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Influence of photosynthetically active radiation and spectral quality on UV-B-induced polyamine accumulation in soybean
003l-9422/,92 $5.00 + 0.00 PergamonPressplc
Vol. 31,No. 4, pp. 11191125,1992 Phytocbemistry, Printedin GreatBntain.
INFLUENCE OF PHOTOSYNTHETICALLY ACTIVE RADIATION AND SPECTRAL QUALITY ON UV-B-INDUCED POLYA~~NE ACCU~ULAT~QN IN SOYBEAN GEORGE F. KRAMER, DONALD T. KRIZEK
and ROMAN M. MXRECKI
Climate Stress Laboratory, Agricultural Research Service, U.S. Department of A~culture,
Beltsville, MD 20705, U.S.A.
(Received in revisedform 10 September 1991)
Key Word Index-Glycine
max; Fabaceae; soybean; polyamines; photosynthetically response; UV-B radiation; photoprotection.
active radiation; stress
Abstract-UV-B-sensitive (Essex) and -insensitive (Williams) cultivars ofsoybean (G&&e mux) were grown in growth chambers at photosynthetically active radiation (PAR) levels of 300 or 600 pmol m-2 see- t provided by either redand far-red-deficient (MH) or blue-deficient (HPS/DX) lamps or a combination of both. The combined treatment provided a balanced output, similar to that provided by fluorescent plus incandescent lighting across the visible spectrum. Under the combined lamps, plants were exposed to 12 kJm-‘day-’ of biologically effective UV-B (UVB,,) with 6-hr irradiance periods centred midway through the photoperiod. This irradiance corresponded to a decrease in stratospheric ozone of ca 20% for clear sky conditions at Beltsville, MD on 21 June. Plant growth was significantly inhibited by UV-B at 300 but not at 600 pmol mm2 set- ’ PAR. No cultivar differences were noted in the UV-B-induced inhibition of growth, although visible injury was less in Williams than in Essex. PAR had a large effect on polyamine levels in leaves, with higher levels of putrescine (Put) and spermidine observed at 600 than at 300 i*molm-* set-’ in both cultivars. ~-B-indu~d polyamine a~umu~ation was observed primarily in Williams. Under MH or HPS/DX lamps alone, plants were exposed to two different UV-B levels, 9.9 and 12 kJm-‘day-“, corresponding to stratospheric ozone reductions of ca 9 and 20%. UV-B inhibited growth at both 300 and 600 ~01 m- ’ set- 1 PAR under either radiation source. There was no effect of PAR on the UV-B-induced growth inhibition with the HPS/DX lamps, but a partial amelioration of this inhibition occurred in Williams at 600 pmol m-* set- ’ PAR under MH lamps. Dose-de~ndent UV-~-indu~d polyamine ~eumulation was also observed in both cultivars. PAR increased Put levels under MH but not HPS/DX lamps. These results indicate that the inhibition of UV-B stress by high PAR may require a balance of red and blue wavelengths and may involve polyamine accumulation.
Human activities which release halogenated hydrocarbons and result in the depletion of the stratospheric ozone layer are expected to increase the amount of solar UV-B (290-320 nm) radiation that reaches the surface of the Earth [ 11. Recent evidence indicates that terrestrial UVB fluxes at high elevations have increased by 1% per year since 1980 [2]. Increased UV-B adversely affects the growth of a number of plant species, with UV-B-dependent reductions in yield having been observed in several ~onomical~y ~~rtant crops [1, 3, 4]. The deleterious effects of UV-B may involve inhibition of photosynthesis F-7$ Photosynthetically active radiation (PAR, 400700 nm) levels have a profound impact on plant sensitivity to UV-B, with high levels decreasing UV-B-de~ndent damage [8-123. Growth under high PAR results in thickening of the epidermis and higher levels of flavonoids in the leaves [S, 10, 111. Such effects would tend to lessen the sensitivity of the plant to UV-B [S, 8, 13-173. However, soybean grown under low PAR and exposed to UV-B under high PAR are less sensitive to UV-B than plants grown under low PAR and then irradiated with UV-B under low PAR [S]. The high PAR must thus
facilitate a biochemical response to UV-B which ameliorates the stress-induced damage. The relative importance of differing wavelengths of radiant energy in eliciting this PAR response has not been investigated. A possible significance of spectral quality is demonstrated by the modulating effect of red and blue wavelengths on UV induction of pigment biosynthesis [18, 193. Polyamines may play a role in protecting plants from UV-B stress 1201. Polyamines possess a number of antisenescent activities and are induced in response to many stressors in plant systems {21,22]. Polyamin~ can bind to membrane surfaces via ionic interactions with phospholipids and act to inhibit lipid peroxidation [23, 243. Membrane damage appears to be involved in the phytotoxicity of enviro~ent~ stressors such as ozone [25] and shilling [26]. Direct app~~ation of polyamines to plant tissue has been shown to inhibit the development of injury induced by ozone [27, 283 and chilling [29, 301. There is also evidence that membrane damage may be involved in UV-B stress [20, 31-331. The inhibition of photosynthesis by UV-B may involve the disruption of chloroplast membrane structure [7,31,34]. Upon UV-B exposure, the total levels of chlorop~ast lipids decrease and the ratio of the individual polar lipids is altered in
1119
G. F. KRAMER et al.
1120
several species [32]. UV-B radiation induces accumulation of lipid peroxidation products in cucumber leaves concomitant with a reduction in the ratio of unsaturated/saturated fatty acids [20]. The magnitude of this effect was correlated with cultivar sensitivity to UV-B. Increased polyamine levels also resulted from UV-B exposure in cucumber [20]. The purpose of the present study was to investigate the influence of UV-B radiation on polyamine levels in two cultivars of soybean found previously to differ in UV-B sensitivity [17, 353 (Essex-W-B sensitive and WilliamsUV-B insensitive) as measured by leaf area and biomass accumulation and to determine whether polyamines may be involved in the enhanced resistance to UV-B resulting from high PAR conditions. The relative importance of red and blue photoreceptors in polyamine and PAR responses to UV-B was investigated by comparing the effects of UV-B under conditions deficient in blue (high pressure sodium deluxe, HPS/DX) and red and far-red radiation (metal halide lamps, MH).
160
B.
r,
RESULTS
1
L.
U V-B effects under a combination of MH and HPS! DX lamps
The first set of experiments involved the use of a combination of MH and HPS/DX lamps as a PAR source and a single dose of 12 kJm-2day-1 of biologically effective UV-B radiation (W-B,,) (which corresponded to a decrease in stratospheric ozone reduction of ca 20%). The plants were grown for two weeks from seeding before the start of UV-B treatment and then irradiated with UV-B for two weeks. Inhibition of mainstem elongation by UV-B was seen only at low levels of PAR (Fig. 1). No significant cultivar differences in the growth responses to UV-B were observed. In the leaves emerging under UV-B, visible injury (in the form of chlorotic flecks) was observed (data not shown). In Williams, this injury was less severe at high PAR than at low PAR. Visible injury in Essex was greater than in Williams and not significantly lessened by high PAR treatment.
600 -1
ISJWillioms +lJV BglBEssex -UV IEssex +UV
400 500
PAR
(pmol
m-*
s-j)
Fig. 1. Effect of UV-B,, radiation (12 kJm_‘day-‘) simulating an ozone depletion of ca 20% at two different PAR levels (300 and 600~molm~2sec~‘) on mainstem length of Williams and Essex soybean plants after two weeks of treatment. PAR source consisted of a combmation of MH and HPS/DX lamps. Error bars represent T s.e.
