Vol. 88, No. 2, 1979 May
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1979
28,
Pages 365-372
CHLOROPLAST BIOGENESIS XXVII DETECTION OF NOVEL CHLOROPHYLL AND CHLOROPHYLL PRECURSORS IN HIGHER PLANTS* Faith Department
Received
March
C. Belanger and Constantin A. Rebeiz** of Horticulture, University of Illinois Urbana, Illinois 61801
22,1979
SUMMARY: In etiolated higher plants, chlorophylls a and b are formed from protochlorophyllide and its divinyl analog via; a) light-induced conversion to distinct chlorophyllide g species, b) dark esterification to distinct chlorophyll a species. Each pair is distinguishable by the Soret excitation maxima in etfier at 77K.
INTRODUCTION
In higher (termed
of solar
of a methyl
group
group
at that
sized
from
the
immediate
with
a long
a differ conversion
as follows:
chain
occurs
group
is
bond
in the
by a photochemical
instead
energy
in Chl b,
to yield
(2); route
a.
(1).
of an ethyl
in the presence
in Chl a and a formyl
Chl fr is
believed
to be synthe-
to the chlorophylls a,-, is
has been
i.e.
Pchlide,
esterified
Protochlorophyllide
and Chlide
of the macrocycle
reaction
(2).
group
at the
This work was supported by a Predoctoral by NSF grant PCM 76-81682 and by funds Experiment Station to C. A. Rebeiz
and the
The Immediate
Pchlide,
i.e.
fourth
is
in the dark
position
to be 2,4-divinyl
species
in the collection
They differ
The latter
Chl a. 7-8
chlorophyll
as participants
3 of the macrocycle
of Chlide
believed
distinct
Mg-2-vinylpheoporphyrin
alcohol
by a double
recognized
The synthetic
precursor
sor of Pchlide
**
at position
same position
(2)
two chemically
to chemical
Chl g (3).
documented
*
only
Chl a and Chl b) are
and conversion
a vinyl
plants,
precur-
a Pchlide
position
with
of the
NSF Fellowship to F. C. Belanger, from the Illinois Agricultural
Reprint requests should be directed to this author Abbreviations: DVPchlide - divinyl protochlorophyllide; chlorophyllide; Pchl - protochlorophyll; Chlide chlorophyll
Pchlide - chlorophyllide;
- protoChl -
0006-291X/79/100365-08$01.00/0 365
Copyright All rights
0 1979
by Academic Press, Inc. in any form reserved
of reproduction
Vol. 88, No. 2, 1979
macrocycle cultures work
BIOCHEMICAL
(4). (5)
This
but was never
we present
etiolated
evidence
tissues
Chlide
compound
which
was detected
before for
the
appear
a, two different
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
detected
in higher
presence
to give
inhibited
plants.
of Pchlide
rise
&. spheroides
and DVPchlide
to at least
Chl a and two different
In this in
two different
Chl b species
in higher
plants. MATERIALS
AND METHODS
k. spheroides mutant MC-7 , that accumulates DVPchlide, kindly supplied by Dr. June Lascelles, was cultured according to Lascelles and Hatch (6). The pigments were isolated from the culture medium by precipitation with ammonium sulfate (6) and the precipitated pigments were dissolved in acetone. The pigments were transferred to ether as described by Koski and Smith (7). Barley (Hordeum vulgare L. var. Beacon Spring) and cucumber (Cucumis sativus L. =Beit Alpha MR) seeds were germinated either in the darkphotoperiodic (14 hr light, 10 hr dark) regime as described before (8). In order to extract the pigments from the etiolated or green tissues, three g of tissue were homogenized in 20 ml acetone-O.1 N NH OH (9:l v/v) at 0-4°C for two min in a Sorvall Omni-mixer (9). After cent$ifugation at 39,000g for ten min, the 80% acetone extract was adjusted to 75% acetone with H 0. The pigments were partitioned into a hexane fraction containing the p ytylated tetrapyrroles and a hexaneextracted acetone fraction containing the unphytylated pigments (9). The unphytylated tetrapyrroles were transferred to ether (9) before chromatography and spectroscopic monitoring. The DVPchlide extracted from R. spheroides in ether was purified by chromatography on thin layers of Silica gel H developed in toluene:ethyl acetate:ethanol (8:2:2 v/v/v) at 4°C. It was eluted in methanol:acetone (4:l v/v) (9), dried under N gas and redissolved in ether for spectroscopic determinations. Chloro$hyll a and b in the hexane fraction were chromatographed on thin layers of cellulose MN 300 developed in ligroin (60~80):n-propanol (9.9:O.l v/v) at room temperature (10) or on thin layers of silica gel H as for DVPchlide. The segregated pigments were eluted in ether, dried under N gas then redissolved in ether for spectroscopic measurements. Photor 6 duction of the unphytylated Pchlide was achieved by2illuminating the etiolated tissues either for two- set ith three mw cm of incandescent light or for one min with 320 VW cm-Y of white fluoresecent light (8). Spectrophotometric and spectrofluorometric determinations were as reported elsewhere by Cohen and Rebeiz (8).
