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Isolation of Dimorphic Chloroplasts from the Single-Cell C4 Species Bienertia Sinuspersici Shiu-Cheung Lung, Makoto Yanagisawa and Simon DX Chuong*

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Isolation of Dimorphic Chloroplasts from the Single-Cell C4 Species Bienertia Sinuspersici Shiu-Cheung Lung, Makoto Yanagisawa and Simon DX Chuong* Lung et al. Plant Methods 2012, 8:8 http://www.plantmethods.com/content/8/1/8 PLANT METHODS METHODOLOGY Open Access Isolation of dimorphic chloroplasts from the single-cell C4 species Bienertia sinuspersici Shiu-Cheung Lung, Makoto Yanagisawa and Simon DX Chuong* Abstract Three terrestrial plants are known to perform C4 photosynthesis without the dual-cell system by partitioning two distinct types of chloroplasts in separate cytoplasmic compartments. We report herein a protocol for isolating the dimorphic chloroplasts from Bienertia sinuspersici. Hypo-osmotically lysed protoplasts under our defined conditions released intact compartments containing the central chloroplasts and intact vacuoles with adhering peripheral chloroplasts. Following Percoll step gradient purification both chloroplast preparations demonstrated high homogeneities as evaluated from the relative abundance of respective protein markers. This protocol will open novel research directions toward understanding the mechanism of single-cell C4 photosynthesis. Keywords: Bienertia sinuspersici, Chloroplast isolation, Dimorphic chloroplasts, Osmotic swelling, Photosynthesis, protoplast, Single-cell C4, Vacuole isolation Background phosphoenolpyruvate carboxylase (PEPC) in mesophyll The majority of terrestrial plants house chloroplasts pri- cells. The C4 acids are broken down by a C4 subtype-spe- marily in one major cell type of leaves (i.e. mesophyll cific decarboxylation enzyme in bundle sheath cells, and cells), and perform C3 photosynthesis to assimilate atmo- the liberated CO2 is subsequently re-fixed by ribulose- spheric CO2 into a 3-carbon product, 3-phosphoglyceric 1,5-bisphosphate carboxylase/oxygenase (Rubisco). The acid. In C4 species, on the other hand, a Kranz-type leaf C4 pathway concentrates CO2 atthesiteofRubiscoand anatomy featuring the second type of chlorenchyma cells minimizes the photorespiration process, an unfavorable surrounding the vascular bundles (i.e. bundle sheath oxygenase activity of Rubisco with O2. cells) was reported as early as in the late 1800’s[1].In The indispensable relationship between the Kranz anat- these species, the initial carbon fixation into 4-carbon omy and C4 photosynthesis has been an accepted feature acids was first documented in the 1960’s [2,3]. The phy- until the discovery of three terrestrial single-cell C4 spe- siological relevance of the Kranz anatomy in relation to cies, Suaeda aralocaspica (formerly called Borszczowia the C4 photosynthetic pathways, however, had not been aralocaspica) [5], Bienertia cycloptera [6,7], and B. sinus- elucidated until the successful separation of the two persici [8] in the Chenopodiaceae family. In chlorench- types of chlorenchyma cells and their respective yma cells of these succulent Chenopodiaceae species, the dimorphic chloroplasts. With the development of various C4 cycles are operational in the absence of Kranz anat- mechanical and enzymatic methods for separating the omy due to the division of cytoplasm into two compart- mesophyll and bundle sheath cells, the biochemistry of ments. The cytoplasmic channels that connect the two C4 cycles has been intensively studied over the past few compartments not only allow metabolite exchange but decades focusing explicitly on characterizing the enzy- also limit inter-compartmental gas diffusion, resembling matic properties and determining their precise subcellu- the function of plasmodesmata traversing the thickened lar locations in these cell types (for review, see [4]), and sometimes suberized bundle sheath cell wall. The leading to the current C4 models. In the C4 model, atmo- two cytoplasmic compartments house two distinct chlor- spheric CO2 is initially converted into C4 acids by oplast types and different subsets of enzymes, respec- tively. Accordingly, the peripheral compartment proximal to the CO entry point is specialized for carboxylation * Correspondence: [email protected] 2 Department of Biology, University of Waterloo, 200 University Avenue West, and regeneration of the initial carbon acceptor, Waterloo, Ontario N2L 3G1, Canada © 2012 Lung et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Lung et al. Plant Methods 2012, 8:8 Page 2 of 13 http://www.plantmethods.com/content/8/1/8 phosphoenolpyruvate, whereas the central compartment and the cytoplasmic channels (Figure 1B). Reducing the distal to the CO2 entry point is responsible for decarbox- osmotic potential of the cell-stabilizing buffer induces ylation