Isolation and Characterization of cDNAs for Differentially Accumulated Transcripts between Mesophyll Cells and Bundle Sheath Strands of Maize Leaves (original) (raw)
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Current Genetics, 2011
The C4 grass Zea mays separates light and light-independent photosynthetic processes into two leaf cell types: bundle sheath (BS) and mesophyll (M). When mature, BS and M cells have anatomically and biochemically distinct chloroplasts that must cooperate to complete the process of photosynthesis. This report compares changes in transcript abundance between young and mature maize BS and M chloroplasts from speciWc segments of the leaf developmental gradient. Representative transcripts encoding components of Photosystem I, Photosystem II, Cytochrome b 6 f, thylakoidal NADH dehydrogenase; and the large subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase as well as nine nuclear-coded transcripts encoding chloroplast proteins were measured using quantitative RT-PCR. In addition, 887 nuclear genes encoding plastidlocalized proteins, as well as 64 chloroplast and 34 mitochondrial genes were assayed utilizing a cDNA microarray. In 9 out of the 18 chloroplast-encoded genes and 84 genes from the 985 element microarray revealed greater than twofold transcript abundance diVerences between developmental stages and/or cell types. Patterns for transcripts associated with operons and gene clusters suggest diVering regulatory mechanisms for particular polycistronic stretches. In summary, this report provides evidence that cell type-speciWc transcript abundance varies more in the young developing chloroplast, and diVerences plateau or subside as chloroplasts mature.
Differential expression of LHCII genes in mesophyll and bundle sheath cells of maize
Carlsberg Research Communications, 1986
The properties and composition of bundle sheath and mesophyl! thylakoids from maize leaves are compared. This was possible because of the isolation of large amounts of purified material obtained by enzymatic digestion of mechanically disrupted leaves. Bundle sheath thylakoids from mature leaves, lack the chlorophyll-proteins and polypeptides associated with the reaction centre ofphotosystem II. They do, however, contain significant amounts of LHCII, which transfers excitation energy to photosystem I. LHCII isolated from bundle sheath thylakoids had a different freeze-fracture ultrastructure and a different polypeptide composition from LHCII isolated from mesophyll thylakoids, indicating a differential expression of the LHCII gene family in mesophyll and bundle sheath cells of maize leaves.
FEBS Letters, 1986
Bundle sheath chloroplasts of maize, a C, plant, lack a functional herbicide-binding site and the 32 kDa-QB thylakoid protein of photosystem II. hileasurements of the amounts of QB-protein mRNA in bundle sheath and mesophyll chloroplasts by hybridization to a cloned psbA probe, and analysis of in vitro translation products of bundle sheath and mesophyll RNA show that during differentiation of maize leaf cells to bundle sheath and mesophyll, the expression of the chloroplast gene, psbA, which encodes the 32 kDa-Qa protein, is controlled at the transcriptional level.
PLANT PHYSIOLOGY, 2012
To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C 4 metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.
Plant Cell, 2005
Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. 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. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway.
The Plant Cell, 1996
Within the maize leaf primordium, coordinated cell division and differentiation patterns result in the development of two morphologically and biochemically distinct photosynthetic cell types, the bundle sheath and the mesophyll. The bundle sheath defective2-mutablel (bsd2-ml) mutation specifically disrupts C4 differentiation in bundle sheath cells in that the levels of bundle sheath cell-specific photosynthetic enzymes are reduced and the bundle sheath chloroplast structure is aberrant. In contrast, mesophyll cell-specific enzymes accumulate to normal levels, and the mesophyll cell chloroplast structure is not perturbed. Throughout mutant leaf development, the large and small subunits of ribulose bisphosphate carboxylase are absent; however, both rbcL and RbcS transcripts accumulate. Moreover, chloroplast-encoded rbcL transcripts accumulate ectopically in mesophyll cells. Although the bundle sheath cell chloroplast structure deteriorates rapidly when plants are exposed to light, this deterioration is most likely a secondary effect resulting from cell-specific photooxidative damage. Therefore, we propose that the Bsd2 gene plays a direct role in the post-transcriptional control of rbcL transcript accumulation and/or translation, both in bundle sheath and mesophyll cells, and an indirect role in the maintenance of bundle sheath cell chloroplast structure.
