Changes in Level and Activity of Phospholipid Transfer Protein during Maturation and Germination of Maize Seeds

The variations of the amounts of phospholipid transfer proteins (PLTP), determined by ELISA and immunoblotting methods, were followed during the maturation and germination of maize (Zea mays L.) seeds. Changes of the amounts of PLTP occur during seed maturation. The levels of PLTP, low in the first 3 weeks after fecondation, strongly raised 3 to 5 weeks after, then reached and maintained a high value (10% of total soluble proteins) during the last steps of maturation. These variations, determined by ELISA, are in accordance with the observations made by immunoblotting. Changes in phospholipid transfer activity were also found when protein extracts prepared from seeds at different stages of maturation were assayed for transfer activity. The levels of PLTP were also determined during the germination of maize seeds and the early growth of the plantlets, both in the endosperm and the aerial parts. While no major change was observed in the endosperm, a high increase in PLTP level was found in the aerial part of the plantlet, both by ELISA and immunoblotting. An enhancement of the phospholipid transfer activity was parallely observed in the protein extracts of plantlets at various stages of germination. These results are consistent with an in vivo correlation between the synthesis of phospholipid transfer protein, observed during the maturation and germination of maize seeds, and the biogenesis of membranes which involves intracellular movements of phospholipids.     

Phytate metabolism in Petunia pollen

Phytic acid has been detected in the anthers of young flower buds of Petunia hybrida, the amount increasing slowly as the flower develops until anther dehydration, when there was a more rapid increase in phytic acid content. In mature pollen, the phytic acid content was found to be 2.0 % by weight, of which 90 % was water soluble, while free myo-inositol was a relatively low 0.06 % by weight. Breakdown of phytic acid was initiated soon after pollen germination began, and its degradation products, myo-inositol and inorganic phosphate, were rapidly mobilized for phospholipid and pectin biosynthesis. Both are in high demand during pollen tube elongation. Utilization of myo-[2-³H]inositol for phospholipid biosynthesis was about five times that for pectin synthesis during the first few hours of pollen germination. The label in the phospholipid was identified as the myo-inositol moiety of phosphaltidylinositol, while the pectin material contained predominantly labelled arabinose, with smaller amounts of label in galacturonic acid, glucose and xylose. A chase experiment showed that the myo-inositol moiety of phosphatidylinositol was subject to a relatively rapid turnover, while the label in pectin was not. Labelling germinating pollen with [³²P]orthophosphate gave label in phosphatidic acid, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine of the phospholipids. Phosphatidylinositol contained 30 % of this label initially, a proportion which declined to 10 % over longer periods of germination.     

Partial characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans

Germination of mung beans (Phaseolus aureus, Roxb.) is accompanied by an increase in the activity of the endopeptidase involved in storage protein metabolism. Enzyme activity in the cotyledons increases 25-fold during the first 5 days of germination. The cotyledons also contain inhibitory activity against the endopeptidase, and this inhibitory activity declines during germination, suggesting that inhibitors may play a role in regulating the activity of the endopeptidase.     

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.     

Fungal spore swelling and germination are restricted by the macrophage phagolysosome

Many species of medically important fungi are prolific in the formation of asexual spores. Spores undergo a process of active swelling and cell wall remodelling before a germ tube is formed and filamentous growth ensues. Highly elongated germ tubes are known to be difficult to phagocytose and pose particular challenges for immune phagocytes. However, the significance of the earliest stages of spore germination during immune cell interactions has not been investigated and yet this is likely to be important for defence against sporogenous fungal pathogens. We show here that macrophages restrict the early phases of the spore germination process of Aspergillus fumigatus and Mucor circinelloides including the initial phase of spore swelling, spore germination and early polarised growth. Macrophages are therefore adept at retarding germination as well as subsequent vegetative growth which is likely to be critical for immune surveillance and protection against sporulating fungi.     

