Using these anatomical data and measured photosynthetic rates in these C4 species, we have now calculated symplastic C4 acid flux per PD across the M-BS interface. Bundle sheath cells constitute ∼15% of chloroplast-containing cells in an Arabidopsis leaf (Kinsman and Pyke, 1998), and they conduct fluxes of compounds both into the leaf, particularly during leaf development, and out of the leaf, during export of photosynthates and during senescence. This position-specific gene expression occurs in the leaf primordium at a time when neither BS nor M cells are histologically distinguishable, suggesting that positional control of BS and M cell photosynthetic development may begins very early in leaf development (Langdale et al., 1988a). Most patches are covered in mesophyll cell debris, and only sites lacking cell debris (asterisk) were used for plasmodesmata quantification. Similarly, a higher PD per µm2 pitfield in the M-M cell interface was found in C4 species, S. viridis and maize, compared with the C3 species, rice and wheat (Figure 6, Table 1). To test the hypothesis that BSEs reduce the hydraulic resistance from the bundle sheath to the epidermis ( r be) and thereby accelerate hydropassive stomatal movements, we compared stomatal responses with reduced humidity and leaf excision among 20 species with heterobaric or … The tissue sections were then embedded in Spurr’s resin at 65°C overnight. The BS surface areas per unit leaf area (Sb) for each species were calculated using the equation described by Pengelly et al. Source for information on bundle sheath cells: A Dictionary of Biology dictionary. In some plant organs, patterns of cell division are sufficiently regular that cell fates can be accurately predicted on the basis of lineage, such as in the Arabidopsis root (Dolan et al., 1993; Dolan et al., 1994). In contrast, the mesophyll is typical of the type of photosynthetic tissue found in leaves of most C 4 plants and comprises thin walled cells with abundant intercellular spaces. Bundle sheath cells, contains starch-rich chloroplasts (agranal) lacking grana in a large amount. Vascular bundle is the isolated unit of the longitudinal strands of conducting tissues consisting essentially of xylem and phloem, frequently with a sheath of thick walled cells or other interspersed cells. (2014) used values between 0.3% and 3% for the percentage of M-BS cell wall interface area occupied by PD. 3A), and PEPCase accumulates only in cytosol of mature M cells (Fig. Head over to the Node to find the details of the next event. However, these data are difficult to compare with the quantitative data derived here from combined scanning electron microscopy and 3D immunolocalization methods. Arrows indicate procambial strands in wild-type (A,B) and late divisions within procambial strands in the tan1 mutant (C,D). Bar, 10 μm (C,D). Quantification of PD at this interface will enable both modeling of C4 metabolic flux and the design of experiments to determine the genetic regulation and evolution of the symplastic transport mechanisms of C4 plants. However, in intact tissues, the absorption and scattering of light by cell walls and cell contents limits detection of PD to the outer layers such as the leaf epidermis and trichomes (Faulkner et al., 2008). It is possible that the lineage-based mechanism by which these cells differentiate as BS might also depend on positional information. The superior photosynthetic performance of C4 crop plants is largely due to the biochemical and anatomical specialization that results in concentration of CO2 at the active site of Rubisco, reducing photorespiration and permitting Rubisco to operate close to its catalytic optimum. The youngest fully expanded leaves from seedlings, 9 d after germination, were used. Separation of the biochemical CO2 pump in the mesophyll (M; where atmospheric CO2 is initially fixed into C4 acids by PEP carboxylase) from the site of decarboxylation and refixation by Rubisco in the bundle sheath (BS) means that C4 acids must move into the BS and C3 products return to the M at rates greater than the net rate of photosynthesis (Hatch and Osmond, 1976; von Caemmerer and Furbank, 2003). In order to determine whether clusters of BS-like cells represent cell clones, we visualized the clonal relationships among BS cells in both wild-type leaves and tan1 leaves using wd sector analysis. © 2020   The Company of Biologists Ltd   Registered Charity 277992, Specification of bundle sheath cell fates during maize leaf development: roles of lineage and positional information evaluated through analysis of the. Guard cell pairs are formed from meristemoids, which are produced through asymmetric cell divisions. It also provided a new and potentially improved method to measure BS and M cell size, an important consideration in quantifying pitfield distribution on a cell interface area basis and important parameters for modeling C4 photosynthesis (von Caemmerer, 