Plant Cell Physiol. 42(7): 784–787 (2001)
JSPP © 2001
Development of UV Defense Mechanisms during Growth of Spinach Seedlings
Megumi Hada 1, 3, 4, Keisuke Hino 2 and Yuichi Takeuchi 1
1
2
Department of Bioscience and Technology, School of Engineering, Hokkaido Tokai University, Sapporo, 005-8601 Japan
Department of Biology, Faculty of Science, Kohnan University, Kobe, 658-8501 Japan
;
(CPDs) into their intact monomer bases is catalyzed by DNA
photolyase, which uses electromagnetic energy of wavelengths
ranging from blue to UV-A (Sancar 1994b). Photorepair has
been reported in many plant species (see Hada et al. 2000b),
and is widely accepted to be the main pathway of the repair of
CPDs induced by UV-B irradiation. In many plant species,
photorepair activity has been reported to be photoregulated
(Buchholz et al. 1995, Hada et al. 1999, Pang and Hays 1991,
Langer and Wellmann 1990, Takayanagi et al. 1994, Taylor et
al. 1997), but few reports are available concerning to the regulation of DNA photolyase in developmental stages (Taylor et
al. 1997, Hidema and Kumagai 1998).
In the present study, in order to better understand the manner in which the two different UV-defense mechanisms function at different growth stages, we examined changes in the
amounts of UV-absorbing substances and the activity of DNA
photolyase during the growth of spinach seedlings.
Spinach seeds (Spinacia oleracea L. cv. Suikou mate No.
14, Sekisui Plastics Co., Ltd, Osaka, Japan) were sown at 2-cm
intervals in rock wool (Nitto Boseki Co., Ltd., Tokyo)
immersed in a hydroponic culture solution (Suikou Mate,
Sekisui Plastics Industry Co., Ltd). The seedlings were grown
in a growth cabinet (LH-300-RDMP, Nippon Medical &
Chemical Instruments Co., Ltd, Tokyo, Japan) maintained at
20°C. Light was supplied by fluorescent lamps (FL40S·BRN;
Toshiba, Tokyo, Japan). The photosynthetic photon flux density at plant height was about 65 mmol m–2 s–1, with a 12-h
light/dark cycle (light cycle: 6:00 a.m.–6:00 p.m.). The white
light used did not contain UV-B light.
The DNA content of spinach leaves was determined using
the method of Naito et al. (1978). Four cotyledons or first
leaves were frozen with liquid nitrogen, ground into powder
using a chilled mortar and pestle, and then homogenized in
10 ml of 10% (w/v) trichloroacetic acid (TCA). After centrifugation of the homogenate at 2,000´g for 10 min, the pellet was
washed with 7% TCA and then with 95% (v/v) ethanol. The
residue was suspended in a mixture of ethanol and diethyl ether
(2 : 1, v/v) and incubated at 50°C for 20 min. This procedure
was performed twice to decolorize the residue completely. The
residue was then suspended in 5 ml of 0.3 M KOH and incubated at 30°C overnight. After neutralization with 3 M HCl,
60% (w/v) perchloric acid (PCA) was added to the mixture to a
Changes in UV defense mechanisms were studied during the growth periods of spinach seedlings grown under
the white light, which did not contain UV-B light. DNA
photolyase activity in the photorepair of cyclobutane pyrimidine dimers in spinach seedlings was high in the early
growth phase (cell division phase) and declined thereafter,
whereas UV-absorbing substances accumulated throughout the growth period acted as a major UV-defense mechanism in the cell expansion phase.
Key words: Cyclobutane pyrimidine dimer — DNA photolyase (EC 4.1.99.3) — Spinach — UV-absorbing substance.
Abbreviation: CPD, cyclobutane pyrimidine dimer.
UV-B radiation present in sunlight induces DNA damage,
such as the formation of pyrimidine dimers, that blocks both
the transcription and the replication of DNA. As plants are
constantly exposed to sunlight, they have evolved defense
mechanisms to limit DNA damage resulting from exposure to
UV-B. Major UV-defense mechanisms in higher plants can be
classified into two main categories (Caldwell 1971): optical
screening of UV radiation by peripheral plant tissue and repair
of damaged DNA. The former defense mechanism involves the
accumulation by most plants of UV-absorbing substances,
notably flavonoids and related phenolic compounds, in the
upper epidermal layers of their leaves (Tevini et al. 1991,
Reuber et al. 1996). These substances attenuate the damaging
effects of solar UV-B radiation, but transmit essential photosynthetically active radiation through the epidermal layers of
the leaves. The contribution of these UV-absorbing pigments to
defense mechanisms against DNA damage by UV-B has been
experimentally demonstrated in cultured cells of Centaurea
cyanus (Takahashi et al. 1991) and in sheath tissue of Zea mays
(Stapleton and Walbot 1994).
