Bot. Bull. Acad. Sin. (1996) 37(4): 229-237

Chao and Lin Adenosine nucleotides of orthodox and recalcitrant seeds

Content of adenylate nucleotides and energy charge in the early stage of germination of orthodox and recalcitrant seeds

Chih-Chang Chao and Tsan-Piao Lin1

Tree Seed Laboratory, Taiwan Forestry Research Institute, 53 Nan-Hai Road, Taipei, Taiwan, Republic of China

(Received April 1, 1996; Accepted July 9, 1996)

Abstract. The content of adenosine phosphates and the energy charges of three species of orthodox seeds, i.e. Phellodendron wilsonii, Michelia compressa, and soybean; three species of recalcitrant seeds, i.e. Podocarpus macrophyllus, Cyclobalanopsis globosa, and Machilus thunbergii; one species of intermediate category, i.e. Neolitsea parvigemma at the early stage of germination were analysed to compare the energy concept established for crop seeds. AMP was the major adenylate in the dry state, and the ATP content in dry orthodox seeds was usually lower than 10 nmol g-1 d.wt resulting in low energy charge values of around 0.20. The highest increase of ATP levels after incubation compared to the level of dry seeds was 167-fold in soybean and 60-fold in Ph. wilsonii. The maximum ATP accumulation coincided with the seed being ready to germinate. A decrease in ATP content was observed after seed germination, and the decline continued throughout the period of radicle emergence. The imbibed dormant seeds of Ph. wilsonii synthesized smaller amounts of ATP than non-dormant ones at the early stage of incubation; the ATP was eventually depleted. The ATP content of recalcitrant seeds, including the intermediate species, ranged from 20 to 220 nmol g-1 d.wt, thereby reflecting energy charge values above 0.6. The ATP levels in the fresh recalcitrant seeds before and after incubation, however, generally remained similar. This suggests that orthodox crop seeds, i.e. soybean synthesize adenosine phosphates extremely efficiently, unlike most of the tree seeds, which are more or less dormant. P. macrophyllus seeds are viviparous and had the highest amount of ATP, ADP and the highest energy charges among the species tested, indicating high metabolic activities in mature seeds. Seeds of C. globosa, another type of recalcitrant seed and N. parvigemma were characterized by low adenylate and energy charges, no significant changes in adenylate in the early stage of incubation, and slow germination. During an artificial drying process, the degradation of adenylates in recalcitrant seeds seemed to continue indefinitely, resulting in lower energy charges. This process must be different from that of orthodox seeds in which AMP is the major adenosine phosphate to accumulate.

Keywords: Adenylates; Artificial dehydration; Energy charge; Orthodox; Recalcitrant; Seed germination.

Abbreviations: EC, energy charges; TA, total adenylate; MC, moisture content.

Introduction

Most crop seeds are in the category of orthodox types, which are tolerant of desiccation. The longevity of orthodox seeds is increased by decreasing storage temperature and moisture content (Roberts, 1973). The accumulation and the synthesis of ATP at the early stage of crop seed germination have been documented since the middle of the 1960s. It was found that AMP is the major adenylate accumulating in mature seeds (Obendorf and Marcus, 1974; Barua et al., 1981). In terms of adenylate energy charge, most orthodox seeds exhibited a value lower than 0.2 (Quebedeaux, 1981; Lunn and Madsen, 1981; Hourmant and Pradet, 1981). During the early stage of seed imbibition and germination, a rapid increase occurs in ATP accumulation, and energy charges increase to a range of 0.6 to 0.9 (Pradet and Raymond, 1983), after which a stationary phase in ATP concentration occurs

(Ching and Ching, 1972; Moreland et al., 1974; Hourmant and Pradet, 1981). Crop seeds like wheat (Ching and Kronstand, 1972) showed strong oscillations of the changes of adenylates during germination probably indicating different growth events.

