Jan Kępczyński

Stimulatory effect of ethephon, ACC, gibberellin A3 and A4+7 on germination of methyl jasmonate inhibited Amaranthus caudatus L. seeds

Methyl jasmonate (JA-Me) inhibited or retarded germination of Amaranthus caudatus seeds in darkness at 24°C, Ethephon, ACC and gibberellins (GA3 or GA4+7) partially or completely reversed this inhibition depending on the concentration of JA-Me applied. Both ethephon and the gibberellins were more effective than ACC. Both GA3 and GA4+7 enhanced the stimulatory effect of ethephon or ACC on germination of seeds inhibited by JA-Me.

Regulation of ethylene biosynthesis at the level of 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene

Ethylene, a gaseous plant hormone regulates essentially all physiological processes during the plant’s life cycle. The practical implications of ethylene biosynthesis regulation for plant improvement have supported the continuous basic research on dissecting the structure of genes encoding ethylene biosynthetic enzymes, their differential expression patterns, and mechanisms underlying their transcriptional activity. ACC oxidase (ACO) is involved in the final step of ethylene production in plant tissues. In various plants several ACO isoforms have been isolated which are encoded by a multigenic family. There is a strong evidence that ACO gene expression is positively correlated to the ethylene production rates and its multiple isoforms are under development and environmental control. Thus, the regulation of ACO gene activity may act either as an additional or in several cases also as a main level for controlling ethylene biosynthesis in higher plants. This review summarizes in detail the knowledge about organization and gene structure, and transcriptional expression of ACO genes from different plant species. The perspectives of manipulating ACO gene as a method in biotechnological modification of ethylene synthesis are also discussed.

Endogenous ethylene in indirect somatic embryogenesis of Medicago sativa L.

Ethylene biosynthesis during different phases of somatic embryogenesis in Medicago sativa L. cv. Rangelander using two regeneration protocols, RPI and RPII, was studied. The highest ethylene production was detected during callus growth on induction medium in both regeneration protocols. Significantly less ethylene was produced by embryogenic suspension than by callus (RPII). Developing embryos synthesized higher amounts of ethylene than mature embryos. Production of ethylene was strongly limited by the availability of 1-aminocyclopropane-1-carboxylic acid and also by ACC-oxidase activity. However, removal of ethylene from culture vessels’ atmosphere using KMnO4 or HgClO4 had no significant effect on callus growth, somatic embryo induction and development. Reducing of ethylene biosynthesis by aminoethoxyvinylglycine substantially decreased somatic embryo production and adversely affected their development, indicating ethylene requirement during proliferation and differentiation but not induction.

Ethylene biosynthesis in Amaranthus caudatus seeds in response to methyl jasmonate

Methyl jasmonate (JA-Me) at 10−3 M completely inhibited Amaranthus caudatus seed germination. Exogenous ethylene could totally reverse this inhibition. The inhibitor of ethylene action, 2,5-norbornadiene (NBD), increased the sensitivity of seeds to JA-Me. Methyl jasmonate inhibited ethylene production and also decreased both 1-aminocyclopropane-1-carboxylic acid (ACC) and malonyl ACC (MACC) content. Likewise, ACC oxidase activity in vivo was decreased by jasmonate. Similarly ACC oxidase activity in vitro isolated from seeds incubated in the presence of JA-Me was lower than that isolated from untreated seeds.The inhibitory JA-Me action on seed germination seems to be mainly associated with the inhibition of ethylene biosynthesis. Both inhibition of ACC synthase and ACC oxidase activity and/or synthesis can be involved.

Regulation of Medicago sativa L. somatic embryogenesis by gibberellins

The influence of exogenous gibberellic acid (GA3) andpaclobutrazol, an inhibitor of gibberellin biosynthesis, on growth of callusandsomatic embryogenesis in petiole-derived tissue cultures of Medicagosativa L. has been investigated. GA3 (0.5–500μM) or paclobutrazol(5–100 μM) were added to either an induction (with 2,4 Dand kinetin) or a differentiation medium (without plant growth regulators).Gibberellin A3, applied during the induction as well as thedifferentiation stage, reduced the weight of callus and increased the number ofsomatic embryos in Medicago sativa L. tissue cultures.Somatic embryo production was increased more by the presence of exogenousGA3 in the differentiation than induction medium. The inclusion ofpaclobutrazol in the induction or differentiation medium caused the inhibitionof callus growth and embryo production. Callus growth was much less affectedthan embryogenesis. These results indicate that gibberellins are beneficial forboth embryoinduction and formation. The level of endogenous gibberellins is presumablysufficient for callus induction and growth. However, it seems not optimal forthe induction and particularly for the differentiation of embryos.

Responsiveness of Amaranthus retroflexus seeds to ethephon, 1-aminocyclopropane 1-carboxylic acid and gibberellic acid in relation to temperature and dormancy

Dormant Amaranthus retroflexus seeds do not germinate in the dark at temperatures below 35°C. Fully dormant seeds germinate only at 35–40°C whereas non-dormant ones germinate within a wider range of temperatures (15 to 40°C). Germination of non-dormant seeds requires at least 10% oxygen, but the sensitivity of seeds to oxygen deprivation increases with increasing depth of dormancy. 10−6 to 10−4M ethephon, 10−3M 1-aminocyclopropane 1-carboxylic acid (ACC) and 10−3M gibberellic acid (GA3) break this dormancy. In the presence of 10−3M GA3 dormant seeds are able to germinate in the same range of temperatures as non-dormant seeds. The stimulatory effect of GA3 is less dependent on temperature than that of ethephon, while ACC stimulates germination only at relatively high temperatures (25–30°C). The results obtained are discussed in relation to the possible involvement of endogenous ethylene in the regulation of germination of A. retroflexus seeds.

