Hydrolytic enzyme activities in germinating spores ofAdiantum capillus-veneris L.

Changes in hydrolytic enzyme activities were investigated during spore germination ofAdiantum capillus-veneris L. The spores were incubated for 3 days in the dark at 25 C for imbibition, and then germination of the spores was induced by continuous irradiation with red light. At day 2 after onset of the red light irradiation, rhizoids appeared out of spore coats and protonemal cells became visible on the following day. Lipase occurred in dry spores and its activity decreased during 3 days of dark incubation. The activity started to increase when the spore germination was induced by red light irradiation. On the other hand, amylolytic and aminopeptidase activities which were also detected in dry spores decreased continuously during the dark incubation and following the germination process. RNase activity also decreased during 3 days of dark incubation but the activity was retained thereafter at a constant level with or without red light irradiation. Developmental patterns of these hydrolytic enzymes were classified into two groups: One decreased during imbibition and dark incubation but increased after red light irradiation and the other continuously decreased during dark incubation and germination. These results are discussed in relation to compositional changes of cell constitutions such as lipid, sugars, proteins and amino acids during spore germination.     

FREE AMINO ACID CHANGES DURING GERMINATION OF TELIOSPORES OF THE WHEAT BUNT FUNGUS,TILLETIA CARIES

Teliospores were aerated and agitated in a mineral salts medium and their free amino acid contents were analyzed at eight different times, from shortly after imbibition of water until just before germ tube emergence. In addition to the common amino acids, eight unidentified ninhydrin-positive components were detected. About 50 % or more of nearly each of the amino acids diffused out of the spores during the initial phase of germination. These released amino acids were actively taken up by the spores during the latter stages of germination. The free amino acids in largest amounts in the dormant spores of T. caries were arginine 15.0, glutamic acid 6.3, and alanine 3.7 μmoles per g dry spores. Together these three amino acids accounted for about 71 % of the total free amino acids in dormant spores of T. caries and T. controversa. The total amounts of free amino acids in spores of common bunt were much higher than in spores of dwarf bunt.     

Biochemistry of fern spore germination: protease activity in ostrich fern spores

Protease activities were detected in quiescent and germinating spores of the ostrich fern (Matteuccia struthiopteris [L.] Todaro). Peak endopeptidase, aminopeptidase, and carboxypeptidase activities were detected 12 to 24 hours after spores began imbibing under light. There was a correlation between activities of proteases, the onset of a decline in levels of soluble protein, and an increase in levels of free amino acids. The earliest visible event of spore germination, breakage of the spore coat and protrusion of a rhizoid cell, was observed after peak protease activity, 48 to 72 hours after the start of imbibition. Results of this study demonstrate similarities in the pattern of protease activities during germination of ostrich fern spores to those of some seeds.     

The solubilization and degradation of pumpkin seed globulin during germination

Pumpkin seed globulin decreased 88% during the first 4 daysof germination. This decrease was concomitant with a 2.5 foldincrease in water soluble protein which arose directly fromthe water insoluble globulin. The sequence of solubilizationand breakdown of the globulin was followed through 12 days ofgermination. Pumpkin seed globulin was determined to have subunitsof 56,000 daltons, while the new water soluble protein consistedof two proteins, as determined by sodium dodecyl sulfate polyacrylamideelectrophoresis; one having a molecular weight of 42,000 daltonsand the other 28,000 daltons. Trypsin mimicked the first stepof the breakdown by solubilizing pumpkin seed globulin to yieldidentical digestion products as were obtained in vitro. Maximumproteolytic acitvity, as measured by the release of ninhydrinpositive, materials occurred at 6 days of germination, at whichtime both the concentration of free amino acids and the incorporationof ¹⁴C into amino acids increased rapidly. A second proteolyticenzyme system which solubilized the pumpkin seed globulin butdid not act on hemoglobin, casein, or bovine serum albumin reachedits maximum activity at two days of germination. (Received April 17, 1978; )     

