Involvement of Polyamine Oxidase-Produced Hydrogen Peroxide during Coleorhiza-Limited Germination of Rice Seeds

Abstract

Seed germination is a complicated biological process that requires regulated enzymatic and non-enzymatic reactions. The action of polyamine oxidase (PAO) produces hydrogen peroxide (H2O2), which promotes dicot seed germination. However, whether and, if so, how PAOs regulate monocot seed germination via H2O2 production is unclear. Herein, we report that the coleorhiza is the main physical barrier to radicle protrusion during germination of rice seed (a monocot seed) and that it does so in a manner similar to that of dicot seed micropylar endosperm. We found that H2O2 specifically and steadily accumulated in the coleorhizae and radicles of germinating rice seeds and was accompanied by increased PAO activity as the germination percentage increased. These physiological indexes were strongly decreased in number by guazatine, a PAO inhibitor. We also identified 11 PAO homologs (OsPAO1-11) in the rice genome, which could be classified into four subfamilies (I, IIa, IIb, and III). The OsPAO genes in subfamilies I, IIa, and IIb (OsPAO1-7) encode PAOs, whereas those in subfamily III (OsPAO8-11) encode histone lysine-specific demethylases. In silico-characterized expression profiles of OsPAO1-7 and those determined by qPCR revealed that OsPAO5 is markedly upregulated in imbibed seeds compared with dry seeds and that its transcript accumulated to a higher level in embryos than in the endosperm. Moreover, its transcriptional abundance increased gradually during seed germination in water and was inhibited by 5 mM guazatine. Taken together, these results suggest that PAO-generated H2O2 is involved in coleorhiza-limited rice seed germination and that OsPAO5 expression accounts for most PAO expression and activity during rice seed germination. These findings should facilitate further study of PAOs and provide valuable information for functional validation of these proteins during seed germination of monocot cereals.

Keywords: Oryza sativa; OsPAO5; gene expression; hydrogen peroxide; in silico analysis; polyamine oxidases; seed germination.

FIGURE 1

Morphologies and germination time courses of rice seeds. (A) Morphologies of rice seeds imbibed in (top) water or in (bottom) 5 mM guazatine. Germination time courses for rice seeds imbibed in (B) water, 10 mM H₂O₂, 5 mM DMTU, 5 mM DMTU plus 10 mM H₂O₂, 5 mM guazatine, or (C) water, 5 mM guazatine or 5 mM guazatine plus 10 mM H₂O₂. Germination number was scored every 6 h for 48 h, and the results are presented as the cumulative germination percentages. Data are the mean ± SE of three biological replicates of 100 seeds each.

FIGURE 2

Histochemical staining and quantification of H₂O₂ and O₂^- content, and peroxidase activity during germination of rice seeds in water or guazatine. Histochemical staining for the location of (A) H₂O₂ (C) O₂^-, and (E) peroxidase activity in whole rice seeds and in (F) embryos of half granule seeds after imbibition of water or 5 mM guazatine. Quantitative determination of (B) H₂O₂ content and (D) O₂^- production in seeds imbibed in water or in 5 mM guazatine. Data are the mean ± SE of three biological replicates of 30 embryos (∼0.1 g total). Means denoted by the same letter did not significantly differ at P < 0.05 according to Fisher’s least significant difference test. FW, fresh weight.

FIGURE 3

Identification of the optimal substrate and absorbance peak for crude PAO activity and measurement of PAO activity during rice seed germination. (A) Substrate specificity and absorbance spectra found for the crude PAO activity in rice seeds after imbibed in water for 12 h. (B) Crude PAO activity in rice seeds, imbibed in water or in 5 mM guazatine at 3, 6, 12, 24, and 48 h, was determined using Spm as the substrate. Data are the mean ± SE of three biological replicates of 60 embryos (∼0.2 g total). Means denoted by the same letter did not significantly differ at P < 0.05 according to Fisher’s least significant difference test.

FIGURE 4

Phylogenetic tree, predicted locations and functions of PAO protein family. (A) Unrooted maximum-likelihood phylogenetic tree for rice and Arabidopsis PAO protein family constructed based on an amino acid sequence alignment of their amino_oxidase domains. Human, maize, and barley sequences are included. HsPAOX (Homo sapiens, ENSP00000278060), HsSMOX (H. sapiens, ENSP00000307252), HsKDMA (H. sapiens, ENSP00000383042), ZmPAO1 (Zea mays, NM_001111636), HvPAO1/2 (Hordeum vulgare, AJ298131 and AJ298132). The associated bootstrap values from 1000 replications are given at their nodes, and the branch lengths are drawn to scale. The subfamily members are bracketed by color, and their subfamily numbers, I, IIa, IIb, and III, are shown to the right of the tree. (B) Domain organizations (left), and predicted functions and locations (right).

FIGURE 5

Expression profiles of OsPAO genes during germination of rice seeds. (A) Expression profiles (heat maps) obtained from rice microarray (Os_51k array) data as reported by GENEVESTIGATOR V3. Expression profiles for OsPAO2 and OsPAO6 were unavailable. The green/red coding reflects the relative expression levels with dark green representing strong downregulation and dark red representing strong upregulation. The corresponding numerical values are shown in Supplementary Table S2. (B) The expression levels of OsPAO1∼7 in embryos assayed by qPCR after seeds had been imbibed in water for 0, 12, or 24 h. Data are the mean ± SE of three biological replicates of 30 embryos (∼0.1 g total). Significant differences for the qPCR data were assessed by the Student’s t-test (^∗P < 0.05; ^∗∗P < 0.01). (C) OsPAO5 expression patterns in the embryo assayed by qPCR after seed imbibition of water or 5 mM guazatine at 0, 3, 6, 12, 24, and 48 h. Data are the mean ± SE of three biological replicates of 30 embryos (∼0.1 g total). Means denoted by the same letter did not significantly differ at P < 0.05 according to Fisher’s least significant difference test.