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.

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 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 stress-responsive 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 μmol 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 μm; 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 0.164±0.025 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 (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).

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.

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.

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.

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.

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).

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 μm2, 340 μm, and 194 μm, 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 μm2) 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).

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).

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).

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 UV-treated 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 μm2 d−1 and UV-treated cells 619 μm2 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.

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.

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.

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).

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.

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.

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.

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 UV-treated 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 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 spatial-dependent 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 wall-loosening xyloglucan endotransglucosylase/hydrolase-encoding 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.

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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Author notes

*
These authors contributed equally to this work.

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