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Carry-over of enhanced ultraviolet-B exposure effects to successive generations of a desert annual: interaction with atmospheric CO2 and nutrient supply

C. F. Musil

Ecology and Conservation, National Botanical Institute, Private Bag X7 Claremont 7735, Cape Town, Republic of South Africa

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G. F. Midgley,

G. F. Midgley

Ecology and Conservation, National Botanical Institute, Private Bag X7 Claremont 7735, Cape Town, Republic of South Africa

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S. J. E. Wand,

S. J. E. Wand

Ecology and Conservation, National Botanical Institute, Private Bag X7 Claremont 7735, Cape Town, Republic of South Africa

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C. F. Musil,

C. F. Musil

Ecology and Conservation, National Botanical Institute, Private Bag X7 Claremont 7735, Cape Town, Republic of South Africa

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G. F. Midgley,

G. F. Midgley

Ecology and Conservation, National Botanical Institute, Private Bag X7 Claremont 7735, Cape Town, Republic of South Africa

Search for more papers by this author

S. J. E. Wand,

S. J. E. Wand

Ecology and Conservation, National Botanical Institute, Private Bag X7 Claremont 7735, Cape Town, Republic of South Africa

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First published: 24 December 2001

https://doi.org/10.1046/j.1365-2486.1999.00236.x

Citations: 29

C.F. Musil, fax + 21/ 7976903, e-mail [email protected]

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Abstract

The performance of fifth generation offspring of a desert annual (Dimorphotheca sinuata DC.) were compared in the absence of UV-B, under variable atmospheric CO₂ and nutrient supply, after four consecutive generations of concurrent exposure of their progenitors to UV-B at ambient (seasonal range: 2.55–8.85 kJ m^–2 d^–1) and enhanced (seasonal range: 4.70–11.41 kJ m^–2 d^–1) levels. Offspring of progenitors grown under elevated UV-B exhibited a diminished photosynthetic rate, a consequence of a reduced leaf density, and diminished foliar levels of carotenoids, polyphenolics and anthocyanins. Conversely, nonstructural carbohydrate and chlorophyll b levels were increased. Altered physiology was accompanied by reduced apical dominance and earlier flowering, features generally considered under photomorphogenic control, increased branching and inflorescence production and greater partitioning of biomass to reproductive structures, but diminished seed production. Many of these changes were magnified under nutrient limitation and intensified under atmospheric CO₂ enriched conditions. The latter disagrees with current opinion that elevated CO₂ may reduce detrimental UV-B effects, at least over the long-term. Observed correlations between seed production and polyphenolic, especially anthocyanin, levels in offspring, and indications of diminished lignification (thinner leaves, less robust stems and fewer lignified seeds set) all pointed to the involvement of the phenylpropanoid pathway in seed formation and plant structural development and its disruption during long-term UV-B exposure. Comparisons with earlier generations revealed trends with cumulative generations of enhanced UV-B exposure of increasing chlorophyll b and nonstructural carbohydrates, decreasing polyphenolics and biomass allocation to vegetative structures, and diminishing seed production despite increasing biomass allocation to reproductive structures. Notwithstanding some physiological compensation (increased chlorophyll b), the accumulation and persistence of these ostensibly inherited changes in physiological and reproductive performance suggest a greater impact of elevated UV-B on vegetation, primary production and regeneration over the long-term than presently envisaged.

Introduction

The changing composition of the earth’s atmosphere is significantly altering the growing environment in which most modern plant species have evolved. Since 1979, there has been a four to five percent decline in mid-latitude stratospheric ozone due to anthropogenic emissions of chlorofluorocarbons and other ozone-destroying trace gases ( Herman & Larko 1994). This has resulted in increased levels of solar ultraviolet-B (UV-B, 280–320 nm) radiation at temperate latitudes ( Kerr & McElroy 1993; Blumthaler et al. 1994 ). Also, global atmospheric CO₂ concentrations have risen as a consequence of fossil-fuel burning and deforestation from 280 μL L^–1 some 150 years ago ( Friedli et al. 1986 ) to current levels of around 360 μL L^–1. These are expected to increase to 700 μL L^–1 between 2030 and 2100 ( Watson et al. 1990 ).

Current understanding is that increases in atmospheric CO₂ may negate or reduce the detrimental effects of elevated UV-B, since there is ample documentation that these two environmental parameters, applied both in isolation and combination, mostly have contrasting effects on plant photosynthesis and growth ( Sullivan 1997). Despite this optimism there are inconsistencies. The magnitude, duration and direction of both UV-B and CO₂ effects are often species specific and modified by the availability of resources, e.g. light, water and nutrients, in the growing environment ( Idso & Idso 1994; Teramura & Sullivan 1994). Also, there is accumulating evidence that photosynthetic stimulation by elevated CO₂ is transient and not fully maintained in the long term (X et al. 1994 ). Notable among plant responses to UV-B is that many occur in the absence of any detectable changes in photosynthetic carbon assimilation ( Beyschlag et al. 1988 ; Ziska et al. 1993 ), chlorophyll fluorescence (Sullivan et alXPP X 1994; Searles et al. 1995 ) and total biomass production ( Barnes et al. 1990 ; Tevini et al. 1991 ). Examples include the inhibition of stem and leaf elongation ( Barnes et al. 1990 ; Ballare et al. 1995b ), altered flowering times ( Tevini & Teramura 1989), branching ( Barnes et al. 1990 ) leaf numbers (Larson et alXPP Barnes et al. 1990 ), leaf thickness ( Cen & Bornman 1993) and flavonoid accumulation ( Beggs & Wellmann 1994). The physiological bases of these photomorphogenic responses is incompletely understood, but may involve various photoreceptors such as phytochrome ( Stapleton 1992), flavins (Ballare et alXPP Searles et al. 1995 ) and auxins ( Ros & Tevini 1995).

Current opinion is that photomorphogenesis induced by UV-B may have a much greater impact on ecosystem structure and composition than direct UV-B damage to the photosynthetic apparatus and genome ( Rozema et al. 1997a ). The rationale is the particular sensitivity of photomorphogenesis to changes in wavelength most affected by stratospheric ozone reduction ( Ensminger 1993), and the low incidence of direct photosynthetic inhibition and biomass reduction observed in field and laboratory studies which have included realistic and balanced levels of visible and UV radiation (X et al. 1994 ; Fiscus & Booker 1995). These rationales are supported by the existence of mechanisms in plants that limit photo-oxidative damage (Foyer et alXPP X 1994), repair damaged DNA ( Britt 1996) and screen sensitive tissues from UV-B damage ( Stapleton & Walbot 1994).

It has been argued that photomorphogenesis induced by UV-B may alter species competitive balance by, for example, differentially affecting species shoot morphology which could lead to changes in canopy structure, light interception and calculated stand photosynthesis ( Barnes et al. 1996 ). Such effects may be pertinent in agricultural communities where harvest yield, quantity and quality are often influenced by interactions with weedy species, but are considered less important in natural communities where competition plays a minor role in maintaining community composition and structure ( Gold & Caldwell 1983). Indeed, in arid ecosystems, the importance, and even existence, of competition among natural plants is questionable ( Fowler 1986). It has been argued that the harsh and unpredictable environmental conditions characterizing arid environments rarely allow densities to increase to levels where competition becomes important ( Shmida et al. 1986 ). Also, in such environments, temporal or spatial refuges, and niche differentiation, may permit coexistence between competitors (Oosthuizen et alXPP Barnes et al. 1996 ).

