Characterization of Rubisco Activase Genes in
Maize: An a-Isoform Gene Functions alongside a
b-Isoform Gene1[W][OPEN]
Zhitong Yin 2, Zhenliang Zhang 2, Dexiang Deng, Maoni Chao, Qingsong Gao, Yijun Wang, Zefeng Yang,
Yunlong Bian, Derong Hao, and Chenwu Xu*
Jiangsu Provincial Key Laboratory of Crop Genetics and Physiology, Key Laboratory of Plant Functional
Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China (Z.Yi., Z.Z., D.D., Q.G.,
Y.W., Z.Ya., Y.B., C.X.); National Center for Soybean Improvement, National Key Laboratory of Crop Genetics
and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China (M.C.); and Jiangsu
Yanjiang Institute of Agricultural Sciences, Nantong 226541, China (D.H.)
ORCID ID: 0000-0003-2482-8681 (C.X.).
Rubisco activase (RCA) catalyzes the activation of Rubisco in vivo and plays a crucial role in regulating plant growth. In maize
(Zea mays), only b-form RCA genes have been cloned and characterized. In this study, a genome-wide survey revealed the
presence of an a-form RCA gene and a b-form RCA gene in the maize genome, herein referred to as ZmRCAa and ZmRCAb,
respectively. An analysis of genomic DNA and complementary DNA sequences suggested that alternative splicing of the
ZmRCAb precursor mRNA (premRNA) at its 39 untranslated region could produce two distinctive ZmRCAb transcripts.
Analyses by electrophoresis and matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry
showed that ZmRCAa and ZmRCAb encode larger and smaller polypeptides of approximately 46 and 43 kD, respectively.
Transcriptional analyses demonstrated that the expression levels of both ZmRCAa and ZmRCAb were higher in leaves and
during grain filling and that expression followed a specific cyclic day/night pattern. In 123 maize inbred lines with extensive
genetic diversity, the transcript abundance and protein expression levels of these two RCA genes were positively correlated with
grain yield. Additionally, both genes demonstrated a similar correlation with grain yield compared with three C4 photosynthesis
genes. Our data suggest that, in addition to the b-form RCA-encoding gene, the a-form RCA-encoding gene also contributes to
the synthesis of RCA in maize and support the hypothesis that RCA genes may play an important role in determining maize
productivity.
Rubisco is the primary regulatory enzyme of photosynthesis and initiates photosynthetic carbon metabolism by combining atmospheric CO2 with ribulose
1,5-bisphosphate to form 3-phosphoglyceric acid. Numerous studies have shown that the activity of Rubisco
is regulated by a protein known as Rubisco activase
(RCA; Portis, 2003). RCA is a soluble chloroplast
ATPase associated with a variety of cellular activities
1
This work was supported by the National Basic Research Program of China (grant no. 2011CB100106 to C.X.), the National Natural
Science Foundation of China (grant nos. 30971846 and 31171187 to
C.X.), the Vital Project of Natural Science of Universities in Jiangsu
Province (grant no. 09KJA210002 to C.X. and grant no. 11KJA210004 to
Z.Yi.), the Priority Academic Program Development of Jiangsu Higher
Education Institutions (to Z.Yi.), and the Innovation of Science and
Technology Development Fund of Yangzhou University (grant no.
2013CXJ049 to Z.Yi.).
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:
Chenwu Xu ([email protected]).
[W]
The online version of this article contains Web-only data.
[OPEN]
Articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.113.230854
2096
(ATPases associated with diverse cellular activities)
that functions as a molecular chaperone (Sánchez de
Jiménez et al., 1995). This protein catalyzes the
activation of Rubisco in vivo by the ATP-dependent
removal of various inhibitory sugar phosphates
(Portis et al., 2008). The activity of RCA is dependent
on the ATP/ADP ratio (Zhang and Portis, 1999;
Carmo-Silva and Salvucci, 2013) and/or the redox
state of the chloroplast (Zhang et al., 2001; Wang and
Portis, 2006) and is extremely sensitive to high
temperature (Salvucci and Crafts-Brandner, 2004).
Thus, RCA can adjust the rate of CO2 fixation to the
rate of electron transport activity and can limit CO2
assimilation during heat stress.
In many plants, there are two forms of RCA: an
a isoform of 45 to 46 kD and a b isoform of 41 to 43 kD.
However, in some species, such as tobacco (Nicotiana
tabacum), cucumber (Cucumis sativus), and mung bean
(Vigna radiata), the a isoform is believed to be absent
(Portis, 2003). The greatest difference between the two
forms of RCA is usually at the carboxy terminus (Salvucci
et al., 1987; Portis, 2003). Compared with the b isoform,
the a isoform has a carboxy-terminal extension that
contains redox-sensitive Cys residues (Zhang and
Portis, 1999; Portis, 2003; Salvucci et al., 2003). Both the
a and b isoforms are capable of activating Rubisco;
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Rubisco Activase Genes in Maize
however, they have slightly different maximal activities (Shen et al., 1991). In rice (Oryza sativa), the
a isoform has been shown to play an important role in
photosynthetic acclimation to moderate heat stress in
vivo, whereas the b isoform has been shown to play
a major role in maintaining the initial activity of
Rubisco under normal conditions (Wang et al., 2010).
More significantly, light modulation of Rubisco in
Arabidopsis (Arabidopsis thaliana) requires a capacity
for redox regulation of the a isoform via thioredoxin-f
(Zhang and Portis, 1999; Zhang et al., 2001, 2002).
Genomic analyses have identified one RCA gene
in spinach (Spinacia oleracea), Arabidopsis, rice, and
wheat (Triticum aestivum; Werneke et al., 1988; To
et al., 1999; Law and Crafts-Brandner, 2001), two RCA
genes in barley (Hordeum vulgare) and cotton (Gossypium
hirsutum; Rundle and Zielinski, 1991; Salvucci et al.,
2003), and more than three RCA genes in tobacco
(Qian and Rodermel, 1993) and soybean (Glycine max;
Yin et al., 2010). In some species, such as spinach,
Arabidopsis, and rice, alternative splicing of RCA
transcripts results in two isoforms of RCA (Werneke
et al., 1989; Rundle and Zielinski, 1991; To et al., 1999),
whereas in other species such as cotton and soybean,
the two RCA isoforms are encoded by different genes
(Salvucci et al., 2003; Yin et al., 2010). In barley, in
addition to one alternatively spliced RCA gene (rcaA)
that produces two RCA isoforms, a second gene (rcaB)
encodes only the b isoform of RCA (Rundle and
Zielinski, 1991). Additionally, in some species such as
tobacco, bean (Phaseolus vulgaris), cucumber, and
mung bean, the RCA gene may only encode the
b isoform of RCA (Portis, 2003).
