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Summary Abscission is predetermined in specialized cell layers called the abscission zone (AZ) and activated by developmental or environmental signals. In the grass family, most identified AZ genes regulate AZ anatomy, which differs among lineages. A YABBY transcription factor,SHATTERING1(SH1), is a domestication gene regulating abscission in multiple cereals, including rice andSetaria. In rice,SH1inhibits lignification specifically in the AZ. However, the AZ ofSetariais nonlignified throughout, raising the question of howSH1functions in species without lignification.Crispr‐Cas9 knockout mutants ofSH1were generated inSetaria viridisand characterized with histology, cell wall and auxin immunofluorescence, transmission electron microscopy, hormonal treatment and RNA‐Seq analysis.Thesh1mutant lacks shattering, as expected. No differences in cell anatomy or cell wall components including lignin were observed betweensh1and the wild‐type (WT) until abscission occurs. Chloroplasts degenerated in the AZ of WT before abscission, but degeneration was suppressed by auxin treatment. Auxin distribution and expression of auxin‐related genes differed between WT andsh1, with the signal of an antibody to auxin detected in thesh1chloroplast.SH1inSetariais required for activation of abscission through auxin signaling, which is not reported in other grass species.
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This content will become publicly available on January 1, 2026
Histological Staining and Hydrogen Peroxide Visualization of the Abscission Zone in Setaria viridis
The abscission zone (AZ) consists of specialized cell layers where cell separation or breakage occurs that result in organ detachment. Microscopic observation of the AZ is crucial for understanding its function. The AZ undergoes cellular and physiological changes prior to abscission, such as cell death, loss of chlorophyll, and the production of reactive oxygen species (ROS). These changes can be visualized using specific dyes and indicators under light or fluorescent microscopes. However, one challenge of using these dyes is their inefficient penetration into the tissue, especially when the epidermal layer has thick secondary cell walls. In this chapter, a detailed protocol to overcome this challenge is described. Using the fruit AZ of Setaria viridis, in which the epidermal cell wall is thick and lignified, we gently fix the dissected tissue, embed it in the Cryomatrix, and trim off the outer cell layers using a cryostat. The tissue with exposed inner cells can then be stained with fluorescent dyes to visualize organelles of interest, or 3,3′-diaminobenzidine (DAB) to visualize hydrogen peroxide accumulated in the tissue.
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- Award ID(s):
- 1938086
- PAR ID:
- 10634524
- Editor(s):
- Tranbarger, Timothy J
- Publisher / Repository:
- Springer US
- Date Published:
- ISSN:
- 1940-6029
- Page Range / eLocation ID:
- 61 to 71
- Subject(s) / Keyword(s):
- Abscission Zone (AZ), Chlorophyll quantification, DAPI staining, Calcofluor white, 3,3′-Diaminobenzidine (DAB), Reactive oxygen species (ROS), Hydrogen peroxide, Cryosection, Setaria Viridis
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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