Additional file 4: of Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7

Forward and reverse primers used to determine gene expression levels via quantitative real-time PCR. E-E-jn, exon-exon junction; E-I-b, exon-intron boundary; YLS, yellow-leaf-specific; UBQ10, ubiquitin; ACT, actin ; EF, elongation factor; APS, ATP sulfurylase; SULTR, sulfate transporter; LAC, laccase; CSD, Cu/Zn superoxide dismutase; FSD, Fe superoxide dismutase. *Pri-miRNA primer concentrations were increased to 900 nM. (DOCX 18 kb

Additional file 5: of Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7

Quantitative real-time PCR parameters according to the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines derived from Bustin et al. [51]. (DOCX 16 kb

Additional file 1: of Toxicity responses of Cu and Cd: the involvement of miRNAs and the transcription factor SPL7

Cq values of 180 pri-miRNAs in leaves and roots measured using an RT-qPCR platform. (XLSX 124 kb

Integration, Disintegration and the Protection of Competition: Of Myths, Stories and Images

OTU distribution across the plant compartments. Venn diagram showing the overlap in operational taxonomic unit (OTU) composition between the different plant compartments.(TIFF 471 kb

Table_2.XLSX

<p>Cadmium (Cd) pollution renders many soils across the world unsuited or unsafe for food- or feed-orientated agriculture. The main mechanism of Cd phytotoxicity is the induction of oxidative stress, amongst others through the depletion of glutathione. Oxidative stress can damage lipids, proteins, and nucleic acids, leading to growth inhibition or even cell death. The plant cell has a variety of tools to defend itself against Cd stress. First and foremost, cell walls might prevent Cd from entering and damaging the protoplast. Both the primary and secondary cell wall have an array of defensive mechanisms that can be adapted to cope with Cd. Pectin, which contains most of the negative charges within the primary cell wall, can sequester Cd very effectively. In the secondary cell wall, lignification can serve to immobilize Cd and create a tougher barrier for entry. Changes in cell wall composition are, however, dependent on nutrients and conversely might affect their uptake. Additionally, the role of ascorbate (AsA) as most important apoplastic antioxidant is of considerable interest, due to the fact that oxidative stress is a major mechanism underlying Cd toxicity, and that AsA biosynthesis shares several links with cell wall construction. In this review, modifications of the plant cell wall in response to Cd exposure are discussed. Focus lies on pectin in the primary cell wall, lignification in the secondary cell wall and the importance of AsA in the apoplast. Regarding lignification, we attempt to answer the question whether increased lignification is merely a consequence of Cd toxicity, or rather an elicited defense response. We propose a model for lignification as defense response, with a central role for hydrogen peroxide as substrate and signaling molecule.</p

Table_1.XLSX

<p>Cadmium (Cd) pollution renders many soils across the world unsuited or unsafe for food- or feed-orientated agriculture. The main mechanism of Cd phytotoxicity is the induction of oxidative stress, amongst others through the depletion of glutathione. Oxidative stress can damage lipids, proteins, and nucleic acids, leading to growth inhibition or even cell death. The plant cell has a variety of tools to defend itself against Cd stress. First and foremost, cell walls might prevent Cd from entering and damaging the protoplast. Both the primary and secondary cell wall have an array of defensive mechanisms that can be adapted to cope with Cd. Pectin, which contains most of the negative charges within the primary cell wall, can sequester Cd very effectively. In the secondary cell wall, lignification can serve to immobilize Cd and create a tougher barrier for entry. Changes in cell wall composition are, however, dependent on nutrients and conversely might affect their uptake. Additionally, the role of ascorbate (AsA) as most important apoplastic antioxidant is of considerable interest, due to the fact that oxidative stress is a major mechanism underlying Cd toxicity, and that AsA biosynthesis shares several links with cell wall construction. In this review, modifications of the plant cell wall in response to Cd exposure are discussed. Focus lies on pectin in the primary cell wall, lignification in the secondary cell wall and the importance of AsA in the apoplast. Regarding lignification, we attempt to answer the question whether increased lignification is merely a consequence of Cd toxicity, or rather an elicited defense response. We propose a model for lignification as defense response, with a central role for hydrogen peroxide as substrate and signaling molecule.</p

