Content of Adenosine Phosphates and Adenylate Energy Charge in Germinating Ponderosa Pine Seeds

Plant Physiol. (1972) 50, 536-540

Received for publication May 18, 1972

TE MAY CHING AND KIM K. CHING Crop Science Department and Forest Research Laboratory, Oregon State University, Corvallis, Oregon 97331

ABSTRACT seed germination; (b) whether the gametophyte tissue (food An average of 540 picomoles of total adenosine phosphates storage) has an energy level and energy charge differing from was found in the embryo of mature seeds of ponderosa pine that in the embryo and seedling that may explain their di- (Pinus ponderosa Laws.) and 1140 picomoles in the gameto- vergent metabolic activities; (c) whether a correlation between phyte. Adenylate energy charges were 0.44 and 0.26, respec- the biogenesis of organelles (11) and energy level and energy tively. After stratification, total adenosine phosphates increased charge exists in the gametophytic tissue; and (d) whether co- 7-fold and 6-fold in embryo and gametophyte, respectively, and niferous seeds follow a pattern of energy metabolism during energy charges rose to 0.85 and 0.75. During germination, total germination similar to the angiosperm seeds. adenosine phosphates increased to a 20-fold peak on the 9th day in gametophytic tissue, parallel with the peak of reserve MATERIALS AND METHODS regradation and organellar synthesis, and then decreased. In embryo and seedling, total adenosine phosphates elevated 80- Uniformly large sized ponderosa pine (Pinus ponderosa fold with two distinct oscillating increases of AMP and ADP. Laws.) seeds were selected from a composite, highly viable lot The oscillating increases occurred before the emergence of of collections for 1970. Seeds were soaked in water at room radicle and cotyledons during which the highest mitotic index temperature for 4 hr, stratified at 5 C for 2 weeks, then germi- prevailed in all tissues. Energy charges fluctuated between 0.65 nated on Sponge Rok at a daily cycle of 15 C for 16 hr in the at the rapid cell dividing stage to 0.85 at the fully differentiated dark and 25 C for 8 hr with 1000 ft-c fluorescent light. stage of the seedling, while energy charges remained around Weight, Size, and Water Content Measurements. Four repli- 0.75 in the gametophyte. These data indicated that the content cations of 10 seeds were selected at each of the 12 develop- of adenosine phosphates of germinating seeds reflects growth, mental stages of germination (Table I). After the seedling organogenesis, and morphogenesis, and that a compartmental- length was measured, the seed coat was removed and the ga- ized energy metabolism must exist in dividing and growing metophyte was dissected. Fresh and dry weights (dried at 100 C plant cells. for 24 hr) were determined on gametophytes and embryos or seedlings for each replication. Water content was calculated from the difference of fresh weight and dry weight. Determination of Adenosine Phosphates. Four seeds of the designated stage of germination were dissected into gametophyte and embryo or seedling in a moist chamber. The dissec- tion of each seed took less than 10 sec in skilled hands. Each Seed germination requires the synthesis of enzymes and part was quickly extracted by 10 ml of boiling, glass-distilled organelles for catabolic degradation of reserve food in the water for 10 min at 100 C. The extract was cooled in an ice storage tissue and for anabolic building up of cellular constitu- bath, and 0.8 ml of the original (early stages) or diluted (later ents in the seedling (12). Because the synthesis of proteins and stages) extract was incubated at 30 C for 15 min in each of the nucleic acids is the major event in embryonic tissue, ATP is in following mixtures (8). great demand during seed germination (12, 19). ATP was A. For ATP determination, 0.1 ml of reaction buffer con- found to increase very rapidly upon imbibition of lettuce seeds taining 0.5 M N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic under optimum temperature and oxygen conditions (22). The acid (HEPES), pH 7.5, and 0.5 M magnesium acetate and 0.1 content of ATP or adenylate energy supply and energy charge ml of glass-distilled water. (EC = ([ATP] + 12 [ADP])/([ATP] + [ADP] + [AMP])) B. For ADP and ATP determination, 0.1 ml of the reaction often control metabolic activity and growth (1, 8, 22). A good buffer and 0.1 ml of solution containing 20 ,ug of pyruvate example of EC'-controlled metabolism was reported in imbibed kinase (EC 2.7.1.40) (Sigma, crystalline), and 500 nmoles of lettuce seeds. Anaerobiosis induced the activity of adenylate trisodium phosphoenolpyruvate (Calbiochem). kinase that in turn regulated the levels of adenosine phosphates C. For TAP, 0.1 ml of the reaction buffer, and 0.1 ml of to an extremely low EC and thus growth activities of the lettuce solution containing 20 ,ug of pyruvate kinase, 500 nmoles of seeds were limited (4). It would, therefore, be of interest to trisodium phosphoenolpyruvate. and 20 ,.tg of dialyzed (against discern (a) whether the energy level and energy charge are 1 mM phosphate buffer, pH 7.0) adenylate kinase (EC 2.7.4.3) regulating metabolic activities under normal conditions during (Sigma). After incubation, the extracts were either stored at 0 C overnight or assayed immediately by the luciferin-luciferase ' Abbreviations: EC: energy charges; TAP: total adenosine phos- method using an Aminco Chem-Glow photometer (24). The phates. procedure was modified from the St. John's method: two 536

