Transcriptome analysis of haploid male gametophyte development in Arabidopsis

Abstract

Background: The haploid male gametophyte generation of flowering plants consists of two- or three-celled pollen grains. This functional specialization is thought to be a key factor in the evolutionary success of flowering plants. Moreover, pollen ontogeny is also an attractive model in which to dissect cellular networks that control cell growth, asymmetric cell division and cellular differentiation. Our objective, and an essential step towards the detailed understanding of these processes, was to comprehensively define the male haploid transcriptome throughout development.

Results: We have developed staged spore isolation procedures for Arabidopsis and used Affymetrix ATH1 genome arrays to identify a total of 13,977 male gametophyte-expressed mRNAs, 9.7% of which were male-gametophyte-specific. The transition from bicellular to tricellular pollen was accompanied by a decline in the number of diverse mRNA species and an increase in the proportion of male gametophyte-specific transcripts. Expression profiles of regulatory proteins and distinct clusters of coexpressed genes were identified that could correspond to components of gametophytic regulatory networks. Moreover, integration of transcriptome and experimental data revealed the early synthesis of translation factors and their requirement to support pollen tube growth.

Conclusions: The progression from proliferating microspores to terminally differentiated pollen is characterized by large-scale repression of early program genes and the activation of a unique late gene-expression program in maturing pollen. These data provide a quantum increase in knowledge concerning gametophytic transcription and lay the foundations for new genomic-led studies of the regulatory networks and cellular functions that operate to specify male gametophyte development.

Figure 1

Spore isolation and numbers of genes expressed throughout Arabidopsis male gametophyte development. (a) Purity of isolated spores in each developmental stage determined by microscopy: UNM, microspores; BCP, bicellular pollen; TCP, tricellular pollen; MPG, mature pollen. (b-e) DAPI-stained populations of developing spores: (b) microspores; (c) bicellular pollen; (d) tricellular pollen; and (e) mature pollen. (f) Total number of genes expressed in developing pollen and their distribution among three relative abundance classes. Gene-abundance classes were defined as follows: high (up to 10-fold less than the maximum signal), medium (10- to 100-fold less) and low (more than 100-fold less).

Figure 2

Scatter-plots comparing relative gene expression in pairs of developmental stages. The expression levels of individual genes were normalized using a logarithmic scale of 0 to 100 and genes coexpressed in pairs of transcriptome datasets were plotted. (a) UNM versus BCP stage; (b) BCP versus TCP stage; (c) TCP versus MPG stage; (d) UNM versus MPG stage. R-value represents the correlation coefficient.

Figure 3

A selection of clusters of genes coexpressed during male gametophyte development. A complete set of all 39 clusters determined using EPCLUST software with a threshold value of 0.05 is available as Additional data files 1 and 2. n, number of genes comprising a cluster.

Figure 4

Expression profiles of regulatory genes throughout male gametophyte development. (a) Core cell-cycle genes; (b) transcription factors; (c-f) selected transcription factor families. (c) R2R3-MYB; (d) WRKY; (e) NAC; (f) C3H. Expression profiles of all transcription factor families analyzed are available as Additional data files 1 and 3. (g) Core translation factors. (h) Poly(A)-binding (PAB) protein genes. Putative male gametophyte-specific (bold) or enhanced (bold dashed) genes are highlighted.

Figure 5

Pollen germination and pollen tube growth in vitro in the presence of inhibitors of (a) transcription and (b) translation. The percentage of germinated pollen and the percentage of pollen capable of extended tube growth were scored independently for each treatment. PT, pollen tube.