Post on 24-Feb-2020
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Short title: MYBs regulate secondary metabolism 1
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Author for Contact details: Qiao Zhao (qzhao@mail.tsinghua.edu.cn) 3
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MYB20, MYB42, MYB43 and MYB85 Regulate Phenylalanine and 5
Lignin Biosynthesis during Secondary Cell Wall Formation 6
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Pan Geng,a Su Zhang,
a,b Jinyue Liu,
a Cuihuan Zhao,
a Jie Wu,
a,b Yingping Cao,
c 8
Chunxiang Fu,c Xue Han,
d Hang He,
d and Qiao Zhao
a,1 9
aCenter for Plant Biology, School of Life Sciences, Tsinghua University, Beijing 10
100084, China 11
bTsinghua University-Peking University, Joint Center for Life Sciences, Beijing 12
100084, China 13
cShandong Technology Innovation Center of Synthetic Biology, Key Laboratory of 14
Biofuels, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese 15
Academy of Sciences, Qingdao, Shandong, 266101, China 16
dState Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center 17
for Life Sciences, School of Advanced Agriculture Sciences and School of Life 18
Sciences, Peking University, Beijing, China 19
1Author for contact: qzhao@mail.tsinghua.edu.cn 20
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One-sentence Summary: MYB proteins regulate carbon flow into the 22
phenylpropanoid pathway for lignin biosynthesis. 23
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Author contributions: P.G. and Q.Z. designed the research. P.G., S.Z., J.L., C.Z., 25
J.W., Y.C. X.H., H.H., and C.F. performed the experiments. P.G. and Q.Z. wrote the 26
manuscript. 27
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Plant Physiology Preview. Published on December 23, 2019, as DOI:10.1104/pp.19.01070
Copyright 2019 by the American Society of Plant Biologists
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ABSTRACT 30
Lignin is a phenylpropanoid-derived polymer that functions as a major 31
component of cell walls in plant vascular tissues. Biosynthesis of the aromatic amino 32
acid phenylalanine provides precursors for many secondary metabolites including 33
lignins and flavonoids. Here, we discovered that MYB transcription factors MYB20, 34
MYB42, MYB43, and MYB85 are transcriptional regulators that directly activate 35
lignin biosynthesis genes and phenylalanine biosynthesis genes during secondary wall 36
formation in Arabidopsis (Arabidopsis thaliana). Disruption of MYB20, MYB42, 37
MYB43, and MYB85 resulted in growth development defects and substantial 38
reductions in lignin biosynthesis. In addition, our data showed that these MYB 39
proteins directly activated transcriptional repressors that specifically inhibit flavonoid 40
biosynthesis, which competes with lignin biosynthesis for phenylalanine precursors. 41
Together, our results provide important insights into the molecular framework for the 42
lignin biosynthesis pathway. 43
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Key words: Lignin biosynthesis, phenylalanine biosynthesis, primary metabolism, 45
secondary metabolism 46
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INTRODUCTION 48
Plant cell walls are commonly classified into primary and secondary based on 49
their composition and cellular location. In general, all plant cells form primary cell 50
walls during cell division, while secondary cell walls are produced after cessation of 51
cell division and expansion only in specific types of cells, such as vessel elements and 52
fibers (Cosgrove and Jarvis, 2012). Secondary cell walls play important roles in 53
providing long-distance water transport, mechanical support, and plant defense (Zhao 54
and Dixon, 2014; Hoson and Wakabayashi, 2015). 55
The main components of typical secondary cell walls are cellulose, 56
hemicellulose, and lignin. Lignins are phenylpropanoid-derived polymers resulting 57
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from oxidative polymerization of three major hydroxycinnamyl alcohols (Bonawitz 58
and Chapple, 2010; Zhao, 2016). Natural lignin polymers are only composed of 59
p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) as basic units, however 60
occasionally some unconventional units such as coniferaldehyde, sinapaldehyde, 61
catechyl (C), and 5-hydroxyl guaiacyl (5-OH-G) units are also incorporated into 62
lignin monomers (Weng et al., 2010; Chen et al., 2012; Chen et al., 2013; Zhao et al., 63
2013). 64
It is widely accepted that lignin biosynthesis is controlled by regulatory 65
hierarchy involving NAC (no apical meristem (NAM), Arabidopsis transcription 66
activation factor (ATAF1/2), and cup-shaped cotyledon (CUC2)) and MYB 67
(myeloblastosis) transcription factors (Zhao, 2016; Ohtani and Demura, 2018). In this 68
network, NAC proteins including VASCULAR-RELATED NAC-DOMAIN 1-7 69
(VND1-7) and NAC SECONDARY WALL THICKNING PROMOTING FACTOR 70
1-3 (NST1-3) were identified in Arabidopsis (Arabidopsis thaliana) to serve as a first 71
layer of transcription factors (Kubo et al., 2005; Mitsuda et al., 2007). These NACs 72
are master regulators in various cell types. NST3, also called SND1, together with 73
NST1 redundantly regulates secondary cell wall formation in fibers (Mitsuda et al., 74
2007) and NST1 and NST2 activate the same process in the endothecium of anthers 75
(Mitsuda et al., 2005). MYB46 and MYB83 in Arabidopsis, the downstream targets of 76
the NAC proteins, are a second layer of the network, redundantly regulating 77
secondary cell wall formation (McCarthy et al., 2009; Ko et al., 2014). Recently two 78
types of cis-element sequences were reported by two different groups, Secondary wall 79
MYB-Responsive Element (SMRE) ACC(A/T)A(A/C)(T/C) and MYB46-Responsive 80
cis-Regulatory Element (M46RE) (T/C)ACC (A/T)A(A/C)(T/C) (Kim et al., 2012; 81
Zhong and Ye, 2012). Comprehensive survey of these cis-elements and DNA-protein 82
binding assays identified the direct targets of MYB46/83 including MYB genes 83
implicated in the regulation of lignin biosynthesis (for instance, MYB58, MYB63, 84
MYB103, and KNAT7), secondary cell wall biosynthesis genes such as cellulose 85
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synthase genes (CesA4, CesA7, and CesA8), xylan biosynthesis genes (IRX7, IRX8, 86
and IRX9), and lignin biosynthesis genes (PAL1, C4H, 4CL1, CCoAOMT, HCT, 87
CCR1, and F5H1) (Kim et al., 2012; Kim et al., 2013; Kim et al., 2013; Kim et al., 88
2014; Wang et al., 2019). 89
It was previously revealed that the majority of promoters of lignin biosynthesis 90
genes contain so-called AC elements, including AC-I (ACCTACC), AC-II 91
(ACCAACC), and AC-III (ACCTAAC), that are recognized by transcription factors 92
(Raes et al., 2003). MYB58 and MYB63 were shown to activate lignin biosynthesis 93
genes by directly targeting the AC elements in Arabidopsis (Zhou et al., 2009). 94
Members from subfamily 4 of the R2R3-MYB family can also target the AC elements 95
to function as transriptional repressors of the lignin biosynthesis pathway. 96
AmMYB308 and AmMYB330 in Antirrhinum majus, EgMYB1 and EgMYB2 in 97
Eucalyptus gunnii, PtMYB1, PtMYB4, PtMYB8, and PtMYB14 in Pinus teada, 98
PgMYB1, PgMYB8, PgMYB14, and PgMYB15 in Picea glauca, ZmMYB31 and 99
ZmMYB42 in maize (Zea mays), and Arabidopsis MYB4, MYB32, and MYB75 were 100
all reported to directly regulate lignin biosynthesis genes (Tamagnone et al., 1998; Jin 101
et al., 2000; Patzlaff et al., 2003; Preston et al., 2004; Goicoechea et al., 2005; 102
