Quantitative proteomics of the tobacco pollen tube secretome identifies novel pollen tube guidance proteins important for fertilization

List of primers used in this study. (XLSX 12 kb

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

<p>Abstract</p> <p>Background</p> <p>Many 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.</p> <p>Results</p> <p>Progression 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.</p> <p>Conclusions</p> <p>The 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.</p

A Plant Germline-Specific Integrator of Sperm Specification and Cell Cycle Progression

The unique double fertilisation mechanism in flowering plants depends upon a pair of functional sperm cells. During male gametogenesis, each haploid microspore undergoes an asymmetric division to produce a large, non-germline vegetative cell and a single germ cell that divides once to produce the sperm cell pair. Despite the importance of sperm cells in plant reproduction, relatively little is known about the molecular mechanisms controlling germ cell proliferation and specification. Here, we investigate the role of the Arabidopsis male germline-specific Myb protein DUO POLLEN1, DUO1, as a positive regulator of male germline development. We show that DUO1 is required for correct male germ cell differentiation including the expression of key genes required for fertilisation. DUO1 is also necessary for male germ cell division, and we show that DUO1 is required for the germline expression of the G2/M regulator AtCycB1;1 and that AtCycB1:1 can partially rescue defective germ cell division in duo1. We further show that the male germline-restricted expression of DUO1 depends upon positive promoter elements and not upon a proposed repressor binding site. Thus, DUO1 is a key regulator in the production of functional sperm cells in flowering plants that has a novel integrative role linking gametic cell specification and cell cycle progression

MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana

<p>Abstract</p> <p>Background</p> <p>New generation sequencing technology has allowed investigation of the small RNA populations of flowering plants at great depth. However, little is known about small RNAs in their reproductive cells, especially in post-meiotic cells of the gametophyte generation. Pollen - the male gametophyte - is the specialised haploid structure that generates and delivers the sperm cells to the female gametes at fertilisation. Whether development and differentiation of the male gametophyte depends on the action of microRNAs and trans-acting siRNAs guiding changes in gene expression is largely unknown. Here we have used 454 sequencing to survey the various small RNA populations present in mature pollen of <it>Arabidopsis thaliana</it>.</p> <p>Results</p> <p>In this study we detected the presence of 33 different microRNA families in mature pollen and validated the expression levels of 17 selected miRNAs by Q-RT-PCR. The majority of the selected miRNAs showed pollen-enriched expression compared with leaves. Furthermore, we report for the first time the presence of trans-acting siRNAs in pollen. In addition to describing new patterns of expression for known small RNAs in each of these classes, we identified 7 putative novel microRNAs. One of these, ath-MIR2939, targets a pollen-specific F-box transcript and we demonstrate cleavage of its target mRNA in mature pollen.</p> <p>Conclusions</p> <p>Despite the apparent simplicity of the male gametophyte, comprising just two different cell types, pollen not only utilises many miRNAs and trans-acting siRNAs expressed in the somatic tissues but also expresses novel miRNAs.</p

Arabidopsis DUO POLLEN3 Is a Key Regulator of Male Germline Development and Embryogenesis

Male germline development in angiosperms produces the pair of sperm cells required for double fertilization. A key regulator of this process in Arabidopsis thaliana is the male germline-specific transcription factor DUO POLLEN1 (DUO1) that coordinates germ cell division and gamete specification. Here, we uncover the role of DUO3, a nuclear protein that has a distinct, but overlapping role with DUO1 in male germline development. DUO3 is a conserved protein in land plants and is related to GON-4, a cell lineage regulator of gonadogenesis in Caenorhabditis elegans. Mutant duo3-1 germ cells either fail to divide or show a delay in division, and we show that, unlike DUO1, DUO3 promotes entry into mitosis independent of the G2/M regulator CYCB1;1. We also show that DUO3 is required for the expression of a subset of germline genes under DUO1 control and that like DUO1, DUO3 is essential for sperm cell specification and fertilization. Furthermore, we demonstrate an essential sporophytic role for DUO3 in cell division and embryo patterning. Our findings demonstrate essential developmental roles for DUO3 in cell cycle progression and cell specification in both gametophytic and sporophytic tissues

A study of mechanisms of cell cycle regulation in the male gametophyte of Arabidopsis thaliana

