Download An investigation into the role of light during desiccation of three

Blackwell Science, LtdOxford, UKPCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2003? 2003
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Original Article
Desiccation and light stress
J. M. Farrant
et al.
Plant, Cell and Environment (2003) 26, 1275–1286
An investigation into the role of light during desiccation of
three angiosperm resurrection plants
J. M. FARRANT1, C. VANDER WILLIGEN1,2 D. A. LOFFELL2, S. BARTSCH2 & A. WHITTAKER1
1
Department of Molecular and Cell Biology and 2Department of Botany, University of Cape Town, Private Bag, Rondebosch,
7701, South Africa
ABSTRACT
Under water-limiting conditions excitation energy harnessed by chlorophyll can lead to the formation of reactive
oxygen species (ROS). Resurrection plants minimize their
formation by preventing the opportunity for light–chlorophyll interaction but also quench them via antioxidants.
Poikilochlorohyllous species such as Xerophyta humilis
break down chlorophyll to avoid ROS formation. Homoiochlorophyllous types retain chlorophyll. We proposed that
leaf folding during drying of Craterostigma wilmsii and
Myrothamnus flabellifolius shades chlorophyll to avoid
ROS (Farrant, Plant Ecology 151, 29–39, 2000). This was
tested by preventing leaf folding during drying in light. As
controls, plants were dried without light, and X. humilis
was included. Craterostigma wilmsii did not survive drying
in light if the leaves were prevented from folding, despite
protection from increased anthocyanin and sucrose and elevated antioxidant enzyme activity. Membranes were damaged, electrolyte leakage was elevated and plastoglobuli
(evidence of light stress) accumulated in chloroplasts.
Restrained leaves of M. flabellifolius survived drying in
light. Leaf folding allows less shading, but the extent of
chemical protection (anthocyanin content and antioxidant
activity) is considerably higher in this species compared
with C. wilmsii. Chemical protection appears to be light
regulated in M. flabellifolius but not in C. wilmsii. Drying
in the dark resulted in loss of viability in the homoiochlorophyllous but not the poikilochlorophyllous species. It is
hypothesized that some of the genes required for protection are light regulated in the former.
Key-words: Craterostigma wilmsii; Myrothamnus flabellifolius; Xerophyta humilis; desiccation tolerance; homoiochlorophyllous; leaf folding; light stress; poikilochlorophyllous.
INTRODUCTION
Resurrection plants are able to survive desiccation to the
air-dry state. Bewley (1979) has proposed that there are
three criteria which must be met for plant tissue to survive
severe loss of protoplasmic water: It must: (1) limit damage
Correspondence: Jill M. Farrant. E-mail: [email protected]
© 2003 Blackwell Publishing Ltd
incurred to a repairable level; (2) maintain its physiological
integrity in the dried state; and (3) mobilize repair mechanisms upon rehydration which effect restitution of damage
suffered during desiccation. Subsequent reports (for example Bewley & Oliver 1992; Oliver, Woods & Mahony 1998)
have suggested that desiccation tolerance is achieved either
by mostly repair of desiccation- (and/or rehydration-)
induced damage, or by of protection of cellular integrity
during drying such that little by way of repair is required.
Furthermore, it is generally held that the lower order resurrection plants (lichens, algae and bryophytes) plants
achieve tolerance using the former mechanism and that
angiosperms tend to protect against desiccation damage
(Gaff 1989; Oliver & Bewley 1997; Oliver et al. 1998; Farrant et al. 1999; Farrant 2000; Cooper & Farrant 2002;
Walters et al. 2002).
There are a number of stresses brought about by, or in
association with, extreme water loss (reviewed by Gaff
1989; Vertucci & Farrant 1995; Oliver et al. 1998; Farrant
2000; Walters et al. 2002), which must be protected against
in angiosperm tissues while they are drying. One of the
associated stresses in photosynthetically active tissues, is
light. Under water-limiting conditions, excitation energy
harnessed by chlorophyll cannot be dissipated via photosynthesis and can lead to the formation of oxygen free
radicals, which, if left unquenched, can cause considerable
subcellular damage (Halliwell 1987; Kaiser 1987; Larson
1988; Smirnoff 1993).
The angiosperm resurrection plants appear to have a
number of mechanisms, which vary among species, to minimize photo-oxidative damage. In all species examined to
date, there is up-regulation of antioxidant systems in leaf
tissues during drying which argues for some amelioration
of free radical damage during dehydration and on subsequent rehydration (Smirnoff 1993; Sgherri et al. 1994a, b;
Kranner & Grill 1997; Navari-Izzo et al. 1997a; Navari-Izzo,
Quartacci & Sgherri 1997b; Sherwin & Farrant 1998; Farrant 2000). In addition to photo-chemical protection, resurrection plants undergo anatomical changes to prevent
light–chlorophyll interaction and thereby put a stasis on
photosynthesis and photo-oxidation reactions. Some species degrade chlorophyll and dismantle thylakoid membranes during dehydration. These plants, examples of which
are the Xerophyta spp., Borya nitidia and Eragrostis nindensis are termed poikilochlorophyllous (Hallum & Luff
1275
1276 J. M. Farrant et al.
1980; Hetherington, Hallum & Smillie 1982; Tuba et al.
