Leaf Senescence Induced by Mild Water Deficit Follows
the Same Sequence of Macroscopic, Biochemical, and
Molecular Events as Monocarpic Senescence in Pea1
Emmanuelle Pic2, Bernard Teyssendier de la Serve, François Tardieu, and Olivier Turc*
Laboratoire de Biochimie and Physiologie Moléculaire des Plantes Unité Mixte de Recherche 5004 Ecole
Nationale Supérieure Agronomique (Montpellier)/Institut National de la Recherche Agronomique/Centre
National de la Recherche Scientifique/Université Montpellier II, 2 place P. Viala, F–34060 Montpellier cedex
1, France (E.P., B.T.d.l.S.); and Laboratoire d’Ecophysiologie des Plantes sous Stress Environnementaux Unité
Mixte de Recherche Ecole Nationale Supérieure Agronomique (Montpellier)/Institut National de la
Recherche Agronomique, 2 place P. Viala, F–34060 Montpellier cedex 1, France (E.P., F.T., O.T.)
We have compared the time course of leaf senescence in pea (Pisum sativum L. cv Messire) plants subjected to a mild water
deficit to that of monocarpic senescence in leaves of three different ages in well-watered plants and to that of plants in which
leaf senescence was delayed by flower excision. The mild water deficit (with photosynthesis rate maintained at appreciable
levels) sped up senescence by 15 d (200°Cd), whereas flower excision delayed it by 17 d (270°Cd) compared with leaves of
the same age in well-watered plants. The range of life spans in leaves of different ages in control plants was 25 d (340°Cd).
In all cases, the first detected event was an increase in the mRNA encoding a cysteine-proteinase homologous to Arabidopsis
SAG2. This happened while the photosynthesis rate and the chlorophyll and protein contents were still high. The 2-fold
variability in life span of the studied leaves was closely linked to the duration from leaf unfolding to the beginning of
accumulation of this mRNA. In contrast, the duration of the subsequent phases was essentially conserved in all studied
cases, except in plants with excised flowers, where the degradation processes were slower. These results suggest that
senescence in water-deficient plants was triggered by an early signal occurring while leaf photosynthesis was still active,
followed by a program similar to that of monocarpic senescence. They also suggest that reproductive development plays a
crucial role in the triggering of senescence.
Senescence is the final phase of leaf development,
during which a large part of leaf nitrogen, carbon,
and minerals is recycled to other organs of the plant
(Noodén, 1988a). It consists of an ordered sequence
of physiological, biochemical, and ultrastructural
changes, involving a decline in functions associated
to carbon assimilation and a massive degradation of
macromolecules (RNAs, proteins, and lipids) and
chlorophylls (Buchanan-Wollaston, 1997; Gan and
Amasino, 1997; Noodén et al., 1997). This process
relies on the execution of a specific genetic program,
which is under the control of a high and complex
regulation by various endogenous and environmental factors (Smart, 1994; Gan and Amasino, 1997). It is
sped up by water or nitrogen deficits (Merrien et al.,
1
This work was supported by grants from the Ministère de
l’Enseignement Supérieur et de la Recherche and from the Institut
National de la Recherche Agronomique (Département Environnement-Agronomie).
2
Present address: Centre Technique Interprofessionnel des Oléagineux Métropolitains, Centre de Grignon, BP 4, F–78850 Thiverval Grignon, France.
* Corresponding author; e-mail [email protected]; fax 33–467–
522–116.
Article, publication date, and citation information can be found
at www.plantphysiol.org/cgi/doi/10.1104/pp.010634.
236
1981; Wolfe et al., 1988a, 1988b) and delayed when
reproductive sinks are removed (Lindoo and
Noodén, 1976; Noodén, 1988b; Wolfe et al., 1988a).
Acceleration of leaf senescence is thought to be
adaptative in plants subjected to water shortage (a)
because it reduces the water demand cumulated over
the whole plant cycle, thereby avoiding water deficit
during seed filling, and (b) because it allows recycling of scarce resources to the reproductive sinks.
However, in crops species, early leaf senescence usually correlates with lower yield because cumulative
photosynthesis is reduced (Fischer and Kohn, 1966;
Merrien et al., 1981; Gifford and Jenkins, 1982; Wolfe
et al., 1988a). Selection based on delayed leaf senescence (“stay-green” plants) under drought conditions
allowed to obtain sorgho hybrids with improved
yields under water deficit (Borrell et al., 2000). Nevertheless, stay-green plants do not necessarily produce higher yields, especially when chlorophyll catabolism and nutrient remobilization are disabled
(for a review, see Thomas and Howarth, 2000). Prediction and manipulation of leaf senescence is therefore crucial to optimize crop management and plant
response to water deficit.