of C4 acids and Rubisco-catalyzed re-fixation of the swelling of the protoplast and its central vacuole, dis- the liberated CO2. In agreement with the immunolocali- rupting the cytoplasmic channels and pushing the CCC zation patterns of the major enzymes involved [5,7,9], the against the plasma membrane (Figure 1C). The hypo- two cytoplasmic compartments appear to be functionally osmotic shock causes the plasma membrane to stretch equivalent to the mesophyll and bundle sheath cells of beyond its extension limit that eventually breaks, releas- Kranz-type C4 plants, respectively. Based on differential ing the intact CCC and vacuole with attached peripheral speed centrifugation for the enrichment of each chloro- chloroplasts (P-Chls; Figure 1D). This is in agreement plast type in subcellular fractions of B. sinuspersici leaves, with the previous observation showing that the two cyto- Offermann et al. [10] have recently examined the protein plasmiccompartmentsareseparatedbyasinglevacuole distribution patterns of dimorphic chloroplasts and con- [9,11]. Eventually, the central vacuole carrying P-Chls on firmed their functional similarities to the mesophyll and the external surface (Figure 1E) and the isolated CCC bundle sheath cells of Kranz-type C4 plants, respectively. containing central chloroplasts (C-Chls; Figure 1F) can Thorough studies of the enzymology of the single-cell C4 be separated into two discrete entities. model, however, requires homogenous preparations of the dimorphic chloroplasts with specific techniques Isolation of chlorenchyma protoplasts from B. Sinuspersici based on the unique cell anatomy, in analogy with the As a first step, we isolated chlorenchyma cells from previous efforts of separation and subcellular fractiona- B. sinuspersici leaves and enzymatically prepared a homo- tion of the dual cell types from Kranz-type C4 plants (for genous population of healthy protoplasts. Interested review, see [4]). readers should refer to our previous report for detailed Here, we present our empirically optimized protocol technical considerations on plant growth conditions and for separating the dimorphic chloroplasts from chlor- chlorenchyma protoplast isolation [12]. Previously, pro- enchyma protoplasts of B. sinuspersici. By reducing the gressive developmental variation has been identified at osmotic potential of culture medium to a suitable level, different stages of B. sinuspersici leaves in terms of the the isolated protoplasts were hypo-osmotically bursted, unique subcellular compartmentation and photosynthetic concomitantly extruding one type of chloroplasts encased gene expression [11,13]. Similar developmental gradients in the central cytoplasmic compartment and another type were also observed across the base-to-tip dimension of of chloroplasts from the peripheral compartment adhered leaves [13]. Thus, these major sources of variability were to the external surface of intact vacuoles. Following a inevitably taken into considerations in order to standar- subsequent centrifugation step, these two structures can dize the degree of C4 functionality of the starting materi- be separated into the sedimented and floating fractions, als for dimorphic chloroplast isolation. To this end, we respectively. Finally, two homogenous populations of routinely propagated B. sinuspersici by vegetative cuttings dimorphic chloroplasts with minimal cross-contamina- and collected entire leaves, 2 cm or longer in length, tion can be further purified by using a Percoll gradient. from healthy branches of 3- to 4-month-old plants Overall, this dimorphic chloroplast isolation protocol can (Figure 2A). At this stage, the single-cell C4 compartmen- be applied in multiple areas of research toward further tation has reached maturity as evident by the presence of understanding the development of single-cell C4 systems. the distinctive subcellular distribution of dimorphic chloroplasts in the majority of chlorenchyma cells (Figure Results 2B). In a mature chlorenchyma cell, the C-Chls are den- Rationale of the isolation procedures in relation to cell sely packed into a large, spherical CCC structure, anatomy whereas the P-Chls are distributed throughout the thin For a better clarification of the each isolation step as layer of cytoplasm, (PCC; Figure 2B). Following cell wall described in the subsequent sections, we first summarize removal by cellulase treatment under our optimized con- the major changes in the complex subcellular organiza- ditions, the resulting protoplasts exhibited no observable tion of chlorenchyma cells throughout the process as illu- changes in their unique chloroplast distribution, which is strated with schematic diagrams (Figure 1). In a mature considered a prerequisite for subsequent preparations of chlorenchyma cell, a large central vacuole (as depicted
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