Arabidopsis Mutants Carrying Chimeric Sigma Factor Genes
Reveal Regulatory Determinants for Plastid Gene Expression
Jennifer Schweer1, Simon Geimer2, Jörg Meurer2 and Gerhard Link1,∗
1Plant
Cell Physiology, University of Bochum, D-44780 Bochum, Germany
Department, Biocenter, Ludwig-Maximilians-University Munich, D-82152 Planegg-Martinsried, Germany
2Botany
Short Communication
Like bacteria, plastids contain sigma factors for promoter
binding and transcription initiation. Accumulating
evidence suggests that members of the plant sigma factor
family can have specialized non-redundant roles in terms
of promoter preference in various developmental
and environmental situations. To specify regulatory
determinants, we have chosen pairwise exchange of
portions of Arabidopsis sigma coding regions, followed by
transformation of the chimeric constructs into a sigma 6
knockout line. The resulting phenotypes and plastid RNA
patterns point to an important though not exclusive role
for the highly variable N-terminal portion of plant sigma
proteins.
Keywords: Arabidopsis • Chloroplast transcription •
Chimeric sigma factors • Domain swap experiments •
Regulatory determinants.
Abbreviations: CaMV, cauliflower mosaic virus; CR,
‘conserved’ C-terminal region; GABI-Kat, German Plant
Genomics Program ‘Genome Analysis in Biological SystemsKnockout Arabidopsis thaliana’; NEP, nucleus-encoded
polymerase, a single-subunit phage-type DNA-dependent
RNA polymerase; PEP, plastid-encoded polymerase, a
multisubunit bacterial-type RNA polymerase; SIG,
transcription initiation factor sigma; sig, sigma knockout
line; UCR, ‘unconserved’ N-terminal region; WT, wild type.
The key role of chloroplasts in plant cellular metabolism is
reflected by the complexity of the gene expression machinery inside these organelles (Sugita and Sugiura 1996, Shiina
et al. 2005, Toyoshima et al. 2005). Two different RNA polymerases, nucleus-encoded polymerase (NEP) and plastidencoded polymerase (PEP), are involved in chloroplast
transcription (Hedtke et al. 1997, Maliga 1998). NEP promoters are defined by GAA/YRTA motifs, PEP recognizes DNA
elements that conform to the –35/–10 consensus sequences
of bacterial promoters (Liere and Maliga 2001). PEP is composed of a prokaryotic core that interacts with regulatory
sigma factor(s) to form the transcription-competent holoenzyme. Whereas the core subunits are products of chloroplast
genes in most plants, plastid sigma factors are nucleus
encoded (Liu and Troxler 1996, Tanaka et al. 1996).
Arabidopsis thaliana contains six sigma genes, AtSig1–
AtSig6 (Isono et al. 1997, Tanaka et al. 1997, Fujiwara et al.
2000, Hakimi et al. 2000, Privat et al. 2003). The derived proteins SIG1–SIG6 have a conserved C-terminal region (CR)
containing the subregions 1.2–4.2 for basic sigma functions
but lacking subregion 1.1 (Gruber and Gross 2003). Instead,
they each contain a long N-terminal region (unconserved
region; UCR) with low sequence conservation and unknown
function. Mutational function studies have been applied to
the CR (e.g. Hakimi et al. 2000) but not yet to the UCR. Here
we have constructed cDNA clones that represent various
UCR/CR combinations of chimeric sigma factors plus a transit peptide for plastid targeting (Fig. 1). These cDNAs were
then introduced into an Arabidopsis sigma 6 knockout line,
sig6-2 (Loschelder et al. 2006), obtained from the GABI-Kat
(German Plant Genomics Program ‘Genome Analysis in Biological Systems-Knockout Arabidopsis thaliana’) collection
(Rosso et al. 2003). Our results suggest a predominant,
though not exclusive, role for the UCR in determining the
visual and molecular phenotype.
Table 1 presents a pairwise sequence comparison of all
six Arabidopsis factors, showing weaker homology within
the UCR as compared with the CR. The actual values (percentage sequence homology for UCR/CR) vary considerably
between the two extremes defined by the pairs SIG1/SIG4
This paper is dedicated to Professor Achim Trebst on the occasion of his 80th birthday.
∗Corresponding author: E-mail, [email protected]; Fax, +49-234-321-4188.
Plant Cell Physiol. 50(7): 1382–1386 (2009) doi:10.1093/pcp/pcp069, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
All rights reserved. For permissions, please email: [email protected]
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Plant Cell Physiol. 50(7): 1382–1386 (2009) doi:10.1093/pcp/pcp069 © The Author 2009.
