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RESEARCH ARTICLE 1 2 Mechanism of auxin and GA crosstalk in diploid strawberry fruit initiation 3 4 Junhui Zhou, John Sittmann, Lei Guo, Xiaolong Huang, Anuhya Pulapaka and Zhongchi 5 Liua 6 7 Dept. of Cell Biology and Molecular Genetics 8 University of Maryland 9 College Park, MD 20742 10 USA 11 12 aCorresponding author: [email protected] 13 ORCID: 0000-0001-9969-9381 14 15
Short title: Mechanism of strawberry fruit initiation 16
One sentence summary: Genome editing and network analysis are applied to 17 investigating the mechanism of accessory fruit initiation in the wild strawberry, revealing 18 conserved as well as novel crosstalk mechanisms. 19
The author responsible for distribution of materials integral to the findings presented in 20 this article in accordance with the policy described in the Instructions for Authors 21 (www.plantcell.org) is: Zhongchi Liu ([email protected]) 22 23 24 25 Abstract 26 Strawberry, a high value fruit crop, has recently become amenable for genetic studies due 27 to genomic resources and CRISPR/CAS9 tools. Unlike ovary-derived botanical fruits, 28 strawberry is an accessory fruit derived from receptacle, the stem tip subtending floral 29 organs. Although both botanical and accessory fruits initiate development in response to 30 auxin and GA released from seeds, the downstream auxin and GA signaling mechanisms 31 underlying accessory fruit development remain unknown. Using wild strawberry, we 32 performed in depth molecular characterizations of accessory fruit development. We show 33 that auxin signaling proteins FveARF8/FveARF6 are bound and hence inhibited by 34 FveIAA4 and FveRGA1, repressors in auxin and GA signaling pathways. This inhibition 35 is relieved post-fertilization or by the application of GA or auxin. Mutants of FveRGA1 36 developed parthenocarpic fruit suggesting a conserved function of DELLA proteins in 37 fruit set. Further, FveARF8 was found to repress the expression of a GA receptor gene 38 GID1c to control fruit’s sensitivity to GA, revealing a novel crosstalk mechanism. We 39 demonstrate that consensus co-expression network provides a powerful tool for non-40 model species in the selection of interacting genes for functional studies. These findings 41 will facilitate the improvement of strawberry fruit productivity and quality by guiding 42 future production of parthenocarpic fruit. 43 44
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
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Introduction 45
Strawberry is a high-value fruit crop immensely important for agriculture and human 46
nutrition. Unlike most fruits developed from the ovary wall, strawberry fruit is derived 47
from receptacle, the stem tip supporting floral organs (Hollender et al., 2012). About 200 48
achenes, ovaries each with a single seed inside, dot the surface of the receptacle, making 49
them highly accessible for manipulation. Nitsch’s 1950 experiment showed that removal 50
of achenes from a strawberry receptacle prevented receptacle fruit enlargement. However, 51
exogenous application of auxin could substitute for the achenes and stimulated receptacle 52
fruit growth, indicating achenes as the source of auxin (Nitsch 1950). Later work showed 53
that emasculated wild strawberry flowers could still develop into fruit if auxin or GA, 54
alone or in combination, was supplied exogenously (Kang et al., 2013; Liao et al., 2018). 55
Transcriptome analyses and reporter gene expression confirm that fertilization-induced 56
biosynthesis of auxin and GA occurs in the embryo and endosperm (Kang et al., 2013; 57
Feng et al., 2019). The phytohormones are then transported to the receptacle to stimulate 58
fruit enlargement. Exactly how auxin and GA interact to coordinately regulate receptacle 59
fruit development is not known. 60
61
The fertilization-induced phytohormone synthesis for “fruit set”, the no return 62
commitment of the floral tissue to enter fruit pathway, is an evolutionarily conserved 63
strategy in angiosperms. The unique strawberry fruit structure as well as prior research 64
provide a strong foundation for further investigations into signal communication between 65
achenes and the receptacle and auxin/GA crosstalk during fruit development. This is 66
further aided by the rich genomic resources including genome sequencing of wild 67
strawberry Fragaria vesca and garden strawberry Fragaria x ananassa (Shulaev et al., 68
2011; Edger et al., 2018; Edger et al., 2019) as well as extensive transcriptome data and 69
network analyses during flower and fruit development (Kang et al., 2013; Hollender et al., 70
2014; Sanchez-Sevilla et al., 2017; Shahan et al., 2018). As garden strawberry is an allo-71
octoploid, wild strawberry F. vesca, a diploid species, is used in this study. 72
73
Prior research in model organisms illuminated auxin and GA signaling cascade. When 74
auxin is absent, IAAs bind to ARFs to prevent ARFs from activating or repressing 75
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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downstream target genes. When auxin is synthesized or supplied, it binds its receptors 76
TIR/AFBs, which are F-box proteins that recruit and add ubiquitins to the Aux/IAA 77
proteins. This leads to the 26S proteasome-mediated degradation of Aux/IAAs and the 78
release of ARFs to execute their functions (Leyser, 2018). Similarly, in the absence of 79
GA, DELLA repressor proteins accumulate and bind transcription factors to prevent their 80
function. When GA is produced, GA binds to its receptor GID1 to facilitate binding of 81
GID1 to DELLA that leads to ubiquitination and proteasome-mediated degradation of 82
DELLA. Transcription factors previously bound by DELLA are now free to act upon 83
downstream genes (Daviere and Achard, 2013). 84
85
In model systems Arabidopsis and tomato (both with botanical fruits), fertilization-86
independent fruit formation (parthenocarpy) could be induced when repressors of auxin 87
or GA signaling response are mutated or reduced as demonstrated by arf8 mutants in 88
Arabidopsis and transgenic knockdowns of tomato ARF7, ARF8, or IAA9 (Wang et al., 89
2005; Goetz et al., 2006; Goetz et al., 2007; de Jong et al., 2009; Wang et al., 2009). 90
These results led to the proposal that the IAA9/ARF7/ARF8 repressor complex inhibits 91
fruit set in the ovary tissue of Arabidopsis and tomato. To investigate the relationship 92
between auxin and GA, several studies were carried out in Arabidopsis, pea, and tomato 93
which showed that auxin acts upstream of GA by activating the biosynthesis of GA 94
(Serrani et al., 2008; Dorcey et al., 2009; Ozga et al., 2009; Fuentes et al., 2012). 95
Recently, direct protein-protein interactions between SlDELLA and SlARF7 and SlIAA9 96
were shown to serve as another mechanism mediating crosstalk between auxin and GA in 97
tomato fruit (Hu et al., 2018). 98
99
In contrast, there has been little functional studies of fruit set in accessory fruits including 100
strawberry. Previously, we employed RNA-seq to profile finely dissected strawberry fruit 101
tissues, embryo, ghost (seed coat + endosperm), ovary wall, and receptacle pith and 102
receptacle cortex, at five early fruit developmental stages (Kang et al., 2013). Based on 103
the RNA-seq data and subsequent reporter GUS studies (Feng et al., 2019), the 104
endosperm within the achene emerges as the site of fertilization-induced auxin and GA 105
biosynthesis targeted for fruit development. However, auxin and GA perception and 106
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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signaling genes, TIR/IAAs and GID1c/RGA1, are highly expressed in the receptacle, 107
indicating receptacle as the site of auxin and GA perception and response (Kang et al., 108
2013). Using the RNA-seq data described above, consensus co-expression network was 109
established (Shahan et al., 2018), which identifies robust co-expression modules in 110
receptacle fruit development. In addition, CRISPR/Cas9 genome editing has been 111
demonstrated in wild and garden strawberries (Zhou et al., 2018; Feng et al., 2019; 112
Martin-Pizarro et al., 2019), providing tools for dissecting gene function. 113
114
Here, we provide an in depth functional dissection of a few key auxin and GA signaling 115
components in the receptacle fruit development in diploid strawberry. We observed both 116
conserved and novel cross-talk mechanisms for GA and auxin signaling, identified 117
FveRGA1 as a key repressor of fruit set in strawberry, and validated the usefulness of 118
consensus co-expression network. The findings lay the foundation for future 119
