Journal of Experimental Botany, Vol. 54, No. 388, pp. 1771-1784,
July 1, 2003
© 2003 Oxford University Press
Response of purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea to ultraviolet-B radiation
Received 23 October 2002; Accepted 11 April 2003
1 Botany Department, University of Cape Town, Private Bag Rhondebosch 7701, South Africa
2 National Botanical Institute, private bag X7, Claremont 7735, Cape Town, South Africa
3 Research Development, Cape Technikon, Room 2.800 Admin. Building Keizersgratch, PO Box 652, Cape Town 8000, South Africa
* To whom correspondence should be addressed. Fax: +27 21 460 3887. E-mail: dakora{at}ctech.ac.za
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Abstract |
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The effects of elevated UV-B radiation on growth, symbiotic function and concentration of metabolites were assessed in purely symbiotic and NO3-fed nodulated plants of Lupinus luteus and Vicia atropurpurea grown outdoors either on tables under supplemental UV-B radiation or in chambers covered with different types of plexi-glass to attenuate solar ultraviolet radiation. Moderately and highly elevated UV-B exposures simulating 15% and 25% ozone depletion as well as sub- ambient UV-B did not alter organ growth, plant total dry matter and N content per plant in both L. luteus and V. atropurpurea. In contrast, elevated UV-B increased (P <0.05) flavonoid and anthocyanin concentrations in roots and leaves of L. luteus, but not of V. atropurpurea. Feeding nodulated plants of L. luteus under elevated UV-B radiation with 2 mM NO3 increased (P <0.05) nodule, leaf and total dry matter, and whole plant N content. With V. atropurpurea, NO3 reduced (P <0.05) nodule activity, root %N and concentrations of flavonoids, anthocyanins in roots and leaves and soluble sugars in roots, in contrast to an observed increase (P <0.05) in nodule dry matter per plant. Similarly, supplying 2 mM NO3 to L. luteus plants exposed to sub-ambient UV-B radiation significantly reduced individual organ growth, plant total biomass, nodule dry matter, nodule %N, and whole plant N content, as well as root concentrations of flavonoids, anthocyanins, soluble sugars, and starch of L. luteus, but not V. atropurpurea plants. These results show no adverse effect of elevated UV-B radiation on growth and symbiotic function of L. luteus and V. atropurpurea plants. However, NO3 supply promoted growth in L. luteus plants exposed to the highly elevated UV-B radiation.
Key words: Anthocyanins, flavonoids, Lupinus luteus, NO3-feeding, nodulation and N2 fixation, soluble sugars, starch, ultraviolet radiation, Vicia atropurpurea.
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Introduction |
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Available data indicate that the ultraviolet-B (UV-B, 290–315) radiation reaching the earth’s surface has increased (Madronich et al., 1998) as a result of depletion of stratospheric ozone, the principal attenuator of UV-B (McKenzie et al., 1999). Ultraviolet-B radiation is damaging to living cells (Mackie and Rycroft, 1988) and, consequently, plants could be at risk with their dependency on solar radiation for photosynthesis. Several reports have shown that UV-B radiation can depress photosynthesis, reduce plant growth and reproduction, and alter species competitive interactions (Teramura and Sullivan, 1994; Rozema et al., 1997; Jansen et al., 1998). However, plant growth and photosynthetic response to elevated UV-B radiation can be species-specific (Jansen et al., 1998; Yuan et al., 2000) and is also modified by environmental and nutritional factors (Murali and Teramura, 1985; Correia et al., 2000). As a result, extrapolation of data between, or even within, species can be difficult.
Numerous studies have demonstrated that the accumulation of flavonoids and anthocyanins by plants provide a defence mechanism against UV-B radiation (Rozema et al., 1997; Bieza and Lois, 2001). However, these molecules also serve as plant signals to symbiotic bacteria in the Rhizobiaceae (Dakora and Phillips, 1996; Phillips, 2000), and their accumulation in root tissues has been shown to promote nodule formation (Muofhe and Dakora, 1999). Thus, an increase in concentration of flavonoids and/or anthocyanins concentration in roots of plants exposed to UV-B radiation could potentially stimulate nodulation and N fixation in legumes. But the exposure of plants to elevated UV-B has also been shown to reduce photosynthesis (Teramura and Sullivan, 1994) and to alter the allocation of biomass to different plant organs (Adamse and Britz, 1992) of sensitive species. The net result was a higher shoot/root ratio in plants exposed to UV-B relative to those receiving no UV-B radiation (Tosserams et al., 1996). An increased allocation of biomass to the shoot would imply low carbohydrate supply to the roots and nodules, with a consequent decrease in the release of root exudate compounds into the rhizosphere. This, in turn, could affect nodulation and N2 fixation in the test species.
