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Abstract Sodiomyces alkalinus is one of the very few alkalophilic fungi, adapted to grow optimally at high pH . It is widely distributed at the plant‐deprived edges of extremely alkaline lakes and locally abundant. We sequenced the genome of S. alkalinus and reconstructed evolution of catabolic enzymes, using a phylogenomic comparison. We found that the genome of S. alkalinus is larger, but its predicted proteome is smaller and heavily depleted of both plant‐degrading enzymes and proteinases, when compared to its closest plant‐pathogenic relatives. Interestingly, despite overall losses, S. alkalinus has retained many proteinases families and acquired bacterial cell wall‐degrading enzymes, some of them via horizontal gene transfer from bacteria. This fungus has very potent proteolytic activity at high pH values, but slowly induced low activity of cellulases and hemicellulases. Our experimental and in silico data suggest that plant biomass, a common food source for most fungi, is not a preferred substrate for S. alkalinus in its natural environment. We conclude that the fungus has abandoned the ancestral plant‐based diet and has become specialized in a more protein‐rich food, abundantly available in soda lakes in the form of prokaryotes and small crustaceans.
Summary Microbial depolymerization of plant cell walls contributes to global carbon balance and is a critical component of renewable energy. The genomes of lignocellulose degrading microorganisms encode diverse classes of carbohydrate modifying enzymes, although currently there is a paucity of knowledge on the role of these proteins in vivo . We report the comprehensive analysis of the cellulose degradation system in the saprophytic bacterium C ellvibrio japonicus . Gene expression profiling of C . japonicus demonstrated that three of the 12 predicted β‐1,4 endoglucanases ( cel5A , cel5B , and cel45A ) and the sole predicted cellobiohydrolase ( cel6A ) showed elevated expression during growth on cellulose. Targeted gene disruptions of all 13 predicted cellulase genes showed that only cel5B and cel6A were required for optimal growth on cellulose. Our analysis also identified three additional genes required for cellulose degradation: lpmo10B encodes a lytic polysaccharide monooxygenase ( LPMO ), while cbp2D and cbp2E encode proteins containing carbohydrate binding modules and predicted cytochrome domains for electron transfer. Cj LPMO10B oxidized cellulose and C bp2 D demonstrated spectral properties consistent with redox function. Collectively, this report provides insight into the biological role of LPMOs and redox proteins in cellulose utilization and suggests that C . japonicus utilizes a combination of hydrolytic and oxidative cleavage mechanisms to degrade cellulose.
The year 2014 marked the 25 th International Conference on Arabidopsis Research. In the 50 yr since the first International Conference on Arabidopsis Research, held in 1965 in Göttingen, Germany, > 54 000 papers that mention Arabidopsis thaliana in the title, abstract or keywords have been published. In this paper, we present herein a citational network analysis of these papers, and touch on some of the important discoveries in plant biology that have been made in this powerful model system, and highlight how these discoveries have then had an impact in crop species. We also look to the future, highlighting some outstanding questions that can be readily addressed in Arabidopsis. Topics that are discussed include Arabidopsis reverse genetic resources, stock centers, databases and online tools, cell biology, development, hormones, plant immunity, signaling in response to abiotic stress, transporters, biosynthesis of cells walls and macromolecules such as starch and lipids, epigenetics and epigenomics, genome-wide association studies and natural variation, gene regulatory networks, modeling and systems biology, and synthetic biology.
Summary Targeted cellular auxin distribution is required for morphogenesis and adaptive responses of plant organs. In Arabidopsis thaliana (Arabidopsis), this involves the prototypical auxin influx facilitator AUX 1 and its LIKE ‐ AUX 1 ( LAX ) homologs, which act partially redundantly in various developmental processes. Interestingly, AUX 1 and its homologs are not strictly essential for the Arabidopsis life cycle. Indeed, aux1 lax1 lax2 lax3 quadruple knock‐outs are mostly viable and fertile, and strong phenotypes are only observed at low penetrance. Here we investigated the Brachypodium distachyon (Brachypodium) AUX 1 homolog Bd AUX 1 by genetic, cell biological and physiological analyses. We report that Bd AUX 1 is essential for Brachypodium development. Bdaux1 loss‐of‐function mutants are dwarfs with aberrant flower development, and consequently infertile. Moreover, they display a counter‐intuitive root phenotype. Although Bdaux1 roots are agravitropic as expected, in contrast to Arabidopsis aux1 mutants they are dramatically longer than wild type roots because of exaggerated cell elongation. Interestingly, this correlates with higher free auxin content in Bdaux1 roots. Consistently, their cell wall characteristics and transcriptome signature largely phenocopy other Brachypodium mutants with increased root auxin content. Our results imply fundamentally different wiring of auxin transport in Brachypodium roots and reveal an essential role of BdAUX1 in a broad spectrum of developmental processes, suggesting a central role for AUX 1 in pooideae.
