Plantacyanin plays a role in reproduction in Arabidopsis

Plantacyanins belong to the phytocyanin family of blue copper proteins. In the Arabidopsis (Arabidopsis thaliana) genome, only one gene encodes plantacyanin. The T-DNA-tagged mutant is a knockdown mutant that shows no visible phenotype. We used both promoter-beta-glucuronidase transgenic plants and immunolocalization to show that Arabidopsis plantacyanin is expressed most highly in the inflorescence and, specifically, in the transmitting tract of the pistil. Protein levels show a steep gradient in expression from the stigma into the style and ovary. Overexpression plants were generated using cauliflower mosaic virus 35S, and protein levels in the pistil were examined as well as the pollination process. Seed set in these plants is highly reduced mainly due to a lack of anther dehiscence, which is caused by degeneration of the endothecium. Callose deposits occur on the pollen walls in plants that overexpress plantacyanin, and a small percentage of these pollen grains germinate in the closed anthers. When wild-type pollen was used on the overexpression stigma, seed set was still decreased compared to the control pollinations. We detected an increase in plantacyanin levels in the overexpression pistil, including the transmitting tract. Guidance of the wild-type pollen tube on the overexpression stigma is disrupted as evidenced by the growth behavior of pollen tubes after they penetrate the papillar cell. Normally, pollen tubes travel down the papilla cell and into the style. Wild-type pollen tubes on the overexpression stigma made numerous turns around the papilla cell before growing toward the style. In some rare cases, pollen tubes circled up the papilla cell away from the style and were arrested there. We propose that when plantacyanin levels in the stigma are increased, pollen tube guidance into the style is disrupted.     

Expression analysis of Arabidopsis thaliana small secreted protein genes

Small proteins secreted to the extracellular matrix in plants regulate many physiological activities, including pathogen response, material transport, and morphogenesis, but the functions of most small secreted proteins have not been elucidated except for some well-known small secreted proteins. To predict the functions and physiological roles of unidentified small secreted proteins, information on their expression patterns is valuable. Here, we report expression analysis of Arabidopsis thaliana small secreted protein (ATSP) genes that encode proteins possessing a signal peptide at N-terminal, and protein sizes were less than 100 amino acid residues. By promoter:reporter experiments, we examined the expression of 122 ATSPs, including 47 unannotated ATSPs that do not have any discernable motifs, in tissues and at the cellular level in Arabidopsis seedlings, and floral organs. As a result, 79 ATSP genes were expressed in various regions of the seedlings, and 37 ATSP genes were specifically expressed.     

Mode of reproduction in Arabidopsis suecica

The breeding system of Arabidopsis suecica was investigated through genetic analysis of microsatellite segregation patterns in five controlled crosses as well as in 16 single-mother families collected in the wild. Analysis of single and two-locus segregations in the F2 generation following a cross clearly shows that A. suecica is reproduces sexually. The single-mother families show a high level of homozygosity corroborating earlier results indicating a high level of inbreeding. The high level of individual homozygosity is due both to a high level of selfing and to the underlying population structure.     

Parthenogenetic reproduction in lizards: Histological evidence

Hermaphroditism and sex reversal have not previously been ruled out adequately as possible explanations for the existence of unisexual species of reptiles. Therefore, we examined serial histological sections of the complete urogenital systems of nine F₂ specimens belonging to two ontogenetic series of Cnemidophorus exsanguis raised in captivity in isolation from males, as well as the urogenital system of the F₁ mother of one of these series. No evidence of spermatozoa or testicular tissue was found in any specimen. Comparative material reveals that the histology of the urogenital tract, including the mesonephric kidney in adults, is similar to that of females of bisexual species of Cnemidophorus. We conclude that C. exsanguis is a unisexual, parthenogenetic species whose normal reproduction does not involve sex reversal, self-fertilization, gynogenesis, hybridogenesis, or spermatozoa in any way whatsoever.     

