Genetic control of generative cell shape by DUO1 in Arabidopsis.

KEY MESSAGE: The main features of generative cell morphogenesis, formation of a cytoplasmic projection and elongation of the GC body, operate through independent genetic pathways. Male gametogenesis in developing angiosperm pollen involves distinctive changes in cell morphogenesis. Re-shaping and elongation of the generative cell (GC) are linked to the formation of a GC cytoplasmic projection connected to the vegetative cell nucleus. Although genetic control of GC morphogenesis is unknown, we suspected the involvement of the germline-specific MYB transcription factor DUO POLLEN1 (DUO1). We used light and fluorescence microscopy to examine male germline development in pollen of wild-type Arabidopsis and in four allelic duo1 mutants expressing introduced cell markers. Our analysis shows that the undivided GC in duo1 pollen forms a cytoplasmic projection, but the cell body fails to elongate. In contrast GCs of cyclin-dependent kinase function mutants, which fail to divide like duo1 mutants, achieve normal morphogenesis. We conclude that DUO1 has an essential role in the elongation of the GC, but DUO1-independent pathways control the development of the GC cytoplasmic projection. The two main features of GC morphogenesis therefore operate through independently regulated genetic pathways.

Regulatory dynamics of gene expression in the developing male gametophyte of Arabidopsis.

Sexual reproduction in angiosperms requires the production and delivery of two male gametes by a three-celled haploid male gametophyte. This demands synchronized gene expression in a short developmental window to ensure double fertilization and seed set. While transcriptomic changes in developing pollen are known for Arabidopsis, no studies have integrated RNA and proteomic data in this model. Further, the role of alternative splicing has not been fully addressed, yet post-transcriptional and post-translational regulation may have a key role in gene expression dynamics during microgametogenesis. We have refined and substantially updated global transcriptomic and proteomic changes in developing pollen for two Arabidopsis accessions. Despite the superiority of RNA-seq over microarray-based platforms, we demonstrate high reproducibility and comparability. We identify thousands of long non-coding RNAs as potential regulators of pollen development, hundreds of changes in alternative splicing and provide insight into mRNA translation rate and storage in developing pollen. Our analysis delivers an integrated perspective of gene expression dynamics in developing Arabidopsis pollen and a foundation for studying the role of alternative splicing in this model.

Comparative transcriptomic analysis reveals conserved programmes underpinning organogenesis and reproduction in land plants.

The appearance of plant organs mediated the explosive radiation of land plants, which shaped the biosphere and allowed the establishment of terrestrial animal life. The evolution of organs and immobile gametes required the coordinated acquisition of novel gene functions, the co-option of existing genes and the development of novel regulatory programmes. However, no large-scale analyses of genomic and transcriptomic data have been performed for land plants. To remedy this, we generated gene expression atlases for various organs and gametes of ten plant species comprising bryophytes, vascular plants, gymnosperms and flowering plants. A comparative analysis of the atlases identified hundreds of organ- and gamete-specific orthogroups and revealed that most of the specific transcriptomes are significantly conserved. Interestingly, our results suggest that co-option of existing genes is the main mechanism for evolving new organs. In contrast to female gametes, male gametes showed a high number and conservation of specific genes, which indicates that male reproduction is highly specialized. The expression atlas capturing pollen development revealed numerous transcription factors and kinases essential for pollen biogenesis and function.

Epigenetic reprogramming rewires transcription during the alternation of generations in Arabidopsis.

Alternation between morphologically distinct haploid and diploid life forms is a defining feature of most plant and algal life cycles, yet the underlying molecular mechanisms that govern these transitions remain unclear. Here, we explore the dynamic relationship between chromatin accessibility and epigenetic modifications during life form transitions in Arabidopsis. The diploid-to-haploid life form transition is governed by the loss of H3K9me2 and DNA demethylation of transposon-associated cis-regulatory elements. This event is associated with dramatic changes in chromatin accessibility and transcriptional reprogramming. In contrast, the global loss of H3K27me3 in the haploid form shapes a chromatin accessibility landscape that is poised to re-initiate the transition back to diploid life after fertilisation. Hence, distinct epigenetic reprogramming events rewire transcription through major reorganisation of the regulatory epigenome to guide the alternation of generations in flowering plants.

