Synthetic Strigolactone GR24 Improves Arabidopsis Somatic Embryogenesis through Changes in Auxin Responses

Abstract

Somatic embryogenesis in Arabidopsis encompasses an induction phase requiring auxin as the inductive signal to promote cellular dedifferentiation and formation of the embryogenic tissue, and a developmental phase favoring the maturation of the embryos. Strigolactones (SLs) have been categorized as a novel group of plant hormones based on their ability to affect physiological phenomena in plants. The study analyzed the effects of synthetic strigolactone GR24, applied during the induction phase, on auxin response and formation of somatic embryos. The expression level of two SL biosynthetic genes, MOREAXILLARY GROWTH 3 and 4 (MAX3 and MAX4), which are responsible for the conversion of carotene to carotenal, increased during the induction phase of embryogenesis. Arabidopsis mutant studies indicated that the somatic embryo number was inhibited in max3 and max4 mutants, and this effect was reversed by applications of GR24, a synthetic strigolactone, and exacerbated by TIS108, a SL biosynthetic inhibitor. The transcriptional studies revealed that the regulation of GR24 and TIS108 on somatic embryogenesis correlated with changes in expression of AUXIN RESPONSIVE FACTORs 5, 8, 10, and 16, known to be required for the production of the embryogenic tissue, as well as the expression of WUSCHEL (WUS) and Somatic Embryogenesis Receptor-like Kinase 1 (SERK1), which are markers of cell dedifferentiation and embryogenic tissue formation. Collectively, this work demonstrated the novel role of SL in enhancing the embryogenic process in Arabidopsis and its requirement for inducing the expression of genes related to auxin signaling and production of embryogenic tissue.

Keywords: 2,4-D; ARFs; MAX3; MAX4; SERK; TIS108; WUS; quantitative PCR.

Figure 1

Schematic representation of the somatic embryogenic process in Arabidopsis. Immature zygotic embryos were cultured for 14 days on a 2,4-D-containing induction medium required for the formation of embryogenic tissue by immature embryos. Continuation of embryo development was achieved by transferring the explants on development medium devoid of 2,4-D. Fully developed somatic embryos were visible after 9 days.

Figure 2

(A) Number of somatic embryos produced by WT tissue and max1, 3–9, and 4 tissues. Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05). (B) Micrographs showing the number of fully developed somatic embryos from tissue in (A). (C) The fold change in expression level of MAX1, 3–9, and 4 at different time points during the induction (I) and developmental (D) stages of embryogenesis. Values are means ± SE of three biological replicates (n = 15).

Figure 3

Requirement of SL for somatic embryogenesis in Arabidopsis. (A) Effects of different concentrations of GR24 and TIS108 applied in the induction medium on the number of somatic embryos. Values are means ± SE of three biological replicates (n = 15). (B) Effects of GR24 (50 nM) and/or TIS108 (10 nM) on the number of somatic embryos produced by the WT tissue. A mock experiment was conducted using water (Control). Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05). (C) Effects of GR24 (50 nM) or TIS108 (10 nM) on the number of somatic embryos produced by max3–9 tissue. Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05). (D) Effects of GR24 (50 nM) or TIS108 (10 nM) on the number of somatic embryos produced by max4 tissue. Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05).

Figure 4

Fold change in expression level of ARF5, 6, 8, 10, 16, and 17 in WT, max3–9, and max4 tissue after 7 days of induction. Tissue was also treated with GR24 (50 nM) and/or TIS108 (10 nM). Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05).

Figure 5

Fold change in expression levels of SERK1 and WUS in WT, max3–9, and max4 tissue after 7 days of induction. Tissue was also treated with GR24 (50 nM) and/or TIS108 (10 nM). Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05).

Figure 6

Effects of different concentration of 2,4-D on the number of somatic embryos generated from WT, max3–9, and max4 explants. Tissue was also treated with GR24 (50 nM). Values are means ± SE of three biological replicates (n = 15). Letters on bars indicate statistically significant differences (p < 0.05).