DOI: 10.5281/zenodo.11078475
title: Supporting Data:A pleiotropic 'stretch' phenotype is associated with metaxylem vessel element length, axial hydraulic conductance, root elongation, water utilization, and drought adaptation in maize.

description: These data were collected to assess how xylem perforation plates affect water use strategies in maize (Zea mays L.) under water deficit through empirical studies in controlled environments and in the field. In this work, our goals were to explore the extent of intraspecific variation for the structure of perforation plates within an annual monocot and assess how this variation affects transport and use of water under drought stress. Specifically, we test the hypotheses that (1) simple perforation plates have a significant effect on water transport, (2) intraspecific variation exists for these features in maize, and (3) this variation affects water use strategies under drought stress. 
description: Anatomical Measurements

To visualize internal anatomy of roots and leaves, samples were preserved, sectioned, and imaged by laser ablation tomography (LAT) as described in Strock et al., (2019, 2022). Analyzed dimensions of brace roots and subterranean roots include cross-sectional and longitudinal images. Analyzed dimensions of leaves include cross-sectional and longitudinal images of the lamina and the midrib, midway along the length of the leaf, approximately 2 cm from the midrib.

Germplasm Selection

For much of this work, we utilized maize recombinant inbred lines (RILs) from the intermated B73 x Mo17 (IBM) population. RILs descend from the same two parents, hence represent distinct genotypes sharing the same genetic background, thereby reducing the risk of confounding effects from genetic interactions, epistasis, and pleiotropy (Zhu et al., 2005, 2006). RILs are especially useful tools in cases in which the genetic basis of a phenotype is complex or unknown, as is the case with perforation plate prominence and metaxylem vessel length in maize, thereby precluding the use of single-gene variants.

To understand the variation that exists for perforation plate prominence and metaxylem vessel length, 234 RILs from the IBM population were grown at the Ukulima Root Biology Center (URBC) in Limpopo Province, South Africa (24.533367°S, 28.123783°E) from February through May 2011. At flowering, root crowns of two representative plants per genotype were extracted and washed as in Trachsel et al., (2011), and the anatomy was analyzed from one segment of nodal root from the fourth node of each plant, 5 cm from the base of the root. Root segments were sectioned and imaged using LAT as described above. The lengths of fifteen representative metaxylem vessels measured in LAT images of each root segment using imageJ (Schneider et al., 2012). From these 234 RILs of the IBM population, four accessions with distinct metaxylem vessel lengths were selected for use in subsequent controlled environment and field studies. The four IBM accessions included IBM015 and IBM111, which were classified as having roots with less prominent (short) perforation plates and long metaxylem vessels, and IBM177 and IBM205, which were classified as having roots with prominent (tall) perforation plates and short metaxylem vessels. Seeds used in all glasshouse and field studies were provided by Dr. Shawn M. Kaeppler from U-Wisconsin, Madison, USA.

Additionally, perforation plate height and MXVL were phenotyped using LAT in 469 genotypes of the Wisconsin Diversity Panel. The Wisconsin Diversity Panel is composed of inbred lines that display uniform vigor and reach physiological grain maturity in the Northern Midwest of the United States. Plants were grown at the Apache Root Biology Center in Wilcox, AZ, USA (32.032079°N, 109.691171°W) in 2016 and samples from forth node roots were collected for anatomical analysis at anthesis. Averages over reps of perforation plate height and MXVL were used for further analysis.

Vulnerability to Cavitation Study  

Vulnerability to cavitation was measured using a modified version of the optical methods described by Gauthy et al., 2020. Three IBM genotypes with contrasting MXVL phenotypes were grown in Hagerstown-Opequon series soil at Mr. Toad’s Glee Club and Research Farm (Boalsburg, PA; 40.794655°N, -77.764351°W). Ten cm of the fifth leaf apex was harvested once fully mature and hydrated in wet paper towels for 2 h before imaging. Leaf segments were affixed to a diffuse LED light source with adhesive tape heated to 37°C while imaging a region of interest 5 cm from the cut leaf edge every 1 min on a Nikon SMZ 1500 stereo microscope. Images were converted to 8-bit grayscale before subtraction of sequential frames in ImageJ to visualize cavitation events by optical refraction/darkening. Due to the parallel venation of maize leaves, medial shrinkage/slippage caused artifacts that prevented automated analysis of cavitation. ImageJ was used to manually measure the length of cavitation events on each frame for comparison between genotypes.