600
300
PAR (Fmol
rnH2
s-‘)
Fig. 2. Effect of UV-BB, radiation (12 kJm--2 day-‘) simulating an ozone depletion of ca 20% at two different PAR levels on polyamine levels in the first (300 and 600~molm-2sec-‘) trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. PAR source consisted of a combination of MH and HPS/DX lamps. Panel A is Put, B is Spd and C is Spn. Error bars represent +s.e.
The effect of elevated UV-B on polyamine levels in the first trifoliolate leaf above the primary leaves is shown in Fig. 2. These leaves were fully expanded at the start of the UV-B treatments. There was a significant effect of PAR on polyamine levels (Table 1A) with higher levels of putrescine (Put) and spermidine (Spd) accumulating in the presence of 600 pmol rn- ’ set- ’ PAR. A significant UV x PAR interaction resulted from the tendency of UVB to decrease polyamine levels at 300 pmol me2 set-’ PAR while at 600 pmol m -2 see- ’ PAR, polyamines increased or remained unchanged. UV-B had a greater positive effect on polyamine levels at high PAR in Williams than in Essex. Polyamine levels were also determined in a leaf which had emerged in the presence of UV-B (fourth fully expanded trifoliolate above primaries, Fig. 3). PAR was again seen to significantly increase the levels of Put and Spd (Table 1B). UV-B radiation-induced increases in Put at 300 and 600 pmol me2 see- ’ PAR and in Spd at 300 pmol mm2 sec.. 1 PAR were seen in Williams, but not in Essex (Fig. 3).
Influence of PAR and UV-B on polyamines
1121
Table 1. F-value and associated probability(p) for polyamines in the first and fourth trifoliolate leaf of soybean grown under MH and HPS/DX lamps combined (data for Williams and Essex cultivars are combined) Putrescine _.___~ Effect
F-value
A. First trifoliolate leaf PAR 15.4 uv ns. cv n.s. PAR x UV 15.6 PAR x CV n.s. uvxcv 10.2 PARxUVxCV n.s. B. Fourth trifoliolate leaf PAR 1.7 uv n.s. cv 7.8 PAR x UV n.s. PAR x CV ns. UVXCV 5.5 PARxUVxCV n.s.
Spermidine -__
Spermine ~
_.~.
p
F-value
p
F-value
p
0.002
5.1 ns. 13.8 8.7 n.s. n.s. n.s.
0.040
5.1 ns. 7.8 19.1 n.s. n.s. ns.
0.038
0.002 0.006
0.010 0.010
0.028 _
38.9 n.s. n.s. n.s. 4.5 n.s. n.s.
0.002 0.010
0.0001 .-. 0.046 _ -
n.s. ns. 8.1 17.4 n.s. ns. 6.5
.~
0.013 0.001
_ 0.009 0.0003
0.018
n.s. = not significant at p < 0.05.
UV-B effects under MH or HPSfDX
lamps alone
The second set of experiments involved the use of either MH or HPS/DX lamps alone as a PAR source and two UV-B,, doses of 9.9 and 12 kJ mm2 day-’ (corresponding to stratospheric ozone reductions of ca 9 and 20%). Treatments were initiated at the time of seeding. Overall effects on vegetative growth were determined by expressing the height, leaf area, fresh weight, and dry weight as a per cent of the control (no UV-B) and averaging these percentages together. The UV-B treatments had a significant inhibitory effect on growth as measured by these parameters under both PAR sources (Fig. 4). Measurement of height alone at this time was inadequate to demonstrate the effects of UV-B, as the shortness of the plants in some treatments made the stress-induced differences in height difficult to detect. After another seven days of elongation, the effects of UV-B on height were consistent with the combined growth parameters measured at 14 days (Krizek et al., in preparation). There was no PAR under HPS/DX lamps, as effect of the inhibition of growth was not greater at 300~olm-2sec-1 than at 600~molm-2sec-‘. With MH lamps, a partial amelioration of UV-B-induced growth reduction was observed in Williams. Plants appeared to be more sensitive to UV-B radiation under MH than under HPS/DX lamps at 300 pmol mm2 set- ’ PAR. Only traces of visible UV-B injury were observed. Few cultivar differences were observed, although the growth of Essex was inhibited by 12 kJm_’ day-’ UV-B,, under MH lamps with 600 pmol m- ’ set- ’ PAR to a slightly greater extent than was Williams. The effects of UV-B irradiation on polyamine levels are shown in Fig. 5 (MH) and Fig. 6 (HPS/DX). Significant UV-B effects were seen in Put, Spd and spermine (Spn) (Table 2) with the highest UV-B level generally eliciting the greatest induction of polyamines. When the data for lamp type were combined for analysis, there were no PAR
effects seen, but the type of lamp did have a significant effect on polyamine levels (Table 2), with Put and Spd levels being higher under HPS/DX and Spn being higher under MH lamps. When the data for each lamp type were analysed separately, a PAR effect was seen only in Put under MH lamps (Table 3B). Cultivar differences were also observed, with higher polyamine levels generally being found in Essex than in Williams, especially in the highest UV-B treatment (Table 2, Figs 5 and 6). DISCUSSION
The effects of UV-B on plant growth and polyamine levels in soybean were greatly influenced by PAR levels when plants were grown under a combination of MH and HPS/DX lamps. This radiation source provided a balanced output across the visible spectrum and was nearly comparable in spectral power distribution to that emitted by a combination of fluorescent and incandescent lamps. Exposure to UV-B radiation significantly reduced mainstem length at 300 pmol me2 set- ‘, while no effect was seen at 600 pm01 m-’ set - I PAR. This result is consistent with findings of other workers who have demonstrated protection from UV-B by high PAR [8-121. This ability to ameliorate UV-B damage has been correlated with the ability of high PAR levels to induce pigment formation and alter leaf morphology such that UV-B transmittance through the epidermis is reduced [8,10,22]. The observation that leaves grown at low PAR were less sensitive to UV-B radiation when exposed under high PAR than under low PAR indicates that biochemical mechanisms of photoprotection and/or photorepair were also activated by high PAR [S, 361. Our results indicate that polyamines could be involved in this photoprotective effect of high PAR. In leaves which expanded in the absence of UV-B and were subsequently exposed for two weeks, the levels of Put and Spd were significantly higher at 600 than
G. F.
1122
KRAMER
et al.
5
60
-5 5
60
V x
40
B. si 300 L; ISI 250 \
:
60
$
0 E
200
i? V
60
=
150
8?
40
a Lr) -
100
7
20 50 0 L.
150
<
125
c F
n. vt
75 50 25
-
0
rn-’
s-l)
Fig. 4. Effect of UV-B,, radiation (9.9 and 12 kJm -*day-‘) simulating an ozone depletion of ca 9 and 20% at two different PAR levels(3OOand 600 pmol m-2sec-‘)ongrowth of Williams and Essex soybean plants after two weeks of treatment. The height, dry weight, leaf area and fresh weight were expressed as a per cent of the nor&V-B-treated controi and then averaged together. UV-1 is 9.9 and UV-2 is 12 kJm-‘day-* UV-Baa. PAR source consisted of MH (panel A) or HPS/DX lamps (panel B). Error bars represent 2s.e.
