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![Images of leaf cells in leaf cell suspension. (a) Spongy mesophyll cell from Arabidopsis (DIC). (b) Palisade parenchyma cell from Heliotropium calcicola, a C 3 photosynthetic species (bright field). (c) Arabidopsis mesophyll cells with 1-2 large plastids isolated from the mutant line pdv1pdv2 generated by Miyagishima et al. [13]. DIC image by Dr. Siddhartha Dutta. (d) Bundle sheath cells of the C 4 photosynthetic species Atriplex rosea stained with aniline blue illustrating callose at pit fields on a lateral wall between two bundle-sheath cells (arrows). Imaged with fluorescence microscope. (e) Bundle-sheath cells of Oryza sativa stained with IKI (brown). Imaged with DIC. Bars, 10 μm. bs bundle sheath; c chloroplast; m mesophyll; s stoma](profile/Roxana-Khoshravesh/publication/326212633/figure/fig1/AS:647640438018048@1531420921433/mages-of-leaf-cells-in-leaf-cell-suspension-a-Spongy-mesophyll-cell-from-Arabidopsis.png)
Images of leaf cells in leaf cell suspension. (a) Spongy mesophyll cell from Arabidopsis (DIC). (b) Palisade parenchyma cell from Heliotropium calcicola, a C 3 photosynthetic species (bright field). (c) Arabidopsis mesophyll cells with 1-2 large plastids isolated from the mutant line pdv1pdv2 generated by Miyagishima et al. [13]. DIC image by Dr. Siddhartha Dutta. (d) Bundle sheath cells of the C 4 photosynthetic species Atriplex rosea stained with aniline blue illustrating callose at pit fields on a lateral wall between two bundle-sheath cells (arrows). Imaged with fluorescence microscope. (e) Bundle-sheath cells of Oryza sativa stained with IKI (brown). Imaged with DIC. Bars, 10 μm. bs bundle sheath; c chloroplast; m mesophyll; s stoma
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Imaging of mesophyll cell suspensions prepared from Arabidopsis has been pivotal for forming our current understanding of the molecular control of chloroplast division over the past 25 years. In this chapter, we provide a method for the preparation of leaf cell suspensions that improves upon a previous method by optimizing cellular preservation and...
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... cell types in C 4 species [8], and control of plastid volume per cell [numbers and size; 9]. Many of the studies characterizing plastid volume per cell have utilized a very easy, rapid, and informative method to prepare mesophyll cell suspen- sions that allows imaging of chloroplasts in intact cells using a compound light microscope (Fig. 1a-c; [10]). This technique has proven to be an essential tool for identification of plastid division , such as those with one or two chloroplasts per cell (Fig. 1c), thereby aiding in transforming our understanding of the molecular control of plastid division over the past 25 years [10][11][12][13][14]. More recently the leaf cell ...
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... have utilized a very easy, rapid, and informative method to prepare mesophyll cell suspen- sions that allows imaging of chloroplasts in intact cells using a compound light microscope (Fig. 1a-c; [10]). This technique has proven to be an essential tool for identification of plastid division , such as those with one or two chloroplasts per cell (Fig. 1c), thereby aiding in transforming our understanding of the molecular control of plastid division over the past 25 years [10][11][12][13][14]. More recently the leaf cell suspension method has been employed to study shifts in chloroplast numbers, size, and position- ing in mesophyll and bundle-sheath cells during the evolution from C 3 to ...
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... a more accurate assess- ment of planar chloroplast size and chloroplast distribution within the cell, a trait important for understanding light perception, CO 2 diffusion, and refixation of photorespired CO 2 [15][16][17]. Chloro- plasts in the isolated cells are easily viewed in the absence of staining with regular bright-field microscopy ( Fig. 1b) as well as by using differential interference contrast imaging (DIC; Fig. 1a, c, e). Thus, this technique is accessible to all researchers and amenable for use with all plant ...
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... within the cell, a trait important for understanding light perception, CO 2 diffusion, and refixation of photorespired CO 2 [15][16][17]. Chloro- plasts in the isolated cells are easily viewed in the absence of staining with regular bright-field microscopy ( Fig. 1b) as well as by using differential interference contrast imaging (DIC; Fig. 1a, c, e). Thus, this technique is accessible to all researchers and amenable for use with all plant ...
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... physiology. Plasmodesmata reside in pit fields and C 4 species have been demonstrated to have a greater pit field area at the mesophyll-bundle sheath interface than C 3 species [8]. Leaf cell suspensions can be utilized to image pit fields using the fluorochrome aniline blue, which stains plasmodesmata associated β-1,3 glucan (callose, Fig. 1d; [18]). This use of the leaf cell suspensions provides either an alternative to time-and labor- intensive techniques for immunolocalization of callose on cleared leaf tissue [8] or an initial rapid screening for pit field/plasmodes- mata phenotypes prior to the use of more time-and labor-intensive techniques. As a second example, leaf ...
