Download Spatial patterns and metabolic regulation of photosynthetic

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Spatial patterns and metabolic regulation of
photosynthetic parameters during leaf senescence
Blackwell Publishing, Ltd.
Astrid Wingler, Magali Marès and Nathalie Pourtau
Department of Biology, University College London, Gower Street, London WC1E 6BT, UK
Summary
Author for correspondence:
Astrid Wingler
Tel: +44 20 7679 7268
Fax: +44 20 7679 7096
Email: [email protected]
Received: 4 September 2003
Accepted: 15 October 2003
doi: 10.1111/j.1469-8137.2003.00996.x
• To prevent premature cell death and to allow efficient nutrient mobilization from
senescing leaves, the photosynthetic apparatus has to be dismantled systematically.
This requires temporal, spatial and metabolic regulation of photosynthetic function
and photoprotection.
• Conventional pulse-modulated fluorometry and chlorophyll fluorescence imaging
were used to study age- and nutrient-dependent senescence patterns in Arabidopsis
thaliana.
• Nonphotochemical quenching (NPQ) rose during leaf maturation, indicating
increased energy dissipation. During later stages of senescence, overall plant NPQ
declined, but NPQ remained high in the base of rosette leaves. Other fluorescence
parameters also showed spatial patterns, for example minimum fluorescence (F0)
was temporarily increased in the tips of inner rosette leaves from where high F0
spread to the base, in a zone preceding cell death. Senescence-dependent changes
in chlorophyll fluorescence characteristics were accelerated by growth on glucosecontaining medium in combination with low, but not with high, nitrogen supply.
• Our experiments revealed distinct spatial patterns of photosynthetic and photoprotective processes in senescing leaves and induction of these processes by high
sugar-to-nitrogen ratios.
Key words: Arabidopsis, chlorophyll fluorescence imaging, leaf senescence,
nonphotochemical quenching, photoinhibition, photoprotection, sugar sensing.
Abbreviations
Minimum fluorescence (F0), maximum fluorescence (Fm), maximum quantum efficiency of photosystem II photochemistry (Fv /Fm), quantum efficiency of excitation
′ ), quantum efficiency of
energy capture by open photosystem II centres ( F v′ /F m
photosystem II electron transport (ΦPSII), nonphotochemical quenching (NPQ).
© New Phytologist (2004) 161: 781–789
Introduction
The main function of leaf senescence is the recycling of
nutrients. For example, > 80% of the nitrogen contained in
Arabidopsis leaves is exported during senescence (Himelblau
& Amasino, 2001). To allow mobilization and transport of
nutrients, cell death has to be prevented until senescence has
been completed. Indeed, membrane integrity and cellular
compartmentalization are maintained until late senescence
© New Phytologist (2004) 161: 781 – 789 www.newphytologist.org
(Lee & Chen, 2002), supporting the view that senescence is a
nonapoptotic transdifferentiation process (Thomas et al.,
2003).
Mobilization of nitrogen from photosynthetic proteins,
such as Rubisco, results in a decline in photosynthetic CO2
assimilation. In Arabidopsis photosynthesis declines early,
before the leaves are fully expanded, whereas the chlorophyll
content remains high until later stages of development (Stessman
et al., 2002). A combination of high chlorophyll content
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and low CO2 assimilation could potentially result in photooxidative damage caused by an imbalance between energy
capture and dissipation. For example, it has recently been
demonstrated that a ‘staygreen’ mutant of soybean exhibits
increased susceptibility to photoinhibition (Guiamét et al.,
2002). Furthermore, oxidative stress in chloroplasts typically
increases with increasing leaf age (Munné-Bosch & Alegre,
2002). To prevent photo-oxidative processes that could lead
to lipid peroxidation and cell death, the photosynthetic apparatus has to be dismantled in an ordered manner. For example,
photosystem II activity declines before photosystem I activity
in Brassica napus cotyledons (Ghosh et al., 2001) and adjustments of the amount of minor light-harvesting complexes
may prevent photo-oxidative damage in senescing barley
leaves (Humbeck & Krupinska, 2003). Protective processes,
such as nonphotochemical quenching (NPQ ) are also likely
to play a role in preventing damage during senescence. In
maize and wheat, NPQ increases in combination with an
accumulation of xanthophyll cycle carotenoids, indicating
increased dissipation of excess excitation energy as heat (Lu &
Zhang, 1998; Lu et al., 2001). By contrast, a decline in NPQ
was found in senescing soybean leaves (Guiamét et al., 2002).
This discrepancy may be caused by different growth conditions or differences in the stage of senescence analysed. In
addition, whether recorded values of photosynthetic parameters, such as NPQ , are increased or decreased may depend
on where within a senescing leaf fluorescence is measured.
