The FBA Motif-Containing Protein AFBA1 Acts As a Novel Positive

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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 eua Paper Regular 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 protein–protein interaction for ubiquitination. F-box proteins contain an F-box motif at their N- terminus, which binds Skp1 to create a link to Cullin, and a C-terminal protein–protein interaction 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 understanding 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 proteins recognizes the substrates for ubiquitination and proteasome-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. 2006, Yu et al. 2007), in which F-box flower than in the root and stem (Fig. 1A). We then

575 Y. Y. Kim et al. | AFBA1 acts as a positive regulator of ABA response

Fig. 2 Expression patterns of the AFBA1 transcript in response to abiotic stresses. Two-week-old Arabidopsis (Col-0) plants were exposed to 300 mM NaCl, 100 mM ABA or dehydration for designated times, and the expression levels of AFBA1 were determined by qRT-PCR. Each bar

represents the expression of the AFBA1 gene transcript relative to that of the eIF4a1 gene as an internal control. The relative level of expression of Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 the AFBA1 gene in untreated seedlings of the WT type was set at 1.0. The data are given as the average value ± SD from three individual experiments.

constructed ProAFBA1:GUS transgenic lines harboring the The elevated expression of AFBA1 in response to the appli- 1,654 bp AFBA1 promoter region upstream of the start codon cation of ABA, to the exposure to abiotic stress (Fig. 2A) and in and observed that b-glucuronidase (GUS) was expressed in all the root tissue at the seedling stage (Fig. 1A, B) implies a role tissues of these transgenic lines. In the seedling stage, GUS ex- for AFBA1 during seed germination under conditions of abiotic pression was mainly observed in the root (Fig. 1B) and the stress. To verify this assumption, we examined the germination stomatal guard cells of the cotyledon (Fig 1C). In the repro- rate (based on radical emergence) according to changes in the ductive stage, strong GUS activity was predominantly observed sensitivity to ABA (Fig. 3D, E). On normal Murashige and Skoog in the guard cells of stem (Fig. 1D), rosette leaf (Fig. 1E), cauline medium (MS medium), the germination rates of the WT, afba1 leaf (Fig. 1F), and the anther and sepal of fully opened flowers mutant and AFBA1-Ox seeds were almost 100%. Following the (Fig. 1G–I), as well as in the anther and filament of the stamen application of ABA, the germination rate of WT seeds dropped (Fig. 1G). significantly with increasing ABA concentration, whereas that Based on the strong expression of AFBA1 in the stomatal of the afba1 seeds was clearly much higher than that of the the guard cells, AFBA1 expression patterns were studied in leaves WT, demonstrating the latter’s insensitivity to ABA stress. The following the application of ABA or exposure to high salinity Ox7-2 and Ox8-4 seeds, however, were more sensitive to the and drought, which are three major abiotic stresses known to ABA stress condition than the WT and afba1 seeds. For ex- trigger stomatal movement. Analysis of the expression pat- ample, on MS medium supplemented with 2 mM ABA, the tern of the AFBA1 transcript in the leaves revealed a rapid seed germination rate of the WT and Ox7-2 and Ox8-4 trans- increase of about 8- and 20-fold, respectively, by the ABA and genic lines was 18, 3 and 9%, respectively, whereas that of the high salt treatments at 3 h post-treatment, followed by a afba1 mutant was 37% (Fig. 3D, E). These results indicate that gradual decrease (Fig. 2). Similarly, the expression of the AFBA1 is able to affect ABA sensitivity during seed germination. AFBA1 transcript under drought stress rapidly increased The germination of Arabidopsis seed is severely inhibited by after 0.5 h and reached about 80-fold at 3 h post-treatment exposure to osmotic stresses (Zhu 2000, Kang et al. 2002, (Fig. 2). Gonzalez-Garcia et al. 2003) and ABA plays a crucial role in this process (Gonzalez-Garcia et al. 2003, Xiong and Zhu Altered expression of AFBA1 affects ABA 2003). To examine whether the observed alteration in sensitiv- sensitivity and plant response to osmotic stresses ity to ABA due to changes in AFBA1 expression also affected during seed germination response to osmotic stresses, we germinated seeds of the WT A T-DNA insertional mutant (SALK_133974C, designated and afba1 and AFBA1-Ox lines on MS medium containing dif- afba1) obtained from the Arabidopsis Biological Resource ferent concentrations of NaCl or mannitol. As shown in Fig. 4, Center was characterized, and a T-DNA insertion in the exon the germination rate of WT seeds decreased significantly with of the AFBA1 gene was identified (Fig. 3A). The homozygous increasing NaCl or mannitol concentrations, while the afba1 loss-of-function afba1 mutant was screened by genomic PCR and AFBA1-Ox lines (Ox7-2 and Ox8-4) were insensitive and and verified by reverse transcription–PCR (RT–PCR) (Fig. 3B). oversensitive, respectively, to NaCl or mannitol. For example, We then constructed transgenic Arabidopsis plants constitu- following exposure to 200 mM NaCl, the seed germination rate tively overexpressing AFBA1 under the control of the of the WT and Ox7-2 and Ox8-4 lines decreased to 21, 8 and Cauliflower mosiac virus (CaMV) 35S promoter, and used two 15%, respectively, of that in the absence of NaCl, whereas the of the independent single-copy transgenic lines, Ox7-2 and seed germination rate of the afba1 line was 55% (Fig. 4A, B). A Ox8-4, for further analysis (Fig. 3C). Under normal growth con- similar response pattern was seen in the mannitol treatment, ditions, both the afba1 mutant and AFBA1-Ox lines did not with the afba1 line showing insensitivity to the mannitol treat- differ morphologically from the wild type (WT) during seed ment and the AFBA1-Ox lines showing an increased sensitivity germination and growth. relative to the WT. Following exposure to 400 mM mannitol,

