Plant Physiol. (1998) 117: 173-181
Ultraviolet-B Radiation Effects on Water Relations, Leaf
Development, and Photosynthesis in Droughted Pea Plants1
Salvador Nogués,
Damian J. Allen,
James I.L. Morison, and
Neil R. Baker*
Department of Biological Sciences, University of Essex, Colchester
CO4 3SQ, United Kingdom
 |
ABSTRACT |
The effects of ultraviolet-B (UV-B)
radiation on water relations, leaf development, and gas-exchange
characteristics in pea (Pisum sativum L. cv Meteor)
plants subjected to drought were investigated. Plants grown throughout
their development under a high irradiance of UV-B radiation (0.63 W
m
2) were compared with those grown without UV-B
radiation, and after 12 d one-half of the plants were subjected to
24 d of drought that resulted in mild water stress. UV-B radiation
resulted in a decrease of adaxial stomatal conductance by approximately
65%, increasing stomatal limitation of CO2 uptake by 10 to
15%. However, there was no loss of mesophyll light-saturated
photosynthetic activity. Growth in UV-B radiation resulted in large
reductions of leaf area and plant biomass, which were associated with a
decline in leaf cell numbers and cell division. UV-B radiation also
inhibited epidermal cell expansion of the exposed surface of leaves.
There was an interaction between UV-B radiation and drought treatments: UV-B radiation both delayed and reduced the severity of drought stress
through reductions in plant water-loss rates, stomatal conductance, and
leaf area.
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INTRODUCTION |
Photosynthetic rate and productivity in many plant species can be
reduced by increased exposure to UV-B radiation (Teramura and Ziska,
1996
). To date there is no consensus on the mechanistic basis of
UV-B-induced inhibition of CO2 assimilation in
mature leaves. A reduction in Rubisco activity has been suggested as a
cause of the reduced CO2 assimilation rate in
leaves exposed to increased UV-B radiation. Prolonged exposure to
elevated levels of UV-B radiation has been demonstrated to result in
decreases of both Rubisco activity and content (Strid et al., 1990;
Jordan et al., 1992; Kulandaivelu and Nedunchezhian, 1993). A
primary cause of the decrease in the light-saturated rate of
CO2 assimilation induced by exposure to elevated
UV-B radiation in leaves of oilseed rape has been shown to be a loss of
Rubisco (Allen et al., 1997), which may also be associated with the
loss of activity of other Calvin cycle enzymes (Baker et al., 1997).
UV-B-induced inhibition of photosynthesis has also been attributed to a
reduction in the activity of PSII photochemistry (Fiscus and Booker,
1995). However, UV-B radiation has been shown to inhibit photosynthesis
without an appreciable effect on PSII photochemistry in pea
(Pisum sativum L.; Nogués and
Baker, 1995), oilseed rape (Allen et al., 1997), soybean (Middleton and
Teramura, 1993), rice (Ziska and Teramura, 1992), and algae (Lesser,
1996). Therefore, it would appear that UV-B inhibition of PSII
photochemistry is not a ubiquitous primary effect on photosynthesis. It
is not clear whether changes in stomatal function play a major role in
the UV-B-induced inhibition of photosynthesis. Exposure to UV-B
radiation can modify the speed of stomatal opening and closing and
reduce the rate of leaf transpiration (Negash, 1987; Middleton and
Teramura, 1993; Day and Vogelmann, 1995), although stomatal effects
have not been found to affect photosynthesis in other studies (Murali and Teramura, 1986; Sullivan and Teramura, 1989; Teramura et al., 1991;
Ziska and Teramura, 1992). It is possible that UV-B-induced effects on
stomata could modify plant-water relations and growth but have
negligible effects on leaf photosynthetic capacity.
The majority of the studies of the mechanistic basis of plant responses
to increased UV-B radiation have been conducted under controlled
environmental conditions on plants grown from seed in low or negligible
flux densities of UV-B radiation and then exposed to UV-B radiation.
