The effect of dormancy release of seeds of Strelitzia juncea by oxygen on their alcohol dehydrogenase activity, cytosolic malate dehydrogenase activity and ethanol content

342

S.-Afr.Tydskr. Plantk., 1990, 56(3): 342-349

The effect of dormancy release of seeds of Strelitzia juncea by oxygen on their alcohol dehydrogenase activity, cytosolic malate dehydrogenase activity and ethanol content S. de Meillon*, H.A. van de Venter and J.G.C. Smal1 1 Margaretha Mes Institute for Seed Research, University of Pretoria, Pretoria, 0002 Republic of South Africa and 1Department of Botany, University of the Orange Free State, Bloemfontein, 9300 Republic of South Africa

Accepted 26 March 1990 The fact that the dormancy of certain types of seeds can be alleviated by incubation in oxygen, had previously been associated with either the oxidation of germination inhibitors or the stimulation of oxygen-dependent respiratory processes. In this respiratory study on Strelitzia juncea Ait. seed dormancy, it was found that ethanolic fermentation of the embryos of dormant normoxically incubated seeds resembled that of hyperoxically incubated seeds. ADH activity was similar over the first four incubation days, and therefore before the beginning of radicle emergence. Ethanol content of the embryos was significantly lower on incubation days 1 and 3. MDH activity was higher on days 3 and 4 as a result of hyperoxic treatment. A close relationship between supernatant ADH and MDH activity was found for individual extracts, and its possible significance is discussed. The overall 4-day average ADH activity (NADH oxidation) was 0.17 ILmol min-1 emb- 1 (4.5 ILmol min- 1 g-1 fresh mass), while the average MDH activity (NADH oxidation) of the embryos was 0.52 ILmol min- 1 emb- 1 (55.3 ILmol min- 1 g-1 fresh mass). The ratio of the ADH forward reaction (NAD reduction) to ADH reverse reaction (NADH oxidation) was 8.30 ± 0.37. The ethanol content of embryos of normoxic incubated seeds was 6.0 ILmol emb- 1 (59.5 ILmol g-1 fresh mass) and of hyperoxic incubated seeds 4.8 ILmol emb- 1 (47.7 ILmol g-1 fresh mass). Die feit dat saaddormansie in sekere gevalle deur suurstofinkubering opgehef kan word, is tot dusver met die oksidering van kiemingsremstowwe of die stimulering van suurstofafhanklike respiratoriese prosesse in verband gebring. In hierdie respiratoriese studie oor die saaddormansie van Strelitzia juncea Ait., is vasgestel dat etanolfermentasie van die embrio's van dormante normoksies-ge',nkubeerde sade soortgelyk aan die van hiperoksies-geYnkubeerde sade was. Die ADH-aktiwiteit was soortgelyk oor die eerste vier inkuberingsdae, dit wil S8 voor die aanvang van radikulaverskyning. Die etanolinhoud van die embrio's was betekenisvol laer op inkuberingsdae 1 en 3, terwyl hiperoksiese inkubering 'n hoer MDH-aktiwiteit op inkuberingsdae 3 en 4 tot gevolg gehad het. 'n Goeie verwantskap tussen bovloeistof ADH- en MDH-aktiwiteit is vir individuele ekstrakte gevind en die moontlike implikasies daarvan word bespreek. Die gemiddelde ADH-aktiwiteit (NADH-oksidasie) oor die geheel geneem, was 0.17 ILmol min- 1 emb- 1 (4.5 ILmol min- 1 g-1 varsmassa), terwyl die gemiddelde MDH-aktiwiteit (NADH-oksidasie) van die embrio's 0.52 ILmol min- 1 emb- 1 (55.3 ILmol min- 1 g-1 varsmassa) was. Die verhouding van die voorwaartse reaksie (NAD-reduksie) tot die terugwaartse reaksie (NADH-oksidasie) was 8.30 ± 0.37. Die etanol-inhoud van die embrio's van normoksies-geYnkubeerde sade was 6.0 ILmol emb- 1 (59.5 ILmol g-1 varsmassa) en vir hiperoksies-ge',nkubeerde sade 4.8 ILmol emb-1 (47.7 ILmol g-1 varsmassa). Keywords: Alcohol dehydrogenase, dormancy, ethanol content, malate dehydrogenase, Strelitzia seed *To whom correspondence should be addressed

Abreviations used ADH: Alcohol dehydrogenase (alcohol: NAD oxidoreductase EC.lolo1.l) ; fr26P 2 : fructose 2,6-bisphosphate; G6PDH: Glucose-6-phosphate dehydrogenase (D-glucose-6phosphate: NADP 1-oxidoreductase, EC.1.1.1.49); LDH: lactate dehydrogenase (L-Iactate: NAD oxidoreductase , EC.1.1.1.28); MDHsup: supernatant malate dehydrogenase (L-malate: NAD oxidoreductase EC.1.1.1.37); NAD: B-nicotinamideadenine dinucleotide NADH: disodium B-nicotinamide adenine dinucleotide (reduced); PCA-EDTA: 3 mol dm-3 perchloric acid - 1 mmol dm- 3 disodium ethylenediamine tetra-acetic acid; PFK: phosphofructokinase (ATP: D-fructose-6-phosphate phosphotransferase, EC.2 .7. 1.11) ; PFP: inorganic pyrophosphate: D-fructose-6-phosphate: 1-

phosphotransferase, EC.2.7.1.90); 6PGDH: 6-phosphogluconate dehydrogenase [6-phospho-Dgluconate: NADP 2-oxidoreduetase (decarboxylating)], EC.1.1.l.44; Tris: tris (hydroxymethyl) aminomethane; DTT: DLdithiothreitol.

