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
Trang 1Prolamellar bodies (PLB) contain two photochemically
active forms of the enzyme protochlorophyllide
oxido-reductase POR-PChlide640and POR-PChlide650(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-PChlide650 to POR-PChlide640 and a
dramatic re-organization of the PLB membrane system In
the absence of excess NADPH, the absorption maximum of
the PORcomplex 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 mMNADPH for
POR-PChlide640reformation The disappearance of
POR-PChlide650and 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-PChlide650 Related spectral
changes are seen in PLB aged at room temperature at pH 7.5
in NADPH-free assay medium The reformation of POR-PChlide650in this system is readily reversible on re-addition
of NADPH with a half-saturation value about 1.0 lM Comparison of the two sets of changes suggest a close link between the stability of the POR-PChlide650, 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 PORcomplex
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 PChlide628)633 and two photoconvertible
forms PChlide640)645and PChlide650)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 referred to here to as POR -PChlide640and POR-PChlide650 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 PORhas been the subject of much discussion A number
of lines of evidence suggest that POR-PChlide640and POR-PChlide650are 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: patrick.williams@kcl.ac.uk
Abbreviations: Chlide, chlorophyllide; PChlide, protochlorophyllide;
PLB, prolamellar body; POR, protochlorophyllide oxidoreductase;
POR-PChlide 635 , POR -PChlide 640 , POR-PChlide 650 , POR
-Chlide676)677and 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)
Trang 2step in the interconversion of different forms of PORcan be
traced back to an early series of experiments by Horton &
Leech [14,15] in which the transformation of
POR-PChlide650to 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-PChlide650on 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-PChlide650was 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 PORand a dissociation of Chlide from
the PORcomplex, 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-PChlide650to POR-PChlide640 These
changes, which take place over an extremely narrow pH
range, are shown to be accompanied by marked decreases in
the ability of PORto 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
M A T E R I A L S A N D M E T H O D S
PLB isolation
Maize seedlings (Zea mays L cv Apache) were grown in
the dark for 9 days at 24C 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 C in 1.3Msucrose, 50 mMKCl, 1 mMMgCl2, 1 mM
EDTA, 0.3 mM NADPH, 20 mM Tricine, 10 mM 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 mMsucrose, 50 mMKCl, 1 mMMgCl2, 1 mMEDTA
and 30 mM 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 (77K) 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 mM
cacodylate buffer, pH 7.4 They were then post-fixed with osmium tetroxide, embedded and sectioned The sections, stained in 2% uranyl acetate followed by lead citrate, were examined using a Philips EM301G electron microscope
R E S U L T S
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 77K 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-PChlide650 and POR-PChlide640, 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 77K fluorescence emis-sion spectrum of the nonphotoconverted PLB is dominated
by the 656-nm emission peak of POR-PChlide650(Fig 1B) Some emission from the nonphotoconvertible species PChlide628is visible at about 630–635 nm but little or no emission is seen from POR-PChlide640, reflecting the efficient excitation energy transfer existing between this species and POR-PChlide650[13] Samples photoconverted
in the presence of excess NADPH are characterized by a maximum at 696 nm typical of the Chlide derivative of POR-PChlide650
The results for maize PLB resuspended at pH 6.5 are strikingly different Under these conditions, there is a rapid conversion of POR-PChlide650 to POR-PChlide640 (Fig 1C) Exposure to bright white light leads to the photoconversion of POR-PChlide to its corresponding
Trang 3Chlide 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-PChlide650 is
completely converted to POR-PChlide640 Following
photo-conversion, the emission peak shifts to 682 nm reflecting the
formation of the Chlide derivative of POR-PChlide640
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-PChlide640to a
nonphotoconverti-ble species POR-PChlide635lacking bound NADPH which
rapidly reconverts to POR-PChlide640 in the presence of
added NADPH POR-PChlide635has a 77K fluorescence
emission peak at 640 nm of similar intensity to the 648 nm
emission peak of POR-PChlide640(data not shown) clearly
distinguishing it from PChlide628, which emits at 633 nm
and remains nonphotoconverted even in the presence of
excess NADPH
The pH-driven conversion of POR-PChlide650to
POR-PChlide640 (POR-PChlide635 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
0C (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 mM 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) after exposure to a second flash of bright white light (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) and the absence (d) of 1.0 m NADPH.
Trang 4640 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-PChlide635to break down to
yield a new PChlide absorption band with a maximum at
653 nm (PChlide653) 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 PChlide653
is easily distinguishable from POR-PChlide650 as it is nonphotoconvertible and gives rise to no obvious low-temperature fluorescence It is probably related to the species PChlide647, 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 PChlide653 formation was avoided
The pH-dependence of the conversion of POR-PChlide650to POR-PChlide640/POR-PChlide635is 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-PChlide650[20] TEM measure-ments were therefore carried out to determine whether or not loss of POR-PChlide650 correlates with loss of PLB structure Typical electron micrographs of PLB samples suspended in pH 7.5 and pH 6.5 assay medium are
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) and the absence (d) of 1.0 m M NADPH.
