Ethylene is integrated into the nitric oxide regulation of Arabidopsis somatic embryogenesis

Abstract

The study confirms the role of the two Arabidopsis hemoglobin genes (Glb1 and Glb2) during somatic embryogenesis and proposes the involvement of ethylene in the regulation of embryo development. Suppression of both Glb1 and Glb2 results in accumulation of nitric oxide (NO) and a different embryogenic response. Compared to WT tissue, down-regulation of Glb1 (Glb1 RNAi line) compromises the embryogenic process, while repression of Glb2 (Glb2-/- line) increases the number of embryos. These differences were ascribed to the differential accumulation of NO in the two lines, as Glb1 is a more effective NO scavenger compared to Glb2. A high elevation of NO level [achieved pharmacologically using the NO donor sodium nitroprusside (SNP), or genetically using the Glb1 suppressing line], activated the two ethylene biosynthetic genes 1-aminocyclopropane-1-carboxylate synthase (ACC synthase) and 1-aminocyclopropane-1-carboxylate oxidase (ACC oxidase). Ethylene accumulation repressed embryogenesis, as shown by the decreased embryo number observed in tissue treated with the ethylene releasing agent Ethephon (ETH), as well as by the increased embryo production obtained with the two ethylene insensitive mutant lines (ein2-1 and ein3-1). A repression in ethylene level increased the expression of many auxin biosynthetic genes and favored the accumulation of the auxin indole-acetic acid (IAA) at the sites of the explants where embryogenic tissue will form. Collectively these data reveal that high levels of NO, generated by the Glb1 suppressing line, but not by the Glb2 suppressing line, might increase the level of ethylene, which represses the production of auxin. Auxin is the inductive signal required for the formation of the embryogenic tissue.

Keywords: Auxin; Ethylene; Hemoglobin; Somatic embryogenesis.

Figure 1

The Arabidopsis somatic embryogenic system. Seed embryos are dissected and placed on an auxin-containing induction medium for 14 days. During this time the embryogenic tissue (arrows) forms from the cotyledons of the explants. Transfer of the tissue on an auxin-free development medium for 9 days results in the formation of fully mature somatic embryos.

Figure 2

Number of somatic embryos [expressed as a percentage of WT control (ctrl)] collected from the WT, Glb1 RNAi and Glb2−/− mutant lines treated with increasing levels of NO donor sodium nitroprusside (SNP) (A), the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) (B), the ethylene releasing agent Ethephon (ETH) (C), and the ethylene biosynthetic inhibitor O-(carboxymethyl)hydroxylamine hemihydrochloride) (AOA) (D). Compounds were added in the induction medium. Values are means ± SE of at least three biological replicates.

Figure 3

(A) Micrographs showing embryo production after 9 days on development medium in the WT, Glb1 RNAi, Glb2−/− mutant, and the two ethylene insensitive mutant lines ein2, ein3. (B) Number of embryos expressed as a percentage of WT control (ctrl) in the different lines subjected to different treatments in the induction medium: SNP (100 μM), ETH (10 μM), cPTIO (50 μM), or AOA (10 μM). Values are means ± SE of at least three biological replicates. ^∗ indicates statistically significant differences (P < 0.005) from the WT (ctrl) value.

Figure 4

Expression level of the two ethylene biosynthetic aminocyclopropane-1-carboxylic acid oxidase (ACC oxidase) and aminocyclopropane-1-carboxylic acid synthase (ACC synthase), and the ethylene responsive factor 1 (ERF1), 10 (ERF10). Treatments were conducted as described in Fig. 3. Values are means ± SE of at least three biological replicates. ^∗ indicates statistically significant differences (P < 0.005) from the WT (ctrl) value set at 1.

Figure 5

GUS localization in the pEBS::GUS and pPDF1.2::GUS reported lines. Embryos were cultured for 7 days on induction medium before being harvested and stained for GUS. Concentrations of the treatments were identical to those reported in Fig. 3.

Figure 6

Expression level by quantitative (q) RT-PCR of the auxin biosynthetic genes anthranilate synthase α-subunit (ASA1), anthranilate isomerase (PAI3), amidase1 (AMI1), cytochrome P450 CYP79B2 (CYP79B2) and YUCCA4 (YUC4). Values are means ± SE of at least three biological replicates and normalized to WT (control, ctrl) set at 1. ^∗ indicates statistically significant differences (P < 0.005) from the WT (control, ctrl) value.

Figure 7

(A) GUS localization in the pASA1::GUS and pYUC4::GUS reported lines. Embryos were cultured for 7 days on induction medium before being harvested and stained for GUS. Concentrations of the treatments were identical to those reported in Fig. 3. (B) Immunolocalization of IAA along the cotyledons of the zygotic explants. Tissue was collected at 7 days on induction medium. Concentrations of the treatments were identical to those reported in Fig. 3.

Figure 8

Proposed model regulating Arabidopsis somatic embryogenesis. Glb1 is a more effective NO scavenger of Glb2, therefore, their suppression would result in a differential accumulation of NO. The Glb1 RNAi line accumulates more NO than the Glb2−/− line. Accumulation of NO activates the transcription of the two ethylene biosynthetic genes (ACC synthase and ACC oxidase) and would possibly increase ethylene level. Ethylene transcriptionally down-regulates many IAA biosynthetic genes and reduces the IAA signal on the zygotic explants. Since IAA is the inductive signal for somatic embryogenesis, a depletion in IAA reduces the ability of the Glb1 RNAi line to produce embryos.