Agric.

BioL Chern.,

54 (10),2629-2634,

1990

2629

Water Deficiency-induced Changes in the Contents ofDefensive Substances against Active Oxygenin Spinach Leaves
Kiyoshi Tanaka,* Ryoichi Masuda,* Toshio Sugimoto,*Kenji Omasa and Takeshi Sakaki
Division of Environmental Biology, the National Institute for Environmental Studies,Tsukuba, Ibaraki 305, Japan*National Food Research Institute, Ministry of Agriculture,Forestry and Fisheries, Tsukuba, Ibaraki 305, Japan
Received April 10, 1990

Changesin the contents of defensive substances against the active oxygenin water-stressedspinach plants were examined. The contents of ascorbate peroxidase (AP; EC 1.ll.1.7), glutathione
reductase (GR; EC 1.6.4.2) and a-tocopherol increased remarkably in water-stressed spinach leaves,
while those of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (EC 1.8.5.1),
ascorbate and glutathionechanged little.

The content of a-tocopherol

in chloroplast

thylakoid

membranesisolated from water-stressed leaves was higher than that from normal leaves. It is, therefore,
conceivable that GR, AP and a-tocopherol might be related to the tolerance of plants to water deficiency.

Water is essential to plant life, and water-deficiency causes various physiologicalandbiochemical
effects of plants. The decreasein photosynthesis,1>2) the closure of stomata3)
and osmotic adjustment4* appear to be typicalplant responses to water deficiency at the firststage. Plant-growth
regulators
have beensuggested to play a role in the adaptive
strategiesof plants to water deficiency, andof environmental
treatmentwithstress suchherbicides12'13)

as aging,11}or heavy

metals,14) airpollutants,15" 17) and a high O218)or low CO2 atmosphere.19) It is also widely

knownthat plants have an intrinsic defensive

system against active oxygen20) and resist thestress-induced production of active oxygen byincreasing

components in this system. In thepresent work, various componentsparticipating
increases in the abscisic acid content have been in the defensive system against active oxygen
reported in a number of plant species were determined in spinach plants, which wereexperiencing water deficiency.5) Changes inwater-stressed for a few days in an environmentprotein and gene levels, which may be related controlled growth cabinet, and it was foundto the tolerance to water stress, have also been that the contents of AP, GRand a-tocopherol
increased.
examinedin water-stressed plants.5 ~ 9)The closure ofstomata by a decrease of water
Materials and Methods
content in plant leaves disturbs the supply of
CO2for photosynthesis. When water-stressedleaves were illuminated,

the reducing

powerfrom the photosystem was not consumedfor
CO2fixation but for the activation of O2.10)
The reduced species of O2 are very toxic toplants and cause plant injury by various kinds

Plant materials. Spinach (Spinacia oleracea L. cv. NewAsia) was grown at 20/15 + 0.5°C day/night temperatureswith a relative humidity of 70+ 5% in pots (the availablesoilcapacity

was 1.8 liter)

in a naturally

lighteden-vironment-controlled glasshouse, as has been previously
described.21) Tabacco (Nicotiana tabacum L. cv. Samsoun)f To whomreprint requests should be addressed.

Abbreviations: AP, ascorbate peroxidase; GR, glutathione reductase; SOD, superoxide dismutase.

2630

K. Tanaka et al.

and petunia (Petunia hybrida) were grown under the same

Results and Discussion
In the analysis by HPLCof the extracts fromKalanchoe rosei and Pelargonium ferulaceum were grownspinach leaves, the retention time of ascorbate
in a greenhouse. The nutritional
conditions for the culconditions, except that the temperature was 25/20 ± 0.5°C.
Polygonumcuspidatum was cultivated in the open air.
tivationof all plants

previously.

