Download Enzymatic Activities for the Synthesis of Chlorophyll in Pigment

Plant Cell Physiol. 37(4): 481-487 (1996)
JSPP © 1996
Enzymatic Activities for the Synthesis of Chlorophyll in Pigment-Deficient
Variegated Leaves of Euonymus japonicus
Tatsuru Masuda 1 , Keiji Takabe 2 , Hiroyuki Ohta 1 , Yuzo Shioi' and Ken-ichiro Takamiya'
1
2
Department of Biological Sciences, Faculty ofBioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midoriku, Yokohama, 226 Japan
Department of Wood Science and Technology, Faculty of Agriculture, Kyoto University, Kyoto, 606-01 Japan
The enzymes involved in the biosynthesis of chlorophyll (Chi) in pigment-deficient variegated leaves of Euonymus japonicus were investigated. Each variegated leaf was
composed of clearly delineated green and white sectors.
The white sectors contained almost no Chls. The rate of
synthesis of 5-aminolevulinic acid (ALA) in the white sectors in vivo was twice that in the green sectors. The level of
glutamate 1-semialdehyde aminotransferase in the white
sectors was much higher than that in the green sectors.
Plastidic tRNAGI° was also present at substantial levels in
the white sectors, indicating that the system for synthesis of
ALA was very active in the white sectors.
The activity of porphobilinogen (PBG) synthase in the
white sectors in vitro was twice that in the green sectors. In
the white sectors the rate of porphyrin synthesis from PBG
was 4- to 6-fold higher than in the green sectors. We measured Mg-chelatase activity indirectly in both sectors by
monitoring the accumulation of Mg-protoporphyrin IX in
the presence of 2,2-dipyridyl, which inhibits isocyclic ring
formation with the resultant accumulation of Mg-protoporphyrin IX. When sectors were incubated in darkness with
2,2-dipyridyl, large amounts of protoporphyrin IX accumulated in the white sectors, whereas Mg-protoporphyrin IX mainly accumulated in the green sectors. These
results suggest that the enzymes for the synthesis of porphyrin that catalyze conversion of ALA to protoporphyrin IX
were very active and that the Mg-insertion step might be
blocked in the white sectors, with the resultant failure to
synthesize Chi. The deficiency is discussed in a comparison
with that in other Chl-deficient plants.
Key words: Chlorophyll biosynthesis — Chlorophyll
deficiency — Euonymus japonicus — Mg-chelatase deficiency — Variegated leaves.
The synthesis of chlorophylls (Chls) is essential for the
biogenesis of the photosynthetic apparatus, which is the
site of all photosynthetic events. Accordingly, inhibition of
Abbreviations: ALA, 5-aminolevulinic acid; GSA, glutamate
1-semialdehyde; PBG, porphobilinogen; Rubisco, ribulose 1,5-bisphosphate carboxylase/oxygenase, TCA, trichloroacetic acid;
TLC, thin layer chromatography.
the synthesis of Chi causes pigment deficiency and alterations in the structure of the chloroplast. The inhibition can
be caused by darkness (etiolation), further differentiation
of chloroplasts to other organelles, such as chromoplasts,
and mutations in the enzymes that catalyze various steps in
the biosynthetic pathway to Chi (as in Chl-deficient plants).
Chl-deficient plants are represented by the albino, chlorina
and xantha mutants of various monocotyledons, such as
wheat, barley and rice (Falbel and Staehelin 1994, Hess et
al. 1992). The leaves and stems of these mutants contain little or no Chi.
Compared with the above-mentioned mutants, variegated plants have leaves that typically contain both green
and white (yellow) sectors. The cells in the green sectors
contain normal chloroplasts, whereas the cells in the
white (yellow) sectors have plastids that lack Chi and/or
carotenoid pigments. The phenotype of the variegated
plants is thought to be induced by hereditary alterations in
the plastids because the color and shape of the plastids are
affected in the mutated sectors of the variegated plants
(Redei 1973, Redei and Plurad 1973). In most variegated
plants, the sectors appear at an early stage of leaf development, indicating that the hereditary alterations are
dominantly expressed in certain leaves. We originally became interested in the variegated leaves of Euonymus
japonicus because the variegation appears only after a
certain stage of leaf development. The young leaves of
E. japonicus are not variegated and contain considerable
amounts of Chi. During leaf maturation, Chls in peripheral regions along the central vein disappear and clearly
delineated white sectors are formed (Fig. 1). The mature
white sectors contain no photosynthetic pigments. The normal and variegated forms of this plant should allow us to
elucidate the mechanisms responsible for the changes in the
levels of Chi, in terms of biosynthesis and degradation, during the dramatic changes in the plastids that occur during
leaf development.
