Tài liệu Báo cáo Y học: The effects of low pH on the properties of protochlorophyllide oxidoreductase and the organization of prolamellar bodies of maize (Zea mays) pot

The effects of low pH on the properties of protochlorophyllide
oxidoreductase and the organization of prolamellar bodies
of maize (
Zea mays
)
Eva Selstam
1
, Jenny Schelin
1
, Tony Brain
2
and W. Patrick Williams
2
1
Umea
˚
Plant Science Center, Department of Plant Physiology, University of Umea
˚
, Sweden;
2
Life Sciences Division, King’s College,
University of London, UK
Prolamellar bodies (PLB) contain two photochemically
active forms of the enzyme protochlorophyllide oxido-
reductase POR-PChlide
640
and POR-PChlide
650
(the spec-
tral forms of POR-Chlide complexes with absorption
maxima at the indicated wavelengths). Resuspension of

maize PLB in media with a pH below 6.8 leads to a rapid
conversion of POR-PChlide
650
to POR-PChlide
640
and a
dramatic re-organization of the PLB membrane system. In
the absence of excess NADPH, the absorption maximum of
the POR complex undergoes a further shift to about 635 nm.
This latter shift is reversible on the re-addition of NADPH
with a half-saturation value of about 0.25 m
M
NADPH for
POR-PChlide
640
reformation. The disappearance of POR-
PChlide
650
and the reorganization of the PLB, however, are
irreversible. Restoration of low-pH treated PLB to pH 7.5
leads to a further breakdown down of the PLB membrane
and no reformation of POR-PChlide
650
. Related spectral
changes are seen in PLB aged at room temperature at pH 7.5
in NADPH-free assay medium. The reformation of POR-
PChlide
650
in this system is readily reversible on re-addition
of NADPH with a half-saturation value about 1.0 l

M
.
Comparison of the two sets of changes suggest a close link
between the stability of the POR-PChlide
650
,membrane
organization and NADPH binding.
The low-pH driven spectral changes seen in maize PLB
are shown to be accelerated by adenosine AMP, ADP and
ATP. The significance of this is discussed in terms of current
suggestions of the possible involvement of phosphorylation
(or adenylation) in changes in the aggregational state of the
POR complex.
Keywords: protochlorophyllide oxidoreductase; prolamellar
body; protochlorophyllide; oxidoreductase; chlorophyllide.
Plant prolamellar bodies (PLB) found in the etioplasts of
dark-grown (etiolated) seedlings, are the precursors of the
chloroplast thylakoid membrane. The PLB membrane is
dominated by the presence of a single protein species,
protochlorophyllide oxidoreductase (EC 1.3.1.33) (POR)
that catalyses the light-driven, NADPH-dependent reduc-
tion of protochlorophyllide (PChlide) to chlorophyllide
(Chlide). Analyses of the absorption spectrum of PLB [1]
and low-temperature fluorescence spectra of etioplast inner
membrane preparations (EPIM) and PLB [2], indicate the
presence of three major pools of PChlide; a nonphotocon-
vertible form PChlide
628)633
and two photoconvertible
forms PChlide

640)645
and PChlide
650)657
. The suffix numbers
relate to the wavelengths of the absorption and emission
maxima, respectively. To emphasize the fact that the two
photoconvertible forms are bound to POR, they will be
referredtoheretoasPOR-PChlide
640
and POR-PChlide
650
.
Under in vivo conditions, exposure of etioplasts to a flash
of bright white light leads to a conversion of the photo-
convertible PChlide pigments to Chlide resulting in a rapid
shift of the main absorption maximum from 650 nm, initially
to about 678 nm and then to 684 nm. Over a period of about
20 min, this absorption maximum shifts back to 672 nm.
This latter shift, referred to as the Shibata shift [3], is
attributed to the release of Chlide from POR. This release is
accompanied by extensive changes both in the composition
and morphology of the PLB eventually leading to its
conversion to the chloroplast thylakoid membrane system.
Isolated PLB show a similar pattern of spectroscopic changes
immediately following illumination. The presence of excess
NADPH, however, is required to ensure the replacement of
the NADP
+
by NADPH and drive the absorption peak shift
from 678 to 684 nm [4,5]. Under these conditions, no Shibata

shift occurs and the PLB lack the ability to undergo the
compositional and morphological changes seen in vivo.
The relationship between the two photoconvertible forms
of POR has been the subject of much discussion. A number
of lines of evidence suggest that POR-PChlide
640
and POR-
PChlide
650
are the less and more aggregated forms, respect-
ively, of the same enzyme [1,6–8] and Ryberg, Sundqvist and
their coworkers [9–11] have recently reported results
suggesting that this aggregation may be favoured by POR
phosphorylation. The idea of a possible phosphorylation
Correspondence to W. P. Williams, Life Sciences Division, King’s
College London, Franklin-Wilkins Building,
150 Stamford Street, London SE1 9NN.
Fax: + 44 20 7848 4450, Tel.: + 44 20 7848 4433,
E-mail:
Abbreviations: Chlide, chlorophyllide; PChlide, protochlorophyllide;
PLB, prolamellar body; POR, protochlorophyllide oxidoreductase;
POR-PChlide
635
,POR-PChlide
640
, POR-PChlide
650
,POR-
Chlide
676)677

