Plant Physiol. (1998) 117: 129-139
The Regulation of Photosynthetic Electron Transport during
Nutrient Deprivation in Chlamydomonas
reinhardtii1
Dennis D. Wykoff*,
John P. Davies,
Anastasios Melis, and
Arthur R. Grossman
The Carnegie Institution of Washington, Department of Plant
Biology, 260 Panama Street, Stanford, California 94305 (D.D.W.,
J.P.D., A.R.G.); Department of Biological Sciences, Stanford
University, Stanford, California 94305 (D.D.W.); and Department of
Plant Biology, 411 Koshland Hall, University of California, Berkeley,
California 94720 (A.M.)
 |
ABSTRACT |
The light-saturated rate of
photosynthetic O2 evolution in Chlamydomonas
reinhardtii declined by approximately 75% on a per-cell basis
after 4 d of P starvation or 1 d of S starvation.
Quantitation of the partial reactions of photosynthetic electron
transport demonstrated that the light-saturated rate of photosystem
(PS) I activity was unaffected by P or S limitation, whereas
light-saturated PSII activity was reduced by more than 50%. This
decline in PSII activity correlated with a decline in both the maximal
quantum efficiency of PSII and the accumulation of the secondary
quinone electron acceptor of PSII nonreducing centers (PSII centers
capable of performing a charge separation but unable to reduce the
plastoquinone pool). In addition to a decline in the light-saturated
rate of O2 evolution, there was reduced efficiency of
excitation energy transfer to the reaction centers of PSII (because of
dissipation of absorbed light energy as heat and because of a
transition to state 2). These findings establish a common suite of
alterations in photosynthetic electron transport that results in
decreased linear electron flow when C. reinhardtii is
limited for either P or S. It was interesting that the decline in the
maximum quantum efficiency of PSII and the accumulation of the
secondary quinone electron acceptor of PSII nonreducing centers were
regulated specifically during S-limited growth by the
SacI gene product, which was previously shown to be
critical for the acclimation of C. reinhardtii
to S limitation (J.P. Davies, F.H. Yildiz, and A.R. Grossman [1996] EMBO J 15: 2150-2159).
| |
INTRODUCTION |
Photosynthesis and plant growth can be limited by macronutrient
(N, P, S, and C) availability. Growth in terrestrial and freshwater ecosystems is often limited for P (Wetzel, 1983
). Even in environments in which P levels are high, much of it is in the form of insoluble phosphate salts of Ca, Al, or Fe, which are not
easily accessible to plants (Raich et al., 1996). S availability may
also limit plant productivity, and the fertilization of soils with
sulfate can result in improved crop yields (Mahler and Maples, 1987;
Samosir et al., 1993; Warman and Sampson, 1994). Free sulfate, the
source of S preferred by plants, is often a minor component of the soil
S; most is in the form of sulfate esters and sulfonates, which may be
difficult for plants to exploit (Stanko-Golden and Fitzgerald, 1991;
Houle and Cargnan, 1992).
We are interested in how photosynthetic organisms respond to
limitations in P and S and, in particular, how the photosynthetic apparatus is controlled as nutrient levels decline. Photosynthesis can
be divided into the light reactions, in which light energy is stored as
ATP and NADPH, and the dark reactions, in which the products of the
light reactions are used to reduce inorganic C. There must be a
coordination of the light and dark reactions for the efficient
utilization of excitation energy. A lack of coordination could result
in the generation of toxic, reduced O2 species
either as a consequence of the generation of triplet excited
chlorophyll molecules or by direct reduction of
O2 by PSI (Asada, 1994). Therefore, the flux of
electrons through the electron-transport chain is modulated by the rate
of ATP and NADPH consumption, which is affected by the physiological
state of the cells. Numerous groups have demonstrated that P
deprivation causes a decline in the in vivo, light-saturated rate of
photosynthesis (Brooks, 1986; Dietz and Heilos, 1990; Jacob and Lawlor,
1993; Plesnicar et al., 1994). This decline has been attributed to a
limitation in the rate of C reduction because of a depletion in the
pool of phosphorylated intermediates of the reductive pentose-phosphate
cycle (Brooks, 1986; Jacob and Lawlor, 1993). Furthermore, S limitation
results in selective degradation of Rubisco in Lemna minor
(Ferreira and Teixeira, 1992).
Studies have demonstrated that the light reactions and photosynthetic
electron transport may also be altered during nutrient limitation.
Spinach leaves have fewer functional PSII centers when the plants are
starved of Mg and S (Godde and Hefer, 1994). Similarly, when the
cyanobacterium Synechococcus sp. strain PCC 7942 is deprived
of S, a decline in O2 evolution probably results from the degradation of PSII reaction centers (Collier et al., 1994).
The efficiency with which absorbed light energy is used is also
modulated during nutrient limitation. Cells starved for nutrients may
convert excess absorbed excitation energy to heat within the LHC (qE)
and/or undergo a state transition in which an increased proportion of
the absorbed light energy is directed away from PSII (qT; Peltier and
Schmidt, 1991; Jacob and Lawlor, 1993; Levy et al., 1993; Collier et
al., 1994; Plesnicar et al., 1994; Braun et al., 1996).
Furthermore, reaction centers that are damaged by excess radiation may
also quench absorbed light energy. Nutrient-deprived cells have been
shown to accumulate the xanthophylls antheraxanthin and zeaxanthin
(Lers et al., 1991; Levy et al., 1993), which correlates with increased
qE (Demmig-Adams and Adams, 1992; Björkman and Demmig-Adams,
1994; Horton et al., 1996). These xanthophylls, plus lutein, appear to
serve a photoprotective role by facilitating the conversion of excess
absorbed light energy to heat (Demmig-Adams and Adams, 1992; Niyogi et
al., 1997). Also contributing to photoprotection during nutrient
starvation is a state transition in which the photosynthetic apparatus
is transformed from state 1 (much of the light energy absorbed by LHCII
is directed into the PSII reaction centers) to state 2 (less of the
light energy absorbed is directed into the PSII reaction centers;
Peltier and Schmidt, 1991; Allen, 1992). There appear to be a number of
different ways in which photosynthetic activity is modulated during
nutrient-limited growth.
The responses of the unicellular green alga Chlamydomonas
reinhardtii to macronutrient limitation (de Hostos et al., 1989; Ball et al., 1990; Davies et al., 1994, 1996; Yildiz et al., 1994, 1996; Quisel et al., 1996) include the secretion of hydrolytic enzymes
(sulfatases and phosphatases), kinetic changes in the uptake of the
limiting nutrient, the cessation of growth, and changes in metabolic
processes (e.g. increased respiration and accumulation of starch, which
include a decrease in the rate of photosynthetic electron transport).
