Light-Dependent and Light-Independent Protochlorophyllide
Oxidoreductases in the Chromatically Adapting
Cyanobacterium Fremyella diplosiphon UTEX 481
1Biology
Department, 701 Moore Avenue, Bucknell University, Lewisburg, PA 17837, USA
of Biology, 1001 East 3rd Street, Indiana University, Bloomington, IN 47405, USA
2Department
The cyanobacterium Fremyella diplosiphon can alternate
its light-harvesting pigments, a process called complementary chromatic adaptation (CCA), allowing it to
photosynthesize in green light (GL) and in fluctuating
light conditions. Nevertheless, F. diplosiphon requires
chlorophylls for photosynthesis under all light conditions.
Two alternative enzymes catalyze the penultimate step of
chlorophyll synthesis, light-dependent protochlorophyllide
oxidoreductase (LPOR) and dark-operative protochlorophyllide oxidoreductase (DPOR). DPOR enzymatic
activity is light independent, while LPOR requires light.
Therefore, we hypothesize that F. diplosiphon up-regulates
DPOR gene expression in GL, so that DPOR is more
abundant when LPOR is less functional. We cloned the
genes encoding the three subunits of DPOR, chlL, chlN and
chlB, and the LPOR gene, por, to determine the abundance
of the transcripts under red light (RL), GL and dark
conditions. We found that F. diplosiphon chlL and chlN
genes are transcribed as parts of a single operon, a gene
structure that is conserved within cyanobacteria. Transcripts levels of all DPOR genes are up-regulated
approximately 2-fold in GL relative to levels in RL, whereas
LPOR transcript levels are reduced in GL. Moreover,
mutations in CCA regulators, RcaE and CpeR, modify
DPOR and LPOR transcript levels under specific light
conditions. Finally, both DPOR and LPOR transcripts are
down-regulated 2- to 5-fold in the dark. These results
provide the first evidence that light quality and CCA affect
the genetic regulation of chlorophyll biosynthesis in
freshwater cyanobacteria, ecologically important
photosynthetic organisms.
∗Corresponding
Keywords: Chlorophyll synthesis • Chromatic adaptation •
Fremyella diplosiphon.
Regular Paper
Jessica Shui1, Eileen Saunders1, Robert Needleman1, Michelle Nappi1, Joseph Cooper1, Lauren Hall1,
David Kehoe2 and Emily Stowe-Evans1,∗
Abbreviations: CCA, complementary chromatic adaptation;
DPOR, dark-operative protochlorophyllide oxidoreductase;
GL, green light; LPOR, light-dependent NADPH:protochlorophyllide oxidoreductase; ORF, open reading frame;
PBS, phycobilisome; PC, phycocyanin; PE, phycoerythrin; RL,
red light; RT–PCR, reverse transcription–PCR.
The nucleotide sequences reported in this paper have
been submitted to GenBank under accession numbers
EU283411 (F. diplosiphon por), GQ217522 (F. diplosiphon chlB)
and AY548448 (F. diplosiphon chlLN).
Introduction
Cyanobacteria comprise a large, ancient and diverse group
of photosynthetic organisms that exploit a wide range of
ecological niches in terrestrial, freshwater and marine habitats. Light wavelength, intensity and variability are significant environmental parameters that determine habitat
suitability for individual species, and much of the diversity in
cyanobacteria is found in their light-harvesting pigments
and light-harvesting strategies (Gantt 1981, Glazer 1982,
Grossman 2003). Except for Prochlorococcus, all known
cyanobacteria can synthesize phycocyanin (PC), a protein
whose attached chromophore (phycocyanobilin) absorbs red
light (RL, λmax ∼620 nm) and reflects green light (GL). Many
aquatic species also produce another protein, phycoerythrin
(PE), whose attached chromophore (phycoerythribilin)
absorbs GL (λmax ∼560 nm) and reflects RL (Bogorad 1975,
author: E-mail, [email protected]; Fax +1-570-577-3537.
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
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J. Shui et al.
Gantt 1981, Glazer 1982, Sidler 1994). The protein allophycocyanin is an important component of the phycobilisome
(PBS) and uses phycocyanobilin to absorb RL (λmax ∼650 nm).
Some species also contain phycoerythocyanin, which, like
PE, absorbs short wavelengths of light (λmax ∼540 nm) (Gantt
1981, Glazer 1982, Sidler 1994). This pigment diversity alone
has allowed a wide range of light-harvesting strategies to
evolve, and within the aquatic cyanobacteria that have both
PC and PE three groups have been identified. Group I
includes species that do not alter their PC or PE composition
in response to changes in environmental light conditions,
Group II increases the ratio of PE to PC in GL, and Group III
species can reversibly switch from producing PC in RL to
producing PE in GL (Tandeau de Marsac 1977). The ability of
Group II and Group III species to alter their pigment composition in response to changes in available ambient light is
referred to as complementary chromatic adaptation (CCA)
(Gaiducov 1903, Bennett and Bogorad 1973, Haury and
Bogorad 1977, Vogelmann and Scheibe 1978, Kehoe and
Gutu 2006). These processes may allow Group II and III species to inhabit GL and variable light color environments
unavailable to RL-restricted species. Moreover, Group II and
Group III species may co-exist with RL-restricted species by
harvesting the GL they reflect (Stomp et al. 2004, Six et al.
2007, Stomp et al. 2007, Stomp et al. 2008).
Fremyella diplosiphon is a Group III, chromatically adapting freshwater, filamentous cyanobacteria that has served as
the model organism for CCA analysis since the 1970s. In this
organism, CCA regulates differential production of PC and
PE and morphological changes to cell shape and filament
length (Bennett and Bogorad 1973, Haury and Bogorad 1977,
Vogelmann and Scheibe 1978). The regulation of CCA in
F. diplosiphon is complex and involves at least two photosensory systems (Seib and Kehoe 2002, Alvey et al. 2003, Kehoe
and Gutu 2006, Alvey et al. 2007, Li et al. 2008). One of these,
the Rca system (regulator for complementary chromatic
adaptation), is controlled by RcaE, a phytochrome class photoreceptor with similarity to two-component sensor kinases
(Kehoe and Grossman 1996), and two response regulators
called RcaF and RcaC (Chaing et al. 1992, Kehoe and Grossman 1997). The Rca system is required for normal PBSrelated CCA responses in RL and GL, particularly in the
regulation of expression of the genes required for PE production (cpeCDESTR and cpeBAYZ) and the RL-inducible form
of PC (cpcB2A2H2I2D2) as well as genes involved in chromophore biosynthesis (Seib and Kehoe 2002, Alvey et al.
