Characterization of the Transition to the Progamic Phase in Zea mays Pollen

approved: ______________________________________________________ John E. Fowler The transition of mature pollen to the progamic phase represents an important step in the regulation of plant sexual reproduction. The progamic phase encompasses the initiation of pollen tube germination, growth of the pollen tube through the stigma (the silk in maize) and concludes with fertilization of the female gametophyte. This action involves interactions between the male gametophyte and female sporophyte and represents possible sites for regulation of reproduction. Due to its location within the sporophytic tissue, analysis of gene expression of the actively growing pollen tube is difficult to conduct in vivo. Two broad, complementary experimental approaches were used to identify genes important in the progamic phase of maize pollen development and to investigate how their products are regulated. The work presented here represents an initial characterization of the transition to the progamic phase of Zea mays (maize) pollen through the use of proteomic and transcriptomic analyses. The first approach was to analyze and characterize changes to the protein profile of maize pollen before and after the initiation of pollen tube germination. Unlike in many plants, germination of the pollen tube in maize occurs rapidly, with no significant changes detected in the transcriptome. This supports a hypothesis where the modulation of protein levels and/or post-transcriptional modification of protein activity, rather than transcription, play critical roles in the developmental transition to the progamic phase in maize. Tests of germination in the presence of drugs that inhibit protein synthesis or degradation indicate that de novo translation and ubiquitin-mediated protein degradation are crucial to successful germination in maize. Consistent with these findings, SDS-PAGE gel silver staining of total protein extracts found evidence of particular proteins increasing and decreasing in abundance at 30 minutes post-germination. Quantitative profiling of the proteome and phosphoproteome identified 393 differentially abundant proteins between mature and germinated pollen. In addition, 103 proteins are associated with a significant change in phosphorylation state largely independent of changes to the total proteome upon germination. Functional analysis by gene ontology of differentially abundant proteins revealed enrichment for several terms associated with translation in the total proteome, as well as significantly increased phosphorylation in several kinases and components of protein degradation machinery. Genes functioning in the jasmonic acid biosynthesis pathway were found to be enriched among those proteins decreasing in abundance with germination suggesting that this pathway is down regulated with the transition to the progamic phase. Male transmission defect analysis (MTDA) was used as a genetic test for biological significance of the results from the quantitative proteome profiling by testing for reduced inheritance of insertions in genes predicted to be important for pollen function. Ten insertions located in exons and with robust PCR genotyping reactions were tested for transmission of the insertions through the male and female. Of these ten, five were found to exhibit reduced transmission though the male supporting the use of proteomic data to identify biologically relevant components affecting pollen function. The second approach presents a pilot study utilizing single nucleotide polymorphisms (SNPs) to distinguish RNA derived from the progamic male gametophyte from that of the female sporophyte during growth of the pollen tube through the silk. This approach takes advantage of the high level of genetic variation found in maize. By using different inbred lines for the pollen (B73) and silk (W22), SNPs known to exist between the two lines were able to identify pollen-derived alleles from those of the silk by RNA-seq. An initial analysis focused on chromosome 8 found 222 genes with alignment of reads to the B73 allele; these putatively represent gene expression of the male gametophyte. Supporting this conclusion is the presence of 23 genes previously found to be associated with enrichment of expression in mature pollen. CAPS (Cleaved Amplified Polymorphic Sequence) can validate the presence of inbred specific transcripts by allele specific endonuclease restriction digest of RT-PCR products. An initial CAPS analysis of the transcripts from one gene was able to detect reads containing the B73 allele in the pollinated W22 silk sample, though the results from the unpollinated W22 silks were inconclusive. ©Copyright by Johanna C. Smyth August 18, 2016 All Rights Reserved Characterization of the Transition to the Progamic Phase in Zea mays Pollen