Such conditions might be expected to increase plant sensitivity to
damage by elevated UV-B radiation (Tevini and Teramura, 1989). In such
experiments, UV-B radiation is generally the only significant
environmental stress, although in natural conditions plants will
experience many other stresses, such as drought, which greatly reduce
plant growth and productivity and may alter the susceptibility to
UV-B-induced damage. Studies of the combined effects of UV-B and water
stress have identified reductions in growth (Teramura et al., 1983,
1984a, 1984b; Tevini et al., 1983; Balakumar et al., 1993), but the
mechanistic bases of such responses have not been
identified.
The objective of this study was to determine the mechanisms by which
UV-B radiation affects water relations, leaf development, and
gas-exchange characteristics in droughted pea plants grown and
developed with UV-B radiation in a greenhouse. To identify the
mechanisms involved in the UV-B response, a high ratio of UV-B to PAR
was used, as is common in such mechanistic studies (He et al., 1993;
Jordan et al., 1994; Liu et al., 1995; Nogués and Baker, 1995;
Jansen et al., 1996; Mackerness et al., 1996; Allen et al., 1997).
Measurements of leaf gas exchange and chlorophyll fluorescence were
made in conjunction with in situ measurements of adaxial and abaxial
gs and estimates of cell frequencies. We demonstrate that long-term growth in UV-B light affects leaf
characteristics, primarily through a reduction in leaf size. UV-B light
was also found to have a direct effect on
gs in the absence of any modification to
photosynthesis.
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MATERIALS AND METHODS |
Pea (Pisum sativum L. cv Meteor) plants were grown from
seed in a mixture of perlite:Levington F2 compost (2:1, v/v) in a greenhouse as described by Nogués and Baker (1995), except that 1.5-dm3 pots (depth of 33.5 cm) were used.
Minimum PPFD during a 16-h photoperiod was maintained at approximately
500 µmol m2 s1 by
supplementary lighting. Temperature and vapor pressure deficit were
maintained at approximately 23/19°C and 1.7/1.3 kPa day/night, respectively. The youngest fully expanded leaves were used for all
measurements unless otherwise stated.
UV-B Radiation and Drought Treatments
After the seeds were sown, pots were placed in a transparent
UV-exposure cabinet within the greenhouse according to the method of
Allen et al. (1997). The UV spectrum at the top of the plants was
measured with a scanning spectroradiometer (model SR 991-PC, Macam
Photometrics, Livingston, UK) and was similar to that previously described (Allen et al., 1997). Greenhouse and cabinet transmission of
UV-A radiation, supplemented by UV fluorescent lamps, ensured that
UV-A exposure was maintained for photorepair and for flavonoid biosynthesis (Teramura and Ziska, 1996). The biologically weighted UV-B
dosages, according to the generalized plant-action spectrum (normalized
to 300 nm; Caldwell, 1971) for the UV-B and control treatments, were
0.63 W m2 (32 kJ m2
d1) and 0.001 W m2,
respectively. The UV-exposure cabinet was divided into four independent
sections (i.e. two without UV-B and two with UV-B radiation),
and these sections were regularly exchanged to minimize any between-section differences other than in the UV-B
treatment.
Plants were watered to saturation on alternate days with Hoagland
solution. After 12 d from sowing one-half of the plants were
subjected to progressive drought by withholding water. Well-watered and
droughted plants were divided equally between all four sections in a
split-plot design. Consequently, plants were subjected to one of four
treatments: (a) without UV-B light and well watered, (b) increased UV-B
light and well watered, (c) without UV-B light and droughted, and (d)
increased UV-B light and droughted. 
w was
measured on alternate days at 8 am using a pressure chamber (PMS Instrument Co., Corvallis, OR), with damp paper at the bottom to
avoid too much evaporation during the measurements. RWC was determined
as: (fresh weight 
dry weight)/(turgid weight dry weight) × 100, where turgid weight is the weight of the leaf after equilibration in distilled water for 24 h. E of the
whole plant was calculated from the change in weight of the pots (water
loss by the soil was minimized by using cellulose film sealed on top of
the pots).
