First investigation into the genetic control of meiosis in sugarcane.

The sugarcane (Saccharum spp.) genome is one of the most complex of all. Modern varieties are highly polyploid and aneuploid as a result of hybridization between Saccharum officinarum and S. spontaneum. Little research has been done on meiotic control in polyploid species, with the exception of the wheat Ph1 locus harboring the ZIP4 gene (TaZIP4-B2) which promotes pairing between homologous chromosomes while suppressing crossover between homeologs. In sugarcane, despite its interspecific origin, bivalent association is favored, and multivalents, if any, are resolved at the end of prophase I. Thus, our aim herein was to investigate the purported genetic control of meiosis in the parental species and in sugarcane itself. We investigated the ZIP4 gene and immunolocalized meiotic proteins, namely synaptonemal complex proteins Zyp1 and Asy1. The sugarcane ZIP4 gene is located on chromosome 2 and expressed more abundantly in flowers, a similar profile to that found for TaZIP4-B2. ZIP4 expression is higher in S. spontaneum a neoautopolyploid, with lower expression in S. officinarum, a stable octoploid species. The sugarcane Zip4 protein contains a TPR domain, essential for scaffolding. Its 3D structure was also predicted, and it was found to be very similar to that of TaZIP4-B2, reflecting their functional relatedness. Immunolocalization of the Asy1 and Zyp1 proteins revealed that S. officinarum completes synapsis. However, in S. spontaneum and SP80-3280 (a modern variety), no nuclei with complete synapsis were observed. Importantly, our results have implications for sugarcane cytogenetics, genetic mapping, and genomics.

Ten Approaches That Improve Immunostaining: A Review of the Latest Advances for the Optimization of Immunofluorescence.

Immunostaining has emerged as one of the most common and valuable techniques that allow the localization of proteins at a quantitative level within cells and tissues using antibodies coupled to enzymes, fluorochromes, or colloidal nanogold particles. The application of fluorochromes during immunolabeling is referred to as immunofluorescence, a method coupled to widefield or confocal microscopy and extensively applied in basic research and clinical diagnosis. Notwithstanding, there are still disadvantages associated with the application of this technique due to technical challenges in the process, such as sample fixation, permeabilization, antibody incubation times, and fluid exchange, etc. These disadvantages call for continuous updates and improvements to the protocols extensively described in the literature. This review contributes to protocol optimization, outlining 10 current methods for improving sample processing in different stages of immunofluorescence, including a section with further recommendations. Additionally, we have extended our own antibody signal enhancer method, which was reported to significantly increase antibody signals and is useful for cervical cancer detection, to improve the signals of fluorochrome-conjugated staining reagents in fibrous tissues. In summary, this review is a valuable tool for experienced researchers and beginners when planning or troubleshooting the immunofluorescence assay.

Costain 1 (ARM19800.1) - The first identified protein of the costa of the pathogenic protozoan Tritrichomonas foetus.

Protists members of the Trichomonadidae and Tritrichomonadidae families include agents of trichomoniasis that constitute important parasitic diseases in humans and in animals of veterinary interest. One of the characteristic features of these eukaryotic microorganisms is that they contain a fibrous structure known as the costa as an important cytoskeleton structure, that differs in several aspects from other cytoskeleton structures found in eukaryotic cells. Previous proteomic analysis of an enriched costa fraction revealed the presence of several hypothetical proteins. Here we describe the localization of one of the most prevalent protein found in this previously made proteomic assay to confirm its presence in the costa of Tritrichomonas foetus. A peptide sequence of the hypothetical protein ARM19800.1 was selected for the production of specific polyclonal antibodies and its specificity was confirmed by Western Blotting using an enriched costa fraction. Next, the specific localization of the selected protein was evaluated by immunofluorescence and electron microscopy immunocytochemistry. Our observations clearly showed that the ARM 19800.1 protein is indeed localized in the costa and displays an almost periodic labeling pattern. Since this is the first protein identified in the costa, it was designated as costain 1. A better understanding of a structure as peculiar as the costa is of great biological and evolutionary importance due to the fact that it contains unique proteins, it may represent a possible chemotherapy target and it may correspond to antigens of interest in immunodiagnosis and/or vaccine development.