and POR-Chlide
684
, spectral forms of POR-PChlide or
POR-Chlide complexes with absorption maxima at the indicated
wavelengths; EPIM, etioplast inner membrane preparations.
(Received 20 December 2001, revised 15 March 2002,
accepted 19 March 2002)
Eur. J. Biochem. 269, 2336–2346 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02897.x
step in the interconversion of different forms of POR can be
traced back to an early series of experiments by Horton &
Leech [14,15] in which the transformation of POR-
PChlide
650
to a short-wavelength form with an absorption
maximum around 630 nm in aged maize etioplasts was
found to be reversed by the addition of ATP. However,
Griffiths [16], working with water-lysed oat etioplasts,
observed the formation of a similar species absorbing at
633 nm that reconverted to POR-PChlide
650
on the addition
of NADPH but was unaffected by the addition of ATP
alone. Similar results were obtained by Brodersen [17]
working with aged barley PLB.
Current suggestions on the involvement of POR-phos-
phorylation are centred around a series of more recent
reports. Wiktorsson et al. [9] reported that the reformation
of photoactive PChlide species in preilluminated etioplast
inner membrane (EPIM) preparations suspended in low pH
media is favoured by ATP. They also found the blue shift in

the absorption maximum of the photoconverted enzyme
occurring under these conditions to be inhibited by ATP
and by the protein phosphatase inhibitor NaF. On the basis
of this study, and subsequent studies on the action of
protein kinase and protein phosphate inhibitors [10,11],
POR-PChlide
650
was suggested to be an aggregated form of
a phosphorylated (or possibly adenylated) ternary complex
of POR, its substrate PChlide, and NADPH. Dephospho-
rylation of the photoconverted form of this complex by an
endogenous phosphatase, it was further suggested, leads to
a disaggregation of POR and a dissociation of Chlide from
the POR complex, giving rise to the Shibata shift.
In this paper, the resuspension of maize PLB in media
with a pH below about pH 6.8, is shown to lead to a rapid
conversion of POR-PChlide
650
to POR-PChlide
640
.These
changes, which take place over an extremely narrow pH
range, are shown to be accompanied by marked decreases in
the ability of POR to bind NADPH and a rapid disassembly
of the PLB. The pH-driven spectral changes are compared
to those seen in aged PLB. The effects of adenosine, AMP,
ADP, ATP and NaF on the pH-driven changes are studied
and discussed in terms of their relevance to the POR
phosphorylation model.
MATERIALS AND METHODS

PLB isolation
Maize seedlings (Zea mays L. cv. Apache) were grown in
the dark for 9 days at 24 °C in a peat-soil mixture
containing fertiliser. PLB, isolated according to the proce-
dure of Widel-Wigge & Selstam [12], were stored at )20 or
)70 °Cin1.3
M
sucrose, 50 m
M
KCl, 1 m
M
MgCl
2
,1m
M
EDTA, 0.3 m
M
NADPH, 20 m
M
Tricine, 10 m
M
Hepes,
adjusted to pH 7.5 with KOH. All experiments, unless
otherwise specified, were carried out at room temperature
on PLB freshly resuspended in assay medium containing
250 m
M
sucrose, 50 m
M
KCl, 1 m

M
MgCl
2
,1m
M
EDTA
and 30 m
M
Hepes adjusted to pH 6.5 or pH 7.5. The
results reported in this paper were normally based on
measurements performed on at least three different PLB
preparations. Minor differences in the rates of the spectral
and structural changes were observed between different
preparations but the overall pattern of changes was
extremely consistent.
Absorption and fluorescence spectrophotometry
Aliquots (50 lL) of freshly thawed samples of PLB
containing  200 lg protein, were thoroughly washed in
cold assay medium at pH 7.5 to remove excess NADPH.
The washed pellet was then re-suspended in 1.0 mL of test
assay medium. Absorption spectra were normally measured
using a Shimadzu MPS 2000 spectrophotometer fitted with
a cuvette holder close to the photomultiplier to reduce light
scattering. A few measurements were made using a Philips
PU8720 spectrophotometer and a computer-generated
baseline used to minimize the effects of light scatter.
Photoconversion was brought about by exposure of the
sample to a defined number of flashes of bright white light
delivered by a Sunpak, Softlite 2000 A (Tocad, Tokyo,
Japan). When required, 40 lL samples were removed for

low-temperature (77 °K) fluorescence emission measure-
ments made using a FluoroMax-2 spectrofluorimeter
(Instrument S.A. Inc. Edison, NJ, USA).
Transmission electron microscopy (TEM)
Samples were fixed in 2.5% gluteraldehyde in 100 m
M
cacodylate buffer, pH 7.4. They were then post-fixed with
osmium tetroxide, embedded and sectioned. The sections,
stainedin2%uranylacetatefollowedbyleadcitrate,were
examined using a Philips EM301G electron microscope.
RESULTS
pH dependence of the spectral properties
of POR pigment complexes
Resuspension of maize PLB in low pH media results in a
rapid irreversible change in spectral properties. The room
temperature absorption and 77 °K fluorescence emission
properties of maize PLB resuspended in assay medium at
pH 7.5 and pH 6.5 are compared in Fig. 1.
The spectral characteristics of maize PLB suspended at
pH 7.5 are very similar to those previously reported [1,2,4].
Prior to photoconversion, the PLB were characterized
by a broad absorption peak with a maximum at about
650 nm and a broad shoulder at around 640 nm, associated
with POR-PChlide
650
and POR-PChlide
640
, respectively
(Fig. 1A). Exposure to a single flash of bright white light
results in formation of the corresponding Chlide derivatives

