Investigations on Heat Resistance of Spinach Leaves* Kurt A. Santarius and Mechthild Mfiller** Botanisches Institut, UniversitS,t Dfisseldorf, Universit/itsstral3e1, D-4000 Dfisseldorf, Federal Republic of Germany
Abstract. Exposure of spinach plants to high temperature (35 ~ C) increased the heat resistance of the leaves by about 3 ~ C. This hardening process occurred within 4 to 6 h, whereas dehardening at 20~176 C required 1 to 2 days. At 5~ dehardening did not take place. Hardening and dehardening occurred in both the dark and the light. The hardiness was tested by exposure of the leaves to heat stress and subsequent measurements of chlorophyU fluorescence induction and light-induced absorbance changes at 535 nm on the leaves and of the photosynthetic electron transport in thylakoids isolated after heat treatment. Heat-induced damage to both heat-hardened and non-hardened leaves seemed to consist primarily in a breakdown of the membrane potential of the thylakoids accompanied by partial inactivation of electron transport through photosystem II. The increase in heat resistance was not due to temperatureinduced changes in lipid content and fatty acid composition of the thylakoids, and no conspicuous changes in the polypeptide composition of the membranes were observed. Prolonged heat treatment at 35~ up to 3 days significantly decreased the total lipid content and the degree of unsaturation of the fatty acids of membrane lipids without further increase in the thermostability of the leaves. Intact chloroplasts isolated from heat-hardened leaves retained increased heat resistance. When the stroma of the chloroplasts was removed, the thermostability of the thylakoids was decreased and was comparable to the heat resistance of chloroplast membranes obtained from non-hardened control plants. Compartmenta-
* Dedicated to Professor Wilhelm Simonis, Wiirzburg, on the occasion of his 70th birthday ** Present address: Fakulffit f/it Biologic, Universit~it Bielefeld, D-4800 Bielefeld,Federal Republic of Germany Abbreviations: DGDG = digalactosyl diglyceride; MGDG = monogalactosyl diglyceride; PG = phosphatidyl glycerol; PGA = 3-phosphoglycericacid
tion studies demonstrated that the content of soluble sugars within the chloroplasts and the whole leaf tissue decreased as heat hardiness increased. This indicated that in spinach leaves, sugars play no protective role in heat hardiness. The results suggest that changes in the ultrastructure of thylakoids in connection with a stabilizing effect of soluble non-sugar stroma compounds are responsible for acclimatization of the photosynthetic apparatus to high temperature conditions. Changes in the chemical composition of the chloroplast membranes did not appear to play a role in the acclimatization. Key words: Absorbance change - Chlorophyll fluorescence - Heat injury - Membrane lipids - Spinacia Sugar compartmentafion.
Although it is well known that heat resistance of plants increases during exposure to supraoptimal, but sublethal temperatures, very little information is available on the molecular mechanism of adaptation of cells to high temperature conditions (Levitt, 1972; Heber and Santarius, 1973 ; Alexandrov, 1977). During recent years it has become well established that in green tissue high temperatures primarily affect the photosynthetic apparatus (Bj6rkman, 1975; Berry et al., 1975). Direct comparisons of various metabolic activities in green plant cells have shown that photosynthesis is inactivated at temperatures which are several degrees below those needed for inactivation of soluble enzymes (Santarius, 1975; Krause and Santaflus, 1975; Bj6rkman et al., 1976), respiration (Bj6rkman, 1975), or for a decrease in the semipermeability of the chloroplast envelope (Krause and Santarius, 1975). Thylakoid membranes proved to be particularly heat-sensitive sites.
