Tree Physiology 24, 181–192
© 2004 Heron Publishing—Victoria, Canada
Responses to ultraviolet-B radiation by purely symbiotic and NO3-fed
nodulated tree and shrub legumes indigenous to southern Africa
SAMSON B. M. CHIMPHANGO,1 CHARLES F. MUSIL2 and FELIX D. DAKORA3,4
1
Botany Department, University of Cape Town, Private Bag, Rhondebosch 7701, South Africa
2
Research and Scientific Services, National Botanic Institute, Private Bag X7, Claremont 7735, Cape Town, South Africa
3
Research Development and Technology Promotion, Cape Technikon, P.O. Box 652, Cape Town 8000, South Africa
4
Author to whom correspondence should be addressed ([email protected])
Received April 15, 2003; accepted July 11, 2003; published online December 15, 2003
Summary Purely symbiotic and NO3-fed nodulated seedlings of Virgilia oroboides (Bergius) T.M. Salter, Cyclopia
maculata (L.) Vent and Podalyria calyptrata Willd. were exposed to biologically effective ultraviolet-B radiation (UV-B)
to assess the effects of above- and below-ambient UV-B on
growth, symbiotic function and metabolite concentrations.
Seedlings were grown outdoors either on tables under ambient
or 34 or 66% above-ambient UV-B conditions (UV-B100 control, UV-B134 and UV-B166, respectively), or in chambers providing below-ambient (22% of ambient) UV-B (UV-B22) along
with a UV-A control and a photosynthetically active radiation
(PAR) control. Exposure of seedlings to UV-B166 radiation reduced (P ≤ 0.05) leaf and stem dry mass by 34 and 39%, respectively, in C. maculata, and reduced leaf nitrogen concentration
(%N) by 12% in V. oroboides. Nodule %N in C. maculata and
stem %N in P. calyptrata also decreased (P ≤ 0.05) in response
to UV-B22 radiation compared with the UV-A control, but not
compared with the PAR control. Concentrations of flavonoids,
soluble sugars and starch were unaltered by the UV-B treatments. Application of 1 mM NO3 to UV-B166-treated seedlings
increased whole-plant dry mass of V. oroboides and P. calyptrata by 47 and 52%, respectively. Dry mass of organs, nodule
%N and total N concentration of these species also increased
with NO3 application. However, NO3 supply decreased (P ≤
0.05) nodule dry mass, stem %N and leaf %N as well as root
and leaf anthocyanin concentrations in C. maculata. In terms
of UV-B × N interactions, dry mass of stems, roots, nodules and
total biomass of NO3-fed C. maculata seedlings were reduced,
and nodule %N, total N and leaf anthocyanins were depressed
by the UV-B134 and UV-B166 treatments relative to UV-B100treated seedlings. Although we found that above-ambient
UV-B had no effects on growth and symbiotic function of
V. oroboides and P. calyptrata seedlings, feeding NO3 to these
species increased (P ≤ 0.05) seedling growth. In contrast,
purely symbiotic C. maculata seedlings were sensitive to the
UV-B166 radiation treatment, and adding NO3 further increased
their sensitivity to both the UV-B134 and UV-B166 treatments.
Keywords: Cyclopia maculata, flavonoids, nodulation and N2
fixation, NO3 supply, Podalyria calyptrata, soluble sugars,
starch, Virgilia oroboides.
Introduction
Reduction in ozone layer thickness in the stratosphere is associated with increased biologically effective ultraviolet-B
radiation (UV-B, 290–315 nm) at ground level (Madronich et
al. 1998) in both the southern and northern hemispheres
(McKenzie et al. 1999). However, destruction of the ozone
layer over Antarctica and the southern hemisphere (Crutzen
1992), as measured by solar UV-B radiation, is about 50%
greater than that at comparable latitudes in the northern hemisphere (Seckmeyer et al. 1995). Consequently, plants in the
southern hemisphere are exposed to more UV-B radiation than
those in the northern hemisphere. However, there have been
many more studies on the effects of UV-B radiation on trees in
the northern hemisphere (Musil et al. 2002b) than in the southern hemisphere (L’Hirondelle and Binder 2002), even though
trees account for up to 80% of global net primary productivity
(Sullivan and Teramura 1989).
Effects of UV-B on plants include reduced biomass accumulation, altered biomass allocation and increased flavonoid content (Schumaker et al. 1997, Lavola 1998, Hofmann et al.
2001, Kolb et al. 2001). Growth responses of tree species to
above-ambient UV-B can be positive or negative (Laakso and
Huttunen 1998, L’Hirondelle and Binder 2002). These differences in UV-B response may be associated with genetic variation, influence of other factors such as drought or nutrient
availability, the presence of protective features like waxy and
reflective layers or a thick epidermis, and the presence of
UV-B-absorbing flavonoids (Murali and Teramura 1985,
Jansen et al. 1998, Correia et al. 2000, Yuan et al. 2000, Bieza
and Lois 2001).
Although the effects of above-ambient UV-B radiation on
the symbiotic performance of legumes have been investigated
(Singh 1997, Chimphango et al. 2003a, 2003b), such studies
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CHIMPHANGO, MUSIL AND DAKORA
have focused mainly on food grain legumes. The few studies
conducted on tree or shrub legumes paid little attention to their
symbiotic function. For example, in studies with African Acacia karroo (Forsk.) Hayne, only nodule dry mass was assessed,
and found to be unaltered by plant exposure to above-ambient
UV-B radiation (Wand et al. 1996, Ernst et al. 1997). More information is needed to assess the effects of above-ambient
UV-B radiation on tree and shrub legume nodulation and nitrogen (N2) fixation. We hypothesized that an increase in aboveambient UV-B radiation will reduce nodulation and N2 fixation if the damaged photosynthetic machinery of legumes results in decreased plant growth and reduced photosynthetic C
supply to nodules or decreased release of biologically functional root exudate compounds into the rhizosphere. Because
N-fed, but not N-depleted, maize (Zea mays L.) and cucumber
(Cucumis sativus L.) plants were sensitive to above-ambient
UV-B radiation (Hunt and McNeil 1998, Correia et al. 2000),
we further hypothesized that NO3 additions will increase seedling sensitivity to UV-B radiation. Our study objective was to
determine the effects of above- and below-ambient UV-B radiation on growth, symbiotic function and concentration of metabolites of purely symbiotic and NO3-fed nodulated tree and
shrub legumes indigenous to southern Africa.