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... fixable fluorescent probes to detect mitochondria, peroxisomes, as well as other cellular fea- tures is tractable for use with plants that are not easily transformed with organelle-specific probes such as GFP or YFP. Finally, Pyke and Leech [10] used potassium iodide (IKI), which stains starch, to enhance chloroplast detection as illustrated in Fig. 1e. IKI staining can also be used in combination with leaf cell suspensions when an investigator wishes to follow the course of starch accumulation in specific leaf cell types during a given time period. Once cells have been imaged, cellular parameters can be quantified to test hypoth- eses of interest as previously described ...
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... this quick and easy procedure can be used for rapid screening of plastid traits in all plant species, the leaf cell suspen- sions are also valuable for characterizing other cellular features important for photosynthetic physiology. Plasmodesmata reside in pit fields and C 4 species have been demonstrated to have a greater pit field area at the mesophyll-bundle sheath interface than C 3 species [8]. Leaf cell suspensions can be utilized to image pit fields using the fluorochrome aniline blue, which stains plasmodesmata associated β-1,3 glucan (callose, Fig. 1d; [18]). This use of the leaf cell suspensions provides either an alternative to time-and labor- intensive techniques for immunolocalization of callose on cleared leaf tissue [8] or an initial rapid screening for pit field/plasmodes- mata phenotypes prior to the use of more time-and labor-intensive techniques. As a second example, leaf suspensions can be employed to image mitochondria and peroxisomes to test hypotheses addres- sing the role(s) of these organelles in C 4 evolution; unfixed leaf sections can be used for live-cell labeling with fixable fluorescent probes that stain mitochondria and peroxisomes. These labeled cells can subsequently be prepared for leaf cell suspensions. The combined use of leaf cell suspensions and fixable fluorescent probes to detect mitochondria, peroxisomes, as well as other cellular fea- tures is tractable for use with plants that are not easily transformed with organelle-specific probes such as GFP or YFP. Finally, Pyke and Leech [10] used potassium iodide (IKI), which stains starch, to enhance chloroplast detection as illustrated in Fig. 1e. IKI staining can also be used in combination with leaf cell suspensions when an investigator wishes to follow the course of starch accumulation in specific leaf cell types during a given time period. Once cells have been imaged, cellular parameters can be quantified to test hypoth- eses of interest as previously described ...
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... this quick and easy procedure can be used for rapid screening of plastid traits in all plant species, the leaf cell suspen- sions are also valuable for characterizing other cellular features important for photosynthetic physiology. Plasmodesmata reside in pit fields and C 4 species have been demonstrated to have a greater pit field area at the mesophyll-bundle sheath interface than C 3 species [8]. Leaf cell suspensions can be utilized to image pit fields using the fluorochrome aniline blue, which stains plasmodesmata associated β-1,3 glucan (callose, Fig. 1d; [18]). This use of the leaf cell suspensions provides either an alternative to time-and labor- intensive techniques for immunolocalization of callose on cleared leaf tissue [8] or an initial rapid screening for pit field/plasmodes- mata phenotypes prior to the use of more time-and labor-intensive techniques. As a second example, leaf suspensions can be employed to image mitochondria and peroxisomes to test hypotheses addres- sing the role(s) of these organelles in C 4 evolution; unfixed leaf sections can be used for live-cell labeling with fixable fluorescent probes that stain mitochondria and peroxisomes. These labeled cells can subsequently be prepared for leaf cell suspensions. The combined use of leaf cell suspensions and fixable fluorescent probes to detect mitochondria, peroxisomes, as well as other cellular fea- tures is tractable for use with plants that are not easily transformed with organelle-specific probes such as GFP or YFP. Finally, Pyke and Leech [10] used potassium iodide (IKI), which stains starch, to enhance chloroplast detection as illustrated in Fig. 1e. IKI staining can also be used in combination with leaf cell suspensions when an investigator wishes to follow the course of starch accumulation in specific leaf cell types during a given time period. Once cells have been imaged, cellular parameters can be quantified to test hypoth- eses of interest as previously described ...