K.A. Santarius and M. Miiller: Heat Resistance of Spinach Leaves
T h e r e are several p o s s i b i l i t i e s to e x p l a i n the increased heat tolerance observed after hardening. Heat i n a c t i v a t i o n o f b i o m e m b r a n e s m a y be p r e v e n t e d by the synthesis or accumulation of protective comp o u n d s s u r r o u n d i n g the m e m b r a n e s , o r by b i o c h e m ical a n d / o r u l t r a s t r u c t u r a l c h a n g e s w i t h i n the m e m b r a n e s . It has b e e n s h o w n t h a t v a r i o u s w a t e r s o l u b l e c o m p o u n d s s u c h as sugars, p r o t e i n s etc. a r e a b l e to p r o t e c t s e n s i t i v e cell s t r u c t u r e s a g a i n s t h e a t i n a c t i v a t i o n in v i t r o ( B e r g e r et al., 1946; F e l d m a n , 1962; M o l o t k o v s k y a n d Z h e s k o v a , 1965; O k u a n d T o m i t a , 1971; S a n t a r i u s , 1973; K r a u s e a n d S a n t a r i u s , 1975 a n d others). H o w e v e r , it is u n k n o w n w h e t h e r these solutes a c c u m u l a t e in t h e cells d u r i n g h e a t h a r d e n i n g . S o m e d a t a s u g g e s t t h a t the t h e r m a l s t a b i l i t y o f the photosynthetic apparatus of plants grown at high t e m p e r a t u r e s i n c r e a s e s as c o m p a r e d to t h a t o f p l a n t s g r o w n at l o w t e m p e r a t u r e s , i.e., t h a t p l a n t s c a n a d a p t t h e i r p h o t o s y n t h e t i c a p p a r a t u s to t h e i r t h e r m a l envir o n m e n t ( A g e e v a a n d L u t o v a , 1971 ; B j 6 r k m a n , 1975 ; S c h r e i b e r , 1976; P e a r c y e t a l . , 1977; S c h r e i b e r a n d Berry, 1977; A r m o n d e t a l . , 1978). It has b e e n s u g g e s t e d t h a t h i g h - t e m p e r a t u r e a c c l i m a t i z a t i o n is d u e to a l t e r a t i o n s in the lipid a n d / o r p r o t e i n c o m p o s i t i o n o f the p h o t o s y n t h e t i c m e m b r a n e s , b u t b i o c h e m ical o r s t r u c t u r a l basis o f s u c h a l t e r a t i o n s is n o t yet known. I n this s t u d y i n v e s t i g a t i o n s o n s p i n a c h leaves h a v e b e e n p e r f o r m e d in o r d e r to o b t a i n m o r e precise i n f o r m a t i o n o n the m e c h a n i s m o f h e a t h a r d e n i n g in l e a f cells o f h i g h e r plants.
at temperatures ranging from 34 to 50~ C (Santarius, 1975). The activities of ferricyanide reduction and noncyclic photophosphorylation were subsequently measm'ed at room temperature in a reaction medium containing 25 mM Tris, 2_2 mM KH2PO4, 2 mM ADP, 4 mM MgCI2, and 1 to 2 mM ferricyanide; the final pH was 7.8. Ferricyanide reduction was recorded with a Zeiss spectrophotometer at 400 nm during illumination with saturating red light (RG 630 cutoff filter, Schott & Gen., Mainz). Noncyclic photophosphorylation which accompanies ferricyanide reduction was measured by enzymatic ATP assay (Santarius, 1975). Polarographic measurement of oxygen evolution during reduction of 3-PGA by intact chloroplasts was performed as outlined by Heber and Krause (1972). For membrane lipid analysis, isolated thylakoids were washed in distilled water (cf. Santarius, 1971). Extraction of lipids, separation by one-dimensional thin-layer chromatography, and quantitative determination of fatty acid methylesters using a gas chromatograph was carried out as described recently (Mtiller and Santarins, 1978). For determination of membrane proteins, isolated thylakoids, washed twice in 50raM Tris-HCl-buffer pH 8.0 containing 350 mM NaCI, were used. Aliquots of the membranes were solubilized with sodium dodecylsulfate and /~-mercaptoethanol (final concentration 1% each) by being heated for 2 min to 10O~ C. The membrane polypeptides were separated by polyacrylamide gel electrophoresis (Shapiro et al., 1967; Weber and Osborne, 1969) and then stained with Coomassie Blue (Koenig et al., 1970). For comparison of band intensities the gels were scanned at 560 nm. Chlorophyll fluorescence of intact leaves was recorded as described by Krause (1973). Before measurements, plants were kept for at least 2 h in the dark and all preparatory operations were carried out either in the dark or in weak green light. For excitation of chlorophyll fluorescence, a broad band of red light was used (half band width ca. 630-680 nm; light intensity 30 W m- 2). Fluorescence induction was measured at room temperature in the farred region at 740 rim. The maximal slope of the chlorophyll fluorescence emission which rises to a peak, as observed between 100 ms and 2 s after onset of illumination, is defined as the rate of the rise of variable fluorescence (Heber et al., 1973 ; Krause and Santarius, 1975). Apparent absorbance changes of intact leaves at 535 nm, induced by a beam of high-intensity red light, were measured according to Heber (1969; see also Krause, 1973). After a dark period of at least 2 h, illumination periods of 2 rain were followed by dark periods of 3 min. For sugar determinations leaves were frozen in liquid nitrogen and freeze-dried. Aliquots of the dry material were pulverized and extracted for 3 min in boiling water. After centrifugation, quantitative determination of glucose, fructose, and sucrose in the supernatant was performed enzymically as described by Bergmeyer (1974). For compartmentation studies, chloroplasts were separated from aliquots of the freeze-dried tissue by a modification of the nonaqueous isolation technique described elsewhere (Heber etal., 1963; Heber and Willenbrink, 1964). Purity of the chloroplast fractions was determined according to Santarius and Stocking (1969) by measuring pyruvate kinase activity under the conditions given by Bergmeyer (1974). The sugar content of the fractions was determined as mentioned above.