Each pollen grain from a flowering plant houses sperm, which contain half of the genes needed to make a new plant, and a companion or vegetative cell (VC) that serves to deliver sperm to the egg. The genes in the vegetative cell and those in the sperm are identical to the genes of the plant they come from, so how can this set of identical genetic information produce such different cells? Both DNA and histones, the proteins that pack and order DNA, can be chemically modified locally through a process called methylation. The location of these modifications can affect how genetic information in the DNA is read to make different types of cells. The use of processes like methylation to regulate whether genes are switched on or off is called epigenetics. So what role does epigenetics play in plant pollen? To answer this question, Borg et al. examined the epigenetics of pollen in Arabidopsis thaliana, a widely studied plant and common weed. In vegetative cells, DNA methylation is lost together with a different methylation mark (H3K9me2), which unlocks several genes needed for pollen to transport sperm. By contrast, sperm loses an entirely different methylation mark, called H3K27me3, which unlocks a different set of genes that help to prepare development of a new plant once sperm fertilizes the egg. Through these different set of epigenetic changes, activity increases at different groups of genes that are important for shaping the function of each pollen cell type. These results reveal how the loss of DNA and histone methylation are important for plants to reproduce sexually via pollen. This offers insights into the evolution of plants and other related life forms. Learning about plant reproduction may also help to increase food production by improving crop yields.

MYB81, a microspore-specific GAMYB transcription factor, promotes pollen mitosis I and cell lineage formation in Arabidopsis.

Sexual reproduction in flowering plants relies on the production of haploid gametophytes that consist of germline and supporting cells. During male gametophyte development, the asymmetric mitotic division of an undetermined unicellular microspore segregates these two cell lineages. To explore genetic regulation underlying this process, we screened for pollen cell patterning mutants and isolated the heterozygous myb81-1 mutant that sheds ~50% abnormal pollen. Typically, myb81-1 microspores fail to undergo pollen mitosis I (PMI) and arrest at polarized stage with a single central vacuole. Although most myb81-1 microspores degenerate without division, a small fraction divides at later stages and fails to acquire correct cell fates. The myb81-1 allele is transmitted normally through the female, but rarely through pollen. We show that myb81-1 phenotypes result from impaired function of the GAMYB transcription factor MYB81. The MYB81 promoter shows microspore-specific activity and a MYB81-RFP fusion protein is only expressed in a narrow window prior to PMI. Ectopic expression of MYB81 driven by various promoters can severely impair vegetative or reproductive development, reflecting the strict microspore-specific control of MYB81. Our data demonstrate that MYB81 has a key role in the developmental progression of microspores, enabling formation of the two male cell lineages that are essential for sexual reproduction in Arabidopsis.

The evolution and patterning of male gametophyte development.

The reproductive adaptations of land plants have played a key role in their terrestrial colonization and radiation. This encompasses mechanisms used for the production, dispersal and union of gametes to support sexual reproduction. The production of small motile male gametes and larger immotile female gametes (oogamy) in specialized multicellular gametangia evolved in the charophyte algae, the closest extant relatives of land plants. Reliance on water and motile male gametes for sexual reproduction was retained by bryophytes and basal vascular plants, but was overcome in seed plants by the dispersal of pollen and the guided delivery of non-motile sperm to the female gametes. Here we discuss the evolutionary history of male gametogenesis in streptophytes (green plants) and the underlying developmental biology, including recent advances in bryophyte and angiosperm models. We conclude with a perspective on research trends that promise to deliver a deeper understanding of the evolutionary and developmental mechanisms of male gametogenesis in plants.

Transcription factor DUO1 generated by neo-functionalization is associated with evolution of sperm differentiation in plants.

Evolutionary mechanisms underlying innovation of cell types have remained largely unclear. In multicellular eukaryotes, the evolutionary molecular origin of sperm differentiation is unknown in most lineages. Here, we report that in algal ancestors of land plants, changes in the DNA-binding domain of the ancestor of the MYB transcription factor DUO1 enabled the recognition of a new cis-regulatory element. This event led to the differentiation of motile sperm. After neo-functionalization, DUO1 acquired sperm lineage-specific expression in the common ancestor of land plants. Subsequently the downstream network of DUO1 was rewired leading to sperm with distinct morphologies. Conjugating green algae, a sister group of land plants, accumulated mutations in the DNA-binding domain of DUO1 and lost sperm differentiation. Our findings suggest that the emergence of DUO1 was the defining event in the evolution of sperm differentiation and the varied modes of sexual reproduction in the land plant lineage.