The sensitivity of plants to UV-B radiation is reported to be affected by the N concentration of the growth medium. This is because a marked effect was observed on N-fertilized, but not N-depleted maize and cucumber plants following exposure to elevated UV-B (Hunt and McNeil, 1998; Correia et al., 2000). Under natural field conditions, legume N nutrition is met by atmospheric N2 fixation and soil N uptake. Although N fertilization is rarely practised due to the N inhibition of nodulation and N2 fixation, legumes grown on N-poor soils can become N-limited before the onset of N2 fixation (Ma et al., 1997). Thus supplementing legumes with mineral N can be beneficial for plant growth and yields. Plant response to environmental stress can also differ depending on their source of nutrients. In one study, pea plants relying entirely on N2-fixation were more tolerant of salt stress compared to their N-fertilized counterparts (Del Pilar Cordovilla et al., 1999). It remains to be known whether legume species depending entirely on symbiotic N2 fixation would be more tolerant, or susceptible to elevated UV-B relative to their N-fertilized counterparts. The aim of this study was to assess the effects of UV-B radiation on growth, symbiotic function and concentration of metabolites of Lupinus luteus and Vicia atropurpurea plants either depending entirely on symbiotic N2 fixation for their nutrition or fed 2 mM NO3.
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Plant species and experimental treatments
Lupinus luteus and Vicia atropurpurea were used in this study because they are important fodder and cover crops grown in the Western Cape of South Africa. Seeds of L. luteus and V. atropurpurea were sown into 20 cm high x 20 cm diameter pots (4 seeds per pot) containing sand. Seeds of Lupinus luteus were inoculated with Bradyrhizobium lupini and V. atropurpurea with Rhizobium leguminosarum biovar viciae, and germinated under different UV-B treatments. There were 12 separate tables with banks of fluorescent sun lamps (Phillips TL/12 40W UV-B, The Netherlands) and 12 chambers (1.5 m2 square x 0.75 m high) constructed of differentially UV-transmitting clear perspex (300 mm thick) inter-dispersed in an open area in the Kirstenbosch National Botanical Gardens, Cape Town (36°56' S, 18°29' E). Each table or chamber had four pots with plants of each test species. For N treatment, two of the four pots received 2 mM NO3 while plants in the other two pots relied entirely on symbiotic N2 fixation for their N nutrition. All pots were irrigated with equal volumes of water, and the seedlings later thinned out to two plants per pot. Beginning with seedling emergence, 400 ml of half-strength N-free Hoagland’s nutrient solution (Hewitt, 1966) was supplied twice weekly to the purely symbiotic plants and the same volume made up to 2 mM NO3 applied to N-fed plants. To avoid accumulation of nutrients in the growth media, the sand was flushed once every week with water.
Supra-ambient UV-B treatments
There were two elevated UV-B treatments and one control each replicated four times. In the ambient UV-B control (UV-B100), lamps in the four alternating banks were filtered with a 0.12 mm thick Mylar-D film (transmission down to 316 nm). In the moderately (UV-B137) and highly (UV-B173) supra-ambient treatments, lamps in intervening banks were filtered with 0.075 mm thick cellulose acetate (transmission down to 290 nm) film (Courtaulds Chemicals, Derby, UK). All filters were replaced weekly to ensure uniformity of UV transmission. Artificial UV-B radiation was supplied daily for an 8 h period. Irradiation was graduated with two-thirds of the total daily UV-B supplement spread over a 4 h photoperiod centred on the solar noon. The remaining one-third was applied equally over the two 2 h early morning and late afternoon photoperiods. This was achieved by switching on fewer lamps in each bank during these photoperiods. This step-wise application of the supplemental UV-B was followed in order to account for alterations in ambient UV-B irradiance intensity due to diurnal changes in solar zenith angle.
