Plant Cell Physiol. 39(6): 665-669 (1998)
JSPP © 1998
Short Communication
Light-Dependent Expression of Protochlorophyllide Oxidoreductase Gene in
the Liverwort, Marchantia paleacea var. diptera
Susumu Takio 1>2, Narumi Nakao 1 , Takanori Suzuki1, Katsuyuki Tanaka1, Isamu Yamamoto 1 and
Toshio Satoh'
1
Department of Biological Science, Faculty of Science, Hiroshima University, Kagamiyama, Higashi-Hiroshima, 739-0046 Japan
A cDNA encoding the NADPH:protochlorophyllide
oxidoreductase (EC 1.6.99.1) was isolated from suspension-cultured cells of the liverwort, Marchantia paleacea
var. diptera. In contrast to the situation in most higher
plants, the liverwort gene was expressed in a light-dependent manner.
Key words: Chlorophyll synthesis — Light regulation —
Liverwort (Marchantia) — Protochlorophyllide oxidoreductase (EC 1.6.99.1).
The reduction of protochlorophyllide to chlorophyllide proceeds in chloroplasts in two different pathways,
light-dependent and light-independent reduction. NADPH:
protochlorophyllide reductase (POR, EC 1.6.99.1), which
catalyzes the light-dependent reduction, is a nuclear-encoded single protein found in angiosperms (Schulz et al.
1989, Darrah et al. 1990, Benli et al. 1991, Spano et al.
1992a, Armstrong et al. 1995, Holtorf et al. 1995, Kuroda
et al. 1995), gymnosperms (Spano et al. 1992b, Forreiter
and Apel 1993), and green algae (Li and Timko 1996).
Three chloroplastic genes, chlL, chlN and chlB, have been
shown to be involved in the light-independent reduction, although the reaction mechanism is still unknown (reviewed
in Fujita 1996). Most green plants other than angiosperms
have chlL/N/B genes and can synthesize chlorophyll in the
dark; angiosperms are reported not to have these genes and
can not synthesize chlorophyll in the dark.
into whole plants, but the green line does not (Takio et al.
1993). Genes chlL/N/B in the yellow line were expressed in
a light-dependent manner (Suzuki et al. 1998). This is quite
different from what is seen in Chlamydomonas, in which expression of chlB is more active in the dark than in the light
(Richard et al. 1994). The question then arises whether the
gene products of por and chlL/N/B function equally well
in the light in the yellow liverwort line. To address this question, we isolated the por gene from the liverwort. To our
knowledge, there have been no reports on the isolation of
por from bryophytes or pteridophytes. Herein we report on
this isolation and its light-dependent expression in cells of
yellow and green lines.
Two M. paleacea var. diptera cell lines, referred to as
the green line (Takio et al. 1988) and the yellow line (Takio
et al. 1993), were grown in N-MS medium (Takio et al.
1988) at 25±1°C with shaking at HOrpm under continuous illumination (7.7 Wm~ 2 ) or in the dark. A cDNA
library was constructed from total RNA from dark-grown
cells of the green line as described elsewhere (Tanaka et
al. 1998). Approximately 50,000 plaques of the liverwort
library in AgtlO were screened with digoxigenin labeled cucumber POR cDNA (Kuroda et al. 1995) as a probe, and
three clones were isolated. These cDNAs were identical in
size and restriction map. We analyzed one of them, which
we referred to as MP1. Both strands of DNA of the clone
were sequenced with a BcaBest Dideoxy Sequencing Kit
(TAKARA Shuzo) and with a DNA autosequencer (model
373A; Applied Biosystems).
For Southern blotting, total DNA was isolated from
light-grown cells of the yellow line as described previously
(Suzuki et al. 1998). The DNA (1 /jg) was digested with various restriction enzymes (10 units), and the resulting fragments were separated electrophoretically and transferred
onto Hybond™-N membranes (Amersham). Hybridization
with the digoxigenin labeled cDNA insert of the clone MP1
was performed as described previously (Suzuki et al. 1998).
