Journal of Experimental Botany, Vol. 52, No. 358, pp. 1063±1070, May 2001
Alterations in carbon and nitrogen metabolism induced by
water deficit in the stems and leaves of Lupinus albus L.
Carla Pinheiro1, Maria Manuela Chaves2,3 and CaÃndido Pinto Ricardo1,2
1
2
Plant Biochemistry, Instituto de Tecnologia QuõÂmica e BioloÂgica, Apartado 127, 2781-901 Oeiras, Portugal
Instituto Superior de Agronomia, Tapada da Ajuda, 1349-017 Lisboa, Portugal
Received 20 October 2000; Accepted 20 December 2000
Abstract
Water deficit (WD) in Lupinus albus L. brings about
tissue-specific responses that are dependent on
stress intensity. Carbohydrate metabolism is very
sensitive to changes in plant water status. Six days
from withholding water (DAW), sucrose, glucose
and fructose levels of the leaf blade had already
increased over 5-fold, and the activities of SS and
INVA had increased c. 1.5 ±2 times. From 9 DAW on,
when stress intensity was more pronounced, these
effects were reversed with fructose and glucose
concentrations as well as INVA activity dropping in
parallel. The stem (specifically the stele) responded
to the stress intensification with striking increases in
the concentration of sugars, N and S, and in the
induction of thaumatin-like-protein and an increase
in chitinase and peroxidase. At 13 DAW, the plants
lost most of the leaves but on rewatering they fully
recovered. Thus, the observed changes appear to
contribute to a general mechanism of survival under
drought, the stem playing a key role in that process.
Key words: L. albus, drought, sugars, sucrose-metabolizing
enzymes, pathogenesis-related proteins.
therefore their metabolism may exhibit some adaptation
to water de®cit (WD).
The speci®c plant responses to WD are dependent on
the amount and rate of water loss, the duration of the
stress and the stage of plant development (Bray, 1997).
Physiological alterations induced by WD in L. albus L.
(mainly considered a drought avoider) have been
described (Henson and Turner, 1991; Ramalho and
Chaves, 1992; Rodrigues et al., 1995), but the modi®cations in plant metabolism remain relatively unexplored.
It was proposed that during WD the lupin stem may
contribute to plant survival by acting as a temporary
storage organ (Rodrigues et al., 1995). If so, leaf nutrients
must be transferred to the stem, which implies changes
in the leaf carbon and nitrogen pools. The expression
pattern of proteins may be altered by WD, as was
observed for both osmotic and salt stresses (Bray, 1993;
Chen et al., 1994; Zhu et al., 1995; Riccardi et al., 1998;
Xiong et al., 1999). Some of the responsive proteins have
a known function, but the role of others (e.g. osmotin,
lipid transfer proteins, and some cell wall structural
proteins) is unknown (Bray, 1993, 1997; Creelman and
Mullet, 1995; Colmenero-Flores et al., 1997).
The changes induced by water de®cit in the proteins
and sugars of the leaf and stem that may be implicated
in a general response strategy to cope with stress have
been studied.
Introduction
Lupins (Lupinus spp.) are important grain legume crops
for animal and human nutrition because of their high
seed protein content and adaptation to dry climates (Hill,
1986; Lopez-Bellido and Fuentes, 1986). Those plants are
frequently subjected to periods of water constraint and
3
Materials and methods
Plant material
Lupinus albus L. plants (cv. Rio Maior) were cultivated on
sterilized 1 : 1 : 1 sand, soil, peat mixture under controlled
conditions of light (240±250 mmol m 2 s 1 PAR), photoperiod
To whom correspondence should be addressed. Fax: q351 213 635031. E-mail: [email protected]
Abbreviations: ChT, chitinase; INV, invertase; PR-10, pathogenesis-related protein of class 10; RWC, relative water content; SS, sucrose synthase;
TL, thaumatin-like protein; WD, water deficit.
ß Society for Experimental Biology 2001
1064
Pinheiro et al.
(12 h), temperature (17u25 8C, nightuday) and relative humidity
(60±70%). Twenty-three days after sowing, WD was induced by
withholding water, the plants being collected 6, 9 and 13 d later.
Watering was then resumed in order to test the plant capacity
for recovery. The leaf water potential was measured with a
Scholander pressure chamber (model 3005 from Soil Moisture
Equipment Crop, Santa Barbara, CA, USA) at predawn (Ypd)
and the relative water content (RWC ) was determined using the
formula (Rodrigues et al., 1995)
RWC ˆ w(FW DW ) 100x u (TW DW )
to Dancer et al. (Dancer et al., 1990) and the fructose
determined by the Somogyi±Nelson method (Nelson, 1944).
