Neochloris oleoabundans cell walls have an altered composition when cultivated under different growing conditions

The impact that environmental factors have on the intracellular components of microalgae has been the focus of research for a number of decades. Despite that, their effects on the cell wall have received very little attention. In this study, we investigated how different growing conditions affect the cell walls of N. oleoabundans. The results revealed that the cell wall composition varied in that the modifications were different in the four cultivation media: freshwater nitrogen-replete (optimum culture) and -depleted conditions, and seawater nitrogen-replete and -depleted conditions. Nitrogen deficiency in freshwater cultivation was the only condition that significantly (p <.05) increased the total content of carbohydrates in the cell wall. The three most abundant components of freshwater-cultivated cell wall polysaccharides were rhamnose, galactose and glucuronic acid whereas in seawater media the main components of cell wall polysaccharides were rhamnose, glucose and galactose. The combined results of the biochemical analyses and monoclonal antibodies epitope-binding revealed that N. oleoabundans cell walls are likely composed of sulphated polysaccharides enriched in mannose, β-(1 → 4)-D-mannans, and glucose as they grow in seawater. Salinity and nitrogen deficiency also had an impact on the nitrogenous components of the cell wall. Under these conditions we observed a decrease in glucosamine in the cell wall. The analysis of specific binding of monoclonal antibodies, revealed that the cell wall of N. oleoabundans is possibly enriched in arabinogalactan proteins (AGPs). Under salinity and nitrogen deficiency N. oleoabundans increased the proportion of the non-polar to polar amino acids in the cell walls. An increase of leucine in the cell walls may suggest that N. oleoabundans contains leucine-rich repeat proteins which are known to play a vital role in stress responses. This report provides new insights into microalgae cell wall biology and how cell walls are remodelled when growing under different conditions.</p

Genotyping data underlying the publication: Investigating the potential of Andean lupin as a lignocellulosic feedstock for Europe: first genome-wide association study on L. mutabilis biomass quality.

This dataset contains the genotyping data obtained from RAD-sequencing of a panel of 223 Lupinus mutabilis accessions and the marker selection used to perform the genome-wide association study described in the publication "Investigating the potential of Andean lupin as a lignocellulosic feedstock for Europe: first genome-wide association study on L. mutabilis biomass quality"

Expression of an (engineered) 4,6-α-glucanotransferase in potato results in changes in starch characteristics

Starch structure strongly influences starch physicochemical properties, determining the end uses of starch in various applications. To produce starches with novel structure and exploit the mechanism of starch granule formation, an (engineered) 4, 6-α-glucanotransferase (GTFB) from Lactobacillus reuteri 121 was introduced into two potato genetic backgrounds: amylose-containing line Kardal and amylose-free mutant amf. The resulting starches showed severe changes in granule morphology regardless of genetic backgrounds. Modified starches from amf background exhibited a significant increase in granule size and starch phosphate content relative to the control, while starches from Kardal background displayed a higher digestibility, but did not show changes in granule size and phosphate content. Transcriptome analysis revealed the existence of a mechanism to restore the regular packing of double helices in starch granules, which possibly resulted in the removal of novel glucose chains potentially introduced by the (engineered) GTFB. This amendment mechanics would also explain the difficulties to detect alterations in starch fine structure in the transgenic lines.</p

Orphan Crops Browser : a bridge between model and orphan crops

Many important crops have received little attention by the scientific community, either because they are not considered economically important or due to their large and complex genomes. De novo transcriptome assembly, using next-generation sequencing data, is an attractive option for the study of these orphan crops. In spite of the large amount of sequencing data that can be generated, there is currently a lack of tools which can effectively help molecular breeders and biologists to mine this type of information. Our goal was to develop a tool that enables molecular breeders, without extensive bioinformatics knowledge, to efficiently study de novo transcriptome data from any orphan crop (http://www.bioinformatics.nl/denovobrowser/db/species/index). The Orphan Crops Browser has been designed to facilitate the following tasks (1) search and identification of candidate transcripts based on phylogenetic relationships between orthologous sequence data from a set of related species and (2) design specific and degenerate primers for expression studies in the orphan crop of interest. To demonstrate the usability and reliability of the browser, it was used to identify the putative orthologues of 17 known lignin biosynthetic genes from maize and sugarcane in the orphan crop Miscanthus sinensis. Expression studies in miscanthus stem internode tissue differing in maturation were subsequently carried out, to follow the expression of these genes during lignification. Our results showed a negative correlation between lignin content and gene expression. The present data are in agreement with recent findings in maize and other crops, and it is further discussed in this paper

