J Müller (2000). 19. We concluded that maceration methods were unsuccessful due to lignification of mesophyll cell walls detected histochemically (Fig. Having no chloroplasts in the spongy mesophyll creates more space for light to be absorbed. The mesophyll layer is made up of the palisade cell and spongy parts. Kubínová Ghannoum 5F, H, I, Supplementary Video S3). Yamasaki question Suppose there were many chloroplast in the cells of the upper epidermis. Terashima Eicke D The chloroplast number per cell represents a frequently examined quantitative anatomical parameter, reflecting various leaf internal and external conditions. Gundersen A . Well-established methods for estimation of the chloroplast number per cell previously used in two-dimensional (2D) space are based on: (i) counting chloroplasts in stomatal guard cells in epidermal peels, (ii) determining the number of chloroplasts in flattened mesophyll cells after maceration, and (iii) counting profiles of chloroplasts in leaf sections. I Coronado at first, cells were counted by the disector with the reference plane and look-up planes in the positions shown in Fig. (C) Sampling frames were superimposed on a needle transverse section using the Rectangles module in Ellipse software. Dinkins The cylindrical shape of palisade cells allows a large amount of light to be absorbed by the chloroplasts. Counting chloroplast profiles in 2D yielded values of chloroplast number per cell that were 10 times underestimated in comparison with the mean number of chloroplasts estimated by the unbiased disector method in 3D. We determined that the systematic shift in an estimated value is inversely related to the ratio of the height of chloroplasts to the height of mesophyll cells. Several stacks were acquired in each cross-section to cover its whole area; altogether, 157 stacks were acquired. To estimate the volume of the spruce needle within each disector probe, the needle area in the middle image of the stack was determined by the point-grid method and multiplied by the disector height. TP SA K Mesophyll cells contain chloroplasts and have an irregular shape with lobed anticlinal cell walls and are prolonged in the direction perpendicular to the needle surface (Fig. A model of a simplified mesophyll cell with 210 chloroplasts was made in the Cortona software (Fig. Leech Chloroplasts are cell organelles where photosynthesis takes place. . Lamppa The acquired images were analysed using Ellipse software (ViDiTo, KoÅ¡ice, Slovakia, http://www.ellipse.sk/, last accessed 21 November 2013), which offers special modules for stereological measurements. for the plant Beneath the palisade mesophyll are the spongy mesophyll cells, which also perform photosynthesis. Chloroplast counting using the virtual 3D disector probe. Chen . The maceration process was further adapted and applied to Arabidopsis thaliana L. and counting was accomplished after chloroplast thresholding using an image analysis program (Pyke and Leech, 1991; Marrison et al., 1999; Stettler et al., 2009). 2F, Supplementary Videos S1 and Supplementary Data, at JXB online). 2B) and observed regularly in our previous study (Soukupová et al., 2000). . M . Palisade cells occur in dicotyledonous plants, and also in the net-veined monocots, the Araceae and Dioscoreaceae. . Kubínová For example, the extreme variability can be seen in the number of chloroplasts in Arabidopsis mesophyll cells estimated by various methods. JV Boffey The probes a, b, and c are located next to each other. . Z 1). within a substack of six optical sections 2 μm apart, the size of the sampling frame was adjusted to cover the whole cross-section area. V (This figure is available in colour at JXB online.). . Thomson Fujita Zechmann Mesophyll is a major tissue for photosynthesis, and contributes about 80% of total RNA from leaves. N Jiang . H Stettler (E) Estimation of needle cross-sectional area by a point counting method using the Point Grid module in Ellipse software (F) Two frames showing the subsequent acquisition of series for counting mesophyll cells. CY Range. Itoh Nicholson Effect of autopolyploidy in sugar beets, Ploidy effects in isogenic populations of alfalfa. CO2 concentration (Wang et al., 2004; Teng et al., 2006). KJ Cell-type-specific transcription factors are key to deducing the distinct functions of specialized cells from gene expression profiles. All our practical and theoretical tests thus clearly showed that the frequently used method for chloroplast number estimation by counting