American Kestrel reproduction: evidence for the selection hypothesis and the role of dispersal

Many hypotheses attempt to explain why younger, less experienced birds have a relatively low reproductive output. We evaluated reproductive patterns of marked American Kestrels Falco sparverius nesting in boxes in southwestern Idaho from 1992 to 2006 to test predictions of these hypotheses. Results were consistent with the selection (differential mortality) hypothesis and did not support the constraint, restraint or recruitment hypotheses. Most known-age Kestrels nested in their first year of life, and there was no apparent short-term or long-term reproductive advantage to delayed breeding. The number of years that Kestrels nested in study area boxes ranged from 1 to 6 years, with most Kestrels nesting in only 1 year. Reproductive rates were higher for birds with at least 1 year of nesting experience than for birds nesting in boxes for the first time. After 2 years of nesting, reproductive rates levelled off; there was no evidence for additional improvement or for senescence. Differences in reproductive output with experience/age were due to variation among and not within individuals. Individuals that nested in more than 1 year had similar reproductive rates in their first and second years. Poor producers either died or dispersed after 1 year of nesting in study area boxes. Successful females that nested early in the season and successful males that had been produced locally had the highest probability of returning to nest in a subsequent year.     

Transposable Element Regulation in Rice and Arabidopsis: Diverse Patterns of Active Expression and siRNA-mediated Silencing

Transposable elements (TEs) are DNA segments that can mediate or cause movement within genomes. We performed a comprehensive, whole-genome analysis of annotated TEs in rice (Oryza sativa L.) and Arabidopsis thaliana, focusing on their expression (mRNA data) and silencing (small RNA data), and we compared these data with annotated genes that are not annotated as transposons. TEs demonstrated higher levels of antisense mRNA expression in comparison to non-TE genes. The majority of the TEs were silenced, as demonstrated by higher levels of small RNAs and a lack of mRNA MPSS data. When TEs were expressed, their activity was usually limited to just one or a few of the mRNA libraries. When we examined TE expression at the whole-genome level and across the complete mRNA dataset, we observed that most activity was contributed by a few highly expressed transposable elements. These TEs were characterized by their low copy number and few matching small RNAs. Our results help define the relationship between gene expression and gene silencing for TEs, and indicate that TE silencing can impact neighboring genes, perhaps via a mechanism of heterochromatin formation and spreading. These data may be used to define active TEs and families of transposable elements that continue to shape plant genomes.     

Driving reproduction: RFamide peptides behind the wheel

The availability of tools for probing the genome and proteome more efficiently has allowed for the rapid discovery of novel genes and peptides that play important, previously uncharacterized roles in neuroendocrine regulation. In this review, the role of a class of neuropeptides containing the C-terminal Arg-Phe-NH(2) (RFamide) in regulating the reproductive axis will be highlighted. Neuropeptides containing the C-terminal Phe-Met-Arg-Phe-NH(2) (FMRFamide) were first identified as cardioregulatory elements in the bi-valve mollusk Macrocallista nimbosa. During the past two decades, numerous studies have shown the presence of structurally similar peptides sharing the RFamide motif across taxa. In vertebrates, RFamide peptides have pronounced influences on opiatergic regulation and neuroendocrine function. Two key peptides in this family are emerging as important regulators of the reproductive axis, kisspeptin and gonadotropin-inhibitory hormone (GnIH). Kisspeptin acts as the accelerator, directly driving gonadotropin-releasing hormone (GnRH) neurons, whereas GnIH acts as the restraint. Recent evidence suggests that both peptides play a role in mediating the negative feedback effects of sex steroids. This review presents the hypothesis that these peptides share complementary roles by responding to internal and external stimuli with opposing actions to precisely regulate the reproductive axis.     

Genome-wide evidence for local DNA methylation spreading from small RNA-targeted sequences in Arabidopsis

Transposable elements (TEs) and their relics play major roles in genome evolution. However, mobilization of TEs is usually deleterious and strongly repressed. In plants and mammals, this repression is typically associated with DNA methylation, but the relationship between this epigenetic mark and TE sequences has not been investigated systematically. Here, we present an improved annotation of TE sequences and use it to analyze genome-wide DNA methylation maps obtained at single-nucleotide resolution in Arabidopsis. We show that although the majority of TE sequences are methylated, ∼26% are not. Moreover, a significant fraction of TE sequences densely methylated at CG, CHG and CHH sites (where H = A, T or C) have no or few matching small interfering RNA (siRNAs) and are therefore unlikely to be targeted by the RNA-directed DNA methylation (RdDM) machinery. We provide evidence that these TE sequences acquire DNA methylation through spreading from adjacent siRNA-targeted regions. Further, we show that although both methylated and unmethylated TE sequences located in euchromatin tend to be more abundant closer to genes, this trend is least pronounced for methylated, siRNA-targeted TE sequences located 5′ to genes. Based on these and other findings, we propose that spreading of DNA methylation through promoter regions explains at least in part the negative impact of siRNA-targeted TE sequences on neighboring gene expression.     