Materials and methods
Plant species and culture
The legumes studied included a shrub, which is the source of a
commercially important herbal beverage in South Africa,
Cyclopia maculata (L.) Vent, and the temperate evergreen
shrub Podalyria calyptrata Willd. and tree Virgilia oroboides
(Bergius) T.M. Salter. Seeds were sown in 20 cm high × 20 cm
diameter pots (four seeds per pot, later thinned to two seedlings per pot) containing sand, and germinated in different
UV-B regimes. At emergence, seedlings were inoculated with
rhizobial isolates from nodules of the same species. The experiment comprised 12 tables with banks of fluorescent sun lamps
(Phillips TL/12 40W UV-B, The Netherlands) and 12 chambers (1.5 m 2 × 0.75 m high) constructed of differentially
UV-transmitting clear Perspex (3 mm thick) inter-dispersed in
an open area in the Kirstenbosch National Botanical Gardens,
Cape Town (36°56′ S, 18°29′ E). Four pots per test species
were assigned to each table and chamber. Seedlings in two of
the four pots received 1 mM NO3, whereas seedlings in the
other two pots relied entirely on symbiotic N2 fixation for N
nutrition. All pots were irrigated with a similar volume of water. Immediately at emergence, 400 ml of quarter-strength
N-free Hoagland’s nutrient solution (Hewitt 1966) was supplied twice weekly to the purely symbiotic seedlings and the
same volume made up to 1 mM NO3 was supplied to the N-fed
seedlings.
Above-ambient UV-B treatments
Two above-ambient UV-B treatments and one control were
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), whereas lamps in the moderately (UV-B134) and
highly (UV-B166) elevated UV-B treatments were filtered with
0.075 mm thick cellulose acetate film (transmission down to
290 nm) (Courtaulds Chemicals, Derby, U.K.). Hence, seedlings serving as UV-B controls were exposed to ambient UV-B
radiation, whereas seedlings in the above-ambient UV-B treatments received extra UV-B from the lamps. 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 centered on
solar noon. The remaining one third was applied equally over
the two 2-h early morning and late afternoon photoperiods.
This stepwise application of supplemental UV-B was followed
to account for alterations in ambient UV-B irradiance caused
by diurnal changes in solar zenith angle.
Spectral irradiances of filtered lamps were measured after
sunset with a computer-interfaced monochromator spectroradiometer (IL-1700, International Light, Newburyport, MA),
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) and 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 seedlings in each bank were adjusted to increase UV-B above modeled clear-sky background
flux (winter to summer range: 0.898 to 8.146 kJ m –2 day –1) by
34% (UV-B134: 1.270 to 10.791 kJ m –2 day –1) and 66%
(UV-B166: 1.626 to 13.263 kJ m –2 day –1). These UV-B increases simulated 15 and 25% depletions, respectively, in the
total ozone column above Cape Town according to a computerized (Musil and Bhagwandin 1992) semi-empirical model
(Green 1983). To avoid overestimating the amount of supplementary UV-B irradiance required for each treatment caused
by local variations in the amount and form of cloud and atmospheric aerosols (Theil et al. 1997), artificial UV-B supplements were applied under predominantly clear-sky conditions
(Musil et al. 2002a). 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 midlatitudes in the southern
hemisphere (Madronich et al. 1995). However, the total daily
UV-B exposure supplied in the UV-B166 treatment was included to exert more stress on the seedlings. Lamps in the Mylar-filtered controls (UV-B100) were fixed at the same distances
above seedlings as in the UV-B134 treatment to provide similar
UV-A exposures (Newsham et al. 1996).
The heights of the lamps were regularly adjusted to accommodate increases in median seedling height in each bank and
seasonal variations in UV-B exposure. Adjustments were
checked with a UV-B biometer sensor (Model 3D-600, Solar
Light, Philadelphia, PA) calibrated against the spectroradiometer for the generalized plant action spectrum, which was
used regularly to check percentage changes in UV-B beneath
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The chamber treatments comprised one UV-B treatment
(UV-B22) and two controls; one for photosynthetically active
radiation (PARcont) and the other for UV-A (UV-Acont) radiation, each replicated four times. Because the UV-B22 treatment
contained both PAR and UV-A wavelengths, both of which are
known to moderate UV-B-induced damage (Middleton and
Teramura 1994), the two controls assisted in separating the effects of these wavelengths from those of UV-B. For instance,
by comparing the results of the UV-B22 treatment and PARcont,
the effect of below-ambient ultraviolet (both UV-B and UV-A)
radiation (Figure 2A) could be assessed. Similarly, comparison of the results of the UV-B22 treatment and UV-Acont permit-
ted determination of the effect of below-ambient UV-B radiation (Figure 2A). For the UV-B22 treatment, chambers were
covered with an extruded grade of Perspex (transmission down
to 250 nm). For the PARcont treatment, chambers were covered
with a cast grade of Perspex (transmission down to 372 nm),
and for the UV-Acont treatment, chambers 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 values
(Figure 2B) with a UV-B biometer sensor (LI-189, Li-Cor,
Lincoln, NE). The small amount of UV-B radiation measured
in the UV-Acont and PARcont chambers (Figure 2B) was due to
diffused scattered UV-B radiation coming from the open
southern side of the chambers. The mean maximum daily air
temperatures for each chamber (28.4 ± 0.58, 27.6 ± 0.62 and
27.7 ± 0.64 °C for PARcont, UV-Acont and UV-B22, respectively), all the chambers (27.9 ± 0.61 °C) and background
(26.4 ± 0.57 °C) were recorded with temperature sensors. The
temperature changes in the chambers against the background
for each radiation treatment are presented in Figure 2B.