Degradation of the Major Storage Protein of Phaseolus vulgaris during Germination : Role of Endogenous Proteases and Protease Inhibitors

Cotyledons from Phaseolus vulgaris L. (var. Improved Tendergreen) were tested for their activity on α-N-benzoyl-dl-arginine-p-nitroanilide (BAPNA) and azocasein during a germination periood of 10 days. Both activities increased throughout germination when activity was expressed on the basis of dry weight or protein. That these two activities were most likely due to the action of different enzymes was indicated by the fact that (a) optimal pH for the hydrolysis of BAPNA and azocasein was 8.2 and 5.5, respectively, and (b) the digestion of azocasein was considerably enhanced by mercaptoethanol and partially inhibited by thiol protease inhibitors, N-ethylmaleimide, and E-64, whereas these same regents caused little change in activity toward BAPNA. The three subunits of the major storage protein, G1, disappeared during germination and were accompanied by the accumulation of lower molecular weight products. The breakdown of G1 by extracts of the germinated beans could be demonstrated in vitro at pH 5 to 6. This activity was enhanced by mercaptoethanol and completely abolished by N-ethylmalemide, leupeptin, and E-64. It is concluded that a thiol protease with an acid pH optimum is primarily responsible for the disappearance of the major storage protein during germination. Although an inhibitor of the plant thiol protease, papain, is present in the mature bean and decreases during germination, its role in the control of the breakdown of the storage protein remains to be elucidated.     

Regulation of Gene Expression in Developing Zea mays Embryos: Protein Synthesis during Embryogenesis and Early Germination of Maize

Polypeptides synthesized in dissected embryos of Zea mays at different stages of embryogenesis and early germination have been characterized by their migration in two-dimensional gel electrophoresis. This analysis has been carried out with in vivo labeled polypeptides from excised embryos and with proteins synthesized in vitro in the rabbit reticulocyte system directed by poly(A⁺) RNA isolated from the different developmental stages. We have identified three main sets of expressed polypeptides: (a) embryonic set: this group of polypeptides is synthesized in young and mature embryos but not in early germination; (b) maturation set: this group of polypeptides is not present in young embryos and appears during the maturation period. Some of these polypeptides are still present in early germination while others disappear from stored mRNAs in dry embryos. One particular group from this set can be induced prematurely in young embryos by incubation with abscisic acid; and (c) germination set: this group of polypeptides is not expressed in the maturation period and appears after brief imbibition of the dry embryos.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

Activation of phosphate metabolism in cotyledons of Pharbitis nil in the early stage of germination as studied by multicompartmental analyis

A new computation method has been introduced into multicompartmentalanalysis, which saves computer time and gives good fitting parameterseven in complicated systems. This method was applied to theanalysis of phosphate metabolism in the cotyledons of Pharbitisnil during the early stage of germination. Soon after the startof imbibition, the metabolic flows of phosphate were initiatedby very small amounts of enzymes. Some enzymes involved in phosphorylation,RNA synthesis, and phospholipid synthesis were indicated asexisting in the seeds and as being activated from nonactiveforms during imbition. The activities of all phosphate metabolisms,except the breakdown of RNA, phospholipids and protein-boundphosphates, were activated about ten to hundred-fold after thestart of protein synthesis. The technique of multicompartmental analysis can be appliedto various physiological problems to estimate the quantitativeand systematic metabolic flows in any tissue. (Received July 5, 1973; )     

Synthesis of macromolecules during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcyst germination in the cellular slime mold Polysphondylium pallidum is a useful model for studying macromolecular changes necessary for or coincident with the transition from one cell type (cyst) to another (amoebae). Protein synthesis starts soon after cysts are incubated under permissive conditions, as evidenced by the incorporation of precursors and the appearance of polysomes. Sodium dodecyl sulfate-polyacrylamide gel analysis of proteins made at intervals during germination shows that protein synthesis is developmentally regulated during this process. RNA synthesis also begins early during germination. Cysts contain polyadenylated RNA that can stimulate the incorporation of radioactive amino acids into protein in an in vitro wheat germ protein synthesizing system. The concentration of poly(A)-containing RNA increases during germination and during inhibition of protein synthesis by cycloheximide.     

Changes in lipid composition during starvation and germ-tube formation inCandida albicans

The changes in the lipid components ofCandida albicans have been determined during growth, starvation, and germ-tube formation.¹⁴C-Labeled cells were used to determine the extent of synthesis and degradation of the different lipid fractions. On a dry weight basis the percentage of total lipid increased from 18% for blastospores to 22% after starvation but decreased to 11% after 4 h of germination. The major components of the lipid fraction were sterols (36–52%) and phospholipids (28–42%). The free and esterified sterol fractions both increased approximately 45% during starvation. The free sterol content continued to increase over the initial stage of germ-tube formation, then decreased with time. The sterol ester fraction decreased throughout germination to the concentration found in growing blastospores. The changes in specific activity (cpm/μg sterol) of these fractions indicated that sterol esters were precursors for free sterols during germination. The total phospholipid fraction increased during starvation but there was a decrease in both the cellular concentration (60%) and the specific activity during germination. There were only minor changes in the relative concentrations of phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine which indicated coordinate synthesis and degradation of these components. Free fatty acids and triacylglycerides are only minor components of the total lipid pool.     

