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Running title: Characterization of Maize rea1 mutant 1
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To whom all the correspondence should be sent: 4
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Rentao Song 6
Address: Shanghai Key Laboratory of Bio-Energy Crops, School of Life 7
Sciences, Shanghai University, 333 Nanchen Road, Shanghai 200444, China 8
Telephone: 86-21-66135182 9
E-mail: [email protected] 10
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Research Area: Genes, Development and Evolution 13
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Plant Physiology Preview. Published on December 8, 2015, as DOI:10.1104/pp.15.01722
Copyright 2015 by the American Society of Plant Biologists
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Title: Maize rea1 mutant stimulates ribosome use efficiency and triggers 35
distinct transcriptional and translational responses 36
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Weiwei Qi 1ab, Jie Zhu 1a, Qiao Wu 1a, Qun Wang a, Xia Li a, Dongsheng Yao a, 38
Ying Jin a, Gang Wang ab, Guifeng Wang ab, and Rentao Song 2ab 39
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1 These authors contributed equally to this study 41
2 Address for correspondence to [email protected] 42
a Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, 43
Shanghai University, Shanghai 200444, China 44
b Coordinated Crop Biology Research Center (CBRC), Beijing 100193, China 45
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Summary: 48
Impaired ribosome biogenesis enhances ribosome use efficiency, triggers 49
distinct transcriptional and translational cellular responses, and affects cell 50
growth and proliferation. 51
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This work was supported by the Major Research plan of the National Natural 68
Sciences Foundation of China (91335208 and 31425019), and the Ministry of 69
Science and Technology of China (2014CB138204). 70
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The author responsible for distribution of materials integral to the findings 73
presented in this article in accordance with the policy described in the 74
Instructions for Authors (www.plantphysiol.org) is: 75
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Maize rea1 mutant stimulates ribosome use efficiency and 98
triggers distinct transcriptional and translational responses 99
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Abstract 101
Ribosome biogenesis is a fundamental cellular process in all cells. Impaired 102
ribosome biogenesis causes developmental defects; however, its molecular 103
and cellular basis is not fully understood. We cloned a gene responsible for a 104
maize small seed mutant dek*, and found it encodes Ribosome export 105
associated 1 (ZmRea1). Rea1 is an AAA-ATPase that controls 60S ribosome 106
export from the nucleus to the cytoplasm after ribosome maturation. dek* is a 107
weak mutant allele with decreased Rea1 function. In dek* cells, mature 60S 108
ribosome subunits are reduced in the nucleus and cytoplasm, but the 109
proportion of actively translating polyribosomes in cytosol is significantly 110
increased. Reduced phosphorylation of eIF2α and the increased eEF1α level 111
indicate an enhancement of general translational efficiency in dek* cells. The 112
mutation also triggers dramatic changes in differentially transcribed genes 113
(DTGs) and differentially translated RNAs (DTRs). Discrepancy was observed 114
between DTGs and DTRs, indicating distinct cellular responses at transcription 115
and translation levels to the stress of defective ribosome processing. DNA 116
replication and nucleosome assembly related gene expression are selectively 117
suppressed at translational level, resulting in inhibited cell growth and 118
proliferation in dek* cells. This study provides insight into cellular responses 119
due to impaired ribosome biogenesis. 120
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Introduction 131
Ribosomes are organelles that translate genetic information into proteins. A 132
great percentage of total RNA transcription is devoted to ribosomal RNA 133
synthesis, and a great part of RNA polymerase Ⅱ transcription and mRNA 134
splicing are devoted to synthesis of ribosomal proteins (Warner, 1999). 135
Ribosome biosynthesis consumes ~80% of a cell's energy (James et al., 2014). 136
In eukaryotes, ribosome biogenesis begins in the nucleolus with the 137
transcription of a large ribosomal precursor RNA that gives rise to the 90S 138
pre-ribosomal particle. Cleavages of the 90S particle generate two subunits: 139
the pre-40S and pre-60S complexes. The pre-40S and pre-60S subunits 140
mature in the nucleolus and nucleoplasm before being exported to the 141
cytoplasm (Venema and Tollervey, 1999; Fromont-Racine et al., 2003; 142
Granneman and Baserga, 2004). Inhibition of ribosome biogenesis causes 143
developmental defects in yeast, humans and plants (Tschochner and Hurt, 144
2003; Galani et al., 2004; Ruan et al., 2012; Weis et al., 2014; Brooks et al., 145
2014). 146
A great deal of research has revealed that hundreds of 147
ribosomal-biogenesis factors contribute to maturation of the ribosome in 148
eukaryotes (Tschochner and Hurt, 2003; Henras et al., 2008), including three 149
essential AAA-ATPases: Ribosome export (Rix) 7, Ribosome export 150
associated (Rea) 1, and Diazaborine resistance gene (Drg) 1 (Pertschy et al., 151
2007; Kressler et al., 2008; Ulbrich et al., 2009; Bassler et al., 2010; Kressler 152
et al., 2012). Rea1 AAA-ATPase is the best-characterized ATPase in ribosome 153
biogenesis and is conserved from yeast to humans (Bassler et al., 2010; 154
Kressler et al., 2012). Rea1 promotes stripping of other biogenesis factors 155
from the pre-60S particle in the nucleolus and nucleoplasm (Ytm1-Erb1-Nop7 156
and Rsa4) prior to the export of the large ribosomal subunit to the cytoplasm 157
(Bassler et al., 2010). However, there is not a comprehensive understanding of 158
cellular responses to the impaired large ribosomal subunit export. 159
The regulation of mRNA translation is a critical feature of gene expression 160
in eukaryotes (Bailey-Serres, 1999). Previous studies highlight the importance 161
of translational control in determining protein abundance, underscoring the 162
value of measuring gene expression at the level of translation. Mechanisms 163
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that underlie differential mRNA translation are likely to involve nucleotide 164
sequence features and the phosphorylation status of initiation factors 165
(Bailey-Serres and Dawe, 1996; Pop et al., 2014). Transcriptome and 166
translatome analyses of the cellular response to heat shock, cell-cycle arrest 167
and mating pheromone in Saccharomyces cerevisiae (Preiss et al., 2003; 168
Serikawa et al., 2003; MacKay et al., 2004), the hypoxia response of HeLa 169
cells (Blais et al., 2004), and the drought and oxygen deprivation responses in 170
Arabidopsis (Kawaguchi et al., 2004; Branco-Price et al., 2005) have shown 171
the importance of translational regulation. These researchers investigated the 172
correlation between total and poly-ribosome (polysome) -bound mRNA 173
accumulation and provided extensive evidence of variation in the translational 174
regulation of individual mRNAs. These studies showed mRNAs differ in their 175
association with polysomes under different circumstances, and gene 176
expression can be regulated at the translational level without a change in 177
mRNA abundance. 178
Maize (Zea mays) is especially well suited for genetic studies, partly 179
because of the feasibility to generate a wide range of easily observable 180
phenotypes (Neuffer and Sheridan, 1980). Many kernel mutants are known 181
(Neuffer et al., 1968), among which one class is defective kernel (dek) mutants 182
(Neuffer and Sheridan, 1980). dek mutants are good resource to investigate 183
seed development. For example, Dek1 encodes a large membrane protein of 184
the calpain gene superfamily (Lid et al., 2002). In dek1 mutants, 185
embryogenesis is blocked, while the endosperm lacks the aleurone layer and 186
is chalky (Becraft et al., 2002). Other dek mutants offer opportunities to 187
investigate many basic biological processes, because embryo formation is the 188
first developmental process after the fertilization. Such defects in basic 189
biological processes create visible phenotypes during kernel development. 190
In this study, we characterized dek*, a novel mutant with small kernels, 191
and delayed developmental of the embryo, endosperm and seedling. We 192
report the map-based cloning of Dek* and demonstrate it encodes Rea1 in 193
maize. dek* is a weak mutant allele that only partly represses the maturation 194
and export of the 60S ribosomal subunit. Taking advantage of this mutant allele, 195
we were able to obtain comprehensive information about the cellular 196
responses to impaired 60S ribosomal subunit biogenesis. 197
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Results 199
dek* produces small kernels with delayed development 200
The dek* mutant was isolated from an opaque mutant stock obtained from the 201
Maize Genetic Stock Center. It was crossed to the W64A inbred line to 202
produce a F2 population that displayed a 1:3 segregation of dek (dek*/ dek*) 203
and wild type (+/+ and dek*/+) phenotypes (Figure 1A&B). At 15 days after 204
pollination (DAP), homozygous dek* kernels exhibited a small, vague 205
phenotype (Figure 1A), and mature kernels were small and shrunken (Figure 206
1B). The 100-kernel weight of dek* was nearly 39.5% less than wild type 207
(Figure 1C), but there was no significant difference in the total protein and zein 208
content (Figure 1D, Supplemental Figure 1), although, there was a slight 209
increase in the amount of non-zeins (13.5%, Figure 1D). Among zein proteins, 210
the 22kD α-zeins were relatively more abundant in dek* endosperms 211
(Supplemental Figure 1). We found no obvious difference in total starch 212
content and the percentage of amylose in dek* and wild type endosperms 213
(Supplemental Figure 2). We analyzed soluble amino acids to determine if the 214
slight increase of non-zeins in dek* altered their composition. The results 215
showed that the amount of lysine was most significantly increased (23.1%) 216
might due to the slight increase of non-zein content (Figure 1E), for zeins lack 217
lysine residues (Mertz et al., 1964). 218