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... imaging of cell architecture has been an essential tool used to unravel the relationship between plant cellular form, physiology, and evolution since the initial observations of plant cells by Robert Hooke published in Micrographia in 1665. Current techniques employed for characterization of plant cell structure are quite sophisticated in comparison to those used during Hooke's time with, for example, the incorporation of confocal microscopy for live-cell imaging to detect proteins labeled with fluorescent probes such as GFP and YFP [1,2]. The integrated use of confocal microscopy with one or more techniques such as light, scanning, and transmission electron microscopy and immunohistochemistry [3] has contributed substantially to our understanding of photo- synthesis. As examples, these aforementioned techniques, used either in combination or alone, have been indispensable for asses- sing pyrenoid formation [4], thylakoid assembly [5,6], formation of dimorphic chloroplasts [7], plasmodesmata distribution between the two photosynthetic cell types in C 4 species [8], and control of plastid volume per cell [numbers and size; 9]. Many of the studies characterizing plastid volume per cell have utilized a very easy, rapid, and informative method to prepare mesophyll cell suspen- sions that allows imaging of chloroplasts in intact cells using a compound light microscope (Fig. 1a-c; [10]). This technique has proven to be an essential tool for identification of plastid division , such as those with one or two chloroplasts per cell (Fig. 1c), thereby aiding in transforming our understanding of the molecular control of plastid division over the past 25 years [10][11][12][13][14]. More recently the leaf cell suspension method has been employed to study shifts in chloroplast numbers, size, and position- ing in mesophyll and bundle-sheath cells during the evolution from C 3 to C 4 photosynthesis in over 12 lineages [15,16] and identifi- cation of candidate genes involved in the evolutionary transition of chloroplast traits between those two cell types [16]. The latter two studies [15,16] underscore the applicability of this technique for use in a broad number of plant ...
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... imaging of cell architecture has been an essential tool used to unravel the relationship between plant cellular form, physiology, and evolution since the initial observations of plant cells by Robert Hooke published in Micrographia in 1665. Current techniques employed for characterization of plant cell structure are quite sophisticated in comparison to those used during Hooke's time with, for example, the incorporation of confocal microscopy for live-cell imaging to detect proteins labeled with fluorescent probes such as GFP and YFP [1,2]. The integrated use of confocal microscopy with one or more techniques such as light, scanning, and transmission electron microscopy and immunohistochemistry [3] has contributed substantially to our understanding of photo- synthesis. As examples, these aforementioned techniques, used either in combination or alone, have been indispensable for asses- sing pyrenoid formation [4], thylakoid assembly [5,6], formation of dimorphic chloroplasts [7], plasmodesmata distribution between the two photosynthetic cell types in C 4 species [8], and control of plastid volume per cell [numbers and size; 9]. Many of the studies characterizing plastid volume per cell have utilized a very easy, rapid, and informative method to prepare mesophyll cell suspen- sions that allows imaging of chloroplasts in intact cells using a compound light microscope (Fig. 1a-c; [10]). This technique has proven to be an essential tool for identification of plastid division , such as those with one or two chloroplasts per cell (Fig. 1c), thereby aiding in transforming our understanding of the molecular control of plastid division over the past 25 years [10][11][12][13][14]. More recently the leaf cell suspension method has been employed to study shifts in chloroplast numbers, size, and position- ing in mesophyll and bundle-sheath cells during the evolution from C 3 to C 4 photosynthesis in over 12 lineages [15,16] and identifi- cation of candidate genes involved in the evolutionary transition of chloroplast traits between those two cell types [16]. The latter two studies [15,16] underscore the applicability of this technique for use in a broad number of plant ...
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... we describe the method for preparation of cell suspen- sions from leaves for imaging of chloroplasts and other cellular features that are important for photosynthetic physiology. We pro- vide slight modifications to the original technique to enhance the ease of cell separation and quality of cell preservation through the use of a lower concentration of tissue fixative and a pectinase treatment. The pectinase treatment enables leaf cell suspensions to be made from a wide range of species that have tightly adherent cells. Enhanced cell preservation provides a more accurate assess- ment of planar chloroplast size and chloroplast distribution within the cell, a trait important for understanding light perception, CO 2 diffusion, and refixation of photorespired CO 2 [15][16][17]. Chloro- plasts in the isolated cells are easily viewed in the absence of staining with regular bright-field microscopy ( Fig. 1b) as well as by using differential interference contrast imaging (DIC; Fig. 1a, c, e). Thus, this technique is accessible to all researchers and amenable for use with all plant ...
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... we describe the method for preparation of cell suspen- sions from leaves for imaging of chloroplasts and other cellular features that are important for photosynthetic physiology. We pro- vide slight modifications to the original technique to enhance the ease of cell separation and quality of cell preservation through the use of a lower concentration of tissue fixative and a pectinase treatment. The pectinase treatment enables leaf cell suspensions to be made from a wide range of species that have tightly adherent cells. Enhanced cell preservation provides a more accurate assess- ment of planar chloroplast size and chloroplast distribution within the cell, a trait important for understanding light perception, CO 2 diffusion, and refixation of photorespired CO 2 [15][16][17]. Chloro- plasts in the isolated cells are easily viewed in the absence of staining with regular bright-field microscopy ( Fig. 1b) as well as by using differential interference contrast imaging (DIC; Fig. 1a, c, e). Thus, this technique is accessible to all researchers and amenable for use with all plant ...
