Journal of Experimental Botany, Vol. 61, No. 15, pp. 4339–4349, 2010
doi:10.1093/jxb/erq235 Advance Access publication 11 August, 2010
RESEARCH PAPER
UV radiation reduces epidermal cell expansion in leaves of
Arabidopsis thaliana
Kathleen Hectors1,2,*, Eveline Jacques1,*, Els Prinsen1, Yves Guisez2, Jean-Pierre Verbelen1,
Marcel A. K. Jansen3 and Kris Vissenberg1,†
1
Department of Biology, Plant Growth and Development, University of Antwerp, Groenenborgerlaan 171, B-2020 Antwerpen, Belgium
Department of Biology, Molecular Plant Physiology and Biotechnology, University of Antwerp, Groenenborgerlaan 171, B-2020
Antwerpen, Belgium
3
Department of Zoology, Ecology and Plant Science, University College Cork, Distillery Field, North Mall, Cork, Ireland
2
* These authors contributed equally to this work.
To whom Correspondence should be addressed. E-mail: [email protected]
y
Received 22 December 2009; Revised 8 June 2010; Accepted 12 July 2010
Abstract
Plants have evolved a broad spectrum of mechanisms to ensure survival under changing and suboptimal
environmental conditions. Alterations of plant architecture are commonly observed following exposure to abiotic
stressors. The mechanisms behind these environmentally controlled morphogenic traits are, however, poorly
understood. In this report, the effects of a low dose of chronic ultraviolet (UV) radiation on leaf development are
detailed. Arabidopsis rosette leaves exposed for 7, 12, or 19 d to supplemental UV radiation expanded less
compared with non-UV controls. The UV-mediated decrease in leaf expansion is associated with a decrease in
adaxial pavement cell expansion. Elevated UV does not affect the number and shape of adaxial pavement cells, nor
the stomatal index. Cell expansion in young Arabidopsis leaves is asynchronous along a top-to-base gradient
whereas, later in development, cells localized at both the proximal and distal half expand synchronously. The
prominent, UV-mediated inhibition of cell expansion in young leaves comprises effects on the early asynchronous
growing stage. Subsequent cell expansion during the synchronous phase cannot nullify the UV impact established
during the asynchronous phase. The developmental stage of the leaf at the onset of UV treatment determines
whether UV alters cell expansion during the synchronous and/or asynchronous stage. The effect of UV radiation on
adaxial epidermal cell size appears permanent, whereas leaf shape is transiently altered with a reduced length/width
ratio in young leaves. The data show that UV-altered morphogenesis is a temporal- and spatial-dependent process,
implying that common single time point or single leaf zone analyses are inadequate.
Key words: Arabidopsis thaliana, cell expansion, chronic UV treatment, leaf development.
Introduction
Because of their sessile lifestyle, plants are frequently
exposed to suboptimal environmental conditions and this
has resulted in the evolution of a wide range of specialized
adaptations and/or developmental plasticity (Potters et al.,
2007; Granier and Tardieu, 2009). Morphological adjustments to unfavourable environmental conditions have been
documented, for example in response to water deficit
(Aguirrezabal et al., 2006; Lechner et al., 2008), anoxia
(Ramonell et al., 2001), suboptimal nutrient supply
(Assuero et al., 2004), enhanced salt levels (Fricke and
Peters, 2002), or ultraviolet (UV)-B radiation (Jansen,
2002). Light is also a major environmental determinant of
plant morphology. Under light-limiting conditions, dicotyledonous seedlings have long hypocotyls and underdeveloped leaves, while under high irradiance hypocotyls are
short with expanded leaves. The effects of light on
ª The Author [2010]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
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4340 | Hectors et al.
development are controlled through day length, light
quality, or light intensity (Morelli and Ruberti, 2002;
Cookson and Granier, 2006; Cookson et al., 2007).
A small fraction of the solar spectrum consists of highly
energetic UV-B (280–315 nm) radiation. UV-B radiation is
a key environmental signal that regulates diverse processes
in a range of organisms (Jenkins, 2009) including plant
morphology (Jansen, 2002). UV-B-irradiated plants show
typically less elongated leaves, stems, and hypocotyls,
increased branching of stems and roots, as well as thicker
leaves (Jansen, 2002). This UV-B ‘dwarfed’ organismal
phenotype is associated with UV-B-mediated alterations in
cell expansion and possibly cell division. Data on the effects
of UV-B on cellular growth are, however, contradictory and
probably reflect differences in experimental conditions.