Analysis of Fluorescent Reporter Activity in the Male Germline During Pollen Development by Confocal Microscopy.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte (pollen). The germline is established by asymmetric division of the microspore to form the generative cell. Mitotic division of the generative cell then produces the two sperm cells required for double fertilization. These differentiate to produce the proteins required for gamete attachment and fusion. An important aspect of understanding germline development is the characterization of germline gene expression. Here, we describe the use of a fluorescent reporter to study germline gene expression in developing pollen to assess the timing and specificity of expression.

Identification of Cis-Regulatory Modules that Function in the Male Germline of Flowering Plants.

The male germline of flowering plants develops within the vegetative cell of the male gametophyte and displays a distinct transcriptional profile. Key to understanding the development of this unique cell lineage is determining how gene expression is regulated within germline cells. This knowledge impacts upon our understanding of cell specification, differentiation, and plant fertility. Here, we describe methods to identify cis-regulatory modules (CRMs) that act as key regulatory regions in the promoters of germline-expressed genes. We detail the complimentary techniques of phylogenetic footprinting and the use of fluorescent reporters in pollen for the identification and verification of CRMs.

Expression of four S. pneumoniae type 2 polysaccharide biosynthetic enzymes utilising the endogenous Kex2 protease activity in tobacco.

In order to express multisubunit proteins, or to manipulate metabolic pathways in plants it is essential to be able to efficiently express multiple proteins within the same plant cell. To increase the efficiency of multi-protein expression, we demonstrate the use of the Golgi localized Kex2 protease activity in tobacco to process a large polyprotein precursor consisting of four individual protein domains into its individual protein constituents. Four genes encoding enzymes involved in the biosynthesis of S. pneumoniae type 2 polysaccharide were assembled into a single expression cassette as a large polyprotein driven by a single cauliflower mosaic virus (CaMV) 35S promoter. Each of the individual protein domains were separated by three sequential Kex2 protease digestion sites. At the N-terminus a Pr1b signal peptide was incorporated for efficient targeting of the polyprotein to the apoplast. Each individual protein domain was tagged with its own immuno-tag. The construct was used for the transformation of Nicotiana tabacum and stable lines were selected. All four processed proteins could be immunologically detected in protein extracts using Western blotting indicating correct expression and Kex2 processing. Utilisation of the Kex2 protease system represents an efficient way of expressing multiple proteins in the same plant. This method simplifies the transformation procedures, and presents a method for expression of multiple proteins within the same plant.

Corrigendum: Transcriptome Analysis of Hamelia patens (Rubiaceae) Anthers Reveals Candidate Genes for Tapetum and Pollen Wall Development.

[This corrects the article on p. 1991 in vol. 7, PMID: 28119704.].

A Conserved cis-Regulatory Module Determines Germline Fate through Activation of the Transcription Factor DUO1 Promoter.

The development of the male germline within pollen relies upon the activation of numerous target genes by the transcription factor DUO POLLEN1 (DUO1). The expression of DUO1 is restricted to the male germline and is first detected shortly after the asymmetric division that segregates the germ cell lineage. Transcriptional regulation is critical in controlling DUO1 expression, since transcriptional and translational fusions show similar expression patterns. Here, we identify key promoter sequences required for the germline-specific regulation of DUO1 transcription. Combining promoter deletion analyses with phylogenetic footprinting in eudicots and in Arabidopsis accessions, we identify a cis-regulatory module, Regulatory region of DUO1 (ROD1), which replicates the expression pattern of DUO1 in Arabidopsis (Arabidopsis thaliana). We show that ROD1 from the legume Medicago truncatula directs male germline-specific expression in Arabidopsis, demonstrating conservation of DUO1 regulation among eudicots. ROD1 contains several short conserved cis-regulatory elements, including three copies of the motif DNGTGGV, required for germline expression and tandem repeats of the motif YAACYGY, which enhance DUO1 transcription in a positive feedback loop. We conclude that a cis-regulatory module conserved in eudicots directs the spatial and temporal expression of the transcription factor DUO1 to specify male germline fate and sperm cell differentiation.

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis.

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin-celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6-1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)-dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6-1 during male meiosis and pollen mitosis I using fluorescent MT-markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6-1 mutants as a valuable tool for the investigation of augmin-dependent MT nucleation and dynamics in plant cells.

Transcriptome Analysis of Hamelia patens (Rubiaceae) Anthers Reveals Candidate Genes for Tapetum and Pollen Wall Development.

