Detection of phosphoenolpyruvate and ribulose 1,5-bisphosphate carboxylase transcripts in maize leaves by in situ hybridization with sulfonated cDNA probes (original) (raw)
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Plant and Cell Physiology, 2000
To characterize novel genes functioning specifically in mesophyll cells (MCs) or bundle sheath cells (BSCs) of C4 plants, differential screening of a maize cDNA library was conducted using 32 P-labeled single-strand cDNAs prepared from MCs and bundle sheath strands (BSS) as probes. Ten genes encoding thylakoid membrane proteins in chloroplasts were identified as MC-abundant genes. These included genes for chlorophyll a/b binding proteins, plastocyanin, PsaD, PsbT, PsbR, PsbO, PsaK, PsaG, PsaN and ferredoxin. Seven genes identified as BSS-abundant genes encoded PEP carboxykinase, salt-inducible SalT homolog, heavy metalinducible metallothionein-like protein, ABA-and droughtinducible glycine-rich protein, and three proteins of unknown function (one of which was named Bss1). In situ hybridization analyses for several selected genes revealed that mRNAs for the metallothionein-like protein and Bss1 were accumulated specifically in BSCs, and that mRNA for the SalT homolog was accumulated in vascular cells around phloem cells. Results suggest that the functional differentiation of MC chloroplasts accompany preferential expression of these small proteins in photosystem complexes and that BSCs are the major site of stress responses.
Plant molecular biology, 2000
C4-type phosphenolpyruvate carboxylase (C4PEPC) acts as a primary carbon assimilatory enzyme in the C4 photosynthetic pathway. The maize C4PEPC gene (C4Ppc1) is specifically expressed in mesophyll cells (MC) of light-grown leaves, but the molecular mechanism responsible for its cell type-specific expression has not been characterized. In this study, we introduced a chimeric maize C4Ppc1 5'-flanking region/beta-glucuronidase (GUS) gene into maize plants by Agrobacterium-mediated transformation. Activity assay and histochemical staining showed that GUS is almost exclusively localized in leaf MC of transgenic maize plants. This observation suggests that the introduced 5' region of maize C4Ppc1 contains the necessary cis element(s) for its specific expression in MC. Next, we investigated whether the 5' region of the maize gene interacts with nuclear proteins in a cell type-specific manner. By gel shift assays with nuclear extracts prepared from MC or bundle sheath cells (BSC...
The Plant Journal, 1995
The distribution of a number of specific mRNAs has been observed in maize growing pollen tubes. Whole-mount in situ hybridization using digoxygenin-labelled RNA probes has been tested. The technique appears to be a simple, rapid and reliable method in this system, in immature anthers and also in other tissues. Results for three probes are presented. They correspond to a hydroxyproline-rich glycoprotein (HRGP), an abundant component of the maize cell wall, to an a-tubulin (encoded by the Tuba 1 gene) highly expressed in the radicular system of the plant and also in pollen, and to an isoform of the malic enzyme, involved in the basic metabolism of the plant. The mRNAs corresponding to these three proteins are differently distributed in the germinating pollen. While HRGP mRNA is only present in the tube, malic enzyme mRNA is only present in the body of the pollen cell, and a-tubulin mRNA is present in both parts of the cell but shows a higher accumulation in the tip of the pollen tube.
The Plant Journal, 2007
A quantitative in situ hybridization technique (quantitative whole-mount in situ hybridization, QISH) for plants is described. It employs direct hybridization of fluorescently labelled gene-specific oligonucleotides in large tissue pieces combined with optical sectioning. It dramatically increases the throughput compared with conventional antibody-and microtome-based in situ mRNA hybridization methods, while simultaneously eliminating artefact-prone preparation steps that prevent reliable quantification in conventional methods. The key feature of this technique is the quantification of gene expression using housekeeping genes (cytosolic GAPDH and 18S RNA) as internal standards. This feature enables a correction of varying cytoplasm/vacuole ratios in different cell types, as well as tissue optical effects and non-specific signals. The quantitative nature of the technique allows for analysis of gene expression in response to different environmental conditions, as well as tissue-and age-dependent differences in gene expression patterns. In addition to testing tissue permeabilization, structural preservation, specificity, linearity and tissue optical effects, we verified the reliability of the technique with three Arabidopsis thaliana genes of known function and distribution. These were the rbcL gene for ribulose 1,5-bisphosphate carboxylase, the developmentally related gene SCARECROW (AtSCR) and PHOT-1, a photoreceptor kinase. As expected, rbcL mRNA was found in all photosynthetic cells, while SCR mRNA was detected mainly in bundle sheath cells and PHOT-1 was found predominantly in epidermal and cortical cells of the apical hook of light-grown seedlings. As an application example, QISH was used to measure transcript abundance for a zinc transporter from the ZIP family of transporters in the Zn/Cd hyperaccumulator model plant, Thlaspi caerulescens, and the related non-accumulator Thlaspi arvense. This showed that QISH can be used to compare differences in mRNA levels between cell types, plant growth conditions and plant species. Messenger RNA for the zinc transporter gene ZNT1 was abundant in photosynthetic cells, but not in the epidermal storage cells where metal hyperaccumulation in T. caerulescens occurs. This indicates that ZNT1 does not directly participate in metal hyperaccumulation within the leaf. Growing T. caerulescens with high zinc levels strongly reduced ZNT1 transcript abundance in the spongy mesophyll cells, but less in the other cell types. In T. arvense, ZNT1 mRNA levels were generally much lower, and were furthermore drastically reduced by growth at increased zinc levels, confirming earlier reports regarding ZNT1 regulation in these two Thlaspi species.