The spectral irradiances of filtered lamps were measured after sunset with a computer-interfaced monochromator spectroradiometer (IL-1700, International Light Inc., Newburyport, USA), calibrated for absolute response and checked for wavelength alignment. Measured irradiances were weighted with the generalized plant response action spectrum (Caldwell, 1971), as mathematically formulated by Green et al. (1974), which was normalized at 300 nm. Weighted irradiances were integrated over wavelength and expressed as a function of distance from the lamp source. Distances between cellulose-acetate filtered lamps and median height of plants in each bank were adjusted to increase UV-B above the modelled clear-sky background flux (winter range: 0.898–3.701 kJ m–2 d–1) by 37% (UV-B137: 1.270–5.017 kJ m–2 d–1) and 73% (UV-B173: 1.626–6.256 kJ m–2 d–1). These UVB increases simulated 15% and 25% depletions, respectively, in total column ozone above Cape Town according to a computerized (Musil and Bhagwandin, 1992) semi-empirical model (Green, 1983). To avoid semi-empirical model overestimation of the level of supplementary UV-B irradiance required for each UV-B treatment due to local variations in the amount and form of cloud and atmospheric aerosols (Thiel et al., 1997), artificial UV-B supplements were applied under predominantly clear-sky conditions (Musil et al., 2002). This was achieved by switching off lamps during the passage of intermittent cold fronts. The experimentally simulated depletion of stratospheric ozone exceeded the predicted 11% for all seasons at southern-hemisphere mid-latitudes (Madronich et al., 1995). However, the total daily UV-B exposures supplied in the UV-B162 was included to exert more stress on the plants. Lamps in the mylar-filtered controls (UV-B100) were fixed at the same distances above plants as in the UV-B137 treatment to provide similar UV-A exposures in both (Newsham et al., 1996).
The height of the lamps was regularly adjusted to accommodate increases in the median height of the plants in each bank and seasonal variations in UV-B exposure. Adjustments were checked with a UV-B biometer sensor (Model 3D-600, Solar Light Company, Philadelphia, USA) calibrated against the spectroradiometer for the generalized plant action spectrum, which regularly checked percentage changes in UV-B beneath the lamps. Measured UV-B exposures over the winter growing period of plants averaged 99.4% (range: 90.4–106.4%) of background in the UV-B100 control, 143.1% (range 132.7–149.9%) of background in the UV-B137 treatment and 176.8% (range: 159.6–193.38%) of background in the UV-B173 treatment (Fig. 1A, B).
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Sub-ambient UV-B treatments
There was one UV-B treatment (UV-B22), and two controls; one for photosynthetically active radiation (PARcontrol) and the other for UV-A (UV-Acontrol) radiation, each replicated three times. Since the UV-B treatment contained both PAR and UVA wavelength, both of which are known to have moderating effects on UV-B induced damage (Middleton and Teramura, 1994), the two controls assisted in separating the effects of these wavelengths from that of UV-B. For instance, by comparing UV-B22 treatment and PAR control, the effect of sub-ambient ultraviolet (both UV-B and UV-A) radiation (Fig. 2A) could be assessed, and the comparison of UV-B22 and the UV-A control permitted the determination of the sole effect of only sub-ambient UV-B radiation (Fig. 2A). For the UV-B22 treatment, chambers were clad with an extruded grade of perspex (transmission down to 250 nm). For the PAR radiation control (PARcontrol), the chambers were covered with a cast grade of perspex (transmission down to 372 nm), and for the UV-A radiation control (UV-Acontrol), they were covered with the extruded grade of perspex coated with a 0.12 mm thick Mylar-D film. Changes in UV-B, UV-A, and PAR radiation inside the chambers were measured against background levels using UV-B biometer sensor, PAR (LI 189, Li-Cor, Lincoln, NE, USA) (Fig. 1B). The average maximum daily air temperatures in the chambers (24.4±0.60 °C) and the background temperature (23.3±0.56 °C) were recorded using temperature sensors, and the changes in the chambers against background for each radiation treatment are presented in Fig. 2B.
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Plant harvest and measurement of biomass
Plants of L. luteus and Vicia atropurpurea were harvested at 108 d and 128 d, respectively, after germination, and separated into nodules, roots, stems, leaves, and pods. At this time, L. luteus was at the pod formation stage of growth, while V. atropurpurea was still at the flowering stage. The plant organs were oven-dried at 60 °C, weighed and ground into a fine powder for the analysis of N and metabolites.
Analysis of N in tissues
Concentrations of N in all plant organs and seeds of the parent material were measured as %N using a Carlo Erba NA 1500 elemental analyser (Fisons Instruments SpA, Strada Rivoltana, Italy) coupled to a Finniggan MAT 252 mass spectrometer (Finnigan MAT GmbH, Bremen, Germany) via a Conflo II open-split device. The amount of N per organ was estimated from the product of %N and the dry matter weight. Total plant N was calculated as the sum of N in the different plant organs, N fixed as total plant N minus seed N, while nodule N2-fixing activity (mg N fixed g–1 nodule weight d–1) was obtained by dividing total plant N by nodule dry weight and length of plant growth period. Amount of N fixed was calculated for plants relying entirely on symbiotic N2 fixation for their N nutrition as total plant N minus seed N.