22)

were the same as those describedno peak other than ascorbate was found (data

Water stress experiments. Spinach plants grown in aglasshouse for three to four weeksweretransferred to anartificially(170x230xlighted(about30,000lux)
growth cabinet190cm),
where the temperatureand therelative humidity weremaintained at 25 ± 1°C and 40 ± 5%,respectively. Half of the plants were provided with about
400ml of water every morning, and the remaining half
were not given water at all during the experiments. At
13:30

was 6.2min at a flow rate of 1.4ml/min, andevery day the leaf segments were sampled

andstored at -80°C. The fresh weight of the water-stressedspinach plants had decreased by about 50% after 5 days.
Extraction of enzymes and reductants from plant leaves.
The leaf segments were homogenized in the presence of asmall amount of liquid N2 with a mortar and pestle innot shown). At a flow rate of 1.4ml/min, a-,/?-, y- and (5-tocopherols showed retention timesof 2.2, 6.6, 7.8 and 10.0min, respectively.
Inthe extracts from spinach leaves the peaks ofa-tocopherol and an unknown substancehaving a retention time of 3.0 min were found,and no peaks corresponding to /?-, y- and(5-tocopherol were apparent in the extracts
from spinach leaves. WhenKalanchoe was usedas the plant material, no peak other thana-tocopherol was found (data not shown).

Spinach plants in pots were grown in anenvironment-controlled
growth cabinet with-out supplying water. The fresh weight of thevarious extraction media and, after centrifuging thehomogenates, the supernatants were subjected to a
determination of such enzymes and reductants as ascorbate
and GSH as described previously.21}
Ascorbate in 5%
metaphosphoric acid was determined by HPLC (Nihon

deficient period (Fig. 2), when several enzymes
and reductants in the leaves were measured

UVIDEC-100II at 258 nm. For tocopherol, 80% ethanol
was used as the extraction medium,because its efficiency
was similar to that of chloroform-methanol (1 : 2). Leaf

(Fig.

Bunko VL-611)
with a column of Inertsil
ODS-2(4.6x250mm)
from Gasukuro Kogyo. The eluting
solution
was 120mM ammonium phosphate(pH 3.2).
Ascorbate was measured by using a Nihon Bunko

segments (36cm2 in leaf area) were homogenized in 8 ml

spinachleaves decreased

duringthe water-(Figs. 1 and 2). Although
there was little
difference in the contents of ascorbate (Fig.
lb), SOD (Fig. lc), protein (Fig. la), andoxidized and reduced glutathione(Fig. Id)
between the water-stressed
and the normal
leaves, a remarkable increase of GR activity2c) and a-tocopherol

content(Fig.

2b),

of80% ethanol and centrifuged at 18,000g for 30min. To and a slight increase of AP activity (Fig. 2a)
5 ml of the clear supernatant, 0.5 ml ofrc-hexane was added, occurred in the water-stressed spinach plants,
and the mixture fully agitated. After standing for 2h, the while the respective contents in normal plants
upper layer of hexane was applied to HPLC.Acolumn changed little. Among the three antioxidants,
offi Bondasphere 5 \x Si-100A (3.9 x 150mm) from Waters
was eluted

with a mixtureof H-hexane-2-propanol-te-

trahydrofuran (96 : 1 : 3). Tocopherol was detected by usinga Shimadzu

RF-530

instrument

by measuring

thefluorescence intensity at 325 nmwhile exciting at 298 nm.
Standard tocopherol from Merck was used.
Preparation of chloroplast thylakoid membranes. Intactchloroplasts were prepared from spinach leaves by usinga Percoll according to the method ofNakano and Asada.23)
After lysis of the intact chloroplasts by fifteen volumes of
10 mMTricine-KOH (pH 7.8) and 4mM MgCl2, thylakoid
membraneswereisolated according to the method ofDouce and Joyard.24) Tocopherol was extracted from thethylakoid membranes with 80%ethanol and determinedby HPLC.

