Promotion of Seed Germination by Nitrate, Nitrite, Hydroxylamine

Plant Physiol. (1974) 54, 304-309

Promotion of Seed Germination by Nitrate, Nitrite,Hydroxylamine, and Ammonium Salts1

Received for publication January 4, 1974 and in revised form March 29, 1974

S. B. HENDRICKS AND R. B. TAYLORSONAgricultural Environmental Quality Institute, Agricultural Research Service, United States Departmenit o]Agriculture, Beltsville, Maryland 20705

ABSTRACT

Action and uptake of azides, nitrates, nitrites, hydroxyla-mines, and ammonium salts were measured on germination ofAmaranthus albus, Lactuca sativa, Phleum pratense, Barbareavulgaris, B. verna, and Setaria glauca seeds. Nitrate and nitritereductase activities were measured in vivo for each of thesekinds of seeds. Activities were measured in vitro for catalase,peroxidase, glycolate oxidase, and pyridine nucleotide quinonereductase on extracts of A. albus and L. sativa seeds before andafter germination. The enzymic activities measured and theresponsiveness of the haemproteins to inhibition by the severalcompounds indicate that nitrites, azides, and hydroxylaminespromote seed germination by inhibition of H202 decompositionby catalase. Ammnonium salts showed pronounced promotiveactivity only for B. verna and B. vulgaris seeds, for which theyserved as metabolic substrates.The promotion of germination is thought to depend on cou-

pling of peroxidase action to NADPH oxidation, which canregulate the pentose pathway of D-glucose 6-phosphate use.Pyridine nucleotide quinone reductase is the possible couplingenzyme. This enzyme and others required for the action arepresent in the seeds before imbibition of water.

Nitrates stimulate germination of many dormant seeds (8,11, 27, 31), while ammonium salts are usually ineffective.Functioning of nitrates in seed germination through reductionto ammonium ions, as occurs in the nutrition of plants (1, 17),is thus open to question. The only analysis of the cause for thiscontrol of germination is by Roberts (24-26), who concludesthat nitrate acts as an oxidizing substrate in a metabolicregulatory process involving NADPH - NADP+ in the pentosepathway of glucose metabolism.We examined the uptake and action of nitrates and other

compounds of various nitrogen oxidation levels on germinationof several kinds of seeds. Evidence is presented for at leasttwo types of action with hvroxylamine and ammonium ions asprobable effective agents. Nitrate action in seed germinationwas found to depend on nitrite or on hydroxylamine or nitricoxide produced from nitrite, rather than on further reductionto ammonium ions or on the process of reduction per se.Promotion of seed germination probably depends on inhibitionby the several compounds of H202 decomposition by catalase.

1Dedicated to Solon A. Gordon.

MATERIALS AND METHODS

Seed Germnation. Seeds showing low germination in dark-ness on water at constant temperatures, but still responsive tosome substrate, were available from lots collected over a periodof 3 to 6 years and held in tight containers at -20 C. Seedstested at the indicated temperatures, which were previouslyfound to be optimum for germination, were those of tumblepigweed (Amaranthus albus L. [30 C]); lettuce (Lactuca sativaL. var. Grand Rapids [23 and 30 C]); yellow rocket (Barbareavulgaris R. Br. [20 C]); winter cress (Barbarea verna [Mill.]Aschers [20 C]); yellow foxtail (Setaria glauca [L.] Beauv. [25C]); timothy [Phleumn pratense L. [25 C]); and barnyardgrass(Echinochloa crusgalli [L.] Beauv. [25 C]). Germination testswere made by placing duplicate lots of 100 seeds on singleWhatman No. 3 filter papers, moistened to somewhat morethan glistening with the appropriate solution, or about 5 ml per100 seeds. The covered Petri dishes were held in darkness in awater-saturated atmosphere. Germination, assessed by emer-gence of the radicle, was determined after 3 to 7 days, withnote of any distortion of seedling growth. All tests were re-peated two or more times.

