Promotion of Cocklebur Seed Germination by Allyl, Sulfur and Cyanogenic Compounds

The effects of allyl, sulfur and cyanogenic compounds on thegermination of upper cocklebur (Xanthium pennsylvanicum Wallr.)seeds were examined. Mercaptoethanol and methylmercaptan aswell as KCN, substrates for rßcyanoalanine synthase(CAS), and H₂S and thiocyanate, the products of the CAS catalyzingreaction, were effective in promoting germination, suggestingthe involvement of CAS in germination. Most of allyl compounds, especially allylthiourea, as well asethylene which activated CAS [Hasegawa et al. (1994) Physiol.Plant. 91: 141], promoted the germination in an abnormal typewhich occurred by the predominant growth of cotyledons as didC₂H₄ [Katoh and Esashi (1975) Plant Cell Physiol. 16: 687].However, they failed to activate CAS unlike ethylene, and toliberate free ethylene during an incubation period. It was thuspossible that an C₂H₄-like double bond within allyl compoundscan act to promote seed germination. (Received June 10, 1996; Accepted August 21, 1996)     

Presence of β-cyanoalanine synthase in unimbibed dry seeds and its activation by ethylene during pre-germination

Evolution of HCN from both rice ( Oryza sativa ) and cocklebur ( Xanthium pennsylvanicum ) seeds increased during a pre-germination period and preceded the evolution of (C₂H₄). These two species were adopted as the representatives of starchy and fatty seeds, respectively. Ethylene promotes seed germination of many species. However, HCN evolution declined abruptly when the radicles emerged and before the peak in C₂H₄ evolution. More-over, both rice and soybean ( Glycine max ) seeds showed some activity of β-cyanoalanine synthase (CAS, EC 4.4.1.9) even in the unimbibed dry state. The activities of CAS in the lower seed of cocklebur and in soybean seeds increased rapidly after emergence of the radicle. However, the CAS of rice seeds, with high activity in the dry state, exhibited a bimodal change, gradually decreasing until radicle emergence had occurred, but then increaing. It is thus likly that HCN evolution during initial imbibition may be derived from cyanogenic reserves and controlled by both pre-existing and subsequently-developing CAS. The exogenous application of C₂H₄ stimulated the activities of CAS in both rice and upper cocklebur seeds and reduced their cyanogen contents. Therefore, the decline of HCN evolution after germination seems to be due to the increased activities of CAS by endogenously produced C₂H₄.     

β-Glucosidase activities and HCN liberation in unimbibed and imbibed seeds, and the induction of cocklebur seed germination by cyanogenic glycosides

In many seed species, the major source of HCN evolved during water imbibition is cyanogenic glycosides. The present investigation was performed to elucidate the role of endogenous cyanogenic glycosides in the control of seed germination and to examine the involvment of β-glucosidase in this process. All seed species used here contained some activities of β-glucosidase already in the dry state before imbibition. in the decreasing order of Malus pumila, Daucus carota, Hordeum vulgare, Chenopodium album and so on. β-Gluosidase activity in upper and lower seeds of cocklebur (Xanthium pennsylvanicum Wallr.) decreased with imbibition, and in lower seeds the activity disappeared when they germinated. On the contrary, in caryopses of rice (Oryza sativa L. cv. Sasanishiki) β-glucosidase increased during imbibition, and this increase continued even after germination. β-Glucosidase in cocklebur seeds was more active in the axial than in the cotyledonary tissue. Amygdalin, prunasin and linamarin could all serve as substrattes for the β-glucosidase(s) from both cocklebur and rice. Amygdalin, prunasin and linamarin as well as KCN, were effective in stimulating the germination of upper cocklebur seeds. The seeds evolved much more free HCN gas when they were exposed to the cyanogenic glycosides than when the glycosides were absent. Moreover, the application of the cyanogenic glycosides or of KCN caused accumulation of bound HCN in the seeds. Carbon monoxide, which stimulated cocklebur seed germination only slightly, did not cause accumulation of bound HCN. We suggest that a balance between the cytochrome and the alternative respiration pathways, which is adequate for germination (Esashi et al. 1987. Plant Cell Physiol. 28: 141–150), may be brought about by the action of endogenous HCN; a large portion of which is liberated from cyanogenic glycosides via the action of β-glucosidase. In addition to the partial suppression of the cytochrome path and unlike carbon monoxide, the HCN thus produced may act to supply cyanide group(s) to unknown compounds necessary for germination.     