The purpose of this study was to determine the effects of S and P
limitation on photosynthetic electron-transport activity.
| |
MATERIALS AND METHODS |
Strains and Growth Conditions
Chlamydomonas reinhardtii strain CC125 (mt+;
wild type), ars 5-4 (mt+ arg7 nit1
sac1::ARG7; the sac1 mutant), and
ars 5-4 C11 (mt+ arg7 nit1 sac1::ARG7 NIT1
SAC1; the complemented sac1 mutant) were grown to the
mid-logarithmic phase (0.5 to 3 × 106 cells
mL
1) in TAP medium (Gorman and Levine, 1966).
To induce P or S starvation, the cells were harvested by centrifugation
at 5000g for 1 min, washed twice with TAP-P medium (Quisel
et al., 1996) or TAP-S medium (Davies et al., 1996), and resuspended to
a final density of 1 × 106 cells
mL1. After 2 d, P-deprived cells were
diluted to 1 × 106 cells
mL1 with fresh TAP-P medium (cells divided
three to four times following the imposition of P starvation). All
cultures were grown in Erlenmeyer flasks with constant shaking and
illumination at 80 µmol photons m2
s1 at 27°C.
Chlorophyll Determination and Cell Viability
Cells were counted using a hemacytometer and the viability of the
cells was assessed by viability staining (Davies et al., 1996). For
chlorophyll determinations cells were suspended in 0.01% Tween 20 (United States Biochemical) and pelleted by centrifugation for 30 s at 16,000g. The cell pellet was extracted in 90% acetone, cellular debris were removed by centrifugation (30 s at
16,000g), and the chlorophyll a and b
levels were determined spectrophotometrically (Jeffrey and Humphrey,
1975).
In Vivo Measurements of O2 Evolution
O2 evolution in the light and dark
respiration was measured with a Clark-type O2
electrode (Hansatech, King's Lynn, UK). Measurements of whole-cell
O2 evolution were conducted at 27°C on
vigorously stirred samples containing 10 µg chlorophyll a
mL1 and 5 mm bicarbonate, pH 8.1, in either TAP-P or TAP-S medium. The photosynthetic rates were
calculated by adding the dark respiration rate to the rate of
O2 evolution at various light intensities. Maximal photosynthetic rates were determined at 800 µmol photons m2 s1 and were not
significantly different from values measured at 400 µmol photons
m2 s1.
In Vitro Measurements of Electron Flow
Five milliliters of cells at 10 µg chlorophyll a
mL1 was sonicated (at a setting of 3 out of 10)
for 6 s in a 10-mL glass beaker using the microtip of the Sonic
Dismembrator 550 (Fisher Scientific). These sonication parameters
yielded optimal rates of ferricyanide-stimulated O2 evolution during both starved and nonstarved
conditions. Electron flow from water to MV,
DCPIPH2 to MV, and DHQ to MV were measured as
O2 uptake (Curtis et al., 1975; White et al.,
1978). All reactions involving MV-stimulated O2
uptake included 2 mm cyanide to inhibit peroxide reduction
and methylamine to uncouple electron transport from ATP synthesis.
The light-driven reduction of DCPIP by DPC was assayed as a decline in
A600; the DCPIP reduction was linear for
approximately 5 min. DPC was prepared in methanol and added to
heat-treated (5 min, 60°C), sonicated cells to a final concentration
of 2.5 mm (Vernon and Shaw, 1969). All of the partial
reactions were shown to be light dependent and inhibited by the
photosynthetic electron-transport inhibitors DCMU or DBMIB (except
electron flow from DCPIPH2 to MV, which is not blocked by
the electron-transport inhibitors used). Furthermore, the assays for
DCPIPH2 to MV and DHQ to MV contained DCMU, and
that for DPC to DCPIP contained DBMIB to prevent light-dependent
alternative electron transport. Because the DHQ-to-MV partial reaction
is not completely inhibited by DBMIB (inhibition is approximately
60%), we set the portion of electron transport sensitive to DBMIB as
100%.
PAM Fluorometry and PSII Characteristics during Nutrient
Limitation
Fluorescence characteristics (Schreiber et al., 1986; van Kooten
and Snel, 1990) were determined with a PAM fluorometer (model OS-100, PP Systems, Haverhill, MA) connected to a personal computer containing the OS-Log program (version 1.5, PP Systems). Samples were
continuously stirred and maintained at 27°C in a water-jacketed chamber. The intensity of the measuring beam was <0.1 µmol photons m2 s1 (setting 70),
which gave a significant signal with cultures containing 10 µg
chlorophyll a mL1. The actinic light
source was an incandescent light of 659 µmol photons
m2 s1, and the
saturating pulse exceeded 3000 µmol photons
m2 s1. The
F0,
Fv/Fmax, and
Fmax of all samples were determined after a
1-min dark adaptation; the
F/Fmax' was determined after
the cells were exposed to actinic light for 10 min. When included in
the assay, nigericin was used at a final concentration of 20 µm (equilibrated for 2 min in the dark).
PSII reaction centers were quantitated by measuring the light-induced
A320 change, which is attributed to the
reduction of QA (Guenther et al., 1990; Neale and
Melis, 1990). This measurement estimates the number of PSII centers
capable of a stable charge separation. The
A320 change was measured with a split-beam
spectrophotometer as previously described (Guenther et al., 1990). The
number of PSII centers was also estimated by western-blot analysis
using a monospecific antibody raised against spinach D1 that was
previously shown to recognize D1 of C. reinhardtii (Guenther
et al., 1988; Kim et al., 1993). Polypeptides that cross-reacted with
the antibody were detected with peroxidase-linked anti-rabbit IgG using
a chemiluminescence kit (Boehringer Mannheim), and the levels of
cross-reacting polypeptides were quantified by densitometry.
Xanthophyll Pigment Analysis
Cells in 1 mL of culture were chilled in liquid N2 and
pelleted by centrifugation at 16,000g for 30 s, and the
pellet was immediately frozen in liquid N2 and
kept at 
80°C until analysis. The levels of zeaxanthin,
antheraxanthin, violaxanthin, and lutein were determined as previously
described (Niyogi et al., 1997).
