Osmosensitivity of Sucrose Uptake by Immature Pea Cotyledons Disappears during Development

Sucrose uptake was studied in isolated, immature pea cotyledons (Pisum sativum L. cv Marzia) in relation to their developmental stage. During the developmental period examined the water content of the cotyledons decreased from ≈80% “stage 1” to ≈55% “stage 2”. When assayed in an isotonic medium (400 osmoles per cubic meter) the influx capacity per gram fresh weight for sucrose was almost constant during this developmental period. The influx could be analyzed into a saturable component (K_m ≈ 9 moles per cubic meter; V_max ≈ 150 nanomoles per minute per gram fresh weight) and an unsaturable component (k_i ≈ 0.5 nanomoles per minute per gram fresh weight [per mole per cubic meter]). Incubation in a hypotonic medium reduced the sucrose influx in stage 1 cotyledons, up to 80% reduction at 0 milliosmole (medium without mannitol), but had no effect on sucrose uptake by stage 2 cotyledons. Reduced uptake in a hypotonic medium (100 osmoles per cubic meter) could be attributed to a lowering of the V_max from 150 to 36 nanomoles per minute per gram fresh weight. During incubation of stage 1 cotyledons and stage 2-cotyledons in a hypotonic medium (200 osmoles per cubic meter) their volume increased by 16% and 5.6%, respectively, while the calculated turgor pressure increased from 0.2 to 0.6 megapascal for cotyledons of both developmental stages. Reduced sucrose influx in hypotonic medium, therefore, seems to be related to cell swelling (membrane stretching) rather than to increased turgor pressure.     

Influence of Cotyledons upon alpha-Amylase Activity in Pea Embryonic Axes

α-Amylase activity remained relatively low in the axes of intact etiolated pea seedlings; the activity was predominantly confined to the epicotyl. Starch accumulated slightly. When the cotyledons were removed and the axes cultured on medium containing no carbon source, the starch reserve in the axes disappeared within a few days. This was accompanied by a 10- to 15-fold increase in α-amylase activity, in the absence of additional epicotyl growth. The phenonemon was observed for axes throughout early growth, although the relative accumulation of α-amylase activity in cultured axes was less for older seedlings. This change was attributed to a reduced response by nongrowing tissues. There was no corresponding change in β-amylase activity. These observations, described for several varieties of peas, demonstrate the control of cotyledons upon the utilization of stored reserves within the axis, with α-amylase as a key enzyme.     

Structural Development during Germination of Different Populations of Mitochondria from Pea Cotyledons

The crude mitochondrial fraction from pea cotyledons can, from days 1 to 7 of germination, be separated into three fractions by sucrose density gradient centrifugation. When seeds were grown in water (control) or cycloheximide (120 micrograms per milliliter of medium) for 4 days, the originally different populations of mitochondria acquired a uniform density and separated together in band 1 (density, 1.205 grams per milliliter). The oxidative and phosphorylative activities of mitochondria obtained from 4-day-old control and 4-day-old cycloheximide-treated pea seeds were the same. However, mitochondria from pea seeds that were grown in d-threo-chloramphenicol (1.5 milligrams per milliliter of medium) or erythromycin (0.5 milligram per milliliter of medium) for 4 days separate into three bands (fully developed mitochondria in the top band [band 1] and partially developed mitochondria in the lower two bands [bands 2 and 3]). Separation patterns and oxidative and phosphorylative activities were the same for mitochondria separated from 4-day-old cotyledons treated with d-threo-chloramphenicol or erythromycin and from 1-day-old cotyledons grown in water. This indicated that these inhibitors prevented the partially developed mitochondria originally in bands 2 and 3 from developing further. In contrast, cycloheximide did not seem to interfere with the mitochondrial structural development. These results along with those obtained from the experiments on the effects of d-threo-chloramphenicol, erthromycin, and cycloheximide on ¹⁴C-leucine incorporation into mitochondrial membrane proteins suggest that the increase in mitochondrial activity during germination may be a result of structural development (membrane synthesis) in pre-existing mitochondria.     

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.     

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.     

Cyanide-insensitive Respiration in Pea Cotyledons

Mitochondria isolated by a zonal procedure from the cotyledons of germinating peas possessed a cyanide-resistant respiration. This respiration was virtually absent in mitochondria isolated during the first 24 hours of germination but thereafter increased gradually until the 6th or 7th day of seedling development. At this time between 15 and 20% of the succinate oxidation was not inhibited by cyanide. The activity of the cyanide-resistant respiration was also determined in the absence of cyanide. Relationships among mitochondrial structure, cyanide-resistant respiration, and seedling development are discussed.     

