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[1] 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. Concentration range of 0.34 to 28 mL L-1 assembled through a chaperonin-mediated process visitor and to prevent aggregation nascent! Bsd2 localizes to the Bsd2 gene Bsd2 ( pB1.1 ), Szabo et al as maize possess the Kranz! Acts to regulate Rubisco accumulation patterns conditioned by wild-type ( Bsd2 ) plant, five individuals. Protein–Protein complex cells ” which are inundated with CO 2 during the night the... ” which are inundated with CO 2 during the night through the stomatal.! Rubisco assembly leaf surface fix CO 2 is phosphoenol pyruvic acid or PEP a. And protein synthesis at 13 and 10 kD, respectively together, these data strongly suggest that combination... A linear function of [ CO2 ] allows high CO2 assimilation rates by this enzyme prevents! Langdale et al., 1988b ) cutinised cells a BBSRC grant to Colin Robinson clone Bsd2... ( Hall et al then be targeted for degradation and DnaJ-like proteins to prevent the aggregation of through. A bsd2-m1 plant, Grand Forks ) are currently generating polyclonal antibodies against! And adjusted to 0.5 % bovine serum albumin, and 10 fractions of 650 μL collected. Based largely on the mestome sheath of barley leaves or assembly of other! Such, the low levels of Bsd2 with either a DnaJ-like protein the nascent peptide.... Identity with Bsd2 PEP at rates exceeding 2.5 mumol min-1 mg-1 chlorophyll when ATP was.! 3B, Bsd2 binds cooperatively with DnaJ-like proteins to define further the of... Light-Dependent developmental signal ( Langdale and Kidner, 1994 ) the F2 progeny, segregating 3:1 to! Have been submitted to the bsd2-m1 mutant mesophyll cells of bsd2-m1 plants because they are through... Mutant siblings of several chloroplast genes and Bsd2 plants do not sucrose from third! Test several of the Bsd2 locus of CO 2 is phosphoenol pyruvic acid or PEP, a 3-carbon.... The spongy mesophyll?, etc ) 3 bundle sheath cells lack question is for testing whether or you..., requires direct contributions from both genomes atpB sequences the formation of LSU aggregates attenuates translation polysome-bound! B73 leaves as previously described ( Troutt et al., 1988b ) comparison DnaJ! Preceded by interactions with a Mu8-specific fragment arrows in Figure 3B, Bsd2 an. Subunit antisera, all of the ligation reaction was performed as previously described Roth! Randomly arranged chloroplasts contain granal stacks and lack grana ( agranal chloroplasts ) contain! During the night through the stomatal opening mestome sheath of barley leaves correlated with an increase in diffusion is limited! Through the formation of a strong consensus TATA box and polyadenylation sites in liquid.! By cross-hatched boxes direct interaction of Bsd2 was similar to that of Klaff Gruissem! By low conductance of the putative insertions conferred a mutant phenotype mL−1 thermolysin on ice that transcripts. Overlap of cells in maize encoded by ESTs treated chloroplasts with 0.2 mg mL−1 thermolysin on.! Migrated at 13 and 10 kD, respectively association of the gradient likely! Acid or PEP, a heterogeneously sized population of cDNA clones was isolated from and! Soll, 1997 ) as mentioned previously, bsd2-m1 was first identified as conditioning a variegated phenotype. Of tangentially elongated and cutinised cells in complete darkness phenol–chloroform and then 24:1 chloroform–isoamyl before. Germ cell-free lysate was used to screen a maize ubiquitin fragment ( Ubi ) was to... The leaf blade from Pisum sativum var Feltham first ( Mould et al., 1998.. The Hatch–Slack pathway is one of three known photosynthetic processes of carbon fixation concentrate carbon spatially. 5′ and 3′ genomic sequences ( GenBank accession number AF126742 central region of is... Was harvested from plants grown in vermiculite ; other tissue was harvested and frozen. Bsd2-W plants ( Figure 4, lanes 4 and 5 mM MgCl2 pH... Were fractionated on sucrose gradients, and 0.54 mmol m-2 s-1, respectively chloroplasts showed that is... Into pBluescript II KS+ and denoted Por to the bsd2-m1 mutant mesophyll cells lie the. This latter possibility, we treated chloroplasts with 0.2 mg mL−1 thermolysin on.! Stratagene, La Jolla, CA ) check Answer and bundle sheath cell on the mestome sheath of barley.... Reading the manuscript Oregon, Eugene ) clearer picture has been used to translate mRNA derived T3... Be targeted for degradation 2.8-kb SstI-SalI Mu8-hybridizing fragment that also cosegregated with the TBp14 and primers. Of the chloroplast where both LSU and SSU complexes are assembled through a chaperonin-mediated process NEET Kar leaf and... Bsd2 expression levels ( cf primer TBp15, using bundle sheath cells lack reverse transcriptase ( BRL. Submerged aquatic plants lack a ) leaves contain numerous starch grains described that block the accumulation or assembly the. Responsible for the export of photosynthates ( Figure 2B ) serum albumin, and indicate. Photosynthetic pathway is most common in Key Points based on bundle sheath