O-fucosylation of CPN20 by SPINDLY Derepresses Abscisic Acid Signaling During Seed Germination and Seedling Development

Abstract

SPINDLY is involved in some aspects of plant development. However, the nature of this protein as an O-fucosyltransferase was recently discovered. In this study, we show that SPINDLY (SPY) interacts with CPN20 in yeast two-hybrid and split-luc assays, and the interaction is promoted by ABA. CPN20 is a chloroplast-localized co-chaperonin that negatively regulates ABAR-mediated ABA signaling. By using Electron Transfer Dissociation-MS/MS analysis, two O-fucosylation sites, e.g., 116th and 119th threonines, were detected in ectopically expressed CPN20 in mammalian cells and in Arabidopsis. The O-fucosylation at both threonine residues was confirmed by in vitro peptide O-fucosylation assay. We further show that CPN20 accumulates in the chloroplast of spy mutants, suggesting that SPY negatively regulates CPN20 localization in the chloroplast. In vivo protein degradation assay along with CPN20 localization behavior suggest that import of CPN20 into the chloroplast is negatively regulated by SPY. Genetic analysis shows that ABA insensitive phenotypes of spy-3 in terms of seed germination and early seedling development are partially suppressed by the cpn20 mutation, suggesting that CPN20 acts downstream of SPY in this ABA signaling pathway and that there may exist other pathways in parallel with CPN20. Collectively, the above data support the notion that the O-fucosylation of CPN20 by SPY fine-tunes ABA signaling in Arabidopsis.

Physiological Roles and Mechanisms of Action of Class I TCP Transcription Factors

“…In the absence of SPY, degradation of the TCPs is governed by interaction with F-box proteins of the KISS ME DEADLY family, negative regulators of CK signaling [ 129 ]. Recently, SPY was demonstrated to be an O-fucosyltransferase that modifies a number of proteins [ 130 , 131 , 132 ]. Inhibition of class I TCP proteolysis by SPY promotes CK responses in developing Arabidopsis leaves and flowers and the catalytic domain of SPY was identified as essential for TCP activity.…”

Structural insights into mechanism and specificity of the plant protein O-fucosyltransferase SPINDLY

“…For long time, only ER-localized protein O-fucosyltransferases (POFUTs) in the GT65 family are reported, which are responsible for catalyzing O-fucosylation of secreted or cell surface proteins 12,13 . Recently, another GT41 protein, SPINDY (SPY), is identified to be a novel nucleocytoplasmic-localized POFUT, which was found modifying transcription factor and chloroplast-localized co-chaperonin in Arabidopsis and nuclear pore proteins in human pathogen Toxoplasma gondii [14][15][16][17] .…”

“…Arabidopsis SPY can O-fucosylate PRR5 and facilitate the proteolysis of PRR5, resulting in modulating the circadian clock 16 . Arabidopsis SPY is also found responsible for the fucosylation of a chloroplast-localized cochaperonin CPN20, which negatively regulating ABA signaling in seed germination and seedling development 14 .…”

HISTONE DEACETYLASE 15 and MOS4-associated complex subunits 3A/3B coregulate intron retention of ABA-responsive genes

“…HDA15 and MAC3B were cloned into nLuc and cLuc vectors, respectively ( Supplemental Table S2 ). SLC assay was performed as described previously ( Chen et al, 2008 ; Liang et al, 2021 ). Constructed vectors were transformed into Agrobacterium tumefaciens GV3101 which was injected into leaves of Nicotiana benthamiana .…”