Trang 5presented 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 PORcomplex, 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-PChlide635, as opposed to
POR-PChlide640, in PLB samples resuspended in low pH assay
medium in the absence of excess NADPH strongly suggests
that POR-PChlide640, under these conditions at least, has a
greatly reduced affinity for NADPH The NADPH binding
capability of POR-PChlide635/POR-PChlide640was
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-PChlide640 and
nonphotoconverti-ble POR-PChlide635 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 mM
NADPH, indicating that POR-PChlide640binds 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-PChlide640at pH 7.5 Attempts to remove NADPH from POR-PChlide640and/or POR-PChlide650by 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.
Trang 6washing 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-PChlide635 complex to form
PChlide653, which interfered with the subsequent spectral
analysis If NADPH is rebound before the restoration of the
pH to pH 7.5, relatively little PChlide653is 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 5C 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 lM 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-PChlide650
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-PChlide650was 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-PChlide635 If excess NADPH is not
removed, POR-PChlide650 is initially converted to
POR-PChlide640, 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-PChlide650
to POR-PChlide635, its reconversion to POR-PChlide650
and its subsequent photoconversion to POR-Chlide684, are
all clearly visible in the difference spectra shown in
Fig 10(C,D) These changes are similar to those reported
by Brodersen [17], who worked with aged barley PLB The NADPH dependence of the reformation process was estimated from measurements of the amounts of reformed POR-PChlide650available for photoconversion from differ-ence spectra of the type shown in Fig 10D The half-saturation value for NADPH binding to POR-PChlide635at
pH 7.5 estimated on this basis is 1 lM (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) or 1 l M NADPH (m) added 7 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.
Trang 7involvement 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-PChlide650 suspended in assay medium at pH 7.5 to a much larger volume (1 mL) of
pH 6.5 assay medium containing 1 mMNADPH, 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 5C 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-Chlide684prior
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.
Trang 8In 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-Chlide684, 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-PChlide650 Here, the photoconvertibility of
POR-PChlide640, 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 0C as
opposed to 5C 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-PChlide640 to undergo
photoconversion was checked by comparing the efficiency
of photoconversion of PLB samples containing excess (1 mM) NADPH in the presence and the absence of 5 mM
adenosine or the adenyl nucleotides Little or no difference was observed, indicating that although they had a marked effect on the stability of POR-PChlide650, they had little effect on the final level of NADPH binding to POR-PChlide640
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-PChlide650, or PLB photoconverted in the presence of excess NADPH to form POR-Chlide684, 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
D I S C U S S I O N
Different spectral forms of POR The sensitivity of the absorption and fluorescence spectra of PChlide and Chlide of PLB to PORorganization 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 PORcomplexes referred to in this paper, based on the generally accepted scheme of Oliver & Griffiths [5], is summarized in Fig 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-PChlide650has long been recognized [20] This rela-tionship is underlined by studies on the etioplasts of mutants such as the cop1 mutant of Arabadopisis [23] and the lip1 mutant of pea [24], which are both deficient in POR-PChlide650and 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.
Trang 9of 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 PORis by far the dominant
component The mechanism by which POR-PChlide650
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-PChlide650to POR-PChlide640,
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 PORand
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-PChlide650, and/or changes in the ionization of groups
associated with POR These changes then lead to the
destabilization of POR-PChlide650and 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 pKafor
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-PChlide650 that triggers its relaxation to POR-PChlide640/POR-PChlide635, which then destabilizes the cubic structure of the membrane This reduced NADPH binding capability of PORis reflected in the large disparity
in the half-saturation concentration for the restoration of PORphotoconvertibility in low pH treated PLB, about 0.25 mM NADPH at pH 6.5 (Fig 8B), as compared to
1 lMNADPH for the reformation of POR-PChlide650in 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-PChlide650in 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-PChlide650 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-PChlide635to POR-PChlide650with 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.
Trang 10inhibitors 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-PChlide650
complex Their effect on NADPH binding to POR,
however, appears to be transitory as the efficiency of
binding of NADPH to POR-Chlide640 at low pH, is not
significantly impaired by the presence of 5 mMATP
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-PChlide650 in wheat PLB
incubated at pH 6.0 for 48 h at 0C This suggests that
wheat PLB may be less pH-sensitive than their maize
counterparts Given the strong connection existing between
PORorganization 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
The measurements on PLB aged at pH 7.5 shown in
Figs 10,11, confirm the finding of Griffiths [16] and
Brodersen [17], who both suggested that the formation of
POR-PChlide650from pre-existing POR-PChlide635(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-PChlide635to POR-Chlide650in 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-Chlide685[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 phototransformnonphototransform-able 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 PORand 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
C O N C L U S I O N S
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-PChlide650in 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
A C K N O W L E D G E M E N T The support of the Swedish Natural Research Council is gratefully acknowledged.
R E F E R E N C E S
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in isolated etioplast inner membranes Photochem Photobiol 52, 83–87.
2 Bo¨ddi, B., Ryberg, M & Sundqvist, C (1993) Analysis of the 77 K fluorescence emission and excitation spectra of isolated etioplast inner membranes J Photochem Photobiol B Biol 21, 125–133.
3 Shibata, K (1957) Spectroscopic studies on chlorophyll formation
in intact leaves J Biochem 44, 147–153.