1 Short title: Flavanoid pathway regulation by miR858...2016/04/27 · 10 Deepika Sharma1, 2,...
Transcript of 1 Short title: Flavanoid pathway regulation by miR858...2016/04/27 · 10 Deepika Sharma1, 2,...
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Short title: Flavanoid pathway regulation by miR858 1
Corresponding author: 2
Prabodh Kumar Trivedi, 3 CSIR-National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, INDIA 4 [email protected]; [email protected] 5 6
Research area: Biochemistry and metabolism 7
MicroRNA858 is a potential regulator of phenylpropanoid pathway and plant 8 development in Arabidopsis 9
Deepika Sharma1, 2, Manish Tiwari1, $, Ashutosh Pandey1, #, Chitra Bhatia1, 2, Ashish Sharma1, 10 Prabodh Kumar Trivedi1, 2,* 11 1CSIR-National Botanical Research Institute, Council of Scientific and Industrial Research 12
(CSIR-NBRI), Rana Pratap Marg, Lucknow-226001, INDIA 13
2Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi 14
Marg, New Delhi-110 001, India 15
Summary: miR858a targets MYB transcriptions factors involved in flavonoid biosynthesis 16
and plant growth and development 17
DS, AP and CB thankfully acknowledge the Council of Scientific and Industrial Research 18
(CSIR), New Delhi for Senior Research Fellowship. MT acknowledges Indian Council of 19
Medical Research for Research Fellowship. Research was supported by Council of Scientific 20
and Industrial Research, New Delhi in the form of Network projects PlaGen (BSC-0107). 21
22 $Present address (MT): Department of Biology, Western Kentucky University Bowling 23
Green, KY, USA. 24
$Present address (AP): National Agri-Food Biotechnology Institute (NABI), Mohali-160071, 25
Punjab, INDIA 26
The authors responsible for distribution of materials integral to the findings presented in this 27
article in accordance with the policy described in the Instructions for Authors 28
(www.plantcell.org) are: Prabodh Kumar Trivedi ([email protected]). 29
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Corresponding author’s e-mail: [email protected]; [email protected] 31
Plant Physiology Preview. Published on April 27, 2016, as DOI:10.1104/pp.15.01831
Copyright 2016 by the American Society of Plant Biologists
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ABSTRACT 32
MicroRNAs (miRNAs) are endogenous, non-coding small RNAs which function as critical 33
regulators of gene expression. In plants, miRNAs have highlighted their potential as 34
regulators of growth, development, signal transduction and stress tolerance. Though miRNA-35
mediated regulation of several processes is known, the involvement of miRNAs in regulating 36
secondary plant product biosynthesis is poorly understood. In this study, we functionally 37
characterized Arabidopsis miR858a which putatively targets R2R3-MYB transcription 38
factors involved in flavonoid biosynthesis. Over-expression of miR858a in Arabidopsis led to 39
down-regulation of several MYB transcription factors regulating flavonoid biosynthesis. In 40
contrast to the robust growth and early flowering of miR858OX plants, reduction of plant 41
growth and delayed flowering was observed in Arabidopsis transgenic lines expressing 42
artificial miRNA target mimic (MIM858). Genome-wide expression analysis using transgenic 43
lines suggested that miR858a targets a number of regulatory factors which modulate 44
expression of down-stream genes involved in plant development, hormonal and stress 45
responses. Further, higher expression of MYBs in MIM858 lines leads to the redirection of 46
the metabolic flux towards the synthesis of flavonoids at the cost of lignin synthesis. 47
Altogether, our study established the potential role of light-regulated miR858a in flavonoids 48
biosynthesis and plant growth and development. 49
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Keywords: miR858, Arabidopsis, gene regulation, transcription factors, development, 51
secondary metabolites 52
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INTRODUCTION 61
MicroRNAs (miRNAs) are small mostly non-coding (22-24 nt) RNAs which modulate gene 62
expression by pairing to corresponding target mRNA and promote mRNA degradation and/or 63
translation inhibition (Llave et al., 2002; Brodersen et al., 2008; Filipowicz et al., 2008). 64
miRNAs are important regulators of many physiological and developmental processes 65
(Voinnet, 2009), and their misexpression leads to a variety of defects in plants (Wu, 2013). 66
Various studies have demonstrated that miRNAs regulate flowering, leaf, seed, root 67
morphology as well as stress and hormonal responses in plants (Leung and Sharp, 2010; Liu 68
and Chen, 2010; Stauffer and Maizel, 2014; Hong and Jackson, 2015; Zhang, 2015). Recent 69
developments in genome-wide experimental approaches involving miRNA over-expression 70
(Wu, 2013), use of miRNA target mimics (Franco-Zorrilla et al., 2007; Eamens and Wang, 71
2011; Meng et al., 2012) and detailed analysis of miRNA promoters (Baek et al., 2013; 72
Sharma et al., 2015) were helpful to understand the physical interactions between miRNAs 73
and their target mRNAs as well as biological significances in plant growth and development 74
(de Lima et al., 2012; Wu, 2013). The majority of miRNAs are known to target transcription 75
factors which regulate development, hormonal regulation and stress responses in plants 76
(Sunkar et al., 2012; Wu, 2013; Zhang, 2015; Zhang and Wang, 2015). 77
Flavonoids are low molecular weight polyphenolic compounds and encompass a 78
diverse class of phenolic compounds including flavonols, flavones, isoflavones, 79
anthocyanins, proanthocyanidins (PAs) and phlobaphene pigments (Taylor and Grotewold, 80
2005; Lepiniec et al., 2006; Buer et al., 2010). The biological functions of flavonoids are as 81
pigments, signalling molecules, UV-protectants, phytoalexins, and regulators of auxin 82
transport in plants (Winkel-Shirley, 2001; Buer and Muday, 2004; Santelia et al., 2008). The 83
phenylpropanoid pathway is the main route for flavonoid biosynthesis, and transcriptional 84
regulation of this pathway is one of the most studied pathways in plants (Quattrocchio et al., 85
2006; Stracke et al., 2007). The synthesis of the most flavonoid compounds shares common 86
enzymes, and sometimes the enzymes of the specific branch are controlled by distinct 87
regulators. MYB11, MYB12 and MYB111 have been demonstrated to regulate biosynthesis 88
of flavonols by targeting genes encoding early pathway enzymes such as CHS, CHI, F3’H 89
and FLS recruited in phenylpropanoid pathway (Mehrtens et al., 2005; Stracke et al., 2007; 90
Stracke et al., 2010). The MYB123 (TT2) controls the biosynthesis of PAs in the seed coat of 91
Arabidopsis (Xu et al., 2014). Similarly, members of the R2R3MYB protein family for 92
instance, MYB58, MYB63 and MYB85, regulate lignin biosynthesis in fibres and vessels 93
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whereas MYB46 and MYB83 act as switch regulators during secondary wall biosynthesis 94
(Zhong et al., 2008; McCarthy et al., 2009; Zhou et al., 2009; Zhong and Ye, 2012). Flavonol 95
biosynthesis is a highly complex, tissue and developmentally regulated process in 96
Arabidopsis which is orchestrated by the group of R2R3-MYB transcription factors, 97
including MYB11, MYB12 and MYB111. These MYBs differentially affect the spatial 98
accumulation of specific flavonols in Arabidopsis (Stracke et al., 2007). 99
In past few years, studies demonstrated that miRNAs may act as master regulators of 100
flavonoid biosynthesis in Arabidopsis. It has been shown that the miR156-SPL9 network 101
directly influence anthocyanin production through targeting genes encoding PAP1 and DFR 102
(Gou et al., 2011; Cui et al., 2014). The miR163 targets S-adenosylmethionine-dependent 103
methyltransferases which methylate secondary metabolites and signalling molecules (Ng et 104
al., 2011). Studies have also revealed that miR397 regulate lignin biosynthesis by altering 105
lacasse expression in Arabidopsis and Populus (Lu et al., 2013; Wang et al., 2014). It has 106
been reported that miR159, miR319 and miR828 families deregulate MYB transcription 107
factors in Malus, Solanum lycopersicum, Vitis and Salvia miltiorrhiza (Xia et al., 2012; Rock, 108
2013; Li et al., 2014; Jia et al., 2015). Similarly, a study demonstrated that miR828 regulate 109
MYB2, which is involved in trichome and cotton fibre development (Guan et al., 2014). 110
In this study, we investigated the role of miR858a in the regulation of the 111
phenylpropanoid pathway and development in Arabidopsis. The genetic, molecular, cellular 112
and biochemical experiments using transgenic lines expressing miR858a (miR858OX) and 113
artificial miR858 target mimic (MIM858) show that miR858a targets flavonol-specific MYBs 114
involved in regulating early steps of the flavonoid biosynthetic pathway. Genome-wide 115
