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Proline responding1 Plays a Critical Role in Regulating
General Protein Synthesis and the Cell Cycle in Maize
C W OPEN
Gang Wang,a,b,1 Jushan Zhang,a,1 Guifeng Wang,a,b,1 Xiangyu Fan,a Xin Sun,a Hongli Qin,a Nan Xu,a Mingyu Zhong,a
Zhenyi Qiao,a Yuanping Tang,a and Rentao Songa,b,2
a Shanghai
Key Laboratory of Bio-Energy Crops, School of Life Sciences, Shanghai University, Shanghai 200444, P.R. China
Crop Biology Research Center, Beijing 100193, P.R. China
b Coordinated
Proline, an important amino acid, accumulates in many plant species. Besides its role in plant cell responses to environmental
stresses, the potential biological functions of proline in growth and development are unclear. Here, we report cloning and
functional characterization of the maize (Zea mays) classic mutant proline responding1 (pro1) gene. This gene encodes a D1pyrroline-5- carboxylate synthetase that catalyzes the biosynthesis of proline from glutamic acid. Loss of function of Pro1
significantly inhibits proline biosynthesis and decreases its accumulation in the pro1 mutant. Proline deficiency results in an
increased level of uncharged tRNApro AGG accumulation and triggers the phosphorylation of eukaryotic initiation factor 2a
(eIF2a) in the pro1 mutant, leading to a general reduction in protein synthesis in this mutant. Proline deficiency also
downregulates major cyclin genes at the transcriptional level, causing cell cycle arrest and suppression of cell proliferation.
These processes are reversible when external proline is supplied to the mutant, suggesting that proline plays a regulatory role
in the cell cycle transition. Together, the results demonstrate that proline plays an important role in the regulation of general
protein synthesis and the cell cycle transition in plants.
INTRODUCTION
In maize (Zea mays), a series of seed mutants with a starchy
endosperm have been identified, and the cause of the opaque/floury kernel phenotype has been investigated through
analyzing the functions of cloned genes. Previous studies
suggested that most of the opaque and floury mutants (opaque2, floury2, Mucronate, and Defective endosperm B30) involve genes that regulate zein synthesis, such as regulatory
genes or structural genes for these storage proteins (Schmidt
et al., 1987; Coleman et al., 1995; Gillikin et al., 1997; Kim
et al., 2004, 2006). Downregulation of zein gene (a-, b-, d-,
and g-zein genes) expression by RNA interference also reproduced an opaque phenotype (Segal et al., 2003; Wu and
Messing, 2010). These results indicate that the opaque/floury
phenotypes of opaque2 (o2), floury2 (fl2), Defective endosperm B30, and Mucronate mutants are caused by either
quantitative or qualitative alterations in zein proteins. However, other opaque/floury mutants, such as o1, o5, and fl1,
show no notable alterations in zein proteins (Holding et al.,
2007; Myers et al., 2011; Wang et al., 2012a), but the phenotypes of these mutants may be related to zein protein body
1 These
authors contributed equally to this work.
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.plantcell.org) is: Rentao Song (rentaosong@
staff.shu.edu.cn).
C
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www.plantcell.org/cgi/doi/10.1105/tpc.114.125559
2 Address
formation. Motto et al. (1996) examined whether other regulatory mechanisms, such as a reduced amino acid supply,
could affect zein synthesis and endosperm texture. The functional
analysis of maize opaque endosperm mutant Mutator-tagged
opaque 140 (Holding et al., 2010) and o7 (Wang et al., 2011)
suggested that amino acid(s) limitation represses zein proteins synthesis.
Proline1 responding1 (pro1), allelic to o6 (Manzocchi et al.,
1986), is a classical recessive opaque mutant. This mutant has
been studied extensively as an important auxotrophic mutant
since it was first reported (Gavazzi et al., 1975; Ma and Nelson,
1975). Mature kernels of homozygous pro1 mutants exhibit
collapsed and starchy endosperm morphology, stunted seedling growth, and seedling lethality. Recovery of the normal
phenotype in mutant seedlings occurs after they are supplemented with external L-proline (Racchi et al., 1978; Tonelli
et al., 1984). Tonelli et al. (1986) predicted that pro1 might be
associated with a defect in proline biosynthesis, but that has
not been validated.
In plants, proline is synthesized from glutamic acid and ornithine. This reaction is catalyzed by the biofunctional D1-pyrroline-5- carboxylate synthetase (P5CS) enzyme and yields
pyrroline-5- carboxylate (P5C) from glutamic acid in a two-step
reaction (Hu et al., 1992). P5C is further reduced to proline by
D1-pyrroline-5-carboxylate reductase. P5CS is a rate-limiting
enzyme in proline biosynthesis. Previous studies have shown
that proline is accumulated in many plants in response to environmental stress, such as drought, high salinity, high light and
UV irradiation, and oxidative stress (Szabados and Savouré,
2010). It is well known that under stress conditions, plants accumulate proline as an adaptive response to adverse conditions.
These data suggest that one primary function of proline is to
protect developing cells from osmotic damage. However, recent
The Plant Cell Preview, www.aspb.org ã 2014 American Society of Plant Biologists. All rights reserved.
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Figure 1. Phenotypic Features of Maize prol1 Mutants.
(A) F2 ear of pro1-ref 3 Chang 7-2. Bar = 1 cm.
(B) Wild-type and mutant kernels randomly selected from F2 ears of pro1-ref 3 Chang 7-2 viewed on a light box. Bar = 1 cm.
(C) and (D) Wild-type (C) and pro1-ref (D) kernels. Bar = 1 cm.
(E) and (F) Phenotype of wild-type and pro1-ref seedlings (14 DAG). Bar = 2 cm.
(G) to (I) Comparison of starch, lipid, and protein (total protein, zein, and nonzein) accumulation in wild-type and pro1-ref kernels. For each sample,
three independent biological replicates were performed. Bars represent average values 6 SD; n = 3 replicates (No refers to P > 0.05, * refers to P < 0.05,
** refers to P < 0.01, Student’s t test). wt, wild type; pro1, pro1-ref.
(J) Comparison of seedling height and root length of the wild type and pro1-ref. Bars represent average values 6 SD (n = 20 seedlings per genotype,
** refers to P < 0.01, Student’s test). wt, wild type; pro1, pro1-ref.
[See online article for color version of this figure.]
data suggest that proline might have certain regulatory functions during protein synthesis and may act as a signaling
molecule during plant development (Szabados and Savouré,
2010). Proline may also play critical roles in cellular metabolism both as a component of proteins and as a free amino
acid. However, the proposed regulatory functions of proline
are not yet well characterized.
In this study, we report the map-based cloning of Pro1 and
demonstrate that it encodes a P5CS. Thus, Pro1 plays an important role in the biosynthesis of proline in the cytosol. Loss of
function of Pro1 represses proline biosynthesis from glutamic
acid and leads to proline deficiency in pro1. Functional characterization of Pro1 demonstrated that proline plays important
regulatory roles in general protein synthesis and the cell cycle
transition in maize.
RESULTS
Maize pro1 Produces a Starchy Endosperm and Causes
Seedling Lethality
The pro1-ref (pro1-NA342) mutant and its six allelic mutants were
obtained from the Maize Genetics Cooperation stock center
(Supplemental Figure 1 and Supplemental Table 1). The pro1-ref
mutant was crossed into the Chang 7-2 genetic background.
Pro1 Regulates Protein Synthesis and Cell Cycle
Table 1. Protein Contents of pro1-ref and Wild-Type Endosperm
Genotype
Total Protein
Zeins
Nonzeins
Wild type
Pro1-ref
P value
11.50 6 0.51
8.67 6 0.69
0.011
8.02 6 0.25
5.73 6 0.47
0.031
3.02 6 1.20
2.94 6 0.91
0.824
Values shown are milligrams of protein per 100 mg of endosperm dry
weight 6 SD. P values are from Student’s t tests, compared to the wild type.
Mature kernels of homozygous pro1-ref exhibit a collapsed and dull
endosperm (Figures 1A to 1D). The F2 ears, with progenies exhibiting 1:3 segregation of opaque (pro1/pro1) and wild-type (+/+and
pro1/+) (vitreous) kernel phenotype, were used for seed component
analysis. We analyzed starch (amylose and total starch), lipid, and
total protein contents of opaque and wild-type endosperms and
found total starch and amylose of opaque endosperms are 81.8 and
62.7% of wild-type endosperms, respectively (obviously decrease,
P < 0.01, Student’s t test) (Figure 1G). The lipid content of opaque
endosperms is only 65.7% of the wild-type endosperms (P < 0.01,
Student’s t test) (Figure 1H). Quantitative analysis showed the
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content of total protein in opaque endosperms is 75.4% of the wild
type (Table 1, Figure 1I). These results indicated that all major seed
components, including starch, lipid, and protein, are decreased
significantly in pro1-ref endosperm.