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Fig. 3 Sensitivity of the afba1 mutant and AFBA1-Ox lines to ABA. Characterization of the T-DNA insertional afba1 mutant and AFBA1-Ox lines (A–C). (A) Location of the T-DNA insertion in the afba1 mutant (white triangle). The black box represents coding sequences, and the inset (area of white box) represents the FBA motif. (B) RT–PCR analysis of AFBA1 expression in the wild type and afba1 mutant. eIF4a1 was used as the internal standard for the cDNA amount. (C) RT–PCR analysis of AFBA1 expression in the wild type and three independent AFBA1-Ox lines. eIF4a1 was used as the internal standard for the cDNA amount. (D) Effect of ABA on germination of the afba1 mutant and AFBA1-Ox lines. Seeds of the afba1 mutant, wild type and two AFBA1-Ox lines (Ox7-2 and Ox8-4) were germinated in MS medium supplemented with various concentrations of ABA. The germination rates were measured on day 3. Data are given as the average value ± SD of three individual experiments (n = 30). (E) Representative photos of the wild type, afba1 mutant and AFBA1-Ox plants germinated in the presence of 0 and 2 mM ABA. Pictures were taken on day 5. the seed germination rate of the WT and Ox7-2 and Ox8-4 lines we measured water loss rates of leaves detached from the 2- decreased to 60, 17 and 33%, respectively, of that in the absence week-old WT, afba1 and AFBA1-Ox plants. Consistent with the of mannitol, whereas seed germination of the afba1 line was phenotypes of the respective plant lines observed under the 85% (Fig. 4C, D). As expected, the alteration of AFBA1 gene drought condition, the detached leaves of afba1 plants lost expression affected sensitivities to osmotic stresses in accord- water faster than those of the WT plants, whereas the detached ance with the altered sensitivity to ABA. leaves from Ox7-2 plants showed the slowest water loss (Fig. 5B). Comparison of the stomatal closure responses of these Overexpression of AFBA1 confers drought three plant lines following a 2 h treatment with ABA revealed tolerance by closing stomata in an ABA- that, as expected, compared with the guard cells of the WT, hypersensitive manner those of the afba1 mutant line were insensitive to the ABA ABA plays a critical role in the regulation of stomatal closure treatment and the stomata remained in the unclosed position, and opening by perceiving the water deficit. As such, the above while the guard cells of the Ox7-2 line showed considerably results may imply that AFBA1 functions in the ABA-mediated increased sensitivity to ABA (Fig. 5C) and a narrower stomatal regulation of stomatal movement. To investigate whether the opening (Supplementary Fig. S1). These results indicate that altered expression of AFBA1 affects the sensitivity of plants to AFBA1 expression is closely correlated with the sensitivity of drought stress, we observed phenotypes of 2-week-old WT, stomatal movement to ABA, i.e. stomatal movement was in- afba1 knock-out (KO) and AFBA1-Ox plants under the drought sensitive in the KO (afba1mutant) line and hypersensitive in condition. Seven days after the start of the dehydration treat- the ABA-overexpressing (AFBA1-Ox) lines. Taken together, ment, afba1 mutant plants were almost completely wilted these data demonstrate that AFBA1 predominantly localized compared with the WT plants, whereas most plants of the in guard cells functions as a positive regulator of ABA-mediated Ox7-2 and Ox8-4 lines remained relatively healthy. Two days stomatal closure in response to water deficit. after the initiation of re-watering, 39% (123/318) of the WT AFBA1 overexpression suppresses the expression plants and only 8% (30/372) of the afba1 plants had recovered, while both AFBA1-Ox lines showed enhanced drought toler- of genes encoding PP2C, a negative regulator of ance and higher recovery rates [93% (318/341) of Ox7-2 plants ABA signaling and 72% (240/355) of Ox8-4 plants] (Fig. 5A). To confirm that To elucidate the regulatory role of AFBA1 on ABA responses in the reduced and enhanced drought tolerance of afba1 and planta, we examined the expression profiles of genes encoding AFBA1-Ox plants, respectively, could be attributed to the PP2C, a negative regulator of ABA signaling, such as AtPP2CA, altered water losses as a result of different transpiration rates, ABI1, ABI2, HAB1 and HAB2, as well as of an ABA-responsive

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Fig. 4 Effect of NaCl and mannitol stresses on germination of the afba1 mutant and AFBA1-Ox lines. Seeds of the afba1 mutant, wild type and two AFBA1-Ox lines (Ox7-2 and Ox8-4) were germinated in MS medium supplemented with various concentrations of (A) NaCl or (B) mannitol. The germination rates were measured on day 3. The data are given as the average value ± SD of three individual experiments (n = 30). Representative photos of the wild type, afba1 mutant and AFBA1-Ox plants germinated in the presence of various concentrations of (C) NaCl or (D) mannitol. Pictures were taken on day 5.

