A Major Role for the Plastid-Encoded RNA
Polymerase Complex in the Expression of
Plastid Transfer RNAs1[W][OPEN]
Rosalind Williams-Carrier, Reimo Zoschke, Susan Belcher, Jeannette Pfalz, and Alice Barkan*
Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403 (R.W.-C., R.Z., S.B., A.B.);
and Department of Plant Physiology, Institute of General Botany and Plant Physiology,
Friedrich-Schiller-University, D–07743 Jena, Germany (J.P.)
Chloroplast transcription in land plants relies on collaboration between a plastid-encoded RNA polymerase (PEP) of cyanobacterial
ancestry and a nucleus-encoded RNA polymerase of phage ancestry. PEP associates with additional proteins that are unrelated to
bacterial transcription factors, many of which have been shown to be important for PEP activity in Arabidopsis (Arabidopsis thaliana).
However, the biochemical roles of these PEP-associated proteins are not known. We describe phenotypes conditioned by transposon
insertions in genes encoding the maize (Zea mays) orthologs of five such proteins: ZmPTAC2, ZmMurE, ZmPTAC10, ZmPTAC12, and
ZmPRIN2. These mutants have similar ivory/virescent pigmentation and similar reductions in plastid ribosomes and photosynthetic
complexes. RNA gel-blot and microarray hybridizations revealed numerous changes in plastid transcript populations, many of which
resemble those reported for the orthologous mutants in Arabidopsis. However, unanticipated reductions in the abundance of
numerous transfer RNAs (tRNAs) dominated the microarray data and were validated on RNA gel blots. The magnitude of the
deficiencies for several tRNAs was similar to that of the most severely affected messenger RNAs, with the loss of trnL-UAA being
particularly severe. These findings suggest that PEP and its associated proteins are critical for the robust transcription of numerous
plastid tRNAs and that this function is essential for the prodigious translation of plastid-encoded proteins that is required during the
installation of the photosynthetic apparatus.
Transcription in land plant chloroplasts involves the
interplay of two RNA polymerases with distinct evolutionary origins (for review, see Liere et al., 2011; Yagi
and Shiina, 2012): a single-subunit, phage-like nucleusencoded RNA polymerase (NEP) and a multisubunit
plastid-encoded RNA polymerase (PEP) derived from
the cyanobacterial enzyme. The core PEP subunits are
encoded by the plastid rpoA, rpoB, rpoC1, and rpoC2
genes; this core polymerase is targeted to specific promoters by interaction with any of several nucleusencoded sigma factors that are derived from bacterial
sigma-70 (for review, see Lerbs-Mache, 2011). In addition, roughly 10 different nucleus-encoded proteins that
are unrelated to bacterial transcription factors consistently copurify with PEP (Suzuki et al., 2004; Pfalz et al.,
2006; Steiner et al., 2011). Genetic analyses in Arabidopsis (Arabidopsis thaliana) have shown that most of
these PEP-associated proteins are important for the accumulation of transcripts derived from PEP promoters
1
This work was supported by the German Research Foundation
(grant no. ZO 302/1–1 to R.Z.) and by the National Science Foundation (grant no. IOS–0922560 to A.B.).
* 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:
Alice Barkan ([email protected]).
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The online version of this article contains Web-only data.
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www.plantphysiol.org/cgi/doi/10.1104/pp.113.228726
and for chloroplast biogenesis (for review, see Yagi and
Shiina, 2012; Pfalz and Pfannschmidt, 2013). That the
transcription of a small genome encoding fewer than 100
products requires such a diversity of novel components
presents numerous evolutionary and mechanistic questions.
Pioneering studies that explored the contributions of
PEP and NEP with a tobacco (Nicotiana tabacum) plastome
mutant lacking RpoB concluded that plastid photosystem
genes are transcribed primarily by PEP, whereas most
other genes can be transcribed by either NEP or PEP
(Allison et al., 1996; Hajdukiewicz et al., 1997). This question was subsequently addressed more comprehensively
by analysis of the plastid transcriptome in a tobacco rpoA
knockout (Legen et al., 2002), by comparison of transcription start sites in normal and ribosome-deficient barley
(Hordeum vulgare) plastids (Zhelyazkova et al., 2012), and
by profiling plastid transcriptomes in the presence of an
inhibitor of PEP (Demarsy et al., 2006, 2012). These studies
demonstrated that most chloroplast genes can be transcribed by either NEP or PEP. That being said, PEP and
NEP preferentially influence mRNA levels from photosystem and genetic system genes, respectively, in a manner
that resembles the original view (for review, see Yagi and
Shiina, 2012).
