Maize reas1 Mutant Stimulates Ribosome Use
Efficiency and Triggers Distinct Transcriptional and
Translational Responses1[OPEN]
Weiwei Qi 2, Jie Zhu 2, Qiao Wu 2, Qun Wang, Xia Li, Dongsheng Yao, Ying Jin, Gang Wang, Guifeng Wang,
and Rentao Song*
Shanghai Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444,
China (W.Q., J.Z., Q.Wu., Q.Wa., X.L., D.Y., Y.J., Ga.W., Gu.W., R.S.); and Coordinated Crop Biology Research
Center, Beijing 100193, China (W.Q., Ga.W., Gu.W., R.S.) and National Maize Improvement Center of China,
China Agricultural University, Beijing, 100193, China (R.S)
ORCID ID: 0000-0003-1810-9875 (R.S.).
Ribosome biogenesis is a fundamental cellular process in all cells. Impaired ribosome biogenesis causes developmental defects;
however, its molecular and cellular bases are not fully understood. We cloned a gene responsible for a maize (Zea mays) small
seed mutant, dek* (for defective kernel), and found that it encodes Ribosome export associated1 (ZmReas1). Reas1 is an AAAATPase that controls 60S ribosome export from the nucleus to the cytoplasm after ribosome maturation. dek* is a weak mutant
allele with decreased Reas1 function. In dek* cells, mature 60S ribosome subunits are reduced in the nucleus and cytoplasm, but
the proportion of actively translating polyribosomes in cytosol is significantly increased. Reduced phosphorylation of eukaryotic
initiation factor 2a and the increased elongation factor 1a level indicate an enhancement of general translational efficiency in dek*
cells. The mutation also triggers dramatic changes in differentially transcribed genes and differentially translated RNAs.
Discrepancy was observed between differentially transcribed genes and differentially translated RNAs, indicating distinct
cellular responses at transcription and translation levels to the stress of defective ribosome processing. DNA replication and
nucleosome assembly-related gene expression are selectively suppressed at the translational level, resulting in inhibited cell
growth and proliferation in dek* cells. This study provides insight into cellular responses due to impaired ribosome biogenesis.
Ribosomes are organelles that translate genetic information into proteins. A great percentage of total
RNA transcription is devoted to ribosomal RNA synthesis, and a great part of RNA polymerase II transcription and mRNA splicing are devoted to the
synthesis of ribosomal proteins (Warner, 1999). Ribosome biosynthesis consumes approximately 80% of a
cell’s energy (James et al., 2014). In eukaryotes, ribosome biogenesis begins in the nucleolus with the transcription of a large ribosomal precursor RNA that gives
rise to the 90S preribosomal particle. Cleavages of the
1
This work was supported by the Major Research Plan of the
National Natural Sciences Foundation of China (grant nos.
91335208 and 31425019) and the Ministry of Science and Technology
of China (grant no. 2014CB138204).
2
These authors contributed equally to the article.
* Address correspondence to [email protected].
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is:
Rentao Song ([email protected]).
R.S., W.Q., and J.Z. designed the experiments; J.Z., W.Q., Q.Wu.,
Q.Wa., X.L., D.Y., and Y.J. performed the experiments; W.Q., J.Z.,
Ga.W., Gu.W., and R.S. analyzed the data; W.Q. and R.S. wrote the
article.
[OPEN]
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90S particle generate two subunits: the pre-40S and pre60S complexes. The pre-40S and pre-60S subunits mature in the nucleolus and nucleoplasm before being
exported to the cytoplasm (Venema and Tollervey,
1999; Fromont-Racine et al., 2003; Granneman and
Baserga, 2004). Inhibition of ribosome biogenesis causes
developmental defects in yeast (Saccharomyces cerevisiae),
humans, and plants (Tschochner and Hurt, 2003; Galani
et al., 2004; Ruan et al., 2012).
A great deal of research has revealed that hundreds of
ribosomal biogenesis factors contribute to maturation of
the ribosome in eukaryotes (Tschochner and Hurt, 2003;
Henras et al., 2008), including three essential AAAATPases: Ribosome export7 (Rix7), Ribosome export
associated1 (Rea1), and Diazaborine resistance gene1
(Pertschy et al., 2007; Kressler et al., 2008, 2012; Ulbrich
et al., 2009; Bassler et al., 2010). The Rea1 AAA-ATPase is
the best-characterized ATPase in ribosome biogenesis
and is conserved from yeast to humans (Bassler et al.,
2010; Kressler et al., 2012). Rea1 promotes the stripping of
other biogenesis factors from the pre-60S particle in the
nucleolus and nucleoplasm (Ytm1-Erb1-Nop7 and Rsa4)
prior to the export of the large ribosomal subunit to the
cytoplasm (Bassler et al., 2010). However, there is not a
comprehensive understanding of cellular responses to
the impaired large ribosomal subunit export.
The regulation of mRNA translation is a critical feature of gene expression in eukaryotes (Bailey-Serres,
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Qi et al.
1999). Previous studies highlight the importance of
translational control in determining protein abundance,
underscoring the value of measuring gene expression at
the level of translation. Mechanisms that underlie differential mRNA translation are likely to involve nucleotide sequence features and the phosphorylation
status of initiation factors (Bailey-Serres and Dawe,
1996; Pop et al., 2014). Transcriptome and translatome
analyses of the cellular response to heat shock, cell cycle
arrest, and mating pheromone in Saccharomyces cerevisiae (Preiss et al., 2003; Serikawa et al., 2003; MacKay
et al., 2004), the hypoxia response of HeLa cells (Blais
et al., 2004), and the drought and oxygen deprivation responses in Arabidopsis (Arabidopsis thaliana; Kawaguchi
et al., 2004; Branco-Price et al., 2005) have shown the
importance of translational regulation. These researchers
investigated the correlation between total and polyribosome (polysome)-bound mRNA accumulation and
provided extensive evidence of variation in the translational regulation of individual mRNAs. These studies
showed that mRNAs differ in their association with
polysomes under different circumstances, and gene
expression can be regulated at the translational level
without a change in mRNA abundance.
Maize (Zea mays) is especially well suited for genetic
studies, partly because of the feasibility to generate a
wide range of easily observable phenotypes (Neuffer
and Sheridan, 1980). Many kernel mutants are known
(Neuffer et al., 1968), among which one class is defective
kernel (dek) mutants (Neuffer and Sheridan, 1980). dek
mutants are a good resource to investigate seed development. For example, Dek1 encodes a large membrane
protein of the calpain gene superfamily (Lid et al.,
2002). In dek1 mutants, embryogenesis is blocked, while
the endosperm lacks the aleurone layer and is chalky
(Becraft et al., 2002). Other dek mutants offer opportunities to investigate many basic biological processes,
because embryo formation is the first developmental
process after fertilization. Such defects in basic biological processes create visible phenotypes during kernel
development.
In this study, we characterized dek*, a novel mutant
with small kernels and delayed development of the
embryo, endosperm, and seedling. We report the mapbased cloning of Dek* and demonstrate that it encodes
Rea1 in maize. dek* is a weak mutant allele that only
partly represses the maturation and export of the 60S
ribosomal subunit. Taking advantage of this mutant
allele, we were able to obtain comprehensive information about the cellular responses to impaired 60S ribosomal subunit biogenesis.
RESULTS
dek* Produces Small Kernels with Delayed Development
The dek* mutant was isolated from an opaque mutant
stock obtained from the Maize Genetic Stock Center. It
was crossed to the W64A inbred line to produce an
F2 population that displayed a 1:3 segregation of dek
(dek*/dek*) and wild-type (+/+ and dek*/+) phenotypes
(Fig. 1, A and B). At 15 DAP, homozygous dek* kernels
exhibited a small, vague phenotype (Fig. 1A), and mature kernels were small and shrunken (Fig. 1B). The
100-kernel weight of dek* was nearly 39.5% less than
that of the wild type (Fig. 1C), but there was no significant difference in the total protein and zein contents
(Fig. 1D; Supplemental Fig. S1), although there was
a slight increase in the amount of nonzeins (13.5%;
Fig. 1D). Among zein proteins, the 22-kD a-zeins
were relatively more abundant in dek* endosperms
(Supplemental Fig. S1). We found no obvious difference
in total starch content and the percentage of amylose in
dek* and wild-type endosperms (Supplemental Fig. S2).
We analyzed soluble amino acids to determine if the
slight increase of nonzeins in dek* altered their composition. The results showed that the amount of Lys was
most significantly increased (23.1%) due to the slight
increase of nonzein content (Fig. 1E), for zeins lack Lys
residues (Mertz et al., 1964).
Wild-type and dek* kernels of 15 and 18 DAP were
analyzed by light microscopy to compare their development. Longitudinal sections of the embryos indicated that
development of the plumule and seminal was delayed
more than 3 d in dek* compared with the wild type (Fig.
1F). To investigate endosperm development, we observed
15- and 18-DAP immature endosperm cells using optical
microscopy. The endosperm cells of dek* kernels were less
cytoplasmic dense with fewer starch granules compared
with wild-type kernels of the same stage, also indicating
more than a 3-d delay in development (Fig. 1G).
At 4 and 7 d after germination (DAG), seedlings of
dek* showed a 3-d developmental delay compared with
the wild type (Fig. 1H). By 4 DAG, wild-type seedlings
had two leaves, one completely expanded and the other
emerging; the dek* seedlings had only one leaf at this
stage. The 7-DAG wild-type seedlings had three leaves,
while the dek* seedlings had only two leaves. The
heading stage of the dek* plant was delayed approximately 15 d compared with the wild type, and its height
was only 50% of the wild type (Supplemental Fig. S3).