We here report that the white sectors of variegated
leaves of E. japonicus contain all of the components for the
synthesis of ALA, as well as highly active enzymes for the
synthesis of protoporphyrin IX. Thus, they are clearly
different from leaves of other pigment-deficient plants that
are deficient in the ALA-synthesizing machinery (Falbel
and Staehelin 1994, Hess et al. 1992, Lotzow and Kleinig
1990). The results further suggest that, in the white sectors,
481
482
T. Masuda et al.
Normal
Variegated
E. japonicus
Fig. 1 Leaves from a normal, wild-type plant (left) and a
variegated plant (right) of E. japonicus.
Bar, 1 cm.
the activity of Mg-chelatase, which catalyzes the insertion
of Mg into protoporphyrin IX, might be blocked. We also
discuss a possible mechanism that allows high levels of activity of the enzymes involved in the early steps in the synthesis of Chi on a background of the total absence of porphyrins and Chls.
Materials and Methods
Materials—Leaves of variegated specimens of Euonymus
japonicus were collected in the Nagatsuta district of Yokohama
City after careful examination. After formation of leaf buds,
young leaves developed variegated peripheral regions along a central vein. After 3-4 weeks, mature white sectors were fully formed. Leaves were then harvested and the green and the white sectors
were obtained by careful sectioning of the leaves such that sectors
were free of contamination by one another.
Assay for the synthesis of ALA—Tissues (0.4 g) were placed
on filter paper that had been moistened with 1.6 ml of 0.1 M
levulinic acid (pH 6.0) and incubated for 6 h under illumination
by white light (43/imol m~ 2 s~'). They were homogenized in 4%
TCA and the homogenate was centrifuged at 10,000 xg for 30
min. The resulting supernatant, containing ALA, was purified on
a column of Dowex 50W-X8 (Muromac, Tokyo; Wang et al.
1984). The volume of the eluate was adjusted to 10 ml. The pH of
the eluate was adjusted to 4.6 by addition of 4.8 ml of 1 M sodium
acetate, and then 0.3 ml of acetylacetone was added. The sample
was heated at 100cC for 10 min and cooled to room temperature.
An aliquot of the sample and the same volume of modified
Ehrlich's reagent (Urata and Granick 1963) were mixed together,
and the absorbance at 553 nm was measured after 15 min.
SDS-PA GE and Western blot analysis—Total proteins were
extracted from each sector by homogenization in a buffer that contained 50 mM Tris-HCl (pH 6.8), 2% SDS, 5% (v/v) /?-mercaptoethanol and 10% (w/v) sucrose. After centrifugation, 10 fig of
total protein in the resulting supernatant were subjected to SDSPAGE as described by Laemmli (1970). Western blot analysis was
performed as described previously by Masuda et al. (1994).
Assay of PBC synthase activity—The formation of PBG by
PBG synthase was measured with ALA as the substrate by the
method of Mauzerall and Granick (1956). Tissues were homogenized in a buffer that contained 20 mM Tris-HCl (pH 8.0), 0.5
mM MgCl2 and 1 mM /?-mercaptoethanol, and the extracts were
centrifuged at 10,000 xg for 15 min at 4°C. Each resulting supernatant was diluted with the buffer to an appropriate concentration of protein. The reaction was started by addition of ALA (final, 3 mM) and it was allowed to proceed at 25°C in darkness. The
amount of PBG formed was calculated from the absorbance of
the product of the reaction with Ehrlich's reagent at 553 nm, using
a molar extinction coefficient of 6.8 xlO 4 (Urata and Granick
1963).
