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. 2019 Mar 25;9:5112. doi: 10.1038/s41598-019-41382-z

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Lignin staining of anthers, NST1/NST2 expression, DAB staining in wild-type and 35S::ADR flowers and the phenotypic analysis of the H₂O₂-treated 35S::ADR flowers. (A–D) Anthers at stage 12 were stained with auramine O and observed by confocal microscopy (488 nm excitation/510–560 nm emission). Secondary thickening is visible in the endothecium (arrow) of the anthers of wild-type plants (A,B) but is absent in 35S::ADR plants (C,D). Red arrowheads indicate pollen grains. (B and D) are close-up images of (A and C), respectively. (E) Detection of NST1/NST2 expression in 35S::ADR transgenic Arabidopsis plants. mRNA accumulation of NST1/NST2 was determined by real-time quantitative PCR. Total RNA was isolated from flowers of one wild-type (WT) plant and one 35S::ADR plant and used as templates. The transcript levels of NST1/NST2 were determined using three replicates and normalized against UBQ10. The NST1/NST2 expression level in 35S::ADR plants is presented relative to wild-type plants, which was set at 1. Error bars represent the standard deviation. Each experiment was repeated twice with similar results. (F–I) At floral stage 11, stronger DAB staining was observed in anther tissue (arrow) of wild-type flowers (G,I) than in 35S::ADR flowers (F,H). (H) is a close-up image for 35S::ADR anther whereas (I) is a close-up image for wild-type anther. Pollen (po) was stained similarly in 35S::ADR and wild-type flowers. (J–L) At floral stage 13, stronger DAB staining was observed in the anther tissue (arrowed) of wild-type flowers (K,L) than in 35S::ADR flowers (J,L). (L) is the close-up images for 35S::ADR (left) and wild-type anthers (right). Pollen (po) was stained similarly in 35S::ADR and wild-type flowers. (M,N) Close-up images for 35S::ADR (M) and wild-type anther tissue (N) from (L). Stronger DAB staining was observed in the anther tissue (arrowed) of wild-type (N) than in 35S::ADR (M) flowers. (O) 7 days after 1 mM H₂O₂ treatment, the 35S::ADR flower developed a well-elongated silique (arrowed), whereas short siliques (s) developed without H₂O₂ treatment. (P) Close-up of a well-elongated silique (H₂O₂ treatment; left) and a short silique (right) without H₂O₂ treatment from (O). (Q) The 35S::ADR ovules developed into normal embryos (arrows) after 10 μM H₂O₂ treatment.