Yukio Morohashi

Molecular cloning of a cDNA encoding a (1-4)-β-mannan endohydrolase from the seeds of germinated tomato (Lycopersicon esculentum)

Abstract. Mannose-containing polysaccharides are widely distributed in cell walls of higher plants. During endosperm mobilization in germinated tomato seeds (1→4)-β-mannan endohydrolases (EC 3.2.1.78) participate in the enzymic depolymerization of these cell wall polysaccharides. A cDNA encoding a (1→4)-β-mannanase from the endosperm of germinated tomato (Lycopersicon esculentum Mill.) seeds has been isolated and characterized. The amino acid sequence deduced from the 5′-region of the cDNA exactly matches the sequence of the 65 NH2-terminal amino acids determined directly from the purified enzyme. The mature enzyme consists of 346 amino acid residues, it has a calculated Mr of 38 950 and an isoelectric point of 5.3. Overall, the enzyme exhibits only 28–30% sequence identity with fungal (1→4)-β-mannanases, but more highly conserved regions, which may represent catalytic and substrate-binding domains, can be identified. Based on classification of the tomato (1→4)-β-mannanase as a member of the family 5 group of glycosyl hydrolases, Glu-148 and Glu-265 would be expected to be the catalytic acid and the catalytic nucleophile, respectively. Southern hybridization analyses indicate that the enzyme is derived from a family of about four genes. Expression of the genes, as determined by the presence of mRNA transcripts in Northern hybridization analyses, occurs in the endosperm of germinated seeds; no transcripts are detected in hypocotyls, cotyledons, roots or leaves.

An Endo-[beta]-Mannanase Develops Exclusively in the Micropylar Endosperm of Tomato Seeds Prior to Radicle Emergence.

A galactomannan-hydrolyzing enzyme that develops pregerminatively in the micropylar region of the endosperm of the tomato (Lycopersicon esculentum [L.] Mill.) seed was characterized. The enzyme was endo-[beta]-mannanase (EC 3.2.1.78), since it hydrolyzed galactomannan into oligosaccharides with no release of galactose and mannose. The mobility of this pregerminative enzyme in sodium dodecyl sulfate and native polyacrylamide gel electrophoresis was not identical to that of any of the three endo-[beta]-mannanases that develop in the same tissue (endosperm) after germination (H. Nonogaki, M. Nomaguchi, Y. Morohashi [1995] Physiol Plant 94: 328–334). There were also some differences in the products of galactomannan hydrolysis between the pregerminative and the postgerminative enzymes, indicating that the action pattern is different between the two types of enzymes. The pregerminative enzyme began to develop in the micropylar region of the endosperm at about 18 h postimbibition and increased up to the time immediately before radicle protrusion (24 h postimbibition). This enzyme was not present in the lateral part of the endosperm at any stage before or after germination. It is proposed that the enzyme develops prior to germination specifically at the micropylar region of the endosperm.

Expression of α-amylase in response to wounding in mung bean

The activity of α-amylase (EC 3.2.1.1) in mung bean (Vigna radiata (L.) Wilczek) cotyledons increased markedly in response to wounding. The changes in enzyme activity were in parallel with those in enzyme content. The level of α-amylase mRNA also notably increased in wounded cotyledons and attained its maximum level during the period between 1 and 2 d after wounding. The level of mRNA for phenylalanine ammonia-lyase, which is one of the well-characterized stress-inducible proteins, also increased after wounding, but the increase in mRNA level was faster than that of α-amylase mRNA. On the other hand, the content of mRNA for actin, a ‘housekeeping’ protein, was almost the same in wounded and unwounded cotyledons. The increase in α-amylase mRNA level in wounded cotyledons was severely inhibited by α-amanitin and cordycepin. α-Amylase expression in the first leaves of mung-bean seedlings was also induced by wounding.

The effect of sodium hypochlorite treatment on the development of α-amylase activity in mung bean cotyledons

Abstract The development of α-amylase activity in mung bean cotyledons was severely retarded, when they were detached from the axis. However, when detached cotyledons were treated with sodium hypochlorite (NaOCl), α-amylase activity was markedly enhanced. The enhancement of the activity was due to a raise in the level of the enzyme protein. The activities of protease and malate dehydrogenase were not affected by the treatment. The enhanced level of α-amylase activity in NaOCl-treated, detached cotyledons was roughly comparable to the level found in attached cotyledons of intact seedlings. However, the localization of α-amylase activity in cotyledon tissues was different between these two kinds of cotyledons. In attached cotyledons, the activity was found in almost all the cells of the tissues. By contrast, in NaOCl-treated, detached cotyledons, the activity developed only in limited regions; mainly in a few cell layers of parenchyma tissues adjacent to the epidermis. The molecular mass (43 kDa) of the enzyme induced in detached cotyledons by NaOCl treatment was the same as that of the enzyme in cotyledons of intact seedlings.