Measurements of Fluorescence Induction
Thylakoid membranes were isolated (Neale and Melis, 1990), diluted
to <20 µg chlorophyll mL1, and placed in a
3-mL quartz cuvette. Ferricyanide was added to a concentration of 500 µm to prevent reduction of the PQ pool and to obtain the
Fpl. The suspension was exposed to 1 s
of green light at 50 µmol photons m2
s1, and the output of the photomultiplier tube
was recorded by a digital voltmeter (model 3437A, Hewlett-Packard) in
2-ms intervals. F0 and
Fpl were obtained from these data. To
determine the Fmax value, DCMU was added to
a final concentration of 20 µm and the measurement was
repeated.
77 K Fluorescence Emission Spectra
Prior to measuring fluorescence emission spectra, cells were
maintained in TAP, TAP-S, or TAP-P medium at 80 µmol photons m2 s1 from fluorescent
tubes or were exposed to far-red light (
> 714 nm, approximately 3 µmol photons m2 s1)
for 20 min. Fluorescence emission spectra were determined with a
single-beam fluorometer (PTI, New Brunswick, NJ), as previously described (Collier et al., 1994). Aliquots of cells containing 5 µg
chlorophyll a mL1 were loaded into a
cuvette that was submerged in liquid N2 (77 K).
The samples were excited at 435 nm (2.74 µmol photons
m2 s1), and the
fluorescence emission was measured between 650 and 750 nm; the
fluorescence of samples containing no cells (just the appropriate
growth medium) was subtracted.
| |
RESULTS |
Photosynthetic Activity Decreases during Nutrient Starvation
When C. reinhardtii was starved of P or S, there was a
dramatic reduction in the level of photosynthesis. After 4 d of P
starvation, the light-saturated rate of photosynthesis as measured by
O2 evolution declined by approximately 75%
compared with the rate measured in cells maintained in complete medium
(Fig. 1; Table
I). P-starved cells remained 100% viable
for up to 7 d after P was eliminated from the medium (data not
shown). C. reinhardtii cultures that were transferred to
medium lacking S also showed a decline in the photosynthetic rate, but
the decline was much more rapid. Twenty-four hours after the transfer
of cells to medium lacking S, photosynthetic O2
evolution decreased by 76%, whereas after 48 h the photosynthetic
rate had declined by nearly 90% (Fig. 1). There was no decrease in
cell viability for at least 4 d following the initiation of S
starvation (Davies et al., 1996). Furthermore, cells deprived of P for
4 d or S for 1 d exhibited no change in their chlorophyll
a/b ratio and a 14 to 20% decrease in the level of chlorophyll per cell (Table I). We have chosen 4 d of P
starvation and 1 d of S starvation for further characterizations
because at these times the cells did not lose viability and showed a
similar reduction in the rate of O2 evolution.
View larger version (17K):
[in this window]
[in a new window]
| Figure 1.
The rate of O2 evolution in saturating
light for P- ( ) and S-deprived ( ) cells expressed as a percentage
of nutrient-replete cells. The percentage of O2 evolution
was calculated by dividing the rate of O2 evolution per
cell in starved cultures by the rate in replete cultures. Each point
represents the mean of at least five separately grown cultures and the
bars indicate the ses. The arrows indicate times at which
the cells were used for the remaining characterizations presented in
this manuscript.
|
|
View this table:
[in this window]
[in a new window]
|
Table I.
Photosynthesis and chlorophyll levels in cells
starved of S and P
All values were calculated from at least five separately grown cultures
and ses are given. Values in parentheses are relative to
unstarved cells. Chl, Chlorophyll.
|
|
To determine the quantum yield of photosynthesis at subsaturating light
intensities for cells deprived of P and S, we measured O2 evolution as a function of light intensity.
Figure 2 shows the light-response curves
of O2 evolution of cells either grown in complete
medium or starved for P and S. Not only was the light-saturated rate of
photosynthesis less, but the rate of increase in
O2 evolution as a function of light intensity was
also less in P- and S-starved cells compared with that of cells grown
in nutrient-replete medium. Therefore, nutrient-deprived cells
exhibited a reduced efficiency of light-energy utilization.
View larger version (20K):
[in this window]
[in a new window]
| Figure 2.
Light-response curves of O2 evolution
after S ( ) and P ( ) starvation and in nutrient-replete ()
cells. Each point represents the mean of three measurements and the
bars indicate the ses. This experiment was performed twice
and the trends were identical.
|
|
PSII Is the Site of Inhibition in Photosynthetic Electron Transport
during Nutrient Starvation
The decline in the light-saturated rate of in vivo
O2 evolution that accompanies nutrient limitation
could be an indirect effect caused by a decline in the activity of the
reductive pentose-phosphate pathway, or it could be a direct effect on
the rate of noncyclic electron flow. To distinguish between these
possibilities, we determined the light-saturated rate of photosynthetic
electron transport in crude cell membrane preparations in which
photosynthetic electron transport was uncoupled from
CO2 fixation and photophosphorylation (Table
II; Curtis et al., 1975). Electron
transport from water to MV for P- and S-starved cells was 30 to 40% of
that of unstarved cells. This decline in electron-transport activity
was similar to the decline in whole-cell O2
evolution (Table I) and the
F/Fmax' (the fluorescence
measurement that reflects the rate of electron transport; Genty et al.,
1989; Table III) observed in
nutrient-deprived cells.
View this table:
[in this window]
[in a new window]
|
Table II.
Summary of partial reactions
Absolute values for partial reactions in unstarved cells (µmol
O2 mg1 Chl h1) range from:
658 to 593 (H2O to MV), +304 to +377 (H2O
to ferricyanide), 869 to 716 (DCPIPH2 to MV), and
1122 to 979 (DHQ to MV). DPC to DCPIP was measured photometrically.
Values are the result of three independent experiments.
|
|
View this table:
[in this window]
[in a new window]
|
Table III.
PAM fluorescence characteristics
Measurements are the means of two independently grown cultures on equal
amounts of chlorophyll. The difference between the mean of the
measurements is less than 5%, except for the F0
and the F/Fmax of replete-grown cells, which
is 6%. Values in parentheses are relative to unstarved wild-type
cells.
|
|
Partial reactions of photosynthetic electron transport were performed
to locate the site at which electron transport was inhibited. PSI
activity, assayed as electron flow from DCPIPH2
(which donates electrons to plastocyanin) to MV, was approximately the
same for unstarved and P- and S-starved cells (Table II). Furthermore, the rate of electron transfer from DHQ (which donates electrons to the
PQ pool) to MV showed little change during nutrient limitation. These
results suggested that nutrient limitation resulted in a decrease in
linear electron transport prior to the PQ pool.
