Short Title: EIL1, SLIM1, and Sulfur Deficiency: Skopriva@uni-Koeln - de

Plant Physiology Preview. Published on October 15, 2020, as DOI:10.1104/pp.20.
01192
1 Short title: EIL1, SLIM1, and sulfur deficiency

2
3 The transcription factor EIL1 participates in the regulation of sulfur-deficiency
4 response
5
6 Christof Dietzen1, Anna Koprivova1, Sarah J. Whitcomb2, Gregor Langen1, Timothy O. Jobe1,
7 Rainer Hoefgen2, Stanislav Kopriva1*
8
1
9 Institute for Plant Sciences, Cluster of Excellence on Plant Sciences, University of Cologne,
10 Zülpicher Str. 47b, 50674 Cologne, Germany
2
11 Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-
12 Golm, Germany
13
14 One-sentence summary:
15 The transcription factor ETHYLENE INSENSITIVE 3-LIKE 1 (EIL1) has an additive role to
16 SULFATE LIMITATION 1 (SLIM1) in the regulation of sulfur-deficiency response.
17
18 Author Contributions:
19 C.D. and S.K. designed the research; C.D., A.K., G.L., and T.O.J. performed the research;
20 C.D., S.J.W., R.H., and S.K. analyzed the data; C.D. and S.K. wrote the article.
21
22 Funding
23 Research in S.K.’s lab is funded by the Deutsche Forschungsgemeinschaft (DFG) under
24 Germany´s Excellence Strategy – EXC 2048/1 – project 390686111. S.J.W. and R.H. are
25 supported by the Max Planck Society.
26
*
27 Corresponding author: skopriva@uni-koeln.de
28
29
30
1
Downloaded from on October 16, 2020 - Published by www.plantphysiol.org
Copyright © 2020 American Society of Plant Biologists. All rights reserved.
Copyright 2020 by the American Society of Plant Biologists
31 Abstract
32 Sulfur, an indispensable component of many cellular components, is a growth-limiting
33 macronutrient for plants. Thus, to successfully adapt to changing sulfur availability and
34 environmental stress, a sulfur-deficiency response helps plants to cope with the limited
35 supply. On the transcriptional level, this response is controlled by SLIM1, a member of the
36 EIL transcription factor family. In this study, we identified EIL1 as a second transcriptional
37 activator regulating the sulfur-deficiency response, subordinate to SLIM1/EIL3. Our
38 comprehensive RNA-Seq analysis in Arabidopsis thaliana allowed us to obtain a complete
39 picture of the sulfur-deficiency response and quantify the contributions of these two
40 transcription factors. We confirmed the key role of SLIM1/EIL3 in controlling the response,
41 particularly in the roots, but showed that in leaves more than 50% of the response is
42 independent of SLIM1/EIL3 and EIL1. The RNA-seq showed an additive contribution of
43 EIL1 to the regulation of the sulfur-deficiency response but also identified genes specifically
44 regulated through EIL1. SLIM1/EIL3 seems to have further functions, e.g., in regulation of
45 genes responsive to hypoxia or mediating defense at both low and normal sulfur supply.
46 These results contribute to the dissection of mechanisms of the sulfur-deficiency response and
47 provide additional possibilities to improve adaptation to sulfur deficiency conditions.
48
2
49 Introduction
50 The assimilation of the macronutrient sulfur (S) (Figure 1) is a growth-limiting process as
51 sulfur is an indispensable active component of many essential metabolites. As an organic
52 thiol, it is incorporated in the amino acids cysteine and methionine, which define protein
53 translation start, activity, and structure. It is also present in several prosthetic groups and
54 cofactors, such as Coenzyme A, iron-sulfur proteins, thiamine pyrophosphate, and biotin
55 (Koprivova and Kopriva, 2014). In plants, sulfur for these metabolites is provided by the
56 assimilation of sulfate, the oxidized, inorganic form of sulfur. The uptake of sulfate through
57 the plasma membrane is achieved by transmembrane transport proteins against a negative
58 membrane potential (Takahashi et al., 2011). Arabidopsis thaliana possesses two high-affinity
59 sulfate transporters in the root cortex, SULTR1;1 and 1;2, which enable the plant to take up
60 sulfate into the roots. Via xylem-based, root–shoot translocation, sulfate moves to leaf cells,
61 where it can be either transported to the plastids for assimilation or stored in the vacuole
62 (Yoshimoto et al., 2002; Kataoka et al., 2004; Cao et al., 2013). In plastids, the inert sulfate is
63 activated to adenosine 5-phosphosulfate (APS) by ATP sulfurylase (ATPS) (Takahashi et al.,
64 2011). APS is a branching point of sulfur metabolism, as it can be further reduced for primary
65 (reductive) assimilation or phosphorylated to 3´-phosphoadenosine 5´-phosphosulfate (PAPS)
66 a sulfate donor for a number of secondary (oxidized) sulfur metabolites (Kopriva et al., 2012).
67 In the primary sulfate assimilation pathway, APS reductase (APR) catalyzes the reduction of
68 APS to sulfite, which subsequently is reduced by the ferredoxin-dependent sulfite reductase
69 (SiR) to sulfide. In addition to the SULTR uptake system, another crucial control point of the
70 sulfate assimilation pathway is the first reduction step driven by three isoforms of APR
71 (Vauclare et al., 2002; Koprivova and Kopriva, 2014). Sulfide is subsequently incorporated
72 into the amino acid skeleton of O-acetylserine (OAS) by O-acetylserine (thiol) lyase (OAS-
73 TL) to form cysteine (Wirtz et al., 2004). Cysteine can be incorporated into proteins and
74 peptides, such as glutathione (GSH) or provide sulfur for the synthesis of methionine and co-
75 enzymes (Takahashi et al., 2011). In the secondary assimilation pathway, PAPS is needed,
76 e.g., for the sulfation of sulfated peptide hormones and glucosinolates (Sonderby et al., 2010;
77 Koprivova and Kopriva, 2016). This group of amino acid-derived secondary metabolites has a
78 crucial function in plant defense against pathogens and herbivores, as they are toxic and
79 deterrent upon breakdown (Halkier and Gershenzon, 2006; Bednarek et al., 2009). Sulfur
80 metabolism, with a broad range of enzyme isoforms in different cellular compartments, is a
81 highly complex process, regulated by sulfur availability and a diversity of environment-
82 dependent signals (Takahashi et al., 2011).
3
83 One of the environmental conditions strongly affecting sulfate assimilation is sulfur
84 deficiency. During sulfur deficiency, the vast majority of enzymes mediating sulfate uptake
85 and assimilation are coordinately regulated (Takahashi et al., 1997). Hereby the acquisition
86 and primary assimilation of sulfate is enhanced, whereas glucosinolate biosynthesis is
87 repressed (Hirai et al., 2003; Maruyama-Nakashita et al., 2003; Nikiforova et al., 2003; Hirai
88 et al., 2005; Nikiforova et al., 2005). This is predominantly achieved via transcriptional
89 regulation (Maruyama-Nakashita et al., 2006). The transcription factor responsible for at least
90 part of this sulfur-deficiency response is SULFUR LIMITATION1 (SLIM1), as it controls the
91 induction of sulfate uptake and acquisition as well as the catabolic recycling of secondary
92 sulfur compounds under low-sulfate conditions (Figure 1; (Maruyama-Nakashita et al.,
93 2006)).
94 SLIM1 is part of the six-member ETHYLENE-INSENSITIVE3-LIKE (EIL) family of
95 transcription factors in Arabidopsis thaliana and, therefore, also referred to as EIL3 (Guo and
96 Ecker, 2004; Maruyama-Nakashita et al., 2006). Other members of the EIL family have been
97 associated with control of ethylene signaling transduction. The eponym EIN3 and its
98 functional homologues EIL1 and EIL2 regulate transcript levels of ethylene-responsive genes
99 upon ethylene signalling transmitted through EIN2, resulting in a further transcriptional
100 cascade through transcription factors such as ERF1 (Chao et al., 1997; Solano et al., 1998;
101 Merchante et al., 2013). SLIM1/EIL3 was assumed to have a distinct function as it did not
102 complement ein3 phenotypes, in contrast to EIL1 and EIL2 (Chao et al., 1997), whereas vice
103 versa the other EILs did not complement the loss of function of SLIM1/EIL3 (Maruyama-
104 Nakashita et al., 2006). However, several recent studies indicated participation of other EIL
105 proteins in regulation of the sulfur-deficiency response. For example, hetero-dimerization of
106 EIN3 and SLIM1, via the highly conserved N-terminus of both transcription factors (EIN3 1–
107 299 aa; SLIM1 75–286 aa), led to impaired DNA binding of SLIM1, suggesting that EIN3
108 acts as an inhibitor of SLIM1 (Wawrzynska and Sirko, 2016). Also, EIL2 was shown to
109 activate the expression of the sulfur deficiency-upregulated gene LSU in tobacco in a sulfur
110 status-dependent manner (Wawrzynska et al., 2010).
111 Despite the importance of SLIM1 for control of the sulfur-deficiency response, the
112 mechanism of this regulation remains poorly understood. SLIM1 transcript is expressed
113 preferentially in the vasculature of leaves, hypocotyl, and roots, particularly in parenchyma
114 and xylem cells, but is not affected by the sulfur status (Maruyama-Nakashita et al., 2006;
115 Aubry et al., 2014). Similarly, SLIM1 protein abundance and localization in nuclei are not
116 altered upon sulfur deficiency (Maruyama-Nakashita et al., 2006). This prompted our attempt
4
117 to understand the molecular mechanisms of sulfur-deficiency response and the wider role of
118 EIL family in this regulation. We reveal here that EIL1 acts as an additional transcription
119 factor regulating the sulfur-deficiency response. We compared the transcriptional response of
120 eil3, eil1, and a double eil1 eil3 mutant to sulfur deficiency using RNA sequencing and show
121 that EIL1 has both overlapping and specific functions compared to SLIM1.
122
123 Results
124 Loss of EIL1 affects the accumulation of sulfur-containing metabolites during sulfur
125 deficiency.
126 Previous studies showed that overexpression of other EIL proteins was not sufficient to rescue
127 the fluorescence signal of pSULTR1;2PRO-GFP reporter in transgenic slim1-1 or slim1-2
128 seedlings (Maruyama-Nakashita et al., 2006). This, however, does not mean that they do not
129 play potential roles in sulfur-deficiency response, such as, e.g., regulation of genes affected by
130 S deficiency independent of SLIM1. Alternatively, the EILs might play a subordinate role in
131 the regulation of sulfur-deficiency response, and their function might be masked by the
132 dominant activity of SLIM1. Therefore, as a first step to assess the importance of the EIL
133 family for sulfur-deficiency response, we generated double mutants eil3 eil1 and tested them
134 and the single mutants for their response to sulfur-deficiency conditions. The eil3 T-DNA line
135 was compared with the original slim1-1 and slim1-2 mutant and showed very similar defects
136 in transcriptional response to S deficiency (Supplemental Figure S1). Seedlings of Col-0
137 wildtype (WT), eil1, eil3, and eil3 eil1 were grown on modified Long Ashton Medium plates
138 with either full (0.75 mM SO42-, +S) or low (0.015 mM SO42-, -S) sulfur supply for 18 days
139 and levels of anions, thiols, and glucosinolates were analyzed. In control plants, sulfate levels
140 in shoots were reduced by ca. 50% in eil3 and eil3 eil1 mutants, but remained the same in the
141 roots (Figure 2A). Sulfate levels strongly diminished in response to sulfate deficiency, except
142 in shoots of eil3 eil1 where the reduction in sulfate accumulation was attenuated compared to
143 WT or the single mutants. Foliar cysteine levels in eil3 and eil3 eil1 were higher than in WT
144 and eil1 in control conditions, but lower in low S (Figure 2B). Thus, no or only small
145 differences in cysteine concentration between control and –S conditions are found in WT and
146 eil1, whereas the genotypes with disrupted SLIM1 exhibit a high difference in cysteine. GSH
147 in the leaves of S-deficient plants shows a similar pattern as cysteine: lower levels in eil3 and
148 eil3 eil1 than in WT (Figure 2B). In control conditions, however, it is the eil1 mutant that
149 accumulates less GSH than the other genotypes. Again, the decrease in GSH is more
150 pronounced in S-deficient eil3 and eil3 eil1 than in WT and eil1. An attenuated –S response
5
151 was also observed for shoot levels of aliphatic glucosinolates (Figure 2C). Under normal
152 sulfur supply, all three mutants had reduced glucosinolate levels compared to WT. Whereas in
153 WT and eil1 the aliphatic glucosinolates were decreased to low levels by S deficiency, this
154 decrease was not as pronounced in eil3 and even less in eil3 eil1. Indolic glucosinolates did
155 not show substantial differences between the genotypes, except a slight reduction in control
156 conditions in mutants with disrupted EIL1: eil1 and eil3 eil1 (Figure 2C). These results
157 suggest that EIL1 participates in the regulation of sulfur metabolism by sulfur deficiency.
158
159 EIL1 is required for maintenance of sulfur uptake and allocation during deficiency
160 conditions
161 To evaluate whether loss of EIL1 also affects the flux through sulfate assimilation, the
162 different genotypes were grown under full and low sulfur supply for 18 days and subsequently
163 fed with [35S] sulfate for 3 hours. The incorporation of [35S] into thiols, glucosinolates, and
164 proteins, as well as the overall uptake and root–shoot translocation, were determined.
165 Arabidopsis normally adapts to sulfur deficiency conditions by increasing uptake of sulfate
166 into the root via the high-affinity sulfate transporters. This response was clearly visible in WT
167 seedlings and was accompanied by enhanced sulfate translocation from root to shoot, the
168 major site of assimilation. In eil1 seedlings, the increases in uptake and translocation were
169 dramatically reduced, whereas in eil3 the S-deficiency response was totally abolished (Figure
170 3A). Interestingly the double mutant seedlings were affected even stronger since their sulfate
171 uptake and translocation were not only the lowest under S-deficiency conditions but were
172 impaired already at normal sulfur supply.
173 A similar pattern was observable for the incorporation of [35S] into GSH (Figure 3B),
174 corresponding to previously observed reduced GSH levels (Figure 2). Although S deficiency
175 leads to a decrease in glucosinolate accumulation, the flux into glucosinolates was unchanged
176 in WT, and slightly increased in eil1 (Figure 3B). This increase was more prominent in eil3
177 and eil3 eil1 (Figure 3B). Indeed, at –S conditions, eil3 and eil3 eil1 seedlings incorporated
178 twice as much [35S] into glucosinolates than eil1 and WT, presumably because of the higher
179 specific activity of GSH as the donor of S for core glucosinolate synthesis and the diminished
180 upregulation of SDI1 (see below). The incorporation of [35S] into proteins did not show any
181 alteration between the mutants and WT under both conditions. The reducing magnitude of S
182 metabolism response to sulfur deficiency from WT, over eil1, to eil3 and to eil3 eil1 clearly
183 indicates that EIL1 plays a role in the control of the S-deficiency response, as shown for
6
184 SLIM1 previously. Furthermore, it indicates that both transcription factors have an additive
185 function for the regulation of acquisition, allocation, and assimilation of sulfate.
186
187 EIL1 contributes to regulation of marker genes for S deficiency
188 Sulfur deficiency leads to profound changes in gene expression in Arabidopsis. A great
189 number of genes, e.g., SULFUR DEFICIENCY-INDUCED 1 (SDI1) or GAMMA-GLUTAMYL
190 CYCLOTRANSFERASE 2;1 (GGCT2;1), are upregulated in a SLIM1-dependent manner,
191 whereas others like the APR isoforms were shown to be SLIM1-independent (Maruyama-
192 Nakashita et al., 2006). We, therefore, tested whether EIL1 contributes to the regulation of
193 these marker genes by S deficiency. Transcript levels of SDI1 and GGCT2;1, involved in the
194 regulation of aliphatic glucosinolate biosynthesis and GSH catabolism, respectively, were
195 highly upregulated in WT seedlings upon low sulfur conditions both in shoots and roots
196 (Figure 4A and B). As expected, this response was significantly decreased in eil3 mutants by
197 ca. 50% in both organs. The same was true for SULTR1;1 in the roots (Figure 4B), but APR3
198 in the leaves was upregulated to the same extent in eil3 and WT (Figure 4A). Loss of EIL1
199 did not affect the S-deficiency response in the leaves; however, in the roots, the upregulation
200 of SDI1 and GGCT2;1 was attenuated to the same degree as in eil3 and for SULTR1;1 it was
201 intermediate between WT and eil3 (Figure 4). Loss of both SLIM1 and EIL1 simultaneously
202 led to an additive effect on gene expression, in leaves SDI1 and GGCT2;1 transcript levels
203 were significantly less affected by S deficiency in eil3 eil1 than in WT and the single mutants.
204 The same was true for the roots, where particularly the upregulation of SULTR1;1 was very
205 low. These findings clearly show that EIL1 is involved in the transcriptional regulation of S-
206 deficiency response, where it seems to regulate gene transcription additively to SLIM1.
207
208 Global transcriptional network of EIL1 and EIL3 in sulfur-deficiency response
209 To obtain insights into the global transcription networks regulated by SLIM1 and EIL1, as
210 well as their functional overlaps, we utilized an RNA sequencing approach (RNA-seq). RNA
211 was isolated from shoots and roots of the four genotypes grown at normal and low-sulfur
212 conditions. The RNA was subjected to RNA-seq using the Illumina platform at The Cologne