Expression analyses of Arabidopsis oligopeptide transporters during seed germination, vegetative growth and reproduction

AtOPT promoter-GUS fusions were constructed for six of the nine known, putative oligopeptide transporters (OPTs) in Arabidopsis thaliana and used to examine AtOPT expression at various stages of plant development. AtOPT1, AtOPT3, AtOPT4, AtOPT6 and AtOPT7 were expressed in the embryonic cotyledons prior to root radicle emergence. Except for AtOPT8, which gave weak expression, all AtOPTs were strongly expressed in post-germinative seedlings with strongest expression in vascular tissues of cotyledons and hypocotyls. Preferential expression of AtOPTs in vascular tissues was also observed in cotyledons, leaves, hypocotyls, roots, flowers, siliques, and seed funiculi of seedlings and adult plants. Differential tissue-specific expression was observed for specific AtOPTs. For example, AtOPT1, AtOPT3 and AtOPT8 were uniquely expressed in pollen. Only AtOPT1 was expressed in growing pollen tubes, while only AtOPT6 was observed in ovules. AtOPT8 was transiently expressed in seeds during early stages of embryogenesis. Iron limitation was found to enhance expression of AtOPT3. These data suggest distinct cellular roles for specific AtOPTs including nitrogen mobilization during germination and senescence, pollen tube growth, pollen and ovule development, seed formation and metal transport.     

Pro-opiomelanocortin-derived peptides in the testis: evidence for a possible role in Leydig and Sertoli cell function

Pro-opiomelanocortin (POMC)-derived peptides such as beta-endorphin, ACTH, and MSHs were identified in the testis where they were exclusively localized in Leydig cells. Examination of testicular extracts by a variety of physicochemical and immunological techniques indicates that the processing of the POMC in the testis is very similar to that in the brain. By using a cDNA probe, the POMC-like mRNA present in total testis and cultured Leydig cells was 150-200 bases shorter than that in the hypothalamus and pituitary. In addition, POMC mRNA was localized to Leydig cells using in situ hybridization. The expression of the POMC-like gene and the accumulation of POMC-derived peptides in Leydig cell were shown to be under the control of gonadotropin. As the testis contains low concentrations of POMC-derived peptides, we suggested that they may be implicated in local regulatory events within this organ. This postulate was supported by results from in vivo and in vitro experiments suggesting that different portions of the POMC-molecule may have opposite effects on Sertoli cell functions. For example, MSHs increased cAMP accumulation and aromatase activity in these cells, while opioids inhibited Sertoli cell proliferation and androgen binding protein (ABP) secretion. Furthermore, following intratesticular administration of opiate antagonists, testosterone production was reduced, suggesting that Leydig cell function may be also modulated by beta-endorphin and/or other related peptides. Taken together, these studies support the hypothesis of a possible role of POMC-derived peptides in testicular function.     

Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.     

The hypocotyl chloroplast plays a role in phototropic bending of Arabidopsis seedlings: developmental and genetic evidence

