Planta (1996)199:118-125 P l m a t a

9 Springer-Verlag 1996

Long-term drought stress induces structural and functional

reorganization of photosystem II
M.T. Giardi 1, A. Cona 1, B. Geiken 1, T. Ku(zera 1., J. Masojidek 1.*, A.K. Mattoo 2
1 Italian National Council of Research (BEV-CNR), Area della Ricerca di Roma, Via Salaria km 29.3, 1-00016 Monterotondo Scalo, Italy
2 Plant Molecular Biology Laboratory, U.S. Department of Agriculture, BARC, Beltsville, MD 20705, USA
Received: 26 April 1995/Accepted: 21 August 1995

Abstract. Long-term drought stress on photosystem II Introduction

(PSII) was studied in pea ( P i s u m s a t i v u m L.) seedlings.
Drought stress (reduction of water content by 35-80%)
led to a considerable depletion of the PSII core, and the
remaining PSII complex appeared to be functional and
reorganized, with a unit size (LHCP/PSII core) twofold
greater than that of well-irrigated plants. By immunoblot-
ting analysis of the PSII proteins from grana and stroma
lamellae, the enhanced degradation of CP43 and D1 pro-
teins was observed in water-stressed plants. Also, water
stress caused increased phosphorylation of the PSII core
and increased D1 protein synthesis. Water-stress-me-
diated increase in D1 synthesis did not occur when
plants were exposed to photoinhibitory light. The de-
pletion of the PSII core was essentially reversed when
water-stressed plants grown at low visible irradiance were
watered. We suggest that the syndrome caused by the
effect of long-term water stress on photosynthesis is
a combination of at least two events: a reduction in the
number of active PSII centres caused by a physical de-
stabilization of the PSII core and a PSII reorganization
with enhanced D1 turnover to counteract the core de-
pletion.
Key words: D1 protein (turnover, modification) -
Drought High irradiance - Photosystem II (core
phosphorylation) - P i s u m s a t i v u m (drought stress) - Stress
syndrome
* Permanent addresses:
Department of Biology, Charles University, Prague, Czech Republic
** Institute of Microbiology, 37981 T~ebofi, Czech Republic
Abbreviations: Chl = chlorophyll; CP43 and CP47 = [3-carotene-
Chla-proteins of PSII core; DCPIP = 2,6-dichlorophenolin-
dophenol; DPC = diphenylcarbazide; Fv/Fm = the ratio of yield of
variable fluorescence to yield of maximal fluorescence when all
reaction centres are closed; LHC(P)= light-harvesting complex
(proteins); Wc = water content
Correspondence to: M.T. Giardi; FAX: 39 (6) 9064492,
E-mail: giardi@nserv.icmat.mlib.cnr.it
Drought is one of the most important environmental
factors limiting photosynthetic CO2 assimilation. It has
been shown that at moderate leaf-water deficits [relative
water content (RWC) down to 70%], photosynthesis is
impaired mainly as a consequence of stomatal closure,
without any significant decline in mesophyll capacity.
Only when drought is prolonged leading to cell dehydra-
tion below 70% RWC, or other stresses are superimposed
(heat, high light), electron transfer and CO2 fixation are
additionally affected (Boyer and Bowen 1970; Chaves
1991; Cornic et a1.1992). Thus, the photosynthetic appar-
atus appears to be relatively resistant to dehydration
(Chaves 1991). However, depending upon which system is
used, water stress may (Kaiser 1987; Masojidek et al. 1991;
Havaux 1992) or may not (Genty et al. 1987; Cornic et al.
1992; Jefferies 1994) predispose photosynthetic mem-
branes to photoinhibition (for a review on photo-inhibi-
tion, see Powles 1984; Critchley 1988). Photosystem II is
believed to play a key role in the response of leaf photo-
synthesis to environmental perturbations (Baker 1991).
Recently, photoinhibition has been proposed to target
various sites associated with the PSII reaction centre,
including components of the primary photochemical reac-
tion sequence between the intermediate electron donor
(Tyrz) and the quinone acceptor QA, and between QA and
the secondary plastoquinone acceptor QB (Eckert et al.
1992; Aro et al. 1993). The PSII reaction center is com-
posed of a D1-D2 heterodimer which carries all of the
components essential for electron transport (Mattoo et al.
1989; Baker 1991). Under natural stress conditions, where
water deficit develops gradually, high light intensities
could impair photosynthetic capacity and cause photo-