100 _
600
300 PAR (pmol
175 2 (i
5 E
HPS
: 300
PAR (pmol
>
600 a
rneL s-t)
Fig. 3. Effect of UV-B,, radiation (12 kJm-2day-1) simulating an ozone depletion of ca 20% at two different PAR levels (300 and 600 pmol m- 2 set- “) on polyamme levels in the fourth trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. PAR source consisted of a combination of MH and HPS/DX lamps. Panel A is Put, B is Spd and C is Spn. Error bars represent &se.
300 pmol mF2sec- r PAR. In leaves which had expanded after the initiation of UV-B treatment, the levels of Put and Spd were also higher at 600 pmol me2 set-’ PAR. The formation of anthocyanins and flavonoids is modulated by red and blue wavelengths, indicating that both phytochrome and the blue/UV photoreceptors are involved in this response [lS, 19-J. Photomorphogenic responses to UV are also regulated by both phytochrome and UV photoreceptors [3’7]. These obse~ations could suggest that the ameliorative effects of PAR on UV-B injury may be modulated by spectral quality. We have tested this hypothesis by comparing the effects of PAR on UV-B sensitivity under MH (red- and far-red-deficient) and HPS/DX (blue-deficient) lamps. Under both radiation sources and at both 300 and 600pmolm-2 set-r PAR, W-B inhibited growth. The higher level of PAR had no protective effect in comparison with the lower PAR level, except for Williams under MH lamps, where partial protection from UV-B injury was observed. Polyamines were induced by UV-B radiation generally in a
dose-dependent fashion under both radiation sources and at both PAR levels. Overall, PAR source had a significant effect on polyamine levels, with Put and Spd being higher under HPS/DX lamps and Spn being higher under MH lamps. This finding is consistent with the reported ability of phytochrome to regulate the activity of the polyamine biosynthetic enzyme arginine decarboxylase [38]. It thus appears that separating the MH and HfS/DX radiation sources abolished or reduced the protective effect of high PAR which was observed when the two lamps were combined. The photoprotective effects of high PAR may require a balanced output throughout the visible spectrum. In these experiments, we also compared the responses of two cultivars of soybean thought to differ in UV-B sensitivity-Williams (UV-B insensitive) and Essex (UV-B sensitive) [17]. In terms of vegetative growth responses, we saw little difference in these cultivars in experiments with either combined or separate lamps. The only major difference in W-B sensitivity observed was in visible injury under the combined PAR source where Williams was injured less than Essex. Cultivar differences in the effect of UV-B on polyamine levels were noted. Under the combined PAR source, a greater induction of polyamines in response to UV-B was observed in Williams than in Essex. The reduced visible injury in Williams as compared to Essex was thus correlated with the ability to induce polyamine accumulation in response to UV-B. Increased polyamine levels have been correlated with the inhibition of the development of
Influence of PAR and UV-B on polyamines
1123
Table 2. F-value and associated probability (p) for polyamines in the first &ifoliolate leaf of soybean grown under MH and HPS/DX lamps alone (data for Williams and E!ssexcultivars are combined; two-, three- and four-way interaction terms which were not significant for any of the polyamines were omitted)
_
Putrescine E&et
F-value
-I_ p
A. MH and HPS/DX data combined 0.002 L=np 14.4 PAR ns. 0.005 uv 8.4 0.0001 cv 15.5 -uvxcv n.s. 0.001 PAR x UV x CV 7.4 B. MH data alone 0.041 PAR 6.7 UV n.s. 0.0001 CV 21.1 0.003 PAR x CV 9.4 wxcv n.s. C. HPS/DX data alone PAR ns. 0.046 uv 5.4 cv ns. 0.010 PAR x CV 7.2 0.046 PARx WxCV 5.4
ozone-de~ndent visible injury in tobacco and barley [28, 391. However, as UV-B radiation affected the growth of both cultivars equally, the amount of UV-B injury sustained by Essex did not appear to have an adverse effect on plant growth, at least in the time frame of this experiment. This difference in IN-B-dependent polyamine accumulation was also dependent on spectral quality as a greater enhancement of polyamine levels was seen in Essex than in Williams under MH or HPS/DX lamps alone. As the combined PAR source more closely resembles natural conditions, the greater UV-B induction of polyamines in Williams under these conditions could be physioiogically relevant. Further experiments are necessary to determine whether the reported differences in UV-B sensitivity in these cultivars could involve differences in polyamine accumulation. The lack of a difference between these cultivars in UVB inhibition of growth is somewhat surprising, as changes in flavonoid content and leaf morphology have been reported [17]. However, in other experiments, there was little difference in the inhibition of vegetative growth by UV-B between Williams and Essex [40, 411. Also,, the relative difference in UV-B sensitivity of these two cultivars, as measured by seed yield, appears to be dependent on microclimatic conditions (i.e. rainfall, temperature, and cloudiness) [35]. Our experiments could have been performed under conditions in which the cultivar differences in UV-B sensitivity were not expressed, or W-B exposures of greater duration may have been required to see these effects. We have shown that polyamine levels are correlated with the ability of high PAR levels to induce resistance to UV-B. Under conditions where a protective effect of high PAR was observed (combined MH and HPS/DX tamps),
_~
Spermidine
__
Spermine -
p
F-value
p
6.5
0.025
I% 8.0 6.4 n.s.
0.0001 0.006 0.002
13.8 n.s. 5.9 45.1 6.4 n.s.
0.003 0.016 0.0001 0.002 -
ns. 8.6 13.1 ns. 4.4
0.017 0.001
ns. 5.7 34.6 ns. 7.9
0.041 0.0001 0.001
n.s. ns. 15.0 n.s. n.s.
.0.0003 -
F-value
n.s. 18.8 ns. ns. n.s.
0.017
0.003 -
PAR increased the levels of po~yamin~. Under conditions where no PAR effect was seen on UV-B sensitivity (HPS/DX lamps alone), polyamine levels were not affected by PAR. Under MH lamps alone, where high PAR provided partial protection against UV-B inhibition of growth in Williams, PAR increased the levels of Put. However, in Essex, photoprotection was not observed even though high PAR increased Put, indicating that lack of photoprotection in Essex is unrelated to Put content. These differences in the effect of PAR on Put between MH and HPS/DX lamps may indicate that blue wavelengths are more important than red wave~en~hs in the induction of Put ~umuIation by high PAR. A possible role of polyamines in protecting against W-B stress is consistent with the apparent ability of these compounds to protect plant cells from ozone [27, 281 and chilling [29, 30, 421 stress. The anti-ozonant activity of polyamines may involve conjugation with hydroxycinnamic acids as these conjugates appear to be effective free radical scavengers [28]. The photoprotective pigments synthesized by plants in response to W-B are derived from cinnamic acid [3]. The induction of polyamine conjugate formation by UV-B is thus a possible response to this stress, as the fo~ation of both precursors is promoted by UV-B. Further studies are necessary to demonstrate conclusively whether polyamines act in uiuo to protect plants from W-B stress.
EXPERIMENTAL
Plant material.Two cultivars of soybean (Glycine mnx (L.) Merr.) reported to differ in sensitivity to UV-B radiation [17, 35-j--Williams, a relatively W-B insensitive cultivar and Essex, a highly sensitive cultivar-were used.
