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Running head: ERF11 promotes GA biosynthesis and GA signaling 1
2
Corresponding author: 3
Tai-ping Sun 4
Department of Biology, Duke University, Durham, NC 27708 5
Email: tps@duke.edu 6
TEL: 919-613-8166 7
8
Research Area: Signaling and Response 9
10
Plant Physiology Preview. Published on June 2, 2016, as DOI:10.1104/pp.16.00154
Copyright 2016 by the American Society of Plant Biologists
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ERF11 Promotes Internode Elongation by Activating Gibberellin 11
Biosynthesis and Signaling Pathways in Arabidopsis 12
13
14
Xin Zhou2, Zhong-Lin Zhang, Jeongmoo Park, Ludmila Tyler3, Jikumaru Yusuke, Kai 15
Qiu, Edward A. Nam4, Shelley Lumba, Darrell Desveaux, Peter McCourt, Yuji Kamiya5, 16
Tai-ping Sun* 17
Department of Biology, Duke University, Durham, NC 27705, U.S.A. (X.Z., Z.-L.Z., 18
L.T., J.P., E.A.N., T.-p.S.); RIKEN Plant Science Center, Yokohama, Kanagawa 230-19
0045, Japan (J.Y., Y.K.); Department of Cell & Systems Biology, University of Toronto, 20
Ontario, Canada M5S 3B2 (S.L., D.D., P.M.); Centre for the Analysis of Genome 21
Evolution and Function, University of Toronto, Ontario, Canada M5S 3B2 (D.D.); State 22
Key Laboratory of Genetic Engineering and Fudan Institute of Plant Biology, School of 23
Life Sciences, Fudan University, Shanghai 200433, China (K.Q.) 24
25
One sentence summary: The transcription factor AtERF11 promotes stem growth by 26
increasing GA biosynthesis and GA response. 27
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1 This work was supported by National Science Foundation (IOS-0641548 and MCB-29
0923723), US Department of Agriculture (2014-67013-21548), National Institutes of 30
Health (R01 GM100051), and Canada Research Chair-National Sciences and 31
Engineering Research Council (grant number 00003714). 32
33
2 Current Address: State Key Laboratory of Genetic Engineering and Fudan Institute of 34
Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China 35
36
3 Current Address: Department of Biochemistry and Molecular Biology, University of 37
Massachusetts, Amherst, MA 01003, U.S.A. 38
39
4 Current Address: Department of Biology, University of St. Thomas, Houston, TX 40
77006, U.S.A. 41
42
5 Current Address: RIKEN Center for Sustainable Resource Science, Wako, Saitama 351-43
0198, Japan 44
45
*Address correspondence to tps@duke.edu. 46
47
The author responsible for distribution of materials integral to the findings presented in 48
this article in accordance with the policy described in the Instructions for Authors 49
(www.plantphysiol.org) is: Tai-ping Sun (tps@duke.edu). 50
51
52
AUTHOR CONTRIBUTIONS 53
X.Z., Z.Z, J.P. and T.S. designed the research; X.Z., Z.Z., L.T., J.Y., K.Q., J.P., E.A.N. 54
performed research; X.Z., Z.Z., J.P., J.Y., Y.K., and T.S. analyzed data; S.L., D.D., and 55
P.M. provided experimental materials and shared unpublished results; X.Z., Z.Z and T.S. 56
wrote the manuscript. 57
58
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ABSTRACT 59
60
The phytohormone gibberellin (GA) plays a key role in promoting stem elongation in 61
plants. Previous studies show that GA activates its signaling pathway by inducing rapid 62
degradation of DELLA proteins, GA signaling repressors. Using an activation-tagging 63
screen in a reduced-GA mutant ga1-6 background, we identified AtERF11 to be a novel 64
positive regulator of both GA biosynthesis and GA signaling for internode elongation. 65
Overexpression of AtERF11 partially rescued the dwarf phenotype of ga1-6. AtERF11 is 66
a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily VIII-B-1a of 67
ERF/AP2 transcription factors in Arabidopsis thaliana. Overexpression of AtERF11 68
resulted in elevated bioactive GA levels by upregulating expression of GA3ox1 and 69
GA20ox genes. Hypocotyl elongation assays further showed that overexpression of 70
AtERF11 conferred elevated GA response, whereas loss-of-function erf11 and erf11 erf4 71
mutants displayed reduced GA response. In addition, yeast two-hybrid, co-72
immunoprecipitation and transient expression assays showed that AtERF11 enhances GA 73
signaling by antagonizing the function of DELLA proteins via direct protein-protein 74
interaction. Interestingly, AtERF11 overexpression also caused a reduction in the levels 75
of another phytohormone ethylene in the growing stem, consistent with recent finding 76
showing that AtERF11 represses transcription of ethylene biosynthesis ACS genes. The 77
effect of AtERF11 on promoting GA biosynthesis gene expression is likely via its 78
repressive function on ethylene biosynthesis. These results suggest that AtERF11 plays a 79
dual role in promoting internode elongation by inhibiting ethylene biosynthesis and 80
activating GA biosynthesis and signaling pathways. 81
82
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INTRODUCTION 83
84
Bioactive GA, a diterpenoid compound, is an allosteric inducer of its nuclear receptor 85
GIBBERELLIN INSENSITIVE DWARF1 (GID1) (Ueguchi-Tanaka et al., 2005; Murase 86
et al., 2008; Shimada et al., 2008). Acting downstream of GID1, the DELLA proteins are 87
transcription regulators that repress GA signaling and restrict plant growth by causing 88
transcriptional reprogramming (Ueguchi-Tanaka et al., 2007). Binding of GA to GID1 89
enhances the interaction between GID1 and DELLA, resulting in rapid degradation of 90
DELLAs via the ubiquitin-proteasome pathway. In Arabidopsis, DELLAs are members 91
of the GRAS [GA INSENSITIVE (GAI), REPRESSOR OF ga1-3 (RGA) and 92
SCARECROW (SCR)] family of regulatory proteins (Tian et al., 2004). Like all GRAS 93
family members, DELLA contains a conserved C-terminal GRAS domain that confers 94
the transcription regulator function. The unique DELLA domain in the N-terminus of the 95
protein is required for GA-induced degradation via GID1 binding (Dill et al., 2001; Itoh 96
et al., 2002; Griffiths et al., 2006; Murase et al., 2008); this domain is absent in other 97
GRAS family members. Among the five DELLAs [RGA, GAI, RGA-LIKE1 (RGL1), 98
RGL2 and RGL3] in Arabidopsis, RGA and GAI are the major DELLAs for regulating 99
