Changes during Germination in Rainforest Seeds with Orthodox and Recalcitrant Viability Characteristics

J.PlantPhysiol. Vol. 134.pp. 9-16(1989}

Changes during Germination in Rainforest Seeds with Orthodox and Recalcitrant Viability Characteristics ANI NKANG 1 2

1

and GRAEME CHANDLER2

Department of Biological Sciences, University of Calabar, Calabar, Nigeria (address for correspondence) School of Biological Sciences, La Trobe University, Bundoora, Victoria, Australia 3083

Received January 27,1988' Accepted July 21,1988

Summary Major differences exist during embryogenesis in the balance of enzyme complements and in the catalytic complex (a term reflecting changes in the quantitative relationships between enzymes in the growing system) in seeds of Erythrina caffra Thunb. (with orthodox viability characteristics) and Guilfoylia monostylis (Bent h) F. Muell (with recalcitrant viability characteristics). Activities of pentose phosphate pathway (PPP) dehydrogenases and glucose-6-phosphate dehydrogenase/succinate dehydrogenase (G6PdH/SDS) and glucose-6-phosphate dehydrogenase/pyruvate kinase (G6PdH/Pk) ratios suggested that PPP activity becomes increasingly important in the embryonic axes in later germination (corresponding to the period after radicle emergence). The cotyledons, unlike the embryonic axes, appear not to be involved in major reductive biosynthesis associated with germination. The rapid activation of Pk and SDH activities and low initial G6PdH/Pk ratios in axes were indicative of glycolysis-TCA being the main operative pathways of glucose metabolism during initial germination (corresponding to the period from imbibition up to radicle emergence). Peroxidase (POD) and catalase activities and G6PdH/POD and G6PdH/ catalase ratios suggested that activities of POD and catalase become increasingly important in later germination. Activity patterns of enzymes and changes in the catalytic complex suggested a correlation between the increase in enzymic activities of PPP dehydrogenases, POD and catalase and the later germination period (which represents growth following germination). The PPP, therefore, may not play a central role in events leading to the germination of seeds in these species unless other pathways (possibly involving POD and catalase) are linked to the PPP in a cooperative manner.

Key words: Erythrina caffra, Guilfoylia monostylis rainforest, germination, enzyme activities. Abbreviations: DW, dry weight; FW fresh weight; H 2 0 2 , hydrogen peroxide; G, Germination fraction; G6PdH glucose-6phosphate dehydrogenase; 6PGdH, 6-phosphogluconate dehydrogenase; PPP, pentose phosphate pathway; Pk pyruvate kinase; PM, physiological maturity; POD, peroxidase; SDH, succinate dehydrogenase; kat, katals (amount of enzyme effecting the conversion of one mol of substrate per sec). Introduction Most germination studies have been restricted to seeds of cultivated or agricultural species. These are the worst possible choice (Mayer and Shain, 1974) for studying events during seed germination because in cultivated species, unlike in wild plants, uniform and rapid germination is sought. The question, therefore, is if the overall outline of the biochem© 1989 by Gustav Fischer Verlag. Stuttgart

ical events postulated using seeds of cultivated species in indeed «universal». Correlative evidence exists, from studies using seeds of cultivated species, that the pentose phosphate pathway (PPP) and glycolysis may be involved in events leading to the breakdown of dormancy and subsequent germination (Roberts, 1973; Taylorson and Hendricks, 1977). Some reports (Kovacs and Simpson, 1976; Swamy et aI., 1980) suggest that