Invertase (INV, EC 3.2.1.26) was incubated at 30 8C in
phosphate-citrate buffer 0.1 M and 0.1 M sucrose at pH 4.5
(acid invertase, INVA) or pH 7.0 (neutral invertase, INVN) and
the reducing sugars determined by the Somogyi±Nelson method.
It was observed that at pH 7.0 activity was detected for both
invertases so, INVN activity was considered as activity at pH
7.0Ð30% activity at pH 4.5.
where FW is the leaf discs fresh weight, TW is their weight after
2 h in H2O and DW is the dry weight after 48 h at 80 8C.
Stomatal conductance was measured with a LI-600 porometer
(LI-COR Inc., Lincoln, NE, USA). For all determinations, fullydeveloped leaves were separated into blade and petiole, and the
stems into vascular (stele) and cortical (cortex) tissue.
Three independent experiments were performed. For each
experiment 15±16 plants were used per treatment (well-watered,
WW and water-stressed, WD). For enzymatic activities and
sugar determinations, samples were collected at the middle of
the photoperiod.
Western blot analysis
Biomass and element analysis
The samples were dried at 80 8C and weighted. For element
analysis (C, N, H, and S), the dried samples were homogenized
and analysed by combustion on a LECO CNHS-932 Micro
Elemental Analyser from LECO Corporation, St Joseph, MI,
USA.
Sugar analysis
The samples were extracted with 80% (vuv) hot ethanol (1 ml per
0.2 g sample fresh weight) for 20 min. Freeze-drying eliminated the remaining ethanol. Then the samples were extracted
twice with water and the supernatants combined and analysed.
Glucose, fructose, sucrose, galactose, and a-galactosides were
quanti®ed enzymatically (Roche Molecular Biochemicals cat.
no. 716260 and 428167), using the Hatterscheid and Willenbrink
modi®cation, which involved the oxidation of NADPH and
reduction of INT ( p-iodonitrotetrazolium violet) catalysed by
diaphorase and measured at 490 nm (Hatterscheid and
Willenbrink, 1991).
Enzymatic activities
The proteins were extracted twice (2.5 ml g 1 fresh weight) with
Tris-HCl 50 mM, pH 7.5 containing 1 mM MgCl2, 5 mM
EDTA (ethylenedinitrilotetraacetic acid), 10 mM DTT (dithiothreitol), 2 mM AEBSF w4-(2-aminoethyl)-benzenesulphonyl
¯uoridex, 20 mM leupeptin, 20 ml ml 1 chymostatin, 1.5 mM
pepstatin A, 10% (wuv) NaCl, and 5% (wuv) PVPP (polyvinylpolypyrrolidone). The extracts were centrifuged at 15 000 g
at 4 8C for 15 min. The supernatant was used after desalting in
PD-10 columns (Amersham, Pharmacia) to Tris-HCl 50 mM,
pH 7.5. After protein and enzyme activity determinations, the
samples were concentrated by centrifugation in Centricon
10 (Amicon) and equilibrated with Tris-HCl 50 mM, pH 7.5,
containing 5 mM DTT, 1 mM AEBSF, 2.5 mM EDTA, 10 mM
leupeptin, 10 ml ml 1 chymostatin, and 1 mM pepstatin A.
Peroxidase activity (EC 1.11.1.7) was measured and isoelectric focusing (IEF) was performed according to the methods
described previously (Jackson and Ricardo, 1994, 1998). Sucrose
synthase (SS, EC 2.4.1.13) was incubated at 30 8C according
Protein was quanti®ed by the Lowry method (Bensadoun and
Weinstein, 1976) and the SDS-PAGE (15% T, 2.5% C) performed according to Laemmli (Laemmli, 1970). Western blots
to detect TL (thaumatin-like protein), ChT (class III chitinase)
and PR 10-like protein were performed as described previously
(Pinto and Ricardo, 1995; Regalado and Ricardo, 1996), on
PVDF (polyvinylidene di¯uoride) membrane with antibodies
previously prepared in the laboratory. The three proteins were
sequentially detected, ®rst ChT, next TL and ®nally PR-10.
Between each detection the membrane was soaked overnight in
TBS containing 0.2% (wuv) Tween 20. No reaction was detected
with the pre-immune sera.
