Heterochromatic silencing is reinforced by ARID1-mediated small RNA movement in Arabidopsis pollen.

In the plant male germline, transposable elements (TEs) are reactivated in the companion vegetative nucleus, resulting in siRNA production and the intercellular movement of these siRNAs to reinforce TE silencing in sperm. However, the mechanism by which siRNA movement is regulated remains unexplored. Here we show that ARID1, a transcription factor which is constitutively expressed in the vegetative nucleus but dynamically accumulates in the generative cell (the progenitor of sperm) to promote the second pollen mitosis, mediates siRNA movement to reinforce heterochromatic silencing in the male germline. We looked for regulators involved in the accumulation of ARID1 in the generative cell, and found that AGO9, a germline-specific AGO in Arabidopsis, is required for the accumulation of ARID1 in the generative cell. Mutations in either ARID1 or AGO9 lead to the interruption of not only the second pollen mitosis but also the movement of siRNA from the vegetative nucleus to the male germline, resulting in the release of heterochromatic silencing in the male germline. Moreover, conditional knockdown of ARID1 in the generative cell causes reduced heterochromatic silencing in both bicellular and mature pollen. This study provides insights into how a spatiotemporal transcription factor coordinates heterochromatic silencing and male germline maturation.

S-Adenosylmethionine Synthetase 3 Is Important for Pollen Tube Growth.

S-Adenosylmethionine is widely used in a variety of biological reactions and participates in the methionine (Met) metabolic pathway. In Arabidopsis (Arabidopsis thaliana), one of the four S-adenosylmethionine synthetase genes, METHIONINE ADENOSYLTRANSFERASE3 (MAT3), is highly expressed in pollen. Here, we show that mat3 mutants have impaired pollen tube growth and reduced seed set. Metabolomics analyses confirmed that mat3 pollen and pollen tubes overaccumulate Met and that mat3 pollen has several metabolite profiles, such as those of polyamine biosynthesis, which are different from those of the wild type. Additionally, we show that disruption of Met metabolism in mat3 pollen affected transfer RNA and histone methylation levels. Thus, our results suggest a connection between metabolism and epigenetics.

Coffee, Cake & Culture: Evaluation of an art for health programme for older people in the community.

Arts for health initiatives and networks are being developed in a number of countries and an international literature is emerging on the evidence of their benefits to people's health, wellbeing and quality of life. Engagement in cultural and creative arts by older people can increase their morale and self-confidence and provides opportunities for social connection. Museums and galleries are increasingly required to justify their expenditure, reach and impact and some are working in partnership with local councils, hospitals, schools and communities to improve access to their collections. There is a body of literature emerging that describes such initiatives but empirical evidence of their benefits is less developed. This article reports an evaluation of an art for health initiative - Coffee, Cake & Culture organised and delivered by Whitworth Art Gallery and Manchester Museum in 2012 for older people living in a care home and a supported living facility. The study has identified the benefits and impacts of the arts for health programme and its feasibility for older people, with or without diagnosed memory loss - dementia, living in a care home or supported living facility and their care staff. The findings demonstrate there were benefits to the older people and their care staff in terms of wellbeing, social engagement, learning, social inclusion and creativity. These benefits were immediate and continued in the short term on their return home. The majority of older people and care staff had not previously been to the art gallery or museum and the programme encouraged creative arts and cultural appreciation which promoted social inclusion, wellbeing and quality of life. The programme is feasible and important lessons were identified for future planning. Further research involving partnerships of researchers, arts for health curators, artists, care staff, older people and their families is warranted.

Intercellular communication in Arabidopsis thaliana pollen discovered via AHG3 transcript movement from the vegetative cell to sperm.

An Arabidopsis pollen grain (male gametophyte) consists of three cells: the vegetative cell, which forms the pollen tube, and two sperm cells enclosed within the vegetative cell. It is still unclear if there is intercellular communication between the vegetative cell and the sperm cells. Here we show that ABA-hypersensitive germination3 (AHG3), encoding a protein phosphatase, is specifically transcribed in the vegetative cell but predominantly translated in sperm cells. We used a series of deletion constructs and promoter exchanges to document transport of AHG3 transcripts from the vegetative cell to sperm and showed that their transport requires sequences in both the 5' UTR and the coding region. Thus, in addition its known role in transporting sperm during pollen tube growth, the vegetative cell also contributes transcripts to the sperm cells.