Fig. 5 Drought stress sensitivity of the afba1 mutant and AFBA1-Ox lines. (A) Response of the afba1 mutant, wild type and two AFBA1-Ox lines (Ox8-4 and Ox7-2) under water-deficit stress conditions. Two-week-old plants (top panel) were dehydrated by withholding water for 7 d (middle panel), and then rewatered for 2 d (bottom panel). (B) Water loss rates of the wild type, afba1 and AFBA1-Ox lines. Leaves of similar developmental stages (from the fifth to the eighth) were excised from 3-week-old plants, kept at room temperature and weighed at various time points after detachment. The data are given as the average value ± SD from three individual experiments (n = 20). (C) Measurements of stomatal aperture in response to ABA. Rosette leaves of 3-week-old plants were detached and floated abaxial-side down on opening solution for 3 h prior to ABA treatment. The data represent the average value ± SD from three individual experiments (n = 30). Different letters above the bars indicate significantly different values between treatments (P < 0.05, t-test).

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Fig. 6 Expression patterns of genes related to ABA signaling in the wild type, afba1 mutant and AFBA1-Ox line. Seedlings grown on MS medium were either untreated or treated with 100 mM ABA. Gene expression of RD22, AtPP2CA, ABI1, ABI2, HAB1 and HAB2 before and after ABA treatment as determined by qRT-PCR analysis is shown. Each bar represents the amount of the transcript of a gene relative to that of the eIF4a1 gene as an internal control. The relative level of expression of each gene in untreated seedlings of the wild type was set at 1.0. The data are given as the average value ± SD of three experiments. gene, RD22, by qRT-PCR in WT, afba1 and AFBA1-Ox seedlings C-terminus by fluorescence microscopy revealed that the exposed to 100 mM ABA. In the absence of exogenous ABA AFBA1:smGFP signal was localized in the nucleus (Fig. 7A). application (i.e. at 0 time point), the expression levels of Searches using the SMART (http://smart.embl-heidelberg. genes encoding PP2C as well as RD22 were not significantly de/) and TAIR (https://www.arabidopsis.org/) databases re- different among the three lines. After exposure to exogenous vealed that AFBA1 possesses only a single FBA motif at its C- ABA, however, the expression of RD22 was induced comparably terminus. FBA motifs are mostly found in the C-terminal region across the three lines as expected, whereas the expression of of the F-box protein subfamily (Winston et al. 1999, Xu et al. genes coding for PP2C was strongly enhanced in the afba1 2009) and are known to bind to other proteins targeted for mutant but diminished in the Ox7-2 line (Fig. 6). This result ubiquitination (Gou et al. 2009, Han et al. 2015). Based on this implies that AFBA1 probably acts as a positive regulator of ABA knowledge as well as our finding that AFBA1 did not contain a responses by virtue of suppressing genes encoding major nega- classical DNA-binding domain, we speculated that AFBA1 tar- tive regulators of ABA signaling. geting in the nucleus interacts with regulatory proteins, such as transcription factors, to regulate ABA-mediated responses. To AFBA1 directly interacts with MYB44 and possibly identify functional target protein(s) of AFBA1, we carried out regulates its stability yeast two-hybrid screening using the cDNA library obtained A subcellular localization study of the AFBA1 protein fused with from ABA-treated WT plants, screening three transcription fac- the soluble modified green fluorescent protein (smGFP) at its tors, namely, MYB44, TCP4 and TCP14, under highly stringent

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Fig. 7 Interaction of AFBA1 with MYB44. (A) Subcellular localization of AFBA1:smGFP protein. Protoplasts were isolated from leaves of 2-week- old transgenic plants and observed by fluorescence microscopy. Bright, bright field image; smGFP, smGFP fluorescence; Chloroplast, chloroplast autofluorescence; Merge, merged smGFP and chloroplast autofluorescence. (B) Interaction between AFBA1 and MYB44 in yeast cells. The growth of transformed cells was monitored on control SD/-L-W medium (bottom panel) or on selective SD/-L-W-H medium (upper panel). BD and AD indicate empty vectors of pGBKT7 and pGADT7, respectively. (C) BiFC analysis of the interaction of AFBA1 with MYB44 in epidermal cells of Nicotiana benthamiana. Each fusion protein was co-expressed in N. benthamiana leaves by agroinfiltration. A yellow-green signal resulted from the physical association of the two indicated proteins in the nuclei. YFP, yellow fluorescent protein fluorescence; Merge, merged YFP image, chloroplast florescence and bright field image. The combination of bZIP63-YN and bZIP63-YC was used for the positive control. Scale bar = 50 mm (D) Western blotting after protoplast transfection assay. Protoplasts isolated from the wild type and AFBA1-Ox plants were transfected with the MYB44:smGFP plasmid and then incubated without ABA (–ABA), with ABA (+ABA) or with a mixture of ABA and MG132 (+ABA +MG132). After 3 h, changes in MYB44:smGFP protein levels were detected by Western blotting using GFP antibody. b-Actin was used as a loading control.

