Conservation of core complex subunits shaped the structure and function of photosystem I in the secondary endosymbiont alga Nannochloropsis gaditana

Photosystem I (PSI) is a pigment protein complex catalyzing the light-driven electron transport from plastocyanin to ferredoxin in oxygenic photosynthetic organisms. Several PSI subunits are highly conserved in cyanobacteria, algae and plants, whereas others are distributed differentially in the various organisms. Here we characterized the structural and functional properties of PSI purified from the heterokont alga Nannochloropsis gaditana, showing that it is organized as a supercomplex including a core complex and an outer antenna, as in plants and other eukaryotic algae. Differently from all known organisms, the N. gaditana PSI supercomplex contains five peripheral antenna proteins, identified by proteome analysis as type-R light-harvesting complexes (LHCr4-8). Two antenna subunits are bound in a conserved position, as in PSI in plants, whereas three additional antennae are associated with the core on the other side. This peculiar antenna association correlates with the presence of PsaF/J and the absence of PsaH, G and K in the N. gaditana genome and proteome. Excitation energy transfer in the supercomplex is highly efficient, leading to a very high trapping efficiency as observed in all other PSI eukaryotes, showing that although the supramolecular organization of PSI changed during evolution, fundamental functional properties such as trapping efficiency were maintained

Going ultra deep to unravel the secret recipe of biofuel

Microalgae are of great importance in ecology, biochemistry, and biotechnology. Nevertheless, just a few genomes have been sequenced so far, most of them by Sanger sequencing. While next generation sequencing techniques have revolutionized genome resequencing, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Microalgae contain fewer repetitive regions in their 30–100 Mb genomes than genomes of mammals or higher plants and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a draft sequence of the Nannochloropsis gaditana genome that was obtained by a combination of SOLiD and Roche 454 sequencing. Mate-Pair SOLiD sequencing of genomic DNA to 250-fold coverage and an additional 7-fold coverage by single-end 454 sequencing resulted in 15 Gb of raw sequence data. Reads were assembled to a 32 Mb draft version (N50 of 50kb) with the a pipeline of tools evolved in our group. 167 scaffolds were produced accounting for 18.7Mb. Our study supports the expectation that for typical microalgae, de novo assembly of genomes from short sequence reads alone is feasible, cheap and efficient; that a mixture of SOLiD and 454 sequencing substantially improves the assembly; and that the resulting data can be used for comparative studies and to provide a valuable framework to plan the application of recombinant techniques. Furthermore a whole transcriptome analyses was carried out to identify, characterize and catalogue all the transcripts expressed, exploiting the great potential of RNA-Seq using the SOLiD platform to determine the correct gene annotation, the identification and characterization of the splicing patterns and to obtain the structure of genes, also defining—at single nucleotide resolution—the transcriptional boundaries of genes and the expressed Single Nucleotide Polymorphisms (SNPs). Important technical innovations were also introduced in this work that allowed a precise mapping of the transcription start to support a robust prediction of the regulatory region on the genome sequence. RNA-Seq was also used to study the differential expression of transcripts in cultures able to accumulate substantially different amounts of lipids, in order to obtain insights on lipid metabolism of N.gaditana. The study was carried on using as input sequences for the SOLiD run both polyadenylated mRNA enriched fractions and ribo-depleted RNA samples that allowed to recovery also the plastidial mRNA. Our results shows how data obtained from a single SOLiD run, applying specific ad hoc variation on the standard protocols, provide enough coverage to support a valuable annotation of a completely new genome and to provide all the information necessary to underline the main features of pathways of interest if different biological samples are compared. The N. gaditana sequencing project fulfilled the two important aims of assembling a draft of the genome sequence using sole next generation short reads and providing a careful genome annotation, with the goal of a better understanding of N. gaditana biology and the idea of improving its value as a model organism for biotechnological applications related to biofuel production.Nonostante le microalghe rivestano una particolare importanza per l’ecologia, la biochimica e le biotecnologie, solo poche specie sono state sequenziate a tutt’oggi e per lo più nell’era del sequenziamento di tipo Sanger. Mentre i sequenziatori di nuova generazione hanno fornito straordinari mezzi al risequenziamento di genomi di singoli individue per cui il genoma della specie di riferimento era già disponibile, il sequenziamento di genomi eucariotici completamente nuovi, utilizzando sequenze corte, presenta ancora grandi difficoltà. Le principali difficoltà si riscontrano in fase di assemblaggio e sono principalmente dovute alle notevoli dimensioni dei genomi eucariotici e alla presenza di regioni ripetute. Le microalghe, nei loro genomi, grandi in genere dalle 30Mb alle 100Mb, contengono poche regioni a bassa complessità e costituiscono pertanto degli interessanti organismi sui quali sperimentare il sequenziamento ex novo utilizzando esclusivamente i sequenziatori di seconda generazione. In questo lavoro, descriviamo il sequenziamento e l’assemblaggio del genoma della microalga Nannochloropsis gaditana, ottenuto utilizzando le sequenze prodotte dal 454 della Roche e dal SOLiD. Il sequenziamento di ‘mate-pairs’ utilizzando il SOLiD ha prodotto una copertura di sequenza di circa 250 volte la grandezza del genoma, mentre il sequenziamento 454 è stato utilizzato per produrre una ulteriore copertura di 7 volte con sequenze di media lunghezza. Le sequenze sono state assemblate in una versione preliminare non del tutto finita di 32Mb, dove 18.7Mb sono state incluse in 167 grandi scaffolds. Il 50% degli scaffolds ottenuti è più grande di 50Kb, mentre un terzo del genoma è stato assemblato in circa 20 contigs. Il nostro lavoro conferma la previsione che le tecniche di nuova generazione sono adatte al sequenziamento del genoma di una microalga e consentono di ottenere risultati utili in un tempo più breve di quelli tradizionali e ad un minor costo. I dati ottenuti potranno esser utilizzati per analisi comparative del genoma e saranno anche un importante prerequisito per l’applicazione di tecniche ricombinati alla microalga di interesse biotecnologico. Inoltre, durante questo progetto, sono state portate avanti anche diverse analisi del trascrittoma tramite sequenziamento, finalizzate alla produzione di una lista di geni utile all’annotazione del genoma e all’identificazione di geni differenzialmente espressi in condizioni di accumulo di lipidi. Vengono presentate, in questo lavoro, anche alcune innovazioni tecniche che hanno permesso di sfruttare al meglio il sequenziamento SOLiD per produrre un’accurata annotazione della struttura del trascrittoma e una più completa analisi dei geni differenzialmente espressi codificati negli organelli. I risultati dimostrano che una sola corsa SOLiD su campioni preparati in modo adeguato, è sufficiente per l’annotazione accurata di un genoma completamente nuovo e per l’individuazione di geni differenzialmente espressi in condizioni di interesse.