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.     

Implications of Reactive Oxygen Species in Heat Shock Induced Germination and Early Growth Impairment in Amaranthus lividus L.

An effort has been made to assess the role of reactive oxygen species in germination and subsequent growth of Amaranthus lividus under elevated temperature. Transfer of A. lividus seeds from 25 to 45 °C for 4, 8 and 12 h, during early imbibitional period reduced percentage of germination, relative germination performance, relative growth index and seedling length. Heat shock during early germination decreased also the activities of free radical scavenging enzymes like catalase, peroxidase and superoxide dismutase, increased the accumulation of superoxide, hydrogen peroxide and induced lipoxygenase mediated membrane lipid peroxidation. Membrane injury index and relative leakage ratio revealed a rise with concomitant reduction in membrane protein thiol content in heat shock raised seedlings. The results indicate that heat shock in A. lividus seeds induced an excessive generation of ROS and led to an oxidative membrane damage, causing early growth impairment.     

Do Conditions During Dormancy Influence Germination of Suaeda maritima?

Background and Aims

Seeds of annual halophytes such as Suaeda maritima experience fluctuating salinity, hydration, hypoxia and temperature during dormancy. Germination then occurs in one flush of 2–3 weeks after about 5 months of winter dormancy during which time the seeds can remain in saline, often waterlogged soil. The aim of this study was to investigate the effect of simulated natural conditions during dormancy on germination and to compare this with germination following the usual conditions of storing seeds dry. The effects of hydration, salinity, hypoxia and temperature regimes imposed during dormancy on germination were investigated. Also looked at were the effects of seed size on germination and the interaction between salinity during dormancy and salinity at the time of germination.

Methods

Various pre-treatments were imposed on samples of seeds that had been stored dry or wet for different periods of time during the 5 months of natural dormancy. Subsequent germination tests were carried out in conditions that simulated those found in the spring when germination occurs naturally. Various salinities were imposed at germination for a test of interaction between storage salinity and salinity at germination.

Key Results

A temperature of about 15 °C was needed for germination and large seeds germinated earlier and better than small seeds. Cold seawater pre-treatment was necessary for good germination; the longer the saline pre-treatment during the natural dormancy period the better the germination. There appeared to be no effect of any specific ion of the seawater pre-treatment on germination and severe hypoxia did not prevent good germination. A short period of freezing stimulated early germination in dry-stored seed. Storage in cold saline or equivalent osmotic medium appeared to inhibit germination during the natural dormancy period and predispose the seed to germinate when the temperature rose and the salinity fell. Seeds that were stored in cold wet conditions germinated better in saline conditions than those stored dry.

Conclusions

The conditions under which seeds of S. maritima are stored affect their subsequent germination. Under natural conditions seeds remain dormant in highly saline, anoxic mud and then germinate when the temperature rises above about 15 °C and the salinity is reduced.Key words: Suaeda maritima, germination, pre-treatment, salinity, temperature     

Isolation and properties of Streptomyces spore membranes.

A simple procedure for the isolation of membranes from Streptomyces spores is described which produces about 12 mg of membrane protein per g of dry weight. The membrane fractions were contaminated by low levels of DNA, RNA, and hexosamines. The functional integrity of the membrane is conserved through the isolation procedure, as evaluated by the presence of several activities of the membrane-bound electron transport chain. This isolation procedure allowed the determination of the biosynthesis of proteins and phospholipids of the membrane. Both biosynthetic processes started in the first 5 min of germination and increased progressively during spore germination. A stable mRNA fraction of the dormant spore encoded 44% of the membrane proteins synthesized early in germination, but most of the phospholipid biosynthesis was not dependent on this fraction.     

Evaluation of Aerated Steam Treatment of Alfalfa and Mung Bean Seeds To Eliminate High Levels of Escherichia coli O157:H7 and O178:H12, Salmonella enterica,and Listeria monocytogenes