The EMBO journal, 1984
We have examined the expression of a member of the multigene family encoding the small subunit (rbcS) of ribulose-1,5-bisphosphate carboxylase in various tissues of pea. The rbcS gene, pPS-2.4, was characterized by DNA sequence analysis and 5' and 3' end mapping of its mRNA transcript. rbcS polypeptides were shown to be differentially present in various tissues of light- and dark-grown plants. Northern analysis shows that compared with green leaves, the level of rbcS mRNA is reduced to approximately 50% in pericarps, 8% in petals and seeds, and 1-3% in etiolated leaves, stems, and roots. 5' S1 nuclease mapping of total rbcS mRNA was used to quantitate the relative amount of pPS-2.4 gene-specific transcripts in each tissue. pPS-2.4 mRNA accounts for approximately 30-35% of total rbcS transcripts in green leaves, but only 5-10% in pericarps, 15-20% in seeds, and is below detection in petals and etiolated leaves. We conclude that the pPS-2.4 gene is expressed in a tissue-sp...
Hybridization to mRNA of Tissue Sections
Methods, 2001
situ work, we have found that fixation conditions In situ hybridization detection of mRNA is an essential tool for undercan strongly influence the sensitivity of an in situ standing regulation of gene expression in cells and tissues of different experiment, especially in Arabidopsis tissues. Also, organisms. Over the years, numerous in situ protocols have been devel-DNA amplified by polymerase chain reaction (PCR) oped ranging from whole-mount techniques that allow fast transcript proved to be a much more efficient template for probe localization in intact organs to high-resolution methods based on the synthesis. Minor variations in other basic steps of electron microscopic detection of mRNAs at the subcellular level. Here, we present a detailed protocol for the detection of mRNAs in plant the protocol did not significantly affect the outcome tissues using radiolabeled single-stranded RNA probes. Hybridizations of the in situ results. Aspects of ISH procedures that are carried out on tissue sections of paraffin-and plastic-embedded are not directly related to Arabidopsis thaliana are plant tissues. Although this in situ protocol is appropriate for plant not discussed. tissues in general, it has been optimized for Arabidopsis thaliana.
Isolation and analysis of mRNAs from specific cell types of plants by ribosome immunopurification
Methods in molecular biology (Clifton, N.J.), 2013
Multiple ribosomes assemble onto an individual mRNA to form a polyribosome (polysome) complex. The epitope tagging of speci fi c ribosomal proteins can enable the immunopuri fi cation of polysomes from crude cell extracts derived from cryopreserved tissue samples. Through expression of the epitope-tagged ribosomal protein in cell-type and regional speci fi c domains of Arabidopsis thaliana and other organisms it is feasible to quantitatively assess the mRNAs that are associated with ribosomes with cell-speci fi c resolution. Here we present detailed methods for development of transgenics that express a FLAG-tagged version of ribosomal protein L18 (RPL18) under the direction of individual promoters with speci fi c domains of expression, the immunopuri fi cation of ribosomes, and bioinformatic analyses of the resultant datasets obtained by microarray pro fi ling. This methodology provides researchers with the opportunity to assess rapid changes at the organ, tissue, regional or cell-type speci fi c level of mRNAs that are associated with ribosomes and therefore engaged in translation.
In situ isolation of mRNA from individual plant cells: creation of cell-specific cDNA libraries
Proceedings of the National Academy of Sciences, 1995
A method for isolating and cloning mRNA populations from individual cells in living, intact plant tissues is described. The contents of individual cells were aspirated into micropipette tips filled with RNA extraction buffer. The mRNA from these cells was purified by binding to oligo(dT)linked magnetic beads and amplified on the beads using reverse transcription and PCR. The cell-specific nature of the isolated mRNA was verified by creating cDNA libraries from individual tomato leaf epidermal and guard cell mRNA preparations. In testing the reproducibility of the method, we discovered an inherent limitation of PCR amplification from small amounts of any complex template. This phenomenon, which we have termed the "Monte Carlo" effect, is created by small and random differences in amplification efficiency between individual templates in an amplifying cDNA population. The Monte Carlo effect is dependent upon template concentration: the lower the abundance of any template, the less likely its true abundance will be reflected in the amplified library. Quantitative assessment of the Monte Carlo effect revealed that only rare mRNAs (<0.04% of polyadenylylated mRNA) exhibited significant variation in amplification at the single-cell level. The cDNA cloning approach we describe should be useful for a broad range of cell-specific biological applications.