Plant Cell Advance Publication. Published on October 21, 2020, doi:10.1105/tpc.20.

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1  RESEARCH ARTICLE

2  Regulation of Aluminum-Resistance in Arabidopsis Involves the

3  SUMOylation of the Zinc Finger Transcription Factor STOP1

4  Qiu Fang1,2,#, Jie Zhang1,#, Yang Zhang1, Ni Fan1,3, Harrold A. van den Burg4, and
5  Chao-Feng Huang1,2 *
6 
1
7  Shanghai Center for Plant Stress Biology & National Key Laboratory of Plant Molecular
8  Genetics, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences,
9  Shanghai 200032, China; 2 State Key Laboratory of Crop Genetics and Germplasm
10  Enhancement, College of Resources and Environmental Sciences, Nanjing Agricultural
11  University, Nanjing 210095, China; 3 University of the Chinese Academy of Sciences, Beijing
12  100049, China; 4 Molecular Plant Pathology, Swammerdam Institute for Life Sciences,
13  University of Amsterdam, 1098 XH Amsterdam, The Netherlands.
14 
15  #These authors contributed equally to this work
16  *Author for correspondence: Chao-Feng Huang, huancf@psc.ac.cn.
17 
18  Short title: STOP1 SUMOylation regulates Al resistance
19 
20  One-sentence summary: The Al-resistance transcription factor STOP1 is
21  mono-SUMOylated at K40, K212, or K395 sites and deSUMOylated by the SUMO protease
22  ESD4, which is involved in the regulation of Al resistance in Arabidopsis.
23 
24  Keywords: aluminum resistance, STOP1, ESD4, SUMOylation, Arabidopsis thaliana.
25 
26  The author responsible for distribution of materials integral to the findings presented in this
27  article in accordance with the policy described in the Instructions for Authors
28  (www.plantcell.org) is: Chao-Feng Huang (huangcf@psc.ac.cn).
29 
30  ABSTRACT
31  Aluminum (Al) is a primary constraint for crop production on acid soils, which make up more
32  than 30% of the arable land in the world. Al resistance in Arabidopsis thaliana is achieved by
33  malate secretion mediated by the AtALMT1 transporter. The C2H2-type transcription factor
34  STOP1 is essential and required for Al resistance, where it acts by inducing the expression of
35  Al-resistance genes, including AtALMT1. In this study, we report that STOP1 protein function
36  is modified by SUMOylation. The SUMO protease ESD4, but not other SUMO proteases,
37  specifically interacts with and deSUMOylates STOP1. Mutation of ESD4 increases the level
38  of STOP1 SUMOylation and the expression of the STOP1-regulated gene AtALMT1, which
39  contributes to the increased Al resistance in esd4. The esd4 mutation does not influence
40  STOP1 protein abundance but increases the association of STOP1 with the AtALMT1
41  promoter, which might explain the elevated expression of AtALMT1 in esd4. We demonstrate
42  that STOP1 is mono-SUMOylated at K40, K212, or K395 sites, and blocking STOP1

©2020 The author(s).

  

43  SUMOylation reduces STOP1 stability and the expression of STOP1-regulated genes, leading
44  to the reduced Al resistance. Our results thus reveal the involvement of SUMOylation in the
45  regulation of STOP1 and Al resistance in Arabidopsis.
46 

47  INTRODUCTION
48  Aluminum (Al) is the most abundant metal and the third most abundant element after
49  oxygen and silicon in the Earth’s crust. Most Al exists as insoluble aluminosilicates or
50  aluminum oxides, which are non-toxic to plant growth. However, as soils acidify to a
51  pH of 5.5 or lower, Al3+ ions are dissolved and become highly toxic to plant root
52  growth (Kochian, 1995; Kochian et al., 2004; Ma, 2007). Al toxicity is, thus, a main
53  constraint for crop production on acid soils. Since 40-50% of the potentially arable
54  lands in the world are acidic, Al toxicity is one of the most serious global problems
55  and the second greatest abiotic restraint to crop production, exceeded only by drought
56  stress (von Uexkull and Mutert, 1995).
57  To survive in Al-toxic environments, plants have evolved various mechanisms to
58  detoxify Al (Kochian et al., 2004; Liu et al., 2014; Kochian et al., 2015). Among them,
59  Al-induced exudation of organic acids, including malate, citrate, and oxalate, is an
60  essential mechanism for Al detoxification in most plant species (Ma et al., 2001; Ryan
61  et al., 2001). Different plant species may secrete distinct organic acids to chelate and
62  detoxify Al. Arabidopsis thaliana (hereafter referred to as Arabidopsis), for instance,
63  mainly secretes malate to detoxify Al, although it also releases citrate in response to
64  Al stress (Hoekenga et al., 2003; Liu et al., 2009). Transporters required for the
65  malate and citrate secretion were first identified in crops (Sasaki et al., 2004;
66  Furukawa et al., 2007; Magalhaes et al., 2007). Based on the sequence similarity and
67  functional analysis, the malate transporter AtALMT1 (Al-activated malate transporter
68  1) and the citrate transporter AtMATE for root malate and citrate exudation,
69  respectively, were subsequently identified in Arabidopsis (Hoekenga et al., 2006; Liu
70  et al., 2009). In addition to the organic acid exudation-based Al-resistance mechanism,
71  Arabidopsis plants also utilize the tonoplast-localized half-size ATP-binding cassette
72  (ABC) transporter ALS1 and the bacterial-type ABC transporter complex

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73  AtSTAR1/ALS3 to sequester Al into the vacuoles or modify the cell wall to detoxify
74  Al (Larsen et al., 2005; Larsen et al., 2007; Huang et al., 2010; Dong et al., 2017).
75  The C2H2-type zinc finger transcription factor SENSITIVE TO PROTON
76  RHIZOTOXICITY 1 (STOP1) plays an essential role in the regulation of Al
77  resistance by modulating the expression of some Al-resistance genes, including
78  AtALMT1, AtMATE, and ALS3 (Iuchi et al., 2007; Sawaki et al., 2009). Although the
79  transcript levels of the STOP1-downstream genes AtALMT1, AtMATE, and ALS3 are
80  induced by Al stress, the mRNA level of STOP1 itself is not influenced by Al (Iuchi et
81  al., 2007; Sawaki et al., 2009), suggesting that STOP1 might be subjected to
82  post-transcriptional and/or post-translational regulation. In line with this idea, we
83  recently found that Al stress can stabilize the STOP1 protein and that the F-box
84  protein REGULATION OF ATALMT1 EXPRESSION 1 (RAE1) acts as an E3
85  ubiquitin ligase to promote STOP1 ubiquitination and degradation (Zhang et al.,
86  2019). More recently, the core component of THO/TREX complex, HPR1, was found
87  to regulate STOP1 at a post-transcriptional level through the modulation of
88  nucleocytoplasmic STOP1 mRNA export (Guo et al., 2020).
89  The SMALL UBIQUITIN-LIKE MODIFIER (SUMO) is a small polypeptide
90  structurally related to ubiquitin. SUMO can be attached to protein substrates in a
91  process called SUMOylation, which can alter protein function, activity, localization,
92  and/or stability (Wilkinson and Henley, 2010; Jentsch and Psakhye, 2013; Augustine
93  and Vierstra, 2018). Like ubiquitin, SUMO is conjugated to its substrates through an
94  E1-E2-E3 cascade (Saitoh et al., 1997). In Arabidopsis, only one heterodimeric E1
95  SUMO activating enzyme (SAE1a/b and SAE2), a single E2 SUMO conjugating
96  enzyme (SCE1), and two E3 ligases (SIZ1 and MMS2/HYP2) have been identified
97  (Miura and Hasegawa, 2010). Recently, two homologous E4-type SUMO ligases
98  PIAL1 and PIAL2 were identified and demonstrated to be involved in SUMO chain
99  formation (Tomanov et al., 2014). SUMOylation is a reversible process:
100  deSUMOylation is catalyzed by SUMO proteases (Yates et al., 2016; Castro et al.,
101  2018). This reversible conjunction of SUMO to target proteins is particularly
102  important for the regulation of responses to abiotic stresses. For instance, SUMO can
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103  target the MYB transcription factor PHR1 through the E3 ligase SIZ1 to regulate
104  phosphate-deficiency responses (Miura et al., 2005). SIZ1-mediated SUMOylation
105  plays an important role in drought stress response through the regulation of
106  drought-responsive genes (Catala et al., 2007). SUMOylation is also involved in the
107  regulation of low-temperature tolerance through targeting the transcription factor
108  ICE1 (Miura et al., 2007).
109  An important class of plant SUMO proteases is that of the ubiquitin-like proteases
110  (ULPs), which belong to the C48 clade of cysteine protease family (Castro et al.,
111  2018). The Arabidopsis genome is presumed to have eight ULPs (Castro et al., 2018).
112  Since there are substantially more ULPs than SUMO E3 ligases in Arabidopsis, it has
113  been suggested that substrate specificity on the SUMOylation process might be
114  conferred by ULPs and that deSUMOylating might be a major regulatory step to
115  control the effect of SUMOylation (Yates et al., 2016; Verma et al., 2018).
116  The ULP member ESD4 was the first demonstrated to be crucial for the regulation
117  of SUMOylation in plants, and mutation of ESD4 induces early flowering and severe
118  pleiotropic effects on plant growth (Reeves et al., 2002; Murtas et al., 2003), which
119  are partially caused by salicylic acid (SA) overaccumulation (Villajuana-Bonequi et
120  al., 2014). Although ESD4 plays a critical role in the regulation of plant development
121  and stress responses, no direct target substrates of ESD4 have been identified. The
122  two ULP members OTS1/ULP1d and OTS2/ULP1c are the best characterized and
123  have been demonstrated to be redundantly involved in the regulation of various
124  biological processes. For instance, OTS1 and OTS2 modulate salt stress responses
125  partly through deSUMOylation and destabilization of growth-repressing DELLA
126  proteins (Conti et al., 2008; Conti et al., 2014) and regulate light-induced signaling by
127  deSUMOylating the photoreceptor phytochrome-B (phyB) (Sadanandom et al., 2015).
128  OTS1 and OTS2 are also reported to regulate jasmonic acid (JA) signaling by
129  modulating JAZ protein SUMOylation and stability (Srivastava et al., 2018). Recently,
130  another ULP member, ULP1a, was demonstrated to mediate the deSUMOylation of
131  BZR1, a key transcription factor of brassinosteroid (BR) signaling, to integrate
132  environmental cues into BR signaling to shape plant growth (Srivastava et al., 2020).
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133  Other ULP members such as SPF1/ULP2b and/or SPF2/ULP2a modulate flowering
134  time and fertility partially through regulating FLC and EDA9 stability, respectively
135  (Kong et al., 2017; Liu et al., 2017).
136  Previously, we generated a transgenic reporter line by fusing the promoter of the
137  STOP1-target gene AtALMT1 to a luciferase (LUC) reporter gene and performed a
138  forward genetic screen to identify genes involved in the regulation of AtALMT1
139  and/or STOP1 (Zhang et al., 2019). In this study, we characterize two independent
140  mutants of the SUMO protease ESD4 that yield enhanced Al resistance. We conclude
141  that ESD4 directly deSUMOylates STOP1 to regulate the expression of
142  STOP1-downstream genes and Al resistance. We also determined that STOP1 can be
143  monoSUMOylated at K40, K212 or K395 sites and that the SUMOylation of STOP1
144  is important for maintaining STOP1 protein levels and Al resistance.
145 

146 

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147  RESULTS
148  rae5 Mutants Show Increased Expression of the pAtALMT1:LUC Reporter and
149  of the Endogenous AtALMT1
150  We previously carried out a forward genetic screen on an EMS-mutagenized
151  population of the AtALMT1 promoter-driven luciferase reporter (pAtALMT1:LUC)
152  line and identified 13 regulation of AtALMT1 expression (rae) mutants with increased
153  LUC signal, which includes eight rae1 mutants (Zhang et al., 2019). In this study, we
154  focused on two other mutants, rae5-1 and rae5-2, which were allelic to each other and
155  showed higher LUC expression than the wild type (WT) (Figure 1A).
156  We compared mRNA expression levels of the LUC reporter gene and the
157  endogenous AtALMT1 gene in roots of WT and rae5 mutants. In accordance with the
158  increased LUC signal in the mutants, expression of both genes was significantly
159  higher in the mutants than in the WT under both –Al and +Al conditions (Figure 1B
160  and 1C). To examine whether the rae5 mutations affect the expression of other
161  Al-resistance genes in the roots, we conducted a reverse transcription-quantitative
162  PCR (RT-qPCR) expression analysis on AtMATE, RAE1, ALS3, STOP1, ALS1, and
163  AtSTAR1 in WT and the rae5 mutants. AtMATE and RAE1 were expressed to lower
164  levels in the mutants than in the WT, while ALS3 was expressed at a similar level in
165  WT and the mutants (Figure 1D-1F). The expression levels of STOP1, ALS1, and
166  AtSTAR1, which are not modulated by the transcription factor STOP1 (Iuchi et al.,
167  2007; Sawaki et al., 2009), were similar between WT and the mutants (Fig. 1G-1I).
168  We also compared the expression of STOP1-regulated Al-resistance genes in shoots.
169  In the shoots, mutation of STOP1 nearly abolished the expression of LUC and
170  AtALMT1 (Supplemental Figure 1A and 1B), indicating that STOP1 is also crucial for
171  AtALMT1 expression in the shoots. Nevertheless, the expression of AtMATE, RAE1
172  and ALS3 was only slightly reduced in stop1-3 mutant under +Al conditions
173  (Supplemental Figure 1C-1E), suggesting that STOP1 does not play a critical role in
174  the regulation of the expression of these genes in the shoots. Mutation of RAE5
175  increased the expression LUC and AtALMT1 but not that of AtMATE, RAE1 and ALS3

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176  (Supplemental Figure 1).