Recalcitrant seeds are desiccation sensitive and have a high moisture content, which is necessary to maintain seed viability for a certain period of time. Most (if not all) are probably metabolically active as reported for mangrove species (Farrant et al., 1992). Respiration of seeds represents the gross metabolic activity, normally quite low in mature orthodox seed but in recalcitrant seed probably in the range of 0.5_1.5 mmol O2 min-1 g-1 f.wt (Lin and Chen, 1995). The adenylates in the early stage of seed germination have received little attention in recalcitrant seeds. It is reasonable to speculate on the different levels of adenylates in these two seed types, but how great the difference is and its significance are presented in this report.

1Corresponding author. Fax: 02-3895531.


Botanical Bulletin of Academia Sinica, Vol. 37, 1996

at 5C. The supernatant was neutralized with 5M KOH as measured by broad-range pH paper. The salt precipitate was removed by centrifugation (13,000 g) at 5C, and the supernatant was transferred to a fresh, preweighed tube where the final volume of the extract was determined by the weight (Oresnik and Layzell, 1994).

The ATP content was measured by the luciferin-luciferase system with a Lumac biocounter MT 2000 (Chapman et al., 1971). The light signal was integrated for 10 sec and compared with the standard for ATP (2.5 to 250 nM). ADP was converted to ATP with phosphoenolpyruvate and pyruvate kinase (EC 2.7.1.40) (Sigma) by incubation at 37C for 15 min. AMP was converted to ADP with endogenous ATP by using myokinase (EC 2.7.4.3) (Sigma); the resulting ADP was converted to ATP (Ching and Ching, 1972). EC was calculated according to Atkinson (1969) : EC = ([ATP] + 1/2 [ADP]) / ([ATP] + [ADP] + [AMP]). Firefly lantern extract (FFT from Sigma) was prepared by ammonium sulphate precipitation (Strehler, 1974), but the precipitates were dissolved in 20 mM glycylglycine (pH 7.4), 50 mM NaAsO2, and 20 mM Mg(CH3COO)2. The limit of sensitivity of this method is 0.1 nM. The conversion rates of AMP to ADP and ADP to ATP were 100%. A known amount of ATP was ground with the sample, and the ATP recovery was generally higher than 90%.

EC values in the early stage of seed germination generally ranged from 0.8 to 0.9 in non-limited supply of oxygen (Rasi-Caldogno and De Michelis, 1978; Mocquot et al., 1981). The slightly lower EC measured in this study, for example, >0.7 for soybean axes, 0.56_0.74 for Ph. wilsonii, 0.55 for M. compressa and 0.78 for N. parvigemma is due probably to partial oxygen deprivation in the polyethylene bags. In a separate experiment, we found that the germination percentages of the seeds incubated in polyethylene bag were the same as in a germination box, even though the germination process may be more or less delayed in the bag. Also the EC of the seeds incubated in the PE bag was generally 0 to 0.1 lower than that obtained in the germination box.

Results

Orthodox Seeds

The changes of ATP, ADP, AMP, TA, and EC in orthodox seeds during the germination process are presented in Tables 1 to 3. Mature seeds have only an embryo in soybeans and Ph. wilsonii (exalbuminous), but a tiny embryo surrounded by endosperm in M. compressa (albuminous). Seeds of Ph. wilsonii are markedly smaller than soybeans, thereby making it difficult to dissect into cotyledon and embryonic axis. The levels of ATP and EC were very low in the dry soybean seeds (Table 1). The axis had 39 pmol ATP and an EC of 0.24; the cotyledon had 426 pmol ATP and an EC of 0.19. Adenylates and EC in the axis and cotyledon of soybean increased to a maximum probably at two days of incubation; with an average of 5792 pmol, 4466 pmol, 6580 pmol for ATP, ADP and