conditions (Supplementary Table S1). Of these, MYB44, which resembled those in the myb44 mutant and MYB44-Ox plants is known to be a regulator of ABA signaling in Arabidopsis (Jung subjected to ABA-mediated stresses. This led us to hypothesize et al. 2008), was selected for further study. that AFBA1 acts as a transcriptional activator for MYB44 or, The interaction between AFBA1 and MYB44 was confirmed alternatively, regulates the stability of MYB44 protein because in the yeast two-hybrid assay, with yeast cells growing only on a the FBA motif is an essential component of the F-box for rec- SD/-L-W-H medium, when AFBA1 was expressed in the pres- ognition of its substrate(s) for degradation. However, our study ence of MYB44 (Fig. 7B, upper panel). To reconfirm the inter- on whether this interaction could lead to activation or repres- action between AFBA1 and MYB44 in vivo, we performed the sion of the reporter gene expression in yeast revealed that the bimolecular fluorescence complementation (BiFC) assay using reporter gene expression appeared to be neither activated nor agro-infiltrated Nicotiana benthamiana leaves. Co-expression of repressed by the interaction of AFBA1 and MYB44 (data now AFBA1-YN and MYB44-YC in N. benthamiana leaf resulted in a shown). Using the protoplast transfection assay, we then strong yellow fluorescence in the nucleus at 36 h post-infiltra- looked at whether overexpression of AFBA1 could increase tion (Fig. 7C, bottom panels). A combination of bZIP63-YN and the stability of the MYB44 protein. Western analysis revealed bZIP63-YC was used as a nuclear-localized BiFC control (Fig. that the amount of MYB44:smGFP protein transfected separ- 7C, upper panels). These results indicate that AFBA1 forms a ately to Ox7-2 and WT protoplasts remained equal in the ab- complex with MYB44 in the nucleus. sence of ABA. In contrast, after 3 h of incubation in the The changes in ABA sensitivity and the expression profiles of presence of 100 mM ABA under the light condition, an essential the genes encoding PP2C (ABI1, ABI2, AtPP2CA, HAB1 and criterion for MYB44 degradation, the Ox7-2 protoplasts con- HAB2) in the afba1 mutant and AFBA1-Ox lines, respectively, tained a considerably higher amount of MYB44:smGFP protein

580 Plant Cell Physiol. 58(3): 574–586 (2017) doi:10.1093/pcp/pcx003

than the WT protoplasts (Fig. 7D). In addition, to determine 2001). Seeds and vegetative tissues of HAB1-overexpressing whether the MYB44 degradation occurred via the UPS, Ox7-2 plants were found to have reduced ABA sensitivity (Saez and WT protoplasts transfected with the MYB44:smGFP pro- et al. 2004), while ABA-hypersensitive phenotypes in the tein were treated with a mixture of ABA (100 mM) and the regulation of seed germination and stomatal closure were proteasome inhibitor MG132 (50 mM). As expected, MG132 observed in a T-DNA mutant of HAB1 (Leonhardt et al. inhibited MYB44 degradation that is accelerated following ex- 2004). The reduced expression of these PP2C-encoding genes posure to ABA (Fig. 7D), suggesting that the MYB44 degrad- observed in plants overexpressing MYB44, which has been sug- ation was dependent on the UPS. These results indicate that the gested to be a positive regulator of ABA signaling, led to an interaction of AFBA1 with MYB44 under ABA-mediated re- enhanced drought-tolerant phenotype in Arabidopsis (Jung sponses increased the stability of MYB44 during UPS-mediated et al. 2008). protein degradation. To summarize, our ﬁndings indicate that AFBA1 is a novel

positive regulator of ABA responses and that changes in AFBA1 Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 expression are sufficient to elicit changes in ABA responses, Discussion including ABA-mediated inhibition of seed germination, stomatal closure and drought tolerance. AFBA1 acts as a novel positive regulator of ABA response in Arabidopsis AFBA1 possibly modulates the stability of positive ABA is an essential mediator in triggering plant responses to regulators of the ABA-mediated response most of the common abiotic stresses, including drought, salin- Based on our study results, AFBA1 possesses a C-terminal FBA ity, high temperature, oxidative stress and cold (Finkelstein motif without any functionally known motif(s) such as the F- et al. 2002, Xiong et al. 2002), and plays a critical role in regulat- box motif and is a novel positive regulator of ABA responses. ing seed germination and stomatal closure under water-deficit Our survey of the Arabidopsis genome (http://www.arabidop- conditions (Zhu 2002, Gonzalez-Garcia et al. 2003, Xiong and sis.org/) identified at least 27 putative genes encoding FBA Zhu 2003). Our results provided several lines of evidence indi- motif-containing proteins that do not belong to the F-box pro- cating that AFBA1 acts as a novel positive regulator of ABA tein family, including AFBA1, as a novel small gene family responses. First, AFAB1 was predominantly expressed in the (Supplementary Table S2), but the functions of all of these guard cells of all vegetative tissues assayed as well as in root genes remain unknown. The FBA motif is generally found in the tissue (Fig. 1), and its expression was strongly induced by the largest subfamily of the F-box proteins, in 206 of the approxi- exogenous application of ABA and exposure to osmotic stres- mately 700 F-box proteins in the Arabidopsis genome (Gagne ses, such as high salt (NaCl) and drought (Fig. 2), suggesting et al. 2002, Kuroda et al. 2002, Schumann et al. 2011). The FBA that AFBA1 is involved in the ABA response. Secondly, seeds of motif in F-box proteins recognizes the target substrates for the afba1 mutant (KO) were insensitive to ABA and osmotic UPS-mediated degradation (del Pozo and Estelle 2000, Gou stress, whereas those of the AFBA1-Ox lines (AFBA1 overexpres- et al. 2009, Han et al. 2015); as such, the FBA motif is essential sors) showed a hypersensitive response to these same stresses for the normal function of F-box proteins (Han et al. 2015). To (Figs. 3, 4), implying that AFBA1 expression affects ABA sensi- date, seven FBA motif-containing F-box proteins have been tivity and positively regulates the ABA-mediated inhibition of functionally characterized in Arabidopsis; these are involved seed germination. Thirdly, the afba1 mutant plants exhibited in pathogen responses (Kim and Delaney 2002, Gou et al. reduced drought tolerance and increased water loss compared 2009, Gou et al. 2012), root development (Dong et al. 2006), with WT plants. Consistent with this result, the guard cells of ethylene signal transduction (Qiao et al. 2009) and ABA re- afba1 mutant plants were more sensitive to ABA-induced sto- sponses (Zhang et al. 2008, Peng et al. 2012). matal closure. In contrast, AFBA1-Ox plants appeared to be The functions of the UPS in modulating ABA responses in more tolerant than WT plants to drought stress and lost plants have been studied by adjusting the abundance of many water rather slowly by promoting stomatal closure (Fig. 5). ABA-responsive regulatory proteins, including transcription Taken together, the extent of drought tolerance was positively factors, as the targets of these proteins have been extensively correlated with the expression level of the AFBA1 gene in two characterized and reviewed (Lyzenga and Stone 2012, Stone overexpressing transgenic lines, suggesting that AFBA1 plays a 2014). The involvement of F-box proteins, TUBBY-like protein positive role in ABA-induced stomatal closure and in the regu- AtTLP9 (Lai et al. 2004), DOR (Drought Tolerance Repressor) lation of drought response. Fourthly, the results of our gene (Zhang et al. 2008) and FOA1 (F-box overexpressed/oppressed expression studies demonstrate that genes encoding a group of ABA signaling) (Peng et al. 2012), in ABA signal transduction serine/threonine PP2Cs, such as ABI1, ABI2, AtPP2CA, HAB1 has also been suggested. Lai et al. (2004) proposed that an F-box and HAB2, all of which are recognized major negative regulators protein AtTLP9 modulates ABA-mediated responses, probably of the ABA signal transduction pathway (Hirayama and by targeting a negative regulator of ABA signaling for degrad- Shinozaki 2007), were up-regulated in the afba1 mutant and ation. These authors performed seed germination assays and down-regulated in the AFBA1-Ox lines following the exogenous observed that AtTLP9 mutant plants and AtTLP9-overexpress- application of ABA (Fig. 6). The Arabidopsis mutants abi1-1 ing plants were ABA insensitive and ABA hypersensitive, re- and abi2-1 exhibit ABA-insensitive seed germination and spectively (Lai et al 2004). The F-box protein DOR functions reduced drought tolerance (Leung et al. 1997, Merlot et al. as a negative regulator of ABA-mediated stomatal closure

