Organ detachment occurs within cell tiers termed abscission zones (AZs). Characterizations of mutants with aberrant abscission behavior showed abscission to involve multiple signaling pathways. How such pathways are regulated is largely unknown. We examined Arabidopsis abscission zone transcriptome changes associated with floral organ detachment to identify novel shedding regulators. For genomic studies, we used laser capture microdissection (LCM) to dissect AZ cells from surrounding tissues. Because the extremely limited number, small size and close proximity of AZs limited immediate use of LCM, we developed new LCM protocols necessary for genomic studies.
Initial work showed that tape-transfer of paraffin sections to adhesive coated slides prior to LCM was superior to conventional slide mounting strategies. Electrophoresis revealed high structural integrity of RNA isolated from multiple types of tape-transferred, laser-captured cells. Optimization of transcriptional profiling methods using RNA from relatively abundant replum cells permitted linking of tape-transfer methods to LCM and GeneChip profiling in a way that allowed detection of rare gene transcripts. Replum transcriptome profiling identified a cellular shift from primary cell wall metabolism to lignin biosynthesis as siliques matured.
Techniques optimized for replums were used to profile laser-captured stamen AZs. Transcriptome changes were monitored at five floral stages from pre-pollination to organ shed. The 554 genes regulated at the highest statistical significance (p-value < = 0.0001) included known abscission regulators related to ethylene and auxin signaling as well as a receptor-like kinase and extracellular ligand thought to act independent of ethylene. Functional categorization of genes showed that cell wall modifying proteins, transcription factors and extracellular regulators were disproportionately represented, and we hypothesized that such genes might encode novel abscission regulators. Functional analyses of one such gene, AtZFP2, were conducted. AtZFP2 encodes a zinc finger protein that is up-regulated in stamen AZs post-anthesis. Transgenic lines overexpressing AtZFP2 show asynchronous and delayed abscission as well as aberrant floral organ development and sterility. These data suggest that AtZFP2 might regulate abscission and other developmental processes. Future functional analyses may identify genes expressed solely or predominantly in AZs and suggest strategies to manipulate undesirable abscission behavior of agriculturally important plants.
|Advisor:||Lashbrook, Coralie C.|
|School:||Iowa State University|
|School Location:||United States -- Iowa|
|Source:||DAI-B 68/12, Dissertation Abstracts International|
|Subjects:||Molecular biology, Genetics|
|Keywords:||Abscission, Arabidopsis, Laser capture microdissection, Replum, Stamen, Zinc finger protein|
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