The recovery of PEP in a large complex whose mass
is dominated by non-PEP proteins (Steiner et al., 2011)
is intriguing, given that bacterial RNA polymerases
form stable complexes with only a few proteins whose
mass is negligible in comparison with the polymerase
itself (for review, see Haugen et al., 2008). Furthermore, the
PEP-associated proteins are not required for PEP-mediated
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Williams-Carrier et al.
transcription in vitro (Hu and Bogorad, 1990), although
their absence disrupts PEP-mediated transcription in
vivo (Pfalz et al., 2006; Garcia et al., 2008; Myouga
et al., 2008; Arsova et al., 2010; Gao et al., 2011, 2012;
Steiner et al., 2011; Jeon et al., 2012; Yagi et al., 2012). A
recent model posits that the PEP-associated proteins
are required to establish a subdomain in the plastid
nucleoid that is required for PEP-mediated transcription (Pfalz and Pfannschmidt, 2013).
In this report, we describe a set of maize (Zea mays)
mutants lacking several PEP-associated proteins whose
orthologs have been genetically characterized in Arabidopsis (PTAC2, PTAC12, MurE, and PRIN2; Pfalz et al.,
2006; Garcia et al., 2008; Kindgren et al., 2012) or tobacco
(PTAC10; Jeon et al., 2012). The phenotypes of these
mutants in maize, both visible and molecular, are similar
to one another and are consistent with those reported
for the orthologous mutants. However, comprehensive
analysis of the plastid transcriptomes in the maize
mutants revealed defects that had not been reported
previously. Most notably, we discovered a deficiency
for numerous plastid tRNAs, several of which were as
severely affected as the “classic” PEP-dependent mRNAs.
Subsequent analysis of the orthologous Arabidopsis
mutants revealed similar effects. These findings strongly
suggest that PEP-associated proteins (and presumably
PEP itself) play an important role in the transcription of
many plastid tRNAs and that this activity is relevant to
the reduction in plastid ribosomes and plastid-encoded
proteins in mutants lacking PEP or its associated proteins. These results highlight the complex division of labor between NEP and PEP and have implications with
regard to the regulatory cascade underlying the initiation
of chloroplast development.
RESULTS
The mutants described here were recovered during
our systematic effort to identify causal mutations
in the Photosynthetic Mutant Library (PML), a large
collection of Mutator (Mu) transposon-induced nonphotosynthetic maize mutants (http://pml.uoregon.
edu/photosyntheticml.html; Stern et al., 2004).
Molecular phenotyping of mutants in this collection
revealed several nonallelic mutants with similar defects in plastid transcript populations that were distinct from the pleiotropic effects commonly observed
in nonphotosynthetic mutants (examples are shown
below). The causal mutations were identified in a twostep process in which (1) cosegregating Mu insertions
were identified by deep sequencing of Mu insertion
sites (Williams-Carrier et al., 2010) and (2) independent
alleles recovered in reverse genetic screens of the PML
collection were used for complementation testing with
the reference alleles. Four of the genes discovered in this
manner proved to encode the maize orthologs of the
PEP-associated proteins PTAC2, PTAC10, PTAC12, and
MurE (Pfalz et al., 2006). The fifth gene encodes the
maize ortholog of Arabidopsis PRIN2 (Kindgren et al.,
2012), which has not been detected as a PEP-associated
protein but which localizes to the plastid nucleoid and
influences plastid transcript profiles in a manner that
is similar to PEP-associated proteins (Kindgren et al.,
2012). The maize PRIN2 ortholog, ZmPRIN2, likewise
localizes to plastid nucleoids (Majeran et al., 2012), and
we show here that the plastid transcriptome in Zmprin2
mutants is similar to that of mutants lacking PEPassociated proteins. For convenience, PRIN2 is referred
to below as a PEP-associated protein, although the
proteomics data suggest that it is not tightly associated
with the PEP complex. The recovery of Zmptac12 mutants was reported previously (Williams-Carrier et al.,
2010) but with little phenotypic data. To our knowledge, the other mutants are reported here for the first
time. In each case, we recovered both strong (likely null)
alleles harboring exon insertions and hypomorphic alleles with insertions in 59 untranslated regions (Fig. 1);
these condition ivory and yellow-green phenotypes,
respectively (Supplemental Fig. S1). The heteroallelic
progeny of allelism crosses exhibit intermediate phenotypes (ivory/yellow leaf blades with greening tips;
Fig. 1A), demonstrating that these mutations fail to
complement and confirming that the insertions in these
genes underlie the chloroplast biogenesis defects.
