DOI: 10.5061/dryad.4b8gthtpf
title: Compensatory photosynthesis in Pseudoroegneria spicata

description: Understanding plant ecophysiological functioning is critical in
 formulating effective ecologically-based strategies to conserve and
 enhance resiliency and resistance in sagebrush steppe, as well as
 improving their restoration following degradation by interactive effects
 of climate change, wildland fire, and invasive annual grasses. 
 Recent research has shown increased reproductive photosynthesis following
 floral defoliation can be important to reproductive potential, yet how
 this is expressed in plant material selected for different functional
 attributes is unknown.  To address this, we measured
 photosynthetic gas exchange in clipped and unclipped basal florets and
 flag leaves of two germplasms of the native perennial bunchgrass,
 bluebunch wheatgrass, var. Anatone and var. Columbia, selected for higher
 reproductive culm production.  Clipping induced a stronger direct
 compensatory reproductive photosynthetic response in basal florets of
 Anatone compared to Columbia germplasm individuals, with no indirect
 compensatory response apparent in unaffected distal florets of either
 germplasm.  Flag-leaf photosynthesis did not differ between the
 germplasm lines, but Columbia flag leaves did show evidence of increased
 photosynthesis on culms with clipped basal florets.  These
 findings suggest selection for increased flowering culms may alter
 reproductive herbivory tolerance, a feature important in the convergence
 of herbivory and drought tolerance traits.  Such information
 could help in planning effective seed mixes to enhance population
 stability across highly variable sagebrush steppe ecosystems, as well as
 directing future plant material selection to improve restoration success
 in these economically important rangelands.  
description: Methods below are provided from the associated manuscript,
 "Variation in reproductive photosynthetic compensation of distinct
 germplasm varieties of a native rangeland grass, <em>Pseudoroegneria
 spicata, </em>following floral defoliation"
 Field-based measurements were made on mature bluebunch wheatgrass
 (<em>Pseudoroegneria spicata</em> [Pursh] A. Lӧve]) plants
 during June and July of 2023. The full ‘training population’ at Evans farm
 spans a 750 m<sup>2</sup> nursery plot consisting of ~1300
 individuals arranged in a 77 x 17 grid, with 0.5 m spacing between
 individuals within a row and 1 m spacing between neighboring rows (Fig
 1b). The training population was developed by breeding three commercially
 available germplasm source populations: Anatone, Columbia, and P-7.
 Maternal parents were determined by pedigree (<em>i.e.</em>
 seed source), and paternity was determined via a genotype-by-sequencing
 approach, allowing us to know the maternal and paternal germplasm source
 of each plant (Poland <em>et al.</em>, 2012; Crain
 <em>et al.</em>, 2020). From this population, we selected 5
 healthy individuals in a similar boot stage for both Anatone x Anatone
 (AA) and Columbia x Columbia (CC) germplasm crosses for inclusion in this
 study. Both Anatone and Columbia germplasm originated from
 low-precipitation sites (MAP &lt; 500 mm) in the western extent of the
 species’ range (Washington, USA), but Columbia germplasm was developed
 through selection cycles specifically for increased spike number (Jones
 and Mott, 2016). In early June of 2023, on each of the
 10 selected plants, we basally tagged 4 flowering culms of similar
 phenological stage and randomly assigned each to one of four treatment
 groups: 1) control (not clipped), 2) flag leaf removed, 3) basal florets
 removed, and 4) flag leaf + basal florets removed. We tracked the length
 of each culm (from flag leaf to base of spike) from the beginning of the
 study until culms were fully expanded (<em>i.e</em>. zero
 change in length between measurement periods). Clipping treatments were
 initiated once plants reached the anthesis stage and took place on June
 21, 2023; flag leaves were removed by clipping fully expanded flag leaves
 at the ligule (Treatment groups 2 and 4), and basal florets were manually
 excised to remove approximately half of the floret biomass occurring below
 the distal 2 cm of each spike (Treatment groups 3 and 4). A visual
 representation of the experimental treatments is provided in Fig 1c.
