Plant & Cell Physiol. 21(8): 1559-1571 (1980)
Effects of ultraviolet irradiation on growth and pigm.entation in seedlings Tohru Hashimoto and Misao Tajima The Institute for Physical and Chemical Research, Wakoshi, Saitama 351, Japan (Received February 22, 1980)
Although a vast amount of work has been devoted to the effects of visible light on higher plants, not many papers deal with those ofUV. Part of the cause seems to be due to the technical difficulty of obtaining proper irradiation systems. Introduction of new irradiation systems has provided new information on this subject. Many experiments conducted with low-pressure mercury lamps which emit solely 254 nm line disclosed mostly phytotoxic effects such as reduced size or distorted growth (29), glazing, bronzing, injury, necrosis and death (4, 5, 7, 19, 27), accelerated senescence (32), inhibition of chlorophyll and protein synthesis (8, 17). Highpressure mercury lamps and xenon lamps made possible investigation of the effects of the middle and near UV (14, 20, 30, 31) but limited availability of filters for use with them restricted selection of wavelengths, particularly when intact plants were to be irradiated. Fluorescent lamps have also been employed. Klein and Edsall (10) used blackAbbreviations: DW, dry weight; FW, fresh weight; 310, 310-0, 350, 395,420 and 660, lights with emission at 290-338, 300-338, 330-370, 384--400, 404--432 and 648-666 nm in halfwidth, respectively; 310-2, secondary emission of the lamps at wavelengths longer than 380 nm.
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Dark-germinated seedlings of maize, radish, soybean, cucumber and eggplant were grown for 2 or 3 days under ultraviolet light, and the differential effects of UV according to wavelength regions were evaluated. Although the effectiveness of UV irradiation differed somewhat depending on the plant species, generally, four wavelength regions having different effects were discerned. The region from 287 to 302 nm was phytotoxic, causing bronzing (radish), epinastyand blazing (cotyledon, cucumber), formation of brown flecks (soybean), and also severe growth inhibition in hypocotyls. The region from 300 to 338 nm greatly promoted anthocyanin formation and inhibited the shoot elongation. The region from 330 to 370 nm inhibited shoot elongation and promoted cotyledon growth as well as chlorophyll formation. The effects of the region from 384 to 400 nm were generally weak, but in maize coleoptile, radish and soybean hypocotyls this region exerted a distinct inhibition, stronger than the region of 330-370 nm, and in chlorophyll formation in maize coleoptile this region was the most effective of those tested. The above-stated shoot growth inhibition and anthocyanin formation caused by UV were generally greater than those caused by blue or red light, but growth promotion in the cotyledons was smaller. Key words: Anthocyanin - Chlorophyll - Cotyledon growth - Phytotoxicity Shoot elongation - Ultraviolet irradiation.
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T. Hashimoto and M. Tajima
Materials and methods
Plants The plant species tested were maize (Zea m~.ys L., cv. Golden Cross Bantam, Watanabe Saishujo, Ltd.), radish (Raphanus sativus L., cv. Akamaru, Tohoku Shubyo, Ltd.), soybean (Glycine max L., obtained locally at a grocery, name of the cultivar not specified), cucumber (Cucumis sativus L., cv. Aonagajibai, Tohoku Shubyo) and eggplant [Solanum esculentum (L.) Nees., cv. Shin B-go, Watanabe Saishujo] . Seeds were imbibed in running tap water for 8 to 16 hr, sown in vermiculite in plastic boxes, 6 X 15 X 10 em (height), and germinated in the dark at 27-30°C. Within 12 hr after emergence of the seedlings, irradiation was started and continued for 48 or 72 hr until harvest. Liquid Hyponex diluted 1,000 times was supplied when seeds were sown and when seedlings emerged.
Irradiation system The lamps used were 20-watt fluorescent tubes which had an emission peak at 310, 350, 395 or 420 nm (specially manufactured for this study, Hitachi, Ltd.) and ones which had an emission peak at 660 nm (Colored Lamp, FI-20-RF, Mitsubishi Electric, Ltd.). For each treatment, 2 or 3 lamps were installed and combined with a sheet of polyvinyl chloride film that cut off unnecessary UV (UVC-O and UVC-2, 0.1 mm thick, Mitsui Toatsu, Ltd.) as follows: three 310 nm lamps with UVC-O, three 310 nm lamps with UVC-2, two 350 nm lamps with UVC0, two 395 nm lamps with UVC-2 and two 420 nm lamps with UVC-2. The lights thus obtained were referred to, respectively, as 310-0, 310-2, 350, 395 and 420 (blue). Lights emitted from unfiltered 310 nm and 660 nm lamps were also used as 310 and 660 (red). These lights had an emission band at 290-338 (halfwidth, 310), 300-338 (310-0), 330-370 (350), 384-400 (395), 404-432 (420) or 648-666
(660) nm.
310-2 was secondary emission due to mercury lines in the region of
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light lamps (General Electric BLB integral filter lamp) to obtain 355-380 nm UV and found that this region of UV inhibited plant growth. Sisson and Caldwell (23) devised a system using a Westinghouse FS-40 'Sunlamp' and filters, which provided radiation of 280-320 nm, to simulate the solar UV irradiance resulting from a decrease of stratospheric ozone. Supplementary use of this with natural solar radiation (1, 21, 22) or artificial light sources (3, 13, 24,25) resulted in suppressed growth of roots, cotylecons and leaves, inhibition of photosynthesis, increased pigmentation or decoloration, phytotoxic damage such as bronzing and glazing of cotyledons, distorted leaves, and finally abscission, and other abnormal curvature of shoots. Most of the results thus far obtained with artificial UV have been based on the effects of a single particular wavelength region and therefore do not provide the entire picture of UV actions at various wavelengths. In an attempt to evaluate the differential effects of UV in relation to wavelengths ranging from 290 through 400 nm, we prepared fluorescent lamps which emit different bands of UV and tested them in experiments on growth and pigmentation of some agricultural plant seedlings.
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UV effects on growth and pigmentation
wavelength longer than 380 nm (Fig. l). The spectral energy distribution curves in the figure were determined with a spectral radiometer (EPS-3T, Hitachi, Ltd.) corrected with a bromine lamp (Quartz Line Lamp DXW 1,000 watts, General Electric, Ltd.).
~
"(i;
c
a>
+-'
..= Q)
:> ~ 0
(i)
n::
I
I
-----l
350
I
I
A
395 420
1\
I
I
I
660
Wavelength (nm)
Fig. 1. Radiation spectra of the lights used. The curve of 660 is from the manufacturer's data, and the others were measured with a spectral radiometer (EPS-3T, Hitachi, Ltd.).
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310-2
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T. Hashimoto and M. Tajima
The irradiance was equalized by adjusting the distance from the lamps and/or inserting a neutral net filter for all treatments except for 310-2, which was kept at the same irradiance level as that of 310-0 in the region of wavelength longer than 380 nm. Light energy was measured with a thermopile (CA-l, Kipp and Zonen, Ltd.). The filter films were renewed as required (almost every two experiments) as their transmittance was changed by UV irradiation. Plants were irradiated in a dark room dimly lit with diffuse daylight (less than 0.1 ftwatt·cm- 2) . The temperature was controlled at 27-30°C.