inhibition. In this regard, it is important to distinguish
between photoinhibition as a mechanism of downregula-
tion~ of PSII function in vivo (Horton and Ruban 1992;
Oquist et al. 1992; Anderson and Aro 1994; Critchley and
Russell 1994), and irreversible damage of PSII structure and
function observed in vitro under strong damaging illumina-
tion of isolated thylakoids (Barber and Anderson 1992).
In the present work, we have examined the effect of
long-term water deficit on PSII activities. We show that
M.T. Giardi et al.: Long-term drought stress
d r o u g h t c a u s e s s t r u c t u r a l r e o r g a n i z a t i o n of P S I I a n d
alters t u r n o v e r of the D1 p r o t e i n .
Material and methods
Plants. Pea (Pisum sativum L. cv. Progress 9; Sementi Dotto S.p.a.,
Rome, Italy) seedlings were grown in a greenhouse, in plastic con-
tainers (25 x 25 x 7 cm), on vermiculite and watered with a Hoag-
land solution. The temperature was maintained at 25~ while the
growth light was kept under 300 Bmol-m- 2.s- 1. Plants were used at
two to three weeks of age when they had six to eight leaves. To
impose drought stress on plants, watering was stopped at day
7 when plants were about 5 cm in height. The drought-stressed
plants were smaller and darker than watered plants and leaves
started to wilt when water content (We) was reduced by about
20%. The amount of water in plant material was measured as Wc
according to the following mathematical expression: 100 (fresh
weight - dry weight)/dry weight (Beadle et al. 1993). The values of
Wc have been expressed as a percentage of the control value.
Isolation of membranes and PSII components. Thylakoids, PSII
grana and stroma-exposed particles were prepared from control and
stressed plants as previously described (Giardi 1993a). For isolation
of grana particles, digitonin was used at a final concentration of
0.4%. Particles (1 mg chlorophyll) of PSII were solubilized with 1 ml
of 1% n-dodecyl-D-maltoside (DM), loaded on a linear sucrose
gradient (0-0.6 M sucrose) in a solution containing Mes (pH 6.3),
15 mM NaC1, 5 mM MgCl2 and 0.1% DM, and centrifuged at
55000"9 for 5 h at 5 ~ (SW56 rotor, Beckman Instruments, Palo
Alto, Calif., USA). The light-harvesting complexes were collected as
a green band at the position of 0.4 M sucrose while the PSII core
preparation was recovered as a pellet at the bottom of the tube.
Alternatively, the PSII membranes were gently solubilized with
0.3% DM and applied to an isoelectrofocusing bed of Ultrodex gel
(pH range 3.5-5; LKB, Bromma, Sweden) to separate light-harvest-
ing complex protein (LHCP) from the remaining PSII cores (Giardi
et al. 1992). The gel bed was cut into sections 1 cm wide and the
polypeptide profile was assessed by SDS-PAGE and immunoblot-
ting as described by Barbato et al. (1991).
Pulse-labelling with [35S]methionine. For pulse experiments, cut
petioles of the seedlings were incubated in a solution containing
50 mM Tricine, pH 7.2, 15 mM NaCI, 5 mM MgCI2 and [35S]me-
thionine (Du Pont Newtown, Conn., USA; 18.5-107 Bq'mmo1-1,
1.11.1014 Bq.mmol- 1) for 30 min in the dark, and then illuminated
for 1.5 h with an irradiance of 300 pmol.m 2.s-1. In some cases,
200 mM NaC1 was used in the incubation medium of control and
stressed plants. Then, PSII particles were isolated as described
above.
Pulse-labelling with [33p]orthophosphate. Pea seedlings were left in
the dark overnight to dephosphorylate phosphorylated proteins.
Petioles were cut in the dark and each incubated in a solution
containing 20 mM Na2 CO3 (pH 8.3), and 1.3.106 Bq of [33p]ortho-
phosphate (Amersham International, Amersham Bucks., UK;
7.4.107 Bq.mmol-1, 1.11.1014 Bq.mmol-1, 33p: tl/2 = 25.4d,
0.246 MeV). The leaves were illuminated for 4 h with an irradiance
of 300 Bmol'm-2"s -1 and then frozen in liquid nitrogen prior to
extraction of grana and stroma-exposed membranes.
Illumination with strong irradiance. High-irradiance treatment of
plants was performed for 7 h using a mercury pressure lamp
(1000 W, Osram, Miinchen, Germany). The light reaching the leaves
was about 3000 txmol.m-2.s -I. To avoid overheating of the leaf
surface, a 10-cm-deep circulating water bath was placed under the
lamp. This set-up also cut off the range of wavelength below 340 nm.
Experiments with isolated thylakoids were performed in
