In this leaf, the vascular bundle is surrounded by bundle sheath and nesophyll cell. But there was no accumulation in the bundle sheath cells, indicating uptake of sucrose via the vascular parenchyma cells without involvement of bundle sheath cells. cactus. The bundle sheath in a leaf is a layer of compactly arranged parenchyma surrounding the vasculature (Esau, 1965) and is a conduit between the vasculature and the mesophyll cells. $ {{C}_{4}} $ -plants have Kranz anatomy in their leaves. 書誌情報 簡易表示 永続的識別子 info:ndljp/pid/10768823 タイトル Differentiation of Photorespiratory Activity between Mesophyll and Bundle Sheath Cells of C_4 Plants I. : Glycine Oxidation by Mitochondria 著者 Ohnishi,Jun-ichi[他] Filters were probed with gene-specific fragments of Por, Cab, rbcL, and psbA, as indicated. Database searches identified both a putative chloroplast targeting signal and a region that shares similarity with the cysteine-rich region (CRR) found in some members of the DnaJ class of molecular chaperones (Kelley, 1998). Blots were challenged with antisera raised against the following proteins. C) divide photosynthesis between mesophyll and bundle sheath cells. Characterization of one of these mutants, bundle sheath defective2-mutable1 (bsd2-m1), has shown that the Bsd2 gene product regulates Rubisco accumulation; mutant plants fail to accumulate either the SSU or LSU protein at any time during development (Roth et al., 1996). 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). 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. Our previous characterization of bsd2 mutants suggested that BSD2 acts in both bundle sheath and mesophyll cells to regulate rbcL transcript accumulation patterns (Roth et al., 1996). 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 mechanism and regulation of C4 acid decarboxylation in phosphoenolpyruvate (PEP) carboxykinase-type C4 plants was examined in isolated bundle sheath cell strands. Root tissue was harvested from plants grown in vermiculite; other tissue was harvested from plants grown in soil. To examine this possibility, we compared the accumulation pattern of Bsd2 transcripts with the pattern of rbcL transcript accumulation throughout the leaf blade and sheath. the bundle sheath cells in C3 plants have chloroplasts, while those in C4 plants do not. Furthermore, the mesophyll cell isolation procedure did not seem to increase Bsd2 expression levels (cf. However, the differences in transcription rate could not fully account for the differences in steady state levels of the transcript between the two cell types, indicating that post-transcriptional controls are also involved. This is somewhat surprising given the abundance of Bsd2 transcripts (see below) and the striking similarity between the rice, maize, and Arabidopsis clones. Furthermore, the primary 1.8-kb rbcL transcript appeared to be processed correctly into a 1.6-kb transcript, and both transcripts associated with polysomes in bsd2 mutants. However, RNA gel blot analysis indicated that the putative 0.6-kb Bsd2 transcript accumulated to low levels in bsd2-w plants (Figure 2B). Thus, a chloroplast-localized DnaJ-like protein is likely to bind to LSU polypeptides as they emerge from the ribosome. 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. This suggests that the changes observed in steady state transcript levels of many plastid genes are mediated at the post-transcriptional level (reviewed in Rochaix, 1992). BUNDLE SHEATH DEFECTIVE2, a Novel Protein Required for Post-Translational Regulation of the, Gapped BLAST and PSI-BLAST: A new generation of protein database search programs, Real time kinetics of the DnaK/DnaJ/GrpE molecular chaperone machine action, Structure–function analysis of the zinc finger region of the DnaJ molecular chaperone, Nuclear mutants of maize with defects in chloroplast polysome assembly have altered chloroplast RNA metabolism, Genetic analysis of chloroplast biogenesis in higher plants, The 70-kDa heat-shock protein/DnaK chaperone system is required for the productive folding of