Wild type and dek* kernels of 15 DAP and 18 DAP were analyzed by light 219
microscopy to compare their development. Longitudinal sections of the 220
embryos indicated development of the plumule and seminal was delayed more 221
than three days in dek* compared to wild type (Figure 1F). To investigate the 222
endosperm development, we observed 15 DAP and 18 DAP immature 223
endosperm cells using optical microscopy. The endosperm cells of dek* 224
kernels were less cytoplasmic dense with fewer starch granules compared to 225
the wild type of the same stage, also indicating more than a three day delay in 226
development (Figure 1G). 227
At 4 and 7 days after germination (DAG), seedlings of dek* showed a 228
three day developmental delay compared to wild type (Figure 1H). By 4 DAG, 229
wild type seedlings had two leaves, one completely expanded and the other 230
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emerging; the dek* seedlings had only one leaf at this stage. The 7 DAG wild 231
type seedlings had three leaves, while the dek* seedlings had only two leaves. 232
The heading stage of the dek* plant was delayed approximately fifteen days 233
compared to wild type, and its height was only 50% of the wild type 234
(Supplemental Figure 3). These results demonstrated that the growth and 235
development of dek* kernels and seedlings is delayed compared to wild type. 236
237
Positional cloning of Dek* 238
Genetic fine mapping of Dek* was carried out with the F2 mapping population 239
and the Dek* gene was placed between the simple sequence repeat (SSR) 240
markers, mmc0241 and umc2162, on the long arm of chromosome 6 (Figure 241
2A). After characterizing a mapping population of 864 individuals, Dek* was 242
mapped between the self-created SSR markers 153.7M-2 (19 recombinants) 243
and 155.1M-1 (29 recombinants). Additional markers InDel438, InDel428, 244
SNP064 and SNP165 were developed, and Dek* gene was eventually placed 245
between SNP064 (1 recombinant) and SNP165 (2 recombinants), a region 246
encompassing a physical distance of 101.6kb (Figure 2A). 247
Nucleotide sequence analysis within this region identified ten predicted 248
open reading frames (ORFs) with gene model information (GRMZM2G405052, 249
GRMZM2G387038, GRMZM5G873561, GRMZM5G807823, 250
GRMZM2G361064, GRMZM5G892685, GRMZM2G059268, 251
GRMZM2G059278, GRMZM2G323939 and GRMZM2G128315). Expression 252
analysis revealed no expression of GRMZM5G892685, GRMZM2G059268 253
and GRMZM2G059278 based on reverse transcription (RT)-PCR and 254
expressed sequence tag (EST) information (http://www.maizegdb.org/); 255
consequently, these three might be pseudogenes. DNA sequence analysis 256
revealed GRMZM2G405052, GRMZM2G387038, GRMZM5G873561, 257
GRMZM5G807823, together with GRMZM2G092001 and GRMZM2G149586 258
which are up-stream of the candidate region, produced one huge transcript 259
that was identified as candidate Gene1. There is a single nucleotide 260
polymorphism in Gene1 resulting in an amino acid replacement between the 261
alleles of dek* and wild type. GRMZM2G361064, GRMZM2G323939 and 262
GRMZM2G128315 were identified as candidate Gene2, Gene3 and Gene4, 263
respectively; however, their consideration for Dek* was eliminated due to no 264
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sequence differences between alleles in dek* and wild type (Figure 2A). 265
Therefore, Gene1 appeared to be the best candidate for the Dek* locus. 266
267
Dek* encodes the 60S-specific ribosome biogenesis factor Rea1 268
The genomic DNA sequence of candidate Gene1 spans ~50 kb and produces 269
a huge transcript containing a 16,278 bp coding sequence (Figure 2B). 270
Sequence data for this gene has been deposited in GenBank 271
(http://www.ncbi.nlm.nih.gov/ ) as accession number KP137367. Gene1 272
encodes a ~600 kD protein of 5,425 amino acids. BLASTP searches of 273
Genbank indicated that Gene1 encodes a ribosome biogenesis factor, 274
AAA-ATPase Rea1, with several conserved domains in maize (Figure 2B). 275
ZmRea1 contains different kinds of molecular domains: a weakly conserved 276
N-terminal region, a dynein-like tandem array of six AAA-type ATPase domains 277
(Neuwald et al., 1999), a large linker, a D/E rich region and a metal ion 278
dependent adhesion site (MIDAS) domain (Figure 2B). Rea1 promotes release 279
of Ytm1 which associates with nucleolar pre-60S particles, and later also 280
promotes release of Rsa4 associates with nucleoplasmic pre-60S particles via 281
the MIDAS-MIDAS interacting domain (MIDO) using the mechanical force 282
created by the ATPase ring domain for the export of the large ribosomal 283
subunit to the cytoplasm (Ulbrich et al., 2009; Bassler et al., 2010). The 284
mutation in the dek* allele of ZmRea1 is a single nucleotide polymorphism in 285
the 2,359th codon of ZmRea1, which results in Ala (GCC) replaced by Val 286
(GTC; Figure 2B). This mutation alters the highly conserved region between 287
the dynein-like array of six AAA-type ATPases and the large linker, which could 288
affect transduction of the mechanical force created by the ATPase ring domain 289
to the large tail for release of the ribosome biogenesis factors. 290
To confirm if ZmRea1 is the Dek* gene, we carried out an allelism test 291
with a Mu induced mutant of ZmRea1 (Figure 2B&C). A UniformMu insertion 292
mutant (rea1-Mu) stock for GRMZM2G092001 was obtained from the Maize 293
Genetics Stock Center. This mutant has a Mutator-8 insertion after the 4th 294
nucleotide of the ZmRea1 coding sequence and is not viable (Figure 2B). The 295
allelism test was done by crossing dek* F1 (dek*/+) and rea1-Mu F1 (rea1-Mu 296
/+). The kernel phenotypes in the F2 ears displayed a 1:3 segregation of 82 297
dek (dek*/rea1-Mu) and 249 wild type phenotype kernels (Figure 2C), 298
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indicating that rea1-Mu can’t complement dek*. Therefore, Gene1 (ZmRea1) is 299
indeed the Dek* gene. We hence named dek* as rea1-ref. 300
Maize endosperm is a triploid tissue with 2 maternal and 1 paternal 301
genomes. The mutant kernels in rea1-ref/+ (maternal) X rea1-Mu/+ (paternal) 302
F2 ear are small and shallow, similar to homozygous rea1-ref kernels. The 303
mutant kernels in rea1-Mu/+ (maternal) X rea1-ref /+ (paternal) F2 ears display 304
an even more severe phenotype with dramatically shrunken kernels. The 305
mutant kernels from rea1-Mu/+ selfing are non-viable (Figure 2C). Thus, 306
results of the allelism test show rea1-ref is a weak allele compared to rea1-Mu, 307
which has a lethal phenotype. 308
To examine Rea1 mRNA expression in rea1-ref, we performed 309
quantitative RT-PCR (qRT-PCR) with the total RNA extracted from 15 and 18 310
DAP mutant and wild type kernels. Surprisingly, mRNA expression of Rea1 311
was significantly up-regulated in rea1-ref (Figure 2D). Because ZmRea1 is too 312
large (~600kD) to perform a regular western blot analysis, we used 313
dot-immunoblot analysis on quantified and gradient diluted total protein 314
samples with Rea1-specific antibody to detect its existence in 15 and 18 DAP 315
rea1-ref and wild type kernels (Figure 2E). The results demonstrated that Rea1 316
is present in rea1-ref and accumulates in rea1-ref at normal levels, but might 317
be only partly functional. 318
319
Rea1 is highly conserved in different organisms and is constitutively 320
expressed in maize 321
Rea1 was first identified as a component of pre-60S ribosome complex in 322
yeast and is conserved from yeast to humans (Bassler et al., 2001; Bassler et 323
al., 2010; Kressler et al., 2012). We constructed a phylogenetic tree on the 324
basis of the ZmRea1 full length protein sequence and Rea1 protein sequences 325
from Brachypodium distachyon, Triticum, Oryza sativa, Setaria, Arabidopsis 326
thaliana, Populus, Glycine-max, Dictyostelium, Monodelphis domestica, 327
Saprelegnia, Mortierella, and Saccharomyces Cerevisiae. The results 328
suggeste that ZmRea1 is highly conserved with the Rea1 proteins in other 329
plants as well as the Rea1 proteins of yeast, mammals and micro-organisms 330
(Figure 3A). 331
Quantitative RT-PCR analysis revealed ZmRea1 is expressed in a broad 332
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range of maize tissues, including silk, tassel, ear, root, husk, stem, leaf and 333
kernel (Figure 3B). During kernel development, expression of Rea1 occurs 334
before 5 DAP and continues later than 25 DAP (Figure 3C). Dot-immunoblot 335
analysis on quantified and gradient diluted total nuclear and cytoplasmic 336
proteins detected Rea1 in these subcellular fractions, and it was predominantly 337
found in the nuclear fraction, consistent with Rea1 localization in the nucleus 338
(Figure 3D). 339
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rea1-ref affects the biogenesis of 60S ribosomal subunits 341
To investigate the effect of rea1-ref on ribosomal subunit biogenesis and the 342
formation of monosome and polysome complexes, polysome profiles of 15 343
DAP rea1-ref and wild type kernel extracts were analyzed by 15-45% (w/v) 344
sucrose gradient centrifugation. Two independent biological replicates were 345
performed. This analysis revealed a significant reduction of 60S ribosomal 346
subunits, as compared to 40S ribosomal subunits, in the mutant (Figure 4A). 347
To compare the levels of monosomes and polysome complexes, calculation of 348
the peak areas of A254 absorbance revealed that about 40.2% of the 349
ribosomes in wild type kernel extracts were in polysomes, while the level of 350
polysome complexes in rea1-ref kernel extracts was 57.2 % (Figure 4A). Thus, 351
there are 1.4-Fold greater polysomes/total ribosomes in rea1-ref. The 352
decrease in 60S subunits and increase in polysomes is consistent with 353
inhibition of large ribosomal subunit export and promotion in initiation of protein 354
synthesis as a consequence of down-regulated ribosome biogenesis in 355
rea1-ref. 356
To confirm that maturation and export of 60S subunits is reduced in 357
rea1-ref, immunoblot analysis with 60S and 40S subunit antibodies was 358
performed on nuclear and cytoplasmic fractions from 15 and 18 DAP rea1-ref 359
or wild-type kernels. Nuclear and cytoplasmic fractions were subjected to 360
immunoblot analysis with antibodies against Bip (cytoplasm marker), and 361
histone (nucleus marker). Tubulin and TATA-box binding protein (TBP) served 362
as sample loading controls of cytoplasmic and nuclear proteins, respectively. 363
The level of eRPL13 was examined using a L13-specific antibody. L13 protein 364
