Exposure of Norway spruce (
) somatic embryos and those of many other conifers to post-maturation desiccation treatment significantly improves their germination. An integration analysis was conducted ...to understand the underlying processes induced during the desiccation phase at the molecular level. Carbohydrate, protein and phytohormone assays associated with histological and proteomic studies were performed for the evaluation of markers and actors in this phase. Multivariate comparison of mature somatic embryos with mature desiccated somatic embryos and/or zygotic embryos provided new insights into the processes involved during the desiccation step of somatic embryogenesis. Desiccated embryos were characterized by reduced levels of starch and soluble carbohydrates but elevated levels of raffinose family oligosaccharides. Desiccation treatment decreased the content of abscisic acid and its derivatives but increased total auxins and cytokinins. The content of phytohormones in dry zygotic embryos was lower than in somatic embryos, but their profile was mostly analogous, apart from differences in cytokinin profiles. The biological processes "Acquisition of desiccation tolerance", "Response to stimulus", "Response to stress" and "Stored energy" were activated in both the desiccated somatic embryos and zygotic embryos when compared to the proteome of mature somatic embryos before desiccation. Based on the specific biochemical changes of important constituents (abscisic acid, raffinose, stachyose, LEA proteins and cruciferins) induced by the desiccation treatment and observed similarities between somatic and zygotic
embryos, we concluded that the somatic embryos approximated to a state of desiccation tolerance. This physiological change could be responsible for the reorientation of Norway spruce somatic embryos toward a stage suitable for germination.
In Norway spruce, as in many other conifers, the germination capacity of somatic embryos is strongly influenced by the desiccation phase inserted after maturation. The intensity of drying during ...desiccation eminently affected the formation of emblings (i.e., seedlings developed from somatic embryos). Compared to non-desiccated embryos, the germination capacity of embryos desiccated at 100% relative humidity was about three times higher, but the reduction of relative humidity to 95 and 90% had a negative effect on the subsequent embryo development. The water loss observed in these embryos did not lead to an increase in lipid peroxidation, as shown by malondialdehyde levels. Another metabolic pathway in plants that mediates a response to abiotic stresses is directed toward the biosynthesis of polyamines (PAs). The activities of PA biosynthetic enzymes increased steadily in embryos during desiccation at 100% relative humidity, whereas they decreased at lower humidity. The total content of free PAs in the embryos gradually decreased throughout desiccation. The increase in free putrescine (Put) and perchloric acid-insoluble Put conjugates was observed in embryos desiccated at lower humidity. These changes were accompanied to some extent by the transcription of the genes for the PA biosynthesis enzymes. Desiccation at 100% relative humidity increased the activity of the cell wall-modifying enzymes β-1,3-glucanases and chitinases; the activities of these enzymes were also significantly suppressed at reduced humidity. The same pattern was observed in the transcription of some β-1,3-glucanase and chitinase genes. Desiccation treatments triggered metabolic processes that responded to water availability, suggesting an active response of the embryo to the reduction in humidity. A positive effect was demonstrated only for desiccation at high relative humidity. Some of the physiological characteristics described can be used as markers of inappropriate relative humidity during somatic embryo desiccation.
Ultraviolet-B (UV-B) radiation is a key environmental signal which initiates diverse responses that affect the metabolism, development, and viability of plants. In keeping with our previous studies, ...we concentrated primarily on how UV-B radiation affects Norway spruce
(L.) Karst. somatic embryo maturation and how phenolics and polyamines (PAs) are linked to the defense response invoked by UV-B irradiation. We treated clusters of Norway spruce embryogenic culture (EC) with UV-B during the five stages of embryo maturation (early, cylindrical, precotyledonary, cotyledonary, and mature embryos). For the first time, we take an advantage of the unique environmental scanning electron microscope AQUASEM II to characterize somatic embryos in their native state. The severity of the irradiation effect on embryonal cell viability was shown to be dependent on the intensity of radiation as well as the stage of embryo development, and might be related to the formation of protoderm. The response of early embryos was characterized by an increase in malondialdehyde (MDA), a marked decrease in PA contents and a decline in phenolics. The reduced ability to activate the defense system seems to be responsible not only for the severe cell damage and decrease in viability but also for the inhibition of embryo development. The significant reduction in spermidine (Spd), which has been reported to be crucial for the somatic embryo development of several coniferous species, may be causally linked to the limited development of embryos. The pronounced decrease in cell wall-bound ferulic acid might correspond to failure of somatic embryos to reach more advanced stages of development. Embryos at later stages of development showed stress defense responses that were more efficient against UV-B exposure.