1996, 1997; Sherwin & Farrant 1998; Tuba, Proctor & Csintalan 1998; Farrant 2000; Vander Willigen et al. 2001). Many
other species (e.g. Boea hydroscopica, Craterostigma spp.,
Myrothamnus flabellifolius and Sporobolus stapfianus)
retain most of their chlorophyll and thylakoid integrity during drying (termed homoiochlorophyllous) (Sherwin &
Farrant 1996, 1998; Quartacci et al. 1997; Farrant et al. 1999;
Farrant 2000). Leaves of C. wilmsii and M. flabellifolius fold
during drying causing shading of the inner surfaces from
light. Those surfaces which are left exposed to light accumulate high levels of anthocyanins (Farrant 2000). These
pigments mask chlorophyll and reflect photosynthetically
active light, and also act as antioxidants (Larson 1988;
Smirnoff 1993).We have proposed that these homoiochlorophyllous plants use leaf folding and anthocyanin accumulation as a means of minimizing light–chlorophyll
interaction and thus photo-oxidative damage (Sherwin &
Farrant 1998; Farrant 2000).
The aim of this study was to determine whether leaf
folding does indeed protect Craterostigma wilmsii Engl.
and Myrothamnus flabellifolius Welw. from light stress during drying. Leaves were tied down in order to prevent
natural folding during drying and the effect of this on plant
viability was assessed using a number of physiological
parameters. In order to determine whether the data
obtained were due to light stress and not a consequence of
restraining the leaves, plants with and without leaf
restraints were also dried in the dark. As a further control
to check for the effect of desiccation in the dark, the
poikilochlorophyllous species Xerophyta humilis (Bak.)
Dur. and Schinz, which does not significantly fold leaves,
was also included in the study.
MATERIALS AND METHODS
Plant material and treatments during
dehydration and rehydration
Plants were collected and maintained in a glasshouse as
previously described (Sherwin & Farrant 1996; Dace et al.
1998) until initiation of the experiments described below,
which were performed under laboratory conditions. Plants
were fully hydrated when the restraints were applied and
were allowed to dry naturally [photosynthetic active radiation (PAR) = 400 mmol m-2 s-1) or in the dark by withholding water. In order to keep drying rates of plants dried in
the light and dark comparable and to minimize time spent
in the dark, plants were placed in the dark only when the
soil had dried to 0.12 g H2O g-1 dry mass (< -1 MPa) but
the plant tissues themselves were still at 100% relative
water content (RWC). Drying to the air-dry state (approximately 5% RWC) took approximately 10 d for C. wilmsii
and X. humilis, the period spent in the dark was only 5 d.
Myrothamnus flabellifolius twigs dried within 48 h (light
and dark treatments). Plants were maintained in the dry
state for 1 week before rehydration. In all treatments plants
were rehydrated in the light. Plants were sampled after 48 h
rehydration as previous studies have shown that by this
stage the tissues were fully rehydrated (100% RWC) (Farrant et al. 1999).
The leaves of C. wilmsii grow in a rosette. During natural
drying, the outer ring of leaves from the rosette fold over
and shade the inner ones, the abaxial surfaces of the outer
leaves being the only surfaces to receive light while the
plant is in the dry state (Sherwin & Farrant 1998; Farrant
2000). For each experiment half of the plants in a tray were
subject to leaf restraints and the other half were not. The
outer leaves of the rosette were restrained by tying them
to the soil with thin plastic (Parafilm; American National
Can, Menasha, WI, USA) strips.
Myrothamnus flabellifolius is a woody shrub and in the
hydrated state the leaves are held perpendicular to the
stem. During drying leaves fold upward through 90∞
becoming parallel to the stem axis with only the abaxial
surface being exposed to light when in the dry state (Farrant 2000). Since it was difficult to restrain leaves from
intact plants, detached branches of more than 10 cm length
were used. Excised twigs and leaves of this species undergo
normal leaf folding during drying and survive desiccation
(Gaff & Loveys 1984; Farrant et al. 1999). Individual leaves
from half of the branches which were allowed to dry were
prevented from folding toward the stem by stapling across
the blade (leaves are small and were not punctured). The
twigs were rehydrated by placing cut ends into water.
The leaves were sampled for the measurements outlined
below from fully hydrated plants prior to drying, from the
variously treated dry (< 5% RWC) plants and upon rehydration of these plants. The RWC was determined as previously described (Sherwin & Farrant 1996). The following
codes were used to denote the various drying treatments:
LU, plants dried in the light (L) the leaves unrestrained
(U); LR, plants dried in the light with leaves restrained (R)
from folding; DU, plants dried in the dark (D); leaves unrestrained; DR, plants dried in the dark, leaves restrained.
