M Owens electron microscope (TEM) or light microscope images Our test showed that the average number of chloroplasts estimated by profile counting was more than 10 times underestimated in comparison with the real number of chloroplasts in the model. 5A–E) and 3D chloroplast arrangement in a mesophyll cell (Fig. . JW I. (D) Stacks of optical sections were acquired at the positions of rectangles in (C) with higher resolution. SM 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. I In palisade cells kept under dim white light (15 µmol m −2 s −1), actin signals were mainly observed like a meshwork surrounding each chloroplast . J J This method enables particles of varying shape, such as chloroplasts in a cell and cells within a tissue, to be sampled interactively and counted in an unbiased way. J EK Zechmann JX L Chloroplasts are important organelles of plant photosynthesizing cells as loci where the photosynthetic processes take place. 3B–D). MV 2A). V J Jiang MJ 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). Katoh T TG Under this condition, 85% of the palisade cells exhibited such a … 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. layer of the leaf is adapted to absorb light efficiently. Pitrat LM Tomato Protein Phosphatase 2C (SlPP2C3) influences fruit ripening onset and fruit glossiness, Fruit presence induces polar auxin transport in citrus and olive stem and represses IAA release from the bud, Leaf apoplastic alkalisation promotes transcription of the ABA synthesising enzyme Vp14 and stomatal closure in, Molecular and functional analysis of a brown planthopper resistance protein with two nucleotide binding site domains, The inverse relationship between solar-induced fluorescence yield and photosynthetic capacity: benefits for field phenotyping, About the Society for Experimental Biology, Receive exclusive offers and updates from Oxford Academic, Copyright © 2020 Society for Experimental Biology. B The average number of chloroplasts per mesophyll cell estimated by profile counting in 2D was 20.92±1.32, while the average number of chloroplasts estimated by the disector probe in 3D was 10 times higher, i.e. Kubínová This method has already been used successfully for estimation of mesophyll cell number (Albrechtová and Kubínová, 1991; Kubínová, 1991, 1993, 1994; Kubínová et al., 2002; Albrechtová et al. 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). The chloroplast number per cell represents a frequently examined quantitative anatomical parameter, reflecting various leaf internal and external conditions. Your comment will be reviewed and published at the journal's discretion. 6). Methods based on these principles were then improved (Possingham and Smith, 1972), and modified protocols have been widely used, e.g. Acquired values were compared using statistical analysis (paired t-test) in NCSS 2000 program (Number Cruncher Statistical Systems, Kaysville, Utah, USA). Molin The probes a, b, and c are located next to each other. B I Photosynthesis, chloroplast number, ribulose-1,5-bisphosphate carboxylase, chlorophyll, and DNA in protoplasts, Chromosome doubling via tuber disc culture in dihaploid potato as determined by confocal microscopy, The use of chloroplast numbers in guard cells as a means of distinguishing the chromosome races of, The study of plant structure: principles and selected methods, Leaf anatomy as a constraint for photosynthetic acclimation: differential responses in leaf anatomy to increasing growth irradiance among three deciduous trees, Changes in chloroplast number per cell during leaf development in spinach (, Factors affecting chloroplast replication in spinach, Plastid replication and development in the life-cycle of higher-plants, Annual Review of Plant Physiology and Plant Molecular Biology, Rapid image analysis screening procedure for identifying chloroplast number mutants in mesophyll cells of, Chloroplast division and expansion is radically altered by nuclear mutations in, Chloroplast number in guard cells as ploidy indicator of, Changes in tomato leaves induced by NaCl stress: leaf organization and cell ultrastructure. Fewer chloroplasts in the spongy mesophyll because most of the light energy is absorbed by the chloroplasts of the palisade mesophyll. Lister In the present study, sample preparation and image acquisition were carefully designed according to the principle of SUR sampling (Gundersen and Jensen, 1987). are column-shaped and arranged closely together; Ellis Niinemets Fojtík NJ Oxford University Press is a department of the University of Oxford. . Comparison of 3D- and 2D-based methods for chloroplast number estimation. (G) 3D reconstruction of the mesophyll cell with chloroplasts from a stack of optical sections from confocal microscopy using Ellipse software. Panneerselvam Jiang Kubínová We hypothesised that the number of chloroplast profiles counted in cell sections in 2D would be different from the number of chloroplasts counted by an unbiased method in 3D. J Login . Kubínová C The spongy mesophyll is found towards the lower epidermis. Boffey Mesophyll cells are found in the plant's leaves. Suppose there were many chloroplasts in the cells of the upper epidermis. sectioned) with a higher probability. Takechi ". Robertson The images were captured during 2009 and analysed during 2009–2012. Most mesophyll cells were too high to be focused through; therefore, a method based on direct counting of chloroplasts within the entire cell could not be used. granules in leaves have been made on a transmission The difference between both estimators tested can be also explained theoretically as shown below. . . M In each position, 20 µm thick stacks comprising 41 serial optical sections 0.5 µm apart were acquired using a plan apochromatic 63× water-immersion objective (NA 1.2) with zoom 2× and resolution 512×512 pixels (119×119 µm) (Fig. 80 to 120 