177 

178  Mutation of RAE5 Increases Malate Secretion and Al Resistance

179  Because of the increased and decreased expression of AtALMT1 and AtMATE in rae5
180  mutants, respectively, we determined whether malate and citrate release was altered in
181  the mutants. The results showed that the mutants secreted more malate than the WT
182  under both –Al and +Al conditions, while the release of citrate was lower in the
183  mutants than in WT under +Al conditions (Figure 2A; Supplemental Figure 2A).
184  To assess the Al resistance phenotype of rae5 mutants, we grew WT, rae5-1, rae5-2,
185  and Atalmt1 plants on agar plates containing different Al concentrations. Under
186  control conditions, root growth of rae5-1 and rae5-2 was slightly slower than that of
187  WT (Figure 2B). rae5-1 showed enhanced resistance to Al than WT at 1.25 mM Al,
188  while rae5-2 was more resistant at both 1.0 and 1.25 mM Al, suggesting that rae5-2 is
189  a stronger allele compared to rae5-1 (Figure 2B and 2C). The Atalmt1 control was
190  more sensitive to Al than WT at all Al concentrations. Consistent with these results,
191  the rae5 mutants accumulated less Al in root tips than the WT, based on staining with
192  the Al indicator Eriochrome Cyanine R (ER) (Figure 2D), while the Atalmt1 mutant
193  control accumulated more Al in the root tips. These results demonstrate that the rae5
194  mutations increase malate secretion and Al resistance.
195  To examine whether rae5 mutants are specifically resistant to Al, we tested the
196  tolerance of rae5 mutants to two other toxic metals Cd and La. The mutants did not
197  show altered tolerance to Cd or La compared to the WT (Supplemental Figure 2B and
198  2C), demonstrating that rae5 mutants are not broadly resistant to all toxic metals.
199  Mutation of RAE5 also induced morphological defects. The mutants showed
200  reduced plant height and organ size, low fertility, and early flowering (Supplemental
201  Figure 2D). rae5-2 had stronger morphological defects than rae5-1.
202 

203  RAE5 Encodes the SUMO Protease ESD4

204  A genetic analysis of the rae5-2 mutation was performed using an F2 population from
205  a cross between rae5-2 and the WT. Among 356 F2 plants, 75 plants showed
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206  increased LUC signal and morphological defects, while the other 281 plants had
207  normal LUC signal and morphology. The ratio of plants with high LUC signal to
208  plants with normal LUC signal fitted to 1:3 (χ2=2.93, P>0.05), suggesting that the
209  increased LUC signal and the morphological defects both were controlled by a single
210  recessive gene.
211  To clone the RAE5 gene, the same F2 population was used and pooled DNA from
212  75 F2 plants with increased LUC signal and morphological defects was subjected to
213  whole-genome sequencing. As a control, we used the WT DNA sequence, which had
214  been sequenced before (Zhang et al., 2019). By MutMap analysis (Abe et al., 2012),
215  we located the candidate gene to a small region of chromosome 4 (Supplemental
216  Figure 3A). To confirm the results of the MutMap analysis, we developed four
217  derived cleaved amplified polymorphic sequence (dCAPS) markers surrounding the
218  candidate region on the basis of mutations in rae5-2 (Supplemental Table 1), and used
219  58 F2 plants with increased LUC expression and defective morphologies to conduct a
220  marker linkage analysis. The four markers displayed, to different extents, some
221  linkage to the mutant phenotype (Supplemental Table 1), confirming that the RAE5
222  gene was indeed located in this region. We mapped the RAE5 gene between D2 and
223  D4 markers with eight and twelve recombinants, respectively, and the D3 marker,
224  developed on a C-to-T substitution at +1459 bp from the start codon of At4G15880
225  (ESD4), was completely linked to the mutant phenotype (Supplemental Table 1). This
226  substitution caused an amino acid change from arginine to stop codon in ESD4 in the
227  mutant. We also sequenced ESD4 in rae5-1 allele and identified a G-to-A substitution
228  at +1369 bp from the start codon in rae5-1, introducing an amino acid change from
229  aspartic acid to asparagine. These results suggest that these mutations in ESD4 are
230  responsible for the increased LUC expression and morphological defects in the rae5
231  mutants.
232  To further confirm that RAE5 is ESD4, we performed a complementation test on
233  rae5-1 by transforming a WT genomic fragment of ESD4 fused with 3Flag tag into
234  the mutant. Quantitative expression analysis showed that the increased expression of
235  LUC and AtALMT1 and reduced expression of AtMATE in rae5-1 was rescued in two
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236  complementation lines (Supplemental Figure 3B). The elevated Al resistance and
237  defective plant growth in the mutant was also recovered (Supplemental Figure 3C-3E).
238  Together, these results demonstrate that RAE5 encodes the SUMO protease ESD4. For
239  clarity, we refer to rae5-1 and rae5-2 as esd4-3 and esd4-4, respectively, in the
240  following sections.
241 

242  ESD4 Protein Accumulation is Increased in Response to Al Stress

243  To investigate whether Al stress affects ESD4 mRNA expression, we exposed WT
244  roots to Al stress for different time and then quantified the mRNA levels of ESD4.
245  Although Al stress could highly induce the expression of AtALMT1 control for all
246  time points, the expression of ESD4 was not responsive to the Al stress (Figure 3A).
247  To examine whether ESD4 protein accumulation is influenced by Al stress, we
248  utilized the complementation line (pESD4:ESD4-3Flag esd4-3) for the detection of
249  ESD4 protein using the anti-Flag antibody and exposed the line to –Al or +Al
250  conditions for 0, 2, 4 or 8 h. Al stress was able to increase the protein accumulation of
251  ESD4-3Flag after 4 h treatment (Figure 3B and 3C).
252 

253  STOP1 is mono-SUMOylated at Multiple Sites and deSUMOylated by ESD4

254  Because ESD4 is involved in the SUMOylation pathway and the regulation of the
255  expression of STOP1-target genes, we hypothesized that ESD4 might regulate the
256  level of STOP1 SUMOylation. We first determined whether STOP1 can be
257  SUMOylated in Arabidopsis. We co-expressed STOP1-2Flag or GFP-2Flag control
258  with 6Myc-tagged SUMO1 precursor into WT Arabidopsis protoplasts and then
259  detected SUMOylated STOP1 using the Myc antibody after Flag immunoprecipitation
260  (IP). We found that STOP1 but not GFP could be SUMOylated, with two major bands
261  (Figure 4A). Nevertheless, when we increased running time of the protein in the gel,
262  we were able to observe SUMOylated STOP1 of three bands (Supplemental Figure
263  4A). Since SUMOylation is a reversible modification that normally occurs in less than
264  5% of the total protein (Creton and Jentsch, 2010), we were unable to detect the
265  SUMOylated forms of STOP1 directly using the anti-Flag antibody. To confirm that
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266  STOP1 was covalently modified by mature SUMO, we co-expressed a mature WT

267  SUMO1GG or a conjugation-deficient mutant SUMO1AA with STOP1 in protoplasts.
268  STOP1 formed higher order adducts with SUMO1GG, but not with SUMO1AA (Figure
269  4B), confirming that STOP1 is SUMOylated by mature SUMO. To investigate
270  whether STOP1 was modified with a SUMO chain at a single site or
271  mono-SUMOylated at multiple acceptor sites, we generated a mutant SUMO1
272  (SUMO14KR) in which four lysines were changed to arginine (K9, K10, K23, K42) to
273  abrogate formation of poly-SUMO chains (Miller et al., 2010). Co-expression of the
274  mutated SUMO14KR with STOP1 did not influence formation of SUMOylated STOP1
275  (Figure 4C). These results suggest that STOP1 is subjected to multiple
276  mono-SUMOylation events.
277  To examine whether the esd4 mutations affected STOP1 SUMOylation, we
278  co-expressed STOP1-2Flag with 6Myc-SUMO1 precursor in WT, esd4-3 or esd4-4
279  protoplasts and then compared the SUMOylation levels of STOP1 between WT and
280  the mutants. STOP1 SUMOylation was increased in the two mutants compared to the
281  WT (Figure 4D). Since ESD4 can be involved in the processing of the SUMO
282  precursor or the deSUMOylation to influence the SUMOylation level of target
283  substrates (Murtas et al., 2003), we co-transformed mature SUMO (6Myc-SUMO1GG)
284  with the STOP1-2Flag into protoplasts to determine whether STOP1 SUMOylation
285  levels are influenced by the esd4 mutations. Like the SUMO precursor, introduction
286  of the mature SUMO1 could also increase the SUMOylation of STOP1 in esd4
287  protoplasts (Figure 4D), suggesting that ESD4 is involved in the deconjugation of
288  SUMO from STOP1.
289  To investigate whether STOP1 can be SUMOylated in planta, we transformed
290  SUMO1 promoter-driven 2Flag-SUMO1 into the previously generated
291  pSTOP1:STOP1-3HA transgenic line (Zhang et al., 2019) and selected a transgenic
292  line with single-locus segregation in the 2Flag-SUMO1 transgene for the detection of
293  SUMOylated STOP1 using the anti-Flag antibody after STOP1-3HA
294  immunoprecipitation. We crossed the transgenic line with esd4-3 mutant to introduce
295  the 2Flag-SUMO1 transgene into the mutant background and also generate
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296  2Flag-SUMO1 control line without pSTOP1:STOP1-3HA transgene. Although the

297  level of SUMOylated protein is low, and normally occurs in less than 5% of the total
298  protein (Creton and Jentsch, 2010), we were able to detect SUMOylated STOP1 with
299  three observable bands (Supplemental Figure 4B; Figure 4E), demonstrating that
300  STOP1 can be SUMOylated in Arabidopsis plants. Because Al stress promotes
301  STOP1 protein accumulation (Zhang et al., 2019), we normalized the total amount of
302  STOP1 to compare the SUMOylation level of STOP1 under –Al and +Al conditions.
303  Al stress decreased the level of SUMOylated STOP1, although the global levels of
304  protein SUMOylation were not affected by the Al stress (Supplemental Figure 4B;
305  Figure 4E and 4F). In esd4-3 mutant background, the level of STOP1 SUMOylation
306  was increased compared to that in WT (Figure 4E and 4F), consistent with the role of
307  ESD4 in the deSUMOylation of STOP1. The level of SUMOylated STOP1 was also
308  reduced by Al stress in the background of esd4-3 mutant (Figure 4E and 4F).
309  Next, we tested whether ESD4 can interact with STOP1 directly. Since the
310  wild-type ESD4 protein is not well expressed in yeast cells (Elrouby and Coupland,
311  2010), we expressed a catalytically inactive form of ESD4 (C448S) to test the
312  interaction between ESD4 and STOP1 in the yeast two-hybrid assay, and found that
313  the inactive variant of ESD4 interacts with STOP1 (Figure 5A). To investigate
314  whether wild-type ESD4 can interact with STOP1 in planta, we carried out split
315  luciferase complementation assays (Chen et al., 2008). We used the combinations of
316  RAE1△F-nLUC (F-box domain-deleted RAE1) and cLUC-STOP1, and RAE1-nLUC
317  and cLUC-STOP1 as positive and negative controls, respectively (Zhang et al., 2019).
318  We detected the LUC signal with the combinations of ESD4-nLUC and cLUC-STOP1
319  and with the positive control, but not with other combinations (Figure 5B), and we
320  also detected protein interaction-dependent LUC signal of cLUC-ESD4 and
321  STOP1-nLUC (Figure 5B). These results indicate that ESD4 can interact with STOP1
322  in planta. To further examine whether ESD4 and STOP1 form protein complexes in
323  Arabidopsis, we conducted co-immunoprecipitation (Co-IP) assays in Arabidopsis
324  protoplasts. ESD4-3HA could co-immunoprecipitate with STOP1-2Flag but not with
325  GFP-2Flag control (Figure 5C). Together, these data indicate that ESD4 can interact
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326  with STOP1 in vivo.