1124
G. F. KRAMER et al.
50
2 IEssex
L;
40
zi
30
+UV2
0
175
2
150
-y
120
-5 E
90
+
60
a 2
30
L;
2 I; < Yi E c _ 7 a v, -
150 125 100 75 50 25 0
0
1c.
180 2 LL: 150 01 2
E c
120 90
x
60
2
30 0
300 PAR (pmol
+. m-*
s-y
Fig. 5. Effect of UV-B,, radiation (9.9 and 12 kJm-*day-‘) simulating an ozone depletion of ca 9 and 20% at two diiTerent levels of PAR (300 and 600 pmol m-‘set-‘) supplied by MH lamps on polyamine levels in the first trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. UV-1 is 9.9 and UV-2 is 12 kJm-‘day-’ UV-Baa. Panel A is Put, B is Spd and C is Spn. Error bars represent fs.e.
Cultural conditions. Experiments were conducted in 1990 in specially designed growth chambers with temp. and relative humidity controlled by Aminco-Aire Environmental Controllers (Black Mountain, NC). Plants were grown from seed in 15 cm diam. green plastic pots containing a peat-vermiculite mix (Jiffy Mix, Jiffy America, West Chicago, IL). Plants were watered l-2 times daily. Nutrient soln (1 g I-’ Peters 20-20-20) was added every other day, beginning on day 9. Environmental conditions consisted of a 14 hr photoperiod, day/night temp. of 25fOS”, relative humidity at 65 f 0.2%. and ambient CO,. Each experiment was performed in duplicate. PAR source. Plants were grown at 300 and 600 mol m-‘set-’ of PAR (40&700 nm) under one of the following three radiation sources: (a) Metal Halide alone to provide a source high in blue irradiance; (b) High Pressure Sodium/Deluxe alone to provide a source high in red and far-red irradiance and (c) MH and HPS/DX lamps combined to provide a balanced spectrum. The spectral composition of these lamps is described elsewhere [43].
PAR (kmol
rn-’
600 1
s- ‘)
Fig. 6. Effect of UV-B,, radiation (9.9 and 12 kJm-*day-‘) simulating an ozone depletion of ca 9 and 20% at two different levels of PAR (300 and 600 pmol m-‘set‘) supplied by HPS/DX lamps on polyamine levels in the first trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. UV-1 is 9.9 and UV-2 is 12 kJ m-* day-’ UV-B,,. Panel A is Put, B is Spd and C is Spn. Error bars represent &s.e.
UV treatment. Each growth chamber was equipped with a UV lamp bank containing 1.2m, 40 W Q-Panel UV-B-313 lamps (Cleveland, OH) located ca 55 cm above the plant canopy. These lamp banks were equipped with dimming ballasts and dimmer switches to allow precise control of the UV-B irradiance level. For the low UV-B treatments, six lamps per bank were used, and for the high UV-B treatments, eight lamps per bank were used. Located between the UV lamps and the growing compartment of the chamber was either a sheet of 0.13 mm (5 mil) polyester (spectral cut-off at ca 318 nm) for the - UV-B experiments or a sheet of 0.13 mm (5 nul) cellulose &acetate (spectra1 cut-off at ca 292 nm, about the same as sunlight reaching the Earth’s surface) for the + UV-B experiments. Filters were obtained from Cadillac Plastics, Baltimore, MD. For uniformity, lamps were aged 100 hr and the plastic filters were aged 5 hr prior to use. The filters were changed every 4-5 days. The plants were UV irradtated for 6 hr daily, centred midway through the photoperiod. In the combined lamp experiments, UV-B irradiation was begun 2 weeks after seeding. In the individual lamp experiments, UV-B irradiation was begun at
Influence of PAR and UV-B on polyamines the time of seeding. The doses used were equivalent to those that would be received at Beltsville, MD (39”N) on a clear day at the summer solstice (21 June) with ca a 9 or 20% reduction in the stratospheric ozone layer based on the model of ref. [44]. They were 460 and 565 mW m-* of biologically effective UV-B (UVBaa) on an instantaneous basis and 9.9 and 12.2 kJ m-a day- 1 UV-Baa when weighted by Caldwell’s [45] generalized plant weighting function normalized to one at 300 nm. UV measurements. UV-B irradiance at the top of the plant canopy was monitored daily by means of a portable UV radiometer [Minimum Erythemal Dose (MED) meter] available from Solar Light Co. (Philadelphia, PA) and did not deviate more than 5% over the growing area. Broad-band UV meter readings obtained on the portable radiometer were converted to W-B,, values by means of a linear regression curve. This curve was drawn by comparing broad-band meter readings with UVBaa measurements obtained with an UV spectroradiometer [46]. The spectroradiometer was equipped with a double monochromator with dual holographic gratings and interfaced with a printing calculator. Polyamine analysis. Samples consisted of the laminae of one trifoliolate leaf which was immediately frozen in liquid N, and stored at -80”. Each harvest consisted of three plants of each cultivar per condition. Free soluble polyamines were determined by dansylation and HPLC separation by methods similar to those reported previously [30]. Two separate extracts were prepared from each sample. Each extract was dansylated and analysed in duplicate. Data analysis. The statistical design was a split-plot with replicates in time. Differences referred to as significant were at least at p ~0.05 as determined by Analysis of Variance using PC SAS version 6.0. Figures were prepared using SigmaPlot software. AcknowZedgetncnts-The authors thank Erdal Adam, Robert Taylor and Randy Rowland for their assistance in this project and Edward Lee for the use of HPLC equipment. REFERENCES 1. Krupa, S. V. and Kickert, R. N. (1989) Environ. Polk. 61, 263. 2. Blumthaler, M. and Ambach, W. (1990) Science 248,206. 3. Tevini, M. and Teramura, A. H. (1989) Photo&em. Photobiol. SO, 419. 4. Teramura, A. H. (1983) Physiol. Plant. Ss, 415.
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23. Kitada, M., Igarashi, K., Hirose, S. and Kitagawa, H. (1979) B&hem.
Biophys. Res. Commun. 87, 388.
24. Tadolini, B. (1988) BZochem. J. 249, 33. 25. Heath, R. L. (1987) Rec. Adu. Phytochem. 21,29. 26. Wang, C. Y. (1990) Chilling Injury of Horticultural Plants. CRC Press, Boca Raton, Florida. 27. Ormrod, D. P. and Beckerson, D. W. (1986) HortScience 21, 1070. 28. Bars, W., Langebartels, C., Michel, C. and Sandermann, Jr., H. (1989) Phytochemistry 28, 1589. 29. Kramer, G. F., Wang, C. Y. and Conway, W. S. (1991) J. Am. Sot. Hort. Sci. 116, 813. 30. Kramer, G. F. and Wang, C. Y. (1989) Physiol. Plant 76,479. 31. Bornman, J. F., Evert, R. F., Mierzwa, R. J. and Bornman, C. H. (1986) in Stratospheric Ozone Reduction, Solar Ultraviolet Radiation and Plant Life (Worrest, R. C. and Caldwell, M. M., eds), pp. 199-209. NATO AS1 Series. Springer, Berlin. 32. Tevini, M., Iwanzik, W. and Thoma, U. (1981) Planta 153, 388. 33. Murphy, T. M. (1983) Physiol. Plant. 58, 381. 34. Mantai, K., Wong, J. and Bishop, N. I. (1970) Biochim. Biopkys. Acta 197,257. 35. Teramura, A. H., Sullivan, J. H. and Lydon, J. (1990) Physiol. Plum. 80, 5. 36. Teramura, A. H. (1986) in Stratospheric Ozone Reduction, Solar Ultraoiolet Radiation and Plant L$t (Worrest, R. C.
