Stanislaw Lewak

Metabolic control of embryonic dormancy in apple seed: seven decades of research

Embryonic dormancy is defined as a set of blocks imposed upon a process(es) cardinal for growth. In apple seeds, all these blocks are removed as a result of cold treatment (stratification), but some of them are also affected by light and/or hormonal treatments. This review summarizes published data related to the modes of action of above factors on the changes in the levels of endogenous hormones and some other plant growth regulators (e.g., hydrogen cyanide), and on certain enzymes involved in mobilization of seed reserves and catabolism of their hydrolysis products. Phytochrome and activities of acid lipase and a protease have been indicated as receptors of light and low temperature, respectively. Several chains of events initiated by these two environmental factors and leading to dormancy removal are proposed, and the sites of their control by hormones and HCN are indicated. These chains are postulated to contribute to the elimination of particular blocks that hinder germination and therefore to be involved in the mechanisms of dormancy breakage.

Jasmonic acid affects dormancy and sugar catabolism in germinating apple embryos

Embryos isolated from dormant seeds of apple (Malus domestica Borb., cv. Antonowka) were cultured in darkness in the presence of jasmonic acid (JA, 20 μM) for 7 d in parallel to control non-treated ones. Soluble sugars were quantified and some sugar-catabolysing enzyme activities were determined in axes and in cotyledons of the embryos during the culture. JA treatment stimulated growth of the axis and sucrose hydrolysis in this organ. In contrast, JA inhibited the growth of isolated cotyledons. In intact embryos, JA treatment inhibited the growth of the lower cotyledon (being in contact with wet medium) thus alleviating the growth asymmetry of cotyledons. In both cotyledons JA stimulated hydrolysis of sucrose during the period preceding growth. The effect persisted in the upper cotyledon for the whole experimental period, whereas in the lower one the treatment provoked a sharp rise in soluble sugar levels observed relatively late during the experiment. The later effect correlated with the stimulation of isocitrate lyase activity in the lower cotyledon by JA. The results suggest the induction of the gluconeogenetic pathway by JA in the lower cotyledon of cultured dormant apple embryos. They also provide evidence for the site of JA action in the regulatory complex controlling embryonic dormancy in apple.

Is the gibberellin A4 biosynthesis involved in the removal of dormancy in apple seeds

Abstract Changes in the gibberellin A4 (GA4) content during culture of isolated apple embryos are dependent on the stage of seed after-ripening. Similarly, the intensity of incorporation of labelled gibberellins precursor into the GA4 fraction depends to a high degree on the physiological state of seeds and correlates with the content of this hormone. The increase of endogenous GA4 level was observed during the culture of embryos isolated from seeds stratified shorter than 40 days. The intensive incorporation of [14C]-mevalonate into a fraction co-chromatographed with GA4 was stated during culture of such embryos. This incorporation was inhibited by 2-isopropyl-4-dimethylamino-5-methylphenyl-piperidinecarboxylate methyl chloride (AMO-1618), an inhibitor of gibberellins biosynthesis. The same treatment inhibits also the germination of embryos from seeds stratified for the same period. The suggestion was drawn concerning the role of the GA4 biosynthesis in the removal of embryonal dormancy of apple seeds.

The role of lipases in the germination of dormant apple embryos

SummaryChanges in the activity of alkaline and acid lipases were studied during the culture of embryos isolated from dormant apple (Pyrus malus L.) seeds. Light and treatment with gibberellins (GAs) and with AMO-1618, an inhibitor of GA biosynthesis, influenced the activity of both enzymes. In dark-grown embryos the activity of the alkaline lipase is stimulated by red irradiation and by treatment with GAs, particularly GA4, but is not increased after far-red irradiation or in the presence of AMO-1618. In light cultured embryos, it is inhibited by the latter compound. Germination of the embryos is affected by the various treatments in a similar manner as alkaline lipase activity. These results suggest that light stimulates germination of dormant apple embryos by promoting GA4 biosynthesis via the phytochrome system; GA4 increases the activity of alkaline lipase, the enzyme hydrolysing the storage lipids. Activation of acid lipase by the same factors is of a more complicated nature, light, GAs, and AMO-1618 generally causing a decrease in the activity of this enzyme.