Quantitation of the rate of porphyrin synthesis—The rate of
porphyrin synthesis was determined by a modified version of the
procedure of Pardo et al. (1980). Tissues were homogenized in the
same buffer as that used for assays of PBG synthase, and extracts
were centrifuged at 10,000xg for 15 min at 4°C. The resulting
pellets were successively extracted with the same buffer supplemented with 0.1, 0.5 and \% Triton X-100, with centrifugation at
10,000xg for 15 min at 4°C. Most of the active enzymes were
extracted with the buffer that contained 1% Triton X-100 (see
Results). The extracts were incubated in 1 ml of a reaction mixture
that consisted of 20 mM Tris-HCl (pH 8.0), 0.5 mM MgCl2, 1 mM
/3-mercaptoethanol, and 0.2 mM PBG at 28°C in darkness. After
a 30-min incubation, 3 ml of a mixture of 2 M perchloric acid and
methanol ( 2 : 1 , v/v) were added to the reaction mixture. After
centrifugation to remove precipitated proteins, the concentrations
of coproporphyrin, uroporphyrin, and protoporphyrin IX in the
perchloric acid/methanol extract were quantitated with a fluorescence spectrophotometer (model 850; Hitachi Co., Tokyo) by the
method of Grandchamp et al. (1980). In some cases, the identity
of the porphyrins was confirmed by TLC with authentic standards
(Henderson 1989). The fluorescence assay allows determinations
of porphyrins but not of porphyrinogens. However, most porphyrinogens can be assayed after conversion to the corresponding porphyrins by incubation with an acidic solvent for several hours
under aerobic conditions (Jacobs and Jacobs 1993).
Extraction and quantitation of tetrapyrroles from 2,2'-dipyridyl-treated tissues—Pigments were extracted as described by
Rebeiz et al. (1984). In 9-cm petri dishes, tissues (0.15 g) were placed on a filter paper that had been moistened with 2 ml of distilled
water or 1 mM 2,2-dipyridyl. The petri dishes were incubated at
27°C for 18 h in darkness. After the homogenization of tissues in
15 ml of a mixture of acetone and 0.1 M NH4OH ( 9 : 1 , v/v), the
extract containing various pigments was centrifuged at 30,000 x g
for 30 min at 0°C to remove lipoproteins and cell debris. Chls
were removed from the aqueous acetone solution by three extractions with hexane. The hexane-washed acetone layer was analyzed
with the fluorescence spectrophotometer.
Other methods—RNA gel blot analysis of tRNA olu was carried out as described by Masuda et al. (1992). Protein concentrations were estimated by a modified version of Lowry's procedure
(Bensadoun and Weinstein 1976). Authentic samples of protoporphyrin IX, Mg-protoporphyrin IX, coproporphyrin III, and uroporphyrin III were purchased from Porphyrin Products (Logan,
UT).
Results
Pigment deficiency in white sectors of E. japonicus—
Figure 2 shows the absorption spectra of extracts in 80%
acetone of both green and white sectors. In the white sec-
Chi synthesis in variegated leaves
(A)
350
400
483
(B)
500
Wavelength (nm)
Fig. 2 Absorption spectra of extracts in 80% (v/v) acetone of
variegated leaves of E. japonicus.
The solid line represents the
extract from green sectors and the dashed line represents that
from white sectors.
tors, the levels of Chls were only 2% of those in green sectors (e.g., 2,350fig (gFW)" 1 in green sectors versus 50
fig in white sectors), indicating that the white sectors of
E. japonicus were almost completely lacking in photosynthetic pigments. The trace of Chls in the white sectors is
considered to be the result of residual contamination after
the disappearance of Chls during leaf development.