Materials and Methods

Seed Collection and Seed Germination

Freshly mature seeds of Podocarpus macrophyllus D. Don, Cyclobalanopsis globosa Lin et Liu, Neolitsea parvigemma (Hay.) Kaneh. et Sasak., and Michelia compressa (Maxim.) Sarg. were collected from natural forests in Taiwan sometime between June and October 1994, depending on the species. Some of the seeds of P. macrophyllus were wet stored (or stratified) at 4C for 4.5 months. Seeds of Machilus thunbergii were collected in June 1995. All fresh mature seeds of the above species were used for the following experiments. Seeds of the soybean cv. "Tainaon no. 15", M. compressa and Phellodendron wilsonii Hay. et Kaneh. collected in October 1993 with a water content of 6.1%, 6.6%, and 7.0%, respectively, were stored at 4C for one year before use. The moisture content of seeds was obtained from a triplicate sample dried at 103C for 17 h and is expressed on a fresh weight basis. The seeds of P. macrophyllus, C. globosa, and Ma. thunbergii are recalcitrant types; and soybean, Ph. wilsonii (Lin et al., 1994) and M. compressa (Lin and Wu, 1995) are recognized as orthodox types. Neolitsea parvigemma was recognized as belonging to an intermediate category (unpublished observation).

The seeds were mixed with moistened clean sphagnum in sealable polyethylene bags (17 11.5 cm, 0.04 mm thick), and the water content of the sphagnum was adjusted to about five times the dry mass (83_85% wet basis), Roughly 2/3 of the space inside the bag was occupied by the sphagnum with seeds. Germination was carried out in growth chambers with 12 h of fluorescent light (60_80 Em-2sec-2 ) at 30/20C. Only seeds of Ph. wilsonii were incubated at 35/10C with 8 h of light to break the deep dormancy (Lin et al., 1994).

Artificial Dehydration

Slow drying was achieved by placing seeds in a desiccator over a series of saturated salt solutions: MgSO47H2O, RH 89% CH3COONa3H2O, RH 75% MgCl26H2O, RH 33% LiCl, RH 13%. Seeds of Ma. thunbergii were first placed over the CH3COONa3H2O solution before proceeding to lower relative humidities. Seeds were transferred to the next saturated salt solution with lower humidity every five days, and the seeds were left over the final salt solution until the desired water content was achieved.

Determination of Adenosine Phosphates

Most of the tree seeds showed a greater or lesser degree of dormancy, taking two weeks to one month to germinate. This probably is associated with the relatively undifferentiated axes in many of the recalcitrant seed species (Berjak et al., 1989). For adenosine phosphates analysis, seeds of three replicates were homogenized with 10% ice-cold perchloric acid. The homogenate was kept on ice for 15 min before being centrifuged (13,000 g) for 10 min


Chao and Lin Adenosine nucleotides of orthodox and recalcitrant seeds

Table 1. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) in the axis and cotyledon of soybean seeds. Each value is the average of three replicates. Standard errors are shown in parentheses. Seeds began germination between 1 and 2 days incubation. Seeds reached 100% germination within 3 days.

Incubation ATP ADP AMP TA EC

(day)

pmol / axis

0 39 (3)c* 193 (25)c 330 (13)c 562 (42)c 0.24 (0.01)c

1 2957 (102)b 1217 (260)b 836 (75)c 5010 (83)b 0.71 (0.01)a

2 5792 (1400)a 4466 (558)a 6580 (787)b 16838 (1221)a 0.48 (0.04)b

3 2156 (102)bc 4265 (111)a 11677 (964)a 18097 (928)a 0.24 (0.02)c

pmol / cotyl

0 426 (44)d + 2203 (143)c 5496 (474)b 8125 (370)c 0.19 (0.02)c

1 56678 (1658)b 32834 (4641)b 14859 (5872)b 104370 (12170)b 0.70 (0.04)a

2 71212 (3237)a 56288 (1693)a 42409 (5092)a 169908 (3548)a 0.58 (0.03)b

3 46759 (4929)c 60028 (5123)a 45292 (1086)a 152079 (1280)a 0.50 (0.02)b

Mean separation for axis (*) or cotyledons (+) within rows by Duncan's multiple range test, 5% level.

Table 2. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) in seeds of Phellodendron wilsonii which were incubated either at 35/10C or 22C. Each value is the average of three replicates. Standard errors are shown in the parentheses. Seeds began germination on the 15th day of incubation at 35/10C. Seeds reached 60% germination after 7 weeks incubation at 35/10C and 8 h light.