a cuvette of 3 mm thickness illuminated with an irradiance intensity
of 1800 ~tmol-m- 2-s - 1.
119
Analysis by SDS-PAGE, immunoblotting and autoradiography. Im-
munoblotting was carried out following electrophoretic transfer of
proteins separated on denaturing 12-17% polyacrylamide gels to
nitrocellulose filters. Polyclonal antibodies against D1 protein were
raised as previously described (Elich et al. 1993). Monoclonal anti-
bodies against phosphothreonine were purchased from Sigma
(St. Louis, Mo., USA). The immuno-complexes were detected using
anti-rabbit or anti-mouse secondary antibodies, coupled to alkaline
phosphatase. For autoradiography, the gels were stained with
Coomassie Blue, dried and then exposed to an X-ray film (Kodak-
Safety light) after a 20-min treatment with amplifier (Amplify,
Amersham).
Chlorophyll fluorescence and measurements of chlorophyll, cyto-
chrome b559, and electron transfer. The photochemical efficiency of
PSII was measured as the ratio of yield of variable fluorescence to
yield of maximal fluorescence when all reaction centres are closed
(Fv/Fm) using the Plant Efficiency Analyzer (Hansatech, King's
Lynn, Norfolk, UK) after 40 min dark adaptation at an irradiance of
3000 lamol-m- 2.s - 1.
Chlorophyll (Chl) content was determined in 80% (v/v) acetone
(Lichtenthaler and Wellburn 1983). Cytochrome b559 was deter-
mined from differential oxidized minus reduced spectra at 559 nm
(Giardi 1993a). Electron transport activity was measured spectro-
photometrically as 2,6-dichlorophenolindophenol (DCPIP) photo-
reduction at 600 nm with diphenylcarbazide (DPC) as an electron
donor (Giardi 1993a). Chlorophyll, cytochrome b559 as well as
electron transfer activity were determined in at least three indepen-
dent experiments each performed with two replicates. Values re-
ported are typical of those obtained.
Herbicide binding. Herbicide binding was performed on isolated
thylakoids as previously reported, using radiolabelled ioxynil
(3.3" 1011 Bq.mmol-i; Giardi et al. 1992; Giardi 1993b), a kind gift
from May and Baker Ltd. (London, UK). The QB content, the
dissociation constant and the number of herbicide-binding sites were
obtained from double reciprocal plots of free versus bound herbicide
concentration.
Results
Photosynthetic activity of drought-stressed pea
plants. L e a v e s a n d t h y l a k o i d s of t w o to three w e e k old
p e a p l a n t s g r o w n at 300 t x m o l - m - Z - s - a at v a r y i n g W c
were a n a l y z e d . Since the t u r g i d - w e i g h t m e a s u r e m e n t s in
y o u n g d r o u g h t - s t r e s s e d p e a seedlings are n o t r e p r o d u c -
ible, the a m o u n t of w a t e r in p l a n t m a t e r i a l was m e a s u r e d
as We.
As s h o w n in Fig. 1A, there was a slight b u t c o n s i s t e n t
decrease in the F v / F m r a t i o p a r a l l e l to t h a t in the P S I I
e l e c t r o n t r a n s p o r t a c t i v i t y of the t h y l a k o i d s as the W c was
decreased. B u t m o r e severe was the r e d u c t i o n i n the elec-
t r o n t r a n s f e r a c t i v i t y f r o m D P C to D C P I P p e r u n i t of
c h l o r o p h y l l as a f u n c t i o n of r e d u c e d W c (Fig. 1A).
I n p l a n t s g r o w n at 3 7 % W c , i n h i b i t i o n of e l e c t r o n t r a n s -
fer of the c o r r e s p o n d i n g t h y l a k o i d s i l l u m i n a t e d w i t h
1 8 0 0 1 a m o l . m - Z . s - 1 ( D C P to D C P I P p h o t o r e d u c t i o n )
o c c u r r e d m u c h earlier in t h o s e i s o l a t e d f r o m d r o u g h t -
stressed p l a n t s t h a n in t h o s e f r o m c o n t r o l p l a n t s (Fig. 1C).
O n e of the c o m m o n t a r g e t s of h i g h - l i g h t a n d u r e a - a n d
t r i a z i n e - t y p e h e r b i c i d e stress i n p h o t o s y n t h e s i s is the r a p -
idly t u r n i n g - o v e r D1 p r o t e i n of the P S I I r e a c t i o n c e n t r e
( D r a b e r et al. 1991). T h e r e f o r e , we tested w h e t h e r the
Q a - b i n d i n g site o n D1 is a l t e r e d b y d r o u g h t stress. T h i s
was d o n e b y m e a s u r i n g the n u m b e r of sites o n D1 for the
h e r b i c i d e i o x y n i l t h a t displaces QB f r o m its b i n d i n g n i c h e
120 M.T. Giardi et al.: Long-term drought stress