Materials and Methods Spinach (Spinacia oleraceaL.) was cultivated in growth chambers under the following standardized conditions : 9 h light at 20~ C/3 h dark at 20~ C/12 h dark at 15~ C; light intensity about 30,000 lx; relative humidity 75%. Plants with fully developed leaves were either kept under these conditions (control material) or heatadapted by increasing the temperature to 35~ C without changes in the light/dark periods and humidity. For dehardening, the temperature was switched back to the 20~176 C regime. Leaves were harvested from heat-adapted, dehardened, and control material and assayed immediately or after 3 min exposures to various temperatures in a water bath. To obtain thylakoid membranes, chloroplasts were isolated in an isotonic NaC1 solution as modified from Heber and Santarius (1964) and Santarius and Heber (1967). Smaller quantities of media and leaf material (5 to 10 g) were used and centrifugation steps for isolation and washing of the chloroplasts were each shortened to 1 min at 1,500 to 2,000 g. After isolation, chloroplasts were ruptured in distilled water and the released thylakoids stored at 0~ C. For the isolation of intact chloroplasts a method similar to that developed by Jensen and Bassham (1966) was used (Heber and Santarius, 1970; Krause, 1971). Chloroplasts were kept in "solution B" (Jensen and Bassham, 1966) in an ice bath until used. Chlorophyll was determined according to Arnon (1949). The tbermostability of isolated thylakoid membranes and intact chloropIasts was tested by incubating small samples for 3 min
Hardening and Dehardening of Intact Leaves When spinach plants grown under standardized cond i t i o n s w e r e e x p o s e d to h i g h e r t e m p e r a t u r e s , their
K,A. Santarius and M. Mfiller: Heat Resistance of Spinach Leaves Table 1. Ferricyanide reduction of thylakoid membranes isolated from heat-adapted spinach leaves and from non-hardened control material after short-term heat stress. Spinach plants were grown at 20~ ~ C, Heat hardening was accomplished at 35 "~C for periods of 4 h to 3 days. After harvesting heat-adapted and control materiaI, leaves were exposed for 3 rain to 43 ~ C in a water bath. ThyIakoids were then isolated from those leaves and activity of photosynthetic electron transport to ferricyanide was measured. Thylakoids isolated from leaves which were not heat treated exhibited ferricyanide reduction activities between 250 and 400 gmol m g - t chlorophyll - h-~, In most cases there were no conspicuous differences between basic activities of heat-hardened and control material Heat hardening at 35 ~ C (h)