Direct damage has not yet been eliminated as an important consequence of elevated UV-B. Indeed, the great majority of studies that have examined UV-B effects on plants have been short-term, i.e. conducted over a single growing season or less ( Rozema et al. 1997a ). A few long-term studies, however, have demonstrated that the relatively small deleterious effects induced by UV-B enhancement over the short-term can accumulate into large effects over prolonged periods. In one multiple year field study of loblolly pine, for example, the reduction in plant biomass observed after three successive years of elevated UV-B irradiation was approximately double that after one years irradiation, ( Sullivan & Teramura 1992). Similarly, in another long-term study of the desert annual Dimorphotheca sinuata DC, it was observed that changes in pigments, biomass and fecundity were cumulative over multiple generations of enhanced UV-B exposure. These changes were not readily explained as indirect UV-B effects of photomorphogenic origin and suggested carry-over of UV-B-induced genetic damage to successive generations ( Musil 1996a). These findings were explored further in this study to ascertain whether these observed changes were: (i) persistent or transient photomorphogenic responses, i.e. did they manifest, or were they reversed, in offspring grown in the absence of UV; (ii) ameliorated in an enriched atmospheric CO₂ environment and modified by nutrient supply; and (iii) intensified with cumulative generations of enhanced UV-B exposure.

Methods and materials

Experimental plants and UV-treatments

The derivation of experimental populations and sequence of UV treatments is illustrated in Fig. 1. Essentially, two ancestral D. sinuata populations, designated ambient UV-B group and enhanced UV-B group were established from the same seed source, and then cultured concurrently under ambient and enhanced levels of UV-B for four consecutive generations (growth cycles). In the third and fourth generations, both population groups were exposed to ambient and elevated levels of UV-B, and their differences in performance (residual UV-B effects) tested irrespective of immediate UV-B supply. The findings have been previously described ( Musil 1996a, b). In each generation, a random sample of seeds set by each population group in the preceding generation were sown during mid-winter (July) into 20 × 20 cm pots containing a medium of coarse sand, leaf mould and loam (2:1:1, v:v). Potted plants were irrigated daily with equivalent volumes of water and fertilized at two weekly intervals with 5.8 mg N, 0.8 mg P and 1.7 mg K per kg dry soil. UV treatments lasted over the full species growth cycle and were given over the natural growing period (mid-winter to late spring). In each generation, plants were harvested when the first visible signs of senescence appeared (c. 20 weeks after germination). Seeds set by each population group were dried for two weeks at 30 °C in a forced draft oven, and them stored at room temperature in sealed sample bottles over the 7-month intervals between growing periods.

Schematic representation of experimental design and sequence of treatments.

The first two generations were cultivated in the absence of UV radiation in a polycarbonate-cladded greenhouse (no transmission below 400 nm) where UV-B radiation at ambient and enhanced levels was supplied exclusively from artificial sources. The experimental design comprised 12 plots (mobile tables), each containing 10 plants from either population group. Table positions in the greenhouse and pot positions on each table were randomized weekly. Air temperatures and relative humidities in the greenhouse were moderated with an extractor fan integrated with a wet-wall. Following reports of the importance of UV-A radiation ( Middleton & Teramura 1993), and total photon flux ratios of UV-B:UV-A and UV-B:PFD (X et al. 1994 ) as mitigating factors in plant responses to UV-B, experimental populations were transferred outdoors into an open, natural setting. The experimental design comprised 12 fixed plots, aligned at different bearings, each containing 8 randomized plants from either population group.

Artificial UV radiation in the greenhouse and the outdoor site was provided daily in a step-wise manner over an 8-h period centred on the solar noon (1300 South African Standard Time) by fluorescent sun lamps (Phillips TL/12 40 W UV-B, The Netherlands) suspended above the plots. In the greenhouse, lamps were filtered with 0.075 mm-thick cellulose acetate (transmission down to 290 nm) film (Courtaulds Chemicals, Derby, UK). At the outdoor site, only lamps in six alternate plots were filtered with cellulose acetate film. Lamps in the intervening plots were filtered with 0.12-mm thick Mylar-D (no transmission below 316 nm) film (DuPont De Nemours, Wilmington, Delaware, USA). Filters were replaced weekly to ensure uniformity of UV-B transmission. Spectral irradiances of the filtered lamps were measured after sunset with a monochromator spectroradiometer (IL-1700, International Light Inc., Newburyport, USA), calibrated for absolute responsitivity and checked for wavelength alignment, which was interfaced with a personal computer. Measured irradiances were weighted with the action spectrum for intact plant DNA ( Quaite et al. 1992 ), normalized at 290 nm, and integrated over wavelength to obtain biologically effective UV irradiances as a function of distance from the lamp source. Solar ultraviolet spectral irradiances were recorded daily at 1200 SAST at the oudoor site with the spectroradiometer. PFD and air temperatures were recorded daily in the greenhouse and the outdoor site with tube solarimeters (Delta-T Devices Ltd, Cambridge, UK) and thermistors interfaced with a data logger.

Distances between the lamps and plant apices in greenhouse and outdoor plots were adjusted to provide the two UV-B treatments. Mylar-filtered lamps (ambient UV-B treatment) in the outdoor site were fixed at the same distance above the plant apices as cellulose-filtered lamps to provide similar UV-A dose levels. Lamp height adjustments were made at weekly intervals to accommodate increases in plant height and seasonal variations in UV-B. During the passage of cold fronts, lamps were switched off so artificial UV-B irradiation was supplied mostly under clear-sky conditions. Weighted doses given in the ambient UV-B treatments (seasonal range: 2.55–8.85 kJ m^–2 d^–1) approximated those received over D. sinuata’s natural growing period at this species southerly distribution limit (33°56′S, 18°29′E: Cape Town, South Africa). Weighted doses given in the enhanced UV-B treatments (seasonal range: 4.70–11.41 kJ m^–2 d^–1) simulated those, according to a computerized empirical model ( Bjorn & Murphy 1985), that would be received simultaneously at this species’ northerly distribution limit (26°38′S, 16°18′E: Aus, Namibia) assuming a 20% ozone depletion scenario. This worst-case scenario represented potential ozone depletion in middle of the next century modelled from satellite ozone data trends for the 13-year period 1979–91. More recent predictions anticipate stratospheric chlorine to peak towards the end of this century, with a slow recovery over the next 50 years, and less (c. 11%) ozone depletion at southern mid-latitudes if there is full compliance to the Montreal Protocol and its amendments ( Madronich et al. 1995 ). Comparable weighted doses given by the generalized plant response action spectrum ( Caldwell 1971), normalized at 300 nm, were: (i) ambient UV-B treatment (seasonal range: 1.51–8.54 kJ m^–2 d^–1), and (ii) enhanced UV-B treatment (seasonal range: 4.40–14.10 kJ m^–2 d^–1). Weighted UV-A and UV-B dose levels supplied by natural and artificial sources, PFD, and photon flux ratios of UV-B against UV-A and PFD received by greenhouse and outdoor populations in the two UV-B treatments are summarized in Table 1. The higher photon flux ratios, especially that of UV-B against UV-A, in the greenhouse than the outdoor site did not present an experimental limitation for UV-B group comparisons, since this ratio exhibited equivalent values in the ambient and enhanced UV-B treatments. Potentially heightened UV-B damage due to this elevated ratio should presumably have been the same in the control (ambient UV-B group) and UV-treated (enhanced UV-B group) populations. Modelled and measured solar UV spectral irradiance levels for clear-sky and partly cloudy conditions in Cape Town are illustrated in Fig. 2. Despite instrumental limitations, e.g. sensitivity of the monochromator spectroradiometer to stray light, the adequate correspondence between modelled and measured irradiances indicated that the modelled supplementary UV dose levels given by the sun lamps were not excessive.