213 Centre for Genomics (Supplemental Dataset S1). In order to shorten the process of transcript
214 abundance quantification without losing accuracy, we performed kallisto pseudo-alignment
215 with subsequent sleuth analysis for determination of differentially expressed genes (DEGs)
216 with FDR adjusted p-value < 0.05 and fold change > 2. Already at control S supply, 179 and
217 98 genes were differentially expressed in leaves of eil3 and eil1, respectively, compared to
7
218 WT (Figure 5A, Supplemental Table S1). In the double mutant, the number of DEGs raised to
219 238. From these, 13 genes were differentially expressed in both parental mutants and the
220 double mutant, but 68 and 64 DEGs were specific to eil3 and eil1, respectively. 128 genes
221 were differentially expressed only in the double mutant. Interestingly, several but not all of
222 the typical sulfur-deficiency markers (LSU1, LSU2, LSU3, SDI1) have been slightly but
223 significantly induced in eil3 mutant, whereas in eil3 eil1 they were induced to a somewhat
224 higher level and additionally APR2 and APR3 were found among the induced DEGs. To
225 identify processes differentially regulated in the mutants, Gene Ontology (GO) over-
226 representation analysis was performed. Under normal sulfur supply, loss of SLIM1 affected
227 genes annotated as being involved in response to hypoxia and oxygen levels, to other
228 organisms, wounding, and salicylic acid, and, interestingly, to shade avoidance (Figure 6A;
229 Supplemental Table S2). Among the DEGs in eil1 genes for response to hypoxia and oxygen
230 levels are also over-represented, in addition to a number of genes involved in response to heat
231 and reactive oxygen species (ROS) and in shoot and flower development, in line with the role
232 of EIL1 in ethylene signaling. In roots, at normal S supply, 103 and 64 DEGs were found in
233 eil3 and eil1, and 117 DEGs in the double mutant, with 16 genes differentially expressed in
234 all three mutants. Most of these genes are affected in an organ-specific way; only 2 and 12
235 genes were differentially expressed in both roots and shoots of eil1 and eil3, respectively,
236 which increased to 16 genes (13.6% of root DEGs) in the eil3 eil1 double mutant. In the roots
237 of single mutants eil1 and eil3, no functional category was over-represented at a stringent p <
238 0.005, However, in eil3 eil1 roots the genes for response to hypoxia and oxygen levels were
239 over-represented again, in addition to new categories, such as response to chitin, to nitrogen
240 compounds, and to cold (Figure 6A). Generally, a high number of the over-represented GO
241 terms in all three genotypes were related to stress response (Figure 6A; Supplemental Table
242 S2).
243 At low S conditions, the number of DEGs between WT and the individual mutants in the
244 leaves significantly increased for genotypes with disrupted SLIM1 expression, namely eil3
245 and eil3 eil1, to 598 and 3,489, respectively (Figure 5A, Supplemental Table S1). On the
246 other hand, in eil1, the number of DEGs decreased in low S compared to control conditions to
247 72. Forty genes were differentially expressed in all three mutants. In roots, 95 DEGs between
248 eil1 and WT were detected under low S, 292 in eil3, and 530 in the double mutant, from
249 which 48 were affected in all three genotypes. The overlap between roots and shoots is
250 somewhat bigger in eil3 and eil3 eil1, where 70 (23.6% of root DEGs) and 188 genes (35.5%
8
251 of root DEGs) were differentially expressed in both roots and shoots, whereas only 4 genes
252 were affected in both organs in eil1.
253 Sulfur deficiency had a large effect on gene expression. The analysis revealed 1,258 and 335
254 sulfur deficiency-regulated genes in shoots and roots of WT, respectively (Figure 5B;
255 Supplemental Table S3). The DEGs with the highest fold change in expression included genes
256 identified by previous microarray analyses (Hirai et al., 2003; Maruyama-Nakashita et al.,
257 2003; Nikiforova et al., 2003), such as MIR395, LSU1, LSU2, SULTR1;1, SULTR1;2,
258 SULTR2;1, SHM7, APS3, APS4, APR2, APR3, SERAT3;2, SDI1, CYP83A1, BCAT4, MAM1,
259 BGLU28, and GGCT2;1 (Supplemental Table S3). These transcripts encode proteins involved
260 in sulfate uptake and allocation, activation and reduction, cysteine biosynthesis, glucosinolate
261 biosynthesis, as well as catabolism of secondary sulfur compounds and thus cover a large part
262 of the sulfate assimilation pathway. Whereas in leaves of eil1 and eil3 similar number of
263 genes were differentially expressed as in WT, specifically 1,210 and 1,414, respectively,
264 almost four times as many DEGs (4,527) were found in eil3 eil1 (Figure 5B). In the roots, the
265 number of DEGs was reduced in all mutants compared to WT, with only 61 DEGs in eil3
266 (Figure 5B; Supplemental Table S3).
267
268 Contribution of SLIM1 and EIL1 to sulfur-deficiency response
269 To identify the contribution of SLIM1 and EIL1 to S-deficiency response, we defined SLIM1-
270 and EIL1-regulated genes as genes that were either differentially expressed in WT and not in
271 the mutants eil3 and eil1, or in which the fold change was at least twofold lower in the
272 mutants than in WT. In the roots, from the 335 DEGs in WT, 211 are regulated by EIL1 and
273 278 by SLIM1 (Figure 7; Supplemental Table S4). From these, 198 genes are regulated by
274 both TFs, 13 by EIL1 but not SLIM1 and 80 by SLIM1 and not EIL1. Thus, SLIM1 is
275 responsible for regulation of 83% of the S-deficiency response in roots, with GO terms related
276 to sulfate assimilation, S-compound transport, and glucosinolate synthesis all highly over-
277 represented (Figure 6B; Supplemental Table S2). Interestingly, none of these GO biological
278 process terms are over-represented among EIL1-dependent, S-deficiency response genes.
279 Among the SLIM1-independent genes are two APR isoforms APR2 and APR3, as shown
280 before (Maruyama-Nakashita et al., 2006), as well as several genes for enzymes of
281 glucosinolate synthesis CYP79B2, FMO_GS-OX3, AOP2, or SOT17 (Supplemental Table
282 S4). Glucosinolate synthesis is also the most highly enriched GO biological process term
283 among the SLIM1-independent genes in roots (Supplemental Table S2). Other SLIM1-
284 independent genes include those for amino acid and organic acid metabolism. Several
9
285 glucosinolate-related genes (CYP79B2, FMO_GS-OX3, IPMI1), which were regulated by S-
286 limitation in the same way in eil3 and WT, however, were among DEGs specific for the
287 double mutant.
288 From 1,258 DEGs in leaves, 372 were under control of EIL1, 553 genes regulated by SLIM1,
289 and 278 by both (Figure 7). Interestingly, from these 278 genes, 59 are regulated normally in
290 the double eil3 eil1 mutant, pointing to opposite regulation of these genes by the two factors
291 (Supplemental Table S4). The loss of both TFs in the double mutant affects 667 genes
292 regulated by sulfur deficiency in leaves, i.e., 53%, with a stronger (44%) contribution of
293 SLIM1 than EIL1 (22%), and so they play a weaker role in leaves than in the roots. These
294 genes include all those previously shown to be regulated by SLIM1 via microarray analysis
295 (Maruyama-Nakashita et al., 2006) (Supplemental Table S4). The 667 genes include 191
296 genes that only differ from WT in the double mutant but not in the single mutants. Among
297 these 191 genes are again many glucosinolate-related genes and also APR1 and APR3, but not
298 APR2 (Supplemental Table S4). The DEG set of eil3 eil1 is enriched with genes involved in
299 the metabolism of sulfur compounds, including glutathione and glucosinolates, and also in
300 response to abiotic and biotic stress (Supplemental Table S2).
301 SLIM1 is considered the key regulator of the sulfate assimilation pathway, but the complete
302 extent of its involvement is unknown. To quantify the contribution of SLIM1 and EIL1 to
303 regulation of the pathway by sulfur deficiency, a heatmap of all genes related to sulfate
304 assimilation and connected pathways was constructed (Supplemental Figure S2). This
305 indicated that both SLIM1 and EIL1 are largely responsible for the regulation of sulfate
306 uptake and the primary assimilation pathway (except APR), and additionally regulate the
307 biosynthesis and catabolism of glucosinolates. On the other hand, both transcription factors
308 seemed to have only a minor function in the biosynthesis of methionine, glutathione, and
309 ethylene (Supplemental Figure S2). Comparing gene expression in all four genotypes revealed
310 that EIL1 often acts as an additive to SLIM1 (Supplemental Figure S2). In addition, this
311 comparison indicated that many genes in sulfur metabolism are not transcriptionally regulated
312 by SLIM1 or EIL1.
313 Analysis of SLIM1-responsive genes revealed that SLIM1 possesses additional functions
314 besides regulation of the S-deficiency response. In shoot and root, 399 and 154 genes,
315 respectively, were differentially expressed under S deficiency in eil3 seedlings compared to
316 WT. The GO enrichment analysis revealed SLIM1 regulation of gene transcripts encoding for
317 proteins involved in response to stress, wounding, and hormones, such as JAZ13, JAZ5, and
318 an Acyl-CoA N-acyltransferase superfamily protein (Supplemental Table S2). Additionally,
10
319 several genes related to abscisic acid (ABA) response, glucosinolate biosynthesis, and nitrate
320 transport were regulated by SLIM1 but not S deficiency. By contrast, the –S response seemed
321 not to be exclusively regulated by SLIM1 and EIL1. 420 genes in shoots (33%) and 23 (6.9%)
322 in roots were regulated by sulfur deficiency to the same extent in all four genotypes
323 (Supplemental Table S4). Among the root transcripts were transcripts involved in
324 glucosinolate biosynthesis like MAM3, SOT17, several GSTs, and CYPs in addition to
325 isoforms of genes directly associated with the primary assimilation pathway of sulfate such as
326 APR3, APK2, ATPS1, SERAT1;1, OAS-TL C, and GSH1. Interestingly, transcripts of several
327 redox-related enzymes like glutaredoxin and thioredoxins were found to be –S responsive but
328 not under SLIM1 and EIL1 control in shoots, and correspondingly the GO category
329 “oxidation-reduction process” was enriched, together with GO terms on stress response and
330 metabolism of tryptophan and indolic compounds (Supplemental Table S2).
331 The overall contribution of SLIM1 and EIL1 to the global –S response showed that whereas
332 the numbers of upregulated and downregulated transcripts are balanced in the roots, the
333 majority of transcripts are upregulated in shoots (Figure 8). DEGs which exceeded a log2FC
334 of 2 or are below –2 formed only a small portion of the DEGs. SLIM1 and also EIL1 seemed
335 to play major functions affecting this fraction of transcripts. By contrast, our data showed the
336 broad range of genes altered by a log2FC between 1 and 2, as well as –1 and –2, are often
337 controlled independently of these transcription factors. The extent of the regulation (black
338 bars) furthermore showed again that SLIM1 is the more important regulator, and EIL1 has
339 mainly a supportive function (Figure 8).
340
341 New genes regulated by sulfur deficiency
342 Since previous investigations of the sulfur-deficiency response were performed using
343 microarrays, another advantage of our RNA-seq approach was an opportunity to identify
344 further genes, those not included in the ATH-1 Array, which are responsive to sulfur
345 deficiency and/or regulated by SLIM1 and EIL1. In shoots and roots, 238 and 67 such genes,
346 respectively, were differentially expressed upon S deficiency (Supplemental Figure S3,
347 Supplemental Table S5). Not surprisingly, these genes included other isoforms of known –S
348 responsive genes, but also many undescribed genes. The latter included, for example,
349 SULPHATE UTILIZATION EFFICIENCY 4 (SUE4, AT3G55880), a gene already associated
350 with sulfur deficiency and heavy metal- and oxidative-stress tolerance (Wu et al., 2010);
351 AT5G24920 encoding GLUTAMINE DUMPER5, one of seven transmembrane proteins
352 involved in amino acid export into the apoplast (Pratelli et al., 2010); AT1G49640, encoding
11
353 alpha/beta-Hydrolases superfamily protein with methyl indole-3-acetate esterase activity;
354 AT3G21351, encoding a putative transmembrane protein expressed in guard cells; and
355 AT2G18193, encoding a protein with possible ATPase activity and metal-ion binding
356 (Supplemental Figure S4). In addition, several unknown genes were highly induced by sulfur
357 deficiency, e.g., AT2G34655, AT1G22065, and AT2G32487 (Supplemental Figure S4), the
358 latter being co-regulated with genes of the OAS cluster (Hubberten et al., 2012). Gene
359 expression analysis via RT-qPCR was used to confirm the regulation of these genes and to
360 validate the RNA-Seq results (Supplemental Figure S5). The two methods showed an
361 excellent correlation (Supplemental Figure S5) and revealed that transcripts levels of
362 AT1G22065 and AT1G49640 were upregulated in response to deficiency conditions in an
363 EIL1- and SLIM1-dependent manner. On the other hand, AT2G18193 was not responsive to
364 sulfur deficiency in WT, and was repressed by EIL1 and SLIM1 under both conditions.
365 Interestingly, transcript levels of AT2G18193 in shoots became –S responsive in all mutants,
366 the highest in eil3 eil1. These genes may represent additional components of the sulfur
367 regulatory network and await further characterization.
368
369 Discussion
370 EIL1 contributes to regulation of the sulfur-deficiency response
371 Several lines of evidence led us to hypothesise that, beside SLIM1, other members of the EIL
372 family may be involved in regulation of the sulfur-deficiency response. EIL proteins share
373 50–80% identity in their amino acid sequences and EIN3, EIL1, and EIL2 share redundant
374 functions in the regulation of gene expression (Chao et al., 1997; Solano et al., 1998; Guo and
375 Ecker, 2004; Merchante et al., 2013). EIL proteins are able to bind the same motif, the
376 tobacco EIN3-binding site (TEBS; A(C/T)G(A/T)A(C/T)CT), which is present in promoters
377 of ethylene-regulated genes (Kosugi and Ohashi, 2000). TEBS are also found in promoters of
378 some sulfur deficiency-induced genes arranged in tandem and forming a UPE-Box, which is
379 bound by SLIM1 homodimers (Wawrzynska et al., 2010; Wawrzynska and Sirko, 2016). In
380 addition, EIN3 forms heterodimers with SLIM1 and modulates its binding to promoters in
381 vitro (Wawrzynska and Sirko, 2016). Overexpression of EILs in transgenic slim1-1 and slim1-
382 2 mutants was not sufficient to recover the fluorescence signal of pSULTR1;2-GFP reporter
383 under sulfur-deficient conditions. However, eil mutants were not tested for response to sulfur
384 deficiency (Maruyama-Nakashita et al., 2006). Thus, it is possible to hypothesise that other
385 EILs contribute to the sulfur-deficiency transcriptional response independently of SLIM1, for
386 example, in regulating the expression of APS reductase. Furthermore, analyses of eil mutants
12
387 under sulfur deficiency might unravel additional mechanisms of this regulatory process. This
388 would be reminiscent of members of the same transcription factor families sharing control of
389 other nutrient-related processes, such as PHR1 and PHL1/PHL2 in phosphate-deficiency
390 response (Bustos et al., 2010; Sun et al., 2016) and NLP7 and NLP6 in nitrate signaling
391 (Konishi and Yanagisawa, 2013). Since we presumed that the contribution of EIL1 in an eil1
392 single mutant might be masked by the strong activity of SLIM1, we also generated and
393 analyzed an eil3 eil1 double mutant.
394 Analyses of metabolite levels indicated a contribution of EIL1 to the maintenance of
395 glutathione and glucosinolate pools already at full sulfur supply (Figure 2B, C). The sulfur-
396 deficiency response was not dramatically affected by the loss of EIL1 in terms of steady-state
397 metabolite levels. However, the sulfate uptake and flux measurements showed the importance
398 of EIL1 in regulation of the assimilatory pathway much more clearly, both at normal and low
399 S supply (Figure 3). In particular, the analysis of the double mutant eil3 eil1 revealed a
400 dramatic effect of the loss of EIL1 in the eil3 background, pointing clearly to a role of EIL1 in
401 regulation of sulfate assimilation and the sulfur-deficiency response. The expression analyses
402 confirmed this assumption. In the shoot, the contribution of EIL1 to the control of sulfur-
403 deficiency marker genes was visible only in the double mutant, but in the root, the genes were
404 less upregulated already in the single eil1 mutants (Figure 4). This was particularly dramatic
405 for SULTR1;1, which in eil3 eil1 was upregulated by sulfur deficiency to a much lesser
406 degree than in eil3. This agrees well with the very low sulfate uptake capacity of the eil3 eil1
407 plants. The expression analysis, however, also refuted our initial hypothesis that EIL1 might
408 control the upregulation of APS reductase. The discovery of EIL1 function in the regulation
409 of sulfur-deficiency response begs the question of whether this response is dependent on the
410 canonical ethylene signaling; however, this is beyond the scope of this study.
411