Development of Mitochondrial Activities in Pea Cotyledons during and following Germination of the Axis

Development of mitochondrial activities in pea cotyledons during early times after the start of imbibition occurred in two phases. In the first phase (0 to 8 hours after the start of imbibition), succinate or NADH oxidation increased rapidly, while malate or α-ketoglutarate oxidation remained low. The latter activities developed only 8 to 12 hours after the start of imbibition (the second phase). Development in the first phase was induced by water uptake, but some development occurred even when the cotyledons were fully imbibed. The presence of the axis was required for the second phase of the development. It is suggested that mitochondrial development in the second phase is brought about by activation of the electron transfer path at a site between the oxidation of endogenous NADH and the reduction of ubiquinone.     

Developmental Regulation of Respiratory Activity in Pea Leaves

The developmental pattern of mitochondrial respiratory activity in pea (Pisum sativum) leaves has been investigated in an attempt to determine changes in mitochondrial function as plant cells mature. NADH and succinate dehydrogenase and cytochrome c oxidase activities remained relatively constant during cell maturation (from d 0 to d 14). Alternative oxidase and glycine decarboxylase activity, however, were low in young leaf tissue (d 0-6) but increased substantially as the tissue matured (d 7-14) and gained photorespiratory activity. Western blot analysis of the alternative oxidase protein revealed that it was primarily in an oxidized state in young leaves (d 0-6) but switched dramatically to the reduced form of the protein as the pea cells matured (d 7-14). The switch to the reduced form of the protein correlated with an increase in alternative oxidase activity. Results are discussed in terms of the changing function of plant mitochondria during leaf development.     

Changes in Properties of Succinate Dehydrogenase during Senescence of Pea Cotyledons

Comparisons were made between succinate dehydrogenases (EC 1.3.99.1 [EC] )from 1-day-old and 5-day-old pea cotyledons. The enzyme wasloosely bound to the mitochondrial inner membrane in 5-day-oldcotyledons, but tightly in 1-day-old cotyledons. In addition,the enzyme partially purified from 5-day-old cotyledons wasmuch more labile than that from 1-day-old cotyledons. Succinaterapidly inactivated partially purified succinate dehydrogenasefrom 1-day-old cotyledons, but not from 5-day-old cotyledons.Dithiothreitol caused a change in the charge of the enzyme proteinfrom either 1- or 5-day-old cotyledons, only when succinatewas present. The enzyme from 5-day-old cotyledons differed fromthe succinate-induced labile form of the enzyme from 1-day-oldcotyledons in electrophoretic properties on a polyacrylamidegel. There was also a difference in the pattern of polyacrylamidegel electrophoresis between succinate dehydrogenases partiallypurified from 1- and 5-day-old cotyleodns. The partially purifiedenzyme from either 1- or 5-day-old cotyledons in the presenceof succinate had a molecular weight of 92,000. The molecularweight of the large subunit was suggested to be 65,000. Thepartially purified enzyme prepared from 1-day-old cotyledonsin the absence of succinate was in a form with a molecular weightof 113,000. (Received August 29, 1980; Accepted December 3, 1980)     

Changes in Free Fatty-acid Content and Respiratory Activity during the Senescence of Cotyledons of Cucumber

During senescence the amount of free fatty acid in the chloroplastdecreases while the level in the tissue as a whole increases.Although this rise is sufficient to bring about an inhibitionof mitochondrial activity during the final stages of senescenceit occurs too late to explain the initial respiratory declinein cucumber cotyledons.     

Effect of Cycloheximide on Development of Mitochondria in Germinating Pea Cotyledons

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 µg protamine sulfate/ml, 0.15 m sucrose, and 10 m m MgSO₄ in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 × 10⁵ PFU/µg DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.     

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.     

胚轴对萌发豌豆子叶中淀粉酶活性表达的影响

萌发豌豆的上、下肢轴均能诱导子叶中淀粉酶活性,外源GA和6—BA具有类似胚轴的作用。离体子叶的淀粉酶凝胶电泳只有一条活性极低的酶带,连生子叶中有两条酶带,其中由胚轴诱导新出现了一条活性很高的同工酶带,它的活性受亚胺环己酮的强烈抑制,而受放线菌素D影响不大。推测豌豆子叶中存在淀粉酶的长寿命mRN—A,胚轴和外源激素的作用在于促进mRNA的翻译。     

Changes in Activities of Enzymes of Nitrogen Metabolism in Seedcoats and Cotyledons during Embryo Development in Pea Seeds

In the seedcoats of developing pea seeds, the maximal activities of asparaginase (EC 3.5.1.1) and aspartate: α-ketoglutarate aminotransferase (EC 2.6.1.1) are attained early in development, before the embryo has expanded to fill the embryo sac. These two enzyme activities could account for the early absence of asparagine and aspartate from the fluid secreted by the seedcoats into the embryo sac.     