Measurement of tissue flavonoid concentrations
Flavonoids and anthocyanins were extracted from oven-dried, ground tissue samples of plant roots and leaves, suspended in 10 ml vols of acidified methanol (methanol:water:HCl, 79:20:1, by vol.), autoextracted at 0 °C for 72 h, centrifuged and absorbances measured at 300, 530 and 657 nm for each supernatant (Mirecki and Teramura, 1984). Flavonoid concentrations were expressed as absorbance (Ab) at 300 nm g–1 dry matter, and anthocyanins calculated as Ab530 nm–1/3 Ab657 nm g–1 dry matter (Lindoo and Caldwell, 1978).
Measurement of non-structural carbohydrates
Total soluble sugars (sucrose, glucose and fructose) were extracted from oven-dried, ground samples of plant roots (0.1 g: 10 ml 80% aqueous ethanol, v/v) and auto-extracted at 0 °C for at least 72 h. The extracts were centrifuged and the supernatant adjusted to 25 ml and measured spectrophotometrically at 490 nm for total soluble sugars as described by Buysse and Merckx (1993). The pellets from centrifugation were oven-dried at 60 °C for 48 h and weighed. Starch was measured by hydrolysing the dried pellet for 3 h in 5 ml 3.6% HCl at 100°C, centrifuging the extract, and adjusting the volume to 25 ml for spectrophotometric determination of the resultant sugars in the extract also at 490 nm (Buysse and Merckx, 1993). Soluble sugar and starch concentrations were expressed as µg mg–1 dry matter.
Statistical analysis
All measurements were loge transformed before statistical analysis to reduce the inequality of variance in the raw data. As a result of the differences in environmental conditions between plants grown on tables and in chambers (e.g. 1.1 °C temperature difference), UV-B treatment effects on all measured parameters were tested separately. Because the number of plants per replicate table or chamber was sometimes unequal (3 or 4), a REML (residual maximum likelihood) variance component analysis (Genstat, 1993) was used to test treatment differences on each species statistically. Here, the Wald 
2 statistic generated by REML tests for significant differences between treatment effects. In this study, the UV-B levels and N sources (UV-B levelsxN sources) were fitted in the fixed model, and plants per table or chamber in the random model. Differences exceeding twice the standard error of differences were used to separate significantly different treatment means at P <0.05. This is based on the fact that for a normal distribution from REML estimates, the 5% two-sided critical value is two. Correlation coefficients and Student’s t-test method were used to test the statistical relationship between root metabolites and nodule dry matter or nodule activity in each species.
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Results |
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Supra-ambient UV-B exposures
Plant growth: Exposing L. luteus and V. atropurpurea plants to elevated UV-B radiation did not change plant total biomass or the dry matter of individual organs (Table 1). Feeding the plants with NO3 did not alter organ or total dry matter in either species, except for leaves of L. luteus which increased with NO3 supply (Table 1).
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Although the interactions between UV-B and NO3 application were not significant (P <0.05) for any plant growth parameter of V. atropurpurea (Table 1), leaf and total dry matter of NO3-fed L. luteus plants increased (P <0.05) with exposure to UV-B173 relative to the control (UV-B100). Total dry matter of purely symbiotic L. luteus plants also increased (P <0.05) relative to the UV-B137, but not the control (Table 1).
Symbiotic performance: Symbiotic components such as nodule dry matter, rates of specific N2-fixing activity and N fixed per plant were not altered in both L. luteus and V. atropurpurea plants with exposure to elevated UV-B radiation (Table 2). However, the concentration of N in L. luteus stems and leaves, unlike other organs was significantly decreased (P <0.05) by UV-B173 relative to only UV-B137. Supplying 2 mM NO3 to L. luteus plants significantly reduced %N in nodules relative to their purely symbiotic counterparts (Table 2). The rates of N2-fixing activity and root %N of V. atropurpurea were also reduced by NO3 application, in contrast to nodule dry matter which increased significantly (P <0.05) (Table 2).