and Caldwell, M. M., eds), pp. 327-343. NATO ASI Series. Springer, Berlin. 37. Lercari, B., Sodi, F. and di Paola, M. L. (1990) Physiol. Plant. 79,668. 38. Dai, Y.-R. and Galston, A. W. (1981) Plant Physiol. 67,266. 39. Rowland-Bamford, A. J., Borland, A. M., Lea, P. J. and Mansfield, T. A. (1989) Environ. Pollut. 61, 95. 40. Lydon, J., Teramura, A. H. and Summers, E. G. (1986) in Stratospheric Ozone Reduction, Solar Ultraviolet Radiation and Plant Life (Worrest, R. C. and Caldwell, M. M., eds),
pp. 314-325. NATO ASI Series, Springer, Berlin. 41. Teramura, A. H. and Murah, N. S. (1986) Environ. Exp. Botany 26, 89. 42. Songstad, D. D., Duncan, D. R. and Widholm, J. M. (1990) J. Exp. Botany 224, 289. 43. Krizek, D. T., Kramer, G. F., Mirecki, R. M. and Taylor, R. L. (1992) Photo&m. Photobiol. (submitted).
44. Green, A. E. S., Cross, K. R. and Smith, L. A. (1980) Photochem. Photobiol. 31, 59. 45. Caldwell, M. M. (1968) Ecol. Monog. 38, 243. 46. Krizek, D. T. and Koch, E. J. (1979) Photo&em. Photobiol. 30,483.
Vol. 31,No. 4, pp. 11191125,1992 Phytocbemistry, Printedin GreatBntain.
INFLUENCE OF PHOTOSYNTHETICALLY ACTIVE RADIATION AND SPECTRAL QUALITY ON UV-B-INDUCED POLYA~~NE ACCU~ULAT~QN IN SOYBEAN GEORGE F. KRAMER, DONALD T. KRIZEK
and ROMAN M. MXRECKI
Climate Stress Laboratory, Agricultural Research Service, U.S. Department of A~culture,
Beltsville, MD 20705, U.S.A.
(Received in revisedform 10 September 1991)
Key Word Index-Glycine
max; Fabaceae; soybean; polyamines; photosynthetically response; UV-B radiation; photoprotection.
active radiation; stress
Abstract-UV-B-sensitive (Essex) and -insensitive (Williams) cultivars ofsoybean (G&&e mux) were grown in growth chambers at photosynthetically active radiation (PAR) levels of 300 or 600 pmol m-2 see- t provided by either redand far-red-deficient (MH) or blue-deficient (HPS/DX) lamps or a combination of both. The combined treatment provided a balanced output, similar to that provided by fluorescent plus incandescent lighting across the visible spectrum. Under the combined lamps, plants were exposed to 12 kJm-‘day-’ of biologically effective UV-B (UVB,,) with 6-hr irradiance periods centred midway through the photoperiod. This irradiance corresponded to a decrease in stratospheric ozone of ca 20% for clear sky conditions at Beltsville, MD on 21 June. Plant growth was significantly inhibited by UV-B at 300 but not at 600 pmol mm2 set- ’ PAR. No cultivar differences were noted in the UV-B-induced inhibition of growth, although visible injury was less in Williams than in Essex. PAR had a large effect on polyamine levels in leaves, with higher levels of putrescine (Put) and spermidine observed at 600 than at 300 i*molm-* set-’ in both cultivars. ~-B-indu~d polyamine a~umu~ation was observed primarily in Williams. Under MH or HPS/DX lamps alone, plants were exposed to two different UV-B levels, 9.9 and 12 kJm-‘day-“, corresponding to stratospheric ozone reductions of ca 9 and 20%. UV-B inhibited growth at both 300 and 600 ~01 m- ’ set- 1 PAR under either radiation source. There was no effect of PAR on the UV-B-induced growth inhibition with the HPS/DX lamps, but a partial amelioration of this inhibition occurred in Williams at 600 pmol m-* set- ’ PAR under MH lamps. Dose-de~ndent UV-~-indu~d polyamine ~eumulation was also observed in both cultivars. PAR increased Put levels under MH but not HPS/DX lamps. These results indicate that the inhibition of UV-B stress by high PAR may require a balance of red and blue wavelengths and may involve polyamine accumulation.
Human activities which release halogenated hydrocarbons and result in the depletion of the stratospheric ozone layer are expected to increase the amount of solar UV-B (290-320 nm) radiation that reaches the surface of the Earth [ 11. Recent evidence indicates that terrestrial UVB fluxes at high elevations have increased by 1% per year since 1980 [2]. Increased UV-B adversely affects the growth of a number of plant species, with UV-B-dependent reductions in yield having been observed in several ~onomical~y ~~rtant crops [1, 3, 4]. The deleterious effects of UV-B may involve inhibition of photosynthesis F-7$ Photosynthetically active radiation (PAR, 400700 nm) levels have a profound impact on plant sensitivity to UV-B, with high levels decreasing UV-B-de~ndent damage [8-123. Growth under high PAR results in thickening of the epidermis and higher levels of flavonoids in the leaves [S, 10, 111. Such effects would tend to lessen the sensitivity of the plant to UV-B [S, 8, 13-173. However, soybean grown under low PAR and exposed to UV-B under high PAR are less sensitive to UV-B than plants grown under low PAR and then irradiated with UV-B under low PAR [S]. The high PAR must thus
facilitate a biochemical response to UV-B which ameliorates the stress-induced damage. The relative importance of differing wavelengths of radiant energy in eliciting this PAR response has not been investigated. A possible significance of spectral quality is demonstrated by the modulating effect of red and blue wavelengths on UV induction of pigment biosynthesis [18, 193. Polyamines may play a role in protecting plants from UV-B stress 1201. Polyamines possess a number of antisenescent activities and are induced in response to many stressors in plant systems {21,22]. Polyamin~ can bind to membrane surfaces via ionic interactions with phospholipids and act to inhibit lipid peroxidation [23, 243. Membrane damage appears to be involved in the phytotoxicity of enviro~ent~ stressors such as ozone [25] and shilling [26]. Direct app~~ation of polyamines to plant tissue has been shown to inhibit the development of injury induced by ozone [27, 283 and chilling [29, 301. There is also evidence that membrane damage may be involved in UV-B stress [20, 31-331. The inhibition of photosynthesis by UV-B may involve the disruption of chloroplast membrane structure [7,31,34]. Upon UV-B exposure, the total levels of chlorop~ast lipids decrease and the ratio of the individual polar lipids is altered in
1119
G. F. KRAMER et al.
1120
several species [32]. UV-B radiation induces accumulation of lipid peroxidation products in cucumber leaves concomitant with a reduction in the ratio of unsaturated/saturated fatty acids [20]. The magnitude of this effect was correlated with cultivar sensitivity to UV-B. Increased polyamine levels also resulted from UV-B exposure in cucumber [20]. The purpose of the present study was to investigate the influence of UV-B radiation on polyamine levels in two cultivars of soybean found previously to differ in UV-B sensitivity [17, 353 (Essex-W-B sensitive and WilliamsUV-B insensitive) as measured by leaf area and biomass accumulation and to determine whether polyamines may be involved in the enhanced resistance to UV-B resulting from high PAR conditions. The relative importance of red and blue photoreceptors in polyamine and PAR responses to UV-B was investigated by comparing the effects of UV-B under conditions deficient in blue (high pressure sodium deluxe, HPS/DX) and red and far-red radiation (metal halide lamps, MH).