Results
When withholding water from the lupin plants a small
decrease in the leaf RWC, Ypd and gs was observed by
day 6. By days 9 and 13 these parameters had markedly
decreased, but the plants maintained the capacity to
recover fully on rewatering. Indeed, 4 d after rewatering
the RWC, Ypd and gs had been equalized in the stressed
and control plants (Fig. 1). As expected, withholding
water led to tissue desiccation that was intensi®ed with
time but in a tissue-speci®c way (Table 1). The leaf blade
and the petiole had the highest water content but it was
the blade that showed the highest water loss. The stem
stele was quite peculiar in having the lowest water content
and showing throughout the experiment the smallest
water loss. It should be pointed out that by day 6 the leaf
blade, the petiole and the stem cortex had lost a similar
percentage of water.
The effects of WD in the lupin plants were studied in
relation to several parameters. The ratios of the stressed
plants to the controls were used for such studies. In order
to judge the changes occurring in the controls with age,
absolute values of different parameters for those plants
are shown in Table 2, for the whole duration of the
experiment.
As a consequence of the water de®cit, the leaf biomass
decreased (up to 50%), but no change was detectable in
the biomass of the stem (Fig. 2). Likely, in what concerns
soluble protein, its concentration decreased in the leaf but
appeared to be unchanged in the stem (Fig. 3). However,
when performing element analysis it was evident that the
increase in stem N and S due to WD (Fig. 4). Despite this,
the total amount of N and S in the blades of remaining
C and N metabolism and water stress
leaves didn't decrease, while in the petiole an increase in
the N content was observed. Presumably the increased
stem N and S was a result of reserve translocation from
senescing leaves before being discarded by the plant.
Considering carbohydrates, changes were detected at
a very early stage of the stress (by day 6 of withholding
water; Fig. 5). As shown in the ®gure, glucose, fructose
1065
and sucrose concentrations of the leaf blades increased
5±7 times relative to the well-watered plants. As the stress
progressed, by day 9, the increments in these sugars were
less evident and by day 13, while sucrose concentration still remained higher, that of reducing sugars had
dropped. Concerning the petiole, the marked increase
in sucrose with stress severity should be emphasized
(c. 18-fold at day 13; Fig. 5) which suggests an increase
in sucrose export out of the leaf. Detected levels of galactose and a-galactosides were very low, and no marked
changes were observed with stress. These sugars, normally
associated with the seed desiccation process (Horbowicz
and Obendorf, 1994; Tabaeizadeh, 1998), apparently are
not involved in the WD responses of lupin vegetative
tissues. The carbohydrate changes in the stem were not
so impressive, but the 2-fold increase in the stele concentrations of glucose, fructose and sucrose, relative to
the control should be noted (Fig. 5). It is striking that
the changes in leaf blade glucose and fructose are paralleled by the changes in invertase activity, particularly of
INVA (Fig. 6). SS activity shows a completely different
pattern, continuously increasing in the blade with stress
severity (Fig. 6) that is consistent with earlier ®ndings
(Wardlaw and Willenbrink, 1994; Tabaeizadeh, 1998),
which described the increase in enzyme activity with WD
and sugar accumulation.
In addition to carbohydrate metabolism, other processes were affected by stress. When lupins were subjected
to WD, the peroxidase level increased in the blade and,
still more drastically, in the stem's stele (Fig. 7). Despite
this large peroxidase increase it was not possible to
ascribe it to any particular isoform, a basic peroxidase
(pI 8.8) being the major form present (results not shown).
TL, ChT and PR-10 like protein were detected in the
apoplastic fractions, but were only barely detectable in
the tissues that were previously extracted for intercellular
¯uid (data not shown). The petiole Western blot for these
proteins was identical to that of the blade and for the
stele identical to that of the cortex (data not shown).
The expression of these proteins was under developmental
control in the leaves (both blade and petiole) and it
seemed that WD intensi®ed the expression of ChT and
Fig. 1. Leaf water status (A, B) and stomatal conductance (C) of L. albus
plants, well watered (k), submitted to drought (m) for 6, 9 and 13 d and
rewatered (shaded area). The water potential of the 5th or 6th leaf was
measured before the beginning of illumination (Ypd), while the relative
water content (RWC) and the stomatal conductance (gs) were measured
2 h after the beginning of illumination. DAW, days after withholding
water. Data are the means"sd of three independent experiments.
