(Received for publication, September 18, 1995; and in revised form, November 6, 1995)
From the
Chlorophyll b is synthesized from chlorophyll a by the oxidation of the methyl group on the ring B of the
tetrapyrrole ring to the formyl group. Previously, we reported that
chlorophyllide b could be converted to chlorophyll a in isolated cucumber etioplasts indicating the conversion of
chlorophyll b to chlorophyll a. To identify the
intermediate molecule, we used barley etioplasts instead of cucumber.
Chlorophyll a and an additional pigment were found after
incubation of chlorophyllide b with isolated barley
etioplasts. The pigment has the same retention time and absorption
spectrum as 7-hydroxymethyl chlorophyll, which has the hydroxymethyl
group on ring B instead of the formyl group of chlorophyll b.
Authentic 7-hydroxymethyl chlorophyll was prepared by reduction of
chlorophyll b by NaBH. Chlorophyll a accumulated during the incubation of 7-hydroxymethyl
chlorophyllide with etioplasts. These findings indicate that
chlorophyll b is converted to chlorophyll a via
7-hydroxymethyl chlorophyll. Chlorophyll b and 7-hydroxymethyl
chlorophyll accumulated within a short period of incubation of
chlorophyllide b with etioplasts. However, chlorophyll a accumulated with a concomitant decrease of chlorophyll b and 7-hydroxymethyl chlorophyll. These observations also suggest
that chlorophyll b is converted to 7-hydroxymethyl chlorophyll
and then to chlorophyll a. Both steps required ATP.
The earliest precursor for chlorophyll (Chl), ()heme,
and bilin synthesis is 5-aminolevulinic acid in higher plants, and the
Chl branch of the pathway begins with insertion of magnesium into the
protoporphyrin. The biosynthetic pathway to Chl is fairly well
understood. Although the activities of many enzymes involved in the
pathway have been demonstrated in crude tissue extracts or intact
organelles, very few have been purified and characterized(1) .
On the other hand, much progress in characterizing bacteriochlorophyll
synthesis has been made especially in genetic analysis(2) . A
46-kilobase region of the Rhodobacter capsulatus contains most
of the genetic loci involved in the magnesium branch of the
bacteriochlorophyll biosynthetic pathway(3, 4) . This
region has been sequenced, revealing the existence of 23 open reading
frames(5) . Bollivar et al.(6) have
undertaken a systematic directed mutational analysis of 12 open reading
frames to evaluate the role in photopigment biosynthesis of individual
open reading frames and identified some genes required for synthesis of
bacteriochlorophyll. Chl and bacteriochlorophyll are produced from the
same precursors by a pathway which is conserved until its later stages.
Therefore, these findings with Rhodobacter are useful for the
study of plant Chl synthesis.
Higher plants and green algae have Chl b as an accessory pigment of light-harvesting Chl a/b-protein complexes for photosystems I and II. Chl b differs from Chl a only by the presence of a 7-formyl group in place of a methyl on ring B of the tetrapyrrole ring. Chl b is believed to be synthesized from Chl a by the following observations. 1) Chl b is synthesized after Chl a accumulation (7) . 2) Many Chl b-less mutants have been reported, but there are no mutants containing only Chl b(8) . 3) Chl b was induced by calcium treatment of the tissues which have only Chl a(7) . The methyl group on pyrrole ring B in Chl a has been reported to be oxidized to an aldehyde group by molecular oxygen and 7-hydroxymethyl chlorophyll (HMChl) was postulated as an intermediate molecule from Chl a to Chl b(9) . However, conversion of Chl a to Chl b has not been demonstrated in isolated plastids, and the regulation and the mechanism of the reaction are not known.
On the other hand, conversion of Chl b to Chl a was not considered to occur, because reduction of a formyl group to a methyl group is a difficult reaction. However, Chl b to Chl a conversion has been suggested by in vivo experiments which showed the increase in Chl a with the concomitant decrease in Chl b in etiolated seedlings illuminated for a short period and then returned to darkness(10) . Recently, we reported the accumulation of Chl a during the incubation of chlorophyllide (Chlide) b with cucumber etioplasts in the dark indicating the conversion of Chl b to Chl a(11, 12) .