Expression and distribution of glutathione transferases in protoscoleces of Echinococcus granulosus sensu lato.

Glutathione transferases (GSTs) belong to a diverse superfamily of multifunctional proteins involved in metabolic detoxification. In helminth parasite, GSTs are particularly relevant since they are also involved in host immunomodulation. Echinococcus granulosus sensu lato (s.l.) is a cestode parasite known to express at least three phylogenetically distant cytosolic GSTs: EgGST1 and EgGST2 previously grouped within Mu and Sigma classes, respectively; and EgGST3 related to both Omega and Sigma classes. To better characterize E. granulosus s.l. GSTs, herein their expression and distribution were assessed in the pre-adult protoscolex (PSC) parasite stage. Potential transcriptional regulatory mechanisms of the corresponding EgGST genes were also explored. Firstly, the transcription of the three EgGSTs was significantly induced during the early stages of the murine model of infection, suggesting a potential role during parasite establishment. EgGST1 was detected in the parenchyma of PSCs and its expression increased after H2O2 exposure, supporting its role in detoxification. EgGST2 was mainly detected on the PSCs tegument, strategically localized for potential immunoregulation functions due to its Sigma-class characteristics. In addition, its expression increased after anthelmintic treatment, suggesting a role in chemotherapy resistance. Finally, the Omega-related EgGST3 was localized throughout the entire PSC body, including suckers and tegument, and since its expression also increased after H2O2 treatment, a potential role in oxidative stress response could also be ascribed. On the other hand, known cis-acting regulatory motifs were detected in EgGST genes, suggesting similar transcription processes to other eukaryotes. The results herein reported provide additional data regarding the roles of EgGSTs in E. granulosus s.l. biology, contributing to a better understanding of its host-parasite interaction.

Trypanosoma cruzi Presenilin-Like Transmembrane Aspartyl Protease: Characterization and Cellular Localization.

The increasing detection of infections of Trypanosoma cruzi, the etiological agent of Chagas disease, in non-endemic regions beyond Latin America has risen to be a major public health issue. With an impact in the millions of people, current treatments rely on antiquated drugs that produce severe side effects and are considered nearly ineffective for the chronic phase. The minimal progress in the development of new drugs highlights the need for advances in basic research on crucial biochemical pathways in T. cruzi to identify new targets. Here, we report on the T. cruzi presenilin-like transmembrane aspartyl enzyme, a protease of the aspartic class in a unique phylogenetic subgroup with T. vivax separate from protozoans. Computational analyses suggest it contains nine transmembrane domains and an active site with the characteristic PALP motif of the A22 family. Multiple linear B-cell epitopes were identified by SPOT-synthesis analysis with Chagasic patient sera. Two were chosen to generate rabbit antisera, whose signal was primarily localized to the flagellar pocket, intracellular vesicles, and endoplasmic reticulum in parasites by whole-cell immunofluorescence. The results suggest that the parasitic presenilin-like enzyme could have a role in the secretory pathway and serve as a target for the generation of new therapeutics specific to the T. cruzi.