Incubation ATP ADP AMP TA EC

(temp. day)

35/10C pmol / seed

0 8.9 (0.9)d* 41.4 (4.2)c 98.7 (2.1)b 149 (3.0)c 0.20 (0.02)c

3 139 (24)c 82 (25)c 28 (4)b 249 (45)c 0.72 (0.02)a

9 175 (7)c 76 (8)c 36 (0)b 287 (9)c 0.74 (0.01)a

15 531 (34)a 438 (66)a 365 (34)a 1334 (91)a 0.56 (0.02)b

24 437 (25)b 317 (20)b 315 (69)a 1069 (114)b 0.56 (0.03)b

22C pmol / seed

0 8.9 (0.9)c+ 41.4 (4.2)c 98.7 (2.1)a 149 (3.0)b 0.20 (0.02)d

3 85 (5)a 71 (8)ab 64 (11)bc 220 (25)a 0.55 (0.02)b

9 69 (5)a 77 (2)a 77 (7)b 223 (9)a 0.48 (0.00)c

15 74 (2)a 59 (7)b 51 (0)c 184 (10)ab 0.56 (0.00)ab

24 38 (8)b 31 (2)c 22 (6)d 91 (15)c 0.59 (0.01)a

*Mean separation for 35/10C (*) or 22C (+) treatment within rows by Duncan's multiple range test, 5% level.

AMP in the axis and 71212, 56288, and 42409 pmol in the cotyledon, respectively.

Similar results for ATP, ADP, TA, and EC were found in Ph. wilsonii seeds (Table 2). ATP, ADP, and AMP in dry seed were 8.9, 41.4, and 98.7 pmol, respectively, with an EC of 0.20. When incubated at alternating temperatures, ATP and ADP increased up to the 15th day, at which time the embryo broke the seed coat and germination became observable. Only the level of AMP decreased in the beginning of incubation. Seeds of Ph. wilsonii maintained dormancy if they were incubated at 22C and the energy metabolism was less dynamic than seeds incubated at 35/10C. ATP and ADP increased initially but decreased continuously later on. AMP decreased slightly in the first several days and became significant at the 24th day of incubation.

Michelia compressa seeds (Table 3) were different from those of soybean or Ph. wilsonii. The seeds had 302, 809, and 1367 pmol of ATP, ADP, and AMP, respectively. ATP increased at the beginning then decreased. ADP and AMP

decreased from the beginning, thus causing an increase of EC from 0.28 to 0.55. Once these seeds started germinating at around the 25th day of incubation, the second peak of adenylate was observed in the endosperm. On the 28th day of incubation, the sizes of the embryo and endosperm of germinating seeds were equal to each other. By the 35th day of incubation, the endosperm was almost exhausted.

Recalcitrant Seeds

ATP was the predominant form in the gametophyte of P. macrophyllus (Table 4) with an average of 113 nmol, as compared with ADP at 37 nmol, and AMP at 16 nmol. Another characteristic of the gametophyte was a high EC value of 0.79. During the incubation period, ATP decreased slightly in the early stage of seed germination. ATP remained roughly similar when radicle emergence occurred on the 10th day of incubation. ATP increased again to 98 nmol on the 20th day of incubation while the cotyledon was beginning to open up from the hypocotyl. In the embryo, ATP content increased significantly after


Botanical Bulletin of Academia Sinica, Vol. 37, 1996

Table 3. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) in seeds of Michelia compressa. The seeds were stored at 4C with water content of 6.6%. Seeds began germination at about the 25th day of incubation and were dissected into the embryo and endosperm at 28 days. Each value is the average of three replicates. Standard errors are shown in parentheses. Seeds reached 41% germination after 7 weeks incubation.

Incubation ATP ADP AMP TA EC

(day)

pmol / seed

0 302 (31)bc* 809 (134)a 1367 (315)a 2478 (467)a 0.28 (0.02)d

7 449 (96)a 536 (15)b 603 (10)bc 1588 (121)b 0.45 (0.03)b

14 279 (10)bc 424 (42)bc 630 (35)b 1333 (87)bc 0.37 (0.00)c

21 258 (28)c 225 (24)de 191 (2)d 674 (54)de 0.55 (0.02)a

pmol / embryo

28 105 (3)d 102 (25)e 85 (4.6)d 292 (27)e 0.53 (0.02)a

35 231 (21)c 322 (36)cd 225 (18)cd 778 (3)cde 0.50 (0.00)a

pmol / endosperm

28 391 (30)ab 457 (3)bc 757 (5)b 1605 (32)b 0.38 (0.01)c

35 73 (8)d 140 (7)e 443 (53)bcd 656 (68)de 0.22 (0.00)e

*Mean separation within rows by Duncan's multople range test, 5% level.

