The Barley Genome Sequence Assembly Reveals Three Additional Members of the <i>CslF </i>(1,3;1,4)-b-Glucan Synthase Gene Family

An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β-glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls

Hv-CBF2A overexpression in barley accelerates COR gene transcript accumulation and acquisition of freezing tolerance during cold acclimation

Abstract C-Repeat Binding Factors (CBFs) are DNAbinding transcriptional activators of gene pathways imparting freezing tolerance. Poaceae contain three CBF subfamilies, two of which, HvCBF3/CBFIII and HvCBF4/CBFIV, are unique to this taxon. To gain mechanistic insight into HvCBF4/CBFIV CBFs we overexpressed Hv-CBF2A in spring barley (Hordeum vulgare) cultivar ‘Golden Promise’. The Hv-CBF2A overexpressing lines exhibited stunted growth, poor yield, and greater freezing tolerance compared to non-transformed ‘Golden Promise’. Differences in freezing tolerance were apparent only upon cold acclimation. During cold acclimation freezing tolerance of the Hv-CBF2A overexpressing lines increased more rapidly than that of ‘Golden Promise’ and paralleled the freezing tolerance of the winter hardy barley ‘Dicktoo’. Transcript levels of candidate CBF target genes, COR14B and DHN5 were increased in the overexpressor lines at warm temperatures, and at cold temperatures they accumulated to much higher levels in the Hv-CBF2A overexpressors than in ‘Golden Promise’. Hv-CBF2A overexpression also increased transcript levels of other CBF genes at FROST RESISTANCE-H2-H2 (FR-H2) possessing CRT/DRE sites in their upstream regions, the most notable of which was CBF12. CBF12 transcript levels exhibited a relatively constant incremental increase above levels in ‘Golden Promise’ both at warm and cold. These data indicate that Hv-CBF2A activates target genes at warm temperatures and that transcript accumulation for some of these targets is greatly enhanced by cold temperatures

The Glycosyltransferase Repertoire of the Spikemoss Selaginella moellendorffii and a Comparative Study of Its Cell Wall

Spike mosses are among the most basal vascular plants, and one species, Selaginella moellendorffii, was recently selected for full genome sequencing by the Joint Genome Institute (JGI). Glycosyltransferases (GTs) are involved in many aspects of a plant life, including cell wall biosynthesis, protein glycosylation, primary and secondary metabolism. Here, we present a comparative study of the S. moellendorffii genome across 92 GT families and an additional family (DUF266) likely to include GTs. The study encompasses the moss Physcomitrella patens, a non-vascular land plant, while rice and Arabidopsis represent commelinid and non-commelinid seed plants. Analysis of the subset of GT-families particularly relevant to cell wall polysaccharide biosynthesis was complemented by a detailed analysis of S. moellendorffii cell walls. The S. moellendorffii cell wall contains many of the same components as seed plant cell walls, but appears to differ somewhat in its detailed architecture. The S. moellendorffii genome encodes fewer GTs (287 GTs including DUF266s) than the reference genomes. In a few families, notably GT51 and GT78, S. moellendorffii GTs have no higher plant orthologs, but in most families S. moellendorffii GTs have clear orthologies with Arabidopsis and rice. A gene naming convention of GTs is proposed which takes orthologies and GT-family membership into account. The evolutionary significance of apparently modern and ancient traits in S. moellendorffii is discussed, as is its use as a reference organism for functional annotation of GTs

Response of cell wall composition and RNA-seq transcriptome to methyl-jasmonate in Brachypodium distachyon callus

Main conclusion: Methyl-jasmonate induces large increases in p-coumarate linked to arabinoxylan in Brachypodium and in abundance of GT61 and BAHD family transcripts consistent with a role in synthesis of this linkage. Jasmonic acid (JA) signalling is required for many stress responses in plants, inducing large changes in the transcriptome, including up-regulation of transcripts associated with lignification. However, less is known about the response to JA of grass cell walls and the monocot-specific features of arabinoxylan (AX) synthesis and acylation by ferulic acid (FA) and para-coumaric acid (pCA). Here, we show that methyl-jasmonate (MeJA) induces moderate increases in FA monomer, > 50% increases in FA dimers, and five–sixfold increases in pCA ester-linked to cell walls in Brachypodium callus. Direct measurement of arabinose acylated by pCA (Araf-pCA) indicated that most or all the increase in cell-wall pCA was due to pCA ester-linked to AX. Analysis of the RNA-seq transcriptome of the callus response showed that these cell-wall changes were accompanied by up-regulation of members of the GT61 and BAHD gene families implicated in AX decoration and acylation; two BAHD paralogues were among the most up-regulated cell-wall genes (seven and fivefold) after 24 h exposure to MeJA. Similar responses to JA of orthologous BAHD and GT61 transcripts are present in the RiceXPro public expression data set for rice seedlings, showing that they are not specific to Brachypodium or to callus. The large response of AX-pCA to MeJA may, therefore, indicate an important role for this linkage in response of primary cell walls of grasses to JA signalling

Better Than Nothing? Limitations of the Prediction Tool SecretomeP in the Search for Leaderless Secretory Proteins (LSPs) in Plants