160
B.
r,
RESULTS
1
L.
U V-B effects under a combination of MH and HPS! DX lamps
The first set of experiments involved the use of a combination of MH and HPS/DX lamps as a PAR source and a single dose of 12 kJm-2day-1 of biologically effective UV-B radiation (W-B,,) (which corresponded to a decrease in stratospheric ozone reduction of ca 20%). The plants were grown for two weeks from seeding before the start of UV-B treatment and then irradiated with UV-B for two weeks. Inhibition of mainstem elongation by UV-B was seen only at low levels of PAR (Fig. 1). No significant cultivar differences in the growth responses to UV-B were observed. In the leaves emerging under UV-B, visible injury (in the form of chlorotic flecks) was observed (data not shown). In Williams, this injury was less severe at high PAR than at low PAR. Visible injury in Essex was greater than in Williams and not significantly lessened by high PAR treatment.
600 -1
ISJWillioms +lJV BglBEssex -UV IEssex +UV
400 500
PAR
(pmol
m-*
s-j)
Fig. 1. Effect of UV-B,, radiation (12 kJm_‘day-‘) simulating an ozone depletion of ca 20% at two different PAR levels (300 and 600~molm~2sec~‘) on mainstem length of Williams and Essex soybean plants after two weeks of treatment. PAR source consisted of a combmation of MH and HPS/DX lamps. Error bars represent T s.e.
600
300
PAR (Fmol
rnH2
s-‘)
Fig. 2. Effect of UV-BB, radiation (12 kJm--2 day-‘) simulating an ozone depletion of ca 20% at two different PAR levels on polyamine levels in the first (300 and 600~molm-2sec-‘) trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. PAR source consisted of a combination of MH and HPS/DX lamps. Panel A is Put, B is Spd and C is Spn. Error bars represent +s.e.
The effect of elevated UV-B on polyamine levels in the first trifoliolate leaf above the primary leaves is shown in Fig. 2. These leaves were fully expanded at the start of the UV-B treatments. There was a significant effect of PAR on polyamine levels (Table 1A) with higher levels of putrescine (Put) and spermidine (Spd) accumulating in the presence of 600 pmol rn- ’ set- ’ PAR. A significant UV x PAR interaction resulted from the tendency of UVB to decrease polyamine levels at 300 pmol me2 set-’ PAR while at 600 pmol m -2 see- ’ PAR, polyamines increased or remained unchanged. UV-B had a greater positive effect on polyamine levels at high PAR in Williams than in Essex. Polyamine levels were also determined in a leaf which had emerged in the presence of UV-B (fourth fully expanded trifoliolate above primaries, Fig. 3). PAR was again seen to significantly increase the levels of Put and Spd (Table 1B). UV-B radiation-induced increases in Put at 300 and 600 pmol me2 see- ’ PAR and in Spd at 300 pmol mm2 sec.. 1 PAR were seen in Williams, but not in Essex (Fig. 3).
Influence of PAR and UV-B on polyamines
1121
Table 1. F-value and associated probability(p) for polyamines in the first and fourth trifoliolate leaf of soybean grown under MH and HPS/DX lamps combined (data for Williams and Essex cultivars are combined) Putrescine _.___~ Effect
F-value
A. First trifoliolate leaf PAR 15.4 uv ns. cv n.s. PAR x UV 15.6 PAR x CV n.s. uvxcv 10.2 PARxUVxCV n.s. B. Fourth trifoliolate leaf PAR 1.7 uv n.s. cv 7.8 PAR x UV n.s. PAR x CV ns. UVXCV 5.5 PARxUVxCV n.s.
Spermidine -__
Spermine ~
_.~.
p
F-value
p
F-value
p
0.002
5.1 ns. 13.8 8.7 n.s. n.s. n.s.
0.040
5.1 ns. 7.8 19.1 n.s. n.s. ns.
0.038
0.002 0.006
0.010 0.010
0.028 _
38.9 n.s. n.s. n.s. 4.5 n.s. n.s.
0.002 0.010
0.0001 .-. 0.046 _ -
n.s. ns. 8.1 17.4 n.s. ns. 6.5
.~
0.013 0.001
_ 0.009 0.0003
0.018
n.s. = not significant at p < 0.05.
UV-B effects under MH or HPSfDX
lamps alone
The second set of experiments involved the use of either MH or HPS/DX lamps alone as a PAR source and two UV-B,, doses of 9.9 and 12 kJ mm2 day-’ (corresponding to stratospheric ozone reductions of ca 9 and 20%). Treatments were initiated at the time of seeding. Overall effects on vegetative growth were determined by expressing the height, leaf area, fresh weight, and dry weight as a per cent of the control (no UV-B) and averaging these percentages together. The UV-B treatments had a significant inhibitory effect on growth as measured by these parameters under both PAR sources (Fig. 4). Measurement of height alone at this time was inadequate to demonstrate the effects of UV-B, as the shortness of the plants in some treatments made the stress-induced differences in height difficult to detect. After another seven days of elongation, the effects of UV-B on height were consistent with the combined growth parameters measured at 14 days (Krizek et al., in preparation). There was no PAR under HPS/DX lamps, as effect of the inhibition of growth was not greater at 300~olm-2sec-1 than at 600~molm-2sec-‘. With MH lamps, a partial amelioration of UV-B-induced growth reduction was observed in Williams. Plants appeared to be more sensitive to UV-B radiation under MH than under HPS/DX lamps at 300 pmol mm2 set- ’ PAR. Only traces of visible UV-B injury were observed. Few cultivar differences were observed, although the growth of Essex was inhibited by 12 kJm_’ day-’ UV-B,, under MH lamps with 600 pmol m- ’ set- ’ PAR to a slightly greater extent than was Williams. The effects of UV-B irradiation on polyamine levels are shown in Fig. 5 (MH) and Fig. 6 (HPS/DX). Significant UV-B effects were seen in Put, Spd and spermine (Spn) (Table 2) with the highest UV-B level generally eliciting the greatest induction of polyamines. When the data for lamp type were combined for analysis, there were no PAR
effects seen, but the type of lamp did have a significant effect on polyamine levels (Table 2), with Put and Spd levels being higher under HPS/DX and Spn being higher under MH lamps. When the data for each lamp type were analysed separately, a PAR effect was seen only in Put under MH lamps (Table 3B). Cultivar differences were also observed, with higher polyamine levels generally being found in Essex than in Williams, especially in the highest UV-B treatment (Table 2, Figs 5 and 6). DISCUSSION
The effects of UV-B on plant growth and polyamine levels in soybean were greatly influenced by PAR levels when plants were grown under a combination of MH and HPS/DX lamps. This radiation source provided a balanced output across the visible spectrum and was nearly comparable in spectral power distribution to that emitted by a combination of fluorescent and incandescent lamps. Exposure to UV-B radiation significantly reduced mainstem length at 300 pmol me2 set- ‘, while no effect was seen at 600 pm01 m-’ set - I PAR. This result is consistent with findings of other workers who have demonstrated protection from UV-B by high PAR [8-121. This ability to ameliorate UV-B damage has been correlated with the ability of high PAR levels to induce pigment formation and alter leaf morphology such that UV-B transmittance through the epidermis is reduced [8,10,22]. The observation that leaves grown at low PAR were less sensitive to UV-B radiation when exposed under high PAR than under low PAR indicates that biochemical mechanisms of photoprotection and/or photorepair were also activated by high PAR [S, 361. Our results indicate that polyamines could be involved in this photoprotective effect of high PAR. In leaves which expanded in the absence of UV-B and were subsequently exposed for two weeks, the levels of Put and Spd were significantly higher at 600 than
G. F.
1122
KRAMER
et al.
