David Honys

Transcriptome analysis of haploid male gametophyte development in Arabidopsis

BackgroundThe 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.ResultsWe 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.ConclusionsThe 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.

Comparative Analysis of the Arabidopsis Pollen Transcriptome

We present a genome-wide view of the male gametophytic transcriptome in Arabidopsis based on microarray analysis. In comparison with the transcriptome of the sporophyte throughout development, the pollen transcriptome showed reduced complexity and a unique composition. We identified 992 pollen-expressed mRNAs, nearly 40% of which were detected specifically in pollen. Analysis of the functional composition of the pollen transcriptome revealed the over-representation of mRNAs encoding proteins involved in cell wall metabolism, cytoskeleton, and signaling and under-representation of mRNAs involved in transcription and protein synthesis. For several gene families, we observed a common pattern of mutually exclusive gene expression between pollen and sporophytic tissues for different gene family members. Our results provide a 50-fold increase in the knowledge of genes expressed in Arabidopsis pollen. Moreover, we also detail the extensive overlap (61%) of the pollen transcriptome with that of the sporophyte, which provides ample potential to influence sporophytic fitness through gametophytic selection.

SETH1 and SETH2, Two Components of the Glycosylphosphatidylinositol Anchor Biosynthetic Pathway, Are Required for Pollen Germination and Tube Growth in Arabidopsis

Glycosylphosphatidylinositol (GPI) anchoring provides an alternative to transmembrane domains for anchoring proteins to the cell surface in eukaryotes. GPI anchors are synthesized in the endoplasmic reticulum via the sequential addition of monosaccharides, fatty acids, and phosphoethanolamines to phosphatidylinositol. Deficiencies in GPI biosynthesis lead to embryonic lethality in animals and to conditional lethality in eukaryotic microbes by blocking cell growth, cell division, or morphogenesis. We report the genetic and phenotypic analysis of insertional mutations disrupting SETH1 and SETH2, which encode Arabidopsis homologs of two conserved proteins involved in the first step of the GPI biosynthetic pathway. seth1 and seth2 mutations specifically block male transmission and pollen function. This results from reduced pollen germination and tube growth, which are associated with abnormal callose deposition. This finding suggests an essential role for GPI anchor biosynthesis in pollen tube wall deposition or metabolism. Using transcriptomic and proteomic approaches, we identified 47 genes that encode potential GPI-anchored proteins that are expressed in pollen and demonstrated that at least 11 of these proteins are associated with pollen membranes by GPI anchoring. Many of the identified candidate proteins are homologous with proteins involved in cell wall synthesis and remodeling or intercellular signaling and adhesion, and they likely play important roles in the establishment and maintenance of polarized pollen tube growth.

ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis

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. Here we find that a member of PIN auxin transporter family, PIN8 is expressed in male gametophyte of Arabidopsis thaliana and has a crucial role in pollen development and functionality. Ectopic expression in sporophytic tissues establishes a role of PIN8 in regulating auxin homoeostasis and metabolism. PIN8 co-localizes with PIN5 to the endoplasmic reticulum (ER) where it acts as an auxin transporter. Genetic analyses reveal an antagonistic action of PIN5 and PIN8 in the regulation of intracellular auxin homoeostasis and gametophyte as well as sporophyte development. Our results reveal a role of the auxin transport in male gametophyte development in which the distinct actions of ER-localized PIN transporters regulate cellular auxin homoeostasis and maintain the auxin levels optimal for pollen development and pollen tube growth.

Expression patterns of a novel AtCHX gene family highlight potential roles in osmotic adjustment and K+ homeostasis in pollen development

A combined bioinformatic and experimental approach is being used to uncover the functions of a novel family of cation/H+ exchanger (CHX) genes in plants using Arabidopsis as a model. The predicted protein (85–95 kD) of 28 AtCHX genes after revision consists of an amino-terminal domain with 10 to 12 transmembrane spans (approximately 440 residues) and a hydrophilic domain of approximately 360 residues at the carboxyl end, which is proposed to have regulatory roles. The hydrophobic, but not the hydrophilic, domain of plant CHX is remarkably similar to monovalent cation/proton antiporter-2 (CPA2) proteins, especially yeast (Saccharomyces cerevisiae) KHA1 and Synechocystis NhaS4. Reports of characterized fungal and prokaryotic CPA2 indicate that they have various transport modes, including K+/H+ (KHA1), Na+/H+-K+ (GerN) antiport, and ligand-gated ion channel (KefC). The expression pattern of AtCHX genes was determined by reverse transcription PCR, promoter-driven β-glucuronidase expression in transgenic plants, and Affymetrix ATH1 genome arrays. Results show that 18 genes are specifically or preferentially expressed in the male gametophyte, and six genes are highly expressed in sporophytic tissues. Microarray data revealed that several AtCHX genes were developmentally regulated during microgametogenesis. An exciting idea is that CHX proteins allow osmotic adjustment and K+ homeostasis as mature pollen desiccates and then rehydrates at germination. The multiplicity of CHX-like genes is conserved in higher plants but is not found in animals. Only 17 genes, OsCHX01 to OsCHX17, were identified in rice (Oryza sativa) subsp. japonica, suggesting diversification of CHX in Arabidopsis. These results reveal a novel CHX gene family in flowering plants with potential functions in pollen development, germination, and tube growth.