The synthesis of ALA— Using treatment with levulinic
acid to inhibit the activity of PBG synthase, we monitored
synthesis of ALA by measuring the amount of accumulated ALA in tissues. The ALA-synthetic activity in
the white sectors was 1.71 ±0.19 nmol (g FW)" 1 h~', being
twice that in the green sectors (0.81 ±0.15 nmol (gFW)" 1
h" 1 ) and indicating that the white sectors contained a
highly active ALA-synthetic system. To investigate the
levels of the components of this system in each sector, we
extracted the protein and the RNA from intact tissues and
analyzed them by immunoblotting and RNA gel blot analysis, respectively. The amount of total extractable protein
from white sectors (23.1 mg (gFW)" 1 ) was substantially
lower than that from green sectors (30.9 mg (g FW)" 1 ). An
equal amount (10j*g) of total protein from both sectors
was fractionated by SDS-PAGE, with subsequent immunoblot analysis of one of the nucleus-encoded enzymes of the
ALA-synthetic system, GSA aminotransferase, which catalyzes the conversion of GSA to ALA, using antibodies
raised against GSA aminotransferase from barley (Grimm
et al. 1989). Proteins of 76 and 57 kDa reproducibly crossreacted with the antibodies in the extracts from E. japonicus (Fig. 3A). The molecular masses of aminotransferases had been reported as follows: 45 kDa for the
enzyme from Chlamydomonas reinhardtii (Wang et al.
1984); 60 kDa for that from Chlorella vulgaris (Avissar and
Beale 1989); and 46 kDa for that from barley (Kannangara
GSA aminotransferase
tRNAGIU
Fig. 3 Expression of the nucleus- and chloroplast-encoded components of the ALA-synthetic system in the white sectors of
E. japonicus.
(A) Immunoblot analysis of nucleus-encoded
GSA aminotransferase from green (G) and white (W) sectors. Ten
fig of total protein from each sector was subjected to SDS-PAGE,
blotted onto a nitrocellulose membrane and probed with antibodies against barley GSA aminotransferase. Bands of proteins of
76 and 57 kDa cross-reacted reproducibly with the antibodies. The
lower band is considered to correspond to the aminotransferase
(see Results). (B) RNA gel blot analysis of plastidic tRNA alu from
green (G) and white (W) sectors. Ten ti$ of total RNA were subjected to electrophoresis on a 4% agarose gel and blotted onto a
nylon membrane. Autoradiography was carried out after hybridization with an oligonucleotide probe which was complementary to
the sequence of tRNAGlu.
and Gough 1978, Grimm et al. 1989). Genomic Southern
hybridization revealed a single structural gene for aminotransferase in Synechococcus PCC6301 and Escherichia
coli (Grimm et al. 1991). From these data, we can assume
that the smaller protein (57 kDa) represents the true GSA
aminotransferase while the larger protein (76 kDa) might
be an artifact. In any case, the results clearly showed that
the levels of GSA aminotransferase were much higher in
the white sectors than in the green sectors (Fig. 3A). RNA
gel blot analysis revealed substantial amounts of plastidic
tRNAGlu, the substrate of glutamyl-tRNA synthetase, in
the white sectors, as well as in the green ones (Fig. 3B). The
levels of expression of tRNAPhe in the white and green sectors, which were measured for a reference, were similar to
those of tRNAGlu (data not shown). These results indicated
that some of the nucleus- and plastid-derived components
of the ALA-synthetic system were present at adequate
levels and active in the plastids in the white sectors.
484
T. Masuda et al.
Synthesis of porphyrins—Since exogenously applied
ALA was not efficiently exploited as a substrate for the synthesis of porphyrins in vivo, we tried to extract an active
fraction that could synthesize porphyrins from each type of
sector. Since PBG deaminase (and subsequent enzymes in
the pathway to porphyrins) was not extractable with the
buffer used for the extraction of PBG synthase, we used a
buffer that contained detergent to obtain the enzymes required for the synthesis of porphyrin. Such activities were
successfully recovered when tissues were homogenized
in a buffer that contained 1%-Triton X-100. Using such
extracts, we monitored the formation of uroporphyrin,
coproporphyrin and protoporphyrin IX. Almost no formation of porphyrin was detected in the absence of PBG
(Table 1). However, upon the addition of 0.2 mM PBG, significant amounts of porphyrin intermediates were formed.
The most abundant intermediate was uroporphyrin. Since
the extracts had no ferrochelatase or Mg-chelatase activities, even in the case of green sectors (data not shown, see
next section), further metabolism of protoporphyrin IX
was negligible. The extracts of white sectors generated
four- to six-times more porphyrins than those of the green
sectors (Table 1). Together with the previous results, these
results indicate that the enzymes for the synthesis of protoporphyrin IX from ALA exist in very active forms in the
white sectors, in spite of the pigment deficiency.