Biochemical Characterization and Allosteric Modulation by Magnesium of (Na+, K+)-ATPase Activity in the Gills of the Red Mangrove Crab Goniopsis cruentata (Brachyura, Grapsidae).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the grapsid crab Goniopsis cruentata. (Na+, K+)-ATPase activity constitutes 95% of total ATPase activity, and sucrose density centrifugation reveals an ATPase activity peak between 25 and 35% sucrose, distributed into two, partially separated protein fractions. The (Na+, K+)-ATPase α-subunit is localized throughout the ionocyte cytoplasm and has an Mr of ≈ 10 kDa and hydrolyzes ATP obeying cooperative kinetics. Low (VM = 186.0 ± 9.3 nmol Pi min-1 mg-1 protein and K0.5 = 0.085 ± 0.004 mmol L-1) and high (VM = 153.4 ± 7.7 nmol Pi min-1 mg-1 protein and K0.5 = 0.013 ± 0.0006 mmol L-1) affinity ATP binding sites were characterized. At low ATP concentrations, excess Mg2+ stimulates the enzyme, triggering exposure of a high-affinity binding site that accounts for 50% of (Na+, K+)-ATPase activity. Stimulation by Mg2+ (VM = 425.9 ± 25.5 nmol Pi min-1 mg-1 protein, K0.5 = 0.16 ± 0.01 mmol L-1), K+ (VM = 485.3 ± 24.3 nmol Pi min-1 mg-1 protein, K0.5 = 0.9 ± 0.05 mmol L-1), Na+ (VM = 425.0 ± 23.4 nmol Pi min-1 mg-1 protein, K0.5 = 5.1 ± 0.3 mmol L-1) and NH4+ (VM = 497.9 ± 24.9 nmol Pi min-1 mg-1 protein, K0.5 = 9.7 ± 0.5 mmol L-1) obeys cooperative kinetics. Ouabain inhibits up to 95% of ATPase activity with KI = 196.6 ± 9.8 µmol L-1. This first kinetic characterization of the gill (Na+, K+)-ATPase in Goniopsis cruentata enables better comprehension of the biochemical underpinnings of osmoregulatory ability in this semi-terrestrial mangrove crab.

Whole-Mount Immunolocalization Procedure for Plant Female Meiocytes.

Here we present an optimized protocol for immunolocalization of meiotic proteins during female meiosis in whole mount tissues. It ensures ovule morphology integrity and homogeneous reagent penetration. The method relies on paraformaldehyde tissue fixation, polyacrylamide embedding, tissue permeabilization, antibody incubation, counterstaining, and confocal microscopy analysis. This protocol has been used in diverse Arabidopsis ecotypes and in the legume Vigna unguiculata.

Distribution of Taenia solium Diagnostic Glycoproteins in the Different Developmental Stages of the Parasite.

Taenia solium is a helminth parasite that causes 2 diseases in humans: cysticercosis and taeniasis. The establishment of T. solium metacestodes in the central nervous system causes neurocysticercosis, while development of the adult tapeworm in the small intestine causes taeniasis. Serological diagnosis of neurocysticercosis is performed by Western blot with an enriched fraction of glycoproteins that has been extensively used for clinical diagnosis and epidemiological surveys. The lectin-bound fraction that is used for this assay contains 7 antigenic glycoproteins. These antigenic proteins are considered to be highly specific for cysticercosis when tested with heterologous parasitic diseases. However, recent studies show that people with taeniasis have cross-reactive antibodies against the neurocysticercosis diagnostic glycoproteins and vice versa. Nevertheless, it is not known if these diagnostic proteins are expressed in the adult stage of the parasite. In this paper, we describe the location of 3 of these glycoproteins in T. solium adults and cysticerci using polyclonal antibodies raised against a synthetic peptide based on the amino acid sequence of TS14, a recombinant protein T24H, and the native GP50. The glycoproteins' distribution was different in invaginated and evaginated cysticerci as well as in adult tapeworms. Specifically, the 3 glycoproteins studied were differentially expressed during embryogenesis. Our findings indicate that expression of the diagnostic glycoproteins is developmentally regulated; this is noteworthy since these glycoproteins are considered specific for the diagnosis of neurocysticercosis but nevertheless are present in different structures throughout the development of T. solium. Here we describe the glycoprotein expression and localization, which can be important in understanding their biological functions. In addition, our results help clarify the cross-reaction observed between people with neurocysticercosis and taeniasis to TS14, T24H, and GP50, which are used as diagnostic antigens for neurocysticercosis.