2000; von Caemmerer and Furbank, 2003; Wang et al., 2014). Mesophyll and bundle sheath cells are connected through numerous cytoplasmic sleeves called plasmodesmata whose permeability at leaf level is called bundle sheath conductance. Examples in which a cell whose fate is already committed or restricted transmits that state to its progeny are more common in the animal developmental biology literature than in the plant literature. Sporadic loss of a ring chromosome uncovering the wd mutation produces clonally derived and non-revertable albino sectors in a green background that can be evaluated in all subepidermal layers of the leaf by conventional microscopy. In C3 species, the estimates give the fluxes per PD needed if a C4 photosynthetic pathway were installed without changes in PD frequency (Table 2). The leaves contain a ring of mesophyll cells, containing a few small chloroplasts concerned with the initial fixing of carbon dioxide, surrounding a sheath of parenchyma cells (the bundle sheath) which has large chloroplasts involved in the Calvin cycle. Assuming that sucrose passes from M to BS cells symplastically in C3 leaves, the sucrose flux across the M-BS interface of the C3 species per PD would be similar to that calculated for C4 metabolite flux through PD in the C4 species (Table 2). The maize leaf vascular pattern consists of repeated longitudinal units of major veins that are separated by varying numbers of smaller minor veins (Sharman, 1942; Esau, 1943; Russell and Evert, 1985). A common method for detecting PD is to use an antibody against callose, a β-1,3-glucan, which is known to be deposited around the PD neck (Turner et al., 1994). In the maize study, however, rare clonal sectors were found in which a subset of the BS cells surrounding a vein was included in a sector with a neighboring M cell. The bundle sheaths regulate the movement of substances between the vascular tissue and the … PD in C3 species were generally spaced farther apart than PD in C4 species (Figure 4). Statistical differences at P value < 0.05 between the M-BS cell interface and M-M cell interface regression lines in Figure 6 were evaluated using SPSS Statistics software. Published June 2016. 3B). We are aware that the COVID-19 pandemic is having an unprecedented impact on researchers worldwide. Before forming a guard cell pair, the meristemoid may undergo additional asymmetric divisions to form non-stomatal cells. 1B with 1A). Please log in to add an alert for this article. Light Micrographs of Transverse Sections of Leaves of the Two C3 Species and Two C4 Species Examined. Click hereto get an answer to your question ️ The bundle sheath cells of C4 plants having Kranz anatomy possess In wild-type leaves of C4 plants, ME accumulates only in chloroplasts of mature BS cells (Fig. The torn patches (arrowheads) are mesophyll cell remnants on the surface of underlying cylindrical bundle sheath cells. In C4 species, the flux rate of C4 acids into the bundle sheath has to equal or slightly exceed the CO2 assimilation rate. That is, these cells might become BS cells through the action of a positional cue for which only procambial derivatives are primed, signaling either an adjacency to M cells or a distance from the vascular tissue. This value is 1/(t + 1.5R), where t is the section thickness and R is the average radius of PD. 1D compared with 1C). PD frequency per µm2 pitfield area was calculated using the linear equation, y = mx + b, where m is the slope, b is the intercept, and y is the PD frequency when pitfield area, x = 1 µm2. Immunolocalizations were performed using antibodies against NADP-dependent malic enzyme (ME) and phosphoenolpyruvate carboxylase (PEPCase). Our new quantitative technique combines scanning electron microscopy and 3D immunolocalization in intact leaf tissues to compare PD density on cell interfaces in leaves of C3 (rice [Oryza sativa] and wheat [Triticum aestivum]) and C4 (maize [Zea mays] and Setaria viridis) monocot species. BS cells occur in a single layer around a vein, making it difficult to distinguish positional effects from lineage effects. Furthermore, the 2D TEM images capture only a fraction of the total PD within a pitfield and do not allow for the mostly noncircular shape of the pitfields. al., 2001). Because there was considerable variation in pitfield area, we plotted the number of PD as a function of pitfield area and found strong linear correlations for all four species (Figure 6). This area is subtracted from the total pitfield area obtained in (E) to obtain the final area of M-BS pitfields. Dawei Sun has just finished his PhD in Emma Rawlins’ lab at The Gurdon Institute. Simultaneously, fluorescence from calcofluor white-stained cell walls was detected at 434 to 445 nm following excitation at 405 nm. Bar, 10 μm. Whole leaves were cut into 10-mm strips and fixed in 4% paraformaldehyde and 0.2% glutaraldehyde in 25 mM sodium phosphate buffer, pH 