Sperm cells of Zea mays have a complex complement of mRNAs

Abstract

SummaryAlthough double fertilization in angiosperm was discovered in 1898, we still know nothing about the proteins that mediate gamete recognition and fusion in plants. Because sperm are small and embedded within the large vegetative cell of the pollen grain, mRNAs from sperm are poorly represented in EST databases. We optimized¯uorescence-activated cell sorting (FACS) in order to isolate Zea mays sperm free of contaminating vegetative cell cytoplasm, and constructed a cDNA library. Sequencing of over 1100 cDNAs from the unampli®ed library revealed that sperm have a diverse complement of mRNAs. Most transcripts were singletons; the most abundant was sequenced only 17 times. About 8% of the sequences are predicted to encode secreted or plasma membrane-localized proteins and are therefore candidates that might mediate gamete interactions. About 8% of the sequences correspond to retroposons. Plant sperm have condensed chromatin and are thought to be transcriptionally inactive. We used RT-PCR and in situ hybridization to determine when selected sperm mRNAs were transcribed. Sperm transcripts encoding proteins involved in general cell functions were present throughout pollen development and were more abundant in tricellular pollen than in sperm cells, suggesting that these transcripts were also present in the larger vegetative cell. However, several transcripts, which encode proteins that are most similar to hypothetical Arabidopsis proteins, appeared to be present exclusively in the sperm cells inside mature pollen, but were already present in unicellular microspores. This suggests that certain transcripts might be transcribed early during pollen development and later partitioned into the sperm cells.

Arabidopsis HAP2(GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

“…We show that a sperm-specific gene, HAP2, is required for optimal ovule targeting by the pollen tube, suggesting that sperm function may impact the growth of the pollen tube. Recently, it has become clear that despite their compact chromatin structure, sperm are dynamic cells that express many genes (Engel et al, 2003;Engel et al, 2005;Xu et al, 1999); HAP2 is the first such gene with a demonstrated function in reproduction (Mori et al, 2006). It is likely that more sperm-expressed genes will be identified that play roles in pollen tube guidance and in fertilization.…”

Truncation of a Protein Disulfide Isomerase, PDIL2-1, Delays Embryo Sac Maturation and Disrupts Pollen Tube Guidance inArabidopsis thaliana

“…Analyses of EST databases obtained from egg, sperm, and embryo sac EST libraries in maize (Zea mays) (Engel et al, 2003;Yang et al, 2006) indicated that several PDI-like genes were represented in these libraries. To determine if Arabidopsis homologs of these maize PDIL proteins played any role in plant reproduction, we analyzed several Arabidopsis T-DNA insertion mutants.…”

Comparative Transcriptomics of Arabidopsis Sperm Cells

“…A cDNA library was constructed from fluorescence-activated cell sorting (FACS)-purified sperm cells of maize (Zea mays), which revealed a diverse complement of mRNAs representing at least 2,560 genes (Engel et al, 2003). These findings were significantly more than the restricted number of transcripts previously described in plant sperm cells (Xu et al, 1999a(Xu et al, , 1999bSingh et al, 2002;Okada et al, 2005).…”

“…Additional sperm-specific genes have been reported more recently (Mori et al, 2006;von Besser et al, 2006;Ingouff et al, 2007), including DUO1, a MYB transcription factor in Arabidopsis that is important for the G2/M transition during PM II (Rotman et al, 2005). Transcriptional profiling of GCs of lily (Lilium longiflorum; Okada et al, 2006Okada et al, , 2007 revealed a considerable overlap with the maize sperm ESTs (Engel et al, 2003) and the Arabidopsis pollen microarray datasets (Becker et al, 2003;Honys and Twell, 2003). Despite this previous work, the male gamete ESTs from maize, Plumbago, and lily were limited, so that testing the potential importance of sperm-expressed transcripts by means of reverse genetics could not be comprehensive, even when an Arabidopsis homolog could be identified for such ESTs.…”