Table 1. Water content on a dry weight basis (g of H2O that correspond to 1 g of tissue dry weight) of L. albus control and stressed
tissues
The changes occurring in the stressed tissues relative to the respective controls are shown as percentages. Data are the means"sd of three independent
experiments.
DAWa
6
9
13
a
Leaf blade
Leaf petiole
Stem cortex
Stem stele
WWa
WDa
(%)
WW
WD
(%)
WW
WD
(%)
WW
WD
(%)
7.4"0.4
6.8"0.3
6.8"0.3
6.3"0.2
4.7"0.4
3.8"0.1
85
69
56
7.3"0.2
6.8"0.3
6.1"0.5
6.3"0.5
5.1"0.1
4.3"0.1
86
75
70
6.3"0.7
6.1"0.4
5.1"0.3
5.4"0.5
4.5"0.3
3.8"0.1
86
74
75
4.6"0.2
3.8"0.1
3.3"0.1
4.5"0.2
3.2"0.2
2.9"0.2
98
84
88
DAW, days after withholding water; WW, well watered; WD, water de®cit.
1066
Pinheiro et al.
Table 2. Biomass, concentration of N, S, protein and sugars and activities of sucrose metabolizing enzymes and peroxidase in
well-watered L. albus (control plants), at three dates during the experiment
Data are the means"sd of a representative experiment.
Daysa
Leaf blade
Leaf petiole
Stem cortex
Stem stele
Biomass (g DW plant 1)
6
9
13
0.80"0.23
1.06"0.01
1.19"0.01
0.15"0.01
0.20"0.03
0.23"0.04
0.04"0.01
0.05"0.01
0.05"0.01
0.03"0.01
0.04"0.01
0.05"0.01
N (g per 100 g DW)
6
9
13
6.1"0.07
6.3"0.11
6.4"0.06
1.9"0.04
1.8"0.07
1.6"0.07
2.0"0.08
2.0"0.07
1.8"0.10
1.0"0.01
1.1"0.11
0.9"0.05
S (g per 100 g DW)
6
9
13
0.27"0.02
0.35"0.04
0.26"0.02
0.07"0.01
0.10"0.01
0.09"0.03
0.28"0.01
0.21"0.02
0.19"0.01
0.13"0.04
0.16"0.02
0.17"0.01
6
9
13
105"16
135"24
138"19
56"10
50"11
54"10
10"0.6
14"0.2
10"0.3
12"1.5
11"0.8
9"0.8
6
9
13
6
9
13
7"1
15"2
27"4
12"2
18"2
59"9
319"29
857"60
883"35
77"8
132"21
135"20
63"2
38"2
74"4
17"1.0
4"0.2
2"0.1
103"6
41"3
67"4
70"3
39"2
60"3
6
9
13
0.3"0.02
0.5"0.04
0.9"0.09
0.5"0.05
0.6"0.05
0.8"0.13
0.4"0.03
0.7"0.11
0.8"0.02
2.3"0.05
1.9"0.08
1.8"0.11
6
9
13
16"2
19"2
42"4
8"1
23"2
25"2
145"17
110"6
116"8
108"2
74"8
122"12
1.6"0.10
0.9"0.14
1.3"0.03
2.2"0.02
1.3"0.08
1.5"0.03
Protein (mg g
1
DW)
Glucose (mmol g
1
1
Fructose (mmol g
Galactose (mmol g
Sucrose (mmol g
DW)
1
DW)
1
DW)
DW)
a-Galactosides (mmol g
1
DW)
6
9
13
5.3"0.15
5.6"0.16
7.4"0.43
1.4"0.13
1.8"0.11
2.2"0.10
INVA (nmol sucrose min
1
g
1
DW)
6
9
13
138"14
153"14
278"28
90"18
99"15
104"8
INVN (nmol sucrose min
1
g
1
DW)
6
9
13
6
9
13
172"19
184"22
193"19
42"16
62"11
67"20
115"8
86"6
97"6
14"3
43"7
37"10
8.2"0.4
6.9"0.3
5.8"0.3
8"2
26"6
22"3
2.2"0.2
1.9"0.2
1.8"0.1
54"9
91"18
76"6
6
9
13
4300"421
4909"302
6895"538
562"90
987"57
1130"103
5189"381
5427"284
7313"455
136"16
202"27
195"17
SS (nmol sucrose min
Peroxidase (nkat g
a
1
1
g
DW)
1
DW)
nd
nd
nd
nd
nd
nd
Equivalent to the days after withholding water in the stressed plants; DW dry weight; nd not detected.
