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
FQR1 is a novel primary auxin-response gene that codes for a flavin mononucleotide-binding flavodoxin-like quinone reductase. Accumulation of FQR1 mRNA begins within 10 min of indole-3-acetic acid application and reaches a maximum of approximately 10-fold induction 30 min after treatment. This increase in FQR1 mRNA abundance is not diminished by the protein synthesis inhibitor cycloheximide, demonstrating that FQR1 is a primary auxin-response gene. Sequence analysis reveals that FQR1 belongs to a family of flavin mononucleotide-binding quinone reductases. Partially purified His-tagged FQR1 isolated from Escherichia coli catalyzes the transfer of electrons from NADH and NADPH to several substrates and exhibits in vitro quinone reductase activity. Overexpression of FQR1 in plants leads to increased levels of FQR1 protein and quinone reductase activity, indicating that FQR1 functions as a quinone reductase in vivo. In mammalian systems, glutathione S-transferases and quinone reductases are classified as phase II detoxification enzymes. We hypothesize that the auxin-inducible glutathione S-transferases and quinone reductases found in plants also act as detoxification enzymes, possibly to protect against auxin-induced oxidative stress.
Assuntos
Proteínas de Arabidopsis/genética , Ácidos Indolacéticos/farmacologia , NAD(P)H Desidrogenase (Quinona)/genética , Quinona Redutases/genética , Adaptação Fisiológica , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cicloeximida/farmacologia , Transporte de Elétrons , Escherichia coli/genética , Mononucleotídeo de Flavina/metabolismo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Dados de Sequência Molecular , NAD(P)H Desidrogenase (Quinona)/metabolismo , NADPH Desidrogenase/metabolismo , Extratos Vegetais/metabolismo , Plantas Geneticamente Modificadas , Quinona Redutases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The generation and analysis of plant chimeras and other genetic mosaics have been used to deduce patterns of cell division and cell fate during plant development and to demonstrate the existence of clonally distinct cell lineages in the shoot meristems of higher plants. Cells derived from these lineages do not have fixed developmental fates but rely on positional information to determine their patterns of division and differentiation. Chimeras with cells that differ genetically for specific developmental processes have been experimentally generated by a variety of methods. This review focuses on studies of intercellular interactions during plant development as well as of the coordination of cells during meristem function and organogenesis. Recent experiments combining mosaic analysis with molecular analysis of developmental mutants have begun to shed light on the nature of the signals involved in these processes and the mechanisms by which they are transmitted and received among cells.
