Dana J. Wohlbach

Analysis of the Arabidopsis histidine kinase ATHK1 reveals a connection between vegetative osmotic stress sensing and seed maturation.

To cope with water stress, plants must be able to effectively sense, respond to, and adapt to changes in water availability. The Arabidopsis thaliana plasma membrane His kinase ATHK1 has been suggested to act as an osmosensor that detects water stress and initiates downstream responses. Here, we provide direct genetic evidence that ATHK1 not only is involved in the water stress response during early vegetative stages of plant growth but also plays a unique role in the regulation of desiccation processes during seed formation. To more comprehensively identify genes involved in the downstream pathways affected by the ATHK1-mediated response to water stress, we created a large-scale summary of expression data, termed the AtMegaCluster. In the AtMegaCluster, hierarchical clustering techniques were used to compare whole-genome expression levels in athk1 mutants with the expression levels reported in publicly available data sets of Arabidopsis tissues grown under a wide variety of conditions. These experiments revealed that ATHK1 is cotranscriptionally regulated with several Arabidopsis response regulators, together with two proteins containing novel sequences. Since overexpression of ATHK1 results in increased water stress tolerance, our observations suggest a new top-down route to increasing drought resistance via receptor-mediated increases in sensing water status, rather than through genetically engineered changes in downstream transcription factors or specific osmolytes.

Comparative genomics of xylose-fermenting fungi for enhanced biofuel production

Cellulosic biomass is an abundant and underused substrate for biofuel production. The inability of many microbes to metabolize the pentose sugars abundant within hemicellulose creates specific challenges for microbial biofuel production from cellulosic material. Although engineered strains of Saccharomyces cerevisiae can use the pentose xylose, the fermentative capacity pales in comparison with glucose, limiting the economic feasibility of industrial fermentations. To better understand xylose utilization for subsequent microbial engineering, we sequenced the genomes of two xylose-fermenting, beetle-associated fungi, Spathaspora passalidarum and Candida tenuis. To identify genes involved in xylose metabolism, we applied a comparative genomic approach across 14 Ascomycete genomes, mapping phenotypes and genotypes onto the fungal phylogeny, and measured genomic expression across five Hemiascomycete species with different xylose-consumption phenotypes. This approach implicated many genes and processes involved in xylose assimilation. Several of these genes significantly improved xylose utilization when engineered into S. cerevisiae, demonstrating the power of comparative methods in rapidly identifying genes for biomass conversion while reflecting on fungal ecology.

An Aspergillus nidulans bZIP response pathway hardwired for defensive secondary metabolism operates through aflR

The eukaryotic bZIP transcription factors are critical players in organismal response to environmental challenges. In fungi, the production of secondary metabolites (SMs) is hypothesized as one of the responses to environmental insults, e.g. attack by fungivorous insects, yet little data to support this hypothesis exists. Here we establish a mechanism of bZIP regulation of SMs through RsmA, a recently discovered YAP‐like bZIP protein. RsmA greatly increases SM production by binding to two sites in the Aspergillus nidulans AflR promoter region, a C6 transcription factor known for activating production of the carcinogenic and anti‐predation SM, sterigmatocystin. Deletion of aflR in an overexpression rsmA (OE:rsmA) background not only eliminates sterigmatocystin production but also significantly reduces asperthecin synthesis. Furthermore, the fungivore, Folsomia candida, exhibited a distinct preference for feeding on wild type rather than an OE:rsmA strain. RsmA may thus have a critical function in mediating direct chemical resistance against predation. Taken together, these results suggest RsmA represents a bZIP pathway hardwired for defensive SM production.

From elements to modules: regulatory evolution in Ascomycota fungi

Regulatory divergence is likely a major driving force in evolution. Comparative transcriptomics provides a new glimpse into the evolution of gene regulation. Ascomycota fungi are uniquely suited among eukaryotes for studies of regulatory evolution, because of broad phylogenetic scope, many sequenced genomes, and facility of genomic analysis. Here we review the substantial divergence in gene expression in Ascomycota and how this is reconciled with the modular organization of transcriptional networks. We show that flexibility and redundancy in both cis-regulation and trans-regulation can lead to changes from altered expression of single genes to wholesale rewiring of regulatory modules. Redundancy thus emerges as a major driving force facilitating expression divergence while preserving the coherent functional organization of a transcriptional response.