The expression patterns of the RCA gene in plants
have been extensively examined. The RCA gene is
expressed almost entirely in the green parts of the
plant in most plant species and is developmentally
regulated by leaf age and light (Watillon et al., 1993;
Liu et al., 1996). In tomatoes (Solanum lycopersicum),
apples (Malus domestica), Arabidopsis, and rice, the
mRNA levels of the RCA gene showed cyclic variations during the day/night period (Martino-Catt and
Ort, 1992; Watillon et al., 1993; Liu et al., 1996; To et al.,
1999). Diversity in gene expression is one of the
mechanisms underlying phenotypic diversity among
individuals. In soybean, positive correlations were
observed between the expression levels of two RCA
genes and the initial activity of Rubisco, photosynthetic rate, and grain yield in a recombinant inbred line
population, and expression quantitative trait loci
(eQTL) mapping revealed four trans-eQTL for the two
genes (Yin et al., 2010).
Maize (Zea mays), a C4 plant, is one of the most
important crops in the world, serving as an essential
source of food, feed, and fuel. Various research groups
have detected different numbers of RCA polypeptides
in this species, varying from one to three; the molecular masses of these polypeptides are approximately
41, 43, and/or 45 to 46 kD (Salvucci et al., 1987; CraftsBrandner and Salvucci, 2002; Vargas-Suárez et al.,
2004; Ristic et al., 2009). Two RCA complementary
DNAs (cDNAs), Zmrca1 and Zmrca2, have been
cloned (Ayala-Ochoa et al., 2004). These two cDNAs
contain identical open reading frames (ORFs) that
encode the 43-kD RCA polypeptide. Based on sequence similarity, this polypeptide appears to correspond to the b isoform of RCA, as reported for
other species (Werneke et al., 1989; To et al., 1999;
Salvucci et al., 2003). Limited proteolysis of the 43-kD
RCA at its amino-terminal region can give rise to a
41-kD RCA (Vargas-Suárez et al., 2004). Although
informative, these data do not clarify the origin of the
45- to 46-kD RCA polypeptide in maize.
Because the entire genome of the maize inbred line
B73 has been sequenced (Schnable et al., 2009), it is
possible to identify maize RCA genes on a genomewide scale. In this study, an a-form RCA gene and a
b-form RCA gene were identified in the maize genome
on the basis of currently available genomic resources,
and the relevance of these two genes to maize RCA
polypeptides was examined. In addition, the expression patterns of these two genes were investigated.
Lastly, the potential relationship between these two
genes and grain yield was analyzed in 123 maize inbred lines with extensive genetic variation. Our results
indicate that an a-form RCA-encoding gene functions
alongside a b-form RCA-encoding gene in maize and
that both genes play a role in determining maize
productivity.
RESULTS
Genomic Analysis and cDNA Cloning Reveal a-Form
and b-Form RCA Genes
In this study, a genome-wide survey of maize RCA
genes was performed. The Arabidopsis RCA gene sequence information (GenBank accession no. 818558)
was used to query the maize genome sequence database (http://www.phytozome.net/). We identified
an a-form RCA-encoding gene and a b-form RCAencoding gene on chromosome 4, designated ZmRCAa
and ZmRCAb, respectively. The identity of these two
genes was corroborated by sequencing the PCR products
amplified from the genomic DNA of four randomly selected inbred lines using gene-specific primers (data not
shown).
Two b-form RCA cDNAs, Zmrca1 (GenBank accession no. AF084478) and Zmrca2 (GenBank accession
no. AF305876), were cloned and characterized in maize
(Ayala-Ochoa et al., 2004). These two cDNAs contain
identical ORFs but have differing 39 untranslated regions (UTRs) with different downstream-like elements.
To investigate the relationship between the two b-form
cDNAs and the ZmRCAb identified in this study, we
cloned the genomic DNA and full-length cDNAs of
Zmrca1 and Zmrca2 from the inbred line JB using genespecific primers (Supplemental Table S1). Alignment
analysis (http://www.ncbi.nlm.nih.gov/spidey/) showed
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Yin et al.
that both the Zmrca1 and Zmrca2 cDNAs precisely
matched the genomic sequences of ZmRCAb, suggesting that the Zmrca1 and Zmrca2 mRNAs could arise
from alternative splicing of the ZmRCAb genomic
DNA (Supplemental Fig. S1). When two splice sites at
the 39 UTRs were utilized, a single 112-nucleotide intron was removed to produce the Zmrca2 mRNA. Alternatively, the first 28 nucleotides of this intron were
retained to produce the Zmrca1 mRNA. Sequence
alignment of the Zmrca1 and Zmrca2 cDNAs identified
from the maize variety Chalqueño (Ayala-Ochoa et al.,
2004) and the ZmRCAb genomic DNA from the inbred
line B73 (http://www.phytozome.net/) also produced
the same result.
Based on the genomic DNA sequence information,
the cDNA of ZmRCAa was also cloned from the inbred
line JB. This ZmRCAa cDNA had not been previously
cloned in maize, and we deposited its sequence in
GenBank under the accession number JX863889. This
cDNA is predicted to encode a protein of 463 amino
acids with a calculated molecular mass of 51.04 kD
(Fig. 1). The first 50 amino acids at the amino terminus
of ZmRCAa were predicted to be chloroplast transit
peptides (ChloroP version 1.1 server; http://www.cbs.
dtu.dk/services/ChloroP/). Thus, the predicted mature
protein encoded by ZmRCAa contains 413 amino acids
and has a calculated molecular mass of 45.96 kD.
Compared with ZmRCAb, the deduced protein sequence of ZmRCAa contains a 30-amino acid extension
at the carboxy terminus (Fig. 1), including two Cys
residues that are known to be involved in redox regulation (Zhang and Portis, 1999; Salvucci et al., 2003).
