Somatic embryogenesis is an artificially induced process that results in the formation of an embryo without the fusion of gametes. Despite its significant potential for plant propagation and conservation of plant genetic resources, many unknowns remain regarding the specific mechanisms and the complexity of genetic, biochemical, and cellular regulations involved. The comparative data on induction of embryogenic tissue and the maturation of somatic embryos, along with additional results, suggest that complex carbohydrate dynamics are among the key factors involved. This review summarizes recent findings focused on early stages of SE, including induction, proliferation, comparison of embryogenic cell lines with contrasting embryogenic capacity, and loss of embryogenic capacity after prolonged cultivation obtained through various methods, including metabolomics, proteomics, transcriptomics, and cytological techniques/histochemical visualization, which highlight the regulation of the energy source starch. This encompasses its synthesis and remobilization, driven by related enzymatic machinery, as crucial for determining embryogenic capacity and prognosis in embryo maturation and germination in many economically important plant species. We also outline potential future directions, critical questions to address, and anticipated advancements in the topic, including the development of new research methodologies.

The accumulation of mRNA transcripts of chitinases class VII and beta-1,3-glucanases class II decreases as embryo development progresses. In later stages, it occurs mainly in actively proliferating meristems. Besides being considered pathogenesis-related proteins, chitinases and β-1,3-glucanases are involved in many physiological and developmental processes. In this study, we investigated the potential role of specific β-1,3-glucanases class II and chitinases class VII in zygotic and somatic embryogenesis using in situ hybridization (ISH). The ISH experiments revealed that the mRNA transcripts of LuChitVII and LuGlucII, along with closely related chitinases and β-1,3-glucanases, accumulated during zygotic embryo development. In the earlier stages of embryo development, both probes showed uniform and relatively strong hybridization signals throughout the embryo. However, as development progressed, the signal intensity decreased. In the cotyledonary stage, the studied mRNA transcripts were more abundant in the meristems, with predominant accumulation in the basal part of the embryo. These structures are known for their high proliferation rates, suggesting the involvement of chitinases and β-1,3-glucanases in cell division. A similar expression pattern of both hydrolases was observed during somatic embryo development.

Somatic embryogenesis is an efficient mean for rapid micropropagation and preservation of the germplasm of valuable coniferous trees. Little is known about how the composition of secretome tracks down the level of embryogenic capacity. Unlike embryogenic tissue on solid medium, suspension cell cultures enable the study of extracellular proteins secreted into a liquid cultivation medium, avoiding contamination from destructured cells. Here, we present proteomic data of the secretome of Pinus nigra cell lines with contrasting embryogenic capacity, accounting for variability between genotypes. Our results showed that cell wall-related and carbohydrate-acting proteins were the most differentially accumulated. Peroxidases, extensin, α-amylase, plant basic secretory family protein (BSP), and basic secretory protease (S) were more abundant in the medium from the lines with high embryogenic capacity. In contrast, the medium from the low embryogenic capacity cell lines contained a higher amount of polygalacturonases, hothead protein, and expansin, which are generally associated with cell wall loosening or softening. These results corroborated the microscopic findings in cell lines with low embryogenic capacity-long suspensor cells without proper assembly. Furthermore, proteomic data were subsequently validated by peroxidase and α-amylase activity assays, and hence, we conclude that both tested enzyme activities can be considered potential markers of high embryogenic capacity.

Embryogenic tissues of Abies alba Mill. were cryopreserved using the slow-freezing approach. Four cell lines were incubated for 24 h on a medium with 0.5 M sorbitol and pre-treated with 5% DMSO. Subsequently, the tissues were frozen at a cooling rate of 1 °C min-1 to -40 °C and transferred to liquid nitrogen for 72 hours. After thawing in a water bath at 40 °C, the tissues were cultivated on a proliferation medium. All tested lines recovered, but variations in regrowth frequencies across cell lines were noticed (91.66 to 100%). The recovered tissues showed similar features to the control 2 (non-pre-treated and non-cryopreserved tissues). In the accumulation of fresh and dry mass, no statistically significant differences were observed between cryopreserved cultures and control 2. The cryopreserved tissues produced cotyledonary somatic embryos capable of germination. Microscopic observations revealed considerable structural changes as a consequence of the cryopreservation procedure. The long vacuolated suspensor cells were disrupted, and mostly the meristematic cells of the embryonal region survived. The typical bipolar structure of early somatic embryos has been regained during the post-thaw period. Differences in cryotolerance across cell lines were also observed.