B
1
540 697
4.1
4.2
UCR
3.0
TP
2.1
2.2
2.3
2.4
A
1.2
Sigma control of plastid transcription
Table 1 Relatedness of members of the Arabidopsis sigma factor
family
948
Sigma factor pair
sig1/6
1
696 541
969
sig6/1
1
762 697
696 763
CR
FL
SIG1
SIG2
13
33
25
SIG1
SIG3
4
20
12
SIG1
SIG4
2
33
26
SIG1
SIG5
5
27
19
SIG1
SIG6
11
27
21
SIG2
SIG3
9
34
24
SIG2
SIG4
16
25
29
948
sig3/6
1
UCR
954
sig6/3
Fig. 1 Architecture of plant sigma factors and chimeric constructs.
(A) Scheme showing the principal regions: transit peptide (TP) of the
precursor and the UCR (left) and CR with subregions 1.2–4.2 (right) of
the mature protein. (B) Chimeric sigma factors derived from Sig1
(At1g64860, gray), Sig3 (At3g53920, yellow) and Sig6 (At2g36990, blue)
cDNAs. Fused sequences code for the TP and UCR of one factor and for
the CR of another. Numbers correspond to sequence positions within
database entries. Constructs were used to retransform the sig6-2
knockout line.
(2/33) and SIG2/SIG4 (16/25). The pairs that were chosen,
SIG1/SIG6 (11/27) as well as SIG3/SIG6 (12/26) (shown in
bold in Table 1), have intermediate values and thus might
result in chimeric sigma lines with a detectable but not too
strong (lethal) phenotype (Fig. 1B).
As shown in Fig. 2 (top row), sig6-2 seedlings reveal a
strong chlorophyll-deficient phenotype, while sig1 and sig3-2
do not. Among the retransformed chimeric lines (bottom
row), a chlorophyll-deficient phenotype is revealed by sig1/6
and sig3/6 but not by sig6/1 and sig6/3, suggesting that the
UCR might be crucial for the observed phenotype.
We next examined plastid transcript levels using macroarrays with RNA from the parental sig6-2 line as well as
from sig3/6 and sig6/3. A summary view, based on percentage of up- versus down-regulated transcript levels in each
functional gene group, is given in Fig. 3 and a more detailed
picture in Supplementary Fig. S1, showing plastid transcript
levels from single genes. All analyzed genes with their average fold expression changes, SD and P-values are listed in
Supplementary Table S2.
Compared with the wild type (WT), the array data for
sig6-2 (Fig. 3 and Supplementary Fig. S2) indicate enhanced
transcript levels of genes for ribosomal proteins, PEP core
subunits, atpH and ndhG, some PSI and PSII genes (psaC, psbI
and psbJ) and several tRNA genes (trnE, trnF and trnL). The
majority of functionally grouped sets of plastid genes, however, revealed a ‘mixed’ pattern of up- or down-regulation
among their individual members. This overall picture is comparable with that previously reported for a different sigma 6
knockout line, termed sig6-1 (Ishizaki et al. 2005).
The summary data (Fig. 3) for the chimeric sigma line
sig6/3 indicate that, on a global scale, the plastid transcript
pattern shares reasonable similarity with that of sig6-2. At
higher resolution (Supplementary Fig. S1 and Table S2),
SIG2
SIG5
7
30
21
SIG2
SIG6
14
38
28
SIG3
SIG4
5
27
23
SIG3
SIG5
6
20
15
SIG3
SIG6
12
26
21
SIG4
SIG5
11
25
21
SIG4
SIG6
18
32
28
SIG5
SIG6
5
29
22
Protein sequences were compared with ClustalW2. Alignments were carried out
with either the conserved (CR) or unconserved region (UCR) alone, or with the
full-length sigma factor (FL).
however, expression details distinct from those of the parental line become more evident, e.g. for ribosomal protein and
ndh transcripts, rRNAs and, in particular, several tRNAs,
which are present at 5–10 times higher levels in sig6/3. One
of them, tRNAGlu, is a known multifunctional molecule with
roles in both gene expression and chlorophyll formation
(Hanaoka et al. 2005). We note that the enhanced tRNA
levels correlate with the green sig6/3 phenotype as
compared with the white cotyledons of the sig6 knockout
(Fig. 2).
Sig3/6, the line containing the converse chimeric construct, reveals a global negative effect on plastid gene expression compared with the parental knockout (Fig. 3). The
more detailed view (Supplementary Fig. S1) shows that
ndhG and trnE, i.e. the most enhanced RNA species in sig6-2,
are still present at higher levels in sig3/6 compared with the
WT. All other transcripts are strongly down-regulated below
WT levels, regardless of whether they were up- or downregulated in the sig6-2 knockout.