Bhargava et al., 2010; Fornale et al., 2010; Legay et al., 2010; Bomal et al., 2013). 103
The synthesis of lignin monomers involves the phenylpropanoid pathway, which 104
is also shared by many other metabolites such as suberin, flavonoids, tannins, and 105
lignans (Fraser and Chapple, 2011). The aromatic amino acid phenylalanine, the 106
end-product of the shikimate pathway, serves as the precursor of downstream 107
phenylpropanoids (Maeda and Dudareva, 2012; Tohge et al., 2013). Lignin 108
biosynthesis is a high energy consuming and irreversible process; therefore, it is 109
critical that lignin deposition is tightly controlled to avoid overconsumption of 110
phenylalanine. Transcription factors have been identified that coordinately regulate 111
the expression of phenylalanine biosynthesis and downstream metabolism of 112
phenylalanine. Ectopic expression of Petunia hybrida transcription factor 113
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ODORANT1 in tomato can induce shikimate biosynthesis genes and 114
phenylpropanoid-associated genes (Dal Cin et al., 2011). Green tea MYB4 negatively 115
regulates the phenylpropanoid and shikimate biosynthesis by directly targeting the AC 116
elements of promoters of the genes involved in the two pathways (Li et al., 2017). 117
L-phenylalanine is an aromatic amino acid required not only for protein 118
biosynthesis, but also for many secondary metabolites such as flavonoids, condensed 119
tannins, lignans, and lignins (Maeda and Dudareva, 2012). In plants, phenylalanine is 120
produced by two alternative pathways. Most phenylalanine is produced via the 121
chloroplast-localized arogenate pathway (Maeda et al., 2010). In addition, a cytosolic 122
post-chorismate phenylalanine biosynthesis pathway has also been functionally 123
characterized (Yoo et al., 2013; Qian et al., 2019). It was proposed that the alternative 124
cytosolic pathway may contribute to produce specialized phenylalanine-derived 125
metabolites in response to biotic and abiotic stress in plants (Qian et al., 2019). 126
MYB20, MYB42, MYB43, and MYB85 were previously identified as secondary 127
cell wall related transcription factors based on a genome-wide transcriptome analysis 128
of NST1 downregulated mutants in Arabidopsis (Zhong et al., 2008). Dominant 129
repression of MYB85, which is a transcriptional activator, substantially reduced 130
secondary wall thickening in fiber cells, and overexpression of MYB85 caused 131
ectopic deposition of lignin in epidermal and cortical cells in stems. However, how 132
these MYBs are involved in secondary cell wall formation remains elusive. In this 133
study, we report that MYB20, MYB42, MYB43, and MYB85 redundantly activate 134
both phenylalanine and lignin biosynthesis. In addition, MYB20, MYB42, MYB43, 135
and MYB85 activate the transcription factor MYB4, which represses flavonoid 136
biosynthesis to optimize phenylalanine flux to the lignin biosynthesis pathway. This 137
discovery uncovers another level of complexity in the regulatory network of plant 138
lignin biosynthesis. 139
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RESULTS 141
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MYB20, MYB42, MYB43, and MYB85 Genes are Regulated by NST3 and MYB46 142
It was previously revealed that several unknown transcription factors are under 143
the control of SND1 and NST1, including two NACs (SND2 and SND3) and eight 144
MYB genes (MYB20, MYB42, MYB43, MYB52, MYB54, MYB69, MYB85, and 145
MYB103) (Zhong et al., 2008). Phylogenetic analysis demonstrated that MYB42 and 146
MYB85 are closely related to each other, while MYB20 and MYB43 are likely more 147
closely related to one another (Supplemental Fig. S1). Furthermore, the two 148
subgroups are also closely related, but are remotely related to the previously reported 149
lignin transcriptional activators MYB58 and MYB63 (Supplemental Fig. S1). 150
Therefore, we selected MYB20, MYB42, MYB43, and MYB85 to functionally 151
characterize their roles in secondary cell wall biosynthesis. Consistent with the fact 152
that MYB20, MYB42, MYB43, and MYB85 are regulated by NST3, the transcript levels 153
of all four MYB genes were reduced in the double mutant of nst1/3 (Fig. 1A). 154
MYB46, which is a direct target of NST3, is also a master regulator of secondary cell 155
wall biosynthesis, potentially controlling the expression of MYB20, MYB42, MYB43, 156
and MYB85. To determine whether MYB46 and NST3 can activate MYB20, MYB42, 157
MYB43, and MYB85 transcription, we performed transient expression assays in 158
Nicotiana benthamiana leaves. The promoters of MYB20, MYB42, MYB43, and 159
MYB85 were respectively fused with Luciferase (LUC) to generate PMYB20-LUC, 160
PMYB42-LUC, PMYB43-LUC, and PMYB85-LUC. MYB46 and NST3 were driven by 35S 161
promoter to act as effectors. Coexpression of MYB46 or NST3 can activate 162
PMYB20-LUC, PMYB42-LUC, PMYB43-LUC, and PMYB85-LUC activity (Fig. 1, B and C). 163
However, neither NST1-3 nor MYB46 and MYB83 were able to bind these promoters 164
in yeast one-hybrid assays (Supplemental Fig. S2). 165
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Simultaneous Disruption of MYB20, MYB42, MYB43, and MYB85 causes Plant 167
Growth Defects 168
To further investigate the function of these genes, the T-DNA insertion lines of 169
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myb20-1 (GK-109C11-012312), myb42-1 (SALKseq_78190.1), myb43-1 170
(SALK_085312), and myb85-1 (SALK_052089) were characterized and the insertion 171
positions were verified by genomic sequencing (Fig. 1D). Reverse transcription PCR 172
(RT-PCR) showed that these T-DNA insertion lines were null mutants (Fig. 1E). . The 173
double mutants myb20/43 and myb42/85 exhibited a normal growth phenotype, 174
similar to the wild type (Fig. 2A). However, the quadruple mutant myb20/42/43/85 175
showed a semi-dwarfism phenotype compared to myb20/43 and myb42/85, indicating 176
genetic redundancy among these genes (Fig. 2A). In addition, the quadruple mutant 177
also showed shorter siliques with compromised fertility (Fig. 2, B-D). 178
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MYB20, MYB42, MYB43, and MYB85 Coordinately Regulate Phenylalanine 180
and Lignin Biosynthesis 181
To determine the possible effects on gene expression of disrupting MYB20, 182
MYB42, MYB43, and MYB85, we used high-throughput mRNA sequencing to identify 183
differentially expressed genes in the quadruple mutant myb20/42/43/85, compared 184
with wild-type plants. In the quadruple mutant myb20/42/43/85, a total of 2,596 genes 185
were expressed at levels significantly different from wild type (Supplemental Datasets 186
S1 and S2). KEGG pathway analysis showed that biosynthesis of secondary 187
metabolites was changed greatly (Supplemental Fig. S3) and genes involved in lignin 188
and phenylalanine biosynthesis showed reduced expression in the quadruple mutant. 189
To further verify the RNA sequencing data, we performed Reverse transcription 190
quantitative PCR (RT-qPCR) to compare gene expression among the double mutants 191
myb20/43 and myb42/85, the quadruple mutant myb20/42/43/85, and wild type. 192
myb20/42/43/85 plants exhibited reduced transcript levels of lignin biosynthesis genes 193
such as PAL1, 4CL1, C4H, CSE, HCT, and CAD4, while the reduction was lesser 194
severe in both double mutants (Fig. 3B). The transcript levels of ADT4, ADT5, and 195
ADT6, which are specifically involved in phenylalanine biosynthesis, were greatly 196
