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.
Embryogenic cultures of Norway spruce (Picea abies) were treated with exogenous putrescine (Put) applied via either proliferation or maturation media to determine how such treatment affected ...endogenous polyamine levels, the histological structures of the embryogenic suspensor mass (ESM), and the yield of mature embryos. Treatment with exogenous Put at 10, 100, or 500 μM significantly increased the endogenous free and conjugated Put contents of the treated ESM in a concentration-dependent manner. All of the Put treatments also reduced endogenous spermidine (Spd) levels. In conjunction with the increased abundance of endogenous Put, this caused a pronounced decrease in the Spd/Put ratios of treated ESMs relative to untreated controls. Exogenous Put stimulated meristem cell division and enlargement. However, single embryos were not readily released from polyembryonic centers and the frequency of development of malformed embryos was high.
To explore poorly understood differences between primary and subsequent somatic embryogenic lines of plants, we induced secondary (2
) and tertiary (3
) lines from cotyledonary somatic embryos (SEs) ...of two Douglas-fir genotypes: SD4 and TD17. The 2
lines exhibited significantly higher embryogenic potential (SE yields) than the 1
lines initiated from zygotic embryos (SD4, 2155 vs 477; TD17, 240 vs 29 g
f.w.). Moreover, we observed similar differences in yield between 2
and 3
lines of SD4 (2400 vs 3921 g
f.w.). To elucidate reasons for differences in embryogenic potential induced by repetitive somatic embryogenesis we then compared 2
vs 1
and 2
vs 3
lines at histo-cytological (using LC-MS/MS) and proteomic levels.
Repetitive somatic embryogenesis dramatically improved the proliferating lines' cellular organization (genotype SD4's most strongly). Frequencies of singulated, bipolar SEs and compact polyembryogenic centers with elongated suspensors and apparently cleavable embryonal heads increased in 2
and (even more) 3
lines. Among 2300-2500 identified proteins, 162 and 228 were classified significantly differentially expressed between 2
vs 1
and 3
vs 2
lines, respectively, with special emphasis on "Proteolysis" and "Catabolic process" Gene Ontology categories. Strikingly, most of the significant proteins (> 70%) were down-regulated in 2
relative to 1
lines, but up-regulated in 3
relative to 2
lines, revealing a down-up pattern of expression. GO category enrichment analyses highlighted the opposite adjustments of global protein patterns, particularly for processes involved in chitin catabolism, lignin and L-phenylalanine metabolism, phenylpropanoid biosynthesis, oxidation-reduction, and response to karrikin. Sub-Network Enrichment Analyses highlighted interactions between significant proteins and both plant growth regulators and secondary metabolites after first (especially jasmonic acid, flavonoids) and second (especially salicylic acid, abscisic acid, lignin) embryogenesis cycles. Protein networks established after each induction affected the same "Plant development" and "Defense response" biological processes, but most strongly after the third cycle, which could explain the top embryogenic performance of 3
lines.
This first report of cellular and molecular changes after repetitive somatic embryogenesis in conifers shows that each cycle enhanced the structure and singularization of EMs through modulation of growth regulator pathways, thereby improving the lines' embryogenic status.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Somatic embryogenesis techniques have been developed for most coniferous species, but only using very juvenile material. To extend the techniques' scope, better integrated understanding of the key ...biological, physiological and molecular characteristics of embryogenic state is required. Therefore, embryonal masses (EMs) and non-embryogenic calli (NECs) have been compared during proliferation at multiple levels. EMs and NECs originating from a single somatic embryo (isogenic lines) of each of three unrelated genotypes were used in the analyses, which included comparison of the lines' anatomy by transmission light microscopy, transcriptomes by RNAseq Illumina sequencing, proteomes by free-gel analysis, contents of endogenous phytohormones (indole-3-acetic acid, cytokinins and ABA) by LC-MS analysis, and soluble sugar contents by HPLC. EMs were characterized by upregulation (relative to levels in NECs) of transcripts, proteins, transcription factors and active cytokinins associated with cell differentiation accompanied by histological, carbohydrate content and genetic markers of cell division. In contrast, NECs were characterized by upregulation (relative to levels in EMs) of transcripts, proteins and products associated with responses to stimuli (ABA, degradation forms of cytokinins, phenols), oxidative stress (reactive oxygen species) and carbohydrate storage (starch). Sub-Network Enrichment Analyses that highlighted functions and interactions of transcripts and proteins that significantly differed between EMs and NECs corroborated these findings. The study shows the utility of a novel approach involving integrated multi-scale transcriptomic, proteomic, biochemical, histological and anatomical analyses to obtain insights into molecular events associated with embryogenesis and more specifically to the embryogenic state of cell in Douglas-fir.