Leaf senescence usually proceeds from the tip to the base of a
leaf, while the veins stay alive until the final stages (Feller
& Fischer, 1994). It is therefore likely that photosynthetic
parameters show heterogeneous spatial patterns.
Spatial patterns of photosystem II processes can be analysed
using chlorophyll a fluorescence imaging. This technique is
now commonly applied for measuring photosystem II processes in heterogeneous systems. Imaging has been used to
study photosynthetic responses to pathogen infection
(Scholes & Rolfe, 1996); ozone-induced perturbations of
photosynthesis (Leipner et al., 2001); photo-oxidative stress
(Fryer et al., 2002); and light adaptation (Lichtenthaler et al.,
2000). The sink–source transition in young leaves has also
been characterized using chlorophyll fluorescence imaging
(Meng et al., 2001), but we do not know of any study where
this technique has been applied to analyse photosynthetic
parameters during leaf senescence.
In addition to allowing spatial analysis of photosynthetic
processes, imaging can be used as a fast and convenient
method for studying photosynthetic changes in a large
number of small plants, e.g. Arabidopsis grown on media with
varied nitrogen and carbon supply. There is increasing evidence that senescence is regulated by the carbon–nitrogen
balance in leaves (Ono et al., 1996; Stitt & Krapp, 1999;
Masclaux et al., 2000; Masclaux-Daubresse et al., 2002).
Whereas it had previously been suggested that leaf senescence
is triggered by an age-dependent decline in photosynthesis
(Hensel et al., 1993), it now seems more likely that the
senescence-related decline in photosynthesis is a consequence of sugar accumulation, especially during early stages of
senescence (Noodén et al., 1997; Wingler et al., 1998; Masclaux
et al., 2000). Sugar sensing has been demonstrated to regulate
a large number of metabolic and developmental processes,
some of which involve hexokinase as a sugar sensor (Jang &
Sheen, 1994; Smeekens, 2000). Hexokinase may also be
responsible for the sugar-dependent regulation of leaf senescence. Tomato plants overexpressing hexokinase-1 from Arabidopsis show accelerated senescence (Dai et al., 1999), while
senescence is delayed in hexokinase-1 mutants of Arabidopsis
(Moore et al., 2003). Further work is required to unravel the
interactions of sugar and nitrogen signalling during the regulation of senescence.
The aim of this study was to analyse spatial and temporal
patterns in photosynthetic function during leaf senescence,
especially with respect to the regulation by sugar and nitrogen
supply.
Materials and Methods
Plant material
Seed of Arabidopsis thaliana (L.) Heinh. (Col-0) was
suspended in 0.5% (w/v) low-melting agarose and pipetted
onto compost (Murphy’s Multi Purpose Compost; Murphy
Garden Products, Ipswich, UK). After cold treatment for
3–4 d at 4°C, the pots were transferred into controlled
environment growth chambers and the plants were grown at
a photon flux density of approximately 100 µmol m−2 s−1 for
12 h d−1 at a temperature of 22°C during the day and 18°C
at night.
For growth on agar medium, seeds were sterilized in commercial bleach, washed, resupended in 0.7% low-melting
agarose, and pipetted on agar (1% w/v) medium. For
high-nitrogen treatments the medium consisted of a halfconcentrated Murashige–Skoog (MS) medium containing
30 m nitrogen (10.3 m NH4+ and 19.7 m NO3–). For lownitrogen treatments the nitrogen concentration was reduced
to 4.7 m (only NO3–). After cold treatment the plates were
transferred to growth chambers and grown in vertical orientation under the same conditions as described for the
compost-grown plants, but with a daylength of 16 h to accelerate plant development and prevent effects caused by drying
of the agar medium.
Determination of chlorophyll content
Relative chlorophyll content was determined using a
Minolta SPAD chlorophyll meter (N-tester, Hydro Agri,
Immingham, UK). Measurements were taken in the middle
of each leaf and values for five outer rosette leaves per plant
were averaged.
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Chlorophyll fluorescence analysis
Chlorophyll a fluorescence was analysed using a pulsemodulated fluorometer (FMS-2, Hansatech, King’s Lynn,
UK) with the fibre optics pointing to the middle of a large
outer rosette leaf (usually leaf 10). Minimum fluorescence
(F0) was measured by exposing leaves of dark-adapted plants
to modulated red light, before a saturating flash of white light
was applied to record maximum fluorescence (Fm). Leaves
were then illuminated with actinic light (225 µmol m−2 s−1)
and saturating flashes of 0.7 s duration were applied every
1.5 min. After 15 min illumination, maximum fluorescence
of light-adapted leaves ( F ′m ), steady-state fluorescence (Fs)
and ground fluorescence ( F ′0) were recorded. The following
equations were used for calculating photosynthetic parameters.