Following germination, the coleoptile of pro1-ref grows normally,
but the seedling becomes necrotic and dies before emergence of
the second leaf. The mutant seedling only yields one or two small
leaves with an abnormal morphology. When necrosis occurs after
emergence of the second leaf, the leaf blade turns white with green
stripes along the veins (Figures 1E and F). At 14 d after germination
(DAG), the average height of pro1-ref seedlings is 6.4 cm, ;18.9%
of the wild type (33.9 cm), and the average length of the primary
root is 5.9 cm, ;26.1% of the wild type (22.6 cm) (Figure 1J). These
results indicated that the growth and development of pro1-ref
seedlings is significantly affected.
The pro1 Mutant Exhibits Impaired Zein Synthesis and
Produces Fewer and Smaller Protein Bodies
To determine the underlying biochemical basis for the opaque
phenotype of the mutant, we examined the zein content in the
Figure 2. Protein Analysis and Transmission Electron Microscopy of Wild-Type and pro1-ref Endosperm.
(A) SDS-PAGE analysis of total protein from pro1-NA342 (pro1-ref), pro1-N1058, pro1-N1533, and their wild-type endosperms. wt, wild type; pro,
pro1-ref.
(B) Comparison of different zeins of wild-type and pro1-ref endosperm by protein gel blot (15, 16, and 50 kD) and SDS-PAGE analysis (19, 22, and 27
kD). wt, wild type; pro, pro1-ref.
(C) and (D) Ultrastructure of 21-DAP endosperms of pro1-ref (C) and the wild type (D) showing smaller and fewer protein bodies in pro1-ref. Bar = 2 mm.
ER, endoplasmic reticulum; pb, protein body; st, starch granules.
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endosperm of pro1-ref and wild-type kernels. Quantitative
analysis showed that the levels of zeins in pro1-ref endosperms
are only 71.4% of wild type (decreased 28.6%) (Table 1, Figure 1 I).
SDS-PAGE analysis indicated that all zein classes (a-, b-, d-,
and g-zein) are reduced in pro1-ref, pro1-N1058, and pro1N1533 (Figures 2A and 2B). The reduction of 15-, 16-, and 50-kD
zeins in pro1-ref was further confirmed by protein gel blotting
analysis (Figure 2B). Using transmission electron microscopy,
we quantified the number and size of protein bodies in the fourth
and fifth endosperm cell layers at 21 d after pollination (DAP).
The average number of protein bodies in pro1-ref was ;84.0%
that in the wild type, and the average size was ;64.0% that of
the wild type (Table 2). There is a significant reduction in protein
body number and size compared with the wild type (Table 2,
Figures 2C and 2D), consistent with the results of seed component analysis.
Positional Cloning of Pro1
An F2 mapping population was generated by selfing the F1
seeds from a cross of pro1-ref and Chang 7-2. After characterizing a population of 6800 individuals using the molecular
markers listed in Supplemental Data Set 1, the Pro1 gene was
placed between molecular markers Single Nucleotide Polymorphism 2 (SNP2) and SNP15, with an interval of 130 kb in
physical distance and contained in one complete BAC clone
(AC199040) (Figure 3A).
Within this 130-kb DNA segment, six candidate genes
were identified to be collinear with rice (Oryza sativa) genes
(Supplemental Table 2). Expression analysis revealed that only
Gene 2 (GRMZM2G375504, collinear to rice Os02g0777700) is
downregulated in pro1-ref (Figure 3B), while five other genes have
unchanged expression in the mutant. Sequence analysis was performed for all six predicted genes in pro1-ref (gene 1 to gene 6), and
a 94-bp deletion 21340 bp upstream of the GRMZM2G375504
transcript start site was found, suggesting that GRMZM2G375504
could be the Pro1 mutant gene. For the other five genes (gene 1
and gene 3 to gene 6), no sequence change was found that could
cause changes in amino acid coding regions between pro1 mutants
and the wild type. Genomic DNA sequence GRMZM2G375504
spans ;10.7 kb and contains 19 exons and 18 introns. The length
of the longest transcript is 2715 bp and contains a 2154 bp coding
sequence and noncoding regions of 228 and 333 bp at the 59 and
39 ends, respectively (Figure 3C). GRMZM2G375504 encodes an
;77-kD protein of 717 amino acids (Figure 3E).
To confirm that GRMZM2G375504 is the Pro1 gene, we examined this gene in six available pro1 allelic mutants (Supplemental
Figure 1 and Supplemental Table 1). Genomic and cDNA sequences
of GRMZM2G375504 from the six pro1 allelic mutants, as well as
wild-type inbreds W22 and B73, were sequenced and analyzed. The
results (Figure 3C) indicated a G/A transition at the first nucleotide of
intron 9 in pro1-N1154A that changes the splice site (GT-AG to ATAG), causing deletion of exon 9 (90 bp) in the mRNA. In pro1-N1530,
a G/A transition at the first nucleotide of intron 14 changes the splice
site (GT-AG to AT-AG), causing a deletion of exon 14 (36 bp) in the
mRNA. In pro1-N1058, a G/A transition at the first nucleotide of
intron 4 changes the splice site (GT-AG to AT-AG), causing a deletion of exon 4 (81 bp) in the mRNA. In pro1-N1121, a G/A transition
at the first nucleotide of intron 17 changes the splice site (GT-AG to
AT-AG), causing a deletion of exon 17 (67bp) and a frameshift
mutation and resulting in a premature stop codon in the mRNA. A G/A
transition at exon 3 causes an amino acid change from Gly (GGG)
to Arg (AGG) in pro1-N1528. In pro1-N1533, a C/T transition in exon
16 changes a Gln (CAA) to a stop codon (TAA) (Figure 3C). RT-PCR
analysis proved that these splice site changes caused deletion of
exons 4, 9, 14, and 17 in pro1-N1058, pro1-N1154A, pro1-N1530,
and pro1-N1528, respectively (Supplemental Figure 2). Therefore, all
six pro1 allelic mutants contain mutations in GRMZM2G375504.
Using antibody raised against the protein encoded by
GRMZM2G375504, we detected a protein with an expected
molecular mass of ;77 kD in developing wild-type kernels (W22
and Chang 7-2), whereas this protein was not detected in the seven
pro1 mutants (Figures 3D). This protein is also present in developing
kernels of other opaque mutants, such as o2/o2 and o7/o7 (Figure
3D). This result indicated the mutations in all seven pro1 mutant
alleles caused dramatic loss of GRMZM2G375504 protein, further
confirming the loss of GRMZM2G375504 function in pro1 mutants.
Pro1 Encodes a P5CS
BLAST searches using the Pro1 protein indicated the gene encodes a P5CS with several conserved domains (Figure 3E;
Supplemental Figure 3). The N terminus (from 61 to 68 amino acids)
is the ATP and glutamate binding site. From 176 to 205 amino acids
and 559 to 586 amino acids are two leucine zipper sequences that
might mediate protein–protein interactions. The P5CS protein is
a bifunctional enzyme that consists of two functional domains, an
N-terminal conserved glu-5-kinase (g-GK) domain (from 233 to 255
amino acids) and a C-terminal glutamic-g-semialdehyde dehydrogenase (GSA-DH) domain (from 650 to 685 amino acids). The
g-GK domain is responsible for phosphorylating glutamate to form
g-glutamyl phosphate, which is reduced to glutamic-g-semialdehyde by GSA-DH. In maize, there are three predicted P5CS
genes; however, only P5CS1 and P5CS2 appear to be expressed.
We constructed a phylogenetic tree on the basis of P5CS fulllength protein sequence from the genomes of Arabidopsis thaliana,
Table 2. Size and Number of Protein Bodies in the Fourth and Fifth Cell Layers from Aleurone of pro1 and Wild-Type Endosperm at 18 and 25 DAP
Genotype
Stage (DAP)
Mean Area (µm2)
SE
Wild type
Pro1-ref
Wild type
Pro1-ref
18
18
21
21
1.27
0.92
1.51
0.97
0.42
0.31
0.40
0.35
P values are from Student’s t tests.