2003, Alvey et al. 2007). Recent work by Bordowitz and
Montgomery (2008) has shown that RcaE also controls cell
shape and filament length. CpeR is a fourth regulatory protein that specifically acts in GL to up-regulate expression
of cpeBA and pebAB (phycoerythribilin synthesis genes;
Cobley et al. 2002, Seib and Kehoe 2002, Alvey et al. 2003).
The second system (Cgi, control of green light induction)
1508
(Kehoe and Gutu 2006) works with the Rca system and regulates GL induction of PE synthesis genes cpeBA and
cpeCDESTR (encoding the linker proteins for PE), though it is
unknown at this time if it regulates any other parts of the
response described by Bennett and Bogorad (1973). Bennett
and Bogorad (1973) also described light-regulated changes
in the development of hormogonia in F. diplosiphon. These
changes are regulated by changes in the oxidation state of
the electron transport chain (Campbell et al. 1993).
Chromatically adapting cyanobacteria that utilize PC and
PE to harvest light energy, like all cyanobacteria, require Chl
a to transform light into stored, chemical energy. Therefore,
regardless of ambient light conditions, Group I, II and III
cyanobacteria require Chl biosynthesis for photosynthetic
activity. All cyanobacteria, like most photosynthetic eukaryotes, including gymnosperms, early seedless plants and
algae, have two distinct and alternative forms of the penultimate enzyme in Chl biosynthesis, protochlorophyllide oxidoreductase (Fig. 1A, reviewed in von Wettstein et al. 1995,
Fujita 1996, Armstrong 1998, Shoefs 2001, Vavilin and Vermaas 2002, Shoefs and Franck 2003, Bollivar 2006, Gomez
Maqueo Chew and Bryant 2007). While the two enzymes
catalyze the same D ring reduction of the protochlorophyllide molecule, they have important structural and mechanistic differences (Rudiger 1997, Fujita and Bauer 2003, Shoefs
and Franck 2003, Masuda and Takamiya 2004). ‘Dark-operative’ protochlorophyllide oxidoreductase (DPOR) does not
require light, and in some species can function in the absence
of light (Fujita et al. 1991, Fujita et al. 1992, Fujita et al. 1993,
Fujita 1996, Fujita et al. 1996, Armstrong, 1998, Fujita
and Bauer 2003, Shoefs and Franck 2003), whereas lightdependent protochlorophyllide oxidoreductase (LPOR)
requires both light (absorption maximum 638–650 nm) and
NADPH for activity (Lebedev and Timko 1998, Masuda and
Takamiya 2004). The LPOR polypeptide binds both NADPH
and protochlorophyllide, but the reduction does not occur
until the protochlorophyllide molecule absorbs light at 628–
630 nm (Suzuki et al. 1997). Analysis of DPOR and LPOR
enzymatic activity in a variety of species has demonstrated
several functional differences in these enzymes, suggesting
that coordination and regulation of DPOR and LPOR activity allow photosynthetic organisms to maintain high levels
of Chl synthesis under distinct and changing environmental
conditions.
Extensive analyses of DPOR and LPOR activity in the
freshwater filamentous cyanobacterium Leptolyngbya boryana (previously identified as Plectonema boryanum) suggest
that the coordination and regulation of these alternative
enzymes are responsive to environmental conditions. Studies of DPOR and LPOR mutants demonstrated that DPOR
is necessary for chlorophyll biosynthesis in the dark and
under low (10–25 mE m–2 s–1) and intermediate (85–130 mE
m–2 s–1) levels of white light. Cells containing a deletion in
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
CCA regulates protochlorophyllide reductase genes
3
A
N
N
B
8
R
R
Mg
N
D
N
N
Mg
N
N
N
C
18
17
COOH
O
COOCH3
COOH
chlN
O
COOCH3
chlL
A9B
chlB
JS1
por
JS3
Fig. 1 (A) The reduction of protochlorophyllide on the Mg branch that
leads to the biosynthesis of chlorophyllide. The double bond of ring D
of the Pchlide molecule is stereospecifically reduced by two different
reductase enzymes: the RL-dependent reductase encoded by the por
gene and the light-independent reductase encoded by three genes:
chlL, chlN and chlB (adapted from Fujita et al. 1996). (B) Arrangement
of protochlorophyllide oxidoreducatse genes in F. diplosiphon. The
ORF A9B is named following the convention of Stowe-Evans et al.
(2004). Designations JS1 and JS3 refer to putative ORFs of unknown
function. Multiple Sau3AI sites are present in the regions containing
por and chlB, so these were left out of the diagram for ease of reading.
H∗: this HincII site represents three sites within 20 nucleotides of each
other.
the genes encoding DPOR subunits failed to synthesize Chl
in the dark (Fujita et al. 1992, Fujita et al. 1993, Fujita 1996).
In contrast, LPOR was required for growth in high (170 mE
m–2 s–1) white light intensities (Fujita et al. 1998). LPOR–
mutant cells that relied solely on DPOR activity did not synthesize Chl or exhibit autotrophic growth in high intensity
white light. More recent studies have indicated that the
inability to grow at high light intensities in the LPOR– mutant
may be due to the oxygen sensitivity of DPOR (Yamazaki
et al. 2006). When grown in anaerobic conditions, LPOR–
mutants were able to grow and synthesize Chl in high light
intensities (Fujita et al. 1998, Yamazaki et al. 2006). In addition, under anaerobic conditions, protein levels of DPOR subunits increased in LPOR– mutants but not in LPOR-containing
cells (Yamazaki et al. 2006). Collectively, these studies of
L. boryana provide the only evidence to date that DPOR and
LPOR carry distinct functional advantages to any photosynthetic organism in response to environmental conditions.
