Publications

Schreiber KJ, Austin RS, Gong Y, Zhang J, Fung P, Wang PW, Guttman DS, Desveaux D

BMC Plant Biol. 2012;12:226

PubMed PMID: 23176361

Abstract

BACKGROUND: The sulfanilamide family comprises a clinically important group of antimicrobial compounds which also display bioactivity in plants. While there is evidence that sulfanilamides inhibit folate biosynthesis in both bacteria and plants, the complete network of plant responses to these compounds remains to be characterized. As such, we initiated two forward genetic screens in Arabidopsis in order to identify mutants that exhibit altered sensitivity to sulfanilamide compounds. These screens were based on the growth phenotype of seedlings germinated in the presence of the compound sulfamethoxazole (Smex).

RESULTS: We identified a mutant with reduced sensitivity to Smex, and subsequent mapping indicated that a gene encoding 5-oxoprolinase was responsible for this phenotype. A mutation causing enhanced sensitivity to Smex was mapped to a gene lacking any functional annotation.

CONCLUSIONS: The genes identified through our forward genetic screens represent novel mediators of Arabidopsis responses to sulfanilamides and suggest that these responses extend beyond the perturbation of folate biosynthesis.

Ness RW, Siol M, Barrett SC

BMC Genomics 2012;13:611

PubMed PMID: 23145563

Abstract

BACKGROUND: Transitions from cross- to self-fertilization are associated with increased genetic drift rendering weakly selected mutations effectively neutral. The effect of drift is predicted to reduce selective constraints on amino acid sequences of proteins and relax biased codon usage. We investigated patterns of nucleotide variation to assess the effect of inbreeding on the accumulation of deleterious mutations in three independently evolved selfing plants. Using high-throughput sequencing, we assembled the floral transcriptomes of four individuals of Eichhornia (Pontederiaceae); these included one outcrosser and two independently derived selfers of E. paniculata, and E. paradoxa, a selfing outgroup. The dataset included ~8000 loci totalling ~3.5 Mb of coding DNA.

RESULTS: Tests of selection were consistent with purifying selection constraining evolution of the transcriptome. However, we found an elevation in the proportion of non-synonymous sites that were potentially deleterious in the E. paniculata selfers relative to the outcrosser. Measurements of codon usage in high versus low expression genes demonstrated reduced bias in both E. paniculata selfers.

CONCLUSIONS: Our findings are consistent with a small reduction in the efficacy of selection on protein sequences associated with transitions to selfing, and reduced selection in selfers on synonymous changes that influence codon usage.

Peek J, Garcia C, Lee J, Christendat D

Biochim. Biophys. Acta 2013 Feb;1834(2):516-23

PubMed PMID: 23142411

Abstract

The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD(+) instead of NADP(+) as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD(+) provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD(+)-dependent member of the SDH family.

Macleod G, Varmuza S

Biochemistry 2012 Dec;51(48):9678-88

PubMed PMID: 23140390

Abstract

Members of the PP1 family of protein phosphatases achieve functional diversity through numerous and varied protein-protein interactions. In mammals, there are four PP1 isoforms, the ubiquitously expressed PPP1CA, PPP1CB, and PPP1CC1, and the testis specific splice isoform PPP1CC2. When the mouse Ppp1cc gene is deleted, the only phenotypic consequence is a failure of spermatogenesis in homozygous males. To elucidate the function of the Ppp1cc gene, we sought to identify novel protein-protein interactions. To this end, we have created SBP-3XFLAG-PPP1CC1 and SBP-3XFLAG-PPP1CC2 knock-in mouse embryonic stem cell lines using a gene-trap-based system. Tandem affinity purification using our knock-in cell lines identified 11 significant protein-protein interactions, including nine known PP1 interacting proteins and two additional proteins (ATP5C1 and DDOST). Reciprocal in vitro sedimentation assays confirmed the interaction between PPP1CC2 and DDOST that may have physiological implications in spermatogenesis. Immunolocalization studies revealed that DDOST localized to the nuclear envelope in dissociated spermatogenic cells and persists throughout spermatogenesis. The knock-in system described in this paper can be applied in creating tandem affinity-tagged knock-in embryonic stem cell lines with any gene for which a compatible gene-trap line is available.