(n
(n
(n
(n
=
=
=
=
212)
230)
315)
287)
P Value
Number (per 100 µm2)
24.60
<0.01
24.10
<0.01
3.37 (n = 15)
20.50
3.17 (n = 19)
20.18
SE
P Value
3.25 (n = 21)
<0.01
3.01 (n = 16)
<0.01
Pro1 Regulates Protein Synthesis and Cell Cycle
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Figure 3. Map-Based Cloning and Identification of Pro1.
(A) The pro1 locus was mapped to a 130-kb region on chromosome 8 containing six candidate genes. See Supplemental Table 1 and Supplemental
Data Set 1 for detailed information.
(B) Candidate gene, gene 2 (GRMZM2G375504), is downregulated in pro1-ref (pro1-NA342). 1, pro1-N1154A; 2, pro1-ref (pro1-NA342); 3, pro1-N1530;
4, pro1-N1058; 5, pro1-N1121.
(C) Structure and mutation sites in the Pro1 gene. Lines represent introns, and green boxes represent exons.
(D) Immunoblot comparing accumulation of Pro1 in endosperms of the wild type, pro1 alleles, and other opaque mutants at 18 DAP. Lane 1, W22; lane
2, o2/o2; lane 3, o7/o7; lane 4, the wild type (Chang 7-2); lane 5, pro1-N1154A; lane 6, pro1-ref (pro1-NA342); lane 7, pro1-N1530; lane 8, pro1-N1058;
lane 9, pro1-N1121; lane 10, pro1-N1528; lane 11, pro1-N1533.
(E) Schematic diagram of Pro1 protein structure. aa, amino acids.
rice, and maize. The tree suggested there are two types of P5CS in
plants, P5CS1 and P5CS2 (Figure 4A), and Pro1 encodes a P5CS2.
To confirm the function of Pro1 as a P5CS enzyme, recombinant
Pro1 was measured for P5CS activity (Parre et al., 2010). In this
assay, Pi is formed after phosphorylation of glutamate to glutamyl
phosphate by the g-GK activity of P5CS. Pi determination is based
on a malachite green colorimetric assay (Parre et al., 2010). The
full-length cDNA of Pro1 (GRMZM2G375504) was expressed using
a glutathione S-transferase (GST) fusion strategy in Escherichia coli
(BL21 expression strain), and the recombinant protein was purified by GSH affinity chromatography and verified by SDS-PAGE
(Figure 4B). GST-Pro1 showed high activity, with glutamate
phosphorylation occurring at 6.87 nmol Pi/min/mg. Using the data
from phosphorylation of up to 350 mM of glutamate provided
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Figure 4. Phylogenetic Analysis of Pro1 and Biochemical Assay of Recombinant P5CS Protein.
(A) Phylogenetic relationships of Pro1 (Zm P5CS) and its homologs. Maize Pro1 and identified P5CS in rice (monocot) and Arabidopsis (dicot) plants
were aligned by MUSCLE method in MEGA 5.2 software package. The phylogenetic tree was constructed using MEGA 5.2, and P5CS of Caenorhabditis elegans, Homo sapiens, and Mus musculus were used as outgroups (see Methods). The numbers at the nodes (100) represent the percentage
of 1000 bootstraps. Zm, Zea mays; Os, Oryza sativa; At, Arabidopsis thaliana; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Mm, Mus musculus.
(B) SDS-PAGE gel of purified GST-P5CS protein stained with Coomassie blue. Left lane, GST-P5CS; right lane, molecular mass markers.
(C) and (D) Standard curve and its control of Pi determination based on a malachite green colorimetric assay.
(E) Kinetic analysis of P5CS was performed using glutamate concentrations from 0 to 350 mM. Km and Vmax were determined from nonlinear regression
to the Michaelis-Menten equation for concentrations of glutamate up to 350 mM from five replicate experiments. For each sample, five independent
biological replicates were performed. Error bars represent SD.
[See online article for color version of this figure.]
a calculated Vmax of 9.8 6 0.7 nmol Pi/min/mg and Km of 25.0 6
1.1 mM, demonstrating that the Pro1 gene indeed encodes an
active P5CS enzyme (Figures 4C to 4E).
Pro1 Is Constitutively Expressed
RT-PCR analysis revealed that Pro1 is expressed in a broad
range of tissues, including roots, stems, leaves, silk, tassels, and
kernels. The transcript level of Pro1 is similar among roots,
stems, leaves, silk, tassels, and kernels (Figure 5A). During
kernel development, the expression of Pro1 occurs before 9
DAP and continues to 33 DAP (Figure 5A). Pro1 accumulation
was consistent with the RNA level (Figure 5B), showing that the
Pro1 is constitutively expressed in all tested tissues and kernel
developmental stages. We analyzed the expression of P5CS1
(GRMZM2G028535) by RT-PCR in leaves, roots, stems, and
kernels of pro1-ref and the wild type. The expression level of
this gene is not different in these tissues between pro1-ref
and the wild type (Figure 5C). These data indicate that P5CS1
does not compensate for the loss of P5CS2 (Pro1) function in
pro1-ref.
Pro1 Is Localized to the Cytosol
Cell fractionation was used to detect the presence of Pro1 in
subcellular fractions using a Pro1 polyclonal antibody. Proteins
extracted from 21-DAP kernels of W22 were separated into
soluble and organelle fractions (contained total membrane,
plasma membrane, and endomembrane) (Figure 6A). Pro1 was
detected only in the soluble fraction, indicating that Pro1 is localized to the cytosol (Figure 6B).
Pro1 Is Required for Proline Biosynthesis
Proline biosynthesis from glutamic acid is catalyzed by a bifunctional P5CS enzyme, which yields P5C in a two-step
reaction (Hu et al., 1992). The activity of P5CS represents a ratelimiting step in proline biosynthesis, which is regulated at the
level of P5CS transcription and through feedback inhibition of
P5CS by proline (Savouré et al., 1995; Yoshiba et al., 1995;
Zhang et al., 1995; Strizhov et al., 1997). P5C is further reduced to
proline by the D1-pyrroline-5-carboxylate reductase (Rayapati
et al., 1989; Delauney and Verma, 1990; Verbruggen et al., 1993).
Pro1 Regulates Protein Synthesis and Cell Cycle
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Figure 5. Expression Analysis of the P5CS Genes.
(A) RNA expression of Pro1 (Zm P5CS2) in various tissues and at different stages of maize kernel development, as determined by RT-PCR.
(B) Immunoblot analysis of Pro1 protein accumulation during kernel development.
(C) RNA expression of P5CS1 in various tissues and RNA expression comparison between the wild type and pro1-ref in various tissues. 1, Wild type; 2,
pro1-ref.
[See online article for color version of this figure.]
To identify the effect caused by the pro1 mutation on proline
biosynthesis, we examined the proline content in protein-bound
(PBAAs) and free amino acids (FAAs) in endosperms of mature
kernels. The result indicated that the proline content decreased
by 35.9% in PBAAs and 56.0% in FAAs in pro1 mutant endosperm compared with the wild type (Figures 7A and 7B). Similarly,
compared with the wild type, the relative quantity of proline in
PBAAs and FAAs in 14-DAG pro1 seedlings decreased by 45.2
and 100%, respectively (Figures 7C and 7D).
half-strength MS medium supplemented with 3 mM L-glutamic
acid (Figures 8B and 8D), the substrate from which P5CS synthesizes proline.
Proline Deficiency Causes Uncharged tRNApro
Accumulation in the pro1 Mutant
Aminoacylated tRNAs read codons in ribosome-bound mRNAs
and transfer their 39-attached amino acids to the growing peptide chain. When the supply of amino acids becomes limiting or
Proline Feeding Rescues the Viability of pro1 Seedlings
Since the maize pro1 mutant was previously demonstrated to
require proline (Manzocchi et al., 1986), we further analyzed the
requirement of this mutant for proline by performing a proline
feeding assay using wild-type and pro1-ref seedlings. On “basic” growth medium (half-strength Murashige and Skoog [MS]
medium without L-proline), wild-type seedlings showed a normal
phenotype, but the mutant seedlings exhibited the described
leaf abnormalities, as well as reduced growth and lethality at the
second leaf stage (Figures 8A and 8C). At 9 d after cultivation in
medium without L-proline, the average height of pro1-ref seedlings was 5.6 cm, or ;17.6% of the wild type (31.8 cm), and the
average length of the pro1-ref primary root was 5.2 cm, or
;25.0% of the wild type (20.7 cm) (Figures 8A and 8C). At 9
d after cultivation in medium supplemented with 3 mM L-proline,
the average height of pro1-ref seedlings was 30.1 cm, or
;97.7% of the wild type (30.8 cm), and the average length of
pro1-ref primary roots was 19.1 cm, or ;91.4% of the wild type
(20.9 cm) (Figures 8A and 8C). These results showed that pro1ref seedlings cultivated on half-strength MS medium supplemented with 3 mM L-proline reverted to the normal phenotype.