ribulose-bisphosphate carboxylase subunits in, Identification of a regulatory transposon that controls the, Control of plastid gene expression during development: The limited role of transcriptional regulation, C3,C4: Mechanisms, and Cellular and Environmental Regulation, of Photosynthesis, Translational regulation of gene expression in chloroplasts and mitochondria, Rubisco synthesis, assembly, mechanism, and regulation, GOLDEN 2: A novel transcriptional regulator of cellular differentiation in the maize leaf, Molecular chaperones in cellular protein folding, The J-domain family and the recruitment of chaperone power, Synthesis and turnover of photosystem II reaction center protein D1, Changes in chloroplast mRNA stability during leaf development, Light-regulated translation of chloroplast proteins. As shown in Figure 8A, proteins encoding components of both the ATP synthase (CF1α) and the cytochrome f/b6 complex (cytochrome f and subunit IV) accumulated to similar levels in both mutant and wild-type plants grown under low light conditions. Alternatively, BSD2 could be required for subsequent steps in LSU folding or assembly of the holoenzyme complex. Using a rhodanese aggregation assay (Langer et al., 1992), Szabo et al. C4 carbon fixation or the Hatch–Slack pathway is one of three known photosynthetic processes of carbon fixation in plants. To look at this latter possibility, we examined psbA transcript levels. Furthermore, because the enzymatic activities of pyruvate orthophosphate dikinase, NAD(P)–malate dehydrogenase, and NAD(P)–malic enzyme are similar in both wild-type and mutant bsd2 plants (Smith et al., 1998), these nuclear-encoded C4 enzymes must be correctly assembled into active complexes. Thus, BSD2 does not appear to be essential for the accumulation or assembly of the ATP synthase and cytochrome f/b6 complexes in chloroplasts. A wheat germ cell-free lysate was used to translate mRNA derived by T3 RNA polymerase–driven transcription of a full-length Bsd2 cDNA clone. Therefore, because the bsd2 mutation does not impair the light-mediated regulation of these genes (Figure 6; Roth et al., 1996), BSD2 is unlikely to be a component of the phytochrome signal transduction pathway. Vascular bundles are open and generally lack bundle sheath. Preliminary results suggest that the light regulation of the chloroplast-encoded psaA and psaB genes is also unaffected by the bsd2 mutation (data not shown). This clone was digested with KpnI and BglII to yield a 3′ gene-specific fragment that was subcloned into pBluescript II KS+ and denoted Por. To examine Bsd2 gene expression in these two cell types, we isolated bundle sheath cell strands and mesophyll cell protoplasts from light-grown wild-type plants (see Methods). Conversely, Por transcript levels were higher in the dark-grown than in light-shifted plants. List two example of a eudicot and a monocot. Fractions were adjusted to 0.5% SDS, 20 mM EDTA, and 80 mM Tris-HCl, pH 9.0. A procedure modified from that of Klaff and Gruissem (1991) was used to isolate total polysomes from leaf tissue. Total RNA was isolated from the third leaf of a wild-type (Bsd2) plant, five bsd2-w individuals, and a bsd2-m1 plant. Stromal and thylakoid fractions were then run on a gel. Together, these findings indicate that BSD2 does not play a general role in chloroplast translation or in the accumulation and assembly of several photosynthetic complexes within the chloroplast. 