Determine tion
ofgrowth andpigment content
Evaluation of UV effects The effects of the UV irradiations were, of course, compared with non-irradiated controls but could be more suitably evaluated by comparison with 310-0, since the UV lamps used also emitted visible light (Fig. 1). Results
Elongation growth Typical results are shown in Tables 1-5. The UV irradiations inhibited the elongation of the coleoptile, the first internode and the first leaf of maize, and the hypocotyl of other plants in comparison with 310-2 as well as with non-irradiated controls. 310 was the most effective followed by 310-0 then 395, and 350 was the least effective. In cucumber and eggplant, 350 was very effective while 395 was not. All these inhibitory effects of UV were greater or not less than those of 420 (blue) and 660 (red), except for soybean in which 660 was very effective. In cucumber, the elongation of hypocotyl which was inhibited by red light was further suppressed by 350 to the level given by the continuous 350 (Table 6). Growth ofcotyledons Although 310 inhibited the growth (increase in FW) of cotyledons, 310-0 and 350 promoted it in cucumber (Table 4) and 395 also did so in eggplant (Table 5). In radish, 310-0 and 395 were promotive, but 350 was not effective (Table 2).
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At the end of a 72-hr (48 hr for soybean) irradiation period, the length and FW of the seedlings were determined, and the seedlings were divided into 2 equal groups, one half was dried at 80°C for 2-3 hr to determine DW, and the other half was extracted to determine pigment content. Seedlings to be extracted were immersed in methanol, kept at 5°C in the dark for 1-2 days, ground in a mortar, and filtered through a teflon filter (Millipore, Ltd.). Total chlorophyll content was determined by the absorbance at 662 nm in a methanol solution. To determine the content of anthocyanin, a dilute hydrochloric acid was added to the methanol extract to make the final concentration 0.5%, and the absorbance at 528 or 530 nm was determined. Since DW of plant materials was not affected greatly by the irradiations (see the Results), pigment contents were compared on a DW basis. For this purpose, the absorbance was measured of the solution containing the extract from plant materials of 1 mg DW per 1 ml of the solution (standard concentration).
UV effects on growth and pigmentation
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Table 1 Effectongrowth of maize seedlings Irradiated with 310
310-0
310-2
350
395
420
660
Nonirradiated control
First internode length Coleoptile length
47±3.8 34±2.6 40±2.7 100±8.1 (14.6 mm)
39±3.7
45±4.4
54±5.7 312±27.3
76±2.9 49±1. 6 65±2.9 100±1. 4 (40.7 mm)
71±4. 1
84±2.2
81±2. 2 114± 6.1
First leaf length
92±3.4 73±1. 8 85±3.4 100±2.8 (85.4mm)
89±3.2 102±3.9 102±2.6
FWof coleoptile
36
60
100 (99.5 mg)
67
49
80
70
108
DW/FWof 172 coleoptile
109
100 (4.4%)
109
120
102
114
109
16
10
13
13
13
13
18
20
Values in the table are the percent of those with 310-2 and s.e, in percent. The observed values with 310-2 are shown in parentheses. Irradiation energy was 190 ,uwatts'cm- 2 except for 310-2 which was 82,uwatts·cm- 2 •
Table 2 Effect on growth of radish seedlings 395
420
660
Nonirradiated control
70±3.1
83±2.7
80±3.4
135±4.2
Irradiated with 310 Hypocotyl Length FW DW/FW Plant height
310-0
310-2
350
50±3.1 65±3.0 100±3.7 82±3.5 (43.5 mm) 42
57
100 (65.0mg)
78
73
78
77
114
163
121
100 (3.8%)
105
108
100
97
87
52±2.7 69±2.9 100±3.4 87±3.3 (52.1 mm)
80±3.3
92±2.7
86±3.2
121 ±3. 6
Cotyledons FW o
DW/FW No of seedlings measured
89
112
100 (25.0 mg)
92
122
112
150
78
105
90
100 (12.2%)
97
77
80
70
120
13
20
20
20
20
20
22
20
For explanation of the figures and experimental conditions, see Table 1. FW per pair of cotyledons.
a
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No of seedlings measured
64± 5.4
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T. Hashimoto and M. Tajima
Table 3 Effect on growth of soybean seedlings Irradiated with 310 Hypocotyl Length FW
DW/FW
No of seedlings measured
395
350
310-2
420
660
Nonirradiated control
60± 4.0 83± 5.3 100± 5.7 101± 3.7 91±5. 0 118± 6.6 67±2.8 133± 7.4 (92. 4 mm) 76
85
145
106
100 (363mg) 100 (3.3%)
101
86
121
70
141
97
97
91
121
91
43±13.1 66±16.6 100±10.8 105± 10.2 54±9.6 (12.9 mm) 13
15
17
15
Irradiation period was 48 hr.
11
91± 13.8 83 ± 7. 5
25±13.4
11
12
14
For other explanations, see Table 1.
The promotive effect of these UV's was far less than that of 660 but greater than that of 420 in cucumber and radish, and greater than that of 660 but less than that of 420 in eggplant. Table 4 Effect on growth of cucumber seedlings Irradiated with 310 Hypocotyl Length FW DW/FW
Nonirradiated control
310-2
350
395
420
660
31±2. 3 60±0.9
100± 1. 5 (112 mm)
70± 1.6
96±1. 5
94±2.1
92± 1. 1
164±3.0
30
62
100 (268 mg)
77
103
103
80
123
248
117
117 100 (2.3%)
100
104
109
104
73
119
128
101
94
170 b
53
135
89
88
93
102
84
153
17
16
15
19
19
310-0
Cotyledons FW a
DW/FW
100 (105 mg) 100 (9. I %)
No of seedlings measured
15
19
18
2 except for 310-2 which was 80,uwatts Irradiation with 173 ,uwatts FW per pair of cotyledons including the plumule. For other explanations, see Table I. b Plumule was well developed. ocm-
a
ocm-
2•
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Epicoty1 Length
310-0
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UV effects on growth and pigmentation Table 5
Effect on growth of eggplant seedlings
Irradiated with 310
310-0
310-2
irradiated control
395
420
660
103± 1. 7
98±2.1
149±3.3
205±2.6
350
Hypocoty1 Length
50± 1. 1 63± 1. 5
FW
53
59
100±2.2 80± 1. 4 (35.3mm) 100
71
106
100
147
206
119
97
106
97
88
114
114
121
107
36
92
95
88
88
151
34
30
32
33
34
(17mg) DW/FW
153
119
100 (3.2%)
71
F\V
107
100 (14mg)
DW/FW
154
100
100 (7.8 %)
No of seedlings measured
37
34
30
Irradiation conditions were the same as in Table 4. For other explanations, see Table 1.
FW of cotyledons was per pair of cotyledons.
Since the growth-promoting effect of 350 on cotyledons was noteworthy, the interaction of 350 with red light was examined using cucumber seedlings. Red light markedly promoted the increase in FW and DW (as well as chlorophyll), and 350 did not promote or suppress the red light-induced growth (chlorophyll formation also) (Table 6) but gave a normal flat shape to the cotyledons, which had an upward-curled blade under the continuous red light.