At the end of the experiment (24 d from the beginning of the drought
treatment) plants were harvested and oven dried at 80°C for 2 d,
and analyses of biomass of shoots and roots were carried out. Total
plant leaf area was estimated prior to drying using a flat-bed ScanJet
scanner (model Iicx, Hewlett-Packard) and analyzed with an IMPIXL
image-processing package. Analysis of variance with Bonferroni adjusted
pairwise comparisons was done using SYSTAT software. All proportional
data were subjected to an arcsine square-root transformation prior to
analysis.
Analysis of Leaf Gas Exchange and Fluorescence
Plants were removed from the cabinet for measurements of
CO2 assimilation in at least six attached leaves
per treatment every 3rd d, using an IR gas analyzer (CIRAS-1, PP
Systems, Hitchin, UK) with a PPFD source of 1200 µmol
m2 s1. Measurements
were made between 10 am and 4 pm. Net
CO2 assimilation rate and intercellular
CO2 concentration were calculated according to
the method of von Caemmerer and Farquhar (1981). l, which is the proportionate decrease in light-saturated net
CO2 assimilation attributable to stomata, was
calculated by the method of Farquhar and Sharkey (1982). Estimations of
Vc,max and Jmax
were made by fitting a maximum likelihood regression below and above
the inflexion of the net CO2 assimilation
rate/intercellular CO2 concentration response
using the method of McMurtrie and Wang (1993).
The adaxial and abaxial leaf conductances were measured in situ at
approximately midday every 3rd d, using an automatic transit-time porometer (AP4, Delta-T Devices, Cambridge, UK) and measurements were
taken from at least six leaves per treatment. In the interest of safety
the UV-B fluorescent lamps were switched off immediately prior to
leaf-conductance measurements.
Steady-state-modulated chlorophyll fluorescence of the adaxial surface
of attached leaves was measured using a fluorimeter (PAM-2000, H. Walz
GmbH, Effeltrich, Germany) following the gas-exchange measurements.
Fluorescence signals were analyzed as described by Andrews et al.
(1993) to provide estimates of the
Fv/Fm, measured after 15 min of dark adaptation, and 
PSII
(Genty et al., 1989), measured at a PPFD of 500 µmol
m2 s1, which was
similar to the minimum mean growth PPFD.
Stomata and Cell Frequency
Determinations of stomata and epidermal cell numbers on both sides
of the sixth pair of leaves were made at different stages of
development, 5 to 12 d after the beginning of the drought treatment using nail-polish impressions (Poole et al., 1996). Three stages of
leaf development were studied: (a) stage I corresponds to leaves immediately prior to emergence from stipules, i.e. before leaves were
exposed to any significant UV-B radiation; (b) stage II corresponds to
leaves immediately prior to unfolding, i.e. before the adaxial surface
was exposed to UV-B radiation; and (c) stage III corresponds to maximum
leaf expansion. Plants were examined daily to determine developmental
stage. A systematic sampling strategy was adopted whereby three pairs
of sites were selected at either side of the main vein at the tip, in
the middle, and at the base of the leaves to account for variation
within leaves (Poole et al., 1996). At each site the numbers of fields
of view chosen were sufficient to count more than 100 epidermal cells
and the associated stomata using a microscope linked to a television
monitor via a video camera (Leica). Mean frequencies of epidermal cells
and stomata per leaf were calculated from the average of the six sites.
Chlorophyll a fluorescence was imaged from the outermost
layer of mesophyll (palisade) cells using a standard fluorescence microscope (model DMRX, Leica) with an attached Peltier-cooled charge-coupled device camera (Wright Instruments, Ltd., Middlesex, UK)
as estimate by Oxborough and Baker (1997), to estimate the number of
palisade mesophyll cells in the leaves. The sampling strategy that we
adopted was similar to that described above, except that only one image
was taken per field of view (six sites per leaf).
Pigment Analysis
Water-soluble pigments (flavonoid and anthocyanin) were extracted
from leaves 24 d after the beginning of the drought treatment using the method of Jordan et al. (1994). Four leaves were ground to a
powder in liquid N2 before extraction in 10 mL of
acidified methanol (HCl:methanol, 1:99, v/v). Absorption spectra of the extracts were determined using a Cary 210 spectrophotometer (Varian, Palo Alto, CA), and the flavonoid and anthocyanin contents were estimated from A300 and
A530, respectively
(Mepsted et al., 1996).