Downloaded from on May 12, 2019 - Published by www.plantphysiol.org Copyright © 1972 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 50, 1972 ATP, ADP, AND AMP IN PINE SEEDS 537 replications of 0.4 ml each of the above mixtures were added meristems were stained in the older seedlings showing localized with 100,ud of firefly lantern extract (Sigma FLE-50; 50 mg of and limited mitotic activity. buffered extract inS ml of water), containing 50 mm potassium arsenate and 20 mm magnesium sulfate, pH 7.4. The instant RESULTS AND DISCUSSION peak height of light emission was used for all three mixtures to avoid endogenous ADP and AMP in the firefly extract inter- The changes in fresh weight, dry weight, and water content fering with the assay of tissue extract, particularly mixtures of the gametophyte and embryo or seedling during the course B and C. The peak height was linear to ATP concentration of of soaking, stratification, and germination are illustrated in 10nM to 10 4LM with the procedure outlined above and the Figure 1 and TableI. These changes are similar to other lots Aminco Chem-Glow photometer. For most batches of the of ponderosa pine seeds (11) and other coniferous tree seeds firefly extracts, a peak height of 20 to 40 equals 1,uM of ATP (10), indicating a normal course of development from an solution or 400 pmoles per 0.4-ml sample. The endogenous embryo enclosed in nutritive gametophyte to an autotrophic ATP in the firefly extract was depleted in the solution at 5 C seedling. Because of the lower (though simulating natural in 16 to 24 hr. Some degradation of the luciferase was observed condition) than usual laboratory germination temperatures during this period, but that did not affect the assay as an excess used, a slower rate of dry weight reduction in the gametophyte of enzyme was used. The step of constant addition of ATP in and a more gradual dry weight increase in the seedling were the original method (24) was not used in this study as the observed than in the previous study (11). quenching effect of various chemicals present in extracts was Energy metabolism is more dynamic in the embryo and minimized by dilution. seedling than in the gametophyte over the whole germination The boiling water extraction method was compared with the period (Fig. 2). In the embryo of mature seed, an average of 60 cold perchloric acid extraction (8) and the boiling ethanol ex- pmoles of ATP, 350 pmoles of ADP, and 130 pmoles of AMP traction (24) in the early stage of this study. The data obtained were found. The 0-day gametophyte contained 60, 480, and by the boiling water method were more reproducible and con- 600 pmoles, respectively. An average energy charge of 0.44 sistently higher than by the other two methods. Stewart and and an ATP concentration of 0.7 mm were found in the em- Guinn (25) also found that hot water extraction resulted in bryo and a lower energy charge of 0.26 and a much lower ATP highest and reproducible ATP quantity from cotton seedlings. concentration of 0.035 mM were observed in the gametophyte Furthermore, the recovery of pure ATP, ADP, and AMP (Fig. 3). A low energy charge is common in quiescent seeds (5, (P-L