Mg-chelatase—It has been reported that the activity of
Mg-chelatase can be measured in isolated, intact plastids
(Fuesler et al. 1984). In the case of E.japonicus, however,
isolation of intact plastids is difficult because of its hard
tissues. We failed to assay successfully the activity of Mgchelatase in extracts with high porphyrin-synthetic activities. Therefore, we monitored the activity of Mg-chelatase
indirectly by examining the effect of an inhibitor. It is well
documented that aromatic chelators of heavy metal ions,
Uro
Copro
Proto
Total
(pmol porphyrin formed (mg protein) ' h ')
Green
None
+ PBG"
<1
49.2
<1
6.7
<1
14.5
<1
70.4
White
None
+ PBG"
<1
227.0
1.2
30.4
1.0
85.1
2.2
342.5
Control
Dpy
Dpy
c
o
Proto
_
.
,
-
„
o
tr
•
-
•
-
8-—-
•
—
Mg-Proto
?>
lat ivefluo
Treatment
White sectors
Control
._ -
O
Table 1 Accumulation of intermediates in the biosynthesis of porphyrins in cell-free extracts of both green and
white sectors of leaves of E. japonicus
Green sectors
intensi
PBG synthase—When ALA was applied exogenously
to the white sectors, almost no formation of PBG or of porphyrin was observed, whereas the green sectors formed significant amounts of PBG (data not shown). Therefore, we
suspected that the blockage or the inactivation of PBG synthase was responsible for the pigment deficiency in the
white sectors. However, substantial amounts of PBG were
formed when an extract of white sectors was incubated
with 3 mM ALA as substrate. The activity of PBG synthase
in a cell-free extract of white sectors was 2.08±0.04 nmol
(mgprotein)"'h~', and it was more than twice that of
the green sectors (0.89±0.02 nmol (mg protein)" 1 h""1. We
note, however, that the PBG formed was not further metabolized in these extracts, as described below. PBG deaminase catalyzes the further metabolism of PBG, and it
is thought to be a water-soluble protein (Leeper 1991).
However, in E.japonicus this enzyme seemed to be extractable only when tissues were homogenized with a buffer
that contained a detergent, such as Triton X-100 (see next
section). Thus, the rate of the further metabolism of PBG
was negligible. These results indicated that PBG synthase
was present in the white sectors in a very active form. It
seems likely that the exogenously applied ALA was not
efficiently incorporated into plastids in the white sectors.
.
1-
--
-
a
—.
\
B-l 1 -J -
r-
-
3
_
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550 S00 650 700
j
.
—
^
_
:
•••
. :
• ••
u
| -
S00 650 700 750 550 600 650 700
SoO 650 700 750
Wavelength (nm)
Uroporphyrin (Uro), coproporphyrin (Copro) and protoporphyrin IX (Proto) were quantitated fluorimetrically after extraction with perchloric acid and methanol. The values are the means
of results from three samples. Data are typical of the results of
two independent experiments.
" 0.2 mM PBG was added to the reaction mixture as a substrate
for the synthesis of porphyrin.
Fig. 4 Fluorescence emission spectra of ammonia-acetone extracts of 2,2'-dipyridyl-treated (Dpy-treated) green and white sectors of E. japonicus.
Both sectors were treated with water (left
side in each panel) or Dpy (right), which inhibits both ferrochelatase activity and isocyclic ring formation. After incubation
of sectors in darkness, pigments were extracted with ammonia and
acetone, and their fluorescence emission spectra (excitation at 419
nm) were recorded. Mg-Proto, Mg-protoporphyrin IX and its
methyl ester; Proto, protoporphyrin IX.
Chi synthesis in variegated leaves
such as 2,2-dipyridyl, inhibit both ferrochelatase and isocyclic ring formation with the resultant accumulation of
Mg-protoporphyrin IX and its methyl ester in treated
tissues in darkness (Duggan and Gassman 1974, Kouji et
al. 1989). If Mg-chelatase in the white sectors were active,
the accumulation of Mg-protoporphyrin IX and its methyl
ester would be expected in the presence of 2,2'-dipyridyl.