Acute, stress-inducing UV-B conditions cause necrosis and
inhibit cell division, while under more ecologically relevant
low doses and/or chronic UV-B treatment, both reductions
and increases in cell expansion and cell division have been
reported (Staxen and Bornman, 1994; Nogués et al., 1998;
Laakso et al., 2000; Hofmann et al., 2001; Hopkins et al.,
2002; Kakani et al., 2003; Rousseau et al., 2004; Wargent
et al., 2009a, b).
Leaf organogenesis is an important feature in plant
development because leaves are essential for photosynthesis
and gas exchange. During leaf development different cell
layers differentiate, including the abaxial and adaxial
epidermis, spongy and palisade mesophyll, and the vascular
system. Cellular development of the epidermis is characterized by the formation of complex puzzle-shaped pavement
cells, stomata, and trichomes (Glover, 2000). The leaf
epidermis is of major importance for controlling plant
growth as epidermal cells both stimulate and restrict growth
of the entire shoot by sending chemical growth signals to
the inner tissues (Scheres, 2007; Savaldi-Goldstein et al.,
2007; Savaldi-Goldstein and Chory, 2008).
Leaf growth in dicotyledonous species is established by
two tightly coordinated processes: cell proliferation and
expansion. Initially there is a uniform proliferation activity
in the young leaf primordium. A proximodistal gradient of
cell expansion then arises, with first expansion at the leaf
top and later in the middle and basal part. Concurrently,
cell division becomes more and more restricted to the leaf
base and ceases when basal cells expand (Donnelly et al.,
1999; Granier and Tardieu, 2009). The regulation of cell
proliferation and expansion determines the shape and size
of the mature leaf and involves the integration of both
external (environmental) and endogenous signals (given by,
for example, phytohormones), resulting in changes in cell
turgor and cell wall extensibility (e.g. Cosgrove, 2005;
Tsukaya, 2006; Wang and Li, 2008; Granier and Tardieu,
2009; Krizek, 2009).
In this report, the influence of UV radiation on leaf
growth and morphology is detailed. In previous work it was
shown that chronic, low doses of UV radiation reduced
expansion of Arabidopsis rosette leaves by up to 25%
(Hectors et al., 2007). This inhibitory effect of UV radiation
on leaf expansion occurs in the absence of photosynthetic
stress and is not linked to induction of typical stressresponsive genes (Hectors et al., 2007). The cellular events
underlying this UV-induced morphogenic response remain,
however, unidentified. As the adaxial epidermis is one of the
most UV-exposed tissues in a plant, the effects of UV on
cell division, cell expansion, cell size distribution, and cell
differentiation of the pavement cells have been examined.
The question was asked whether UV-mediated morphogenesis involves alterations in the proximodistal gradient of
adaxial epidermal cell development during Arabidopsis leaf
growth, a determinant of shape and size of mature leaves.
Materials and methods
Plant material and growth conditions
Arabidopsis thaliana Col-0 seeds were vernalized during 1 week
(at 4 C) and germinated on compost (Tref EGO substrates,
Moerdijk, The Netherlands). One week after germination, seedlings were transferred to AraSystem trays (Betatech, Gent,
Belgium) and grown for one additional week in a growth chamber
(10 h light, 14 h dark), under Cool White (Philips, Eindhoven, The
Netherlands), Fluora (Osram, Munich, Germany), and GRO-LUX
(Sylvania, Denvers, MA, USA) bulbs (1:1:1), with a light intensity
of 60–80 lmol m 2 s 1 (QRT1 Quantitherm light meter, Hansatech, King’s Lynn, UK) and at a temperature of 22 C.
UV exposure conditions
UV radiation effects on the rosette and leaf morphology of
Arabidopsis plants grown in a glasshouse under supplemental UV
have previously been described (Hectors et al., 2007). For this
study, UV exposure conditions were selected that induce a similar
phenotype to that observed in these earlier glasshouse studies.
