Knockdown of CELL DIVISION CYCLE16 Reveals an Inverse Relationship between Lateral Root and Nodule Numbers and a Link to Auxin in Medicago truncatula

The postembryonic development of lateral roots and nodules is a highly regulated process. Recent studies suggest the existence of cross talk and interdependency in the growth of these two organs. Although plant hormones, including auxin and cytokinin, appear to be key players in coordinating this cross talk, very few genes that cross-regulate root and nodule development have been uncovered so far. This study reports that a homolog of CELL DIVISION CYCLE16 (CDC16), a core component of the Anaphase Promoting Complex, is one of the key mediators in controlling the overall number of lateral roots and nodules. A partial suppression of this gene in Medicago truncatula leads to a decrease in number of lateral roots and a 4-fold increase in number of nodules. The roots showing lowered expression of MtCDC16 also show reduced sensitivity to phytohormone auxin, thus providing a potential function of CDC16 in auxin signaling.As in all eukaryotic organisms, cell division in plants is strictly controlled by a concerted action of several key regulators, such as cyclin-dependent kinases and cyclins (De Veylder et al., 2007). The progression of the cell cycle from one phase to another requires the targeted degradation of selected cyclin molecules mediated by two ubiquitin-mediated proteolytic pathways. The SKP1-Cullin/F-Box protein (SCF) pathway acts in the G1-to-S phase transition by degrading the D-type cyclins and other substrate proteins (Yanagawa and Kimura, 2005). The second pathway, mediated by Anaphase Promoting Complex/Cyclosome (APC/C), regulates the sequential destruction of A- and B-type cyclins in a D-box or a KEN-box-dependent manner, resulting in chromosome segregation and exit from mitosis (Genschik et al., 1998; Pfleger and Kirschner, 2000). Evidence of the role of the APC/C in plant development comes from studies of the Arabidopsis (Arabidopsis thaliana) hobbit (hbt) mutant that shows defects in root growth. The HBT gene is required for both cell division and cell differentiation in root meristems and encodes CDC27, a core subunit of APC/C (Blilou et al., 2002; Perez-Perez et al., 2008). Cebolla et al. (1999) used the root nodule system of the model legume Medicago truncatula to study the function of an APC/C activator, CCS52, which is homologous to the yeast APC/C activator CDH1. A nodule-specific homolog of CCS52, CCS52A, was found to be required to initiate endoreduplication in the dividing cells, and its down-regulation affected nodule development, resulting in lower ploidy, reduced cell size, and inefficient rhizobial invasion and nodule maturation (Vinardell et al., 2003; Kondorosi et al., 2005). T-DNA insertions in the Arabidopsis CELL DIVISION CYCLE16 (CDC16) and APC2 genes result in gametophytic lethality due to the failure to degrade mitotic cyclins (Capron et al., 2003b; Kwee and Sundaresan, 2003). Although the completed Arabidopsis genome has allowed the identification of homologs of almost all vertebrate APC/C subunits in plants (Capron et al., 2003a), the functions of most of these subunits still remains largely unexplored.Direct links between root growth and auxin signaling have been well documented. Several Arabidopsis mutants with decreased auxin sensitivity often exhibit an overall defect in both primary and lateral root development (Hellmann and Estelle, 2002; Casimiro et al., 2003; Hellmann et al., 2003; Vanneste et al., 2005). A number of these auxin-resistant mutants belong to the SCF proteolysis pathway, supporting a role for the SCF pathway in auxin signaling (Teale et al., 2006; Benjamins and Scheres, 2008). Auxin appears to control lateral root development by promoting G1-to-S transition in selected xylem pericycle cells, perhaps by targeting KRP2, a cyclin-dependent kinase inhibitor, and E2F, an S phase inhibitor to SCF-mediated proteolysis (del Pozo et al., 2002; Himanen et al., 2002). Unlike SCF, the role of APC/C in auxin-mediated plant development is not clear. The only report that has so far integrated APC/C with auxin signaling pertains to the hbt mutant, which shows an increased resistance to exogenous auxin due to accumulation of Aux/IAAs in the roots (Blilou et al., 2002).As in lateral roots, auxin is an important player in the development of nodules on the roots of leguminous plants (Beveridge et al., 2007). Studies with auxin-responsive reporter gene constructs have shown auxin''s participation in cortical cell reactivation and initiation of nodule primordia (Mathesius et al., 1998). The exogenous application of Nod factor results in a transient inhibition of auxin transport capacity in roots of Vicia sativa (Boot et al., 1999) and Trifolium repens (Mathesius et al., 1998). Consistent with this, localized application of synthetic auxin transport inhibitors on alfalfa (Medicago sativa) roots induces pseudonodules (Hirsch et al., 1989). Complementing these findings, a more recent study in M. truncatula has demonstrated that increased auxin transport, caused by silencing the flavonoid pathway, results in reduced nodule formation in response to rhizobia (Wasson et al., 2006). Finally, hypernodulating mutants like sunn and skl show defective long-distance transport of auxin, further suggesting the importance of polar auxin transport, not only in regulating nodule induction but also in controlling nodule numbers (Prayitno et al., 2006; van Noorden et al., 2006).In this report, we investigated the role of the APC/C component CDC16 in root and nodule development in M. truncatula. CDC16 was identified via microarray analysis as a gene that was significantly induced in roots of M. truncatula following inoculation by Sinorhizobium meliloti and in nodules relative to uninoculated roots (Kuppusamy, 2005), thus encouraging further functional analysis of this gene. To overcome the problem of the gametophytic lethality resulting from CDC16 knockout, as seen from analysis of an insertional mutation in Arabidopsis (Kwee and Sundaresan, 2003) we undertook an RNA interference (RNAi) approach to partially suppress the expression of CDC16 gene in Agrobacterium rhizogenes-transformed roots of M. truncatula. We report that roots transformed with the CDC16 RNAi construct (hereafter called Mtcdc16i roots) displayed a decreased sensitivity to auxin, defective primary root growth, and fewer lateral roots. Interestingly, in response to S. meliloti, the Mtcdc16i roots showed almost 4-fold increase in number of nodules, suggesting that decreased sensitivity to auxin leads to a hypernodulation phenotype. Thus, this work highlights the importance of CDC16 in root and nodule development and indicates a possible role for this gene in auxin signaling.     