327  To investigate whether the interaction of STOP1 with ESD4 is specific, we first
328  used the yeast two-hybrid method to test the interaction of STOP1 with four other
329  ULP SUMO proteases including ELS1, ELS2, OTS1 and OTS2 and observed that
330  STOP1 did not interact with the four SUMO proteases (Supplemental Figure 5A). We
331  then used split-LUC assays to test the interaction of STOP1 with all seven other ULP
332  SUMO proteases. ESD4 but not the other seven ULP SUMO proteases were able to
333  interact with STOP1 (Supplemental Figure 5B). These data indicate that the
334  interaction between STOP1 and ESD4 is specific.
335  To further test whether ESD4 deSUMOylates STOP1, STOP1-2Flag was
336  co-expressed with WT or mutated ESD4-3HA and SUMO precursor (6Myc-SUMO1)
337  or mature SUMO (6Myc-SUMO1GG) in WT protoplasts. The results showed that WT
338  ESD4-3HA deSUMOylated STOP1-2Flag in both 6Myc-SUMO1 and
339  6Myc-SUMO1GG expressing cells (Figure 5D and 5E), whereas mutated versions of
340  ESD4 including esd4-3-3HA, esd4-4-3HA and catalytically inactive ESD4-3HA
341  (ESD4C448S-3HA) were unable to deSUMOylate STOP1-2Flag. Together, these data
342  indicate that ESD4 interacts with and deSUMOylates STOP1.
343 

344  Mutation of ESD4 does not Affect STOP1 Protein Accumulation, but Alters its
345  Association with the Promoters of Different STOP1-Target Genes
346  To investigate whether mutation of ESD4 influences STOP1 protein levels, we
347  crossed pSTOP1:STOP1-3HA and pSTOP1:STOP1-GUS transgenic lines, which have
348  been generated previously (Zhang et al., 2019), with esd4-3 to introduce the
349  transgenes into the mutant background. Immunoblot and GUS staining analysis
350  revealed that the STOP1-3HA/GUS protein levels were not significantly different in
351  the esd4-3 background compared to WT under both -Al and +Al conditions (Figure
352  6A and 6B). To examine whether mutation of ESD4 affects STOP1 stability, we
353  treated plant roots with the protein synthesis inhibitor cycloheximide (CHX) and Al
354  stress for different time. Although CHX treatment was able to inhibit STOP1 protein
355  synthesis in WT and esd4-3, the decrease in STOP1 levels did not exhibit a clear
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356  difference between WT and the mutant (Figure 6C), which suggested that the esd4-3
357  mutation did not affect STOP1 stability under Al stress conditions. Nevertheless,
358  when the plant roots were first treated with Al to induce STOP1 accumulation and
359  then transferred the plants to Al-free conditions with the addition of (CHX), the
360  STOP1 protein levels decreased more slowly in the mutant than in WT (Figure 6D),
361  suggesting that mutation of ESD4 can increase the stability of STOP1 once the Al
362  stress is removed.
363  ESD4 localizes to the nuclear rim and can interact with the nuclear pore component
364  NUA (Murtas et al., 2003; Xu et al., 2007), which suggests that EDS4 might regulate
365  nuclear accumulation of STOP1. To test this hypothesis, we generated a single-locus
366  35S:STOP1-GFP transgenic line in the WT Arabidopsis background and then
367  introduced the transgene into the esd4-3 mutant background through crossing. We
368  compared the ratio of GFP fluorescence in the nucleus versus the cytosol to determine
369  whether the esd4-3 mutation alters the subcellular localization of STOP1-GFP. The
370  results revealed that mutation of ESD4 did not affect the subcellular localization of
371  STOP1 under either –Al and +Al conditions (Supplemental Figure 6). Interestingly,
372  Al stress promoted STOP1 accumulation in the nucleus in both WT and the mutant as
373  indicated by the increased nuclear cytoplasmic (N/C) ratio of STOP1 protein
374  (Supplemental Figure 6), although the total STOP1 protein levels were also increased
375  by Al stress (Supplemental Figure 6).
376  Because mutation of ESD4 increases AtALMT1 expression while concomitantly
377  reducing the expression of AtMATE and RAE1, we performed chromatin
378  immunoprecipitation (ChIP) assay to determine whether the association of
379  STOP1-3HA to the promoters of these genes was altered in the esd4-3 background.
380  The binding regions of STOP1 in the promoters of AtALMT1 and RAE1 were
381  previously identified (Tokizawa et al., 2015; Zhang et al., 2019), but those in the
382  promoter of AtMATE had not yet been determined. Therefore, we performed ChIP
383  assays using the pSTOP1:STOP1-3HA transgenic line to screen for the
384  STOP1-binding region in the AtMATE promoter. STOP1-3HA preferentially
385  associated with the fragments 6 and 7 of the AtMATE promoter (Supplemental Figure
  13
  

386  7). We then performed ChIP assays in the WT and esd4-3 backgrounds under –Al or
387  +Al conditions. The association of STOP1-3HA with the AtALMT1 promoter was
388  more pronounced in the mutant than in WT under both –Al and +Al conditions,
389  whereas the opposite was observed for the AtMATE and RAE1 promoters (Figure
390  6E-6H). These results demonstrate that increased SUMOylation had different effects
391  on the association of STOP1 with the promoters of different STOP1-target genes.
392 

393  STOP1 can be SUMOylated at K40, K212 or K395 Sites

394  To identify the SUMO acceptor sites in STOP1, we mutated six predicted SUMO sites
395  of STOP1-3HA (Figure 7A) and co-expressed this mutated STOP1-3HA construct
396  with 6Myc-SUMO1 in protoplasts to determine whether mono-SUMOylation still
397  occurred. SUMOylation was fully blocked for the STOP1-3HA mutant, in which
398  these six lysine residues were substituted for arginines (Figure 7B). We then generated
399  six mutant versions of STOP1-3HA in which only five of the six lysine residues were
400  replaced by arginine (leaving each time a different lysine intact) and co-expressed
401  each of them with 6Myc-SUMO1 to examine whether specific lysines were required
402  for SUMOylation. We found that three residues (K40, K212 and K395) could be
403  SUMOylated (Figure 7B). To confirm that these three sites are sufficient and required
404  for STOP1 SUMOylation, we constructed a series of mutated versions of STOP1 with
405  single, double or triple lysine-to-arginine substitutions, and then co-transformed each
406  construct with 6Myc-SUMO1 in the protoplasts. Consistently, the combined mutation
407  of all three lysines eliminated STOP1 SUMOylation (Figure 7C). Mutating K40 or
408  K395 reduced STOP1 SUMOylation level at the lower band, while the residue K212
409  controlled SUMOylation of the upper band of STOP1 (Figure 7B and 7C). These
410  results indicate that STOP1 is mono-SUMOylated at three sites: K40, K212, and
411  K395.
412 
413  Blocking of STOP1 SUMOylation Reduces STOP1 Accumulation and Associated
414  Phenotypes of Al Resistance and Low Phosphate Response
415  To determine the biological function of STOP1 SUMOylation in Arabidopsis, we

  14
  

416  conducted complementation assays on stop1-2 by transforming

417  pSTOP1:STOP1K40R-3HA, pSTOP1:STOP1K212R-3HA or pSTOP1:STOP1K395R-3HA
418  into the mutant background, respectively. We selected single-locus homozygous
419  transgenic lines that showed similar mRNA accumulation levels of STOP1 to a
420  previously generated wild-type STOP1-3HA complementation line (Supplemental
421  Figure 8A-8C), and then compared the expression of STOP1-regulated genes, STOP1
422  protein levels and Al resistance in these lines. Mutation of K40 reduced the expression
423  of AtALMT1 and ALS3, while increased AtMATE expression (Supplemental Figure
424  8A). Intriguingly, the STOP1 protein level was not significantly affected by this
425  mutation (Figure 8A), although decreased Al resistance was observed in the
426  complementation line of pSTOP1:STOP1K40R-3HA (Figure 8D and 8E). The K212R
427  mutation did not affect the expression of STOP1-regulated genes, STOP1
428  accumulation and Al resistance (Supplemental Figure 8B; Figure 8B, 8D and 8E).
429  Unlike the K40R and K212R mutations, the K395R mutation decreased the
430  expression of both AtALMT1 and AtMATE, and the STOP1 protein level
431  (Supplemental Figure 8C; Figure 8C). Consistent with those results, Al resistance was
432  decreased in the complementation line of pSTOP1:STOP1K395R-3HA (Figure 8D and
433  8E).
434  We also mutated all three sites of STOP1 to conduct a complementation test of
435  stop1. We selected two complementation lines with the triple mutation 3KR (K40R,
436  K212R and K395R), which showed similar mRNA levels of STOP1 to the WT
437  complementation line (Supplemental Figure 8D). The results revealed that mutating
438  all the three SUMO acceptor sites reduced the expression of STOP1-target genes,
439  including AtALMT1, AtMATE, and ALS3 in the presence of Al (Supplemental Figure
440  8D). Consistent with the reduced expression of its target genes, STOP1 protein levels
441  were decreased in both 3KR complementation lines (Figure 8F). To examine the
442  stability of STOP13KR in vivo, we first exposed the plants with Al stress to increase
443  STOP1 levels and then transferred the plants to –Al conditions with the addition of
444  CHX to inhibit protein synthesis. STOP13KR levels decreased more quickly than the
445  STOP1WT protein levels (Figure 8G), which suggests that SUMOylation of STOP1
  15
  

446  stabilizes STOP1 protein. We then evaluated Al resistance in these lines and the
447  results showed that the Al resistance was reduced in the STOP13KR complementation
448  lines compared to the STOP1WT line (Figure 8H and 8I). Together, these results
449  indicate that a block of STOP1 SUMOylation destabilizes STOP1 and reduces Al
450  resistance.
451  Since STOP1 is required for both Al resistance and proton tolerance (Iuchi et al.,
452  2007), we also compared the proton tolerance in these complementation lines. The
453  results showed that K40R, K212R or K395R single mutations did not affect the
454  tolerance to proton toxicity (Supplemental Figure 9A and 9B). Nevertheless, the triple
455  mutation 3KR reduced the proton tolerance (Supplemental Figure 9C and 9D).
456  Recently, AtALMT1-mediated malate secretion was reported to promote apoplastic Fe
457  toxicity and consequently inhibit root growth under low phosphate (Pi) conditions,
458  and STOP1 is involved in the low Pi-induced root growth inhibition through the
459  modulation of AtALMT1 expression (Balzergue et al., 2017; Mora-Macias et al.,
460  2017). In accordance with the reduced AtALMT1 expression, the K40R and K395R
461  complementation lines were less sensitive to low Pi-induced inhibition of root growth
462  compared to the wild-type STOP1 complementation lines (Supplemental Figure 10A
463  and 10B), while K212 mutation did not affect the low Pi response. Similarly, the 3KR
464  complementation lines also showed reduced sensitivity to the low Pi-induced
465  inhibition of root growth (Supplemental Figure 10C and 10D). These results
466  demonstrate that a block of STOP1 SUMOylation also decreases proton tolerance and
467  low Pi response.
468 

469 

  16
  

470  DISCUSSION
471  The transcription factor STOP1 is crucial for Al resistance in Arabidopsis (Iuchi et al.,
472  2007). Previous work has demonstrated that STOP1 is regulated by Al stress at
473  post-translational levels and the F-box protein RAE1 is involved in the regulation of
474  STOP1 ubiquitination and degradation (Zhang et al., 2019). In this study, we unravel
475  another layer of post-translational regulation of STOP1 by SUMOylation. We show
476  that Al resistance can be modulated via a SUMO conjugation switch on the STOP1
477  transcription factor (Figure 9). STOP1 is mono-SUMOylated at the sites K40, K212,
478  and K395, and deSUMOylated by the SUMO protease ESD4. This dynamic
479  SUMOylation of STOP1 is involved in the control of the expression of downstream
480  genes and Al resistance. Prevention of the ESD4-mediated deSUMOylation of STOP1
481  elevates STOP1 SUMOylation levels, which leads to altered association to the
482  promoters of downstream genes, increasing binding to the promoter of AtALMT1 and
483  decreasing binding to the promoters of AtMATE. These differences in physical
484  association to the promoters in turn lead to the increased expression of AtALMT1 and
485  the reduced expression of AtMATE, which contributes to the enhanced Al resistance.
486  Blocking of STOP1 SUMOylation at K395 or all the three sites (K40, K212, and
487  K395), on the other hand, destabilizes STOP1 and causes reduced expression of
488  STOP1-regulated genes and decreased Al resistance (Figure 9). Interestingly, contrary
489  to the effect of increased STOP1 SUMOylation on the expression of STOP1-regulated
490  genes, blocking of STOP1 SUMOylation at K40 decreases the expression of
491  AtALMT1 and ALS3 while increases AtMATE expression. SUMO modification has
492  been suggested to affect the assembly of transcription factors and the recruitment of
493  chromatin-modifying proteins (Hay, 2005; Wilkinson and Henley, 2010), which can
494  cause transcriptional activation as well as transcriptional repression. For instance,
495  SUMOylation of heat shock transcription factors HSF1 and HSF2 increases DNA
496  binding activity (Goodson et al., 2001; Hong et al., 2001), while SUMO modification
497  on Elk-1 transcription factor can cause the recruitment of histone deacetylases to
498  repress transcription (Yang and Sharrocks, 2004). Likewise, SUMOylation of some