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RESULTS |
The changes in plant water status during 24 d of drought
treatment are shown in Figure 1. The
plants were grown throughout their development without UV-B or under
UV-B irradiation. Well-watered leaves had an RWC of about 90% and a
w of approximately 0.5 MPa throughout the
treatment period (data not shown). No major changes in the RWC of
plants irradiated with UV-B light were observed for more than 15 d
of drought, but after 24 d RWC had decreased to about 70% (Fig.
1a). In the non-UV-B-treated plants, RWC decreased from the 7th d of
drought treatment, and after 24 d RWC had decreased by about 20%
to a level similar to that in UV-B-treated leaves. No changes in
w were observed in plants irradiated or not
irradiated with UV-B for 13 d, but subsequently,
w decreased rapidly (from approximately 0.5
to 1.3 MPa over 10 d) in the non-UV-B-treated plants. In the
UV-B-treated plants, w remained at
approximately 0.6 MPa until 17 d from the beginning of the
drought treatment, and then decreased to approximately 1.0 MPa. The
increase in E as the plants grew was substantially smaller
in UV-B-irradiated plants (Fig. 1c).
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| Figure 1.
Changes in the RWC, w, and
E during 24 d of drought treatment. The plants were
grown throughout their development without UV-B radiation ( ) or with
0.63 W m2 UV-B ( ). Leaves of well-watered plants had
an RWC of about 90% and a w of about 0.5 MPa
throughout the treatment period. Measurements began on the fourth pair
and were finished on the eighth pair of fully mature leaves, as
indicated at the top of the figure. Data are the means of six
replicates and the ses are shown when larger than the
symbols.
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The effects of UV-B radiation from sowing and from drought for 24 d on gs are shown in Figure
2. Growth of well-watered pea plants
under UV-B radiation greatly reduced adaxial
gs by approximately 65% (Fig.
2a), but no significant effect was found on abaxial gs (Fig. 2b). Therefore, total
gs (adaxial plus abaxial
gs, Fig. 2c) decreased, and the ratio of adaxial
to total gs (Fig. 2d) was decreased from
approximately 50 to 30% by UV-B radiation. After 24 d of drought
treatment, adaxial gs of
non-UV-B-irradiated plants had decreased by approximately 85%, to a
level similar to that in UV-B-treated plants (Fig. 2e). Drought
decreased abaxial gs in both non- and
UV-B-irradiated plants, but the magnitude of the decrease was greater
in the non-UV-B-treated plants after 24 d (Fig. 2f). As with
well-watered plants, the ratio of adaxial to total
gs was maintained at approximately 50% for
non-UV-B-treated plants, but UV-B radiation reduced it to about 30%
(Fig. 2h).
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| Figure 2.
Changes in the adaxial (a and e), abaxial (b and
f), and total (adaxial plus abaxial; c and g) gs, and the
ratio of adaxial to total gs (d and h)
during 24 d of well-watered (a-d) or droughted (e-h) treatments.
The plants were grown throughout their development without UV-B
radiation () or with 0.63 W m2 UV-B (). Data are
the means of six replicates and the ses are shown when
larger than the symbols.
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To evaluate the effects of UV-B irradiation and drought on
photosynthesis, the changes in the Asat,
Vc,max, Jmax,
and l were measured on attached leaves of plants from all
treatments throughout the droughting period (Fig.
3). There were no significant effects of
UV-B irradiation or drought on the Asat,
Vc,max, or Jmax
(Fig. 3, a-g). The only photosynthetic parameter that was
significantly affected by UV-B was l, which was increased by
about 10 to 15% throughout the measurement period in both well-watered
and droughted plants (Fig. 3, d and h). Drought also increased
l by 10 to 15% after 15 d in plants grown with and
without UV-B (Fig. 3h). Furthermore, at no time during the experiment
was there a significant effect of UV-B or drought on the
Fv/Fm or on the
PSII at a PPFD of 500 µmol
m2 s1.
