The FBA motif-containing protein AFBA1 acts as a novel positive regulator of ABA response in Arabidopsis Yun Young Kim1, Mei Hua Cui1,2, Min Soo Noh1, Kwang Wook Jung1,3,* and Jeong Sheop Shin1,* 1Division of Life Sciences, Korea University, Seoul 136-701, Korea Regular Paper 2Present address: School of Life Sciences and Biotechnology, Shanghai JiaoTong University, Shanghai 200240, China. 3Present address: DuPont Pioneer Hi-Bred, DuPont (Korea) Inc., Gangnam-gu, Seoul 135-719, Korea. *Corresponding authors: Jeong Sheop Shin, E-mail, [email protected]; Fax, +82-2-927-9028; Kwang Wook Jung, E-mail, Kwang- [email protected]; Fax, +82-2-2222-5484. (Received July 21, 2016; Accepted January 4, 2017) Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 ABA plays a critical role in regulating seed germination and Introduction stomatal movement in response to drought stress. Screening ABA-responsive genes led to the identification of a novel The phytohormone ABA regulates many important processes Arabidopsis gene encoding a protein which contained a con- in plants, including seed germination, developmental processes served F-box-associated (FBA) domain, subsequently named and tolerance to abiotic stresses, especially to drought ABA-responsive FBA domain-containing protein 1 (AFBA1). (Finkelstein et al. 2002, Himmelbach et al. 2003). Under water-deficit conditions, ABA triggers a signaling cascade in Expression of ProAFBA1:GUS revealed that this gene was mainly expressed in guard cells. Expression of AFBA1 guard cells, resulting in stomatal closure, thereby preventing increased following the application of exogenous ABA and water loss through transpiration (Schroeder et al. 2001, exposure to salt (NaCl) and drought stresses. Seed germin- Israelsson et al. 2006). To date, analysis of mutant lines has ation of the loss-of-function mutant (afba1) was insensitive to led to the characterization of many negative and positive regu- ABA, salt or mannitol, whereas AFBA1-overexpressing (Ox) lators of ABA signaling in response to abiotic stresses seeds were more sensitive to these stresses than the wild- (Nakashima and Yamaguchi-Shinozaki 2006, Hirayama and type seeds. The afba1 plants showed decreased drought tol- Shinozaki 2007, Jung et al. 2008, Ding et al. 2009, Kim et al. erance, increased water loss rate and ABA-insensitive stoma- 2011, Yoshida et al. 2014). Of these, the serine/threonine pro- tal movement compared with the wild-type. In contrast, tein phosphatases (PP2Cs), such as AtPP2CA, ABI1, ABI2, HAB1 AFBA1-Ox plants exhibited enhanced drought tolerance and and HAB2, are well studied as the negative regulators of ABA- a rapid ABA-induced stomatal closure response. The expres- mediated signaling (Merlot et al. 2001, Tahtiharju and Palva sion of genes encoding serine/threonine protein phosphatases 2001, Saez et al. 2004, Yoshida et al. 2006, Hirayama and that are known negative regulators of ABA signaling increased Shinozaki 2007). For example, the Arabidopsis mutants abi1-1 in afba1 plants but decreased in AFBA1-Ox plants. AFBA1 was and abi2-1, which have defects in phosphatases ABI1 and ABI2, also found to be localized in the nucleus and to interact with respectively, were found to be insensitive to ABA during seed an R2R3-type transcription factor, MYB44, leading to the sug- germination and to stomatal regulation under drought stress gestion that it functions in the stabilization of MYB44. Based conditions (Leung et al. 1997, Merlot et al. 2001), and the loss- on these results, we suggest that AFBA1 functions as a novel of-function mutants of HAB1 and HAB2 exhibit ABA-hypersen- positive regulator of ABA responses, regulating the expression sitive inhibition of seed germination (Saez et al. 2004, Yoshida of genes involved in ABA signal transduction in Arabidopsis et al. 2006). Positive regulators of ABA signaling include the through its interaction with positive regulators of ABA signal- serine/threonine protein kinase OST1 (Mustilli et al. 2002) ing including MYB44, and increasing their stability during and R2R3-type transcription factor MYB44 (Jung et al. 2008). ABA-mediated responses. OST1-overexpressing lines are hypersensitive to ABA and ac- celerate stomatal closure in response to water limitation Keywords: ABA response Abiotic stress Arabidopsis thali- (Acharya et al. 2013), and MYB44-overexpressing lines are ana Drought tolerance FBA motif Stomatal movement. also sensitive to ABA and show the rapid ABA-induced stoma- Abbreviations: AFBA1, ABA-insensitive; BiFC, bimolecular tal closure response (Jung et al. 2008). fluorescence complementation; FBA motif-containing protein Regulation of the ABA signaling pathway is often associated 1; GUS, b-glucuronidase; MS medium, Murashige and Skoog with the ubiquitin–proteasome system (UPS), an important medium; ORF, open reading frame; PP2C, protein phosphat- regulatory mechanism for the selective degradation of abnormal ase 2C; qRT-PCR, quantitative real-time PCR; RT–PCR, re- proteins and of most short-lived regulatory proteins during plant verse transcription–PCR; smGFP, soluble modified green growth, developmental processes and abiotic stress tolerance fluorescent protein; UPS, ubiquitin–proteasome system; (Liu and Stone 2011). The UPS consists of ubiquitin, a highly WT, wild type. conserved 76 amino acid polypeptide, as a molecular tag and Plant Cell Physiol. 