Oceani e clima

Il percorso didattico che presentiamo si compone di quattro esperienze nelle quali si utilizzano conoscenze e competenze di chimica di diverso livello per studiare fenomeni naturali interessanti e rilevanti per l’educazione alla cittadinanza. Nelle varie esperienze le reazioni chimiche, l’acidità, il pH e gli equilibri chimici sono messi al servizio dello studio della ripartizione dell’anidride carbonica tra atmosfera e oceani in diverse condizioni, dei meccanismi di feedback climatico che coinvolgono gas serra e oceani, degli effetti dell’aumento della disponibilità di anidride carbonica sulla chimica degli oceani e sui viventi che li abitano. Le esperienze che presentiamo sono molto intuitive, possono essere usate in contesti didattici diversi e consentono di lavorare anche sul concetto di modello sperimentale

Acclimation of Nannochloropsis gaditana to different illumination regimes: effects on lipids accumulation

Algae are interesting potential sources of biodiesel, although research is still needed to develop efficient large scale productions. One major factor affecting productivity is light use efficiency. The effect of different light regimes on the seawater alga Nannochloropsis gaditana was accessed monitoring growth rate and photosynthetic performances. N. gaditana showed the capacity of acclimating to different light intensities, optimizing its photosynthetic apparatus to illumination. Thanks to this response, N. gaditana maintained similar growth rates under a wide range of irradiances, suggesting that this organism is a valuable candidate for outdoor productions in variable conditions. In the conditions tested here, without external CO(2) supply, light intensity alone was not found to be a major signal affecting lipids accumulation showing the absence of a direct regulatory link between the light stress and lipids accumulation. Strong illumination can nevertheless indirectly influences lipid accumulation if combined with other stresses or in the presence of excess CO(2)

ScaMPI: a program for genome Scaffolding using Mate Paired Information

Motivation: the revolution in sequencing technologies referred to as "Next Generation Sequencing" has enabled rapid genome sequencing at reduced costs. While it has become easier to obtain a \u201cdraft\u201d of a genome (that is usually highly fragmented into small contigs), producing a high quality genome assembly, with scaffolds spanning entire chromosomes, still presents hurdles and a lack of dedicated tools. Methods: ScaMPI is a comprehensive suite of programs to perform genome scaffolding using Mate Paired reads (in particular SOLiD color-space encoded reads). ScaMPI provides a greedyalgorithm for scaffolding with mate paired reads, a web - based interface to assist manual scaffolding and refinements of the assembly, and a set of tools for complementary tasks like contig consistency validation via physical coverage check, primer design, gap - closure, BAC - ends validation of the assembly and de novo telomere identification (TRAP, Telomeric Repeat Analysis Program). Results: the ScaMPI suite has been used to scaffold the contigs of a genome project of an oil - producing microalga (N. gaditana) sequenced with the 454 (N50: 40 kbp). ScaMPI automatically produced a set of scaffold (N50: 600 kbp) using two libraries of SOLiD mate pairs. The web interface has been used for manual refinements to produce a set of 58 scaffolds (N50: 1 Mbp). The telomere - identification module has been used to find telomere, thus discovering that 21 scaffolds were complete chromosomes (out of 30 estimated). Sequencing a set of 528 BAC - ends we found that 97% of them confirmed the assembly of 32 large scaffolds (accounting for 20 Mbp), while the remainder 3% did not disprove it