Altered resource availability and the population dynamics of tree species in Amazonian secondary forests

Despite research demonstrating that water and nutrient availability exert strong effects on multiple ecosystem processes in tropical forests, little is known about the effect of these factors on the demography and population dynamics of tropical trees. Over the course of 5 years, we monitored two common Amazonian secondary forest species—Lacistema pubescens and Myrcia sylvatica—in dry-season irrigation, litter-removal and control plots. We then evaluated the effects of altered water and nutrient availability on population demography and dynamics using matrix models and life table response experiments. Our results show that despite prolonged experimental manipulation of water and nutrient availability, there were nearly no consistent and unidirectional treatment effects on the demography of either species. The patterns and significance of observed treatment effects were largely dependent on cross-year variability not related to rainfall patterns, and disappeared once we pooled data across years. Furthermore, most of these transient treatment effects had little effect on population growth rates. Our results suggest that despite major experimental manipulations of water and nutrient availability—factors considered critical to the ecology of tropical pioneer tree species—autogenic light limitation appears to be the primary regulator of tree demography at early/mid successional stages. Indeed, the effects of light availability may completely override those of other factors thought to influence the successional development of Amazonian secondary forests.     

Cortical Reorganization after Hand Immobilization: The beta qEEG Spectral Coherence Evidences

There is increasing evidence that hand immobilization is associated with various changes in the brain. Indeed, beta band coherence is strongly related to motor act and sensitive stimuli. In this study we investigate the electrophysiological and cortical changes that occur when subjects are submitted to hand immobilization. We hypothesized that beta coherence oscillations act as a mechanism underlying inter- and intra-hemispheric changes. As a methodology for our study fifteen healthy individuals between the ages of 20 and 30 years were subjected to a right index finger task before and after hand immobilization while their brain activity pattern was recorded using quantitative electroencephalography. This analysis revealed that hand immobilization caused changes in frontal, central and parietal areas of the brain. The main findings showed a lower beta-2 band in frontal regions and greater cortical activity in central and parietal areas. In summary, the coherence increased in the frontal, central and parietal cortex, due to hand immobilization and it adjusted the brains functioning, which had been disrupted by the procedure. Moreover, the brain adaptation upon hand immobilization of the subjects involved inter- and intra-hemispheric changes.     

Bacterial biosurfactant increases ex situ biodiesel bioremediation in clayey soil

The contamination of soils by oily compounds has several environmental impacts, which can be reversed through bioremediation, using biosurfactants as auxiliaries in the biodegradation process. In this study, we aimed to perform ex situ bioremediation of biodiesel-contaminated soil using biosurfactants produced by Bacillus methylotrophicus. A crude biosurfactant was produced in a whey-based culture medium supplemented with nutrients and was later added to biodiesel-contaminated clayey soil. The produced lipopeptide biosurfactant could reduce the surface tension of the fermentation broth to 30.2 mN/m. An increase in the microbial population was observed in the contaminated soil; this finding can be corroborated by the finding of increased CO₂ release over days of bioremediation. Compared with natural attenuation, the addition of a lower concentration of the biosurfactant (0.5% w/w in relation to the mass of diesel oil) to the soil increased biodiesel removal by about 16% after 90 days. The added biosurfactant did not affect the retention of the contaminant in the soil, which is an important factor to be considered when applying in situ bioremediation technologies.

