Studies on energy status and mitochondrial respiration during growth and senescence of mung bean cotyledons

Several characteristics of mitochondrial respiration and energy status have been studied during growth and senescence of mung bean ( Phaseolus radiatus L.) cotyledons. The results showed that mitochondrial oxygen consumption, respiratory control, ADP:O ratios, and energy charge changed in the cotyledons during germination and growth of the seedlings. The respiration rate of intact cotyledons approximately reflected the trend of the oxidative activities of the isolated mitochondria. An increase was observed in both whole cotyledon respiration and mitochondrial oxygen uptake at the onset of senescence of mung bean cotyledons (day 3 after germination), which thereafter declined gradually. The capacity and activity of the alternative pathway increased markedly in mitochondria isolated from senescent cotyledons. After the onset of senescence, the mung bean cotyledon mitochondria exhibited a decrease both in the respiratory control ratios and ADP:O ratios, and the cotyledons exhibited a gradual decline in energy charge. All these results showed an irreversible deterioration of energy conservation in mung bean cotyledons. The role(s) of the alternative pathway in senescent mung bean cotyledons is discussed.     

萌发绿豆子叶衰老期间蛋白质代谢的变化

萌发绿豆子叶自然衰老过程中可溶性蛋白质含量一直下降;从衰老开始到衰老前期,总游离氨基酸含量明显上升;但游离氨基酸各组分在子叶衰老期间的变化趋势并不相同。~3H-亮氨酸掺入蛋白质试验和多聚核糖体的相对量及其与总核糖体的比值(P/T)测定都证明在子叶衰老前期有蛋白质的新合成。子叶衰老期间。氨肽酶活性明显降低;而以酪蛋白为底物的蛋白水解酶活性却急剧上升,承担着催化蛋白质降解的主要功能。     

ATP Synthesis in Cotyledons of Cucumber and Mung Bean Seeds during the First Hours of Imbibition

ATP concentration increased rapidly during the first 6h of imbibitionin cotyledons of cucumber and mung bean seeds. The increasewas strongly inhibited by 1-h treatment of tissues with cyanidein both species. Carbonylcyanide m-chlorophenylhydrazone, anuncoupler of oxidative phosphorylation, showed little effectduring the first hour of the treatment, but its inhibitory effecton ATP synthesis became significant after 3 h. Mitochondrialfractions prepared from 6-h-old cucumber cotyledons were capableof phosphorylating ADP to ATP. These results suggest that mitochondrialoxidative phosphorylation may be involved in ATP synthesis duringthe early hours of imbibition in both cucumber and mung beanseeds. (Received December 7, 1987; Accepted April 9, 1988)     

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.     

萌发绿豆子叶衰老期间的呼吸作用与线粒体功能的改变

暗中培养的绿豆幼苗子叶在萌发后3—4天时,外观出现衰老征状,6天后子叶凋落。随子叶日龄的增加,子叶的呼吸强度一直下降,呼吸商始终小于1。当外加L—苹果酸、a—酮戊二酸、琥珀酸和NADH为底物测定离体线粒体氧化活性时,衰老子叶的线粒体对上述四种底物的氧化活性有不同程度的增加;抗氰呼吸也有所升高。子叶衰老时,线粒体的ADP／O和呼吸控制(RC值均降低);线粒体ATPase水解ATP的活性升高。衰老绿豆子叶线粒体氧化磷酸化偶联效率的降低和ATPase水解活性的增强是与线粒体结构改变相联系的一种功能变化,它导致能量亏缺,并进一步加速了衰老的恶化进程。     

Disappearance of desiccation tolerance of imbibed crop seeds is not associated with the decline of oligosaccharides