10

ANI NKANG and GKAEME CHANDLER

the ability to maintain, or increase initial levels in activities of PPP enzymes following imbibition is obligatory for germination. There are reports, however, which indicate no obvious correlation between the activity of the PPP and dormancy breaking/germination (Adkins and Ross, 1981; Upadhyaya et aI., 1981). The use of C 6/C 1 ratios, an inadequate measure of pathway efficiency (Bewley, 1979; Ap Rees, 1980) has provided most of the available evidence for PPP participation in germination regulation (Simmonds and Simpson, 1971; Nicolas and Aldasoro, 1979). An alternative, and more valid approach to estimate the relative participation of the PPP and glycolytic pathway is to make direct comparisons of the extractable activities of enzymes involved in the particular pathways (Kovacs and Simpson, 1976; Adkins et aI., 1980; Swamy et aI., 1980). The latter approach is adopted in this study. The after-ripening problem evident in the majority of germination studies (Adkins and Ross, 1981) has been sidetracked by working with seeds of rainforest species (Eryh· trina caffra and Guilfoylia monostylis) which have no endogenous dormancy (see also Nikolaeva et aI., 1978). Seeds of G. monostylis, unlike those of E. caffra, exhibit unorthodox or recalcitrant seed viability characteristics; that is, the seeds cannot be dried to low moisture contents (5-7%) like orthodox seeds, without loss of viability (King and Roberts, 1979; Roberts and Ellis, 1982). Studies during embryogenesis in these species (Nkang and Chandler, 1986) had indicated that major differences which exist in the catalytic complex and in the patterns of enzyme activities at physiological maturity (PM) may be indicative of differences in germination strategies. We define PM as the «rundown» state of enzymic activities after organizing the metabolism of seed-filling [synthesis of storage compounds (Nkang, 1986; Nkang and Chandler, 1986)]. The present report shows the modulation of enzymic activities of succinate dehydrogenase (SDH), pyruvate kinase (Pk), PPP dehydrogenases, peroxidases (POD) and catalase in relation to germination in seeds of E. caffra and G. monostylis.

Materials and Methods Plant Material Physiologically mature seeds of Erythrina ca/fra Thunb. and Guilfoylia monostylis (Benth) F. Muell, were harvested directly from the parent trees. Seeds of E. ca/fra and G. monostylis had an average moisture content of 8.1 % and 56 % respectively, on collection. Seeds of G. monostylis were decoated. In order to eliminate hard· seededness, E. ca/fra seeds were scarified for 60 min in concentrated sulphuric acid. Seeds were then rinsed several times with deionized water before putting out to germinate in darkness at 25°C on wet vermiculite. Germination fractions (G) were calculated only on seeds whose radicles had emerged by more than 2 mm. Seeds were removed periodically from the germination trays for analysis. Fresh weight determinations were made on axes, cotyle. dons or intact seeds and dry weights similarly, after oven drying at 105°C for 24 h. Values shown represent means of duplicate determinations on batches of 40 seeds.

Extraction 0/ cytoplasmic (soluble) enzymes and isolation 0/ mitochondria All procedures were carried out as previously described (Nkang and Chandler, 1986). Catalase, unlike other cytoplasmic enzymes which appeared only in the B fraction, appeared in both A and B fractions in partially purified enzyme preparations. Catalase activity is consequently expressed as total activity in both fractions. Enzyme data shown represent means of duplicate enzyme determinations from two independent experiments.

Enzyme assay Assays for succinate dehydrogenase (SDH-EC 1.3.99.1), glucose6-phosphate dehydrogenase (G6PdH-EC 1.1.1.49), 6-phosphogluconate dehydrogenase (6PdH-EC 1.1.1.44), pyruvate kinase (PkEC 2.7.1.40) and peroxidases (POD-EC 1.11.1.7) were carried out spectrophotometrically as previously described (Nkang and Chandler, 1986). Assays for catalase (EC 1.11.1.6) were based on the method of Bergmeyer et al. (1983). The assay mixture contained 300 JLmol mixed phosphate buffer (pH 7.0) and 100 JLmol enzyme preparation in a final volume of 3.10 ml. The reaction (at 25°C) was initiated with the addition of 16 JLmol H 20 2• The decrease in extinction was followed spectrophotometrically at 240 nm for 40 sec.

Protein estimations Mitochondrial and soluble proteins were estimated in duplicate using the method of Bradford (1976).