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 transition from vegetative growth to reproduction is the essential commitment in plant life. It is triggered by environmental cues (day length, temperature, nutrients) and regulated by the very ...complex signaling gene network and by phytohormones. The control of flowering is well understood in Arabidopsis thaliana and in some crops, much less is known about the other angiosperms. We performed the detailed transcriptomic survey of the course of floral induction in seedlings of Chenopodium ficifolium accession 459, a close relative of the important crop Chenopodium quinoa. It flowers earlier under short days (6 hours light) than under long days (18 hours light). Plants were sampled at the age 14, 18, 21 and 24 days in the morning and afternoon, both at long and short day, for RNA-Sequencing, and also for phytohormone analyses. We employed Illumina NovaSeq6000 platform to generate raw reads, which were cleaned and mapped against the de novo constructed transcriptome of C. ficifolium. The global gene expression levels between long and short days were pairwise compared at each time points. We identified differentially expressed genes associated with floral induction in C. ficifolium 459. Particular attention was paid to the genes responsible for phytohormone metabolism and signaling. The datasets produced by this project contributed to better understanding of the regulation of growth and development in the genus Chenopodium.
Maritime pine somatic embryos (SEs) require a reduction in water availability (high gellan gum concentration in the maturation medium) to reach the cotyledonary stage. This key switch, reported ...specifically for pine species, is not yet well understood. To facilitate the use of somatic embryogenesis for mass propagation of conifers, we need a better understanding of embryo development. Comparison of both transcriptome (Illumina RNA sequencing) and proteome two‐dimensional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis with mass spectrometry (MS) identification of immature SEs, cultured on either high (9G) or low (4G) gellan gum concentration, was performed, together with analysis of water content, fresh and dry mass, endogenous abscisic acid (ABA; gas chromatography‐MS), soluble sugars (high‐pressure liquid chromatography), starch and confocal laser microscope observations. This multiscale, integrated analysis was used to unravel early molecular and physiological events involved in SE development. Under unfavorable conditions (4G), the glycolytic pathway was enhanced, possibly in relation to cell proliferation that may be antagonistic to SE development. Under favorable conditions (9G), SEs adapted to culture constraint by activating specific protective pathways, and ABA‐mediated molecular and physiological responses promoting embryo development. Our results suggest that on 9G, germin‐like protein and ubiquitin‐protein ligase could be used as predictive markers of SE development, whereas protein phosphatase 2C could be a biomarker for culture adaptive responses. This is the first characterization of early molecular mechanisms involved in the development of pine SEs following an increase in gellan gum concentration in the maturation medium, and it is also the first report on somatic embryogenesis in conifers combining transcriptomic and proteomic datasets.
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.
•Both latrunculin B and cytochalasin D depolymerized actin filaments in different way.•Latrunculin B targeted primarily suspensor cells of embryogenic culture.•Cytochalasin D damaged both suspensor ...and meristematic cells.•Damage of suspensors (lat B) accelerates and synchronizes embryo development.•Damage both suspensors and meristems (cyt D) resulted in lower embryo yield.
The role of the actin cytoskeleton in somatic embryo development was investigated using latrunculin B and cytochalasin D. Brief treatments (1h) with either drug at the start of maturation fragmented the actin in suspensor cells and/or depolymerized actin filaments in meristematic cells. The drugs targeted different cells: latB primarily affected the suspensor cells, but cchD damaged both suspensor and meristematic cells. Lethal damage to the meristematic and suspensor cells was observed when the drugs were applied throughout the maturation period, although the severity of this effect depended on their concentrations.
The drugs’ effects on the yield of mature somatic embryos were investigated by applying them to embryo cultures throughout the maturation period or for one week at three different points in the maturation process: immediately prior to the start of maturation, during the first week of maturation, and during the fourth week of maturation. The strongest effects were observed when the drugs were applied at the start of maturation. Under these conditions, latB destroyed the suspensors, eliminating the underdeveloped embryos that depend on them. This accelerated the development of embryos that were capable of separating from the suspensors. Thus, while the total number of embryos at the end of the maturation period was lower than in untreated control cultures, the surviving mature embryos were of high quality. cchD treatment at the start of maturation strongly inhibited embryo development. Drug treatment at the end of the maturation period did not significantly affect embryo development: latB caused no change in the yield of somatic embryos, but cchD treatment increased the number of malformed embryos compared to untreated controls.
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