expression and detailed metabolite analyses of transgenic lines suggest that miR858a plays an 116
important role in plant growth and development. We conclude that the miR858a-MYB 117
network is an additional regulatory sector of the module that coordinates flavonoid and lignin 118
biosynthesis in plants. 119
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RESULTS 122
Identification of miRNAs Regulating Flavonoid Biosynthesis 123
As a first step to identify the potential miRNAs regulating MYB11, MYB12 and MYB111 124
transcription factors, nucleotide sequences of the mRNAs were used for BLAST searches 125
against the Sanger miRNA database (http://microrna.sanger.ac.uk). Ath-miR858 showed 126
significant sequence homology with these MYB transcription factors. Besides, analysis using 127
the WMD3 miRNA target identification tool (Schwab et al., 2006) identified a number of 128
putative targets of miR858. The result suggested that MYB11, MYB12 and MYB111 could be 129
the best potential targets of miR858 with hybridization energy of the duplex structure ≤ −35 130
kcal/mol and average homology ≥ 80% (Figure 1A). The detailed bioinformatic analysis 131
revealed that miR858 sequences are evolutionarily conserved in dicot species such as 132
Arabidopsis, Gossypium, Solanum and Cucumis (Supplementary Table S1). Similar to our 133
observation, Xia et al., (2012) reported that miR858 and its targets are well conserved in 134
diverse dicot species. There are two members of the miR858 family (ath-miR858a/b) present 135
in Arabidopsis genome as listed in miRBase, release 21. The mature sequence of miR858a 136
differs from miR858b by the single nucleotide (Supplementary Table S1). However, 137
miR858a and miR858b are transcribed from different loci, but target similar sets of MYB 138
transcription factors. Therefore, we focused on studying the role of miR858a as a putative 139
regulator of MYB transcription factors in this study. 140
Widespread Expression of miR858a in Arabidopsis 141
To investigate the spatio-temporal expression of endogenous miR858a, quantitative RT-PCR 142
analysis of mature miR858a was carried out at different stages of Arabidopsis development. 143
The analysis suggested that miR858a is expressed in rosette, root, stem, flower, silique and 144
seedling (Figure 1B). Comparative expression analysis revealed that miR858a is 145
predominantly found in the silique, at least, an order of magnitude higher compared to 146
seedling, root, stem, rosette leaves and flower of the mature plant. These results revealed that 147
miR858a is expressed extensively in all the studied tissues and indicated that it may have an 148
important role in the plant growth and development. 149
To study the tissue-specific expression of miR858a, a promoter-reporter 150
(ProMIR858a:GUS) expressing transgenic lines of Arabidopsis were developed. 151
Histochemical GUS analysis in tissues of various developmental stages revealed that 152
ProMIR858a is active in seedling, rosette leaf, stem, flower buds and mature silique (Figure 153
1C). It is important to mention that an intense GUS staining was observed in the vascular 154
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tissues of hypocotyl, root and leaf veins of the seedling (Figure 1C, b and c) as well as in 155
rosette leaf, stem, flower and silique of mature plants (Figure 1C, d to h). Though GUS 156
staining patterns (Figure 1C) are in general consistent with the miR858a transcript abundance 157
(Figure 1B), differences in relative GUS staining between rosette, flower and silique tissues 158
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were observed. These differences might be due to the limitation of the promoter:GUS 159
approach in studying the spatial expression of genes. Altogether, these results suggested that 160
miR858a is expressed in all the developmental stages and might be one of the key regulators 161
of the plant growth and development. 162
The search for cis-regulatory motifs in the promoter region of pre-miR858a using the 163
Plant-CARE database revealed the assortment of putative light responsive elements (LREs) 164
(Supplementary Table S2). Light is one the most important factors affecting MYB 165
transcription factor and thereby flavonoid biosynthesis in Arabidopsis (Stracke et al., 2010). 166
Based on the abundance of LREs in the miR858a promoter, we assumed that light may be an 167
important factor for transcriptional activation of miR858a. To verify this, we measured the 168
expression of miR858a and the ProMIR858a:GUS reporter gene under light and dark cycles. 169
In the light-to-dark transition assay, miR858a precursor transcript level was considerably 170
reduced in the WT (Col-0) background (Figure 1D). We further confirmed the transcriptional 171
activation of miR858a precursor gene in response to light (Figure 1E). A marked decrease in 172
GUS staining and GUS gene expression was observed when seedlings were grown under 173
continuous dark for 5 days as compared to standard growth conditions (Supplementary Figure 174
S1A, a, b, S1B). Interestingly, GUS staining and GUS gene expression increased after 175
transfer of dark-grown seedlings to light for different time periods (Supplementary Figure 176
S1A, c to e, S1C). The pattern of GUS staining was comparable to GUS gene expression 177
under light-dark cycle (Supplementary Figure S1). Together, our results demonstrate that 178
miR858a is extensively expressed in Arabidopsis, and its transcript levels are highly 179
dependent on light. 180
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miR858a Targets R2R3-MYB Transcription Factors 182
To predict possible targets of miR858a in Arabidopsis, we used the online prediction 183
algorithm WMD3 and psRNAtarget. The analysis identified more than 40 putative targets of 184
miR858a including MYB11, MYB12 and MYB111 (Supplementary Table S3). Most of the 185
identified targets were members of Arabidopsis R2R3 domain containing MYB family 186
including MYB3, MYB4, MYB6, MYB11, MYB12, MYB32, MYB63, MYB85, MYB111, 187
MYB123, MYB305 and MYBL2. Our analysis suggests that miR858a may target these 188
transcription factors as it has a sequence complementary to the mRNA sequences encoding a 189
conserved part of the R2R3MYB DNA binding domain. To correlate expression of miR858a 190
and their putative targets, the abundance of the MYB11, MYB12 and MYB111 genes in 191
different tissues of Arabidopsis was analysed through qRT-PCR. Results demonstrated 192
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differential expression of miR858a as well as MYB11, MYB12 and MYB111 in rosette, stem, 193
flower, silique and seedling. The maximum expression of miR858a was observed in siliques 194
and the least in rosettes (Figure 1B). Interestingly, the expression of MYB11, MYB12 and 195
MYB111 overlapped with the abundance of miR858a in the stem and flower (Figure 1B). 196
These results correlate with previous reports where both miRNA and target mRNAs are 197
predominantly detected in the same tissues in Arabidopsis (Llave et al., 2002; Zhang and Li, 198
2013). The highest expression of miR858a was observed in the seedling with least expression 199
of MYB11 and MYB12. The observations clearly suggest that miR858a may target flavonol-200
specific MYBs in Arabidopsis. 201
Moreover, to experimentally confirm the target of miR858a, we analysed cleavage 202
products of RISCmiR858a action through RNA ligase-mediated 5' amplification of cDNA ends 203
(RLM-RACE). MYB11 and MYB111 were confirmed as targets of miR858a (Figure 1F). 204
Although, during RLM-RACE analysis, we were unable to get cleavage product of MYB12, 205
however, an earlier study has shown MYB12 to be a direct target of miR858 (Fahlgren et al., 206
2007). In addition, cleavage products for MYB6, MYB13, MYB20, MYB42, MYB48, MYB83, 207
TT2 and MYBL2 were also identified through RLM-RACE (Supplementary Figure S2). The 208
analysis confirmed MYB13 as a direct target of miR858a (Supplementary Figure S2B). A 209
majority of the cleavage products for MYB6, MYB20, MYB42, MYB48, MYB83, TT2 and 210
MYBL2 were detected outside of the canonical miRNA cleavage sites (Supplementary Figure 211
S2). It is likely that such cleavage might be mediated by variants of the mature miRNA, 212
abundant miRNA* sequences or miRNA/miRNA* duplexes from the same hairpin as 213
suggested by Addo-Quaye et al., (2008). Overall, the results showed that flavonol-specific 214
MYBs namely MYB11, MYB12 and MYB111 are direct targets of miR858a. 215
miR858a Modulates Phenylpropanoid Pathway in Arabidopsis 216
To investigate the function of miR858a, we developed miR858a over-expressing transgenic 217
lines (miR858OX) under the control of CaMV35S promoter in Arabidopsis. To further 218
strengthen the study, we generated the artificial target mimic (MIM858) expressing transgenic 219
lines that supposed to nullify the effect of the endogenous miR858a activity. Homozygous 220