  17
  

499  transcription factors such as Smad4 can activate transcription on some promoters and
500  repress transcription on other promoters (Long et al., 2004), which is dependent on a
501  complex interplay between promoter context and intrinsic capability of SUMO to
502  regulate transcription (Hay, 2005). Therefore, the different effects of SUMOylated
503  and unSUMOylated STOP1 on the expression of AtALMT1 and AtMATE might also
504  be attributed to different assemblies of STOP1 and its associated proteins and/or the
505  recruitment of different chromatin-modifying proteins on the promoters of AtALMT1
506  and AtMATE, although the exact underlying mechanism remains to be demonstrated
507  in the future.
508  Mutation of ESD4 causes reduced plant growth and increased accumulation of
509  stress hormone SA (Reeves et al., 2002; Villajuana-Bonequi et al., 2014). Here we
510  found that dysfunction of ESD4 could enhance Al resistance (Figure 2). These
511  observations suggest that ESD4 might be involved in balancing plant growth and
512  stress tolerance. Intriguingly, Al stress increases instead of decreases ESD4 protein
513  accumulation (Figure 3), although ESD4 plays a negative role in Al resistance. The
514  increased ESD4 accumulation under Al stress conditions did not affect global protein
515  SUMOylation (Figure 4E), suggesting that under Al stress conditions, the elevated
516  ESD4 protein might be inefficient in the deSUMOylation of ESD4-target proteins.
517  Thus, we speculate that the increased ESD4 accumulation might help plants to rapidly
518  deSUMOylate target proteins to attenuate Al resistance responses and ensure growth
519  robustness once the Al stress is removed.
520  The current work establishes STOP1 as a bona fide ESD4 target substrate. We
521  demonstrate that ESD4 directly interacts with and deSUMOlates STOP1. Mutation of
522  RAE5/ESD4 increases STOP1 SUMOylation (Figure 4), which causes the exudation
523  of more malate but less citrate in the mutants than in WT (Figure 2; Supplemental
524  Figure S2A). Since AtALMT1-mediated malate secretion plays a predominant role in
525  the detoxification of Al compared to AtMATE-mediated citrate exudation (Hoekenga
526  et al., 2003; Hoekenga et al., 2006; Liu et al., 2009), the increased malate secretion
527  might contribute significantly to the enhanced Al resistance in the esd4 mutants, while
528  the negative effect of reduced citrate secretion on the Al resistance is negligible. As
  18
  

529  mutation of ESD4 also causes pleiotropic development phenotypes under control
530  conditions (Figure 2C; Supplemental Figure 2D), we cannot exclude the possibility
531  that the esd4 mutations affect the SUMOylation and function of other unidentified
532  proteins, which in turn affects plant development and also contributes to the increased
533  Al resistance in the mutants.
534  Since two major bands were observed for SUMOylated STOP1, STOP1 might be
535  modified with a poly-SUMO chain at a single acceptor site or mono-SUMOylated at
536  multiple sites. Given that mutation of four lysines in SUMO1 (SUMO14KR), which are
537  required for the formation of poly-SUMO chains (Miller et al., 2010), does not affect
538  the two major bands of SUMOylated STOP1 (Figure 4C), STOP1 is likely to be
539  mono-SUMOylated at multiple sites. Further site-directed mutagenesis revealed that
540  SUMOylation at the K212 site of STOP1 is responsible for the upper band, while
541  SUMOylation at the K40 or K395 sites is required for the lower band (Figure 7B and
542  7C). Although the topology of SUMOylated STOP1 is still unknown, the apparent
543  larger size of SUMOylated STOP1 than predicted could be attributed to the
544  anomalous running of branched SUMO-modified proteins in SDS-PAGE, which has
545  been documented before (Chupreta et al., 2007; Park-Sarge and Sarge, 2009). The
546  SUMOylation at the K212 site, which is in the middle of STOP1, may be particularly
547  inclined to such anomalous behavior since the SUMOylated STOP1 at this site
548  migrates substantially slower than would correspond to its predicted size (Figure 7B
549  and 7C). Despite the unknown reason underpinning extremely slow migration in
550  SDS-PAGE, the SUMOylated STOP1 at K212 site does not influence STOP1
551  function and Al resistance (Figure 8B and 8E).
552  Previous work has demonstrated that Al and iron can trigger STOP1 accumulation
553  in the nucleus under phosphate deficient conditions (Balzergue et al., 2017; Godon et
554  al., 2019). Nevertheless, it is not clear whether the increased nuclear STOP1
555  accumulation is solely caused by the increase in total STOP1 levels. We found that Al
556  stress increases the nuclear cytoplasmic (N/C) ratio of STOP1 as well as the total
557  STOP1 levels (Supplemental Figure 6), which suggests that Al stress might promote
558  STOP1 movement from the cytoplasm to the nucleus or preferentially inhibit the
  19
  

559  STOP1 degradation in the nucleus. Intriguingly, although elimination of

560  SUMOylation destabilizes STOP1, Al stress reduces rather than increases this
561  post-translational modification in STOP1 (Supplemental Figure 4B; Figure 4E and
562  4F). While Al-induced ESD4 accumulation might contribute to the decreased STOP1
563  SUMOylation (Figure 2), Al stress likely also influences other components to reduce
564  SUMO modification on STOP1 since the SUMOylation levels of STOP1 were also
565  decreased in esd4-3 mutant under Al stress conditions (Figure 4E and 4F). In addition,
566  although loss of STOP1 SUMOylation reduces STOP1 protein levels (Figure 8), the
567  increased SUMOylation in esd4 did not significantly influence STOP1 accumulation
568  and stability under Al stress conditions (Figure 6A-6C). Nevertheless, when
569  Al-stressed roots were transferred to Al-free solution, STOP1 protein stability was
570  higher in esd4 than in WT (Figure 6D). Together, these results suggest that Al might
571  affect other post-translational modifications of STOP1, which can interfere with and
572  override the STOP1 SUMOylation to trigger the increased nuclear accumulation of
573  this transcription factor at a similar level in WT and esd4, and that once Al stress is
574  removed, the SUMO modification on STOP1 can exert an effect on stabilizing STOP1
575  protein. Our results also imply that the Al-induced reduction of STOP1 SUMOylation
576  might enable plants to accelerate the degradation of over-accumulated STOP1 to
577  alleviate Al-resistance responses when the Al stress is absent. Uncovering additional
578  layers of post-translational regulation and dissecting the interactions among these
579  potential regulatory mechanisms will be a stepping stone to engineer Al resistance in
580  crops in the future.
581 

582 

  20
  

583  METHODS
584  Plant Materials and Growth Conditions
585  Wild-type plants (WT) (Columbia ecotype genetic background; Col-0) carrying a
586  homozygous AtALMT1 promoter-driven luciferase transgene (pAtALMT1:LUC)
587  (Zhang et al., 2019) were treated with ethyl methanesulfonate (EMS) to generate a
588  mutation M2 population. The rae5-1 (esd4-3), rae5-2 (esd4-4) and stop1-3 mutants
589  with increased LUC signal were identified from this mutagenized seed population
590  previously (Zhang et al., 2019). The T-DNA insertion line Atalmt1 (SALK_00962)
591  and stop1-2 (SALK_114108) was obtained from the Arabidopsis Biological Resource
592  Center (ABRC).
593  Seeds were grown on a 1/2 MS medium with 1.2% agar and 1% sucrose for 7 d and
594  then the seedlings were observed for luminescence signal by a CCD imaging
595  apparatus (Lumazone P1300B; Roper Scientific). Plants were grown on 1/10 strength
596  Hoagland hydroponic culture or soil culture conditions in a growth chamber
597  (CU-36L4, Perivical) or growth room at 22°C with 14 h of light (100 μmol/m2/s,
598  Philips TLD26W865 cool daylight tubes) and 10 h of darkness.
599 

600  Measurement of Malate and Citrate Secretion in Roots

601  Ten-day-old seedlings of WT, rae5-1 and rae5-2 grown on 1.2% agar plates with 1/2
602  MS nutrient medium and 1% sucrose were pretreated with a 2% MGRL solution
603  (Fujiwara et al., 1992) containing 1% sucrose for 2 h at pH 4.8 and then treated with
604  the same solution containing 0 or 10 μM AlCl3 at pH 4.8 for 12 h in a 12-well plate.
605  The root exudates from 15 seedlings in each of four biological replicates in the treated
606  solution were collected and then concentrated by lyophilizing (CHRIST ALPHA 1-2
607  LDplus, Germany). The NAD/NADH enzymatic cycling method (Hampp et al., 1984)
608  was used to determine the malate and citrate concentration in the root exudates.
609 

610  Evaluation of Al resistance, Low Pi Response and Low pH Tolerance

611  A modified soaked gel medium method was adopted to assess Al resistance according

  21
  

612  to Larsen et al. (2005) (Larsen et al., 2005). A gel medium consisting of 50 ml (pH 5.0)
613  of 0.25 mM (NH4)2SO4, 1 mM KNO3, 0.2 mM KH2PO4, 2 mM MgSO4, 1 mM
614  Ca(NO3)2, 1 mM CaSO4, 1 mM K2SO4, 1 µM MnSO4, 5 µM H3BO3, 0.05 µM CuSO4,
615  0.2 µM ZnSO4, 0.02 µM NaMoO4, 0.1 µM CaCl2, 0.001 µM CoCl2, 1% sucrose, and
616  0.4% Gellan gum (G1910; Sigma-Aldrich) was prepared firstly. The solidified  gel
617  medium was then soaked with 25 ml of the same nutrient solution without Gellan gum
618  containing 0, 0.5, 0.75, 1.0 or 1.25 mM AlCl3 at pH 3.6. After 2 d soaking, the
619  solution was discarded, and the soaked gel medium was dried and used for seedling
620  growth. After 7 d, the seedlings grown on the soaked gel medium were imaged and
621  root lengths were measured by Image J software. Relative root length was used to
622  assess Al resistance, which was determined as a percentage (root length with Al
623  treatment / root length without Al × 100). For Eriochrome Cyanine R staining of Al
624  accumulation, 7-day-old seedlings of WT, rae5-1, rae5-2 and Atalmt1 grown on the
625  soaked gel medium containing 1.0 mM Al were stained with 0.1% Eriochrome
626  Cyanine R for 30 seconds. Roots were then washed with distilled water for 1 min for
627  three times and photographed using a stereo microscope.
628  For the evaluation of low-Pi response, seeds were grown on 0.35% Gellan gum
629  medium (pH5.7) containing 1% sucrose and 1×Hoagland nutrient solution with 40
630  µM Fe(III)-EDTA, 0.5 mM (NH4)2SO4 and 0, 15 μM or 1 mM NH4H2PO4 (Pi). After
631  growth for 7 d, the seedlings were subjected for picture taken and root length
632  measurement. Relative root length normalized to 1 mM Pi condition was used to
633  evaluate the response to low Pi-induced root growth inhibition.
634  To evaluate the tolerance to low pH, a nutrient medium consisting of full-strength
635  Hoagland nutrient solution, 1% sucrose, and 0.4 % gellan gum was prepared with pH
636  at 5.6 or 4.2 after autoclaving. Seedlings were grown on the nutrient medium plate for
637  7 d and then root length was measured. Relative root length normalized to pH 5.6
638  condition was used for the evaluation of low pH tolerance.
639 

640  Evaluation of the Tolerance to Cd and La stresses

641  To compare the Cd and La tolerance in WT, rae5-1 and rae5-2, seeds were grown on
  22
  

642  a plastic mesh floating on 1/30 strength Hoagland nutrient solution with 1 mM CaCl2
643  and 0, 2 µM CdCl2 or 0.6 µM LaCl3 at pH 5.0 in a growth chamber at 22°C. After
644  growth for 7 d, root lengths of 25 to 33 seedlings in each treatment were measured by
645  Image J and relative root growth as described above was used to assess their tolerance
646  to Cd or La.
647 

