F-box protein RAE1 regulates the stability of the aluminum-resistance transcription factor STOP1 in Arabidopsis Yang Zhanga,b,1, Jie Zhanga,1, Jinliang Guoa,b,1, Fanglin Zhoua, Somesh Singha, Xuan Xub, Qi Xiec, Zhongbao Yangd, and Chao-Feng Huanga,b,2 aNational Key Laboratory of Plant Molecular Genetics, Shanghai Center for Plant Stress Biology, Center of Excellence for Molecular Plant Sciences, Chinese Academy of Sciences, 200032 Shanghai, China; bCollege of Resources and Environmental Sciences, Nanjing Agricultural University, 210095 Nanjing, China; cState Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 100101 Beijing, China; and dKey Laboratory of Plant Cell Engineering and Germplasm Innovation, Ministry of Education, College of Life Science, Shandong University, 250100 Jinan, China Edited by Luis Herrera-Estrella, Center for Research and Advanced Studies, Irapuato, Mexico, and approved November 19, 2018 (received for review August 21, 2018) Aluminum (Al) toxicity is a major factor limiting crop production expression of STOP1-downstream Al-resistance genes, including on acid soils, which represent over 30% of the world’s arable land. AtALMT1, AtMATE, and ALS3, is induced by Al (8, 12). These Some plants have evolved mechanisms to detoxify Al. Arabidopsis, results suggest the possibility that STOP1 might be regulated by for example, secretes malate via the AtALMT1 transporter to che- Al at posttranscriptional or posttranslational levels. late and detoxify Al. The C2H2-type transcription factor STOP1 plays a Ubiquitin-mediated protein degradation is an important post- crucial role in Al resistance by inducing the expression of a set of translational mechanism that regulates numerous biological processes genes, including AtALMT1. Here, we identify andcharacterizeanF- (13). Ubiquitin conjugation is mediated through the sequential box protein-encoding gene regulation of Atalmt1 expression 1 (RAE1) action of three enzyme complexes: ubiquitin-activating (E1), ubiquitin- that regulates the level of STOP1. Mutation and overexpression of conjugating (E2), and ubiquitin ligase (E3) enzymes. The E3 li- RAE1 increases or decreases the expression of AtALMT1 and other gases are responsible for substrate recognition and ubiquitin STOP1-regulated genes, respectively. RAE1 interacts with and pro- transfer. Among various E3 families, the SKP1–CUL1–F-box (SCF) PLANT BIOLOGY motes the degradation of STOP1 via the ubiquitin-26S proteasome is the largest and best characterized and is composed of four major pathway, while Al stress promotes the accumulation of STOP1. We subunits: SKP1, Cullin1, RBX1, and an F-box protein (13, 14). find that STOP1 up-regulates RAE1 expression by directly binding to Cullin functions as a scaffold and interacts with SKP1 and RBX1 to the RAE1 promoter, thus forming a negative feedback loop between form a complex that generates the core ligase activity. F-box pro- STOP1 and RAE1. Our results demonstrate that RAE1 influences Al teins selectively interact with target proteins and deliver the targets resistance through the ubiquitination and degradation of STOP1. to the complex (15). The Arabidopsis genome contains nearly 700 genes that potentially encode F-box proteins (16). The F-box pro- aluminum toxicity | AtALMT1 | E3 ligase | low phosphate response | ubiquitination teins can be further divided into subgroups based on C-terminal protein-interaction domains. Members of the leucine-rich repeat – (LRR) subgroup are involved in the regulation of auxin, ethylene, pproximately 30 40% of the world arable land consists of jasmonate, and strigolactone hormone signaling (17–20), but no soils with a pH of 5.5 or lower (1). In these acidic soils, A roles in metal stress tolerance or signaling have been reported. aluminum (Al) is solubilized and becomes highly toxic to plants. Consequently, Al toxicity is a severe global problem for crops Significance growing on acid soils (2). To chelate and detoxify Al, some plants secrete organic acids, including malate, citrate, and oxalate (3–5). Arabidopsis plants, Aluminum (Al) toxicity is a major constraint of crop production for example, release both malate and citrate in response to Al on acid soils. Arabidopsis can secrete malate via the AtALMT1 stress, with malate being essential for Al detoxification (6–8). transporter to chelate and detoxify Al. The transcription factor The gene responsible for Al-activated secretion of malate in STOP1 is essentially required for Al resistance, mainly through roots, Al-activated malate transporter 1 (ALMT1), was first iden- the control of AtALMT1 expression. Here, we report an F-box tified in wheat and encodes an anion transporter/channel (9). protein RAE1 that regulates STOP1 stability. RAE1 interacts There are 12 ALMT members in Arabidopsis, and only AtALMT1 with STOP1 to promote STOP1 degradation via the ubiquitin- 26S proteasome pathway, whereas Al stress stabilizes STOP1. is indispensable for root malate secretion and Al detoxification Together, our results reveal an important role for RAE1 in the (7). Unlike TaALMT1, whose expression is largely constitutive and regulation of Al resistance. unaffected by Al stress (9), AtALMT1 expression is induced by Al in all Arabidopsis ecotypes (7). In addition to Al, plant hormones Author contributions: Y.Z., J.Z., J.G., and C.