Results

(1) Hydrational patterns (fresh weight changes) during germination of Erythrina caffra and Guilfoylia monostylis The hydrational pattern in germinating seeds of E. caffra was triphasic (Fig. 1). During the initial germination period (-corresponding to the period from imbibition up to radicle emergence) there was no change in axial dry weight (DW) but axial fresh weight (FW) increased slowly and exponentially. Thereafter, both axial DW and FW increased exponentially during the later germination period (-corresponding to the period after radicle emergence). There were no DW changes in the cotyledons of E. caffra except in the later germination period when DW decreased (Fig. 1). These FW and DW changes follow the generally reported trend (Bewley and Black, 1978) and indicate that the exponential increase in axial DW and the second exponential increase in axial and seedling FW is a consequence of axis growth. In germinating seeds of G. monostylis there was no appreciable increase in FW except in the last 2 - 3 days when slow FW increases, induced by the now slowly growing axis, became apparent (Fig. 1). Seeds of E. caffra started germinating by the third day after sowing with a germination value of 20 % (G = 0.20; Fig. 1) while G. monostylis seeds started germinating by the sixth day after sowing (G = 0.30; Fig. 1). Therefore, seeds of G. monostylis a late secondary and mature phase rainforest species germinated much later and had a relatively slower rate of seedling growth (based on FW increments) than E. caffra, an early secondary species.

11

Germination in rainforest seeds

Table 1: Changes in soluble and mitochondrial proteins during seed germination in E. caffra and G. monostylis. Soluble proteins Time after sowing Cotyledons Axes (mg axis-I) (mg coty. pair-I) (days) E. cal£a G. monostylis E. caffia

3·2

2h 7h 12h 2

3

·s ~

C>

ou'"

....

'" .,...." E

.,

'x

'x

g

2·0 ~

~

~

" 2·0 ~

...

o

'"o ...J

'"o ...J ,.0

'·0

0·0 Time after sowing (days)

Fig. 1: Semi-Log plots showing fresh weight changes in axis of E. caJ Ira (0) and whole seedlings of E. caffra (6) and G. monostylis (.&) as well as dry weight changes in axes (.) and cotyledons (0) of E. caJ

Ira during germination. The open and solid arrows on the abscissa represent, respectively, germination fractions (G.) of O' 20 and 0·30 for E. caffra and G. monostylis.

4 6

11.55±0.5 40.60± 1.7

OA4±0.04 N.D. 41.37± 5.0 OA4±0.04 11.20±1.0 40.00±0.5 043±0.01 11.89±1.0 N.D. 0048 ±0.01 11.06±0.9 41.12±0.7 0.54±0.05 40.12± 1.7 N.D. N.D. 7.70±0.4 39.02±0.6 0.55±0.05

N.D. N.D.

N.D.

Mitochondrial proteins Cotyledons Axes (mg axis-I) (mg coty. pair - I) E. caffia G. monostylis E. caffra

OA4±0.04 0040 ± 0.Q1 0.53±0.04 0.56±0.0 0.65±0.04 0.71 ±0.06 N.D. 0.50±0.02

1.80±0.2 1.90±0.2 1.96±0.2 2.24±0.3 N.D. 2.37±0.2 2.50±0.3 2.35±0.2

0.06±0.0 0.06±0.0 0.09±0.0 O.IO±O.O

N.D. O.IO±O.O

0.11±0.0 0.13±0.0

Proteins were estimated as described in the text. N. D., not determined. Values shown are means ± standard deviations (SO).

(ii) Changes in soluble and mitochondrial proteins Soluble protein (cytoplasmic or enzymic protein; Bewley and Black, 1978) content was relatively constant in axes of E. caffra and in cotyledons of both species during the initial germination period (Table 1). Similar observations have been made with other species (Davies and Slack, 1981; Marcus and Rodaway, 1982) and suggested that before elongation the embryonic axis relied predominantly on reserves stored within the axis, the various changes in enzymic activity being due to transformation in cytoplasmic proteins (Brown, 1972). Axis growth (germination) was accompanied by an increase in soluble protein content (Table 1). The decline in cotyledonary soluble protein content in later germination may be associated (Basha and Beevers, 1975; Bewley and Black, 1978) with its mobilization to the germinating axis. Mitochondrial protein levels in both axes and cotyledons of E. caffra were relatively constant following steeping (Table 1). Since succinate dehydrogenase activity increased rapidly during the period (Table 2) these results agree with previous reports (Bewley and Black, 1978; Morohashi et al.,

Table 2: Changes insuccinate dehydrogenase (SDH) and pyruvate kinase (Pk) activities in E. caffra and G. monostylis. Time after sowing (days)

