Polarized extension of the pollen tube to deliver twin sperm cells to the female gametophyte is a fundamental process required for successful sexual reproduction and preservation of the floristic dominance of flowering plants. Understanding sexual reproduction is at the forefront of plant research and has benefited immensely from the development of genomic, transcriptomic and proteomics techniques in recent decades. Advances in microarray technology and development of gene chips for Arabidopsis, rice and other species have allowed studies on large-scale transcriptional profiling from diverse tissues, which has emerged as a key tool for identification of novel targets for functional genomics.
As in other angiosperms, the tobacco (Nicotiana tabacum
) male gametophyte is formed within the anthers following meiotic division of the archesporial sporophytic cells to produce haploid microspores [1
]. Thereafter, only two mitotic divisions occur. The first pollen mitosis (PMI) involves division of the haploid microspore to produce a gametic germ cell and a vegetative cell. These two cell types differ not only in the quantity and diversity of expressed transcripts [2
], but also, crucially, in their fate. Whereas after pollen tube germination the germ cell undergoes a second mitotic division (PMII) to produce two male gametes (sperm cells), the vegetative cell adopts a different fate and acquires the critical task of ensuring growth of the pollen tube. At the stigma surface, the pollen tube germinates and penetrates the female stylar tissues to deliver the two sperm cells to the proximity of the egg and central cell for double fertilization. Among flowering plants, the production of sperm cells after pollen germination represents the ancestral pattern. However, over 30% of angiosperms, including Arabidopsis thaliana
, complete the second division before pollen maturation [4
]. No explanation has been proposed for the mechanisms that have imposed this regulon prompting differential timing in sperm cell proliferation in the male gametophyte of seed plants, nor have the evolutionary benefits of early gamete production been elucidated. Independent analysis of sperm cell DNA content revealed five patterns of sperm-cell development that differ with respect to relative timing of sperm-cell formation and maturation prior to fusion with the female gametes [5
Pollen-tube germination represents a unique cellular phenomenon. The pollen tube grows through the female tissues in a polarized fashion similar to root-hair outgrowth, trichome specification, hyphal growth in fungi and extension of neuronal dendrites in the animal nervous system [7
]. Thus, common factors are likely to be involved in the raw molecular cascades that initiate and control the single cell-tip expansion and morphogenesis, which are subsequently modulated by cell- or tissue-specific morphological demands. Studies using pollen tubes and root hairs as model systems have revealed a number of signaling molecules and regulatory pathways that promote and maintain the tip-growth characteristic. Recently, a study by Qin et al. [9
] revealed a specific subset of genes that are induced in pollen tubes during growth through the pistil, but not when grown in vitro. Among this set of genes are potential receptor proteins that may respond to female cues for guidance of the pollen-tube tip growth. Collectively, these experiments demonstrated the direct dependence of cell-tip expansion on polarized exocytosis of vesicles at the apical growth region of the cell. Vesicular trafficking to the tip region is a structural requirement for tip growth and plays a significant role in feedback regulation of the tip-localized signaling to maintain the apical cell expansion [10
Until recently, no major studies had analyzed the bicellular pollen transcriptome, with the sole exception of soybean (Glycine max
]. A first glimpse into the tobacco pollen transcriptome was reported by Xin and co-workers [12
], who constructed an EST library from isolated tobacco sperm cells. This project aimed to identify proteins involved in sperm-egg recognition and fusion and identified over 1,864 EST sequences distributed in more than 1,050 clusters. This analysis discovered paternal candidate genes that could play a role during fertilization and in the early stages of embryogenesis. However, the sperm cell transcriptome alone represents only a small proportion of the total pollen-expressed transcripts and, although EST sequencing is sensitive, it provides only a limited list of expressed genes and thus minimizes comprehensive functional downstream analyses [3
In the present study, we capitalized on the recent development of the Agilent 44 K tobacco microarray to present the first insight into tobacco pollen and pollen-tube transcriptomes, which represents a contrary experimental model from the tricellular pollen of Arabidopsis to a bicellular model of scientific, medical and commercial interest. In addition to broad-spectrum gene expression profiling, we performed a comparative analysis of the transcriptomes of tobacco pollen tubes and Arabidopsis root-hair trichoblasts [15
] to uncover common pathways involved in polar cell expansion. To investigate their potential role in tobacco pollen-tube growth, we used gene ontology (GO) tools to allocate their biological roles, and applied an antisense-transfection approach to verify the function of selected candidate genes.
We further explored our data set to elucidate the control of male germ-cell proliferation and sperm-cell formation from a molecular perspective. In angiosperms, heterochronic alterations in cell cycle activity led to the developmental shift in the timing of germ-cell division and resulted in diversified patterns of spermatogenesis and gamete fusion during fertilization [4
]. To elucidate the genetic network responsible for this shift among angiosperms, we investigated the expression of core cell-cycle repressors and activators at five stages of pollen development--namely, mature pollen and in vitro-germinated pollen tubes grown for 4, 13, 24 and 48 h - following completion of the germ cell mitotic division. On the basis of these results, we postulated a genetic model in which the male germ cell-cycle progression is coordinated with 'time to fertilization'.
The tobacco pollen and pollen-tube gene expression profiles generated in this study together with our analyses provide an ideal platform for future research into aspects of polarized cell expansion and control of spermatogenesis. In addition, cataloguing the expressed genes will provide an opportunity for development of gametophytic fluorescent markers with which cell behaviour and cell fate can be studied. The present study represents a step towards a comprehensive understanding of male gametogenesis, knowledge of which will provide an effective framework to improve current farming and breeding programs to sustain food requirements and for investigation of tobacco health implications.