1,4
100_ =
~ 0.9
<1 80 -- E 1.2
o c
v

=0.8 80 o
~
o

5 A "
40

20
o
~
~ D.8
A (9

0.7 \.
Z o.e
, i i i
0 uJ
800
B ~ 0.4
8.0 c
v 700 o 0 Confrol K=3OnM r=0.96

600 m ().2 W5 K=14nM r=0.94

~_ 5.0 @
t.j 500 "~
..c 0.0
10 20 30 40 50

rj
= 4.o
~ 400

300
o
Free herbicide (1/p~M)

i , i , i i , r

100 80 60 40 20
Fig. 2. Double reciprocal plots for the binding of the herbicide
Wc, % of control vcllue ionyxil to thylakoids from leaves of control (control) and water-
stressed (WS) pea plants. The Wc of stressed plants was 37%. K,
dissociation constant for the herbicide binding; r, correlation coeffic-
_~ 1oo ~C ient. Experiments were performed three times, each with two repli-
cates. The plots represent a typical result
>o 8o

~ 6o
9- ~ . Con Control Drought
20 e~ 559 nm 5 5 9 nrn
e~ e W S
o A
0 20 40 60 80
- Pt
IlluminotTon time (min)
r

Fig. 1A-C. Characteristics of pea leaves and isolated thylakoids 0 0

during drought stress. A The Fv/Fm ratio of leaf samples (A) and
electron transfer activity (&), measured as DPC to DCPIP photo-
reduction at 600 nm, of isolated thylakoids from plants at different 0 |1

Wc stages. B The Chl/QB ratio (O), measured by the herbicide-

o ~
binding method, and the ChlLnc/Chlps, ratio (0) of thylakoids from
I | _ J
plants at different Wc stages. Chlorophyll associated with LHCII
and the PSII core was determined after fractionation of PSI! mem-
brane fragments followed by solubilization with 1% digitonin in
a sucrose gradient. The light-harvesting complexes were collected as
a green band in the position of 0.4 M sucrose while the PSII core t I

preparation was recovered as a pellet at the bottom of the tube. 25 nm

C Inhibition of electron transfer (ET), measured as DCP to DCPIP
photoreduction at 600 nm, in thylakoids from control (Cont) and
drought-stressed (WS) plants (Wc 37%) under high light conditions
(1800 gmol-m 2.s-t) as a function of illumination time. Experi-
ments were performed three times each with two replicates, and
reproducible results were obtained of which the plots represent
a typical result. The error bars in A represent the +SE (four
replicates)
Wavelength
Fig. 3. Cytochrome b559 signal (measured spectroscopically as the
difference of the oxidized and reduced spectra at 559 nm) in PSII
particles from leaves of water-stressed (Wc 37%) and control pea
plants. The measurements were performed at room temperature and
the suspensions of thylakoids used for the measurements contained
the same amount of chlorophyll. Experiments were performed three
times each with two replicates. Reproducible results were obtained
of which the plots represent a typical result