To measure PSII activity we used ferricyanide and
p-benzoquinone. The electron flow from water to ferricyanide
(Table II) or to p-benzoquinone (data not shown) was reduced
by approximately 50% in starved cells. Electron transfer from DPC
(which donates electrons to the PSII reaction center) to DCPIP (which
accepts electrons from the PQ pool) was also inhibited by approximately 50%. These results demonstrated that S and P starvation caused a
reduction in the rate of electron transfer between the site of DPC
electron donation (TyrZ) and the PQ pool and
could reflect a decline in the rate of electron transfer from
QA to the PQ pool and/or a reduction in the total
number of active PSII reaction centers.
Loss of Functional PSII Reaction Centers during Nutrient
Starvation
To examine changes in the maximum quantum yield of PSII that
occurred during P and S starvation, we measured
Fv/Fmax (Krause and Weis, 1991; Lavergne and Briantais, 1996; Table III). Limitation of
either P or S resulted in a 20 to 25% decrease in the
Fv/Fmax, suggesting that some of the PSII centers were damaged. However, the
Fv/Fmax is a
relative measure of the maximal PSII quantum yield and does not provide
information about the absolute number of functional PSII reaction
centers present in nutrient-limited cells. To determine the absolute
number of functional PSII reaction centers, we spectrophotometrically
measured the amount of QA that could be reduced
(Table IV). Cells starved of either P or
S exhibited a 30% decline in the amount of functional
QA relative to unstarved cells. In addition, we
quantitated the D1 protein content in thylakoid membrane preparations
from starved and unstarved cells (Fig.
3). Quantitation from three separate
preparations (one representative experiment is shown in Fig. 3)
indicated that there was also a reduction in the absolute level of the
D1 polypeptide by approximately 25% during nutrient limitation.
Together, these results suggested that less than half of the decline in
O2 evolution that occurred during S and P
limitation reflected a loss of functional PSII reaction centers
(centers devoid of the D1 protein).
View this table:
[in this window]
[in a new window]
|
Table IV.
Measurement of PSII centers using change of
A320
Values are the means ± ses of three thylakoid
membrane preparations.
|
|
View larger version (29K):
[in this window]
[in a new window]
| Figure 3.
Western-blot analysis using D1 antibody against
thylakoid membrane preparations from nutrient-replete (TAP), P-starved
(TAP-P), and S-starved (TAP-S) cells. Three different concentrations of thylakoids (in micrograms of chlorophyll) were electrophoresed in 15%
polyacrylamide-urea gels: a, 5.12; b, 2.56; and c, 1.28.
|
|
Accumulation of PSII Centers Unable to Reduce PQ during Nutrient
Starvation
In addition to damage to PSII, reduced electron flow through PSII
may occur as a consequence of the retardation of the transfer of
electrons from the reaction center to the PQ pool. This can be assessed
by the analysis of fluorescence-induction curves that provide
information about the reduction state of QA, the
primary quinone electron acceptor from P680
(Melis, 1991). When QA is reduced (i.e. in the
presence of DCMU, which blocks the transfer of electrons from
QA to QB), the fluorescence
rapidly reaches a maximum. When QA is oxidized
(i.e. in low light in the presence of electron acceptors), the
fluorescence approaches F0. Under our
illumination conditions, in the presence of DCMU, the variable fluorescence yield increases with characteristic biphasic kinetics (Melis, 1991; Fig. 4). In the absence of
DCMU and in the presence of ferricyanide, the variable fluorescence
yield gradually attained the intermediate
Fpl level.
View larger version (26K):
[in this window]
[in a new window]
| Figure 4.
Fluorescence induction upon illumination with 50 µmol photons m2 s1 of green light.
Ferricyanide was added to the membrane preparation to oxidize all of
the functional PSII reaction centers. The
Fmax was determined in the presence of DCMU.
These curves were normalized to Fmax = 100 units. The inset shows the F0 of each curve.
The F0 values are 0.46, 0.44, 0.55, 0.56, 0.53 for TAP, TAP plus DCMU, TAP-P, TAP-S, and
sac1-TAP-S, respectively. The data presented are from
one experiment, but numerous replicates gave nearly identical results.
|
|
The increase from F0 to
Fpl in the presence of ferricyanide is
attributed to the reduction of QA present in a
small pool of PSII reaction centers (Govindjee, 1990; Melis, 1991).
Such centers, termed PSII QB-nonreducing centers
(Chylla and Whitmarsh, 1989), oxidize
QA at a rate that is
approximately 1000 times slower than that of QB-reducing centers (Ort and Whitmarsh, 1990) and
are essentially nonproductive in O2 evolution
(Melis, 1991). In both S- and P-starved cells, the
Fpl was significantly higher than in
unstarved cells, indicating an increase in the level of
QB-nonreducing centers under both conditions.
Because the thylakoid membranes used in these experiments were washed
and the assays were performed in the presence of ferricyanide, the
apparent increase in QB-nonreducing centers in
nutrient-deprived cells was not a consequence of partial PQ reduction
by cellular metabolites.
Calculations of (Fpl F0)/(Fmax F0) allowed for an estimate of the
percentage of PSII reaction centers that are QB
nonreducing. As shown in Table V, during
nutrient-replete growth these centers made up approximately 29% of all
PSII reaction centers. In thylakoid preparations from P- and S-starved
cells the (Fpl F0)/(Fmax F0) was greater by 83 and 65%,
respectively, than for thylakoid preparations from unstarved cells.
Light Energy Is Directed Away from PSII during Nutrient
Starvation
The decrease in quantum yield of O2
evolution at subsaturating light levels suggested that less of the
absorbed light energy was being directed to functional PSII centers.
Previously, the decrease in quantum yield of O2
evolution at subsaturating light levels was correlated with a decline
in Fv/Fmax
(Krause and Weis, 1991); however, a decline in the slope of the
light-saturation curves during nutrient starvation (Fig. 2) can result
from any process that causes the dissipation of excitation energy
before it reaches the PSII reaction center.
Utilization of absorbed light energy was examined in more detail via
measurements of chlorophyll fluorescence. Room-temperature chlorophyll
fluorescence, primarily a measure of fluorescence from the chlorophyll
a of LHCII (Krause and Weis, 1991), was analyzed by PAM
fluorometry (Schreiber et al., 1986). Fluorescence measurements on
light-grown cells containing equal amounts of chlorophyll demonstrated that the Fmax of P- and S-starved cells was
83 and 76%, respectively, of cells grown in complete medium (Table
III). This reduction in the Fmax suggested
that more of the light energy absorbed by LHCII of nutrient-deprived
cells relative to unstarved cells was (a) dissipated as heat within the
LHCII (quenching of fluorescence of singlet excited chlorophyll, qE);
(b) dissipated as a consequence of the formation of damaged PSII
reaction centers (qI); and/or (c) directed away from PSII because of a
state transition (transition from state 1 to state 2, qT).