reduced in myb20/42/43/85 plants, but were not significantly changed in the myb20/43 197
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and myb42/85 mutants, indicating genetic redundancy among the MYB genes (Fig. 198
3A). On the other hand, expression of ADH2, TSA1, and TSB1, involved in tyrosine 199
and tryptophan biosynthesis was not significantly changed in myb20/42/43/85 mutants 200
even though these genes are also related to the shikimate biosynthesis pathway (Fig. 201
3A). 202
The thioacidolysis method was used to determine the lignin composition of 203
double mutants myb20/43 and myb42/85, and the quadruple mutant myb20/42/43/85. 204
As shown in Fig. 3D, the G and S subunits were significantly reduced in 205
myb20/42/43/85 plants, while the reduction was less severe in the two double mutants, 206
which is consistent with the growth phenotypes and gene expression analysis (Fig. 2A 207
and Fig. 3B). To further confirm the impact on lignin biosynthesis of disrupting 208
MYB20, MYB42, MYB43, and MYB85, the total lignin content of mutants and 209
control plants was quantified by Klason analysis. As shown in Supplemental Fig. S4, 210
myb20/42/43/85 plants produced significantly reduced lignin, while double mutants 211
myb20/43 and myb42/85 contained similar lignin content as wild-type plants. 212
Phenylalanine content was also quantified using the same mature stem tissues 213
used for lignin measurements. Interestingly, both myb20/43 and myb42/85 214
accumulated greatly elevated levels of phenylalanine, and myb20/42/43/85 plants 215
contained even more phenylalanine than the double mutants (Fig. 3C). These results 216
were not consistent with the fact that ADT genes were downregulated in the mutants. 217
However, the increase of the free form of phenylalanine may be due to the reduction 218
of downstream phenylalanine-derived lignin biosynthesis. 219
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MYB20, MYB42, MYB43, and MYB85 Activate Phenylalanine and Lignin 221
Biosynthesis Gene Expression 222
To investigate the molecular mechanism underlying the roles of the MYB 223
transcription factors in lignin and phenylalanine biosynthesis pathways, yeast 224
one-hybrid assay was performed. We found that full-length MYB proteins had very 225
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low expression levels in the EGY48 yeast strain, but truncated MYB proteins could be 226
easily expressed (Supplemental Fig. S5). Therefore, we used truncated proteins in the 227
yeast one-hybrid assay. ADT6 and HCT were selected as representative phenylalanine 228
and lignin biosynthesis genes, respectively. MYB20, MYB42, and MYB85 were able 229
to activate the LacZ reporter genes driven by the ADT6 and HCT promoters, while 230
MYB43 could only activate the LacZ reporter gene driven by the HCT promoter (Fig. 231
4A and Fig. 5A). We utilized the electrophoretic mobility shift assay (EMSA) to 232
further verify the direct binding to the ADT6 and HCT promoters. All four 233
recombinant MBP-MYB fusion proteins caused an up-shift of the labeled ADT6 234
promoter or HCT promoter. By contrast, MBP alone did not generate the same 235
mobility shift (Fig. 4, C-F and Fig. 5, C-F). The schematic diagrams of the promoters 236
in yeast one-hybrid assays and EMSA assays are shown in Fig. 4B and Fig. 5B. 237
To further confirm that MYB20, MYB42, MYB43, and MYB85 were able to 238
activate the expression of ADT6 and HCT, we performed a transient expression assay 239
in Arabidopsis protoplasts. Coexpression of MYB20, MYB42, MYB43, or MYB85 240
dramatically activated PADT6-LUC and PHCT-LUC activity in Arabidopsis Col-0 241
protoplasts (Fig. 4G and Fig. 5G). To further examine whether each MYB is able to 242
activate the genes independently, a similar transient expression assay was carried out 243
in Arabidopsis myb20/42/43/85 mutant protoplasts. It turned out that each MYB was 244
able to activate downstream targets alone (Fig. 4H and Fig. 5H), suggesting that these 245
four MYBs are not interdependent. 246
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MYB20, MYB42, MYB43, and MYB85 Optimize Phenylalanine Distribution in 248
the Phenylpropanoid Biosynthesis Pathway 249
Our RNA sequencing data indicated that the previously identified 250
phenylpropanoid pathway transcription factor MYB7 was downregulated in 251
myb20/42/43/85 compared with the wild type (Supplemental Datasets S2). Through 252
RT-qPCR analysis, we showed that not only MYB7 but also two homologous genes, 253
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MYB4 and MYB32, showed lower transcript levels in the myb20/42/43/85 mutant 254
compared with the wild type (Fig. 6A). Transient expression assays in N. 255
benthamiana leaves indicated that MYB20, MYB42, MYB43, and MYB85 activate 256
the promoters of MYB4 (Fig. 6B). EMSA was performed to investigate whether MYB4 257
was directly targeted, as described previously (Zhao et al., 2007). EMSA showed that 258
all four MBP-MYB proteins could bind the MYB4 promoter, but not the MBP tag (Fig. 259
6, C-F). 260
It has been reported that MYB4 can repress the expression of Chalcone Synthase 261
(CHS), encoding the enzyme that catalyzes the first step of flavonoid biosynthesis, 262
which competes with lignin biosynthesis for phenylalanine as a precursor (Jin et al., 263
2000). Consistent with this finding, the transcript level of CHS was significantly 264
enhanced in myb20/42/43/85 compared with the wild type, and increased even more 265
in the myb4/7/32 mutant (Fig. 7A). The results suggest that MYB20, MYB42, 266
MYB43, and MYB85 activate MYB4, MYB7, and MYB32, resulting in downregulation 267
of CHS expression. To investigate the possible effects on the phenylpropanoid 268
biosynthesis pathway of disrupting MYB20, MYB42, MYB43, and MYB85, we 269
compared flavonoid accumulation among myb20/42/43/85, myb4/7/32, and wild-type 270
plants. As shown in Fig. 7B, kaempferol 3-O-[6"-O-(rhamnosyl) glucoside] 271
7-O-rhamnoside (K1), kaempferol 3-O-glucoside 7-O-rhamnoside (K2), and 272
kaempferol 3-O-rhamnoside 7-O-rhamnoside (K3) accumulated at significantly 273
higher amounts in myb20/42/43/85 and myb4/7/32. In addition, myb20/43, myb42/85, 274
and myb20/42/43/85 also accumulated enhanced levels of anthocyanin (Supplemental 275
Fig. S6). These data suggest that downregulation of the transcription repressors MYB4, 276
MYB7, and MYB32 in myb20/42/43/85 leads to enhanced flavonoid biosynthesis due 277
to elevated expression of CHS. 278
279
DISCUSSION 280
During the last decade, the transcriptional regulation of secondary cell wall 281
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formation has been extensively studied. Lignin biosynthesis has been shown to be 282
under the control of the NAC-MYB-based transcriptional regulatory network (Ohtani 283
and Demura, 2018). NAC proteins as master switches coordinately regulate the 284
entirety of secondary cell wall formation including cellulose, hemicellulose, and 285
lignin (Zhong et al., 2010). Lignin biosynthesis is derived from the aromatic amino 286
acid phenylalanine and the deposition process is believed to be irreversible. Therefore, 287
lignin biosynthesis is not only coordinately regulated along with other secondary cell 288
wall components, but also interacts with primary metabolic pathways related to 289
phenylalanine biosynthesis and other branches of the phenylpropanoid pathway (Li et 290
al., 2016; Ohtani et al., 2016). 291