THE EFFECT OF LIGHT ON THE USE OF THE NITROGEN RESERVES OF GERMINATING SOYBEAN SEEDS

Light affects the mobilization and distribution of several of the storage components of the cotyledons of germinating soybean seeds. The nitrogen content of the cotyledons began to decrease during the first day of germination, continued through day 12 for plants in the light, and day 14 for those in the dark. Cotyledons from both treatments had lost about the same amount of nitrogen by day 14. Plants from both treatments lost about the same amount of dry weight by day 8, but those in the light had taken up nitrogen from the nutrient solution; while those in the dark showed no increase. The plants in the light had higher concentrations of soluble amino nitrogen in the cotyledons than did those in the dark, but the opposite was true for the seedling axis. Aspartate and its amide accounted for half or more of the total free amino acids in all parts of dark-grown plants at 6 and 14 days. In the light-grown plants aspartate and asparagine usually accounted for less than half of the total free amino acids in all plant parts except the cotyledons at 6 and 14 days. Total soluble amino acids were much lower in these plants than those in the dark, excepting the cotyledons.     

The involvement of amino acids in latex lipid synthesis in Euphorbia lathyris seedlings

The breakdown of triglycerides and proteins in the endosperm of Euphorbia lathyris was assayed in a 14 day germination period. Six days after germination, the average daily production was 2.7 μmol of amino acids. Arginine, glutamine, asparagine and glutamic acid accounted for 53% of the total amino acids. Excised cotyledons with 1 cm hypocotyls were used for amino acid uptake and their involvement in terpenoid synthesis was studied. Glutamine and aspartate were hardly involved in apolar lipid synthesis. Leucine, isoleucine, valine and threonine were mainly incorporated into the triterpenes in the laticifers. Alanine and serine were also involved in phytosterol synthesis in the adjacent tissue. In the 14 day germination period, ca3% of the daily yield of latex triterpenes may be synthesized from a variety of amino acids.     

Carbohydrate and Lipid Metabolism During Germination of Uredospores of Puccinia graminis tritici

Uredospores of Puccinia graminis (Pers.) tritici (Eriks. and Henn.) were uniformly labeled with ¹⁴C by permitting the host (Triticum aestivum L.) to carry out photosynthesis in ¹⁴CO₂ during the process of spore production by the obligate parasite. The use of ¹⁴C labeled spores provided advantages in a study of the utilization of endogenous substrates at frequent intervals with small amounts of spores under conditions conducive to germination.

Because of previous uncertainties about the nature of the substrates of importance to germination, a detailed study of carbohydrate and lipid components, both in the spores and in the germination medium, was made during the first 7 hours after placing the spores on aqueous media. Diethyl ether and 80% ethanol soluble metabolites each constituted approximately 20% of the total spore carbon. During the first hour nearly 60% of the 80% alcohol solubles disappeared from the spores while the total ether soluble material did not change appreciably. A significant part of the 80% ethanol soluble materials appeared in the germination medium.

During germination and germ tube extension, there was rapid utilization of trehalose, arabitol and mannitol even though appreciable amounts of these materials were present as exogenous pools in the germination medium. Although the total amounts of ether soluble components did not change as drastically as the carbohydrate fraction, there was extensive utilization of palmitic, oleic, linolenic and 9,10-epoxyoctadecanoic acids.

The results indicate that the germination process in spores of obligate parasites is not based solely on the utilization of lipids and some possible roles of the changes in internal and external pools of soluble carbohydrates are discussed.

     

Growth and some metabolic activities ofScenedesmus obliquus cultivated under different NaCl concentrations

The physiological response ofScenedesmus obliquus to salinity (NaCl concentration of 40, 80, 120, 60 and 200 mM) for 7 d (long-term experiments) or 2 h (short-term experiments) was followed. Cell number, dry matter and the content of photosynthetic pigments decreased with the rise of NaCl concentration. However, the photosynthetic O₂ evolution mostly increased with the increase of NaCl concentration up to 80 mM, and respiration (dark O₂ uptake) was markedly promoted. Photosynthesis/respiration ratio went in concomitance with the cell number, dry matter or chlorophyll content. Contents of soluble saccharides and soluble proteins increased with the rise of salinization, while the content of insoluble and total saccharides or proteins decreased. Proline content increased greatly with salinization, whereas of other free amino acids were mostly reduced, especially at higher salinities. Similarly, the lipoid contents of salinizedScenedesmus obliquus were obviously higher than those of the control cultures.     