Experiments were performed to determine the role of qE and qT in the
depression of Fmax in nutrient-deprived
cultures (maintained at 80 µmol photons m2
s1). Nigericin, which dissipates the pH
necessary for maintaining qE, had no effect on the
Fmax of cells maintained in complete medium. In contrast, the addition of nigericin to P- and S-starved cells caused a 6 and 13% increase in the
Fmax, respectively (Table III). Both P- and
S-starved cells treated with nigericin had an Fmax that was 89% of the value determined
for unstarved cells. Therefore, at least part of the reduction in
Fmax (between 30 and 50%) observed for
nutrient-deprived cells appeared to result from qE.
Previous work correlated the accumulation of de-epoxidated xanthophylls
with the development of qE in nutrient-deprived photosynthetic organisms (Plesnicar et al., 1994; Braun et al., 1996; Niyogi et al.,
1997). Using HPLC analysis (Table VI), we
found that both P- and S-starved cells accumulated zeaxanthin (Z),
antheraxanthin (A), and violaxanthin (V), the pigments of the
xanthophyll cycle (Demmig-Adams and Adams, 1992; Björkman and
Demmig-Adams, 1994), even during growth in relatively low light.
Furthermore, the de-epoxidation state of the xanthophylls
([Z + 0.5A]/[Z + A + V]) also increased following
exposure of the cells to P or S limitation, suggesting that the
xanthophyll cycle and the dissipation of excess absorbed light energy
as heat contributes to the Fmax depression
observed in starved cells. In addition to the xanthophyll cycle
pigments, nutrient-deprived cells accumulated lutein, which may also
function in the dissipation of excess absorbed light energy (Niyogi et al., 1997).
View this table:
[in this window]
[in a new window]
|
Table VI.
HPLC analysis
All values are normalized to moles of chlorophyll a and are
the means of two independent measurements. The deviation from the mean
for all measurements was 10% or less.
|
|
Because the addition of nigericin to nutrient-deprived cells did not
completely restore the Fmax to that of
nutrient-replete cells (Table III), qE did not entirely explain the
lower Fmax observed in P- and S-starved
cells. Exposing P- and S-starved cells to 20 min of far-red light
(far-red light preferentially excites PSI, oxidizes the PQ pool, and
promotes a transition to state 1) also resulted in a substantial
increase in the Fmax, indicating that the
cells were originally in state 2 and that qT was also important during
the acclimation of C. reinhardtii to nutrient limitation
(Table III). Furthermore, the simultaneous addition of nigericin and
exposure to far-red light brought the Fmax
of S-starved cells to the level observed for nutrient-replete cells, suggesting that qE and qT were essentially the only processes contributing to the reduction in the Fmax.
In contrast, nigericin and far-red light treatments were not able to
fully restore the Fmax in P-deprived cells,
suggesting a contribution of qI. To further test whether
nutrient-deprived cells were in states 1 or 2, we measured fluorescence
emission from whole cells at 77 K, both before and after exposure of
the cells to 20 min of far-red light. The relative ratio of
PSII-associated fluorescence emission (684 nm) to PSI-associated
fluorescence emission (714 nm) was less for P- and S-starved cells than
for unstarved cells (Table VII). However,
when the starved cells were exposed to 20 min of far-red light prior to
the measurement, the relative fluorescence emission from PSII increased
dramatically; far-red light treatment had only a small effect on
fluorescence emission from unstarved cells. Furthermore, dark
adaptation for 20 min or treatment with nigericin led to relatively
small increases in the 684/714 ratio (10-30% change relative to that
observed following far-red light treatment). Therefore, much of the
change in the 684/714 ratio during nutrient starvation appears to be a
consequence of a state transition, which is consistent with the
increase in Fmax observed in P- and
S-starved cultures after a 20-min exposure to far-red light (Table
III). Together the results presented above indicate that cells starved
of P or S are in state 2, while unstarved cells are primarily in state
1.
View this table:
[in this window]
[in a new window]
|
Table VII.
PSII fluorescence/PSI fluorescence emission
measured at 77 K
Values are means ± sd of at least three measurements.
|
|
Photosynthetic Parameters of the sac1 Mutant during
S Limitation
Using the sac1 mutant of C. reinhardtii, we
were able to determine which responses to S starvation were actively
regulated by the SacI polypeptide. The rate of
O2 evolution in the sac1 mutant does
not decline significantly (based on the number of viable cells) after
24 h of S starvation (Davies et al., 1996), whereas photosynthesis
in wild-type cells declines by more than 70%. Furthermore, the
sac1 mutant showed little reduction in the F/Fmax' (Table III) and in
the light-driven transfer of electrons from water to MV (data not
shown) following 24 h of S deprivation. Chlorophyll levels
declined by 10% (data not shown), whereas the Fv/Fmax
declined by 16% (Table III) in the sac1 strain during S starvation. However, more than 10% of the cells in the S-starved culture of sac1 were dead within 24 h of starvation,
suggesting that the decline in the
Fv/Fmax (and
perhaps in chlorophyll levels) was not part of the same mechanism
controlling the reduction of PSII activity in wild-type cells during S
starvation.
Furthermore, unlike wild-type cells, the sac1 mutant did not
accumulate PSII QB-nonreducing centers during S
starvation (Table V; Fig. 4). This effect was specific for S
deprivation (as expected), since an increase in
(Fpl F0)/(Fmax F0) was observed when the sac1
mutant was starved for P (Table V). The complemented sac1
mutant strain (ars5-4 C11) exhibited an increase in
(Fpl F0)/(Fmax F0) during S starvation that was similar to
that of wild-type cells (Table V). Therefore, the accumulation of PSII
QB-nonreducing centers is part of the
S-acclimation response and is governed by the SacI gene
product.