Capron A, Chang XF, Hall H, Ellis B, Beatson RP, Berleth T

J. Exp. Bot. 2013 Jan;64(1):185-97

PubMed PMID: 23136168

Abstract

Fibre properties and the biochemical composition of cell walls are important traits in many applications. For example, the lengths of fibres define the strength and quality of paper, and lignin content is a critical parameter for the use of biomass in biofuel production. Identifying genes controlling these traits is comparatively difficult in woody species, because of long generation times and limited amenability to high-resolution genetic mapping. To address this problem, this study mapped quantitative trait loci (QTLs) defining fibre length and lignin content in the Arabidopsis recombinant inbred line population Col-4 × Ler-0. Adapting high-throughput phenotyping techniques for both traits for measurements in Arabidopsis inflorescence stems identified significant QTLs for fibre length on chromosomes 2 and 5, as well as one significant QTL affecting lignin content on chromosome 2. For fibre length, total variation within the population was 208% higher than between parental lines and the identified QTLs explained 50.58% of the observed variation. For lignin content, the values were 261 and 26.51%, respectively. Bioinformatics analysis of the associated intervals identified a number of candidate genes for fibre length and lignin content. This study demonstrates that molecular mapping of QTLs pertaining to wood and fibre properties is possible in Arabidopsis, which substantially broadens the use of Arabidopsis as a model species for the functional characterization of plant genes.

Weadick CJ, Chang BS

BMC Evol. Biol. 2012;12:206

PubMed PMID: 23078361

Abstract

BACKGROUND: Gene duplications play an important role in the evolution of functional protein diversity. Some models of duplicate gene evolution predict complex forms of paralog divergence; orthologous proteins may diverge as well, further complicating patterns of divergence among and within gene families. Consequently, studying the link between protein sequence evolution and duplication requires the use of flexible substitution models that can accommodate multiple shifts in selection across a phylogeny. Here, we employed a variety of codon substitution models, primarily Clade models, to explore how selective constraint evolved following the duplication of a green-sensitive (RH2a) visual pigment protein (opsin) in African cichlids. Past studies have linked opsin divergence to ecological and sexual divergence within the African cichlid adaptive radiation. Furthermore, biochemical and regulatory differences between the RH2a? and RH2a? paralogs have been documented. It thus seems likely that selection varies in complex ways throughout this gene family.

RESULTS: Clade model analysis of African cichlid RH2a opsins revealed a large increase in the nonsynonymous-to-synonymous substitution rate ratio (?) following the duplication, as well as an even larger increase, one consistent with positive selection, for Lake Tanganyikan cichlid RH2a? opsins. Analysis using the popular Branch-site models, by contrast, revealed no such alteration of constraint. Several amino acid sites known to influence spectral and non-spectral aspects of opsin biochemistry were found to be evolving divergently, suggesting that orthologous RH2a opsins may vary in terms of spectral sensitivity and response kinetics. Divergence appears to be occurring despite intronic gene conversion among the tandemly-arranged duplicates.

CONCLUSIONS: Our findings indicate that variation in selective constraint is associated with both gene duplication and divergence among orthologs in African cichlid RH2a opsins. At least some of this variation may reflect an adaptive response to differences in light environment. Interestingly, these patterns only became apparent through the use of Clade models, not through the use of the more widely employed Branch-site models; we suggest that this difference stems from the increased flexibility associated with Clade models. Our results thus bear both on studies of cichlid visual system evolution and on studies of gene family evolution in general.

Campitelli BE, Stinchcombe JR

Mol. Ecol. 2013 Feb;22(3):552-64

PubMed PMID: 23061399

Abstract

Clines in phenotypic traits with an underlying genetic basis potentially implicate natural selection. However, neutral evolutionary processes such as random colonization, spatially restricted gene flow, and genetic drift could also result in similar spatial patterns, especially for single-locus traits because of their susceptibility to stochastic events. One way to distinguish between adaptive and neutral mechanisms is to compare the focal trait to neutral genetic loci to determine whether neutral loci demonstrate clinal variation (consistent with a neutral cline), or not. Ivyleaf morning glory, Ipomoea hederacea, exhibits a latitudinal cline for a Mendelian leaf shape polymorphism in eastern North America, such that lobed genotypes dominate northern populations and heart-shaped genotypes are restricted to southern populations. Here, we evaluate potential evolutionary mechanisms for this cline by first determining the allele frequencies at the leaf shape locus for 77 populations distributed throughout I. hederacea’s range and then comparing the geographical pattern at this locus to neutral amplified fragment length polymorphism (AFLP) loci. We detected both significant clinal variation and high genetic differentiation at the leaf shape locus across all populations. In contrast, 99% of the putatively neutral loci do not display clinal variation, and I. hederacea populations show very little overall genetic differentiation, suggesting that there is a moderate level of gene flow. In addition, the leaf shape locus was identified as a major F(ST) outlier experiencing divergent selection, relative to all the AFLP loci. Together, these data strongly suggest that the cline in leaf shape is being maintained by spatially varying natural selection.