7.2, overnight at 4°C. As shown in Table 1 and Fig. Imaging was done under 10× and 40× objectives using Nikon Eclipse 50i upright microscope (Nikon Instruments). Our observations on tan1 mutants suggest that BS cells and any subsequent daughter cells are committed to BS fate at this time. The values in Table 2 will actually underestimate the malate/aspartate and pyruvate/alanine fluxes required to support these net rates of photosynthesis by ∼20%. It may be that when a cell is stimulated to divide but daughter cells of appropriate shape or volume are not produced because the new cell wall is misoriented, one or both daughters can respond again to the same stimulus and re-enter the cell cycle. For rice leaf, 5× enzyme cocktail was used. They are seen around leaf veins surrounding the vascular bundles. To provide insight into the role of the BS in the C 3 species Arabidopsis thaliana,we This generates high metabolic fluxes between these cells, through interconnecting plasmodesmata (PD). This demonstrates that the ectopic BS cells in tan1 leaves are more closely related to normally positioned BS cells than they are to the M cells with which they share a vein-distal location. Calculations using the Gunning constant (Gunning, 1978) and the plasmodesmogram method (Botha and Evert, 1988; Botha, 1992; Botha and van Bel, 1992) are common in the literature. We used measurements of CO2 assimilation rates to calculate the flux of C4 and C3 metabolites across the M-BS interface for C4 species, the sucrose flux across the M-BS interface in the C3 species, as well as the hypothetical flux that would need to cross the M-BS interface in C3 species if a C4 photosynthetic pathway were installed without anatomical changes (Table 2). They protect the “veins” of the leaf. The box and whisker represent the 25 to 75 percentile and minimum-maximum distributions of the data, respectively. Pitfields are in green (Alexa Fluor 488 fluorescence). This allowed visualization of pitfields on the outer surface of BS cells, parallel to the field of view. In this study, we report a new method that combines scanning electron microscopy and 3D immunolocalization by confocal microscopy to better quantify PD connections in the leaf. If the properties of PD are similar between C3 and C4 plants (physical cross sectional areas of individual C3 and C4 PDs measured here were similar; 0.006 to 0.008 μm2), one would predict that the PD density at this interface would need to increase by up to 12-fold for effective exploitation of a C4 mechanism in rice. 8 μm sections of the embedded material were made using a rotary microtome. Microscope observations were made and photographs taken using a Zeiss Axiophot light microscope. Scanning electron microscopy quantification of PD density revealed that C4 species had approximately twice the number of PD per pitfield area compared with their C3 counterparts. This study revealed that the C4 species, S. viridis and maize, have PD densities per M-BS cell interface area around 9-fold higher than the C3 species, rice and wheat. Rice (Oryza sativa cultivar Kitaake), wheat (Triticum aestivum cultivar Yecora 70), Setaria viridis cultivar A10, and maize (Zea mays cultivar B73) were grown in a growth cabinet (High Resolution Plant Phenomics Centre, CSIRO Black Mountain) equipped with white fluorescent tubes (Philips TL5 HO) and maintained at 28°C day/22°C night temperatures, 60% relative humidity, and 16 h light/8 h dark with peak at 1000 µmol quanta m−2 s−1 light intensity and ambient CO2 concentration. Due to the high degree of secondary thickening and often suberization of the BS walls, metabolite movement is limited to the symplasm and abundant plasmodesmata (PD) at this cell interface have been demonstrated (Hatch, 1987). A third interpretation, that the marked BS cells were generated from a non-procambial lineage, is unlikely because the sectors included, in cross section, several of the BS cells surrounding the vein, but only a single M cell. However, this idea is challenged by the observation, investigated here, that in the maize tangled1 (tan1) mutant, clusters of BS-like cells extend various distances from veins. Fluxes of sucrose (a 12-carbon sugar) will be an order of magnitude less than the rate of photosynthesis if sucrose is continuously exported from the M tissues in the light. Confocal Micrograph Background Controls for 3D Immunolocalization. This possibility might be investigated by examining the expression in tan1 leaves of cell cycle regulators such as cyclin-dependent kinases, which reflect the competency of the cell to divide (Hemerly et al., 1993; Shaul et al., 1996) and mitotic cyclins, which appear to be expressed only in actively dividing cells (Ferriera et al., 1994; Shaul et al., 1996). ab150117, lot no. No suberized layer was found between M cells in either C3 or C4 species (Figures 2E to 2H). This suggests that the cells in the ectopic cell clusters are functional C4-type