FW and DW The change In FW of hypocotyls and coleoptiles caused by the irradiations Table 6
Effects ofjoint irradiation with 350 and 660 in cucumberseedlings
Non -irradiated control Hypocotyl Length (mm) Chlorophyll
l84±3.4 0
Irradiated with 350 78± 1.B 0.076
660
350/660
103± 1. 2 0.051
BO±1. 3 0.064
Cotyledons FW(mg)
56
DW(mg)
7.84
Chlorophyll
0
134 10. 7
1.08
178 13.5
1.56
173 12.8
1.56
Irradiation was done alternately with 12 hr each of350 and 660 for 350/660, and continuously with the indicated lights for other treatments, totalling 72 hr. Chlorophyll content represents the absorbance at 662 nm (in methanol) at the standard concentration. The number of seedlings used in each treatment was 16.
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Cotyledons
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T. Hashimoto and M. Tajima
generally paralleled the change of length in all the plants tested. In contrast, the DW was not affected as much by the irradiation as the FW, thus the ratio ofDWjFW increased, particularly with 310 (Tables 1-5). A similar tendency of the DWjFW
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Fig. 2. Epinasty qj a cucumber cotyledon caused ky 287-302 nm UV. Seedlings were grown for 3 days from their emergence under 310 (190 I1watts'cm-2), 310-0 (190 /1watts'cm- 2) and 310-2 (82I1watts·cm-2).
1567
UV effects on growth and pigmentation
ratio was observed with the cotyledons (Tables 2, 4 and 5), but discussion of their DW is meaningless because cotyledons have their original storage matter.
Phytotoxicity
Chlorophyll content In the cotyledons and hypocotyl of eggplant, cucumber, and soybean, 350 and 310-0 promoted chlorophyll formation compared with 310-2 (Table 7), but 310 inhibited it markedly, except in eggplant hypocotyl where the content of chlorophyll was greatest with 310 of all treatments including 660 and 420. In maize coleoptile, 395 followed by 420 then 350 caused large production of chlorophyll. 310-0 was slightly inhibitory. These promotive effects of UV, i.e., 395 and 350 in maize coleoptile, 350 in Table 7 Effict on chlorophyll content in seedlings
310-0
310-2
350
395
420
660
Nonirradiated control
82
100 (0.073)
155
223
218
82
17
Irradiated with
310 Maize Coleoptile
Eggplant Hypocotyl
159
112
100 (0.0674)
128
108
89
77
3
Cotyledons
70
104
100 (0.776)
111
88
96
140
0
Soybean Hypocotyl
66
153
100 (0.0074)
130
116
47
183
0
Hypocotyl
77
106
100 (0.049)
133
94
106
90
2
Cotyledons
53
113
100 (0.636)
109
104
77
159
0
Cucumber
Values in the table represent chlorophyll content as the percent of that in 310-2 and those in parentheses the absorbances at 662 nm of 31 0-2 (in methanol) at the standard concentration.
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Irradiation with 310 caused various abnormalities in the appearance of the seedlings. Radish seedlings had a weak brown pink color (bronzing) and were slightly hyaline. In cucumber, one of the cotyledons showed epinasty and the surfaces of both cotyledons were blazed in the 3-day experiment (Fig. 2). When the irradiation was prolonged for 3 more days the seedlings gradually became necrotic from the base toward the top. Soybean had brown flecks on the cotyledons and hypocotyl. However, 310-0 and the longer wavelengths of UV did not have such toxicity. Maize and eggplant were resistant to 310, i.e., they showed no signs of abnormality.
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T. Hashimoto and M. Tajima
Table 8 Effecton anthocyanin content ofseedlings 310
310-0
310-2
350
395
420
660
Nonirradiated control
Soybean hypocotyl
148
307
100 (0.0163)
174
134
62
201
26
Eggplant hypocotyl
376
418
100 (0.0136)
182
100
71
71
29
Irradiated with
Values in the table represent anthocyanin content as the percent of that with 310-2. Values in parentheses are the absorbances at 530 nm of 310-2 (in HC1-methanol) at the standard concentration.
Anthocyanin formation Promotion of anthocyanin formation by UV was striking in hypocotyls (Table 8). In soybean and eggplant, 310, 310-0 and 350 were very effective and 395 was also promotive in soybean but without effect in eggplant. The effectiveness of 310-0 in soybean and of 310-0, 310 and 350 in eggplant was greater than that of red light. The effect of red light was very weak in eggplant. In cucumber, no anthocyanin was detected in all treatments.
Discussion The effects of UV on seedlings described in this paper varied from injury or lethality to promotion of growth depending on the wavelength of the irradiation given and the plant parts irradiated. From the types of action, four wavelength regions were discerned: 1) the region from 287 to 302 nm was phytotoxic, 2) the region from 300 to 338 nm greatly promoted anthocyanin formation and inhibited the shoot elongation, 3) the region from 330 to 370 nm inhibited hypocotyl elongation and promoted the growth of cotyledons as well as chlorophyll formation, and 4) the region from 384 to 400 nm showed a species-specific inhibition of hypocotyl elongation. The phytotoxic effects of 254 nm emitted from a germicidal lamp have been reported in many papers as reviewed by Lockhart and Franzgrote (16). Recently, the information that UV-B radiation (290-315 nm) also causes toxicity is accumulating (1, 3, 13, 21, 22). In the present study, blazing (cucumber cotyledons), bronzing (radish), necrosis (cucumber), epinasty of a cotyledon (cucumber) and the formation of brown flecks (soybean hypocotyl and cotyledons) were observed with 310, whereas such phytotoxicities were absent with 310-0 and other lights. With 310, anthocyanin formation and chlorophyll synthesis as well as growth (hypocotyl and cotyledons) were less than with 310-0. The reduced pigment formation and severe growth inhibition with 310 are regarded as indications of the phytotoxicity. These results suggest that the phytotoxicity is ascribed to the region from 287 to 302 nm (difference in emission of 310 from 310-0), although it is not clear
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cucumber hypocotyl, surpassed that of red light, whereas in cotyledons of the tested plants, the action of UV was inferior to that of red light.
