Proc. Nat. Acad. So. USA Vol. 72, No. 1, pp. 306-309, January 1975
Breaking of Seed Dormancy by Catalase Inhibition (seed germination/hydrogen peroxide action/metabolic control)
S. B. HENDRICKS AND R. B. TAYLORSON Agricultural Environmental Quality Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20706
Contributed by S. B. Hendricks, November 7, 1974 Germination of some dormant seeds is ABSTRACT promoted by solutions of thiourea, sodium nitrite, and hydroxylamine salts. The promotions are accompanied by irreversible inhibition of catalase (EC 1.11.1.6) in extracts from the seeds. The seeds are also promoted in germination by catechol and pyrogallol solutions. These effects are recorded for lettuce (Lactuca sativa L. cv. Grand Rapids) and pigweed (Amaranthus albus L.) seeds. The results indicate that metabolically derived hydrogen peroxide, spared from decomposition by catalase inhibition, oxidizes reduced NADPH required as the oxidant in the pentose pathway of glucose use. The metabolic system for such use of H202 involves the enzymes, peroxidase (EC 1.11.1.7) and pyridine nucleotide quinone oxidoreductase (EC 1.6.99.2), which are present in the dormant seed prior to imbibition of water.
Some viable seeds fail to germinate at favorable temperatures when they imbibe water and are held in darkness. This arrest of development, or dormancy, is a major factor in survival of plant species and is a main reason for persistence of weeds in cultivated fields. The dormancy can hold for millennia, but it can often be broken in a few moments when there is a change in the form of phytochrome by light (1). Sometimes it can be broken when the seeds imbibe solutions of any of several compounds, notably nitrates, nitrites, hydroxylamines, and thiourea. Their effectiveness has long raised questions about how they act to break dormancies. Previous work by others (2) indicates that change from dormant to germinating seeds is often accompanied by an increased functioning of the pentose-phosphate pathway of glucose use. The uptake and properties of the several promotive compounds, particularly their interaction with hemoproteins in vitro, suggest that inhibition of catalase (EC 1.11.1.6; hydrogen-peroxide: hydrogen-peroxide oxidoreductase) activity favors the pentose-phosphate pathway (3). This possibility was supported by the presence of several enzymes in the dry seeds that could serve to couple H202, spared by catalase inhibition, to D-glucose-6-phosphate oxidation. We extend the observations to include action of thiourea on two kinds of seeds, and demonstrate that catalase is irreversibly inhibited in vivo by the several compounds. This work and current observations by others suggest operation of the metabolic network shown in Fig. 1. This network affords a way for eventual reoxidation by oxygen of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+ required in the pentose-phosphate pathway.
(3) in common use. Duplicate lots of 100 seeds were held saturated at a favorable temperature in darkness, except for a 10-min exposure to radiation >690 nm after 3-5 hr of imbibition by the pigweed seeds in order to reduce the germination in darkness. To enhance comparabilities of enzymic assays, 0.25-g lots of treated seeds were wet-ground for 2 min with 100 mesh A1203 and 3 ml of 20 mM phosphate buffer (pH 6.8) in a single Ten Broeck unit and then transferred to centrifuge tubes with three successive 1-ml lots of buffer. First-order rate constants for loss of H202 by catalase or peroxidase (EC 1.11.1.7) activity were measured on the centrifuged extracts of seeds saturated with water or solutions of thiourea, hydroxylamine hydrochloride, or sodium nitrite at several concentrations. Initial rates of H202 108s per mg of protein-min-' are reported for activity in 8 mM H202 solutions for catalase and 0.9 mM H202 for peroxidase. Protein was determined by fluorescence of conjugated fluorescamine (4-phenyl spiro [furon-2(3H),l'-phthalan]3,3'-dione) (4). Free amino acids, which react with fluorescamine, were not removed because they were approximately constant and low n amount relative to free amino groups of extracted protein. i RESULTS Extracted catalase activities of seeds saturated for 0-24 hr with water were approximately constant with values of 8.3 ± 0.14 and 10.6 4 0.30 itmol of H202 lost per mg of protein-min-' for lettuce and pigweed, respectively. PerH20+'/202 Ir~bitorsz4tf Substrate
Oxidation
NAD++ATP3
Substates
-H202\'~'c NADP F~D-GIuosP H20 A
tki Quinones
PADRIos5P 1+1C02 4A+
Gemination
FIG. 1. A part of the postulated reaction network involved in promotion of dormant seed germination by thiourea, hydroxylamines, nitrites, and other inhibitors of catalase enzymic activities. The several designated enzymes are (a) EC 1.1.1.49, nglucose-6-phosphate :NADP+ 1-oxidoreductase; (b) EC 3.1.1.31,66-phospho-D-gluconate-5-lactone hydrolase; (c) EC 1.1.1.44, phospho-i-gluconate:NADP+ 2-oxidoreductase (decarboxylating); (d) EC 1.6.99.2, NAD(P)H:quinone oxidoreductase; (e) EC 1.11.1.7, peroxidase; (f) EC 1.11.1.6, catalase; (g) EC 2.7.1.23, ATP:NAD + 2'-phosphotransferase. The endogenous quinone substrates of (d) and (e) are unknown. Dotted arrows indicate materials supplied in the ambient media.