For Northern blotting, total RNA was isolated from
both dark-grown and light-grown cells of the yellow and
green lines, as described previously (Suzuki et al. 1998).
Denatured total RNA (15/ig) was fractionated by electrophoresis and blotted onto a nylon membrane (Hybond™N + ; Amersham). Hybridization was performed with the
We previously reported on chlorophyll synthesis in the
dark in suspension-cultured cells of two cell lines from the
liverwort, Marchantia paleacea var. diptera; the green line
synthesized chlorophyll in the dark at the same level as in
the light (Takio et al. 1988); the yellow line synthesized it at
much reduced level in the dark (Takio et al. 1993). The
yellow line of the liverwort has the ability to redifferentiate
The nucleotide sequence reported in this paper has been submited to the DDBJ, EMBL, and GenBank under accession number AB007321.
2
Present address: Department of Biological Science, Faculty of
Science, Kumamoto University, Kurokami, Kumamoto, 860-8555
Japan.
665
666
Protochlorophyllide reductase gene in Marchantia
same probe used for Southern hybridization. The filters
were washed twice for 15 min in 2 x SSC, 0.1% (w/v) SDS
at room temperature, twice for 15 min in 0.1 xSSC, 0.1%
SDS at 50°C for Northern hybridization or at 55°C
for Southern hybridization. The hybridized product was detected by a nonradioactive digoxigenin luminescent detection system (Boehringer Mannheim). DNA fragments to
be used as hybridization probes were excised from the
plasmids containing cucumberpor (Kuroda et al. 1995) and
the liverwort por (this study) and 25S rDNA (DDBJ accession number D89524), gel-purified, and digoxigenin-labeled by the random primer method (Boehringer Mannheim). The probes used were as follows: for cucumber
por, 1.4 kbp Notl fragment; for liverwort por, 1.7 kbp
Notl fragment; for liverwort 25S rDNA, 275 bp EcoRl
fragment.
TCAATTTCCGAGGTATGCCCAAACGTTCAAATGGTTCTCTCGTAGTCAGGTGCGCTGTCAGCGTTGTTCGI1111CAAAAGAGAACGTTT
M P K R S N G S L V V R C A V S V V R F S K E N V S
90
CnGTGATTTGGCTTCTGAAMTTTTACATTCTCTCGTGACTCTTTTCCAGTGGTTTCAACCGTCTTGAGAGTGTCTGAAGCCGTCTATC
C O L A S E N F T F S R D S F P V V S T V L R V S E A V Y R
180
GAATGGCTGCTGTCGCTAGTCTCGGTTCGGCTTTGTCCGTGAGTTCGGCTGCCCTGAGCCAGAATGTGAGTGTCTCCAACAATGCTACCA
M A A V - A S L G S A L S V S S A A L S Q N V S V S N N A T K
270
AGGAATCTGCCTTCCrCGGTCTGCGCATGGGAGAGGTTGCCAAATTCGGCGGAGCGCTTCTCTCTGTGTCGACGGTTGCTGCGAACCTTA
E S A F L G L R M G E V A K F G G A L L S V S T V A A N L K
360
AGTCGAAGCCCGGTGTCTTATCAGTGAATGCCGTGACTGCTCCAGCAGAGACCATGAACAAGCCCTCGTCCAAGAAGACTGCCACCAAGA
S K P G V L S V N A V T A P A E T M N K P S S K K T A T K S
