Proton Gradient Regulation5-Like1-Mediated Cyclic Electron Flow Is Crucial for Acclimation to Anoxia and Complementary to Nonphotochemical Quenching in Stress Adaptation

International audienceTo investigate the functional importance of Proton Gradient Regulation5-Like1 (PGRL1) for photosynthetic performances in the moss Physcomitrella patens, we generated a pgrl1 knockout mutant. Functional analysis revealed diminished nonphotochemical quenching (NPQ) as well as decreased capacity for cyclic electron flow (CEF) in pgrl1. Under anoxia, where CEF is induced, quantitative proteomics evidenced severe down-regulation of photosystems but up-regulation of the chloroplast NADH dehydrogenase complex, plastocyanin, and Ca(2+) sensors in the mutant, indicating that the absence of PGRL1 triggered a mechanism compensatory for diminished CEF. On the other hand, proteins required for NPQ, such as light-harvesting complex stress-related protein1 (LHCSR1), violaxanthin de-epoxidase, and PSII subunit S, remained stable. To further investigate the interrelation between CEF and NPQ, we generated a pgrl1 npq4 double mutant in the green alga Chlamydomonas reinhardtii lacking both PGRL1 and LHCSR3 expression. Phenotypic comparative analyses of this double mutant, together with the single knockout strains and with the P. patens pgrl1, demonstrated that PGRL1 is crucial for acclimation to high light and anoxia in both organisms. Moreover, the data generated for the C. reinhardtii double mutant clearly showed a complementary role of PGRL1 and LHCSR3 in managing high light stress response. We conclude that both proteins are needed for photoprotection and for survival under low oxygen, underpinning a tight link between CEF and NPQ in oxygenic photosynthesis. Given the complementarity of the energy-dependent component of NPQ (qE) and PGRL1-mediated CEF, we suggest that PGRL1 is a capacitor linked to the evolution of the PSII subunit S-dependent qE in terrestrial plants

Photophysics and Chemistry of Nitrogen-Doped Carbon Nanodots with High Photoluminescence Quantum Yield

Fluorescent carbon nanodots (CNDs) are very promising nanomaterials for a broad range of applications because of their high photostability, presumed selective luminescence, and low cost at which they can be produced. In this respect, CNDs are superior to well-established semiconductor quantum dots and organic dyes. However, reported synthesis protocols for CNDs typically lead to low photoluminescence quantum yield (PLQY) and low reproducibility, resulting in a poor understanding of the CND chemistry and photophysics. Here, we report a one-step synthesis of nitrogen-doped carbon nanodots (N-CNDs) from various carboxylic acids, Tris, and ethylenediaminetetraacetic acid resulting in high PLQY of up to 90%. The reaction conditions in terms of starting materials, temperature, and reaction time are carefully optimized and their influence on the photophysical properties is characterized. We find that citric acid-derived N-CNDs can result in a very high PLQY of 90%, but they do not show selective luminescence. By contrast, acetic acid-derived N-CNDs show selective luminescence but a PLQY of 50%. The chemical composition of the surface and core of these two selected N-CND types is characterized among others by high-resolution synchrotron X-ray photoelectron spectroscopy using single isolated N-CND clusters. The results indicate that photoexcitation occurs in the N-CND core, whereas the emission properties are determined by the N-CND surface groups

Deciphering the Cryptic Genome: Genome-wide Analyses of the Rice Pathogen <i>Fusarium fujikuroi</i> Reveal Complex Regulation of Secondary Metabolism and Novel Metabolites

