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RESEARCH ARTICLE 1
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MRF Family Genes Are Involved in Translation Control, Especially Under 3
Energy-Deficient Conditions, and Their Expression and Functions Are 4
Modulated by the TOR Signaling Pathway 5
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Du-Hwa Lee, Seung Jun Park, Chang Sook Ahn, and Hyun-Sook Pai*7
Department of Systems Biology, Yonsei University, Seoul 120-749, Korea 8
*Corresponding author: E-mail: [email protected]; Fax: 82-2-312-56579
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Short title: MRF family genes in plant translation control 11
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One-sentence summary: MRF family genes encode translation regulatory factors, with functions that are 13
important under energy-deficient conditions, and the TOR signaling pathway modulates MRF expression 14
and functions. 15
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The author responsible for distribution of materials integral to the findings presented in this article and in 17
accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Hyun-Sook 18
Pai ([email protected]). 19
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ABSTRACT 21
Dynamic control of protein translation in response to the environment is essential for the survival of plant 22
cells. Target of rapamycin (TOR) coordinates protein synthesis with cellular energy/nutrient availability 23
through transcriptional modulation and phosphorylation of the translation machinery. However, 24
mechanisms of TOR-mediated translation control are poorly understood in plants. Here, we report that 25
Arabidopsis thaliana MRF (MA3 DOMAIN-CONTAINING TRANSLATION REGULATORY 26
FACTOR) family genes encode translation regulatory factors under TOR control, and their functions are 27
particularly important in energy-deficient conditions. Four MRF family genes (MRF1–MRF4) are 28
transcriptionally induced by dark and starvation (DS). Silencing of multiple MRFs increases susceptibility 29
to DS and treatment with a TOR inhibitor, while MRF1 overexpression decreases susceptibility. MRF 30
proteins interact with eIF4A and co-fractionate with ribosomes. MRF silencing decreases translation 31
activity, while MRF1 overexpression increases it, accompanied by altered ribosome patterns, particularly 32
in DS. Furthermore, MRF deficiency in DS causes altered distribution of mRNAs in sucrose gradient 33
fractions, and accelerates rRNA degradation. MRF1 is phosphorylated in vivo, and phosphorylated by S6 34
kinases in vitro. MRF expression, and MRF1 ribosome association and phosphorylation are modulated by 35
cellular energy status and TOR activity. We discuss possible mechanisms of the function of MRF family 36
proteins under normal and energy-deficient conditions and their functional link with the TOR pathway. 37
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INTRODUCTION 39
Translation, a fundamental cellular process that is highly conserved in eukaryotes, occurs in four 40
stages: initiation, elongation, termination, and ribosome recycling (Sonenberg and Hinnebusch, 41
Plant Cell Advance Publication. Published on October 30, 2017, doi:10.1105/tpc.17.00563
©2017 American Society of Plant Biologists. All Rights Reserved
2
2009). Initiation is the rate-limiting step, and is controlled by eukaryotic translation initiation 42
factors (eIFs) and many other accessory proteins (Holcik and Sonenberg, 2005). During the 43
initiation step, the eIF2-GTP-Met-tRNAiMet
ternary complex binds to the eukaryotic small 44
ribosomal subunit (40S) to form the 43S pre-initiation complex (PIC). The 43S PIC attaches to 45
the 5′-end of mRNA via the eIF4F complex composed of eIF4E (5’ cap-binding protein) and 46
eIF4G (scaffold). The 5’ cap-bound eIF4F complex recruits eIF4A (DEAD-box RNA helicase), 47
eIF4B (eIF4A enhancer) and PABPs [poly (A)-binding proteins] (Muench et al., 2012; Browning 48
and Bailey-Serres, 2015; Merchante et al., 2017). A second form of eIF4F, eIFiso4F, exists only 49
in plants and is composed of eIFiso4G and eIFiso4E; the eIFiso4F form shows differential 50
translation-promoting activities on mRNAs (Allen et al., 1992; Patrick and Browning, 2012; 51
Browning and Bailey-Serres, 2015). The 43S PIC including eIF4F or eIFiso4F scans along the 52
5′-untranslated region of the mRNA to select the AUG codon, at which point it is joined with the 53
60S subunit via eIF5B to form a functional 80S ribosome (Jackson et al., 2010; Browning and 54
Bailey-Serres, 2015). 55
Control of global translation activity is critical for cellular adaptation to fluctuating growth 56
conditions and environmental stimuli (Sonenberg and Hinnebusch, 2009; Sengupta et al., 2010). 57
Translation initiation that determines the overall rate of translation is the primary target for 58
regulation under stress conditions; two key points of the regulation are ternary complex 59
formation and 5’-cap recognition (Jackson et al., 2010). Many stress conditions trigger 60
phosphorylation of eIF2α by eIF2α kinases, inhibiting ternary complex formation in mammals; 61
phosphorylation of eIF2α inhibits the eIF2B-catalyzed exchange of GDP for GTP, required for 62
regeneration of active eIF2-GTP (Jackson et al., 2010; Silvera et al., 2010). Arabidopsis and rice 63
(Oryza sativa) have a single eIF2α kinase (GCN2) that phosphorylates eIF2α in response to 64
nutrient starvation, UV, cold shock, and wounding (Browning, 2004; Lageix et al., 2008). A 65
recent study revealed that Arabidopsis GCN2 requires an interaction partner GCN1 for eIF2α 66
phosphorylation (Wang et al., 2017). Since plant eIF2 has much less affinity to GDP than its 67
mammalian counterparts, the eIF2B activity as a guanine nucleotide exchange factor may not be 68
absolutely necessary in plants (Shaikhin et al., 1992; Browning and Bailey-Serres, 2015). Further 69
studies will be required to determine the roles of eIF2α phosphorylation and eIF2B in global 70
translation control in plants. The 5’-cap recognition step is the major regulation point of 71
3
translation initiation in mammals and yeast, but how the step is regulated according to cell 72
environments is largely unknown in plants. Arabidopsis eIF4A is phosphorylated by cyclin-73
dependent kinase A (CDKA) on Thr-164 residue, and a phosphomimetic mutant (T164E) of 74
eIF4A-1 lacks ATPase and helicase activities and cannot support protein translation, suggesting 75
that CDKA may repress translation during cell proliferation through eIF4A phosphorylation 76
(Hutchins et al., 2004). The eIF4E-binding protein (4E-BP) is the main regulator of the 5’-cap 77
recognition step in mammals. Stress signaling activates 4E-BP, which suppresses translation 78
initiation by interfering with the interaction between eIF4E and eIF4G (Sonenberg and 79
Hinnebusch, 2009; Jackson et al., 2010). Plants lack 4E-BP, and it remains to be discovered 80
whether plants have evolved an equivalent system for global regulation of translation (Lageix et 81
al., 2008; Xiong et al., 2013). 82
The target of rapamycin (TOR) signaling pathway plays a vital role in sensing and 83
responding to external signals to control cell growth and metabolism in all eukaryotes. Such 84
signals include nutrient availability, energy status, growth factors, and environmental conditions 85
(Dobrenel et al., 2016a). The TOR pathway regulates myriad biological processes, including 86
transcription, translation, ribosome biogenesis, protein trafficking, and autophagy (Wullschleger 87
et al., 2006). The mammalian TOR complex 1 (mTORC1) regulates the activity of the translation 88
initiation machinery, including eIF4G, eIF4B, and 4E-BP, through direct or indirect 89
phosphorylation (Ma and Blenis, 2009). In plants, inactivation of TOR causes a significant 90
decrease in polysome accumulation (Deprost et al., 2007; Ahn et al., 2011). Schepetilnikov et al. 91
(2013) suggested that TOR and S6K1 stimulate translation re-initiation of upstream open reading 92
frame (uORF)-containing mRNAs via phosphorylation of eIF3h in Arabidopsis. Considering that 93
uORFs are found in more than 30% of Arabidopsis full-length mRNAs, the aforementioned 94
mechanism of translation control may play an important role in plant development (Zhou et al., 95
2010). As with yeast and mammalian S6Ks, Arabidopsis S6Ks phosphorylate the 40S ribosomal 96
protein S6 (RPS6) (Turck et al., 2004; Mahfouz et al., 2006; Dobrenel et al., 2016b). Additional 97
substrates of mammalian S6Ks have been identified, including eIF4B, elongation factor 2 kinase, 98
and cap-binding protein 80 (Magnuson et al., 2012). However, downstream targets of plant S6Ks 99
related to plant mRNA translation are largely unknown, apart from RPS6 and eIF3h. 100
Throughout their lifespan, plants are exposed to diverse stresses, which disrupt cellular 101
4
energy homeostasis. Protein synthesis demands a large amount of energy, and is tightly regulated 102
according to the cellular energy status in yeast and mammals (Ma and Blenis, 2009). Similarly, a 103
tight correlation between translation activity and cellular sugar levels has been observed in plants 104
(Pal et al., 2013; Lastdrager et al., 2014). The key players of plant energy signaling are sucrose 105
non-fermenting 1-related protein kinase 1 (SnRK1) and TOR, which act in an antagonistic 106
crosstalk in the plant’s response to energy deprivation (Lastdrager et al., 2014; Mair et al., 2015; 107
Nukarinen et al., 2016). TOR silencing mimics energy starvation conditions, and activates 108
catabolic processes and autophagy while repressing global translation (Deprost et al., 2007; 109
Moreau et al., 2012; Ren et al., 2012; Caldana et al., 2013; Xiong et al., 2013). However, the 110
detailed mechanisms of TOR’s control of stress responses, particularly regarding global mRNA 111
translation, are largely unclear in plants. 112
Programmed cell death 4 (PDCD4) is a tumor suppressor that has been implicated in the 113
development of multiple cancers (Lankat-Buttgereit and Goke, 2009). Human PDCD4 (hPDCD4) 114
binds to eIF4A through its two MA3 domains, inhibiting the eIF4A helicase activity and the 115
eIF4A-eIF4G interaction, leading to a decrease in translation initiation rates (Loh et al., 2009). 116
Homologs of hPDCD4 are found in animals, plants, and lower eukaryotes, but not in yeast. Only 117
the homologs of higher plants contain four MA3 domains in tandem, instead of two in the other 118
systems (Cheng et al., 2013). The Arabidopsis thaliana genome contains four genes encoding 119
PDCD4 homologs, and one of them was recently reported to interact with the ethylene signaling 120
protein EIN2, hence it was designated ECIP1 (EIN2 C-TERMINUS-INTERACTING PROTEIN 121
1; AT4G24800). Loss-of-function mutations in ECIP1 have been shown to result in ethylene 122
hypersensitivity (Lei et al., 2011). Apart from these findings, we lack evidence of the cellular 123
functions of these homologs. Here, we investigated protein characteristics and in planta 124
functions of four PDCD4 homologs in Arabidopsis. Our results suggested that these proteins 125
positively regulate protein translation in plants, particularly under dark and starvation conditions; 126
we thus designated them MA3-containing translation regulatory factor (MRF) 1 to 4. We also 127
found that the transcription of the MRF genes, and ribosome association and phosphorylation of 128
MRF1 are modulated by TOR activity, suggesting a functional link with the TOR signaling 129
pathway. 130
131
5
RESULTS 132
MRF Family Proteins Have Four MA3 Domains 133
The Arabidopsis MRF gene family consists of four genes, MRF1, MRF2, MRF3 (ECIP1), and 134
MRF4; each encodes a protein with four tandem MA3 domains (Figure 1A; Supplemental Table 135
1; Cheng et al. (2013). The MA3 domain functions as a protein-protein interaction module 136
(Yang et al., 2003; Yang et al., 2004). Notably, the N-terminal region of MRF4 is smaller than 137
that of the other MRFs. Arabidopsis MRF proteins exhibit less than 30% protein sequence 138
identity to human PDCD4. Arabidopsis MRF gene family is divided into two clades: one 139
including MRF1, MRF3, and MRF4, and the other including MRF2 (Cheng et al., 2013). 140
141
MRF Family Genes Are Transcriptionally Induced by Dark and Starvation 142
We examined expression patterns of each MRF gene using reverse transcription quantitative PCR 143
(RT-qPCR) with gene-specific primers (Supplemental Table 2). All MRF genes were expressed in 144
all major plant organs, but exhibited different expression profiles. MRF1 and MRF3 transcripts 145
were more abundant in vegetative tissues, such as rosette leaves, cauline leaves, and stems, than 146
in reproductive tissues, such as flower buds and flowers (Figure 1B). MRF4 transcript levels 147
were highest in rosette leaves and flower buds, although the overall mRNA level was much 148
lower for MRF4 than for the other MRF genes. MRF2, which belongs to a different subgroup 149
within the MRF gene family, displayed the highest transcript levels in the reproductive organs, in 150
contrast to the other MRF genes (Figure 1B). According to the Genevestigator database 151
(https://genevestigator.com), MRF1 and MRF3 transcript levels increased 2-fold in the dark, and 152
decreased 4-fold upon glucose feeding after starvation. Since MRF4 was not included in the 153
Genechip Affymetrix ATH1 genome array, we examined expression patterns of all MRF genes in 154
response to darkness and starvation using Arabidopsis seedlings grown in liquid culture for 12 155
days (Figure 1C, D). RT-qPCR revealed that transcript levels of all MRF genes dramatically 156
increased after 30 min of darkness. During the next 24 hours, MRF1 transcripts stayed at the high 157
level and MRF3 transcript levels increased further, while MRF2 and MRF4 transcript levels 158
decreased after reaching a peak (Figure 1C). 159
We next examined MRF gene expression upon glucose feeding following a period of 160
starvation (Figure 1D). All MRF genes were highly induced after 24 h starvation (no glucose in 161
6
medium). Subsequent glucose feeding in the following 4 h did not affect MRF2 expression, but 162
significantly decreased the transcript levels of MRF1, MRF3, and MRF4, at all glucose 163
concentrations (10-180 mM). Mannitol feeding (10-180 mM) for 4 h after starvation had little 164
effect on the transcript levels, suggesting that osmotic changes were not responsible for the 165
observed transcriptional repression by glucose (Supplemental Figure 1). These results suggest 166
that MRF family genes are transcriptionally induced by darkness and starvation, i.e. low-energy 167
conditions. Finally, transcript levels were compared between the four MRF genes after 24 h 168
starvation and subsequent 4 h glucose feeding (10-180 mM; Supplemental Figure 2). Although 169
the primer efficiency may differ between the MRF genes, the results clearly suggest that MRF1 is 170
a dominantly expressed gene among MRF family members after 24-h starvation. 171
172
MRF Family Proteins Predominantly Localize in the Cytosol 173
To investigate the subcellular localization of MRF family proteins, we generated green 174
fluorescent protein (GFP) fusion constructs of MRF1 to MRF4 under the control of the CaMV 175
35S promoter. The constructs were transiently expressed in Nicotiana benthamiana leaves via 176
agroinfiltration. Confocal laser scanning microscopy using protoplasts isolated from the 177
infiltrated leaves showed that MRF1 to MRF4 are mainly localized in the cytosol, but also found 178
near the nucleus (Figure 2A). The green fluorescence signals partially overlapped with the red 179
fluorescence of the nucleus marker (histone H2B:mRFP), but did not overlap with the 180
chloroplast autofluorescence signal. To verify the localization of the MRFs, we prepared total 181
(T), nuclear (N), and cytosolic (C) protein fractions from the infiltrated N. benthamiana leaves, 182
and performed immunoblotting using anti-GFP antibody and anti-histone H3 antibody as nuclear 183
markers (Figure 2B). All of the MRF:GFP proteins were predominantly associated with the 184
cytosolic fraction. Finally, we generated transgenic Arabidopsis lines that expressed GFP-fusion 185
proteins of each MRF under the control of the CaMV35S promoter. Confocal microscopy of the 186
leaf epidermal cells confirmed the cytosolic localization of all MRFs (Figure 2C). GFP signals 187
were rarely observed near the nucleus. 188
189
Altered MRF Expression Affects Flowering Time under Long-Day Conditions 190
7
Three T-DNA insertion mutant lines were available for MRF3 (Supplemental Figure 3A); mrf3-3 191
and mrf3-2 alleles had been previously identified and designated ecip1-1 and ecip1-2, 192
respectively (Lei et al., 2011). RT-PCR analyses showed that mrf3-2 (ecip1-1) is a null allele, 193
while mrf3-3 (ecip1-2) produces a truncated MRF3 mRNA (Supplemental Figure 3D, E; Lei et 194
al., 2011). mrf3-1 has a T-DNA insertion in the promoter region, resulting in a slight decrease in 195
MRF3 mRNA. The single T-DNA insertion mutant of MRF4, mrf4-1, has no full-length 196
