Histone Deacetylase Inhibitors Increase the Embryogenic Potential and Alter the Expression of Embryogenesis-Related and HDAC-Encoding Genes in Grapevine (Vitis vinifera L., cv. Mencía)

Abstract

The low induction rates of somatic embryogenesis are one of the main limitations in its routine application in the grapevine (Vitis vinifera L.). The use of an induction medium containing histone deacetylase inhibitors (trichostatin A and, mainly, sodium butyrate) resulted in an improvement of the embryogenic responses in grapevine (cv. Mencía) cotyledonary and recently germinated somatic embryos. The relative expression of several grapevine genes related to embryogenic competence or encoding histone deacetylase enzymes was studied in cotyledonary somatic embryos that were cultured in the presence of 0.5 mM sodium butyrate. The results showed a significant overexpression of the BBM and VvSERK2 genes after 24 h of culture, whereas the VvWOX2 gene was underexpressed less in treated versus untreated explants. The results suggest that the inhibitor may trigger a molecular response related to an increase in embryogenic competence and changes in the expression of associated genes. The treatment with sodium butyrate also produced significant variations in the expression of several histone deacetylase enzyme-encoding genes. These results may enhance the possibility of obtaining somatic embryos, reducing the seasonal constraints associated with the use of floral explants in grapevines.

Keywords: BABY BOOM; SERK2; Vitis vinifera; gene expression; histone deacetylase-encoding genes; sodium butyrate; somatic embryogenesis; trichostatin A.

Figure 1

Responses observed in grapevine cv. Mencía cotyledonary somatic embryos. (A) Cotyledonary somatic embryo with no response and signs of necrosis after eight weeks of culture in an induction medium with 5 mM NaB. (B) Non-embryogenic callus originating from cotyledonary somatic embryos that were cultured in an induction medium without HDAC inhibitors for eight weeks. (C) Proembryos formed on the surface of cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB for eight weeks. (D) Somatic embryo aggregate formed by secondary embryogenesis of proembryos formed from cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB for eight weeks. (E) Grapevine microplant regenerated from somatic embryos induced in cotyledonary somatic embryos that were cultured in an induction medium with 0.5 mM NaB. Bars: 1 mm (A,B); 0.5 mm (C,D); 1 cm (E).

Figure 2

Histological analysis of non-embryogenic calli (A,C,E) and somatic embryo aggregates (B,D,F) formed from grapevine cv. Mencía cotyledonary somatic embryos that were cultured in an induction medium without HDAC inhibitors or supplemented with 0.5 mM NaB. Sections were stained with calcofluor (A,B), toluidine blue (C,D), or DAPI (E,F). Bars: 200 μm.

Figure 3

Effect of HDAC inhibitors (NaB and TSA) on embryogenic responses in grapevine cv. Mencía cotyledonary somatic embryos. The percentage of explants with an embryogenic response (mean ± standard error) after (A) 4 and (B) 8 weeks of culture is shown. Different letters indicate statistically significant differences (p < 0.05).

Figure 4

Effect of HDAC inhibitors (NaB and TSA) on the embryogenic response in grapevine cv. Mencía recently germinated somatic embryos. The percentage of explants with an embryogenic response (mean ± standard error) after (A) 4 and (B) 8 weeks of culture is shown. There were no statistically significant differences between any of the treatments (p < 0.05).

Figure 5

Relative expression of genes related to somatic embryogenesis in grapevine cv. Mencía cotyledonary somatic embryos that were cultured in the induction medium supplemented with 0.5 mM NaB (blue) or without NaB (green) for 24 (A) and 48 (B) h. The mean values of two independent experiments ± standard errors are shown. Asterisks indicate statistically significant differences (p < 0.05) between the calibrator group (cotyledonary somatic embryos used as starting material) and the analyzed group, while red squares framing genes indicate statistically significant differences (p < 0.05) between the NaB and no NaB treatments.

Figure 6

Relative expression of HDAC-encoding genes in grapevine cv. Mencía cotyledonary somatic embryos that were cultured in the induction medium supplemented with 0.5 mM NaB (blue) or without NaB (green) for (A) 24 and (B) 48 h. The mean values of the two independent experiments ± standard errors are shown. Asterisks indicate statistically significant differences (p < 0.05) between the calibrator group (cotyledonary somatic embryos used as starting material) and the analyzed group, while the red squares framing genes indicate statistically significant differences (p < 0.05) between the NaB and no NaB treatments.

Figure 7

Grapevine (Vitis vinifera L., cv. Mencía) explants used in this work. (A) Somatic embryo in the cotyledonary morphological stage, (B) recently germinated somatic embryo, (C) shoot apex, and (D) leaf from a grapevine plant maintained in vitro. Bars: 1 mm (A,C); 1 cm (B); 0.5 cm (D).