2
RESULTS The unesterified plants
has always
namely
Pchlide
ether,
at 621-623
absorption/excitation
(Z),
protochlorophyll
been considered with
to consist
a red absorption
nm, an emission
fraction
of one chemical
366
higher
species,
maximum at room temperature,
maximum at about
maximum at about
of etiolated
432-433
629-630
nm (7,
in
nm and a Soret
11, 12).
The DVPchlide
Vol. 88, No. 2, 1979
BIOCHEMICAL
of E. spheroides
was distinguishable
Soret
absorption/excitation
ether
solutions
reported
leaves,
exhibited
in ether
an emission
at about that
mutant
(12)
fraction
significant
the
Soret
amounts
room temperature properties were
emission
In all
(Fig.
Surprisingly,
three
1.
Both maxima
shoulder
at 438 nm
tissues of the
by other
barley
barley
and cucumber excitation
Pchlide
The Soret
DVPchlide
standard
excitation
(Fig.
and the and will
shoulders cucumber
extract
be discussed
367
of several the
fluorescence
2, the fluorescence Pchl
Soret
standard
DVPchlide
2B).
In addition,
also
im-
emission
of etiolated
to those
divinyl
of standard at 627 nm excitation was detected
a blue-shifted
the
Pchlide
present
clearly
shown at 451 nm in the ascribed
elsewhere.
in the
both
was not
are
at
atiolated
and peak resolution
a distinct
exhibited
is masked
maximum was found
of the
to
the spectroscopic
temperature
are compared
and cucumber extracts
fraction
unesterified
the emission
to that
which
of Pchlide,
In Figure of the
however,
ether
extract
significantly
maximum at 437 nm which
profile.
of Chl biosynthesis
barley
Pchl
cotyledons cases
at 438 nm may correspond
At this
narrow
in ether,
corresponded
at 444 nm in both
Soret
excitation of etiolated
concentrations
1, 2).
and cucumber
DVPchlide.
barley
corre-
of etiolated
excitation
shoulder
at 77°K.
(Fig.
spectra,
maximum which
Pchlide
spectrum
the
and dif-
627 nm and Soret
unesterified
bands
considerably
2A).
nm in
from
that
in Fig.
in the
higher
extracted,
and excitation
leaves
spectrum
depicted
in extracts
excitation
reinvestigated
and excitation
are
in the excitation
of DVPchlide
by the
of the
tissues
437-438
purified
and the
A Soret
overlooked obvious
at about
The similarities
DVPchlide
at room temperature
red-shifted
1).
Since
barley
11).
maximum at about
was also
(Fig.
proves
(MC-7)
by its
The DVPchlide
(5,
438 and 432 nm, respectively,
workers
only
an absorption
by others
had been previously
RESEARCH COMMUNICATIONS
was found
(11).
exhibited
of the E. spheroides
barley
Pchlide
maximum which
(MC-7)
to those
ferences
from
at room temperature
8. spheroides sponded
AND BIOPHYSICAL
DVPchlide
to an intermediate The barley
unestri-
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
‘T-----
loor
0,
600
650
I
700
I
L
L
1.
I
X(nm1
Fluorescence emission (A) and excitation unesterified Pchl of etiolated barley (a) DVPchlide (b), in ether at room temperature. attenuation is indicated on the spectra, least attenuation possible. The emission cited by the E-wavelength indicated. The were recorded at the F-wavelength indicated. to wavelengths of interest.
f 4
I
450
X hm) Figure
I
400
loo
(B) spectra of the and of standard Ordinate scale 350X being the spectra were eliexcitation spectra Arrows point
-437
8
444
ii ii
$ we
-
0
_
444
ii
it ii’ !I !I
i
2
633 Xhm)
Figure
2.
