Pollen World
Pollen Biology Lab, Czech Academy of Sciences

Pollen Transcription Group


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Regulation of Arabidopsis pollen development


Arabidopsis pollen developmental transcriptomics


Blizna
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 and its dynamics throughout pollen development (Honys and Twell 2003, 2004). 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 considered 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. 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 provided 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.


Screen and functional analyses of male gametophytic transcription factors


Male gametophyte development leading to the formation of a mature pollen grain is precisely controlled at various levels, including transcriptional, post-transcriptional and post-translational, during its whole progression.
Here we focused on the identification of
pollen-expressed transcription factors (TF) involved in the regulation of male gametophyte development by thorough screening of 74 T-DNA insertion lines representing 49 genes of 21 TF families active in either early or late pollen development. 29 screened lines showed strong phenotypic changes (i.e., ≥ 25% aberrant pollen) including four lines that produced a remarkably high proportion (70-100%) of disturbed pollen. Our results served as a basal information resource for future functional characterization of specific TFs in male gametophyte development This phenotype screen was partly enabled by the optimization of our previously published protocol for large-scale separation of developing spores.

bZIP pyl2
bZIP TFs having critical roles in plants, animals and other kingdoms were selected for more detailed analyses. We reported the functional characterization of Arabidopsis thaliana AtbZIP34 that is expressed in both gametophytic and surrounding sporophytic tissues during flower development. T-DNA insertion mutants in AtbZIP34 show pollen morphological defects that result in reduced pollen germination efficiency and slower pollen tube growth both in vitro and in vivo. Light and fluorescence microscopy revealed misshapen and misplaced nuclei with large lipid inclusions in the cytoplasm of atbzip34 pollen. Scanning and transmission electron microscopy revealed defects in exine shape and micropatterning and a reduced endomembrane system. Several lines of evidence, including the AtbZIP34 expression pattern and the phenotypic defects observed, suggest a complex role in male reproductive development that involves a sporophytic role in exine patterning, and a sporophytic and/or gametophytic mode of action of AtbZIP34 in several metabolic pathways, namely regulation of lipid metabolism and/or cellular transport. We further functionally analysed the regulatory network of bZIP transcription factors in long-term collaboration with D. Twell, University of Leicester, UK. That extended our previous study of AtbZIP34 by the inclusion of its interactors AtbZIP18, AtbZIP52.

Another selected TF was early male gametophytic gene
AtREN1, a close homolog of HSFA5 gene, a member of the heat shock transcription factor (HSF) gene family., their role in male gametophyte development is largely unknown. The atren1 mutation causes multiple defects in male gametophyte development in both structure and function including defective pollen heat stress response, pollen phenotype abnormalities and pollen germination defects associated with the limited transmission via male gametophyte. We localized the AtREN1 protein specifically to the nucleolus that suggests its likely involvement in ribosomal RNA biogenesis therefore linking heat stress response with translation.


Role of auxin in pollen development


Auxin is a key coordinative signal required for many aspects of plant development and its levels are controlled by auxin metabolism and intercellular auxin transport. Within the multilateral network lead by J. Friml (IST Austria), we found that the non-canonical member of PIN auxin transporter family,
PIN8 was active in Arabidopsis pollen and played a crucial role in pollen development and function by regulating auxin homoeostasis and metabolism. Our results revealed a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters maintained the auxin levels optimal for pollen development and pollen tube growth. Our results of double mutant functional tests were also the first indication of the possible antagonistic role of PIN8 and PIN5 on the ER.


arabidopsisGFP database and toolbox


aGFP
Microarray technologies now belong to the standard functional genomics toolbox and have undergone massive development leading to increased genome coverage, accuracy and reliability. The number of experiments exploiting microarray technology has markedly increased in recent years. In parallel with the rapid accumulation of transcriptomic data, on-line analysis tools are being introduced to simplify their use. We presented a curated gene family-oriented gene expression database, Arabidopsis Gene Family Profiler (aGFP), which gives the user access to a large collection of normalised Affymetrix ATH1 microarray datasets. The database currently contains NASC Array and AtGenExpress transcriptomic datasets for various tissues at different developmental stages of wild type plants. The Arabidopsis GFP database has been designed as an easy-to-use tool for users needing an easily accessible resource for expression data of single genes, pre-defined gene families or custom gene sets, with the further possibility of keyword search. Arabidopsis Gene Family Profiler presents a user-friendly web interface using both graphic and text output. Data are stored at the MySQL server and individual queries are created in PHP script. Arabidopsis GFP gives users the possibility to analyze current Arabidopsis developmental transcriptomic data starting with simple global queries that can be expanded and further refined to visualize comparative and highly selective gene expression profiles.




Tobacco pollen as a bicellular model for –omic studies


The majority of 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.


Tobacco pollen developmental transcriptomics


We applied the Agilent 44K tobacco gene chip to conduct the first comprehensive developmental transcriptomic analysis of the tobacco male gametophyte representing the first plant species shedding bicellular pollen annualized this way. These transcriptomic datasets presented a benchmark for future functional studies using developing pollen as a model. In addition, we performed a comparative study of the Arabidopsis root hair trichoblast transcriptome to evaluate genetic factors and common genes and regulatory pathways involved in polarized cell-tip expansion. 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) were 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. 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. We further showed the stable and even slightly increasing complexity of tobacco male gametophyte transcriptome over long period of progamic phase - 24 h of pollen tube growth. We demonstrated the ongoing transcription activity and specific transcript accumulation in post-pollen mitosis II pollen tubes cultivated in vitro. In all, we have identified 320 genes that were newly transcribed as late as at least after 4h of pollen tube cultivation in vitro. This represented the first evidence for such late transcriptional activity in pollen tubes. As pollen tube growth and competition of pollen tubes in female pistil can be viewed as a race of the fittest, there is an apparent evolutionary trend among higher plants to store large material reserves and nutrients during pollen maturation. This supply ensures that after pollen germination, the pollen tube utilizes its resource predominantly for its rapid elongation in the female pistil. Previous transcriptomic data from Arabidopsis showed massive expression of genes encoding proteins forming both ribosomal subunits that were accumulated in developing pollen, whereas their expression was not detectable in growing pollen tubes. We observed a similar phenomenon in less advanced bicellular tobacco pollen.