Characterization of bacterial communities associated with <i>Brassica napus</i> L. growing on a Zn-contaminated soil and their effects on root growth

<p>The interaction between plant growth-promoting bacteria (PGPB) and plants can enhance biomass production and metal tolerance of the host plants. This work aimed at isolating and characterizing the cultivable bacterial community associated with <i>Brassica napus</i> growing on a Zn-contaminated site, for selecting cultivable PGPB that might enhance biomass production and metal tolerance of energy crops. The effects of some of these bacterial strains on root growth of <i>B. napus</i> exposed to increasing Zn and Cd concentrations were assessed. A total of 426 morphologically different bacterial strains were isolated from the soil, the rhizosphere, and the roots and stems of <i>B. napus</i>. The diversity of the isolated bacterial populations was similar in rhizosphere and roots, but lower in soil and stem compartments. <i>Burkoholderia, Alcaligenes, Agrococcus, Polaromonas, Stenotrophomonas, Serratia, Microbacterium</i>, and <i>Caulobacter</i> were found as root endophytes exclusively. The inoculation of seeds with <i>Pseudomonas</i> sp. strains 228 and 256, and <i>Serratia</i> sp. strain 246 facilitated the root development of <i>B. napus</i> at 1,000 µM Zn. <i>Arthrobacter</i> sp. strain 222, <i>Serratia</i> sp. strain 246, and <i>Pseudomonas</i> sp. 228 and 262 increased the root length at 300 µM Cd.</p

Phylum-level PCR bias assessed using qPCR.

<p>Average PCR efficiencies were calculated for each phylum using 5 basidiomycetes, 5 ascomycetes, 2 glomeromycetes and 3 zygomycetes. Error bars represent standard errors. No significant differences between primer pairs and phyla were found at the 95% significance level.</p

Primers used in the current study.

<p>Primers are indicated as forward (F) or reverse (R). ITS86R contains a wrong base at the 3′ end. The <u>G</u> should be replaced by a C (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097629#s4" target="_blank">Discussion</a>).</p

Comparison and Validation of Some ITS Primer Pairs Useful for Fungal Metabarcoding Studies

<div><p>Current metabarcoding studies aiming to characterize microbial communities generally rely on the amplification and sequencing of relatively short DNA regions. For fungi, the internal transcribed spacer (ITS) region in the ribosomal RNA (rRNA) operon has been accepted as the formal fungal barcode. Despite an increasing number of fungal metabarcoding studies, the amplification efficiency of primers is generally not tested prior to their application in metabarcoding studies. Some of the challenges that metabarcoding primers should overcome efficiently are the amplification of target DNA strands in samples rich in non-target DNA and environmental pollutants, such as humic acids, that may have been co-extracted with DNA. In the current study, three selected primer pairs were tested for their suitability as fungal metabarcoding primers. The selected primer pairs include two primer pairs that have been frequently used in fungal metabarcoding studies (ITS1F/ITS2 and ITS3/ITS4) and a primer pair (ITS86F/ITS4) that has been shown to efficiently amplify the ITS2 region of a broad range of fungal taxa in environmental soil samples. The selected primer pairs were evaluated in a 454 amplicon pyrosequencing experiment, real-time PCR (qPCR) experiments and <i>in silico</i> analyses. Results indicate that experimental evaluation of primers provides valuable information that could aid in the selection of suitable primers for fungal metabarcoding studies. Furthermore, we show that the ITS86F/ITS4 primer pair outperforms other primer pairs tested in terms of <i>in silico</i> primer efficiency, PCR efficiency, coverage, number of reads and number of species-level operational taxonomic units (OTUs) obtained. These traits push the ITS86F/ITS4 primer pair forward as highly suitable for studying fungal diversity and community structures using DNA metabarcoding.</p></div