and a shift in absorption maximum, in the presence of
excess NADPH, to 684 nm. The 77 °K fluorescence emis-
sion spectrum of the nonphotoconverted PLB is dominated
by the 656-nm emission peak of POR-PChlide
650
(Fig. 1B).
Some emission from the nonphotoconvertible species
PChlide
628
is visible at about 630–635 nm but little or no
emission is seen from POR-PChlide
640
, reflecting the
efficient excitation energy transfer existing between this
species and POR-PChlide
650
[13]. Samples photoconverted
in the presence of excess NADPH are characterized by a
maximum at 696 nm typical of the Chlide derivative of
POR-PChlide
650
.
The results for maize PLB resuspended at pH 6.5 are
strikingly different. Under these conditions, there is a rapid
conversion of POR-PChlide
650
to POR-PChlide
640
(Fig. 1C). Exposure to bright white light leads to the
photoconversion of POR-PChlide

640
to its corresponding
Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2337
Chlide derivative with an absorption maximum at about
675 nm (Fig. 1C). Corresponding changes are seen in low-
temperature fluorescence emission (Fig. 1D). The main
emission peak prior to photo-conversion is now centred at
648 nm. There is no sign of the 656 nm emission peak seen
at pH 7.5, reflecting the fact that POR-PChlide
650
is
completely converted to POR-PChlide
640
. Following photo-
conversion, the emission peak shifts to 682 nm reflecting the
formation of the Chlide derivative of POR-PChlide
640
.
The presence of excess NADPH is an important factor at
low pH. If PLB are resuspended in pH 6.5 assay medium in
the absence of excess NADPH, the PChlide absorption
maximum shifts to about 635 nm rather than 640 nm, as
illustrated in Fig. 2. Under these conditions, there is
minimal photoconversion of the sample on exposure to
light. On addition of NADPH, however, the absorption
maximum shifts back to 640 nm and photoconvertibility is
restored. These changes are attributed to the conversion of
photoconvertible POR-PChlide
640
to a nonphotoconverti-

ble species POR-PChlide
635
lacking bound NADPH which
rapidly reconverts to POR-PChlide
640
in the presence of
added NADPH. POR-PChlide
635
has a 77 °K fluorescence
emission peak at 640 nm of similar intensity to the 648 nm
emission peak of POR-PChlide
640
(data not shown) clearly
distinguishing it from PChlide
628
, which emits at 633 nm
and remains nonphotoconverted even in the presence of
excess NADPH.
The pH-driven conversion of POR-PChlide
650
to POR-
PChlide
640
(POR-PChlide
635
in the absence of excess
NADPH) is very rapid, taking less than a minute at room
temperature and is complete in less than 10 min even at
0 °C (Fig. 3). As illustrated in Fig. 4, the process is
irreversible. Samples exposed to pH 6.5 were resuspended

in pH 7.5 assay medium containing 3 m
M
NADPH. The
PChlide absorption maximum, however, remained close to
Fig. 2. Absorption spectra of maize. (a) PLB freshly resuspended
sample in pH 6.5 assay medium lacking NADPH; (b) after exposure to
one flash of bright white light; (c) the same sample plus 1 m
M
NADPH;(d)afterexposuretoasecondflashofbrightwhitelight(e)
after a 60-s exposure to full room light.
Fig. 1. Light-driven changes in maize PLB. (A) Room-temperature
absorption changes at pH 7.5. (B) Absorption changes corresponding
to low-temperature fluorescence emission changes. (C) Room-tem-
perature absorption changes at pH 6.5. (D) Corresponding low-tem-
perature fluorescence emission changes. All samples contained 1.0 m
M
NADPH. Fluorescence excitation wavelength was 440 nm. Solid lines
and dashed lines correspond to nonphotoconverted and photocon-
verted forms, respectively.
Fig. 3. Plots of the time dependence of the blue-shift in the absorption
maximum of PChlide following resuspension of PLB in assay medium
pH 6.5. Measurements were made at 5 °C in the presence (j)andthe
absence (d)of1.0m
M
NADPH.
2338 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
640 nm even after 20 min incubation at room temperature.
Multiple flashes of saturating white light were required for
full photoconversion of the incubated sample and led to the
formation of a Chlide peak centred at about 675 nm

characteristic of the low pH form (cf. Figs 2 and 4).
A potential complicating factor in these latter measure-
ments is the tendency of POR-PChlide
635
to break down to
yield a new PChlide absorption band with a maximum at
653 nm (PChlide
653
). Traces of this species are detectable in
the spectra shown in Fig. 4. This breakdown is more clearly
illustrated in Fig. 5, which shows the effects of ageing on the
absorption spectrum of maize PLB suspended in pH 6.5
assay medium in the absence of excess NADPH. PChlide
653
is easily distinguishable from POR-PChlide
650
as it is
nonphotoconvertible and gives rise to no obvious low-
temperature fluorescence. It is probably related to the
species PChlide
647
, attributed to aggregated protochloro-
phyllide, reported in dried etioplast membrane preparations
[19]. Care was taken in all measurements to restrict the time
of exposure of PLB samples to low pH in media lacking
excess NADPH to ensure that PChlide
653
formation was
avoided.
The pH-dependence of the conversion of POR-

