Gene Family Analysis of the Arabidopsis Pollen Transcriptome Reveals Biological Implications for Cell Growth, Division Control, and Gene Expression Regulation

Abstract

Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of one-third of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/M-associated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components.

A reference map of the Arabidopsis thaliana mature pollen proteome

“…The latter either originate from experiments performed in our laboratory (cf. Materials and methods) or publicly available datasets ( [12] and unpublished datasets available at the NASC website; http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl; slides Honys_MPG1_SLD; ATGE_73A and DT001_AG_pollen, respectively). In total, transcripts of proteins unequivocally identified in 14 spots (corresponding to 10 distinct AGI loci) have been reproducibly classified as absent (based on MicroArray Suite 5.0 software) in our own transcriptome analyses (At2g07698; At2g33210; At3g18190; At3g23990; At3g59480; At4g10260; At5g03630; At5g20080; At5g20720; At5g42740, see Table 1; please note that our transcriptome studies were carried out by applying the same plant growth conditions and techniques for pollen mining as for the proteome analysis).…”

“…In total, transcripts of proteins unequivocally identified in 14 spots (corresponding to 10 distinct AGI loci) have been reproducibly classified as absent (based on MicroArray Suite 5.0 software) in our own transcriptome analyses (At2g07698; At2g33210; At3g18190; At3g23990; At3g59480; At4g10260; At5g03630; At5g20080; At5g20720; At5g42740, see Table 1; please note that our transcriptome studies were carried out by applying the same plant growth conditions and techniques for pollen mining as for the proteome analysis). Likewise, nine of the ten transcripts were declared as absent in the study of Pina et al [12], while the remaining datasets deposited on the NASC webpage provide ambiguous results for these proteins. The latter might be due to differences in plant growth and/or pollen mining as compared to our techniques.…”

“…In the model plant Arabidopsis thaliana, the extremely reduced, tricellular male gametophyte constitutes an ideal experimental system for analyses of important biological processes in higher plant reproduction. In addition, it represents a very useful model for studying fundamental aspects of plant biology such as cell fate determination, cell-cell interactions, cell polarity, and tip-growth [5][6][7].The availability of the full genome sequence of Arabidopsis [8] has made genome-wide, microarray-based analyses of the male gametophyte transcriptome of this model plant possible [9][10][11][12]. 13,977 male gametophyte-expressed mRNAs were recently identified using the ATH1 Genome Array, which covers $81% of the currently estimated 28,000 protein-coding genes in Arabidopsis.…”

“…The availability of the full genome sequence of Arabidopsis [8] has made genome-wide, microarray-based analyses of the male gametophyte transcriptome of this model plant possible [9][10][11][12]. 13,977 male gametophyte-expressed mRNAs were recently identified using the ATH1 Genome Array, which covers $81% of the currently estimated 28,000 protein-coding genes in Arabidopsis.…”

“…The majority of the respective genes appear to be expressed during the two earliest developmental stages of sporogenesis (i.e., in microspores and bicellular pollen). Indeed, 8788 transcripts were found to be expressed in tricellular pollen [11] and either 7235 [11] or 6587 [12] in mature pollen. Compared to the uninucleate microspore stage, male-gametophyte-specific genes identified from the mature pollen grain stage exhibit an increase in the collective proportion of cell wall-, cytoskeleton-, signaling-, and transport-related genes [11].…”