Cotyledonary SDH activity /-Ikat /-Ikat Coty. pair - I mg protein

Axis SDH activity /-Ikat /-Ikat mg protein axis- I

PM 2h 7h 12h 2 3¢ 4 S

N.D. 3.90±0.0 7.SS±0.6S 7.29±0.20 7.04±0.lS S.12±0.60 7.13±0.46 7.40±0.20

N.D. O.7S±O.O 2.0S±0.04 2.02±0.06 1.97±0.04 2.64±0.20 2.S3±0.02 2.22±0.04

N.D. 2.S3±0.0 S.S3±0.49 S.40±0.23 S.60±0.S6 7.00±0.lS 7.S0±0.04 S.OS±0.30

PM 12h 2 4 6" S

3.22±0.22 3.26±0.3 3.S4±0.3 3.40±0.3 2.50±0.2 2.62±0.2 2.36±O.1

S.20±0.3 6.S0±0.6 7.2S±0.7 7.62± 1.0 S.92±0.5 6.S0±0.S 5.75±0.S

N.D. N.D. N.D. N.D. N.D. N.D. N.D.

A. Erythrina caffra N.D. O.lS±O.OO 0.3S±0.01 O.SO±O.Ol 0.S6±0.01 0.70±0.OS O.S3±O.OS 0.96±0.03

Cotyledonary Pk activity /-Ikat /-Ikat Coty. pair- I mg protein

Axis Pk activity /-Ikat /-Ikat axis- I mg protein

2.74±0.2 N.D. N.D. 2.64±0.2S 3.00±0.12 3.32±0.13 2.91±0.10 3.60±0.16

37.2±0.3 N.D. N.D. 30.60±0.7 34.0± 1.6 39.5± 1.7 32.2± 1.7 27.6±0.7

S.49±0.S N.D. N.D. S.12±0.4 6.1S±0.17 9.74±0.21 S.S2±0.6S 10.55 ± 1.01

2.2±0.2 N.D. N.D. 2.2S±0.2 2.64±0.1 3.70±0.1 4.60±0.4 S.SO±O.S

4S.20± 1.5 S2.S4± 1.9 62.17± 1.7 69.4 ± 1.4 91.7 ±2.7 79.3 ±2.6 69.5 ± 1.7

N.D. N.D. N.D. N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D. N.D. N.D. N.D.

B. Gui/foylia monostylis

10

N.D. N.D. N.D. N.D. N.D. N.D. N.D.

US±O.l 1.27±0.1 1.S7±0.1 1.73±0.3 2.3S±0.2 LSI ±0.2 1.69±0.2

Enzymes were assayed as described in the text. Values shown are means ± SD; N.D., not determined; PM, activity at physiological maturity; ¢, G = 0.20;", G = 0.30.

12

ANI NKANG and GRAEME CHANDLER 1·7

1981) that in some species the development of mitochondrial activity is not associated with the accumulation (synthesis) of additional mitochondrial proteins. In the slowly germinating recalcitrant seeds of G. monostylis, however mitochondrial protein levels increased gradually with higher levels being associated with visible germination (Table 1).

~

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1·4

:I:

'0

<.!)

(iii) Changes in enzymic activity

a.


(a) Succinate dehydrogenase (SDH)

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,

II

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o

(b) Pyruvate kinase (Pk)

_--'0'--- 0---

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:' ,,

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The activity pattern of SDH was triphasic and closely followed the triphasic hydrational pattern of the germinating seeds. Activity of SDH in both axes and cotyledons increased rapidly following steeping (Table 2). This was followed by a phase of relatively constant SDH activity (7h-day 2) and thereafter, another phase of increased SDH activity marked by visible germination. Succinate dehydrogenase activity in the cotyledons thereafter declined. In the cotyledons of G. monostylis there were slow increases in SDH activity during the initial germination period but activity declined in later germination (Table 2).

0----

2

if t

4

6

8

10

Time after sowing (days)

Preliminary investigations indicated that Pk actlVlty in axes of E. cafJra increased exponentially following steeping (Fig. 2). In the cotyledons of both E. cafJra and G. monostylis Pk activity also increased from the first few hours of sowing (Table 2).

Fig. 2: Results of preliminary experiments showing Semi-Log plots of changes in axial Pk activity (.6) and 6 PGdH activity in axes (0) and cotyledons (e) of E. ca/fra during seed germination. The open and solid arrows represent, respectively, germination fractions of o.35 and 0 . 70. Seeds used were of a different provenance to those used in subsequent experiments (Tables 1- 4).

