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Association of antioxidative enzymes with 0

the synergistic effect of selenium and


UV irradiation in enhancing plant growth
Tailin Xue
Institute of Geography, Chinese Academy of Sciences, Beijing 100101, China
Helin Hartikainen
Department of Applied Chemistry and Microbiology, PO Box 27, FIN-00014 University of Helsinki,
Finland, e-mail: helina.hartikainen@helsinki.fi

Selenium (Se) is able to defend human and animal cells against UV(B) stress. Higher plants are gener-
ally considered not to require Se but to have a low tolerance to it. However, recently it has been dem-
onstrated that Se is able to protect also plants against UV-induced oxidative stress and even to pro-
mote the growth of plants subjected to high-energy light. In the present study the effects of Se on anti-
oxidative enzymes possibly associated with this synergistic effect were investigated. Ryegrass and
lettuce were grown in soil supplemented with Se at 0, 0.1 or 1.0 mg kg-1 under normal light or sub-
jected to UV episodes. Lipid peroxidation and the changes of antioxidative enzymes were measured at
two growing stages. The positive synergistic effect of the lower Se dosage and UV was found to be at
least partly associated with the antioxidative role of Se through increased glutathione peroxidase
(GSH-Px) and catalase (CAT) activity, whereas ascorbate peroxidase (APX) responded negatively to
both factors. The contribution of the other enzymes studied seemed to be plant-specific: glutathione
S-transferase (GST) increased in both ryegrass assays and superoxide dismutase (SOD) in the first let-
tuce assay. At the higher addition level Se acted as a pro-oxidant and diminished fresh weight yields.
UV irradiation alleviated the toxicity coincidently with increase of CAT in ryegrass and SOD in let-
tuce.
Key words: ascorbate peroxidase, catalase, enzymes, glutathione peroxidase, glutathione S-trans-
ferase, lipid peroxidation, selenium, superoxide dismutase, ultraviolet radiation

Introduction
100 100

essential component of the glutathione peroxi-


95
dase (GSH-Px) family, which has been shown to 95

75 The upward trends of UV(B) radiation linked to be able to resist UV radiation damages in human 75
ozone depletion may be harmful to living organ- beings (Ruche and Cesarini 1991, Burke et al.
isms (Kerr and McElroy 1993). Selenium is an 1992a, Leccia et al. 1993), experimental ani-

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mals (Burke et al. 1992b, Pence et al. 1994, cipally in chloroplasts, and scavenges H 2O2 pro-
Stewart et al. 1996) and even a human dermato- duced during photosynthesis (Nakano and
tropic poxvirus (Shisler et al. 1998). Agricul- Asada 1981, Mittler and Zilinskas 1991). The
25
tural plants are generally considered not to re- subcellular localization of plant GST is unclear, 25

5
quire Se and to have usually a low tolerance to but in animal cells the substrates of GSH-Px and 5
it. Its toxicity mechanisms have been discussed GST are similar, as both can scavenge organic
0 extensively (for references see e.g. Luchli hydroperoxides (Habig et al. 1974). In plants, 0

