Biogenesis of Mitochondria in Imbibed Peanut Cotyledons: Influence of the Axis

Development of mitochondrial activities (state 3 respiration,respiratory control ratio, ADP/O ratio) in peanut cotyledonsoccurs over the first 5 d from the start of imbibition. Mitochondriain cotyledons with the axis attached develop better than inthose from which the axis has been removed. Initially, mitochondriaare deficient in cytochrome c, but after 2 d from the startof imbibition this deficiency is overcome. Mitochondrial developmentin attached cotyledons, as measured by state 3 respiration,respiratory control ratio, ADP/O ratio, and succinate dehydrogenaseand cytochrome oxidase activities, is severely impaired by cycloheximide.This indicates that de novo synthesis of proteins is necessaryfor mitochondria and their enzymes to develop, a situation whichis in sharp contrast to the situation in pea cotyledons. Electronmicroscope studies also show that there is an increase in thenumbers of mitochondria in peanut cotyledons with time afterthe start of imbibition. Two patterns of mitochondrial developmentexist in legumes: in imbibed peanut cotyledons respiratory activitiesincrease due to biogenesis of mitochondria, whereas in pea cotyledonsthe increases are due to improvement of pre-existing organelles     

Development of Mitochondrial Activity in Pea Cotyledons Following Imbibition; Influence of the Embryonic Axis

The development of mitochondria in cotyledons during the initialstages following imbibition and the subsequent degenerationwere faster when the embryonic axis was attached. This was moreclearly demonstrated when mitochondrial activity was determinedusing malate or -oxoglutarate as substrate rather than NADHor succinate. Cycloheximide did not inhibit the initial developmentof mitochondria in attached cotyledons, although it inhibitedthe incorporation of ¹⁴C-amino acid into protein. Abscisic acidinhibited almost completely the initial increase in mitochondrialactivities of attached cotyledons. The activities of severalenzymes in mitochondrial fractions were almost the same in attachedand detached cotyledons during 4 d after, imbibition. The degenerationof mitochondria was not accompanied by the loss of enzymes.It was inferred that the initial development and the subsequentdegeneration of mitochondria in cotyledons during 4 d afterimbibition was brought about by the structural improvement anddisorganization of mitochondria, respectively. The initial differencesin the development of mitochondrial activities between attachedand detached cotyledons were suggested to be attributable todifferences in the development of the activities of electrontransfer pathway from endogenous NADH to ubiquinone.     

Appearance and Disappearance of Cyanide-Resistant Respiration in Vigna mungo Cotyledons during and following Germination of the Axis

Mitochondrial preparations isolated from black gram (Vigna mungo L.) cotyledons exhibited cyanide-resistant respiration which was of mitochondrial origin. The appearance and the disappearance of this alternative respiration took place during and following imbibition. During the first 6 hours of imbibition, the respiration was completely inhibited by cyanide, but after this time the alternative respiration markedly developed, reaching a maximal cyanide-resistance 12 to 16 hours after the start of imbibition. Subsequently, the alternative respiration gradually disappeared. The actions of cycloheximide and chloramphenicol indicated that the appearance was dependent on cytoplasmic protein synthesis and that the disappearance depended on both cytoplasmic and mitochondrial protein synthesis. The alternative pathway contributed to state 4 respiration, but not to state 3 respiration, in mitochondria from 1-day-old cotyledons. On day 3, it contributed to neither state 3 nor state 4.     

Studies on the Development of Mitochondria of Mung Bean Cotyledons During Imbibition

