Download Characterization of the Arabidopsis thaliana Mutant pcb2 which

Plant Cell Physiol. 46(3): 467–473 (2005)
doi:10.1093/pcp/pci053, available online at www.pcp.oupjournals.org
JSPP © 2005
Characterization of the Arabidopsis thaliana Mutant pcb2 which Accumulates
Divinyl Chlorophylls
Hiromitsu Nakanishi 1, Hatsumi Nozue 1, Kenji Suzuki 1, Yasuko Kaneko 2, Goro Taguchi 1 and Nobuaki
Hayashida 1, 3
1
Division of Gene Research, Department of Life Science, Research Center for Human and Environmental Sciences, Shinshu University, Ueda,
386-8567 Japan
2
Department of Regulation Biology, Faculty of Science, Saitama University, Saitama, 338-8570 Japan
;
We characterized the pcb2 (pale-green and chlorophyll b reduced 2) mutant. We found through electron
microscopic observation that chloroplasts of pcb2 mesophyll cells lacked distinctive grana stacks. High-performance liquid chromatography (HPLC) analysis showed that
the pcb2 mutant accumulated divinyl chlorophylls, and the
relative amount of divinyl chlorophyll b was remarkably
less than that of divinyl chlorophyll a. The responsible gene
was mapped in an area of 190 kb length at the upper arm of
the 5th chromosome, and comparison of DNA sequences
revealed a single nucleotide substitution causing a nonsense mutation in At5g18660. Complementation analysis
confirmed that the wild-type of this gene suppressed the
phenotypes of the mutation. Antisense transformants of the
gene also accumulated divinyl chlorophylls. The genes
homologous to At5g18660 are conserved in a broad range
of species in the plant kingdom, and have similarity to
reductases. Our results suggest that the PCB2 product is
divinyl protochlorophyllide 8-vinyl reductase.
phyll a, and the peripheral light-harvesting antenna complexes
contain chlorophyll a and chlorophyll b (Grossman et al.
1995). There are some other kinds of chlorophyll found in various photosynthetic organisms, such as bacteriochlorophylls in
photosynthetic bacteria. Prochlorococcus, a group of marine
cyanobacteria, possesses divinyl chlorophylls in place of chlorophylls (Chisholm et al. 1992). All chlorophylls are believed
to be synthesized from 5-aminolevulinic acid through similar
biosynthetic pathways (Willows 2003). However, these pathways are not understood completely.
Chlorophyll a is synthesized in 15 steps from L-glutamate
in higher plants (Lange and Ghassemian 2003). Two glutamates form a porphobilinogen after the conversion to 5-aminolevulinic acids, and then four porphobilinogens make up a
tetrapyrol ring that leads to protoporphyrin IX after some
modifications. Insertion of a Mg ion into protoporphyrin IX by
Mg-chelatase is the specific step to chlorophyll formation at
the branch point of chlorophyll and heme biosynthesis (Beale
1999). Then, Mg-protoporphyrin IX becomes chlorophyll a
after five steps of enzymatic reactions. Chlorophyll b is derived
from chlorophyll a by conversion of a methyl group to a formyl
group catalyzed by chlorophyllide a oxygenase (CAO) (Tanaka
et al. 1998). It has also been suggested that precursor molecules
of chlorophyll are involved in ‘plastid signal’, which is a communication system assumed to exist between the chloroplast
and the nucleus (Mochizuki et al. 2001).
In a previous study, we collected the color mutants of Arabidopsis thaliana in order to analyze the biogenesis of the chloroplast (Nakanishi et al. 2004). It is highly likely that color
mutants have defects in chlorophyll biosynthesis steps or in the
development of the chloroplast, where chlorophyll is accumulated. The pcb2 (pale-green and chlorophyll b reduced 2)
mutant line isolated from the ethyl methanesulfonate (EMS)
mutant library has a reduced amount of chlorophylls resulting
in pale-green leaves, and the reduction of chlorophyll b content is especially remarkable. Allelism tests and mapping
showed that pcb2 is not an allele of any known similar mutants
such as ch1-1 (Oster et al. 2000), ch42 or cch (Mochizuki et al.
2001). In this study, we describe the novel mutant pcb2.
Keywords: Arabidopsis thaliana — Chloroplast — Complementation — Divinyl protochlorophyllide 8-vinyl reductase —
Grana stack — pcb2 mutant.
Abbreviations: CaMV, cauliflower mosaic virus; CAO, chlorophyllide a oxygenase; Col, Columbia; dCAPS, derived cleaved amplified polymorphic sequence; EMS, ethyl methanesulfonate; ESI,
electrospray ionization; EST, expressed sequence tag; LC, liquid chromatography; Ler, Landsberg erecta; MS, mass spectrometry; ODS,
octadecasilyl; RT–PCR, reverse trascription–polymerase chain reaction; SSLP, simple sequence length polymorphism.