This prediction is made difficult because the first
events that trigger senescence may occur long before
chlorophyll degradation and associated leaf yellow-
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Time Course of Leaf Senescence in Water-Deficient Pea Plants
ing, which seems to be one of the latest events of leaf
senescence (Hensel et al., 1993; Bernhard and Matile,
1994; Humbeck et al., 1996). The senescence program
may therefore be triggered by an environmental constraint occurring before any detectable symptom,
making difficult to ascribe it to a particular environmental event. Decreases in net photosynthesis and
stomatal conductance are not good indicators of senescence initiation because they exhibit rapid fluctuations unrelated to the senescence program. Monitoring changes in gene expression might give a more
reliable indication.
The aim of this work was therefore to identify
early events in the sequence of macroscopic, biochemical, and molecular events associated with leaf
senescence of plants experiencing water deficiency.
We tested whether this sequence of events differed
in water-deficient plants and in “normal” monocarpic senescence. To avoid confusion of effects, this
sequence of events in well-watered plants was established in leaves inserted at three positions on the
stem, which had markedly different life spans. We
also compared it with that observed in plants in
which leaf senescence was delayed by flower excision. The water deficit imposed in this study corresponded to the mild stresses commonly observed in
field conditions, i.e. which affect plant architecture
and water flux without appreciably affecting leaf
water status (Lecoeur et al., 1995; Tardieu and
Simonneau, 1998). In this respect, it differed in intensity and duration from those imposed in other
studies by dehydrating detached leaves or by withholding irrigation (Becker and Apel, 1993; Weaver et
al., 1998; He et al., 2001).
We simultaneously analyzed changes with time in
net photosynthesis, chlorophyll and protein contents,
and relative abundances of several mRNAs in pea
(Pisum sativum L. cv Messire) leaves. The relative
abundance of a mRNA encoding a chlorophyll a/b
(CAB) protein was measured because the corresponding gene was shown to be down-regulated during leaf senescence (Hensel et al., 1993; Lohman et al.,
1994). We also monitored the relative abundances of
mRNAs encoding a Cys-proteinase and a ferritin,
which correspond to senescence up-regulated genes
(Hensel et al., 1993; Smart et al., 1995; Drake et al.,
1996; Buchanan-Wollaston and Ainsworth, 1997).
Measuring the abundance of the proteinase mRNA
necessitated the cloning of a cDNA that we named
PsELSA (GenBank accession no. AJ278699). The
mode of expression of time as it is sensed by plants is
essential in studies of the time course of leaf development. We have expressed it in two ways: (a) physical time, which is the most intuitive, and (b) thermal
time, which allows more precise comparisons when
temperature undergoes fluctuations and is a necessary expression for modeling (Turc and Lecoeur,
1997; Granier and Tardieu, 1998).
Plant Physiol. Vol. 128, 2002
RESULTS
A Mild Water Deficit, Compatible with an Appreciable
Photosynthesis Rate, Caused an Acceleration of
Senescence by 13 d
In the treatment with water deficit, soil water potential decreased from ⫺10 to ⫺80 kPa during the
first 24 d (336°Cd) of the experiment, and was then
maintained at that level until the end of the experiment. The stabilization of soil water potential at
about ⫺80 kPa, therefore, occurred 22 d (270°Cd)
before unfolding of leaf 14 (Fig. 1). In the other two
treatments, soil water potential remained above ⫺15
kPa. Predawn leaf water potential decreased to ⫺0.5
Mpa in the water deficit treatment, whereas it remained above ⫺0.25 Mpa in the other two treatments
(data not shown). Net photosynthesis in leaf 14 (Table I) was halved in water-deficient plants compared
with well-watered plants at leaf unfolding, but the
value of maximum net photosynthesis was nearly
unaffected by the water deficit. These results show
that the water deficit experienced by plants was very
mild. However, the decrease in net photosynthesis
was much faster (net photosynthesis was close to
zero 27 d [390°Cd] after leaf unfolding in waterdeficient plants, whereas it was still close to its maximum in the other two treatments [Table I]), and leaf
yellowing occurred 13 d (180°Cd) earlier compared
with controls (Table II).
Figure 1. Change with time in soil water potential in the treatment
with excised flowers, control treatment, and treatment with water
deficit. Soil water potential was measured daily at depths of 0.20 (E)
and 0.30 m (F). For better legibility, means and SDs (12 measurements) are given every 4 d.
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237
Pic et al.
Table I. Net photosynthesis in leaf 14 of plants with excised flowers, control plants, and water-deficient plants at different critical stages
The dates of measurements are expressed in thermal time (°Cd) after leaf unfolding (corresponding days in parentheses). Measurements were
made at saturating photosynthetic photon flux density (PPFD greater than 700 ␮mol m⫺2 s⫺1). Means and SD of at least four measurements.