Similar to the ZmRCAb protein, two conserved ATPbinding domains, GGKGQGKS and LFIND (Shen and
Ogren, 1992), were also identified in the ZmRCAa
protein. Based on its size and its long carboxy terminal
region, the ZmRCAa protein appears to correspond to
the a-form RCA reported in other species (Werneke
et al., 1989; To et al., 1999; Salvucci et al., 2003). We
also cloned the cDNAs of ZmRCAa from three other
maize inbred lines, XD053, Y6, and Y53, and obtained
similar results. A sequence alignment of the ZmRCAa
ORFs from the different inbred lines is shown in
Supplemental Figure S2.
ZmRCAa and ZmRCAb Encode Two Different Maize
RCA Polypeptides
Protein extracts from Escherichia coli transformed with
pET-30a expressing the truncated ORF of either ZmRCAa
or ZmRCAb and several other protein extracts used as
controls were separated using SDS-PAGE and probed
with polyclonal cotton RCA antibodies (AS10700,
Agrisera; Fig. 2A). Two polypeptides of approximately
47 and 43 kD were detected in the positive control,
Arabidopsis (lane 6), which is consistent with previous
findings (Salvucci et al., 1987), whereas the negative
controls showed no specific bands (lanes 4 and 5).
Both the ZmRCAa and ZmRCAb recombinant proteins were recognized by the anti-cotton RCA antibody
(lanes 2 and 3). As expected, the molecular mass of
the recombinant His-tag ZmRCAa was larger than that
of the recombinant His-tag ZmRCAb. Because the two
Figure 1. Alignment of the amino acid sequences deduced from the ZmRCAa and ZmRCAb cDNAs. The sequences were
aligned using the ClustalX program (version 1.81) and viewed using the GeneDOC program (version 2.6). The numbers to the
right of the alignment indicate the amino acid positions in the ZmRCAa or ZmRCAb putative protein sequences. Identical
amino acids (single-letter code) are shown in white characters on a black background. The arrow indicates the putative
cleavage site of the putative transit peptide. The numbers in the middle of the sequences indicate the two conserved ATPbinding domains (1 and 2). The diamonds indicate the conserved Cys residues involved in redox regulation in ZmRCAa. The
dashes represent gaps that were introduced into ZmRCAa or ZmRCAb to optimize the alignment. The black line and the double
lines indicate the sequences used to develop the polyclonal antibodies against ZmRCAa and ZmRCAb.
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Rubisco Activase Genes in Maize
Figure 2. Immunological detection of RCA polypeptides. Protein extracts were separated using SDS-PAGE and probed with an anti-cotton
RCA antibody. Immunoreactive proteins were detected by chemiluminescence using an anti-rabbit IgG-HRP conjugate. A, Protein
markers (lane 1), recombinant His-tag ZmRCAa expressed in E. coli
using the vector pET-30a (lane 2), recombinant His-tag ZmRCAb
expressed in E. coli using the vector pET-30a (lane 3), untransformed
E. coli (lane 4), E. coli transformed with the empty pET-30a vector
(lane 5), and protein extracts from Arabidopsis leaves (lane 6). B, Protein
markers (lane 1), purified maize leaf ZmRCAb (lane 2), purified maize
leaf ZmRCAa (lane 3), and protein extracts from maize leaves (lane 4).
The purified maize leaf ZmRCAa and ZmRCAb were prepared by
immunoaffinity chromatography using their specific antibodies.
recombinant proteins contain amino-terminal His-tags,
their molecular masses were larger than those of the
RCA polypeptides detected in the Arabidopsis leaves
(lane 6). These results confirm that ZmRCAa and
ZmRCAb do encode RCA proteins.
To examine the relationship of ZmRCAa and
ZmRCAb to maize RCA polypeptides, two synthesized antigen peptides corresponding to the predicted
sequences of ZmRCAa and ZmRCAb, respectively
(Fig. 1), were used to generate specific polyclonal antibodies. In two independent western-blot experiments, both antibodies reacted with one maize leaf
RCA polypeptide (data not shown). We purified maize
leaf ZmRCAa and ZmRCAb using the two specific
antibodies. The purified maize leaf RCAs and maize
leaf protein extracts were probed with polyclonal
cotton RCA antibodies (Fig. 2B). Both the purified
leaf ZmRCAa and ZmRCAb showed specific bands
(lanes 2 and 3). Three polypeptides were detected in
maize (lane 4). The two larger polypeptides had molecular masses similar to those of the purified leaf
ZmRCAa and ZmRCAb, suggesting that the maize
polypeptides might correspond to the a- and b-RCA
isoforms. The smallest maize polypeptide appeared to be
a degradation product, possibly from proteolysis. Different numbers of RCA polypeptides, varying from one
to three, were previously detected in maize (Salvucci
et al., 1987; Crafts-Brandner and Salvucci, 2002; Vargas-Suárez et al., 2004; Ristic et al., 2009). The variability in these observations might be due to the use of
different affinities of the antibodies, different experimental conditions, and/or proteolysis (Salvucci et al.,
1993; To et al., 1999).
We used matrix-assisted laser desorption/ionization
(MALDI)-tandem time-of-flight (TOF) mass spectrometry
(MS) analysis to precisely determine the molecular masses
of maize RCA proteins. As shown in Supplemental
Figure S3, the molecular masses of purified recombinant His-tag ZmRCAa, purified recombinant His-tag
ZmRCAb, purified leaf ZmRCAa, and purified leaf
ZmRCAb were 52.8, 50.1, 46.1, and 43.3 kD, respectively. The determined molecular mass of the purified
leaf ZmRCAa was 0.14 kD larger than its predicted
molecular mass. This deviation is within the error limits
(60.2 kD) of the MALDI-TOF instrument. Together
with the observations made by SDS-PAGE analysis
(Fig. 2), these results show that, similar to the previously
characterized b-form gene ZmRCAb (Ayala-Ochoa et al.,
2004; Vargas-Suárez et al., 2004), the a-form gene
ZmRCAa also encodes an RCA polypeptide, but a
larger one.