Introduction
Chlorophyll is the main component of the photosynthetic
pigments and it has the function of absorbing light energy and
transferring that energy to the reaction centers to produce
chemical energy. In all land plants, green algae and prochlorophytes, the photosynthetic reaction centers contain only chloro3
Corresponding author: E-mail, [email protected]; Fax, +81-268-21-5810.
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468
Characterization of the pcb2 mutant
Fig. 1 Photograph of the whole plant. (A) Wild-type, (B) pcb2
mutant and (C) pcb2 mutant bearing the PCB2 genomic copy under the
control of the 35S promoter in the sense direction. (D) Wild-type plant
bearing the PCB2 genomic copy under the control of the 35S promoter in the antisense direction. All plants are 14 d old. Bar = 0.5 cm
Results
Phenotype of the pcb2 mutant
The pcb2 mutant has a pale-green phenotype (Fig. 1). In
the F2 populations of a backcross with wild-type Columbia
(Col), normal and mutant plants segregated in a ratio very close
to 3 : 1 (85 : 32; χ2 = 0.34; P = 0.56). This result indicates that
the pcb2 mutation was inherited in a single recessive manner.
The growth rate of the pcb2 mutant was severely reduced compared with the wild-type.
The thylakoid membranes of the pcb2 chloroplasts were
arranged in a disorderly fashion and did not develop distinct
grana stacks; extensive adhesion of two to a few thylakoid
membranes was observed instead (Fig. 2). There were no distinct differences in the size and the number of chloroplasts
between the wild-type and the mutant. Starch granules were not
found in the mutant chloroplasts, suggesting the reduction of
photosynthetic activity in the mutant.
Analysis of chlorophylls
The chlorophylls in leaf extracts were separated chromatographically and the peaks that were assumed to be chlorophylls
a and b from their retention time and absorption spectrum were
compared. The comparative amounts of chlorophylls between
wild-type and pcb2 were calculated from the peak areas at
660 nm. The amounts of chlorophylls a and b extracted from
the pcb2 plants were approximately one-half and one-quarter of
that from the wild-type, respectively (Fig. 3A, B).
Fig. 2 Electron microscopic analysis of the pcb2 mutant. Electron
micrographs of chloroplasts in mesophyll cells from wild-type (A) and
pcb2 (B). Portions of (A) and (B) are shown in (C) and (D), respectively, with higher magnification to reveal the detailed structure of
thylakoid membranes. C, chloroplast; S, starch; V, vacuole; IS, intercellular space; M, mitochondria.
The peaks corresponding to chlorophylls a and b of the
pcb2 mutant were eluted a little earlier than those of the wild
type (Fig. 3C). Peaks 1′ and 2′ in Fig. 3B are recovered, and
their spectra in diethyl ether are compared with those of the
chlorophyll a and b standards. The result showed that the peaks
shifted from 430 to 435 nm (chlorophyll vs. peak 2′, Fig. 3D)
and from 455 to 462 nm (chlorophyll b vs. peak 1′, Fig. 3E).
The profiles of these spectra are consistent with those of divinyl chlorophylls (Shedbalkar and Rebeiz 1992).
The mass spectra obtained for chlorophyll a and b standards showed major ions at m/z 912 [M + NH4]+ and m/z 926 [M
+ NH4]+, whereas the corresponding peaks (peaks 1′ and 2′,
Fig. 3) from the pcb2 mutant showed major ions at m/z 910 [M
+ NH4]+ and m/z 924 [M + NH4]+, respectively. The mass of
2 Da shifted from the standards is equivalent to the mass
changing in the reduction of a –CH=CH– double bond to a
–CH2–CH2– single bond.
Candidate for the pcb2 gene
In a previous study, we mapped pcb2 to a locus at the
upper arm of chromosome 5 (Nakanishi et al. 2004). Further
analysis using 442 F2 seedlings revealed that PCB2 is located
Characterization of the pcb2 mutant
469
Fig. 3 HPLC analysis of chlorophylls. The
elution profiles of wild-type (A), pcb2 (B) and
a mixture of wild-type (1/4) and pcb2 (3/4)
(C). Peak 1, chlorophyll b; peak 2, chlorophyll a; peak 1′, chlorophyll b-like pigment;
peak 2′, chlorophyll a-like pigment. The
absorption spectra of the peaks 2 and 2′ (D),
and peaks 1 and 1′ (E) in diethyl ether are
compared (continuous line, peaks 1 and 2;
dashed line, peaks 1′ and 2′).
between PAT1.2 [simple sequence length polymorphism
(SSLP) marker, AGI position; 5,397 kb] and dPCB2 [derived
cleaved amplified polymorphic sequence (dCAPS) marker,
AGI position; 6,335 kb]. Three recombinations were observed
in 884 chromosomes between PAT1.2 and the pcb2 locus, one
in 884 chromosomes between the pcb2 locus and dPCB2.