transgenic lines for both, miR858OX and MIM858, were selected for further analysis. 221
Significant modulation in the expression of the miR858a was observed in miR858OX and 222
MIM858 transgenic lines (Figure 2A, 2B). As expected, expression of miR858a was 223
significantly higher in miR858OX lines (Figure 2A). Surprisingly, expression of miR858a 224
was significantly compromised in transgenic lines expressing MIM858 compared to WT 225
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plants (Figure 2B). Previous studies on Arabidopsis and bacteria have also described 226
decreased levels of the targeted miRNA after MIM expression. These studies suggested that 227
unproductive interaction of RISCmiRNA with MIM affects miRNA stability (Overgaard et al., 228
2009; Todesco et al., 2010). We suggest that a similar mechanism might be the reason for the 229
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decreased levels of miR858a in MIM858 transgenic lines. Mature miR858a accumulated in 230
miR858OX lines at significantly higher levels in all the tissues as compared to the WT 231
(Supplementary Figure S3) indicating the successful biogenesis of ectopic miR858a 232
precursors. 233
To study whether over-expression and sequestration of miR858a affected the 234
expression of MYB11, MYB12 and MYB111 genes, the transcript levels of these MYBs were 235
analyzed in the rosette tissue. Expression of MYB11, MYB12 and MYB111 was significantly 236
reduced in miR858OX lines (Figure 2C) which suggests a negative correlation between 237
miR858a and target gene expression. On the other hand, as a result of the sequestration of the 238
endogenous miR858a by the expression of MIM858, elevated levels of the MYB11, MYB12 239
and MYB111 transcripts were observed (Figure 2D). Detailed study of the expression patterns 240
of miR858a targets revealed that most of these genes were down-regulated in different tissues 241
in miR858OX lines (Supplementary Figure S4). 242
To study whether modulation of miR858a levels affects flavonoid pathway genes, 243
expression of CHS, CHI, F3’H and FLS1 was studied in transgenic lines. Analysis revealed 244
that expression of these genes was reduced in miR858OX lines in comparison to WT (Figure 245
2E). Contrasting results were obtained in MIM858 transgenic plants with a significant 246
enhanced expression of CHS, CHI, F3’H and FLS1 as compared to WT (Figure 2F). This 247
suggests that miR858a expression negatively affects the flavonoid biosynthetic pathway by 248
repressing activities of the MYB11, MYB12 and MYB111 transcription factors. Moreover, 249
we also performed comparative expression analysis between WT and miR858OX lines using 250
Affymetrix microarray platform. Analysis revealed that expression of the putative targets 251
(MYB40, MYB65, MYB83, MYB86 and MYB111) and flavonoid biosynthesis genes (CHS, 252
CHI and FLS1) are negatively correlated with the miR858a expression levels (Supplementary 253
Table S4, Supplementary Figure S5A, S5B). Altogether, the data implies that miR858a 254
controls the phenylpropanoid pathway by regulating expression of the flavonol-specific MYB 255
transcription factors in Arabidopsis. 256
miR858a is Required for Plant Growth and Development 257
Our genome-wide expression analysis and in silico prediction along with the RLM-RACE 258
analysis confirmed MYB20, MYB42, MYB83 as targets of miR858a (Supplementary Data Set 259
S1, Supplementary Figure S2). These MYBs are known to be associated with plant growth 260
and development (Zhong et al., 2008; McCarthy et al., 2009; Dubos et al., 2010; Zhong and 261
Ye, 2012). Therefore, we studied growth parameters of the transgenic and WT plants 262
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throughout the life cycle. The results suggested that ectopic expression of miR858a and 263
MIM858 changes the various morphological characters with contrasting phenotypes between 264
miR858OX and MIM858 transgenic lines. Transgenic plants over-expressing miR858a 265
showed an elevated vegetative growth phenotype while MIM858 exhibited reduced 266
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vegetative growth as compared to WT plants just after germination. Significantly longer root 267
length, cotyledonary growth and seedling fresh weight were observed in miR858OX lines as 268
compared to the WT. In addition, shorter primary roots with smaller cotyledonary growth and 269
lower seedling fresh weight were observed in MIM858 lines as compared to miR858OX lines 270
and WT (Figure 3A-3C, Supplementary Table S5, Supplementary Figure S6A). 271
Rapid growth and more serrate margins of rosette leaves were observed in mature 272
miR858OX transgenic lines as compared to WT (Figure 3D, 3E). However, no significant 273
difference was seen in the leaf number, rosette diameter, rosette fresh weight and plant height 274
in mature miR858OX transgenic plants (Figure 3E-3G, Supplementary Table S5, S6, 275
Supplementary Figure S6B). Concomitantly, MIM858 lines displayed smaller rosette 276
phenotype during the early developmental stage. Even at the time of bolting, the rosette 277
diameter is significantly reduced as compared to WT (Figure 3D, Supplementary Table S5). 278
The rosette leaves of mature MIM858 plants displayed elongated with sinuate margins 279
compared to WT and miR858OX (Supplementary Figure S6B). Marked differences in the 280
rosette fresh weight and plant height were also observed (Supplementary Table S6, 281
Supplementary Figure S6). These results suggest that miR858a is one of the crucial regulators 282
of plant growth and development. 283
Altered Reproductive Phase Parameters in Transgenic Lines 284
The variation in the flowering time and flower size of miR858OX and MIM858 transgenic 285
lines was observed as compared to WT plants (Figure 3, 4). Under standard growth 286
conditions, early bolt formation and flowering were observed in the miR858OX plants in 287
comparison to WT whereas bolt formation and flowering time was significantly delayed in 288
the MIM858 plants relative to the WT (Figure 3D, 3F). More rosette leaves as compared to 289
WT were observed in the MIM858 transgenic lines though no significant change was 290
observed in the miR858OX lines. Unlike miR858OX lines, a significantly reduced rosette 291
diameter was observed in the MIM858 transgenic lines as compared to the WT (Figure 3D, 292
3G). Aside from flowering time, a significant reduction in the length and width of flowers 293
was observed in MIM858 transgenic lines compared to the WT (Figure 4A, 4B, 294
Supplementary Table S7). However, no such difference was observed in the miR858OX 295
lines. These results suggested that modulation in the expression of miR858a affected 296
flowering time and flower size which might be controlled co-ordinately by miR858 in 297
flowering plants. 298
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Furthermore, observations also suggest that miR858OX lines have an increased 299
number and length of siliques per plant (Figure 4C-4E, Supplementary Table S8, 300
Supplementary Figure S6C) as well as enlarged seed size (Figure 4F-4G) as compared to 301
WT. In contrast, MIM858 lines produced a lower number but longer siliques per plant with 302
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seeds significantly smaller in size (Figure 4C-4F, Supplementary Table S8, Supplementary 303
Figure S6C). Additionally, miR858OX lines had a smaller number of seeds per silique while 304
MIM858 produced more seeds per silique as compared to WT (Figure 4H, Supplementary 305
Table S8). We have also counted the number of seeds per unit (5 mg) weight and observed a 306
significant increase in seed count in MIM858 as compared to miR858OX and WT plants 307
(Figure 4I, Supplementary Table S8) indicating larger seed size in miR858OX and smaller 308
seed size in MIM858 transgenic lines. Enhanced vegetative growth with increased seed size 309
and weight in miR858OX suggested an important role of miR858a during seed development 310
and plant yield. 311
To identify differentially expressed genes in miR858OX lines as compared to WT, 312
microarray analysis was carried out. Analysis revealed differential expression of genes 313
involved in jasmonate, ethylene and gibberellin signalling pathways and regulatory 314
mechanisms. In addition, expression of many developmental- and stress-responsive genes 315
was modulated in the transgenic lines (Supplementary Data Set S1). The expression of 316
significantly up-regulated genes identified through microarray analysis was validated using 317
qRT-PCR (Supplementary Figure S7) and showed a similar expression trend. Altogether, 318
significant changes in the phenotypes and microarray expression analysis suggested that 319
miR858a plays a versatile role in plant growth and development of Arabidopsis. 320
Metabolite profiling in miR858OX and MIM858 Transgenic Plants 321