ZmRCAa and ZmRCAb Transcripts Are Predominantly
Expressed in Leaves and Show Cyclic Day/Night
Expression Patterns
At the 16-leaf stage, samples of the roots, stems,
leaves, tassel spikelets, and immature ears of the maize
plants were collected, and the transcript abundance
of ZmRCAa and ZmRCAb in these tissues was investigated using semiquantitative reverse transcription
(RT)-PCR (Supplemental Fig. S4A) and real-time quantitative RT-PCR assays (Fig. 3A). Because alternative
splicing of the ZmRCAb precursor RNA (premRNA) at
its 39 UTR created two transcripts with identical ORFs
as mentioned above, we measured the expression of
this gene by designing primers that detect the two
transcripts simultaneously (Supplemental Table S1).
The ZmRCAa and ZmRCAb transcripts accumulated
primarily in the leaves and, to a lesser extent, in the
stems, tassel spikelets, and immature ears of the plants,
whereas no or very low signal was detected in the roots.
We also examined the protein expression levels of
ZmRCAa and ZmRCAb and obtained a similar result
(Fig. 3B).
At 32 d after anthesis (DAA), the leaves closest
to the ear were harvested at 4-h intervals during a
48-h span from maize plants grown under a 12-hlight/12-h-dark cycle (Fig. 3C). Both semiquantitative
RT-PCR (Supplemental Fig. S4B) and real-time quantitative RT-PCR (Fig. 3D) assays showed that transcripts of ZmRCAa and ZmRCAb gradually increased
during the dark period, with relatively higher levels
detected at 8:30 AM, 2.5 h after the beginning of the
light period. Following this peak in expression, the
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Yin et al.
Figure 3. Expression profiles of ZmRCAa and ZmRCAb in different maize tissues (A and B) and during day/night cycles (C and E).
A and D, Transcript expression level measured by real-time quantitative RT-PCR. B and E, Protein expression level determined by
an ELISA quantitative sandwich technique using specific antibodies. C, The 12-h-light and 12-h-dark periods over 2 d of leaf
sampling are represented by white and black bars, respectively. At the 16-leaf stage, the indicated samples of the inbred line JB at
32 DAA were used to determine the expression profiles. The error bars represent the SEs of three independent repetitions.
levels of these transcripts gradually declined, reaching
relatively lower levels at 8:30 PM, 2.5 h after the end of
the light period. Similar results were observed previously for the two b-form RCA transcripts (Ayala-Ochoa
et al., 2004). By contrast, no abrupt changes in the
protein expression levels of ZmRCAa and ZmRCAb
within the 12-h-light/12-h-dark periods were observed
(Fig. 3E).
The Expression Levels of ZmRCAa and ZmRCAb Are
Correlated with Grain Yield in Maize
The transcript abundance of ZmRCAa and ZmRCAb
was determined in maize plants at various stages of
growth. Both semiquantitative RT-PCR (Supplemental
Fig. S4C) and real-time quantitative RT-PCR (Fig. 4A)
assays showed that the ZmRCAa and ZmRCAb mRNAs
were expressed at a relatively low level in the early
growth stages of maize (up to tasselling) but accumulated to higher levels during the grain-filling stages
(16–40 DAA), with maximum values at approximately
32 DAA. Measurement of the protein expression levels
of ZmRCAa and ZmRCAb showed that the proteins
encoded by the two genes were expressed at higher
levels during the grain-filling stages (Fig. 4B). Because
grain filling is crucial for grain formation, the higher
expression levels of ZmRCAa and ZmRCAb during
this stage suggested that these two genes might play a
role in determining grain yield.
To further investigate the relationship between the
two RCA genes ZmRCAa and ZmRCAb and grain
yield, we determined the transcript abundance and
protein expression levels of these two genes, as well as
the grain yield, in 123 maize inbred lines. Pedigree
information revealed that these lines are genetically
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Rubisco Activase Genes in Maize
2010). The transcript abundance of these genes was
also significantly correlated with grain yield (Table I),
and ZmRCAa and ZmRCAb expression showed a
similar correlation.
DISCUSSION
An a-Form Large RCA Polypeptide Gene Is Expressed
in Maize
Figure 4. Expression profiles of ZmRCAa and ZmRCAb at different
growth stages. A, Transcript expression level measured by real-time
quantitative RT-PCR. B, Protein expression level determined by an
ELISA quantitative sandwich technique using specific antibodies. The
mature upper third leaves of the inbred line JB were collected at the
indicated growth stages for expression profiling. The error bars represent the SEs of three independent repetitions.
diverse and that they originated from diverse germplasm
resources that are currently used in China (e.g. Lancaster, Reid, Tangsipingtou, P, Lvdahonggu, and
Waxy maize; Supplemental Table S2). All of the measured traits varied widely among the different inbred
lines (Supplemental Tables S2 and S3). The transcript
abundance of ZmRCAa and ZmRCAb in the 123 maize
lines was positively correlated with their protein expression levels, and both the transcript abundance and
protein expression levels of these two genes were
significantly correlated with grain yield (Table I).
In the 123 maize lines, we also determined the transcript
abundance of three C4 photosynthesis genes, Nicotinamide
Adenine Dinucleotide Phosphate-Malic Enzyme (NADP-ME),
Pyruvate Orthophosphate Dikinase (PPDK), and Phosphoenolpyruvate Carboxylase (PEPC), which are widely
thought to play an import role in determining grain
yield (Hibberd et al., 2008; Hibberd and Covshoff,
In maize under nonstress conditions, only b-form
RCA polypeptides were previously detected by westernblot analysis (Salvucci et al., 1987; Vargas-Suárez et al.,
2004). In this study, we used different antibodies than
those used previously, i.e. an antibody against cotton
RCA and a ZmRCAa-specific antibody, and detected
an a-form RCA polypeptide of approximately 46 kD
(Fig. 2; Supplemental Fig. S3). By contrast, when we
used an antibody against Arabidopsis RCA (aA-18; sc15864; Santa Cruz Biotechnology), we did not detect
this a-form RCA polypeptide but only a polypeptide
of approximately 43 kD, possibly a b-form RCA (data
not shown). This variability might be due to the different affinities of the antibodies employed. Heat stress
conditions might induce the expression of the a-form
RCA in maize. During heat stress, a large RCA polypeptide of 45 to 46 kD, possibly the a-form RCA, was
detected in maize by western-blot analysis (Sánchez de
Jiménez et al., 1995; Crafts-Brandner and Salvucci,
2002; Ristic et al., 2009).