4 61/2 24 48 72
1 O0 - ' - ' - -.--.'-'--- - o ~ .-.
Ferricyanide reduction in gmol rag- ~ chlorophyll - h - ~ of thylakoids isolated from Heat-adapted leaves (leaves at 35 ~ C)
Control material (leaves at 20~ ~ C)
136 207 193 204 214
103 I28 106 108 92
20 heat resistance was increased. As can be seen f r o m Table 1, during short-term heat stress such heat-hardened leaves showed higher thermostability than nonhardened material. In n u m e r o u s experiments it was f o u n d that heat hardening occurred within 4 to 6 h after increasing the temperature to 35 ~ C ; extended exposure to 3 5 ~ up to 3 days caused no further rise in the degree o f hardiness. H e a t hardening took place in the light as well as in the dark. It is k n o w n that the chlorophyll fluorescence yield is a sensitive indicator of ultrastructural changes acc o m p a n y i n g changes o f the energy state o f thylakoid m e m b r a n e s (Papageorgiou, 1975). Figure 1 shows the rise o f chlorophyll fluorescence of leaves illuminated with short-wavelength red light as a function o f stress temperature. Stress was imposed by a 3 rain treatment of the leaves at various temperatures. Prior to this treatment, some plants had been hardened at 3 5 ~ while others had first been hardened and then subsequently dehardened at 20~176 C (see methods). As can be seen, a d a p t a t i o n at 35 ~ C increased heat resistance by a b o u t 3 ~ C. The increase in the heat resistance was reversible. In the experiment presented in Fig. 1, dehardening for 2 2 h at n o r m a l growing conditions (20~ ~ C) resulted in the loss o f a b o u t the half o f the heat resistance obtained during heat hardening. Even after a dehardening period o f 46 h, part o f the acquired heat stability remained, indicating that m o r e time was necessary for dehardening than hardening. The same degree o f dehardening t o o k place in both the light and the dark. On the other hand, temperature condi-
0 . . . . .40.
\"',,.\:,,'o )\ \\\ ,: 4'2 . . 44. . 46 temperature in ~
Fig. 1. The rise in chlorophyll fluorescence of heat-hardened and dehardened intact spinach leaves and non-hardened control material as a function of the temperature of short-term heat stress. Fluorescence measurements were performed following a dark period of ca. 2 h and exposure of single leaves for 3 rain to various temperatures. Rates of the maximum rise in variable fluorescence (cf. the insert of the signals of chlorophyll a fluorescence induction) in percent of the signal of the respective leaves which were not exposed to high temperatures are plotted against temperature of short-term heat stress. - x - control material grown at 20~176 -e-heat hardening at 35~ for 24h; . . . . o dehardening of those heat-hardened leaves for 22 h at 20~176 ..... ~ ..... dehardening for 46 h at 20~ ~C .
tions drastically influenced the dehardening process. As can be seen from the fluorescence data given in Fig. 2, maintainance o f heat-adapted spinach plants for 2 2 h at 5 ~ did not result in the loss o f the heat resistance of the leaves, whereas at 2 0 ~ 1 7 6 dehardening occurred, as already d e m o n s t r a t e d in Fig. 1, a l t h o u g h it was more complete in the experim e n t o f Fig. 2. Similar to fluorescence measurements, lightinduced changes in apparent absorbance at 535 n m are rapidly responding, sensitive probes o f the energy state o f photosynthetic m e m b r a n e s (Heber, 1969; Krause, 1973, 1974). In Fig. 3 characteristic absorb-
K.A. Santarius and M. M~ller: Heat Resistance of Spinach Leaves
ero u utO e~
I/I L. E 6G =
_EEx ,/,0 "6
(/1 JO O
\ \ \
\xo \ \ \
4/, /,6 temperature in "C
r 2 rain
t e m p e r a t u r e in ~
Fig. 2. The rise in chlorophyll fluorescence of heat-hardened and dehardened intact spinach leaves and non-hardened control material as a function of the temperature of short-term heat stress. Chlorophyll fluorescence measurements took place as outlined in Fig. 1. - x control material grown at 20~176 --e-heat hardening at 35~ for 24h; . . . . o .... dehardening of those heat-hardened leaves for 22h at 20~176 ..... zx..... "dehardening" for 22 h at 5~
Fig. 3. Light-induced changes in apparent absorbance at 535 nm of intact spinach leaves after short-term heat stress. Spinach plants were grown at 20~ ~ C. After a dark period of at least 2h, light-induced absorbance changes were measured in the absence of CO2 either before (control) or after heat treatment for 3 min at 41, 44 and 47 ~ C, respectively. Light on: upward arrows; light off: downward arrows
Fig. 4. Fast changes in the light-induced absorbance at 535 nm in heat-hardened intact spinach leaves and in non-hardened control material as a function of the temperature of short-term heat stress. Following a dark period of at least 2 h, light-induced absorbance changes were measured in the absence of CO2 after exposure of single leaves for 3 rain to various temperatures (abscissa). The fast decrease in the apparent absorbance immediately after turning off the light (cf. Fig. 3) was calculated in percent of those changes in leaves which were not exposed to a 3 min heat treatment ( = 100%). - x control material grown at 20~176 --e-heat hardening at 35~ for 24 h
ance changes at 535 nm are depicted. In a control leaf, illumination produced a fast increase in absorbance followed by a large slow absorbance change; when the light was turned off, a fast and then a slow decrease in the absorbance was observed. The difference spectrum of the fast absorbance change has a positive peak at 518 nm and a negative peak at 475 nm. An absorbance change with these characteristics is believed to indicate a light-generated thylakoid membrane potential (7/). The difference spectrum of the slow absorbance changes has a m a x i m u m at 535 nm and a minimum around 420 nm, indicating light-induced changes in light-scattering of chloroplast thylakoids in the leaves (Heber, 1969). Shortterm heat stress resulted in drastic changes of the leaf response : increasing temperatures caused a reduction in the fast signal (indicating a decrease in A 7j)
K.A. Santarius and M. Mtiller: Heat Resistance of Spinach Leaves
the control material (Figs. 4 and 5). This again reflects an increase in thermostability of the photosynthetic apparatus in heat-adapted leaves.