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To investigate the impact of miR858a on synthesis of metabolites via phenylpropanoid 322
pathway, Q-Trap mass spectrometric analysis of methanolic extracts of rosette and stem of 5-323
week-old WT, miR858OX and MIM858 plants was carried out (Table 1). The modulation in 324
contents of molecules of various steps of the phenylpropanoid pathway in miR858OX and 325
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MIM858 transgenic plants as compared to the WT is summarized in Figure 5. The analysis 326
revealed higher accumulation of the major flavonoids such as kaempferol, quercetin and rutin 327
hydrate in MIM858 as compared to the WT plants. In contrast, a significant decrease in these 328
flavonoids was observed in the miR858OX rosette tissue (Table 1, Figure 5). Interestingly, 329
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these compounds are amongst the most abundant flavonol derivatives in WT plants (Stracke 330
et al., 2007). Kaempferol was significantly higher (10199.6 ± 161.0 µg/100 g DW) in rosette 331
tissues of MIM858 as compared to WT (4808.3 ± 93.3 µg/100 g DW) and miR858OX 332
(2320.4 ± 36.6 µg/100 g DW). In addition, other flavonols like quercetin-3-o-galactoside (Q-333
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3-O-G) and metabolites belonging to different classes of flavonoids such as flavanones 334
(naringenin and naringin) and flavan-3-ols (gallocatechin) showed similar accumulation 335
patterns (Table 1, Figure 5). 336
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Other phenylpropanoids such as p-coumaric acid, caffeic acid, ferulic acid, sinapic 337
acid and syringic acid were significantly increased in MIM858 while their accumulation 338
decreased in miR858OX line in comparison to the WT (Table 1, Figure 5). The accumulation 339
of metabolites in stem significantly varied as compared to rosette samples in the transgenic 340
lines. Surprisingly, higher metabolite accumulation was detected in stem tissue of miR858OX 341
as well as in MIM858 in contrast to the WT (Supplementary Table S9). These differences 342
were indicative of the existence of a differential regulation of metabolic pathways by 343
miR858a in different tissues. Collectively, higher accumulation of flavonoids in MIM858 and 344
their reduced levels in the miR858OX in rosette tissue of transgenic lines indicates that 345
miR858a regulates the biosynthesis of flavonoids through transcriptional regulation of 346
upstream transcription factors which controls the expression of genes involved in the 347
flavonoid biosynthesis pathway. 348
miR858a Affects Lignin Biosynthesis in Transgenic Plants 349
Lignin accumulation analysis suggested modulated lignin content in the vascular and inter-350
fascicular tissues of transgenic lines as compared to the WT. Lignin staining and 351
quantification of stem cross-section of the WT, miR858OX and MIM858 lines revealed an 352
enhanced lignification in the vascular and interfascicular tissues of the miR858OX lines 353
(Figure 6A, Supplementary Figure S8). Although, miR858a over-expression caused ectopic 354
deposition of lignin no prominent secondary wall thickening was observed in the epidermal 355
cells. These results are distinct from the observations of the MIM858 lines that showed less 356
lignification of cells (Supplementary Figure S8). The examination of vascular bundles and 357
epidermal cells showed that secondary wall thickness of both vessels and fibres were reduced 358
in the MIM858 line as compared to WT. Surprisingly, these results are inconsistent with 359
phenotypic observations with weaker inflorescence stems of MIM858 lines than the WT 360
plants (Figure 6B). Consistent with the histological data, the enhanced expression of several 361
secondary wall promoting and lignin biosynthetic genes including CESA4, SND1, MYB58, 362
HCT, CCoAOMT1, CCR1 and CAD6 was observed in the miR858OX stem tissue of 363
transgenic lines (Figure 6C). At the same time, reduced expression of these genes was 364
observed in the MIM858 lines. These observations explicitly suggest that miR858a-MYBs 365
interaction maintains the metabolic flux balance between the flavonoid and lignin 366
biosynthesis via activating a subset of the secondary wall and lignin biosynthetic genes. 367
368
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369
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DISCUSSION 370
The regulation of gene expression in specific cells and tissues is essential for normal growth 371
of the plant. Over the past few years, large number of experiments have shown that a sub-372
group of R2R3-MYBs such as MYB11, MYB12 and MYB111 apparently influence the 373
spatial accumulation of flavonols via activating a branch of the phenylpropanoid pathway 374
(Mehrtens et al., 2005; Czemmel et al., 2009; Misra et al., 2010; Stracke et al., 2010; Liu et 375
al., 2015). These MYBs participate in fine-tuning of transcriptional activation of a set of 376
genes engaged in flavonol biosynthesis, and maintain a subtle balance with other branches of 377
the phenylpropanoid pathway (Xu et al., 2015). Studies suggest that the organ and temporal 378
distribution of flavonols are the consequence of differential expression of these MYBs and 379
environmental factors in Arabidopsis (Stracke et al., 2007). The fundamental question arises 380
about whether only cis-acting elements present in promoters of these MYBs are responsible 381
for differential expressions or any other trans-factors also determine their regulatory 382
mechanisms. Numerous past studies have suggested that miR858 can target R2R3-MYBs in 383
apple, grape and Salvia miltiorrhiza (Xia et al., 2012; Rock, 2013; Li et al., 2014). In a quest 384
for such factor, we have shown that miR858a controls the expression of flavonol-specific 385
MYBs for instance, MYB11, MYB12 and MYB111, in addition to other members of MYB 386
family, and studied its detailed role further, in Arabidopsis. 387
miR858 is transcribed from two genomic loci in Arabidopsis and targets similar 388
genes. The additional targets of miR858a identified in this study might be involved in 389
regulating diverse developmental aspects in the plant which confirmed the significance of 390
miR858a at specific developmental programs in Arabidopsis. The earlier function of miR858 391
has been recognised in trichome fibre development via regulating MYB2 in Arabidopsis 392
(Guan et al., 2014) and anthocyanin biosynthesis in tomato (Jia et al., 2015). These reports 393
are in agreement with the idea that miR858 might target a number of R2R3-MYB genes and 394
may be involved in the regulatory responses leading to the secondary metabolite production 395
and specific developmental programs. The expression of miR858a was found to be 396
widespread in Arabidopsis as evident from qRT-PCR results and promoter activity (Figure 397
1). The GUS staining observed in the different developmental stages throughout the life cycle 398
of Arabidopsis suggests an active participation of miR858a mediated regulation in plant 399
growth and development. Despite the in planta expression, miR858a was specifically active 400
in vascular tissues of the seedling. 401
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Light serves as a critical factor of the transcriptional activation of miR858a precursor 402
which indicates that similar to that of MYBs, this sector of flavonol biosynthesis regulation is 403
also under the tight control of light. In silico analysis of the promoter of miR858a revealed 404
the presence of many LREs, specifically HY5 binding sites indicating the putative light-405
mediated regulation of miR858a expression (Supplementary Table S2). HY5 is a member of 406
the bZIP/bHLH transcription factor family which plays a pivotal role in light signalling and 407
photomorphogenesis processes by interacting with various pathways (Lee et al., 2007; Delker 408
et al., 2014). It has been shown that HY5-mediated transcriptional activation of MYB12 and 409
MYB111 genes are the under control of UV-B and visible light (Stracke et al., 2010). Our 410
light-dark transition experiment revealed that miR858a is substantially reduced under dark 411
condition in Col-0 (Figure 1D). The expression of miR858a is induced so rapidly that it 412
reached to the maximum within 0.5 hr exposure of light showing the relevance of light in its 413
regulation (Figure 1E). Therefore, it would be concluded that light is the major factor that 414
controls the expression of miR858a and various genes of the phenylpropanoid pathway. 415
miR858a cleaves the flavonol-specific MYBs such as MYB11, MYB12 and MYB111 416
which are predicted as the potential target using online target prediction tools (Supplementary 417
Table S3). The MYB11 and MYB111 have been verified as targets through 5’- RLM-RACE 418