In previous studies, two b-form maize RCA cDNAs
that encode the same polypeptide but show different
sequences at their 39 UTRs were cloned and characterized (Ayala-Ochoa et al., 2004; Vargas-Suárez et al.,
2004). In this study, on the basis of the genomic sequence of the inbred line B73, we cloned an a-form
RCA gene, ZmRCAa, of maize for the first time. PCR
amplification and sequence analysis confirmed that
this gene is present in the genomes of diverse maize
inbred lines, and both transcript and protein expression measurements showed that it is expressed in
maize. The presence of an a-form RCA gene sequence
in maize genome was also inferred in a recent study
(Carmo-Silva and Salvucci, 2013).
Similar to the phenomenon previously observed
for b-form RCA genes (Ayala-Ochoa et al., 2004),
ZmRCAa transcripts also show cyclic variations related to day/night period (Fig. 3), consistent with the
reported involvement of the circadian clock in the accumulation of RCA mRNA (Pilgrim and McClung,
1993; Watillon et al., 1993). However, compared with
the b-form RCA gene ZmRCAb, the transcript and
protein expression levels of ZmRCAa were approximately 10 and 2 times lower, on average, in the 123
inbred lines (Supplemental Tables S2 and S3), indicating that the a-form RCA is not as abundant as the
b-form RCA in maize under nonstress conditions.
Under heat stress conditions in maize, limited proteolysis of the 43-kD b-form RCA gene product(s)
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Yin et al.
Table I. Correlation coefficients among the transcript abundance of ZmRCAa, ZmRCAb, NADP-ME, PPDK, and PEPC, the protein expression levels
of ZmRCAa and ZmRCAb, and grain yield in 123 maize inbred lines
Single and double asterisks represent significance at P , 0.05 and P , 0.01, respectively.
Traits
ZmRCAb
Transcript
Abundance
ZmRCAa
Transcript
Abundance
ZmRCAa transcript abundance
ZmRCAb protein expression level
ZmRCAa protein expression level
NADP-ME transcript abundance
PPDK transcript abundance
PEPC transcript abundance
Grain yield
0.360**
0.599**
0.364**
0.493**
0.750**
0.391**
0.489**
0.214*
0.634**
0.487**
0.408**
0.406**
0.214*
ZmRCAb Protein
Expression Level
ZmRCAa Protein
Expression Level
0.339**
0.273**
0.390**
0.244**
0.299**
0.377**
0.366**
0.310**
0.222*
occurs at its amino-terminal region, resulting in a
smaller RCA polypeptide of 41 kD (Vargas-Suárez
et al., 2004). However, no information regarding the
origin of the larger heat-induced maize RCA polypeptide of 45 to 46 kD (Sánchez de Jiménez et al., 1995;
Crafts-Brandner and Salvucci, 2002; Ristic et al., 2009)
is available. The polypeptide encoded by the a-form
RCA gene ZmRCAa identified in this study is larger
than the 43-kD polypeptide encoded by the ZmRCAb
gene (Fig. 2); its molecular mass is 46.1 kD, as determined by MALDI-TOF-MS analysis (Supplemental
Fig. S3). It is thus very possible that this gene contributes to the synthesis of the larger heat-induced
RCA polypeptide in maize. Additional studies are required to investigate the potential role of ZmRCAa in
the heat stress response of maize.
The regulation of Rubisco activity by light depends
on the response of RCA to the chloroplast redox state
and/or the ADP/ATP ratio (Zhang and Portis, 1999;
Zhang et al., 2002; Carmo-Silva and Salvucci, 2013).
RCA sensitivity is conferred by two Cys residues in the
carboxy terminus of the a-isoform (Zhang et al., 2002).
The gene product of ZmRCAa contains these two Cys
residues (Fig. 1), suggesting that this protein could
play a role in the light modulation of Rubisco in maize.
However, Rubisco in maize is not extensively regulated in response to changes in light intensity (Sage
and Seemann, 1993), a finding that might be due to the
low expression level of the a-form gene in maize, as
mentioned above. Additionally, we cannot exclude the
possibility that the regulatory properties of the a-form
RCA in maize differ from those of RCA in other species. The activities of b-form RCAs from different
species have been reported to vary when the proteins are subjected to the same physiological ratios
of ADP/ATP (Carmo-Silva and Salvucci, 2013); a similar
situation might also apply to the RCA a-isoform.
Posttranscriptional Regulation of the RCA Gene
Posttranscriptional regulation of gene expression
occurs at the levels of premRNA processing (capping,
splicing, and polyadenylation) and/or mRNA translation. In many species, alternative splicing of one
NADP-ME
Transcript
Abundance
PPDK
Transcript
Abundance
0.706**
0.874**
0.328**
0.594**
0.427**
PEPC Transcript
Abundance
0.306**
premRNA can produce a- and b-form RCA transcripts
(Portis, 2003), whereas in soybean and cotton, the two
types of transcripts arise from separate genes (Salvucci
et al., 2003; Yin et al., 2010). Additionally, under heat
stress conditions, RCA gene transcripts can be regulated by alternative polyadenylation of their 39 UTR
(DeRidder and Salvucci, 2007). Although a previous
study suggested that the two b-form RCA cDNAs
Zmrca1 and Zmrca2 arise from separate genes in the
maize genome (Ayala-Ochoa et al., 2004), in the light
of the new evidence, knowledge, and technologies
available, our study shows that these two cDNAs appear to arise from alternative splicing of the same gene.
The alternative splicing sites are located in the 39 UTR
of the ZmRCAb premRNA (Supplemental Fig. S1);
thus, splicing at these sites would not change the
amino acid sequence of the resulting protein products.
Alternative splicing of premRNA at its 39 UTR is not a
rare phenomenon in plants. In Arabidopsis, it was
reported that approximately 6.4% of all alternativesplicing events occur in 39 UTRs (Reddy, 2007).
