DOI: 10.5061/dryad.44j0zpcn6
title: Data from: Does stomatal patterning in amphistomatous leaves minimize the CO2 diffusion path length within leaves?

description: Photosynthesis is co-limited by multiple factors depending on the plant
 and its environment. These include biochemical rate limitations, internal
 and external water potentials, temperature, irradiance, and carbon dioxide
 (CO2). Amphistomatous leaves have stomata on both abaxial and adaxial leaf
 surfaces. This feature is considered an adaptation to alleviate CO2
 diffusion limitations in productive environments as the diffusion path
 length from stomate to chloroplast is effectively halved in amphistomatous
 leaves. Plants may also reduce CO2 limitations through other aspects of
 optimal stomatal anatomy: stomatal density, distribution, patterning, and
 size. A number of studies have demonstrated that stomata are overdispersed
 compared to a random distribution on a single leaf surface; however,
 despite their prevelance in nature and near ubiquity among crop species,
 much less is known about stomatal anatomy in amphistomatous leaves,
 especially the coordination between leaf surfaces. Here we use novel
 spatial statistics based on simulations and photosynthesis modeling to
 test hypotheses about how amphistomatous plants may optimize CO2 diffusion
 in the model angiosperm Arabidopsis thaliana grown in different light
 environments. We find that 1) stomata are overdispersed, but not ideally
 dispersed, on both leaf surfaces across all light treatments; 2) the
 patterning of stomata on abaxial and adaxial leaf surfaces is independent;
 and 3) the theoretical improvements to photosynthesis from abaxial-adaxial
 stomatal coordination are miniscule (≪ 1%) across the range of feasible
 parameter space. However, we also find that 4) stomatal size is correlated
 with the mesophyll volume that it supplies with CO2, suggesting that
 plants may optimize CO2 diffusion limitations through alternative pathways
 other than ideal, uniform stomatal spacing. We discuss the developmental,
 physical, and evolutionary constraits which may prohibit plants from
 reaching this theoretical adaptive peak of uniform stomatal spacing and
 inter-surface stomatal coordination. These findings contribute to our
 understanding of variation in the anatomy of amphistomatous leaves.
description: Plants from the Columbia (Col-0) ecotype of <em>Arabidopsis
 thaliana</em> (L.) Heynh. were grown in three different light
 environments: low light (PAR = 50 μmol m<sup>-2 
 </sup>s<sup>-1</sup>), medium light (100 μmol
 m<sup>-2  </sup>s<sup>-1</sup>), and high light
 (200 μmol m<sup>-2  </sup>s<sup>-1</sup>). PAR
 stands for photosynthetically active radiation. Seeds were
 surface-sterilized and stratified at 4 °C for 3–5 d in 0.15% agarose
 solution and then sown directly into Pro-Mix HP soil (Premier
 Horticulture; Quakerstown, PA, USA) and supplemented with Scott's
 Osmocote Classic 14-14-14 fertilizer (Scotts-Sierra, Marysville, OH, USA).
 At 10–14 d, seedlings were thinned so only one seedling per container
 remained. Plants were grown to maturity in growth chambers where the
 conditions were as follows: 16 : 8 h, 22 : 20°C, day : night cycle.
 Imaging of the epidermis and internal leaf structures was performed using
 a Leica SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany)
 with the protocol developed by Wuyts et al. (2010) with additional
 modification described in Dow et al. (2017). We captured 132 images in
 total, making 66 abaxial-adaxial image pairs. Images were square with an
 area of 0.386 mm<sup>2</sup>. We measured stomatal position
 and length using ImageJ (Schneider et al. 2012). 
 References 
 G. J.
 Dow, J. A. Berry, and D. C. Bergmann. Disruption of stomatal lineage
 signaling or transcriptional regulators has differential effects on
 mesophyll development, but maintains coordination of gas exchange.
 <em>New Phytologist</em>, 216(1):69–75, Oct. 2017. ISSN
 0028-646X, 1469-8137. doi: 10.1111/nph.14746. URL https://nph.onlinelibrary.wiley.com/doi/10.1111/nph.14746. C. A. Schneider, W. S. Rasband, and K. W. Eliceiri. NIH Image to ImageJ: 25 years of image analysis. <em>Nature Methods</em>, 9(7):671–675, July 2012. ISSN 1548-7091, 1548-7105. doi: 10.1038/nmeth.2089. URL http://www.nature.com/articles/nmeth.2089. N. Wuyts, J.-C. Palauqui, G. Conejero, J.-L. Verdeil, C. Granier, and C. Massonnet. High-contrast three-dimensional imaging of the <em>Arabidopsis</em> leaf enables the analysis of cell dimensions in the epidermis and mesophyll. <em>Plant Methods</em>, 6(1):17, Dec. 2010. ISSN 1746-4811. doi: 10.1186/1746-4811-6-17. URL https://plantmethods.biomedcentral.com/articles/10.1186/1746-4811-6-17.
description: # Data from: Does stomatal patterning in amphistomatous leaves minimize
 the CO2 diffusion path length within leaves?
 [https://doi.org/10.5061/dryad.44j0zpcn6](https://doi.org/10.5061/dryad.44j0zpcn6) The dataset contains the x and y coordinates (in pixels), angle, and length (in pixels) of each stomate on each leaf surface from 66 *Arabidopsis thaliana* (Col-0 ecotype) plants grown under low, medium, and high light treatments. ## Description of the data and file structure | Variable | Type | Description | | :-------- | :-------- | :------------------------------------- | | `light` | character | light treatment (low, medium, high) | | `surface` | character | leaf surface (abaxial or adaxial) | | `leaf_id` | character | integer leaf id within light treatment | | `x` | numeric | x position of stomate in pixels | | `y` | numeric | y position of stomate in pixels | | `length` | numeric | guard cell length in pixels | Each pixel is 0.824 um. ## Sharing/Access information The raw data and code to analyze it are also publicly accessible from these locations: * [https://github.com/cdmuir/stomata-spacing](https://github.com/cdmuir/stomata-spacing) * [https://doi.org/10.5281/zenodo.10775962](https://doi.org/10.5281/zenodo.10775962)