Computational protocol: Humidity-dependent wound sealing in succulent leaves of Delosperma cooperi – An adaptation to seasonal drought stress

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[…] Nawaschin’s fluid (mixture of equal portions of solutions A and B; A: 5 mL 10% chromic oxide, 3.5 mL acetic acid, 42 mL distilled water; B: 15 mL 37% formaldehyde, 35 mL distilled water) was used for the fixation of leaf samples. After dehydration with increasing ethanol concentrations, the samples were embedded in 2-hydroxymethacrylate (Technovit 7100, Kulzer, Wehrheim/Ts, Germany) according to the manufacturer’s instructions. A custom-built rotary microtome (Technical Workshop, Institute of Biology II/III, University of Freiburg, Germany) was used to cut thin sections between 5 µm and 10 µm in thickness. Overview staining with toluidine blue (0.05% toluidine blue in distilled water) was used to give contrast to the various leaf tissues. Unlignified primary cell walls appeared dark blue and lignified secondary cell walls were light blue. Permanent slides were prepared by using the mounting medium Entellan (Merck KGaA). Sections were examined with an Olympus BX61 microscope (Olympus Corporation, Tokyo, Japan) equipped with a DP71 camera module. Parameters of the cells and tissues were determined with the image analysis software ImageJ 1.46h. [...] The biomechanical properties of entire leaves and of single tissue layers (epidermis, central strand of vascular bundles) were studied in tensile tests performed on a modified custom-made micro-tensile-testing device (Technical Workshop, Institute of Biology II/III, University of Freiburg, Germany, for details see []). The device was equipped with microstep motors with an accuracy of ±3 µm, a high precision linear table, a compression–tension load cell with a maximum load of 10 N and a resolution of ±10 mN (model 31E, Honeywell, Columbus, OH, USA) and two sample holders (aluminium platelets) on opposite sides. The platelets were fitted with holes for easy mounting onto the tensile apparatus via a pinhole assembly. Experimental control and the reading of data were performed by a measurement amplifier (Spider 8, Hottinger Baldwin Messtechnik GmbH).The ends of the samples were stuck to the sample holders with a rapid cyanoacrylate adhesive (Uhu Sekundenkleber blitzschnell Pipette, UHU GmbH and Co. KG Bühl, Germany). The samples were carefully arranged parallel to the tension forces to ensure an even strain field over the diameter of the sample. During hardening the adhesive glued samples were stored in a humidity chamber (>95% relative air humidity) in order to prevent or slow down dehydration (storage time for leaves: 2–4 h, storage time for strands of vascular bundles and epidermis samples: a few minutes). After glue hardening, the platelets were mounted onto the tensile apparatus and the displacement tests were conducted. During the tensile tests, images were taken at a frame rate of one image per second by use of an Olympus SZX9 dissecting microscope via a Color View II digital camera (Soft Imaging Systems GmbH, Münster, Germany) and the software cell D. Measured values of time t, force F and displacement ΔL were captured at five readings per second. The strain rate was 0.002 s−1.During the testing of whole leaves, images were taken from two sides in order to allow the measurement of the original length L 0, height (2b) and width (2a). The height and width were used to calculate the cross-sectional area. Before the start of the tensile tests, the leaves were marked with a black line at mid-length to ensure that transverse strains and axial strains were always calculated from the same point for the subsequent calculation of Poisson’s ratio. For the testing of isolated tissues, such as central vascular bundles or epidermis, adjacent tissues were removed carefully and as completely as possible. Thin-sections were prepared from epidermis samples and from the basal and apical region of the tapered strands and cross-sectional areas were calculated. The epidermis was tested in a longitudinal and in a transversal direction to test possible anisotropy in this tissue layer. To reduce dehydration effects during tensile tests, epidermis samples and strands of vascular bundles were sprayed with finely dispersed water vapour from an ultrasound nebuliser. The images were taken with an Olympus SZX9 dissecting microscope via a Color View II digital camera (Soft Imaging Systems