Differential accumulation of soluble proteins in roots of metallicolous and nonmetallicolous populations of Agrostis capillaris L. exposed to Cu.

Differential expression of soluble proteins was explored in roots of metallicolous (M) and non-M (NM) plants of Agrostis capillaris L. exposed to increasing Cu to partially identify molecular mechanisms underlying higher Cu tolerance in M plants. Plants were cultivated for 2 months on perlite with a CuSO4 (1-30 µM) spiked-nutrient solution. Soluble proteins extracted by the trichloroacetic acid/acetone procedure were separated with 2DE (linear 4-7 pH gradient). After Coomassie Blue staining and image analysis, 19 proteins differentially expressed were identified using LC-MS/MS and Expressed Sequence Tag (ESTs) databases. At supra-optimal Cu exposure (15-30 µM), glycolysis was likely altered in NM roots with increased production of glycerone-P and methylglyoxal based on overexpression of triosephosphate isomerase and fructose bisphosphate aldolase. Changes in tubulins and higher expressions of 5-methyltetrahydropteroyltriglutamatehomocysteine methyltransferase and S-adenosylmethionine synthase underpinned impacts on the cytoskeleton and stimulation of ethylene metabolism. Increased l-methionine and S-adenosylmethionine amounts may also facilitate production of nicotianamine, which complexes Cu, and of l-cysteine, needed for metallothioneins and GSH. In M roots, the increase of [Cu/Zn] superoxide dismutase suggested a better detoxification of superoxide, when Cu exposure rose. Higher Cu-tolerance of M plants would rather result from simultaneous cooperation of various processes than from a specific mechanism.

Proteomic plasticity of two Eucalyptus genotypes under contrasted water regimes in the field.

Water deficit affects tree growth and limits wood production. In an attempt to identify the molecular triggers of adaptation mechanisms to water deficit in Eucalyptus, we investigated protein expression patterns of two ecophysiologically contrasted Eucalyptus genotypes. They were grown in the field in either natural conditions or irrigated for 7 weeks during the dry season in the Republic of Congo. At the phenotypic level, genotype (G), treatment (T) and/or G × T interaction effects were observed for above- and below-ground biomass-related traits. At the molecular level, changes in protein abundance were recorded in leaves (acidic pH 4-7, and basic pH 7-11, proteomes) and stems (acidic proteome) using two-dimensional gel electrophoresis (2-DE). One third of the detected protein spots displayed significant G, T and/or G × T effects, and 158 of them were identified by tandem mass spectrometry (LC-MS/MS) analysis. Thus, several proteins whose molecular plasticity was genetically controlled (i.e. G × T effect) were revealed, highlighting adaptive mechanisms to water deficit specific to each genotype, namely cell wall modification, cell detoxification and osmoregulation. Transcript abundances corresponding to G × T proteins were also investigated by quantitative RT-PCR. These proteins represent relevant targets to improve drought resistance in this ecologically and economically important forest tree genus.

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine.

BACKGROUND: Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (Pinus pinaster Ait.), the main conifer used for commercial plantation in southwestern Europe. RESULTS: We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 in vitro SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 in silico SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for in silico and in vitro SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a Pinus taeda linkage map, made it possible to align the 12 linkage groups of both species. CONCLUSIONS: Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.

Chloroplast DNA variation in a hyperdiverse tropical tree community.

We investigate chloroplast DNA variation in a hyperdiverse community of tropical rainforest trees in French Guiana, focusing on patterns of intraspecific and interspecific variation. We test whether a species genetic diversity is higher when it has congeners in the community with which it can exchange genes and if shared haplotypes are more frequent in genetically diverse species, as expected in the presence of introgression.We sampled a total of 1,681 individual trees from 472 species corresponding to 198 genera and sequenced them at a noncoding chloroplast DNA fragment.Polymorphism was more frequent in species that have congeneric species in the study site than in those without congeners (30% vs. 12%). Moreover, more chloroplast haplotypes were shared with congeners in polymorphic species than in monomorphic ones (44% vs. 28%).Despite large heterogeneities caused by genus-specific behaviors in patterns of hybridization, these results suggest that the higher polymorphism in the presence of congeners is caused by local introgression rather than by incomplete lineage sorting. Our findings suggest that introgression has the potential to drive intraspecific genetic diversity in species-rich tropical forests.

R-Syst::diatom: an open-access and curated barcode database for diatoms and freshwater monitoring.

Diatoms are micro-algal indicators of freshwater pollution. Current standardized methodologies are based on microscopic determinations, which is time consuming and prone to identification uncertainties. The use of DNA-barcoding has been proposed as a way to avoid these flaws. Combining barcoding with next-generation sequencing enables collection of a large quantity of barcodes from natural samples. These barcodes are identified as certain diatom taxa by comparing the sequences to a reference barcoding library using algorithms. Proof of concept was recently demonstrated for synthetic and natural communities and underlined the importance of the quality of this reference library. We present an open-access and curated reference barcoding database for diatoms, called R-Syst::diatom, developed in the framework of R-Syst, the network of systematic supported by INRA (French National Institute for Agricultural Research), see http://www.rsyst.inra.fr/en. R-Syst::diatom links DNA-barcodes to their taxonomical identifications, and is dedicated to identify barcodes from natural samples. The data come from two sources, a culture collection of freshwater algae maintained in INRA in which new strains are regularly deposited and barcoded and from the NCBI (National Center for Biotechnology Information) nucleotide database. Two kinds of barcodes were chosen to support the database: 18S (18S ribosomal RNA) and rbcL (Ribulose-1,5-bisphosphate carboxylase/oxygenase), because of their efficiency. Data are curated using innovative (Declic) and classical bioinformatic tools (Blast, classical phylogenies) and up-to-date taxonomy (Catalogues and peer reviewed papers). Every 6 months R-Syst::diatom is updated. The database is available through the R-Syst microalgae website (http://www.rsyst.inra.fr/) and a platform dedicated to next-generation sequencing data analysis, virtual_BiodiversityL@b (https://galaxy-pgtp.pierroton.inra.fr/). We present here the content of the library regarding the number of barcodes and diatom taxa. In addition to these information, morphological features (e.g. biovolumes, chloroplasts…), life-forms (mobility, colony-type) or ecological features (taxa preferenda to pollution) are indicated in R-Syst::diatom. Database URL: http://www.rsyst.inra.fr/.