Excised Flowers
Approximate stage
Leaf unfolding
Maximum photosynthesis
Leaf yellowing water deficit
Leaf yellowing control
a
Control
Water Deficit
Date
Photosynthesis
Date
Photosynthesis
Date
Photosynthesis
°Cd (d)
␮mol m⫺2 s⫺1
°Cd (d)
␮mol m⫺2 s⫺1
°Cd (d)
␮mol m⫺2 s⫺1
44 (4)
372 (26)
416 (29)
664 (46)
16.9 (⫾ 4.5)
28.8 (⫾ 3.0)
22.7 (⫾ 5.5)
16.8 (⫾ 8.3)
41 (4)
369 (26)
413 (29)
661 (46)
19.9 (⫾ 3.2)
27.3 (⫾ 2.7)
22.4 (⫾ 4.8)
0.3 (⫾ 0.3)
20 (2)
348 (24)
393 (27)
–a
9.7 (⫾ 3.8)
24.8 (⫾ 2.1)
5.8 (⫾ 2.1)
–a
–, Water-deficient plants were dead at that time.
Three Independent Sources of Variations Caused Large
Differences in Leaf Duration
In leaf 14, phytomere initiation and leaf unfolding
occurred synchronously in the three treatments (Table II). In contrast, the duration from leaf unfolding
to yellowing largely differed between treatments,
and was 27 (390°Cd), 42 (600°Cd), and 59 d (870°Cd),
respectively in water-deficient plants, control plants,
and plants with excised flowers (Table II). These
differences are consistent with the measurements of
photosynthesis, which decreased before yellowing in
the three treatments and was close to zero 46 d
(660°Cd) after leaf unfolding in controls, i.e. 19 d
(270°Cd) later than in water-deficient plants, whereas
it was still 17 ␮mol m⫺2 s⫺1 in plants with excised
flowers (Table I). The duration from leaf yellowing to
complete desiccation was approximately the same in
water-deficient plants and in controls (7–9 d; 100°Cd–
145°Cd), whereas desiccation had not yet occurred in
plants with excised flowers at the end of the experiment (Table II).
In leaves 10 and 20 of control plants, phytomere
initiation and leaf unfolding occurred, respectively,
sooner and later than in leaf 14, but visible yellowing
and complete desiccation were nearly simultaneous
at the three positions on the stem (Table II). As a
consequence, the life span of leaf 10 exceeded by 10 d
(130°Cd) that of leaf 14, and that of leaf 20 was 15 d
(200°Cd) shorter. It is noteworthy that the life span of
leaf 14 of water-deficient plants equaled that of leaf
20 of control plants, thereby providing an adequate
system to compare monocarpic and stress-induced
senescence.
Declines in Chlorophyll and Protein Contents Began
Slightly before Yellowing in All Studied Cases
Chlorophyll content per unit leaf area was similar
in the five studied cases at leaf unfolding (except in
leaf 10 of control plants, in which it was halved). It
remained roughly constant afterward and declined at
a time that greatly varied across leaf positions and
treatments, from 20 d (290°Cd) after leaf unfolding in
leaf 14 of plants with water deficit to at least 50 d
(730°Cd) in leaf 14 of plants with excised flowers
(Fig. 2). The decline in chlorophyll content always
began shortly before visible yellowing. It lasted
about 11 d (170°Cd) in all cases until chlorophyll
content reached values close to zero (lower than 0.2
␮g mm⫺2), except in leaf 14 of plants with excised
flowers where chlorophyll content was still 35% of its
maximum level at the end of the experiment.
Protein content closely paralleled chlorophyll content in all studied cases (Fig. 2). It first decreased
slowly, and then dropped rapidly at the same date as
chlorophyll content. It reached values close to zero
(lower than 2 ␮g mm⫺2, again with the exception of
leaf 14 of plants with excised flowers) approximately
at the same date as chlorophyll content.
Table II. Initiation, unfolding, visible yellowing, life span, and desiccation of the studied leaves
The dates of phytomere initiation, leaf unfolding, leaf visible yellowing, and leaf desiccation are expressed in thermal time (°Cd) after
emergence (corresponding days in parentheses). The life span of the leaves was calculated as the time interval between unfolding and visible
yellowing.
Treatment
Phytomere
Position
Phytomere
Initiation
Leaf
Unfolding
Leaf Visible
Yellowing
Excised flowers
Control
Control
Control
Water deficit
14
10
14
20
14
151 (10)
46 (3)
151 (10)
344 (25)
149 (10)
580 (44)
436 (33)
583 (44)
804 (60)
604 (46)
1,450 (103)
1,165 (85)
1,180 (86)
1,196 (87)
996 (73)
Leaf Life
Span
Leaf
Desiccation
870 (59)
729 (52)
597 (42)
392 (27)
392 (27)
–a
1,311 (94)
1,311 (94)
1,311 (94)
1,094 (80)
°Cd (d)
a
238
–, Dessication had not yet occurred in leaf 14 of plants with excised flowers at the last sampling date (1,643 °Cd [115 d] after emergence).