To determine whether the transition of light-grown cells to state 2 during S-limited growth is an active process also controlled by the
SacI protein, we determined the state of the light-grown (80 µmol photons m2 s1),
S-starved sac1 strain (Table VII). Like wild-type cells, the sac1 mutant was in state 2 during S starvation in the light
and shifted to state 1 upon exposure to far-red light, demonstrating that the SacI protein is not required for this aspect of the
S-stress response. In addition, the de-epoxidation state of the
xanthophylls (Table VI) and nigericin-sensitive quenching of
Fmax in the sac1 mutant during S
starvation were approximately the same as in wild-type cells (Table
III). The overall pool size of the xanthophyll cycle pigments were
somewhat lower in the S-starved sac1 mutant than in
S-starved wild-type cells.
| |
DISCUSSION |
This study demonstrates that P and S deprivation cause similar
alterations to photosynthetic electron transport in C. reinhardtii, which exhibited a 75% decrease in maximal in vivo
O2 evolution within 4 d of P deprivation or
1 d of S deprivation. The longer time required to observe the
effects of P limitation may reflect the accumulation of much larger
intracellular reserves of P than of S (Wetzel, 1983). The decline in
photosynthetic O2 evolution was shown to occur at
the level of photosynthetic electron transport using fluorescence
measurements (F/Fmax') and
in vitro quantitation of the activities of specific sections of the
electron-transport chain. The latter analyses demonstrated that
electron flow was inhibited at PSII, whereas PSI activity was
essentially unchanged in starved cells. The continued operation of PSI
during nutrient-limited growth would provide energy for cell
maintenance and for transporting the limiting nutrient into the
cell when it becomes available.
The decreased maximal electron flow through PSII is a consequence of at
least two processes. First, between 20 and 30% of the PSII reaction
centers appear to be inactivated because of photodamage. Quantitation
of photodamage is complicated by the inherent inaccuracies in
quantitating protein levels by western analysis and the potential
contribution of PSII QB-nonreducing centers to
the loss of Fv (which can result from an
apparent increase in the F0 value).
However, analyses of fluorescence using a very low-intensity measuring
beam (<0.01 µmol photons m2
s1) had little effect on either
F0 or
Fv/Fmax
(<5%), and the measurements of QA levels (Table
IV) demonstrated an absolute loss of functional PSII (by approximately
30%). These results indicate that nutrient limitation results in a
loss of active PSII centers.
Nutrient limitation also promoted the formation of PSII
QB-nonreducing centers, which cannot rapidly
transfer electrons from QA to
QB (Chylla and Whitmarsh, 1989). Others have
demonstrated an increase in the percentage of PSII
QB-nonreducing centers during nutrient limitation
or exposure to excessive light (Falk et al., 1992; Godde and Hefer,
1994). These centers may result from the reduction and subsequent loss
of the quinone from the QB-binding site (Godde
and Hefer, 1994), although it is possible that other alterations of
PSII may also result in centers that are unable to reduce
QB. It has been postulated that PSII
QB-nonreducing centers serve as intermediates in
the D1 repair cycle and constitute a PSII reservoir that can be rapidly
activated (Guenther and Melis, 1990). Furthermore, these centers are
resistant to photoinhibition and may facilitate the dissipation of
excess absorbed light energy (Neale and Melis, 1990; Falk and
Samuelsson, 1992). Therefore, increased photodamage and increased
formation of QB-nonreducing centers in cells
deprived of S or P may be directly linked.
There is still little mechanistic understanding of the modifications to
the photosynthetic machinery that occur during nutrient-limited growth.
In previous reports it was suggested that P deprivation caused a
decline in the levels of reductive pentose-phosphate cycle
intermediates, which led to decreased levels of terminal energy
acceptors and consequently depressed photosynthetic electron-transport activity (Brooks, 1986; Dietz and Heilos, 1990; Jacob and Lawlor, 1993). The accumulation of specific reductive pentose-phosphate cycle
metabolites may also serve to inhibit electron transport, which is
consistent with the finding that the rate of electron flow from water
to MV in vitro was unaffected by P starvation in spinach (Brooks,
1986).
In addition, some studies have shown a direct effect of nutrient
deprivation on photosynthetic electron transport. Both C. reinhardtii and Hematococcus pluvialis exhibit a
significant decrease in the level of the Cyt
b6-f complex as the cells become
starved for specific nutrients (Peltier and Schmidt, 1991; Bulte and
Wollman, 1992; Tan et al., 1995). During N deprivation vegetative cells differentiate into gametes and the loss of the Cyt
b6-f complex parallels the
reduction in O2 evolution. Other studies have
demonstrated that nutrient limitation can directly affect PSII activity
(Herzig and Falkowski, 1989; Collier et al., 1994; Godde and Dannehl, 1994), which is consistent with the results presented in this manuscript.
In addition to a reduction in the rate of light-saturated
photosynthetic electron flow, nutrient limitation caused a decline in
the quantum yield of O2 evolution at
subsaturating light levels. Such a decline can result from a less-
efficient transfer of excitation energy to the PSII reaction centers.
Mechanistically, the decrease in the quantum yield of
O2 evolution at subsaturating light could be a
consequence of nonphotochemical quenching, which is composed of qE, qI,
and qT. A nigericin-induced increase in
Fmax and increased levels of xanthophyll
cycle pigments in nutrient-deprived cells suggested that qE was at
least partially responsible for the decrease in the quantum yield of
O2 evolution. However, the observation that
nutrient-deprived cells were primarily in state 2, and cells maintained
on complete medium were primarily in state 1, demonstrated a role for
qT as well. Although probably less important than qE or qT, qI may
become important to some extent during P deprivation and in more
severely starved cells. Cultures starved of P for 4 d were unable
to return to state 1 as readily as cultures starved of S for 1 d,
suggesting that P limitation causes alterations in the structure of
LHCII (or PSII) that are not readily reversible by far-red
illumination.
The reduction in photosynthetic electron flow that develops during
nutrient limitation of C. reinhardtii cells is an active process and is necessary for survival. During S deprivation the reduction in photosynthetic electron flow appears to be controlled by
the SacI gene product, which may be involved in sensing the S status of the environment (Davies et al., 1996). A sac1
mutant strain becomes light sensitive during S deprivation because it cannot alter photosynthetic electron transport. S starvation of sac1, as in wild-type cells, results in the induction of
both qE and qT. These processes may be triggered by metabolic changes in nutrient-starved cells that are independent of the SacI
signal-transduction pathway. However, in contrast to wild-type cells,
the level of damaged PSII centers in the sac1 mutant
reflects cell death and the mutant strain is unable to form PSII
QB-nonreducing centers. DCMU, which phenocopies
the formation of QB-nonreducing centers, rescues
the lethal phenotype (Davies et al., 1996). This suggests that either
the formation of O2 radicals generated by
photosynthetic electron transport or the hyperreduction of electron
transport components downstream of the QB-binding
site, or both, leads to a loss of viability in the sac1
mutant.