581 Y. Y. Kim et al. | AFBA1 acts as a positive regulator of ABA response

(Zhang et al. 2008). These authors demonstrated that plants test this hypothesis, we examined whether the overexpression of carrying a null mutation in DOR had enhanced drought toler- MYB44 in the afba1 mutant was sufﬁcient to confer on this KO ance and accumulated more ABA than WT plants in response mutant normal ABA sensitivity in germination. We constructed to drought, while the transgenic plants overexpressing DOR 35S:MYB44/afba1 (T2) transgenic lines (Supplementary Fig. S3A) were more susceptible to drought stress (Zhang et al. 2008). and monitored the germination rates of WT, afba1 mutant and The F-box protein FOA1 also functions as a negative regulator two independent 35S:MYB44/afba1 lines on MS medium supple- in ABA signal transduction (Peng et al. 2012), with the foa1 mented with 0 or 2 mM ABA over 5 d. When germinated in the mutant showing a lower germination rate, increased stomatal absence of ABA, there were no obvious differences during seed opening and relative hypersensitivity to ABA in comparison germination among all tested lines (Supplementary Fig. S3B). In with the WT plants in response to the application of ABA, thepresenceof2mMABA,the35S:MYB44/afba1 lines maintained while its overexpression lines showed a lower sensitivity to ABA-insensitive phenotypes relative to the WT, while their ger-