The other defense mechanism against UV-B-induced damage to DNA is repair of damaged DNA itself. Nucleotide excision repair involves endonucleolytic steps, release of damaged
oligonucleotides and DNA resynthesis (Sancar 1994a). Photorepair that directly reverses cyclobutane pyrimidine dimers
3
4
Corresponding author: E-mail, [email protected]; Fax, +81-774-38-3512.
Present address: Institute of Advanced Energy, Kyoto University, Gokanosho, Uji, Kyoto, 611-011 Japan.
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UV-defense mechanisms in spinach seedlings
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Fig. 2 Changes in the content of UV-absorbing substances (A) and
the activity of DNA photolyase (B) in the cotyledons (open circles)
and the first leaves (closed circles) of spinach seedlings during growth.
(A) Four cotyledons or first leaves were used for the extraction of UVabsorbing substances, and the values of two separate samples were
plotted. (B) Two g of the cotyledons or first leaves were used for the
enzyme extraction, and the average and SD of eight separate samples
were plotted. Relative activity at 1.0 was the ability to repair 4.04 fmol
dimers h–1 (mg protein)–1.
Fig. 1 Changes in length (A), fresh weight (B) and DNA content (C)
in the cotyledons (open circles) and the first leaves (closed circles) of
spinach seedlings during growth. (A) and (B) n = 16. SD was smaller
than the size of the circles. (C) Four cotyledons or first leaves were
used for DNA extraction, and the values of two separate samples were
plotted.
final concentration of 5% (w/v). The mixture was centrifuged
to remove RNA, and the resulting residue was digested with
3 ml of 5% PCA at 90°C for 10 min. The DNA in the supernatant obtained from the above centrifugation was quantitated
using the method of Giles and Myers (1965), with calf thymus
DNA as the standard.
To determine the amount of UV-absorbing substances
present, four cotyledons or first leaves were crushed into powder after freezing with liquid nitrogen, and then homogenized
in 10 ml of methanol containing 1% (v/v) HCl. After heating
for 30 min at 95°C, the homogenate was centrifuged at 2,000´g
for 10 min. The supernatant was diluted 1 : 8 with methanol–
HCl and the absorbance of the diluted sample at 325 nm was
measured.
To determine the extent of DNA photolyase activity, cotyledons or first leaves (2.0 g fresh weight) were homogenized
with 3.2 ml of an extraction buffer, 200 mM Tris-HCl (pH 7.6)
containing 100 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol
(DTT), 10% (v/v) glycerol and 1.33 M ammonium sulfate. The
homogenate was filtered through two layers of gauze to
remove leaf debris. Solid ammonium sulfate was added to the
filtrate to 40% saturation, and the mixture was stirred for
20 min on ice. After centrifugation at 9,000´g for 10 min, the
supernatant was fractionated by adding solid ammonium sulfate to 60% saturation. This mixture was subsequently stirred
for 20 min on ice and centrifuged at 9,000´g for 15 min. The
resulting pellet was dissolved in a reaction buffer, 100 mM
Tris-HCl buffer (pH 7.6) containing 100 mM NaCl, 1 mM
EDTA, and 1 mM DTT in a total volume of 1.6 ml, and
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UV-defense mechanisms in spinach seedlings
desalted using a Sephadex G-25 spin column (f0.7´5 cm,
Amersham Pharmacia Biotech AB, Uppsala, Sweden) equilibrated with the reaction buffer. To remove intrinsic DNA, the
desalted solution was passed through a pre-packed Toyopak
DEAE-S column (f5.5´7 mm, Tosoh Co., Tokyo, Japan) equilibrated with the reaction buffer, and was used as the enzyme
preparation.
The activity of DNA photolyase was determined as
described previously (Hada et al. 2000a, Hada et al. 2000b).
The enzyme preparation was mixed with the substrate DNA
(salmon sperm DNA, Sigma Chemical Co.) that had been irradiated with UV light. The assay mixture was incubated at 25°C
for 30 min under the repair light, and control samples were
kept in darkness. The substrate DNA was extracted from the
reaction mixture, and the remaining amount of CPDs in the
DNA was measured by enzyme-linked immunosorbent assay.
Fig. 1 shows the time course in relation to leaf length,
fresh weight and DNA content of spinach seedling growth.
DNA content in the cotyledons increased until the 10th day
after sowing, and thereafter remained at an almost constant
level. In contrast, length and fresh weight of the cotyledons
continued to increase at an approximately constant rate until
the 13th day.