Because albino phenotypes are associated with a suite
of pleiotropic effects that can mask mutant-specific defects (Williams and Barkan, 2003), we used the hypomorphic progeny of complementation crosses (Fig. 1)
for all molecular analyses described below. Heteroallelic
mutants derived from strong and weak alleles of Zmwhy1
(Prikryl et al., 2008) were analyzed in parallel for comparative purposes; ZmWHY1 is an abundant plastid
nucleoid protein (Majeran et al., 2012) that is not tightly
associated with PEP and that is required for the biogenesis of the plastid translation machinery (Prikryl et al.,
2008; Maréchal et al., 2009; Melonek et al., 2010; Steiner
et al., 2011). The severities of the chlorophyll, photosynthetic protein, and plastid ribosome deficiencies are similar in the Zmwhy1, Zmptac2, Zmptac10, Zmptac12,
ZmmurE, and Zmprin2 heteroallelic mutants used for
the experiments described here (Fig. 1A; see below).
Therefore, molecular defects observed in mutants lacking
PEP-associated proteins but not in the Zmwhy1 mutants
are not simply secondary effects of defects in photosynthesis, chlorophyll deficiency, or plastid gene expression.
Zmptac2, Zmptac10, Zmptac12, ZmmurE, and Zmprin2
Mutants Have Reduced Levels of Plastid Ribosomes and
Share Characteristic Defects in Plastid mRNA Metabolism
As an initial assessment of plastid gene expression in
these mutants, the abundance of photosynthetic complexes that harbor plastid-encoded subunits was examined by immunoblot analysis of one core subunit of each
complex (Fig. 2A). All of the mutants exhibit a more
than 10-fold loss of each marker protein. This type of
“global” protein deficiency is typical of mutants with
defects in the biogenesis of the plastid translation machinery (Barkan, 1993). In fact, staining of gel-resolved
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Plastid-Encoded Polymerase and tRNA Expression
increased levels in Zmptac2, Zmptac10, Zmptac12, ZmmurE,
and Zmprin2 mutants (Fig. 2A), consistent with the
increased levels of rpoB mRNA detected on microarrays
(Supplemental Data Set S1). Two different antibodies
were used to detect RpoB, and both indicate that RpoB
accumulates to lower levels in Zmptac12 mutants than
in the other mutants examined. This hints at a distinct
function for PTAC12, but our use of hypomorphic
rather than null alleles in this experiment precludes firm
conclusions.
RNA gel-blot assays detected various plastid transcript
defects that are shared by Zmptac2, Zmptac10, Zmptac12,
ZmmurE, and Zmprin2 mutants (Fig. 3). For example, the
rbcL, psaB, and psbA RNAs accumulate to reduced levels
in these mutants but are less affected in the Zmwhy1
control, consistent with the known role of PEP in mediating rbcL, psaB, and psbA transcription in other species
(for review, see Lerbs-Mache, 2011; Liere et al., 2011).
However, whereas atpB RNA levels are not strongly reduced in mutants lacking PEP or its associated proteins
in Arabidopsis and tobacco (Hajdukiewicz et al., 1997;
Garcia et al., 2008) or in the Zmwhy1 mutant control
(Fig. 3), atpB RNA is severely reduced in the maize mutants lacking PEP-associated proteins (Fig. 3). In addition, the blots revealed a loss of specific transcript
isoforms from the polycistronic transcription units
encoding psaC, psaJ, and petG. A similar effect at psaJ
was reported for an Arabidopsis sigma factor sig2 mutant
(Nagashima et al., 2004) and was suggested to result from
a SIG2-dependent promoter. However, the Zmwhy1
mutant exhibited a similar change in the psaJ transcript
pattern, suggesting that the loss of monocistronic psaJ
RNA in the maize mutants may be an indirect effect of
compromised plastid biogenesis or translation. The
isoform-specific defects for psaC and petG are consistent
with the discovery of operon-internal PEP promoters
for these genes in barley (Zhelyazkova et al., 2012).
Genome-Wide Microarray Analysis Reveals a Major
Role for PEP-Associated Proteins in the Expression
of Plastid tRNAs
Figure 1. Mutants used in this study. A, Plants were grown for 7 d in soil.
The mutants shown are the heteroallelic progeny of complementation
crosses involving the two alleles diagrammed in B. The Zmwhy1 mutant
is heteroallelic for an exon and a 59 untranslated region insertion
(Zmwhy1-1 and Zmwhy1-2) described previously (Prikryl et al., 2008).