transcripts based on RT-PCR (Supplemental Figure 3A, F, G). There were no T-DNA insertion 197
mutants available for MRF1. Homozygote mrf2 seeds could not be obtained from two T-DNA 198
insertion alleles, both carrying the insertion in the 3rd
exon. Since no usable mutant lines were 199
available, we generated artificial microRNA (amiRNA) lines for MRF1 and MRF2 under the 200
control of the CaMV 35S promoter in Arabidopsis (Col-0 ecotype). Three unique target sites 201
were designed for both MRF1 and MRF2 using an online designer tool (Schwab et al., 2006), 202
and more than 10 independent amiRNA transgenic lines were generated per target site (Figure 203
3A). RT-qPCR showed that significant gene silencing occurred in target a and c lines of MRF1, 204
and target d and f lines of MRF2 amiRNA lines (Supplemental Figure 3B, C). However, MRF1 205
target b and MRF2 target e sites were not effective in inducing target gene silencing. 206
To circumvent possible redundancy of MRF functions, we also generated multiple-target 207
amiRNA (Ami-m) lines against MRF1, MRF3, and MRF4, which belong to the same subgroup 208
and show similar expression patterns (Figure 3A; Cheng et al., 2013). The Ami-m target 209
sequence was designed to silence all three genes. In addition, we generated constitutive 210
overexpression (OE) lines of Flag tag-fused MRF1 (Flag:MRF1) under the control of a 211
CaMV35S promoter (Figure 3A), since MRF1 is most highly expressed among the MRF family 212
genes, particularly under starvation conditions (Supplemental Figure 2). Based on RT-qPCR, 213
MRF1 and MRF3 transcript levels in all three independent Ami-m lines (#3, #10, and #14) 214
decreased to 17-34% of the wild-type (WT) levels (Figure 3B). MRF4 transcript level was 215
reduced to at most ~60% of the WT levels under dark and starvation conditions, possibly due to 216
low target efficiency (Supplemental Figure 4A). Selected MRF1 OE lines, #1 and #2, produced 217
~34- and ~15.5-fold higher levels of MRF1 transcripts than the WT, respectively (Figure 3C). 218
We observed overall growth and development of amiRNA lines of MRF1 and MRF2, and 219
T-DNA insertion mutants of MRF3 and MRF4 (Supplemental Figure 4B, C). Under normal long-220
8
day conditions, these plants did not show any visible developmental abnormalities, except for 221
slightly early flowering in mrf3-2 and mrf3-3 T-DNA mutants, and MRF1 amiRNA lines, which 222
was measured by counting numbers of rosette and cauline leaves, consistent with previous 223
reports on ecip1 (mrf3) (Lei et al., 2011). MRF2 amiRNA lines and mrf4 T-DNA mutant did not 224
show early flowering phenotype (Supplemental Figure 4B, C). Both the Ami-m and MRF1 OE 225
lines exhibited no gross abnormalities during vegetative growth. However, the Ami-m lines 226
clearly showed early flowering phenotype under long-day conditions, while in MRF1 OE #1 and 227
#2 lines flowering was significantly delayed (Figure 3D, E). Thus, altered MRF gene expression 228
affects the transition from vegetative to reproductive growth. 229
230
MRF Modulates Plant Resistance to Darkness and Starvation Stress 231
Since expression of all MRF genes was induced by darkness and starvation, we investigated 232
plant phenotypes under these conditions and after subsequent re-illumination and glucose 233
feeding, using a liquid culture system (Figure 4). WT, MRF Ami-m (#3, #10, and #14), and 234
MRF1 OE (#1 and #2) seedlings at 12 days after germination (DAG), which were grown under 235
light and glucose (LG) conditions (control), were exposed to darkness and starvation (DS; no 236
sugar in the medium) for 5 days, and then transferred to LG conditions (ReLG) for 5 days to 237
observe re-greening. The seedlings had shown no differences in growth or total chlorophyll 238
content before treatment (Figure 4A, top; Supplemental Figure 5A). All seedlings started to lose 239
chlorophyll after 2 days of DS, progressing to complete chlorosis within 5 days (Figure 4A). 240
ReLG treatment after DS induced regeneration of green leaves at the shoot apex. MRF1 OE 241
seedlings re-greened somewhat earlier than WT, while Ami-m seedlings showed markedly 242
delayed re-greening after 5 days of ReLG treatment (Figure 4A). Correspondingly, total 243
chlorophyll contents in Ami-m and OE lines were lower and higher, respectively, than those in 244
the WT after 3 days of ReLG treatment (Supplemental Figure 5A, B). MRF1 amiRNA line (ami 245
c-#12) exhibited slightly delayed re-greening, but no visible DS-induced senescence and re-246
greening phenotypes were found in MRF2 amiRNA lines and T-DNA insertion mutants mrf3 and 247
mrf4 (Supplemental Figure 6). 248
Using RT-qPCR, we determined time-course expression profiles of CHLOROPHYLL A/B-249
BINDING PROTEIN 2 (CAB2), SENESCENCE 4 (SEN4), DARK-INDUCED 6 (DIN6), DIN10, 250
9
and PROLINE DEHYDROGENASE (PRODH) mRNAs during the DS and ReLG treatments 251
(Figure 4B, D; Supplemental Figure 7A, C, E). In addition, we plotted relative transcript levels 252
of the genes at specific time points when the samples showed the biggest differences (Figure 4C, 253
E; Supplemental Figure 7B, D, F). CAB2 is a representative marker for cellular photosynthetic 254
activity and SEN4 (encoding xyloglucan endotransglucosylase/hydrolase) is a marker for natural 255
and dark-induced senescence. Expression of DIN6 (encoding asparagine synthethase), DIN10 256
(encoding raffinose synthase), and ProDH are up-regulated in darkness. The MRF1 OE and Ami-257
m lines maintained higher and lower CAB2 mRNA levels than WT, respectively, after 3 days of 258
ReLG treatment (Figure 4B, C). In contrast, mRNA levels of SEN4, DIN6, DIN10, and ProDH 259
were higher than WT in the Ami-m lines and lower in the OE lines after 5 days of DS (Figure 4D, 260
E; Supplemental Figure 7). Detached leaf senescence assays using the 6-7th
leaf from soil-grown 261
plants resulted in similar findings: measured after 4 days in the dark, Ami-m leaves were more 262
susceptible to dark-induced senescence than WT with lower total chlorophyll levels, while MRF1 263
OE leaves exhibited delayed senescence with higher chlorophyll contents (Supplemental Figure 264
5C, D). Collectively, these results suggest that MRF deficiency and MRF1 overexpression lead 265
to decreased and increased resistance to DS-induced senescence, respectively. 266
267
MRF Proteins Interact with eIF4A in Vivo 268
MRF proteins have four tandem MA3 domains (Figure 1A). eIF4G and PDCD4 have one and 269
two MA3 domains, respectively, and these domains function in eIF4A binding (Yang et al., 2003; 270
Yang et al., 2004; Loh et al., 2009). There is moderate sequence similarity between the MA3 271
domains of human eIF4G and PDCD4, and between the MA3 domains of Arabidopsis eIF4G 272
and MRFs (Supplemental Figure 8). To determine whether MRF proteins interact with eIF4A in 273
planta, we performed bimolecular fluorescence complementation (BiFC). MRFs and eIF4A-1 of 274
Arabidopsis were expressed in combination as yellow fluorescent protein (YFP)N- and YFP
C-275
fusion proteins in N. benthamiana leaves by agroinfiltration. Confocal microscopy revealed 276
yellow fluorescence in the cytosol of the leaf cells in every combination, suggesting interaction 277
between all members of the MRF family and eIF4A-1, occurring mostly in the cytosol (Figure 278
5A; Supplemental Figure 9). However, BiFC of MRFs with eIF4E-1, the 5′ cap-binding protein, 279
10
did not result in yellow fluorescence in any combination, suggesting a lack of interaction, despite 280
the protein expression in leaf cells shown by immunoblotting with polyclonal anti-GFP antibody 281
(Figure 5A; Supplemental Figures 9, 10). As the control, no interaction was observed between 282
eIF4A-1 and Gle1 (mRNA export factor localized in both nuclear envelope and cytosol; 283
Supplemental Table 1; Lee et al. (2015)), or between the YFPN and YFP
C vector control. 284
Next, we performed co-immunoprecipitation assays (Figure 5B). Flag-tagged MRFs and 285
Myc-tagged eIF4A-1 were expressed in N. benthamiana leaves using agroinfiltration. eIF4A-286
1:Myc was immunoprecipitated from leaf extracts using anti-Myc antibody-conjugated resin, 287
followed by immunoblotting with anti-Flag antibody to detect co-immunoprecipitated Flag:MRF 288
proteins. All MRF proteins were detected in the immunoprecipitates, supporting the interactions 289
between MRF family proteins and eIF4A-1 in vivo (Figure 5B). Furthermore, after extensive 290
washing, Flag:MRF1 co-immunoprecipitated with eIF4A-1, but not with eIF4A-2 or eIF4A-3, 291
suggesting that MRF1 interacts most strongly with eIF4A-1 among eIF4A family proteins 292
(Supplemental Figure 11). 293
Finally, we performed yeast two-hybrid assays to measure the relative binding affinity of 294
each MRF for eIF4A (Figure 5C). GAL4 activating domain (AD)-fused eIF4A-1 and GAL4 295
promoter binding domain (BD)-fused MRFs were expressed together in yeast, and α-296
galactosidase assays were performed and the activity was statistically analyzed with biological 297
replications. Immunoblotting with anti-Myc and anti-HA antibodies showed that the expression 298
levels of BD- and AD-fusion proteins were similar in these yeast cells (Supplemental Figure 12). 299
The α-galactosidase activity of MRF1 and MRF3 appeared to be somewhat higher than that of 300
MRF2 and MRF4, suggesting higher binding affinity for eIF4A-1. 301
302
MRF1 Co-Sediments with Monosomes 303
Since MRF proteins interact with eIF4A, it is possible that they are involved in protein 304
translation control in plants. We first investigated the co-fractionation of MRFs with ribosomes 305
(Figure 6A). GFP-fused MRFs, and Myc-tagged eIF4A-1 and eIF4E-1 were expressed in leaves 306
of soil-grown N. benthamiana plants via agroinfiltration. The leaf extracts were then fractionated 307
on a 15-50% sucrose density gradient. After ultracentrifugation, fractions were collected, and 308
11
subjected to immunoblot analyses with anti-GFP and anti-Myc antibodies. As a control for 309
fractionation, immunoblotting was performed with an antibody against the 60S ribosomal protein 310
L10a (RPL10a), which is associated with 60S large subunits, 80S monosomes, and polysomes. 311
All MRF proteins were distributed up to the fractions in which 60S/monosomes occurred, similar 312
to the pattern of eIF4E-1, suggesting a possibility of ribosomal association of MRFs (Figure 6A). 313
eIF4A-1 was broadly distributed in the fractions including the polysomal fractions. 314
Next, we tested whether RNA is involved in MRF co-sedimentation with ribosomes 315
(Figure 6B). Cell extracts were prepared from Arabidopsis MRF1 OE seedlings grown in liquid 316
culture under LG conditions, and briefly treated with RNase A or RNase-free water (control) 317
before sucrose gradient sedimentation (15-50%). After ultracentrifugation, fractions were 318
collected for immunoblotting with anti-Flag and anti-L10a antibodies while performing 319
polysome analyses. Without RNase treatment, Flag:MRF1 was co-fractionated with ribosome 320
subunits and light polysomes (Figure 6B, left). The observed differences in MRF distribution 321
patterns between N. benthamiana and Arabidopsis samples may be caused by their differences in 322
plant developmental stages and cellular energy/nutrient status. A brief RNase treatment resulted 323
in a large increase in 60S/80S amounts and disruption of polysomes by digesting ribosome-324
associated mRNAs (Figure 6B, right). Accordingly, RPL10a mostly accumulated in the 60S/80S 325
region based on immunoblotting. Interestingly, the RNase treatment shifted MRF1 to less dense 326
fractions, demonstrating that MRF1 co-sedimentation with ribosomes partially depends on RNA. 327
To explore further the relationship between MRF function and RNA, we performed 328
complementation assays using an E. coli mutant lacking cold-shock proteins (Figure 6C). RNA 329
chaperones, such as bacterial cold-shock proteins (CSPs), are required for adaptation to low 330
temperatures, because cellular RNAs tend to form stable nonfunctional secondary structures 331
under low temperature conditions (Kang et al., 2013). The E. coli BX04 strain is a quadruple 332
mutant of cold-shock proteins (ΔcspA, ΔcspB, ΔcspE, and ΔcspG), and cannot grow at low 333
temperatures (Xia et al., 2001). Plasmids containing MRFs, E. coli CspA encoding a cold-shock 334
protein, and Arabidopsis LOW EXPRESSION OF OSMOTICALLY RESPONSIVE GENES 4 335
(LOS4) (Gong et al., 2005; Lee et al., 2015), which encodes an RNA helicase/mRNA export 336
factor, were introduced into the BX04 strain, followed by IPTG (isopropyl β-D-1-337
12
thiogalactopyranoside) treatment to induce protein expression. All of the transformants in the 338
BX04 background, including the vector control, grew well at 37 °C. However, at 18 °C, growth 339
of the vector control was defective, while CspA strongly complemented the cold-sensitive 340
growth defect of the BX04 strain. All MRF proteins partially rescued the cold-sensitive 341
phenotype of the mutant strain, similar to the activity of the RNA helicase LOS4 (Figure 6C). 342
Collectively, these results suggest that RNA interaction may play a role in MRF functions in 343
plants. 344
345
MRF Deficiency Causes Reduced Cellular Translation Activity under DS Conditions 346
To investigate the role of MRFs in protein translation, we first determined cellular translation 347
activity in WT, MRF Ami-m (#3, #10, and #14), and MRF1 OE (#1 and #2) seedlings using 35
S-348
methionine labeling (Figure 7A, B; Supplemental Figure 13). Seedlings were grown for 12 days 349
under long-day conditions in liquid culture. For pre-treatment, they were then transferred to fresh 350
1/2 MS medium with 30 mM glucose for 30-minute incubation in the light (LG), or to 1/2 MS 351
medium without glucose for 30-minute incubation in the dark (DS). After 30 min, 35
S-352
methionine was added to the medium, and the seedlings were incubated further for 2.5 h under 353
the same conditions. After labeling, protein extracts from the seedlings were subjected to SDS-354
PAGE and analyzed by a phosphorimager. Radioactive bands indicated newly synthesized 355
proteins after incorporation of 35
S-methionine. Based on the intensity of radioactivity, nascent 356
protein synthesis was lower in all of the lines under DS conditions than under LG, as previously 357
reported (Juntawong and Bailey-Serres, 2012). Comparing radioactive band intensity between 358
the lines under LG conditions, we found a slightly decreased signal from Ami-m (#3) lines, but 359
no other meaningful differences (Supplemental Figure 13B). However, under DS conditions, 360
nascent protein synthesis in all of the Ami-m lines decreased to 60~75% of the WT levels, 361
suggesting a significant reduction in global translation (Figure 7B). MRF1 OE lines showed 362
slightly higher cellular translation activity than that in the WT. These results suggest that MRFs 363
are required for efficient protein translation under low-energy conditions. 364
365
Polyribosome Profiles in MRF-Deficient and MRF1-OE Plants under Normal and Energy-366
13
Deficient Conditions 367
Next, we performed polysome analyses in WT, MRF Ami-m (#3), and MRF1 OE (#1) lines using 368
sucrose density gradient sedimentation (Figure 7C). The seedlings were grown for 12 days under 369
long-day conditions in liquid culture, and then transferred either to fresh 1/2 MS medium with 30 370
mM glucose, followed by a 2-hour incubation in the light (LG); or to 1/2 MS medium without 371
glucose, followed by a 1-day incubation in the dark (DS). The seedlings were treated with 372
cycloheximide (50 μg/ml) for 5 min before harvest to preserve the polyribosome profile. 373
Ribosome profiles at LG (blue line) and DS (red line) were superimposed, and the average ratio 374
of polysomes to nonpolysomes (P/NP) was calculated based on four independent sedimentation 375
experiments. Under LG conditions, the Ami-m line exhibited consistently lower 60S and 80S 376
peaks than the WT without significant difference in polysome profiles, while the overall profiles 377
of the MRF1 OE line were similar to those of WT (Figure 7C; blue line). The P/NP ratio in the 378
Ami-m lines (4.1 ± 0.3) appeared to be slightly higher than that of the WT (4.0 ± 0.1) and MRF1 379
OE lines (3.9 ± 0.3) in LG (2 h). 1-day DS treatment reduced the amount of polysomes and 380
increased the amount of 60S/80S in WT seedlings, implying repression of global translation 381
(Figure 7C; red line). Polysome peaks of the Ami-m and MRF1 OE lines were almost 382
indistinguishable from those of the WT. However, the 60S/80S peak was significantly lower in 383
the Ami-m line and slightly higher in the MRF1 OE line, respectively, than in the WT. Under DS 384
conditions, the P/NP ratio in the Ami-m (1.6 ± 0.1) and MRF1 OE lines (1.1 ± 0.1) was higher 385
and lower than that of the WT (1.3 ± 0.1), respectively. Thus, the P/NP ratio tended to differ 386
more significantly between the lines under DS than under LG, albeit not statistically significant. 387
These results suggest that MRF deficiency may cause decreased 60S/80S amounts under LG (2 h) 388
