Rapporti Tra Assunzione D'acqua,Metabolismo e Sintesi di Enzimi Nell'endosperma Di Semi Germinanti di Ricinus Communis

Abstract

Water uptake, activation of metabolism and enzyme synthesis in germinating castor bean seeds. — During the first days of germination water uptake by the castor bean seed endosperm is accompanied by a rapid rise of respiratory activity and of the « in vitro » detectable activity of a number of enzymes. The finding that the increase of enzyme activity is strongly inibited by protein synthesis inhibitors suggests an « ex novo » synthesis of enzymes in the endosperm of the germinating seed. The present investigation on the relationship between water uptake, metabolic activity and enzyme activity level lead to the following conclusions:

I - The increase of enzyme activity is strictly dependent on the availability of water, and on the rate of water uptake. When water uptake is depressed by incubation of the seed in high osmolarity media, enzyme activation is also severely depressed.

This is also observed when the seeds are germinating in contact with an amount of water consistently lower then the one they would taken up, in a given time (24 h), under conditions of unlimeted water availability.

II - The temperature coefficient of water uptake is close to 1.5 during the first 24 h, higher than 2 in the following 3 days. Low temperature almost completely inhibits the increase of enzyme activities in the endosperm.

III - Anaerobiosis inhibits the rate of water uptake by about 50%, in the first 24 h, and almost completely, in the following 3 days. Also the rise of enzyme activities is severely inhibited by lack of oxygen. The effect of protein synthesis inhibitors on water uptake is somewhat smaller, and the one on enzyme activity is somewhat larger than that of anaerobiosis.

These results are interpreted as indicating that during the early period of germination water uptake becomes more and more dependent on the metabolic activities of the endosperm cells, in as much the latter lead to the appearance of osmotically active substances and, possibly, to changes of the cell wall properties.

On the other hand, the level of hydration of the cytoplasm represents a limiting factor for the development of the mechanism involved in enzyme synthesis and metabolic activation.     

Inattivazione dei Mitocondri Negli Endospermi di Ricino Durante la Maturazione

Abstract

inactivation of mitochondria in the castor bean seed endosperm during ripening. — The present research deals with the behaviour of mitochondria from the endosperm of castor bean seeds, during the last phases of seed maturation. The activities of citochrome oxidase, of malate dehydrogenase, of the succinate-citochrome c reductase system, and the phosphorylating activity, were chosen as tests of the state of mitochondria.

The results obtained show an increase of the first two activities up to the moment when some ovular tissues are still present, and, successively, a more or less rapid inactivation of the three enzymes investigated, which fall to extremely low values in the dry seed. Also the phosphorylating capacity, high during the first phases, drops quickly as the seed approches to dormancy.

A certain amount of citochrome oxidase is revealed in the supernatant from 20000xg centrifugation made to prepare the mitochondrial fraction; its activity gradually increases as the seed advances to ripeness. A further fractionation of the activity not sedimenting at 20000xg reveals that approximately one half of it sediments when centrifugated for 1 hour at 50000xg, while the other half remains in the supernatant. The particles sedimenting between 20000 and 50000xg show very little, if any, phosphorylating capacity (with succinate).

It is suggested that the gradual inactivation of mitochondrial efficiency during the ripening phase is due to a degradation of mithochondrial structures.     

Variazioni di Metaboliti Nel Seme di Ricino in Fase di Maturazione

Abstract

Changes of glycolytic substrates level during ripening of the castor bean seed. — The changes of the concentration of carbohydrates and of the main glycolytic substrates in the castor bean seed during the ripening phase were investigated. The following results were obtained:

The level of unphosphorylated sugars and of acid hydrolysable polysaccharides remains almost unchanged, with a tendency to a rise during the ripening phase. The slight increase of these compounds, together with the transition of the R. Q. from high to low values, might be interpreted as an indication of a shift of the seed from the a metabolism of fat synthesis to one of conversion of lipids into sugars, such as is observed in the germinating castor bean seeds.

Hexose monophosphate level sharply decreases during the last period of maturation. However, the level of these substrates does not fall so low as to suggest a severe limitation for the pentose-P pathway activity.

