Microarray Analysis of Siberian Ginseng Cyclic Somatic Embryogenesis Culture Systems Provides Insight into Molecular Mechanisms of Embryogenic Cell Cluster Generation

<div><p>Four systems of cyclic somatic embryogenesis of Siberian ginseng (<i>Eleutherococcus senticosus</i> Maxim) were used to study the mechanism of embryonic cell cluster generation. The first, direct somatic embryo induction (DSEI), generates secondary embryos directly from the primary somatic embryos; the second, direct embryogenic cell cluster induction (DEC)), induces embryogenic cell clusters directly from somatic embryos in agar medium. Subsequently, we found that when DEC-derived somatic embryos are transferred to suspension culture or a bioreactor culture, only somatic embryos are induced, and embryogenic cell clusters cannot form. Therefore, these new lines were named DEC cultured by liquid medium (ECS) and DEC cultured by bioreactor (ECB), respectively. Transmission electron microscopy showed that DEC epidermal cells contained a variety of inclusions, distinct from other lines. A cDNA library of DEC was constructed, and 1,948 gene clusters were obtained and used as probes. RNA was prepared from somatic embryos from each of the four lines and hybridized to a microarray. In DEC, 7 genes were specifically upregulated compared with the other three lines, and 4 genes were downregulated. <i>EsXTH1</i> and <i>EsPLT1</i>, which were among the genes upregulated in DEC, were cloned using the rapid amplification of cDNA ends (RACE). Real-time quantitative PCR showed <i>EsXTH1</i> was more highly expressed in DEC than in other lines throughout the culture cycle, and <i>EsPLT1</i> expression in DEC increased as culture duration increased, but remained at a low expression level in other lines. These results suggest that <i>EsXTH1</i> and <i>EsPLT1</i> may be the essential genes that play important roles during the induction of embryogenic cell clusters.</p></div

<b>Differentially regulated genes in DEC compared with DSEI, ECS and ECB.</b>

<p><b>Differentially regulated genes in DEC compared with DSEI, ECS and ECB.</b></p

Amino acid sequence alignment of <i>EsXTH1</i> with XTH from other plant species.

<p>Black represents 100% homology; gray represents ≥ 50% homology. Amino acid XTH from cucumber (<i>Cucumis sativus</i>, XM 004165650.1), grape (<i>Vitis vinifera</i>, XM 002270146.2), soybean (<i>Glycine max</i>, XM 003542108.1), peach (<i>Prunus persica</i>, KB 638913.1), kiwifruit (<i>Actinidia hemsleyana</i>, EU 494954.1), apple (<i>Malus domestica</i>, EU 494967.1), castor bean (<i>Ricinus communis</i>, XM 002526182.1), poplar (<i>Populus trichocarpa</i>, XM 002324444.1), and alfalfa (<i>Medicago truncatula</i>, XM 003625289.1) revealed the existence of conserved functional domains of Glyco_hydro_16 (<i>lined section</i>) and XET_C (<i>double lined section</i>).</p

Schematic diagram of the process used to generate four lines (DEC, DSEI, ECS and ECB) of somatic embryos.

<p><b>A</b> Mature seed, <b>B</b> somatic embryos were induced from mature seed directly through heat-stressed culture on hormone-free 1/3 MS medium, <b>C</b> secondary embryos were developed directly after transfer somatic embryos to the original culture environment cultured on hormone-free medium for 3–4 weeks, <b>D</b> somatic embryos were developed when secondary embryos were subcultured on hormone-free medium, <b>E</b> somatic embryos, <b>F</b> embryogenic cell clusters were induced from somatic embryos through culture on 1/3 MS medium with 1 mg·L⁻¹ 2,4-D, <b>G</b> mature embryos were developed when embryogenic cell clusters were cultured on hormone-free medium, <b>H</b> embryogenic cell clusters were generated when embryos were cultured on 1/3 MS medium with 1 mg·L⁻¹ 2,4-D, <b>I</b> embryogenic cell clusters, <b>J</b> secondary embryos were developed directly when mature embryos (DEC line) were transferred into 1/3 MS liquid medium without PGR for suspension culture, <b>K</b> secondary embryos in suspension culture, <b>L</b> secondary embryos were developed directly when mature embryos (DEC line) were transferred into 1/3 MS liquid medium without PGR for bioreactor culture, <b>M</b> secondary embryos in bioreactor. <b>1.</b> DSEI process. <b>2.</b> DEC process <b>3.</b> ECS process <b>4.</b> ECB process.</p

TEM observation of epidermal cells in DEC, DSEI, ECS and ECB of Siberian ginseng somatic embryos.

<p>Epidermal cells in DEC on the first day of culture (A); epidermal cells in DEC (B, C), DSEI (D, E), ECS (F, G) and ECB (H, I) on the 5th days of culture. <i>a</i> amyloplast, <i>cw</i> cell wall, <i>g</i> glyoxysome, <i>Ga</i> Golgi apparatus, <i>lb</i> lipid body, <i>m</i> mitochondria, <i>n</i> nucleus, <i>nu</i> nucleous, <i>pb</i> protein body, <i>s</i> starch grain, <i>v</i> vacuole.</p

<b>Differentially regulated genes in DEC compared with DSEI.</b>

<p><b>Differentially regulated genes in DEC compared with DSEI.</b></p

Pie charts showing functional categories assigned using COG.

<p>490 clusters predicted to encode characterized proteins classified into 23 different functional classifications.</p

Supplementary Figures

Supplementary Figure 1 Anthocyanins improve liver fibrosis (A) Mouse model of liver fibrosis intervention with anthocyanins. H&E, Masson staining and immunofluorescence staining were used to evaluate liver fibrosis in mouse models. Scale bar, 50 μm. (B) Effects of anthocyanins on liver injury markers AST and ALT. Data are presented as the mean ± SEM (n = 6). *P Supplementary Figure 2 Selection of effective cells and verification of anthocyanins' regulation on ferroptosis in AML-12 and RAW246.7 cells. (A) Western blotting was used to detect the expression of GPX4 in AML-12 and RAW246.7 cells after anthocyanin treatment. (B) Fe2+ content in AML-12 and RAW246.7 cells after anthocyanin treatment. (C) The GSH content in AML-12 and RAW246.7 cells after anthocyanin treatment. (D) The ROS content in AML-12 and RAW246.7 cells after anthocyanin treatment. (E) Isolated HC cells from different treatment groups display differences in Fe2+ concentration, ROS levels, and GSH activity. (F) Isolated KC cells from different treatment groups display differences in Fe2+ concentration, ROS levels, and GSH activity.</p