/
il
oE%c,
700
I DV Pchlide \,b
,40X ,
I
I
I
I
450
400 x (nm)
Fluorescence emission (A) and excitation (B) spectra of standard DVPchTide (a), and of the unesterified Pchl of cucumber (b) and barley (c) in ether at 77°K. All symbols are as in Figure 1.
368
Vol. 88, No. 2, 1979
fied
Pchlide
profile
was similar contrary lated
BIOCHEMICAL
was similar
to that
of corn.
to previous higher
DVPchlide
to that
plants
is
tissues
species,
etiolated
the
heterogeneous
to find
above data
that
of bean
indicated
unesterified
Pchl
and appears
to consist
were
out whether
cucumber
extracted
and transferred
illumination a emitting
terified
Pchl
pigment
that
fraction
of etio-
of both
maximum at 673 nm.
627 nm (Fig.
3A);
and Pchlide. fraction
it
recorded
two Soret
a fraction
3B). that
Pchl
excitation emission
Altogether,
unesterified
were
was produced
Pchl
species Pchl
by photoreduction
unes-
an emisat
of DVPchlide
formed
Chlide
a
a at 673 nm exhibited
indicated of the
and appeared
a
is observable
of the newly
results
that
into
with
the same proportions
maximum of Chlide
heterogeneous
then
3A, most of this
unesterified
the above
a
one min with
one at 438 nm, and a red-shifted
maxima:
was also
Chlide for
a emitting
spectrum
of
It was shown earlier
in Fig.
a Chlide
The unreacted
at the
excitation
445 nm (Fig.
of the
Pchlide
The pigments
to ether.
was made up of about
The Soret
illuminated
As depicted
into
and the
two distinct
at 28°C.
most
(8).
was converted
were
light
photoconverted
Chlide
DVPchlide into
cotyledons
fluorescent
in acetone
the
phototransformable
320 uw cm-2 of white
geneous
the
while
and Pchlide.
etiolated
sion
of cucumber
Altogether,
assumptions,
In order
this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
maximum at that
the
unesterified
to consist
Chlide hetero-
of two distinct
components. The conversion next
about
two Chlide
The unesterified
investigated.
was converted
of the
to heterogeneous
converted in the to ether
to Chl --~ a in vivo dark
at 28°C (13).
and purified
The Chl 3, eluted
of etiolated
5 by illumination
light
(8)
by incubating
and the
the irradiated
The Chl a was extracted
on thin in ether,
Pchl
Chlide
3.0 mw cm-2 of incandescent
a components
layers
of silica
exhibited
an emission
369
gel
into
Chl a was
cucumber for Chlide
cotyledons
two set 5 fraction
tissue in acetone, H as described
for
with was 45 min
transferred in Methods.
maximum at 673 nm, simi-
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100 A
673 1
E440
C I” EjO( 1
I 7cc
650
400
450
Xhm)
Figure
lar
3.
to that
recorded Soret
Fluorescence Chlide a (a) ber, in-ether of Chlide
at the
putative
wavelength
of the red-shifted
depicted
Chlide
in Fig.
38,
Chl a that
the Soret
i.e.
that
was also
38).
of the
excitation
in the
wavelength Finally, green
tissues
the
thus
to the
short possible
was also
any
concentrations
was also
recorded
at 666 nm.
As
recorded
red-shifted from the
exhibited component.
heterogeneous
and was made up of at least
to the occurred
Rough calculations to the
Chl a band,
a single
to uncover
spectrum
the Chl 5 synthesized
Chl a fractions
one.
In order
spectrum
heterogeneous
spectrum
at 673 nm, exhibited
excitation
tail
in contrast
excitation
may be masked by higher
a Soret
the Soret
However,
blue-shifted
mature
maximum,
emission
indicated
a fraction
components.
long
However,
maximum at 435 nm in addition
results
component
3A).
component,
wavelength
a blue-shifted These
5 (Fig.
maximum at 446 nm (Fig.
short
on the short
emission (A) and excitation (B) spectrd of the and newly formed Chl a (b,c) fractions of cucumat 77°K. All symbols are as in Fig. 1.