Introduction The external barriers to oxygen diffusion and dense internal structure of seeds probably gives rise to a hypoxic atmosphere in many seeds . The earliest stages of germination has been cited as the most widespread type of natural anaerobiosis among plants, 'and is taken by the seeds in their stride', although 'longer deprivation may stop germination altogether' (James 1953; p 139). Spragg & Yemm (1959) also concluded that partial anaerobiosis is an important feature during the first

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S.Afr.l. Bot., 1990,56(3)

phase of germination in air. More recently it was reported that the muskmelon seed coat exerts an inhibiting influence on germination by promoting anaerobiosis (Pesis & Ng 1986). In contrast, Raymond et ai. (1985) concluded from their results with 12 fatty and starchy types of seeds that natural anaerobiosis is not a general phenomenon in imbibing and germinating seeds. Quiescent or nondormant seeds differ in their ability to germinate under external hypoxic conditions (Morinaga 1926; Rumpho & Kennedy 1981; AI-ani et ai. 1985; Small et al. 1989; Rabie et ai. 1989). Consequential to the internal anaerobic conditions of some types of seeds, ADH activity was shown to peak during the germination process (Leblova 1978a), and a maximum ethanol concentration was characteristically obtained between 30 and 60 hours of swelling for seeds of agricultural crops (Leblova 1978b). ADH activity of quiescent pea seeds declined upon rupture of the seed coat and ADH activity declined upon transfer of the seeds from anaerobic to aerobic conditions (Kolloffel 1968). The eventual decline in ADH activity may possibly be related to the formation of an ADH inhibitor under aerobic conditions (Shimumura & Beevers 1983), although the in vivo interaction between ADH and the inhibitor is unclear. With regard to the dormant condition of seeds, little information is available as to whether the dormancyrelieving effect which hyperoxia has in some cases may possibly be attributed to its effect on anaerobic/aerobic respiration. The main approach to ascertain whether the dormant condition results from restrictive oxygen supply, was to determine oxygen diffusion coefficients for seed-covering structures and comparing it with oxygen requirements of the embryo (Edwards 1969; Porter & Wareing 1974). Although the constants for the mathematical models used were criticized (CollisGeorge & Mellville 1974), the fact remains that substances such as GA3 may alleviate dormancy, thereby implying that oxygen diffusion through the seedcovering structures was sufficient for germination (Edwards 1972). According to Roberts (1969) there is little evidence for the view that the covering structures of dormant seed impose a limitation on oxygen uptake, and that germination is prevented by a restriction of respiration. Yet, many hypotheses on seed dormancy were based on the restricted availability of oxygen as discussed by Roberts (1969). From the above and other literature, Bewley & Black (1985) concluded that for seeds with coat-imposed dormancy, hyperoxic conditions probably cause the oxidation of endogenous germination inhibitors, thereby allowing germination to occur. However, information on the effect which hyperoxic treatment of dormant seeds has on enzyme activity and products of anaerobic metabolism is difficult to find in literature surveys (Amen 1968; Bewley & Black 1982, 1985; Khan 1977; Mayer & Poljakoff-Mayber 1982). Should the dormancy-alleviating effect of hyperoxia primarily be related to the abolishment of an internal hypoxic atmosphere, oxygen treatment could be expected to have an effect on ethanol content and ADH activity, as reported for

quiescent seeds. This paper reports on the effect of hyperoxic treatment on ethanol content and ADH activity of Strelitzia juncea seeds of which dormancy can be alleviated by incubation in a 100% oxygen atmosphere (van de Venter 1978; Ybema et al. 1984). The work forms part of a comprehensive study of the respiratory metabolism of S. juncea seeds (De Meillon 1985) in which the activity of various respiratory enzymes was determined. Pentose phosphate pathway activity was also studied (De Meillon et ai. 1990a) with regard to Roberts' (1969) hypothesis on dormancy alleviation, and in this context it is also of importance to assess fermentative activity. The work of Ybema (1983) indicated that the incubation of S. juncea seeds in oxygen caused a drop in average RQ values from 2 .3 to 1.5 over the first 4 days when compared to air-incubated seeds. The drop in RQ values may reflect a reduction of anaerobic conditions upon oxygen incubation . In conclusion, many of the extracts used for determining ADH activity, were also used for MDH determinations, and a close relationship between the two enzyme activities for individual extracts was noticed, as reported here. Materials and Methods Incubation of seeds Strelitzia juncea seeds were obtained from plants growing in the Uitenhage district in the Cape Province. The arils were removed and the seeds kept in closed, opaque glass jars at 5°C until used. All experiments were conducted within a year of harvest. Seeds were surface sterilized as described previously (van de Venter & Small 1974). They were then aseptically transferred to sterile 500-cm 3 conical flasks containing 20 cm 3 water. For oxygen incubation, the flasks were fitted with vaccine caps and purged with medical oxygen for 10 min. Control flasks were plugged with non-absorbent cottonwool. The flasks were kept in the dark in a germination cabinet at a constant temperature of 25 ± 0.5°C. Seeds were regarded as germinated at the first visible sign of radicle protrusion. After 1, 2, 3 and 4 days of incubation in air or oxygen, flasks were removed for extraction of the seed. Each replicate consisted of 25 seeds per flask. Extractions for enzyme activity as well as for ethanol determination were replicated at least three times. Enzyme extraction