Interaction between environmental factors affects the accumulation of root proteins in hydroponically grown Eucalyptus globulus (Labill.).

Eucalyptus globulus (Labill.) is used for pulp and paper production worldwide. In this report we studied changes in protein expression in one osmotically stressed elite clone widely used in industrial plantations in Spain. High molecular weight polyethylene glycol (PEG) was used as an osmoticum in the growing medium. Roots of rooted cuttings were sampled after 3 and 36 h of treatment. Water potential and abscissic acid content were measured in shoot and root apices to characterize the physiological states of the plants. Total soluble proteins from roots were extracted and separated using two-dimensional gel electrophoresis (2-DE). Gels were stained with Coomassie brillant blue for quantitative analysis of protein accumulation. From a total of 406 reproducible spots, 34 were found to be differentially expressed depending on treatment (osmotic versus control condition) and/or stress duration (3 h versus 36 h), and were further characterized by tandem mass spectrometry. Several proteins were reliably identified including adenosine kinase, actin, stress-related proteins as well as proteins associated to cellular processes, among which some residents of the endoplasmic reticulum. This study constitutes the first investigation of the root proteome in this important forest tree genus.

A micromethod for high throughput RNA extraction in forest trees.

A large quantity of high quality RNA is often required in the analysis of gene expression. However, RNA extraction from samples taken from woody plants is generally complex, and represents the main limitation to study gene expression, particularly in refractory species like conifers. Standard RNA extraction protocols are available but they are highly time consuming, and not adapted to large scale extraction. Here we present a high-throughput RNA extraction protocol. This protocol was adapted to a micro-scale by modifying the classical cetyltrimethylammonium (CTAB) protocol developed for pine: (i) quantity of material used (100-200 mg of sample), (ii) disruption of samples in microtube using a mechanical tissue disrupter, and (iii) the use of SSTE buffer. One hundred samples of woody plant tissues/organs can be easily treated in two working days. An average of 15 \ig of high quality RNA per sample was obtained. The RNA extracted is suitable for applications such as real time reverse transcription polymerase chain reaction, cDNA library construction or synthesis of complex targets for microarray analysis.

Automated SNP detection in expressed sequence tags: statistical considerations and application to maritime pine sequences.

We developed an automated pipeline for the detection of single nucleotide polymorphisms (SNPs) in expressed sequence tag (EST) data sets, by combining three DNA sequence analysis programs: Phred, Phrap and PolyBayes. This application requires access to the individual electrophoregram traces. First, a reference set of 65 SNPs was obtained from the sequencing of 30 gametes in 13 maritime pine (Pinus pinaster Ait.) gene fragments (6671 bp), resulting in a frequency of 1 SNP every 102.6 bp. Second, parameters of the three programs were optimized in order to retrieve as many true SNPs, while keeping the rate of false positive as low as possible. Overall, the efficiency of detection of true SNPs was 83.1%. However, this rate varied largely as a function of the rare SNP allele frequency: down to 41% for rare SNP alleles (frequency < 10%), up to 98% for allele frequencies above 10%. Third, the detection method was applied to the 18498 assembled maritime pine (Pinus pinaster Ait.) ESTs, allowing to identify a total of 1400 candidate SNPs, in contigs containing between 4 and 20 sequence reads. These genetic resources, described for the first time in a forest tree species, were made available at http://www.pierroton.inra/genetics/Pinesnps. We also derived an analytical expression for the SNP detection probability as a function of the SNP allele frequency, the number of haploid genomes used to generate the EST sequence database, and the sample size of the contigs considered for SNP detection. The frequency of the SNP allele was shown to be the main factor influencing the probability of SNP detection.

A micromethod for high throughput RNA extraction in forest trees

A large quantity of high quality RNA is often required in the analysis of gene expression. However, RNA extraction from samples taken from woody plants is generally complex, and represents the main limitation to study gene expression, particularly in refractory species like conifers. Standard RNA extraction protocols are available but they are highly time consuming, and not adapted to large scale extraction. Here we present a high-throughput RNA extraction protocol. This protocol was adapted to a micro-scale by modifying the classical cetyltrimethylammonium (CTAB) protocol developed for pine: (i) quantity of material used (100-200 mg of sample), (ii) disruption of samples in microtube using a mechanical tissue disrupter, and (iii) the use of SSTE buffer. One hundred samples of woody plant tissues/organs can be easily treated in two working days. An average of 15 /ig of high quality RNA per sample was obtained. The RNA extracted is suitable for applications such as real time reverse transcription polymerase chain reaction, cDNA library construction or synthesis of complex targets for microarray analysis.