Chl a emission
excitation
X(nm)
Chlide
in higher
indicated
wavelength occurrence investigated.
370
a fraction,
red-shifted
concentrations
that
component
the
the
proportions
was about
of heterogeneous To this
effect,
three
two
than
the
of the to one.
Chl a and b in the
Chl of
Vol. 88, No. 2, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
htnm)
Figure
4.
green
fully
tally
for
X(nm)
Fluorescence emission purified Chl 3 (a,b) cucumber cotyledons, in Fig. 1. expanded
14 days
cucumber
(8)
cotyledons
was extracted
The emission
spectra
of the
are
in
4A.
depicted
(A) and excitation (B) spectra of the and b (c,d) fractions from green mature in ether at 77°K. All symbols are as
Fig.
from
and purified
purified Chlorophyll
recorded (Fig.
48).
In this
same proportion geneous
as in the
Chlide
a.
respectively.
Likewise,
tail
however,
with
by recording of the
the
Chl b species.
the
Soret
4B).
The Soret of the
that
of the
(Fig.
component
excitation
of the
The same Chl profile
371
in ether
maximum at spectrum
excitation
of
spectrum
red-shifted
component
occurred
was formed also
at 77°K
in the
from
the
appeared
hetero-
to be made
maxima at 475 nm and 487 nm,
red-shifted
48).
of cellulose,
maximum at about
two Chl a components
excitation
the Soret
layers
excitation
a blue-shifted
the Chl b fraction
Chl b band
blue-shifted
The Soret
the Soret
photoperiodi-
an emission
both
Chl a fraction
The detection
was enhanced length
only
case too,
up of two components
nant
one (Fig.
at 674 nm exhibited
on thin
5 exhibited
at 666 nm exhibited
475 nm and a red-shifted
grown
Chl a and b fractions
674 nm and Chl b a maximum at 660 nm. Chl 2 recorded
seedlings
Chl b component spectrum
In contrast
at the to the
Chl b fraction was also
observed
at 487 nm long
wave-
Chl a fraction,
was the predomiin an extract
Vol. 88, No. 2, 1979
of green
spinach
BIOCHEMICAL
leaves.
Altogether,
Chl a and Chl b of a green proposed
that
two distinct
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tissue
the above are
also
Chl a and b of higher
chemical
results
heterogeneous.
plants
are
indicated It
that is
the
therefore
each made up of at least
species.
Acknowledgment: The authors a culture of the E. spheroides
wish to thank mutant.
Dr.
June Lascelles
for
donating
REFERENCES 1. 2. i: i: i: 9. 10. 11. K:
Govindjee and Govindjee, R. (1975) In Bioenergetics of Photosynthesis (Govindjee, ed.) pp. 2-43, Academic Press, New York. Rebeiz, C. A. and Castelfranco, P. A. (1973) Ann. Rev. Plant Physiol. 24, 129-172. Shlyk, A. A. (1971) Ann. Rev. Plant Physiol. 22, 169-184. Jones, O.T.G. (1966) Biochem. J. 101, 153-160. Jones, O.T.G. (1963) Biochem. J. 89, 182-189. Lascelles, J. and Hatch, T. P. (1972) In Methods in Enzymology, Vol. XXIV (San Pietro, ed.) pp. 407-411, Academic Press, New York. Koski, V. M. and Smith, J.H.C. (1948) J. Am. Chem. Sot. 70, 3558-3562. Cohen, C. E. and Rebeiz, C. A. (1978) Plant Physiol. 61, 824-829. Rebeiz, C. A., Mattheis, J. R., Smith, B. B., Rebeiz, C. C., and Dayton, D. F. (1975) Arch. Biochem. Biophys. 171, 549-567. Rebeiz, C. A. (1968) Magon Serie Scientifique 21, l-25. Houssier, C. and Sauer, K. (1969) Biochim. Biophys. Acta 172, 476-491. Stanier, R. Y. and Smith, J.H.C. (1959) Yearb. Carneg. Instn. 58, 336-338. Wolff, J. B. and Price, L. (1957) Arch. Biochem. Biophys. 72, 293-301.