The strong negative effect on plastid gene expression in
sig3/6 cannot be inferred from the transcript patterns in
sig6 mutants (Ishizaki et al. 2005, this work). Likewise, it does
not reflect that of a sigma 3 knockout, which (with the
exception of psbN) was shown to be largely unaffected compared with the WT (Zghidi et al. 2007). Taken together,
sig3/6 has a plastid transcript pattern indicative of a global
negative effect on plastid transcription, probably as a severe
Plant Cell Physiol. 50(7): 1382–1386 (2009) doi:10.1093/pcp/pcp069 © The Author 2009.
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J. Schweer et al.
Fig. 2 Phenotype (10 d) of the wild type, sig6-2, sig1, sig3-2 and chimeric sigma lines: sig6/1 and sig6/3 resemble the wild type (green cotyledons),
while sig1/6 and sig3/6 resemble the parental sig6-2 knockout (white).
Numbers of genes (%)
100
50
0
−50
sig 6-2
sig 6/3
As
tRN
As
rRN
r ps
rpl
pol RNA
ym
era
se
deh NAD
ydr H
oge
nas
e
Cyt
o
c
h
b6/
f co rome
mp
lex
pho
tos
yst
em
I
pho
tos
yst
em
II
ATP
syn
tha
se
−100
sig 3/6
Fig. 3 Overview of plastid transcript levels in sig6-2 and the chimeric sigma lines sig6/3 and sig3/6 (color-coded bars). The percentage of upand down-regulated plastid genes (compared with the WT) within each functional class indicated at the bottom is given (percentage scale on
the left).
consequence of imbalances among the other members of
the sigma family.
To complement the global array data, Northern blot
hybridizations were carried out for two regions of Arabidopsis
chloroplast DNA containing the psbA and atpB genes, respectively (Fig. 4). The psbA probe (top panel) detects the
monocistronic 1.2 kb transcript synthesized under the control
1384
of the single PEP promoter (Liere et al. 1995). Using RNA samples from 5-day-old seedlings, the sig1 and sig3-2 knockouts
contain WT levels of psbA transcript, whereas sig6-2 has
reduced amounts at this time point (Loschelder et al. 2006).
Of the retransformed chimeric lines, sig6/1 and sig6/3 have
WT transcript levels. In contrast, sig1/6 and sig3/6 each show
reduced levels, thus resembling the parental sigma 6 knockout.
Plant Cell Physiol. 50(7): 1382–1386 (2009) doi:10.1093/pcp/pcp069 © The Author 2009.
psbA
6/
3
si
g
3/
6
si
g
6/
1
si
g
1/
6
si
g
2
6si
g
3si
g
1
si
g
W
kb
T
2
Sigma control of plastid transcription
1.2
actin-2
atpB
4.8
aspects, including (i) the identity and location of the critical
determinant(s) within the UCR; (ii) the expression of introduced sigma gene variants as well as the localization and
function of their proteins; and (iii) the control circuits involving the sigma family in a ‘natural’ (non-mutant) situation in
response to environmental cues.
Materials and Methods
2.6
2.0
1.6
Fig 4 Northern blot analysis of plastid transcripts in the wild type,
sigma knockouts and chimeric lines. Total RNA (1 µg) was hybridized
with digoxigenin-labeled RNA probes for the psbA, atpB and actin-2
genes. Left margin: transcript size (kb) determined by RNA markers
(Promega). Bottom: portions of the stained gel at the position of 25S
rRNA as loading control.
The atpB probe (Fig. 4, second panel) detects three different transcripts. Only one of them (2.6 kb) originates from a
PEP promoter, while those at 2.0 and 4.8 kb have been
assigned to NEP transcription (Schweer et al. 2006, SwiateckaHagenbruch et al. 2007). Whereas all lines show the 2.0 kb
NEP transcript, the 2.6 kb PEP-related signal is visible only in
the WT as well as in the sig1 and sig3-2 (but not sig6-2)
knockouts. None of the retransformed chimeric lines show
this latter transcript, confirming previous findings that functionally intact SIG6 is required for efficient transcription
from this promoter (Schweer et al. 2006).