648  Cloning of RAE5

649  The rae5-2 mutant was crossed with the WT background to construct an F2
650  population for genetic analysis and mapping-by-sequencing. A total of 356 F2 plants
651  were used and 75 plants showing both increased LUC expression and morphological
652  defects were pooled for high throughput DNA sequencing using the Illumina
653  HiSeq4000 system that produces 150-bp paired-end reads, which was carried out by a
654  commercial company (Shanghai Hanyu Biotech lab, Shanghai, China) (accession
655  number in NCBI: SRR9290163). The sequencing data had a depth of around 41-fold
656  coverage of the Arabidopsis genome. Clean reads were mapped to the Arabidopsis
657  Col-0 reference genome (TAIR 10) by using bwa software (version: 0.7.10) with
658  default parameters. The aligned reads were processed by GATK (version: 3.5, Broad
659  institute) for calling SNPs. The MutMap method was adopted to search the candidate
660  region of rae5-2 (Abe et al., 2012). To confirm the candidate region of RAE5, four
661  dCAPS polymorphic markers were designed based on single-nucleotide
662  polymorphisms in the RAE5 candidate region of chromosome 4 (Supplemental Table
663  1). Fifty-eight F2 plants with increased LUC signal and morphological defects were
664  subjected for linkage analysis by using the four dCAPS markers. The dCAPS maker
665  D3 based on the C-to-T substitution in At4g15880 (ESD4) was completely linked to
666  the F2 mutant phenotype (Supplemental Table 1), suggesting that RAE5 encodes the
667  SUMO protease ESD4.
668  For the complementation test of rae5-1, a DNA fragment consisting of a 1.7-kb
669  promoter and the gene of RAE5/ESD4 (At4g15880) without stop codon was amplified
670  by a primer pair (Supplemental Table 2) and cloned in frame (HindIII and DraIII
671  restriction sites) with 3Flag tag into the pCAMBIA3301 vector by using ClonExpress
  23
  

672  II One Step Cloning Kit ( C112-02; Vazyme, China). The construct was then
673  introduced into rae5-1 mutant by Agrobacterium tumefaciens (strain
674  GV3101)-mediated transformation, and T3 plants harboring the homozygous
675  transgene were subjected to expression analysis and phenotypic analysis for the
676  complementation test.
677 

678  RNA Isolation and RT-qPCR Analysis

679  To conduct expression analysis of Al-resistance genes, seeds were sown on agar plates
680  containing 1/2 MS medium and 1% sucrose and allowed to grow for 10 d. The
681  seedlings were then exposed to a 0.5 mM CaCl2 solution with 0 or 30 µM AlCl3 at pH
682  4.7. After 8 h treatment, roots and/or shoots of around 24 seedlings were excised and
683  pooled for RNA extraction in each of three sets of seedlings. Total RNA of each root
684  sample was extracted using the TaKaRa M iniBEST plant RNA Extraction Kit (Cat #
685  9769) and then digested with DNase I to remove contaminating DNA. Approximately
686  1 µg of total RNA was used for first-strand cDNA synthesis by the HiScript® 1st
687  Strand cDNA Synthesis Kit (Vazyme Biotech Co., Ltd., Nanjing, China).
688  One-twentieth of the cDNA products were used for real-time PCR analysis by the
689  SYBR® Green Master Mix kit (Vazyme Biotech Co., Ltd., Nanjing, China) and
690  gene-specific primers (Supplemental Table 2). UBQ10 was used as an internal control
691  for sample normalization (reference gene) in the real-time RT-PCR analysis (2ΔΔCT
692  method). Real-time RT-PCR data were recorded and analyzed using the CFX96 Touch
693  real-time PCR detection system (BioRad).
694 

695  Yeast Two-Hybrid Assay and Split-LUC Complementation Analysis

696  For yeast two-hybrid experiments, the CDS of STOP1 was introduced into pGADT7
697  vector, while the CDS of catalytically inactive SUMO proteases ESD4C448S, ELS1C461S,
698  ELS2C300S, OTS2C512S, and OTS1C525S were cloned into pGBKT7 vector, respectively.
699  STOP1 and each SUMO protease were, respectively, co-expressed into yeast strain
700  AH109 and then the yeast cells were grown on SD (-Leu/Trp) and SD (-Leu/Trp/His)
701  media to determine the protein-protein interaction.
  24
  

702  For split-LUC complementation analysis, the CDS of STOP1 was cloned into
703  pCAMBIA1-nLUC or pCAMBIA1-cLUC vectors, and the CDS of ESD4, ELS1,
704  ELS2, OTS2, OTS1, FUG1, SPF1, and SPF2 were also introduced into
705  pCAMBIA1-nLUC or pCAMBIA1-cLUC (Chen et al., 2008). STOP1-nLUC and
706  cLUC-STOP1 was, respectively, co-transformed with cLUC and nLUC fused ESD4,
707  ELS1, ELS2, OTS1, OTS2, FUG1, SPF1, or SPF2 into fully expanded N.
708  benthamiana leaves by Agrobacterium-mediated transformation, respectively. The
709  transformed plants were grown in the dark for 1 d and in the long-day conditions for 2
710  d, and then subjected for LUC signal detection to determine whether the two proteins
711  interacted with each other. The primers used for vector construction are listed in
712  Supplemental Table 2.

713 

714  Observation of STOP1-GFP Subcellular Localization in WT and esd4-3 roots

715  The coding sequence of STOP1 was amplified and fused in frame with GFP gene and
716  then inserted into pCAMBIA1301 vector (SpeI and PstI restriction sites) harboring
717  35S promoter and hygromycin resistance marker using ClonExpress II One Step
718  Cloning Kit ( C112-02; Vazyme, China). The resultant vector was transformed into
719  Arabidopsis wild-type plants. A T2 line showing single-locus segregation on
720  hygromycin resistance was screened out and crossed with esd4-3 to introduce the
721  35S:STOP1-GFP transgene into the mutant background. To compare the subcellular
722  localization of STOP1-GFP in WT and esd4-3 under different Al conditions,
723  35S:STOP1-GFP seedlings of WT and esd4-3 were grown on 1/2 MS medium plates
724  for 5 d and then transferred to a soaked gel medium described above with or without 1
725  mM AlCl3 overnight. Roots were first stained with 10 μM propidium iodide (PI)
726  solution for 10 s and then subjected to GFP and PI fluorescence observation by using
727  a Leica confocal microscope (Leica SP8). ImageJ software was used to quantify GFP
728  fluorescence intensity, which was integrated from all pixels in selected areas. To
729  measure the ratio between the nuclear and cytoplasmic signals for each cell, the entire
730  cellular and nuclear area was selected to quantify fluorescence intensity. The

  25
731  cytoplasmic intensity was calculated by subtracting the value for the nuclear area
732  from the whole cell. Thirteen root tip cells of each of five roots were used for the
733  calculation of the ratio of the GFP signal in nucleus to cytosol.
734 

735  Transient Expression in Protoplasts

736  Arabidopsis protoplasts were isolated from 14-day-old seedlings according to the
737  method described by Zhai et al. (2009). To determine the SUMOylation status of
738  STOP1, 2 mL protoplasts (~2x106 cells/ml) of WT or esd4 mutants was co-transfected
739  with 50 μg 35S:6Myc-SUMO1 (CDS of the SUMO1 precursor, mature (GG) or
740  mutated SUMO1 (4KR, AA) constructed into pHBT vector) and with 50 μg
741  35S:STOP1-2Flag (WT or mutated STOP1) and with or without WT or mutated
742  ESD4-3HA under the 35S promoter control. The total protein fraction was extracted
743  by macerating frozen tissue in protein extraction buffer (20 mM Tris-HCl pH 7.4, 150
744  mM NaCl, 1 mM MgCl2, 0.25% NP-40, 0.25% Triton X-100, 0.05% SDS, 1 mM DTT,
745  50 μM MG132 (A2585; APExBIO, USA), 20 mM NEM, 50 μM PR-619, and 1×
746  complete protease inhibitor mixture (4693132001, Sigma-Aldrich) and were
747  immunoprecipitated with 20 μL of anti-FLAG M2 magnetic beads (M8823,
748  Sigma-Aldrich). Next, the beads were washed with 1 mL protein extraction buffer
749  three times, and the immunoprecipitated proteins were eluted with 1× SDS loading
750  buffer (50 mM Tris-HCl pH 6.8, 2% SDS, 10% glycerol, 0.1% bromophenol blue and
751  1% β-Mercaptoethanol) for immunoblot analysis. The SUMOylated form of STOP1
752  was detected with anti Myc-HRP antibody (A00704-100; Genscript Biotech Co., Ltd.,
753  China).
754  For the prediction of SUMOylation sites in STOP1, Joined Advanced
755  SUMOylation site and SIM Analyser (JASSA) and GPS-SUMO online services were
756  both used, and the predicted sites were combined. As a result, six potential
757  SUMOylation sites of STOP1 were predicted. We then generated mutated versions of
758  STOP1 with different number of mutation sites on the six potential SUMOylation
759  sites by site-directed mutagenesis using WT 35S:STOP1-2Flag as a template and
760  oligonucleotide primers listed in Supplemental Table 2. The resultant different
26
761  versions of 35S:STOP1-2Flag vectors were individually co-expressed with
762  35S:6Myc-SUMO1 in the protoplasts and subjected to the detection of STOP1
763  SUMOylation sites as described above.
764  For Co-IP assays, 2 ml of protoplasts (~2x106 cells/ml) were co-transfected with 50
765  μg 35S:ESD4-2HA and with 50 μg 35S:STOP1-2Flag or 35S:GFP-2FLAG. The total
766  protein fraction was extracted using the protein extraction buffer (100 μL), and 20 μL
767  of the protein extract solution was used as the input control. The remaining cell
768  extracts were diluted to 1 ml and incubated with 20 μL of anti-FLAG M2 magnetic
769  beads for 3 h at 4°C with gentle rotation. The extracts were then washed three times
770  with 1 mL protein extraction buffer, and the bound proteins were eluted with 1× SDS
771  loading buffer for immunoblot analysis using anti Flag-HRP (A8592; Sigma-Aldrich)
772  or HA-HRP (12013819001, Lot 44323100; Roche) antibodies. The primers used for
773  the vector construction are listed in Supplemental Table 2.
774 

775  Determination of STOP1 SUMOylation and Protein Levels in Roots

776  To detect STOP1 SUMOylation in planta, ten-day-old transgenic seedlings harboring
777  pSUMO1:2Flag-SUMO1 and/or pSTOP1:STOP1-3HA transgenes were exposed to a
778  0.5 mM CaCl2 solution containing 0 or 30 μM AlCl3 at pH 4.7 for 8 h. The roots
779  (~600 mg) were excised and frozen for protein extraction using the extraction buffer
780  composed of 50 mM HEPES (pH 7.5), 100 mM NaCl, 5 mM EDTA, 10 % Glycerol,
781  0.25% NP-40, 0.5% Triton X-100, 0.05% SDS, 50 μM MG132, 20 mM
782  N-Ethylmaleimide, 50 μM PR-619, and 1× complete protease inhibitor mixture. The
783  extracted proteins were immunoprecipitated with anti-HA magnetic beads (B26202,
784  Biomake), and the immunoprecipitates were then washed with 1 mL protein
785  extraction buffer for five times and subsequently eluted with 1× SDS loading buffer
786  for immunoblot analysis. The SUMOylated form of STOP1 was detected with anti
787  Flag-HRP antibody. The ACTIN protein detected by anti-ACTIN antibody
788  (CW0264M; CoWin Biosciences Co., Ltd., China) was used as the loading control.
789  For the determination of ESD4 and STOP1 protein levels in roots, ten-day-old
790  seedlings were exposed to a 0.5 mM CaCl2 solution containing 0 or 30 μM AlCl3 at
27
791  pH 4.7 for 0, 2, 4, or 8 h. To examine the effect of esd4-3 mutation or STOP13KR on
792  the STOP1 stability, seedlings were treated with 100 μM cycloheximide (A8244,
793  ApexBio) and with 0 or 30 μM AlCl3 for different time. Total proteins were extracted
794  from the roots (~100 mg) using the extraction buffer (150 μL per 100 mg tissue)
795  composed of 20 mM Tris-HCl (pH 7.4), 300 mM NaCl, 5 mM MgCl2 (pH 8.0), 5 mM
796  DTT, 0.5% NP40, 50 μM MG132, and 1× Complete Protease inhibitor tablets
797  EDTA-free (5892791001; Roche). The total proteins were separated on 8%
798  SDS-PAGE and the resolved ESD4-3Flag and STOP1-3HA proteins were analyzed by
799  standard immunoblot using anti Flag-HRP (A8592; Sigma-Aldrich) and HA-HRP
800  (12013819001, Lot 44323100; Roche) antibodies, respectively. Protein bands on the
801  membranes were captured by ChemiDoc Touch Imaging System (1708370; Bio-Rad,
802  USA). The ACTIN or TUBULIN proteins were used as the loading control, which
803  were detected by anti-ACTIN or anti-TUBULIN (M0267-1-HRP; Abiocode, USA)
804  antibodies.
805  To examine the effect of mutation of STOP1 SUMOylation sites on STOP1
806  accumulation, Constructs of pSTOP1:STOP1K40R-3HA, pSTOP1:STOP1K212R-3HA,
807  pSTOP1:STOP1K395R-3HA, and pSTOP1:STOP1K40R, K212R, K395R
-3HA were made by
808  site-directed mutagenesis using WT pSTOP1:STOP1-3HA as a template and
809  oligonucleotide primers listed in Supplemental Table 2 and the constructs were then,
810  respectively, transformed in stop1-2 mutant background. Single-locus homozygous
811  transgenic lines showing similar expression level of STOP1 were screened.
812  Ten-day-old seedling of these transgenic lines were exposed a 0.5 mM CaCl2 solution
813  containing 0 or 30 μM AlCl3 at pH 4.7 for 8 h and then STOP1 protein levels in roots
814  were determined by using anti HA-HRP antibody.
815  For quantification of STOP1-GUS levels, 7-day-old seedlings of WT and esd4-3
816  harboring the pSTOP1:STOP1-GUS transgene were treated with 0.5 mM CaCl2
817  solution containing 0 or 30 μM AlCl3 at pH 4.7. After 8 h treatment, the roots were
818  collected and ground to a fine powder, and then mixed with GUS extraction buffer (50
819  mM sodium phosphate at pH 7.0, 10 mM EDTA, 0.1% SDS, 0.1% Triton X-100, 10
820  mM 2-mercaptoethanol and 25 μg/ml PMSF). The mixed solution was centrifuged at
28
821  15,000 rpm for 10 minutes at 4℃, and subsequently a portion of the supernatant was
822  used for the reaction with the GUS extraction buffer containing 1 mM
823  4-Methylumbelliferyl-β-D-glucuronide (A602251; Sangon Biotech Co., Ltd.,
824  Shanghai, China) at 37°C. After 1 h, Na2CO3 solution (0.2 M) was added to stop the
825  reaction. The fluorochrome 4-methyl umbelliferone (4-MU) reaction product was
826  detected for its fluorescence with an excitation and emission wavelength of 350 nm
827  and 455nm, respectively. GUS activity in each sample was normalized to the total
828  protein.
829 