Calculating the recombination ratio, the presumed area was
narrowed to a range of about 190 kb where 49 genes are predicted (Fig. 4A, B). According to TargetP (Emanuelsson et al.
2000) and the PSORT program (Nakai and Horton 1999), products of four genes are suggested to localize in the chloroplast.
By means of DNA sequence analysis of these four genes, one
of them, At5g18660, had a single nucleotide substitution at the
85th residue (cytosine to thymine) resulting in a stop codon in
the pcb2 mutant, instead of glutamine in the wild-type (Fig. 4C).
Gene cloning and complementation analysis
In order to confirm that At5g18660 is the gene causing the
pcb2 mutation, we carried out complementation and antisense
analysis. A genomic copy of At5g18660 under the control of
the cauliflower mosaic virus (CaMV) 35S promoter was introduced into the pcb2 mutant (sense) and wild-type (antisense) by
the floral dipping method (Fig. 5A). The resultant transgenic
plants bearing the 35S-At5g18660 sense gene had green leaves
(Fig. 1C) and normal chlorophylls (Fig. 5B). On the other
hand, the transgenic plants suppressed with antisense DNA
turned out to show the same phenotype as the pcb2 mutant
(Fig. 1D, 5B). The reverse transcription–polymerase chain
reaction (RT–PCR) analysis of At5g18660 mRNA in leaves
showed that PCB2 was expressed in the wild-type and the pcb2
complemented plants, but not in the pcb2 mutant, nor in antisense suppressed plants (Fig. 5C, D). Thus, a mutation in
At5g18660 seems to be responsible for the pcb2 phenotype.
Expression pattern and localization of PCB2
We examined the tissue-specific expression pattern of
PCB2 in various organs by RT–PCR. PCB2 was expressed
highly in leaves, stems and flower buds, and slightly in roots
(Fig. 6A). To determine the subcellular localization of the
PCB2 protein, the sGFP gene was fused to the 3′ end of the
PCB2 gene, generating the PCB2-sGFP chimeric gene under
the control of the CaMV 35S promoter (Fig. 6B). Confocal
laser scanning microscopic observation revealed that the
PCB2–sGFP protein localized in chloroplasts in mesophyll
cells (Fig. 6C).
Fig. 4 Chromosome mapping of the PCB2 gene. (A) A partial
genetic map of the A. thaliana chromosome 5 between nga106 and
ciw8 with a summary of genetic recombinants. (B) Enlarged map of
the pcb2 locus. Predicted genes are shown by arrowheads. Closed
arrowheads indicate genes whose products are predicted to target chloroplasts. (C) Schematic drawing of At5g18660. The positions of the
initiation codon, mutation in pcb2, termination codon and primers used
in this study are indicated.
470
Characterization of the pcb2 mutant
Fig. 5 Complementation and antisense analysis of PCB2. (A) Structure of sense (upper) and antisense (lower) constructs. (B) HPLC
chromatograms of chlorophylls from wild-type, complemented pcb2,
pcb2 and antisense plants. (C) RT–PCR products using total RNA
isolated from wild-type (lane 1), pcb2 mutant (lane 2) and pcb2 complemented (lane 3). PCR products using genomic DNA isolated from
wild-type as a control (lane 4). PCB2-specific primers At5g18660-F2
and At5g18660-R2 were used (Fig. 4C). The actin2 gene was used as a
standard. (D) RT–PCR products using total RNA isolated from wildtype (lane 1), pcb2 mutant (lane 2) and antisense plants with pcb2
phenotype (lane 3–5). Primers At5g18660-F3 and At5g18660-R2
(Fig. 4C), corresponding to positions outside of the introduced antisense At5g18660 fragment, were used. The actin2 gene was used as a
standard.
Comparison with PCB2 homologs
The PCB2 gene encodes an uncharacterized protein with a
transit peptide to chloroplast (Fig. 7). A BLAST search
revealed that there are no other genes significantly similar to
PCB2 in the genome of A. thaliana. Referring to the DDBJ
database, expressed sequence tags (ESTs) that encode proteins
highly homologous (>55% identities) to PCB2 were found in
various species of dicots (Solanum tuberosum, Medicago
truncatula, Lycopersicon esculentum, etc.), monocots (Oryza
sativa, Triticum turgidum, etc.), a moss (Physcomitrella
patens), a cyanobacterium (Synechococcus sp. WH8102) and
Fig. 6 Expression pattern and localization of PCB2. (A) Tissuespecific expression pattern of PCB2 in various organs revealed by RT–
PCR. RT–PCR products using total RNA isolated from leaf (lane 1),
inflorescence stem (lane 2), root (lane 3) and flower bud (lane 4) of
wild-type. PCR products using genomic DNA isolated from wild-type
as a control (lane 5). PCB2-specific primers At5g18660-F2 and
At5g18660-R2 are used. (B) Construction of a sGFP fusion to the Cterminus of PCB2. (C) Fluorescent microscopic images of sGFP
(green) and autofluorescence image of chlorophyll (red) in mesophyll
cells. The non-fused sGFP transformant nA5-4 was analyzed as a control. Bar = 10 µm.