analysis; however, we were unable to detect the cleavage product of MYB12 in our 419
experimental conditions, although MYB12 was confirmed to be an actual target of miR858 in 420
an earlier study (Fahlgren et al., 2007). MYB transcription factors are considered as potential 421
targets of miR858 in several plant species (Addo-Quaye et al., 2008; Xia et al., 2012; Jeong 422
et al., 2013; Guan et al., 2014; Jia et al., 2015). In addition, our in silico prediction and 423
cleavage products analysis for a set of other MYBs (Supplementary Figure S2) suggest that 424
miR858a offers complex regulation in Arabidopsis and is not only confined to flavonol-425
specific MYBs. 426
miR858a Expression is Important for Arabidopsis Growth and Development 427
miR858a caused a decrease in the expression of target MYBs and affected growth and 428
morphology in Arabidopsis in miR858OX and MIM858 lines. To nullify the effect of 429
endogenous miR858a, we employed the sponge decoy strategy and developed MIM858 in 430
such a way that it contains two repeats of the exact complementary sequence of miR858a, 431
and three repeats have 3 nt bulge between 9-11 position (Ebert et al., 2007). As expected, 432
MIM858 lines successfully delivered the knockdown phenotype in Arabidopsis. A significant 433
reduction in miR858a and increase in target gene expression was observed in MIM858 lines 434
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(Figure 2A-2D). Phenotypic examination of both types of transgenic lines revealed that 435
miR858OX and MIM858 lines produced contrasting features of various growth parameters 436
(Figure 3, 4, Supplementary Figure S6, Supplementary Table S5, S6). Though expression of 437
miR858a was least in rosette tissue (Figure 1B), transgenic lines overexpressing miR858a 438
significantly changed rosette size, flowering time and metabolite content. This indicates that 439
even a slight variation of miR858a expression in rosette tissue could lead to the much bigger 440
impact on morphology of the plant where this miRNA is lowly abundant. It is well known 441
that MYB transcription factors play an important role in many developmental processes 442
(Ishida et al., 2007; Reyes and Chua, 2007; McCarthy et al., 2009; Dubos et al., 2010; Cui et 443
al., 2013; Chaonan et al., 2014). Previous studies have found that AtMYB11 modulates overall 444
growth in the plants by reducing the proliferation activity of the meristematic cells and 445
delaying plant development whereas mutant showed a faster development of the 446
inflorescence and initiate more lateral inflorescences and fruits (Petroni et al., 2008). 447
Similarly, contrasting phenotypes were obtained in miR858OX and MIM858 transgenic lines 448
in which expression of the MYB11 is significantly altered (Figure 3, 4, Supplementary Figure 449
S6, Supplementary Table S5, S6). 450
The miRNAs such as miR156, miR172 and miR159 regulate key genes involved in 451
flowering related processes (Hong and Jackson, 2015; Teotia and Tang, 2015). The 452
contrasting observations of bolt formation, flowering and flower size between miR858OX 453
and MIM858 lines might be due to perturbation of flowering related MYBs. MYBs and 454
flavonoids play a very crucial role in the Arabidopsis seed development (Routaboul et al., 455
2006; Doughty et al., 2014; Xu et al., 2014), therefore, a significant change in seed size in 456
transgenic lines may be an outcome of varied flavonoids content brought by miR858a. On 457
other hand, validation of several other MYBs, participating in various developmental 458
processes, as targets of miR858a (Supplementary Table S3) could be another probable reason 459
for such developmental alteration in the transgenic lines. The relation of these transcription 460
factors with miR858a and the downstream cascade could be a subject of future study. 461
miR858a-mediated Regulation Affects the Phenylpropanoid Pathway 462
The regulation of the flavonol biosynthesis mainly takes place at transcriptional level and 463
feedback inhibition by the end products. The flavonol-specific MYBs induced expression of 464
the structural genes such as CHS, CHI, F3H and FLS1 of the phenylpropanoid pathway 465
(Stracke et al., 2007; Dubos et al., 2010; Xu et al., 2015). Previously, a plethora of reports 466
suggested that expression of such MYBs in tobacco and tomato enhances the expression of 467
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structural genes of the pathway leading to the enhanced flavonoid content, nutritional quality 468
and insect resistance (Luo et al., 2008; Misra et al., 2010; Pandey et al., 2012; Pandey et al., 469
2014; Pandey et al., 2014; Pandey et al., 2015; Pandey et al., 2015; Zhang et al., 2015; 470
Choudhary et al., 2016). Here, we describe the participation of miR858a in transcription 471
factor regulon responsible for biosynthesis of flavonoids and development in Arabidopsis. 472
Our analysis suggested that the expression of CHS, CHI, F3H and FLS1 was down-regulated 473
in miR858OX whereas induced in MIM858 lines (Figure 2E, 2F). These results indicate that 474
MIM858 titrates the endogenous miR858a activity in vivo and enhances expression of genes 475
encoding enzymes of flavonoid biosynthesis. 476
Metabolic profiling demonstrated a significant rise in major flavonoids such as 477
kaempferol and rutin (Table 1) and other phenylpropanoids in the MIM858 transgenic lines. 478
Previous studies suggested that flavonoids influence plant architecture (Taylor and Grotewold 479
2005; Buer and Djordjevic 2009; Buer, 2010) mainly through their effects on auxin transport. 480
Flavonoid-defective mutants display a wide range of alterations in a myriad of developmental 481
processes such as root and shoot development flower organ number, overall architecture and 482
stature, and seed organ density (Buer and Djordjevic 2009). Modulation in major flavonoids, 483
namely, kaempferol and quercetin in our metabolic profiles (Table 1, Supplementary Table 484
S9) clearly provide the justification for diverse alteration in phenotype in the transgenic lines. 485
The notion was also supported by the microarray and RT-PCR analyses which revealed 486
down-regulation of flavonoid-specific MYBs and early pathway genes (Supplementary Data 487
Set S1, Supplementary Table S4 and Supplementary Figure S5). 488
CHS and HCT share a common substrate (p-coumaroyl CoA) leading to the 489
biosynthesis of flavonoids and lignin in the phenylpropanoid pathway (Besseau et al., 2007). 490
The repression of the lignin biosynthesis via HCT-silencing impacts flavonoid accumulation 491
significantly in Arabidopsis (Hoffmann et al., 2004; Besseau et al., 2007; Li et al., 2010). We 492
quantified lignin in the stem of the WT and transgenic lines and measured expression of 493
genes such as CESA4, SND1, MYB58, HCT, CoOAMT, CCR1 and CAD6. The results clearly 494
showed a high lignin content in the stem of miR858OX in contrast to lower lignin content in 495
MIM858 transgenic lines (Figure 6). This might be due to the atypical lignification of the 496
mechanical tissues in the stem of MIM858 lines. These results indicate that miR858a 497
mediated perturbation of flavonoid branch of the phenylpropanoid pathway can affect the 498
lignin biosynthesis branch of the pathway. The same interpretation could be applied to 499
explain the rosette diameter and fresh weight phenotype of MIM858 transgenic plants 500
(Supplementary Table S6). Together, flavonoid profiling and transcriptome data from 501
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combinatorial analysis of miR858OX and MIM858 plants suggest that miR858a responses 502
affect the flavonoid biosynthetic pathway. Our results provide new insights into the 503
regulation of the phenylpropanoid pathway by miR858a besides various transcription factors. 504
505
MATERIALS AND METHODS 506
Bioinformatic Analyses 507
To identify miRNAs targeting A. thaliana MYB11, MYB12 and MYB111 genes, BLASTn 508
searches were performed on the Sanger database for miRNAs (http://microrna. sanger.ac.uk; 509
release 15) and for available genomic sequences on the National Centre for Biotechnology 510
Information (http://www.ncbi.nlm.nih.gov/BLAST). Sequences of the miR858a precursor 511
and mature isoforms in Arabidopsis and other plant species were retrieved from the miRBase 512
database. To evaluate the hybridization energies and percentage homology between miR858a 513
and their putative targets, alignments of 21 bp between the mature miRNA and the target 514
mRNAs were carriedout using psRNAtarget server (http://plantgrn.noble.org/psRNATarget) 515
(Dai and Zhao, 2011) and WMD3 (http://wmd3.weigelworld.org) (Schwab et al., 2006). 516
Plant Material and Growth Conditions 517
A. thaliana (ecotype Col-0) was used as WT plant in this study. For germination, seeds were 518
surface-sterilized and placed on half-strength Murashige and Skoog (MS) medium (Sigma, 519