These studies suggest that L. boryana depends on DPOR
activity in low light and low oxygen, while it uses LPOR in
high light or aerobic conditions. However, L. boryana produces only PC and it is not capable of CCA. Therefore, studies of this organism cannot determine if Group I, II and III
cyanobacteria regulate the ratio of DPOR to LPOR activity in
order to maintain Chl a synthesis during CCA. Moreover, the
studies in L. boryana focused on enzyme activity and did not
determine if DPOR and LPOR RNA accumulation is an
important environmentally responsive regulatory point in
Chl a synthesis.
There are few published studies systematically examining
the RNA and protein expression patterns of the genes that
encode the DPOR and LPOR enzymes in any cyanobacterial
species. DPOR is composed of three subunits encoded by
the chlL, chlN and chlB genes. LPOR is composed of a single
peptide encoded by por. The expectation that cyanobacteria
express chlL, chlN and chlB in the dark was established based
on the fact that Synechocystis sp. PCC6714, PCC6702 and
PCC6805, Chlorogloea sp. PCC6712, Chlorogloea fritschii,
Plectonema boryanum and Anabaena sp. ATCC 29413 all
contain Chl when grown heterotrophically in the dark (Peat
and Whitton 1967, Rippka 1972, Raboy et al. 1976, Mannan
and Pakrasi 1993). In P. boryanum (L. boryana) the production of Chl in the dark is lost when the chlL gene is mutated
(Kada et al. 2003). The only data available on the expression
of any candidate chlL, chlN, chlB or por genes are indirect,
coming from DNA microarray studies of the unicellular
freshwater species Synechocystis sp. PCC6803. Ultraviolet
and high intensity white light (the spectral distributions of
these light sources were not defined) repressed the expression of the chlN, chlL and chlB genes (Hihara et al. 2001,
Huang et al., 2002, Hihara et al. 2003). The high intensity
white light treatment also transiently decreased por levels,
but the expression of many other genes encoding photosynthetic proteins was also affected and may reflect a general
response to the stress of such high intensity irradiance
(Hihara et al. 2001), and these experiments do not assess the
regulation of the abundance of DPOR and LPOR transcript
in response to CCA.
Previous genomic microarray analysis of the response of
F. diplosiphon to growth in RL vs. GL suggested that in this
species, Chl a biosynthesis, in addition to PC and PE protein
synthesis, is a target of CCA-induced regulation (StoweEvans et al. 2004). We found that transcripts of a potential
homolog of chlL, a DPOR gene, are more abundant in cells
acclimated to GL (Stowe-Evans et al. 2004). The increase in
chlL transcript accumulation in GL suggested that F. diplosiphon compensates for the reduction of LPOR activity in GL
by increasing the expression of the genes encoding DPOR,
presumably leading to greater DPOR activity.
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Here we test the hypothesis that Chl a biosynthesis is regulated and maintained during CCA via the light-responsive
regulation of DPOR- and LPOR-encoding genes with changes
to transcript accumulation. Our results provide the first
demonstration that Chl a biosynthesis is controlled by CCA
in cyanobacteria.
Results
Identification of the genes encoding DPOR, chlLN
and chlB, in F. diplosiphon.
We previously identified the F. diplosiphon chlL gene as part
of a microarray study designed to identify genes that are
regulated in response to CCA (Stowe-Evans et al. 2004, GenBank accession No. AY548448.1). In that study we observed
that the chlL transcript is 2.2-fold more abundant in cells
grown in GL than in those grown in RL (Stowe-Evans et al.
2004). The predicted F. diplosiphon ChlL protein has 93%
amino acid identity to the predicted Anabaena PCC 7120
ChlL protein and 91% amino acid identity to the L. boryana
homolog (Fig. 2A). Both Anabaena PCC 7120 and L. boryana, like F. diplosiphon, are freshwater, filamentous cyanobacteria, but neither is subject to CCA regulation. We have used
these two cyanobacteria for comparison with F. diplosiphon
in our current study for two reasons: (i) Anabaena PCC 7120
has served as a model organism for cyanobacterial photosynthesis; and (ii) the biochemistry of DPOR and LPOR has
been studied in detail with L. boryana. Our current Southern
analysis of the F. diplosiphon chlL homolog indicates that
this gene is found as a single copy in the F. diplosiphon
genome (Fig. 2B).
The F. diplosiphon genomic DNA insert containing chlL
within the plasmid identified in our microarray study was
sequenced, and it also carried approximately 300 bp of a
putative chlN gene downstream of the chlL open reading
frame (ORF). This 300 bp sequence was used to search for
the F. diplosiphon chlN gene in a database of partially annotated F. diplosiphon genomic sequence (D. Kehoe unpublished data). As expected from the analysis of the plasmid
containing chlL, the full-length F. diplosiphon chlN gene was
identified downstream of chlL (Fig. 1B). The predicted
F. diplosiphon ChlN protein is 95% identical to the predicted
Anabaena PCC 7120 protein and 86% identical to the
L. boryana protein (Fig. 3A). Southern analysis using a PCR
product amplified from the chlN gene as a probe indicated
that the chlN gene is present in a single copy in the F. diplosiphon genome (Fig. 3B).
Located between chlL and chlN in the F. diplosiphon
genome is an ORF of unknown function identified as A9B in
Fig. 1, which was also identified in Stowe-Evans et al. (2004).
The chlL–ORF–chlN structure of this operon is conserved in
many freshwater, filamentous cyanobacteria, including Anabaena PCC 7120 (all5077) and L. boryana (ORF133, GenBank
accession No. Q00247.1). Interestingly, the corresponding
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center ORFs from all three species are similar to each other.
However, the similarity is much greater between F. diplosiphon and Anabaena PCC 7120 (62% identity) than it is
between F. diplosiphon and L. boryana (34%). ORF133 is predicted to be approximately 100 amino acids shorter than the
corresponding predicted proteins in the other species. Most
of the difference is due to a truncation of the C-terminal end
of the L. boryana protein, though there is an additional internal stretch of 30 amino acids in the F. diplosiphon protein
absent in the L. boryana protein (data not shown). Currently
there is no known function for this protein and it contains
no known conserved domains.