1993), but recently Se has been demonstrated to SOD is so far the only enzyme found to specifi-
exert beneficial effects on plants. At proper con- cally scavenge O2-, an initiator in a series of
centrations it may increase their antioxidative radical chain reactions. This activated oxygen
capacity (Hartikainen et al. 1997). radical is produced in various reactions and
Since some of the detrimental effects of UV metabolic processes involving oxygen, e.g. pho-
irradiation in plants are due to the oxidative tosynthesis (Elstner 1991) and respiration (Werf
stress it produces (Takeuchi et al. 1995, Hideg et al. 1991). In order to find out possible
and Vass 1996), Se can be hypothesized to be changes of antioxidative capacity in response to
able to increase the tolerance of plants against prolonged stress, the oxidative status of the
UV. In fact, a previous study of Hartikainen and plants and the enzyme activities were deter-
Xue (1999) demonstrated that Se is able to pro- mined at two growing stages.
tect plants against UV-induced oxidative stress
and even to promote plant growth under high-
energy light. The positive interaction between
Se and UV light was manifested e.g. as in-
creased concentrations of nucleic acids and
Material and Methods
soluble proteins. The antioxidative function of
Se was associated with the promotion of glu- Pot Experiment
tathione peroxidase (GSH-Px) activity. The ele-
Plant material and cultivation methods are de-
ment analysis proved that the positive interac- scribed in our previous report (Hartikainen and
tion was not assisted by other nutrients (P, K, Xue 1999). Briefly, ryegrass (Lolium perenne
Mg, Ca, Mn, Fe, Zn, Cu). var. Prego) and lettuce (Lactuca sativa var. Aus-
The objective of the present supplementary tralischer gelber) were cultivated in pot experi-
study was to gain more detailed information ments in a greenhouse at 2033C with a
about the role of antioxidative enzymes in their coarse-textured soil. For both plants two sets of
possible association with the synergistic air-dried, sieved (35 mm) and homogenized
growth-promoting effect of Se and UV irradia- soil samples weighing 4 kg each were treated
tion. In addition to glutathione peroxidase with H2SeO4 to create Se addition levels of 0, 0.1
(GSH-Px), superoxide dismutase (SOD), cata- -1
and 1.0 mg kg soil, respectively. Then 4.5 g of
lase (CAT), glutathione S-tranferase (GST) and ground and homogenized NPK fertilizer (16-
ascorbate peroxidase (APX) were chosen as 16-16, Se background 0.1 mg kg-1) was mixed
100 candidate enzymes due to their importance in with a 3.5 kg portion of soil, and this soil mix- 100

cell defence systems. They are normally local- ture was put into plastic pots (4 l) and covered
95
ised in different cell parts and scavenge differ- with the remaining soil. The seeds (2.5 g of rye-
95

75
ent oxygen radicals. GSH-Px scavenges H 2O2 grass and 15 seeds of lettuce per pot) were sown 75
and converts the toxic lipid hydroperoxides to onto the upper layer of the soil. After sowing,
less toxic lipid hydroxide. CAT exists in peroxi- 100 g of soil without application of Se and NPK
somes and catalyzes H2O2 decomposition, while was added to cover the seeds. The experiment
25 25
APX exists in chloroplasts and cytosol, but prin- was performed in quadruplicate.
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During the experiment, daylight was supple- and ascorbate peroxidase (APX, EC 1.11.1.7)
mented with Osram Vialox Nav-T 400W lamps by the method described by Mittler and Zil-
to provide a photoperiod of 16 hours and a quan- inskas (1991). Superoxide dismutase (SOD, EC
25
tum flux density at the top of the pots of 160 1.15.1.1) was analyzed by the method of Gian- 25

5
-2 -1
mmol m s . Two weeks after sprouting, one pot nopolitis and Ries (1977), where the nitro- 5
set was illuminated every second day for one blue-tetrazolium (NBT) reaction mixtures were
0 min by Philips TL-40W/12 Ultraviolet-B (Hol- illuminated for 3 min with an Osram Vialox 0

land) lamps for two weeks and then for three Nav-T 400 W lamp. In addition to the enzyme
min daily for the following two weeks. The UV activities, the fresh plant material was also ana-
intensity at the top of the pots (measured by lyzed for thiobarbituric reactive substances
Lighting Laboratory, Helsinki University of (TBARS) by a modified method of Yagi (1982)
Technology) was 0.177 (280320 nm) and in order to monitor lipid peroxidation. The de-
-2
0.077 (321360 nm) mW cm . According to cal- termination of dry weight (DW), Se and nutrient
culations based on the radiation spectrum, a concentrations, and proteins used in the calcula-
small amount of UV(C) light included in the ir- tions of enzyme activities are given in Hartika-
radiation episodes amounted to about 4% of the inen and Xue (1999).
total energy. During the irradiation treatment
the control pots without UV treatment were pro-
tected by isolating them with black plastic cur-
tains. The experimental plants were irrigated
from below with deionized water applied to the Results
plates.
As expected, the effect of added Se and UV irra-
diation on FW yields (Table 1) followed the
same patterns reported previously for DW (Har-
Plant analyses tikainen and Xue 1999). Under normal light
After the first 2-week round of UV irradiation, conditions, the lower Se addition did not affect
three small subsamples were randomly taken plant growth, but the higher one exerted a toxic
from the shoots in each pot and and used imme- effect on both plants. Without added Se, UV ir-
diatedly for biochemical analysis as the first as- radiation did not affect the growth of ryegrass
say After the second 2-week round of UV treat- but diminished the lettuce yield. However, in
ment, the whole shoot yield in each pot was har- combination with the high-energy light the
vested. The fresh weight (FW) of the yield was lower Se dosage significantly increased FW
measured and three subsamples were then ran- yields, and the toxicity of the higher addition
domly taken for biochemical analysis as the sec- was alleviated to some extent (Table 1). UV did
ond assay. In statistical analyses of the results, not affect the Se concentration in ryegrass but
the mean of three subsamples was taken to rep- reduced it significantly in lettuce cultivated
resent the result of each pot. The fresh weight without added Se (Table 1).
100 yields of the subsamples used for the first assay In both plants cultivated under normal light, 100