improvement of strawberry fruit. 120
121
122
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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Results 123
arf8 mutants develop larger and rounder fruits 124
Previously, we reported successful genome editing of FveARF8, and the arf8 mutants 125
showed faster seedling growth (Zhou et al., 2018). However, arf8 adult plants are similar 126
to WT plants in stature. To determine the defect of arf8 mutants in flower and fruit 127
development, we first characterized four independent CRISPR lines of arf8, two lines are 128
arf8 (-2, -2), one line is arf8 (-2+1, -2+1), and one line is arf8 (-8, -23+5) (Figure S1). 129
Second, we established that fruits derived from primary and secondary flowers are 130
similar in size (Figure S2, allowing us to collect data from both primary and secondary 131
fruits. Third, to remove impact of environment on plant vigor and hence fruit size, the 132
height and width of fruits were normalized respectively to the height and width of petals 133
of the same flower. 134
135
arf8 mutant fruit is larger and rounder than wild type (Figure 1). Significant increase in 136
the width and the height of arf8-1 (-2, -2) fruit is found when compared with wild type 137
(Figure 1A, B); the higher increase of fruit width results in rounder shape in developing 138
fruit. The other three CRISPR-induced arf8 lines exhibited similar phenotype as arf8-1 139
with increased fruit height and width (Figure 1C). Hence, arf8-1 is used for the 140
subsequent analyses. In ripe fruit, the width remains larger in arf8-1 mutants, but the 141
height becomes similar to WT (Figure 1A, D) probably due to compensatory mechanisms 142
at late stages of fruit development. arf8 mutants also exhibited a mild floral phenotype; 143
they showed an increase in petal number (Figure S2C). While 80% wild type flowers 144
have 5 petals, only 40% arf8 mutants produce 5 petals and the remaining flowers produce 145
6 or 7 petals (Figure S2D). The average petal size however appears the same between 146
WT and arf8-1 mutants (Figure S2E). 147
148
Using the Arabidopsis Ubiquitin 10 promoter, FveARF8 was over-expressed in F. vesca; 149
the resulting transgenic lines (#1, #5, and #26), which exhibit a significantly higher 150
FveARF8 transcript level than the non-transgenic control, showed retarded seedling and 151
plant growth (Figure S3). Transgenic lines #1 and #26 showed a reduction of fruit size 152
when normalized to petal (Figure S3C, D), while transgenic line #4 with a wild type 153
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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expression level of FveARF8 exhibited normal plant statue (Figure S3A, B). Together, 154
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our data suggest that FveARF8 is a negative regulator of plant growth and fruit 155
enlargement. 156
157
Mechanism of auxin and GA signaling in fruit set 158
Fertilization-induced phytohormone synthesis and signaling are critical for “fruit set”, a 159
decision to proceed with fruit development upon successful fertilization (Gillaspy et al., 160
1993). In F. vesca, we previously showed that auxin and GA each substituted for 161
fertilization to stimulate fruit set, yet each hormone alone stimulated fruit development to 162
a size smaller than the pollinated control fruit (Kang et al., 2013). Only when both auxin 163
and GA were applied at the same time, did parthenocarpic strawberry develop to the 164
same size as the pollinated control, suggesting the requirement of both auxin and GA for 165
strawberry fruit set. Nevertheless, the molecular mechanism underlying this double 166
requirement is not known. 167
168
To test if FveARF8 could act to block fruit set pre-fertilization, we examined arf8-1 fruit 169
formation in the absence of fertilization. Wild type and arf8-1 mutant flowers were 170
emasculated to prevent fertilization. Fruit size at 0DPA (pre-fertilization) was compared 171
to fruit size at 7 DPA (post-emasculation) within the same genotype. No significant 172
change in fruit size was observed at 7DPA for arf8-1 as well as WT (Figure 2), 173
suggesting that fertilization-induced hormone production is still required for arf8 mutants 174
to develop fruit and that FveARF8 is unlikely involved in preventing fruit set. 175
176
During GA signaling, the GA-GID1 receptor complex targets the DELLA repressor 177
protein for proteasome-mediated degradation, resulting in the release of downstream 178
transcription factors (TFs) normally sequestered by the DELLA protein (Hartweck, 2008; 179
Sun, 2011). In F. vesca, gene06210 (FveRGA1) encodes one of the five annotated 180
probable DELLA proteins and is the only one with a full DELLA motif (Caruana et al., 181
2018). A loss-of-function mutation rga1-1 (also named srl-1) was identified in FveRGA1 182
during a chemical mutagenesis screen (Caruana et al., 2018) (Figure S1B). This mutant 183
offers an opportunity to determine if the DELLA repressor encoded by FveRGA1 184
regulates fruit set in strawberry. rga1-1 receptacle size was measured at Stage 1 (1DPA), 185
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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stage 4 (9DPA), and stage 5 (11-12 DPA). While the receptacle size difference between 186
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WT and rga1-1 is not obvious at 1DPA, the receptacle becomes significantly larger and 187
taller at post-fertilization stages 4-5 (9DPA and 11-12 DPA) (Figure 2C, D), suggesting 188
that constitutive GA response in the rga1-1 receptacle may underlie the larger fruit 189
phenotype. To determine if rga1-1 mutant could develop parthenocarpic fruit, 190
emasculated WT and rga1-1 flowers were marked and the receptacle size measured. 191
While WT fruit remains unchanged when measured at stage 1 (0DPA) and stage 3 192
(7DPA), rga1-1 mutants develop significantly larger receptacle at stage 3 (7DPA) when 193
compared with stage 1 (0DPA) (Figure 2E, F). The data suggests that FveRGA1 encodes 194
a key negative regulator of fruit set, perhaps by binding and inhibiting the activity of 195
unknown TFs. 196
197
To investigate if the increased fruit size in arf8 or rga1-1 is due to increased cell division 198
or cell expansion, histological sections were conducted. Histological sections of wild type, 199
arf8-1 and rga1-1 fruits at 7DPA reveal an increased number of cell files across the width 200
of the pith in arf8 and rga1-1 receptacle (Figure S4); WT (YW5AF7) has an average of 201
28 cell files, arf8-1 has an average of 40 cell files, and rga1-1 has an average of 46.5 cell 202
files across the width of the pith (Figure S4J). In addition, arf8-1 and rga1-1 appear to 203
have dense cell files near the vasculatures (Figure S4F, I). Thus, increased cell division 204
might have contributed to the increased fruit width in both arf8 and rga1-1 mutants. 205
206
FveRGA1, a key crosstalk node and regulator of fruit set 207
To investigate the mechanism of how auxin and GA coordinate their effect on fruit set 208
and fruit growth and explain distinct effects of arf8-1 and rga1-1 on parthenocarpy, we 209
investigated if FveRGA1 interacts with FveARF8 and FveARF6. FveARF6 was included 210
as it was previously shown in Arabidopsis to interact with and inhibited by RGA1(Oh et 211
al., 2014). In BiFC, FveRGA1 interacts positively with FveARF6 and FveARF8 (Figure 212
3). In Y2H, full length FveRGA1 self-activates, thus a N-terminal 564 bp deletion was 213
made leading to FveRGA1-C (similar to the M5 version of AtRGA in Arabidopsis). 214
Consistent with the BiFC result, FveRGA1-C interacts positively with FveARF6 and 215
FveARF8 (Figure 3B), suggesting FveRGA1 as a key crosstalk node for hormone 216
signaling. 217
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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218
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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To determine which domain of FveARF8 interacts with FveRGA1, FveARF8 is divided 219
into three segments: DBD (DNA-binding), MR (Middle Region), and PB1 (Figure S5). 220
While each domain of FveARF8 fails to interact with FveRGA1-C, the combined DBD-221
MR domains of FveARF8 positively interact with FveRGA1-C (Figure 3C). The work 222
also indicates that the C-terminal portion of FveRGA1 is sufficient for the interaction 223
with these TFs. Since rga1-1 is a W601X mutation that deletes the last 10 amino acids 224
from the C-terminal SAW domain (Figure S1B), we tested if removing the SAW domain 225
in FveRGA1-C affect the interaction with FveARF8 or FveARF6 in Y2H. FveRGA1-C (-226
