Yohji Esashi

A mechanism of seed deterioration in relation to the volatile compounds evolved by dry seeds themselves

Some of the seed-evolved volatiles, which were mainly composed of methanol, acetaldehyde, ethanol and acetone, caused the loss of seed germinability during storage. In general, the deleterious effects of the volatiles increased with increasing RH and temperature during storage. Acetaldehyde had the strongest deleterious effect regardless of RH and temperature, while ethanol caused seed deterioration only at high RHs. Acetone was slightly deleterious to some species, while methanol was almost inert in most seeds. Various aldehydes applied during storage showed some toxicity to seed germinability, which decreased, except for 3-methylbutanal, with increasing molecular size, suggesting that the endogenous volatile compounds with an aldehyde group cause seed deterioration. On the other hand, the contents of volatile compounds in seeds were higher when they were stored at 44% RH (water sorption zone 2), than when stored at 12% RH (water sorption zone 1). Acetaldehyde, the most deleterious volatile, was more abundantly accumulated within the seeds stored at −3.5°C than at 23°C. Based on these facts, it is suggested that endogenous volatiles, especially acetaldehyde, may be an important factor that accelerates seed deterioration which often occurs under lower RHs and/or temperatures throughout long-term storage.

A Competitive Enzyme-Linked Immunosorbent Assay to Quantify Acetaldehyde-Protein Adducts That Accumulate in Dry Seeds during Aging.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to quantify endogenous acetaldehyde-protein adducts (APAs) produced in plant seeds at low acetaldehyde concentrations without exogenous reducing agents. The key point of this technique is the use of a gelatin-acetaldehyde adduct, which is synthesized under 1 mM acetaldehyde and 10 mM NaCNBH3, to pre-coat plate wells to obtain the proper binding parameters for the quantification of APA in seed proteins. Compared with the traditional, direct ELISA method, the competitive one has higher sensitivity and less background. Using competitive ELISA, we determined the accumulation of endogenous APAs in seeds in relation to the loss of seed viability. Lettuce seeds were exposed to 2 mM gaseous acetaldehyde during storage for 30 or 45 d; the relative humidity and temperature of storage were studied independently. Viability decreased only in acetaldehyde-treated seeds, as either the temperature or the relative humidity increased. A loss in viability was accompanied by an increase in the accumulation of APA. The APA content also increased as viability decreased in five species of seeds, which were aged naturally without exposure to acetaldehyde. It is suggested that the modification of functional seed proteins with endogenously evolved acetaldehyde may be an important cause of seed aging.

Possible participation of β-cyanoalanine synthase in increasing the amino acid pool of cocklebur seeds in response to ethylene during the pre-germination period

The amino acid content in cocklebur (Xanthium pennsylvanicum Wallr.) seeds was increased by ethylene, which stimulated their germination, regardless of whether they were non-dormant or secondarily dormant. This increase in amino acid content coincided with the increased activities of β-cyanoalanine synthase (CAS, EC 4.4.1.9) in response to ethylene. KCN and/or cysteine, the substrates of CAS, also increased the amino acid content in both non-dormant and secondarily dormant cocklebur seeds. The degrees of the increased amino acid content corresponded roughly to the germination rates of the seeds reported previously. The actual involvement of CAS in the germination process in cocklebur seeds was demonstrated by incorporation into asparagine and aspartate from 14CN which was fed to the cotyledon segments of both non-dormant and secondarily dormant cocklebur seeds. In this case, the incorporation of 14CN was augmented by ethylene, and incorporated more abundantly in the cotyledons of secondarily dormant seeds. Moreover, ethylene decreased the cysteine + cystine content in both the axial and cotyledon tissues, but increased asparagine and aspartate regardless of whether they were non-dormant or secondarily dormant. This suggests that CAS responsiveness to ethylene participates in supplying asparagine and aspartate and in increasing the amino acid pool of cocklebur seeds during the pre-germination period.

D-Amino-acid-stimulated ethylene production in seed tissues

Ethylene production by axial and cotyledonary tissues excised from Xanthium pennsylvanicum Wallr. seeds was markedly (up to 5-fold) stimulated by the D-isomers of phenylalanine, valine, leucine, threonine, methionine and eithionine while the L-isomers caused no such effect. Responsiveness of these seed tissues to D-methionine appeared soon after the beginning of imbibition, reached a maximum after 6–12 and 12–24 h for the axial and cotyledonary tissues, respectively, and then decreased sharply. D-Phenylalanine and D-methionine also stimulated ethylene production in seed tissues of X. canadense Mill. and in cotyledonary segments from seeds of Helianthus annuus L., Cucurbita moschata Duch. and Vigna radiata (L.) Wilczek. The endogeneous ethylene production and the D-amino-acid-stimulated ethylene production by the seed segments was strongly inhibited by aminoethoxyvinyl glycine, a potent inhibitor of ethylene synthesis from L-methionine.

Induction of cocklebur seed germination by anaerobiosis: A question about the “inhibitor hypothesis” of seed dormancy

SummaryGermination of non-dormant small cocklebur (Xanthium pennsylvanicum Wallr.) seeds was improved by immersing them in water, suggesting that during their germination endogenous germination inhibitors are leached out. However, the same effect could be obtained by the quasi-anaerobic pre-incubation of the seeds. When seeds were fully imbibed, moreover, water immersion could no longer potentiate them to germinate, and only anaerobiosis increased the germination potential, thus raising a question against the “inhibitor hypothesis” of seed dormancy.

d-amino-acid-stimulated ethylene production: Molecular requirements for the stimulation and a possible receptor site

Abstract Various kinds of d -amino acids enhanced ethylene production in the cotyledonary segments of Xanthium pennsylvanicum seeds. To be effective they required not only their d -configuration but also the presence of α-NH 2 , α-COOH and a hydrophobic as well as a bulky side chain. Moreover, they exhibited almost the same dissociation constant in kinetic analysis. On the other hand, l -phenylalanine and d -serine, which themselves were ineffective in the stimulation of ethylene production, competitively inhibited d -phenylalanine-stimulated ethylene production. Thus, the d -amino-acid-stimulated ethylene production was explained by assuming the existence in cells of d -amino-acid receptor site associated with ethylene synthesis.