Maximum quantum efficiency of photosystem II
photochemistry, Fv/Fm = (Fm − F0)/Fm; quantum efficiency
of excitation energy capture by open photosystem II centres,
F ′v/F ′m = (F ′m − F ′0)/F ′m ; quantum efficiency of photosystem
II electron transport, ΦPSII = (F m
′ − Fs )/F m
′ ; photochemical
quenching, qP = (F m
′ − Fs )/(F m
′ − F 0′ ); nonphotochemical
quenching, NPQ = (Fm − F m
′ )/F ′m.
Chlorophyll fluorescence images were captured with a pulsemodulated imaging fluorometer (FluorCam 700MF, Photon
Systems Instruments, Brno, Czech Rebublic) as described by
Nedbal et al. (2000). After measuring Fv /Fm in dark-adapted
plants, the plants were illuminated with actinic light
(100 µmol m−2 s−1) and saturating flashes of 0.8 s duration
Fig. 1 Development of senescence in rosettes of Arabidopsis plants.
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were applied every 2 min to determine quenching parameters.
For plants grown in compost, data were analysed for
individual leaves (usually leaf 10) and averaged for the whole
rosette. For plants grown on agar medium, data were averaged
for the whole rosette.
Results
Development of senescence in Arabidopsis rosettes
Senescence proceeded from the old (outer) to young (inner)
rosette leaves (Fig. 1). In the Col-0 accession of Arabidopsis
studied here, but not in Ws-2 plants (not shown), loss of
chlorophyll was accompanied by an accumulation of
anthocyanins. Cell death (visible collapse of cells) proceeded
from tip to base of outer rosette leaves (Fig. 1). The inner
rosette leaves stayed alive until late development.
Overall changes in chlorophyll fluorescence parameters
during leaf senescence
Chlorophyll content (Table 1) increased while the outer
rosette leaves were still expanding (Fig. 1). At the same time,
the quantum efficiency of photosystem II electron transport,
ΦPSII, already started to decline, indicating that electron
transport rates were reduced before full leaf expansion. At day
54, ΦPSII was reduced by 41% compared with leaves of
young plants (day 34), while chlorophyll content was 5%
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Table 1 Changes in chlorophyll content and fluorescence characteristics during leaf senescence in Arabidopsis
Day
Chlorophyll
(relative units)
Fv /Fm
ΦPSII
F ′v /F m
′
NPQ
34
41
46
49
54
60
63
67
314 ± 18
392 ± 51
389 ± 18
330 ± 49
329 ± 73
225 ± 83
198 ± 31
93 ± 41
0.824 ± 0.012
0.832 ± 0.012
0.804 ± 0.015
0.782 ± 0.023
0.728 ± 0.062
0.646 ± 0.106
0.454 ± 0.155
0.337 ± 0.215
0.653 ± 0.014
0.623 ± 0.030
0.528 ± 0.130
0.450 ± 0.058
0.388 ± 0.166
0.180 ± 0.082
0.183 ± 0.020
0.173 ± 0.120
0.767 ± 0.005
0.753 ± 0.014
0.699 ± 0.082
0.649 ± 0.072
0.534 ± 0.150
0.363 ± 0.109
0.336 ± 0.057
0.303 ± 0.188
0.40 ± 0.05
0.52 ± 0.08
0.67 ± 0.33
0.72 ± 0.24
0.77 ± 0.28
1.23 ± 0.52
0.95 ± 0.73
0.37 ± 0.20
Chlorophyll was measured in five outer rosette leaves per plant. Maximum quantum efficiency of photosystem II photochemistry (Fv /Fm),
quantum efficiency of photosystem II electron transport (ΦPSII), quantum efficiency of excitation energy capture by open photosystem II centres
( F ′v /F m
′ ) and nonphotochemical quenching (NPQ) were determined by conventional pulse-modulated fluorometry in one outer rosette leaf
(usually leaf 10) per plant. Data are means ± SD of five plants.
higher. This initial decline in ΦPSII was mainly caused by a
decrease in F ′v /F m
′ and thus was likely to be caused by a
reduced efficiency of excitation capture, not by a change in the
concentration of open photosystem II reaction centres (Genty
et al., 1989). Nonphotochemical quenching (NPQ) increased
until day 60, but declined during the final stages (Table 1).
The maximum quantum efficiency of photosystem II
photochemistry in dark-adapted leaves, Fv /Fm, was clearly
reduced from day 49 onwards. The large standard deviation of
fluorescence parameters during late senescence (Table 1) was
probably caused by variations in the extent of senescence
between and within individual leaves of a plant (Fig. 1).
Spatial patterns in photosynthetic parameters
To study how the changes in photosynthetic parameters were
distributed over individual leaves and the whole leaf rosette,
photosynthesis was analysed in a separate experiment by
imaging of chlorophyll fluorescence. Although the exact time
course of fluorescence changes in this experiment was not
identical with the experiment presented in Table 1, the overall
changes were the same: ΦPSII declined early, accompanied by
a temporary increase in NPQ , while Fv /Fm declined later (Fig. 2).