SAW) failed to interact with FveARF8 and FveARF6 (Figure 3D). rga1-1 may thus fail 227
to inhibit fruit set due to its inability to bind and inhibit FveARF6, ARF8, and perhaps 228
other unidentified transcription factors. 229
230
The emerging theme from above studies is that FveRGA1 acts to prevent fruit set 231
(transition from stage 1 to stage 2). Fertilization induced GA production may remove 232
FveRGA1 through GA-GID1-induced degradation, permitting fruit set to occur 233
accompanied by the activation of a number of TFs. 234
235
FveARF8 represses the expression of GA receptor FveGID1c 236
Since arf8-1 fruit could not develop in the absence of fertilization (Figure 2A), the larger 237
fruit of arf8-1 post-fertilization (Figure 1A) may reflect an increased sensitivity or 238
enhanced response to fertilization-induced hormonal signals, namely auxin and/or GA. 239
To test this hypothesis, emasculated flowers of arf8-1 were treated with 1mM NAA (1-240
naphthaleneacetic acid) or 1mM GA3. Mock treated fruits of the same genotype served as 241
the control. The ratio of fruit size between hormone treated vs. mock-treated fruit at 242
7DPA was compared; the ratio between treated and mock for arf8-1 mutants is 243
significantly higher than that of WT under GA treatment as well as under auxin (NAA) 244
treatment (Figure 4). Therefore, arf8-1 mutant may exhibit higher sensitivity or response 245
to GA and NAA than wild type, suggesting that one normal function of FveARF8 is to 246
dampen the receptacle’s sensitivity or response to GA and NAA perhaps in a feedback 247
regulatory loop. 248
249
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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While it is understandable that arf8-1, a mutant gene in the auxin signaling pathway, 250
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affects sensitivity or response to auxin, why does arf8-1 also exhibit increased sensitivity 251
or response to GA? One possibility is that FveARF8 regulates the expression of GA 252
receptors to affect the sensitivity to GA. Three FveGID1 genes were identified from the F. 253
vesca genome (gene22353, gene20092, and gene27756) as the candidates of GA receptor 254
(Figure S6), only gene22353, named as either FveGID1a (Kang et al., 2013) or 255
FveGID1c (Li et al., 2019) and thereafter, is specifically expressed in the receptacle at 256
early fruit stages (Figure S6B-D). If FveGID1c encodes a GA receptor for fruit 257
development, FveGID1c should bind FveRGA1 when GA is present. Indeed, FveGID1c 258
directly interacts with FveRGA1 in yeast only when GA is added to the medium (Figure 259
4D) and this interaction requires the DELLA domain at the N-terminus as FveRGA1-C 260
(missing N-terminal 564 bp) fails to interact with FveGID1c even when GA is added 261
(Figure 4D). The interaction between FvGID1c and FveRGA1 is also demonstrated in 262
tobacco cells via BiFC (Figure 4E). CRISPR/Cas9 was used to knockout GID1c; a 263
biallelic mutant gid1c (-149; -166+15) was obtained (Figure S1C), which exhibited 264
strong dwarf but never flowered (Figure 4F). Although the GID1c sgRNA1 and sgRNA2 265
show 5 and 9 mismatches respectively to the other two GID1 genes (gene20092 and 266
gene27756) (Figure S6E), we still sequenced the other two GID1 genes (gene20092 and 267
gene27756) from the gid1c (-149; -166+15) plant and found no mutations in them. The 268
strong dwarf phenotype indicates that FveGID1c encodes an important GA receptor. 269
270
Next, we tested if FveGID1c expression is altered in arf8-1 at the early fruit stages 1-3. 271
FveGID1c expression level is similar in arf8-1 and WT at stage 1; at stages 2-3, 272
FveGID1c expression is reduced in WT but remains high in arf8-1 mutant receptacle, 273
significantly higher in arf8-1 when compared with WT (Figure 5), indicating that 274
FveARF8 may limit the sensitivity to GA by down-regulating FveGID1c at stages 2-3. 275
Given that FveARF8 is no longer inhibited by FveRGA1 at stages 2-3 due to fertilization-276
induced GA production and subsequent FveRGA degradation, FveARF8 may then be 277
free to repress FveGID1c expression at stage 2-3 to dampen sensitivity to GA in feedback 278
regulation. 279
280
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We then tested FveGID1c transcript level in rga1-1 mutant background. In rga1-1 mutant, 281
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a reduction of FveGID1c transcript is observed at stage 1 (Figure 5B), consistent with 282
that de-repressed FveARF8 in rga1-1 could act to repress FveGID1c transcription. Given 283
that FveARF8’s function may still be constrained by IAAs at stage 1, the repression of 284
FveGID1c is moderate (Figure 5B). At stage 2-3 (post-fertilization), freed from inhibition 285
by FveIAAs and FveRGA1, FveARF8 can repress FveGID1c expression in WT as well 286
as in rga1-1 (Figure 5B). The above qRT-PCR data are consistent with a negative 287
regulatory relationship of FveRGA1 to FveARF8 and to FveGID1c. 288
289
To test if FveARF8 directly represses FveGID1c, three segments of FveGID1c promoters, 290
P1, P2, and P3 (Figure S6F), were respectively fused to the AUR1-C reporter and 291
integrated into the yeast strain in a Y1H system. Self-activating activity of each segment 292
was examined at two Abi concentrations, 500 µM and 1000 µM (Figure 5C). Promoter 293
fragment 2 (P2), which does not self-activate and contains two potential AuxREs and two 294
potential G-boxes (Figure S6F), was used in the Y1H against transcription factor 295
FveARF6-AD or FveARF8-AD (Figure 5C). FveARF6-AD and FveARF8 (DBD)-AD 296
readily activate the FveGID1c (P2)::AUR1-C reporter, indicating direct binding of 297
FveARF8 and FveARF6 to the FveGID1c promoter. However, full length FveARF8 or a 298
truncated FveARF8 (DBD + MR) fail to activate the FveGID1c (P2) reporter (Figure 5C), 299
indicating that the MR domain of FveARF8 may possess strong repressor activity that 300
could counter Gal4-AD. 301
302
A transient expression reporter system in tobacco cells is also used to test direct effect of 303
FveARF8 and FveARF6 on the FveGID1c promoter. A 1kb promoter fragment of 304
FveGID1c was fused to the firefly luciferase (LUC) and integrated into vector PLAH-305
LARM (Taylor-Teeples et al., 2015), which also contains 35S::REN (Renilla luciferase) 306
as a control and 35S::FveARF8 or 35S::FveARF6 (Figure 5D). 1 µM GA was applied to 307
the tobacco cells to cause robust expression from pGID1c::LUC perhaps due to 308
endogenous transcription factors mediating GA responses. Compared with 35S::YFP 309
control; 35S::FveARF8 significantly reduces the expression from pGID1c::LUC (Figure 310
5D), suggesting that FveARF8 harbors strong repressor activity and binds directly to the 311
promoter of FveGID1c. FveARF6 appears to possess very weak repressor activity (Figure 312
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5D). Together, these data suggest that FveARF8, and perhaps FveARF6, may directly 313
bind and repress FveGID1c to limit sensitivity to GA at post-fertilization stage. 314
315
Consensus co-expression network identifies FveARF8, FveARF6, and FveIAA4 in 316
the same co-expression module 317
Our data revealed a previously unknown mechanism of GA-auxin crosstalk where auxin 318
represses the sensitivity to GA via activation of FveARF8. In auxin signaling pathway, 319
AUX/IAA proteins are known to bind and repress ARFs in the absence of auxin 320
(Guilfoyle, 2015; Salehin et al., 2015). Which FveIAAs could bind and inhibit FveARF8? 321
F. vesca genome V4.0a2 has about 34 FveARFs and 25 FveIAAs (Edger et al., 2018; Li et 322
al., 2019). To identify candidate FveIAAs, we mined the consensus co-expression 323
network generated previously (Shahan et al., 2018) (http://159.203.72.198:3838/fvesca/). 324
FveARF8 resides in module 80 of the Consensus_90 Fruit Network; this module 80 325
correlates positively in expression with early stage receptacle fruit tissues. Interestingly, 326
the two enriched GO terms for this module are “auxin activating signaling pathway” and 327
“cellular response to auxin stimulus”. FveARF6 and FveIAA4 are found in this module 328
with a high correlation co-efficient (>0.91) with FveARF8 (Figure 6; Data S1). Based on 329
RNA-seq data (Kang et al., 2013; Hollender et al., 2014; Hawkins et al., 2017), all three 330
genes are highly and specifically expressed in the receptacle, ovary wall and style during 331
early fruit development at stage 1-5 (Figure 6B). To validate their expression, qRT-PCR 332
was conducted using receptacle at fruit stages 1, 2, and 3; all three genes are expressed in 333