412 Global effect of loss of EIL1 on the Arabidopsis transcriptome
413 The finding that EIL1 is involved in regulation of the sulfur-deficiency response triggered the
414 question of the global extent of its contribution. Whereas the Arabidopsis transcriptomic
415 response to sulfur deficiency was analyzed numerous times (Hirai et al., 2003; Maruyama-
416 Nakashita et al., 2003; Nikiforova et al., 2003; Hirai et al., 2005; Higashi et al., 2006;
417 Bielecka et al., 2014), each of these analyses were based on microarray technology. In
418 addition, the information on SLIM1-dependent expression is limited to roots, based on ATH1
419 microarrays and therefore missing analysis of ca. 13,000 genes, and was determined with only
420 two biological replicates (Maruyama-Nakashita et al., 2006). Therefore, a new comprehensive
13
421 analysis using RNA-Seq from roots and shoots separately was expected to yield substantial
422 further understanding regarding both the global contribution of EIL1 to the sulfur-deficiency
423 response and the function of SLIM1 beyond this response.
424 Indeed, the RNA-Seq analysis confirmed an important role for EIL1 in the regulation of the
425 sulfur-deficiency response. Although transcript levels of many genes were affected by the loss
426 of EIL1 alone, the loss of EIL1 was particularly large in the eil3 background, as almost four-
427 fold more DEGs were found in leaves of eil3 eil1 mutants than in leaves of the individual
428 ones (Figure 5). Most of the EIL1-regulated transcripts are also regulated by SLIM1,
429 particularly in the roots (Figure 6); however, presumably because of the subordinate function
430 in the sulfur-deficiency response, fewer GO functional terms are over-represented among the
431 EIL1-regulated genes at the stringent significance threshold (Figure 7). Thus, when SLIM1 is
432 impaired, EIL1 might partially fulfill the role of SLIM1 under sulfate limitation to prevent a
433 complete collapse of the response. Nonetheless, this function of EIL1 seems to be only
434 supportive since EIL1 is not sufficient for the full induction of the sulfur-deficiency response
435 and the eil3 mutant still shows a strongly deficient response to sulfate limitation. It can be
436 hypothesized that this insufficiency is due to different expression patterns of EIL1 and
437 SLIM1. However, this does not seem to be the case because: (1) based on the TPM values in
438 our RNA-seq dataset, EIL1 transcript levels are 4–5-fold more abundant than those of SLIM1
439 (Supplemental Dataset 1); (2) the tissue specific expression patterns of EIL1 and SLIM1 in
440 eFP Browser (Winter et al., 2007) are very similar; and (3) both genes did not show any
441 enrichment in bundle sheath cells (Aubry et al., 2014).
442 Yet, a small number of prominent S-deficiency marker genes are strongly regulated by EIL1
443 in roots, affecting essential parts of the sulfate assimilation pathway. For example, EIL1
444 regulates the ability to enhance catabolism of various S-containing compounds via regulation
445 of BGLU28/30, TGG1, and NSP5 for glucosinolates, as well as GGCT2;1 for degradation of
446 glutathione (Maruyama-Nakashita, 2017). EIL1 also contributes to enhanced sulfate uptake
447 and root–shoot translocation upon sulfate demand (Figure 3, Supplemental Figure S2). EIL1
448 has minor but distinct effects on several sulfate transporters (SULTR1;1, -1;2, -2;2, -4;1, and -
449 4;2) particularly when SLIM1 is impaired (Supplementary Figure S2). In particular, the well
450 described upregulation of SULTR1;1 and SULTR2;1 transcripts is also partially controlled by
451 EIL1. Additionally, EIL1 contributes to control of xylem-based, root–shoot transport via
452 regulation of the low-affinity SULTR2;1 but without altering mRNA levels of its facilitator
453 SULTR3;5, or SULTR1;3 responsible for phloem-based, source-to-sink sulfate translocation
454 (Yoshimoto et al., 2002; Kataoka et al., 2004). This might be due to the EIL1 contribution to
14
455 the upregulation of miR395 (Supplemental Figure S2). Previous studies showed that an
456 increase in miR395 is strongly dependent on SLIM1 (Kawashima et al., 2009; Kawashima et
457 al., 2011). Phloem-specific expression of miR395 confines its target mRNA SULTR2;1 to
458 xylem parenchyma cells and results in enhanced sulfate translocation to the shoot
459 (Kawashima et al., 2009). All isoforms of miR395 transcripts are highly upregulated by S-
460 deficiency in shoot and root tissue of WT plants, with miR395d and -f showing slightly
461 weaker regulation. In shoots, miR395f is the only isoform affected by the loss of EIL1,
462 whereas in roots, miR395c, -d, -e, and -f are all induced to a lesser extent in eil1.
463
464 The sulfur-deficiency response is not entirely controlled by SLIM1 and EIL1
465 The RNA-seq analysis allowed us to obtain the first complete picture of the transcriptional
466 response to sulfur deficiency, including the full gene families (Supplemental Figure S2).
467 Although sulfate assimilation is the major pathway regulated by sulfur deficiency, some
468 transcripts in the primary and secondary sulfate assimilation pathways were not regulated
469 under these conditions. This is somewhat unexpected, since the pathway was previously
470 shown to be coordinately regulated, e.g., by jasmonate (Jost et al., 2005), or in its spatial
471 expression in bundle sheath cells of Arabidopsis (Aubry et al., 2014). Some of the genes
472 found not to be regulated by S deficiency are key for the sulfate assimilation pathway.
473 SULTR3;1 encodes a sulfate transporter localized to the plastid envelope, providing sulfate for
474 the reduction in plastids (Cao et al., 2013). ATPS1 is the major isoform of ATP sulfurylase
475 responsible for ca. 50% of the activity in leaves (Koprivova et al., 2013). However, ATPS1 is
476 a target of miR395, and two processes maintain its transcript levels—cleavage by the
477 microRNA and increased transcription—which are well-coordinated, so that the steady-state
478 transcript levels do not change (Kawashima et al., 2011). Likewise, although SLIM1 is
479 considered the key regulator of the sulfur-deficiency response, many genes associated with
480 the sulfate assimilation pathway (Supplemental Figure S2) are regulated independently of
481 SLIM1 (and EIL1). Some of these genes have been known for a long time, such as the three
482 isoforms of APS reductase (Maruyama-Nakashita et al., 2006; Hubberten et al., 2012), but
483 some have been added in this study, such as SULTR2;2, 12-oxophytodienoic acid reductase
484 OPR2, or BAT5 and some glucosinolate synthesis genes.
485 The RNA-seq allowed us to quantify the contribution of the two transcription factors to the
486 control of the sulfur-deficiency response. Quantifying this contribution is important, as
487 SLIM1 has been considered a key regulator of this response (Maruyama-Nakashita et al.,
15
488 2006; Koprivova and Kopriva, 2014). Whereas this is clearly true for the roots, where 83% of
489 sulfur deficiency-regulated genes are under the control of SLIM1, in leaves, the contribution
490 of SLIM1 is much lower and reaches only 44% (Figure 7). EIL1 modulates the expression of
491 63% of the sulfur deficiency-responsive genes in roots and 30% in shoots. However, very few
492 genes with a fold change greater than 5 are independent of SLIM1 and EIL1, among which
493 are APS reductase and some glucosinolate synthesis genes. Most of the SLIM1-independent
494 genes are only moderately affected by sulfur deficiency, and their contribution to the
495 physiological response needs to be established. Similarly, it needs to be shown whether these
496 genes are controlled by one or a few transcription factors, or whether their regulation depends
497 on a large number of regulators.
498
499 Pathways regulated by sulfur deficiency beyond sulfur metabolism
500 Obviously, cellular processes other than sulfate uptake and metabolism are affected by sulfur
501 deficiency, as evidenced in the RNA-seq analysis. For example, several DEGs belong to other
502 nutrient and phytohormone pathways. Transcripts of phosphate, nitrate, and molybdenum
503 transporters were upregulated under sulfur deficiency conditions in shoots and roots
504 (Supplemental Table S3). This regulation was largely independent of SLIM1 and EIL1.
505 Another big group of genes regulated by sulfur deficiency are genes involved in redox
506 signaling (Supplemental Table S3). This is not surprising, since the connection between
507 ROS/redox and hormone signaling seems to optimize plant physiology under normal and
508 stress conditions, and sulfur deficiency results in a lower concentration of the redox buffer
509 glutathione (Rouhier et al., 2015). Changes in the transcript levels of redox genes indicate
510 alterations of the redox state during sulfur deficiency. Several peroxidases and oxidases are
511 upregulated during sulfur deficiency conditions in a SLIM1-independent manner, such as
512 PEROXIDASE 2 (PA2), PEROXIDASE CB (PRXCB), RESPIRATORY BURST OXIDASE
513 HOMOLOG B (RBOHB), -C, and -D. Indeed, NADP(H) oxidases and peroxidases were
514 shown to produce superoxide and H2O2, which subsequently are second messengers in many
515 processes associated with plant growth and development (Mhamdi and Van Breusegem,
516 2018). Interestingly, these oxidases were also upregulated upon phosphorus, nitrogen, and
517 potassium deficiency (Shin et al., 2005). Thus, (transient) ROS pulses might be the initial
518 response to general nutrient imbalance as these genes are controlled independently of SLIM1.
519 Altered expression of genes encoding for ROS scavenging enzymes like COPPER/ZINC
520 SUPEROXIDE DISMUTASE 1 (CSD1), FE SUPEROXIDE DISMUTASE 1 (FSD1),
521 ACONITASE 2 (ACO2), ACO3, and GLUTATHIONE PEROXIDASE 6 (GPX6) might indicate
16
522 contribution of the cellular ROS homeostasis to the initiation of the sulfur-deficiency response
523 (Mittler, 2002). In addition, several members of CC-type glutaredoxins (ROXY4, -12, -13, -15,
524 -16, -17) were downregulated during deficiency conditions in a SLIM1-dependent manner.
525 Whether their repression might act as a secondary response of SLIM1 regulation, either as a
526 negative feedback loop or an all-or-nothing principle, needs further elucidation. Both are
527 possible since ROS plays a dual function in abiotic stress by elevating the damage but also
528 mediating the initial signal for the activation of defense responses (reviewed by (Dat et al.,
529 2000).
530 It was shown that persistent increase of H2O2 levels leads to transcriptional expression
531 profiles similar to the response to biotic and abiotic stress, including upregulation of marker
532 genes associated with ethylene signaling (Vandenabeele et al., 2003). Moreover, transient
533 ROS burst is involved in plant hormone signaling (Noctor et al., 2018). Correspondingly,
534 response to stress and response to ROS were among the functional categories affected by
535 sulfur deficiency (Figure 6, Supplemental Table S2) and a number of jasmonic acid and ABA-
536 responsive genes, such as ALLENE OXIDE CYCLASE 2 (AOC2), CORONATINE INDUCED
537 1 (CORI3), VEGETATIVE STORAGE PROTEIN 1 and -2 (VSP1 and -2), COPPER AMINE
538 OXIDASE (CUAO), OPEN STOMATA 1 (OST1), and HIGHLY ABA-INDUCED PP2C GENE
539 2 (HAI2), were upregulated under sulfur-deficiency conditions, which indicates the
540 involvement of ROS-mediated hormone signaling (Jones et al., 2003; Stenzel et al., 2012;
541 Kim et al., 2013; Mittler and Blumwald, 2015). In fact, sulfur deficiency seems to mimic or
542 induce JA and ABA signaling.
543 GO-enrichment analysis of DEGs regulated by SLIM1 also indicated its function in the
544 control of defense response to pathogens, primarily through jasmonate signaling (Figure 6).
545 This is not surprising, as links between jasmonate signaling and sulfate assimilation have been
546 long known. Jasmonate treatment induces genes involved in sulfate reduction, glutathione
547 biosynthesis, and glucosinolate metabolism, which leads to glucosinolate accumulation
548 (Xiang and Oliver, 1998; Harada et al., 2000; Jost et al., 2005; Frerigmann and Gigolashvili,
549 2014). The effect of sulfur deficiency on jasmonate-related processes, however, might occur
550 at different levels of regulation; the SLIM1/EIL1-dependent regulation of JAZ proteins in
551 response to sulfur deficiency is similar to the strong upregulation of SDI1 protein, which
552 binds MYB28 and inhibits its function (Aarabi et al., 2016). Interestingly, JAZ1, -5, -7, and -
553 8, which were differentially regulated in the RNA-Seq, are target genes of two other
554 transcription factors, WRKY40 and WRKY18 (Pandey et al., 2010). Several other WRKY-
555 associated, jasmonate-responsive genes like LIPOXYGENASE2 (LOX2) or PLANT
17
556 DEFENSIN1.2 (PDF1.2) are regulated in a SLIM1- and/or EIL1-dependent manner.
557 Induction of jasmonate biosynthesis in response to sulfur deficiency might serve the
558 enhancement of defense capabilities of the plants when sulfur-containing defense compounds
559 are less available (Koprivova and Kopriva, 2016).
560
561 SLIM1 and EIL1 function independent of sulfur deficiency
562 Although EIL1 and particularly SLIM1 have been mainly discussed to control the sulfur-
563 deficiency response, they affect sulfate metabolism and other processes also during normal
564 sulfur supply. Indeed, the contents of glucosinolates are decreased in leaves of both eil3 and
565 eil1 mutants under normal conditions, as is sulfate in eil3 and glutathione in eil1 (Figure 2).
566 RNA-seq data confirmed their regulatory function towards several genes associated with
567 glucosinolate biosynthesis in a mild manner (–0.6 > log2FC > 0.6) at full sulfur supply.
568 Breakdown of glucosinolates into glucose and an unstable aglycone intermediate that can be
569 further rearranged into highly bioactive isothiocyanate, nitrile, thiocyanate, or cyclic
570 compounds is mediated and catalyzed by myrosinases (Halkier and Gershenzon, 2006). EIL1
571 has a distinct function in the control of thioglucosidases TGG1/2 and BGLU30 under normal
572 conditions. Also, under adequate sulfur supply, transcript levels of SULTR2;1, SULTR4;2,
573 GGCT2;1, and surprisingly APR1 and APR3 are higher in eil3 and to a lesser extent in eil1.
574 This suggests a repressor function of SLIM1, as has been discussed previously (Wawrzynska
575 and Sirko, 2014). Upregulation of mRNA levels for GGCT2;1 and APR3 in eil3 and eil3 eil1
576 seedlings might also explain the decrease in sulfate and increase of cysteine levels (Figure 2
577 and 4). Interestingly, also several LSU transcripts are upregulated in eil3 in roots and shoots
578 under normal conditions.
579 GO-Enrichment analysis of eil3 and eil1 under normal sulfur conditions, moreover, revealed
580 the function of SLIM1 and EIL1 in various other processes, such as hormone signaling, stress
581 response, and ROS and redox signaling (Supplemental Table S2). The upregulation of
582 INDOLE-3-ACETIC ACID INDUCIBLE gene transcripts (IAA6, -19, -20, -29, -30), SMALL
583 AUXIN REGULATED RNAs (SAUR10, -46, -68, -76), as well as 1-AMINO-
584 CYCLOPROPANE-1-CARBOXYLATE SYNTHASE 8 (ACS8) in leaves points to a repressive
585 function of SLIM1 in auxin signaling. Interestingly, we could also identify SLIM1 regulation
586 of several ERFs, among them ERF11, 13, 71, 98. However, as ERFs can also be activated by
587 jasmonate or ABA signaling, it needs to be shown whether SLIM1 is able to directly
588 transduce ethylene perception (Chao et al., 1997; Lorenzo et al., 2003).
589
18
590 Additional genes regulated by sulfur deficiency
591 Our RNA-seq data further detail the sulfur-deficiency response with the identification of up to
592 20% of additional sulfur deficiency-responsive DEGs: 238 in shoots and 67 in roots. Whereas
593 this gene set includes several new isoforms of known sulfur deficiency-responsive genes, 50–
594 60% of the newly identified genes have not been previously described in this context. It can
595 be assumed that a large proportion of these genes are involved in sulfur metabolism, oxidative
596 stress response, and redox or phytohormone signaling. This is supported, e.g., by confirming
597 the upregulation of SUE4, a gene previously associated with sulfur deficiency, heavy metal,
598 and oxidative stress tolerance (Wu et al., 2010). A gain-of-function mutant showed improved
599 tolerance to low-sulfur conditions and improved root establishment. SUE4, a small protein
600 with four transmembrane domains, might act in GSH homeostasis-related processes since
601 GSH levels were enhanced under both conditions compared to WT (Wu et al., 2010).
602 Whereas the function of SUE4 has not been shown, it is similar to several alpha/beta-
603 Hydrolases, with AT2G40095 as the closest homologue. Interestingly, we identified a further
604 9 genes encoding alpha/beta-Hydrolases superfamily proteins as upregulated under sulfur-
605 deficiency conditions (AT1G68620, AT4G39955, AT4G10955, AT3G23570, AT1G49640,
606 AT1G72620, AT1G56630, AT1G73480, AT3G02410). As many proteins of this superfamily
607 function as peroxidases, these proteins might be involved in redox signaling. Therefore, they
608 might constitute interesting proteins that contribute to signaling within the sulfur-deficiency
609 response (Nardini and Dijkstra, 1999; Lenfant et al., 2013).
610 Another gene of interest is GLUTAMINE DUMPER5 (GDU5), a member of a family of 7