WT, Wild type. B, Positions of Mu insertions. Protein-coding regions
are indicated by black rectangles, and transcribed but untranslated
regions are indicated by white rectangles. The target site duplications
flanking each Mu insertion are underlined.
total leaf RNA revealed a reduction in plastid ribosomal
RNAs (rRNAs) in these mutants (Fig. 2B, bands marked
16S and 23S*), which implies a corresponding deficiency
for plastid ribosomes. Despite this ribosome defect,
the RpoB subunit of the PEP complex accumulates to
To obtain a genome-wide perspective on the effects
of PEP-associated proteins on the maize plastid transcriptome, total leaf RNA from the Zmptac2 and ZmmurE
mutants was compared with that in their phenotypically
normal siblings by hybridization to tiling microarrays
that include strand-specific probes for every annotated
maize chloroplast gene. Each protein-coding gene was
represented across its length by overlapping 50-mers,
each rRNA was represented by several 50-mers, and each
tRNA was represented by a single 50-mer (Supplemental
Data Set S1). The results are presented as the ratio of
signal in the wild type versus the mutant (Fig. 4B) or as
separate plots of the signal in the wild-type and mutant
samples (Fig. 4C). The transcriptome profiles for the
Zmptac2 and ZmmurE mutants were quite similar and
included a set of dominating “peaks” representing severe
deficiencies for a subset of RNAs. However, although
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Williams-Carrier et al.
Figure 2. Chloroplast protein and ribosome deficiencies caused by mutations in maize genes encoding PEP-associated proteins. A, Immunoblots
of total leaf extract (5 mg of protein and the indicated dilutions) were
probed with antibodies against one core subunit of each photosynthetic
enzyme complex: AtpB (ATP synthase), D1 (PSII), PsaD (PSI), and PetD
(cytochrome b6f). Duplicate blots were probed with antibodies to RpoB, a
plastid-encoded subunit of PEP: the blot labeled RpoB1 was probed with an
antibody raised to Arabidopsis RpoB, and the blot labeled RpoB2 was
probed with an antibody raised to rice RpoB. The Ponceau S-stained blot at
bottom served as a loading control and demonstrates the abundance of
RbcL, the large subunit of Rubisco. B, Seedling leaf RNA (3 mg) was resolved on a denaturing agarose gel, transferred to a nylon membrane, and
stained with methylene blue. 28S and 18S rRNAs are cytosolic rRNAs; 16S
and 23S* rRNAs are plastid rRNAs; 23S* is a fragment of 23S rRNA that is
found in native chloroplast ribosomes. This image is the same as that shown
for the petG-probed blot in Figure 3. WT, Wild type.
PEP is generally described as being particularly important for the transcription of genes encoding photosystem
proteins (Hajdukiewicz et al., 1997; Pfalz et al., 2006; Gao
et al., 2011; Kindgren et al., 2012; Yagi and Shiina, 2012),
the psbA mRNA was the only such RNA among the
dominating peaks. Other known PEP-dependent mRNAs
(e.g. psaA/psaB and rbcL) were revealed in the microarray data, but the magnitude of the difference between
wild-type and mutant samples appeared relatively
small. It is important to note, however, that these plots
underrepresent the magnitude of plastid mRNA deficiencies in the mutants due to the normalization
method that was employed: values are represented as
a fraction of total chloroplast RNA rather than total
leaf RNA because the array contained only chloroplast
probes, and the highly abundant plastid rRNAs and
tRNAs are substantially reduced in the mutants. In any
case, the major differences between the wild-type and
mutant profiles were largely restricted to tRNAs and
rRNAs. PEP is known to contribute to rRNA transcription
(Sriraman et al., 1998; Suzuki et al., 2003) and to the
transcription of several tRNAs (Kanamaru et al., 2001;
Legen et al., 2002; Ishizaki et al., 2005). However, the most
severe tRNA defects suggested by our microarray data
(e.g. trnL-UAA, trnF-GAA, trnL-CAA, and trnL-UAG)
as well as the widespread effects on tRNA abundance
have not been highlighted previously. Microarray
analysis of plastid transcriptomes in apical versus basal leaf tissue (representing mature and immature
chloroplasts, respectively) yielded profiles that were very
similar to those of the wild type versus Zmptac2 or
ZmmurE mutants (Supplemental Fig. S3). These results
support the prevailing view that NEP-mediated transcription dominates in immature chloroplasts whereas
PEP-mediated transcription dominates in mature chloroplasts and provide additional evidence that PEP
stimulates the expression of many plastid tRNAs.
Quantification of abundant RNAs such as tRNAs and
rRNAs by microarray hybridization can be problematic.
Therefore, 12 plastid tRNAs were further assayed by
RNA gel-blot hybridization (Fig. 5). The RNA gel-blot
data showed that trnL-UAA is almost absent in the
ZmmurE, Zmptac2, and ptac10 mutants and that it is
severely reduced in Zmptac12 and Zmprin2 mutants. By
contrast, trnL-UAA accumulates to much higher levels
in the Zmwhy1 mutant, indicating that its loss in mutants lacking PEP-associated proteins does not result
solely from a deficiency for chlorophyll, plastid ribosomes, or photosynthesis. The trnL-UAA gene includes
a group I intron; both the unspliced precursor and
spliced product are reduced, as would be expected for a
defect in transcription.
