Summary Genomic assay of sperm cell RNA provides insight into functional control, modes of regulation, and contributions of male gametes to double fertilization. Sperm cells of rice (Oryza sativa) were isolated from field‐grown, disease‐free plants and RNA was processed for use with the full‐genome Affymetrix microarray. Comparison with Gene Expression Omnibus (GEO) reference arrays confirmed expressionally distinct gene profiles. A total of 10 732 distinct gene sequences were detected in sperm cells, of which 1668 were not expressed in pollen or seedlings. Pathways enriched in male germ cells included ubiquitin‐mediated pathways, pathways involved in chromatin modeling including histones, histone modification and nonhistone epigenetic modification, and pathways related to RNAi and gene silencing. Genome‐wide expression patterns in angiosperm sperm cells indicate common and divergent themes in the male germline that appear to be largely self‐regulating through highly up‐regulated chromatin modification pathways. A core of highly conserved genes appear common to all sperm cells, but evidence is still emerging that another class of genes have diverged in expression between monocots and dicots since their divergence. Sperm cell transcripts present at fusion may be transmitted through plasmogamy during double fertilization to effect immediate post‐fertilization expression of early embryo and (or) endosperm development.

BROTHER OF LUX ARRHYTHMO (BOA) is a GARP family transcription factor in Arabidopsis thaliana and is regulated by circadian rhythms. Transgenic lines that constitutively overexpress BOA exhibit physiological and developmental changes, including delayed flowering time and increased vegetative growth under standard growing conditions. Arabidopsis circadian clock protein CIRCADIAN CLOCK ASSOCIATED1 (CCA1) binds to the evening element of the BOA promoter and negatively regulates its expression. Furthermore, the period of BOA rhythm was shortened in cca1-11, lhy-21 (for LATE ELONGATED HYPOCOTYL), and cca1-11 lhy-21 genetic backgrounds. BOA binds to the promoter of CCA1 through newly identified promoter binding sites and activates the transcription of CCA1 in vivo and in vitro. In transgenic Arabidopsis lines that overexpress BOA, the period length of CCA1 rhythm was increased and the amplitude was enhanced. Rhythmic expression of other clock genes, including LHY, GIGANTEA (GI), and TIMING OF CAB EXPRESSION1 (TOC1), was altered in transgenic lines that overexpress BOA. Rhythmic expression of BOA was also affected in mutant lines of toc1-1, gi-3, and gi-4. Results from these studies indicate that BOA is a critical component of the regulatory circuit of the circadian clock.

SUMMARYPlumbago zeylanica produces cytoplasmically dimorphic sperm cells that target the egg and central cell during fertilization. In mature pollen, the larger sperm cell contains numerous mitochondria, is associated with the vegetative nucleus (S vn ), and fuses preferentially with the central cell, forming endosperm. The other, plastidenriched sperm cell (S ua ) fuses with the egg cell, forming the zygote and embryo. Sperm expressed genes were investigated using ESTs produced from each sperm type; differential expression was validated through suppression subtractive hybridization, custom microarrays, real-time RT-PCR and in situ hybridization. The expression profiles of dimorphic sperm cells reflect a diverse and broad complement of genes, including high proportions of conserved and unknown genes, as well as distinct patterns of expression. A number of genes were highly up-regulated in the male germ line, including some genes that were differentially expressed in either the S ua or the S vn . Differentially up-regulated genes in the egg-targeted S ua showed increased expression in transcription and translation categories, whereas the central cell-targeted S vn displayed expanded expression in the hormone biosynthesis category. Interestingly, the up-regulated genes expressed in the sperm cells appeared to reflect the expected post-fusion profiles of the future embryo and endosperm. As sperm cytoplasm is known to be transmitted during fertilization in this plant, sperm-contributed mRNAs are probably transported during fertilization, which could influence early embryo and endosperm development.

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Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25+/-2 degrees C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.

Flowering plant reproduction is characterized by double fertilization, in which two diminutive brother sperm cells initiate embryo and endosperm. The role of the male gamete, although studied structurally for over a century at various levels, is still being explored on a molecular and cellular level. The potential of the male to influence development has been historically underestimated and the reasons for this are obvious: limitations provided by maternal imprinting, the much greater cellular volume of female gametes and the general paucity of paternal effects. However, as more is known about molecular expression of chromatin-modifying proteins, ubiquitin pathway proteins and transcription factors in sperm cells, as well as their ability to achieve effect by intaglio expression, passing transcripts directly into translation, the role of the male is likely to expand. Much of the expression in the male germline that appears to be distinct from patterns of pollen vegetative cell expression may be the result of chromosomal level regulation of transcription.

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