Electrolyte leakage
Membrane integrity of leaves from the variously treated
plants was assessed by measuring the extent of electrolyte
leakage from the tissue as described previously (Farrant
et al. 1999). Five replicates of individual leaves of each of
the species were used per treatment. Following each measurement the maximum leakage of the tissue was determined by snap freezing and thawing the leaves. The results
are expressed at a percentage of maximum.
Pigment analysis
The chlorophyll and carotenoids were extracted from three
replicates of leaf tissue from each of the the variously
treated plants according to Lichtenthaler (1987). Material
was cut into fine segments and pigments extracted in 100%
acetone at 4 ∞C in the dark. Pigment concentrations in the
supernatant was determined spectrophotmetrically (DU
650; Beckman, Fullerton, CA, USA) at 661.6, 644.8 and
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
Desiccation and light stress 1277
470 nm. Chlorophyll (a + b) and carotenoid (x + c) contents
were calculated using extinction coefficients provided by
Lichtenthaler (1987). Total anthocyanin content of leaves
(three replicates per treatment) were determined as
described in Sims & Gamon (2002). Extraction was in acidified methanol and the pigment in the supernatant determined spectrophotmetrically at 530 and 657 nm.
Sucrose analysis
Leaves from various treatments were ground in liquid
nitrogen and sugars were extracted in 100 mM NaOH in
50% ethanol. The extract was neutralized by addition of
100 mM HEPES in 100 mM acetic acid, centrifuged at
16 000 g for 20 min and the extract retained. The pellet was
re-extracted using the above procedure, and supernatants
combined. Quantitation of sucrose was by use of the Dglucose/D-fructose sugar assay kit (Boehringer Mannheim,
Mannheim, Germany) based on the methodology of Bergmeyer & Bernt 1974). The production of NADPH was
determined spectrophotometrically at 340 nm (Beckman
DU 6500) and used to calculate the quantity of sucrose in
each sample.
Antioxidant enzyme assays
Our previous studies (Farrant 2000; Sherwin & Farrant
1998) have shown that ascorbate peroxidase activity (AP)
increased markedly in all three species, and glutathione
reductase (GR) activity increased in M. flabellifolius and
X. humilis, during drying. Thus the activities of these antioxidant enzymes were measured in leaves of the variously
treated plants.
Leaf material (0.04–0.07 g) was ground in a mortar with
liquid nitrogen. Insoluble polyvinylpyrrolidone (twice the
weight of leaf material) was added and the enzyme
extracted in 25 mM KH2PO4 (pH 7.5), 1 mM ethylenediaminetetraacetic acid, and 5 mM dithiothreitol. Following
centrifugation (20 mins, 12 000 g), 2 mL of extract was
desalted on 5 mL sephadex G25 (medium bead) columns
and the enzyme measurements made on desalted extract.
Ascorbate peroxidase was measured as described by Wang,
Jaio & Fuast (1991) and glutathione reductase was
measured as described by Sgherri et al. (1994b). Enzyme
activities were calculated per mg protein [measured using
the Bradford (1979) assay]. Due to large discrepancies
among species, for comparative purposes the enzyme activities are expressed as a percentage of that typical of control,
hydrated (pretreated) leaves.
Ultrastructural studies
Mesophyll leaf segments (approximately 5 mm2) were processed for transmission electron microscopy (TEM) using
the method previously reported for these species (Farrant
et al. 1999; Farrant 2000). Tissues were sectioned using a
Reichert Ultracut-S microtome (Vienna, Austria), stained
with uranyl acetate and lead citrate (Reynolds 1963) and
viewed with a Jeol CX TEM (Jeol, Tokyo, Japan).
RESULTS
Table 1 gives the final water content (RWC) of leaves from
plants subjected to the various treatments and summarizes
the ability of the three species used in this study to recover
and resume growth following the various treatments
imposed. All treatments resulted in drying of the respective
species to similar water contents, these being £ 5% RWC.
Craterostigma wilmsii survived only if dried normally
(leaves unrestrained) in the light. Plants dried in the light
with leaves restrained died, although any leaves not
restrained folded normally and survived. Myrothamnus flabellifolius survived drying in the light, regardless of
whether leaves were restrained or not. Neither of these two
species survived if dried in the dark, regardless of whether
leaves were restrained or not. Xerophyta humils survived
drying both in the light and dark.
