Evidence of two enzymes performing the de-N-glycosylation of proteins in barley: expression during germination, localization within the grain and set-up during grain formation

The occurrence of two enzymes performing de-N-glycosylation of glycoproteins, namely, endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety). The dry grain showed both activities according to the HPLC detection of the hydrolysis of fluorescent resorufin-labelled substrates. However, PNGase activity was 16-fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5-fold compared to nearly 4.8-fold for ENGase over the 4 d following imbibition. The localization of these activities within the grain showed that the major contribution of PNGase was due to the endosperm, typically representing over 90% of the whole grain activity. In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10-fold higher than in the endosperm), particularly in the rootlets and scutellum. In developing spikes, PNGase activity was 5.6-fold higher than in the leaves, but similar ENGase activity was measured in both organs. During grain formation, PNGase activity followed dry matter increase together with endosperm development. In contrast, ENGase activity dropped by 66% at the beginning of grain filling before stabilizing until harvest. The occurrence of de-N-glycosylation-performing enzymes throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could represent the first evidence of specific functions for each enzyme.     

Amylolytic Activities in Cereal Seeds under Aerobic and Anaerobic Conditions

An adequate carbohydrate supply contributes to the survival of seeds under conditions of limited oxygen availability. The amount of soluble, readily fermentable carbohydrates in dry cereal seeds is usually very limited, with starch representing the main storage compound. Starch breakdown during the germination of cereal seeds is the result of the action of hydrolytic enzymes and only through the concerted action of [alpha]-amylase (EC 3.2.1.1), [beta]-amylase (EC 3.2.1.2), debranching enzyme (EC 3.2.1.41), and [alpha]-glucosidase (EC 3.2.1.20) can starch be hydrolyzed completely. We present here data concerning the complete set of starch-degrading enzymes in three cereals, rice (Oryza sativa L.), which is tolerant to anaerobiosis, and wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.), which are unable to germinate under anoxia. Among the cereal seeds tested under anoxia, only rice is able to degrade nonboiled, soluble starch, reflecting the ability to degrade the starch granules in vivo. This is explained by the presence of the complete set of enzymes needed to degrade starch completely either as the result of de novo synthesis ([alpha]-amylase, [beta]-amylase) or activation of preexisting, inactive forms of the enzyme (debranching enzyme, [alpha]-glucosidase). These enzymes are either absent or inactive in wheat and barley seeds kept under anaerobic conditions.     

Contribution of the Pentose Phosphate Pathway and Glycolytic Pathway to Dormancy Breakage and Germination of Peanut (Arachis hypogaea L.) Seeds

Levels of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADPH oxidoreductase, E.C. 1.1.1.49 [EC] ) 6-phosphogluconate dehydrogenase(6-phospho-D-gluconate : NADP⁺ oxidoreduc tase, E.C. 1.1.1.44 [EC] )and aldolase (fructose 1, 6-diphosphate, D-glyceraldehyde, 3-phosphatelyase, E.C. 4.1.2.13 [EC] ) were assayed in the seeds of geneticallydormant and non-dormant pure lines of groundnut. In dormantlines cotyledons showed increased levels of activity of G-6-PDHand 6-PGDH during dry storage after-ripening. While the embryonicaxis did not exhibit detectable levels of enzyme activitiesimmediately after harvest, the activity started after a lapseof time during dry storage. When seeds of dormant lines wereincubated with kinetin (6-furfurylaminopurine) a distinct increasein the levels of both the enzymes was observed. The levels ofaldolase activity gradually decreased in the cotyledons andincreased in the embryonic axis of both control and kinetintreated seeds during the period of after-ripening. Comparedto control, kinetin treatment increased the aldolase activityin the embryonic axis and decreased it in the cotyledons. In non-dormant lines the activity of both the enzymes of PPpathway increased sharply both in the cotyledons and embryonicaxis while aldolase activity decreased in the cotyledons andincreased in the embryonic axis during germination i.e., from24 h to 96 h of germination. Abscisic acid caused inhibitionof enzyme activities to a large extent. Key words: PP pathway, dormancy breakage, germination, peanut     

