Jana Krajňáková

Mitochondrial bioenergetics linked to the manifestation of programmed cell death during somatic embryogenesis of Abies alba

The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial KATP+ channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the KATP+ channel activity could induce an entry of K+ into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.

Long-term cryopreservation of Greek fir embryogenic cell lines: Recovery, maturation and genetic fidelity

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.

Activity of a KATP+ channel in Arum spadix mitochondria during thermogenesis

This report demonstrates that mitochondria isolated from thermogenic Arum spadices possess an ATP-sensitive potassium channel--responsible for electrical potential (DeltaPsi) collapse and mitochondrial swelling--whose characteristics are similar to those previously described in pea and wheat mitochondria. In order to study the relationship between this K(ATP)(+) channel and the uncoupled respiration, linked to thermogenesis, K(+) transport activities were compared with those of mitochondria that were isolated from pea stems, soybean suspension cell cultures and Arum tubers. The channel from Arum spadices is highly active and its major features are (i) potassium flux is performed primarily in an inward-rectifying manner; (ii) the influx of K(+) is associated with a matrix volume increase in both energized and non-energized mitochondria; and (iii) its activity depends on the redox state of electron transport chain (ETC) and oxygen availability. In particular, this paper shows that the K(ATP)(+) channel is inwardly activated in parallel with the alternative oxidase (AO). The activation is linked to an ETC-oxidized state and to high oxygen consumption. The putative role of this K(ATP)(+) channel is discussed in relation to flowering of thermogenic Arum spadices.

Isolation of mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud.: characterization of a K+(ATP) channel.

A valuable method to isolate and purify mitochondria from embryonal masses of two coniferous species (Picea abies [L.] Karst. and Abies cephalonica Loud.) is described. Crude mitochondria from both species were shown to be intact, oxygen consuming (with malate plus glutammate, succinate and NADH as substrates) and well coupled (respiratory control ratio ca. 4). The oxidation of the substrates was only partially KCN-insensitive (alternative oxidase) in some cases. However, these fractions were contaminated by membranes (e.g. plasmalemma, tonoplast, Golgi and endoplasmic reticulum). After purification by a discontinuous Percoll gradient (18, 23, 40%, v/v), three mitochondrial populations were separated. The 0/18 interface fraction was composed mainly of broken and uncoupled mitochondria, while the other two (18/23 and 23/40 interface fractions) contained intact and coupled mitochondria, but only 23/40 interface fraction revealed to be better purified starting from both coniferous embryonal masses. In the latter purified fraction, the presence of a cyclosporin A-sensitive KATP+ channel was demonstrated. These findings were discussed in the light of the potential use of these mitochondrial fractions in bioenergetic studies, or in the involvement of these organelles to stress response in conifers.

Fulvic acid affects proliferation and maturation phases in Abies cephalonica embryogenic cells.

Embryogenic cell masses (ECM) of Abies cephalonica were grown on proliferation media in the presence and absence of fulvic acid (FA), whose molecular composition and conformational rigidity were evaluated by CPMAS-¹³C NMR spectroscopy. To assess the physiological effects of this humic material during proliferation and maturation stages of somatic embryogenesis (SE), proliferation rate, proportion of consecutive developmental stages of pro-embryogenic masses (PEM), cellular ATP and glucose-6-phosphate were evaluated at regular intervals. FA increased the proliferation rate, especially during the early sampling days, and the percentage of PEM in their advanced developmental stage. Cellular ATP and glucose-6-phospahte were increased by FA pre-treatment during the maturation phase. Furthermore, the effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), such as a decrease of growth and the enhancement of PEM III induction, were inverted by FA. Proton pumping ATPase and PPase activities were decreased in microsomes from PCIB-treated ECM, while they increased in the presence of FA. This fulvic matter also induced a delay in somatic embryo formation during the maturation phase. Both the improvement of the PEM proliferation and the reduction of the subsequent maturation process of A. cephalonica are explained by a release from the complex humic structure of low molecular-weight molecules, which may interact with the plant hormonal signaling pathway. These effects appear to be related to the hydrophilic and conformationally labile nature of FA. The structure-activity relationship observed here suggests that the influence of FA on ECM may be attributed to specific bioactive molecules that are preferentially released from the FA loose superstructure.

Changes in ATP, glucose-6-phosphate and NAD(P)H cellular levels during the proliferation and maturation phases of Abies alba Mill. embryogenic cultures

The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phosphate (glu-6P) and reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle, four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 μM) applied together (Experiments A and B) or with addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.

Somatic Embryogenesis of Greek Fir ( Abies cephalonica Loud.)

Euro-Mediterranean firs (the genus Abies Mill.) belong to ecologically and commercially most important tree genera in Europe. Fir forests represent a major component of Central European, Alpine and Mediterranean mountain forests. Their distribution ranges from 6°W to 44°E in longitude, from 35°N to 52°N in latitude and from 135 to 2900 m in altitude.