Ewa Kępczyńska

A Medicago truncatula ABC transporter belonging to subfamily G modulates the level of isoflavonoids

Full-sized ATP-binding cassette (ABC) transporters of the G subfamily (ABCG) are considered to be essential components of the plant immune system. These proteins have been proposed to be implicated in the active transmembrane transport of various secondary metabolites. Despite the importance of ABCG-based transport for plant-microbe interactions, these proteins are still poorly recognized in legumes. The experiments described here demonstrated that the level of Medicago truncatula ABCG10 (MtABCG10) mRNA was elevated following application of fungal oligosaccharides to plant roots. Spatial expression pattern analysis with a reporter gene revealed that the MtABCG10 promoter was active in various organs, mostly within their vascular tissues. The corresponding protein was located in the plasma membrane. Silencing of MtABCG10 in hairy roots resulted in lower accumulation of the phenylpropanoid pathway-derived medicarpin and its precursors. PCR-based experiments indicated that infection with Fusarium oxysporum, a root-infecting pathogen, progressed faster in MtABCG10-silenced composite plants (consisting of wild-type shoots on transgenic roots) than in the corresponding controls. Based on the presented data, it is proposed that in Medicago, full-sized ABCG transporters might modulate isoflavonoid levels during the defence response associated with de novo synthesis of phytoalexins.

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.

Priming of seeds with methyl jasmonate induced resistance to hemi-biotroph Fusarium oxysporum f.sp. lycopersici in tomato via 12-oxo-phytodienoic acid, salicylic acid, and flavonol accumulation.

Methyl jasmonate (MeJA) was tested by seed treatment for its ability to protect tomato seedlings against fusarium wilt caused by the soil-borne fungal pathogen Fusarium oxysporum f.sp. lycopersici. Isolated from Solanum lycopersicon L. seeds, cv. Beta fungus was identified as F. oxysporum f.sp. lycopersici Race 3 fungus by using phytopathological and molecular methods. MeJA applied at 0.01, 0.1 and 1 mM reduced spore germination and mycelial growth in vitro. Soaking of tomato seeds in MeJA solution at 0.1 mM for 1 h significantly enhanced the resistance level against the tested fungus in tomato seedlings 4 weeks after inoculation. The extracts from leaves of 15-day-old seedlings obtained from previously MeJA soaked seeds had the ability to inhibit in vitro spore germination of tested fungus. In these seedlings a significant increase in the levels phenolic compounds such as salicylic acid (SA), kaempferol and quercetin was observed. Up-regulation of phenylalanine ammonia-lyase (PAL5) and benzoic acid/salicylic acid carboxyl methyltransferase (BSMT) genes and down-regulation of the isochorysmate synthase (ICS) gene in response to exogenous MeJA application indicate that the phenylalanine ammonia-lyase (PAL), not the isochorismate (IC) pathway, is the primary route for SA production in tomato. Moreover, the increased accumulation of the flavonols quercetin and kaempferol appears closely related to the increase of PAL5, chalcone synthase (CHS) and flavonol synthase/flavanone 3-hydroxylase-like (FLS) genes. Elevated levels of salicylic acid in seedlings raised from MeJA-soaked seeds were simultaneously accompanied by a decrease of jasmonic acid, the precursor of MeJA, and an increase of 12-oxo-phytodienoic acid (OPDA), the precursor of jasmonic acid. The present results indicate that the priming of tomato seeds with 0.1mM MeJA before sowing enables the seedlings grown from these seeds to reduce the attack of the soil-borne fungal pathogen F. oxysporum f.sp. lycopersici, so it can be applied in practice.

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.

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.

Effects of matriconditioning on onion seed germination, seedling emergence and associated physical and metabolic events

The effect of matriconditioning, the physiological presowing seed technique, using Micro-Cel E on Allium cepa L. cv. Czerniakowska seed quality was studied. Several ratios of seeds, carrier, water and time of priming were tested. The most effective treatment for improving onion seed germination at most tested temperatures was priming to a ratio of 2 g seed:1 g Micro-Cel:3 g water for 5 days in light at 15 °C. Matriconditioning greatly improved the germination and emergence percentage, seedling fresh and dry weight and reduced electrolyte leakage compared to that of untreated seeds; this beneficial effect was especially evident at suboptimal temperatures. Matriconditioning improved the germinability of aged seeds, the effect being more pronounced in the more aged seeds. No significant differences in ethylene production by primed and non-primed seeds were observed in the absence of its precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), but its presence during imbibition caused an increase in ethylene production; an enhanced activity of in vivo ACC oxidase in Allium cepa matriconditioned seeds in comparison to untreated seeds, indicates that the endogenous level of ACC is a limiting factor of ethylene production. Likewise, the activity of ACC oxidase isolated from matriconditioned seeds was higher than that from untreated seeds. Higher endo-β-mannanase and total dehydrogenase activities were observed in primed air-dried seeds in comparing to non-primed seeds.

Implication of Ethylene in the Release of Secondary Dormancy in Amaranthus caudatus L. Seeds by Gibberellins or Cytokinin

Ethephon (Eth), gibberellin A3, A4 + 7 (GA3, GA4 + 7), and 6-benzyladenine (BA) removed secondary dormancy of Amaranthus caudatus seeds. The GAs and BA potentiated the effect of ethephon or 1-aminocyclopropane-1-carboxylic acid (ACC), an ethylene biosynthesis precursor, in terms of the rate or final percent of germination. Aminoethoxyvinylglycine (AVG), an ACC synthase activity inhibitor, was observed to simultaneously inhibit the release from dormancy effected by GA3 or BA as well as the ethylene production stimulated by these regulators. Breaking of secondary dormancy by GA3, GA4 + 7 or BA was prevented by 2,5-norbornadiene (NBD), an inhibitor of ethylene binding. Ethylene completely or markedly reversed the inhibitory effect of NBD. We thus conclude that the removal of secondary dormancy in Amaranthus caudatus seeds by gibberellin or benzyladenine involves ethylene biosynthesis and action.