Metabolism of Inositol Phosphates: I. Phytase Synthesis during Germination in Cotyledons of Mung Beans, Phaseolus aureus

The degradation of phytin in germinating mung bean seeds has been found to be associated with the increased activity of phytase in the cotyledon. In the differentiated embryo the increase of this activity is very low all throughout the growth periods studied. Phytase appears in the cotyledon during germination. No activity has been detected in the cotyledons of unsoaked seeds. Cycloheximide (10⁻⁶ M) inhibits the appearance of phytase by 61% during 24 and 48 hours after the start of germination. This phytase increase is dependent on the synthesis of new RNA in the cotyledon. Synthesis of DNA is not detected in the cotyledon during germination.     

Changes in Nicotinic Acid Content and Its Nucleotide Derivatives of Rice and Wheat Seeds during Germination

Relative distribution of bound and free forms of nicotinic acid in rice and wheat seeds and their metabolism during germination were the subject of the present investigation. Measurement of the levels of NAD (nicotinamide adenine dinucleotide) and NADP (nicotinamide adenine dinucleotide phosphate) formed another part of the work. Total nicotinic acid in both rice and wheat increased with germination and was maximum at 72 hours. From this time onwards, it began to decline rapidly and at the end of experiment, i.e., after 120 hours, it was lower than that for ungerminated seeds on per seedling basis, although it was slightly higher on per g dry weight basis. Ungerminated seeds of wheat and rice contained about 89 per cent and 80 per cent respectively of their total nicotinic acid in bound form which became partially free in course of germination. Total nucleotides (oxidised plus reduced forms) increased progressively up to 96 hours followed by a slight fall at 120 hours. NAD reached a maximum at 24 hours and fell gradually thereafter. The depletion of NAD was associated with a progressive accumulation of NADH. NADP decreased from the peak value at 72 hours. Formation of NADP and its maintenance at high level depend on NAD in the oxidised form and the content slowed down in tissues with higher NADU/NAD ratio. A relatively large amount of NADPH was present throughout the experiment.     

Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.

The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.

     

The dynamics of indole-3-acetic acid in Acer platanoides seeds during stratification and germination

During stratification at 5°C indole-3-acetic acid (IAA) levels in embryos of Acer platanoides decreased during the early stages but subsequently increased again throughout the remainder of a 144 day period. The reduction in IAA levels in embryos of fruits stored at 17°C was even more pronounced, and in addition, no increase was observed after longer storage periods at this temperature, the levels of IAA remaining very low. Germination in seeds maintained at 5°C was not observed until after 120 days or longer, but germination potential increased at an earlier stage, as shown by the fact that seeds transferred to 20°C gave appreciable increases in germination after much shorter chilling periods. Endogenous IAA levels in embryos from seeds transferred to 20°C after a chilling period, long enough to break dormancy, increased within 24 h, i.e. before visible germination, to levels similar to those observed in embryos from seeds chilled continuously for 144 days. Embryos from seeds chilled for 120 days, i.e. when the samples already showed visible germination and when the endogenous IAA content was already high, showed no further increase in endogenous IAA during a three day incubation at 20°C. None of the treatments employed was effective in inducing germination of seeds or embryos from fruits stored at 17°C.     

Seed dormancy in red rice : v. Response to azide, hydroxylamine, and cyanide

The activity of NaN₃ (0.5 millimolar), hydroxylamine-HCl (10-18 millimolar), and potassium cyanide (1 millimolar) as dormancy-breaking agents of dehulled red rice (Oryza sativa) is pH-dependent such that medium pH values favoring formation of the uncharged chemical species resulted in the highest germination percentages. There was no promotive effect of pH itself in the range of 3 to 10. The minimum contact times for maximum response (≥90% germination) to NaN₃, KCN, and NH₂OH-HCl are 8 hours at pH 4, 24 hours at pH 8, and 72 hours at pH 6 or 7, respectively, for exposure commencing at the start of imbibition. Dehulled seeds, imbibed first in water, show only slightly reduced germination when subsequently transferred to solutions of dormancy-breaking chemicals.