Chloroplasts/cell. J O For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Light absorption happens in the palisade mesophyll. M Hirose JV We concluded that maceration methods were unsuccessful due to lignification of mesophyll cell walls detected histochemically (Fig. ), Scheme of the hypothetic spatial mosaic of 3D disector sampling probes illustrating that particles are unambiguously sampled by only one disector sampling probe. Lhotáková Thomson Messerli Zechmann 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. Teng . Wazuddin Tholen Chloroplasts lying within this block or intersecting its planes are counted, except those intersecting the exclusion planes. Rotino of sections through mesophyll cells." L The disector probe is a 3D block. one and about 30 chloroplasts per mesophyll Bendich GR 1). YT They are a type of ground tissue that's actually found as two distinct types in the leaves. Possingham L Pazourek, 1966; Possingham and Saurer, 1969). . HJG Y Lin However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. Sung (I) 2D sections through the model cell shown in (F) and (H). Leech Hortensteiner Dinkins 2. To eliminate the possible shrinkage of a tissue caused by cutting, the dry stem pith of Sambucus nigra L. was used to fix the sample during cutting. In this scheme, in probe a eight chloroplasts are counted (labelled a), in probe b eight chloroplasts are counted (labelled b), and in probe c nine chloroplasts are counted (labelled c). M Kubínová Rutherford Counted particles are those within the probe not intersecting the exclusion planes: point, counted particle; cross, particle is not counted. 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). BE . Possingham current-year needles were collected from crowns of 18-year-old trees planted on an experimental site of the Global Change Research Centre, Academy of Sciences, Czech Republic, the Bílý Kříž in Moravskoslezské Beskydy mountains, in 2004 (Urban et al., 2001). . . Please check for further notifications by email. Wang I 5F, H, Supplementary Video S3 at JXB online) in order to demonstrate comparison of the 3D disector and the 2D profile counting methods. J Marrison Pokorný The studies were conducted on various plant species, considering herbaceous species in the majority of cases. Chloroplasts that lie fully inside the probe are always counted. Lamppa Moreover, it is known that anatomical parameters often exhibit gradients along the leaf blade (e.g. The 3D model also showed that neglecting the 3D appearance of the cell led to the underestimation of the number of chloroplasts (Fig. The palisade mesophyll consists of chloroplasts with chlorophyll that absorb the light energy. C McCully Ellis . The Ellipse Disector module (Tomori et al., 2001) enables application of a virtual 3D probe to the stack of serial optical sections. C Koffler GL Janáček Weibel Comparison of ploidy level screening methods in watermelon: High resolution imaging of temporal and spatial changes of subcellular ascorbate, glutathione and H, Histochemical and biochemical approaches to the study of phenolic compounds and peroxidases in needles of Norway spruce (, The unbiased estimation of number and sizes of arbitrary particles using the disector, Blocking the metabolism of starch breakdown products in Arabidopsis leaves triggers chloroplast degradation, Changes in photosynthesis and other chloroplast traits in lanceolate leaflet isoline of soybean, Leaf functional anatomy in relation to photosynthesis, DISECTOR program for unbiased estimation of particle number, numerical density and mean volume, Glass domes with adjustable windows: a novel technique for exposing juvenile forest stands to elevated CO, Chloroplast numbers, mitochondrion numbers and carbon assimilation physiology of, Practical methods for biological morphometry, Leaf structural characteristics are less important than leaf chemical properties in determining the response of leaf mass per area and photosynthesis of, A vertical gradient of the chloroplast abundance among leaves of, Cytological modifications in zucchini yellow mosaic virus (ZYMV)-infected Styrian pumpkin plants. Itoh J H . Fewer chloroplasts in the spongy mesophyll because most of the light energy is absorbed by the chloroplasts of the palisade mesophyll. Maslova AJ Mesophyll is a major tissue for photosynthesis, and contributes about 80% of total RNA from leaves. That's why the top of the leaf is darker than the bottom of the leaf. MJ The chloroplasts in these cells absorb a major portion of the light energy used by the leaf. The cells that form palisade tissue are the two upper layers of mesophyll cells, the cells that contain many chloroplasts to maximise photosynthesis. FJM Gopi * To whom correspondence should be addressed. Charles University in Prague, Faculty of Science, Department of Experimental Plant Biology. Zellnig Ramirez Sung and Chen (1989) extracted chloroplasts from macerated leaves of Glycine max L. Merrill and counted chloroplasts per leaf using a haemocytometer—a special microscope slide with an engraved graticule. Gundersen It was found that some cells might have been pulled out of the specimen during cutting (Lhotáková et al., 2008), and this is why the disector probe was placed in the middle of a stack. 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. 3A). Albrechtová T Terashima . (This figure is available in colour at JXB online. The mesophyll layer is made up of the palisade cell and spongy parts. . The key difference between mesophyll and bundle sheath cells is that in C4 plants, light-dependent reactions of photosynthesis take place in the mesophyll cells, while light-independent reactions or Calvin cycle take place in the bundle sheath cells.. C4 plants are a group of plants that carry out C4 photosynthesis or C4 carbon fixation. B Scott Iqbal UK Cells compactly arranged at right angle to the epidermis. Cvikrová The present 3D model of mesophyll cell (Fig. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. Zubkova The particles are sampled without bias if each particle has the same probability of being sampled, i.e. Srinivasa Reddy Fixation and maceration of Spinacia oleracea L. leaves enabled the separation of mesophyll cells, which were then flattened into a single plane of focus, allowing the chloroplasts to be viewed in a single layer and thus to be easily counted under phase contrast in an optical microscope with an eyepiece graticule. 1. Thale cress Arabidopsis thaliana. Soukupová 2B), as proved in a histochemical study by Soukupová et al. The cells possess chloroplast but three to four times lesser than the palisade tissue. L . (F) Face view of a model of a mesophyll cell with 210 chloroplasts made in Cortona 3D software. Numerous maceration methods were tested in order to count chloroplasts directly in separated spruce mesophyll cells. . CA Where is gluconse made in plants? JR . Eicke Z J 4). The bottom look-up plane belongs to exclusion planes together with two side planes. Nevertheless, sampling design is crucial for yielding unbiased results, as anatomical gradients within a leaf blade were demonstrated also for the number of chloroplasts per cell (Dean and Leech, 1982). The number of chloroplasts cannot be directly estimated by counting profiles in the section, because the areal density of profiles is related not only to density Nv but also to height h: . G Possingham A double disector (Gundersen, 1986) was used: the probe was used in both directions, i.e. Müller In general, plant mesophyll cells contain a large central vacuole and, in vivo, chloroplasts are usually located in the peripheral cytoplasm, close to the cell wall. Matsum. In plants, chloroplasts are concentrated particularly in the parenchyma cells of the leaf mesophyll (the internal cell layers of a leaf). . CY Mesophyll cells are specialized for photosynthesis. Kubínová © The Author 2013. There are more in the palisade cells than in the spongy mesophyll. One of the most frequently used methods for estimation of chloroplast number per mesophyll cell in herbaceous plants in 2D is counting chloroplasts in separated mesophyll cells obtained by maceration procedures, as described by Possingham and Saurer (1969). From these images, the areas of needle cross-sections and the proportions of mesophyll were estimated by the point counting method (Weibel, 1979) using the Ellipse Point Grid software module (Fig. O L In simpler terms, they are known as leaf cells. Palisade and spongy mesophyll cells are sub-tissues that have different morphologies and physiologies. This is clearly demonstrated in Fig. Spruce needles were collected from south- and south-west-facing branches from the middle crown part and were stored in a deep freeze until processing (Lhotáková et al., 2008). (C) Sampling frames were superimposed on a needle transverse section using the Rectangles module in Ellipse software. However, the distribution of peroxisomes in palisade mesophyll cells of chup1 differed from that of the wild type, because they remained at the bottom of the cells, along with the chloroplasts. (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. The number of chloroplasts per cell was calculated as the ratio of the chloroplast profile number per needle area to the cell profile number per needle area, while the needle area was estimated previously by the point counting method (Weibel, 1979). Y K The vascular tissue of leaves is comparable in many respects to that of stems with the conspicuous exception that in leaves there is great variation in the size of the vascular bundles. N 6). N Wang A leaf (plural leaves) is the principal lateral appendage of the vascular plant stem, usually borne above ground and specialized for photosynthesis.The leaves and stem together form the shoot. 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. Wang 4. 5) do not correspond to in vivo chloroplast arrangement; however, they illustrate well the size of Norway spruce mesophyll cells, their variable and irregular shape, as well as the number and size of chloroplasts within a cell. Garvey The estimated number of chloroplasts per mesophyll cell was calculated by the ratio of the chloroplast number and the mesophyll cell number per needle volume: where estNNcell(chl) is the estimated number of chloroplasts per mesophyll cell, estNVneedle(cell) is the estimated number of mesophyll cells per needle volume, and estNVneedle(chl) is the estimated number of chloroplasts per needle volume. Key to deducing the distinct functions of specialized cells from gene expression profiles mesophyll layer of spherical or ovoid with... Times lesser than the palisade tissue, 2000 ) obtained in 2D 3D... B–D ) three optical sections acquired by confocal microscopy using Ellipse software factor…. Mesophyll are the two upper layers of a Norway spruce needle in histochemical... Cells and the method used for counting chloroplasts by applying the disector the... Many chloroplast in the results yielded by both methods, we are aware that 3D... 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Autopolyploidy in sugar beets, ploidy effects in isogenic populations of alfalfa are... Or purchase an annual subscription Feb158 ( 2 ):905-16. doi: 10.1104/pp.111.186957 STANDS4 Network... palisade of... 3D model of a mesophyll cell chosen for 3D reconstruction was a smaller one, so that it possible... 1955 ) and ( H ) number of chloroplasts with chlorophyll that absorb the light is. Can be seen in the Cortona software ( Fig, right below the cuticle and the method for. Was statistically significantly different ( P < 0.001 ) cells … 1 of optical sections the. From the spongy mesophyll because most of the light energy used by the chloroplasts in the leaves, below.

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