Biochemicals, Inc.) averaged about 97, 85, and 87% by 22); ponderosa pine appears to be no exception. After 4 hr of the method outlined above. soaking, TAP doubled in the gametophyte, but little change Calculation of the Content of Adenosine Phosphates and was observed in the embryo. This difference is probably at- Energy Charge. Based on the peak height of light intensity pro- tributable to the slowness of water penetration, which resulted duced from the three reaction mixtures, ATP concentrations in a lower degree of hydration in cells of the embryo. At a were read directly from a standard curve of freshly prepared 6.5% water content (Table I), the activation of organelles and ATP solution and the particular batch of enzyme-substrate enzymes in the embryo was clearly limited (12). The three preparation used for the assay. ATP content of the extract was species of adenosine phosphates changed, however, at different obtained from reaction mixture A, ADP content was the rates in thle soaked gametophyte with ATP to 310 pmoles, ADP difference of B and A, and AMP was the difference of C and B. to 1580 pmoles, and AMP down to 340 pmoles. These changes The adenylate energy level of the tissue was calculated to indicate a rapid phosphorylation of ADP to ATP by oxidative molar concentration by the total ATP and water content in respiration, an active conversion of AMP and ATP to ADP each gametophyte or seedling. The adenylate energy charge is calculated according to Atkinson (1): RADICLE COTYLEDON TOTALI Energy charge = ([ATP] + 1/2 [ADP])/([ATP] + [ADP] + [AMP]) 200K EMERGING EMERGING According to Atkinson (1), the energy charge modulates the activity of various metabolic sequences related to energy I20_ / / SEEDLING utilization and regeneration. When the energy charge is greater than 0.5, ATP-utilizing systems increase their activities. At a lower energy charge than 0.5 in cells, ATP-regeneration systems are dominant. Growing and multiplying cells maintain a high energy charge around 0.8, but senescent cells have low EC of 0.5 (8). Estimation of Mitotic Index in Different Tissue. Ten seeds for each germination stage were dissected, fixed in 95% ethanol-chloroform-glacial acetic acid (6:3:1 v/v) overnight, 10 hydrolyzed in 1 N HCI for 10 min at 60 C, and stained in Feul- gen for 3 hr (29). No cell division was ever observed in gameto- o -0;~ tSTANDARD 100- ~~~~~~~~\S phytic tissue, but the whole embryo was stained purple indicat- ,o ,,\DEVIATION ing its meristemic characteristics. To discern their sequence and rate of cell division, the embryo as well as seedling were di- vided into cotyledon tips, cotyledons, shoot meristem, hypocotyl, and root tip. Each portion was macerated and made into slide (15). Four radian areas containing at best O 2 4 6 8 10 12 0 2 4 6 8 10 12 14 a permanent 1-STRATIFICATION AT 5%C-C - GERMINATION AT 15-25-C 100 cells each per slide were counted for total number of cells DAYS and the number of dividing cells. Mitotic index was the per- FIG. 1. Changes in fresh and dry weight in gametophyte and centage of dividing cells in each area. Only shoot and root embryo or seedling during stratification and germination.