Integrating membrane transport with male gametophyte development and function through transcriptomics

Male fertility depends on the proper development of the male gametophyte, successful pollen germination, tube growth, and delivery of the sperm cells to the ovule. Previous studies have shown that nutrients like boron, and ion gradients or currents of Ca2+, H+, and K+ are critical for pollen tube growth. However, the molecular identities of transporters mediating these fluxes are mostly unknown. As a first step to integrate transport with pollen development and function, a genome-wide analysis of transporter genes expressed in the male gametophyte at four developmental stages was conducted. Approximately 1,269 genes encoding classified transporters were collected from the Arabidopsis (Arabidopsis thaliana) genome. Of 757 transporter genes expressed in pollen, 16% or 124 genes, including AHA6, CNGC18, TIP1.3, and CHX08, are specifically or preferentially expressed relative to sporophytic tissues. Some genes are highly expressed in microspores and bicellular pollen (COPT3, STP2, OPT9), while others are activated only in tricellular or mature pollen (STP11, LHT7). Analyses of entire gene families showed that a subset of genes, including those expressed in sporophytic tissues, was developmentally regulated during pollen maturation. Early and late expression patterns revealed by transcriptome analysis are supported by promoter∷β-glucuronidase analyses of CHX genes and by other methods. Recent genetic studies based on a few transporters, including plasma membrane H+ pump AHA3, Ca2+ pump ACA9, and K+ channel SPIK, further support the expression patterns and the inferred functions revealed by our analyses. Thus, revealing the distinct expression patterns of specific transporters and unknown polytopic proteins during microgametogenesis provides new insights for strategic mutant analyses necessary to integrate the roles of transporters and potential receptors with male gametophyte development.

Enrichment techniques employed in phosphoproteomics

Rapid changes of protein phosphorylation play a crucial role in the regulation of many cellular processes. Being post-translationally modified, phosphoproteins are often present in quite low abundance and tend to co-exist with their unphosphorylated isoform within the cell. To make their identification more practicable, the use of enrichment protocols is often required. The enrichment strategies can be performed either at the level of phosphoproteins or at the level of phosphopeptides. Both approaches have their advantages and disadvantages. Most enriching strategies are based on chemical modifications, affinity chromatography to capture peptides and proteins containing negatively charged phosphate groups onto a positively charged matrix, or immunoprecipitation by phospho-specific antibodies.In this article, the most up-to-date enrichment techniques are discussed, taking into account their optimization, and highlighting their advantages and disadvantages. Moreover, these methods are compared to each other, revealing their complementary nature in providing comprehensive coverage of the phosphoproteome.

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

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.

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 thalianaAtbZIP34 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.

The translationally repressed pollen-specific ntp303 mRNA is stored in non-polysomal mRNPs during pollen maturation

Abstract A tobacco pollen tube glycoprotein, p69 is encoded by the pollen-specific gene ntp303 that is transcribed during pollen development and pollen tube growth, but it is abundantly translated only after pollen germination. To investigate the translational repression of ntp303 mRNA during pollen development the compartmentation of ntp303 mRNA was examined and compared against another transcript (ntp52), which is efficiently translated during pollen maturation. Three subcellular fractions were isolated: a post-polysomal fraction enriched with messenger ribonucleoprotein particles, a polysomal fraction and a novel fraction of EDTA/ puromycin-resistant particles co-sedimentating with polysomes (EPP). At all developmental stages studied, ntp303 mRNA was found to be present in all fractions. Surprisingly, most of the translationally inactive ntp303 mRNA was localised in the polysomal fraction and EPPs, whereas ntp52 mRNA was distributed between the post-polysomal fraction and polysomes but was virtually undetectable in EPPs. This differential mRNA distribution pattern may help to explain the developmentally regulated translational repression of the ntp303 gene during pollen maturation, highlighting a potential role of EPPs. A model of how this differential mRNA compartmentation pattern regulates ntp303 mRNA translation is proposed.