a photosynthetic bacterium (Chlorobium tepidum TLS), but not
in yeast and animals. None of these homologous proteins have
been characterized yet. The search also yielded the noteworthy
information that PCB2 has similarity to some reductases such
as NADH dehydrogenase/oxidoreductase and isoflavone
reductase (27 and 26% identities, respectively). Another
feature of the PCB2 protein is the presence of a stretch of
hydrophobic amino acid residues that constitute a putative
transmembrane domain near its C-terminus (Fig. 7, asterisks).
Discussion
In this report, we demonstrated the replacement of chlorophylls a and b by other pigments in the pcb2 mutant (Fig. 3),
together with a severe reduction of grana stacks in chloroplasts
(Fig. 2). We found a single nucleotide substitution in
At5g18660 in the pcb2 mutant (Fig. 4), which is probably
responsible for the pcb2 phenotype. This hypothesis is strongly
supported by transformation experiments using sense and antisense constructs of At5g18660 (Fig. 1, 5). At5g18660 encodes
Characterization of the pcb2 mutant
471
Fig. 7 Comparison of PCB2 homologs. Alignment of the deduced amino acid sequence of
the PCB2 protein and its homologs in Oryza
sativa (AC135257), Chlorobium tepidum TLS
(AE012869–9) and Synechococcus sp. WH 8102
(BX569691–252). Black boxes indicate residues with a degree of conservation of three-quarters. An arrowhead indicates the position of the
mutation found in the pcb2 mutant. Underlines
indicate predicted signal peptides (see text). Asterisks indicate the putative transmembrane domain (predicted by the SOSUI program,
Hirokawa et al. 1998)
a chloroplast-localized protein that has low homology to a certain category of reductases, and that seems to be conserved in a
broad range of species of photosynthetic organisms.
Chlorophyll a is synthesized in 15 steps from L-glutamate.
The genes coding for the proteins which catalyze 14 of these
steps have been cloned in Arabidopsis (Lange and Ghassemian
2003, Tottey et al. 2003). Divinyl protochlorophyllide 8-vinyl
reductase, which catalyzes the conversion of divinyl protochlorophyllide a to monovinyl protochlorophyllide a (Parham and
Rebeiz 1992, Parham and Rebeiz 1995), is the last one to be
identified. Electrospray ionization (ESI) mass spectrometry
analysis of the pcb2 mutant detected the chlorophyll-like substances with masses of 892 and 906 Da, corresponding to divinyl chlorophylls a and b respectively. The absorption spectra in
diethyl ether and the elution time of HPLC also indicated that
the pcb2 mutant accumulates divinyl chlorophylls instead of
monovinyl (normal) chlorophylls (Fig. 3, Shedbalkar and
Rebeiz 1992, Zapata et al. 2000). Considering this together
with the similarity of the PCB2 protein to some reductases,
divinyl protochlorophyllide 8-vinyl reductase is a potent candidate to be the PCB2 product. This is supported by the absence
of a homologous gene in the completely sequenced genome of
Prochlorococcus, a photosynthetic prokaryote using divinyl
chlorophylls as photosynthetic pigments (Chisholm et al.
1992). Enzymatic study should be able to confirm this aspect.
We isolated the pcb2 mutant as a chlorophyll b reduced
mutant at first. The result of HPLC analysis in this study
showed that divinyl chlorophyll b was accumulated at a very
low level compared with divinyl chlorophyll a. The answer to
the question as to why the disruption of divinyl protochlorophyllide 8-vinyl reductase caused a reduction of divinyl chlorophyll b may lie in their chemical structure. In divinyl
chlorophyll a, an ethyl group is replaced by a vinyl group at the
C8 site of chlorophyll a. CAO catalyzes a reaction that converts a methyl group to a formyl group at the C7 site of chlorophyll a. It is thought that the replacement of an ethyl group
with a vinyl group at the C8 site of divinyl chlorophyll a probably interferes with the CAO reaction. Prochlorococcus which
accumulates the divinyl chlorophyll b carries no CAO homologous gene in the genome (CyanoBase; http://www.kazusa.or.jp/
cyano/, Nakamura et al. 2000), suggesting that another
enzyme(s) which carries out the reaction of divinyl chlorophyll a to divinyl chlorophyll b may exist in the bacteria. The
amount of divinyl chlorophyll a is also reduced to almost half
of that of monovinyl chlorophyll a in wild-type. This fact may
support the idea that the divinyl chlorophyllide a is not exactly
suited as a substrate to fill the enzymatic pathway for chlorophyll a synthesis in Arabidopsis. We cannot deny another possibility that the trouble in assembly or stability of divinyl
chlorophylls in the thylakoid membrane are brought in the
pcb2 mutant.