Table 4. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) of the embryo and gametophyte in seeds of Podocarpus macrophyllus. Each value is the average of three replicates. Standard errors are shown in parentheses. Seeds without radicle emergence when mature were selected for the experiment. Seeds began germination on the 10th day of incubation. Seeds reached 92% germination within 21 days.

Incubation ATP ADP AMP TA EC

(day)

nmol / embryo

0 1.78 (0.30)b* 1.11 (0.32)bc 1.54 (0.08)b 4.43 (0.48)b 0.53 (0.03)b

3 1.69 (0.15)b 1.12 (0.17)bc 1.41 (0.53)b 4.22 (0.41)b 0.53 (0.07)b

6 3.77 (0.26)b 0.96 (0.36)c 0.55 (0.28)b 5.28 (0.82)b 0.81 (0.06)a

15 2.59 (0.02)b 1.75 (0.02)b 0.88 (0.28)b 5.22 (0.07)b 0.66 (0.05)b

20 9.77 (2.16)a 4.56 (0.23)a 4.99 (1.49)a 19.32 (3.89)a 0.62 (0.01)b

nmol g-1 f.wt gametophyte

0 113 (13)a + 37 (12)ab 16 (2)ab 166 (26)a 0.79 (0.01)a

3 91 (1)ab 17 (14)b 21 (2)ab 129 (10)a 0.77 (0.07)ab

6 99 (9)ab 31 (3)ab 13 (1)b 143 (11)a 0.80 (0.01)a 15 84 (5)b 30 (5)ab 30 (2)a 144 (1)a 0.68 (0.03)b

20 98 (2)ab 48 (4)a 25 (5)ab 171 (11)a 0.71 (0.02)ab

*Mean separation for embryo (*) or gametophyte (+) within rows by Duncan's multiple range test, 5% level.

Table 5. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) in seeds of Cyclobalanopsis globosa. Each value is the average of three replicates. Standard errors are shown in parentheses. Only seed germinating by the 15th day were further dissected into the axis and cotyledon. Seeds reached 58.7% germination after 17 weeks incubation.

Incubation ATP ADP AMP TA EC

(day)

nmol / seed

0 3.80 (0.59)a* 4.77 (0.91)a 1.57 (0.24)ab 10.14 (1.23)a 0.61 (0.02)b

3 7.16 (2.84)a 3.65 (0.94)a 1.39 (0.44)ab 12.20 (2.44)a 0.74 (0.06)ab

6 4.75 (1.22)a 4.43 (0.44)a 2.20 (0.56)a 11.38 (1.97)a 0.61 (0.02)b

nmol / axis

15 1.43a 0.36b 0.15b 1.93b 0.83a

nmol / cotyl

15 7.66a 3.21a 0.84ab 11.71a 0.79a

*Mean separation within rows by Duncan's multiple range test, 5% level.


Chao and Lin Adenosine nucleotides of orthodox and recalcitrant seeds

the seeds had been incubated for 20 days. The quantities of ADP and AMP ranged from 1.11 to 4.56 and 1.54 to 4.99 nmol, respectively, during the 20 days of incubation. EC values varied from 0.53 to 0.81.

The axes of the other two species of recalcitrant types were too small to be dissected from their cotyledons, so the whole seeds were used for analysis. ATP, ADP, and AMP of C. globosa seeds up to the 15th day of germination (Table 5) maintained roughly the same levels during the germination period. On the 15th day of incubation, EC values increased from an initial 0.61 to 0.83 and 0.79 in the axis and in the cotyledon, respectively.