ABA than the WT. Interestingly, two F-box proteins, DOR mination rates were considerably but not significantly lower than Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 and FOA1, contain the FBA motif at their C-terminus for the that of the afba1 mutant (Supplementary Fig. S3C). Moreover, in protein–protein interaction domain, but their targets and regu- an additional effort to identify more interactors with AFBA1, we latory mechanisms during ABA responses have not been tested whether AFBA1 could interact with each of the other three characterized. R2R3 MYB transcription factors (MYB70, MYB73 and MYB77) AFBA1 was found to be localized in the nucleus of leaf that form the S22 subfamily of R2R3MYB transcription factors protoplasts (Fig. 7A) and not to retain transcriptional activa- together with MYB44 (Persak and Pitzschke 2013). We confirmed tion activity in yeast cells (data not shown), leading us to infer the direct interaction of AFBA1 with MYB70, MYB73 and MYB77 that AFBA1 modulates responses to ABA through its inter- by BiFC assays (data not shown), although protein functions of action with the transcription factors that could regulate the these R2R3 MYB transcription factors during the ABA response expression of genes encoding PP2C under the ABA-mediated have yet to be elucidated. These data indicate that the overexpres- stress condition. Based on the results of the yeast two-hybrid sion of MYB44 partially recovered ABA-insensitive phenotypes of screening assay, we identified three putative interacting target the afba1 mutant, and the target transcription factors including transcription factors, TCP4, TCP14 and MYB44. MYB44 was MYB44, MYB70, MYB73, MYB77, TCP4 and TCP14 could be chosen for further analysis, and the results of yeast two- involved in the function of AFBA1 during ABA-mediated re- hybrid screening and the BiFC assays confirmed the direct inter- sponses in plants. However, further studies are needed to elucidate action between AFBA1 and MYB44 (Fig. 7B, C). Subsequent their detailed functions in the ABA response. protoplast transfection assays demonstrated that MYB44 deg- On the other hand, in the absence of exogenous ABA appli- radation via the UPS is prevented more in the AFBA1-Ox proto- cation, the expression levels of PP2C-encoding genes in the plasts than in WT protoplasts (Fig. 7D), suggesting that AFBA1 AFBA1-Ox and afba1 mutant were similar to that in the WT stabilizes the MYB44 protein and prevents its proteolysis by the (Fig. 6). These data indicated that AFBA1 would not affect the UPS under ABA-mediated responses. stabilization of MYB44 protein in the absence of exogenous Interestingly, whether the R2R3-type transcription factor ABA application, and thereafter led us to hypothesize that MYB44/MYBR1 acts as a positive or negative regulator of ABA ABA-inducible MYB44 protein might not be a target for UPS- signaling remains controversial to date. Jung et al. (2008) and mediated protein degradation under normal growth conditions Persak and Pitzschke (2013) suggested that MYB44 functioned due to its lower expression level, or that the possible destabil- as a positive regulator of ABA-mediated responses, while other ization of MYB44 protein in the afba1 mutant in the absence of research teams demonstrated that MYB44 negatively regulated exogenous ABA application might be minimized by the com- ABA responses (Huang et al. 2007, Jaradat et al. 2013, Li et al. pensation by other FBA motif-containing protein(s) having a 2014). However, AFBA1-Ox plants showed similar responses to redundant function with the AFBA1. MYB44-overexpressing plants reported by Jung et al. (2008), Taken together, our findings suggest that AFBA1 is a positive such as hypersensitivity to ABA during seed germination, drought regulator in ABA-mediated responses, probably by modulating tolerance through the acceleration of stomatal closure, reduced the stability of transcription factors acting as positive regula- expression of PP2C-encoding genes (ABI1, ABI2, AtPP2CA, HAB1 tor(s) of ABA signal transduction. Based on our results as well as and HAB2) under the ABA-mediated stress condition and no on those of earlier studies, we infer that AFBA1 as well as many altered expressions of major ABA-responsive genes including as yet uncharacterized F-box protein(s) containing a functional RD29A, RAB18 (Supplementary Fig. S2)andRD22. Likewise, the FBA motif share their target transcription factors, such as posi- KO mutant of MYB44 and the afba1 mutant also show similar tive regulators of ABA signaling (e.g. MYB44) and can competi- responses, such as susceptibility to drought. However, MYB44- tively combine with these to modulate their degradation during overexpressing plants showed growth retardation and delayed ABA-mediated responses (Fig. 8). More precisely, the targets flowering, and the myb44 mutant did not exhibit the ABA-insensi- forming a complex with AFBA1 cannot be recognized by the tive response during seed germination, in contrast to AFBA1-Ox FBA-containing F-box proteins for UPS-mediated protein deg- plants and the afba1 mutant, respectively (Fig. 3), suggesting that radation. However, further investigations are needed to deter- other target transcription factors in addition to MYB44 may also mine the exact regulatory mechanism(s) of AFBA1 in ABA be involved during the function of AFBA1 in response to ABA. To signaling.

582 Plant Cell Physiol. 58(3): 574–586 (2017) doi:10.1093/pcp/pcx003

Identification of the T-DNA insertion line and construction of the AFBA1-overexpressing lines The T-DNA insertion mutant (SALK_133974C) was obtained from the Salk Institute via the Nottingham Arabidopsis Stock Center (NASC) for mutant analysis. The homozygous lines were identified by genomic PCR using primers LBa1 (50-TGG TTC ACG TAG TGG GCC ATC G-30), AFBA1-S (50-ATC TTC ACA CTG AAA CCT TTC GAG T-30) and AFBA1-AS (50-TTA TGA AAT AGA GAT TAA ACT CTG GAA ATA ACA-30). Once the homozygous lines were identified, leaves were collected and stored at –70C for RNA isolation and gene expression analysis. To generate AFBA1-overexpressing transgenic Arabidopsis plants, we cloned the AFBA1 full-length (GenBank accession No. KY084522) open reading frame (ORF; 915 bp) into the binary plant transformation vector pCAMBIA Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 1300 (Cambia) and then transformed the construct into A. tumefaciens strain GV3101 using the freeze–thaw method as described by Xie et al. (2001). Agrobacterium tumefaciens-mediated in planta transformation of Arabidopsis was performed according to the method of Clough and Bent (1998). Seeds from these plants were harvested, and transgenic plants were screened on 1 MS medium containing 1% (w/v) sucrose and 50 mg l1 hygro-

Fig. 8 Proposed model of AFBA1 during ABA-mediated responses in mycin, transferred to soil and grown to maturity. Three independent T3 homo- Arabidopsis. Dashed lines indicate the proposed actions of AFBA1 by zygous lines were selected for further study. direct intermolecular competition with the uncharacterized F-box protein containing an FBA motif for access to transcription factors Subcellular localization of the smGFP fusion (TFs), such as MYB44, that function as positive regulators of ABA proteins signaling, as based on the results of this study. Solid lines represent Transgenic Arabidopsis plants expressing the AFBA1:smGFP chimeric protein previously deﬁned pathways. T-shaped lines and arrows refer to in- or smGFP alone (control) were generated as described above. Protoplasts iso- hibition and promotion, respectively. lated from 2-week-old transgenic plant leaves were observed by ﬂuorescence microscopy (model BX-51; Olympus Corp.).