beta]-Tubulin Accumulation and DNA Replication in Imbibing Tomato Seeds

The activation of the cell cycle in embryo root tips of imbibing tomato (Lycopersicon esculentum Mill. cv Lerica) seeds was studied by flow cytometric analyses of the nuclear DNA content and by immunodelection of [beta]-tubulin. With dry seeds, flow cytometric profiles indicated that the majority of the cells were arrested at the G1 phase of the cell cycle. In addition, [beta]-tubulin was not detectable on western blots. Upon imbibition of water, the number of cells in G2 started to increase after 24 h, and a 55-kD [beta]-tubulin signal was detected between 24 and 48 h. Two-dimensional immunoblots revealed at least three different [beta]-tubulin isotypes. Thus, [beta]-tubulin accumulation and DNA replication were induced during osmotic priming. These processes, as well as seed germination rate, were enhanced upon subsequent imbibition of water, compared with control seeds that imbibed but were not primed. By contrast, when aged seeds imbibed, DNA replication, [beta]-tubulin accumulation, and germination were delayed. In all cases studied, both DNA replication and [beta]-tubulin accumulation preceded visible germination. We suggest that activation of these cell-cycle-related processes is a prerequisite for tomato seed germination. Furthermore, [beta]-tubulin expression can be used as a parameter for following the initial processes that are activated during seed imbibition.     

Identification of two discrete peptide: N-glycanases in Oryzias latipes during embryogenesis.

Two different types of peptide:N-glycanase (PNGase) were identified in developing embryos of medaka fish ( Oryzias latipes ). Because the optimum pH values for their activities were acidic and neutral, they were designated as acid PNGase M and neutral PNGase M, respectively. The acid PNGase M corresponded to the enzyme that had been partially purified from medaka embryos (Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1991) J. Biol. Chem., 266, 22110-22114). The apparent molecular weight of this enzyme was 150 K, and the optimal pH was 3.5-4.0, and the K m for L-hyosophorin was 44 microM. L-Hyosophorin is a cortical alveolus-derived glycononapeptide with a large N-linked glycan chain present in the perivitelline space of the developing embryo. Acid PNGase M was competitively inhibited by a free de-N-glycosylated nonapeptide derived from L-hyosophorin. This enzyme was expressed in ovaries and embryos at all developmental stages after gastrulation, but activity was not detected in embryos at developmental stages between fertilization and gastrula. Several independent lines of evidence suggested that acid PNGase M may be responsible for the unusual accumulation of free N-glycans derived from yolk glycoproteins (Iwasaki,M., Seko,A., Kitajima,K., Inoue,Y. and Inoue,S. (1992) J. Biol. Chem., 267, 24287-24296). In contrast, the neutral PNGase M was expressed in blastoderms from the 4-8 cell stage and in cells up to early gastrula. The general significance of these findings is that they show a developmental stage-dependent expression of the two PNGase activities, and that expression of the neutral PNGase M activity occurs concomitantly with the de-N-glycosylation of L-hyosophorin. These data thus support our conclusion that the neutral PNGase M is responsible for the developmental-stage-related de-N-glycosylation of the L-hyosophorin.     

Changes in trigonelline (N-methylnicotinic acid) content and nicotinic acid metabolism during germination of mungbean (Phaseolus aureus) seeds

Changes in trigonelline content and in biosynthetic activity were determined in the cotyledons and embryonic axes of etiolated mungbean (Phaseolus aureus) seedlings during germination. Accumulation of trigonelline (c. 240 nmol per pair of cotyledons) was observed in the cotyledons of dry seeds; trigonelline content decreased 2 d after imbibition. Trigonelline content in the embryonic axes increased with seedling growth and reached a peak (c. 380 nmol per embryonic axis) at day 5. Trigonelline content did not change significantly during the differentiation of hypocotyls, and the concentration was greatest in the apical 5 mm. Nicotinic acid and nicotinamide were better precursors for pyridine nucleotide synthesis than quinolinic acid, but no great differences were found in the synthesis of trigonelline from these three precursors. Trigonelline synthesis was always higher in embryonic axes than in cotyledons. Activity of quinolinate phosphoribosyltransferase (EC 2.4.2.19), nicotinate phosphoribosyltransferase (EC 2.4.2.11), and nicotinamidase (EC 3.5.1.19) was found in cotyledons and embryonic axes, but no nicotinamide phosphoribosyltransferase (EC 2.4.2.12) activity was detected. It follows that quinolinic acid and nicotinic acid were directly converted to nicotinic acid mononucleotide by the respective phosphoribosyltransferases, but nicotinamide appeared to be converted to nicotinic acid mononucleotide after conversion to nicotinic acid. Trigonelline synthase (nicotinate N-methyltransferase, EC 2.1.1.7) activity increased in the embryonic axes, but decreased in cotyledons during germination. [14C]Nicotinic acid and trigonelline absorbed by the cotyledons were transported to the embryonic axes during germination. Trigonelline had no effect on the growth of seedlings, but nicotinic acid and nicotinamide significantly inhibited the growth of roots. Based on these findings, the role of trigonelline synthesis in mungbean seedlings is discussed.     