The haploid male gametophyte of angiosperms has an integral role in the production of twin sperm cells necessary for the double fertilization, the essence of flowering plants. However, the mechanisms regulating sperm cell formation and cell fate specification has yet to be identified. In this study, the thesis investigates the function of key cell cycle regulators and presents characterisation of a novel pollen division mutant of Arabidopsis (duo3) that fails to produce twin sperm cells. In addition, the project also examines the activity of small RNA (smRNA) pathways as a potential mechanism that modulates native gene expression. Pollen cell-specific vectors were constructed to drive the expression of hairpin double stranded RNA (hp-dsRNA) as tools for investigating the activity of smRNA pathways, and their efficacy was tested by manipulating expression of key cell cycle regulators in Arabidopsis. Indeed, expression of hp-dsRNA intended to knockdown transcripts of Cyclin B1 members, revealed a putative role Cyclin B1 in microspore and germ cell division. Furthermore, analysis of a Cyclin B1;1 reporter led to the identification of DUO1 (a pollen specific R2R3 MYB protein) but not DUO3 as a germ cell-specific regulator of Cyclin B1;1 expression. This interaction was further verified by rescuing mutant duo1 plants with Cyclin B1;1. Analysis of DUO3 expression revealed restricted patterns confined predominantly in dividing tissues. Moreover, study of Cyclin B1;1 reporter revealed mutant duo3 cells to be impaired in degrading Cyclin B1;1 protein, suggesting a role in modulating Cyclin B1;1 activity. In summary, this work has highlighted a potential role of the Cyclin B1 family in the development of the male gametophyte. Use of Cyclin B1;1 marker has demonstrated a first example of germ cell specific integrator of cell division and cell differentiation and a putative role of DUO3 in germ cell division. A significant progress has been achieved in understanding smRNA pathways and the vectors generated will be exploited to gain more insight into the development of the male gametophyte

A study of mechanisms of cell cycle regulation in the male gametophyte of Arabidopsis thaliana

The haploid male gametophyte of angiosperms has an integral role in the production of twin sperm cells necessary for the double fertilization, the essence of flowering plants. However, the mechanisms regulating sperm cell formation and cell fate specification has yet to be identified. In this study, the thesis investigates the function of key cell cycle regulators and presents characterisation of a novel pollen division mutant of Arabidopsis (duo3) that fails to produce twin sperm cells. In addition, the project also examines the activity of small RNA (smRNA) pathways as a potential mechanism that modulates native gene expression. Pollen cell-specific vectors were constructed to drive the expression of hairpin double stranded RNA (hp-dsRNA) as tools for investigating the activity of smRNA pathways, and their efficacy was tested by manipulating expression of key cell cycle regulators in Arabidopsis. Indeed, expression of hp-dsRNA intended to knockdown transcripts of Cyclin B1 members, revealed a putative role Cyclin B1 in microspore and germ cell division. Furthermore, analysis of a Cyclin B1;1 reporter led to the identification of DUO1 (a pollen specific R2R3 MYB protein) but not DUO3 as a germ cell-specific regulator of Cyclin B1;1 expression. This interaction was further verified by rescuing mutant duo1 plants with Cyclin B1;1. Analysis of DUO3 expression revealed restricted patterns confined predominantly in dividing tissues. Moreover, study of Cyclin B1;1 reporter revealed mutant duo3 cells to be impaired in degrading Cyclin B1;1 protein, suggesting a role in modulating Cyclin B1;1 activity. In summary, this work has highlighted a potential role of the Cyclin B1 family in the development of the male gametophyte. Use of Cyclin B1;1 marker has demonstrated a first example of germ cell specific integrator of cell division and cell differentiation and a putative role of DUO3 in germ cell division. A significant progress has been achieved in understanding smRNA pathways and the vectors generated will be exploited to gain more insight into the development of the male gametophyte.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Transcriptome profiling of male gametophyte development in Nicotiana tabacum

Pollen, an extremely reduced bicellular or tricellular male reproductive structure of flowering plants, serves as a model for numerous studies covering wide range of developmental and physiological processes. The pollen development represents a fragile and vital phase of plant ontogenesis and pollen was among the first singular plant tissues thoroughly characterized at the transcriptomic level (Honys and Twell [5]). Arabidopsis pollen developmental transcriptome has been published over a decade ago (Honys and Twell, 2004) and transcriptomes of developing pollen of other species have followed (Rice, Deveshwar et al. [2]; Triticeae, Tran et al. [11]; upland cotton, Ma et al. [8]). However, the transcriptomic data describing the development of tobacco pollen, a bicellular model for cell biology studies, have been missing. Here we provide the transcriptomic data covering three stages (Tupý et al., 1983) of wild type tobacco (Nicotiana tabacum, cv. Samsun) pollen development: uninucleate microspores (UNM, stage 1), early bicellular pollen (eBCP, stage 3) and late bicellular pollen (lBCP, stage 5) as a supplement to the mature pollen (MP), 4 h-pollen tube (PT4), 24 h-pollen tubes (PT24), leaf (LF) and root (RT) transcriptomic data presented in our previous studies (Hafidh et al., 2012a; Hafidh et al., 2012b). We characterized these transcriptomes to refine the knowledge base of male gametophyte-enriched genes as well as genes expressed preferentially at the individual stages of pollen development. Alongside updating the list of tissue-specific genes, we have investigated differentially expressed genes with respect to early expressed genes. Pollen tube growth and competition of pollen tubes in female pistil can be viewed as a race of the fittest. Accordingly, there is an apparent evolutionary trend among higher plants to store significant 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 (Honys and Twell, 2004). We observed a similar phenomenon in less advanced bicellular tobacco pollen. Here, we describe in detail how we obtained and analyzed validated microarray dataset deposited in Gene Expression Omnibus (GSE62349)