GA-induced vegetative growth (Dill and Sun, 2001; King et al., 2001). 100
101
Recent studies also show that DELLAs integrate GA and other signaling 102
pathways by antagonizing or enhancing functions of key regulators in other pathways via 103
direct protein-protein interactions (Xu et al., 2014; Daviere and Achard, 2015). Most of 104
the DELLA-interacting proteins are transcription factors or transcription regulators. 105
Examples of DELLA-inhibited transcription factors/regulators include bHLH 106
transcription factors, PIFs, in light signaling (de Lucas et al., 2008; Feng et al., 2008); the 107
jasmonic acid (JA) signaling repressors, JAZs (Hou et al., 2010; Yang et al., 2012); 108
ETHYLENE INSENSITIVE3 (EIN3), an ethylene signaling activator (An et al., 2012); 109
and BRASSINAZOLE-RESISTANT1 (BZR1), a brassinosteriod signaling activator (Bai 110
et al., 2012). DELLA-activated transcription factors/regulators include type-B 111
ARABIDOPSIS RESPONSE REGULATORS (ARRs) (Marin-de la Rosa et al., 2015), 112
ABSCISIC ACID INSENSITIVE 3 (ABI3) and ABI5 (Lim et al., 2013). Other types of 113
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DELLA interactors include chromatin-remodeling complexes [Switch (SWI)/Sucrose 114
Nonfermenting (SNF), and a Chromodomain-Helicase-DNA-binding domain-containing 115
protein PICKLE](Sarnowska et al., 2013; Zhang et al., 2014), RING domain proteins 116
BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI) and BOI-RELTED GENEs (BRG1, 117
BRG2 and BRG3) (Park et al., 2013), and subunits of the prefoldin complex for tubulin 118
folding (Locascio et al., 2013). These findings indicate that protein-protein interaction is 119
a central regulatory mechanism in DELLA-modulated plant development. Although a 120
number of DELLA-interacting proteins have been reported, our current knowledge on 121
how DELLAs regulate plant growth and development is still limited. 122
123
To uncover new regulators of the GA pathway, we performed an activation-124
tagging mutant screen and identified AtERF11, a member of the ERF (ETHYLENE 125
RESPONSE FACTOR)/AP2 (APETALA2) family (Nakano et al., 2006), as a novel 126
regulator of GA pathway. Our results show that AtERF11 promotes cell elongation by 127
increasing bioactive GA accumulation. ERF11 also enhances GA responses by directly 128
antagonizing DELLA function through protein-protein interaction. Interestingly, ERF11 129
has been reported to repress genes encoding ethylene biosynthetic enzymes (ACC 130
synthases, ACSs) (Li et al., 2011). Therefore, ERF11 may provide a molecular link 131
between GA and ethylene pathways in modulating internode elongation. 132
133
134
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RESULTS 136
137
Identification of ERF11 as a Positive Regulator of Internode Elongation through an 138
Activation-Tagging Approach 139
To identify positive components of the GA signaling pathway, an activation-tagging 140
mutant screen was employed to isolate mutants that grow taller than parental ga1-6 141
plants. The ga1-6 mutant is a GA-deficient semi-dwarf because of a missense mutation in 142
GA1 (AtCPS) that encodes ent-copalyl diphosphate synthase (CPS) for GA biosynthesis 143
(Sun et al., 1992; Sun and Kamiya, 1994). We reasoned that enhanced expression of 144
positive regulators of the GA pathway would lead to a taller phenotype in this semi-dwarf 145
mutant background. The ga1-6 plants (backcrossed four times to Col-0) were transformed 146
with Agrobacterium tumefaciens that carried four copies of 35S transcriptional enhancers 147
linked to a constitutively expressed BASTA-resistance gene (Weigel et al., 2000). 148
Approximately 12,500 T1 transformants were screened for increased final height. Among 149
the mutants identified, mutant #279-2 was dramatically taller than the parental ga1-6 150
plants -- an average height of 28.5 cm vs. 18.3 cm (Figs 1A and 1B). 151
The #279-2 mutant contains a single T-DNA insertion site, as the linked BASTA-152
resistance gene segregated 3:1 in the T2 generation. Thermal asymmetric interlaced PCR 153
(TAIL-PCR) revealed that the activation tag in #279-2 is inserted in chromosome 1 154
between At1g28360 and At1g28370, which encode two members of the ETHYLENE 155
RESPONSE FACTOR family, AtERF12 and AtERF11, respectively (Supplemental Fig. 156
1A). RT-qPCR further showed that the transcript levels of AtERF11 (At1g28370) were 157
18.3-fold higher in the mutant #279-2 than in the ga1-6 control (Fig.1C). In contrast, the 158
expression levels of the other two adjacent genes At1g28360 (AtERF12) and At1g28375 159
(an expressed endomembrane protein) were unchanged or only 3-fold higher, 160
respectively, in #279-2 compared to ga1-6 (Supplemental Fig. 1A). To verify whether 161
overexpression of ERF11 causes the mutant phenotype of #279-2, we generated 162
transgenic ga1-6 plants carrying CaMV 35S promoter:HA-ERF11-GFP (ERF11-OE 163
lines). Indeed, the final heights of these ERF11-OE lines correlated with ERF11 protein 164
levels in these plants (Figs. 1A and 1D). The line with the highest ERF11 protein 165
expression (#1-1) reached a similar final height as #279-2 (Fig 1A), indicating that 166
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overexpression of ERF11 is responsible for the observed tall phenotype. Mutant #279-2 167
will be referred to as erf11-1D ga1-6 in the rest of this report; the homozygous erf11-1D 168
ga1-6 double mutant in the T5 generation was used for all data presented here. 169
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To understand better the effect of overexpression of ERF11 on stem elongation, 170
the inflorescence stem of erf11-1D ga1-6 was characterized in more detail. erf11-1D ga1-171
6 produced a similar number of siliques as ga1-6 (Supplemental Fig. 1B), but with 82.3% 172
longer internodes which contributed to the increased final height of the mutant (Figs. 1E 173
and 1F). Scanning electron microscopy analysis revealed that the longer internode in 174
erf11-1D ga1-6 is caused by increased cell length (Figs. 1G and 1H), but not greater cell 175
numbers (Supplemental Fig. 1C). A previous study had indicated that ERF11 is expressed 176