An increase in pectin methyl esterase activity accompanies dormancy breakage and germination of yellow cedar seeds

Pectin methyl esterase (PME) (EC 3.1.1.11) catalyzes the hydrolysis of methylester groups of cell wall pectins. We investigated the role of this enzyme in dormancy termination and germination of yellow cedar (Chamaecyparis nootkatensis [D. Don] Spach) seeds. PME activity was not detected in dormant seeds of yellow cedar but was induced and gradually increased during moist chilling; high activity coincided with dormancy breakage and germination. PME activity was positively correlated to the degree of dormancy breakage of yellow cedar seeds. The enzyme produced in different seed parts and in seeds at different times during moist chilling, germination, and early post-germinative growth consisted of two isoforms, both basic with isoelectric points of 8.7 and 8.9 and the same molecular mass of 62 kD. The pH optimum for the enzyme was between 7.4 and 8.4. In intact yellow cedar seeds, activities of the two basic isoforms of PME that were induced in embryos and in megagametophytes following dormancy breakage were significantly suppressed by abscisic acid. Gibberellic acid had a stimulatory effect on the activities of these isoforms in embryos and megagametophytes of intact seeds at the germinative stage. We hypothesize that PME plays a role in weakening of the megagametophyte, allowing radicle emergence and the completion of germination.     

Studies on peroxisomes. VI. Relationship between the peroxisomal core and urate oxidase.

The peroxisomal core from the liver of rats was purified 450-fold as a marker of urate oxidase [EC 1.7.3.3.] activity. This preparation has a high specific activity of urate oxidase but not of other peroxisomal enzymes: D-amino acid oxidase [EC 1.4.3.3.], L-alpha-hydroxy acid oxidase [EC 1.1.3.15], or catalase [EC 1.11.1.6]. No activity of marker enzymes for other subcellular particles; cytochrome c oxidase [EC1.9.3.1] (mitochondria), acid phosphatase [EC 3.1.3.2] (lysosomes), or glucose-6-phosphatase [EC 3.1.3.9] (microsomes), was detected in this preparation. The core obtained showed a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the position of the band was found to correspond to a molecular weight 35,000. When the peroxisomal core was subjected to treatment at various pH's with 0.1 M carbonate buffer, urate oxidase was almost completely solubulized at pH 11.0, although approximately 35% of the core protein still remained in the pellet After solubilization of the core at pH 11.0, the specific activity of urate oxidase in the supernatant increased about 1.6 times; the density of the insoluble protein remaining in the pellet was identical with the that of the original core on sucrose density gradient centrifugation.     

Changes in structural features of free N-glycan and endoglycosidase activity during tomato fruit ripening

In developing plants, free N-glycans occur ubiquitously at micromolar concentrations. Such oligosaccharides have been proposed to be signaling molecules in plant development. As a part of a study to elucidate the physiological roles of de-N-glycosylation machinery involved in fruit ripening, we analyzed changes in the amounts and structural features of free N-glycans in tomato fruits at four ripening stages. The amount of high-mannose type free N-glycans increased significantly in accordance with fruit ripening, and the relative amounts of high-molecular size N-glycans, such as Man(8-9)GlcNAc(1), became predominant. These observations suggest that the de-N-glycosylation machinery, including endo-beta-N-acetylglucosaminidase (ENGase) activity, is stimulated in the later stages of fruit ripening. But contrary to expectation, we found that total ENGase activities in the tomato fruits did not vary significantly with the ripening process, suggesting that ENGase activity must be maintained at a certain level, and that the expression of alpha-mannosidase involved in the clearance of free N-glycans decreases during tomato fruit ripening.     

Some biochemical parameters of rice varieties of Primorskii krai

The content of protein, ascorbic acid, starch, amylose, amylopectin and glucose in seeds and seedlings of six varieties of rice cultivars released in Primorskii krai was investigated. The possibility of determining the quality of the grain based on the activity of enzymes of carbohydrate metabolism amylase [EC 3.2.1.1] and phosphorylase [EC 2.4.1.1] in caryopsis was demonstrated.     