Fig. 2. Biomass ratio (drought tissuesucontrol tissues) of L. albus leaves
(blade and petiole) and stems (cortex and stele), 6 d (empty bars), 9 d
(partly ®lled bars) and 13 d (®lled bars) after withholding water. For the
calculation of the biomass ratios, g of dry weight was the unit used. Data
are the means"sd of three independent experiments.
Fig. 3. Ratio of protein concentration (drought tissuesucontrol tissues)
of L. albus leaves (blade and petiole) and stems (cortex and stele), 6 d
(empty bars), 9 d (partly ®lled bars) and 13 d (®lled bars) after
withholding water. For the calculation of the protein concentration
ratio, mg of protein g 1 tissue dry weight was the unit used. Data are the
means"sd of three independent experiments.
C and N metabolism and water stress
1067
Fig. 4. Ratios of nitrogen concentration (A) and sulphur concentration
(B) (drought tissuesucontrol tissues) of L. albus leaves (blade and petiole)
and stems (cortex and stele), 6 d (empty bars), 9 d (partly ®lled bars) and
13 d (®lled bars) after withholding water. For the calculation of the N
concentration and S concentration ratios, mg of element g 1 tissue dry
weight was the unit used. Data are the means"sd of three independent
experiments.
TL (Fig. 8A). In the stem, PR-10 and ChT, but not TL,
were present in the unstressed plants and WD strongly
induced TL and ChT and barely affected PR-10 (both in
the cortex and stele; Fig. 8B).
Discussion
Studies of water stress have been mainly directed to
the physiology of the whole plant and to leaf blade metabolism, namely photosynthesis (Schulze, 1986; Chaves,
1991; Tabaeizadeh, 1998; Teraza et al., 1999; Cornic,
2000), and less attention has been paid to other organs.
In these studies with L. albus tissue-speci®c reactions
dependent on stress intensity were detected, which appear
as distinct strategies to cope with WD. Typical stress
avoidance mechanisms occurred such as stomata closure
and leaf senescence while the metabolic changes observed
in the stem cortex and stele could be indicative of some
tolerance to dehydration. Indeed, despite the massive loss
of leaf biomass, plants were able to survive for up to 13 d
and regrow on rewatering.
When withholding water, the ®rst signs of stress in
L. albus involved pronounced changes in sugar metabolism despite small variations in the water status
parameters. Losses in water content of the same order
of magnitude (10±15%) have been found to cause large
changes in plant growth and metabolism (Cheng et al.,
1993; Mullet and Whitstitt, 1996). The observed increase
in the concentration of soluble sugars may be the result of
growth being more inhibited by WD than photosynthesis,
Fig. 5. Sugar concentration ratios (drought tissuesucontrol tissues) of
L. albus leaves (blade and petiole) and stems (cortex and stele), 6 d
(empty bars), 9 d (partly ®lled bars) and 13 d (®lled bars) after withholding water. (A) Glucose; (B) fructose; (C) sucrose; (D) galactose; (E)
a-galactosides. For the calculation of the sugar concentration ratios,
mmol sugar g 1 tissue dry weight was the unit used. Data are the
means"sd of three independent experiments.
as well as an increased partitioning of ®xed carbon to
sucrose, as shown for various species (including lupins)
under WD (Chaves, 1991; Quick et al., 1992). This accumulation of soluble sugars may be related to osmoregulation and desiccation tolerance (Morgan, 1984; Hare et al.,
1998) contributing to plant survival.
In addition, it is known that sucrose and other sugars
regulate the expression of many genes involved in
photosynthesis, respiration, N and secondary metabolism
1068
Pinheiro et al.
Fig. 6. Activity ratios of sucrose metabolizing enzymes (drought
tissuesucontrol tissues) of L. albus leaves (blade and petiole) and stems
(cortex and stele), 6 d (empty bars), 9 d (partly ®lled bars) and 13 d
(®lled bars) after withholding water. (A) INVA; (B) INVN; (C) SS. For
the calculation of the enzymatic activity ratio, nmol min 1 g 1 tissue dry
weight was the unit used. nd, Activity not detected; nd1, activity not
detected in the WD tissue. Data are the means"sd of three independent
experiments.
