Sperm cells of Zea mays have a complex complement of mRNAs

Although 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 fluorescence-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 unamplified 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.