The somatic embryogenesis of conifers is a process susceptible to exogenous phytohormonal treatments. We report the effects of the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) and the auxin ...inhibitor p-chlorophenoxyisobutyric acid (PCIB) on the endogenous level of the auxin indole-3-acetic acid (IAA) and on the anatomical composition of early somatic embryos of Abies alba (European silver fir). The embryogenic suspensor mass (ESM) of Abies alba proliferated on a medium supplemented by 2,4-D as well as on an auxin-free medium. The endogenous level of IAA was significantly higher in the ESM cultivated on a medium supplemented by 2,4-D. The decrease in the endogenous level of IAA in the first week of maturation is one of the most important stimuli responsible for the subsequent development of embryos. However, suppression of IAA synthesis by an auxin inhibitor did not stimulate the development of embryos. The maturation of somatic embryos from the globular to the cotyledonary stage occurs when the concentration of endogenous auxin in the ESM (including the embryos) increases. Early somatic embryos proliferating on a medium supplemented by auxin had an increased probability of maturing successfully. Exogenous auxin treatment during maturation did not compensate for the auxin deficiency during proliferation.
The polyamine (PA) contents and activities of PA biosynthetic enzymes in Norway spruce somatic embryos Picea abies L. (Karst.), genotype AFO 541 were studied in relation to anatomical changes during ...their development, from proliferation to germination, and changes in these variables associated with the germination of mature somatic and zygotic embryos were compared. Activities of PA biosynthetic enzymes steadily increased during the development of somatic embryos, from embryogenic suspensor mass until early cotyledonary stages. In these stages, the spermidine (Spd) level was significantly higher than the putrescine (Put) level, and the increases coincided with the sharp increases in S-adenosylmethionine decarboxylase activity in the embryos. The biosynthetic enzyme activity subsequently declined in mature cotyledonary embryos, accompanied by sharp reductions in PA contents, especially in cellular Put contents in embryos from 6 weeks old through the desiccation phase (although the spermine level significantly increased during the desiccation phase), resulting in a shift in the Spd/Put ratio from ca. 2 in early cotyledonary embryos to around 10 after 3 weeks of desiccation. In mature zygotic embryos, Spd contents were twofold lower, but Put levels were higher, than in mature somatic embryos, hence their Spd/Put ratio was substantially lower (ca. 2, in both embryos and megagametophytes). In addition, the PA synthesis activity profiles in the embryos differed (ornithine decarboxylase and arginine decarboxylase activities predominating in mature somatic and zygotic embryos, respectively). The start of germination was associated with a rise in PA biosynthetic activity in the embryos of both origins, which was accompanied by a marked increase in Put contents in somatic embryos, resulting in the decline of Spd/Put ratio to about 2, similar to the ratio in mature and germinating zygotic embryos. The accumulation of high levels of PAs in somatic embryos may be causally linked to their lower germinability than in zygotic embryos.
Seed dormancy is an adaptive mechanism that allows seed germination under suitable environmental conditions. Germination of stored dormant seeds proceeds after dormancy breaking induced by ...stratification. To improve understanding of dormancy breaking in beechnuts, we: investigated effects of moisture content and temperature during storage; analysed contents of abscisic acid, abscisic acid metabolites and indole-3-acetic acid in embryonic axes during storage and stratification; and histochemically localized storage proteins in embryogenic axes and cotyledons of beechnut embryos. The results show that storage conditions can affect the nuts’ abscisic acid contents, but not the stratification process. In addition, dormancy breaking is linked to a reduction in abscisic acid contents and depth of dormancy is not influenced by either of these storage factors. Detected changes that correlated most strongly with dormancy breaking were a big increase in abscisic acid-glucosyl ester levels and accompanying reduction in the abscisic acid-glucosyl ester to abscisic acid ratio. We detected extremely low concentrations of other abscisic acid metabolites-neophaseic, phaseic and dihydrophaseic acids—in non-dormant stored beechnuts, but somewhat higher concentrations during dormancy. No relationships between changes in indole-3-acetic acid levels with either storage conditions or dormancy breaking were detected. Changes in distributions of storage proteins were related to the seeds’ moisture content during storage and stratification rather than seed dormancy. We conclude that increases in endogenous abscisic acid-glucosyl ester levels and abscisic acid-glucosyl ester to abscisic acid ratios are good markers of depth of dormancy and/or effectiveness of stratification in beechnuts.