An ARID domain-containing protein within nuclear bodies is required for sperm cell formation in Arabidopsis thaliana.

In plants, each male meiotic product undergoes mitosis, and then one of the resulting cells divides again, yielding a three-celled pollen grain comprised of a vegetative cell and two sperm cells. Several genes have been found to act in this process, and DUO1 (DUO POLLEN 1), a transcription factor, plays a key role in sperm cell formation by activating expression of several germline genes. But how DUO1 itself is activated and how sperm cell formation is initiated remain unknown. To expand our understanding of sperm cell formation, we characterized an ARID (AT-Rich Interacting Domain)-containing protein, ARID1, that is specifically required for sperm cell formation in Arabidopsis. ARID1 localizes within nuclear bodies that are transiently present in the generative cell from which sperm cells arise, coincident with the timing of DUO1 activation. An arid1 mutant and antisense arid1 plants had an increased incidence of pollen with only a single sperm-like cell and exhibited reduced fertility as well as reduced expression of DUO1. In vitro and in vivo evidence showed that ARID1 binds to the DUO1 promoter. Lastly, we found that ARID1 physically associates with histone deacetylase 8 and that histone acetylation, which in wild type is evident only in sperm, expanded to the vegetative cell nucleus in the arid1 mutant. This study identifies a novel component required for sperm cell formation in plants and uncovers a direct positive regulatory role of ARID1 on DUO1 through association with histone acetylation.

RNA-seq of Arabidopsis pollen uncovers novel transcription and alternative splicing.

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5' and 3' untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser.

The anaphase-promoting complex is a dual integrator that regulates both MicroRNA-mediated transcriptional regulation of cyclin B1 and degradation of Cyclin B1 during Arabidopsis male gametophyte development.

The anaphase-promoting complex/cyclosome (APC/C), an essential ubiquitin protein ligase, regulates mitotic progression and exit by enhancing degradation of cell cycle regulatory proteins, such as CYCB1;1, whose transcripts are upregulated by DUO POLLEN1 (DUO1). DUO1 is required for cell division in male gametophytes and is a target of microRNA 159 (miR159) in Arabidopsis thaliana. Whether APC/C is required for DUO1-dependent CYCB1;1 regulation is unknown. Mutants in both APC8 and APC13 had pleiotrophic phenotypes resembling those of mutants affecting microRNA biogenesis. We show that these apc/c mutants had reduced miR159 levels and increased DUO1 and CYCB1;1 transcript levels and that APC/C is required to recruit RNA polymerase II to MIR159 promoters. Thus, in addition to its role in degrading CYCB1;1, APC/C stimulates production of miR159, which downregulates DUO1 expression, leading to reduced CYCB1;1 transcription. Both MIR159 and APC8-yellow fluorescent protein accumulated in unicellular microspores and bicellular pollen but decreased in tricellular pollen, suggesting that spatial and temporal regulation of miR159 by APC/C ensures mitotic progression. Consistent with this, the percentage of mature pollen with no or single sperm-like cells increased in apc/c mutants and plants overexpressing APC8 partially mimicked the duo1 phenotype. Thus, APC/C is an integrator that regulates both microRNA-mediated transcriptional regulation of CYCB1;1 and degradation of CYCB1;1.

Proper regulation of a sperm-specific cis-nat-siRNA is essential for double fertilization in Arabidopsis.

Natural cis-antisense siRNAs (cis-nat-siRNAs) are a recently characterized class of small regulatory RNAs that are widespread in eukaryotes. Despite their abundance, the importance of their regulatory activity is largely unknown. The only functional role for eukaryotic cis-nat-siRNAs that has been described to date is in environmental stress responses in plants. Here we demonstrate that cis-nat-siRNA-based regulation plays key roles in Arabidopsis reproductive function, as it facilitates gametophyte formation and double fertilization, a developmental process of enormous agricultural value. We show that male gametophytic kokopelli (kpl) mutants display frequent single-fertilization events, and that KPL and a inversely transcribed gene, ARIADNE14 (ARI14), which encodes a putative ubiquitin E3 ligase, generate a sperm-specific nat-siRNA pair. In the absence of KPL, ARI14 RNA levels in sperm are increased and fertilization is impaired. Furthermore, ARI14 transcripts accumulate in several siRNA biogenesis pathway mutants, and overexpression of ARI14 in sperm phenocopies the reduced seed set of the kokopelli mutants. These results extend the regulatory capacity of cis-nat-siRNAs to development by identifying a role for cis-nat-siRNAs in controlling sperm function during double fertilization.