and DS (1 d) conditions, while MRF1 overexpression may cause increased 60S/80S amounts 389
only under DS (1 d) conditions. 390
We also performed polysome analyses without cycloheximide pretreatment after LG (2 h), 391
DS (2 h), and DS (1 d) treatments (Supplemental Figure 14). Removal of the pretreatment step 392
changed the shape of the ribosome profiles particularly in LG; it increased the 60/80S peaks and 393
decreased polysomes. Nevertheless, these experiments led to similar conclusions to those of 394
Figure 6C. The Ami-m lines showed decreased 60S/80S peaks under LG (2 h), DS (2 h), and DS 395
(1 d) conditions, while the MRF1 OE lines showed increased 60S/80S peaks only under DS (1 d) 396
14
conditions. 397
398
MRF Deficiency Decreases Cellular Ribosomal RNA Contents under DS Conditions 399
To determine whether MRF deficiency or MRF1 OE affects cellular rRNA levels, we examined 400
total RNA contents in the WT, Ami-m (#3, #10, and #14), and MRF1 OE (#1 and #2) lines after 401
prolonged DS and ReLG treatments, because total RNA is a useful proxy for rRNA (Figure 7D). 402
Seedlings grown under LG conditions (control) were subjected to 2 and 5 days of DS treatment 403
(DS+2 and DS+5), followed by 3 and 5 days of LG treatment (ReLG+3 and ReLG+5) as 404
described in Figure 4A. Total RNA contents in the seedlings of equal weight were measured at 405
each stage. There were no significant differences in the contents among these seedlings before 406
treatment (control). In all the lines, however, the RNA contents progressively decreased during 407
prolonged DS, and then increased upon subsequent supply of light and glucose (ReLG). Total 408
RNA contents were consistently lower in the Ami-m lines throughout DS and ReLG treatments, 409
suggesting that MRF deficiency accelerates rRNA degradation upon prolonged DS, considering 410
the stability of rRNAs, and delays rRNA recovery during ReLG. Notably, the Ami-m (#3) line 411
that showed the most severe phenotype during prolonged DS and ReLG treatments (Figure 4A) 412
also exhibited the most significant decrease in total rRNA contents during the treatments (Figure 413
7D). In contrast, the MRF1 OE lines contained higher RNA contents, particularly during ReLG 414
treatment, suggesting faster rRNA recovery. 415
416
MRF Deficiency Causes Altered Distribution of mRNAs in Sucrose Gradient Fractions 417
To analyze the effects of MRF deficiency on protein translation in detail, we examined the 418
distribution patterns of selected mRNAs in sucrose gradient fractions of WT and Ami-m (#3) 419
seedlings after LG (2 h) and DS (1 d) treatment (Figure 8A; Supplemental Figure 15A). Then we 420
quantified the abundance of the mRNAs in polysomal (P; fractions 9-15) and nonpolysomal 421
fractions (NP; fractions 1-8), as a percentage of their total amount in all fractions (Figure 8B; 422
Supplemental Figure 15B). The selected genes were previously used in translation studies 423
(Nicolai et al., 2006; Juntawong and Bailey-Serres, 2012; Perrella et al., 2013; Schepetilnikov et 424
al., 2013; Juntawong et al., 2014). After LG (2 h) or DS (1 d) treatment, cell extracts from the 425
seedlings were subjected to sucrose density gradient sedimentation using the same protocol as in 426
15
Figure 7C, and 15 fractions were collected from each tube. Total RNA was precipitated from 427
each fraction, followed by cDNA synthesis and RT-qPCR for calculation of the average mRNA 428
content in each fraction. 429
Distribution patterns of PP2AA3 (PP2A regulatory subunit A3), GAPC (cytosolic 430
glyceraldehyde-3-phosphate dehydrogenase), HDA19 (histone deacetylase), and HDC1 (histone 431
deacetylation complex1) mRNAs in sucrose gradients were very different between LG and DS 432
conditions in both WT and Ami-m lines (Figure 8A). While these mRNAs accumulated mostly in 433
heavy polysomes in LG, their accumulation shifted to less dense fractions including monosomes 434
in DS, suggesting a decrease in global translation under DS conditions. This result is consistent 435
with the report that HDA19 mRNA translation is repressed by sucrose starvation (Nicolai et al., 436
2006). For all four mRNAs, there was no significant difference between the WT and Ami-m lines 437
regarding the mRNA percentage in P and NP fractions under LG (Figure 8B). However, under 438
DS, the percentage of polysome-associated mRNAs were higher in the Ami-m lines than in the 439
WT samples for a significant portion of tested mRNAs, based on statistical analyses of three 440
biological replicates (Figure 8B; Supplemental Figure 15B). Those mRNAs include PP2AA3, 441
HDA19, HDC1, MCCA (3-methylcrotonyl-CoA carboxylase), CAB2, and RHIP1 (RGS1 and 442
HXK1 interacting protein 1), with particularly significant effects on HDA19 and HDC1. 443
However, there was no statistically significant difference between two samples for GAPC, bZIP1 444
(basic-leucine zipper transcription factor 1), and bZIP11 mRNAs. There is a possibility that the 445
higher polysomal loading of these particular mRNAs indicates more robust translation of the 446
mRNAs under DS. However, considering that Ami-m lines exhibited a decrease in 60S/80S 447
amounts, global translation activity, and cellular rRNA contents under DS conditions, it may 448
suggest that MRF deficiency delays/impairs normal progression of translation for the mRNAs. 449
Translation of some mRNAs such as HDA19 and HDC1 may be more sensitive to MRF 450
deficiency. Future studies including genome-wide translatome analyses would provide a clear 451
picture. 452
453
Phosphorylation and Ribosome Association of MRF1 Are Regulated by Cellular Energy 454
Availability 455
In animals and yeast, the rapid response to cellular nutrient availability is generally controlled by 456
16
phosphorylation of several regulatory proteins (Sonenberg and Hinnebusch, 2009; Jackson et al., 457
2010). Therefore, we examined whether MRF proteins are phosphorylated in vivo. Each MRF 458
protein was expressed as a Myc fusion in N. benthamiana leaves. Then the leaf extracts were 459
subjected to Zn2+
-Phos-tag SDS-PAGE, and also to normal SDS-PAGE, with or without 460
treatment with recombinant lambda phosphatase (λPP), a non-specific protein phosphatase. The 461
more common Mn2+
-Phos-tag SDS-PAGE system did not clearly separate phosphorylated MRF1 462
from its unphosphorylated form. Immunoblotting with anti-Myc antibody showed most visible 463
mobility shift in MRF1 after λPP treatment in Phos-tag SDS-PAGE; normal SDS-PAGE failed to 464
detect it (Figure 9A). Thus, Zn2+
-Phos-tag gels detected phosphorylation of MRF1 in vivo. 465
To test the possibility that MRF1 phosphorylation depends on cellular energy conditions, 466
we performed Phos-tag and normal SDS-PAGE using seedling extracts from MRF1 OE (#1) 467
lines, in which MRF1 is fused with the Flag-tag sequence (Figure 9B). We failed to make 468
specific antibodies against the endogenous MRF1. Twelve-day-old seedlings grown in liquid 469
culture were kept in DS conditions for 1-48 h, and then re-illuminated and fed with 30 mM 470
glucose (LG) for 0.5-2 h. Immunoblotting with anti-Flag antibody showed that the upper band, 471
representing phosphorylated MRF1, started to decrease after 2 h of DS treatment, almost 472
disappeared after 48 h, and reappeared after 30 min of LG treatment (Figure 9B). These results 473
suggest that MRF1 is de-phosphorylated under energy deprivation conditions, but becomes 474
rapidly phosphorylated in vivo when energy is reintroduced. 475
We examined MRF1 co-sedimentation with ribosomes under different energy conditions 476
(Figure 9C). Twelve-day-old MRF1 OE (#1) seedlings grown in liquid culture were harvested 477
without treatment (control), after 24-h DS treatment, or after 1-h LG treatment following DS, 478
and the extracts were loaded on a 10-55% sucrose gradient for ultracentrifugation. Inactive 479
monosomes were expected to accumulate in DS conditions, which decrease protein synthesis. As 480
expected, 24-h DS treatment led to a higher monosome peak and lower polysomal peaks than 481
observed for the control (Figure 9C, upper). After 1-h LG treatment following DS, the 482
monosome amount decreased and polysome amounts were partially restored. The ribosomal 483
fractions were then subjected to immunoblotting with anti-Flag antibody to detect Flag:MRF1, 484
and with anti-RPL10a antibody as control (Figure 9C, lower). In normal conditions, MRF1 was 485
broadly detected up to the 13th
fraction (light polysomes). After DS treatment, MRF1 was 486
17
distributed only up to the 9th
fraction (monosomes). Interestingly, despite the large increase in the 487
monosome peak upon DS, the amount of MRF1 found in the monosome fractions (fractions 6 488
and 7) did not increase proportionally (Figure 9C, center). This result suggests that MRF1 has 489
low affinity towards the inactive 80S ribosomes. 1-h LG treatment following the DS expanded 490
MRF1 distribution up to the 10th
fraction (light polysomes). These results suggest that MRF1-491
ribosome association is correlated with cellular translation activity, which depends on the energy 492
status of the cell. 493
To explore the mechanism of the condition-dependent MRF1-ribosome association, we 494
examined MRF1-eIF4A interactions under LG and DS conditions using co-immunoprecipitation 495
(Figure 9D). eIF4A-1 was broadly distributed in the ribosome fractions up to the polysome 496
fraction (Figure 6A). Leaf disks were prepared from N. benthamiana plants expressing 497
Flag:MRF1 and eIF4A-1:Myc, and then were floated for 3 h either in the light on MS medium 498
with 30 mM glucose (LG), or in the dark on MS medium without glucose (DS). Interestingly, 499
MRF1 was co-immunoprecipitated with eIF4A-1 more efficiently under LG, rather than DS, 500
conditions (Figure 9D). A stronger MRF1-eIF4A-1 interaction might have contributed to the 501
more robust MRF1-ribosome association under LG conditions. Collectively, these results suggest 502
that MRF1 phosphorylation and ribosome association are modulated by cellular energy 503
availability. 504
505
TOR Regulates MRF Gene Expression in Response to Starvation and Sugar Feeding 506
Our results suggest that MRF transcription is modulated by cellular energy status (Figure 1), and 507
that MRFs are involved in protein translation control (Figures 7, 8). TOR kinase is an important 508
regulator of protein translation and plays a role in linking nutrient signaling to cellular 509
adaptations (Dobrenel et al., 2016a). To explore a possible relationship between MRFs and the 510
TOR signaling pathway, we first tested whether the abundance of MRF transcripts is changed by 511
TOR activity, using estradiol-inducible TOR RNAi (es-tor1) lines (Figure 10A). Estradiol 512
treatment reduced the TOR mRNA levels (Supplemental Figure 16A). Nine-day-old seedlings 513
grown in liquid culture were treated for 24 h with either ethanol (-ES) or estradiol (10 µM; +ES). 514
Then they were transferred to fresh medium containing ethanol or estradiol, but lacking sugar, 515
and incubated for 24 h for starvation treatment. After 24 h of starvation, glucose, mannitol, or 516
18
sucrose was added to the medium (final concentration 30 mM) for further incubation for 4 h. RT-517
qPCR of (-)ES samples revealed that feeding with glucose or sucrose, but not with mannitol, led 518
to significantly lower transcript levels of MRF1, MRF3, and MRF4 than in starvation conditions 519
(Figure 10A), consistent with the previous results (Figure 1D, Supplemental Figure 1). However, 520
TOR silencing by estradiol treatment disrupted these transcript level changes of the three MRF 521
genes, with a particularly strong effect on MRF1 and MRF4. MRF2 gene expression remained 522
unchanged upon estradiol treatment. These results suggest that TOR regulates MRF gene 523
expression in response to the cellular energy status, except for MRF2. 524
525
Phosphorylation and Ribosome Association of MRF1 Are Regulated by TOR Activity 526
TOR inhibition reduces translation in eukaryotes, mainly through repression of both translation 527
initiation and new ribosome synthesis (Powers and Walter, 1999; Cherkasova and Hinnebusch, 528
2003). Recently, it was reported that TOR inhibition by rapamycin triggers a rapid decrease (40-529
60%) in ribosome content in yeast, through rapid cytoplasmic turnover of the existing ribosomes 530
(Pestov and Shcherbik, 2012). We peformed virus-induced gene silencing (VIGS) using the 531
tobacco rattle virus (TRV) system against TOR in MRF1 OE (#1) Arabidopsis plants, to analyze 532
phosphorylation status and ribosome association of Flag:MRF1 in TOR-silenced leaf cells 533
(Figure 10B, C). The 6-8th
leaves were collected from the TRV control and from the TOR VIGS 534
plants grown in soil at 10 days after infiltration (DAI), when morphological phenotypes were not 535
yet fully visible in the TOR VIGS plants. TOR VIGS caused defective plant growth and 536
excessive starch accumulation at 14 DAI, associated with reduced TOR mRNA levels 537
(Supplemental Figure 16B-D). At ~21 DAI, TOR VIGS plants exhibited growth arrest and severe 538
chlorosis. Immunoblotting of the Phos-tag gel with anti-Flag antibody revealed that the upper 539
phosphorylated form of Flag:MRF1 was significantly reduced in TOR-silenced cells, while the 540
lower unphosphorylated form was increased, suggesting that reduced TOR activity leads to 541
MRF1 dephosphorylation (Figure 10B). Ribosome fractionation was performed using the same 542
gram-fresh-weight of leaves from TRV control and TOR VIGS plants in a 10-55% sucrose 543
gradient, followed by immunoblotting with anti-Flag and anti-RPL10a antibodies (Figure 10C). 544
RPL10a distribution patterns in the ribosomal fractions suggested reduced translation activity in 545
TOR VIGS plants. Furthermore, MRF1 cofractionation with ribosomes was diminished in TOR-546
19
silenced cells, suggesting that MRF1 distribution is modulated by TOR activity either directly or 547
indirectly (Figure 10C). 548
Since TOR activity regulated MRF gene expression, MRF1 phosphorylation, and MRF1-549
ribosome association, we next examined the sensitivity of MRF Ami-m (#3, #10, and #14) and 550
MRF1 OE (#1 and #2) seedlings to Torin-1 (TOR inhibitor) compared with WT seedlings (Figure 551
10D, E). Since root growth is sensitive to TOR inhibition, we measured root length of the 552
seedlings after 3 and 5 days of Torin-1 (2 μM) or control DMSO treatment. Torin-1 caused 553
reduced root growth and premature senescence in all seedlings with respect to the DMSO 554
controls; the Ami-m and MRF1 OE seedlings appeared to be more susceptible and more resistant 555
to Torin-1 than WT seedlings, respectively (Figure 10D, E). Consistent with their senescence 556
symptoms, the Ami-m and OE seedlings showed the respective lower and higher CAB2 mRNA 557
levels than that of the WT at 3 days after Torin-1 treatment (Figure 10D; Supplemental Figure 558
17). Collectively, these results suggest that active TOR promotes MRF1 phosphorylation and 559
ribosome association, and MRF mutants show altered Torin-1 sensitivity. 560
561
MRF1 Is Phosphorylated by S6K1 and S6K2 in Vitro 562
Since MRF1 phosphorylation is regulated by the TOR signaling pathway, and S6 kinase (S6K) is 563
a major effector of TOR (Mahfouz et al., 2006; Schepetilnikov et al., 2013), we examined 564
whether MRF proteins are phosphorylated by S6K in vitro. MRF proteins were purified from E. 565
coli as MBP fusion proteins. S6K1:Myc and S6K2:Myc were expressed in N. benthamiana 566
leaves and immunoprecipitated using anti-Myc antibody-conjugated resin. In vitro protein kinase 567
assays showed that only MRF1 was phosphorylated by the immunoprecipitated S6K1 and S6K2 568
(Figure 11A, B), which is consistent with MRF1 being mainly phosphorylated in vivo (Figure 569
9A). MRF1 phosphorylation did not occur without the addition of S6K1 (Supplemental Figure 570
18A). Arabidopsis S6K1 has a conserved site, T449, which is phosphorylated by the TOR kinase 571
(Xiong and Sheen, 2012; Ahn et al., 2015). We determined MRF1 phosphorylation activities of 572
two mutant forms of S6K1, a phospho-null mutant (T449A) and a phospho-mimetic mutant 573
(T449D), and compared them to normal S6K1 (Figure 11C). The T449A mutation reduced 574
MRF1 phosphorylation activity of S6K1, while T449D had no effect. These results suggest that 575
phosphorylation at the T449 site in S6K1 is important for MRF1 phosphorylation in vitro (Figure 576
20
11C). The MBP control was not phosphorylated by either mutant form (Supplemental Figure 577
18B). 578
BiFC demonstrated that MRF1 interacts with both S6K1 and S6K2; co-expression of 579
YFPN-fused MRF1 and YFP
C-fused S6K1 or S6K2 in N. benthamiana leaves resulted in yellow 580
fluorescence in epidermal cells, observed by confocal microscopy (Figure 11D). MRF1 581
interactions with S6K1 and S6K2 were further confirmed by using co-immunoprecipitation 582
(Figure 11E, F). Flag:MRF1 and S6K1/2:Myc were co-expressed in N. benthamiana leaves. 583
S6K1:Myc and S6K2:Myc were immunoprecipitated from leaf extracts using anti-Myc antibody-584