UV effects on growth and pigmentation
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whether this wavelength region exerts the action solely or jointly with the region of wavelength longer than 302 nm in the main emission of 310. Tolerance to the harmful wavelength region varied from species to species. The tolerant plants in this study were maize and eggplant in which no toxicity was observed at all. Particularly in eggplant, the anthocyanin formation in hypocotyls was not suppressed, i.e., the value with 310 was not significantly less than that with 310-0, and chlorophyll content conversely increased (Table 7). Cucumber was very sensitive to light of this region. Cline and Salisbury (7) and Basiouny et al. (3) have pointed out that maize is tolerant and eggplant sensitive to 254 nm UV. In our experiment, eggplant was tolerant to 287-302 nm UV. The wavelength region 300-338 nm is characterized by its high effectiveness for anthocyanin formation and for intense inhibition of elongation. That these responses to the UV region were observed only in a hypocotyl or a coleoptile but not in cotyledons deserves attention. High effectiveness of the UV region has also been reported with anthocyanin formation in apple skin (2) and Spirodela oligorhiza (18). Characteristic features of the region at 330-370 nm are inhibition of hypocotyl elongation and promotion of the growth of cotyledons as well as chlorophyll synthesis. Schonbohm (20) has reported that a brief period of irradiation with 370 nm UV induced unfolding of dark-grown wheat leaves. Wagne (28) has also obtained a similar result with UV at 313-334 nm. Our result that irradiation with 330370 nm (350) promoted the growth of cotyledons is along the same line as these authors' results. Klein and Edsall (10) and Klein et al. (11) have reported that irradiation with UV at 355-380 nm suppresses the growth of some species of plants when given together with white light. Although the authors did not mention it, the photographs in the former paper (10) show that the development of the leaves as well as the stems was inhibited by this UV radiation. The result with joint irradiation by 350 and 660 (Table 6) showed that this wavelength region of UV did not inhibit the red light-induced growth of cotyledons but gave a normal flat shape to the cotyledons. Thus, we consider that irradiation with UV at 330-370 nm promotes the growth of cotyledons and leaves when given solely or in combination with red light. The region from 384 to 400 nm was generally the least effective of those tested. In maize coleoptile and the hypocotyl of radish and soybean, however, it exerted distinct inhibitory actions which were greater than those of the neighboring light regions, i.e., 330-370 and 404-432 nm. Especially, maize coleoptile formed the greatest amount of chlorophyll in this UV region among those tested. On the other hand, cucumber and soybean hypocotyls did not greatly respond to this region. I ts effect seems to vary with the plant species. The above discussion on the effects of UV is based on the postulate that the main emission from the UV lamps is solely responsible for the observed effects. However, the UV fluorescent lamps used in the present study had an additional emission (mostly mercury lines) from the UV through the visible regions of the spectrum, and the observed effects might have been caused by a joint action of the main and additional emissions. Thus far, the effect of pure monochromatic UV light on the growth of higher plants has not been reported except for a few papers on
1570
T. Hashimoto and M. Tajima
anthocyanin formation (14, 18), and further improvement of irradiation systems is required to study this. We thank Dr. Y. Togari, Professor of Nippon University and president of Nokoken Research Union, and Dr. K. Inada, Agricultural Research Institute, Ministry of Agriculture, Forestry and Fishery, for their encouragement during this work. The generous help from Hitachi, Ltd. and Mitsui Toatsu, Ltd. with supplies of the materials is gratefully acknowledged.
References
(11) Klein, R. M., P. C. Edsall and A. C. Gentile:
(12)
(13) (14) (15)
(16)
(17) (18)
(19)
Effects of near ultraviolet and green radiations on
plant growth. Plant Physiol. 40: 903-906 (1965). Klein, R. M. and]. Wansor: Effects of non-ionizing radiation on expansion of disks from leaves of dark-grown bean plants. ibid. 38: 5-10 (1963). Krizek, D. T.: Influence of ultraviolet radiation on germination and early seedling growth. Physiol. Plant. 34: 182-186 (1975). Lackmann, I.: Wirkungsspektren der Anthocyansynthese in Gewebekulturen und Keimlingen von Haplopappus gracilis. Planta 98: 258-269 (1971). Lindoo, S.]. and M. M. Caldwell: Ultraviolet-B radiation-induced inhibition of leaf expansion and promotion of anthocyanin production. Lack of involvement of the low irradiation phytochrome system. Plant Physiol. 61: 278-282 (1978). Lockhart,]. A. and U. B. Franzgrote: The effects of ultraviolet radiation on plants. In Encyclopedia ofPlant Physiology 16. Edited by W. Ruhland. p. 532-554. Springer-Verlag, Berlin, 1961. Murphy, T. M., L. A. Wright,]r. and]. B. Murphy: Inhibition of protein synthesis in cultured tobacco cells by ultraviolet radiation. Photochem. Photobiol. 21: 219-225 (1975). Ng, Y. L., K. V. Thimann and S. A. Gordon: The biogenesis of anthocyanins X. The action spectrum for anthocyanin formation in Spirodela oligorhiza. Arch. Biochem. Biophys. 107: 550--558 (1964). Owen, P. C.: Effect of ultra-violet radiation on the respiration-rates of tobacco leaves, and its reversal by visible light. Nature 180: 610-611 (1957).
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( 1) Ambler,]. E., P. T. Krizek and P. Semeniuk: Influence of UV-B radiation on early seedling growth and translocation of 65Z n from cotyledons in cotton. Physiol. Plant. 34: 177-181 (1975). ( 2) Arthur,]. M.: Red pigment production in apples by means of artificial light sources. Contr. Boyce Thompson Inst. 4: 1-18 (1932). ( 3) Basiouny, F. M., T. K. Van and R. H. Biggs: Some morphological and biochemical characteristics of C s and C 4 plants irradiated with UV-B. Physiol. Plant. 42: 29-32 (1978). ( 4) Bawden, F. C. and F. R. S. Kleczkowski: Ultra-violet injury to higher plants counteracted by visible light. Nature 169: 90-91 (1952). ( 5) Benda, G. T. H.: Some effects of ultra-violet radiation on leaves of French bean (Phaseolus vulgaris L.). Ann. Appl. Biol. 43: 71-85 (1955). (6) Butler, W. L., S. B. Hendricks and H. W. Siegelman: Action spectra of phytochrome in vitro. Photochem. Photobiol. 3: 521-528 (1964). ( 7) Cline, M. G. and F. B. Salisbury: Effects of ultraviolet radiation on the leaves of higher plants. Rad. Bot. 6: 151-163 (1966). ( 8) EI-Mansy, H. I. and F. B. Salisbury: Biochemical responses of Xanthium leaves to ultraviolet radiation. ibid. 11: 325-328 (1971). ( 9) Klein, R. M.: Influence of ultraviolet radiation on auxin-controlled plant growth. Amer. J. Bot. 54: 904-914 (1967). (10) Klein, R. M. and P. C. Edsall: Interference by near ultraviolet and green light with growth of animal and plant cell cultures. Photochem. Photobiol. 6: 841-850 (1967).
