CAND1 is required for pollen viability in Arabidopsis thaliana-a test of the adaptive exchange hypothesis.

The dynamic assembly of SKP1•CUL1•F-box protein (SCF) ubiquitin ligases is important for protein ubiquitination and degradation. This process is enabled by CAND1, which exchanges F-box proteins associated with the common CUL1 scaffold, and thereby, recycles the limited CUL1 core and allows diverse F-box proteins to assemble active SCFs. Previous human cell biological and computational studies have led to the adaptive exchange hypothesis, which suggests that the CAND1-mediated exchange confers plasticity on the SCF system, allowing cells to tolerate large variations in F-box protein expression. Here, we tested this hypothesis using Arabidopsis thaliana, a multicellular organism expressing hundreds of F-box protein genes at variable levels in different tissues. The cand1 null mutant in Arabidopsis is viable but produce almost no seeds. Bioinformatic, cell biological, and developmental analyses revealed that the low fertility in the cand1 mutant is associated with cell death in pollen, where the net expression of F-box protein genes is significantly higher than any other Arabidopsis tissue. In addition, we show that the transmission efficiency of the cand1 null allele was reduced through the male but not the female gametophyte. Our results suggest that CAND1 activity is essential in cells or tissues expressing high levels of F-box proteins. This finding is consistent with the proposed adaptive exchange hypothesis, demonstrating the necessity of the evolutionarily conserved CAND1-mediated exchange system in the development of a multicellular organism.

Infective Aortic Valve Endocarditis in a Patient With Mixed Connective Tissue Disease.

Mixed connective tissue disease (MCTD) is a rare autoimmune disorder that is characterized by overlapping clinical features of systemic lupus erythematosus (SLE), scleroderma, and myositis. Both SLE and mixed connective tissue disease patients are more prone to have acute endocarditis, and immunosuppression is a risk factor for recurrence of infective endocarditis. We present the case of a 53-year-old female with mixed connective tissue disease presenting with interstitial lung disease and precapillary pulmonary hypertension. The patient was chronically medicated with prednisolone, mycophenolate mofetil, and hydroxychloroquine. She was admitted for Enterococcus faecalis infective endocarditis and was treated with a four-week course of ceftriaxone and ampicillin. Immunosuppressive chronic medication was maintained due to severe lung involvement. One month later, the patient was re-admitted due to respiratory infection with identification of influenza B virus. However, fever persisted for over one week, and subsequent relapse of the E. faecalis infective endocarditis was found. The diagnosis was made based on blood cultures and a transoesophageal echocardiogram. No other focus of infection was identified. She completed a six-week course of vancomycin and gentamicin and underwent cardiac surgery with success. This case highlights the difficulty of the management of immunosuppressed patients in the presence of serious infections.

Bone Marrow Granulomatosis in Acute Q Fever.

Reported cases of Q fever in people living in urban areas after occasional contact with farm animals or infected pets such as dogs and cats have been increasing. The diagnosis of Q fever is usually laborious due to unspecific and variable clinical manifestations. The most common clinical presentation is an influenza-like illness with varying degrees of pneumonia and hepatitis. Acute hepatitis is more frequent than pneumonia in countries where the disease is endemic, such as in Portugal. We report a case of acute Q fever with hepatic and bone marrow involvement presented as fever of unknown origin (FUO) in a 56-year-old sportive hunter man. Typical fibrin ring granulomas (doughnut granulomas) were found in the bone marrow biopsy and were essential for the diagnosis. Bone marrow involvement is considered a rare manifestation of Q fever. Coxiella infection activates a granulomatous inflammatory response that can lead to persistent immune cell activation. Doughnut granulomas are not pathognomonic but they are highly specific for the diagnosis of Q fever.

Copper-induced Haemolytic Anaemia.

Acute copper toxicity is uncommon in Western countries and is often the result of accidental consumption or a suicide attempt. We report the case of a 65-year-old man presenting to the accident and emergency department after a suicide attempt with ingestion of Bordeaux mixture, ibuprofen, acetaminophen and bleach. Primary evaluation showed caustic oesophagitis, toxic hepatitis and acute renal injury, which were treated with supportive care. During admission, he developed a non-immune haemolytic anaemia associated with high levels of copper in urine and blood. Chelation treatment with penicillamine was started and evolution was favourable after 1 month of treatment. Copper poisoning can be lethal. Prompt diagnosis and treatment are key for a favourable prognosis. LEARNING POINTS: Acute copper intoxication is rare and early clinical suspicion and diagnosis are essential to reduce mortality.The diagnosis of copper poisoning should be based on clinical presentation and measurement of urine and blood copper levels in addition to serum ceruloplasmin levels.Treatment includes reduction of absorption, supportive measures, management of complications and chelation therapy.