were added to the final yields. TBARS increased upon prolonged cultivation
95
The fresh plant samples were analyzed for (Table 2), which indicates enhanced lipid per- 95

enzyme activities as follows: glutathione per- oxidation. In the control plants not supplied
75 75
oxidase (GSH-Px, EC 1.11.1.9) by a modified with Se, UV raised TBARS in both assays, dem-
method of Flohe and Gunzler (1984), catalase onstrating that the short-wavelength light pro-
(CAT, EC 1.11.1.6) by the method of Aebi duced an extra oxidative stress. The added Se re-
25
(1983), glutathione S-transferase (GST, EC duced the lipid peroxidation irrespective of light 25

5 2.5.1.18) by the method of Habig et al. (1974), conditions, except the higher dosage, which ex- 5

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Table 1. The fresh weight (FW, g pot ) and Se concentration (mg kg dry weight) of the yields at various Se dosages
without or with UV irradiation. Data are means of four replicates, values followed by a common letter do not differ at
P0.05 with Duncan test. Each plant and irradiation treatment is tested seperately. The effect of UV irradiation at
25 each Se level is tested by the t-test: *= difference significant at P0.05, **= difference significant at P0.01. 25

5 5

0 0

-1
Table 2. Thiobarbituric acid reactive substances (nmol malondialdehyde g fresh weight) in shoots in two assays at
various Se dosages without or with UV irradiation. Data are means of four replicates, values followed by a common
letter do not differ at P0.05 with Duncan test. Each plant and irradiation treatment is tested seperately. The effect of
UV irradiation at each Se level is tested by the t-test: *= difference significant at P0.05, **= difference significant
at P0.01.

erted a pro-oxidative effect on the second let- not appear to reverse these decreasing trends.
tuce assay under normal light. Actually, in the The growth-promoting effect of Se as triggered
young seedlings (the first assay) supplied with by UV irradiation was associated at least partly
100 Se, UV did not have any effect on TBARS. In with the increased activity of GSH-Px in both 100

the second assay, the antioxidative effect of Se assays (Tables 3 and 4). Irrespective of the light
95
was relative more pronounced in the plants sub- conditions, the lower Se addition increased the 95

75
jected to external UV stress. The pro-oxidative activity of GSH-Px in both plants, but the higher 75
effect of the higher Se addition on lettuce disap- dosage depressed GSH-Px in the first lettuce as-
peared under UV. say. In ryegrass, also UV enhanced GSH-Px ac-
When no Se was added, the activity of all en- tivity in all treatments. In lettuce, on the con-
25 25
zymes analyzed decreased upon progressive trary, this response was found in the first assay
5
plant growth (Tables 3 and 4). The added Se did in all Se-supplied plants but in the second assay 5

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Table 3. Activities of antioxidative enzymes in two ryegrass assays at various Se levels without or with UV treat-
ment. Data are means of four replicates, values followed by a common letter do not differ at P0.05 with Duncan test.
Each plant and irradiation treatment is tested seperately. The effect of UV irradiation at each Se level is tested by the
25 t-test: *= difference significant at P0.05, **= difference significant at P0.01. 25

5 5

0 0

only at the lower Se level.