When data were averaged for the whole leaf rosette, Fv /Fm
decreased to a much smaller extent than for individual outer
rosette leaves (Fig. 2a). ΦPSII was highest in young rosettes
and declined before the onset of senescence was detectable as
a decline in Fv /Fm or as visible leaf yellowing. Until day 61,
ΦPSII values were the same for whole rosettes and for outer
rosette leaves (Fig. 2b). After this time point, ΦPSII declined
drastically in outer rosette leaves but remained constant averaged over the whole rosette, probably because of the formation of additional young rosette leaves. NPQ initially rose
during leaf development, both in the whole rosette and in
outer rosette leaves (Fig. 2c), but declined when senescence
became visible. Again the decline in outer rosette leaves was
more pronounced than for the whole rosette.
Images revealed local changes in F0 (Fig. 3a) from day 61
onwards. While F0 declined in the outer rosette, it rose from
tip to base of inner rosette leaves. F0 was highest in a band of
cells separating green from dead areas of the leaves, shortly
before cell death occurred. By contrast, Fm mainly paralleled
the distribution of chlorophyll and reached highest values in
the inner rosette leaves during late senescence (Fig. 3b). As
expected, Fv /Fm declined in senescing leaves, showing the
lowest values on the tips of the outer rosette leaves (Fig. 3c).
Images of NPQ showed that values remained low in the cotyledons but increased almost uniformly in the rosette leaves
until day 47 (Fig. 3d). During later stages of senescence, NPQ
mainly declined in the leaf tips, but high NPQ was maintained the base of individual rosette leaves until day 74.
Effect of nitrogen and sugar supply on photosynthetic
parameters during leaf senescence
To analyse the metabolic regulation of photosynthetic
function during leaf senescence, plants were grown on agar
medium with varied nitrogen and glucose supply. In this
system the plants remained small, but flowered and produced
seeds (Fig. 4). Compared with high nitrogen supply, growth
at low nitrogen supply led to anthocyanin accumulation in
the petioles and stems, but did not accelerate visible senescence.
This may be because the chosen lower concentration of 4.7 m
nitrate was still quite high. However, when glucose was added
to the low-nitrogen medium, senescence was clearly accelerated
and anthocyanins accumulated in the leaf blades. It is possible
that addition of glucose accelerated nitrogen utilization by
the plants and thus led to faster nitrogen depletion from the
medium. By contrast, plants grown on high-nitrogen medium
plus glucose did not show accelerated senescence, but were
darker green than plants grown in the absence of sugar.
Addition of sorbitol and mannitol in combination with low
nitrogen supply did not induce senescence, demonstrating
that the effect of glucose was not merely osmotic (not shown).
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a combination of glucose with low nitrogen supply accelerated senescence (Fig. 5a,b). A low concentration of 2% glucose was equally as effective as 4% glucose, without causing
the delay in early photosynthetic development that was
detectable with 4% glucose. At high nitrogen supply the addition of sugar, especially 4% glucose, prevented the decline in
ΦPSII that occurs in the absence of an external carbon source
(Fig. 5c). At low nitrogen supply the fall in ΦPSII was initially
also delayed by glucose, but only until day 20, after which glucose accelerated the decline in ΦPSII (Fig. 5d). NPQ rose
until day 20 at high nitrogen supply (Fig. 5e). This increase
was delayed in the presence of 4% glucose. At low nitrogen
supply the increase in NPQ was not as pronounced (Fig. 5f),
but NPQ declined earlier in the 2% glucose treatment.
Discussion
To allow efficient reallocation of nutrients in senescing plants,
photosynthetic processes have to be regulated at the following
levels: (i) temporal regulation is required to activate photoprotective processes while photosynthetic proteins are being
degraded; (ii) spatial regulation is required to protect those parts
of the leaves that are essential for enzymatic and transport processes; (iii) metabolic regulation is required to integrate senescencedependent nutrient recycling with environmental factors.
Temporal regulation of photosynthetic parameters
Fig. 2 Changes in chlorophyll fluorescence characteristics during leaf
senescence as determined by fluorescence imaging. (a) Maximum
quantum efficiency of photosystem II photochemistry (Fv /Fm);
(b) quantum efficiency of photosystem II electron transport (ΦPSII);
and (c) nonphotochemical quenching (NPQ) were analysed for
whole rosettes and individual outer rosette leaves (usually leaf 10).
Data are means ± SD of four plants.
In plants grown on the 2% glucose/low-nitrogen medium, Fv /
Fm first declined in the cotyledons followed by the rosette
leaves. In the siliques, Fv /Fm remained high when the leaf rosette
had already senesced (Fig. 4e), indicating that nutrients were
exported out of the leaves and used for reproduction.
