Carlos Takeshi Hotta

Calcium-dependent modulation by melatonin of the circadian rhythm in malarial parasites.

he development of malarial parasites is a complex process involving both intracellular and extracellular phases. Intraerythrocytic maturation proceeds through well-defined stages, termed rings, trophozoites and schizonts. In vivo, transition to a new stage and invasion of new erythrocytes are highly synchronous. The timing of these processes varies between Plasmodium species, but is always a multiple of 24 h. The simultaneous appearance of billions of individual parasites in the bloodstream may represent an efficient evolutionary strategy to escape the defence mechanisms of the host. The synchronicity of these processes is rapidly lost in culture, indicating the possible involvement of a host-derived signal, although the nature of this signal is presently unknown. Here we show that the hormone melatonin modifies the development of malarial parasites in vitro, that in vivo surgical ablation of the pineal gland leads to reduced synchronicity in the maturation process of Plasmodium, an effect that is reversed upon treatment with melatonin, and that in vivo inhibition of melatonin receptors mimics the effect of pinealectomy. We also demonstrate that melatonin, through activation of specific receptors coupled to phospholipase C activation, causes release of Ca from the intracellular stores of Plasmodium grown in vitro. We therefore propose that circadian changes in melatonin concentration in the host represent a key signal that controls synchronous maturation of Plasmodium in vivo. In search of a host-derived signal that undergoes circadian changes in mammals (and other vertebrates), we considered melatonin as a potential candidate. Melatonin is synthesized and released by the pineal gland during darkness, and this hormone is thought to participate in regulation of circadian rhythms in many eukaryotes, including vertebrates, invertebrates, higher plants and dinoflagellates. Not only does melatonin release exhibit a circadian rhythm, but the molecule is also sufficiently hydrophobic to cross T

Sugarcane improvement: how far can we go?

In recent years, efforts to improve sugarcane have focused on the development of biotechnology for this crop. It has become clear that sugarcane lacks tools for the biotechnological route of improvement and that the initial efforts in sequencing ESTs had limited impact for breeding. Until recently, the models used by breeders in statistical genetics approaches have been developed for diploid organisms, which are not ideal for a polyploid genome such as that of sugarcane. Breeding programs are dealing with decreasing yield gains. The contribution of multiple alleles to complex traits such as yield is a basic question underlining the breeding efforts that could only be addressed by the development of specific tools for this grass. However, functional genomics has progressed and gene expression profiling is leading to the definition of gene networks. The sequencing of the sugarcane genome, which is underway, will greatly contribute to numerous aspects of research on grasses. We expect that both the transgenic and the marker-assisted route for sugarcane improvement will contribute to increased sugar, stress tolerance, and higher yield and that the industry for years to come will be able to rely on sugarcane as the most productive energy crop.

Building the sugarcane genome for biotechnology and identifying evolutionary trends.

BackgroundSugarcane is the source of sugar in all tropical and subtropical countries and is becoming increasingly important for bio-based fuels. However, its large (10 Gb), polyploid, complex genome has hindered genome based breeding efforts. Here we release the largest and most diverse set of sugarcane genome sequences to date, as part of an on-going initiative to provide a sugarcane genomic information resource, with the ultimate goal of producing a gold standard genome.ResultsThree hundred and seventeen chiefly euchromatic BACs were sequenced. A reference set of one thousand four hundred manually-annotated protein-coding genes was generated. A small RNA collection and a RNA-seq library were used to explore expression patterns and the sRNA landscape. In the sucrose and starch metabolism pathway, 16 non-redundant enzyme-encoding genes were identified. One of the sucrose pathway genes, sucrose-6-phosphate phosphohydrolase, is duplicated in sugarcane and sorghum, but not in rice and maize. A diversity analysis of the s6pp duplication region revealed haplotype-structured sequence composition. Examination of hom(e)ologous loci indicate both sequence structural and sRNA landscape variation. A synteny analysis shows that the sugarcane genome has expanded relative to the sorghum genome, largely due to the presence of transposable elements and uncharacterized intergenic and intronic sequences.ConclusionThis release of sugarcane genomic sequences will advance our understanding of sugarcane genetics and contribute to the development of molecular tools for breeding purposes and gene discovery.