The activities of CAT, SOD, GST and APX Discussion
were not affected or even diminished by the UV
irradiation when no Se was added (Tables 3 and The present study was carried out under low
4). Under normal light conditions, in most cases background light conditions and some UV(C)
Se addition did not promote the activity of these light was present, which explains that the im-
enzymes, except for the higher dosage, which pacts of UV irradiation were significant despite
100
increased GST in both ryegrass assays (Table 3) short episodes. Nevertheless, the results ob- 100

and CAT in the second lettuce assay (Table 4). tained in both assays agree with those obtained
95 95
In combination with UV in ryegrass, both Se ad- by Takeuchi et al. (1995) with UV(B) showing
75 ditions promoted CAT in the second assay and that the high-energy light enhances the accumu- 75

the lower dosage GST in both assays. In lettuce, lation of lipid peroxidation products. In their
in turn, UV treatment enhanced in the first assay study, this reaction pattern was reversed by ap-
the activity of SOD at both Se levels, and in the plication of exogenous scavengers of free radi-
25 25
second assay CAT at the lower Se level. cals, such as hydroquinone. In the present study,
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Xue, T. & Hartikainen, H. Antioxidative enzymes with synergistic effect of Se and UV irradiation 95

75 1)
75
Table 4. Activities of antioxidative enzymes in two lettuce assays at various Se levels without or with UV treatment.
Data are means of four replicates, values followed by a common letter do not differ at P0.05 with Duncan test. Each
plant and irradiation treatment is tested seperately. The effect of UV irradiation at each Se level is tested by the t-test:
25 *= difference significant at P0.05, **= difference significant at P0.01. 25

5 5

0 0

the added Se took this role and diminished the the activity of all enzymes, except that of GSH-
UV-induced increase in TBARS. However, it Px was higher than in lettuce.
reduced the lipid peroxidation irrespective of Willekens et al. (1994) found that ozone,
light conditions, even though in the second as- SO2 and UV(B) had similar effects on mRNA
say the antioxidative effect was relatively more accumulation of antioxidant genes in Nicotiana
pronounced in the plants subjected to the short- plumbaginifolia L. The effects of the different
wavelenght light. stresses were characterized by a decline in
100 100
Jansen et al. (1996) have reported that plants CAT1, a moderate increase in CAT3, and a
95 can defend themselves against UV irradiation strong increase in CAT2 and GSH-Px, whereas 95
damage by strengthening their antioxidative SODs and cyt APX were not affected. However,
75 systems. The present study suggests that various in the present study the only enzyme increasing 75

antioxidative enzymes can be plant-specific and in response to UV without Se application in rye-


their responses to external stress can vary at dif- grass was GSH-Px, the scavenger of lipid hy-
25 ferent growing stages (Tables 3 and 4). In rye- droperoxides and H2O2. In lettuce where UV di- 25

grass which showed a higher tolerance to UV, minished the Se concentration and reduced
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plant growth, all antioxidative enzymes re- scavenged by GSH-Px. The rest can penetrate
sponded indifferently or negatively to UV. The further into peroxisomes and be decomposed by
antioxidative role of Se was confirmed by the CAT (Lawrence and Burk 1978, Fang and Li
25
observation that irrespective of light conditions 1989). When Se is applied, GSH-Px increases 25

5
it enhanced GSH-Px activity in ryegrass and and more H2O2 can be scavenged in the cytosol. 5
strengthened the antioxidative capacity of let- Thus the quantity of H2O2 penetrating into the
0 tuce when added at non-toxic levels. peroxisomes is reduced and, accordingly, the 0