The relationship between sugar content and loss of desiccation tolerance of hydrated crop seeds (tomato, okra, snow pea, mung bean, and cucumber) was evaluated by imbibing seeds with or without ABA, followed by dehydration and germination. During the process of hydration, but before the seeds lost desiccation tolerance, monosaccharide content increased only slightly, sucrose increased in snow peas, mung bean and cucumber, but maintained its original level in other species and the oligosaccharides declined dramatically. At the time of losing desiccation tolerance, the sucrose content of imbibed seeds was 2-3 times higher than the original level in most species. Positive significant correlation coefficients (r) were found in many, but not all crop seeds between desiccation tolerance and the oligosaccharide mass, or oligo/sucrose ratio. The ratio of oligo/sucrose in intact seeds at the time of losing desiccation tolerance, however, was not a fixed value and varied among species. Oligosaccharides declined significantly in different seed parts of imbibed cucumber seeds while sucrose increased to a higher level in the radicle than in the hypocotyl. Radicles were far more sensitive to desiccation than hypocotyls. The same observation was found for cucumber seeds imbibed in 100 M ABA, yet desiccation tolerance was largely maintained in hypocotyls and cotyledons. It is concluded that sucrose and oligosaccharides are not the determinants of the loss of desiccation tolerance in hydrated seeds.Imbibed seeds did not show any differences between seed parts in their ability to resynthesize sugars during the process of slow dehydration. Differences in sensitivity to desiccation among seed parts were not due to differences in the initial water content or to the rate of water content increase among seed parts. Physiological regulation of the loss of desiccation tolerance in crop seeds during hydration is discussed.     

Pullulanase in mung bean cotyledons. Purification, some properties and developmental pattern during and following germination

Starch debranching enzyme was purified from mung bean ( Vigna radiata ) cotyledons to investigate its properties and developmental pattern during and following germination. A debranching enzyme was purified up to the step where only a doublet of polypeptides with molecular masses of 99 and 101 kDa, respectively, was detected by SDS-PAGE. The enzyme is thought to be a single chain monomer, as the molecular mass of the enzyme determined by gel filtration was 72 kDa. Monoclonal antibodies raised against the purified preparation recognized the doublet, indicating that the two polypeptides have immunological homology to each other. The enzyme preparation showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with glycogen. These substrate specificities indicate that the debranching enzyme from mung bean cotyledons is of the pullulanase type. Immunoblotting profiles revealed that the enzyme is present in dry seeds and decreases gradually after imbibition, suggesting the possibility that the pullulanase plays a role in developing mung bean cotyledons.     

Effect of embryonic axis removal and exogenous calcium on carboxypeptidase I of mung bean seedling cotyledons

Removal of the embryonic axis prevents the normal decline of carboxypeptidase (Cpase) I in mung bean seedling cotyledons. Cpase I activity and protein, the latter manifested on western blots, almost completely disappear about 24 h before the cotyledon abscises. Of the 3 proteolytic enzyme patterns, only that of Cpase I can be restored by an exogenous supply of 10 m M CaCl₂ in the agar growth medium. The calcium effect is dependent on [CaCl₂] and is not manifested in the presence of chelators and calcium channel blockers. For detached cotyledons to show the normal low level of Cpase I by the eighth day of growth, calcium had to be supplied during seed imbibition and throughout the entire time from removal of the axis. The difference between detached cotyledons in the absence and presence of calcium was greatest when the cotyledons were detached 4–6 days after seed imbibition. Loss of Cpase I activity and protein can be demonstrated in vitro, with the maximum level of Cpase I-degrading activity measured 4 days after seed imbibition under the same growth conditions used to study the calcium effect. It is sensitive to pepstatin and has a pH optimum of 3, suggesting that this Cpase I-degrading activity is due to an aspartic protease.     

Inhibitory effect of polyamines on the activity of endopeptidase in mung bean cotyledons

The activity of cysteine endopeptidase (EP) in the cotyledons of mung bean seeds increased with time after germination. When cotyledons were excised from the embryonic axis in the course of seedling growth, the activity of EP in the excised cotyledon markedly dropped during the following incubation of 1 d. However, the level of EP protein in excised cotyledons, as examined by immunoblotting, was similar to that in axis-attached cotyledons at the corresponding stage. Thus, it seems that the low activity of EP in excised cotyledons is not due to a decrease in the content of EP protein, but due to a loss of the activity of existing EP. Treatment of attached cotyledons with polyamines (PAs; putrescine and spermidine [Spd]) resulted in a decrease in EP activity, while the same PA-treatment brought about little alteration in the level of EP protein. This indicates that PAs somehow produce an inhibitory effect on the activity of EP. Axis-removal resulted in an accumulation of Spd in the cotyledon. The possibility is suggested that PA, especially Spd, is involved in the inhibition of EP activity in excised mung bean cotyledons.     

