Check Answer and Other antisera were as previously described (Roth et al., 1996). Electron micrographs of third leaf sections (Figure 2A) indicated that this grainy appearance in the leaf is due to the presence of both phenotypically wild-type and bsd2-m1–like mutant bundle sheath cell chloroplasts in these intermediate or bsd2-weak (bsd2-w) plants. Chloroplast Import and Processing of the in Vitro–Synthesized BSD2 Protein in Isolated Pea Chloroplasts. This 1.8-kb fragment was cloned into a plasmid vector (pTBP6), and sequences 5′ to the Mu element were amplified by polymerase chain reaction (PCR) (Bsd2.1; Figure 1C). What purpose are the air spaces in the spongy mesophyll?, etc) 3. Conserved residues in the CXXCXGXG motif repeated four times in DnaJ are highlighted. Thus, the ectopic accumulation of rbcL transcripts in mesophyll cells of bsd2-m1 mutants may be due to an increase in polysome-associated transcripts relative to the wild type. The proportion of polysome-associated to unassociated transcripts provides a means to examine the efficiency of translation initiation and elongation (Barkan, 1993). Thus, it is possible that in the absence of functional BSD2 protein, stalled ribosomes protect rbcL transcripts from degradation. The putative processing site of the chloroplast transit peptide is marked by an arrow. It owes the names to the discovery by Marshall Davidson Hatch and Charles Roger Slack that some plants, when supplied with 14CO 2 , incorporate the 14C label into four-carbon molecules first. The dimorphic bundle sheath and mesophyll cells of C4 leaves exhibit specific ultrastructural features. Plants that use crassulacean acid metabolism (CAM) photosynthesis fix CO 2 at night, when their stomata are open. NH 3 and serine would be assimilated in both cell types, but because of the high activity of glycine decarboxylase in bundle-sheath cells, a considerable fraction of these metabolites would have to be transported back to mesophyll cells. Because Rubisco accounts for more than half of the soluble protein in the chloroplast (Ellis, 1979), it is reasonable to envision the need for additional factors to prevent aggregation. In contrast, bundle sheath cell–specific Rubisco accumulation in A. rosea, another C4 dicot, occurs very early in leaf development, before the maturation of bundle sheath cells (Dengler et al., 1995). In contrast to wild-type seedlings, which showed a light-induced increase in rbcL transcript levels, the levels of rbcL transcript in bsd2 mutants were similar in both the dark-grown and light-shifted seedlings (Figure 6). Some sheath cells have thin secondary walls. After inhibition, CO2 uptake was a linear function of [CO2] over much of the range tested. The Ppc1 (pTN1), RbcS (pJL10), rbcL (pJL12), psbA (pSD7), Cab (LHCP 1020), and ubiquitin (pSkuB1) cDNA clones have been described previously (Roth et al., 1996; Hall et al., 1998). (d) Chloroplasts in the bundle sheath cells are larger and lack grana (agranal chloroplasts) but contain numerous starch grains. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Arabidopsisbundle sheath cells 1817 the vascular axis, with either perpendicular or oblique end walls (Fig. Furthermore, nuclear run-on experiments have demostrated low-level RbcS transcriptional activity in mesophyll cell protoplasts (Schäffner and Sheen, 1991). The C4 photosynthetic pathway is most common in Arundinella hirta L. is a C 4 plant having an unusual C 4 leaf anatomy. This results in a reduced photorespiration rate. Cyt, cytochrome. Amino acids conserved between BSD2 and the putative rice (Os.est; GenBank accession number D48303) and Arabidopsis proteins (At.est) are shown in capital letters, and similar residues are shown in lowercase letters. Because the Mu family of transposable elements is extremely diverse (Chandler and Hardeman, 1992), several gene-specific Mu fragments were used in DNA gel blot analyses to identify a band that cosegregated with the bsd2-m1 mutant phenotype (see Methods). This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein–protein interactions. Assuming a similar pattern of expression in maize, the decreased levels of NDH-H in bsd2 mutants may reflect the severe disruption of bundle sheath cell chloroplast ultrastructure seen when plants are grown under moderate light conditions (Roth et al., 1996). Maharashtra CET 2007: IN C4 planes, chloroplasts of bundle