Fructose diphosphate, DOAP, GAP, 3 PGA, 2 PGA, PEP and pyruvate levels consistently increase during the ripening process. This indicates that the drop of oxygen uptake observed in this phase cannot be due to a lack of glycolytic substrates. On the other hand, the ratios between some substrates are shifted, during ripening, from values close to the theoretical equilibrium constants to quite different values. This finding, when correlated with the one of the strong decrease of the glycolytic flow, strongly suggests a severe inactivation of the glycolyting enzymes during ripening.

The increase of pyruvate in tissues showing a decreasing respiratory activity indicates a fall of the oxidative capacity of mitochondria. This might be due to a lack of ADP, or other high energy bond acceptor, following a block of synthetic processes. However, no decrease of ADP level, and an increase of the ADP/ATP ratio during ripening is observed, Among the alternative hypothesis: a) lack or excess of oxalacetate; b) increase of concentration of some Krebs cycle inhibitor; c) inactivation of mitochondrial enzymes, the latter is thought most probable, in view of the finding of a sharp decrease of some other enzyme activities during ripening, of the above mentioned interpretation of the shift of the ratios between glycolytic substrates, and of the very low level of mitochondrial activity in preparation from the mature castor bean seed. These results when correlated with those from parallel investigations on the biochemistry of castor bean seed maturation and germination suggest, as a working hypothesis, that the respiratory metabolic inactivation accompanyng seed repening is due to a general block of the metabolism of ribonucleic acid and thus protein synthesis.     

Effetto di Inibitori Della Sintesi Proteica Sull'aumento di Attività Enzimatiche e sul Risveglio del Metabolismo Nell'Endosperma di Semi di Ricino in Germinazione

Abstract

Effects of inhibitors of protein synthesis on the development of metabolic activity in the endosperm during the germination of castor bean seeds. — The effect of chloramphenicol, streptomycin and actinomycin-C on the increase of the activities of glyceroaldehyde-phosphate dehydrogenase, aldolase, glucose-6-phosphate dehydrogenase, fructose 1–6 diphosphate-1-phosphatase, phosphomonoesterase, in the endosperm of germinating castor bean seeds was investigated.

In all cases, the protein synthesis inhibitors depressed the activation of the enzymes tested: in particular, actinomycin (50 μg/ml) completely suppressed the increase of the activities.

The development of the rate of oxygen uptake and the conversion of fats to sugars was strongly affected by the inhibitors.

These data suggest that the increase of the activities of several enzymes in the germinating endosperm is dependent on enzyme synthesis rather than on the conversion from the inactive to the active form of the enzymes.     

Rapporti Fra Endosperma ed Embrione Nella Germinazione di Pinus Pinea L.

Abstract

Endosperm embryo interrelationships during germination of PINUS PINEA L. (2nd Note). — Experiments have been performed with unripe fresh seeds paralleled with unripe stored seeds (50 days of storage).

The effect of storage is very similar to the effect of further permanence on the tree.

It is confirmed the importance of the endosperm in the germination process already put in evidence in the 1rst note.

The relationships existing between endosperm and embryo during the germination of the seed become more and more strict as the seed ripens, so that in a ripe seed the embryo develops in a normal seedling only in connection of its own endosperm.

In an unripe seed the embryo is uncompletely controlled by its endosperm.

Some remarks have been made about the degree of maturity of the different parts of the embryo.     

Aspetti Enzimatici Della Maturazione e Germinazione dei Semi di Ricino

Abstract

Enzyme levels during ripening and germination of castor bean seeds. — During the development of the endosperm of castor bean seeds two distinct phases can be recognized: pre-maturation and germination. The former is characterized, metabolically, by the rapid conversion of carbohydrates into lipids, and storage proteins. The latter is characterized by the reconversion of these storage materials into sugars. Both these processes are dependent upon the activity of the glycolytic pathway; for this reason the behaviour of some enzymes of this pathway and, in general, of the carbohydrate metabolism have been studied during the two phases. The changes (during the evolution of the seeds) of the following enzymes have been studied:

Gl-6-P-dehydrogenase, 6-P-gluconate dehydrogenase, P-glucomutase, Hexokinase Hexoseisomerase, Aldolase, alcaline and acid Phosphatase, Pyrophosphatase and ATP-ase.

All these activities have been measured in the 20.000 × g supernatant fraction of cell homogenates.