Muscle Infarction Following Rapid Glycemic Control in a Patient With Diabetes-Associated Microvascular Disease.

We report a case of a 40-year-old African male with a history of diabetes mellitus with multiple microvascular complications, having recently initiated insulin treatment with a rapid decline in glycosylated hemoglobin (HbA1c) concentration. The patient presented with a sudden onset of right thigh pain and swelling not associated with trauma. Blood work revealed elevated inflammatory markers. A presumptive diagnosis of pyomyositis was made and the patient was treated with intravenous antibiotics with no improvement. Diabetic muscle infarction was then considered and confirmed by magnetic resonance imaging of the affected thigh. As with retinopathy and neuropathy deterioration that have been described as secondary to an aggressive glycemic control, it is possible that muscle myonecrosis may have been consequent to the rapid HbA1c normalization.

Native Valve Aspergillus Endocarditis in a Non-Neutropenic Immunocompromised Patient on Anti-TNF α Blockers Therapy.

Invasive aspergillosis is a rare opportunistic infection mainly occurring in patients with a well-established risk such as neutropenia or conditions that lead to chronically impaired cellular immune responses. Systemic corticosteroids are a well-known risk factor for fungal infections. Recently, reports of invasive aspergillosis in patients treated with monoclonal biologic agents, such as tumor necrosis factor-alpha inhibitors, have been increasing. We present the case of a 47-year-old female patient with seronegative spondyloarthropathy treated with infliximab and corticosteroids. The patient presented classical symptoms of an acute lower respiratory infection, and she was treated with a ß-lactam antibiotic. Infliximab administration was deferred until nine days after clinical recovery. Fourteen days after drug administration, she was admitted with a symptomatic subcortical hematoma in the left parietal region. There was a rapid neurological recovery, and there were no risk factors for haemorrhagic stroke detected. The chest X-ray revealed an oval mass with an air crescent sign, and the CT scan was suggestive of aspergilloma. Bronchoalveolar lavage cytology identified Aspergillus spp. Voriconazole was initiated and, after one month of treatment, the patient was readmitted with a left facial palsy associated with hemiparesis and dysarthria. Laboratory evaluation showed leukocytosis and elevated C-reactive protein. A severe right middle cerebral artery stroke was present on the brain CT scan. Transesophageal echocardiogram revealed large mitral valve vegetation, and the diagnosis of Aspergillus endocarditis with cerebral embolization was made. Fungal infections are challenging due to the diagnosis infrequency and paucisymptomatic natural history. Despite being crucial in the treatment of autoimmune diseases, immunosuppressive drugs increase the risk of fungal infection. It is extremely important to consider Aspergillus infection in immunosuppressed patients, and the need for prophylaxis in non-neutropenic patients with risk factors should be clarified.

AtNOT1 is required for gametophyte development in Arabidopsis.

In flowering plants, pollen development is under a dynamic and well-orchestrated transcriptional control, characterized by an early phase with high transcript diversity and a late post-mitotic phase skewed to a cell-type-specific transcriptome. Such transcriptional changes require a balance between synthesis and degradation of mRNA transcripts, the latter being initiated by deadenylation. The CCR4-NOT complex is the main evolutionary conserved deadenylase complex in eukaryotes, and its function is essential during germline specification in animals. We hypothesized that the CCR4-NOT complex might play a central role in mRNA turnover during microgametogenesis in Arabidopsis. Disruption of NOT1 gene, which encodes the scaffold protein of the CCR4-NOT complex, showed abnormal seed set. Genetic analysis failed to recover homozygous progeny, and reciprocal crosses confirmed reduced transmission through the male and female gametophytes. Concordantly, not1 embryo sacs showed delayed development and defects in embryogenesis. not1 pollen grains exhibited abnormal male germ unit configurations and failed to germinate. Transcriptome analysis of pollen from not1/+ mutants revealed that lack of NOT1 leads to an extensive transcriptional deregulation during microgametogenesis. Therefore, our work establishes NOT1 as an important player during gametophyte development in Arabidopsis.