Electrolyte leakage
Electrolyte leakage, which gives an indication of the degree
of integrity of membranes, from leaves of variously treated
plants is given in Fig. 1. There was no difference in extent
of leakage, compared to hydrated material, in leaves of C.
wilmsii which had been dried unrestrained in the light, nor
upon their rehydration, suggesting little membrane damage
occurred during these treatments (Fig. 1a). However, when
leaves were restrained during drying in the light there was
Table 1. Survival of Craterostigma wilmsii, Myrothanmus flabellifolius and Xerophyta humilis when dried normally or with leaves restrained
under light or dark conditions
Light unrestrained
Light restrained
Dark unrestrained
Dark restrained
Species
Survival
RWC
Survival
RWC
Survival
RWC
Suvival
RWC
C. wilmsii
M. flabellifolius
X. humilis
Yes
Yes
Yes
4.1% (±1.2)
3.0% (±0.95)
5% (±1.0)
No
Yes
4.0% (±0.99)
3.0% (±0.99)
No
No
Yes
4.0% (±0.96)
3.1% (±1.0)
5% (±0.99)
No
No
4.0% (±1.1)
3.0% (±0.95)
The final air-dry water contents [expressed as relative water content (RWC)] of leaves from plants at each treatment are given. Standard
deviations are given in parentheses.
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
1278 J. M. Farrant et al.
considerable increase in leakage in leaves from both dried
and rehydrated plants, suggesting that this treatment
caused a change in membrane configuration, probably
damage, which was not repaired upon rehydration. Leaves
from plants dried in the dark, whether restrained or not,
also had increased electrolyte leakage in the dry and rehydrated state. The extent of leakage from plants dried in the
dark was similar regardless of whether leaves were
restrained or not, suggesting that lack of light rather than
the restraining treatment, was the primary cause of membrane damage in these plants.
Leaves of M. flabellifolius dried in the light, whether
restrained or not, had a similar extent of electrolyte leakage
compared to leaves from hydrated and rehydrated plants
(Fig. 1b). Thus prevention of leaf folding during drying in
the light did not appear to cause membrane damage as was
the case for C. wilmsii. However, as for C. wilmsii, leaves
from plants dried in the dark, both restrained and unrestrained, had increased electrolyte leakage compared with
those from hydrated plants and this was exacerbated in
leaves from rehydrated plants. Drying in the dark thus also
appeared to have allowed some membrane damage in this
species, which was not repaired upon rehydration.
There was no change in electrolyte leakage in leaves of
X. humilis dried in the light or in the dark, nor upon rehydration of the plants. Unlike the previous two species, drying in the dark did not appear to cause membrane damage
in this species.
Pigments
Figure 1. Electrolyte leakage, expressed as a percentage of maximum possible leakage, in leaves of C. wilmsii (a), M. flabellifolius
(b) and X. humilis (c). Black bars, control hydrated leaves; white
bars, dry leaves; hatched bars, rehydrated leaves. LU, light unrestrained; LR, light restrained; DU, dark unrestrained; DR, dark
restrained. Error bars indicate standard deviation (n = 5).
The exposure of chlorophyll to light during dehydration can
lead to formation of free radicals which can be extremely
damaging to tissues. In C. wilmsii, the chlorophyll content
of leaves was reduced during drying in all treatments except
those plants with leaves dried restrained in the dark
(Fig. 2a). Restrained leaves dried in the light lost somewhat
more chlorophyll than unrestrained ones. Only plants dried
normally in the light were able to resynthesize chlorophyll
during rehydration. In all other treatments, further chlorophyll breakdown occurred after prolonged rehydration
(not shown) as the plants senesced. In M. flabellifolius,
some loss of chlorophyll also occurred during drying in all
treatments, but, as for C. wilmsii, those dried in the light
with leaves restrained had greatest loss of chlorophyll
(Fig. 2d). Upon rehydration, plants dried in the light were
able to resynthesize chlorophyll. Plants dried in the dark
lost chlorophyll as they senesced. Xerophyta humilis leaves
Figure 2. Pigment contents of leaves of C. wilmsii (a, b, c), M.
flabellifolius (d, e, f) and X. humilis (g, h, i). Black bars, control
hydrated leaves; white bars, dry leaves; hatched bars, rehydrated
leaves. LU, light unrestrained; LR, light restrained; DU, dark unrestrained; DR, dark restrained. Chlorophyll contents are shown in
the left-hand panel (a, d, g); carotenoid contents in the middle
panel (b, e. h) and anthocyanin content in the right-hand panel (c,
f, i). Error bars indicate standard deviation (n = 3).
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
Desiccation and light stress 1279
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
1280 J. M. Farrant et al.
lost virtually all chlorophyll when dried in the light (as is
normal, since the plant is poikilochlorophyllous), but only
half when dried in the dark (Fig. 2g). Chlorophyll was completely recovered during rehydration in both treatments.
Carotenoids can protect against light-induced free radical formation (Larson 1988; Smirnoff 1993). The carotenoid
content of leaves of C. wilmisii followed the same trend as
that of chlorophyll, with some loss occurring during drying
in all treatments except plants with restrained leaves dried
in the dark, and only plants dried normally in the light being
able to maintain carotenoids at original levels when rehydrated (Fig. 2b). Similarly, the carotenoid content of M.
flabellifolius followed the same trends as occurred in the
chlorophyll content in this species (Fig. 2e). Xerophyta
humilis leaves lost 50% of their carotenoids in the light but
only 10% in the dark. These pigments were recovered upon
rehydration in both treatments (Fig. 2h).
