DOI: 10.5061/dryad.jsxksn0h1
title: Grass veins are leaky pipes: Vessel widening in grass leaves explain variation in stomatal conductance and vessel diameter among species

description: The widening of xylem vessels from tip-to-base of trees is an adaptation
 to minimize the hydraulic resistance of a long pathway.  Given
 that parallel veins of monocot leaves do not branch hierarchically,
 vessels should also widen basipetally but, in addition to minimizing
 resistance, should also account for water volume lost to transpiration
 since they supply water to the lamina along their lengths, i.e.
 “leakiness”. We measured photosynthesis, stomatal conductance, and vessel
 diameter at 5 locations along each leaf of 5 perennial grass species. We
 found that the rate of conduit widening in grass leaves was larger than
 the widening exponent required to minimize pathlength resistance (0.35 vs.
 ~0.22).  Furthermore, variation in the widening exponent among
 species was positively correlated with maximal stomatal conductance (r2 =
 0.20) and net CO2 assimilation (r2 = 0.45). These results suggest that
 faster rates of conduit widening (&gt;0.22) were associated with
 higher rates of water loss. Taken together, our results show that the
 widening exponent is linked to plant function in grass leaves and that
 natural selection has favored parallel vein networks that are constructed
 to meet transpiration requirements while minimizing hydraulic resistance
 within grass blades.
description: <em>Plant Material </em> Six
 common grass species native to the tallgrass prairie were selected for
 this study: Andropogon gerardii Vitman (Ag, C4 NADP-ME), Panicum virgatum
 L. (Pv, C4 NAD-ME), Sorghastrum nutans Nash (Sn,
 C<sub>4</sub> NADP-ME), Schizachyrium scoparium Nash (Ss,
 C<sub>4 </sub>NADP-ME), Elymus canadensis L. (Ec,
 C<sub>3</sub>) and Bromus inermis Leyss. (Bi,
 C<sub>3</sub>).  C<sub>3</sub> and
 C<sub>4 </sub>species were included in this study to
 investigate the generality of the patterns across a range of grass species
 rather than an attempt to characterize differences between these
 functional groups.  Seeds were collected from Konza Prairie Biological
 Station, Kansas, USA (KPBS) and stored at 4°C until the initiation of the
 study.  Seeds of each species were germinated on wet filter paper and then
 five individuals were transplanted into 1.65 L pots (“Short One” Treepots;
 Stuewe & Sons, Inc., Tangent, OR, USA) filled with soil taken from
 KPBS.  This resulted in five replicates per species, which were then
 randomly distributed within a growth chamber (Conviron PGV 36; Conviron
 Environments Limited, Winnipeg, Manitoba, Canada).  Air temperature in the
 growth chamber was maintained at 30°C with a 16 h photoperiod.  The light
 intensity at the top of the canopy was maintained at ∼1000 µmol m−2.s−1 by
 adjusting the height of the lights as the canopy grew.  Plants were
 watered daily to ensure that soil moisture remained near pot-holding
 capacity.  Plants were grown until they had four to six mature leaves
 before beginning measurements.  5-6 plants of each species were used to
 measure rates of gas-exchange and anatomy.  The length of the leaf blade
 of each individual was measured and gas exchange and anatomy measurements
 (described below) were centered at 10, 25, 50, 75, and 90% of the leaf
 length.  The distance from the tip of the leaf to each of these
 measurement points is reported as dtip.  The gas-exchange measurements
 were made over 5 days, and 2-3 individuals of each species were randomly
 selected and measured each day.  After gas-exchange measurements were
 completed, plant tissue was placed in a chemical fixative and stored until
 further processing for microscopic analysis.
