Plant Cell Advance Publication. Published on September 28, 2016, doi:10.1105/tpc.16.00612
RESEARCH PAPER
Transcriptional Activation of Two Palmitoyl-ACP ∆9 Desaturase Genes by MYB115 and MYB118 is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acid in the Endosperm of Arabidopsis Seeds Manuel Adrián Troncoso-Ponce1‡, Guillaume Barthole1,2‡, Geoffrey Tremblais1, Alexandra To1, Martine Miquel1, Loïc Lepiniec1, and Sébastien Baud1 1
Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles, France 2 Univ. Paris-Sud, Université Paris-Saclay, 91405 Orsay, France ‡ Both authors contributed equally to this work Corresponding author e-mail:
[email protected] Short title Regulation of ω-7 fatty acid synthesis in seeds One-sentence summary The MYB115 and MYB118 transcription factors activate two palmitoyl-ACP desaturases, AAD2 and AAD3, which are responsible for omega-7 fatty acid biosynthesis in the endosperm of Arabidopsis seeds.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Sébastien Baud (
[email protected]). Contact information: Institut Jean-Pierre Bourgin, UMR1318 INRA-AgroParisTech, INRA Centre de Versailles-Grignon, Route de Saint-Cyr (RD10), 78026 Versailles Cedex, France. Tel. +33 1 30 83 33 25 ABSTRACT In angiosperms, double fertilization of the embryo sac initiates the development of the embryo and the endosperm. In Arabidopsis thaliana, an exalbuminous species, the endosperm is reduced to one cell layer during seed maturation and reserves such as oil are massively deposited in the enlarging embryo. Here, we consider the strikingly different fatty acid (FA) compositions of the oils stored in the two zygotic tissues. Endosperm oil is enriched in ω-7 monounsaturated FAs, that represent more than 20 Mol% of total FAs, whereas these molecular species are ten-fold less abundant in the embryo. Two closely related transcription factors, MYB118 and MYB115, are transcriptionally induced at the onset of the maturation phase in the endosperm and share a set of transcriptional targets. Interestingly, the endosperm oil of myb115 myb118 double mutants lacks ω-7 FAs. The identification of two Δ9 palmitoyl-ACP desaturases responsible for ω-7 FA biosynthesis, which are activated by MYB115 and MYB118 in the endosperm, allows us to propose a model for the transcriptional control of oil FA composition in this tissue. In addition, an initial characterization of the structure-function relationship for these desaturases reveals that their particular substrate specificity is conferred by amino acid residues lining their substrate pocket that distinguish them from the archetype Δ9 stearoyl-ACP desaturase.
©2016 American Society of Plant Biologists. All Rights Reserved
1
INTRODUCTION
2 3
In spermatophyta, also known as seed plants, the double fertilization of the
4
embryo sac initiates the development of zygotic tissues, namely the embryo and the
5
endosperm. They are protected by the seed coat, which comprises several cell layers
6
of maternal origin derived from the ovular integuments. Seed formation therefore
7
requires the coordinated growth of tissues of distinct origins that undergo two
8
successive developmental phases: morphogenesis and maturation (Vicente-
9
Carbajosa and Carbonero, 2005). Maturing seeds accumulate reserve compounds
10
that are remobilized to fuel post-germinative seedling establishment. Depending on
11
the species considered, the nature, relative proportion, and tissue localization of
12
these reserve components vary greatly. Exalbuminous seeds of Arabidopsis store
13
approximately equivalent amounts of oil (triacylglycerols, TAGs) and storage proteins
14
(2S albumins and 12S globulins), these compounds being mostly deposited in a large
15
embryo structure acquired at the expense of the endosperm (Baud et al., 2002). In
16
mature dry seeds, the residual endosperm consists of a thin peripheral cell layer that
17
contains no more than 10% of total seed reserves (Li et al., 2006). The fine
18
biochemical characterization of the endosperm has revealed a reserve composition
19
clearly different from that of the embryo, with a strongly decreased abundance of
20
globulins (Barthole et al., 2014) and a unique oil fatty acid (FA) composition (Penfield
21
et al., 2004). If all the FAs detected in the endosperm are also present in the embryo,
22
the former contains ten-fold higher proportions of ω-7 monounsaturated FAs, like
23
vaccenic acid (cis-ω-7 C18:1) and paullinic acid (cis-ω-7 C20:1), that account for
24
more than 50% of the total ω-7 FAs present in the whole seed.
25
In plants, de novo synthesis of FAs occurs in plastids (Harwood, 1996).
26
Production of 16- or 18-carbon saturated FAs is catalyzed by the type II fatty acid
27
synthase. Stromal ∆9 acyl-ACP desaturases (AADs) can introduce a carbon-carbon
28
double bond (also called unsaturation) within these saturated acyl chains to form cis-
29
monoenes (Lindqvist et al., 1996). AAD isoforms with different substrate specificities
30
catalyze the formation of distinct monoenes differing by the position of the
31
unsaturation within their aliphatic chains (referred to as ω-x). For instance, ∆9
32
stearoyl-ACP desaturases (SADs) efficiently desaturate C18:0 to form cis-ω-9 C18:1
33
(oleic acid). SADs represent the predominant AAD isoforms in most seed plants.
34
Accordingly, the majority of the FAs found in embryo oil of Arabidopsis consists of 2
35
oleic acid and of its derivatives. However, other AAD isoforms prefer C16:0 instead of
36
C18:0 as a substrate. These ∆9 palmitoyl-ACP desaturases (PADs) catalyze the
37
formation of cis-ω-7 C16:1 (palmitoleic acid), which can be further elongated to cis-ω-
38
7 C18:1 (vaccenic acid), then to cis-ω-7 C20:1 (paullinic acid). Monoenes of the ω-7
39
FA series occur infrequently in most seed plants, with the noticeable exception of a
40
few plant species that produce unusual oils enriched in these ω-7 monounsaturated
41
FAs, such as cat’s claw vine (Doxantha unguis-cati) or sea buckthorn (Hippophae
42
rhamnoides) (Bondaruk et al., 2007; Fatima et al., 2012).
43
The genome of the model plant Arabidopsis thaliana contains seven closely
44
related genes coding for AADs (Kachroo et al., 2007). FAB2, the best-characterized
45
member of the family, encodes a SAD (Lightner et al., 1994), whereas the other
46
members of the family have been poorly characterized. The PAD(s) responsible for
47
the production ω-7 monounsaturated FAs that accumulate at high levels in the
48
endosperm oil of Arabidopsis seeds remain to be identified.
49
Over the last decade, our knowledge on the regulation of storage compound
50
metabolism in maturing seeds has increased tremendously. This knowledge has
51
arisen mostly from genetic analyses carried out in Arabidopsis. Transcriptional
52
regulators ensuring that maturation-related programs, such as oil biosynthesis, are
53
correctly deployed during the transition phase between embryogenesis and seed
54
maturation have been identified (Santos Mendoza et al., 2008). These transcription
55
factors (TFs) participate in a complex network essential for completion of seed filling
56
(Roscoe et al., 2015). Master regulators of the maturation program include members
57
of the AFL (ABSCISIC ACID INSENSITIVE3/FUSCA3/LEAFY COTYLEDON2)
58
network. These TFs belong to the B3 domain superfamily of DNA binding proteins
59
and cooperate with LEAFY COTYLEDON1 (LEC1), a protein homologous to the
60
HAP3 subunit of CCAAT-box binding proteins (Lotan et al., 1998; Suzuki and
61
McCarty, 2008). Next to these master regulators, other TFs like basic leucine zippers
62
bZIP53 or bZIP67 confer correct expression patterns to maturation genes (Mendes et
63
al., 2013). Several genes encoding storage proteins or actors involved in TAG
64
assembly and storage were shown to be direct targets of the above-mentioned TFs.
65
By contrast, transcriptional activation of many glycolytic and FA biosynthetic genes,
66
which is essential to support sustained rates of oil production, is indirectly mediated
67
by WRINKLED1 (WRI1), a TF of the AP2-EREBP family (Cernac et al., 2004; Baud
3
68
and Lepiniec, 2010). All together, these studies have led to a significant breakthrough
69
in our understanding of the activation of reserve compound synthesis in the embryo,
70
while the regulation of endosperm metabolism has scarcely been investigated in
71
Arabidopsis. The recent characterization of MYB118, a TF transcriptionally induced in
72
the maturing endosperm and repressing storage compound accumulation in this seed
73
compartment, has shed some new light on the differential regulation of reserve
74
partitioning between the embryo and endosperm (Barthole et al., 2014). However, the
75
regulatory mechanisms explaining the contrasting compositions of these reserves
76
remain completely unknown.
77
To isolate new regulators of endosperm maturation and elucidate the peculiar
78
composition of endosperm reserves, new screening procedures have been
79
undertaken. Here, we report the functional characterization of MYB115 (At5g40360),
80
a close homolog of MYB118 also induced in the endosperm at the onset of seed
81
maturation. We provide evidence that the master regulator LEC2 positively regulates
82
the two genes. This regulation and the negative feedback exerted by MYB118 on
83
LEC2 expression suggest a partial compensation of the myb118 mutation by an
84
overexpression of MYB115 in this genetic background. This hypothesis was
85
confirmed by the thorough characterization of maturing myb115 myb118 mutant
86
seeds demonstrating that the two MYBs are positive regulators of ω-7
87
monounsaturated FA synthesis in the endosperm. We finally describe the
88
identification of two targets of MYB115 and MYB118 belonging to the AAD multigene
89
family. We show that these targets, namely AAD2 and AAD3, encode two ∆9 PADs
90
responsible for the biosynthesis of ω-7 FA in the maturing endosperm. Taken
91
together, these results allow us to establish a model for a transcriptional activation
92
cascade participating in the control of oil FA composition within the maturing
93
endosperm of Arabidopsis seeds.
94 95 96
RESULTS
97 98
MYB115 is induced in the endosperm of maturing seeds
99
Functional redundancy between related TFs has been previously documented in
100
Arabidopsis and recent studies suggest that MYB118 and MYB115, two close
101
relatives of the MYB family (Wang et al., 2009; Zhang et al., 2009; Dubos et al.,
102
2010), share transcriptional targets involved in glucosinolate biosynthesis (Zhang et 4
103
al., 2015). To determine whether overlaps in function between MYB118 and close
104
homologs occur in the maturing endosperm, we first examined the expression
105
patterns of the three closest paralogs of MYB118, namely MYB115, MYB22, and
106
MYB100 (Dubos et al., 2010) by reverse transcription quantitative PCR (RT-qPCR)
107
on a set of cDNAs prepared from a range of plant organs of the wild-type accession
108
Columbia-0 (Col-0). In all analyzed tissues, the accumulation of MYB22 and MYB100
109
transcripts was below detectable levels; this was consistent with previously published
110
transcriptomic analyses (Schmid et al., 2005; Le et al., 2010). By contrast, MYB115
111
appeared to be expressed at very low levels in vegetative organs and induced in
112
reproductive organs (flowers and developing siliques) (Figure 1A). To further
113
characterize the expression pattern of MYB115, a time course analysis of MYB115
114
mRNA abundance was carried out in developing seeds excised from siliques, which
115
revealed a peak of transcript accumulation at the onset of seed maturation (Figure
116
1B). Maturing seeds were then dissected and the two fractions obtained, namely
117
embryo and endosperm fraction (comprising the endosperm and the seed coat; see
118
methods), were independently analyzed. MYB115 mRNA abundance was high in the
119
endosperm fraction during early maturation and hardly detected in the embryo
120
(Figure 1C).
121
To gain complementary information about the expression pattern of MYB115, the
122
spatiotemporal activity of the MYB115 promoter was investigated. A 1-kb promoter
123
fragment was transcriptionally fused to the uidA reporter gene. The corresponding
124
construct was assayed for the resulting uidA expression pattern in transgenic
125
Arabidopsis lines (Figure 1D-N). β-Glucuronidase (GUS) activity was observed in
126
pollen grains (Figure 1D-E) and in seeds. A closer examination of developing seeds
127
showed that the endosperm was stained (Figure 1G-J), whereas the seed coat and
128
the embryo (Figure 1K-N) were not. The intense staining observed in the chalazal
129
endosperm 12 days after anthesis (DAA; Figure 1J) was consistent with previous
130
results of laser-capture microdissection of maturing seeds followed by mRNA
131
quantification using stringent analyses of Affymetrix ATH1 GeneChip hybridization
132
data (Le et al., 2010). MYB115 therefore appears to be co-expressed with MYB118 in
133
the endosperm of early-maturing seeds (Barthole et al., 2014).