Lipid and Protein Composition of Thylakoid Membranes
There is a question whether the increase in heat resistance of the leaves is a result of changes in the biochemical composition and/or ultrastructure of the heat-sensitive chloroplast membranes, or of the synthesis and accumulation of protective compounds within the leaf cells. In order to determine whether biochemical changes in the membranes during heat adaptation are responsible for differences in the heat stability, thylakoids were isolated from leaves grown at different temperatures and the composition of membrane lipids and membrane proteins was investigated. Table 2 shows that following a heat hardening period of 7 h at 35 ~ C (sufficient for a considerable increase in heat resistance of the leaves, cf. Table 1) and on a chlorophyll basis, there were no significant differences in the total lipid content nor in fatty acid composition of thylakoid membranes isolated from leaves which differ in heat hardiness. Differences in the fatty acid composition of the chloroplast membranes were significant only after extended exposure to 35 ~ C, e.g., for 24 h. On a chlorophyll basis, the total lipid content decreased at the higher temperature which was due mainly to a decrease in tipids containing highly unsaturated fatty acids such as linolenic (18:3) and hexadecatrienic acid (16:3). In contrast, the amount of more highly saturated fatty acids for instance palmitic acid (16:0) and linolic acid (18:2) - increased. This was even more pronounced in thylakoids isolated from leaves which were heat hardened for 3 days. As outlined in Table 3, these
O U I
l,'/, ' ~'6 ' temperature in ~
Fig. 5. Dark decay of light-scattering in heat-hardened intact spinach leaves and in non-hardened control material as a function of the temperature of short-term heat stress. Conditions were outlined in Fig. 4. The decrease in the apparent absorbance during the first 10 s after turning off the light, reduced by the fast decline in the signal, was measured in relation to the dark decay of leaves which were not exposed to a 3 rain heat treatment. The latter was taken as 100%. - x control material grown at 20~176 - - 9 - - heat hardening at 35 ~ C for 24 h
and faster kinetics of the light-scattering signal. The extent of light-scattering increased in response to 41 and 44~ temperatures and then decreased with 47 ~ C. The stress-induced decrease in A ~ and the increase in light-scattering occurred in heat hardened material at considerably higher temperatures than in
Table 2. Fatty acid composition of total lipids from thylakoid membranes isolated from heat-hardened spinach leaves and from nonhardened control material. Adaptation of plants to 35 ~ C was performed for 7 h in the light and for 24 h and 72 h during light-dark periods of 9 and 15 h, respectively; control material was kept for the same time period at 20~ ~ C. Fatty acid content of the control material and differences between thylakoids isolated from heat-hardened and non-hardened leaves is given in gmol rag-1 chlorophyll. Results are from 3 single experiments Temperature treatment
Fatty acids 16:0
Controls 7 h at 35 ~ C
Controls 24 h at 35 ~ C
Controls 72 ta at 35 ~ C
K.A. Santarius and M. Miiller: Heat Resistance of Spinach Leaves
Table 3. Fatty acid composition of MGDG, DGDG and PG from thylakoid membranes isolated from heat-hardened spinach leaves and from non-hardened control material. Adaptation of plants to 35~ C was performed for 24 h during light-dark change of 9 and 15 h, respectively; control material was kept for the same period at 20~ ~ C. Major acyl groups are given in % of the total amount of fatty acids within the three main lipids of the chloroplast lamellae. Traces of 16:2 are not shown. Average of 5 experiments (MGDG and DGDG) and 4 experiments (PG), respectively Lipid
Temperature Fatty acids treatment 16:0
within the statistic deviation of the material. This demonstrates that no drastic changes in proteins of thylakoid membranes occurred during heat hardening.