The indifferent or negative responses of demand for CAT is relatively decreased. Since
APX, CAT, GST and SOD to added Se under GSH-Px and CAT do not compete for H 2O2 in
normal light conditions in the first assay indi- the same compartment and H2O2 scavenged by
cate that the antioxidative role of Se diminish- GSH-Px is only a fraction of the H2O2 scavenged
ing TBARS was not mediated through these en- by CAT, an increase in GSH-Px activity cannot
zymes (Tables 2, 3 and 4). Nevertheless, the completely replace the function of CAT.
added Se may have diminished their demand. As Although UV irradiation generally dimin-
result of increased Se concentration GSH-Px ac- ished the activities of enzymes other than GSH-
tivity was enhanced, and this made it possible to Px irrespective of Se addition, in combination
scavenge more lipid hydroperoxide and H 2O2. with Se it increased CAT in the second assays of
Consequently, the need for enzymes using these both plants, SOD in the first lettuce assay and
radicals as substrate (APX, CAT, GST) may GST in the second ryegrass assay. This suggests
have diminished. Takeda et al. (1997) reported that Se may increase the antioxidative capacity
for Chlamydomonas cells that without Se- of plants by multiple systems that act alone or
enrichment 40% of external H 2 O 2 was scav- synergistically and are likely different at differ-
enged by APX and the residual H2O2 by CAT, ent growing stages. For instance, in the second
while in Se-containing medium GSH-Px func- assay the antioxidative capacity of ryegrass
tioned primarily in the removal of external against UV was strengthened by simultanoeus
H2O2. Similar processes may also exist in higher increase in CAT, GST and GSH-Px.
plants. On the other hand, Se might indirectly On the other hand, depressed GSH-Px activ-
decrease SOD that catalyses the dismutation of ity due to Se toxicity in the first lettuce assay co-
superoxide radicals (O2-) to 2O2. Superoxide incided under UV with enhanced SOD activity.
radicals can be used in spontaneous dispropor- Although GSH-Px and SOD do not share com-
tion reactions producing H2O2 and singlet oxy- mon substrates and GSH-Px cannot scavenge
gen (Thompson et al. 1987). Thus, it can be hy- superoxide anion radicals (O2-) directly, the en-
pothesized that at the same time that Se pro- hancing influence of Se addition on SOD might
motes the scavenging of produced H 2O2 through be caused by increased demand for antioxida-
increased GSH-Px, it also enhances the sponta- tive capacity to counteract Se toxicity. It is pos-
neous disproportion of superoxide radicals and sible that the spontaneous disproportion of su-
thus reduces the need for SOD, their scavenger. peroxide radicals producing H2O2 and singlet
Drotar et al. (1985) have hypothesized that oxygen (Thompson et al. 1987) was retarded
100 GSH-Px scavenges hydrogen peroxide in cyto- when the scavenger of H2O2, GSH-Px, was di- 100

plasmic compartment. There is, however, no minished. Therefore, more SOD activity was
95 95
agreement about the occurrence of Se-dependent needed to detoxify superoxide radicals and to
75 GSH-Px in higher plants. On the other hand, it prevent their accumulation. 75

has been shown for animal cells, where GSH-Px Similarly, GST and CAT were enhanced as
and CAT are localized in different compart- response to increased demand of antioxidative
ments, that H2O2 produced, e.g. by mitochondria capacity to counteract Se toxicity. It is notewor-
25 25
can diffuse into cytosol, where part of it can be thy that APX did not increase in order to resist
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75 75
UV-induced oxidative stress and always re- of GSH-Px. The contribution of CAT and GST
sponded negatively to added Se, although it to the synergistic growth-promoting effect can
shares a common substrate H2O2 with GSH-Px be concluded from their analogous behaviour;
25
and CAT. Analysis of SOD and APX expression under normal light they responded indifferently 25

5
carried out by Willekens et al. (1994) in ozone- or even negatively to Se but increased under UV 5
sensitive Nicotiana tabacum L. cv PBD6 re- light. In addition, the slight toxicity-alleviating
0 vealed that the induction of cytosolic cop- effect of UV at the high Se addition level 0

per/zinc SOD and cyt APX under ozone stress seemed to be associated with antioxidative en-
occurs only with the onset of visible damage. In zymes: the high-energy light increased CAT in
the present study, the Se toxicity resulted in ryegrass and SOD in lettuce.
visible symptoms, whereas those caused by UV
remained less distinct.
The results show that the synergistic effect Acknowledgements. We express our thanks to Ms. Mar-
of UV and Se added at non-toxic levels on plant jatta Koivisto for skillfull technical assistance and to
growth is at least partly associated with the anti- the Academy of Finland and Kemira Ltd for financial
support.
oxidative role of Se through increased activity

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tioxidant nutrients protect against UVB-induced oxi- 106: 10071014.
dative damage to DNA of mouse keratinocytes in Yagi, K. 1982. Lipid peroxides in biology and medicine.
New York: Academic Press. p. 223230.