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The interactions between UV-B and NO3 feeding was significant (P <0.05) for only nodule dry matter and total N of L. luteus (Table 2) as both parameters increased (P <0.05) in NO3-fed, but not purely symbiotic plants with exposure to elevated UV-B radiation. A comparison of NO3 versus symbiotic nutrition in V. atropurpurea at each level of UV-B showed an increase (P <0.05) in nodule dry matter with NO3 feeding, but with a decrease in rates of fixation at both UV-B100 and UV-B137, but not UV-B173 (data not shown). By contrast, NO3 nutrition decreased nodule dry matter in L. luteus relative to purely symbiotic plants at UV-B100 (data not shown).
Tissue concentrations of flavonoids and metabolites: Exposure of L. luteus plants to the highly elevated UV-B level significantly (P <0.05) increased the concentration of flavonoids in leaves (Table 3), but not in those of V. atropurpurea (Table 3). With N supply, the concentrations of flavonoids and anthocyanins decreased markedly (P <0.05) in the leaves of both L. luteus and V. atropurpurea, and also in the roots of the latter (Table 3). The levels of flavonoids, anthocyanins and soluble sugars were similarly decreased (P <0.05) in roots of V. atropurpurea plants supplied with N (Table 3).
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As with their plant growth parameters, UV-B and NO3 interacted significantly (P <0.05) only in L. luteus with root anthocyanin and leaf flavonoids, but not in V. atropurpurea (Table 3). Relative to the control and UV-B137, the highly elevated UV-B radiation significantly increased the concentration of root anthocyanins in purely symbiotic, but not in NO3-fed, plants of L. luteus (Table 3). Conversely, only leaf flavonoids of NO3-fed, but not purely symbiotic plants, increased with UV-B exposure of this species. Applying NO3 to L. luteus plants grown at each UV-B level decreased root anthocyanins at UV-B173 and leaf flavonoids at UV-B137, but not at the other UV-B level (data not shown).
Sub-ambient UV-B exposures
Plant growth: Growing L. luteus and V. atropurpurea plants in chambers under sub-ambient UV-B radiation did not alter overall plant growth or organ development (Table 4). However, supplementing the nodulated plants with NO3 reduced (P <0.05) dry matter accumulation in stems, leaves, pods and whole plants of the two species (Table 4). But root dry matter was only reduced in V. atropurpurea with NO3 supply (Table 4). There was also a significant (P <0.05) interaction between sub-ambient UV-B and N source (Table 4). Stem, leaf and total dry matter of purely symbiotic V. atropurpurea plants increased (P <0.05) with plant exposure to UV-B22, but not visible or UV-A controls. By contrast, NO3 supply decreased (P <0.05) stem dry matter of V. atropurpurea plants receiving UV-B22 and UV-A relative to visible control (Table 4).
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Symbiotic performance: Nodule dry matter, %N, N fixed, and nodule N2-fixing activity were all unaltered in both L. luteus and V. atropurpurea plants exposed to sub-ambient UV-B radiation (Table 5). However, supplying NO3 to roots of L. luteus increased (P <0.05) the rate of N2-fixing activity and concentration of N in leaves and stems, but reduced (P <0.05) nodule dry matter, %N in nodules, and total plant N (Table 5). With V. atropurpurea, however, the NO3 application significantly increased (P <0.05) %N in stems and leaves (Table 5).
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The interaction between sub-ambient UV-B and N sources was significant for only %N in leaves of L. luteus (Table 5), in that exposure to UV-B22 of purely symbiotic plants increased (P <0.05) leaf %N relative to UV-A, but not visible controls. With V. atropurpurea, UV-B22 increased %N in roots of purely symbiotic plants, but decreased that of NO3-fed plants relative to visible but not UV-A controls (Table 5).
Tissue concentrations of flavonoids and metabolites: The concentration of flavonoids, anthocyanins, soluble sugars, and starch were unchanged in root and leaf organs of both L. luteus and V. atropurpurea plants exposed to sub-ambient UV-B radiation (Table 6). However, supplying NO3 to L. luteus plants markedly reduced (P <0.05) the concentration of flavonoids, soluble sugars and starch in leaves and roots as well as anthocyanins in roots only (Table 6). The concentration of flavonoids in leaves of V. atropurpurea plants was also reduced (P <0.05) with NO3 supply (Table 6). As shown for other parameters, there was a significant interaction between sub-ambient UV-B and N nutrition. In NO3-fed, but not in purely symbiotic plants, exposure to UV-B22 decreased (P <0.05) anthocyanins and soluble sugars in roots of L. luteus and V. atropurpurea, respectively, when compared to the visible control (Table 6). Comparing NO3 and symbiotic N nutrition at each level of sub-ambient UV-B showed a large reduction (P <0.05) in root anthocyanins of NO3-fed L. luteus plants grown at UV-B22 (data not shown). With V. atropurpurea, NO3 supply more than halved root soluble sugars of plants grown under UV-A relative to their symbiotic counterparts (data not shown).