Heat Treatment of Isolated Chloroplasts and Thylakoids
distance from the origin
Fig. 6. Electrophoretic analysis of polypeptide composition of thylakoid membranes isolated from heat-hardened spinach leaves and from non-hardened control material. - - control material kept at 20~ ~C; ...... heat hardening at 35~ for 24 h
shifts did occur in all of the major lipids of the thylakoid membranes: M G D G , D G D G and PG. However, it must be emphasized that changes in the lipid composition of thylakoids observed in the course of prolonged adaptation to 35 ~ C were not accompanied by a significant increase in heat resistance of the leaves (cf. Table 1). According to Levitt (1972), Alexandrov (1977) and others, changes in membrane proteins might be responsible for changes in temperature resistance. Investigations on the polypeptide composition of thylakoid membranes in relation to heat hardening led to a similar result as lipid analysis: the increase in the heat resistance of spinach leaves after exposure of the whole plants to 35~ for 2 4 h was not accompanied by significant changes in the polypeptide composition of membrane proteins (Fig. 6). Parallel experiments showed that the small differences in the absorbance at 560 nm between thylakoid polypeptides isolated from heat-adapted and control leaves are
The finding that heat hardening of spinach leaves was relatively fast and occurred within 4 to 6 h (Table 1), but was not accompanied by significant biochemical changes within the thylakoid membranes, suggests that heat adaptation might be due to ultrastructural changes within these membranes. Therefore, intact chloroplasts and thylakoids were isolated from heat-adapted spinach leaves and from non-hardened control material and subsequently exposed to temperature stress. As can be seen in Fig. 7, 3-PGA reduction by intact chloroplasts showed significant differences in thermostability when chloroplasts from leaves which differ in heat resistance were compared. In contrast, thylakoid membranes isolated from heatadapted and non-hardened spinach leaves did not show differences in the heat-sensitivity of electron transport (Fig. 8) and noncyclic photophosphorylation (Table 4). F r o m these results it can be concluded that differences in the heat resistance of thylakoid membranes are lost when the membranes are separated from the stroma phase which contains soluble chloroplast components.
Sugar Content and Compartmentation in Intact Leaves As previously mentioned, sugars stabilize biomembranes during heat exposure (Santarius, 1973). In ad-
K,A. Santarius and M, Mfdler: Heat Resistance of Spinach Leaves
Table 4, Thermostability of noncyclic photophosphorylation of thylakoid membranes isolated from heat-hardened spinach leaves and from non-hardened control material 9 Heat hardening of plants took place for 19 h at 35 ~ C; control material was kept for the same period at 20~ ~ C, Heat treatment of isolated thyIakoids was performed for 3 rain at the temperatures listed below. The activity of the isolated membranes ( - 100%) were 206 gmol ATP synthesized per m g chlorophyll per h for the controls and 230 ~mol for the heat-adapted thylakoids
1. Spinach was harvested once a week, beginning five weeks after sowing. The leaves were divided into three categories, according to their height of insertion. For every category, Hill-activity and manganese content (by atomic absorption) were measur
Heat hardening of leaves which leads to an increase in the heat resistance of their cells, also increases the heat resistance of their enzymes (urease, acid phosphatase, ATPase). As judged by the temperature reducing enzyme activity by 50%, the heat
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Spinach is one of the highly consumed vegetable, with significant nutritional, and beneficial properties. This study revealed for the first time, the effects of high temperature frying on the carotenoids, chlorophylls, and tocopherol contents of spin
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Glutathione reductase (EC 188.8.131.52) was purified from spinach (Spinacia oleracea L.) leaves by affinity chromatography on ADP-Sepharose. The purified enzyme has a specific activity of 246 enzyme units/mg protein and is homogeneous by the criterion of