Table 6 displays the ATP, ADP, and AMP contents of N. parvigemma seeds at 4.37, 3.63, and 2.67 nmol, respec

tively. An increase in ATP and total adenylate levels in the beginning of incubation was not found. ATP decreased to 1.03 nmol per seed on the 6th day of incubation and increased to 9.27 nmol in the cotyledon on the 31st day of incubation, which coincided with seed germination. ADP and AMP decreased after an initial increase for 3 days of incubation. EC values increased from an initial 0.58 to 0.78 for the axis and 0.70 for the cotyledon by the 31st day of incubation.

The contents of adenylates on a dry weight basis in various fresh recalcitrant and dry orthodox seeds are summarized in Table 7. The orthodox species had moisture contents in the range of 6.1% to 7.0%, and EC values varied from 0.20 to 0.28. The ATP level was markedly lower

Table 6. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) in seeds of Neolitsea parvigemma. Each value is the average of three replicates. Standard errors are shown in parentheses. Only seeds germinating by the 31st day were further dissected into the axis and cotyledon. Seeds reached 93.3% germination after 15 weeks incubation.

Incubation ATP ADP AMP TA EC

(day)

nmol / seed

0 4.37 (1.70)b* 3.63 (0.68)ab 2.67 (1.20)ab 10.67 (3.36 )ab 0.58 (0.06)bc

3 3.38 (0.63)b 4.23 (0.58)a 6.07 (1.77)a 13.68 (2.94)ab 0.40 (0.02)cd

6 1.03 (0.22)b 2.27 (0.48)bc 5.31 (0.82)a 8.61 (0.25)bc 0.25 (0.06)d

15 2.62 (1.05)b 2.76 (0.62)b 3.11 (0.47)ab 8.49 (1.22)b 0.47 (0.0.9)c

nmol / axis

31 1.63b 1.04c 0.07b 2.74c 0.78a

nmol / cotyl

31 9.27a 4.16a 2.70ab 16.13a 0.70ab

* Mean separation within rows by Duncan's multiple range test, 5% level.

Table 7. Comparison of ATP, ADP, AMP, total adenylates (TA), energy charge (EC) and moisture content (MC) in various seeds. Each value is the average of three replicates. Standard errors are shown in parentheses.

ATP ADP AMP TA EC MC (%)

1. Orthodox seeds nmol g-1 d.wt

Soybean

Axis 3.3 (0.2) 16.8 (0.9) 38.2 (1.2) 58.3 (0.5) 0.19 (0.02) 6.1

Cotyl 9.9 (0.9) 48.5 (6.8) 83.1 (4.1) 141.5 (11.7) 0.24 (0.01) 6.1

Phellodendron wilsonii

Seed 1.3 (0.2) 6.3 (0.7) 15.0 (0.1) 23.6 (0.7) 0.20 (0.02) 7.0

Michelia compressa

Seed 10 (1) 28 (3) 47 (8) 85 (12) 0.28 (0.02) 6.6

2. Recalcitrant seeds nmol g-1 d.wt

Podocarpus macrophyllus

Fresh seed

Embryo 243 (43) 149 (32) 213 (21) 605 (27) 0.53 (0.03) 61.5

Gametophyte 220 (27) 73 (24) 31 (4) 324 (51) 0.79 (0.01) 48.5

Stratified seed

Embryo 491 (62) 114 (14) 78 (13) 642 (38) 0.80 (0.04) 61.5

Gametophyte 272 (6) 48 (6) 38 (9) 385 (9) 0.83 (0.03) 48.9

Machilus thunbergii

Fresh seed 91.1 (18.2) 42.7 (6.7) 9.2 (0.3) 143.0 (24.6) 0.79 (0.02) 44.7

Cyclobalanopsis globosa

Fresh seed 23.6 (2.6) 29.7 (3.9) 9.9 (1.8) 63.2 (3.5) 0.61 (0.02) 49.0

3. Intermediate category

Neolitsea parvigemma

Fresh seed 23 (6) 19 (2) 14 (5) 56 (11) 0.58 (0.06) 26.0


Botanical Bulletin of Academia Sinica, Vol. 37, 1996

Table 8. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) in seeds of Machilus thunbergii during artificial dehydration. Each value is the average of three replicates. Standard errors are shown in parentheses. Percentage of germination (G) was calculated after 35d of incubation at fluctuating temperatures of 30/20C, and 8 h light.