Chl b stabilizes Chl a/b-binding protein together with Chl a by making a folded structure, resulting in regulation of the accumulation of Chl a/b-binding protein(13, 14) . Accumulation of Chl b must be controlled also in plants growing under different light conditions. Plants grown under low intensity light have a low Chl a/b ratio, and the Chl a/b ratio increases after transfer of these plants to a high intensity light condition(15) .
As well as conversion of Chl a to Chl b, conversion of Chl b to Chl a would play an important role in adjusting the Chl a/b ratio. To determine the mechanism of the regulation of Chl b to Chl a conversion, the metabolic pathway from Chl b to Chl a must be elucidated. Previously, we could not find a candidate for the intermediate molecule from Chl b to Chl a on high performance liquid chromatography (HPLC) profiles (12) . Herein, we found that HMChl was accumulated during the incubation of Chlide b with barley etioplasts and also showed that 7-hydroxymethyl chlorophyllide (HMChlide) was converted to Chl a.
Previously, we could not detect HMChl after incubation of Chlide b with cucumber etioplasts, although we observed an accumulation of Chl a and Chl b(12) . If HMChl is an intermediate molecule from Chl b to Chl a, it should accumulate in the reaction mixture. In the present study, we used barley etioplasts and, as described below, observed HMChl accumulation. Unhydrogenated Chl (esterified with geranylgeraniol, dihydrogeranylgeraniol, tetrahydrogeranylgeraniol) accumulated as well as Chl esterified with phytol because Chlide b was esterified with endogenous geranylgeraniol and the side chain was hydrogenated to a phytyl group successively. These Chl derivatives interfered with the detection of the intermediate molecule from Chl b to Chl a. To reduce the accumulation of these Chl derivatives, Chlide b was incubated in the presence of phytyl pyrophosphate because Chlide b was preferentially esterified with phytyl pyrophosphate.
Fig. 1shows the HPLC elution profile of Chls accumulated after incubation of Chlide b with barley etioplasts in the presence of phytyl pyrophosphate. Chl a and Chl b accumulated indicating that Chl b was converted to Chl a (Fig. 1, A and B). In addition to Chl a and Chl b, we found a new pigment with a retention time on HPLC different from those of unhydrogenated Chls (Fig. 1B, peak 1). We compared the retention time of the pigment with authentic HMChl to show that the pigment corresponding to peak 1 is HMChl. Both chemically synthesized HMChl and peak 1 have a retention time of 5.67 min (Fig. 1, B and C).
Figure 1:
HPLC
elution profiles of Chls. A and B, 100 pmol of Chlide b or HMChlide was incubated with isolated barley etioplasts in
the dark for 30 min. The pigments were extracted and eluted from an
octadecyl silica column with methanol at a flow rate of 1.5 ml/min at
40 °C and detected by absorption at 663 nm. A, before
incubation; B, after incubation. C, authentic HMChl
was prepared by reduction of Chl b with NaBH in
methanol and analyzed by HPLC. D, HMChlide was incubated and
Chls were analyzed as for A. 1, HMChl; 2,
Chl b; 3, Chl b`; 4, Chl a.
Peak 1 was collected, and its absorption spectrum in diethyl ether was determined (Fig. 2). The absorption spectrum was identical with that of authentic HMChl (Fig. 5). We concluded from these experiments that the pigment corresponding to peak 1 is HMChl.
Figure 2: Absorption spectrum of the pigment appearing after incubation of Chlide b. Fraction of peak 1 in Fig. 1B was collected. The fraction was dried and the pigments were separated by thin layer chromatography on a cellulose plate with hexane/2-propanol (20:1) to remove carotenoid which co-eluted with peak 1 on HPLC. The green pigment was eluted from the plate, and the absorption spectrum of the pigment was determined in diethyl ether.
Figure 5:
Absorption spectra of Chls in diethyl
ether. Chls were extracted with acetone from spinach leaves, and Chl a and Chl b were purified by HPLC. Purified Chl b was reduced by NaBH, and HMChl was purified by HPLC.