The Biotechnology Roadmap for Sugarcane Improvement

Due to the strategic importance of sugarcane to Brazil, FAPESP, the main São Paulo state research funding agency, launched in 2008 the FAPESP Bioenergy Research Program (BIOEN, http://bioenfapesp.org). BIOEN aims to generate new knowledge and human resources for the improvement of the sugarcane and ethanol industry. As part of the BIOEN program, a Workshop on Sugarcane Improvement was held on March 18th and 19th 2009 in São Paulo, Brazil. The aim of the workshop was to explore present and future challenges for sugarcane improvement and its use as a sustainable bioenergy and biomaterial feedstock. The workshop was divided in four sections that represent important challenges for sugarcane improvement: a) gene discovery and sugarcane genomics, b) transgenics and controlled transgene expression, c) sugarcane physiology (photosynthesis, sucrose metabolism, and drought) and d) breeding and statistical genetics. This report summarizes the roadmap for the improvement of sugarcane.

Melatonin and N-acetyl-serotonin cross the red blood cell membrane and evoke calcium mobilization in malarial parasites

The duration of the intraerythrocytic cycle of Plasmodium is a key factor in the pathogenicity of this parasite. The simultaneous attack of the host red blood cells by the parasites depends on the synchronicity of their development. Unraveling the signals at the basis of this synchronicity represents a challenging biological question and may be very important to develop alternative strategies for therapeutic approaches. Recently, we reported that the synchrony of Plasmodium is modulated by melatonin, a host hormone that is synthesized only during the dark phases. Here we report that N-acetyl-serotonin, a melatonin precursor, also releases Ca2+ from isolated P. chabaudi parasites at micro- and nanomolar concentrations and that the release is blocked by 250 mM luzindole, an antagonist of melatonin receptors, and 20 mM U73122, a phospholipase C inhibitor. On the basis of confocal microscopy, we also report the ability of 0.1 microM melatonin and 0.1 microM N-acetyl-serotonin to cross the red blood cell membrane and to mobilize intracellular calcium in parasites previously loaded with the fluorescent calcium indicator Fluo-3 AM. The present data represent a step forward into the understanding of the signal transduction process in the host-parasite relationship by supporting the idea that the host hormone melatonin and N-acetyl-serotonin generate IP3 and therefore mobilize intracellular Ca2+ in Plasmodium inside red blood cells.

Co-expression network analysis reveals transcription factors associated to cell wall biosynthesis in sugarcane

Sugarcane is a hybrid of Saccharum officinarum and Saccharum spontaneum, with minor contributions from other species in Saccharum and other genera. Understanding the molecular basis of cell wall metabolism in sugarcane may allow for rational changes in fiber quality and content when designing new energy crops. This work describes a comparative expression profiling of sugarcane ancestral genotypes: S. officinarum, S. spontaneum and S. robustum and a commercial hybrid: RB867515, linking gene expression to phenotypes to identify genes for sugarcane improvement. Oligoarray experiments of leaves, immature and intermediate internodes, detected 12,621 sense and 995 antisense transcripts. Amino acid metabolism was particularly evident among pathways showing natural antisense transcripts expression. For all tissues sampled, expression analysis revealed 831, 674 and 648 differentially expressed genes in S. officinarum, S. robustum and S. spontaneum, respectively, using RB867515 as reference. Expression of sugar transporters might explain sucrose differences among genotypes, but an unexpected differential expression of histones were also identified between high and low Brix° genotypes. Lignin biosynthetic genes and bioenergetics-related genes were up-regulated in the high lignin genotype, suggesting that these genes are important for S. spontaneum to allocate carbon to lignin, while S. officinarum allocates it to sucrose storage. Co-expression network analysis identified 18 transcription factors possibly related to cell wall biosynthesis while in silico analysis detected cis-elements involved in cell wall biosynthesis in their promoters. Our results provide information to elucidate regulatory networks underlying traits of interest that will allow the improvement of sugarcane for biofuel and chemicals production.