Assays. Nitrate was assayed, after the method of Wooleyet al. (35), by reduction to nitrite with zinc. The nitrite pro-duced was measured spectrometrically after conversion to anazo compound. Nitrite was similarly determined without re-duction (29). Cyanide was determined by treatment of solu-tions with chloramine-T to form CNC1, which was coupledwith a pyridine-pyrazolone reagent, and the absorbance wasdetermined at 630 nm (30). Hydroxylamine was assayed byoxidation with iodine to nitrite after the method of Czaky (5).Ammonia was determined by the Conway microdiffusionmethod (4).Enzymic Activities. Nitrate reductase activity of seeds was

assayed in vivo by the method of Ferrari and Varner (6). Thismethod involved cell leakage of nitrate and nitrite in thepresence of 1% (v/v) propanol under anaerobic conditions.Nitrite reductase was measured in vivo by disappearance fromthe solution ambient to the seed, followed by determinationof nitrite in the seeds by leakage under anaerobiosis. An at-tempt to measure hydroxylamine reductase by aerobic in vivouptake failed because of nonenzymatic reaction of hydroxyl-amine with seeds.

Peroxidase, catalase, glycolate oxidase, and pyridine nucleo-tide quinone reductase activities were measured at 25 C inaliquots from extracts of imbibed, washed seeds, ground with100 mesh A1203 at ice-bucket temperature in a Ten-Broeck2

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304

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PROMOTION OF SEED GERMINATION

unit, using 6 ml/0.25 g seeds of 10 mm K phosphate buffer atpH 6.8. Extracts were centrifuged at 12,000g at 3 C for 15min, followed, in some instances, by millipore filtration tolower the starch content of supernatants. Catalase activity ofaliquots was measured by iodometric assay of added H20,remaining after various times (10). Peroxidase activity wasmeasured by the rate of loss of H202 in the presence of 0-anisidine as hydrogen donor, with appearance of absorbance at460 nm (36). Both activities were expressed in units of ptmolesof H202 decomposed/min at 25 C/g of seed extracted. Glycolateoxidase was assayed by following the rate of reduction of 2,6-dichlorophenolindophenol with glycolate as a substrate, in thepresence of FMN and the presence or absence of 02 (23).Pyridine nucleotide quinone reductase was measured by follow-ing the rate of oxidation of NADH or NADPH by p-benzo-quinone, with correction for the nonenzymic rate (37). Proteinin the extracts was measured by the method of Lowry et al.(20) and in seeds by microkjeldahl methods.

Inhibitor Actions. Effects at various concentrations of KN3,NH20H HCl, NaNO2, KNO3, NO, CH3NHOHCl, and [(C2H4),-NOH]2- [COOH]2 on catalase and peroxidase activities weremeasured on the seed extracts, as well as on commercial ly-ophilized liver catalase and horseradish peroxidase.

Uptake of Substrates. Four to 20 ml of the required salt solu-tions of known concentrations were placed on 0.25 or 0.5 g ofseed in 5-cm diameter Petri dishes and held at the appropriateconstant temperatures in darkness in a water-saturated at-mosphere for 20 to 72 hr. The seeds thus were covered withsolution to a depth of several mm. Some of the indicated testswere made in the presence of 1 mm streptomycin sulfate and0.5 mM mycostatin to suppress growth of microorganisms. Theinitial pH values were 7.0, except for KCN solutions, whichwere >9.0. Initial and final assays were made on aliquots ofthe solutions. Salts used were commercial ones or were pre-pared from the appropriate commercial base.Oxygen consumption. A Clark electrode was used to measure

O2 consumption of 0.3 g of seed, which had been maintainedin 4 ml of the appropriate solution at 30 C for the indicatedperiods. Initial values were obtained from the slopes of the 0,consumption versus time curves.

RESULTS

Seed Germination. Percentage of germination for the variouskinds of seeds incubated with the several substrates are shownin Table I. Concentrations listed are near optimum values formaximum germination, as shown by tests over concentrationranges. Variation of A. albus seed germination with concentra-tion of the several substrates are shown for a typical lot(Fig. 1). Some lots of A. albus seeds were somewhat promotedin germination by 10 mM NH,Cl (Table I).

All lots of B. verna and B. vulgaris seeds tested were mostresponsive to NH4Cl among the substrates used (Table I, Fig.2). Germination of P. pratense seeds was inhibited by 10 mMNH4C1 at 30 C, relative to values with water (Fig. 3). Theeffect was negligible at 1 mm NH4C1. Germinations of E.crusgalli and A. albus seeds were less responsive to [(C2H5)2-NOH]12 (COOH)2 than to NH20H HCI (Fig. 4).Damage to emergent seedlings was noted at substrate con-

centrations corresponding to the dotted portions of the curvesin the figures. Seedlings of A. albus and E. crusgalli were dam-aged by hydrazine and its 1-methyl, 1 , 1-methyl, and 1, 2-methyl derivatives (results not shown), when used at >0.32

Department of Agriculture, and does not imply its approval to theexclusion of other products that may also be suitable.