(c) Peroxidases (POD) Rapid increases in axial and cotyledonary POD activity of E. cafJra were associated with radicle emergence; that is, visible germination (Table 3). The increase in POD activity was by far greater in axes than in cotyledons of E. cafJra. Per-

oxidative activity increased gradually in the cotyledons of G. monostylis with higher levels of activity being associated with visible germination (Table 3). Initial levels in activities

Table 3: Changes in peroxidase (POD) and activities in E. ca/fra and G. monostylis during germination. Time after sowing (days)

Cotyledonary POD activity J.lkat J.lkat mg- 1 protein coty. pair- 1

PM 12h 2 3¢ 4 8

3.60±0.10 3.72±0.18 4.43±0.16 8.60±0.50 15.02±0.65 38.40±2.57

PM 12h 2 4 6" 8 10

13.0±0.4 14.l±0.5 19.2±2.0 20.6±0.1 25.5± 1.0 26.3±0.2 26.4±0.3

48.94± 0.5 43.04± 1.7 49.62± 2.2 102.3 ± 8.6 166.12± 13.5 295.7 ± 8.4 529.9 ± 17.1 583.8 ±42.8 760.32±29.1 826.9 ±31.2 995.7 ±38.6 1081.4 ±39.8 1150.7 ±39.9

Axis POD activity J.lkat J.lkat mg - 1 protein axis - 1

Cotyledonary catalase activity J.lkat J.lkat mg - 1 protein coty pair- 1

A. Erythrina ca/fra 4.22± 0.35 1.69±0.04 3.28±0.14 5.65± 0.40 2.49±0.11 3.40±0.14 9.65± 0.69 4.15±0.14 3.14±0.09 159.2 ± 3.39 60.5 ±0.80 8.90±0.60 233.6 ± 5.97126.1 ±5.84 10.81±0.23 263.9 ± 19.7 145.2 ±7.9 26.76±2.1 N.D. N.D. N.D. N.D. N.D. N.D. N.D.

B. Guilfoylia monostylis N.D. 8.99±0.3 N.D. 9.5l±0.4 N.D. N.D. N.D. 10.56±0.1 N.D. 11.25±0.4 N.D. 11.78±0.1 N.D. 12.77±0.2

44.6 39.3 35.2 105.8 119.6 206.9

± 1.9 ± 0.9 ± 1.5 ± 4.4 ± 5.4 ± 10.7

371.49± 13.6 393.44± 14.4 N.D. 423.7 ± 8.5 439.0 ±13.0 484.4 ±1l.5 556.4 ± 9.8

Axis catalase activity J.lkat J.lkat mg - 1 protein axis- 1 1.68±0.15 1.50±0.11 1.68±0.03 3.88±0.30 5.16±0.34 9.98±0.37

0.65±0.06 0.66±0.03 0.74±0.02 1.46±0.04 2.79±0.20 5.49±0.20

N.D. N.D. N.D. N.D. N.D. N.D. N.D.

N.D. N.D. N.D. N.D. N.D. N.D. N.D.

Enzymes were assayed as described in the text. Values shown are means ± SD; N.D., not determined; PM, activity at physiological maturity; ¢, G = 0.20; .. , G = 0.30.

Germination in rainforest seeds

13

Table 4: Changes in glucose-6-phosphate dehydrogenase (G6PdH) and 6-phosphogluyconate dehydrogenase (6PGdH) activity in E. ca/fra and G. monostylis during germination. Time after sowing (days)

PM 12h 2 3<> 4 8

Cotyledonary 6PGdH activity /Lkat /Lkat Coty. pair - I mg - I protein

Cotyledonary G6PdH activity /Lkat /Lkat mg - I protein Coty. pair- 1

Axis G6PdH activity /Lkat /Lkat mg - I protein axis- 1

0.60±0.02 0.69±0.04 0.7HO.07 0.77±0.03 0.72±0.06 0.96±0.OS

A. Erythrina ca/fra 0.8HO.OS 0.3HO.02 1.SHO.11 0.8HO.08 0.37±0.02 1.Sl±0.07 0.8S±o.o9 0.37±0.02 1.68±0.07 2.30±0.20 0.87±0.02 1.60±0.13 2.88±O.lS l.S3±0.10 1.Sl±0.06 4.2HO.20 2.33±0.21 1.98±0.OS