绿豆子叶衰老期间核酸代谢的变化

萌发绿豆的子叶自然衰老期间,核酸含量降低,RNA降低的幅度比DNA大。电泳分析结果表明,子叶衰老期间细胞核主带DNA明显降低;而迁移慢的卫星带DNA变化不大。在RNA各组分中,18S rRNA从衰老前期就开始降低;25S rRNA和4～5S小分子RNA到衰老后期才缓慢下降。DNase和RNase活性在子叶整个衰老期间都明显升高,是导致核酸含量下降的主要原因。~3H-核苷掺入试验表明,核酸的合成速率在子叶衰老前期有所上升,到衰老后期又降低。poly(A)~ -mRNA含量在子叶开始衰老时明显上升。     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

Changes of Adenine Nucleotide in the Course of Mitochondrial Development of Mung Bean Cotyledon and Their Effect on Cellular Energy Status

The changes of adenine nucleotide and adenylate energy charge (AEC) during the development of mitochondria in imbibed mung bean cotyledons and the relationship between these changes and cellular energy status are studied. After cotyledons were imbibed in water for two hours, mitochondrial cristae were not observed, but for 12 hours, they appeared obviously on the inner membrane. With the structural integrity of the mitochondria, the functional mitochondria were graduately shown. For instance, the activity of H+-ATPase of cotyledons imbibed for 24 hours was about twice higher than that of 2 hours. The ATP content and the AEC value in the cotyledons imbibed for 24 hours increased sharply and the AMP decreased, but these were not observed in the mitochondria of the cotyledons imbibed either for 24 hours or 2 hours. When the cotyledons were imbibed in 1 × 10-4 mol/l or 5 × 10-4 mol/l DNP solution for 24 hours, the ATP and the AEC in the Cells exhibited a rapid decrease, but in the mitochondria they remained canstant. In the same DNP solution with cotyledons for 24 hours, the activity of mitochondrial adenylate kinase (AK) not only was not decreased but also increased by about 50% over the control. This result shows that the energy equilibration in the mitochondria seems likely to be regulated by adenylate kinase locating between inner and out membranes of the mitochondria.     

Effects of spermidine on the synthesis of α-amylase in cotyledons of mung bean seedlings

When cotyledons of mung bean [ Vigna radiata (L.) Wilczek] were treated with spermidine (3 m M ) during the first 6 h of imbibition, the development of α-amylase activity in cotyledons during the following 3 days was severely inhibited (75%) This inhibition was due to a slower accumulation of α-amylase protein, which in turn resulted from an inhibition of α-amylase synthesis. The rise in the level of α-amylase mRNA in cotyledons was also inhibited by spermidine treatment. However, the degree of inhibition of mRNA accumulation (40%) was not so marked as that of the activity of α-amylase synthesis (80%). These results are discussed in relation to the mode of action of spermidine on α-amylase expression.     

Modulation of Chlorophyll, Carotene and Xanthophyll Formation by Penicillin, Benzyladenine and Embryonic Axis in Mung Bean (Phaseolus aureus L.) Cotyledons

Penicillin stimulated the synthesis of pigments in the cotyledonsof intact embryos and excised cotyledons of mung bean (Phaseolusaureus L.) and enhanced benzyladenine-induced accumulation ofchloroplast pigments. The presence of the embryonic axis duringlight exposure proved to be beneficial for chlorophyll synthesisby the cotyledons whereas its presence in dark germination producedan adverse effect. The possible involvement of nucleic acidand protein synthesis in light-regulated chlorophyll formationis suggested. The stimulating effect on pigment synthesis providedby penicillin in this system seems to involve a maintenanceof nucleic acid and protein synthesis. Phaseolus aureus L., mung bean, pigment synthesis, cotyledons     