Intact seeds remain dormant in the presence of NaN₃, KCN, or NH₂OH-HCl unless partially dry-afterripened. The pH dependence of these chemicals is reduced in intact, afterripening seeds.

     

The effects of phosphate supply on uptake of potassium ions, 2,4-D and atrazine by wheat and maize

The germination percentage of peach [ Prunus persica (L.) Batsch cv. Halford] seeds at 20°C was low (< 20%) after incubation at 5°C for as long as 35 days, but then increased considerably (> 40%) when the seeds were maintained at 5°C for longer than 42 days. Four zones of gibberellin-like activity were found in partially purified seed extracts. Gibberellin-like activity remained low in seeds incubated at 5°C for as long as 28 days, but increased significantly in three of these zones after 35 days, and in the fourth zone after 49 days. The increase in gibberellin-like activity was evident prior to the transfer of the seeds to 20°C. Moreover, seeds maintained at 5°C germinated at this temperature after 63 days. For seeds incubated and germinated at 20°C, both the germination percentage and the gibberellin-like activity remained low throughout the experimental period. Application of the growth retardant paclobutrazol to seeds after 28 days of a 49 day total incubation period at 5°C did not substantially reduce seed germination, although the increase in gibberellin-like activity was prevented. Seeds did, however, require a longer time to germinate after transfer to 20°C and were dwarfed in appearance. Application of GA₃ to seeds prior to stratification increased the percentage germination of seeds only when they had been incubated at 5°C for at least 35 days. The major changes in gibberellin-like activity are, therefore, associated not so much with the processes which allow germination to take place in peach, but more with those processes which allow normal growth and development of the seedling.     

Respiratory activity during germination of seeds of Cicer arietinum L. II. Effect of some metabolic inhibitors and mitochondrial activities

Changes in respiration rate during the early period of germination of Cicer arietinum L. seeds were studied. Three phases were distinguished. During phases I and II the RQ was higher than unity. Sodium fluoride showed no effect on gaseous exchange during phase I while in phases II and III a progressive increase in the inhibition of the respiratory activity was obtained. Iodoacetate inhibited oxygen uptake and carbon dioxide evolution from the beginning of germination. The activities of mitochondrial enzymes increased during the germination period, especially after the first 24 hours. The respiratory control and ADP/O ratio of the isolated mitochondria increased gradually during germination, reaching maximum values at 48 hours, lower, however than the expected theoretical ones.     

Cadaverine, an Essential Diamine for the Normal Root Development of Germinating Soybean (Glycine max) Seeds

When the polyamine content of soybean (Glycine max) seeds was examined during the early stages of germination, the major polyamine in the cotyledons was found to be spermidine, followed by spermine; while very low concentrations of cadaverine were found. In the embryonic axes, however, cadaverine was the main polyamine and its content markedly increased 24 hours after the start of germination. When the germination of the seeds was performed in the presence of 1 millimolar α-difluoromethylornithine (DFMO), a marked decrease in the cadaverine content was found, while the other polyamines were not affected. This decrease of the cadaverine content was already noticeable after the first hours of germination. In the presence of DFMO, a pronounced elongation in the roots of the seedlings and a marked decrease in the appearance of secondary roots as compared with controls, was observed. This abnormal rooting of the seedlings caused by DFMO was almost completely reverted by the addition of 1 millimolar cadaverine. The latter also increased the appearance of secondary roots in the seedlings. The decrease in the cadaverine content produced by DFMO could be traced to a strong inhibition of lysine decarboxylase. A temporal correlation between the increase in cadaverine content and the increase in lysine decarboxylase activity was found. Both reached a maximum at the second day of germination. The activity of diamine oxidase, the cadaverine degrading enzyme, started to increase at the third day and reached a maximum between the fourth and fifth day of germination. DFMO increased the activity of diamine oxidase by about 25%. Hence, the large decrease in cadaverine content produced by DFMO has to be attributed to the in vivo suppression of lysine decarboxylase activity. Ornithine decarboxylase activity was also suppressed by DFMO, but putrescine and spermidine contents were not affected, except in the meristematic tissues. The obtained results suggest an important role for cadaverine in the normal rooting process of soybean seedlings.     