Quantification of the RNA gel-blot data (Fig. 5B;
Supplemental Table S1) revealed that trnK-UUU, trnQUUG, trnS-UGA, trnL-UAA, trnF-GAA, and trnL-UAG
were decreased at least 2-fold more in all of the mutants
lacking PEP-associated proteins than in the Zmwhy1
mutant control. By contrast, the loss of trnS-GGA and
trnL-CAA may be secondary effects, as these were reduced to a similar extent in Zmwhy1 mutants as in
mutants lacking PEP-associated proteins. Strong defects
(in comparison with Zmwhy1) were also observed for
trnfM, trnE-UUC, trnW-CCA, and trnA-UGC but only in
a subset of the mutants lacking PEP-associated proteins.
The differential effect of PEP-associated proteins on trnE
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Plastid-Encoded Polymerase and tRNA Expression
Figure 3. Examples of chloroplast mRNA defects caused by mutations in genes encoding PEP-associated proteins. Seedling leaf
RNA (3 mg) was analyzed by RNA gel-blot hybridization using probes for the indicated genes. Size markers (kb) are shown to
the left. The psaB probe detects a polycistronic mRNA that also includes psaA. Quantification of the results for psbA, psaB, rbcL,
and atpB/atpE are shown in Supplemental Table S1. WT, Wild type.
abundance is intriguing in light of the role for trnE in
tetrapyrrole biosynthesis, which impacts retrograde signaling pathways that connect nuclear gene expression
with chloroplast physiology (Woodson et al., 2012).
To determine whether these functions for PEPassociated proteins are conserved in Arabidopsis, several tRNAs were assayed in Arabidopsis mutants
lacking PEP-associated proteins or the PEP sigma factor
SIG6 (Fig. 5C; Supplemental Fig. S2B). As in maize, both
spliced and unspliced trnL-UAA are severely reduced
in the Arabidopsis ptac2, ptac10, and ptac12 mutants.
Effects on the abundance of trnS-UGA, trnF-GAA, and
trnE-UUC were also similar between the two species.
SIG6 was shown previously to be important for transcription of the trnYED operon in Arabidopsis (Ishizaki
et al., 2005). Our results confirm that observation and
show further that SIG6 is important for the expression of
trnF-GAA (Supplemental Fig. S2B).
DISCUSSION
Genetic analyses have shown that many of the proteins
that copurify with PEP are important for chloroplast
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Williams-Carrier et al.
Figure 4. Analysis of plastid transcriptomes in Zmptac2 and ZmmurE mutants by hybridization to a high-density microarray.
Total leaf RNA was fragmented, labeled with Cy5 (the wild type [WT]) or Cy3 (mutant), and competitively hybridized to a highdensity, strand-specific synthetic oligonucleotide microarray covering all annotated plastid genes. For comparison, several RNA
gel-blot hybridizations using these same RNA samples are shown in Supplemental Figure S2A. A, Map of the maize chloroplast
genome created with OGDRAW (Lohse et al., 2013). Asterisks mark genes that correspond to peaks in the microarray data,
which reflect RNAs that are more abundant in the wild type than in the mutant. B, Ratio of signal in the wild type relative to the
mutant samples. The average median of ratios (F635:F532) in each analysis was normalized to 1, such that values reflect relative
signal (wild type to mutant) for each array element as a fraction of the total chloroplast RNA in each sample. C, Normalized
fluorescence intensities from each individual genotype (red, the wild type; green, mutant). The signal intensities for each
channel were normalized to one another based on the average signal for both channels (F635 and F532) in the two assays. The
underlying data are the same as those used in B.
development and for the accumulation of PEP-dependent
mRNAs (Garcia et al., 2008; Myouga et al., 2008; Arsova
et al., 2010; Gao et al., 2011, 2012; Steiner et al., 2011; Jeon
et al., 2012; Kindgren et al., 2012; Yagi and Shiina, 2012;
Pfalz and Pfannschmidt, 2013). It is intriguing that these
proteins promote the activity of an enzyme that is closely
related to its cyanobacterial ancestor, yet they are neither
derived from bacterial transcription factors nor do they
have apparent functional homologs in bacteria. Many
fundamental questions about the PEP-associated proteins
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Plastid-Encoded Polymerase and tRNA Expression
Figure 5. RNA gel-blot hybridizations validating plastid tRNA deficiencies in mutants lacking PEP-associated proteins. A, Total
leaf RNA (3 mg) was analyzed by RNA gel-blot hybridization using probes specific for the indicated tRNAs. Blots were stained
with methylene blue to visualize rRNAs; a representative stained blot is shown to illustrate equal loading. B, Quantified signals
from the blots shown in A are graphed as a fraction of signal in the wild-type sample (WT). C, RNA gel-blot analysis of the
indicated plastid tRNAs in Arabidopsis ptac2, ptac10, and ptac12 mutants. The stained gels used for blotting are shown at
bottom. Additional analyses of plastid tRNAs in these and other Arabidopsis mutants are shown in Supplemental Figure S2.