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Plant Physiol. Vol. 128, 2002
Time Course of Leaf Senescence in Water-Deficient Pea Plants
PsELSA Cys-proteinase and a ferritin are presented
in Figure 3. The curves present the results obtained
with the CAB and the proteinase probes after quantification of the signals and normalization relative to
the most intense signal. The last lane of the blots (first
date of measurement after leaf yellowing) was not
taken into account for quantifications because
ethidium bromide staining of the electrophoresis gels
showed that RNA deposition was much lower in this
lane (data not shown).
Maximum accumulation of the CAB mRNA occurred at unfolding in all cases, except in leaf 14 of
water-deficient plants in which it was delayed (Fig. 3,
second or third sampling date according to the considered blot). The subsequent decrease was the slowest in leaves having the longest life spans: 30% of the
initial level was reached about 50 d (715°Cd) after
leaf unfolding in leaf 14 of plants with excised flowers and in leaf 10 of control plants (Fig. 3). It was the
most rapid in leaves having the shortest life spans:
30% of the initial level was reached, respectively, 23
(330°Cd) and 20 d (290°Cd) after leaf unfolding in
leaf 20 of control plants and in leaf 14 of waterdeficient plants (Fig. 3).
The mRNA coding for the PsELSA Cys-proteinase
first accumulated slowly and began to accumulate
rapidly 11 to 21 d (150°Cd–330°Cd) before leaf yellowing according to the studied case (Fig. 3). The
onset of rapid accumulation occurred the latest in
leaf 14 of plants with excised flowers and in leaf 10 of
control plants (38 and 41 d, 540°Cd and 570°Cd, after
leaf unfolding, respectively). It occurred the soonest
in leaf 20 of control plants and in leaf 14 of waterdeficient plants (about 14 d, 200°Cd, after leaf unfolding in both cases). In contrast, the ferritin mRNA was
detected at low levels at leaf unfolding, nearly undetectable afterward, and accumulated markedly after
leaf yellowing in all studied cases (Fig. 3).
DISCUSSION
Synchrony of the Progression of Senescence in the Five
Studied Cases
Figure 2. Change with time in chlorophyll and protein contents
(black and white symbols, respectively) in leaf 14 of plants with
excised flowers, in leaves 10, 14, and 20 of control plants, and in leaf
14 of water-deficient plants. Means and SDs of at least three measurements. Dotted lines represent the dates of leaf visible yellowing.
Rapid Changes with Time in Relative Abundances of
mRNAs Encoding a CAB Protein and PsELSA
Cys-Proteinase Were Early Events in the Time
Course of Senescence
RNA-blot analysis of changes with time in relative
abundance of mRNAs coding for a CAB protein, the
Plant Physiol. Vol. 128, 2002
The time course of senescence-associated events in
water-deficient plants displayed striking similarities
with those observed in leaves of control plants, whatever the ages of these leaves, and with that observed
in plants with excised flowers. This is illustrated in
Figure 4. The beginning of the decline in chlorophyll
content, which occurred at different leaf ages between treatments and leaf positions, was taken as a
time reference. Certain events were simultaneous in
all cases and marked the beginning of the degradation
of the photosynthetic apparatus. These were the onset
of the rapid declines in chlorophyll and protein contents, and the time when the relative abundance of the
CAB mRNA was reduced to 30% of its initial level.
The decreases in chlorophyll and protein contents and
in the abundance of the CAB mRNA were also simul-
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239
Pic et al.
Figure 3. Change with time in relative abundance of mRNAs coding for a CAB protein, the PsELSA Cys-proteinase and a
ferritin in leaf 14 of plants with excised flowers, in leaves 10, 14, and 20 of control plants, and in leaf 14 of water-deficient
plants. The blots (10 ␮g of total RNA per lane) were successively hybridized with 32P-labeled probes corresponding to the
PEACAB66 CAB protein, the PsELSA Cys-proteinase, and the PSFERRI ferritin cDNAs (for GenBank accession nos., see
“Materials and Methods”). These results were obtained with two independent RNA extracts and at least three blots for each
extract. For each blot, the intensity of the signals obtained with the CAB and the Cys-proteinase probes was normalized
relatively to the most intense signal among those that were quantified (see text). The curves present means and SDs of all the
results obtained for each leaf with the CAB and the Cys-proteinase probes (black and white symbols, respectively). Dotted
lines represent the dates of leaf visible yellowing.