sheath cells lack (A) Stroma (B) Grana (C) Chlorophylls (D) All the above. These sequence data have been submitted to the GenBank database under accession number AF126742. Chloroplasts containing processed BSD2 protein were fractionated into stromal (lane 4) and thylakoid (lane 5) compartments. (1998). Without the inhibitor, CO2 uptake increased steeply at low [CO2] and saturated at about 1 mL L-1. Chloroplasts (50 μg of chlorophyll in a final volume of 125 μL) in 50 mM Hepes-KOH, pH 8.0, and 330 mM sorbitol, were preincubated with 8 mM MgATP for 10 min at 25°C in the light (60 μmol m−2 sec−1). Until to CO2 runs out completely-Why are not all plants C4? Published December 1993. The Bundle sheath defective2 (Bsd2) gene is required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Although transcription rates of individual chloroplast genes can vary greatly, the relative rates of transcription of most are maintained throughout chloroplast development (Deng and Gruissem, 1987). Thus, a chloroplast-localized DnaJ-like protein is likely to bind to LSU polypeptides as they emerge from the ribosome. Transverse Section . An example of C3 are Sunflower, Spinach, Beans, Rice, Cotton, while the example of C4 plants is Sugarcane, Sorghum, and Maize, and Cacti, orchids are the example of CAM plants. Sets of filters were hybridized with Mu family–specific fragments for Mu1, Mu1.7, Mu3, Mu5, Mu8, and MuDr (Chandler and Hardeman, 1992) kindly provided by V. Chandler (University of Arizona, Tucson). One explanation for the heterogeneous transcript start sites is the absence of a strong consensus TATA box motif in the Bsd2 gene. (B) Protein alignment (MSA 2.1, http://www.ibc.wustl.edu/msa/man.html) of BSD2 with putative protein products encoded by ESTs. is supported by a BBSRC David Phillips Fellowship. our model, the absence of BSD2 would result in the aggregation of the nascent peptide chain. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Bundle sheath chloroplasts of maize, a C4 plant, lack a functional herbicide-binding site and the 32 kDa-QB thylakoid protein of photosystem II. o. f Sunflower Stem (Hellanthus Annuus) Epidermis: This is the outermost uniseriate layer consisting of tangentially elongated and cutinised cells. To examine the role of BSD2 in mediating light-induced changes in chloroplast gene expression, we compared levels of rbcL and psbA transcripts between wild-type and mutant plants (Figure 6). Transcripts were fractionated on 15 to 45% sucrose gradients, on the basis that transcripts associated with polysomes have higher sedimentation constants than do monosomes or free RNA. The ~3500 plants generated from these crosses were screened in a sand bench, and no pale green seedlings were identified. … For example, in RbcS antisense tobacco plants, the decrease in LSU accumulation is proportional to the decrease in SSU levels (Rodermel et al., 1996). These findings suggest that rbcL transcripts accumulate ectopically in mesophyll cells of bsd2-m1 plants because they are stabilized through an association with ribosomes. Ribosome pausing has been well documented in plant chloroplasts as a mechanism of translational control (Klein et al., 1988; Kim et al., 1994) that results in the accumulation of polysome-associated transcripts without accumulation of corresponding peptides (reviewed in Gillham et al., 1994). The bundle sheath cells have RuBisCO but lack PEPcase Carbon Fixation in CAM Plants CAM pathway of carbon fixation or Crassulacean acid metabolism is present in plants present in arid conditions, e.g. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. Protein was isolated from plants grown under moderate (100 μmol m−2 sec−1) or low (10 μmol m−2 sec−1) light conditions. (C) Hybridization to RNA isolated from purified bundle sheath strands (BS) and mesophyll cell protoplasts (M). PCR amplification of the ligation reaction was performed with the TBp14 and T3 primers. Similarly, multiple polyadenylation sites were identified at the 3′ end of the gene (Figure 3A, underlined sequences), suggesting that different polyadenylation signals are recognized during Bsd2 transcriptional processing. As mentioned previously, bsd2-m1 was first identified as conditioning a variegated leaf phenotype. In C3 plant both Mesophyll cells and Bundle sheath cells have Rubisco while in C4 plant only Bundle sheath (B) Mesophyll cell–specific C4 