UV effects on growth and pigmentation
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(20) Schonbohm, E.: Die Wirkung kurzwelliger Strahlung aufden Hellrot-Dunkelrot-Antagonismus bei einigen Photomorphosen an Triticum vulgare und Lactuca sativa. Z. Bot. 52: 335-345 (1964). (21) Semeniuk, P. and R. N. Stewart: Seasonal effect of UV-B radiation on Poinsettia cultivars. J. Amer. Soc. Hort. Sci. 104: 246-248 (1979). (22) Semeniuk, P. and R. N. Stewart: Comparative sensitivity of cultivars of Coleus to increased UV-B radiation. ibid. 471-474 (1979). (23) Sisson, W. B. and M. M. Caldwell: Lamp/filter systems for simulation of solar UV irradiance under reduced atmospheric ozone. Photochem. Photohiol. 21: 453-456 (1975). (24) Sisson, W. B. and M. M. Caldwell: Photosynthesis, dark respiration, and growth of Rumex patientia L. exposed to ultraviolet irradiance (288 to 315 nanometers) simulating a reduced atmospheric ozone column. Plant Physiol. 58: 563-568 (1976). (25) Sisson, W. B. and M. M. Caldwell: Atmospheric zoone depletion: reduction of photosynthesis and growth of a sensitive higher plant exposed to enhanced U.V.-B radiation. J. Exp, Bot. Downloaded from http://pcp.oxfordjournals.org/ at UQ Library on April 29, 2015
28: 691-705 (1977). (26) Skokut, T. A.,]. H. Wu and R. S. Daniel: Retardation of ultraviolet light accelerated chlorosis by visible light or by benzyladenine in Nicotiana glutinosa leaves: Changes in amino acid content and chloroplast ultrastructure. Photochem. Photohiol. 25: 109-118 (1977). (27) Tanada, T. and S. B. Hendricks: Photoreversal of ultraviolet effects in soybean leaves. Amer. J. Bot. 40: 634-637 (1953). (28) Wagne, C.: Effect ofUV light on lettuce seed germination and on the unfolding of grass leaves. Physiol. Plant. 19: 128-133 (1966). (29) Wang, H. C., R. H. Hamilton and R. A. Deering: Ultraviolet light as an inhibitor of auxininduced growth in Avena coleoptiles. In Biochemistry and Physiology of Plant Growth Substances. Edited by F. Wightman and G. Setterfield. p. 685-697. The Runge Press, 1968. (30) Wellmann, E.: UV dose-dependent induction of enzymes related to flavonoid biosynthesis in cell suspension cultures of parsley. FEBS Lett. 51: 105-107 (1975). (31) Wellmann, E., G. Hrazdina and H. Grisebach: Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis. Phytochemistry' 15: 913-915 (1976). (32) Wu,]. H., T. Skokut and M. Hartman: Ultraviolet-radiation-aceclerated leaf chlorosis: prevention of chlorosis by removal of epidermis or by floating leaf discs on water. Photochem. Photobiol. 18: 71-77 (1973).
Effects of ultraviolet irradiation on growth and pigm.entation in seedlings Tohru Hashimoto and Misao Tajima The Institute for Physical and Chemical Research, Wakoshi, Saitama 351, Japan (Received February 22, 1980)
Although a vast amount of work has been devoted to the effects of visible light on higher plants, not many papers deal with those ofUV. Part of the cause seems to be due to the technical difficulty of obtaining proper irradiation systems. Introduction of new irradiation systems has provided new information on this subject. Many experiments conducted with low-pressure mercury lamps which emit solely 254 nm line disclosed mostly phytotoxic effects such as reduced size or distorted growth (29), glazing, bronzing, injury, necrosis and death (4, 5, 7, 19, 27), accelerated senescence (32), inhibition of chlorophyll and protein synthesis (8, 17). Highpressure mercury lamps and xenon lamps made possible investigation of the effects of the middle and near UV (14, 20, 30, 31) but limited availability of filters for use with them restricted selection of wavelengths, particularly when intact plants were to be irradiated. Fluorescent lamps have also been employed. Klein and Edsall (10) used blackAbbreviations: DW, dry weight; FW, fresh weight; 310, 310-0, 350, 395,420 and 660, lights with emission at 290-338, 300-338, 330-370, 384--400, 404--432 and 648-666 nm in halfwidth, respectively; 310-2, secondary emission of the lamps at wavelengths longer than 380 nm.
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Dark-germinated seedlings of maize, radish, soybean, cucumber and eggplant were grown for 2 or 3 days under ultraviolet light, and the differential effects of UV according to wavelength regions were evaluated. Although the effectiveness of UV irradiation differed somewhat depending on the plant species, generally, four wavelength regions having different effects were discerned. The region from 287 to 302 nm was phytotoxic, causing bronzing (radish), epinastyand blazing (cotyledon, cucumber), formation of brown flecks (soybean), and also severe growth inhibition in hypocotyls. The region from 300 to 338 nm greatly promoted anthocyanin formation and inhibited the shoot elongation. The region from 330 to 370 nm inhibited shoot elongation and promoted cotyledon growth as well as chlorophyll formation. The effects of the region from 384 to 400 nm were generally weak, but in maize coleoptile, radish and soybean hypocotyls this region exerted a distinct inhibition, stronger than the region of 330-370 nm, and in chlorophyll formation in maize coleoptile this region was the most effective of those tested. The above-stated shoot growth inhibition and anthocyanin formation caused by UV were generally greater than those caused by blue or red light, but growth promotion in the cotyledons was smaller. Key words: Anthocyanin - Chlorophyll - Cotyledon growth - Phytotoxicity Shoot elongation - Ultraviolet irradiation.
1560
T. Hashimoto and M. Tajima
Materials and methods
Plants The plant species tested were maize (Zea m~.ys L., cv. Golden Cross Bantam, Watanabe Saishujo, Ltd.), radish (Raphanus sativus L., cv. Akamaru, Tohoku Shubyo, Ltd.), soybean (Glycine max L., obtained locally at a grocery, name of the cultivar not specified), cucumber (Cucumis sativus L., cv. Aonagajibai, Tohoku Shubyo) and eggplant [Solanum esculentum (L.) Nees., cv. Shin B-go, Watanabe Saishujo] . Seeds were imbibed in running tap water for 8 to 16 hr, sown in vermiculite in plastic boxes, 6 X 15 X 10 em (height), and germinated in the dark at 27-30°C. Within 12 hr after emergence of the seedlings, irradiation was started and continued for 48 or 72 hr until harvest. Liquid Hyponex diluted 1,000 times was supplied when seeds were sown and when seedlings emerged.
Irradiation system The lamps used were 20-watt fluorescent tubes which had an emission peak at 310, 350, 395 or 420 nm (specially manufactured for this study, Hitachi, Ltd.) and ones which had an emission peak at 660 nm (Colored Lamp, FI-20-RF, Mitsubishi Electric, Ltd.). For each treatment, 2 or 3 lamps were installed and combined with a sheet of polyvinyl chloride film that cut off unnecessary UV (UVC-O and UVC-2, 0.1 mm thick, Mitsui Toatsu, Ltd.) as follows: three 310 nm lamps with UVC-O, three 310 nm lamps with UVC-2, two 350 nm lamps with UVC0, two 395 nm lamps with UVC-2 and two 420 nm lamps with UVC-2. The lights thus obtained were referred to, respectively, as 310-0, 310-2, 350, 395 and 420 (blue). Lights emitted from unfiltered 310 nm and 660 nm lamps were also used as 310 and 660 (red). These lights had an emission band at 290-338 (halfwidth, 310), 300-338 (310-0), 330-370 (350), 384-400 (395), 404-432 (420) or 648-666
(660) nm.
310-2 was secondary emission due to mercury lines in the region of
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light lamps (General Electric BLB integral filter lamp) to obtain 355-380 nm UV and found that this region of UV inhibited plant growth. Sisson and Caldwell (23) devised a system using a Westinghouse FS-40 'Sunlamp' and filters, which provided radiation of 280-320 nm, to simulate the solar UV irradiance resulting from a decrease of stratospheric ozone. Supplementary use of this with natural solar radiation (1, 21, 22) or artificial light sources (3, 13, 24,25) resulted in suppressed growth of roots, cotylecons and leaves, inhibition of photosynthesis, increased pigmentation or decoloration, phytotoxic damage such as bronzing and glazing of cotyledons, distorted leaves, and finally abscission, and other abnormal curvature of shoots. Most of the results thus far obtained with artificial UV have been based on the effects of a single particular wavelength region and therefore do not provide the entire picture of UV actions at various wavelengths. In an attempt to evaluate the differential effects of UV in relation to wavelengths ranging from 290 through 400 nm, we prepared fluorescent lamps which emit different bands of UV and tested them in experiments on growth and pigmentation of some agricultural plant seedlings.