830  ChIP Assay

831  Eight-day-old plants of WT and esd4-3 harboring pSTOP1:STOP1-3HA were exposed
832  to a 0.5 mM CaCl2 solution with 0 or 30 µM AlCl3 at pH 4.7 for 8 h. The roots (~1.2 g)
833  were excised for the ChIP analysis. The ChIP method was according to Saleh et al.
834  (2008) (Saleh et al., 2008) with slight modifications for the sonication step in which
835  isolated nuclei were sonicated for 55 cycles by a Bioruptor (Diagenode) with 15s on
836  and 15s off on high power set. Protein immunoprecipitation was carried out by using
837  the anti-HA antibody (H3663; Sigma-Aldrich) or IgG control (B3001S; Abmart Co.,
838  Ltd., China) followed by adding 100 μl Salmon DNA pre-equilibrium beads
839  (SM00405; Smart Life Science). The protein bound DNA was quantified by real-time
840  PCR using gene-specific primers (Supplemental Table 2).
841 

842  Statistical analysis

843  Two-tailed student’s t test was used to do statistical analysis on two lines, while for
844  more than two lines one-way ANOVA followed by Tukey test was used for the
845  statistical analysis. The results of statistical analyses and the list of individual data for
846  the statistical analyses are shown in Supplemental Data set 1 and Supplemental Data
847  set 2, respectively.
848 

849  Accession Numbers

850  Gene sequence data in this article can be found in the Arabidopsis Information
29
851  Resource (TAIR) database under the following accession number: At1g34370 for
852  STOP1; At4g15880 for ESD4. Whole genome sequence data of pooled F2 rae5-2
853  mutant plants can be found in NCBI under the accession number: SRR9290163.
854 

855  SUPPLEMENTAL DATA

856  Supplemental Figure 1. Effect of rae5 Mutations on the Expression of

857  STOP1-Regulated Genes in Shoots (Supports Figure 1).

858  Supplemental Figure 2. Effect of rae5 Mutations on Citrate Exudation, Cd and La
859  Tolerance, and Plant Morphology (Supports Figure 2).
860  Supplemental Figure 3. Mutmap Analysis and Complementation Test of rae5
861  Mutants (Supports Figures 1 and 2).
862  Supplemental Figure 4. STOP1 is SUMOylated with Three Bands and Al Stress
863  Reduces STOP1 SUMOylation (Supports Figure 4).
864  Supplemental Figure 5. Interaction analysis of STOP1 with SUMO proteases
865  (Supports Figure 5).
866  Supplemental Figure 6. Effect of the esd4-3 mutation and Al treatment, alone and in
867  combination, on the subcellular localization of STOP1 (Supports Figure 6).
868  Supplemental Figure 7. ChIP analysis of the association of STOP1 with different
869  regions of AtMATE promoter (Supports Figure 6E).
870  Supplemental Figure 8. Effect of Mutation of STOP1 SUMOylation Sites on the
871  Expression of STOP1-Regulated Genes (Supports Figure 8).
872  Supplemental Figure 9. Effect of Mutation of STOP1 SUMOylation Sites on
873  Low-pH Tolerance (Supports Figure 8).
874  Supplemental Figure 10. Effect of Mutation of STOP1 SUMOylation Sites on
875  Low-Phosphate (Pi) Response (Supports Figure 8).
876  Supplemental Table 1 List of mutations in rae5-2 and linkage analysis.
877  Supplemental Table 2 Primers used in this study.
878  Supplemental Data set 1. Statistics Summary of Quantitative Data in all the Figures.
879  Supplemental Data set 2. List of Individual Data for the Statistical Analyses.
880 

30
881  AUTHOR CONTRIBUTIONS
882  Q.F., J.Z. and C.-F.H. designed the experiments. Q.F., J.Z. N.F., Y.Z. performed the
883  experiments. H.A.B. commented on the research and the manuscript. C.-F.H. wrote
884  the manuscript.
885 

886  ACKNOWLEDGEMENTS
887  We thank Dr. Rosa Lozano-Duran for commenting on the manuscript, and we also
888  thank Dr. Yan Guo for providing us the 6Myc-SUMO1 plasmid. This work was
889  supported by Shanghai Natural Science Foundation (Grant No. 20ZR1466500),
890  National Natural Science Foundation of China (Grant No. 31570253 and 31870223
891  to C.-F. H.), the funding from Shanghai Center for Plant Stress Biology, Chinese
892  Academy of Sciences, and National Key Laboratory of Plant Molecular Genetics.

893  FIGURE LEGENDS

894  Figure 1. Mutation of RAE5 Alters the Expression of STOP1-Regulated Genes.
895  (A) Increased LUC signal of pAtALMT1:LUC in rae5 mutants and F1 plants from a
896  cross between rae5-1 and rae5-2. Scale bar=1 cm. (B-I) Effect of rae5 mutations on
897  the mRNA accumulation levels of Al-resistance genes. Seedlings of WT, rae5-1 and
898  rae5-2 were exposed to 0 or 30 µM Al for 8 h and then the roots were excised for
899  mRNA isolation and expression analysis. RT-qPCR analysis was carried out to
900  determine the expression of LUC (B), AtALMT1 (C), AtMATE (D), RAE1 (E), ALS3
901  (F), STOP1 (G), ALS1 (H), and AtSTAR1 (I). Data shown are means ± SD of three
902  biological replicates. Different letters at each treatment indicate values significantly
903  different (P<0.05, ANOVA followed by Tukey test). At least three independent
904  experiments were performed with similar results.
905 

906  Figure 2. The rae5 Mutations Increase Malate Secretion and Al Resistance. (A)
907  Increased malate release in rae5 mutants. Seedlings of WT, rae5-1 and rae5-2 were
908  treated with 0 or 10 μM AlCl3 for 12 h and the root exudates were analyzed for the
909  malate concentrations (pmol plant-1 hour-1). Values are means ± SD of four biological
31
910  replicates. (B-D) Al-resistance (B and C) and Al-accumulation phenotype (D) of WT,
911  rae5-1, rae5-2 and Atalmt1. Seedlings were grown on a soaked gel medium
912  containing 0, 0.75, 1.0 or 1.25 mM Al for 7 d and the relative root growth was used to
913  evaluate Al resistance. The Al indicator Eriochrome Cyanine R was used to stain the
914  roots exposed to 0 or 1.0 mM Al (D). Scale bar=1 cm. Values are means ± SD of
915  relative root length of 48 to 60 seedlings. Means with different letters are significantly
916  different (P<0.05, ANOVA test followed by Tukey test). At least three independent
917  experiments were performed with similar results.
918 

919  Figure 3. Al stress Promotes ESD4 Protein Accumulation. (A) Effect of Al on

920  ESD4 mRNA expression. WT seedlings were treated without Al or with 30 μM Al for
921  2, 4, 6 or 8 h and roots were sampled for RT-qPCR analysis of AtALMT1 and ESD4.
922  (B and C) Representative pictures (B) and relative protein level of ESD4-3Flag (C)
923  under –Al or +Al conditions. Seedlings of an esd4-3 complementation line
924  (pESD4:ESD4-3Flag) were treated without Al or with 30 μM Al for 2, 4 or 8 h and
925  roots were sampled for immunoblot analysis of ESD4-3Flag and ACTIN control.
926  ESD4-3Flag protein levels were first normalized to their respective ACTIN controls
927  and then normalized to the protein level under the –Al condition. Data shown are
928  means ± SD of three biological replicates. Asterisks indicate values statistically
929  different (Student’s t test, **P<0.01). Two and three independent experiments were
930  performed with similar results for (A) and (B and C), respectively.
931 

932  Figure 4. SUMOylation of STOP1 in WT and esd4 Mutants. (A and B) Detection

933  of STOP1 SUMOylation with the expression of Myc-tagged SUMO precursor
934  (6Myc-SUMO1) (A), mature SUMO (6Myc-SUMO1GG) or conjugation-deficient
935  SUMO (6Myc-SUMO1AA) (B). GFP-2Flag served as a negative control. (C) STOP1
936  is mono-SUMOylated. Expression of SUMO14KR variant where four different lysines
937  are substituted for arginine, which prevents the formation of poly-SUMO chains, does
938  not affect STOP1 SUMOylation pattern. (D) SUMOylation levels of STOP1 were
939  increased in the esd4-3 and esd4-4 mutants. For detection of STOP1 SUMOylation in
32
  

940  protoplasts, the protoplasts of WT or esd4 mutants were co-transfected with

941  constructs for STOP1-2Flag and different forms of 6Myc-SUMO1. Crude total
942  protein lysates (input) were used for STOP1 immunoprecipitation (IP) with
943  anti-FLAG magnetic beads and 6Myc-SUMO1 modified STOP1 was detected using
944  an anti-Myc antibody. (E and F) Al stress reduces the level of STOP1 SUMOylation
945  in Arabidopsis plants. Plants of WT and/or esd4-3 harboring different transgenes were
946  treated with or without 30 μM Al for 8 h. Total root proteins (input) were
947  immunoprecipitated with anti-HA magnetic beads and SUMOylated STOP1 was
948  detected using anti-Flag antibody. The total STOP1 proteins were adjusted to a similar
949  level between –Al and +Al treatments for the immunoprecipitation of STOP1-3HA.
950  STOP1 SUMOylation was normalized to the WT level under the –Al condition. Data
951  shown are means ± SD of three biological replicates. Asterisks indicate values
952  statistically different (Student’s t test, *P<0.05). Three independent experiments were
953  performed with similar results.
954 

955  Figure 5. ESD4 Interacts with and deSUMOylates STOP1. (A) Interaction of
956  STOP1 and ESD4 in a yeast two-hybrid assay. The CDS for STOP1 and catalytically
957  inactive (C448S) ESD4 were introduced into pGADT7 and pGBKT7 vectors,
958  respectively. Yeast cells co-expressing STOP1 and ESD4C448S were grown on SD
959  (-Leu/Trp) and SD (-Leu/Trp/His) mediums. (B) Interaction of ESD4 with STOP1 as
960  indicated by split luciferase complementation assays. CaMV 35S promoter-driven
961  construct pairs indicated in the figure were co-expressed in N. benthamiana leaves,
962  and then the luciferase activity due to LUC reconstitution was measured for the
963  different combinations. (C) Coimmunoprecipitation of ESD4 with STOP1.
964  ESD4-3HA was co-expressed with STOP1-3Flag or GFP-2Flag in Arabidopsis
965  protoplasts. Crude protein extracts were immunoprecipitated with anti-HA magnetic
966  beads and then detected with anti-Flag antibody. (D and E) ESD4 mediates the
967  deSUMOylation of STOP1. STOP1-2Flag was co-expressed with ESD4-3HA,
968  esd4-3-3HA, esd4-4-3HA, or ESD4C448S-3HA and SUMO precursor (6Myc-SUMO1)
969  (D) or mature SUMO (6Myc-SUMO1GG) (E) in Arabidopsis protoplasts. Crude total
  33
970  protein extracts were used to immunoprecipitate STOP1 with anti-FLAG magnetic
971  beads and the SUMOylated form of STOP1 was detected with anti-Myc antibody.
972  Two and three independent experiments were performed with similar results for (A)
973  and (B-E), respectively.
974 