Materials and Methods Seed germination assays under stress conditions Seeds of the WT, afba1 mutant and AFBA1-Ox lines (Ox7-2 and Ox8-4) were Plant growth conditions surface-sterilized and then placed at 4C for 3 d in the dark prior to germination. Germination assays were carried out in three replicates of 30 seeds each. Arabidopsis thaliana ecotype Columbia (Col) was used in this work. Seeds of the The effect of ABA, NaCl or mannitol on germination was determined in MS WT and all transgenic plants were germinated on MS medium (Murashige and medium supplemented with various concentrations of ABA (1, 2 and 5 mM), Skoog 1962) supplemented with 1% (w/v) sucrose and 1% (w/v) agar, pH 5.8. NaCl (100, 150 and 200 mM) or mannitol (100, 200 and 300 mM). Seeds were Seedlings were transferred to Sunshine potting soil #5 (Sun Gro Horticulture) considered to have germinated when the radicle protruded through the seed and grown under fluorescent light (intensity 2,500 lux), 40% relative humidity coat; all seeds were scored daily for 7 d. The germination rate was calculated as a and a long-day photoperiod (16 h light/25C; 8 h dark/22C). percentage of the total number of seeds plated. GUS histochemical staining analysis Assessment of drought tolerance and water loss To construct ProAFBA1:GUS transgenic plants, we first amplified a 1,654 bp frag- rate ment of the promoter region upstream from the start codon of the AFBA1 gene by PCR using Arabidopsis genomic DNA and then cloned the fragments into For the drought treatment, 2-week-old seedlings of the WT, afba1 mutant and the pBI101 binary vector (Clontech). The binary plasmids were then trans- AFBA1-Ox lines (Ox7-2 and Ox8-4) planted side by side in a pot to reduce formed into Agrobacterium tumefaciens strain GV3101 using the freeze–thaw experimental variations were exposed to the dehydration condition by with- method as described by Xie et al. (2001). The construct was introduced into holding of water for 10 d, and then rewatered for 2 d. The experiment was Arabidopsis plants by the floral dip method (Clough and Bent 1998) with minor carried out in triplicate, and the number of surviving plants was scored as the modifications. Transgenic plants were screened on 1 MS medium containing percentage of the total plants treated. To measure the water loss rates of the 1% (w/v) sucrose and 50 mg l1 kanamycin, transferred to soil and grown to WT, afba1 and AFBA1-Ox (Ox7-2) plants, we removed 20 rosette leaves at maturity. For GUS histochemical staining, samples were incubated overnight similar developmental stages from 3-week-old plants and placed the leaves with X-Gluc solution [1 mM 5-bromo-4-chloro-3-indolyl-b-D-glucuronate, on filter paper with the abaxial side facing downwards, at room temperature;

10 mM Na2EDTA, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyan- the detached leaves were then weighed at 0, 15, 30, 60 and 120 min. The ide, 0.1% (v/v) Triton X-100, 50 mM NaPO4 buffer, pH 7.0] at 37 C. After experiment was carried out in triplicate, and the water loss was calculated as staining, the Chl-containing tissues were cleared in an ascending series of etha- the percentage of initial fresh weight at each time point. nol (50, 70 and 100%; 30 min at each concentration) and then observed under a microscope (model BX-51; Olympus Corp.). Measurement of stomatal aperture Thirty rosette leaves of 3-week-old WT, afba1 and AFBA1-Ox (Ox7-2) plants Plant treatments were detached and ﬂoated on opening solution (5 mM MES-KOH, 20 mM KCl, For salt and ABA treatments, 2-week-old plants were sprayed with 300 mM pH 6.15) under light for 1 h to open the stomata fully; ABA was then added to NaCl and 100 mM ABA solutions. For drought treatment, leaves detached from the solution and stomatal closing was assessed. After 2 h of exposure to ABA, 2-week-old plants were put on a ﬁlter paper under light at 25C. Samples taken stomatal apertures in epidermal peels were observed and measured under a at different time points after treatments were frozen in liquid nitrogen and microscope (model BX-51; Olympus Corp.). Control experiments (no ABA) stored at 80C for further use. were performed in parallel. The experiment was carried out in triplicate.

583 Y. Y. Kim et al. | AFBA1 acts as a positive regulator of ABA response

Analysis of RNA expression by exposing the ﬁlm to ECL Western blotting reagent (Pharmacia

Õ Biotechnology). Total RNA was isolated from plants using TRI Reagent (Molecular Research Center) according to the manufacturer’s instructions. The amount and quality of total RNA was checked by spectrophotometry (OD: 260/280) and 5 mgof total RNA was reverse transcribed using the Transcriptor First Strand cDNA Supplementary data Synthesis kit (Roche Diagnostics). The cDNA product was diluted 20-fold, and 1 ml of diluted cDNA was used in a 20 ml PCR. The RT–PCR analysis was per- Supplementary data are available at PCP online. Õ formed using the LightCycler 480 II system (Roche Diagnostics) with the KAPA SYBR FAST qPCR kit (Kapa Biosystems). The thermal cycling conditions con- sisted of 26–35 cycles at 95C for 20 s, 53C for 20 s and 72C for 40 s, followed Funding by a 3 min ﬁnal extension at 72C. Relative amounts of transcripts were calcu-