For ADH and MDHsup extraction, the seeds from a single flask were rinsed with distilled water and blotted dry before excising the embryos. The embryos were ground in a chilled mortar for 1 min with ice-cold buffer solution (the composition of the buffer solutions used is supplied in the section on results). In some experiments, DTT (5 mmol dm- 3 final concentration) was included in the extraction medium. The extract was made up to 40 cm 3 with buffer and centrifuged at 23 000 g for 20 min at 2°C. Using a syringe fitted with a 0.8 x 90-mm needle an aliquot of the supernatant was removed without

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S.-Afr.Tydskr. Plantk. , 1990,56(3)

disturbing the top fatty layer. This aliquot was used for enzyme assays. The time taken from the removal of the first embryo to the first enzyme assay was 60 min.

ADH assay Assay conditions of Leblova (1978a), Grondal et al. (1983) and Shimomura & Beevers (1983) were initially investigated but the following assay medium was found suitable and gave linear response of enzyme activity with extract concentration. For the forward reaction (ethanol production from acetaldehyde), the 3 cm 3 reaction mixture consisted of 0.1 mol dm- 3 potassium phosphate buffer pH 6.9, 0.27 mmol dm-3 NADH and 7 mmol dm- 3 acetaldehyde . The reaction was initiated by addition of acetaldehyde. For the reverse reaction (acetaldehyde production from ethanol), the 3-cm 3 reaction mixture consisted of 0.1 mol dm-3 potassium phosphate buffer pH 8.9 , 1 mmol dm- 3 NAD, and 100 mmol dm- 3 ethanol. The reaction was initiated by addition of ethanol. MDHsup assay The assay of Rustin et al. (1980) for mitochondrial MDH was found to be optimal for the MDHsup determination. The 3-cm 3 reaction mixture consisted of 0.1 mol dm- 3 potassium phosphate buffer pH 7.'5, 0.2 mmol dm- 3 NADH and 0.5 mmol dm- 3 oxaloacetate. The reaction was initiated by adding oxaloacetate . Dilutions of the crude extract were made with extraction buffer. For both ADH and MDHsup it was found that the activity increased linearly with enzyme concentration. In all cases formation or oxidation of NADH was monitored at 340 nm in a Cary 219 spectrophotometer at 25 ± 0.2°C. Enzyme activity was calculated by C x cot( Y) x E x G x I E"

fLmol min-l emb- l

x F x J

where J amount (or mass) of embryos was extracted in I cm 3 medium, and an aliquot F cm 3 diluted G times was used in a total assay volume of E cm 3 , and E" is the molar extinction coefficient for NADH. C x cot(Y) expresses the gradient of the graph in A minot as explained (De Meillon 1990). Ethanol extraction and determination Ethanol extraction was based on the method of Lowry & Passonneau (1972) for metabolite extraction as described (De Meillon et al. 1990b). Ethanol was determined enzymatically with iII 2 weeks after extraction, using the standard Boehringer kit (cat. no. 176290) by following NADH formation at 340 nm. Results and Discussion The effect of oxygen incubation on germination

Strelitzia juncea seeds are released from dormancy by incubation of the seeds in an atmosphere of 100% oxygen at 25°C with germination commencing on the fifth day of incubation (De Meillon et al. 1990a). Similar results were reported for Strelitzia reginae (van de

Venter & Small 1974) and S. juncea (Ybema et al. 1984) . The data subsequently reported in this paper were collected on days 1, 2, 3 and 4 of incubation and therefore reflect enzyme activity and ethanol content prior to the commencement of radicle emergence.