(Draber et al. 1991 and references therein). T h y l a k o i d s

were isolated from control and water-stressed (Wc 37%)
than in control plants. Together, these data provide
plants and incubated with radiolabelled ioxynil. Both the
evidence for the reorganization of PSII c o m p l e x under
n u m b e r of i o x y n i l - b i n d i n g sites and the dissociation con-
drought stress, which, in turn, results in a greater PSII
stant were reduced twofold in stressed versus control
unit size in these plants than in the controls.
plants (Fig. 2). The ratios of Chl/QB and ChleHc/ChlpsH
increased with the decrease in W c content of the plants
(Fig. 1B), suggesting that the content of PSII core is re- Composition and organization of PSII. The preceeding
duced under drought stress. This is further supported by results implicated PSII as a site for drought-stress dam-
the data in Fig. 3 s h o w i n g a high difference in the age. Therefore, we tested PSII particle c o m p o s i t i o n and
a m o u n t of c y t o c h r o m e b559 in the PSII m e m b r a n e s from organization in detail. T h y l a k o i d s were subfractionated
control plants c o m p a r e d to drought-stressed plants. Thus, into grana and stroma membranes and the polypeptide
at the same chlorophyll concentration the Chl/cyto- profiles of the isolated m e m b r a n e s were analyzed. The
c h r o m e b559 ratio was higher in drought-stressed plants yields of grana and stroma m e m b r a n e s were significantly
M.T. Giardi et al.: Long-term drought stress
Fig. 4A, B. Characteristics of particles isolated from leaves of con-
trol and stressed pea plants. A Yield of grana and stroma-exposed
membranes isolated from leaves of drought-stressed (WS, Wc 22%)
and control plants, using the digitonin method as described in
Material and methods. B SDS-PAGE (12-17% polyacrylamide-
gradient gels with 5 M urea) analysis of grana and stroma-exposed
regions of thylakoids isolated from leaves of control (C) and water-
stressed plants (WS, Wc 37%). M, molecular-weight standard in
kilodaltons (kDa). The position of specified proteins is indicated on
the left
lower in drought-stressed than in control plants, the re-
duction in yield being particularly severe for stroma mem-
branes (Fig. 4A).
The protein profiles of grana and stroma membranes
in control (C) and water-stressed (WS) plants are shown in
Fig. 4B. Distinct separation of the two membrane types is
shown by their distinct protein profiles, as seen before
(Callahan et al. 1989). Grana isolated from water-stressed
plants appeared to have lower contents of CP43 (a 13-
carotene-Chla-protein of the PSII core), D1, D2 and a
20-kDa protein (Fig. 4B, left panel, compare lanes C with
lanes WS). Stroma membranes from water-stressed plants,
on the other hand, showed a lower content of the 13-
subunit of the proton-ATPase and a slight increase in
some proteins between 27 kDa and 36 kDa compared
121
to the controls (stroma, lanes marked WS versus C).
Steady-state levels of CP47 (a [3-carotene-Chla-protein of
the PSII core) and the light-harvesting chlorophyll a/b
apoproteins (LHCII) remained more or less unchanged in
grana and stroma membranes isolated from control and
water-stressed plants. Some of the results obtained by
protein staining were further confirmed by immunoblot-
ting analysis (see corresponding immunoblot panels for
CP43, D1, CP47 and L H C I I in Fig. 5). The decrease in D2
observed in water-stressed samples on stained gels was
not corroborated by immunoblot analysis (Fig. 5C). The
decrease in the contents of CP43 and D1 in grana mem-
branes from the water-stressed plants seems to be due, in
part, to their enhanced degradation rates as shown by the
accumulation of their degradation products identified by
antibodies (see 22 kDa band in Fig. 5A for D1 and several
bands in the region of 20 kDa in Fig. 5B for CP43). The
immunoblots further confirm the dual location of PSII
proteins in chloroplast membranes (Callahan et al. 1989)
and show that the steady-state levels of CP43, CP47 and
L H C I I are higher in the grana than in the stroma mem-
branes of pea plants. On the other hand, almost equal
proportions of D1 and D2 are seen distributed in these
membranes. A protein band of 18 kDa in stroma mem-
branes of control plants immunoreacts with the anti-D1
antibody and is missing in the corresponding sample from
water-stressed plants (Fig. 5A), suggesting that it might be
a degradation product of D1 which has a shorter half-life
in water-stressed plants.
Enhanced PSII protein phosphorylation and D1 synthesis in
water-stressed plants. Proteins of PSII undergo light-