The results show that all the enzymes activities measured increase rapidly during the period of accumulation of storage materials. In the following period all of these activities decrease until the stage of ripeness of the seed. During the first few days of germination the activities increase again rapidly. A particular behaviour is the one of Fr-1-6-P-phosphatase (the enzyme cleaving the phosphate bond in C ¹ position). This enzyme reaches during germination a level much higher than the maximal observed during the ripening process. This might be an important fact correlated with the inversion of the glycolytic reactions during germination.     

Biosynthesis of Starch in Proplastids of Germinating Ricinus communis Endosperm Tissue

Electron photomicrographs of endosperm tissue from germinating seed of Ricinus communis L. cv. Hale show proplastids which contain prominent starch grains. The content of starch in endosperm tissue increased from 500 micrograms per seed, in imbibed seed, to 1,100 micrograms per seed in 5-day-old seedlings. The maximum net rate of starch deposition was 1.1 nanomoles glucose incorporated per minute per seed. About 200 micrograms of starch remained in the endosperm 9 days after imbibition. Starch content followed the same developmental pattern as the content of sucrose, free reducing sugars, and other metabolic processes found in this tissue. Two key enzymes of starch synthesis, adenosine diphosphoglucose (ADPG) pyrophosphorylase and ADPG-starch glucosyl transferase (starch synthetase) exhibited maximum activities at 4 and 5 days after germination, respectively. The maximum activity of ADPG pyrophosphorylase was 8.17 nanomoles ADPG formed per minute per seed, whereas starch synthetase exhibited an activity of 125 nanomoles glucose incorporated per minute per seed. These levels of enzyme activity are sufficient to account for the starch synthesis observed. Other enzymes which may be involved in starch synthesis include 3-phosphoglycerate kinase which showed an activity of 8.76 units per seed, triose-P isomerase (2.56 units per seed), fructose-1,6-bisphosphate aldolase (0.99 units per seed), fructose-1,6-bisphosphatase (0.23 units per seed), phosphoglucose isomerase (12.6 units per seed), and phosphoglucomutase (9.72 units per seed). The activities of these enzymes were similar to previously reported values.

Starch synthetase was found in association with the fraction containing proplastids isolated from endosperm tissue. Of the total starch synthetase activity in the endosperm, 38% was particulate. Forty-four% of the total particulate activity of starch synthetase placed on sucrose gradients was associated with the band containing proplastids. The proplastids contained 98% of the ribulose 1,5-bisphosphate carboxylase carboxylase activity placed on the gradient.

     

Primi Processi di Germinazione in Pinus Pinea L. var. Fragilis Du Hamel

Abstract

Early stages of germination in PINUS PINEA L. var. FRAGILIS Du Hamel. — The main stages of the hydration process preceeding germination and accompaning root elongation have been observed in Pinus pinea L. seeds, by means of vital colours (Congo red, neutral red, acid fuchsine).

The results are as follows:

a) water penetrates easily through the outer shell of the seed reaching its deepest layer which is less permeable to water. Two or three days were required in our experience for water could overcome this barrier.

b) The inner shell (known as « soft shell ») is almost water-proof and seems to draw water towards the micropilar pole of the seed, so that the first region of the seed which sucks up water is the micropile.

c) Through the micropile water enters the seed and imbibes the column, the pericolumn and the endosperm cells.

The endosperm swells with water until the seed shell blows up, because of the inside pressure. At this time water freely penetrates the seed everywhere.

In natural conditions we may infer that the first tissues which take contact with the soil water are the column and pericolumn. As a certain amount of time is required for penetration of water as far as the column (two or three days in experience conditions) germination starts only after a given amount of water is available in the soil for a certain period of time.

When seed hydration is performed the embryo root starts elongating and gets out of the seed.

The behaviour of the column, the pericolumn and the root cap during the early stages of germination are dexcribed.     

Regulation of development of leucine uptake activity by glutamine in the scutellum of germinating barley grain

Scutella from ungerminated grains of barley (Hordeum vulgare L. cv Pirkka) take up leucine at a slow rate, which increases rapidly during germination. When endosperms were removed from the grains after imbibition for 4 hours or after germination for 12 or 72 hours, the increase in the rate of leucine uptake was greatly accelerated during subsequent incubation of the embryos or scutella. These increases were rapidly inhibited by cordycepin and cycloheximide, suggesting that protein synthesis, probably synthesis of the carrier protein, was required for the development of the uptake activity.