Spore Germination and Rhizoid Differentiation in Onoclea sensibilis: A Two-Dimensional Electrophoretic Analysis of the Extant Soluble Proteins

Following a geometrically asymmetrical cell division during germination of spores of the fern Onoclea sensibilis L., the small cell differentiates into a rhizoid and the large cell divides to form the protonema. Using silver-staining of two-dimensional gels, we have examined the soluble proteins of spores during germination and of separated rhizoid protoplasts and protonemal cells. Of over 500 polypeptides followed, nearly 25% increased or decreased in prominence during spore germination and the initial phases of rhizoid elongation. Soluble proteins from purified protoplasts of young rhizoids were quantitatively different from those of protonemal cells and germinated spores. Nine polypeptides which appeared after cell division were substantially more prominent in rhizoid protoplasts than in whole germinated spores and have been putatively designated rhizoid-specific polypeptides. The differences in the soluble protein composition of young rhizoids and protonemal cells probably reflect the differential organelle distribution between the two cells as well as differential net protein synthesis in the cytoplasms of the two cells.     

Translocation and Metabolic Conversion of C-Labeled Assimilates in Detached and Attached Leaves of Phaseolus vulgaris L. in Different Phases of Leaf Expansion

Leaf excision greatly affected the actual levels of ¹⁴C-assimilates in laminas and petioles of primary bean leaves (Phaseolus vulgaris L.) following a transport period. However, it did not affect the percentage of starch in the insoluble residue; starch decreased from 20% of the insoluble residue after exposure to ¹⁴CO₂ to 3% after 5 hr in both attached and detached leaves. The transition from import to export of attached and detached leaves was at the same stage, i.e., when the cotyledons were 63 to 85% depleted. The composition of the ¹⁴C-assimilates in importing leaves was different from that in exporting leaves. In the former, only 5% of the soluble label was free sugar, while 74% was free sugar in the latter. The failure of importing leaves to export was not due to the labeled substances being nontransferable. Extracts from importing leaves applied to exporting leaves were exported; these extracts were high in amino acids and organic acids but low in free sugar. However, exporting leaves exposed to ¹⁴CO₂ appeared to export sugars more readily than amino acids. Cotyledon excision did not delay transition of leaves from import to export. Actually, excision seemed to enhance slightly the transition of the primary leaves from import to export.     

Effect of sulfite on compositional changes of cell constituents in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L., which were preincubated at 25 C for three days in the dark, were suspended in 1 mM potassium phosphate buffer, pH 6.0, and incubated for four days under continuous red light in the presence or absence of 3 mM sulfite. At day 0, 2 and 4 of the incubation, contents of cell constituents were determined. Total lipid content decreased continuously over four days of incubation in the absence of sulfite or in the presence of 3 mM sulfate. In contrast, when sulfite was added to the medium, the decrease stopped after day 2. The content of insoluble glucan increased markedly between day 2 and 4 in the medium without sulfite, whereas it decreased continuously for four days in the medium containing sulfite. The protein content decreased promptly by day 2, but its decrease was delayed when 3 mM sulfite was added to the medium. The content of amino acids also decreased by day 2, but it increased thereafter in the absence of sulfite or in the presence of 3 mM sulfate. In the presence of sulfite, however, the content continued to decrease until day 4. The results indicate that 3 mM sulfite in the incubation medium depressed the utilization of reserve lipid and protein, the synthesis of insoluble glucan and the increase of amino acid pool sizes in fern spores.     