remain, including the biochemical contributions of each
protein to PEP-mediated transcription, whether they have
additional functions beyond transcription, and how they
contribute to the regulation of plastid gene expression by
environmental, physiological, and developmental cues.
The results presented here highlight a function for
PEP-associated proteins that has been hinted at in
prior literature (see below) but that has not been fully
appreciated: PEP-associated proteins are essential for
the robust expression of numerous plastid tRNAs. The
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Williams-Carrier et al.
expression of trnL-UAA is particularly sensitive to the
loss of PEP-associated proteins: very little expression remains even in the hypomorphic alleles analyzed here.
trnL-UAA and several other strongly affected tRNAs
(e.g. trnF-GAA and trnL-UAG) are essential for plastid
translation (Alkatib et al., 2012). These tRNA deficiencies,
therefore, are expected to contribute to the reduction in
plastid ribosomes in mutants lacking PEP-associated
proteins: limiting tRNAs will reduce the synthesis of
plastid-encoded ribosomal proteins, which, in turn, are
needed to assemble a stable ribosomal structure. Thus,
the reduced content of rRNA (and ribosomes) in mutants
lacking PEP-associated proteins is likely due to a combination of reduced transcription from the PEP promoter
upstream of the rRNA genes and increased rRNA degradation due to limiting ribosomal proteins. The relative
contributions of these two effects cannot currently be
assessed.
To our knowledge, prior studies of mutants lacking
PEP-associated proteins have not assayed the expression
of plastid tRNAs. “Genome-wide” array or quantitative
reverse transcription-PCR assays were used in several
cases (Nagashima et al., 2004; Kindgren et al., 2012; Yagi
et al., 2012), but tRNAs were excluded. That being said,
our results were foreshadowed by genetic analyses of
PEP itself. A mutation in the Arabidopsis SIG2 gene
(encoding a PEP sigma factor) was shown to reduce
the expression of trnE-UUC, trnD-GUC, trnV-UAC, and
trnM-CAU but to have no effect on trnG-GCC or trnWCCA (Kanamaru et al., 2001). Mutation of the Arabidopsis SIG6 gene reduced the expression of trnQ-UUG
and the trnYED transcription unit but did not affect
trnV-UAC (Ishizaki et al., 2005). In addition, the PEPassociated protein PTAC3 was shown to associate in
vivo with the trnE/Y/D promoter (Yagi et al., 2012).
However, the tRNAs we found to be most severely affected in mutants deficient for PEP-associated proteins
(trnL-UAA, trnF-GAA, trnL-CAA, and trnL-UAG) appear not to have been assayed in these studies. Importantly, a broad role for PEP in the expression of plastid
tRNAs was detected in a thorough macroarray survey of
plastid transcription and RNA levels in a tobacco plastome rpoA knockout (Legen et al., 2002). Although reduced expression of many tRNAs was observed, the
magnitude of these effects appeared small in comparison
with the effects on PEP-dependent mRNAs. Perhaps it is
for this reason that these widespread effects on tRNAs
have not been incorporated into the generally accepted
view of the repertoire of PEP functions.
A deep analysis of transcription start sites in barley
plastids detected PEP but not NEP promoters for trnLUAA, trnS-UGA, trnQ-UUG, trnM-CAU, trnN-GUU, and
trnT (Zhelyazkova et al., 2012), but the assay employed
did not address the possibility that these tRNAs can
be transcribed from distal NEP promoters. However, a
very recent study demonstrated a physical association
between the RpoA subunit of PEP and numerous tRNA
genes in tobacco chloroplasts (Finster et al., 2013). Those
results in conjunction with our data and prior evidence
that SIG2 and SIG6 enhance the expression of several
plastid tRNAs (Kanamaru et al., 2001; Ishizaki et al.,
2005) provide strong evidence that many plastid tRNAs
are highly dependent on PEP and its associated proteins
for their transcription. Notably, trnL-UAA goes almost
unexpressed in the absence of PEP-associated proteins.
The PEP promoter inferred for trnL-UAA in barley
(Zhelyazkova et al., 2012) is conserved in dicots
(Supplemental Fig. S4), in accord with our finding that
trnL-UAA expression is severely reduced in Arabidopsis
mutants lacking PEP-associated proteins (Fig. 5C).