Purification and partial characterization of an aminopeptidase from mung bean cotyledons

An aminopeptidase (EC 3.4.11.-) was purified to homogeneity, as judged by SDS-PAGE. from mung bean ( Vigna radiata ) cotyledons. The molecular mass of this peptidase was estimated as 75 kDa by gel filtration. When an oligopeptide consisting of 5 amino acid residues was used as substrate, amino acids were released in the order of the N-terminal sequence of the oligopeptide chain. This enzyme apparently requires free sulfhydryl for its activity, as judged by the effects of various proteinase inhibitors. Among aminoacyl- p -nitroanilides examined for the availability as substrates of the enzyme, p -nitroanilides with hydrophobic amino acids were preferred substrates. According to western immunoblot profiles, the enzyme level in cotyledons was high at the early stage of imbibition and declined rapidly after germination.     

Promotive effect of chrysanthemic acids on adventitious root formation in some plants

Adventitious root formation in excised cucumber (Cucumis sativus L.) cotyledons was significantly promoted by (±)-cis-chrysanthemic acid at 0.006–1.8 mM. The effect of (±)-cis-chrysanthemic acid on indole-3-acetic acid (IAA)-induced rooting was additive. Rooting in excised cucumber cotyledons was significantly promoted by several isomers of chrysanthemic acid and sodium (±)-cis-chrysanthemate at 0.18 mM. Rooting in mung bean (Phaseolus radiatus L.) hypocotyls was also stimulated by the sodium salt at 0.06–0.6 mM. Rooting of kidney bean (Phaseolus vulgaris L.) hypocotyls was also clearly enhanced by sodium (±)-cis-chrysanthemate at 0.18–6 mM.     

Chloroplast Biogenesis: XXII. Contribution of Short Wavelength and Long Wavelength Protochlorophyll Species to the Greening of Higher Plants

The contribution of short and long wavelength membrane-bound fluorescing protochlorophyll species to the over-all process of chlorophyll formation was assessed during photoperiodic growth. Protochlorophyll forms were monitored spectrofluorometrically at 77 K during the first six light and dark cycles in homogenates of cucumber (Cucumis sativus L.) cotyledons grown under a 14-hour light/10-hour dark photoperiodic regime, and in cotyledons developing in complete darkness. In the etiolated tissue, short wavelength protochlorophyll having a broad emission maximum between 630 and 640 nm appeared within 24 hours after sowing. Subsequently, the long wavelength species fluorescing at 657 nm appeared, and accumulated rapidly. This resulted in the preponderance of the long wavelength species which characterizes the protochlorophyll profile of etiolated tissues. The forms of protochlorophyll present in etiolated cucumber cotyledons resembled those in etiolated bean leaves in their absorption, fluorescence, and phototransformability. A different pattern of protochlorophyll accumulation was observed during the dark cycles of photoperiodic greening. The short wavelength species appeared within 24 hours after sowing. Subsequently, the long wavelength form accumulated and disappeared. The long wavelength to short wavelength protochlorophyll emission intensity ratio reached a maximum (~3:1) during the second dark cycle, then declined during subsequent dark cycles. Short wavelength species were continuously present in the light and dark. Primary corn and bean leaves exhibited a similar pattern of protochlorophyll accumulation. In cucumber cotyledons, both the short and long wavelengths species appeared to be directly phototransformable at all stages of photoperiodic development. It thus appears that whereas the long wavelength protochlorophyll species is the major chlorophyll precursor during primary photoconversion in older etiolated tissues, both long wavelength and short wavelength species seem to contribute to chlorophyll formation during greening under natural photoperiodic conditions.     