Anthocyanins are believed to mask chlorophyll from
light, so preventing free radical formation, and also act as
antioxidants (Larson 1988). These pigments were accumulated during drying in leaves of C. wilmsii regardless of the
light regime given (Fig. 2c). Although the greatest levels
accummulated in restrained leaves dried in the light, the
fact that these pigments accummulated in plants dried in
the dark suggests that it is in response to drying, rather than
light. Anthocyanin content declined during rehydration in
all treatments, a normal response in healthy plants (Farrant
2000) but probably due to senescence in those treatments
(LR, DU, DR) which killed the plants. In M. flabellifolius
anthocyanin content increased only in leaves of plants dried
in the light, with slightly higher levels being recorded in
those with unrestrained leaves (Fig. 2f). The amount of
anthocyanin accummulated in this species was greater than
in C. wilmsii and it would appear that in M. flabellifolius
(compare Fig. 2c & f), anthocyanin accummulation might
well be a light-mediated response. Levels dropped upon
rehydration in all treatments. In X. humilis, low levels
(compared with the other species) of anthocyanins accummulated in leaves dried in the light, although leaves dried
in the dark also accummulated some anthocyanin. Like the
other two species, anthocyanin content declined again upon
rehydration (Fig. 2i).
Antioxidants
Changes in activity of the antioxidant enzymes ascorbate
peroxidase (AP) and glutathione reductase (GR), are presented in Fig. 3 as a percentage of their activity in control,
hydrated leaf tissues (where control = 100%). In C. wilmsii,
AP activity increased during drying by 80–100% in all treat-
Figure 3. Activity of the antioxidant
enzymes ascorbate peroxidase (AP) and
glutathione reductase (GR) as a percentage of activity in control, hydrated leaves,
of C. wilmsii (a, b), M. flabellifolius (c, d)
and X. humilis (e, f). White bars, dry leaves;
hatched bars, rehydrated leaves. LU, light
unrestrained; LR, light restrained; DU,
dark unrestrained; DR, dark restrained.
Error bars indicate standard deviation
(n = 5).
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
Desiccation and light stress 1281
ments, but only plants dried normally in the light reduced
activity to that of control levels upon rehydration. Activity
remained high (with considerable variation in the data) in
restrained leaves from plants dried in the light and in leaves
from dark-treated plants. GR activity increased markedly
(to 275% of control) during drying in leaves of plants dried
normally in the light, although some increase (< 40%)
occurred in other treatments. On rehydration, the activity
of this enzyme dropped to control levels (light unrestrained
leaves) or below (Fig. 3b). Although protection afforded by
AP was similar among treatments, GR activity was reduced
in plants given treatments which killed them (LR, DR,
DU). The lack of sufficient antioxidant protection would
have allowed free radical damage to accrue, probably contributing to the death of plants from those treatments.
In M. flabellifolius AP activity increased only in plants
dried in the light, with significantly greater increases in
activity occurring in leaves which had been restrained. On
rehydration, activity remained high for longer periods in
plants dried normally in the light; activity in restrained
leaves dried in the light returned rapidly to control levels
(Fig. 3c). Similar trends were observed for GR activity
(Fig. 3d), although plants dried normally in light had higher
GR activity on drying than those with restrained leaves.
Plants dried in the dark had no measurable activity on
drying nor on rehydration. The lack of increased antioxidant activity in plants which died (DU and DR treatments)
could have allowed for accrual of free radical-associated
damage, which in turn contributed to their death.
AP activity increased markedly in leaves of X. humilis
dried in the light, but there was no significant change in
activity in leaves of plants dried in the dark (Fig. 3e). The
activity of this enzyme returned to control levels during
rehydration of plants dried in the light and became elevated
during rehydration of plants dried in the dark. This pattern
of activity may suggest that light is required for activation
of the enzyme (which could occur during rehydration of
plants dried in the dark, since rehydration occurred in the
light). Such elevated activity during rehydration might have
been required to ameliorate against ROS formation due to
the presence of chlorophyll (Fig. 2c) and partially intact
thylakoid membranes (Fig. 7b) present in tissues dried in
the dark. GR activity was elevated during drying of plants
dried both in the light and dark, although considerably
more so in plants dried in the dark. Activity declined to
control levels during rehydration in both treatments
(Fig. 3e).
Sucrose content
Non-reducing sugars, sucrose in particular (Ingram et al.