 During each measurement date, we also took 3 readings of volumetric soil
 moisture in the upper 5-cm of topsoil (Theta Probe ML2x meter and HH2
 datalogger, Delta-T Devices, Cambridge, UK) under the canopy of each plant
 to track changes in soil moisture throughout the study. These values,
 along with precipitation data from the Utah Climate Center, are presented
 in Figure S1. <em>Ecophysiological
 measures</em> Physiological measurements were
 recorded on flag leaves and the basal and distal portions of spikes of
 each of the four culms on each selected plant using a portable
 photosynthesis system (LI-6800, LiCoR Instruments, Lincoln NE, USA) at
 five timepoints: one pre-anthesis (June 8), and four subsequent
 measurements spanning anthesis and post-anthesis (June 23, June 29, July
 5, and July 11). Sampling occurred between 0800 and 1300 local time and
 measurement order was randomized each sampling day to minimize any diurnal
 effects. Each spike section or leaf was enclosed inside of a round leaf
 chamber with a 2 cm<sup>2</sup> aperture while maintaining its
 natural orientation and using the following chamber conditions: 1500 µmol
 m<sup>-2</sup> s<sup>-1</sup> (40 µmol blue light,
 1460 red) photosynthetic photon flux density (PPFD) supplied by an LED
 light array, 400 ppm CO<sub>2 </sub>supplied at a flow rate of
 200 µmol sec<sup>-1</sup>, 40 % relative humidity, and 25 °C
 Peltier-exchange block temperature at a 0.1 kPa pressure differential.
 Light-adapted PSII photochemical yield (f<sub>PSII</sub>) was
 determined by measuring chlorophyll fluorescence (F) with a multi-phase
 fluorimeter integrated with the cuvette. A beam of 5.0 mmol
 m<sup>-2</sup> s<sup>-1</sup> intensity modulated
 at 50 kHz was applied for 5 sec to determine steady-state fluorescence
 yield (Fs) under the incident PPFD of 1500 mmol m<sup>-2</sup>
 s<sup>-1</sup>. This was followed by exposure to three
 successive flashes of a saturating actinic beam of 10,000 mmol
 m<sup>-2</sup> s<sup>-1</sup>, each of 300 ms
 pulse width and a ~97% red/3% blue light balance, modulated at 250 kHz
 with data gathered at 100 Hz to determine maximum light-adapted
 fluorescence yield (Fm’); f<sub>PSII</sub> was calculated as
 f<sub>PSII</sub> = (Fm’-Fs)/Fm’. Before initiating a point
 measurement, each sample was allowed to equilibrate to chamber conditions
 and reach steady-state for net photosynthesis
 (A<sub>net</sub>), stomatal conductance
 (g<sub>sw</sub>), and intercellular
 [CO<sub>2</sub>], as indicated by stability parameters. The
 widths of each leaf blade and spike were estimated to the nearest mm at
 the exact measurement location prior to enclosure in the chamber using
 electronic calipers, allowing the instrument to calculate basic area-based
 gas exchange measurements. Further adjustments were made to the basal and
 distal spike area calculations assuming a basic open-ended cylindrical
 geometry. For clipped basal florets, the cylindrical area was reduced by
 half to account for the 50 % biomass removal in those treatment groups.
 This approach assumes negligible contribution of the rachis to the
 photosynthetic surface area and has been applied in previous studies of
 rangeland grass physiology (Hamerlynck <em>et al.</em>, 2019).
 Response variables of interest included net photosynthesis
 (A<sub>net</sub>), stomatal conductance to water
 (g<sub>sw</sub>), and φ<sub>PSII</sub>. Due to
 sample loss resulting from broken culms and damaged flag leaves, our final
 sample size for floret measurements was n = 9 individuals (5 Columbia and
 4 Anatone), and the final sample size for flag leaves was n = 5
 individuals (3 Columbia and 2 Anatone).  Within the
 clipped treatment groups (2 and 4), flag leaves were returned to the lab,
 scanned on a flatbed scanner (Epson Expression 12000XL), and digital
 images were analyzed for leaf area using analytical software (WinRhizo Pro
 2021, Regent Instruments Inc., Quebec, CA); leaves were then oven-dried at
 65 °C to constant mass and weighed to the nearest 0.1 mg using a Metler
 Toledo XSR64 microbalance. At the end of the study, all culms were
 collected for analysis of remaining flag leaves and spikes. Each sample
 was assessed for biomass and projected area, and spike and leaf specific
 mass were calculated as the mass per unit area (g
 cm<sup>-2</sup>).
