. BiFC analysis further revealed that EIN2 directly interacts with LecRK-IV.1, LecRK-IV.2 and LecRK-IV.4 in planta.

Further in vitro protein kinase assays demonstrated that LecRK-IV.1, -IV.2 and -IV.4 serve as negative regulators of CTR1 in modulating EIN2 phosphorylation by preferentially tight binding to EIN2 to form EIN2-LecRK-IV.1/2/4 multicomplex. LecRK-IV.1, -IV.2 and -IV.4 protein level also showed accumulation in response to ethylene. Western blotting results also demonstrated that LecRK-IV.1, -IV.2 and LecRK-IV.4 negatively regulate EIN2 phosphorylation at Ser⁶⁴⁵ and Ser⁹²⁴ in-planta in the ethylene signaling cascade. Altogether, our findings provide insight that LecRK-IV.1, LecRK-IV.2 and LecRK-IV.4 kinases play invincible roles to act as redundant modulators in ethylene signaling cascade in Arabidopsis thaliana.

Date of Award	2022
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Ning LI (Supervisor)