Two-week-old Arabidopsis rosettes (growth stage 1.04; Boyes
et al., 2001) were exposed to supplemental UV-B radiation using
Philips TL12 tubes (Philips, Eindhoven, The Netherlands) suspended ;70 cm above the plants. UV-C was blocked using one
cellulose acetate filter (95 lm; Kunststoff-Folien-Vertrieb GmbH,
Hamburg, Germany). A digital dimmable ballast (PCA 2/36 T8
EXCEL combined with winDIM V4.0 software; TridonicAtco
GmbH & Co KG, Dornbirn, Austria) was used to regulate the
intensity of the TL12 tubes without changing the UV-B spectrum
[verified
with
an
Ocean
Optics
Spectroradiometer
(USB2000+RAD) (Ocean Optics, Dunedin, FL, USA); data not
shown]. The output of the lamps was set to generate
0.16460.025 W m 2 in the UV-B part of the spectrum and at
plant level. Plants were exposed for 7, 12, or 19 d, receiving 2 h of
UV radiation each day at around noon (except the first day, when
the exposure time was 1 h). The spectral output of the TL12 tubes
was weighted using the biological spectral weighting function
described by Flint and Caldwell (2003). Although a single action
spectrum might not accurately reflect induction of a specific plant
response, a calculated biologically effective daily dose may
facilitate comparison with natural light conditions. The calculated
biologically effective daily dose (280–315 nm) was 0.59 kJ m 2
(76 mW m 2 for 2 h).
TL12 UV-B bulbs also emit a small amount of UV-A radiation,
therefore observed morphological effects were due to either UV-B
or UV-A exposure. Control plants were moved to a compartment
without UV-B bulbs.
Macroscopic morphological analysis
Leaf morphology was analysed after 7, 12, or 19 d of UV
acclimation in 10 different plants. Rosettes were dissected and
leaves were arranged in developmental order. Pictures were taken
UV radiation reduces leaf cell expansion | 4341
(Nikon D50, Tokyo, Japan) and petiole length and leaf blade
length, width, and area were measured using ImageJ software
(Abramoff et al., 2004). Only the leaves with a considerable petiole
(>2 mm) were retained for morphological analysis.
Microscopic morphological analysis
The fifth rosette leaf was stained with propidium iodide (1.5 mM)
for 20 min before images were made with a Nikon C1 confocal
microscope (Tokyo, Japan). Pictures were taken from cells located
at the base, middle, and top region of the adaxial epidermis of leaf
5 at day 7, 12, and 19. Up to 200 cells were analysed per region.
All experiments were performed five times. The area and convexity
of puzzle-shaped pavement cells were measured using Cell^P
software (Olympus, Japan). Cells close to the leaf margin or in the
vicinity of veins and trichomes were excluded from analysis.
The total number of adaxial epidermal pavement cells was
estimated by dividing the total leaf area by the mean area of
pavement cells. To check whether cell division was still present in
the early stages of UV exposure, the cell number of leaf 5 at day 0
was determined five times (100 cells per leaf).
The stomatal index was determined by dividing the number of
stomata by the number of pavement cells in a specific area.
Statistical analysis
SPSS version 16 (SPSS Inc., Chicago, IL, USA) was used for
statistical analysis. UV effects on leaf morphology, stomatal index,
cell numbers, cell size, and cell shape were tested using two-sided
t-tests. Pavement cell sizes and the stomatal index in three different
regions of the leaf (base, middle, and top region) and the cell
number over time were compared using one-way analysis of
variance (ANOVA). Two-way ANOVA was used to test UV
response differences between different regions. Cell area distributions were analysed using whisker box plots.
Results
UV radiation impairs leaf expansion
Arabidopsis plants were grown either in the absence of UV
or under a low dose rate of supplemental UV radiation.
Rosettes developing under low dose rates of UV were
smaller compared with rosettes developing under control
conditions. This effect is clearly visible at each time point
analysed (7, 12, and 19 d of irradiance) (Fig. 1). Rosette
leaves were dissected in order of emergence and morphometric parameters were determined. Plants treated for
7 d with UV radiation showed a significantly reduced
petiole length (–29%), blade length (–15%), maximal
lamina width (–14%), and lamina area (–25%) in leaves 1–4
(Fig. 2A).