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。     

Hydrolysis of lipid and protein reserves in loblolly pine seeds in relation to protein electrophoretic patterns following imbibition

The correlation between changes in seed protein electrophoretic patterns and the hydrolysis of lipid and protein reserves of loblolly pine ( Pinus taeda L.) seed was studied. Seeds were incubated at 30°C for up to 12 days following stratification, then megagametophytes and embryos were assayed for lipid and protein content after each day of imbibition. The megagametophyte of mature seed was found to contain 20% lipid and 12% storage protein on a fresh weight basis. The embryo contained 26% lipid and 15% protein. Both lipid and protein reserves were depleted constantly following imbibition. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of soluble and insoluble protein fractions showed a 60 kDa protein that was representative of crystalloid-like proteins. These crystalloid-like proteins comprised 85% of the insoluble protein storage reserves. A small number of insoluble storage proteins, including a 47 kDa protein, were distinct in that they were unaffected by 2-mercaptoethanol treatment. The soluble fractions from both tissues were labelled with [³⁵S]-methionine, and incorporation was visualized by two-dimensional electrophoresis. Proteins were found to belong to one of three categories, those synthesized constitutively (comprising the bulk of newly synthesized proteins), those synthesized during germination or those synthesized after radicle emergence. Accompanying seed reserve hydrolysis were developmental shifts in protein pattern and synthesis, suggesting the possibility that control of hydrolysis is at the level of enzyme accumulation.     

Metabolic Study of Douglas-fir Pollen Germination in vitro

Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] pollen was germinated and grown in mass in a sterile mineral medium supplemented with 0.3 M mannitol as osmotic stabilizer. During the 4-day period, pollen elongated threefold; free sugar and amino acid contents did not change significantly; soluble protein, insoluble protein, and RNA decreased; starch reduced to 30%; and DNA doubled in quantity. Respiration rate remained high and constant during the first 36 h and then increased, but the respiratory quotient averaged about 1.0 throughout the period. The pool size of adenine nucleotides remained constant, whereas ATP content and energy charge increased rapidly during the first 8 h and remained high for the remainder of the period. ¹⁴C-glucose was rapidly metabolized within 2 h at 25°C to amino acids (33%), organic acids (22%), sugars (20%), CO₂ (15%), lipids (5%), and insoluble components (3%). Experimental data indicated that the cultural conditions provided a suitable environment for rapid germination and active metabolism of Douglas-fir pollen.     

Bacillus cereus Spores Release Alanine that Synergizes with Inosine to Promote Germination

Background

The first step of the bacterial lifecycle is the germination of bacterial spores into their vegetative form, which requires the presence of specific nutrients. In contrast to closely related Bacillus anthracis spores, Bacillus cereus spores germinate in the presence of a single germinant, inosine, yet with a significant lag period.

Methods and Findings

We found that the initial lag period of inosine-treated germination of B. cereus spores disappeared in the presence of supernatants derived from already germinated spores. The lag period also dissipated when inosine was supplemented with the co-germinator alanine. In fact, HPLC-based analysis revealed the presence of amino acids in the supernatant of germinated B. cereus spores. The released amino acids included alanine in concentrations sufficient to promote rapid germination of inosine-treated spores. The alanine racemase inhibitor D-cycloserine enhanced germination of B. cereus spores, presumably by increasing the L-alanine concentration in the supernatant. Moreover, we found that B. cereus spores lacking the germination receptors gerI and gerQ did not germinate and release amino acids in the presence of inosine. These mutant spores, however, germinated efficiently when inosine was supplemented with alanine. Finally, removal of released amino acids in a washout experiment abrogated inosine-mediated germination of B. cereus spores.

Conclusions

We found that the single germinant inosine is able to trigger a two-tier mechanism for inosine-mediated germination of B. cereus spores: Inosine mediates the release of alanine, an essential step to complete the germination process. Therefore, B. cereus spores appear to have developed a unique quorum-sensing feedback mechanism to monitor spore density and to coordinate germination.     

Variations de la composition azotee de Pinus canariensis au cours de la germination

The study of the different nitrogen fractions of Pinus canariensis during seed germination and seedling development indicates a progressive hydrolysis of the insoluble fraction. Arginine is quantitatively one of the most important amino acids in seed proteins.     