134 135
Transcriptional activation of MYB115 by LEC2
5
136
The master regulator LEC2 plays a key role in the transcriptional activation of
137
MYB118 (Barthole et al., 2014). To test whether LEC2 also influences the
138
transcriptional activation of MYB115, analysis of MYB115 mRNA abundance was first
139
carried out in lec2 mutants. MYB115 transcripts were analyzed by RT-qPCR on
140
cDNA prepared from 10-DAA-old seeds. MYB115 transcript steady-state levels were
141
significantly reduced in lec2 alleles, suggesting the down-regulation of MYB115
142
expression in the absence of LEC2 (Figure 1O). To test the transcriptional activation
143
of MYB115 by LEC2, we used a dexamethasone (DEX; a synthetic glucocorticoid
144
that activates the rat glucocorticoid receptor GR) inducible system (Santos Mendoza
145
et al., 2005). The relative expression level of MYB115 was quantified by RT-qPCR in
146
leaves of Pro35S:LEC2:GR plants. In transgenic plants treated for two weeks with
147
DEX, a specific and significant accumulation of MYB115 mRNA was observed
148
(Figure 1P). A time course analysis of MYB115 mRNA accumulation in rosette leaves
149
treated with DEX revealed a marked increase of MYB115 mRNA levels from four
150
days after induction onwards (Figure 1Q). Finally, the ProMYB115:uidA construct
151
was introduced into transgenic Pro35S:LEC2:GR lines. The seedlings obtained were
152
grown for 14 days on DEX-containing medium and were then assayed for the
153
resulting uidA expression pattern (Figure 1R). GUS staining was detected in rosette
154
leaves of these seedlings, confirming the ability of the LEC2:GR fusion protein to
155
trigger MYB115 transcription.
156 157
Transcriptional repression of MYB115 by MYB118 is LEC2-dependent
158
MYB115 transcripts were analyzed by RT-qPCR on cDNA prepared from 10-
159
DAA-old myb118 mutant seeds. MYB115 transcript steady-state levels were
160
significantly increased both in myb118-1 seeds and in the OE3 line (a
161
Pro35Sdual:MYB118 transgenic line exhibiting a strong repression of MYB118
162
expression; Barthole et al., 2014), suggesting the up-regulation of MYB115
163
expression in the absence of MYB118 (Figure 1S). To further evaluate the effect of
164
MYB118 on MYB115 promoter activity, the ProMYB115:uidA construct was
165
introduced into the myb118-1 mutant background and the resulting uidA expression
166
pattern assayed in seeds aged 10 DAA and 12 DAA (Supplemental Figure 1). The
167
proportion of stained seeds and the intensity of GUS staining were drastically
168
increased in myb118-1 seeds at both developmental stages, showing the importance
169
of MYB118 for repressing MYB115 promoter activity in maturing seeds. 6
170
Considering the antagonistic regulation exerted by LEC2 and MYB118 on
171
MYB115 expression, we tested whether these regulations were independent of each
172
other (Supplemental Figure 2). For this purpose, MYB115 transcript levels were
173
quantified by RT-qPCR on cDNA prepared from 10-DAA-old lec2 myb118-1 seeds
174
and compared to that of single mutants. MYB115 transcripts levels in seeds of the
175
double mutants were not significantly different from that measured in lec2 single
176
mutants, demonstrating that the de-repression of MYB115 observed in response to
177
the myb118-1 mutation is LEC2-dependent.
178 179
MYB115 and MYB118 are transcriptional regulators that share common targets
180
To firmly establish that MYB115 is a functional TF, we first investigated the in
181
vivo subcellular localization of the protein with the aid of the green fluorescent protein
182
(GFP). A derivative of GFP, mGFP6, was fused to MYB115 cDNA and placed under
183
the control of the CaMV dual35S promoter for ubiquitous and high expression. The
184
Pro35S:MYB115:GFP construct was transfected into leaves of transgenic Nicotiana
185
benthamiana stably expressing RFP fused to histone 2B (RFP-H2B, used as nuclear
186
marker; Martin et al., 2009). The Pro35S:MYB118:GFP construct was used as a
187
positive control. Confocal imaging of transfected cells showed a co-localization of the
188
GFP and RFP signals, demonstrating the nuclear targeting of MYB-GFP fusions
189
(Figure 2A).
190
In order to determine whether MYB115, like MYB118, possesses transcriptional
191
activity, coding regions of the TFs were individually cloned in frame with the GAL4
192
DNA binding domain (GAL4-DBD). The constructs thus obtained were introduced into
193
the yeast strain AH109, which carries the HIS3 and ADE2 reporter genes under the
194
control of heterologous GAL4-responsive upstream activating sequences and
195
promoter elements. The expression of these two reporters could be activated in the
196
presence of MYB115 or MYB118 fused to GAL4-DBD, thus establishing their ability
197
to activate transcription (Figure 2B).
198
The predicted DNA-binding domains of MYB115 and MYB118 share 73% amino
199
acid identity. In order to test whether MYB115 and MYB118 share common
200
transcriptional targets, myb115-1, myb118-1 and myb115-1 myb118-1 lines were
201
grown together with wild-type controls and the expression level of target genes of
202
MYB118 (Barthole et al., 2014) was measured by RT-qPCR on cDNA prepared from
203
10-DAA-old seeds of each line. Repressed targets of MYB118 were first considered 7
204
(Figure 2C). Whereas no de-repression of these genes could be detected in myb115-
205
1, a significant over-accumulation of corresponding cDNA was measured in the
206
double mutant as compared to the myb118-1 single mutant, showing that MYB115
207
and MYB118 redundantly repress this set of genes. Conversely, the mRNA level of
208
the 2-OXOGLUTARATE-DEPENDENT DIOXYGENASE (ODD) gene, a direct
209
inducible target of MYB118, was further decreased in the double mutant as
210
compared to myb118-1, suggesting that inducible targets of MYB118 can also be
211
shared by the two TFs (Figure 2D). To validate the response of the ODD gene to
212
MYB115 deregulation, Pro35Sdual:MYB115 transgenic lines were generated (OE11
213
and OE19; Supplemental Figure 3). However, these lines exhibited altered vegetative
214
development and were partially sterile, preventing us from analyzing the effect of
215
MYB115 overexpression in seeds. Measurements were consequently performed on
216
rosette leaves that demonstrated the ability of MYB115 to ectopically activate ODD
217
(Figure 2E).
218
To test the ability of MYB115 to directly activate ODD expression, we used a
219
ProODD:uidA reporter construct in transactivation assays in N. benthamiana (Figure
220
2F). The ProBCCP2:uidA construct was used as a negative control. Reporter
221
constructs were infiltrated alone or in combination with a vector allowing the
222
expression of MYB115, MYB118 (positive control), or MYB107 (negative control) in
223
young leaves of N. benthamiana. MYB115, like MYB118, was able to specifically
224
activate the ProODD:uidA reporter construct, showing a strong increase in GUS
225
activity compared with the reporter alone or the reporter cotransfected with MYB107.
226
A similar result was obtained with a reporter construct made of four repeats of the
227
TAACGG element fused to the 35S cauliflower mosaic virus minimal promoter
228
upstream of the uidA reporter gene. This element was proposed to be the cis-
229
regulatory element bound by MYB118 in the promoter sequence of ODD (Barthole et
230
al., 2014). All together, these results establish the ability of MYB115 and MYB118 to
231
activate common target genes, possibly through the same cis-regulatory element.
232
The binding of MYB115 to the ODD promoter sequence was examined in vitro by
233
electrophoretic mobility shift assay (EMSA). Purified recombinant MYB115 was
234
incubated with a 40-bp promoter fragment containing the TAACGG element and
235
binding was determined using a biotin-labeled DNA probe. Addition of MYB115
236
resulted in the formation of shifted bands (Figure 2G). The signal intensity increased
237
with the concentration of MYB115 in the assay, indicating that the protein binds to the 8
238
DNA fragment. The binding was specific since addition of the recombinant WRI1 TF
239
did not result in the appearance of shifted bands. Furthermore, in competition
240
experiments, addition of increasing amounts of unlabeled promoter fragments
241
decreased the binding of MYB115 to the labeled probe. Using EMSA, we finally
242
demonstrated the binding of MYB115 to three additional promoter fragments
243
containing the TAACGG element and previously shown to be bound by MYB118
244
(At5g01030, At3g62230, and At3g12880 promoters; Figure 2H). These results
245
confirm that the two TFs bind common cis-regulatory elements.
246 247
Impact of myb115 and myb118 mutations on ω-7 monoene accumulation in
248
seeds
249
To investigate the function of MYB115 during endosperm maturation and to test
250
its redundancy with MYB118, the myb115-1, myb118-1 and myb115-1 myb118-1
251
mutants were grown under controlled conditions. Vegetative development of the
252
mutants was unaffected. Whole-mount clearing of developing seeds was carried out
253
during embryo morphogenesis and early maturation. The structure and early
254
development of the three tissues composing the seed were unaffected in the various
255
mutant backgrounds considered (Supplemental Figure 4). Likewise, observation of
256
14-DAA-old peeled endosperms suggested that the organization of the monolayer of
257
endosperm cells was unmodified in the mutant lines. During the course of seed
258
maturation, a slight delay could be observed in the elongation and enlargement of
259
myb115-1 myb118-1 embryos, that was associated with a moderate decrease of
260
mature seed DW with respect to the other genotypes (Supplemental Figure 4).
261
Since MYB115 and MYB118 are strongly induced in the maturing endosperm, we
262
then evaluated the effects of their mutations on endosperm filling (Figure 3).
263
Dissected endosperm and embryo fractions were collected separately during the
264
course of seed maturation and total FAs were quantified by gas chromatography.
265
Whereas the myb118 mutation led to a significantly increased FA content in the
266
endosperm fraction from 12 DAA onward (Figure 3B; Barthole et al., 2014)
267
compensated by an equivalent decrease of the embryo FA content (Supplemental
268
Figure 5A), no effect of the myb115 mutation on the overall amount of FAs stored in
269
the endosperm could be detected.
270
The effect of the myb mutations on the FA composition of the oil stored in the
271
endosperm fraction was then examined. A focus was put on monoenes of the ω-7 9
272
series that were previously reported to be highly abundant in the endosperm oil of
273
mature Arabidopsis seeds (Penfield et al., 2004; Li et al., 2006). Before addressing
274
the role of the MYB TFs in the control of oil FA composition, we first characterized the
275
accumulation of ω-7 FAs in the two zygotic tissues of wild-type seeds (Supplemental
276
Figure 6). In the endosperm fraction, ω-7 FAs were massively deposited between 9
277
and 14 DAA. In mature seeds, they accounted for more than 20 Mol% of total FAs,
278
with paullinic acid representing the more abundant species of the ω-7 series of FAs.
279
The pattern of ω-7 FA accumulation was strikingly different in the embryo, with a later
280
storage of ω-7 FA species, a decreased abundance of these monoenes (they
281
represent only 2 Mol% of total FAs in dry embryos), and a predominance of vaccenic
282
acid over paullinic acid. We then evaluated the effect of the myb mutations on the
283
accumulation of ω-7 FAs in the endosperm (Figure 3C). The myb118 mutation
284
yielded a sharp decrease in the proportion of ω-7 FAs stored in this compartment.
285
Reversion of this phenotype could be obtained by introgression of a wild-type copy of
286
the MYB118 gene into the mutant background (Figure 3D). Whereas the single
287
myb115 mutation did not affect the accumulation of ω-7 FAs, an aggravated
288
phenotype could be detected in the double myb115-1 myb118-1 mutant with respect
289
to the myb118-1 background, denoting partially redundant functions of MYB115 and
290
MYB118 in the control of ω-7 FA biosynthesis in the endosperm (Figure 3C). The
291
endosperm tissue comprises different territories and the expression patterns of
292
MYB115 and MYB118 were not similar in this tissue. MYB118 exhibited a high and
293
homogeneous induction throughout the endosperm (Barthole et al., 2014), whereas
294
the induction level of MYB115 was weaker and less homogeneous, the promoter
295
activity of MYB115 being particularly intense in the chalazal endosperm (see above).
296
We therefore measured the ω-7 FA contents of subfractions of the endosperm
297
compartment in mature dry seeds. The peripheral endosperm was separated from
298
the basal endosperm (comprising both the chalazal and micropylar endosperms) and
299
the collected subfractions were analyzed separately by gas chromatography (Figure
300
3E-G). The results obtained suggest that MYB115 and MYB118 redundantly control
301
ω-7 FA production in all endosperm territories, with the action of MYB118
302
predominating over that of MYB115 everywhere. A slight but reproducible negative
303
effect of the myb mutations on ω-7 FA synthesis was also observed in the embryo
304
(Supplemental Figure 5). 10
305 306
Identification of two acyl-ACP desaturases transcriptionally activated by
307
MYB115 and MYB118
308
In order to identify the ∆9 PADs responsible for ω-7 FA biosynthesis in seeds of
309
Arabidopsis, we used an RT-qPCR approach and examined the expression profiles
310
of the seven Arabidopsis genes predicted to encode AADs (named AAD1/SAD1 to
311
AAD6/SAD6, plus FAB2) in the search for positively regulated targets of MYB115 and
312
MYB118. Target mRNAs were quantified in maturing myb mutant seeds (10 DAA)
313
and in rosette leaves of transgenic lines overexpressing MYB118 (lines OE1 and
314
OE2) or MYB115 (lines OE11 and OE19). Two AAD genes, namely AAD2 and AAD3,
315
were strongly downregulated in myb mutant seeds with corresponding mRNA levels
316
correlating with the reduced ω-7 FA contents in these seeds (Figure 4A). Conversely,
317
MYB115 and MYB118 were able to ectopically activate these two putative target
318
genes (Figure 4B,C).