PChlide
650
to POR-PChlide
640
/POR-PChlide
635
is reflected
in the measurements of the pH dependence of the
wavelengths of the absorption maxima of PChlide and
Chlide shown in Fig. 6. In both cases, there is a dramatic
reduction in wavelength maximum over a narrow pH range
spanning about 0.5 pH units centred at about pH 6.9.
Structural studies
The formation of PLB is generally believed to be associated
with the presence of POR-PChlide
650
[20]. TEM measure-
ments were therefore carried out to determine whether or
not loss of POR-PChlide
650
correlates with loss of PLB
structure. Typical electron micrographs of PLB samples
suspendedinpH7.5andpH6.5assaymediumare
Fig. 4. Changes in absorption spectrum of maize PLB. (a) Spectrum of
sample initially suspended in pH 6.5 assay medium containing 3 m
M
NADPH and then pelleted, resuspended and incubated for 20 min at
room temperature in pH 7.5 assay medium containing 3 m
M
NADPH. The sample was then exposed to one (b), two (c) and four (d)

flashes of bright white light followed by (e) a 60-s exposure to full room
light.
Fig. 5. Absorption spectra showing the formation of PChlide
653
in PLB
suspended in pH 6.5 assay medium incubated at room temperature for
(a) 22, (b) 37, (c) 46, (d) 67, (e) 80, (f) 100 and (g) 125 min.
Fig. 6. Plots showing the pH dependence of the absorption maxima of
(A) PChlide in nonphotoconverted PLB samples (B) Chlide formed after
photoconversion by two flashes of bright white light measured 2 min after
conversion. Measurements were made in the presence (j)andthe
absence (d)of1.0m
M
NADPH.
Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2339
presented in Fig. 7. At pH 7.5 (Fig. 7A), the majority of the
PLB are in the form of highly ordered paracrystalline arrays
based on networks of interconnected tubular tetrapodal
membrane units forming a bicontinuous diamond cubic
(Fd3m) lattice [21,22]. Resuspension at pH 6.5 (Fig. 7B),
however, leads to complete loss of such ordered structures
and their replacement by highly disordered arrays of
entangled tubes. Parallel X-ray diffraction measurements
(data not shown) confirmed that resuspension of PLB in
low pH media leads to a complete loss of long-range order
in the samples.
This breakdown in PLB structure, like the conversion of
the POR complex, is irreversible. There is no evidence of the
reformation of organized PLB if the pH of the low pH
sample is returned to pH 7.5 by the addition of small

amounts of KOH. In contrast, the turbid PLB suspensions
rapidly clarify and become optically clear, suggesting a
further breakdown of the tubular structures into small
vesicles.
NADPH binding studies
The formation of POR-PChlide
635
, as opposed to POR-
PChlide
640
, in PLB samples resuspended in low pH assay
medium in the absence of excess NADPH strongly suggests
that POR-PChlide
640
, under these conditions at least, has a
greatly reduced affinity for NADPH. The NADPH binding
capability of POR-PChlide
635
/POR-PChlide
640
was estima-
ted by measuring the NADPH dependence of the photo-
conversion of PLB at pH 6.5. Plots of the extent of
photoconversion in response to a single saturating flash, and
to a series of such flashes separated by a dark time of 30 s,
are presented in Fig. 8A. The response to a single flash
reflects the position of the equilibrium existing between
photoconvertible POR-PChlide
640
and nonphotoconverti-

ble POR-PChlide
635
. The higher yield achieved by multiple
flashes, indicates the re-establishment of this equilibrium in
the dark period between flashes. The equilibrium between
the two forms is also reflected in the measurements of the
NADPH dependence of the red-shift in the PChlide
absorption maximum shown in Fig. 8B. In both cases,
half-saturation of these changes occurs at  0.25 m
M
NADPH, indicating that POR-PChlide
640
binds NADPH
comparatively weakly at pH 6.5. Interestingly, even at high
levels of NADPH, a single saturating flash was unable to
photoconvert all the pigment present at low pH. The
reasons for this are unclear.
This contrasts strongly with the binding of NADPH to
POR-PChlide
640
at pH 7.5. Attempts to remove NADPH
from POR-PChlide
640
and/or POR-PChlide
650
by repeated
Fig. 7. Typical electron micrographs of PLB
samples suspended in assay medium at (A)
pH 7.5 and (B) pH 6.5. Magnification bar
200 nm.

Fig. 8. NADPH concentration dependence of the photoconversion of
PChlide to Chlide of PLB suspended in pH 6.5 assay medium. (A)
Changes in percentage photoconversion in response to one, two, and
four saturating flashes of white light. (B) Variation of the wavelength
of the absorption maximum of PChlide with NADPH concentration.
2340 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
washing in cold NADPH-free media were unsuccessful,
suggesting that NADPH is effectively irreversibly bound to
both forms of the enzyme under these conditions. The
alternative approach of first resuspending the PLB in low
pH media to dissociate bound NADPH and then restoring
the suspension to pH 7.5 by addition of small amounts of
KOH, prior to re-addition of NADPH was attempted. This,
however, invariably led to a breakdown of an appreciable
fraction of the POR-PChlide
635
complex to form
PChlide
653
, which interfered with the subsequent spectral
analysis. If NADPH is rebound before the restoration of the
pH to pH 7.5, relatively little PChlide
653
is formed indica-
ting the ability of NADPH to stabilize the enzyme.
The NADPH binding properties of the photoconverted
enzyme at pH 7.5 were investigated using the red-shift in the
Chlide absorption maximum accompanying the displace-
ment of bound NADP
+