Lignin is the major phenolic polymer in plant secondary cell walls and is polymerized from monomeric subunits, the monolignols. Eleven enzyme families are implicated in monolignol biosynthesis. In this report, we examined the functions of members of the cinnamyl alcohol dehydrogenase (CAD) and cinnamoyl-CoA reductase (CCR) families in wood formation in Populus trichocarpa , including the regulatory effects of their transcripts and protein activities on monolignol biosynthesis. Enzyme activity assays from stem-differentiating xylem (SDX) proteins showed that RNAi suppression of PtrCAD1 in P. trichocarpa transgenics caused a reduction in SDX CCR activity. RNAi suppression of PtrCCR2 , the only CCR member highly expressed in SDX, caused a reciprocal reduction in SDX protein CAD activities. The enzyme assays of mixed and coexpressed recombinant proteins supported physical interactions between PtrCAD1 and PtrCCR2. Biomolecular fluorescence complementation and pull-down/co-immunoprecipitation experiments supported a hypothesis of PtrCAD1/PtrCCR2 heterodimer formation. These results imply evidence for the formation of PtrCAD1/PtrCCR2 protein complexes in monolignol biosynthesis in planta.
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Plants have developed tissue-specific defense strategies in response to various herbivores with different feeding habits. Although defense responses to leaf-chewing insects have been well studied, little is known about stem-specific responses, particularly in the pith, to stem-boring herbivores. To understand the stem-specific defense, we first conducted a comparative transcriptomic analysis of the wild tobacco Nicotiana attenuata before and after attack by the leaf-chewing herbivore Manduca sexta and the stem borer Trichobaris mucorea. When the stem-boring herbivore attacked, lignin-associated genes were upregulated specifically in the inner parenchymal cells of the stem, the pith; lignin also accumulated highly in the attacked pith. Silencing the lignin biosynthetic gene cinnamyl alcohol dehydrogenase enhanced the performance of the stem-boring herbivore but had no effect on the growth of the leaf-chewing herbivore. Two-dimensional nuclear magnetic resonance results revealed that lignified pith contains feruloyltyramine as an unusual lignin component in the cell wall, as a response against stem-boring herbivore attack. Pith-specific lignification induced by the stem-boring herbivore was modulated by both jasmonate and ethylene signaling. Furthermore, these results suggest that lignin provides a stem-specific inducible barrier, protecting plants against stem-boring insects.
Summary We aimed to identify genomic traits of transitions to ectomycorrhizal ecology within the Boletales by comparing the genomes of 21 symbiotrophic species with their saprotrophic brown‐rot relatives. Gene duplication rate is constant along the backbone of Boletales phylogeny with large loss events in several lineages, while gene family expansion sharply increased in the late Miocene, mostly in the Boletaceae. Ectomycorrhizal Boletales have a reduced set of plant cell‐wall‐degrading enzymes (PCWDEs) compared with their brown‐rot relatives. However, the various lineages retain distinct sets of PCWDEs, suggesting that, over their evolutionary history, symbiotic Boletales have become functionally diverse. A smaller PCWDE repertoire was found in Sclerodermatineae. The gene repertoire of several lignocellulose oxidoreductases (e.g. laccases) is similar in brown‐rot and ectomycorrhizal species, suggesting that symbiotic Boletales are capable of mild lignocellulose decomposition. Transposable element (TE) proliferation contributed to the higher evolutionary rate of genes encoding effector‐like small secreted proteins, proteases, and lipases. On the other hand, we showed that the loss of secreted CAZymes was not related to TE activity but to DNA decay. This study provides novel insights on our understanding of the mechanisms influencing the evolutionary diversification of symbiotic boletes.