450
GTACCTGCATTATCACCGGTGCCTCATCTGGACTCGGTCTGGCAACCGCGAAGGCCCTTGCTGACACCGGCGAATGGCATGTCATCATGG
T C I I T G A S S G L G L A T A K A L A D T G E W H V I M A
540
CCTGCCGTGACTTTTTAAAGGCAGAAAGAGCTGCCAGGTCTGTTGGAATTCCCAAGGATAGTTATACCGTGATTCACTGTGATCTCGCGT
C R D F L K A E R A A R S V G I P K O S Y T V I H C D L A S
630
CATTCGATAGTGTTCGTGCATTCGTAGATAACTTTAGAAGAACCGAGAGACAGCTGGACGTGTTGGTGTGCAATGCCGCTGTCTACTTCC
F D S V R A F V D N F R R T E R Q L D V L V C N A A V Y F P
720
CTACTGACAAGGAACCCAAATTCTCAGCTGAGGGTTTCGAGaGAGTGTGGGAACAAACCACATGGGTCACTTCCTCTTGGaCGTTTGC 810
T D K E P K F S A E G F E L S V G T N H M G H F L L A R L L
TGATGGAAGACCTCCAAAAGGCTAAAGACAGCTTGAAGAGAATGATCATTGTGGGATCTATCACTGGTAATTCTAACACCGTTGCCGGTA
M E D L Q K A K D S L K R M I I V G S I T G N S N T V A G N
900
ACGTACCCCCGAAGGCCAATCTGGGCCACTTAAGAGGTCTTGCCGGAGGGTTGAACGGAGTAAACTCTTCTTCCATGATCGATGGAGGTG
V P P K A N L G H L R G L A G G L N G V N S S S M I D G G E
990
AATTTGACGGTGCGAAGGCTTACAAAGACAGCAAGGTCTGCAACATGTTCACAATGCAAGAGTTTCACAGGCGTTATCACGCAGAGACAG 1080
F D G A K A Y K D S K V C N M F T M Q E F H R R Y H A E T G
GMTTACCTTCTCTTCTTTGTATCCCGGATGTATCGCAGAGACTGGCCTCTTCCGAAACCACGTCACTCTCTTCCGAACGCTGTTTCCCC 1170
I T F S S L Y P G C I A E T G L F R N H V T L F R T L F P P
CGTTTCAGAAGTACATTACCAAGGGATACGTCTCAGAGGAAGAAGCCGGCAAGCGCATGGCCCAGGTCGTTAGTGATCCCAAGTTGAGCA 1260
F Q K Y I T K G Y V S E E E A G K R M A Q V V S D P K L S K
AGTaGGGGTGTACTGGAGCTGGAACAAAGACTaGGaCCTTCGAAAACGAGTTGTCTGAAGAAGCTAGCAACCCCGAGAAGGCGAAGA 1350
S G V Y W S W N K D S G S F E N E L S E E A S N P E K A K R
GACTCTGGGAGCTGAGCGAGAGACTTTCCGGACTCGTTTAGMTGCTGTaGTAACMTTTTGCAGTGTTTGATCCAAGCAAGCTCGCAC 1440
L W E L S E R L S G L V *
TTTAGACGCAGGATATGCGAGaATaaCAGAAGTTCTTCCGACCATATTCCTAGCTTaATATGGGCTATGGATATGACTAGGTGACC 1530
GGAAGATGGTATGAATGGTATTTTCAnCATCTACGAaTCTTGAAGGGGGTTTAGAGAAGGACGTTGTAATACTACCAGGTTTGTCAGT 1620
Fig. 1
ATTAGTCTnGMTCTAQVAACMTnunACTAWGTTATCTGATCCGAACCCTCAAGAGGATGAAAGCCACGAGCTTATGTATATAT
1710
ATTGCATTGTAATGTAAAAGGTTTGGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
1774
Nucleotide and deduced amino acid sequences of the liverwort POR cDNA.
Protochlorophyllide reductase gene in Marchantia
Figure 1 shows the nucleotide and deduced amino acid
sequences of the cDNA for liverwort POR. The cDNA had
1,774 bps containing a 5'-untranslated region of 14 bps, an
open reading frame of 1,377 bps, and a 3'-untranslated
region of 383 bps. The deduced amino acid sequence showed that the open reading frame encoded 458 amino acid residues.
In Arabidopsis (Armstrong et al. 1995) and barley
(Holtorf et al. 1995) two por genes have been reported.