Drought tolerance of Periploca sepium during seed germination: antioxidant defense and compatible solutes accumulation

Periploca sepium Bunge is a native and widespread shrub on the Loess Plateau, an arid and semi-arid region in China. To understand the adaptability of its seed germination to dry environments, we investigated the germination rate, water relations, lipid peroxidation, antioxidant capacity and accumulation of major organic solutes during seed germination under water deficit conditions. Results showed that seeds pre-treated by hydration–dehydration or ?0.9 MPa PEG germinated faster than control seeds, indicating strong resistance of P. sepium to drought condition. The re-dried seeds showed higher proline, total free amino acids (TFAA) and soluble proteins (SP) contents than control dry seeds, indicating the maintenance of physiological advancement when dehydrated. Osmotic stress made seed germination stay on the plateau phase (phase II). However, germinating seeds moved into phase III immediately once transferred into distilled water. Large increases in SP and soluble sugars (SS) of both re-dried and osmotic stressed seeds help themselves to resist drought stress. The re-hydrated seeds showed significantly higher levels of proline, TFAA, SP and SS than control seeds. The largely accumulated SS during osmotic stress declined sharply when transferred into distilled water. Our data demonstrate that P. sepium’s tolerance to drought stresses during germination is associated with enhanced activity of antioxidant enzymes and accumulation of some compatible solutes. Seed physiological advancement progressed slowly under low water conditions and it was maintained when seeds were air dried. This strategy ensures high and more rapid seed germination of P. sepium under drying and wetting conditions in drought-prone regions.     

Comparative biochemical profiling during the stages of acquisition and development of somatic embryogenesis in African oil palm (Elaeis guineensis Jacq.)

In this study, the different stages of somatic embryogenesis (SE) of the African oil palm (Elaeis guineensis Jacq.) were characterized biochemically. The total soluble sugars, starch, total free amino acids, and total proteins were extracted, identified and quantified at various stages of embryogenesis: zygotic embryos (initial explants), primary calluses, embryogenic calluses, calluses with pro-embryos, globular embryos, differentiated somatic embryos, and regenerated plants. It was found that at the onset of induction of SE, the level of soluble sugars in the tissues of the explants fell by half. During this period, the total soluble sugars present in the cultures consisted basically of glucose and fructose. In the process of regeneration and maturation, the concentrations of soluble sugars gradually increased, reaching the highest values in the last two stages of development. At this stage, the disaccharide sucrose accounts for more than 80 % of the composition of total soluble sugars in the explants. Compared to starch, we found that the concentrations thereof in developing tissues are inversely proportional to that of soluble sugars virtually throughout embryogenic development. As for free amino acids, we found that after 30 days of induction until formation of the embryogenic calluses, there is an accentuated synthesis of total free amino acids in the explant tissue. In this stage, there was a significant increase in the levels of alanine and serine in the tissues. However, after the formation of the embryogenic calluses, the levels of total free amino acids present in the cultures become stabile and remain constant until the end of cultivation. Similar results were found for total protein, which also showed a significant increase at the onset of induction, undergoing slight changes during the remainder of the cultivation.     

Effects of salinity and heat-shock on wheat seedling growth and content of carbohydrates,proteins and amino acids

The effects of salinity (0, 50, 100, 150, and 200 mM NaCl) and heat-shock (42°C) and their interactions on germination, seedling growth, and some relevant metabolic changes of two cultivars (cv. Giza 155 and cv. Stork) of wheat (Triticum vulgaris L.) were studied. Germination studies indicate that plants tolerated salinity up to 100 mM NaCl. The lengths of roots and shoots and their water content, as well as fresh and dry matter yield of cv. Giza 155 seedlings remained more or less unchanged up to 100 mM NaCl and of cv. Stork up to 50 mM NaCl. Salinity induced progressive increase in soluble carbohydrates, soluble proteins and proline in cv. Giza 155 and in soluble proteins, proline and other free amino acids in cv. Stork. However, under the higher salinity levels, in cv. Giza 155 increase in soluble carbohydrates was accompanied by lose in other free amino acids, whereas in cv. Stork an opposite effect was obtained. Heat-shock treatment (42°C for 24 h) induced a significant decrease in the final germination percentage, the shoot and root lengths, fresh matter yield and the water content. The dry matter yield of the two cultivars was considerably increased as compared with the corresponding treatments with NaCl only. Heat-shock treatment resulted in a significant increase, in the amount of soluble carbohydrates and proline in salt treated seedlings of both cultivars. The pattern of changes in amino acids was opposite to that of soluble proteins, indicating that the increase in soluble proteins was at the expense of other amino acids in cv. Giza 155 andvice versa in cv. Stork.