Renewed interests in the development of bioenergy, biochemicals, and biomaterials have elicited new strategies for engineering the lignin of biomass feedstock plants. Furthermore, this study shows, for the first time, that 3,4-dihydroxybenzoate (DHB) is compatible with the radical coupling reactions that assemble polymeric lignin in plants. We introduced a bacterial 3-dehydroshikimate dehydratase into hybrid poplar (Populus alba × grandidentata) to divert carbon flux away from the shikimate pathway, which lies upstream of lignin biosynthesis. Transgenic poplar wood had up to 33% less lignin with p-hydroxyphenyl units comprising as much as 10% of the lignin. Mild alkaline hydrolysis of transgenic wood released fewer ester-linked p-hydroxybenzoate groups than control trees, and revealed the novel incorporation of cell-wall-bound DHB, as well as glycosides of 3,4-dihydroxybenzoic acid (DHBA). Two-dimensional nuclear magnetic resonance (2D-NMR) analysis uncovered DHBA-derived benzodioxane structures suggesting that DHB moieties were integrated into the lignin polymer backbone. In addition, up to 40% more glucose was released from transgenic wood following ionic liquid pretreatment and enzymatic hydrolysis. This work highlights the potential of diverting carbon flux from the shikimate pathway for lignin engineering and describes a new type of ‘zip-lignin’ derived from the incorporation of DHB into poplar lignin.
Here, we discover a player in root development. Recovered from a forward-genetic screen in Brachypodium distachyon, the buzz mutant initiates root hairs but they fail to elongate. In addition, buzz roots grow twice as fast as wild-type roots. Also, lateral roots show increased sensitivity to nitrate, whereas primary roots are less sensitive to nitrate. Using whole-genome resequencing, we identified the causal single nucleotide polymorphism as occurring in a conserved but previously uncharacterized cyclin-dependent kinase (CDK)-like gene. The buzz mutant phenotypes are rescued by the wild-type B. distachyon BUZZ coding sequence and by an apparent homolog in Arabidopsis thaliana. Moreover, T-DNA mutants in A. thaliana BUZZ have shorter root hairs. BUZZ mRNA localizes to epidermal cells and develops root hairs and, in the latter, partially colocalizes with the NRT1.1A nitrate transporter. Based on qPCR and RNA-Seq, buzz overexpresses ROOT HAIRLESS LIKE SIX-1 and -2 and misregulates genes related to hormone signaling, RNA processing, cytoskeletal, and cell wall organization, and to the assimilation of nitrate. Overall, these data demonstrate that BUZZ is required for tip growth after root hair initiation and root architectural responses to nitrate.
Summary Our study utilized genome‐wide association studies (GWAS) to link nucleotide variants to traits in Populus trichocarpa , a species with rapid linkage disequilibrium decay. The aim was to overcome the challenge of interpreting statistical associations at individual loci without sufficient biological context, which often leads to reliance solely on gene annotations from unrelated model organisms. We employed an integrative approach that included GWAS targeting multiple traits using three individual techniques for lignocellulose phenotyping, expression quantitative trait loci (eQTL) analysis to construct transcriptional regulatory networks around each candidate locus and co‐expression analysis to provide biological context for these networks, using lignocellulose biosynthesis in Populus trichocarpa as a case study. The research identified three candidate genes potentially involved in lignocellulose formation, including one previously recognized gene (Potri.005G116800/VND1, a critical regulator of secondary cell wall formation) and two genes (Potri.012G130000/AtSAP9 and Potri.004G202900/BIC1) with newly identified putative roles in lignocellulose biosynthesis. Our integrative approach offers a framework for providing biological context to loci associated with trait variation, facilitating the discovery of new genes and regulatory networks.
Summary The leaf economics spectrum (LES) describes a suite of functional traits that consistently covary at large spatial and taxonomic scales. Despite its importance at these larger scales, few studies have examined the major drivers of intraspecific variation in the LES – phenotypic plasticity and standing genetic variation. Using experimental precipitation manipulations, we examined whether covariation among leaf economics traits and selection on leaf economics traits and trait combinations change as diverse genotypes of the widespread perennial grass Panicum virgatum are exposed to differences in precipitation. We also used RNA‐Seq to examine whether groups of co‐expressed genes that align with leaf economics traits function in processes hypothesized to underlie the LES. Water availability impacted leaf economics trait covariation in important ways – covariation between leaf economics traits and selection on covariation between traits (i.e. correlational selection) tended to be strongest when water availability was high. Additionally, many genes associated with leaf economics traits functioned in processes that may explain how the LES originates, such as chloroplasts, cell walls, and nitrogen metabolism. Water availability is likely an important modulator of selection and evolution of the LES in P. virgatum that can be better understood by examining gene expression.