Table I. Age, Average Size, Morphological Characteristics, and Water Content of Seed or Seedling at Different Developmental Stages of Germination

Water Content Germination Stage Seedling Length Morphological Characteristics Gametophyte Embryo or seedling

0-Day 0.3 Mature, air-dried seed 6.1 + 0.5 3.2 + 0.1 4-Hr soaked 0.3 Imbibing 15.2 i4 1.1 6.5 + 0.5 4-Day stratified 0.4 Imbibed 25.4 + 0.8 40.2 + 1.0 10-Day stratified 0.4 Imbibed 33.5 i 1.0 53.6 + 0.8 14-Day stratified 0.4 Imbibed 40.2 4 1.0 62.3 ± 0.2 1-Day germinated 0.5 Swollen 48.5 4 0.3 65.8 4 0.8 3-Day germinated 0.5 Seed coat splitting 50.6 i 0.2 70.5 + 1.1 5-Day germinated 0.6-2 Radicle emerged 53.0 + 1.0 77.7 + 1.2 7-Day germinated 3-5 Radicle elongating 57.8 i 1.8 83.6 i 1.1 9-Day germinated 5-7 Hypocotyl crooked 66.9 + 1.8 85.4 ± 0.9 12-Day germinated 8-10 Cotyledons emerging 76.5 + 2.2 85.5 ± 0.4 15-Day germinated 12-14 Cotyledons elongating 84.3 4 2.0 87.0 i 1.1 18-Day germinated 15-18 Cotyledons emerged 94.0 4 2.5 86.9 ± 0.8

terials. Rapid increase of TAP upon hydration further indicated that the synthesizing machinery of these nucleotides is pre-existing in mature seeds. During stratification at 5 C, TAP increased gradually in the gametophyte to 6-fold and rather rapidly in the embryo to 7-fold of the original quantity (Fig. 2). ATP content rose to 2580 pmoles per embryo (0.675 mM) and to 3640 pmoles per gametophyte (0.266 M) after 2 weeks stratification. The energy charge also increased to 0.85 in the embryo and 0.75 in the gametophyte (Fig. 3). This energy supply permits synthetic sequences or energy-requiring pathways to occur when temperature becomes more adequate for growth and substrates are available; thus, a state of readiness for germination is created during this period. The beneficial effects of stratification or chilling of seeds were known to be increased soluble sugars

O 2 4 6 8 10 12 0 2 4 6 8 10 12 14 16 I8 H-STRATIFICATION AT 5%C---GERMINATION AT 15-25%C-.i DAYS FIG. 2. Changes in adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), and total adenosine phosphate (Total) in gametophyte and embryo or seedling during stratification and germination. by adenylate kinase, and a less active synthesis or a rapid utilization of AMP. Adenylate kinase was reported to be very active in imbibed lettuce seeds (4, 22). Adenosine kinase and 5-phosphoribosyl pyrophosphate synthetase were found in mature wheat embryo (23). Phytate, as phosphate reserve (14), and nucleosides are common in seeds (5, 17). These enzymes and i-STRATIFICATION AT 5C-C---- GERMINATION AT 15-25 -C substrates may possibly be present in pine gametophyte as well DAYS as embryonic cells. mitochondria are present Furthermore, in FIG. 3. Changes in adenylate energy charge and adenosine tri- mature seeds (11) and respiration usually takes place immedi- phosphate (ATP) concentration in gametophyte and embryo or ately after water imbibition in coniferous seeds (9). Nuclei are seedling during stratification and germination. ATP concentration also present in mature seeds and nuclear phosphorylation is in each part at different stages was calculated by dividing water possible (2) even though little has been explored in plant ma- content into the ATP content of the part.