Table 1. Weighted UV doses from natural (sun) and artificial (lamps) sources, PFD, photon flux ratios and air temperatures under ambient and enhanced UV-B treatments in greenhouse and outdoor experimental systems. Photon flux ratios are expressed as weighted UV-B and/or UV-A irradiance (mW m–2 s–1) against PFD (mmol m–2 s–1)

GREENHOUSE		OUTDOORS
Seasonal range	Ambient UV-B	Elevated UV-B	Ambient UV-B	Elevated UV-B
UV-A kJ m–2 d–1
Sun	. 0	. 0	1.25–3.42	1.25–3.42
Lamps	0.13–0.45	0.24–0.57	0.11–0.13	0.11–0.13
Total	0.13–0.45	0.24–0.57	1.36–3.55	1.36–3.55
UV-B kJ m–2 d–1
Sun	. 0	. 0	2.55–8.85	2.55–8.85
Lamps	2.55–8.85	4.70–11.41	. 0	2.15–2.56
Total	2.55–8.85	4.70–11.41	2.55–8.85	4.70–11.41
PFD umol m–2 s–1	606–1240	606–1240	877–1795	877–1795
Photon flux ratios
UV-B/UV-A	19.6–19.7	19.6–20.0	5.9–7.1	3.2–3.5
UV-B/PFD	145.8–247.8	269.4–319.6	100.8–171.2	186.1–220.7
UV – B + UV – A/PFD	153.3–260.4	283.1–335.5	154.4–239.8	239.8–289.4
Air temperature °C	18.4–23.9	18.4–23.9	15.2–19.0	15.2–19.0

Biologically weighted solar spectral irradiances measured under clear and partly cloudy skies at 1200 SAST (South African Standard Time) in Cape Town compared with those computed from an empirical model.

Atmospheric CO₂ and nutrient treatments

Fifth generation offspring, established from seeds set by the preceding generation, were grown in a mixture of sterile sand and perlite contained in 10 cm diameter × 50 cm deep pots (1 plant per pot). The experimental design was a split-plot. It comprised eight hexagonal open-top chambers (58 cm in diameter, 50 cm high) constructed from clear polycarbonate mounted on tables in the polycarbonate-cladded greenhouse. Pots were suspended beneath the tables and held in position by plastic collars. Air was drawn into the chambers from outside the greenhouse by fans and circulated in plenums surrounding the chamber bases. Plenum inner walls were perforated to allow equal distribution of air within the chambers. In four alternate chambers, the circulating air was enriched with pure CO₂ to give a CO₂ concentration of 700 μL L^–1. The circulating air in the intervening chambers remained at ambient CO₂ concentration (360 μL L^–1). Elevated CO₂ concentrations were controlled by needle valve flow controllers and calibrated with an infra-red gas analyser (LI-6200, Li-Cor, Lincoln, NE, USA). Each chamber contained 16 randomized plants (8 plants from each population group), which received equivalent volumes of water daily through an automated irrigation system. Four randomly selected individuals from each population group in each chamber were fertilized twice weekly with 150 mL of complete Rorison nutrient solution ( Booth et al. 1993 ), and the remaining plants with a 20% strength Rorison nutrient solution.

Photosynthesis

Gas exchange measurements were performed with a portable photosynthesis system (LI-6400, Lincoln, NE, USA) on mid-axial leaves of 12-week-old plants growing under conditions of high nutrient supply only. The response of net CO₂ assimilation rate to PFD was measured under over a range of PFD (0–2000 μ mol m^–2 s^–1) provided by an adjustable LED light source at a constant leaf temperature of 28 °C. All measurements were carried out under ambient atmospheric CO₂ conditions. These represented examination for acclimation rather than gas exchange changes induced by growth CO₂ conditions. Light response curves were fitted individually with the aid of a nonlinear regression using the mono-molecular function ( Causton & Dale 1990). The fitted curve gave the light-saturated rate of net CO₂ assimilation (A_max). The apparent quantum efficiency (AQE) was derived from the slope of the CO₂ flux between a PFD of 0 and 200 μmol m^–2 s^–1 computed with the aid of an ordinary least-squares regression. Photosynthetic CO₂ response (A:c_i) curves were determined at a saturating PFD of 2000 μmol m^–2 s^–1 using atmospheric CO₂ (c_a) concentrations ranging between 60 and 360 μ L L^–1. The apparent carboxylation efficiency (ACE) was determined as the initial slope of the response of net CO₂ assimilation rate (A) to internal leaf CO₂ concentration (c_i) between 60 and 200 μL L^–1. The stomatal limitation to photosynthesis was estimated from c_i:c_a ratios at a c_a of 360 μL L^–1.

Pigments and metabolites

Photosynthetic pigments, secondary compounds and nonstructural carbohydrates (soluble sugars and starch) were determined in fresh leaves sampled at mid-day from mid-axial regions of 10-week-old plants.

Photosynthetic pigments were extracted in 10 mL of 100% methanol at 2 °C in the dark. Absorbances of filtrates were measured with a spectrophotometer (Beckman DU 640, Beckman Instruments Inc., Fullerton, USA) at specified wavelengths required for computation of chlorophyll a, chlorophyll b, and total carotenoid concentrations from published formulae ( Lichtenthaler 1987) Precipitates were dried at 60 °C in a forced draft oven and weighed. Concentrations were expressed per unit leaf dry mass.

Secondary compounds (total polyphenolics and anthocyanins) were extracted in 10 mL of acidified methanol (79:20:1, v:v, methanol:water:HCl). Absorbances of filtrates were measured at 657 nm and 530 nm, and at 300 nm after appropriate dilution. Precipitates were dried at 60 °C in forced draft oven and weighed. Total polyphenolics (methanol extractable UV-B absorbing compounds) were computed from measured absorbances (A_b) at 300 nm ( Mirecki & Teramura 1984). Anthocyanins were computed from A_b530 nm – 1/3 A_b657 nm ( Lindoo & Caldwell 1978). Absorbance values were expressed per unit leaf dry mass.

Total soluble sugars (sucrose, glucose and fructose) were extracted in two 10 mL volumes of 80% ethanol (80:20, v:v, ethanol:water) and filtrates adjusted to 25 mL in volumetric flasks. For starch determinations, the precipitates were dried at 60 °C in a forced draft oven, weighed, hydrolysed for 3 hs in 5 mL of 3.6% HCl at 100 °C, and filtrates also adjusted to 25 mL in volumetric flasks. After appropriate dilution, soluble sugar and starch concentrations were determined spectrophotometrically according to the method of Buysse & Merckx (1993), and expressed per unit leaf dry mass.

Plant growth and reproduction

Flowering plants within each population group were artificially cross-pollinated and inspected regularly for presence of seed during the course of each experiment, primarily to prevent seed loss from natural dispersal. The effectiveness of cross-pollinations was indeterminate, since D. sinuata does set seed in the absence of pollinators. The degree of inbreeding due to self-pollination or apomixis, a feature of some Asteraceae, was uncertain. Mature seed-bearing inflorescences were removed from plants, dried at 30 °C in a forced draft oven, and stored in sealed, labelled sample bottles.