Studies of the anther transcriptome on non-model plants without a known genome are surprisingly scarce. RNA-Seq and digital gene expression (DGE) profiling provides a comprehensive approach to identify candidate genes contributing to developmental processes in non-model species. Here we built a transcriptome library of developing anthers of Hamelia patens and analyzed DGE profiles from each stage to identify genes that regulate tapetum and pollen development. In total 7,720 putative differentially expressed genes across four anther stages were identified. The number of putative stage-specific genes was: 776 at microspore mother cell stage, 807 at tetrad stage, 322 at uninucleate microspore stage, and the highest number (1,864) at bicellular pollen stage. GO enrichment analysis revealed 243 differentially expressed and 108 stage-specific genes that are potentially related to tapetum development, sporopollenin synthesis, and pollen wall. The number of expressed genes, their function and expression profiles were all significantly correlated with anther developmental processes. Overall comparisons of anther and pollen transcriptomes with those of rice and Arabidopsis together with the expression profiles of homologs of known anther-expressed genes, revealed conserved patterns and also divergence. The divergence may reflect taxon-specific differences in gene expression, the use RNA-seq as a more sensitive methodology, variation in tissue composition and sampling strategies. Given the lack of genomic sequence, this study succeeded in assigning putative identity to a significant proportion of anther-expressed genes and genes relevant to tapetum and pollen development in H. patens. The anther transcriptome revealed a molecular distinction between developmental stages, serving as a resource to unravel the functions of genes involved in anther development in H. patens and informing the analysis of other members of the Rubiaceae.

Organelles maintain spindle position in plant meiosis.

Accurate positioning of spindles is a critical aspect of cell division as it ensures that each daughter cell contains a single nucleus. In many flowering plants, two meiotic chromosome separations occur without intervening cytokinesis, resulting in two spindles in one cell during the second division. Here we report a detailed examination of two mutants, jason (jas) and parallel spindle1 (ps1), in which disturbed spindle position during male meiosis II results in the incorporation of previously separated chromosome groups into a single cell. Our study reveals that an organelle band provides a physical barrier between the two spindles. The loss of a single protein, JAS, from this organelle band leads to its disruption and a random movement of the spindles. JAS is largely associated with vesicles in the organelle band, revealing a role for vesicles in plant meiosis and that cytoplasmic events maintain spindle position during the chromosome division.

A decade of pollen transcriptomics.

KEY MESSAGE: Overview of pollen transcriptome studies. Pollen development is driven by gene expression, and knowledge of the molecular events underlying this process has undergone a quantum leap in the last decade through studies of the transcriptome. Here, we outline historical evidence for male haploid gene expression and review the wealth of pollen transcriptome data now available. Knowledge of the transcriptional capacity of pollen has progressed from genetic studies to the direct analysis of RNA and from gene-by-gene studies to analyses on a genomic scale. Microarray and/or RNA-seq data can now be accessed for all phases and cell types of developing pollen encompassing 10 different angiosperms. These growing resources have accelerated research and will undoubtedly inspire new directions and the application of system-based research into the mechanisms that govern the development, function and evolution of angiosperm pollen.

Young Genes out of the Male: An Insight from Evolutionary Age Analysis of the Pollen Transcriptome.

The birth of new genes in genomes is an important evolutionary event. Several studies reveal that new genes in animals tend to be preferentially expressed in male reproductive tissues such as testis (Betrán et al., 2002; Begun et al., 2007; Dubruille et al., 2012), and thus an "out of testis" hypothesis for the emergence of new genes has been proposed (Vinckenbosch et al., 2006; Kaessmann, 2010). However, such phenomena have not been examined in plant species. Here, by employing a phylostratigraphic method, we dated the origin of protein-coding genes in rice and Arabidopsis thaliana and observed a number of young genes in both species. These young genes tend to encode short extracellular proteins, which may be involved in rapid evolving processes, such as reproductive barriers, species specification, and anti-microbial processes. Further analysis of transcriptome age indexes across different tissues revealed that male reproductive cells express a phylogenetically younger transcriptome than other plant tissues. Compared with sporophytic tissues, the young transcriptomes of the male gametophyte displayed greater complexity and diversity, which included a higher ratio of anti-sense and inter-genic transcripts, reflecting a pervasive transcription state that facilitated the emergence of new genes. Here, we propose that pollen may act as an "innovation incubator" for the birth of de novo genes. With cases of male-biased expression of young genes reported in animals, the "new genes out of the male" model revealed a common evolutionary force that drives reproductive barriers, species specification, and the upgrading of defensive mechanisms against pathogens.