Degenerate PCR primers based on highly conserved
amino acids from the predicted sequence of several
cyanobacterial ChlB proteins were used to amplify a 350 bp
DNA fragment containing sequences from the 5′ region of a
putative chlB gene from F. diplosiphon genomic DNA. The
350 bp PCR product was sequenced and used to generate
non-degenerate primers to amplify a segment of the chlB
gene from the F. diplosiphon genome. The resulting 300 bp
sequence was used to search for the F. diplosiphon chlB
homolog in a database of partially annotated F. diplosiphon
genomic sequence (D. Kehoe unpublished data). Analysis of
the genomic sequence confirmed that a conceptual translation product of one predicted ORF was highly homologous
to ChlB proteins of other cyanobacteria. The F. diplosiphon
ChlB predicted protein is 91% identical to the predicted
Anabaena PCC 7120 protein and 83% identical to the ChlB
protein in L. boryana (Figs. 1, 4A). Southern analysis using a
PCR product containing the full-length chlB gene as probe
indicates that chlB is found as a single-copy gene in the
F. diplosiphon genome (Fig. 4B). As in many other filamentous cyanobacteria, chlB of F. diplosiphon is not located adjacent to chlL and chlN (Fujita et al. 1991, Fujita et al. 1992,
Fujita et al. 1993, Fujita et al. 1996). To date, only a single
copy of any of the genes encoding subunits of DPOR have
been identified in any of the sequenced cyanobacterial
genomes (Raymond et al. 2002, Cyanobase database).
Co-transcription of chlL and chlN
Our analysis of the genomic arrangement of chlL and chlN in
F. diplosiphon suggests that these genes are within the same
operon and that they are co-transcribed. In F. diplosiphon
chlL and chlN are separated by a single ORF of unknown
function (Fig. 1B); this arrangement is similar to the arrangement in other filamentous cyanobacterial species, particularly L. boryana (Fujita et al. 1991). Fujita et al. (1991) have
previously shown that chlL and chlN are in an operon and
co-transcribed in L. boryana. It is possible that these three
ORFs form an operon in F. diplosiphon and are transcribed as
a unit. RNA blot analysis using chlL as a probe revealed three
bands of approximately 4.4, 2.4 and 1.4 kb containing all
three genes, just chlL and 3109A9B, and just chlL, respectively
(data not shown). However, when chlN was used as a probe
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
CCA regulates protochlorophyllide reductase genes
Fig. 2 F. diplosiphon contains a single chlL gene. (A) Protein sequence alignment of ChlL from F. diplosiphon, Anabaena PCC7120 and L. boryana.
The sequences exhibit 91% identity. (B) Southern blot analysis indicates that chlL is a single gene in F. diplosiphon. F. diplosiphon genomic DNA
digested with HindIII (lane 2), HincII (lane 3) and Sau3A (lane 4) Lane 1: Roche DIG-labeled DNA molecular weight marker III.
in Northern blot analysis, a single band of approximately
4.4 kb was seen (data not shown). We used reverse transcription–PCR (RT–PCR) to determine if the chlL and chlN genes
are co-transcribed in F. diplosiphon. Using a primer internal
to chlL and another internal to chlN we are able to amplify a
PCR product of the expected 2,000 bp from cDNA generated
from DNase-treated total RNA from both RL- and GL-grown
cells and from genomic DNA (Fig. 5, lanes 1, 2 and 5). In contrast, no product was amplified when the DNase-treated
total RNA that was used to make the cDNA was used directly
as template for PCR without prior reverse transcription
(Fig. 5, lanes 3 and 4). This analysis suggests that chlL, ORF
A9B and chlN are co-transcribed in F. diplosiphon. We will
refer to this as the chlLN transcript.
Identification of the gene encoding LPOR, por,
in F. diplosiphon
To identify the por gene of F. diplosiphon, the full-length
por gene was PCR amplified from Anabaena PCC 7120
genomic DNA and this 990 bp sequence was used to probe a
F. diplosiphon genomic DNA plasmid library. Screening the
genomic library identified several candidate plasmids, and
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J. Shui et al.
Fig. 3 F. diplosiphon contains a single chlN gene. (A) Protein sequence alignment of ChlN from F. diplosiphon, Anabaena PCC7120 and L. boryana.
ChlN from F. diplosiphon is 93% identical to Anabaena PCC7120 and 86% identical to L. boryana. (B) Southern blot analysis indicates that chlN is
a single gene in F. diplosiphon. Lanes 1 and 5: Roche Dig-labeled DNA ladders III and VII, respectively. F. diplosiphon genomic DNA digested with
HincII (lane 2), NciI (lane 3) and XmnI (lane 4). The blot was hybridized with a probe corresponding to the first 300 nucleotides of the chlN gene
of F. diplosiphon.
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CCA regulates protochlorophyllide reductase genes
Fig. 4 F. diplosiphon contains a single chlB gene. (A) Protein sequence alignment of ChlN from F. diplosiphon, Anabaena PCC7120 and L. boryana.
ChlB from F. diplosiphon is 93% identical to that from Anabaena PCC7120 and 86% identical to that from L. boryana. (B) Southern blot analysis
indicates that chlB is a single gene in F. diplosiphon. F. diplosiphon genomic DNA digested with XmnI (lane 2), HincII (lane 3) and NciI (lane 4).