In proteomic analyses of the plant secretome, the presence of putative leaderless secretory proteins (LSPs) is difficult to confirm due to the possibility of contamination from other sub-cellular compartments. In the absence of a plant-specific tool for predicting LSPs, the mammalian-trained SecretomeP has been applied to plant proteins in multiple studies to identify the most likely LSPs. This study investigates the effectiveness of using SecretomeP on plant proteins, identifies its limitations and provides a benchmark for its use. In the absence of experimentally verified LSPs we exploit the common-feature hypothesis behind SecretomeP and use known classically secreted proteins (CSPs) of plants as a proxy to evaluate its accuracy. We show that, contrary to the common-feature hypothesis, plant CSPs are a poor proxy for evaluating LSP detection due to variation in the SecretomeP prediction scores when the signal peptide (SP) is modified. Removing the SP region from CSPs and comparing the predictive performance against non-secretory proteins indicates that commonly used threshold scores of 0.5 and 0.6 result in false-positive rates in excess of 0.3 when applied to plants proteins. Setting the false-positive rate to 0.05, consistent with the original mammalian performance of SecretomeP, yields only a marginally higher true positive rate compared to false positives. Therefore the use of SecretomeP on plant proteins is not recommended. This study investigates the trade-offs of using SecretomeP on plant proteins and provides insights into predictive features for future development of plant-specific common-feature tools

Characterisation of Cellulose Synthase Like F6 (CslF6) Mutants Shows Altered Carbon Metabolism in beta-D-(1,3;1,4)-Glucan Deficient Grain in Brachypodium distachyon

Brachypodium distachyon is a small, fast growing grass species in the Pooideae subfamily that has become established as a model for other temperate cereals of agricultural significance, such as barley (Hordeum vulgare) and wheat (Triticum aestivum). The unusually high content in whole grains of β-D-(1,3;1,4)-glucan or mixed linkage glucan (MLG), considered a valuable dietary fibre due to its increased solubility in water compared with cellulose, makes B. distachyon an attractive model for these polysaccharides. The carbohydrate composition of grain in B. distachyon is interesting not only in understanding the synthesis of MLG, but more broadly in the mechanism(s) of carbon partitioning in cereal grains. Several mutants in the major MLG synthase, cellulose synthase like (CSL) F6, were identified in a screen of a TILLING population that show a loss of function in vitro. Surprisingly, loss of cslf6 synthase capacity appears to have a severe impact on survival, growth, and development in B. distachyon in contrast to equivalent mutants in barley and rice. One mutant, A656T, which showed milder growth impacts in heterozygotes shows a 21% (w/w) reduction in average grain MLG and more than doubling of starch compared with wildtype. The endosperm architecture of grains with the A656T mutation is altered, with a reduction in wall thickness and increased deposition of starch in larger granules than typical of wildtype B. distachyon. Together these changes demonstrate an alteration in the carbon storage of cslf6 mutant grains in response to reduced MLG synthase capacity and a possible cross-regulation with starch synthesis which should be a focus in future work in composition of these grains. The consequences of these findings for the use of B. distachyon as a model species for understanding MLG synthesis, and more broadly the implications for improving the nutritional value of cereal grains through alteration of soluble dietary fibre content are discussed

Seismological Experiment across the Wilkes Subglacial Basin and Adjacent Transantactic Mountains

During the Itali-Antartide XXI Antarctic expedition (October 2005 – February 2006), an experiment of passive seismology was carried out in the framework of the WISE Project. The main targets of the experiment, devoted to the recording of teleseismic events, were to: - reconstruct the deep structure (Moho and other possibile seismic discontinuities) of the Wilkes Subglacial Basin (WSB); - single out the presence and the tickness of a possibile sedimentary layer in the Wilkes Subglacial Basin (Ferraccioli et al., 2001; Studinges et al., 2004), thereby also providing independent constraints for the interpretation of new aerogeophysical data over the study region (Ferraccioli et a., this volume). Installation and maintenance was made in conjunction with the GDS (Geomagnetic Depth Sounding) team (Armadillo et al., this volume), as in each site there was a seismic and a GDS station. The transportation required the use of helicopters for the locations on the Transantactic Mountains (TAM) (S01 and S02) and of a Twin Otter for the other three sites (S03, S04 and S05). More than three hours flight were required to reach S05 from Mario Zuccheri Station (MZS). On the basis of the seismological results of the previous 2003-2004 WIBEM project, we decided to extend the exploration of the WSB further to the West, while repeating the data acquisition across the adjacent TAM, where these results were more uncertain (Piana Agostinetti et al., 2005). The data analysis is based on the Receiver Function (RF) tecnique (Langston, 1979; Piana Agostinetti et al., 2004) that allows deep crustal and upper mantle structure exploration on the basis of teleseismic data received by single stations. The RF is composite of P to S converted waves that reverberate in the structure beneath the seismometer (Ammon et al., 1990). The arrival times and the amplitudes of the converted phase depend on the depth of the velocity contrast under the frequency deconvolution of vertical from radial and transverse components. For estimating the sedimentary layer thickness, we will follow the approach developed by Anandakrishnan & Winberry (2004