Plants were harvested from pots when the first visible signs of senescence appeared (c. 20 weeks after germination). Each plant was separated into leaf, stem and reproductive material. Leaf areas were determined with an image analyser (Delta T Devices Ltd, Cambridge, UK). The numbers of stems, buds, immature and seed-bearing inflorescences present on each plant were counted. Seeds were dissected from mature inflorescences, collected during the course of the experiment and at harvest. They were separated into winged disc and lignified ray seed morph fractions, counted and weighed. The remaining reproductive and vegetative material (stems, leaves and roots) were dried separately in a forced draft oven at 60 °C to a constant mass and weighed. Specific leaf mass (SLM) was calculated as the ratio of leaf dry mass to leaf area, leaf area ratios (LAR) as the ratio of total plant leaf area to total plant dry mass. Fruiting success was calculated as the fraction of total inflorescences that developed seed.

Statistical analyses

A 3-way split-plot anova (2-way split-plot anova for gas exchange measurements taken only under high nutrient supply) tested for significant main-plot differences due to atmospheric CO₂ supply and subplot differences due to UV-B group (residual UV-B effects) and nutrient supply, and their interactions. Percentage values were arc-sine transformed prior to statistical analysis. Ordinary least squares regressions examined correlations between ostensibly related parameters. The statistical significances of the correlations were tested with students t-tests on the slopes and intercepts of the regression lines.

Results

Physiology

Light and CO₂ response curves showed an ostensibly diminished net CO₂ assimilation rate in enhanced UV-B group offspring ( Fig. 3). However, an outlier was evident among members of this group in the elevated CO₂ treatment, which transgressed the normal-data distribution requirements for an analysis of variance ( Fig. 3). Elimination of this outlier from the statistical analysis reduced the overall magnitude of reduction in A_max from 27% to 19.3%, but the latter was still significant at P≤ 0.05 ( Table 2). Reduced A_max in enhanced UV-B group offspring was not associated with any significant (P≤ 0.05) changes in stomatal conductance, dark respiration, AQE or ACE ( Table 2). Also, there were no significant (P≤ 0.05) UV-B group–CO₂ interactions for any measured gas exchange parameters, although elevated atmospheric CO₂ did decrease (P≤ 0.05) dark respiration ( Table 2).

Light and CO₂ response curves (leaf area basis) for fifth generation D. sinuata offspring grown under two different levels of atmospheric CO₂ supply. Ambient and enhanced UV-B groups represent offspring of progenitors grown concurrently for four consecutive generations under ambient and elevated levels of UV-B, respectively.

Table 2. Statistics for the main effects of UV-B group (residual UV-B effects), atmospheric CO₂ and nutrient supply and their interactions on the physiology of D. sinuata offspring. d.f. = degrees of freedom. Significant (P 0.05) contrasts presented in bold type

Main effects						Interactions
UV-B Group (Residual UV-B effects)		Atmospheric CO₂		Nutrients		UV-B Group × CO₂			UV-B Group × nutrients			CO₂× nutrients			UV-B Group × CO₂× nutrient
Factor	d.f. F-ratio	% change UV-B Group	d.f. F-ratio	% change CO₂ rise	d.f. F-ratio	% change nutrient decline	d.f. F-ratio	% change UV-B Group Low CO₂	% change UV-B Group High CO₂	d.f. F-ratio	% change UV-B Group Low Nut	% change UV-B Group High Nut	d.f. F-ratio	% change CO₂ rise Low Nut	% change CO₂ rise High Nut	d.f. F-ratio
	1, 14		1, 6				1, 14
Photosynthetic gas exchange
Amax	10.9 *	–19.3	0.9	–4.8	–	–	<0.1	–19.8	–19.6	–	–	–	–	–	–	–
gs	5.7	–25.6	0.8	–18.4	–	–	<0.1	–27.4	–25.9	–	–	–	–	–	–	–
Dark respiration	0.7	10.5	7.3 *	–28.1	–	–	0.9	16.2	–4.0	–	–	–	–	–	–	–
AQE	0.2	–2.5	4.1	–9.3	–	–	<0.1	–4.8	–1.3	–	–	–	–	–	–	–
ACE	0.6	–9.4	< 0.1	–0.9	–	–	0.3	–13.7	–4.3	–	–	–	–	–	–	–
	1, 114		1, 6		1, 114		1, 114			1, 114			1, 114			1, 114
Photosynthetic pigments
Chlorophyll a	0.2	–2.3	89.5 ***	–34.4	4.2 *	–8.3	0.6	0.4	–5.4	0.9	2.0	–7.1	5.4 *	–27.8	–39.7	1.5
Chlorophyll b	20.3 ***	28.3	80.7 ***	–30.9	14.3 ***	–17.9	3.0	41.8	28.8	10.6 **	63.6	7.0	1.9	–26.8	–32.7	0.1
Chlorophyll a/b	51.9 ***	–24.9	6.0 *	–7.7	33.6 ***	25.5	0.3	–23.3	–25.2	15.5 ***	–34.2	–14.4	0.1	–5.3	–10.9	3.0
Carotenoids	11.4 **	–25.5	83.3 ***	–38.0	5.6 *	23.8	0.4	–23.0	–26.2	5.0 *	–37.0	–12.1	1.1	–28.0	–47.4	1.3
Secondary compounds
Polyphenolics	22.7 ***	–20.9	42.6 ***	–26.3	60.2 ***	50.1	0.9	–23.2	–21.6	1.5	–24.9	–19.9	1.0	–22.2	–32.2	0.4
Anthocyanins	25.2 ***	–93.9	<1.1	–16.6	29.2 ***	>1000	0.9	–47.5	–57.4	24.7 ***	–95.5	–9.4	1.4	–33.3	>100	1.0
Non-structural carbohydrates
Souble sugar	13.0 ***	26.4	0.5	3.2	0.6	5.1	0.4	32.8	21.1	2.6	39.4	14.4	2.2	12.9	–5.0	1.4
Starch	6.0 *	17.5	16.7	39.5	4.6 *	–13.1	0.9	14.1	25.3	4.8 *	37.6	1.8	<0.1	42.4	35.9	1.1

* significant at
* P 0.05,
** P 0.01 and
*** P 0.001

Reduced A_max in enhanced UV-B group offspring was accompanied by decreased (P≤ 0.01) foliar carotenoid, polyphenolic and anthocyanin levels, and a reduced chlorophyll a/b ratio, but increased (P≤ 0.05) foliar chlorophyll b, soluble sugar and starch levels ( Table 2, Fig. 4). However, there were no significant (P≥ 0.05) UV-B group–CO₂ interactions ( Table 2), although elevated atmospheric CO₂ reduced (P≤ 0.05) foliar chlorophyll a and b, carotenoid and polyphenolic levels, and the chlorophyll a/b ratio, but increased (P≤ 0.01) foliar starch levels ( Table 2, Fig. 4). In contrast, there were several significant (P≥ 0.05) UV-B group–nutrient interactions ( Table 2). Foliar chlorophyll b and starch levels exhibited proportionately greater increases in enhanced UV-B group offspring under low nutrient conditions. Also, proportionately greater reductions in foliar carotenoid and anthocyanin levels, and chlorophyll a/b ratio, occurred in these offspring under low nutrient conditions ( Table 2). One significant (P≤ 0.05) CO₂–nutrient interaction was evident for foliar chlorophyll a, which showed a proportionately greater reduction in concentration under high nutrient conditions in an enriched atmospheric CO₂ environment ( Table 2).