In separated embryos or scutella, the increases in the leucine uptake activity were inhibited by glutamine. The inhibitions caused by glutamine and cycloheximide were not additive, suggesting that glutamine did not interfere with the function of the carrier but repressed its synthesis. Glutamine did not inhibit the simultaneous increase in peptide uptake; in this respect, its effect was specific for leucine uptake, which appears to be due to a general amino acid uptake system.

Some other protein amino acids also inhibited the increase in leucine uptake without inhibiting the increase in peptide uptake. However, these effects were smaller than that of glutamine.

These results suggest that the transfer of leucine (and other amino acids) from the endosperm to the seedling in a germinating barley grain is regulated at the uptake step by repression of the synthesis of the amino acid carrier protein by glutamine and—possibly to a lesser extent—by some other amino acids taken up from the endosperm.

     

Alcuni Aspetti Citologici Dell'Endosperma di Phaseolus Coccineus L.

Abstract

Some cytological aspects of Phaseolus coccineus L. endosperm. — Cytological observations were made on the endosperm of Phaseolus coccineus, in a limited stage of development of the seed, using different dyes and labelling with ³H-thymidine and ³H-uridine. The results seem to indicate that, contrary to suspensor cells, in the endosperm of Phaseolus, at least in the considered stage, extra DNA synthesis is not present. Chromosomes in these nuclei, however, undergo several endoreduplication cycles. Nucleoli may be either one or more in a cell, and show a characteristic structure. They are often eccentric in the nucleus and extrusions of nucleolar content in the nucleoplasm or cytoplasm are seen. An apparent alternate activity of RNA synthesis in the nucleoli of the suspensor and in those of endosperm cells is discussed.     

Ricerche Comparate Sulla Morfologia e Sulla Fisiologia di Larix e di Picea

Abstract

Comparative researches on morphology and physiology of PICEA and LARIX. The chlorophyll content of seeds and seedlings during germinations in darkness. — Very different are the chlorophyll contents of the seeds and the seedlings of Picea excelsa and Larix decidua grown in darkness.

In Picea chlorophyll is abundantly synthetized in cotyledons in the passage from seed to seedling stages. In Larix however this synthesis is very poor. These differences are more evident after the outgrowth of cotyledons from the primary endosperm and its exhaustion.

If seedlings of both species are exposed to 3.000 lux of light intensity for 24 hours, after development in darkness, one can observe much stronger chlorophyll synthesis in Larix cotyledons than icea.

These figures quanti atively express the different light-dipendence of chlorophyll synthesis in these two plants and offer a more adherent interpretation of the different ecological behaviours that are caracteristic of these two plants.     

Characterization of asparaginyl endopeptidase activity in endosperm of developing and germinating castor oil seeds

A spectrophotometric assay was devised to characterize the asparaginyl (Asn) endopeptidase activity from the endosperm of castor oil seeds. (Ricinus communis L. var. Baker 296). The assay measures the release of p-nitroaniline from the hydrolysis of benzoyl-l-Asn-p-nitroanilide. Assay sensitivity was improved through diazotization of the reaction product with N(]-napthy])-ethylenediamine dihydrochloride: diazotized p-nitroaniline was determined spectrophotometrically at 548 nm (?₅₄₈= 1.64 × 10^?1M^?1 cm^?2). By using this assay. Asn endopeptidase activity was detected in endosperm extracts of developing, mature and germinating castor seeds. Comparison of the Asn endopeptidase activities of developing and germinating castor endosperms revealed that they: 1) have identical pH-activity profiles with optimal activity occuring at pH 5.4: 2) are heat-labile proteins displaying comparable thermal stability profiles, and 3) are activated and inhibited by dithiothreitol and thiol modifying reagents, respectively. Thus, the Asn endopeptidases of developing and germinating castor seeds are very similar, if not identical, cysteine proteases. The most significant increase in the activity of endosperm Asn endopeptidase occurs during the full coryledon to maturation stage of seed development, this period coincides with the most active phase of reserve protein accumulation by ripening castor oil seeds. Asn endopeptidase activity of fully mature (dry) castor seeds was about 2-fold lower than that of muturation stage ripening castor oil seed. Asn endopeptidase activity showed a slight reduction over the inicial 2-day period following seed imbibition, and then rapidly decreased over the next several days of germination. The results are compatible with the proposal that Asn endopeptidase functions both to process storage preproteins following their import into protein bodies of developing seeds, as well as to participate in the mobilization of storage proteins during the early phase of seed germination.     