     

Increased Oxidative Parameters and Decreased Cytokine Levels in an Animal Model of Attention-Deficit/Hyperactivity Disorder

Attention-deficit/hyperactivity disorder (ADHD) is a highly heterogeneous disorder characterized by impairing levels of hyperactivity, impulsivity and inattention. Oxidative and inflammatory parameters have been recognized among its multiple predisposing pathways, and clinical studies indicate that ADHD patients have increased oxidative stress. In this study, we aimed to evaluate oxidative (DCFH oxidation, glutathione levels, glutathione peroxidase, catalase and superoxide dismutase activities) and inflammatory (TNF-α, IL-1β and IL-10) parameters in the most widely accepted animal model of ADHD, the spontaneously hypertensive rats (SHR). Prefrontal cortex, cortex (remaining regions), striatum and hippocampus of adult male SHR and Wistar Kyoto rats were studied. SHR presented increased reactive oxygen species (ROS) production in the cortex, striatum and hippocampus. In SHR, glutathione peroxidase activity was decreased in the prefrontal cortex and hippocampus. TNF-α levels were reduced in the prefrontal cortex, cortex (remaining regions), hippocampus and striatum of SHR. Besides, IL-1β and IL-10 levels were decreased in the cortex of the ADHD model. Results indicate that SHR presented an oxidative profile that is characterized by an increase in ROS production without an effective antioxidant counterbalance. In addition, this strain showed a decrease in cytokine levels, mainly TNF-α, indicating a basal deficit. These results may present a new approach to the cognitive disturbances seen in the SHR.     

Dynamics of the Leaf-Litter Arthropod Fauna Following Fire in a Neotropical Woodland Savanna

Fire is an important agent of disturbance in tropical savannas, but relatively few studies have analyzed how soil-and-litter dwelling arthropods respond to fire disturbance despite the critical role these organisms play in nutrient cycling and other biogeochemical processes. Following the incursion of a fire into a woodland savanna ecological reserve in Central Brazil, we monitored the dynamics of litter-arthropod populations for nearly two years in one burned and one unburned area of the reserve. We also performed a reciprocal transplant experiment to determine the effects of fire and litter type on the dynamics of litter colonization by arthropods. Overall arthropod abundance, the abundance of individual taxa, the richness of taxonomic groups, and the species richness of individual taxa (Formiciade) were lower in the burned site. However, both the ordinal-level composition of the litter arthropod fauna and the species-level composition of the litter ant fauna were not dramatically different in the burned and unburned sites. There is evidence that seasonality of rainfall interacts with fire, as differences in arthropod abundance and diversity were more pronounced in the dry than in the wet season. For many taxa the differences in abundance between burned and unburned sites were maintained even when controlling for litter availability and quality. In contrast, differences in abundance for Collembola, Formicidae, and Thysanoptera were only detected in the unmanipulated samples, which had a lower amount of litter in the burned than in the unburned site throughout most of our study period. Together these results suggest that arthropod density declines in fire-disturbed areas as a result of direct mortality, diminished resources (i.e., reduced litter cover) and less favorable microclimate (i.e., increased litter desiccation due to reduction in tree cover). Although these effects were transitory, there is evidence that the increasingly prevalent fire return interval of only 1–2 years may jeopardize the long-term conservation of litter arthropod communities.     

Dual Immune Modulatory Effect of Vitamin A in Human Visceral Leishmaniasis

Vitamin A supplementation has shown to prevent mortality by diarrheal and respiratory diseases in several countries. Nevertheless, there are few studies investigating the effect of vitamin A in visceral leishmaniasis (VL), although there are reports of its deficiency in children with symptomatic VL in Brazil and Bangladesh. This study analyzed the effect of vitamin A on a subset of Treg cells and monocytes isolated from symptomatic VL and from healthy children residing in an endemic area for VL in Northeast Brazil. Serum retinol concentrations correlated inversely with IL-10 and TGF-β productions in CD4⁺CD25^highFoxp3⁺ T cells isolated from children with VL stimulated with leishmanial antigens. All-trans retinoic acid in vitro induced IL-10 in CD4⁺CD25^highFoxp3⁺ T cells; IL-10 and TGF-β production in CD4⁺CD25⁻Foxp3⁻ T cells, and IL-10 in monocytes isolated from healthy children. However, the use of all-trans retinoic acid together with leishmanial antigens in vitro prevented increases in IL-10 production in Treg cells and monocytes isolated from VL children. Strikingly, those results show a potential dual role of vitamin A in the immune system: improvement of a regulatory profile in cells from healthy children after leishmanial stimulation and down modulation of IL-10 in Treg cells and monocytes during symptomatic VL. Therefore, the use of vitamin A concomitant to VL therapy might be useful in improving recovery from disease status caused by Leishmania infantum infection and warrants additional study.     

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.