However, pro1-ref seedlings did not recover when cultivated on
Figure 6. Pro1 Is Localized to the Cytosol.
(A) Immunoblot of Pro1 in fractionated maize endosperm proteins probed with anti-Pro1 antibody.
(B) Immunoblot showing that Pro1 is predominantly associated with the
soluble protein fraction. Fractions were subjected to immunoblot analysis with antibodies against Pro1, Bip (endomembrane marker), and
ATPase (plasma membrane marker).
[See online article for color version of this figure.]
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The Plant Cell
Kimball, 2003; Zaborske et al., 2009). GCN2 phosphorylates
eukaryotic initiation factor 2a (eIF2a) to regulate translation. We
measured the phosphorylation levels of eIF2a in pro-ref endosperm (12, 18, and 24 DAP) and seedlings by protein gel blot
analysis with eIF2a-P and eIF2a (total eIF2a as control) antibodies. Compared with the wild type, eIF2a in pro1-ref endosperms (18 and 24 DAP) and seedlings was significantly
phosphorylated, while the total eIF2a protein level was not altered (Figures 9D and 9E). When we measured the phosphorylation level of eIF2a in pro1-ref seedlings supplied with 3 mM
external L-proline, we found that the phosphorylation level of
eIF2a had decreased drastically (Figure 9E).
Proline Deficiency Affects Protein Accumulation in
pro1 Mutant
Figure 7. Analysis of Total and Free Proline from Mature Endosperms
and 14-DAG Seedling of the Wild Type and pro1-ref.
(A) and (B) Comparison of total and free proline from mature endosperm
of the wild type and pro1-ref. wt, wild type; pro1, pro1-ref.
(C) and (D) Comparison of total and free proline from 14-DAG seedlings
of the wild type and pro1-ref. wt, wild type; pro1, pro1-ref.
For each sample, three independent biological replicates were performed.
Bars represent average values 6 SD, n = 3 replicates (** refer to P < 0.01,
Student’s t test).
deficient for protein synthesis, the level of cognate charged
tRNA will rapidly decline (Morris and DeMoss, 1965; Böck et al.,
1966; Yegian and Stent, 1969; Sørensen, 2001; Dittmar et al.,
2005). Amino acid analysis showed that the relative amounts of
free proline in 14-DAG seedlings and 12-, 18-, and 24-DAP
endosperms of pro1-ref were 0 and 54.6%, and 6.2 and 15.4%,
respectively, of the wild type (Figures 7D and 9A). To examine
the levels of charged and uncharged tRNApro in pro1-ref, we
used RNA gel blot analysis to measure tRNApro charging, with
a 59-digoxigenin-labeled probe complementary to nucleotides
20 to 47 nucleotides of the major proline isoacceptor tRNAAGG
(tRNApro AGG). The results showed that in 12-DAP endosperm, the
detected tRNApro AGGs were mostly present in the charged form,
in both the wild type and pro1-ref. However, in 18- and 24-DAP
endosperms, the detected tRNApro AGGs in the wild type were
mostly charged but were mostly uncharged in pro1-ref (Figure
9B). Thus, there is a good correlation between uncharged tRNApro
AGG accumulation and the deficiency in available free proline in
the endosperm. Similarly, tRNApro AGGs were mostly uncharged in
pro1-ref seedlings at 14 DAG without supplemental proline but
were mostly charged when external L-proline was supplemented
at 1 and 3 mM (Figure 9C).
Proline Deficiency Leads to Eukaryotic Initiation Factor 2a
Phosphorylation in pro1 Mutant
General Control Non-derepressing kinase-2 (GCN2), which is
conserved from yeast to mammals, is activated in response to
amino acid starvation (Natarajan et al., 2001; Jefferson and
We analyzed the total proteins in leaf tissues with the same fresh
weight from 14-DAG seedlings of pro1-ref and the wild type by
SDS-PAGE. Compared with the wild type, the levels of total proteins in 14-DAG seedlings of pro1-ref were generally reduced in
response to proline deficiency (Figure 10A). However, the amount
of total proteins in pro1-ref seedlings (14 DAG) was similar to the
wild type (14 DAG) when cultivated on half-strength MS medium
supplemented with 3 mM L-proline (Figure 10A).
Quantitative analysis showed that total protein levels decreased by 24.6% and zeins decreased by 28.6% in the pro1-ref
kernels (Table 1). SDS-PAGE analysis indicated that all types of
zeins and some nonzeins in mature endosperm of pro1-ref are
reduced compared with the wild type (Figures 2A and 2B). Zein
gene expression was examined during kernel development at
the transcriptional level by quantitative real-time RT-PCR (qRTPCR), and the results indicated that the transcription of a- (19
and 22 kD), b- (15 kD), d- (10 kD), and g-zein (27 and 50 kD)
classes is downregulated in pro1-ref, especially at 18 DAP
(Figure 10B). O2, the transcription factor that regulates 22- and
19-kD a-zein expression, was analyzed at the transcript and
protein levels. The results showed that the levels of the O2 RNA
transcript do not differ between pro1-ref and the wild type, but
O2 is downregulated at the protein level in pro1-ref, especially at
18 DAP (Figure 10C). Bip was also analyzed at the transcript and
protein levels. Bip transcript levels are upregulated in pro1-ref;
however, Bip is downregulated at the protein level in mutant
endosperms at 18 DAP (Figure 10D).
Proline Deficiency Also Affects the Cell Cycle in pro1 Mutant
Seedlings of the pro1 mutant exhibit a slow growth phenotype.
To determine if there is an aberrant cell cycle in pro1-ref seedlings, the mitotic cell cycle was assessed by flow cytometry
(FCM). The result showed that 83.3% of the nuclei are at stage
G1 (2C DNA content) in pro1-ref seedlings, while 75.2% of the
nuclei are at stage G1 (2C DNA content) in the wild type (Figure
11A). The fraction of G1 (2C DNA content) nuclei shifted to
76.1% in pro1-ref seedlings supplemented with 3 mM L-proline
(Figure 11A).
Endoreduplication, a common feature of endosperm development, involves replication of the nuclear genome in the
absence of cell division and leads to elevated nuclear gene
Pro1 Regulates Protein Synthesis and Cell Cycle
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Figure 8. Proline Feeding Rescue Assay.
(A) and (B) Phenotypes of wild-type and pro1-ref seedlings cultivated in half-strength MS culture medium supplemented with various concentrations of
L-proline (A) and L-glutamate (B). wt, wild type; pro1, pro1-ref. Bar = 2 cm.
(C) and (D) Seedling height and root length of wild-type and pro1-ref seedlings cultivated in half-strength MS culture medium supplemented with
various concentrations of L-proline and L-glutamate. Bars represent average values 6 SD (n = 20 seedlings per genotype; No refers to P > 0.05, ** refers
to P < 0.01, Student’s test). wt, wild type; pro1, pro1-ref.
[See online article for color version of this figure.]
content and polyploidy. Endoreduplication is a variant of the
mitotic cell cycle (G1-S-G2-M) with only G1 and S phase. FCM
analysis showed that endoreduplicated nuclei (at stage S) with
C-values of 12C or greater account for 28.09% of the DNA in 18
DAP endosperm of pro1-ref, but 33.85% of the DNA in 18-DAP
wild-type endosperm (Figure 11B). At 24 DAP, there are 30.83
and 37.58% endoreduplicated nuclei (at stage S) with C-values
of 12C or greater in pro1-ref and wild-type endosperm, respectively (Figure 11C). Other allelic mutants, such as pro1N1058 and pro1-N1533, also had fewer endoreduplicated nuclei
(at stage S) with C-values of 12C or greater than their corresponding wild types (Supplemental Figure 4).
significantly in pro1-ref seedlings (Supplemental Data Set 2). RTPCR and qRT-PCR were used to validate downregulation of these
genes (Figures 12A and 12B). These analyses revealed that CDC6
and MCM7, two essential genes encoding proteins involved in the
formation of the prereplicative complex (pre-RC), are downregulated in pro1-ref seedlings (Figure 12A). These analyses also
showed that exogenous L-proline (3 mM) induces not only the
expression of A, B, and D cyclins, but also some histones (Histone
H2, H3, and H4) and DNA replication-related genes in pro1-ref
seedlings (Figures 12A and 12B).