 <em>Gas exchange</em> The most
 recently mature leaf blade of each plant was identified and divided into
 five equal longitudinal sections.  The middle of each section was marked
 (using a black marker) and all subsequent measurements were centered on
 this mid-point; the distance from the leaf tip to the middle of each
 section was measured (d<sub>tip</sub>).  Photosynthetic rate
 (<em>A</em>) and stomatal conductance to water vapor
 (gs*wv) were measured using an infrared gas exchange system (Li-6400,
 Li-Cor, Inc., Lincoln, NE, USA) with a small (2
 cm<sup>2</sup>) fluorometer cuvette (6400-40, Li-Cor, Inc.,
 Lincoln, NE, USA), which was chosen to maximize the proportion of the
 cuvette occupied by leaf.  Conditions inside the cuvette were maintained
 to match the condition of the growth chamber [∼400 µmol
 mol<sup>−1</sup> CO2, 30 °C, 50 ± 5% relative humidity, 1000
 µmol m<sup>−2</sup> s<sup>−1</sup>
 photosynthetically active radiation (PAR)].  Gas exchange measurements
 were first made on the basal section and then sequentially along the
 length of the blade toward the apex.  Data from the Li-6400 was logged
 every 5 s until both A and g<sub>s*wv</sub> were stable for
 &gt;1 min (typically ∼5–10 min of logged data).  Data were imported
 into Matlab (Mathworks, Inc., Natick, MA, USA), visually inspected to
 ensure stability and then the mean of the last minute of data (12 logged
 points) was calculated and corrected for the leaf area in the chamber
 during the measurement. <em>Leaf
 Anatomy</em> Leaf blade tissue was vacuum
 infiltrated with a fixative (formalin-acetic acid-alcohol) and placed in
 4°C storage for further processing. Each blade section was embedded with
 paraffin and then double stained with Safranin-O and Fast Green at the
 Kansas State University Histology Lab.  Images were taken with a digital
 camera (Leica DFC 290; Leica Microsystems GmbH, Wetzlar, Germany) coupled
 to a light microscope (Leica DM1000; Leica Microsystems GmbH) and analysed
 using ImageJ.  We measured xylem lumen diameter of the largest vessel in 3
 different vascular bundles: the center bundle in the midrib (there can be
 multiple bundles in a midrib), the major bundle closest to the leaf edge,
 and the small vascular bundle that was next to, but closer to, the leaf
 center.  These measurements were made in the specified vascular bundle in
 each of the 5 blade sections.  For the midrib, we could ensure that we
 were measuring the same vessel in all 5 sections.  For the 2 other
 vascular bundles selected, it was not always possible to identify the same
 vascular bundle and so some of the measurements were likely made on
 vessels on opposite sides of the leaf. 
description: # Grass veins are leaky pipes: vessel widening in grass leaves explain
 variation in stomatal conductance and vessel diameter among species
 [https://doi.org/10.5061/dryad.jsxksn0h1](https://doi.org/10.5061/dryad.jsxksn0h1) Stomatal conductance, photosynthesis, and vessel diameter at multiple points along the length of grass leaves. Data from the six following species were collected:L *Andropogon gerardii* Vitman (*Ag*, C<sub>4 </sub>NADP-ME), *Panicum virgatum* L. (Pv, C<sub>4</sub> NAD-ME), *Sorghastrum nutans* Nash (*Sn*, C<sub>4 </sub>NADP-ME), *Schizachyrium scoparium* Nash (*Ss*, C<sub>4 </sub>NADP-ME), *Elymus canadensis* L. (*Ec*, C<sub>3</sub>) and *Bromus inermis* Leyss. (*Bi*, C<sub>3</sub>). Each of these measurements was made on a different section along a grass blade. ## Description of the data and file structure There are two files in this dataset. The file named "Ocheltree\_Gleason\_vessel\_widening\_data.csv" contains the anatomical data on each of 5 leaf segments along the leaf blade. The file named "Ocheltree\_Gleason\_vessel\_widening\_data\_wholeleafdata.csv contains data on the entire leaf blade and so only has one value for each leaf blade. Below is a description of each column in the data: File: Ocheltree\_Gleason\_vessel\_widening\_data\_dryad\_leafsectiondata.csv Sample - The sample ID includes the species code followed by the replicate number. The species code is a two letter code described above. Species - This column includes only the species code from the 'Sample' column. Segment Number - The segment code is a single digit indicating which segment, of 5, on the leaf blade. '1' indicates the basal most section and '5' the most apical. length from ligule (cm) - This is the distance from the ligule of the leaf blade (the division between leaf sheath and blade) to the center of the leaf segment in cm. Leaf Length (cm) - The total length of the leaf blade in cm. distance from leaf tip (cm) - The distance from the leaf tip to the leaf segment in cm. max\_vessel\_ID\_midrib (micrometer) - The inner diameter of the largest vessel in the centermost vascular bundle of the midrib. The diameter measurement is in units of micrometer. max vessel ID - major vein near leaf edge (micrometer) - The inner diameter of the largest vessel in the major vein closest to the leaf edge. The diameter measurement is in units of micrometer. max vessel ID - minor vein near leaf edge (micrometer) - The inner diameter of the largest vessel in the minor vein next to the vein measured for 'max vessel ID - major vein near leaf edge (micrometer)' above. The minor vein selected was always toward the center of the leaf, and the diameter measurement is in units of micrometer. NA - indicate that the data is Not Available for the particular sample/variable. File: Ocheltree\_Gleason\_vessel\_widening\_data\_wholeleafdata.csv Sample - The sample ID includes the species code followed by the replicate number. The species code is a two letter code described above. Species - This column includes only the species code from the 'Sample' column. Segment Number - The segment code is a single digit indicating which segment, of 5, on the leaf blade. '1' indicates the basal most section and '5' the most apical. stomatal\_conductance\_mmol\_m2\_s photosynthetic\_rate\_umol\_m2\_s transpiration\_mmol\_m2\_s NA - indicate that the data is Not Available for the particular sample/variable.