Therefore, we treated tissues with 2,2'-dipyridyl and examined the accumulation of Mg-porphyrins in darkness.
Figure 4 shows the fluorescence emission spectra of ammonia-acetone extracts from both sectors, after excitation
at 419 nm. The fluorescence intensity was normalized by
reference to fresh weights. In the green sectors, a peak due
to a trace of Chi was observed at 663 nm. In 2,2'-dipyridyltreated green sectors, the main accumulated compounds
were Mg-protoporphyrin IX and its methyl ester, which
have the same fluorescence emission peak at 595 nm. Lowlevel accumulation of protoporphyrin IX, visible as a
shoulder at 633 nm, was also observed. In the case of white
sectors, only protoporphyrin IX was detected in 2,2'dipyridyl-treated tissues, indicating that Mg-chelatase was
absent or inactivated in the white sectors. The estimated
amount of accumulated protoporphyrin IX was 7.52 nmol
(g FW)" 1 , which was approximately four-fold higher than
the amount of Mg-porphyrin accumulated in the green sectors. This result is in harmony with the previous observation that the porphyrin-synthetic activity in the white sectors was much higher than that in the green sectors.
Discussion
In the present study, we analyzed the activities of
the enzymes involved in the synthesis of porphyrins in the
pigment-deficient variegated leaves of E.japonicus. Our
results revealed a new, distinct feature of the white sectors
of E.japonicus: they contain an intact ALA-synthetic system, and most of the enzymes for the biosynthesis of porphyrins (from ALA to protoporphyrin IX) are present in
very active forms in the Chl-free plastids.
In higher plants, ALA is formed via the C5 pathway,
which consists of three chloroplast tRNA0lu-dependent
steps. In pigment-deficient mutants, it is generally considered that the synthesis of ALA in plastids is easily interrupted because the ALA-synthetic system requires
both nucleus- and plastid-derived enzymes and substrates.
Kusumi et al. (1994) reported the transcriptional suppression of chloroplast-encoded genes, such as rbcL andpsbA,
in a pigment-deficient virescent mutant of rice. They
suggested that the Chi deficiency in the virescent mutant
was caused by blockage of the expression of chloroplast
tRNAGlu. Hess et al. (1992) also reported that a lack of
tRNAGIU in the plastid-ribosome-deficient albostrains mutant of barley caused the pigment deficiency. In contrast to
these mutants, the white sectors of E.japonicus contained
485
considerable amounts of tRNAGlu (Fig. 3B). Furthermore,
substantial levels of the large subunit of Rubisco, which is
encoded by the chloroplast genome, were detected in the
white sectors (Masuda et al., in preparation). It is likely,
therefore, that the systems for transcription and translation in plastids are active in the white sectors of
E. japonicus.
The levels of GSA aminotransferase, which catalyzes
the third step in the synthesis of ALA, were much higher in
the white sectors than in the green sectors (Fig. 3A). We
have also observed such increases in levels of aminotransferase in the white sectors of other variegated plants, such
as Tradescantia sp. and Schefflera sp. (Masuda et al. unpublished results). Thus the increase is not specific to
E.japonicus but could be a rather general feature of pigment-deficient variegated tissues. A similar result was reported by Hess et al. (1992), who showed that the mRNA
for GSA aminotransferase accumulated to higher levels
in a white mutant of barley than in the green wild type,
despite the absence of formation of ALA. They speculated
that ALA deficiency in plastids might induce the expression
of the gene for GSA aminotransferase. In the white sectors
of E.japonicus, however, the synthesis of ALA was not
restricted. Therefore, the induction of GSA aminotransferase in these plants seems not to be related to the ALA
deficiency.
The increases in enzymatic activities seemed not to be
limited to GSA aminotransferase because the enzymes in
the biosynthetic pathway from ALA to protoporphyrin IX
had also higher activities in the white sectors (Table 1). A
similar finding has also been made in Chl-free chromoplasts from daffodil, which contain most of the enzymes
for Chi synthesis in very active forms (Lotzow and Kleinig
1990). In contrast to the white sectors of E.japonicus,
daffodil chromoplasts have no ALA-synthetic activity, but
they do contain active enzymes on the pathway from ALA
to Mg-protoporphyrin IX monomethyl ester.