611 transmembrane proteins involved in amino acid export into the apoplast (Pratelli et al., 2010).
612 Upon sulfur deficiency, GDU5 was downregulated in leaves and roots, whereas another
613 member of the family, GDU6, was upregulated. Both genes were found to be controlled by
614 SLIM1 and EIL1. Their relative GDU1 is the best studied glutamine dumper and regulates
615 amino acid export from plant cells (Pratelli et al., 2012). As the GDU proteins act in amino
616 acid transport, they might play a role in GSH biosynthesis and homeostasis (Pilot et al., 2004;
617 Pratelli et al., 2010). In the plasma membrane, GDU1 interacts with RING E3 ubiquitin ligase
618 LOG2 (Pratelli et al., 2012), which was shown to be a positive regulator of ABA-mediated
619 drought- and salt-stress tolerance mechanisms (Kim and Kim, 2013). Interestingly, LOG2
620 transcript levels were upregulated upon low S conditions as well. Further experiments need to
621 identify the specific function of these identified genes.
622 Altogether, with EIL1 we identified a second transcriptional activator regulating the sulfur-
623 deficiency response in Arabidopsis. EIL1 possesses a subordinate and additive function to
19
624 SLIM1 in control of sulfur-deficiency response, particularly in the roots, but also in fine-
625 tuning sulfur homeostasis at normal S supply. We performed a comprehensive RNA-Seq
626 analysis to obtain a complete picture of the transcriptional response to sulfur deficiency and
627 the role of SLIM1 and EIL1 within and beyond this response. We confirmed that SLIM1 is
628 the main regulator of the sulfate assimilation pathway and showed that it has additional
629 functions in the regulation of phytohormone-, defense-, and redox-related processes at both
630 low and normal S supply. However, we also showed that more than 50% of the transcriptional
631 response to sulfur deficiency in leaves is independent of SLIM1 and EIL1 and that further
632 components of the regulatory circuit await identification.
633
634 Methods
635 Plant Material
636 Arabidopsis thaliana plants of the wildtype ecotype Col-0 and the T-DNA insertion lines (for
637 schemes of T-DNA insertion see Supplemental Figure S1) eil1 (AT2G27050;
638 SALK_042113), eil3 (AT1G73730; SALK_089129), and the double mutant eil3 eil1 were
639 used for experiments.
640 Seeds were surface-sterilized with chlorine gas for 4 h. Under sterile conditions, seeds were
641 placed on modified Long Ashton Medium agarose plates with either full-sulfur supply (+S;
642 0.75 mM MgSO4) or low-sulfur supply (-S; 0.75 mM MgCl2 + 0.015 mM MgSO4), and
643 stratified for 3 days at 4°C in the dark. Afterward the plates were incubated in Sanyo light
644 chambers at 22°C in long-day conditions with a 16/8 h light cycle and 100 µmol photons m-2
645 s-1. After 18 days, shoot and root samples were collected and immediately frozen in liquid
646 nitrogen. Each biological replicate was collected from seedlings grown on different plates.
647 Results were obtained from at least two independent experiments.
648
649 Sulfate Analysis
650 For sulfate measurement, plant material was homogenized in 200 μl deionized H2O and
651 transferred to a screw cap tube with another 800 µl dH2O. The whole extract was shaken for 1
652 h at 4°C and afterward heated at 95°C for 15 min. After 15 min centrifugation at 4°C and
653 16,000 g, 100 μL supernatant was transferred into an IC vial with 900 µl of H2O. Inorganic
654 anions were measured with the Dionex ICS-1100 chromatography system and separated on a
655 Dionex IonPac AS22 RFIC 4x 250 mm analytic column (Thermo Scientific, Darmstadt,
656 Germany). 4.5 mM NaCO3/1.4 mM NaHCO3 was used as running buffer. A standard curve of
657 sulfate was generated by using the external standards of 0.5 mM, 1 mM, and 2 mM K2SO4.
20
658
659 Isolation and Quantification of Low-Molecular-Weight Thiols
660 For thiols measurement, approx. 20 mg plant material was extracted in exactly a 10-fold
661 volume of 0.1 M HCl and afterward centrifuged for 10 min at 4°C and 16,000 g. In a new
662 tube, 25 μl supernatant was incubated with 25 µl of 0.1 M NaOH and 1 µl of freshly prepared
663 100 mM dithiothreitol (DTT) for 15 min at 37°C in the dark. Subsequently, 10 μl 1 M
664 Tris/HCl pH 8.0, 35 μl water, and 5 μl 100 mM monobromobimane, (Thiolyte® MB,
665 Calbiochem) in acetonitrile were added to conjugate SH groups of Cys and GSH for 15 min at
666 37°C in the dark. 100 μl of 9% (v/v) acetic acid was used to stabilize bimane conjugates. 180
667 µl supernatant was transferred into HPLC vials. The thiol conjugates were separated and
668 measured via high-performance liquid chromatography (HPLC; Spherisorb™ ODS2, 250 x
669 4.6 mm, 5 µm, Waters, Eschborn, Germany) and detected fluorimetrically with 474 detector
670 (excitation: 390 nm, emission: 480 nm). Two solvents were used in a linear gradient of 95–
671 82% A (10% (v/v) methanol, 0.25% (v/v) acetic acid, pH 3.9) in B (90% (v/v) methanol,
672 0.25% (v/v) acetic acid, pH 3.9) with a constant flow rate of 1 mL/min.
673
674 Isolation and Quantification of Glucosinolates
675 Glucosinolate content was determined by reverse phase HPLC via UV detection (Burow et
676 al., 2006) after isolation as described previously (Aghajanzadeh et al., 2015). Right before
677 isolation, specific columns were prepared using 1-mL tips plugged with non-absorbent cotton
678 wool and a layer of 0.5 mL DEAE Sephadex A-25. The samples were extracted twice with
679 250 µl hot 70% (v/v) MeOH, with the addition of 10 µl sinigrin as internal standard and
680 incubated at 70°C for 45 min. The samples were cooled and spun down at 16,000 g. The
681 supernatants were transferred onto the prepared columns, washed twice with 0.5 ml dH2O,
682 and subsequently twice with 0.5 mL 0.02 M sodium acetate buffer. With a new tube placed
683 underneath each column, 75 µl sulfatase solution was added directly on the surface of the
684 column. After incubation at room temperature overnight, the produced desulfo-glucosinolates
685 were eluted twice with 0.5 mL H2O and finally with 0.25 mL dH2O, vortexed and centrifuged.
686 350 µl of the supernatant was added into a HPLC vial. Desulfo-glucosinolates were measured
687 via HPLC (Spherisorb™ ODS2, 250 x 4.6 mm, 5 µm, Waters, Eschborn, Germany) by UV
688 absorption at 229 nm and identified by retention time of the peaks, and quantified with the
689 help of the internal standard sinigrin. For separation a gradient of acetonitrile in water (5–30%
690 (v/v) in 8 min, 30–50 % (v/v) in 7 min) was used.
691
21
692 Sulfate Uptake and Flux Analyses
693 Sulfate uptake and flux through the assimilation pathway were measured in seedlings grown
694 for 18 days under sulfur-sufficient or -deficient conditions. After short incubation of two
695 seedlings in 2 mL nonradioactive Long Ashton nutrient solution containing 0.2 mM sulfate in
696 24-well plates, the medium was exchanged with 1 mL of the same solution supplemented with
697 12 µCi [35S]sulfuric acid and incubated for 3 h in the light. Whole seedlings were washed
698 thoroughly, blotted dry, and shoot and root samples were stored separately in liquid nitrogen
699 until further processing on the same day. Samples were extracted in a 10-fold volume of 0.1
700 M HCl. Ten microliters of extract were used to determine sulfate uptake, and 50 µL aliquots
701 of each extract were collected for quantification of [35S] incorporation into thiols, proteins,
702 and glucosinolates exactly as previously described (Mugford et al., 2011).
703
704 Gene Expression Analyses
705 Total RNA was isolated by phenol-chlorophorm-isoamylalcohol extraction and LiCl
706 precipitation. DNase treatment and first-strand cDNA synthesis was performed with
707 QuantiTect® Reverse Transcription with 800 ng RNA in a 6-µl reaction according to the
708 manufacturer’s instruction. RT-qPCR reactions were performed using SYBR Green in a
709 CFX96 Touch™ Real-Time PCR Detection System (Bio Rad, Munich, Germany). Transcript
710 levels were normalized to TIP41 using the 2-ΔΔCT method. RT-qPCR primers (Supplemental
711 Table S6) were designed via Universal ProbeLibrary Assay Design Center (Roche
712 Diagnostics, Mannheim, Germany).
713
714 RNA-Seq Experiment
715 RNA-Seq experiment was performed by the Cologne Center for Genomics (portal.ccg.uni-
716 koeln.de) using Illumina Polymerase-based sequencing-by-synthesis obtaining a read length
717 of 150 bp (paired-end with 75 bp each) and coverage of approx. 40 Mio reads per sample.
718 Data from 3 biological replicates were processed with trimmomatic v.0.36 trimming tool to
719 remove adapters and low-quality reads (http://www.usadellab.org/cms/?page=trimmomatic)
720 (Bolger et al., 2014). Trimmed, paired files underwent a second quality control via FastQC.
721 FastQ files were further processed with kallisto v.0.43.1 (Bray et al., 2016) to quantify
722 transcript abundances in pseudo-alignment to the Arabidopsis reference transcriptome
723 (www.arabidopsis.org/, TAIR10_seq_20110103_representative_gene_model_updated.fas) as
724 TPM (Transcripts Per Kilobase Million). 96–98% of reads reached processability and could
725 be pseudo-aligned. Differentially expressed genes (qval<0.05) were determined from
22
726 transcript abundance files using the packages “sleuth” and compared to DESeq2 in R
727 environment (Love et al., 2014; Pimentel et al., 2017). Highmeans and lowmeans from the
728 sleuth table were used to manually calculate fold-changes and log2FC with Windows Excel.
729
730 Enrichment Analysis
731 Enrichment analyses of GO biological process terms (GO database release 2019-12-09) was
732 performed with PANTHER (http://www.pantherdb.org) (Mi et al., 2019) using Fisher’s exact
733 test and Bonferroni correction for multiple testing. Significantly over-represented GO terms
734 (P<0.005) in each list of differentially expressed genes were scored and clustered based on
735 their semantic similarity (simRel calculation) using REVIGO (http://revigo.irb.hr, GO
736 mapping file goa_uniprot_gcrp.gaf.gz 15 Mar 2017) (Supek et al., 2011).
737
738 Statistical Analysis
739 Statistical difference of two populations was tested by two-tailed, unpaired Student’s t-test.
740 When results were tested for hypotheses based on knowledge about SLIM1 function and
741 physiological changes under sulfur-deficiency conditions, samples of two populations were
742 statistically compared by one-tailed, unpaired Student’s t-test in Excel (Microsoft Office 365).
743 To test equality of variances, Levene’s test was performed in advance. With accepted null
744 hypothesis (P<0.05, Levene’s test) homoscedastic Student’s t-test was performed. Different
745 letters represent significant differences between values of two genotypes within one treatment
746 (P<0.05, Student’s t-test). Capital letters are assigned to normal, small letters to S-deficiency
747 conditions. Asterisks indicate significant differences between values of one genotype at
748 control or low S (*P<0.05, **P<0.01 and ***P<0.001, Student’s t-test). All experiments,
749 except the RNA-Seq, were independently repeated at least twice.
750
751 Accession Numbers
752 The RNA-seq data from this article can be found in the NCBI GEO data repository under
753 accession number GSE157765.
754
755 Supplemental Data
756 Supplemental Figure S1. Comparison of eil3 mutant with slim1-1 and slim1-2.
757 Supplemental Figure S2 Differentially expressed genes (DEGs), regulated by –S response,
758 SLIM1, and EIL1
23
759 Supplemental Figure S3. Newly identified differentially expressed genes (DEGs), regulated
760 by –S response, in a SLIM1- and EIL1-dependent manner
761 Supplemental Figure S4. Expression analyses of new genes regulated by sulfur deficiency
762 Supplemental Figure S5. Comparison of RNA-seq and RT-qPCR results
763 Supplemental Table S1. Differentially expressed genes between WT Col-0 and eil1, eil3,
764 and eil3 eil1 mutants at control and low S
765 Supplemental Table S2. Enrichment analyses
766 Supplemental Table S3. Differentially expressed genes in the four genotypes and two organs
767 between control and low S
768 Supplemental Table S4. Dependence of differentially expressed genes in WT Col-0 on
769 SLIM1 and EIL1.
770 Supplemental Table S5. Newly identified sulfur deficiency-responsive genes
771 Supplemental Table S6. Primers used in the study
772 Supplemental Dataset S1. RNA-seq data
773
774 Acknowledgements
775 We thank the Cologne Centre for Genomics for performing the RNA sequencing. Research in
776 SK’s lab is funded by the Deutsche Forschungsgemeinschaft (DFG) under Germany´s
777 Excellence Strategy – EXC 2048/1 – project 390686111.
778
779
24
780 Figure legends
781 Figure 1. SLIM1/EIL3 in regulation of sulfur-deficiency response and a link to
782 EIN3/EIL1 function in ethylene signal transduction
783 APS, adenosine 5’-phosphosulfate; PAPS, 3’-phosphoadenosine-5’-phosphosulfate; OAS, O-
784 acetylserine, Cys, cysteine; GSH, glutathione; Met, methionine; Trp, tryptophan; SLIM1,
785 SULFUR LIMITATION 1; CTR1, CONSTITUTIVE TRIPLE RESPONSE 1; EIN2,
786 ETHYLENE INSENSITIVE 2; EBF1/2, EIN3-BINDING F BOX PROTEIN 1/2; EIN3/EIL1,
787 ETHYLENE-INSENSITIVE3/ETHYLENE-INSENSITIVE3-LIKE 1
788 Figure 2. Metabolite analysis of eil1, eil3, and eil3 eil1
789 The mutants and WT Col-0 plants were grown for 18 days on either 0.75 mM SO42- (grey) or
790 0.015 mM SO42- (green). A Sulfate content was determined via ion chromatography (IC),
791 whereas B thiols and C glucosinolate content were measured by HPLC. Data from one
792 representative example of at least two independent experiments are presented as box plots of
793 four biological replicates, with lines marking medians. Different letters represent values
794 significantly different at P < 0.05 (Student’s t-test). Asterisks indicate significant differences
795 between values of one genotype at control or low S (*P<0.05, **P<0.01, and ***P<0.001,
796 Student’s t-test).
797 Figure 3. Flux through sulfate assimilation in eil1, eil3, and eil3 eil1
798 Plants were grown for 18 days on either 0.75 mM SO42- (grey) or 0.015 mM SO42- (green) and
799 subsequently fed with [35S]sulfate. A Uptake and root–shoot translocation were determined
800 via scintillation counter. B [35S] incorporation to glutathione and glucosinolates was measured
801 via high-performance liquid chromatography (HPLC) with a radiodetector or after elution
802 from DEAE Sephadex by scintillation counting, respectively. Data from one representative
803 example of at least two independent experiments are presented as box plots of four biological
804 replicates. Different letters represent values significantly different at P < 0.05 (Student’s t-
805 test). Asterisks indicate significant differences between values of one genotype at control or
806 low S (*P<0.05, **P<0.01, and ***P<0.001, Student’s t-test).
807 Figure 4. Expression analysis of S-deficiency marker genes in eil1, eil3, and eil3 eil1.
808 Plants were grown for 18 days on either 0.75 mM SO42- (grey) or 0.015 mM SO42- (green).
809 Relative gene expression (2-ΔΔCt) of –S marker genes A APR3, SDI1, and GGCT2;1 in the
810 leaves and B SULTR1;1, SDI1, and GGCT2;1 in the roots was determined via RT-qPCR. A
811 representative example of at least 2 independent experiments is presented. Box plots of four
812 biological replicates are given, lines represent medians. The values in Col-0 at control
813 conditions were set to 1. Different letters represent values significantly different at P < 0.05
25
814 (Student’s t-test). Asterisks indicate significant differences between values of one genotype at
815 control or low S (*P<0.05, **P<0.01, and ***P<0.001, Student’s t-test).
816 Figure 5. Venn diagrams of differentially expressed genes.
817 Plants were grown for 18 days on either 0.75 mM SO42- or 0.015 mM SO42-, and the RNAs
818 from shoots and roots were subjected to transcriptome analysis by RNA-seq. Differentially
819 expressed genes (qval > 0.05; –1 > log2FC > 1) were determined via kallisto pseudo-
820 alignment, and sleuth analysis. A Differentially expressed genes between the mutants and
821 Col-0 at control and low S. B Differentially expressed genes between control and low S in
822 individual genotypes.
823 Figure 6. Functional enrichment.
824 Over-representation analysis of GO terms annotated to lists of DEGs was performed with
825 PANTHER using Fisher’s exact test. Biological processes terms with Bonferroni corrected p-
826 values < 0.005, with semantic similarity < 0.7 (as determined by REVIGO), and with fewer
827 than 10% of Arabidopsis genes annotated to them are visualized. GO terms are sorted on the
828 y-axis by size, with the most specific terms on top. The number of Arabidopsis genes
829 annotated to each term is provided in parenthesis after the term name. Fold enrichment of the
830 GO term in the indicated gene list is represented by the point fill color. The number of
831 differentially expressed genes in the gene list that are annotated to each term is represented by
832 point size. The shape of the point specifies the tissue for the differentially expressed gene list,
833 and the size of the gene list is shown in parentheses. A GO terms over-represented in eil3,
834 eil1, and eil3 eil1 at 0.75 mM SO42-. B GO terms over-represented among SLIM1- and EIL1-
835 dependent genes responsive to sulfur deficiency.
836 Figure 7. Contribution of SLIM1 and EIL1 to control of the sulfur-deficiency response.