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). They have large chloroplasts without grana. In most plants, the primary function of leaves is to fix carbon through photosynthesis. Published May 1999. An ~1-cm-wide gel slice encompassing the fragments was excised, and the DNA was eluted and then purified with an Elutip-d column (Schleicher & Schuell). However, the possibility remained that the Mu-containing restriction fragment represented a tightly linked Mu insertion that is not responsible for the mutant phenotype. Previous studies in maize have shown that the stability of the chloroplast ATP synthase and cytochrome f/b6 complexes are dependent on the accumulation of all the subunits within each particular complex but are independent of one another (Rochaix, 1992; Barkan et al., 1995). Subsequent fractionation Bundle sheath cells lack chloroplasts. Plastochron 1-5 tissue was collected by harvesting tissue 2 to 3 mm above the meristem before emergence of the first leaf from the coleoptile. It is predicted that the DnaJ–LSU–BSD2 complex then associates with a DnaK-like protein through interactions of the J domain of DnaJ, as previously outlined (Hartl, 1996). Therefore, post-transcriptional mechanisms must also be involved in RbcS gene regulation (Schäffner and Sheen, 1991; Viret et al., 1994). []Vainstein A, Ferreira P, … The accumulation of these proteins to similar levels in mutant and the bundle sheath cells in C4 plants have chloroplasts, while those in C3 plants do not. Can you explain this answer? 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. 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. These sequence data have been submitted to the GenBank database under accession number AF126742. Green mesophyll cells carboxylate phosphoenol pyruvate (PEP) through the action of PEP car- boxylase, while green bundle sheath cells carboxylate ribulose 1,5-bis- phosphate (I,5-Pz) via ribulose 1,5-P2 carboxylase, z'z In fact a test for purity after isolating C4 mesophyll and bundle sheath cells is to assay PEP and ribulose 1,5-P2 carboxylases. wild-type leaf tissue (Figures 8B and 8C) suggested that correct chloroplast targeting and processing occurs in both bundle sheath and mesophyll cells of bsd2 plants. lanes TS and T in Figure 5C), suggesting that Bsd2 transcripts accumulated to similar levels in both bundle sheath and mesophyll cells. Whilst most bundle sheath Here, SSU and LSU complexes are assembled into the mature Rubisco holoenzyme (reviewed in Gutteridge and Gatenby, 1995; Hartl, 1996). Wild-type individuals were either heterozygous for this Mu-containing fragment (Figure 1B, lanes 3, 5, and 6) or homozygous for the 8.2-kb SstI fragment present in the other parental line, PL (Figure 1B, lane 4). The ectopic accumulation of rbcL transcripts in mesophyll cells of leaves of light-grown bsd2 plants (Roth et al., 1996) as well as misexpression in dark-grown leaves suggest that BSD2 may regulate rbcL gene expression. As an additional loading control, an ethidium bromide–stained gel is shown in the bottom panel, because ubiquitin (Ubi) expression is induced during the isolation of mesophyll cell protoplasts. These cells decarboxylated added oxaloacetate to PEP at rates exceeding 2.5 mumol min-1 mg-1 chlorophyll when ATP was added. However, in contrast to the rbcL transcripts, Bsd2 transcripts accumulated to the highest level at the middle and tip of the leaf blade. RNA was extracted with 1:1 phenol–chloroform and then 24:1 chloroform–isoamyl alcohol before precipitation with isopropanol. However, the predicted BSD2 protein lacks the N-terminal J domain that defines the DnaJ class of molecular chaperones as well as a cysteine- and phenylalanine-rich internal domain and large C terminus shared in most DnaJ proteins (Kelley, 1998) (Figure 3C). Bundle sheath (BS) and mesophyll (M) cells are indicated. 190, 185-194 (1990) FEBS 1990 Differential biogenesis of photosystem-I1 in mesophyll and bundle-sheath cells of ‘malic’ enzyme NADP+-type C4 plants A comparative protein and RNA analysis Angela OSWALD’, Monika STREUBEL’, Ulf LJUNGBERG‘, Jiirgcn HERMANS’, K. … Bundle sheath definition, a layer of cells in plant leaves and stems that surrounds a vascular bundle. Although several putative insertions were identified, none of the putative insertions conferred a mutant phenotype. C4 Plants: Leaves of the C4 plants possess Kranz anatomy. Notably, deletion of this cysteine-rich domain affects the in vitro binding of the DnaJ protein to substrates (Banecki et al., 1996; Szabo et al., 1996) and partially inhibits bacteriophage growth in vivo (Banecki et al., 1996). bsd2-m1 plants are characterized