To the best of our knowledge, this study is the first
to show that alternative splicing can produce two
different b-form RCA transcripts. Although it does not
alter the encoded amino acid sequence, alternative
splicing in the 39 UTR can produce transcripts with
different sequence lengths and/or structures. In a previous study, under nonstress conditions, the Zmrca1
and Zmrca2 transcripts showed cyclic variations during
a day/night period, with the Zmrca2 transcript exhibiting greater amplitude in its steady-state levels than
Zmrca1 (Ayala-Ochoa et al., 2004). The authors of that
study suggested that the downstream-like elements in
the 39 UTRs may regulate the expression of these two
transcripts. In this study, we further observed that Zmrca2
was more highly expressed than Zmrca1 in six randomly
selected maize inbred lines under nonstress conditions
(data not shown). In cotton and Arabidopsis plants acclimated to heat stress, stabilization of the RCA transcript levels was linked to the production of transcripts
with shorter 39 UTRs (DeRidder and Salvucci, 2007;
DeRidder et al., 2012). The Zmrca1 transcript has a
shorter 39 UTR than the Zmrca2 transcript. Future studies
aimed at investigating whether heat stress conditions
induce Zmrca1 transcript expression are necessary.
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Rubisco Activase Genes in Maize
Genome Duplication Could Potentially Be Related to the
Origin of Two RCA Genes in Maize
In most plants studied to date, only one single
alternatively spliced RCA gene has been identified
(Werneke et al., 1988; To et al., 1999; Portis, 2003). In
this study, two RCA genes, including an a-form and a
b-form, were identified in the maize genome. Polyploidy is a crucial force in plant evolution, and many
angiosperms have undergone one or more polyploidization events (Adams and Wendel, 2005). Compared with old polyploid plant species such as rice
and Arabidopsis, which contain only one RCA gene
(Werneke et al., 1989; To et al., 1999), maize experienced
a recent genome duplication 5 to 12 million years ago
(Schnable et al., 2011). Thus, we guess that this recent
genome duplication could potentially be related to the
origin of the two RCA genes identified in maize. Consistent with this idea, cotton and soybean, the two
polyploid species that have most recently undergone
genome duplication within the past 5 million years
(Adams and Wendel, 2005), also contain at least two
RCA genes (Salvucci et al., 2003; Yin et al., 2010).
The Expression Levels of ZmRCAa and ZmRCAb Could
Modulate Grain Yield in Maize
By transforming the C4 plant Flaveria bidentis with
an antisense RCA gene, von Caemmerer et al. (2005)
showed that RCA activity was essential for the proper
functioning of the C4 photosynthetic pathway. In
maize, which is also a C4 plant, the RCA protein
content of leaves during grain filling was higher in
high-yield populations than in low-yield populations
(Martı’nez-Barajas et al., 1997; Morales et al., 1999). In
this study, we used a new strategy to investigate the
effect of RCA on maize grain yield by determining both
the transcript abundance and protein expression levels
of two RCA genes in diverse maize inbred lines.
The two RCA genes ZmRCAa and ZmRCAb were
more highly expressed at both the transcript and
protein levels during grain filling than at any other
growth stages (Fig. 4; Supplemental Fig. S4). The
transcript abundance and protein expression levels of
these genes during grain filling were positively correlated with grain yield in 123 inbred lines (Table I).
Additionally, both genes demonstrated a similar
correlation with grain yield compared with three C4
photosynthesis genes. Lastly, our preliminary eQTL
analysis revealed that each of the two RCA genes
had one eQTL that coincided with the quantitative
trait loci for grain yield in the 123 inbred lines
(Supplemental Table S4). These data support the hypothesis that RCA genes play an important role in
determining plant productivity. A similar result was
observed in our recent studies in soybean, in which
the transcript abundance of two RCA genes was
positively correlated with grain yield (Yin et al., 2010;
Chao et al., 2014).
It was recently reported that RCA plays an important role in regulating non-steady-state photosynthesis
(Yamori et al., 2012). Both the maize in this study and
the soybean in the previous studies (Yin et al., 2010;
Chao et al., 2014) were grown under natural conditions
and thus were exposed to a highly variable light environment each day. Plants with higher levels of RCA
gene expression are thought to exhibit a more rapid
increase in photosynthesis following an increase in
light intensity, which could have resulted in increased
grain yield. However, considering that the two RCA
genes ZmRCAa and ZmRCAb showed similar correlations with grain yield as the three C4 photosynthesis
genes NADP-ME, PPDK, and PEPC (Table I), it is more
likely that the relationship between RCA gene expression and grain yield is a general response reflecting greater photosynthetic capacity than that it involves
a more rapid increase in photosynthesis during light
transients.
CONCLUSION
In maize, an a-form RCA gene, ZmRCAa, functions
alongside a b-form RCA gene, ZmRCAb. ZmRCAa
encodes a larger maize RCA polypeptide. Similar to
ZmRCAb, ZmRCAa transcripts accumulate to higher
levels in leaves than in other tissues and show cyclic
variation during a day/night period. Both ZmRCAa
and ZmRCAb may play important roles in determining maize productivity.
MATERIALS AND METHODS
Plant Material and Plant Growth Conditions
The following four maize (Zea mays) inbred lines, which have different
genetic backgrounds, were used for cloning, western blotting, and/or expression pattern analysis of RCA: JB, XD053, Y6, and Y53. These four inbred
lines are all members of the popular heterotic groups used in China. JB and
Y53 are the parents of the commercial hybrid Suyu 16, which is currently
grown in the Jiangsu province of China. XD053 and Y6 differ in multiple traits
and are the parents of a recombinant inbred line population developed in our
laboratory.
To investigate the relationship between RCA genes and grain yield, a total of
123 maize inbred lines from various geographic locations in China or from other
origins were chosen for this study. These inbred lines included the parents of
the commercial hybrids widely used in China as well as lines derived from
Chinese landraces and waxy maize lines. Details of the pedigrees of these
inbred lines are provided in Supplemental Table S2.
The four inbred lines JB, XD053, Y6, and Y53 were grown under field
conditions at the experimental farm of Yangzhou University, Yangzhou, China.
Sowing was performed on July 10, 2010. The 123 maize inbred lines were grown
under field conditions at the experimental farm of Jiangsu Yanjiang Institute of
Agricultural Sciences, Nantong, China. All of the lines were planted in a
randomized complete block design with two replications. Each plot included a
single row 2.5 m long and 0.60 m wide, with a total of 10 plants and a density of
60,000 plants ha–1. Sowing was performed on March 25, 2011. The cultivation
management protocol followed local standard practices at each location.