8.2 8.0 8.3 9.2 8.0 704

± ± ± ± ± ±

0.8 0.3 0.7 0.8 0.6 004

20.2S± 17A4± 18.82± 19.02± 16.70± lS.2S±

1.47 004 0.8 0.8 0.7 004

Axis 6PGdH activity /Lkat /Lkat mg - I protein axis - I 2.2l±0.13 2.07±0.20 2.13±0.20 S.SS±OA9 7AO±0.28 10.84±0.33

0.88±0.OS 0.91±0.04 0.92±0.02 2.10±0.06 3.2HO.30 S.8S±0.S4

B. Guilfoylia monostylis N.D. N.D. 341.02± 10.2 3.10±0.1 12S.8S± 3.8 N.D. 8AO±0.3 N.D. N.D. N.D. 3S9.S2± 6.7 136.S3± 2.S N.D. 8.69±0.3 N.D. 3.30±0.3 N.D. N.D. 390.8S±2S.8 3.80±0.2 lS0A8 ± 11.4 N.D. 9.90±0.1 N.D. N.D. 8.6S±0.S 347.73± 1.0 N.D. 3.3l±0.S 133.06± 0.3 N.D. N.D. 6~ 2.94±0.1 114.7l± 2.2 N.D. N.D. 8.0HO.3 313.69± 604 N.D. N.D. N.D. 360.20± 1.3 N.D. 8 N.D. 8.76±0.1 N.D. 3.12±0.1 128.29± 0.5 N.D. N.D. 10 3SS.9l± 1.8 2.90±0.1 122.73 ± 0.6 N.D. 8AO±0.1 N.D. Enzymes were assayed as described in the text. Values shown are means ± SD; N.D. not determined; PM, activity at physiological maturity; <>, G = 0.20; ~, G = 0.30.

PM 12h 2 4

of POD were much higher in the recalcitrant seeds of G. monostylis than in the orthodox seeds of E. cafJra.

(d) Catalases In E. cafJra rapid increases in axial and cotyledonary catalase activity were coincidental with radicle emergence (Table 3). Unlike POD, catalase activity increased proportionately in both axes and cotyledons. In the cotyledons of G. monostylis activity increased gradually during germination. Initial levels in activities of catalase, just as with POD, were much higher in G. monostylis than in E. cafJra. Their low initial levels notwithstanding, POD and catalase activities increased remarkably faster and to much higher levels during germination in E. cafJra.

(e) Glucose-6-phosphate dehydrogenase (G6PdH) and 6-phosphogluconate dehydrogenase (6-PGdH) In E. cafJra cotyledonary activity of PPP dehydrogenases increased exponentially during initial germination (Fig. 2) but activity may decline in the later germination period (Fig. 2, Table 4). Activity of PPP dehydrogenases in the embryonic axes may decline or remain relatively constant during the initial germination period (Fig. 2, Table 4). Increased PPP activity in the axes, over and above levels established at physiological maturity, was coincidental with radicle emergence (Fig. 2, Table 4). In G. monostylis cotyledons, activities of PPP dehydrogenases increased slowly during the initial germination period but declined in later germination (Table 4).

Discussion The initial exponential increase in Pk and SDH activities in axes and cotyledons and in PPP activities in cotyledons

(Fig. 2, Table 2) occurred at a time of relatively low moisture content and was not associated with any increase in DW (Fig. 1) or in the content of soluble or mitochondrial proteins (Table 1). Such intensification of metabolic activity that is a feature of progress in germination cannot be due to progressive activation (Brown, 1972), as then the rate of particular reactions might be expected to increase with increasing water content but with the rate of increase diminishing with time. Results of some enzymic activities supported this contention. Axial Pk activity of E. cafJra, for example, continued to increase long after seeds were fully hydrated (on the first day) and prior to radicle emergence (on the third day). Results suggested that the initial activation of enzyme activities (a consequence of the hydration of reaction sites and the improvement and restitution of pre-existing enzymes and mitochondria) was brief and was replaced by one of enzyme synthesis. Increased activity of the PPP enzyme (6PGdH) in the axis was exponential but coincidental with radicle emergence (Fig. 2, Table 4) and thus may be associated with synthesis in the growing seedling. Succinate dehydrogenase activity of G. monostylis cotyledons increased slightly but did not exhibit any phasic pattern as in E. cafJra. This is thought to reflect the fact that at maturation G. monostylis seeds maintained a relatively high moisture content and loss of mitochondrial integrity associated with desiccation was lacking (Nkang and Chandler, 1986). The subsequent decline in SDH activity and in mitochondrial protein content in the cotyledons of both species (Tables 1, 2) agreed with previous reports (Marbach and Mayer, 1976; Bewley and Black, 1978; Sahai et aI., 1978) and may be a consequence of the deterioration in mitochondrial structure at the later stages of germination. Changes in the catalytic complex (Brown, 1972; Nkang and Chandler, 1986) further highlighted the metabolic differences between axes and cotyledons and between the two species under investigation. In E. cafJra the sharp drop in