240
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Plant Physiol. Vol. 128, 2002
Time Course of Leaf Senescence in Water-Deficient Pea Plants
Figure 4. Chronology of events in leaf 14 of plants with excised flowers, leaves 10, 14, and 20 of control plants, and leaf
14 of plants with water deficit. 1, Leaf unfolding and low accumulation of the ferritin mRNA. 2, Beginning of rapid
accumulation of the mRNA coding for the PsELSA Cys-proteinase. 3, End of the period with stable chlorophyll content. 4,
End of the period with stable protein content. 5, Abundance of the CAB mRNA reduced to 30% of its initial level. 6, Leaf
visible yellowing. 7, Strong accumulation of the ferritin mRNA. 8, Chlorophyll content close to zero. 9, Protein content close
to zero. 10, Leaf desiccation. Graphs are positioned to align event 3 (end of the period with stable chlorophyll content) in
the five studied cases, irrespective of leaf age at that time.
taneous in senescing leaves of Arabidopsis in the analysis of Lohman et al. (1994), whereas the decline in
chlorophyll content occurred slightly later than the
other two events in the study of Hensel et al. (1993).
Leaf yellowing shortly followed these events at the
three studied positions on the stem in control plants
Plant Physiol. Vol. 128, 2002
and was slightly delayed in plants subjected to water
deficit. Leaf desiccation occurred approximately 1
week after yellowing in these four cases (Fig. 4). The
delay before leaf yellowing was longer in plants with
excised flowers, in which chlorophyll and protein
contents were still at appreciable levels 12 d (190°Cd)
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241
Pic et al.
after leaf yellowing, consistent with observations on
soybeans (Wittenbach, 1983).
The beginning of rapid accumulation of the mRNA
encoding the PsELSA Cys-proteinase preceded the
beginning of chlorophyll and protein degradation by
a constant delay of about 9 d (125°Cd), regardless of
the source of variation of senescence, either environmental or due to leaf position on the stem (Fig. 4). In
all studied cases, this first event of senescence was
observed while the photosynthesis rate and the chlorophyll and protein contents were still high. It was
not related to leaf age, which ranged from 14 to 41 d
(200°Cd–575°Cd) after unfolding at that time. The
ferritin mRNA exhibited a contrasting behavior, as it
accumulated only at leaf unfolding and, more markedly, at late stages of senescence; in all cases, that late
accumulation occurred after leaf yellowing, at a time
when chlorophyll and protein contents were close to
zero and leaf desiccation was under way (Fig. 4).
These results are consistent with those obtained by
Buchanan-Wollaston and Ainsworth (1997).
The sequence of events was therefore common to
the five studied cases. The 2-fold variability in the life
span of the leaves was essentially linked to the duration from unfolding to the beginning of rapid accumulation of the Cys-proteinase mRNA (Fig. 4). In
contrast, the duration of the subsequent phases until
complete desiccation was essentially conserved in all
studied cases, except in plants with excised flowers,
where the degradation of chlorophylls and proteins
and leaf desiccation were delayed, probably due to
the lack of sinks. This suggests that the life span
variability was determined by early events, whereas
the time course of events occurring during protein
depletion was largely insensitive to environmental
conditions. The synchrony of the progression of senescence once it was triggered at different dates by
different promoting factors is consistent with the
concept of regulatory network (Gan and Amasino,
1997; He et al., 2001), in which multiple pathways
responding to various autonomous and environmental factors are interconnected to control senescence.
Accumulation of the PsELSA Cys-Proteinase mRNA
Marked an Early Step of Senescence
Accumulation of the PsELSA Cys-proteinase
mRNA during pea leaf senescence is consistent with
the results obtained in other plant species (Hensel et
al., 1993; Smart et al., 1995; Drake et al., 1996;
Buchanan-Wollaston and Ainsworth, 1997; Weaver et
al., 1998). The originality of this work was to show (a)
that this mRNA began to accumulate rapidly at a
time when the degradation processes (especially protein degradation) had not yet begun, and (b) that the
kinetics of the subsequent sequence of events was
conserved through various leaf positions and experimental conditions (Fig. 4).
High accumulation of mRNAs encoding the SAG2
and SAG12 Cys-proteinases occurred later in Arabi242
dopsis than that of PsELSA mRNA in our study at a
time when yellowing and protein degradation were
under way and CAB mRNA had dropped down
(Hensel et al., 1993; Lohman et al., 1994; Weaver et
al., 1998). Our results do not necessarily contradict
these observations. The most marked difference is
that the duration from leaf unfolding to yellowing
was considerably longer in pea (up to 59 d in plants
with excised flowers) compared with Arabidopsis
(5–6 d). This gives a better time definition to analyze
sequence of events, and makes it easier to distinguish
short-term events after a stress from longer term
developmental processes. Another consequence is
that two events may appear concomitant in Arabidopsis (within 1 or 2 d) and distinct in pea (by several
days). It is also possible that kinetics slightly differ in
Arabidopsis and in pea. The PsELSA cDNA is highly
homologous to that of barley (Hordeum vulgare) aleurain (Rogers et al., 1985). Our observation of an early
accumulation of the corresponding mRNA, before
the drop in leaf protein content, rises again the question of whether this class of Cys-proteinases plays a
direct role in protein dismantling or rather activates
enzymes involved in massive protein degradation
(Holwerda and Rogers, 1992).