Fv/Fm and
PSII remained constant at about 0.78 ± 0.03 and 0.52 ± 0.04, respectively, throughout the measurement
period. Imaging of
Fv/Fm and
PSII from leaves from any treatment did not
show any heterogeneity of these PSII quantum efficiencies at the
palisade mesophyll cellular level (data not shown).
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| Figure 3.
Changes in the Asat,
Vc,max, Jmax, and
l during 24 d of well-watered (a-d) or droughted
(e-h) treatments. The plants were grown throughout their development
without UV-B radiation () or with 0.63 W m2 UV-B
(). Leaf temperature was maintained at 25 ± 0.5°C, with 1200 µmol m2 s1 incident PPFD. Data are the
means of six replicates and the ses are shown when larger
than the symbols.
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Table I gives the stomatal frequencies
and indices of the adaxial and abaxial surfaces of the sixth leaf pair
at three stages of development. Stomatal index is the frequency of
stomata expressed as a percentage of the frequency of all epidermal
cells. At the time of sampling, after only 5 to 12 d of drought,
there was no significant effect of this treatment on any of the
parameters studied, because water stress had not yet developed (Fig.
1b); therefore, results from well-watered and droughted plants were pooled. Stomatal development progressed at different rates on adaxial
and abaxial surfaces, with the abaxial stomatal index at stage I being
approximately 7 times that of the adaxial surface (Table I). At this
stage, stomata were poorly differentiated on the adaxial surface,
whereas one-third of the stomata were already differentiated on the
abaxial surface. By stage II the stomatal index was similar on both
surfaces and subsequently showed little change at full leaf expansion
(stage III). At stage II all stomata were differentiated, although cell
frequencies were changing because of cell expansion (only 60-70% of
the cell expansion was completed as judged from epidermal cell
frequencies at stage III). Palisade mesophyll cell frequencies changed
little after stage II. Data from spongy mesophyll cell frequencies are
not presented because of the difficulties in counting accurately the number of cells interspersed with air spaces. At stage II, UV-B irradiation significantly increased the adaxial stomatal frequency and
index by about 14%, and this effect may also have occurred at stage
III. However, no effects of UV-B treatment on the frequencies of
abaxial stomata or other cell types were found at any stage. UV-B
irradiation significantly reduced leaf area at stage II (by 19%) and
stage III (by 39%) as a result of 21 to 39% reductions in the
estimated number of stomatal, epidermal, and palisade mesophyll cells
per leaf (the product of cell frequency and leaf area) at stage III
(Table I). The same effects of UV-B irradiation are evident at stages I
and II, but only the numbers of palisade cells and abaxial stomata at
stage II were significantly affected (Table I).
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Table I.
The effects of 0.63 W m2 UV-B
radiation on the frequencies and number per leaf of stomata and
epidermal and mesophyll (palisade) cells of the sixth leaf pair at
different stages of development
Results from well-watered and droughted plants were pooled, because
after only 5 to 12 d of drought, water stress had not yet
developed, and there were no significant effects of this treatment on
any of the parameters studied. Growth stages I, II, and III correspond
to leaves immediately prior to emergence from stipules (i.e. before
leaves were exposed to direct UV-B), immediately prior to unfolding
(i.e. before the adaxial surface was exposed to direct UV-B), and at
maximum leaf expansion, respectively. Leaf area is also given. Values
are the means ± SE of eight replicate leaves. * indicates
significant difference at P < 0.05 between UV-B treatments (UVB+)
and controls (UVB) at each growth stage.
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The effects of increased UV-B exposure for over 36 d and after
24 d of drought on several plant growth characteristics are shown
in Table II. UV-B exposure alone
significantly reduced plant height, leaf area (by 47%), total dry
weight (by 43%), leaf dry weight, and root dry weight, but did not
significantly affect the number of leaves. Drought alone significantly
reduced plant height, leaf area (by 57%), number of leaves per plant,
specific leaf area, leaf area ratio, total dry weight (by 33%), leaf
dry weight, and plant and soil water content. The combination of UV-B and drought produced an additive effect on most of the parameters studied, although only the decrease in soil water content was significantly different (Table II), confirming that the reduced leaf
area in the UV-B treatment resulted in reduced plant water loss (Fig.