Figure 2 compares the amino acid sequence of POR derived from the nucleotide sequence of the liverwort cDNA
with those of two PORs from Arabidopsis and that of the
cucumber POR (Kuroda et al. 1995). In the N-terminal
regions which include the plastid transit peptides, the sequence and length of the liverwort POR was quite different
from those of other plants. The first four amino acids of
the POR transit peptides, MALQ, are conserved in PORs
liverwort
-
667
of Arabidopsis (Armstrong et al. 1995), barley (Schulz et
al. 1989), cucumber (Kuroda et al. 1995), and pea (Spano et
al. 1992a). The N-terminal sequences of PORs in mountain
pine (Forreiter and Apel 1993) and Chlamydomonas (Li
and Timko 1996) are MGTLLQ and MALTS, respectively,
indicating that the ALQ motif is partially conserved. However, the ALQ motif was absent in the liverwort POR.
The N-terminal residues of the mature POR purified
from pea (Spano et al. 1992a) and barley (Benli et al. 1991)
were determined to be Glu65 and Gly68, respectively, indicated by the black arrowhead in Fig. 2. Therefore, amino
acid sequences around the cleavage sites of PORs from pea
and barley were also aligned. As shown in the boxes in
Fig. 2, conserved sequences including or neighbouring the
cleavage site were found, i.e., the (I/V)RA(Q/E) and KKT
motifs. In liverwort POR protein, the (I/V)RA(Q/E) motif
was absent, but the KKT motif was present. Therefore,
** « * *
MPKRSNGSLWRSAVSWRFSKENVSBDLASENFTFSRDSFPWSTVLRVSEAVYRMAAVASLGSALSVSSAALSQNVSV
Arabidopsis (porA) MAL-QAASL-VSSAFSV-R--KDGKLNA--SASSSF-KES
Arabidopsis (porB) MAL-QAASL-VSSAFSV-R--KDAKLNA--S-SSSF-KDS
cucumber
MAL-QAASL-VSPALSI-P--KEGK
S-SVHL-KDS
SLFG-ISL--SLFG-ASI
SLF-GISF---
so
SEQSKADFVSSS-LR
TDQIKSEHGSSS-LR
SDHLKSEFSSST-LR
55
54
51
SNNATKESAFLGLRMGEVAKFGGALLSVSTVAANLKSKPGVLSVNAVTAPAETMNKPSSfOCiiATKSTBlITGASSGLGLA 160
liverwort
STPSVTK
SSLDR <K1 LRKGNWVTGASSGLGLA 107
Arabidopsis CporA) ---HKREQ---SLRNN-KAI-I
---HKREQ---SLRNN-KAI-IIRAQ'AIAT
Arabidopsis (porfi) ---FKREQ---SLRN--LA--IRAQ TAAT
SSPTV
TK
SVDO <K1 LRKGNWVTGASSGLGLA 103
- - -HKRE
LNQQIGA- - IRAQ
TTAT
ES-PAV
NK
ATPDO OCILRKGSWTTGASSGLGLA 101
cucumber
pea
barley
(porA)
---BK-E
KA--KKA
LRQKVGA--VRAE ---TAAPAT---PAVSLA--VRTQ -VATAPVTT-S-PGS
NK
TA
SSSEG <K1 LRKGNWITGASSGLGLA 102 ,
SSPSGKKJLRQGVWITGASSGLGLA 89 |
liverwort
TAKALADTGEWHV:IMARI| R O F L K A E R A A R S V G I P K D S Y T V I ^ D L A S F D S V R A F V D N F R R T E R Q L D V L V S N A A V Y F P T D K E
Arabidopsis CporA') TAKALAETGKWHV:IMA I!RDFLKAERAAQSAGMPKDSYTVMHLDLASLDSVRQFVDNFRRAEMPLDVLVHNAAVYQPTANQ
Arabidposis (porB) TAKALAETGKWNVIMA!