Germination induction of dormant Avena fatua caryopses by KAR1 and GA3 involving the control of reactive oxygen species (H2O2 and O2−) and enzymatic antioxidants (superoxide dismutase and catalase) both in the embryo and the aleurone layers

Avena fatua L. caryopses did not germinate at 20 °C in darkness because they were dormant. However, they were able to germinate in the presence of karrikinolide (KAR1), a key bioactive compound present in smoke, and also in the presence of gibberellin A3 (GA3), a commonly known stimulator of seed germination. The aim of this study was to collect information on a possible relationship between the above regulators and abscisic acid (ABA), reactive oxygen species (ROS) and ROS scavenging antioxidants in the regulation of dormant caryopses germination. KAR1 and GA3 caused complete germination of dormant A. fatua caryopses. Hydrogen peroxide (H2O2), compounds generating the superoxide (O2(·-)), i.e. menadione (MN), methylviologen (MV) and an inhibitor of catalase activity, aminotriazole (AT), induced germination of dormant caryopses. KAR1, GA3, H2O2 and AT decreased ABA content in embryos. Furthermore, KAR1, GA3, H2O2, MN, MV and AT increased α-amylase activity in caryopses. The effect of KAR1 and GA3 on ROS (H2O2, O2(·-)) and activities of the superoxide dismutase (SOD) and catalase (CAT) were determined in caryopses, embryos and aleurone layers. SOD was represented by four isoforms and catalase by one. In situ localization of ROS showed that the effect of KAR1 and GA3 was associated with the localization of hydrogen peroxide mainly on the coleorhiza. However, the superoxide was mainly localized on the surface of the scutellum. Superoxide was also detected in the protruding radicle. Germination induction of dormant caryopses by KAR1 and GA3 was related to an increasing content of H2O2, O2(·-)and activities of SOD and CAT in embryos, thus ROS homeostasis was probably required for the germination of dormant caryopses. The above regulators increased the content of ROS in aleurone layers and decreased the activities of SOD and CAT, probably leading to the programmed cell death. The presented data provide new insights into the germination induction of A. fatua dormant caryopses by KAR1 and also by GA3. In A. fatua, KAR1 or GA3 is included in the induction germination of dormant caryopses through regulation level of ABA in embryos and ROS-antioxidant status both in embryos and aleurone layers.

The influence of the jasmonates and abscisic acid on callus growth and somatic embryogenesis in Medicago sativa L. tissue culture

The jasmonates as well as abscisic acid were found to be inhibitors of callus growth and somatic embryogenesis in Medicago sativa L. tissue cultures. An exposure to these inhibitors during the induction as well as the differentiation stage reduced the number of somatic embryos obtained. The jasmonates showed to be less active in the inhibition of callus growth and somatic embryo production than abscisic acid.

The release of secondary dormancy by ethylene in Amaranthus caudatus L. seeds

Neither ethylene nor 1-aminocyclopropane-1-carboxylic acid (ACC) was able to prevent the induction of secondary dormancy of Amaranthus caudatus at 45°C. Both ethylene (4.5 × 10 -9 –4.5 × 10 -7 M) and ACC (10 -3 –10 -2 M) removed secondary dormancy at 25°C, although ethylene was much more effective. The presence of ethylene for only 10 h was sufficient to remove secondary dormancy in almost all seeds. Incubation of secondary dormant seeds for up to 5 d at 25°C did not change sensitivity to ethylene. The breaking of secondary dormancy by ethylene was prevented by 2,5-norbornadiene (NBD; 1.5 × 10 -5 –3 × 10 -4 M), indicating the physiological action of ethylene. Abscisic acid (ABA; 10 -4 –10 -3 M) increased the requirement for exogenous ethylene. It is suggested that secondary dormancy in A. caudatus seeds might be related to insufficient ethylene production associated with an insufficient amount of ACC.

The involvement of ethylene in the release of primary dormancy in Amaranthus retroflexus seeds

Primary dormancy in A. retroflexus seeds wascompletely broken by dry storage or ethylene treatment and partially removedwith GA3. Norbornadiene counteracted the dormancy breaking action ofethylene and GA3. The GA3 effect was lowered bycobaltous ions. ABA increased the ethylene requirement in primary dormant seeds.Dormant seeds had a similar or different ability to produce ethylene and ACCoxidase in vivo activity than did non-dormant seeds,depending on the period of incubation. Dormant seeds contained less endogenousACC than non-dormant seeds. Thus, ethylene seems to play an essential role inthe release of primary dormancy in A. retroflexus seeds.Ethylene also participates in the release of dormancy achieved by GA3treatment. The results indicate that both ethylene biosynthesis and action isinvolved in the control of primary dormancy in Amaranthusretroflexus seeds.