372
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
1979
28,
Pages 365-372
CHLOROPLAST BIOGENESIS XXVII DETECTION OF NOVEL CHLOROPHYLL AND CHLOROPHYLL PRECURSORS IN HIGHER PLANTS* Faith Department
Received
March
C. Belanger and Constantin A. Rebeiz** of Horticulture, University of Illinois Urbana, Illinois 61801
22,1979
SUMMARY: In etiolated higher plants, chlorophylls a and b are formed from protochlorophyllide and its divinyl analog via; a) light-induced conversion to distinct chlorophyllide g species, b) dark esterification to distinct chlorophyll a species. Each pair is distinguishable by the Soret excitation maxima in etfier at 77K.
INTRODUCTION
In higher (termed
of solar
of a methyl
group
group
at that
sized
from
the
immediate
with
a long
a differ conversion
as follows:
chain
occurs
group
is
bond
in the
by a photochemical
instead
energy
in Chl b,
to yield
(2); route
a.
(1).
of an ethyl
in the presence
in Chl a and a formyl
Chl fr is
believed
to be synthe-
to the chlorophylls a,-, is
has been
i.e.
Pchlide,
esterified
Protochlorophyllide
and Chlide
of the macrocycle
reaction
(2).
group
at the
This work was supported by a Predoctoral by NSF grant PCM 76-81682 and by funds Experiment Station to C. A. Rebeiz
and the
The Immediate
Pchlide,
i.e.
fourth
is
in the dark
position
to be 2,4-divinyl
species
in the collection
They differ
The latter
Chl a. 7-8
chlorophyll
as participants
3 of the macrocycle
of Chlide
believed
distinct
Mg-2-vinylpheoporphyrin
alcohol
by a double
recognized
The synthetic
precursor
sor of Pchlide
**
at position
same position
(2)
two chemically
to chemical
Chl g (3).
documented
*
only
Chl a and Chl b) are
and conversion
a vinyl
plants,
precur-
a Pchlide
position
with
of the
NSF Fellowship to F. C. Belanger, from the Illinois Agricultural
Reprint requests should be directed to this author Abbreviations: DVPchlide - divinyl protochlorophyllide; chlorophyllide; Pchl - protochlorophyll; Chlide chlorophyll
Pchlide - chlorophyllide;
- protoChl -
0006-291X/79/100365-08$01.00/0 365
Copyright All rights
0 1979
by Academic Press, Inc. in any form reserved
of reproduction
Vol. 88, No. 2, 1979
macrocycle cultures work
BIOCHEMICAL
(4). (5)
This
but was never
we present
etiolated
evidence
tissues
Chlide
compound
which
was detected
before for
the
appear
a, two different
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in
detected
in higher
presence
to give
inhibited
plants.
of Pchlide
rise
&. spheroides
and DVPchlide
to at least
Chl a and two different
In this in
two different
Chl b species
in higher
plants. MATERIALS
AND METHODS
k. spheroides mutant MC-7 , that accumulates DVPchlide, kindly supplied by Dr. June Lascelles, was cultured according to Lascelles and Hatch (6). The pigments were isolated from the culture medium by precipitation with ammonium sulfate (6) and the precipitated pigments were dissolved in acetone. The pigments were transferred to ether as described by Koski and Smith (7). Barley (Hordeum vulgare L. var. Beacon Spring) and cucumber (Cucumis sativus L. =Beit Alpha MR) seeds were germinated either in the darkphotoperiodic (14 hr light, 10 hr dark) regime as described before (8). In order to extract the pigments from the etiolated or green tissues, three g of tissue were homogenized in 20 ml acetone-O.1 N NH OH (9:l v/v) at 0-4°C for two min in a Sorvall Omni-mixer (9). After cent$ifugation at 39,000g for ten min, the 80% acetone extract was adjusted to 75% acetone with H 0. The pigments were partitioned into a hexane fraction containing the p ytylated tetrapyrroles and a hexaneextracted acetone fraction containing the unphytylated pigments (9). The unphytylated tetrapyrroles were transferred to ether (9) before chromatography and spectroscopic monitoring. The DVPchlide extracted from R. spheroides in ether was purified by chromatography on thin layers of Silica gel H developed in toluene:ethyl acetate:ethanol (8:2:2 v/v/v) at 4°C. It was eluted in methanol:acetone (4:l v/v) (9), dried under N gas and redissolved in ether for spectroscopic determinations. Chloro$hyll a and b in the hexane fraction were chromatographed on thin layers of cellulose MN 300 developed in ligroin (60~80):n-propanol (9.9:O.l v/v) at room temperature (10) or on thin layers of silica gel H as for DVPchlide. The segregated pigments were eluted in ether, dried under N gas then redissolved in ether for spectroscopic measurements. Photor 6 duction of the unphytylated Pchlide was achieved by2illuminating the etiolated tissues either for two- set ith three mw cm of incandescent light or for one min with 320 VW cm-Y of white fluoresecent light (8). Spectrophotometric and spectrofluorometric determinations were as reported elsewhere by Cohen and Rebeiz (8).