These results demonstrated that the growth and development of dek* kernels and seedlings is delayed
compared with the wild type.
Positional Cloning of Dek*
Genetic fine-mapping of Dek* was carried out with
the F2 mapping population, and the Dek* gene was
placed between the simple sequence repeat markers
mmc0241 and umc2162 on the long arm of chromosome
6 (Fig. 2A). After characterizing a mapping population
of 864 individuals, Dek* was mapped between the
self-created simple sequence repeat markers 153.7M-2
(19 recombinants) and 155.1M-1 (29 recombinants).
Additional markers InDel438, InDel428, SNP064, and
SNP165 were developed, and the Dek* gene was eventually placed between SNP064 (one recombinant) and
SNP165 (two recombinants), a region encompassing a
physical distance of 101.6 kb (Fig. 2A).
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Characterization of the Maize reas1 Mutant
Figure 1. Phenotypic features of maize dek*/reas1-ref mutants. A, A 15-d after pollination (DAP) F2 ear of dek* 3 W64A and randomly
selected 15-DAP dek* and wild-type (WT) kernels in a segregated F2 population. The red arrow identifies the dek* kernel. Bar = 5 mm. B,
Mature F2 ear of dek* 3 W64A and randomly selected mature dek* and wild-type kernels in a segregated F2 population. The red arrow
identifies the dek* kernel. Bar = 5 mm. C, Comparison of 100-grain weight of randomly selected mature dek* and wild-type kernels in a
segregated F2 population. Values are means with SE; n = 3 individuals (***, P , 0.001, Student’s t test). D, Comparison of total, zein, and
nonzein proteins from dek* and wild-type mature endosperm. The measurements were done per mg of dried endosperm. Values are means
with SE; n = 3 individuals (ns, not significant; **, P , 0.01, Student’s t test). E, Soluble amino acids with different contents in dek* and wildtype mature endosperm. Values are means with SE; n = 3 individuals (*, P , 0.05; **, P , 0.01; ***, P , 0.001, Student’s t test). F, Paraffin
sections of 15- and 18-DAP dek* and wild-type embryos. Bars = 200 mm. G, Microstructure of developing endosperms of dek* and the
wild type (15 and 18 DAP). SG, Starch granule. Bars = 100 mm. H, Phenotypes of dek* and wild-type seedlings (4 and 7 DAG). Bars = 5 cm.
Nucleotide sequence analysis within this region identified 10 predicted open reading frames with gene model
information (GRMZM2G405052, GRMZM2G387038,
GRMZM5G873561, GRMZM5G807823, GRMZM2G361064,
GRMZM5G892685, GRMZM2G059268, GRMZM2G059278,
GRMZM2G323939, and GRMZM2G128315). Expression
analysis revealed no expression of GRMZM5G892685,
GRMZM2G059268, and GRMZM2G059278 based
on reverse transcription-PCR and EST information
(http://www.maizegdb.org/); consequently, these
three might be pseudogenes. DNA sequence analysis
revealed that GRMZM2G405052, GRMZM2G387038,
GRMZM5G873561, and GRMZM5G807823, together
with GRMZM2G092001 and GRMZM2G149586,
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Qi et al.
Figure 2. Map-based cloning and identification of Reas1 A, The Dek* locus was mapped to a 101.6-kb region between molecular
markers SNP064 and SNP165 on chromosome 6, which contained four candidate genes. For primer information, see
Supplemental Table S3. B, Protein structure of ZmReas1 and mutation sites in the ZmReas1 gene. C, Heterozygous reas1-ref/dek*
and reas1-Mu were used in an allelism test because homozygous reas1-Mu was lethal. Top, heterozygous reas1-ref 3 heterozygous
reas1-Mu; middle, heterozygous reas1-Mu 3 heterozygous reas1-ref; bottom, heterozygous reas1-Mu 3 heterozygous reas1-Mu. The
red arrows identify the reas1 kernel. D, qRT-PCR comparing the expression levels of ZmReas1 in 15- and 18-DAP reas1-ref and wildtype (WT) kernels. Ubiquitin was used as an internal control. Values are means with SE; n = 3 individuals (***, P , 0.001, Student’s t
test). E, Dot immunoblot comparing the accumulation of ZmReas1 protein in 15- and 18-DAP reas1-ref and wild-type kernels. The
720-, 360-, and 180-ng 15- and 18-DAP reas1-ref and wild-type kernel proteins were subjected to immunoblot analysis with antibodies against ZmReas1.
which are upstream of the candidate region, produced
one huge transcript that was identified as candidate
Gene1. There is a single-nucleotide polymorphism in
Gene1 resulting in an amino acid replacement between
the alleles of dek* and the wild type. GRMZM2G361064,
GRMZM2G323939, and GRMZM2G128315 were identified as candidate Gene2, Gene3, and Gene4, respectively; however, their consideration for Dek* was
eliminated due to no sequence differences between alleles in dek* and the wild type (Fig. 2A). Therefore,
Gene1 appeared to be the best candidate for the Dek*
locus.
Dek* Encodes the 60S-Specific Ribosome Biogenesis
Factor Rea1
The genomic DNA sequence of candidate Gene1
spans approximately 50 kb and produces a huge
transcript containing a 16,278-bp coding sequence
(Fig. 2B). Sequence data for this gene have been deposited in GenBank (http://www.ncbi.nlm.nih.gov/)
as accession number KP137367. Gene1 encodes an
approximately 600-kD protein of 5,425 amino acids.
BLASTP searches of GenBank indicated that Gene1
encodes a ribosome biogenesis factor, AAA-ATPase
Rea1, with several conserved domains in maize (Fig.
2B). And we named it ZmReas1. ZmReas1 contains
different kinds of molecular domains: a weakly conserved N-terminal region, a dynein-like tandem array
of six AAA-type ATPase domains (Neuwald et al.,
1999), a large linker, a D/E-rich region, and a metal
ion-dependent adhesion site (MIDAS) domain (Fig.
2B). Rea1 promotes the release of Ytm1, which associates with nucleolar pre-60S particles, and later also
promotes the release of Rsa4, which associates with
nucleoplasmic pre-60S particles via the MIDAS-MIDASinteracting domain using the mechanical force created
by the ATPase ring domain for the export of the large
ribosomal subunit to the cytoplasm (Ulbrich et al., 2009;
Bassler et al., 2010). The mutation in the dek* allele of
ZmReas1 is a single-nucleotide polymorphism at codon
2,359 of ZmReas1, which results in Ala (GCC) being
replaced by Val (GTC; Fig. 2B). This mutation alters the
highly conserved region between the dynein-like array of
six AAA-type ATPases and the large linker, which could
affect the transduction of the mechanical force created by
the ATPase ring domain to the large tail for release of the
ribosome biogenesis factors.
To confirm if ZmReas1 is the Dek* gene, we carried out
an allelism test with a Mu-induced mutant of ZmReas1
(Fig. 2, B and C). A UniformMu insertion mutant (reas1Mu) stock for GRMZM2G092001 was obtained from
the Maize Genetics Stock Center. This mutant has a
Mutator-8 insertion after the fourth nucleotide of the
ZmReas1 coding sequence and is not viable (Fig. 2B).
The allelism test was done by crossing dek* F1 (dek*/+)
and reas1-Mu F1 (reas1-Mu/+). The kernel phenotypes
in the F2 ears displayed a 1:3 segregation of 82 dek
(dek*/reas1-Mu) and 249 wild-type phenotype kernels
(Fig. 2C), indicating that reas1-Mu cannot complement
dek*. Therefore, Gene1 (ZmReas1) is indeed the Dek*
gene. We hence named dek* as reas1-ref.
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Characterization of the Maize reas1 Mutant
Maize endosperm is a triploid tissue with two maternal and one paternal genomes. The mutant kernels in
reas1-ref/+ (maternal) 3 reas1-Mu/+ (paternal) F2 ear
are small and shallow, similar to homozygous reas1-ref
kernels. The mutant kernels in reas1-Mu/+ (maternal) 3
reas1-ref /+ (paternal) F2 ears display an even more severe phenotype with dramatically shrunken kernels. The
mutant kernels from reas1-Mu/+ selfing are nonviable
(Fig. 2C). Thus, results of the allelism test show that reas1ref is a weak allele compared with reas1-Mu, which has a
lethal phenotype.
To examine Reas1 mRNA expression in reas1-ref, we
performed quantitative reverse transcription (qRT)PCR with the total RNA extracted from 15- and 18-DAP
mutant and wild-type kernels. Surprisingly, mRNA
expression of Reas1 was significantly up-regulated in
reas1-ref (Fig. 2D). Because ZmReas1 is too large (approximately 600 kD) to perform a regular western-blot
analysis, we used dot-immunoblot analysis on quantified and gradient-diluted total protein samples with
Reas1 specific antibody to detect its existence in 15- and
18-DAP reas1-ref and wild-type kernels (Fig. 2E). The
results demonstrated that Reas1 is present in reas1-ref
and accumulates in reas1-ref at normal levels, but it
might be only partly functional.
Reas1 Is Highly Conserved in Different Organisms and Is
Constitutively Expressed in Maize
Rea1 was first identified as a component of pre-60S
ribosome complex in yeast and is conserved from yeast
to humans (Bassler et al., 2001, 2010; Kressler et al.,
2012). We constructed a phylogenetic tree on the basis
of the ZmReas1 full-length protein sequence and Rea1
protein sequences from Brachypodium distachyon, Triticum urata, Oryza sativa, Setaria italica, Arabidopsis,
Populus trichocarpa, Glycine max, Dictyostelium discoideum, Monodelphis domestica, Saprelegnia diclina,
Mortierella verticillata, and Saccharomyces cerevisiae. The
results suggest that ZmReas1 is highly conserved with the
Rea1 proteins in other plants as well as the Rea1 proteins
of yeast, mammals, and microorganisms (Fig. 3A).