profiles of particles from 2D sections (e. g. Boffey et al., 1979; Sam et al., 2003; Wang et al., 2004; Hayashida et al., 2005; Teng et al., 2006; Gopi et al., 2008) yielded biased estimates and that the results may be one order of magnitude different from the real chloroplast numbers. JE Sampling of 0.2mm thick needle cross-sections cut off using a hand microtome and placed into a drop of water was done in a systematic uniform random (SUR) way at intervals of 3mm (Fig. NJ 2A). The mesophyll cell density (number of cells per needle volume) was estimated by the formula (Sterio, 1984; Gundersen, 1986): where estNVneedle(cell) is the estimated number of mesophyll cells per needle volume, is the sum of all sampled cells in all disector probes within a needle, ∑Pi is the sum of all points falling within a needle in all disector probes (for calculation needle volume in each probe) used for cell counting, p is the number of test points in a grid in a sampling frame used for cell counting, a is the area of the disector sampling frame (the base plane of the 3D probe) used for cell counting, and h is the height of the disector probe used for cell counting. 3B–D). Leech ME . the number of chloroplasts per cell, also exist in dependence on distance of a leaf from the root system (Possingham and Saurer, 1969). K SP J A double disector (Gundersen, 1986) was used: the probe was used in both directions, i.e. However, a SUR sampling, along with both assumptions—the unbiased method for counting particles and the sufficient number of measurements—are essential for getting unbiased and accurate results (Sterio, 1984). For maceration of needle mesophyll samples, in order to count chloroplasts directly in separated cells, different methods were tested: a 1M aqueous solution of HCl (Possingham and Saurer, 1969); a 3.5% aqueous solution of glutaraldehyde and 0.1M aqueous solution of Na2EDTA (Boffey et al., 1979); and a solution of 10% aqueous CrO3 and 10% aqueous HNO3 (O’Brian and McCully, 1981). Palisade cells are plant cells located on the leaves, right below the epidermis and cuticle. JV For mesophyll cell counting, 30 µm thick stacks comprising 16 serial optical sections 2 µm apart were acquired by using a plan apochromatic 20× water-immersion objective (NA 0.7) with resolution 2048×2048 pixels (750×750 µm) (Fig. . G This result was statistically significantly different (P<0.001). We are grateful to Drahomíra Bartáková for technical assistance, Barbora Radochová for assistance with confocal microscopy, and Marianna Dubánková for help with sample collection. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. Slow development of leaf photosynthesis in an evergreen broad-leaved tree, Genetic studies on the number of plastid in stomata. Zhu Crumpton-Taylor M, Grandison S, Png KM, Bushby AJ, Smith AM. . Marek TG To visualize differences in the results yielded by both methods, we used the 3D model of a mesophyll cell (Fig. The positions of chloroplasts do not correspond to the in vivo state. Thank you for submitting a comment on this article. The Ellipse Disector module (Tomori et al., 2001) enables application of a virtual 3D probe to the stack of serial optical sections. S . We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. WW Particles lying within this block or intersecting its planes, except the exclusion ones, are counted (Fig. 3. In the 3D method, we summed the number of chloroplasts per volume as estimated by the disector method in each cross-section and divided it by the sum of the estimated number of cells in each cross-section. The particles are sampled without bias if each particle has the same probability of being sampled, i.e. . Charles University in Prague, Faculty of Science, Department of Experimental Plant Biology. LE (A) Scheme of the disector probe with chloroplasts. Adachi Katoh Owens Schrader Particles are labelled according to the label of the probe they are counted in. Applying the maceration method to coniferous needles is problematic or even impossible, as shown in our study, as they contain phenolic compounds and lignin in cell walls (Fig. M In mesophyll cells, chloroplasts are usually located next to the cytoplasmic membrane adjacent to intercellular spaces to decrease the resistance to CO2 diffusion (Terashima et al., 2011). Iqbal Chloroplasts labelled by crosses are not counted by any of the three probes shown here, but they would be unambiguously sampled by adjacent hypothetical probes. 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