319
To confirm the ability of the two MYBs to directly activate AAD2 and AAD3
320
expression, we used reporter ProAAD2:uidA and ProAAD3:uidA constructs in
321
transactivation assays in N. benthamiana leaves. The ProBCCP2:uidA construct was
322
used as a negative control. Reporter constructs were infiltrated alone or in
323
combination with vectors allowing the expression of MYB115, MYB118, or MYB107
324
(negative control) in young leaves (Figure 4D). MYB115 and MYB118 were able to
325
specifically activate ProAAD2:uidA and ProAAD3:uidA reporter constructs, showing a
326
strong increase in GUS activity compared with the reporters alone or the reporters
327
cotransfected with MYB107.
328
We finally characterized the expression patterns of AAD2 and AAD3 in
329
developing seeds. The reporter constructs previously used for transient activation
330
assays in N. benthamiana were stably introduced in Arabidopsis and resulting uidA
331
expression patterns were assayed. For each construct tested, GUS staining was
332
specifically observed in the maturing endosperm (Figure 4E,F). Then, RT-qPCR
333
experiments carried out with cDNA prepared from dissected seed fractions confirmed
334
the tissue specificity of AAD2 and AAD3 expression, with corresponding mRNA
335
levels peaking 14 DAA in the endosperm fraction (Figure 4G). Expression patterns of
336
AAD2 and AAD3 were consistent with the positive transcriptional regulation exerted
337
both by MYB115 and MYB118 on these genes.
338 11
339
Role of AAD2 and AAD3 in the biosynthesis of ω-7 monoenes in seeds
340
In order to demonstrate that transcriptional activation of AAD2 and AAD3 is
341
sufficient to trigger the biosynthesis of ω-7 monoenes, we transiently overexpressed
342
these desaturases in leaves of N. benthamiana. FA composition was analyzed in
343
transformed leaves five days after infiltration and revealed a significant enrichment in
344
ω-7 monoenes in this material (Figure 5A). This ectopic stimulation of ω-7 FA
345
synthesis was specific since overexpression of other Arabidopsis AAD isoforms
346
(AAD1 or AAD6) had no effect on the biosynthesis of these FA species. To confirm
347
these results, Pro35Sdual:AAD2 and Pro35Sdual:AAD3 constructs were stably
348
introduced in Arabidopsis transgenic lines. First, RT-qPCR experiments carried out
349
with cDNAs prepared from rosette leaves demonstrated the efficient overexpression
350
of the transgenes in the selected lines (Figure 5B). Then, total FA analyses carried
351
out with leaf material confirmed the ability of AAD2 and AAD3 to ectopically induce ω-
352
7 FA biosynthesis.
353
To firmly establish that transcriptional activation of AAD2 and AAD3 is
354
responsible for ω-7 FA biosynthesis in the endosperm of Arabidopsis seeds, a
355
collection of aad2 and aad3 T-DNA insertion alleles (all in Col-0 background) was
356
obtained and characterized at the molecular level (Figure 5C). The mutants were
357
grown under controlled conditions. Their mature dry seeds were dissected and seed
358
fractions were subjected to biochemical analyses. Determinations of total FA
359
compositions demonstrated that the aad mutations negatively affected ω-7 FA
360
accumulation in the endosperm fraction of seeds (Figure 5D). For each pair of
361
mutants considered, an allelism test was carried out. No complementation could be
362
observed in the F1 hybrid seeds (Supplemental Figure 7), demonstrating that the
363
mutated aad alleles of each pair were allelic and ascertaining the link between the T-
364
DNA insertions and the oil phenotype characterized. Ultimately, the aad2-3 aad3-3
365
double mutant was obtained. Only traces of ω-7 FAs could be detected in the
366
endosperm fraction of the double mutant (Figure 5D), demonstrating that AAD2 and
367
AAD3 are the major contributors to ω-7 FA biosynthesis in this tissue, the relative
368
contributions of the two isoforms being proportional to their respective induction
369
levels (Figure 4G).
370 371
Molecular determination of substrate specificity of AAD2 and AAD3
12
372
Previous studies have established the importance of the side chains of the eight
373
amino acid residues lining the bottom part of the substrate channel of AADs for
374
determining the substrate specificity of the enzymes (Cahoon et al., 1997). The
375
channel of the archetype ∆9 SAD is deep enough to accommodate C18:0 substrates,
376
thus forming ω-9 monoenes (Supplemental Figure 8). A shorter channel is more
377
adapted to C16:0 substrates, yielding a ∆9 PAD activity and the production of ω-7
378
monoenes. These eight amino acid residues were first identified thanks to the crystal
379
structure of a ∆9 SAD from R. communis (Lindqvist et al., 1996). They appear to be
380
well conserved among the SAD isoforms characterized so far in higher plants, as in
381
the Arabidopsis FAB2 protein (Supplemental Figure 8). Alignments of amino acid
382
sequences
383
(http://swissmodel.expasy.org/) (Arnold et al., 2006; Bordoli et al., 2009) and the SAD
384
from R. communis (pdb code: 1AFR; Lindqvist et al., 1996) as a template allowed
385
identification of the corresponding amino acid residues in the AAD2 and AAD3
386
sequences and a proposed model for the substrate channel of these enzymes
387
(Supplemental Figure 8). These analyses revealed three divergent residues with
388
respect to the SAD archetype. To test the importance of these residues in
389
determining the specific PAD activity of AAD2 and AAD3, amino acid substitutions
390
were realized by site-directed mutagenesis in the sequence of FAB2 so as to
391
introduce these residues, alone or in combination, in a SAD sequence. Modified
392
versions of FAB2 were then transiently expressed in leaves of N. benthamiana and
393
the production of ω-7 FAs was monitored in order to detect PAD activities. Plasmids
394
allowing the expression of the Arabidopsis plastidial enolase or that of AAD2 were
395
used
396
overexpression of FAB2 was not sufficient to trigger a significant accumulation of ω-7
397
FAs in transformed leaves, mutagenized versions of FAB2 harboring a T219F
398
mutation, alone or in combination with other substitutions, significantly stimulated the
399
biosynthesis of ω-7 FAs, denoting a PAD activity (Figure 5E). These results strongly
400
suggested that the Phe residue lining the bottom part of the substrate channel of
401
AAD2 and AAD3 (at position 226 or 216, respectively) plays a key role in determining
402
the substrate specificity of these isoforms, most probably by reducing the depth of
403
this channel thanks to its bulky lateral chain.
as
and
3D
negative
structure
and
modeling
positive
using
controls,
404
13
the
SWISS-MODEL
respectively.
Whereas
server
ectopic
405 406 407
DISCUSSION
408 409
Reserve accumulation in maturing seeds is finely regulated. Developmental
410
regulators ensure that the corresponding biosynthetic pathways are activated during
411
the transition phase between embryogenesis and seed maturation, then shut down in
412
late maturing and germinating seeds. Superimposed on this temporal pattern of
413
regulation, spatial control elements regulate the differential partitioning of reserves
414
between seed compartments. Arabidopsis seeds indeed consist of two compartments
415
accumulating storage compounds, namely the endosperm and the embryo, the latter
416
accounting for about 90% of total seed reserves. Early during seed formation,
417
developmental programs establish the embryo as the preponderant tissue within
418
these exalbuminous seeds. Then, transcriptional repressors of the maturation
419
program expressed in the endosperm during the maturation phase further reinforce a
420
differential partitioning of reserves between zygotic tissues (Barthole et al., 2014).
421
Beyond this differential partitioning, the fine characterization of Arabidopsis seeds
422
has revealed the strikingly different compositions of the reserves stored in the two
423
compartments, with endosperm oil exhibiting for instance dramatically increased
424
proportions of the economically important ω-7 monounsaturated FAs (Penfield et al.,
425
2004). Here we demonstrate that the two closely related MYB115 and MYB118 TFs,
426
which are transcriptionally induced by LEC2 in the endosperm at the onset of seed
427
maturation, activate the transcription of two PAD genes, namely AAD2 and AAD3, in
428
this tissue. Transcriptional activation of these two isoforms is necessary and sufficient
429
to promote ω-7 FA biosynthesis in the endosperm. Taken together, these data allow
430
a better understanding of how FA metabolism is developmentally regulated at the
431
spatiotemporal level, adding another level of complexity to the regulatory network
432
controlling reserve accumulation in maturing Arabidopsis seeds.
433 434
Transcriptional activation of MYB115 in the maturing endosperm
435
Detailed characterization of the MYB115 expression pattern based on
436
complementary approaches like RT-qPCR and promoter:GUS analyses established
437
the endosperm-specific induction of this gene in early maturing seeds. These data
438
are fully consistent with the results of transcriptome analyses of developmental series 14
439
of seeds and seed tissues microdissected by laser-capture (Le et al., 2010; Day et
440
al., 2008; Belmonte et al., 2013). They suggest that the closely related MYB115 and
441
MYB118 genes, both induced in the maturing endosperm, may have similar cis-
442
regulatory elements in their promoter sequences. Their respective expression
443
patterns are not strictly identical though. If accumulation of the two mRNA
444
populations dramatically increases at the onset of seed maturation, before
445
decreasing during the course of seed maturation, induction level of MYB115 is much
446
weaker. Then, expression of MYB115 is not restricted to seeds since the activity of
447
the MYB115 promoter is also detected in pollen grains. These observations reflect
448
the ongoing divergent evolution of the promoter sequences of the two paralogs.
449
The pattern of MYB115 promoter activity matches MYB115 mRNA accumulation
450
both at the spatial and temporal levels; therefore MYB115 tissue-specific expression
451
is probably largely controlled at the transcriptional level. Interestingly, the data
452
presented in this study establish that MYB115, like MYB118, is a target of the master
453
regulator LEC2. This common transcriptional activation of the two TFs by LEC2,
454
together with the negative feedback regulation exerted by MYB118 on LEC2
455
expression (Barthole et al., 2014) provides an interesting mechanism explaining how
456
the myb118 mutation can be partially compensated by overexpression of MYB115 in
457
the myb118 mutant background (Figure 6). At the temporal level, the examination of
458
the expression profiles of LEC2, MYB118, and MYB115 is consistent with a
459
transcriptional activation of the MYB TFs by the master regulator. At the spatial level
460
however, the lack of induction of the two MYBs in the embryo is striking considering
461
that LEC2 is expressed in the embryo too (Kroj et al., 2003; Barthole et al., 2014). To
462
reconcile these apparent discrepancies, one has to postulate the existence of
463
endosperm-specific factors directing the expression of MYB115 and MYB118 in this
464
tissue, or that of embryo specific repressors counteracting LEC2 in the embryo.
465
Endosperm development appears to be predominantly under epigenetic control
466
(Berger, 2003; Sun et al., 2010); therefore, it would be interesting to further
467
investigate how these controls affect oil metabolism in the maturing endosperm
468
(Fatihi et al., 2013), possibly through dedicated transcriptional regulators such as
469
MYB115 and MYB118.
470 471
MYB115 and MYB118 have common transcriptional targets
15
472
MYB118 was previously shown to antagonistically regulate distinct gene
473
networks. Whereas several maturation-related genes are repressed by the TF in the
474
maturing endosperm, MYB118 directly triggers the expression of several endosperm-
475
induced genes (Barthole et al., 2014). The data presented in this study demonstrate
476
that sets of target genes are shared by MYB115 and MYB118 in each of the two
477
subcircuits (e.g., GRP19, SM3, and CRUL, or ODD, AAD2, and AAD3). These
478
observations are consistent with a recent report by Zhang et al. (2015) depicting how
479
the two conserved TFs co-control expression of genes encoding enzymes of the
480
benzoyloxy glucosinolate pathway in seeds. Expression analyses supported by yeast
481
one-hybrid assays have shown that several actors (AOP3, BZO1, SCPL17) of this
482
newly evolved biosynthetic pathway are transcriptionally repressed by the two MYBs.
483
Interestingly, the TAACGG element present as a part of the in vivo MYB118-binding
484
site, that may consequently belong to type 1 MYB-binding sites (pAACnG, where p
485
indicates T or C, and n indicates any nucleotide; Romero et al., 1998; Prouse and
486
Campbell, 2012) was identified both in positively and in negatively regulated targets
487
of the MYB TFs under study (Barthole et al., 2014; Zhang et al., 2015). These
488
observations raise the question of (i) the molecular mechanisms specifying the type
489
of regulation exerted by the TFs and of (ii) the identity of the unknown actors
490
participating in these regulations. In the same line, a detailed structure-function study
491
using in vitro techniques like SELEX or Biacore, or in vivo chromatin
492
immunoprecipitation experiments, would be useful to fully characterize the respective
493
DNA-binding matrixes of MYB115 and MYB118 in order to (i) better characterize the
494
spectrum of putative targets of the TFs and (ii) to determine whether these matrixes
495
are identical or not. The amino acid identity shared by the predicted DNA-binding
496
domains of MYB115 and MYB118 (73%) falls within the range of values obtained for
497
other MYBs previously shown to act redundantly. For instance, DNA-binding domains
498
of TT2/MYB123 (At5g35550) and MYB5 (At3g13540) share 72% amino acid identity
499
(Xu et al., 2014). Some of the data presented in this article (e.g., induction levels of
500
target genes in stable Pro35Sdual:MYB lines) indicate that the two MYBs exhibit
501
different behaviors in the presence of certain targets, implying that their binding
502
specificity may have started diverging. The hypothesis according to which these
503
divergences are such that MYB115 and MYB118 only share a subset of targets could
504
explain why the effects of the myb115 and myb118 mutations are additive for some
505
phenotypes (e.g., production of ω-7 monounsaturated FAs or some glucosinolates) 16
506
and not for some others (e.g., total amount of oil stored in the endosperm). However,
507
diverging binding specificities are not the only driving force for differentiating sets of
508
targets between related TFs: evolution of protein-protein interactions between protein
509
partners of a transcriptional complex can also play a key role. In this regard,
510
complementary studies would now be required to characterize the transcriptional
511
complexes involving MYB115 and MYB118.