The effect of glucose and nitrogen supply on senescence of
leaf rosettes was quantified by imaging of fluorescence parameters. Fv /Fm values confirmed the optical impression that only
© New Phytologist (2004) 161: 781 – 789 www.newphytologist.org
Based on our results, it is possible to divide senescence into an
early phase that is characterized by a decrease in ΦPSII, increase
in NPQ and high Fv /Fm values; and a late phase during which
NPQ declines in parallel with Fv /Fm (Table 1; Figs 2, 3, 5).
The early decline in photosynthetic electron transport, as
indicated by a decrease in ΦPSII, is in agreement with the
early reduction in CO2 assimilation reported by Stessman
et al. (2002) for Arabidopsis. Reduced energy consumption by
CO2 assimilation, in combination with the high chlorophyll
content during this phase, is potentially dangerous. An imbalance between energy capture and utilization can result in an
over-reduction of the electron transport chain, photoinhibition
and oxidative stress caused by photoreduction of oxygen to
superoxide in the Mehler reaction (Badger, 1985). In addition, reactive singlet oxygen could be formed through reaction of oxygen with triplet chlorophyll, especially when
chlorophyll is released by the breakdown of the chlorophyll–
protein complexes in photosynthetic membranes (Merzlyak
& Hendry, 1994). Formation of reactive oxygen species can
lead to lipid peroxidation in senescing tissues (Berger et al.,
2001; Leverentz et al., 2002), resulting in decompartmentalization and cell death.
To prevent damage, excess energy has to be dissipated, for
example by NPQ (Horton et al., 1994; Müller et al., 2001).
The results presented here confirm that NPQ is involved in
photoprotection during early senescence. This is in agreement
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Fig. 3 Images of chlorophyll fluorescence characteristics in senescing Arabidopsis plants. (a) False colour images of minimum fluorescence
of dark-adapted plants (F0); (b) maximum fluorescence (Fm); (c) maximum quantum efficiency of photosystem II photochemistry (Fv /Fm);
(d) nonphotochemical quenching (NPQ).
with results obtained by Lu & Zhang (1998) and Lu et al.
(2001) for senescing leaves of maize and wheat. We also found
a temporary increase in NPQ in glasshouse-grown tobacco
plants (data not shown). Given the low growth photon flux
density in this study, it may be surprising that NPQ did
increase, and the response may have been stronger under high
light conditions. As NPQ relies on xanthophyll cycle activity
and formation of a proton gradient across the thylakoid
membrane (Horton et al., 1994), it is not surprising that it declines
during later stages of senescence. Analysis of senescence in
Arabidopsis mutants with reduced NPQ, for example mutants
lacking PsbS (Li et al., 2000), would show the extent to which
NPQ is involved in preventing premature cell death.
In addition to NPQ, anthocyanin formation, as seen in
plants grown in compost and on agar medium with a combination of glucose and low nitrogen supply (Figs 1, 4), could be
involved in the protection of senescing leaves. Based on experiments with ‘red-senescing’ compared with ‘yellow-senescing’
leaves of Cornus stolonifera, Feild et al. (2001) suggested that
optical masking of chlorophyll by anthocyanins may reduce
the risk of photo-oxidative damage during leaf senescence. By
contrast to the Col-0 accession of Arabidopsis studied here,
anthocyanins did not accumulate during senescence in Ws-2
plants, which showed an earlier decline in Fv /Fm and accelerated cell death (data not shown).
Spatial regulation of photosynthetic parameters
Senescence usually proceeds from leaf tip to base, while the
vascular bundles stay intact until the final stages (Feller &
Fischer, 1994). During the early stages photosynthetic parameters
were evenly distributed over the leaf rosette. However, spatial
patterns became apparent later during the senescence process
(Fig. 3). Whereas overall plant NPQ declined during late
senescence, high NPQ was maintained in the base of individual
rosette leaves. Photoprotection in the leaf base is probably
important to allow nutrient export.
The decline in Fv /Fm in outer rosette leaves and from tip to
base of inner rosette leaves shows that these parts of the rosette
were no longer sufficiently protected against photoinhibition.
A decrease in Fv /Fm is usually caused by a decrease in Fm, in
combination with an increase in F0 (Ögren, 1991). In the
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Fig. 4 Effect of sugar and nitrogen supply on senescence of Arabidopsis plants. Plants were grown for 3 d on agar medium with high nitrogen
(a,c) or low nitrogen (b,d) supply, without sugar (a,b) or with addition of 2% glucose (c,d). (e) False colour image of Fv /Fm in a plant grown at
low nitrogen supply plus 2% glucose.
outer rosette leaves, which had already lost most of their chlorophyll, the drastic decline in Fv /Fm was caused by lower Fm
values in combination with reduced F0 values. In a band
between the tip and base of inner rosette leaves, Fv /Fm
decreased because of a strong increase in F0, combined with a
decrease in Fm, whereas the leaf bases exhibited increased F0
in combination with increased Fm. This shows that at least
two different factors independently affecting F0 and Fm are
responsible for the decline in Fv /Fm in senescing leaves.