A nonsense mutation close to the N-terminus of PCB2
provided a null phenotype, the pcb2 mutant (Fig. 4). It is
unlikely that other proteins compensate the lack of PCB2,
because there are no homologs of PCB2 in the genome of A.
thaliana and because the monovinyl chlorophylls were
replaced by divinyl chlorophylls in the pcb2 mutant. Why
could the pcb2 mutant survive? The answer may be related to
the accumulation of divinyl chlorophylls. The pcb2 mutant
may survive by using divinyl chlorophylls as photosynthetic
pigments as Prochlorococcus does. Additionally, it was
reported that a Zea mays mutant accumulating only divinyl
chlorophyll instead of monovinyl chlorophyll was also capable
of photosynthetic growth with divinyl chlorophylls (Bazzaz
and Brereton 1982).
The pcb2-related genes are found in higher plants, Chlorobium tepidum TLS and Synechococcus sp. WH8102, but not in
other sequenced genomes of photosynthetic prokaryotes. In
Rhodobacter capsulate and C. tepidum TLS, bchJ was identified as divinyl protochlorophyllide 8-vinyl reductase by genetic
methods (Suzuki and Bauer 1995), but the bchJ homologous
gene was not found in higher plants. As there is no homology
472
Characterization of the pcb2 mutant
between pcb2 and bchJ, they seem to be distantly related to
each other or evolutionarily coming from different origins.
There are some other organisms that utilize monovinyl chlorophylls but have neither pcb2 nor bchJ, suggesting the existence of a third gene(s) that encodes enzyme(s) acting as divinyl
protochlorophyllide 8-vinyl reductase. Further analysis of
PCB2 and mono/di-vinyl chlorophylls from the standpoint of
evolution of photosynthetic efficiency may be worthwhile.
Electron microscopic observation of the chloroplast in the
pcb2 mutant showed a remarkable decrease in the number of
grana stacks. There would seem to be three possibilities. First,
the thylakoid membrane may have become immature because
of poor accumulation of chlorophylls, since the total amount of
chlorophyll in the pcb2 mutant is only 40% of that of the wild
type. The second possibility is that accumulations of divinyl
chlorophylls disturb the formation of grana stacks, and that perhaps divinyl chlorophylls are not available for the composition
of grana stacks. The third possibility focuses on the (divinyl)
chlorophyll a/chlorophyll b ratio. It is possible that the balance
of (divinyl) chlorophyll a vs. b contents is important for the
composition of the grana stack as discussed in Murray and
Kohorn (1991). Further research is required to understand the
morphogenesis of thylakoid membranes including grana stacks,
and the pcb2 mutant analyzed here should provide good material for this research.
Materials and Methods
Plant materials and growth condition
Arabidopsis thaliana ecotype Columbia (Col) and Landsberg
erecta (Ler) were used. The pcb2 mutant was isolated from the EMS
mutant library of Col as a pale-green mutant (Nakanishi et al. 2004).
Seeds were sterilized in 0.2% sodium hypochlorite with 0.05% Tween20, then kept at 4°C in the dark for 2 days. The seeds were germinated
on a 1% sucrose agar plate containing 1/1,000 volume of liquid fertilizer (Hanakoujo; Sumika-Takeda-Engei, Tokyo, Japan) at 40%
humidity and in a 14 h light/10 h dark cycle at 22°C. Seedlings 2 to 4
weeks old were transferred to rock wool (NICHIAS, Tokyo, Japan)
and grown under the same conditions.
Mapping and DNA sequencing of the pcb2 gene
To fine map PCB2, an F2 population was generated from a cross
between pcb2 and Ler wild-type. Markers used for PCR-based mapping were nga106 (TAIR Accession No. Genetic Marker: 1945512),
ciw8 (TAIR Accession No. Genetic Marker: 2005439) and PAT1.2
(TAIR Accession No. Genetic Marker: 1945631). These are SSLP
markers (Bell and Ecker 1994). dPCB2B was designed to distinguish
Ler from Col by the unique restriction site of BamHI as a dCAPS
marker (Neff et al. 1998). Primers used for creating dPCB2B on PCR
were dPCB2B-F (5′-agttcttgtctcttgatgcctggat-3′) and dPCB2B-R (5′aagttcgttaaagcagcagcaccaa-3′).