5
60
-5 5
60
V x
40
B. si 300 L; ISI 250 \
:
60
$
0 E
200
i? V
60
=
150
8?
40
a Lr) -
100
7
20 50 0 L.
150
<
125
c F
n. vt
75 50 25
-
0
rn-’
s-l)
Fig. 4. Effect of UV-B,, radiation (9.9 and 12 kJm -*day-‘) simulating an ozone depletion of ca 9 and 20% at two different PAR levels(3OOand 600 pmol m-2sec-‘)ongrowth of Williams and Essex soybean plants after two weeks of treatment. The height, dry weight, leaf area and fresh weight were expressed as a per cent of the nor&V-B-treated controi and then averaged together. UV-1 is 9.9 and UV-2 is 12 kJm-‘day-* UV-Baa. PAR source consisted of MH (panel A) or HPS/DX lamps (panel B). Error bars represent 2s.e.
100 _
600
300 PAR (pmol
175 2 (i
5 E
HPS
: 300
PAR (pmol
>
600 a
rneL s-t)
Fig. 3. Effect of UV-B,, radiation (12 kJm-2day-1) simulating an ozone depletion of ca 20% at two different PAR levels (300 and 600 pmol m- 2 set- “) on polyamme levels in the fourth trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. PAR source consisted of a combination of MH and HPS/DX lamps. Panel A is Put, B is Spd and C is Spn. Error bars represent &se.
300 pmol mF2sec- r PAR. In leaves which had expanded after the initiation of UV-B treatment, the levels of Put and Spd were also higher at 600 pmol me2 set-’ PAR. The formation of anthocyanins and flavonoids is modulated by red and blue wavelengths, indicating that both phytochrome and the blue/UV photoreceptors are involved in this response [lS, 19-J. Photomorphogenic responses to UV are also regulated by both phytochrome and UV photoreceptors [3’7]. These obse~ations could suggest that the ameliorative effects of PAR on UV-B injury may be modulated by spectral quality. We have tested this hypothesis by comparing the effects of PAR on UV-B sensitivity under MH (red- and far-red-deficient) and HPS/DX (blue-deficient) lamps. Under both radiation sources and at both 300 and 600pmolm-2 set-r PAR, W-B inhibited growth. The higher level of PAR had no protective effect in comparison with the lower PAR level, except for Williams under MH lamps, where partial protection from UV-B injury was observed. Polyamines were induced by UV-B radiation generally in a
dose-dependent fashion under both radiation sources and at both PAR levels. Overall, PAR source had a significant effect on polyamine levels, with Put and Spd being higher under HPS/DX lamps and Spn being higher under MH lamps. This finding is consistent with the reported ability of phytochrome to regulate the activity of the polyamine biosynthetic enzyme arginine decarboxylase [38]. It thus appears that separating the MH and HfS/DX radiation sources abolished or reduced the protective effect of high PAR which was observed when the two lamps were combined. The photoprotective effects of high PAR may require a balanced output throughout the visible spectrum. In these experiments, we also compared the responses of two cultivars of soybean thought to differ in UV-B sensitivity-Williams (UV-B insensitive) and Essex (UV-B sensitive) [17]. In terms of vegetative growth responses, we saw little difference in these cultivars in experiments with either combined or separate lamps. The only major difference in W-B sensitivity observed was in visible injury under the combined PAR source where Williams was injured less than Essex. Cultivar differences in the effect of UV-B on polyamine levels were noted. Under the combined PAR source, a greater induction of polyamines in response to UV-B was observed in Williams than in Essex. The reduced visible injury in Williams as compared to Essex was thus correlated with the ability to induce polyamine accumulation in response to UV-B. Increased polyamine levels have been correlated with the inhibition of the development of
Influence of PAR and UV-B on polyamines
1123
Table 2. F-value and associated probability (p) for polyamines in the first &ifoliolate leaf of soybean grown under MH and HPS/DX lamps alone (data for Williams and E!ssexcultivars are combined; two-, three- and four-way interaction terms which were not significant for any of the polyamines were omitted)
_
Putrescine E&et
F-value
-I_ p
A. MH and HPS/DX data combined 0.002 L=np 14.4 PAR ns. 0.005 uv 8.4 0.0001 cv 15.5 -uvxcv n.s. 0.001 PAR x UV x CV 7.4 B. MH data alone 0.041 PAR 6.7 UV n.s. 0.0001 CV 21.1 0.003 PAR x CV 9.4 wxcv n.s. C. HPS/DX data alone PAR ns. 0.046 uv 5.4 cv ns. 0.010 PAR x CV 7.2 0.046 PARx WxCV 5.4
ozone-de~ndent visible injury in tobacco and barley [28, 391. However, as UV-B radiation affected the growth of both cultivars equally, the amount of UV-B injury sustained by Essex did not appear to have an adverse effect on plant growth, at least in the time frame of this experiment. This difference in IN-B-dependent polyamine accumulation was also dependent on spectral quality as a greater enhancement of polyamine levels was seen in Essex than in Williams under MH or HPS/DX lamps alone. As the combined PAR source more closely resembles natural conditions, the greater UV-B induction of polyamines in Williams under these conditions could be physioiogically relevant. Further experiments are necessary to determine whether the reported differences in UV-B sensitivity in these cultivars could involve differences in polyamine accumulation. The lack of a difference between these cultivars in UVB inhibition of growth is somewhat surprising, as changes in flavonoid content and leaf morphology have been reported [17]. However, in other experiments, there was little difference in the inhibition of vegetative growth by UV-B between Williams and Essex [40, 411. Also,, the relative difference in UV-B sensitivity of these two cultivars, as measured by seed yield, appears to be dependent on microclimatic conditions (i.e. rainfall, temperature, and cloudiness) [35]. Our experiments could have been performed under conditions in which the cultivar differences in UV-B sensitivity were not expressed, or W-B exposures of greater duration may have been required to see these effects. We have shown that polyamine levels are correlated with the ability of high PAR levels to induce resistance to UV-B. Under conditions where a protective effect of high PAR was observed (combined MH and HPS/DX tamps),
_~
Spermidine
__
Spermine -
p
F-value
p
6.5
0.025
I% 8.0 6.4 n.s.
0.0001 0.006 0.002
13.8 n.s. 5.9 45.1 6.4 n.s.
0.003 0.016 0.0001 0.002 -
ns. 8.6 13.1 ns. 4.4
0.017 0.001
ns. 5.7 34.6 ns. 7.9
0.041 0.0001 0.001
n.s. ns. 15.0 n.s. n.s.
.0.0003 -
F-value
n.s. 18.8 ns. ns. n.s.