Foliar pigment and metabolite concentrations in fifth generation D. sinuata offspring grown under two different levels of atmospheric CO₂ and nutrient supply. Ambient and enhanced UV-B groups represent offspring of progenitors grown concurrently for four consecutive generations under ambient and elevated levels of UV-B, respectively. The overall changes (%) due to UV-B exposure history, CO₂ and nutrient supply (*P≤ 0.05, **P≤ 0.01, ***P≤ 0.001) are shown. These and all interaction terms and statistics are detailed in Table 2.

Growth and development

Enhanced UV-B group offspring displayed reduced apical dominance, which was visibly evident in about 20% of all individuals ( Fig. 6). They produced greater (P≤ 0.01) numbers of branches (stems per plant), but their stems and leaves were less (P≤ 0.05) robust (decreased mass per stem and reduced SLM). Also, they accumulated more (P≤ 0.001) biomass into their floral structures ( Table 3, Fig. 5). However, there were no significant (P≥ 0.05) UV-B group–CO₂ interactions, although elevated CO₂ reduced (P≤ 0.01) LAR, but increased (P≤ 0.01) SLM and masses of individual stems. Conversely, there were several significant (P≤ 0.05) UV-B group–nutrient interactions ( Table 3). Indeed, root and leaf fractions of enhanced UV-B group offspring displayed contrasting changes in mass under low (increased masses) and high (decreased masses) nutrient conditions. However, stem numbers and reproductive dry mass in these offspring showed proportionately greater increases under high nutrient conditions. In contrast, masses of individual stems in these offspring exhibited a proportionately greater reduction under high nutrient conditions ( Table 3). Also, masses of individual stems displayed a significant (P≤ 0.05) CO₂–nutrient interaction ( Table 3). These showed a proportionately greater increase under high nutrient conditions in an enriched atmospheric CO₂ environment.

Visibly reduced apical dominance and earlier reproductive development in fourth generation D. sinuata offspring. Plant to the left is an offspring of progenitors grown under elevated UV-B. The plant to the right is an offspring of progenitors grown concurrently under ambient UV-B (Control). These developmental changes persisted in fifth generation offspring re-grown in the absence of UV.

Table 3. Statistics for the main effects of UV-B group (residual UV-B effects), atmospheric CO₂ and nutrient supply and their interactions on the growth of D. sinuata offspring. d.f. = degrees of freedom. Significant (P 0.05) contrasts presented in bold types

Main effects						Interactions
UV-B Group (Residual UV-B effects)		Atmospheric CO₂		Nutrients		UV-B Group × CO₂			UV-B Group × nutrients			CO₂× nutrients			UV-B Group × CO₂× nutrient
Factor	d.f. F-ratio	% change UV-B Group	d.f. F-ratio	% change CO₂ rise	d.f. F-ratio	% change nutrient decline	d.f. F-ratio	% change UV-B Group Low CO₂	% change UV-B Group High CO₂	d.f. F-ratio	% change UV-B Group Low Nut	% change UV-B Group High Nut	d.f. F-ratio	% change CO₂ rise Low Nut	% change CO₂ rise High Nut	d.f. F-ratio
	1, 114		1, 6		1, 114		1, 114			1, 114			1, 114			1, 114
Photosynthetic area
Leaves/plant	0.9	–13.0	2.2	24.9	65.9 ***	–75.2	<0.1	6.3	–9.9	1.5	14.9	–18.6	1.1	20.5	27.1	<0.1
Leaf area	2.8	12.8	0.4	5.1	330.0 ***	–78.8	0.5	21.5	28.7	<0.1	42.9	7.4	0.4	1.8	5.8	0.4
SLM	6.4 *	–10.5	22.7 **	19.7	1.2	–4.6	1.5	–13.6	–4.3	1.6	–2.0	–15.9	1.1	26.2	16.0	8.2 **
LAR	2.8	8.3	18.2 **	–13.4	9.1 **	15.3	0.1	8.3	8.3	<0.1	8.2	8.4	2.4	17.9	–7.3	2.9
Branching
Stems/plant	11.2 ***	38.6	0.6	6.6	175.9 ***	–78.1	0.7	41.0	17.7	8.8 *	12.0	46.8	<0.1	17.4	6.9	0.3
Mass/stem	7.1 **	–23.0	14.1 **	48.9	99.4 ***	–65.3	3.6	–5.5	–22.6	6.2 *	–2.8	–25.3	5.7 *	36.9	51.6	2.7
Biomass accumulation
Root mass	0.1	–2.3	3.7	18.9	172.1 ***	–70.5	0.4	6.3	19.8	4.2 *	38.3	–12.1	2.1	11.4	21.4	<0.1
Stem mass	0.5	–4.7	2.3	14.2	452.8 ***	–84.5	0.1	0.9	15.8	2.5	25.1	–8.4	1.2	23.3	12.7	1.0
Leaf mass	0.5	–4.8	5.5	25.2	364.2 ***	–78.8	<0.1	2.2	19.2	4.9 *	32.6	–11.2	3.4	31.3	23.6	0.8
Floral mass	11.9 ***	31.4	<0.1	1.3	275.7 ***	–79.1	0.3	32.5	27.3	5.7 *	27.3	32.6	<0.1	0.6	2.4	0.4
Total plant mass	0.1	1.4	3.5	15.0	597.3 ***	–80.6	<0.1	7.9	18.7	1.6	29.7	–3.0	2.2	18.7	14.1	0.9

* significant at
* P 0.05,
** P 0.01 and
*** P 0.001

Changes in biomass and fecundity in fifth generation D. sinuata offspring grown under two different levels of atmospheric CO₂ and nutrient supply. Ambient and enhanced UV-B groups represent offspring of progenitors grown concurrently for four consecutive generations under ambient and elevated levels of UV-B, respectively. The overall changes (%) due to UV-B exposure history, CO₂ and nutrient supply (*P≤ 0.05, **P≤ 0.01, ***P≤ 0.001) are shown. These and all interaction terms and statistics are detailed in Tables 3 and 4.

Reproduction

Enhanced UV-B group offspring exhibited earlier reproductive development ( Fig. 6), and produced greater (P≤ 0.01) numbers of inflorescences ( Table 4). However, a smaller (P≤ 0.001) fraction of their inflorescences developed seeds (decreased fruiting success). Also, these offspring set fewer (P≤ 0.05) seeds per inflorescence, and per plant (reduced total seed mass also), of which a smaller (P≤ 0.001) fraction were ray seed morphs of smaller (P≤ 0.001) mass (reduced mass of individual disc seeds also). Elevated CO₂ had no significant effect (P≥ 0.05) on any measured reproductive parameters, although significant (P≤ 0.01) UV-B group–CO₂ interactions were evident for masses of individual seeds. Indeed, masses of both disc and ray seeds set by enhanced UV-B group offspring were proportionately smaller in an enriched atmospheric CO₂ environment ( Table 4). However, several significant (P≤ 0.05) UV-B group–nutrient interactions were evident ( Table 4). A proportionately greater increase in inflorescence production occurred in enhanced UV-B group offspring under high nutrient conditions, and masses of disc seeds set by these offspring were also proportionately smaller under high nutrient conditions. However, masses of ray seeds set by these offspring were proportionately smaller under low nutrient conditions ( Table 4).