ubiquitously in different tissues of wild-type plants; however, the highest transcript levels 177
were detected in leaves and stems (Yang et al., 2005). Consistent with this ERF11 178
expression pattern, the rosette leaves of erf11-1D ga1-6 were 22.7% larger than ga1-6 179
(Figs. 1I and 1J). In addition to the longer internode length and larger rosette size, erf11-180
1D ga1-6 flowered slightly earlier and displayed increased fertility compared to ga1-6 181
(Supplemental Table 1). 182
AtERF11 is a member of the ERF (ETHYLENE RESPONSE FACTOR) subfamily 183
VIII-B-1a of ERF/AP2 transcription factors. There are eight members in this subfamily 184
(ERF3, 4, 7-12); each of them contains an ERF/AP2 domain and a transcription 185
repression EAR motif [DLNxxP; (McGrath et al., 2005; Nakano et al., 2006)]. Recently, 186
AtERF11 was shown to inhibit ethylene biosynthesis by binding to the promoters of two 187
ACC synthase genes ACS2 and ACS5 to repress their expression (Li et al., 2011). 188
Furthermore, overexpression of another VIII-B-1a ERF member, AtERF4, confers 189
reduced ethylene sensitivity in hypocotyl growth (Yang et al., 2005). Similar to erf11-1D 190
ga1-6, overexpression of AtERF4 or AtERF8 (another close homolog of ERF11 in the 191
same subfamily) also resulted in increased final height in the ga1-6 background 192
(Supplemental Fig. 1D). We also generated the erf11-1D single mutant by backcrossing 193
erf11-1D ga1-6 to the wild-type Col-0, and found that erf11-1D is taller with 26.5% 194
longer internodes than wild-type (WT) (Figs. 1K and 1L). Moreover, the erf11 knockout 195
mutant showed slightly shorter final height and internode length compared with WT Col-196
0; the erf11 erf4 double homozygous mutant displayed even shorter stems compared to 197
Col-0 and the erf11 single mutant (Figs. 1K and 1L). Consistent with the shorter stem 198
phenotype, the rosette leaf length of the erf11 erf4 double mutant was slightly reduced 199
comparing with WT (Supplemental Fig. 1E). These results indicated that ERF11 and its 200
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close homologs share redundant function in promoting stem elongation and rosette leaf 201
expansion. 202
203
Overexpression of ERF11 Causes Elevated Bioactive GA4 and Reduced Ethylene 204
Levels in the Growing Internodes 205
To determine whether the longer internodes of erf11-1D ga1-6 are caused by increased 206
levels of bioactive GAs, we first analyzed the transcript levels of GA biosynthesis and 207
catabolism genes that are important for vegetative growth (Mitchum et al., 2006; Rieu et 208
al., 2008; Rieu et al., 2008) by RT-qPCR. We found that expression of several GA 209
biosynthesis genes, including GA20ox1, GA20ox2 and GA3ox1, was up-regulated in the 210
internodes of erf11-1D ga1-6 compared with ga1-6, while a GA catabolism gene, 211
GA2ox6 was down-regulated in erf11-1D ga1-6 (Fig 2A). In contrast, GA3ox2 mRNA 212
levels were not altered (Fig. 2A). GA analysis further showed that the GA4 level in the 213
rosette leaves of erf11-1D ga1-6 was about 2-fold higher than that in ga1-6 (Fig. 2B). 214
Our previous study showed that a two-fold reduction in GA4 levels in the Arabidopsis 215
rosette leaves could lead to a two-fold reduction in the final plant height (Mitchum et al., 216
2006). 217
Consistent with the elevated GA levels in erf11-1D ga1-6, the amounts of RGA 218
protein (an Arabidopsis DELLA) in the internodes of erf11-1D ga1-6 were much lower 219
than in ga1-6 (Fig 2C), even though RGA mRNA levels were not altered by erf11-1D 220
(Fig. 2D). Taken together, overexpression of ERF11 in erf11-1D ga1-6 caused elevated 221
bioactive GA4 levels through up-regulation of GA biosynthesis genes and down-222
regulation of a GA catabolism gene, which subsequently led to DELLA degradation and 223
internode growth. 224
Being an EAR-containing transcription repressor, ERF11 is unlikely to up-regulate 225
expression of GA biosynthesis genes directly. Instead, the elevated GA levels and longer 226
internodes of erf11-1D ga1-6 may be due to reduced ethylene levels because ERF11 is 227
known to inhibit ethylene biosynthesis by down-regulating ACS2 and ACS5 transcription 228
(Li et al., 2011) and enhanced ethylene signaling decreases bioactive GA levels in 229
Arabidopsis rosette plants (Achard et al., 2007). To test this possibility, the transcript 230
levels of ACS2 in the internodes of erf11-1D ga1-6 and ga1-6 were analyzed by RT-231
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qPCR. As predicted, expression of ACS2 was approximately 5-fold lower in the 232
internodes of erf11-1D ga1-6 than in ga1-6 (Fig 2E). Moreover, the ethylene production 233
in internodes of erf11-1D ga1-6 was 60% lower than in ga1-6 (Fig 2F). Consistently, 234
erf11-1D displayed reduced ethylene response (Fig. S1F), whereas erf11 and erf4 erf11 235
showed increased ethylene response (Fig. S1G). These results indicated that the taller 236
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phenotype caused by overexpression of ERF11 is due to elevated bioactive GA4 levels 237
and decreased ethylene levels. 238
239
ERF11 Positively Regulates GA Responses 240
The above data indicated that ERF11 promotes bioactive GA accumulation. 241
Interestingly, our hypocotyl elongation assays showed that ERF11 also enhances GA 242
response (Fig. 3 and Supplemental Fig. 2A). The erf11 single mutant displayed slightly 243
reduced GA response compared to WT, and the erf11 erf4 double mutant showed a 244
further reduction in GA response (Figs. 3A and 3C). Consistent with these results, 245
overexpression of ERF11 (due to erf11-1D) caused an elevated GA response; both the 246
double mutant erf11-1D ga1-6 and the single erf11-1D mutant displayed increased GA 247
response compared to ga1-6 and Col-0, respectively (Figs. 3B, 3D and Supplemental Fig. 248
2A). We also examined RGA protein levels in seedlings of ga1-6 and erf11-1D ga1-6 in 249
response to GA treatments. Supplemental Figure 2B shows that RGA levels decreased in 250
both lines in response to GA treatments, although RGA accumulated to lower levels in 251
erf11-1D ga1-6 than in ga1-6 when untreated or with 0.01 µM GA4. This is consistent 252