Table I. Germiniation in Darkntess of Several Kinids of Seedson Various Salt Substrates anid Water

Salt Concn A. L: B. B. S. P. E.albus sativa verna vuilgaris glauca pratense crusgalli

mM %KNO3 10.0 77 34* 14 31* 46 82 65

(44) (30) (1) (30) (26) (56) (46)NaNO2 1.0 92 30* 6* 262* 58 68 66

(56) (30) (0) (30) (35) (31) (46)NH2OH*HCI 0.32 99 34* 2* 1 * 39 88 68

(44) (30) (1) (1) (20) (77) (46)NH4Cl 10.0 65 28* 62 40 30* 34 39

(44) (30) (1) (30) (26) (56) (46)KCN 0.1 81 74 24 54* 90*

(32) (15) (1) (58) (92)KN3 0.01 86 21 24 57 81

(47) (11) (0) (47)1 (68)

1 Values shown in parentheses are for germination in water.2 Concentration was 10 mM.* Only results so indicated fail to differ from the water controls

at the 95% level of significance.

-4 -3Concentration (Log M)

FIG. 1. Germination of A. albus seeds after 3 days at 30 C, as afunction of concentrations of various salts. Dotted portions ofcurves indicate concentrations causing seedling injury.

8 NH4NO3

6Co NH4CIC

FIG. 2. Germination of B. verna seeds after 6 days at 20 C, asa function of concentration of NH4Cl. Results for seeds in thepresence of 0.01 M NaNO3 or NH4NO3 are indicated. The dottedportion indicates concentrations causing seedling injury.

Plant Physiol. Vol. 54, 1974 305



HENDRICKS AND TAYLORSON

03lv (b)

gO75 A K 30C

H~~ ~ ~ ~~

0

20 25 30 ( )10 1 3.2 10

Temperature Concentration mM

FIG. 3. Germination of P. pratense seeds after 6 days: on H20,

10 mM NH4C1, or 10 mrv KNO3 at various temperatures (a), and at

30 C as a function of concentration for NH4C1 and KNO2 (b).

iOOr

from the several integrations over time and the various con-ditions of measurement.

Catalase and Peroxidase Activities. Seeds of A. albus at 30 Cand L. sativa at 23 C (>600 nm irradiated) are just beginningto germinate after 20 hr imbibition. After 44 hr under theseconditions, germination is >60%, and the first internodes ofthe seedlings are about 1 cm in length. Peroxidase activity in-creases concomitantly (Table IV) with a smaller increase incatalase activity. It is important to note that L. sativa seeds do

Table I1. Disappear-anice of Several Salts from Sollitionisoni Variious Kinids of Seeds in Darkniess

I Amount A. L, S. B P.Salt Concn Amount alb;s sativa gla;ca -er,ta pralenseApp led (20 hr) (20 hr) (72 hr) (72 hr) (72 hr)

| M Pzmoles/ ntomsles/g seedg seed

KNO3' 10.0 40 12; < 12 52 62 1 2NaNO,' 1.0 10 0.3 3.0 .42 52 22NH2OH'*HCI 0.1 2 1.6 1.6 1.5 1.7 1.5NH 4'Cl 10.0 40 14 102 142 332 1KCNi 0.1 8 1.6 2.7i 0.4 0.6 0

IIon measured.2 In the presence of 1.2 ml

mycostatin.streptomycin sulfate and 2 mNI

080

0

-

a. _

en 6

-c -5 -4Concentration (Log M)

-3

FiG. 4. Germination of E. crusgalli and A. albus on NH20H-HC1 and [(C2H5) NOH]2 [COOH]2 solutions of various concen-trations. Dotted portions of curves indicate concentrations causingseedling injury.

mM, where germination was on the order of 15%. They did notpromote germination significantly at 0.1 mM.

Uptake of Salts. Salt disappearance from ambient solutionswith all the seeds tested indicated that values for nitrate andnitrite were of similar magnitude, but much less than those forammonium salts (Table II). A nonmetabolic component of up-take was probably predominant with NH20H - HCI, as evi-denced by reaction with dead seeds. A. albus seeds changedfrom deep purple to red, and B. vulgaris seeds turned red after24 hr in NH20H * HCI solutions > 1 mm at pH 7.