1c) and a slower reduction in soil water content.
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Table II.
The effects of 0.63 W m2 UV-B
exposure from sowing for over 36 d of growth and for 24 d of
drought on several plant-growth and leaf-pigment characteristics
Anthocyanin and flavonoid contents are expressed as absorbance per gram
fresh weight of tissue at 530 and 300 nm, respectively. Leaf area
values include stipules. Values are the means of four replicates and
those in each row with the same letter are not significantly different
according to analysis of variance pairwise comparisons (Bonferroni
adjustment) at P < 0.05.
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Pigments that absorb UV-B radiation strongly are considered to play a
major role in protecting plants from UV-B damage. Flavonoid and
anthocyanin concentrations of plants subjected to UV-B radiation and
drought are given in Table II. Flavonoid concentration was increased 43 and 45% by UV-B radiation and drought, respectively, and the
combination of UV-B radiation and drought produced an additive and
significant increase (112%) compared with the well-watered controls.
Anthocyanin was also significantly increased by UV-B radiation and
drought. Differences in the pigment concentrations with UV-B radiation
or drought treatments were similar whether expressed on the basis of
leaf area (data not shown) or fresh weight.
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DISCUSSION |
Large UV-B-induced reductions in plant biomass, similar to those
seen in Table II, have been widely reported (Teramura et al., 1991;
Barnes et al., 1993; Caldwell et al., 1994; Ålenius et al., 1995;
Mepsted et al., 1996). However, the mechanisms involved in this
response have not been identified. The amount of photosynthesis that
can contribute to biomass is a product of both the area of leaf and the
photosynthetic rate per unit area. Reductions in leaf area have been
recorded for crops as diverse as rice, sunflower, rhubarb, and brussels
sprouts (Teramura and Ziska, 1996), although few studies have made a
distinction between leaf size and leaf number as components of leaf
area. The mechanisms responsible for the reduction of leaf area have so
far received comparatively little attention, particularly in
dicotyledonous plants. In this study leaf number was not significantly
affected, whereas leaf size was (Tables I and II), indicating UV-B
inhibition of either cell division or cell expansion.
UV-B inhibition of cell expansion has been observed in cucumber
cotyledons (Ballaré et al., 1991), tomato hypocotyls
(Ballaré et al., 1995), and wheat (Hopkins, 1997) and barley
leaves (Liu et al., 1995). UV-B irradiation could reduce cell expansion
by changing turgor pressure or cell wall extensibility, and Tevini and
Iwanzik (1986) suggested that direct oxidation of indole acetic acid by
UV-B irradiation reduces cell wall expansion. The effects of UV-B
irradiation on cell size at full expansion were small, at least in the
sixth leaf, with cell frequencies largely unchanged in the mesophyll
and lower epidermis (Table I). Stomatal frequency did increase in the
upper surface in response to UV-B radiation without changes in stomatal
index, which indicates a small (although not statistically significant
at P = 0.05) reduction in cell expansion (Table I). This may be
the cause of the typical slight curling of the leaf surface, which is
often seen under high UV-B irradiances (Teramura and Ziska, 1996).
However, it is clear that in the palisade mesophyll and both epidermal
layers the largest contributor to the 39% reduction in leaf size was
fewer cells per leaf (26-38% lower than control plants) rather than
smaller cells (2-13% reduction in size, Table I). These results show
that the primary cause of the reduced individual area at all three
stages of the development of the sixth leaf was UV-B-induced inhibition
of cell division. This observation was confirmed by the overall
approximately 25% reduction in mean leaf size in either well-watered
or droughted plants at the end of the experiment (Table II).
