NOTES

The photodamage process of

pigments and proteins of PSI

complexes from Spinacia

Oleracea L.

WEI Jie1, YU Hui

1, LI Liangbi

1, KUANG Tingyun

1,

WANG Jushuo2, GONG Yandao

2 & ZHAO Nanming

2

1. Laboratory of Photosynthesis Basic Research, Institute of Botany,

Chinese Academy of Sciences, Beijing 100093, China;

2. Department of Biological Science and Biotechnology, Tsinghua Uni-

versity, Beijing 100084 China

Correspondence should be addressed to Kuang Tingyun.

Abstract Purified PSI complexes from Spinacia Oleracea

L. were exposed to the strong light (PFD=2300 mol m2s

1)

for various period. Along with the illumination the photo-

damage process of pigments and proteins of PSI complexes

was investigated using absorption, fluorescence, circular

dichroism (CD) spectroscopy and SDS-PAGE. It was found

from the optical absorption spectra that the maximal ab-

sorbance of PSI complexes decreased and maximal peaks

blue-shifted during the illumination, and the forth derivative

spectra demonstrated that the absorbance decreasing at red

region mainly resulted from the aborbance decreasing of the

long wavelength Chla, implying that the long-wavelength

Chla was readily to be bleached. The CD signals contributed

by LHCI decreased more rapidly than other CD signals con-

tributed by Chla and Carotenoid, indicating that the LHCI

was more sensitive to light than core complexes. It was ob-

served by SDS-PAGE that some small polypeptides of PSI

complexes were damaged earlier than reaction center pro-

teins PsaA and PsaB. Lhca3, Lhca2 and PsaD were the early

degraded proteins during illumination. In addition, it is also

observed that the insoluble-cohesive-denatured proteins ap-

peared after prolonged illumination.

Keywords: PSI pigment-protein complex, photodamage, LHCI,

absorption spectra, CD spectra.

The early considerable studies suggested PSII as the

main site of lesion in photoinhibition. Powles concluded

in his review that the degree of inhibition of PSI was less

than that of PSII, or in some cases be negligible compared

with that of PSII[1]

. Terashima et al. discovered PSI is

much more susceptible to aerobic photoinhibition than

PSII in chilling-sensitive plants[2]

. Then the physiological

significance of the photoinhibition of PSI was recognized.

Havaux et al. suggested that PSI is the rate-limiting step in

electron transport rather than PSII in chilling-sensitive

plants[3]

. Recent findings demonstrated significant inacti-

vation of PSI by low light treatment at low temperatures

also happened in cold-tolerant barley[4]

and rye[5]

.

Photoinhibition of PSI at chilling temperature thus ap-

pears to be a general phenomenon in higher plants[6]

. Re-

cent observations showed that the active oxygen generated

by PSI also led to the inactivation of PSII[7]

. Sonoike[8]

concluded in his review that some mechanisms exist to

protect PSI against photoinhibition in vivo; and the

mechanism is inactivated during the isolation of thy-

lakoid membranes; in addition, the protective mechanism

is chilling-sensitive factor in chilling-sensitive plants.

Baba et al.[9, 10]

observed the bleaching of chlorophyll,

degradation of reaction center proteins and decreasing of

electron transfer activity when PSI preparations were ex-

posed to strong light irradiation. It has been found that

PsaB protein, one of the reaction center subunits of PSI,

degraded by light illumination of spinach thylakoid mem-

branes[11]

. In the present report, the photodamage process

of the subunits and chlorophyll of PSI particles during

illumination was investigated. It was found that the LW

Chla was readily to be bleached. PsaD and the subunits of

LHCI degraded more rapidly than reaction center proteins

PsaA/B. The results are discussed, associated with the

structure and function of PS I complex.

1 Materials and methods

( ) The PSI-200 particles were isolated and purified

from fresh thylakoid membranes of Spinach Oleracea L.

leaves as described by Mullet et al.[12]

. Chl concentration

was determined according to the method of Arnon[13]

.