In summation, similar alterations in photosynthetic electron transport
occur during both P and S starvation. First, the levels of chlorophyll
a and b decline by approximately 15%. Second,
there is a 20 to 30% decrease in functional PSII reaction centers.
Third, fewer of the functional centers can rapidly reduce the PQ pool. Fourth, more of the light harvested by LHCII is dissipated as heat or
directed away from PSII reaction centers, possibly toward PSI, via a
transition to state 2. These alterations result in a decline in linear
electron flow and a reduction in the efficiency of energy transfer to
PSII, while possibly allowing PSI cyclic electron flow to continue. If
nutrient-starved cells maintain PSI cyclic electron flow with little
PSII activity, the cells would produce less reductant (which is no
longer required at high levels) but would continue to generate a pH
across the thylakoid membrane, allowing for significant
nonphotochemical quenching and the production of ATP for the
maintenance of vital cellular processes.
| |
FOOTNOTES |
1
D.D.W. was supported as a predoctoral trainee by
the National Institutes of Health (grant no. GM07276). This work was
also supported by the Carnegie Institution of Washington, the U.S. Department of Agriculture (grant no. 9302076), and the National Science
Foundation (grant no. IBN950-6254). This is Carnegie Institution of
Washington Department of Plant Biology publication no. 1327.
*
Corresponding author; e-mail dwykoff{at}andrew.stanford.edu; fax
1-650-325-6857.
Received October 17, 1997;
accepted January 29, 1998.
| |
ABBREVIATIONS |
Abbreviations:
DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-1,4-benzoquinone.
DCPIP, 2,6-dichlorophenol-indo-phenol.
DCPIPH2, DCPIP (reduced
form).
F, Fmax', steady-state
fluorescence during actinic light.
DHQ, durohydroquinone.
DPC, sym-diphenylcarbazide.
ferricyanide, potassium ferricyanide.
Fmax, maximal fluorescence when
QA is fully reduced.
Fmax', Fmax during exposure to actinic light.
Fpl, fluorescence plateau in the presence of
ferricyanide.
F0, initial fluorescence when
QA is fully oxidized.
Fv, variable fluorescence (Fmax F0).
LHC, light-harvesting complex.
MV, methyl viologen.
PAM, pulse-amplitude-modulated.
PQ, plastoquinone.
qE, energy-dependent chlorophyll fluorescence quenching.
qI, chlorophyll fluorescence quenching due to photodamage.
qT, chlorophyll fluorescence quenching due to a state transition.
TAP, Tris-acetate-P.
TAP-P, TAP without P.
TAP-S, TAP without S.
| |
ACKNOWLEDGMENTS |
We thank Krishna Niyogi and Klaas van Wijk for critically
reading the manuscript.
| |
LITERATURE CITED |
Allen JF
(1992)
Protein phosphorylation in regulation of photosynthesis.
Biochim Biophys Acta
1098:
275-335
[Medline]
Asada K
(1994)
Production and action of active oxygen species in photosynthetic tissues.
In
PM Foyer Mullineaux,
eds, Causes of Photooxidative Stress and Amelioration of Defense Systems in Plants.
CRC Press, Boca Raton, FL, pp 77-104
Ball SG,
Dirick L,
Decq A,
Martiat J-C,
Matagne RF
(1990)
Physiology of starch storage in the monocellular alga Chlamydomonas reinhardtii.
Plant Sci
66:
1-9
Björkman O,
Demmig-Adams B
(1994)
Regulation of photosynthetic light energy capture, conversion, and dissipation in leaves of higher plants.
In
E-D Schulze,
MM Caldwell,
eds, Ecophysiology of Photosynthesis.
Springer-Verlag, Berlin, pp 17-47
Braun P,
Banet G,
Tal T,
Malkin S,
Zamir A
(1996)
Possible role of Cbr, an algal early-light-induced protein, in nonphotochemical quenching of chlorophyll fluorescence.
Plant Physiol
110:
1405-1411
[Abstract]
Brooks A
(1986)
Effects of phosphorus nutrition on ribulose-1,5-bisphosphate carboxylase activation, photosynthetic quantum yield and amounts of some Calvin-cycle metabolites in spinach leaves.
Aust J Plant Physiol
13:
221-237
Bulte L,
Wollman FA
(1992)
Evidence for a selective destabilization of an integral membrane protein, the cytochrome b6/f complex, during gametogenesis in Chlamydomonas reinhardtii.
Eur J Biochem
204:
327-336
[ISI][Medline]
Chylla RA,
Whitmarsh J
(1989)
Inactive photosystem II complexes in leaves.
Plant Physiol
90:
765-772
[Abstract/Free Full Text]
Collier JL,
Herbert SK,
Fork DC,
Grossman AR
(1994)
Changes in the cyanobacterial photosynthetic apparatus in response to macronutrient deprivation.
Photosynth Res
42:
173-183
[CrossRef]
Curtis VA,
Brand JJ,
Togasaki RK
(1975)
Partial reactions of photosynthesis in briefly sonicated Chlamydomonas.
Plant Physiol
55:
183-186
[Abstract/Free Full Text]
Davies JP,
Yildiz F,
Grossman AR
(1994)
Mutants of Chlamydomonas with aberrant responses to sulfur deprivation.
Plant Cell
6:
53-63
[Abstract]
Davies JP,
Yildiz FH,
Grossman AR
(1996)
Sac1, a putative regulator that is critical for survival of Chlamydomonas reinhardtii during sulfur deprivation.
EMBO J
15:
2150-2159
[ISI][Medline]
de Hostos EL,
Schilling J,
Grossman AR
(1989)
Structure and expression of the gene encoding the periplasmic arylsulfatase of Chlamydomonas reinhardtii.
Mol Gen Genet
218:
229-239
[CrossRef][ISI][Medline]
Demmig-Adams B,
Adams WW III
(1992)
Photoprotection and other responses of plants to high light stress.
Annu Rev Plant Physiol Plant Mol Biol
43:
599-626
[CrossRef][ISI]
Dietz K-J,
Heilos L
(1990)
Carbon metabolism in spinach leaves as affected by leaf age and phosphorus and sulfur nutrition.
Plant Physiol
93:
1219-1225
[Abstract/Free Full Text]
Falk S,
Leverenz JW,
Samuelsson G,
Oquist G
(1992)
Changes in photosystem II fluorescence in Chlamydomonas reinhardtii exposed to increasing levels of irradiance in relationship to the photosynthetic response to light.
