The broad scientific aim of our research group is to study regulatory mechanisms underlying the flawless progress of male gametophyte development and function. We are interested in both regulatory levels: transcription and translation. Our model systems are pollen and pollen tubes of Arabidopsis thaliana and Nicotiana tabacum.
Research topics
1) Regulation of transcription. Our broad scientific aim is to extend the field traditionally studied in the IEB AS CR, the regulation of male gametophyte development, by opening the area of global regulation of gametophytic developmental program. The particular focus is on transcription factors and their regulatory networks that control haploid male gametophyte development in particular phases. Moreover, we are interested in identification of regulatory cis-elements in promoters of male gametophyte-expressed genes controlling their specific expression pattern.

2) Regulation of translation. This traditional field denotes the investigation of mechanisms of translational repression of stored pollen-specific transcripts in developing pollen and their controlled activation after pollen germination in tobacco. Stored mRNAs were shown to be sequestered in a form of so-called EPP particles (Honys et al. 2009). Surprisingly, these particles contain whole translational machinery and are involved also in mRNA activation and protein maturation and localisation. We are now studying the dynamic structure and precise function of EPP particles throughout various phases of pollen development and pollen tube growth.

3) Pollen proteomics. We are also interested in tobacco pollen proteomics and its dynamics at the level of proteome and its subfractions (phosphoproteome, cell wall proteome, membrane proteome) in developing pollen and growing pollen tubes.
Key results
Discovery and characterisation of EPP particles
The progamic phase of male gametophyte development involves activation of synthetic and catabolic processes required for the rapid growth of the pollen tube. It is well-established that both transcription and translation play an important role in global and specific gene expression patterns during pollen maturation. On the contrary, germination of many pollen species has been shown to be largely independent of transcription but vitally dependent on translation of stored mRNAs. We published the first structural and proteomic data about large ribonucleoprotein particles (EPPs) in tobacco male gametophyte (Honys et al. 2009). These complexes are formed in immature pollen where they contain translationally silent mRNAs. Although massively activated at the early progamic phase, they also serve as a long-term storage of mRNA transported along with the translational machinery to the tip region. Moreover, EPPs were shown to contain ribosomal subunits, rRNAs and a set of mRNAs. Presented results extend our view of EPP complexes from mere RNA storage and transport compartment in particular stages of pollen development to the complex and well-organized machinery devoted to mRNA storage, transport and subsequent controlled activation resulting in protein synthesis, processing and precise localization. Such an organization is extremely useful in fast tip-growing pollen tube. There, massive and orchestrated protein synthesis, processing, and transport must take place in accurately localized regions. Moreover, presented complex role of EPPs in tobacco cytoplasmic mRNA and protein metabolism makes them likely to be active in another plant species too. Expression of vast majority of the closest orthologues of EPP proteins also in Arabidopsis male gametophyte further extends this concept from tobacco to Arabidopsis, the model species with advanced tricellular pollen.

Role of bZIP transcription factors in pollen development
Sexual plant reproduction depends on the production and differentiation of functional gametes by the haploid gametophyte generation. Currently, we have a limited understanding of the regulatory mechanisms that have evolved to specify the gametophytic developmental programs. To unravel such mechanisms, it is necessary to identify transcription factors (TF) that are part of such haploid regulatory networks. We focused on bZIP TFs that have critical roles in plants, animals and other kingdoms. We reported the functional characterization of Arabidopsis thaliana AtbZIP34 that is expressed in both gametophytic and surrounding sporophytic tissues during flower development (Gibalova et al. 2009). 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.

arabidopsisGFP database and toolbox
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; http:// agfp.ueb.cas.cz), which gives the user access to a large collection of normalised Affymetrix ATH1 microarray datasets (Dupžáková et al. 2007). The database currently contains NASC Array and AtGenExpress transcriptomic datasets for various tissues at different developmental stages of wild type plants gathered from nearly 350 gene chips. 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. The most distinguishable features of Arabidopsis Gene Family Profiler database are: 1) the presentation of normalized datasets (Affymetrix MAS algorithm and calculation of model-based gene-expression values based on the Perfect Match-only model); 2) the choice between two different normalization algorithms (Affymetrix MAS4 or MAS5 algorithms); 3) an intuitive interface; 4) an interactive "virtual plant" visualizing the spatial and developmental expression profiles of both gene families and individual genes. 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.

