For scanning electron microscopy, immature barley spikes were fixed in 50 mM cacodylate buffer (pH 7.2) containing 2% glutaraldehyde and 2% formaldehyde at 4°C. Samples were washed with distilled water and dehydrated in an ascending ethanol series and point-dried in a Bal-Tec critical point dryer (https://leica-microsystems.com ). Dried specimens were gold-coated in an Edwards S150B sputter coater (http://edwardsvacuum.com ) and examined in a Zeiss Gemini30 scanning electron microscope (https://zeiss.de ) at an acceleration voltage of 10 kV. For TEM, samples were embedded in Spurr’s resin. Ultrathin sections (70 nm) were cut with a microtome (Leica Ultracut, Leica Microsystems, Bensheim, Germany) and subjected to TEM (Tecnai Sphera G2, FEI, Eindhoven, The Netherlands) at 120 kV. For confocal laser scanning microscopy (CLSM) observation of chlorophyll, immature spikes were dissected and embedded in 8% agarose placed in a flat-bottomed mold. After concreting, agarose blocks were removed from the mold and cut into 80 μm with a microtome (Leica VT1000 S vibrating blade microtome). Autofluorescence was recorded under the LSM780 CLSM (Carl Zeiss MicroImaging) with a 640-nm laser line and 650- to 720-nm emission. For measuring the vascular area, transverse sections through all rachis of a barley spike were recorded by CLSM with autofluorescence of a 405-nm laser line and 406- to 715-nm emission. The surface area of rachis internodes and vasculature was measured with the open source Fiji software (67 (link)). The cross-sectional surface area of individual veins was determined as the area surrounded by a bundle sheath. The distinction between central, lateral, and peripheral bundles followed (68 ). Because of their aberrant distribution and different functions in metabolite transportation (68 , 69 ), peripheral bundles were not included in the present investigation, which thus involves lateral and central bundles only.
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