The PCR-amplified fragments of the four genes which had a
transit peptide into the chloroplast from the pcb2 genome were
sequenced directly with a DSQ-2000L (Shimadzu, Kyoto, Japan) and
an ABI PRISM 310 (Applied Biosystems, CA, U.S.A.). Primers used
for PCR were At5g18570-F1 (5′-tttatcagagaaacaaatggcttcc-3′),
At5g18570-R1 (5′-cggttcaatccaatgaccga-3′), At5g18660-F1 (5′-atgtcactttgctcttccttcaacg-3′), At5g18660-R1 (5′-ctacacagcaaactcatttcgc-3′),
At5g18820-F1 (5′-ccggttccaaaaactcggtc-3′), At5g18820-R1 (5′-cac-
cactttgtgagcaaagg-3′), At5g18910-F1 (5′-cttctctctttctctctctttccc-3′) and
At5g18910-R1 (5′-tcatctccaatcctcagcag-3′).
Construction of expression vectors
Full-length PCB2 genomic DNA was amplified by PCR with a
set of primers, At5g18660-F1 and At5g18660-R1. The fragment was
cloned into a T-vector pXcmKn12 by TA-cloning (Cha et al. 1993) to
create the plasmids having the insert in both orientations. Two kinds of
HincII–SacI fragments containing PCB2 were released from each plasmid and subcloned into the SmaI–SacI site of the pSMAK251 vector
generating pKS-PCB2 and pKA-PCB2. pKS-PCB2 encodes sense
mRNA under the control of the CaMV 35S promoter and pKA-PCB2
encodes antisense mRNA (Fig. 5A). The vector pSMAK251 was a gift
of Dr. H. Ichikawa, National Institute of Agrobiological Sciences,
Tsukuba, Japan.
To create pGWB5-PCB2 (Fig. 6B), we used GATEWAY™ Technology (Invitrogen, CA, U.S.A.) according to the manufacturer’s
instruction. The entire coding region of PCB2 was amplified by PCR
using attB1-At5g18660-Fw (5′-aaaaagcaggctggatgtcactttgctcttccttc-3′)
and attB2-At5g18660-Rv (5′-agaaagctgggtagaagaactgttcaccgagtt-3′) as
primers. The genomic DNA of wild-type (Col) was used as a template. The fragment was amplified again with ‘adapter primers’ and
then introduced into pDONR201 (Invitrogen) using ‘BP recombination
reaction’ to generate the ‘entry clone’ of At5g18660. The binary vector was constructed using an ‘LR recombination reaction’ between the
entry clone and the destination vector pGWB5. The vector pGWB5
was a gift of Dr. T. Nakagawa, Shimane University.
Suppression and expression of PCB2 in transgenic plants
All plasmids were transformed into the Agrobacterium tumefaciens C58 strain by electroporation (GENE PULSER II; Bio-Rad,
Tokyo, Japan). The pKA-PCB2 and pGWB5-PCB2 plasmids were
introduced into wild-type (Col) by floral dipping as described by
Clough and Bent (1998). The pKS-PCB2 plasmid was introduced into
PCB2/pcb2 heterozygous plants. Seeds from transformed plants were
selected on 1% sucrose agar plates containing 1/1,000 liquid fertilizer
and 20 µg ml–1 kanamycin. To select the pcb2/pcb2 homozygous
plants from the (T1) plants, the PCR-amplified genomic DNAs were
sequenced directly. Primers used for PCR and sequencing were
At5g18660-F3 (5′-ggatcttcactttctcgcac-3′) and At5g18660-R2 (5′-taatgccttgcctggtccac-3′), corresponding to positions outside of the introduced sense At5g18660 fragment (Fig. 4C). T1 and T2 progeny were
used for phenotype analysis.
RT–PCR analysis
Total RNA was extracted from leaves using a PURESCRIPT Cell
and Tissue RNA Isolation Kit (Gentra System, MN, U.S.A.). After
DNase I treatment, complementary DNA was synthesized from 500 ng
of total RNA using an oligo(dT) primer with ReverTraAce (TOYOBO,
Osaka, Japan). The cDNA synthesized from 10 ng of total RNA was
used for PCRs with Ex-Taq polymerase (TaKaRa, Kyoto, Japan).
At5g18660-F2 (5′-gagaagagtgggattagagg-3′), At5g18660-F3 and
At5g18660-R2 were used as PCB2-specific primers; actin2-F (5′ggaaggatctgtacggtaac-3′) and actin2-R (5′-tgtgaacgattcctggacct-3′)
were used as a control for constant expression (Andersson et al. 2003).
The temperature cycles were: 94°C for 4 min; 25 cycles of 94°C for
30 s, 55°C for 30 s, 72°C for 1 min; and finally 72°C for 7 min.