Conifer somatic embryogenesis (SE) is a process driven by exogenously supplied plant growth regulators (PGRs). Exogenous PGRs and endogenous phytohormones trigger particular ontogenetic events. ...Complex mechanisms involving a number of endogenous phytohormones control the differentiation of cells and tissues, as well as the establishment of structures and organs. Most of the mechanisms and hormonal functions in the SE of conifers have not yet been described. With the aim to better understand these mechanisms, we provided detailed analysis of the spectrum of endogenous phytohormones over the course of SE in Norway spruce
. Concentrations of endogenous phytohormones including auxins, cytokinins (CKs), abscisic acid (ABA), jasmonates, and salicylic acid (SA) in somatic
embryos were analyzed by HPLC-ESI-MS/MS. The results revealed that the concentrations of particular phytohormone classes varied substantially between proliferation, maturation, desiccation, and germination. Endogenous ABA showed a maximum concentration at the maturation stage, which reflected the presence of exogenous ABA in the medium and demonstrated its efficient perception by the embryos as a prerequisite for their further development. Auxins also had concentration maxima at the maturation stage, suggesting a role in embryo polarization. Endogenous jasmonates were detected in conifer somatic embryos for the first time, and reached maxima at germination. According to our knowledge, we have presented evidence for the involvement of the non-indole auxin phenylacetic acid,
-zeatin- and dihydrozeatin-type CKs and SA in SE for the first time. The presented results represent the currently most comprehensive overview of plant hormone levels in embryos throughout the whole process of conifer SE. The differences in concentrations of various classes of phytohormones over the proliferation, maturation, desiccation, and germination in somatic
embryos clearly indicate correlations between endogenous phytohormone profiles and particular developmental stages of the SE of conifers.
The time courses of the contents of free, soluble and insoluble polyamine (PA) conjugates, PA biosynthetic and catabolic enzyme activities and mRNA levels of PA biosynthetic genes were monitored ...during the cell cycle of synchronized tobacco BY-2 cell line (
Nicotiana tabacum L. cv. Bright Yellow 2). Progression through the cell cycle was characterized by specific biphasic changes of PA levels. The first, moderate peak in the amount of free PAs coincided with the S-phase. After a transient decline in G2 phase the contents of free PAs increased rapidly and peaked again during G2/M interface. Then sharply decreased with the minimum at the end of mitosis and during M/G1 transition and started to rise again with the next replication phase. Levels of PA soluble conjugates paralleled those of the free forms. Biosynthetic enzyme activities followed the biphasic manner analogous to the levels of free PAs and seemed to be regulated on both transcriptional and (post)translational level. PA cellular levels were further controlled by both catabolic degradation and conjugation of PAs. PA catabolism played an important role in the PA down-regulation during G2 phase and late mitosis, while the decline in free PAs in G2/M interface and during the whole mitosis resulted mainly from PA conjugation. This study's results demonstrate that during the cell cycle of tobacco BY-2 cells endogenous PA levels are intricately controlled, involving regulation of activities of biosynthetic, catabolic and conjugation enzymes.
5. CONCLUSIONS . The development of somatic embryos on maturation meďum can be divided at anatomical level into fow developmental stages, differing in the inner struďure: stage of early somatic ...embryo, cylindricď stage, precotyledonary stageand cotyledonary stage. o The prolonged cultivation of coýledonary errrbryos on maturation medium leads to disintegration of the root cap, connected with starch and phenolic cornpormdsdeposition. o The time course of disctete developmental stagesis affected by PEG 4000; use of 3,75 % PBG speeds up the process of maturation by tvro weeks. Increased level of PEG (7,5%o)leads to the formation of nrpturesin ernbryohypocotyls. The content of non-structural saccharides increases during the cultivation of embryogenic culture on maturation medium due to increasing level of sucrose in developing embryos. The content of hexoses in embryos is low. The addition of 3,75 % PEG 4000 into maturation medium increases the sucrose to hexoses ratio in somatic embryos, the total amotmt of saccharides slightly decreases.7,5 %PEG leadsto hexosesaccumulationin embryos. Insertion of pre-maturation phase (without growth regulators) between proliferation and maturation increases total amount of developed embryos in a majority lines tested. cultivation of embryogenic cultues of rafts with...
Maturation of Norway spruce (Picea abies L.) somatic embryos is induced by abscisic acid (ABA). Several proteins were proven to be involved in ABA sensing including ABI3/VP1 transcription factors and ...their orthologue PaVP1 was characterized in spruce. To evaluate the role of PaVP1 both in embryogenic potential and in the process of embryo maturation, we studied PaVP1 expression in lines with contrast embryogenic capacities in parallel with detailed anatomical characterization. PaVP1 expression was determined by northern blot hybridisation, which revealed presence of two differentially regulated VP1-like B3-domain transcripts. Full-length PaVP1 transcript level was negligible in all lines on the proliferation media, but it differed strongly on the maturation media containing ABA. In non-embryogenic line, lacking any differentiated structures, the transcript remained undetectable. In contrast, in embryogenic lines with meristematic centres attached to suspensor cells, PaVP1 expression increased strongly after transition onto the maturation media. In highly embryogenic lines, it kept on a high level until the embryos reached cotyledonary stage, while in developmentally arrested line incapable to form mature embryos, the expression dropped down in connection with advanced disintegration of the meristematic centres. Removal of ABA from the maturation media after 2 weeks of maturation resulted in aberrant embryo development and rapid decrease in PaVP1 expression, indicating the impact of exogenously supplemented ABA on both initiation and maintenance of PaVP1 expression and proper embryo development. Since permanently high or increasing PaVP1 transcript levels accompanied proper embryo development in all experiments, it could be regarded as a good marker of this process.
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