Starch phosphorylation plays an important role in starch biosynthesis

Starch phosphate esters are crucial in starch metabolism and render valuable functionality to starches for various industrial applications. A potato glucan, water dikinase (GWD1) was introduced in tubers of two different potato genetic backgrounds: an amylose-containing line Kardal and the amylose-free mutant amf. In both backgrounds, this resulted in two contrasting effects, a number of plants showed higher phosphate content compared to the respective control, while others lines exhibited lower phosphate content, thereby generating two series of starches with broad-scale variation in phosphate content. The results of systematic analyses on these two series of starches revealed that starch phosphate content strongly influenced starch granule morphology, amylose content, starch fine structure, gelatinization characteristics and freeze-thaw stability of starch gels. Further analyses on the expression level of genes involved in starch metabolism suggested that starch phosphorylation regulates starch synthesis by controlling the carbon flux into starch while simultaneously modulating starch-synthesizing genes

Different levels of gene expression in transgenic lines.

<p>(a) Protein accumulation level and gene expression level in KDSGB transformants. The protein accumulation and gene expression level were determined using Western dot blot with an anti-SBD antibody and qRT-PCR analysis, respectively. The number above each dot stands for the different line, while UT represents the control. The intensity of the dots shows the various protein levels. The qRT-PCR analysis was performed in triplicate for each line and the relative expression level of target genes was transformed as described in the Materials and Methods. The resulting value (v) was used to divide transformants to different categories: undetectable (N, v = 0), low (L, 0 < v < 2), medium (M, 2≤ v < 2.5) and high (H, v ≥ 2.5) expressors. (b) Distribution of the individual transformants over the classes of gene expression level in KDGB, a<i>mf</i>GB and <i>amf</i>SGB transgenic series. SGB and GB represent SBD-GTFB fusion protein and GTFB alone, respectively. KD stands for the Kardal background, while <i>amf</i> stands for the <i>amf</i> background. The striped columns indicate the lines selected for further characterization. Differences between individual transformants are due to differences in the copy number and location of the genome where the transgene was inserted.</p

Schematic depiction of two different binary vector constructs (a) pBIN19/GB and (b) pBIN19/SGB.

<p>Genes were cloned in frame with GBSSI transit peptide to allow amyloplast targeting and were driven by the potato GBSSI promoter for tuber expression. RB and LB represent right and left borders, respectively. SBD, LK, Kan and 3’NOS stand for starch binding domain of cyclodextrin glycosyltransferase from <i>Bacillus circulans</i>, linker, kanamycin resistant gene and NOS terminator, respectively. <i>Sal</i>I, <i>Xba</i>I, <i>Hpa</i>I, <i>Nco</i>I and <i>Xba</i>I are restriction enzymes.</p

Starch granule size of starches from transgenic lines and control lines.

<p>(a) Boxplot presenting median granule size of modified starches and their respective controls. The measurements were performed on all transformants except N-expressors (lines with undetectable expression of target gene), which are 31, 23, 25 and 17 lines for KDGB, KDSGB, <i>amf</i>GB and <i>amf</i>SGB series, respectively. Boxes in the plot include values in the 25%–75% interval. Internal lines, filled circles, unfilled circles and bars represent the median, the mean, outliers and extremes, respectively. Statistical significance was analysed using one-way ANOVA. Different letters indicate statistically significant differences between means at <i>p</i> < 0.01. (b) Average particle size distribution of starches from <i>amf</i>SGB H-expressors (lines with high expression of target gene) and the control UT-<i>amf</i>. Each starch sample was analysed in duplicate.</p

The expression level of the genes encoding key enzymes involved in starch metabolism in (a) <i>amf</i> and (b) KD potatoes.

<p>Include: glucan, water-dikinase (<i>GWD</i>), starch phosphorylase (<i>SP</i>), isoamylase (<i>ISA1</i>, <i>ISA2</i> and <i>ISA3</i>), starch branching genes (<i>SBEI and SBEII</i>), starch synthase (<i>SSIII</i>), β-amylase (<i>BAM</i>) and ADP-glucose pyrophosphorylase (<i>AGPase</i>). Three/Four H-expressors (lines with high expression of target gene) of each series and the control lines were selected and subjected to qRT-PCR analysis. Statistical significance was analysed using t-test (*<i>p</i> < 0.05; **<i>p</i> < 0.01). The values are expressed as the mean ± SD from three independent measurements.</p