The first leaves emerged on approximately the 10th day,
and their DNA content increased up to the 18th day. Length
and fresh weight of the first leaves continued to increase until
the 25th day.
From these results, two distinct growth phases were distinguishable: (a) a cell division phase (cotyledons, until the
10th day; first leaves, from the 10th to the 18th day); and (b) a
cell expansion phase (cotyledons, after the 10th day; first
leaves, after the 18th day).
Accumulation of UV-absorbing substances during growth
was observed in both the cotyledons and the first leaves (Fig.
2A). In the cotyledons, total amount of UV-absorbing substances per leaf increased until the 12th day and then remained
at an almost constant level. The rate of accumulation of UVabsorbing substances in the first leaves was relatively low until
the 18th day, but subsequently increased. The contents of UVabsorbing substances per fresh weight were estimated to be
almost constant during growth both in the cotyledons and the
first leaves.
Changes in the activity of DNA photolyase in the cotyledons and the first leaves during growth are shown in Fig. 2B.
Photolyase activity in the cotyledons peaked at the 10–13th
day, and then gradually decreased, whereas that in the first
leaves was highest immediately after emergence, and decreased
after the 16th day.
In the spinach seedlings, two different mechanisms of
defense against damage of DNA by UV radiation were
observed to act at different times during growth: DNA photolyase activity was high in the early growth phase (cell division
phase), before decreasing to a low level in the second growth
phase (cell expansion phase). Conversely, the amounts of UV-
absorbing substances in the cotyledons and the first leaves
were constant during growth. These results suggest that two
UV-defense mechanisms, photorepair and screening UV by
UV-absorbing substances, act in cells that actively replicate
DNA. In non-dividing cells in the cell expansion phase, photorepair activity declines and the screening UV by UV-absorbing substances acts as a major defense mechanism.
Phenolic compounds are suggested to contribute to the
UV-B adaptation of plants not only by absorbing UV but also
by their other functions including radical scavenging and antioxidative ability (Markham et al. 1998, Olsson et al. 1998,
Kawashima et al. 2000). Phenolic compounds have also been
reported to defend the plant against microorganisms and herbivores (Nicholson and Hammerschmidt 1992, Hatcher and
Paul 1994, Grantpetterson and Renwick 1996). Although the
physiological functions of phenolic compounds in spinach are
not yet clear, our present findings that accumulation of UVabsorbing substances occurs in the leaf expansion phase suggests that they might defend against the other environmental
stress besides that of UV-damage in spinach leaves.
DNA photolyase is reportedly induced by UV irradiation
(Hada et al. 1999, Pang and Hays 1991), and UV-absorbing
substances were found to accumulate in the upper epidermal
layers of leaves after UV-B irradiation (Bornman et al. 1997).
Therefore, it is important to investigate the effects of various
growth conditions including that of UV-B irradiation on the
activity of these two UV-defense mechanisms if we are to characterize the likely effects on plants of the increased UV-B radiation that will result from depletion of the ozone layer.
Acknowledgements
This work was supported in part by a research grant from the
Japan Society for the Promotion of Science (Research for the Future
Program, JSPS-RFTF96L00602), and a grant from the Global
Environment Research Fund of the Environment Agency of Japan. We
thank Mr Kozo Sakai, of Sekisui Plastics Co., Ltd, for his gift of
spinach seeds and hydroponic culture solution.
References
Bornman, J.F., Reuber, S., Cen, Y.-P. and Weissenboeck, G. (1997) Ultraviolet
radiation as a stress factor and the role of protective pigments. In Plants and
UV-B: Response to Environmental Change. Edited by Lumsden, P.J. pp. 157–
168. Cambridge University Press, Cambridge.
Buchholz, G., Ehmann, B. and Wellmann, E. (1995) Ultraviolet light inhibition
of phytochrome-induced flavonoid biosynthesis and DNA photolyase formation in mustard cotyledons (Sinapis alba L.). Plant Physiol. 108: 227–234.
Caldwell, M.M. (1971) Solar UV irradiation and the growth and development of
higher plants. In Photophysiology. Edited by Giese, A.C. pp. 131–177. Academic Press, New York.
Giles, K.W. and Myers, A. (1965) An improved diphenylamine method for estimation of deoxyribonucleic acid. Nature 206: 93.
Grantpetterson, J. and Renwick, J.A.A. (1996) Effects of ultraviolet-B exposure
of Arabidopsis thaliana on herbivory by two crucifer feeding insects (Lepidoptera). Environ. Entomol. 25: 135–142.