What Triggered Senescence?
In the water-deficit treatment, the beginning of the
senescence program was probably not a direct consequence of a water stress sensed at the cellular level,
in opposition with cases where water deficit was
imposed by dehydrating detached leaves or withholding irrigation (Becker and Apel, 1993; Oh et al.,
1996; Weaver et al., 1998). More than 1 month elapsed
from the beginning of water deficit to the increase in
the Cys-proteinase mRNA, and photosynthesis rate
was still high when this increase occurred. Furthermore, a mild water deficit does not appreciably alter
the day-time leaf water status in plants such as maize
(Zea mays) or pea, in which a combination of abscisic
acid and hydraulic signalings allows stomatal control
to avoid any leaf dehydration by fine-tuning transpiration (Tardieu and Davies, 1993; Tardieu and
Simonneau, 1998). Finally, the life span and the time
course of events in leaf 14 of water-deficient plants
were very similar to those observed in leaf 20 of
control plants. These three arguments suggest that
the early senescence observed under mild water deficit followed a program similar to that of monocarpic
senescence, after it was triggered by a signal, which
was linked to soil water deficit rather than to a stressing leaf water status.
The onset of monocarpic senescence was probably
not caused in our case by an age-related decline in
photosynthetic processes, as proposed by Hensel et
al. (1993). First, the rapid increase in the Cysproteinase mRNA began at least 1 week before the
declines in photosynthesis and in chlorophyll and
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Plant Physiol. Vol. 128, 2002
Time Course of Leaf Senescence in Water-Deficient Pea Plants
protein contents in all studied cases. It is therefore
difficult to consider that the decline in photosynthesis could have a causal effect. Second, the life span of
lately initiated leaves was much shorter than that of
early initiated ones, and all leaves of a plant senesced
within a short time (Table II). This suggests that the
onset of senescence was linked to a developmental
control at the whole plant level, rather than to the age
of individual leaves. Third, excision of flowers
greatly delayed senescence. Taken together, these
data suggest that source-sink relationships and relationships between reproductive and vegetative development may have a crucial role in the onset of
senescence. This is consistent with analyses on soybean (Lindoo and Noodén, 1976) and maize (Wolfe et
al., 1988a), in opposition to the case of Arabidopsis
where mutants with sterile flowers senesced at the
same rate as wild-type plants (Hensel et al., 1993).
MATERIALS AND METHODS
Plant Material, Growth Conditions, and
Environmental Measurements
Pea (Pisum sativum L. cv Messire) plants were grown in
a greenhouse in Montpellier (France). Seeds were sown in
36 pots (0.35-m diameter and 0.35-m height) filled with a
1:1 (v/v) mixture of loamy soil:organic compost complemented with 130 g m⫺3 of P2O5:K2O (10:25, w/w). Twenty
seeds were sown at 25-mm depth in each pot. All the pots
were fully irrigated after sowing and allowed to drain
freely for 24 h. Plants were thinned to 12 per pot at emergence (8 d after sowing) and to eight per pot at the beginning of flowering. Lateral branches were removed as soon
as they became visible, so each studied plant consisted of
one main stem only.
Air temperature and relative humidity were measured
with a capacitive hygrometer (HMP35A Vaisala Oy, Helsinki) protected from direct radiation. Photosynthetic photon flux density (PPFD) was measured using silicon cells
calibrated in situ using a PPFD sensor (LI-190SB, LI COR,
Inc., Lincoln, NE). Air temperature, relative humidity, and
PPFD were measured at 1.5 m from the soil. Temperature
of the apical bud was measured with a fine copperconstantan thermocouple (0.2 mm) inserted in the apical
bud of two plants per treatment. Data were collected every
20 s, and means were stored every 1,800 s in a data logger
(Campbell Scientific, LTD-CR10 Wiring Panel, Shepshed,
Leicestershire, UK). Air temperature was regulated to
maintain a day/night amplitude of 20°C/10°C, and was
never allowed to exceed 25°C. Natural light was supplemented with sodium lamps (250 ␮mol m⫺2 s⫺1) to obtain a
constant photoperiod of 16 h. Soil water potential was
measured daily with two tensiometers (DTE 1000, Nardeux, Saint-Avertin, France) per pot placed at depths of
0.20 and 0.30 m.
Experimental Treatments
Three treatments were imposed to 12 randomly chosen
pots each. Control plants were watered manually every
Plant Physiol. Vol. 128, 2002
day either with water or, once a month, with a 1:10 (v/v)
Hoagland nutritive solution corrected for minor elements.