Chloroplasts of guard cells and coleoptiles have been implicated in the sensory transduction of blue light. The present study was aimed at establishing whether the chloroplast of the hypocotyl from Arabidopsis, another blue light-responding organ, has similar characteristics to that of sensory-transducing guard cell and coleoptile chloroplasts. Results showed that the phototropic curvature and arch length induced by blue light in Arabidopsis seedlings matched the distribution of mature chloroplasts in the bending hypocotyl. The bending arch consistently included the region of the hypocotyl containing mature chloroplasts, and never extended beyond that region. Manipulation of the extent of greening of dark-grown hypocotyls by varying red light pretreatments elicited blue light-stimulated curvatures and arch lengths that depended on the duration of the red light pretreatment and on the distribution of mature chloroplasts in the hypocotyl. Albino psd2 mutants of Arabidopsis, which lack mature chloroplasts, are devoid of phototropic sensitivity under conditions in which wild-type seedlings show large curvatures. The star mutant of Arabidopsis has a delayed greening and a delayed phototropic response as compared with wild type. Measurements of photosynthetic oxygen evolution and carbon fixation, dark respiration, and light-dependent zeaxanthin formation in the hypocotyl showed features similar to those of guard cells and coleoptiles, and distinctly different from those of mesophyll tissue. These results indicate that the hypocotyl chloroplast has characteristics similar to those associated with guard cell and coleoptile chloroplasts, and that phototropic bending of Arabidopsis hypocotyls appears to require mature chloroplasts.     

Hormonal control of reproduction in small ruminants

Reproduction of small ruminants can be controlled by several methods developed in recent decades. Some of these involve administration of hormones that modify the physiological chain of events involved in the sexual cycle. Methods which utilise progesterone or its analogues are based on their effects in the luteal phase of the cycle, simulating the action of natural progesterone produced by the corpus luteum after ovulation, which is responsible for controlling LH secretion from the pituitary. Use of prostaglandins is an alternative method for controlling reproduction by eliminating the corpus luteum and inducing a subsequent follicular phase with ovulation. Finally, the discovery of the properties of melatonin in photoperiod-dependent breeding animals opened up a new methodology to control reproduction in these species, inducing changes in the perception of photoperiod and the annual pattern of reproduction. Use of hormones to induce oestrus has allowed increased use of artificial insemination in small ruminants, a very useful management tool, considering the difficulty of detecting oestrus in these species. At commercial level, synchronisation of oestrus allows control of lambing and kidding, with subsequent synchronisation of weaning of young animals for slaughter. Also, it allows more efficient use of labour and animal facilities. Multiple ovulation and embryo transfer programmes are also possible with the use of oestrus synchronisation and artificial insemination. Finally, hormonal treatments have also been used to induce puberty in ewe-lambs and doelings.     

Oxygen binding to Arabidopsis thaliana AHb2 nonsymbiotic hemoglobin: evidence for a role in oxygen transport

Nonsymbiotic hemoglobins AHb1 and AHb2 discovered in Arabidopsis thaliana are likely to carry out distinct physiological roles, in consideration of their differences in sequence, structure, expression pattern, and tissue localization. Despite a relatively fast autoxidation in the presence of O(2) , we were able to collect O(2) -binding curves for AHb2 in the presence of a reduction enzymatic system. AHb2 binds O(2) noncooperatively with a p50 of 0.021 ± 0.003 Torr, a value consistent with a recently proposed role in O(2) transport. The analysis of the internal cavities derived from the structures sampled in molecular dynamics simulations confirms strong differences with AHb1, proposed to work as a NO deoxygenase in vivo. Overall, our results are consistent with a role for AHb2 as an oxygen carrier, as recently proposed on the basis of experiments on AHb2-overexpressing mutants of A. thaliana.     

Anorexia in cancer: role of feeding-regulatory peptides

Anorexia is one of the most common symptoms in advanced cancer and is a frequent cause of discomfort for cancer patients and their families. The pathogenesis of cancer anorexia is multi-factorial and involves most of the hypothalamic neuronal signalling pathways modulating energy homeostasis. It is considered to be the result of a failure of usual appetite and satiety signals. Loss of appetite can arise from decreased taste and smell of food, as well as from dysfunctional hypothalamic signalling pathways and cytokine production. Cytokines in particular, appear to play a key role in energy balance through persistent activation of the melanocortin system and inhibition of the neuropeptide Y pathway. The imbalance between anorexigenic and orexigenic peptides leads to suppression of appetite, and increased satiety and satiation associated with marked weight loss and decline in physical performance. High levels of serotonin also appear to contribute to these effects and recent findings implicate corticotropin-releasing factor in the pathogenesis of cancer anorexia as well. Despite significant advances in our understanding of the regulation of food intake and energy expenditure, few effective therapies are available. A better appreciation of the molecular and neuronal mechanisms that control body weight homeostasis may lead to the development of new therapies for improving the survival and quality of life of these patients.