1997; Ghasempour et al. 1998; Oliver, Hincha & Crowe
2001; Whittaker et al. 2001), are thought to protect the subcellular milieu against desiccation damage in a number of
different ways. As some treatments involved dehydration
in the dark, photosynthesis and thus sucrose accummulation might have been compromised, and so sucrose content
during drying and rehydration was determined (Fig. 4). The
Figure 4. Sucrose content of leaves of C. wilmsii (a), M. flabellifolius (b) and X. humilis (c). Black bars, control hydrated leaves;
white bars, dry leaves; hatched bars, rehydrated leaves. LU, light
unrestrained; LR, light restrained; DU, dark unrestrained; DR,
dark restrained. Error bars indicate standard deviation. (n = 3).
sucrose content of leaves of C. wilmsii dried in the light
increased markedly, regardless of whether leaves were
restrained or not (Fig. 4a) to levels previously reported for
this species (Cooper & Farrant 2002). Leaves dried in the
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1282 J. M. Farrant et al.
dark accummulated only half the amount of sucrose accummulated by those plants dried in the light. This indicates
that photosynthesis, and/or a light-regulated switch for
genes necessary for sucrose storage upon drying, is required
for maximual accumulation of this protectant. On rehydration, sucrose declined in plants dried normally in the light,
but not in those with restrained leaves dried in the light,
nor unrestrained dried in the dark. This suggests that
enzymes for utilization of sucrose might have been compromised by these dyring treatments. Plants with restrained
leaves in the dark lost all sucrose, possibly being leached
from the damaged tissues.
In M. flabellifolius, sucrose accummulated during drying
in leaves from all treatments (Fig. 4b) indicating that neither restraining, nor lack of light, interfered with accumulation of this protectant sugar. Upon rehydration, sucrose
declined only in plants dried in the light indicating its utilization in metabolism in these plants but not in those dried
in the dark.
Sucrose content also increased in X. humilis plants
regardless of whether they were dried in the light or dark
and declined upon rehydration in plants dried in the light
but not in those dried in the dark (Fig. 4c). Thus like M.
flabellifolius, this species did not need to photosynthesize
to produce adequate sucrose levels. It is unlikely that lack
of mobilization of sucrose during rehydration of dark-dried
plants was due to damage of enzymes required for this
process. The plants had retained much of their chlorophyll
and thus photosynthesis could be initiated almost immediately on full rehydration, supplementing the sucrose content and precluding measurable decline in this sugar.
However, it is also possible that the enzymes for sucrose
a
c
b
d
metabolism required de novo transcription and/or translation which delayed sucrose mobilization upon rehydration.
Ultrastructural status
Mesophyll cells from leaves of C. wilmsii dried unrestrained
in the light had subcellular organization typical of that
reported previously (Farrant et al. 1999; Farrant 2000) and
the data are not shown. Cells from dry leaves had folded
walls and the plasmalemma was closely adhered to them.
Thylakoid membranes were intact, albeit displaced to one
side of the chloroplast. There were no obvious signs of
damage in the dried or rehydrated state.
Mesophyll cells from restrained leaves dried in the light
and those dried in the dark (whether restrained or not) had
signs of subcellular damage (Fig. 5a & b). Although wall
folding was evident in all treatments (arrowed in Fig. 5a)
drying in the dark resulted in considerable plasmalemma
withdrawal (more than what could be ascribed to conventional fixation, if one compares tissue dehydrated unrestrained in the light) and loss of integrity (arrowed in
Fig. 5b). Chloroplasts from tissues dried in the light did not
appear damaged and had well-organized thylakoid membranes, but numerous plastoglobuli were present in some
(Fig. 5a). Chloroplasts from plants dried in the dark had
blistered thylakoid membranes (Fig. 5b). All damage was
considerably exacerbated when these plants were rehydrated (Fig. 5c & d). Complete loss of plasmalemma and
organellar integrity occurred.
Mesophyll cells from leaves of M. flabellifolius dried in
the light showed no sign of damage, regardless of whether
the leaves were restrained or not and only those from
Figure 5. Subcellular organization of
mesophyll cells of Craterostigma wilmsii
dehydrated (a) in the light (LR), and (b) in
the dark (DR); and rehydrated after drying
with leaves restrained (c) in the light and
(d) in the dark. Note the plasmalemma
withdrawal and folded walls (arrowed),
small vacuoles (v), plastoglobuli (p) in chloroplasts (c) in (a). In (b), vacuoles were
absent, plasmalemma withdrawal and rupture (arrowed) was evident, thylakoids
were blistered. Rehydrated tissue (c & d)
showed total disruption of plasmalemma
and general cytoplasmic dissolution.
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Desiccation and light stress 1283
a
b
c
d
restrained tissues are shown. There was no plasmalemma
withdrawal from the cell wall and vacuoles containing some
relatively electron dense material were evident (Fig. 6a) as
previously described for tissues dried under normal conditions (Farrant 2000; Farrant & Kruger 2001). Thylakoid
membranes were blistered, but this too is normal for this
species (Koonjul et al. 2000). On rehydration subcellular
organization was typical of hydrated unrestrained leaves
(Sherwin & Farrant 1996; Farrant & Kruger 2001). A single
large central vacuole had reformed in each cell and thylakoid membranes had become restacked in the typical ‘staircase’ formation, with plastoglobuli present (Fig. 6b).