Lanes 1 and 5: Roche Dig-labeled DNA ladders III and VII, respectively.
each plasmid was screened via Southern blot. One positive
plasmid was fully sequenced, and all putative ORFs of at least
300 nucleotides were identified. The conceptual translation
product of one ORF of 962 nucleotides was found to have
significant similarity to known LPOR sequences and identified as the putative F. diplosiphon por gene. The predicted
F. diplosiphon LPOR protein is 87% identical to the predicted
Anabaena PCC 7120 protein and 69% identical to the
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A
MW
1
2
3
4
5
B
1 kb
ch1NF
chlN
ch1LR
A9B
chlL
Fig. 5 FdchlL and FdchlN are co-transcribed. (A) RT–PCR analysis with
primers located within chlL and chlN indicate that the genes are
co-transcribed. Lane 1, GL cDNA; lane 2, RL cDNA; lane 3, DNasetreated GL RNA; lane 4, DNase-treated RL RNA; lane 5, F. diplosiphon
DNA. MW, molecular weight marker. (B) Arrangement of chlL and
chlN genes in F. diplosiphon. The ORF A9B is named following the
convention of Stowe-Evans et al. (2004). Arrows indicate primer
location for RT–PCR.
L. boryana protein (Fig. 6A). Fremyella diplosiphon por gene
sequences were PCR amplified from genomic DNA, and the
950 bp PCR product was used to generate a probe for Southern blot analysis. A single 1 kb NciI fragment hybridizes to
the probe, indicating that there is a single copy of the por
gene in the F. diplosiphon genome (Fig. 6B). While many
plant species have multiple copies of por, to date, all
cyanobacteria genomes that have been searched, including
the F. diplosiphon genome, contain only a single copy of the
por gene (Kaneko et al., 1996, Masuda and Takamiya 2004,
Cyanobase database).
Chromatic regulation of protochlorophyllide
reductase genes
RT–PCR is a semi-quantitative procedure and the chlLN
RT–PCR product from GL-grown cells (Fig. 5, lane 1) is more
abundant that the product from RL-grown cells (Fig. 5, lane
2), suggesting that the chlLN transcript is more abundant in
cells grown in GL than cells grown in RL. This observation is
consistent with our previous observation that the transcript
of chlL is 2.2-fold more abundant in GL than in RL (StoweEvans et al. 2004). To investigate further the possibilities that
the wavelength of light affects the accumulation of transcript from the genes encoding DPOR subunits, and that this
regulation allows cells to increase the amount of active
DPOR to compensate for the loss of LPOR activity, we conducted quantitative Northern blot analysis of chlLN and chlB
transcript accumulation in wild-type cells grown in GL and
RL. For each DPOR transcript analyzed the amount of transcript in wild-type cells grown in GL is set as a standard of
100% accumulation. The transcript of chlLN is approximately
twice as abundant in cells grown in GL than cells grown in RL
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(100% vs. 60%, respectively) (0.002 > P > 0.001)) (Fig. 7A, B).
The abundance of chlB transcript in GL is slightly more than
twice that seen in RL (100% in GL vs. 42% in RL) (0.05 > P
> 0.02) (Fig. 7A, C). Therefore, it appears that the transcripts
that encode the three DPOR subunits are more abundant in
cells grown in GL, the very cells in which LPOR activity is limited, than cells grown in RL.
We also observed a statistically significant reduction in
por transcript accumulation in cells grown in GL vs. those
grown in RL. In wild-type cells grown in GL the transcript
accumulation of por is approximately 88% of that seen in
wild-type cells grown in RL (0.05 > P > 0.02) (Fig. 7A, C). This
observation suggests that por gene activity is only slightly
higher in RL-grown cells than in GL-grown cells. Thus, while
wild-type cells appear to increase the amount of DPOR transcripts by ≥2-fold, in GL the cells reduce the amount of por
transcript only slightly, possibly as little as 10% in GL, where
the LPOR protein product is presumably inactive.
CCA regulatory mutations affect DPOR and LPOR
transcript accumulation
To determine if known regulators of CCA affect DPOR and
LPOR transcripts we analyzed chlLN, chlB and por transcript
accumulation in cells with mutations in genes encoding CCA
regulatory components using quantitative Northern blots.
In F. diplosiphon, RcaE and CpeR are two CCA-regulatory
proteins with distinct roles in regulating the transcript abundance of several GL-induced genes including those encoding
PE and related proteins. The mutant BK14 lacks functional
RcaE (Terauchi et al. 2004), and TQ1 is a double mutant lacking functional RcaE and CpeR (Seib and Kehoe 2002). The
levels of chlLN transcript in the RL- and GL-grown BK14 and
TQ1 mutant cells are both approximately 51–55% of the
level observed in GL-grown wild-type cells (0.05 > P > 0.02)
(Fig. 7B). There is no statistical difference between the
expression of chlLN in BK14 and TQ1 in either GL (P > 0.5) or
RL (P > 0.5). These observations suggest that the absence of
functional RcaE prevents increased accumulation of chlLN
transcript in GL-grown cells, but the additional loss of CpeR
function has no effect on this operon’s expression in either
light condition. In contrast, the accumulation of chlB transcript in the GL-grown BK14 mutant cells is approximately
97% of that observed in wild-type cells grown in GL, while
the accumulation of chlB transcript in the GL-grown TQ1
mutant cells is reduced to 65% of that observed in wild-type
cells grown in GL (Fig. 7C). However, these reductions are
not statistically significant and therefore may represent
alterations in transcript abundance due to changes in photosynthetic efficiency in the mutants and could indicate that
the RL/GL differential of chlB is regulated in a manner different from chlLN.
The levels of por transcript in cells subjected to CCA were
also altered by the mutations in rcaE and cpeR. In both
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
CCA regulates protochlorophyllide reductase genes
Fig. 6 F. diplosiphon contains a single por gene. (A) Protein sequence alignment of POR from F. diplosiphon, Anabaena PCC7120 and L. boryana.
(B) Southern blot analysis indicates that por is a single gene in F. diplosiphon. F. diplosiphon genomic DNA digested with HincII (lane 2), NciI
(lane 3) and XmnI (lane 4). Lanes 1 and 5: Roche Dig-labeled DNA ladders III and VII, respectively.
mutants, transcripts levels in GL-grown cells remain relatively similar to wild-type levels. However, when grown in RL,
the level of por transcript increased in both the BK14 and the
TQ1 mutant cells as compared with levels seen in wild-type
cells when all are grown in RL. With the level of por transcript
in RL-grown wild-type cells set as a standard of 100%, por
transcript are found at 124% in Bk14 cells and 155% in TQ1
cells (0.02 > P > 0.01). The increase in BK14 is large but not
statistically significant. However, the difference seen in TQ1
compared with the wild type is statistically significant.