Sugar metabolism as related to the cyanide-mediated elimination of dormancy in apple embryos

Embryos isolated from dormant seeds of apple (Malus domestica Borb., cv. Antonowka) were treated by gaseous HCN (1 mM) for 6 h and then cultured in the light for 9 d in parallel to control non-treated embryos. Soluble sugars were quantified, and oligosaccharide hydrolysing enzyme activities were determined in axes and in cotyledons of such embryos during culture. HCN pre-treatment stimulated germination and hydrolysis of oligosaccharides in embryonic axes. Hydrolysis of sucrose in the lower cotyledon (in contact with wet medium and therefore growing and greening faster) was affected by HCN to a small extent. On the other hand, growth, greening and sucrose hydrolysis in the upper cotyledon were stimulated by HCN pre-treatment to the levels observed in lower cotyledon. It is postulated that endogenous HCN in apple seeds controls the removal of embryonic dormancy in parallel to the earlier described control by light and gibberellin. Gibberellin was demonstrated to act on the hydrolysis and further transformations of storage lipids, including gluconeogenesis, but not β-oxidation of fatty acids. It appears that hydrolysis of oligosaccharides and their catabolism is the target for cyanide.

Interaction of jasmonic acid with some plant growth regulators in the control of apple (Malus domestica) embryo germination

Embryos isolated from dormant apple seeds were treated with jasmonic acid (JA), gibberellin A3 (GA3), abscisic acid (ABA) and hydrogen cyanide in darkness and in light. The chemicals were present in the culture medium continuously and simultaneously or applied for 2 days and in different sequences. All treatments stimulated embryo germination except ABA, which was strongly inhibitory. Additive effects of JA with light and with GA3 on embryo germination were observed, whereas ABA interacted synergically with JA, HCN and light. ABA and GA3 were most effective when applied early during embryo incubation, but the late JA treatment was more stimulatory. It is concluded that JA does not act on the regulatory pathway that is initiated by light and which leads to embryo germination through gibberellin accumulation and alkaline lipase activation. ABA and HCN appear to be involved in the control of this pathway. JA and ABA may be involved in the control of alkaline lipase activity, independently of this regulatory chain.

Stimulation of L-phenylalanine ammonia-lyase (PAL) activity by D-phenylalanine in germinating seeds

Abstract D-Phenylalanine, contrary to its L-stereoisomer causes an increase in L-phenylalanine ammonia-lyase (PAL) activity during the culture of dormant and stratified apple embryos and during the germination of mustard, rape and oat seeds. None of the other six pairs of L- and D-aminoacids tested caused a comparable effect. It was demonstrated that the increase of PAL activity follows the normal germination and that the stimulation of this activity by D-phenylalanine is not directly related either to germination or to the growth processes. The possible mechanism of PAL stimulation in vivo is discussed.

Hormone Interactions in the Formation of the Photosynthetic Apparatus in Dormant and Stratified Apple Embryos

Summary The following criteria for embryonal dormancy in nonstratified as compared with stratified apple embryos were used: diminished germinability, lowered chlorophyll accumulation, diminished ribulose-bis-phosphate carboxylase activity and enhanced phosphoenolpyruvate carboxylase activity. All these symptoms occurred in stratified embryos in culture as a result of abscisic acid (ABA) treatment. In non-stratified embryos all these symptoms could be removed, at least partly, by gibberellin and/or kinetin treatment. On the contrary, only few of them, when induced by ABA in non-dormant, stratified embryos were reversed by growth stimulators; in some cases gibberellin or kinetin increased the effect of ABA. Thus it was concluded that ABA is not the main factor in the maintenance of dormancy and the possibility of involvement in embryonal dormancy of mechanisms different from a simple hormonal balance was discussed.

Growth regulators differentially affect photosensitivity induced by low temperature and osmotic stresses in germinating white clover seeds

Negative photoblastism induced in white clover seeds at 5°C or by lowered water potential (−0.3 MPa, polyethylene glycol) was affected by ethrel, gibberellin A3, benzylaminopurine and kinetin treatments. The effects were different for water and temperature stressed seeds. The observed synergistic and additive effects of light and growth regulators confirm the earlier suggestion that there are two different mechanisms involved in the light inhibition of white clover seed germination induced by various adverse environmental conditions.

Non-decarboxylating transformation of indol-3-ylacetic acid in apple seeds

An enzyme extract from apple(Pyrus malus Borb.) seeds which causes the disappearance of free indol-3-ylacetic acid (IAA) requires the presence of oxygen, but is not inhibited by cyanide. Using 1-14C-IAA it has been demonstrated that the IAA transformation is not accompanied by its decarboxylation. Decarboxylating IAA oxidase is absent during the whole period of apple seed cold stratification. Free IAA has not been detected in dormant apple seeds and in seeds stratified at low temperature. It appears during stratification at 25 °C. Ethyl ester of IAA and indol-3-ylacetyl aspartate have been identified in dormant and after-ripened seeds. Exogenous 1-14C IAA taken up by apple embryos is converted into conjugates with aspartate and short peptides containing an aspartate moiety.