Figure 1. (A) Measured changes in UV-B, UV-A and PAR beneath
lamp systems over the growing period, and (B) measured and modeled UV-B radiation changes at 15 and 25% ozone loss for the entire
year. Abbreviations: UV-B = ultraviolet-B radiation; UV-A = ultraviolet-A radiation; and PAR = photosynthetically active radiation.
Figure 2. (A) Spectral characteristics of the extruded and cast grades
of Perspex and Mylar film used in chamber construction, and (B)
measured changes in UV-B, UV-A, PAR and maximum daily air temperatures in the chambers. Abbreviations: UV-B = ultraviolet-B radiation; UV-A = ultraviolet-A radiation; and PAR = photosynthetically
active radiation.
the lamps. Measured UV-B exposures over the winter to summer growing period averaged 98.3% (winter to summer range:
90.4–114.3%) of background in the UV-B100 control, 142.0%
(range: 130.8–154.5%) of background in the UV-B134 treatment and 171.5% (range: 139.4–193.4%) of background in
the UV-B166 treatment (Figures 1A and 1B).
Below-ambient UV-B treatments
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CHIMPHANGO, MUSIL AND DAKORA
Seedling harvest and biomass measurements
Seedlings of V. oroboides, P. calyprata and C. maculata were
harvested at 167, 184 and 194 days after germination, respectively, and separated into nodules, roots, stems, and leaves.
Plant organs were oven-dried at 60 °C, weighed and ground to
a fine powder for analysis of N and metabolites.
Analysis of N in tissues
Concentrations of N (%N) in all plant organs and seeds of the
parent material were measured with a Carlo Erba NA 1500 elemental analyzer (Fisons Instruments SpA, Strada Rivoltana,
Italy) coupled to a Finnigan MAT 252 mass spectrometer
(Bremen, Germany) through a Conflo II open-split device.
Amount of N per organ was estimated from the product of %N
and organ dry mass. Total plant N was calculated as the sum of
N in the different plant organs.
for a normal distribution from REML estimates, the 5% twosided critical value is two, differences exceeding twice the
standard error of differences were used to separate significantly different treatment means at P ≤ 0.05.
Results
Components of seedling growth (leaf, stem, root, nodule or
whole-plant dry mass), symbiotic function (leaf, stem, root
and nodule %N and total N content) and concentrations of metabolites (flavonoids, anthocyanins, soluble sugars and starch
in the root, and flavonoids and anthocyanins in the leaves) that
were not significantly (P ≤ 0.05) influenced by either UV-B
exposure, NO3 supply, or their interaction are not presented in
the tables or figures.
Above-ambient UV-B effects
Measurement of tissue flavonoid concentrations
Flavonoids and anthocyanins were extracted from oven-dried,
ground tissue samples of seedling roots and leaves, suspended
in 10 ml of acidified methanol (79:20:1, v/v, methanol:water:
HCl), autoextracted at 0 °C for 72 h and centrifuged, and absorbances were measured at 300, 530 and 657 nm for each
supernatant (Mirecki and Teramura 1984). Flavonoid concentrations were expressed as absorbance at 300 nm per gram dry
mass (A300 g –1 DM), and anthocyanins were calculated as A530
– 0.333A657 g –1 DM (Lindoo and Caldwell 1978).
Measurement of nonstructural carbohydrates
Total soluble sugars (sucrose, glucose and fructose) were extracted from oven-dried, ground samples of seedling roots
(0.1 g in 10 ml of 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 hydrolyzing the dried pellet for 3 h in 5 ml of
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 at 490 nm (Buysse and Merckx
1993). Soluble sugar and starch concentrations were expressed as µg mg –1 DM.
Statistical analysis
To reduce inequality of variance in the raw data, all measurements were loge transformed before statistical analysis. Because the number of seedlings per replicate table or chamber
was sometimes unequal (3 or 4), an REML (restricted maximum likelihood) variance component analysis (Genstat 1993)
was used to statistically test treatment differences within each
species for tables and chambers separately. The Wald χ2 statistic was used to test for significant differences between treatment effects. The UV-B treatments and N sources (UV-B
treatments × N sources) were fitted in the fixed model, and
seedlings per table or chamber in the random model. Because,
Seedling growth Exposing C. maculata to UV-B166 radiation
reduced (P ≤ 0.05) stem and leaf dry mass relative to values in
the UV-B100 treatment; however, root and total biomass were
unchanged (Table 1). Total biomass and dry mass of individual
organs of V. oroboides and P. calyprata were unaltered by exposure to UV-B166 radiation (Table 1). Supplying 1 mM NO3 to
C. maculata seedlings increased (P ≤ 0.001) root dry mass but
not stem, leaf or total biomass (Table 1). The dry mass yield of
individual organs as well as plant total biomass of V. oroboides
and P. calyprata seedlings also increased (P ≤ 0.001) with NO3
feeding (Table 1). There were significant interactions between
above-ambient UV-B and N source on seedling growth, including root, stem and total dry mass, only in C. maculata (Table 1).
Mean separation of the effect of UV-B at each N source in this
species showed that stem, root and total dry mass of NO3-fed
seedlings were reduced (P ≤ 0.05) by UV-B134 and UV-B166 radiation (Figures 3A, 3B and 3D). In contrast, stem, root and total dry mass of purely symbiotic C. maculata were reduced
(P ≤ 0.05) by UV-B166 radiation only (Figures 3A, 3B and 3D).
Symbiotic performance Components of symbiotic performance, such as nodule dry mass and plant N content, were
unaffected by UV-B166 radiation in seedlings of any species
(data not shown). However, leaf %N of V. oroboides seedlings
was decreased (P ≤ 0.05) by the UV-B166 treatment (Table 1).