by NADPH [4,5]. The samples
were first photoconverted in the absence of excess NADPH
to form the NADP
+
-bound enzyme. The extent of the red-
shift following the addition of different concentrations of
NADPH was then used to estimate the efficiency of
NADPH binding. The results of these measurements,
performed at 5 °C to slow down other possible changes in
the photoconverted enzyme, are shown in Fig. 9. In the
absence of added NADPH, the wavelength of the absorp-
tion maximum measured 10–20 s after photoconversion
was at  680–681 nm falling to  677–676 nm after about
5 min. The full red-shift was obtained even if the addition of
the NADPH was delayed until the wavelength had stabil-
ized at this shorter wavelength, indicating that this initial
decline is not linked to a loss of Chlide (Fig. 9A).
Approximately 2 l
M
NADPH was sufficient to drive the
full shift (Fig. 9B). This approach, unfortunately, cannot be
adopted at low pH as the enzyme is essentially nonphoto-
convertible in the absence of excess NADPH and the red-
shift, if one exists at all, is negligibly small.
Comparison with the effects of ageing
A similar, but much slower, conversion of POR-PChlide
650
to shorter wavelength forms is seen in aged etioplasts and
PLB [14–17]. Maize PLB prewashed in NADPH-free assay
medium (pH 7.5) were aged in the dark at room tempera-

ture for five hours. After this time,  50% of the POR-
PChlide
650
was converted to a shorter wavelength form with
an absorption maximum close to 635 nm. This species
(referred to in the early literature as P-630) is nonphoto-
convertible, but is converted to a photoconvertible form in
the presence of NADPH It is clearly very closely related to,
if not identical to, POR-PChlide
635
. If excess NADPH is not
removed, POR-PChlide
650
is initially converted to POR-
PChlide
640
, which then slowly converts to the shorter
wavelength form. Following their dark incubation, the aged
samples were exposed to room light for one minute to
convert all the photoconvertible PChlide present to Chlide
(Fig. 10A). NADPH was then added to the samples to
convert the remaining nonphotoconvertible PChlide to a
photoconvertible form. To check that the product was
indeed photoconvertible, the PLB were re-exposed to room
light (Fig. 10B). The original conversion of POR-PChlide
650
to POR-PChlide
635
, its reconversion to POR-PChlide
650

and its subsequent photoconversion to POR-Chlide
684
,are
all clearly visible in the difference spectra shown in
Fig. 10(C,D). These changes are similar to those reported
byBrodersen[17],whoworkedwithagedbarleyPLB.
The NADPH dependence of the reformation process was
estimated from measurements of the amounts of reformed
POR-PChlide
650
available for photoconversion from differ-
ence spectra of the type shown in Fig. 10D. The half-
saturation value for NADPH binding to POR-PChlide
635
at
pH 7.5 estimated on this basis is  1 l
M
(Fig. 11). In
agreement with the findings of Griffiths [16] for water-lysed
etioplasts, we found no requirement for ATP in these
changes. TEM measurements (not shown) indicated a
decrease in the overall degree of order of the PLB with
ageing but no dramatic structural changes of the type seen
on exposure to low pH. Re-addition of NADPH had no
obvious effects on structure.
Adenyl nucleotide and fluoride sensitivity
Ryberg & Sundquist and their coworkers [9–11] have
presented a number of lines of evidence suggesting the
Fig. 9. Changes in the wavelength of the Chlide absorption maximum
following photoconversion of washed PLB samples suspended in pH 7.5

assay medium. (A) Plots of the time dependence in samples contained
no added NADPH (d), 2 l
M
NADPH added directly after photo-
conversion (j), 2 l
M
NADPH (r)or1 l
M
NADPH (m)added7 min
after photoconversion. (B) Plot showing the NADPH concentration
dependence of the red shift in the wavelength maximum of Chlide
following the addition of NADPH to PLB photoconverted in the
absence of excess NADPH.
Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2341
involvement of a kinase/phosphatase system in the POR
system. One line of evidence of particular relevance to the
present study is their observation that ATP inhibited the low
pH-induced blue-shift in the wavelength of the low-
temperature fluorescence maximum of Chlide seen in wheat
EPIM photoconverted in the presence of excess NADPH
[9]. They also demonstrated that ATP and NaF (presum-
ably acting as phosphatase inhibitors) inhibited the loss of
the long-wavelength form of Chlide following photocon-
version of reformed phototransformable PChlide in this
system.
The results of measurements of the effects of ATP, ADP,
AMP and adenosine on the corresponding low pH-induced
blue-shift in the absorption maximum of maize PLB, made
in the presence and absence of NaF, are presented in
Fig. 12A. The measurements were made by adding small

aliquots (70 lL) of POR-PChlide
650
suspended in assay
medium at pH 7.5 to a much larger volume (1 mL) of
pH 6.5 assay medium containing 1 m
M
NADPH, immedi-
ately photoconverting the sample by exposure to a satur-
ating flash of white light and then monitoring the changes in
wavelength of the Chlide absorption maximum. All meas-
urements were performed at 5 °C to reduce the rate of the
pH-driven conversion between the long- and short-wave-
length forms of the enzyme. Additions of NaF, adenyl
nucleotides and adenosine were made 2 min after photo-
conversion to ensure the formation of POR-Chlide
684
prior
to their addition. Measurements were restricted to the
changes seen directly after initial photoconversion as no
reformation of photoconvertible PChlide occurs in the
maize PLB system. There is, however, no obvious reason
why the stability of the reformed pigment complex should
differ from that originally present.
Fig. 10. Regeneration of POR-PChlide
650
in PLB aged in NADPH-
free assay medium at pH 7.5. (A) The initial sample (thin line); aged
sample before (thick line) and after (medium line) exposure to light.
(B) Illuminated sample before (thin line) and after (thick line) dark
incubation with 50 l

