Samuel P. Hazen

Network Discovery Pipeline Elucidates Conserved Time-of-Day–Specific cis-Regulatory Modules

Correct daily phasing of transcription confers an adaptive advantage to almost all organisms, including higher plants. In this study, we describe a hypothesis-driven network discovery pipeline that identifies biologically relevant patterns in genome-scale data. To demonstrate its utility, we analyzed a comprehensive matrix of time courses interrogating the nuclear transcriptome of Arabidopsis thaliana plants grown under different thermocycles, photocycles, and circadian conditions. We show that 89% of Arabidopsis transcripts cycle in at least one condition and that most genes have peak expression at a particular time of day, which shifts depending on the environment. Thermocycles alone can drive at least half of all transcripts critical for synchronizing internal processes such as cell cycle and protein synthesis. We identified at least three distinct transcription modules controlling phase-specific expression, including a new midnight specific module, PBX/TBX/SBX. We validated the network discovery pipeline, as well as the midnight specific module, by demonstrating that the PBX element was sufficient to drive diurnal and circadian condition-dependent expression. Moreover, we show that the three transcription modules are conserved across Arabidopsis, poplar, and rice. These results confirm the complex interplay between thermocycles, photocycles, and the circadian clock on the daily transcription program, and provide a comprehensive view of the conserved genomic targets for a transcriptional network key to successful adaptation.

Brachypodium as a model for the grasses: Today and the future

Over the past several years, Brachypodium distachyon (Brachypodium) has emerged as a tractable model system to study biological questions relevant to the grasses. To place its relevance in the larger context of plant biology, we outline here the expanding adoption of Brachypodium as a model grass and compare this to the early history of another plant model, Arabidopsis thaliana. In this context, Brachypodium has followed an accelerated path in which the development of genomic resources, most notably a whole genome sequence, occurred concurrently with the generation of other experimental tools (e.g. highly efficient transformation and large collections of natural accessions). This update provides a snapshot of available and upcoming Brachypodium resources and an overview of the community including the trajectory of Brachypodium as a model grass.

A morning-specific phytohormone gene expression program underlying rhythmic plant growth.

Most organisms use daily light/dark cycles as timing cues to control many essential physiological processes. In plants, growth rates of the embryonic stem (hypocotyl) are maximal at different times of day, depending on external photoperiod and the internal circadian clock. However, the interactions between light signaling, the circadian clock, and growth-promoting hormone pathways in growth control remain poorly understood. At the molecular level, such growth rhythms could be attributed to several different layers of time-specific control such as phasing of transcription, signaling, or protein abundance. To determine the transcriptional component associated with the rhythmic control of growth, we applied temporal analysis of the Arabidopsis thaliana seedling transcriptome under multiple growth conditions and mutant backgrounds using DNA microarrays. We show that a group of plant hormone-associated genes are coexpressed at the time of day when hypocotyl growth rate is maximal. This expression correlates with overrepresentation of a cis-acting element (CACATG) in phytohormone gene promoters, which is sufficient to confer the predicted diurnal and circadian expression patterns in vivo. Using circadian clock and light signaling mutants, we show that both internal coincidence of phytohormone signaling capacity and external coincidence with darkness are required to coordinate wild-type growth. From these data, we argue that the circadian clock indirectly controls growth by permissive gating of light-mediated phytohormone transcript levels to the proper time of day. This temporal integration of hormone pathways allows plants to fine tune phytohormone responses for seasonal and shade-appropriate growth regulation.

Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana

We used hybridization to the ATH1 gene expression array to interrogate genomic DNA diversity in 23 wild strains (accessions) of Arabidopsis thaliana (arabidopsis), in comparison with the reference strain Columbia (Col). At <1% false discovery rate, we detected 77,420 single-feature polymorphisms (SFPs) with distinct patterns of variation across the genome. Total and pair-wise diversity was higher near the centromeres and the heterochromatic knob region, but overall diversity was positively correlated with recombination rate (R2 = 3.1%). The difference between total and pair-wise SFP diversity is a relative measure contrasting diversifying or frequency-dependent selection, similar to Tajimas D, and can be calibrated by the empirical genome-wide distribution. Each unique locus, centered on a gene, has a diversity and selection score that suggest a relative role in past evolutionary processes. Homologs of disease resistance (R) genes include members with especially high levels of diversity often showing frequency-dependent selection and occasionally evidence of a past selective sweep. Receptor-like and S-locus proteins also contained members with elevated levels of diversity and signatures of selection, whereas other gene families, bHLH, F-box, and RING finger proteins, showed more typical levels of diversity. SFPs identified with the gene expression array also provide an empirical hybridization polymorphism background for studies of gene expression polymorphism and are available through the genome browser http://signal.salk.edu/cgi-bin/AtSFP.

Cellulose Synthase-Like Genes of Rice

Identification of the biosynthetic enzymes involved in cell wall biosynthesis remains one of the major unsolved problems of plant biology. Of the major polysaccharides of the plant cell wall, pectins and hemicelluloses are synthesized in the Golgi, and callose and cellulose are synthesized at the

Population Structure in the Model Grass Brachypodium distachyon Is Highly Correlated with Flowering Differences across Broad Geographic Areas

Genotyping diverse Brachypodium accessions expands research tools for grasses. The B. hybridum genome is a mosaic of B. distachyon‐ and B. stacei‐like sequences. Three distinct, genetically defined populations of B. distachyon were identified. Flowering time, more than geography, distinguishes B. distachyon populations. Results support the feasibility of genome‐wide association studies in a model grass.

