Ascorbic acid and reactive oxygen species are involved in the inhibition of seed germination by abscisic acid in rice seeds

Abstract

The antagonism between abscisic acid (ABA) and gibberellin (GA) plays a key role in controlling seed germination, but the mechanism of antagonism during this process is not known. The possible links among ABA, reactive oxygen species (ROS), ascorbic acid (ASC), and GA during rice seed germination were investigated. Unlike in non-seed tissues where ROS production is increased by ABA, ABA reduced ROS production in imbibed rice seeds, especially in the embryo region. Such reduced ROS also led to an inhibition of ASC production. GA accumulation was also suppressed by a reduced ROS and ASC level, which was indicated by the inhibited expression of GA biosynthesis genes, amylase genes, and enzyme activity. Application of exogenous ASC can partially rescue seed germination from ABA treatment. Production of ASC, which acts as a substrate in GA biosynthesis, was significantly inhibited by lycorine which thus suppressed the accumulation of GA. Consequently, expression of GA biosynthesis genes was suppressed by the low levels of ROS and ASC in ABA-treated seeds. It can be concluded that ABA regulates seed germination in multiple dimensions. ROS and ASC are involved in its inhibition of GA biosynthesis.

Fig. 1.

Time course of germination of dehulled rice seeds. Seeds were directly sown on sterile filter paper in 5 cm Petri dishes and placed in a growth chamber in continuous darkness at 28 °C to facilitate germination. Seeds were weighed at various time points. Values are means ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 2.

Effects of ABA and diniconazole on seed germination (a) and ABA contents (b). Rice seeds were imbibed at 28 °C in the presence of water, diniconazole, and ABA solutions. Seed samples were collected at different time intervals and stored at –80 °C for ABA content determination. ABA was detected by RIA as described in the Materials and methods. Values are means ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 3.

Effects of ABA treatment on MDA content (a, b), H₂O₂ content (c, e), and superoxide anion (d, f). Seeds were sown on filter paper with ABA and Cu solutions for imbibition. Seed samples were collected at various times and used for measurements of MDA, H₂O₂, and superoxide anion as rapidly as possible. For concentration experiments, seeds imbibed for 36 h were used for the measurements. Error bars show ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 4.

Changes in germination rate and ROS content in imbibing seeds under different treatments. (a) Effects of the ROS scavengers, DPI and Tiron, on seed germination. (b) Changes of H₂O₂ and O₂⁻ contents in imbibing seeds treated with DPI and Tiron. (c) H₂O₂ fluorescent images of seeds imbibed for 24 h under different treatments. The seeds which were cut straight across were loaded with H₂DCF-DA for 30 min and then cut into very thin sections. The embryo-containing section was detected by confocal laser scanning microscopy. (d) Localization of O₂⁻ by NBT staining. The seeds which were cut straight across were stained by NBT solution for 5 min and viewed by light microscopy. Error bars show ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 5.

Effects of ABA on SOD activity (a), GR activity (b), CAT activity (c), and APx activity (d) during seed imbibition. Seed samples were collected at different times and stored at –80 °C for these antioxidant enzymes assays. Values are means ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 6.

Expression analyses of CAT and APx gene families. (a, b) Expression of the CAT gene family and the APx gene family during seed germination. (c, d) Changes in expression of OsCATA and OsAPx1 genes during seed germination under treatment with ABA and DPI. Seeds samples were collected at different times and stored at –80 °C for expression analysis by qRT-PCR. Error bars show ±SD (n=3). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 7.

Changes of expression of ASC biosynthesis genes and ASC contents in seeds under treatment with ABA, DPI, Tiron, and diniconazole. (a) ASC content during imbibition under ABA treatment. (b) Effect of ABA at different concentrations on ASC content and APx activity. (c, d) Changes in xpression of ASC biosynthesis gene in seeds treated with ABA and DPI for 36 h. (e) Change of ASC content in seeds treated with ABA, DPI, Tiron, and diniconazole for 36 h. Means are values ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 8.

Effects of ASC on germination of rice seeds in water and ABA. (a) Inhibition of seed germination by different concentrations of ASC. (b) Endogenous ASC level in seeds treated with different concentrations of ASC. (c) Effect of ASC on ABA during seed germination. Seeds were sown on filter paper with solutions of ABA at 5 μM plus different concentrations of ASC for imbibition. Seeds imbibed for 36 h were used for the ASC measurement. Error bars here show ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 9.

Effects of ABA and DPI treatments on expression of GA biosynthesis genes (a), amylase gene expression (c), and amylase activity (b). Seeds were sampled after imbibition for 36 h in water, 5 μM ABA, and 10 μM DPI solutions. Samples were stored at –80 °C for further experiments. Error bars here show ±SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.

Fig. 10.

Effect of lycorine at different concentrations on seed germination (a), ASC content (b), amylase activity (d), and amylase gene expression (e). Inhibition of expression of GA biosynthesis genes by ABA and lycorine (b). Seeds for ASC content, RNA extraction, and amylase activity were sampled after imbibition in lycorine solution for 36 h and stored at –80 °C for various experiments. Values are means ± SD (n=5). Means denoted by the same letter did not differ significantly at P < 0.05 according to Duncan’s multiple range test.