conjugated resin. Immunoblotting with anti-Flag antibody detected Flag:MRF1 proteins co-585
immunoprecipitated with both S6K1:Myc and S6K2:Myc. Collectively, these results suggest that 586
MRF1 is a potential substrate of S6K1 and S6K2, and TOR-S6K1 signaling regulates MRF1 587
phosphorylation. 588
589
DISCUSSION 590
Low-energy stress induces massive transcriptional, translational, and metabolic reprogramming 591
in plants (Tome et al., 2014). The stress decreases global protein translation rates, but the 592
repression is rapidly reversible upon energy supply (Juntawong and Bailey-Serres, 2012). Recent 593
studies using global translation analyses suggested paradigms of plant translation control in 594
response to hypoxia, daily light-dark cycle, carbon deprivation, and extended darkness 595
(Juntawong and Bailey-Serres, 2012; Liu et al., 2012; Pal et al., 2013; Gamm et al., 2014; 596
Juntawong et al., 2014). Despite the general repression under unfavorable conditions, a basal 597
level of translation is essential for maintaining cell homeostasis during the nighttime and coping 598
with stress. However, the regulatory mechanisms of basal translation and the related control 599
factors are largely unknown in plants. Here, we identified MRF family proteins as translation 600
regulators in plants, with functions that are particularly important for translation under energy-601
deficient conditions. 602
We investigated the gene expression, protein characteristics, and cellular functions of four 603
MRF genes in Arabidopsis. Expression of all four MRF genes was induced in dark and starvation 604
(DS) conditions, but with different profiles (Figure 1C, D). MRF Ami-m lines designed to 605
simultaneously silence MRF1, MRF3, and MRF4 exhibited early flowering and early DS-606
21
induced senescence (Figures 3, 4). MRF1 OE lines exhibited the opposite phenotypes: late 607
flowering and delayed senescence in DS conditions. However, T-DNA insertion mutants and 608
amiRNA lines targeted for a single MRF gene did not show any significant phenotypic 609
differences from the WT, suggesting that three MRF genes, MRF1, MRF3, and MRF4, are 610
functionally redundant (Supplemental Figures 4, 6). It has been proposed that MRF family genes 611
divergently evolved through gene duplication events from the ancestral algal linage that 612
contained two MA3 domains (Cheng et al., 2013). It is possible that MRF proteins have 613
individual functions in other conditions through their differences in gene expression and 614
biochemical activity. 615
MA3 is known to be a protein-protein interaction domain, acting as an eIF4A-binding 616
module (Yang et al., 2003; Yang et al., 2004; Loh et al., 2009). eIF4A may be an archetypal 617
DEAD-box RNA helicase (Andreou and Klostermeier, 2013). Possessing RNA-dependent 618
ATPase activity and weak RNA helicase activity, eIF4A catalyzes the unwinding of mRNA 619
secondary structures at the 5′-UTR region to facilitate ribosome scanning through the region for 620
the initiation codon (Hemerly et al., 1995; Jackson et al., 2010). Moreover, eIF4A promotes the 621
dissociation of 5′-UTR-bound proteins and release of the 43S initiation complex from the 5′-cap, 622
using its helicase activity (Jankowsky et al., 2001; Andreou and Klostermeier, 2013). 623
Arabidopsis plants express three eIF4A proteins: eIF4A-1, eIF4A-2, and eIF4A-3. eIF4A-1 and 624
eIF4A-2 are involved in translation, but eIF4A-3, a nuclear protein, participates in RNA 625
processing by incorporating into the exon junction complex (Koroleva et al., 2009). Here, we 626
found that MRF proteins are associated with eIF4A and co-fractionated with ribosomes (Figures 627
5, 6A). MRF1 appears to interact with eIF4A-1 most strongly, and our data are consistent with 628
the possibility that MRF1 associates with ribosomes partially depending on RNA, although 629
additional experiments are required to confirm the finding (Figure 6B; Supplementary Figure 11). 630
MRFs can also rescue the BX04 mutant phenotype, albeit partially. Collectively, these results 631
suggest that MRFs are functionally linked to eIF4A and RNA. Interactions with both mRNA and 632
the translation apparatus may contribute to ribosome association of MRFs. 633
The human homolog of the MRFs is hPDCD4, which contains two MA3 domains. 634
hPDCD4 binding to eIF4A inhibits eIF4A helicase activity and interferes with eIF4A binding to 635
the MA3 domain of eIF4G, suggesting that hPDCD4 suppresses the translation initiation of 636
22
mRNAs with structured 5′-UTRs (Yang et al., 2003; Yang et al., 2004). Indeed, p53 mRNA with 637
a highly structured 5′-UTR has been identified as an endogenous target mRNA of hPDCD4 638
(Wedeken et al., 2011). hPDCD4 also appears to have an additional mechanism of translational 639
suppression; it binds to a secondary structure located in the coding region of c-myb mRNA and 640
blocks translation elongation through interaction with the poly(A)-binding protein (Fehler et al., 641
2014). Thus, the mechanisms of hPDCD4 action in translation control appear to be complex. 642
MRF silencing and MRF1 overexpression in DS conditions caused reduced and slightly 643
increased translation activity, respectively; these effects were not clear under LG conditions 644
(Figure 7; Supplemental Figure 13). Thus, normal MRF activity was required for efficient 645
translation in energy-deficient conditions. Interestingly, MRF silencing triggered a visible 646
decrease in 60S/80S amounts under DS conditions (Figure 7C; Supplemental Figure 14). In 647
contrast, 1-day DS slightly increased the 60S/80S peak in the MRF1 OE lines. Polysome profiles 648
were largely unaffected in all conditions. The average ratio of polysomes to nonpolysomes (P/NP) 649
in MRF deficient and MRF1 OE lines tended to be higher and lower than that of WT, 650
respectively, particularly in DS (Figure 7C). Furthermore, MRF deficiency increased the 651
percentage of polysome-associated mRNAs under DS conditions (for a significant portion of 652
mRNAs tested), despite the decrease in translation rates upon MRF silencing (Figure 8; 653
Supplemental Figure 15). 654
A high P/NP ratio usually represents efficient translation initiation, and mutations 655
impairing translation initiation generally decrease the polysome peak with a concomitant 656
increase in the monosome peak in eukaryotes (Kim et al., 2004; Jao and Chen, 2006; Bolger et 657
al., 2008; Saini et al., 2009; Juntawong and Bailey-Serres, 2012). If MRFs were repressors of 658
translation initiation, like hPDCD4, MRF deficiency would lead to a high P/NP ratio. However, 659
this hypothesis is not consistent with the reduction in 35
S-incorporation found after MRF-660
silencing in DS. On the other hand, specific defects in translation termination/ribosome recycling, 661
initiation, or elongation might have increased the P/NP ratio, simultaneously reducing the 662
translation rate. An MRF function in termination/ribosome recycling seems unlikely, because 663
MRFs were seldom detected in heavy polysomal fractions (Figures 6B, 9C), and were not co-664
immunoprecipitated with poly(A)-binding proteins, PAB2 and PAB8 (data not shown). 665
Furthermore, MRF interaction with eIF4A suggests MRF involvement in initiation control. 666
23
MRFs may act in concert with eIF4A to facilitate ribosome scanning through the 5′-UTR, and 667
MRF deficiency may lead to a reduction in active monosomes in DS. Finally, MRFs may 668
promote translation elongation. As described earlier, the Ami-m lines showed reduced protein 669
translation activity, accompanied by reduced total rRNA contents under DS. The Ami-m lines 670
also showed a higher P/NP ratio and an increase in polysome loading of specific mRNAs under 671
DS. Collectively, these results are most consistent with the hypothesis that MRF deficiency 672
delays translation elongation and subsequent ribosome run-off during DS. Interestingly, the 673
aforementioned molecular phenotypes of the Ami-m (#3) plants under DS mimicked those of the 674
yeast eIF5A mutants defective in translation elongation; disruption of eIF5A activity resulted in 675
reduced monosome amounts without affecting polysomes, and reduced 35
S-methionine 676
incorporation in yeast (Saini et al., 2009). Yeast eIF5A was broadly distributed in sucrose 677
gradients up to the polysome fractions in logarithmic phase cells, but less eIF5A was detected, 678
and only up to the monosome fractions, in stationary phase cells (Jao and Chen, 2006). These 679
ribosome association patterns are analogous with those of MRF1 under LG and DS conditions 680
(Figures 6B, 9C). Interestingly, eIF4A has been identified as a potential interacting partner of 681
pumpkin eIF5A isoforms (Ma et al., 2010). Taken together, our results suggest that MRFs play a 682
positive role in plant translation, possibly modulating translation initiation and/or elongation, and 683
are particularly important in low-energy conditions such as DS. Future studies will address 684
molecular mechanisms of MRF function in plant translation. 685
The dramatic decrease in rRNAs during prolonged DS in plants (Figure 7D) is reminiscent 686
of rapid rRNA degradation in E. coli cells during starvation or at the entry into the stationary 687
phase (Zundel et al., 2009; Piir et al., 2011). The authors proposed that ribosomes not engaged in 688
translation, and consequently present as subunits, are sensitive to endoribonuclease cleavage and 689
subsequent degradation (Zundel et al., 2009). Furthermore, TOR inactivation by rapamycin 690
rapidly decreased the cellular ribosome numbers by 40-60% in yeast, correlated with rRNA 691
degradation by cytoplasmic nucleases (Pestov and Shcherbik, 2012). These results suggest that 692
TOR controls both new ribosome biosynthesis and degradation of mature ribosomes, in order to 693
adjust the size of the translation machinery to changing environmental conditions. In this study, 694
MRF deficiency accelerated rRNA degradation during DS, while MRF1 OE accelerated rRNA 695
recovery during ReLG (Figure 7D). We speculate that the translation defect by MRF deficiency 696
24
under DS may lead to premature dissociation of the translating ribosomes or stalled ribosomes, 697
which then become a target for degradation via cytosolic ribonucleases, ribophagy, or both 698
(Zundel et al., 2009; Floyd et al., 2015). In contrast, MRF1 OE may boost mRNA translation 699
during ReLG, resulting in faster rRNA recovery. Alternatively, higher cell viability of MRF1 OE 700
seedlings during prolonged DS may be an underlying cause of faster rRNA recovery, 701
accomplished by higher rates of rDNA transcription and ribosome biogenesis. 702
Plant TOR kinase connects photosynthesis-driven nutrient availability to comprehensive 703
growth programs through signal transduction and transcription networks (Xiong et al., 2013). 704
TOR downregulation causes wide-reaching transcriptional changes for metabolic reprogramming, 705
accompanied by the accumulation of amino acids and organic acids, which mimics starvation 706
conditions (Moreau et al., 2012; Caldana et al., 2013). Here, we found that TOR modulates the 707
transcript abundance of MRF genes, except MRF2, in response to starvation and sugar feeding 708
(Figure 10A). Thus, MRF genes belong to the TOR-regulated transcriptional networks, which 709
fluctuate according to nutrient/energy availability. MRF silencing and MRF1 overexpression 710
caused hypersensitivity and resistance, respectively, to both DS and Torin-1 treatments (Figures 711
4, 10D, 10E). Furthermore, MRF1 phosphorylation was positively regulated by the TOR 712
pathway, possibly through the action of S6K1/2 (Figures 10B, 11). Mammalian PDCD4 is 713
phosphorylated by S6K1 following mTORC activation, but PDCD4 phosphorylation leads to its 714
dissociation from eIF4A and subsequent ubiquitylation and degradation (Dorrello et al., 2006; 715
Dennis et al., 2012). Transition from DS to LG conditions led to rapid phosphorylation and 716
incorporation of MRF1 into light polysomal fractions (Figure 9B, C), while TOR silencing 717
significantly inhibited MRF1 phosphorylation and decreased MRF1 co-fractionation with 718
ribosomes (Figure 10B, C). Combined with the fact that the affinity between MRF1 and eIF4A-1 719
is higher in LG than in DS (Figure 9D), these results suggest that MRF1 phosphorylation 720
positively correlates with active translation under LG conditions. Yet MRF1 becomes 721
dephosphorylated in DS, but is still co-sedimented with ribosomes. Thus, unphosphorylated 722
forms of MRF1 also appear to be active in translation, particularly under energy-deficient 723
conditions when their abundance increases. There is a possibility that MRFs may interact with 724
different helicases or initiation factors to promote translation in DS. Considering that MRF1 is 725
the most abundant MRF member under starvation conditions, phosphorylation of MRF1 by the 726
25
TOR-S6K signaling pathway during the transition from DS to LG may play a role in rapid 727
rebooting of active translation when the cell environment becomes favorable to growth. 728
Our data suggest that translation of some mRNAs is more sensitive to MRF deficiency: e.g. 729
HDA19 (histone deacetylase) and HDC1 (histone deacetylation complex1) (Figure 8; 730
Supplemental Figure 15). HDA19 and HDC1 are components of the histone deacetylase complex, 731
which epigenetically controls gene expression through repressive function of histone 732
deacetylation (Perrella et al., 2013). HDA19 and HDC1 are involved in plant’s response to ABA, 733
abiotic stresses, and in seed germination, among other functions (Perrella et al., 2013; Mehdi et 734
al., 2016). Altered translation of HDA19 and HDC1 mRNAs might have contributed to the MRF-735
deficient phenotypes in DS. Additionally, MRF functions may be connected to the ethylene 736
signaling pathway. MRF3 was initially identified as EIN2 C-terminus interacting protein 1 737
(ECIP1), and loss-of-function of ECIP1 caused an enhanced ethylene response (Lei et al., 2011). 738
The EIN2 C-terminus after cleavage was found to suppress the translation of EIN3-BINDING F-739
BOX 1 (EBF1) and EBF2 mRNA, both encoding negative regulators of the ethylene pathway, 740
through direct binding to the multiple poly-uridylate motifs in their 3′-UTR and forming 741
processing bodies with ETHYLENE INSENSITIVE 5 (EIN5) and poly(A)-binding proteins (Li 742
et al., 2015; Merchante et al., 2015). In this scenario, MRF3 may inhibit the translation 743
repression activity of EIN2 via a protein-protein interaction near the 3′-UTR of EBF1/2 mRNA. 744
It is unclear whether the EIN2-interacting activity is confined to MRF3 among the four MRF 745
members. Further studies are required to uncover the detailed mechanisms of MRF action in 746
translation and their effects on mRNA translation at a global scale. 747
748
METHODS 749
750
Plant Materials and Growth Conditions 751
Arabidopsis thaliana (ecotype Columbia-0) plants were grown in a growth chamber [23 °C, 100-752
120 μmol m–2
s–1
light intensity using light bulbs (Philips TLD36W/865/FL40SS/36/EX-D), and 753
16 h light:8 h dark cycle]. For the liquid culture, Arabidopsis seeds were surface sterilized and 754
sown in six-well plates containing 1 mL of liquid medium (0.5× Murashige-Skoog [MS] medium 755
[Duchefa], pH 5.7 adjusted with KOH). After germination, seedlings were grown in 0.5× MS 756
26
medium containing 30 mM glucose, changed every other day. 757
758
Generation of Arabidopsis Transgenic Lines 759
AmiRNAs targeting MRF genes were designed by using the Web MicroRNA Designer 760
(http://wmd3.weigelworld.org). Seven different amiRNA PCR products were generated using 761
primer pairs (Supplemental Table 2) and the amiRNA cloning vector pRS300, containing the 762
miR319a backbone as a template, as previously described (Schwab et al., 2006). PCR products 763
were cloned into the pGEM-T-easy vector (Promega), and then transferred to the 764
pCAMBIA1390 vector using the SalI and BamHI sites. To generate MRF1 overexpression lines, 765
the MRF1 protein-coding sequence was cloned into the pCAMBIA-Flag vector using the SalI 766
and EcoRI sites. Arabidopsis (Col-0) plants were transformed by the floral dip method (Clough 767
and Bent, 1998), using Agrobacterium GV3101 strain. More than 30 independent T1 lines were 768
generated for each construct, from which 5-7 T2 lines were selected for T3 propagation, based on 769
gene expression levels. The seed batch that showed 100% hygromycin resistance was selected as 770
the homozygous T3 generation. T3 and T4 homozygous seeds were used for the analyses. 771
772
Virus-Induced Gene Silencing (VIGS) 773
VIGS was performed in Arabidopsis as described previously (Burch-Smith et al., 2006), using 774
soil-grown seedlings at two- to four-leaf stages. A 649-bp cDNA fragment of Arabidopsis TOR 775
was PCR-amplified using TOR-specific primers (Supplemental Table 2). The cDNA fragment 776
was then cloned into the pTRV2 vector (Burch-Smith et al., 2006) using the EcoRI and XhoI 777
sites. pTRV1 containing the viral RNA-dependent RNA polymerase, pTRV2-TOR, and pTRV2 778