837 DEGs of the sulfur-deficiency response (grey), which are regulated by SLIM1 (blue) and
838 EIL1 (yellow), defined as either being differentially expressed in WT and not in the
839 mutants eil3 and eil1, or with the fold change at least two-fold lower in the mutants than in
840 WT.
841 Figure 8. Summary of SLIM1/EIL1 contribution to the –S response
842 Plants were grown for 18 days on either 0.75 mM SO42- or 0.015 mM SO42-. Differentially
843 expressed genes (qval > 0.05; –1 > log2FC > 1) of RNA-Seq (Illumina Sequencing) were
844 determined via kallisto pseudo-alignment, and sleuth analysis. Differentially expressed genes
845 regulated by –S response (Col-0 +S vs. Col –S; red line), SLIM1 (Col -S vs. eil3 -S; inverted,
846 black bars), EIL1 (Col -S vs. eil1 -S; inverted, black bars), and SLIM1+EIL1 (Col -S vs. eil3
847 eil1 -S; inverted, black bars) in shoot and root tissue are compared.
26
848
27
849 Literature cited
850
851 Aarabi F, Kusajima M, Tohge T, Konishi T, Gigolashvili T, Takamune M, Sasazaki Y,
852 Watanabe M, Nakashita H, Fernie AR, Saito K, Takahashi H, Hubberten HM,
853 Hoefgen R, Maruyama-Nakashita A (2016) Sulfur deficiency-induced repressor
854 proteins optimize glucosinolate biosynthesis in plants. Sci Adv 2: e1601087.
855 Aghajanzadeh T, Kopriva S, Hawkesford MJ, Koprivova A, De Kok LJ (2015) Atmospheric
856 H2S and SO2 as sulfur source for Brassica juncea and Brassica rapa: impact on the
857 glucosinolate composition. Front Plant Sci 6: 924.
858 Aubry S, Smith-Unna RD, Boursnell CM, Kopriva S, Hibberd JM (2014) Transcript
859 residency on ribosomes reveals a key role for the Arabidopsis thaliana bundle sheath
860 in sulfur and glucosinolate metabolism. Plant J 78: 659-673.
861 Bednarek P, Pislewska-Bednarek M, Svatos A, Schneider B, Doubsky J, Mansurova M,
862 Humphry M, Consonni C, Panstruga R, Sanchez-Vallet A, Molina A, Schulze-Lefert P
863 (2009) A glucosinolate metabolism pathway in living plant cells mediates broad-
864 spectrum antifungal defense. Science 323: 101-106.
865 Bielecka M, Watanabe M, Morcuende R, Scheible WR, Hawkesford MJ, Hesse H, Hoefgen R
866 (2014) Transcriptome and metabolome analysis of plant sulfate starvation and
867 resupply provides novel information on transcriptional regulation of metabolism
868 associated with sulfur, nitrogen and phosphorus nutritional responses in Arabidopsis.
869 Front Plant Sci 5: 805.
870 Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina
871 sequence data. Bioinformatics 30: 2114-2120.
872 Bray NL, Pimentel H, Melsted P, Pachter L (2016) Near-optimal probabilistic RNA-seq
873 quantification. Nat Biotechnol 34: 525-527.
874 Burow M, Muller R, Gershenzon J, Wittstock U (2006) Altered glucosinolate hydrolysis in
875 genetically engineered Arabidopsis thaliana and its influence on the larval
876 development of Spodoptera littoralis. J Chem Ecol 32: 2333-2349.
877 Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Perez-Perez J, Solano R, Leyva A, Paz-
878 Ares J (2010) A central regulatory system largely controls transcriptional activation
879 and repression responses to phosphate starvation in Arabidopsis. PLoS Genet 6:
880 e1001102.
881 Cao MJ, Wang Z, Wirtz M, Hell R, Oliver DJ, Xiang CB (2013) SULTR3;1 is a chloroplast-
882 localized sulfate transporter in Arabidopsis thaliana. Plant J 73: 607-616.
883 Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the
884 ethylene gas response pathway in Arabidopsis by the nuclear protein ETHYLENE-
885 INSENSITIVE3 and related proteins. Cell 89: 1133-1144.
886 Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van Breusegem F (2000) Dual
887 action of the active oxygen species during plant stress responses. Cell Mol Life Sci 57:
888 779-795.
889 Frerigmann H, Gigolashvili T (2014) Update on the role of R2R3-MYBs in the regulation of
890 glucosinolates upon sulfur deficiency. Front Plant Sci 5: 626.
891 Guo H, Ecker JR (2004) The ethylene signaling pathway: new insights. Curr Opin Plant Biol
892 7: 40-49.
893 Halkier BA, Gershenzon J (2006) Biology and biochemistry of glucosinolates. Annual Review
894 of Plant Biology 57: 303-333.
895 Harada E, Kusano T, Sano H (2000) Differential expression of genes encoding enzymes
896 involved in sulfur assimilation pathways in response to wounding and jasmonate in
897 Arabidopsis thaliana. Journal of Plant Physiology 156: 272-276.
898 Higashi Y, Hirai MY, Fujiwara T, Naito S, Noji M, Saito K (2006) Proteomic and
899 transcriptomic analysis of Arabidopsis seeds: molecular evidence for successive
28
900 processing of seed proteins and its implication in the stress response to sulfur
901 nutrition. Plant J 48: 557-571.
902 Hirai MY, Fujiwara T, Awazuhara M, Kimura T, Noji M, Saito K (2003) Global expression
903 profiling of sulfur-starved Arabidopsis by DNA macroarray reveals the role of O-
904 acetyl-l-serine as a general regulator of gene expression in response to sulfur nutrition.
905 Plant Journal 33: 651-663.
906 Hirai MY, Klein M, Fujikawa Y, Yano M, Goodenowe DB, Yamazaki Y, Kanaya S,
907 Nakamura Y, Kitayama M, Suzuki H, Sakurai N, Shibata D, Tokuhisa J, Reichelt M,
908 Gershenzon J, Papenbrock J, Saito K (2005) Elucidation of gene-to-gene and
909 metabolite-to-gene networks in arabidopsis by integration of metabolomics and
910 transcriptomics. Journal of Biological Chemistry 280: 25590-25595.
911 Hubberten HM, Klie S, Caldana C, Degenkolbe T, Willmitzer L, Hoefgen R (2012)
912 Additional role of O-acetylserine as a sulfur status-independent regulator during plant
913 growth. Plant J 70: 666-677.
914 Jones PR, Manabe T, Awazuhara M, Saito K (2003) A new member of plant CS-lyases. A
915 cystine lyase from Arabidopsis thaliana. J Biol Chem 278: 10291-10296.
916 Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, Hahnel U, Hansch R, Hartmann T,
917 Kopriva S, Kruse C, Mendel RR, Papenbrock J, Reichelt M, Rennenberg H, Schnug E,
918 Schmidt A, Textor S, Tokuhisa J, Wachter A, Wirtz M, Rausch T, Hell R (2005)
919 Expression profiling of metabolic genes in response to methyl jasmonate reveals
920 regulation of genes of primary and secondary sulfur-related pathways in Arabidopsis
921 thaliana. Photosynthesis Research 86: 491-508.
922 Kataoka T, Hayashi N, Yamaya T, Takahashi H (2004) Root-to-shoot transport of sulfate in
923 Arabidopsis. Evidence for the role of SULTR3;5 as a component of low-affinity
924 sulfate transport system in the root vasculature. Plant Physiology 136: 4198-4204.
925 Kataoka T, Watanabe-Takahashi A, Hayashi N, Ohnishi M, Mimura T, Buchner P,
926 Hawkesford MJ, Yamaya T, Takahashi H (2004) Vacuolar sulfate transporters are
927 essential determinants controlling internal distribution of sulfate in Arabidopsis. Plant
928 Cell 16: 2693-2704.
929 Kawashima CG, Matthewman CA, Huang SQ, Lee BR, Yoshimoto N, Koprivova A, Rubio-
930 Somoza I, Todesco M, Rathjen T, Saito K, Takahashi H, Dalmay T, Kopriva S (2011)
931 Interplay of SLIM1 and miR395 in the regulation of sulfate assimilation in
932 Arabidopsis. Plant Journal 66: 863-876.
933 Kawashima CG, Yoshimoto N, Maruyama-Nakashita A, Tsuchiya YN, Saito K, Takahashi H,
934 Dalmay T (2009) Sulphur starvation induces the expression of microRNA-395 and
935 one of its target genes but in different cell types. Plant Journal 57: 313-321.
936 Kim JH, Kim WT (2013) The Arabidopsis RING E3 ubiquitin ligase AtAIRP3/LOG2
937 participates in positive regulation of high-salt and drought stress responses. Plant
938 Physiol 162: 1733-1749.
939 Kim W, Lee Y, Park J, Lee N, Choi G (2013) HONSU, a protein phosphatase 2C, regulates
940 seed dormancy by inhibiting ABA signaling in Arabidopsis. Plant Cell Physiol 54:
941 555-572.
942 Konishi M, Yanagisawa S (2013) Arabidopsis NIN-like transcription factors have a central
943 role in nitrate signalling. Nat Commun 4: 1617.
944 Kopriva S, Mugford SG, Baraniecka P, Lee BR, Matthewman CA, Koprivova A (2012)
945 Control of sulfur partitioning between primary and secondary metabolism in
946 Arabidopsis. Front Plant Sci 3: 163.
947 Koprivova A, Giovannetti M, Baraniecka P, Lee BR, Grondin C, Loudet O, Kopriva S (2013)
948 Natural Variation in the ATPS1 Isoform of ATP Sulfurylase Contributes to the
949 Control of Sulfate Levels in Arabidopsis. Plant Physiol 163: 1133-1141.
29
950 Koprivova A, Kopriva S (2014) Molecular mechanisms of regulation of sulfate assimilation:
951 first steps on a long road. Front Plant Sci 5: 589.
952 Koprivova A, Kopriva S (2016) Hormonal control of sulfate uptake and assimilation. Plant
953 Mol Biol 91: 617-627.
954 Koprivova A, Kopriva S (2016) Sulfation pathways in plants. Chem Biol Interact 259: 23-30.
955 Kosugi S, Ohashi Y (2000) Cloning and DNA-binding properties of a tobacco Ethylene-
956 Insensitive3 (EIN3) homolog. Nucleic Acids Res 28: 960-967.
957 Lenfant N, Hotelier T, Bourne Y, Marchot P, Chatonnet A (2013) Proteins with an alpha/beta
958 hydrolase fold: Relationships between subfamilies in an ever-growing superfamily.
959 Chem Biol Interact 203: 266-268.
960 Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano R (2003) ETHYLENE RESPONSE
961 FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense.
962 Plant Cell 15: 165-178.
963 Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for
964 RNA-seq data with DESeq2. Genome Biol 15: 550.
965 Maruyama-Nakashita A (2017) Metabolic changes sustain the plant life in low-sulfur
966 environments. Curr Opin Plant Biol 39: 144-151.
967 Maruyama-Nakashita A, Inoue E, Watanabe-Takahashi A, Yamaya T, Takahashi H (2003)
968 Transcriptome profiling of sulfur-responsive genes in Arabidopsis reveals global
969 effects of sulfur nutrition on multiple metabolic pathways. Plant Physiology 132: 597-
970 605.
971 Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K, Takahashi H (2006) Arabidopsis
972 SLIM1 is a central transcriptional regulator of plant sulfur response and metabolism.
973 Plant Cell 18: 3235-3251.
974 Merchante C, Alonso JM, Stepanova AN (2013) Ethylene signaling: simple ligand, complex
975 regulation. Curr Opin Plant Biol 16: 554-560.
976 Mhamdi A, Van Breusegem F (2018) Reactive oxygen species in plant development.
977 Development 145
978 Mi H, Muruganujan A, Ebert D, Huang X, Thomas PD (2019) PANTHER version 14: more
979 genomes, a new PANTHER GO-slim and improvements in enrichment analysis tools.
980 Nucleic Acids Res 47: D419-D426.
981 Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 405-
982 410.
983 Mittler R, Blumwald E (2015) The roles of ROS and ABA in systemic acquired acclimation.
984 Plant Cell 27: 64-70.
985 Mugford SG, Lee BR, Koprivova A, Matthewman C, Kopriva S (2011) Control of sulfur
986 partitioning between primary and secondary metabolism. Plant Journal 65: 96-105.
987 Nardini M, Dijkstra BW (1999) Alpha/beta hydrolase fold enzymes: the family keeps
988 growing. Curr Opin Struct Biol 9: 732-737.
989 Nikiforova V, Freitag J, Kempa S, Adamik M, Hesse H, Hoefgen R (2003) Transcriptome
990 analysis of sulfur depletion in Arabidopsis thaliana: interlacing of biosynthetic
991 pathways provides response specificity. Plant Journal 33: 633-650.
992 Nikiforova VJ, Kopka J, Tolstikov V, Fiehn O, Hopkins L, Hawkesford MJ, Hesse H,
993 Hoefgen R (2005) Systems rebalancing of metabolism in response to sulfur
994 deprivation, as revealed by metabolome analysis of Arabidopsis plants. Plant
995 Physiology 138: 304-318.
996 Noctor G, Reichheld JP, Foyer CH (2018) ROS-related redox regulation and signaling in
997 plants. Semin Cell Dev Biol 80: 3-12.
998 Pandey SP, Roccaro M, Schon M, Logemann E, Somssich IE (2010) Transcriptional
999 reprogramming regulated by WRKY18 and WRKY40 facilitates powdery mildew
1000 infection of Arabidopsis. Plant J 64: 912-923.
30
1001 Pilot G, Stransky H, Bushey DF, Pratelli R, Ludewig U, Wingate VP, Frommer WB (2004)
1002 Overexpression of GLUTAMINE DUMPER1 leads to hypersecretion of glutamine
1003 from Hydathodes of Arabidopsis leaves. Plant Cell 16: 1827-1840.
1004 Pimentel H, Bray NL, Puente S, Melsted P, Pachter L (2017) Differential analysis of RNA-
1005 seq incorporating quantification uncertainty. Nat Methods 14: 687-690.
1006 Pratelli R, Guerra DD, Yu S, Wogulis M, Kraft E, Frommer WB, Callis J, Pilot G (2012) The
1007 ubiquitin E3 ligase LOSS OF GDU2 is required for GLUTAMINE DUMPER1-
1008 induced amino acid secretion in Arabidopsis. Plant Physiol 158: 1628-1642.
1009 Pratelli R, Voll LM, Horst RJ, Frommer WB, Pilot G (2010) Stimulation of nonselective
1010 amino acid export by glutamine dumper proteins. Plant Physiol 152: 762-773.
1011 Rouhier N, Cerveau D, Couturier J, Reichheld JP, Rey P (2015) Involvement of thiol-based
1012 mechanisms in plant development. Biochim Biophys Acta 1850: 1479-1496.
1013 Shin R, Berg RH, Schachtman DP (2005) Reactive oxygen species and root hairs in
1014 Arabidopsis root response to nitrogen, phosphorus and potassium deficiency. Plant
1015 Cell Physiol 46: 1350-1357.
1016 Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a
1017 transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-
1018 RESPONSE-FACTOR1. Genes Dev 12: 3703-3714.
1019 Sonderby IE, Geu-Flores F, Halkier BA (2010) Biosynthesis of glucosinolates--gene
1020 discovery and beyond. Trends in Plant Science 15: 283-290.
1021 Stenzel I, Otto M, Delker C, Kirmse N, Schmidt D, Miersch O, Hause B, Wasternack C
1022 (2012) ALLENE OXIDE CYCLASE (AOC) gene family members of Arabidopsis
1023 thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization. J
1024 Exp Bot 63: 6125-6138.
1025 Sun L, Song L, Zhang Y, Zheng Z, Liu D (2016) Arabidopsis PHL2 and PHR1 Act
1026 Redundantly as the Key Components of the Central Regulatory System Controlling
1027 Transcriptional Responses to Phosphate Starvation. Plant Physiol 170: 499-514.
1028 Supek F, Bosnjak M, Skunca N, Smuc T (2011) REVIGO summarizes and visualizes long
1029 lists of gene ontology terms. PLoS One 6: e21800.
1030 Takahashi H, Buchner P, Yoshimoto N, Hawkesford MJ, Shiu SH (2011) Evolutionary
1031 relationships and functional diversity of plant sulfate transporters. Front Plant Sci 2:
1032 119.
1033 Takahashi H, Kopriva S, Giordano M, Saito K, Hell R (2011) Sulfur Assimilation in
1034 Photosynthetic Organisms: Molecular Functions and Regulations of Transporters and
1035 Assimilatory Enzymes. Annual Review of Plant Biology, Vol 62 62: 157-184.
1036 Takahashi H, Yamazaki M, Sasakura N, Watanabe A, Leustek T, Engler JA, Engler G, Van
1037 Montagu M, Saito K (1997) Regulation of sulfur assimilation in higher plants: a
1038 sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis
1039 thaliana. Proc Natl Acad Sci U S A 94: 11102-11107.
1040 Vandenabeele S, Van Der Kelen K, Dat J, Gadjev I, Boonefaes T, Morsa S, Rottiers P,
1041 Slooten L, Van Montagu M, Zabeau M, Inze D, Van Breusegem F (2003) A
1042 comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco.
1043 Proc Natl Acad Sci U S A 100: 16113-16118.
1044 Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ballmoos P, Krahenbuhl U, den Camp
1045 RO, Brunold C (2002) Flux control of sulphate assimilation in Arabidopsis thaliana:
1046 adenosine 5 '-phosphosulphate reductase is more susceptible than ATP sulphurylase to
1047 negative control by thiols. Plant Journal 31: 729-740.
1048 Wawrzynska A, Lewandowska M, Sirko A (2010) Nicotiana tabacum EIL2 directly regulates
1049 expression of at least one tobacco gene induced by sulphur starvation. J Exp Bot 61:
1050 889-900.
31
1051 Wawrzynska A, Lewandowska M, Sirko A (2010) Nicotiana tabacum EIL2 directly regulates
1052 expression of at least one tobacco gene induced by sulphur starvation. Journal of
1053 Experimental Botany 61: 889-900.
1054 Wawrzynska A, Sirko A (2014) To control and to be controlled: understanding the
1055 Arabidopsis SLIM1 function in sulfur deficiency through comprehensive investigation
1056 of the EIL protein family. Front Plant Sci 5: 575.
1057 Wawrzynska A, Sirko A (2016) EIN3 interferes with the sulfur deficiency signaling in
1058 Arabidopsis thaliana through direct interaction with the SLIM1 transcription factor.
1059 Plant Sci 253: 50-57.
1060 Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An "Electronic
1061 Fluorescent Pictograph" browser for exploring and analyzing large-scale biological
1062 data sets. PLoS One 2: e718.
1063 Wirtz M, Droux M, Hell R (2004) O-acetylserine (thiol) lyase: an enigmatic enzyme of plant
1064 cysteine biosynthesis revisited in Arabidopsis thaliana. Journal of Experimental
1065 Botany 55: 1785-1798.
1066 Wu Y, Zhao Q, Gao L, Yu XM, Fang P, Oliver DJ, Xiang CB (2010) Isolation and
1067 characterization of low-sulphur-tolerant mutants of Arabidopsis. J Exp Bot 61: 3407-
1068 3422.
1069 Xiang C, Oliver DJ (1998) Glutathione metabolic genes coordinately respond to heavy metals
1070 and jasmonic acid in Arabidopsis. Plant Cell 10: 1539-1550.
1071 Yoshimoto N, Takahashi H, Smith FW, Yamaya T, Saito K (2002) Two distinct high-affinity
1072 sulfate transporters with different inducibilities mediate uptake of sulfate in
1073 Arabidopsis roots. Plant Journal 29: 465-473.
1074
1075
1076
32
Figure 1. SLIM1/EIL3 in regulation of sulfate deficiency response and a link to EIN3/EIL1
function in ethylene signal transduction
APS, adenosine 5’-phosphosulfate; PAPS, 3’-phosphoadenosine-5’-phosphosulfate; OAS, O-
acetylserine, Cys, cysteine; GSH, glutathione; Met, methionine; Trp, tryptophan; SLIM1, SULFUR
LIMITATION 1; CTR1, CONSTITUTIVE TRIPLE RESPONSE 1; EIN2, ETHYLENE INSENSITIVE
2; EBF1/2, EIN3-BINDING F BOX PROTEIN 1/2; EIN3/EIL1, ETHYLENE-
INSENSITIVE3/ETHYLENE-INSENSITIVE3-LIKE 1