Expression profiling of rice segregating for drought tolerance QTLs using a rice genome array

Plants alter their gene expression patterns in response to drought. Sometimes these transcriptional changes are successful adaptations leading to tolerance, while in other instances the plant ultimately fails to adapt to the stress and is labeled as sensitive to that condition. We measured the expression of approximately half of the genes in rice (∼21,000) in phenotypically divergent accessions and their transgressive segregants to associate stress-regulated gene expression changes with quantitative trait loci (QTLs) for osmotic adjustment (OA, a trait associated with drought tolerance). Among the parental lines, a total of 662 transcripts were differentially expressed. Only 12 genes were induced in the low OA parent, CT9993, at moderate dehydration stress levels while over 200 genes were induced in the high OA parent, IR62266. The high and low OA parents had almost entirely different transcriptional responses to dehydration stress suggesting a complete absence of an appropriate response rather than a slower response in CT9993. Sixty-nine genes were up-regulated in all the high OA lines and nine of those genes were not induced in any of the low OA lines. The annotation of four of those genes, sucrose synthase, a pore protein, a heat shock and an LEA protein, suggests a role in maintaining high OA and membrane stability. Of the 3,954-probe sets that correspond to the QTL intervals, very few had a differential expression pattern between the high OA and low OA lines that suggest a role leading to the phenotypic variation. However, several promising candidates were identified for each of the five QTL including a snRNP auxiliary factor, a LEA protein, a protein phosphatase 2C and a Sar1 homolog.

Rapid Array Mapping of Circadian Clock and Developmental Mutations in Arabidopsis

Classical forward genetics, the identification of genes responsible for mutant phenotypes, remains an important part of functional characterization of the genome. With the advent of extensive genome sequence, phenotyping and genotyping remain the critical limiting variables in the process of map-based cloning. Here, we reduce the genotyping problem by hybridizing labeled genomic DNA to the Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 GeneChip. Genotyping was carried out on the scale of detecting greater than 8,000 single feature polymorphisms from over 200,000 loci in a single assay. By combining this technique with bulk segregant analysis, several high heritability development and circadian clock traits were mapped. The mapping accuracy using bulk pools of 26 to 100 F2 individuals ranged from 0.22 to 1.96 Mb of the mutations revealing mutant alleles of EARLY FLOWERING 3, EARLY FLOWERING 4, TIMING OF CAB EXPRESSION 1, and ASYMMETRIC LEAVES 1. While direct detection of small mutations, such as an ethyl-methane sulfonate derived single base substitutions, is limited by array coverage and sensitivity, large deletions such as those that can be caused by fast neutrons are easily detected. We demonstrate this by resolving two deletions, the 77-kb flavin-binding, kelch repeat, f-box 1 and the 7-kb cryptochrome2-1 deletions, via direct hybridization of mutant DNA to ATH1 expression arrays.

Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays

BackgroundOrganisms are able to anticipate changes in the daily environment with an internal oscillator know as the circadian clock. Transcription is an important mechanism in maintaining these oscillations. Here we explore, using whole genome tiling arrays, the extent of rhythmic expression patterns genome-wide, with an unbiased analysis of coding and noncoding regions of the Arabidopsis genome.ResultsAs in previous studies, we detected a circadian rhythm for approximately 25% of the protein coding genes in the genome. With an unbiased interrogation of the genome, extensive rhythmic introns were detected predominantly in phase with adjacent rhythmic exons, creating a transcript that, if translated, would be expected to produce a truncated protein. In some cases, such as the MYB transcription factor AT2G20400, an intron was found to exhibit a circadian rhythm while the remainder of the transcript was otherwise arrhythmic. In addition to several known noncoding transcripts, including microRNA, trans-acting short interfering RNA, and small nucleolar RNA, greater than one thousand intergenic regions were detected as circadian clock regulated, many of which have no predicted function, either coding or noncoding. Nearly 7% of the protein coding genes produced rhythmic antisense transcripts, often for genes whose sense strand was not similarly rhythmic.ConclusionsThis study revealed widespread circadian clock regulation of the Arabidopsis genome extending well beyond the protein coding transcripts measured to date. This suggests a greater level of structural and temporal dynamics than previously known.

Gene expression profiling of plant responses to abiotic stress

Expression profiling has become an important tool to investigate how an organism responds to environmental changes. Plants, being sessile, have the ability to dramatically alter their gene expression patterns in response to environmental changes such as temperature, water availability or the presence of deleterious levels of ions. Sometimes these transcriptional changes are successful adaptations leading to tolerance while in other instances the plant ultimately fails to adapt to the new environment and is labeled as sensitive to that condition. Expression profiling can define both tolerant and sensitive responses. These profiles of plant response to environmental extremes (abiotic stresses) are expected to lead to regulators that will be useful in biotechnological approaches to improve stress tolerance as well as to new tools for studying regulatory genetic circuitry. Finally, data mining of the alterations in the plant transcriptome will lead to further insights into how abiotic stress affects plant physiology.