Targeted reprogramming of H3K27me3 resets epigenetic memory in plant paternal chromatin.

Epigenetic marks are reprogrammed in the gametes to reset genomic potential in the next generation. In mammals, paternal chromatin is extensively reprogrammed through the global erasure of DNA methylation and the exchange of histones with protamines1,2. Precisely how the paternal epigenome is reprogrammed in flowering plants has remained unclear since DNA is not demethylated and histones are retained in sperm3,4. Here, we describe a multi-layered mechanism by which H3K27me3 is globally lost from histone-based sperm chromatin in Arabidopsis. This mechanism involves the silencing of H3K27me3 writers, activity of H3K27me3 erasers and deposition of a sperm-specific histone, H3.10 (ref. 5), which we show is immune to lysine 27 methylation. The loss of H3K27me3 facilitates the transcription of genes essential for spermatogenesis and pre-configures sperm with a chromatin state that forecasts gene expression in the next generation. Thus, plants have evolved a specific mechanism to simultaneously differentiate male gametes and reprogram the paternal epigenome.

Plant Evolution: What Does It Take To Be an Egg?

The genetic regulation of cell patterning within plant gametophytes remains poorly understood. Now, two new studies in the liverwort Marchantia polymorpha shed light on the conserved function of an RKD transcription factor as a key regulator of egg cell fate in the land plant lineage.

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.

Arabidopsis tetraspanins are confined to discrete expression domains and cell types in reproductive tissues and form homo- and heterodimers when expressed in yeast.

Tetraspanins are evolutionary conserved transmembrane proteins present in all multicellular organisms. In animals, they are known to act as central organizers of membrane complexes and thought to facilitate diverse biological processes, such as cell proliferation, movement, adhesion, and fusion. The genome of Arabidopsis (Arabidopsis thaliana) encodes 17 members of the tetraspanin family; however, little is known about their functions in plant development. Here, we analyzed their phylogeny, protein topology, and domain structure and surveyed their expression and localization patterns in reproductive tissues. We show that, despite their low sequence identity with metazoan tetraspanins, plant tetraspanins display the typical structural topology and most signature features of tetraspanins in other multicellular organisms. Arabidopsis tetraspanins are expressed in diverse tissue domains or cell types in reproductive tissues, and some accumulate at the highest levels in response to pollination in the transmitting tract and stigma, male and female gametophytes and gametes. Arabidopsis tetraspanins are preferentially targeted to the plasma membrane, and they variously associate with specialized membrane domains, in a polarized fashion, to intercellular contacts or plasmodesmata. A membrane-based yeast (Saccharomyces cerevisiae) two-hybrid system established that tetraspanins can physically interact, forming homo- and heterodimer complexes. These results, together with a likely genetic redundancy, suggest that, similar to their metazoan counterparts, plant tetraspanins might be involved in facilitating intercellular communication, whose functions might be determined by the composition of tetraspanin complexes and their binding partners at the cell surface of specific cell types.

FACS-based purification of Arabidopsis microspores, sperm cells and vegetative nuclei.

BACKGROUND: The male germline in flowering plants differentiates by asymmetric division of haploid uninucleated microspores, giving rise to a vegetative cell enclosing a smaller generative cell, which eventually undergoes a second mitosis to originate two sperm cells. The vegetative cell and the sperm cells activate distinct genetic and epigenetic mechanisms to control pollen tube growth and germ cell specification, respectively. Therefore, a comprehensive characterization of these processes relies on efficient methods to isolate each of the different cell types throughout male gametogenesis. RESULTS: We developed stable transgenic Arabidopsis lines and reliable purification tools based on Fluorescence-Activated Cell Sorting (FACS) in order to isolate highly pure and viable fractions of each cell/nuclei type before and after pollen mitosis. In the case of mature pollen, this was accomplished by expressing GFP and RFP in the sperm and vegetative nuclei, respectively, resulting in 99% pure sorted populations. Microspores were also purified by FACS taking advantage of their characteristic small size and autofluorescent properties, and were confirmed to be 98% pure. CONCLUSIONS: We provide simple and efficient FACS-based purification protocols for Arabidopsis microspores, vegetative nuclei and sperm cells. This paves the way for subsequent molecular analysis such as transcriptomics, DNA methylation analysis and chromatin immunoprecipitation, in the developmental context of microgametogenesis in Arabidopsis.

Whole genome analysis of gene expression reveals coordinated activation of signaling and metabolic pathways during pollen-pistil interactions in Arabidopsis.