Effect of laser light treatment on some biochemical and physiological processes in seeds and seedlings of white lupine and faba bean

The aim of present study was to determine the changes of some biochemical and physiological processes, which occurred in seeds and seedlings of white lupine and faba bean after pre-sowing treatment with laser beams. It was found this treatment of seeds considerably increased the activity of amylolytic enzymes in seeds of both plants. The greatest differentiation of the enzymatic activity was noticed after 120?h from the time of sowing but the activity of these enzymes in the seeds of both tested plants was similar and it had the same course in time. The irradiated seeds of white lupine and faba bean had higher fresh weight at the time of imbibition than the seeds which were not treated with laser beams. It resulted in earlier and more uniform germination. The concentration of free radicals increased considerably in the seeds pre-treated with laser beams and the largest increase in seeds of both plant species was noticed after five exposures to laser beams. Treating seeds with laser beams considerably increased the amount of indole-3-acetic acid (IAA) in the germinating seeds. Three exposures of seeds caused the largest increase of this plant hormone content in the seeds. The activity of IAA in faba bean was slightly higher than in white lupine seeds. Pre-sowing stimulation with laser had a positive influence on the growth and development of seedlings, which had longer hypocotyl and roots in comparison to seedlings which grew from non irradiated seeds.     

Isoenzyme Changes during Seed Germination of Saguaro Cactus (Carnegiea gigantea)

Esterases, acid phosphatases, leucine aminopeptidases, peroxidases and glutamic-oxalacetic transaminases from extracts made from seeds of Saguaro cactus at different stages of germination were studied using starch gelelectrophoretic techniques. In general, four of the five enzyme systems showed significant increase in the number of isoenzymes and their activity after 72 hours of germination. It is suggested that Saguaro cactus seeds reach their enzymatically most active state between 48 to 72 hours after being placed at optimum moisture conditions at 30°C in continuous light.     

Changes in lipoxygenase properties and activity related to postgerminative growth and senescence in oat (Avena sativa L cv. Victory 1 )

Lipoxygenase (LOX), one of the main oxidative catalysts in plants, is involved in the regulation of growth and senescence. We investigated changes in LOX activity or its properties as they related to the development of oat plants at four stages (germination, growth, natural senescence, and dark-incubated senescence). LOX activity was high during early growth and at senescence. At pH 4.5, activity showed an abrupt surge compared with a normal enzyme reaction pattern at pH 6.5. The optimum reaction temperature was 25°C; LOX and peroxidase exhibited similar activity patterns. Polyacrylamide gel electrophoresis revealed that the purified LOX consisted of three isoenzymes in germinating seeds, two in growing seedlings, and three during both natural and dark-induced senescence. As determined by isoelectric focusing, the isoelectric points (pl) of LOX ranged from 3.6 to 6.5 throughout the four developmental stages; for natural or dark-induced senescence, the pl was 9.0.     

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8 kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.     

Localization and Activity of Naphthylamidases in Germinating Seeds of Scots Pine, Pinus sylvestris

Extracts prepared from endosperms of germinating seeds of Scots pine, Pinus sylvestris L., rapidly hydrolysed the β-naphthylamides of L-phenylalanine and L-leucine optimally at pH 6.5 and that of L-arginine at pH 7.7. Disc electrophoresis followed by activity staining showed that the activities were due to two naphthylamidases (aminopeptidases) with different substrate specificities. Seeds were allowed to germinate at 20°C on agar gel in the dark and the activities on the three substrates were assayed from separated endosperms and seedlings at various stages of germination. The activities in the endosperm of resting seeds were relatively high and they remained unchanged throughout the period of reserve protein mobilization (seedling length up to 50 mm), after which they began to decrease. The activities of the naphthylamidases are rather small compared with those of the two alkaline peptidases of pine, contributing about 17% of the total amino-peptidase activity in the endosperm of germinating seeds. The total aminopeptidase activity is sufficient to account for the rate of storage protein mobilization during germination. In the seedlings the naphthylamidase activities (per seedling) increased continuously during germination, and activities per g dry weight were higher than those in the endosperm.     

Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis)

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.     