description: # Compensatory photosynthesis in Pseudoroegneria spicata
 [https://doi.org/10.5061/dryad.4b8gthtpf](https://doi.org/10.5061/dryad.4b8gthtpf) ## Description of the data and file structure Field-based measurements were made on mature bluebunch wheatgrass (*Pseudoroegneria spicata* [Pursh] A. Lӧve]) plants during June and July of 2023. The full ‘training population’ at Evans farm spans a 750 m2 nursery plot consisting of ~1300 individuals arranged in a 77 x 17 grid, with 0.5 m spacing between individuals within a row and 1 m spacing between neighboring rows (Fig 1b). The training population was developed by breeding three commercially available germplasm source populations: Anatone, Columbia, and P-7. Maternal parents were determined by pedigree (*i.e.* seed source), and paternity was determined via a genotype-by-sequencing approach, allowing us to know the maternal and paternal germplasm source of each plant (Poland *et al.*, 2012; Crain *et al.*, 2020). From this population, we selected 5 healthy individuals in a similar boot stage for both Anatone x Anatone (AA) and Columbia x Columbia (CC) germplasm crosses for inclusion in this study. Both Anatone and Columbia germplasm originated from low-precipitation sites (MAP &lt; 500 mm) in the western extent of the species’ range (Washington, USA), but Columbia germplasm was developed through selection cycles specifically for increased spike number (Jones and Mott, 2016). In early June of 2023, on each of the 10 selected plants, we basally tagged 4 flowering culms of similar phenological stage and randomly assigned each to one of four treatment groups: 1) control (not clipped), 2) flag leaf removed, 3) basal florets removed, and 4) flag leaf + basal florets removed. We tracked the length of each culm (from flag leaf to base of spike) from the beginning of the study until culms were fully expanded (*i.e*. zero change in length between measurement periods). Clipping treatments were initiated once plants reached the anthesis stage and took place on June 21, 2023; flag leaves were removed by clipping fully expanded flag leaves at the ligule (Treatment groups 2 and 4), and basal florets were manually excised to remove approximately half of the floret biomass occurring below the distal 2 cm of each spike (Treatment groups 3 and 4). A visual representation of the experimental treatments is provided in Fig 1c. During each measurement date, we also took 3 readings of volumetric soil moisture in the upper 5-cm of topsoil (Theta Probe ML2x meter and HH2 datalogger, Delta-T Devices, Cambridge, UK) under the canopy of each plant to track changes in soil moisture throughout the study. These values, along with precipitation data from the Utah Climate Center, are presented in Figure S1. *Ecophysiological measures* Physiological measurements were recorded on flag leaves and the basal and distal portions of spikes of each of the four culms on each selected plant using a portable photosynthesis system (LI-6800, LiCoR Instruments, Lincoln NE, USA) at five timepoints: one pre-anthesis (June 8), and four subsequent measurements spanning anthesis and post-anthesis (June 23, June 29, July 5, and July 11). Sampling occurred between 0800 and 1300 local time and measurement order was randomized each sampling day to minimize any diurnal effects. Each spike section or leaf was enclosed inside of a round leaf chamber with a 2 cm2 aperture while maintaining its natural orientation and using the following chamber conditions: 1500 µmol m-2 s-1 (40 µmol blue light, 1460 red) photosynthetic photon flux density (PPFD) supplied by an LED light array, 400 ppm CO2 supplied at a flow rate of 200 µmol sec-1, 40 % relative humidity, and 25 °C Peltier-exchange block temperature at a 0.1 kPa pressure differential. Light-adapted PSII photochemical yield (fPSII) was determined by measuring chlorophyll fluorescence (F) with a multi-phase fluorimeter integrated with the cuvette. A beam of 5.0 mmol m-2 s-1 intensity modulated at 50 kHz was applied for 5 sec to determine steady-state fluorescence yield (Fs) under the incident PPFD of 1500 mmol m-2 s-1. This was followed by exposure to three successive flashes of a saturating actinic beam of 10,000 mmol m-2 s-1, each of 300 ms pulse width and a ~97% red/3% blue light balance, modulated at 250 kHz with data gathered at 100 Hz to determine maximum light-adapted fluorescence yield (Fm’); fPSII was calculated as fPSII = (Fm’-Fs)/Fm’. Before initiating a point measurement, each sample was allowed to equilibrate to chamber conditions and reach steady-state for net photosynthesis (Anet), stomatal conductance (gsw), and intercellular [CO2], as indicated by stability parameters. The widths of each leaf blade and spike were estimated to the nearest mm at the exact measurement location prior to enclosure in the chamber using electronic calipers, allowing the instrument to calculate basic area-based gas exchange measurements. Further adjustments were made to the basal and distal spike area calculations assuming a basic open-ended cylindrical geometry. For clipped basal florets, the cylindrical area was reduced by half to account for the 50 % biomass removal in those treatment groups. This approach assumes negligible contribution of the rachis to the photosynthetic surface area and has been applied in previous studies of rangeland grass physiology (Hamerlynck *et al.