Comprehensive analysis of tobacco pollen transcriptome unveils common pathways in polar cell expansion and underlying heterochronic shift during spermatogenesis

Abstract

BackgroundMany flowering plants produce bicellular pollen. The two cells of the pollen grain are destined for separate fates in the male gametophyte, which provides a unique opportunity to study genetic interactions that govern guided single-cell polar expansion of the growing pollen tube and the coordinated control of germ cell division and sperm cell fate specification. We applied the Agilent 44 K tobacco gene chip to conduct the first transcriptomic analysis of the tobacco male gametophyte. In addition, we performed a comparative study of the Arabidopsis root-hair trichoblast transcriptome to evaluate genetic factors and common pathways involved in polarized cell-tip expansion.ResultsProgression of pollen grains from freshly dehisced anthers to pollen tubes 4 h after germination is accompanied with > 5,161 (14.9%) gametophyte-specific expressed probes active in at least one of the developmental stages. In contrast, > 18,821 (54.4%) probes were preferentially expressed in the sporophyte. Our comparative approach identified a subset of 104 pollen tube-expressed genes that overlap with root-hair trichoblasts. Reverse genetic analysis of selected candidates demonstrated that Cu/Zn superoxide dismutase 1 (CSD1), a WD-40 containing protein (BP130384), and Replication factor C1 (NtRFC1) are among the central regulators of pollen-tube tip growth. Extension of our analysis beyond the second haploid mitosis enabled identification of an opposing-dynamic accumulation of core regulators of cell proliferation and cell fate determinants in accordance with the progression of the germ cell cycle.ConclusionsThe current study provides a foundation to isolate conserved regulators of cell tip expansion and those that are unique for pollen tube growth to the female gametophyte. A transcriptomic data set is presented as a benchmark for future functional studies using developing pollen as a model. Our results demonstrated previously unknown functions of certain genes in pollen-tube tip growth. In addition, we highlighted the molecular dynamics of core cell-cycle regulators in the male gametophyte and postulated the first genetic model to account for the differential timing of spermatogenesis among angiosperms and its coordination with female gametogenesis.

Dynamic Adaption of Metabolic Pathways during Germination and Growth of Lily Pollen Tubes after Inhibition of the Electron Transport Chain

“…So far, the majority of transcriptome studies did not consider time-dependent aspects during germination and tube growth but were focused on the differences between pollen and sporophytic tissue or emphasized specific biological processes in pollen physiology and development or described differences between pollen grains and tubes of various species (Becker et al, 2003;Honys and Twell, 2003;Bock et al, 2006;Haerizadeh et al, 2009;Qin et al, 2009;Hafidh et al, 2012). A metabolic network was presented for Arabidopsis pollen using the transcriptome data of Pina et al (2005) and the MAPMAN software (Thimm et al, 2004).…”

De novo post-pollen mitosis II tobacco pollen tube transcriptome

“…In our original work, 1 we applied the Agilent 44K tobacco gene chip to conduct the first thorough transcriptomic analysis of the tobacco male gametophyte development and the dynamics of gene expression in the first four hours of pollen germination and tube growth. We presented four independent data sets-two male gametophytic mature pollen (MPG) and 4 h pollen tubes (PT4) and two sporophytic leaves (L) and roots (R) as a reference.…”

Phosphoproteomics Profiling of Tobacco Mature Pollen and Pollen Activated in vitro

“…Such a fact was already demonstrated for Arabidopsis mitochondrial phosphoproteome where phosphopeptide enrichment led to the identification of novel phosphorylation sites that were not previously identified by the alternative approaches (38). As mentioned above, active transcription in activated pollen grain as long as 24 h of pollen tube growth has been shown (54,55). Here, we identified several transcription factors, most of which contained a zinc finger motif.…”

“…Tobacco pollen activation and subsequent pollen tube growth was originally shown to be vitally dependent on translation but almost independent of transcription (53). Although our recent microarray transcriptomic analyses revealed a number of mRNAs being synthesized during pollen tube growth even after 24 h of cultivation (54,55), many of the transcripts in the desiccated mature pollen are stored in EDTA/puromycine-resistant particles (EPPs). These particles contain parts of ribosomes and translation apparatus together with mRNAs (56,57) and the translation of EPP-stored mRNAs starts after pollen activation.…”

“…MAPKs play a key regulatory role in many physiological processes including stress reactions and pollen hydration (62). CDKs were originally shown to regulate cell cycle and their activity in male gametophyte was expected because both pollen mitoses are precisely regulated (54). The alternative function of CDKs is for example the regulation of pre-mRNA splicing of callose synthase in pollen tube that influences cell wall formation (66).…”