Controlled Environment Study

This study was conducted in a glasshouse located at Pennsylvania State University in University Park (40.801955°N, 77.862544°W). Plants were grown from April through June 2021 under a 16/8-h (light/dark) photoperiod, 40% relative humidity, and maximum/minimum temperatures of 28°C/26°C. Midday photosynthetic active radiation was 900 to 1,000 μmol photons m-2s-1. Natural light was supplemented from 06:00 to 22:00 with approximately 500 μmol photons m-2s-1 from metal-halide lamps. Seeds were surface sterilized in a 25% (v/v) NaOCl in water for 2 min, rinsed in deionized water, and germinated in 0.5 mM CaSO4 in the dark at 28°C for 72 h. Uniform seedlings were transplanted to the glasshouse in opaque, 30 L mesocosms 15 cm in diameter and 155 cm in height and lined with transparent 6 mm high-density polyethylene film to facilitate root sampling. Mesocosms were filled with a mixture of 4% (w/w) coarse grade A perlite (Whittemore), 50% (w/w) medium-grade sand (US Silica), 26% (w/w) D3 coarse grade A vermiculite (Whittemore), and 20% (w/w) field soil (Ap2 Hagerstown silt loam [fine, mixed, semiactive, mesic Typic Hapludalf]) sieved through 6 mm mesh. The soil was incorporated to replicate features found under field conditions, such as the presence of organic matter, soil biota, and oxide surfaces that serve to buffer nutrient availability. Mesocosms were fertilized with 5 g kg-1 Osmocote (15-9-12; 5-6 mo.) (The Scotts Co., Marysville, OH) incorporated into the media at the time of mixing and consisting of (%): NO3 (8) NH4 (7), P (9), K (12), S (2.3), B (0.02) Cu (0.05), Fe (0.68), Mn (0.06), Mo (0.02), and Zn (0.05).

A Randomized Complete Block Design was utilized with two irrigation levels; water stress (WS) and well-watered (WW). Irrigation was supplied through drip rings, with pots being brought to field capacity daily. Irrigation was halted on pots assigned to the WS treatment at 17 days after planting (DAP). The experiment was run for a total of 42 d with destructive measurements taken from all genotypes in all treatments at 17, 31, and 42 DAP. Each genotype had four replications at each time point and treatment.

To determine net water loss from pots in the WS treatment, 5 pots from each phenotypic group were weighed hourly from 17 to 42 DAP using Adam CPWplus 75 floor scales (Adam Equipment, Oxford, CT). Gravimetric soil moisture was also determined at 31 and 42 DAP at 20 cm increments by depth from the soil surface to the bottom of the container. Leaf relative water content and specific leaf area were determined from 5, 2.5 cm leaf discs collected at 16:00 on 41 DAP as in Smart and Bingham (1974). Predawn water potential of plants in the WS treatment was determined using the PMS model 615 Scholander pressure bomb at 42 DAP from 03:00 to 06:00 (PMS Instruments, Albany, OR).

At 17, 31, and 42 DAP destructive measurements were taken including leaf number, leaf area, internode distance, plant height, as well as dry root and shoot biomass. Leaf area was determined using the Li-Cor LI-3100C leaf area meter (Li-Cor, Lincoln, NE). Dry mass was determined from tissues dried at 65°C for 7 d.

At 31 DAP, two, 10 cm segments of nodal roots from the most recently emerging node were collected 5 cm from the base of the root for measurement of root respiration. Root respiration rates were determined immediately after excavation and washing using a Li-Cor 6400 gas-exchange system with a modified respiration chamber (Li-Cor, Lincoln, NE). Lateral roots were removed from these segments with a razor prior to respiration measurements. Measurements were performed under ambient glasshouse conditions, with the sealed chamber being kept at a temperature of 28°C and baseline sample chamber and reference chamber CO2 concentration of 400 μmol mol-1.

42 DAP, mesocosms were cut into 20 cm increments by depth from the soil surface to the bottom of the container. Roots were washed, collected, and imaged from each 20 cm segment using an EPSON Perfection V700 PHOTO scanner and total length was quantified with WinRhizo software (WinRhizo Pro; Reagent Instruments). The scanned roots were then dried and weighed to determine specific root length, calculated by dividing the total root length by the total root dry weight.