Immunolocalization to Study ARGONAUTE Proteins in Developing Ovules of the Brassicaceae.

Determining the in situ pattern of protein expression is crucial to accurately establish regulatory function and mode of action of any plant developmental program. Here, we describe two immunolocalization procedures that are consistently used to determine subcellular localization of ARGONAUTE proteins in the ovule of the Brassicaceae. The first is performed in resin-embedded semi-thin sections of developing ovules that can be observed under bright-field microscopy. The second is based in polyacrylamide immersion of complete (whole-mounted) gynoecia or ovules that are observed under confocal microscopy. Both procedures have been successfully performed to localize proteins involved in RNA-directed DNA methylation during the development of the anatropous bitegmic ovule in Arabidopsis, Brassica, or Boechera species.

Contrasting pectin polymers in guard cell walls of Arabidopsis and the hornwort Phaeoceros reflect physiological differences.

BACKGROUND AND AIMS: In seed plants, stomata regulate CO2 acquisition and water relations via transpiration, while minimizing water loss. Walls of guard cells are strong yet flexible because they open and close the pore by changing shape over the substomatal cavity. Pectins are necessary for wall flexibility and proper stomata functioning. This study investigates the differences in pectin composition in guard cells of two taxa that represent key lineages of plants with stomata: Arabidopsis, an angiosperm with diurnal stomatal activity, and Phaeoceros, a bryophyte that lacks active stomatal movement. METHODS: Using immunolocalization techniques in transmission electron microscopy, this study describes and compares the localization of pectin molecule epitopes essential to stomata function in guard cell walls of Arabidopsis and Phaeoceros. KEY RESULTS: In Arabidopsis, unesterified homogalacturonans very strongly localize throughout guard cell walls and are interspersed with arabinan pectins, while methyl-esterified homogalacturonans are restricted to the exterior of the wall, the ledges and the junction with adjacent epidermal cells. In contrast, arabinans are absent in Phaeoceros, and both unesterified and methyl-esterified homogalacturonans localize throughout guard cell walls. CONCLUSIONS: Arabinans and unesterified homogalacturonans are required for wall flexibility, which is consistent with active regulation of pore opening in Arabidopsis stomata. In contrast, the lack of arabinans and high levels of methyl-esterified homogalacturonans in guard cell walls of Phaeoceros are congruent with the inability of hornwort stomata to open and close with environmental change. Comparisons across groups demonstrate that variations in guard cell wall composition reflect different physiological activity of stomata in land plants.

Localization and phosphorylation of Plasmodium falciparum nicotinamide/nicotinate mononucleotide adenylyltransferase (PfNMNAT) in intraerythrocytic stages.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD+) is an essential molecule in the energy metabolism of living beings, and it has various cellular functions. The main enzyme in the biosynthesis of this nucleotide is nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) because it is the convergence point for all known biosynthetic pathways. NMNATs have divergences in both the number of isoforms detected and their distribution, depending on the organism. METHODS: In the laboratory of basic research in biochemistry (LIBBIQ: acronym in Spanish) the NMNATs of protozoan parasites (Leishmania braziliensis, Plasmodium falciparum, Trypanosoma cruzi, and Giardia duodenalis) have been studied, analysing their catalytic properties through the use of proteins. Recombinants and their cellular distribution essentially. In 2014, O'Hara et al. determined the cytoplasmic localization of NMNAT of P. falciparum, using a transgene coupled to GFP, however, the addition of labels to the study protein can modify several of its characteristics, including its sub-cellular localization. RESULTS: This study confirms the cytoplasmic localization of this protein in the parasite through recognition of the endogenous protein in the different stages of the asexual life cycle. Additionally, the study found that PfNMNAT could be a phosphorylation target at serine, tyrosine and threonine residues, and it shows variations during the asexual life cycle. CONCLUSIONS: These experiments confirmed that the parasite is situated in the cytoplasm, fulfilling the required functions of NAD+ in this compartment, the PfNMNAT is regulated in post-transcription processes, and can be regulated by phosphorylation in its residues.