Expression of protochlorophyllide genes in the dark
Previous studies have determined that the transcript levels
of protochlorophyllide genes show some responsiveness to
changes in light intensity and growth in darkness. For example, in Chlamydomonas, the transcript levels for chlLN and
chlB are 2- to 4-fold higher in cells grown in the light relative
to those grown in the dark (Cahoon and Timko 2003).
In contrast, our initial experiments in F. diplosiphon showed
that cells acclimated to GL and then incubated in the
dark overnight had substantially reduced levels of chlLN
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
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J. Shui et al.
Fig. 7 Red- and green light-regulated transcript accumulation patterns of protochlorophyllide oxidoreductase genes in F. diplosiphon.
(A) Representative images from Northern analysis of the data presented in B–D. (B–D) Mean relative expression values from 3–5 experiments of
protochlorophyllide reductase gene expression in the wild type (WT), rcaE null mutants (BK14) and rcaE/cpeR double mutants (TQ1) grown in
RL and GL (15 µmol m–2 s–1). (B) chlLN, n = 5. (C) chlB, n = 4. (D) por, n = 4. Measurements were standardized by using ribosomal values prior to
calculation of the means. P-values are given in the text; standard errors are shown. An asterisk indicates a statistical difference 0.05 > P > 0.01
when compared with WT GL-grown cells (B and C) or WT RL-grown cells (D).
transcript (data not shown). A more detailed kinetic analysis
demonstrated that after a brief increase at 30 min the transcript level of chlLN drops precipitously, and within 4 h in the
dark the chlLN transcript levels are just under 20% of those
of GL-grown cells (0.005 > P > 0.002) (Fig. 8A, B). Slightly
different patterns were seen for chlB and por transcript accumulation. Within 30 min in the dark the chlB transcript
abundance drops to approximately 70% that of GL-grown
cells and remains at that level before dropping to approximately 40% that of GL-grown cells at 4 h (0.02 > P > 0.01)
(Fig. 8A, C). The por transcript, however, quickly drops to
approximately 20% of GL levels by 1 h of dark incubation
and while there is some variability in expression at 2 h of
incubation, by 4 h in the dark expression levels are still at
20% that of GL (0.005 > P > 0.002) (Fig. 8A, D). Future studies are required to address two important questions: (i) is
the regulation of chlL and chlB gene expression transcriptional or post-transcriptional; and (ii) do transcript accumulation patterns positively correlate with the levels of protein
accumulation and activity?
1516
Discussion
In cyanobacteria, the reduction of protochlorophyllide to
chlorophyllide can be accomplished through the activity of
either of two structurally unrelated protochlorophyllide
reductases, DPOR and LPOR (Fujita 1996, Yamazaki et al.
2006). The activity of DPOR is light independent while LPOR
requires RL for activity. The RL requirement of LPOR is probably not limiting to cyanobacteria except in aquatic environments, where RL may be attenuated by depth, turbidity or
competition (Stomp et al. 2004, Six et al. 2007, Stomp et al.
2007, Stomp et al. 2008). In these environments, cyanobacteria utilize PE for light harvesting, but also must maintain sufficient Chl levels for functional reaction centers (Campbell
1996). CCA and its associated sensory and regulatory proteins regulates the production of PE is some species of
cyanobacteria. Our current findings demonstrate that CCA
also regulates expression of the genes encoding the DPOR
subunits and thus may compensate for the loss of LPOR
activity as RL becomes attenuated.
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
CCA regulates protochlorophyllide reductase genes
Fig. 8 Light-regulated transcript accumulation patterns of
protochlorophyllide oxidoreductase genes in F. diplosiphon. Changes
in the transcript levels of protochlorophyllide reductase genes after
GL-illuminated cells were transferred to darkness for 0.5, 1, 2 or 4 h.
(A) Representative images from Northern blot analysis of the data
presented in B. (B) Mean relative expression values from three
experiments of protochlorophyllide reductase gene expression after
transfer of the culture to the dark. Measurements were standardized
by using ribosomal values prior to calculation of the means. P-values
are given in the text; standard errors are shown. n = 4; an asterisk
indicates a statistical difference 0.05 > P > 0.01 when compared with
GL-grown cells at time = 0.
Previous work in cyanobacteria has shown some functional differentiation between DPOR and LPOR based on
light intensity (Fujita et al. 1996, Fujita et al. 1998) and oxygen
levels (Yamazuki et al. 2006). In anaerobic conditions a por–
mutant accumulated more ChlL and ChlN protein than
wild-type cells, while levels of ChlB were unaffected by the
absence of LPOR. Yamazaki et al. (2006) have shown that
altering the abundance of just the ChlL and ChlN proteins
can lead to an increase in active DPOR because, typically,
ChlN protein is limiting. In anaerobically grown cells of
L. boryana, a por– mutant accumulated three times more
functional DPOR protein than the wild type, simply by
increasing the abundance of the ChlL and ChlN proteins
(Yamazuki et al. 2006). While this increase in anaerobic conditions could be caused by the oxygen sensitivity of DPOR,
similar protein level increases were not seen in anaerobically
grown wild-type cells. However, this study did not address
transcript accumulation patterns, and thus it is not known
whether protein stability or changes in gene expression
patterns are responsible for the changes in protein
accumulation.
Previous studies with the liverwort Marchantia palacaea
have shown that chlL expression is increased after pulses of
RL, and this expression exhibits RL/FR (far red) photoreversibility characteristic of phytochrome-regulated genes (Suzuki
et al. 2001). While an as yet undescribed phytochrome may
play a role in chlLN, chlB and por gene expression in F. diplosiphon, the data in Fig. 7 A and B demonstrate that the
differential accumulation of chlLN transcript in GL compared with RL is regulated by the CCA photoreceptor RcaE,
since the rcaE mutant BK14 fails to exhibit differential GL
accumulation of the chlLN transcript. The complete loss of
chlLN light responsiveness in this mutant also suggests that
the Rca system is probably the only photosensory system
controlling this operon.