Volatile Compounds and Accumulation of Acetaldehyde-Protein Adducts in Relation to Seed Quality and Storage Conditions

ABSTRACT Volatile compounds were studied from soybean (Glycine max L. Merrill) and snap bean (Phaseolus vulgaris L.) seeds in relation to seed quality and controlled aging treatments. Qualitative and semiquantitative analysis revealed more than 30 compounds from dry seeds as measured by gas chromatography and a thermal desorption method, including aldehydes, alcohols, ketones, alkanes, alkenes and furans. Ethanol, acetaldehyde and methanol were the major volatiles related to aging and further studied. Evolution profiles were similar for both species; during aging, but snap bean produced ca. two times the concentration of volatiles as soybeans. Ethanol evolution increased while acetaldehyde decreased as soybean seed aged in an open storage condition. Methanol and ethanol evolution increased with aging period conducted with a range of aging conditions from 0.60 to 0.75 water activity (Aw) and 45 to 35_C in closed packets. However, ethanol generally decreased with aging at 0.80 Aw, which was attributed to mitochondrial respiration at the higher moisture level. Acetaldehyde can non-enzyma-tically react with proteins, and acetaldehyde-protein adducts (APA) formed in intact seeds were quantified using a competitive ELISA. APA formation coincided with the three phases of germination loss in storage, and was negatively correlated with the percent standard germination (r = 0.989). APA formation may be one mechanism responsible for the loss of germinability in storage.

Mechanism of Action of C2H4 in Promoting the Germination of Cocklebur Seeds. III. A Further Enhancement of Priming Effect With Nitrogenous Compounds and C2H4 Responsiveness of Seeds

Efficiency of organic or inorganic osmotica for seed priming of cocklebur (Xanthium pennsylvanicum Wallr.) revealed that KNO3 was the most promising, and was more effective than mannitol or other salts at the same concentration (200 mM) and was independent of the C2H4 action. However, KNO3 applied as a priming reagent enhanced the effect of C2H4 or that of the water stress imposed with mannitol. Unlike the action of mannitol, both KNO3M and C2H4 augmented the pool size of amino acids in seed cells. However, below 50 mM KNO3 imposing no stress only slightly, though insignificantly, affected the germinability as well as the levels of total cyanogen. On the other hand, at a high concentration which imposed water stress on the seeds, 200 mM KNO3 remarkably elevated the contents of both cyanogenic glycosides and lipids in the excised cotyledons. When C2H4 was added with KNO3, the level of cyanogenic compounds significantly increased but when added without KNO3, the contrary effect was shown. Hence the enhancement of the mannitol-induced priming effect by nitrogenous reagents in cocklebur seeds could be implicated in the accumulation of cyanogenic compounds. Unlike cocklebur, both common chickweed and barnyard grass seeds are very responsive to 30 mM KNO3 on germination, and such species abundantly contain cyanogen. The amount of cyanogen was further augmented by contact with KNO3 at only 30 mM. The role of NO-3 -dependent cyanogenesis is highlighted in relation to germination response of seeds.

In vivo formation of 1-malonylaminocyclopropane-1-carboxylic acid and its relationship to ethylene production in cocklebur seed segments: a tracer study with 1-amino-2-ethylcyclopropane-1-carboxylic acid

Abstract The in vivo formation of 1-malonylaminocyclopropane-1-carboxylic acid (malonyl-ACC) and its relationship to ethylene production in the axial tissue of cocklebur ( Xanthium pennsylvanicum ) seeds were investigated using the stereoisomers of the 2-ethyl derivative of ACC (AEC), as tracers of ACC. Of the four AEC isomers, the (1 R , 2 S )-isomer was converted most effectively to a malonyl conjugate as well as to 1-butene. Malonyl-AEC, once formed, was not decomposed, supporting the view that malonyl-ACC does not liberate free ACC for ethylene production in this tissue. d -Phenylalanine inhibited the formation of malonyl-AEC and, at the same time, promoted the evolution of 1-butene, whereas l -phenylalanine did not. Possibly, the d -amino-acid-stimulated ethylene production in cocklebur seed tissues is due to an increase in the amount of ACC available for ethylene production which results from the decrease of ACC malonylation in the tissues treated with d -amino acid. 2-Aminoisobutyric acid, a competitive inhibitor of ACC-ethylene conversion, did not affect the malonylation of AEC.

Two C2H4-producing systems in cocklebur seeds

SummaryC2H4 production of the embryonic axes and cotyledons excised from dormant and non-dormant cocklebur (Xanthium pennsylvanicum Wallr.) seeds was examined in relation to ambient O2 tensions. There were two kinds of C2H4-producing systems, quasi-anaerobic and aerobic, in both organs. Regardless of the organ, the former activity was high in the dormant state and, particularly in axes, declined with after-ripening. On the other hand, the latter activity was almost insignificant in the dormant state, but increased with release from dormancy and the non-dormant axes exclusively produced C2H4 through this system. In the cotyledons, however, the former was still predominant even after they were fully after-ripened. Thus, the C2H4-producing systems were different in the seed organ and in the dormancy state.