was markedly decreased in both the nuclear and cytoplasmic fractions of 365
rea1-ref compared to wild-type kernels (Figure 4B). The level of eRPS14 was 366
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also examined using a specific antibody, and its content was the same in both 367
the nuclear and cytoplasmic fractions of rea1-ref and wild-type kernels (Figure 368
4B). Meanwhile, we also observed slight decrease of histone protein in 369
rea1-ref nuclear fraction (Figure 4B). 370
A reduction of 60S ribosomal subunits in the cytoplasm of 15 and 18 DAP 371
rea1-ref and wild-type endosperms was also observed by Transmission 372
Electron Microscopy (TEM) analysis. There were fewer ribosomes on rough 373
ER (RER), and the RER around protein bodies (PBs, Supplemental Figure 4). 374
Nucleolus stress due to ribosomal failure alters the morphology and increases 375
the surface area of nucleolus in humans (Bailly et al., 2015). This stress, which 376
misshapes and expands nucleolus, was also observed in rea1-ref endosperm 377
by TEM (Figure 4C). All these data are consistent with a biogenesis defect of 378
60S subunits and demonstrates it is specific to the 60S maturation and export 379
pathway. 380
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rea1-ref affects transcription of ribosome biogenesis, translational 382
elongation and nucleosome-related genes 383
We compared the transcript profile of 15 DAP rea1-ref and wild type 384
endosperm using RNA sequencing (RNA-seq). Among the 45,730 gene 385
transcripts detected by RNA-seq, significantly differentially transcribed genes 386
(DTGs) were identified as those with a threshold fold change>2 and 387
p-value<0.05. Based on this criterion, 2,076 genes showed significant altered 388
expression between rea1-ref and the wild type. There were 1,518 genes with 389
increased transcription, while 558 genes showed decreased transcription. 390
Within the 2,076 DTGs, 39.9% could be functionally annotated 391
(annotations were found using BLASTN and BLASTX analyses against the 392
Genbank (http://www.ncbi.nlm.nih.gov/) database). Gene Ontology (GO; 393
http://bioinfo.cau.edu.cn/agriGO/) and the Kyoto Encyclopedia of Genes and 394
Genomes (http://www.genome.jp/kegg/) pathway analysis indicated that 828 395
DTGs were mostly related to four GO terms: GO: 0005840 (ribosome, 396
p-value=2.34E-130); GO: 0006414 (translational elongation, 397
p-value=2.30E-17); and GO: 0000786 (nucleosome, p-value= 8.22E-29); GO: 398
0045735 (nutrient reservoir activity, p-value=6.47E-33). This analysis is 399
illustrated in Figure 5A and Supplemental Table 1. 400
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Ninety-eight DTGs classified to GO: 0005840 (ribosome) could be divided 401
into three categories: small ribosomal subunit proteins, e.g., eRPS6 402
(GRMZM5G851698), eRPS13 (GRMZM2G130544); large ribosomal subunit 403
proteins, e.g. eRPL14 (GRMZM2G168330), eRPL18 (GRMZM2G030731); 404
and ribosome biogenesis factors. Transcription of all the genes related to 405
ribosome biogenesis was increased in rea1-ref endosperm. rea1-ref also has a 406
strong impact on translational elongation. The twenty DTGs involved in GO: 407
0006414 (translational elongation) could be divided into two categories: 60S 408
acidic ribosomal proteins, e.g. eRPLP0 (GRMZM2G066460), eRPLP1 409
(GRMZM2G157443); and translation elongation factors, e.g. eEF1α 410
(GRMZM2G151193), eEF1β (GRMZM2G122871). These genes were also 411
up-regulated. Fifty-two DTGs related to GO: 0000786 (nucleosome) could be 412
divided into two categories: histones, e.g., H2A (GRMZM2G056231), H2B 413
(GRMZM2G401147); and nucleosome assembly protein (NAP, 414
GRMZM2G176707). Transcription of these genes, which are related to 415
nucloesome assembly and cell cycle, was markedly induced in rea1-ref 416
endosperm. DTGs involved in GO: 0045735 (nutrient reservoir activity) were 417
storage proteins, including 22kD α-zein (GRMZM2G346897), and 19kD α-zein 418
(GRMZM2G059620), e.g. these genes were down-regulated. To validate the 419
differences observed by RNA-Seq, we performed qRT-PCR on the most 420
significant DTGs from each GO category, and the results confirmed similar 421
differences of mRNA accumulation (Figure 5B). 422
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rea1-ref exhibits uncoordinated expression of distinct groups of genes at 424
the translational level 425
The increase in polysomes in rea1-ref indicated promotion in initiation of 426
protein synthesis in response to down-regulated ribosome biogenesis. The 427
mechanisms that underlie differences of mRNA translation involve sequence 428
features of individual mRNAs and the phosphorylation status of translation 429
initiation factors (Bailey-Serres and Dawe, 1996). General Control 430
Non-derepressing kinase-2 (GCN2) was reported to phosphorylate eukaryotic 431
initiation factor 2α (eIF2α) to down-regulate translation (Zhang et al., 2008). 432
We firstly measured the phosphorylation levels of eIF2α in 15 and 18 DAP 433
rea1-ref and wild-type cytoplasms by protein gel blot analysis with P-eIF2α and 434
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eIF2α (total eIF2α as control) antibodies. Compared with wild type, eIF2α in 435
rea1-ref was significantly less phosphorylated, while the eIF2α protein level 436
was not altered (Figure 6A). The level of eEF1α protein in rea1-ref and 437
wild-type cytoplasms was examined using specific antibody. eEF1α was 438
markedly increased in rea1-ref (Figure 6A). These results indicated that 439
initiation and elongation of translation are promoted in rea1-ref. 440
The level of an mRNA in polysomes reflects its translation (Branco-Price 441
et al., 2005). To examine the effects of rea1-ref on translational regulation of 442
individual mRNAs, we evaluated the amount of RNA in polysomes, relative to 443
the total amount of transcript in 15DAP rea1-ref and wild type kernels. Kernel 444
extracts were centrifuged (170,000 g) to obtain a polysome pellet for 445
comparing total extract and polysome-bound RNA samples by RNA-Seq 446
analysis. The polysome-bound/total RNA ratios in 15DAP rea1-ref and wild 447
type kernels were 36.4% and 25.6%, respectively. Consequently, there was a 448
1.4-Fold increase of polysome-bound/total RNA in rea1-ref, which is consistent 449
with the polysome complexes/total ribosomes A254 absorbance by ribosome 450
profile analysis. 451
Within the 30,188 gene transcripts detected by RNA-seq, significantly 452
differentially translated RNAs (DTRs) were identified as those with a 2nP/T 453 (normalized polysome-bound/total)×100% (see Methods) between rea1-ref and wild type, fold 454
change>2.0 or <0.5. Based on this criterion, 1,802 genes showed significantly 455
increased translation in rea1-ref compared to the wild type, while 2,959 genes 456
showed decreased translation. To confirm the differences between wild type 457
and rea1-ref endosperm observed by RNA-Seq, we performed qRT-PCR on 458
the most significantly increased or decreased DTRs selected from each 459
category and the results were consistent (Figure 6B&C). We also performed 460
puromycin treatment for the releasing of polysome as negative control 461
(Supplemental Figure 5). No significant difference of sequence features was 462
observed between the up-regulated and down-regulated DTRs (Supplemental 463
Table 2). 464
Within the increased DTRs, 687 could be functionally annotated. GO 465
analysis indicated these RNAs are mostly related to three GO terms, GO: 466
0045449 (Regulation of transcription, p-value=1.33E-03), GO: 0045735 467
(Nutrient reservoir activity, p-value=3.51E-12) and GO: 0006414 (Translational 468
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15
elongation, p-value= 1.17E-07). For the decreased DTRs, 601 could be 469
functionally annotated, belonging to GO: 0006334 (Nucleosome assembly, 470
p-value=6.59E-10), GO: 0006260 (DNA replication, p-value=1.18E-03) and 471
GO: 0033279 (Ribosomal subunits, p-value= 1.16E-03). Ten most strongly 472
up-regulated or down-regulated DTRs of each classification are illustrated in 473
Table 1 and all DTRs are shown in Supplemental Table 2. 474
Transcriptional factors (e.g. NAC domain transcription factors, MADS-box 475
transcription factors) and translation elongation-related genes had a markedly 476
higher translation level in rea1-ref. Although the expression of zeins was 477
down-regulated in rea1-ref (Figure 5), surprisingly their translation level is 478
significantly increased (Table 1& Supplemental Table 2). There is significant 479
overlap (p-value=2.23E-16, chi-sq test) for zeins between transcriptional 480
down-regulated genes and translational up-regulated genes (Figure 6E). 481
Meantime, although the expression of histone RNAs was up-regulated in 482
rea1-ref (Figure 5), their translation level was dramatically reduced. There is 483
also significant overlap (p-value=1.57E-14, chi-sq test) for histones between 484
transcriptional up-regulated genes and translational down-regulated genes 485
(Figure 6E). DNA replication related genes (e.g. DNA polymerase subunits, 486
minichromosome maintenance proteins (MCMs)) had lower translation level in 487
rea1-ref. Histones and DNA replication related genes are both related to 488
nucloesome assembly and cell cycle. Although the transcription of ribosomal 489
subunit proteins is up-regulated in rea1-ref (Figure 5), their translation level is 490
dramatically down-regulated. There is overlap between transcriptional 491
up-regulated genes and translational down-regulated genes for ribosomal 492
proteins (Figure 6E). These results demonstrate that the transcriptional and 493
translational regulation of individual genes responding to reduced 60S 494
ribosome exportation is not always consistent. 495
We measured the level of 22kD α-zeins in 470 ng and 1,190 ng total 496
proteins of 15 DAP rea1-ref and wild-type kernels, respectively, by protein gel 497
blot analysis with 22kD α-zein antibodies. Compared with the wild type, 22kD 498
α-zeins in rea1-ref are significantly increased. Meanwhile, there is no effect on 499
19kD α-zein content (Figure 6D). Increased eEF1α protein content (Figure 6A) 500
and lower protein content of histone (Figure 4B) in rea1-ref also confirmed 501
their increased or decreased translation level. 502
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16
503
rea1-ref inhibits cell proliferation and cell growth 504
The synthesis of nucloesome assembly proteins that related to cell cycle 505
transition is markedly reduced in rea1-ref (Table1&Figure 6). This mutant 506