Nitrate and Nitrite Reductase Activities. Both reductaseswere present in all the seed lots tested. Induction of nitratereductase activity was evident in A. albus, L. sativa, and P.pratense seeds, but not in B. verna, B. vulgaris, and S. glaucaseeds (Table III). The activity of nitrite reductase was adequateto use the nitrite formed by nitrate reductase action. Measuredreductase activities were compatible with the values for disap-pearance of the salts from solutions (Table II) on A. albus. L.sativa, and P. pratense seeds. They were low by a factor ofthree for B. verna and S. glauca, which could well have arisen

Table III. Nitr-ate andcS Nitr-ite Redctclase Activities Assayed in Vivofor Several Kinzds of Seeds

Nitrate reductase was measured in I mm KNO3 after the indi-cated hours imbibition in H20 or 10 mm KNO3 by the method ofFerrari and Varner (6). Nitrite reductase activities were taken asthe nmoles NaNO2/g seed hr disappearing from 1 mNi NaNO2,averaged over the indicated imbibition time.

Nitrate Reductase NitriteActivity Reductase

Seed Time Activity

1120 KNO31 NaNO22

h r wlnoles/g seedlkr

A. albuts 21 60 255 25L. sativa 20 51 84 150P. pratenise 44 13 53 603

72 53 303B. vernla 44 93 93 553B. vulgaris 44 163 213 753S. glantca 44 293 273 10,

1 pH 4.5. (Results were not significantly different at pH 7.0.)2 pH 7.0.3 In the presence of 1.2 mm streptomycin sulfate and 2 mM

mycostatin.

Table IV. E,izymic Activities of Catalase anid Peroxidase at 25 Cin E.xtracts of A. albus anid L. sativa Seeds Imbibed in Water

Imbibition Germina- Catalase Activity' Peroxidase Activity,Seed Tempera- tion

ture (44 hr) O hr 20 hr 44 hr o hr 20 hr 44 hr

C p.'inotes H202/g seedl nin

A. albius 30 65 10 20 56 0.4 0.2 6.2L. sativa 23 99 20 23 180 1.6 2.4 3.4L. sativa 30 0 20 9 29 1.6 1.2 2.4

I20 to 28%/a of seed protein was present in the extracts.

306 Plant Physiol. Vol. 54, 1974



PROMOTION OF SEED GERMINATION

not germinate at 30 C. Catalase and peroxidase activities inextracts from these seeds increased 2- to 3-fold between the20th and 44th hr for seeds imbibed in water. The measuredextracted activities were not significantly changed by imbibitionin 1 mM NH3OHCI or 0.1 mim KN3 (results not detailed).

Inhibitor Actions. Catalase activities in lettuce seed extractswere inhibited 50% by 1.7 4tyM KN. and by 10 uM NH2OH HCI.Activities of lyophilized liver catalase in pH 6.8 K-orthophos-phate buffer was inhibited 50% by 0.3 /tM KN3 or by 1.0 IMNH3OHCI. The inhibition was 30% in 1 mm CH3NHOHCI,15% in 1 mM [(C2HJ)2NOH]*[COOH]}, and >10% in 10 mMKNO3.

Peroxidase activities in A. albus seed extracts were inhibited20% by 0.1 mm KN8, 40% by 0.1 mi NH2OH HCl, and by<10% by 0.1 mM NaNO2 and 1 mM KNO.. Horseradishperoxidase activity was inhibited 10% by 0.2 mM KN3 or 0.2mM NH3OHCI.

Glycolate Oxidase and Pyridine Nucleotide Quinone Reduc-tase Activities. Glycolate oxidase activity was not detected inseeds of A. albus and L. sativa after 20 hr imbibition in waterat 23 C or 30 C, respectively. A. albus seeds were just beginningto germinate. The oxidase was present after 3 days in seedlingsexposed to diffuse sunlight for 10 hr each day. Activities, ex-pressed as moles of 2,6-dichlorophenolindophenol reduced/gseed min in the seed extracts were 3 X 10' and 1 X 10-7 molesfor A. albus and L. sativa, respectively. Pyridine nucleotidequinone reductase activities in the seed extracts after 0 or 20hr imbibition were 1300 or 3200 units/g seed-min for both A.albus and L. sativa seeds, with NADH as hydrogen donor and6,400 units/g seed min with NADPH after 20 hr. These valuescorrespond to 55 and 110 units/mg extracted protein-min,respectively. A unit is AA = 0.001 at 340 nm (29). Endogenousrates in the absence of the p-benzoquinone substrate, for whichcorrection was made, were 10 to 20% of these values.Oxygen Consumption. A. albus seeds after 20 hr imbibition