MATERIALS AND METHODS Germination tests and enzymic activity measurements on
seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) and pigweed (Amaranthus alblus L.) followed described procedures 306
Proc. Nat. A cad. Sci. USA 72
(1975)
Dormancy Breaking by Catalase Inhibition
307
1i r 1%
8C
c
60
-
0
0
a.
40
Catalase
_
Inhibilion
,,,, 20
Om
.....
msales.
20
-
& ...... to
-CC
I
-4
II
I
-3
-2
Concentration log M
FIG. 2. Effects of various concentrations of ambient thiourea solutions on germination of lettuce seeds after 48 hr at 250 and inhibition of extracted catalase activities after 24 hr at 250. Catalase activities of controls that had imbibed water were 8.50 ± 0.10 /Amol of H202 lost per mg of protein-min-. Peroxidase activities were constant at 52 1 4 nmol of H202-lost per mg of protein -min-' for all concentrations.
oxidase values in the 0- to 24-hr period were constant for imbibition in
Breaking of Seed Dormancy by Catalase Inhibition (seed germination/hydrogen peroxide action/metabolic control)
S. B. HENDRICKS AND R. B. TAYLORSON Agricultural Environmental Quality Institute, Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland 20706
Contributed by S. B. Hendricks, November 7, 1974 Germination of some dormant seeds is ABSTRACT promoted by solutions of thiourea, sodium nitrite, and hydroxylamine salts. The promotions are accompanied by irreversible inhibition of catalase (EC 1.11.1.6) in extracts from the seeds. The seeds are also promoted in germination by catechol and pyrogallol solutions. These effects are recorded for lettuce (Lactuca sativa L. cv. Grand Rapids) and pigweed (Amaranthus albus L.) seeds. The results indicate that metabolically derived hydrogen peroxide, spared from decomposition by catalase inhibition, oxidizes reduced NADPH required as the oxidant in the pentose pathway of glucose use. The metabolic system for such use of H202 involves the enzymes, peroxidase (EC 1.11.1.7) and pyridine nucleotide quinone oxidoreductase (EC 1.6.99.2), which are present in the dormant seed prior to imbibition of water.
Some viable seeds fail to germinate at favorable temperatures when they imbibe water and are held in darkness. This arrest of development, or dormancy, is a major factor in survival of plant species and is a main reason for persistence of weeds in cultivated fields. The dormancy can hold for millennia, but it can often be broken in a few moments when there is a change in the form of phytochrome by light (1). Sometimes it can be broken when the seeds imbibe solutions of any of several compounds, notably nitrates, nitrites, hydroxylamines, and thiourea. Their effectiveness has long raised questions about how they act to break dormancies. Previous work by others (2) indicates that change from dormant to germinating seeds is often accompanied by an increased functioning of the pentose-phosphate pathway of glucose use. The uptake and properties of the several promotive compounds, particularly their interaction with hemoproteins in vitro, suggest that inhibition of catalase (EC 1.11.1.6; hydrogen-peroxide: hydrogen-peroxide oxidoreductase) activity favors the pentose-phosphate pathway (3). This possibility was supported by the presence of several enzymes in the dry seeds that could serve to couple H202, spared by catalase inhibition, to D-glucose-6-phosphate oxidation. We extend the observations to include action of thiourea on two kinds of seeds, and demonstrate that catalase is irreversibly inhibited in vivo by the several compounds. This work and current observations by others suggest operation of the metabolic network shown in Fig. 1. This network affords a way for eventual reoxidation by oxygen of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to NADP+ required in the pentose-phosphate pathway.