M
NADPH and subsequent reillumination
(medium line) (C) difference spectra showing conversion of POR-
PChlide
650
to POR-PChlide
635
during ageing (thick line) and the
photoconversion of remaining photo-transformable pigment (medium
line) (D) difference spectra showing the regeneration of POR-
PChlide
650
in the presence of NADPH and (thick line) its subsequent
photoconversion on reillumination (medium line).
Fig. 11. NADPH dependence of regeneration of POR-PChlide
650
from
POR-PChlide
635
estimated from difference spectra of type shown in
Fig. 10D.
Fig. 12. Plots of the time dependence of the pH-driven blue-shift in the
absorption maxima of (A) Chlide and (B) PChlide (measured at 5° and
0 °C, respectively) following resuspension in assay medium pH 6.5 of
PLB initially suspended in assay medium pH 7.5. All samples contained
1.0 m
M
NADPH with either no other additions (j), 10 m
M
NaF alone

(d). 5 m
M
ATP (h), 5 m
M
ADP (s), 5 m
M
AMP (e), or 5 m
M
adenosine (n) in the presence or absence of 10 m
M
NaF as indicated.
2342 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
In contrast to the study on wheat EPIM [9], the addition
of ATP (or adenosine and the other adenyl nucleotides)
accelerated rather than inhibited the blue shift. An inhibi-
tion was observed if both ATP and NaF were present. A
similar inhibition, however, was also observed for ADP,
AMP and adenosine under these conditions indicating that
in maize PLB at least this inhibition is not ATP-specific. In
contrast to the study on wheat EPIM, no significant
difference was seen between the rate of the blue shift in the
presence or absence of NaF alone.
The NADPH-binding efficiency of POR-Chlide
684
,at
low pH is unknown. It is thus hard to disentangle the effects
of a possible loss of bound NADPH (leading to a reversal of
the NADPH-dependent red shift seen in Fig. 11) from those
of a physical dissociation of Chlide and/or conformational
changes associated with the pH-dependent conversion of

the enzyme from a more aggregated long-wavelength form
to a less-aggregated short-wavelength form. In an attempt
to isolate the contribution of the pH-driven conformational
changes from the other effects, a parallel study was carried
out on the low-pH induced blue shift in the PChlide
absorption maximum of nonphotoconverted POR-
PChlide
650
. Here, the photoconvertibility of POR-
PChlide
640
, the final product, indicates that both the
pigment and NADPH remain bound.
The rate of the changes in absorption for the nonpho-
toconverted enzyme was faster than those for the photo-
converted form, necessitating measurement at 0 °Cas
opposed to 5 °C. However, the general pattern of the
results, presented in Fig. 12B, is very similar to that for the
photoconverted enzyme, indicating that it is the conform-
ational changes that predominate in both cases. Minor
differences were seen in the rates of change seen for the
adenyl nucleotides in the presence of NaF, with ATP
showing the greatest inhibitory effect. To simplify presen-
tation, only those changes seen for ATP and NaF + ATP
are shown in Fig. 12B. The effect of the presence of the
adenylates on the ability of POR-PChlide
640
to undergo
photoconversion was checked by comparing the efficiency
of photoconversion of PLB samples containing excess

(1 m
M
) NADPH in the presence and the absence of 5 m
M
adenosine or the adenyl nucleotides. Little or no difference
was observed, indicating that although they had a marked
effect on the stability of POR-PChlide
650
, they had little
effect on the final level of NADPH binding to POR-
PChlide
640
.
Control measurements indicated that the effects of
adenosine and the adenyl nucleotides were limited to the
low pH range. No significant changes on the absorption
spectra of nonphotoconverted PLB containing POR-
PChlide
650
, or PLB photoconverted in the presence of
excess NADPH to form POR-Chlide
684
, were observed at
pH 7.5. However, as shown in Fig. 13, changes were seen, if
the measurements on the photoconverted enzyme were
made in the absence of excess NADPH. Under these
conditions, the absorption maximum of the freshly photo-
converted Chlide shows the usual decline from  679 to
676–677 nm. Addition of ATP leads to a rapid decrease in
the wavelength to 673–674 nm. If NADPH is then added,

the red-shift associated with the replacement of NADP
+
by
NADPH is not observed indicating that the pigment has
already dissociated from the parent enzyme. Similar, but
smaller, blue shifts were seen for ADP and AMP but no
discernible shift with respect to the adenylate-free control
was seen in the case of adenosine. In all cases, the results
were uninfluenced by the presence or absence of NaF.
DISCUSSION
Different spectral forms of POR
The sensitivity of the absorption and fluorescence spectra of
PChlide and Chlide of PLB to POR organization is well
documented. Griffiths and his coworkers [4,5] successfully
used this sensitivity to establish the basic framework of
relationships existing between the different ternary com-
plexes formed between POR, PChlide/Chlide and
NADP(H). The proposed relationship between the different
POR complexes referred to in this paper, based on the
generally accepted scheme of Oliver & Griffiths [5], is
summarizedinFig.14.
The relationship between spectral changes
in POR and structural changes in PLB
The existence of a correlation between the presence of the
cubic membrane structure of the PLB and the presence of
POR-PChlide
650
has long been recognized [20]. This rela-
tionship is underlined by studies on the etioplasts of mutants
such as the cop1mutantofArabadopisis [23] and the lip1

