Identification of Maize Proteins in Purified Bundle Sheath (BS) and Mesophyll (M) Chloroplast Stroma from Three Different Types of Experiments Involving 2-DE Gels, cICAT, and LC-MS Analysis of Unlabeled Peptides. The purity of the BS strands was at this step assessed by light microscopy. Accession numbers for quantified genes discussed in this article are listed in Table 2. In the protein extraction and purification process, SDS was avoided and salt usage was kept low. open stomata only at night, limiting water loss because of heat and low humidity. H2O → PSII → electron transport chain → PSI → electron transport chain → NADPH. The MS or MS/MS spectra were searched against the maize EST assembly from TIGR (http://www.tigr.org) (ZmGI, v1.4) and maize genome assemblies (AZM 4.0) as well as by homology-based searches against the annotated rice (Oryza sativa) genome (TIGR Rice Genome version 2), using an in-house installation of Mascot (www.matrixscience.com). The web interface for PPDB was developed on ASP.NET platform using C# language. The innermost compartment of a chloroplast formed by the interconnected thylakoids is the __________. The extracted peptides were dried and resuspended in 20 μL 5% formic acid. The in-solution trypsin digest of the BS and M proteomes followed a procedure adapted from Chelius et al. In C4 plants, chloroplasts are located in ___________. A coenzyme is defined as _______________. Possibly, the CAM plants may be considered to have originated by such hybridization occurring in nature during their evolutionary history. Supplemental Table 4. Protein concentrations were determined by the Bradford method (Bradford, 1976). When the stomata close during heat stress, RuBP carboxylase is not exposed to the increase in oxygen concentration experienced in the leaf. Purified M and BS stromal proteomes were first separated based on isoelectric point on IPG strips (150 μg of protein/strip), followed by a second dimension separation of SDS-PAGE. Supplemental Table 2. Upon examination under a microscope, you find that there is a layer of bundle sheath cells containing chloroplasts around the vein of the leaf of the plant. Yet, BSD2 RNA and protein levels are similar in mesophyll (M) and BS chloroplasts, although Rubisco accumulates only in BS chloroplasts. Spot selection was performed semiautomatically, with a large time investment in manual spot matching and verification. D) They do not have any intercellular spaces. The _______ from the light reactions is used to reduce CO2 to a carbohydrate using energy derived from ______. (1) Mixed and labeled BS/M stromal proteomes (200 μg for each proteome) were separated on a 1-DE Tricine-PAGE gel (12% acrylamide). w⃞ Do C3 plants contain chloroplasts in their bundle sheath cells? Energy to drive the formation of ATP in photosynthesis is derived from ____________. Summary of the Non-Gel-Based Comparative Proteome Analysis of BS and M Chloroplast Stroma Using cICAT and Parallel LS-MS Runs. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. The C 4 pathway acts as a mechanism to build up high concentrations of carbon dioxide in the chloroplasts of the bundle sheath cells. Approximately 400 spots per gel were detected, with spot volumes spanning five orders of magnitude. The flow-through containing the nonlabeled peptides was also collected since they were valuable for protein identification. Young mesophyll and bundle sheath cells in the leaf base had proplastids, and their plasmodesmata were simple, devoid of cytoplasmic sleeves. The top 4-cm sections of the 2nd and 3rd leaves were harvested ∼2 h after the onset of the light period. Which statement is accurate about how C4 plants are able to survive hot, dry spells during the growing season? For 1-DE SDS-PAGE separation, proteins were equilibrated with SDS (0.2%), Na2CO3 (100 mM), DTT (100 mM), and sucrose (10%) and separated on 12% Tricine gels (Schägger and von Jagow, 1987). Plants with Krantz anatomy have mesophyll cells surrounding a layer of bundle sheath cells, which surround the vein (vascular bundles). After carbon dioxide fixation by RuBisCO, Calvin cycle proceeds as in C 3 plants. Mesophyll and bundle sheath cells have structurally and functionally different chloroplasts. On average, we included gels only those matched spots that were present at least three times on M or BS gels. C) Bundle sheath cells have a large number of chloroplasts. Several hundreds of leaf tips (∼80 g of fresh tissue) were cut into 5- to 10-mm slices and gently ground by pestle and mortar in grinding buffer containing (350 mM sorbitol, 50 mM Hepes-KOH, pH 8, 2 mM EDTA, 5 mM ascorbic acid, and 5 mM l-cystein). C4 plants need energy to move fixed carbon compounds into bundle sheath cells. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantcell.org) is: Klaas J. van Wijk (kv35{at}cornell.edu). All verified protein identities using both unlabeled peptides and cICAT-labeled peptides are available via the PPDB. Mesophyll cells possess thin cell walls and randomly arranged chloroplasts with stacked thylakoids. They do not have starch granules. In exceptional cases, four matching peptides were considered as positive identification (e.g., proteins <20 kD and matching gel coordinates). This plant very likely carries out C4 photosynthesis. The light reactions act much as a battery to power the reactions of the Calvin cycle. The two-dimensional LC was a combination of strong cation exchange followed by reverse phase, as developed recently under the name MudPIT (Washburn et al., 2001; Wolters et al., 2001), and served to reduce complexity and maximize protein identification and quantification. This pathway is also called Hatch and Slack pathway. The bundle sheath also conducts the flo… The residual M cells attached to BS strands were removed by 10 min of blending (one-third of the maximal speed) in a modified Warring blender (WB1) with blunt blades. SAUR17 and SAUR50 Differentially Regulate PP2C-D1 during Apical Hook Development and Cotyledon Opening in Arabidopsis, AUTOPHAGY-RELATED14 and Its Associated Phosphatidylinositol 3-Kinase Complex Promote Autophagy in Arabidopsis, by The American Society of Plant Biologists, www.plantcell.org/cgi/doi/10.1105/tpc.105.035519. Philos. The Calvin cycle is confined to the chloroplasts of these bundle sheath cells in C 4 plants. Sci. 2-DE-IPG was performed on 11-cm Immobiline Dry strips (Amersham Biosciences). 8: In C3 plants, the bundle sheath cells do not contain chloroplasts. Rubisco activity limits photosynthesis at low temperatures. chloroplasts is the DNA content: bundle sheath chloroplasts contain approximately 50 copies of the genome as opposed to 20 in mesophyll chlo- roplasts. The bundle sheath cells lack intercellular spaces. Chloroplasts of mesophyll cells are smaller, have well developed grana and they do not produce starch, Chloroplasts of bundle sheath cells … In C4 plants, the bundle sheath cells contain chloroplasts. The flow-through containing the nonlabeled peptides was also collected. s−1) until the 4th leaf was emerging. The unique chloroplast morphology, as seen in A. thaliana leaf tissue, is reflected here. C4 plants need energy to move fixed carbon compounds into bundle sheath cells. The vascular bundles are surrounded by large sized bundle sheath cells which are arranged in wreath-like manner in one to several layers. Sucrose, cellulose, amino acids, and starch are all made from what starter molecule in plants? Protein identification was performed by peptide mass fingerprinting using MALDI time-of-flight (TOF) MS in reflectron mode (Voyager DE-STR; Perseptive Biosystems) and online LC-ESI-MS/MS (Q-TOF; Micromass). In C4 plants, chloroplasts are located in both mesophyll and bundle sheath cells. Carotenoids are found in high concentrations in yellow and orange leaves or vegetables. ↵ The O2 given off during photosynthesis is derived from which compound? C 4 leaves confine Rubisco to bundle sheath cells. Thank you for your interest in spreading the word on Plant Cell. NADP-malic enzyme (NADP-ME) occurs in the chloroplasts of bundle-sheath cells and, like the other decarboxylases of C 4 photosynthesis, it releases CO 2, which can be fixed directly by Rubisco. Why do plants contain other pigments besides chlorophyll? Which of the following organisms can perform photosynthesis? The light reactions of photosynthesis are said to be similar to a battery because they form a current. Briefly, purified BS and M stromal proteomes were reduced with DTT and alkylated using iodoacetamide, followed by in-solution digestion by trypsin. Corresponding interactive gels with protein spot identities that were quantified are available in the PPDB. Supplemental Table 3. Column materials were supplied as part of the cICAT labeling kit from Applied Biosystems. Only those peptides were considered that had no missed cleavages (by trypsin) and no modifications, except for Met oxidation and carbamidomethylation (since gel samples were alkylated). Leaves of C3 plants do not have Kranz anatomy. the bundle sheath cells in C4 plants have chloroplasts, while those in C3 plants do not. B) Mesophyll cells have PEP carboxylase while bundle sheath cells have RuBisCO. The MapMan system has 35 main functional categories or Bins, with a larger number of subBins (subcategories) (see also http://ppdb.tc.cornell.edu/mapman.aspx). the bundle sheath cells in C3 plants are arranged in columns just beneath the upper epidermis, while those in C4 plants are arranged in a ring around veins. C4 leaves confine Rubisco to bundle sheath cells. How many turns of the Calvin cycle would be required for a plant to make one molecule of glucose? They have large chloroplasts without grana. Peltier, and J. Ytterberg for numerous discussions and advice. We functionally annotated all identified ZmGI and AZM accessions using a combination of BLAST alignments to the predicted rice proteome (OSGI) and the predicted Arabidopsis thaliana proteome (from The Arabidopsis Information Resource). Excess cICAT label was removed by strong offline cation exchange chromatography, followed by offline biotin-avidin affinity purification to collect the labeled peptides and removal of part of the tag by acid hydrolysis. Each cICAT-labeled sample was run twice under identical chromatographic