Quantitative RT-PCR analysis revealed that ZmReas1
is expressed in a broad range of maize tissues, including
silk, tassel, ear, root, husk, stem, leaf, and kernel (Fig.
3B). During kernel development, expression of Reas1
occurs before 5 DAP and continues later than 25 DAP
(Fig. 3C). Dot-immunoblot analysis on quantified and
gradient-diluted total nuclear and cytoplasmic proteins
detected Reas1 in these subcellular fractions, and it was
predominantly found in the nuclear fraction, consistent
with Reas1 localization in the nucleus (Fig. 3D).
reas1-ref Affects the Biogenesis of 60S Ribosomal Subunits
To investigate the effect of reas1-ref on ribosomal
subunit biogenesis and the formation of monosome and
polysome complexes, polysome profiles of 15-DAP
reas1-ref and wild-type kernel extracts were analyzed
by 15% to 45% (w/v) Suc gradient centrifugation. Two
independent biological replicates were performed. This
analysis revealed a significant reduction of 60S ribosomal subunits, as compared with 40S ribosomal subunits, in the mutant (Fig. 4A). To compare the levels of
monosomes and polysome complexes, calculation of
the peak areas of A254 revealed that about 40.2% of the
ribosomes in wild-type kernel extracts were in polysomes, while the level of polysome complexes in reas1ref kernel extracts was 57.2% (Fig. 4A). Thus, there are
1.4-fold greater polysomes/total ribosomes in reas1-ref.
The decrease in 60S subunits and the increase in polysomes are consistent with the inhibition of large ribosomal subunit export and the promotion in initiation of
protein synthesis as a consequence of down-regulated
ribosome biogenesis in reas1-ref.
To confirm that maturation and export of 60S subunits are reduced in reas1-ref, immunoblot analysis with
60S and 40S subunit antibodies was performed on nuclear and cytoplasmic fractions from 15- and 18-DAP
reas1-ref or wild-type kernels. Nuclear and cytoplasmic
fractions were subjected to immunoblot analysis with
antibodies against Bip (cytoplasm marker) and histone
(nucleus marker). Tubulin and TATA box-binding
protein (TBP) served as sample loading controls of cytoplasmic and nuclear proteins, respectively. The level
of eukaryotes 60S ribosomal protein L13 eRPL13 was
examined using an L13-specific antibody. L13 protein
was markedly decreased in both the nuclear and cytoplasmic fractions of reas1-ref compared with wild-type
kernels (Fig. 4B). The level of eRPS14 was also examined using a specific antibody, and its content was the
same in both the nuclear and cytoplasmic fractions of
reas1-ref and wild-type kernels (Fig. 4B). Meanwhile, we
also observed a slight decrease of histone protein in the
reas1-ref nuclear fraction (Fig. 4B).
A reduction of 60S ribosomal subunits in the cytoplasm of 15- and 18-DAP reas1-ref and wild-type endosperms was also observed by transmission electron
microscopy (TEM) analysis. There were fewer ribosomes on rough endoplasmic reticulum and the
rough endoplasmic reticulum around protein bodies
(Supplemental Fig. S4). Nucleolus stress due to ribosomal failure alters the morphology and increases the
surface area of nucleolus in humans (Bailly et al., 2015).
This stress, which misshapes and expands the nucleolus, was also observed in reas1-ref endosperm by TEM
(Fig. 4C). All these data are consistent with a biogenesis
defect of 60S subunits and demonstrates that it is specific to the 60S maturation and export pathway.
reas1-ref Affects the Transcription of Ribosome Biogenesis,
Translational Elongation, and Nucleosome-Related Genes
We compared the transcript profiles of 15-DAP reas1ref and wild-type endosperm using RNA sequencing
(RNA-seq). Among the 45,730 gene transcripts detected
by RNA-seq, significantly differentially transcribed
genes (DTGs) were identified as those with a threshold
fold change greater than 2 and P , 0.05. Based on this
criterion, 2,076 genes showed significantly altered
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Qi et al.
Figure 3. Phylogenetic analysis, expression pattern, and subcellular localization of ZmReas1 A, Phylogenetic relationships of
ZmReas1 and its homologs. Maize Reas1 and identified Rea1 proteins in B. distachyon, T. urata, O. sativa, S. italica, Arabidopsis,
P. trichocarpa, G. max, D. discoideum, M. domestica, S. diclina, M. verticillata, and S. cerevisiae were aligned by the MUSCLE
method in the MEGA 5.2 software package. The phylogenetic tree was constructed using MEGA 5.2. The numbers at the nodes
represent the percentage of 1,000 bootstraps. B, RNA expression level of ZmReas1 in various tissues. Ubiquitin was used as an
internal control. Representative results from two biological replicates are shown. For each RNA sample, three technical replicates
were performed. Values are means with SE; n = 6 individuals. C, Expression profiles of ZmReas1 during maize kernel development. Ubiquitin was used as an internal control. Representative results from two biological replicates are shown. For each RNA
sample, three technical replicates were performed. Values are means with SE; n = 6 individuals. D, Dot-immunoblot analysis of
ZmReas1 protein accumulation. Reas1 is predominantly associated with the nuclear protein fraction. The 720-, 360-, and 180-ng
nuclear (histone as nuclear marker) and cytoplasmic (Bip as cytoplasm marker) fraction proteins were subjected to immunoblot
analysis with antibodies against ZmReas1.
expression between reas1-ref and the wild type. There
were 1,518 genes with increased transcription, while
558 genes showed decreased transcription.
Within the 2,076 DTGs, 39.9% could be functionally
annotated (annotations were found using BLASTN and
BLASTX analyses against the GenBank (http://www.
ncbi.nlm.nih.gov/) database. Gene Ontology (GO;
http://bioinfo.cau.edu.cn/agriGO/) and Kyoto Encyclopedia of Genes and Genomes (http://www.
genome.jp/kegg/) pathway analysis indicated that 828
DTGs were mostly related to four GO terms: GO: 0005840
(ribosome; P = 2.34E-130), GO: 0006414 (translational
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Characterization of the Maize reas1 Mutant
Figure 4. The production of mature 60S subunits is
reduced in reas1-ref kernels. A, Analysis of ribosome
profiles (A254) was performed by sedimentation centrifugation in 15% to 45% Suc density gradients: 40S,
60S, and 80S ribosomes and polysomes are indicated. B, Immunoblot analysis of ribosome proteins
accumulated in nuclear and cytoplasmic fractions.
Nuclear and cytoplasmic fraction proteins of 15- and
18-DAP reas1-ref and wild type (WT) kernels were
subjected to immunoblot analysis with antibodies
against eRPL13 (60S ribosomal subunit marker),
eRPS14 (40S ribosomal subunit marker), Bip (cytoplasm marker), Histone (nuclear marker), Tubulin
(cytoplasm sample loading control), and TBP (nuclear
sample loading control). C, Ultrastructure of developing endosperms of the wild type and reas1-ref (15
DAP) for nucleus observation. There was nucleolus
stress, as shown by the expended nucleolus in reas1-ref.
NL, Nucleolus; NP, nucleoplasm; SG, starch granule.
The nucleolus size/nucleus size measurements were
done on TEM results. Values are means with SE; n = 10
individuals (**, P , 0.01, Student’s t test). Bars = 2 mm.
elongation; P = 2.30E-17), GO: 0000786 (nucleosome;
P = 8.22E-29), and GO: 0045735 (nutrient reservoir activity; P = 6.47E-33). This analysis is illustrated in Figure 5A
and Supplemental Table S1.
Ninety-eight DTGs classified to GO: 0005840 (ribosome) could be divided into three categories: small ribosomal subunit proteins (e.g. eRPS6 [GRMZM5G851698]
and eRPS13 [GRMZM2G130544]), large ribosomal subunit proteins (e.g. eRPL14 [GRMZM2G168330] and
eRPL18 [GRMZM2G030731]), and ribosome biogenesis
factors. Transcription of all the genes related to ribosome
biogenesis was increased in reas1-ref endosperm. reas1-ref
also has a strong impact on translational elongation.
The 20 DTGs involved in GO: 0006414 (translational
elongation) could be divided into two categories: 60S
acidic ribosomal proteins (e.g. eRPLP0 [GRMZM2G066460]
and eRPLP1 [GRMZM2G157443]) and translation elongation factors (e.g. eEF1a [GRMZM2G151193] and eEF1b
[GRMZM2G122871]). These genes were also up-regulated.
Fifty-two DTGs related to GO: 0000786 (nucleosome) could
be divided into two categories: histones (e.g. H2A
[GRMZM2G056231] and H2B [GRMZM2G401147]) and
nucleosome assembly protein (GRMZM2G176707). Transcription of these genes, which are related to nucleosome
assembly and the cell cycle, was markedly induced in reas1ref endosperm. DTGs involved in GO: 0045735 (nutrient
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Qi et al.