Twelve days of UV exposure resulted in a substantially
reduced petiole length (–25%), blade length (–12%), blade
width (–9%), and blade area (–17%) in leaves 1–9 and
a reduced leaf length/width ratio in leaves 6–9. Statistical
significance is indicated in Fig. 2. In the youngest leaves
(leaves 8 and 9) the leaf blade width and area are unaltered
(Fig. 2B).
Nineteen days of UV acclimation showed a substantially
reduced petiole length (–25%), blade length (–16%), blade
width (–13%), and blade area (–25%) in leaves 1–13 (Fig.
2C).
Fig. 1. Effect of UV on Arabidopsis rosette morphology.
Arabidopsis plants grown for 7, 12, or 19 d under control
conditions (upper panel) or chronically UV-supplemented conditions (lower panel). The diameter of the pots is 5 cm.
Strikingly, the length/width ratio of young UV-treated
leaves is significantly reduced. This reduction is, however,
transient. When these leaves grow further, the initial effect
of UV on leaf symmetry disappears (Figure 2A–C).
UV radiation affects the proximodistal gradient of cell
expansion during leaf development
To analyse the effect of UV radiation at the cellular level,
different cellular parameters of the fifth leaf were scored
using confocal microscopy. This leaf was in the primordial
cell proliferation stage when the UV radiation started. Cell
division ceased before the first sampling point (7 d). At day
0, the area, length, and width of leaf 5 are 54 823 lm2,
340 lm, and 194 lm, respectively, on average, with
a length/width ratio of 1.75. The average adaxial epidermal
cell area is the same at the base, middle, and top region
(36 lm2) and the mean cell convexity is 0.92. There are no
stomata formed yet.
The effects of UV on the size of the adaxial pavement
cells in leaf 5 are already detectable after 7 d of UV
treatment (Fig. 3A). The effect on cell size was investigated
in the proximal (base), middle, and distal (top) zones of
a leaf to reflect the proximodistal axis of leaf development.
On day 7, both in control (one-way ANOVAregion;
P <0.001) and in the UV-treated plants (one-way
ANOVAregion; P <0.001), a clear base-to-top gradient of
average cell area can be seen (Fig. 3A). This gradient is
most prominent in the control plants, where cells in the top
region are on average 3.8-fold larger than in the basal
region, whereas in UV-treated plants, the increase in size
from top to base is only 2.9-fold (Fig. 3A). The decrease in
cell size gradient upon UV exposure is due to a significantly
decreased expansion of cells in the top zone compared with
the base (two-way ANOVA; P <0.05). After 7 d of UV
irradiation, the average pavement cell size was reduced by
20% in the top region (t-test; P <0.05). No significant UV
effects were measured on the cell size in the basal (t-test;
P >0.05) or middle zone of the leaf (t-test; P >0.05)
(Fig. 3A).
4342 | Hectors et al.
Fig. 2. Leaf morphological parameters (petiole length, leaf blade length, maximal leaf blade width, blade length/width ratio, and blade
area) of Arabidopsis plants. Plants were grown under standard growth conditions (filled circles) or exposed for 7 (A), 12 (B), or 19 d (C) to
UV radiation (open circles). Leaves 1 and 2 are the first real leaves produced (cotyledons excluded) and leaf 13 is the newest one.
Statistically significant differences in morphology between the UV-irradiated and the non-treated Col-0 plants are indicated by asterisks
(t-tests; *P <0.05; **P <0.01; ***P <0.001). Error bars indicate the standard error of the means of 10 individual plants and might be
smaller than the symbol.
UV radiation reduces leaf cell expansion | 4343
Fig. 3. Pavement cell area in the adaxial epidermis after 7, 12, or 19 d of UV treatment. Cell areas were measured in three different
zones along the proximal–distal axis of the fifth rosette leaf. Black bars represent the non-UV-exposed control leaves, and grey bars the
UV-treated leaves. (A–C) Average cell area after 7 (A), 12 (B), or 19 d (C) of irradiation. Error bars represent the standard error based on
five leaves. In each leaf region, 50–200 cells were measured at every time point. Statistically significant differences in morphology
between the UV-irradiated and the non-treated Arabidopsis plants are indicated by asterisks (t-tests; *P <0.05; **P <0.01; ***P <0.001).