( ) White light illumination (PFD = 2300 mol

m2s

1), measured by the luminometer (Model LI-189,

L1-COR, Lincoln, NE, USA), was provided from a 50W

tungsten lamp at 25 with stirring, and the water filter

was used for heat insulation. PSI-200 particles were re-

suspended in 0.1 mol/L sorbitol, 10 mmol/L NaCl,

50 mmol/L Tricine-NaOH (pH 7.8) and 0.05% (W/V)

Triton X-100 at 50 gChl/mL (200 gChl/mL for SDS-

PAGE).

( ) Absorption spectra were measured with a Con-tron UV-943 spectrophotometer at room temperature. The

samples were scanned at 100 nm/min with a 0.5 nm reso-

lution at 50 gChl/mL.

( ) CD spectra were measured with a Jasco J-500cs

spectro-polarimeter at a scanning speed of 100 nm min1,

a bandwidth of 2 nm, a response time of 1 s and an accu-

mulation of four times. The samples were scanned at 30

gChl/mL. The corresponding absorption spectra were

obtained from the optical density conversion of the high

tension voltage, which was recorded simultaneously with

the CE data, using the Standard Analysis program pro-

vided by Jasco.

( ) SDS-PAGE was performed as the method of Yu

et al.[14]

. The gels were stained with Coomassie Brilliant

Blue R-250 and scanned with a CS-910 densitometer at

591 nm.

2 Results

( ) The degradation of polypeptides of PSI pig-

1812 Chinese Science Bulletin Vol. 46 No. 21 November 2001

ment-protein complexes. Fig. 1 shows the polypeptides

of the PS I complexes after different periods of illumina-

tion. It was found that PsaD of PS I complex as well as

Lhca2 and Lhca3 (23, 25ku) of LHCI began degrading

after 10 min illumination, and degraded thoroughly after

80 min. However, other two subunits of LHCI, Lhca1 and

Lhca4 (20.5, 21 ku), degraded only 30% after 100 min

illumination. The reaction center subunits (66, 67 ku) be-

gan to degrade after 60 min, and degraded 15% after 100

min illumination (lane 9). A 220 ku insoluble-cohesive-

denatured protein band appeared at the upper of the sepa-

ration gel after 100 min illumination (lane 9), by which it

was suggested that some proteins were polymerized by

illumination.

was destroyed. It was considered that the positive peak at

668 nm and negative peak at 684 nm, as well as positive

peak at 446 nm were the characteristic peaks of Chla; the

negative peaks at 650 nm and 461.4 nm and the positive

peak at 480 nm generally have been considered to be con-

tributed by Chlb of PSI complex; the positive peak at 500

nm has been suggested to be contribution of Carote-

noids[15—17]

. By analysis of decrease percentage of CD

signals, it was found that two negative peaks at 650 nm

and 461.4 nm contributed by LHCI decreased most rap-

idly, at the same time a pair of positive-negative peaks at

668 nm and 683.4 nm, suggested to be the result of exci-

ton interaction between Chla, decreased most slowly (fig

2(b)). The decreasing difference of CD signals increased

along the prolonged illumination, which implied that the

different destroy degree of regular interaction between

chromophores. The CD signal at 500 nm decreased more

rapidly than at 480 nm at the beginning 40 min illumina-

tion, but more slowly than at 480 nm after 40 min pro-

longed illumination. It was possibly indicated that the

Chlb had been destroyed more than Carotenoids after 40

min illumination.

( ) Spectra analysis of pigments photodamage

process of PSI pigment-protein complex. A CD spec-

trum is the difference spectra between levorotatory polar-

ized light and dextrorotatory polarized light through sam-

ples. A CD spectrum sensitively reports the microenvi-

ronmental changes of the chromophore. It was shown that

CD signals of PSI particles decreased along with the illu-

mination and peaks shifted (fig. 2(a)), by which it was

indicated that the regular arrangement of pigments of PSI As the absorption spectra of PSI complexes shown

Fig. 1. SDS-PAGE profile of the

polypeptide compositions of PSI com-

plexes after various periods of illumina-

tion. 1, Marker; 2, control (0 min); 3,

5 min; 4, 10 min; 5, 20 min; 6, 40 min;

7, 60 min; 8, 80 min; 9, 100 min.

Fig. 2. The circular dichroism (CD) spectra (a) and the decrease percentage of CD signal intensities (b) of PSI complexes after various

periods of illumination (PSI complex without illumination as control).