These results have implications for the regulatory cascade that activates the plastid gene expression machinery
early in chloroplast development. The transcription of the
rpoB, rpoC1, and rpoC2 genes, which encode PEP core
subunits, is mediated primarily by NEP (Demarsy et al.,
2012, and refs. therein). However, PEP synthesis also
requires a functioning translation apparatus, including
trnL-UAA and the other tRNAs whose transcription
relies primarily on PEP itself. This predicts that a basal
level of PEP must be maintained in all cells that will
give rise to photosynthetic tissues. Consistent with this
view, it has been shown that PEP is present in the
seeds of several dicot species (Demarsy et al., 2006,
2012). In addition, these findings predict that a small
increase in PEP will initiate a positive feedback loop by
increasing the concentration of limiting tRNAs and
thereby accelerating the synthesis of PEP by plastid ribosomes. This may be of physiological importance, as
the effect is predicted to be a responsive genetic switch
to rapidly trigger chloroplast development upon a small
burst in NEP-mediated transcription of the rpo genes.
On a technical note, apparent levels of RNAs analyzed
by both microarray and RNA gel-blot hybridization
generally mirrored one another, but there were some
major exceptions (Supplemental Table S1). For example, the microarray data suggested that trnA-UGC and
the psaB mRNA accumulate to near-normal levels in
the ZmmurE and Zmptac2 mutants, but the RNA gelblot data showed them to be strongly reduced. Differences of this nature could arise from artifacts due to
signal saturation on the microarrays or to the fact that
the microarray integrates transcripts of all sizes whereas
the RNA gel blots do not. Although microarray data on
their own should be interpreted with caution, it is striking
that the microarray data for the wild-type samples span a
much larger dynamic range than do the microarray data
for the mutant samples (Fig. 4C). These results support
the view that NEP promoters have rather uniform, low
activity and that PEP activity at specific promoters is required to boost the transcription of a subset of genes to
the exceptional levels required for the biogenesis and
maintenance of the photosynthetic apparatus.
The direct biochemical functions of PEP-associated
proteins remain unknown. Elucidation of their roles
would be fostered by the ability to study biochemical
defects in mutants that lack them. However, these
mutants are nonphotosynthetic, and it is challenging
to generate substantial quantities of nonphotosynthetic
mutant leaf tissue in Arabidopsis. By contrast, growth
of nonphotosynthetic mutants in large seeded plants
246
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Plastid-Encoded Polymerase and tRNA Expression
like maize is supported for several weeks by seed reserves; during this period, such mutants grow rapidly
to substantial size (Fig. 1A), simplifying the recovery
of nonphotosynthetic tissue for biochemical analysis.
The gradient of chloroplast development along the
length of the maize seedling leaf (Leech et al., 1973) is
useful for developmental studies, and the fact that
maize leaves develop in the absence of light can be used
to discriminate the effects of light from the effects of
developmental cues. Therefore, the functional analysis
of PEP-associated proteins will be facilitated by their
parallel study in maize and Arabidopsis, exploiting the
advantages offered by each system.
MATERIALS AND METHODS
(Mycroarray) that included all annotated genes in the maize chloroplast, using
the methods described previously (Zoschke et al., 2013). Each microarray data
set came from one competitive hybridization (i.e. one mutant and one wild-type
sample combined to probe the same array), but all conclusions are based on
results that were validated by RNA gel-blot hybridization. The elements on the
array and a summary of the data are provided in Supplemental Data Set S1.
Corrections to tRNA Annotations
During the course of this work, we discovered errors and ambiguities in the
annotations of several tRNAs in the reference plastid genome for maize (Maier
et al., 1995). Therefore, the identity of each tRNA was checked with tRNAscan
(http://lowelab.ucsc.edu/tRNAscan-SE/). For clarity, the residue number of
the first nucleotide of each tRNA in the reference maize plastid genome (Maier
et al., 1995) is included in figures and tables, where relevant. The corrections to
annotations are indicated in the footnotes to Supplemental Table S1.
Supplemental Data
The following materials are available in the online version of this article.