Possible involvement of ethylene-activated {beta}-cyanoalanine synthase in the regulation of cocklebur seed germination

A possible involvement of ß-cyanoalanine synthase(CAS: EC 4.4.1.9 [EC] ) in germination processes of seeds was demonstratedusing pre-soaked upper seeds of cocklebur (Xanthium pennsylvanicumWallr.). Pretreatment in anoxia not only with KCN but also cysteine,as the substrates for CAS, stimulated the subsequent germinationof cocklebur seeds in air. However, the effect of cysteine wasmanifested even in air when applied together with C₂H₄, andits effect was further enhanced in combination with KCN. Thegermination-stimulating effect of KCN was intensified by C₂H₄only when 0₂ was present. In contrast, serine, another substrateof CAS, was effective in air only when combined with C₂H₄ and/orKCN. The addition of cysteine greatly reduced the cyanogenicglycoside content of seeds, but increased HCN evolution. Onthe other hand, glutathione did not have any effect on cockleburseed germination, HCN evolution or bound cyanogen content, suggestingthat cysteine is not acting as a reducing reagent. It is suggestedthat CAS regulates the process of cocklebur seed germinationby the dual action of enlarging the pool of amino acids andsupplying sulphydryl bases, the latter being more determinatelyimportant. Serine is effective only via the former action, whilecysteine would act via both. Key words: Cyanide, cyanogenic glycoside, ß-cyanoalanine synthase, seed germination, Xanthium pennsylvanicum     

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.     

{alpha}-Aminoisobutyric acid : A probable competitive inhibitor of conversion of 1-aminocyclopropane-1-carboxylic acid to ethylene

Of 16 compounds related to 1-aminocyclopropane-1-carboxylicacid (ACC), aminoisobutyric acid (AIB) inhibited the productionof endogenous ethylene in the cotyledonary segments of cocklebur(Xanthium pennsylvanicum Wallr.) seeds most strongly. AIB at4 mM inhibited the formation of ethylene by about 50%, althoughthe O₂ uptake of the segments was not affected even at 20 mM.AIB also inhibited ethylene formation in the stem segments ofetiolated pea (Pisum sativum L. cv. Alaska) seedlings. Kineticanalysis with cell free extracts from etiolated pea shoots revealedthat AIB competitively inhibits the conversion of ACC into ethylene. (Received May 26, 1980; )     

Involvement of acetaldehyde in seed deterioration of some recalcitrant woody species through the acceleration of aerobic respiration

The rate of acetaldehyde (Ald) evolution in the deterioration of recalcitrant woody seeds was investigated. Four plant species, Ligustrum japonicum, Quercus serrata, Quercus myrsinaefolia and Camellia japonica, were used for the experiments. Similar to orthodox seeds, all of the recalcitrant seeds used contained Ald in addition to methanol and ethanol, although the amount of Ald in Camellia, a typical oil seed, was very small. These volatiles were accumulated in a container in which Ligustrum and Q. serrata seeds were stored for a short period. Moreover, all of the seeds that had been previously exposed to Ald for only 6 d at 3 or 13 degrees C lost their vigor rapidly in proportion to the concentration of Ald. The occasional removal by decompression of Ald accumulated in the container prolonged the life span of Q. serrata seeds from 4 to 6 months. These findings suggest that a short life span of the hydrated recalcitrant seeds may involve Ald synthesis as in the orthodox seeds. However, the action mechanism of Ald in Ligustrum and Quercus seeds in which storage substances were polysaccharides seems to differ slightly from that in orthodox seeds, because their aerobic respiration was significantly stimulated by exposure to exogenously applied Ald. It was, therefore, thought that the rapid deterioration of some recalcitrant seeds in woody species may result from a decline in vigor, not only due to the denaturation of functional proteins by Ald as in the orthodox seeds but also due to the rapid consumption of direct substrates for the Ald-stimulated aerobic respiration and related co-enzymes within seeds. In contrast, in the oil-bearing Camellia seeds, Ald was slightly produced and their aerobic respiration was not enhanced by Ald, although they were very sensitive to Ald. Desiccation storage of Camellia seeds caused the deterioration of their outer part, which was accelerated by exogenously applied Ald, which suggests that in Camellia Ald acts only to denature the functional proteins as in orthodox seeds. Thus, the short longevity of these woody recalcitrant seeds is discussed in relation to the actions of Ald produced endogenously.     

