Abstract
BackgroundPollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. Cellular and regulatory mechanisms underlying pollen tube elongation are under intense investigation, which stands to greatly benefit from a comprehensive understanding of global gene expression profiles in pollen and pollen tubes. Here, RNA sequencing technology was applied to de novo assemble a Nicotiana tabacum male gametophytic transcriptome and to compare transcriptome profiles at two different stages of gametophyte development: mature pollen grains (MPG) and pollen tubes grown for six hours in vitro (PT6).ResultsDe novo assembly of data obtained by 454 sequencing of a normalized cDNA library representing tobacco pollen and pollen tube mRNA (pooled mRNA isolated from mature pollen grains [MPG] and from pollen tubes grown in vitro for 3 [PT3] or 6 [PT6] hours) resulted in the identification of 78,364 unigenes. Among these unigenes, which mapped to 24,933 entries in the Sol Genomics Network (SGN) N. tabacum unigene database, 24,672 were predicted to represent full length cDNAs. In addition, quantitative analyses of data obtained by Illumina sequencing of two separate non-normalized MPG and PT6 cDNA libraries showed that 8979 unigenes were differentially expressed (differentially expressed unigenes: DEGs) between these two developmental stages at a FDR q-value of <0.0001. Interestingly, whereas most of these DEGs were downregulated in PT6, the minor fraction of DEGs upregulated in PT6 was enriched for GO (gene ontology) functions in pollen tube growth or fertilization.ConclusionsA major output of our study is the development of two different high-quality databases representing the tobacco male gametophytic transcriptome and containing encompassing information about global changes in gene expression after pollen germination. Quantitative analyses of these databases 1) indicated that roughly 30% of all tobacco genes are expressed in the male gametophyte, and 2) support previous observations suggesting a global reduction of transcription after pollen germination. Interestingly, a small number of genes, many of which predicted to function in pollen tube growth or fertilization, were found to be upregulated in elongating pollen tubes despite globally reduced transcription.
Highlights
Pollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development
454 sequencing and de novo assembly To sequence the tobacco male gametophytic transcriptome, equal amounts of mRNA isolated at the mature pollen grains (MPG), pollen tubes grown for 3 (PT3) and Pollen tube grown for six hours (PT6) stages of development were pooled
A normalized cDNA library was synthesized from the pooled mRNA by Eurofins MWG GmbH and subjected to a full plate run on the 454 GS FLX platform
Summary
Pollen tube growth is essential for plant reproduction and represents a widely employed model to investigate polarized cell expansion, a process important for plant morphogenesis and development. By a process known as tip growth, pollen tubes elongate extremely rapidly at rates of several μm per minute [1, 2] This process is widely employed as an experimental model to investigate cellular and molecular mechanisms underlying polar cell expansion, which is essential for morphogenesis during plant development [3]. Cell and molecular biological as well as biochemical research has identified a large number of proteins with important functions in pollen germination and/or pollen tube growth, in many cases the precise molecular identity and/or functions of these proteins remain to be clarified To address these open questions and to generally enhance our understanding of the genetic and molecular control of different male gametophyte functions (pollen germination, pollen tube growth, fertilization), it is essential to establish highquality databases representing the transcriptome of the male gametophyte and providing information about global changes in gene expression during its development
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