Figure 2. Metabolite analysis of eil1, eil3 and eil3 eil1
The mutants and WT Col-0 plants were grown for 18 days on either 0.75 mM SO42- (grey) or 0.015 mM
SO42- (green). A Sulfate content was determined via ion chromatography (IC), B thiols and C glucosinolate
content was measured by HPLC. Data from one typical of at least 2 independent experiments are presented
as box plots of 4 biological replicates, with lines marking medians. Different letters represent values
significantly different at P < 0.05 (Student’s t test). Asterisks indicate significant differences between values
of one genotype at control or low S (*P<0.05, **P<0.01 and ***P<0.001, Student’s t-test).

Figure 3. Flux through sulfate assimilation in eil1, eil3 and eil3 eil1
Plants were grown for 18 days on either 0.75 mM SO42- (grey) or 0.015 mM SO42- (green) and
subsequently fed with [35S]sulfate. A Uptake and root-shoot translocation were determined via
scintillation counter. B [35S] incorporation to glutathione and glucosinolates was measured via
high-performance liquid chromatography (HPLC) with a radiodetector or after elution from DEAE
Sephadex by scintillation counting, respectively. A typical of at least 2 independent experiment are
presented. Data from one typical of at least 2 independent experiments are presented as box plots
of 4 biological replicates. Different letters represent values significantly different at P < 0.05
(Student’s t test). Asterisks indicate significant differences between values of one genotype at
control or low S (*P<0.05, **P<0.01 and ***P<0.001, Student’s t-test).