with the elevated GA content in erf11-1D ga1-6. These results indicate that ERF11 253
functions as a positive regulator of both GA production and GA signaling. 254
255
ERF11 Antagonizes DELLA Function via Protein-Protein Interaction 256
To place ERF11 in the GA signaling pathway, genetic interactions between ERF11 257
and RGA were examined by double mutant analysis. rga-
Δ
17 is a transgenic line that 258
expresses a dominant active form of RGA, which lacks a 17 amino-acid motif within the 259
DELLA domain that is required for GA-induced degradation (Dill et al., 2001). rga-
Δ
17 260
displays a severe dwarf phenotype, while erf11-1D has longer internodes than WT. The 261
use of rga-
∆
17 in the double mutant analysis allowed us to uncouple the GA-response 262
activity from GA biosynthesis so that we could examine the direct role of ERF11 in the 263
GA response. Because homozygous rga-
Δ
17 plants are sterile and grow extremely 264
slowly, we compared phenotypes of the semi-dwarf hemizygous rga-
Δ
17 in the 265
homozygous erf11-1D background (referred to as erf11-1D rga-
Δ
17) to the hemizygous 266
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rga-
Δ
17. We found that the final height of erf11-1D rga-
Δ
17 was 30% taller than rga-267
Δ
17 (Fig. 4A and Supplemental Fig. 3A), and the internode length of erf11-1D rga-
Δ
17 268
was 46% longer than that of rga-
Δ
17 (Supplemental Fig 3B), whereas the average 269
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number of siliques of erf11-1D rga-
Δ
17 was similar to that of erf11-1D (Supplemental 270
Fig. 3C). These results indicated that erf11-1D partially rescued the dwarf phenotype of 271
rga-
Δ
17, suggesting that erf11-1D either inhibits rga-Δ17 protein accumulation or 272
activity, or acts downstream of RGA. However, rga-Δ17 protein levels in the internodes 273
of rga-
Δ
17 and erf11-1D rga-
Δ
17 were similar (Fig 4B), indicating that the longer 274
internodes in erf11-1D rga-
Δ
17 were not caused by reduced rga-Δ17 accumulation. 275
We then tested whether erf11-1D inhibits RGA activity by direct protein-protein 276
interaction, a known regulatory mechanism for DELLA and its interactors (Xu et al., 277
2014; Daviere and Achard, 2015). Our yeast two-hybrid assays showed that the GRAS 278
domain of RGA directly interacts with ERF11 minus the EAR motif (Fig. 4C). No 279
interaction was detected between RGA and the full-length ERF11, presumably because 280
the transcription repression mediated by the EAR motif interfered with reporter gene 281
expression. We also observed interactions between RGA and three ERF11 close 282
homologues ERF4, ERF8 and ERF10 (in subgroup VIII-B-1a). However, RGA did not 283
interact with ERF88, which belongs to a different subgroup VIII-B-1b (Fig. 4C), 284
suggesting that RGA specifically interacts with ERFs in the VIII-B-1a subfamily. RGA 285
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and GAI are the major DELLA proteins that control stem elongation (Dill and Sun, 2001; 286
King et al., 2001). We found that GAI also interacted with ERF11, ERF4, ERF8 and 287
ERF10 in yeast two-hybrid assays (Supplemental Fig. 4). 288
To confirm ERF11-RGA interaction in planta, we performed co-289
immunoprecipitation (co-IP) assays by transiently co-expressing 35S:cMyc-ERF11 (or 290
ERF8) and 35S:HA-RGA constructs in leaves of Nicotiana benthamiana through 291
Agrobacterium-mediated transformation. Tissues infiltrated with 35S:HA-RGA alone or 292
co-infiltrated with 35S:cMyc-GUS-NLS served as negative controls. Immunoprecipitation 293
was performed using anti-cMyc antibody-conjugated agarose beads. Fig. 4D shows that 294
HA-RGA was co-immunoprecipitated when it was co-expressed with cMyc-ERF11 (–295
EAR) or cMyc-ERF8 (–EAR), but not when it was expressed alone or co-expressed with 296
cMyc-GUS-NLS. These co-IP assays further support the idea that ERF11 and its close 297
homologs directly interact with RGA. To test whether overexpression of ERF11 inhibits 298
DELLA function, we then examined transcript levels of DELLA target genes (Zentella et 299
al., 2007) in rga-
Δ
17 and erf11-1D rga-
Δ
17 by RT-qPCR analysis (Fig. 4E). We found 300
that expression of three of the DELLA target genes (bHLH137, bHLH154 and Exp-PT1) 301
was down-regulated in the internodes of erf11-1D rga-
Δ
17, suggesting that ERF11 and 302
DELLA interfere with each other's function by direct protein-protein interaction. 303
To examine further the direct antagonistic interaction between ERF11 and DELLA, 304
these proteins were expressed alone or co-expressed in tobacco leaves by agroinfiltration 305
to test whether they antagonistically modulate transcription of bHLLH137 and bHLH154 306
promoters using the dual luciferase (LUC) reporter assay (Fig. 5). The reporter constructs 307
contain promoter sequences of bHLH137, bHLH154 and SCL3 genes, respectively, which 308
were fused to the firefly LUC gene (fLUC) (Fig. 5A). The SCL3 promoter was included 309
as a control; SCL3 is an RGA-induced target gene (Zentella et al., 2007), but its 310
expression was not altered by erf11-1D (Fig. 4E). The 35S:Renilla LUC (rLUC) was used 311
as an internal standard in the assay. Two effectors are 35S:HA-RGA and 35S:myc-ERF11. 312
As expected, expression of RGA alone induced all three promoters of RGA target genes 313
(4-fold for bHLH137, 3-fold for bHLH154 and 8-fold for SCL3) compared to the empty 314
effector control (Fig. 5B). ERF11 alone repressed expression of bHLH137 by 5-fold, and 315
co-expression of RGA and ERF11 displayed antagonistic effects on the expression of this 316
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promoter (Fig. 5B). In contrast, ERF11 did not significantly repress expression of SCL3, 317
indicating that the inhibitory effect of ERF11 on the bHLH137 promoter is specific. We 318
also found that bHLH154 expression was not affected by ERF11 in the dual luciferase 319
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assay (Fig. 5B), suggesting that this gene may not be a direct target of ERF11. The 320
optimal cis-element for ERFs has been identified to be the GCC box (AGCCGCC), 321