At 42 DAP, two, 10 cm segments of nodal roots from the most recently emerging node were collected 5 cm from the base of the root. Lateral roots were then removed with a razor and in situ measures of axial conductance were then performed on these root segments as in Strock et al., (2021). Prior to measurement, each 10 cm root segment was soaked in a de-gassed 20 mM KCl solution for 30 min. Paraffin wax was melted and painted on the surface of the root to preclude radial losses of flow through lateral root junctions across the segment. A 0.0093 MPa hydraulic head of degassed 20 mM KCl solution was attached to one end of the root segment and flow out the opposite end of the segment was quantified over a 1 min period using an Adventurer Pro AV13C analytical balance (Ohaus Corporation, Pine Brook, NJ). Following measurement of axial conductance across the 10 cm segment, 2.5 cm increments of root were subsequently excised from the end of the segment and the measurement was repeated with 7.5, 5, and 2.5 cm lengths of root to determine the effect of perforation plated on axial flow.

Following these in situ conductance measurements, the wax coating was removed, and the root segments were preserved in 75% (v/v) ethanol in water. Preserved segments were sectioned in both the longitudinal and cross-sectional dimensions with laser ablation tomography (Strock et al., 2019). Length of metaxylem vessels was measured in the longitudinal dimensions while the number and area of metaxylem vessels was measured from the cross-sectional dimension at both ends of the segment. Theoretical axial metaxylem conductance (kh; kg m MPa-1 s-1) was calculated for each cross-sectional image using the modified Hagen-Poiseuille law, where d is the diameter of the vessel in meters, r is the fluid density (equal to water at 20°C; 1000 kg m-3), and h is the viscosity of the fluid (equal to water at 20°C; 1 x 10-9MPs s-1; Tyree and Ewers, 1991). The mean theoretical conductance estimate calculated from images at each end of the segment were used for comparison with the in situ measure of conductance across that segment.

USA Field Trials

The PA19 and PA20 field trials was conducted at the Russell E. Larson Agricultural Research Farm at Rock Springs, PA, USA (40.711365°N, 77.953089°W) from June through September 2019 and 2020, respectively. The soil at this site is a Hagerstown silt loam (fine, mixed, mesic Typic Hapludalf). A split plot design was utilized with two irrigation levels; two, 0.02 ha rainout shelters were split into two, 0.01 ha blocks each, and two 0.02 ha irrigated fields split into two, 0.01 ha blocks. In 2019, sixteen genotypes and in 2020, four genotypes were randomized within each block. To manage fungal pathogens, seed were treated with Captan 50W fungicide solution (0.2g/ L) at a rate of 0.5 ml/100 seeds prior to planting. All fields were fertilized to meet the nutrient requirements of maize as determined by soil tests at the beginning of the season. Each genotype was planted in a single row, 4.6 m long plot with 76 cm row spacing at a density of 73,300 plants ha-1. During periods of inadequate rainfall, irrigation was supplied to the well-watered treatment. Drought treatment was initiated at 20 DAP, after which water-stressed plots experienced no rainfall or irrigation through the time of yield harvest. Each genotype had four replications within each irrigation treatment.

At anthesis, leaf, brace root, and forth node root samples were collected for anatomical analysis as described above. Dry masses were determined from tissues dried at 60°C for 7 d.

Graneros, Chile Field Trial

The CL20 field trial was conducted at the Tuniche Research Farm near Graneros, Chile (-34.108279°S, -70.748495°W, soil order is Inceptisol) from November 2019 through May 2020. A split plot design was utilized with two irrigation levels; one field where irrigation was limited was split into two water-stressed blocks, and one irrigated field split into two blocks. Thirty hybrid genotypes known to contrast in water use efficiency (15 drought tolerant genotypes, 15 drought sensitive genotypes) were randomized within each block. All fields were fertilized to meet the nutrient requirements of maize. Each genotype was planted in a two row, 4.6 m long plot with 76 cm row spacing. During periods of inadequate rainfall, irrigation was supplied to the well-watered treatment. Each genotype had four replications within each irrigation treatment.

At anthesis, destructive measurements were taken including dry shoot biomass, leaf samples and forth node root samples for anatomical analysis as described above. Dry masses were determined from tissues dried at 60°C for 7 d.

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