Absorption spectra of purified Chl a, Chl b, and
HMChl were determined in diethyl ether.
If HMChl is an intermediate molecule from Chl b to Chl a, HMChl would be converted to Chl a in isolated etioplasts. To show whether HMChl can be converted to Chl a, HMChlide was prepared from HMChl by chlorophyllase treatment and incubated with etioplasts. After incubation, HMChl and Chl a accumulated (Fig. 1D), indicating that HMChlide was esterified and converted to Chl a. From these observations, we conclude that Chl b is converted to Chl a via HMChl.
Next we
investigated whether Chlide b is converted to Chl a by chloroplasts as well as etioplasts.
[C]Chlide b was incubated with
chloroplasts prepared from 12-h illuminated seedlings. The
radioactivity was incorporated into Chl a by chloroplast,
indicating that Chlide b was converted to Chl a by
chloroplasts (Fig. 3). The radioactivity was observed at
fraction 13, whose retention time was identical with that of HMChl,
suggesting that Chlide b was converted to Chl a via
HMChl in chloroplasts.
Figure 3:
Accumulation of labeled Chl.
[C]Chlide b was incubated with
chloroplasts prepared from 12-h illuminated seedlings. Pigments were
analyzed by HPLC at room temperature, and fractions were collected
every 30 s. The radioactivity of each fraction was measured with a
liquid scintillation counter.
Many reports showed the conversion of Chl a to Chl b. These indicate the interconversion between Chl a and Chl b. We call this metabolic pathway the Chl cycle, and the enzymes reducing Chl b to HMChl and HMChl to Chl a as Chl b reductase and HMChl reductase, respectively (Fig. 4).
Figure 4: Interconversion of Chl a and Chl b.
To carry out
the quantitative analysis, we determined the molar extinction
coefficient of HMChl. Chl b was reduced to HMChl by NaBH treatment, and HMChl was purified by HPLC. Purified HMChl, 355
(± 5) µg, was dissolved in 30 ml of diethyl ether, and the
absorbance at 655.5 nm was measured (0.795). The molar extinction
coefficient of HMChl at 655.5 nm was determined from these values, and
the molecular weight of HMChl (909.5). The molar extinction coefficient
of HMChl in diethyl ether was 6.11
10
(M
cm
), and the amount
of HMChl in diethyl ether was determined by the following equation,
HMChl (nmol/ml) = 16.4A
.
Fig. 5shows the absorption spectra of Chl a, Chl b, and HMChl in diethyl ether.
Figure 6:
Time course of Chl accumulated after
incubation of Chlide. Chlide b (,
,
) or
HMChlide (
,
), 100 pmol, were incubated with etioplasts
for various periods in the dark. After incubation, Chl a (
,
), Chl b (
), and HMChl (
,
) were identified and quantified by
HPLC.
We used Chlide b instead of Chl b as the precursor of Chl a. Chl is lipophilic due to the presence of a prenyl side chain and does not dissolve in the incubation mixture, but Chlide is soluble and can be incubated with intact plastids without detergent(21) . We also used etioplasts instead of chloroplasts because etioplasts have no Chls, and a small amount of Chls (below 1 pmol) which accumulated during incubation can be detected by HPLC. Chl synthesis starts after onset of illumination in angiosperms, and some enzymes responsible for Chl synthesis are induced by light treatment. However, our preliminary experiments showed that high conversion activity existed in etiolated tissues and that the activity did not change during greening (data not shown). It is reasonable for the plants to have a high conversion activity before the accumulation of Chl b.
Chl a and HMChl accumulated during the incubation of Chlide b with barley etioplasts. HMChlide was also converted to Chl a by isolated etioplasts. From these observations, we concluded that Chl b was converted to Chl a via HMChl. Previously, HMChl could not be detected by HPLC in the reaction mixture after incubation of Chlide b with cucumber etioplasts. To examine whether cucumber has the same pathway from Chl b to Chl a, HMChlide was used as a substrate. Incubation of HMChlide with cucumber etioplasts resulted in the accumulation of Chl a indicating that Chl b is converted to Chl a via HMChl in cucumber cotyledons. HMChl did not accumulate probably due to the high activity of HMChl reductase in cucumber etioplasts.