ADH activity For most of the determinations , 0.1 mol dm- 3 Tris-HCl containing 0.4 mol dm- 3 sucrose was used as extractant (Table 1). Results similar to those found with the above extractant were also obtained by using 0.01 mol dm- 3 Tris-HCI pH 7.4 (Shimomura & Beevers 1983) and 0.1 mol dm- 3 potassium phosphate buffer pH 8.9 (Leblova 1978a) as extraction medium. Therefore, no chloride inhibition of ADH (Leblova 1978a) was observed by using Tris-HCI. As in vitro ADH activity may be impaired by the presence of an ADH inhibitor in the extracts (Shimumura & Beevers 1983) , embryos were also extracted with Tris-sucrose medium containing 5 mmol dm- 3 DTT (Table 1) and activity was determined either keeping extracts for 12 h at 4°C or 5 days at 12°C (Table 1). The rapid loss in ADH activity of extracts not containing DTI may be indicative of the presence of a DTTantagonizable ADH inhibitor. However, the presence/ absence of DTT does not differentiate between embryo ADH activity of day 1 air- or oxygen-incubated seeds . The average ADH activity of Strelitzia juncea embryos over the 4-day period of seed incubation , was 0.17 fLmol N~DJ-:I min-] embryo-l (4.5 fLmol min-' g-' fresh mass) whIch IS much lower than the 7 fLmol min-l seed-! activity found for Erythrina caffra under normoxic and anoxic conditions, though similar values (7.1 fLmol min-l g-l dry mass) were found for Cucumis sativus seeds during incubation (Rabie et al. 1989) . Based on in vitro ADH activity measurements alone (Table 1), it may provisionally be concluded that, relative to hyperoxic treated seeds, the internal atmosphere of air-incubated seeds is not anaerobic to such an extent that ADH is induced with concomitant ethanol fermentation over the first 4 incubation days. Recent findings on ADH activity of Cucumis sativus (Rabie et al. 1989) and Erythrina caffra (Small et al. 1989) also showed that the activity of extractable ADH from seeds was similar upon incubation under aerobic or anaerobic conditions . Flooding of maize seeds on the other hand , caused increased ADH activity , and new ADH isoenzymes were synthesized upon prolonged flooding (VanToai et al. 1985). The embryo ADH activity of imbibing soybean seeds peaked, while for Populus delta ides seeds activity remained high during the whole incubation period (Kimmerer 1987). In barley seeds and young seedlings, ADH activity was induced by hypoxia (Good & Crosby 1989) . The ratio of the forward (NADH oxidation) to the reverse ADH reaction (NADH formation) was 8.30 ± 0.37 for both air- and oxygen-incubated seeds and the ratio remained constant with time of incubation. This constant ratio is not indicative of an increased ADH capability of embryo extracts to oxidize ethanol as a

S.Afr.l. Bot., 1990, 56(3)

345

Table 1 Effect of various extractants on ADH activity of embryos of Strelitzia juncea seeds incubated in air or oxygen AOH activity (fJ.mol NAOH formed min " 50 emb") with extractant Incubation period (days)

Seed treatment

o (dry seed)

Tris" 7.95

±

0.17"

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±

8.95

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Air

1.95 Oxygen

9.90 1.95

2

3

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8.93

8.22

9.19

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0.82

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0.23

8.27

Air

8.26

±

0.61

8.56

Oxygen

4

8.18

Air

8.73

Oxygen

8.18

± ±

Tris-suc

+

OTT c

0.17

Pid 6.87

9.95 8.95 8.90 7.95

±

0.49

± 0.59' ± 0.65" ± 0.73' ± 0.67"

0.70 0.71

± 0.76 ± 0.72 ± 0.54 ± 0.51

"0.01 mol dm,3 Tris-HCI pH 7.5 "0.10 mol dm ,3 Tris-HCI pH 7.4 containing 0.4 mol dm ,3 sucrose cmedium b containing 5.0 mmol dm '" OTT

dO.lO mol dm ,3 potassium phosphate buffer pH 8.5 containing 0.4 mol dm ,3 sucrose "average ± standard deviation (n = 3) 'activity determined after extract was kept at 4°C for 12 h "activity determined after extract was ke pt at 12°C for 5 days

result of hyperoxic seed treatment. From the literature review by Davies (1980) it may be concluded that plant tissues do not contain ADH isoenzymes primarily effective for ethanol ~ acetaldehyde conversion. Ethanol metabolization in seeds as reviewed by Cossins (1978) may therefore possibly be effected through favorable mass action ratios. Kinetic data obtained for ADH of crude barley root extracts were interpreted as evidence for adaptation to short-term oxygen deficiency (Fagerstedt & Crawford 1986). However, their ADH kinetic data obtained for the recovery period (anoxia ~ normoxia) are not indicative of an adaptation to metabolize ethanol, although ADH activity of the seminal roots decreased to control levels. The ADH profile of Strelitzia juncea and S. reginae (Figure 1) differs from that obtained for G6PDH and 6PGDH (De Meillon et al. 1990a) and fr26P r stimulated PFP (De Meillon et al. 1990b) for which oxygen incubation caused a definite increase in enzyme activity during the pre-germination period. Aldolase and PK activity , like ADH, was similar for both air and oxygen incubation.

tends to be lower , no large decrease in ethanol content over the incubation period is obvious for either air- or oxygen-treated seeds, and the data is not indicative of a marked difference in ethanolic fermentation between Strelitzia juncea seeds incubated in air or oxygen.

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Ethanol content The ethanol content of embryos from oxygen-treated seeds was lower than that of embryos from air-incubated seeds on days 1 and 3 of incubation (Figure 2). Although the ADH profile (Figure 1) does not indicate any difference in ethanol fermentation resulting from oxygen incubation, the lower ethanol content after one incubation day may indicate a change in metabolic flux. Although the ethanol content for oxygen incubation

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346

S.-Afr.Tydskr. Plantk., 1990,56(3)

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Figure 2 Ethanol content of embryos from Stre/itzia juncea seed incubated in air and oxygen. A, 8: Ethanol content per 50 embryos (use left hand ordinate). C, D: Values for A, 8 recalculated as per gram fresh mass (use right hand ordinate).