Chinese Science Bulletin Vol. 46 No. 21 November 2001 1813

NOTES

(fig. 3(a)), the maximal absorbance of PSI complexes de-

creased along with the illumination, and the maximal ab-

sorbance at the red region decreased more rapidly than

that at blue region, also the maximal peak at the red re-

gion blue-shifted more than that at blue region (blue shift

7 nm at the red region and 2 nm at the blue region after

100 min illumination). The forth derivative spectra dem-

onstrated that the absorbance decrease at the red region

mainly resulted from the absorbance decrease of the

longer wavelength Chla (fig. 3(a), inserted). The absorb-

ance at 682 nm decreased more quickly than that at 667

nm during PSI complexes exposed to the strong light, and

the absorbance at 667 nm became higher than that at 682

nm after 100 min illumination. So it was possible that the

bleaching of the long wavelength Chla resulted in the

blue-shift of the maximal peak at the red region. The ab-

sorption difference spectra of PSI complexes (control mi-

nus illuminated) contained the maximal peaks at 682 nm,

485 nm, 448 nm and 425 nm (data not shown). At the red

region the peak of the absorption difference spectra was at

682 nm but not at 679 nm at which just was the maximal

absorbance of the absorption spectra at the red region.

Also it was indicated by which that longer wavelength

Chla were destroyed more easily during illumination. By

comparatively analyzing the aborbance decrease percent-

age of the PSI complexes from absorption difference

spectra (fig. 3(b)), it was found that the absorbance at 485

nm and at 682 nm decreased at a close rate. In the mean-

while the absorbance at 448 nm decreased more slowly

and the absorbance at 425 nm decreased most slowly. In

addition, it was found that the peak at 485 nm of absorp-

tion difference spectra of PSI complexes illuminated 5

min shifted to 477 nm after PSI complexes illuminated

100 min. It was demonstrated that the maximal absorb-

ance of Chlb of PSI complexes was at about 473 nm and

Car of PSI complexes was at about 500 nm (data not

shown). So the peak shift from 485 nm to 477 nm during

the prolonged illumination implied that more Chlb than

Carotenoids were destroyed during the later stage of illu-

mination. The result is consistent with that of CD.

3 Discussion

LHCI contains four aproproteins, namely Lhca1,

Lhca2, Lhca3 and Lhca4. The isolated LHCI can be fur-

ther fractionated into LHCI-680 and LHCI-730, which

emit at 680 nm and 730 nm, respectively[18, 19]

. LHCI-730

contains Lhca1 and Lhca4 with a Chla/b ratio about 2.3,

LHCI-680 contains Lhca2 and Lhca3 with a Chla/b ratio

about 1.4[20]

. The structure of PSI complex was studied by

electron microscopy and it was found that PSI core com-

plex surrounded by a monolayer molecules, it was con-

cluded that a shell of about eight light-harvesting complex

(LHCI) subunits attached to the PSI-100 complex[21]

. Thus

it is possible that the proteins of LHCI degraded more

rapidly than reaction center protein PsaA and PsaB re-

sulted from the fact that they were located outside of PSI

complex and received more excessive illumination. How-

ever, it was obvious that the observed significant degrada-

tion difference between LHCI-680 and LHCI-730 could

not be explained by their locations. The previous studies

showed that LHCI-730 is closer to PSI core complex, and

LHCI-680 is easier than LHCI-730 to be separated from

PSI core complex[15]

. It has also been suggested that

LHCI-680 directly connected LHCI to the PSII

light-harvesting complex (LHCII)[14]

. Thus, it is suggested

that the instability and sensitivity of LHCI-680 to strong

light makes it possibly play a role in adjusting the light

energy balance between two photosystems. It needs to be

Fig. 3. The absorption spectra of PSI complexes after various period of illumination (a), and the decrease percentage of absorbance at

682 nm, 448 nm, 485 nm and 425 nm at which the maximal peaks in the absorption difference spectra (control minus illuminated) were

during the illumination (b). The line indication in (a) is the same as in fig. 2(a), PSI complex without illumination as control, the inserted

in (a) is the forth derivative spectra.