GRactivity rose rapidly at an earlier time thanthe others did. The fact that the length of timerequired to bring about the increase ofantioxidants
in water-stressed plants varies

with the antioxidant species may be due to thedifferent

time for the biosynthesisof anti-oxidants in water-stressed plants. There havebeen several reports of evidence that plants tooxygen-rich air, 1 8) the herbicide acifluorfen25)
and the air pollutant
O3,26) which mightenhance the production of active oxygen inplant cells, increased the GR activity.
Infield-grown wheat, GRactivity was increasedin dry lands more than in irrigated lands.27)

Water-deficiency

Change in Defensive Substances against Active Oxygen263 1Fig. 1. Changes in Active Oxygen-defensive Substances (Protein, Superoxide Dismutase, Ascorbate andGlutathione) in Water-stressed Spinach Plants.
One group of spinach plants was supplied once a day with water (open symbols: O-O, A-A), and theother group was not (closed symbols: #-#, A-A)- At the indicated times, leaf segments (1.5 x 4cm) were
sampled by scissors and stored at -80°C.

2632

K. Tanaka etal.

Fig. 2. Changes in Active Oxygen-defensive Substances (Ascorbate Peroxidase, Tocopherol and Glutathione
Reductase) in Water-stressed Spinach Leaves.
The experimental conditions and the symbols are the same as those in Fig. 1.

Here, the increase of GRactivity was observed that active oxygen may increase in waterin spinach plants water-stressed for a few days stressed plants and that active oxygen may
in a glasshouse.
It is, therefore,
conceivablestimulate the increase ofGR activity. Similarly,

2633

Water-deficiency Change in Defensive Substances against Active Oxygen
since an increase of AP activity has been
observed in a low cooncentrationof O3-tocopherol,

the greater

partof which is

localized in thylakoids, may be important for
fumigated spinach leaves,28) the rise of AP the plant tolerance to water stress. It has been
activity in water-stressed spinach plants may confirmed that the main site for the biosynshow plant detoxification
to water stress- thesis of tocopherol is localized in the chloroplast envelope.29'30)(It is, therefore,possible
induced active oxygen production.
Chloroplast

thylakoid

membranes were that the increase of a-tocopherol content inisolated both from water-stressed (5 days) and the thylakoid membranes was caused by anormal spinach plants, and their tocopherol
content was determined. The content ofa-tocopherol in the thylakoid membranes ofwater-stressed plants was about twice that of
the normal plants (Table I). It is, therefore,
conceivable that the plants might protect
themselves against active oxygen toxicity byincreasing the a-tocopherol content in chloro-

plast thylakoid membranes when plants sufferfrom water stress. Since active oxygen is mainly
produced in chloroplast thylakoid membranes,
it is also conceivable that the increase of
Table I. Content of <z-Tocopherol in Chloroplast
Thylakoid Membranesfrom Water-Stressed(5 days) Spinach Plants and Normal Plants
Valuesindicate

the mean±standard

deviation

oftriplicate determinations.
Treatmenta-Tocopherol content(jugà"mg of protein~ *)Water-stressed

0.86 ± 0. 14

Normal0.42+ 0.05stimulation of the biosynthesis of tocopherol
in the chloroplast envelope, or by a rise of thetransport rate of tocopherol from the chloroplast envelope to the thylakoid membranes.
The content of a-tocopherol in mature leaves
was higher in the water stress-tolerant plants(Polygonum

cuspidatum,Kalanchoe rosei andPelargoniumferulaceum)
than intobacco
the waterstress-sensitive
ones (spinach,andpetunia)(TableII). This resultthat tocopherol may play an important role indefenseagainstwater stress.

Chloroplast thylakoid membranes both from water-Table II. Content of oc-Tocopherol in
Six Species of Plants
Each value is the mean+ standard deviation of threeplants. Mature leaves from each plants were used.

have an outstanding

ability

SpinachTobaccoPetunia
Polygonumcuspidatum
Kalanchoe rosei

Pelargon ium ferulaceum

a-Tocopherol content

for conserving

water. Here, it was confirmed that they wereequipped with a physiological defensive systemagainst damagecaused by water stress. The
increases of GRactivity and of the content ofa-tocopherol in the water-stressed plants, andthe fact thattocopherol

is present

abundantly in water stress-tolerant

possibility

moreplants thanplants, suggests thethat tocopherol, AP and GRarecandidates for physiologicalwater stress-tolerance.

indicesof plant

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