Date of Graduation

2007

Document Type

Dissertation/Thesis

Abstract

The plant hormone ethylene plays an important role in numerous plant growth and developmental processes, including flower development and senescence. Once perceived by ethylene receptors, the ethylene signal is transduced through a series of components until it reaches its ultimate targets (e.g. senescence-related (SR) genes). EIN3, a transcription factor and positive regulator of the ethylene signaling pathway, most likely affects these SR genes. Using a combination of approaches, three EIN3-like (EIL) cDNAs, DC-EIL1/2 (AY728191), DC-EIL3 (AY728192) and DC-EIL4 (AY728193), were isolated from carnation (Dianthus caryophyllus L.) petals. The cloned cDNAs share a high amino acid identity among each other and with previously cloned EILs. DC-EILs transcript analysis performed on vegetative and flower tissues (petals, ovaries and styles) during growth, development, and senescence (natural and ethylene-induced) indicated that the mRNA accumulation of the DC-EIL family of genes in carnation is regulated developmentally and by ethylene. Especially DC-EIL3 mRNA showed considerable accumulation upon ethylene exposure, during flower development, and upon pollination in petals and styles. Interestingly, decreasing levels of DC-EIL3 mRNA were found in wounded leaves and ovaries of senescing flowers whenever ethylene levels increased. Flowers treated with sucrose showed a two-day delay in the accumulation of DC-EIL3 transcripts when compared to control flowers. These observations suggest an important role for DC-EIL3 during growth and development. Changes in DC-EIL1/2 and DC-EIL4 mRNA levels during flower development, and upon ethylene exposure and pollination were very similar, but less dramatic than changes in DC-EIL3 transcript levels. mRNA levels of the DC-EILs in styles of pollinated flowers showed a clear relationship with ethylene production after pollination. The characterization of the EIN3-like genes in carnation indicated in the present study showed transcriptional regulation not previously observed for EILs in other plant species. CEBP, a nuclear encoded chloroplast protein and putative repressor of ethylene signaling, most likely regulates the transcription of SR genes containing an Ethylene Response Element (ERE) by preventing transcription until certain developmental conditions are met. The characterization of changes in CEBP mRNA levels in flowers (petals, ovaries and styles) during flower development and senescence (natural and ethylene induced), as well as in leaves following wounding, indicated that CEBP is down-regulated developmentally and by ethylene. Interestingly, during flower development and senescence (both natural and ethylene-induced), DC-EIL3 transcript started to accumulate at the same point CEBP transcript levels decreased. CEBP transcript levels decreased dramatically after anthesis, as opposed to the gradual decrease throughout development of the petal chlorophyll content and chloroplast number. Furthermore, transient transformation of carnation petals by particle bombardment with GFP-tagged CEBP indicates that CEBP can be localized to both chloroplasts and nuclei. Based on the above-mentioned results, as well as previous findings, a novel mechanism that regulate ethylene signaling regulation was proposed. In this model, early in petal development, when chloroplasts are numerous, chloroplast-located CEBP plays a role in processing and/or stabilizing chloroplast RNA, whereas nucleus-located CEBP acts as a repressor of SR genes. When developmental changes initiate chloroplast degradation as part of developmental processes associated with aging, CEBP levels decrease, possibly through a negative feedback loop. Lower levels of CEBP allow the promoters of the SR genes to become available for activation by DC-EIL3, initiating the first steps of the petal senescence process.

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