the receptacle at stage 1 (pre-fertilization) and show downward expression trend post-334
fertilization at stage 2 and 3 (Figure 6C). 335
336
To test if FveIAA4 in the same network module may interact and repress FveARF6/8 in 337
the absence of auxin, Y2H shows that FveIAA4 interacts with both FveARF8 and 338
FveARF6 (Figure 7). The C-terminal PB1 domain of FveARF8 is shown to be both 339
necessary and sufficient for the interaction with FveIAA4 (Figure 7B). BiFC confirms 340
these interactions (Figure 7C). The data suggest that fertilization-induced auxin may 341
cause degradation of FveIAA4, releasing FveARF8/6 to exert positive or negative 342
regulatory effects on downstream genes. 343
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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344
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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To test if FveARF6 and FveARF8 could work together as heterodimers, we first 345
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performed BiFC (Figure 7D) which showed that while FveARF6 and FveARF8 do not 346
interact with each other, FveARF6, but not FveARF8, interacts with itself (Figure 7D). 347
FveARF8’s inability to interact with FveARF6 is subsequently confirmed in co-348
immunoprecipitation assay (Figure 7E) using protein extracts from yeast containing 349
MYC-tagged Gal4BD-FveARF6 and HA-tagged Gal4AD-FveARF8 or Gal4AD-350
FveARF6. 351
352
The different MR sequence between FveARF8 and FveARF6 protein (Figure S5) 353
combined with their distinct ability to hormodimerize suggests that FveARF6 and 354
FveARF8 may not be functionally redundant. CRISPR construct was made for FveARF6 355
and transformed into WT F. vesca as well as arf8-1 (-2, -2) line# 156 that has segregated 356
away the original CRISPR-sgARF8 transgene. While we failed to obtain an arf6 single 357
mutant, arf6-1 (-1, -1) mutation was identified in arf8-1 (-2, -2) background in two 358
different transgenic lines (Figure S1D). These arf6-1; arf8-1 double mutant lines 359
developed fruits similar in size to WT and significantly smaller than arf8-1 single 360
mutants (Figure S7). The phenotype supports that FveARF6 and FveARF8 may play 361
different roles during fruit initiation and enlargement. 362
363
Discussion 364
Strawberry has long been a model to study fruit development, especially fruit set, when 365
Nitsch (1950) first observed the effect of auxin in stimulating receptacle fruit fleshy 366
formation when achenes were removed (Nitsch, 1950). However, the molecular 367
mechanisms underlying this elegant communication between seed and fruit flesh are not 368
well understood. Further, being the accessory fruit, where the fruit fleshy is derived from 369
receptacle instead of ovary wall, it poses additional questions concerning if the conserved 370
fertilization signals, auxin and GA, activate downstream fruit program in a similar or 371
distinct mechanism from botanical fruits. The work presented here provides basic 372
molecular frame work on how the GA and auxin signaling components interact and 373
coordinate fleshy fruit program in strawberry receptacle using both conserved and novel 374
mechanisms. The work laid the foundation for future engineering of parthenocarpic 375
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strawberry for enhancing fruit productivity when pollinators are scarce. 376
377
FveRGA1 is a key regulator of fruit set in diploid strawberry, but FveARF8 is 378
involved in downstream regulation of fruit development post-fertilization 379
rga1-1 is the only strawberry mutant so far reported to develop parthenocarpic fruit, 380
indicating FveRGA1 as a key regulator of fruit set in strawberry. FveRGA1 normally 381
prevents fruit sets by inhibition of a large number of TFs through protein-protein 382
interaction. In rga1-1 mutants, the inhibition is absent, and the TFs are free to activate 383
downstream genes for fruit development even in the absence of plant hormone, leading to 384
parthenocarpic fruit. This mutational effect of rga1-1 mimics the application of GA in 385
emasculated flowers, where GA-GID1c induced degradation of FveRGA1 lead to 386
parthenocarpic fruit (Kang et al., 2013). Our study identifies FveRGA1 as a target gene 387
for genetic manipulation to produce parthenocarpic strawberry. 388
389
The larger fruit size of arf8 mutants appears caused by different reasons from rga1-1. 390
Further, the effect of arf8 on receptacle size can be divided into pre- and post-fertilization 391
stages. At the pre-fertilization stage, the larger receptacle size is likely due to a defect on 392
flower development, which is also reflected by increased petal numbers in arf8 mutants 393
(Figure S2). It is important to note that the arf8-1 mutant receptacle size stays the same 394
from stage 1 to stage 3 when fertilization is prevented (Figure 2A-B). In contrast, at the 395
post-fertilization stages when auxin and GA are produced, the faster increase in fruit size 396
of arf8-1 mutants reflects an enhanced sensitivity to auxin and GA (Figure 4A-C). In 397
other words, same concentrations of phytohormones could mount a stronger response in 398
arf8-1 mutants. This is why arf8-1 mutants do not develop parthenocarpic fruit as GA 399
and auxin are still required for the arf8-1 mutants to develop fruit. 400
401
A model on the mechanism of GA-auxin crosstalk on fruit initiation 402
Figure 8 is a model that both summarizes previous understanding of GA and auxin 403
signaling pathways and incorporates our findings here. First, auxin and GA signaling 404
pathways act in parallel to promote fruit set and fruit development (thick green curved 405
arrows). At pre-fertilization (stage 1) receptacle (Figure 8B), RGA1 and IAA4, 406
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respectively bind and inhibits a large number of TFs including ARF8 (red bars), blocking 407
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Zhou et al.,
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fruit development. At post-fertilization (stage 2) receptacle (Figure 8C), GA and auxin 408
are produced from the fertilized seed and transported to the receptacle, where GA and 409
auxin interact with their respective receptors, FveGID1c and FveTIR, which subsequently 410
facilitate ubiquitin-mediated degradation of FveRGA1 and FveIAA4. Therefore, upon 411
fertilization, TFs in auxin and GA pathways are activated to promote fruit set and 412
subsequent fruit development (blue arrows). 413
414 We show that FveARF8’s DBD-MR and PB1 domains interact respectively with 415
FveRGA1 and FveIAA4 (Figure 3 and 7), indicating that FveARF8 activity could be 416
simultaneously inhibited by FveIAA4 and FveRGA1 prior to fertilization (Figure 8B). At 417
stage 2, when both auxin and GA are produced and FveIAA4 and FveRGA1 are both 418
degraded, FveARF8 and other TFs are now freed from both inhibitions to regulate the 419
transcription of downstream genes (blue lines) (Figure 8C). This explains why combined 420
treatment of auxin and GA is required to cause emasculated fruit to develop to the same 421
size as pollinated fruit (Kang et al., 2013). 422
423
One of the downstream targets of FveARF8 is the FveGID1c gene, and FveARF8 424
represses FveGID1c transcription to dampen sensitivity to GA in a feedback regulatory 425
loop (Figure 8C). This transcriptional down-regulation of FveGID1c by FveARF8 at 426
stage 2 is detected by qRT-PCR (Figure 5A) and appears to be direct (Figure 5C-D). As 427
ARFs normally have a large number of target genes in a genome (Oh et al., 2014), 428
FveARF8 may also promote fruit development indirectly through repressing other 429
negative regulators of fruit enlargement. This is indicated by a dotted blue arrow from 430
FveARF8 in Figure 8C. 431
432
FveGID1c (gene22353) encodes a GA receptor in F. vesca 433
Previous gene expression analysis (Kang et al., 2013), current Y2H assay in the presence 434
or absence of GA (Figure 4D), and CRISPR/CAS9-induced knockout of FveGID1c 435
(Figure 4F) all support that FveGID1c is an important GA receptor. While there are three 436
FveGID1 genes (Figure S6A), FveGID1c (gene22353) not only exhibits significantly 437
higher expression than the other two FveGID1b genes (gene20092 and gene27756) but 438
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Zhou et al.,
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also is more specifically expressed in the receptacle (Figure S6B-D). The severe 439
phenotype of gid1c (-149, -166+15) prevented the mutant plant to produce flowers. 440
Future work using inducible RNAi maybe necessary to allow the examination of gid1c 441