Some Mg-chelatase-defective mutants accumulate
protoporphyrin IX (Gough 1972, Mascia 1978, Falbel and
Staehelin 1994). In the white sectors of E.japonicus, by
contrast, no accumulation of this porphyrin analog was detected when the sectors were incubated in the absence of
2,2'-dipyridyl in darkness (Fig. 4), although they retained
high porphyrin-synthetic activity in vitro. The synthesis of
ALA is a key regulatory and rate-limiting step in the formation of hemes and Chls (Beale and Weinstein 1990), and it
is controlled through feedback regulation by heme in vivo
(Hoober and Stegeman 1973). In tissues, light is required
for the continuous synthesis of ALA (Fluhr et al. 1975)
and, thus the rate of synthesis of ALA is very low in darkness. Duggan and Gassman (1974) reported, however, that
2,2'-dipyridyl depressed the steady-state concentration of
heme by inhibiting its synthesis and, thus, released the inhibition of ALA synthesis even in darkness. It is possible,
486
T. Masuda et al.
therefore, that the dramatic accumulation of porphyrin
caused by 2,2-dipyridyl in the white sectors was due to the
release from inhibition of ALA synthesis. In fact, the levels
of protoheme in the white sectors were about 30% of those
in the green sectors (data not shown). Although it is unknown whether the levels of heme are sufficient to inhibit
the synthesis of ALA in the white sectors, we can speculate
that the pathway for the synthesis of Chi in the white sectors might be blocked during the normal growth phase,
while treatment with 2,2'-dipyridyl, might stimulate the synthesis of ALA via the release of the feedback inhibition by
heme.
Inactivation of Mg-chelatase in the white sectors could
be considered as a possible reason for the 2,2'-dipyridyl-dependent accumulation of protoporphyrin IX. However,
the possibility of the absence of the Chl-biosynthetic pathway after the Mg-insertion step in the white sectors cannot
be ruled out. Hess et al. (1992) reported that chlorophyll
synthetase and NADPH:protochlorophyllide oxidoreductase, which catalyze the final steps in the biosynthesis of
Chi are present in the white albostrains mutant of barley.
However, in the present experiments, we did not determine
the levels of these enzymes in the variegated leaves of
E. japonicus.
It seems likely that the various types of Chl-deficient
chloroplast (e.g., those in the albino or chlorina mutants of
wheat and barley and the chromoplasts of daffodil flower)
are caused by blockage of a specific step(s) in the biosynthetic pathway to Chi in the individual plants, while other
steps remain unaffected in spite of the total absence of porphyrins or Chls. As in these cases, in the variegated leaves
of E. japonicus, the blockage of the reaction catalyzed by
Mg-chelatase (and subsequent steps) might cause the pigment deficiency in the white sectors. In contrast to most of
mutant plants, however, the young leaves of E. japonicus
are not variegated; the variegated white sectors are formed
in the peripheral regions along to a central vein during
leaf maturation. Although genetic information is currently
unavailable, we can speculate that, in variegated leaves of
E. japonicus, some unknown factor(s) triggers the transition of plastids during leaf maturation in a strictly localized
manner. During this transition, we have observed dramatic
changes in the membrane structures in plastids by transmission electron microscopy, namely, the disappearance of
thylakoid membranes and subsequent formation of convoluted membrane-like structures (Masuda et al., in preparation), as reported in the chromoplasts of daffodil flowers
(Lotzow and Kleinig 1990). This structural change might
cause the degradation of Chls and simultaneous interruption of the biosynthesis of Chi (or vice versa) as a consequence of dramatic changes in the activities of the enzymes
involved in the biosynthesis and degradation of Chi.
The authors are deeply grateful to Dr. C. Gamini Kannangara of Carlsberg Laboratory, Denmark, for valuable advice
and for the antibodies against GSA aminotransferase. This study
was supported financially by a grant from the Kihara Memorial
Yokohama Foundation for the Advancement of Life Sciences.
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(Received November 15, 1995; Accepted March 21, 1996)