exhibits a slow growth phenotype for both kernels and seedlings. There was a 507
more than three days’ delay in endosperm development (Figure 1). 508
Endoreduplication is a general feature of endosperm development in maize, 509
involving replication of the nuclear genome without cell division, and leading to 510
elevated nucleic acid content (Sabelli and Larkins, 2009). Endoreduplication 511
includes only G1 and S phase, which is different from the mitotic cell cycle 512
(G1-S-G2-M). 513
Flow cytometry (FCM) analysis of 15 DAP rea1-ref and wild type 514
endosperms showed endoreduplicated nuclei with C-values of 12C or greater, 515
accounting for 18.1% of the DNA in 15 DAP endosperm of rea1-ref, and 22.2% 516
of the DNA in 15 DAP wild-type endosperm (Figure 7A). At 18 DAP, there are 517
19.3 and 24.2% endoreduplicated nuclei with C-values of 12C or greater in 518
rea1-ref and wild-type endosperm, respectively (Figure 7B). The mitotic cell 519
cycle was also assessed in 7 DAG seedlings by FCM. The result showed that 520
63.3% of the nuclei are with 2C DNA content in rea1-ref 7 DAG seedlings, 521
while 45.1% of the nuclei in the wild type seedlings are with 2C DNA content 522
(Figure 7C). These results demonstrate that mutation of rea1-ref affects the 523
cell proliferation. The first leaf of 7 DAG rea1-ref and wild-type seedlings was 524
analyzed by scanning electron microscopy (SEM) to observe the cell size of 525
lower epidermis (Figure 7D). There was significantly smaller cell size in 526
rea1-ref seedling than wild type; with cell width and cell length both decreased 527
(Figure 7E). These results demonstrate that cell growth and cell proliferation 528
are suppressed as a result of impaired 60S ribosome maturation, resulting in a 529
developmental delay. 530
531
Discussion 532
rea1-ref/dek* is a weak mutant allele that functionally suppresses 533
ZmRea1 and affects 60S ribosome biogenesis 534
Rea1 is a highly conserved ribosome biogenesis factor first identified in the 535
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17
Nug1-purified pre-60S subunit in yeast, which is the pre-ribosomal particle at 536
the export from the nucleolus to the nucleoplasm (Bassler et al., 2001). The six 537
ATPase modules of Rea1 create a ring domain, while the large linker and the 538
MIDAS domain compose the tail structure (Ulbrich et al., 2009). Rea1 attaches 539
the pre-ribosome at the Rix1 complex (Rix1-Ipi3-Ipi1) via the ATPase ring 540
domain (Nissan et al., 2002; Nissan et al., 2004; Galani et al., 2004). The tail of 541
Rea1 containing the MIDAS domain contacts the pre-ribosome at other 542
positions where the pre-60S factor, Rsa4 or Ytm1, is located. Ytm1 associates 543
with nucleolar pre-60S particles, while Rsa4 associates with nucleoplasmic 544
particles (Bassler et al., 2001; Miles et al., 2005; De la Cruz et al., 2005; 545
Ulbrich et al., 2009). The MIDAS domain of Rea1 interacts with the MIDO 546
domain of Ytm1or Rsa4; this interaction is essential for 60S unit maturation 547
and export from the nucleolus to the nucleoplasm, or from the nucleoplasm to 548
cytoplasm, respectively (Bassler et al., 2001; Ulbrich et al., 2009). Rea1 is 549
bound to the pre-60S ribosome at two distinct sites: one is mediated via the 550
motor ring domain and the other via MIDAS interaction with Ytm1 or Rsa4, 551
creating a mechanochemical device to release Ytm1 or Rsa4 for 60S ribosome 552
export (Kressler et al., 2012). 553
Compared to dek1, which creates severe effects on kernel development 554
(Becraft et al., 2002), dek* causes only mild effects. The mutant kernels have 555
an obvious small phenotype and decreased seed weight (Figure 1), with a 556
delay of embryo, endosperm and seedling development (Figure 1). 557
rea1-ref/dek* is a weak, non-lethal mutant allele, where the 2,359th Ala (GCC) 558
of ZmRea1 is replaced by Val (GTC, Figure 2). The rea1 mutant allele derived 559
from a Mutator transposon insertion in the coding region has a defective 560
phenotype and is lethal. rea1-ref is defective at the highly conserved linkage 561
region and might suppress function of ZmRea1 by affecting the mechanical 562
force of the ATPase ring domain to the MIDAS tail that releases Rsa4 or Ytm1 563
factors. The expression of Rea1 is increased at the transcript level in rea1-ref, 564
which might be a response to its functional suppression (Figure 2). 565
The characterization of dek* provides the first description of Rea1 in 566
maize. A significant reduction of mature 60S subunits were observed in yeast 567
rea1 mutants at restrictive conditions (Garbarino and Gibbons, 2002; Galani et 568
al., 2004). Rea1 homologous gene in arabidopsis is essential for female 569
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18
gametophyte development (Chantha et al., 2010). Rea1 AAA-ATPase is 570
conserved from yeast to humans (Bassler et al., 2010). Phylogenetic analysis 571
of ZmRea1 suggests Rea1 protein is highly conserved in plants, and is 572
homologous to proteins in yeast, mammals and micro-organisms (Figure 3). A 573
significant reduction of mature 60S ribosomal subunits is observed in the 574
rea1-ref mutant, consistent with the effects observed in yeast rea1, indicating 575
an biogenesis defect specific to the 60S subunit maturation pathway; 576
maturation and export of 40S subunits is not affected (Figure 4). This indicates 577
conserved function on 60S subunit biogenesis of Rea1 in yeast and plants. 578
There is reduction of mature 60S subunits in cytoplasm and pre-60S subunits 579
in nucleus (Figure 4&8), indicating the nucleus detained pre-60S subunits 580
might be degraded, for pre-mature ribosomal particles with biogenesis failure 581
would be dispersed and degraded in the nucleoplasm (Andersen et al., 2005; 582
Lam et al., 2007). 583
584
Impaired ribosome biogenesis enhances ribosome use efficiency 585
Regulation of protein synthesis is a key control point in cellular responses to 586
distinct stresses (Faye et al., 2014). The proportion of actively translating 587
ribosomes is reflected by the percentage of polysome complexes/total 588
ribosomes (Branco-Price et al., 2005; Faye et al., 2014). We observed a 589
1.4-Fold increase of polysome complexes/total ribosomes in rea1-ref (Figure 590
4). This increase in polysomes in rea1-ref is indicative of a promotion in the 591
initiation of protein synthesis. The proportion of actively translating ribosomes 592
might be in response to the down-regulation of ribosome biogenesis. 593
The results of our study of rea1-ref suggest suppressed ribosome export 594
enhances ribosome use efficiency and cellular translational efficiency. mRNA, 595
the 40S ribosomal subunit and eIF2α comprise the 43S pre-initiation complex, 596
termed “half-mer”, before attachment of the 60S ribosomal subunit (Helser et 597
al., 1981; Moy et al., 2002). Multiple eukaryotic protein kinases each of which 598
responds to different signals are known to phosphorylate eIF2α and 599
down-regulate general translation initiation, (Chen and London, 1995; Harding 600
et al., 1999; Williams, 1999; Harding et al., 2000; Huizen et al., 2003). GCN2 is 601
the only eIF2α kinase found in all eukaryotes, including plants like Arabidopsis 602
(Berlanga et al., 1999; Zhang et al., 2008). Phosphorylation of eIF2α is 603
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19
significantly reduced in rea1-ref (Figure 6), indicating increased formation of 604
pre-initiation complexes for protein synthesis, consistent with an increased 605
proportion of actively translating ribosomes. Further more, the level of eEF1α 606
protein is markedly increased in rea1-ref (Figure 6), suggesting a promotion of 607
both initiation and elongation of translation in rea1-ref. Consequently, there is 608
evidence for increased efficiency of ribosome usage during translation in 609
rea1-ref to ensure normal rates of protein synthesis (Figure 8). 610
611
Impaired ribosome biogenesis triggers distinct transcriptional and 612
translational cellular responses 613
We have found the suppressed ribosome maturation associated with rea1-ref 614
brings about global changes in transcription and translation. Translation of 615
individual mRNAs is regulated, producing discrepancies between mRNA and 616
protein levels. mRNAs have a distinct pattern of ribosome loading under 617
certain conditions, resulting in altered translational efficiencies (Branco-Price 618
et al., 2005; Gawron et al., 2014). Thus, analysis of transcript level is 619
insufficient to completely describe cellular responses under different conditions. 620
There is also alternative translation that contributes to the complexity of 621
proteomes (Preiss et al., 2003; Serikawa et al., 2003; MacKay et al., 2004; 622
Blais et al., 2004; Kawaguchi et al., 2004; Branco-Price et al., 2005; Lin et al., 623
2014). According to our transcriptome and translatome analysis, there is 624
evidence for consistent and inconsistent transcriptional and translational 625
regulation of genes in rea1-ref endosperm (Figure 8). The large amount of 626
transcriptionally up-regulated genes is not the consequence of developmental 627
delay, according to the expression data for developing maize kernels (Chen et 628
al., 2014). Our transcriptome analysis revealed immediate cellular responses, 629
while the translatome revealed specific protein changes that are independent 630
of transcriptional regulation for efficient use of limited ribosomes and energy. 631
For nucleus located proteins, transcription of small and large ribosomal 632
subunit proteins is increased in rea1-ref endosperm (Figure 5), whereas their 633
translation is down-regulated (Table 1). The transcriptional up-regulation of 634
ribosomal proteins might be a response to a decreased level of mature 635
ribosomes in the cytoplasm in rea1-ref (Figure 4). But increased transcription 636
is incapable of rescuing 60S ribosome export in rea1-ref. Consequently, the 637
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20
percentage of polysome-bound RNA of these genes might be reduced for 638
more efficient usage of limited ribosomes. Similar to ribosomal proteins, there 639
is also a discrepancy in expression of nucleosome assembly-related genes. 640
Transcription of histones is increased in rea1-ref (Figure 5), while their 641
translation is down-regulated (Table 1). DNA replication-related genes also 642
have reduced translation in rea1-ref (Table 1). The polysome-bound RNA of 643
histone- and DNA replication- related genes are markedly decreased (Figure 644
