Proanthocyanidins in seed coat tegmen and endospermic cap inhibit seed germination in Sapium sebiferum

Abstract

Sapium sebiferum, an ornamental and bio-energetic plant, is propagated by seed. Its seed coat contains germination inhibitors and takes a long time to stratify for germination. In this study, we discovered that the S. sebiferum seed coat (especially the tegmen) and endospermic cap (ESC) contained high levels of proanthocyanidins (PAs). Seed coat and ESC removal induced seed germination, whereas exogenous application with seed coat extract (SCE) or PAs significantly inhibited this process, suggesting that PAs in the seed coat played a major role in regulating seed germination in S. sebiferum. We further investigated how SCE affected the expression of the seed-germination-related genes. The results showed that treatment with SCE upregulated the transcription level of the dormancy-related gene, gibberellins (GAs) suppressing genes, abscisic acid (ABA) biosynthesis and signalling genes. SCE decreased the transcript levels of ABA catabolic genes, GAs biosynthesis genes, reactive oxygen species genes and nitrates-signalling genes. Exogenous application of nordihydroguaiaretic acid, gibberellic acid, hydrogen peroxide and potassium nitrate recovered seed germination in seed-coat-extract supplemented medium. In this study, we highlighted the role of PAs, and their interactions with the other germination regulators, in the regulation of seed dormancy in S. sebiferum.

Keywords: ABA; Endospermic cap; GA; Proanthocyanidins; Sapium sebiferum; Seed dormancy; Tegmen.

Figure 1. Sulphuric acid (SA) scarification significantly promoted the seed germination of Sapium sebiferum.

(A), Effect of SA scarification time on seed coat; red arrows indicate the bruises, scars and cracks caused by SA. Bars 1 mm. (B), SA impacts on water uptake in the seed. (C), SA-induced seed germination of S. Sebiferum. (D and E), Impact of SA on shoot and root length of seedlings respectively. Shoot and root length were measured after 45 days of imbibition. Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Duncan’s multiple range HSD post hoc test. The photographs were taken by Shah Faheem Afzal and Jun Ni.

Figure 2. Impact of sulphuric acid scarification on PA contents of S. Sebiferum seed coat.

Seeds of S. Sebiferum were dipped in concentrated sulphuric acid for 10, 20, 30, 40, 50 and 60 min separately. PA contents of acid-scarified seeds were determined by vanillin assay. Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Tukey’s HSD post hoc test.

Figure 3. Impacts of exogenous application of SCE and PA on seed germination.

(A), PA contents in SCE, tegmen and testa of S. Sebiferum seed coat. (B), Impact of different concentrations of SCE and PAs on seed germination. Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Tukey’s HSD post hoc test.

Figure 4. PAs in the endospermic cap affected the seed germination.

(A), Accumulation of PAs in endospermic cap of dormant seed. (B and C), Decaping of endospermic cap significantly promoted seed germination as compared to control (with endospermic cap). (D), Dynamic changes of PAs in the endospermic cap of non-dormant seed. Bars in (A) and (D) 2 mm, (B) 1 cm. The photographs were taken by Shah Faheem Afzal.

Figure 5. Effect of SCE on the expression of seed dormancy-related gene (SsDOG1) and ABA-related genes.

(A), SsDOG1 (B), SsNCED6 (C), SsCYP707A2 and (D), SsABI3. The expression of SsDOG1, SsNCED6, SsCYP707A2 and SsABI3 were determined by qRT-PCR on third and sixth day after treatment. SsACTIN was used as the reference gene. Control, seed grown in half MS medium. PAs, proanthocyanidins-supplemented half MS medium. SCE, half MS medium supplemented with seed-coat extract. Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Tukey’s HSD post hoc test.

Figure 6. Effect of SCE on the expression of GA-related genes.

(A), SsGA3OX1 (B), SsGAOX (C), SsRGL2 and (D), SsGAI. The expression of SsGA3OX1, SsGAOX, SsRGL2 and SsGAI was determined by qRT-PCR on third and sixth days after treatment. SsACTIN was used as the reference gene. Control, seed grown in half MS medium. PAs, proanthocyanidins-supplemented (0.1%) half MS medium. SCE, half MS medium supplemented with seed-coat extract (0.3%). Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Tukey’s HSD post hoc test.

Figure 7. Impact of SCE on expression levels of ROS- and nitrates-signalling genes.

(A), SsMPK6 (B), SsNLP8 and (C), SsCIPK23. The expression of SsMPK6, SsNLP8 and SsCIPK23 were determined by qRT-PCR on the third and sixth days after treatment. SsACTIN was used as the reference gene. Control, seeds grown in half MS medium. PAs, proanthocyanidins-supplemented half MS medium. SCE, seeds cultivated on half MS medium supplemented with seed-coat extract. Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Tukey’s HSD post hoc test.

Figure 8. Inhibitory effects of SCE on seed germination was alleviated by GA₃, NDGA, H₂O₂ and KNO₃.

Seeds were primed in double distilled water (Control), 50 μΜ GA₃, 50 μM NDGA, 20 mM H₂O₂ and 0.4% KNO₃ and were sowed in 0.3% SCE-supplemented half MS medium. The seed germination rate was recorded seven days after imbibition. Data shown are means ± SD (n = 3). Means with different letters are significantly different at P < 0.05 using Tukey’s HSD post hoc test.