MYB58 and MYB63 were first reported in Arabidopsis as lignin-specific 292
transcriptional activators (Zhou et al., 2009). These MYBs are able to directly activate 293
lignin biosynthesis genes, including early genes such as PAL, C4H, and 4CL that are 294
shared by other branches of the phenylpropanoid pathway such as flavonoid 295
biosynthesis. MYB4 was shown to directly target lignin biosynthesis genes as 296
repressors (Shen et al., 2012; Li et al., 2017). Currently, there is no evidence that 297
MYB58/63 and MYB4 dynamically regulate lignin biosynthesis. MYB103, a 298
NST1-regulated transcription factor, was recently suggested to be involved in syringyl 299
lignin biosynthesis (Ohman et al., 2013). However, MYB103 did not directly target 300
FERULATE-5-HYDROXYLASE (F5H), which is required for syringyl lignin 301
formation (Ohman et al., 2013). 302
Phenylalanine derived from the shikimate pathway, which also gives rise to the 303
other two aromatic amino acids tyrosine and tryptophan, serves as a precursor for 304
many secondary metabolites such as lignins, flavonoids, coumarins, and lignans 305
(Maeda and Dudareva, 2012). Since lignin deposition is a very energy-consuming and 306
non-reversible process, it is critical that phenylalanine distribution among the 307
phenylpropanoid pathway and protein synthesis is tightly controlled. AtMYB12 was 308
previously shown to not only increase flavonoid biosynthesis, but also to enhance the 309
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supply of precursors and carbon from primary metabolism (Zhang et al., 2015). It was 310
also reported that a MYB transcription factor ODO1 from petunia was able to activate 311
genes involved in the shikimate pathway and the biosynthesis of 312
phenylalanine-derived volatile compounds in tomato (Dal Cin et al., 2011). However, 313
it remained unclear how plants coordinately regulate phenylalanine formation in 314
response to lignin biosynthesis. 315
It was previously shown that MYB85 was able to activate the expression of a 316
lignin biosynthesis gene, 4CL1, in a transient assay in Arabidopsis protoplasts (Zhong 317
et al., 2008). In this study, we provided evidence that MYB85, MYB42, along with 318
MYB20 and MYB43, function as regulators of lignin and shikimate biosynthesis 319
pathways. MYB20, MYB42, MYB43, MYB85, MYB58, and MYB63 belong to a 320
monophyletic group in the tree of MYB transcription factors, indicating they have 321
high similar protein sequences (Supplemental Fig. S1). Among them, AtMYB20 and 322
AtMYB43 have the highest similarity with AtMYB42 and AtMYB85 in Arabidopsis, 323
forming a monophyletic clade with their putative orthologs in Amborella trichopoda 324
and Oryza sativa (Supplemental Fig. S1). 325
Disruption of MYB20, MYB42, MYB43, and MYB85 significantly reduced the 326
expression of genes involved in lignin biosynthesis (Fig. 3B). Consistent with gene 327
reduction, lignin content was significantly reduced in myb20/42/43/85 plants, 328
indicating that MYB20, MYB42, MYB43, and MYB85 are regulators of lignin 329
biosynthesis (Fig. 3D and Supplemental Fig. S4). Interestingly, ADT genes that are 330
specifically involved in phenylalanine biosynthesis were also downregulated in 331
myb20/42/43/85 plants (Fig. 3A). However, free phenylalanine content was increased 332
despite of the fact that ADT genes were downregulated (Fig. 3C). This may be a 333
consequence of downregulated flux toward lignin biosynthesis. Even though 334
phenylalanine must be shunted toward enhanced flavonoid biosynthesis, it seems that 335
lignin biosynthesis is downregulated to a higher extent. Our data suggested that 336
MYB20, MYB42, and MYB85 could directly target both lignin biosynthesis genes 337
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and ADT genes (Fig. 4A and 5A). However, MYB43 was only able to bind to the 338
promoter of lignin biosynthesis genes, and not ADT6, in our yeast one-hybrid assay. 339
EMSA data indicated that all four recombinant MBP-MYB fusion proteins caused an 340
up-shift of the labeled ADT6 or HCT promoters. Interestingly, each MYB protein 341
could activate ADT or lignin biosynthesis genes independently (Fig. 4G and 5G). 342
Like lignin, flavonoids are derived from phenylalanine. Studies in Arabidopsis, 343
as well as in maize, have pointed out that R2R3-MYBs have dual 344
activation/repression effects on genes from flavonoid and lignin pathways that 345
compete for carbon. For example, MYB32 and MYB4 in Arabidopsis were shown to 346
influence the composition of the pollen wall by changing the flux through 347
phenylpropanoid pathways (Preston et al., 2004). Similarly, Arabidopsis MYB75, a 348
positive regulator of anthocyanin biosynthesis, was shown to regulate lignin 349
accumulation negatively in secondary cell walls, which reflects carbon flux 350
redistribution within the branches of phenylpropanoid metabolism (Bhargava et al., 351
2010). Overexpression of ZmMYB31 from Z. mays downregulated several genes 352
involved in monolignol biosynthesis and upregulated flavonoid biosynthesis in 353
Arabidopsis (Fornalé et al., 2010). However, it remains unclear how these MYBs 354
fine-tune the biosynthesis of lignins and flavonoids. The channeling of carbon toward 355
lignin and flavonoid pathways is thought to be highly coordinated by crosstalk 356
between these pathways. It was reported that AtMYB75 can interact with AtKNAT7, 357
which functions as a negative regulator of secondary cell wall formation in 358
interfascicular fibers (Bhargava et al., 2010; Wang et al., 2019). A recent study further 359
demonstrated that AtMYB4, a transcriptional repressor of lignin biosynthesis, along 360
with its homologs MYB7 and MYB32, can attenuate the transcriptional function of 361
MYB75-TT8-TTG1 complexes in flavonoid metabolism by interacting with the TT8 362
transcription factor (Wang et al., 2019). The coordinated action of MYB repressors 363
and activators has been proposed as a fine-tuning mechanism for the regulation of 364
plant secondary metabolism (Bomal et al., 2013). In this study, we found that MYB20, 365
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15
MYB42, MYB43, and MYB85 redundantly activate MYB4, which was reported to 366
repress the expression of CHS, which encodes the enzyme that catalyzes the first step 367
of flavonoid biosynthesis. Consistent with the finding that the transcript level of CHS 368
is enhanced in myb20/42/43/85, flavonoid biosynthesis is upregulated in the mutant. 369
In addition, MYB20 was shown to positively regulate the CHS promoter (Ohman et 370
al., 2013), indicating that multiple levels of transcriptional control by MYB regulators 371
can allow fine-tuned regulation of carbon flux along the phenylpropanoid pathway. In 372
conclusion, our results showed that MYB20, MYB42, MYB43, and MYB85, in 373
addition to positively regulating lignin biosynthesis by directly activating lignin and 374
phenylalanine biosynthesis genes, repress flavonoid accumulation through 375
MYB4-mediated regulation of flavonoid biosynthesis. This suggests that the 376
transcription factors MYB20, MYB42, MYB43, and MYB85 control carbon flow into 377
the phenylpropanoid pathway by activating phenylalanine biosynthesis to ensure 378
precursor supply, while at the same time repressing other competing metabolic 379
pathways to optimize lignin biosynthesis. This discovery uncovers another level of 380
complexity in the regulatory network of plant lignin biosynthesis pathway. 381
382
MATERIALS AND METHODS 383