6); and the histone protein content is down-regulated in rea1-ref (Figure 4). 645
Up-regulation of histone transcription might be a response to decreased 646
growth vigor of rea1-ref (Figure 1), in order to accelerate growth rate. But 647
accelerated growth might be lethal due to ribosome shortage. Different kinds of 648
transcriptional factors have markedly higher translation in rea1-ref (Table 1), 649
including auxin-signaling related genes (GRMZM2G081930 and 650
GRMZM5G809195, Zhang et al., 2014). Among cytosol-located proteins, 651
transcription and translation of translation elongation-related genes are both 652
up-regulated in rea1-ref (Figure 5&Table 1). The final polysome-bound RNA 653
content of eEF1α and eEF-Tu, as well as the protein level of eEF1α are 654
markedly increased in rea1-ref (Figure 6). There is also inconsistent 655
transcriptional and translational levels of Rea1 itself (Figure 2), and it might be 656
a developmental stage-dependent translational regulation 657
In maize kernel, the most abundant protein is zein storage protein, that 658
accounts for 50%-70% of the total protein (Holding and Larkins, 2009), and 659
α-zein is the largest class of zein protein (Heidecker and Messing, 1986). The 660
transcriptionally down-regulated zeins might be the consequence of 661
developmental delay. α-zeins have an increased translation level (Table 1), 662
especially the 22kD α-zeins show markedly increased protein in both 663
SDS-PAGE and immunoblot analysis (Figure 1&Figure 6). The percentage of 664
α-zein polysome-bound RNA might be increased to ensure basic protein 665
storage in rea1-ref. 666
The mechanisms that underlie variation in translation of individual genes 667
are likely to involve features of the mRNA sequence (Bailey-Serres and Dawe, 668
1996). Evaluation of highly translated genes under hypoxia in Arabidopsis 669
showed mRNA sequences with a low GC nucleotide content in the 670
5’-untranslated region (UTR) (Branco-Price et al., 2005). RNA 5’- UTR GC 671
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21
content, 5’- UTR length, and ORF length were also observed to affect 672
ribosome loading (Jiao and Meyerowitz, 2010; Yángüez et al., 2013). When 673
we analyzed sequence features that might affect ribosome loading of 674
individual gene transcripts affected by shortage of 60S ribosome subunits 675
(Supplemental Table 3), no significantly different features were observed 676
between the 1,802 up-regulated DTRs and 2,959 down-regulated DTRs, 677
compared to 3,000 randomly selected control genes. However, there might be 678
other feedback pathways for independent regulation at the transcriptional and 679
translational levels. 680
681
Impaired ribosome biogenesis affects cell growth and proliferation 682
Cell growth and proliferation are tightly linked, as enhanced protein synthesis 683
is required for cell proliferation (Thomas, 2002). The increase in protein 684
synthesis is accomplished by an enhanced rate of ribosome biogenesis in 685
support of the metabolic effort for cell proliferation (Sollner-Webb and Tower, 686
1986). Normal mitosis includes four successive phases: G1 (postmitotic 687
interphase), S (DNA synthesis phase), G2 (postsynthetic phase), and M 688
(mitosis) (Fowler et al., 1998; Riou-Khamlichi et al., 2000), where as 689
endoreduplication of endosperm includes only G1 and S phases (Sabelli and 690
Larkins, 2009). rea1-ref exhibits slower growth and cell proliferation (Figure 7), 691
indicating an intrinsic link between ribosome biogenesis and cell cycle 692
transition. Based on our analysis, this linkage is through regulation of DNA 693
replication and nucleosome assembly. 694
In mammalian cells, the tumor suppressor, p53, has been shown to arrest 695
the cell cycle at the G1–S transition in response to impaired ribosome 696
biogenesis, while p53-independent cell cycle arrest in response to alteration of 697
ribosome biogenesis has also been described (Mayer and Grummt, 2005; 698
Zhang and Lu, 2009; Deisenroth and Zhang, 2010; Grimm et al., 2006; Donati 699
et al., 2011). p53 stabilization leads to cell cycle arrest through the regulation 700
of cyclins and cyclin-dependent kinases (CDKs) (Sherr and McCormick, 2002). 701
But the expression level of cyclins does not appear to be affected in rea1-ref 702
according to our trancriptome and translatome analysis. The target of 703
rapamycin (TOR) kinase is evolutionarily conserved among plant, yeast, and 704
animal cells, and is reported to integrate nutrient and energy signaling partly 705
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22
through the phosphorylation of RPS6 to promote ribosome biogenesis, 706
polysome accumulation, translation initiation, cell growth and cell proliferation 707
(Xiong and Sheen, 2014). The transcription and translation level of TOR 708
signaling-related genes does not appear to be affected in rea1-ref, but there 709
might be an altered phosphorylation level of RPS6 or other post-translational 710
level regulation that is responsible for immediate transcriptional up-regulation 711
of genes in rea1-ref. The underlying mechanisms merit further explorations. 712
S phase is the period for DNA replication, histone synthesis and 713
nucleosome assembly. Nucleosome assembly is essential for a variety of 714
biological processes, such as cell cycle progression, development and 715
senescence (Gal et al., 2015). The synthesis of nucleosome assembly-related 716
proteins (histones and DNA replication-related enzymes) might be reduced to 717
decelerate the growth rate for survival under the suppressed ribosome 718
biogenesis condition in rea1-ref (Table 1&Figure 6). As a result, the 719
nucleosome assembly process during S phase would be dramatically 720
suppressed. The cell proliferation is slowed in rea1-ref (Figure 7), thus 721
together with impaired cell growth kernel and seedling development are 722
slowed for more than three days (Figure 1). 723
724
rea1-ref/dek* as a non-lethal maize small kernel mutant offered an 725
opportunity to explore comprehensive cellular responses to impaired 60S 726
ribosome biogenesis. Based on our results, we propose reduced 60S 727
ribosome biogenesis lead to differentially regulating the transcription and 728
translation of distinct groups of genes that affect translation efficiency and cell 729
proliferation (Figure 8). Firstly, there is increased efficiency of translation 730
initiation and ribosome usage. Secondly, there is selective translational 731
regulation of different groups of genes for intensive usage of quantitatively 732
limited mature ribosome. Finally, there is inhibited cell proliferation, leading to 733
slower growth and survival. 734
735
Materials and Methods 736
Plant Materials 737
The o*-N1117 mutant stock was obtained from the Maize Genetics Cooperation stock 738
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23
center and identified as an EMS-induced allele of the opaque15 mutant. The dek* 739
mutation was separated from the o*-N1117 stock as an independent mutant. The dek* 740
mutant was crossed to the W64A inbred line and a F1 and F2 was produced to generate a 741
mapping population. All plants were cultivated in the field at the campus of Shanghai 742
University. Seeds were harvested at 5, 9, 13, 15, 17, 18, 21, 25 and 36 DAP. 743
744
Paraffin and resin sections 745
The 15 and 18 DAP embryos were fixed at 4°C overnight in FAA [ethanol 50% (v/v), acetic 746
acid 5.0% (v/v) and formaldehyde 3.7% (v/v)]. After embedding in paraffin, 10 μm 747
microtome sections on glass slides were dewaxed in xylene, rehydrated, and stained with 748
fuchsin. The 15 and 18 DAP endosperm tissues were fixed at 4°C overnight in FAA 749
[ethanol 50% (v/v), acetic acid 5.0% (v/v) and formaldehyde 3.7% (v/v)]. After embedding 750
in Spurr’s epoxy resin, thin sections (1μm) were heat fixed to glass slides and stained with 751
fuchsin. Stained sections were rinsed in water three times and air dried. Bright-field 752
photographs of the sections were taken using a Leica microscope. 753
754
Transmission Electron Microscopy 755
The 15 and 18 DAP kernels of rea1-ref and wild type were prepared according to Lending 756
and Larkins, (1992), with some modifications: kernels were fixed in paraformaldehyde and 757
post-fixed in osmium tetraoxide. After dehydration in an ethanol gradient, samples were 758
transferred to a propylene oxide solution and gradually embedded in acrylic resin (London 759
Resin Company). Sections (70 nm) were made with a diamond knife microtome (Reichert 760
Ultracut E). Sample sections were stained with uranyl acetate, post-stained with lead 761
citrate, and observed with a Hitachi H7600 transmission electron microscope. 762
763
Scanning Electron Microscopy 764
The first leaf mature region of 7 DAG rea1-ref and wild-type seedlings was fixed at 4°C 765
overnight in FAA [ethanol 50% (v/v), acetic acid 5.0% (v/v) and formaldehyde 3.7% (v/v)]. 766
Samples were critically dried and spray coated with gold. Gold-coated samples were then 767
observed with a scanning electron microscope (S3400N; Hitachi). 768
769
Protein Quantification 770
Mature kernels of rea1-ref and wild type were soaked in water and endosperm was 771
separated from the embryo and pericarp. Endosperm samples were critically dried to 772
constant weight, powdered in liquid N2, and measured according to (Wang et al., 2011). 773
Proteins were extracted from 50 mg of 3 pooled endosperm flour samples. Extracted 774
proteins were measured using a bicinchoninic acid protein assay kit (Pierce) according to 775
instructions. Measurements of all samples were replicated three times. 776
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24
777
Measurement of starch 778
Mature kernels of rea1-ref and wild type were soaked in water, and endosperm was then 779
separated from the embryo and pericarp. Endosperm samples were dried to constant 780
weight, pulverized in liquid N2, and starch was extracted and measured using an 781
amyloglucosidase /α- amylase method starch assay kit (Megazyme) according to the 782
instructions adapted as: add 0.2 mL of aqueous ethanol (80 % v/v) to 100 mg sample and 783
aid dispersion; immediately add 3 mL of thermostable α-amylase. Incubate the tube in a 784
boiling water bath for 6 min; place the tube in a bath at 50°C; add 0.1 mL 785
amyloglucosidase; stir the tube on a vortex mixer and incubate at 50°C for 30 min; 786
transfer duplicate aliquots (0.1 mL) of the diluted solution to the bottom, add 3.0 mL of 787
GOPOD reagent and incubate the tubes at 50°C for 20 min; read the absorbance for each 788