although AtERF3 and AtERF4 can also bind similar sequences with single nucleotide 322
substitutions (Ohme-Takagi and Shinshi, 1995; Fujimoto et al., 2000). Interestingly, 323
using pDRAW32 DNA analysis software (http://www.acaclone.com), we found that the 324
bHLH137 promoter contains 2 modified GCC elements (Fig. 5A), whereas the promoters 325
of bHLH154 and SCL3 genes lack any putative ERF binding sequences. Taken together, 326
our results show that ERF11 directly represses RGA-induced bHLH137 expression. 327
328
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DISCUSSION 330
331
The data in this report revealed that ERF11 enhances GA responses by two 332
mechanisms: (1) increasing bioactive GA levels by inducing expression of the GA 333
biosynthesis genes GA3ox and GA20ox; (2) promoting GA responses by antagonizing the 334
activity of the GA signaling repressor DELLA via direct protein-protein interaction (Fig. 335
6). Our transient expression assay showed that ERF11 represses whereas DELLA induces 336
transcription of the target gene bHLH137 (Fig. 5), indicating that ERF11 and DELLA 337
interfere with each other's function. The second mechanism should reduce DELLA 338
function immediately, whereas the first mechanism acts slower in regulating DELLA 339
activity as its effect on DELLA is via alteration in GA biosynthesis. ERF11 is unique in 340
that it promotes both GA biosynthesis and GA signaling. All of the previously reported 341
elevated GA-signaling mutants down-regulate GA biosynthesis via the negative feedback 342
mechanism (Sun and Gubler, 2004). Our data further suggest that induction of GA 343
biosynthesis gene expression by ERF11 is likely an indirect effect mediated by 344
decreasing ethylene levels (Fig. 6). erf11-1D conferred a reduction in the amounts of 345
ethylene in the inflorescence stems, consistent with a recent report showing ERF11 346
represses ACS transcription (Li et al., 2011). The exact molecular mechanism of how 347
ethylene inhibits GA biosynthesis gene expression requires further investigation. 348
349
In Arabidopsis, there are 122 ERF/AP2 family members. AtERF11 belongs to the 350
subfamily VIII-B-1a (McGrath et al., 2005; Nakano et al., 2006). All eight members in 351
this subfamily (ERF3, 4, 7-12) contain a transcription repressor EAR motif near their C-352
terminus. Interestingly, our study and previous reports reveal that three of the EAR-353
containing ERFs (ERF4, ERF7 and ERF11) regulate multiple hormone pathways. 354
Overexpression of AtERF4 confers reduced sensitivity to ethylene, abscisic acid (ABA) 355
and JA in hypocotyl or root growth, whereas the loss-of-function erf4-1 mutant displays 356
increased JA response (McGrath et al., 2005; Yang et al., 2005). Overexpression or 357
silencing of AtERF7 resulted in reduced or increased ABA response in stomatal closure, 358
respectively, indicating that AtERF7 negatively regulates ABA responses (Song et al., 359
2005). Our data showed that overexpression of ERF11, ERF4 or ERF8 partially rescued 360
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21
the dwarf phenotype of the GA-deficient ga1-6 mutant. Phenotype analyses of the loss-361
of-function erf11 and erf4 single and double mutants further confirmed that ERF4 and 362
ERF11 positively regulate GA response in hypocotyl elongation. Similar to ERF11, 363
ERF4, 8 and 10 interact with DELLA in co-IP and/or yeast two-hybrid assays. Future 364
studies will determine whether all eight VIII-B-1a subfamily ERFs regulate GA and/or 365
other hormone pathways by a similar mechanism as illustrated for ERF11. Our yeast two-366
hybrid results indicate that RGA does not interact with ERF88 in the VIII-B-1b 367
subfamily. Interestingly, a group-VII ERF/AP2 (RELATED TO APETALA2.3) was 368
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22
shown recently to be a DELLA-interacting protein, playing a role in GA and ethylene-369
regulated apical hook development (Marin-de la Rosa et al., 2014). Future studies will 370
address whether any of the other ERF/AP2 subfamily members are DELLA-interacting 371
proteins. 372
373
In rice, group VII ERFs (OsSUB1A, SNORKEL1 and SNORKEL2) that lack the 374
EAR motif, have been shown to regulate internode elongation that is modulated by 375
ethylene and GA (Xu et al., 2006; Hattori et al., 2009). Interestingly, these OsERFs 376
(transcription activators) function differently from AtERF11. In submergence-tolerant 377
rice, ethylene inhibits internode growth under submergence conditions. In this case, 378
ethylene induces OsSUB1A expression (Xu et al., 2006), which in turn inhibits GA 379
response by promoting SLR1 (rice DELLA) and SLRL1 transcript and protein 380
accumulation (Fukao and Bailey-Serres, 2008). Ectopic expression of OsSUB1A in 381
Arabidopsis leads to reduced GA response and increased ABA response (Fukao et al., 382
2011). In contrast to the submergence-tolerant rice, deepwater rice responds to 383
submergence by rapid internode growth. In this case, ethylene promotes internode 384
elongation by increasing GA levels via induction of SNORKEL1 and SNORKEL2 385
expression, although it is unclear how SNORKEL1 and SNORKEL2 promote GA 386
accumulation (Hattori et al., 2009). Therefore, different members of the group VII ERFs 387
play distinct roles in mediating ethylene and GA responses in controlling internode 388
elongation. 389
390
In summary, increasing numbers of ERFs have been shown to regulate plant 391
development in response to hormonal signals or abiotic stresses. Our work reveals that 392
AtERF11 promotes internode elongation by promoting both GA biosynthesis and 393
signaling pathways. 394
395
396
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METHODS 398
399
Plant Materials and Growth Conditions 400
The ga1-6 semi-dwarf mutant plant used for activation tagging was generated by 401
crossing the original ga1-6 in the Landsberg erecta (Ler) background (Koornneef and 402
van der Veen, 1980; Sun and Kamiya, 1994) four times into Columbia-0 (Col-0). The 403
activation-tagging mutant pools were generated by transforming ga1-6 (4x Col-0) with 404