in solutions gave values of 1.47 to 1.57 [d °2/g seed-min with3.17 mm KNO3, 0.1 and 1 mm NH.OHCl and HD0. Values forS. glauca seeds after 20 hr in the respective solutions were 1.58to 1.65 ,ul 02/g seed-min with 10 mm KNO,, 1 mm NaNO2,and H20 and 1.26 td 02/g seed-min with 0.1 mm NH.OHC1.After 44 hr solution imbibition, B. vulgaris seeds consumed4.2 to 4.5 PI 02/g seed-min on 0.1 or 1 mm NH3OHC1 andHDO. This value was reduced by 88% on 10 mm NH,OHCIand by greater than 75% at 1 mm for several seed kinds.Presence of antibiotics did not alter the values, but it shouldbe noted that hydroxylamine forms oximes with both strepto-mycin and mycostatin. Cyanide at 10 mm reduced 02 con-sumption of A. albus seeds after 18 hr imbibition only to80% of the value on water. This is suggestive of some cyanide-insensitive 02 consumption.

DISCUSSION

Germination in darkness of A. albus, S. glauca, P. pratense,and E. crusgalli seed is enhanced in the order NH3OHX >>XNO2> XNO3>> NH4X (where X is the balancing cation oranion) in terms of reciprocal concentrations for equal effec-tiveness (Table I). Disappearance of the salts from the ambientsolutions is in the reverse order (Table II). Failure of NH4Xsalts to promote germination despite high uptake (Tables I andII) indicates that the compounds of higher nitrogen oxidationlevels are not effective by reduction to NHX. The fact thatnitrites are as effective as nitrates, and uptake of both is low,indicates that the process of reduction is not determinative forgermination, other than to produce nitrite.

Measured in vivo activities of nitrate and nitrite reductases(Table III) are adequate, with concentrations of XNO3 and

XNO2 promotive for germination, to produce sufficientNH2OH HCI for the germination effect, provided reductant isadequate and the hydroxylamine is an intermediate. While thecourse of nitrite reduction in plant tissue is not well under-stood, particularly with respect to possible free or bound inter-mediates at the hydroxylamine level of reduction (12), NH4, isgenerally thought to be the end product (1).Ammonium salts inhibited germination of P. pratense seeds,

particularly as the temperature was increased to 30 C (Fig. 3).Inhibition was minor or absent in other cases. An inhibition isan expected possibility because of the considerable uptake ofNH4X and the known actions of ammonium salts as inhibitorsof L-amino acid oxidase and as uncouplers of oxidative phos-phorylation (18). Findings for B. verna seed germination, andB. vulgaris to a lesser extent, contrast sharply with the responseto the nitrogen compounds by the other seeds. Only NH4X ishighly effective for promoting germination in darkness (TableI, Fig. 2). It also is avidly taken up (Table II). These are ex-pected results if ammonium ion serves as a reduced nitrogensource (28) for metabolic processes limiting germination inthese seeds.

Neither NOa- nor NO2- can be acting to any considerableextent as substrates to spare oxygen consumption. This is evi-dent in that the amounts of XNO3 and XNO2 taken up (in theconcentration ranges promoting germination) are on the orderof 1 ,umole/g seed-day, while oxygen consumptions are about100 /tmoles 02/g seed *day.Two extreme classes of germination dependence on nitrogen

are thus evident at (a) the nitrite or hydroxylamine or (b) theammonium oxidation level. A. albus seeds possibly have bothdependencies, while L. sativa seeds are deficient in both re-spects, but respond to azide. Two cautions are raised aboutpossible actions of hydroxylamine in seed germination. Its dis-appearance from solution is in part non-metabolic as shownby reaction with dead seed and seedling injury is evident at con-centrations exceeding 0.3 mm (Fig. 1). Hydroxylamine salts,when effective, maximally promote germination at 0.1 mm orless and inhibit oxygen consumption only at greater concen-trations. The respiration and possible injury effects thus areprobably not significant at 0.1 mm where germination re-ponses are pronounced.

Hydroxylamine is a strong reductant and a strong chelatingagent. It reacts to form oximes with aldehydes and ketones,or nitrogen ethers with aldehydes, when mono-N substituted(32). In a metabolic sense it can be reduced to NH2( andcan probably form an oxime with the formyl group of cyto-chrome a to limit electron flow to oxygen or associated phos-phorylation (18) in respiration. Formation of oximes, fol-lowed by reduction, has sometimes been considered in aminoacid formation (34, 38). Hydroxylamine is an acceptor withlow Km value for the glutamyl radical in transamination re-actions (7).