512 513
Transcriptional control of ω-7 monounsaturated FA biosynthesis
514
The most common monoenes in land plants are of the ω-9 series, as oleic acid.
515
Unlike the ω-9, ω-7 monoenes like palmitoleic acid and its elongation products
516
vaccenic acid and paullinic acid are relatively rare. Plant oils containing ω-7
517
monoenes, though uncommon, are enriched in seeds or fruits of non-crop species
518
like cat’s claw vine (Doxantha unguis-cati), macadamia, or sea buckthorn (Hippophae
519
rhamnoides) (Bondaruk et al., 2007; Fatima et al., 2012). Oils enriched in ω-7 FA
520
were also described in seeds of the Brassicaceae family, but this enrichment solely
521
concerned the endosperm (Penfield et al., 2004; Li et al., 2006). The transcriptional
522
activation cascade described in this study (Figure 6) allows us to understand how two
523
∆9 PADs specifically induced in the maturing endosperm of Arabidopsis seeds,
524
namely AAD2 and AAD3, confer its peculiar FA composition to the oil stored in this
525
tissue. Further work will be required to fully elucidate the molecular mechanisms
526
underpinning the transcriptional activation of these two desaturases by MYB115 and
527
MYB118 and to precisely identify the cis-elements required for this activation.
528
Interestingly, MYB118 exerts antagonistic control on different actors of oil
529
metabolism: while repressing the overall amount of oil stored in the endosperm, this
530
TF promotes the biosynthesis of ω-7 monoenes over that of ω-9, raising the question
531
of the biological function of these molecular species in the endosperm. As for the ω-7
532
FAs present at low levels in the embryo, their biosynthesis may result from a very
533
limited, though detectable, activation of the above-mentioned transcriptional
534
regulatory cascade in this tissue.
535
The position of the unsaturation within the aliphatic chains of monounsaturated
536
FAs contributes to the physicochemical properties of acyl lipids derived from these
537
FAs. The specific functions of ω-7 containing lipids in the plant cell, if any, remain
538
unknown. Under standard growth conditions, development of the aad2 aad3 double
17
539
mutant is unaffected, but the remnant ω-7 FAs present in this genetic background
540
may impair the detection of phenotypes. Complementary studies, possibly requiring
541
the preparation of new multiple mutants for the AAD genes, will be essential to
542
address this question. Regardless of their function in plants, these ω-7 have uses for
543
a number of industrial applications. Biodiesel s produced from plant oils with high ω-7
544
content have superior functional properties (Wu et al., 2012). The ω-7 FAs also have
545
considerable potential as a feedstock for the production of 1-octene by metathesis
546
chemistry (Meier, 2009), 1-octene representing a high-demand feedstock mainly
547
used to make linear low-density polyethylene. Vegetable oils enriched in ω-7 FAs
548
have finally been ascribed a number of beneficial health properties. In animals,
549
adipose tissues use lipokines such as palmitoleic acid to communicate with distant
550
organs and regulate systemic metabolic homeostasis (Cao et al., 2008). There is
551
growing evidence that palmitoleic acid plays a key role in the pathophysiology of
552
insulin resistance in humans, increasing muscle response to insulin (Stefan et al.,
553
2010). Palmitoleic acid is also a candidate anti-melanogenic agent (Yoon et al.,
554
2010). Aside from above-mentioned nutritional functions, palmitoleic acid also has
555
anti-oxidant,
556
Pharmaceutical companies have already developed foods and nutraceuticals for
557
health purposes enriched in ω-7 FAs, often sourced from species exhibiting low
558
yields and poor agronomic properties such as sea buckthorn. In recent years,
559
biotechnical approaches have been implemented to develop specialized high-yielding
560
platforms through the metabolic engineering of oilseed crops (Bondaruk et al., 2007;
561
Nguyen et al., 2015). Findings regarding the control of ω-7 FA biosynthesis in the
562
plant cell may provide new interesting tools for the development of new strategies for
563
ω-7 FA production.
anti-microbial,
and
anti-aging
properties
(Wu
et
al.,
2012).
564 565
AAD2 and AAD3 are ∆9 palmitoyl-ACP desaturases
566
The Arabidopsis thaliana genome contains seven related genes coding for
567
predicted acyl-ACP desaturases, previously named FAB2 and SAD1 to SAD6
568
(Kachroo et al., 2007). FAB2, the best-characterized member of the family, encodes
569
a ∆9 SAD (producing ω-9 FAs) essential for plant development and defense signaling
570
(Lightner et al., 1994; Kachroo et al., 2001). Despite recent advances in the
571
characterization of this multigene family (Klinkenberg et al., 2014), the functions of
18
572
most members of the family have long remained elusive. In the light of the research
573
presented in this article, it appears that two isoforms have diverged from the
574
archetype SAD and exhibit a ∆9 PAD activity responsible for the biosynthesis of ω-7
575
monoenes. We therefore propose to adopt a new nomenclature and to rename the
576
already published SAD1-6 genes ACYL-ACP DESATURASE1-6 (AAD1-6), this
577
general designation encompassing the variety of in planta activities exhibited by the
578
corresponding isoforms. The ∆9 PAD activities of AAD2 and AAD3 are responsible
579
for the production of ω-7 monoenes stored in the endosperm of Arabidopsis seeds.
580
Using a quantitative trait loci (QTL) approach followed by genetic analyses, Bryant
581
and coworkers (2016) have also identified AAD2 and AAD3 as the major desaturases
582
synthesizing ω-7 monoenes in the endosperm. These observations are consistent
583
with previous results from Kachroo et al. (2007), who assayed the enzymatic
584
activities and substrate specificities of several Arabidopsis ∆9 AADs produced in E.
585
coli. These in vitro data already pointed out the preference of DES3/AAD3 for C16:0
586
substrates, yielding a PAD activity. The enzymatic characterization of AAD from
587
Arabidopsis (Kachroo et al., 2007) and other plant species (Cahoon et al., 1998;
588
Rodriguez Rodriguez et al., 2015) then unraveled that, beyond marked preferences
589
of the desaturases for substrates of a given chain length, the enzymes can also
590
desaturate slightly shorter or longer chains, although with a reduced affinity. The
591
traces of ω-7 FAs detected in aad2 aad3 seeds may consequently originate from the
592
activity of SADs like FAB2, which is also expressed in maturing seeds.
593
A group of eight residues lining the bottom part of the substrate channels of
594
AADs was previously shown to set constraints on the chain lengths of FA substrates,
595
thus determining the substrate specificity of the enzymes (Cahoon et al., 1997;
596
Cahoon et al., 1998). Despite the functional divergence of some of its founding
597
members, the Arabidopsis AADs still share a high degree of amino acid sequence
598
similarity and a common structural fold (Kachroo et al., 2007). Interestingly, three of
599
the eight amino acid residues determining substrate-specificity distinguish AAD2 and
600
AAD3 from the archetype SAD structure. The results of our site-directed mutagenesis
601
experiments point out the importance of one of the divergent amino acids identified,
602
namely Phe-226 in AAD2 or -216 in AAD3, for conferring a PAD activity to the
603
desaturases. The bulky lateral chain of this Phe residue may reduce the depth of the
604
substrate pocket, thus favoring the binding of shorter C16:0-ACP substrate. Beyond
19
605
the predominant role played by this residue, the two other divergent residues
606
identified in AAD2 and AAD3 may also contribute to create the substrate profile
607
displayed by these isoforms. A ∆9 PAD from cat’s claw vine was previously cloned
608
and characterized (Cahoon et al., 1998). Despite similar enzymatic activities, the ∆9
609
PAD from Arabidopsis and cat’s claw vine do not have identical substrate channels.
610
In the case of cat’s claw vine, only one residue diverged from the archetype SAD
611
sequence: replacement of a Leu by a Trp residue at the extremity of the channel was
612
presented as the major determinant of substrate specificity for this isoform (Cahoon
613
et al., 1998). During the evolution of AADs, the diversification of which was probably
614
favored by the emergence of multigene families within the genome of higher plants,
615
∆9 PAD activities may therefore have appeared independently in different species.
616
The prevalence of structural rules (e.g., presence of a hydrophobic residue with a
617
bulky lateral chain lining the bottom part of the substrate channel) over highly
618
conserved consensus sequences for determining the substrate specificity of PAD
619
enzymes was further established by metabolic engineering based on combinatorial
620
saturation mutagenesis and logical redesign of desaturases (Whittle and Shanklin,
621
2001; Cahoon et al., 1997; Cahoon and Shanklin, 2000). These approaches have
622
allowed tailoring several original PAD enzymes by reducing the ability of the
623
substrate pocket of SAD enzymes to accommodate the longer 18:0-ACP substrate.
624
Most of the combinations of amino acid substitutions yielding such a result involved
625
the replacement of a hydrophobic residue lining the pocket by a bulkier and still
626
hydrophobic residue.
627
In conclusion, these results exemplify how transcriptional regulations significantly
628
contribute to the differential regulation of FA and oil metabolism in the two zygotic
629
tissues comprising maturing seeds of Arabidopsis, leading to different oil
630
compositions in these adjacent compartments. In the maturing endosperm, two
631
closely related MYB TFs activated by LEC2, namely MYB118 and MYB115, trigger in
632
turn the expression of the AAD2 and AAD3 genes, that code for ∆9 PADs responsible
633
for the biosynthesis of the ω-7 monounsaturated FAs stored at high levels in the
634
endosperm oil. Specialization of oil metabolism in the endosperm arose both from the
635
emergence of an original structure of the substrate channel of one of the members of
636
the AAD family, and from the concomitant set up of a complex transcriptional
637
regulatory cascade able to precisely control the spatiotemporal expression of this
20
638
desaturase. Further work will be required to decipher the upstream molecular
639
mechanisms that allow this cascade to be specifically activated in the endosperm.
640 641
METHODS
642 643
Plant material and growth conditions
644
Arabidopsis thaliana seeds of the Col-0 accession were obtained from the
645
Arabidopsis thaliana resource center for genomics at the Institut Jean-Pierre Bourgin
646
(http://www-ijpb.versailles.inra.fr/) and T-DNA mutant lines (aad2-3, N670942; aad2-
647
4, N584160; aad3-3, N567280; aad3-4, N825777) were ordered from the NASC
648
(http://arabidopsis.info). Lec2-10, lec2-11, myb115-1 and myb118-1 T-DNA mutant
649
lines were described previously in Barthole et al. (2014). Plants were cultured as
650
described in Baud et al. (2007a). Dexamethasone induction experiments using the
651
Pro35S:LEC2:GR construct were carried out as described by Santos Mendoza et al.
652
(2005). To sample embryo and endosperm fractions, seeds excised from siliques
653
were dissected using a scalpel and dissecting tweezers under an optical glass
654
binocular magnifier. Material used for RNA extraction was frozen in liquid nitrogen
655
immediately after harvest, and then stored at -80°C.
656 657
Molecular characterization of T-DNA mutants
658
Plant genomic DNA flanking the T-DNA border of the mutants were amplified by PCR
659
(Supplemental Table 1) and sequenced to confirm the flanking sequence tags
660
identified. Homozygous lines were then isolated for further characterization. RT-PCR
661
analyses were ultimately carried out to analyze gene expression in mutant
662
backgrounds (Supplemental Table 2).
663 664
Constructs and plant transformation
665
The sequences of primers used for DNA amplification are indicated in Supplemental
666
Table 3.
667
Construction of the ProMYB115:uidA transgene: region -998 to -1 bp relative to the
668
MYB115 translational start codon was amplified with the proofreading Pfu Ultra DNA
669
polymerase (Stratagene) from Col-0 genomic DNA. The PCR product was introduced
670
by BP recombination into the pDONR207 entry vector (Invitrogen) and transferred
671
into the destination vector pBI101-R1R2-GUS (Baud et al., 2007b) by LR 21
672
recombination. The resulting binary vector was electroporated into Agrobacterium
673
tumefaciens C58C1 strain and used for agroinfiltration of flower buds of Arabidopsis
674
(Bechtold et al., 1993). Primary transformants were selected on MS medium
675
containing kanamycin (50 mg.l-1) and transferred to soil for further characterization:
676
23 independent transgenic lines were analyzed.
677
Construction of the ProAAD3:uidA transgene: a similar procedure was adopted.
678
Region -2,022 to -1 bp relative to the AAD3 translational start codon was cloned; 12
679
independent transgenic lines were analyzed.
680
Construction of the ProAAD2:uidA transgene: a similar procedure was adopted.
681
Region -1,000 to -1 bp relative to the AAD2 translational start codon was cloned into
682
the pGWB3 vector (Nakagawa et al., 2007); 19 independent transgenic lines were
683
analyzed.
684
Construction of the Pro35Sdual:MYB115, Pro35Sdual:AAD, and Pro35Sdual:FAB2
685
transgenes: the procedure adopted was similar to that described for the construction
686
of Pro35Sdual:MYB107 and Pro35Sdual:MYB118 transgenes (Barthole et al., 2014).