1814 Chinese Science Bulletin Vol. 46 No. 21 November 2001

further studied to reveal the difference between LHCI-680

and LHCI-730.

PsaD is one of the extrinsic proteins of PSI complex.

It is now clear that PsaC, PsaD and PsaE are in intimate

contact with each other on the stromal side of PSI com-

plex[21,22]

. It was demonstrated by reconstitution in vitro

that PsaD is required for the stable binding of PsaC to the

PSI core protein and PsaC also is absolutely necessary for

the integrating of Psa D and PsaE to the PSI core pro-

tein[22,23]

. In this note, it was found that PsaE is more sta-

ble than PsaD as PsaD is easier to be degraded than PsaE

during illumination. It was reported that three iron-sulfur

center, FA, FB and FX were destroyed by illumination[24,25]

.

Denatuartion of FA/FB could result in the unfolding of the

PsaC, and further affect the stability of PsaD and PsaE. So

it is possible that the degradation of PsaD and PsaE during

light treatment is related to the denaturation of iron-sulfur

centers. As PsaE is much more stable during illumination

than PsaD, it implied that PsaD is closer than PsaE to

PsaC. SDS-PAGE analysis of the polypeptide composition

of PSI complex showed that there was polyreaction be-

tween proteins and no new fragments were observed.

Baba et al.[10]

have not detected the fragments of the reac-

tion center proteins of PSI complex after light treatment

either. So this suggests that there occurred polyreaction

instead of degradation of proteins of PSI complex in vitro

with strong light treatment.

The absorption spectra and forth-derivative spectra

demonstrated that Chla in PSI complex followed the prin-

ciple that Chls absorbing longer wavelength light would

be early destroyed during excess light treatment. Thus, the

long-wavelength Chla of PSI absorbing long wavelength

light more than 700 nm, were destroyed firstly and prac-

tically took a role of protection to P700. According to the

same pronciple, Chlb of PSI complex should be bleached

after Chla. However, both of absorption difference spectra

and CD spectra showed that Chlb of PSI complex was

also easy to be destroyed. As the proteins of LHCI were

easier to be degraded than proteins of reaction center by

light treatment, it is reasonable that Chlb of PSI only as-

sociated with LHCI was readily to be impaired during

illumination. Bleached Chlb could not absorb and funnel

light energy to Chla, and practically protected the reaction

center against absorbing excess light.

77 K fluorescence spectra showed that the fluores-

cence emission peak blue-shifted (blue-shift 5 nm after 40

min illumination) and decreased quickly during illumina-

tion (data not shown). The previous papers reported that

the long-wavelength Chla has a maximum fluorescence at

735 nm associated with LHCI, and the long-wavelength

Chla has a maximum fluorescence at 720 nm associated

with PSI core complex[26]

. The blue-shift of fluorescence

peak of PSI complex indicated that the Chls associated

with LHCI were easier to be bleached by strong light

treatment than those associated with core complex.

The spectra analysis showed that the CD signals and 77 K fluorescence emission of PSI complex decreased

more rapidly than absorbance of PSI complex during

strong light illumination. There was no fluorescence of

PSI complex excited at 440 nm detected after 60 min il-

lumination (data not shown). Meanwhile absorbance of

PSI at the blue region only decreased by 20%. It indicated that the energy transfer of light-harvesting system of PSI

was blocked. So it was concluded that the excess light

treatment could not destroy and bleach the Chls of PSI

complex thoroughly in a short time, but it could affect the

structure of Chls and block the normal energy transfer in

PSI complex. Chls are indispensable for the constitution of the normal function-structure of PSI pigment-protein

complex. A great damage of chlorophylls could result in

the degradation of the subunits of PSI complex. In addi-

tion, the sensitivity of PSI complex to illumination is also

related to the structure and the arrangement of proteins,

membrane-lipids and characteristics of proteins them-selves.

Acknowledgements This work was supported by the National Natural

Science Foundation of China (Grant No. 39890390), the State Key Basic

Research and Development Program (Grant No. G1998010100), and the

Innovative Foundation of Laboratory of Photosynthesis Basic Research,

Institute of Botany, the Chinese Academy of Sciences.

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(Received June 7, 2001)

1816 Chinese Science Bulletin Vol. 46 No. 21 November 2001