In considering the possibility of oxime involvement in thegermination effects, tests were made with a number of sub-stituted hydroxylamines and hydrazines. Results are reportedonly for NH2OH HCl, CH3NH,OHX, and (C2H5)2 NHOHX(Figs. 1 and 4). If the cause for promotive action of thesethree types of compounds is similar, it cannot be by oximeformation or reduction to the ammonium ion, which cannotdirectly occur for R2NHOHX. The role of oximes in nitrogenmetabolism has recently been reviewed by Mahadevan (21).In the light of his review, we tested for HCN formationthrough reactions 11 and 12 of Mahadevan's Figure 2, aspossible evidence of involvement of oximes. Cyanide was notdetected by the exceedingly sensitive pyridine-pyrazolonereagent (30) (data not reported) when A. albus were imbibedin NHI3OHCI solutions.

307Plant Physiol. Vol. 54, 1974



HENDRICKS AND TAYLORSON

The marked chelating capacities of hydroxylamine for the

iron atoms of haemproteins (19) and the definite but lower

capacities for N-aliphatic substituted hydroxylamines indicate

the presence of this type of action in seeds. The promotion of

germination at concentrations that do not significantly affect

respiration, however, show that the haemprotein involved is

not a cytochrome component of the respiratory chain. The

two other prominent haemproteins in plant tissues are cata-

lase and peroxidase. Results in Table IV show these enzy-

matic activities at various germination stages in A. albus and

L. sativa seeds and in nongerminating L. sativa seeds. The de-

grees of inhibition of catalase and peroxidase by hydroxyl-

amines and azide in vitro indicate that the primary inhibitory

action can be on catalase in the seeds.

Keilen and Hartree (15, 16) found that catalase, in the

absence of H202, chelates with azides. In the presence of

H202, the azide in the chelated compound is oxidized to nitric

oxide, and the iron is reduced to the ferrous form. The result-

ing NO-ferrocatalase has a low dissociation constant (<10-6

M) and has lost its catalytic effectiveness for decomposition

of H202. A model system is formation of nitrosylferricyanide

(nitroprusside) and other nitrosyl complex salts of transition

metals by the classical hydroxylamine method (14). Hy-

droxylamines, nitrites, and other nitrogenous compounds

yielding NO under strong oxidation have actions similar to

those of aZides. These are the striking promoters for germina-

tion of some kinds of seeds as found in this work. N-Diethyl-

hydroxylamine is neither oxidized readily to NO, nor does it

chelate avidly with the iron in catalase, as shown by the high

concentrations necessary for inhibition of catalase action.

Higher concentrations are required than for N-methylhy-

droxylamine and hydroxylamine for equivalent enhancement

of germination (Fig. 4). Nitric oxide gas, at 1 mm if fully

absorbed as such, completely inhibited catalase, as also did

XNO2 (data not presented). Peroxidase, in contrast to cata-

lase, probably does not oxidize the several compounds to

nitric oxide. We find that horseradish peroxidase is less in-

hibited by KN5 and NH2OH* HCl than is catalase.

Action of nitrate on seed germination thus is ascribed to

inhibition of catalase either by nitrite or hydroxylamine

formed upon reduction, or by nitric oxide. The central ques-

tion accordingly is, How does inhibition of catalase serve to

promote seed germination? A suggested answer is that in-

hibition of catalase spares some H202 for peroxidase action.

We now consider a probable way in which peroxidase action

might promote seed germination.

Roberts (24-26) and his associates have accumulated evi-

dence for control of barley and rice seed germination by a

shift from the Embden-Meyerhof-Parnas pathway of glucose

utilization to the pentose phosphate pathway. Breaking of

dormancy is associated with decrease in the C6/Cl ratio of

CO, evolved from exogenous D-glucose, indicative of the

change in metabolic pathway. The pentose phosphate path-

way utilizes NADP+ as an oxidant, which can be limited by

the rate of reoxidation of NADPH. We suggest that nitrate

and the several other compounds considered here could have

a regulatory function in this reoxidation.