Figure 3 MDH slIp activity of embryos from Strelitzia juncea seed incubated in air and oxygen. A, 8: MDH slIp activity per 50 embryos (use left hand ordinate). C, D: Values for A, 8 recalculated as per gram fresh mass (use right hand ordinate).

Starchy as well as fatty seeds had high ethanol production under anoxia (Raymond el al. 1985) as did seeds of Erythrina caffra (Small el al. 1989). For Cucumis sativus a large increase in ethanol content of seeds and surrounding medium was found under hypoxic conditions (Rabie el al. 1989). One or more of the following factors may be responsible for the ethanol content of plant tissues: fermentation rate, conversion of ethanol to other compounds, and/or excretion to the surrounding medium (see Cossins ] 978; Davies 1980). No conclusion as to which of these factors may have led to the lower ethanol content in the embryos of oxygen-treated Slrelitzia juncea seeds could be drawn from the available data. No obvious relationship exists between ADH activity (Figure 1) and ethanol content (Figure 2). This is in agreement with recent findings for Cucumis sativus seeds (Rabie et al. 1989) , Erythrina caffra seeds (Small et al. 1989) and Urtica roots (Smith et al. 1986). For maize root tips subjected to anoxia the flux to ethanol was unrelated to changes in normal levels of ADH activity according to Roberts el al. 1989. Possible explanations offered for this lack of correlation are (loc. cit.) that the flux through a metabolic pathway is controlled by several enzymes (Westerhoff el al. 1984) and that the mechanisms for hypoxic ADH induction may act independently of existing ADH levels. The production of fermentation products other than ethanol cannot be ruled out, although Raymond et al. (1985) found ethanol to be the main fermentative product by far of the types of seeds investigated. Good & Crosby (1989) concluded that LDH does not appear to be important in anaerobic glycolysis in seeds. The average ethanol content of embryos over the 4-day period was 0.60 f-Lmol embryo-I (59.5 f-Lmol g-I fresh mass) for air-incubated seeds and 0.48 f-Lmol embryo-I

(47.7 f-Lmol g-I fresh mass) for oxygen-incubated seeds. These values for the starchy Strelitzia juncea seeds are much lower than that reported for the five starchy seeds (rice, maize, sorghum, pea , wheat) after 48 hours of anoxia as calculated from the data of Raymond et al . (1985), but is far in excess when compared with nine of the 12 types of seeds held under aerobic conditions. Scarified Erythrina caffra seeds had 27 and 60 f-Lmol ethanol seed-I under normoxic and anoxic conditions respectively (Small et al. 1989). For soybean seeds a value corresponding to 37 f-Lmol g-I fresh mass was found (Leblova 1978b). Based on ethanol content it would seem that incubated Strelitzia juncea seeds have a fermentation rate higher than would be expected for seeds such as maize, soybean and pea (Raymond et ai. 1985). It should be kept in mind that ethanol may be excreted to high values into the surrounding medium (Rabie et al. 1989) . MDH activity Malate dehydrogenase was extracted with Tris-sucrose medium and it is assumed that most of the activity recovered in the supernatant is that of the cytosolic MDH enzyme, although organelle breakage may contribute to the observed supernatant activity especially after short periods of seed incubation . The profile for MDH activity obtained with the 18 OOO-g supernatant (Figure 3), is similar to the MDH activity of the 39 OOO-g supernatant obtained during isolation of mitochondria (unpublished data). The drop in supernatant MDH activity from day 1 to day 4 is far greater than the increase in MDH activity of the mitochondrial fractions. The supernatant MDH represents between 95% (day 1) and 80% (day 4) of the total MDH activity (unpublished data). The in vitro MDHsup activity (as malate formation)

347

S.Afr.l. Bot., 1990,56(3)