Fig. 7. Peroxidase activity ratio (drought tissuesucontrol tissues) of L.
albus leaves (blade and petiole) and stems (cortex and stele), 6 d (empty
bars), 9 d (partly ®lled bars) and 13 d (®lled bars) after withholding
water. For the calculation of the enzymatic activity ratio, nkat g 1 tissue
dry weight was the unit used. Data are the means"sd of three
independent experiments.
as well as defence processes (Koch, 1996; Jang and Sheen,
1997; Hare et al., 1998; Halford et al., 1999). Thus, sugarregulated genes are a means of integrating cellular
responses (sugar transport, allocation and utilization)
affecting plant development and stress response (Koch,
1996; Yu, 1999). The large alterations observed in
L. albus sugar metabolism preceded the drastic decrease
of soluble leaf protein, the accumulation of N and S in the
stem and petiole and, also, the increase in stem ChT,
TL and peroxidase. These proteins are typically related to
stress responses, such as freezing, osmotic and salt stress
and pathogen attack (Chen et al., 1994; Yun et al., 1996;
Moons et al., 1997; Riccardi et al., 1998; Tabaeizadeh,
1998; Trudel et al., 1998). Thus the WD response of
lupins seems to have characteristics in common to other
adverse conditions in agreement with suggestions made
for other species (Shinozaki and Shinozaki, 1996;
Tabaeizadeh, 1998).
Some reports suggest that the pathway for PR-gene
induction (chitinase and turgor-responsive PR-proteins)
is related to soluble sugars (Herbers et al., 1996) or to
alterations in INVA activity (Herbers and Sonnewald,
1998). In this context, the particular response of L. albus
INVA activity that seems to be related to WD intensity
should be noted, increasing under mild stress and
dramatically decreasing with severe WD. The con¯icting
data in the literature concerning invertase behaviour
under WD could therefore be explained by the degree of
stress. INVA may have a role not only in the sucrosesensing pathway but also in the integration of signals for
defence responses and could be considered as a central
modulator of these processes (Kingston-Smith et al.,
1999; Roitsch, 1999).
The physiological signi®cance of the increase in L.
albus peroxidase activity and in TL and ChT under WD
could be related to changes in the cell wall properties
potentially important for the stem in order to cope with
the stress. TL can participate in the cell wall metabolism,
due to the ability of some of its forms to bind cell wall
polymers (Trudel et al., 1998) and to hydrolyse watersoluble and -insoluble complex b-1,3-glucans (Grenier
et al., 1999). Peroxidase can also alter the cell wall properties by promoting the cross-linking between molecules
like lignin, suberin, proteins, hemicelluloses, and ferulic
acid (Espelie et al., 1986; Fry, 1986; BarceloÂ, 1995;
Krishnamurthy, 1999). The fact that in the stele N and S
content and peroxidase activity markedly increased could
suggest that the stele is a region of protein insolubilization. Such insolubilization of cell wall proteins had
already been observed as a result of osmotic stress and
was considered to contribute to the adjustment of cell
wall elasticity despite the water loss (Marshall et al.,
1999). Since WD causes the formation of active oxygen
species, an additional function of the increased peroxidase
activity could be the protection against oxidative damage
(Tabaeizadeh, 1998).
The fact that lupins subjected to WD recovered after
rewatering, despite a massive loss of leaves, suggests that
WD triggered a defenceuresistanceuadaptation mechanism
in these plants, particularly in the stems. Sugars explain
C and N metabolism and water stress
1069
Fig. 8. Immunodetection of TL, ChT and PR-10 in leaves (A) and stems (B) of L. albus plants, well-watered (control) and submitted to water de®cit
(drought) for 6, 9 and 13 d. Blade protein (15 mg) and stem cortex protein (5 mg) were separated by SDS-PAGE and TL, ChT and PR-10
immunodetected (Regalado and Ricardo, 1996; Pinto and Ricardo, 1995).
plant desiccation tolerance only in part (Farrant et al.,
1993) and water stress-responsive proteins (ChT, TL and
peroxidase) may play an additional important role in
tolerating WD (Ingram and Bartels, 1996; Pelah et al.,
1995, 1997). Thus, the stem appears to play a central
role in the strategy to overcome WD being a transient
reservoir of nutrients, a survival structure able to withstand the stress and allowing plant reconstruction when
water is available.
Acknowledgements
This work was ®nanced by PRAXIS XXI programme
(2u2.2uBIAu227u94 and BDu16137u98). We would like to thank
Ana Paula Regalado and Maria Paula Pinto for the antibodies,
and Leonor OsoÂrio for her help on water parameters assays.
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