empty vector (control) were introduced into Agrobacterium tumefaciens GV3101 strain. The 779
recombinant Agrobacterium strains were cultured overnight in Luria-Bertani medium containing 780
10 mM MES, 20 μM acetosyringone, Kanamycin (50 μg/mL), and Rifampicin (50 μg/mL), and 781
then harvested and resuspended in infiltration medium (10 mM MgCl2, 10 mM MES, and 200 782
μM acetosyringone) to OD600=1.5. After incubation at 23°C for 4 h, the Agrobacterium culture 783
was infiltrated into the largest true leaf using a needless syringe. 784
785
Agrobacterium tumefaciens-Mediated Transient Expression 786
27
Agrobacterium-mediated transient expression was performed using Agrobacterium C58C1 strain 787
as described (Voinnet et al., 2003), except for BiFC experiments that used the GV3101 strain. 788
Overnight-grown Agrobacterium culture was resuspended in infiltration medium (10 mM MES-789
KOH, pH 5.7, 10 mM MgSO4, and 500 μM acetosyringone) to different OD600 depending on 790
experiments (described below), and incubated for 2 h at room temperature, before infiltration 791
into N. benthamiana leaves. In all experiments, Agrobacterium strain carrying the 35S:p19 792
construct (Voinnet et al., 2003) was co-infiltrated at different OD600 ratio depending on 793
experiments (described below), in order to achieve maximum levels of protein expression. 794
Expressed proteins were analyzed at 48-72 h post-infiltration. 795
796
Analysis of the Re-Greening Phenotypes 797
The re-greening assay was performed using 12-day-old seedlings grown in liquid culture. After 798
washing five times with the 0.5X MS medium without glucose, seedlings were incubated for 5 799
days in the dark in the medium lacking glucose for dark/starvation (DS) treatment. After DS, the 800
seedlings were supplied with fresh medium containing 30 mM glucose and incubated in the light 801
for 5 days under the long-day conditions. 802
803
Detached Leaf Senescence Assay 804
The 5th
and 6th
leaves from 3-week-old Arabidopsis plants grown in soil were used for the assay. 805
Detached leaves were floated adaxial side up on the surface of sterilized water in petri dishes. 806
Plates were placed at 23oC in the dark for the indicated times. 807
808
Chlorophyll Measurement 809
Chlorophyll was extracted from four seedlings or individual leaves in aqueous 80% acetone. 810
Absorbance of the extract was measured at 663.6 and 646.6 nm using VersaMax Absorbance 811
Microplate Reader (Molecular Devices). The total chlorophyll contents were calculated based on 812
the absorbance as previously described (Porra and Scheer, 2000), and normalized by fresh weight. 813
814
RT-qPCR 815
Total RNA was extracted using the IQeasy Plus Plant RNA Extraction Mini Kit (iNtRON 816
28
Biotechnology, Korea) according to the manufacturer’s instructions. 2 μg of total RNA was used 817
for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher 818
Scientific) with oligo-dT primers according to the manufacturer’s instructions. Real-time 819
quantitative RT-PCR was performed with diluted cDNAs (1:100) in 96-well plates using the 820
SYBR Premix Ex Taq (TAKARA) and the StepOnePlus Real-Time PCR System (Applied 821
Biosystems), as previously described (Ahn et al., 2011). PP2AA3 (protein phosphatase 2A 822
subunit A3) mRNA was used as control for normalization. To determine relative expression 823
levels of four MRF genes, the Ct (threshold cycle) value of each MRF gene was compared with 824
that of PP2AA3 mRNA as control. 825
826
Subcellular Localization 827
Protein coding sequences of the MRF genes were PCR-amplified using primers listed in 828
Supplemental Table 2, and then cloned into the pCAMBIA1390-sGFP vector using SalI and 829
SmaI sites for MRF1, MRF2, and MRF3, and SalI and EcoRI sites for MRF4. These GFP 830
constructs were agro-infiltrated into N. benthamiana leaves. Agrobacterium strains containing 831
the GFP construct and 35S:p19 construct were co-infiltrated into N. benthamiana leaves at the 832
OD600 ratio of 1.5:0.8. Expression of the GFP fusion proteins was monitored 48 h post-833
infiltration in protoplasts prepared from the infiltrated leaves by a confocal laser scanning 834
microscope (Carl Zeiss LSM 510). 835
836
Subcellular Fractionation 837
Nuclear and cytosolic fractionation was performed using the CelLytic PN Plant Nuclei 838
Isolation/Extraction Kit (Sigma-Aldrich) according to the manufacturer’s protocol. We followed 839
the protocol for semi-pure nuclei preparation for Arabidopsis plants. After fractionation, each 840
fraction was mixed with 2X SDS sample buffer, and subjected to SDS-PAGE using 10-15% 841
gradient gel and immunoblotting with the mouse monoclonal anti-GFP antibody (Clontech, cat. 842
no. 632381, lot no. A0042539; 1:10,000) and the rabbit polyclonal anti-Histone H3 antibody 843
(Santa Cruz, cat. no. Sc10809, lot no. G0110; 1:2000). Signals were detected by Imagequant 844
LAS 4000 (GE Healthcare Life Sciences). 845
846
29
BiFC 847
BiFC was performed with the pSPYNE vector containing the N-terminal region of YFP (amino 848
acid residues 1-155) and the pSPYCE vector containing the C-terminal region of YFP (residues 849
156-239) (Walter et al., 2004). MRF1 and MRF4 coding sequences (CDS) were cloned into 850
pSPYNE using SalI and KpnI sites, while MRF2 and MRF3 CDSs were cloned into pSPYNE 851
using SalI and SmaI sites. Arabidopsis eIF4A-1 and eIF4E-1 CDSs were cloned into pSPYCE 852
using SpeI/SmaI and XbaI/XhoI sites, respectively. Construction of pSPYCE-S6K1 and 853
pSPYCE-S6K2 was previously described (Ahn et al., 2015). Agrobacterium strains containing 854
the pSPYNE, pSPYCE, and 35S:p19 construct was co-infiltrated at the OD600 ratio of 1:1:1.5 855
into leaves of 3 week-old N. benthamiana plants. BiFC signals were monitored 48 h post-856
infiltration in the abaxial side of leaf epidermis using a confocal laser scanning microscope 857
(Zeiss LSM510). To detect protein expression, 50 μg of protein extract was subjected into SDS-858
PAGE using 10-15% gradient gel and immunoblotting was performed using the goat polyclonal 859
anti-GFP antibody (ABM, cat. no. G095; 1:5,000). Signals were detected by Imagequant LAS 860
4000 (GE Healthcare Life Sciences). 861
862
Yeast Two Hybrid Assay 863
The Matchmarker Gold Yeast Two-Hybrid System (Clontech) was used for the analysis. 864
Arabidopsis eIF4A-1 CDS was cloned into the pGADT7 vector (Clontech) containing the GAL4 865
activation domain using EcoRI and SmaI sites. CDSs of the MRF genes were cloned into the 866
pGBKT7 vector (Clontech) containing the GAL4 DNA-binding domain using EcoRI and SalI 867
sites. Yeast two hybrid assays were performed according to the manufacturer’s manual 868
(Clontech). Alpha-galactosidase activity was measured by using the 200-μL scale assay with 48-869
h incubation time, according to the manufacturer’s instruction (Clontech). The activity was 870
statistically analyzed with three biological replications. To examine protein expression in yeast 871
cells, proteins were extracted from 800 μL of yeast cell cultures (OD600 = 1.5) using the NaOH/β-872
mercaptoethanol extraction method. Then the protein extracts (5 μL) were subjected to 8% SDS-873
PAGE and immunoblotting using the anti-c-Myc-Peroxidase-conjugated antibody (Sigma-874
Aldrich, cat. no. A5598, lot no. 045M4854; 1:5,000) and the anti-HA-Peroxidase-conjugated 875
antibody (Roche, cat. no. 12013819001; 1:10,000). Signals were detected by Imagequant LAS 876
30
4000 (GE Healthcare Life Sciences). 877
878
Complementation of E. coli BX04 Strains 879
Bacterial complementation using the pINIII vector was performed as previously described 880
(Nakaminami et al., 2006). The pINIII-CspA plamid was obtained from Dr. Hunseung Kang 881
(Chonnam National University, Korea). Protein coding sequences of the MRF genes and 882
Arabidopsis LOS4 were cloned into the pINIII vector using EcoRI and EcoRI/BamHI sites, 883
respectively. The recombinant plasmids were transformed into E. coil BX04 strain (ΔcspA, 884
ΔcspB, ΔCspE, and ΔcspG) (Xia et al., 2001). The transformed BX04 cell culture (OD600=1) was 885
diluted and spotted onto LB plates containing ampicillin (50 μg/mL), kanamycin (50 μg/mL), 886
and IPTG (0.1 mM) for induction of protein expression. Spotted plates were incubated at 37°C 887
for 1 day or at 18°C for 5 days. 888
889
Co-Immunoprecipitation 890
Arabidopsis eIF4A-1 CDS was cloned into the pCAMBIA1390-6xMyc vector using PstI and 891
EcoRI sites. CDSs of the MRF genes were cloned into the pCAMBIA1390-3xFlag vector using 892
SalI and EcoRI sites. Construction of pCAMBIA1390-S6K1:Myc and pCAMBIA1390-893
S6K2:Myc was previously described (Ahn et al., 2015). Agrobacterium strains containing each 894
pCAMBIA construct and 35S:p19 construct was co-infiltrated into 4-week-old N. benthamiana 895
leaves at the OD600 ratio of 1.0:1.0:1.5. At 72 h post-infiltration, leaves were ground and mixed 896
with an equal volume of ice-cold immunoprecipitation buffer [50 mM sodium phosphate buffer, 897
pH 7.4, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM DTT, 1% Triton X-100, 50 mM 898
MG132, 2 mM Na3VO4, 2 mM NaF, 20 mM β-glycerophosphate, and cOmplete protease 899
inhibitor cocktail (Roche)]. After brief centrifugation to remove cell debris, the supernatant 900
containing 1 mg of total protein was incubated with EZview Red anti-c-Myc affinity gel (10 μL 901
gel per 1 mg of total proteins; Sigma-Aldrich) at 4oC for 4 h. After incubation, the affinity gel 902
was washed four times with IP washing buffer (50 mM sodium phosphate buffer, pH 7.4, 150 903
mM NaCl, 10% glycerol, 5 mM EDTA, 1 mM DTT, and 0.1% Triton X-100), and then 904
resuspended in 50 μL 2X SDS sample buffer. 10 μg of INPUT protein (1%) and 15 μL of IP elute 905
were subjected into 10 % SDS-PAGE. Immunoblotting was performed using the mouse 906
31
monoclonal anti-D tag antibody (ABM, cat. no. G191; 1:5,000) that recognizes Flag epitope, and 907
the mouse monoclonal anti-Myc antibody (ABM, cat. no. G019; 1:5,000). Signals were detected 908
by Imagequant LAS 4000 (GE Healthcare Life Sciences). 909
910
35S-Methionine Labeling 911
35S-methionine labeling was performed as described previously (Ahn et al., 2011). Twelve-day-912
old seedlings grown in liquid culture were pre-treated in LG or DS conditions for 30 min, and 913
then treated with 50 µCi of 35
S-methionine for 2.5 h under the same conditions. After two washes 914
with the culture medium, total proteins were extracted, normalized by Bradford assay, and 915
subjected to 8% Bis-Tris NuPAGE (Thermo Fisher Scientific). The gel was stained with Sun-gel 916
staining solution (LPS Solution, Korea) and dried using a gel drier. Radioactive signal was 917
detected by a phosphorimager (BAS-2500; Fujifilm). Intensity of Coomassie Brilliant Blue 918
staining and radioactive signal was measured by ImageJ software from same area (25-100 kDa) 919
of the gel (https://imagej.nih.gov/ij/). 920
921
Sucrose Density Gradient Sedimentation and Polysome Analysis 922
Frozen tissues (0.2 g) were mixed with 1 mL of polysome isolation buffer (200 mM Tris-HCl, 923
pH 8.4, 50 mM KCl, 1% sodium deoxycholate, 25 mM MgCl2, 2% polyethylene [10] tridecyl 924
ether, 400 U/mL RNasin [Promega], and 50 μg/mL cycloheximide). Cell debris was removed by 925
brief centrifugation. Cell extracts (500 μL) were loaded onto an 11.5 mL sucrose gradient and 926
spun in a Beckman SW41Ti rotor at 38,000 rpm for 3.5 h at 4 °C. Fifteen fractions of 0.8 mL 927
were collected using the BioLogic low-pressure liquid chromatography system (Bio-Rad) with a 928
fraction collector. Absorbance was automatically detected at 254 nm for polysome analysis. One-929
half volume of all fractions was precipitated using the methanol precipitation method. After 930
precipitation, the pellet was resuspended in 50 μL of 1X protein sample buffer (200 mM Tris-931
HCl, pH 6.8, 3% SDS, 15% β-mercaptoethanol, 30% glycerol, 0.09% bromophenol blue, and 932
100 mM DTT) and incubated in 70℃ for 10 min. 10-20 μL of protein samples were loaded onto 933
8% Bis-Tris NuPAGE gels. Immunoblotting was performed with mouse monoclonal antibodies 934
against GFP (Clontech; cat. no. 632381, lot no. A0042539; 1:10,000), Myc (ABM, cat. no. G019; 935
1:5,000), and RPL10a (Santa Cruz, cat. no. Sc-100827, lot no. H2212; 1:4,000). 936
32
937
RNase A Treatment 938
Cell extracts (500 μL) in polysome isolation buffer (except RNase inhibitor) were incubated with 939
RNAse A at the final concentration of 1 mg/mL (Sigma-Aldrich) for 5 min on ice. After 5 min, 940
200 U of RNase inhibitor (RNasin) was added into the reaction mix to stop the RNAse action. 941
Then the reaction mix was loaded onto sucrose density gradients for polysome analysis and 942
fractionation as described above. 943
944
Analyses of mRNA Distribution Patterns and Quantification 945
To purify RNA, each sucrose gradient fraction (800 μL) was mixed with 300 μL of QIAzol Lysis 946
Reagent (QIAGEN) and 300 μL of chloroform, followed by vortexing and brief centrifugation. 947
To precipitate RNA, the upper aqueous layer was mixed with 700 μL of isopropanol and 70 μL 948
of 3 M sodium acetate (pH 5.7), and then centrifuged for 20 min at 4℃ at the maximum speed. 949
The pellet was washed with 75% ethanol and resuspended in 20 μL of RNase-free water. 7.5 μL 950
of RNA was used for cDNA synthesis using the RevertAid First Strand cDNA Synthesis Kit 951
(Thermo Fisher Scientific) with oligo-dT primers according to the manufacturer’s instructions. 952
The cDNA was diluted 20 times in water and analyzed by real-time quantitative PCR using 953
SYBR Premix Ex Taq (TAKARA) and the StepOnePlus Real-Time PCR System (Applied 954
Bioscience). After PCR, the obtained Ct values were converted to transcript amounts. The 955
abundance of mRNA in each fraction was calculated as a percentage of their total amount in all 956
fractions. 957
958
Zn2+
-Phos-Tag SDS-PAGE and Lambda Phosphatase Treatment 959
Zn2+
-Phos-tag SDS-PAGE was carried out under neutral pH conditions as previously described 960
(Kinoshita and Kinoshita-Kikuta, 2011), with some modifications. Homogenized samples were 961
resuspended in the same volume of extraction buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 962
mM DTT, and cOmplete protease inhibitor cocktail [Roche]). For the λ-phosphatase treatment, 963
10 mM MnCl2 and either 400 U (1 μL) λ-phosphatase (New England Biolabs), or enzyme 964
storage buffer (100 mM NaCl, 50 mM HEPES, pH 7.5, 0.1 mM MnCl2, 0.1 mM EGTA, 2 mM 965
DTT, 0.01% Brij35, and 50% glycerol) was added to aliquots (21.5 μL) of protein extracts for 966
33
incubation at 30 °C for 1 h. The reaction was stopped by adding 3× protein sample buffer (200 967
mM Tris-HCl, pH 6.8, 3% SDS, 15% β-mercaptoethanol, 30% glycerol, 0.09% bromophenol 968
blue, and 100 mM DTT), followed by incubation at 70°C for 10 min. Then the samples were 969
subjected to 8% Zn2+
-Phos-tag Bis-Tris NuPAGE with 50 μM ZnCl2 and 50 μM Phos-taq system 970
(Wako). After electrophoresis, the Phos-tag gels were washed twice for 10 min each with transfer 971
buffer (10 mM CAPS, pH 11) containing 10 mM EDTA, followed by 10 min washing with 972
transfer buffer without EDTA. Immunoblotting was performed with the rabbit polyclonal anti-973
Myc antibody (ABM, cat. no. G019; 1:5,000) and anti-Flag M2-HRP-conjugated antibody 974
(Sigma-Aldrich, cat. no. A8592, lot no. 059K6059; 1:10,000), according to the manufacturer’s 975
instructions. 976
977
Purification of Recombinant Proteins 978
CDSs of MRF genes were cloned into pMal-C2X vector (New England Biolabs) for MBP fusion, 979
using EcoRI and SalI sites. The constructs were transformed into E. coli BL21 (DE3) strain. 980
Cells were grown in 1% glucose-NZCYM medium containing ampicillin (50 µg/mL) at 37°C to 981
an OD600 of 0.4, and induced by 0.25 mM IPTG for 16 h. MBP fusion proteins were purified 982
using Amylose Resin (New England Biolabs), following the manufacturer’s instruction with 983
minor modification. We used a single buffer (20 mM Tris-HCl, pH 7.5, and 200 mM NaCl) 984
throughout the purification procedure, and added 10 mM maltose into the buffer for elution. 985
After purification, proteins were concentrated using Amicon Ultracel 30K (Millipore) according 986
to the manufacturer’s instruction. Purified proteins were stored at -80oC until use. 987
988
In Vitro Kinase Assay Using Immunoprecipitated S6K1 and S6K2 989
Kinase assay was performed as previously described by Xiong et al. (2013) with minor 990
modifications. S6K1:Myc or S6K2:Myc proteins were transiently expressed in N. benthamiana 991
leaves via Agrobacterium infiltration. Infiltrated leaves were ground on liquid nitrogen and 992
mixed with extraction buffer (50 mM Sodium phosphate buffer, pH 7.4, 150 mM NaCl, 10% 993
Glycerol, 5 mM EDTA, 1 mM DTT, 1% Triton X-100, 50 mM MG132, 2 mM Na3VO4, 2 mM 994
NaF, 20 mM β-glycerophosphate, and cOmplete protease inhibitor cocktail [Roche]). After brief 995
centrifugation, the supernatant was mixed with EZview Red Anti-c-Myc affinity gel (10 μL gel 996
34
per 1 mg of total proteins; Sigma-Aldrich) for incubation at 4 °C for 4 h. After incubation, the 997
affinity gel was washed twice with low-salt buffer 1 (40 mM HEPES, pH 7.4, 150 mM NaCl, 2 998
