A study of programmed cell death during xylem differentiation in Zinnia elegans L.
Abstract
Programmed cell death (PCD) is a genetically active and selective process of cell elimination. During normal development of multicellular organisms, execution and regulation of PCD are used as a fine control mechanism for balancing cell replication and differentiation. Over the past several years, research on PCD has exploded in relation to the hypothesis that abnormally controlled PCD may be an underlying cause of some diseases such as Alzheimer's, AIDS, and cancer. Although little is known about programmed death or survival in plant cells, PCD appears to be an essential component of plant development. In this study, an in vitro system for tracheary element (TE) differentiation from mesophyll cells isolated from Zinnia elegans L. was used to establish the occurrence of the programmed death of these cells. Stereotypical characteristics associated with apoptotic cell death were observed during the differentiation process. Nuclear fragmentation into nucleosomal pieces was detected by gel electrophoresis as a nucleosomal ladder. The presence of nucleolytically cleaved DNA was confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end-labeling (TUNEL) staining. Transient expression of the C. elegans PCD gene, ced-3, along with the reporter gene, gusA in maize protoplasts resulted in decreased levels of GUS as compared to controls. This suggested that ced-3 functions in maize protoplasts. Differential gene expression in differentiating TE was observed by reverse transcription polymerase chain reaction (RT-PCR) procedures, and several unique DNAs were characterized. Two of these cDNAs were cloned and characterized via putative amino acid sequence and RNA gel blot analyses. Deduced amino acid sequence analysis revealed that a cDNA Z05 encodes a 217 amino acid protein highly homologous to ubiquitin-ribosomal proteins from a variety of species. The deduced amino acid sequence analysis of cDNA J41 exhibited a 300 amino acid peptide sequence sharing strong identity to a protein member of the ubiquinol-cytochrome C reductase complex (Wilms' tumor suppressor gene). In summary, the machinery for apoptotic PCD appears to be present during the differentiation of TEs. Furthermore, utilization of RT-PCR technologies allowed the identification of cDNAs in Zinnia elegans participating in this important developmental process.
Degree
Ph.D.
Advisors
Hodges, Purdue University.
Subject Area
Molecular biology|Botany
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