was 15 to 30 times lower than the ADH activity (ethanol formation), but higher than ADH activity expressed as ethanol oxidation (Figure 1). From 0 to 3 days of incubation, a decrease in MDHsup activity occurred which was more prominent when expressed on a fresh mass basis. From day 3 to 4 the increase in MDH slIp activity was more distinct in the case of embryos of oxygen-incubated seeds (Figure 3) . Perl (1978) reported three phases in the MDH activity of Gossypium hirsutum seeds during incubation, namely a decrease, a period of constant activity, and finally, an increase in MDH activity which coincided with the emergence of the radicle. Possibly the rise in MDH activity of oxygen-incubated Strelitzia juncea seeds on day 4 is related to imminent radicle emergence (commencing on day 5). A good linear relationship was found between the in vitro MDH (malate formation) and ADH activities (NADH formation) of individual S. juncea embryo extracts (Figure 4), the significance of which can only be speculated upon. A possible connection between ADH and MDH activity may be inferred from the literature review of Morohashi (1978) . He concluded for Phaseolus mungo seeds that mitochondrial activity develops relatively slowly during imbibition , while glycolytic activity is high from the start of imbibition. The imbalance between glycolytic and mitochondrial activity may lead to high pyruvate concentrations which may be depleted by conversion to ethanol by ADH catalysis and to alanine by transamination (Morohashi 1978). MDH is involved in the latter process when OAA produced by transamination is converted to malate . AI-ani et al. (1985) concluded that the high glycolytic flux observed during the early imbibitional phase for Phaseolus mungo seeds (Morohashi 1978) was rather the exception than the rule for imbibing seeds. Another possible relation between ADH and MDH may be inferred for rice seedlings grown hypoxicallly (Avadhani et al. 1978). Ethanol production increased sharply, under hypoxic conditions, and malate formation by CO 2 dark fixation also was much higher under hypoxia. Similarly, for seedlings of Echinochloa crusgalli held anaerobically for 5 days and then pulsed with NaH I4 C0 3 , malate comprised 60% of the total soluble metabolites as a result of CO 2 dark fixation in comparison to aerobically grown seedlings where the percentage malate was 35% (Rumpho & Kennedy 1983). In addition to malate accumulation, anaerobiosis of E. crus-galli seedlings led to high ethanol content and ADH activity (Kennedy et al. 1983). On the other hand, Menegus et al. (1989) found that malate concentration decreased during anoxia in shoots of the above species. It follows from Hochachka & Mommsen (1983) that for the successful survival from anoxia, proton production per A TP molecule synthesized by the organism must be minimised. In this regard, fermentation to ethanol or succinate is most effective. Succinate production in shoot tissue was correlated with ability to withstand anaerobiosis (Menegus et al. 1989). MDH or ME is necessary for the production of succinate from glycolytic metabolites. Crawford & McMannon (1968) reported that ADH

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ADH ACTIVITY (Jlmol NADH produced . min:-' g-' fresh mass)

Figure 4 Relationship between MDH slIp and ADH activities of embryo extracts of Strelitzia juncea seeds incubated for various periods in either air or oxygen.

and MDH activities In the roots of flood-intolerant species increased under hypoxic conditions, and they found similar profiles for the two enzymes. According to the theory of flood tolerance (Crawford 1978), species tolerant of flooding have low fermentation rates and low ethanol production. For seeds, it was reported that tolerant species had lower ADH activities and lower ethanol but higher lactate content than seeds of intolerant species under hypoxic conditions (Crawford 1977). At the start of the project, (1983), it was thought that oxygen alleviation of Strelitzia juncea dormancy could possibly be explained by the flood theory in that hyperoxic treatment may prevent a presumed internal anaerobic atmosphere and an associated uncontrolled ethanol fermentation. The results reported here do not sustain such an assumption. In addition, the reported correlation between ADH activity and flood intolerance for seeds (Crawford 1977) was commented upon by Davies (1980). The conclusions reached by VanToai et ai. (1985) with maize caryopses and by Raymond et al. (1985) using seeds of 12 species, were that the metabolic flood theory could not be supported by their results. Researchers using root tissue (Smith et ai. 1986) also rejected the theory on account of their findings. The major contribution would seem to indicate that high fermentative activity of certain starchy seeds is related to their ability to germinate under hypoxic conditions

348

(Raymond et ai. 1985). It is becoming accepted that the synthesis of anaerobic proteins (i .e . ADH) are induced in root tissues by hypoxic conditions (Rivoal et ai. 1989 and references quoted therein) , and that such synthesis is associated with tolerance to anoxia. The presented work on fermentative activity of Streiitzia juncea seeds indicates that dormancy alleviation by oxygen is not primarily related to the abolishment of ethanol fermentation . The possibility of other fermentative activity resulting in malate or succinate formation needs to be investigated. Acknowledgements The authors wish to thank Orma Badenhorst for valued laboratory assistance and Erna Snyman and Rachel Potgieter for assisting with preparation of the manuscript. References AL-ANI , A . , BRUZAU, F., RAYMOND , P. , SAINT-GES , V. , LEBLANC , 1.M. & PRADET, A. 1985. Germination, respiration and adenylate energy charge of seeds at various oxygen partial pressures. Pl. Physiol. 79: 885- 890. AME N, R.D. 1968. A model of seed dormancy. Bot. Rev. 34: 1-30. AVADHANI , P .N ., GREENWAY H. , LE FROY , R . & PRIOR , L. 1978. Alcoholic fermentation and malate metabolism in rice germinating at low oxygen concentrations. Aust. 1. Pl. Physiol. 5: 15- 25. BE WLEY , J.D . & BLACK, M. 1982. Physiology and biochemistry of seeds in relation to germination. Viability, dormancy , and environmental control , Vol. 2, SpringerVerlag, Berlin . BEWLEY , 1.D . & BLACK, M. 1985. Seeds. Physiology of development and germination , Plenum, New York. COLLIS-GEORGE, N. & MELVILLE , M.D. 1974. Models of oxygen diffusion in respiring seed. 1. Exp. Bot. 25 : 1053- 1069. COSSINS , E. A. 1978. Ethanol metabolism in plants. In: Plant life in anaerobic environments, eds Hook , D.D. & Crawford , R.M.M. , Ch. 7, Ann Arbor Science Publishers, Michigan . CRAWFORD , R.M.M. 1977. Tolerance of anoxia and ethanol metabolism in germinating seeds. New Phytol. 79: 511-517. CRAWFORD , R.M.M . 1978. Metabolic adaptations to anoxia . In: Plant life in anaerobic environments , eds Hook , D.D. & Crawford , R.M.M., Ch . 4, Ann Arbor Science Publishers, Michigan. CRAWFORD , R.M.M. & McMANNON , M . 1968. Inductive responses of alcohol and malic dehydrogenase in relation to flooding tolerance in roots. 1. Exp . Bot. 19: 435-441. DAVIES, D.D. 1980. Anaerobic metabolism and the production of organic acids. In: The biochemistry of plants. A comprehensive treatise , eds Stumpf, P.K. & Conn, E.E . , Vol. 2, Ch . 14, Academic Press, New York. DE MEILLON , S. 1985. Invloed van rusverbreking deur suurstof op sekere aspekte van die respiratoriese metabolisme van saad van Strelitzia juncea. (The effect of dormancy release through oxygen treatment on certain aspects of the respiratory metabolism of Strelitzia juncea seeds .) M .Sc. thesis , Univ. of Pretoria , Pretoria. DE ME ILLON, S. 1990. Simple determination of gradients on recorder paper and conversion to appropriate units .