pSKI015 (Weigel et al., 2000), and the mutant #279-2 was identified in the T1 generation 405
as a BASTA-resistant plant that was taller than the parental plant. #279-2 displayed a 3:1 406
segregation ratio of the BASTA resistance in the T2 generation; homozygous lines were 407
identified by screening in the T3 generation (renamed erf11-1D ga1-6). The erf11 408
(SALK_116053) T-DNA insertion mutant was requested from the Arabidopsis Stock 409
Center, and the erf4-1 (Salk_073394) mutant was provided by Dr. Kemal Kazan 410
(McGrath et al., 2005). The homozygous double mutant erf4 erf11 was generated by 411
crossing erf11 to erf4. The erf11-1D rga-∆17 double mutant was generated by crossing a 412
rga-∆17 transgenic line (#18-2-1) in the Col-0 background (Dill et al., 2001) to erf11-1D. 413
Genotyping primers are listed in Supplemental Table 2. 414
For growth on media, seeds were plated on 1x or 0.5x Murashige and Skoog (MS) 415
medium containing 2% or 1% sucrose and 0.7% agar, and incubated at 22°C under 416
constant light (50-70 µmol m-2 sec-1). For growth on soil, seeds were sown on MetroMix 417
200 (Sun Gro Horticulture, Bellevue, WA), and incubated at 22°C under 16h light. The 418
procedure for the hypocotyl elongation assay was described previously (Zhang et al., 419
2011). For ethylene-mediated seedling triple-response assay, the detailed procedure was 420
described before (Zhou et al., 2007). Seeds were stratified at 4°C for 72 h, and then 421
germinated on half strength MS at 22°C for 80 h in the dark with or without 20 µL/L 422
ethylene gas. Dark-grown seedlings were photographed and hypocotyl length was 423
measured using software Image J. Transgenic Arabidopsis lines were generated by the 424
floral dip method (Clough and Bent, 1998). For selection, 10 μg/mL of BASTA or 50 425
μg/mL kanamycin was included in the MS medium. 426
All statistical analyses were performed using Student’s t-tests with the statistical 427
package JMP Pro 10.0.2 (SAS Institute Inc., Cary, NC). 428
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429
Plasmid Constructs 430
All the primers used in this study are listed in Supplemental Table 2. The PCR-431
amplified fragments in all constructs were sequenced to ensure that no mutations were 432
introduced. Detailed information on plasmid construction is described in Supplemental 433
Methods. 434
435
TAIL-PCR 436
Genomic DNA was extracted by using the CTAB method (Weigel and Glazebrook, 437
2002). TAIL-PCR was carried out as described previously (Liu et al., 1995). The 438
degenerate primers used for TAIL-PCR were AD1, AD2, and AD3 (Liu and Whittier, 439
1995) and AD4 and AD6 (Liu et al., 1995). The T-DNA specific primers LB8, LB7, and 440
JL-202 (Alonso et al., 2003) were used in the primary, secondary, and tertiary TAIL-PCR 441
reactions, respectively. Specific TAIL-PCR products were gel-purified and sequenced 442
and BLAST searched against the National Center for Biotechnology Information (NCBI) 443
database to identify the T-DNA insertion site. 444
445
Quantitative Real Time RT-PCR Analysis 446
The real-time RT-qPCR analyses were performed with an Eppendorf realplex2 447
Mastercycler ep gradient S (Eppendorf, Hamburg, Germany). Total RNA isolation, 448
cDNA synthesis and qPCR analyses were performed as described previously (Zentella et 449
al., 2007). At4g33380 whose expression remains constant (Rieu et al., 2008), was used as 450
the control to normalize the qPCR data. Unless specified otherwise, the qPCR data are 451
the means of 4 repeats (2 biological repeats and 2 technical replicates of each set of 452
samples). 453
454
GA Measurements 455
Rosette leaves of 33d-old soil-grown ga1-6 and erf11-1D ga1-6 plants were 456
harvested and immediately frozen in liquid nitrogen and lyophilized. GAs were purified 457
and quantified according to (Plackett et al., 2012) by using a 6410 Triple Quad LCMS 458
(Agilent Technologies, Santa Clara, CA, USA) with an Agilent 1200 series rapid 459
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26
resolution liquid chromatography system fitted with a ZORBAX SB-Phenyl column (1.8 460
µm, 2.1 x 50 mm). 461
462
Immunoblot Analyses 463
Total proteins from seedlings or internodes were extracted as described before 464
(Silverstone et al., 2001). The extracted proteins were fractionated by 8% SDS-PAGE 465
and analyzed by immunoblot analysis using anti-HA antibodies (Covance, Philadelphia, 466
PA), or crude anti-RGA antibodies (Silverstone et al., 2001). 467
468
Yeast Two-Hybrid Assays 469
The yeast two-hybrid assay was performed using the ProQuest system (Invitrogen) 470
in the yeast strain pJ69-4A (James et al., 1996). Varying concentrations of 3-AT (0, 1, 471
2.5, 5, 10, and 25 mM) were included in medium lacking Trp, Leu, and His. For each 472
combination, 2 μL yeast cells with OD600 values of 0.25 and 0.025, respectively, were 473
spotted on media plates. 474
475
Co-immunoprecipitation of ERFs and RGA, and Dual Luciferase Assay 476
Transient expression and co-immunoprecipitation (co-IP) assays were performed as 477
described previously (Zhang et al., 2011) with the following modifications: After cells 478
were resuspended in infiltration media, Agrobacterium GV3101 strains carrying 479
individual expression constructs were mixed to make a final OD600 of about 0.8 for each 480
strain and infiltrated into 28-d-old Nicotiana benthamiana (tobacco) leaves by needle-less 481
syringe; 40 h transiently transformed tobacco leaves were harvested for co-IP assay 482
without crosslinking. Two grams leaves were ground into a fine powder in liquid 483
nitrogen, followed by resuspension in 5 mL extraction buffer [50 mM Tris pH 7.5, 150 484
mM NaCl, 1% Triton X-100, 5 mM β-mercaptoethanol, 1xProtease Inhibitors (Sigma P-485
9599)]. Keep grinding the powder in extraction buffer for 10 min on ice, until there is no 486
visible debris. The homogenates were centrifuged at 20,000 x g at 4o C for 20 min. co-IP 487
was performed using 10 µL anti-cMyc agarose-conjugated beads (A7470; Sigma) by 488
incubating with supernatant for 2 h at 4⁰C. 489
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The dual luciferase assays were also performed using the transient expression 490