Applying NO3 to V. oroboides seedlings increased (P ≤ 0.01)
nodule dry mass (Table 1). In C. maculata, only root %N markedly increased (P ≤ 0.05) in response to NO3 feeding, whereas
stem and leaf %N were reduced (P ≤ 0.05) (Table 1). Only in
C. maculata were significant (P ≤ 0.05) interactions apparent
between above-ambient UV-B and N source on components of
symbiotic function such as nodule dry mass, nodule %N and
total plant N concentration (Table 1). In NO3-fed C. maculata,
nodule dry mass, nodule %N and total plant N were reduced
(P ≤ 0.05) in seedlings exposed to UV-B134 and UV-B166 (Figures 3C, 3E and 3F), whereas in their purely symbiotic counterparts, nodule %N and total plant N concentration were reduced
only in seedlings exposed to UV-B166 (Figures 3E and 3F).
Nodule dry mass of purely symbiotic C. maculata seedlings
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Table 1. Effects of above-ambient UV-B radiation on seedling growth, symbiotic parameters and concentrations of metabolites in purely symbiotic and NO3-fed nodulated seedlings of tree and shrub legumes. Within UV-B and N source treatments, means that are significantly different are
followed by different letters at: * = P ≤ 0.05, ** = P ≤ 0.01 and *** = P ≤ 0.001. Abbreviations: UV-B100 = ambient ultraviolet-B radiation;
UV-B134 = 34% above-ambient ultraviolet-B radiation; UV-B166 = 66% above-ambient ultraviolet-B radiation; and DM = dry mass. Measurements of components not listed under various parameters in this table were not significantly different.
Parameter
Above-ambient treatments
N source
Wald χ2 statistic
UV-B100
UV-B134
UV-B166
Symbiotic-N NO3-N
(df = 2)
UV-B
(df = 1)
N
(df = 2)
UV-B × N
9.854
8.125
10.495
1.884
30.359
9.110
7.572
9.830
1.804
28.317
8.626
6.654
9.000
1.727
26.007
7.777 b
5.447 b
8.000 b
1.618 b
22.842 b
10.617 a
9.454 a
11.550 a
1.993 a
33.613 a
0.83
2.11
0.75
0.78
1.06
13.57 ***
27.03 ***
19.62 ***
9.09 **
22.64 ***
1.46
2.45
1.11
0.50
1.69
Symbiotic parameters
Leaf %N
Total plant N (mg g –1)
2.551 a
635.08
2.668 a
598.27
2.253 b
509.60
2.388
476.52 b
2.594
685.44 a
6.54 *
2.34
2.69
14.74 ***
2.59
1.60
Metabolite concentration
Root soluble sugars (mg g –1 )
Leaf anthocyanins (Abs g –1 )
20.988
94.601
25.437
96.670
25.162
95.850
21.634 b
91.235 b
26.090 a
100.18 a
2.21
0.08
4.61 *
7.01 **
0.63
10.12 **
10.284 a
7.087 a
11.081
1.278
29.73
8.325 ab
5.109 ab
9.059
1.318
23.81
6.809 b
4.320 b
9.143
1.160
21.43
8.139
5.146
8.543 b
1.375 a
23.20
8.862
5.900
10.954 a
1.138 b
26.85
5.93 *
8.10 *
1.25
0.50
4.84
0.87
2.09
12.43 ***
5.19 *
3.42
3.08
7.69 *
6.83 **
6.84 *
8.16 *
Symbiotic parameters
Nodule %N
Leaf %N
Total plant N (mg g –1)
4.144
2.686
632.7
3.734
2.603
522.07
3.536
2.581
451.45
3.651 b
2.880 a
537.40
3.963 a
2.380 b
545.46
5.18
0.78
4.85
3.99 *
16.61 ***
0.00
8.57 *
0.61
8.68 *
Metabolite concentration
Root flavonoids (Abs g –1 )
Root anthocyanins (Abs g –1 )
Leaf anthocyanins (Abs g –1 )
152.31
1.376
0.388
163.26
1.529
0.386
154.56
1.327
0.321
159.62
1.528 a
0.420 a
156.79
1.302 b
0.291 b
0.17
2.01
4.51
0.80
8.31 *
6.65 *
11.42 **
0.90
6.64 *
Podalyria calyptrata
Seedling growth (g DM plant –1)
Leaf
Stem
Root
Total plant
10.963
6.160
10.836
29.580
9.535
5.592
10.759
27.220
10.696
6.061
10.302
28.594
8.565 b
4.469 b
8.092 b
22.576 b
12.230 a
7.406 a
13.173 a
34.353 a
1.09
0.69
0.33
0.64
1348 ***
18.45 ***
38.91 ***
21.06 ***
0.17
0.11
0.86
0.28
Symbiotic parameter
Total plant N (mg g –1)
552.6
506.92
542.65
450.94 b
612.94 a
0.51
11.15 ***
0.24
Metabolite concentration
Root soluble sugars (mg g –1 )
Leaf flavonoids (Abs g –1 )
17.952
179.05
15.339
179.49
19.500
182.64
17.222
189.00 a
17.971
171.75 b
3.48
0.08
0.00
3.89 *
6.28 *
3.75
Virgilia oroboides
Seedling growth (g DM plant –1)
Leaf
Stem
Root
Nodule
Total plant
Cyclopia maculata
Seedling growth (g DM plant –1)
Leaf
Stem
Root
Nodule
Total plant
was unaltered by exposure to either UV-B134 or UV-B166
(Figure 3C).