Plant Materials and Growth Conditions 384
The Arabidopsis (Arabidopsis thaliana) mutants myb20-1 (GK-109C11-012312), 385
myb42-1 (SALKseq_78190.1), myb43-1 (SALK_085312), myb85-1 (SALK_052089), 386
nst1 (SALK_120377), and nst3 (SALK_149909) were obtained from the Arabidopsis 387
Biological Resource Center at Ohio State University. Seeds were sterilized with 3% 388
(v/v) sodium hypochlorite (NaClO), sown on half-strength Murashige and Skoog (1/2 389
MS) plates, stored at 4℃, and then transferred into a chamber under long-day 390
conditions at 22℃ (day/night cycle 16/8 h). The primers used for genotyping are 391
listed in Supplemental Table S1. 392
393
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16
Phylogenetic Tree Analysis 394
Arabidopsis MYB20 was used as query to perform a BLASTP search against all 395
genes annotated on 19 published genomes, covering all main Archaeplastida clades 396
(Supplemental Table S2). Then, similar sequences with E-values less than 1e-3 and 397
an annotation term “MYB transcription factors” assigned by InterProScan (v5.24) 398
were selected (Jones et al., 2014). Multiple protein sequence alignments of MYB 399
transcription factors were performed using MAFFT (v7), positions with more than 50% 400
gaps were removed using Phyutility program (v2.2.6) (Smith and Dunn, 2008; 401
Katoh and Standley, 2013). Phylogenetic analyses were performed with a maximum 402
likelihood method by FastTree (v2.1) with optimal model of amino acid substitution 403
(LG+G) chosen based on the result of ProtTest (v3.4.2), and the bootstrap analysis 404
with 500 replicates were performed (Price et al., 2010; Darriba et al., 2011). 405
Sequences belonging to the monophyletic group of Arabidopsis MYB20, MYB43, 406
MYB42, MYB85, MYB58, and MYB63 were identified and selected for another 407
round of phylogenetic analyses as described above. Only sequences annotated from 408
Arabidopsis thaliana, Oryza sativa, Amborella trichopoda, Salvinia cucullata, Azolla 409
filiculoides, Sphagnum fallax, and Physcomitrella patens genomes were displayed on 410
the final tree. Sequences with very atypical sizes and extremely long braches were 411
manually removed. 412
Anthocyanin Measurement 413
Seedlings were grown on 1/2 MS medium containing 100 μM norflurazon. The 414
seedlings were then grown for 4 days in a growth chamber. Photographs of the 415
seedlings were taken by a microscope. To analyze anthocyanin content, the seeds 416
were grown on plates with 1/2 MS medium containing 1% (w/v) sucrose for 14 days 417
and then transferred to 1/2 MS medium containing 12% (w/v) sucrose for 3 days 418
(Yonekura-Sakakibara et al., 2012). Plants were harvested and frozen with liquid 419
nitrogen immediately. Anthocyanins were extracted with methanol containing 0.2% 420
(v/v) formic acid and 30% (v/v) water, and then placed at -20℃ overnight to remove 421
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17
chlorophyll. The determination of anthocyanin content was carried out by 422
spectrophotometry at 530 nm. The anthocyanin level of wild type Col-0 (1.13 mg 423
cyanidin 3-O-glucoside equivalence g–1
) was set to 100. 424
425
RNA Analyses 426
Total RNA was extracted from the inflorescence stems of 33-day-old Arabidopsis 427
wild type Col-0 and myb20/42/43/85 mutants using the RNeasy Plant Mini Kit 428
(QIAGEN). Three biological replicates of each sample were sent to BGI Genomics 429
Co., Ltd (The Beijing Genomics Institute) for construction of libraries and RNA-seq. 430
Data was analyzed on the Dr. Tom system from BGI. 431
The double mutants, myb20/43 and myb42/85, were planted and harvested at the 432
same time as wild type Col-0 and the myb20/42/43/85 mutant. Total RNA was 433
reverse-transcribed by Superscript Reverse Transcriptase III (Invitrogen). Reverse 434
transcription quantitative PCR (RT-qPCR) was performed with SYBR Premix Ex Taq 435
(Takara). The average levels of transcripts were from three biological replicates, and 436
each biological replicate was from triplicate cDNA. The Arabidopsis Tubulin gene 437
was used as a reference. The primers are listed in Supplemental Table S1. 438
439
KEGG (Kyoto Encyclopedia of Genes and Genomes) Pathway Analysis. 440
KEGG pathway enrichment analysis was performed using the Dr. Tom system from 441
BGI. According to KEGG pathway annotation, phyper function in the R project was 442
used to calculate P value and false discovery rates (FDRs). Q value was the correction 443
of P value. Terms with Q values ≤ 0.05 were considered as enriched. 444
445
Dual-luciferase Assay 446
To test transcriptional activation, the dual-luciferase assay was performed in 447
Nicotiana benthamiana leaves. About 1kb of the promoters of MYB20, MYB42, 448
MYB43, MYB85, and MYB4 were cloned into the pGreenII 0800-LUC vector to 449
generate P-LUC reporter constructs (Hellens et al., 2005). Each promoter in pGreenII 450
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18
0800-LUC was transformed into Agrobacterium tumefaciens strain GV3101 451
containing the helper plasmid pSoup-P19 (Hellens et al., 2005). The full-length 452
coding sequences (without stop codons) of NST3, MYB46, MYB20, MYB42, MYB43, 453
MYB85, and GUS were cloned into the pEarleyGate 101 vector (pEG101). These six 454
constructs were transformed into Agrobacterium tumefaciens strain GV3101. 455
Overnight cultures of Agrobacterium strains were re-suspended in infiltration buffer 456
(10mM MgCl2, 10mM MES and 0.1 mM acetosyringone) to OD600 ~ 0.6 and 457
incubated at room temperature for 2 hours. The reporter:effector ratio was 2:8. 458
Different combinations of Agrobacterium suspension were infiltrated onto N. 459
benthamiana leaves. After 48 hours, leaf samples were collected and ground in liquid 460
nitrogen for the dual-luciferase assay. Luciferase activities were detected using the 461
Dual-luciferase Reporter Assay System (Promega). Each sample contained three 462
biological repeats. 463
To examine whether each MYB was able to activate the genes independently, a 464
transcriptional activation assay was conducted in Arabidopsis Col-0 and 465
myb20/42/43/85 protoplasts. About 1kb promoters of ADT6 and HTC were cloned 466
into the pGreenII 0800-LUC. The coding sequences of MYB20, MYB42, MYB43, and 467
MYB85 were inserted into p2GW7 under the control of the 35S promoter. After the 468
protoplast preparation and subsequent transfection were completed (Yoo et al., 2007), 469
samples were collected. The test of luciferase activities was described above. The raw 470
data was in the Supplemental Table S3. 471
472
Yeast One-hybrid Assays 473
GAD fusion constructs were co-transformed with the LexAop::LacZ (Clontech) 474
reporter gene plasmid into the EGY48 yeast strain. Transformants were grown on 475
appropriate drop-out plates containing X-gal 476
(5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) for blue color test. 477
478
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19
EMSA 479
The DNA fragments of MYBs were cloned into pMAL-c2x vector, and 480
expressed in the Escherichia coli BL21 (DE3) cell line, which were cultured in LB 481
culture at 37°C to an OD600 between 0.6–0.8 was reached. To induce protein 482
expression, a final concentration of 0.5 mM isopropyl β-D-1-thiogalactopyranoside 483
was added to the cultures, after which the cells were incubated for 8 h at 28°C. Cells 484
were harvested and resuspended in buffer containing 20 mM Tris-HCl pH 7.4, 200 485
mM NaCl, and 1 mM EDTA pH 8.0. After sonication and centrifugation, the MBP-TF 486