14

ANI NKANG and GKAEME CHANDLER

Table 5: Changes in the catalytic complex (quantitative relations between any two enzymes) using G6PdH as reference. Values were determined using enzyme activities expressed on a mg- 1 protein basis (Mean t SD). G6PdH/POD

Time after sowing (days) (i) Cotyledons PM 2h l2h 2 3¢ 4 8

G6PdH/SDH

G6PdH/Pk

N.D. 0.15tO.Ol O.lOtO.OO O.lOtO.Ol O.lOtO.OO O.lOtO.Ol O.lHO.OO

0.21 to.Ol N.D. 0.26tO.Ol 0.25tO.Ol 0.2HO.00 0.25tO.Ol 0.27±0.00

A. Erythrina caffra 0.17tO.Ol N.D. 0.19tO.00 0.17tO.Ol 0.09tO.00 0.05tO.00 O.OHO.OO

(ii) Axes PM 2h l2h 2 3¢ 4 8

N.D. 0.30tO.03 0.16±0.01 0.15tO.00 0.3HO.02 0.38tO.02 0.52±0.01

0.15tO.Ol N.D. 0.16tO.00 0.14±0.01 0.24±0.02 0.34±0.01 OAOtO.02

0.20tO.Ol N.D. 0.15tO.00 0.09tO.00 0.01±0.00 0.01±0.00 0.02tO.00

Cotyledons PM l2h 2 4 68 10 N.D., not determined;

B. Guilfoylia monostylis 2.70tO.15 0.24±0.00 0.96tO.03 2.60tO.03 0.2HO.Ol 1.01±0.00 0.20tO.Ol 2A2±0.03 0.99tO.03 1.91±0.04 0.16±0.02 0.97±0.06 0.12±0.00 U8tO.05 1.25±0.06 U9tO.05 1.7HO.14 0.12±0.00 1.72±0.14 0.11±0.00 l.2HO.Ol PM, activity at physiological maturity; ¢, G, = 0,20; -, G = 0.30.

G6PdH/SDH ratios in the first few hours of hydration is a consequence of the rapid activation of mitochondrial enzymes. Ratios increased in later germination in the axes (Table 5) and is suggestive of an increasingly important PPP which supplies NADPH in the cytoplasm for use in biosynthetic processes. This contrasted with the situation in the cotyledons where ratios declined and thereafter remained relatively constant (Table 5). The cotyledons, therefore, appear not to be invovled in major reductive biosynthesis in the later germination period. Low initial G6PdH/Pk ratios in the axes of E. ca./fra (Table 5) and the rapid activation of Pk activity (Fig. 2, Table 2) suggested the dominance of glycolysis over the PPP during the initial germination period. Pyruvate or its appropriate amino acid, which may accumulate in the axes during seed maturation (Nkang and Chandler, 1986), could serve as substrate in such glycolytic activity. Increasing ratios in later germination (Table 5) suggested that the PPP becomes increasingly important over glycolysis. These findings corroborated previous reports (Yamamoto, 1963; Bewley and Black, 1978, 1982) that in germinating embryos glycolytic activity predominated early in germination and that PPP activity increased in later germination. In the cotyledons in contrast, G6PdH/Pk ratios declined (G. monostylis) or remained relatively constant (E. ca./fra) and was suggestive of a greater role for glycolysis in cotyledonary respiration in the later germination period. Major increases in activities of POD and catalase were coincidental with «visible» germination (Table 3) and thus in