Pollen developmental transcriptomics
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 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.
Collaborative links
Prof. David Twell lab, Dept. of Botany, University of Leicester, Leicester, UK;
http://www.le.ac.uk/bl/research/pollen/pollen.html

Dr. Hans-Peter Mock lab, Dept. of Applied Biochemistry, IPK Gatersleben, Germany;
http://www.ipk-gatersleben.de/Internet/Forschung/PhysiologieZellbiologie/Biochemie

Prof. Heven Sze lab, Cell Biology & Molecular Genetics, University of Maryland, College Park, MD, USA;
http://cbmg.umd.edu/

Prof. Jiří Friml lab, VIB Department of Plant Systems Biology, Ghent University, Belgium;
http://www.psb.ugent.be/auxin-projects
 
MALE GAMETOPHYTE (Adapted from Honys and Twell 2004)
Pollen development: from pollen mother cell to mature pollen grain
             Both gametophytes, the male and the female, represent the haploid generative phase of the life cycle of higher plants (Fig. 1). There has been an evolutionary tendency towards reduction of the male gametophyte and its increasing functional dependence on the sporophyte. This trend is most acute within flowering plants, such that the male gametophyte consists of just two or three cells when shed as pollen grains. Despite its diminutive form the functional specialisation of the haploid male gametophyte and the closed carpel are thought to be key factors in the evolutionary success of flowering plants through mechanisms that promote rigorous selection of superior haploid genotypes and outbreeding.

Fig. 1.
Figure 1. Life cycle of a flowering plant (from Chasan R and Walbot V (1993) Mechanisms of plant reproduction: questions and approaches. Plant Cell 5: 1139-1146).

            Angiosperm pollen development consists of two sequential phases, microsporogenesis and microgametogenesis. Both of them take place inside the anther loculi that are lined by the tapetal cell layer (tapetum). Microsporogenesis is initiated upon meiotic division of the diploid pollen mother cell (microsporocyte) that produces four haploid microspores comprising a tetrad (Fig. 2). During microgametogenesis, microspores released from the tetrads undergo cell expansion, cell wall synthesis, asymmetric division and differentiation of the vegetative and generative cells before partial desiccation and release from the anther. The tapetal cells play a major role in pollen development through their contribution to microspore release, nutrition, pollen wall synthesis and pollen coat deposition. Disturbance of tapetal cell functions usually results in reduced pollen fertility or male sterility through a variety of mechanisms including arrest of microgametogenesis at the microspore stage or altered pollen hydration through modified pollen coat composition.

Fig. 2.
Figure 2. Schematic diagram illustrating pollen development.
Microsporogenesis
             A unique feature of the walls surrounding the microsporocytes and newly formed microspores within the tetrad is that they consist largely of callose, a ß(1,3)glucan. The callose wall is secreted by microsporocytes before meiosis I and separates the microspores within the tetrad following meiosis II (Fig. 2). Microspores begin to synthesize the first elements of the sculptured outer pollen wall layer (exine) starting with primexine that functions as a template for subsequent exine elaboration. When young microspores are still developing the exine within the tetrad, an enzyme complex (callase) is secreted by the tapetal cells allowing individual microspores to be released from the tetrads. Correct timing of callase secretion is critical since premature or delayed dissolution of the callose wall results in male sterility.
Microgametogenesis
             Once released free microspores increase in size, their multiple small vacuoles enlarge and fuse into a single large vacuole, occupying most of the volume of the cell. In concert the microspore nucleus migrates to a peripheral position that is required for the subsequent asymmetric division at pollen mitosis I. (PMI, Fig. 2). From this moment on, the male gametophyte is called young pollen grain. PMI results in two morphologically and functionally distinct cells, a large vegetative cell and a small generative cell. The generative cell subsequently becomes engulfed within a membrane bound compartment in the cytoplasm of its vegetative sister. This involves dissolution of the hemispherical callose wall separating the vegetative and generative cells, inward migration and membrane fusion events. The asymmetric division at PMI is a key determinative event in generative cell fate.

            In microspores and immature pollen cultivated in vitro, gametophytic development can be switched to a sporophytic pathway by heat stress and/or starvation treatment, leading to microspore embryogenesis and haploid plant formation. Such techniques are routinely applied to accelerate breeding programmes through the rapid generation of double haploid plants and selection among large numbers of homozygous lines.

            The generative cell undergoes further mitotic division at pollen mitosis II (PMII) to produce the two sperm cells. In tricellular pollen this division occurs within the anther, whereas in bicellular pollen it occurs within the growing pollen tube. Although the majority of flowering plants produce bicellular pollen, many plants such as rice, wheat and maize produce advanced, but often short-lived, tricellular pollen grains (Fig. 2, 3).

