Arabidopsis CPL4 is an essential C‐terminal domain phosphatase that suppresses xenobiotic stress responses

Abstract

). Accession number: DQ503426, KF545094, KF545095 KF545096. SUMMARYEukaryotic gene expression is both promoted and inhibited by the reversible phosphorylation of the C-terminal domain of RNA polymerase II (pol II CTD). More than 20 Arabidopsis genes encode CTD phosphatase homologs, including four CTD phosphatase-like (CPL) family members. Although in vitro CTD phosphatase activity has been established for some CPLs, none have been shown to be involved in the phosphoregulation of pol II in vivo. Here we report that CPL4 is a CTD phosphatase essential for the viability of Arabidopsis thaliana. Mass spectrometry analysis identified the pol II subunits RPB1, RPB2 and RPB3 in the affinity-purified CPL4 complex. CPL4 dephosphorylates both Ser2-and Ser5-PO 4 of the CTD in vitro, with a preference for Ser2-PO 4 . Arabidopsis plants overexpressing CPL4 accumulated hypophosphorylated pol II, whereas RNA interference-mediated silencing of CPL4 promoted hyperphosphorylation of pol II. A D128A mutation in the conserved DXDXT motif of the CPL4 catalytic domain resulted in a dominant negative form of CPL4, the overexpression of which inhibited transgene expression in transient assays. Inhibition was abolished by truncation of the phosphoprotein-binding Breast Cancer 1 C-terminal domain of CPL4, suggesting that both catalytic function and protein-protein interaction are essential for CPL4-mediated regulation of gene expression. We were unable to recover a homozygous cpl4 mutant, probably due to the zygotic lethality of this mutation. The reduction in CPL4 levels in CPL4 RNAi plants increased transcript levels of a suite of herbicide/ xenobiotic-responsive genes and improved herbicide tolerance, thus suggesting an additional role for CPL4 as a negative regulator of the xenobiotic detoxification pathway.

Herbicide-related signaling in plants reveals novel insights for herbicide use strategies, environmental risk assessment and global change assessment challenges

“…The efficiency of atrazine toxicity could therefore be envisaged as an inclusive toxicity resulting from the action on several processes. Moreover, in the case of chlorsulfuron, the mutation of CPL4 (Table 1) can increase the IC50 in Arabidopsis seedlings from less than 10 nM (Singh et al, 1992) to over 40 nM (Fukudome et al, 2014), thus suggesting that the CPL4 regulatory protein contributes to herbicide toxicity. It can thus be hypothesized that variable amounts or activity levels of regulatory proteins may act as adjustable cursors, i.e.…”

“…The involvement of master regulatory or signaling proteins that control the coordinated gene expression of cell stress, xenobiotic stress and xenobiotic detoxification responses (Ramel et al, 2012;Xiong et al, 2013) enhances the importance of such a threat. Independent studies (Fukudome et al, 2014;Ramel et al, 2012;Zwack et al, 2013) show that common regulatory proteins can be involved in the responses to herbicides (atrazine, chlorsulfuron, paraquat) of different classes and modes of action. Since adaptive evolution of weed populations is due to processes of selection pressure and to situations that reduce plant mortality (Neve and Powles, 2005;Yu and Powles, 2014), it can be speculated that genes involved in cell stress and xenobiotic stress cross-tolerance can be under the selection pressure not only of recurrent applications of a given herbicide, but also of carryover and accumulation of persistent herbicides, herbicide residues and other pesticides in agricultural soils (Cui et al, 2010;Zhang et al, 2016) or in field margins .…”

“…Independent studies have revealed that the At3g61630 gene, which encodes the AP2/ERF family cytokinin-response factor 6 (CRF6) that is directly connected to the cytokinin signaling pathway (Cutcliffe et al, 2011), can be induced by the triazine atrazine (Ramel et al, 2007), by paraquat (methyl viologen) (Zwack et al, 2013) and by the sulfonylurea chlorsulfuron (Fukudome et al, 2014). Moreover, Ramel et al (2012) have shown that mutation of At3g61630 led to significant and complex modifications in the responses to atrazine with enhanced tolerance under conditions of carbohydrate limitation and decreased tolerance under conditions of sucrose feeding.…”

“…Recent developments using mutation approaches have revealed unexpected links between herbicide tolerance and plant regulatory genes that are unrelated to herbicide targets or herbicide metabolisms (Faus et al, 2015;Fukudome et al, 2014;Kurepa et al, 1998;Ramel et al, 2012;Sanchez-Villarreal et al, 2013;Sharkhuu et al, 2014;Smeets et al, 2013). It is therefore timely to reassess the novel mechanisms that are involved in plant-herbicide interactions (Section 2), to re-consider whether the unilinear herbicide-target-disruption-death string of events is still relevant or should be replaced with an inclusive-toxicity regulatory model (Section 3), and to evaluate how novel models of plant-herbicide interactions are important to deal with pressing agro-environmental issues such as herbicide use strategies, environmental risk assessment and global change assessment challenges (Section 4).…”

“…Moreover, involvement of circadian clock modules was independently confirmed by the effects of the chemically-distinct herbicide imazethapyr on flowering (Qian et al, 2014a). Table 1 shows that mutant analysis studies have now uncovered more signaling components and more signaling pathways that are linked to herbicide responses and whose impairment modulates herbicide action: a stress-responsive transcriptional regulator (Rama Devi et al, 2006), the ethylene signaling CTR1 kinase , the protein kinase of eukaryotic translation initiation factor-2α (eIF2α) (Faus et al, 2015;Zhang et al, 2008), GRAS-family transcription factors (Fode et al, 2008), the ClpR4 subunit of the chloroplast-localized Clp protease complex (Saini et al, 2011), the cytokinin response factor 6 (CRF6) transcription factor (Ramel et al, 2012), the jasmonate signaling COI1 component of ubiquitin-ligase complexes (Köster et al, 2012), the circadian clock regulator TIME FOR COFFEE (Sanchez-Villarreal et al, 2013), the TOR kinase carbon and energy sensor (Xiong et al, 2013), the protein phosphatase of C-terminal domain RNA polymerase II (Fukudome et al, 2014), and the red/far-red photoreceptor phytochrome B (Sharkhuu et al, 2014). All of these herbicide-signaling interactions differ from the direct interactions that necessarily occur between hormone-analogue herbicides, such as auxinic analogues, and corresponding hormone-binding proteins, such as auxin receptors or transporters (Gleason et al, 2011).…”