In the treatment with flower excision, watered as control,
newly opening flowers were excised as soon as they appeared on successive phytomeres. In the water deficit treatment, water supply was withheld after sowing until soil
water potential reached approximately ⫺70 kPa at the two
depths of measurement. The pots were then watered daily
to maintain soil water potential between ⫺70 and ⫺80 kPa
at the two depths.
Follow-Up of Leaf Development and Leaf Sampling
The studied leaves were those inserted on phytomeres
10, 14, and 20 in control plants and that on phytomere 14 in
the other two treatments. Phytomere 14 was the first reproductive phytomere in all sampled plants (the few plants
in which this was not the case were excluded from sampling). The number of initiated phytomeres was determined once a week on six to eight plants per treatment as
described in Turc and Lecoeur (1997). The number of fully
unfolded leaves was determined on the same plants plus,
two or three times a week, on 12 marked plants per treatment (one per pot) followed non-destructively during the
whole experiment. The dates of phytomere initiation and
leaf unfolding (Table II) were deduced from those countings as described in Turc and Lecoeur (1997). A leaf was
considered to reach visible yellowing when its leaflets
exhibited visual symptoms equivalent to those that characterize stage S2 of Arabidopsis leaves in the study of
Lohman et al. (1994). Leaf complete desiccation was also
recorded visually. The progressions of leaf yellowing and
desiccation from the bottom to the top of the plant were
followed twice or three times a week in each treatment to
determine the dates of yellowing and desiccation of the
studied leaves (Table II).
The final area of each leaf was measured after completion of leaf expansion with an image analyzer (BioscanOptimas V 4.10, Edmonds, WA) on six to 14 plants. On the
first sampling date (leaf unfolding), at which leaf area was
still increasing, leaf area was calculated from the developmental stage of the leaf and from its final area (Turc and
Lecoeur, 1997).
Time courses were expressed in thermal time calculated
by daily integration of temperature minus a base temperature of 3°C. However, day-to-day variations in temperature were not very large, so physical time was still acceptable and was also provided.
Leaves were sampled every week (twice a week as soon
as photosynthesis began to decrease) from unfolding to
complete desiccation, i.e. on six to 11 sampling dates depending on treatment and leaf position. Ten whole leaves
(stipules ⫹ leaflets ⫹ tendrils) were collected on randomly
chosen plants and immediately frozen in liquid nitrogen.
They were pooled in a calibrated plastic flask, and total
fresh matter was weighed. They were then kept at ⫺50°C.
Specific leaf area was calculated at each sampling date as
the ratio between mean leaf area and mean fresh weight
per leaf at that date.
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243
Pic et al.
Photosynthesis Measurements and Determination of
Chlorophyll and Protein Contents
Photosynthesis of leaf 14 was measured on leaflets with
a Photosynthesis Portable System LI-6200 (LI-COR, Inc.,
Lincoln, NE) according to Leuning and Sands (1989) and
Pearcy et al. (1991). Only measurements with saturating
PPFD greater than 700 ␮mol m⫺2 s⫺1 were taken into
account.
For the determination of chlorophyll and protein contents, the samples (250 mg of fresh matter) were homogenized (Ultra-Turrax T25, shaft type N-10G, IKA Labortechnik, Staufen, Germany) at 15,000 rpm in cold 80% (v/v)
acetone. After centrifugation at 5,000g for 5 min at 4°C, the
supernatant was collected, and acetone extraction was repeated once on the pellet. The second supernatant was
added to the first one, and chlorophyll (a⫹b) content was
determined by spectrophotometry according to Vernon
(1960). The pellet was resuspended in 10 mL of 0.1 n
NaOH, and the tubes were placed under shaking overnight
at 4°C. After centrifugation at 6,000g for 15 min at 4°C, the
supernatant was used for protein content determination
according to Bradford (1976), using bovine serum albumin
as a standard. Titration was performed in microtitration
plates (POLYSORP, Flat-Bottom, Nunc, Naperville, IL). In
each well, proteins (0–1.5 ␮g) were solubilized in 50 ␮L of
0.02 n NaOH, and 200 ␮L of Bradford reagent was then
added. Absorbance was measured at 620 nm with a Titertek Multiskan apparatus (MCC/340, Flow Laboratories,
Irving, UK).
Chlorophyll and protein contents were expressed on a
fresh weight basis, or on a leaf area basis by dividing the
former by specific leaf area. In leaf 14 of plants with excised
flowers, chlorophyll and protein contents remained
roughly constant until 50 d (730°Cd) after leaf unfolding
when expressed per unit leaf area (Fig. 2), whereas they
continuously declined with time when expressed per unit
fresh weight (data not shown). This was probably due to an
accumulation of other compounds in the leaves (in particular carbohydrates, as the main sink for plant carbon was
removed in these plants), which diluted chlorophyll and
protein contents expressed per unit fresh weight. We therefore expressed all concentrations per unit leaf area in further analyses.