Figure 4. Expression analysis of S deficiency marker genes in eil1, eil3 and eil3 eil1.
Plants were grown for 18 days on either 0.75 mM SO42- (grey) or 0.015 mM SO42- (green). Relative
gene expression (2-ΔΔCt) of –S marker genes A APR3, SDI1, and GGCT2;1 in the leaves and B
SULTR1;1, SDI1, and GGCT2;1 in the roots was determined via qRT-PCR. A typical of at least 2
independent experiment are presented. Box plots of 4 biological replicates are given, lines represent
medians. The values in Col-0 at control conditions were set to 1. Different letters represent values
significantly different at P < 0.05 (Student’s t test). Asterisks indicate significant differences
between values of one genotype at control or low S (*P<0.05, **P<0.01 and ***P<0.001, Student’s
t-test).

A B
0.75 mM 0.015 mM
eil3
eil1 eil3 eil1
eil1 eil3 eil1 eil3
Col-0 eil1
eil3 eil1 eil3 eil1
eil3 eil3 eil1

eil1 eil3 eil1 eil3
Col-0 eil1
eil3 eil1 eil3 eil1
Figure 5. Venn diagrams of differentially expressed genes.
Plants were grown for 18 days on either 0.75 mM SO42- or 0.015 mM SO42-, and the RNAs from
shoots and roots was subjected to transcriptome analysis by RNAseq. Differentially expressed genes
(qval > 0.05; -1 > log2FC > 1) were determined via kallisto pseudo-alignment, and sleuth analysis. A
Differentially expressed genes between the mutants and Col-0 at control and low S. B Differentially
expressed genes between control and low S in individual genotypes.

Figure 6. Functional enrichment.
Over-representation analysis of GO terms annotated to lists of DEG was performed with PANTHER using
Fisher’s exact test. Biological processes terms with Bonferroni corrected p-values < 0.005, with semantic
similarity < 0.7 (as determined by REVIGO), and with fewer than 10% of Arabidopsis genes annotated to
them are visualized. GO terms are sorted on the y-axis by size, with the most specific terms on top. The
number of Arabidopsis genes annotated to each term is provided in parenthesis after the term name. Fold
enrichment of the GO term in the indicated gene list is represented by the point fill color. The number of
differentially expressed genes in the gene list that are annotated to each term is represented by point size.
The shape of the point specifies the tissue for the differentially expressed gene list, and the size of the gene
list is shown in parentheses. A GO terms©over-represented
Copyright 2020 American Societyinof eil3, eil1, andAlleil3
Plant Biologists. rightseil1 at 0.75 mM SO42-. B GO
reserved.
terms over-represented among SLIM1- and EIL1-dependent genes responsive to sulfur deficiency.
shoot root
Figure 7. Contribution of SLIM1 and EIL1 to control of sulfur deficiency response.

DEGs of the sulfur deficiency response (grey), which are regulated by SLIM1 (blue) and
EIL1 (yellow), defined as either being differentially expressed in WT and not in the
mutants eil3 and eil1, or with the fold change at least two-fold lower in the mutants than
in WT.

Figure 8. Summary of SLIM1/EIL1 contribution to the –S response
Plants were grown for 18 days on either 0.75 mM SO42- or 0.015 mM SO42-. Differentially expressed
genes (qval > 0.05; -1 > log2FC > 1) of RNA-Seq (Illumina Sequencing), were determined via kallisto
pseudo-alignment, and sleuth analysis. Differential expressed genes regulated by –S response (Col-0 +S
vs. Col –S; red line), SLIM1 (Col -S vs. eil3 -S; inverted, black bars), EIL1 (Col -S vs. eil1 -S; inverted,
black bars), and SLIM1+EIL1 (Col -S vs. eil3 eil1 -S; inverted, black bars) in shoot and root tissue is
compared.

Parsed Citations
Aarabi F, Kusajima M, Tohge T, Konishi T, Gigolashvili T, Takamune M, Sasazaki Y, Watanabe M, Nakashita H, Fernie AR, Saito K,
Takahashi H, Hubberten HM, Hoefgen R, Maruyama-Nakashita A (2016) Sulfur deficiency-induced repressor proteins optimize
glucosinolate biosynthesis in plants. Sci Adv 2: e1601087.
Google Scholar: Author Only Title Only Author and Title
Aghajanzadeh T, Kopriva S, Hawkesford MJ, Koprivova A, De Kok LJ (2015) Atmospheric H2S and SO2 as sulfur source for
Brassica juncea and Brassica rapa: impact on the glucosinolate composition. Front Plant Sci 6: 924.
Aubry S, Smith-Unna RD, Boursnell CM, Kopriva S, Hibberd JM (2014) Transcript residency on ribosomes reveals a key role for
the Arabidopsis thaliana bundle sheath in sulfur and glucosinolate metabolism. Plant J 78: 659-673.
Bednarek P, Pislewska-Bednarek M, Svatos A, Schneider B, Doubsky J, Mansurova M, Humphry M, Consonni C, Panstruga R,