Pyridine nucleotide quinone reductase, which has been

concentrated from imbibed nongerminating pea seeds (37),

might be the coupling enzyme for NADPH oxidation. We

find it in extracts of A. albus and L. sativa seeds at the same

order of activity as found in extracts of pea seeds. It has a

somewhat lower Kin(0.8 ltM) for NADPH than for

NADH(2. 1,uM) and is not particularly specific for the

quinone substrate (37). Catalase, peroxidase, and pyridine

nucleotide quinone reductase are present before imbibition in

both A. albus and L. sativa seeds, as well as in seeds of L.sativa held at 30 C (Table IV), which prevents germination.The values of Km for the several catalyzed reactions are low(on the order of scM), and the values of k, and k, in the dif-ferent steps are probably adequate to maintain reasonablereaction rates with endogenous substrates.An NADPH-oxidizing enzyme system was observed in

water extracts of wheat germ by Conn et al. (3) in 1952. Theyestablished that peroxidase was involved and that the oxidationwas inhibited by catalase, apparently through H202 destruc-tion. They realized that the oxidizing system involved en-zymes other than peroxidase and catalase.

Production of H202 in the germinating seeds probably in-volves a flavin-sensitized nonrespiratory chain reduction ofoxygen. Two sources to be considered for H202 generation arein actions of enzymes of peroxisomes and glyoxysomes. Gly-colate oxidase action would be likely if peroxisomes were in-volved, but these are predominantly associated with chlorophyl-lous tissue and develop chiefly upon greening of seedlings (33).Action of the glyoxysomal enzymes is the most likely sourceof the H202. The early utilization of fat in germination ofseeds having high oil contents, with conversion of fat to car-bohydrate, was noted by Murlin in 1933 (22). Grbhne in1952 (9) reported formation of starch from fat in Digitalispuirputrea L. seeds within 3 hr of initiation of germination bylight. The discovery of the glyoxysomes (2) and elaboration ofknowledge about enzymes of the glyoxylate cycle, as well asknowledge of fat utilization in seeds (13), established the basisfor understanding the fat to carbohydrate conversion. It islikely that fat degradation is an early step in the germinationof many kinds of seeds, other than those having high fatcontents.

LITERATURE CITED

1. BEES ERS, L. AND R. H. HAGENIAN. 1969. Nitrate reduction in higher plants.Annu. Rev. Plant Physiol. 20: 495-522.

2. BREIDENBACII,R. W., A. KAHN, AN-D H. BEEVERS. 1968. Characterization oflglyoxysomes from castor bean endosperm. Plant Physiol. 43: 705-713.

3. CONN, E. E., L. 'M. KRAEMER, P. N. Liu, AND B. VENNESLAND. 1952. Theaerobic oxicdation of reduced triphosphopyridineinucleotide by a wbeat germenzymne system. J. Biol. Chem. 194: 143-151.

4. CONWNAY, E. J. 1963.AMicrodiffusion Analyses and Volumetric Error. ChemicalPublislhers Co., New York. pp. 9-13.

5. CZAKY, T. Z. 1948. On the estimation of bound hydroxylamine in biologicalmaterial. Acta Cheiin. Scanid. 2: 450-454.

6. FERRARI, T. E. AND J. E. VARNER. 1970. Control of nitrate reductase ac-tivity inbaraley aleurone layers. Proc. Nat. Acad. Sci. U.S.A. 65: 720-736.

7. FOWADEN, L. 1967. Aspects of amino acid metabolism in plants. Annu. Rev.Plant Pllysiol. 18: 85-106.

8. GASSNER, G. 1915. Uber die keirnungsausl6sende Wirkung der Stickstoffsalzeauflichltempfindliche Samesi. Jahrb. Wissen. Bot. 55: 259-342.

9. GR1SHNE, U. 1952. Untersuclitibugen ztur Frage der Liclitkeinung von Digitalispuirpurea L. Biol. Zentralbl. 71:1-42.

10. HERBERT, D. 1955. Catalase fromn bacteria. Methods Enzymol. 2: 785.11. HESSE, 0. 1924. Uslitersucliungentuber dieEinwirkung cliemische Stoffe atif

die Keimung lieltempfindlieler Samen. Bot. Arch. 5:133-171.12. HULCKLESBY, D. P. AND E. J. HEWITr. 1970. Nitrite and hydroxylamiaine

reduction inhigher plants. Biocbem. J. 119:615-627.13. HUTTON, D. AND N. K. STUNIPF. 1969. Fat metabolism in higher plants.

XXXVI. Characterization ofthe/3-oxidation systems from maturing andgerminating castor bean seeds. Plant Physiol. 44: 508-516.

