The ARRE RING-Type E3 Ubiquitin Ligase Negatively Regulates Cuticular Wax Biosynthesis in Arabidopsis thaliana by Controlling ECERIFERUM1 and ECERIFERUM3 Protein Levels.

Shuang Liu,Ljerka Kunst,Xin Li,Lifang Zhao,Meixuezi Tong

doi:10.3389/fpls.2021.752309

Abstract

The outer epidermal cell walls of plant shoots are covered with a cuticle, a continuous lipid structure that provides protection from desiccation, UV light, pathogens, and insects. The cuticle is mostly composed of cutin and cuticular wax. Cuticular wax synthesis is synchronized with surface area expansion during plant development and is associated with plant responses to biotic and abiotic stresses. Cuticular wax deposition is tightly regulated by well-established transcriptional and post-transcriptional regulatory mechanisms, as well as post-translationally via the ubiquitin-26S proteasome system (UPS). The UPS is highly conserved in eukaryotes and involves the covalent attachment of polyubiquitin chains to the target protein by an E3 ligase, followed by the degradation of the modified protein by the 26S proteasome. A large number of E3 ligases are encoded in the Arabidopsis genome, but only a few have been implicated in the regulation of cuticular wax deposition. In this study, we have conducted an E3 ligase reverse genetic screen and identified a novel RING-type E3 ubiquitin ligase, AtARRE, which negatively regulates wax biosynthesis in Arabidopsis. Arabidopsis plants overexpressing AtARRE exhibit glossy stems and siliques, reduced fertility and fusion between aerial organs. Wax load and wax compositional analyses of AtARRE overexpressors showed that the alkane-forming branch of the wax biosynthetic pathway is affected. Co-expression of AtARRE and candidate target proteins involved in alkane formation in both Nicotiana benthamiana and stable Arabidopsis transgenic lines demonstrated that AtARRE controls the levels of wax biosynthetic enzymes ECERIFERUM1 (CER1) and ECERIFERUM3 (CER3). CER1 has also been confirmed to be a ubiquitination substrate of the AtARRE E3 ligase by an in vivo ubiquitination assay using a reconstituted Escherichia coli system. The AtARRE gene is expressed throughout the plant, with the highest expression detected in fully expanded rosette leaves and oldest stem internodes. AtARRE gene expression can also be induced by exposure to pathogens. These findings reveal that wax biosynthesis in mature plant tissues and in response to pathogen infection is controlled post-translationally.

Highlights

The primary aerial surfaces of land plants are covered with a cuticle, a continuous lipidic layer that restricts transpirational water loss, reflects harmful UV light, and prevents organ fusions during development (Reicosky and Hanover, 1978; Sieber et al, 2000; Riederer and Schreiber, 2001; Riederer, 2006)
The AtARRE/At5g66070 gene encoding a RINGtype E3 ubiquitin ligase was expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter (Supplementary Figures S1A,B)
VLC alkane production is catalyzed by CER1 and CER3 enzymes, which together with the cytochrome B5 form a multiprotein endoplasmic reticulum (ER)-membrane-associated complex (Bourdenx et al, 2011; Bernard et al, 2012) Not surprisingly, both enzymes act as key control points for wax biosynthesis

Summary

Introduction

The primary aerial surfaces of land plants are covered with a cuticle, a continuous lipidic layer that restricts transpirational water loss, reflects harmful UV light, and prevents organ fusions during development (Reicosky and Hanover, 1978; Sieber et al, 2000; Riederer and Schreiber, 2001; Riederer, 2006). The cuticle is mostly composed of cutin and cuticular wax (Samuels et al, 2008). Cutin is a polymer of oxidized 16- and 18-carbon (C16 and C18) fatty acids and glycerol (Beisson et al, 2012), which forms the structural scaffold of the cuticle. Cuticular wax embeds and overlays this cutin matrix and is composed of very long-chain fatty acids (VLCFAs; C20-C38) and their derivatives, including alkanes, aldehydes, primary and secondary alcohols, ketones, and esters. Wax composition varies among plant species, as well as between different organs, tissues, and developmental stages of the same plant species. These variations in wax composition affect the biochemical and physical properties of the plant surface, which helps the plant adapt to different environments

Results

Discussion

Conclusion