DOI: 10.5061/dryad.p5hqbzkz3
title: Data from: Salicaceae endophyte inoculation alters stomatal patterning and improves the intrinsic water-use efficiency of Populus trichocarpa after a water-deficit

description: Microorganisms may enhance plant resilience to water stress by influencing
 their hosts’ physiology and anatomy at the leaf-level. Bacterial and yeast
 endophytes, isolated from wild poplar and willow, can improve the
 intrinsic water-use efficiency (iWUE) of cultivated poplar (Populus) under
 water-deficits by lowering stomatal conductance (gsw). However, the
 relevance of stomatal anatomy underlying this reduction remains unclear.
 We hypothesized endophyte inoculation could change host stomatal anatomy,
 and this would relate to decreases in gsw. We subjected Salicaceae
 endophyte-inoculated and uninoculated Populus trichocarpa to well-watered
 and water-deficit treatments in greenhouse studies. We examined the
 changes of individual stomatal traits and related the composition of these
 parameters, termed stomatal patterning, to leaf gas-exchange under light
 saturation. After a water-deficit, inoculation improved iWUE at light
 saturation from preserving carbon assimilation (Anet) and lowering gsw,
 but these changes were independent of soil-moisture status. Drops in gsw
 corresponded to underlying shifts in stomatal patterning. Inoculated
 plants had smaller, more compact stomata and greater anatomical maximum
 stomatal conductance (gsmax) relative to the control. Salicaceae
 endophytes may alter stomatal density and size, lowering gsw and
 increasing iWUE. Future efforts may quantify endophyte colonization of the
 host to draw direct relationships between microbes and stomatal traits.
description: # Data from: Salicaceae endophyte inoculation alters stomatal patterning
 and improves the intrinsic water-use efficiency of Populus trichocarpa
 after a water-deficit
 [https://doi.org/10.5061/dryad.p5hqbzkz3](https://doi.org/10.5061/dryad.p5hqbzkz3) ## Description of the data and file structure Microorganisms may enhance plant resilience to water stress by influencing their hosts’ physiology and anatomy at the leaf-level. Bacterial and yeast endophytes, isolated from wild poplar and willow, can improve the intrinsic water-use efficiency (iWUE) of cultivated poplar (Populus) under water-deficits by lowering stomatal conductance (gsw). However, the relevance of stomatal anatomy underlying this reduction remains unclear. We hypothesized endophyte inoculation could change host stomatal anatomy, and this would relate to decreases in gsw. We subjected Salicaceae endophyte-inoculated and uninoculated Populus trichocarpa to well-watered and water-deficit treatments in greenhouse studies. We examined the changes of individual stomatal traits and related the composition of these parameters, termed stomatal patterning, to leaf gas-exchange under light saturation. To collect this data, we examined stomatal morphology and leaf gas-exchange prior to the water-deficit and at three separate timepoints after initiating the abiotic stress. We made leaf impressions at every timepoint and imaged each under a microscope. We collected data from the photos using both automated image recognition models (i.e., convolutional neural networks) and manual measurements in ImageJ. We gathered the leaf gas-exchange variables using LI6400s. See the Material & Methods section in the corresponding manuscript for a more detailed description for data collection and analysis. ### Files and variables #### File: epgh2204\_zone3\_rh\_temp\_april\_december\_2022.csv **Description:** Hourly relative humidity and temperature data from April - December within the greenhouse where the experiment took place. Contains maximum, minumums, and averages. The experiment took place between 05/31/2022 to 08/20/2022. 5881 rows x 7 columns ##### Variables * Date and Time: date (month/day/year) and time (12 hr interval) * Air Temperature - Zone 3-Zone 3 Temp&RH (Avg) (: average temperature (Fahrenheit) * Air Temperature - Zone 3-Zone 3 Temp&RH (Min) (: Minumum temperature (Fahrenheit) * Air Temperature - Zone 3-Zone 3 Temp&RH (Max) (: Maximum temperature (Fahrenheit) * Humidity - Zone 3-Zone 3 Temp&RH (Avg) (% RH): Average relative humidity (%) * Humidity - Zone 3-Zone 3 Temp&RH (Min) (% RH): Minimum relative humidity (%) * Humidity - Zone 3-Zone 3 Temp&RH (Max) (% RH): Maximum relative humidity (%) #### File: epgh2204\_irrigation\_calibration.csv **Description:** Dataset (11 rows x 8 columns) of ten pots used to generate a calibration curve that estimated pot water-holding capacity for the experimental pots/plants ##### Variables * test_id: A unique identifier for each calibration pot * empty_pot_weight_g: Mass of the empty pot (grams) * air_dry_g: Mass of the potting media at room temperature prior to oven-drying or water saturation (grams) * WHC_100: 100% water-holding capacity assumed from the Mass of the potting media after saturating it with water. Media that is at 100% its water-holding capacity is the threshold that it can physically hold water against the force of gravity. Pots were covered with plastic wrap to limit the amount of water lost to evaporation. (grams) * WHC_0: 0 % water-holding capacity assumed from the mass of the potting media after drying it in a forced air oven at 70 degrees Celsius. Pots were reweighed periodically until the mass reached a consistent value. (grams) * est_80: Media mass at 80% water-holding capacity. This value was estimated from generating a calibration curve from the measured 0% and 100% water-holding capacity. (grams) * est_35: Media mass at 35% water-holding capacity. This value was estimated from generating a calibration curve from the measured 0% and 100% water-holding capacity. (grams) * est_WHC_airdry: Estimated water-holding capacity of the airdry media. This was generated from using point-slope equations. (%) #### File: epgh2204\_summarized\_stomata\_morph\_data.csv **Description:** a data file (127 rows x 23 columns) containing all the leaf stomatal traits collected for each plant in the experiment.  