A comparative study of glutathione and ascorbate metabolism during germination of Pinus pinea L. seeds

The ascorbate and glutathione systems have been studied during the first stages of germination in orthodox seeds of the gymnosperm Pinus pinea L. (pine). The results indicate that remarkable changes in the content and redox balance of these metabolites occur in both the embryo and endosperm; even if with different patterns for the two redox pairs. Dry seeds are devoid of the ascorbate reduced form (ASC) and contain only dehydroascorbic acid (DHA). By contrast, glutathione is present both in the reduced (GSH) and in the oxidized (GSSG) forms. During imbibition the increase in ASC seems to be mainly caused by the reactivation of its biosynthesis. On the other hand, the GSH rise occurring during the first 24 h seems to be largely due to GSSG reduction, even if GSH biosynthesis is still active in the seeds. The enzymes of the ascorbate--glutathione cycle also change during germination, but in different ways. ASC peroxidase (EC 1.11.1.11) and glutathione reductase (EC 1.6.4.2) activities progressively rise both in the embryo and in endosperm. These changes are probably required for counteracting production of reactive oxygen species caused by recovery of oxidative metabolism. The two enzymes involved in the ascorbate recycling, ascorbate free radical (AFR) reductase (EC 1.6.5.4) and DHA reductase (EC 1.8.5.1), show different behaviour: the DHA reductase activity decreases, while that of AFR reductase remains unchanged. The relationship between ascorbate and glutathione metabolism and their relevance in the germination of orthodox seeds are also discussed.     

In Vivo and in Vitro Phosphorylation of the Phosphoenolpyruvate Carboxylase from Wheat Seeds during Germination

Phosphoenolpyruvate carboxylase (PEPC) activity was detected in the aleurone endosperm of wheat (Triticum aestivum cv Chinese Spring) seeds, and specific anti-Sorghum C4 PEPC polyclonal anti-bodies cross-reacted with 103- and 100-kD polypeptides present in dry seeds and seeds that had imbibed; in addition, a new, 108-kD polypeptide was detected 6 h after imbibition. The use of specific anti-phosphorylation-site immunoglobulin G (APS-IgG) identified the presence of a phosphorylation motif equivalent to that found in other plant PEPCs studied so far. The binding of this APS-IgG to the target protein promoted changes in the properties of seed PEPC similar to those produced by phosphorylation, as previously shown for the recombinant Sorghum leaf C4 PEPC. In desalted seed extracts, an endogenous PEPC kinase activity catalyzed a bona fide phosphorylation of the target protein, as deduced from the immunoinhibition of the in vitro phosphorylation reaction by the APS- IgG. In addition, the major, 103-kD PEPC polypeptide was also shown to be radiolabeled in situ 48 h after imbibition in [32P]orthophosphate. The ratio between optimal (pH 8) and suboptimal (pH 7.3 or 7.1) PEPC activity decreased during germination, thereby suggesting a change in catalytic rate related to an in vivo phosphorylation process. These collective data document that the components needed for the regulatory phosphorylation of PEPC are present and functional during germination of wheat seeds.     

Activities of de-N-glycosylation are ubiquitously found in tomato plant

Activities of two de-N-glycosylation enzymes, PNGase (peptide N ⁴(N-acetyl-glucosaminyl)asparagine amidase) and ENGase (endo N-acetyl-β-D-glucosaminidase), involved in the release of N-glycans from N-glycoproteins, were monitored in several organs of tomato plants (Lycopersicon esculentum, Mill., cv. Dombito) with a fluorescence-HPLC procedure using a resofurin-labelled N-glycopeptide substrate. PNGase and ENGase activities were detected in every organ assayed but with quantitative differences. The highest activities were found in the youngest parts of the plant, i.e. apical buds, flowers and leaf blades. PNGase activities were consistently higher than ENGase activities (three-fold in average). Both de-N-glycosylation activities were associated with high levels of proteins and protease activities. During fruit growth and ripening, these three parameters decreased notably. The ubiquitous detection of these enzyme activities in the different organs is probably associated with the previously characterized unconjugated N-glycans in tomato. The possible role of PNGase and ENGase degradation products (i.e. unconjugated N-glycans) are discussed in relation with their biological functions in plant development.     