phenotype in fruit development. 442
443
Both conserved and novel GA-auxin crosstalk mechanisms exist in strawberry 444
While both auxin and GA act to promote fruit set in several organisms including 445
Arabidopsis, tomato, and strawberry, their interaction and crosstalk mechanism may vary. 446
In the botanical fruit of Arabidopsis and tomato, auxin was shown to act upstream of GA 447
by promoting GA biosynthesis (Serrani et al., 2008; Dorcey et al., 2009; Fuentes et al., 448
2012). Recent work in tomato revealed another crosstalk point through direct protein-449
protein interactions between SlARF7 and SlDELLA as well as SlIAA9 via SlARF7’s 450
MR2 and PB1 domains respectively (Hu et al., 2018). However, it is not known if 451
strawberry employs similar or novel crosstalk mechanisms to induce fruit formation 452
given the distinct floral tissue used by strawberry to form fruit flesh. 453
454
Our finding of direct interaction of FveIAA4 with FveARF8/FveARF6 and of RGA1 455
with FveARF8/FveARF6 illustrates conservation of crosstalk mechanism in a different 456
spatial context (ie. in accessary fruit). However, our data also reveal a previously 457
unknown crosstalk mechanism where FveARF8 represses FveGID1c transcription to 458
reduce sensitivity to GA in a feedback regulatory loop (Figure 8). The enhanced 459
sensitivity to auxin in arf8-1 (Figure 4A) also suggests that FveARF8 may act in a 460
feedback loop of auxin signaling. Proper regulation of FveARF8 protein activity through 461
FveIAA4 and FveRGA1 may serve to ensure that auxin and GA are limiting each other’s 462
signaling pathway to balance each other’s impact on fruit enlargement. One interesting 463
outcome of this balance is reflected in fruit shape. Previously, exogenous application of 464
GA to F. vesca fruit was shown to impact fruit shape differently from exogenous 465
application of NAA (Liao et al., 2018). Similarly, we showed that treatment of 466
emasculated WT or arf8-1 with NAA significantly increases fruit width and hence 467
rounder fruit shape, while treatment with GA significantly increases fruit length and 468
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Zhou et al.,
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hence longer fruit shape (Figure 4A). Hence, a balance between the two hormonal 469
pathways may impact fruit shape as well as size. 470
471
FveARF8 and FveARF6 exhibit differences in protein sequence, dimerization, 472
repression strength, and phenotype 473
Most ARF proteins can be divided into three domains, a N-terminal domain for DNA-474
binding and dimerization, a variable middle region (MR) that confers activation or 475
repression activity, and a C-terminal PB1 domain for interaction with ARFs as well as 476
Aux/IAA inhibitors (Tiwari et al., 2003; Boer et al., 2014; Guilfoyle, 2015; Powers et al., 477
2019). A recent report indicates that Arabidopsis activator ARF7 and ARF19 form 478
cytoplasmic assemblies as a mechanism to sequester them to modulate cellular 479
competence to respond to auxin (Powers et al., 2019). Sequence divergence in all three 480
domains contribute to distinct functional properties of ARFs within and between species. 481
482
Arabidopsis ARF6 and ARF8 genes are classified as activator ARFs that promote both 483
vegetative and reproductive development (Ulmasov et al., 1999; Tiwari et al., 2003). 484
Arabidopsis arf6; arf8 double mutants show more severe floral phenotypes than either 485
single mutants, suggesting partial redundancy between AtARF6 and AtARF8 (Nagpal et 486
al., 2005). In Arabidopsis and tomato, over-expression of miR167, which targets both 487
ARF6 and ARF8, also caused similar phenotypes as the arf6; arf8 double mutants (Wu et 488
al., 2006; Liu et al., 2014; Zheng et al., 2019). Most relevant to this work, Arabidopsis 489
arf8 mutants develop parthenocarpic fruit, and the expression of an “dominant negative” 490
AtARF8 causes parthenocarpy in Arabidopsis and tomato (Goetz et al., 2007). Thus, 491
ARF8 possesses a conserved role in preventing fruit set in Arabidopsis and tomato. 492
493
In this study, FveARF8 (gene31631) and FveARF6 (gene30394) are identified and named 494
based on their blast best hit with their Arabidopsis counterparts. A second F. vesca ARF6 495
genes (gene22728) is not studied here since it is not in the same co-expression module as 496
FveARF8. Sequence alignment among AtARF6/AtARF8/FveARF6/FveARF8 reveals 497
high levels of sequence conservation at the N-terminal DBD and C-terminal PB1 498
domains but poor sequence similarity at the MR domain (Figure S5). When compared 499
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Zhou et al.,
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with the MR of AtARF6, MR of FveARF6 has a large 40 amino acid deletion and a 500
reduced number of polyQ tracks (Figure S5), which may weaken the activation function 501
of FveARF6. Together, the poor sequence conservation in the MR domain, different 502
strength of repression on FveGID1c promoter, and different ability to homodimerize 503
suggest that FveARF6 and FveARF8 may function differently. This is supported by the 504
fruit phenotypes where a loss-of-FveARF6 led to fruit size reduction in arf8-1 505
background and a loss-of-FveARF8 caused fruit size increase (Figure S7). 506
507
Consensus co-expression network is a powerful tool 508
Previously, a consensus-clustering approach was applied to the standard WGCNA 509
(Weighted Gene Co-expression Network Analysis) to generate robust and reproducible 510
modules (Shahan et al., 2018). Since ARF and IAA genes are from large gene families, 511
the consensus co-expression network led us to a co-expression module consisting of 512
FveIAA4, FveARF6 and FveARF8 (Figure 6A). Our results support their protein-protein 513
interaction and functional roles in early fruit development and demonstrate the predictive 514
power of consensus co-expression network analysis. Such network is especially valuable 515
in guiding selection of genes belonging to large gene families as well as studies in non-516
model species where transformation is resource-intensive and challenging. 517
518
519
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Zhou et al.,
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Methods 520
Plant materials and growth condition 521
The Fragaria vesca cultivar Yellow Wonder 5AF7 (YW5AF7) was used in most of this 522
study. Cultivar Hawaii 4 (H4) was used in generating BZR1-RNAi lines. Plants were 523
grown in a growth chamber at a temperature of 25 °C/16 hour light followed by 22 °C/8 524
hour darkness with a relative humidity of 50%. The F. vesca genes studied here are 525
indicated by their gene IDs (genome version 2.0.a2; genome version 4.0.a2): FveARF8 526
(gene31631; FvH4_1g27650), FveARF6 (gene30394; FvH4_3g05010), FveIAA4 (gene 527
16569; FvH4_6g02870), FveGID1c (gene22353; FvH4_6g04960), and FveRGA1 (gene 528
06210; FvH4_4g34110). The names FveARF8, FveARF6, and FveIAA4 are assigned 529
based on their best blast hit with their Arabidopsis counterparts. 530
531
arf8 mutants were generated using CRISPR/CAS9 (Zhou et al., 2018). Four independent 532
arf8 transgenic lines #100 (-2, -2), #156 (-2, -2), #391-3 (-8, -23+5), and #391-31 (-2+1, -533
2+1) were detailed in Figure S1. Line #100 (-2, -2) and line #156 (-2, -2) are referred to 534
as arf8-1 in the text. T1 generation arf8 mutant plants were analyzed, and about 20% of 535
them have segregated away the CRISPR-sgARF8 transgene. 536
537
The rga1-1 (u230) mutant was previously generated by ENU chemical mutagenesis in a 538
ga20ox4 loss of function mutant background (Tenreira et al., 2017; Caruana et al., 2018). 539
This rga1-1; ga20ox4 was backcrossed with WT (H4) to yield rga1-1 single mutant, 540
which was used in all analyses. H4 was used as WT control whenever rga1-1 was used. 541
542
CRISPR/Cas9 genome editing and over-expression 543
To generate the arf8; arf6 double mutants, two seed gRNA sites (gRNA1: 544
TGCTTCAACCAACATGGAAG and gRNA2: GCCAGTGACACTAGTACCCA) 545
targeting an FveARF6 (gene30394) exon were inserted into the JH4 entry vector and then 546
incorporated into binary vector JH19 via gateway cloning (Zhou et al., 2018). Cotyledon 547
of arf8 mutant line #156 (-2, -2), which has segregated away its prior sgARF8-Cas9 548
construct, was used for transformation with the CRISPR-sgARF6 construct. Four 549
transgenic lines were generated. To detect mutation, genomic sequences spanning two 550
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Zhou et al.,
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target sites of FveARF6 were amplified by primers FvARF6-F5 and FvARF6-R5 (Data 551