Figure 5. GO classification for genes with altered expression in reas1-ref kernels. A, The most significantly related GO terms of
the 828 functional annotated DTGs. The significance and number of genes classified within each GO term are shown. B, qRT-PCR
confirmation of DTGs associated with each category, including small ribosomal subunit proteins (GRMZM5G851698,
GRMZM2G120432, GRMZM2G130544, GRMZM2G156110, and GRMZM2G151252); large ribosomal subunit proteins
(GRMZM2G132968, GRMZM2G100403, GRMZM2G168330, GRMZM2G030731, and GRMZM2G010991); ribosome biogenesis factors (GRMZM2G063700, GRMZM2G110233, and GRMZM2G468932); 60S acidic ribosomal proteins
(GRMZM2G157443 and GRMZM2G077208); translation elongation factors (GRMZM2G151193, GRMZM2G153541,
GRMZM2G122871, and GRMZM2G029559); histones (GRMZM2G056231, GRMZM2G401147, GRMZM2G078314,
GRMZM2G479684, and GRMZM2G164020); nucleosome assembly protein (GRMZM2G176707); and nutrient reservoir activity (GRMZM2G346897, GRMZM2G059620, and GRMZM2G138727). Ubiquitin was used as an internal control. Values are
means with SE; n = 6 individuals (***, P , 0.001, Student’s t test). WT, Wild type.
reservoir activity) were storage proteins, including
22-kD a-zein (GRMZM2G346897) and 19-kD a-zein
(GRMZM2G059620); that is, these genes were downregulated. To validate the differences observed by RNAseq, we performed qRT-PCR on the most significant
DTGs from each GO category, and the results confirmed
similar differences of mRNA accumulation (Fig. 5B).
reas1-ref Exhibits Uncoordinated Expression of Distinct
Groups of Genes at the Translational Level
The increase in polysomes in reas1-ref indicated promotion in the initiation of protein synthesis in response
to down-regulated ribosome biogenesis. The mechanisms that underlie differences of mRNA translation
involve sequence features of individual mRNAs and
the phosphorylation status of translation initiation
factors (Bailey-Serres and Dawe, 1996). General Control
Nonderepressing kinase2 (GCN2) was reported to
phosphorylate eukaryotic initiation factor 2a (eIF2a) to
down-regulate translation (Zhang et al., 2008). We first
measured the phosphorylation levels of eIF2a in 15and 18-DAP reas1-ref and wild-type cytoplasm by
protein gel-blot analysis with P-eIF2a and eIF2a (total
eIF2a as a control) antibodies. Compared with the wild
type, eIF2a in reas1-ref was significantly less phosphorylated, while the eIF2a protein level was not altered (Fig. 6A). The level of eEF1a protein in reas1-ref
and wild-type cytoplasm was examined using specific
antibody. eEF1a was markedly increased in reas1-ref
(Fig. 6A). These results indicated that the initiation and
elongation of translation are promoted in reas1-ref.
The level of an mRNA in polysomes reflects its
translation (Branco-Price et al., 2005). To examine the
effects of reas1-ref on the translational regulation of individual mRNAs, we evaluated the amount of RNA
in polysomes, relative to the total amount of transcript
in 15-DAP reas1-ref and wild-type kernels. Kernel extracts
were centrifuged (170,000g) to obtain a polysome pellet
for comparing total extract and polysome-bound RNA
samples by RNA-seq analysis. The polysome-boundto-total RNA ratios in 15-DAP reas1-ref and wild-type
kernels were 36.4% and 25.6%, respectively. Consequently, there was a 1.4-fold increase of polysomebound-to-total RNA in reas1-ref, which is consistent
with the polysome complex/total ribosome A254 by ribosome profile analysis.
Within the 30,188 gene transcripts detected by RNA-seq,
significantly differentially translated RNAs (DTRs) were
identified as those with a 2nP/T (normalized polysome-bound/total) 3
100% (see “Materials and Methods”) between reas1-ref
and the wild type, fold change greater than 2 or P , 0.5.
Based on this criterion, 1,802 genes showed significantly
increased translation in reas1-ref compared with the wild
type, while 2,959 genes showed decreased translation. To
confirm the differences between wild-type and reas1-ref
endosperm observed by RNA-seq, we performed qRTPCR on the most significantly increased or decreased
DTRs selected from each category, and the results were
consistent (Fig. 6, B and C). We also performed
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Characterization of the Maize reas1 Mutant
Figure 6. Induced general translation efficiency and specific regulation of translation of individual mRNAs in reas1-ref kernels.
A, Immunoblot comparing the phosphorylated (P) eIF2a accumulation in wild-type (WT) and reas1-ref kernels (15 and 18 DAP).
Anti-eIF2a was used as a control immunoblot comparing the accumulation of eEF1a in wild-type and reas1-ref kernels at the
same stage. Anti-TUB was used as a sample loading control. B and C, qRT-PCR confirmation of DTRs with increased
(GRMZM2G081930, GRMZM2G007038, and GRMZM2G110509) or decreased (GRMZM2G084195, GRMZM2G154267, and
GRMZM2G021069) translation levels. Samples were mRNA preparations from the polysome fractions. Ubiquitin was used as an
internal control. Values are means with SE; n = 6 individuals (*, P , 0.05; **, P , 0.01, Student’s t test). D, Immunoblot comparing
the accumulation of 22- and 19-kD a-zein in 470 and 1,190 ng of total protein of 15-DAP reas1-ref and wild-type kernels by
protein gel-blot analysis with 22- and 19-kD a-zein-specific antibodies. E, Overlap between transcriptional up-regulated genes
and translational down-regulated genes for ribosomal proteins and histones, and overlap between transcriptional down-regulated
genes and translational up-regulated genes for zeins.
puromycin treatment for the release of polysome as
a negative control (Supplemental Fig. S5). No significant difference of sequence features was observed between the up-regulated and down-regulated DTRs
(Supplemental Table S2).
Within the increased DTRs, 687 could be functionally
annotated. GO analysis indicated that these RNAs are
mostly related to three GO terms, GO: 0045449 (regulation of transcription; P = 1.33E-03), GO: 0045735
(nutrient reservoir activity; P = 3.51E-12), and GO:
0006414 (translational elongation; P = 1.17E-07). For the
decreased DTRs, 601 could be functionally annotated,
belonging to GO: 0006334 (nucleosome assembly; P =
6.59E-10), GO: 0006260 (DNA replication; P = 1.18E-03),
and GO: 0033279 (ribosomal subunits; P = 1.16E-03).
Ten most strongly up-regulated or down-regulated
DTRs of each classification are illustrated in Table I,
and all DTRs are shown in Supplemental Table S2.
Transcriptional factors (e.g. NAC domain transcription factors and MADS box transcription factors) and
translation elongation-related genes had markedly
higher translation levels in reas1-ref. Although the expression of zeins was down-regulated in reas1-ref (Fig.
5), surprisingly, their translation level was increased
significantly (Table I; Supplemental Table S2). There
was significant overlap (P = 2.23E-16, x 2 test) for zeins
between transcriptional down-regulated genes and
translational up-regulated genes (Fig. 6E). Meanwhile,
although the expression of histone RNAs was up-
regulated in reas1-ref (Fig. 5), their translation level
was dramatically reduced. There is also significant
overlap (P = 1.57E-14, x 2 test) for histones between
transcriptional up-regulated genes and translational
down-regulated genes (Fig. 6E). DNA replicationrelated genes (e.g. DNA polymerase subunits and
minichromosome maintenance proteins [MCMs]) had
lower translation levels in reas1-ref. Histones and DNA
replication-related genes are both related to nucleosome assembly and the cell cycle. Although the transcription of ribosomal subunit proteins is up-regulated
in reas1-ref (Fig. 5), their translation level is dramatically
down-regulated. There is overlap between transcriptional up-regulated genes and translational downregulated genes for ribosomal proteins (Fig. 6E). These
results demonstrate that the transcriptional and translational regulation of individual genes responding
to reduced 60S ribosome exportation is not always
consistent.
We measured the level of 22-kD a-zeins in 470 and
1,190 ng of total proteins of 15-DAP reas1-ref and wildtype kernels, respectively, by protein gel-blot analysis
with 22-kD a-zein antibodies. Compared with the wild
type, 22-kD a-zeins in reas1-ref were increased significantly. Meanwhile, there was no effect on 19-kD a-zein
content (Fig. 6D). Increased eEF1a protein content (Fig.
6A) and lower protein content of histone (Fig. 4B) in
reas1-ref also confirmed their increased or decreased
translation level.
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Qi et al.