photosynthetic enzymes: phosphoenolpyruvate carboxylase (PEPCase), pyruvate orthophosphate dikinase (PPdK), and NAD(P)–malate dehydrogenase (MDH). Scientists wanted to find out how C4 crops are able to express several important enzymes inside bundle sheath cells instead of the mesophyll. To identify components of these signaling pathways, mutagenized maize populations were screened for mutations that specifically disrupt photosynthetic enzyme accumulation patterns in either bundle sheath or mesophyll cells (Langdale et al., 1995). The bundle-sheath cells are the photosynthetic cells arranged into a tightly packed sheath around the vein of a leaf. Genomic clones are shown in Figure 1. In the maize leaf, a developmental gradient exists such that older tissue near the tip of the leaf differentiates before younger tissue located near the base. In the CAM pathway, plants take CO 2 during the night through the stomatal opening. (1989) with permission of Wiley-Blackwell. A 3H-labeled BSD2 translation mixture (12.5 μL) was mixed with an equal volume of unlabeled leucine (5 mM final concentration) and added to the chloroplast suspension. When these plants were outcrossed to several different inbred lines and selfed, only stable mutant phenotypes segregated in the F2 progeny. Light-grown seedling tissue (0.5 g) was ground in liquid nitrogen to a fine powder and added to 1.5 mL of buffer (Klaff and Gruissem, 1991). Differences between Mesophyll and Bundle Sheath Cells and Chloroplasts in C4 Plants (Mesophyll vs Bundle Sheath Chloroplasts: A Comparison Table)The C4 cycle or Hatch and Slack pathway of dark reaction of photosynthesis are characterized by two structurally and functionally different chloroplasts in their leaves. The tight linkage of a Mu transposon with the bsd2-conferred phenotype and the absence of the 0.6-kb transcript in mutant plants suggested that Bsd2 sequences had been cloned. Mutant plants died soon after seed reserves were exhausted; therefore, seedling or third leaf tissue was used for DNA, RNA, and protein gel blot analyses. Sites of polyadenylation are underlined. C) stomata. Furthermore, measurements of rbcL transcription rates in wild-type maize plants by using in vitro run-on assays suggest that the differential expression of rbcL in bundle sheath and mesophyll cells is mediated, in part, by post-transcriptional processes (Kubicki et al., 1994). A maize ubiquitin fragment (Ubi) was used as a loading control. Although increased transcription rates could account for the increased accumulation of rbcL transcripts in bsd2 mutants relative to the wild type, it seems more likely that a defect in post-transcriptional regulation mediates these changes. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. One possible explanation is that the formation of LSU aggregates attenuates translation of polysome-bound mRNA. Introns are denoted with open triangles. Thank you for your interest in spreading the word on Plant Physiology. from the Biotechnology and Biological Sciences Research Council (BBSRC) and the Gatsby Charitable Foundation. Our previous phenotypic characterization of the bsd2 mutant suggested that the BSD2 product regulates rbcL gene expression. We do not capture any email address. Restriction mapping of these fragments together with gel blot analysis of genomic DNA under low-stringency conditions indicated that Bsd2 is a single-copy gene in maize (data not shown). In DnaJ, these cysteines coordinate two Zn(II) ions (Banecki et al., 1996; Szabo et al., 1996). Although BSD2 only shows similarity to DnaJ-like proteins over a limited domain, the functionality of this domain has been demonstrated in vitro (Szabo et al., 1996) and in vivo (Banecki et al., 1996). Chloroplasts of maize ( Zea mays ) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. These species-specific differences in Rubisco accumulation patterns may reflect different underlying genetic mechanisms that were exploited during the polyphyletic evolution of the C4 pathway (Moore, 1982). Ten fractions of 650 μL were collected corresponding to the central region of the gradient. These results suggest that BSD2 does 1.In C3 plants only rubisco is functional and only mesophyll cells are present while in C4 plants both pepcase and rubisco are present nd here both mesophyll and bundle sheath cells are present. 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