The values of the equilibrium constant of alcohol dehydrogenases are, in general, negative, and these enzymes do not require ATP. However, conversion of Chl b to HMChl occurred only in the presence of ATP. These findings suggest that Chl b reductase required ATP for its activation. Activation of the enzymes responsible for Chl synthesis was reported for magnesium chelatase(22) . Magnesium insertion, which is the first step unique to Chl synthesis, is a two-step reaction activated by ATP.
Chl b is considered to be synthesized from Chl a,
but the mechanism of formation of the formyl group of Chl b is
not known. Experiments using O
showed that
molecular oxygen is the precursor of 7-formyl oxygen of Chl b in higher plants(23, 24) , and HMChl was proposed
as a hypothetical intermediate molecule from Chl a to Chl b(9, 25) . However, we could not detect HMChl
or Chl b by HPLC after incubation of Chlide a with
etioplasts or chloroplasts (data not shown). This indicates that
conversion from Chl a to HMChl does not occur in our system.
We also incubated HMChlide with etioplasts or chloroplasts under
various conditions, but HMChl was preferentially converted to Chl a, and Chl b could not be detected. One possible
explanation is that the value of the equilibrium constant of HMChl
reductase (enzyme responsible for the conversion from HMChl to Chl b) is very negative similar to other alcohol dehydrogenases,
and Chl b is not accumulated without removing Chl b from the reaction mixture by some mechanism. However, at present,
we cannot exclude the possibility that another molecule is an
intermediate from Chl a to Chl b.
Plants respond to various intensities of light under which they are grown by adjusting the composition and structure of their photosynthetic apparatus(15, 26) . Growth under low intensity illumination induces an increase in the number of light-harvesting complexes associated with each photosystem to absorb enough light, whereas growth under high intensity light reduces the number of light-harvesting complexes and increases the number of core complexes. During adaptation to high light intensity, Chl b which is released from light-harvesting complexes would be converted to Chl a by the Chl cycle and used for the formation of core complexes of the photosystems. This hypothesis is supported by the observation that increases in Chl a and core complexes were accompanied by a decrease in Chl b and light-harvesting Chl a/b-protein complexes(15) . The Chl cycle would also play an important role in constructing photosystems during greening. If more Chl b is synthesized than required, Chl b would be converted to Chl a and would bind to Chl a-protein complexes. Previously, we reported that light-harvesting Chl a/b-protein complexes act as a temporary pool of Chl when Chl synthesis is raised(27) . Chl b which is pooled in light-harvesting Chl a/b-protein complexes would be converted to Chl a and used for the formation of other Chl-protein complexes. As described above, the Chl cycle enables flexible regulation of the accumulation of Chls and Chl protein complexes and plays an important role in the formation and reorganization of photosystems.
Reduction of the hydroxymethyl group to a methyl group is difficult due to the strong bond of carbon and the hydroxyl group. The mechanism of cleavage of the carbon-hydroxyl bond is well known for ribonucleotide reductase, which plays a central role in DNA biosynthesis catalyzing the reduction of ribonucleotides to their corresponding deoxyribonucleotides by replacing the C-2 hydroxyl group with a hydrogen atom (for review, see (28) ). This enzyme contains a free tyrosyl radical at its catalytic site. Hydroxyurea inhibits the ribonucleotide reductase by quenching free radicals, but HMChl reductase was not inhibited by hydroxyurea (data not shown). Furthermore, HMChl reductase requires ATP for synthesis of Chl a, but ribonucleotide reductase requires it only when the feedback inhibition of dATP is reversed. These observations indicate that HMChl reductase and ribonucleotide reductase replace the hydroxyl group with a hydrogen atom via different mechanisms. Analysis of HMChl reductase would be useful in elucidating the mechanisms of reduction of the hydroxy group as well as chloroplast biogenesis.