The development of mitochondria in the course, of imbibition ofmung bean cotyledons was studied. Mitochondria were prepared by differential centrifugation, and purified by discontinuous sucrose, density gradient centrifugation. Theelectron micrographs revealed that the mitochondria isolated from dry seeds cotyledonslook like small vesicles, when cotyledons were imbibed for two hours, the mitochondrial cristae were not observed, but after four hours, a few cristae appeared on the innermembrane. Till 12 hours, the inner membrane systems wore well-developed. Al this time.all the space in mitochondria are filled with cristae. With the structural integrity of the mitochondria, the functions of oxidation and phosphorylation were graduallyshown, For instance, ADP/O ratio and RCR were not able to be measured in the imbibition of 2 and 4 hours, but at 6th hours, ADP/O Was increased by 0.6, RCR nearly 2.0, After 24 hours imbibition, ADP/O and RCR were increased to 1.5 and 3.5 respectively. The activity of cytochrome oxidase reached the highest after imbibition for 3 hours (2.54 OD/mg protein/min). If cotyledons were imbibed continuously, the activityof this enzyme in mitochondria remained constant. The activity of ATPase located onthe inside of the mitochondrial inner membrane was gradually enhanced from the beginnning of imbibition. These results suggest that the assembly of cytochrome oxidaseand ATPase on mitochondrial membrane may not be synchronous.     

Influence of the axis on the development of mitochondrial activity in imbibed black gram cotyledons

The respiration rate of mitochondria from detached Vigna mungo L. cotyledons (without the embryonic axis) doubled during the first day after imbibition, whereas that of mitochondria from attached ones increased by only 50%. Contrary to the respiration rate, the respiratory control ratio was higher in attached cotyledons. The activities of enzymes in the mitochondrial fraction from detached cotyledons increased by about 30% during the first day, while those in mitochondria from attached ones changed little. Cycloheximide did not inhibit the development of mitochondrial respiration in either attached or detached cotyledons, although it almost completely inhibited the incorporation of [¹⁴C]-leucine into mitochondrial proteins. Cycloheximide did not retard the increase in the activities of mitochondrial enzymes in detached cotyledons. It is inferred that the development of mitochondria in black gram cotyledons is brought about by repair or activation of mitochondria present in dry seeds and that the axis affects this repair process.     

Development of Mitochondrial Activities in Pea Cotyledons: INFLUENCE OF DESICCATION DURING AND FOLLOWING GERMINATION OF THE AXIS

Mitochondria in 4-hour imbibed and desiccated pea cotyledons develop in a similar manner upon rehydration to those in cotyledons hydrated only once. As a consequence of desiccation during imbibition, mitochondria revert to their original state as in the mature dry cotyledon, although limited damage occurs. This damage is more evident when the initial imbibition time before desiccation is longer. The presence of the axis does not protect cotyledonary mitochondria from damage, nor does it influence mitochondrial development upon rehydration of desiccated cotyledons. During the early hours after the start of imbibition mitochondrial development is dependent both upon hydration levels of the cotyledon and upon other metabolic processes, as shown by sensitivity to conditions that prevent ATP synthesis.     

Rapid Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination

Rapid increases in activities and components of mitochondrial particles isolated from cotyledons of Pisum sativum var. Alaska during the early stage of germination are described. Respiratory rate of the cotyledons increased rapidly as hydration proceeded. A similar but slightly delayed increase in respiratory activity of the isolated mitochondrial fraction was observed. The respiratory control ratio and adenosine 5′-pyrophosphate/oxygen ratio rose during imbibition. Cytochrome oxidase and malate dehydrogenase activities in the mitochondrial fraction increased during the initial phase of imbibition. The increase seemed to precede that in respiratory activity. A significant activity of cytochrome oxidase and most of the malate dehydrogenase activity in the cotyledons were present in the postmitochondrial fraction in the case of the dry seeds. Mitochondrial protein and phospholipid also increased during imbibition, and the rise in the components seemed to concur with that in respiratory activity. The mechanism of mitochondrial development during imbibition is discussed.     

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons : II. DEVELOPMENT OF LIGHT AND HEAVY MITOCHONDRIA

There are two types of mitochondria present in imbibed peanut cotyledons: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known.     