975  Figure 6. Effect of the esd4-3 Mutation on STOP1 Level and Stability and on the
976  Association of STOP1 with the Promoters of STOP1-Regulated Genes. (A)
977  Immunoblot analysis of STOP1-3HA in WT and esd4-3 under –Al or +Al conditions.
978  TUBULIN protein was used as an internal control. (B) Quantification of STOP1-GUS
979  expression in WT and esd4-3 under –Al or +Al conditions. (C and D) Effect of esd4-3
980  mutation on STOP1 stability. (C) Roots of WT and esd4-3 harboring the
981  pSTOP1:STOP1-3HA transgene were treated without Al or with 30 μM Al and 100
982  μM cycloheximide (CHX) for different time as indicated in the figure. STOP1-3HA
983  protein levels were detected at each time point with anti-HA antibody. ACTIN protein
984  was served as an internal control. (D) Roots were treated with 30 μM Al for 4 h and
985  then transferred to –Al conditions plus 100 μM CHX for different time as indicated in
986  the figure and then the protein levels of STOP1-3HA at each time point were detected.
987  (E-H) ChIP assay was performed to determine the association of STOP1-3HA with
988  the promoters of STOP1-regulated genes including AtALMT1 (E), AtMATE (F) and
989  RAE1 (G), and with the promoter of ACTIN7 control (H). Values are means ± SD of
990  three technical replicates. Asterisks indicate values statistically different (Student’s t
991  test, *P<0.05, **P<0.01). N.S. indicates non-significantly different P values. Three
992  independent experiments were performed with similar results.
993 

994  Figure 7. STOP1 is SUMOylated at K40, K212 or K395 Sites. (A) Domain
995  structure of STOP1 with the predicted SUMO acceptor sites. Six predicted SUMO
996  acceptor sites with the cognate motif are shown, including the lysine acceptor. The
997  green boxes indicate the four C2H2 zinc finger domains of STOP1. (B) Effect of the
998  combined replacement of five of the six SUMO acceptor lysines in STOP1-2Flag with
999  arginines on STOP1 SUMOyation. Red arrows indicate the bands with
34
  

1000  SUMO-modified STOP1. (C) Effect of single, double or triple K-to-R mutations of
1001  K40, K212 and K395 on STOP1 SUMOylation. Crude protein extracts of protoplasts
1002  co-expressing 6Myc-SUMO1 and mutated STOP1-2Flag were used to
1003  immunoprecipitate STOP1 with anti-FLAG antibody and the SUMOylated forms of
1004  STOP1 were detected using anti-Myc antibody. At least three independent
1005  experiments were performed with similar results.
1006 

1007  Figure 8. Effect of mutation of STOP1 SUMOylation Sites on STOP1 Protein

1008  Levels and Al Resistance. WT and mutated versions of STOP1 were transformed
1009  into stop1-2 mutant, respectively, to generate various complementation lines. (A-C)
1010  Immunoblot analysis of STOP1-3HA in STOP1WT (WT), STOP1K40R (K40R) (A),
1011  STOP1K212R (K212R) (B), and STOP1K395R (K395R) (C) complementation lines
1012  exposed to 0 (–Al) or 30 μM Al (+Al) for 8 h. (D and E) Al-resistance phenotype of
1013  WT, K40R, K212R, and K395R complementation lines. Seedlings were grown on a
1014  soaked gel medium containing 0, 0.75 or 1.0 mM Al for 7 d. Values are means ± SD
1015  of root lengths of 21 to 35 seedlings. (F) Immunoblot analysis of STOP1-3HA in one
1016  STOP1WT (WT) and two STOP1 K40R , K212R, K395R (3KR-1 and 3KR-2) complementation
1017  lines under –Al and +Al conditions. (G) STOP1 stability was increased in the 3KR
1018  line. Roots of WT and 3KR lines were treated with 30 μM Al for 4 h and then
1019  transferred to –Al conditions with 100 μM cycloheximide (CHX) for different time as
1020  indicated in the figure. STOP1-3HA protein levels were detected at each time point
1021  with anti-HA antibody. (H and I) Al-resistance phenotype of WT and 3KR
1022  complementation lines. Values are means ± SD of relative root length of 32 to 43
1023  seedlings. Means with different letters are significantly different (P<0.05, ANOVA
1024  test followed by Tukey test). Scale bar= 1 cm. Three independent experiments were
1025  performed with similar results.
1026 

1027  Figure 9. Model for Regulation of STOP1 Function and Stability by

1028  SUMOylation. STOP1 is mono-SUMOylated at the three sites K40, K212 or K395,
1029  presumably through the SUMOylation pathway. The STOP1 SUMOylation is
  35
  

1030  reversible, and it can be deSUMOylated by the SUMO protease ESD4, which is
1031  increased in response to Al stress. Mutation of ESD4 elevates STOP1 SUMOylation
1032  levels and consequently increases and decreases the association of STOP1 with the
1033  promoters of AtALMT1 and AtMATE, respectively, which causes the increased
1034  AtALMT1 expression and the reduced expression of AtMATE and RAE1, and finally
1035  contributes to the increased Al resistance. Mutation of STOP1 SUMOylation site at
1036  K40 increases AtALMT1 and decreases AtMATE expression, which leads to reduced
1037  Al resistance. Mutation at K212 site does not affect STOP1 function and Al resistance.
1038  Blocking of STOP1 SUMOylation at K395 or all the three (K40, K212 and K395)
1039  sites reduces STOP1 protein level and the expression of AtALMT1 and AtMATE,
1040  leading to the decreased Al resistance.
1041 

1042 

  36
 A B C
LUC AtALMT1
15 25

Relative expression

Relative expression
WT rae5-1 rae5-2 F1 WT WT A
rae5-1 A 20 rae5-1
A B
10 rae5-2 rae5-2
15
C
B 10
5
b a 5
1 cm c b a
c
0 0
-Al +Al -Al +Al
D E F
AtMATE RAE1 ALS3
8 6 6
Relative expression

Relative expression

Relative expression
WT WT WT
rae5-1 rae5-1 rae5-1
6 rae5-2 rae5-2 A
4 4 rae5-2
A
A
4 B
A A A
B B
2 2 a
2 a a
b b a a
ab b
0 0 0
-Al +Al -Al +Al -Al +Al
G H I
STOP1 ALS1 STAR1
3 3 3
Relative expression

Relative expression

Relative expression

WT WT WT
rae5-1 rae5-1 rae5-1
2 rae5-2 2 rae5-2 2 rae5-2
A a A
a a A A a
a a A A a
a
1 b 1 1 A A A

0 0 0
-Al +Al -Al +Al -Al +Al

Figure 1. Mutation of RAE5 Alters the Expression of STOP1-Regulated Genes. (A) Increased LUC
signal of pAtALMT1:LUC in rae5 mutants and F1 plants of rae5-1 and rae5-2. Scale bar=1 cm. (B-I)
Effect of rae5 mutations on the mRNA accumlation levels of Al-resistance genes. Seedlings of WT,
rae5-1 and rae5-2 were exposed to 0 or 30 µM Al for 8 h and then the roots were excised for mRNA
isolation and expression analysis. Real-time RT-PCR analysis was carried out to determine the
expression of LUC (B), AtALMT1 (C), AtMATE (D), RAE1 (E), ALS3 (F), STOP1 (G), ALS1 (H), and
AtSTAR1 (I). Data shown are means ± SD of three biological replicates. Different letters at each
treatment indicate values significantly different (P<0.05, ANOVA followed by Tukey test). At least three
independent experiments were performed with similar results.
 A D

50 WT A

(pmol plant-1 h-1 )

40 rae5-1
Malate secretion B
30

Al concentration (mM)
rae5-2 C 0
20
10
6
4 a
c b
2 1.0
0
- Al +Al

B C WT
WT
150 rae5-1
rae5-2
length
root length a almt1
Atalmt1
a
(% of control)
control) 100 b ab a
c b
c
Relative root

b
c
of
Relative

50
d
(%

d
1 cm

0
0 1.25 0 0.75 1.0 1.25
Al concentration (mM) Al concentration (mM)

Figure 2. The rae5 Mutations Increase Malate Secretion and Al Resistance. (A) Increased
malate release in rae5 mutants. Seedlings of WT, rae5-1 and rae5-2 were treated with 0 or 10 μM
AlCl3 for 12 h and the root exudates were analyzed for the malate concentrations (pmol plant-1
hour-1). Values are means ± SD of four biological replicates. (B-D) Al-resistance (B and C) and Al
accumulation phenotype (D) of WT, rae5-1, rae5-2 and Atalmt1. Seedlings were grown on a
soaked gel medium containing 0, 0.75, 1.0 or 1.25 mM Al for 7 d and the relative root growth was
used to evaluate Al resistance. The Al indicator Eriochrome Cyanine R was used to stain the roots
exposed to 0 or 1.0 mM Al (D). Scale bar=1 cm. Values are means ± SD of relative root length of
48 to 60 seedlings. Means with different letters are significantly different (P<0.05, ANOVA test
followed by Tukey test). At least three independent experiments were performed with similar
results.
 A C
8 AtALMT1 -Al 3 -Al **
Relative expression

Relative protein level

AtALMT1 +Al **
ESD4 -Al
+Al
6
ESD4 +Al
2

of ESD4
4
1
2

0 0
0 2 4 6 8 0 2 4 6 8
Time (h) Time (h)

B pESD4:ESD4-3Flag/esd4-3 pESD4:ESD4-3Flag/esd4-3
0 2 4 8 0 0 2 4 8 (h)
(kDa)
70 ESD4-3Flag

40 Actin

-Al +Al

Figure 3. Al stress Promotes ESD4 Protein Accumulation. (A) Effect of Al on ESD4 mRNA
expression. WT seedlings were treated without Al or with 30 μM Al for 2, 4, 6 or 8 h and roots
were sampled for RT-qPCR analysis of AtALMT1 and ESD4. (B and C) Representative pictures
(B) and relative protein level of ESD4-3Flag (C) under –Al or +Al conditions. Seedlings of an
esd4-3 complementation line (pESD4:ESD4-3Flag) were treated without Al or with 30 μM Al for 2,
4 or 8 h and roots were sampled for immunoblot analysis of ESD4-3Flag and Actin control.
ESD4-3Flag protein levels were first normalized to their respective Actin controls and then
normalized to the protein level under the –Al condition. Data shown are means ± SD of three
biological replicates. Asterisks indicate values statistically different (Student’s t test, **P<0.01).
Two and three independent experiments were performed with similar results for (A) and (B and
C), respectively.
 A B
GFP-2Flag + - + - STOP1-2Flag + - + - +
STOP1-2Flag - + - + 6Myc-SUMO1AA - + + - -
6Myc-SUMO1 + + + +
6Myc-SUMO1GG - - - + +
(kDa) (kDa)
180
135 α Myc
180 180 IP: Flag
100
130 130 SUMO-STOP1 70 α Flag
100 100
IP: Flag (kDa) 70
75 α Flag
75
α Myc Input
26 α Myc
17
C
25 Input IP
25
STOP1-2Flag + + + +
α Flag
Input

25 α Myc 6Myc-SUMO1 + - + -
6Myc-SUMO14KR - + - +
180
26 α Myc
130
17 100
E
2Flag-SUMO1/STOP1-3HA α Flag
70 70
WT esd4-3 (kDa)
(kDa) - - + - + - + Al
180 D STOP1-2Flag STOP1-2Flag
130 + 6Myc-SUMO1 + 6Myc-SUMO1GG
100
75 α-Flag (kDa)
180
Input

30 α-Myc
IP:Flag

130
100
α-Flag
75 70
STOP1-3HA
α-Flag
70
Input

45 Actin 26
α-Myc
17
180
Longer
to unmodifed

130
100 exposure F 5
STOP1 SUMOylation

α-Flag -Al
signal intensity

*
of

Shorter 4 +Al
180 **
level

exposure
130 3
relative

100 SUMOylated-STOP1
IP：HA

Relative

180 2
**
STOP1

130
SUMOylated

Reprobed with HA
100 1
75 STOP1-3HA
63 0
WT esd4-3

Figure 4. SUMOylation of STOP1 in WT and esd4 Mutants. (A and B) Detection of STOP1 SUMOylation with the
expression of Myc-tagged SUMO precursor (6Myc-SUMO1) (A), mature SUMO (6Myc-SUMO1GG) or conjugation-
deficient SUMO (6Myc-SUMO1AA) (B). GFP-2Flag was served as a negative control. (C) STOP1 is mono-
SUMOylated. Expression of SUMO14KR variant where four different lysines are substituted for arginine, which prevents
the formation of poly-SUMO chains, does not affect STOP1 SUMOylation pattern. (D) SUMOylation levels of STOP1
were increased in the esd4-3 and esd4-4 mutants. For detection of STOP1 SUMOylation in protoplasts, the
protoplasts of WT or esd4 mutants were co-transfected with constructs for STOP1-2Flag and different forms of 6Myc-
SUMO1. Crude total protein lysates (input) were used for STOP1 immunoprecipitation (IP) with anti-FLAG magnetic
beads and 6Myc-SUMO1 modified STOP1 was detected using an anti-Myc antibody. (E and F) Al stress reduces the
level of STOP1 SUMOylation in Arabidopsis plants. Plants of WT and/or esd4-3 harboring different transgenes were
treated with or without 30 μM Al for 8 h. Total root proteins (input) were immunoprecipitated with anti-HA magnetic
beads and SUMOylated STOP1 was detected using anti-Flag antibody. The total STOP1 proteins were adjusted to a
similar level between –Al and +Al treatments for the immunoprecipitation of STOP1-3HA. STOP1 SUMOylation was
normalized to the WT level under the –Al condition. Data shown are means ± SD of three biological replicates.
Asterisks indicate values statistically different (Student’s t test, *P<0.05). Three independent experiments were
performed with similar results.
 A 10-1 10-2 10-3 10-1 10-2 10-3 B
nLUC nLUC
+ cLUC-STOP1 + cLUC