Ionizing radiation is a genotoxic anthropogenic stressor. It can cause heritable changes in the plant genome, which can be either adaptive or detrimental. There is still considerable uncertainty about the effects of chronic low-intensity doses since earlier studies reported somewhat contradictory conclusions. Our project focused on the recovery from the multiyear chronic ionizing radiation stress. Soybean (Glycine max) was grown in field plots located at the Chernobyl exclusion zone and transferred to the clean ground in the subsequent generation. We profiled proteome of mature seeds by two-dimensional gel electrophoresis. Overall, 15 differentially abundant protein spots were identified in the field comparison and 11 in the recovery generation, primarily belonging to storage proteins, disease/defense, and metabolism categories. Data suggested that during multigenerational growth in a contaminated environment, detrimental heritable changes were accumulated. Chlorophyll fluorescence parameters were measured on the late vegetative state, pointing to partial recovery of photosynthesis from stress imposed by contaminating radionuclides. A plausible explanation for the observed phenomena is insufficient provisioning of seeds by lower quality resources, causing a persistent effect in the offspring generation. Additionally, we hypothesized that immunity against phytopathogens was compromised in the contaminated field, but perhaps even primed in the clean ground, yet this idea requires direct functional validation in future experiments. Despite showing clear signs of physiological recovery, one season was not enough to normalize biochemical processes. Overall, our data contribute to the more informed agricultural radioprotection.

Initiation of somatic embryogenesis from immature zygotic embryos, long-term maintenance of embryogenic tissue in vitro or by cryopreservation, as well as maturation, of somatic embryos of Abies alba Mill. are reported in this study. For the initiation of embryogenic tissues, a DCR medium containing different types of cytokinins (1 mg.L−1) were tested. During three consecutive years, 61 cell lines were initiated out of 1308 explants, with initiation frequencies ranging between 0.83 and 13.33%. The type of cytokinin had no profound effect on the initiation frequency within one given year. Microscopic observations revealed presence of bipolar somatic embryos in all initiated embryogenic tissues. Besides the typical bipolar somatic embryos, huge polyembryonal complexes, as well as “twin” embryos, were observed. Maturation of somatic embryos occurred on a DCR medium supplemented by abscisic acid (10 mg.L−1), polyethylene glycol (PEG-4000, 7.5%) and 3% maltose. The maturation capacity was cell-line dependent. All of the four tested cell lines produced cotyledonary somatic embryos, though at different quantities, of 16 to 252 per g of fresh weight. After germination, seedlings developed, but their further growth soon stopped after the formation of a resting bud. Altogether, seven cell lines were cryopreserved, using the slow-freezing technique. After rewarming, all tested cell lines showed regrowth rates between 81.8 and 100%.

Champagne, Antoine ; Boutry, Marc. A comprehensive proteome map of glandular trichomes of hop (Humulus lupulus L.) female cones: One shot for the entire biosynthetic pathways of terpenoidrelated compounds and possible transport proteins.2nd INPPO World Congress (Bratislava, Slovakia, du 04/09/2016 au 08/09/2016). In: Martin Hajduch; Katarina Klubicova; Ludovit Skultety (eds), Bookf of abstracts of the 2nd INPPO World Congress in Bratislava, 2016, p. 13 http://hdl.handle.net/2078.1/176746