BS cells and that C4 enzyme accumulation in these cells is independent of cell position relative to the vein. Maize had the highest CO2 assimilation rate per leaf area at 38.6 ± 1.14 µmol CO2 m−2 s−1, but this is not statistically different from wheat at 35.0 ± 1.48 µmol CO2 m−2 s−1 (Table 2). In both C3 and C4 species studied, chloroplasts in the M cells were distributed along the cell periphery. The PD density per cell interface area was obtained from the product of PD frequency per µm2 pitfield area (scanning electron microscopy) and pitfield area per cell interface area (3D immunolocalization). Field Emission Scanning Electron Micrograph of Cell Interfaces in S. viridis Leaf. The Tan1 gene encodes a highly basic protein with microtubule-binding activity, and with a localization pattern consistent with its inferred role in the orientation of cytoskeletal structures in dividing cells (Smith et. Our observations here suggest that once the procambial lineage is established, its most peripheral derivatives always differentiate as BS cells. We thank the ANU Centre for Advanced Microscopy, Australian Microscopy and Microanalysis Research Facility (AMMRF) and CSIRO Microscopy Centre for providing support and technical assistance. Thus, the two C4 species had up to nine times more PD per M-BS interface area (S. viridis, 9.3 PD µm−2; maize, 7.5 PD µm−2; rice 1.0 PD µm−2; wheat, 2.6 PD µm−2). The middle portion of each leaf was cut into 1-mm2 strips and fixed in 2.5% glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.3, overnight at 4°C. Sections for plastic-embedding were rinsed 3 times in 100% polypropylene oxide after ethanol dehydration and infiltrated overnight at room temperature in a 1:1 mixture of polypropylene oxide:Spurr’s resin (Electron Microscope Sciences). In early work, assumptions of the proportion of M-BS interface area populated by PD varied from 1 to 10% (Osmond, 1971); subsequently, these values and those of Hatch and Osmond (1976), based on median literature values for C3 plant cell PD data, have been routinely used. The transmission of a lineage-committed state from a mother cell to one or both of its daughters may be uncommon in plants because of the nature of the plant body. We do not capture any email address. Each vascular bundle consists of phloem and xylem tissues surrounded by a bundle sheath. In a C3 system like rice, the M-BS interface is most likely a conduit for delivery of sucrose to the phloem for export from the leaf (Aoki et al., 2012). Cleared tissue was digested in an enzyme cocktail containing 1 μL mL−1 β-xylanase M6 (Megazyme), 1 μL mL−1 α-L-arabinofuranosidase (Megazyme), 1 μL mL−1 pectate lyase (Megazyme), 0.5 μL mL−1 of 2 mg mL−1 cellulase (Sigma-Aldrich), 1 μL mL−1 α-amylase (Sigma-Aldrich), and 1 μL mL−1 pullulanase (Sigma-Aldrich) for at least 8 d at 37°C with gentle shaking. F.R.D. Transmission electron microscopy (TEM) has been routinely used to study details of PD structure (Robards, 1976; Evert et al., 1977; Ding et al., 1992; Overall and Blackman, 1996), but extracting quantitative data requires careful serial sectioning and reconstruction. Immunolocalizations of BS and M cell-specific antibodies. Then, the CO2 assimilation rate per PD was calculated by dividing CO2 assimilation per BS surface area by the PD density per M-BS cell interface area. in vasculature or bundle sheath (BS) cells rather than the mes-ophyll (M) cells where the mutant phenotype is manifested. This suggests that in the evolution of C4 plants, a general increase in foliar symplastic connections may have occurred, not specific to the C4 mechanism, and a phylogenetic analysis of this hypothesis is currently underway. The absence of reliable estimates of this parameter has substantively hindered modeling studies. For light microscopy, semithin sections (0.5- to 1.0-µm thick) were cut with glass knives using a Reichert Jung ultramicrotome and stained with hot aqueous 0.05% Toluidine blue, pH 5.0. Suc and fructan accumulated in both mesophyll and parenchymatous bundle-sheath (PBS) cells because of the reduced export of sugars from leaves under cooling and to … However, the simplest interpretation is that, once specified as BS, cells remain committed to the BS fate while they continue to divide, and therefore differentiate as BS cells regardless of their final positions. Plasmodesmograms provide semiquantitative data on symplastic connectivity between cells, based on PD that can be detected using TEM and yield values in relative units (Botha and Evert, 1988; Botha, 1992; Botha and van Bel, 1992). ME antibodies were as described previously (Rothermel and Nelson, 1989) and PEPCase antibodies were a gift from Dr James Berry (SUNY; Buffalo). All tissue was embedded in Paraplast Plus for this experiment. (F) Area from (E) corresponding to M-M cell interface pitfields detected at the edges of certain focal planes. 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