S2) and analyzed by Sanger sequencing. 552
553
The FveGID1c CRISPR construct was created using JH4 and JH17 vectors (Zhou et al., 554
2018). Two seed guide RNA sequences (gRNA1: GCAACTGAGACACGCCTGG and 555
gRNA2: GTTCCCAAGGAGCCTTGTGG) targeting FveGID1c were inserted into the 556
entry vector JH4 and incorporated into binary vector JH17 via gateway cloning. In total, 557
3 transgenic lines were generated. The genomic region spanning the two target sites in 558
FveGID1c was amplified by primers FvGID1a-F1 and FvGID1a-R1 (Data S2) and 559
sequenced. Only one transgenic line, line #7 gid1c (-149, -166+15), has a clear bi-allelic 560
mutation (Figure 4F). 561
562
To test if the other two FveGiD1b genes (gene20092 and gene27756) were also mutated 563
in gid1c line #7, primer pairs gene20092-F&gene20092-R and gene27756-564
F&gene27756-R (Data S2) were used to respectively amplify the genome sequence of the 565
two FveGiD1b genes. The PCR products were then sequenced. 566
567
To generate the FveARF8 over-expression (OX) construct, full-length FveARF8 CDS 568
was PCR amplified from YW5AF7cDNA library with primers FvARF8-SalI-F and 569
FvARF8-Kpn-R (Data S2) and inserted into pENTR-2b vector via SalI/KpnI. The 570
pENTR-FveARF8 was incorporated into JH23 destination vector through gateway 571
cloning. In total, eight over-expression transgenic lines were generated and analyzed. 572
573
To construct the over-expression vector JH23 (Figure S6), the OCS terminator was 574
amplified from JH19 (Zhou et al., 2018) and inserted into pMDC99(Curtis and 575
Grossniklaus, 2003) at PacI and SacI sites; the AtUBQ10 promoter was amplified from 576
JH19 (Zhou et al., 2018) and then inserted into above PMDC99-OCS at HindIII and KpnI 577
sites to yield JH23. 578
579
Most above constructs were transformed in F. vesca (YW5AF7 or arf8-1 mutant in 580
YW5AF7 background) using previously published protocols (Kang et al., 2013; Caruana 581
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Zhou et al.,
28
et al., 2018). 582
583
Receptacle size measurement 584
The receptacle of arf8 mutant and WT (YW5AF7 or Hawaii 4) were cut in half and 585
imaged under Stemi SV 6 dissecting microscope (Zeiss. Inc). The height and width were 586
then measured using the ZEN software. Flowers that just open with anthers not yet 587
dehiscent were considered stage 1 (0DPA). Flowers at 5-7 DPA were considered stage 3 588
usually with only 1 petal left. Receptacle measurements were normalized to petal size 589
(height and width of receptacle divided respectively by height and width of petal) to yield 590
relative receptacle size in Figure 1. To measure the receptacle size of rga1-1 (Figure 2) 591
the newly opened but non-dehiscent flowers were labeled as 1DPA (stage 1), the 592
receptacle length and width were measured at 9 DPA (stage 4) and 11-12 DPA (stage 5). 593
To determine parthenocarpy, arf8-1 or rga1-1 flowers were emasculated to prevent self-594
fertilization. Fruit size at 0DPA (pre-fertilization) was compared to fruit size at 7 DPA 595
(post-emasculation) within the same genotype. 596
597
For measuring the receptacle size of GA3- and NAA-treated fruits, WT (YW5AF7) or 598
arf8 flowers were emasculated at 0DPA under Stemi SV 6 dissecting microscope (Zeiss. 599
Inc), 1mM GA3 or 1mM NAA mixed with 0.1% Tween 20 was applied to the 600
emasculated receptacle. This treatment was repeated every two days until day 7 when the 601
receptacle size was measured. The relative GA- and NAA-treated receptacle height and 602
width were normalized to mock treated fruit of the same genotype. 603
604
For arf8-1, 8-19 fruits derived from 5 to 10 plants of the same genotype were measured; 605
and then combined to yield one bar in bar graph (or one box in box plot). For YW5AF7 606
(WT) control, fruit measurement was similarly made on fruits from 12 plants. 607
Measurement of other arf8 CRISPR lines were similarly conducted. For hormone 608
treatment experiments, the same groups of arf8-1 and YW5AF7 plants above were used 609
in fruit measurement. For rga1-1, twelve mutant plants were grown from the same 610
original mother plant through vegetative propagation via runner. Ten H4 (WT) control 611
plants were similarly propagated through an original H4 mother plant. The same 612
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Zhou et al.,
29
measurement and analysis strategy described for arf8-1 was used. For arf8-1; arf6-1 613
double mutants, two transgenic lines were used, and each line has 3-4 plants derived from 614
the same calli. 10 YW5AF7 (WT) plants were grown together and used as controls. 615
616
Statistical analysis 617
Data from fruit size measurements, qRT-PCR, and LUC assay were all analyzed using 618
unpaired, unequal variance, two-tailed student's t test. Significant difference (P <0.01) 619
and difference (P <0.05) between treatments or genotypes are denoted by **and *, 620
respectively. Data are expressed as the means ± standard deviation for different 621
biological replicates. 622
623
BiFC and Y2H to test protein-protein interaction 624
For the BiFC, full-length CDS of FveARF8, FveARF6 were PCR amplified from cDNA 625
and ligated into PXY105 (cYFP) at BamHI and SalI sites, full-length CDS of FveRGA1 626
was ligated into PXY105 through Gibson cloning by using primers N-termless 627
Bamh1BIfc_FWP & Gib_Pxy105_06210_Xba1_R. Next, full-length CDS of FveARF8, 628
FveARF6, and FveIAA4, were amplified from cDNA and ligated into PXY106 (nYFP) at 629
BamHI and SalI sites. BiFC constructs were transformed into Agrobacterium EHA105. 630
Transformants were harvested when OD600 reached 1.0, and resuspended in 1/2 MS 631
medium (suppled with 50uM acetosyringone) to a final OD600 of 0.5. The agrobacterial 632
cells containing the cYFP fusion vector were mixed with agrobacterial cells containing 633
the nYFP fusion vector at 1:1 ratio. The mixture was infiltrated into young Nicotiana 634
benthamiana leave. After 48 hours, the YFP florescence signal was visualized and 635
photographed using Leica SP5X Confocal microscope (Leica Co. USA). 636
637
For the yeast two-hybrid (Y2H), full-length CDS of FveARF8, FveARF6, FveGID1c 638
were PCR amplified and ligated into GAL4 AD vector PGADT7 (Clontech Inc.) at the 639
BamHI/XhoI, BamHI/XhoI, EcoRI/XhoI double digestion sites, respectively. Full-length 640
CDS of FveIAA4 and FveGID1c were ligated into GAL4 BD vector PBGK T7 (Clontech 641
Inc.) through EcoRI/SalI double digestion sites. FveRGA1-C was cloned into the Y2H 642
vector pGBTK7 by Gibson assembly using primer pair GBTK7_M5_06210_F/ 643
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Zhou et al.,
30
GBTK7_BamH1_06210_R (Data S2). To delete the SAW domain from FveRGA1-C, 644
primer pair RGA1-564-BglII-F & RGA1-564-SalI-R were used to amplify FveRGA1-C 645
(-SAW) fragment and inserted into pGBK T7 at BamHI-SalI sites. The auto activity of 646
each BD construct was examined by co-transformation with an empty AD vector PGAD 647
T7. 648
649
To make truncated FveARF8 for Y2H, FveARF8 (DBD), FveARF8 (MR), FveARF8 650
(PB1), and FveARF8 (MR+PB1) fragments were amplified respectively by primer pairs 651
ARF8-CDS-F1/ARF8-DBD-XhoI-R; FveARF8-MD-F/ARF8-MD-R; FveARF8-PBI-652
F/ARF8-CDS-R1, FveARF8-MD-F/ARF8-CDS-R1 (Data S2) and inserted into PGAD 653
T7 via EcoRI-XhoI/SalI sites. FveARF8 (DBD+MR) was amplified by ARF8-CDS-654
F1/ARF8-MD-R and inserted into PGAD T7 via BamHI/BglII-XhoI/SalI sites. 655
656
For Y2H essay, yeast constructs were transformed into PJ69-4A yeast strain using 657
LiAc/PEG method (Gietz and Schiestl, 2007). The transformed yeast cells were plated 658
onto -LW two drop-out or -HLWA four drop-out medium; -HLWA medium is often 659
supplemented with 5mM 3-AT to reduce background. Yeast cells were grown at 30 ℃ 660
for 3-4 days. 661
662
Each BiFC, Y1H, and Y2H experiment was performed three times. All repeats yielded 663
the same result. 664
665
Co-immunoprecipitation of proteins from yeast 666
The co-IP assay was performed following a published protocol (Gerace and Moazed, 667
2014) with minor modifications. The yeast strains containing PGBK T7-ARF6-Myc and 668
PGAD T7-ARF8-HA (or PGBK T7-ARF6-Myc and PGAD T7-ARF6-HA) were grown 669
to an OD600=1 at 28 0C. After centrifugation, the yeast cells were suspended in 1×lysis 670
buffer (100mM Na-HEPES, pH7.5, 400mM NaoAc, 2mM EDTA, 2mM EGTA, 10mM 671
MgOAc, and 10% Glycerol) and lysed by Mini-Beadeater (model: 2412PS-12W-B30), 672
Minebea Co., LTD. For each immunoprecipitation, 30μl Magnetic Dynabeads with 673
protein A (Invitrogen) were resuspend in 1×lysis buffer and incubated with 5μg c-Myc 674
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
31