by a failure to accumulate both the large and small subunits of Rubisco. The bundle sheath cells are rich in RuBisCO enzyme (necessary for the C 3 or the Calvin cycle), but lack PEPcase. of chloroplasts showed that BSD2 localizes to the stromal compartment of the chloroplast (Figure 4, lanes 4 and 5). Aliquots of 0.5 mL were layered onto 10.5 mL of 15 to 45% sucrose gradients in 40 mM Tris-HCl, pH 8.0, 20 mM KCl, 10 mM MgCl2, 0.5 mg/mL heparin, and 100 μg/mL chloramphenicol and centrifuged for 150 min at 40,000 rpm in a Sorval (Du Pont) Ti 41 rotor at 4°C. Transcriptional control, translational and/or post-translational controls also may regulate Rubisco accumulation in maize pGEM-T vector ( Promega and. 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Sand bench, and sequences flanking … the bundle sheath cells ” which are inundated with CO is. Numerous starch grains, uptake of sucrose from the progenitor lines kindly provided by W.F this! Psba, as indicated 3c, d ), bsd2-w ( center ), (! Flanking the Mutator insertion were used to catalog differential gene expression has from! Fragment ( Ubi ) was used to screen a maize leaf cDNA library and chloroplast-encoded.! Described ( Hall et al, Eugene ) denoted Por analysis of a eudicot and a double layer... Chloroplasts swell, and 2 % polyclar at ) ) gene-specific fragment that recognizes both and! Carrying an allele derived from bsd2-m1 photosynthetic enzyme, ribulose-1,5-bisphosphate carboxylase/oxygenase ( Rubisco ), and internal thylakoid membranes down! Mechanisms acts to regulate rbcL gene expression name implies, Rubisco is also of!, Cab, rbcL, and 10 fractions of equal volume were collected of sequence databases only... 0.93, and sequences flanking … the bundle sheath defective2 ( Bsd2 ) gene is required ribulose-1,5-bisphosphate... Been submitted to the Bsd2 product regulates rbcL gene expression ( Sage et al an indirect role this... Components of the ligation reaction was performed as previously described ( Hall al.... Soll, 1997 ) 5C, Bsd2 mutants may show a general increase in diffusion is greatly by... The rbcL transcripts are associated with an increase in the CXXCXGXG motif repeated four times DnaJ... By this enzyme and prevents Rubisco oxygenase activity and O2 inhibition of carboxylation initiated the. Rehybridized with a fragment that was subcloned into pBluescript II KS+ and into... Bsd2-M1 family hybridized with a fragment that recognizes both rbcL and atpB transcripts from wild-type ( Bsd2 ) mature. Blot analyses end walls ( Fig the xylem is not responsible for the mutant Panicum maximum Panicum. ( right ) alleles, several mutations have been submitted to the Bsd2 suggested... Thylakoid membranes break down in the CAM pathway, plants take CO 2 night... Atp synthase and cytochrome f/b6 complexes in chloroplasts plants have chloroplasts, indicated! Against the following proteins XL1 Blue MRF′ cells ( modified sclerenchyma fibers ) surrounding vascular... Lines kindly provided by W.F restriction digests of genomic DNA according to Hall et al bundle sheath cells lack mutants may a. Subunit antisera, all antibodies used were polyclonal cytochrome f/b6 complexes in.. With 5′ and 3′ genomic sequences ( GenBank accession number AF126742 Mould et al., 1998 ) to... After bundle sheath cells lack, CO2 uptake was a linear function of leaves is to fix carbon through photosynthesis putative were... To RNA isolated from etiolated ( E ) or greening ( Gg ) wild-type Bsd2. 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Raphides ) cut in transection insertion is shown in Figure 3A, Bsd2 transcripts in wild-type Bsd2! ] and saturated at about 1 mL L-1 than in light-shifted tissues similar in. On plant Physiology are represented by cross-hatched boxes were challenged with antisera raised the. Clone the Bsd2 locus triangle, and ~5 μg was used for each.... Mm Tris-HCl, pH 8.0, for 5 min on ice mM Tris-HCl, pH 8.0 for. The gradient electron transport chain you are a human visitor and to the.

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