Phytochrome Control of the Development of Ascorbate Oxidase Activity in Mustard (Sinapis alba L.) Cotyledons

The activity of ascorbate oxidase (AOX) in mustard (Sinapis alba L.) cotyledons was markedly increased by irradiation with continuous far-red light. The involvement of phytochrome in this light-mediated response was demonstrated by red/far-red reversibility experiments. To determine immunochemically the contents of AOX in cotyledons, the antibody against the enzyme was raised in a rabbit. However, the antiserum was not monospecific to AOX; it also recognized glycoproteins. To remove antibodies that are specific to a carbohydrate moiety of glycoproteins, the anti-AOX antiserum was applied to a horseradish peroxidase-conjugated Sepharose column. By using the antibodies that were not retained in the column, the changes in the content of AOX were followed. Western immunoblot profiles revealed that the content of AOX protein in cotyledons notably increased after continuous far-red light treatment. Pulse-labeling experiments indicated that the synthesis of AOX protein occurred in the cotyledons. These results are in good agreement with the hypothesis that phytochrome-mediated increase in AOX activity is accompanied by the synthesis of the enzyme.

Isolation and properties of a carboxypeptidase from cotyledons of germinated mung bean seeds

Abstract A serine-type carboxypeptidase (EC 3.4.16.1) present in cotyledons of mung bean ( Vigna radiata ) seedlings was purified up to the step where only a doublet of polypeptides with molecular mass of 43 and 45 kDa, respectively, was detected by SDS-PAGE. The enzyme is thought to be a single chain monomer, as the molecular mass of the enzyme determined by gel filtration is 55 kDa. Although the isolation of each peptide in native form was not successful, the SDS-treated peptides could be separately isolated by preparative electrophoresis. These two polypeptides are immunologically homologous. When proangiotensin is used as a substrate, the enzyme releases amino acid residues in the order of the C-terminal sequence of this oligopeptide chain. This enzyme belongs to the class of serine-type carboxypeptidases, judging from the effects of various protease inhibitors on the enzyme activity. The enzyme preferentially catalyzes the hydrolysis of the peptide bonds formed by the carboxyl group of hydrophobic amino acids. Western immunoblot profiles reveal that the enzyme is absent in cotyledons during the early stage after imbibition and that it begins to increase only after 2–3 days.

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

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

Effect of the Embryonic Axis on the Development of Glyoxysomes, Plastids and Mitochondria in Cotyledons of Germinated Cucumber Seeds

Summary The effect of the presence of the embryonic axis was examined on the following parameters of activities of cellular organelles in dark-grown cucumber cotyledons: activities of malate synthase, isocitrate lyase and 3-hydroxy-acyl-CoA dehydrogenase with glyoxysomes, ribulose bis-phosphate carboxylase activity and rate of chlorophyll formation with plastids, and activities of some respiratory enzymes (succinate and malate dehydrogenase and cytochrome oxidase) and respiratory activities (O 2 uptake and its cyanide-sensitivity, and respiratory control) with mitochondria. The development of these parameters depends upon the presence of the axis, and benzyladenine can bring about a full replacement of the action of the axis on the development of all the parameters except the two, respiratory control and cyanide-sensitivity of mitochondrial respiration. As to these two parameters, benzyladenine showed little effect on their development. On the basis of these results, the role of the axis in the development of the organelles in cotyledons is discussed.

Effect of benzyladenine on the development of mitochondrial activities in imbibed black gram cotyledons

The development of mitochondria was followed in black gram cotyledons cultured on filter paper wetted with water or benzyladenine solution. Treatment with benzyladenine brought about an increase in activities of some mitochondrial enzymes and in respiratory activities of mitochondria. Cycloheximide did not inhibit the increase, although it almost completely inhibited the incorporation of (14)C-leucine into mitochondrial protein. It was inferred that the increase in mitochondrial activity by benzyladenine-treatment was not due to biogenesis of mitochondria but due to an acceleration of activation processes in pre-existing mitochondria. The respiratory control ratio of mitochondria from benzyladenine-treated cotyledons was lower than that of mitochondria from untreated ones. It was also suggested that benzyladenine brought about some qualitative changes in mitochondrial activities.

Induction of amylase in pea cotyledons by a factor present in lactalbumin hydrolysate

Abstract Enzymatically hydrolysed lactalbumin was found to contain some substance whose treatment removed the inhibition of amylase formation in excised pea cotyledons and restored the amylase activity to a level similar to that in attached cotyledons. This factor was dialysable. An amino acid mixture with the same composition as that of lactalbumin showed no activity. The R F value of this substance in thin layer chromatography was 0.09 in the solvent system BuOH/HOAc/water (4 : 1 : 1) and 0.55 in pyridine/EtOAc/HOAc/water (36 : 36 : 7 : 21).