UV-B-induced inhibition of cell division has been reported in cucumber
cotyledons (Tevini and Iwanzik, 1986), petunia leaf protoplasts
(Staxén et al., 1993), and parsley (Logemann et al., 1995) and
wheat leaves (Hopkins, 1997). Repair of UV-B damage to DNA before
replication and direct UV-B-induced oxidation of tubulin, delaying
microtubule formation, have been suggested as mechanisms for direct
slowing of cell division (Staxén et al., 1993). UV-B radiation
may also affect the key stages of cell division through transcriptional
repression of the genes encoding for a mitotic cyclin and a
p34cdc2 protein kinase (Logemann et al., 1995).
Reductions in leaf area and cell division in all measured cell types
were observed at all three stages of leaf development, with the effects
becoming more pronounced approaching full expansion (Table I). This
indicates that it is unlikely that UV-B radiation acts directly on the
dividing cells because leaves at stage I were still enclosed by the
folded bracts and, therefore, would have experienced a very low UV-B radiation exposure.
Indirect UV-B effects on leaf area could be a result of reduced
photosynthate supply; however, we found no effects on
Asat (Fig. 3). The lack of effect of UV-B
radiation on Asat and other photosynthetic
parameters contrasts with reports in which short-term exposure of
mature leaves to similar UV-B irradiances in pea (Nogués and
Baker, 1995) and oilseed rape (Allen et al., 1997) resulted in a rapid
loss of photosynthetic competence primarily through effects on Rubisco.
Clearly, when plants develop from seed, even under these high UV-B
doses, they are protected from the loss of photosynthetic ability. A
likely protective mechanism is the 43% increase in flavonoid content
found here in UV-B-treated plants (Table II), as has been observed
before in peas (Strid and Porra, 1992; Jordan et al., 1994) and many
other species. Whereas Asat was not
significantly affected by UV-B radiation throughout plant development
(Fig. 3a), UV-B radiation did increase stomatal limitation by about
10% (Fig. 3d). This differs from reports that UV-B radiation can
inhibit photosynthesis without changes in stomatal function (Ziska et
al., 1992; Middleton and Teramura, 1993). The absence of a reduction in
Asat in association with the increase in
stomatal limitation (Fig. 3, a and d) suggests that there may have been a biochemical compensation within the photosynthetic apparatus. There
is no evidence that this involved Rubisco, because there was no UV-B
effect on Vc,max at any stage of plant
development (Fig. 3b). However, there was a small but consistent
increase in Jmax, indicating an increase in
the rate of ribulose 1,5-bisphosphate regeneration in UV-B-treated
plants (Fig. 3c). Given the complexity of the feedback in the control
of photosynthesis, it is not possible from these gas-exchange data to
identify the underlying mechanism for such a compensation. In addition,
it is possible that in situ photosynthetic rates may have been affected
by the UV-B radiation, even if the effects were not detected by the
Asat assessment.
In situ measurements throughout the growth of the plants revealed an
approximately 65% inhibition of adaxial gs
by UV-B radiation, with no effect on the abaxial surface (Fig. 2, a and
b), which reduced total gs by approximately
30% (Fig. 2c). This inhibitory effect on
gs was clearly through changes in stomatal
aperture, since stomatal frequency was either unchanged or increased
(Table I). The adaxial stomata are the cells that are most exposed to UV-B radiation, because they are not screened by flavonoids in the
epidermal layer. The reduction in stomatal aperture must be a direct
response to UV-B radiation that was not mediated through photosynthesis, because this was unchanged and
ci was slightly (1.5-2.0 Pa) lower in
UV-B-treated plants than in the controls. Furthermore, the in situ
inhibition of gs persisted when plants were
temporarily removed from the UV-B cabinet for assessment of stomatal
limitation. Wright and Murphy (1982) have shown that UV-B radiation can
induce stomatal closure directly by inhibiting K+
accumulation by guard cells, and the mechanisms underlying the stomatal
effect of UV-B warrant further investigation.