lated by the comparative CT (threshold cycle number at the cross-point be- This work was supported by the National Research Foundation tween the amplification plot and threshold) method, and values were of Korea (NRF) funded by the Ministry of Science and normalized to the internal eIF4al control. Each RNA sample was run in triplicate Technology [grant No. NRF-2014R1A2A2A01005974]; the Downloaded from https://academic.oup.com/pcp/article/58/3/574/2981967 by guest on 28 September 2021 and repeated in two independent sets. All gene-specific primers used are presented in Supplementary Table S3. Rural Development Administration, Republic of Korea [a grant (PJ011065) from the Next-Generation BioGreen 21 Yeast two-hybrid interaction assay Program]; and Korea University. The yeast two-hybrid MATCHMAKER system (Clontech) was used for the analysis of protein–protein interactions according to the manufacturer’s proto- cols. The coding sequence of AFBA1 was cloned into the pGBKT7 vector as a Disclosures bait, and each coding sequence of MYB44 was cloned into the pGADT7 vector to generate preys. All gene-specific primers used to amplify each gene are The authors have no conflicts of interest to declare. presented in Supplementary Table S3. For the co-expression assays, AFBA1 and MYB44 were co-transformed into yeast strain AH109, and successfully co- transformed cells were then screened on the synthetic dropout (SD) medium References lacking tryptophan and leucine (SD/-L-W). To detect a specific interaction between two proteins, yeast cells were streaked on control SD/-L-W medium or Acharya, B.R., Jeon, B.W., Zhang, W. and Assmann, S.M. (2013) Open on selective SD medium lacking tryptophan, leucine and histidine (SD/-L-W-H) Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis supplemented with 50 mM 3-amino-1,2,4-triazole (3-AT). The yeast cells were guard cells. New Phytol. 200: 1049–1063. incubated for 5 d at 30 C and growth was monitored. Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Bimolecular fluorescence complementation assay Plant J. 16: 735–743. using agroinfiltration del Pozo, J.C. and Estelle, M. (2000) F-box proteins and protein degradation: an For the BiFC assay, AFBA1 full-length coding sequences were cloned into the emerging theme in cellular regulation. Plant Mol. Biol. 44: 123–128. pUC-SPYNE vector (YN) and MYB44 full-length coding sequences were cloned Dharmasiri, N., Dharmasiri, S., Weijers, D., Lechner, E., Yamada, M., Hobbie, into the pUC-SPYCE vector (YC) (Walter et al. 2004) to make constructs L., et al. (2005) Plant development is regulated by a family of auxin AFBA1-YN and MYB44-YC, respectively. receptor F box proteins. Dev. Cell 9: 109–119. For agroinfiltration, all plasmids were transformed into A. tumefaciens Dill, A., Thomas, S.G., Hu, J., Steber, C.M. and Sun, T.-p. (2004) The strain GV3101. Each single colony was grown in YEP medium overnight at Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repres- 28C, and then cells were harvested by centrifugation and resuspended in sors for gibberellin-induced degradation. Plant Cell 16: 1392–1405.

infiltration buffer (10 mM MgCl2, 10 mM MES and 100 mM acetosyringone, Ding, Z., Li, S., An, X., Liu, X., Qin, H. and Wang, D. (2009) Transgenic pH 5.7) to adjust the cell concentration to 0.4 at OD600. Agrobacterium cells expression of MYB15 confers enhanced sensitivity to abscisic acid harboring constructs were mixed in an equal ratio with Agrobacterium cells and improved drought tolerance in Arabidopsis thaliana. J. Genet. expressing the p19 silencing suppressor to enhance the transgene expression. Genomics 36: 17–29. Prepared agrobacterium cells were infiltrated into N. benthamiana leaves using Dong, L., Wang, L., Zhang, Y.e., Zhang, Y., Deng, X. and Xue, Y. (2006) An a needleless syringe. After 3 d, signals in agroinfiltrated leaves were observed by auxin-inducible F-box protein CEGENDUO negatively regulates auxin- confocal microscopy (model LSM510 META, Carl Zeiss MicoImaging). mediated lateral root formation in Arabidopsis. Plant Mol. Biol. 60: 599–615. Protoplast transfection assays and Western blot Finkelstein, R.R., Gampala, S.S. and Rock, C.D. (2002) Abscisic acid signaling analysis in seeds and seedlings. Plant Cell 14: S15–S45. Gagne, J.M., Downes, B.P., Shiu, S.-H., Durski, A.M. and Vierstra, R.D. (2002) For transfection, we cloned the MYB44 cDNA full-length ORF (918 bp) into the binary plant transformation vector pCAMBIA 1300 (Cambia). The construct The F-box subunit of the SCF E3 complex is encoded by a diverse was then transfected into protoplast cells of WT and AFBA1-Ox (Ox7-2) plants superfamily of genes in Arabidopsis. Proc. Natl. Acad. Sci. USA 99: by the polyethylene glycol-mediated method (Yoo et al. 2007). After transfec- 11519–11524. tion, protoplasts cells were divided into three aliquots and incubated in W5 Gonza´lez-Garcıá, M.P., Rodrı´guez, D., Nicola´s, C., Rodrı´gu‘ez, P.L., Nicola´s, solution without ABA, with 100 mM ABA or with 100 mM ABA plus 50 mM G. and Lorenzo, O. (2003) Negative regulation of abscisic acid signaling MG132, at 22C for 3 h, following which the protoplasts were harvested and by the Fagus sylvatica FsPP2C1 plays a role in seed dormancy regulation resuspended in sonication buffer [25 mM HEPES, pH 7.5, 1 mM dithiothreitol and promotion of seed germination. Plant Physiol. 133: 135–144.

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