sample, and the D-glucose control at 510 nm against the reagent blank. The percentage 789
of starch content in per mg dried sample was analyzed. 790
791
Soluble amino acids analysis 792
Soluble amino acids were analyzed according to (Holding et al., 2010): 3 mg samples 793
were refluxed for 24 hr in 6N HCl. Samples were hydrolyzed at 110℃ for 24 hr. Sample 794
hydrolysates were critically dried and dissolved in 10 ml of citrate buffer. The amino acids 795
were analyzed with a Hitachi-L8900 amino acid analyzer at Shanghai Jiaotong University. 796
On the wt and dek kernels analyses were replicated three times. 797
798
Map-Based Cloning 799
The Dek* locus was mapped using 864 individuals from a F2 mapping population of the 800
cross between the dek* and the W64A inbred line. For preliminary mapping, molecular 801
markers distributed throughout maize chromosome 6 were used. SSR 155.1M-1, 802
SSR153.7M-2, SSR154.7M-3, InDel438, InDel428, SNP064 and SNP165 (see 803
Supplemental Table 4 online) as additional molecular markers for fine mapping, were 804
developed to localize the Dek* locus to a 101.6 kb region. 805
806
RNA Extraction and real-time PCR Analysis 807
Total RNA was extracted with TRIzol reagent (Tiangen) and DNA was removed by a 808
treatment with RNase free DNase I (Takara). Using ReverTra Ace reverse transcriptase 809
(Toyobo), RNA was reverse transcribed to cDNA. Quantitative real-time PCR was 810
performed with SYBR Green Real-Time PCR Master Mix (Toyobo) using a Mastercycler 811
ep realplex 2 (Eppendorf) according to the standard protocol. Specific primers were 812
designed (see Supplemental Table 4 online) and the experiments were performed with 813
two independent RNA samples sets with ubiquitin as the reference gene. From a pool of 814
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25
kernels collected from three individual plants, a RNA sample was extracted, for which 815
three technical replicates were performed. A final volume of 20 mL contained 1 mL 816
reverse transcribed cDNA (1 to 100 ng), 10 mL 23 SYBR Green PCR buffer, and 1.8 mL 817
10 mM/L forward and reverse primers for each sample. Relative quantifiable differences in 818
gene expression were analyzed as described previously (Livak and Schmittgen, 2001). 819
820
Fractionation of cytoplasmic and nuclear proteins 821
Pulverized tissue was hydrated in cold harvest buffer [10mM HEPES (PH 7.9), 50mM 822
NaCl, 0.5M sucrose, 0.1mM EDTA, 0.5% Triton X-100, 1mM DTT, 10mM tetratsodium 823
pyrophosphate, 100mM NaF, 17.5mM beta-glycrophosphate, 1mM PMSF, 4μg/ml 824
Aprotinin, 2μg/ml Pepstatin A ] incubated on ice for 5 min, and nuclei was pelleted (1,000 825
rpm, 10 min). After transfer of the supernatant to a new tube for extracting cytoplasmic 826
proteins, the pellet of nucleic was washed and resuspended by Buffer A [10mM HEPES 827
(PH7.9), 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT, 1mM PMSF, 4μg/ml 828
Aprotinin, 2μg/ml Pepstatin A] and pelleted again (1,000 rpm, 10 min). Nuclei were 829
washed and resuspended in Buffer C [10mM HEPES (PH7.9), 500mM NaCl, 0.1mM 830
EDTA, 0.1mM EGTA, 0.1% NP-40, 1mM DTT, 1mM PMSF, 4μg/ml Aprotinin, 2μg/ml 831
Pepstatin A] and vortexed (4℃, 15 min), pelleted again (14,000 rpm, 4℃, 10 min), and 832
transfered to a new tube for extracting nuclear proteins. 833
834
Polyclonal Antibodies 835
For anti-Rea1 antibody production, the 12,478-16,038 bp cDNA fragment was cloned into 836
pGEX-4T-1 (Amersham Biosciences), and GST–tagged fusion protein was purified with 837
the AKTA purification system (GE Healthcare) using a GSTrap FF column. Protein 838
expression and purification followed established procedures. Antibodies were produced in 839
rabbits according to standard protocols of Shanghai ImmunoGen Biological Technology. 840
For 22kD α-zein and 19kD α-zein antibody production, regions of low similarity of 22kD 841
α-zein and 19kD α-zein were selected according to a previous study (Woo et al., 2001). 842
The cDNAs responsible for selected polypeptides were cloned into pGEX-4T-1 843
(Amersham Biosciences), and GST–tagged fusion protein was purified with the AKTA 844
purification system (GE Healthcare) using a GSTrap FF column. Protein expression and 845
purification followed established procedures. Antibodies were produced in rabbits 846
according to standard protocols of Shanghai ImmunoGen Biological Technology. 847
848
Immunoblot Analysis 849
Proteins extracted from rea1-ref and wild type mature kernels were separated by 850
SDS-PAGE. Separated protein samples were then transferred to polyvinylidene difluoride 851
membrane (0.45 mm; Millipore). The membrane with a protein sample attached on it was 852
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26
incubated with primary and secondary antibodies. Using the Super Signal West Pico 853
chemiluminescent substrate kit (Pierce) the signal was visualized according to the 854
manufacturer’s instructions. The purified anti-Rea1 antibody was used at 1: 500; the 22kD 855
α-zein and 19kD α-zein antibodies were used at 1: 5000; the α-tubulin antibody 856
(Sigma-Aldrich) was used at 1: 5000; the BIP (at-95) antibody (Santa Cruz Biotechnology), 857
Histone antibody (Cell signaling), eRPL13 antibody (Agrisera), eIF2α antibody (Cell 858
signaling), and P- eIF2α antibody (Cell signaling) were used at 1: 1000, and the TBP 859
antibody (Santa Cruz Biotechnology), eRPS14 antibody (Santa Cruz Biotechnology), and 860
eEF1α antibody (Santa Cruz Biotechnology) were used at 1:500. 861
862
Phylogenetic Analysis 863
Related sequences were identified in the NCBI nr (nonredundant protein sequences) 864
database by performing a BLASTp search with ZmRea1 protein sequences. Amino acid 865
sequences were aligned with the MUSCLE method in the MEGA5.2 software package 866
using their default settings for protein multiple alignment. A rooted phylogenetic tree of 867
Rea1 from maize, Brachypodium distachyon, Triticum, Oryza sativa, Setaria, Arabidopsis 868
thaliana, Populus, Glycine-max, Dictyostelium, Monodelphis domestica, Saprelegnia, 869
Mortierella, and Saccharomyces Cerevisiae was constructed by the neighbor-joining 870
method using the MEGA5.2 software package. The evolutionary distances were 871
computed using the Poisson correction analysis. 872
873
RNA-seq Analysis 874
Total RNA (10 μg) was extracted from endosperm of rea1-ref and wild type kernels 875
harvested at 15 DAP, and three rea1-ref or wild type biological samples were pooled 876
together. The poly-A selected RNA-Seq library was prepared according to Illumina 877
standard instruction (TruSeq Stranded RNA LT Guide). Library DNA was checked for 878
concentration and size distribution in an Agilent2100 bioanalyzer before sequencing with 879
an Illummina HiSeq 2500 system according to the manufacturer’s instructions (HiSeq 880
2500 User Guide). Single-end reads were aligned to the maize B73 genome build Zea 881
mays AGPv2.15 using TopHat 2.0.6 (Langmead et al., 2009). Data were normalized as 882
fragments per kilobase of exon per million fragments mapped (FPKM), for the sensitivity 883
of RNA-seq depends on the transcript length. Significant differentially transcribed genes 884
(DTGs) were identified as those with a fold change and P-value of differential expression 885
above the threshold (Fold change>2.0, P<0.05). 886
887
Ribosome profile and isolation of polysomal RNA 888
For the ribosome profile, approximately 2.5ml of pulverized tissue (approx. twenty 15 DAP 889
kernels) was hydrated in two volumes of polysome extraction buffer (PEB) [200mM 890
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27
Tris–HCl (pH 9.0), 200mM KCl, 25mM EGTA, 35mM MgCl2, 1% (w/v) Brij-35, 1% (v/v) 891
Triton X-100, 1 % (v/v) Tween-20, 1% (v/v) Igepal CA–630, 1% (w/v) deoxycholic acid, 1% 892
(v/v) polyethylene-10-tridecylether, 1mM Phenylmethylsulfonyl fluoride (PMSF), 0.5mg/ml 893
heparin, 5mM dithiothreitol (DTT), 50 mg/ml cycloheximide, 50 mg/ml chloramphenicol] 894
(Kawaguchi et al., 2004), homogenized, filtered through two layers of sterile Miracloth 895
(Calbiochem, La Jolla, CA), and cleared by centrifugation (16,000 g, 4℃, 15 min). Four 896
hundred A260 units of the supernatant was layered over a 15–45% (w/v) sucrose density 897
gradient, centrifuged (237,000 g, 2.5h, at 4℃, Beckman Optima™ L-100 XP) and the 898
A254 nm absorbance profile was recorded with chart recorder by using a gradient 899
fractionator connected to a UA-5 detector (BIOCOMP,Canada) as described previously 900
(Kawaguchi et al., 2003, 2004). Two independent biological replicates were performed. 901
For isolation of polysomal RNA, approximately 5 ml of pulverized tissue powder 902
(approx. forty 15 DAP kernels) was hydrated in two volumes of PEB, homogenized, 903
filtered through two layers of sterile Miracloth (Calbiochem, La Jolla, CA), and cleared by 904
centrifugation (16,000 g, 4℃, 15 min). The supernatant was layered over a 1.75 M 905
sucrose cushion [400mM Tris–HCl (pH 9.0), 200mM KCl, 30mM MgCl2, 1.75 M sucrose, 906
5mM DTT, 50 mg/ml chloramphenicol, 50 mg/ml cycloheximide], and centrifuged at 907
170,000 g, at 4 ℃ for 3 h (modified from Fennoy and Bailey-Serres, 1995). The polysome 908
pellet was washed with sterile water and resuspended in 700 μl PEB lacking heparin and 909
detergents. Total or polysome-bound RNA was precipitated from total supernatant or the 910
ribosome fraction of the same amount of sample powder, by addition of 2.5 vol of 8 M 911
guanidine chloride and 3.5 vol of 99% (v/v) ethanol, and extracted by use of Qiagen Plant 912
RNeasy mini kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. 913
For negative control, same amount of pulverized tissue powder was hydrated in two 914
volumes of PEB buffer with 2mM puromycin. 915
The polysome-bound/total RNA value for individual genes was determined from the 916
ratio of the signal in the polysome RNA sample divided by the signal in the total RNA 917
sample. Due to the required use of an equal RNA quantity in each RNA-Seq reaction, in 918
spite of the unequal proportion of RNA in the polysome fraction under the two conditions, 919
it was necessary to normalize the signal values obtained for polysome RNA. 920
Normalization was performed according to Branco-Price et al., 2005. Polysomes 921
accounted for 57.2% and 40.2% of the total absorbance in rea1-ref and wild type kernels, 922
respectively. The percentage of an individual mRNA species in polysomes was calculated 923
as, 2nP/T (normalized polysome-bound/total)×100%. 924