Downloaded from on May 12, 2019 - Published by www.plantphysiol.org Copyright © 1972 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 50, 1972 ATP, ADP, AND AMP IN PINE SEEDS 539 and nitrogenous compounds (10), catalase activity, nucleic phyte of germinating seed showed a different pattern than the acids, mineral elements (18), and growth hormones or pro- embryo (Fig. 2). Apparently only two cycles of synthesis were moters (3, 27), elevated hydrolytic activity of starch and lipids evident. One is the result of activation of pre-existing enzymes, (18), and reduced growth inhibitors (3, 18, 27). The elevation mitochondria, and nuclei in the cell. The other is derived from of energy supply and energy charge as observed for the first the newly synthesized enzymes and mitochondria (1 1, 12). The time in this study could be considered as another beneficial second rise and fall of TAP and ATP concentration during effect of stratification of seeds. germination paralleled the developmental pattem of mito- As a result of the warm temperature stimulation on the pre- chondria and glyoxysomes in that tissue (1 1), indicating a con- existing systems, the embryo of stratified seeds quickly built up certed coordination of metabolic activity. A similar parabolic the content of TAP under germination conditions (Fig. 2). Ap- pattern of ATP changes in the endosperm of germinating rice parently, synthesis of RNA and proteins began simultaneously, grain has been reported recently (20), indicating a temporal and utilization of ATP exceeded biosynthesis, which resulted rise of energy supply to provide the need of enzyme production in a reduction of energy charge (Fig. 3) to 0.71 in 1 day. On the in catabolic breakdown of reserves. 3rd day, when the radicle was emerging, a distinct reduction of The energy charge increased from 0.26 in the gametophyte the TAP was again noted. In view of extremely active mitosis of dry seed to 0.75 in stratified seed, remained around 0.75 observed at this stage (Table II) the reduction probably is re- during germination, then lowered to 0.6 at the end of germina- lated to the active synthesis of cellular constituents. This tem- tion (Fig. 3). The maintenance of a relatively constant energy poral reduction had been observed in germinating peas (5). charge in nondividing gametophytic cells probably is a general A net loss of ATP may be attributed partly to the protrusion of characteristic of cells in storage tissue. Imbibed lettuce seeds radicles, which require energy for the mechanical work. (22) composed mainly of cotyledonous tissue and a very small The young seedling rapidly synthesized all three species of embryo axis are another example. Perhaps only at this steady adenosine phosphates after radicle emergence (Fig. 2). The state stage of determined metabolism can anaerobiosis alter TAP reached a peak on the 9th day of germination, reduced the energy charge in lettuce seeds and regulate their metabolism somewhat during cotyledon emergence, and then accumulated (4). again to a new plateau of 40 nmoles per seedling. This pattern In bacterial culture (8), the energy charge remained at 0.80 of increase follows the change in total nucleotides during seed during the growth period, even though cell number and total germination (10), and it appears to be the general pattern of adenylates increased at an exponential rate. During the sta- development with concerted events of temporal increase of en- tionary phase after cessation of growth, or during carbon- zymes, mitochondria, chloroplasts, nuclei, and other cellular source starvation, the energy charge of bacteria declined to 0.5 constituents accompanying the multiplication of cell number and then fell rapidly accompanying cell death. The authors, and the enlargement of cell size. The temporal control of en- therefore, suggested that growth can occur only at energy zyme synthesis during development is known (13), and the charge values above 0.8, that viability is maintained at values oscillation of different nucleosides triphosphate pools during between 0.8 and 0.5, and that cells die at values below 0.5. In cell cycles of synchronous cultures of bacteria has been re- sycamore cell culture (6), the energy charge was 0.73 at the ported (16). Therefore, the changes in the content of adenosine lag phase, 0.66 at the exponentially growing stage, and 0.81 at phosphates appear to be controlled by a genetically pro- the stationary phase. In germinating ponderosa pine seedlings, grammed pattern of development in embryonic tissue. a situation parallel to that of the sycamore cell culture is shown. The over-all EC was lower than 0.7 after radicle emergence During stratification, cell number was not increased (Table II), and during the rapid growth period of the seedlings, then in- but the EC was 0.85 (Fig. 3). During early stages of germina- creased to 0.9 and remained high during the period of cotyle- tion, all tissues were growing in cell number as evidenced by don expansion. Owing partly to the continuous increase of the high mitotic index (Table II), and the EC was 0.65 to 0.75 water content in seedlings and vacuolation in differentiated (Fig. 3). During the later stages of germination, the EC was cells, the over-all ATP concentration in tissue reduced con- high again (0.8 to 0.9), but the increase of cell number became tinuously from 0.7 mm in soaked seeds to 0.2 mM in com- limited to the meristemic tissues, which comprised only about pletely germinated seedlings. Differential concentrations of one-thousandth of the volume of the seedling. A distinct differ- ATP in various cell types and organs probably exist, but were ence in the energy status between bacteria cells and higher not studied in this work. plant cells is well illustrated in these studies. In cultured rat Changes in the content of adenosine phosphates in gameto- hepatoma cells, two independent adenine nucleotide pools

Table II. Mitotic Index in Embryo anid Seedlinzg of Germinatintg Ponzderosa Pinte Seeds