687
Construction of the Pro35Sdual:FAB2m1-7 transgenes: mutations on the FAB2 cDNA
688
cloned in pDONR207 were performed with the QuickChange Site Directed
689
Mutagenesis kit (Agilent) according to the manufacturer’s instructions. Primers used
690
are presented in Supplemental Table 4. Mutagenized cDNAs were then transferred in
691
pMDC32 as described above.
692
Construction
693
DBD:MYB118 transgenes: MYB115 and MYB118 cDNAs previously cloned into the
694
pDONR207 entry vector were transferred into a modified pDEST32 (carrying a
695
kanamycin-resistance gene) for GAL4-DBD fusion (Invitrogen).
696
Construction of the Pro35Sdual:MYB115:mGFP6 and Pro35Sdual:MYB118:mGFP6
697
transgenes: cDNAs without STOP codon were amplified with the proofreading Pfu
698
Ultra DNA polymerase (Stratagene) from a mixture of seed cDNA (Col-0 accession).
699
The PCR products were introduced by BP recombination into the pDONR207 entry
700
vector (Invitrogen) and transferred into the destination vector pMDC83 (Curtis and
701
Grossniklaus, 2003) by LR recombination.
702
Construction of the ProMYB118:MYB118 transgene was previously described in
703
Barthole et al. (2014).
of
the
ProADH1:GAL4-DBD:MYB115
704 705
RNA analyses 22
and
ProADH1:GAL4-
706
RNA extraction, reverse transcription, RT-PCR and real-time RT quantitative PCR
707
were carried out as previously described (Baud et al., 2004). The sequences of
708
primers used for RT-PCR and real-time RT-qPCR are indicated in Supplemental
709
Tables 2 and 5. Purity of the different seed fractions sampled was assessed as
710
described in Barthole et al. (2014). Briefly, marker genes for each of the fractions
711
sampled, namely ZHOUPI (endosperm-specific) and At2g23230 (embryo-specific)
712
were quantified on cDNA prepared from these fractions, thus confirming that no
713
significant contamination occurred between fractions.
714 715
Lipid analyses
716
Total FA analyses were performed as previously described (Li et al., 2006) on leaf
717
disks from agroinfiltrated N. benthamiana, or on pools of Arabidopsis seeds or seed
718
fractions. The endosperm tissue was analyzed with the seed coat attached as in
719
Penfield et al. (2004). However, this procedure did not bias our evaluation of the
720
endosperm oil content since the integuments of the seed do not accumulate storage
721
compounds and in fact undergo programmed cell death early during maturation
722
(Beeckman et al., 2000; Li et al., 2006).
723 724
Electrophoretic mobility shift assays
725
The expression plasmid was constructed by transferring MYB115 cDNA from the
726
pDONR207 to the expression vector pETG10A (http://www.embl-hamburg.de/). The
727
resulting vector was electroporated into E. coli RosettaBlue(DE3)pLysS strain
728
(Novagen)
729
thiogalactopyranoside (IPTG) in LB buffer, cells were grown over night at 20°C. Cell
730
lysis and protein purification were performed as previously described (Baud et al.,
731
2009). To prepare DNA probes, complementary biotin-labeled (at the 5’ end)
732
oligonucleotides (Eurofins MWG Operon) were annealed. For DNA-binding assays,
733
MYB115 recombinant protein was incubated with 30 fmol probe in binding buffer (20
734
mM Tris-HCl pH 8, 250 mM NaCl, 2 mM MgCl2, 1% glycerol (v/v), 1 mg.ml-1 BSA, 1
735
mM DTT, 85 ng µl-1 poly(dI-dC)). For competition assays, the unbiotinylated
736
competitor was incubated briefly with the recombinant protein before the biotinylated
737
probe was added. After addition of the biotinylated probe, reactions were incubated
738
30 min at room temperature, then fractionated at 4°C by 6% PAGE. Electrophoretic
739
transfer to nylon membrane and detection of the biotin-labeled DNA were carried out
for
expression.
After
induction
23
by
1
mM
isopropyl-b-D-
740
according to the manufacturer’s instructions (Chemiluminescent Nucleic Acid
741
Detection Module, PIERCE) using an ImageQuant LAS 4000 system (GE
742
Healthcare).
743 744
Microscopy
745
Histochemical detection of GUS activity and bright-field microscopy observations
746
were carried out as described in Baud et al. (2007a). Leaves of N. benthamiana were
747
imaged with a Zeiss LSM710 confocal microscope as described in Miart et al. (2014).
748 749
Accession Numbers
750
Sequence data from this article can be found in the EMBL/GenBank data libraries
751
under accession numbers: AAD1/SAD1, At5g16240; AAD2/SAD2, At3g02610;
752
AAD3/SAD3,
At5g16230;
AAD4/SAD4,
753
AAD6/SAD6,
At1g43800;
BCCP2,
754
At5g60390; FAB2/SSI2, At2g43710; GRP19, At5g07550; LEC2, At1g28300;
755
MYB107, At3g02940; MYB115, At5g40360; MYB118, At3g27785; ODD, At1g04380;
756
SM3, At2g25890.
At3g02620;
At5g15530;
AAD5/SAD5,
CRUL,
At3g02630;
At1g03890;
EF1αA4,
757 758
Supplemental Data
759
Supplemental Figure 1. Characterization of the regulation of MYB115 by MYB118.
760
Supplemental Figure 2. Characterization of the LEC2-dependent de-repression of
761
MYB115 in myb118-1.
762
Supplemental Figure 3. Characterization of MYB115 overexpressing lines.
763
Supplemental Figure 4. Characterization of myb115-1, myb118-1, and myb115-1
764
myb118-1 seed development.
765
Supplemental Figure 5. Impact of myb115 and myb118 mutations on ω-7 fatty acid
766
accumulation in the embryo of developing seeds.
767
Supplemental Figure 6. Time course analysis of ω-7 monounsaturated fatty acid
768
accumulation in Arabidopsis seeds.
769
Supplemental Figure 7. Complementation tests among the aad mutants.
770
Supplemental Figure 8. Complementary information for the sequences, structures,
771
and functions of acyl-ACP desaturases in Arabidopsis.
772
Supplemental Table 1. Primers used for molecular characterization of T-DNA
773
insertions. 24
774
Supplemental Table 2. Primers used for characterizing gene expression by RT-PCR
775
(as displayed in Figure 5C).
776
Supplemental Table 3. Primers used for construct preparation.
777
Supplemental Table 4. Primers used for site-directed mutagenesis experiments.
778
Supplemental Table 5. Primers used for quantitative RT-PCR.
779 780 781
ACKNOWLEDGMENTS
782
We thank D. Kosma for critical reading of the article, D. de Vos, C. Boulard, A. Wilch,
783
and O. Grandjean (Observatoire du Végétal, INRA-IJPB) for their technical
784
assistance. This work was supported by the French National Research Agency
785
(SOLAR, grant no. ANR-10-GENM-009) and by the Research Executive Agency
786
(TRIANON, grant no. PIEF-GA-2013-625204). The IJPB benefits from the support of
787
the Labex Saclay Plant Sciences-SPS (ANR-10-LABX-0040-SPS). The confocal
788
equipment used in this study was partly financed by the Ile-de-France Region.
789 790 791
AUTHORS CONTRIBUTIONS
792
A.T., G.T., and M.M. performed the research and analyzed the data, L.L. designed
793
the research and analyzed the data, M.A. T.-P., G.B. and S.B. designed and
794
performed the research, analyzed the data, and wrote the article.
795 796
25
797
REFERENCES
798
Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL
799
workspace: a web-based environment for protein structure homology modelling.
800
Bioinformatics 22,195-201.
801
Barthole, G., To, A., Marchive, C., Brunaud, V., Soubigou-Taconnat, L., Berger,
802
N., Dubreucq, B., Lepiniec, L., and Baud, S. (2014). MYB118 represses
803
endosperm maturation in seeds of Arabidopsis. Plant Cell 26: 3519-3537.
804 805
Baud, S., Boutin, J.P., Miquel, M., Lepiniec, L., and Rochat, C. (2002). An
806
integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant
807
Physiol. Biochem. 40: 151-160.
808 809
Baud, S., Vaultier, M.-N., and Rochat, C. (2004). Structure and expression profile of
810
the sucrose synthase multigene family in Arabidopsis. J. Exp. Bot. 55: 397-409.
811 812
Baud, S., Wuillème, S., Dubreucq, B., de Almeida, A., Vuagnat, C., Lepiniec, L.,
813
Miquel, M., and Rochat, C. (2007a). Function of plastidial pyruvate kinase in seeds
814
of Arabidopsis thaliana. Plant J. 52: 405-419.
815 816
Baud, S., Santos Mendoza, M., To, A., Harscoët, E., Lepiniec, L., and Dubreucq,
817
B. (2007b). WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2
818
towards fatty acid metabolism during seed maturation in Arabidopsis. Plant J. 50:
819
825-838.
820 821
Baud, S., Wuillème, S., To, A., Rochat, C., and Lepiniec, L. (2009). Role of
822
WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic
823
genes in Arabidopsis. Plant J. 60: 933-947.
824 825
Baud, S., and Lepiniec, L. (2010). Physiological and developmental regulation of
826
seed oil production. Prog. Lipid Res. 49: 235-249.
827 828
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta infiltration of adult
26
829
Arabidopsis plants. C. R. Acad. Sci. Paris Life Sci. 316: 1194-1199.
830 831
Beeckman, T., De Rycke, R., Viane, R., and Inze, D. (2000). Histological study of
832
seed coat development in Arabidopsis thaliana. J. Plant Res. 113: 139-148.
833 834
Belmonte, M.F., Kirkbride, R.C., Stone, S.L., Pelletier, J.M., Bui, A.Q., Yeung,
835
E.C., Hashimoto, M., Fei, J., Harada, C.M., Munoz, M.D., Le, B.H., Drews, G.N.,
836
Brady,
837
developmental profiles of gene activity in regions and subregions of the Arabidopsis
838
seed. Proc. Natl. Acad. Sci. USA 110: 435-444.
S.M.,
Goldberg,
R.B.,
and
Harada,
J.J.
(2013).
Comprehensive
839 840
Bryant, F.M., Munoz-Ascarate, O., Kelly, A.A., Beaudoin, F., Kurup, S., and
841
Eastmond, P.J. (2016) ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 are
842
responsible for making omega-7 fatty acids in the Arabidopsis aleurone. Plant
843
Physiol. 172: 154-162.
844 845
Berger, F. (2003). Endosperm: the crossroad of seed development. Curr. Opin. Plant
846
Biol. 6: 42-50.
847 848
Bondaruk, M., Johnson, S., Degafu, A., Boora, P., Bilodeau, P., Morris, J.,
849
Wiehler, W., Foroud, N., Weselake, R., and Shah, S. (2007). Expression of a cDNA
850
encoding palmitoyl-acyl carrier protein desaturase from cat’s claw (Doxantha unguis-
851
cati L.) in Arabidopsis thaliana and Brassica napus leads to accumulation of unusual
852
unsaturated fatty acids and increased stearic acid content in the seed oil. Plant
853
Breed. 126: 186-194.
854 855
Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., and Schwede, T. (2009).
856
Protein structure homology modelling using SWISS-MODEL workspace. Nat. Protoc.
857
4: 1-13.
858 859
Cahoon, E.B., Lindqvist, Y., Schneider, and G., Shanklin, J. (1997). Redesign of
860
soluble fatty acid desaturases from plants for altered substrate specificity and double
861
bond position. Proc. Natl. Acad. Sci. USA 94: 4872-4877.
862 27
863
Cahoon, E.B., Shah, S., Shanklin, J., and Browse, J. (1998). A determinant of
864
substrate specificity predicted from the acyl-acyl carrier protein desaturase of
865
developing cat’s claw seed. Plant Physiol. 117: 593-598.
866 867
Cahoon,
E.B.,
and
Shanklin,
J.
(2000).
Substrate-dependent
mutant
868
complementation to select fatty acid desaturase variants for metabolic engineering of
869
plant seed oils. Proc. Natl. Acad. Sci. USA 97: 12350-12355.
870 871
Cao, H.M., Gerhold, K., Mayers, J.R., Wiest, M.M., Watkins, S.M., and
872
Hotamisligil, G.S. (2008). Identification of a lipokine, a lipid hormone linking adipose
873
tissue to systemic metabolism. Cell 134: 933-944.
874 875
Cernac, A., and Benning, C. (2004). WRINKLED1 encodes an AP2/EREB domain
876
protein involved in the control of storage compound biosynthesis in Arabidopsis.
877
Plant J. 40: 575-585.
878 879
Curtis, M., and Grossniklaus, U. (2003). A Gateway TM cloning vector set for high-
880
throughput functional analysis of genes in plants. Plant Physiol. 133: 462-469.
881 882
Day, R.C., Herridge, R.P., Ambrose, B.A., and Macknight, R.C. (2008).
883
Transcriptome analysis of proliferating Arabidopsis endosperm reveals biological
884
implications for the control of syncytial division, cytokinin signaling, and gene
885
expression regulation. Plant Physiol. 148: 1964-1984.
886 887
Dubos, C., Stracke, R., Grotewold, E., Weisshaar, E., Martin, C., and Lepiniec, L.
