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The RING-type E3 ligase RIE1 sustains leaf longevity by specifically targeting AtACS7 to fine-tune ethylene production in Arabidopsis
Friday, 2024/11/29 | 08:25:03

Xianglin TangYuanyuan MeiKaixuan HeRan LiuXiaoyan LvYujia ZhaoWenjing LiQian WangQinshan GongShengnan LiChang XuXu ZhengQingyu CaoDan Wang, and Ning Ning Wang.

PNAS; November 20, 2024; 121 (48) e2411271121; https://doi.org/10.1073/pnas.2411271121

Significance

Ethylene is a major “accelerator” of leaf senescence. This study uncovers a regulatory mechanism controlling ethylene production during leaf development. RIE1, a RING-type E3 ubiquitin ligase, was identified as a “brake” on the progression of leaf senescence. By specifically targeting and facilitating the degradation of AtACS7, the key ACS isoform responsible for “senescence ethylene” biosynthesis, RIE1 ensures an optimal level of ethylene production, contributing to normal leaf vitality and longevity. Overexpression of RIE1 reduces ethylene biosynthesis and delays leaf senescence, while its loss of function increases ethylene emission and accelerates leaf senescence. Collectively, this study provides crucial insights into the intricate balance of ethylene homeostasis essential for the proper progression of leaf development.

Abstract

Ethylene is widely recognized as a positive regulator of leaf senescence. However, how plants coordinate the biosynthesis of ethylene to meet the requirements of senescence progression has not been determined. The rate-limiting enzyme in the ethylene biosynthesis pathway is ACC synthase. AtACS7 was previously considered one of the major contributors to the synthesis of “senescence ethylene” in Arabidopsis. However, the “brake signal” that fine-tunes the expression of AtACS7 to ensure optimal ethylene production during leaf development has yet to be identified. In the present study, the RING-H2 zinc-finger protein RIE1 was found to specifically interact with and ubiquitinate AtACS7, among all functional ACSs in Arabidopsis, to promote its degradation. Overexpression of RIE1 markedly decreased ethylene biosynthesis and delayed leaf senescence, whereas loss of function of RIE1 significantly increased ethylene emission and accelerated leaf senescence. The ethylene-related phenotypes of RIE1 overexpressing or knockout mutants were effectively rescued by the ethylene precursor ACC or the competitive inhibitor of ACS, respectively. In particular, AtACS7-induced precocious leaf senescence was strongly enhanced by the loss of RIE1 but was significantly attenuated by the overexpression of RIE1. The specific regions of interaction between AtACS7 and RIE1, as well as the major ubiquitination sites of AtACS7, were further investigated. All results demonstrated that RIE1 functions as an important modulator of ethylene biosynthesis during leaf development by specifically targeting AtACS7 for degradation, thereby enabling plants to produce the optimal levels of ethylene needed.

 

See https://www.pnas.org/doi/10.1073/pnas.2411271121

 

Figure 1: In vitro self-ubiquitination assay and in planta subcellular localization analysis of RIE1. (A) Schematic diagram of the E3 ligase RIE1, which contains four transmembrane domains (pink boxes) and one RING-H2 domain (green box). The numbers indicate the amino acid positions. The detailed amino acid sequence of the RING-H2 domain is displayed below the diagram, in which the conserved cysteine and histidine residues are highlighted in red. The yellow box indicates the cysteine residue (C) at position 347 that was mutated to alanine (A) to generate the RIE1C347A variant. RING, really interesting new gene. (B) In vitro self-ubiquitination assay for RIE1. Purified GST-tagged wild-type RIE1, RIE1C347A, or free GST protein was incubated with E1, E2, and ubiquitin (Ub). Ubiquitination of RIE1 was analyzed via immunoblot analysis with an anti-GST (Upper) or anti-Ub (Lower) antibody. The protein ubiquitination bands generated by GST-RIE1 are indicated on the Right. (C) Subcellular localization of RIE1 protein. The GFP-RIE1 and ER-mCherry plasmids were transiently coexpressed in tobacco leaves by agroinfiltration. GFP and RFP fluorescence signals were detected under a laser-scanning confocal microscope. The white arrows specifically indicate the localization signal of GFP-RIE1 within the cytoplasm that is distinct and separate from the endoplasmic reticulum (ER). (Scale bar, 10 µm.) (D) Subcellular distributions of RIE1 (a) and AtACS7 (b). Protein extracts from 35S:6Myc-RIE1 (RIE1ox) or 35S:AtACS7-GFP (AtACS7ox) seedlings were separated into soluble (S) and membrane (M) fractions by ultracentrifugation and analyzed by western blot. UGP and VHA were used as controls for soluble and membrane fractions, respectively. T, total protein extracts. The experiments in B, C, and D were all biologically repeated three times, and similar results were obtained.

 

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