Plasticity in plant hydraulic traits: An evaluation of a common-taxa experiment across a climatic gradient in the Western U.S.

Drought impacts plant productivity, ecosystem function, and the global carbon cycle, with many species-level responses remaining unknown. Variable responses to water availability exist among plant species and across biomes. This research utilized a coordinated water deficit experiment of horticultural taxa across three sites in the Western U.S. to assess taxa-level plasticity to water availability and location. Four taxa (*Cercis occidentalis, Cercis canadensis, Physocarpus ‘*Diabolo’, and *Physocarpus ‘*Little Devil’) were measured for physiological and morphological traits affecting plant hydraulic conductivity under two water deficit treatments. Full gas exchange, specific leaf area, vessel diameter, theoretical hydraulic conductance, and ΦPSII were collected, and water use efficiencies calculated for each taxon at each location. Impacts of site, treatment, and taxa were analyzed on this suite of traits. Results show differences in taxa performance by climatic location (p < 0.001) and between closely related species and cultivars. However, the irrigation treatments had limited significant impacts on physiological performance. These findings highlight the merits of common-taxa trials over multiple geographic locations to evaluate and identify climate suitability of plants. Our results provide evidence that landscape irrigation can be reduced substantially and utilize a unique research framework for filling plant-water knowledge gaps critical to address in the face of anthropogenic climate change.

Methods
Leaf physiology

End of season measurements of stomatal conductance to water vapor (gsw, mol m-2 s-1) and ΦPSII were taken with a LI-600 Porometer/Fluorometer (LI-COR Biosciences, Lincoln, NE, USA) during midday (11:30-1:30pm, to reduce risk of variable weather influence) at the three sites. The LI-600 configuration was set to automode with fast gs, fluorescence and flowrate, with matching to ambient conditions set to every 10 minutes. Midday instantaneous gas exchange survey measurements were taken with a portable photosynthesis system (LI-6400XT at OSU and LI6800 at UW and USU; LI-COR Biosciences, Lincoln, NE, USA) with a 2 cm2 chamber head and parameters set to 420 mmol mol -1 CO2; flow rate 500 mmol s-1, PAR at 2000 mmol m-2 s-1. Full gas exchange survey measurements were used to calculate instantaneous water use efficiency (WUE; mmol mol-1) as photosynthetic rate over transpiration rate (A/E) and intrinsic water use efficiency (iWUE; mmol mol-1) calculated as photosynthetic rate over stomatal conductance (A/gs). All leaf physiology measurements were conducted on full sun, undamaged, new but fully expanded leaves, with one representative leaf per plant selected for full gas exchange measurements, and four leaves measured for LI-600 measurements, of which the lowest value of gsw was excluded and data were averaged to act as a per plant measurement. Gas exchange measurements were timed to occur prior to irrigation treatments to capture the effects of the deficit treatment.

Specific leaf area

Leaf punches of known areas were collected at the end of the growing season at all three sites from new, fully expanded leaves at each time. Samples were dried in an oven overnight and weighed to calculate specific leaf area (SLA, cm2/g).

Xylem anatomy

Stem segments from the top 5 cm of actively growing branches were collected from target taxa and stored frozen at -20 °C prior to being hand sectioned with a double-edge razor blade, stained with a 0.1% safranin solution for 40 seconds, cleared with DI water, and imaged under a microscope (E200 Eclipse, Nikon Instruments Inc., Melville, New York, USA) with a camera attachment (MU-1803 HS, AMScope, Irvine, California, USA). Image analysis was conducted using ImageJ-Fiji version: 2.14.0/1.54f (Schindelin, 2012).  For images with good contrast, vessel area was measured with an autothresholding function, and for the rest, vessels were manually traced, totaling a minimum of 50 vessels per individual. Since vessels were not circular, diameter was estimated as the mean diameter of an ellipse: D = (D1 + D2)/2, with D1 as the maximum vessel diameter and D2 as the minimum vessel diameter. Vessel density (VD, n µm-2) was calculated as number of vessels per set area from which mean vessel hydraulic diameter (Dmh;* µ*m) was calculated as: Dmh = (/ n)1/4 (Fontes et al., 2022). The theoretical specific conductivity (Kp; kg m MPa-1s-1) was calculated using the Hagen-Poiseuille equation:  where  is the density of water (998.23 kg/m3) and  is the viscosity of water (1.002 x10-9 MPa/s), both at 20°C (Santiago et al., 2004; Tyree & Ewers, 1991).