RAE1-nLUC ESD4-nLUC
+ cLUC-STOP1 +cLUC

B
-L-T -L-T- H

C Input IP: HA
RAE1ΔF-nLUC ESD4-nLUC
+ cLUC-STOP1 + cLUC-STOP1
ESD4-3HA + + + + cLUC-ESD4 cLUC
STOP1-2Flag + - + - + STOP1-nLUC +cLUC
STOP1-nLUC
GFP-2Flag - + - +

70
α-Flag
25

70 α-HA
cLUC-ESD4
(kDa) cLUC + nLUC
+ nLUC
D E
STOP1-2Flag + + + + + STOP1-2Flag+ + + + +
ESD4-3HA - + - - - ESD4-3HA - + - - -
esd4-3-3HA - - + - - esd4-3-3HA - - + - -
esd4-4-3HA - - - + - esd4-4-3HA - - - + -
ESD4 C448S-3HA - - - - +
ESD4C448S-3HA - - - - +
6Myc-SUMO1 + + + + + 6Myc-SUMO1GG + + + + +
180 180
130 α Myc 130 α Myc
IP: Flag IP: Flag 100
100
70 α Flag α Flag
70
70 α Flag 70 α Flag
Input 70 α HA Input α HA
70
α Myc 17 α Myc
17
(kDa) (kDa)

Figure 5. ESD4 Interacts with and deSUMOylates STOP1. (A) Interaction of STOP1 and ESD4 in a yeast
two-hybrid assay. The CDS for STOP1 and catalytically inactive (C448S) ESD4 were introduced into pGADT7
and pGBKT7 vectors, respectively. Yeast cells co-expressing STOP1 and ESD4C448S were grown on SD (-
Leu/Trp) and SD (-Leu/Trp/His) mediums. (B) Interaction of ESD4 with STOP1 as indicated by split luciferase
complementation assays. CaMV 35S promoter-driven construct pairs indicated in the figure were co-expressed
in N. benthamiana leaves, and then the luciferase activity due to LUC reconstitution was measured for the
different combinations. (C) Coimmunoprecipitation of ESD4 with STOP1. ESD4-3HA was co-expressed with
STOP1-2Flag or GFP-2Flag in Arabidopsis protoplasts. Crude protein extracts were immunoprecipitated with
anti-HA magnetic beads and then detected with anti-Flag antibody. (D and E) ESD4 mediates the
deSUMOylation of STOP1. STOP1-2Flag was co-expressed with ESD4-3HA, esd4-3-3HA, esd4-4-3HA, or
ESD4C448S-3HA and SUMO precursor (6Myc-SUMO1) (D) or mature SUMO (6Myc-SUMO1GG) (E) in
Arabidopsis protoplasts. Crude total protein extracts were used to immunoprecipitate STOP1 with anti-FLAG
magnetic beads and the SUMOylated form of STOP1 was detected with anti-Myc antibody. Two and three
independent experiments were performed with similar results for (A) and (B-E), respectively.
 A B
pSTOP1:STOP1-3HA 8 WT

(mmol 4-MU/mg/h)
WT esd4-3 WT esd4-3 esd4-3
6

GUS activity
(kDa) -Al -Al +Al +Al

70 STOP1-3HA 4

50 Tubulin 2

0
-Al +Al

C D
transfer to + Al, + CHX transfer to – Al, + CHX
- Al +Al 1 2.5 4 6 8 9 10 Time (h) - Al +Al 1 2.5 4 6 8 9 10 Time (h)
(kDa) (kDa)
70 STOP1-3HA 70 STOP1-3HA

WT
WT

40 Actin 40 Actin

70 STOP1-3HA
esd4-3
70 STOP1-3HA
esd4-3

40 Actin 40 Actin

E F G H
AtALMT1 AtMATE RAE1 Actin7
100
100 ** 20
20 30
30 55
WT
WT WT
WT WT
WT WT
WT
esd4-3
esd4-3 esd4-3
esd4-3 ** esd4-3
esd4-3 esd4-3
esd4-3 N.S
25
Foldenrichment
enrichment

enrichment

25
enrichment

Fold enrichment
Fold enrichment
Foldenrichment

80
Foldenrichment

80 44
IgG
IgG 15
15 IgG
IgG IgG
IgG IgG
IgG
* 20
20
60
60 * ** 33
10
10 15
15
40
40 22
10
10
Fold

Fold
Fold

55
20
20 55 11
00 00 00 00
- Al + Al - Al + Al - Al + Al - Al + Al

Figure 6. Effect of the esd4-3 Mutation on STOP1 Level and Stability and on the Association of STOP1
with the Promoters of STOP1-Regulated Genes. (A) Imummnoblot analysis of STOP1-3HA in WT and esd4-3
under –Al or +Al conditions. Tubulin protein was used as an internal control. (B) Quantification of STOP1-GUS
expression in WT and esd4-3 under –Al or +Al conditions. (C and D) Effect of esd4-3 mutation on STOP1
stability. (C) Roots of WT and esd4-3 harboring the pSTOP1:STOP1-3HA transgene were treated without Al or
with 30 μM Al and 100 μM cycloheximide (CHX) for different time as indicated in the figure. STOP1-3HA protein
levels were detected at each time point with anti-HA antibody. Actin protein served as an internal control. (D)
Roots were treated with 30 μM Al for 4 h and then transferred to –Al conditions plus 100 μM CHX for different
time as indicated in the figure and then the protein levels of STOP1-3HA at each time point were detected. (E-H)
ChIP assay was performed to determine the association of STOP1-3HA with the promoters of STOP1-regulated
genes including AtALMT1 (E), AtMATE (F) and RAE1 (G), and with the promoter of ACTIN7 control (H). Values
are means ± SD of three technical replicates. Asterisks indicate values statistically different (Student’s t test,
*P<0.05, **P<0.01). N.S. indicates non-significantly different P values. Three independent experiments were
performed with similar results.
 A
QKWE MKDE ETKP
K40 K212 K395

499 aa
K91 K235 K254
EQKI EKEE GFKR

B
K40R - - + + + + + +
K91R - + - + + + + +
STOP1-2Flag K212R - + + - + + + +
K235R - + + + - + + +
K254R - + + + + - + +
K395R - + + + + + - +
6Myc-SUMO1 + + + + + + + +
180
IP:Flag

130 α-Myc
100
70 α-Flag

70 α-Flag
Input

26
α-Myc
17
(kDa)
C
K40R - + - - + + - +
STOP1-2Flag K212R - - + - + - + +
K395R - - - + - + + +
6Myc-SUMO1 + + + + + + + +
180
IP:Flag

130 α-Myc
100
70 α-Flag

70 α-Flag
Input

26
17 α-Myc
(kDa)

Figure 7. STOP1 is SUMOylated at K40, K212 or K395 Sites. (A) Domain structure of STOP1 with
the predicted SUMO acceptor sites. Six predicted SUMO acceptor sites with the cognate motif are
shown including the lysine acceptor. The green boxes indicate the four C2H2 zinc finger domains of
STOP1. (B) Effect of the combined replacement of five of the six SUMO acceptor lysines in STOP1-
2Flag with arginines on STOP1 SUMOyation. Red arrows indicate the bands with SUMO-modified
STOP1. (C) Effect of single, double or triple K-to-R mutations of K40, K212 and K395 on STOP1
SUMOylation. Crude protein extracts of protoplasts co-expressing 6Myc-SUMO1 and mutated STOP1-
2Flag were used to immunoprecipitate STOP1 with anti-FLAG antibody and the SUMOylated forms of
STOP1 were detected using anti-Myc antibody. At least three independent experiments were performed
with similar results.
 A B C
WT K40R WT K212R WT K395R
(kDa) -Al +Al -Al +Al (kDa) -Al +Al -Al +Al (kDa) -Al +Al -Al +Al

70 STOP1-HA 70 STOP1-HA 70 STOP1-HA

50 Tubulin 50 Tubulin 50 Tubulin

D E 150
150 WT
K40R

length
125
125

rootlength
a K212R
a

control)
(% of control)
100
100 b K395R
b

root
75
75 a a

of
Relative
b b

Relative
(%
50
50

25
25

1 cm
- Al 1.0 mM Al
00 0 0.75 1.0
Al concentration (mM)

transfer to – Al, + CHX

F G (kDa) - Al +Al 2 3 4 5 6 7 8 Time (h)
WT 3KR-1 3KR-2 STOP1WT-HA
70
(kDa) -Al +Al -Al +Al -Al +Al
- + 40 Actin
70 STOP1-HA
70 STOP13KR-HA
50 Tubulin
40 Actin
H
I 150
150 WT
WT
rae5-1
3KR-1
root length
length

a rae5-2
3KR-2
(% of control)
control)

100
100
b b
a
Relativeroot

b
of
Relative

50
50 b
(%
1 cm

0 1.0
00 0 0.75 1.0
Al concentration (mM) Al concentration (mM)

Figure 8. Effect of mutation of STOP1 SUMOylation Sites on STOP1 Protein Levels and Al Resistance. WT
and mutated versions of STOP1 were transformed into stop1-2 mutant, respectively, to generate various
complementation lines. (A-C) Immunoblot analysis of STOP1-3HA in STOP1WT (WT), STOP1K40R (K40R) (A),
STOP1K212R (K212R) (B), and STOP1K395R (K395R) (C) complementation lines exposed to 0 (–Al) or 30 μM Al (+Al)
for 8 h. (D and E) Al-resistance phenotype of WT, K40R, K212R, and K395R complementation lines. Seedlings
were grown on a soaked gel medium containing 0, 0.75 or 1.0 mM Al for 7 d. Values are means ± SD of root
lengths of 21 to 35 seedlings. (F) Immunoblot analysis of STOP1-3HA in one STOP1WT (WT) and two STOP1 K40R ,
K212R, K395R (3KR-1 and 3KR-2) complementation lines under –Al and +Al conditions. (G) STOP1 stability was

increased in the 3KR line. Roots of WT and 3KR lines were treated with 30 μM Al for 4 h and then transferred to –
Al conditions with 100 μM cycloheximide (CHX) for different time as indicated in the figure. STOP1-3HA protein
levels were detected at each time point with anti-HA antibody. (H and I) Al-resistance phenotype of WT and 3KR
complementation lines. Values are means ± SD of relative root length of 32 to 43 seedlings. Means with different
letters are significantly different (P<0.05, ANOVA test followed by Tukey test). Scale bar= 1 cm. Three independent
experiments were performed with similar results.
Figure 9. Model for Regulation of STOP1 Function and Stability by SUMOylation.
STOP1 is mono-SUMOylated at the three sites K40, K212 or K395, presumably through
the SUMOylation pathway. The STOP1 SUMOylation is reversible, and it can be
deSUMOylated by the SUMO protease ESD4, which is increased in response to Al
stress. Mutation of ESD4 elevates STOP1 SUMOylation levels and consequently
increases and decreases the association of STOP1 with the promoters of AtALMT1 and
AtMATE, respectively, which causes the increased AtALMT1 expression and the
reduced expression of AtMATE and RAE1, and finally contributes to the increased Al
resistance. Mutation of STOP1 SUMOylation site at K40 increases AtALMT1 and
decreases AtMATE expression, which leads to reduced Al resistance. Mutation at K212
site does not affect STOP1 function and Al resistance. Blocking of STOP1 SUMOylation
at K395 or all the three (K40, K212 and K395) sites reduces STOP1 protein level and
the expression of AtALMT1 and AtMATE, leading to the decreased Al resistance.
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 Regulation of Aluminum Resistance in Arabidopsis Involves the SUMOylation of the Zinc Finger
Transcription Factor STOP1
Qiu Fang, Jie Zhang, Yang Zhang, Ni Fan, Harrold A. van den Burg and Chao-Feng Huang
Plant Cell; originally published online October 21, 2020;
DOI 10.1105/tpc.20.00687
This information is current as of October 21, 2020

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