Combination of maleic hydrazide and coumarin inhibits rice seed germination involving reactive oxygen species accumulation, ABA metabolism and starch degradation

Abstract

Pre-harvest sprouting (PHS) in cereal crops is a prevalent phenomenon that impacts grain yield and quality. Several PHS inhibitory compounds were screened and identified in previous studies, such as eugenol (EUG), maleic hydrazide (MH), coumarin (COU), etc. However, few studies have focused on the combination of PHS inhibitors, and the inhibitory mechanism remains unclear. Here, through combination tests of EUG, MH, and COU, the optimal combination of PHS inhibitors was selected as MH 20 mg L⁻¹ + COU 100 mg L⁻¹, which presented the lowest germination percentages. The optimal combination treatment significantly decreased the germination rate, α-amylase activity, content of soluble sugar and soluble protein, enhanced ABA content and the activity of superoxide dismutase (SOD) and peroxidase (POD), inhibited the production of superoxide anion (O₂⁻) and hydrogen peroxide, and reduced the content of malondialdehyde (MDA); conversely, this trend is precisely the opposite in normal germination. Furthermore, gene expression analysis revealed that the optimal combination of MH and COU significantly decreased the expression level of OsAmy1A and OsAmy3D at 12 and 48 h after imbibition (HAI); and promoted the expression of OsRbohs (OsRbohA, OsRbohC, OsRbohD, OsRbohE, OsRbohH) and ABA biosynthetic genes OsNCED1, OsNCED2, and OsNCED5, especially OsNCED2 at 12 HAI, but down-regulated expression of OsRbohs and ABA catabolic genes OsABA8ox1-3 at 48 HAI. These results demonstrated that the delay in seed germination induced by MH and COU involved in ROS, ABA, and sugars; the optimal combination of MH and COU inhibited the germination process by promoting ABA biosynthesis and reducing ABA catabolism, and restraining the α-amylase activity to lower soluble sugar content. Intriguingly, although the expression of OsRbohs, which play a crucial role in generating ROS, increased in early imbibition (12h), the activity of the antioxidant enzymes SOD and POD also increased with the optimal combination treatment of MH and COU, which lead to the delay in ROS accumulation and inhibition of germination. These results have deepened our understanding of the regulatory mechanism of PHS inhibitors and provided theoretical support for the application of MH and COU in preventing sprouting before crop harvesting.