Rapid separation of Arabidopsis male gametophyte developmental stages using a Percoll gradient.

Research investigating the dynamics of male gametophyte (MG) development has proven to be challenging for the plant science community. Here we describe our protocol for separating Arabidopsis MG developmental stages, which is based on the centrifugation of pollen through a discontinuous Percoll concentration gradient. This Percoll gradient can be formed using a pipette, and it does not require a gradient maker. The purity of the isolated developing spores is as high as 70%, and in most separations it is well above 80%. Using this protocol, we can separate four different stages of pollen development-uninucleate microspore (UNM), bicellular pollen (BCP), tricellular immature pollen (TCP) and mature pollen grain (MPG). The duration of the separation procedure, excluding the cutting of flower inflorescences, is 6 h. This is reduced to 4 h when using a vacuum cleaning method to remove the MPGs before the Percoll density separation.

A new link between stress response and nucleolar function during pollen development in Arabidopsis mediated by AtREN1 protein.

Heat shock transcription factors (Hsfs) are involved in multiple aspects of stress response and plant growth. However, their role during male gametophyte development is largely unknown, although the generative phase is the most sensitive and critical period in the plant life cycle. Based on a wide screen of T-DNA mutant lines, we identified the atren1 mutation (restricted to nucleolus1) in early male gametophytic gene At1g77570, which has the closest homology to HSFA5 gene, the member of a heat shock transcription factor (HSF) gene family. The mutation causes multiple defects in male gametophyte development in both structure and function. Because the mutation disrupts an early acting (AtREN1) gene, these pollen phenotype abnormalities appear from bicellular pollen stage to pollen maturation. Moreover, the consequent progamic phase is compromised as well as documented by pollen germination defects and limited transmission via male gametophyte. In addition, atren1/- plants are defective in heat stress (HS) response and produce notably higher proportion of aberrant pollen grains. AtREN1 protein is targeted specifically to the nucleolus that, together with the increased size of the nucleolus in atren1 pollen, suggests that it is likely to be involved in ribosomal RNA biogenesis or other nucleolar functions.

Wide-scale screening of T-DNA lines for transcription factor genes affecting male gametophyte development in Arabidopsis.

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. Transcriptomic studies exploiting genome-wide microarray technologies revealed the uniqueness of pollen transcriptome and the dynamics of early and late successive global gene expression programs. However, the knowledge of transcription regulation is still very limited. In this study, we focused on the identification of pollen-expressed transcription factor (TF) genes involved in the regulation of male gametophyte development. To achieve this, the reverse genetic approach was used. Seventy-four T-DNA insertion lines were screened, representing 49 genes of 21 TF families active in either early or late pollen development. In the screen, ten phenotype categories were distinguished, affecting various structural or functional aspects, including pollen abortion, presence of inclusions, variable pollen grain size, disrupted cell wall structure, cell cycle defects, and male germ unit organization. Thirteen lines were not confirmed to contain the T-DNA insertion. Among 61 confirmed lines, about half (29 lines) showed strong phenotypic changes (i.e., ≥ 25% aberrant pollen) including four lines that produced a remarkably high proportion (70-100%) of disturbed pollen. However, the remaining 32 lines exhibited mild defects or resembled wild-type appearance. There was no significant bias toward any phenotype category among early and late TF genes, nor, interestingly, within individual TF families. Presented results have a potential to serve as a basal information resource for future research on the importance of respective TFs in male gametophyte development.

Cytoskeleton-associated large RNP complexes in tobacco male gametophyte (EPPs) are associated with ribosomes and are involved in protein synthesis, processing, and localization.

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. Here, we report the first structural and proteomic data about large ribonucleoprotein particles (EPPs) in tobacco male gametophyte. 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.

AtbZIP34 is required for Arabidopsis pollen wall patterning and the control of several metabolic pathways in developing pollen.

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. Here we focus on bZIP TFs that have critical roles in plants, animals and other kingdoms. We report 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.

Arabidopsis Gene Family Profiler (aGFP)--user-oriented transcriptomic database with easy-to-use graphic interface.

BACKGROUND: 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. Global statistical data analysis methods contribute to the development of overall concepts about gene expression patterns and to query and compose working hypotheses. More recently, these applications are being supplemented with more specialized products offering visualization and specific data mining tools. We present 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. 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. RESULTS: 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. CONCLUSION: 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.

Identification of microspore-active promoters that allow targeted manipulation of gene expression at early stages of microgametogenesis in Arabidopsis.

BACKGROUND: The effective functional analysis of male gametophyte development requires new tools enabling the spatially and temporally controlled expression of both marker genes and modified genes of interest. In particular, promoters driving expression at earlier developmental stages including microspores are required. RESULTS: Transcriptomic datasets covering four progressive stages of male gametophyte development in Arabidopsis were used to select candidate genes showing early expression profiles that were male gametophyte-specific. Promoter-GUS reporter analysis of candidate genes identified three promoters (MSP1, MSP2, and MSP3) that are active in microspores and are otherwise specific to the male gametophyte and tapetum. The MSP1 and MSP2 promoters were used to successfully complement and restore the male transmission of the gametophytic two-in-one (tio) mutant that is cytokinesis-defective at first microspore division. CONCLUSION: We demonstrate the effective application of MSP promoters as tools that can be used to elucidate gametophytic gene functions in microspores in a male-specific manner.