Gaba shunt in developing soybean seeds is associated with hypoxia

In the present study we investigated the proposal that the γ-aminobutyrate (Gaba) shunt in developing soybean (Glycine max [L.] Merr.) seeds is associated with hypoxia. The ontogeny and pH profile of enzymes associated with glutamate metabolism (glutamate decarboxylase [EC 4.1.1.15]. Gaba transaminase [EC 2.6.1.19], succinic semialdehyde dehydrogenase [EC 1.2.1.16], glutamate dehydrogenase [EC 1.4.1.2], glutamate:oxaloacetate transaminase [EC 2.6.1.1], glutamate:pyruvate transaminase [EC 2.6.1.2] and 2-oxoglutarate dehydrogenase complex [EC 1.2.4.2]) and hypoxia (alcohol dehydrogenase [ADH, EC 1.1.1.1] and pyruvate decarboxylase [PDC, EC 4.1.1.1]) were determined in cotyledons, nucellus and seed-coat tissues. Gaba-shunt enzymes were ubiquitous in the developing seed. Activities of enzymes catalyzing glutamate-C entry into the Krebs cycle via 2-oxoglutarate were generally greater than those of Gaba-shunt enzymes. In cotyledons, the activity of ADH increased throughout seed development (up to 72 days after anthesis [DAA]), whereas PDC was static during early development, then increased. In contrast, the activities of ADH and PDC in maternal tissues (nucellus and seed coat) were initially high, then declined dramatically after 37 DAA. The adenylate energy charge (AEC) = ([ATP]+0.5 [ADP])/ ([ATP] + [ADP] + [AMP]) of soybean seeds from fruits (37 DAA) frozen in situ was low (0.67±0.01) compared to the AEC of adjacent pod tissue (0.82 ± 0.04) and cotyledons exposed to air (0.84 ± 0.01). A 60-min time-course study showed that the rate of [U-¹⁴C]-glutamate catabolism by an intact excised cotyledon at 37 DAA was markedly lower at 8 and 0% O₂ than at 21%; the pool size of [¹⁴C]-Gaba was unaffected. The data indicated that: (1) Gaba-shunt activity is not a response to limited glutamate deamination/transamination: (2) the soybean seed is hypoxic; and (3) the relative partitioning of glutamate-C through glutamate decarboxylase is increased by hypoxia.     

Control of galactosyl-sugar metabolism in relation to rate of germination

The storage sugars stachyose and raffinose (galactosyl derivatives of sucrose) are metabolized early during germination of soybean [ Glycine max (L.) Merr.] seeds. The activities of four enzymes involved in the catabolism of these sugars were monitored in soybean cotyledons and embryonic axes during a 7-day germination period. An increase in enzyme activities correlated with a decline in galactosyl sugars. In embryonic axes, uridine diphosphate glucose (UDPglc)-hexose-l-P uridyltransferase (EC 2.7.7.12), an enzyme characteristic of the Leloir pathway, predominated over galactose-1-phosphate uridyltransferase (EC 2.7.7.10), an enzyme characteristic of the pyrophosphorylase pathway; whereas in cotyledons, the situation was reversed. There were differences between two cultivars. Ransom and Amsoy, in the levels of UDPglc-4-epimerase (EC 5.1.3.2); but not in glucose-1-phosphate uridyltransferase (EC 2 7.7.9). An accelerated aging treatment had a significant effect on the development of embryonic axes, as measured by dry weight. In vitro aging of seeds reduced the rate of growth and resulted in higher levels of galactose-containing sugars and significantly lower levels of UDPglc-hexose-l-P uridyltransferase. Thus, reduced development may be related to inability to mobilize or utilize stored carbon reserves. However, it has not been proved that the reduced enzyme activity is responsible for the effects of accelerated aging on growth and sugar metabolism.     

Enzyme activities during imbibition and germination of seeds of tamarack (Larix laricina)

Activities of six enzymes from extracts of separated embryos and gametophytes of tamarack [ Larix laricina (Du Roi) K. Koch] seeds were assayed at various stages of imbibition and germination. On a per seed part basis, activities of 6-phosphogluconate dehydrogenase (6-PGD, EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G-6-PD, EC 1.1.1.49), malate dehydrogenase (NAD⁺–MDH, EC 1.1.1.37), isocitrate dehydrogenase (NADP⁺–IDH, EC 1.1.1.42), soluble peroxidase (PER, EC 1.11.1.7), and acid phosphatase (ACP, EC 3.1.3.2) from both the embryo and gametophyte tissues generally increased slowly, following cold stratification for 30 days and imbibition under germinating conditions for 5 days, but then increased at a faster rate with emergence of the radicle and subsequent growth of the seedling. The rate of increase of enzyme activity was highest for PER. Soluble protein levels also increased with imbibition and germination, with about 3 times greater levels present in the gametophyte than in the embryo. Heat inactivation experiments showed that, except for G-6-PD, activities were stable up to 40°C. Inactivation occurred at lower temperatures for G-6-PD, while higher temperatures were required for PER. Incubation of extracts for 7 days at 4°C indicated that loss of enzyme activity was greatest for G-6-PD (3.9% remaining) and least for PER and ACP (94 and 95% remaining, respectively).     