2
RESULTS The unesterified plants
has always
namely
Pchlide
ether,
at 621-623
absorption/excitation
(Z),
protochlorophyll
been considered with
to consist
a red absorption
nm, an emission
fraction
of one chemical
366
higher
species,
maximum at room temperature,
maximum at about
maximum at about
of etiolated
432-433
629-630
nm (7,
in
nm and a Soret
11, 12).
The DVPchlide
Vol. 88, No. 2, 1979
BIOCHEMICAL
of E. spheroides
was distinguishable
Soret
absorption/excitation
ether
solutions
reported
leaves,
exhibited
in ether
an emission
at about that
mutant
(12)
fraction
significant
the
Soret
amounts
room temperature properties were
emission
In all
(Fig.
Surprisingly,
three
1.
Both maxima
shoulder
at 438 nm
tissues of the
by other
barley
barley
and cucumber excitation
Pchlide
The Soret
DVPchlide
standard
excitation
(Fig.
and the and will
shoulders cucumber
extract
be discussed
367
of several the
fluorescence
2, the fluorescence Pchl
Soret
standard
DVPchlide
2B).
In addition,
also
im-
emission
of etiolated
to those
divinyl
of standard at 627 nm excitation was detected
a blue-shifted
the
Pchlide
present
clearly
shown at 451 nm in the ascribed
elsewhere.
in the
both
was not
are
at
atiolated
and peak resolution
a distinct
exhibited
is masked
maximum was found
of the
to
the spectroscopic
temperature
are compared
and cucumber extracts
fraction
unesterified
the emission
to that
which
of Pchlide,
In Figure of the
however,
ether
extract
significantly
maximum at 437 nm which
profile.
of Chl biosynthesis
barley
Pchl
cotyledons cases
at 438 nm may correspond
At this
narrow
in ether,
corresponded
at 444 nm in both
Soret
excitation of etiolated
concentrations
1, 2).
and cucumber
DVPchlide.
barley
corre-
of etiolated
excitation
shoulder
at 77°K.
(Fig.
spectra,
maximum which
Pchlide
spectrum
the
and dif-
627 nm and Soret
unesterified
bands
considerably
2A).
nm in
from
that
in Fig.
in the
higher
extracted,
and excitation
leaves
spectrum
depicted
in extracts
excitation
reinvestigated
and excitation
are
in the excitation
of DVPchlide
by the
of the
tissues
437-438
purified
and the
A Soret
overlooked obvious
at about
The similarities
DVPchlide
at room temperature
red-shifted
1).
Since
barley
11).
maximum at about
was also
(Fig.
proves
(MC-7)
by its
The DVPchlide
(5,
438 and 432 nm, respectively,
workers
only
an absorption
by others
had been previously
RESEARCH COMMUNICATIONS
was found
(11).
exhibited
of the E. spheroides
barley
Pchlide
maximum which
(MC-7)
to those
ferences
from
at room temperature
8. spheroides sponded
AND BIOPHYSICAL
DVPchlide
to an intermediate The barley
unestri-
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
‘T-----
loor
0,
600
650
I
700
I
L
L
1.
I
X(nm1
Fluorescence emission (A) and excitation unesterified Pchl of etiolated barley (a) DVPchlide (b), in ether at room temperature. attenuation is indicated on the spectra, least attenuation possible. The emission cited by the E-wavelength indicated. The were recorded at the F-wavelength indicated. to wavelengths of interest.
f 4
I
450
X hm) Figure
I
400
loo
(B) spectra of the and of standard Ordinate scale 350X being the spectra were eliexcitation spectra Arrows point
-437
8
444
ii ii
$ we
-
0
_
444
ii
it ii’ !I !I
i
2
633 Xhm)
Figure
2.