58(3): 574–586 (2017) doi:10.1093/pcp/pcx003, Advance Access publication on 10 February 2017, available online at www.pcp.oxfordjournals.org ! The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: [email protected] Plant Cell Physiol. 58(3): 574–586 (2017) doi:10.1093/pcp/pcx003 protein recognizes specific substrates and mediates the pro- tein–protein interaction for ubiquitination. F-box proteins con- tain an F-box motif at their N- terminus, which binds Skp1 to create a link to Cullin, and a C-terminal protein–protein inter- action domain, such as leucine-rich repeats, kelch repeats or an F- box associated (FBA) motif, which targets multiple substrates for ubiquitination (Gou et al. 2009, Xu et al. 2009). Nearly 700 F-box proteins have been predicted in the Arabidopsis genome (Gagne et al. 2002, Kuroda et al. 2002), but the functions of most of the F- box proteins have yet to be elucidated. F-box proteins have been shown to play essential roles in plant growth, developmental processes and abiotic stress tolerance through the regulation Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 of multiple phytohormone signaling pathways, such as those mediated by auxin (Dharmasiri et al. 2005), gibberellin (McGinnis et al. 2003, Dill et al. 2004), ethylene (Potuschak et al. 2003), salicylic acid (Gou et al. 2009) and ABA (Zhang et al. 2008). This has led to the suggestion that a better under- standing of the function of F-box proteins and their regulatory roles in controlling the stability of target proteins will further our understanding of the in-depth regulatory mechanisms of nearly every aspect of plant growth and development. Winston et al. (1999) reported on a family of mammalian F- box proteins and identified a highly conserved domain which they named the FBA motif. It has since been determined that F- box proteins carrying FBAs represent the largest known F-box protein subfamily, with 206 members in Arabidopsis (Schumann et al. 2011). The FBA motif within the F-box pro- teins recognizes the substrates for ubiquitination and prote- asome-mediated degradation (del Pozo and Estelle 2000, Gou et al. 2009, Han et al. 2015) and is essential for normal function of the F-box protein (Han et al. 2015). However, the roles of proteins containing only the FBA motif and no other known motif(s), including the F-box motif, have not been functionally characterized in plants, despite there being approximately 27 putative proteins containing solely the FBA motif in the Arabidopsis genome. Fig. 1 Tissue specificity of AFBA1 expression. (A) Spatial expression of In our previous study, the AFBA1 gene was screened as a novel AFBA1 by qRT-PCR. Each bar represents the amount of the transcript ubiquitous redox regulator gene by a full genome microarray ana- of a gene relative to that of the eIF4a1 gene as an internal control. lysis (ATH1 Affymetrix chips) using transgenic plants of CBSX2 Total RNAs were obtained from the rosette leaves (RL), cauline leaves (Jung et al. 2013) and selected for further functional characteriza- (CL), stem (ST), flower (FL) and root (RO). (B–I) Promoter activities of tion in this study. In the study reported here, we characterized the the AFBA1 gene. Histochemical GUS analysis of transgenic function of the AFBA1 gene in Arabidopsis using a reverse genetic lines in: (B) 4-day-old seedling; (C) cotyledon; (D) ProAFBA1:GUS approach. Our results support the role of AFBA1 as a positive stem; (E) rosette leaf; (F) cauline leaf; (G) fully open flower; (H) anther; and (I) sepal. Scale bar = 100 mm. Insets at bottom right of regulator of the ABA signaling pathway in Arabidopsis. (C), (D), (E), (F), (H) and (I) are an enlarged image of representative guard cells. An, anther; Fi, filament; Se, sepal. Results sequential enzymatic reactions modulated by the E1 (ubiquitin- AFBA1 is predominantly expressed in stomatal activating enzyme), E2 (ubiquitin-conjugating enzyme) and E3 guard cells, and ABA, salinity and drought ubiquitin ligases that provide the required substrate specificity. increase its expression Recently, a number of E3 ubiquitin ligases have been reported to regulate the ABA-mediated stress response by modulating a large The spatial expression pattern of AFBA1 was examined in five number of regulatory proteins, including transcription factors different plant tissues (rosette leaf, cauline leaf, stem, flower and (reviewed by Liu and Stone 2011, Lyzenga and Stone 2012). root) by quantitative real-time PCR (qRT-PCR). The expression One major type of E3 ligases is the Skp1–Cullin–F-box (SCF) level of AFBA1 was higher in the rosette leaf, cauline leaf and complex (Lechner et al.