Vegetative propagation opens opportunities for the multiplication of elite tree progeny for forest regeneration material. For conifers such as Norway spruce (
Picea abies
) the most efficient ...vegetative propagation method is seed multiplication through somatic embryogenesis. Efficient culture methods are needed for somatic embryogenesis to be commercially viable. Compared to culturing as clumps, filter disc cultures can improve the proliferation of embryogenic tissue (ET) due to more even spread and better developmental synchronization. In this study, ET proliferation on filter discs was compared to proliferation as clumps. The study comprised 28 genotypes in four trials. The benefits of adding a pre-maturation step and the selection of fresh ET for the subculture were evaluated. Pre-maturation on hormone-free media before maturation did not significantly improve embryo yield but improved greenhouse survival from 69% to 80%, although there was high variation between lines. Filter disc cultivation of ET did result in better growth than in clumps but was more dependent on ET selection and the amount of ET than the clump cultivation method. Filter proliferation also favors certain lines. Post-maturation storage can be used to change the storage compound composition of the produced mature embryos. The embryo storage compound profile was analyzed after post-maturation cold storage treatments of 0, 4, 8, 31, and 61 weeks and compared to that of the zygotic embryos. Cold storage made the storage compound profile of somatic embryos closer to that of zygotic embryos, especially regarding the raffinose family oligosaccharides and storage proteins. Sucrose, hexose, and starch content remained higher in somatic embryos even through cold storage. Prolonged storage appeared less beneficial for embryos, some of which then seemed to spontaneously enter the germination process.
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.
The Douglas fir (Pseudotsuga menziesii) is a conifer native to North America that has become increasingly popular in plantations in France due to its many advantages as timber: rapid growth, quality ...wood, and good adaptation to climate change. Tree genetic improvement programs require knowledge of a species’ genetic structure and history and the development of genetic markers. The very slow progress in this field, for Douglas fir as well as the entire genus Pinus, can be explained using the very large size of their genomes, as well as by the presence of numerous highly repeated sequences. Proteomics, therefore, provides a powerful way to access genomic information of otherwise challenging species. Here, we present the first Douglas fir proteomes acquired using nLC-MS/MS from 12 different plant organs or tissues. We identified 3975 different proteins and quantified 3462 of them, then examined the distribution of specific proteins across plant organs/tissues and their implications in various molecular processes. As the first large proteomic study of a resinous tree species with organ-specific profiling, this short note provides an important foundation for future genomic annotations of conifers and other trees.
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.
The metabolism of apigenin, a weak estrogenic flavonoid phytochemical, was investigated in the rat. After a single oral administration of radiolabeled apigenin, 51.0% of radioactivity was recovered ...in urine, 12.0% in feces, 1.2% in the blood, 0.4% in the kidneys, 9.4% in the intestine, 1.2% in the liver, and 24.8% in the rest of the body within 10 days. Sex differences appear with regard to the nature of compounds eliminated via the urinary route: immature male and female rats, like mature female rats, excreted a higher percentage of the mono-glucuronoconjugate of apigenin than the mono-sulfoconjugate of apigenin (10.0-31.6% versus 2.0-3.6%, respectively). Mature male rats excreted the same compounds in an inverse ratio (4.9% and 13.9%, respectively). Radioactivity appeared in the blood only 24 h after oral administration. Blood kinetics showed a high elimination half-time (91.8 h), a distribution volume of 259 ml, and a plasmatic clearance of 1.95 ml/h. All of the parameters calculated from these experiments suggested a slow metabolism of apigenin, with a slow absorption and a slow elimination phase. Thus, a possible accumulation of this flavonoid in the body can be hypothesized.
The metabolism of apigenin, a low estrogenic flavonoid phytochemical, was investigated in rat using liver models both in vitro (subcellular fractions) and ex vivo (isolated perfused liver). In vitro, ...phase I metabolism led to the formation of three monohydroxylated derivatives: luteolin which was the major metabolite (K(m) = 22.5 +/- 1.5 microM; V(max) = 5.605 +/- 0.090 nmol/min/mg protein, means +/- S.E.M.), scutellarein, and iso-scutellarein. These oxidative pathways were mediated by cytochrome P450 monooxygenases (P450s). The use of P450 inhibitors and inducers showed that CYP1A1, CYP2B, and CYP2E1 are involved. In vitro studies of phase II metabolism indicated that apigenin underwent conjugation giving three monoglucuronoconjugates and one monosulfoconjugate. Luteolin led to the formation of four monoglucuronoconjugates, two sulfoconjugates, and one methylconjugate identified as diosmetin. Ex vivo during the apigenin perfusion of an isolated rat liver, none of the phase I metabolites could be recovered. In contrast, two monoglucuronoconjugates and one of the sulfoconjugates of apigenin already identified in vitro were recovered. Moreover, two new derivatives were isolated and identified as a diglucuronoconjugate and a glucuronosulfoconjugate. This work provides new data about the metabolism of apigenin and shows the interest value of using various experimental models in metabolic studies.