Localization of Chromatin Marks in Arabidopsis Early Embryos.

During early embryo development, profound changes in chromatin structure and regulation take place. It is difficult to study these changes in plant embryos however, largely because of their relative inaccessibility, which impedes the application of current epigenomic and biochemistry protocols. To circumvent this issue and to analyze the epigenetic status of the embryo at both the cellular and subcellular level, we describe here a simple method to immunolocalize chromatin marks in whole mount early Arabidopsis embryos, either within maternal tissues or isolated from seeds. We show that this protocol can be combined with fluorescent protein markers, allowing for the simultaneous detection of several chromatin components and/or cell fate markers. This new protocol will facilitate deciphering the epigenetic circuits controlling early embryogenesis in plants.

Imunolocalização de receptores de leptina no ovário de preás (Galea spixii Wagler, 1831) / Immunolocalization of leptin receptor of Spix's Yellow-toothed Cavy (Galea spixii Wagler, 1831) ovary

A leptina, uma citocina produzida pelas células adiposas, é alvo da comunidade científica por acreditarem que ela apresente impacto sobre a reprodução dos animais promovendo a puberdade, foliculogênese e oogênese, ciclo estral e auxiliando na fecundação. A compreensão dos mecanismos que controlam a atividade reprodutiva de preás (Galea spixii) possui papel relevante para a preservação da espécie. Desta forma, o presente trabalho propôs analisar a imunolocalização dos receptores de leptina (Ob-R) no ovário de preás. Coletaram-se os ovários de 20 fêmeas adultas, não prenhes e saudáveis. As amostras foram fixadas em paraformaldeído a 4% em tampão fosfato, incluídas em parafina e seccionadas para a realização de imunohistoquímica (IHC). As secções foram fotomicrografadas e avaliadas quanto à intensidade da reação. Observou-se forte imunorreação no oócito e nas células da teca, moderada nas células do estroma ovariano e nas células luteínicas grandes e fracamente coradas nas células da granulosa, endoteliais, perivasculares e células luteínicas pequenas. Quando comparado a expressão de receptores ao longo do desenvolvimento folicular foi observado que o oócito e as células da teca se mantiveram com expressão na mesma intensidade. Entretanto, as células da granulosa apresentaram forte marcação nos estádios pré-antrais enquanto que nos folículos antrais apresentou fraca intensidade. Concluímos que em ovários de Galea spixii existe a presença de Ob-R nas principais estruturas do ovário sugerindo que este hormônio desempenhe papel fundamental na reprodução desta espécie.

Leptin, a cytokine produced by adipose cells, is the target of the scientific community for believing that it has an impact on the reproduction of the animals promoting puberty, folliculogenesis and oogenesis, estrous cycle and aiding in fertilization. The understanding of the mechanisms controlling the reproductive activity of Spix's Yellow-toothed Cavy (Galea spixii) plays a relevant role in the preservation of the species. Thus, the present study proposed to analyze the immunolocalization of leptin receptors (Ob-R) in the ovary of cavies. Ovaries from 20 adult, non-pregnant, healthy females were collected. The samples were fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and sectioned for immunohistochemistry. The sections were photomicrographs and intensity of the reaction was measured. Strong immunoreaction was observed in oocyte and theca cells, moderate in ovarian stromal cells and large luteal cells and weak stained in granulosa, endothelial, perivascular and small luteal cells. When compared to receptor expression along follicular development it was observed that the oocyte and the theca cells remained with expression at the same intensity. However, the granulosa cells presented strong stained in the preantral stages, whereas in the antral follicles it presented low intensity. We conclude that in the ovaries of Galea spixii there is the presence of Ob-R in the main structures of the ovary sugesting that this hormone plays a fundamental role in the reproduction of this species.