dependent reversible phosphorylation in vivo (Giardi et al.
1991, 1994; Elich et al. 1992, 1993). Phosphorylation of the
D1 protein was shown to result in lowered electrophoretic
mobility of D1 (Callahan et al. 1990), thus allowing visual
distinction between the phosphorylated and non-phos-
phorylated D1 (Elich et al. 1992, 1993). A perusal of the
D1 immunoblot (Fig. 5A) indicated the presence of
a higher amount of phosphorylated D1 (D1-P) in the
grana membranes of the water-stressed plants than in the
controls. We therefore surmised that water stress may
result in an enhancement of phosphorylation of PSII
proteins. To test this, we first incubated control and
water-stressed pea plants overnight in the dark to allow
turnover of phosphate on phosphorylated proteins, and
then radiolabelled them in vivo with [33p]orthophos-
phate for 4 h under 300 ~tmol.m-Z.s -1. In preliminary
experiments, we found that the radiolabel was taken
up by the plants within the labelling period, with the
water-stressed plants having only a slightly lower rate of
uptake than the c o n t r o l s . Following labelling, grana-
associated proteins were isolated, fractionated by
SDS-PAGE and radiolabel associated with D1, D2,
CP43, L H C I I and the psbH gene-product quantified
by densitometric scanning. Figure 6A shows that, with
the exception of the psbH gene-product, PSII core
proteins had a higher level (ca. average increase of 50%) of
phosphorylation in water-stressed plants than in the
control plants. Little or insignificant phosphorylation
of stroma-membrane-associated proteins was seen (data
not shown), consistent with the observation that stroma
122
Fig. 5A-C. Immunoblot analysis of proteins associated with grana
and stroma-exposed membranes isolated from leaves of control (C)
and water-stressed (WS, Wc 37%) pea plants. Polyclonal antibodies
against the D1 (A), CP43 (B), and D2, CP47 and LHCII (C) were
used in conjunction with the alkaline-phosphatase detection system.
In order to see differences in the PSII polypeptides of stroma-
exposed membranes isolated from control and drought-stressed
plants, the amount of chlorophyll loaded on the gel was twice as
much as for grana membranes
membranes lack protein kinase activity (Elich et al. 1992).
Using antibodies that recognize either D1 or D1 plus
phosphorylated-D 1 on immunoblots (data not shown), we
estimated the ratio of phosphorylated-D1 to D1 to be 0.35
in control plants compared to 1.15 in water-stressed
plants. Similarly, using monoclonal antibodies against
phosphothreonine (threonine is the residue phos-
phorylated in PSII core proteins, see Allen 1992), we
determined that phosphorylation of D2, CP43 and L H C I I
in water-stressed plants was higher than in the control
plants (data not shown). Together, these data provide
strong evidence that drought increases the extent of phos-
phorylation of the PSII core proteins D1, D2, CP43 and
L H C I I in pea plants.
Although it was apparent that drought caused less
accumulation of the PSII core proteins, perhaps due to
enhancement in the rates of their degradation, we tested
whether D1 synthesis was also affected, since this could be
a limiting factor as well. Plants were radiolabelled for 2 h
with [35S]methionine. To avoid rehydration and recovery
during the pulse-chase experiments, in some cases,
2 0 0 m M NaC1 was used in the incubation medium of
control and stressed plants. The PSH particles were iso-
lated, fractionated on S D S - P A G E and autoradiographed.
Figure 6B showed that, surprisingly, drought-stressed
plants were able to synthesize D1 several-fold more than
the control (WS/C = 2.5-4.0). Experiments conducted at
a higher irradiance showed that under these conditions in
M.T. Giardi et al.: Long-term drought stress
Fig. 6. A Phosphorylation index of PSII core proteins under water-
stress conditions, expressed as the ratio of phosphorylation per unit
protein in water-stressed versus control plants. Plants were labelled
in vivo with [3~P]orthophosphate for 4 h at 300 gmol'm-2"s-t.
Grana-associated proteins were isolated and fractionated by SDS-
PAGE. The amount of each protein on the gel and that of labelled
protein (after autoradiography) were determined densitometrically.
Relative ratios (labelled protein/total protein) are shown.
B Autoradiograph of an SDS-polyacrylamide gel analysis of PSII
membranes from leaves incubated with [3SS]methionine for 1.5 h.
C, control treated with light of 300 gmol-m 2.s ~; C + HL, control
treated with light of 3000gmol.m-2.s-1; WS, water-stressed
(Wc 37%); WS + HL, water stressed (Wc 37%) treated with light
of 3000gmol.m Z.s-~. The samples were loaded on an equal-