Tissue concentrations of flavonoids and other metabolites
Exposure of seedlings to above-ambient UV-B radiation did
not alter the concentrations of flavonoids and anthocyanins in
roots and leaves, or the concentrations of soluble sugars and
starch in roots of any of the species tested (Table 1). However,
application of NO3 to seedlings reduced (P ≤ 0.05) root and leaf
concentrations of anthocyanins in C. maculata (Table 1), and
decreased leaf flavonoids in P. calyptrata (Table 1). In contrast,
V. oroboides increased the concentration of flavonoids in
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phenolics such as root flavonoids and leaf anthocyanins in
C. maculata (Table 1), leaf flavonoids in V. oroboides (Table 1),
and soluble sugars in roots of P. calyptrata (Table 1). In purely
symbiotic seedlings, UV-B134 radiation increased (P ≤ 0.05) the
concentrations of leaf flavonoids in V. oroboides (Figure 3J)
and root flavonoids in C. maculata (Figure 3G), but reduced
root soluble sugars in P. calyptrata (Figure 3I). In NO3-fed
C. maculata, the concentration of root flavonoids was
unaltered by UV-B (Figure 3G), but the concentration of leaf
anthocyanins was reduced by both UV-B134 and UV-B166 (Figure 4H).
Below-ambient UV-B effects
Seedling growth Growing seedlings in chambers in belowambient UV-B radiation did not alter organ growth or total biomass in any of the study species (Table 2). Application of 1 mM
NO3 to the seedlings increased stem, root and total dry mass of
V. oroboides and P. calyptrata (Table 2). The interactions between UV-B22 radiation and N source on growth parameters
were significant (P ≤ 0.05) for dry mass of leaves and whole
seedlings of C. maculata and P. calyptrata, and root and nodule
dry mass of C. maculata and P. calyptrata, respectively (Table 2). Separating the UV-B22 treatment effect on each N source
showed that, relative to UV-Acont but not PARcont, UV-B22 radiation decreased (P ≤ 0.05) leaf dry mass of purely symbiotic
C. maculata and P. calyptrata, and total dry mass of the latter
species (Figures 4A, 4F and 4H). None of these growth parameters was altered in either purely symbiotic or NO3-fed V. oroboides exposed to UV-B22 radiation (data not shown).
However, symbiotically dependent seedlings of C. maculata
increased (P ≤ 0.05) root and total dry mass in response to exposure to UV-Acont relative to exposure to UV-B22, whereas
their NO3-fed counterparts were unchanged (Figures 4B and
4C).
Figure 3. Interactive effects of above-ambient UV-B and N on seedling growth, symbiotic function and metabolite concentrations in
purely symbiotic and NO3-fed nodulated seedlings of tree and shrub
legumes. Vertical lines on bars represent the standard error of mean.
Within an N source, different letters on bars indicate significantly different means at P ≤ 0.05. Abbreviations: ns = not significant;
UV-B100 = ambient ultraviolet-B radiation; UV-B134 = 34% aboveambient ultraviolet-B; and UV-B166 = 66% above-ambient ultraviolet-B.
leaves and the concentration of soluble sugars in roots in response to NO3 supply (Table 1). Above-ambient UV-B radiation and N source interacted significantly (P ≤ 0.05) for tissue
Symbiotic performance Exposure of C. maculata, V. oroboides and P. calyprata seedlings to UV-B22 radiation had no effect on components of symbiotic function, including nodule
dry mass and total N concentration (Table 2). However, leaf
%N increased (P ≤ 0.01) in C. maculata seedlings in the
UV-B22 treatment compared with the PARcont treatment, but not
relative to the UV-Acont treatment (Table 2). This was in contrast to nodule %N, which decreased (P ≤ 0.05) in C. maculata
relative to the UV-Acont treatment but not the PARcont treatment
(Table 2). Stem %N in P. calyptrata was also decreased (P
0.05) by UV-B22 compared with the UV-Acont treatment. Feeding seedlings with NO3 decreased nodule dry mass of
C. maculata and P. calyptrata. The %N in nodules and roots of
C. maculata as well as total N of V. oroboides increased with
NO3 supply (Table 2).
The interactions between UV-B22 radiation and N source on
symbiotic parameters were significant (P ≤ 0.05) for root %N
and total N in V. oroboides seedlings, total N concentration in
C. maculata seedlings, and nodule dry mass in P. calyptrata
seedlings (Table 2). Symbiotically dependent P. calyptrata
showed decreased nodule dry mass with UV-B22 exposure relative to exposure to UV-Acont but not to PARcont, whereas their
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187
Table 2. Effects of below-ambient UV-B radiation on seedling growth, symbiotic parameters and concentration of metabolites in purely symbiotic
and NO3-fed nodulated seedlings of tree and shrub legumes. Within UV-B and N source treatments, means that are significantly different are followed by different letters at: * = P ≤ 0.05, ** = P ≤ 0.01 and *** = P ≤ 0.001. Abbreviations: UV-B22 = 22% of ambient ultraviolet-B radiation;
UV-Acont = ultraviolet-A control; PARcont = photosynthetically active radiation control; and DM = dry mass. Measurements of components not
listed under various parameters in this table were not significantly different.