The drought treatment used here resulted in water stress developing
slowly, with the first effects on RWC evident only after 8 d and
on other parameters after 13 to 15 d (Figs. 1 and 2). Photosynthetic competence was maintained throughout this mild drought
experiment (when RWC decreased to about 70% and
w decreased to about 1.3 MPa) with no change
in Asat,
Vc,max, or Jmax
being observed (Fig. 3, e-g). This supports previous results (Cornic, 1994) that mild water stress does not result in inhibition of photosynthetic capacity. However, the drought treatment was sufficient to result in 33 and 57% reductions in plant biomass and leaf area, respectively, at the end of the experiment (Table II).
Field (Teramura et al., 1990; Petropoulou et al., 1995) and controlled
environment studies (Balakumar et al., 1993) have suggested an
interaction between UV-B irradiation and water stress, and this was
confirmed in this study with the reduction in final biomass due to UV-B
irradiation being only 23% in the droughted plants compared with 43%
in the well-watered ones (Table II). As discussed above, UV-B radiation
exposure reduced both leaf area and gs. Under drought these effects reduced the total plant transpiration rate
(Fig. 1c) and therefore slowed soil drying. At the final harvest this
difference in plant transpiration rate was fully accounted for by the
reduced leaf area, with daily transpiration per unit leaf area being
very similar (2.3 and 2.5 g cm2
d1 for droughted controls and UV-B plants,
respectively), even though UV-B plants had a slightly higher soil water
content (Table II). The slower soil drying under UV-B irradiation
resulted in a delay in the development of drought stress with declines
in leaf RWC, w (Fig. 1, a and b), and
gs (Fig. 2, e-g) occurring 5 to 12 d after those observed in the droughted controls. It is tempting to
conclude that drought reduced the effect of UV-B irradiation on biomass
accumulation, because UV-B irradiation had little effect in droughted
conditions (only a 10% decline) compared with a 33% decline in
biomass in well-watered conditions (Table II). However, in the
droughted plants the soil water status in control and UV-B treatments
was not the same; therefore, such a conclusion should not be drawn.
Water stress can induce accumulation of UV-B-absorbing compounds
(Murali and Teramura, 1986), which is likely to offer some increased
protection from UV-B. In this study both UV-B and drought
increased the concentration of flavonoids when imposed alone and had a
synergistic effect when imposed together (UV-B radiation increased
flavonoid concentration by 43%, drought by 45%, and UV-B radiation
with drought by 112%; Table II).
Summary
This study has shown that irradiation with UV-B resulted
in a decrease in adaxial gs by about 65%,
increasing stomatal limitation of CO2 uptake by
10 to 15%. There was no loss of mesophyll photosynthetic activities
with UV-B irradiation. The reductions in leaf area and plant biomass
under long-term exposure to UV-B irradiation were associated with a
decline in leaf cell numbers and cell division. There was an
interaction between UV-B radiation and drought treatments: UV-B
radiation delayed and reduced the severity of drought stress through a
reduction in plant water-loss rates and through reductions in
gs and leaf area.
| |
FOOTNOTES |
1
This research was supported by a research grant
to N.R.B. from the UK Natural Environment Research Council and to S.N.
from the Generalitat de Catalunya (1996BEAI300222). D.J.A. was the recipient of a research studentship from the UK Biotechnology and
Biological Sciences Research Council.
*
Corresponding author; e-mail baken{at}essex.ac.uk; fax
44-1206-873416.
Received November 5, 1997;
accepted February 2, 1998.
| |
ABBREVIATIONS |
Abbreviations:
Asat, light-saturated
net CO2 assimilation rate.
E, daily
evaporation rate.
Fv/Fm, ratio of
variable to maximal fluorescence yield.
gs, stomatal conductance.
Jmax, maximum
potential rate of electron transport contributing to ribulose
1,5-bisphosphate regeneration .
l, stomatal limitation to
Asat.
PSII, relative quantum
efficiency of PSII photochemistry.
w, leaf water
potential.
RWC, relative leaf water contentVc,.
max, maximum carboxylation velocity of
Rubisco.
| |
ACKNOWLEDGMENTS |
We are grateful to Andy McLeod (Institute of
Terrestrial Ecology, Monks Wood) for the use of a spectroradiometer and
to Kevin Oxborough for assistance with the fluorescence imaging system.
| |
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Top
Abstract
Introduction