Purification and characterization of a cysteine endopeptidase in cotyledons of germinated mung bean seeds

A cysteine endopeptidase (EC 3.4.22.-) present in cotyledons of mung bean (Vigna radiata) seedlings was purified to homogeneity, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). This proteinase has an apparent molecular mass of 33 kilodaltons as estimated by SDS-PAGE and belongs to the class of cysteine proteinases as judged by the effects of various proteinase inhibitors on the activity of the enzyme. When proangiotensin is used as a substrate, the enzyme preferentially hydrolyzes the peptide bonds formed by the amino group of Leu or lle in this oligopeptide chain; for the enzyme to cleave those bonds, peptide sequences consisting of at least three amino acid residues on the amino side of Leu or lle must be present. The proteinase readily digests globulin present in mung bean cotyledons to smaller polypeptides.     

Low temperature effects on ubiquinone content, respiration rates and lipid peroxidation levels of etiolated seedlings of two differentially chilling-sensitive species

Ubiquinone functions primarily in the electron transport chain of the mitochondria of plants and animals. Secondary roles in plant tissue, such as antioxidant activity, have also been proposed. The effect of low temperature exposure on etiolated seedling embryonic axes of two differentially chilling-sensitive species, mung bean ( Vigna radiata L.) (chilling-sensitive) and pea ( Pisum sativum L. cv. Lincoln) (chilling-tolerant) with respect to respiration rate, lipid peroxidation and ubiquinone content was examined. Whole seedlings (embryonic axis and cotyledon) of both species were exposed to control temperatures (20°C) (6 days) or an acclimatory low temperature treatment of 10°C (3 days) followed by exposure at 5°C (3 days). Measurements were initiated 3 days after seedlings had reached 50% germination (D0). Prior to measurements the cotyledons were removed and only the embryonic axis was used in these experiments. Ubiquinol (UQH₂), ubiquinone (UQ) and total ubiquinone (UQ_tot) content decreased in mung bean in response to the temperature treatment and UQH₂ and UQ_tot remained stable in the more chilling-tolerant pea. The reduction of the total Q-pool was approximately 85–92%, suggesting a high degree of saturation of the respiration pathways. Respiration declined and the RQ ratio increased in both species in response to low temperature. Cytochrome c oxidase (COX) (EC 1.9.3.1) activity was higher in pea than in mung bean but decreased during low temperature exposure in both species. Considering that levels of MDA (lipid peroxidation) did not increase in either species in response to chilling, decreased levels of UQH₂ and UQ observed in chilling-sensitive mung bean may indicate that these compounds were damaged prior to other membrane lipids during low temperature treatment and rendered undetectable.     

The biosynthesis of ribonuclease and its accumulation in protein bodies in the cotyledons of mung bean seedlings

Germination and seedling growth of mung bean are accompanied by a 7- to 10-fold increase in the ribonuclease content of the cotyledons. The increase occurs during the first 4 days of seedling growth and precedes the senescence of the cotyledons. Separation of the RNases in the cotyledons by polyacrylamide gel electrophoresis indicates the presence of several minor bands in seeds imbibed for 24 hr. On the second day of seedling growth a new major band with an R_f of 0.76 is present. In 4- to 5-day old seedlings this major band accounts for nearly all the RNase activity in the tissue. The characteristics of this RNase show that it is a plant ribonuclease I (pH optimum of 5.0; MW 16,000; activity preferentially inhibited by purine nucleotides; no activity toward DNA; no phosphodiesterase activity). When the seedlings are grown in 66% D₂O the RNase activity undergoes a density shift of 0.61% indicating that the increase in enzyme activity is due to the de novo synthesis of the enzyme molecules. A method is described for the isolation of protein bodies from protoplasts of storage parenchyma cells. Fractionation of protoplast lysates on Ficoll gradients results in the recovery of a high proportion (75%) of intact protein bodies. On these gradients RNase activity comigrates with α-mannosidase, a protein body marker enzyme indicating that the newly synthesized RNase accumulates in the protein bodies. We suggest that the synthesis of RNase in the cotyledons and its accumulation in the protein bodies indicates that protein bodies may function in the degradation of cellular macromolecules other than the reserves stored within them.