Abscisic acid controls embryo growth potential and endosperm cap weakening during coffee (<Emphasis Type="Italic">Coffea arabica</Emphasis> cv. Rubi) seed germination

The mechanism and regulation of coffee seed germination were studied in Coffea arabica L. cv. Rubi. The coffee embryo grew inside the endosperm prior to radicle protrusion and abscisic acid (ABA) inhibited the increase in its pressure potential. There were two steps of endosperm cap weakening. An increase in cellulase activity coincided with the first step and an increase in endo--mannanase (EBM) activity with the second step. ABA inhibited the second step of endosperm cap weakening, presumably by inhibiting the activities of at least two EBM isoforms and/or, indirectly, by inhibiting the pressure force of the radicle. The increase in the activities of EBM and cellulase coincided with the decrease in the force required to puncture the endosperm and with the appearance of porosity in the cell walls as observed by low-temperature scanning electronic microscopy. Tissue printing showed that EBM activity was spatially regulated in the endosperm. Activity was initiated in the endosperm cap whereas later during germination it could also be detected in the remainder of the endosperm. Tissue printing revealed that ABA inhibited most of the EBM activity in the endosperm cap, but not in the remainder of the endosperm. ABA did not inhibit cellulase activity. There was a transient rise in ABA content in the embryo during imbibition, which was likely to be responsible for slow germination, suggesting that endogenous ABA also may control embryo growth potential and the second step of endosperm cap weakening during coffee seed germination.     

Osservazioni Sull'effetto Inibente di Elevate Pressioni Osmotiche su Diversi Tipi di Crescita

Abstract

On the inhibition of seeds germination and of growth by cell enlargement by the osmotic pressure of the medium. — The mechanism of inhibition by osmotic pressure (O.P.) of the medium on growth and respiration of germinating wheat, castor bean and lettuce sèeds and of etiolated pea internode segments was investigated.

The following results were obtained:

1 - External osmotic pressure (up to 0.3 M) of various substances such as mannitol, urea, glucose, NaCl, was shown to inhibit the germination and growth of lettuce, wheat and castor bean seeds.

2 - a) A remarkable decrease of the development of respiration during the first 48 h of germination was demonstrated in embryos of wheat seeds germinated and maintained in mannitol solutions at concentration from 0,2 to 0,3 M.

b) A slight but reproducible inhibition of óxygen uptake by O.P. was also observed in embryos isolated from wheat seeds germinated in water for 24 and 34 h and transported respectively in water or into 0,2 M mannitol solutions.

This is interpreted as indicating that high external O.P. inhibits both the respiratory metabolism and the development with time of enzyme systems supporting respiration.

3 - Mannitol solutions (0,2–0,3 M) inhibited completely growth by cell enlargement in pea internode sections, while they did not at all affect oxygen uptake and protein synthesis ( ¹⁴ C - leucine incorporation). The stimulatory effect of auxin on pea elongation was almost completely suppressed by mannitol, whereas the hormone stimulation of respiration remained unchanged.

These data are interpreted as indicating that in tissues, presenting an advanced differentiation, high external O.P. inhibits growth by a direct physico-chemical mechanism; while the inhibitory effect in embrional tissues seems to comprehend, besides this direct effect, a complicated metabolic component, apparently influencing protein synthesis.     

Immunolocalization of endo-β-mannanases in Phacelia tanacetifolia seeds during early phases of germination

ABSTRACT

Endosperm weakening is a key event for completion of seed germination in plants such as tomato and tobacco. Weakening is related to the action of endo-β-mannanases able to hydrolyse the mannose polymers typically stored in the wall of the endosperm cells. In this study, we determined the presence and the localisation of endo-β-mannanases in Phacelia tanacetifolia seeds during the early phases of germination. In endosperm cells of dry seeds, and of seeds incubated in the light for 16 h, a similar distribution of endo-β-mannanases, mainly localised in protein bodies, was revealed by immunolocalization. In contrast, under conditions of permissive germination (seeds incubated for 16 h in the dark), these enzymes appeared localised near the cell walls, and were no longer detectable in protein bodies. Western blot analyses showed the presence of three isoforms of endo-β-mannanases in the endosperm and one isoform in the embryo. All these isoforms had similar molecular weights (approx 38 kDa). A possible role of endo-β-mannanases during early phases of germination is suggested.     

