Protein Phosphatases Type 2C Group A Interact with and Regulate the Stability of ACC Synthase 7 in Arabidopsis

Abstract

Ethylene is an important plant hormone that controls growth, development, aging and stress responses. The rate-limiting enzymes in ethylene biosynthesis, the 1-aminocyclopropane-1-carboxylate synthases (ACSs), are strictly regulated at many levels, including posttranslational control of protein half-life. Reversible phosphorylation/dephosphorylation events play a pivotal role as signals for ubiquitin-dependent degradation. We showed previously that ABI1, a group A protein phosphatase type 2C (PP2C) and a key negative regulator of abscisic acid signaling regulates type I ACS stability. Here we provide evidence that ABI1 also contributes to the regulation of ethylene biosynthesis via ACS7, a type III ACS without known regulatory domains. Using various approaches, we show that ACS7 interacts with ABI1, ABI2 and HAB1. We use molecular modeling to predict the amino acid residues involved in ABI1/ACS7 complex formation and confirm these predictions by mcBiFC-FRET-FLIM analysis. Using a cell-free degradation assay, we show that proteasomal degradation of ACS7 is delayed in protein extracts prepared from PP2C type A knockout plants, compared to a wild-type extract. This study therefore shows that ACS7 undergoes complex regulation governed by ABI1, ABI2 and HAB1. Furthermore, this suggests that ACS7, together with PP2Cs, plays an essential role in maintaining appropriate levels of ethylene in Arabidopsis.Cells 2020, 9, 978 2 of 20 also abbreviated as ACC synthase) to 1-aminocyclopropane 1-carboxylate (ACC). In the second step, ACC is converted to ethylene with release of carbon dioxide and cyanide by 1-aminocyclopropane 1-carboxylate oxidase (ACO). ACS is thus a crucial enzyme in ethylene biosynthesis and in all plant species is encoded by a multigene family consisting of nine members in Lycopersicon esculentum, six in Oriza sativa and 11 in poplar [7,8]. In Arabidopsis, the ACS gene family comprises 12 members, including eight genes encoding functional enzymes (ACS2, ACS4-9 and ACS11), a single inactive form of ACS1 and a pseudogene ACS3 [9].The available domain structures of the ACS isozymes allow their classification based on the presence or absence of non-catalytic C-terminal phosphorylation motifs [10]. Type I ACSs contain a C-terminal fragment that includes phosphorylation sites for both mitogen-activated protein kinases (MAPKs) and calcium-dependent protein kinases (CDPKs). In Arabidopsis thaliana, ACS2 and ACS6 belong to type I. Type II isozymes carry only the CDPK target site, and type III isozymes lack both MAPK and CDPK sites [11,12]. In Arabidopsis, ACS4, ACS5, ACS8, ACS9 and ACS11 are type II ACSs, while there is only a single type III isozyme, ACS7.Although ACS7 lacks known regulatory domains, it still undergoes ubiquitin-dependent proteasomal degradation. The E3 ubiquitin ligase, XBAT32, initiates this process by attachment of ubiquitin to ACS7 [13,14]. XBAT32 is member of the RING domain-containing ankyrin repeat E3 ligase subfamily and was first characterized as a positive regulator of lateral root develo...

The regulation of ethylene biosynthesis: a complex multilevel control circuitry

“…While the C‐terminal domain of type 1 and type 2 ACS is certainly important for regulation of protein stability, a short region of the N‐terminal domain of the Arabidopsis type 3 ACS7 fulfils this role, as it can be marked for degradation via ubiquitination by XB3 orthologue 2 in Arabidopsis (XBAT32; Lyzenga et al ., 2012; Xiong et al ., 2014). ACS7 can also be stabilised via the interaction with several members of the Protein Phosphatase 2C family (PP2C’s; Marczak et al ., 2020). Another mechanism of ACS stabilisation occurs via the interaction with 14‐3‐3 proteins that protects them from degradation (Yoon & Kieber, 2013).…”

Identification of Novel Inhibitors of a Plant Group A Protein Phosphatase Type 2C Using a Combined In Silico and Biochemical Approach

“…To further investigate the effect of ZINC59151964 and ZINC05273880 on PP2C function, we tested the stability of the ABI1/2 PP2C-interacting protein, ACC synthase 7 (ACS7) ( Marczak et al., 2020 ), using a cell-free degradation assay ( Figure 9 ). Recombinant GST-ACS7 was incubated with total protein extract prepared from WT Arabidopsis seedlings treated with 300 µM ZINC59151964, 300 µM ZINC05273880, 100 µM MG132 (a proteasome inhibitor), or an equivalent mock-treated control.…”

“…The cell-free degradation assay was performed as described in Marczak et al (2020). In brief, 7-day-old WT Arabidopsis seedlings were treated with 100 µM MG132, 300 µM ZINC59151964, or 300 µM ZINC05273880.…”

“…Simultaneous treatment with ABA and ZINC05273880 decreased germination rate of the double mutant after seven to eight days (p < 0.01) compared to ABA ( Figure 8D) suggesting that, in contrast to ZINC59151964, ZINC05273880 increases sensitivity to ABA. To further investigate the effect of ZINC59151964 and ZINC05273880 on PP2C function, we tested the stability of the ABI1/2 PP2C-interacting protein, ACC synthase 7 (ACS7) (Marczak et al, 2020), using a cell-free degradation assay (Figure 9). Recombinant GST-ACS7 was incubated with total protein extract prepared from WT Arabidopsis seedlings treated with 300 µM ZINC59151964, 300 µM ZINC05273880, 100 µM MG132 (a proteasome inhibitor), or an equivalent mock-treated control.…”

Data_Sheet_2.PDF

“…We first analyzed the expression pattern of genes involved in ethylene biosynthesis in 0-4 DAE stigmas of Chinese cabbage. The rate-limiting factor in ethylene production is considered to be the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene (Yamagami et al, 2003;Lee and Yoon, 2018;Marczak et al, 2020). Genes in B. rapa were named based on their homologs in A. thaliana.…”