Spatially regulated genes expressed during seed germination and postgerminative development are activated during embryogeny

Summary We investigated the control of genes expressed primarily during seed germination and postgerminative development in Brassica napus L. We identified cloned mRNA sequences which became prevalent within 1 day after the start of imbibition and were at low or undetectable levels in immature embryos, dry seeds, and leaves. Most postgermination-abundant mRNAs accumulated primarily, though not exclusively, in different parts of the seedling. Of the 14 cloned mRNAs, 8 were prevalent in cotyledons, 2 were abundant in seedling axes, and 4 were approximately equally distributed in both parts. We showed that although these mRNAs reached maximal levels in seedlings, the spatially regulated mRNAs were also detected at distinct embryonic stages; mRNAs prevalent in seedling axes accumulated primarily during early embryogenesis while cotyledon-abundant mRNA concentration increased during late embryogeny. We conclude that the temporal and spatial regulation of gene expression in seedlings reflects similarities and differences in the physiological functions of cotyledons and axes. Furthermore, the regulated expression of cotyledon-abundant genes during late embryogeny suggests that the mRNAs and possibly proteins may accumulate in preparation for subsequent seedling growth. Similarities in the accumulation of cotyledon-abundant mRNAs may indicate coordinate regulation of this gene set.Abbreviations DAF days after flowering - DAI days after the start of imbibition - HAI hours after the start of imbibition - kb kilobase(pairs)     

Biochemical Studies on Development of Mitochondria in Pea Cotyledons during the Early Stage of Germination: Effects of Antibiotics on the Development

l-Leucine-U-¹⁴C was incorporated into mitochondrial protein in pea (Pisum sativum var. Alaska) cotyledons during the imbibing stages. Incorporation was almost completely inhibited by cycloheximide but not by chloramphenicol. Both antibiotics did not affect increases in mitochondrial activities and components of the cotyledons during imbibition. Therefore, mitochondrial development seems to be achieved by a transfer of protein pre-existing in the cytoplasm into the mitochondria rather than by de novo synthesis of mitochondrial protein. Cycloheximide stimulated an increase in bile saltsoluble protein of mitochondria in imbibing pea cotyledons. The recovery of cytochrome oxidase activity after sucrose density gradient centrifugation was enhanced, and the morphological properties of mitochondria were altered by cycloheximide.     

Development of Pyrimidine-metabolizing Enzymes in Cotyledons of Germinating Peas

Mechanisms controlling conversion of orotic acid-6-¹⁴C to uridine-5′-phosphate in cotyledons of germinating Alaska peas (Pisum sativum L.) were investigated. The content of 5-phosphoribosyl-1-pyrophosphate was very low in dry seeds, increased to a maximum after about 12 hours of imbibition, and then rapidly declined. Orotidine-5′-phosphate pyrophosphorylase and orotidine-5′-phosphate decarboxylase activities more than doubled during the first 24 hours of germination and then also decreased. These results do not account for the continuous increases of orotate anabolism in such cotyledons as we observed previously. The initial increases in activities of these two enzymes were unaffected by cycloheximide, while the subsequent decreases were less rapid in the presence of this inhibitor. Activities of cotyledonary cytidine deaminase and uridine hydrolase also increased during imbibition, but the activity of only the latter showed a decrease after imbibition was completed. Cycloheximide inhibited the initial rapid increase in uridine hydrolase activity but had little effect on its subsequent decline. Cycloheximide had only slight inhibitory effects on the development of cytidine deaminase activity during the first 62 hours. The evidence suggests that uridine hydrolase might be synthesized de novo during the first few days of germination, but that the other three enzymes might not be.     

Presence in Dry Pea Cotyledons of Soluble Succinate Dehydrogenase That Is Assembled into the Mitochondrial Inner Membrane during Seed Imbibition

SUCCINATE DEHYDROGENASE (SUCCINATE: phenazine methosulfate oxidoreductase, EC 1.3.99.1) activity in crude mitochondrial fraction from pea (var. Alaska) cotyledons increased during seed imbibition to reach a maximum after about 12 hours. The increase was not inhibited by either cycloheximide or d(-)threo-chloramphenicol. The postmicrosomal fraction from dry cotyledons, but not that from fully imbibed ones, contained a soluble form of succinate dehydrogenase. The soluble enzyme was partially purified by ammonium sulfate fractionation and diethylaminoethyl-cellulose and Sepharose 6B column chromatography. The enzyme showed no succinate-coenzyme Q oxidoreductase activity and had a molecular mass of about 100,000 daltons. The soluble enzyme seemed to differ only slightly from succinate dehydrogenase solubilized from the mitochondrial inner membrane from fully imbibed cotyledons by a detergent. It is proposed that the soluble succinate dehydrogenase is associated with an inert mitochondrial inner membrane in dry cotyledons to form an active one during seed imbibition.     