chlorophyll-concentration basis
unstressed plants D1 synthesis was increased compared
to the low-light control. However, higher light (HL) in-
tensity was detrimental to water-stressed plants in that
the drought-stimulated D1 synthesis was prevented
(WS + HL/C + HL = 0.6-0.8). The presence of NaC1 did
not alter the results. These data suggest that D1 degrada-
tion rather than D1 synthesis caused a decreased steady-
state level of D1 in water-stressed plants and that
high-light stress superimposed on water stress is deleterious
for the plant.
Recovery studies. To ascertain the severeness of water
stress imposed on the pea plants, variously stressed plants
were re-watered. The PSII particles were then isolated and
analyzed for Fv/Fm ratio, electron transfer activity and
cytochrome b559 content (Table 1). Results show that
water-stressed plants (Wc 40%) recovered 90% of the
initial cytochrome b559 content after 2d, whereas the
desiccated plants (Wc 23%) took 6 d following re-water-
ing to recover 80% of cytochrome b559 content. Plants
that were water-stressed at the high light intensity were
not able to recover.
Finally, if the stressed plants were rewatered with
30 gg.l- 1 of the herbicide atrazine (that is known to bIock
D1 degradation and phosphorylation as well; Mattoo
M.T. Giardi et al.: Long-term drought stress
Table 1. Fv/Fm ratio, electron transfer activity (ET; Control value was 455 txmol'm- 2"s- x of reduced DCPIP), and cytochrome b559content
of leaves from control, water-stressed (WS; Wc 40% and 23%), and water-stressed/strong illumination (WS + HL; Wc 40%) pea plants before
and after re-watering (recovery). Recovery time was 2 d from rewetting at initial Wc 40% and 6 d from rewetting at initial Wc 23%.
Experiments were performed three times each with two replicates. Data are means (_ SE) for Fv/Fm from three replicates
Wc Fv/Fm
before after before
recovery recovery
Control 100% 100% 0.870 + 0.005
WS1 40% 80% 0.810 + 0.006
WS2 23% 41% 0.798 + 0.018
WS1 + HL 40% 35% 0.654 + 0.038
et al. 1989; Allen 1992; Elich et al. 1993) the recovery in
the first few days was found to be accelerated (data not
shown).
Discussion
In the present work we demonstrated that drought (the
decrease of Wc by 40%) affects photosynthesis at the level
of PSII, causing a considerable depletion of the PSII core,
enhanced CP43 and D1 protein degradation, and en-
hanced phosphorylation of PSII core proteins, most prob-
ably by activating the kinase activity. This reduction in
the PSII core complex is accompanied by a reorganiza-
tion of the remaining PSII to a functional form since the
F v / F m ratio was high as well as the electron transfer from
D P C to D C P I P , when considered per unit of protein.
Thus, the "surviving" PSII core seems functionally reor-
ganized. These observations are in accordance with pre-
vious results (Bj6rkmann and Powles 1984; Ogreen and
Oquist 1984; Masojidek et al. 1991) showing that drought
halved the active PSII reaction centres but are at variance
with the conclusion drawn by Meyer and Kouchkovsky
(1993) that the inactivation is not due to a physical de-
struction of the PSII core. Our results further show that
thylakoids from water-stressed plants are highly suscep-
tible to high irradiance, as was also observed previously
(Masojidek et al. 1991; Masojidek and Hall 1992). It seems
possible that this synergism of light and water stress is the
reason for the reduced content of PSII core. Thus, the
drought-stress syndrome is a combination of two compo-
nents. One, a water-stress effect, enhanced by illumina-
tion, that leads to a disassembly of a part of PSII core.
Second, a reorganization process that rebuilds and main-
tains the remaining PSII functional to counteract the
depletion of PSII core. The importance of these two
drought consequences depends on specific growth
conditions and the intensity of light, offering an explana-
tion for the conflicting results seen in water-stress experi-
ments carried out in a growth chamber versus those car-
ried out under field conditions (Kaiser 1987; Cornic et al.
1992; Jefferies 1994).
Phosphorylation of PSII polypeptides as well as D1
turnover have been proposed as two protective mecha-
nisms for the structural and functional integrity of PSII
(Mattoo et al. 1989; G o d d e et al. 1991; Giardi et al. 1991,
123
ET (% of control) Chl/Cyt b559
after before after before after
recovery recovery
0.870 + 0.005 100 100 314 314
0.800 + 0.013 35 65 488 335
0.762 _ 0.033 22 43 500 351
0.633 +_0.041 0.5 0.5 480 500
1993, 1994; H o r t o n and Ruban 1992; Giardi 1993a, b;