Parameter
Below-ambient treatments
N source
Wald χ2 statistic
PARcont
UV-Acont
UV-B22
Symbiotic-N NO3-N
UV-B
(df = 2)
N
(df = 1)
UV-B × N
(df = 2)
5.411
6.665
18.416
5.292
7.851
19.283
4.553
6.253
16.207
4.083 b
6.099 b
15.529 b
6.089 a
7.747 a
20.408 a
1.22
2.72
1.90
8.44 **
7.12 **
9.05 **
3.92
1.01
4.59
Symbiotic parameters
Root %N
Total plant N (mg g –1)
1.603
407.46
1.745
442.50
1.601
344.43
1.608
339.68 b
1.692
456.68 a
2.71
2.97
2.22
9.20 **
11.73 **
8.43 *
Metabolite concentration
Root flavonoids (Abs g –1 )
Root soluble sugars (mg g –1 )
61.307
13.800
52.929
13.492
50.588
15.036
52.320 b
12.275 b
57.562 a
15.937 a
2.10
0.14
5.53 *
7.54 **
8.74 *
1.28
Cyclopia maculata
Seedling growth (g DM plant –1)
Leaf
Root
Nodule
Total plant
4.480
4.949
0.463
12.618
5.009
4.992
0.496
14.120
3.959
4.707
0.469
12.370
4.467
4.749
0.564 a
13.118
4.497
5.002
0.394 b
13.012
1.15
0.14
0.40
0.78
0.31
0.31
8.90 **
0.03
11.02 *
5.92 *
0.09
6.45 *
Symbiotic parameters
Nodule %N
Root %N
Stem %N
Leaf %N
Total plant N (mg g –1)
4.265 b
1.749
0.969 b
2.229 b
229.11
4.859 a
1.847
1.117 a
2.813 a
295.97
4.278 b
1.908
1.061 ab
2.626 a
254.51
4.249 b
1.760 b
1.087
2.837 a
271.29
4.703 a
1.917 a
1.020
2.325 b
252.94
8.87 *
1.54
6.16 *
11.54 **
1.98
5.31 *
6.01 *
3.33
14.77 ***
0.05
0.88
3.82
1.35
1.92
6.60 *
Metabolite concentration
Root flavonoids (Abs g –1 )
Root anthocyanins (Abs g –1 )
212.86
35.546
189.24
33.351
206.44
36.132
220.00 a
37.010 a
185.14 b
33.070 b
2.43
2.07
7.84 *
4.33 *
6.93 *
0.43
Podalyria calyptrata
Seedling growth (g DM plant –1)
Leaf
Root
Nodule
Total plant
4.872
5.708
0.673
13.532
6.208
7.183
0.697
17.474
4.440
5.706
0.564
12.943
4.976
5.618 b
0.743 a
13.998
5.371
6.779 a
0.546 b
15.302
1.69
2.40
0.52
1.95
0.46
3.69
6.07 *
0.89
6.81 *
4.12
6.77 *
7.15 *
Symbiotic parameter
Stem %N
1.188 b
1.348 a
1.187 b
1.253
1.229
6.02 *
0.07
2.14
Metabolite concentration
Root anthocyanins (Abs g –1 )
Root soluble sugars (mg g –1 )
Leaf flavonoids (Abs g –1 )
0.494
13.592
119.35 b
0.500
13.158
114.51 b
0.560
13.975
139.19 a
0.567 a
15.789 a
123.48
0.469 b
11.361 b
123.14
1.46
0.19
31.60 ***
5.37 *
8.84 **
1.10
1.27
0.34
1.10
Virgilia oroboides
Seedling growth (g DM plant –1)
Stem
Root
Total plant
NO3-fed counterparts remained unaltered (Figure 4G). Root
%N of V. oroboides seedlings relying solely on symbiotic N increased (P ≤ 0.05) with exposure to UV-B22 relative to exposure to PARcont but not to UV-Acont. This was in contrast to
NO3-fed seedlings, which showed reduced root %N relative to
both UV-Acont and PARcont (Figure 4I). Total plant N of symbiotically fed C. maculata seedlings increased with exposure to
UV-Acont relative to exposure to PARcont, but not to UV-B22
(Figure 4D). Virgilia oroboides seedlings showed reduced (P ≤
0.05) total plant N with exposure to UV-B22 relative to both
UV-Acont and PARcont, unlike their symbiotically dependent
counterparts, which remained unchanged (Figure 4J).
Tissue concentrations of flavonoids and metabolites
Concentrations of flavonoids and anthocyanins in roots and leaves
of the test species were unchanged by UV-B22 radiation, except
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188
CHIMPHANGO, MUSIL AND DAKORA
for leaf flavonoids in P. calyptrata, which increased (P ≤ 0.001)
(Table 2). Root concentrations of soluble sugars and starch in
the test species were unaffected by exposure to UV-B22 radiation (Table 2). However, NO3 supply decreased root concentrations of anthocyanins in C. maculata and P. calyptrata
(Table 2). The concentration of flavonoids in roots of C. maculata also decreased with NO3 provision in contrast to NO3-fed
V. oroboides where root flavonoid concentration increased
considerably (Table 2). Leaf concentrations of flavonoids and
anthocyanins were unaltered by NO3 provision in all species
tested (data shown only for P. calyptrata). Concentrations of
soluble sugars increased (P ≤ 0.05) with NO3 application in
roots of V. oroboides, but decreased in roots of P. calyptrata
(Table 2). The interaction between UV-B22 radiation and N
source was significant (P ≤ 0.05) only for root flavonoid concentrations in C. maculata and V. oroboides (Table 2). Relative
to PARcont but not UV-Acont, UV-B22 decreased root flavonoid
concentrations in both C. maculata and V. oroboides seedlings
relying solely on symbiotic N, whereas root flavonoid concentrations of their NO3-fed counterparts were unaltered (Figures 4E and 4K).
Discussion
Above-ambient UV-B effects on seedling growth and
symbiotic function
Figure 4. Interactive effects of below-ambient UV-B and N on seedling growth, symbiotic function and metabolite concentrations in
purely symbiotic and NO3-fed nodulated seedlings of tree and shrub
legumes. Vertical lines on bars represent the standard error of mean.
Within an N source, different letters on bars indicate significantly different means at P ≤ 0.05. Abbreviations: ns = not significant; UV-B22
= 22% of ambient ultraviolet-B radiation; UV-Acont = ultraviolet-A
control; and PARcont = photosynthetically active radiation control.
Exposure to above-ambient UV-B radiation decreased leaf and
stem dry mass of C. maculata seedlings by 34 and 39%, respectively, but had no effect on these parameters in P. calyptrata or V. oroboides (Table 1). At the whole-plant level,
however, there were no major differences in response to
above-ambient UV-B among the test species. Several studies
(Laakso and Huttunen 1998, L’Hirondelle and Binder 2002)
have shown that plants differ in their UV-B sensitivity and that
species’ tolerance of UV-B may be genetically determined.