Cytosolic r{beta}-Cyanoalanine Synthase Activity Attributed to Cysteine Synthases in Cocklebur Seeds. Purification and Characterization of Cytosolic Cysteine Synthases

The activity of rß-cyanoalanine synthase (CAS, EC4.4.1.9 [EC] ) in cotyledons of cocklebur seeds (Xanthium penn-sylvanicumWallr.) was detected both in the soluble and particulate fractions.The CAS activity of the soluble fraction (cytosolic CAS activity)was 10 times higher than that of the particulate fraction. TheCAS activity of the particulate fraction was confirmed to belocalized in the mitochondria. Both enzymatic activities wereclearly separated by non-denaturing PAGE. The enzyme with cytosolicCAS activity has been extensively purified and separated intothree different forms designated as cyt-1, cyt-2, and cyt-3.According to the SDS-PAGE analysis, the three enzymes are estimatedto be a homodimer composed of 35-kDa sub-units. The purifiedenzymes showed CS activity. Partial amino acid sequences ofcyt-1 were determined and had a high homology with cysteinesynthases (CS, EC 4.2.99.8 [EC] ) from other plant sources. The catalyticaction of the purified CSs in converting cyanide and cysteineinto H₂S and rß-cyanoalanine was confirmed by thedetection of significant ¹⁴CN incorporation into rß-cyanoalanine.These results indicated that cytosolic CAS activity is due tocytosolic CS and suggested that the CAS activity of CS is likelyto be involved in cyanide metabolism in plant tissues. (Received January 7, 1998; Accepted March 16, 1998)     

Possible involvement of volatile compounds in the after-ripening of cocklebur seeds

The mechanism of emergence from primary dormancy, the process of after-ripening, in cocklebur (Xanthium pennsylvanicum) seeds was examined in relation to the involvement of volatile compounds and to the relative humidity (RH) in which the seeds were stored. The after-ripening of these seeds proceeds only at water contents between 7 and 14% which are conditioned under RHs of 33% to 53% and are identified with water-binding region II. After-ripening of cocklebur seeds occurred even in water-binding region I. imposed by 12% RH. when exposed to HCN gas during the storage period. Exposure of dormant seeds to acetaldehyde (ethanal) retarded after-ripening. even in water-binding region II. thus decreasing germinability. This decrease of germinability by ethanal was found also in the after-ripened seeds, suggesting that ethanal accelerates seed deterioration rather than retarding the after-ripening. The contents of ethanal. ethanal and HCN were high only in the dormant seeds held at 12% RH. Regardless of RH. a possible conversion of ethanal to ethanol. perhaps via alcohol dehydrogenase. was far larger in dormant than in non-dormant seeds. In contrast, the reverse conversion of ethanol to ethanal was more profound in non-dormant seeds. Pre-exposure of both types of seeds to HCN reduced the contents of both ethanal and ethanol at 12% RH. The contents of various adenylales including ATP in seed tissues were higher in dormant seeds stored at 12% RH than in non-dormant seeds after-ripened at 44% RH. It is suggested that emergence of cocklebur seeds from primary dormancy by HCN treatment at 12% RH may result from the reduction in the contents of ethanal via an unknown mechanism incurring the consumption of ATP. This implies involvement of volatile compound metabolism at the water-binding region II in the after-ripening process of cocklebur seeds.     

The Time Course of Dynamic Computed Tomographic Appearance of Radiation Injury to the Cirrhotic Liver Following Stereotactic Body Radiation Therapy for Hepatocellular Carcinoma

This study aimed to evaluate the dynamic computed tomographic (CT) appearance of focal radiation injury to cirrhotic liver tissue around the tumor following stereotactic body radiation therapy (SBRT) for hepatocellular carcinoma (HCC). Seventy-seven patients with 92 HCCs were observed for >6 months. Sixty-four and 13 patients belonged to Child–Pugh class A and B, respectively. The median SBRT dose was 48 Gy/4fr. Dynamic CT scans were performed in non–enhanced, arterial, portal, and venous phases. The median follow-up period was 18 months. Dynamic CT appearances were classified into 3 types: type 1, hyperdensity in all enhanced phases; type 2, hypodensity in arterial and portal phases; type 3, isodensity in all enhanced phases. Half of the type 2 or 3 appearances significantly changed to type 1, particularly in patients belonging to Child–Pugh class A. After 3–6 months, Child–Pugh class B was a significant factor in type 3 patients. Thus, dynamic CT appearances were classified into 3 patterns and significantly changed over time into the enhancement group (type 1) in most patients belonging to Child–Pugh class A. Child–Pugh class B was a significant factor in the non–enhancement group (type 3).