mutant of pea [24], which are both deficient in POR-
PChlide
650
and have been shown to be characterized by a
parallel deficiency in organized PLB.
The ability of the PLB to form a bicontinuous cubic
phase is linked to the high proportion of the nonbilayer
forming lipid monogalactosyldiacylglycerol (MGDG) pre-
sent in the membranes. MGDG normally accounts for
 50 mol% of the membrane lipids in the PLB membrane
[25]. The presence of such a high content of nonbilayer
forming lipid, however, is a necessary but not a sufficient
cause for cubic phase formation. Whilst cubic structures can
be formed in model systems containing high proportions
Fig. 13. Plots of the time dependence of the blue-shift in the absorption
maxima of Chlide following photoconversion of washed PLB suspended
in assay medium pH 7.5 containing no excess NADPH. Samples con-
tained no additions (j), or 5 m
M
adenosine or adenyl nucleotide in the
presence (d)or(s) absence of 10 m
M
NaF.
Ó FEBS 2002 pH-dependent changes in PLB organization (Eur. J. Biochem. 269) 2343
of MGDG, they are stable under only very limited ranges
of composition and hydration [26]. The existence of stable
cubic structure in the PLB is dependent on the membrane
protein content where POR is by far the dominant
component. The mechanism by which POR-PChlide
650

stabilizes the cubic structure of the PLB is not fully
understood but probably reflects its accumulation in, and
subsequent stabilization of, membrane regions of a local
curvature important to the overall stability of the cubic
phase. Conversion of POR-PChlide
650
to POR-PChlide
640
,
possibly by disaggregation, removes these constraints
destabilizing the cubic phase, resulting in the relaxation
of the membrane into the planar configuration characteris-
tic of the prothylakoid region of the etioplast membrane
(within the intact etioplast, at least). In the absence of
attached prothylakoids, PLB are unable to undergo such a
reorganization, possibly accounting for their high pH
sensitivity.
Driving force for pH-dependent changes
Parallel changes in the spectral properties of POR and
structural properties of PLB similar to those studied in this
paper have been reported to occur in salt-washed PLB
[12,27]. The two phenomena are clearly related and strongly
point to the importance of changes in the surface properties
of the PLB membrane. The obvious candidates for the
driving forces in the case of the pH-dependent changes are
either changes in the ionization of the membrane lipid
headgroups, leading to a destabilization of the lipid–protein
interactions that normally stabilize the cubic structure of the
membrane, and hence to a destabilization of POR-
PChlide

650
, and/or changes in the ionization of groups
associated with POR. These changes then lead to the
destabilization of POR-PChlide
650
and membrane reorgan-
ization.
The suspension of total polar lipid extracts of chloroplast
membranes, which have a very similar lipid composition to
the PLB membrane, in low pH media favours membrane
fusion and formation of nonlamellar structures. The pK
a
for
this process is  pH 4.5 [28], indicating that it reflects the
protonation of the acidic lipids present in the mixture. The
critical pH for the changes reported in the present study is
close to pH 7, suggesting that the initial changes are unlikely
to be directly related to changes in lipid headgroup
ionization.
Our results can be explained by attributing the effects of
low pH to a reduction in the strength of NADPH binding to
POR-PChlide
650
that triggers its relaxation to POR-
PChlide
640
/POR-PChlide
635
, which then destabilizes the
cubic structure of the membrane. This reduced NADPH

binding capability of POR is reflected in the large disparity
in the half-saturation concentration for the restoration of
POR photoconvertibility in low pH treated PLB, about
0.25 m
M
NADPH at pH 6.5 (Fig. 8B), as compared to
 1 l
M
NADPH for the reformation of POR-PChlide
650
in
PLB aged at pH 7.5 (Fig. 10). The importance of mem-
brane morphology in these processes is underlined by the
failure of added NADPH to reform POR-PChlide
650
in low
pH-treated PLB on restoration to pH 7.5 (Fig. 4). This is
almost certainly a direct reflection of PLB membrane
disruption linked with the pH-cycling. Once the membrane
has reorganized into a tubular form, there is essentially no
way back to a cubic structure under these conditions, hence
the lack of reformation of POR-PChlide
650
. Addition of low
concentrations of NADPH to PLB aged at pH 7.5 which
show only limited structural reorganization, in contrast,
results in a rapid reconversion of POR-PChlide
635
to POR-
PChlide

650
with no obvious effect on membrane organiza-
tion.
Adenylate-sensitivity of pH effects
POR, in common with many NADPH-binding enzymes,
contains the characteristic motif Gly-X-X-X-Gly-X-Gly
associated with the bA-aA-bB binding domain known as
the Rossmann fold [29]. The detailed organization of the
NADPH-binding pocket has yet to be established for POR
but it has been determined in other members of the short-
chain dehydrogenase/reductase family [30]. In common
with Rossmann folds in general, these sites contain two
mononucleotide binding sites; one for the nicotinamide and
one for the adenosine moiety [31].
2¢-Adenyl nucleotides, of the type found in NADPH, and
the 5¢-nucleotides used in this study, are both known to bind
within the adenosine site of such folds and can act as
Fig. 14. Model illustrating the relationship
between the different POR-PChlide and
POR-Chlide complexes studied in this paper.
2344 E. Selstam et al. (Eur. J. Biochem. 269) Ó FEBS 2002
inhibitors interfering with NAD(P)
+
binding [32,33]. A
possible explanation of the acceleration of the low-pH
driven spectral changes seen for the photoconverted and
nonphotoconverted enzymes on addition of the adenyl
nucleotides or adenosine (Fig. 12) is that these compounds
are able to compete for the NADPH binding site under low
pH conditions destabilizing the aggregated POR-PChlide