888
(2010). MYB transcription factors in Arabidopsis. Trends Plant Sci. 15: 573-581.
889 890
Fatihi, A., Zbierzak, A.M., and Dörmann, P. (2013). Alterations in seed
891
development gene expression affect size and oil content of Arabidopsis seeds. Plant
892
Physiol. 163: 973-985.
893 894
Fatima, T., Snyder, C.L., Schroeder, W.R., Cram, D., Datla, R., Wishart, D.,
895
Weselake, R.J., and Krishna, P. (2012). Fatty acid composition of developing sea
896
buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature 28
897
seed. PLOS ONE 7: e34099.
898 899
Harwood, J. (1996). Recent advances in the biosynthesis of plant fatty acids.
900
Biochim. Biophys. Acta 1301: 7-56.
901 902
Kachroo, P., Shanklin, J., Shah, J., Whittle, E.J., and Klessig, D.F. (2001). A fatty
903
acid desaturase modulates the activation of defense signaling pathways in plants.
904
Proc. Natl. Acad. Sci. USA 98: 9448-9453.
905 906
Kachroo, A., Shanklin, J., Whittle, E., Lapchyk, L., Hildebrand, D., and Kachroo,
907
P. (2007). The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the
908
accumulation of leaf isoforms to oleic acid synthesis. Plant Mol. Biol. 63: 257-271.
909 910
Klinkenberg, J., Faist, H., Saupe S., Lambertz, S., Krischke, M., Stingl, N.,
911
Fekete, A., Mueller, M.J., Feussner, I., Hedrich, R., and Deeken, R. (2014). two
912
fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN ∆9-DESATURASES6
913
and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress
914
signaling in Arabidopsis crown galls. Plant Physiol. 164: 570-583.
915 916
Kroj, T., Savino, G., Valon, C., Giraudat, J., and Parcy, F. (2003). Regulation of
917
storage protein gene expression in Arabidopsis. Development 130: 6065-6073.
918 919
Le, B.H., Cheng, C., Bui, A.Q., Wagmaister, J.A., Henry, K., Pelletier, J., Kwong,
920
L., Belmonte, M., Kirkbride, R., Horvath, S., Drews, G.N., Fischer, R.L.,
921
Okamuro, J.K., Harada, J.J., and Goldberg, R.B. (2010). Global analysis of gene
922
activity during Arabidopsis seed development and identification of seed-specific
923
transcription factors. Proc. Natl. Acad.Sci. USA 107: 8063-8070.
924 925
Li, Y., Beisson, F., and Ohlrogge, J. (2006). Oil content of Arabidopsis seeds: the
926
influence of seed anatomy, light and plant-to-plant variation. Phytochemistry 67: 904-
927
915.
928 929
Lightner, J., Wu, J., and Browse, J. (1994). A mutant of Arabidopsis with increased
930
levels of stearic acid. Plant Physiol. 106: 1443-1451. 29
931 932
Lindqvist, Y., Huang, W., Schneider, G., and Shanklin, J. (1996). Crystal structure
933
of Δ9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to
934
other di-iron proteins. EMBO J. 15: 4081-4092.
935 936
Lotan, T., Ohto, M., Yee, K.M., West, M.A., Lo, R., Kwong, R.W., Yamagishi, K.,
937
Fischer, R.L., Goldberg, R.B., and Harada, J.J. (1998). Arabidopsis LEAFY
938
COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell,
939
93: 1195-1205.
940 941
Martin, K., Kopperud, K., Chakrabarty, R., Banerjee, R., Brooks, R., and Goodin,
942
M.M. (2009). Transient expression in Nicotiana benthamiana fluorescent marker lines
943
provides enhanced definition of protein localization, movement and interactions in
944
planta. Plant J. 59: 150-162.
945 946
Meier, M.A.R. (2009). Metathesis with oleochemicals: new approaches for the
947
utilization of plant oils as renewable resources in polymer science. Macromol. Chem.
948
Phys. 210: 1073-1079.
949 950
Mendes, A., Kelly, A.A., van Erp, H., Shaw, E., Powers, S.J., Kurup, S., and
951
Eastmond, P.J. (2013). bZIP67 regulates the omega-3 fatty acid content of
952
Arabidopsis seed oil by activating FATTY ACID DESATURASE3. Plant Cell 25:
953
3104-3116.
954 955
Miart, F., Desprez, T., Biot, E., Morin, H., Belcram, K., Höfte, H., Gonneau, M.,
956
and Vernhettes, S. (2014). Spatio-temporal analysis of cellulose synthesis during
957
cell plate formation in Arabidopsis. Plant J. 77: 71-84.
958 959
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y.,
960
Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007). Development of
961
series of Gateway binary vectors, pGWBs, for realizing efficient construction of fusion
962
genes for plant transformation. J. Biosci. Bioeng. 104: 34-41.
963
30
964
Nguyen, H.T., Park, H., Koster, K.L., Cahoon, R.E., Nguyen, H.T.M., Shanklin, J.,
965
Clemente, T.E., and Cahoon, E.B. (2015). Redirection of metabolic flux for higher
966
levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds. Plant
967
Biotech. J. 13: 38-50.
968 969
Penfield, S., Rylott, E.L., Gilday, A.D., Graham, S., Larson, T.R., and Graham,
970
I.A. (2004). Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl
971
elongation
972
PHOSPHOENOLPYRUVATE CARBOXYKINASE1. Plant Cell 16: 2705-2718.
in
the
dark,
is
dependent
of
abscisic
acid,
and
requires
973 974
Prouse, M.B., and Campbell, M.M. (2012). The interaction between MYB proteins
975
and their target DNA binding sites. Bioch. Biophys. Acta 1819: 67-77.
976 977
Rodríguez Rodríguez M.F., Sánchez-Garcia, A., Salas, J.J., Garcés, R., and
978
Martínez-Force, E. (2015). Characterization of soluble acyl-ACP desaturases from
979
Camelina sativa, Macadamia tetraphylla and Dolichandra unguis-cati. J. Plant
980
Physiol. 178: 35-42.
981 982
Romero, I., Fuertes, A., Benito, M., Malpica, J., Levya, A., and Paz-Ares, J.
983
(1998). More than 80 R2R3-MYB regulatory genes in the genome of Arabidopsis
984
thaliana. Plant J. 14: 273-284.
985 986
Roscoe, T.T., Guilleminot, J., Bessoule, J.-J., Berger, F., and Devic, M. (2015).
987
Complementation of seed maturation phenotypes by ectopic expression of ABSCISIC
988
ACID INSENSITIVE3, FUSCA3 and LEAFY COTYLEDON2 in Arabidopsis. Plant Cell
989
Physiol. 56: 1215-1228.
990 991
Santos Mendoza, M., Dubreucq, B., Miquel, M., Caboche, M., and Lepiniec, L.
992
(2005). LEAFY COTYLEDON2 activation is sufficient to trigger the accumulation of
993
oil and seed specific mRNAs in Arabidopsis leaves. FEBS Lett. 579: 4666-4670.
994 995
Santos Mendoza, M., Dubreucq, B., Baud, S., Parcy, F., Caboche, M., and
996
Lepiniec, L. (2008). Deciphering gene regulatory networks that control seed
997
development and maturation in Arabidopsis. Plant J. 54: 608-620. 31
998 999
Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M.,
1000
Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005). A gene expression map of
1001
Arabidopsis thaliana development. Nat. Genet. 37, 501-506.
1002 1003
Stefan, N., Kantartzis, K., Celebi, N., Staiger, H., Machann, J., Schick, F., Cegan,
1004
A., Elcnerova, M., Schleicher, E., Fritsche, A., and Häring, H.U. (2009).
1005
Circulating palmitoleate strongly and independently predicts insulin sensitivity in
1006
humans. Diabetes Care 33: 405-407.
1007 1008
Sun, X., Shantharaj, D., Kang, X., and Ni, M. (2010). Transcriptional and hormonal
1009
signaling control of Arabidopsis seed development. Curr. Opin. Plant Biol. 13: 611-
1010
620.
1011 1012
Suzuki, M., and McCarty, D.R. (2008). Functional symmetry of the B3 network
1013
controlling seed development. Cur. Opin. plant Biol. 11: 548-553.
1014 1015
Vicente-Carbajosa, J., and Carbonero, P. (2005). Seed maturation: developing an
1016
intrusive phase to accomplish a quiescent state. Int. J. Dev. Biol. 49: 645-651.
1017 1018
Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, S. (2003). An enhanced
1019
transient expression system in plants based on suppression of gene silencing by the
1020
p19 protein of tomato bushy stunt virus. Plant J. 33: 949-956.
1021 1022
Wang, X., Niu, Q.-W., Teng, C., Li, C., Mu, J., Chua, N.-H., and Zuo, J. (2008).
1023
Overexpression of PGA37/MYB118 and MYB115 promotes vegetative-to-embryonic
1024
transition in Arabidopsis. Plant Cell Res. 19: 224-235.
1025 1026
Whittle, E., and Shanklin, J. (2001). Engineering ∆9-16:0-acyl carrier protein (ACP)
1027
desaturase specificity based on combinatorial saturation mutagenesis and logical
1028
redesign of the castor ∆9-18:0-ACP desaturase. J. Biol. Chem. 276: 21500-215005.
1029
32
1030
Wu, Y., Li, R., and Hildebrand, D.F. (2012). Biosynthesis and metabolic engineering
1031
of palmitoleate production, an important contributor to human health and sustainable
1032
industry. Prog. Lipid Res. 51: 240-249.
1033 1034
Xu, W., Grain, D., Bobet, S., Le Gourrierec, J., Thévenin, J., Kelemen, Z.,
1035
Lepiniec, L., and Dubos, C. (2014) Complexity and robustness of the flavonoid
1036
transcriptional regulatory network revealed by comprehensive analyses of MYB-
1037
bHLHL-WDR complexes and their targets in Arabidopsis seed. New Phytol. 202: 132-
1038
144.
1039 1040
Yoon, W.J., Kim, M.J., Moon, J.Y., Kang, H.J., Kim, G.O., Lee, N.H., and Hyun,
1041
C.G. (2010). Effect of palmitoleic acid on melanogenic protein expression in murine
1042
b16 melanoma. J. Oleo. Sci. 59: 315-319.
1043 1044
Zhang, Y., Cao, G., Qu, L.-J., and Gu, H. (2009). Involvement of an R2R3-MYB
1045
transcription factor gene AtMYB118 in embryogenesis in Arabidopsis. Plant Cell Rep.
1046
28: 337-346.
1047 1048
Zhang, Y., Li, B., Huai, D., Zhou, Y., and Kliebenstein, D.J. (2015). The conserved
1049
transcription factors, MYB115 and MYB118, control expression of the newly evolved
1050
benzoyloxy glucosinolate pathway in Arabidopsis thaliana. Front. Plant Sci. 13: 343.
33
Figure 1. Expression pattern and transcriptional regulation of MYB115. (A-C) Analysis of relative mRNA accumulation of MYB115 was performed in different plant organs (A), in developing seeds (B), and in developmental series of endosperm and embryo fractions (C). The results obtained are standardized to the EF1αA4 (EF) gene expression level. Values are the means and SE of three to six replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. Cl, cauline leaves; Fl, flowers; ND, not detected; Rl, rosette leaves; Ro, roots; St, stems.(D-N) Pattern of activity of the ProMYB115:uidA cassette in flowers (D), in stamens (E), in pollen grains (F), in developing seeds harvested 6 (G), 8 (H), 10 (I) or 12 (J) DAA, and in early maturing embryos harvested 6 (K), 8 (L), 10 (M) or 12 (N) DAA. For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 0.2 mM each of potassium ferrocyanide and potassium ferricyanide. The results for GUS activity were observed on whole-mounted inflorescences; microscopy observations of stamens, pollen grains, seeds and excised embryos were performed using Nomarski optics. Bars = 1 mm in (D), 50 μm in (E), 20 μm in (F), 100 μm in (G-J), and 50 μm in (K-N). (O) Accumulation of MYB115 mRNA in lec2 mutant seeds was quantified 10 DAA by RT-qPCR and presented as the percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three to six replicates carried out on three independent cDNA preparations obtained from batches of seeds dissected from four to five siliques. The three silique sets were harvested on distinct individuals. ***, Significant difference from WT according to t-test, P < 0.001. (P) Accumulation of MYB115 mRNA in leaves of transgenic Pro35S:LEC2:GR, Pro35S:TTG1:GR (negative control), or wild-type 10-day-old plants cultured in vitro on a medium with (+ DEX) or without (-DEX) 10-5 M dexamethasone for two additional weeks, was quantified by RT-qPCR and presented as percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three to four replicates carried out on three independent cDNA preparations. (Q) Time-course analysis of MYB115 mRNA accumulation in leaves of 10-day-old Pro35S:LEC2:GR plants transferred to a growth medium containing 10-5 M dexamethasone (induction) and grown two weeks in vitro. Accumulation of mRNA was determined by RT-qPCR and presented as the percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three to six replicates carried out on three independent cDNA preparations. (R) Transgenic ProMYB115:uidA x Pro35S:LEC2:GR seedlings were transferred 10 days after germination on a dexamethasone-containing medium (10-5 M; + DEX). Rosette leaves were analyzed two weeks after induction. For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 0.2 mM each of potassium ferrocyanide and potassium ferricyanide. Bars = 0.2 cm. (S) Accumulation of MYB115 mRNA in myb118 mutant seeds (myb118-1 and OE3) was quantified 10 DAA by RTqPCR and presented as the percentage of the EF1αA4 (EF) gene expression. Values are the means and SE of three replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. OE3, Pro35Sdual:MYB118 transgenic line exhibiting a strong repression of MYB118 expression (Barthole et al., 2014); WT, wild type (Col-0). ***, **: Significant difference from WT according to t-test at P < 0.001 and 0.01, respectively. DAA, days after anthesis; WT, wild type.