India) containing 1% sucrose. After stratification for 2-3 days at 4°C in the dark, seeds were 520
transferred to the growth chamber (Conviron, USA) under controlled conditions of 16 hr 521
light-8 hr dark photoperiod cycle, 22ºC temperature, 150–180 µm E m-2 s-1 light intensity and 522
60% relative humidity for 10 days unless otherwise mentioned. Measurements of the root 523
length and fresh weight were performed on 10-day-old seedlings. Rosette fresh weight and 524
diameter were determined for 20-day-old and 1-month-old plants. Plant height and silique 525
data were taken from the mature plants. For light-to-dark transition assay, WT (Col-0) and 526
ProMIR858a:GUS transgenic seeds were transferred to standard light and complete dark 527
conditions for 5 days. After dark treatment, seedlings were exposed to light for different time 528
periods (0.5 hr, 2 hr and 6 hr) and used for further analysis. 529
Construct Preparation and Transformation 530
To over-express miR858a, the miR858a precursor (187 bp) (AT1G71002) was amplified 531
from the single stranded cDNA library prepared from Arabidopsis rosette leaves through 532
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26
PCR using specific primers (Supplementary Table S10). Amplicon was cloned into pT/Z sub-533
cloning vector (Fermentas, USA), and then transferred into plant expression vector, pBI121, 534
containing CaMV35S promoter. To reduce miR858a activity, a target mimicry approach 535
(Franco-Zorrilla et al., 2007) was used in the study. To synthesise and clone the miR858a 536
target mimic, forward and reverse overlapping primers were designed (Supplementary Table 537
S10). The amplified PCR product was cloned into pT/Z sub-cloning vector (Fermentas, 538
USA), and then transferred into plant expression vector pBI121. All the constructs were 539
transformed into the Agrobacterium tumefaciens (strain GV3101) and used to transform A. 540
thaliana (Col-0) using floral dip method (Clough and Bent, 1998). Transformants were 541
screened on half-strength MS agar plates containing kanamycin (50 µg/mL). For promoter 542
analysis, 1847 bp upstream region from the transcription start site of miR858a precursor 543
(Figure 1C, a) was PCR amplified using High Fidelity enzyme mix (Fermentas, USA) from 544
genomic DNA. Amplicon was cloned into pCAMBIA1303 plant binary vector resulting in 545
the construct ProMIR858:GUS. For identification of the transcription start site of miR858a, 546
plant promoter analysis navigator (PlantPAN; http://PlantPAN2.itps.ncku.edu.tw) resource 547
was used. Transgenic plants were generated and screened on hygromycin (10 mg/mL). Out of 548
minimum six transgenic lines generated using different constructs, three independent lines for 549
each of miR858OX, MIM858 and ProMIR858:GUS were selected for further analysis. All the 550
studies were performed in T4 homozygous lines. 551
Expression Analysis 552
For expression analysis, microRNA858a TaqMan PCR assays were used following the 553
recommendations of the manufacturer (Applied Biosystems, USA). A TaqMan PCR assay for 554
the small nuclear RNA snoR41Y was used as normalization control. For qRT-PCR, total 555
RNA was treated with TURBO DNAfree (Ambion, USA), and 2 µg was reverse transcribed 556
using RevertAid H minus first strand cDNA synthesis Kit (Fermentas, USA) according to the 557
manufacturers’ instructions. The cDNA was diluted 20 times with nuclease free water and 1 558
µl was used as template into qPCR reactions performed using Fast Syber Green mix (Applied 559
Biosystems, UK) in Fast 7500 Thermal Cycler instrument (Applied Biosystems, UK). 560
Expression was normalized using Tubulin and analysed through comparative ∆∆CT method 561
(Livak and Schmittgen, 2001). Primer sequences used for expression analysis are listed in 562
Supplementary Table S10. 563
Microarray Analysis 564
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Genome-wide expression analysis was performed using Affymetrix GeneChip Arabidopsis 565
Genome ATH1 microarrays (Affymetrix, USA) and as per MIAME guidelines (Brazma et 566
al., 2001). Total RNA was extracted from 3-week-old rosette leaves of Col-0 and miR858OX 567
plants using Spectrum Plant Total RNA Kit (Sigma, USA). Briefly, total RNA (250 ng) of 568
each sample was taken as starting material and strictly followed the Affymetrix protocol 569
during RNA amplification, hybridization, washing-staining and scanning of the arrays. After 570
hybridization, raw data from CEL files were initially analyzed using Affymetrix Expression 571
Console with library files supplied by Affymetrix. Analyses were performed using the RMA 572
for gene levels without normalization. The log2 transformed data were exported for analysis 573
using Affymetrix Transcriptome Analysis Console (TAC) 2.0 software. Quantitative Real-574
Time PCR (qRT-PCR) was carried out to validate microarray data. 575
RNA Ligase-mediated (RLM) 5’ RACE 576
To map cleavage sites in the candidate targets of miR858a in planta, modified RLM 5’ 577
RACE was performed using First Choice RLM-RACE kit (Ambion, USA). Total RNA was 578
extracted from WT and miR858OX rosette leaves and directly ligated to RNA oligo adapter 579
without CIP and TAP treatments. cDNA synthesis was performed using oligo(dT) primer 580
using RevertAid H minus first strand cDNA synthesis Kit (Fermentas, USA) according to the 581
manufacturers’ instructions. RACE and nested-RACE PCR were subsequently carried out 582
with adaptor-specific forward primers and gene-specific reverse primers for the detection of 583
cleavage products of the target mRNAs. PCR products were cloned into the pT/Z sub-cloning 584
vector (Fermentas, USA) and sequenced to locate miRNA cleavage sites. 585
586
Histochemical Assays 587
GUS staining was performed using standard method (Jefferson, 1989). Different tissues of 588
ProMIR858:GUS transgenic lines were incubated in a solution containing 100 mM Na 589
phosphate buffer, pH 7.2; 10 mM EDTA; 0.1% Triton X-100; 2 mM potassium ferricyanide; 590
2 mM potassium ferrocyanide; and 1 mg/mL 5-bromo-4-chloro-3-indolyl-b-D-glucuronide at 591
37ºC overnight for 4 to 6 hr. The chlorophyll was removed by incubation and multiple 592
washes using 70% ethanol. 593
Flavonoid Accumulation Analysis 594
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Dried tissues (100-500 mg rosette and stem from mature plants) were ground into the fine 595
powder in liquid N2 and were extracted in 80% methanol using an Ultra-Turrax T-25 Digital 596
Homogenizer (IKA, Germany) at 16000 RPM for 2 min. The homogenate was centrifuged at 597
20,000xg for 10 min at 4°C and the supernatant was filtered through a 0.22 µm PTFE syringe 598
filter (Millipore, USA). The Filtrate was collected in a clear glass HPLC vial (Agilent 599
Technologies, Singapore) and inject into HPLC-MS. All the samples were analysed on 1260 600
infinity series HPLC system (Agilent Technologies, Singapore) equipped with 1260 infinity 601
series pump, autosampler, column compartment and thermostat using Zorbax Eclipse Plus 602
column (4.6 x 100 mm, 3.5 µm) maintained at 40°C. The mobile phase consisted aqueous 603
solution of 0.1% formic acid LCMS grade (Solution A) and 0.1% formic acid in LCMS grade 604
acetonitrile (Solution B). The gradient was programmed as follows: 0-2 min, solution B 605
(2%); 2-22 min, Solution B (2-98%); 22.01-25 min, Solution B (98-2%). Other 606
chromatographic parameters included a constant flow of 600 µl/min (injection volume 5 µl), 607
autosampler temperature 10°C and a run time of 25 min including equilibration. Before 608
analysis, all of the samples were filtered through a 0.22 µm syringe membrane filters 609
(Millipore, USA). 610
The detection of different molecules was performed using HPLC system (Agilent 611
Technologies 1260 infinity series) coupled to a triple quadrupole system QTRAP5500 612
(ABSciex, Singapore) using ESI probe in both the mode of ionisation. The voltage was set at 613
5500V for positive ionisation and -4500V for negative ionisation. The values of Gas1 and 614
Gas2 (50), curtain gas (CUR; 30), collision assisted dissociation (CAD; medium), and 615
temperature of the source (TEM; 550°C) were used. The mass spectrometer was used in 616
Multiple Reaction Monitoring (MRM) mode for the identification and quantification analysis. 617
Analytical standards of phenolics were purchased from Sigma-Aldrich (India). The 618
identification and quantitative analysis was carried out using Analyst software (version 1.5.2) 619
and MultiQuant software (version 2.0.2). The Signal finder algorithm was used in Multiquant 620
software for quantitative analysis. 621
Lignin Analysis 622
To visualize lignified cell in stems, hand cut sections were stained for 2 min using Toluidine 623
blue O (Sigma-Aldrich, USA) and visualized on Leica DM2500 microscope (Leica 624
Microsystems, Germany). Lignin quantification was carried out using method described by 625