proteins were purified using amylose resin from the supernatant. 487
The oligonucleotide probes were labeled with TAMRA or FAM and synthesized 488
from Sangon Biotech. Then, the oligonucleotides were diluted to 10 μM, and heated 489
to 95℃ for 5 min, and slowly cooled to room temperature. Briefly, 1 pmol of labeled 490
probe was incubated with 0.1 μg of the indicated protein in 20-μL reaction buffer (25 491
mM HEPES, pH 8.0, 40 mM KCl, 5 mM MgCl2, 1 mM DTT, 1 mM EDTA, 8% v/v 492
glycerol, 1 μg poly (dI-dC)) for 20 min at room temperature. Then, to the reaction 493
mixture was added 5 μL of 5X loading buffer (12.5% w/v Ficoll-400, 0.2% w/v 494
bromophenol blue, 0.2% w/v xylene cyanol FF) and loaded onto as 4.5% 495
polyacrylamide gel in 0.5X TBE. The labeled DNAs were detected using a Typhoon 496
FLA 9500 Instrument (General Electric Company). 497
498
Amino Acid Extraction and Analysis 499
Amino acids were extracted from 50 mg (dry weight) of 5-week-old Arabidopsis 500
stem samples with 1 mL of 0.5 M HCl solution. The supernatants were subsequently 501
filtered using a 0.22 μm nylon membrane and 250 μL of the extraction supernatant 502
was transferred into autosamper vials. Finally, the mixture was diluted to 1 mL with 503
20% (v/v) water in ACN prior to further analysis. Ultra-performance liquid 504
chromatography-electrospray ionization tandem mass spectrometric 505
(UPLC-ESI-MS/MS) analysis was carried out on an Agilent 1290 LC system coupled 506
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20
to an Ultivo Triple Quadrupole mass spectrometer by means of an electrospray 507
ionization (ESI) probe. Eluent A was 20 mM ammonium formate in water (pH 3), 508
while eluent B was 20 mM aqueous ammonium formate (pH 3) in 9:1 509
acetonitrile/water. The separation gradient was: 100% B at 0 min, 70% B at 5 min, 510
and 100% B at 8 min. The column temperature was maintained at 25°C. The injection 511
volume of each sample was 1 μL. The MRM-MS method was employed for the 512
quantitation of amino acids. Data for phenylalanine was acquired in positive-ion (PI) 513
mode under the following operating conditions: desolvation temperature: 330°C; 514
desolvation gas (N2) rate, 13 L/min; collision energy, 13 V for quantitative ion (m/z 515
120.1) and 29 V for qualitative ion (m/z 103), respectively. 516
517
Determination of Lignin Composition 518
Thioacidolysis analysis was performed to determine lignin composition. Briefly, 519
30 mg samples were reacted with 3 mL of 0.2 M BF3-etherate in an 8.75:1 520
dioxane/ethanethiol mixture at 100°C for 4 h. After adding 4 mL of water, the mixture 521
was extracted using 4 mL of dichloromethane (three times). The organic layer was 522
separated and dried under a stream of nitrogen. After derivatization with 523
N,O-bis-(trimethylsily)trifluoroacetamide (BSTFA) + 1% (w/v) trimethylchlorosilane 524
(TMCS) (Sigma-Aldrich), lignin-derived monomers were identified by GC-MS and 525
quantified by GC as their trimethylsilyl derivatives. GC-MS was performed on an 526
Agilent Intuvo 9000 GC system with a 5977B series mass-selective detector (column: 527
HP-5MS; 30 m × 0.25 mm; 0.25-μm film thickness), and mass spectra were recorded 528
in electron impact mode (70 eV) with a 50–650 m/z scanning range. 529
530
Quantification of Flavonoids 531
Samples of three replicates of Arabidopsis stems were collected and ground into 532
a fine powder with a mortar and pestle under liquid nitrogen. Twenty microgram dried 533
samples were extracted with 1 mL 80% (v/v) methanol (containing 100 μM 534
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21
naringenin as an internal reference), and then ultrasonic processing was conducted for 535
30 min at room temperature. After that, the samples were centrifuged at 14000 g for 536
10 min, and the supernatant was transferred to a sterile 1.5 mL Eppendorf tube for 537
another centrifugation as above. 300 μL sterile milli-Q water was added to each 1 mL 538
supernatant solution and stored at -20℃ overnight to precipitate chlorophyll. The 539
following day, 5 μL of the supernatant solution after another centrifugation was 540
injected for UPLC analysis. The identification of soluble flavonoid compounds was 541
described previously (Yonekura-Sakakibara et al., 2008) and quantification of 542
detected compounds was based on the standard curves of a kaempferol standard 543
detected in the same UPLC runs. 544
545
Statistical Analyses 546
The SPSS 20.0 statistical package was used for the statistical data analysis. 547
Statistical analysis was done with univariate analysis and Scheffe post-hoc testing at 548
the 5% level. Samples included in the analysis were arranged based on at least three 549
biological replicates. 550
551
Data Availability 552
RNA-seq data have been uploaded to the SRA (http://www.ncbi.nlm.nih.gov/sra) 553
with the accession number PRJNA578015. 554
555
Accession Numbers 556
The Arabidopsis Information Resource accession numbers of genes mentioned 557
are as follows: AT2G46770 (NST1), AT3G61910 (NST2), AT1G32770 (NST3), 558
AT5G12870 (MYB46), AT1G66230 (MYB20), AT4G12350 (MYB42), AT5G16600 559
(MYB43), AT4G22680 (MYB85), AT4G38620 (MYB4), AT2G16720 (MYB7), 560
AT4G34990 (MYB32), AT2G37040 (PAL1), AT2G30490 (C4H), AT1G15710 561
(ADH2), AT3G44720 (ADT4), AT5G22630 (ADT5), AT1G08250 (ADT6), 562
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22
AT1G51680 (4CL1), AT3G19450 (CAD4), AT1G52760 (CSE), AT5G48930 (HCT), 563
AT3G54640 (TSA1), AT5G54810 (TSB1), AT5G13930 (CHS). 564
565
SUPPLEMENTAL DATA 566
The following supplemental materials are available. 567
Supplemental Figure S1. Phylogenetic tree analysis of MYBs in 19 published 568
genomes in main archaeplastida clades. 569
Supplemental Figure S2. NST1/2/3 and MYB46/83 could not bind the promoters of 570
MYB20/42/43/85 directly. 571
Supplemental Figure S3. KEGG pathway enrichment analysis. 572
Supplemental Figure S4. Lignin content Klason analysis of wild type (Clo-0), 573
myb20/43, myb42/85, and myb20/42/43/85. 574
Supplemental Figure S5. Truncated MYB proteins have higher expression in yeast. 575
Supplemental Figure S6. Disruption of MYB20/42/43/85 promotes anthocyanin 576
accumulation. 577
Supplemental Table S1. Primers used for vector construction and gene expression 578
analysis. 579
Supplemental Table S2. Resources of 19 representative genomes used in 580
phylogenetic tree analysis. 581
Supplemental Table S3. Source data of the firefly and Renilla luciferase signal in the 582
dual-luciferase assay. 583
Supplemental Dataset S1. A full list of the genes that are upregulated in 584
myb20/42/43/85 (FDR < 0.05, log2FC > 1). 585
Supplemental Dataset S2. A full list of the genes that are downregulated in 586
myb20/42/43/85 (FDR < 0.05, log2FC < -1). 587
588
Acknowledgments: This work was supported by grants from the Youth Thousand 589
Scholar Program of China, National Natural Science Foundation of China (grant 590
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23
number 31522008 and 31570296) and the School of Life Sciences, Tsinghua 591
University. 592
593
Figure Legends 594
595
Figure 1. Description of myb20, myb42, myb43, and myb85 mutants. A, Relative 596
transcript levels of MYB20, MYB42, MYB43, and MYB85 in the inflorescence stems of 597
33-day-old wild-type (Col-0) and nst1/3 mutant plants were determined by reverse 598
transcription quantitative PCR (RT-qPCR). The expression level in Col-0 was set to 1. 599
Tubulin was used as the internal control. B and C, Transient expression assays showed 600