S.-Afr.Tydskr. Plantk., 1990, 56(3) Biochem. Ed. 18: 46-47 . DE MEILLON , S., SMALL, J.G.c. & VAN DE VENTER, H .A . 1990a. The effect of dormancy release through oxygen incubation of the seeds on the activity of glucose-6phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. 1. Exp . Bot . 42(227) (in press). DE MEILLON , S., VAN DE VENTER , H.A . & SMALL, J.G .c. 1990b . The respiratory metabolism of Strelitzia juncea Ait seeds. The effect of dormancy release by oxygen on certain glycolytic enzyme activities and metabolite concentrations. 1. Exp. Bot 41(228) (in press). E DWARDS, M .M. 1969. Dormancy in seeds of charlock 4. Interrelationships of growth, oxygen supply and concentration of inhibitor. 1. Exp . Bot. 20: 876--894. EDWARDS , M .M. 1972. Seed dormancy and seed environment - internal oxygen relationships. In: Seed ecology, ed . Heydecker W . , Proceedings of the nineteenth Easter School in Agricultural Science , University of Nottingham , Butterworths , London. FAGERSTEDT, K.V . & CRAWFORD, R.M.M . 1986. Changing kinetic properties of barley alcohol dehydrogenase during hypoxic conditions. 1. Exp. Bot. 37 : 857-864. GOOD, A.G. & CROSBY, W.L. 1989. Induction of alcohol dehydrogenase and lactate dehydrogenase in hypoxically induced barley. Pl. Physiol. 90: 860-866. GRONDAL, E.J .M., BETZ, A. & KREUZBERG, K. 1983 . Partial purification and properties of alcohol dehydrogenase from the unicellular green alga Chlamydomonas moewusii. Phytochemistry 22: 1695- 1699. HOCHACHKA , P.W. & MOMMSE N , T .P . 1983. Protons and anaerobiosis. Science 219: 1391- 1397. JAMES, W.O. 1953. Plant respiration. Oxford University Press, London. KENNEDY , R.A. , RUMPHO, MARY E. & VAN DER ZEE , DELMA. 1983. Germination of Echinochloa crusgalli (Barnyard Grass) seeds under anaerobic conditions. Respiration and response to metabolic inhibitors. PI. Physiol. 72: 787- 794. KHAN , A.A . 1977. The physiology and biochemistry of seed dormancy and germination. North-Holland Publishing Company, Amsterdam . KIMMERER, T.W. 1987. Alcohol dehydrogenase and pyruvate decarboxylase activity in leaves and roots of Eastern Cottonwood (Populus deltoides Bartr.) and Soybean (Glycine max L.) PI. Physiol. 84: 1210-1213 . KOLLOFFEL, C. 1968. Activity of alcohol dehydrogenase in the cotyledons of peas germinated under different environmental conditions. Acta Bot. Neerl. 17: 70-77. LEBLOV A, S. 1978a. Isolation and identification of alcohol dehydrogenases (ADH) from germinating seeds . In: Plant life in anaerobic environments, eds Hook D .D. & Crawford R .M.M. , Ch . 14, Ann Arbor Science Publishers, Michigan. LEBLOV A, S. 1978b . Pyruvate conversions in higher plants during natural anaerobiosis. In: Plant life in anaerobic environments, eds Hook D.D. & Crawford R.M.M. , Ch. 6, Ann Arbor Science Publishers , Michigan. LOWRY, O.H. & PASSONNEAU, J.V. 1972. A flex ible system of enzymatic analysis . Academic Press, New York. MAYER , A .M . & POLJAKOFF-MA YBER, A . 1982. The germination of seeds, 3rd edn , Pergamon Press , Oxford. MENEGUS, F ., CATTARUZZA, L., CHERSI, A. & FRONZA , G . 1989. Differences in the anaerobic lactate-succinate production and in the changes of cell sap pH for plants with high and low resistance to anoxia. Pl. Physiol. 90: 29-32.