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Correlating root metabolites with symbiotic parameters and leaf flavonoids: There was a positive correlation (P <0.05) between root concentrations of flavonoids or anthocyanins and nodule dry matter in L. luteus grown under elevated UV-B radiation (Table 7). Positive correlations (P <0.05) were also observed between flavonoids or anthocyanins in the roots and the rate of N2-fixing activity, leaf flavonoids and leaf anthocyanins of L. luteus (Table 7). In V. atropurpurea, however, the concentration of root anthocyanins correlated negatively with nodule dry matter. Except for the negative correlation between root soluble sugars and N2-fixing activity in nodules of L. luteus, the levels of non-structural carbohydrates in roots were unrelated to symbiotic parameters (Table 7).
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With sub-ambient UV-B exposure, nodule dry matter was positively correlated (P <0.05) with both flavonoid and anthocyanin concentration in roots of L. luteus (Table 7). By contrast, root soluble sugars correlated negatively with the rate of nodule activity in this species. There were no significant (P <0.05) correlations between any parameters following exposure of V. atropurpurea plants to sub-ambient UV-B (Table 7).
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Discussion |
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Supra-ambient UV-B effects on plant growth and symbiotic function
Whether measured on the basis of whole plants, or individual organs, plant growth response was unaltered following exposure of L. luteus and V. atropurpurea to elevated UV-B radiation simulating 15% and 25% ozone depletion. Similarly, there was no change in nodulation and N2 fixation of both L. luteus and V. atropurpurea with exposure to elevated UV-B radiation (Table 2). This lack of growth and symbiotic response of L. luteus and V. atropurpurea to elevated UV-B radiation is consistent with data obtained for Vicia faba (Al-Oudat et al., 1998), Pisum sativum plants (Allen et al., 1999) and three tropical legumes (Chimphango et al., 2003). These findings, however, differ from those of Singh (1996) and Hofmann et al. (2001) who found major reductions in plant dry matter of Vigna radiata, Phaseolus mungo, Glycine max, Trifolium repens, and cultivars of Pisum sativum grown under elevated UV-B radiation. Singh (1997) and Van de Staaij et al. (1999) also found large decreases in nodule numbers, nodule mass, nodule diameter, and nitrogenase activity in symbiotic legumes exposed to elevated UV-B. Although these variations in growth and symbiotic response of nodulated legumes to elevated UV-B could be attributed to genotypic differences in plant sensitivity (Jansen et al., 1998), and/or nutritional status of the plant (Hunt and McNeil, 1998; Correia et al., 2000; Murali and Teramura, 1985), it appears that the intensity of UV-B radiation applied could be a major factor (Fiscus and Booker, 1995). For example, although the total daily UV-B exposure used by Singh (1996) was below this study’s highly elevated UV-B, but similar to the moderately elevated UV-B, the reduction in plant growth and symbiotic function was huge, and would be more likely due to the high intensity of UV-B applied over a short 2 h time period compared to the 8 h spread in this study.
To mimic field situation where N2-fixing plants often depend on both soil-N and symbiotic-N for their N nutrition, 2 mM NO3 was applied to nodulated plants of L. luteus and V. atropurpurea. Interestingly, the extra N from NO3 did not affect the overall plant growth relative to purely symbiotic material, although leaf dry matter increased with reduced nodule %N in L. luteus (Tables 1, 2). Supplying 2 mM NO3 to V. atropurpurea also showed markedly (P <0.05) increased nodule dry matter, but repressed N2-fixing activity in root nodules (Streeter, 1988), possibly due to the formation of nitrosyl-leghaemoglobin and/or an increased O2 diffusion barrier (Dakora and Atkins, 1989). Root %N was also reduced with NO3 feeding of V. atropurpurea (Table 2). The overall lack of growth response by the two species to NO3 supply is consistent with earlier reports by Ma et al. (1997) and Hill-Cottingham and Lloyd-Jones (1980).