1561
UV effects on growth and pigmentation
wavelength longer than 380 nm (Fig. l). The spectral energy distribution curves in the figure were determined with a spectral radiometer (EPS-3T, Hitachi, Ltd.) corrected with a bromine lamp (Quartz Line Lamp DXW 1,000 watts, General Electric, Ltd.).
~
"(i;
c
a>
+-'
..= Q)
:> ~ 0
(i)
n::
I
I
-----l
350
I
I
A
395 420
1\
I
I
I
660
Wavelength (nm)
Fig. 1. Radiation spectra of the lights used. The curve of 660 is from the manufacturer's data, and the others were measured with a spectral radiometer (EPS-3T, Hitachi, Ltd.).
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310-2
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T. Hashimoto and M. Tajima
The irradiance was equalized by adjusting the distance from the lamps and/or inserting a neutral net filter for all treatments except for 310-2, which was kept at the same irradiance level as that of 310-0 in the region of wavelength longer than 380 nm. Light energy was measured with a thermopile (CA-l, Kipp and Zonen, Ltd.). The filter films were renewed as required (almost every two experiments) as their transmittance was changed by UV irradiation. Plants were irradiated in a dark room dimly lit with diffuse daylight (less than 0.1 ftwatt·cm- 2) . The temperature was controlled at 27-30°C.
Determine tion
ofgrowth andpigment content
Evaluation of UV effects The effects of the UV irradiations were, of course, compared with non-irradiated controls but could be more suitably evaluated by comparison with 310-0, since the UV lamps used also emitted visible light (Fig. 1). Results
Elongation growth Typical results are shown in Tables 1-5. The UV irradiations inhibited the elongation of the coleoptile, the first internode and the first leaf of maize, and the hypocotyl of other plants in comparison with 310-2 as well as with non-irradiated controls. 310 was the most effective followed by 310-0 then 395, and 350 was the least effective. In cucumber and eggplant, 350 was very effective while 395 was not. All these inhibitory effects of UV were greater or not less than those of 420 (blue) and 660 (red), except for soybean in which 660 was very effective. In cucumber, the elongation of hypocotyl which was inhibited by red light was further suppressed by 350 to the level given by the continuous 350 (Table 6). Growth ofcotyledons Although 310 inhibited the growth (increase in FW) of cotyledons, 310-0 and 350 promoted it in cucumber (Table 4) and 395 also did so in eggplant (Table 5). In radish, 310-0 and 395 were promotive, but 350 was not effective (Table 2).
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At the end of a 72-hr (48 hr for soybean) irradiation period, the length and FW of the seedlings were determined, and the seedlings were divided into 2 equal groups, one half was dried at 80°C for 2-3 hr to determine DW, and the other half was extracted to determine pigment content. Seedlings to be extracted were immersed in methanol, kept at 5°C in the dark for 1-2 days, ground in a mortar, and filtered through a teflon filter (Millipore, Ltd.). Total chlorophyll content was determined by the absorbance at 662 nm in a methanol solution. To determine the content of anthocyanin, a dilute hydrochloric acid was added to the methanol extract to make the final concentration 0.5%, and the absorbance at 528 or 530 nm was determined. Since DW of plant materials was not affected greatly by the irradiations (see the Results), pigment contents were compared on a DW basis. For this purpose, the absorbance was measured of the solution containing the extract from plant materials of 1 mg DW per 1 ml of the solution (standard concentration).
UV effects on growth and pigmentation
1563
Table 1 Effectongrowth of maize seedlings Irradiated with 310
310-0
310-2
350
395
420
660
Nonirradiated control
First internode length Coleoptile length
47±3.8 34±2.6 40±2.7 100±8.1 (14.6 mm)
39±3.7
45±4.4
54±5.7 312±27.3
76±2.9 49±1. 6 65±2.9 100±1. 4 (40.7 mm)
71±4. 1
84±2.2
81±2. 2 114± 6.1
First leaf length
92±3.4 73±1. 8 85±3.4 100±2.8 (85.4mm)
89±3.2 102±3.9 102±2.6
FWof coleoptile
36
60
100 (99.5 mg)
67
49
80
70
108
DW/FWof 172 coleoptile
109
100 (4.4%)
109
120
102
114
109
16
10
13
13
13
13
18
20
Values in the table are the percent of those with 310-2 and s.e, in percent. The observed values with 310-2 are shown in parentheses. Irradiation energy was 190 ,uwatts'cm- 2 except for 310-2 which was 82,uwatts·cm- 2 •
Table 2 Effect on growth of radish seedlings 395
420
660
Nonirradiated control
70±3.1
83±2.7
80±3.4
135±4.2
Irradiated with 310 Hypocotyl Length FW DW/FW Plant height
310-0
310-2
350
50±3.1 65±3.0 100±3.7 82±3.5 (43.5 mm) 42
57
100 (65.0mg)
78
73
78
77
114
163
121
100 (3.8%)
105
108
100
97
87
52±2.7 69±2.9 100±3.4 87±3.3 (52.1 mm)
80±3.3
92±2.7
86±3.2
121 ±3. 6
Cotyledons FW o
DW/FW No of seedlings measured
89
112
100 (25.0 mg)
92
122
112
150
78
105
90
100 (12.2%)
97
77
80
70
120
13
20
20
20
20
20
22
20
For explanation of the figures and experimental conditions, see Table 1. FW per pair of cotyledons.
a
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No of seedlings measured
64± 5.4
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T. Hashimoto and M. Tajima
Table 3 Effect on growth of soybean seedlings Irradiated with 310 Hypocotyl Length FW
DW/FW
No of seedlings measured
395
350
310-2
420
660
Nonirradiated control
60± 4.0 83± 5.3 100± 5.7 101± 3.7 91±5. 0 118± 6.6 67±2.8 133± 7.4 (92. 4 mm) 76
85
145
106
100 (363mg) 100 (3.3%)
101
86
121
70
141
97
97
91
121
91
43±13.1 66±16.6 100±10.8 105± 10.2 54±9.6 (12.9 mm) 13
15
17
15
Irradiation period was 48 hr.
11
91± 13.8 83 ± 7. 5
25±13.4
11
12
14
For other explanations, see Table 1.
The promotive effect of these UV's was far less than that of 660 but greater than that of 420 in cucumber and radish, and greater than that of 660 but less than that of 420 in eggplant. Table 4 Effect on growth of cucumber seedlings Irradiated with 310 Hypocotyl Length FW DW/FW
Nonirradiated control
310-2
350
395
420
660
31±2. 3 60±0.9
100± 1. 5 (112 mm)
70± 1.6
96±1. 5
94±2.1
92± 1. 1
164±3.0
30
62
100 (268 mg)
77
103
103
80
123
248
117
117 100 (2.3%)
100
104
109
104
73
119
128
101
94
170 b
53
135
89
88
93
102
84
153
17
16
15
19
19
310-0
Cotyledons FW a
DW/FW
100 (105 mg) 100 (9. I %)
No of seedlings measured
15
19
18
2 except for 310-2 which was 80,uwatts Irradiation with 173 ,uwatts FW per pair of cotyledons including the plumule. For other explanations, see Table I. b Plumule was well developed. ocm-
a
ocm-
2•
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Epicoty1 Length
310-0
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UV effects on growth and pigmentation Table 5
Effect on growth of eggplant seedlings
Irradiated with 310
310-0
310-2
irradiated control
395
420
660
103± 1. 7
98±2.1
149±3.3
205±2.6
350
Hypocoty1 Length
50± 1. 1 63± 1. 5
FW
53
59
100±2.2 80± 1. 4 (35.3mm) 100
71
106
100
147
206
119
97
106
97
88
114
114
121
107
36
92
95
88
88
151
34
30
32
33
34
(17mg) DW/FW
153
119
100 (3.2%)
71
F\V
107
100 (14mg)
DW/FW
154
100
100 (7.8 %)
No of seedlings measured
37
34
30
Irradiation conditions were the same as in Table 4. For other explanations, see Table 1.