system in tobacco. The control constructs, and the reporter and effector constructs were 491
individually transformed into agrobacterium strain GV3101. Then each reporter- and 492
rLUC-containing strains were co-infiltrated into tobacco leaves with various 493
combinations of effector strains. Forty hr after infiltration, tobacco leaves were harvested 494
for protein extraction, and luciferase activities were measured using the dual-luciferase 495
reporter assay system (Promega). Relative promoter activity was calculated as the ratio of 496
fLUC to rLUC activities for each sample. Six biological repeats were conducted for each 497
effector combination. 498
499
Ethylene Measurement 500
The top 10 internodes of the main stems were detached, and the flower clusters and 501
siliques were removed. For each set of measurements, 15 stems (per genotype) were 502
placed in a 22-mL gas chromatography vials containing 0.5 mL of 0.5x liquid MS media. 503
The vials were capped and then placed in a 16h/8h light/dark cycle incubator at 22°C for 504
5h. The accumulated ethylene was measured and calculated based on comparison to a 1 505
mL/L ethylene standard (Woeste et al., 1999). Data were from three replicates and each 506
experiment was repeated at least once with comparable results. 507
508
Cell Length Measurement by Scanning Electron Microscopy 509
The 10th-20th internodes from the bottom on the main stem of 70d-old ga1-6 and 510
erf11-1D ga1-6 plants were fixed overnight with FAA solution (5% acetic acid, 45% 511
ethanol, and 5% formaldehyde) and dehydrated with a graded ethanol series (30%, 50%, 512
70%, 90%, 100%) (Tsukaya et al., 1993). After dehydration, the samples were chemically 513
dried with HMDS (hexamethyldisilazane), and then were imaged on an FEI XL30 ESEM 514
(FEI, Oregon, USA). 515
Accession Numbers 516
Arabidopsis Genome Initiative locus identifiers for the genes mentioned in this 517
article are as follows: ERF11 (At1g28370), ERF4 (At3g15210), ERF8 (At1g53170), 518
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ERF10 (At1g03800), ERF12 (At1g28360), ERF88 (At1g12890), ACS2 (At1g01480), 519
GA1 (At4g02780), RGA (At2g01570), GAI (At1g14920), GA3ox1 (At1g15550), GA3ox2 520
(At1g80340), GA20ox1 (At4g25420), GA20ox2 (At5g51810), GA2ox6 (At1g02400), 521
AtGID1A (At3g05120), MYB (At3g11280), bHLH137 (At5g50915), bHLH154 522
(At2g31730), WRKY27 (At5g52830), SCL3 (At1g50420), EXP-PT1 (At2g45900), 523
XERICO (At2g04240), RING (At4g19700), EXP-PT (At4g33380), and unknown protein 524
(At1g28375). 525
526
Supplemental Data 527
The following materials are available. 528
529
Supplemental Methods, Figure Legends and References. 530
Supplemental Figure 1. Characterization of ERF Overexpression Lines and erf Loss-Of-531
Function Mutants. 532
Supplemental Figure 2. erf11-1D Caused an Elevated GA Response and Reduced 533
Levels of RGA Protein. 534
Supplemental Figure 3. erf11-1D Partially Rescued rga-∆17 Phenotypes. 535
Supplemental Figure 4. Interactions between ERFs and GAI in Yeast Two-Hybrid 536
Assays. 537
Supplemental Table 1. Silique and Flowering Time Phenotypes of ga1-6 and erf11-1D 538
ga1-6 539
Supplemental Table 2. List of Primers and Their Uses. 540
541
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29
ACKNOWLEDGMENTS 542
We are grateful to Joe Kieber and Gyeong Mee Yoon for their advice with 543
ethylene analyses, and for providing the facility for ethylene measurements. We thank 544
Benke Kuai for his generous support of some of the ethylene-related studies, and Tomoe 545
Nose and Yumiko Takebayashi for technical support in LC-MS/MS hormone analysis. 546
We also thank Kemal Kazan for erf4-1, Jian-Min Zhou for ein3 and EIN3-OE lines, Chi-547
Kuang Wen for helpful discussions, and Michelle Gignac at the SEM facility at Duke for 548
help with SEM analysis. 549
550
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30
FIGURE LEGENDS 551
Figure 1. Overexpression of ERF11 Increased Plant Height by Promoting Cell 552
Elongation. 553
(A) The tall phenotype of mutant #279-2 (renamed erf11-1D ga1-6) was recapitulated by 554
expressing 35S:HA-ERF11-GFP (ERF11-OE) in the ga1-6 background. Plant image was 555
taken 50d after planting. #1-1, #1-2 and #2-8 are 3 independent ERF11-OE ga1-6 556
transgenic lines (T2 generation). 557
(B) The final height of erf11-1D ga1-6 is taller than ga1-6. n=24. 558
(C) The ERF11 transcript levels are significantly higher in erf11-1D ga1-6 than in ga1-6. 559
Data represent RT-qPCR results using RNA isolated from 8d-old seedlings. A GA non-560
responsive gene (At4g33380) was used to normalize different samples. Means ± SE of 4 561
repeats (from 2 biological replicas, 2 technical repeats each) are shown. The level in ga1-562
6 was set to 1. 563
(D) HA-ERF11-GFP protein levels correlated with the plant heights in (A). The 564
immunoblot contains proteins from 8d-old seedlings of three transgenic ERF11-OE ga1-6 565
lines as described in (A), and was probed with an anti-HA antibody. The bottom panel 566
shows equal loading by Ponceau staining. 567
(E) and (F) Internodes in erf11-1D ga1-6 were longer than in ga1-6. Average internode 568
lengths of 70d-old ga1-6 and erf11-1D ga1-6 were calculated by dividing the length of 569
the primary stem from the apex to the last secondary inflorescence with the total numbers 570
of nodes. n=24. 571
(G) and (H) The average epidermal cell length in the primary stem of erf11-1D ga1-6 is 572
longer than in ga1-6. In (G), n≥600. In (H), scanning electron microscope (SEM) 573
images of the internodes of 70d-old plants. A representative cell in each line was 574
outlined. 575
(I) and (J) erf11-1D ga1-6 and ERF11-OE ga1-6 double homozygous mutants have larger 576
rosette leaves than ga1-6. Images in (I) showed 33d-old plants. In (J), n=20. 577
(K) and (L) The final heights (K) and internode lengths (L) of erf11 and erf4 erf11 578
mutants are shorter than those of the WT. n=24. 579
In (B-C), (F-G), (J-L), data are means ± SE. * p<0.05; ** p<0.01. 580
581
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31
Figure 2. Elevated Bioactive GA Levels and Reduced Ethylene Levels in erf11-1D ga1-582
6. 583