Photosynth Res
31:
31-40
Falk S,
Samuelsson G
(1992)
Recovery of photosynthesis and photosystem II fluorescence in Chlamydomonas reinhardtii after exposure to three levels of high light.
Physiol Plant
85:
61-68
[CrossRef]
Ferreira RMB,
Teixeira ARN
(1992)
Sulfur starvation in Lemna leads to degradation of ribulose-bisphosphate carboxylase without plant death.
J Biol Chem
267:
7253-7257
[Abstract/Free Full Text]
Genty B,
Briantais J-M,
Baker NR
(1989)
The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence.
Biochim Biophys Acta
990:
87-92
Godde D,
Dannehl H
(1994)
Stress-induced chlorosis and increase in D1-protein turnover precede photoinhibition in spinach suffering under magnesium/sulphur deficiency.
Planta
195:
291-300
Godde D,
Hefer M
(1994)
Photoinhibition and light-dependent turnover of the D1 reaction centre polypeptide of photosystem II are enhanced by mineral-stress conditions.
Planta
193:
290-299
Gorman DS,
Levine RP
(1966)
Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardtii.
Proc Natl Acad Sci USA
54:
1665-1669
Govindjee
(1990)
Photosystem-II heterogeneity
the acceptor side.
Photosynth Res
25:
151-160
[CrossRef]
Guenther JE,
Melis A
(1990)
The physiological significance of photosystem II heterogeneity in chloroplasts.
Photosynth Res
23:
105-109
Guenther JE,
Nemson JA,
Melis A
(1988)
Photosystem stoichiometry and chlorophyll antenna size in Dunaliella salina (green algae).
Biochim Biophys Acta
934:
108-117
Guenther JE, Nemson JA, Melis A (1990) Development of photosystem
II in dark grown Chlamydomonas reinhardtii. A
light-dependent conversion of PSII
,
QB-nonreducing centers to the
PSII
, QB-reducing form.
Photosynth Res 24: 35-46
Herzig R,
Falkowski PG
(1989)
Nitrogen limitation in Isochrysis galbana (haptophyceae). I. Photosynthetic energy conversion and growth efficiencies.
J Phycol
25:
462-471
[CrossRef][ISI]
Horton P,
Ruban AV,
Walters RG
(1996)
Regulation of light harvesting in green plants: indication by nonphotochemical quenching of chlorophyll fluorescence.
Annu Rev Plant Physiol Plant Mol Biol
47:
655-684
[CrossRef][ISI]
Houle D,
Cargnan R
(1992)
Sulfur speciation and distribution in soils and above ground biomass of a boreal coniferous forest.
Biogeochemistry
16:
63-82
Jacob J,
Lawlor DW
(1993)
In vivo photosynthetic electron transport does not limit photosynthetic capacity in phosphate-deficient sunflower and maize leaves.
Plant Cell Environ
16:
785-795
[CrossRef]
Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations
for determining chlorophylls a, b, c1 and c2 in higher plants, algae and natural
phytoplankton. Biochem Physiol Pflanz 167:
191-194
Kim JH,
Nemson JA,
Melis A
(1993)
Photosystem II reaction center damage and repair in Dunaliella salina (green alga).
Plant Physiol
103:
181-189
[Abstract]
Krause GH,
Weis E
(1991)
Chlorophyll fluorescence and photosynthesis: the basics.
Annu Rev Plant Physiol Plant Mol Biol
42:
313-349
[CrossRef][ISI]
Lavergne J,
Briantais JM
(1996)
Photosystem II heterogeneity.
In
DR Ort,
CF Yocum,
eds, Oxygeneic Photosynthesis: The Light Reactions.
Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 265-287
Lers A,
Levy H,
Zamir A
(1991)
Co-regulation of a gene homologous to early light-induced genes in higher plants and 
-carotene biosynthesis in the alga Dunaliella bardawil.
J Biol Chem
266:
13698-13705
[Abstract/Free Full Text]
Levy H,
Tal T,
Shaish A,
Zamir A
(1993)
Cbr, an algal homolog of plant early light-induced proteins, is a putative zeaxanthin binding protein.
J Biol Chem
268:
20892-20896
[Abstract/Free Full Text]
Mahler RJ,
Maples RL
(1987)
Effect of sulfur additions on soil and the nutrition of wheat.
Commun Soil Sci Plant Anal
18:
653-673
Melis A
(1991)
Dynamics of photosynthetic membrane composition and function.
Biochim Biophys Acta
1058:
87-106
[CrossRef]
Neale PJ,
Melis A
(1990)
Activation of a reserve pool of photosystem II in Chlamydomonas reinhardtii counteracts photoinhibition.
Plant Physiol
92:
1196-1204
[Abstract/Free Full Text]
Niyogi KK,
Björkman O,
Grossman AR
(1997)
Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching.
Plant Cell
9:
1369-1380
[Abstract]
Ort DR,
Whitmarsh J
(1990)
Inactive photosystem II centers.
Photosynth Res
23:
101-104
Peltier G,
Schmidt GW
(1991)
Chlororespiration: an adaptation to nitrogen deficiency in Chlamydomonas reinhardtii.
Proc Natl Acad Sci USA
88:
4791-4795
[Abstract/Free Full Text]
Plesnicar M,
Kastori R,
Petrovic N,
Pankovic D
(1994)
Photosynthesis and chlorophyll fluorescence in sunflower (Helianthus annuus L.) leaves as affected by phosphorus nutrition.
J Exp Bot
45:
919-924
[Abstract/Free Full Text]
Quisel JQ,
Wykoff DD,
Grossman AR
(1996)
Biochemical characterization of the extracellular phosphatases produced by phosphorus-deprived Chlamydomonas reinhardtii.
Plant Physiol
111:
839-848
[Abstract]
Raich JW,
Russell AE,
Crews TE,
Farrington H,
Vitousek PM
(1996)
Both nitrogen and phosphorus limit plant production on young Hawaiian lava flows.
Biogeochemistry
32:
1-14
Samosir SSR,
Blair GJ,
Lefroy RDB
(1993)
Effects of placement of elemental S and sulfate on the growth of two rice varieties under flooded conditions.
Aust J Agric Res
44:
1775-1788
Schreiber U,
Schliwa U,
Bilger W
(1986)
Continuous recording of photochemical and non-photochemical chlorophyll fluorescence quenching with a new type of modulation fluorometer.
Photosynth Res
10:
51-62
Top
Abstract
Introduction