DNA Probes Used for RNA Hybridization
The degenerate primers THP5⬘, 5⬘-TTTTCARAMITGYTCNGCNAC-3⬘, and THP3⬘, 5⬘-TTTTNAYIGGGTAGGANGCRCA-3⬘ (IUPAC ambiguity code “I” stands for
inosine) were made from the alignment of the deduced
amino acid sequences of five Cys-proteinases (Tournaire et
al., 1996). These primers were used to reverse transcriptasePCR amplify a partial cDNA from pea leaf RNA. This
partial cDNA was further used to screen a pea leaf cDNA
library provided by D. Macherel (Commissariat à l’Energie
Atomique, Grenoble). The insert of the longest polyadenylated clone was fully sequenced on both strands and named
PsELSA (GenBank accession no. AJ278699). Database
search revealed that this cDNA is nearly 100% identical to
244
that of the PSRNACP Cys-proteinase isolated from germinating seeds of pea by Jones et al. (1996). It shares a high
degree of derived amino acid sequence similarity with
Arabidopsis SAG2 (Hensel et al., 1993), petunia (Petunia
hybrida) PeTh3 (Tournaire et al., 1996), barley aleurain (Rogers et al., 1985), and rice (Oryza sativa) oryzain-␥ (Watanabe
et al., 1991). The probe used for RNA hybridization corresponded to a 636-bp internal fragment of the PsELSA cDNA
obtained by PCR amplification with the primers PsELSA 1,
5⬘-CCGATGCTAATCTTCCTGACGAGA-3⬘, and PsELSA 2,
5⬘-CAACACCGCACATATTCTTCCCCATT-3⬘.
The CAB protein probe was the PstI insert of the pAB96
plasmid (Broglie et al., 1981), which corresponds to the
PEACAB66 cDNA (GenBank accession no. M64619). The
ferritin probe was a 636-bp internal fragment of the
PSFERRI cDNA (GenBank accession no. X73369) generated
by Sau 3A I digestion of the EcoRI insert fragment from the
cDNA clone PeSd1 (Lobréaux et al., 1992).
RNA Purification and Hybridization
Total RNA extract was prepared from leaf tissue using a
modified phenol-SDS method (Teyssendier de la Serve and
Jouanneau, 1979) and further purified by centrifugation
through a 5.7 m CsCl cushion (Chirgwin et al., 1979). RNA
concentration of the extract was estimated assuming that
A260 ⫽ 1 corresponds to 40 ng ␮L⫺1 RNA. For the northernblot analyses, RNA (10 ␮g) was separated by electrophoresis (1.25% [w/v] agarose) under denaturating conditions
according to Sambrook et al. (1989a). Equivalent RNA deposition in all the lanes of the electrophoresis gel was
checked by ethidium bromide staining and observation of
the gel under UV light. Transfer to Hybond N membrane
was carried out as described in Sambrook et al. (1989b).
Probes were 32 P-labeled by the random primer method (T7
Quick Prime Kit, Pharmacia Biotech, Piscataway, NJ) and
purified on Sephadex columns (NICK Column, Pharmacia
Biotech). Hybridization was performed overnight at 42°C
in the following buffer: 750 mm NaCl, 50 mm Na2 HPO4
(pH 7), 5 mm Na2 EDTA, 50% (v/v) formamide, 1% (w/v)
sarkosyl, and 10% (w/v) dextran sulfate. Final wash was
carried out in 0.1⫻ SSC, 0.1% (w/v) SDS, at temperatures
depending on the probe, i.e. 48°C, 45°C, and 42°C for the
CAB, the Cys-proteinase, and the ferritin probes, respectively. Intensity of the signals was measured using an
optical scanner (STORM, Molecular Dynamics, Sunnyvale,
CA) and quantified with Image QuaNT software (Molecular Dynamics). Autoradiographs (X-OMAT AR films,
Kodak, Rochester, NY) were then exposed in the presence
of a fluorescent screen (Cronex, DuPont, Wilmington, DE)
at ⫺80°C, for 3 h in the case of hybridization with the CAB
probe, 8 h for the Cys-proteinase probe, and 2 weeks for the
ferritin probe.
ACKNOWLEDGMENTS
We thank Philippe Naudin for technical assistance during the greenhouse experiment and Gérard Vansuyt for
useful advice in biochemical measurements. Thanks are
also due to Jean-François Briat and David Macherel for
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Copyright © 2002 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 128, 2002
Time Course of Leaf Senescence in Water-Deficient Pea Plants
providing the PeSd1 clone and the pea leaf cDNA library,
respectively. Special thanks to Frédéric Gaymard for skilful
help during reverse transcriptase-PCR and screening experiments, and for careful reading of the manuscript. The
authors also thank Claude Grignon and Gilles Lemaire for
their contribution to this project and support during the
writing of the manuscript.
Received July 17, 2001; returned for revision September 6,
2001; accepted October 12, 2001.
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