Table 4. Statistics for the main effects of UV-B group (residual UV-B effects), atmospheric CO₂ and nutrient supply and their interactions on the reproduction of D. sinuata offspring. d.f. = degrees of freedom. Significant (P 0.05) contrasts presented in bold type

Main effects						Interactions
UV-B Group (Residual UV-B effects)		Atmospheric CO₂		Nutrients		UV-B Group × CO₂			UV-B Group × nutrients			CO₂× nutrients			UV-B Group × CO₂× nutrient
Factor	d.f. F-ratio	% change UV-B Group	d.f. F-ratio	% change CO₂ rise	d.f. F-ratio	% change nutrient decline	d.f. F-ratio	% change UV-B Group Low CO₂	% change UV-B Group High CO₂	d.f. F-ratio	% change UV-B Group Low Nut	% change UV-B Group High Nut	d.f. F-ratio	% change CO₂ rise Low Nut	% change CO₂ rise High Nut	d.f. F-ratio
	1, 114		1, 6		1, 114		1, 114			1, 114			1, 114			1, 114
Flowering and seed production
Inflorescences/plant	9.5 **	42.2	0.2	4.3	131.5 ***	–78.6	1.4	47.5	12.1
Fruiting success	20.4 ***	–60.4	0.3	–8.5	0.5	–12.5	<0.1	–59.5	–51.8
Seeds/inflorescence	15.8 ***	–66.5	2.6	–33.9	3.5	–37.9	0.5	–65.4	–68.9
Seeds/plant	4.9 *	–56.6	1.3	–30.7	14.4 ***	–80.7	1.3	–71.4	–58.6
Ray seed fraction	20.1 ***	–86.0	0.5	–10.9	<0.1	34.1	0.3	–86.3	–76.2	1.4	–92.4	–70.1	<0.1	–41.0	38.3	2.5
Seed mass
Seed mass/plant	5.0 *	–53.5		1.8	–26.3	16.4 ***	–79.6	0.7	–66.8	–62.4	0.4	–88.5	–40.7	0.2	–60.9	–15.6	0.6
Mass/disc seed	7.8 **	–10.0		3.1	9.9	3.7	7.0	6.8 **	–0.4	–16.6	7.0 **	–0.3	–16.7	0.1	9.3	10.2	1.8
Mass/ray seed	12.0 ***	–26.6		0.2	–4.3	4.5 *	–17.2	36.7 ***	25.8	–51.6	27.6 ***	–44.3	18.5	1.7	–11.4	10.1	14.9 ***

* significant at
* P 0.05,
** P 0.01 and
*** P 0.001

Correlations

Significant (P≤ 0.05) correlations were observed between measured seed production parameters (fruiting success, fractions and numbers of ray and disc seed morphs set) and foliar levels of secondary metabolites, especially anthocyanins in offspring ( Table 5). Total numbers of seeds set per inflorescence were also positively correlated (P≤ 0.05) with foliar levels of carotenoids in offspring ( Table 5).

Table 5. Statistics for correlations between foliar pigment composition and fecundity in D. sinuata. d.f. = degrees of freedom. Regressions with significant (P ≤ 0.05) slopes presented in bold type

Factor	Polyphenolics t-statistic (slope)	Anthocyanins t-statistic (slope)	Carotenoids t-statistic (slope)	Error (residual d.f.)
Fruiting success	3.32 **	2.27 *	1.07	1, 69
Total seeds/inflorescence	1.97	0.2	2.02 *	1, 69
Ray seed morph fraction	1.93	2.61 *	1.60	1, 43
Disc seeds/inflorescence	2.84 **	2.85 **	1.19	1, 69
Ray seeds/inflorescence	1.81	2.47 *	1.17	1, 43

* significant at
* P≤ 0.05,
** P≤ 0.01 and
*** P≤ 0.001

Inter-generation comparisons

UV-B group differences (residual UV-B effects) previously reported for third- and fourth-generation offspring ( Musil 1996a, b) are compared with those measured in fifth-generation offspring in Fig. 7. Visible trends with cumulative generations of enhanced UV-B exposure were: (i) increasing foliar levels of chlorophyll b and non structural carbohydrates (soluble sugars and starch), (ii) diminishing foliar levels of polyphenolics, (iii) decreasing magnitude of vegetative biomass reduction associated with increasing allocation of biomass to reproductive structures, and (iv) diminishing fruiting success, seed production and fraction of lignified ray seed morphs set.

Changes in (a) physiology, (b) growth and (c) reproduction of D. sinuata offspring with cumulative generations of enhanced UV-B exposure. Values are a synopsis of those reported in this and two earlier studies ( Musil 1996a, b). They are expressed as a percentage change in offspring of progenitors grown under elevated UV-B (enhanced UV-B group) relative to offspring of progenitors grown concurrently under ambient UV-B (ambient UV-B group). Values beyond the broken lines are significant at P≤ 0.05.

Discussion

Physiology

The measured 19% reduction in A_max in enhanced UV-B group offspring was an unprecedented finding. However, it was not associated with any corresponding changes in AQE, ACE and primary photosynthetic pigments, and there was no evidence of any significant stomatal limitation to CO₂ assimilation (less than 1% change in c_i:c_a ratio). Also, increased foliar starch levels, manifested only in offspring under low nutrient conditions ( Table 2, Fig. 4), so potential feed-back inhibition of photosynthesis by starch accumulation did not apply ( Stitt 1991). However, there did exist a trend of increasing foliar starch levels in offspring with cumulative generations of enhanced UV-B exposure ( Fig. 7). Such progressive starch accumulation could potentially inhibit photosynthesis in future generations through several routes. These include: physical disruption of chloroplast function through formation of large starch granules ( Cave et al. 1981 ), feed-back inhibition of photosynthesis due to reductions in Rubisco activity or concentration, and inorganic phosphate limitation ( Stitt 1991). Expression of A_max on a unit leaf dry mass basis to compensate for the 10.5% reduction in SLM measured in enhanced UV-B group offspring revealed a much smaller (4.2%) decrease in A_max, which was not statistically significant (F_1,14 = 1.31, P≥ 0.05). However, the insignificant 4.8% reduction in A_max measured under enriched atmospheric CO₂ conditions was magnified when expressed on a leaf dry mass basis (19.4% decline), and this was statistically significant (F_1,6 = 8.50, P≤ 0.05). Notable was that this reduction was of similar magnitude to the change in SLM (19.7% increase) measured in offspring under these conditions ( Table 3). It indicated that the measured changes in A_max merely reflected alterations in leaf density (SLM), i.e. the amount of photosynthetic pigments and enzymes per unit leaf area. Similarly, the significant 28.1% reduction in dark respiration measured in offspring under elevated CO₂ was also magnified (38.8% decline) when expressed on a leaf dry mass basis (F_1,6 = 15.02, P≤ 0.01). This suggested acclimation, but the mechanism is unclear, since respiratory repression under elevated growth CO₂ is due to the inhibition of enzymes of the mitochondrial electron transport system, possibly through carbamylation of key enzymes such as succinate dehydrogenase and cytochrome c oxidase ( Drake et al. 1997 ).