Tissue Preparation
All leaf samples were obtained from the mature upper third of the leaf
unless otherwise noted. For each leaf sample, the middle portions of the leaves
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Yin et al.
were cut into small pieces and then pooled. At the four-leaf stage, leaf samples
were collected from maize plants for cloning and/or western-blot analysis. At
the 16-leaf stage, the leaves, roots, stems, immature ears, and tassel spikelets
were individually collected for use in the analysis of RCA gene expression
levels in different tissues. At 32 DAA, leaf samples obtained at different times
(12:30 AM, 4:30 AM, 8:30 AM, 12:30 PM, 4:30 PM) on two successive days were
collected to determine RCA gene expression levels during a day/night cycle.
At nine developmental stages (four-, eight-, 12-, and 16-leaf and 16, 24, 32, 40,
and 46 DAA), leaf samples were collected at approximately 8:30 AM to determine RCA gene expression at the different growth stages. To determine the
RCA gene expression levels in the 123 inbred lines, at approximately 30 DAA,
the leaves closest to the ear were collected from the plants in each plot in the
morning (8:00 AM to 9:30 AM) on a sunny day.
All of the samples were collected from three plants of each corresponding
inbred line in each plot. After collection, the samples were immediately frozen
in liquid nitrogen and subsequently stored at –80°C until further use.
Cloning of the Maize RCA Gene and Sequence Analysis
To identify RCA genes in maize, BLASTP searches targeting the maize genome sequence were performed with the Arabidopsis (Arabidopsis thaliana) RCA
protein (GenBank accession no. 818558) as the query using the Phytozome
Search Tools program (http://www.phytozome.net). The deduced nucleotide
sequences of the target genes were downloaded. Consequently, two maize RCA
genes were obtained; these were designated ZmRCAa (for the sequence with the
longer ORF) and ZmRCAb (for the sequence with the shorter ORF).
Based on the sequence information, the genomic DNA and full-length cDNA
sequences of ZmRCAa and ZmRCAb were amplified from maize leaves using
gene-specific primers (Supplemental Table S1). The amplified products were
purified, cloned into the pGEM-T vector (Promega), and subsequently sequenced (Beijing Genomics Institute). Sequence analysis was performed using
DNAMAN software (http://www.lynnon.com) and the ChloroP version 1.1
server (http://www.cbs.dtu.dk/services/ChloroP/). The molecular mass of the
predicted protein was calculated using the BioXM program (version 2.6; http://
www.bio-soft.net/format/bioxm.htm).
Expression of Recombinant RCA Proteins
Using the primers listed in Supplemental Table S1, the ZmRCAa and
ZmRCAb cDNAs were used as templates to amplify the truncated ORF
lacking a signal sequence. PCR products were digested with BamHI, HindIII,
and/or EcoRI restriction enzymes and inserted into the pET-30a expression
vector (Novagen). The resulting constructs were introduced into Escherichia
coli strain BL21 (DE3; Novagen). The expression of these two recombinant
RCA isoforms was performed as previously described (Yin et al., 2010).
Leaf Protein Extraction and Western-Blot Analysis of
RCA Protein
Leaf protein was extracted using previously described methods (Yin et al.,
2010). Protein extracts were subjected to SDS-PAGE using a 12.5% (w/v) acrylamide resolving gel. The separated proteins were then transferred onto
polyvinylidene difluoride membranes, and nonspecific antibody binding was
blocked with 5% (w/v) nonfat dried milk in phosphate-buffered saline (PBS;
pH 7.4) for 1 h at room temperature. The membranes were then incubated
overnight at 4°C with polyclonal cotton anti-RCA antibodies (AS10700,
Agrisera) diluted 1:10,000 in PBS plus 1% (w/v) nonfat milk. Immune complexes were detected using goat anti-rabbit IgG-horseradish peroxidase (HRP;
sc-2004; Santa Cruz Biotechnology). Color was developed with a solution
containing 3,39-diaminobenzidine tetrahydrochloride as the peroxidase substrate, and the membranes were scanned.
Relative Quantification of Transcript Expression
We used semiquantitative RT-PCR and/or real-time quantitative RT-PCR
assays to determine the transcript abundance of ZmRCAa, ZmRCAb, NADP-ME,
PPDK, and PEPC. The constitutively expressed Actin gene (GenBank accession
no. J01238) was used as an endogenous reference. For the semiquantitative RTPCR assays, 178- and 153-bp fragments of the ZmRCAa and ZmRCAb cDNAs,
respectively, were amplified using Pfu DNA polymerase (Promega). Real-time quantitative RT-PCR was performed according to previously described procedures (Yin
et al., 2010). A mixture of cDNA from different inbred lines was used to calibrate each
RT-PCR plate. The normalized expression for each line was calculated as delta-delta
cycle threshold (DDCT) = (CT, Target – CT, Actin)genotype – (CT, Target – CT, Actin)calibrator.
The gene-specific primers used for semiquantitative RT-PCR and real-time
quantitative RT-PCR are listed in Supplemental Table S1.
Peptide Synthesis and Polyclonal Antibody Generation
Two antigen peptides, N-F-D-P-T-A-R-S-D-D-G-S and A-K-E-V-D-E-T-KQ-T-D, corresponding to the carboxy terminus of predicted ZmRCAa and the
amino terminus of predicted ZmRCAb, respectively, were synthesized, and
polyclonal antibodies against these peptides were generated in male New
Zealand rabbits (GL Biochem). The antibodies were affinity purified on protein A-Sepharose, and their specificity for the corresponding peptides was
determined by ELISA. The antibody titers were 1:512,000 and 1:128,000 for the
antibodies corresponding to ZmRCAa and ZmRCAb, respectively.
Purification of RCA Polypeptides from Leaves and
Recombinant RCA from E. coli
After induction with isopropyl-1-thio-b-D -galactopyranoside, E. coli
containing recombinant RCA were harvested by centrifugation, resuspended in 8 mL of buffer (50 mM NaH2PO4 and 300 mM NaCl, pH 8.0)
containing 2 mM phenylmethylsulfonyl fluoride and disrupted using a
sonicator (Tianmei) at 40 W for 3-s bursts on ice for a total time of 10 min.