<div><p>The fungus <i>Fusarium fujikuroi</i> causes “bakanae” disease of rice due to its ability to produce gibberellins (GAs), but it is also known for producing harmful mycotoxins. However, the genetic capacity for the whole arsenal of natural compounds and their role in the fungus' interaction with rice remained unknown. Here, we present a high-quality genome sequence of <i>F. fujikuroi</i> that was assembled into 12 scaffolds corresponding to the 12 chromosomes described for the fungus. We used the genome sequence along with ChIP-seq, transcriptome, proteome, and HPLC-FTMS-based metabolome analyses to identify the potential secondary metabolite biosynthetic gene clusters and to examine their regulation in response to nitrogen availability and plant signals. The results indicate that expression of most but not all gene clusters correlate with proteome and ChIP-seq data. Comparison of the <i>F. fujikuroi</i> genome to those of six other fusaria revealed that only a small number of gene clusters are conserved among these species, thus providing new insights into the divergence of secondary metabolism in the genus <i>Fusarium</i>. Noteworthy, GA biosynthetic genes are present in some related species, but GA biosynthesis is limited to <i>F. fujikuroi</i>, suggesting that this provides a selective advantage during infection of the preferred host plant rice. Among the genome sequences analyzed, one cluster that includes a polyketide synthase gene (<i>PKS19</i>) and another that includes a non-ribosomal peptide synthetase gene (<i>NRPS31</i>) are unique to <i>F. fujikuroi</i>. The metabolites derived from these clusters were identified by HPLC-FTMS-based analyses of engineered <i>F. fujikuroi</i> strains overexpressing cluster genes. <i>In planta</i> expression studies suggest a specific role for the <i>PKS19</i>-derived product during rice infection. Thus, our results indicate that combined comparative genomics and genome-wide experimental analyses identified novel genes and secondary metabolites that contribute to the evolutionary success of <i>F. fujikuroi</i> as a rice pathogen.</p></div

Expression pattern^a of the secondary metabolite biosynthetic gene clusters under four growth conditions.

a<p>+++, >90% of the genes belonging to the cluster are expressed under the condition indicated. ++, 50–90% of the genes belonging to the cluster are expressed under the condition indicated. +, 25–50% of the genes belonging to the cluster are expressed under the condition indicated. −, 0–25% of the genes belonging to the cluster are expressed under the condition indicated.</p>b<p>DTC and STC indicate diterpene synthase and sesquiterpene synthase, respectively.</p><p>Key enzymes of which the respective product is known are indicated in bold letters and the respective metabolites are listed; n/k indicates that the corresponding metabolite is not yet known. Red labeled key enzymes and corresponding metabolites are <i>Fusarium fujikuroi</i>-specific.</p

Changes in levels of selected proteins encoded by SM biosynthetic genes in <i>F. fujikuroi</i> as determined by comparative (−N/+N) quantitative proteomics.

<p><b>A</b>: Increased (blue) and decreased (yellow) protein levels in response to nitrogen availability. Protein levels shown in columns A and B are data from independent experiments. Values are shown for only proteins quantified in both experiments. A log₂ ratio>0 (−N/+N) indicates an increase in abundance in the low-nitrogen condition; a log₂ ratio<0 indicates a decrease in abundance in the low-nitrogen condition; and a log₂ ratio of zero indicates no changes in protein levels. Boxes with an asterisk indicate, that this protein could only be quantified in one nitrogen condition. The numbers in the far right column indicate how many proteins could be quantified within a cluster; value to the left of the slash is from replicate A, and value after the slash is from replicate B. <b>B</b>: Key to heat map showing Log₂ values that correspond to different shades of blue and yellow. Standard deviation of a ratio is reflected in the size of the blue and yellow boxes.</p

Infection of roots of rice plants by GA-producing wild-type (IMI58289) and GA-deficient (SG139) strains of <i>F. fujikuroi</i>.