The elevated foliar chlorophyll b levels in enhanced UV-B group offspring ( Table 2, Fig. 4), and the observed trend of increasing foliar chlorophyll b levels with cumulative generations of enhanced UV-B exposure ( Fig. 7), suggests some physiological compensation or complementary light control mechanism. A relatively high proportion of chlorophyll b could partly compensate for the greater sensitivity of this pigment to high UV-B irradiance levels, reported in both laboratory (Tevini et alXPP Cave et al. 1981 ) and field studies ( Gonzalez et al. 1993 ). Indeed, wavelengths between 450 and 480 nm are absorbed by chlorophyll b, which would enable more efficient light utilization. This could also compensate for the reduced carotenoid levels measured in enhanced UV-B group offspring, since carotenoids are known to function as accessory light-harvesting pigments absorbing light energy in the 400–500 nm range. However, the decreased foliar carotenoid levels in these offspring did imply increased sensitivity to photo-oxidative damage by UV-B (Rau et alXPP Tevini et al. 1991 ). Indeed one group of carotenoids (xanthophyll cycle components) have been implicated in the photoprotection of photosystems through a mechanism of radiationless dissipation of excessive excitation energy, termed antenna quenching ( Young 1991). The mechanism involves the formation of the xanthophyll, zeaxanthin, through light-induced de-epoxidation of violaxanthin ( Demmig-Adams & Adams 1992). Conspicuous also among enhanced UV-B group offspring were decreased foliar anthocyanin and polyphenolic levels. Also, a trend of decreasing foliar polyphenolic levels was evident in these offspring with cumulative generations of enhanced UV-B exposure ( Fig. 7). This progressive decline in foliar polyphenolic levels could negatively impact on plant UV-B protection and adaptation mechanisms, since polyphenolic derivatives of the phenylpropanoid pathway have important UV-screening and antioxidant properties ( Caldwell et al. 1983 ). Indeed, plant mutants deficient in either the general phenylpropanoid or flavonoid pathway display hypersensitivity to UV-B ( Li et al. 1993 ; Stapleton & Walbot 1994), and increased production of various phenylpropanoid derivitives in response to UV-irradiation have been widely reported ( Alenius et al. 1995 ; van de Staaij et al. 1995 ), and associated with inter- and intra- specific differences in UV-B sensitivity (Day et alXPP X 1994; Rozema et al. 1997b ).

The observed physiological changes in enhanced UV-B group offspring were not ameliorated in an enriched atmospheric CO₂ environment or under low nutrient conditions, as indicated in other studies ( Teramura & Sullivan 1994). In fact, the converse was generally apparent. For example, the reduced foliar carotenoid and anthocyanin levels, and increased foliar starch levels, in enhanced UV-B group offspring were magnified under low nutrient conditions due to a diminished responsiveness by these offspring to nutrient limitation ( Fig. 4). Also, under enriched atmospheric CO₂ conditions, elevated foliar starch levels in these offspring were increased further, a feature commonly observed in many different taxa grown in elevated CO₂ ( Drake et al. 1997 ). Also, decreased foliar polyphenolic levels in these offspring were reduced further in elevated CO₂ ( Fig. 4), which contrasted with expectations of the C/N balance hypothesis (Bryant et alXPP Caldwell et al. 1983 ), and recent reviews ( Penuelas & Estiarte 1998). The latter indicate a general trend of increasing concentrations of carbon-based secondary and structural compounds (CBSSC) in elevated CO₂, although this trend is restricted to only some kinds of CBSSC ( Penuelas & Estiarte 1998). In addition, atmospheric CO₂ enrichment further reduced foliar carotenoid levels in offspring, and also decreased foliar chlorophyll a and b levels ( Fig. 4). Such responses have been reported in several other taxa grown in elevated CO₂ in the absence of UV light (e.g. Oberbauer et al. 1985 ; Houpis et al. 1988 ; Wullschleger et al. 1992 ).

Growth and development

Enhanced UV-B group offspring displayed certain developmental features generally considered reversible photomorphogenic responses. The most clearly visible features were reduced apical dominance, i.e. decreased elongation of the primary axis ( Fig. 6). This is thought to be induced either by the destruction of auxins, such as indoleacetic acid, in apical meristems by UV-B ( Ros & Tevini 1995) or initiated through a UV photoreceptor system involving a flavin chromophore ( Ballare et al. 1995a ). In fact, flavonoids may be involved in the transduction of the light signal that interfere with auxin transport causing elongation inhibition ( Jones et al. 1991 ). However, the persistence of this developmental feature in enhanced UV-B group offspring was irreconcilable with its anticipated reversal under UV-deficient conditions. It suggested a genetic basis to this developmental change, i.e. altered molecular signals resulting from DNA damage ( Beggs & Wellmann 1994). Indeed, it is known that DNA damage by UV does evoke specific cellular responses in bacteria ( Walker 1984), and that the action spectrum for inhibition of hypocotyl elongation by UV does resemble that for DNA damage ( Steinmetz & Wellmann 1986).

Despite a reduced apical dominance, overall biomass production was unaffected in enhanced UV-B group offspring, because reductions in their vegetative biomass were only observed under high nutrient conditions ( Table 3). Notable also was that the measured 11.2% to 12.1% reduction in vegetative (root and leaf) biomass in these offspring under high nutrient conditions was of smaller magnitude than that observed in fourth (19% to 24% decline) and third (27% to 35% decline) generation offspring grown under similar conditions of nutrient supply, but ambient atmospheric CO₂ conditions ( Musil 1996a, b). Indeed, a trend of declining magnitude of vegetative biomass reduction was apparent in offspring with cumulative generations of enhanced UV-B exposure ( Fig. 7). However, this declining trend was an unlikely consequence of greater UV-B damage in earlier generations caused by the considerably elevated UV-B:UV-A ratios in the greenhouse, since both population groups were grown under equivalent UV-B:UV-A ratios in the greenhouse ( Table 1). Also, there was no evidence that the slightly higher temperatures (3–5 °C) in the greenhouse aggravated UV-B damage in earlier generations. Indeed, Mark & Tevini (1996) reported that an increase in temperature of 4 °C ameliorated growth reduction in sunflower and maize seedlings caused by increased UV-B levels. Also, other studies show that organisms with induced or inherent resistance to oxidative stress are often thermotolerant (e.g. Caldwell 1994). Furthermore, there was no indication that the enriched atmospheric CO₂ conditions in this study ameliorated any negative changes in vegetative biomass induced by long-term enhanced UV-B exposure. In fact, there was a poor overall growth response of offspring to elevated CO₂ (only 15% increase in total biomass). This conformed with similar findings in a close relative D. pluvialis ( Wand et al. 1996 ), and other herbaceous species with a ruderal life-history strategy ( Hunt et al. 1991 ), although it did contrast with a positive growth enhancement of about 35% reported in other wild herbaceous C3 species ( Poorter 1993). The only definitive effects of atmospheric CO₂ enrichment were to increase SLM (19.7% increase) and stem robustness (48.9% increase in mass per stem), which may alleviate the risk of leaf injury and breakage of flower-bearing stems in natural situations. It seems that the declining trend in vegetative biomass reduction observed in offspring with cumulative generations of enhanced UV-B exposure reflect progressively altered development, i.e. greater branching and biomass partitioning to reproductive structures.

Reproduction

Another developmental feature of ostensibly photomorphogenic origin clearly evident in enhanced UV-B group offspring was earlier reproductive effort. This feature has also been observed in other species ecotypes from naturally high UV-irradiance environments (Ziska et alXPP Quaite et al. 1992 ), and both observations imply a genetic basis to this response. Earlier reproductive effort was accompanied by increased inflorescence production ( Fig. 5), but reduced synthesis of polyphenolics and anthocyanins ( Fig. 4). This contrasted with the reported stimulation of flower formation by polyphenolic compounds ( Nakanishi et al. 1995 ). Indeed, increased flavonoid synthesis in response to UV-B enhancement ( Chappell & Hahlbrock 1984) may explain several reports of increased floral production in response to elevated UV-B ( Demchik & Day 1996; Wand et al. 1996 ; Grammatikopoulos et al. 1998 ), although disparities do exist. For example, some Brassica species display decreased flowering under elevated UV-B ( Fendheim & Conner 1996).