In contrast, the abundance of the chlB transcript appears
to be unaffected by the absence of functional RcaE. Recent
studies have indicated that RcaE is not the only photoreceptor with a regulatory effect on PBS-related genes in F. diplosiphon (Seib and Kehoe 2002, Kehoe and Gutu 2006). All of
the GL-up-regulated operons studied thus far in this organism continue to show 2- to 3-fold GL activation in the rcaE
mutant (Seib and Kehoe 2002, Alvey et al. 2003). This regulatory mechanism for GL induction, the Cgi system, may also
be responsible for the GL induction of the chlB gene, which
is nearly the same in wild-type cells and the rcaE mutant
BK14 but appears to be lost in the double rcaE/cpeR mutant
TQ1 (Fig. 7A, C). Since CpeR expression is under the dual
control of both the Rca and Cgi systems (Kehoe and Gutu
2006), this implicates the Cgi system in the CCA regulation
of chlB expression.
While LPOR activity is reduced when cells are grown in
GL, we detected only a slight, though significant, difference
in por transcript accumulation between GL- and RL-grown
cells. If the same is true at the protein level, the cell seems to
maintain a steady-state level of LPOR when grown in the
light, regardless of whether or not the wavelengths that can
be absorbed by LPOR-bound protochlorophyllide are present. Surprisingly, we measured an increase in transcript
abundance in the RL-grown BK14 and TQ1 cells. It is possible
that the BK14 mutant is not as photosynthetically efficient
as the wild type given that their PBSs are not able to acclimate to changing light color conditions, and thus the cell
may compensate by producing more Chl. As the increase is
only seen in RL, where LPOR is most active, raising the
amount of por in this situation may help maintain photosynthetic efficiency while raising the amount of por in
GL, where LPOR is less active, would not be able to increase
photosynthetic efficiency. Future studies characterizing the
amount of Chl in this combination of mutants and light conditions must be conducted to test this hypothesis further.
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
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J. Shui et al.
One interesting commonality among all four genes is the
requirement of light for transcript accumulation. All four
genes showed a decrease in transcript abundance within 4 h
of being moved from constant light incubation to dark incubation (Fig. 8). The transient increase of chlL transcript seen
at 0.5 h may be a short-term response to the loss of active
LPOR in the dark. The decrease in transcript abundance at
later times probably represents an acclimation to extended
incubation in the dark—reminiscent of the normal diurnal
light cycle. In Chlamydomonas a similar pattern is seen in
transcript accumulation, with higher chlL, chlN and chlB
transcript abundance in the light than in the dark (Cahoon
and Timko 2003a, Cahoon and Timko 2003b). However, a
different pattern is seen in M. palacaea for chlL, with higher
transcript levels seen in the dark than in the light (Eguchi
et al. 2002). In gymnosperms, chlL and chlN are constitutively
expressed in both light- and dark- grown cotyledons, but
show light-induced expression in the stem (Skinner and
Timko 1999). In M. palacae, por transcripts are nearly undetectable in the dark but abundant in the light, and the stability of the transcript is also affected by the redox status of the
photosynthetic electron transport chain (Takio et al. 1998,
Eguchi et al. 2002). Many organisms lacking DPOR have multiple copies of LPOR and individual homologs have varying
expression patterns (Spano et al. 1992, Reinbothe et al. 1995,
Masuda et al. 2002, Masuda and Takamiya 2004). For example, in Arabidopsis, PorA is more abundant in the dark than
in the light while PorB exhibits the reverse expression pattern and is found in continuously illuminated seedlings and
adult tissues, and is diurnally regulated (Armstrong et al.
1995, Masuda et al. 2003). PorC in Arabidopsis is not detected
in dark-grown plants but is detected in light-grown plants,
with expression increasing as the light intensity increased
(Oosawa et al. 2000, Su et al. 2001). In many eukaryotes,
accumulation of LPOR in the dark occurs for photoprotective purposes (Buhr et al. 2008) and presumably LPOR (and
potentially DPOR) could perform this role in cyanobacteria
as well. The decrease in protochlorophyllide gene expression
may result from an overall decrease in the metabolism of the
cells while sufficient levels of the proteins remain for photoprotective purposes and to supply Chl for photosynthesis to
recommence at dawn. Further experiments will need to be
conducted to determine whether the dark reduction of
expression is driven by a photoreceptor or by changes in the
redox state of the photosynthetic electron transport chain.
These genes probably experience many levels of regulation, and the presence or absence of light and the wavelength of available light are both strong regulators of their
expression. While known CCA regulatory proteins RcaE and
CpeR appear to be important for regulation of chlLN and
possibly chlB, they have no obvious impact on the differential accumulation of the por transcript in RL and GL except
when they are absent. Future studies are required to
1518
elucidate fully the regulation of these four genes in response
to light conditions and to determine what portions of this
regulation are mediated by photoreceptors or the redox
status of the photosynthetic machinery.
Materials and Methods
Cell growth and light conditions
Fremyella diplosiphon UTEX 481 (also called Calothrix sp.
PCC 7601 and Tolypothrix sp PCC 7601) shortened filament
mutant strain SF33 was used as the wild-type strain (Colby
et al. 1993). This strain forms discrete colonies on solid media
but retains the CCA responses as seen in true wild-type
F. diplosiphon. The rcaE null mutant strain Black 14 (BK14,
Kehoe and Grossman 1996, Kehoe and Grossman 1997) and
the cpeR null mutant strain Turquoise 1 (TQ1, Seib and Kehoe
2002) were also used. All cultures were completely acclimated
to light conditions by growing from low cell density in 15 µmol
m–2 s–1 of continuous RL (General Electric F20T12/R/24/CVG,
λmax 618–622 nm) or GL (General Electric F20T12/G/89/CVG,
λmax ∼540 nm) for a minimum of 5 d at 27°C. All cells were
grown in BG11 plus 10 mM HEPES (pH 8.0) and bubbled with
air. Cultures for kinetic analysis of transcript accumulation in
the dark were grown for 5 d in GL and transferred to dark
conditions with bubbling for 0.5–4 h.