Desynchronizing Plasmodium Cell Cycle Increases Chloroquine Protection at Suboptimal Doses

We have previously shown that in vivo and in vitro the hormone melatonin is responsible for the synchronous development of Plasmodia. Melatonin can also mobilize calcium from internal stores in these parasites and this response is abolished by luzindole, a melatonin antagonist. We here demonstrate that in vivo alteration of parasite synchronous de- velopment, using luzindole, partially improves survival of infected mice and dramatically increases the antimalarial ac- tivity of chloroquine. The data presented may lead to a conceptually new paradigm for malaria infection therapy and pro- vide novel evidence suggesting that the malaria parasite uses the cell cycle synchrony as one of the strategies to evade the host immune system.

NO-Mediated [Ca2+]cyt Increases Depend on ADP-Ribosyl Cyclase Activity in Arabidopsis

ADP-ribosyl cyclase activity in Arabidopsis is up-regulated by nitric oxide to increase the cellular concentration of cyclic ADP ribose and free Ca2+. Cyclic ADP ribose (cADPR) is a Ca2+-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca2+ release from intracellular stores to increase the concentration of cytosolic free Ca2+ in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or that a homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca2+ signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca2+.

Circadian Rhythms of Sense and Antisense Transcription in Sugarcane, a Highly Polyploid Crop

Commercial sugarcane (Saccharum hybrid) is a highly polyploid and aneuploid grass that stores large amounts of sucrose in its stem. We have measured circadian rhythms of sense and antisense transcription in a commercial cultivar (RB855453) using a custom oligoarray with 14,521 probes that hybridize to sense transcripts (SS) and 7,380 probes that hybridize to antisense transcripts (AS).We estimated that 32% of SS probes and 22% AS probes were rhythmic. This is a higher proportion of rhythmic probes than the usually found in similar experiments in other plant species. Orthologs and inparalogs of Arabidopsis thaliana, sugarcane, rice, maize and sorghum were grouped in ortholog clusters. When ortholog clusters were used to compare probes among different datasets, sugarcane also showed a higher proportion of rhythmic elements than the other species. Thus, it is possible that a higher proportion of transcripts are regulated by the sugarcane circadian clock. Thirty-six percent of the identified AS/SS pairs had significant correlated time courses and 64% had uncorrelated expression patterns. The clustering of transcripts with similar function, the anticipation of daily environmental changes and the temporal compartmentation of metabolic processes were some properties identified in the circadian sugarcane transcriptome. During the day, there was a dominance of transcripts associated with photosynthesis and carbohydrate metabolism, including sucrose and starch synthesis. During the night, there was dominance of transcripts associated with genetic processing, such as histone regulation and RNA polymerase, ribosome and protein synthesis. Finally, the circadian clock also regulated hormone signalling pathways: a large proportion of auxin and ABA signalling components were regulated by the circadian clock in an unusual biphasic distribution.

Reference genes for transcript quantification in Gracilaria tenuistipitata under drought stress

Gracilaria tenuistipitata is a red macroalga often found in intertidal environments, where it is frequently under drought stress due to tidal water scarcity. In order to be able to study the molecular mechanisms involved in G. tenuistipitata resistance to dehydration, we have done a dehydration assay, tested different RNA extraction protocols, and selected new reference genes for reverse transcription quantitative PCR (RT-qPCR). After testing four different methods, we found that an adapted TRIzol method resulted in the highest quantities of pure RNA from less biomass. This method yielded an average of 783.6xa0ngxa0mg−1 of clean RNA from 25xa0mg of fresh tissue in only 1.5xa0h. Additionally, DNAse I treatment and silica spin column purification were done prior to RT-qPCR. Of the nine putative reference genes tested for expression stability under control and drought-like conditions, a combination of four genes: β-GALACTOSIDASE B (GLB), TRANSCRIPTION INITIATION FACTOR IIB (GTF2B), EUKARYOTIC ELONGATION FACTOR 2 (EEF2), and β-TUBULIN (TUB) was selected. This allowed us to measure the transcription level of THIOREDOXIN (TRX), OXYGEN-EVOLVING ENHANCER 1 (OEE), and NITRATE REDUCTASE 2 (NIA2). TRX was upregulated after 1-h dehydration and kept higher transcriptional levels even 4xa0h later. In contrast, OEE1 transcriptional levels were slowly downregulated, and NIA2 transcriptional levels were downregulated 1xa0h after dehydration but returned to regular levels 4xa0h after. This suggests that 40xa0% dehydration of G. tenuistipitata induced transcriptional responses without changing the relative growth rate or affecting the thalli mortality.