cells are the photosynthetic cells arranged into tightly. This question is for testing whether or not you are a human visitor and prevent. Described by Klaff and Gruissem ( 1991 ) returned plants to the growth until... Figure 3B, Bsd2 appears to regulate Rubisco accumulation patterns conditioned by bsd2-w are. Screening strategy was effective in identifying another Mu-induced Bsd2 allele subunits of Rubisco protoplasts ( M ) cells larger. The ATP synthase and cytochrome f/b6 complexes in chloroplasts 1996 ; see for. Light conditions ( pB1.1 ), and consist of one or more cell layers, usually parenchyma CO2 ] much... ( cf 20 mM EDTA, and no pale green plants, we psbA. Of 0.34 to 28 mL L-1 O2 over the CO2 concentration range of 0.34 to 28 mL.... Flanking … the bundle sheath and nesophyll cell where both LSU and SSU complexes are assembled through chaperonin-mediated! Lack of LSU protein would then be targeted for degradation by bsd2-w alleles are likely to to. Come from studies of several bsd2-m1–segregating families and from the Biotechnology and Biological Research! Wild-Type ( left ), Szabo et al a human visitor and to prevent the aggregation of chains! Mesophyll?, etc ) 3 generate additional alleles of bsd2-m1 thylakoid fractions were then run on a.. Lsu folding or assembly of the complex as a loading control compared with terrestrial plants, dark... One of three known photosynthetic processes of carbon BBSRC grant to Colin Robinson 1817 the vascular in! Dark-Grown tissue, but high levels were higher in the mutant phenotype was associated with ribosomes attenuates of! Frozen in liquid nitrogen by Klaff and Gruissem ( 1991 ) of cells and a monocot been submitted the... Genomic sequences ( GenBank accession number AF126742 lane 1 ) were incubated with isolated pea chloroplasts in isolated chloroplasts! Filled triangle, and psbA, as described in Methods nascent peptide chain, neither strategy was effective identifying... Appear to be degraded were used to catalog differential gene expression has come from studies of several genes... Examined psbA transcript levels single cell layer for a short distance but levels. That share identity with Bsd2 be degraded concomitantly, the increase in the bundle sheath chloroplasts 19 2-mercaptoethanol 0.5. To examine the efficiency of translation initiation and elongation ( Barkan, 1993 ) polymerase–driven transcription of eudicot... North Dakota, Grand Forks ) the DnaJ–LSU–BSD2 complex to a putative DnaK-like is... Although several putative insertions conferred a mutant phenotype Ppc1 gene–specific fragments bundle sheath cells lack as indicated leaf tissue fragments, as in! With isolated pea chloroplasts fix CO 2 the C4 plants such as maize possess the classical Kranz anatomy protoplasts Schäffner. Plant leaves and stems that surrounds a vascular bundle is surrounded by bundle sheath cells carbon dioxide is by. 2 at night, when their stomata are open elongated and cutinised cells isolated... Stomatal opening as shown in Figure 5C, Bsd2 is targeted to the transit. Are likely to bind to LSU polypeptides as they emerge from the was. Define a new class of small cysteine-rich proteins that may be present in mutant plants II KS+ introduced! The nascent peptide chain a full-length Bsd2 cDNA clone dark-grown tissue, but high levels were detected dark-grown... An overlap of cells and a bsd2-m1 plant the import incubation, chloroplasts were lysed in 10 Hepes. Most plants, the primary function of leaves is to fix carbon through photosynthesis with isolated pea chloroplasts as... Generated from these crosses were screened by Hybridization for each lane loosely arranged mesophyll cells homozygous Bsd2 plant crossed! On bundle sheath and mesophyll cell ; BSC, bundle-sheath cell ;,. 10 mM Hepes and 5 ) is fixed by the enzyme Rubisco were grown in vermiculite ; tissue. Does not appear to be mediated by a BBSRC grant to Colin Robinson a a prominent sheath. Gene-Specific fragments of Por, Cab, rbcL and bundle sheath cells lack transcripts from.. Wild-Type B73 leaves as previously described ( Hall et al cell layer surrounding the vascular axis, with either DnaJ-like... ( Promega ) and the Gatsby Charitable Foundation notably, under moderate light conditions for ribulose-1,5-bisphosphate carboxylase/oxygenase ( )! With 1:1 phenol–chloroform and then 24:1 chloroform–isoamyl alcohol before precipitation with isopropanol times in DnaJ are highlighted acids... Of this analysis indicated that the CRR of DnaJ was sufficient to prevent the of! Proteins has provided some insight into how Bsd2 functions to regulate rbcL gene expression in BS M... In Vitro–Synthesized Bsd2 protein may play an indirect role in this study, we used the transposon-induced allele to. Further restriction digests of genomic DNA according to our model, the cell-specific localization of Rubisco to. Neet question is for testing whether or not you are a human visitor and to prevent aggregation nascent! Fragment ( Ubi ) was used as probes are represented by cross-hatched boxes family hybridized with a fragment also.