RNA isolation and analysis
RNA was isolated and assessed essentially as described
in Stowe-Evans et al. (2004) and Seib and Kehoe (2002). Purified PCR products were used as probes and labeled with [α32P]dCTP using the Amersham RediPrimeII Random Prime
Labeling System (GE Healthcare). Blots were imaged using a
Storm 860 Molecular Imager, and phosphorimages were
quantified using ImageQuant analysis tools (GE Healthcare).
The relative expression of each gene was normalized to that
of the16S ribosomal control. Three to five independent biological replicates were analyzed. Statistical significance was
determined as previously described (Stowe-Evans et al.
2004). Semi-quantitative RT–PCR was performed on cDNA
generated using the Ambio RETRO-script kit and RNA
treated with RNase-free DNase I (Promega). RT–PCR analyses were carried out by using total RNA and gene-specific
primers (chlNF: 5′-CGTTTAATTTGTAAGCACAACCG-3′
and chlLR: 5′-GAAAAGGTGGAATCGGCA-3′). A 1 µg aliquot
of total RNA was used for each reverse transcription in a
20 µl volume, and 1 µl of the reaction mixture was subsequently used for PCR amplification using the following conditions: 95°C for 5 min, followed by 30 cycles of 95°C for
1 min, 52°C for 40 s, 65°C for 2 min and a final extension at
65°C for 4 min. To detect DNA contamination, control PCRs
were performed on DNase I-treated RNA not subjected to
reverse transcription. The identities of all PCR products were
confirmed via DNA sequencing.
Plant Cell Physiol. 50(8): 1507–1521 (2009) doi:10.1093/pcp/pcp095 © The Author 2009.
CCA regulates protochlorophyllide reductase genes
DNA isolation, library construction and Southern
blotting
Genomic DNA was isolated from F. diplosiphon using the
Fungal/Bacterial DNA kit from Zymo Research. Standard
Southern blotting techniques were used to detect chlB, chlL,
chlN and por in the F. diplosiphon genome. The Dig High
Prime DNA labeling and Detection kit (Roche catalog No. 1
745 832) was used for non-isotopic detection of probes for
Southern and colony hybridization. A genomic library was
constructed essentially as described in Stowe-Evans et al.
(2004) except that the vector pZERO (Invitrogen) was used
instead of pGem7Zf(+). The library was plated onto Luria–
Bertani (LB) plates containing 50 µg ml–1 kanamycin and
grown overnight at 37°C. Approximately 5,000 colonies were
screened to identify the por gene. Cells were transferred to
NytranSPC nylon by replica plating, lysed by soaking in 0.5 N
NaOH/1.5 M NaCl and neutralized in 1.0 M Tris pH 7.6, 1.5 M
NaCl. DNA was fixed to the membrane by UV cross-linking.
Identification of the por gene in F. diplosiphon
The por gene of Anabaena PCC 7120 was PCR amplified
(POR3 5′-GGCACGTCGTCATGGCTTGC-3′ and POR4 5′GGAATTTTACCACCTAGCTC-3′) from genomic DNA and
used to probe the F. diplosiphon genomic library created as
described above. Several plasmids were identified, rescreened
via Southern blot and sequenced at Pennsylvania State University DNA Core facilities. ORF sequences were identified
via ORFinder at NCBI, and potential homology was identified via BLAST (Altschul et al. 1990). After the F. diplosiphon
por gene was identified, F. diplosiphon por-specific primers
were created (Fpor 5′-CCACTAACTTCTCACTTAAATCCC-3′
and Rpor 5′-CAAAATCGGAAGTCAACGGT-3′). The fulllength F. diplosiphon por PCR fragment was labeled using the
Roche Dig High Prime Labeling and Detection kit and used
for Southern blot analysis. This same fragment was used for
Northern blot analysis. The final coding sequence of por was
submitted to GenBank under accession No. EU283411.
Funding
Identification of the chlB gene in F. diplosiphon
The ChlB sequences from several cyanobacteria were
retrieved from NCBI and aligned using ClustalW. Two highly
conserved regions in the protein sequence were chosen and
degenerate PCR primers were generated based on the preferred codon usage of F. diplosiphon. A 350 bp fragment was
amplified using the chlBdU (GAA RTT RGC NTA YCT GAT
GT) and chlBdL (TCT AAN AGR TGR TCY TCC AT) primers,
the FailSafe PCR system from Epicentre and the following
amplification conditions: 95°C for 5 min, followed by 35
cycles of 95°C for 1 min, 50°C for 40 s, 65°C for 2 min and a
final extension at 65°C for 4 min. The PCR product was purified using Qiagen PCR clean up columns and sequenced
using the chlBdU and chlBdL primers at the DNA Facility at
Pennsylvania State University, State College, PA, USA. We
confirmed that the sequence we obtained with the above
primers was chlB by blasting the fragment against the nonredundant library at GenBank. From this sequence, chlBF
(5′-CCCTAGATGACTATGTTA-3′) and chlBR (5′-TAGAA
GTGCAGGTGGGAGTTA-3′) non-degenerate primers were
made to amplify a 300 bp fragment. This fragment was used
as a probe for Northern analysis and to screen the genomic
library. Ultimately this fragment was used to screen the partially annotated genome to identify a putative homolog of
the chlB gene. Gene-specific primers were developed to
amplify the full length of chlB (chlBF 5′-CTACAGCTT
CTTTTGCAGCGTAA-3′ and chlBR 5′-TTGGCTTACTGGAT
GTATGCAG-3′). This fragment was labeled with the Roche
Dig High Prime Labeling kit and used for Southern analysis.
The final coding sequence of chlB was submitted to GenBank under accession No. GQ217522. The chlL and chlN
sequences can be found at GenBank accession No.
AY548448.
Bucknell University start-up funds; the National Science
Foundation (0527672 to E.S.-E); Bucknell University
Undergraduate Research Program (J. S., E. S., M. N., R. N., J. C.
and L. H.).
Acknowledgments
We thank M. Howe and W. Stowe for their thoughtful
reviews and comments.
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(Received April 30, 2009; Accepted June 25, 2009)
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