/
il
oE%c,
700
I DV Pchlide \,b
,40X ,
I
I
I
I
450
400 x (nm)
Fluorescence emission (A) and excitation (B) spectra of standard DVPchTide (a), and of the unesterified Pchl of cucumber (b) and barley (c) in ether at 77°K. All symbols are as in Figure 1.
368
Vol. 88, No. 2, 1979
fied
Pchlide
profile
was similar contrary lated
BIOCHEMICAL
was similar
to that
of corn.
to previous higher
DVPchlide
to that
plants
is
tissues
species,
etiolated
the
heterogeneous
to find
above data
that
of bean
indicated
unesterified
Pchl
and appears
to consist
were
out whether
cucumber
extracted
and transferred
illumination a emitting
terified
Pchl
pigment
that
fraction
of etio-
of both
maximum at 673 nm.
627 nm (Fig.
3A);
and Pchlide. fraction
it
recorded
two Soret
a fraction
3B). that
Pchl
excitation emission
Altogether,
unesterified
were
was produced
Pchl
species Pchl
by photoreduction
unes-
an emisat
of DVPchlide
formed
Chlide
a
a at 673 nm exhibited
indicated of the
and appeared
a
is observable
of the newly
results
that
into
with
the same proportions
maximum of Chlide
heterogeneous
then
3A, most of this
unesterified
the above
a
one min with
one at 438 nm, and a red-shifted
maxima:
was also
Chlide for
a emitting
spectrum
of
It was shown earlier
in Fig.
a Chlide
The unreacted
at the
excitation
445 nm (Fig.
of the
Pchlide
The pigments
to ether.
was made up of about
The Soret
illuminated
As depicted
into
and the
two distinct
at 28°C.
most
(8).
was converted
were
light
photoconverted
Chlide
DVPchlide into
cotyledons
fluorescent
in acetone
the
phototransformable
320 uw cm-2 of white
geneous
the
while
and Pchlide.
etiolated
sion
of cucumber
Altogether,
assumptions,
In order
this
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
maximum at that
the
unesterified
to consist
Chlide hetero-
of two distinct
components. The conversion next
about
two Chlide
The unesterified
investigated.
was converted
of the
to heterogeneous
converted in the to ether
to Chl --~ a in vivo dark
at 28°C (13).
and purified
The Chl 3, eluted
of etiolated
5 by illumination
light
(8)
by incubating
and the
the irradiated
The Chl a was extracted
on thin in ether,
Pchl
Chlide
3.0 mw cm-2 of incandescent
a components
layers
of silica
exhibited
an emission
369
gel
into
Chl a was
cucumber for Chlide
cotyledons
two set 5 fraction
tissue in acetone, H as described
for
with was 45 min
transferred in Methods.
maximum at 673 nm, simi-
Vol. 88, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
100 A
673 1
E440
C I” EjO( 1
I 7cc
650
400
450
Xhm)
Figure
lar
3.
to that
recorded Soret
Fluorescence Chlide a (a) ber, in-ether of Chlide
at the
putative
wavelength
of the red-shifted
depicted
Chlide
in Fig.
38,
Chl a that
the Soret
i.e.
that
was also
38).
of the
excitation
in the
wavelength Finally, green
tissues
the
thus
to the
short possible
was also
any
concentrations
was also
recorded
at 666 nm.
As
recorded
red-shifted from the
exhibited component.
heterogeneous
and was made up of at least
to the occurred
Rough calculations to the
Chl a band,
a single
to uncover
spectrum
the Chl 5 synthesized
Chl a fractions
one.
In order
spectrum
heterogeneous
spectrum
at 673 nm, exhibited
excitation
tail
in contrast
excitation
may be masked by higher
a Soret
the Soret
However,
blue-shifted
mature
maximum,
emission
indicated
a fraction
components.
long
However,
maximum at 435 nm in addition
results
component
3A).
component,
wavelength
a blue-shifted These
5 (Fig.
maximum at 446 nm (Fig.
short
on the short
emission (A) and excitation (B) spectrd of the and newly formed Chl a (b,c) fractions of cucumat 77°K. All symbols are as in Fig. 1.