IMA IIRDFLKAERAAKSVGMPKDSYTVMHLDLASLDSVRQFVDNFRRTETPLDVLV»IAAVYFPTAKE
cucumber
TAKALAETGKWHVIMAHII R D F L K A E R A A K S A G I T K E N Y T V M H L O L A S L D S V R Q F V D N F R Q S G R P L D V L V H N A A V Y L P T A K E
187
183
liverwort
Ababidopsis (porA)
Arabidopsis (.porB~)
cucumber
320
267
263
260
PKFSAEGFELSVGTNWGHFLLARLLMEDLQKAKDSLKRMIIVGSITGNSNTVAGNVPPKANLGHLRGLAGGLNGVNSSS
PTFTAEGFELSVGINHLGHFLLSRLLIDDLKNSDYPSKRLIIVGSITGNTNTLAGNVPPKANLGDLRGLAGGLNGLNSSA
PTYSAEGFELSVATNHLGHFLLARLLLDDLKKSDYPSKRLIIVGSITGNTNTLAGNVPPKANLGDLRGLAGGLNGLNSSA
PTFTAEGFELSVGTNHLGHFLLSRLLLEDLNKSSYPSKRLIIVGSITGNTNTLAGNVPPKANLGDLRGLAGGLNGLKSS-
240
181
• • ** ***** *
liverwort
MIOGGEFDGAKAYKDSKVINMFTMQEFHRRYHAETGITFSSLYP :IIAETGLFRNHVTLFRTLFPPFQKYITKGYVSEEE 400
Arabidopsis CporA') MIDGGDFVGAKAYKDSKV jNMLTMQEFHRRFHEDTGITFASLYP :IIATTGLFREHIPLFRTLFPPFQKYITKGYVSESE 347
Arabidopsis (.porB) MIDGGDFDGAKAYKDSKVIWLTMQEFHRRFHEETGVTFASLYP :|IASTGLFREHIPLFRALFPPFQKYITKGYVSETE 343
cucumber
MIDGGEFDGAKAYKOSKVHNMLTMQEFHKRYHEETGITFASLYPGalATTGLFREHIPLFRILFPPFQKFITQGYVSEDE 340
liverwort
AGKRMAQWSDPKLSKSGVYWSWNKDSGSFENELSEEASNPEKAKRLWELSERLSGLV
Arabidopsis (.porA~) AGKRLAQWADPSLTKSGVYWSWNKTSASFENQLSQEASDVEKARRVWEVSEKLVGLA
Arabidopsis CporB) SGKRLAQWSDPSLTKSGVYWSWNNASASFENQLSEEASDVEKARKVWEISEKLVGLA
cucumber
AGKRLAQWSEPSLTKSGVYWSWNKNSASFENQLSQEASDAEKARKVWELSEKLVGLA
458
405
401
398
Fig. 2 Comparison of the deduced amino acid sequences of POR DNA clones of liverwort (this study), Arabidopsis (Armstrong et al.
1995), and cucumber (Kuroda et al. 1995). Asterisks indicate residues conserved in the four plant POR proteins. To elucidate the cleavage site of the transit peptides, alignments of POR from pea (Spano et al. 1992a) and PORA from barley (Schulz et al. 1989) are also
shown in the box with the broken line. The black arrowheads indicate the N-terminal residues of mature POR proteins from pea and
barley. The motifs around the putative cleavage site in PORs are boxed. The white arrowhead indicates the probable site for cleavage of
transit peptide of the liverwort POR. The positions of cysteine residues are indicated by white letters on a black background.
668
(A)
Protochlorophyllide reductase gene in Marchantia
(B)
ii
B
kbp
23.4
9.4
6.64A132.0-
0.56
0.56 -
Fig. 3 Southern blot hybridization of total DNA from the liverwort cells. Total DNA from yellow line cells grown in the light was
isolated. The DNA was digested with restriction enzymes with no
restriction site, Hindlll, Kpnl, Sphl, Pstl, Sad, andflamHI, (A)
or one restriction site, Hindi, Dral, Xbal, and EcoKl, (B) in the
liverwort POR cDNA and hybridized with the liverwort POR
cDNA probe.