-F.H. designed research; Y.Z., J.Z., J.G., F.Z., S.S., and other stresses such as indole-3-acetic acid (IAA), abscisic acid and X.X. performed research; Q.X. and Z.Y. contributed new reagents/analytic tools; Y.Z., (ABA), low pH, and hydrogen peroxide trigger AtALMT1 tran- J.Z., J.G., and C.-F.H. analyzed data; and C.-F.H. wrote the paper. scription (10), which suggests that the regulation of AtALMT1 The authors declare no conflict of interest. expression is complex. The mechanisms that underlie the regula- This article is a PNAS Direct Submission. tion of AtALMT1 expression, however, are not fully understood. Published under the PNAS license. Through a forward genetic screen of mutants exhibiting in- Data deposition: The sequences reported in this paper have been deposited in the NCBI creased sensitivity to proton toxicity, a zinc-finger transcription Sequence Read Archive database (accession nos. SRR6781578 and SRR6781579). factor sensitive to proton rhizotoxicity 1 (STOP1) was identified 1Y.Z., J.Z., and J.G. contributed equally to this work. and found to be critical for both proton and Al tolerance (11). 2To whom correspondence should be addressed. Email: [email protected]. Mutation of STOP1 greatly suppresses AtALMT1 transcription (11), This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. demonstrating that STOP1 is a crucial regulator of AtALMT1 ex- 1073/pnas.1814426116/-/DCSupplemental. pression. STOP1 transcription is not affected by Al stress, but the Published online December 17, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1814426116 PNAS | January 2, 2019 | vol. 116 | no. 1 | 319–327 Downloaded by guest on September 24, 2021 To gain insights into how AtALMT1 or STOP1 are modulated promoter to the uidA gene, which encodes β-glucuronidase at transcriptional or posttranscriptional levels, we conducted a (GUS) and selected a transgenic line that had a one-locus seg- forward genetic screen on an EMS-mutagenized population in regation pattern for GUS expression and introduced the trans- an AtALMT1 promoter-driven luciferase reporter (LUC) line. gene into the rae1-1 background by crossing. GUS staining Through this screen, we identified an F-box protein-encoding showed that GUS was expressed at higher levels in rae1-1 than in gene RAE1 that interacts with STOP1 to regulate its stability the WT in both the absence and presence of Al (SI Appendix, via the ubiquitin-26S proteasome pathway. Fig. S1 C and D). To determine whether rae1 mutations influence the expression Results of other Al-resistance genes, we analyzed the expression levels of Identification of Mutants with Altered AtALMT1 Expression. To AtMATE, ALS3, AtSTAR1, ALS1, and STOP1 in the WT and the identify components involved in the regulation of AtALMT1 rae1 mutants. The results showed that AtMATE and ALS3, two expression, we fused the AtALMT1 promoter to the luciferase additional target genes of STOP1 (Fig. 1 D and E) (8, 12), were reporter gene (pAtALMT1:LUC) and then introduced the con- also expressed at higher levels in the rae1 mutants than in the struct into the wild-type (WT) [Columbia-0 (Col-0)] seedlings. WT under both −Al and +Al conditions (Fig. 1 D and E). In We screened for single-copy transgenic lines displaying high lu- contrast, the expression levels of AtSTAR1 and ALS1, which are minescence and then performed thermal asymmetric interlaced not regulated by STOP1 (SI Appendix, Fig. S2 A and B) (12, 21), (TAIL)-PCR on these lines to pinpoint the location of the re- did not differ between the WT and the rae1 mutants (SI Ap- porter gene in the genome. With this procedure, we identified a pendix, Fig. S2 A and B). The expression of STOP1 was also not line in which the reporter gene was inserted in the intergenic affected in rae1 (SI Appendix, Fig. S2C). In addition, introduc- region between At4g23000 and At4g23010. We generated an tion of the stop1-3 mutation into the rae1-1 mutant background EMS-mutagenized library in the background of this transgenic fully suppressed the increased expression of LUC in the rae1-1 line for mutant screening. After screening ∼10,000 M2 lines, we mutant (SI Appendix, Fig. S1E). These results suggest that RAE1 finally identified 17 regulation of AtALMT1 expression (rae) mu- acts upstream of STOP1 to regulate the expression of Al- tants with altered luminescent signal. Two mutants with ex- resistance genes. tremely low luminescence had different mutations in the key Al- Because the expression of AtALMT1 was increased in rae1 resistance gene STOP1 (SI Appendix, Fig. S1A), which indicated mutants, we compared malate secretion between the WT and that our screening system was effective.