Because tissue accumulation of UV-B absorbing flavonoids
reduces UV-B damage (Bieza and Lois 2001), the increased
concentration of flavonoids in leaves of C. maculata seedlings
exposed to UV-B166 radiation may constitute a mechanism that
overcomes the damaging effects of UV-B radiation. Cyclopia
maculata was more sensitive to UV-B than V. oroboides and
P. calyptrata, suggesting that, in this species, the greater tissue
concentration of flavonoids was not directly linked to defense
against UV-B radiation (Hunt and Kelliher 1996). Although
leaf and stem growth in C. maculata were altered by UV-B exposure, root and total dry mass were unchanged, indicating altered biomass allocation in response to UV-B treatment
(Sullivan et al. 1994, Schumaker et al. 1997). The lack of
growth response by V. oroboides (a tree) and P. calyptrata (a
shrub) to above-ambient UV-B is consistent with data obtained
for Acacia karroo (Wand et al. 1996, Ernst et al. 1997), a
nodulating tree legume common to the African savanna.
Symbiotic performance of V. oroboides and P. calyptrata
seedlings was unaffected by exposure to above-ambient
UV-B. For example, nodule dry mass and total plant N were
unchanged by elevated UV-B (Table 1), as observed for other
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UV-B EFFECTS ON AFRICAN TREE AND SHRUB LEGUMES
African tree legumes (Wand et al. 1996, Ernst et al. 1997). In
contrast, Singh (1997) observed depressed symbiotic function
in several species of tropical grain legumes exposed to elevated UV-B. Although the inconsistencies in symbiotic response could be attributed to genotypic differences in UV-B
sensitivity (Jansen et al. 1998), we speculate that it was more
likely associated with the high UV-B irradiance used by Singh
(1997)—a daily dose of 10.28 kJ m –2 day –1 over a 2-h daily
exposure period versus our daily dose of 4.50 or 8.05 kJ m –2
day –1 over an 8-h daily exposure period.
Supplying nodulated seedlings with 1 mM NO3 mimicked
field conditions where legumes often depend on soil N and
symbiotic N for their N nutrition. The extra N provided as NO3
to nodulated tree and shrub species exposed to above-ambient
UV-B in our study significantly (P ≤ 0.05) increased overall
growth by 47% in V. oroboides and 52% in P. calyptrata, and
insignificantly increased overall growth by 15% in C. maculata compared with purely symbiotic seedlings (Table 1). Although plant components such as leaves, stems and nodules
contributed to total biomass, root growth was consistently
much greater (P ≤ 0.05) in NO3-fed seedlings than in their
purely symbiotic counterparts (Table 1). This difference may
reflect the signaling function of NO3 (Crawford 1995). Exposure of seedlings to low NO3 concentrations (e.g., 1.0 mM) increases root biomass and lateral root development (Drew et al.
1973). The symbiotic response to NO3 supply differed among
the study species. For example, NO3 application depressed
nodule development in C. maculata, increased it significantly
(P ≤ 0.05) in V. oroboides, but had no affect on nodule development in P. calyptrata. These results are inconsistent with the
findings of several other studies where supplemental N had little effect on plant growth and symbiotic performance
(Hill-Cottingham and Lloyd-Jones 1980, Ma et al. 1997).
The increase in growth of NO3-fed seedlings of V. oroboides
and P. calyptrata was accompanied by a significant (P ≤ 0.05)
increase in total N concentration (Table 1). However, in
C. maculata, where NO3 had no effect on growth (Table 1),
leaf and stem N concentrations decreased (P ≤ 0.05) with NO3
feeding (Table 1). There was also a decrease in concentrations
of flavonoids and anthocyanins in leaves and roots of NO3-fed
seedlings of C. maculata and P. calyptrata (Table 1). Such reductions in tissue flavonoid concentrations with NO3 provision have been observed previously in soybean (Glycine max
(L.) Merr. (Cho and Harper 1991) and cucumber (Hunt and
McNeil 1998), and are apparently caused by a generalized
down-regulation of the phenylpropanoid pathway, which results in decreased concentrations of defense molecules
(Bryant et al. 1983). Based on the results of these studies, we
suggest that the decreased nodulation in NO3-fed seedlings of
C. maculata is associated with NO3 inhibition of nodule formation (Streeter 1988) as a result of a lowered synthesis of nod
gene inducers by roots (Cho and Harper 1991). Although the
mechanism of NO3 inhibition of the phenylpropanoid pathway
remains unknown, it is probably related to a reduction in C and
N supply to cells. For example, if C and N metabolism competes for ATP, NADPH and C skeletons needed for synthesis
189
of organic acids and carbohydrates on the one hand, and amino
acids on the other, it will influence the shikimate pathway,
thereby affecting the formation of phenylalanine lyase (PAL).
Because PAL is the enzyme catalyzing the first step of the
phenylpropanoid pathway, a reduction in PAL synthesis will
decrease tissue concentration of phenolics such as flavonoids
and anthocyanins.
Below-ambient UV-B effects on seedling growth and
symbiotic function
The UV-B22 treatment did not alter biomass accumulation, either on a whole-plant or individual-organ basis in any of the
test species (Table 2). The lack of response to UV-B22 radiation
by C. maculata, which showed reduced leaf and stem growth
in response to above-ambient UV-B, suggests growth adaptation at below-ambient to ambient thresholds. These results are
consistent with data obtained for UV-B exclusion and attenuation studies on trees and other species (Tosserams et al. 1996,
Schumaker et al. 1997), but contrast with results of increased
biomass when UV-B radiation was excluded from the solar
spectrum (Bogenrieder and Klein 1982, Sharma et al. 1991).
These inconsistencies in plant growth response to below-ambient UV-B radiation could be purely genetic, or attributable to
differences in materials used in UV-B attenuation studies, differences in the period of exposure to UV-B (Schumaker et al.
1997), or differences in ambient UV-B among study sites
(Tosserams et al. 1996).