0.017
0.003 -
PAR increased the levels of po~yamin~. Under conditions where no PAR effect was seen on UV-B sensitivity (HPS/DX lamps alone), polyamine levels were not affected by PAR. Under MH lamps alone, where high PAR provided partial protection against UV-B inhibition of growth in Williams, PAR increased the levels of Put. However, in Essex, photoprotection was not observed even though high PAR increased Put, indicating that lack of photoprotection in Essex is unrelated to Put content. These differences in the effect of PAR on Put between MH and HPS/DX lamps may indicate that blue wavelengths are more important than red wave~en~hs in the induction of Put ~umuIation by high PAR. A possible role of polyamines in protecting against W-B stress is consistent with the apparent ability of these compounds to protect plant cells from ozone [27, 281 and chilling [29, 30, 421 stress. The anti-ozonant activity of polyamines may involve conjugation with hydroxycinnamic acids as these conjugates appear to be effective free radical scavengers [28]. The photoprotective pigments synthesized by plants in response to W-B are derived from cinnamic acid [3]. The induction of polyamine conjugate formation by UV-B is thus a possible response to this stress, as the fo~ation of both precursors is promoted by UV-B. Further studies are necessary to demonstrate conclusively whether polyamines act in uiuo to protect plants from W-B stress.
EXPERIMENTAL
Plant material.Two cultivars of soybean (Glycine mnx (L.) Merr.) reported to differ in sensitivity to UV-B radiation [17, 35-j--Williams, a relatively W-B insensitive cultivar and Essex, a highly sensitive cultivar-were used.
1124
G. F. KRAMER et al.
50
2 IEssex
L;
40
zi
30
+UV2
0
175
2
150
-y
120
-5 E
90
+
60
a 2
30
L;
2 I; < Yi E c _ 7 a v, -
150 125 100 75 50 25 0
0
1c.
180 2 LL: 150 01 2
E c
120 90
x
60
2
30 0
300 PAR (pmol
+. m-*
s-y
Fig. 5. Effect of UV-B,, radiation (9.9 and 12 kJm-*day-‘) simulating an ozone depletion of ca 9 and 20% at two diiTerent levels of PAR (300 and 600 pmol m-‘set-‘) supplied by MH lamps on polyamine levels in the first trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. UV-1 is 9.9 and UV-2 is 12 kJm-‘day-’ UV-Baa. Panel A is Put, B is Spd and C is Spn. Error bars represent fs.e.
Cultural conditions. Experiments were conducted in 1990 in specially designed growth chambers with temp. and relative humidity controlled by Aminco-Aire Environmental Controllers (Black Mountain, NC). Plants were grown from seed in 15 cm diam. green plastic pots containing a peat-vermiculite mix (Jiffy Mix, Jiffy America, West Chicago, IL). Plants were watered l-2 times daily. Nutrient soln (1 g I-’ Peters 20-20-20) was added every other day, beginning on day 9. Environmental conditions consisted of a 14 hr photoperiod, day/night temp. of 25fOS”, relative humidity at 65 f 0.2%. and ambient CO,. Each experiment was performed in duplicate. PAR source. Plants were grown at 300 and 600 mol m-‘set-’ of PAR (40&700 nm) under one of the following three radiation sources: (a) Metal Halide alone to provide a source high in blue irradiance; (b) High Pressure Sodium/Deluxe alone to provide a source high in red and far-red irradiance and (c) MH and HPS/DX lamps combined to provide a balanced spectrum. The spectral composition of these lamps is described elsewhere [43].
PAR (kmol
rn-’
600 1
s- ‘)
Fig. 6. Effect of UV-B,, radiation (9.9 and 12 kJm-*day-‘) simulating an ozone depletion of ca 9 and 20% at two different levels of PAR (300 and 600 pmol m-‘set‘) supplied by HPS/DX lamps on polyamine levels in the first trifoliolate leaf above the primary leaves of Williams and Essex soybean after two weeks of treatment. UV-1 is 9.9 and UV-2 is 12 kJ m-* day-’ UV-B,,. Panel A is Put, B is Spd and C is Spn. Error bars represent &s.e.
UV treatment. Each growth chamber was equipped with a UV lamp bank containing 1.2m, 40 W Q-Panel UV-B-313 lamps (Cleveland, OH) located ca 55 cm above the plant canopy. These lamp banks were equipped with dimming ballasts and dimmer switches to allow precise control of the UV-B irradiance level. For the low UV-B treatments, six lamps per bank were used, and for the high UV-B treatments, eight lamps per bank were used. Located between the UV lamps and the growing compartment of the chamber was either a sheet of 0.13 mm (5 mil) polyester (spectral cut-off at ca 318 nm) for the - UV-B experiments or a sheet of 0.13 mm (5 nul) cellulose &acetate (spectra1 cut-off at ca 292 nm, about the same as sunlight reaching the Earth’s surface) for the + UV-B experiments. Filters were obtained from Cadillac Plastics, Baltimore, MD. For uniformity, lamps were aged 100 hr and the plastic filters were aged 5 hr prior to use. The filters were changed every 4-5 days. The plants were UV irradtated for 6 hr daily, centred midway through the photoperiod. In the combined lamp experiments, UV-B irradiation was begun 2 weeks after seeding. In the individual lamp experiments, UV-B irradiation was begun at
Influence of PAR and UV-B on polyamines the time of seeding. The doses used were equivalent to those that would be received at Beltsville, MD (39”N) on a clear day at the summer solstice (21 June) with ca a 9 or 20% reduction in the stratospheric ozone layer based on the model of ref. [44]. They were 460 and 565 mW m-* of biologically effective UV-B (UVBaa) on an instantaneous basis and 9.9 and 12.2 kJ m-a day- 1 UV-Baa when weighted by Caldwell’s [45] generalized plant weighting function normalized to one at 300 nm. UV measurements. UV-B irradiance at the top of the plant canopy was monitored daily by means of a portable UV radiometer [Minimum Erythemal Dose (MED) meter] available from Solar Light Co. (Philadelphia, PA) and did not deviate more than 5% over the growing area. Broad-band UV meter readings obtained on the portable radiometer were converted to W-B,, values by means of a linear regression curve. This curve was drawn by comparing broad-band meter readings with UVBaa measurements obtained with an UV spectroradiometer [46]. The spectroradiometer was equipped with a double monochromator with dual holographic gratings and interfaced with a printing calculator. Polyamine analysis. Samples consisted of the laminae of one trifoliolate leaf which was immediately frozen in liquid N, and stored at -80”. Each harvest consisted of three plants of each cultivar per condition. Free soluble polyamines were determined by dansylation and HPLC separation by methods similar to those reported previously [30]. Two separate extracts were prepared from each sample. Each extract was dansylated and analysed in duplicate. Data analysis. The statistical design was a split-plot with replicates in time. Differences referred to as significant were at least at p ~0.05 as determined by Analysis of Variance using PC SAS version 6.0. Figures were prepared using SigmaPlot software. AcknowZedgetncnts-The authors thank Erdal Adam, Robert Taylor and Randy Rowland for their assistance in this project and Edward Lee for the use of HPLC equipment. REFERENCES 1. Krupa, S. V. and Kickert, R. N. (1989) Environ. Polk. 61, 263. 2. Blumthaler, M. and Ambach, W. (1990) Science 248,206. 3. Tevini, M. and Teramura, A. H. (1989) Photo&em. Photobiol. SO, 419. 4. Teramura, A. H. (1983) Physiol. Plant. Ss, 415.
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