HPLC analysis
Chlorophylls were extracted by soaking approximately 5 mg of
fresh 16-day-old whole plants in 100% acetone at 4°C in complete
darkness. The extract was centrifuged at 15,000 rpm for 15 min, and
the supernatants were separated on an octadecylsilyl (ODS) column
[4.6 mm i.d.×150 mm: LUNA 5u C18 (2), Phenomenex, Torrance,
Characterization of the pcb2 mutant
CA, U.S.A.] with solvent (methanol : acetonitrile : acetone = 1 : 3 : 1)
at a flow-rate of 1.0 ml min–1 at 40°C (Zapata et al. 2000) using an
LC10Avp system (Shimadzu). Elution profiles were monitored by
absorbance at 660 nm using a diode array detector (SPD-M10Avp,
Shimadzu). The spectrum graph of eluted peaks was developed from
records in the detector. Two fractions corresponding to divinyl chlorophylls a and b were separated, then the eluate was blown dry using
nitrogen gas and dissolved in diethyl ether. Their absorption spectra
were measured using a spectrophotometer (DU7400, BECKMAN)
between 350 and 700 nm.
LC/ESI-MS analysis
Liquid chromatography (LC)-mass spectometry (MS) was carried out using an LCMS-2010A system (Shimadzu) equipped with an
STR ODS II column (15 cm length×2.0 mm i.d., Shinwa Chemical
Industries, Ltd) and an ESI source. Materials extracted by acetone
from pcb2 leaves and the standard chlorophylls a and b (from spinach,
Sigma, St. Louis, U.S.A.) were separated with methanol at a flow rate
of 0.2 ml min–1 at 40°C. The mass spectra between 200 and 1,000 m/z
were obtained with positive ion mode.
Electron microscopy
Leaf segments were fixed with 2% glutaraldehyde in 50 mM
potassium phosphate (pH 7.0) for 2 h at room temperature, and washed
with the same buffer for 10 min at room temperature. The washing
step was repeated six times. They were post-fixed with 2% osmium
tetroxide in 50 mM potassium phosphate (pH 7.0) for 2 h at room temperature. The fixed samples were dehydrated through a series of acetone solutions. Ultrathin sections were cut with a diamond knife on a
Sorvall MT2-B ultramicrotome and transferred onto copper grids. The
sections were stained with 2% uranyl acetate for 10 min followed by
lead citrate for 2 min at room temperature. The specimens were
observed on a Hitachi H-7500 transmission electron microscope at an
accelerating voltage of 100 kV.
Monitoring of sGFP expression by microscopy
T2 progeny of the pGWB5-PCB2 transformant were subjected to
confocal microscopic analysis (Radiance 2000, Bio-Rad). The nonfused sGFP transformant nA5-4 was used as a control (Niwa et al.
1999). Green fluorescence and red autofluorescence from chlorophyll
were monitored by Kr/Ar laser excitation as described (Niwa et al.
1999).
Acknowledgments
We thank Dr. H. Ichikawa (National Institute of Agrobiological
Sciences, Tsukuba, Japan) for the kind gift of pSMAK251; Dr. T. Nakagawa (Shimane University) for the kind gift of pGWB5; Dr. Y. Niwa
(University of Shizuoka) for the kind gift of nA5-4; and Drs. M. Okazaki, M. Kojima, M. Shimosaka, M. Nozue and M. Nogawa (Shinshu
University) for their useful discussions.
References
Andersson, J., Wentworth, M., Walters, R.G., Howard, C.A., Ruban, A.V.,
Horton, P. and Jansson, S. (2003) Absence of the Lhcb1 and Lhcb2 proteins
of the light-harvesting complex of photosystem II—effects on photosynthesis,
grana stacking and fitness. Plant J. 35: 350–361.
Bazzaz, M.B. and Brereton, R.G. (1982) 4-Vinyl-4desethyl chlorophyll a: a new
naturally occurring chlorophyll. FEBS Lett. 128: 104–108.
Beale, S.I. (1999) Enzymes of chlorophyll biosynthesis. Photosynth. Res. 60:
43–73
473
Bell, C.J. and Ecker, J.R. (1994) Assignment of 30 microsatellite loci to the
linkage map of Arabidopsis. Genomics 19: 137–144.
Cha, J., Bishai, W. and Chandrasegaran, S. (1993) New vectors for direct cloning of PCR products. Gene 136: 369–370.
Chisholm, S.W., Frankel, S.L., Goericke, R., Olson, R.J., Palenik, B., Waterbury, J.B., West-Johnsrud, L. and Zettler, E.R. (1992) Prochlorococcus marinus nov. gen. nov. sp.: a marine prokaryote containing divinylchlorophyll a
and b. Arch. Microbiol. 157: 297–300.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16: 735–743.
Emanuelsson, O., Nielsen, H., Brunak, S. and Heijne, G.V. (2000) Predicting
subcellular localization of proteins based on their N-terminal amino acid
sequence. J. Mol. Biol. 300: 1005–1016.