Leaf concentrations of flavonoids and anthocyanins decreased in L. luteus, just as root concentrations of flavonoids, anthocyanins and soluble sugars were decreased with NO3 supply to V. atropurpurea (Table 3). These changes in flavonoid concentrations with N application have been observed previously (Cho and Harper, 1991; Hunt and Mc Neil, 1998; Khanna et al., 1999). Apparently, N fertilization of plants appears to have a generalized effect in down-regulating the phenylpropanoid pathway, usually leading to decreased tissue concentrations of most defence molecules such as phenols, phenol glucosides and condensed tannins (Bryant et al., 1987). It is possible that competition between C and N metabolism for ATP, NADPH as well as C skeletons needed in the synthesis of organic acids and carbohydrates on the one hand, and amino acids on the other, influences the shikimate pathway, thereby affecting the synthesis of phenylalanine-ammonia lyase (PAL). Being the enzyme that catalyses the first step of the phenylpropanoid pathway, a reduction in PAL would decrease tissue concentration of phenolics. But how NO3 specifically inhibits the phenylpropanoid pathway, remains to be unravelled. By contrast, a recent finding has also shown that nitrite oxide (NO) formed during NO3 reduction can increase radical formation with a potential to increase the synthesis of phenolics/antioxidants under NO3 fertilization (Stöhr and Ullrich, 2002).
Sub-ambient UV-B effects on plant growth and symbiotic function
Plants of L. luteus and V. atropurpurea showed no changes in growth, symbiotic function and concentration of metabolites when cultured under sub-ambient UV-B, UV-A control or visible control (Tables 4, 5, 6). This suggests that the species involved are probably well adapted to growth in the presence of both UV-A and UV-B radiation. The lack of plant response to sub-ambient UV-B observed in this study agrees with reports by Tosserams et al. (1996) and Becwar et al. (1982). However, similar studies that attenuated or excluded UV-B and/or the UV-A component of natural solar radiation showed decreased biomass production in lettuce and cucumber cultivars (Krizek et al., 1997, 1998). These inconsistencies in plant response to ambient levels of UV-B and/or UV-A could be attributed to genotypic differences.
However, feeding 2 mM NO3 to L. luteus plants exposed to sub-ambient UV-B radiation markedly (P <0.05) decreased total biomass and growth of individual organs, including root nodules (Table 4). Poor nodule development as a consequence of nitrate nutrition is a common feature of symbiotic legumes (Streeter, 1988) which can lower nodule N concentration, as observed in this study. Patterns of N allocation to organs were also altered with NO3 supply as manifested by the increased concentration of N in leaves and stems of the two legumes relative to those of purely symbiotic plants (Table 5). Symbiotically speaking, rhizobial inoculation of legumes and nitrate feeding tend to produce directly opposite effects on tissue concentration of nod gene inducers. Whereas inoculating legumes with infective rhizobial cells increases the synthesis and release of nod gene inducers (Dakora et al., 1993) for increased nodule formation (Phillips et al., 1994), nitrate application decreases tissue levels of nod gene inducers (Cho and Harper, 1991) and thus limits the legume’s nodulation potential. So, relative to purely symbiotic plants, NO3 supply to L. luteus probably decreased nod gene inducers with the consequent reduction in nodule formation, nodule biomass and total N content, as obtained here (Table 5). With V. atropurpurea, however, nodulation was not affected by NO3 application (Table 5), possibly suggesting species differences in the nitrate reduction of nod gene inducers in root tissues. This argument is supported by the fact that, with NO3 supply, the concentrations of flavonoids, anthocyanins, soluble sugars, and starch were all significantly (P <0.05) reduced in roots and to some extent leaves of L. luteus, but not in V. atropurpurea plants (Table 6). Although leaf anthocyanins were the only metabolites that decreased with NO3 provision to V. atropurpurea plants grown in sub-ambient UV-B radiation (Table 6), being leaf-based, they were less likely to affect root nodulation. However, leaf and stem N concentrations were significantly (P <0.05) increased in both legumes with NO3 supply under sub-ambient UV-B conditions, suggesting changes in allocation patterns.
UV-BxN interactions
In this study, there were significant interactions between UV-B radiation and sources of N nutrition. When the effects of ambient and the two levels of elevated UV-B radiation were compared, it was apparent that in purely symbiotic L. luteus plants, total biomass and root anthocyanins were increased (P <0.05) under UV-B173 compared to UV-B137 or ambient control (Tables 1, 3). With NO3-feeding, the leaf, nodule and total dry matter of L. luteus, as well as leaf flavonoids and plant total N were also markedly (P <0.05) greater under UV-B173 relative to ambient or UV-B137 radiation (Tables 1, 2, 3). From these interactions, it is probably fair to suggest that elevated UV-B, especially UV-B173, up-regulates the accumulation of biomass, flavonoids and anthocyanins in organs of NO3-fed L. luteus plants. Chamber-grown L. luteus plants receiving sub-ambient UV-B and dependent on symbiosis for their N nutrition showed reduced leaf %N under UV-A compared to UV-B22 and visible control (Table 5). Their NO3-fed counterparts also exhibited lowered root anthocyanins at UV-B22 compared with the visible control (Table 6).
