Kateřina Eliášová

Early molecular events involved in Pinus pinaster Ait. somatic embryo development under reduced water availability: transcriptomic and proteomic analyses.

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 role of actin isoforms in somatic embryogenesis in Norway spruce

BackgroundSomatic embryogenesis in spruce is a process of high importance for biotechnology, yet it comprises of orchestrated series of events whose cellular and molecular details are not well understood. In this study, we examined the role of actin cytoskeleton during somatic embryogenesis in Norway spruce line AFO 541 by means of anti-actin drugs.ResultsApplication of low doses (50-100 nM) of latrunculin B (Lat B) during the maturation of somatic embryos predominantly killed suspensor cells while leaving the cells in meristematic centres alive, indicating differential sensitivity of actin in the two cell types. The treatment resulted in faster development of more advanced embryos into mature somatic embryos and elimination of insufficiently developed ones. In searching for the cause of the differential actin sensitivity of the two cell types, we analysed the composition of actin isoforms in the culture and isolated four spruce actin genes. Analysis of their expression during embryo maturation revealed that one actin isoform was expressed constitutively in both cell types, whereas three actin isoforms were expressed predominantly in suspensor cells and their expression declined during the maturation. The expression decline was greatly enhanced by Lat B treatment. Sequence analysis revealed amino-acid substitutions in the Lat B-binding site in one of the suspensor-specific actin isoforms, which may result in a different binding affinity for Lat B.ConclusionsWe show that manipulating actin in specific cell types in somatic embryos using Lat B treatment accelerated and even synchronized the development of somatic embryos and may be of practical use in biotechnology.

The role of auxins in somatic embryogenesis of Abies alba

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.

Cryotolerance in Norway spruce and its association with growth rates, anatomical features and polyamines of embryogenic cultures

Our study focused on the possible association between the cryotolerance of Norway spruce (Picea abies (L.) Karst.) embryogenic cultures and the anatomical structures of their embryogenic suspensor mass (ESM), their growth rate and their content of endogenous polyamines (PAs). The anatomical characteristics and PA content during cryopreservation and regrowth were studied in the ESMs of AFO 541 and C110 cultures, which have comparable ESM anatomy but diverse growth rates, PA content and regeneration abilities after cryopreservation. Different levels of tolerance to exogenous treatment were already apparent after transfer of the ESMs to liquid media. The endogenous free PAs were maintained at high levels, with spermidine being the predominant PA in the ESM of AFO 541, while in the ESM of C110 the content of putrescine and spermidine was almost identical and rather low, the content of spermidine being approximately one-third that in the ESM of AFO 541. Osmotic pretreatment, using a double application of sorbitol followed by an application of dimethyl sulfoxide (DMSO) resulted in the continual disintegration of polyembryogenic centers and suspensors in both cell lines. A continual decrease in the level of PAs was observed during the cell osmotic pretreatment. The cells that retained their viability and regrowth ability after cryopreservation were the meristematic cells inside the embryonal heads and the cells in the intermediate area between suspensor and meristems. Restoration of AFO 541 growth after cryopreservation was almost immediate; however, the C110 ESM culture regrew with difficulty, often exhibiting callogenesis. High levels of PA-soluble conjugates and an increase in the amount of PAs bound to high-molecular-mass substances was observed in cells of AFO 541 on Day 6 after thawing and also to some extent on Day 11. On Day 21 after thawing, the amount of free putrescine and spermidine in the AFO 541 cells reached the level observed in the suspension culture before the cryotreatment. The extremely low level of PAs determined in the ESM of C110 3 weeks after thawing agreed with the cell viability and rate of regrowth observed in this culture. The possible role of PAs in the process of cryopreservation of Norway spruce cultures is discussed.