Pressure-Driven Extrusion of Intracellular Substances from Bean and Pea Cotyledons during Imbibition

Intracellular substances leak from imbibing cotyledons of grain legumes during imbibition. This work reports the discovery of a biophysical process by which intracellular substances are driven from cotyledons during imbibition. Light and scanning electron microscopy were used to examine bean (Phaseolus vulgaris L.) and pea (Pisum sativum L.) cotyledons and the material released from them into imbibition water. A large fraction of the visible materials released from excised bean and pea cotyledons during the first 30 minutes of imbibition consisted of convoluted or helical streams of material which rapidly emerged from the cotyledons surfaces. Large streams of material from bean cotyledons contained starch grains and protein bodies, and smaller streams from bean and pea cotyledons probably contained protein bodies. The forms of streams were characteristic of a viscous fluid which had been forced by pressure through irregular orifices. The sites of extrusion from bean cotyledons were multicellular blisters which formed on the surfaces of imbibing cotyledons. In 6 hours, pea and bean cotyledons leaked from 1 to 11 micrograms protein per milligram of seed dry weight. The quantities of protein leaked primarily depended on cultivar.     

Gibberellic Acid and Starch Breakdown in Pea Cotyledons

The stimulation of starch breakdown in the cotyledons of dwarfpea cultivars (Progress No. 9 and Dark Skin Perfection) whentreated with gibberellic acid (GA³) is mediated through theremoval of metabolites by the axis. When applied to excisedcotyledons, GA³ had only a minor effect on starch breakdown,as it did in the cotyledons of a tall pea (cv. Alaska) irrespectiveof whether they were attached to the plant or excised. Enzyme activity appears to be controlled by the level of solublesugars in the cotyledons, and GA³delays the increase in amylolyticactivity in the cotyledons through a direct effect on cotyledonmetabolism.     

Changes in mRNA of Vigna mungo Cotyledons during Seed Germination

Quantitative and qualitative changes of mRNA in Vigna mungocotyledons during seed germination have been investigated. TotalRNA is higher in dry cotyledons and declines during germination.Poly(A)⁺ RNA also is present at a relatively high level in drycotyledons, increases slightly during the first day of germination,and then decreases. Polysomal RNA is very low in dry cotyledonsbut increases rapidly during the first day of germination, andthen declines. The translational activity of the mRNA in a wheatgerm cell-free system is low on day 0 but increases rapidlyon day 1 of germination. Two-dimensional gel electrophoresisof in vitro translation products reveals that many new peptidesare synthesized on day 1 of germination. Synthesis of most ofthese polypeptides continue throughout 5 days of germination. Change in the mRNA population during germination has been investigatedusing cDNA against poly(A)⁺ RNA from 3-day-old cotyledons. Withtotal RNA of day 3 and 5, the cDNA strongly hybridized withRNA similar in size to 25 S ribosomal RNA, but no specific bandsare detected with samples of day 0 or 1. With poly(A)⁺ RNA ofday 5 or 1, the cDNA tends to hybridize with RNAs of relativelysmall molecular size. Cordycepin and -amanitin prevent the increasein poly (A)⁺ RNA content and the appearance of new mRNAs duringthe first day of germination. ¹Present address: Division of Regulation of Macromolecular Function,Institute for Protein Research, Suita City, Osaka 565, Japan. (Received January 13, 1986; Accepted June 10, 1986)     

Respiratory Transition during Seed Germination

Experiments with germinating seeds of Wayne soybean (Glycine max Merr.) show that between the 4th and the 8th hour of germination, respiration experiences a transition from predominantly “alternate” respiration, which is sensitive to salicylhydroxamic acid, to a cyanide-sensitive respiration. The dependence of early germination stages on alternate respiration is reflected in several types of seed functions, including subsequent root growth rate, chlorophyll synthesis, and germination itself. The early period of germination is shown to require a normal O₂ tension, which is no longer a requirement at later stages. The changing sensitivity to cyanide and to salicylhydroxamic acid is found to be common to seven different types of germinating seeds. It is proposed that the alternate pathway of respiration provides something essential for the completion of the earliest stages of seed germination.