Imunolocalização de receptores de leptina no ovário de preás (Galea spixii Wagler, 1831) / Immunolocalization of leptin receptor of Spix's Yellow-toothed Cavy (Galea spixii Wagler, 1831) ovary

A leptina, uma citocina produzida pelas células adiposas, é alvo da comunidade científica por acreditarem que ela apresente impacto sobre a reprodução dos animais promovendo a puberdade, foliculogênese e oogênese, ciclo estral e auxiliando na fecundação. A compreensão dos mecanismos que controlam a atividade reprodutiva de preás (Galea spixii) possui papel relevante para a preservação da espécie. Desta forma, o presente trabalho propôs analisar a imunolocalização dos receptores de leptina (Ob-R) no ovário de preás. Coletaram-se os ovários de 20 fêmeas adultas, não prenhes e saudáveis. As amostras foram fixadas em paraformaldeído a 4% em tampão fosfato, incluídas em parafina e seccionadas para a realização de imunohistoquímica (IHC). As secções foram fotomicrografadas e avaliadas quanto à intensidade da reação. Observou-se forte imunorreação no oócito e nas células da teca, moderada nas células do estroma ovariano e nas células luteínicas grandes e fracamente coradas nas células da granulosa, endoteliais, perivasculares e células luteínicas pequenas. Quando comparado a expressão de receptores ao longo do desenvolvimento folicular foi observado que o oócito e as células da teca se mantiveram com expressão na mesma intensidade. Entretanto, as células da granulosa apresentaram forte marcação nos estádios pré-antrais enquanto que nos folículos antrais apresentou fraca intensidade. Concluímos que em ovários de Galea spixii existe a presença de Ob-R nas principais estruturas do ovário sugerindo que este hormônio desempenhe papel fundamental na reprodução desta espécie.(AU)

Leptin, a cytokine produced by adipose cells, is the target of the scientific community for believing that it has an impact on the reproduction of the animals promoting puberty, folliculogenesis and oogenesis, estrous cycle and aiding in fertilization. The understanding of the mechanisms controlling the reproductive activity of Spix's Yellow-toothed Cavy (Galea spixii) plays a relevant role in the preservation of the species. Thus, the present study proposed to analyze the immunolocalization of leptin receptors (Ob-R) in the ovary of cavies. Ovaries from 20 adult, non-pregnant, healthy females were collected. The samples were fixed in 4% phosphate buffered paraformaldehyde, embedded in paraffin and sectioned for immunohistochemistry. The sections were photomicrographs and intensity of the reaction was measured. Strong immunoreaction was observed in oocyte and theca cells, moderate in ovarian stromal cells and large luteal cells and weak stained in granulosa, endothelial, perivascular and small luteal cells. When compared to receptor expression along follicular development it was observed that the oocyte and the theca cells remained with expression at the same intensity. However, the granulosa cells presented strong stained in the preantral stages, whereas in the antral follicles it presented low intensity. We conclude that in the ovaries of Galea spixii there is the presence of Ob-R in the main structures of the ovary sugesting that this hormone plays a fundamental role in the reproduction of this species.(AU)

A Kinetic Characterization of the Gill (Na+, K+)-ATPase from the Semi-terrestrial Mangrove Crab Cardisoma guanhumi Latreille, 1825 (Decapoda, Brachyura).