Dehydration ATP ADP AMP TA EC MC (%) G (%)

(day)

nmol g-1 d.wt

0 91a* 43a 9.2a 143a 0.79a 44.7 99.0

(18) (6.7) (0.3) (25) (0.02)

10 48b 34a 16a 98ab 0.66b 39.3 56.7

(3) (4) (4) (9) (0.04)

19 19bc 31ab 13a 63bc 0.55c 31.3 3.0

(6) (10) (1) (17) (0.02)

29 13c 12b 13a 38c 0.50c 22.5 0.0

(1) (1) (4) (4) (0.04)

*Mean separation within rows by Duncan's multiple range test, 5% level.

Table 9. Contents of ATP, ADP, AMP, and total adenylates (TA) and energy charge (EC) of the embryo and gametophyte in seeds of Podocarpus macrophyllus during artificial dehydration. Each value is the average of three replicates. Standard errors are shown in parentheses. Percentage of germination (G) was calculated after 21d of incubation at fluctuating temperatures of 30/20C, and 8 h light.

Dehydration ATP ADP AMP TA EC MC (%) G (%)

(day)

nmol / embryo

0 3.59a* 0.85a 0.56a 5.00a 0.80a _ 99.0

(0.61) (0.06) (0.40) (0.95) (0.04)

14 1.95ab 0.67a 0.45a 3.07b 0.74ab _ _

(0.37) (0.02) (0.09) (0.26) (0.06)

20 1.32b 0.91a 0.80a 3.03b 0.59b _ 0.0

(0.19) (0.09) (0.14) (0.42) (0.00)

nmol g-1 d.wt gametophyte

0 272a + 48b 38a 358a 0.83a 48.9

(6) (6) (10) (9) (0.03) (1.4)

14 177b 54b 27a 258b 0.79a 39.7

(1.4) (23) (2) (2) (23) (0.02)

20 168b 119a 31a 318ab 0.72a 33.9

(2.1) (25) (34) (12) (21) (0.07)

* Mean separation for embryo (*) or gametophyte (+) within rows by multiple range test, 5% level.

than ADP and AMP. Phellodendron wilsonii seeds were especially low in adenylates. The moisture contents of recalcitrant seeds ranged from 44% to 49%, except for the embryo of P. macrophyllus (61.5%). The quantities of adenylates were different in various species and the TA varied from 63 nmol g-1 d.wt in C. globosa to 324 nmol g-1 d.wt in P. macrophyllus. EC values varied from 0.61 in C. globosa to 0.79 in P. macrophyllus and Ma. thunbergii. ATP was the major form of adenylates with the exception of ADP in C. globosa.

Artificial Dehydration

The correlation between water content and adenylates in seeds of Ma. thunbergii and P. macrophyllus was presented in Tables 8 and 9. After drying treatment, the water content of Ma. thunbergii seeds decreased from 44.7% to 22.5%. Diminishing quantities of ADP and especially ATP resulted in the decline of EC values from 0.79 to 0.50 and TA values from 143 to 38 nmol g-1 d.wt. AMP remained the same. The germination percentage of seeds

dehydrated for 19 days decreased from 99% to 3%. The water content of P. macrophyllus seeds declined from 48.9% to 33.9% during the 20 days of dehydration. Germinability was totally lost at the 20th day of dehydration. ATP decreased from 272 to 168 nmol, ADP increased from 48 to 119 nmol, and AMP maintained similar levels. These factors resulted in a slight decline of TA and EC values.

Discussion

The maximum ATP accumulation in orthodox seeds coinciding with seed germination agrees with previous investigations (Ching and Ching, 1972; Moreland et al., 1974; Hourmant and Pradet, 1981). However, the time period required to reach the maximum ATP may be different depending on the species considered (Moreland et al., 1974; Lunn and Madsen, 1981). The increase in the quantity of ATP in tree seeds may not be as large as that observed in soybeans. The slow germinating seeds of M. compressa exhibited a pattern dissimilar to soybeans. The


Chao and Lin Adenosine nucleotides of orthodox and recalcitrant seeds

total adenylates in the process of germination were lower than those in the seeds before incubation. Further investigation is needed to disclose the energy metabolism of this type of orthodox seed.