Serl39 may be the N-terminus of the mature protein of the
liverwort, which would result in 320 amino acids in the
mature protein. The deduced amino acid sequence of liverwort POR showed strong similarity to those of PORA
(A)
Green
kb
7.7-
Yellow
(76.9%) and PORB (79.4%) of Arabidopsis, and POR of
cucumber (78.1%), indicating that this cDNA very likely encodes liverwort POR. The four cysteine residues almost
completely conserved in the higher plant enzymes are also
present in the liverwort enzyme (Fig. 2).
To determine the copy number of the por gene in the
liverwort, Southern blot analysis was performed (Fig. 3).
When total DNA from the yellow line of the liverwort was
digested with restriction enzymes with no restriction site in
the POR cDNA, one restriction fragment was generated
(Fig. 3A). In the case of digestion with enzymes with one
restriction site in POR cDNA, two bands were detected
(Fig. 3B). These results indicated that the genome of the
liverwort contains only one por gene.
Figure 4 shows the expression of the por gene indicated by Northern blot hybridization. In both the green
and yellow lines, a transcript of 1.8 kb was detected in cells
grown in the light, but the expression of por was much reduced in the dark (Fig. 4A). These experiments were performed with cells of the yellow and green lines which had
been maintained in the dark or in the light, respectively, for
more than one year. To understand the photp-responsivity
of the yellow line, cells which had been subcultured in the
dark for more than one year were transferred to incubation
in the light, and the level of the por transcript was determined every day (Fig. 4). When transferred to the light, the
por transcript level of the yellow line increased day by day
(Fig.4B). The same result was obtained in the green line
(data not shown). These results indicated that por was ex-
(B)
L D L D
Light
Dark
Days 0
4A>
por
1.35-
0.24'
25SrRNA
m++mmm
25SrRNA
Fig. 4 Light-dependent expression of por in liverwort cells. (A) Total RNA was isolated from dark-grown (D) or light-grown (L) cells
of the green (Green) or yellow line (Yellow) and hybridized with the liverwort POR cDNA or 25S rDNA probe. (B) Cells of the yellow
line in the stationary phase, which had been subcultured in the dark for more than one year, were transferred to fresh medium and incubated in the dark. After 2 d, the cells were transferred into the light or the dark and incubated. Total RNA was extracted from the cells
grown in the light every day or in the dark for 3 d and hybridized with the liverwort POR cDNA or 25S rDNA probe.
Protochlorophyllide reductase gene in Marchantia
pressed in a light-dependent manner in both the yellow line
and the green line. Expression patterns of por genes can be
divided into three groups. In one, the mRNA for por is
abundant in the dark, and the amount rapidly decreases
upon illumination for pors such as porAs in Arabidopsis
(Armstrong et al. 1995) and barley (Holtorf et al. 1995). In
the second, the mRNA is present in the dark, and the
amount does not change upon illumination, as reported for
porBs in these plants. In the third, the amount of the
mRNA is low in the dark but increases upon illumination.
Such light-dependent expression of a por gene has been
reported only in cucumber (Kuroda et al. 1995). The expression pattern of por in the liverwort cells appears to be similar (Fig. 4).
Thus, to the question of whether the gene products of
por and chlL/N/B are all produced in the light in the
yellow line, we can answer yes, and we can predict that
their gene products are involved in chlorophyll synthesis.
With regard to the expression patterns of the nuclear gene
por (present study) and the chlorop.lastic gene chlL/N/B
(Suzuki et al. 1998), the expression of both por and chlL/
N/B is suppressed in the dark in the yellow line. However,
in the green line, por is suppressed in the dark but chlL/N/
B is not. These results indicate that por and chlL/N/B are
controlled by light via different regulatory pathways.
We thank Professor Ken-ichiro Takamiya of Tokyo Institute
of Technology for a gift of cucumber POR cDNA.
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(Received January 5, 1998; Accepted March 17, 1998)