14. JOHNSON-, B. F. G. AND J. A. MCCLEVERTY. 1966. Nitric oxide conmpound(Is oftransition metals. Prog. Inorg. Chem. 7: 276-359.

15. KEILEN, D. AND E. F. HARTREE. 1936. On some properties of catalasehaematin. Proc. Roy. Soc. (London) B. 121:173-191.

16. KEILEN, D. AND E. F. HARTREE. 1954. Reactions of methaemoglobin an(dcatalase with peroxides and hydrogen donors. Nature 407: 720-723.

17. KESSLER, E. 1964. Nitrate assimilation by plants. Annu. Rev. Plant Physiol.15: 57-72.

18. LEHNINGER, A. L. 1964. The Mlitochondrion. W. A. Benjamin, New York. p.124.

19. LE'MBERG, R. AND J. W. LEGGE. 1949. Hematin Compounds and Bile Pigmients.Interscience Publishers. New York. 409-410.

308 Plant Physiol. Vol. 54, 1974



PROMOTION OF SE]

20. LoWRY, 0. H., N. J. RoSEBROUGH, A. L. FARR, AND R. J. RANDALL. 1951.Protein measurement with the Folin reagent. J. Biol. Chem. 193: 265-275.

21. MAHADEVAN, S. 1973. Role of oximes in nitrogen metabolism in plants. Annu.Rev. Plant Physiol. 24: 69-88.

22. MURLIN, J. C. 1933. The conversion of fat to carbohydrate in the germinatingcastor bean. I. The respiratory mechanism. J. Gen. Physiol. 17: 283-302.

23. NELSON, E. B. AND N. E. TOLBERT. 1970. Glycolate dehydrogenase in greenalgae. Arch. Biochem. Biophys. 141: 102-110.

24. ROBERTS, E. H. 1963. The effect of inorganic ions on dormancy in rice seed.Physiol. Plant. 16: 732-755.

25. ROBERTS, E. H. 1969. Seed dormancy and oxidation processes. Symp. Soc.Expt. Biol. 33: 161-192.

26. ROBERTS, E. H. 1973. Oxidative processes and the control of seed germination.In: W. Heydecker, ed., Seed Ecology. Pennsylvania State Ulniversity Press,University Park. pp. 189-218.

27. SCHWENDINIAN, A. AND H. L. SHAN'DS. 1943. Delayed germination on seeddormancy in Vicland oats. J. Amer. Soc. Agron. 35: 681-688.

28. SHOZo, T. 1929. The action of nitrates and ammonium salts on some plants. II.The action of nitrates and ammoniumn salts on germination. Bot. Mag.(Tokyo) 43: 295-305.

ED GERMINATION 309

29. Standard Methods for the Examination of Water and Waste Water, Ed. 13.1971. American Public Health Association, New York. pp. 240-243.

30. Standard Methods for the Examination of Water and Waste Water, Ed. 13.1971. American Public Health Association, New York. pp. 404-406.

31. STEINBAIER, G. P. AND B. GRISBY. 1957. Interaction of temperature, light,and moistening agent in germination of weed seeds. Weeds 5: 175-182.

32. TAYLOR, T. W. J. AND W. BAKER. 1937. Sidgwich's Organic Chemistry ofNitrogen. Oxford University Press, New York. p. 166-169.

33. TOLBERT, N. E. 1971. Microbodies-peroxisomes and glyoxysomes. Annu.Rev. Plant Physiol. 22: 45-74.

34. VIRTANE-N, A. I. 1947. The biology and chemistry of nitrogen fixation bylegume bacteria. Biol. Res. 22: 239-269.

35. WOOLEY, J. T., G. P. HICKS, AND R. H. HAGEMAN. 1960. Rapid determina-tion of nitrate and nitrate in plant material. Agric. Food Chem. 8: 481-482.

36. WORTHINGTON, 1972. Enzymes, Enzyme Reagents, Related Biochemicals.Worthington Biochemical Corp., Freehold, N. J. pp. 43-44.

37. WASILAIT, W. D. AND A. NASON. 1955. Pyridine nucleotide quinone reductasefrom pea seeds. Methods Enzymol. 2: 725-729.

38. YAMAFrGI, K., M. SHIMAMURA, AND H. TAKABASHI. 1954. Oximase andtransoximase in green algae. Enzymologia 17: 110-112.

Plant Physiol. Vol. 54, 1974



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Promotion of Seed Germination by Nitrate, Nitrite, Hydroxylamine