Figure 2. Functional characterization of MYB118 and MYB115. (A) Confocal micrographs showing localization of MYB115:GFP and MYB118:GFP fusion proteins in transgenic Nicotiana benthamiana plants expressing RFP:H2B (Martin et al., 2009). Plants were co-infiltrated with the Pro35Sdual:MYB:GFP construct and a vector allowing the expression of the p19 protein of tomato bushy stunt virus (TBSV) that prevents the onset of post-transcriptional gene silencing (Voinnet et al., 2003). GFP was observed 4 days after infiltration. Bar = 50 µm. (B) Transcriptional activity of MYB115 and MYB118. MYB115 and MYB118 coding sequences were cloned in frame with the GAL4 DNA-binding domain (DBD). The fusion constructs were introduced into reporter yeast containing the HIS3 and ADE2 reporter genes, before being plated on appropriate media to maintain the expression of the vectors (SD-Leu) and to test the activation of the HIS3 (SD-Leu-His) or HIS3 and ADE2 reporter genes (SD-Leu-His-Ade). Data presented are representative from the results obtained for eight independent colonies. SD, synthetic drop-out medium. (C,D) RT-qPCR analysis of transcript abundance for negatively (C) and positively (D) regulated targets of MYB118 in cDNA prepared from wild-type (WT; Col-0) and mutant seeds harvested 10 days after anthesis. Values are the means and SE of 12 replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. ***, **, and *: Significant difference from wild type (WT) according to t-test at P < 0.001, 0.01, and 0.05, respectively. (E) RT-qPCR analysis of transcript abundance in cDNA prepared from rosette leaves of Pro35Sdual:MYB115 lines (OE11 and OE19). Values are the means and SE of nine replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. ** and * indicate significant difference from wild type (WT) according to ttest at P < 0.01 and 0.05, respectively. (F) Transactivation assay in leaves of N. benthamiana. Schematic representations of the reporter constructs used are presented. Open boxes indicate TAACGG elements and the closed box represents the 35S cauliflower mosaic virus minimal promoter. Pro:uidA reporter constructs alone or in combination with a vector allowing the expression of MYB115, MYB118 or MYB107 (negative control) were co-infiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs were assayed for GUS activity three days after infiltration. Tissues were incubated 17 h in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Representative discs (diameter = 0.8 cm) are presented. TAACGG concatemer, promoter sequence made of a concatemer of TAACGG elements separated by ten nucleotides (Barthole et al., 2014). (G) Binding of MYB115 to the proximal upstream region of ODD. Electrophoretic mobility shift assay (EMSA) of a probe covering a region from -240 to -200 upstream from the ATG codon of ODD with increasing amounts of MYB115 (‘+’ = 0.5 µg, ‘++’ = 1.5 µg). WRI1 was used as a negative control. Competition of MYB115 binding was carried out in the presence of 75-, 100-, and 200-fold amounts of the unlabeled ProODD(-240 to -200 bp) fragment. Position of free probe (open arrowhead) and the shifted bands (closed arrowhead) are indicated. (H) Binding of MYB115 to the proximal upstream regions of targets of MYB118, namely At5g01030, At3g62230, and At3g12880. The promoter sequence of the ODD gene (At1g04380) was used as a positive control. For each gene considered, the promoter region covered by the probe is indicated between brackets.
Figure 3. Impact of myb115 and myb118 mutations on the accumulation of ω-7 fatty acids in the endosperm fraction of developing seeds. (A) Schematic representation of mature Arabidopsis seeds. (B,C) Time-course analysis of total fatty acid (FA) content (A) and relative proportions of ω-7 FAs (cis-ω-7 C18:1 and cis-ω-7 C20:1) (B) in whole endosperm fractions dissected from wild-type and mutant seeds. Values are the means and SE of five replicates performed on batches of 20 to 40 seeds from five distinct plants. ***, **, and *: Significant difference according to t-test at P < 0.001, 0.01, and 0.05, respectively. (D) ω-7 FA (cis-ω-7 C18:1 and cis-ω-7 C20:1) content of whole endosperm fractions dissected from wild-type, myb118-1, or complemented mature dry seeds. Values are the means and SE of five replicates performed on batches of 20 seeds from five distinct plants. (E-G) ω-7 fatty acid content (cis-ω-7 C18:1 and cis-ω-7 C20:1) of whole endosperm fractions (E), peripheral endosperm fractions (F), and basal endosperm fractions (G) dissected from mature seeds. Values are the means and SE of five replicates performed on batches of 40 seeds from five distinct plants. . ***: Significant difference according to t-test at P < 0.001.
Figure 4. AAD genes induced by MYB115 and MYB118. (A-C) RT-qPCR analysis of transcript abundance in cDNA prepared from myb115-1, myb118-1, and myb115-1 myb118-1 mutant seeds harvested 10 DAA (A), from rosette leaves of Pro35Sdual:MYB118 lines (OE1 and OE2) (B), or from rosette leaves of Pro35Sdual:MYB115 lines (OE11 and OE19) (C). Values are the means and SE of 3 to 9 replicates carried out on cDNA dilutions obtained from three (B,C) or four (A) independent mRNA extractions. ***, **, and *: Significant difference from wild type (WT) according to t-test at P < 0.001, 0.01, and 0.05, respectively. (D) Transactivation assay in leaves of Nicotiana benthamiana. Pro:uidA reporter constructs alone or in combination with a vector allowing the expression of MYB115, MYB118 or MYB107 (negative control) were coinfiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs were assayed for GUS activity three days after infiltration. Tissues were incubated 4 h in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Representative discs (diameter = 0.8 cm) are presented. (E,F) Pattern of activity of the ProAAD2:uidA (E) and ProAAD3:uidA (F) cassettes in developing seeds harvested 8, 10 or 12 DAA (main pictures, from left to right), and in early maturing embryos harvested 8, 10 or 12 DAA (secondary small pictures, from left to right). For histochemical detection of GUS activity, tissues were incubated overnight in a buffer containing 2 mM each of potassium ferrocyanide and potassium ferricyanide. Microscopy observations of seeds and excised embryos were performed using Nomarski optics. Bars = 100 µm. (G) Analysis of relative mRNA accumulation of AAD2 and AAD3 was performed in developmental series of endosperm and embryo fractions. The results obtained are standardized to the EF1αA4 (EF) gene expression level. Values are the means and SE of three to six replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. Endo., endosperm fraction. DAA, days after anthesis.
Figure 5. Functional characterization of AAD2 and AAD3. (A) Transient expression assay in leaves of Nicotiana benthamiana. Pro35Sdual:AAD constructs were coinfiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs harvested five days after infiltration were subjected to total fatty acid (FA) analyses to determine the relative proportion of ω-7 FAs (cis-ω-7 C18:1) in this material. Values are the means and SE of ten replicates performed on batches of 2 disks from two to three distinct plants. ***: Significant difference from control according to ttest at P < 0.001. (B) Stable overexpression of AAD2 and AAD3 in transgenic A. thaliana lines. RT-qPCR analysis of transcript abundance in cDNA prepared from rosette leaves of the transgenic lines were first carried out to assess efficient overexpression of the transgenes (upper panel). Values are the means and SE of three replicates carried out on cDNA dilutions obtained from three independent mRNA extractions. Rosette leaves were then subjected to total FA analyses to determine the relative proportion of ω-7 FAs (cis-ω-7 C18:1) in this material (lower panel). Values are the means and SE of ten replicates performed on batches of two leaf tips from ten distinct plants. ***, **: Significant difference from WT according to t-test at P < 0.001 and 0.01, respectively. (C) Molecular characterization of aad2 and aad3 mutants. Structure of the AAD2 and AAD3 genes showing the position of T-DNA insertions in aad2-3, aad2-4, aad3-3, and aad3-4 are presented. For each T-DNA insertion considered, confirmed flanking sequence tag(s) are anchored in the gene structure and represented by vertical bar(s). Closed boxes represent exons and open boxes untranslated regions (UTRs). Accumulation of AAD2 and AAD3 mRNA in wild-type and corresponding mutant backgrounds was studied by reverse transcriptase polymerase chain reaction (RT-PCR) on developing seeds harvested 16 days after anthesis. EF1αA4 (EF) gene expression was used as a constitutive control. Primers used for this study are indicated by arrows (see Supplemental Table 2). (D) Relative proportion of ω-7 FAs (cis-ω-7 C18:1 and cis-ω-7 C20:1) in endosperm fractions dissected from wildtype and aad mature dry seeds. Values are the means and SE of five replicates performed on batches of 20 seeds from five distinct plants. ***: Significant difference from WT according to t-test at P < 0.001. (E) Site-directed mutagenesis experiments followed by transient expression assays in leaves of N. benthamiana. Constructs allowing the expression of the plastidial enolase (At1g74030; negative control), FAB2, AAD2, and mutagenized versions of FAB2 (FAB2m1-7) were co-infiltrated in young leaves of N. benthamiana with a vector allowing the expression of the p19 protein. Leaf discs harvested five days after infiltration were subjected to total FA analyses to determine the relative proportion of ω-7 FAs (cis-ω-7 C18:1) in this material. Values are the means and SE of 20 replicates performed on batches of 2 disks from four distinct plants. Statistical analyses of the data were performed using a variance analysis (ANOVA), followed by a comparison of means using the Newman–Keuls (SNK) test (P < 0.05). A schematic view of the eight residues lining the bottom part of the substrate pockets of the desaturases assayed is presented (see also Supplemental Figure 8). WT, wild type (Col-0).
Figure 6. Model for the regulation of ω-7 monoene fatty acid production in Arabidopsis seeds. MYB115 and MYB118 are induced in the endosperm at the onset of the maturation phase. The master regulator LEC2 activates their transcription, and MYB118 exerts negative feedback regulation on LEC2. MYB115 and MYB118 coordinately trigger the transcription of two soluble ∆9 palmitoyl-ACP desaturases, namely AAD2 and AAD3. These enzymes catalyze the synthesis of ω-7 monounsaturated fatty acids, which are accumulated at high levels in the oil stored within the endosperm. The transcriptional negative regulatory loop involving LEC2 and MYB118 was previously described in Barthole et al. (2014).
Parsed Citations Arnold, K., Bordoli, L., Kopp, J., and Schwede, T. (2006). The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22,195-201. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Barthole, G., To, A., Marchive, C., Brunaud, V., Soubigou-Taconnat, L., Berger, N., Dubreucq, B., Lepiniec, L., and Baud, S. (2014). MYB118 represses endosperm maturation in seeds of Arabidopsis. Plant Cell 26: 3519-3537. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Baud, S., Boutin, J.P., Miquel, M., Lepiniec, L., and Rochat, C. (2002). An integrated overview of seed development in Arabidopsis thaliana ecotype WS. Plant Physiol. Biochem. 40: 151-160. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Baud, S., Vaultier, M.-N., and Rochat, C. (2004). Structure and expression profile of the sucrose synthase multigene family in Arabidopsis. J. Exp. Bot. 55: 397-409. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Baud, S., Wuillème, S., Dubreucq, B., de Almeida, A., Vuagnat, C., Lepiniec, L., Miquel, M., and Rochat, C. (2007a). Function of plastidial pyruvate kinase in seeds of Arabidopsis thaliana. Plant J. 52: 405-419. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Baud, S., Santos Mendoza, M., To, A., Harscoët, E., Lepiniec, L., and Dubreucq, B. (2007b). WRINKLED1 specifies the regulatory action of LEAFY COTYLEDON2 towards fatty acid metabolism during seed maturation in Arabidopsis. Plant J. 50: 825-838. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Baud, S., Wuillème, S., To, A., Rochat, C., and Lepiniec, L. (2009). Role of WRINKLED1 in the transcriptional regulation of glycolytic and fatty acid biosynthetic genes in Arabidopsis. Plant J. 60: 933-947. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Baud, S., and Lepiniec, L. (2010). Physiological and developmental regulation of seed oil production. Prog. Lipid Res. 49: 235-249. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Bechtold, N., Ellis, J., and Pelletier, G. (1993). In planta infiltration of adult Arabidopsis plants. C. R. Acad. Sci. Paris Life Sci. 316: 1194-1199. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Beeckman, T., De Rycke, R., Viane, R., and Inze, D. (2000). Histological study of seed coat development in Arabidopsis thaliana. J. Plant Res. 113: 139-148. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Belmonte, M.F., Kirkbride, R.C., Stone, S.L., Pelletier, J.M., Bui, A.Q., Yeung, E.C., Hashimoto, M., Fei, J., Harada, C.M., Munoz, M.D., Le, B.H., Drews, G.N., Brady, S.M., Goldberg, R.B., and Harada, J.J. (2013). Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed. Proc. Natl. Acad. Sci. USA 110: 435-444. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Bryant, F.M., Munoz-Ascarate, O., Kelly, A.A., Beaudoin, F., Kurup, S., and Eastmond, P.J. (2016) ACYL-ACYL CARRIER PROTEIN DESATURASE2 and 3 are responsible for making omega-7 fatty acids in the Arabidopsis aleurone. Plant Physiol. 172: 154-162. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Berger, F. (2003). Endosperm: the crossroad of seed development. Curr. Opin. Plant Biol. 6: 42-50. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Bondaruk, M., Johnson, S., Degafu, A., Boora, P., Bilodeau, P., Morris, J., Wiehler, W., Foroud, N., Weselake, R., and Shah, S.