DISCUSSION
Proline Deficiency Affects Expression of Cell Cycle–Related
Genes in pro1 Mutant
Pro1 Encodes a P5CS Protein in Maize
To further investigate the link between proline synthesis and cell
division, we analyzed the expression profile of 14 DAG seedlings
by RNA-seq (Supplemental Figure 5). These data showed that, as
compared with the wild type, the expression of cyclin genes (six
genes), nucleosome and nucleosome assembly genes (55 genes),
and DNA replication-related genes (five genes) are downregulated
In plants, proline is synthesized mainly from glutamate, but not
arginine or ornithine (Funck et al., 2008). P5CS, which catalyzes the
first two reactions of proline biosynthesis, is a bifunctional enzyme
containing both the g-GK and GSA-DH domains. The activity of
P5CS is a rate-limiting step in proline biosynthesis. In most plant
species, P5CS is encoded by two genes (P5CS1 and P5CS2) that
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Figure 9. Uncharged tRNApro Accumulation Caused by Proline Deficiency Triggers the Phosphorylation of eIF2a.
(A) Free proline content analysis of 12 DAP (I), 18 DAP (II), and 24 DAP (III) endosperm from the wild type and pro1-ref. For each sample, three
independent biological replicates were performed. Bars represent average values 6 SD; n = 3 replicates (** refers to P < 0.01, Student’s t test). wt, wild
type; pro, pro1-ref.
(B) RNA gel blot analysis of charged and uncharged tRNApro accumulation in developing endosperm. wt, wild type; pro, pro1-ref.
(C) RNA gel blot analysis of charged and uncharged tRNApro accumulation in 14-DAG seedlings of the wild type and pro1-ref cultivated with 0, 1, and 3
mM L-proline. wt, wild type; pro, pro1-ref.
(D) Immunoblot analysis of eIF2a phosphorylation during kernel development. wt, wild type; pro, pro1-ref.
(E) Immunoblot analysis of eIF2a phosphorylation in 14-DAG seedlings of the wild type and pro1-ref cultivated with 0, 1, and 3 mM L-proline. wt, wild
type; pro, pro1-ref.
have different expression patterns and perform nonredundant
functions during development. In Arabidopsis, P5CS1 is inducible
by salt, abscisic acid, and drought; however, P5CS2 is apparently
a housekeeping gene (Strizhov et al., 1997; Székely et al., 2008).
In rice, P5CS1 and P5CS2 are upregulated by different stresses
(Igarashi et al., 1997; Hur et al., 2004). In Sorghum bicolor, Su et al.
(2011) found that P5CS2 is a housekeeping gene that mainly
functions in basic proline metabolism; the P5CS1 gene could play
a major role in stress responses. In this study, RNA expression
analysis showed that Zm P5CS2 (Pro1) is constitutively expressed
and not inducible by drought and acid stresses (Supplemental
Figure 6). In Arabidopsis, P5CS1-GFP (green fluorescent protein) is
localized in the cytosol of leaf mesophyll cells in normal growth
condition, whereas P5CS1-GFP accumulates in chloroplasts under
salt or osmotic stress, and P5CS2-GFP has been shown to be
localized predominantly in the cytosol. Expression pattern analysis
and subcellular localization showed Pro1 encodes the housekeeping gene Zm P5CS2, likely an ortholog of At P5CS2, whose
main function is basic proline synthesis in the cytosol.
Uncharged tRNApro Accumulation Caused by Proline
Deficiency Triggered the Phosphorylation of eIF2a through
the GCN2 Pathway
The eukaryotes use four different eIF2a-kinases to respond to
various stresses, and each kinase has a predominant role in the
response to a specific cellular stress condition. For example,
GCN2 is the primary eIF2a-kinase that functions in the response
to nutrient limitation (Harding et al., 2000), while the PKR-like
endoplasmic reticulum kinase (PERK or PEK) functions in response to protein misfolding in the endoplasmic reticulum
(Harding et al., 1999), the double-stranded RNA-dependent
protein kinase (PKR) participates in the response to viral infection (Williams, 1999), and the heme-regulated inhibitor functions in the response to the lack of heme in the cell (Chen and
London, 1995). To date, GCN2 is the only eIF2a kinase found in
all eukaryotes (Berlanga et al., 1999). GCN2 is activated by
amino acid deprivation, purine and glucose deprivation, oxidative and osmotic stress, and UV irradiation. In Arabidopsis,
Zhang et al. (2008) confirmed that GCN2 is required for phosphorylation of eIF2 in response to treatment with herbicides that
interfere with amino acid biosynthesis.
The phosphorylation level of eIF2a is significantly increased in
pro1-ref endosperms (18 and 24 DAP; Figure 9D) and seedlings
(14 DAG; Figure 9E). Correspondingly, free proline levels are
reduced by 93.8, 84.6, and 100%, respectively, in 18- and 24DAP endosperm and 14-DAG seedlings of pro1-ref compared
with the wild type (Figures 7D and 9A). Phosphorylation of eIF2a
is reduced drastically when pro1-ref seedlings were cultivated
on half-strength MS medium supplied with 3 mM L-proline
(Figure 9E). This indicated that proline deficiency causes eIF2a
Pro1 Regulates Protein Synthesis and Cell Cycle
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Figure 10. Proline Deficiency Affects Protein Synthesis.
(A) SDS-PAGE analysis of total proteins extracted from 14-DAG seedlings treated with various concentrations of L-proline. wt, wild type; pro, pro1-ref.
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The Plant Cell
phosphorylation. GCN2 can bind different uncharged tRNAs
with similar affinities, but it binds to uncharged tRNAs more
tightly than charged tRNAs (Dong et al., 2000). GCN2 is activated to phosphorylate eIF2a by binding uncharged tRNAs.
RNA gel blot analysis of uncharged tRNApro AGG showed that it
significantly accumulates in pro1-ref endosperm and seedling
cells (Figures 9B and 9C), and the amount of uncharged tRNApro
AGG of pro1-ref seedlings decreases to the wild-type levels
when 3 mM L-proline is supplied (Figure 9C). Therefore, GCN2dependent eIF2a phosphorylation is activated by uncharged
tRNApro AGG accumulation in the pro1 mutant in response to
proline deficiency. Multiple uncharged tRNAs can also activate
GCN2 during starvation of different amino acids (Zaborske et al.,
2009). This suggests that there could be a cellular monitoring
system which senses uncharged tRNAs accumulation during
starvation of certain amino acids, such as proline (this study),
histidine, leucine, and tryptophan (Zaborske et al., 2009, 2010).
Phosphorylation of eIF2a Represses Zein Levels and
General Protein Synthesis
eIF2 is a heterotrimer of a-, b-, and g-subunits (Preiss and W
Hentze, 2003). Before translation initiation, eIF2 binds GTP and
initiator Met-tRNAMet to form a ternary complex (TC). The 40S
ribosomal subunit-linked TC moves along the mRNA until it
reaches the AUG start codon, then eIF2-GTP is hydrolyzed to
eIF2-GDP and released from the TC. Recycling inactive eIF2GDP to active eIF2-GTP is pivotal to reintegrating the TC for the
next round of translation. Phosphorylation of eIF2a inhibits the
recycling and represses initiation of protein synthesis. Although
there are some differences between the fungal and plant translation initiation complexes, the three subunits that make up eIF2
are conserved (Browning, 1996, 2004; Zhang et al., 2008). In
yeast (Saccharomyces cerevisiae) cells, GCN2-dependent eIF2a
phosphorylation leads to a general downregulation of protein
synthesis under amino acid starvation (Hinnebusch, 2005). In
Arabidopsis, Lageix et al. (2008) provided evidence that GCN2
represses the protein synthesis under stress conditions. Compared with Arabidopsis seedlings on complete media, the amount
of newly synthesized proteins in purine-deprived seedlings is
reduced by 80%.