Table I. Ten most strongly up-regulated or down-regulated DTRs of each GO classification
GO Identifier
P
Gene
Description
Polysome/Total in Polysome/Total
Fold
the Wild Type
in reas1
Change
% of total
Genes with increased ratio of polysome-bound mRNA
GO: 0045449, regulation of 1.33E-03 GRMZM2G081930
transcription
GRMZM2G167018
GRMZM2G134717
GRMZM2G170079
GRMZM5G812272
NAC1
0.1437
0.6978
4.86
0.0336
0.0291
0.0165
0.0106
0.3461
0.2145
0.1979
0.1038
10.31
7.36
12.03
9.76
0.0316
0.0158
0.0142
0.2861
0.1272
0.2754
9.05
8.07
19.38
0.0480
0.0451
0.3018
0.4536
6.29
10.06
3.51E-12 GRMZM2G346897
NAC domain transcription factor
NAC domain transcription factor
BZIP-type transcription factor
WRKY DNA-binding domain
superfamily protein
BEL1-related homeotic protein
Leu zipper domain protein
Homeobox-Leu zipper protein
ATHB-4
Homeobox protein liguleless3
IAA14-auxin-responsive Aux/IAA
family member
22-kD a-zein
0.2875
0.7691
2.68
GRMZM2G353272
GRMZM2G044152
GRMZM2G397687
GRMZM2G053120
GRMZM2G008341
GRMZM2G353268
AF546188.1_FG003
AF546188.1_FG007
AF546187.1_FG007
1.17E-07 GRMZM5G859846
22-kD a-zein
22-kD a-zein
22-kD a-zein
22-kD a-zein
Zein-a 19-kD z1A
Zein-a 19-kD z1A
Zein-a 19-kD z1B
Zein-a 19-kD z1B
Zein-a 19-kD z1D
Elongation factor Tu
0.2442
0.2126
0.2153
0.2127
0.2862
0.2504
0.2120
0.2578
0.2429
0.0476
0.9829
0.7821
0.8910
0.9571
0.9428
0.8631
0.7470
0.8318
0.9263
0.1744
4.03
3.68
4.14
4.51
3.29
3.45
3.52
3.23
3.81
3.67
Elongation
Elongation
Elongation
Elongation
Elongation
0.2158
0.3131
0.1984
0.2001
0.2248
0.6634
0.6392
0.4218
0.4162
0.4732
3.07
2.04
2.13
2.08
2.11
Histone H2A
0.5549
0.0717
0.13
Histone H3.2
Histone H3.2
Histone H3.2
Histone H4
Histone H4
Histone H4
Histone H4
Histone H4
Histone H4
Origin recognition complex
subunit 6
DNA polymerase
DNA polymerase «-subunit 2
DNA replication licensing factor
MCM3 homolog 2
Origin recognition complex
subunit 2
Minichromosome maintenance
complex protein family
Replication protein A 70-kD
DNA-binding subunit
Minichromosome maintenance
protein
0.9458
0.8418
0.6460
0.7069
0.5894
0.9550
0.3357
0.9435
0.5823
0.4301
0.0945
0.1126
0.0847
0.0850
0.0784
0.1012
0.0266
0.1030
0.0750
0.1093
0.09
0.13
0.13
0.12
0.13
0.11
0.08
0.11
0.13
0.25
0.4674
0.7544
0.6059
0.1236
0.0657
0.0796
0.26
0.09
0.13
0.6233
0.1285
0.21
0.8304
0.0891
0.11
0.5513
0.1185
0.22
0.7020
0.0859
0.12
GRMZM2G327059
GRMZM2G021339
GRMZM2G126239
GRMZM2G087741
GRMZM5G809195
GO: 0045735, nutrient
reservoir activity
GO: 0006414, translational
elongation
GRMZM2G007038
GRMZM2G407996
GRMZM2G110509
GRMZM2G151193
GRMZM2G001327
Genes with reduced ratio of polysome-bound mRNA
GO: 0006334, nucleosome 6.59E-10 GRMZM5G883764
assembly
GRMZM2G355773
GRMZM2G447984
GRMZM2G130079
GRMZM2G349651
GRMZM2G073275
GRMZM2G479684
GRMZM2G084195
GRMZM2G421279
GRMZM2G149178
GO: 0006260,
1.18E-03 GRMZM5G825512
DNA replication
GRMZM5G872710
GRMZM2G154267
GRMZM2G100639
GRMZM2G117238
GRMZM2G162445
GRMZM2G086934
GRMZM2G021069
factor
factor
factor
factor
factor
Tu
Tu
1a
1a
1a
(Table continues on following page.)
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Characterization of the Maize reas1 Mutant
Table I. (Continued from previous page.)
GO Identifier
GO: 0033279, ribosomal
subunits
P
Polysome/Total in Polysome/Total
Fold
the Wild Type
in reas1
Change
Gene
Description
GRMZM2G108712
GRMZM2G304362
Proliferating cell nuclear antigen
Ribonucleoside-diphosphate
reductase
40S ribosomal protein S3
0.8651
0.6543
0.1292
0.0827
0.15
0.13
0.3527
0.1547
0.44
40S
40S
40S
40S
40S
40S
60S
60S
60S
0.3491
0.3811
0.4542
0.2834
0.4064
0.4259
0.5257
0.3371
0.5604
0.1649
0.1484
0.1796
0.1151
0.1659
0.1402
0.2523
0.1421
0.2650
0.47
0.39
0.39
0.41
0.41
0.33
0.48
0.42
0.48
1.16E-03 GRMZM2G099352
GRMZM2G078985
GRMZM2G064640
GRMZM2G170336
GRMZM2G110952
GRMZM2G140609
GRMZM2G163561
GRMZM5G868433
GRMZM2G119169
GRMZM2G091921
reas1-ref Inhibits Cell Proliferation and Cell Growth
The synthesis of nucleosome assembly proteins related
to the cell cycle transition is markedly reduced in reas1-ref
(Table I; Fig. 6). This mutant exhibits a slow-growth
phenotype for both kernels and seedlings. There was a
more than a 3-d delay in endosperm development (Fig. 1).
Endoreduplication is a general feature of endosperm development in maize, involving replication of the nuclear
genome without cell division and leading to elevated
nucleic acid content (Sabelli and Larkins, 2009). Endoreduplication includes only G1 and S phases, which is different from the mitotic cell cycle (G1-S-G2-M phases).
Flow cytometry analysis of 15-DAP reas1-ref and wildtype endosperms showed endoreduplicated nuclei with
C values of 12C or greater, accounting for 18.1% of the
DNA in 15-DAP endosperm of reas1-ref and 22.2% of the
DNA in 15-DAP wild-type endosperm (Fig. 7A). At 18
DAP, there were 19.3% and 24.2% endoreduplicated nuclei with C values of 12C or greater in reas1-ref and wildtype endosperm, respectively (Fig. 7B). The mitotic cell
cycle was also assessed in 7-DAG seedlings by flow cytometry. The results showed that 63.3% of the nuclei have
2C DNA content in reas1-ref 7-DAG seedlings, while
45.1% of the nuclei in the wild-type seedlings have 2C
DNA content (Fig. 7C). These results demonstrate that the
mutation of reas1-ref affects cell proliferation. The first leaf
of 7-DAG reas1-ref and wild-type seedlings was analyzed
by scanning electron microscopy to observe the cell size of
lower epidermis (Fig. 7D). There was significantly smaller
cell size in reas1-ref than in wild-type seedlings, with cell
width and cell length both decreased (Fig. 7E). These results demonstrate that cell growth and cell proliferation
are suppressed as a result of impaired 60S ribosome
maturation, resulting in a developmental delay.
DISCUSSION
reas1-ref/dek* Is a Weak Mutant Allele That Functionally
Suppresses ZmReas1 and Affects 60S Ribosome Biogenesis
Rea1 is a highly conserved ribosome biogenesis factor first identified in the Nug1-purified pre-60S subunit
ribosomal
ribosomal
ribosomal
ribosomal
ribosomal
ribosomal
ribosomal
ribosomal
ribosomal
protein
protein
protein
protein
protein
protein
protein
protein
protein
S5
S9
S20
S23
S23
S23
L7-2
L17
L32
in yeast, which is the preribosomal particle at the export
from the nucleolus to the nucleoplasm (Bassler et al.,
2001). The six ATPase modules of Rea1 create a ring
domain, while the large linker and the MIDAS domain
compose the tail structure (Ulbrich et al., 2009). Rea1
attaches the preribosome at the Rix1 complex (Rix1Ipi3-Ipi1) via the ATPase ring domain (Nissan et al.,
2002, 2004; Galani et al., 2004). The tail of Rea1 containing the MIDAS domain contacts the preribosome at
other positions, where the pre-60S factor, Rsa4 or Ytm1,
is located. Ytm1 associates with nucleolar pre-60S particles, while Rsa4 associates with nucleoplasmic particles (Bassler et al., 2001; de la Cruz et al., 2005; Miles
et al., 2005; Ulbrich et al., 2009). The MIDAS domain
of Rea1 interacts with the MIDAS-MIDAS-interacting
domain of Ytm1 or Rsa4; this interaction is essential for
60S unit maturation and export from the nucleolus to
the nucleoplasm or from the nucleoplasm to the cytoplasm, respectively (Bassler et al., 2001; Ulbrich et al.,
2009). Rea1 is bound to the pre-60S ribosome at two
distinct sites: one is mediated via the motor ring domain and the other via MIDAS interaction with Ytm1 or
Rsa4, creating a mechanochemical device to release
Ytm1 or Rsa4 for 60S ribosome export (Kressler et al.,
2012).
Compared with dek1, which creates severe effects on
kernel development (Becraft et al., 2002), dek* causes
only mild effects. The mutant kernels have an obvious
small phenotype and decreased seed weight (Fig. 1),
with a delay of embryo, endosperm, and seedling development (Fig. 1). reas1-ref/dek* is a weak, nonlethal
mutant allele, where the 2,359th Ala (GCC) of ZmReas1
is replaced by Val (GTC; Fig. 2). The reas1 mutant allele
derived from a Mutator transposon insertion in the
coding region has a defective phenotype and is lethal.
reas1-ref is defective at the highly conserved linkage
region and might suppress the function of ZmReas1 by
affecting the mechanical force of the ATPase ring domain to the MIDAS tail that releases Rsa4 or Ytm1
factors. The expression of Reas1 is increased at the
transcript level in reas1-ref, which might be a response
to its functional suppression (Fig. 2).
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Figure 7. Evidence of inhibited cell proliferation and cell growth in reas1-ref kernels. A and B, Cell proliferation analysis of 15and 18-DAP endosperms of the wild type (WT) and reas1-ref. The inset shows the cell cycle diagrams analyzed by flow cytometry.
3C and 6C are DNA contents of the nuclei at G1 and S phase of 15- and 18-DAP endosperms, and 12C and 24C are DNA contents
of endoreduplicated nuclei at S phase of 15- and 18-DAP endosperms. For each sample, three independent biological replicates
were performed. Values are means with SE; n = 3 individuals (**, P , 0.01; ***, P , 0.001, Student’s t test). C, Cell proliferation
analysis of 7-DAG seedlings of the wild type and reas1-ref. The inset shows the cell cycle diagrams analyzed by flow cytometry.