Dehydration in the dark resulted in no obvious subcellular
damage in neither restrained nor unrestrained leaves
(Fig. 6c). However, upon rehydration there was general dissolution of subcellular membranes with few organelles
remaining intact (Fig. 6d).
Leaf tissues of X. humilis dried in the light had no subcellular damage and were typical of that previously
reported (Farrant et al. 1999; Farrant 2000). Several smaller
vacuoles (compared to the one central vacuole, typical of
hydrated material) were evident in the cytoplasm and thylakoid membranes were no longer present within chloroplasts (Fig. 7a). When dried in the dark, thylakoid
membranes were only partially dissembled (Fig. 7b) but all
other organelles were intact and resembled those of plants
dried in the light. Upon rehydration, subcellular orgaization was fully recovered in both treatments (Fig. 7c).
DISCUSSION
It is well documented that light energy harnessed by chlorophyll, which cannot be dissipated via photosynthesis as
Figure 6. Subcellular organization of
mesophyll cells of Myrothamnus flabellifolius on dehydration (a) and then rehydration (b) of leaves restrained and dried in
the light. (c) Mesophyll cells of leaves
restrained dried in the dark and (d) following rehydration. Note blistered thylakoid
membranes in chloroplasts [c] and vacuole
[v] with relatively electron dense material
in (a). Thylakoids had reformed and plastoglobuli were evident in chloroplasts in
(b). Numerous small vacuoles and chloroplasts with blistered thylakoids were evident in (c) and plasmalemma damage and
general dissolution of cytoplasm was evident in (d).
occurs under water-limiting conditions, can lead to the formation of oxygen free radicals (Halliwell 1987; Kaiser 1987;
Larson 1988; Smirnoff 1993; Navari-Izzo et al. 1997b). We
have previously proposed that the homoiochlorophyllous
resurrection plants C. wilmsii and M. flabellifolius are able
to minimize such free radical formation, in part, by leaf
folding which minimizes light–chlorophyll interaction in
inner leaves of the former and adaxial surfaces of the latter
(Sherwin & Farrant 1998; Farrant 2000). In the present
study we attempted to test this suggestion by preventing
leaf folding during drying of these species. Such treatment
did indeed lead to loss of viability in restrained leaves of C.
wilmsii but not in the case of M. flabellifolius.
Although not unequivocal, it is likely that light stress
during dehydration contributed significantly to the loss of
viability in restrained leaves of C. wilmsii. All leaves that
were prevented from folding, either the outermost layer or
any of the inner leaves, died when dried in the light.
Restrained leaves dried in the dark did not suffer any more
(or different) damage to unrestrained leaves, so it was
unlikely to be the restaining per se that caused viability loss.
Evidence of light stress in restrained leaves are the
increased number of plastoglobuli in chloroplasts (not usually present in chloroplasts from dry tissue in this species)
(Fig. 5a), reduced chlorophyll and elevated anthocyanin
content of restrained leaves (Fig. 2c). Interestingly,
restrained leaves had either similar, or elevated levels of
putative protectants [anthocyanins (Fig. 2c), sucrose
(Fig. 4) and AP activity (Fig. 3a)] than in plants dried normally in the light. Presumably this protection was insufficient to prevent lethal damage [measured here as increased
electrolyte leakage (Fig. 1a) and loss of subcellular compartmentation (Fig. 5a & c)] and loss of viability. We have
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
1284 J. M. Farrant et al.
a
b
Figure 7. Subcellular organization of
mesophyll cells of Xerophyta humilis dried
in the light (a), in the dark (c) and following rehydration after drying in the dark (c).
Note dismantled thylakoid membranes and
plastoglobuli [p] in chloroplasts [c] and
small vacuoles [v] in (a). In (b), thylakoid
membranes had not totally dismantled. On
rehydration (c) thylakoids reformed and
starch [s] was present.
c
shown using metabolic inhibitors of transcription and translation that this species relies predominantly on establishing
protection during drying, rather than repair on rehydration
(Cooper & Farrant 2002). Although repair can occur, it was
presumably also insufficient to prevent loss of viability
here. Two explainations can be advanced to account for loss
of viability in restrained leaves:
1 Protective mechanisms are only up-regulated in abaxial
cells which normally get exposed to light during drying,
and thus were not (sufficiently) present in adaxial tissues
to adequately protect them from light stress in restrained
leaves. Since whole leaves were sampled for the biochemical studies, we cannot differentiate between the
protection content of abaxial versus adaxial surfaces. In
partial support of this theory, abaxial surfaces of
restrained leaves (light- and dark-dried) were always
noticeably more purple, reflecting increased anthocyanin
content.