(3) in common use. Duplicate lots of 100 seeds were held saturated at a favorable temperature in darkness, except for a 10-min exposure to radiation >690 nm after 3-5 hr of imbibition by the pigweed seeds in order to reduce the germination in darkness. To enhance comparabilities of enzymic assays, 0.25-g lots of treated seeds were wet-ground for 2 min with 100 mesh A1203 and 3 ml of 20 mM phosphate buffer (pH 6.8) in a single Ten Broeck unit and then transferred to centrifuge tubes with three successive 1-ml lots of buffer. First-order rate constants for loss of H202 by catalase or peroxidase (EC 1.11.1.7) activity were measured on the centrifuged extracts of seeds saturated with water or solutions of thiourea, hydroxylamine hydrochloride, or sodium nitrite at several concentrations. Initial rates of H202 108s per mg of protein-min-' are reported for activity in 8 mM H202 solutions for catalase and 0.9 mM H202 for peroxidase. Protein was determined by fluorescence of conjugated fluorescamine (4-phenyl spiro [furon-2(3H),l'-phthalan]3,3'-dione) (4). Free amino acids, which react with fluorescamine, were not removed because they were approximately constant and low n amount relative to free amino groups of extracted protein. i RESULTS Extracted catalase activities of seeds saturated for 0-24 hr with water were approximately constant with values of 8.3 ± 0.14 and 10.6 4 0.30 itmol of H202 lost per mg of protein-min-' for lettuce and pigweed, respectively. PerH20+'/202 Ir~bitorsz4tf Substrate
Oxidation
NAD++ATP3
Substates
-H202\'~'c NADP F~D-GIuosP H20 A
tki Quinones
PADRIos5P 1+1C02 4A+
Gemination
FIG. 1. A part of the postulated reaction network involved in promotion of dormant seed germination by thiourea, hydroxylamines, nitrites, and other inhibitors of catalase enzymic activities. The several designated enzymes are (a) EC 1.1.1.49, nglucose-6-phosphate :NADP+ 1-oxidoreductase; (b) EC 3.1.1.31,66-phospho-D-gluconate-5-lactone hydrolase; (c) EC 1.1.1.44, phospho-i-gluconate:NADP+ 2-oxidoreductase (decarboxylating); (d) EC 1.6.99.2, NAD(P)H:quinone oxidoreductase; (e) EC 1.11.1.7, peroxidase; (f) EC 1.11.1.6, catalase; (g) EC 2.7.1.23, ATP:NAD + 2'-phosphotransferase. The endogenous quinone substrates of (d) and (e) are unknown. Dotted arrows indicate materials supplied in the ambient media.
MATERIALS AND METHODS Germination tests and enzymic activity measurements on
seeds of lettuce (Lactuca sativa L. cv. Grand Rapids) and pigweed (Amaranthus alblus L.) followed described procedures 306
Proc. Nat. A cad. Sci. USA 72
(1975)
Dormancy Breaking by Catalase Inhibition
307
1i r 1%
8C
c
60
-
0
0
a.
40
Catalase
_
Inhibilion
,,,, 20
Om
.....
msales.
20
-
& ...... to
-CC
I
-4
II
I
-3
-2
Concentration log M
FIG. 2. Effects of various concentrations of ambient thiourea solutions on germination of lettuce seeds after 48 hr at 250 and inhibition of extracted catalase activities after 24 hr at 250. Catalase activities of controls that had imbibed water were 8.50 ± 0.10 /Amol of H202 lost per mg of protein-min-. Peroxidase activities were constant at 52 1 4 nmol of H202-lost per mg of protein -min-' for all concentrations.
oxidase values in the 0- to 24-hr period were constant for imbibition in