Large areas polluted with toxic heavy metals or radionuclides were formed as a side product of rapid industrial development of human society. Plants, due to their sessile nature, should adapt to these challenging genotoxic environmental conditions and develop resistance. Herein, we evaluated the response of three natural ecotypes of Arabidopsis thaliana (L.) Heynh (Oasis, Columbia-0, and Chernobyl-07) to cadmium, using discovery gel-based proteomics. These accessions are differing by level of tolerance to heavy metal probably achieved by various exposure to chronic ionizing radiation. Based on the pairwise comparison (control versus cadmium-treated) we recognized 5.8–13.4% of identified proteins as significantly altered at the presence of cadmium. Although the majority of photosynthesis-related proteins were found to be less abundant in all ecotypes it was noted that in contrast to the sensitive variants (Col and Oas), the tolerant Che accession may activate the mechanism preserving photosynthesis and energy production. Also, proteins modulating energy budget through alternative route and mediating higher resistance to heavy metals were upregulated in this ecotype. Although we suggest that regulation of enzymes acting in peptide and protein synthesis, protection of the plants against various abiotic stresses, or those neutralizing the effects of reactive oxygen species are rather associated with general response to cadmium, they were found to be altered more intensively in the Che accession. Thus, the identified affected proteins may represent good candidate molecules for molecular breeding to improve tolerance of crops to heavy metal stress.

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba ×A. cephalonica and Abies alba ×A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 μM 2,4-D and 2.2 μM BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 μM BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 μM abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG-4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.

Somatic embryogenesis was achieved in the conifers Pinus nigra Arn. and in the hybrids Abies alba x A. cephalonica and Abies alba x A. numidica. For initiation of embryogenic tissue in P. nigra, immature zygotic embryos enclosed in megagametophytes were used. The initiated embryogenic cultures were maintained and proliferated on solid culture medium DCR supplemented with 9 mu M 2,4-D and 2.2 mu m M BA. Microscopic investigations revealed the presence of bipolar early somatic embryos in proliferating tissue. Suspension cultures have also been established by resuspending the embryogenic tissue in liquid culture medium. Experimentation with abscisic acid concentration resulted in successful somatic embryo maturation. Besides abscisic acid, the carbohydrate content or higher concentration of gelling agent in maturation medium were also important requirements for somatic embryo maturation. Germination of cotyledonary somatic embryos occurred on hormone-free medium and terminated in somatic seedlings regeneration. The regenerated somatic seedlings were transferred to soil and were capable of successful development. For initiation of embryogenic tissue in Abies hybrids juvenile explants as immature or mature zygotic embryos as well as cotyledons were used and 4.4 m M BA as sole plant growth regulator was sufficient. Medium of the same composition was also suitable for their long-term maintenance. Maturation of somatic embryos was achieved on solid DCR medium supplemented with 38 m M abscisic acid, polyethylene glycol (0, 5, 7.5, and 10% PEG4000) and different carbohydrates such as maltose, sucrose and glucose (each 3%). PEG-4000 stimulated somatic embryo development depending on the carbohydrate source used. Cotyledonary somatic embryos germinated after desiccation treatment and the regenerated somatic seedlings were transferred to soil. Cryopreservation of embryogenic tissue could be an alternative method for long-term maintenance. For cryopreservation the slow-freezing method was used with success. Tissue regeneration in the post thaw period was relatively high and the regenerated tissue produced mature somatic embryos and subsequent plantlets. The embryogenic tissue was also used in experiments focused on genetic transformation either by biolistic (P. nigra) or Agrobacterium-mediated (Abies hybrids) methods. A proteomic study was performed to gain a deeper insight into the early stages of P. nigra somatic embryogenesis.

The somatic embryogenesis in conifers represents a suitable model of plant regeneration system facilitating studies of fundamental aspects of an early development as well as in vitro micropropagation. The aim of our study was to deeper understand the somatic embryogenesis in the conifer tree Pinus nigra Am. Comparative proteomic analysis based on 2D-PAGE in 1) proliferating embryogenic tissues (E) initiated from immature zygotic embryos, 2) non-embryogenic calli (NEC) initiated from cotyledons of somatic seedlings of the same genotypes, 3) embryogenic tissues that lost the maturation capacity (E-L) of two cell lines (E362, E366). Investigated pine tissues showed distinct structural features. The 24 protein spots were altered in both cell lines in comparison of embryo genic and non-embryogenic tissues. These proteins are involved in disease and defence mechanism, energy metabolism and biosynthesis of cell wall components. Two of three protein spots detected only in embryogenic form of both cell lines are similar to water deficit inducible protein LP3, the third remains uncharacterised. The loss of the maturation capacity was accompanied by changes in 35 and 38 protein spots in 362 and 366 cell lines, respectively. Only two of them were altered in both cell lines, suggesting non-uniform process of ageing. Biological significance: Somatic embryogenesis in conifers represents an experimental system for the study of early plant development as well as a biotechnological tool for large-scale micropropagation. The obtained results give a new insight into the process of somatic embryogenesis of a conifer Pinus nigra Am. by revealing differences at proteomic levels among in vitro cultured tissues characterised by different embryogenic potential. Microscopic investigations have also shown differences in the structural organisation of studied tissues. (C) 2017 Elsevier B.V. All rights reserved.