An integrated physiological and proteomic approach was used to investigate the effects of high gellan gum concentration in the medium during maturation of somatic embryos (SE) of hybrid larch, by ...comparing embryos incubated in media with a high gellan gum concentration (8 g l⁻¹) and the standard concentration (4 g l⁻¹) after 1, 3, 6 and 8 weeks of maturation. Because of the reduced availability of water in the 8 g l⁻¹ medium, the cultured embryos had a lower osmotic water potential (Ψπ) and water contents, but higher dry weights (DWs), at 8 weeks compared with embryos cultured on the standard medium. The high gellan gum concentration induced a desiccation that is characteristic in zygotic embryo maturation. Total soluble proteins were extracted from SE with trichloroacetic acid (TCA)-acetone after 1 and 8 weeks of maturation on media with 4 and 8 g l⁻¹ of gellan gum, and separated by two-dimensional gel electrophoresis (2-DE) at pH 4-7. More than 1100 proteins were reproducibly detected on each gel. At 1 and 8 weeks respectively, the abundances of 62 and 49 spots detected in analyses of embryos matured at the two gellan gum concentrations, significantly differed. Among 62 significantly differing spots at 1 week of maturation, the corresponding proteins of 56 were reliably identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), and were found to be mainly involved in ‘carbohydrate metabolism', ‘genetic information processing' or ‘environmental information processing' according to kegg taxonomy. Both physiological parameters and the proteins identified suggested that the embryos were stressed when they were cultured on 4 g l⁻¹ of gellan gum.
The morphology of somatic embryos (SE) is not a sufficient criterion to determine the level of maturation and the optimal stage to transfer embryos for germination, unlike the biochemical components. ...This composition characterization in the laboratory is too restrictive to be considered at each maturation cycle, as would be necessary. It is, therefore, essential to consider alternative methods. The objectives of this work were to achieve a complete biochemical characterization of the embryos during their development, to serve as a reference and develop a characterization based on infrared spectrometry and chemometrics. During the precotyledonary stage (0–3 weeks of maturation), water content and glucose and fructose levels were high, which is consistent with SE development. After 4 weeks, the cotyledonary SE had a metabolism oriented towards the storage accumulation of lipids, proteins and starch, whereas raffinose only appeared from 8 weeks. Mid‐infrared calibration models were developed for water, proteins, lipids, carbohydrates, glucose, fructose, inositols, raffinose, stachyose and starch contents with an r2 average of 0.84. A model was also developed to discriminate the weeks of SE maturation. Different classes of age were discriminated with at least 72% of accuracy. Infrared analysis of the SE based on their full biochemical spectral fingerprint revealed a very slight variation in composition between 7 and 9 weeks, information that is very difficult to obtain by conventional analysis methods. These results provide novel insights into the maturation of conifer SE and indicate that mid‐infrared spectrometry could be an easy and effective method for SE characterization.
The metabolism of diallyl disulfide (DADS), a garlic sulfur compound, was investigated in human liver microsomes. Diallyl thiosulfinate (allicin) was the only metabolite observed and its formation ...followed Michaelis-Menten kinetics with a Km = 0.61 +/- 0.2 mM and a Vmax = 18.5 +/- 4.2 nmol/min/mg protein, respectively (mean +/- S.E. M., n = 4). Both flavin-containing monooxygenase and the cytochrome P-450 monooxygenases (CYP) were involved in DADS oxidation, but the contribution of CYP was predominant. The cytochrome P-450 isoforms involved in this metabolism were investigated using selective chemical inhibitors, microsomes from cells expressing recombinant CYP isoenzymes, and studying the correlation of the rate of DADS oxidation with specific monooxygenase activities of human liver microsomes. Diethyldithiocarbamate and tranylcypromine inhibited allicin formation, whereas other specific inhibitors had low or no effect. Most of the different human microsomes from cells expressing CYP were able to catalyze this reaction, but CYP2E1 showed the highest affinity with a substantial activity. Furthermore, allicin formation by human liver microsomes was correlated with p-nitrophenol hydroxylase activity, a marker of CYP2E1, and tolbutamide hydroxylase activity, a marker of CYP2C9. Among these approaches only CYP2E1 was identified in each case, which suggested that DADS is preferentially metabolized to allicin by CYP2E1 in human liver. However the minor participation of other CYP forms and flavin-containing monooxygenases is likely.
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