Harnessing Genetic Diversity in Saccharomyces cerevisiae for Fermentation of Xylose in Hydrolysates of Alkaline Hydrogen Peroxide-Pretreated Biomass

ABSTRACT The fermentation of lignocellulose-derived sugars, particularly xylose, into ethanol by the yeast Saccharomyces cerevisiae is known to be inhibited by compounds produced during feedstock pretreatment. We devised a strategy that combined chemical profiling of pretreated feedstocks, high-throughput phenotyping of genetically diverse S. cerevisiae strains isolated from a range of ecological niches, and directed engineering and evolution against identified inhibitors to produce strains with improved fermentation properties. We identified and quantified for the first time the major inhibitory compounds in alkaline hydrogen peroxide (AHP)-pretreated lignocellulosic hydrolysates, including Na+, acetate, and p-coumaric (pCA) and ferulic (FA) acids. By phenotyping these yeast strains for their abilities to grow in the presence of these AHP inhibitors, one heterozygous diploid strain tolerant to all four inhibitors was selected, engineered for xylose metabolism, and then allowed to evolve on xylose with increasing amounts of pCA and FA. After only 149 generations, one evolved isolate, GLBRCY87, exhibited faster xylose uptake rates in both laboratory media and AHP switchgrass hydrolysate than its ancestral GLBRCY73 strain and completely converted 115 g/liter of total sugars in undetoxified AHP hydrolysate into more than 40 g/liter ethanol. Strikingly, genome sequencing revealed that during the evolution from GLBRCY73, the GLBRCY87 strain acquired the conversion of heterozygous to homozygous alleles in chromosome VII and amplification of chromosome XIV. Our approach highlights that simultaneous selection on xylose and pCA or FA with a wild S. cerevisiae strain containing inherent tolerance to AHP pretreatment inhibitors has potential for rapid evolution of robust properties in lignocellulosic biofuel production.

Transcriptional changes in response to growth of Arabidopsis in high external calcium

Transcriptional responses to growth in high environmental calcium concentrations were characterized and compared between wild‐type and mutant Arabidopsis plants containing a knockout mutation in the gene encoding a cyclic nucleotide‐gated channel (CNGC2). We show that the transcriptional profile of cngc2 plants grown in normal media resembled that from wild‐type plants grown under elevated exogenous calcium conditions. The mutant grown in high‐calcium media exhibited transcriptional changes not seen in the wild‐type. The pattern of transcription suggests that adaptation to high external calcium overlaps with responses towards various biotic and abiotic stresses.

Comparative Genomics of Saccharomyces cerevisiae Natural Isolates for Bioenergy Production

Lignocellulosic plant material is a viable source of biomass to produce alternative energy including ethanol and other biofuels. However, several factors—including toxic byproducts from biomass pretreatment and poor fermentation of xylose and other pentose sugars—currently limit the efficiency of microbial biofuel production. To begin to understand the genetic basis of desirable traits, we characterized three strains of Saccharomyces cerevisiae with robust growth in a pretreated lignocellulosic hydrolysate or tolerance to stress conditions relevant to industrial biofuel production, through genome and transcriptome sequencing analysis. All stress resistant strains were highly mosaic, suggesting that genetic admixture may contribute to novel allele combinations underlying these phenotypes. Strain-specific gene sets not found in the lab strain were functionally linked to the tolerances of particular strains. Furthermore, genes with signatures of evolutionary selection were enriched for functional categories important for stress resistance and included stress-responsive signaling factors. Comparison of the strains’ transcriptomic responses to heat and ethanol treatment—two stresses relevant to industrial bioethanol production—pointed to physiological processes that were related to particular stress resistance profiles. Many of the genotype-by-environment expression responses occurred at targets of transcription factors with signatures of positive selection, suggesting that these strains have undergone positive selection for stress tolerance. Our results generate new insights into potential mechanisms of tolerance to stresses relevant to biofuel production, including ethanol and heat, present a backdrop for further engineering, and provide glimpses into the natural variation of stress tolerance in wild yeast strains.

Identification of an in vitro transcription-based artifact affecting oligonucleotide microarrays

This study identified the widely used T7 in vitro transcription system as a major source of artifact in the tiling array data from nine eukaryotic genomes. The most affected probes contained a sequence motif complementary to the +1 to +9 initial transcribed sequence (ITS) of the T7‐(dT)24 primer. The abundance of 5′ ITS cRNA fragments produced during target preparation was sufficient to drive undesirable hybridization. A new T7‐(dT)24 primer with a modified ITS was designed that shifts the artifactual motifs as predicted and reduces the effect of the artifact. A computational algorithm was generated to filter out the likely artifactual probes from existing whole‐genome tiling array data and improve probe selection. Further studies of Arabidopsis thaliana were conducted using both T7‐(dT)24 primers. While the artifact affected transcript discovery with tiling arrays, it showed only a minor impact on measurements of gene expression using commercially available ‘gene‐only’ expression arrays.

Democratization and Integration of Genomic Profiling Tools

Systems biology is a comprehensive means of creating a complete understanding of how all components of an organism work together to maintain and procreate life. By quantitatively profiling one at a time, the effect of thousands and millions of genetic and environmental perturbations on the cell, systems biologists are attempting to recreate and measure the effect of the many different states that have been explored during the 3 billion years in which life has evolved. A key aspect of this work is the development of innovative new approaches to quantify changes in the transcriptome, proteome, and metabolome. In this chapter we provide a review and evaluation of several genomic profiling techniques used in plant systems biology as well as make recommendations for future progress in their use and integration.