Patterns of mitochondrial development in reserve tissues of germinated seeds: A survey

Patterns of mitochondrial development in reserve tissues of several species of seeds were examined during 3 to 4 days after the start of imbibition. In starch-storing seeds (cowpea, kidney bean, Egyptian kidney bean, mung bean, black gram and soybean), mitochondrial activities (state 3 respiration rate, respiratory control ratio) increased during the first 1 to 2 days after imbibition and leveled off thereafter. The initial increase in the activities was little affected by cycloheximide and chloramphenicol. Activities of mitochondrial enzymes (succinate dehydrogenase, EC 1.3.99.1; NAD⁺-malate dehydrogenase, EC 1.1.1.37; cytochrome oxidase, EC 1.9.3.1) did not change much during the experimental period. This suggests that mitochondrial development in starch-storing seeds is primarily due to improvement of pre-existing mitochondria. On the other hand, in the lipid-storing seeds examined (pumpkin, cucumber, okra and castor bean), the rate of mitochondrial respiration and activities of mitochondrial enzymes continued to increase markedly during the experimental period. These increases were strongly inhibited by cycloheximide and chloramphenicol. This indicates that active de novo synthesis of mitochondrial proteins is primarily responsible for the development of organelle activities. The possibility was suggested that the patterns of mitochondrial development in reserve tissues of imbibed seeds might be determined by the kinds of reserve substances.     

Mobilisation of the major stored reserves in the embryo of fenugreek (Trigonella foenum-graecum L., Leguminosae), and correlated enzyme activities

Changes in total nitrogen, soluble amino nitrogen, lipid and phytate contents, and in the activities of proteinase (pH 7.0), isocitrate lyase and phytase were followed in the endosperm, cotyledons, and axis during germination of fenugreek seeds and subsequent growth of the seedlings. The endosperm is comprised largely of cell-wall galactomannans: the majority of the seed total nitrogen, lipid and phytate (5%, 8%, 0.44% of seed dry weight respectively) is localised within the cotyledons as stored reserves. Germination is completed after 10–14 h from the start of imbibition, but the major reserves are not mobilised during the first 24 h. Then the total nitrogen content of the cotyledons starts to decrease and that of the axis increases; there is a concomitant accumulation of soluble amino nitrogen in both cotyledons and axis. An increase in proteinase activity in the cotyledons correlates well with the depletion of total nitrogen therein. Depletion of lipid and phytate reserves in the different seed tissues constitutes a late event, occurring after 50 h from the start of imbibition, and is coincident with the final disintegration of the endosperm tissue. The depletion of phytate and stored lipids is accompanied by an increase in phytase and isocitrate lyase activity. It appears that the products of lipid hydrolysis are converted by gluconeogenesis to serve as the major source of sugars for the growing axis after the endosperm galactomannan has been completely mobilised.     

Uptake of benzyladenine by excised watermelon cotyledons

The uptake of 8-[¹⁴C]N⁶-benzyladenine (BA) was studied in excised watermelon (Citrullus vulgaris Schrad.) cotyledons 24 hours after the start of imbibition. The passive nature of this uptake is suggested by the following evidence: (a) no sign of saturation on increasing external concentration of BA; (b) no decrease in uptake under conditions that inhibit ATP synthesis; (c) no change in amount of radioactivity absorbed when cotyledons are frozen and thawed before the uptake test. About two-thirds of the radioactivity taken up is released after 12 hours of washing. If the washing is performed at 2 C very little radioactivity is released.     