FW of cotyledons was per pair of cotyledons.
Since the growth-promoting effect of 350 on cotyledons was noteworthy, the interaction of 350 with red light was examined using cucumber seedlings. Red light markedly promoted the increase in FW and DW (as well as chlorophyll), and 350 did not promote or suppress the red light-induced growth (chlorophyll formation also) (Table 6) but gave a normal flat shape to the cotyledons, which had an upward-curled blade under the continuous red light.
FW and DW The change In FW of hypocotyls and coleoptiles caused by the irradiations Table 6
Effects ofjoint irradiation with 350 and 660 in cucumberseedlings
Non -irradiated control Hypocotyl Length (mm) Chlorophyll
l84±3.4 0
Irradiated with 350 78± 1.B 0.076
660
350/660
103± 1. 2 0.051
BO±1. 3 0.064
Cotyledons FW(mg)
56
DW(mg)
7.84
Chlorophyll
0
134 10. 7
1.08
178 13.5
1.56
173 12.8
1.56
Irradiation was done alternately with 12 hr each of350 and 660 for 350/660, and continuously with the indicated lights for other treatments, totalling 72 hr. Chlorophyll content represents the absorbance at 662 nm (in methanol) at the standard concentration. The number of seedlings used in each treatment was 16.
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Cotyledons
1566
T. Hashimoto and M. Tajima
generally paralleled the change of length in all the plants tested. In contrast, the DW was not affected as much by the irradiation as the FW, thus the ratio ofDWjFW increased, particularly with 310 (Tables 1-5). A similar tendency of the DWjFW
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Fig. 2. Epinasty qj a cucumber cotyledon caused ky 287-302 nm UV. Seedlings were grown for 3 days from their emergence under 310 (190 I1watts'cm-2), 310-0 (190 /1watts'cm- 2) and 310-2 (82I1watts·cm-2).
1567
UV effects on growth and pigmentation
ratio was observed with the cotyledons (Tables 2, 4 and 5), but discussion of their DW is meaningless because cotyledons have their original storage matter.
Phytotoxicity
Chlorophyll content In the cotyledons and hypocotyl of eggplant, cucumber, and soybean, 350 and 310-0 promoted chlorophyll formation compared with 310-2 (Table 7), but 310 inhibited it markedly, except in eggplant hypocotyl where the content of chlorophyll was greatest with 310 of all treatments including 660 and 420. In maize coleoptile, 395 followed by 420 then 350 caused large production of chlorophyll. 310-0 was slightly inhibitory. These promotive effects of UV, i.e., 395 and 350 in maize coleoptile, 350 in Table 7 Effict on chlorophyll content in seedlings
310-0
310-2
350
395
420
660
Nonirradiated control
82
100 (0.073)
155
223
218
82
17
Irradiated with
310 Maize Coleoptile
Eggplant Hypocotyl
159
112
100 (0.0674)
128
108
89
77
3
Cotyledons
70
104
100 (0.776)
111
88
96
140
0
Soybean Hypocotyl
66
153
100 (0.0074)
130
116
47
183
0
Hypocotyl
77
106
100 (0.049)
133
94
106
90
2
Cotyledons
53
113
100 (0.636)
109
104
77
159
0
Cucumber
Values in the table represent chlorophyll content as the percent of that in 310-2 and those in parentheses the absorbances at 662 nm of 31 0-2 (in methanol) at the standard concentration.
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Irradiation with 310 caused various abnormalities in the appearance of the seedlings. Radish seedlings had a weak brown pink color (bronzing) and were slightly hyaline. In cucumber, one of the cotyledons showed epinasty and the surfaces of both cotyledons were blazed in the 3-day experiment (Fig. 2). When the irradiation was prolonged for 3 more days the seedlings gradually became necrotic from the base toward the top. Soybean had brown flecks on the cotyledons and hypocotyl. However, 310-0 and the longer wavelengths of UV did not have such toxicity. Maize and eggplant were resistant to 310, i.e., they showed no signs of abnormality.
1568
T. Hashimoto and M. Tajima
Table 8 Effecton anthocyanin content ofseedlings 310
310-0
310-2
350
395
420
660
Nonirradiated control
Soybean hypocotyl
148
307
100 (0.0163)
174
134
62
201
26
Eggplant hypocotyl
376
418
100 (0.0136)
182
100
71
71
29
Irradiated with
Values in the table represent anthocyanin content as the percent of that with 310-2. Values in parentheses are the absorbances at 530 nm of 310-2 (in HC1-methanol) at the standard concentration.
Anthocyanin formation Promotion of anthocyanin formation by UV was striking in hypocotyls (Table 8). In soybean and eggplant, 310, 310-0 and 350 were very effective and 395 was also promotive in soybean but without effect in eggplant. The effectiveness of 310-0 in soybean and of 310-0, 310 and 350 in eggplant was greater than that of red light. The effect of red light was very weak in eggplant. In cucumber, no anthocyanin was detected in all treatments.
Discussion The effects of UV on seedlings described in this paper varied from injury or lethality to promotion of growth depending on the wavelength of the irradiation given and the plant parts irradiated. From the types of action, four wavelength regions were discerned: 1) the region from 287 to 302 nm was phytotoxic, 2) the region from 300 to 338 nm greatly promoted anthocyanin formation and inhibited the shoot elongation, 3) the region from 330 to 370 nm inhibited hypocotyl elongation and promoted the growth of cotyledons as well as chlorophyll formation, and 4) the region from 384 to 400 nm showed a species-specific inhibition of hypocotyl elongation. The phytotoxic effects of 254 nm emitted from a germicidal lamp have been reported in many papers as reviewed by Lockhart and Franzgrote (16). Recently, the information that UV-B radiation (290-315 nm) also causes toxicity is accumulating (1, 3, 13, 21, 22). In the present study, blazing (cucumber cotyledons), bronzing (radish), necrosis (cucumber), epinasty of a cotyledon (cucumber) and the formation of brown flecks (soybean hypocotyl and cotyledons) were observed with 310, whereas such phytotoxicities were absent with 310-0 and other lights. With 310, anthocyanin formation and chlorophyll synthesis as well as growth (hypocotyl and cotyledons) were less than with 310-0. The reduced pigment formation and severe growth inhibition with 310 are regarded as indications of the phytotoxicity. These results suggest that the phytotoxicity is ascribed to the region from 287 to 302 nm (difference in emission of 310 from 310-0), although it is not clear
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cucumber hypocotyl, surpassed that of red light, whereas in cotyledons of the tested plants, the action of UV was inferior to that of red light.