S.Afr.J. Bot. , 1990, 56(3) MORINAGA , T. 1926. Germination of seeds under water. Am. 1. Bot. 13: 126-140. MOROHASHI, Y. 1978. Development of respiratory metabolism in seeds during hydration. In: Dry biological systems , eds Crowe J.H . & Clegg J.S ., Academic Press , New York . PERL , M. 1978. A possible role of malate dchydrogenase activity in seedling development of cotton (Gossypium hirsutum) . Isr. 1. Bot. 27: 54--61 . PESIS , E . & NG , T.J. 1986. The effect of seedcoat removal on germination and respiration of muskmelon seeds. Seed Sci. Technol. 14: 117- 125. PORTER, N .G. & WAREING , P.F. 1974. Thc role of thc oxygen permeability of the seed coat in the dormancy of seed of Xanthium pennsylvanicum Wallr. J. Exp. Bot . 25: 583- 594. RABIE , A.e. , SMALL, J.G.e. & BOTHA , F .e. 1989. The effect of submergence on gcrmination and some aspects of the respiratory metabolism of Cucumis sativus L. seeds. Pl. Sci. 63: 7- 13. RAYMOND , P ., AL-ANI , A. & PRADET, A . 1985. ATP production by respiration and fermentation , and energy charge during aerobiosis and anaerobiosis in twelve fatty and starchy germinating seeds. Pl. Physiol. 79: 879- 884. RIVOAL , J., RICARD, B. & PRADET, A. 1989 . Glycolytic and fermentative enzyme induction during anaerobiosis in rice seedlings. Pl. Physiol. Biochem. 27: 43- 52. ROBERTS , E.H . 1969. Secd dormancy and oxidation processes. Symp . Soc. Exp. Biol. 23: 161- 192. ROBERTS, J.K.M., CHANG , K., WE BSTE R , e. , CALLIS, J. & WALBOT, V. 1989. Dependence of ethanolic fermentation , cytoplasmic pH regulation , and viability on the activity of alcohol dehydrogenase in hypoxic maize root tips. PI. Physiol. 89: 1275- 1278. RUMPHO , M .E . & KENNEDY , R.A . 1981. Anaerobic metabolism in germinating seeds of Echinochloa crus-galli (Barnyard Grass). Metabolite and enzyme studies. PI. Physiol. 68: 165- 168 . RUMPHO, M.E. & KENNEDY, R.A . 1983. Anaerobiosis in Echinochloa crus-galli (Barnyard Grass) seedlings. Intermediary metabolism and ethanol tolerance . Pl. Physiol. 72: 44--49 .

349 RUSTIN , P ., MOREAU , F. & LANCE , e. 1980. Malate oxidation in plant mitochondria via malic enzyme and the cyanide insensitive electron transport pathway. Pl. Physiol. 66: 457--462. SHTMUMURA , S. & BEEVERS, H. 1983 . Alcohol dehydrogenase and an inactivator from rice seedlings. Pl. Physiol. 71: 736-741. SMALL, J.G.e. , POTGIETER, G.P. & BOTHA , F.e. 1989. Anoxic seed germin ation of Erythrina caffra: Ethanol fermentation and response to metabolic inhibitors. 1. Exp. Bot. 40: 375-381. SMITH , A.M., HYLTON, e.M ., KOCH , L. & WOOLHOUSE , H.W. 1986 . Alcohol dehydrogenase activity in the roots of marsh plants in naturally waterlogged soils. Planta 168: 130-138. SPRAGG , S.P. & YEMM, E .W. 1959. Respiration mechanisms and the changes of glutathione and ascorbic acid in germinating peas. 1. Exp . Bot. 10: 409--425. V AN DE VENTER , H.A. 1978. Effect of various treatments on germination of dormant seeds of Strelitzia reginae Ait. 1. S. Afr. Bot. 44: 103- 110. VAN DE VENTER , H .A. & SMALL, J.G.C. 1974. Dormancy in seeds of Strelitzia Ait. S. Afr. 1. Sci. 70: 216-217. VANTOAI , T.T. , FAUSEY, N.R. & McDONALD , M.B. 1985. Alcohol dehydrogenase and pyruvate decarboxylase activiti es in flood-tolerant and susceptible corn seeds during flooding. Agron. J. 77: 753- 757. WESTERHOFF, H .V., GROEN, A .K. & WANDERS , R.J.A. 1984. Modern theories of metabolic control and their applications. Bioscience Rep. 4: 1- 22. YBEMA , S.G . 1983. Invloed van suurstof op rusverbreking , respirasie en aspekte van styselafbraak in Strelitzia junceasaad. (Effect of oxygen on dormancy, respiration and aspects of starch degradation in Strelitzia juncea seed). M .Sc. thesis , Univ. of Pretoria , Pretoria . YBEMA , S.G. , SMALL, J.G.e., ROBBERTSE , P.J. & GROBBELAAR , N. 1984. Oxygen and gibberellic acid effects on germination , amylase activity and reducing substance levels of Strelitzia juncea seeds. Seed Sci. Technol. 12: 597-612.