H u r r y et al. 1993; for a recent review on phosphorylation,
see Allen 1992). In line with this idea, it has been suggested
that D1 turnover has a physiological significance leading
to downregulation of PSII activity under high light stress
(Mattoo and Edelman 1985; Oquist et al. 1992; Critchley
and Russell 1994; O s m o n d 1994; Russell et al. 1995) be-
sides constituting a damage-repair cycle (for review, see
Barber and Anderson 1992). In-vitro phosphorylation of
PSII proteins has been shown to protect against irrevers-
ible light inactivation of PSII function (photoinhibition,
H o r t o n and Lee 1985); however, the contribution of each
PSII core polypeptide seems to be different (Giardi et al.
1994).
The lower steady-state level of D1 and CP43 caused by
water stress parallels their enhanced phosphorylation,
suggesting that phosphorylation of these proteins, but not
of D2 and L H C I I , m a y be a committed step for their
degradation. The role of in-vivo phosphorylation in the
dynamics or function of any of the PSII core proteins is
not well defined. D1 phosphorylation has been linked to
D1 degradation (Callahan et al. 1990; Elich et al. 1992),
protection of D1 against photoinhibition (Aro et al. 1993)
and to chromatic adaptation of photosystems (Elich et al.
1993).
The results presented here indicate that altering the
phosphorylation state of PSII core proteins could have
direct consequences on D1 turnover and on the half-lives
of the PSII core proteins as well. These indications are
consistent with other reports. For instance: (i) In-vitro
phosphorylation of the peripheral antenna proteins and
psbH protein protects electron transfer from photoinhibi-
tion (Horton and Lee 1985; Giardi et al. 1993, 1994). (ii) A
heterogeneity of PSII core phosphorylation has been
shown. Four differently phosphorylated PSII core popu-
lations, which are interconvertible, are present in grana
membranes (Giardi et al. 1991, 1994). Thylakoids with
highly phosphorylated forms of PSII core result in PSII
disassembly within a few minutes of illumination (Giardi
1993a). (iii) In-vivo phosphorylation of D1 is an integral
part of light-mediated D1 metabolism (Elich et al. 1992).
Recently, it has been proposed that D1 turnover is regu-
lated by a light-dependent kinase activity (Bracht and
Trebst 1994).
Greatly enhanced synthesis of D1 in water-stressed
leaves and yet a lower D1 content suggest that a decreased
124
a m o u n t of D1 is due to its e n h a n c e d degradation. The
higher rate of synthesis m a y reflect preferential t r a n s l a t i o n
of psbA message in response to greater depletion of the
p r o t e i n as seen in senescing leaves (Droillard et al. 1992).
This response m a y be a n a t t e m p t of water-stressed plants
to avoid complete depletion of the core. G i v e n the de-
pletion of the PSII core u n d e r water-stress conditions, it
seems reasonable to suggest that u n d e r a d d i t i o n a l stress
due to p h o t o i n h i b i t o r y light, D1 d e g r a d a t i o n m a y be
altered a n d thus, synthesis, being d e p e n d e n t o n the rate of
d e g r a d a t i o n , is reduced, as has also been shown by G e i k e n
et al. (1992), S u n d b y et al. (1993), a n d Russell et al. (1995).
Similar results have been recently observed u n d e r min-
eral stress, where also loss of active PSII centres (observed
by fast fluorescence decay), a n increased sensitivity to
i l l u m i n a t i o n , a n d e n h a n c e d D1 protein t u r n o v e r were
observed (Godde a n d Hefer 1994). O u r results o n the
effect of water stress o n the d y n a m i c s of D1 m e t a b o l i s m
a n d P S I I core p h o s p h o r y l a t i o n indicate that D1 metabol-
ism is very i m p o r t a n t for stress a d a p t a t i o n of plants. This
suggestion is s u p p o r t e d by the o b s e r v a t i o n that P S I I core
depletion is reversed in a relatively short time after re-
watering, more so when b o t h D1 d e g r a d a t i o n a n d phos-
p h o r y l a t i o n are decreased by the presence of the herbicide
atrazine. Thus, we suggest that the biochemical response
at the level of D1 t u r n o v e r could act as a general a d a p t a -
tion signal for the p l a n t in response to e n v i r o n m e n t a l
stress.
This work was supported by the Italian National Council of Re-
search special grant RAISA, subproject 2 (paper No. 2179) on water
stress B. Geiken was supported by the European program "Human
Capital and Mobility". We thank Dr. Roberto Barbato (Department
of Biology, University of Padua, Italy) for generous gifts of various
PSII antibodies.
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