2C and 4C are DNA contents of the nuclei at G1/S phase and G2/M phase of 7-DAG seedlings. For each sample, three independent biological replicates were performed. Values are means with SE; n = 3 individuals (**, P , 0.01, Student’s t test). D,
Scanning electron microscopy analysis of the lower epidermis of the first leaf mature region of 7-DAG reas1-ref and wild-type
seedlings. S, Stoma. Bars = 50 mm. E, Comparison of cell width and cell length of lower epidermis in wild-type and reas1-ref
7-DAG seedlings. The measurements were done on scanning electron microscopy results. Values are means with SE; n = 50
individuals (***, P , 0.001, Student’s t test).
To our knowledge, the characterization of dek* provides the first description of Rea1 in maize. A significant
reduction of mature 60S subunits was observed in yeast
rea1 mutants at restrictive conditions (Garbarino and
Gibbons, 2002; Galani et al., 2004). The Rea1 homologous
gene in Arabidopsis is essential for female gametophyte
development (Chantha et al., 2010). Rea1 AAA-ATPase is
conserved from yeast to humans (Bassler et al., 2010).
Phylogenetic analysis of ZmReas1 suggests that the Rea1
protein is highly conserved in plants and is homologous
to proteins in yeast, mammals, and microorganisms (Fig.
3). A significant reduction of mature 60S ribosomal
subunits is observed in the reas1-ref mutant, consistent
with the effects observed in yeast rea1, indicating a biogenesis defect specific to the 60S subunit maturation
pathway; the maturation and export of 40S subunits are
not affected (Fig. 4). This indicates a conserved function
in 60S subunit biogenesis of Rea1 in yeast and plants.
There are reductions of mature 60S subunits in cytoplasm
and pre-60S subunits in nucleus (Fig. 4), indicating that
the nucleus-detained pre-60S subunits might be degraded, for premature ribosomal particles with biogenesis failure would be dispersed and degraded in the
nucleoplasm (Andersen et al., 2005; Lam et al., 2007).
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Characterization of the Maize reas1 Mutant
Impaired Ribosome Biogenesis Enhances Ribosome
Use Efficiency
The regulation of protein synthesis is a key control
point in cellular responses to distinct stresses (Faye
et al., 2014). The proportion of actively translating ribosomes is reflected by the percentage of polysome
complexes/total ribosomes (Branco-Price et al., 2005;
Faye et al., 2014). We observed a 1.4-fold increase of
polysome complexes/total ribosomes in reas1-ref (Fig. 4).
This increase in polysomes in reas1-ref is indicative of a
promotion in the initiation of protein synthesis. The
proportion of actively translating ribosomes might be in
response to the down-regulation of ribosome biogenesis.
The results of our study of reas1-ref suggest that
suppressed ribosome export enhances ribosome use
efficiency and cellular translational efficiency. mRNA,
the 40S ribosomal subunit, and eIF2a constitute the 43S
preinitiation complex, termed half-mer, before attachment of the 60S ribosomal subunit (Helser et al., 1981;
Moy et al., 2002). Multiple eukaryotic protein kinases,
each of which responds to different signals, are known
to phosphorylate eIF2a and down-regulate general
translation initiation (Chen and London, 1995; Harding
et al., 1999, 2000; Williams, 1999; van Huizen et al.,
2003). GCN2 is the only eIF2a kinase found in all eukaryotes, including plants like Arabidopsis (Berlanga
et al., 1999; Zhang et al., 2008). Phosphorylation of
eIF2a is significantly reduced in reas1-ref (Fig. 6), indicating increased formation of preinitiation complexes
for protein synthesis, consistent with an increased
proportion of actively translating ribosomes. Furthermore, the level of eEF1a protein is markedly increased
in reas1-ref (Fig. 6), suggesting a promotion of both the
initiation and elongation of translation in reas1-ref.
Consequently, there is evidence for increased efficiency
of ribosome usage during translation in reas1-ref to ensure normal rates of protein synthesis (Fig. 8).
Impaired Ribosome Biogenesis Triggers Distinct
Transcriptional and Translational Cellular Responses
We have found that the suppressed ribosome maturation associated with reas1-ref brings about global
changes in transcription and translation. The translation of individual mRNAs is regulated, producing
discrepancies between mRNA and protein levels.
mRNAs have a distinct pattern of ribosome loading
under certain conditions, resulting in altered translational efficiencies (Branco-Price et al., 2005; Gawron
et al., 2014). Thus, analysis of transcript level is insufficient to completely describe cellular responses under
different conditions. There is also alternative translation
that contributes to the complexity of proteomes (Preiss
et al., 2003; Serikawa et al., 2003; Blais et al., 2004;
Kawaguchi et al., 2004; MacKay et al., 2004; BrancoPrice et al., 2005; Lin et al., 2014). According to our
transcriptome and translatome analysis, there is
evidence for consistent and inconsistent transcriptional and translational regulation of genes in reas1-ref
endosperm (Fig. 8). The large amount of transcriptionally
up-regulated genes is not the consequence of developmental delay, according to the expression data for
developing maize kernels (Chen et al., 2014). Our
transcriptome analysis revealed immediate cellular responses, while the translatome revealed specific protein
changes that are independent of transcriptional regulation for the efficient use of limited ribosomes and energy.
For nucleus-located proteins, the transcription of
small and large ribosomal subunit proteins is increased
in reas1-ref endosperm (Fig. 5), whereas their translation
is down-regulated (Table I). The transcriptional upregulation of ribosomal proteins might be a response
to a decreased level of mature ribosomes in the cytoplasm in reas1-ref (Fig. 4). But increased transcription is
incapable of rescuing 60S ribosome export in reas1-ref.
Consequently, the percentage of polysome-bound RNA
of these genes might be reduced for the more efficient
usage of limited ribosomes. Similar to ribosomal proteins, there is also a discrepancy in the expression of
nucleosome assembly-related genes. The transcription
of histones is increased in reas1-ref (Fig. 5), while
their translation is down-regulated (Table I). DNA
replication-related genes also have reduced translation
in reas1-ref (Table I). The polysome-bound RNA of
histone- and DNA replication- related genes is markedly decreased (Fig. 6), and the histone protein content
is down-regulated in reas1-ref (Fig. 4). The up-regulation
of histone transcription might be a response to decreased
growth vigor of reas1-ref (Fig. 1) in order to accelerate
growth rate. But accelerated growth might be lethal due
to ribosome shortage. Different kinds of transcriptional
factors have markedly higher translation in reas1-ref
(Table I), including auxin signaling-related genes
(GRMZM2G081930 and GRMZM5G809195; Zhang
et al., 2014). Among cytosol-located proteins, the transcription and translation of translation elongationrelated genes are both up-regulated in reas1-ref (Fig. 5;
Table I). The final polysome-bound RNA content of
eEF1a and eEF-Tu, as well as the protein level of eEF1a,
are markedly increased in reas1-ref (Fig. 6). There are
also inconsistent transcriptional and translational levels
of Reas1 itself (Fig. 2), and it might be a developmental
stage-dependent translational regulation.
In maize kernel, the most abundant protein is zein
storage protein, which accounts for 50% to 70% of the
total protein (Holding and Larkins, 2009), and a-zein is
the largest class of zein protein (Heidecker and Messing, 1986). The transcriptionally down-regulated zeins
might be the consequence of developmental delay.
a-Zeins have an increased translation level (Table I);
especially the 22-kD a-zeins show markedly increased
protein in both SDS-PAGE and immunoblot analyses
(Figs. 1 and 6). The percentage of a-zein polysomebound RNA might be increased to ensure basic protein storage in reas1-ref.
The mechanisms that underlie variation in the
translation of individual genes are likely to involve
features of the mRNA sequence (Bailey-Serres and
Dawe, 1996). Evaluation of highly translated genes
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Qi et al.
Figure 8. Suppressed 60S ribosome
biogenesis promotes translation initiation and ribosome usage as well
as inconsistently regulates the transcription and translation of individual
genes that affect general translation
efficiency and cell proliferation. WT,
Wild type.
under hypoxia in Arabidopsis showed mRNA sequences with a low GC nucleotide content in the 59
untranslated region (UTR; Branco-Price et al., 2005).
RNA 59 UTR GC content, 59 UTR length, and open
reading frame length were also observed to affect ribosome loading (Jiao and Meyerowitz, 2010; Yángüez
et al., 2013). When we analyzed sequence features that
might affect the ribosome loading of individual gene
transcripts affected by the shortage of 60S ribosome
subunits (Supplemental Table S3), no significantly different features were observed between the 1,802 upregulated DTRs and 2,959 down-regulated DTRs,
compared with 3,000 randomly selected control genes.
However, there might be other feedback pathways
for independent regulation at the transcriptional and
translational levels.
Impaired Ribosome Biogenesis Affects Cell Growth
and Proliferation
Cell growth and proliferation are tightly linked, as
enhanced protein synthesis is required for cell proliferation (Thomas, 2000). The increase in protein synthesis is accomplished by an enhanced rate of ribosome
biogenesis in support of the metabolic effort for
cell proliferation (Sollner-Webb and Tower, 1986).
Normal mitosis includes four successive phases: G1
(postmitotic interphase), S (DNA synthesis phase), G2
(postsynthetic phase), and M (mitosis; Fowler et al.,
1998; Riou-Khamlichi et al., 2000), whereas endoreduplication of endosperm includes only G1 and S phases
(Sabelli and Larkins, 2009). reas1-ref exhibits slower
growth and cell proliferation (Fig. 7), indicating an
intrinsic link between ribosome biogenesis and cell
cycle transition. Based on our analysis, this linkage is
through the regulation of DNA replication and nucleosome assembly.