Development of cytosol and chloroplast aldolases during germination of spinach seeds

The total activity of aldolase (EC 4.1.2.13) and the activities of cytosol and chloroplast aldolase were determined in seeds, cotyledons, primary leaves and secondary leaves of spinach (Spinacia oleracea L., cv. Monopa) during germination. Total aldolase activity in cotyledons increased from low levels to a low maximum in the dark after one week and to a high maximum in white light after three to four weeks and declined thereafter. The activity in primary and secondary leaves started to rise strongly from the 18th and 26th days, respectively, up to the 42nd day of germination. The levels of aldolase activity paralleled the development of leaf area, chlorophyll content and protein content per leaf except that the leaf area of cotyledons continued to increase steadily up to the 42nd day after the maximum of aldolase activity was reached. Resolution of cytosol- and chloroplast-specific isoenzymes by chromatography on diethylaminoethylcellulose indicated that in the light the cytosol enzyme represented approx. 8% of the total activity in cotyledons, primary and secondary leaves throughout germination, and the chloroplast enzyme represented the remaining 92%. Only in cotyledons of dark-grown seedlings was the cytosol aldolase between 25 and 50% of the total activity. Seeds contained almost exclusively a cytosol aldolase. In cotyledons the increase of total activity in the light was specifically the consequence of an increase in chloroplast aldolase while the cytosol aldolase was little affected by light. The light effect was mediated by phytochrome as demonstrated by classical induction and reversion experiments with red and far-red light and by continuous far-red light treatment.Abbreviation DEAE-cellulose diethylaminoethylcellulose     

The Characteristics of Low Temperature Tolerance during Seed Germination of the Ephemeral Plant Lepidium apetalum (Cruciferae)

Lepidium apetalum was used to explore the characteristics and mechanismes of low temperature tolerance during seed germination. The results showed that pre treatment the seeds at 4℃ would improve the seeds viability after stressed by low temperature at -5℃ and -10℃ and the capability of seeds tolerance to cold temperature became weaker along with germination development. Although the growth of radicle was inhibited significantly at 4℃ during germination, the seeds could still survive and grow. With appropriate time of stratified at 4℃, germination pace would be improved and germination rate would not change, while both of which decreased if stratification lasted too long. Moreover, Lapetalum seeds were very sensible to the temperature during germination. It showed no germination at 4℃, while if seeds were stratificated for 48 hours at 4℃ and subsequently treated with 25℃ for more than 6 hours, seeds would germinate at 4℃ and the germination rate increased with the time of 25℃ treatment prolonged. If treatment at 25℃ was less than 5 houes, it showed no germinating. In addition, seeds with stratification for 10 days following 1 h treatment at 25℃ could also germinated at 4℃. The present study indicated that some pivotal genes that related with a key physiological stage before the onset of radicle protrusion might inhibite at 4℃, which resulted in Lapetalum seed could not germinate at 4℃. Except that physiological stage, the Lapetalum seed had a strong tolerance to low temperature at other germination stages. This experiment provided a new way to explore the germination mechanism of ephemerals plants seeds at low temperature.     

Cytologische und stoffwechselphysiologische Untersuchungen am aktiven Meristem der Wurzelspitze von Vicia faba L

Summary Seeds ofVicia faba L. var.minor (2n=12) were submerged in tap water for 12 to 96 hours. After submersion and recovery times of 48 to 144 hours root tips were fixed in intervals of 24 hours. The presoaking of seeds before germination led to an increase of the spontaneous chromosomal aberration rate. Up to 65% of all cells in the root tips showed fragments, chromatid-translocations and triradials after 72 hours of presoaking and up to 12% of all chromosomes showed breaks or participated in chromatidal reunions. Submersion of seeds for 12 hours already caused a statistically significant increase of chromosomal aberrations in comparison to the controls. The maximum of aberrations was found after a recovery period of 120 hours, afterwards the aberration rate dropped. Parallel experiments with the N₂-atmosphere showed that the increase in aberration rate is caused by metabolic changes produced by anaerobiosis. Probably the changed metabolic conditions led to the formation of automutagenic substances. The distribution of breaks between the mand SAT-chromosomes was not at random (theoretical expectation 5∶2); more breaks appeared in the mthan in the SAT chromosomes (actual relation 5∶1,1). The breaks in the mand SAT chromosomes were localized in a few special regions.      