We demonstrated that uncharged tRNA pro accumulation
caused by proline deficiency triggers the phosphorylation of
eIF2a through the GCN2 pathway in kernels of pro1-ref. Transcript and protein analysis of zeins suggested that eIF2a
phosphorylation could repress zeins protein accumulation in
pro1 (Figure 10B). The analysis of O2 and Bip reveled that, at the
transcript level, O2 was not changed and Bip was upregulated in
pro1. However, at the protein level, both O2 and Bip proteins
were reduced in the pro1 mutant (Figures 10C and 10D). These
results suggested that protein synthesis is repressed in pro1 in
response to eIF2a phosphorylation due to proline deficiency.
The decrease in O2 levels could also explain the downregulation
of 22- and 19-kD a-zeins transcriptional level in pro1. To further
understand the relationship between eIF2a phosphorylation and
protein synthesis, we analyzed the protein content and the
status of eIF2a phosphorylation in seedlings of pro1-ref and the
wild type. Compared with the wild type, the eIF2a in pro1-ref
seedlings was obviously phosphorylated and proteins synthesis
was reduced (Figures 9C and 10A). This suggested that proline
deficiency in pro1-ref seedlings induces phosphorylation of
eIF2a, which reduces protein synthesis efficiency. Indeed, an
external L-proline supply could reduce phosphorylation of eIF2a
and promote protein synthesis (Figure 10A).
GCN2 and Cyclins Coordinately Regulate the Cell Cycle
Transition from G1 to S Phase in Response to
Proline Deficiency
In order to proliferate, cells pass through a series of distinct
stages of the cell cycle, which is separated into four successive
phase: G1 (postmitotic interphase), S phase (DNA synthesis
phase), G2 (postsynthetic phase), and M phase (mitosis). Many
plant cells arrest in G1 in response to nutrient limitation (Fowler
et al., 1998). G1 is the major part of the cell cycle and can be
extended in response to alterations in environmental conditions,
such as temperature, CO2 level, light level, and sugar level (RiouKhamlichi et al., 2000). There is some evidence that proline has
certain regulatory functions, such as regulating plant development
and acting as a signaling molecule. In Arabidopsis, high levels of
expression of P5CS2 in leaf primordia and callus indicated that
rapidly dividing and growing cells have a high demand for proline
(Strizhov et al., 1997; Székely et al., 2008), suggesting that proline
may participate in the regulation of cell division.
FCM was used to assess endoreduplication in endosperms
and the mitotic cycle in pro1 seedlings. The results confirmed
that the G1/S transition is arrested in pro1 endosperm and
seedlings. But how does proline deficiency lead to G1/S transition delay? GCN2 is known to regulate the cell cycle transition
in response to starvation, thus mediating slower growth as
a consequence of a reduced nutrient supply. GCN2 can also
play a central role in regulating the cell cycle transition from G1
to S phase (Tvegård et al., 2007; Grallert and Boye, 2007). In the
fission yeast Schizosaccharomyces pombe, Tvegård et al.
(2007) showed that the G1/S transition delay is dependent upon
GCN2 kinase. In S. cerevisiae, Menacho-Marquez et al. (2007)
provided evidence that GCN2 regulates the G1/S cell cycle
transition. Our results suggested that the G1/S transition could
Figure 10. (continued).
(B) RNA expression profiles of representative of zein genes. Ubiquitin was used as an internal control. For each sample, three technical and two
independent biological replicates were performed. Error bars represent SD. wt, wild type, pro1, pro1-ref.
(C) RNA and protein expression analysis of O2 during endosperm development. wt, wild type; pro, pro1-ref.
(D) RNA and protein expression analysis of Bip during endosperm development. wt, wild type; pro, pro1-ref. Lanes: 1, W22; 2, wild type; 3, pro1-ref; 4,
pro1-N1058; 5, pro1-N153.
Pro1 Regulates Protein Synthesis and Cell Cycle
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be regulated by GCN2 through eIF2a phosphorylation and
translation repression in response to proline deficiency.
Cell cycle–related genes, especially cyclins, participate in the
regulation of the G1/S transition at the transcriptional level in
response to proline deficiency. Cyclins are a family of proteins
that regulate progression of the cell cycle by activating cyclindependent kinase (Cdk) enzymes. In plants, A-, B-, and D-type
cyclins are assumed to play a major role in cell cycle regulation
(de Jager et al., 2005). The D-type cyclin is assumed to be
a sensor of external signals and to play an essential role in cell
cycle progression from G1 to S phase (Harbour and Dean, 2000;
Oakenfull et al., 2002; Nelsen et al., 2003; Menges et al., 2006).
Nelsen et al. (2003) proved that deprivation of a set of nonessential amino acids downregulated cyclin D1 mRNA and
transcription of a cyclin D1 promoter-reporter gene in mammalian cells. However, the mechanisms by which amino acids
regulate the cell cycle are not well characterized. In plants, it is
not clear whether a single amino acid deficiency can inhibit
cyclin expression and cell cycle progression. In our study, the
gene expression profile showed that transcripts of cell cycle
related genes, including cyclins (A-, B-, and D-type), nucleosome assembly genes, and DNA replication-related genes, are
generally downregulated in pro1-ref seedlings (Supplemental
Data Set 2; Figures 12A and 12B). However, their expression
recovered to normal levels when pro1-ref seedlings were cultivated on medium supplemented with 3 mM L-proline (Figures
12A and 12B). These results suggested that proline could regulate cell cycle progression by repressing transcription of cell cycle–related genes. RT-PCR and qRT-PCR analysis also revealed
that CDC6 and MCM7, essential genes encoding proteins involved in the formation of the pre-RC, were transcriptionally
downregulated in pro1-ref seedlings (Figures 12A and 12B). The
delay in formation of the pre-RC indicated that the cell cycle was
arrested in G1, which is further supported by reduced endoreduplication in pro1 endosperm.
Based on these results, we propose a model for how Pro1
(P5CS2) affects general protein synthesis and the cell cycle
transition by affecting proline biosynthesis (Figure 13). In plants,
Pro1 (P5CS2), a rate-limiting enzyme, is responsible for proline
synthesis from glutamic acid in cytosol. When Pro1 (P5CS2)
function is disrupted, proline biosynthesis is suppressed and
proline becomes deficient. Due to this deficiency, uncharged
tRNApro is accumulated and triggers GCN2 activation. Activated
Figure 11. Evidence of Aberrant Cell Cycle in pro1-ref.
(A) Cell cycle analysis of 14-DAG seedlings of the wild type, pro1-ref, and
pro1-ref cultivated on medium supplemented with 3 mM L-proline. wt,
wild type; pro1, pro1-ref.
(B) Cell cycle analysis of 18-DAP endosperms of the wild type and pro1ref. wt, wild type; pro1, pro1-ref.
(C) Cell cycle analysis of 24-DAP endosperms of the wild type and pro1ref. wt, wild type; pro1, pro1-ref.
The small graphs (I to III) inserted in (A) to (C) are the cell cycle diagrams
of 14-DAG seedlings and 18- and 24-DAP endosperms analyzed by flow
cytometry. The 2C and 4C are DNA contents of the nuclei at stage G1
and S phase of 14-DAG seedlings, respectively. The 3C and 6C are DNA
contents of the nuclei at stage G1 and S phase of 18- and 24-DAP endosperms. The 12C and $24C are DNA contents of endoreduplicated
nuclei at stage S phase of 18- and 24-DAP endosperms. For each
sample, three independent biological replicates were performed. Bars
represent average values 6 SD, n = 3 replicates (No refers to P > 0.05,
* refers to P < 0.05, ** refers to P < 0.01, Student’s t test).
[See online article for color version of this figure.]
Figure 12. Cell Cycle–Related Gene Expression Analysis in the Wild Type and pro1-ref.
(A) RNA expression of cell cycle–related genes based on RT-PCR analysis of 14-DAG wild-type and pro1-ref seedlings cultivated in half-strength MS
culture medium supplemented with various concentrations of L-proline and L-glutamate. wt, wild type; pro1, pro1-ref.
(B) Expression of various cell cycle-related genes in 14-DAG wild-type and pro1-ref seedlings cultivated in half-strength MS culture medium supplemented with various concentrations of L-proline. Ubiquitin was used as an internal control. For each sample, three technical and two independent
biological replicates were performed. Error bars represent SD. wt, wild type; pro1, pro1-ref.