Chl a emission
excitation
X(nm)
Chlide
in higher
indicated
wavelength occurrence investigated.
370
a fraction,
red-shifted
concentrations
that
component
the
the
proportions
was about
of heterogeneous To this
effect,
three
two
than
the
of the to one.
Chl a and b in the
Chl of
Vol. 88, No. 2, 1979
8lOCHEMlCAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
htnm)
Figure
4.
green
fully
tally
for
X(nm)
Fluorescence emission purified Chl 3 (a,b) cucumber cotyledons, in Fig. 1. expanded
14 days
cucumber
(8)
cotyledons
was extracted
The emission
spectra
of the
are
in
4A.
depicted
(A) and excitation (B) spectra of the and b (c,d) fractions from green mature in ether at 77°K. All symbols are as
Fig.
from
and purified
purified Chlorophyll
recorded (Fig.
48).
In this
same proportion geneous
as in the
Chlide
a.
respectively.
Likewise,
tail
however,
with
by recording of the
the
Chl b species.
the
Soret
4B).
The Soret of the
that
of the
(Fig.
component
excitation
of the
The same Chl profile
371
in ether
maximum at spectrum
excitation
of
spectrum
red-shifted
component
occurred
was formed also
at 77°K
in the
from
the
appeared
hetero-
to be made
maxima at 475 nm and 487 nm,
red-shifted
48).
of cellulose,
maximum at about
two Chl a components
excitation
the Soret
layers
excitation
a blue-shifted
the Chl b fraction
Chl b band
blue-shifted
The Soret
the Soret
photoperiodi-
an emission
both
Chl a fraction
The detection
was enhanced length
only
case too,
up of two components
nant
one (Fig.
at 674 nm exhibited
on thin
5 exhibited
at 666 nm exhibited
475 nm and a red-shifted
grown
Chl a and b fractions
674 nm and Chl b a maximum at 660 nm. Chl 2 recorded
seedlings
Chl b component spectrum
In contrast
at the to the
Chl b fraction was also
observed
at 487 nm long
wave-
Chl a fraction,
was the predomiin an extract
Vol. 88, No. 2, 1979
of green
spinach
BIOCHEMICAL
leaves.
Altogether,
Chl a and Chl b of a green proposed
that
two distinct
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
tissue
the above are
also
Chl a and b of higher
chemical
results
heterogeneous.
plants
are
indicated It
that is
the
therefore
each made up of at least
species.
Acknowledgment: The authors a culture of the E. spheroides
wish to thank mutant.
Dr.
June Lascelles
for
donating
REFERENCES 1. 2. i: i: i: 9. 10. 11. K:
Govindjee and Govindjee, R. (1975) In Bioenergetics of Photosynthesis (Govindjee, ed.) pp. 2-43, Academic Press, New York. Rebeiz, C. A. and Castelfranco, P. A. (1973) Ann. Rev. Plant Physiol. 24, 129-172. Shlyk, A. A. (1971) Ann. Rev. Plant Physiol. 22, 169-184. Jones, O.T.G. (1966) Biochem. J. 101, 153-160. Jones, O.T.G. (1963) Biochem. J. 89, 182-189. Lascelles, J. and Hatch, T. P. (1972) In Methods in Enzymology, Vol. XXIV (San Pietro, ed.) pp. 407-411, Academic Press, New York. Koski, V. M. and Smith, J.H.C. (1948) J. Am. Chem. Sot. 70, 3558-3562. Cohen, C. E. and Rebeiz, C. A. (1978) Plant Physiol. 61, 824-829. Rebeiz, C. A., Mattheis, J. R., Smith, B. B., Rebeiz, C. C., and Dayton, D. F. (1975) Arch. Biochem. Biophys. 171, 549-567. Rebeiz, C. A. (1968) Magon Serie Scientifique 21, l-25. Houssier, C. and Sauer, K. (1969) Biochim. Biophys. Acta 172, 476-491. Stanier, R. Y. and Smith, J.H.C. (1959) Yearb. Carneg. Instn. 58, 336-338. Wolff, J. B. and Price, L. (1957) Arch. Biochem. Biophys. 72, 293-301.
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