Excessive leaf oil modulates the plant abiotic stress response via reduced stomatal aperture in tobacco (Nicotiana tabacum).

axial side of the leaf rather than a cross section. While small lipid droplets were present in the WT stomatal guard cells and epidermis, large lipid droplets were present in the HLP guard cells under both control and after 7 days of treatment (representative control images in Figure 8A–D , complete dataset available on Zenodo, https://zenodo.org/records/10711864 ). In addition, while HLP oil appeared to form spherical droplets, it did not “line” the stomatal opening as in WT (Figure 8C,D ). Figure 8 High lipid producing (HLP) had excessive oil droplets in stomatal guard cells. Representative confocal microscopy images, shown as focused Z‐stack, of tobacco leaf tissue fixed in paraforma

mated marginal means (LSMEANS) to determine which sample types were significantly different from others. Means are reported in text with standard error. Plots were made using ggplot2 package (v.3.5.0) in R. Jupyter notebooks associated with PlantCV analyses and R scripts associated with this manuscript are available on Github ( https://github.com/danforthcenter/tobacco‐heat‐paper ). AUTHOR CONTRIBUTIONS DKA, MAG, PDB, BSJ and KMM designed experiments. KMM and BSJ performed experiments and data analysis. KJC designed and aided KMM in confocal and brightfield microscopy experiments and advised TEM experiments. JW performed TEM experiments, and KG‐O and SK performed data analysis of TEM images.

tification was used to isolate only individual plants in each mask. Then, the mask was applied to the registered thermal image to calculate the average plant temperature, as well as a histogram of pixel temperatures for each plant. A PlantCV workflow was used to analyze the images in parallel. A tutorial is available on GitHub: https://github.com/danforthcenter/plantcv‐tutorial‐thermal?tab=readme‐ov‐file (Acosta‐Gamboa et al., 2024 ). Scripts for this project are available at https://github.com/danforthcenter/tobacco‐heat‐paper . Raw image data are available on Zenodo, https://zenodo.org/records/10711864 . Stomatal aperture measurements To measure stomatal number and aperture, leaf impressio

package was then used to calculate the number of stomata and the area of the aperture. A limitation of this method is that it does not provide the width and length of stomata, or measurements of the guard cells themselves; instead, it provides the aperture area (a result of length and width). A tutorial is available on GitHub: https://github.com/danforthcenter/plantcv‐stomata‐tutorial‐pcv4 (Murphy, 2024 ). Scripts for this project are available at https://github.com/danforthcenter/tobacco‐heat‐paper . Raw image data are available on Zenodo, https://zenodo.org/records/10711864 . Photosynthesis and gas exchange

PlantCV (Gehan et al., 2017 ) using the photosynthesis package; the chlorophyll fluorescence image was used to mask the image for only plant pixels, and average F v / F m , F q ′ / F m ′ , NPQ, chlorophyll index, and anthocyanin index were calculated as an average per plant at each timepoint. A tutorial is available on GitHub: https://github.com/danforthcenter/plantcv‐tutorial‐photosynthesis?tab=readme‐ov‐file (Schuhl et al., 2024 ). Scripts for this project are available at https://github.com/danforthcenter/tobacco‐heat‐paper . Raw image data are available on Zenodo, https://zenodo.org/records/10711864 . Microscopy imaging of lipids Leaf samples analyzed for lipid content were taken from th