Hada, M., Buchholz, G., Hashimoto, T., Nikaido, O. and Wellmann, E. (1999)
Photoregulation of DNA photolyase in broom Sorghum seedlings. Photochem. Photobiol. 69: 681–685.
UV-defense mechanisms in spinach seedlings
Hada, M., Hino, K., Buchholz, G., Goss, J., Wellmann, E. and Shin, M. (2000a)
Assay of DNA photolyase activity in spinach leaves in relation to cell compartmentation – Evidence for lack of DNA photolyase in chloroplasts. Biosci. Biotechnol. Biochem. 64: 1288–1291.
Hada, M., Iida, Y. and Takeuchi, Y. (2000b) Action spectra of DNA photolyases
for photorepair of cyclobutane pyrimidine dimers in sorghum and cucumber.
Plant Cell Physiol. 41: 644–648.
Hatcher, P.E. and Paul, N.D. (1994) The effect of elevated UV-B radiation on
herbivory by Autographa gamma. Entmol. Exp. Appl. 71: 227–233.
Hidema, J. and Kumagai, T. (1998) UVB-induced cyclobutyl pyrimidine dimer
and photorepair with progress of growth and leaf age in rice. J. Photochem.
Photobiol. B 43: 121–127.
Kawashima, M., Takeda, K. and Kondo, N. (2000) Enhancement of oxidative
stress tolerance and antioxidative systems in UV-B irradiated cucumber
(Cucumis sativus L.) seedlings. Environ. Sci. 13: 539–548.
Langer, B. and Wellmann, E. (1990) Phytochrome induction of photoreactivating enzyme in Phaseolus vulgaris L. seedlings. Photochem. Photobiol. 52:
861–863.
Markham, K.R., Tanner, G.J., Caasi-Lit, M., Whitecross, M.I., Nayudu, M. and
Mitchell, K.A. (1998) Possible protective role for 3¢, 4¢-dihydroxyflavones
induced by enhanced UV-B in a UV-tolerant rice cultivar. Phytochemistry 49:
1913–1919.
Naito, K., Tsuji, H. and Hatakeyama, I. (1978) Effects of benzyladenine on
DNA, RNA, protein, and chlorophyll contents in intact bean leaves: differential responses to benzyladenine according to leaf age. Physiol. Plant. 43:
367–371.
Nicholson, R.L. and Hammerschmidt, R. (1992) Phenolic compounds and their
role in disease resistance. Annu. Rev. Phytopathol. 30: 369–389.
Olsson, L.C., Veit, M., Weissenböck, G. and Bornman, J.F. (1998) Differential
787
flavonoid response to enhanced UV-B radiation in Brassica napus. Phytochemistry 49: 1021–1028.
Pang, Q. and Hays, J. (1991) UV-B-inducible and temperature sensitive photoreactivation of cyclobutane pyrimidine dimers in Arabidopsis thaliana. Plant
Physiol. 95: 536–543.
Reuber, S., Bornman, J.F. and Weissenböck, G. (1996) Phenylpropanoid compounds in primary leaf tissues of rye (Secale cereale). Light response of their
metabolism and the possible role in UV-B protection. Physiol. Plant. 97:
160–168.
Sancar, A. (1994a) Mechanisms of DNA excision repair. Science 266: 1954–
1956.
Sancar, A. (1994b) Structure and function of DNA photolyase. Biochemistry 33:
2–9.
Stapleton, A.E. and Walbot, V. (1994) Flavonoids can protect maize DNA from
the induction of ultraviolet radiation damage. Plant Physiol. 105: 881–889.
Takahashi, A., Takeda, K. and Ohnishi, T. (1991) Light-induced anthocyanin
reduces the extent of damage to DNA in UV-irradiated Centaurea cyanus
cells in culture. Plant Cell Physiol. 32: 541–547.
Takayanagi, S., Trunk, J.G., Sutherland, J.C. and Sutherland, B.M. (1994)
Alfalfa seedlings grown outdoors are more resistant to UV-induced DNA
damage than plants grown in a UV-free environmental chamber. Photochem.
Photobiol. 60: 363–367.
Taylor, R.M., Tobin, A.K. and Bray, C.M. (1997) DNA damage and repair in
plants. In Plants and UV-B: Response to Environmental Change. Edited by
Lumsden, P.J. pp. 53–76. Cambridge University Press, Cambridge.
Tevini, M., Braun, J. and Fieser, G. (1991) The protective function of the epidermal layer of rye seedlings against ultraviolet-B radiation. Photochem. Photobiol. 53: 329–333.
(Received December 22, 2000; Accepted May 10, 2001)