Plants continue to flourish around the site of the Chernobyl Nuclear Power Plant disaster. The ability of plants to transcend the radio-contaminated environment was not anticipated and is not well understood. The aim of this study was to evaluate the proteome of flax (Linum usitatissimum L.) during seed filling by plants grown for a third generation near Chernobyl. For this purpose, seeds were harvested at 2, 4, and 6 weeks after flowering and at maturity, from plants grown in either non-radioactive or radio-contaminated experimental fields. Total proteins were extracted and the two-dimensional gel electrophoresis (2-DE) patterns analyzed. This approach established paired abundance profiles for 130 2-DE spots, e.g., profiles for the same spot across seed filling in non-radioactive and radio-contaminated experimental fields. Based on Analysis of Variance (ANOVA) followed by sequential Bonferroni correction, eight of the paired abundance profiles were discordant. Results from tandem mass spectrometry show that four 2-DE spots are discordant because they contain fragments of the cupin superfamily-proteins. Most of the fragments were derived from the N-terminal half of native cupins. Revisiting previously published data, it was found that cupin-fragments were also involved with discordance in paired abundance profiles of second generation flax seeds. Based on these observations we present an updated working model for the growth and reproductive success of flax in a radio-contaminated Chernobyl environment. This model suggests that the increased abundance of cupin fragments or isoforms and monomers contributes to the successful growth and reproduction of flax in a radio-contaminated environment.

The paper discusses different methodological approaches to the study of transgenerational alterations of metabolic pathways in soybean and flax seeds in the process of adaptation to chronic irradiation in the Chernobyl alienation zone. A combination of general biological methods and novel approaches, such as genomics, proteomics, cytogenetics, and mutagenesis, allows researchers to analyze an organism’s systemic response and identify the latent chronic irradiation effects in plants from the Chernobyl zone. The proteomic approaches are especially efficient, since they range from the identification of changes in abundance and folding of individual proteins to the characterization of posttranslational modifications, trends of qualitative changes during seed maturation, or protein-protein interactions during plant growth and development under permanent impacts of stress factors. The application of proteomics opens new horizons in the understanding of the hidden mechanisms behind the impact of chronic low-dose radiation on living cells and makes it possible to visualize metabolic network alterations regardless of their transcriptional, translational, or epigenetic nature.

Amaranth, a staple food source in ancient Aztec, Maya and Inca cultures, has been recognised as a 21st century crop. This superfood, known as Inca wheat, attracts the worldwide attention of researchers and farmers for its superior agronomical and technological properties but especially because of its exceptional nutritive value. A combination of two-dimensional electrophoresis (2-DE) with MS facilitating the effective differentiation of 13 classes of Amaranthus cruentus L. “Ficha” mature seed proteins was used in the current study. The best resolution results in the 2-DE were obtained using immobilised pH gradients strips with a pH range of 5–8. Out of the 461 spots detected, 249 were successfully identified by LC-MS/MS analysis, making this the most inclusive protein profile of mature amaranth seed. Unknown proteins represented the most abundant class of proteins (59), the second most abundant category was related to energy (46) and then to seed storage proteins (43). These results expand the knowledge of the amaranth seed proteome and it is expected that the data presented here will contribute to further in-depth characterisation of Amaranthus seeds.