G6PdH/catalase

G6PdH/6PGdH

0.18tO.00 N.D. 0.20tO.00 0.24±0.02 0.09tO.00 0.07±0.00 0.04tO.00

OAOtO.Ol N.D. OA6tO.Ol

0.50tO.02 N.D. 0.56tO.Ol 0.51 to.04 0.59tO.Ol 0.56±0.01

0.38±0.03 N.D. OAOtO.OO OAOtO.OO

OA4±0.02 OA8±0.02

OA8±O.Ol OA8tO.Ol

OA1±O.OO

OA2±O.OO

0.39tO.Ol 0.39tO.Ol

0.34±0.00 0.35tO.02 N.D. 0.31±0.00 0.26tO.00 0.26tO.Ol 0.2HO.00

0.37tO.00 0.38tO.02 0.38tO.02 0.38tO.00 0.37tO.00 0.36tO.Ol 0.35±0.01

agreement with previous reports (Lewak et al., 1975; Thevenot et al., 1977) that increased POD and catalase activities are associated with growth of the germinating embryo. Ratios of G6PdH/POD and G6PdH/catalase (Table 5) suggested also that peroxidative and catalase activities become increasingly important in later germination. Relative to POD, activity of catalase (an enzyme associated with the elimination of harmful metabolic products; Halder et al., 1983) in axes acquired predominance much later in germination. Studies during embryogenesis (Nkang and Chandler, 1986) had indicated that high POD levels are maintained in C. monostylis but in E. ca./fra POD activities declined with maturation. The partial adaptive advantage of higher initial levels of POD and catalase activities in the recalcitrant seeds (Table 3) may be that the higher metabolism, resulting from the high moisture content, militates against any germination delay on the forest florr. There is, presumably, the necessity for seeds to posses adequate levels of enzymes capable of eliminating harmful metabolic products and/or capable of creating a «favourable hormonal balance for germination». Nikolaeva et al. (1978) suggest that a germination blocking mechanism consists of a combination of high auxin and abscisic acid contents and further, POD (which may function in parallel as IAA oxidase; Gaspar et al., 1982; Kevers et al., 1984) is capable of reducing high auxin to levels favourable for germination. In the orthodox seeds of E. ca./fra (an early secondary species) basic survival strategies dictate rapid germination/seedling establishment and hence the

Germination in rainforest seeds rapid increases in POD and catalase activities with germination (Table 3). Results with E. cafJra corroborated other reports which indicate that during the initial germination period PPP activity may increase slowly and exponentially in the cotyledons (Fig. 2; Nicolas and Aldasoro, 1979; Umezurike and Numfor, 1979) while remaining relatively constant in the axes (Fig. 2; Table 4). Increased axial PPP activity in the later germination period corroborated other reports (Yamamoto, 1963; Nicolas and Aldasoro, 1979) that the PPP plays an increasingly important role in the development of seedling tissues during germination. Reports of increased PPP activity during initial germination in studies in which whole seeds had been utilized (Kovacs and Simpson, 1976; Swamy et aI., 1980) should, therefore, be viewed with caution as such studies may reflect events in the dominant storage tissues rather than those of the germinating axes. Lower G6PdH acitivity as indicated by G6PdH/6PGdH ratios suggested that G6PdH participated in PPP control (Table 5). Results indicate, therefore, that a correlation exists between seed germination and the increase in activities of G6PdH, 6PGdH, POD and catalase in the embryonic axes of E. cafJra (Table 3, 4). That is, the rapid increase in their activities in the axes is coincidental with the rapid increase in axial FW and DW which represents growth following germination. Results with Pk and SDH (Fig. 2, Table 2) appear consistent with previous reports (Burguillo and Nicolas, 1974; Nicolas and Aldasoro, 1979) of glycolysis - TeA cycle being the main operative pathways of glucose metabolism during initial germination. Although these results to some extent, corroborated the view that the ability to maintain or increase initial levels of PPP enzymes (established at PM) is obligatory for germination (Kovacs and Simpson, 1976; Swamy et aI., 1980), it is felt that evidence is insufficient to give the PPP a central role in germination, unless of course other pathways (possibly involving POD and catalase activities) are linked to the PPP in a cooperative manner. This is examined further in our subsequent paper regarding cyanide stimulation of germination in G. monostylis seeds.

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