Hydrogen peroxide signaling integrates with phytohormones during the germination of magnetoprimed tomato seeds

Abstract

Seeds of tomato were magnetoprimed at 100 mT for 30 min followed by imbibition for 12 and 24 h, respectively, at 20 °C, to examine the biochemical and molecular changes involved in homeostasis of hydrogen peroxide (H₂O₂) and its signaling associated with hormone interactions for promoting vigor. The relative transcript profiles of genes involved in the synthesis of H₂O₂ like Cu-amine oxidase (AO), receptor for activated C kinase 1 (RACK1) homologue (ArcA2) and superoxide dismutase (SOD1 and SOD9) increased in magnetoprimed tomato seeds as compared to unprimed ones with a major contribution (21.7-fold) from Cu-amine oxidase. Amongst the genes involved in the scavenging of H₂O₂ i.e, metallothionein (MT1, MT3 and MT4), catalase (CAT1) and ascorbate peroxidase (APX1 and APX2), MT1 and MT4 exhibited 14.4- and 15.4-fold increase respectively, in the transcript abundance, in primed seeds compared to the control. We report in our study that metallothionein and RACK1 play a vital role in the reactive oxygen species mediated signal transduction pathway to enhance the speed of germination in magnetoprimed tomato seeds. Increased enzymatic activities of catalase and ascorbate peroxidase were observed at 12 h of imbibition in the magnetoprimed seeds indicating their roles in maintaining H₂O₂ levels in the primed seeds. The upregulation of ABA 8'-hydroxylase and GA3 oxidase1 genes eventually, lead to the decreased abscisic acid/gibberellic acid (ABA/GA₃) ratio in the primed seeds, suggesting the key role of H₂O₂ in enhancing the germination capacity of magnetoprimed tomato seeds.

Figure 1

Effect of magnetic field exposure on speed of germination in primed and unprimed tomato seeds. Data represent mean ± S.E. of three biological replications replicated four times. Significant difference between treatments is indicated with the p value according to Student’s t test.

Figure 2

Effect of magnetic field exposure on generation of reactive oxygen species in germinating tomato seeds at different duration of imbibition. Data represent mean ± S.E. of three biological replications. Significant difference between treatments is indicated with the p value according to Student’s t test.

Figure 3

Photomicrographs showing localization of superoxide anion (NBT staining) and hydrogen peroxide (DAB staining) in tomato seeds. (A) superoxide anion in 12 h unprimed seeds; (B) superoxide anion in 12 h primed seeds; (C) superoxide anion in 24 h unprimed seeds; (D) superoxide anion in 24 h primed seeds. The formation of insoluble blue formazan precipitates in the endosperm of primed seeds indicate the strong accumulation of O₂^•− as compared to unprimed seeds. (E) hydrogen peroxide in 12 h unprimed seeds; (F) hydrogen peroxide in 12 h primed seeds; (G) hydrogen peroxide in 24 h unprimed seeds; (H) hydrogen peroxide in 24 h primed seeds. Increased brown coloration in outer part of the endosperm of primed seeds shows higher production of H₂O₂ as compared to unprimed seeds.

Figure 4

Effect of magnetic field exposure on total antioxidant capacity (% DPPH radical scavenging activity) in germinating tomato seeds at different duration of imbibition. Data represent mean ± S.E. of three biological replications. Significant difference between treatments is indicated with the p value according to Student’s t test. ns = non significant.

Figure 5

Effect of magnetic field exposure on antioxidant enzymes in germinating tomato seeds at different duration of imbibition [superoxide dismutase (units mg⁻¹ protein), catalase (nmol H₂O₂ reduced min⁻¹ mg⁻¹ protein), ascorbate peroxidase (µmol ascorbate oxidized min⁻¹ mg⁻¹ protein)]. Data represent mean ± S.E. of four biological replications. Significant difference between treatments is indicated with the p value according to Student’s t test. ns = non significant.

Figure 6

(A–C). Bar graphs depicting the effect of magnetic field treatment on the relative expression of various genes in germinating tomato seeds at 12 h of imbibition. (A) Relative expression of genes involved in the synthesis of H₂O₂, i.e. AO (Amine oxidase), NOX (NADPH oxidase), SOD1 and SOD9 (Superoxide dismutase 1 and 9), and ArcA2 (receptor for activated C kinase 1/RACK1 homologue)^#; scavenging of H₂O₂, i.e. CAT1 (Catalase 1), APX1 and APX2 (Ascorbate peroxidase 1 and 2), MT1 (Metallothionein 1); signaling of H₂O₂, i.e. ArcA2 and MT1. (B) Relative expression of isoforms of Metallothionein (MT1, MT3 and MT4)^#. (C) Relative expression of ABA-H (ABA 8′-hydroxylase) and GA3ox1 and GA3ox2 (Gibberellic acid 3 oxidase1 and 2). Data represent mean ± S.E. of three biological replicates with two technical replications. GAPDH gene was used as internal control to normalize the expression level. Fold change was calculated using 2^−ΔΔCT method that represents the changes in expression levels in the primed seeds as compared to the unprimed ones. Significant difference between treatments is indicated with the p value, according to Student’s t test. ns = non significant. qRT-PCR primers for all genes are provided in Table 2. ^#ArcA2 gene is involved in synthesis as well as signaling processes of H₂O₂, while MT1 gene is involved in signaling as well as scavenging processes of H₂O₂.

Figure 7

Schematic model of regulation of H₂O₂ for oxidative signaling in magnetoprimed tomato seeds. Bold letters show major contribution of genes involved in the metabolic process. Primimg of tomato seeds with magnetic field of 100 mT (30 min) followed by imbibition for 12 h led to increased production of hydrogen peroxide in the primed seeds. Amongst the genes involved in H₂O₂ production (amine oxidase, NADPH oxidase and superoxide dismutase), amine oxidase is the key player for H₂O₂ synthesis. Metallothionein and receptor for activated C kinase 1 are proposed to be responsible for signaling process, besides scavenging and production of H₂O_2, respectively. Thus, H₂O₂ acts as pivotal signaling molecule by integrating with phytohormones through reduction in ABA and increase in GA levels for increasing the speed of germination in magnetoprimed tomato seeds.