mM EDTA, 10 mM pyrophosphate, 10 mM β-glycerophosphate, and 0.3% CHAPS), followed 999
by washing twice with low-salt buffer 2 (25 mM HEPES, pH 7.4, and 20 mM KCl). Kinase 1000
reaction was carried out with 2 μCi [γ-32
P]-ATP, immunoprecipitated S6K1/2, and 10 μg1001
recombinant substrate proteins in 20 μL kinase buffer (20 mM HEPES, pH 7.4, 125 mM NaCl, 1 1002
mM DTT, 10 mM MgCl2, 5 mM MnCl2, and 10 μM ATP) for 30 min at 30 °C. The reaction was 1003
stopped by adding 2× SDS sample buffer and boiling for 1 min. After SDS-PAGE, the gel was 1004
stained with Sun-Gel staining solution (LPS Solution, Korea), and dried using a gel drier. 1005
Radioactivity within the gel was detected using a phosphorimager (BAS-2500; Fujifilm). 1006
Immunoblotting of input samples was performed with the mouse monoclonal anti-Myc antibody 1007
(ABM, cat. no. G019; 1:5,000). 1008
1009
Accession Numbers 1010
All accession numbers can be found in Supplemental Table 1. 1011
1012
1013
SUPPLEMENTAL DATA 1014
Supplemental Figure 1. RT-PCR Analyses of MRF Expression in Response to Mannitol 1015
Feeding after Starvation (Related to Figure 1D). 1016
Supplemental Figure 2. Relative Transcript Levels of the MRF Genes (Related to Figure 1D). 1017
Supplemental Figure 3. Analyses of T-DNA Insertion Mutants of MRF3 and MRF4, and 1018
AmiRNA Lines of MRF1 and MRF2 (Related to Figure 3). 1019
Supplemental Figure 4. MRF4 mRNA Levels in Ami-m Lines and Flowering Phenotypes in T-1020
DNA Insertion Mutants and AmiRNA Lines of the MRF Genes (Related to Figure 3). 1021
Supplemental Figure 5. Measurement of Chlorophyll Contents and Detached Leaf Senescence 1022
Assay (Related to Figure 4A). 1023
Supplemental Figure 6. Re-greening Phenotypes of AmiRNA Lines and T-DNA Insertion 1024
Mutants (Related to Figure 4A). 1025
Supplemental Figure 7. Gene Expression of Ami-m and MRF1 OE Lines (Related to Figure 4B, 1026
35
C). 1027
Supplemental Figure 8. Protein Sequence Alignment of MA3 Domains in Diverse Proteins 1028
(Related to Figure 5). 1029
Supplemental Figure 9. BiFC Assays for Interactions between MRFs and eIF4A (Related to 1030
Figure 5A). 1031
Supplemental Figure 10. Immunoblotting to Determine Protein Expression in BiFC 1032
Experiments Shown in Figure 5A. 1033
Supplemental Figure 11. Co-immunoprecipitation of MRFs with the eIF4A Family Proteins 1034
(Related to Figure 5B). 1035
Supplemental Figure 12. Immunoblotting to Determine Protein Expression in Yeast Two-1036
Hybrid Experiments Shown in Figure 5C. 1037
Supplemental Figure 13. 35
S-Methionine Labeling under Light/Glucose Conditions (Related to 1038
Figure 7A, B). 1039
(Related to Figure 7A, B). 1040
Supplemental Figure 14. Polysome Analyses without a Brief Cycloheximide Treatment Step 1041
before Sample Harvest (Related to Figure 7C). 1042
Supplemental Figure 15. Distribution Patterns and Quantifications of Specific mRNAs in 1043
Sucrose Gradient Fractions (Related to Figure 8). 1044
Supplemental Figure 16 Gene Silencing Phenotypes and Reduced mRNA Levels of TOR in 1045
TOR RNAi and VIGS Plants (Related to Figure 10A-C). 1046
Supplemental Figure 17. CAB2 Transcript Levels in Seedlings after Torin-1 Treatment (Related 1047
to Figure 10D). 1048
Supplemental Figure 18. Control Experiments for in Vitro Kinase Assay Shown in Figure 11. 1049
Supplemental Table 1. Information on the Genes Used in This Study. 1050
Supplemental Table 2. Primers Used in This Study. 1051
1052
ACKNOWLEDGEMENTS 1053
1054
The authors wish to thank Drs. Masayori Inouye (Rutgers University, USA) and Sangita Phadtare 1055
(Rowan University, USA) for providing the E. coli BX04 mutant strain and pINIII vector, Dr. Jen 1056
36
Sheen (Harvard Medical School, USA) for providing seeds of the estradiol-inducible TOR RNAi 1057
lines, Dr. Detlef Weigel (Max Planck Institute for Developmental Biology, Germany) for the 1058
pRS300 vector, and Dr. Hunseung Kang (Chonnam National University, Korea) for helpful 1059
discussions. This research was supported by the Cooperative Research Program for Agriculture 1060
Science & Technology Development (Project numbers PJ01114701 [PMBC] and PJ01118901 1061
[SSAC]) from the Rural Development Administration, and the Mid-Career Researcher Program 1062
(NRF-2016R1A2B4013180) from the National Research Foundation (NRF) of the Republic of 1063
Korea. 1064
1065
AUTHOR CONTRIBUTIONS 1066
1067
D.-H.L. performed most of the experiments and analyzed the results together with S.J.P. and 1068
C.S.A. D.-H.L. and H.-S.P. designed the experiments and wrote the manuscript. All authors 1069
discussed the results and commented on the manuscript. 1070
1071
FIGURE LEGENDS 1072
1073
Figure 1. Predicted Protein Structure and Expression Patterns of Four MRF Genes. 1074
(A) Schematic representation of four Arabidopsis MRF proteins with four MA3 domains 1075
arranged in tandem. Residue numbers are marked. aa, amino acids. 1076
(B) RT-qPCR analyses of MRF gene expression in plant organs. Three organ pieces were 1077
collected from three different 6-week-old Arabidopsis plants: the 7th
and 8th
rosette leaves (RL), 1078
the 1st cauline leaves (CL), stems (~1 cm from the bottom; St), and the primary roots (R). Ten 1079
pieces of buds (stages 11-12; B) and open flowers (F) were also collected from the three plants. 1080
The collected tissues were combined for RNA extraction and RT-qPCR. Transcript levels are 1081
expressed relative to those in rosette leaf (RL). 1082
(C) RT-qPCR analyses of MRF gene expression in response to darkness. Twelve seedlings grown 1083
in three different sets in liquid culture were incubated in the dark for the indicated times. 1084
Transcript levels are expressed relative to those at 0 h. 1085
37
(D) RT-qPCR analyses of MRF gene expression in response to starvation and glucose feeding. 1086
Twelve seedlings grown in three different sets in liquid culture were incubated in glucose-free 1087
medium for 24 h (starvation; S), and then fed with the indicated concentrations of glucose for 4 h. 1088
Transcript levels are expressed relative to those before starvation (BS). 1089
For (B) to (D), transcript levels are normalized by PP2AA3 mRNA, and error bars represent 1090
standard errors (SE) calculated from triplicate technical replications. 1091
1092
Figure 2. Cytosolic Localization of MRF Proteins. 1093
(A) Subcellular localization of MRF:GFP fusion proteins in leaf protoplasts. Each MRF:GFP1094
was transiently expressed together with histone H2B:mRFP as a nuclear marker in N. 1095
benthamiana leaves via agro-infiltration. Protoplasts were prepared from the infiltrated leaves, 1096
and observed by confocal microscopy. Chlorophyll autofluorescence was pseudo-colored blue. 1097
More than 20 cells showing green fluorescence were observed for each construct. Scale bars = 1098
10 µm. 1099
(B) Subcellular fractionation. N. benthamiana leaf extracts expressing MRF:GFP proteins were1100
fractionated and subjected to SDS-PAGE using 10-15% gradient gel, followed by 1101
immunoblotting with anti-GFP antibody. Total (T), nuclear (N), and cytosolic (C) fractions were 1102
indicated. Histone H3 was detected as a nuclear marker protein using anti-H3 antibody. Two 1103
independent experiments yielded similar results. 1104
(C) Confocal microscopy of GFP fluorescence in epidermal cells of the Arabidopsis transgenic1105
plants that express each MRF gene under the CaMV35S promoter. Multiple independent 1106
transgenic lines were analyzed for each MRF gene, which similarly suggested cytosolic 1107
localization of MRF proteins. More than three independent observations were made for each 1108
transgenic line. Scale bars = 10 µm. 1109
1110
Figure 3. Generation of MRF Artificial miRNA and MRF1 Overexpression Lines, and Analysis 1111
of their Flowering Phenotypes. 1112
(A) Description of MRF artificial miRNA (amiRNA) and MRF1 overexpression (OE) lines (left),1113
and target sites for the amiRNA lines (right). The target sites (arrowheads) were designed for 1114
38
silencing of MRF1, MRF2, or multiple genes (MRF1, MRF3, and MRF4). “Ami-m” represents 1115
amiRNA lines with multiple targets. 1116
(B) RT-qPCR to determine MRF1 and MRF3 mRNA levels in the Ami-m lines. Transcript levels 1117
in the Ami-m lines are expressed relative to those in the WT. Values represent the means ± S.E. 1118
of N = three biological replicates of 10-day-old seedlings grown in different sets in liquid culture. 1119
Asterisks denote statistical significance of the differences between the WT and the transgenic 1120
lines, calculated using Student’s t-test (***, P ≤ 0.001). 1121
(C) RT-qPCR to determine MRF1 mRNA levels in the MRF1 OE lines, compared with those in 1122
WT. Error bars represent SE from triplicate biological replications using 10-day-old seedlings 1123
grown in different sets of liquid culture (***, P ≤ 0.001). 1124
(D) Flowering phenotypes of the Ami-m and MRF1 OE lines. Plants were grown for 4 weeks 1125
under long-day conditions. 1126
(E) Quantification of rosette and cauline leaf numbers at the bolting stage with the first open 1127
flower. Values represent means SE of 40 plants per sample (***, P ≤ 0.001). 1128
1129
Figure 4. Phenotypes and Gene Expression of the MRF Ami-m and MRF1 OE Lines after DS 1130
and ReLG treatments. 1131
(A) Seedlings at 12 days after germination were incubated in the dark/starvation (DS) for 5 days, 1132
and then re-illuminated and fed with 30 mM glucose (ReLG) for 5 days. Photos were taken 1133
periodically during the process. d, days. 1134
(B, C) Time-course RT-qPCR analyses of CAB2 mRNA levels. Seedlings grown under LG 1135
conditions were subjected to 2 and 5 days of DS treatment (DS+2 and DS+5), followed by 3 and 1136
5 days of LG treatment (ReLG+3 and ReLG+5). RT-qPCR was performed for CAB2 mRNAs at 1137
the indicated time points (B). The relative CAB2 transcript levels in different lines at ReLG+3 1138
[boxed with dotted line in (B)] were plotted (C). The transcript level is normalized by PP2AA3 1139
mRNA, and expressed relative to those in WT. Values represent the means ± S.E. of N = three 1140
biological replicates of seedlings grown in different sets in liquid culture. Asterisks denote 1141
statistical significance of the differences between WT and the transgenic lines, calculated using 1142
Student’s t-test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). 1143
39
(D, E) Time-course RT-qPCR analyses of SEN4 mRNA levels. The SEN4 transcript levels at the 1144
indicated time points are shown (D), and the values at DS+5 [boxed with dotted line in (D)] were 1145
plotted (E). 1146
1147
Figure 5. Interactions between MRFs and Eukaryotic Translation Initiation Factor 4A. 1148
(A) Bimolecular fluorescence complementation (BiFC). YFPN- and YFP
C-fusion proteins were 1149
co-expressed in N. benthamiana leaves by agroinfiltration. Leaf epidermal cells were observed 1150
by confocal microscopy. More than 20 leaf cells showing yellow fluorescence were observed for 1151
each BiFC experiment. Bars = 20 µm. 1152
(B) Co-immunoprecipitation. Each MRF protein in Flag fusion (Flag:MRF) was expressed alone 1153
or together with eIF4A-1:Myc in N. benthamiana leaves. Total leaf proteins were 1154
immunoprecipitated with anti-Myc antibody-conjugated resin, and the co-immunoprecipitate was 1155
detected using the anti-Flag antibody. 1156
(C) Yeast two-hybrid assay. GAL4 activation domain (AD)-fused to eIF4A-1 and GAL4 DNA 1157
binding domain (BD)-fused MRF proteins were co-expressed in yeast. Alpha-galactosidase 1158
activity indicates protein-protein interaction affinity. Error bars represent SE from triplicate 1159
biological replications using three individual colonies. Asterisks denote the statistical 1160
significance of the differences between the control (AD:eIF4A-1/BD vector) and other samples 1161
(*, P ≤ 0.05; **, P ≤ 0.01). 1162
1163
Figure 6. Ribosome Association of MRF Proteins and BX04 Complementation Assays. 1164
(A) Co-fractionation of MRFs with ribosome subunits and translation initiation factors. 1165
MRFs:GFP, eIF4E-1:Myc, and eIF4A-1:Myc were expressed in N. benthamiana leaves. After 1166
sucrose density gradient sedimentation, the fractions were subjected to immunoblotting with 1167
anti-GFP, anti-Myc, and anti-60S ribosomal protein L10a (RPL10a) antibodies. Lanes indicated 1168
the fractions from top (15%) to bottom (50%). 1169
(B) Distribution of MRFs in sucrose gradient fractions after RNAse A treatment. Total cell 1170
extract was prepared from Flag-MRF1 OE seedlings (#1) grown under light/glucose conditions. 1171
The cell extract was treated with 1 mg/ml of RNase A on ice for 15 min (+RNAse A) or with 1172
RNase-free water (control), prior to sucrose density gradient sedimentation (15%-50%). The UV 1173
40
absorbance at 254 nm was monitored for gradient fractions to produce the absorbance profiles 1174
(top). The collected fractions were subjected to immunoblotting with anti-Flag and anti-L10a 1175
antibodies (bottom). 1176
(C) BX04 complementation assays. The E. coli BX04 strain is a quadruple mutant of cold-shock 1177
proteins, which cannot grow at low temperature. The BX04 strain was transformed with 1178
plasmids carrying MRFs, E. coli CspA (cold-shock protein; positive control), Arabidopsis LOS4 1179
(RNA helicase), and vector control. The transformants were grown overnight, and then serially 1180
diluted and spotted onto media plates. The plates were incubated at 37 °C (left) and 18 °C (right). 1181
1182
Figure 7. 35
S-Methionine Labeling under Dark/Starvation Conditions, Polysome Analyses, and 1183
Total RNA Contents 1184
(A) Autoradiography images of 35
S-Met incorporation. Seedlings grown in liquid culture were 1185
pre-incubated without glucose in the dark for 30 min, followed by 35
S-Met labeling for 2.5 h 1186
under the same conditions. After SDS-PAGE of protein extracts from the labeled seedlings, the 1187
gel was stained with Coomassie brilliant blue (CBB) and dried. The radioactive signal within the 1188
gel was detected by a phosphorimager. Four independent experiments yielded similar results, and 1189
representative images are shown. 1190
(B) Relative band intensity. The radioactive intensity of 35
S-Met-labeled proteins was normalized 1191
by CBB band intensity, and the ratio was expressed relative to the WT. Error bars represent SE 1192
from four biological replications based on four independent experiments (*, P ≤ 0.05; **, P ≤ 1193
0.01). 1194
(C) Polysome analyses. Seedlings were incubated with LG for 2 h or DS for 1 day (1 d). The 1195
seedlings were treated with cycloheximide (50 μg/ml) for 5 min before harvest, and total cell 1196
extracts from the seedlings were subjected to sucrose density gradient sedimentation (15%-50%). 1197
The UV absorbance at 254 nm was monitored for gradient fractions to produce the absorbance 1198
profiles. The absorbance profiles of LG (blue lines) and DS (red lines) samples were 1199
superimposed for comparison. An average ratio (P/NP) of polysomes to 60S/80S ribosomes was 1200
calculated for each sample using Image J program, from four biological replications based on 1201
four independent experiments. 1202
41
(D) Total RNA contents after prolonged DS and ReLG treatments. Seedlings grown under LG 1203
conditions (control; CTL) were subjected to 2 and 5 days of DS treatment (DS+2 and DS+5), 1204
followed by 3 and 5 days of LG treatment (ReLG+3 and ReLG+5) as described in Figure 3A. 1205
Total RNA was extracted from an equal weight of the seedlings at each stage, and measured by 1206
absorbance at 260 nm using a spectrophotometer. Error bars represent SE from triplicate 1207
biological replications using seedlings grown in different sets of liquid culture. Asterisks denote 1208
statistical significance of the differences between WT and the transgenic lines, calculated using 1209
Student’s t-test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). 1210
1211
Figure 8. Distribution Patterns and Quantifications of Specific mRNAs in Sucrose Gradient 1212
Fractions. 1213
(A) Distribution of PP2AA3, GAPC, HDA19, and HDC1 mRNAs in sucrose gradient fractions.1214
WT and Ami-m (#3) seedlings were incubated under LG conditions for 2 h (left) or under DS 1215
conditions for 1 d (right). Total cell extracts prepared from the seedlings were subjected to 1216
sucrose density gradient sedimentation (15%-50%), and total 15 fractions were collected from 1217
each tube. Total RNA was extracted from each fraction, followed by cDNA synthesis and RT-1218
qPCR using gene-specific primers. The abundance of mRNA in each fraction was quantified as a 1219
percentage of their total amount in all fractions. Similar results were obtained in three 1220
independent experiments, and a representative result is shown. Error bars represent SE from 1221
three technical replications. 1222
(B) The abundance of mRNA in polysomal (P; fractions 9-15) and nonpolysomal fractions (NP;1223
fractions 1-8), quantified as a percentage of their total amount. Ami3 represents Ami-m (#3). 1224
Error bars represent SE from three biological replications based on three independent 1225
experiments (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). 1226
1227