2 Outer leaves suffer light stress during normal drying, but
are ‘sacrificed’ in order to protect the inner leaves. In C.
plantigineum, it is reported that outer leaves rarely resurrect and die back after each cycle of drying and rehydration (Norwood et al. 1999, 2000). Usually, in C.
wilmsii, the outer leaves will resurrect for two to three
cycles of drying and rehydration before dying (personal
observation), suggesting some degree of protection and/
or ability to repair damage in these leaves.
We propose that a combination of above explanations is
true. That is, abaxial surfaces have a greater ability to protect and repair against light stress, but these leaves become
increasingly damaged with repeated cycles of drying and
rehydration and are ultimately lost. The advantage of this
strategy of leaf folding is that chloroplast structure and
chlorophyll of inner, shaded leaves can be retained (Sherwin & Farrant 1996, 1998; Farrant 2000) and photosynthesis
can start quickly upon rehydration in inner leaves and
recovery of the plant is rapid (Sherwin & Farrant 1996). In
the poikilochlorophyllous resurrection plants such as X.
humilis, where thylakoids are dismantled and chloropyll is
broken down (Fig. 2g), recovery is far slower (Sherwin &
Farrant 1998; Tuba et al. 1998) but all leaves recover.
In M. flabellifolius, restraining does not cause subcellular
damage and loss of viability of leaves dried in the light. In
this species, leaf folding normally shades only the adaxial
surface of each leaf; our experiments served only to swop
the surface exposed to light during drying. Thus in this
species it appears that both ad- and abaxial cells are able
to protect against light stress. Furthermore, abaxial surfaces, at least, must be able to undergo repeated cycles of
drying and rehydration without accrual of damage such that
leaves are lost. Indeed the degree of protection in leaves of
this species is considerable; AP and GR activity (Fig. 3c &
d), and anthocyanin levels (Fig. 2f) are far higher in M.
flabellifolius than in C. wilmsii or X. humilis. In addition,
thylakoid membranes become unstacked during drying
(Fig. 6a) a feature suggested to put a stasis on photosynthesis and minimize ROS formation (Koonjul et al. 2000; Farrant 2000). Although this strategy slows the rate of recovery
of photosynthesis during rehydration (Sherwin & Farrant
1996; Farrant & Kruger 2001) relative to C. wilmsii, the
trade-off is in retention of all leaves during drying and
rehydration, at least for more cycles of drying and rehydration than observed for C. wilmsii. It is also possible that this
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286
Desiccation and light stress 1285
species has better ability to repair light-induced damage on
rehydration (Farrant & Kruger 2001).
The intriguing aspect of this work is that the homoiochlorophyllous species did not survive drying in the dark,
whereas the poikilochlorophyllous X. humilis did. The
plants were not held in the dark for long periods, so it is
unlikely that death was due to ‘starvation’ [indeed, M. flabellifolius synthesized as much sucrose in the dark as plants
dried in the light (Fig. 4b)]. Many genes, nuclear and chloroplastic, are light regulated (Terzaghi & Cashmore 1995)
and it is possible that at least some of the genes responsible
for laying down of protection against desiccation in
homoichlorophyllous species examined here, require light,
in combination with water deficit, for their transcription/
regulation. Indeed Early Light Inducable Genes (ELIP)
have been shown to be up-regulated in Craterostigma plantigineum (Alamillo & Bartels 2001; Phillips et al. 2002) and
in the resurrection moss Tortula ruralis (Zeng, Chen &
Woods 2002), and are believed to play a role in protection
against photoinhibition during early stages of dehydration.
Since the poikilochlorophyllous X. humilis survived drying
in the dark it is possible that light-induced regulation of
protection mechanisms does not occur in this species. Furthermore, since the major difference between homoiochlorphylly and poikilochlorophylly is the maintenance of
chlorophyll and intact photosystems during drying in the
former, it is tempting to speculate that these might be
important in signal transduction of desiccation-related protection mechanisms in the homoiochlorophyllous types.
Our data suggests that light is necessary for increased
activity of antioxidant enzymes and anthocyanin accumulation in M. flabellifolius but not in C. wilmsii. Although it is
not possible to distinguish here whether light does indeed
act as a signal for up-regulation of these protectants, we
propose that their increased levels in leaves of M. flabellifolius dried in the light is necessary because this species is
afforded less protection against light–chlorophyll interaction by leaf folding than is C. wilmsii. In the latter, all the
inner leaves (> 90% of the plant) are shaded from light,
with only the abaxial surfaces of outer leaves requiring
antioxidant and anthocyanin protection and a light-driven
regulation of such systems may not be as vital.
ACKNOWLEDGMENTS
The authors thank John and Sandy Burrows for the collection of C. wilmsii and Borakalalo National Park for allowing collection of X. humilis. The work was supported by
grants to J.M.F. by the National Research Foundation and
University of Cape Town.
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Received 10 December 2002; received in revised form 7 March 2003;
accepted for publication 13 March 2003
© 2003 Blackwell Publishing Ltd, Plant, Cell and Environment, 26, 1275–1286