短命植物独行菜种子萌发过程对低温的耐受特性

以独行菜（Lepidium apetalum）为材料,研究了其种子在萌发过程中耐受低温的特性,并对耐受低温的机制作了初步的探讨。结果表明：1.萌发至I、II、III期的独行菜种子经过冷诱导处理后,对零下低温-5℃、-10℃胁迫具有较强的耐受性,这种耐受性随萌发发展相对有所降低。2.独行菜种子萌发过程中胚根生长速度明显受到低温抑制,但也仍然能够生长。3.适当时间的低温层积能使独行菜萌发势显著提高,对终萌发率影响不大;过长时间的低温层积会使独行菜种子萌发势和最终萌发率降低。4.独行菜种子在4℃条件下不能萌发,但4℃层积2d的种子经25℃处理6h以上后,可耐受4℃低温而萌发,且萌发率随25℃处理时间增加而升高。如果25℃处理时间少于5h则不能在4℃低温下萌发。经4℃层积10d、再经25℃萌发处理1h的独行菜种子就能在4℃低温下萌发。综合分析认为独行菜种子不能耐受4℃低温萌发,原因可能是在露白前存在一个关键的生理阶段,在4℃胁迫逆境中不能越过这个阶段,该阶段之前与之后的萌发过程都能耐受4℃低温,因此对低温胁迫有良好的耐受性。这为探索早春短命植物耐受低温萌发的机制提供了新的思路。     

Putrescine and Acid Stress : Induction of Arginine Decarboxylase Activity and Putrescine Accumulation by Low pH

Incubation of peeled oat Avena sativa L. var Victory leaf segments on media of pH 5.0 or below leads to a rapid and massive increase in the titer of putrescine while incubation at pH values above 5.0 causes little or no change. The low pH effect is independent of the buffer system employed. Putrescine levels rise within 3 hours and reach their peak 8 to 9 hours after acidification. At this time, putrescine titer is eight times greater at pH 3.5 than at 6.0. None of the other polyamines shows a response to changes in external pH. The increase in putrescine is blocked by the addition of cycloheximide or dl-alpha-difluoromethylarginine, a specific inhibitor of the putrescine biosynthetic enzyme, arginine decarboxylase. In one experiment, arginine decarboxylase activity was 110% greater at pH 4.0 than at 6.0 after a 4-hour incubation, although the average increase over many experiments was 47%. The activity of the other possible putrescine biosynthetic enzyme, ornithine decarboxylase, falls throughout the incubation period and is virtually equal at pH 4.0 and 6.0.     

The effect of stratification on the endogenous levels of gibberellins and cytokinins in seeds of douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and sugar pine (Pinus lambertiana Dougl.)

Abstract Seeds of Douglas-fir and sugar pine which had been exposed to various periods of moist chilling (stratification) were extracted and bioassayed for gibberellin and cytokinin activity. In Douglas-fir a peak of gibberellin activity increased progressively during stratification and was seven times higher after 7 weeks, but declined subsequently. No change in the level of cytokinin activity was observed during stratification. However, a slight increase (1.7-fold) occurred in stratified seeds placed at 22°C for 3 days. In sugar pine a high level of gibberellin activity, approximately 20-fold higher than in unstratified controls, was present in extracts of seeds which had been stratified for 8 weeks. After 10 weeks of stratification cytokinin levels had increased approximately 200-fold. Four cytokinin-like substances were detected in seeds. Three of the substances are similar in their chromatographic properties to zeatin glucoside, zeatin riboside and zeatin, respectively. Both gibberellin and cytokinin activity declined rapidly after reaching maximum levels. In seeds with the coats removed, germination was rapid and complete in the absence of stratification. Prior to radicle emergence in coatless seeds cytokinin activity increased to a maximum (approximately a 1-7-fold increase) within 24 h. Significant levels of gibberellin-like substances were present after 8 h. Gibberellin activity could not be detected in coatless seeds which were imbibed in AMO-1618, and germination was inhibited significantly.