(D–F) Whisker box plots representing the distribution of cell areas after 7 (D), 12 (E), or 19 d (F) of irradiation. Boxes represent the
interquartile distance (IQD) from the first quartile (lower boundary; 25% of the cells have a size smaller than this value) to the third quartile
(upper boundary; 75% of the cell sizes are smaller than this value). The white line in the boxes indicates the median. The bars indicate
the range of cells (minimum and maximum cell size), excluding outliers and extremes (values >1.5 times the IQD).
4344 | Hectors et al.
After 12 d of irradiance, the average size of adaxial
pavement cells is significantly smaller upon UV irradiance,
irrespective of the zonation (i.e. base, middle, and top
region; t-tests; P <0.05). The proximodistal cell size gradient
is absent in both UV-irradiated and control leaves harvested
at day 12 (Fig. 3B) (both one-way ANOVAregion; P >0.05)
and in fully expanded leaves (day 19) (both one-way
ANOVAregion; P >0.05). Yet, the average size of the
pavement cells is significantly reduced in plants treated with
UV for 19 d compared with the control leaves, irrespective
of the cell’s location in the leaf epidermis (t-tests; P <0.05)
(Fig. 3C).
Adaxial pavement cells vary in size at any given location
within the leaf epidermis (e.g. Donnelly et al., 1999;
Cookson and Granier, 2006; Cookson et al., 2007). To
visualize potential variations in cell size distribution that are
associated with the statistically significant changes in
average cell size, data were plotted using whisker box plots
(Fig. 3D–F). Figure 3D–F shows how variation in cell size
increases during leaf expansion in control as well as in
UV-treated leaves.
After 7 d of UV exposure, the 25th, 50th, and 75th
percentile values of control and UV-exposed cells at the leaf
base are comparable, and this confirms the absence of UV
effects on the average cell size (Fig. 3A). The UV-induced
reduction in average cell size at day 7 in the top region
(Fig. 3A) and in all regions at day 12 (Fig. 3B) is reflected
by the lower percentile values in UV-treated leaves in
Fig. 3D and E. Nineteen days after the start of the UV
exposure, the absence of a proximodistal gradient of
average cell size (Fig. 3C) is associated with homogenous
cell size distributions with similar interquartile distances
(distance between the 25th and 75th percentile) in base,
middle, and top zones in control leaves (Fig. 3F, black
bars). This is also the case in UV-treated leaves (Fig. 3F,
grey bars). Yet, the distribution range is shifted to lower
area values in UV-treated leaves, indicating that across the
full cell size distribution range, cells remain smaller under
UV treatment (Fig. 3F).
Cell expansion starts at the top of the leaf and progresses
towards the leaf base. Consistently, it was found that the
proximodistal gradient of adaxial epidermal cell size is most
prominent in the early stage of leaf development (Fig. 3A).
It appears that cell expansion in leaves is biphasic. Between
day 7 and 12, cell expansion rates in top and basal zones are
different. During this period, the area of cells increased
12-fold (control) or 9-fold (UV treated) in the basal region
but only 4-fold in the top zone (in both control and UVtreated cells) (Fig. 3A, B). Between days 12 and 19, cell size
increased 2-fold in all zones (Fig. 3B, C), although the
absolute increase in cell size is 20% smaller in UV-treated
leaves (i.e. control cells increase on average 774 lm2 d 1
and UV-treated cells 619 lm2 d 1). The observed reduction
in cell expansion is thus mainly due to decreased expansion
rates in the early stages of expansion in UV-exposed leaves
(between day 7 and 12). Later expansion rates are comparable between treated and untreated plants, but the extent is
proportional to the initial cell area.
UV radiation does not affect cell numbers and stomata
formation
To examine possible effects of UV on cell division, the total
number of adaxial pavement cells was estimated. At the
start of the UV treatment, leaf 5 consisted of on average
1500 adaxial pavement cells. At day 7 the cell number was
12-fold higher (t-test; P <0.001). During UV treatment the
cell number showed no statistically significant difference
between UV-treated and control plants or between the three
time points (Fig. 4A; t-tests and one-way ANOVAs,
respectively; P >0.05). These data reveal that UV has no
effect on pavement cell division and that cell division in
leaf 5 has ceased before the first sampling point at day 7.