primary antibody (sc-40, Santa Cruz Biotechnology) for 10min at room temperature. The 675
protein A/c-Myc antibody slurry were dispensed into each cell lysate and rotate for 2h at 676
40C. Then, the Dynabeads- cMyc antibody-protein complex were washed 5 times with 677
wash buffer (100mM Na-HEPES, pH7.5, 400mM NaoAc, 2mM EDTA, 2mM EGTA, 678
10mM MgOAc, 0.25% NP-40, 3mM DTT, 1mMPMSF, and 10% Glycerol), followed by 679
elution with 4×SDS-PAGE Protein Loading Buffer (10% SDS, 500Mm DTT, 50% 680
Glycerol, 500mM Tris-HCL and 0.05% bromophenol blue dye) and loaded onto SDS-681
PAGE gel. 10% lysate was used as the “Input” loading. The western blots were 682
performed following General V3 Western Workflow Blotting Protocol (Bio-rad, 683
Laboratories, Inc). The anti-HA primary antibody (SKU: H6908, Sigma-Aldrich, Inc. 684
USA) was used to detect the pull-down products in western. The co-IP experiment was 685
repeated three times with the same result. 686
687
Yeast one-hybrid and the luciferase reporter assays 688
For yeast one hybrid assay, a 1077bp promoter fragment upstream of FveGiD1c 689
(gene22353) was amplified with primer FvGID1a-P-KpnI-F and FvGID1a-P-SalI-R 690
(TableS2) and ligated into pAbAi vector (Clontech Inc.) at KpnI and SalI sites. The 691
resulting GiD1c reporter vector was linearized by BstBI, and then introduced into 692
competent yeast cell Y1HGold to integrate at the ura3-52 locus. All procedures were 693
based on the protocol Matchmaker Gold Yeast One-Hybrid Library Screening System 694
User Manual (PT4087-1). The AD fusions in pGAD T7 with FveARF8 (and various 695
truncations) and FveARF6 are the same as those described for Y2H. 696
697
The pENTR vectors LEI02 and LEI04 (Figure S9) for LUC experiments are modified 698
from pLAH-R4R3-VP64Ter (Taylor-Teeples et al., 2015; Zhan et al., 2018). Specifically, 699
full length VP64-35S terminator was PCR-amplified from pLAH-R4R3-VP64Ter with 700
primers VP64-F and T35S-R (Data S2), and inserted into pCR™8/GW/TOPO™ vector 701
(Invitrogen™) via EcoRI, to generate LEI01 vector. The LEI01 vector was digested by 702
PacI and then self-ligated to drop off the VP64 sequence to produce LEI02. To construct 703
the negative control vector LEI04, YFP was amplified from pLAH-L1L4-Citrine and 704
then inserted into LEI02 vectors via EcoRI and AscI. Full length FveARF8 or FveARF6 705
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
32
was PCR amplified and inserted into LEI02 at AscI and PacI to yield 35S::FveARF8 and 706
35S::FveARF6 respectively. The expression cassettes of 35S::FveARF8 (or 707
35S::FveARF6), pGiD1c::LUC, and 35S::REN were simultaneously incorporated into the 708
destination vector pLAH-LARm(Taylor-Teeples et al., 2015) through gateway cloning, 709
resulting in the final vectors 35S::FveARF8_pGiD1c::LUC_35S::REN and 710
35S::FveARF6_pGiD1c::LUC_35S::REN. The 35S::Citrine_pGiD1c::LUC_35S::REN 711
vector served as a control. 712
713
The luciferase reporter assay was performed following the instruction of Dual-714
Luciferase®Reporter Assay System (Promega, Inc, USA). Various constructs were 715
transformed into Agrobacterium strain EHA105 and infiltrated into tobacco Nicotiana 716
benthamiana leaves. 1µM GA was also applied during infiltration to increase the 717
autoactivity of GiD1c. After two days, a small leaf disc from infection site was collected 718
and finely ground in the passive lysis buffer (1×). 10 µl supernatant was mixed with LAR 719
solution (from the Promega kit), luciferase activities were measured using TD-20/20 720
Luminometer (Turner Designs, Inc, USA). The LUC readout was divided by the REN 721
readout to provide a normalized expression level. The LUC experiment was performed 722
three times with same results. The data shown is one of the three experiments. 723
724
RNA extraction and qRT-PCR 725
For testing FveGID1c expression (Figure 5), total RNA was extracted from YW5AF7, 726
arf8-1, Hawaii 4 (H4), and rga1-1 receptacles at stages indicated. Achenes were removed 727
from the receptacle, and 3 to 5 receptacles from several plants of the same genotype were 728
combined as one biological replicate. The CTAB RNA extraction method was 729
used(Gambino et al., 2008). The CTAB RNA extraction solution (2% CTAB, 2% PVPk-730
30, 10mM Tris-Hcl(pH8), 25mM EDTA, 2M Nacl, 0.5g/L Spermidine) was added to the 731
flesh tissues. The tissues were ground immediately following by the addition of 2/3 732
volume of 10M LiCl to precipitation RNA. In total, 1 ug total RNA was used for cDNA 733
synthesis with SuperScript IV VILO Master Mix (Invitrogen, USA). The cDNA product 734
was diluted 4 times and ~5 ul was used as a template in qRT-PCR using BioRad CFX96 735
Real-time system and SYBR Green PCR MasterMix (Applied Biosystems). The 736
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
33
expression levels of four F.vesca genes FveARF8, FveARF6, FveIAA4, and FveGiD1c 737
were quantified via 4 primer pairs ARF8-F1-2/ARF8-R1-2, FveARF6-F1/FveARF6-R1, 738
FveIAA4-F1 /FveIAA4-XhoI-R and Fv GID1c-F2/ Fv GID1c-R2 (Data S2), respectively. 739
F. vesca gene03773 was used as the internal control with primers gene03773-740
F/gene03773-R (Caruana et al., 2018). The experiment was performed three times each 741
with RNA extracted from newly collected samples (ie. biological replicates). 742
743
Mining consensus co-expression network and other bioinformatics tools 744
The expression pattern of each F. vesca gene in wild type (YW5AF7) was visualized via 745
strawberry eFP browser (Hawkins et al., 2017) (http://mb3.towson.edu/efp/cgi-746
bin/efpWeb.cgi). The consensus co-expression network analysis of YW5AF7 floral and 747
fruit tissues(Shahan et al., 2018) was mined using the network website 748
(http://159.203.72.198:3838/fvesca/). The co-expression cluster/module was visualized 749
using Cytoscape (cytoscape.org). Protein alignments were performed using clustalw 750
(https://www.genome.jp/tools-bin/clustalw). Heatmap was generated with morpheus 751
(https://software.broadinstitute.org/morpheus/). 752
753
Tissue section and histological staining 754
Tissue section was performed following the protocol (Retamales and Scharaschkin, 2014) 755
with minor modification. YW5AF7, arf8-1, rga1-1 fruit (receptacle and achene) of each 756
stage was fixed in FAA (50% ethanol, 5% acetic acid, 3.7% Formaldehyde) for 3 days 757
and went through an ethanol series 50%, 60%, 70%, 80%, 90%, 95% (containing 0.1% 758
Eosin), and 100%. The tissues went through a series of changes in 25% xylenes/75% 759
ethanol, 50% xylenes/50% ethanol, 75% xylenes/25% ethanol, and finally 100% xylenes. 760
After two more changes of 100% xylene, paraffin chips were added into the xylene. 761
Afterwards, the tissue in xylene/paraffin mix is exchanged with 100% paraffin wax. After 762
4 changes of 100% paraffin in a duration of 4-5 days, a wax boat was poured. The tissues 763
were sectioned at 12 μm thickness on a microtome, and stained by Safranin-O/Fast Green 764
based on a protocol (http://microscopy.berkeley.edu/Resources/instruction/staining.htm). 765
Slides were mounted with Permount (Fisher) and dried overnight. 766
767
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
34
Acknowledgements 768
This work has been supported by a grant from the National Science Foundation (IOS 769
1444987). We thank Dr. Ramin Yadegari at University of Arizona, Samuel Hazen at 770
UMass Amherst, Dr. Yanhai Yin from Iowa State University, and Dr. Kunsong Chen 771
from Zhejiang University for vectors. 772
773
Author Contributions 774
J. Z. and Z. L. designed experiments. J. Z., J. S., and L. G. performed experiments. X. H. 775
and A. P. assisted experiments. J. Z. and Z. L. analyzed and discussed the data, Z. L. and 776
J. Z. wrote the paper. 777
778
779
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
35
Figure legend 780
781
Figure 1. arf8 mutants develop larger and rounder fruit 782
A. Images of bisected receptacle at stage 1 (0DPA, before fertilization), stage 3 (7DPA, 783
post-fertilization), stage 5 (15 DPA), and ripe stage in WT and arf8-1 (-2, -2). Note the 784
rounder fruit shape in arf8-1. Bars; 1 mm. 785
B. Average size of receptacle fruit (height and width, normalized to petal size) at stage 1 786
and stage 3. ** above bars represent significant difference (P < 0.01) between WT and 787
arf8-1 mutant. 788
C. Relative receptacle fruit size (height and width, normalized to petal size) in WT and 789
three additional arf8 mutant lines at stage 3 (7DPA). These arf8 mutant lines, #156 (-2, -790
2), #391-3 (-8, -23+5), and #391-31 (-2+1, -2+1) are illustrated in Figure S1. 791
D. WT and arf8 mutant receptacle fruits at ripe stage. Since petal has senesced, the 792