that the promoters of MYB20, MYB42, MYB42, and MYB85 can be activated by NST3 601
(B) and MYB46 (C). The PMYB20-LUC, PMYB42-LUC, PMYB43-LUC, and 602
PMYB85-LUC reporters were respectively cotransformed with the indicated 603
constructs in Nicotiana benthamiana leaves. The LUC/REN ratio represents the LUC 604
activity relative to the REN activity. CK was the vector of GUS-pEarleyGate 101, 605
used as a control, and was set to 1. Data are means ± SD of three biological replicates. 606
D, Schematic diagrams of the structures of MYB20, MYB42, MYB43, MYB85, and 607
T-DNA insertions. Solid boxes represent exons, black lines represent introns, and 608
hollow boxes represent UTRs. E, RT-qPCR analysis of MYB20, MYB42, MYB42, and 609
MYB85 transcripts in the inflorescence stems of 33-day-old plants. Tubulin was used 610
as a positive control. Data are means ± SD of three biological replicates. Asterisks 611
indicate significant differences by the Student’s t test (*P <0.05, **P <0.01, ***P 612
<0.001, and ****P <0.0001). 613
614
Figure 2. Disruption of MYB20/42/43/85 causes growth and fertility defects. A, 615
Five-week-old plants of Col-0, the double mutants myb20/43 and myb42/85, and the 616
quadruple mutant myb20/42/43/85. B, The inflorescence stems of six-week-old plants 617
of Col-0 and myb20/42/43/85. C, The upper inflorescence stems as in (B). D, The 618
middle inflorescence stems as in (B). 619
620
Figure 3. MYB20, MYB42, MYB43, and MYB85 redundantly regulate 621
phenylalanine and lignin biosynthesis. A and B, Relative transcript levels of 622
aromatic amino acid (A) and lignin (B) biosynthesis genes in the inflorescence stems 623
of 33-day-old Arabidopsis wild type, myb20/43, myb42/85, and myb20/42/43/85 624
mutants as determined by RT-qPCR. C, Phenylalanine level (Phe) in the inflorescence 625
stems of plants with the same genotypes and period as in (A). DW, dry weight. D, 626
Lignin composition analysis of the inflorescence stems of plants with the same 627
genotypes and period as in (A). Gas chromatographic quantification of H, G, and S 628
lignin units by thioacidolysis. Data are means ± SD of three biological replicates. 629
Based on Tukey’s test, columns with the same letters are not significantly different 630
and columns with the different letters are significantly different (P < 0.05; one-way 631
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24
ANOVA). 632
633
Figure 4. MYBs activate phenylalanine biosynthesis genes. A, GAD-MYBs 634
activate the expression of the LacZ reporter gene driven by the ADT6 promoter in 635
yeast. GFP was used as the negative control. B, The upper schematic diagram and the 636
lower one show the promoter regions of ADT6 in (A) and in (C-F) separately. C-F, 637
EMSA showed that MYB20 (C), MYB42 (D), MYB43 (E), and MYB85 (F) bind to 638
the promoter of ADT6. The cold probes used as competitors were with the same 639
sequence as the TAMRA-labeled probes. Truncated MYB20 (Δ166-282 a.a.), MYB42 640
(Δ138-286 a.a.), MYB43 (Δ201-327 a.a.), and MYB85 (Δ168-266 a.a.) were used for 641
yeast one-hybrid assay (A) and EMSA assay (C-F). G and H, Transient expression 642
assays showed that the ADT6 promoter can be activated by MYB20/42/43/85 in 643
Arabidopsis Col-0 protoplasts (G) and in Arabidopsis myb20/42/43/85 mutant 644
protoplasts (H). CK was empty vector as control and was set to 1. Data are means ± 645
SD of three biological replicates. Based on Tukey’s test, columns with the same letters 646
are not significantly different and columns with the different letters are significantly 647
different (P < 0.05; one-way ANOVA). 648
649
Figure 5. MYBs activate lignin biosynthesis genes. A, GAD-MYBs activate the 650
expression of the LacZ reporter gene driven by the HCT promoter in yeast. GFP was 651
used as the negative control. B, The upper schematic diagram and the lower one show 652
the promoter regions of HCT in (A) and in (C-F) separately. C-F, EMSA showed that 653
MYB20 (C), MYB42 (D), MYB43 (E), and MYB85 (F) bind to the promoter of HCT. 654
The cold probes used as competitors were with the same sequence as the 655
FAM-labeled probes. Truncated MYB20 (Δ166-282 a.a.), MYB42 (Δ138-286 a.a.), 656
MYB43 (Δ201-327 a.a.), and MYB85 (Δ168-266 a.a.) were used for yeast one-hybrid 657
assay (A) and EMSA assay (C-F). G and H, Transient expression assays showed that 658
the HCT promoter can be activated by MYB20/42/43/85 in Arabidopsis Col-0 659
protoplasts (G) and in Arabidopsis myb20/42/43/85 mutant protoplasts (H). CK was 660
empty vector as control and was set to 1. Data are means ± SD of three biological 661
replicates. Based on Tukey’s test, columns with the same letters are not significantly 662
different and columns with the different letters are significantly different (P < 0.05; 663
one-way ANOVA). 664
665
Figure 6. MYBs activate MYB4/7/32 genes directly. A, MYB4, MYB7, and MYB32 666
transcript levels in the inflorescence stems of 33-day-old wild type, myb20/43, 667
myb42/85, and myb20/42/43/85 mutants as determined by RT-qPCR. B, Transient 668
expression assays showed that the MYB4 promoter can be activated by MYB20, 669
MYB42, MYB43, and MYB85 in N. benthamiana leaves. CK was the vector of 670
GUS-pEarleyGate 101, used as a control, and was set to 1. C-F, EMSA showing that 671
MYB20 ©, MYB42 (D), MYB43 (E), and MYB85 (F) bind to the promoter of MYB4. 672
The cold probes used as competitors were with the same sequence as the 673
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25
FAM-labeled probes. Truncated MYB20 (Δ166-282 a.a.), MYB42 (Δ138-286 a.a.), 674
MYB43 (Δ201-327 a.a.), and MYB85 (Δ168-266 a.a.) were used for EMSA assay 675
(C-F). Data are means ± SD of three biological replicates. Based on Tukey’s test, 676
columns with the same letters are not significantly different and columns with the 677
different letters are significantly different (P < 0.05; one-way ANOVA). 678
679
Figure 7. Disruption of MYB20/42/43/85 promotes soluble flavonoids 680
accumulation. A, RT-qPCR analysis of CHS in the inflorescence stems of 5-week-old 681
wild type, myb20/42/43/85, and myb4/7/32 mutants. B, Quantification of the three 682
major flavonols in the inflorescence stems of the same plants as (A). K1, kaempferol 683
3-O-[6"-O-(rhamnosyl)glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside 684
7-O-rhamnoside; K3, kaempferol 3-O-rhamnoside 7-O-rhamnoside. Data are means ± 685
SD of three biological replicates. Based on Tukey’s test, columns with the same letters 686
are not significantly different and columns with the different letters are significantly 687
different (P < 0.05; one-way ANOVA). 688
689
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Figure 1. Description of myb20, myb42, myb43, and myb85 mutants. A, Relative transcript levels of MYB20,
MYB42, MYB43, and MYB85 in the inflorescence stems of 33-day-old wild type (Col-0) and nst1/3 mutant
plants were determined by reverse transcription quantitative PCR (RT-qPCR). The expression level in Col-0
was set to 1. Tubulin was used as the internal control. B and C, Transient expression assays showed that the
promoters of MYB20, MYB42, MYB42, and MYB85 can be activated by NST3 (B) and MYB46 (C). The
PMYB20-LUC, PMYB42-LUC, PMYB43-LUC, and PMYB85-LUC reporters were respectively cotransformed with the
indicated constructs in Nicotiana benthamiana leaves. The LUC/REN ratio represents the LUC activity
relative to the REN activity. CK was the vector of GUS-pEarleyGate 101, used as a control, and was set to 1.