(2007). Expression of a cDNA encoding palmitoyl-acyl carrier protein desaturase from cat's claw (Doxantha unguis-cati L.) in Arabidopsis thaliana and Brassica napus leads to accumulation of unusual unsaturated fatty acids and increased stearic acid content in the seed oil. Plant Breed. 126: 186-194. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Bordoli, L., Kiefer, F., Arnold, K., Benkert, P., Battey, J., and Schwede, T. (2009). Protein structure homology modelling using SWISS-MODEL workspace. Nat. Protoc. 4: 1-13. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Cahoon, E.B., Lindqvist, Y., Schneider, and G., Shanklin, J. (1997). Redesign of soluble fatty acid desaturases from plants for altered substrate specificity and double bond position. Proc. Natl. Acad. Sci. USA 94: 4872-4877. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Cahoon, E.B., Shah, S., Shanklin, J., and Browse, J. (1998). A determinant of substrate specificity predicted from the acyl-acyl carrier protein desaturase of developing cat's claw seed. Plant Physiol. 117: 593-598. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Cahoon, E.B., and Shanklin, J. (2000). Substrate-dependent mutant complementation to select fatty acid desaturase variants for metabolic engineering of plant seed oils. Proc. Natl. Acad. Sci. USA 97: 12350-12355. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Cao, H.M., Gerhold, K., Mayers, J.R., Wiest, M.M., Watkins, S.M., and Hotamisligil, G.S. (2008). Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism. Cell 134: 933-944. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Cernac, A., and Benning, C. (2004). WRINKLED1 encodes an AP2/EREB domain protein involved in the control of storage compound biosynthesis in Arabidopsis. Plant J. 40: 575-585. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Curtis, M., and Grossniklaus, U. (2003). A Gateway TM cloning vector set for high-throughput functional analysis of genes in plants. Plant Physiol. 133: 462-469. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Day, R.C., Herridge, R.P., Ambrose, B.A., and Macknight, R.C. (2008). Transcriptome analysis of proliferating Arabidopsis endosperm reveals biological implications for the control of syncytial division, cytokinin signaling, and gene expression regulation. Plant Physiol. 148: 1964-1984. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Dubos, C., Stracke, R., Grotewold, E., Weisshaar, E., Martin, C., and Lepiniec, L. (2010). MYB transcription factors in Arabidopsis. Trends Plant Sci. 15: 573-581. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Fatihi, A., Zbierzak, A.M., and Dörmann, P. (2013). Alterations in seed development gene expression affect size and oil content of Arabidopsis seeds. Plant Physiol. 163: 973-985. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Fatima, T., Snyder, C.L., Schroeder, W.R., Cram, D., Datla, R., Wishart, D., Weselake, R.J., and Krishna, P. (2012). Fatty acid composition of developing sea buckthorn (Hippophae rhamnoides L.) berry and the transcriptome of the mature seed. PLOS ONE 7: e34099. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Harwood, J. (1996). Recent advances in the biosynthesis of plant fatty acids. Biochim. Biophys. Acta 1301: 7-56. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Kachroo, P., Shanklin, J., Shah, J., Whittle, E.J., and Klessig, D.F. (2001). A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Proc. Natl. Acad. Sci. USA 98: 9448-9453. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Kachroo, A., Shanklin, J., Whittle, E., Lapchyk, L., Hildebrand, D., and Kachroo, P. (2007). The Arabidopsis stearoyl-acyl carrier protein-desaturase family and the accumulation of leaf isoforms to oleic acid synthesis. Plant Mol. Biol. 63: 257-271. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Klinkenberg, J., Faist, H., Saupe S., Lambertz, S., Krischke, M., Stingl, N., Fekete, A., Mueller, M.J., Feussner, I., Hedrich, R., and Deeken, R. (2014). two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN (9-DESATURASES6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls. Plant Physiol. 164: 570-583. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Kroj, T., Savino, G., Valon, C., Giraudat, J., and Parcy, F. (2003). Regulation of storage protein gene expression in Arabidopsis. Development 130: 6065-6073. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Le, B.H., Cheng, C., Bui, A.Q., Wagmaister, J.A., Henry, K., Pelletier, J., Kwong, L., Belmonte, M., Kirkbride, R., Horvath, S., Drews, G.N., Fischer, R.L., Okamuro, J.K., Harada, J.J., and Goldberg, R.B. (2010). Global analysis of gene activity during Arabidopsis seed development and identification of seed-specific transcription factors. Proc. Natl. Acad.Sci. USA 107: 8063-8070. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Li, Y., Beisson, F., and Ohlrogge, J. (2006). Oil content of Arabidopsis seeds: the influence of seed anatomy, light and plant-to-plant variation. Phytochemistry 67: 904-915. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Lightner, J., Wu, J., and Browse, J. (1994). A mutant of Arabidopsis with increased levels of stearic acid. Plant Physiol. 106: 14431451. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Lindqvist, Y., Huang, W., Schneider, G., and Shanklin, J. (1996). Crystal structure of ?9 stearoyl-acyl carrier protein desaturase from castor seed and its relationship to other di-iron proteins. EMBO J. 15: 4081-4092. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Lotan, T., Ohto, M., Yee, K.M., West, M.A., Lo, R., Kwong, R.W., Yamagishi, K., Fischer, R.L., Goldberg, R.B., and Harada, J.J. (1998). Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell, 93: 1195-1205. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Martin, K., Kopperud, K., Chakrabarty, R., Banerjee, R., Brooks, R., and Goodin, M.M. (2009). Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced definition of protein localization, movement and interactions in planta. Plant J. 59: 150-162. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Meier, M.A.R. (2009). Metathesis with oleochemicals: new approaches for the utilization of plant oils as renewable resources in polymer science. Macromol. Chem. Phys. 210: 1073-1079. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Mendes, A., Kelly, A.A., van Erp, H., Shaw, E., Powers, S.J., Kurup, S., and Eastmond, P.J. (2013). bZIP67 regulates the omega-3 fatty acid content of Arabidopsis seed oil by activating FATTY ACID DESATURASE3. Plant Cell 25: 3104-3116. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Miart, F., Desprez, T., Biot, E., Morin, H., Belcram, K., Höfte, H., Gonneau, M., and Vernhettes, S. (2014). Spatio-temporal analysis of cellulose synthesis during cell plate formation in Arabidopsis. Plant J. 77: 71-84. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Nakagawa, T., Kurose, T., Hino, T., Tanaka, K., Kawamukai, M., Niwa, Y., Toyooka, K., Matsuoka, K., Jinbo, T., and Kimura, T. (2007). Development of series of Gateway binary vectors, pGWBs, for realizing efficient construction of fusion genes for plant transformation. J. Biosci. Bioeng. 104: 34-41. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Nguyen, H.T., Park, H., Koster, K.L., Cahoon, R.E., Nguyen, H.T.M., Shanklin, J., Clemente, T.E., and Cahoon, E.B. (2015). Redirection of metabolic flux for higher levels of omega-7 monounsaturated fatty acid accumulation in camelina seeds. Plant Biotech. J. 13: 38-50. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Penfield, S., Rylott, E.L., Gilday, A.D., Graham, S., Larson, T.R., and Graham, I.A. (2004). Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is dependent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1. Plant Cell 16: 2705-2718. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Prouse, M.B., and Campbell, M.M. (2012). The interaction between MYB proteins and their target DNA binding sites. Bioch. Biophys. Acta 1819: 67-77. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Rodríguez Rodríguez M.F., Sánchez-Garcia, A., Salas, J.J., Garcés, R., and Martínez-Force, E. (2015). Characterization of soluble acyl-ACP desaturases from Camelina sativa, Macadamia tetraphylla and Dolichandra unguis-cati. J. Plant Physiol. 178: 35-42. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Romero, I., Fuertes, A., Benito, M., Malpica, J., Levya, A., and Paz-Ares, J. (1998). More than 80 R2R3-MYB regulatory genes in the genome of Arabidopsis thaliana. Plant J. 14: 273-284. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Roscoe, T.T., Guilleminot, J., Bessoule, J.-J., Berger, F., and Devic, M. (2015). Complementation of seed maturation phenotypes by ectopic expression of ABSCISIC ACID INSENSITIVE3, FUSCA3 and LEAFY COTYLEDON2 in Arabidopsis. Plant Cell Physiol. 56: 1215-1228. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Santos Mendoza, M., Dubreucq, B., Miquel, M., Caboche, M., and Lepiniec, L. (2005). LEAFY COTYLEDON2 activation is sufficient to trigger the accumulation of oil and seed specific mRNAs in Arabidopsis leaves. FEBS Lett. 579: 4666-4670. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Santos Mendoza, M., Dubreucq, B., Baud, S., Parcy, F., Caboche, M., and Lepiniec, L. (2008). Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis. Plant J. 54: 608-620. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Schmid, M., Davison, T.S., Henz, S.R., Pape, U.J., Demar, M., Vingron, M., Scholkopf, B., Weigel, D., and Lohmann, J.U. (2005). A gene expression map of Arabidopsis thaliana development. Nat. Genet. 37, 501-506. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Stefan, N., Kantartzis, K., Celebi, N., Staiger, H., Machann, J., Schick, F., Cegan, A., Elcnerova, M., Schleicher, E., Fritsche, A., and Häring, H.U. (2009). Circulating palmitoleate strongly and independently predicts insulin sensitivity in humans. Diabetes Care 33: 405-407. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Sun, X., Shantharaj, D., Kang, X., and Ni, M. (2010). Transcriptional and hormonal signaling control of Arabidopsis seed development. Curr. Opin. Plant Biol. 13: 611-620. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Suzuki, M., and McCarty, D.R. (2008). Functional symmetry of the B3 network controlling seed development. Cur. Opin. plant Biol. 11: 548-553. Pubmed: Author and Title
CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Vicente-Carbajosa, J., and Carbonero, P. (2005). Seed maturation: developing an intrusive phase to accomplish a quiescent state. Int. J. Dev. Biol. 49: 645-651. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, S. (2003). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33: 949-956. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Wang, X., Niu, Q.-W., Teng, C., Li, C., Mu, J., Chua, N.-H., and Zuo, J. (2008). Overexpression of PGA37/MYB118 and MYB115 promotes vegetative-to-embryonic transition in Arabidopsis. Plant Cell Res. 19: 224-235. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Whittle, E., and Shanklin, J. (2001). Engineering (9-16:0-acyl carrier protein (ACP) desaturase specificity based on combinatorial saturation mutagenesis and logical redesign of the castor (9-18:0-ACP desaturase. J. Biol. Chem. 276: 21500-215005. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Wu, Y., Li, R., and Hildebrand, D.F. (2012). Biosynthesis and metabolic engineering of palmitoleate production, an important contributor to human health and sustainable industry. Prog. Lipid Res. 51: 240-249. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Xu, W., Grain, D., Bobet, S., Le Gourrierec, J., Thévenin, J., Kelemen, Z., Lepiniec, L., and Dubos, C. (2014) Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-bHLHL-WDR complexes and their targets in Arabidopsis seed. New Phytol. 202: 132-144. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Yoon, W.J., Kim, M.J., Moon, J.Y., Kang, H.J., Kim, G.O., Lee, N.H., and Hyun, C.G. (2010). Effect of palmitoleic acid on melanogenic protein expression in murine b16 melanoma. J. Oleo. Sci. 59: 315-319. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Zhang, Y., Cao, G., Qu, L.-J., and Gu, H. (2009). Involvement of an R2R3-MYB transcription factor gene AtMYB118 in embryogenesis in Arabidopsis. Plant Cell Rep. 28: 337-346. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Zhang, Y., Li, B., Huai, D., Zhou, Y., and Kliebenstein, D.J. (2015). The conserved transcription factors, MYB115 and MYB118, control expression of the newly evolved benzoyloxy glucosinolate pathway in Arabidopsis thaliana. Front. Plant Sci. 13: 343. Pubmed: Author and Title CrossRef: Author and Title Google Scholar: Author Only Title Only Author and Title
Transcriptional Activation of Two Palmitoyl-ACP ∆9 Desaturase Genes by MYB115 and MYB118 is Critical for Biosynthesis of Omega-7 Monounsaturated Fatty Acid in the Endosperm of Arabidopsis Seeds Manuel Adrian Troncoso-Ponce, Guillaume Barthole, Geoffrey Tremblais, Alexandra To, Martine Miquel, Loic Lepiniec and Sébastien Baud Plant Cell; originally published online September 28, 2016; DOI 10.1105/tpc.16.00612 This information is current as of September 28, 2016 Supplemental Data
http://www.plantcell.org/content/suppl/2016/09/28/tpc.16.00612.DC1.html
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