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After entering the leaf, CO2 faces an intricate pathway to the site of photosynthetic fixation embedded within the chloroplasts. The efficiency of CO2 flux is hindered by a number of structural and biochemical barriers which, together, define the ease of flow of the gas within the leaf, termed mesophyll conductance. Previous authors have identified...
Citations
... Increasing measurement throughput is also important to properly screen transgenic plants carrying multigene constructs for increased photosynthesis in replicated field trials. Measuring in vivo photosynthetic parameters in tandem with other techniques, e.g., including those discussed herein such as quantifying photosynthesis-related enzymes [104], purifying RuBisCO for determining catalytic constants and [105] quantifying RuBisCO activity and activation state [106], evaluating thylakoid lipid content [107], and determining high-resolution ultrastructure [108] and chloroplast structure [109], would be useful to more fully understand the physiological and molecular effects of gene function or environmental changes, as determined by the experiment. Fortunately, there are several emergent approaches that may help increase throughput, in addition to the PhotosynQ platform mentioned previously. ...
Measurements of in vivo photosynthesis are powerful tools that probe the largest fluxes of carbon and energy in an illuminated leaf, but often the specific techniques used are so varied and specialized that it is difficult for researchers outside the field to select and perform the most useful assays for their research questions. The goal of this chapter is to provide a broad overview of the current tools available for the study of in vivo photosynthesis so as to provide a foundation for selecting appropriate techniques, many of which are presented in detail in subsequent chapters. This chapter also organizes current methods into a comparative framework and provides examples of how they have been applied to research questions of broad agronomical, ecological, or biological importance. The chapter closes with an argument that the future of in vivo measurements of photosynthesis lies in the ability to use multiple methods simultaneously and discusses the benefits of this approach to currently open physiological questions. This chapter, combined with the relevant methods chapters, could serve as a laboratory course in methods in photosynthesis research or as part of a more comprehensive laboratory course in general plant physiology methods.
In the leaves of C3 species such as rice (Oryza sativa), mesophyll cells contain the largest compartment of photosynthetically active chloroplasts. In contrast, plants that use the derived and more efficient C4 photosynthetic pathway have a considerable chloroplast compartment in both bundle sheath and mesophyll cells. Accordingly, the evolution of C4 photosynthesis from the ancestral C3 state required an increased chloroplast compartment in the bundle sheath. Here, we investigated the potential to increase chloroplast compartment size in rice bundle sheath cells by manipulating brassinosteroid signalling. Treatment with brassinazole, a brassinosteroid biosynthesis inhibitor, raised leaf chlorophyll content and increased the number but decreased the area of chloroplasts in bundle sheath cells. Ubiquitous overexpression of the transcription factor-encoding BRASSINAZOLE RESISTANT 1 (OsBZR1) increased bundle sheath chloroplast area by up to 45%, but these plants became chlorotic. However, when OsBZR1 expression was driven by a bundle sheath-specific promoter, the negative effects on growth and viability were alleviated whilst chloroplast area still increased. In summary, we report a role for brassinosteroids in controlling chloroplast area and number in rice and conclude that cell-specific manipulation of brassinosteroid signalling can be used to manipulate the chloroplast compartment in rice bundle sheath cells.
Plant leaves contain multiple cell types which achieve distinct characteristics whilst still coordinating development within the leaf. The bundle sheath possesses larger individual cells and lower chloroplast content than the adjacent mesophyll, but how this morphology is achieved remains unknown. To identify regulatory mechanisms determining bundle sheath cell morphology we tested the effects of perturbing environmental (light) and endogenous signals (hormones) during leaf development of Oryza sativa (rice). Total chloroplast area in bundle sheath cells was found to increase with cell size as in the mesophyll but did not maintain a 'set-point' relationship, with the longest bundle sheath cells demonstrating the lowest chloroplast content. Application of exogenous cytokinin and gibberellin significantly altered the relationship between cell size and chloroplast biosynthesis in the bundle sheath, increasing chloroplast content of the longest cells. Delayed exposure to light reduced the mean length of bundle sheath cells but increased corresponding leaf length, whereas premature light reduced final leaf length but did not affect bundle sheath cells. This suggests that the plant hormones cytokinin and gibberellin are regulators of the bundle sheath cell-chloroplast relationship and that final bundle sheath length may potentially be affected by light-mediated control of exit from the cell cycle.