Because seedling growth was not altered by exposure to
UV-B22 radiation, symbiotic performance was also unaffected
by UV-B22 (Table 2). However, relative to the UV-Acont treatment but not the PARcont treatment, nodule %N of C. maculata
seedlings and stem %N of P. calyptrata seedlings were markedly reduced (Table 2). In contrast, %N in leaves of C. maculata seedlings in the UV-B22 treatment was significantly
increased relative to the PARcont treatment but not the UV-Acont
treatment, suggesting that N uptake, translocation or storage in
different plant organs was affected by the attenuation of UV-B
radiation in the chambers. These changes may be associated
with several factors in our experimental system such as the
decreased UV-B and UV-A radiation, the 0.8 °C decrease in
temperature between the PARcont and UV-Acont treatments, or
the altered spectral composition of light in the chambers (Figure 2B). However, because air temperatures in the UV-B22 and
UV-Acont chambers were similar, and changes in %N of nodules in C. maculata and %N of stems in P. calyptrata were different in the UV-Acont treatment relative to both the PARcont and
UV-B22 treatments, and because %N in stems and leaves of
C. maculata were different in the UV-Acont treatment relative
to the PARcont treatment but not the UV-B22 treatment (Table 2), changes in temperature and PAR could not have accounted for the observed differences. It is therefore likely that
the observed effects were a result of UV-A radiation. However,
the increase in leaf flavonoid concentration in P. calyptrata
seedlings exposed to UV-B22, relative to both UV-Acont and
PARcont, could be a direct effect of UV-B22 radiation, a result
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CHIMPHANGO, MUSIL AND DAKORA
consistent with other UV-B attenuation and exclusion studies
(Lovelock et al. 1992, Searles et al. 1995).
Application of 1 mM NO3 to the test species in the UV-B22
treatment increased (P ≤ 0.05) root dry mass of V. oroboides
and P. calyptrata seedlings. Furthermore, NO3 supply to
V. oroboides under below-ambient UV-B radiation significantly (P ≤ 0.05) increased whole-plant N (Table 2), leading to
an increase in stem, root and total dry mass (Table 2) and an increase in the concentrations of flavonoids and soluble sugars
in roots (Table 2). These increases with NO3 supply show that
nodule function in purely symbiotic seedlings was unable to
meet the different metabolic demands of seedlings exposed to
UV-B radiation. However, the decrease in nodule dry mass of
P. calyptrata fed with NO3 suggests inhibition of nodulation
by exogenous N (Del Pilar Cordovilla et al. 1999). Root concentrations of anthocyanins decreased (P ≤ 0.05) with NO3
feeding in both C. maculata and P. calyptrata. Flavonoids
were similarly decreased in NO3-fed C. maculata but increased (P ≤ 0.05) in roots of V. oroboides. The reduction of
flavonoids or anthocyanins, or both, with NO3 feeding is consistent with down-regulation of the phenylpropanoid pathway
(Bryant et al. 1983).
UV-B × N interactions
Stem, root and total dry mass, nodule %N and total plant N of
purely symbiotic C. maculata were all depressed by treatment
with UV-B166 relative to UV-B134 and UV-B100. Nitrate application also resulted in consistently decreased amounts of dry
mass in stems, roots, nodules and whole plants, as well as reductions in nodule %N and total plant N in the UV-B166 and
UV-B134 treatments relative to the control. Although purely
symbiotic C. maculata seedlings were sensitive to UV-B166 radiation, adding NO3 further increased their sensitivity to both
UV-B134 and UV-B166, an observation consistent with the results of N and P supply to plants exposed to above-ambient
UV-B (Murali and Teramura 1985, Hunt and McNeil 1998,
Correia et al. 2000). Root flavonoids of purely symbiotic seedlings were also low in the UV-B166 treatment relative to the
UV-B134 treatment, but increased (P ≤ 0.05) with NO3 feeding
in seedlings exposed to UV-B166 relative to UV-B134 or
UV-B100. It is not known if this is a result of PAL induction by
NO from NO3 (Wendehenne et al. 2001, Stöhr and Ullrich
2002). In contrast, leaf anthocyanins in NO3-fed C. maculata
seedlings were markedly decreased by UV-B166 relative to
UV-B134, which in turn was decreased compared with the
UV-B100 treatment. In contrast to above-ambient UV-B conditions, NO3 feeding under below-ambient UV-B had no effect
on leaf, root and total plant mass. However, purely symbiotic
C. maculata seedlings increased their leaf and total dry mass
and total N in the UV-Acont treatment relative to the UV-B22 and
PARcont treatments. Leaf, nodule and total dry mass of P. calyptrata seedlings were similarly increased in response to
UV-Acont radiation compared with the UV-B22 and PARcont
treatments. Root flavonoids of C. maculata and V. oroboides,
however, were decreased by UV-Acont and UV-B22 relative to
PARcont.
Because flavonoids protect plant cells against UV-B damage (Mazza et al. 2000, Bieza and Lois 2001), their reduced
concentrations in tissues from NO3 feeding would be expected
to increase sensitivity to UV-B (Sheahan 1996). Although
flavonoid concentrations in the leaves of P. calyptrata seedlings decreased with NO3 feeding (Table 3), a result consistent
with reports by Khanna et al. (1999) and Cho and Harper
(1991), the species was not adversely affected by above-ambient UV-B radiation. This suggests that other mechanisms are
involved in plant sensitivity to UV-B radiation, such as increased UV-B degradation of photosystem II proteins (Greenberg et al. 1989), which are reported to increase with N supply
(Kolber et al. 1988).
In conclusion, exposing V. oroboides and P. calyptrata seedlings to above-ambient UV-B radiation did not affect their
growth and symbiotic function. However, feeding 1 mM NO3
to these two species increased seedling growth. Purely symbiotic seedlings of C. maculata were sensitive to above-ambient
UV-B radiation, a response that increased with NO3 supply.
Our findings suggest that the impact of anticipated increases in
UV-B radiation on legume growth and symbiotic function of
sensitive species will be more severe in agricultural ecosystems subjected to NO3 application and in natural ecosystems
subjected to N deposition from anthropogenic activities.
Acknowledgments
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 National Research Foundation for grants to FFD. We
also thank Ms. J. Arnolds and Mr. S. Snyders for technical assistance.
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