Here, lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin-degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the β-aryl ether bond. In this study, we expressed the LigD , LigF and LigG ( LigDFG ) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized β-aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG , showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.
Caffeic acid O-methyltransferase (COMT), the lignin biosynthesis gene modified in many brown-midrib high-digestibility mutants of maize and sorghum, was targeted for downregulation in the small grain temperate cereal, barley (Hordeum vulgare), to improve straw properties. Phylogenetic and expression analyses identified the barley COMT orthologue(s) expressed in stems, defining a larger gene family than in brachypodium or rice with three COMT genes expressed in lignifying tissues. RNAi significantly reduced stem COMT protein and enzyme activity, and modestly reduced stem lignin content while dramatically changing lignin structure. Lignin syringyl-to-guaiacyl ratio was reduced by ~50%, the 5-hydroxyguaiacyl (5-OH-G) unit incorporated into lignin at 10-–15-fold higher levels than normal, and the amount of p-coumaric acid ester-linked to cell walls was reduced by ~50%. No brown-midrib phenotype was observed in any RNAi line despite significant COMT suppression and altered lignin. The novel COMT gene family structure in barley highlights the dynamic nature of grass genomes. Redundancy in barley COMTs may explain the absence of brown-midrib mutants in barley and wheat. The barley COMT RNAi lines nevertheless have the potential to be exploited for bioenergy applications and as animal feed.
Summary Rich ecotype collections are used for several plant models to unravel the molecular causes of phenotypic differences, and to investigate the effects of environmental adaption and acclimation. For the model moss Physcomitrella patens collections of accessions are available, and have been used for phylogenetic and taxonomic studies, for example, but few have been investigated further for phenotypic differences. Here, we focus on the Reute accession and provide expression profiling and comparative developmental data for several stages of sporophyte development, as well as information on genetic variation via genomic sequencing. We analysed cross‐technology and cross‐laboratory data to define a confident set of 15 mature sporophyte‐specific genes. We find that the standard laboratory strain Gransden produces fewer sporophytes than Reute or Villersexel, although gametangia develop with the same time course and do not show evident morphological differences. Reute exhibits less genetic variation relative to Gransden than Villersexel, yet we found variation between Gransden and Reute in the expression profiles of several genes, as well as variation hot spots and genes that appear to evolve under positive Darwinian selection. We analyzed expression differences between the ecotypes for selected candidate genes in the GRAS transcription factor family, the chalcone synthase family and in genes involved in cell wall modification that are potentially related to phenotypic differences. We confirm that Reute is a P. patens ecotype, and suggest its use for reverse‐genetics studies that involve progression through the life cycle and multiple generations.
Due to a large and growing collection of genomic and experimental resources, Brachypodium distachyon has emerged as a powerful experimental model for the grasses. To add to these resources we sequenced 21 165 T-DNA lines, 15 569 of which were produced in this study. This increased the number of unique insertion sites in the T-DNA collection by 21 078, bringing the overall total to 26 112. Thirty-seven per cent (9754) of these insertion sites are within genes (including untranslated regions and introns) and 28% (7217) are within 500 bp of a gene. Approximately 31% of the genes in the v.2.1 annotation have been tagged in this population. To demonstrate the utility of this collection, we phenotypically characterized six T-DNA lines with insertions in genes previously shown in other systems to be involved in cellulose biosynthesis, hemicellulose biosynthesis, secondary cell wall development, DNA damage repair, wax biosynthesis and chloroplast synthesis. In all cases, the phenotypes observed supported previous studies, demonstrating the utility of this collection for plant functional genomics. The Brachypodium T-DNA collection can be accessed at http://jgi.doe.gov/our-science/science-programs/plant-genomics/brachypodium/brachypodium-t-dna-collection/.