Data are means ± SD of three biological replicates. D, Schematic diagrams of the structures of MYB20,
MYB42, MYB43, MYB85, and T-DNA insertions. Solid boxes represent exons, black lines represent introns,
and hollow boxes represent UTRs. E, RT-qPCR analysis of MYB20, MYB42, MYB42, and MYB85 transcripts
in the inflorescence stems of 33-day-old plants. Tubulin was used as a positive control. Data are means ± SD
of three biological replicates. Asterisks indicate significant differences by the Student’s t test (*P <0.05, **P
<0.01, ***P <0.001, and ****P <0.0001).
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Figure 2. Disruption of MYB20/42/43/85 causes growth and fertility defects. A, Five-week-old plants of
Col-0, the double mutants myb20/43 and myb42/85, and the quadruple mutant myb20/42/43/85. B, The
inflorescence stems of six-week-old plants of Col-0 and myb20/42/43/85. C, The upper inflorescence stems
as in (B). D, The middle inflorescence stems as in (B).
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Figure 3. MYB20, MYB42, MYB43, and MYB85 redundantly regulate phenylalanine and lignin
biosynthesis. A and B, Relative transcript levels of aromatic amino acid (A) and lignin (B) biosynthesis
genes in the inflorescence stems of 33-day-old Arabidopsis wild type, myb20/43, myb42/85, and
myb20/42/43/85 mutants as determined by RT-qPCR. C, Phenylalanine level (Phe) in the inflorescence
stems of plants with the same genotypes and period as in (A). DW, dry weight. D, Lignin composition
analysis of the inflorescence stems of plants with the same genotypes and period as in (A). Gas
chromatographic quantification of H, G, and S lignin units by thioacidolysis. Data are means ± SD of three
biological replicates. Based on Tukey’s test, columns with the same letters are not significantly different and
columns with the different letters are significantly different (P < 0.05; one-way ANOVA).
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Figure 4. MYBs activate phenylalanine biosynthesis genes. A, GAD-MYBs activate the expression of the
LacZ reporter gene driven by the ADT6 promoter in yeast. GFP was used as the negative control. B, The
upper schematic diagram and the lower one show the promoter regions of ADT6 in (A) and in (C-F)
separately. C-F, EMSA showed that MYB20 (C), MYB42 (D), MYB43 (E), and MYB85 (F) bind to the
promoter of ADT6. The cold probes used as competitors were with the same sequence as the
TAMRA-labeled probes. Truncated MYB20 (Δ166-282 a.a.), MYB42 (Δ138-286 a.a.), MYB43 (Δ201-327
a.a.), and MYB85 (Δ168-266 a.a.) were used for yeast one-hybrid assay (A) and EMSA assay (C-F). G and
H, Transient expression assays showed that the ADT6 promoter can be activated by MYB20/42/43/85 in
Arabidopsis Col-0 protoplasts (G) and in Arabidopsis myb20/42/43/85 mutant protoplasts (H). CK was
empty vector as control and was set to 1. Data are means ± SD of three biological replicates. Based on
Tukey’s test, columns with the same letters are not significantly different and columns with the different
letters are significantly different (P < 0.05; one-way ANOVA). www.plantphysiol.orgon February 29, 2020 - Published by Downloaded from
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Figure 5. MYBs activate lignin biosynthesis genes. A, GAD-MYBs activate the expression of the LacZ
reporter gene driven by the HCT promoter in yeast. GFP was used as the negative control. B, The upper
schematic diagram and the lower one show the promoter regions of HCT in (A) and in (C-F) separately. C-F,
EMSA showed that MYB20 (C), MYB42 (D), MYB43 (E), and MYB85 (F) bind to the promoter of HCT.
The cold probes used as competitors were with the same sequence as the FAM-labeled probes. Truncated
MYB20 (Δ166-282 a.a.), MYB42 (Δ138-286 a.a.), MYB43 (Δ201-327 a.a.), and MYB85 (Δ168-266 a.a.)
were used for yeast one-hybrid assay (A) and EMSA assay (C-F). G and H, Transient expression assays
showed that the HCT promoter can be activated by MYB20/42/43/85 in Arabidopsis Col-0 protoplasts (G)
and in Arabidopsis myb20/42/43/85 mutant protoplasts (H). CK was empty vector as control and was set to 1.
Data are means ± SD of three biological replicates. Based on Tukey’s test, columns with the same letters are
not significantly different and columns with the different letters are significantly different (P < 0.05;
one-way ANOVA). www.plantphysiol.orgon February 29, 2020 - Published by Downloaded from
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Figure 6. MYBs activate MYB4/7/32 genes directly. A, MYB4, MYB7, and MYB32 transcript levels in the
inflorescence stems of 33-day-old wild type (Col-0), myb20/43, myb42/85, and myb20/42/43/85 mutants as
determined by RT-qPCR. B, Transient expression assays showed that the MYB4 promoter can be activated
by MYB20, MYB42, MYB43, and MYB85 in N. benthamiana leaves. CK was the vector of
GUS-pEarleyGate 101, used as a control, and was set to 1. C-F, EMSA showing that MYB20 (C), MYB42
(D), MYB43 (E), and MYB85 (F) bind to the promoter of MYB4. The cold probes used as competitors were
with the same sequence as the FAM-labeled probes. Truncated MYB20 (Δ166-282 a.a.), MYB42 (Δ138-286
a.a.), MYB43 (Δ201-327 a.a.), and MYB85 (Δ168-266 a.a.) were used for EMSA assay (C-F). Data are
means ± SD of three biological replicates. Based on Tukey’s test, columns with the same letters are not
significantly different and columns with the different letters are significantly different (P < 0.05; one-way
ANOVA).
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Figure 7. Disruption of MYB20/42/43/85 promotes soluble flavonoids accumulation. A, RT-qPCR analysis
of CHS in the inflorescence stems of 5-week-old wild type (Col-0), myb20/42/43/85, and myb4/7/32 mutants.
B, Quantification of the three major flavonols in the inflorescence stems of the same plants as (A). K1,
kaempferol 3-O-[6"-O-(rhamnosyl)glucoside] 7-O-rhamnoside; K2, kaempferol 3-O-glucoside
7-O-rhamnoside; K3, kaempferol 3-O-rhamnoside 7-O-rhamnoside. Data are means ± SD of three biological
replicates. Based on Tukey’s test, columns with the same letters are not significantly different and columns
with the different letters are significantly different (P < 0.05; one-way ANOVA).
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