*, 2019). Response variables of interest included net photosynthesis (Anet), stomatal conductance to water (gsw), and φPSII. Due to sample loss resulting from broken culms and damaged flag leaves, our final sample size for floret measurements was n = 9 individuals (5 Columbia and 4 Anatone), and the final sample size for flag leaves was n = 5 individuals (3 Columbia and 2 Anatone).  Within the clipped treatment groups (2 and 4), flag leaves were returned to the lab, scanned on a flatbed scanner (Epson Expression 12000XL), and digital images were analyzed for leaf area using analytical software (WinRhizo Pro 2021, Regent Instruments Inc., Quebec, CA); leaves were then oven-dried at 65 °C to constant mass and weighed to the nearest 0.1 mg using a Metler Toledo XSR64 microbalance. At the end of the study, all culms were collected for analysis of remaining flag leaves and spikes. Each sample was assessed for biomass and projected area, and spike and leaf specific mass were calculated as the mass per unit area (g cm-2). ### Files and variables #### File: QuigleyEtAl*ConservationPhysiology2024\_FINAL*.xlsx **Description:** This file contains 4 sheets. The first two sheets contain physiological data recorded with the LI-6800, and the final two sheets contain trait data. All variables contained within are described below. ##### **LICOR\_FlagLeaf *and* LICOR\_Floret** ·         Date: measurement date, as month/day/year ·         Germ: germplasm variety; AA = Anatone, CC = Columbia ·         Row: position within the row by range matrix, as described in methods ·         Range: position within the row by range matrix, as described in methods ·         TrtID: Numeric treatment ID, where 1 = control, 2 = clipped flag leaf, 3 = clipped florets, and 4 = clipped flag leaf and florets ·         SampleType: Type of tissue measured; FlagLeaf, BasalFloret, or DistalFloret ·         Treatment: Descriptive experimental treatment corresponding with “TrtID” above ·         Time: measurement time, as hour:minute:seconds ·         A_net: area-corrected net assimilation rate (µmol m⁻² s⁻¹) ·         Gsw: area-corrected stomatal conductance to water vapor (mol m⁻² s⁻¹) ·         RHcham_%: Relative humidity in the chamber (%) ·         PhiPSII: quantum efficiency of photosystem-2 efficiency (unitless) ·         ETR_umol…: electron transport rate (µmol s⁻¹) ·         Qin_umol…: PPFD incident on the leaf (µmol m⁻² s⁻¹) ·         CO2_s_umol…: sample cell CO2 concentration (µmol mol⁻¹) ·         CO2_r_umol…: reference cell CO2 concentration (µmol mol⁻¹) ·         H2O_s_mmol…: sample cell H2O concentration (mmol mol⁻¹) ·         H2O_ref_mmol…: reference cell H2O concentration (mmol mol⁻¹) ·         Flow_umol…: flow rate into chamber (µmol s⁻¹) ·         Pa_kPA: Atmospheric pressure (kPa) ·         Pcham_kPa: Chamber overpressure (kPa) ·         Tair_C: chamber air temperature (°C) ·         Tleaf_C: leaf thermocouple temperature (°C) ·         Fan_speed_rpm: chamber fan rotation rate (rpm) ·         Qamb_in…: in-chamber ambient PPFD (µmol m⁻² s⁻¹) ·         Qamb_out…: external ambient PPFD (µmol m⁻² s⁻¹) ·         F_red: fraction of red light (unitless) ·         F_blue: fraction of blue light (unitless) ·         F_farred: fraction of far red light (unitless) ·         F: demodulated fluorescence (unitless) ·         Q_red…: red actinic contribution to Q (µmol m⁻² s⁻¹) ·         Q_blue…: blue actinic contribution to Q (µmol m⁻² s⁻¹) ·         period_s: time period for fluorescence statistics (seconds) ·         Txch_C: heat exchanger temperature (°C) ·         Tirga_C: IRGA block temperature (°C) ·         WUE_i: intrinsic water use efficiency (unitless)   **TRAITS_FlagLeaf** ·         Row: position within the row by range matrix, as described in methods ·         Range: position within the row by range matrix, as described in methods ·         Trt: numeric treatment ID, where 1 = control, 2 = clipped flag leaf, 3 = clipped florets, and 4 = clipped flag leaf and florets ·         PrjArea (cm2): projected area (cm-2); for spikes, this value does not include detached floral parts ·         Sample type: tissue type measured (flag leaf or spike) ·         Dry mass (g): sample dry mass (grams) ·         LMA: leaf mass per area (g cm-2) ·         Germ: germplasm variety; AA = Anatone, CC = Columbia ·         Treatment: Descriptive experimental treatment corresponding with “TrtID” above **Traits_Spike** *As above, plus the following variables:* ·         Floret count: number of florets present ·         WholeSpikeMass.g: dry mass of the entire spike (grams) ·         FloretMass.g: dry mass of florets (grams) ·         SpikeSpecMass: mass per unit area of spike (g cm-2 ) ## Code/software Data may be viewed using Microsoft Excel or similar program. Data analysis was completed in the R Statistical programming language; scripts and codes for specific analyses are available upon request.