Pro1 Regulates Protein Synthesis and Cell Cycle
15 of 19
ethanol gradient up to 100% and then transferred to a propylene oxide
solution and slowly embedded in acrylic resin (London Resin Company),
where they were allowed to polymerize for at least 48 h. Thin sections (70
nm) were made using a diamond knife microtome (Reichert Ultracut E).
Sections were placed on 100-mesh copper grids and stained for 30 min
with uranyl acetate and for 15 min with lead citrate. Sections were visualized using a Hitachi H7600 transmission electron microscope.
Measurement of Starch, Lipids, Zeins, Nonzeins, and Total Protein
Twenty mature kernels of the wild type or mutant were collected from wellfilled, mature ears. The starch, lipid, zein, nonzein, and total protein
measurements were performed according to Wang et al. (2011), and
measurements were replicated three times for the pro1-ref and wild-type
endosperm.
Proline Feeding Assay
Figure 13. Proposed Model for Proline Function in General Protein
Synthesis and Cell Division.
Loss of function of Pro1 significantly inhibits proline biosynthesis and
decreases its accumulation in the pro1 mutant. Proline deficiency results
in an increased level of uncharged tRNApro AGG accumulation and triggers the phosphorylation of eIF2a. This causes a general reduction in
global protein synthesis in the mutant. Proline deficiency also downregulates major cyclin genes at the transcriptional level, causing cell
cycle arrest and suppression of cell proliferation.
GCN2 further phosphorylates eIF2a, resulting in general repression of protein synthesis. This directly affects zein protein
synthesis, explaining the small size and small number of protein
bodies and the subsequent opaque endosperm phenotype in
pro1. Proline deficiency also downregulates the expression of
cyclins and other cell cycle–related genes. The translational repression and downregulation of cyclins and cell cycle–related
genes inhibit pre-RC formation and DNA replication, causing cell
cycle arrest at the transition from G1 to S phase. Consequently,
the seedling lethality phenotype of pro1 is caused by general
protein translation repression and G1 phase cell cycle delay.
Seeds of pro1-ref and wild type were surface-sterilized by 10 min in 70%
ethanol, followed by 0.1% HgCl2 for 10 min. The seeds were rinsed three
times in sterile water and sown on a piece of sterile filter paper (Whatman
Grade No. 3MM filter paper) wetted with sterile water. After germination
for 5 d, the seedlings were transferred onto half-strength MS culture
medium containing different concentration L-proline or L-glutamate (0, 1,
and 3 mM) and placed in a growth chamber with a photoperiod of 16 h
light and 8 h dark at 25°C.
RT-PCR, Quantitative Real-time RT-PCR, and Gene
Expression Profiling
The maize (Zea mays) mutants pro1-ref (pro1-NA342 828D), pro1-N1154A
(828C), pro1-N1530 (828E), pro1-N1058 (827F), pro1-N1121 (827G), pro1N1528 (827H), and pro1-N1533 (827I) were obtained from the Maize Genetics
Cooperation stock center (additional information is provided in Supplemental
Table 1). The mutants were crossed into the Chang 7-2 genetic background to
produce the F2 populations. Maize plants were cultivated in the field at the
campus of Shanghai University, Shanghai, China.
Root, stem, the third leaf, silk, and tassel tissues were collected from at
least three W22, pro1, and wild-type plants at the V12 stage, and immature seeds were harvested at 9, 12, 15, 18, 21, 24, 27, 30, 33, and 36
DAP (Wang et al., 2012b)
RNA extraction, purification, and quantification were performed according to
Wang et al. (2012b). RT-PCR was performed as described previously (Wang
et al., 2012b). The qRT-PCR experiments were performed with two independent sets of RNA samples. Each RNA sample was extracted from
a pool of seedlings/kernels collected from at least three individual pro1-ref and
wild-type plants. For each RNA sample, three technical replicates were
performed in a final volume of 20 mL containing 10 mL 23 SYBR Green PCR
buffer, 1 mL reverse-transcribed cDNA (1 to 100 ng), and 1.8 mL 10 mM/L
forward and reverse primers. The qRT-PCR was performed using a Mastercycler ep realplex 2 (Eppendorf) with SYBR Green Real-Time PCR Master Mix
(Toyobo) according to the manufacturer’s protocol. Representative results
from two biological replicates are shown. The specificity of the PCR amplification procedures was checked with a heat dissociation curve protocol.
Quantification of the relative changes in gene expression was performed using
the 22DDcycle threshold method as described previously (Wang et al., 2012a), and
Ubiquitin was used as a reference gene. The RT-PCR and qRT-PCR reaction
was performed with the primers listed in Supplemental Data Set 1.
Three pro1-ref or wild-type biological repeats were pooled together.
For RNA-seq, total RNA of pro1-ref and the wild type was extracted using
an RNeasy Micro Kit (Qiagen) and provided to the ShanghaiBio Corporation
for quality check, library construction, and transcriptome sequencing.
Library construction was performed according to Illumina standard
instructions. Reads were aligned to the maize B73 genome using
TopHat2 (Langmead et al., 2009). Data were normalized as fragments
per kilobase of exon per million fragments mapped, as the sensitivity
of RNA-seq depends on the transcript length. Significant differentially
expressed genes were identified as genes with at least a 1.3-fold
change in expression and P value < 0.05.
Transmission Electron Microscopy Analysis
Phylogenetic Analysis
For transmission electron microscopy analysis, 18- and 21-DAP kernels
of the wild type and pro1-ref were fixed with paraformaldehyde and
postfixed in osmium tetraoxide. Fixed samples were dehydrated in an
Related sequences were identified in the NCBI nr (nonredundant protein
sequences) database by performing a BLASTp search (Camacho et al.,
2009) with Pro1 protein sequences. Amino acid sequences were aligned
METHODS
Plant Materials
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The Plant Cell
with MUSCLE method in MEGA5.2 software package using their default
settings for protein multiple alignment (Supplemental Data Set 3). A rooted
phylogenetic tree of P5CS genes of maize, rice (Oryza sativa), and Arabidopsis thaliana was constructed by the neighbor-joining method using the
MEGA5.2 software package. The evolutionary distances were computed
using the Poisson correction analysis. The bootstrap method with 1000
replicates (Tamura et al., 2007) for phylogeny test shows that all branch
nodes are 100% support. P5CS of Caenorhabditis elegans, Homo sapiens,
and Mus musculus as reference sequences.
Determining P5CS Enzyme Activity and Kinetics
The full-length Pro1 open reading frame was amplified from cDNA of W22
endosperm using primers FL Pro1-ORF F and FL Pro1-ORF R, digested
with BamHI and EcoRI, and cloned into pGEX-4T-1 (Amersham Biosciences) to create an in-frame fusion with GST. The GST-Pro1 fusion
protein was expressed in Escherichia coli host strain BL21 (Invitrogen) and
purified with a GSTrap FF column using an AKTA protein purification
system (GE Healthcare). P5CS activity measurement was according to
a previously published method (Parre et al., 2010).
Amino Acid Analysis
PBAAs were analyzed according to the method of Holding et al. (2010)
with some modification. Briefly, 3 mg aliquots of each sample were refluxed for 24 h in 6 N HCl. Samples were typically hydrolyzed at 110°C for
24 h in a vacuum or inert atmosphere to prevent oxidation. Hydrolysates
were evaporated to dryness. The residue was dissolved in 10 mL of pH 2.2
citrate buffer, and the amino acids were profiled on a Hitachi-L8900 amino
acid analyzer at the Instrumental Analysis Center of Shanghai Jiaotong
University. The FAAs were analyzed according to the method of Holding
et al. (2010). PBAA and FAA analyses were replicated three times for wildtype and opaque kernels.
Polyclonal Antibody Production
For Pro1 antibody, the purified GST fusion Pro1 protein produced as
described above was used as antigen. Production of 15-kD b-zein, 16-kD
g-zein, and 50-kD g-zein antibodies was according to Woo et al. (2001).
Antibodies were produced in white rabbits at the Shanghai ImmunoGen
Biological Technology. The polyclonal antibodies were affinity purified
using their respective bacterially expressed GST fusion proteins according to standard procedures (Harlow and Lane, 1988).