Increased F0 could be caused by the release of free chlorophyll
from protein–pigment complexes; while Fm is affected by the
capacity to reduce the electron acceptor Q A. It would not
have been possible to detect these changes using conventional
pulse-modulated fluorometry.
Metabolic regulation of leaf senescence
Our results demonstrate that the metabolic regulation of
senescence can be studied by growing plants on agar medium
(Fig. 4) in combination with simple and rapid monitoring of
senescence using chlorophyll fluorescence imaging (Fig. 5).
Although the plants grown on agar medium were cultivated
under longer days than the plants grown in compost (16 h
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compared with 12 h), they showed similar fluorescence
characteristics: an early decline in ΦPSII combined with a rise
in NPQ and a late decline in Fv /Fm. There was no indication
that senescence could be triggered by glucose in the presence
of high nitrogen supply. Electron transport rates, as indicated
by high ΦPSII values, even remained higher than in the
absence of sugars, indicating that sugars may extend the
lifespan of leaves in nitrogen-sufficient plants. By contrast, in
combination with low nitrogen supply, glucose triggered
visible senescence which was most closely associated with an
early decline in Fv /Fm.
Senescence-like symptoms can also be triggered by feeding
glucose to leaf discs (Wingler et al., 1998). Although external
supply of sugars may seem artificial, there is evidence that it
reflects processes occurring during natural leaf senescence.
Sugar contents increase during senescence in Arabidopsis,
tobacco and a range of other plant species (Noodén et al.,
1997; Wingler et al., 1998; Quirino et al., 2001; Stessman
et al., 2002), and a glucose-insensitive hexokinase-1 mutant
has recently been shown to exhibit delayed senescence (Moor
et al., 2003). Regulation of metabolic and developmental
processes by sugars often depends on nitrogen supply, suggesting that the sugar and nitrogen signalling pathways interact
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Fig. 5 Effect of sugar and nitrogen supply
on chlorophyll fluorescence characteristics in
senescing Arabidopsis plants as determined
by chlorophyll fluorescence imaging.
(a,b) Maximum quantum efficiency of
photosystem II photochemistry (Fv /Fm);
(c,d) quantum efficiency of photosystem
II electron transport (ΦPSII); and (e,f)
nonphotochemical quenching (NPQ) were
determined in Arabidopsis plants grown on
agar medium containing 30 mM nitrogen
(HN, filled symbols) or 4.7 mM nitrogen (LN,
open symbols) with or without addition of 2
or 4% glucose. Data are means ± SD of at
least 10 plants.
(Paul & Driscoll, 1997; Nielsen et al., 1998; Martin et al.,
2002). Our results clearly show that the regulation of senescence by sugars is nitrogen-dependent, supporting the view
that senescence is regulated by the carbon–nitrogen balance in
leaves (Ono et al., 1996; Stitt & Krapp, 1999; Masclaux et al.,
2000; Masclaux-Daubresse et al., 2002). This could also explain
accelerated senescence in plants grown in elevated CO2 (Nie
et al., 1995; Miller et al., 1997). In this scenario a high sugarto-nitrogen ratio would signal a reduced requirement for
investment in Rubisco and other photosynthetic proteins in
the old leaves, releasing nitrogen that would become available for
the growth of young leaves and for fruit and seed formation.
Using Arabidopsis as a model species, significant progress has already been made in understanding the control
of leaf senescence (Buchanan-Wollaston et al., 2003). Work
with mutants in sugar or nitrogen signalling could help
unravel the interactions between sugar and nitrogen signalling pathways during senescence. The development of
a simple Petri dish system in combination with monitoring of
senescence using chlorophyll fluorescence imaging makes it
possible to isolate mutants that are affected in sugar-regulated
senescence.
Acknowledgements
This work was supported by research grants from the BBSRC
(31/P16341) and the Royal Society.
References
Badger MR. 1985. Photosynthetic oxygen exchange. Annual Review of Plant
Physiology 36: 27–53.
Berger S, Weichert H, Porzel A, Wasternack C, Kühn H, Feussner I. 2001.
Enzymatic and non-enzymatic lipid peroxidation in leaf development.
Biochimica et Biophysica Acta 1533: 266–276.
Buchanan-Wollaston V, Earl S, Harrison E, Mathas E, Navabpour S,
Page T, Pink D. 2003. The molecular analysis of leaf senescence – a
genomics approach. Plant Biotechnology Journal 1: 3–22.