Normalized polysome-bound/total in rea1-ref: 925
926
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28
Normalized polysome-bound/total in wild type: 927
928 929
FCM Detection 930
For extraction of nuclei, endosperm and seedling tissues were finely chopped with a sharp 931
razor blade in Beckman lysis buffer. The resulting slurry was filtered through a 30 μm 932
nylon filter to eliminate cell debris and the suspension containing nuclei was immediately 933
measured using an Accuri C6 (BD) flow cytometer equipped with an argon-ion laser tuned 934
at a wavelength of 488 nm. For each sample, at least 15,000 nuclei were collected and 935
analyzed using a logarithmic scale display. Each flow cytometric histogram was saved 936
and analyzed using BD Accuri C6 Software 1.0.264.21. 937
938
Accession numbers 939
Sequence data from this article can be found in the GenBank/EMBL data libraries under 940
the following accession numbers: Zm Rea1, KP137367; Zm BIP1, NM_001112423, 941
GRMZM2G114793; Zm eRPS6, NM_001112164, GRMZM5G851698; Zm eEF1α, 942
NM_001112117, GRMZM2G153541; Zm Histone H4, NM_001138113, 943
GRMZM2G084195; Zm DNA polymerase epsilon subunit 2, NM_001153609, 944
GRMZM2G154267, Zm MCM6, NM_001111819, GRMZM2G021069. RNA-seq data is 945
available from the National Center for Biotechnology Information Gene Expression 946
Omnibus (www.ncbi.nlm. nih.gov/geo) under the series entry GSE67103. 947
948
Supplemental data 949
The following materials are available in the online version of this article. 950
Supplemental Figure 1, SDS–PAGE analysis of total (A), zein (B), and nonzein (C) 951
proteins from dek*/ rea1-ref and WT mature endosperm. 952
Supplemental Figure 2, Comparison of total starch content and the percentage of 953
amylose in wild type and rea1-ref mature endosperm. 954
Supplemental Figure 3, Phenotype of rea1-ref and WT plants (90 DAG). 955
Supplemental Figure 4, Ultrastructure of developing endosperms of wild type and 956
rea1-ref (15 DAP and 18 DAP). 957
Supplemental Figure 5, qRT-PCR confirmation of DTRs with increased or decreased 958
translation level. 959
Supplemental Table 1, Gene ontology classifications of DTGs with functional annotation. 960
Supplemental Table 2. Gene ontology classifications of DTRs with functional annotation. 961
Supplemental Table 3, Sequence feature analysis of DTRs. 962
Supplemental Table 4, Primers used in these experiments. 963
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29
964
Acknowledgements 965
This work was supported by the Major Research plan of the National Natural Sciences 966
Foundation of China (91335208 and 31425019), and the Ministry of Science and 967
Technology of China (2014CB138204). We thank Dr.Yuanyuan Ruan (Fudan University) 968
for technical support on ribosome profile experiment and Dr. Brian A. Larkins (University 969
of Nebraska, Lincoln) for critical reading the article. 970
971
Author contributions 972
R.S., W.Q., and J.Z. designed the experiment. J.Z., W.Q., Qiao.W., Qun.W., X.L., D.Y., 973
and Y.J. performed the experiments. W.Q., J.Z. Gang. W., Gui. W. and R.S. analyzed the 974
data. W.Q. and R.S. wrote the article. 975
976
Tables 977
Table 1. Ten most strongly up-regulated or down-regulated DTRs of each Gene 978
ontology classification. 979
GO ID P-value Gene Description Polysome/Total
in WT Polysome/Total
in rea1 Fold
change Genes with increased ratio of polysome-bound mRNA (% of total)
GO:0045449
Regulation of
transcription
1.33E-03 GRMZM2G081930 NAC1 0.1437 0.6978 4.86
GRMZM2G167018 NAC domain transcription factor 0.0336 0.3461 10.31
GRMZM2G134717 NAC domain transcription factor 0.0291 0.2145 7.36
GRMZM2G170079 BZIP-type transcription factor 0.0165 0.1979 12.03
GRMZM5G812272 WRKY DNA-binding domain
superfamily protein 0.0106 0.1038 9.76
GRMZM2G327059 BEL1-related homeotic protein 0.0316 0.2861 9.05
GRMZM2G021339 leucine zipper domain protein 0.0158 0.1272 8.07
GRMZM2G126239 Homeobox-leucine zipper protein
ATHB-4 0.0142 0.2754 19.38
GRMZM2G087741 Homeobox protein liguleless 3 0.0480 0.3018 6.29
GRMZM5G809195 IAA14-auxin-responsive Aux/IAA
family member 0.0451 0.4536 10.06
GO:0045735
Nutrient reservoir
activity
3.51E-12 GRMZM2G346897 22kD alpha zein 0.2875 0.7691 2.68
GRMZM2G353272 22kD alpha zein 0.2442 0.9829 4.03
GRMZM2G044152 22kD alpha zein 0.2126 0.7821 3.68
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30
GRMZM2G397687 22kD alpha zein 0.2153 0.8910 4.14
GRMZM2G053120 22kD alpha zein 0.2127 0.9571 4.51
GRMZM2G008341 Zein-alpha 19kD z1A 0.2862 0.9428 3.29
GRMZM2G353268 Zein-alpha 19kD z1A 0.2504 0.8631 3.45
AF546188.1_FG003 Zein-alpha 19kD z1B 0.2120 0.7470 3.52
AF546188.1_FG007 Zein-alpha 19kD z1B 0.2578 0.8318 3.23
AF546187.1_FG007 Zein-alpha 19kD z1D 0.2429 0.9263 3.81
GO:0006414
Translational
elongation
1.17E-07 GRMZM5G859846 Elongation factor Tu 0.0476 0.1744 3.67
GRMZM2G007038 Elongation factor Tu 0.2158 0.6634 3.07
GRMZM2G407996 Elongation factor Tu 0.3131 0.6392 2.04
GRMZM2G110509 Elongation factor 1-alpha 0.1984 0.4218 2.13
GRMZM2G151193 Elongation factor 1-alpha 0.2001 0.4162 2.08
GRMZM2G001327 Elongation factor 1-alpha 0.2248 0.4732 2.11
Genes with reduced ratio of polysome-bound mRNA (% of total) GO:0006334
Nucleosome
assembly
6.59E-10 GRMZM5G883764 Histone H2A 0.5549 0.0717 0.13
GRMZM2G355773 Histone H3.2 0.9458 0.0945 0.09
GRMZM2G447984 Histone H3.2 0.8418 0.1126 0.13
GRMZM2G130079 Histone H3.2 0.6460 0.0847 0.13
GRMZM2G349651 Histone H4 0.7069 0.0850 0.12
GRMZM2G073275 Histone H4 0.5894 0.0784 0.13
GRMZM2G479684 Histone H4 0.9550 0.1012 0.11
GRMZM2G084195 Histone H4 0.3357 0.0266 0.08
GRMZM2G421279 Histone H4 0.9435 0.1030 0.11
GRMZM2G149178 Histone H4 0.5823 0.0750 0.13
GO:0006260
DNA replication 1.18E-03 GRMZM5G825512 Origin recognition complex subunit 6 0.4301 0.1093 0.25
GRMZM5G872710 DNA polymerase 0.4674 0.1236 0.26
GRMZM2G154267 DNA polymerase epsilon subunit 2 0.7544 0.0657 0.09
GRMZM2G100639 DNA replication licensing factor
MCM3 homolog 2 0.6059 0.0796 0.13
GRMZM2G117238 Origin recognition complex subunit 2 0.6233 0.1285 0.21
GRMZM2G162445 mini-chromosome maintenance
(MCM) complex protein family 0.8304 0.0891 0.11
GRMZM2G086934 Replication protein A 70 kDa
DNA-binding subunit 0.5513 0.1185 0.22
GRMZM2G021069 Minichromosome maintenance
protein 0.7020 0.0859 0.12
GRMZM2G108712 Proliferating cell nuclear antigen 0.8651 0.1292 0.15
GRMZM2G304362 Ribonucleoside-diphosphate
reductase 0.6543 0.0827 0.13
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31
GO:0033279
Ribosomal subunits 1.16E-03 GRMZM2G099352 40S ribosomal protein S3 0.3527 0.1547 0.44
GRMZM2G078985 40S ribosomal protein S5 0.3491 0.1649 0.47
GRMZM2G064640 40S ribosomal protein S9 0.3811 0.1484 0.39
GRMZM2G170336 40S ribosomal protein S20 0.4542 0.1796 0.39
GRMZM2G110952 40S ribosomal protein S23 0.2834 0.1151 0.41
GRMZM2G140609 40S ribosomal protein S23 0.4064 0.1659 0.41
GRMZM2G163561 40S ribosomal protein S23 0.4259 0.1402 0.33
GRMZM5G868433 60S ribosomal protein L7-2 0.5257 0.2523 0.48
GRMZM2G119169 60S ribosomal protein L17 0.3371 0.1421 0.42
GRMZM2G091921 60S ribosomal protein L32 0.5604 0.2650 0.48
980
Figure legends 981
Figure 1. Phenotypic features of maize dek*/rea1-ref mutants. 982
A. A 15 DAP F2 ear of dek* ×W64A and randomly selected 15 DAP dek* and wild type 983
(WT) kernels in segregated F2 population, red arrow identifies the dek* kernel, Bar = 984
5mm. 985
B. Mature F2 ear of dek*×W64A and randomly selected mature dek* and WT kernels in 986
segregated F2 population, red arrow identifies the dek* kernel, Bar = 5mm. 987
C. Comparison of 100-grain weight of randomly selected mature dek* and WT kernels in 988
segregated F2 population. Values are the mean values with standard errors, n= 3 989
individuals (***P<0.001, Student’s t-test). 990
D. Comparison of total, zein, and nonzein proteins from dek* and WT mature endosperm. 991
The measurements were done on per mg of dried endosperm. Values are the mean 992
values with standard errors, n= 3 individuals (ns refers to not significant, **P<0.01, 993
Student’s t-test). 994
E. The soluble amino acids with different content in dek* and WT mature endosperm. 995
Values are the mean values with standard errors, n= 3 individuals (*P<0.05, **P<0.01, 996
***P<0.001, Student’s t-test). 997
F. Paraffin sections of 15 DAP and 18 DAP dek* and WT embryo. Bars = 200μm. 998
G. Microstructure of developing endosperms of dek* and WT (15 DAP and 18 DAP), SG, 999
starch granule. Bars = 100μm. 1000
H. Phenotype of dek* and WT seedlings (4 DAG and 7 DAG). Bar = 5 cm. 1001
1002
Figure 2. Map-based cloning and identification of Rea1. 1003
A. The Dek* locus was mapped to a 101.6kb region between molecular markers SNP064 1004
and SNP165 on chromosome 6, which contained four candidate genes. See 1005
Supplemental Table 3 online for primer information. 1006
B. Protein structure of ZmRea1 and mutation sites in the ZmRea1 gene. 1007
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32
C.Heterozygous rea1-ref / dek* and rea1-Mu were used in allelism test because 1008
homozygous rea1-Mu were lethal. Top, heterozygous rea1-ref X heterozygous rea1-Mu; 1009
Middle, heterozygous rea1-Mu X heterozygous rea1-ref; Bottom, heterozygous rea1-Mu X 1010
heterozygous rea1-Mu. Red arrows identify the rea1 kernel. 1011
D. qRT-PCR comparing expression level of ZmRea1 gene in the 15 DAP and 18 DAP 1012
rea1-ref and WT kernels. Ubiquitin was used as internal control. Values are the mean 1013
values with standard errors, n= 3 individuals (***P<0.001, Student’s t-test). 1014
E. Dot-immunoblot comparing accumulation of ZmRea1 protein in the 15 DAP and 18 1015
DAP rea1-ref and WT kernels. 720 ng, 360 ng and 180 ng 15 DAP and 18 DAP rea1-ref 1016
and WT kernel proteins were subjected to immunoblot analysis with antibodies against 1017
ZmRea1. 1018
1019
Figure 3. Phylogenetic analysis, expression pattern and sub-cellular localization of 1020
ZmRea1. 1021
A. Phylogenetic relationships of ZmRea1 and its homologs. Maize Rea1 and identified 1022
Rea1 proteins in Brachypodium distachyon, Triticum, Oryza sativa, Setaria, Arabidopsis 1023
thaliana, Populus, Glycine-max, Dictyostelium, Monodelphis domestica, Saprelegnia, 1024
Mortierella, and Saccharomyces Cerevisiae were aligned by MUSCLE method in MEGA 1025
5.2 software package. The phylogenetic tree was constructed using MEGA 5.2. The 1026
numbers at the nodes represent the percentage of 1000 bootstraps. 1027
B. RNA expression level of ZmRea1 in various tissues. Ubiquitin was used as an internal 1028
control. Representative results from two biological replicates are shown. For each RNA 1029