The stomatal index (ratio of the number of stomata to
the number of pavement cells in a specific leaf area) is
identical in base, middle, and top zones at the three time
points, irrespective of UV exposure (one-way ANOVAs and
t-tests, respectively; P >0.05; Fig. 4B–D).
UV radiation does not alter epidermal cell shape
UV radiation effects on pavement cell shape were determined using cell convexity as a quantitative parameter.
Convexity is the measured cell area relative to the area of its
convex hull, whereby more complex shapes have a lower
convexity value. Representatives of the different categories
are shown in Fig. 5A. Epidermal pavement cells were
subdivided into these 11 categories of decreasing convexity
and the average cell size within each category was determined (Fig. 5B–D, H–J, N–P) and the relative abundance
of cells within each category was scored (Fig. 5E–G, K–M,
Q–S). Regardless of the age, location, or even the treatment
of the leaf, the average cell area and the category of
convexity are linked. Lower categories are presented by
smaller cells, indicating a relatively simple polygonal shape
(i.e. high convexity value) and, vice versa, the biggest cells
are scored in the higher categories, reflecting a highly lobed
shape (Fig. 5B–D, H–J, N–P).
After 7 d of irradiation, cells with the highest convexity
values (category 1) are only present in the base and middle
zone of the leaf, whereas the top and middle region also
display cells in higher categories (category 7), regardless of
treatment (Fig. 5B–D). Moreover, a shift in maximum
relative abundance of cells within each category occurs in
a base-to-top manner from category 3 to 5 for UV-exposed
and from category 3 to 6 for control leaves (Fig. 5E–G).
This indicates a proximodistal gradient in cell shape that
mirrors the observed cell size gradient (Fig. 3A, D).
At the second time point, the gradient in cell shape
diminishes in both control and UV conditions, but, after
UV irradiation, more cells remain in lower categories or
display higher convexity values (Fig. 5H–M).
After 19 d, again, cells from UV-treated leaves tend to
exhibit a lower degree of complexity (Fig. 5N–S). Thus,
cells from UV-treated leaves are in general less complex,
and this is associated with a smaller average size rather than
a UV effect on cell shape differentiation per se.
UV radiation reduces leaf cell expansion | 4345
Fig. 4. Number of adaxial pavement cells and the stomatal index.
(A) The total number of adaxial epidermal pavement cells within the
fifth rosette leaf was estimated by dividing the leaf area by the
average cell size. One-way ANOVA and t-test analysis did not
reveal statistically significant differences. (B–D) Stomatal index
(i.e. the number of stomata per pavement cell) after 7 (B), 12 (C), or
19 d (D) of UV treatment. t-Tests did not reveal statistically
significant differences in stomatal index upon UV treatment. Bars
represent the average estimation of five different leaves (black
bars, control plants; grey bars, UV-irradiated plants). Error bars
indicate the standard error of the mean of the five replicates.
Discussion
Chronic UV radiation negatively regulates cell expansion
UV-B radiation is a key environmental signal stimulating
diverse metabolic or developmental responses in plants
(Jansen, 2002; Jenkins, 2009). UV-B irradiance induces
a range of morphogenic alterations, including the inhibition
of hypocotyl, stem, and leaf expansion, stimulation of
axillary branching in roots and shoots, and redirection of
growth along the adaxial–abaxial axes (Jansen, 2002).
In this report, it is shown that rosette leaves of UVtreated Arabidopsis plants remain smaller with a shorter
petiole. As growth results from the formation of cells
followed by their expansion and differentiation, UV effects
could be expected in either process. The present data clearly
indicate that UV treatment did not affect pavement cell
number, cell shape, cell area variation, or stomata formation, but that the reduction in leaf size was solely due to
smaller pavement cells.