Included in this are the two treatment factors, blocking level, sampling time point, and stomata features.  ##### Variables * Pot_ID: unique identifier for the experimental unit (i.e., plant) * block: blocking factor for the experimental design, ordinal 1-9 * treatment: treatment (i.e., interaction) factor that includes both the inoculation and abiotic factor separated by an underscore; categorical; inoculated_WW is an inoculated, well-watered (80% water-holding capacity) plant; inoculated_WD is an inoculated, water-deficit (35% water-holding capacity) plant; control_WW is an uninoculated, well-watered (80% water-holding capacity) plant; control_WD is an uninoculated, water-deficit (35% water-holding capacity) plant. * endo: inoculation factor; categorical; inoculated represents and inoculated plant, control is an uninoculated plant * abiotic: abiotic factor; categorical; WW represents a well-watered plant, WD is a plant from the water-deficit group * Timepoint: time point of sample collection; ordinal; t1 represents the baseline measurement before starting the water-deficit treatment; t2, t3, t4 are subsequent sampling dates after starting the water-deficit * Bottom_gcl: average guard cell length (major axis) of 10 individually measured abaxial stomata; um * Bottom_pl: average pore length (major axis) of 10 individually measured abaxial stomata; um * Top_gcl: average guard cell length (major axis) of 10 individually measured adaxial stomata; um * Top_pl: average pore length (major axis) of 10 individually measured adaxial stomata; um * Bottom_Density: average abaxial stomatal density from 4 images each of size 1.104 mm2; mm-2 * Top_Density: average adaxial stomatal density from 4 images of size 1.104 mm2; mm-2 * Ratio: ratio between the adaxial and abaxial stomata (i.e., top/bottom); unitless * Bottom_Index: ratio of the number of abaxial stomata to the total number of abaxial cells. It is a parameter that normalizes the number of stomata from epidermal cell expansion, which depends on environmental conditions. Stomatal Index = Stomata/((Stomata+Pavement Cells))  ∗100; unitless * Top_Index: ratio of the number of adaxial stomata to the total number of adaxial cells. It is a parameter that normalizes the number of stomata from epidermal cell expansion, which depends on environmental conditions. Stomatal Index = Stomata/((Stomata+Pavement Cells))  ∗100; unitless * Bottom_gcw: NOT USED; average guard cell width (minor axis) of abaxial stomata taken from CNN model; um * Top_gcw: NOT USED; average guard cell width (minor axis) of adaxial stomata taken from CNN model; um * Bottom_pore_area: NOT USED; estimated average abaxial pore area taken from CNN model; um^2 * Top_pore_area: NOT USED; estimated average adaxial pore area taken from CNN model; um^2 * Bottom_pw: average pore width (minor axis) of abaxial stomata taken from CNN model; um * Top_pw: average pore width (minor axis) of adaxial stomata taken from CNN model; um * Top_Gsmax: anatomical gsmax of the adaxial leaf surface, see equation below for its calculation; mol m-2 s-1 * Bottom_Gsmax: anatomical gsmax of the abaxial leaf surface, see equation below for its calculation; mol m-2 s-1  Anatomical gsmax = (SD x Ap x D/V)/((dp) + (pi/4) + sqrt(Ap/pi)) where SD is the average stomatal density *D* is the diffusivity of water in air (2.82 × 10−5 m2 s−1 at 25°C) and *V* is the molar volume of air (m3 mol−1, at 25°C and 101.3 kPa). Pore depth (dp; m) was equal to guard cell width, represented as a quarter of the guard cell length (dp = gcl/4). The mean maximum stomatal pore area (Ap, m2) was calculated assuming stomatal pores were circular, with the diameter equal to pore length (Ap = (pl/2)2) See Franks & Beerling (2009) and Wall et al. (2022) for further information about the geometric assumptions used in this calculation. #### File: epgh2204\_irrigation\_records.csv **Description:** dataset (1189 rows x 10 columns) containing the mass of the experimental units (pot + media + plant) weighed every 2-3 days before and after fertigation; these values were used to estimate water-holding capacity for each individual experimental unit. "Null" values under the "fertigation_supplied_ml" and the "net_wt_after_g" indicate that no fertigation was supplied at the corresponding dates. ##### Variables * date: date (month/day/year) * pot_id: unique identifier for the experimental unit * block: blocking factor for the experimental design, ordinal 1-9 * endo: inoculation factor; categorical; inoculated represents and inoculated plant, control is an uninoculated plant * abiotic : abiotic factor; categorical; WW represents a well-watered plant, WD is a plant from the water-deficit group * gross_wt_g: mass of experimental unit ( ;grams * net_wt_before_g: mass of media/plant prior to fertigation; grams * fertigation_supplied_ml: amount of fertigation added to the experimental unit; ml * net_wt_after_g: mass of media/plant after fertigation; grams * air_dry_wt_g: mass of the airdry media for the experimental unit; grams #### File: epgh2204\_summarized\_spot\_gas\_exchange\_data.csv **Description:** dataset (94 rows x 111 columns) containing the leaf gas-exchange data collected at light saturation (2000 μmolphotons m-2 s-1) for each plant in the experiment.  