In mammalian cells, the tumor suppressor p53 has
been shown to arrest the cell cycle at the G1-S transition
in response to impaired ribosome biogenesis, while
p53-independent cell cycle arrest in response to alteration of ribosome biogenesis has also been described
(Mayer and Grummt, 2005; Grimm et al., 2006; Zhang
and Lu, 2009; Deisenroth and Zhang, 2010; Donati et al.,
2011). p53 stabilization leads to cell cycle arrest through
the regulation of cyclins and cyclin-dependent kinases
(Sherr and McCormick, 2002). But the expression level
of cyclins does not appear to be affected in reas1-ref
according to our transcriptome and translatome analysis. The target of rapamycin (TOR) kinase is evolutionarily conserved among plant, yeast, and animal
cells and is reported to integrate nutrient and energy
signaling partly through the phosphorylation of RPS6
to promote ribosome biogenesis, polysome accumulation, translation initiation, cell growth, and cell proliferation (Xiong and Sheen, 2014). The transcription and
translation levels of TOR signaling-related genes do not
appear to be affected in reas1-ref, but there might be an
altered phosphorylation level of RPS6 or some other
posttranslational level regulation that is responsible for
the immediate transcriptional up-regulation of genes in
reas1-ref. The underlying mechanisms merit further
explorations.
S phase is the period for DNA replication, histone
synthesis, and nucleosome assembly. Nucleosome assembly is essential for a variety of biological processes,
984
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Characterization of the Maize reas1 Mutant
such as cell cycle progression, development, and senescence (Gal et al., 2015). The synthesis of nucleosome
assembly-related proteins (histones and DNA replicationrelated enzymes) might be reduced to decelerate the
growth rate for survival under the suppressed ribosome biogenesis condition in reas1-ref (Table I; Fig. 6).
As a result, the nucleosome assembly process during
S phase would be dramatically suppressed. The cell
proliferation is slowed in reas1-ref (Fig. 7); thus, together
with impaired cell growth, kernel and seedling development are slowed for more than 3 d (Fig. 1).
reas1-ref/dek*, as a nonlethal maize small kernel mutant, offered an opportunity to explore comprehensive
cellular responses to impaired 60S ribosome biogenesis.
Based on our results, we propose that reduced 60S ribosome biogenesis leads to differentially regulated
transcription and translation of distinct groups of genes
that affect translation efficiency and cell proliferation
(Fig. 8). First, there is increased efficiency of translation
initiation and ribosome usage. Second, there is selective
translational regulation of different groups of genes for
intensive usage of quantitatively limited mature ribosome. Finally, there is inhibited cell proliferation,
leading to slower growth and survival.
MATERIALS AND METHODS
Plant Materials
The o*-N1117 mutant stock of maize (Zea mays) was obtained from the Maize
Genetics Cooperation stock center and identified as an ethyl methanesulfonateinduced allele of the opaque15 mutant. The dek* mutation was separated from
the o*-N1117 stock as an independent mutant. The dek* mutant was crossed to
the W64A inbred line, and F1 and F2 were produced to generate a mapping
population. All plants were cultivated in the field at the campus of Shanghai
University. Seeds were harvested at 5, 9, 13, 15, 17, 18, 21, 25, and 36 DAP.
Paraffin and Resin Sections
The 15- and 18-DAP embryos were fixed at 4°C overnight in 50% (v/v)
ethanol, 5% (v/v) acetic acid, and 3.7% (v/v) formaldehyde (FAA). After embedding in paraffin, 10-mm microtome sections on glass slides were dewaxed in
xylene, rehydrated, and stained with fuchsin. The 15- and 18-DAP endosperm
tissues were fixed at 4°C overnight in FAA. After embedding in Spurr’s epoxy
resin, thin sections (1 mm) were heat fixed to glass slides and stained with
fuchsin. Stained sections were rinsed in water three times and air dried. Brightfield photographs of the sections were taken using a Leica microscope.
Transmission Electron Microscopy
The 15- and 18-DAP kernels of reas1-ref and the wild type were prepared
according to Lending and Larkins (1992), with some modifications: kernels
were fixed in paraformaldehyde and postfixed in osmium tetraoxide. After
dehydration in an ethanol gradient, samples were transferred to a propylene
oxide solution and gradually embedded in acrylic resin (London Resin). Sections (70 nm) were made with a diamond knife microtome (Reichert Ultracut E).
Sample sections were stained with uranyl acetate, poststained with lead citrate,
and observed with a Hitachi H7600 transmission electron microscope.
Scanning Electron Microscopy
The first leaf mature region of 7-DAG reas1-ref and wild-type seedlings was
fixed at 4°C overnight in FAA. Samples were critically dried and spray coated
with gold. Gold-coated samples were then observed with a scanning electron
microscope (S3400N; Hitachi).
Protein Quantification
Mature kernels of reas1-ref and the wild type were soaked in water, and
endosperm was separated from the embryo and pericarp. Endosperm samples
were critically dried to constant weight, powdered in liquid N2, and measured
according to Wang et al. (2011). Proteins were extracted from 50 mg of three
pooled endosperm flour samples. Extracted proteins were measured using a
bicinchoninic acid protein assay kit (Pierce) according to instructions. Measurements of all samples were replicated three times.
Measurement of Starch
Mature kernels of reas1-ref and the wild type were soaked in water, and
endosperm was then separated from the embryo and pericarp. Endosperm
samples were dried to constant weight and pulverized in liquid N2, and starch
was extracted and measured using an amyloglucosidase/a-amylase method
starch assay kit (Megazyme) according to the instructions with some modifications: add 0.2 mL of aqueous 80% (v/v) ethanol to a 100-mg sample and aid
dispersion; immediately add 3 mL of thermostable a-amylase. Then incubate
the tube in a boiling water bath for 6 min; place the tube in a bath at 50°C; add
0.1 mL of amyloglucosidase; stir the tube on a vortex mixer and incubate at 50°C
for 30 min; transfer duplicate aliquots (0.1 mL) of the diluted solution to the
bottom; add 3 mL of GOPOD reagent and incubate the tubes at 50°C for 20 min;
and read the absorbance for each sample and the D-Glc control at 510 nm against
the reagent blank. The percentage of starch content in mg of dried sample was
analyzed.
Soluble Amino Acid Analysis
Soluble amino acids were analyzed according to Holding et al. (2010): 3-mg
samples were refluxed for 24 h in 6 N HCl. Samples were hydrolyzed at 110°C for
24 h. Sample hydrolysates were critically dried and dissolved in 10 mL of citrate
buffer. The amino acids were analyzed with a Hitachi-L8900 amino acid analyzer at Shanghai Jiaotong University. Wild-type and dek kernel analyses were
replicated three times.
Map-Based Cloning
The Dek* locus was mapped using 864 individuals from an F2 mapping
population of the cross between the dek* and the W64A inbred line. For preliminary mapping, molecular markers distributed throughout maize chromosome 6 were used. SSR155.1M-1, SSR153.7M-2, SSR154.7M-3, InDel438,
InDel428, SNP064, and SNP165 (Supplemental Table S4), as additional molecular markers for fine-mapping, were developed to localize the Dek* locus to a
101.6-kb region.
RNA Extraction and Real-Time PCR Analysis
Total RNA was extracted with TRIzol reagent (Tiangen), and DNA was
removed by treatment with RNase-free DNase I (Takara). Using ReverTra Ace
reverse transcriptase (Toyobo), RNA was reverse transcribed to complementary
DNA (cDNA). Quantitative real-time PCR was performed with SYBR Green
Real-Time PCR Master Mix (Toyobo) using a Mastercycler ep realplex 2
(Eppendorf) according to the standard protocol. Specific primers were designed
(Supplemental Table S4), and the experiments were performed with two independent RNA sample sets with ubiquitin as the reference gene. From a pool
of kernels collected from three individual plants, an RNA sample was extracted,
for which three technical replicates were performed. A final volume of 20 mL
contained 1 mL of reverse transcribed cDNA (1–100 ng), 10 mL of 23 SYBR
Green PCR buffer, and 1.8 mL of 10 mM L21 forward and reverse primers for
each sample. Relative quantifiable differences in gene expression were analyzed
as described previously (Livak and Schmittgen, 2001).
Fractionation of Cytoplasmic and Nuclear Proteins
Pulverized tissue was hydrated in cold harvest buffer (10 mM HEPES, pH 7.9,
50 mM NaCl, 0.5 M Suc, 0.1 mM EDTA, 0.5% Triton X-100, 1 mM dithiothreitol
[DTT], 10 mM tetratsodium pyrophosphate, 100 mM NaF, 17.5 mM b-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride [PMSF], 4 mg mL21 aprotinin,
and 2 mg mL21 pepstatin A) incubated on ice for 5 min, and nuclei was pelleted
(1,000 rpm for 10 min). After transfer of the supernatant to a new tube for
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Qi et al.
extracting cytoplasmic proteins, the pellet of nucleic acid was washed and
resuspended in buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA,
0.1 mM EGTA, 1 mM DTT, 1 mM PMSF, 4 mg mL21 aprotinin, and 2 mg mL21 pepstatin A) and pelleted again (1,000 rpm for 10 min). Nuclei were washed and
resuspended in buffer C (10 mM HEPES, pH 7.9, 500 mM NaCl, 0.1 mM EDTA,
0.1 mM EGTA, 0.1% Nonidet P-40, 1 mM DTT, 1 mM PMSF, 4 mg mL21 aprotinin,
and 2 mg mL21 pepstatin A) and vortexed (4°C for 15 min), pelleted again
(14,000 rpm, 4°C for 10 min), and transferred to a new tube for extracting nuclear proteins.
