DOI: 10.5061/dryad.dv41ns265
title: Comparative histology of abscission zones reveals the extent of convergence and divergence in seed shattering in weedy and cultivated rice

description: The modification of seed shattering has been a recurring theme in rice
 evolution. The wild ancestor of cultivated rice disperses its seeds, but
 reduced shattering was selected during multiple domestication events to
 facilitate harvesting. Conversely, selection for increased shattering
 occurred during the evolution of weedy rice, a weed invading cultivated
 rice fields that has originated multiple times from domesticated
 ancestors. Shattering requires formation of a tissue known as the
 abscission zone (AZ), but how the AZ has been modified throughout rice
 evolution is unclear. We quantitatively characterized the AZ
 characteristics of relative length, discontinuity, and intensity in 86
 cultivated and weedy rice accessions. We reconstructed AZ evolutionary
 trajectories and determined the degree of convergence among different
 cultivated varieties and among independent weedy rice populations. AZ
 relative length emerged as the feature best distinguishing high and low
 shattering rice. Cultivated varieties differed in average AZ morphology,
 revealing lack of convergence in how shattering reduction was achieved
 during domestication. In contrast, weedy rice populations typically
 converged on complete AZs, irrespective of origin. By examining AZ
 population-level morphology, our study reveals its evolutionary
 plasticity, and suggests that the genetic potential to modify the
 ecologically and agronomically important trait of shattering is plentiful
 in rice lineages.
description: Rice florets were fixed in an FAA solution (37% formaldehyde:
 ethanol: water: acetic acid=10:50:35:5), and dehydrated in a graduated
 ethanol series (50%, 70%, 85%, 95%, and 2 changes of 100% ethanol),
 followed by clearing with an ethanol HistoClear solution (25%, 50%, 75%,
 and 3 changes of 100% Histoclear). The HistoClear was drained and
 paraplast was added and changed every 6 hours six times at 60℃. Florets
 were embedded in tissue cassettes with paraplast, serially sectioned into
 10 𝜇m longitudinal slices with a Leica RM2125 microtome, mounted on
 slides, and placed on a slide warmer at 42℃ for at least 24 hours. The
 slides were stained with 1% toluidine blue solution for 30 minutes. Excess
 paraplast was cleared with two xylene washes. A glass cover slip was
 affixed to the slide with Permount. A Leica DM750 LED biological
 microscope with a 4X or 10x objective was used to image florets. Images
 were captured with an AmScope MU1000-HS camera and AmLite
 software.   Images with a clear intact vascular bundle
 and symmetric flanking AZ were selected for quantification. RGB color
 images were first adjusted to 8-bit grayscale in ImageJ. Under grayscale,
 darkly stained regions are converted to pixels with low gray values while
 lightly stained regions are converted to pixels with high gray values. The
 AZ was identified as the darkly stained cells that span from the edge of
 the floret toward the vascular bundle. For each image, a square region was
 selected in the non-AZ region near the pedicel side and its minimum gray
 value was set as a threshold. Line scans were drawn, with the plot profile
 tool in ImageJ, from the edge of the floret to the vascular bundle
 following the natural curve of the AZ. Pixels along the line scan with
 gray values greater than the threshold were classified as belonging to
 non-AZ tissue and pixels with lower values were classified as AZ tissue.
 The resulting profiles were used to produce three measures: relative
 length, discontinuity, and intensity. AZ relative length was calculated by
 dividing the number of pixels below the threshold by the total number of
 pixels in the line scan. AZ discontinuity was calculated by summing the
 number of AZ pixels clusters, where these refer to a series of contiguous
 pixels below the threshold bounded by non-AZ tissue on one or both sides.
 AZ intensity was calculated by dividing the threshold value by the mean
 grey value of the AZ tissue, which provided a scaled measure of the
 staining contrast between AZ and non-AZ tissues. The three measures were
 performed on the left and right side of each section and averaged for
 subsequent analyses.
description: # Comparative histology of abscission zones reveals the extent of
 convergence and divergence in seed shattering in weedy and cultivated rice
 [https://doi.org/10.5061/dryad.dv41ns265](https://doi.org/10.5061/dryad.dv41ns265) **Background introduction**  Seed shattering requires the formation of one or more specialized cell layers, collectively called an abscission zone (AZ), where an organ separates from the plant. The cells that constitute the AZ are often distinct from surrounding cells, and are generally small in size, have dense cytoplasm, lack secondary cell wall, and have highly branched plasmodesmata. AZ location and anatomy vary among grass species. In rice, the abscission zone is formed in the rachilla, between the pedicel and floret, and consists of cells that lack lignin.  Here, we take a population-level approach to characterize AZ morphology in different cultivated and weedy rice groups. We develop measures to quantify aspects of the AZ and use this comparative histology to understand the morphological basis for changes in seed shattering during cultivated and weedy rice evolution. **Description of the data and file structure** We produced toluidine blue-stained, longitudinally sectioned images of rice florets for each rice accession. The AZ was identified as the darkly stained cells that span from the edge of the floret toward the vascular bundle. To quantitatively characterize AZ, RGB color images were adjusted to 8-bit grayscale in ImageJ (Schneider et al., 2012). Under grayscale, darkly stained regions are converted to pixels with low gray values while lightly stained regions are converted to pixels with high gray values. For each image, a square region was selected in the non-AZ region near the pedicel side and its minimum gray value was set as a threshold. Line scans were drawn, with the plot profile tool in ImageJ, from the edge of the floret to the vascular bundle following the natural curve of the AZ. Both left and right sides of the AZ were characterized separately. Pixels along the line scan with gray values greater than the threshold were classified as belonging to non-AZ tissue and pixels with lower values were classified as AZ tissue. As a result, we have uploaded the following files for each rice accession (one folder for each accession): 1. One original image displaying the AZ highlighted in dark blue color. The file is named with the accession name (.jpg).  2. One processed image indicating the threshold of gray value used to determine the AZ. The file is named with the accession name_threshold (.jpeg). One purple square was selected in the non-AZ area and the value of min was chosen as the threshold for each AZ image.  3. Two processed images featuring drawn line scans for each side of the AZ. The files are named with the accession name left/right_line_scan (.jpeg). One purple line scan was drawn along the AZ of left or right side.  4. Two Excel files containing the gray values along the two line scans obtained in step 2. The files are named with accession name left/right (.csv) Additionally, we uploaded an excel file (accessions.xlsx) with the description of all the accessions measured in this study. This file includes the information of accession name (corresponding to the folder names for each accession), seed source ID, oryza type, crop/weed, country.  **Reference** **Schneider CA**, **Rasband WS**, **Eliceiri KW**. **2012**. NIH Image to ImageJ: 25 years of image analysis. *Nature methods* **9**: 671–675.