Dai N, Schaffer A, Petreikov M, Shahak Y, Giller Y, Ratner K, Levine A,
Granot D. 1999. Overexpression of Arabidopsis hexokinase in tomato
plants inhibits growth, reduces photosynthesis, and induces rapid
senescence. Plant Cell 11: 1253–1266.
www.newphytologist.org © New Phytologist (2004) 161: 781 – 789
Research
Feild TS, Lee DW, Holbrook NM. 2001. Why leaves turn red in autumn.
The role of anthocyanins in senescing leaves of red-osier dogwood. Plant
Physiology 127: 566–574.
Feller U, Fischer A. 1994. Nitrogen metabolism in senescing leaves. Critical
Reviews in Plant Sciences 13: 241–273.
Fryer MJ, Oxborough K, Mullineaux PM, Baker NR. 2002. Imaging of
photo-oxidative stress responses in leaves. Journal of Experimental Botany
53: 1249–1254.
Genty B, Briantais JM, Baker NR. 1989. The relationship between the
quantum yield of photosynthetic electron transport and quenching of
chlorophyll fluorescence. Biochimica et Biophysica Acta 990: 87–92.
Ghosh S, Mahoney SR, Penterman JN, Peirson D, Dumbroff EB. 2001.
Ultrastructural and biochemical changes in chloroplasts during Brassica
napus senescence. Plant Physiology and Biochemistry 39: 777–784.
Guiamét JJ, Tyystjärvi E, Tyystjärvi T, John I, Kairavuo M, Pichersky E,
Noodén LD. 2002. Photoinhibition and loss of photosystem II reaction
centre proteins during senescence of soybean leaves. Enhancement of
photoinhibition by the ‘stay-green’ mutation cyt6. Physiologia Plantarum
115: 468–478.
Hensel LL, Grbiaç V, Baumgarten DA, Bleecker AB. 1993. Developmental
and age-related processes that influence the longevity and senescence of
photosynthetic tissues in Arabidopsis. Plant Cell 5: 553 –564.
Himelblau E, Amasino RM. 2001. Nutrients mobilized from leaves of
Arabidopsis thaliana during leaf senescence. Journal of Plant Physiology 158:
1317–1323.
Horton P, Ruban AV, Walters RG. 1994. Regulation of light harvesting in
green plants. Indication by nonphotochemical quenching of chlorophyll
fluorescence. Plant Physiology 106: 415 – 420.
Humbeck K, Krupinska K. 2003. The abundance of minor chlorophyll
a /b-binding proteins CP29 and LHCI of barley (Hordeum vulgare L.)
during leaf senescence is controlled by light. Journal of Experimental
Botany 54: 375–383.
Jang JC, Sheen J. 1994. Sugar sensing in higher plants. Plant Cell 6: 1665–
1679.
Lee RH, Chen SCG. 2002. Programmed cell death during rice leaf
senescence is nonapoptotic. New Phytologist 155: 23 –32.
Leipner J, Oxborough K, Baker NR. 2001. Primary sites of ozone-induced
perturbations of photosynthesis in leaves: identification and
characterization in Phaseolus vulgaris using high resolution chlorophyll
fluorescence imaging. Journal of Experimental Botany 52: 1689–1696.
Leverentz MK, Wagstaff C, Rogers HJ, Stead AD, Chanasut U,
Silkowski H, Thomas B, Weichert H, Feussner I, Griffiths G. 2002.
Characterization of a novel lipoxygenase-independent senescence
mechanism in Alstroemeria peruviana floral tissue. Plant Physiology 130:
273–283.
Li XP, Björkman O, Shih C, Grossman AR, Rosenquist M, Jansson S,
Niyogi KK. 2000. A pigment-binding protein essential for regulation of
photosynthetic light harvesting. Nature 403: 391–395.
Lichtenthaler HK, Babani F, Langsdorf G, Buschmann C. 2000.
Measurement of differences in red chlorophyll fluorescence and
photosynthetic activity between sun and shade leaves by fluorescence
imaging. Photosynthetica 38: 521–529.
Lu C, Zhang J. 1998. Modifications in photosystem II photochemistry in
senescent leaves of maize plants. Journal of Experimental Botany 49:
1671–1679.
Lu C, Lu Q, Zhang J, Kuang T. 2001. Characterization of photosynthetic
pigment composition, photosystem II photochemistry and thermal energy
dissipation during leaf senescence of wheat plants grown in the field.
Journal of Experimental Botany 52: 1805 –1810.
Martin T, Oswald O, Graham IA. 2002. Arabidopsis seedling growth,
storage lipid mobilization, and photosynthetic gene expression are
regulated by carbon : nitrogen availability. Plant Physiology 128:
472–481.