Plant Molecular Biology, 1984
We have investigated the molecular basis of differential localization of enzyme activities in mesophyll (M) and bundle-sheath (B) cells of maize leaves. M protoplasts and B strands were prepared by enzymatic digestions and mechanical treatment of secondary leaves. Soluble and thylakoid membrane proteins from the two cell types were compared by one-and two-dimensional gel electrophoresis and quantitative rocket immunoelectrophoresis. In addition, several thylakoid polypeptides were identified by crossed immunoelectrophoresis using monospecific antibodies. M and B thylakoids show quantitative and qualitative differences in their polypeptide compositions. While the M thylakoids contain the normal complement of polypeptides, the B thylakoids are deficient in ferredoxin-NADP + reductase, photosystem II reaction center polypeptides, and the light-harvesting chlorophyll a/b-protein complex. Comparison of the soluble proteins by two-dimensional gel electrophoresis revealed marked differences between M and B cells. The major proteins of one cell type are clearly absent from the other. These differences are paralleled by differences in the in vitro translation products of poly A + RNA isolated from the two cell types. Immunoprecipitation experiments showed that mRNA encoding the small subunit of ribulose-l,5-bisphosphate carboxylase (rbcS) is localized exclusively in B cells, whereas mRNA encoding phosphoenolpyruvate carboxylase is detected only in M cells. cDNA clones encoding the carboxylase rbcS and the chlorophyll a/b binding protein were used as probes in Northern blot analysis. M ceils contain no detectable RNA encoding rbcS but have a higher steady state level of RNA encoding the chlorophyll a/b-binding polypeptide compared to B cells. Taken together, our results demonstrate that differential gene expression in the two leaf cell types is regulated at the level of translatable mRNA, and, for at least two proteins, at the level of steady-state RNA.
Organization and activity of photosystems in the mesophyll and bundle sheath chloroplasts of maize
Journal of Plant Physiology, 2006
Photosystem I and Photosystem II activities, as well as polypeptide content of chlorophyll (Chl)-protein complexes were analyzed in mesophyll (M) and bundle sheath (BS) chloroplasts of maize (Zea mays L.) growing under moderate and very low irradiance. This paper discusses the application of two techniques: mechanical and enzymatic, for separation of M and BS chloroplasts. The enzymatic isolation method resulted in depletion of polypeptides of oxygen evolving complex (OEC) and aCF1 subunit of coupling factor; D1 and D2 polypeptides of PSII were reduced by 50%, whereas light harvesting complex of photosystem II (LHCII) proteins were still detectable. Loss of PSII polypeptides correlated with the decreasing of Chl fluorescence measured at room temperature. Using mechanical isolation of chloroplasts from BS cells, all tested polypeptides could be detected. We found a total lack of O 2 evolution in BS chloroplasts, but dichlorophenolindophenol (DCPIP) was photoreduced. PSI activity of chloroplasts isolated from 14-and 28-day-old plants was similar in BS chloroplasts in moderate light (ML), but in low light (LL) it was reduced by about 20%. PSI and PSII activities in M chloroplasts of plants growing in ML decreased with aging of plants. In older LL-grown plants, activities of both photosystems were higher than those observed in chloroplasts from ML-grown plants.
Molecular & cellular proteomics : MCP, 2008
Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I a...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2009
Photoinhibition is caused by an imbalance between the rates of the damage and repair cycle of photosystem II D1 protein in thylakoid membranes. The PSII repair processes include (i) disassembly of damaged PSII-LHCII supercomplexes and PSII core dimers into monomers, (ii) migration of the PSII monomers to the stroma regions of thylakoid membranes, (iii) dephosphorylation of the CP43, D1 and D2 subunits, (iv) degradation of damaged D1 protein, and (v) co-translational insertion of the newly synthesized D1 polypeptide and reassembly of functional PSII complex. Here, we studied the D1 turnover cycle in maize mesophyll and bundle sheath chloroplasts using a protein synthesis inhibitor, lincomycin. In both types of maize chloroplasts, PSII was found as the PSII-LHCII supercomplex, dimer and monomer. The PSII core and the LHCII proteins were phosphorylated in both types of chloroplasts in a light-dependent manner. The rate constants for photoinhibition measured for lincomycin-treated leaves were comparable to those reported for C3 plants, suggesting that the kinetics of the PSII photodamage is similar in C3 and C4 species. During the photoinhibitory treatment the D1 protein was dephosphorylated in both types of chloroplasts but it was rapidly degraded only in the bundle sheath chloroplasts. In mesophyll chloroplasts, PSII monomers accumulated and little degradation of D1 protein was observed. We postulate that the low content of the Deg1 enzyme observed in mesophyll chloroplasts isolated from moderate light grown maize may retard the D1 repair processes in this type of plastids.