Polyclonal Antibodies
For anti-Reas1 antibody production, the 12,478- to 16,038-bp cDNA fragment
was cloned into pGEX-4T-1 (Amersham Biosciences), and glutathione
S-transferase (GST)-tagged fusion protein was purified with the AKTA purification system (GE Healthcare) using a GSTrap FF column. Protein expression
and purification followed established procedures. Antibodies were produced in
rabbits according to standard protocols of Shanghai ImmunoGen Biological
Technology. For 22- and 19-kD a-zein antibody production, regions of low
similarity of 22- and 19-kD a-zein were selected according to a previous study
(Woo et al., 2001). The cDNAs responsible for selected polypeptides were
cloned into pGEX-4T-1 (Amersham Biosciences), and GST-tagged fusion protein was purified with the AKTA purification system (GE Healthcare) using a
GSTrap FF column. Protein expression and purification followed established
procedures. Antibodies were produced in rabbits according to standard protocols of Shanghai ImmunoGen Biological Technology.
Immunoblot Analysis
Proteins extracted from reas1-ref and wild-type mature kernels were separated by SDS-PAGE. Separated protein samples were then transferred to polyvinylidene difluoride membrane (0.45 mm; Millipore). The membrane with a
protein sample attached on it was incubated with primary and secondary antibodies. Using the Super Signal West Pico chemiluminescent substrate kit
(Pierce), the signal was visualized according to the manufacturer’s instructions.
The purified anti-Reas1 antibody was used at 1:500; the 22- and 19-kD a-zein
antibodies were used at 1:5,000; the a-tubulin antibody (Sigma-Aldrich) was
used at 1:5,000; the Bip (at-95) antibody (Santa Cruz Biotechnology), histone
antibody (Cell Signaling), eRPL13 antibody (Agrisera), eIF2a antibody (Cell
Signaling), and P-eIF2a antibody (Cell Signaling) were used at 1:1,000; and the TBP
antibody (Santa Cruz Biotechnology), eRPS14 antibody (Santa Cruz Biotechnology), and eEF1a antibody (Santa Cruz Biotechnology) were used at 1:500.
Phylogenetic Analysis
Related sequences were identified in the National Center for Biotechnology
Information nonredundant protein sequences database by performing a
BLASTP search with ZmReas1 protein sequences. Amino acid sequences were
aligned with the MUSCLE method in the MEGA5.2 software package using their
default settings for multiple protein alignment. A rooted phylogenetic tree of
Rea1 from maize, Brachypodium distachyon, Triticum urata, Oryza sativa, Setaria
italica, Arabidopsis (Arabidopsis thaliana), Populus trichocarpa, Glycine max,
Dictyostelium discoideum, Monodelphis domestica, Saprelegnia diclina, Mortierella
verticillata, and Saccharomyces cerevisiae was constructed by the neighbor-joining
method using the MEGA5.2 software package. The evolutionary distances were
computed using Poisson correction analysis.
RNA-seq Analysis
Total RNA (10 mg) was extracted from endosperm of reas1-ref and wild-type
kernels harvested at 15 DAP, and three reas1-ref or wild-type biological samples
were pooled together. The poly(A) selected RNA-seq library was prepared
according to Illumina standard instructions (TruSeq Stranded RNA LT Guide).
Library DNA was checked for concentration and size distribution in an Agilent2100 bioanalyzer before sequencing with an Illumina HiSeq 2500 system
according to the manufacturer’s instructions (HiSeq 2500 User Guide). Singleend reads were aligned to the maize B73 genome build Zea mays AGPv2.15
using TopHat 2.0.6 (Langmead et al., 2009). Data were normalized as fragments
per kilobase of exon per million fragments mapped, because the sensitivity of
RNA-seq depends on the transcript length. Significant DTGs were identified as
those with a fold change and P value of differential expression above the
threshold (fold change greater than 2 and P , 0.05).
Ribosome Profile and Isolation of Polysomal RNA
For the ribosome profile, approximately 2.5 mL of pulverized tissue (approximately 20 15-DAP kernels) was hydrated in 2 volumes of polysome extraction buffer (PEB; 200 mM Tris-HCl, pH 9, 200 mM KCl, 25 mM EGTA, 35 mM
MgCl2, 1% [w/v] Brij-35, 1% [v/v] Triton X-100, 1% [v/v] Tween 20, 1% [v/v]
Igepal CA-630, 1% [w/v] deoxycholic acid, 1% [v/v] polyethylene-10tridecylether, 1 mM PMSF, 0.5 mg mL21 heparin, 5 mM DTT, 50 mg mL21
cycloheximide, and 50 mg mL21 chloramphenicol; Kawaguchi et al., 2004),
homogenized, filtered through two layers of sterile Miracloth (Calbiochem),
and cleared by centrifugation (16,000g, 4°C for 15 min). Four hundred A260
units of the supernatant was layered over a 15% to 45% (w/v) Suc density
gradient, centrifuged (237,000g, 2.5 h at 4°C; Beckman Optima L-100 XP),
and the A254 profile was recorded with a chart recorder using a gradient
fractionator connected to a UA-5 detector (BIOCOMP) as described previously (Kawaguchi et al., 2003, 2004). Two independent biological replicates
were performed.
For the isolation of polysomal RNA, approximately 5 mL of pulverized tissue
powder (approximately 40 15-DAP kernels) was hydrated in 2 volumes of PEB,
homogenized, filtered through two layers of sterile Miracloth (Calbiochem),
and cleared by centrifugation (16,000g, 4°C for 15 min). The supernatant was
layered over a 1.75 M Suc cushion (400 mM Tris-HCl, pH 9, 200 mM KCl, 30 mM
MgCl2, 1.75 M Suc, 5 mM DTT, 50 mg mL21 chloramphenicol, and 50 mg mL21
cycloheximide) and centrifuged at 170,000g at 4°C for 3 h (modified from
Fennoy and Bailey-Serres, 1995). The polysome pellet was washed with
sterile water and resuspended in 700 mL of PEB lacking heparin and detergents. Total or polysome-bound RNA was precipitated from total supernatant or the ribosome fraction of the same amount of sample powder by the
addition of 2.5 volumes of 8 M guanidine chloride and 3.5 volumes of 99%
(v/v) ethanol and extracted by use of the Qiagen Plant RNeasy mini kit
according to the manufacturer’s protocol. For the negative control, the same
amount of pulverized tissue powder was hydrated in 2 volumes of PEB with
2 mM puromycin.
The polysome-bound/total RNA value for individual genes was determined from the ratio of the signal in the polysome RNA sample divided by the
signal in the total RNA sample. Due to the required use of an equal RNA
quantity in each RNA-seq reaction, in spite of the unequal proportion of RNA
in the polysome fraction under the two conditions, it was necessary to normalize the signal values obtained for polysome RNA. Normalization was
performed according to Branco-Price et al. (2005). Polysomes accounted for
57.2% and 40.2% of the total absorbance in reas1-ref and wild-type kernels,
respectively. The percentage of an individual mRNA species in polysomes
was calculated as 2nP/T (normalized polysome-bound/total) 3 100%.
Normalized polysome-bound/total in reas1-ref:
ðgene in polysome RNA signalÞ
þ log2 0:5717
nP Tðreas1Þ ¼ log2
ðgene in total RNA signalÞ
Normalized polysome-bound/total in the wild type:
ðgene in polysome RNA signalÞ
þ log2 0:4017
nP TðWTÞ ¼ log2
ðgene in total RNA signalÞ
Flow Cytometry Detection
For extraction of nuclei, endosperm and seedling tissues were finely chopped
with a sharp razor blade in Beckman lysis buffer. The resulting slurry was filtered
through a 30-mm nylon filter to eliminate cell debris, and the suspension containing nuclei was immediately measured using an Accuri C6 flow cytometer
(BD Biosciences) equipped with an argon-ion laser tuned at a wavelength of 488
nm. For each sample, at least 15,000 nuclei were collected and analyzed using a
logarithmic scale display. Each flow cytometric histogram was saved and analyzed using Accuri C6 software 1.0.264.21 (BD Biosciences).
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: ZmReas1 KP137367; ZmBip1,
NM_001112423, GRMZM2G114793; ZmeRPS6, NM_001112164, GRMZM5G851698;
ZmeEF1a, NM_001112117, GRMZM2G153541; ZmHistone H4, NM_001138113,
GRMZM2G084195; ZmDNA polymerase «-subunit2, NM_001153609,
GRMZM2G154267; and ZmMCM6, NM_001111819, GRMZM2G021069. RNA-seq
data are available from the National Center for Biotechnology Information
Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo) under the series entry
GSE67103.
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Characterization of the Maize reas1 Mutant
Supplemental Data
The following supplemental materials are available.
Supplemental Figure S1. SDS-PAGE analysis of total, zein, and nonzein
proteins from dek*/reas1-ref and wild-type mature endosperm.
Supplemental Figure S2. Comparison of total starch content and the percentage of amylose in wild-type and reas1-ref mature endosperm.
Supplemental Figure S3. Phenotypes of reas1-ref and wild-type plants
(90 DAG).
Supplemental Figure S4. Ultrastructure of developing endosperms of the
wild type and reas1-ref (15 and 18 DAP).
Supplemental Figure S5. qRT-PCR confirmation of DTRs with increased
or decreased translation levels.
Supplemental Table S1. GO classifications of DTGs with functional annotations.
Supplemental Table S2. GO classifications of DTRs with functional annotations.
Supplemental Table S3. Sequence feature analysis of DTRs.
Supplemental Table S4. Primers used in these experiments.
ACKNOWLEDGMENTS
We thank Dr. Yuanyuan Ruan (Fudan University) for technical support on
the ribosome profile experiment and Dr. Brian A. Larkins (University of
Nebraska, Lincoln) for critical reading of the article.
Received November 13, 2015; accepted December 7, 2015; published December
8, 2015.
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