Figure 9. Phosphorylation and Ribosome Association of MRF1 According to Cellular Energy 1228
Availability. 1229
(A) Phosphorylation of MRF1 in vivo. Total protein extracts from N. benthamiana leaves, which1230
express MRF:Myc proteins, were treated with the lambda phosphatase (λPP). After treatment, 1231
the samples were subjected to Zn2+
-Phostag SDS-PAGE (top) and to normal SDS-PAGE (bottom)1232
42
for immunoblotting with anti-Myc antibody. The phosphorylated form of MRF1 was marked 1233
with the asterisk. 1234
(B) Phosphorylation of MRF1 under different energy conditions. Flag:MRF1 OE (#1) seedlings1235
were incubated under dark/starvation conditions for 1-48 h, and then re-illuminated and fed with 1236
30 mM glucose (light/glucose) for 0.5-2 h. Protein extracts from the seedlings harvested at 1237
different time points were separated by Zn2+
-Phostag SDS-PAGE (top) and by normal SDS-1238
PAGE (middle), followed by immunoblotting with anti-Flag antibody. The Rubisco large subunit 1239
was stained with CBB as loading control (bottom). 1240
(C) Ribosome association of Flag:MRF1 under different energy conditions. Flag:MRF1 OE (#1)1241
seedlings were incubated in the dark/starvation for 24 h, followed by re-illumination and 1242
glucose-feeding for 1 h. Polysome analysis was performed by ultracentrifugation through a 10-1243
55% sucrose gradient. Then the fractions were precipitated and analyzed by immunoblotting with 1244
anti-Flag and anti-RPL10a antibodies. Lanes indicate the fractions from top (10%) to bottom 1245
(55%). Arrowheads indicate the final positions of MRF1 detection. 1246
(D) Co-immunoprecipitation. Flag:MRF1 was expressed alone or together with eIF4A-1:Myc in1247
N. benthamiana leaves. Leaf disks were prepared for treatment with light/glucose (LG) or1248
dark/starvation (DS) for 3 h. Total leaf proteins were immunoprecipitated with anti-Myc 1249
antibody-conjugated resin, and the co-immunoprecipitate was detected using the anti-Flag 1250
antibody. 1251
1252
Figure 10. TOR-Modulated MRF Gene Expression and MRF1 Phosphorylation, and Seedling 1253
Phenotypes upon Torin-1 Treatment. 1254
(A) Altered MRF gene expression in TOR-silenced seedlings in response to starvation and sugar1255
feeding. Estradiol-inducible TOR RNAi seedlings (es-tor1) were treated with ethanol (-ES) or 10 1256
μM estradiol (+ES) for gene silencing. Twelve seedlings grown in three different sets of liquid 1257
culture were incubated in glucose-free medium for 24 h (Stv), and then fed with 30 mM glucose 1258
(Glc), mannitol (Man), and sucrose (Suc) for 4 h. RT-qPCR was performed with gene-specific 1259
primers. Transcript levels are normalized by PP2AA3 mRNA, and expressed relative to those of 1260
Stv samples. Error bars represent SE from triplicate technical replications. 1261
43
(B) MRF1 phosphorylation in TOR-silenced plants. TOR VIGS was performed in Flag:MRF1 1262
OE (#1) lines. Protein extracts from TRV control or TOR VIGS leaves (10 DAI) were separated 1263
by Phostag SDS-PAGE (top) and by normal SDS-PAGE (middle), followed by immunoblotting 1264
with anti-Flag antibody. The Rubisco large subunit was stained with CBB as loading control 1265
(bottom). 1266
(C) Ribosome association of Flag:MRF1 in TOR-silenced plants. TOR VIGS was performed in1267
Flag:MRF1 OE (#1) lines. Protein extracts from TRV control and TOR VIGS leaves (10 DAI) 1268
were fractionated by sucrose density gradient sedimentation (10-55%). The fractions were 1269
precipitated and analyzed by immunoblotting with anti-Flag and anti-RPL10a antibodies. Lanes 1270
indicate the fractions from top (10%) to bottom (55%). Arrowheads indicate the final positions of 1271
MRF1 detection. 1272
(D) Phenotypes of the Ami-m and MRF1 OE seedlings after Torin-1 treatment. Seven-day-old1273
seedlings grown in liquid culture were treated with Torin-1 (2 μM) or control DMSO for 3 days. 1274
(E) Root length of the seedlings was measured after treatment with Torin-1 (2 μM) or control1275
DMSO for 3 and 5 days. Each data point represents the mean SE (n > 14 seedlings). Asterisks 1276
denote statistical significance of the differences between Torin-1-treated samples and DMSO-1277
treated samples, calculated using Student’s t-test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). 1278
1279
Figure 11. In Vitro Phosphorylation of MRF1 by S6K kinases. 1280
(A) In vitro kinase assay of immunoprecipitated S6K1:Myc with the recombinant MBP:MRF1281
proteins as substrates. After the kinase assay with [γ-32
P]-ATP, SDS-PAGE was performed. 1282
Phosphorylated MBP:MRF proteins were detected by a phosphorimager (top); the MBP:MRF 1283
protein in the reaction was detected by CBB staining (middle); immunoprecipitated S6K1:Myc 1284
was detected by immunoblotting with anti-Myc antibody (bottom). 1285
(B) In vitro kinase assay of immunoprecipitated S6K2:Myc with the recombinant MBP:MRF1286
proteins as substrates. 1287
(C) In vitro kinase assay with S6K1 mutant forms that carry a mutation in the TOR1288
phosphorylation site T449. In vitro kinase assay was performed with S6K1:Myc, 1289
S6K1(T449A):Myc, and S6K1(T449D):Myc proteins. 1290
44
(D) BiFC analyses for MRF1 interactions with S6K1 and S6K2. MRF1:YFPN was expressed 1291
together with S6K1:YFPC or S6K2:YFP
C in N. benthamiana leaves using agroinfiltration. Leaf 1292
epidermal cells were observed by confocal microscopy. More than 20 leaf cells showing yellow 1293
fluorescence were observed for each BiFC experiment. As a negative control, MRF1:YFPN and 1294
eIF4E-1:YFPC were co-expressed in N. benthamiana leaves, which resulted in little yellow 1295
fluorescence. Bars = 20 µm. 1296
(E), (F) Co-immunoprecipitation of MRF1 with S6K1 and S6K2. Flag:MRF1 was expressed 1297
alone or together with S6K1:Myc (E) or S6K2:Myc (F) in N. benthamiana leaves. Total leaf 1298
proteins were immunoprecipitated with anti-Myc antibody-conjugated resin, and the co-1299
immunoprecipitate was detected using the anti-Flag antibody. 1300
1301
45
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1535
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
RL CL St R B F
Rela
tive
mR
NA
le
vels
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
RL CL St R B F
0
0.2
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0.8
1.0
1.2
1.4
RL CL St R B F
0
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1.0
1.2
1.4
RL CL St R B F
B MRF1 MRF2 MRF3 MRF4
Figure 1
0
5
10
15
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25
0 0.5 2 4 24
Re
lative m
RN
A le
vels
0
1
2
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5
0 0.5 2 4 24
0
2
4
6
8
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12
14
16
0 0.5 2 4 24
0
1
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3
4
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0 0.5 2 4 24
Dark (h)
C MRF1 MRF2 MRF3 MRF4
Dark (h) Dark (h) Dark (h)
A MA3 MA3 MA3 MA3
MA3 MA3 MA3 MA3
MA3 MA3 MA3 MA3
MA3 MA3 MA3 MA3
57 168 222 332 352 462 624
633 aa
516
702 aa
693 aa
702 aa
117 228 281 392 415 525 579 688
91 202 255 366 390 500 561 672
123 234 288 398 421 531 585 692
MRF1
MRF2
MRF3
MRF4
D
Glucose (mM)
MRF1 MRF2 MRF3 MRF4
Glucose (mM) Glucose (mM) Glucose (mM)
0
2
4
6
8
10
12
14
BS S 10 30 60 180
Rela
tive m
RN
A levle
ls
0
2
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BS S 10 30 60 180
0
5
10
15
20
25
30
35
BS S 10 30 60 180
0
5
10
15
20
25
BS S 10 30 60 180
Figure 1. Predicted Protein Structure and Expression Patterns of Four MRF Genes.
(A) Schematic representation of four Arabidopsis MRF proteins with four MA3 domains arranged
in tandem. Residue numbers are marked. aa, amino acids.
(B) RT-qPCR analyses of MRF gene expression in plant organs. Three organ pieces were collected
from three different 6-week-old Arabidopsis plants: the 7th and 8th rosette leaves (RL), the 1st cauline
leaves (CL), stems (~1 cm from the bottom; St), and the primary roots (R). Ten pieces of buds
(stages 11-12; B) and open flowers (F) were also collected from the three plants. The collected
tissues were combined for RNA extraction and RT-qPCR. Transcript levels are expressed relative to
those in rosette leaf (RL). For (B) to (D), transcript levels are normalized by PP2AA3 mRNA, and
error bars represent standard errors (SE) calculated from triplicate technical replications.
(C) RT-qPCR analyses of MRF gene expression in response to darkness. Twelve seedlings grown in
three different sets of liquid culture were incubated in the dark for the indicated times. Transcript
levels are expressed relative to those at 0 h.
(D) RT-qPCR analyses of MRF gene expression in response to starvation and glucose feeding.
Twelve seedlings grown in three different sets of liquid culture were incubated in glucose-free
medium for 24 h (starvation; S), and then fed with the indicated concentrations of glucose for 4 h.
Transcript levels are expressed relative to those before starvation (BS).
100
140
10
15
kDa
MRF1:GFP
MRF2:GFP
MRF3:GFP
MRF4:GFP
GFP Histone
H2B:mRFP Chlorophyll Merged
A
B
C
α-GFP
α-H3
Total C N
MRF1:GFP
Total C N
MRF2:GFP
Total C N
MRF3:GFP
Total C N
MRF4:GFP
MRF1:GFP MRF2:GFP MRF3:GFP MRF4:GFP
Tra
nsg
enic
lin
es
Figure 2
Figure 2. Cytosolic Localization of MRF Proteins.
(A) Subcellular localization of MRF:GFP fusion proteins in leaf protoplasts. Each MRF:GFP was
transiently expressed together with histone H2B:mRFP as a nuclear marker in N. benthamiana
leaves via agro-infiltration. Protoplasts were prepared from the infiltrated leaves, and observed by
confocal microscopy. Chlorophyll autofluorescence was pseudo-colored blue. More than 20 cells
showing green fluorescence were observed for each construct. Scale bars = 10 µm.
(B) Subcellular fractionation. N. benthamiana leaf extracts expressing MRF:GFP proteins were
fractionated and subjected to SDS-PAGE using 10-15% gradient gel, followed by immunoblotting
with anti-GFP antibody. Total (T), nuclear (N), and cytosolic (C) fractions were indicated. Histone
H3 was detected as a nuclear marker protein using anti-H3 antibody. Two independent experiments
yielded similar results.
(C) Confocal microscopy of GFP fluorescence in epidermal cells of the Arabidopsis transgenic
plants that express each MRF gene under the CaMV35S promoter. Multiple independent transgenic
lines were analyzed for each MRF gene, which similarly suggested cytosolic localization of MRF
proteins. More than three independent observations were made for each transgenic line. Scale bars =
10 µm.
D WT
Ami-m
#3
Ami-m
#10
MRF1
OE #2
MRF1
OE #1
A
MRF1 amiRNA lines
(a, b, c)
MRF2 amiRNA lines
(d, e, f)
Flag:MRF1
overexpression lines
(OE #1, #2)
0
0.2
0.4
0.6
0.8
1.0
1.2
WT Ami-m#3
Ami-m#10
Ami-m#14
Rela
tive
mR
NA
le
vels
(M
RF
1 o
r M
RF
3/P
P2
AA
3) MRF1 MRF3
B
2256 2915 3009 2451
m
MRF1 (At5G63190.1)
1733 2278 2651
MRF2 (At1G22730.1)
MRF4 (At3G48390.1)
2398
MRF3 (At4G24800.1)
m
1547
Figure 3
Transgenic lines
MRF1, MRF3, and MRF4
multi-target amiRNA lines
(Ami-m #3, #10, #14)
m
Target sites for artificial miRNAs
m b a c
d e f
0
2
4
6
8
10
12
14
16
18
WT Ami-m#3
Ami-m#10
MRF1OE#1
MRF1OE#2
Leaf num
ber
(Rosett
e+
Caulin
e) Rosette CaulineE
0
5
10
15
20
25
30
35
40
WT MRF1OE #1
MRF1OE #2
Rela
tive m
RN
A levels
(M
RF
1/P
P2A
A3
)
C
Figure 3. Generation of MRF Artificial miRNA and MRF1 Overexpression Lines, and Analysis of
their Flowering Phenotypes.
(A) Description of MRF artificial miRNA (amiRNA) and MRF1 overexpression (OE) lines (left),
and target sites for the amiRNA lines (right). The target sites (arrowheads) were designed for
silencing of MRF1, MRF2, or multiple genes (MRF1, MRF3, and MRF4). “Ami-m” represents
amiRNA lines with multiple targets.
(B) RT-qPCR to determine MRF1 and MRF3 mRNA levels in the Ami-m lines. Transcript levels in
the Ami-m lines are expressed relative to those in the WT. Values represent the means ± S.E. of N =
three biological replicates of 10-day-old seedlings grown in different sets of liquid culture. Asterisks
denote statistical significance of the differences between the WT and the transgenic lines, calculated
using Student’s t-test (***, P ≤ 0.001).
(C) RT-qPCR to determine MRF1 mRNA levels in the MRF1 OE lines, compared with those in WT.
Error bars represent SE from triplicate biological replications using 10-day-old seedlings grown in
different sets of liquid culture (***, P ≤ 0.001).
(D) Flowering phenotypes of the Ami-m and MRF1 OE lines. Plants were grown for 4 weeks under
long-day conditions.
(E) Quantification of rosette and cauline leaf numbers at the bolting stage with the first open flower. Values represent
means SE of 40 plants per sample (***, P ≤ 0.001).
WT Ami-m (#3) Ami-m (#10) MRF1 OE (#1) MRF1 OE (#2) Ami-m (#14)
Da
rk+
Sta
rvation
(DS
)
+2d
Lig
ht+
Glu
cose
(Re
LG
)
+3d
+3d
+2d
A Figure 4
Seedlings
(CTL)
CAB2 (at ReLG+3)
SEN4 (at DS+5) E
C B
D Ami-m
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
CTL DS+2 DS+5 ReLG+3 ReLG+5
Rela
tive m
RN
A levels
(C
AB
2/P
P2A
A3)
WT #3 #10 #14 OE1 OE2
Ami-m
0
5
10
15
20
25
30
35
40
45
CTL DS+2 DS+5 ReLG+3 ReLG+5
Rela
tive m
RN
A levels
(S
EN
4/P
P2A
A3)
WT #3 #10 #14 OE1 OE2
Ami-m
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
WT #3 #10 #14 OE1 OE2
Rela
tive m
RN
A levels
(C
AB
2/P
P2A
A3)
Ami-m
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
WT #3 #10 #14 OE1 OE2
Rela
tive m
RN
A levels
(S
EN
4/P
P2A
A3)
CAB2
SEN4
Figure 4. Phenotypes and Gene Expression of the MRF Ami-m and MRF1 OE Lines after DS and
ReLG treatments.
(A) Seedlings at 12 days after germination were incubated in the dark/starvation (DS) for 5 days,
and then re-illuminated and fed with 30 mM glucose (ReLG) for 5 days. Photos were taken
periodically during the process. d, days.
(B, C) Time-course RT-qPCR analyses of CAB2 mRNA levels. Seedlings grown under LG
conditions were subjected to 2 and 5 days of DS treatment (DS+2 and DS+5), followed by 3 and 5
days of LG treatment (ReLG+3 and ReLG+5). RT-qPCR was performed for CAB2 mRNAs at the
indicated time points (B). The relative CAB2 transcript levels in different lines at ReLG+3 [boxed
with dotted line in (B)] were plotted (C). The transcript level is normalized by PP2AA3 mRNA, and
expressed relative to those in WT. Values represent the means ± S.E. of N = three biological
replicates of seedlings grown in different sets of liquid culture. Asterisks denote statistical
significance of the differences between WT and the transgenic lines, calculated using Student’s t-
test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
(D, E) Time-course RT-qPCR analyses of SEN4 mRNA levels. The SEN4 transcript levels at the
indicated time points are shown (D), and the values at DS+5 [boxed with dotted line in (D)] were
plotted (E).
A B Flag:MRF1
eIF4A-1:Myc
IB : α-Flag
IB : α-Myc
INPUT IP : α-Myc
Flag:MRF3
eIF4A-1:Myc
IB : α-Flag
IB : α-Myc
INPUT IP : α-Myc
Flag:MRF2
eIF4A-1:Myc
IB : α-Flag
IB : α-Myc
INPUT IP : α-Myc
C
Figure 5
eIF4A-1:YFPC
YF
PN:G
le1
eIF4E-1:YFPC
MR
F1:Y
FP
NM
RF
2:Y
FP
NM
RF
3:Y
FP
NM
RF
4:Y
FP
N
YFPN / YFPC
0
10
20
30
40
50
60
70
80
90
100
AD:eIF4A-1BD
AD:eIF4A-1BD:MRF1
AD:eIF4A-1BD:MRF2
AD:eIF4A-1BD:MRF3
AD:eIF4A-1BD:MRF4
α-G
ala
cto
sid
ase a
ctivity (
un
it)
Flag:MRF4
eIF4A-1:Myc
IB : α-Flag
IB : α-Myc
INPUT IP : α-Myc
kDa
75
100
kDa
75
75
60
75
60
100
kDa
75
75
60
100
kDa
75
75
60
+ +
− ++ +
− +
+ +
− ++ +
− +
+ +
− ++ +
− +
+ +
− ++ +
− +
Figure 5. Interactions between MRFs and Eukaryotic Translation Initiation Factor 4A.
(A) Bimolecular fluorescence complementation (BiFC). YFPN- and YFPC-fusion proteins were co-
expressed in N. benthamiana leaves by agroinfiltration. Leaf epidermal cells were observed by
confocal microscopy. More than 20 leaf cells showing yellow fluorescence were observed for each
BiFC experiment. Bars = 20 µm.
(B) Co-immunoprecipitation. Each MRF protein in Flag fusion (Flag:MRF) was expressed alone or
together with eIF4A-1:Myc in N. benthamiana leaves. Total leaf proteins were immunoprecipitated
with anti-Myc antibody-conjugated resin, and the co-immunoprecipitate was detected using the anti-
Flag antibody.
(C) Yeast two-hybrid assay. GAL4 activation domain (AD)-fused to eIF4A-1 and GAL4 DNA
binding domain (BD)-fused MRF proteins were co-expressed in yeast. Alpha-galactosidase activity
indicates protein-protein interaction affinity. Error bars represent SE from triplicate biological
replications using three individual colonies. Asterisks denote the statistical significance of the
differences between the control (AD:eIF4A-1/BD vector) and other samples (*, P ≤ 0.05; **, P ≤
0.01).
Figure 6
15% 50%
A Sucrose gradient
MRF4:GFP
eIF4A-1:Myc
eIF4E-1:Myc
MRF3:GFP
MRF1:GFP
MRF2:GFP α-GFP
α-Myc
RPL10a α-L10a
60S/80S Polysomes
C
CspA
Vector
MRF1
MRF2
MRF3
MRF4
LOS4
37oC / 0.1 mM IPTG
10-1 10-2 10-3 10-4 10-5
10-1 10-2 10-3 10-4 10-5
18oC / 0.1 mM IPTG
E. coli BX04 mutant strain
CspA
Vector
MRF1
MRF2
MRF3
MRF4
LOS4
B
Flag:MRF1
(α-Flag)
α-L10a α-L10a
Flag:MRF1
(α-Flag)
2 3 4 5 6 7 8 9 10 11 12 13 14 2 3 4 5 6 7 8 9 10 11 12 13 14
0.080
0.100
0.120
0.140
0.160
0.080
0.100
0.120
0.140
0.160
15% 50% 15% 50%
Control (+) RNase A
OD
25
4
OD
25
4
polysome
60S 80S
60S/80S
100
kDa
100
100
100
75
45
35
75
60
35
25
100
75
kDa kDa
35
25
35
25
100
75
Figure 6. Co-Sedimentation of MRFs with Ribosomes and BX04 Complementation Assays.