Shoot Stage Root !Meristem Cotyledon Tip Cotyledon MAeristem Hypocotyl % dividing cells 0-Day 0 0 0 0 0 7-Day stratified 0 0 0 0 0 14-Day stratified 0 0 0 0 0 1 -Day germinated 0 0 0 0 0.1 2-Day germinated 7.1 4 0.8 6.4 i 0.6 16.1 i 1.9 6.8 + 1.2 12.3 i 2.1 4-Day germinated 7.8 ± 1.1 4.3 i 0.6 11.2 =1= 2.3 3.6 i 0.3 11.8 ± 1.9 7-Day germinated 5.6 i 1.3 1.8 + 0.3 5.0 4 1.1 0.1 7.5 ± 1.5 9-Day germinated 5.1 i 0.2 0 7.2 + 1.8 0 4.3 ± 1.1 12-Day germinated 1.1 4 0.1 0 0.8 4 0.1 0 1.6 ± 0.3 18-Day germinated 0 0 1.4 + 0.3 0 1.5 i 0.2

were observed, one of which is cytoplasmic and the other 3. BLACK, M. 1970. Seed germination and dormancy. Sci. Progr. 58: 379-393. nuclear (21). Compartmental synthesis and utilization of vari- 4. BOMSEL, J. AND A. PRADET. 1968. Study of adenosine 5'-mono, di- and tri- ous adenosine in phosphates in plant tissues. IV. Regulation of the level of nucleotides, in phosphates nuclei, chloroplasts, and mito- vitro, by adenylate kinase: theoretical and experimental study. Biochim. chondria could occur in plant cells. Particularly, if one accepts Biophys. Acta 162: 230-242. the hypothesis that the immediate precursor of DNA in vivo 5. BROWN, E. G. 1965. Changes in free nucleotides and nucleoside pattern of be pea seeds in relation to germination. Biochem. J. 95: 509-614. may deoxyribonucleoside monophosphate (or diphosphate) 6. BROWN, linked with a carrier and that a E. G. AND K. C. SHORT. 1969. 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Compositional changes of Douglas-fir seeds during germi- sycamore support speculation. nation. Plant Physiol. 41: 1313-1319. The vacuoles commonly present in plant cells may cause 11. CHING, T. M4. 1970. Glyoxysomes of ponderosa pine seed. Plant Physiol. 46: further interference in energy charge values, because vacuoles 475-482. accumulate soluble compounds among which nucleotides are 12. CHING, T. M. 1972. Metabolism of germinating seeds. In: T. T. Kozlowlski, candidates. Even the identification of ed., Seed Biology, Vol. 2. Academic Press, New York. pp. 103-218. likely though nucleotides 13. FILNER, P., J. L. WRAY, AND J. E. V7ARNER. 1969. Enzyme induction in high in vacuoles has not been reported (7), an accumulation of plants. Science 165: 358-367. newly synthesized AMP in vacuoles is possible for the mainte- 14. HALL, J. R. AND T. K. HODGES. 1966. Phosphorus metabolism of germinating nance of a high energy charge in the cytosol. This possible in- oat seeds. 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Ribonuclease exists in Tatarian maple and little-leaf linden during stratification. Izv. Vlyssh. mature and germinating seeds (5, 12, 23, 26) and it is not linked Ucheb. Zaxved. Les. Zh. 12: 24-27. with metabolism. 19. MIARCUS, A. 1969. Seed germination and the capacity for protein synthesis. energy Furthermore, in germinating ponder- Symp. Soc. Exp. Biol. 23: 143-160. osa pine seedlings, transport of adenosine phosphates from 20. PALMIIANO, E. P. A_ND B. 0. JULIAN_O. 1972. Biochemical changes in the rice gametophyte to seedling could occur, and the meristematic cells grain during germination. Plant Physiol. 49: 751-756. may have different ATP levels and EC values than that of 21. PLAGEMANN, P. G. WV. 1971. Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture: II. Independent nucleotide pools for nucleic elongating (e.g., hypocotyl) and differentiating (e.g., greening acid synthesis. J. Cell Physiol. 77: 241-285. cotyledons) cells. 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