Included in this are the two treatment factors, blocking level, sampling time point, and physiological features collected.  ##### Variables * Pot_ID: unique identifier for the experimental unit * block: blocking factor for the experimental design, ordinal 1-9 * endo: inoculation factor; categorical; inoculated represents and inoculated plant, control is an uninoculated plant * abiotic: abiotic factor; categorical; WW represents a well-watered plant, WD is a plant from the water-deficit group * Timepoint: time point of sample collection; ordinal; t1 represents the baseline measurement before starting the water-deficit treatment; t2, t3, t4 are subsequent sampling dates after starting the water-deficit * Obs: log number * HHMMSS: time of the internal * FTime: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * EBal.: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * A: Assimilation rate; μmol m-2 s-1 * gs: stomatal conductance to water vapor; mol m-2 s-1 * Ci: Intercellular CO2; µmol mol-1 * FCnt: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * DCnt: NOT USED; 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see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * SVTleaf: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * h2o_i: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * h20diff: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * CTair: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * SVTair: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * CndTotal: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * vp_kPa: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * VpdA: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * CndCO2: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * Ci_Pa: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * Ci.Ca: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * RHsfc: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * C2sfc: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * AHs.Cs: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * Fv: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * PARabs: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * Fv.: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * qP_Fo: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * qN_Fo: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * CsMchSD: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * HsMchSD: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * CrMchSD: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * HrMchSD: NOT USED; see LICOR website for summary of symbols: [https://www.licor.com/env/support/LI-6800/topics/symbols.html](https://www.licor.com/env/support/LI-6800/topics/symbols.html) * iWUE: intrinsic water-use efficiency; ratio of carbon assimilation (A) over stomatal conductance to water (gsw); A/gsw; μmol mol-1 * WUE: extrinsic water-use efficiency; ratio of carbon assimilation (A) over leaf transpiration; A/E; μmol mol-1 #### File: Daily\_Data\_AgWeatherNet\_at\_Washington\_State\_University.csv **Description:** Daily weather data outside of the greenhouse from March - December where the experiment took place. Contains maximum, minumums, and averages. The experiment took place between 05/31/2022 to 08/20/2022. 277 rows x 16 columns ##### Variables * Date: date (month/day/year) * Date: day * MinF: NOT USED; Minimum air temperature in °F * AvgF: NOT USED; Average air temperature over the 24 h in °F * MaxF: NOT USED; Maximum air temperature in °F * Avg1.5m DPF: NOT USED; Average dewpoint temperature in °F at 1.5 m height * Avg1.5m RH%: NOT USED; Average relative humidity at 1.5 m height * 2 in.F: NOT USED; Soil temperature at two inches depth in °F * MinF: NOT USED; Minimum soil temperature at eight inches depth in °F * AvgF: NOT USED; Average soil temperature at eight inches depth in °F * Avg 2in.SWPkPa: NOT USED; Average stem water potential at two inches depth * Avg 8in.SWPkPa: NOT USED; Average stem water potential at eight inches depth * TotPrecin: NOT USED; total precipitation; inches * TotalSolarRadMJ/m: Total solar radiation in a day; MJ/m^2 * EToin: NOT USED; ASCE Grass reference evapotranspiration * ETrin: NOT USED; ASCE Alfalfa reference evapotranspiration ## Code/software All analyses were performed in R v4.2.2 (R Core Team, 2023), using tidyverse functions (Wickham et al., 2019). PERMANOVA was run with the adonis2 function from vegan v2.6-4; univariate parametric and permutational ANOVAs were performed with the aovperm function from permuco v1.1.2; PCA biplots were created using ggbiplot v0.55; correlograms were generated with the ggcorrplot v0.1.4 package; model selection and exploration was done with MuMIn v1.47.1, lmerTest v3.1-3, and lme4 v1.1-33; confidence intervals and bootstraps were generated with boot v1.3-28; nonlinear least squares fitting was performed using nls.multstart v1.2.0; violin plots and response curves were created using cowplot v1.1.1 and ggh4x v0.2.4.Inkscape v1.2.0, a vector-based imaging editor, was used to increase text size after exporting figures from R.