Year of Publication

2013

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture

Department

Plant and Soil Science

First Advisor

Dr. Arthur G. Hunt

Second Advisor

Dr. A. Bruce Downie

Abstract

Seed germination has many impacts on the uses of seeds, and is an important subject for study. Seed germination is regulated at both transcriptional and post-transcriptional levels. Therefore, it is important to study how polyadenylation regulates gene expression during seed germination. To this end, a modified Illumina GAIIx sequencing protocol (described in Chapter Two) was developed that allows deep coverage of poly(A) site position and distribution.

Alternative polyadenylation (APA) regulates gene expression by choosing one potential poly(A) site on a precursor RNA consequentially shortening/lengthening the mRNA relative to other possible sites. To further explore this phenomenon, genes affected by APA during seed germination and other developmental stages were identified (Chapter Three). These genes were categorized based on the location of poly(A) sites. Several genes were chosen to demonstrate how APA, especially that occurring in the coding regions and 5’ untranslated regions, might down regulate gene expression by generating truncated transcripts.

In animal oocytes, maternally-derived mRNAs are stored with short poly(A) tails and reactivated by the cytoplasmic polyadenylation complex. It has been reported that seeds also contain stored mRNAs. Moreover, germination and its completion are less sensitive to de novo transcription inhibitors than to poly(A) polymerase inhibitors. Together, these considerations suggest that stored RNA without or with a short poly(A) tail (stored, unadenylated RNA) may be present in dry seed and function in seed germination upon reactivation by cytoplasmic polyadenylation. To further explore this, in Chapter Four, mRNA polyadenylation was studied through the course of germination using a combination of transcriptional inhibitors and the modified sequencing protocol described in Chapter Two. 273 putative stored, unadenylated RNAs were identified. Gene ontology analysis revealed that genes whose products are involved in translation are overrepresented; these genes encode 21 60S- and 10 40S-ribosomal proteins. These results indicate that transcripts whose products are involved in translation might be a major component of the stored, unadenylated RNA pool and, more importantly, translation might be the first cellular process to be activated during seed germination.

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