Fig. 3.
Figure 3. Bicellular and tricellular pollen. (A) Bicellular tomato and (B) tricellular oilseed rape pollen stained with DAPI. Nuclear DNA within the vegetative (V) and generative (G) or sperm (S) cells are highlighted.

            During pollen maturation the vegetative cell accumulates considerable carbohydrate and/or lipid reserves that are transient or which are stored in the mature pollen grain. Transient reserves are thought to provide metabolites for energy demanding developmental events such as asymmetric division and pollen cell wall (intine) synthesis. Osmoprotectants including proline also accumulate in mature pollen grains to protect vital membrane and proteins from damage. In mature pollen grains the extensive stores of lipids and polysaccharides are required to supply the extensive demands for plasma membrane and pollen tube wall synthesis.

            During dehydration, the final phase of pollen maturation, pollen grains are finally prepared for release from the anthers. This represents an adaptation to survive exposure to the hostile terrestrial environment. The extent of dehydration varies widely in different species, for example in poplar the water content is reduced to only 6%, maize loses 50%, whereas cucumber pollen remains fully hydrated. The degree of dehydration as well as the levels of cytoplasmic reserves positively correlates with pollen fitness and viability. Hydrated pollen is very susceptible to dehydration stress and generally survives only few hours, whereas fully dehydrated pollen may survive for months or even years under certain conditions.
The unique pollen wall
             The unique activities and biological role of the pollen grain are reflected in the unique composition of the pollen wall. The pollen wall and its coatings isolate and protect the male gametophyte and its associated gametes and mediate the complex communication with the stigma surface. The pollen wall consists of an inner intine and outer exine layer. Its synthesis begins at the microspore stage, when the pectocellulosic intine and the primexine are formed. The primexine serves as a matrix for subsequent deposition of sporopollenin. Sporopollenin is a highly resistant biopolymer containing fatty acids and phenylpropanoids and its synthesis involves tight cooperation between microspore cytoplasm and tapetal cells. The exine is not evenly distributed over the pollen grain surface and regions lacking sporopollenin form apertures that are used as sites for pollen tube emergence. The number and size of apertures and exine patterning are under strict sporophytic control.

            The formation of pollen coatings is completed at later stages of microgametogenesis. Remnants of degenerating tapetal cells are deposited onto the pollen grain surface creating the pollen coat. The pollen coat is involved in pollen-pistil signalling, self incompatibility, pollen hydration, adhesivity, colour and odour. The yellow or purple colours of mature pollen grain results from the presence of both carotenoid and phenylpropanoid compounds. These features as well as the elaborate patterning of sporopollenin are highly variable among different plant species. In animal-pollinated species pollen is often decorated with elaborate structures that facilitate vector adhesion, whereas in wind pollinated species pollen lacks such sculpturing or may be decorated with air sacs to increase buoyancy.
Pollen gene expression
             Development of the male gametophyte is associated with an extensive haploid gene expression programme. Until recently, approximately 150 pollen-specific genes falling into 50 functional classes have been cloned from various species. Our recent genome-wide studies using microarray hybridisation technology has comprehensively demonstrated the scale and diversity of haploid gene expression in Arabidopsis thaliana (click here for detailed information). Mature Arabidopsis pollen grains express over 7,000 different mRNA species out of more than 27,000 predicted from the Arabidopsis genome. Approximately 10% of these transcripts are predicted to be preferentially or specifically expressed in pollen. Moreover, there is significant overlap between sporophytic and male gametophytic gene expression that reflects the large proportion of genes that are required for basic cellular functions. The most abundant classes of pollen-specific genes are predicted to have functions associated with transcriptional regulation, signal transduction, cytoskeleton organisation and cell wall synthesis. This highlights the importance of these functions for the unique cellular specialisation that is required for pollen differentiation and function.

            Interestingly, some of these pollen-specific genes encode proteins representing major allergens, the cause of hayfever and allergic asthma. Recent work has shown that the expression of one class of allergens from ryegrass pollen can be reduced without significant impact on plant fertility, indicating that the genetic engineering of hypoallergenic cultivars is possible.

            Pollen-expressed genes have been also classified according to their temporal regulation. In addition to constitutively expressed genes, groups of early genes active in microspores and late genes acting after PMI have been identified. The late pollen genes and their regulation have been most intensively studied which has resulted in the functional dissection of several pollen-specific transcriptional and translational regulatory elements.