Tài liệu Báo cáo Y học: The effects of low pH on the properties of protochlorophyllide

The effects of low pH on the properties of protochlorophyllide

oxidoreductase and the organization of prolamellar bodies

of maize (Zea mays)

Eva Selstam1

, Jenny Schelin1

, Tony Brain2 and W. Patrick Williams2

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-PChlide640 and 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 mM NADPH for

POR-PChlide640 reformation. The disappearance of POR￾PChlide650 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-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￾PChlide650 in 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)645 and 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 -PChlide640 and 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 themain absorptionmaximum 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-PChlide640 and POR￾PChlide650 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: [email protected]

Abbreviations: Chlide, chlorophyllide; PChlide, protochlorophyllide;

PLB, prolamellar body; POR, protochlorophyllide oxidoreductase;

POR-PChlide635, POR -PChlide640, POR-PChlide650, POR -

Chlide676)677 and POR-Chlide684, 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)
FEBS 2002 doi:10.1046/j.1432-1033.2002.02897.x