Grossman, A.R., Bhaya, D., Apt, K.E. and Kehoe, D.M. (1995) Light-harvesting
complexes in oxygenic photosynthesis: diversity, control, and evolution.
Annu. Rev. Genet. 29: 231–288
Hirokawa, T., Boon-Chieng, S. and Mitaku, S. (1998) SOSUI: classification and
secondary structure prediction system for membrane proteins. Bioinformatics
14: 378–379.
Lange, B.M. and Ghassemian, M. (2003) Genome organization in Arabidopsis
thaliana: a survey for genes involved in isoprenoid and chlorophyll metabolism. Plant Mol. Biol. 51: 925–948.
Mochizuki, N., Brusslan, J.A., Larkin, R., Nagatani, A. and Chory, J. (2001)
Arabidopsis genomes uncoupled 5 (GUN5) mutant reveals the involvement of
Mg-chelatase H subunit in plastid-to-nucleus signal transduction. Proc. Natl
Acad. Sci. USA 98: 2053–2058.
Murray, D.L. and Kohorn, B.D. (1991) Chloroplasts of Arabidopsis thaliana
homozygous for the ch-1 locus lack chlorophyll b, lack stable LHCPII and
have stacked thylakoids. Plant Mol. Biol. 16: 71–79.
Nakai, K. and Horton, P. (1999) PSORT: a program for detecting sorting signals
in proteins and predicting their subcellular localization. Trends Biochem. Sci.
24: 34–36.
Nakamura, Y., Kaneko, T. and Tabata, S. (2000) CyanoBase, the genome database for Synechocystis sp. strain PCC6803: status for the year 2000. Nucleic
Acids Res. 28: 72.
Nakanishi, H., Suzuki, K., Jouke, T., Kodaira, R., Taguchi, G., Okazaki, M. and
Hayashida, N. (2004) Collection analysis of Arabidopsis color mutants.
Endocytobiosis Cell Res. 15: 328–338.
Neff, M.M., Neff, J.D., Chory, J. and Pepper, A.E. (1998) dCAPS, a simple
technique for the genetic analysis of single nucleotide polymorphisms: experimental applications in Arabidopsis thaliana genetics. Plant J. 14: 387–392.
Niwa, Y., Hirano, T., Yoshimoto, K., Shimizu, M. and Kobayashi, H. (1999)
Non-invasive quantitative detection and applications of non-toxic, S65T-type
green fluorescent protein in living plants. Plant J. 18: 455–463.
Oster, U., Tanaka, R., Tanaka, A. and Rudiger, W. (2000) Cloning and functional expression of the gene encoding the key enzyme for chlorophyll b biosynthesis (CAO) from Arabidopsis thaliana. Plant J. 21: 305–310.
Parham, R. and Rebeiz, C.A. (1992) Chloroplast biogenesis: [4-vinyl] chlorophyllide a reductase is a divinyl chlorophyllide a-specific, NADPH-dependent enzyme. Biochemistry 31: 8460–8464.
Parham, R. and Rebeiz, C.A. (1995) Chloroplast biogenesis 72: a [4-vinyl] chlorophyllide a reductase assay using divinyl chlorophyllide a as an exogenous
substrate. Anal. Biochem. 231: 164–169.
Shedbalkar, V.P. and Rebeiz, C.A. (1992) Chloroplast biogenesis: determination
of the molar extinction coefficients of divinyl chlorophyll a and b and their
pheophytins. Anal. Biochem. 207: 261–266.
Suzuki, J.Y. and Bauer, C.E. (1995) Altered monovinyl and divinyl protochlorophyllide pools in bchJ mutants of Rhodobacter capsulatus. J. Biol. Chem.
270: 3732–3740.
Tanaka, A., Ito, H., Tanaka, R., Tanaka, N.K., Yoshida, K. and Okada, K. (1998)
Chlorophyll a oxygenase (CAO) is involved in chlorophyll b formation from
chlorophyll a. Proc. Natl Acad. Sci. USA 95: 12719–12723.
Tottey, S., Block, M.A., Allen, M., Westergren, T., Albrieux, C., Scheller, H.V.,
Merchant, S. and Jensen, P.E. (2003) Arabidopsis CHL27, located in both
envelope and thylakoid membranes, is required for the synthesis of protochlorophyllide. Proc. Natl Acad. Sci. USA 100: 16119–16124.
Willows, R.D. (2003) Biosynthesis of chlorophylls from protoporphyrin IX.
Nat. Prod. Rep. 20: 327–341.
Zapata, M., Rodriguez, F. and Garrido J.L. (2000) Separation of chlorophylls
and carotenoids from marine phytoplankton: a new HPLC method using
reversed phase C8 column and pyridine-containing mobile phases. Mar. Ecol.
Prog. Ser. 195: 29–45.
(Received October 29, 2004; Accepted December 25, 2004)