Plant Material
The recovery of maize (Zea mays) Zmwhy1 and Zmptac12 mutants was described previously (Prikryl et al., 2008; Williams-Carrier et al., 2010). The other
mutants employed here were initially recognized as recessive mutations that
condition chlorotic, seedling-lethal phenotypes and distinctive plastid RNA profiles during the systematic phenotyping of mutants in the PML mutant collection
(http://pml.uoregon.edu/photosyntheticml.html). Mu insertions that cosegregate
with these phenotypes were identified with an Illumina-based method
(Williams-Carrier et al., 2010). Candidate genes were then validated by the
recovery of second alleles in reverse-genetic screens of the PML collection,
followed by complementation tests involving crosses among heterozygous
plants harboring each allele. The progeny of the allelism crosses segregated
mutant progeny (of intermediate phenotype when the parental alleles differed
in strength), demonstrating a lack of complementation and validating the gene
identifications. The maize genes were named according to nomenclature
established for their Arabidopsis (Arabidopsis thaliana) orthologs (evidence for
orthology is summarized at the POGs2 Database; http://cas-pogs.uoregon.
edu/#/): ZmmurE, maize locus GRMZM2G009070, orthologous to At1g63680;
Zmptac2, maize locus GRMZM2G122116, orthologous to At1g74850; Zmptac10, maize
locus GRMZM2G091419, orthologous to At3g48500; Zmptac12, maize locus
GRMZM5G897926, orthologous to At2g34640; and Zmprin2, maize locus
GRMZM2G119906, orthologous to At1g10522. Maize tissue for RNA and
protein analysis was harvested from plants grown for 1 week under the following
conditions: 16 h of light (400 mE m22 s21) at 28°C, 8 h of dark at 26°C. Phenotypically
normal siblings from the same plantings were used as the wild-type samples.
Transfer DNA insertion lines for the Arabidopsis mutants were obtained from
the Nottingham Arabidopsis Stock Centre (http://arabidopsis.info): Salk_075736,
ptac2; Salk_025099, ptac12; CS16115, ptac10; GK-443A08, fln1; GK 242G06, sig6.
Arabidopsis was grown on Murashige and Skoog medium with 1.37% Suc at
21°C under continuous light (20–30 mE m22 s21; Osram LW30) after seeds
were stratified at 4°C. Total RNA was extracted from the cotyledons of 6-d-old
plants (sig6 mutant and wild-type control) or from leaves of 14-d-old plants
(the other mutants) as indicated in the figures.
Supplemental Figure S1. Phenotypes conditioned by the individual mutant alleles used to generate the heteroallelic plants shown in Figure 1.
Supplemental Figure S2. Additional RNA gel-blot analyses of plastid RNAs
in maize and Arabidopsis mutants lacking PEP-associated proteins.
Supplemental Figure S3. Comparative analysis of the plastid transcriptome in apical (mature) versus basal (immature) maize leaf tissue.
Supplemental Figure S4. Multiple sequence alignment showing the similarity between the barley PEP promoter for trnL-UAA (Zhelyazkova
et al., 2012) and orthologous sequences in other species.
Supplemental Table S1. Quantification of northern and microarray data
for those RNAs assayed by both methods.
Supplemental Table S2. Probes used for RNA gel-blot hybridization.
Supplemental Data Set S1. Plastid tiling microarray design and transcriptome data.
ACKNOWLEDGMENTS
Antibody to rice RpoB was generously provided by Congming Lu (Chinese
Academy of Sciences). We are grateful to Bobby Coalter and Dylan Udy (formerly
of the University of Oregon; currently at the University of California [Davis and
San Francisco, respectively]) for contributing to surveys of RNA defects in PTAC
mutants by RNA gel-blot hybridization, to Tiffany Kroeger (University of
Oregon) and Nick Stiffler (University of Oregon) for expert technical assistance,
and to David Stern and his former laboratory members (Boyce Thompson
Institute) for the survey RNA gel blots that originally brought to our attention
several of the mutants described here.
Received September 16, 2013; accepted November 16, 2013; published
November 18, 2013.
RNA and Protein Analyses
RNA was extracted from the second leaf of 1-week-old seedlings. For the
developmental analysis in Supplemental Figure S3, RNA was extracted from the
base (1.5–3.5 cm above the basal nodule; referred to as “young”) or the tip (apical
3 cm; referred to as “mature”) of the second seedling leaf. RNA gel-blot hybridizations were performed as described previously (Barkan, 1998) using the
probes listed in Supplemental Table S2. RNA gel-blot signals were quantified
with a Storm phosphorimager and QuantityOne software. Immunoblot analysis
employed extracts of the apical half of the second seedling leaf, using the methods
and antibodies described by Prikryl et al. (2008). Two RpoB antibodies were
employed: an antibody raised against Arabidopsis RpoB was purchased from
Uniplastomic (catalog no. AB018b; www.uniplastomic.com); an antibody raised
against rice (Oryza sativa) RpoB was a generous gift of Congming Lu. Microarray
transcriptome assays used 3 mg of RNA per sample. RNA was fragmented, directly labeled with Cy5 (wild-type or apical tissue) or Cy3 (mutant or basal tissue)
dye, combined, and hybridized to custom oligonucleotide tiling microarrays
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