With V. atropurpurea, however, the UV-BxN interactions occurred mainly in the chamber-grown plants receiving sub-ambient UV-B radiation. Leaf and stem dry matter as well as root %N were increased (P <0.05) in purely symbiotic V. atropurpurea plants grown under UV-B22 relative to those in the visible control (Table 4). With NO3 supply, stem dry matter together with root %N and root soluble sugars were markedly decreased (P <0.05) under UV-B22 and UV-A compared to visible control (Tables 4, 5, 6). So relative to UV-A and visible light, UV-B22 seems to up-regulate biomass accumulation in leaves and stems, and %N in roots of purely symbiotic V. atropurpurea plants; however, supplying NO3 down-regulates these parameters. This suggests that NO3 supply reduced resistance of V. atropurpurea plants to UV-B damage under the chamber conditions. Increased plant sensitivity to UV-B radiation with N application has also been observed in several studies (Hunt and McNeil, 1998; Correia et al., 2000). It has been reported that both UV-A and PAR have moderating effects on UV-B damage by inducing photo-reactivating processes that repair DNA lesions resulting from UV-B radiation (Jagger et al., 1969), and by stimulating biosynthesis of UV-B absorbing phenolics (Middleton and Teramura, 1994). However, in UV-B attenuation studies, Krizek et al. (1997, 1998) showed that ambient UV-A reduced biomass production of Lactuca sativa (lettuce) and Cucumis sativus (cucumber) over and above that caused by ambient UV-B. It is therefore possible that the presence of UV-B and higher levels of UV-A radiation in the UV-Acontrol than the PARcontrol chamber (Fig. 2B) adversely affected stem dry matter, root %N and root soluble sugars of the NO3-fed V. atropurpurea plants.
This study also tested the effects of the two modes of N nutrition (purely symbiotic versus symbiotic+NO3) on the plant’s response to elevated UV-B radiation. A comparison at each level of elevated UV-B radiation revealed marked differences in species response. Relative to purely symbiotic plants, NO3-fed L. luteus showed reduced (P <0.05) nodule dry matter at UV-B100, but greater leaf flavonoids and root anthocyanins at UV-B137 and UV-B173, respectively (data not shown). In the chamber-grown sub-ambient UV-B plants, nodule mass and root anthocyanins of NO3-fed L. luteus were also lower (P <0.05) at UV-B22 relative to their purely symbiotic counterparts (data not shown). However leaf %N of NO3-fed plants was greater (P <0.05) than that of purely symbiotic plants under UV-A control (data not shown). With V. atropurpurea, however, nodule dry matter was increased (P <0.05) in NO3-fed compared to purely symbiotic plants grown under UV-B100 or UV-B137. As to be expected, rates of N2 fixation were lower (P <0.05) in nodules of NO3-fed plants cultured at UV-B100 and UV-B137. Plants grown in the visible control showed greater (P <0.05) root %N with NO3 feeding compared with the purely symbiotic counterparts. However, plants receiving only UV-A radiation had less (P <0.05) soluble sugars in roots of NO3-fed plants relative to those dependent on symbiotic N nutrition (data not shown). These interactive responses between elevated UV-B and NO3 supply are consistent with data obtained by Musil and Wald (1994) which showed increased shoot biomass of Dimorphotheca pluvialis plants with higher UV-B radiation under high, but not low, nutrient conditions. However, these results are in contrast with several reports on the increased sensitivity of plants to elevated UV-B radiation with high nutrient feeding (Correia et al., 2000; Hunt and McNeil, 1998; Murali and Teramura, 1985).
In conclusion, the results of this study show no adverse effect of elevated UV-B radiation (simulating 15% and 25% ozone depletion) on growth, and symbiotic function of L. luteus and V. atropurpurea plants. However, feeding NO3 to nodulated plants of L. luteus under the highly elevated UV-B radiation promoted plant growth and total N content.
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Acknowledgements |
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We are grateful to the Association of African Universities and Deutscher Akademischer Austauach Dienst for a fellowship awarded to SBMC, to the National Botanical Institute for financial support to CFM, and to the National Research Foundation for grants to FFD. We also thank Ms J Arnolds and Mr S Snyders for technical assistance.
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