absolute fruit size was measured. arf8-1 mutants show a significant increase in width but 793
not in height at the ripe stage as shown in A. 794
795
796
Figure 2. rga1-1, but not arf8-1, develop parthenocarpic fruit 797
A. Photos of bisected receptacle of WT and arf8-1 at 0DPA and 7DPA. The flowers were 798
emasculated at the 0DPA. Bars; 1 mm 799
B. Quantitative measurement of receptacle size in emasculated fruit shown in A. No 800
significant difference in fruit size between 0DPA and 7DPA was observed in emasculated 801
WT and arf8 receptacle. 802
C. Images of bisected WT and rga1-1 receptacle at stage 1 (1DPA), Stage 4 (9DPA), and 803
stage 5 (11-12 DPA) without emasculation. rga1-1 receptacle fruit is larger and longer 804
than WT at stages 4-5. Bar: 1 mm. 805
D. Quantitative measurement of WT and rga1-1 receptacles at stage 1 and stage 4. rga1-1 806
develops significantly larger fruit, particularly in receptacle height. 807
E. Image of emasculated WT and rga1-1 receptacles at stage 1 (1DPA) and stage 3 808
(7DPA). Red arrows highlight the width of the receptacle. Fertilization-independent fruit 809
enlargement is evident in rga1-1 mutant. Bar, 1 mm. 810
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
36
F. Quantitative measurements of emasculated WT and rga1-1 receptacles at stage 1 811
(0DPA) and stage 3 (7DPA). Emasculated WT receptacles at stage 1 and stage 3 are 812
similar in size. Emasculated rga1-1 receptacles are significantly larger and taller at stage 813
3 when compared with stage 1. 814
815
816
Figure 3. BIFC and Y2H revealed direct interaction of FveRGA1 with FveARF8, 817
and FveARF6 818
A. BiFC in tobacco leaf showing positive interactions between FveRGA1 and 819
respectively FveARF8 and FveARF6. 820
B. Y2H assay confirming the interactions between FveRGA1-C (fused to BD) and 821
respectively FveARF8 and FveARF6. Because of self-activating activity, a truncated 822
FveRGA1-C with its N-terminal DELLA domain removed is used in the Y2H. 823
C. Dissection of FveARF8 protein domains reveals the requirement of both DBD and MR 824
for the interaction with RGA1-C. 825
D. Removing the SAW domain from RGA1-C eliminates its ability to interact with 826
FveARF6 and FveARF8 in Y2H assay. 827
828
829
Figure 4. arf8-1 exhibits enhanced response to GA and auxin 830
A. Photos of bisected stage 3 receptacles of WT and arf8-1. The flowers were first 831
emasculated and then applied with NAA, GA3, or mock solution (see Methods) until 832
7DPA. Bars; 1 mm. 833
B. Relative receptacle size of WT and arf8-1 at stage 3 (7DPA) after GA treatment of 834
emasculated flowers. The height and width of hormone GA-treated receptacles are 835
divided respectively by the height and width of mock treated receptacles of the same 836
genotype. ** above bar represents significant difference (P < 0.01) between WT and 837
arf8-1. 838
C. Relative receptacle size of WT and arf8-1 at stage 3 (7DPA) after auxin (NAA) 839
treatment of emasculated flowers. Data is similarly measured and analyzed as B. 840
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
37
D. Yeast two-hybrid assay showing the interaction between FveGID1c and FveRGA1 841
only in the presence of GA3 (200 M). The interaction is dependent on the DELLA 842
domain, which, when deleted in RGA1-C, could no longer support the interaction even in 843
the presence of GA. 844
E. BIFC assay in tobacco leaf cells confirming positive interaction between FveRGA1 845
and FveGID1c. The tobacco cells may produce GA to enable the interaction. 846
F. A CRISPR/CAS9-induced gid1c (-149, -166+15) biallelic mutant exhibiting severe 847
dwarf. 848
849
850
Figure 5. FveARF8 and FveARF6 directly represses the expression of FveGID1c 851
A. qRT-PCR showing relative expression of FveGID1c in WT (YW5AF7) and arf8-1 852
mutants at stage 1-3. 853
B. qRT-PCR showing relative expression of FveGID1c in WT (H4) and rga1-1 mutants 854
at stage 1-3. 855
C. Y1H assay showing activation of GID1c Promote fragment 2 (P2) when FveARF6 or 856
ARF8-DBD domain fused to GAL4 AD is introduced into the yeast. On the right, three 857
different promoter fragments (P1, P2, and P3) are tested for self-activation at two 858
different concentrations of antibiotics Abi500 and Abi1000. P2 does not have any self-859
activation activity. 860
D. Diagram of the LUC reporter system. The expression of LUC driven by the GID1c 861
promoter is repressed strongly by 35S::FveARF8 and weakly by 35S::FveARF6. Robust 862
self-activation in the absence of transacting factor (with YFP serving as a negative 863
control) is shown when 1 m GA3 is added in all essays. Y-axis shows relative LUC 864
expression normalized against 35S::REN expressed from the same vector. 865
866
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
38
867
868
869
Figure 6. Network analysis identifies FveARF8, FveARF6 and FveIAA4 as potential 870
regulators of fruit initiation 871
A. Consensus Network analysis identifies FveARF8, FveARF6, and FveIAA4 in module 872
80 of Consensus 90 Fruit Network. The five-digit ID numbers for all other genes in the 873
same module are shown and also listed in Table S1. This module correlates with young 874
receptacle fruit. The consensus score and correlation score between genes are indicated 875
by color and thickness of lines respectively. 876
B. Heatmap of FveARF6, FveARF8 and FveIAA4 based on prior RNA-seq data, revealing 877
specific expression in young fruit and style. 878
C. qRT-PCR of FveARF8, FveARF6, and FveIAA4 in developing receptacle (pith and 879
cortex) at stages 1-3. Data represents mean ± SE of gene expression from three biological 880
replicates of YW receptacle tissues. The Y-axis indicates relative expression level 881
compared to reference gene PP2a (gene03773) derived using formula 2-ΔCt. Different 882
assaying techniques and tissue sampling methods may contribute to some differences 883
between qRT-PCR result and the RNA-seq data in (B). 884
885
886
Figure 7. Yeast two-hybrid, BiFC, and co-immunoprecipitation assays testing the 887
interaction between FveIAA4 and FveARF8/FveARF6 888
A. Yeast two-hybrid assay revealing the positive interaction between FveIAA4 and 889
FveARF6/FveARF8. 890
B. Domain dissection of FveARF8 showing that the PB1 domain is necessary and 891
sufficient for the interaction with FveIAA4. A diagram of ARF8 is drawn beneath, 892
showing DNA binding (DBD), Middle Region (MR), and PB1 domains. Numbers 893
beneath the diagram indicate amino acid residues. 894
C. FveARF8 and FveARF6 respectively interact with FveIAA4 shown by BiFC in 895
tobacco cells. 896
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
39
D. BiFC in tobacco cells showing a lack of interaction between FveARF8 and FveARF6. 897
While FveARF8 does not homodimerize, FveARF6 does. 898
E. Co-immunoprecipitation using protein extracts from yeast cells containing c-Myc-899
tagged Gal4BD-FveARF6 and HA-tagged Gal4AD fusion constructs. INPUT lanes show 900
western blots of yeast protein extracts containing indicated constructs. Co-IP lanes are 901
western blots of immunoprecipitated proteins with anti-HA antibody. 902
903
904
Figure 8. A model that summarizes auxin/GA crosstalk in strawberry fruit initiation. 905
A. A diagram illustrating spatial separation of hormone synthesis in the achene from 906
hormonal signal perception and transduction in the receptacle. 907
B. In receptacle at stage 1 (pre-fertilization), when auxin and GA are absent, IAA4 and 908
RGA1 bind and repress ARF8 and other TFs in the auxin and GA signaling pathway. 909
Fruit set is blocked. 910
C. In the receptacle at stage 2 (post-fertilization), auxin and GA are transported to the 911
receptacle (from achenes) to induce signal transduction. Specifically, GA/GID1c and 912
auxin/TIR receptor complex caused degradation of FveRGA1 and FveIAA4, releasing 913
FveARF8 and TFs which promote fruit set and subsequent development. FveARF8 at the 914
same time represses the transcription of FveGID1c in a feedback regulation. 915
916
In B and C, the red lines describe negative regulation at post-translational level via 917
protein degradation or binding of repressors to transcription factors (TFs). Blue lines 918
describe positive (arrows) or negative (bar) regulation at transcription level. Light 919
shaded lines or names indicate “inactive” or “absent”. Dotted blue line from FveARF8 920
indicates FveARF8’s putative and indirect role in promoting fruit development. 921
922
923
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
Zhou et al.,
40
924
was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted April 1, 2020. ; https://doi.org/10.1101/2020.03.30.016188doi: bioRxiv preprint
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