The cellular processes underlying UV-B-mediated morphogenic changes are poorly understood. This is - in part due to the fact that leaf growth and development are highly
complex and dynamic processes (Barkoulas et al., 2007;
Wang and Li, 2008; Krizek, 2009). In young Arabidopsis
leaves, adaxial epidermal cell expansion displays a proximodistal gradient from apex to base (Donnelly et al., 1999)
(Fig. 3), which is defined here as the ‘asynchronous’ growth
phase. It is shown that further cell expansion after this
asynchronous phase is synchronously along the proximodistal axis of the leaf (referred to as the ‘synchronous growth
phase’) (Fig. 3A compared with B and C). Inhibitory effects
of UV on cell size are clearly visible during both the
asynchronous expansion and subsequent synchronous phase
in leaf 5. During the latter growth stage, UV decreases the
absolute cell size but does not alter the relative increase as
both UV and control cells increase 2-fold between day 12
and 19. This means that leaves which are acclimated to
chronic UV during the asynchronous growth phase are
modified and UV has no further impact on the synchronous
growth in relative values. However, morphological changes
occur not only in leaves that are initiated during the UV
exposure experiment (e.g. leaf 5) but also in leaves which
had emerged but were not yet mature before UV exposure
started (e.g. leaves 1–4) (Fig. 2). Leaf 1 and 2 had passed
the asynchronous expansion phase (data not shown) when
the exposure period started but still became smaller upon
UV treatment, indicating that the synchronous expansion
phase can also be affected by UV.
The strongest relative reduction in leaf size was noted for
leaves 6–10 (Fig. 2). It can therefore not be excluded that
there is a UV effect on cell division in young apices that are
not yet macroscopically discernible.
The substantial variation in individual cell size is not
affected by UV treatment. The range of cell size distribution
of leaves treated for 19 d with UV resembles that of control
leaves, but is shifted to lower values, which is in accordance
with the decreased average cell size. UV effects on cell
expansion are independent of the location along the
proximodistal axis, and expansion of small and large cells
is proportionally impeded.
Dynamic UV-mediated alterations in growth can also be
seen in leaf shape. Young leaves have a stronger reduction
in expansion along the longitudinal axis compared with the
4346 | Hectors et al.
Fig. 5. UV effects on adaxial pavement cell shape. (A) Overview of the categories of convexity used in (B–S). Two examples of shape are
shown per category, corresponding to the minimum and maximum value of convexity. (B–S) Pavement cell area per category of convexity
after 7 (B–D), 12 (H–J), or 19 d (N–P) of UV treatment in the base (B, H, N), middle (C, I, O), and top (D, J, P) region of the fifth rosette leaf.
The number of cells (%) present in each category after 7 (E–G), 12 (K–M), or 19 d (Q–S) of UV treatment in the base (E, K, Q), middle (F, L,
R), and top (G, M, S) region are shown. Black bars and filled circles represent non-UV-exposed control cells; grey bars and open circles
represent UV-treated cells. In each leaf region, 50–200 cells were measured at every time point. Error bars represent the standard error
based on the means (bars) of five leaves. Statistically significant differences between the UV-irradiated and the non-treated Arabidopsis
plants are indicated by asterisks (t-test; *P <0.05; **P <0.01) for both cell area and number of cells per category of convexity.
UV radiation reduces leaf cell expansion | 4347
transverse axis, leading to a smaller length/width ratio. In
contrast, when these leaves grow older, this difference in
length/width ratio disappears. This implies that UV transiently alters the developmentally regulated growth pattern
in the early stages of leaf growth.
Cellular mechanisms of UV effects
Conflicting observations about the effects of UV-B on
cell division and/or cell expansion have resulted in contradictory hypotheses about the cellular mechanisms underlying UV-B-driven morphogenesis. Several authors conclude
that UV-B-reduced leaf expansion is exclusively due to
UV-B-mediated inhibition of cell division in Rumex
patientia (Dickson and Caldwell, 1978), Vicia faba (Visser
et al., 1997), Pisum sativum (Gonzalez et al., 1998), Lactuca
sativa and Avena sp. (Rousseaux et al., 2004), and
A. thaliana (Lake et al., 2009). Yet, UV was also found to
stimulate cell division in Petunia hybrida (Staxen and
Bornman, 1994). Besides contradictory effects on division,
other authors have concluded that UV-B decreases
cell expansion without affecting cell division in Solanum
lycopersicum [formerly Lycopersicon esculentum (Ballaré
et al., 1995)], Hordeum vulgare (Liu et al., 1995), Liquidambar styraciflua and Pinus taeda (Sullivan et al., 1996), and
L. sativa (Wargent et al., 2009b). It was also described that
in Arabidopsis rosette leaves UV-B increases cell expansion,
while inhibiting the cell division process (Wargent et al.,
2009b). Finally, some authors have concluded that both cell
division and cell expansion are reduced by UV-B treatment
in P. sativum (Nogués et al., 1998), Triticum aestivum
(Hopkins et al., 2002), and Trifolium repens (Hofmann
et al., 2003).