Interdependence of endomembrane trafficking and actin dynamics during polarized growth of Arabidopsis pollen tubes.

During polarized growth of pollen tubes, endomembrane trafficking and actin polymerization are two critical processes that establish membrane/wall homeostasis and maintain growth polarity. Fine-tuned interactions between these two processes are therefore necessary but poorly understood. To better understand such cross talk in the model plant Arabidopsis (Arabidopsis thaliana), we first established optimized concentrations of drugs that interfere with either endomembrane trafficking or the actin cytoskeleton, then examined pollen tube growth using fluorescent protein markers that label transport vesicles, endosomes, or the actin cytoskeleton. Both brefeldin A (BFA) and wortmannin disturbed the motility and structural integrity of ARA7- but not ARA6-labeled endosomes, suggesting heterogeneity of the endosomal populations. Disrupting endomembrane trafficking by BFA or wortmannin perturbed actin polymerization at the apical region but not in the longitudinal actin cables in the shank. The interference of BFA/wortmannin with actin polymerization was progressive rather than rapid, suggesting an indirect effect, possibly due to perturbed endomembrane trafficking of certain membrane-localized signaling proteins. Both the actin depolymerization drug latrunculin B and the actin stabilization drug jasplakinolide rapidly disrupted transport of secretory vesicles, but each drug caused distinct responses on different endosomal populations labeled by ARA6 or ARA7, indicating that a dynamic actin cytoskeleton was critical for some steps in endomembrane trafficking. Our results provide evidence of cross talk between endomembrane trafficking and the actin cytoskeleton in pollen tubes.

A collection of Ds insertional mutants associated with defects in male gametophyte development and function in Arabidopsis thaliana.

Functional analyses of the Arabidopsis genome require analysis of the gametophytic generation, since approximately 10% of the genes are expressed in the male gametophyte and approximately 9% in the female gametophyte. Here we describe the genetic and molecular characterization of 67 Ds insertion lines that show reduced transmission through the male gametophyte. About half of these mutations are male gametophytic-specific mutations, while the others also affect female transmission. Genomic sequences flanking both sides of the Ds element were recovered for 39 lines; for 16 the Ds elements were inserted in or close to coding regions, while 7 were located in intergenic/unannotated regions of the genome. For the remaining 16 lines, chromosomal rearrangements such as translocations or deletions, ranging between 30 and 500 kb, were associated with the transposition event. The mutants were classified into five groups according to the developmental processes affected; these ranged from defects in early stages of gametogenesis to later defects affecting pollen germination, pollen tube growth, polarity or guidance, or pollen tube-embryo sac interactions or fertilization. The isolated mutants carry Ds insertions in genes with diverse biological functions and potentially specify new functions for several unannotated or unknown proteins.

The pollen receptor kinase LePRK2 mediates growth-promoting signals and positively regulates pollen germination and tube growth.

In flowering plants, the process of pollen germination and tube growth is required for successful fertilization. A pollen receptor kinase from tomato (Solanum lycopersicum), LePRK2, has been implicated in signaling during pollen germination and tube growth as well as in mediating pollen (tube)-pistil communication. Here we show that reduced expression of LePRK2 affects four aspects of pollen germination and tube growth. First, the percentage of pollen that germinates is reduced, and the time window for competence to germinate is also shorter. Second, the pollen tube growth rate is reduced both in vitro and in the pistil. Third, tip-localized superoxide production by pollen tubes cannot be increased by exogenous calcium ions. Fourth, pollen tubes have defects in responses to style extract component (STIL), an extracellular growth-promoting signal from the pistil. Pollen tubes transiently overexpressing LePRK2-fluorescent protein fusions had slightly wider tips, whereas pollen tubes coexpressing LePRK2 and its cytoplasmic partner protein KPP (a Rop-GEF) had much wider tips. Together these results show that LePRK2 positively regulates pollen germination and tube growth and is involved in transducing responses to extracellular growth-promoting signals.

