Regulation of Arabidopsis photoreceptor CRY2 by two distinct E3 ubiquitin ligases

Abstract

Cryptochromes (CRYs) are photoreceptors or components of the molecular clock in various evolutionary lineages, and they are commonly regulated by polyubiquitination and proteolysis. Multiple E3 ubiquitin ligases regulate CRYs in animal models, and previous genetics study also suggest existence of multiple E3 ubiquitin ligases for plant CRYs. However, only one E3 ligase, Cul4COP1/SPAs, has been reported for plant CRYs so far. Here we show that Cul3LRBs is the second E3 ligase of CRY2 in Arabidopsis. We demonstrate the blue light-specific and CRY-dependent activity of LRBs (Light-Response Bric-a-Brack/Tramtrack/Broad 1, 2 & 3) in blue-light regulation of hypocotyl elongation. LRBs physically interact with photoexcited and phosphorylated CRY2, at the CCE domain of CRY2, to facilitate polyubiquitination and degradation of CRY2 in response to blue light. We propose that Cul4COP1/SPAs and Cul3LRBs E3 ligases interact with CRY2 via different structure elements to regulate the abundance of CRY2 photoreceptor under different light conditions, facilitating optimal photoresponses of plants grown in nature.

Signaling Mechanisms by Arabidopsis Cryptochromes

“…After light-induced phosphorylation, CRY2 is rapidly degraded through polyubiquitination by the COP1/SPA and the Light-Response Bric-a-Brack/Tramtrack/Broad (LRB) E3 ubiquitin ligases. While the COP1/SPA complex particularly mediates the rapid response after 1–2 h of blue light exposure, LRBs are important for CRY2 degradation after more prolonged blue light irradiation ( Shalitin et al, 2002 ; Weidler et al, 2012 ; Chen et al, 2021 ; Figure 2 ). Interestingly, CRY2 degradation is enhanced by low ambient temperature (16°C) through the polyubiquitination by LRBs ( Ma et al, 2021 ).…”

The Function and Photoregulatory Mechanisms of Cryptochromes From Moso Bamboo (Phyllostachys edulis)

“…2 The sequences subcloned into plasmids were verified by Sanger sequencing. The human cell expression vectors used in this study were pQCMV-Flag-GFP, pQCMV-GFP, and pCMV-Myc, which were described previously ( Liu et al, 2017 ; Chen et al, 2021 ). pQCMV-YFP and pQCMV-mTagRFP-T vectors were modified from pQCMV-Flag-GFP by replacing GFP with YFP or mTagRFP-T ( Supplementary Figure 1 ).…”

“…pFGFP binary vectors was used for creating overexpression transgenic lines ( Chen et al, 2021 ). The pFGFP binary vector was modified from pCambia3301, which allows the interested genes to express under control of ACTIN 2 promoter and fuse with 2 × Flag and GFP tags.…”

“…The wild-type Arabidopsis in this study is rdr6-11 , which suppresses gene silencing ( Peragine et al, 2004 ). The cry1cry2rdr6 was described previously ( Chen et al, 2021 ). Arabidopsis transgenic lines were generated via Agrobacterium tumefaciens -mediated the floral dip method ( Clough, 2005 ).…”

“…The prototype plant CRYs are Arabidopsis CRY1 and CRY2, which mediate primarily blue light inhibition of cell elongation and photoperiodic promotion of floral initiation ( Ahmad and Cashmore, 1993 ; Mockler et al, 2003 ). It is previously reported that Arabidopsis CRY1 and CRY2 undergo these photoreactions: light-induced photo-oligomerization that is inhibited by the Blue-light Inhibitor of Cryptochromes (BICs), blue light-induced phosphorylation that is catalyzed by Photoregulatory Protein Kinases (PPKs), and polyubiquitination that is catalyzed by the E3 ubiquitin ligase Cul4 COP1/SPAs and Cul3 LRBs ( Shalitin et al, 2002 , 2003 ; Wang et al, 2016 ; Liu et al, 2017 , 2020 , 2022 ; Ma et al, 2020 , 2021 ; Shao et al, 2020 ; Chen et al, 2021 ; Miao et al, 2021 ). Biological functions of CRYs have been reported in various plant species, including tomato ( Ninu et al, 1999 ), soybean ( Zhang et al, 2008 ; Lyu et al, 2021 ), Brassica ( Chatterjee et al, 2006 ), pea ( Platten et al, 2005 ), poplar ( Mao et al, 2014 ), rice ( Matsumoto et al, 2003 ), barley ( Szucs et al, 2006 ), sorghum ( Zhou et al, 2018 ), apple ( Li et al, 2013a , b ), and wheat ( Xu et al, 2009 ).…”