Masclaux C, Valadier MH, Brugière N, Morot-Gaudry JF, Hirel B. 2000.
Characterization of the sink /source transition in tobacco (Nicotiana
© New Phytologist (2004) 161: 781 – 789 www.newphytologist.org
tabacum L.) shoots in relation to nitrogen management and leaf
senescence. Planta 211: 510–518.
Masclaux-Daubresse C, Valadier MH, Carrayol E, Reisdorf-Cren M,
Hirel B. 2002. Diurnal changes in the expression of glutamate
dehydrogenase and nitrate reductase are involved in the C/N balance
of tobacco source leaves. Plant, Cell & Environment 25: 1451–1462.
Meng Q, Siebke K, Lippert P, Baur B, Mukherjee U, Weis E. 2001.
Sink–source transition in tobacco leaves visualized using chlorophyll
fluorescence imaging. New Phytologist 151: 585–595.
Merzlyak MN, Hendry GAF. 1994. Free radical metabolism, pigment
degradation and lipid peroxidation in leaves during senescence. Proceedings
of the Royal Society of Edinburgh 102B: 459–471.
Miller A, Tsai CH, Hemphill D, Endres M, Rodermel S, Spalding M. 1997.
Elevated CO2 effects during leaf ontogeny. A new perspective on
acclimation. Plant Physiology 115: 1195–1200.
Moore B, Zhou L, Rolland F, Hall Q, Cheng WH, Liu YX, Hwang I,
Jones T, Sheen J. 2003. Role of the Arabidopsis glucose sensor HXK1
in nutrient, light, and hormonal signaling. Science 300: 332–336.
Müller P, Li XP, Niyogi KK. 2001. Non-photochemical quenching. A
response to excess light energy. Plant Physiology 125: 1558–1566.
Munné-Bosch S, Alegre L. 2002. Plant aging increases oxidative stress in
chloroplasts. Planta 214: 608–615.
Nedbal L, Soukupová J, Kaftan D, Whitmarsh J, Trtílek M. 2000. Kinetic
imaging of chlorophyll fluorescence using modulated light. Photosynthesis
Research 66: 3–12.
Nie GY, Long SP, Garcia RL, Kimball BA, Lamorte RL, Pinter PJ,
Wall GW, Webber AN. 1995. Effects of free-air CO2 enrichment on
the development of the photosynthetic apparatus in wheat, as indicated
by changes in leaf proteins. Plant, Cell & Environment 18: 855– 864.
Nielsen TH, Krapp A, Röper-Schwarz U, Stitt M. 1998. The sugarmediated regulation of genes encoding the small subunit of Rubisco
and the regulatory subunit of ADP glucose pyrophosphorylase is
modified by phosphate and nitrogen. Plant, Cell & Environment 21:
443–454.
Noodén LD, Guiamét JJ, John I. 1997. Senescence mechanisms. Physiologia
Plantarum 101: 746–753.
Ögren E. 1991. Prediction of photoinhibition of photosynthesis from
measurements of fluorescence quenching components. Planta 184:
538–544.
Ono K, Terashima I, Watanabe A. 1996. Interaction between nitrogen
deficit of a plant and nitrogen content in the old leaves. Plant and Cell
Physiology 37: 1083–1089.
Paul MJ, Driscoll SP. 1997. Sugar repression of photosynthesis: the role of
carbohydrates in signalling nitrogen deficiency through source : sink
imbalance. Plant, Cell & Environment 20: 110–116.
Quirino BF, Reiter WD, Amasino RD. 2001. One of two tandem
Arabidopsis genes homologous to monosaccharide transporters is
senescence-associated. Plant Molecular Biology 46: 447–457.
Scholes JD, Rolfe SA. 1996. Photosynthesis in localised regions of oat leaves
infected with crown rust (Puccinia coronata): quantitative imaging of
chlorophyll fluorescence. Planta 199: 573–582.
Smeekens S. 2000. Sugar-induced signal transduction in plants.
Annual Review of Plant Physiology and Plant Molecular Biology 51:
49–81.
Stessman D, Miller A, Spalding M, Rodermel S. 2002. Regulation of
photosynthesis during Arabidopsis leaf development in continuous light.
Photosynthesis Research 72: 21–37.
Stitt M, Krapp A. 1999. The interaction between elevated carbon dioxide
and nitrogen nutrition: the physiological and molecular background.
Plant, Cell & Environment 22: 583–621.
Thomas H, Ougham HJ, Wagstaff C, Stead AD. 2003. Defining senescence
and death. Journal of Experimental Botany 54: 1127–1132.
Wingler A, von Schaewen A, Leegood RC, Lea PJ, Quick WP. 1998.
Regulation of leaf senescence by cytokinin, sugars, and light. Effects on
NADH-dependent hydroxypyruvate reductase. Planta 116: 329–335.
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