Biphasic oxidation of mitochondrial NAD(P)H

The redox state of mitochondrial pyridine nucleotides is known to be important for structural integrity of mitochondria. In this work, we observed a biphasic oxidation of endogenous NAD(P)H in rat liver mitochondria induced by tert-butylhydroperoxide. Nearly 85% of mitochondrial NAD(P)H was rapidly oxidized during the first phase. The second phase of NAD(P)H oxidation was retarded for several minutes, appearing after the inner membrane potential collapse and mitochondria swelling. It was characterized by disturbance of ATP synthesis and dramatic permeabilization of the inner membrane to pyridine nucleotides. The second phase was completely prevented by 0.5 microM cyclosporin A or 0.2 mM EGTA or was significantly delayed by 25 microM butylhydroxytoluene or trifluoperazine. The obtained data suggest that the second phase resulted from oxidation of the remaining NADH via the outer membrane electron transport system of permeabilized mitochondria, leading to further oxidation of the remaining NADPH in a transhydrogenase reaction.     

Endoplasmic Reticulum of Mung Bean Cotyledons: ACCUMULATION DURING SEED MATURATION AND CATABOLISM DURING SEEDLING GROWTH

Homogenates of mung bean cotyledons were subjected to equilibrium density centrifugation on linear sucrose gradients and the positions of the various organelles determined by assay of marker enzymes. Measurement of phospholipid distribution on such gradients showed that the major peak of phospholipid at a density of 1.11 to 1.13 grams per cubic centimeter coincided with the position of the endoplasmic reticulum (ER), confirming ultrastructural evidence that storage parenchyma cells are rich in ER. Germination and seedling growth were accompanied by a rapid decline in ER-associated phospholipid but a marked increase in the ER marker enzyme NADH cytochrome c reductase. Similar experiments with developing seeds indicated that the amount of ER-associated phospholipid increases during cotyledon expansion reaching a maximum during seed maturation. There was no subsequent decline during seed desiccation, instead ER-associated phospholipid levels were maintained in the dry seed until germination when catabolism was initiated 12 to 24 hours after the start of imbibition. This timing indicates that the observed ER breakdown is not an expression of the overall senescence of the cotyledons, but may represent the dismantling of the extensive rough ER used for reserve protein synthesis during cotyledon development.     

In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA

Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.     

Root Formation by Detached White Mustard (Sinapis alba) Cotyledons

Rooting is shown to occur in excised cotyledons of Sinapis alba when grown in petri dishes on moist filter paper. Cotyledons were excised at intervals from 6 hours after the start of imbibition, when they were yellow, unexpanded and enclosed within the testa, to 27 days after sowing when the cotyledons were green and fully expanded and on plants possessing up to 3 foliage leaves. Rooting generally began 5 or 6 days after excision and was completed dining the following 5 days. The age of cotyledons at t ho time of excision had three effects on rooting: the lag period be-fore rooting began and the period during which rooting took place both increased with age: but the most marked effect was on the total number of cotyledons which were able to form roots, which increased until cotyledon expansion was almost complete, then decreased as the mature cotyledons became older. Optimal rooting was shown by cotyledons detached 8 days alter sowing, when they were half expanded. At this age S5 % of them formed roots between 6 days and 8 days after excision.     

The development of ribonuclease and acid phosphatase during germination of Pisum arvense

1. Development of ribonuclease activity in the cotyledons of germinating peas is biphasic, the time of appearance of the two phases depending on the conditions of growth. 2. Acid phosphatase exhibits a single phase of development. 3. Cycloheximide inhibits development of ribonuclease activity in phase II but not in phase I. 4. (14)C-labelled amino acids are not incorporated into ribonuclease isolated during phase I. 5. The buoyant density of ribonuclease isolated during phase I is not affected by imbibition of the seed in 80% deuterium oxide. 6. Acid phosphatase was isolated from the supernatant fraction of the cotyledons of germinating peas and partially purified. 7. Development of acid phosphatase activity during germination is inhibited by treatment of the seed with cycloheximide or actinomycin D. 8. Partial purification of acid phosphatase from peas germinated in the presence of (14)C-labelled amino acids suggests that the enzyme is radioactively labelled. 9. Germination of peas in the presence of 80% deuterium oxide results in an increase in the buoyant density of acid phosphatase. 10. The results suggest that increase in ribonuclease activity during the first 4 days of germination does not result from synthesis of protein de novo, but that the corresponding increase in acid phosphatase activity does result from synthesis de novo.