(A) Co-fractionation of MRFs with ribosome subunits and translation initiation factors. MRFs:GFP,
eIF4E-1:Myc, and eIF4A-1:Myc were expressed in N. benthamiana leaves. After sucrose density
gradient sedimentation, the fractions were subjected to immunoblotting with anti-GFP, anti-Myc,
and anti-60S ribosomal protein L10a (RPL10a) antibodies. Lanes indicated the fractions from top
(15%) to bottom (50%).
(B) Distribution of MRFs in sucrose gradient fractions after RNAse A treatment. Total cell extract
was prepared from Flag-MRF1 OE seedlings (#1) grown under light/glucose conditions. The cell
extract was treated with 1 mg/ml of RNase A on ice for 15 min (+RNAse A) or with RNase-free
water (control), prior to sucrose density gradient sedimentation (15%-50%). The UV absorbance at
254 nm was monitored for gradient fractions to produce the absorbance profiles (top). The collected
fractions were subjected to immunoblotting with anti-Flag and anti-L10a antibodies (bottom).
(C) BX04 complementation assays. The E.coli BX04 strain is a quadruple mutant of cold-shock
proteins, which cannot grow at low temperature. The BX04 strain was transformed with plasmids
carrying MRFs, E. coli CspA (cold-shock protein; positive control), Arabidopsis LOS4 (RNA
helicase), and vector control. The transformants were grown overnight, and then serially diluted and
spotted onto media plates. The plates were incubated at 37 °C (left) and 18 °C (right).
Figure 7
Ami-m
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
WT #3 #10 #14 OE1 OE2
Rela
tive b
and
in
ten
sity
(Auto
rad
iogra
ph/C
BB
)
B
C
0.075
0.090
0.105
0.120
0.135
LG (2 h) DS (1 d)
40S
60S/80S
60S 80S
P/NP = 1.3±0.1
P/NP = 4.0±0.1
WT
0.075
0.090
0.105
0.120
0.135
LG (2 h) DS (1 d)
P/NP = 4.1±0.3
P/NP = 1.6±0.1
MRF Ami-m (#3)
0.075
0.090
0.105
0.120
0.135
LG (2 h) DS (1 d)
P/NP = 1.1±0.1
P/NP = 3.9±0.3
MRF1 OE (#1)
OD
25
4
polysome
15% 50% 15% 50% 15% 50%
A Dark / Starvation (3 h)
D
0
20
40
60
80
100
120
140
160
CTL DS+2 DS+5 ReLG+3 ReLG+5
Tota
l R
NA
co
nte
nts
(μ
g/g
FW
)
WT Ami-m #3 Ami-m #10 Ami-m #14 OE #1 OE #2
***
**
* *
*
*
*
*
** **
** **
Dark / Starvation (3 h)
35S
-Meth
ionin
e
Auto
rad
iogra
ph
C
BB
25
35
45
60
75
100
25
35
45
60
75
100
kDa
Figure 7. 35S-Methionine Labeling under Dark/Starvation Conditions, Polysome Analyses, and
Total RNA Contents
(A) Autoradiography images of 35S-Met incorporation. Seedlings grown in liquid culture were pre-
incubated without glucose in the dark for 30 min, followed by 35S-Met labeling for 2.5 h under the
same conditions. After SDS-PAGE of protein extracts from the labeled seedlings, the gel was
stained with Coomassie brilliant blue (CBB) and dried. The radioactive signal within the gel was
detected by a phosphorimager.
(B) Relative band intensity. The radioactive intensity of 35S-Met-labeled proteins was normalized by
CBB band intensity, and the ratio was expressed relative to the WT. Error bars represent SE from
four biological replications based on four independent experiments (*, P ≤ 0.05; **, P ≤ 0.01).
(C) Polysome analyses. Seedlings were incubated with LG for 2 h or DS for 1 day (1 d). The
seedlings were treated with cycloheximide (50 μg/ml) for 5 min before harvest, and total cell
extracts from the seedlings were subjected to sucrose density gradient sedimentation (15%-50%).
The UV absorbance at 254 nm was monitored for gradient fractions to produce the absorbance
profiles. The absorbance profiles of LG (blue lines) and DS (red lines) samples were superimposed
for comparison. An average ratio (P/NP) of polysomes to 60S/80S ribosomes was calculated for
each sample using Image J program, from four biological replications based on four independent
experiments.
(D) Total RNA contents after prolonged DS and ReLG treatments. Seedlings grown under LG
conditions (control; CTL) were subjected to 2 and 5 days of DS treatment (DS+2 and DS+5),
followed by 3 and 5 days of LG treatment (ReLG+3 and ReLG+5) as described in Figure 3A. Total
RNA was extracted from an equal weight of the seedlings at each stage, and measured by
absorbance at 260 nm using a spectrophotometer. Error bars represent SE from triplicate biological
replications using seedlings grown in different sets of liquid culture. Asterisks denote statistical
significance of the differences between WT and the transgenic lines, calculated using Student’s t-
test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
PP2AA3 GAPC HDA19 HDC1
WT(LG)
Ami3(LG)
WT(DS)
Ami3(DS)
WT(LG)
Ami3(LG)
WT(DS)
Ami3(DS)
WT(LG)
Ami3(LG)
WT(DS)
Ami3(DS)
WT(LG)
Ami3(LG)
WT(DS)
Ami3(DS)
B
WT
Dark / Starvation (1 d) Light / Glucose (2 h)
WT Ami-m (#3) PP2AA3
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
GAPC
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
HDA19
HDC1 1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
1 3 5 7 9 11 13 15 1 3 5 7 9 11 13 15
A Figure 8
Ami-m (#3)
Polysome (P) Nonpolysome (NP)
*** **
*
% m
RN
A
% m
RN
A
% m
RN
A
% m
RN
A
% o
f to
tal
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
20
15
10
5
0
25
20
15
10
5
0
25
20
15
10
5
0
30
25
20
15
10
5
0
30
25
20
15
10
5
0
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
100
75
50
25
0
Figure 8. Distribution Patterns and Quantifications of Specific mRNAs in Sucrose Gradient
Fractions.
(A) Distribution of PP2AA3, GAPC, HDA19, and HDC1 mRNAs in sucrose gradient fractions. WT
and Ami-m (#3) seedlings were incubated under LG conditions for 2 h (left) or under DS conditions
for 1 d (right). Total cell extracts prepared from the seedlings were subjected to sucrose density
gradient sedimentation (15%-50%), and total 15 fractions were collected from each tube. Total RNA
was extracted from each fraction, followed by cDNA synthesis and real-time qRT-PCR using gene-
specific primers. The abundance of mRNA in each fraction was quantified as a percentage of their
total amount in all fractions. Similar results were obtained in three independent experiments, and a
representative result is shown. Error bars represent SE from three technical replications.
(B) The abundance of mRNA in polysomal (P; fractions 9-15) and nonpolysomal fractions (NP;
fractions 1-8), quantified as a percentage of their total amount. Ami3 represents Ami-m (#3). Error
bars represent SE from three biological replications based on three independent experiments (*, P ≤
0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
Figure 9
B CTL 1 2 4 24 48 0.5 1 2 (h)
Phostag
α-Flag
α-Flag
CBB
Dark + Starvation Light + Glucose
Flag:MRF1
MRF4 :Myc
A
λPP
MRF1 :Myc
MRF2 :Myc
MRF3 :Myc
Phostag
α-Myc
α-Myc
*
C Dark + Starvation (24 h) Control Light + Glucose (1 h)
10% 55% 10% 55% 10% 55%
D
0.075
0.085
0.095
0.105
0.115
0.125
0.135
0.075
0.085
0.095
0.105
0.115
0.125
0.135
0.075
0.085
0.095
0.105
0.115
0.125
0.135
INPUT IP : α-Myc
α-Flag
α-Myc
Flag:MRF1
eIF4A-1:Myc
LG DS LG DS LG DS LG DS
OD
25
4
2 3 4 5 6 7 8 9 10 11 12 13 14
Flag:
MRF1
(α-Flag)
RPL10a
2 3 4 5 6 7 8 9 10 11 12 13 14 2 3 4 5 6 7 8 9 10 11 12 13 14
100
kDa
100
75
kDa
60
45
kDa
35
25
100
75
kDa
35
25
100
75
kDa
35
25
100
75
100
kDa
75
75
60
+ + + +
− − + +
+ + + +
− − + +
− + − + − + − +
Figure 9. MRF1 Phosphorylation and Ribosome Association According to Cellular Energy
Availability.
(A) Phosphorylation of MRF1 in vivo. Total protein extracts from N. benthamiana leaves, which
express MRF:Myc proteins, were treated with the lambda phosphatase (λPP). After treatment, the
samples were subjected to Zn2+-Phostag SDS-PAGE (top) and to normal SDS-PAGE (bottom) for
immunoblotting with anti-Myc antibody. The phosphorylated form of MRF1 was marked with the
asterisk.
(B) Phosphorylation of MRF1 under different energy conditions. Flag:MRF1 OE (#1) seedlings
were incubated under dark/starvation conditions for 1-48 h, and then re-illuminated and fed with 30
mM glucose (light/glucose) for 0.5-2 h. Protein extracts from the seedlings harvested at different
time points were separated by Zn2+-Phostag SDS-PAGE (top) and by normal SDS-PAGE (middle),
followed by immunoblotting with anti-Flag antibody. The Rubisco large subunit was stained with
CBB as loading control (bottom).
(C) Distribution of Flag:MRF1 in sucrose gradient fractions of Flag:MRF1 under different energy
conditions. Flag:MRF1 OE (#1) seedlings were incubated in the dark/starvation for 24 h, followed
by re-illumination and glucose-feeding for 1 h. Polysome analysis was performed by
ultracentrifugation through a 10-55% sucrose gradient. Then the fractions were precipitated and
analyzed by immunoblotting with anti-Flag and anti-RPL10a antibodies. Lanes indicate the
fractions from top (10%) to bottom (55%). Arrowheads indicate the final positions of MRF1
detection.
(D) Co-immunoprecipitation. Flag:MRF1 was expressed alone or together with eIF4A-1:Myc in N.
benthamiana leaves. Leaf disks were prepared for treatment with light/glucose (LG) or
dark/starvation (DS) for 3 h. Total leaf proteins were immunoprecipitated with anti-Myc antibody-
conjugated resin, and the co-immunoprecipitate was detected using the anti-Flag antibody.
TRV TOR VIGS
Phostag
α-Flag
α-Flag
CBB
B
Flag:MRF1
D
DM
SO
WT #3 #10 #14 OE#1 OE#2
Tori
n-1
(2 µ
M)
E Ami-m
C
TRV
control
TOR
VIGS
2 3 4 5 6 7 8 9 10 11 12 13 14
α-Flag
10% 55%
0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
DMSO(3 days)
DMSO(5 days)
Torin-1(3 days)
Torin-1(5 days)
Ro
ot le
ng
th (
cm
)
WT #3 #10 #14 OE1 OE2
Ami-m
100
75
kDa
60
45
100
kDa
75
100
75
A MRF1 MRF2 MRF3 MRF4
0
0.5
1.0
1.5
2.0
Stv
Glc
Ma
nS
uc
Stv
Glc
Ma
nS
uc
0
0.5
1.0
1.5
2.0
Stv
Glc
Ma
nS
uc
Stv
Glc
Ma
nS
uc
0
0.5
1.0
1.5
2.0
Stv
Glc
Ma
nS
uc
Stv
Glc
Ma
nS
uc
es-tor1
(RNAi) :
0
0.5
1.0
1.5
2.0
Stv
Glc
Man
Su
c
Stv
Glc
Man
Su
c
Rela
tive
mR
NA
le
vels
−ES +ES −ES +ES −ES +ES −ES +ES
Figure 10
25
25
α-Flag
α-RPL10a
α-RPL10a
Flag:MRF1
Figure 10. TOR-Modulated MRF Gene Expression and MRF1 Phosphorylation, and Seedling
Phenotypes upon Torin-1 Treatment.
(A) Altered MRF gene expression in TOR-silenced seedlings in response to starvation and sugar
feeding. Estradiol-inducible TOR RNAi seedlings (es-tor1) were treated with ethanol (-ES) or 10
μM estradiol (+ES) for gene silencing. Twelve seedlings grown in three different sets of liquid
culture were incubated in glucose-free medium for 24 h (Stv), and then fed with 30 mM glucose
(Glc), mannitol (Man), and sucrose (Suc) for 4 h. RT-qPCR was performed with gene-specific
primers. Transcript levels are normalized by PP2AA3 mRNA, and expressed relative to those of Stv
samples. Error bars represent SE from triplicate technical replications.
(B) MRF1 phosphorylation in TOR-silenced plants. TOR VIGS was performed in Flag:MRF1 OE
(#1) lines. Protein extracts from TRV control or TOR VIGS leaves (10 DAI) were separated by
Phostag SDS-PAGE (top) and by normal SDS-PAGE (middle), followed by immunoblotting with
anti-Flag antibody. The Rubisco large subunit was stained with CBB as loading control (bottom).
(C) Ribosome association of Flag:MRF1 in TOR-silenced plants. TOR VIGS was performed in
Flag:MRF1 OE (#1) lines. Protein extracts from TRV control and TOR VIGS leaves (10 DAI) were
fractionated by sucrose density gradient sedimentation (10-55%). The fractions were precipitated
and analyzed by immunoblotting with anti-Flag and anti-RPL10a antibodies. Lanes indicate the
fractions from top (10%) to bottom (55%). Arrowheads indicate the final positions of MRF1
detection.
(D) Phenotypes of the Ami-m and MRF1 OE seedlings after Torin-1 treatment. Seven-day-old
seedlings grown in liquid culture were treated with Torin-1 (2 μM) or control DMSO for 3 days.
(E) Root length of the seedlings was measured after treatment with Torin-1 (2 μM) or control
DMSO for 3 and 5 days. Each data point represents the mean SE (n > 14 seedlings). Asterisks
denote statistical significance of the differences between Torin-1-treated samples and DMSO-treated
samples, calculated using Student’s t-test (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
B
CBB
Kinase assay
α-Myc
MBP
MBP:MRF1
MBP:MRF2
MBP:MRF3
MBP:MRF4
[IP-Myc] S6K2:Myc
+
−
−
−
−
+
−
+
−
−
−
+
−
−
+
−
−
+
−
−
−
+
−
+
−
−
−
−
+
+
MBP:MRF1
[IP-Myc] S6K1:Myc
[IP-Myc] S6K1(T449A):Myc
[IP-Myc] S6K1(T449D):Myc
+
−
−
−
+
+
−
−
+
−
+
−
+
−
−
+
Kinase assay
CBB
α-Myc
C
A MBP
MBP:MRF1
MBP:MRF2
MBP:MRF3
MBP:MRF4
[IP-Myc] S6K1:Myc
+ − − − − +
−
+
−
−
−
+
−
−
+
−
−
+
−
−
−
+
−
+
−
−
−
−
+
+
CBB
Kinase assay
α-Myc
Figure 11
D
S6K
1:Y
FP
C
MRF1:YFPN MRF1:YFPN
S6K
2:Y
FP
C
INPUT IP : α-Myc
IB : Myc
IB : Flag
Flag:MTR1
S6K2:Myc
F
IB : Myc
INPUT IP : Myc
IB : Flag
Flag:MTR1
S6K1:Myc
E
140
100
kDa
140
100 100
75
140
100
kDa
140
100 100
75
140
100
kDa
140
100
100
75
100
75
kDa
100
75
100
75
kDa
100
75
+ +
− +
+ +
− +
+ +
− +
+ +
− +
Figure 11. In Vitro Phosphorylation of MRF1 by S6K kinases.
(A) In vitro kinase assay of immunoprecipitated S6K1:Myc with the recombinant MBP:MRF
proteins as substrates. After the kinase assay with [γ-32P]-ATP, SDS-PAGE was performed.
Phosphorylated MBP:MRF proteins were detected by a phosphorimager (top); the MBP:MRF
protein in the reaction was detected by CBB staining (middle); immunoprecipitated S6K1:Myc was
detected by immunoblotting with anti-Myc antibody (bottom).
(B) In vitro kinase assay of immunoprecipitated S6K2:Myc with the recombinant MBP:MRF
proteins as substrates.
(C) In vitro kinase assay with S6K1 mutant forms that carry a mutation in the TOR phosphorylation
site T449. In vitro kinase assay was performed with S6K1:Myc, S6K1(T449A):Myc, and
S6K1(T449D):Myc proteins.
(D) BiFC analyses for MRF1 interactions with S6K1 and S6K2. MRF1:YFPN was expressed
together with S6K1:YFPC or S6K2:YFPC in N. benthamiana leaves using agroinfiltration. Leaf
epidermal cells were observed by confocal microscopy. More than 20 leaf cells showing yellow
fluorescence were observed for each BiFC experiment. As a negative control, MRF1:YFPN and
eIF4E-1:YFPC were co-expressed in N. benthamiana leaves, which resulted in little yellow
fluorescence. Bars = 20 µm.
(E), (F) Co-immunoprecipitation of MRF1 with S6K1 and S6K2. Flag:MRF1 was expressed alone
or together with S6K1:Myc (E) or S6K2:Myc (F) in N. benthamiana leaves. Total leaf proteins were
immunoprecipitated with anti-Myc antibody-conjugated resin, and the co-immunoprecipitate was
detected using the anti-Flag antibody.
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DOI 10.1105/tpc.17.00563; originally published online October 30, 2017;Plant Cell
Du-Hwa Lee, Seung Jun Park, Chang Sook Ahn and Hyun-Sook PaiConditions, and Their Expression and Functions Are Modulated by the TOR Signaling Pathway
MRF Family Genes Are Involved in Protein Translation Control, Especially under Energy-Deficient
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