650
complex. Their effect on NADPH binding to POR,
however, appears to be transitory as the efficiency of
binding of NADPH to POR-Chlide
640
at low pH, is not
significantly impaired by the presence of 5 m
M
ATP.
In contrast to the present results, Wiktorsson et al.[9]
working with wheat EPIM observed a reduction in the rate
of the low pH-induced blue shift in the Chlide fluorescence
maximum in the presence of ATP. This could in principle
reflect a species difference or a difference in the nature of the
preparations. Measurements of Grevby et al. [18] indicate
the retention of significant levels of low-temperature fluor-
escence emission from POR-PChlide
650
in wheat PLB
incubated at pH 6.0 for 48 h at 0 °C. This suggests that
wheat PLB may be less pH-sensitive than their maize
counterparts. Given the strong connection existing between
POR organization and membrane morphology seen in the
present study, the use of EPIM with their increased scope
for membrane organization would only serve to enhance
these differences.
Relation to POR phosphorylation
ThemeasurementsonPLBagedatpH7.5shownin
Figs 10,11, confirm the finding of Griffiths [16] and
Brodersen [17], who both suggested that the formation of

POR-PChlide
650
from pre-existing POR-PChlide
635
(P-630),
is solely dependent upon the presence of NADPH and does
not appear to require ATP. This contrasts the earlier studies
of Horton & Leech on aged maize etioplasts [14,15] where
this conversion appeared to be ATP-driven. The possibility
that the preparations employed by Griffiths and ourselves
have lost the putative kinase during the course of prepar-
ation cannot be excluded, but that still leaves unanswered
the question of how the addition of micromolar concentra-
tions of NADPH suffice to drive the conversion of POR-
PChlide
635
to POR-Chlide
650
in its absence. It is noteworthy
that the preparations used in the earlier studies contained
sufficient excess NADPH to allow NADP
+
/NADPH
exchange in the photoconverted enzyme leading to the
formation of POR-Chlide
685
[15] raising the possibility that
these results might reflect an ATP-dependent perturbation
in NADPH binding efficiency rather than a direct POR-
phosphorylation step.

Kovacheva et al. [11] have recently reported ADP to
inhibit the blue shift of the fluorescence emission of the
Chlide peak of phototransformed wheat EPIM prepar-
ations from 695 nm to 680 nm. A small amount of reformed
phototransformable PChlide with an emission peak at
655 nm was observed at the same time. This inhibition was
not seen if ADP was replaced by ATP. The reformed
PChlide formed under these conditions was nonphototrans-
formable and emitted at 651 nm rather than 655 nm. In the
same report, the protein kinase inhibitor, K252a, was
observed to inhibit the reformation of nonphototransform-
able PChlide and no phototransformable PChlide was
formed following subsequent addition of ATP and
NADPH. The presence of a 695-nm emission peak indicates
that the samples forming phototransformable PChlide
contained sufficient residual NADPH to allow NADP
+
/
NADPH exchange in the photoconverted pigment and,
presumably, in the reformed PChlide complex. The above
results could thus reflect a role for an ADP-dependent
kinase step in the initial loading of PChlide on to POR.
The absence of any apparent ATP (or ADP) requirement
for the regeneration of photoconvertible PChlide in model
systems supplemented with exogenous pigment [34] or for
the mobilization of endogenous pigment in isolated etioplast
membranes [35] would, however, seem to argue against a
need for such a kinase. Klement et al. [36], have recently
reported the formation of a complex between pigment-free
POR and Zn-protopheide a in the presence of etioplast

lipids. Again, no phosphorylation step is involved in
pigment loading. The precise role of any possible POR
kinase therefore still remains unclear.
The main line of evidence for the existence of a POR-
phosphatase is the observation by Wiktorsson et al. [9] of an
inhibition, by NaF and ATP, of the loss of the long-
wavelength form of Chlide following photoconversion of
reformed phototransformable PChlide in wheat EPIM. In
agreement with these findings, we observed related inhibi-
tions of the pH-driven blue shift in Chlide and PChlide
absorption in the presence of ATP and NaF (Fig. 12). The
presence of NaF clearly perturbs the PLB system in some
way. However, it must be emphasized that these effects, in
maize PLB at least, are only observed under low-tempera-
ture conditions and when adenosine or adenyl nucleotides
are present. The possibility of other explanations of these
effects cannot therefore be excluded.
CONCLUSIONS
The present study emphasizes the close relationship existing
between the local organization of the PLB membrane and
the stability of different POR-pigment complexes. It dem-
onstrates the extreme sensitivity of these complexes, in PLB
at least, to small changes in pH. It also confirms the central
role of NADPH in the reformation of POR-PChlide
650
in
aged PLB and raises the question that the sensitivity of
spectral changes to the presence of adenyl nucleotides
may reflect their effects on NADPH binding rather than
their effects on specific phosphorylation (or adenylation)

steps.
ACKNOWLEDGEMENT
The support of the Swedish Natural Research Council is gratefully
acknowledged.
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