The 4.8 kb (NEP) transcript of sig6 is virtually absent from
the WT as well as from sig1 and sig3-2. It is also absent from
the sig1/6 and sig6/3 chimeric lines but is present in sig6/1
and sig3/6. This large transcript has been implicated in rescue
transcription by the NEP from a far upstream promoter cluster (Schweer et al. 2006). Our current data suggest that NEP
rescue transcription is not activated in any of the chimeric
sigma lines (sig1/6, 6/1, 3/6 or 6/3) regardless of whether SIG6
CR or UCR sequences were part of the fusion construct. In
any case, as for the global plastid transcript patterns (Fig. 3),
no simple correlation seems to exist in these chimeric lines
between atpB gene expression and the visual phenotype
(Fig. 2). This differs from the situation seen for psbA transcript levels (Fig. 4, top panel), which are reduced only in
those lines that reveal a chlorophyll-deficient phenotype
(Fig. 2).
In conclusion, the current data provide initial clues to the
involvement of the UCR in specifying the visual and/or
molecular phenotype. It can be anticipated that more
detailed studies will provide insights into several important
The cDNA for each chimeric sigma factor was generated by
fusion PCR using PfuUltra II Fusion HS DNA Polymerase
(Stratagene, La Jolla, CA, USA). In a first PCR step, the CR and
UCR were separately amplified. In the second step, the chimeric product was generated by using primers for full-length
hybrid sigma factors (including the transit sequence for plastid import) and was then cloned into vector pBSKS(–)
(Stratagene). The insert was sequenced, cut out, and ligated
behind the cauliflower mosaic virus (CaMV) 35S promoter
of pBINAR (Höfgen and Willmitzer 1990). Constructs were
then introduced into Rhizobium radiobacter, followed
by floral dip transformation (Clough and Bent 1998) of the
Arabidopsis sig6-2 mutant. The transformed plants were
selected by kanamycin resistance and tested by PCR and
reverse transcription–PCR (RT–PCR). At least three independent lines were maintained and regularly retested.
Arabidopsis T-DNA insertion lines were obtained from
GABI-Kat (Rosso et al. 2003). To distinguish these lines from
published knockouts from different sources (Ishizaki et al.
2005, Zghidi et al. 2007), the suffix ‘-2’ was added where
appropriate. The insertion site is in exon 8 (of nine) of AtSig1
in the sig1 line (758B02) and in exon 7 (of nine) of AtSig3 in
the sig3-2 line (238A06) (http://www.gabi-kat.de). The site is
in exon 5 (of nine) of AtSig6 in sig6-2 (242G06), which would
place it within the UCR (Loschelder et al. 2006). WT, knockout and chimeric mutant seedlings were grown (8 h short
day at 24°C and 60 mmol m−2 s−1), and cotyledons were harvested and powdered in liquid nitrogen for preparation of
total RNA (Loschelder et al. 2006).
Digoxigenin-labeled RNA probes were synthesized by in
vitro transcription using phage polymerases (Promega, Madison, WI, USA) and cloned DNA templates. Total plant RNA
samples (1 µg) were subjected to gel blot analysis with the
probes according to the Roche DIG manual (Loschelder et al.
2006, Schweer et al. 2006).
PCR-amplified DNA probes representing 94 Arabidopsis
chloroplast genes were spotted onto nylon membranes. A
20 µg aliquot of total RNA was reverse transcribed using
[α-32P]dCTP and SuperScript III reverse transcriptase (Invitrogen, Karlsruhe, Germany). The macroarray filters were
hybridized with the labeled cDNA, washed and then exposed
to phosphoimaging plates, followed by scanning in an FLA3000 phosphoimager (Fuji, Tokyo, Japan). Images were analyzed using TotalLab software (Phoretix International) and
Plant Cell Physiol. 50(7): 1382–1386 (2009) doi:10.1093/pcp/pcp069 © The Author 2009.
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J. Schweer et al.
an online normalization tool (http://nbc11.biologie.uni-kl.
de). For each RNA sample, macroarray experiments were
performed in triplicate and each gene was represented in
four spots at two different concentrations. Based on the
hybridization signals obtained, intensity ratios and P-values
were calculated and data were further evaluated using
scatter plots (Cho et al. 2008).
Supplementary data
Supplementary data mentioned are available at PCP online.
Funding
The Deutsche Forschungsgemeinschaft (SFB 480/B7 to G.L.
and SFB TR1/B2 to J.M.).
Acknowledgments
We gratefully acknowledge the generous supply of the sig1,
sig3-2 and sig6-2 knockout mutant lines by Professor B. Weisshaar, University of Bielefeld, and the GABI-Kat team at the
Max-Planck-Institute für Zuechtungsforschung, Cologne.
We would like to thank Brigitte Link for expert technical
assistance.
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(Received March 6, 2009; Accepted May 10, 2009)
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