Exportin1 genes are essential for development and function of the gametophytes in Arabidopsis thaliana.

Gametes are produced in plants through mitotic divisions in the haploid gametophytes. We investigated the role of EXPORTIN1 (XPO1) genes during the development of both female and male gametophytes of Arabidopsis. Exportins exclude target proteins from the nucleus and are also part of a complex recruited at the kinetochores during mitosis. Here we show that double mutants in Arabidopsis XPO1A and XPO1B are gametophytic defective. In homozygous-heterozygous plants, 50% of the ovules were arrested at different stages according to the parental genotype. Double-mutant female gametophytes of xpo1a-3/+; xpo1b-1/xpo1b-1 plants failed to undergo all the mitotic divisions or failed to complete embryo sac maturation. Double-mutant female gametophytes of xpo1a-3/xpo1a-3; xpo1b-1/+ plants had normal mitotic divisions and fertilization occurred; in most of these embryo sacs the endosperm started to divide but an embryo failed to develop. Distortions in male transmission correlated with the occurrence of smaller pollen grains, poor pollen germination, and shorter pollen tubes. Our results show that mitotic divisions are possible without XPO1 during the haploid phase, but that XPO1 is crucial for the maternal-to-embryonic transition.

Comparative transcriptomics of Arabidopsis sperm cells.

In flowering plants, the two sperm cells are embedded within the cytoplasm of the growing pollen tube and as such are passively transported to the embryo sac, wherein double fertilization occurs upon their release. Understanding the mechanisms and conditions by which male gametes mature and take part in fertilization are crucial goals in the study of plant reproduction. Studies of gene expression in male gametes of maize (Zea mays) and Plumbago and in lily (Lilium longiflorum) generative cells already showed that the previously held view of transcriptionally inert male gametes was not true, but genome-wide studies were lacking. Analyses in the model plant Arabidopsis (Arabidopsis thaliana) were hindered, because no method to isolate sperm cells was available. Here, we used fluorescence-activated cell sorting to isolate sperm cells from Arabidopsis, allowing GeneChip analysis of their transcriptome at a genome-wide level. Comparative analysis of the sperm cell transcriptome with those of representative sporophytic tissues and of pollen showed that sperm has a distinct and diverse transcriptional profile. Functional classifications of genes with enriched expression in sperm cells showed that DNA repair, ubiquitin-mediated proteolysis, and cell cycle progression are overrepresented Gene Ontology categories. Moreover, analysis of the small RNA and DNA methylation pathways suggests that distinct mechanisms might be involved in regulating the epigenetic state of the paternal genome. We identified numerous candidate genes whose involvement in sperm cell development and fertilization can now be directly tested in Arabidopsis. These results provide a roadmap to decipher the role of sperm-expressed proteins.

A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana.

Rop/Rac small GTPases are central to diverse developmental and cellular activities in plants, playing an especially important role in polar growth of pollen tubes. Although it is established that a class of plant-specific RopGEFs promotes the activity of Rop/Rac through the catalytic PRONE (Plant-specific Rop nucleotide exchanger) domain, not much is known about how RopGEF function is controlled to allow a spatiotemporally regulated Rop activity. To understand such a process in pollen, we performed functional analysis with a pollen-specific RopGEF, AtRopGEF12. Overexpression of AtRopGEF12 had minimal phenotypic effects, whereas overexpression of a C-terminally truncated version disturbed tube growth, suggesting that the C terminus was inhibitory to GEF function. In contrast to non-pollen-expressed RopGEFs, pollen-expressed RopGEFs have conserved C termini. A phospho-mimicking mutation at an invariant serine within the C terminus of AtRopGEF12 resulted in loss of the C-terminal inhibition, suggesting that phosphorylation regulates GEF activity in vivo. The PRONE domain of AtRopGEF12 (PRONE12) was not sufficient to induce isotropic tube growth. We used mbSUS to show that AtRopGEF12 interacts with an Arabidopsis pollen receptor kinase AtPRK2a through its C terminus, and BiFC to show that they interact in pollen tubes. Coexpression of AtRopGEF12 and AtPRK2a caused isotropic growth reminiscent of that seen upon overexpression of a constitutively active (CA) Rop. Coexpression of AtPRK2a with an N-terminally truncated AtRopGEF12 did not induce isotropic growth, indicating a positive role for the N-terminal domain. Our results suggest a mechanism by which the noncatalytic domains of pollen-specific/enriched RopGEFs regulate PRONE function, leading to polarized pollen tube growth.

Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana.

Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen has been elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 80% and pollen tube lengths of hundreds of microns, with both Columbia and Landsberg erecta (Ler) ecotypes. We found that pollen germination and tube growth were dependent on pollen density in both liquid and solid medium. Pollen germination rates were not substantially affected by flower or plant age. The quartet1 mutation negatively affected pollen germination, especially in the Ler ecotype. This protocol will facilitate functional analyses of insertional mutants affecting male gametophyte function, and should allow detailed gene expression analyses during pollen tube growth. Arabidopsis thaliana can now be included on the list of plant species that are suitable models for physiological studies of pollen tube elongation and tip growth.

Proteome mapping of mature pollen of Arabidopsis thaliana.

The male gametophyte of Arabidopsis is a three-celled pollen grain that is thought to contain almost all the mRNAs needed for germination and rapid pollen tube growth. We generated a reference map of the Arabidopsis mature pollen proteome by using multiple protein extraction techniques followed by 2-DE and ESI-MS/MS. We identified 135 distinct proteins from a total of 179 protein spots. We found that half of the identified proteins are involved in metabolism (20%), energy generation (17%), or cell structure (12%); these percentages are similar to those determined for the pollen transcriptome and this similarity is consistent with the idea that in addition to the mRNAs, the mature pollen grain contains proteins necessary for germination and rapid pollen tube growth. We identified ten proteins of unknown function, three of which are flower- or pollen-specific, and we identified nine proteins whose RNAs were absent from the transcriptome, seven of which are involved in metabolism, energy generation, or cell wall structure. Our work complements and extends recent analyses of the pollen transcriptome.

Green sperm. Identification of male gamete promoters in Arabidopsis.

Previously, in an effort to better understand the male contribution to fertilization, we completed a maize (Zea mays) sperm expressed sequence tag project. Here, we used this resource to identify promoters that would direct gene expression in sperm cells. We used reverse transcription-polymerase chain reaction to identify probable sperm-specific transcripts in maize and then identified their best sequence matches in the Arabidopsis (Arabidopsis thaliana) genome. We tested five different Arabidopsis promoters for cell specificity, using an enhanced green fluorescent protein reporter gene. In pollen, the AtGEX1 (At5g55490) promoter is active in the sperm cells and not in the progenitor generative cell or in the vegetative cell, but it is also active in ovules, roots, and guard cells. The AtGEX2 (At5g49150) promoter is active only in the sperm cells and in the progenitor generative cell, but not in the vegetative cell or in other tissues. A third promoter, AtVEX1 (At5g62850) [corrected] was active in the vegetative cell during the later stages of pollen development; the other promoters tested (At1g66770 and At1g73350) did not function in pollen. Comparisons among GEX1 and GEX2 homologs from maize, rice (Oryza sativa), Arabidopsis, and poplar (Populus trichocarpa) revealed a core binding site for Dof transcription factors. The AtGEX1 and AtGEX2 promoters will be useful for manipulating gene expression in sperm cells, for localization and functional analyses of sperm proteins, and for imaging of sperm dynamics as they are transported in the pollen tube to the embryo sac.

Is there more than one way to attract a pollen tube?

ZmEA1 (Zea mays egg apparatus 1) is expressed only in the egg and synergid cells. Embryo sacs with presumed reduced expression of ZmEA1 fail to attract pollen tubes. Together with data from Arabidopsis mutants and from elegant laser ablation experiments in Torenia fournieri, these results indicate that embryo sacs send signals to the incoming pollen tubes. We need to decipher how such signals are perceived and determine if the signals are species-specific.