Sprouts contaminated with human pathogens are able to cause food-borne diseases due to the favorable growth conditions for bacteria during germination and because of minimal processing steps prior to consumption. We have investigated the potential of hot humid air, i.e., aerated steam, to treat alfalfa and mung bean seeds which have been artificially contaminated with Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Weltevreden, and Listeria monocytogenes Scott A. In addition, a recently collected E. coli O178:H12 isolate, characterized by a reduced heat sensitivity, was exposed to the treatment described. Populations of E. coli O157:H7 and S. enterica on alfalfa and mung bean seeds could be completely eliminated by a 300-s treatment with steam at 70 ± 1°C as revealed by enrichment studies. L. monocytogenes and E. coli O178:H12 could not be completely eliminated from artificially inoculated seeds. However, bacterial populations were reduced by more than 5 log CFU/g on alfalfa and by more than 4 log CFU/g on mung bean seeds. The germination rate of mung beans was not affected by the 300-s treatment compared to the germination rate of untreated seeds whereas that of alfalfa seeds was significantly lower by 11.9%. This chemical-free method is an effective alternative to the 20,000-ppm hypochlorite treatment presently recommended by the U.S. Food and Drug Administration (FDA).     

Analysis of the Loss in Heat and Acid Resistance during Germination of Spores of Bacillus Species

A major event in the nutrient germination of spores of Bacillus species is release of the spores'' large depot of dipicolinic acid (DPA). This event is preceded by both commitment, in which spores continue through germination even if germinants are removed, and loss of spore heat resistance. The latter event is puzzling, since spore heat resistance is due largely to core water content, which does not change until DPA is released during germination. We now find that for spores of two Bacillus species, the early loss in heat resistance during germination is most likely due to release of committed spores'' DPA at temperatures not lethal for dormant spores. Loss in spore acid resistance during germination also paralleled commitment and was also associated with the release of DPA from committed spores at acid concentrations not lethal for dormant spores. These observations plus previous findings that DPA release during germination is preceded by a significant release of spore core cations suggest that there is a significant change in spore inner membrane permeability at commitment. Presumably, this altered membrane cannot retain DPA during heat or acid treatments innocuous for dormant spores, resulting in DPA-less spores that are rapidly killed.     

Nucleic acid and protein synthesis and loss of vigour in germinating wheat embryos

A study has been made of the RNA and protein synthesising systems of wheat embryos isolated from seed lots having high viability but differing in vigour. The rate of RNA and protein synthesis in wheat embryos during the early hours of germination is related to the vigour of the seed lot. The imposition of a stress factor, in the nature of a sub-optimal germination temperature, during germination of isolated wheat embryos magnifies the differences in rates of protein and RNA synthesis between high and low vigour seed. Using cell-free protein synthesising systems it has been demonstrated that an important difference between high and low vigour embryos lies in the relative levels of messenger RNA in the embryo. High vigour embryos contain relatively higher levels of poly A⁺-RNA (i.e. potential mRNA species) than lower vigour embryos and furthermore the level of poly A⁺-RNA in high vigour embryos increases during early germination whilst in lower vigour embryos the level decreases. The difference in poly A⁺-RNA levels accounts, at least partially, for the differences in rates of protein synthesis observed between embryos from high and low vigour wheat seed during early germination at both optimal and sub-optimal germination temperatures.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - poly A⁺-RNA polyadenylated RNA - GM germination medium - PMS post-mitochondrial supernatant fraction     

Ethylene antagonizes the inhibition of germination in Arabidopsis induced by salinity by modulating the concentration of hydrogen peroxide

The capacity of plants to achieve successful germination and early seedling establishment under high salinity is crucial for tolerance of plants to salt. The gaseous hormone ethylene has been implicated in modulating salt tolerance, but the detailed role of how ethylene modulates the response of early seedling establishment to salt is unclear. To better understand the role of the ethylene signal transduction pathway during germination and seedling establishment, an ethylene insensitive mutation (ein2-5) and an ethylene sensitive mutation (ctr1-1) of Arabidopsis were analyzed under saline conditions and compared with the wild type plant (Col-0) as control. High salinity (>100?mM NaCl) inhibited and delayed germination. These effects were more severe in the ethylene insensitive mutants (ein2-5) and less severe in the constitutive ethylene sensitive plants (ctr1-1) compared with Col-0 plants. Addition of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or inhibitors of ethylene action implied that ethylene was essential for early seedling establishment under normal and saline conditions. Salt stress increased the endogenous concentration of hydrogen peroxide (H₂O₂) in germinating seeds and ACC reduced its concentration. Our results suggest that ethylene promotes germination under salinity by modulating the endogenous concentration of H₂O₂ in germinating seeds. These findings demonstrate that ethylene is involved in regulating germination as an initiator of the process rather than consequence, and that ethylene promotes germination by modulating the endogenous concentration of H₂O₂ in germinating seeds under salinity.     

Control of storage protein metabolism in the cotyledons of germinating mung beans: role of endopeptidase

The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either α-mannosidase or N-acetyl-β-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.