We provide a kinetic characterization of (Na+, K+)-ATPase activity in a posterior gill microsomal fraction from the semi-terrestrial mangrove crab Cardisoma guanhumi. Sucrose density gradient centrifugation reveals two distinct membrane fractions showing considerable (Na+, K+)-ATPase activity, but also containing other microsomal ATPases. The (Na+, K+)-ATPase, notably immuno-localized to the apical region of the epithelial pillar cells, and throughout the pillar cell bodies, has an M r of around 110 kDa and hydrolyzes ATP with V M = 146.8 ± 6.3 nmol Pi min-1 mg protein-1 and K M = 0.05 ± 0.003 mmol L-1 obeying Michaelis-Menten kinetics. While stimulation by Na+ (V M = 139.4 ± 6.9 nmol Pi min-1 mg protein-1, K M = 4.50 ± 0.22 mmol L-1) also follows Michaelis-Menten kinetics, modulation of (Na+, K+)-ATPase activity by MgATP (V M = 136.8 ± 6.5 nmol Pi min-1 mg protein-1, K 0.5 = 0.27 ± 0.04 mmol L-1), K+ (V M = 140.2 ± 7.0 nmol Pi min-1 mg protein-1, K 0.5 = 0.17 ± 0.008 mmol L-1), and NH4+ (V M = 149.1 ± 7.4 nmol Pi min-1 mg protein-1, K 0.5 = 0.60 ± 0.03 mmol L-1) shows cooperative kinetics. Ouabain (K I = 52.0 ± 2.6 µmol L-1) and orthovanadate (K I = 1.0 ± 0.05 µmol L-1) inhibit total ATPase activity by around 75%. At low Mg2+ concentrations, ATP is an allosteric modulator of the enzyme. This is the first study to provide a kinetic characterization of the gill (Na+, K+)-ATPase in C. guanhumi, and will be useful in better comprehending the biochemical underpinnings of osmoregulatory ability in a semi-terrestrial mangrove crab.

Arabidopsis phosphatidylinositol-phospholipase C2 (PLC2) is required for female gametogenesis and embryo development.

MAIN CONCLUSION: AtPLC2 is an essential gene in Arabidopsis, since it is required for female gametogenesis and embryo development. AtPLC2 might play a role in cell division during embryo-sac development and early embryogenesis. Phosphoinositide-specific phospholipase C (PI-PLC) plays an important role in signal transduction during plant development and in the response to various biotic- and abiotic stresses. The Arabidopsis PI-PLC gene family is composed of nine members, named PLC1 to PLC9. Here, we report that PLC2 is involved in female gametophyte development and early embryogenesis. Using two Arabidopsis allelic T-DNA insertion lines with different phenotypic penetrations, we observed both female gametophytic defects and aberrant embryos. For the plc2-1 mutant (Ws background), no homozygous plants could be recovered in the offspring from self-pollinated plants. Nonetheless, plc2-1 hemizygous mutants are affected in female gametogenesis, showing embryo sacs arrested at early developmental stages. Allelic hemizygous plc2-2 mutant plants (Col-0 background) present reduced seed set and embryos arrested at the pre-globular stage with abnormal patterns of cell division. A low proportion (0.8%) of plc2-2 homozygous mutants was found to escape lethality and showed morphological defects and disrupted megagametogenesis. PLC2-promoter activity was observed during early megagametogenesis, and after fertilization in the embryo proper. Immunolocalization studies in early stage embryos revealed that PLC2 is restricted to the plasma membrane. Altogether, these results establish a role for PLC2 in both reproductive- and embryo development, presumably by controlling mitosis and/or the formation of cell-division planes.

Protein Localization in the Cyanobacterium Anabaena sp. PCC7120 Using Immunofluorescence Labeling.

Techniques such as immunoflorescence are widely used to determine subcellular distribution of proteins. Here we report on a method to immunolocalize proteins in Anabaena sp. PCC7120 with fluorophore-conjugated antibodies by fluorescence microscopy. This method improves the permeabilization of cyanobacterial cells and minimizes the background fluorescence for non-specific attachments. In this protocol, rabbit antibodies were raised against the synthetic peptide of CyDiv protein ( Mandakovic et al., 2016 ). The secondary antibody conjugated to the fluorophore Alexa488 was used due to its different emission range in comparison to the autofluorescence of the cyanobacterium.

Biochemical characterization and immunolocalization studies of a Capsicum chinense Jacq. protein fraction containing DING proteins and anti-microbial activity.