Genome Physical Mapping with Next Generation Sequencing

Next generation sequencing technology has considerably improved over the past few years, making easier and more affordable the shotgun sequencing approach. Short reads are particularly popular as they are very easy and cheap to produce. On the other hand, their assembly results in the generation of a vast number of relatively short contigs that would require suitable physical maps and scaffolding procedures to be further assembled in a draft genomic sequence. Unfortunately, physical maps are still very difficult to produce and take little advantage of the next generation sequencing technology. The aim of our project is to investigate the possibility to overcome this problem. The organism we chose as field test is Nannochloropsis gaditana a unicellular algae that could be very useful in biofuel production, because of its capacity to accumulate high amount of lipids under particular growth conditions, and because its genome is relatively small (32Mb). We obtained a BAC library of more than 11,000 clones with an average insert size of 120 kb. This BAC library is the starting point of our method: by selecting random clones from the library we produced 32 pools, each representing about 40% of the genome. Each pool was fragmented by sonication and sequenced with a SOLiD 5500XL. A high-coverage of the genome was also produced by an independent shotgun project. Non-repeated sequences (tags) can be identified taking into consideration their coverage and each of them can be considered as a genetic marker. The presence or absence of a tag in each pool can then be analyzed: the more two tags are close in the genome, the more they are expected to be present together in each pool. Analyzing the profiles of presence/absence of each tag in each pool it is possible to sort the tags according to their relative position, producing a high density and high quality physical map. We are currently in the process of analyzing 32 pools of 96 BAC clones randomly fragmented. A computer simulation indicated that 32 pools should be sufficient to produce a complete physical map of a genome equivalent to that of N. gaditana. If necessary, we could easily produce more pools. Larger genomes could also benefit from this approach, although the number of BACs per pool and the number of pools must be adjusted accordingly. The perspective of producing physical maps at a low cost is very important for the improvement of de novo assembly, and in particular for the scaffolding procedures that are now the limiting step of the entire process

Chromosome scale genome assembly and transcriptome profiling of Nannochloropsis gaditana in nitrogen depletion

Nannochloropsis is rapidly emerging as a model organism for the study of biofuel production in microalgae. Here, we report a high-quality genomic assembly of Nannochloropsis gaditana, consisting of large contigs, up to 500 kbp long, and scaffolds that in most cases span the entire length of the chromosomes. We identified 10646 complete genes and characterized possible alternative transcripts. The annotation of the predicted genes and the analysis of cellular processes revealed traits relevant for the genetic improvement of this organism such as genes involved in DNA recombination, RNA silencing, and cell wall synthesis. We also analyzed the modification of the transcriptional profile in nitrogen deficiency-a condition known to stimulate lipid accumulation. While the content of lipids increased, we did not detect major changes in expression of the genes involved in their biosynthesis. At the same time, we observed a very significant down-regulation of mitochondrial gene expression, suggesting that part of the Acetyl-CoA and NAD(P)H, normally oxidized through the mitochondrial respiration, would be made available for fatty acids synthesis, increasing the flux through the lipid biosynthetic pathway. Finally, we released an information resource of the genomic data of N. gaditana, available online at www.nannochloropsis.org

A deep survey of alternative splicing in grape reveals changes in the splicing machinery related to tissue, stress condition and genotype

Alternative splicing (AS) significantly enhances transcriptome complexity. It is differentially regulated in a wide variety of cell types and plays a role in several cellular processes. Here we describe a detailed survey of alternative splicing in grape based on 124 SOLiD RNAseq analyses from different tissues, stress conditions and genotypes.Results: We used the RNAseq data to update the existing grape gene prediction with 2,258 new coding genes and 3,336 putative long non-coding RNAs. Several gene structures have been improved and alternative splicing was described for about 30% of the genes. A link between AS and miRNAs was shown in 139 genes where we found that AS affects the miRNA target site. A quantitative analysis of the isoforms indicated that most of the spliced genes have one major isoform and tend to simultaneously co-express a low number of isoforms, typically two, with intron retention being the most frequent alternative splicing event.Conclusions: As described in Arabidopsis, also grape displays a marked AS tissue-specificity, while stress conditions produce splicing changes to a minor extent. Surprisingly, some distinctive splicing features were also observed between genotypes. This was further supported by the observation that the panel of Serine/Arginine-rich splicing factors show a few, but very marked differences between genotypes. The finding that a part the splicing machinery can change in closely related organisms can lead to some interesting hypotheses for evolutionary adaptation, that could be particularly relevant in the response to sudden and strong selective pressures