To understand these often contradictory data, it is
important to compare experimental conditions. Species
specificity and morphology are potential determinants of
divergent UV responses. For example, the leaf primordium
and the young developing leaf can either be shielded by
older leaves (e.g. monocots) or directly exposed to UV-B
photons (e.g. Arabidopsis). Furthermore, experimental conditions (field, semi-field, or growth chamber), the presence
or absence of UV-A, the duration of the treatment (long
term or short term), and UV-B intensity (exposure to
ambient or below ambient supplemented UV-B) all determine plant morphological responses. The importance of
the irradiation conditions for UV morphogenic responses is
well illustrated (e.g. Wargent et al., 2009b).
Moreover, the dynamics of leaf growth further complicate
the analysis. It is shown in this study that the behaviour of
cells in the different leaf zones varies in time, resulting in
a biphasic expansion with different responses to UV (e.g. leaf
5). These dynamics of cell development during leaf growth
are often disregarded when studying UV-induced morphogenesis. UV-B morphogenic effects that are dependent on the
stage of leaf development were noted previously in conifers
(Laakso et al., 2000). Only a few studies, for example on
Antarctic species (Ruhland and Day, 2000) and wheat
(Hopkins et al., 2002), reported UV-mediated differences in
epidermis cell area along the proximodistal axis. It is
concluded that single time point, single leaf zone analyses of
UV morphogenesis may not visualize temporal- and spatialdependent UV effects on cell and leaf development.
Potential mechanisms for the UV-mediated decrease in
cell expansion
Several genes that are known to influence cell wall loosening
and cell expansion, such as expansins (Cosgrove, 2000), are
repressed by short-term UV-B exposure (Ulm et al., 2004;
Brown and Jenkins, 2008; Favory et al., 2009) and are
either up- or down-regulated under long-term UV-B
treatment (Hectors et al., 2007). The expression of cell wallloosening
xyloglucan
endotransglucosylase/hydrolaseencoding genes (XTHs; Nishitani and Vissenberg, 2007;
Van Sandt et al., 2007) varies depending on the UV-B
treatment (Ulm et al., 2004; Hectors et al., 2007; Brown and
Jenkins, 2008; Favory et al., 2009). Changes in peroxidase
activity, possibly acting on UV-induced phenolics forming
cross-links inside the cell walls (Fry, 1986; Schopfer, 1996),
could account for the reduced cell expansion. UV-B
radiation stimulates the expression of gene products that
are involved in synthesis of UV-B screening phenylpropanoids (Ulm et al., 2004; Hectors et al., 2007; Brown and
Jenkins, 2008; Favory et al., 2009).
UV radiation also has an impact on the phytohormone
auxin (Hectors et al., 2007), which is a key regulator of cell
division and expansion and plays an important role in
leaf development (Scarpella et al., 2006), leaf initiation
(Kuhlemeier, 2007), and leaf expansion (Ljung et al., 2001;
Keller et al., 2004). Typically, auxin levels are high in leaf
regions with high division activity and lower in areas of cell
expansion, resulting in a proximodistal auxin gradient
throughout young, developing leaves (Ljung et al., 2001).
The future challenge is to highlight the molecular
mechanisms underlying the complex and dynamic process
of UV-B-mediated morphogenic responses.
The present data emphasize the importance of well-chosen
sampling time points and locations within the leaf, during leaf
development, and during the UV-B acclimation process. A
better appreciation of the dynamic character of leaf development and the effect of UV thereupon may well resolve
some of the contradictions that are present in the literature.
Acknowledgements
This work was supported by grants from the Research
Foundation Flanders (FWO; Project G.0382.04N), the
FWO Research Community (W0.038.04N), and the University of Antwerp. The authors also acknowledge the use of the
Cell^P software provided by the department of Veterinary
Sciences (University of Antwerp; Professor Adriaensen).
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