The supernatants obtained after centrifugation (12,000g, 30 min, 4°C) were
used for His-tag affinity purification of the recombinant protein. The purification was performed using a Ni2+ column (GenScript) according to the
manufacturer’s protocol.
Antibodies against ZmRCAa or ZmRCAb (15 mg) were immobilized on
2.5 mL of CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s instructions. This resin was used to pour a 1- 3 5-cm column.
Pulverized leaf samples were homogenized with extraction buffer and
centrifuged for 20 min at 20,000g. Proteins in the supernatant were precipitated with ammonium sulfate at 35% (w/v) saturation, and the precipitate
was collected. The resulting pellet was then dissolved in 13 PBS and loaded
onto an immunoaffinity column equilibrated with PBS buffer. After washing
with 5 column volumes of PBS, the bound RCA was eluted with 50 mM GlyHCl (pH 2.5). Fractions containing RCA were pooled, neutralized by dropwise
addition of 250 mM Na2HPO4 (pH 10.3), and stored at –80°C.
Quantification of RCA Polypeptide Expression in
Maize Leaves
The protein expression levels of ZmRCAa and ZmRCAb were determined
on the basis of the total soluble protein content. The total soluble protein
concentration of each leaf protein extract was determined using the Bradford
assay, and bovine serum albumin (BSA) was used as a standard.
The concentration of ZmRCAa or ZmRCAb in each leaf extract sample was
measured using an ELISA quantitative sandwich technique modified from
Leitao et al. (2003). Nunc Immuno Plates were coated with 100 mL of specific
ZmRCAa or ZmRCAb antibodies (1 g mL–1 in 0.1 M Na2CO3/NaHCO3 buffer,
pH 9.5). The plates were incubated for 1 h at 37°C and subsequently washed
once with PBS-Tween (0.1% [v/v] Tween 20 diluted in PBS [103, Interchim]
single strength). To ensure specific fixation of ZmRCAa or ZmRCAb, each
well was blocked with 100 mL of 1% (w/v) BSA in PBS-Tween. The plates
were incubated for 30 min at 37°C and washed three times with PBS-Tween.
Triplicate 100-mL aliquots of each leaf protein extract (each of which had been
diluted or concentrated to contain 10 mg of total soluble protein) were dispensed into the wells. After a 1-h incubation at 37°C followed by five washes
with PBS-Tween, 100 mL of polyclonal cotton anti-RCA antibodies (5 mg mL–1
in PBS-Tween containing 1% [w/v] BSA) was added to each well. The plates
were then incubated for 1 h at 37°C. After five additional washes with PBSTween, immune complexes were detected using goat anti-rabbit IgG-HRP. For
color development, 200 mL of a solution containing 3,39,5,59-tetramethylbenzidine liquid substrate (Sigma-Aldrich) was dispensed into each well.
The plates were then placed in the dark at room temperature for 30 min. The
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Rubisco Activase Genes in Maize
reaction was stopped by the addition of 100 mL HCl (1 mol L–1). Lastly, the
absorbance was read at 405 nm using a Titertek microplate reader.
MALDI-TOF-MS Analysis of RCA Polypeptides
An AB 4800 MALDI-TOF Analyzer (Applied Biosystems, MDS SCIEX)
equipped with a 200-Hz neodymium-doped yttrium aluminum garnet laser
(355 nm) was utilized to determine the molecular masses of RCA polypeptides.
ZipTip C4 was used for the desalting and purification of RCA polypeptide
extracts. One microliter of RCA polypeptide extract was spotted onto the
MALDI target plate and allowed to air dry. Then, 0.6 mL sinapinic acid matrix
was added to the same position on the plate. After the target plate was air
dried, MS spectra (20,000–100,000 mass-to-charge ratio [m/z]) were acquired in
positive linear ion mode with 7,000 laser shots intensity (25 shots per subspectrum for 500 total shots per spectrum). Mass calibration of MALDI-TOF
was achieved using a standard mixture containing aldolase (39,212 m/z) and
BSA (66,430 m/z). The parameters for mass assignments were set as follows:
minimum signal-to-noise ratio = 20, mass tolerance = 200 m/z, minimum peaks
to match = 2, and maximum outlier error = 10 ppm. The 4000 Series Explorer
software (V3.5.2) and the Data Explorer software (V4.9; both from Applied
Biosystems/MDS SCIEX) were used to perform the MS, data acquisition, and
processing.
Grain Yield Measurement
Grain yield was estimated using the average yield of five plants in the
middle of each row. At maturity, the ears of the corresponding plants were
hand harvested, dried to a constant weight, and threshed, and the mean grain
yield per plant was recorded.
Statistical Analysis
Transcript abundance data for ZmRCAa, ZmRCAb, NADP-ME, PPDK, and
PEPC, protein expression data for ZmRCAa and ZmRCAb, and grain yield
data for the inbred lines were analyzed using the SAS system (9.0 for Windows). ANOVA was performed using SAS PROC GLM. The mean values of
each trait for each inbred line were calculated using SAS PROC MEANS. The
Pearson phenotypic correlations among the traits were calculated using SAS
PROC CORR.
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession number JX863889.
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Alternative splicing of ZmRCAb.
Supplemental Figure S2. Alignment of ZmRCAa ORFs from four inbred
lines.
Supplemental Figure S3. MALDI-TOF-MS spectrum of the purified recombinant and leaf RCA proteins.
Supplemental Figure S4. Transcript expression levels of ZmRCAa and
ZmRCAb determined by semiquantitative RT-PCR.
Supplemental Table S1. Primer pairs used in this study.
Supplemental Table S2. List of 123 inbred lines and their mean values for
different traits.
Supplemental Table S3. Descriptive statistics and variance analysis of different traits in 123 lines.
Supplemental Table S4. Marker loci associated with ZmRCAa and ZmRCAb
transcript abundance and grain yield in 123 lines.
ACKNOWLEDGMENTS
We thank Dr. Yuyang Wang of the Testing Center of Yangzhou University
for technical assistance with experiments and anonymous reviewers for
valuable comments and discussions.
Received October 20, 2013; accepted February 5, 2014; published February 7,
2014.
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