Plant reproduction depends on the concerted activation of many genes to ensure correct communication between pollen and pistil. Here, we queried the whole transcriptome of Arabidopsis (Arabidopsis thaliana) in order to identify genes with specific reproductive functions. We used the Affymetrix ATH1 whole genome array to profile wild-type unpollinated pistils and unfertilized ovules. By comparing the expression profile of pistils at 0.5, 3.5, and 8.0 h after pollination and applying a number of statistical and bioinformatics criteria, we found 1,373 genes differentially regulated during pollen-pistil interactions. Robust clustering analysis grouped these genes in 16 time-course clusters representing distinct patterns of regulation. Coregulation within each cluster suggests the presence of distinct genetic pathways, which might be under the control of specific transcriptional regulators. A total of 78% of the regulated genes were expressed initially in unpollinated pistil and/or ovules, 15% were initially detected in the pollen data sets as enriched or preferentially expressed, and 7% were induced upon pollination. Among those, we found a particular enrichment for unknown transcripts predicted to encode secreted proteins or representing signaling and cell wall-related proteins, which may function by remodeling the extracellular matrix or as extracellular signaling molecules. A strict regulatory control in various metabolic pathways suggests that fine-tuning of the biochemical and physiological cellular environment is crucial for reproductive success. Our study provides a unique and detailed temporal and spatial gene expression profile of in vivo pollen-pistil interactions, providing a framework to better understand the basis of the molecular mechanisms operating during the reproductive process in higher plants.

The pollen tube journey in the pistil and imaging the in vivo process by two-photon microscopy.

The process of pollen germination and tube growth in the pistil involves a series of cell-cell interactions, some facilitating fertilization while others prohibiting pollen tube access to the female gametophyte, either because of incompatibility or as a result of mechanisms to avert polyspermy and to ensure reproductive success. Understanding pollen tube growth and guidance to the female gametophyte has long been a pursuit among plant biologists, and observations indicate that diverse strategies may be adopted by different plant species. Recent studies in Arabidopsis, maize, and Torenia fournieri suggest that low molecular weight secretory molecules probably play major roles in the short-range attraction of pollen tubes to the female gametophyte. The process of pollen tube growth in the pistil occurs beneath several cell layers so much of the information that conveys the intimate partnership between penetrating pollen tubes and the female tissues has come from fixed samples and observations of in vitro pollen tube growth responses to female factors. A unique glimpse of the in vivo pollen germination and tube growth process is provided here by intra-vital two-photon excitation (TPE) microscopy of pollinated Arabidopsis pistils that remained on intact plants. Further discoveries of critical factors of male or female origins and how they control the pollen tube growth and fertilization process will broaden our understanding of the common themes and diverse strategies that plants have evolved to ensure reproductive success. The advancement of imaging technology to monitor pollination and fertilization and the development of probes to monitor various aspects of the pollen tube growth process, including pollen intracellular dynamics, will allow us to superimpose details obtained from studying pollen tube growth in culture conditions to interpret and understand the in vivo events.

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.

Truncation of a protein disulfide isomerase, PDIL2-1, delays embryo sac maturation and disrupts pollen tube guidance in Arabidopsis thaliana.

Pollen tubes must navigate through different female tissues to deliver sperm to the embryo sac for fertilization. Protein disulfide isomerases play important roles in the maturation of secreted or plasma membrane proteins. Here, we show that certain T-DNA insertions in Arabidopsis thaliana PDIL2-1, a protein disulfide isomerase (PDI), have reduced seed set, due to delays in embryo sac maturation. Reciprocal crosses indicate that these mutations acted sporophytically, and aniline blue staining and scanning electron microscopy showed that funicular and micropylar pollen tube guidance were disrupted. A PDIL2-1-yellow fluorescent protein fusion was mainly localized in the endoplasmic reticulum and was expressed in all tissues examined. In ovules, expression in integument tissues was much higher in the micropylar region in later developmental stages, but there was no expression in embryo sacs. We show that reduced seed set occurred when another copy of full-length PDIL2-1 or when enzymatically active truncated versions were expressed, but not when an enzymatically inactive version was expressed, indicating that these T-DNA insertion lines are gain-of-function mutants. Our results suggest that these truncated versions of PDIL2-1 function in sporophytic tissues to affect ovule structure and impede embryo sac development, thereby disrupting pollen tube guidance.

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.