RESUMO
Laser capture microdissection (LCM) is a technique by which individual cells can be harvested from tissue sections while they are viewed under the microscope, by tacking selected cells to an adhesive film with a laser beam. Harvested cells can provide DNA, RNA, and protein for the profiling of genomic characteristics, gene expression, and protein spectra from individual cell types. We have optimized LCM for a variety of plant tissues and species, permitting the harvesting of cells from paraffin sections that maintain histological detail. We show that RNA can be extracted from LCM-harvested plant cells in amount and quality that are sufficient for the comparison of RNAs among individual cell types. The linear amplification of LCM-captured RNA should permit the expression profiling of plant cell types.
Assuntos
Técnicas de Preparação Histocitológica/métodos , Lasers , Plantas/genética , DNA de Plantas/genética , DNA de Plantas/metabolismo , Perfilação da Expressão Gênica , Técnicas de Preparação Histocitológica/instrumentação , Células Vegetais , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas/química , RNA de Plantas/genética , RNA de Plantas/metabolismo , Sensibilidade e Especificidade , Inclusão do Tecido , Fixação de TecidosRESUMO
NADP-dependent malic enzymes (NADP-ME; EC1.1.1.40) have been implicated in a wide range of metabolic pathways in the plastids and cytosol of plant cells. In maize, an NADP-ME type C4 plant, the most abundant NADP-ME form is the chloroplastic leaf isoform that delivers CO2 intracellularly to ribulose bisphosphate carboxylase (RuBPCase). A second NADP-ME isoform predominates in maize roots and exhibits distinct C3-like enzymatic characteristics. We show that the C3-like isoform is encoded by a pair of nearly identical genes that encode precursor proteins with functional chloroplast transit peptides. Using RT-PCR, we also show that the messages encoding the C4 and C3-like NADP-ME isoforms are differentially regulated with respect to the developmental stage of the leaf, light conditions, and tissue type. Based on these characteristics and on sequence comparison of ME families in other species, we propose a scheme for the origin of the C4-specific NADP-ME gene.
Assuntos
Malato Desidrogenase/genética , Plastídeos/genética , Zea mays/genética , Sequência de Aminoácidos , Celulose/farmacologia , Ciclopentanos/farmacologia , Evolução Molecular , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos da radiação , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Malatos/metabolismo , Dados de Sequência Molecular , Oxilipinas , Filogenia , Folhas de Planta/genética , Folhas de Planta/efeitos da radiação , Plastídeos/enzimologia , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Plântula/enzimologia , Plântula/genética , Plântula/microbiologia , Homologia de Sequência de Aminoácidos , Estresse Mecânico , Zea mays/enzimologiaRESUMO
A cytosolic NADP-malic enzyme (CYTME) has been described previously in several plants, all C3 species. CYTME is distinct from the chloroplastic NADP-malic enzyme (CHLME) that is highly active in C4 species. We show that at least one CytMe gene is present in all Flaveria spp., including C3, C4, and C3-C4 intermediate types. Based on the CytMe expression patterns in Flaveria pringlei (C3) and Flaveria trinervia (C4), we suggest CYTME has several distinct roles, including the supplying of NADPH for cytosolic metabolism, the supporting of wound response or repair, and the balancing of cellular pH in illuminated leaves. These three roles are likely correlated with CytMe mRNAs of apparent sizes 2.0, 2.2, and 2.4 kb, respectively, which differ in the length of the 5' untranslated regions. Various regulatory mechanisms involving RNA processing and translational efficiency are discussed.