SELOSTUS
Seleenin ja UV-steilyn synergistinen kasvua edistv vaikutus
ja siihen liittyvt antioksidatiiviset entsyymit kasveissa
Tailin Xue ja Helin Hartikainen
Kiinan tiedeakatemia ja Helsingin yliopisto

-1
Seleeni (Se) on ihmisille ja elimille vlttmtn al- kg (H2SeO4:n) tai jtetty ilman Se-lisyst. Puolet
kuaine, jonka tiedetn mm. puolustavan soluja koekasveista kasvoi normaaleissa valaistusolosuh-
UV(B)-steilyn aiheuttamia vaurioita vastaan. Val- teissa, puolet altistettiin 13 minuutin episodeina an-
litsevan ksityksen mukaan korkeammat kasvit eivt netulle UV-steilylle. Kaksi ja nelj viikkoa kasva-
tarvitse Se:, vaan pikemminkin ovat sille varsin ar- neista kasveista mitattiin lipidien hapettumista m-
koja. skettin on kuitenkin osoitettu, ett sopivan rittmll tiobarbituurihapporeaktiivisten aineiden
100 alhaisina pitoisuuksina Se pystyy suojaamaan mys pitoisuudet (TBARS). Lisksi niist mritettiin glu- 100

kasveja UV-steilyn aiheuttamalta hapetusstressilt tationiperoksidaasin (GSH-Px), superoksididismu-


95 ja jopa edistmn steily saaneiden kasvien kas- taasin (SOD), katalaasin (CAT), glutationi- S-trans- 95

vua. feraasin (GST) ja askorbaattireduktaasin (APX) ak-


75 75
Tss tutkimuksessa selvitettiin Se:n vaikutuksia tiivisuudet.
antioksidatiivisiin entsyymeihin, jotka mahdollisesti Ilman Se-lisyst UV-steilytys pienensi salaatti-
liittyvt havaittuun synergismiin. Kasvihuoneesssa satoa (22 %), mutta ei vaikuttanut raiheinn kasvuun.
25
toteutetussa kokeessa kasvatettiin raihein ja salaat- Pienempi Se-lisys toimi antioksidatiivisesti ja v- 25
tia maassa, johon oli listty Se: 0,1 mg tai 1,0 mg hensi kummassakin kasvissa valaistusolosuhteista
5 5

0 0

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100
AGRICULTURAL AND FOOD SCIENCE IN FINLAND 100

95
Tuorila, H. Consumer perceptions and acceptance of nutritionally modified foods 95

75 75
riippumatta lipidien hapettumista (TBARS pieneni). nss se liittyi GST:n aktiivisuuden lisntymiseen
Yhdess UV:n kanssa Se-lisys edisti molempien kummassakin kasvuvaiheessa, sen sijaan nuoressa
kasvien kasvua. Ainakin osittain tm positiivinen salaatissa nousi SOD. Suurempi Se-lisys oli toksi-
25 synergistinen vaikutus liittyi lisntyneeseen GSH- nen ja pienensi kuiva-ainesatoja. Mielenkiintoista oli 25
Px:n ja CAT:n aktiivisuuteen, mutta APX reagoi ne- kuitenkin havaita, ett UV-steilylle altistuneissa
5 gatiivisesti sek Se-lisykseen ett UV-steilyyn. kasveissa toksisuus lieveni samalla kun CAT nousi 5

Muiden tutkittujen entsyymien yhteys havaittuun sy- raiheinss ja SOD salaatissa.


0
nergismiin nytti olevan kasvilajikohtaista: raihei- 0

100 100

95 95

75 75

25 25

5 5

0 0

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