Maughan H, Wang PW, Diaz Caballero J, Fung P, Gong Y, Donaldson SL, Yuan L, Keshavjee S, Zhang Y, Yau YC, Waters VJ, Tullis DE, Hwang DM, Guttman DS

PLoS ONE 2012;7(10):e45791

PubMed PMID: 23056217

Abstract

The characterization of bacterial communities using DNA sequencing has revolutionized our ability to study microbes in nature and discover the ways in which microbial communities affect ecosystem functioning and human health. Here we describe Serial Illumina Sequencing (SI-Seq): a method for deep sequencing of the bacterial 16S rRNA gene using next-generation sequencing technology. SI-Seq serially sequences portions of the V5, V6 and V7 hypervariable regions from barcoded 16S rRNA amplicons using an Illumina short-read genome analyzer. SI-Seq obtains taxonomic resolution similar to 454 pyrosequencing for a fraction of the cost, and can produce hundreds of thousands of reads per sample even with very high multiplexing. We validated SI-Seq using single species and mock community controls, and via a comparison to cystic fibrosis lung microbiota sequenced using 454 FLX Titanium. Our control runs show that SI-Seq has a dynamic range of at least five orders of magnitude, can classify >96% of sequences to the genus level, and performs just as well as 454 and paired-end Illumina methods in estimation of standard microbial ecology diversity measurements. We illustrate the utility of SI-Seq in a pilot sample of central airway secretion samples from cystic fibrosis patients.

Suzuki H, MacDonald J, Syed K, Salamov A, Hori C, Aerts A, Henrissat B, Wiebenga A, VanKuyk PA, Barry K, Lindquist E, LaButti K, Lapidus A, Lucas S, Coutinho P, Gong Y, Samejima M, Mahadevan R, Abou-Zaid M, de Vries RP, Igarashi K, Yadav JS, Grigoriev IV, Master ER

BMC Genomics 2012;13:444

PubMed PMID: 22937793

Abstract

BACKGROUND: Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus, Phanerochaete carnosa, has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by a white-rot fungus, the present study reports the P. carnosa genome sequence and its comparative analysis with the previously reported P. chrysosporium genome.

RESULTS: P. carnosa encodes a complete set of lignocellulose-active enzymes. Comparative genomic analysis revealed that P. carnosa is enriched with genes encoding manganese peroxidase, and that the most divergent glycoside hydrolase families were predicted to encode hemicellulases and glycoprotein degrading enzymes. Most remarkably, P. carnosa possesses one of the largest P450 contingents (266 P450s) among the sequenced and annotated wood-rotting basidiomycetes, nearly double that of P. chrysosporium. Along with metabolic pathway modeling, comparative growth studies on model compounds and chemical analyses of decomposed wood components showed greater tolerance of P. carnosa to various substrates including coniferous heartwood.

CONCLUSIONS: The P. carnosa genome is enriched with genes that encode P450 monooxygenases that can participate in extractives degradation, and manganese peroxidases involved in lignin degradation. The significant expansion of P450s in P. carnosa, along with differences in carbohydrate- and lignin-degrading enzymes, could be correlated to the utilization of heartwood and sapwood preparations from both coniferous and hardwood species.

Tsai AY, Canam T, Gorzsás A, Mellerowicz EJ, Campbell MM, Master ER

Plant Biotechnol. J. 2012 Dec;10(9):1077-87

PubMed PMID: 22924998

Abstract

A family 15 carbohydrate esterase (CE15) from the white-rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col-0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl-4-O-methyl-d-glucopyranuronate and could target ester linkages that contribute to lignin-carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf-yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT-IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross-linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross-linking within plant cell walls.