Sanchez-Vallet A, Molina A, Schulze-Lefert P (2009) A glucosinolate metabolism pathway in living plant cells mediates broad-
spectrum antifungal defense. Science 323: 101-106.
Bielecka M, Watanabe M, Morcuende R, Scheible WR, Hawkesford MJ, Hesse H, Hoefgen R (2014) Transcriptome and
metabolome analysis of plant sulfate starvation and resupply provides novel information on transcriptional regulation of
metabolism associated with sulfur, nitrogen and phosphorus nutritional responses in Arabidopsis. Front Plant Sci 5: 805.
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114-2120.
Bray NL, Pimentel H, Melsted P, Pachter L (2016) Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 34: 525-527.
Burow M, Muller R, Gershenzon J, Wittstock U (2006) Altered glucosinolate hydrolysis in genetically engineered Arabidopsis
thaliana and its influence on the larval development of Spodoptera littoralis. J Chem Ecol 32: 2333-2349.
Bustos R, Castrillo G, Linhares F, Puga MI, Rubio V, Perez-Perez J, Solano R, Leyva A, Paz-Ares J (2010) A central regulatory
system largely controls transcriptional activation and repression responses to phosphate starvation in Arabidopsis. PLoS Genet
6: e1001102.
Cao MJ, Wang Z, Wirtz M, Hell R, Oliver DJ, Xiang CB (2013) SULTR3;1 is a chloroplast-localized sulfate transporter in
Arabidopsis thaliana. Plant J 73: 607-616.
Chao Q, Rothenberg M, Solano R, Roman G, Terzaghi W, Ecker JR (1997) Activation of the ethylene gas response pathway in
Arabidopsis by the nuclear protein ETHYLENE-INSENSITIVE3 and related proteins. Cell 89: 1133-1144.
Dat J, Vandenabeele S, Vranova E, Van Montagu M, Inze D, Van Breusegem F (2000) Dual action of the active oxygen species
during plant stress responses. Cell Mol Life Sci 57: 779-795.
Frerigmann H, Gigolashvili T (2014) Update on the role of R2R3-MYBs in the regulation of glucosinolates upon sulfur deficiency.
Front Plant Sci 5: 626.
Guo H, Ecker JR (2004) The ethylene signaling pathway: new insights. Curr Opin Plant Biol 7: 40-49.
Halkier BA, Gershenzon J (2006) Biology and biochemistry of glucosinolates. Annual Review of Plant Biology 57: 303-333.
Harada E, Kusano T, Sano H (2000) Differential expression of genes encoding enzymes involved in sulfur assimilation pathways
in response to wounding and jasmonate in Arabidopsis thaliana. Journal of Plant Physiology 156: 272-276.
Higashi Y, Hirai MY, Fujiwara T, Naito S, Noji M, Saito K (2006) Proteomic and transcriptomic analysis of Arabidopsis seeds:
molecular evidence for successive processing of seed proteins and its implication in the stress response to sulfur nutrition.
Plant J 48: 557-571.
Google Scholar: Author Only Title Only Author and
Downloaded Titleon October 16, 2020 - Published by www.plantphysiol.org
from
Hirai MY, Fujiwara T, Awazuhara M, Kimura T, Noji M, Saito K (2003) Global expression profiling of sulfur-starved Arabidopsis by
DNA macroarray reveals the role of O-acetyl-l-serine as a general regulator of gene expression in response to sulfur nutrition.
Plant Journal 33: 651-663.
Hirai MY, Klein M, Fujikawa Y, Yano M, Goodenowe DB, Yamazaki Y, Kanaya S, Nakamura Y, Kitayama M, Suzuki H, Sakurai N,
Shibata D, Tokuhisa J, Reichelt M, Gershenzon J, Papenbrock J, Saito K (2005) Elucidation of gene-to-gene and metabolite-to-
gene networks in arabidopsis by integration of metabolomics and transcriptomics. Journal of Biological Chemistry 280: 25590-
25595.
Hubberten HM, Klie S, Caldana C, Degenkolbe T, Willmitzer L, Hoefgen R (2012) Additional role of O-acetylserine as a sulfur
status-independent regulator during plant growth. Plant J 70: 666-677.
Jones PR, Manabe T, Awazuhara M, Saito K (2003) A new member of plant CS-lyases. A cystine lyase from Arabidopsis thaliana. J
Biol Chem 278: 10291-10296.
Jost R, Altschmied L, Bloem E, Bogs J, Gershenzon J, Hahnel U, Hansch R, Hartmann T, Kopriva S, Kruse C, Mendel RR,
Papenbrock J, Reichelt M, Rennenberg H, Schnug E, Schmidt A, Textor S, Tokuhisa J, Wachter A, Wirtz M, Rausch T, Hell R
(2005) Expression profiling of metabolic genes in response to methyl jasmonate reveals regulation of genes of primary and
secondary sulfur-related pathways in Arabidopsis thaliana. Photosynthesis Research 86: 491-508.
Kataoka T, Hayashi N, Yamaya T, Takahashi H (2004) Root-to-shoot transport of sulfate in Arabidopsis. Evidence for the role of
SULTR3;5 as a component of low-affinity sulfate transport system in the root vasculature. Plant Physiology 136: 4198-4204.
Kataoka T, Watanabe-Takahashi A, Hayashi N, Ohnishi M, Mimura T, Buchner P, Hawkesford MJ, Yamaya T, Takahashi H (2004)
Vacuolar sulfate transporters are essential determinants controlling internal distribution of sulfate in Arabidopsis. Plant Cell 16:
2693-2704.
Kawashima CG, Matthewman CA, Huang SQ, Lee BR, Yoshimoto N, Koprivova A, Rubio-Somoza I, Todesco M, Rathjen T, Saito K,
Takahashi H, Dalmay T, Kopriva S (2011) Interplay of SLIM1 and miR395 in the regulation of sulfate assimilation in Arabidopsis.
Plant Journal 66: 863-876.
Kawashima CG, Yoshimoto N, Maruyama-Nakashita A, Tsuchiya YN, Saito K, Takahashi H, Dalmay T (2009) Sulphur starvation
induces the expression of microRNA-395 and one of its target genes but in different cell types. Plant Journal 57: 313-321.
Kim JH, Kim WT (2013) The Arabidopsis RING E3 ubiquitin ligase AtAIRP3/LOG2 participates in positive regulation of high-salt
and drought stress responses. Plant Physiol 162: 1733-1749.
Kim W, Lee Y, Park J, Lee N, Choi G (2013) HONSU, a protein phosphatase 2C, regulates seed dormancy by inhibiting ABA
signaling in Arabidopsis. Plant Cell Physiol 54: 555-572.
Konishi M, Yanagisawa S (2013) Arabidopsis NIN-like transcription factors have a central role in nitrate signalling. Nat Commun 4:
1617.
Kopriva S, Mugford SG, Baraniecka P, Lee BR, Matthewman CA, Koprivova A (2012) Control of sulfur partitioning between
primary and secondary metabolism in Arabidopsis. Front Plant Sci 3: 163.
Koprivova A, Giovannetti M, Baraniecka P, Lee BR, Grondin C, Loudet O, Kopriva S (2013) Natural Variation in the ATPS1 Isoform
of ATP Sulfurylase Contributes to the Control of Sulfate Levels in Arabidopsis. Plant Physiol 163: 1133-1141.
Koprivova A, Kopriva S (2014) Molecular mechanisms of regulation of sulfate assimilation: first steps on a long road. Front Plant
Sci 5: 589.
Koprivova A, Kopriva S (2016) Hormonal control of sulfate uptake and assimilation. Plant Mol Biol 91: 617-627.
Koprivova A, Kopriva S (2016) Sulfation pathways in plants. Chem Biol Interact 259: 23-30.
Kosugi S, Ohashi Y (2000) Cloning and DNA-binding properties of a tobacco Ethylene-Insensitive3 (EIN3) homolog. Nucleic Acids
Res 28: 960-967.
Lenfant N, Hotelier T, Bourne Y, Marchot P, Chatonnet A (2013) Proteins with an alpha/beta hydrolase fold: Relationships
between subfamilies in an ever-growing superfamily. Chem Biol Interact 203: 266-268.
Lorenzo O, Piqueras R, Sanchez-Serrano JJ, Solano R (2003) ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene
and jasmonate pathways in plant defense. Plant Cell 15: 165-178.
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome
Biol 15: 550.
Maruyama-Nakashita A (2017) Metabolic changes sustain the plant life in low-sulfur environments. Curr Opin Plant Biol 39: 144-
151.
Maruyama-Nakashita A, Inoue E, Watanabe-Takahashi A, Yamaya T, Takahashi H (2003) Transcriptome profiling of sulfur-
responsive genes in Arabidopsis reveals global effects of sulfur nutrition on multiple metabolic pathways. Plant Physiology 132:
597-605.
Maruyama-Nakashita A, Nakamura Y, Tohge T, Saito K, Takahashi H (2006) Arabidopsis SLIM1 is a central transcriptional regulator
of plant sulfur response and metabolism. Plant Cell 18: 3235-3251.
Merchante C, Alonso JM, Stepanova AN (2013) Ethylene signaling: simple ligand, complex regulation. Curr Opin Plant Biol 16:
554-560.
Mhamdi A, Van Breusegem F (2018) Reactive oxygen species in plant development. Development 145
Mi H, Muruganujan A, Ebert D, Huang X, Thomas PD (2019) PANTHER version 14: more genomes, a new PANTHER GO-slim and
improvements in enrichment analysis tools. Nucleic Acids Res 47: D419-D426.
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7: 405-410.
Mittler R, Blumwald E (2015) The roles of ROS and ABA in systemic acquired acclimation. Plant Cell 27: 64-70.
Mugford SG, Lee BR, Koprivova A, Matthewman C, Kopriva S (2011) Control of sulfur partitioning between primary and
secondary metabolism. Plant Journal 65: 96-105.
Nardini M, Dijkstra BW (1999) Alpha/beta hydrolase fold enzymes: the family keeps growing. Curr Opin Struct Biol 9: 732-737.
Nikiforova V, Freitag J, Kempa S, Adamik M, Hesse H, Hoefgen R (2003) Transcriptome analysis of sulfur depletion in Arabidopsis
thaliana: interlacing of biosynthetic pathways provides response specificity. Plant Journal 33: 633-650.
Nikiforova VJ, Kopka J, Tolstikov V, Fiehn O, Hopkins L, Hawkesford MJ, Hesse H, Hoefgen R (2005) Systems rebalancing of
metabolism in response to sulfur deprivation, as revealed by metabolome analysis of Arabidopsis plants. Plant Physiology 138:
304-318.
Noctor G, Reichheld JP, Foyer CH (2018) ROS-related redox regulation and signaling in plants. Semin Cell Dev Biol 80: 3-12.
Pandey SP, Roccaro M, Schon M, Logemann E, Somssich IE (2010) Transcriptional reprogramming regulated by WRKY18 and
WRKY40 facilitates powdery mildew infection of Arabidopsis. Plant J 64: 912-923.
Downloaded
Google Scholar: Author Only Title Only from
Author and Titleon October 16, 2020 - Published by www.plantphysiol.org
Pilot G, Stransky H, Bushey DF, Pratelli R, Ludewig U, Wingate VP, Frommer WB (2004) Overexpression of GLUTAMINE
DUMPER1 leads to hypersecretion of glutamine from Hydathodes of Arabidopsis leaves. Plant Cell 16: 1827-1840.
Pimentel H, Bray NL, Puente S, Melsted P, Pachter L (2017) Differential analysis of RNA-seq incorporating quantification
uncertainty. Nat Methods 14: 687-690.
Pratelli R, Guerra DD, Yu S, Wogulis M, Kraft E, Frommer WB, Callis J, Pilot G (2012) The ubiquitin E3 ligase LOSS OF GDU2 is
required for GLUTAMINE DUMPER1-induced amino acid secretion in Arabidopsis. Plant Physiol 158: 1628-1642.
Pratelli R, Voll LM, Horst RJ, Frommer WB, Pilot G (2010) Stimulation of nonselective amino acid export by glutamine dumper
proteins. Plant Physiol 152: 762-773.
Rouhier N, Cerveau D, Couturier J, Reichheld JP, Rey P (2015) Involvement of thiol-based mechanisms in plant development.
Biochim Biophys Acta 1850: 1479-1496.
Shin R, Berg RH, Schachtman DP (2005) Reactive oxygen species and root hairs in Arabidopsis root response to nitrogen,
phosphorus and potassium deficiency. Plant Cell Physiol 46: 1350-1357.
Solano R, Stepanova A, Chao Q, Ecker JR (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by
ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev 12: 3703-3714.
Sonderby IE, Geu-Flores F, Halkier BA (2010) Biosynthesis of glucosinolates--gene discovery and beyond. Trends in Plant
Science 15: 283-290.
Stenzel I, Otto M, Delker C, Kirmse N, Schmidt D, Miersch O, Hause B, Wasternack C (2012) ALLENE OXIDE CYCLASE (AOC)
gene family members of Arabidopsis thaliana: tissue- and organ-specific promoter activities and in vivo heteromerization. J Exp
Bot 63: 6125-6138.
Sun L, Song L, Zhang Y, Zheng Z, Liu D (2016) Arabidopsis PHL2 and PHR1 Act Redundantly as the Key Components of the
Central Regulatory System Controlling Transcriptional Responses to Phosphate Starvation. Plant Physiol 170: 499-514.
Supek F, Bosnjak M, Skunca N, Smuc T (2011) REVIGO summarizes and visualizes long lists of gene ontology terms. PLoS One 6:
e21800.
Takahashi H, Buchner P, Yoshimoto N, Hawkesford MJ, Shiu SH (2011) Evolutionary relationships and functional diversity of
plant sulfate transporters. Front Plant Sci 2: 119.
Takahashi H, Kopriva S, Giordano M, Saito K, Hell R (2011) Sulfur Assimilation in Photosynthetic Organisms: Molecular Functions
and Regulations of Transporters and Assimilatory Enzymes. Annual Review of Plant Biology, Vol 62 62: 157-184.
Takahashi H, Yamazaki M, Sasakura N, Watanabe A, Leustek T, Engler JA, Engler G, Van Montagu M, Saito K (1997) Regulation of
sulfur assimilation in higher plants: a sulfate transporter induced in sulfate-starved roots plays a central role in Arabidopsis
thaliana. Proc Natl Acad Sci U S A 94: 11102-11107.
Vandenabeele S, Van Der Kelen K, Dat J, Gadjev I, Boonefaes T, Morsa S, Rottiers P, Slooten L, Van Montagu M, Zabeau M, Inze
D, Van Breusegem F (2003) A comprehensive analysis of hydrogen peroxide-induced gene expression in tobacco. Proc Natl Acad
Sci U S A 100: 16113-16118.
Vauclare P, Kopriva S, Fell D, Suter M, Sticher L, von Ballmoos P, Krahenbuhl U, den Camp RO, Brunold C (2002) Flux control of
sulphate assimilation in Arabidopsis thaliana: adenosine 5 '-phosphosulphate reductase is more susceptible than ATP
sulphurylase to negative control by thiols. Plant Journal 31: 729-740.
Wawrzynska A, Lewandowska M, Sirko A (2010) Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene
induced by sulphur starvation. J Exp Bot 61: 889-900.
Wawrzynska A, Lewandowska M, Sirko A (2010) Nicotiana tabacum EIL2 directly regulates expression of at least one tobacco gene
induced by sulphur starvation. Journal of Experimental Botany 61: 889-900.
Wawrzynska A, Sirko A (2014) To control and to be controlled: understanding the Arabidopsis SLIM1 function in sulfur deficiency
through comprehensive investigation of the EIL protein family. Front Plant Sci 5: 575.
Wawrzynska A, Sirko A (2016) EIN3 interferes with the sulfur deficiency signaling in Arabidopsis thaliana through direct
interaction with the SLIM1 transcription factor. Plant Sci 253: 50-57.
Winter D, Vinegar B, Nahal H, Ammar R, Wilson GV, Provart NJ (2007) An "Electronic Fluorescent Pictograph" browser for
exploring and analyzing large-scale biological data sets. PLoS One 2: e718.
Wirtz M, Droux M, Hell R (2004) O-acetylserine (thiol) lyase: an enigmatic enzyme of plant cysteine biosynthesis revisited in
Arabidopsis thaliana. Journal of Experimental Botany 55: 1785-1798.
Wu Y, Zhao Q, Gao L, Yu XM, Fang P, Oliver DJ, Xiang CB (2010) Isolation and characterization of low-sulphur-tolerant mutants of
Arabidopsis. J Exp Bot 61: 3407-3422.
Xiang C, Oliver DJ (1998) Glutathione metabolic genes coordinately respond to heavy metals and jasmonic acid in Arabidopsis.
Plant Cell 10: 1539-1550.
Yoshimoto N, Takahashi H, Smith FW, Yamaya T, Saito K (2002) Two distinct high-affinity sulfate transporters with different
inducibilities mediate uptake of sulfate in Arabidopsis roots. Plant Journal 29: 465-473.


Short Title: EIL1, SLIM1, and Sulfur Deficiency: Skopriva@uni-Koeln - de

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Short Title: EIL1, SLIM1, and Sulfur Deficiency: Skopriva@uni-Koeln - de

Uploaded by

Copyright:

Available Formats

Plant Physiology Preview. Published on October 15, 2020, as DOI:10.1104/pp.20.

1 Short title: EIL1, SLIM1, and sulfur deficiency

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

eil3 eil1 eil3 eil1

eil3 eil3 eil1

eil3 eil1 eil3 eil1

Figure 5. Venn diagrams of differentially expressed genes.

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

Figure 7. Contribution of SLIM1 and EIL1 to control of sulfur deficiency response.

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

Bednarek P, Pislewska-Bednarek M, Svatos A, Schneider B, Doubsky J, Mansurova M, Humphry M, Consonni C, Panstruga R,

Downloaded from on October 16, 2020 - Published by www.plantphysiol.org

You might also like