Bruce and West (1989). Briefly, samples (600 mg) were pulverized in liquid N2 and 626
suspended in 2 ml ethanol. Following centrifugation at 12,000xg (30 min, 4ºC), the pellet was 627
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29
dried at 25ºC for 12 hr. Dried pellet was resuspended in 5 ml HCl (2N) and 0.5 ml 628
thioglycolic acid and incubated at 95ºC for 8 hr followed by cooled to room temperature. The 629
suspension was centrifuged at 12000xg for 30 min, and the pellet was washed with 2.5 ml 630
double distilled H2O. After recentrifuging at 12000xg (4ºC) for 5 min, the resulting pellet was 631
suspended in 5 ml of 1N NaOH and agitated gently at 25ºC for 18 hr. After centrifugation at 632
12000xg (4ºC) for 30 min, 1 ml HCl was added to the supernatant, and the solution was 633
allowed to precipitate at 4ºC overnight. Following centrifugation at 12000xg (4ºC) for 30 634
min, the pellet was dissolved in 3 ml of 1N NaOH, and the absorbance at 280 nm was 635
recorded as lignin content. 636
Statistical Analysis 637
The experiments were arranged in a completely randomized design. Each sample time point, 638
for each treatment, was comprised of three independent biological replicates. Data were 639
plotted as means ± standard errors (SE) in figures. Phenotypic observations were monitored 640
up to 2–3 generations for consistency. One-way ANOVA followed by post hoc Newman–641
Keuls was used for multiple comparison test of significance by using GraphPad Prism 6.05 642
software. 643
644
Acknowledgements 645
DS, AP and CB thankfully acknowledge the Council of Scientific and Industrial Research 646
(CSIR), New Delhi for Senior Research Fellowship. MT acknowledges Indian Council of 647
Medical Research for Research Fellowship. Research was supported by Council of Scientific 648
and Industrial Research, New Delhi in the form of Network projects PlaGen (BSC-0107). 649
650
651
Author contributions 652
P.K.T. designed and supervised this study. D.S., M.T., A.P., C.B., and A.S. performed the 653
experiments. D.S., M.T., and P.K.T. performed the data analysis. D.S. and P.K.T. prepared 654
the article. All authors read, contributed, and approved the article. 655
656
Competing financial interest statements 657
All the authors declare that they have no conflict of interest. 658
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30
659
FIGURE LEGENDS 660 661 Figure 1. Identification and characterization of the miR858a in Arabidopsis. (A) Sequences 662
of MYB11, MYB12 and MYB111 mRNAs (5’ to 3’) aligned with the mature sequence of 663
miR858a (3’ to 5’). Dashed line and space represent perfect match and G:U wobble pairing, 664
respectively. The free energy of binding (ΔG) is indicated. (B) Expression of miR858a, 665
MYB11, MYB12 and MYB111 in rosette, root, stem, flower and silique from the 5-week-old 666
plant and 10-day-old seedling of Arabidopsis thaliana Col-0 ecotype. For miR858a, 667
SnoR41Y RNA and for MYB11, MYB12 and MYB111, tubulin was used as the endogenous 668
control to normalize the relative expression levels. Error bars represents ±SE (n = 3). (C) 669
Histochemical staining showing GUS activity in different developmental organs in the 670
ProMIR858:GUS transgenic lines. (a) Schematic representation of miR858a promoter 671
upstream of the precursor (b) 5-day-old seedling, (c) 12-day-old seedling, (d) rosette leaf, (e) 672
stem, (f) inflorescence, (g) silique and (h) seed. Bars indicate 1 cm for a, b, c, d, e, f and 500 673
µm for g image. (D) qRT-PCR analysis of the mRNA level of miR858a in WT (Col-0) 674
seedlings grown in the light and dark conditions for 5 days. Error bars indicate ±SE (n=3) 675
from three biological replicates. (E) qRT-PCR analysis of the mRNA level of miR858a in 676
WT (Col-0) upon exposure to light (150 µmol m-2 s-1) at the indicated times. Error bars 677
indicate ±SE (n=3) from three biological replicates. (F) Target validation for miR858a by 678
RLM-RACE. Cleavage products of MYB11 and MYB111 mRNA are shown. The fraction of 679
clones with the expected 5’ end among all sequenced products is indicated above the target 680
site. Arrows indicate the cleavage sites detected by RLM-RACE. 681
Figure 2. Characterization of miR858OX and MIM858 transgenic lines. (A) qRT-PCR 682
analysis showing expression of mature miR858a in rosette tissue of different miR858OX 683
transgenic lines. (B) qRT-PCR analysis showing expression of mature miR858a in rosette 684
tissue of different MIM858 transgenic lines. (C) Expression of MYB11, MYB12 and MYB111 685
in miR858OX transgenic lines. (D) Expression of MYB11, MYB12 and MYB111 in the 686
MIM858 transgenic lines. (E) Expression of CHS, CHI, F3’H and FLS1 in miR858OX 687
transgenic lines. (F) Expression of CHS, CHI, F3’H and FLS1 in the MIM858 transgenic 688
lines. For miR858a, SnoR41Y RNA and for MYBs and pathway genes, tubulin was used as 689
the endogenous control to normalize the relative expression levels. Error bars in each figure 690
represents ±SE (n=3). 691
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31
Figure 3. Alteration of morphology in the miR858a transgenic lines. (A) The seedling 692
phenotype of WT, miR858OX (L1) and MIM858 (MIM1) transgenic lines grown on ½ MS 693
media for 10 days. (B) Root length of the seedlings of WT, miR858OX (L1) and MIM858 694
(MIM1) after growth of 10 days on ½ MS media. (C) Seedling fresh weight (mg) of the WT, 695
miR858OX (L1) and MIM858 (MIM1) after growth of 10 days on ½ MS media. Phenotype 696
(A) and other data included in B and C for other transgenic lines (L2, L3, MIM2 and MIM3) 697
are shown in Supplementary Figure S6. (D) Rosette phenotypes of WT, miR858OX and 698
MIM858 transgenic lines. (E) Leaf sizes and pattern of each rosette leaf beginning from the 699
oldest one of WT, miR858OX and MIM858 transgenic plants. (F) Number of rosette leaves 700
(black bars) and days at bolting (gray bars) of WT and transgenic lines of the miR858OX and 701
MIM858 grown under LD conditions (16 hr light-8 hr dark cycles). (G) Rosette diameter of 702
transgenic lines and WT. Plants were grown for 3 weeks under 16 hr light-8 hr dark 703
photoperiod before photograph. Error bars represent +SE of 8-10 individual plants. The level 704
of significance was evaluated by One-way ANOVA (Newman-Keuls used as post hoc test). 705
***Significantly different from WT (P < 0.0001). White line indicates the scale bar (1 cm). 706
L1, L2 and L3 are miR858OX transgenic lines whereas MIM1, MIM2 and MIM3 represent 707
MIM858 transgenic lines. 708
Figure 4. Variation of flower, silique and seed parameters in the transgenic lines. (A) Flower 709
size, both length and width, was reduced in MIM858 transgenic plants while no significant 710
change was observed in miR858OX flowers. Photograph of flowers were taken after 5 week 711
growth under standard conditions. White line indicates the scale bar (1 cm). (B) Bars are 712
mean of length and width of independent transgenic line and variation represent as +SE (n = 713
8-12). (C) The phenotype of transgenic lines producing longer silique than WT. Photographs 714
of 6-week-old grown plants were taken. (D) Bars represented transgenic line produced longer 715
silique per plant than WT. (E) Bars represented transgenic lines produced more silique per 716
plant than WT. (F) Representative photograph of seed size of transgenic and WT plants. Seed 717
size of miR858OX was bigger, and that of MIM858 was significantly smaller as compared to 718
WT. White line indicates the scale bar (500 µm). (G) Seed size of different transgenic lines 719
and WT plants. (H) Seed number per silique was higher in MIM858 transgenic line while no 720
difference was observed in miR858OX line. (I) Number of seeds per 5 mg was counted less 721
for miR858OX while more in MIM858 transgenic lines. Data of one transgenic line presented 722
as mean and +SE (n=8-12). Data was recorded from 15 siliques per plant and 8-12 plant for 723
each line and WT. L1, L2 and L3 are miR858OX transgenic lines whereas MIM1, MIM2 and 724
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32
MIM3 represent MIM858 transgenic lines. Significance was determined by One-way 725
ANOVA (Newman-Keuls used as post hoc test). ***Significantly different from WT (P < 726
0.0001). 727
Figure 5. Metabolic profiling of miR858OX and MIM858 transgenic lines. Schematic 728
diagram of the phenylpropanoid pathway. Heat maps indicate log2 transformed values of the 729
concentrations of metabolites in rosette tissue of WT, miR858OX and MIM858 transgenic 730
lines (for values, refer Table 1). LC-MS-MS analysis was performed with the methanolic 731
extracts of dried rosette tissues. Arrows indicate the order of steps in the pathway; dashed 732
arrows represent multiple enzymatic steps. The main enzymes included PAL, phenylalanine 733
ammonia lyase; C4H cinnamate 4-hydroxylase; 4CL, 4-coumaroyl CoA ligase; CHS, 734
chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3’H, flavonoid 735
3’ hydroxylase; FLS, flavonol synthase; DFR, dihydro flavonol 4-reductase; LDOX, 736
Leucoanthocyanidin dioxygenase; LAR, leucoanthocyanidin reductase; UGT78D1, flavonol 737
3-O-rhamnosyltransferase; UGT78D2, flavonoid 3-O-glucosyltransferase; 3GRT, flavonol-3-738
O-glucoside L-rhamnosyltransferase; HCT, p-hydroxycinnamoyl-CoA:quinate shikimate p–739
hydroxycinnamoyltransferase; C3H, p-coumaroyl ester 3-hydroxylase; COMT I, caffeic acid 740
O-methyltransferase of class I; CAD, cinnamyl alcohol dehydrogenase; CCoAOMT, 741
caffeoyl-CoA O-methyltransferase; CCR, cinnamoyl-CoA reductase; F5H, ferulic acid 5-742
hydroxylase. MYB transcription factors are shown in green and red colours (Green for 743
activation and red for inhibition). Likewise green arrow is for activation and red arrow for 744
inhibition. 745
Figure 6. miR858a expressions affects lignin depositions in Arabidopsis. (A) Quantification 746
of total lignin content in WT and transgenic lines. Mature stems of WT, miR858OX and 747
MIM858 transgenic lines were equally weighed and used for quantification. Bars indicate 748
standard error of three biological replicates at each sampling time-point. Significance was 749
determined by One-way ANOVA (Newman-Keuls used as post hoc test). ***Significantly 750
different from WT (P < 0.0001). (B) Growth phenotypes of 5-week-old WT, miR858OX and 751
MIM858 transgenic plants. Rectangular boxes representing close-ups of stems of WT, 752
miR858OX and MIM858 transgenic stems (representative of 1 line of each transgenic). Bar = 753
1 cm. (C) Expression profiles of CESA4, SND1, MYB58, HCT, CCoAOMT1, CCR1 and 754
CAD6 in the stem of WT, miR858X and MIM858 lines. Transcript abundance of each gene 755
was normalized by the level of tubulin gene. Bars indicate ±SE (n=3). 756
757
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33
SUPPLEMENTARY INFORMATION 758
Supplementary Figure S1. Expression of the miR858a is regulated by light and requires 759
HY5. (A) GUS activity in ProMIR858:GUS transgenic driven by light. (a) GUS activity 760
driven by a 1.8 kbp upstream promoter sequence of pre-miR858a in the ProMIR858:GUS 761
expressing seedlings after 5 days of germination in light. Bar = 1 mm (b) GUS activity in 762
ProMIR858:GUS expressing seedlings grown in complete dark condition for 5 days. (c) GUS 763
activity in dark-grown seedlings exposed to light for 0.5 hr. (d) GUS activity in dark-grown 764
seedlings exposed to light for 2 hr. (e) GUS activity in dark-grown seedlings exposed to light 765
for 6 hr. For b to e, Bar = 0.5 mm. (B) Relative expression of GUS and Chlorophyll A/B 766
Binding (CAB) genes in ProMIR858:GUS expressing seedling grown under light and dark 767
conditions for 5 days. CAB is taken as positive control. Transcript abundance of each gene 768
was normalized by the level of tubulin gene. (C) Relative expression of GUS gene in 769
ProMIR858:GUS transgenic lines (Pro-1, Pro-2 and Pro-3) at different times of exposure to 770
light after 5 days of growth in dark. Error bars indicate ±SE (n=3) from three biological 771
replicates (each with three technical replicates). 772
773
Supplementary Figure S2. miR858a-mediated cleavage of the MYB genes. Cleavage 774
analysis was performed using RLM-RACE. Amplified products were sequenced for (A) 775
MYB6, (B) MYB13, (C) MYB20, (D) MYB42, (E) MYB48, (F) MYB83, (G) TT2 and (H) 776
MYBL2. miR858a mature sequence is written in 3’ to 5’ direction while target gene sequence 777
in 5’ to 3’. The identified cleavage sites are indicated by black arrows with the frequency of 778
cleavage showing on the bottom of the arrows and position of cleavage showing on the top of 779
the target sequence. 780
Supplementary Figure S3. Expression analysis of miR858a expression in Arabidopsis. 781
Expressions of mature miR858a gene in different tissues of WT and miR858OX transgenic 782
lines. Tubulin was taken as internal control for normalization. Error bars represents ±SE 783
(n=3). 784
Supplementary Figure S4. Relative expression of MYB transcription factors in miR858OX 785
transgenic lines. A to H, Expression of MYB6, MYB13, MYB20, MYB48, MYB63, MYB111, 786
TT2, MYBL2 in seedling, rosette, root, stem, flower and silique tissue of miR858OX lines. 787
For MYB111 (F) no expression was obtained in the root. Tubulin was taken as internal control 788
for normalization. Error bars represents ±SE (n=3). 789
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34
Supplementary Figure S5. Modulation of gene expression in transgenic line expressing 790
miR858a. (A) Relative expression of MYB genes identified through microarray analysis. (B) 791
Relative expression of structural genes of flavonoid pathway. Tubulin was taken as 792
endogenous control. Data given are fold expression log2 (transgenic/WT). Error bars 793
represents ±SE (n=3). 794
Supplementary Figure S6. Phenotypic variations in miR858a transgenic lines. (A) The 795
seedling phenotype of WT, miR858OX (L2 and L3) and MIM858 (MIM2 and MIM3) 796
transgenic lines. Phenotypic changes in WT, miR858OX and MIM858 transgenic lines grown 797
on ½ MS media for 10 days are shown. Bars = 1 cm. (B) Growth phenotypes of 5-week-old 798
WT, miR858OX and MIM858 transgenic plants. Bar = 1 cm. (C) Phenotype of transgenic 799
lines producing longer silique than WT. Photograph of 6 week grown plants was taken. 800
Supplementary Figure S7. Expression analysis of significantly up-regulated genes in 801
transgenic line expressing miR858a. Relative expression of significantly up-regulated genes 802
in miR858OX line. Tubulin was taken as endogenous control. Data given are fold expression 803
log2 (transgenic/WT). Error bars represents ±SE (n=3). 804
Supplementary Figure S8. miR858a affects lignin depositions in Arabidopsis stem. 805
Histochemical staining of stem sections of mature Arabidopsis stem sections with toluidine 806
blue O (TBO) for detection of lignin. i, interfascicular fiber; x, xylem. Bars = 500 µm. (A) 807
TBO staining (blue colour) of a WT stem section showing the normal lignin deposition in the 808
walls of interfascicular fibers and xylem cells. (B) TBO stained miR858OX stem section 809
showing ectopic lignin deposition in the walls of interfascicular fibers and xylem cells. (C) 810
TBO stained MIM858 stem section showing reduced lignin deposition in the walls of 811
interfascicular fibers and xylem cells. 812
813
Supplementary Table S1. Alignment of miR858a mature sequence among different plant 814
species 815
Supplementary Table S2. Putative cis-acting elements in miR858a promoter 816
Supplementary Table S3. Putative targets of Arabidopsis miR858a 817
Supplementary Table S4. Differentially expressed MYB transcription factors and flavonoid 818
structural genes 819
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35
Supplementary Table S5. Comparisons of seedling root length, fresh weight and rosette 820
diameter in WT and miR858a transgenic lines 821
Supplementary Table S6. Comparisons of plant height, rosette fresh weight and rosette 822
diameter in mature WT and miR858a transgenic lines 823
Supplementary Table S7. Comparisons silique, seed number and morphology in WT and 824
miR858a transgenic lines 825
Supplementary Table S8. Comparisons of flower size in WT and miR858a transgenic lines 826
Supplementary Table S9. Metabolite content in stem tissues of wild-type, miR858OX and 827
MIM858 transgenic lines 828
Supplementary Table S10. Information of primers used 829
Supplementary Data Set S1. List of differentially expressed genes in miR858OX identified 830
through microarray analysis. 831
832
833
834
835
836
Table 1. Metabolite content in rosette tissues of wild-type, miR858OX and MIM858 transgenic lines 837
MOLECULE WT miR858OX MIM858
Naringenin 108.6 ± 1.4 34.91 ± 0.4*** 276.89 ± 3.7***
Naringin 77.4 ± 1.2 19.5 ± 0.2** 104.73 ± 1.4**
Kaempferol 4861.65 ± 85.6 2331.8 ± 38.9** 10249.41 ± 171.2***
Quercetin 701.59 ± 13.6 285.84 ± 3.9*** 1995.2 ± 33.3***
Q-3-O-G 110.71 ± 2.7 92.8 ± 1.5* 430.84 ± 7.1***
Rutin hydrate 3898.99 ± 65.1 3540.58 ± 59.1* 6683.54 ± 111.6**
Gallocatechin 431.4 ± 7.2 306.93 ± 4.0** 763.9 ± 10.2***
p-Coumaric acid 14434.29 ± 194.1 7120.55 ± 118.9** 32506.44 ± 444.3***
Caffeic acid 1961 ± 34.2 1062.07 ± 17.7* 4516.59 ± 75.4***
Ferulic acid 3793.51 ± 66.8 679.3 ± 11.3*** 1333.91 ± 18.2**
Sinapic Acid 32940.7 ± 550.3 8701.96 ± 145.3** 66980.51 ± 915.5***
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36
Syringic acid 15325.29 ± 269.9 3901.08 ± 36.7** 19290.53 ± 252.7*
Rosette tissue samples from plants were taken and metabolite contents analyzed by LC/MS/MS. Values presented are means ±SE (n=6) (µg 100 g-1 dry weight) of measurements from at least five biological replicates per genotype. Statistically significant values from the WT is based on One-way ANOVA (*P < 0.01, **P < 0.001 and ***P < 0.0001). 838
839
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