Subcellular Fractionation, Total Endosperm Protein Extraction, and
Immunoblot Analysis
For subcellular fractionation, 21-DAP kernels of W22 were ground into
a powder in liquid nitrogen and dissolved in resuspension buffer (25 mM
Tris-HCl, pH 7.5, 0.25 mM Suc, 2 mM EDTA, 2 mM DTT, 15 mM
b-mercaptoethanol, 10% glycerol, and proteinase inhibitor cocktail). After
removing cell debris by centrifuging at 500g and 10,000g, total proteins
were separated into soluble and total cell organelle fractions, the latter
containing total membrane, plasma membrane, and endomembrane,
according to Wang et al. (2012a).
Total protein from wild-type and mutant endosperms was extracted
according to the method of Bernard et al. (1994). Protein separation,
transfer, and immunoblotting were according to Wang et al. (2012a).
Immunoblot analyses with the Pro1 antibody (1:500 dilution), BiP antibody
(Santa Cruz Biotechnology) (1:1000 dilution), and ATPase antibody
(Sigma-Aldrich) (1:1000 dilution), 15-, 16-, and 50-kD zein antibody (1:500
dilution), a-tubulin antibody (Sigma-Aldrich) (1:1000 dilution), and O2
antibody (Santa Cruz Biotechnology) (1:1000 dilution) were performed
according to the manufacturer’s instructions.
tRNA Gel Blot Analysis
Developing endosperms (12, 18, and 24 DAP) and 14-DAG seedlings
(supplemented with 0, 1, and 3 mM, respectively) of pro1 and the wild
type were dissected and ground in liquid N2 and homogenized in 0.3 mL
of 0.3 M sodium acetate, 10 mM EDTA, pH 4.5. Phenol:chloroform (0.3
mL, pH 4.7) was added to each tube. Each Eppendorf tube was vortexed four to five times for 30 s each time, with incubation on ice in
between. Samples were centrifuged for 15 min, 4°C, 13,200 rpm, and
the top aqueous phase was transferred to new tubes and the phenol
extraction was repeated by adding 300 mL phenol:chloroform (pH 4.7).
The samples were centrifuged for 15 min, 4°C, 13,200 rpm, and the
aqueous phase was transferred to a fresh Eppendorf tube. The RNA
was precipitated by adding three volumes of cold 100% ethanol and
centrifuged at 13,200 rpm for 25 min at 4°C. The RNA pellet was resuspended in 60 mL of cold sodium acetate (0.3 M, pH 4.5) and the RNA
reprecipitated by adding 400ul 100% ethanol; the RNA pellet was airdried on ice. The RNA was resuspended in 25 mL of cold sodium
acetate (10 mM, pH 4.5) and quantitated spectrophotometrically. Total
RNA (5 µg) was separated on a 12% polyacrylamide gel (48 3 17 cm
gel). RNA gel electrophoresis and electroblotting was performed according to Jester (2011), Jester et al. (2003), and Huang et al. (2012).
Oligonucleotide probes complementary to nucleotides 21 to 47 of
maize tRNApro AGG were labeled with digoxigenin at the 59 end. The
membranes were prehybridized, hybridized, and washed according to
Huang et al. (2012).
eIF2 Phosphorylation Analysis
Developing endosperms of pro1 and wild type (12, 18, and 21 DAP) and
14-DAG seedlings (supplemented with 0, 1, and 3 mM, respectively) were
dissected. Total proteins were extracted in an ice-cold extraction buffer
containing 25 mM Tris-HCl (pH 7.5), 75 mM NaCl, 5% (v/v) glycerol,
0.05% (v/v) Nonidet P-40, 0.5 mM EDTA, 0.5 mM EGTA, 2 mM DTT, and
2% (w/v) insoluble PVP, supplemented with protease inhibitor cocktail VII
(CalBiochem) and a protein phosphatase inhibitor mix of 50 mM sodium
fluoride, 25 mM b-glycerophosphate, 10 mM sodium pyrophosphate, and
2 mM sodium orthovanadate (Na3VO4). Insoluble cell debris were removed
from the crude extracts by centrifugation for 15 min at 15,000 rpm and
4°C. The cleared protein extracts were quantified by Bradford assay
(Bradford, 1976).
Immunoblot analyses with the phosphor-eIF2a (S51) (Cell Signaling)
(1:1000 dilution) and eIF2a antibody (1:1000 dilution) (Cell Signaling) were
performed according to Harding et al. (2000), with some modification.
Briefly, the membranes were treated with blocking solution containing 2%
Amersham’s ECL advance blocking reagent in TBST buffer (20 mM TrisHCl, 137 mM NaCl, pH 7.6, and 0.1% [v/v] Tween 20) for 1 h at room
temperature with gentle shaking. Then, the membranes were incubated
with antibodies and visualized using the Super Signal West Pico
chemiluminescent substrate kit (Pierce) according to the manufacturer’s
instruction.
FCM 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
a Facscan (Becton Dickinson) laser flow cytometer equipped with an
argon-ion laser tuned at a wavelength of 448 nm. For each sample, at least
20,000 nuclei were collected and analyzed using a logarithmic scale
display. Each flow cytometric histogram was saved using PARTEC CA3
software and analyzed with WinMDI 2.8 software (http://www.cyto.
purdue.edu/flowcyt/software/Winmdi.htm).
Pro1 Regulates Protein Synthesis and Cell Cycle
17 of 19
Accession Numbers
REFERENCES
Sequence data from this article can be found in the GenBank/EMBL
data libraries under the following accession numbers: 10-kD d-zein,
AF371266 (GRMZM2G100018); 15-kD b-zein, M12147 (GRMZM2G086294);
16-kD g-zein, AF371262 (GRMZM2G060429); 27-kD g-zein, AF371261
(GRMZM2G138727); 50-kD g-zein, BT062750 (GRMZM2G138689); Zm BIP,
NM_001112423 (GRMZM2G114793); and Zm UBQ, BT018032. The transcriptome sequence data can be found in GenBank (http://www.ncbi.nlm.nih.
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Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure 1. Allelism Test among Other Mutants and
pro1-ref.
Supplemental Figure 2. Evidence of Exon Deletions Caused by Splice
Site Changes in pro1-N1154A, pro1-N1530, pro1-N1058, and pro1N1121.
Supplemental Figure 3. Sequence Alignment of P5CS Proteins.
Supplemental Figure 4. Aberrant Cell Cycles in pro1-N1058 and
pro1-N1533.
Supplemental Figure 5. Gene Expression Profiling Graphs of Wild
Type and pro1-ref.
Supplemental Figure 6. P5CS1 and P5CS2 Expression Analysis in
Maize Inbred Lines under Drought and Acid Stress during the Seedling
Stage.
Supplemental Table 1. Information about pro1 Alleles.
Supplemental Table 2. Summary of pro1 Candidate Genes (Collinear
to Rice) in a 130-kb Interval.
Supplemental Data Set 1. List of Primers Used in This Study.
Supplemental Data Set 2. Differentially Expressed Genes in 14-DAG
pro1 and Wild-Type Seedlings.
Supplemental Data Set 3. Text File of P5CS Protein Sequence
Alignment Data.
ACKNOWLEDGMENTS
This work was supported by the Ministry of Science and Technology of
China (2012AA10A305 and 2014CB138204), the National Natural Foundation of China (31171559, 91335208, and 31370035), the Shanghai
Key Basic Research Program (13JC1405000), and the Industry-StudyResearch Program of Shanghai High Education Commission. We thank
Brian A. Larkins (University of Arizona) for critically reading the article,
Qian Huang, Renfu Shang, and Ligang Wu (Institute of Biochemistry and
Cell Biology) for giving advice on tRNA analysis, Yanyun He (Shanghai
University) for flow cytometry assay, and Fei Wang (Shanghai University)
for helping with plant materials.
AUTHOR CONTRIBUTIONS
G.W. and R.S. designed the experiment. G.W., J.Z., X.F., G.F.W., X.S.,
H.Q., N.X., M.Z., Z.Q., and Y.T. performed the experiments. G.W., J.Z.,
G.F.W., and R.S. analyzed the data. G.W. and R.S. wrote the article.
Received March 21, 2014; revised May 19, 2014; accepted May 29,
2014; published June 20, 2014.
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Proline responding1 Plays a Critical Role in Regulating General Protein Synthesis and the Cell
Cycle in Maize
Gang Wang, Jushan Zhang, Guifeng Wang, Xiangyu Fan, Xin Sun, Hongli Qin, Nan Xu, Mingyu
Zhong, Zhenyi Qiao, Yuanping Tang and Rentao Song
Plant Cell; originally published online June 20, 2014;
DOI 10.1105/tpc.114.125559
This information is current as of June 17, 2017
Supplemental Data
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