Non-optimal temperatures could not stimulate the germination of seeds, but metabolism of reserves, including synthesis of adenylates, could be induced (Ching, 1975). The imbibed dormant seeds of Ph. wilsonii accumulated smaller amounts of ATP than non-dormant seeds. This may be a result of decreasing ATP synthesis and the level of adenylates during incubation eventually becoming depleted. The initial increase in ATP during incubation is definitely not related to seed germination as has been observed in wild oat seeds (Adkins and Ross, 1983).

An increase of ATP and TA in the cotyledon of soybean and the endosperm of M. compressa indicated biosynthesis of adenylates in the storage tissue as has been reported in the endosperm of germinating rice grain (Palmiano and Juliano, 1972) and in the gametophyte of germinating ponderosa pine seeds (Ching and Ching, 1972). This is a temporal rise of the energy supply to provide for the needs of enzyme production in the catabolic breakdown of reserves. A decrease in ATP content was found following seed germination and was continuous throughout the period of radicle emergence, an energy-consuming process (Ching and Ching, 1972).

High moisture content in freshly mature seeds characterizes recalcitrant species. The generally high content of ATP and ADP in mature recalcitrant seeds are due in part to the moderately high rate of respiration (Lin and Chen, 1995). Seeds of P. macrophyllus are viviparous, and roughly 25% of the mature seeds show an emergence of radicles, indicating a continuous metabolic activity. ATP was the most abundant energy component as reflected in a high EC value of about 0.8 in mature seeds. However, stratified P. macrophyllus seeds produced even more total adenylates, ATP, and energy charge than mature seeds (Table 7). This is similar to recalcitrant seeds, Quercus rubra, in which the seeds before and after stratification had a energy charge of 0.5 and around 0.8, respectively (Hopper et al., 1985). High adenylates in mature seeds of P. macrophyllus are probably sufficient for the consumption of germinating seeds, especially as no higher rate of adenylate production was observed as in other recalcitrant seeds.

Seeds of C. globosa may be considered another type of recalcitrant seed in terms of energy metabolism. Cyclobalanopsis globosa and also N. parvigemma both were characterized by much lower ADP and ATP contents and EC in mature seeds (Table 7), and slower germination than P. macrophyllus. It took 16 weeks and 15 weeks to complete germination of the seeds of C. globosa and N. parvigemma, respectively. The slight increase (if it exists) in the levels of total adenylates in seeds during the early stage of incubation, as in orthodox seeds, is not directly related to seed germination.

Differences in ATP contents between orthodox and recalcitrant seeds are obvious. The ATP contents in ortho

dox seeds are normally lower than 10 nmol g-1 d.wt while in recalcitrant seeds they range from 20 to 220 nmol g-1 d.wt depending on the species. ATP increased 167-fold in the first 2 days of incubation in soybean seeds, and almost 60-fold in 15 days of incubation in Ph. wilsonii seeds. The ATP quantities before and at seed germination in recalcitrant seeds remain similar. This finding indicates that recalcitrant tree seeds do not have to change the energy balance significantly as the orthodox crop seeds do. The increase in ATP levels in orthodox seeds during the early stages of germination is considered to be due to the onset of respiration and concomitant oxidative phosphorylation in the mitochondria as soon as seeds are hydrated (Hourmant and Pradet, 1981; Attucci et al., 1991). Recalcitrant seeds have not been examined in terms of energy metabolism in the early stage of seed germination.

During an artificial drying process, the degradation of adenylates, especially ATP, resulted in a decrease in total adenylates and energy charge in recalcitrant seeds. Whether the degradation of adenylates resulted from either enzymatic or non-enzymatic regulation remains unclear. Since AMP did not obviously accumulate in the seeds of Ma. thunbergii and P. macrophyllus during the drying process as has been observed in orthodox seeds (Obendorf and Marcus, 1974; Barua et al., 1981), a different mechanism for the degradation of ATP from that of orthodox seeds must be involved.

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