(A) Relative mRNA levels of GA biosynthesis genes (left panel) and GA catabolism gene 584
(right panel) in the internodes of ga1-6 and erf11-1D ga1-6. Data represents means ± SE 585
of 4 repeats. **p<0.01. The levels in ga1-6 were set to 1. 586
(B) GA4 levels (ng/g dry weight) in erf11-1D ga1-6 were elevated comparing to ga1-6. 587
GA4 contents in rosette leaves of 33d-old plants were analyzed using two biological 588
repeats. Due to very low levels of bioactive GA4 content in the ga1-6 background, we 589
were unable to measure GA4 accurately in one of the ga1-6 repeats (test 2), which had 590
trace amounts of GA4 estimated to be < 0.27 ng/g dry weight 591
(C) RGA protein levels were reduced in the internodes of erf11-1D ga1-6 compared to 592
ga1-6. Proteins were extracted from internodes of 50-d-old plants, and immunoblotting 593
was performed with affinity-purified anti-RGA antibodies. The bottom panel shows equal 594
loading by Ponceau staining. 595
(D) The RGA transcript levels were similar in the internodes of ga1-6 and erf11-1D ga1-596
6. Data represents means ± SE of 4 repeats. 597
(E) The ACS2 transcript levels in the internodes of erf11-1D ga1-6 were reduced in 598
comparison to ga1-6. Data represents means ± SE of 4 repeats. **p<0.01. 599
(F) Ethylene production (picoL/mg dry weight/h) from the internodes of 50d-old plants. 600
Data represent means ± SE of 4 repeats. Similar results were obtained using another set 601
of samples. **p<0.01. 602
603
Figure 3. Loss-Of-Function erf Mutants Displayed Reduced GA Response, Whereas 604
Overexpression of ERF11 increased GA Response. 605
(A) and (B) Hypocotyl elongation assays of 4d-old seedlings grown in different GA 606
concentrations under continuous light (50 µmol m-2 sec-1). * p<0.05; ** p<0.01. 607
(C) erf11 and erf11 erf4 displayed shorter hypocotyl length in the presence of 0.05 µM 608
GA4, but not in the untreated control (– GA). Image shows 4d-old seedlings. 609
(D) erf11-1D ga1-6 showed longer hypocotyl length with 0.05 and 1µM GA4 treatment, 610
but not in the untreated control. Image showed 4d-old seedlings. 611
612
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32
Figure 4. ERF11 Interacts with RGA and Inhibits Its Function. 613
(A) erf11-1D partially rescued the dwarf phenotype of rga-∆17. Image showed 60d-old 614
plants. 615
(B) Similar rga-∆17 protein levels were present in the upper growing internodes of 70d-616
old rga-∆17 and erf11-1D rga-∆17. The immunoblot was probed with affinity-purified 617
anti-RGA antibodies. The bottom panel shows equal loading by Ponceau staining. 618
(C) Interactions between ERFs and RGA in yeast two-hybrid assays. A truncated RGA 619
protein (aa187-aa587) that contains the C-terminal GRAS domain was used as the bait. 620
ERFs were used as the prey. FL, full-length; –EAR, EAR motif deleted. For each strain, 2 621
μL yeast cells with OD600 values of 0.25 and 0.025 were spotted on control media (+ 622
His) and – His media +1mM 3-AT (a competitive inhibitor of His3 enzyme). Empty prey 623
and bait vectors were included as negative controls. 624
(D) co-IP of ERFs and RGA in planta. HA-RGA was transiently expressed alone or co-625
expressed with cMyc-tagged GUS, ERF11 (–EAR) or ERF8 (–EAR) in N. benthamiana. 626
The total protein extracts were immunoprecipitated with anti-cMyc antibody-conjugated 627
agarose beads; the input and IP samples were analyzed by immunoblotting using 628
antibodies for HA and cMyc, separately. 629
(E) erf11-1D reduced the transcript levels of DELLA target genes bHLH137, bHLH154 630
and Exp-PT1 in the internodes of 70d-old rga-∆17 plants. RT-qPCR data represents 631
means ± SE of 4 repeats. ** p<0.01. 632
In (A, B and E), rga-∆17 is hemizygous in both rga-∆17 and erf11-1D rga-∆17.
633
634
Figure 5. ERF11 Repressed, whereas RGA Induced Transcription of the bHLH137 635
Promoter in Tobacco Transient Expression Assay by Agro-Infiltration. 636
(A) Schematics of the normalization control, reporter and effector constructs. 35S:Renilla 637
LUC (rLUC) served to normalize transformation efficiency. In the reporter constructs, the 638
firefly LUC gene (fLUC) was placed under the control of different promoters of RGA 639
target genes (bHLH137, bHLH154 and SCL3). 35S:RGA and 35S:ERF11 served as two 640
effector constructs, respectively. The positions of 2 modified GCC box sequences in 641
bHLLH137 promoter are labeled by asterisks: AGCCGCT at –2 kb and ACCCGCC at 642
–0.2 kb. The empty effector vector was used as a negative control. (B) RGA induced 643
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33
expression of all 3 target gene promoters, whereas ERF11 only repressed bHLH137 644
expression. Each reporter construct and the 35S:rLUC construct were introduced into 645
tobacco leaves in the presence of the empty effector constructs (Control) or 35S:RGA 646
and/or 35S:ERF11 (with the same molar ratios) by agro-infiltration. The relative fLUC 647
activity (normalized by rLUC activity) in the empty effector control was set to 1. Data 648
represent the average value ± SE of 8 biological replicas. Different letters above the bars 649
indicate significant difference (P < 0.01). 650
651
Figure. 6. Model for Antagonistic Interaction between ERF11 and DELLA in Regulating 652
GA and Ethylene Pathways and Internode Elongation. 653
The arrows and T-bars highlighted in blue represent new links that are revealed in this 654
study. ERF11 promotes internode elongation by enhancing GA responses through two 655
mechanisms: (1) increasing GA accumulation (and therefore DELLA degradation) 656
indirectly via its inhibitory effect on ethylene biosynthesis; (2) inhibiting DELLA 657
function by direct protein-protein interaction. DELLA and ERF11 antagonize each other's 658
function in regulating downstream gene expression, as shown in the transient expression 659
assay. In (1), ERF11-induced GA accumulation is likely an indirect effect of reduction of 660
ethylene production via inhibition of ACS2 expression by ERF11. The reduced ethylene 661
levels then result in repression of GA3ox and GA20ox expression, likely through the 662
ethylene signaling pathway. 663
664
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