Glyoxysomal malate dehydrogenase of watermelon cotyledons: De novo synthesis on cytoplasmic ribosomes

The development of glyoxysomal malate dehydrogenase (gMDH, EC 1.1.1.37) during early germination of watermelon seedlings (Citrullus vulgaris Schrad.) was determined in the cotyledons by means of radial immunodiffusion. The active isoenzyme was found to be absent in dry seeds. By density labelling with deuterium oxide and incorporation of [¹⁴C] amino acids it was shown that the marked increase of gMDH activity in the cotyledons during the first 4 days of germination was due to de novo synthesis of the isoenzyme. The effects of protein synthesis inhibitors (cycloheximide and chloramphenicol) on the synthesis of gMDH indicated that the glyoxysomal isoenzyme was synthesized on cytoplasmic ribosomes. Possible mechanisms by which the glyoxysomal malate dehydrogenase isoenzyme reaches its final location in the cell are discussed.Abbreviations mMDH mitochondrial malate dehydrogenase - gMDH glyoxysomal malate dehydrogenase - D₂O deuterium oxide - EDTA ethylenediaminetetraacetic acid, disodium salt     

Thiamine binding and metabolism in germinating seeds of selected cereals and legumes.

The basic characteristics of thiamine metabolism in germinating seeds of maize (Zea mays), oat (Avena sativa), faba bean (Vicia faba) and garden pea (Pisum sativum) are presented with a special emphasis of a possible thiamine storage function of seed thiamine-binding proteins (TBPs). Seeds were germinated for 6 d in the dark. Thiamine-binding activity in seeds decreased during germination by 50% in cereals and by 30% in legumes. The degradation of TBPs was also detected by polyacrylamide gel electrophoresis. The total thiamine content decreased rapidly to 20-40% of the initial value in cereal seeds during first 3 d of germination while in legume seeds thiamine content started changing from the fourth day and dropped by 50% at the sixth day. A composite pattern was found for the changes in thiamine pyrophosphate (TPP) contribution to total thiamine during seed germination. A peak of the coenzyme percentage was usually detected at the second day of germination. Another gain of TPP was often seen toward the sixth day of germination. The activity of thiamine pyrophosphokinase (EC 2.7.6.2) was high in resting legume seeds and did not significantly change during germination. In contrast, the low activity of this thiamine-activating enzyme in cereal seeds progressively increased during germination. Thiamine phosphate synthase (EC 2.5.1.3) was also detected in seeds and was shown to contribute significantly to the balance of thiamine compounds during seed germination.     

A Single-Seed Assay for Endo-[beta]-Mannanase Activity from Tomato Endosperm and Radicle Tissues

Completion of germination (radicle emergence) is an all-or-none developmental event for an individual seed. Variation in germination timing among seeds in a population therefore reflects variation among seeds in the rates or extents of physiological or biochemical processes prior to radicle emergence. For tomato (Lycopersicon esculentum Mill.) seeds, correlative evidence suggests that endo-[beta]-mannanase activity weakens the endosperm cap tissue opposite the radicle tip to permit radicle emergence. To test whether endo-[beta]-mannanase activity is causally related to germination rates, we have developed a sensitive assay suitable for use with individual radicle tips or endosperm caps. We show that endo-[beta]-mannanase activity varies at least 100-fold and often more than 1000-fold among individual inbred tomato seeds prior to radicle emergence. Other sources of variation (tissue size and experimental error) were evaluated and cannot account for this range of activity. Endo-[beta]-mannanase activity was generally 10-fold greater in leachates from endosperm caps than from radicle tips. Release of reducing sugars from individual endosperm caps also varied over a considerable (9-fold) range. These extreme biochemical differences among individual tomato seeds prior to radicle emergence indicate that results obtained from bulk samples could be misleading if it is assumed that all seeds exhibit the "average" behavior.