Two serious nuclear accidents during the past quarter of a century contaminated large agricultural areas with radioactivity. The remediation and possible recovery of radio-contaminated areas for agricultural purposes require comprehensive characterization of plants grown in such places. Here we describe the quantitative proteomics method that we use to analyze proteins isolated from seeds of plants grown in radioactive Chernobyl zone.

Starting in 2007, we have grown soybean (Glycine max [L.] Merr. variety Soniachna) and flax (Linum usitatissimum, L. variety Kyivskyi) in the radio-contaminated Chernobyl area and analyzed the seed proteomes. In the second-generation flax seeds, we detected a 12% increase in oil content. To characterize the bases for this increase, seed development has been studied. Flax seeds were harvested in biological triplicate at 2, 4, and 6 weeks after flowering and at maturity from plants grown in nonradioactive and radio-contaminated plots in the Chernobyl area for two generations. Quantitative proteomic analyses based on 2-D gel electrophoresis (2-DE) allowed us to establish developmental profiles for 199 2-DE spots in both plots, out of which 79 were reliably identified by tandem mass spectrometry. The data suggest a statistically significant increased abundance of proteins associated with pyruvate biosynthesis via cytoplasmic glycolysis, l-malate decarboxylation, isocitrate dehydrogenation, and ethanol oxidation to acetaldehyde in early stages of seed development. This was followed by statistically significant increased abundance of ketoacyl-[acylcarrier protein] synthase I related to condensation of malonyl-ACP with elongating fatty acid chains. On the basis of these and previous data, we propose a preliminary model for plant adaptation to growth in a radio-contaminated environment. One aspect of the model suggests that changes in carbon assimilation and fatty acid biosynthesis are an integral part of plant adaptation.

Over the last several decades, there have been a large number of studies done on the all aspects of legumes and bacteria which participate in nitrogen-fixing symbiosis. The analysis of legume-bacteria interaction is not just a matter of numerical complexity in terms of variants of gene products that can arise from a single gene. Bacteria regulate their quorum-sensing genes to enhance their ability to induce conjugation of plasmids and symbiotic islands, and various protein secretion mechanisms; that can stimulate a collection of chain reactions including species-specific combinations of plant-secretion isoflavonoids, complicated calcium signaling pathways and autoregulation of nodulation mechanisms. Quorum-sensing systems are introduced by the intra- and intercellular organization of gene products lead to protein-protein interactions or targeting of proteins to specific cellular structures. In this study, an attempt has been made to review significant contributions related to nodule formation and development and their impacts on cell proteome for better understanding of plant-bacterium interaction mechanism at protein level. This review would not only provide new insights into the plant-bacteria symbiosis response mechanisms but would also highlights the importance of studying changes in protein abundance inside and outside of cells in response to symbiosis. Furthermore, the application to agriculture program of plant-bacteria interaction will be discussed.

Plants grow and reproduce in the radioactive Chernobyl area, however there has been no comprehensive characterization of these activities. Herein we report that life in this radioactive environment has led to alteration of the developing soybean seed proteome in a specific way that resulted in the production of fertile seeds with low levels of oil and β-conglycinin seed storage proteins. Soybean seeds were harvested at four, five, and six weeks after flowering, and at maturity from plants grown in either non-radioactive or radioactive plots in the Chernobyl area. The abundance of 211 proteins was determined. The results confirmed previous data indicating that alterations in the proteome include adaptation to heavy metal stress and mobilization of seed storage proteins. The results also suggest that there have been adjustments to carbon metabolism in the cytoplasm and plastids, increased activity of the tricarboxylic acid cycle, and decreased condensation of malonyl-acyl carrier protein during fatty acid biosynthesis.

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.

Two serious nuclear accidents during the last quarter century (Chernobyl, 1986 and Fukushima, 2011) contaminated large agricultural areas with radioactivity. The database "Seeds in Chernobyl" (http://www.chernobylproteomics.sav.sk) contains the information about the abundances of hundreds of proteins from on-going investigation of mature and developing seed harvested from plants grown in radioactive Chernobyl area. This database provides a useful source of information concerning the response of the seed proteome to permanently increased level of ionizing radiation in a user-friendly format.