The DING protein family consists of proteins of great biological importance due to their ability to inhibit carcinogenic cell growth. A DING peptide with Mr ∼7.57 kDa and pI ∼5.06 was detected in G10P1.7.57, a protein fraction from Capsicum chinense Jacq. seeds. Amino acid sequencing of the peptide produced three smaller peptides showing identity to the DING protein family. G10P1.7.57 displayed a phosphatase activity capable of dephosphorylating different phosphorylated substrates and inhibited the growth of Saccharomyces cerevisiae cells. Western immunoblotting with a custom-made polyclonal antibody raised against a sequence (ITYMSPDYAAPTLAGLDDATK), derived from the ∼7.57 kDa polypeptide, immunodetected an âˆ¼ 39 kDa polypeptide in G10P1.7.57. Purification by electroelution followed by amino acid sequencing of the ∼39 kDa polypeptide yielded seven new peptide sequences and an additional one identical to that of the initially identified peptide. Western immunoblotting of soluble proteins from C. chinense seeds and leaves revealed the presence of the ∼39 kDa polypeptide at all developmental stages, with increased accumulation when the organs reached maturity. Immunolocalization using Dabsyl chloride- or Alexa fluor 488-conjugated antibodies revealed a specific fluorescent signal in the cell cytoplasm at all developmental stages, giving support to the idea that the ∼39 kDa polypeptide is a soluble DING protein. Thus, we have identified and characterized a protein fraction with a DING protein from C. chinense.

New observations on gametogenic development and reproductive experimental tools to support seed yield improvement in cowpea [Vigna unguiculata (L.) Walp].

KEY MESSAGE: Cowpea reproductive tools. Vigna unguiculata L. Walp. (cowpea) is recognized as a major legume food crop in Africa, but seed yields remain low in most varieties adapted to local conditions. The development of hybrid cowpea seed that could be saved after each generation, enabling significant yield increases, will require manipulation of reproductive development from a sexual to an asexual mode. To develop new technologies that could support the biotechnological manipulation of reproductive development in cowpea, we examined gametogenesis and seed formation in two transformable, African-adapted, day-length-insensitive varieties. Here, we show that these two varieties exhibit distinct morphological and phenological traits but share a common developmental sequence in terms of ovule formation and gametogenesis. We present a reproductive calendar that allows prediction of male and female gametogenesis on the basis of sporophytic parameters related to floral bud size and reproductive organ development, determining that gametogenesis occurs more rapidly in the anther than in the ovule. We also show that the mode of megagametogenesis is of the Polygonum-type and not Oenothera-type, as previously reported. Finally, we developed a whole-mount immunolocalization protocol and applied it to detect meiotic proteins in the cowpea megaspore mother cell, opening opportunities for comparing the dynamics of protein localization during male and female meiosis, as well as other reproductive events in this emerging legume model system.

Ultrastructural insights into tomato infections caused by three different pathotypes of Pepino mosaic virus and immunolocalization of viral coat proteins.

This paper presents studies on an ultrastructural analysis of plant tissue infected with different pathotypes of Pepino mosaic virus (PepMV) and the immunolocalization of viral coat proteins. Because the PepMV virus replicates with a high mutation rate and exhibits significant genetic diversity, therefore, isolates of PepMV display a wide range of symptoms on infected plants. In this work, tomato plants of the Beta Lux cultivar were inoculated mechanically with three pathotypes representing the Chilean 2 (CH2) genotype: mild (PepMV-P22), necrotic (PepMV-P19) and yellowing (PepMV-P5-IY). The presence of viral particles in all infected plants in the different compartments of various cell types (i.e. spongy and palisade mesophyll, sieve elements and xylem vessels) was revealed via ultrastructural analyses. For the first time, it was possible to demonstrate the presence of crystalline inclusions, composed of virus-like particles. In the later stage of PepMV infection (14 dpi) various pathotype-dependent changes in the structure of the individual organelles (i.e. mitochondria, chloroplasts) were found. The strongest immunogold labeling of the viral coat proteins was also observed in plants infected by necrotic isolates. A large number of viral coat proteins were marked in the plant conductive elements, both xylem and phloem.