Phosphoenolpyruvate carboxylase activity and concentration in the endosperm of developing and germinating castor oil seeds

Monospecific polyclonal antibodies against maize leaf phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were utilized to examine the subunit composition and developmental profile of endosperm PEPC in developing and germinating castor oil seeds (Ricinus communis L. cv Baker 296). PEPC from developing endosperm consists of a single type of 100-kilodalton subunit, whereas the enzyme from 2- to 5-day germinated endosperm appears to contain equal proportions of immunologically related 103- and 108-kilodalton subunits. The maximal activity of PEPC in developing endosperms (2.67 micromoles oxaloacetate produced per minute per gram fresh weight) is approximately 20-fold and threefold greater than that of fully mature (dry seed) and germinating endosperms, respectively. The most significant increase in the activity and concentration of endosperm PEPC occurs during the middle cotyledon to full cotyledon stage of seed development; this period coincides with the most active phase of storage oil accumulation by ripening castor oil seeds. The data are compatible with the recent proposal (RG Smith, DA Gauthier, DT Dennis, DH Turpin [1992] Plant Physiol 1233-1238) that PEPC plays a fundamental role in vivo in the cytosolic production of an important substrate (malate) for fatty acid biosynthesis by developing castor oil seed leucoplasts. Immediately following seed imbibition, PEPC activity and concentration increase in parallel, with the greatest levels attained by the third day of germination. It is suggested that during this early phase of seed germination PEPC has a critical function to build up cellular dicarboxylic acid pools required to initiate significant activities of both the tricarboxylic acid and glyoxylate cycles.     

Nuove Prospettive Nello Studio dei Fattori che Controllano la Germinazione Dei Semi

Abstract

New perspectives in the study of factors which control seed germination. — Seedlings of Triticum durum, cv. « Cappelli », coming from unirradiated embryos grafted on to irradiated endosperms (EM(u)/EN(i) of presoaked seeds (in distilled water for 24 h. at 20[ddot]C.) (treatment: X-rays, doses, 2,4,6,8,10 and 20 Kr), grow more than seedlings of control EM(u)/EN(u) (dose 0) (fig. 1). To have this reaction, it is necessary that the used seeds be after-ripe; at the various stages of seed ripening, be ginning from the milk stage, the phenomenon is not present.

On this basis, the author has thought that a natural inhibitor occurs in the after-ripe endosperm of « Cappelli », which is neutralized or destroyed by X-rays.

As an experimental demonstration, some trials have been made of growing wheat seedlings in Petri dish, on moistened (distilled water) filter paper, together with excised embryos or isolated endosperms (fig. 2): the after-ripe endosperm is able to depress the seedling growth (fig. 3). In the same experimental conditions, X-rays, dose 6 Kr, neutralize the inhibition effect given by the endosperm. (fig. 4).

A completely different situation occurs in wheat seed, during its ripening: endosperm is inactive, embryo produces inhibition effects on the seedling growth, which, also in this case, are reduced by X-rays.

These phenomena, put in relation with dormancy in Triticum durum, cv. « Cappelli », which is a relative dormancy, having its maximum at the milk stage, have led the author to the general conclusion that, during dormancy, a germination inhibitor occurs in the embryo of wheat seed; when dormancy is finished, the inhibitor appears in the endosperm, in a situation which becomes stable and definitive.     

Localizzazione intracellulare della clorogenico ossidasi in tessuti di tubero di patata in fase di attivazione

Abstract

INTRACELLULAR LOCALIZATION OF CHLOROGENIC ACID OXIDASE IN AGED POTATO TUBER DISCS. — The localization in the cells of chlorogenic acid oxidase has been investigated in potato tubers and discs of potato tuber. It has been ascertained that the rise of activity per gram of tissue, after preparation of discs, is not due to bacterial or fungal growth. The activity is widely distributed among cell fractions. Some activity is found in mitochondria, while most of the activity is distributed among soluble fraction and a « microsomal » fraction sedimented by centrifugation in the range 15.000–50.000 x g. This fraction appears to contain mitochondrial fragments, fragments of the endoplasmic reticulum and ribosomes. When tuber discs are aged, the rise of chlorogenic acid oxidase activity is much larger in the soluble than in particulate fraction.