Several studies also point to an active role of some polyphenolics, especially flavonoids, in pollen metabolism and growth, apart from their function in passive protection or as attractants of pollinating vectors ( Stanley & Linskens 1974). In-vitro stimulation of pollen germination and tube growth by the flavonoids quercetin, isorhamnetin, rutin and isoquercitin have been reported ( Stanley & Linskens 1974). Also, some hypotheses implicate specific flavonoids in pollen tube growth in the pistil, e.g. by their interaction with boron or by their stimulation of functional endogenous levels of indoleacetic acid, which are known to influence pollen tube growth ( Stanley & Linskens 1974). Conspicuous in this study was the large decline in fruiting success (fraction of inflorescences developing seed) and comparable reduction in total numbers of seeds set, expressed on an inflorescence and whole plant basis, by enhanced UV-B group offspring ( Table 4). Indeed, diminished fruiting success and seed production were positively correlated with reduced foliar polyphenolic, especially anthocyanin, levels in offspring ( Table 5). Carotenoids have also been implicated as UV-screening substances, growth stimulators and germination control substances in pollen ( Stanley & Linskens 1974). However, foliar carotenoid levels in offspring were positively correlated only with total numbers of seeds set per inflorescence ( Table 5). Apparently, the role of carotenoids in pollen is mainly as an inhibitor of pollen germination, which is reversed by naturally occurring growth substances such as indoleacetic acid ( Stanley & Linskens 1974).

There are several other derivatives of the phenylpropanoid pathway, e.g. hydroxycinnamic acid derivatives, tannins and lignins, which have been implicated in UV-B protection. Sinapate esters, for example, attenuate UV-B more effectively than flavonoids ( Sheahan 1996), and increased lignification of epidermal cell walls may also protect sensitive underlying tissues from UV-B damage, since UV-B transmission is considerably greater through anticlinal walls than through protoplasts (Day et alXPP Gonzalez et al. 1993 ). In this study, there were also indications of diminished lignification in enhanced UV-B group offspring. Notable were the large reductions in the numbers and masses of heavily lignified ray seeds set by these offspring. These reductions, like those of foliar secondary metabolites, particularly anthocyanins, were of greater magnitude under enriched atmospheric CO₂ and nutrient limiting conditions ( Tables 2 and 4). Furthermore, these offspring produced leaves with less mass per unit area (decreased SLM) and less robust stems (decreased mass per stem), which indicated diminished lignification. All of these relationships pointed to the involvement of the phenylpropanoid pathway in plant structural development and seed formation, and its disruption during long-term UV-B exposure. There are few morphological descriptions of plant mutants deficient in flavonoid biosynthesis, although reduced leaf masses and decreases in thickness of the leaf mesophyll layer have been reported in one barley mutant ( Reuber et al. 1996 ).

Diminished fecundity in enhanced UV-B group offspring was not ameliorated in an enriched atmospheric CO₂ environment. Indeed, the poor responsiveness of the reproductive phase to elevated atmospheric CO₂ corroborate previous findings in a close relative D. pluvialis ( Wand et al. 1996 ), and other species of annuals ( Garbutt & Bazzaz 1984; Garbutt et al. 1990 ). However, like these species and several short-day plants (X et al. 1994 ), the transition to the reproductive phase was slightly delayed under elevated atmospheric CO₂ conditions. Striking, was the extremely low level of fruiting success and total numbers of seeds set by offspring under atmospheric CO₂ enriched and nutrient limiting conditions. Indeed, no heavily lignified ray seeds were set at all by enhanced UV-B group offspring under these conditions ( Fig. 5). This has important ecological implications for D. sinuata, since possession of dimorphic seeds is an important means of ensuring survival in highly variable environments ( Beneke et al. 1993 ). It enables the species to adopt two survival strategies ( Venable & Lawlor 1980), namely escape in space, e.g. winged pericarp in disc seed morphs, or escape in time, e.g. dormancy enforced by the greater mechanical and chemical resistance of heavily lignified pericarps in ray seed morphs ( Beneke et al. 1992 ). The potential elimination of the second survival strategy with future climate change, and the limited out-crossing opportunities due to habitat fragmentation in arid regions, may greatly increase the risk of localized extinction of D. sinuata in a region world-renowned for its spectacular spring-time floral displays ( Lovegrove 1993).

Conclusions

Observed changes in the physiology, development and fecundity of D. sinuata offspring after repeated exposure of their progenitors to elevated UV-B suggest an unlikely UV-B effect of photomorphogenic origin, but conceivable genetic inheritance of UV-B damage. These findings are corroborated by evidence of a progressive alteration in offspring performance with cumulative generations of enhanced UV-B exposure. Also, they are supported by previously published findings of a simple linear dose–response relationship between the number of enhanced UV-B exposure iterations in seed parentage of offspring and their leaf fluctuating asymmetry, an indirect measure of developmental instability indicating genetic damage ( Midgley et al. 1998 ). The apparent intensification of many of the observed changes under enriched atmospheric CO₂ conditions disagrees with current opinion that elevated CO₂ may reduce detrimental UV-B effects, at least over the long-term. Albeit some physiological compensation, e.g. increased chlorophyll b, the accumulation and persistence of these ostensibly inherited changes in physiological and reproductive performance indicate a much greater impact of elevated UV-B on vegetation primary production and regeneration over the long-term than presently envisaged. Such impacts may be particularly pertinent to arid-environment populations isolated by habitat fragmentation with limited out-crossing opportunities.

Acknowledgements

We thank B.J. de Jagger and S. Snyders for assistance with maintenance of CO₂-enrichment systems and plants. Greenhouse facilities were supplied by the Hill Trust administered by the World Wildlife Fund (South Africa)

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Citing Literature

Volume5, Issue3

March 1999

Pages 311-329

Carry-over of enhanced ultraviolet-B exposure effects to successive generations of a desert annual: interaction with atmospheric CO2 and nutrient supply

Abstract

Introduction

Methods and materials

Experimental plants and UV-treatments

Atmospheric CO₂ and nutrient treatments

Photosynthesis

Pigments and metabolites

Plant growth and reproduction

Statistical analyses

Results

Physiology

Growth and development

Reproduction

Correlations

Inter-generation comparisons

Discussion

Physiology

Growth and development

Reproduction

Conclusions

Acknowledgements

References

Citing Literature

Figures

References

Information

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Carry-over of enhanced ultraviolet-B exposure effects to successive generations of a desert annual: interaction with atmospheric CO2 and nutrient supply

Abstract

Introduction

Methods and materials

Experimental plants and UV-treatments

Atmospheric CO2 and nutrient treatments

Photosynthesis

Pigments and metabolites

Plant growth and reproduction

Statistical analyses

Results

Physiology

Growth and development

Reproduction

Correlations

Inter-generation comparisons

Discussion

Physiology

Growth and development

Reproduction

Conclusions

Acknowledgements

References

Citing Literature

Figures

References

Related

Information

Atmospheric CO₂ and nutrient treatments