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Membralin is required for maize development and defines a branch of the endoplasmic reticulum–associated degradation pathway in plants
Saturday, 2024/06/22 | 06:33:33

Baiyu LiuChangzheng XuQiuxia HeKe ZhangShoumei QiZhe JinWen ChengZhaohua Ding

Donghua Chen, Xiangyu Zhao, Wei Zhang, Kewei Zhang, and Kunpeng Li.

PNAS; June 12, 2024; 121 (25) e2406090121; https://doi.org/10.1073/pnas.2406090121

Significance

Endoplasmic reticulum (ER)-associated degradation (ERAD) machinery enforces protein homeostasis control by eliminating aberrant or unnecessary proteins during protein folding, which requires ER membrane-embedded complexes assembled around E3-ubiquitin ligases. We discovered that ZmNLD1 encodes the ER membrane–localized protein membralin and specifically interacts with RING domain-containing E3-ubiquitin ligases RNF185 and RNF5, forming a branch of ERAD pathway essential for maize plant development, independent of the classical HRD1 and Doa10 ERAD complexes. Membralin deficiency results in impaired medial–lateral leaf expansion, retarded root morphogenesis, and plant dwarfism. Multiple lines of evidence indicate that ZmNLD1 has similar modes of action in ERAD to its mammalian homologs, revealing the evolutionary conservation of membralin-mediated ERAD branch between the animal and plant kingdoms.

Abstract

Endoplasmic reticulum (ER)–associated degradation (ERAD) plays key roles in controlling protein levels and quality in eukaryotes. The Ring Finger Protein 185 (RNF185)/membralin ubiquitin ligase complex was recently identified as a branch in mammals and is essential for neuronal function, but its function in plant development is unknown. Here, we report the map-based cloning and characterization of Narrow Leaf and Dwarfism 1 (NLD1), which encodes the ER membrane–localized protein membralin and specifically interacts with maize homologs of RNF185 and related components. The nld1 mutant shows defective leaf and root development due to reduced cell number. The defects of nld1 were largely restored by expressing membralin genes from Arabidopsis thaliana and mice, highlighting the conserved roles of membralin proteins in animals and plants. The excessive accumulation of β-hydroxy β-methylglutaryl-CoA reductase in nld1 indicates that the enzyme is a membralin-mediated ERAD target. The activation of bZIP60 mRNA splicing–related unfolded protein response signaling and marker gene expression in nld1, as well as DNA fragment and cell viability assays, indicate that membralin deficiency induces ER stress and cell death in maize, thereby affecting organogenesis. Our findings uncover the conserved, indispensable role of the membralin-mediated branch of the ERAD pathway in plants. In addition, ZmNLD1 contributes to plant architecture in a dose-dependent manner, which can serve as a potential target for genetic engineering to shape ideal plant architecture, thereby enhancing high-density maize yields.

 

See https://www.pnas.org/doi/10.1073/pnas.2406090121

 

 

Figure 2: Mapping, cloning, and functional identification of ZmNLD1. (A) The ZmNLD1 locus was mapped to a 77.54-kb interval between molecular markers SSR57 and SSR59 on chromosome 5, which contains six candidate genes. The red arrow indicates the causal gene of nld1. The structure of ZmNLD1 and mutation sites of the nld1 (the last base of the 10th exon) and nld2 (the last base of the 11th intron) alleles are indicated in red font. The two transcripts produced by ZmNLD1 in the wild-type background are shown. (B) Sequence alignment (partial) of ZmNLD1 in the wild type, nld1, and nld2. The black boxes indicate the mutation sites in nld1 and nld2. (C) PCR-amplified products of ZmNLD1 from Qi319 and nld1, and illustrations of splice variants (ZmNLD1-M1 and ZmNLD1-M2). The positions marked in red differ from the ZmNLD1-T01 transcript. (D and E) Photographs of plants (D) and leaves (E) used for the allelism test of nld1 and nld2. (F–H) ZmNLD1-T01 and ZmNLD1-T02 partially rescued the nld1 (F), ear-leaf width (G), and grain yield per ear (H) phenotypes. Three ZmNLD1-T01 and three ZmNLD1-T02 transgenic lines were examined, each with 11 individual plants. Values are means ± SD (n = 11). Different letters indicate significant differences (P < 0.01) according to an unpaired Student’s t test. (Scale bar: 5 cm.)

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