sample, three technical replicates were performed. Values are the mean values with 1030
standard errors, n= 6 individuals. 1031
C. Expression profiles of ZmRea1 during maize kernel development. Ubiquitin was used 1032
as an internal control. Representative results from two biological replicates are shown. For 1033
each RNA sample, three technical replicates were performed. Values are the mean 1034
values with standard errors, n= 6 individuals. 1035
D, Dot-immunoblot analysis of ZmRea1 protein accumulation. Rea1 is predominantly 1036
associated with the nuclear protein fraction. 720 ng, 360 ng and 180 ng nuclear (Histone 1037
as nuclear marker) and cytoplasmic (Bip as cytoplasm marker) fraction proteins were 1038
subjected to immunoblot analysis with antibodies against ZmRea1. 1039
1040
Figure 4. Production of mature 60S subunits is reduced in rea1-ref kernels. 1041
A. Analysis of ribosome profiles (A254 nm) was performed by sedimentation 1042
centrifugation in 15-45% sucrose density gradients: 40 S, 60 S, 80 S ribosomes and 1043
polysomes are indicated. 1044
B. Immunoblot analysis of ribosome proteins accumulated in nuclear and cytoplasmic 1045
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33
fractions. Nuclear and cytoplasmic fraction proteins of 15 and 18 DAP rea1-ref and wild 1046
type kernels were subjected to immunoblot analysis with antibodies against eRPL13 (60S 1047
ribosomal subunit marker), eRPS14 (40S ribosomal subunit marker), Bip (cytoplasm 1048
marker), Histone (nuclear marker), Tubulin (cytoplasm sample loading control), and TBP 1049
(nuclear sample loading control). 1050
C. Ultrastructure of developing endosperms of wild type and rea1-ref (15 DAP) for nucleus 1051
observation. There was nucleolus stress, as shown by the expended nucleolus in rea1-ref. 1052
Bars = 2μm. NL, Nucleolus; NP, Nucleoplasm; SG, Starch granule. The nucleolus size/ 1053
nucleus size measurements were done on TEM results. Values are the mean values with 1054
standard errors, n= 10 individuals (**P<0.01, Student’s t-test). 1055
1056
1057
Figure 5, GO classification for genes with altered expression in rea1-ref kernels. 1058
A. The most significantly related GO terms of the 828 functional annotated DTGs. The 1059
significance and number of genes classified within each GO term is shown. 1060
B. qRT-PCR confirmation of DTGs associated with each category, including small 1061
ribosomal subunit proteins (GRMZM5G851698, GRMZM2G120432, GRMZM2G130544, 1062
GRMZM2G156110, GRMZM2G151252); large ribosomal subunit proteins 1063
(GRMZM2G132968, GRMZM2G100403, GRMZM2G168330, GRMZM2G030731, 1064
GRMZM2G010991); ribosome biogenesis factors (GRMZM2G063700, 1065
GRMZM2G110233, GRMZM2G468932); 60S acidic ribosomal proteins 1066
(GRMZM2G157443, GRMZM2G077208); translation elongation factors 1067
(GRMZM2G151193, GRMZM2G153541, GRMZM2G122871, GRMZM2G029559); 1068
histones (GRMZM2G056231, GRMZM2G401147, GRMZM2G078314, 1069
GRMZM2G479684, GRMZM2G164020); nucleosome assembly protein 1070
(GRMZM2G176707); nutrient reservoir activity (GRMZM2G346897, GRMZM2G059620, 1071
GRMZM2G138727). Ubiquitin was used as an internal control. Values are the mean 1072
values with standard errors, n= 6 individuals (***P<0.001, Student’s t-test). 1073
1074
Figure 6. Induced general translation efficiency and specific regulation of 1075
translation of individual mRNAs in rea1-ref kernels. 1076
A. Immunoblot comparing the phosphorylated eIF2α accumulation in wild type and 1077
rea1-ref kernels (15 DAP and18 DAP). Anti- eIF2α was used as control. And immunoblot 1078
comparing the accumulation of eEF1αin wild type and rea1-ref kernels at the same stage. 1079
Anti-Tub was used as sample loading control. 1080
B and C. qRT-PCR confirmation of DTRs with increased or decreased translation level: 1081
DTRs with increased translation level (GRMZM2G081930, GRMZM2G007038, 1082
GRMZM2G110509) and DTRs with decreased translation level (GRMZM2G084195, 1083
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34
GRMZM2G154267, GRMZM2G021069). Samples were mRNA preps from the polysome 1084
fractions. Ubiquitin was used as an internal control. Values are the mean values with 1085
standard errors, n= 6 individuals (*P<0.05, **P<0.01, Student’s t-test). 1086
D. Immunoblot comparing the accumulation of 22kD and 19kD α-zein in 470 ng and 1,190 1087
ng total protein of 15 DAP rea1-ref and wild-type kernels by protein gel blot analysis with 1088
22kD and 19kD α-zein specific antibodies. 1089
E. The overlap between transcriptional up-regulated genes and translational 1090
down-regulated genes for ribosomal proteins and histones, and the overlap between 1091
transcriptional down-regulated genes and translational up-regulated genes for zeins. 1092
1093
Figure 7. Evidence of inhibited cell proliferation and cell growth in rea1-ref kernels. 1094
A and B. Cell proliferation analysis of 15 DAP and 18 DAP endosperms of the wild type 1095
and rea1-ref. The small graph inserted is the cell cycle diagrams analyzed by flow 1096
cytometry. The 3C and 6C are DNA contents of the nuclei at stage G1 and S phase of 15 1097
DAP and 18 DAP endosperms. The 12C and 24C are DNA contents of endoreduplicated 1098
nuclei at stage S phase of 15 DAP and 18 DAP endosperms. For each sample, three 1099
independent biological replicates were performed. Values are the mean values with 1100
standard errors, n= 3 individuals (**P<0.01, ***P<0.001, Student’s t-test). 1101
C. Cell proliferation analysis of 7 DAG seedlings of the wild type and rea1-ref. The small 1102
graph inserted is the cell cycle diagrams analyzed by flow cytometry. The 2C and 4C are 1103
DNA contents of the nuclei at stage G1/S phase and G2/M phase of 7 DAG seedlings. For 1104
each sample, three independent biological replicates were performed. Values are the 1105
mean values with standard errors, n= 3 individuals (**P<0.01, Student’s t-test). 1106
D. Scanning electron microscopy analysis of the lower epidermis of the first leaf mature 1107
region of 7 DAG rea1-ref and wild-type seedlings. Bars = 50μm. S,stoma. 1108
E. Comparison of cell width and cell length of lower epidermis in wild type and rea1-ref 7 1109
DAG seedlings. The measurements were done on SEM results. Values are the mean 1110
values with standard errors, n= 50 individuals (***P<0.001, Student’s t-test). 1111
1112
Figure 8. Suppressed 60S ribosome biogenesis promotes translation initiation and 1113
ribosome usage, as well as inconsistently regulates the transcription and 1114
translation of individual genes that affects general translation efficiency and cell 1115
proliferation. 1116
1117
References 1118
Andersen JS, Lam YW, Leung AK, Ong SE, Lyon CE, Lamond AI, Mann M (2005) 1119
Nucleolar proteome dynamics. Nature. 433:77–83 1120
Bailey-Serres J, Dawe RK (1996) Both 50 and 30 sequences of maize adh1 mRNA are 1121
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required to enhance translation under low oxygen. Plant Physiology 112: 685–695 1122
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Galani K, Nissan TA, Petfalski E, Tollervey D, Hurt E (2004) Rea1, a dynein-related nuclear AAA-ATPase, is involved in late rRNAprocessing and nuclear export of 60S subunits, J. Biol. Chem. 279: 55411-55418
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Granneman S, Baserga SJ (2004) Ribosome biogenesis: of knobs and RNA processing, Exp. Cell Res. 296:43-50Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Gawron D, Gevaert K, Van Damme P (2014) The proteome under translational control. Proteomics 14: 2647-62Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Grimm T, Holzel M, Rohrmoser M, Harasim T, Malamoussi A, Gruber-Eber A et al (2006) Dominant-negative Pes1 mutants inhibitribosomal RNA processing and cell proliferation via incorporation into the PeBoWcomplex. Nucleic Acids Res 34:3030-43
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Harding HP, Zhang Y, Ron D (1999) Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase.Nature 397: 271-274
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Harding HP, Novoa I, Zhang Y, Zeng H, Wek R, Schapira M, Ron D (2000) Regulated translation initiation controls stressinducedgene expression in mammalian cells. Mol. Cell 6: 1099-1108 https://plantphysiol.orgDownloaded on March 12, 2021. - Published by
Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Heidecker G, Messing J (1986). Structural analysis of plant genes. Annual review of plant physiology 37: 439-466Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Helser TL, Baan RA, Dahlberg AE (1981) Characterization of a 40S ribosomal subunit complex in polyribosomes of Saccharomycescerevisiae treated with cycloheximide. MolCell Biol 1: 51-57
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Henras AK, Soudet J, Gerus M, Lebaron S, Caizergues-Ferrer M, Mougin A, Henry Y (2008) The post-transcriptional steps ofeukaryotic ribosome biogenesis, Cell. Mol. Life Sci. 65:2334-2359
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Holding DR, Larkins BA (2009). Zein storage proteins. Molecular Genetic Approaches to Maize Improvement: 269-286Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Holding DR, Meeley RB, Hazebroek J, Selinger D, Gruis F, Jung R, Larkins BA (2010) Identification and characterization of themaize arogenate dehydrogenase gene family. J. Exp. Bot. 61: 3663-3673
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Huizen R, Martindale JL, Gorospe M, Holbrook NJ (2003) P58IPK, a Novel Endoplasmic Reticulum Stress-inducible Protein andPotential Negative Regulator of eIF2a Signaling. The journal of biological chemistry 278: 15558-15564
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
James A, Wang Y, Raje H, Rosby R, DiMario P (2014) Nucleolar stress with and without p53. Nucleus. 5:402-26Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Jiao Y, Meyerowitz EM (2010) Cell-type specific analysis of translating RNAs in developing flowers reveals new levels of control.Mol Syst Biol. 6:419
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kawaguchi R, Bray EA, Bailey-Serres J (2003) Water-deficit induced translational control in Nicotiana tabacum. Plant, Cell andEnvironment, 26: 221-229
Pubmed: Author and TitleCrossRef: Author and TitleGoogle Scholar: Author Only Title Only Author and Title
Kawaguchi R, Girke T, Bray EA, Bailey-Serres J (2004) Differential mRNA translation contributes to gene regulation under non-stress and dehydration stress conditions in Arabidopsis thaliana. The Plant Journal 38: 823-839
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