GmbH, Münster, Germany) and the software cell D. The measurements were conducted with the software ImageJ 1.46h.The recorded force–displacement data of the tested leaves and tissues were used to assess the tensile strength and elastic modulus. On the basis of the images taken, the height (2b) and width (2a) of each leaf were measured at five points along the longitudinal axis of the leaf. Every cross-sectional area (A leaf) was calculated () assuming an elliptical shape of the leaf; this holds true as a good approximation in this species.For further calculations, the mean cross-sectional area () of the leaf was determined as the mean value of the five calculated cross-sectional areas along each leaf. The cross-sectional area of the tapered strands of vascular bundles was calculated from the basal radius achieved from thin sections. Because of the large diameter of the window cells, the cross-sectional area of the epidermis was difficult to assess. The height and width were determined based on a rectangle, from thin sections.Stress (σ) was calculated () as the force (F) per mean cross-sectional area () of a tested sample.The tensile strength (σmax) is given by () the maximum force (F max) per mean cross-sectional area ().The strain (ε) was calculated () as the fraction of the displacement (ΔL) divided by the original length (L 0) of a tested sample.The elastic modulus, a measure of material stiffness, was calculated () from the slope of the initial (in good approximation) linear (i.e., elastic) part of the respective stress–strain curve (σ/ε).The Poisson’s ratio (ν) of a leaf is calculated as the quotient () of transverse strain (εtrans) and axial strain (εaxial), where d = 2a. The first image taken in the linear-elastic range was used for measuring L 0 and d 0. The linear-elastic range ΔL and Δd were determined from the last image taken. [...] The measurement of self-sealing efficiency in terms of the relative bending angle γ was carried out on leaves of potted plants. The upper side of the leaf was injured in a transversal direction by a razor blade. The cutting depth was chosen so that in each experiment, the parenchyma was injured with certainty. On average, the parenchyma starts at 55% of the radius relative to the leaf centre []. Beginning with an image of the undamaged leaf and then at every 30 sec after the injury, an image was taken with an Olympus SZX9 dissecting microscope via a Color View II digital camera (Soft Imaging Systems GmbH, Münster, Germany) and the software cell D. The images were recorded over a period of 55 min. The movement of the leaf was determined by measuring three points on the leaf (). The first point was selected in the vicinity of the leaf base, the second point close to the injury, and the third one at the leaf tip. The movement of the three points was analysed with the software ImageJ 1.46h and the aid of the Plugin MTrackJ.In each image, the second point was chosen to be the origin of the coordinate system. Starting from this zero point, the two angles α and β were calculated relative to a horizontal line and the straight lines between points 2 and 1 and between points 2 and 3, respectively. The variations in the angle α represent the relative movement of the leaf base whereas variations in angle β were a measure of the rotation of the leaf tip with respect to point 2. The additional movement of the leaf base could be eliminated by the subtraction of angle α from angle β. This led to the actual bending angle δ, representing the actual closing movement of a leaf after an injury. The relative bending angle γ n was calculated from the difference of the actual bending angle (δn) at time t = n min and the actual bending angle before injury, that is, at the beginning of the movement (δ0) at time t = 0 min. Positive values of the relative bending angle represented a closure of the lesion, whereas negative values indicated an opening of the wound ( and ). The experiments were carried out at 24, 33, 49 and 100% relative air humidity and after a water droplet was applied on the wound within times spans of 1, 5, 10, 20, 40 and 55 minutes.The temperature and relative humidity were measured by using a data logger 175-H2 (Testo, Lenzkirch, Germany). At a temperature of 22.95 ± 2.26 °C, the absolute amount of water vapour in the air varies with the humidity as follows: 24% = 4.66 g/m³, 33% = 6.40 g/m³, 49% = 9.51 g/m³, and 100% = 19.41 g/m³. […]

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