<p>Rice plants were co-cultivated with either strain of <i>F. fujikuroi</i> for 7 days at 28°C and 80% humidity. Images of the corresponding whole plants following the incubation period are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g001" target="_blank"><b>Figure 1Bf</b></a>. Both the wild-type strain and GA-deficient mutant were engineered to express the dsRed fluorescent protein. Stars indicate the position of cells that have been penetrated by and are filled with hyphae of <i>F. fujikuroi</i>. <b>A</b>: Microscopic overviews (10-fold magnification) of a root infected with the mutant SG139 (above) or wild-type. Note the absence of invaded cells in the root infected with the GA-deficient mutant. Images are an overlay of images from brightfield and Texas Red filter, which captures fluorescence emitted by the DsRed protein. <b>B/C</b>: 40-fold (B) and 63-fold (C) magnified images of roots infected with GA-deficient mutant (left) or wild-type strain (right). In most cases, hyphae of the mutant strain were observed between cells, in ‘intercellular’ spaces, whereas wild-type hyphae were often observed inside the cells (indicated by stars; see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g001" target="_blank">Figure 1Bd</a>) as well as in intercellular spaces. White lines in the images are scale bars corresponding to 200 µm (B) or 50 µm (C). <b>D</b>: Quantification of penetration events per rice root infected with wild-type or SG139, respectively. In addition, the total number of shots per root taken for analysis as well as penetration events of the mutant compared to the wild-type are shown.</p

Occurrence of selected gene families and other genetic elements in genome sequences of seven <i>Fusarium</i> species.

a<p>SM gene predictions are based on InterPro domains, manually validated and corrected based on reports in the literature and on comparative analysis of fusaria. Values are the number of genes per genome; values in brackets are the numbers of genes unique to a genome among the species examined. Predicted genes were regarded as unique when they had either no match or an e-value≥10⁻¹⁰ in BLASTN analysis against available <i>Fusarium</i> genome sequences. PKS - polyketide synthase, NRPS - non-ribosomal peptide synthetase, DMATS - dimethylallyl tryptophan synthase, TC – terpene cyclase.</p

Location of the NRPS/APS biosynthetic gene cluster on <i>F. fujikuroi</i> chromosome I, levels of histone modifications and gene expression within and flanking the cluster, and production of metabolites following overexpression of cluster genes <i>APS2</i> and <i>APS8</i>.

<p><b>A</b>: Synteny between the apicidin gene cluster in <i>F. semitectum </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475-Han2" target="_blank">[105]</a> and the apicidin-like gene cluster in <i>F. fujikuroi</i>. <b>B</b>: Histone modifications and gene expression in and flanking the cluster. Histone marks are described in the legend to <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat-1003475-g003" target="_blank">Figure 3</a>. Expression data were derived from microarray experiments in low and high nitrogen and are plotted as the changes in log₂ expression values in high-nitrogen medium compared to low-nitrogen medium. H3K9ac and gene expression are overall correlated, as both are increased under high nitrogen conditions. In some genes increased H3K4me2 was observed, also suggesting transcription. <b>C</b>: Chemical analysis of the product of the unique PKS19 gene cluster. The traces show the extracted ion chromatograms for [C₃₄H₄₈O₆N₅+H]⁺ (first line) and [C₃₅H₄₂O₇N₅+H]⁺ (second to fourth line) determined by HPLC-FTMS of an apicidin standard (first line) and of culture fluids from <i>F. fujikuroi</i> IMI58289, OE::APS8 and the OE::APS2/OE::APS8 mutant. <b>D</b>: UV spectra of apicidin and the apicidin-like compound. The similar spectra suggest a structural similarity.</p

Whole genome comparison of <i>F. fujikuroi</i> with<i>F. verticillioides</i>.

<p>Dotplot of <i>F. fujikuroi</i> chromosomes and scaffolds against <i>F. verticillioides</i> calculated using MUMer <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003475#ppat.1003475-Delcher1" target="_blank">[121]</a> highlights overall collinearity. Orthologous DNA is represented by red dots, inverted segments are shown as blue dots. Inset magnifies <i>F. fujikuroi</i> chromosome XII, which has no homologue in the <i>F. verticillioides</i> scaffold set. The missing subtelomeric regions of chromosome IV in <i>F. fujikuroi</i> are highlighted by vertical purple lines. Dots that are located above or below the line indicating collinearity represent largely repetitive DNA.</p