UV effects on growth and pigmentation
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whether this wavelength region exerts the action solely or jointly with the region of wavelength longer than 302 nm in the main emission of 310. Tolerance to the harmful wavelength region varied from species to species. The tolerant plants in this study were maize and eggplant in which no toxicity was observed at all. Particularly in eggplant, the anthocyanin formation in hypocotyls was not suppressed, i.e., the value with 310 was not significantly less than that with 310-0, and chlorophyll content conversely increased (Table 7). Cucumber was very sensitive to light of this region. Cline and Salisbury (7) and Basiouny et al. (3) have pointed out that maize is tolerant and eggplant sensitive to 254 nm UV. In our experiment, eggplant was tolerant to 287-302 nm UV. The wavelength region 300-338 nm is characterized by its high effectiveness for anthocyanin formation and for intense inhibition of elongation. That these responses to the UV region were observed only in a hypocotyl or a coleoptile but not in cotyledons deserves attention. High effectiveness of the UV region has also been reported with anthocyanin formation in apple skin (2) and Spirodela oligorhiza (18). Characteristic features of the region at 330-370 nm are inhibition of hypocotyl elongation and promotion of the growth of cotyledons as well as chlorophyll synthesis. Schonbohm (20) has reported that a brief period of irradiation with 370 nm UV induced unfolding of dark-grown wheat leaves. Wagne (28) has also obtained a similar result with UV at 313-334 nm. Our result that irradiation with 330370 nm (350) promoted the growth of cotyledons is along the same line as these authors' results. Klein and Edsall (10) and Klein et al. (11) have reported that irradiation with UV at 355-380 nm suppresses the growth of some species of plants when given together with white light. Although the authors did not mention it, the photographs in the former paper (10) show that the development of the leaves as well as the stems was inhibited by this UV radiation. The result with joint irradiation by 350 and 660 (Table 6) showed that this wavelength region of UV did not inhibit the red light-induced growth of cotyledons but gave a normal flat shape to the cotyledons. Thus, we consider that irradiation with UV at 330-370 nm promotes the growth of cotyledons and leaves when given solely or in combination with red light. The region from 384 to 400 nm was generally the least effective of those tested. In maize coleoptile and the hypocotyl of radish and soybean, however, it exerted distinct inhibitory actions which were greater than those of the neighboring light regions, i.e., 330-370 and 404-432 nm. Especially, maize coleoptile formed the greatest amount of chlorophyll in this UV region among those tested. On the other hand, cucumber and soybean hypocotyls did not greatly respond to this region. I ts effect seems to vary with the plant species. The above discussion on the effects of UV is based on the postulate that the main emission from the UV lamps is solely responsible for the observed effects. However, the UV fluorescent lamps used in the present study had an additional emission (mostly mercury lines) from the UV through the visible regions of the spectrum, and the observed effects might have been caused by a joint action of the main and additional emissions. Thus far, the effect of pure monochromatic UV light on the growth of higher plants has not been reported except for a few papers on
1570
T. Hashimoto and M. Tajima
anthocyanin formation (14, 18), and further improvement of irradiation systems is required to study this. We thank Dr. Y. Togari, Professor of Nippon University and president of Nokoken Research Union, and Dr. K. Inada, Agricultural Research Institute, Ministry of Agriculture, Forestry and Fishery, for their encouragement during this work. The generous help from Hitachi, Ltd. and Mitsui Toatsu, Ltd. with supplies of the materials is gratefully acknowledged.
References
(11) Klein, R. M., P. C. Edsall and A. C. Gentile:
(12)
(13) (14) (15)
(16)
(17) (18)
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Effects of near ultraviolet and green radiations on
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( 1) Ambler,]. E., P. T. Krizek and P. Semeniuk: Influence of UV-B radiation on early seedling growth and translocation of 65Z n from cotyledons in cotton. Physiol. Plant. 34: 177-181 (1975). ( 2) Arthur,]. M.: Red pigment production in apples by means of artificial light sources. Contr. Boyce Thompson Inst. 4: 1-18 (1932). ( 3) Basiouny, F. M., T. K. Van and R. H. Biggs: Some morphological and biochemical characteristics of C s and C 4 plants irradiated with UV-B. Physiol. Plant. 42: 29-32 (1978). ( 4) Bawden, F. C. and F. R. S. Kleczkowski: Ultra-violet injury to higher plants counteracted by visible light. Nature 169: 90-91 (1952). ( 5) Benda, G. T. H.: Some effects of ultra-violet radiation on leaves of French bean (Phaseolus vulgaris L.). Ann. Appl. Biol. 43: 71-85 (1955). (6) Butler, W. L., S. B. Hendricks and H. W. Siegelman: Action spectra of phytochrome in vitro. Photochem. Photobiol. 3: 521-528 (1964). ( 7) Cline, M. G. and F. B. Salisbury: Effects of ultraviolet radiation on the leaves of higher plants. Rad. Bot. 6: 151-163 (1966). ( 8) EI-Mansy, H. I. and F. B. Salisbury: Biochemical responses of Xanthium leaves to ultraviolet radiation. ibid. 11: 325-328 (1971). ( 9) Klein, R. M.: Influence of ultraviolet radiation on auxin-controlled plant growth. Amer. J. Bot. 54: 904-914 (1967). (10) Klein, R. M. and P. C. Edsall: Interference by near ultraviolet and green light with growth of animal and plant cell cultures. Photochem. Photobiol. 6: 841-850 (1967).
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28: 691-705 (1977). (26) Skokut, T. A.,]. H. Wu and R. S. Daniel: Retardation of ultraviolet light accelerated chlorosis by visible light or by benzyladenine in Nicotiana glutinosa leaves: Changes in amino acid content and chloroplast ultrastructure. Photochem. Photohiol. 25: 109-118 (1977). (27) Tanada, T. and S. B. Hendricks: Photoreversal of ultraviolet effects in soybean leaves. Amer. J. Bot. 40: 634-637 (1953). (28) Wagne, C.: Effect ofUV light on lettuce seed germination and on the unfolding of grass leaves. Physiol. Plant. 19: 128-133 (1966). (29) Wang, H. C., R. H. Hamilton and R. A. Deering: Ultraviolet light as an inhibitor of auxininduced growth in Avena coleoptiles. In Biochemistry and Physiology of Plant Growth Substances. Edited by F. Wightman and G. Setterfield. p. 685-697. The Runge Press, 1968. (30) Wellmann, E.: UV dose-dependent induction of enzymes related to flavonoid biosynthesis in cell suspension cultures of parsley. FEBS Lett. 51: 105-107 (1975). (31) Wellmann, E., G. Hrazdina and H. Grisebach: Induction of anthocyanin formation and of enzymes related to its biosynthesis by UV light in cell cultures of Haplopappus gracilis. Phytochemistry' 15: 913-915 (1976). (32) Wu,]. H., T. Skokut and M. Hartman: Ultraviolet-radiation-aceclerated leaf chlorosis: prevention of chlorosis by removal of epidermis or by floating leaf discs on water. Photochem. Photobiol. 18: 71-77 (1973).
