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Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

ABSTRACT: KEY MESSAGE:In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways. In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell division pattern yet develop into histodifferentiated embryos that can be converted into seedlings. We used the Brassica napus microspore embryogenesis system, in which the male gametophyte is reprogrammed in vitro to form haploid embryos, to identify the developmental fates of the different types of embryogenic structures found in culture. Using time-lapse imaging of LEAFY COTYLEDON1-expressing cells, we show that embryogenic cell clusters with very different morphologies are able to form haploid embryos. The timing of surrounding pollen wall (exine) rupture is a major determinant of cell fate in these clusters, with early exine rupture leading to the formation of suspensor-bearing embryos and late rupture to suspensorless embryos. In addition, we show that embryogenic callus, which develops into suspensor-bearing embryos, initially expresses transcripts associated with both basal- and apical-embryo cell fates, suggesting that these two cell fates are fixed later in development. This study reveals the inherent plasticity of in vitro embryo development and identifies new pathways by which embryo cell fate can be established.

SUBMITTER: Corral-Martinez P

PROVIDER: S-EPMC7648746 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

Corral-Martínez Patricia P Siemons Charlotte C Horstman Anneke A Angenent Gerco C GC de Ruijter Norbert N Boutilier Kim K

Plant reproduction 20200710 3-4

<h4>Key message</h4>In vitro embryo development is highly plastic; embryo cell fate can be re-established in tissue culture through different pathways. In most angiosperms, embryo development from the single-celled zygote follows a defined pattern of cell divisions in which apical (embryo proper) and basal (root and suspensor) cell fates are established within the first cell divisions. By contrast, embryos that are induced in vitro in the absence of fertilization show a less regular initial cell ...[more]

PMID: 32651727

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Project description:Microspore embryogenesis (ME) is the most powerful tool for creating homozygous lines in plant breeding and molecular biology research. It is still based mainly on the reprogramming of microspores by temperature, osmotic and/or nutrient stress. New compounds are being sought that could increase the efficiency of microspore embryogenesis or even induce the formation of haploid embryos from recalcitrant genotypes. Among these, the mitogenic factor phytosulfokine alpha (PSK-α) is promising due to its broad spectrum of activity in vivo and in vitro. The aim of our study was to investigate the effect of PSK-α on haploid embryogenesis from microspores of oilseed rape (Brassica napus L., DH4079), one of the most important oil crops and a model plant for studying the molecular mechanisms controlling embryo formation. We tested different concentrations (0, 0.01, 0.1 and 1 µM) of the peptide and evaluated its effect on microspore viability and embryo regeneration after four weeks of culture. Our results showed a positive correlation between addition of PSK-α and cultured microspore viability and a positive effect also on the number of developed embryos. The analysis of transcriptomes across three time points (day 0, 2 and 4) with or without PSK-α supplementation (15 RNA libraries in total) unveiled differentially expressed genes pivotal in cell division, microspore embryogenesis, and subsequent regeneration. PCA grouped transcriptomes by RNA sampling time, with the first two principal components explaining 56.8% variability. On day 2 with PSK, 45 genes (15 up- and 30 down-regulated) were differentially expressed when PSK-α was added and their number increased to 304 by day 4 (30 up- and 274 down-regulated). PSK, PSKR, and PSI gene expression analysis revealed dynamic patterns, with PSK2 displaying the highest increase and overall expression during microspore culture at days 2 and 4. Despite some variations, only PSK1 showed significant differential expression upon PSK-α addition. Of 16 ME-related molecular markers, 3 and 15 exhibited significant differential expression in PSK-supplemented cultures at days 2 and 4, respectively. Embryo-specific markers predominantly expressed after 4 days of culture, with higher expression in medium without PSK, while on day 0, numerous sporophyte-specific markers were highly expressed.

Project description:Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4x10(-6) M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.

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Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

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Live Imaging of embryogenic structures in Brassica napus microspore embryo cultures highlights the developmental plasticity of induced totipotent cells.

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