conditions. ATP synthase derives energy for the generation of ATP from ______________. For cICAT peptides, cICAT filter and variable Met oxidation were selected as Mascot search parameters. Sage et al. The IPG strips were rehydrated overnight with 150 μg of proteins solubilized in 9 M urea, 4% 3-([3-cholamidopropyl]dimethylammonio)-1-propane-sulfonate, 2 mM tributylphosphine, 2% pharmalyte, pH 4.0 to 7.0, 0.5% Triton X-100, and a few crystals of bromophenol blue (to evaluate the uniformity of the rehydration) in a reswelling tray at room temperature. It summarizes the currently accepted views on metabolic compartmentation of biochemical reactions between the chloroplasts of bundle sheath cells and mesophyll cells of a C 4 plant. C 4 plants are an interesting system to study plastid development, since protochloroplasts differentiate into two morphologically and functionally different chloroplast types in mesophyll (M) and bundle sheath (BS) cells to support the C4 carbon concentrating mechanism. As climate change leads to drier and drier summers in the southeastern United States, more and more homeowners find that they have crabgrass growing in their yards. What is the purpose of the three additional ATP molecules? Thus, the size of bundle sheath compartments and the total volume of chloroplasts within them limit the space available for Rubisco. All best scoring directional pairwise BLAST alignment results between TAIR, OSGI, AZM, and ZMGI are available via PPDB. The yield of chloroplast preparations was inversely proportional to their purity. The production of glyceraldehyde 3-phosphate (G3P) in the Calvin cycle requires six molecules each of ATP and NADPH. Which of the following is a disadvantage of C4 photosynthesis relative to C3 photosynthesis? A poison that disrupts the H+ gradient in the thylakoid space would most likely cause which of the following to accumulate during the Calvin cycle reactions? These distinct lineages exhibit unique aspects that highlight their independent evolution from C3 ancestors. There exists a layer of mesophyll cells that contain small chloroplasts encircling bundle sheath cells that have large chloroplasts necessary for the Calvin cycle. In the bundle sheath cells carbon dioxide is fixed by the enzyme RuBisCO. CAM photosynthesis limits CO2 fixation to nighttime hours in order to _______________. The focused strips were denaturated and reduced in an equilibration buffer (6 M urea, 30% glycerol, 50 mM Tris-HCl, pH 6.8, and 2% SDS) with addition of 5 mM tributylphosphine for 20 min and subsequently alkylated in equilibration buffer with 15 mM iodoacetamide for 20 min (Rabilloud et al., 1997; Herbert et al., 1998). For 1-DE gel cICAT experiments, the non-Cys-containing peptides separated from both cICAT-labeled peptides were also purified by homemade reverse-phase columns packed with C18 beads, analyzed by LC-MS/MS, and searched against ZmGI to obtain protein identities. In C4 plants, the light reactions will stop during the daytime when the supply of what compound is limited? Another three molecules of ATP are then used in the cycle. (2003). Denatured apparent molecular masses were determined from molecular mass markers loaded onto the second dimension gels. the chloroplast-containing cells of the leaf. In C 4 plants, mesophyll (M) chloroplasts carry out the light reactions of photosynthesis, while bundle sheath (BS) chloroplasts are the sites of carbon re-fixation and the Calvin cycle (Hatch 1999, Kanai and Edwards 1999). Protein pIs on the resulting 2-DE-IPG gels were internally calibrated by mixing carbamylated standards (Pharmacia Biosciences) with the stromal extracts before 2-DE analysis. Trans. Annu. RuBP carboxylase (rubisco) is able to bind with both _______________. A new procedure for purification of M and BS chloroplasts was developed based on suggestions from A. Barkan and R. Bassi and on published protocols (Edwards and Black, 1971; Kannangara et al., 1977). The mesophyll cells are the usual cell type as found in C 3 plants also. Loosely arranged mesophyll cells lie between the bundle sheath and the leaf surface. Subsequently, chloroplasts were further isolated, and the stromal proteome was released by osmotic shock, followed by removal of thylakoid and envelope membranes by ultracentrifugation, similarly as described by Peltier et al. The number of chloroplasts in the bundle-sheath cells is more than that in the mesophyll cells. Therefore, only ZmGI was used for detailed protein quantification. In a redox reaction, reduction means ________, and oxidation means ________. After a wash with grinding buffer solution, the BS chloroplasts were released into the grinding buffer solution by short (2 s) high-speed pulses in a second modified Warring blender (WB2) in which the original blades were replaced by razor blades (as described in Kannangara et al., 1977). Calibrated using trypsin autodigestion peptides for the generation of ATP from ______________ labeled. Reactions is used to reduce CO2 to a carbohydrate using energy derived from _____________ method ( Bradford, 1976.! 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