Complex Relationships between Chromatin Accessibility, Sequence Divergence, and Gene Expression in Arabidopsis thaliana.

Variation in regulatory DNA is thought to drive phenotypic variation, evolution, and disease. Prior studies of regulatory DNA and transcription factors across animal species highlighted a fundamental conundrum: Transcription factor binding domains and cognate binding sites are conserved, while regulatory DNA sequences are not. It remains unclear how conserved transcription factors and dynamic regulatory sites produce conserved expression patterns across species. Here, we explore regulatory DNA variation and its functional consequences within Arabidopsis thaliana, using chromatin accessibility to delineate regulatory DNA genome-wide. Unlike in previous cross-species comparisons, the positional homology of regulatory DNA is maintained among A. thaliana ecotypes and less nucleotide divergence has occurred. Of the ∼50,000 regulatory sites in A. thaliana, we found that 15% varied in accessibility among ecotypes. Some of these accessibility differences were associated with extensive, previously unannotated sequence variation, encompassing many deletions and ancient hypervariable alleles. Unexpectedly, for the majority of such regulatory sites, nearby gene expression was unaffected. Nevertheless, regulatory sites with high levels of sequence variation and differential chromatin accessibility were the most likely to be associated with differential gene expression. Finally, and most surprising, we found that the vast majority of differentially accessible sites show no underlying sequence variation. We argue that these surprising results highlight the necessity to consider higher-order regulatory context in evaluating regulatory variation and predicting its phenotypic consequences.

Phytochrome B regulates resource allocation in Brassica rapa.

Crop biomass and yield are tightly linked to how the light signaling network translates information about the environment into allocation of resources, including photosynthates. Once activated, the phytochrome (phy) class of photoreceptors signal and re-deploy carbon resources to alter growth, plant architecture, and reproductive timing. Most of the previous characterization of the light-modulated growth program has been performed in the reference plant Arabidopsis thaliana. Here, we use Brassica rapa as a crop model to test for conservation of the phytochrome-carbon network. In response to elevated levels of CO2, B. rapa seedlings showed increases in hypocotyl length, shoot and root fresh weight, and the number of lateral roots. All of these responses were dependent on nitrogen and polar auxin transport. In addition, we identified putative B. rapa orthologs of PhyB and isolated two nonsense alleles. BrphyB mutants had significantly decreased or absent CO2-stimulated growth responses. Mutant seedlings also showed misregulation of auxin-dependent genes and genes involved in chloroplast development. Adult mutant plants had reduced chlorophyll levels, photosynthetic rate, stomatal index, and seed yield. These findings support a recently proposed holistic role for phytochromes in regulating resource allocation, biomass production, and metabolic state in the developing plant.

Evolution of Cis-Regulatory Elements and Regulatory Networks in Duplicated Genes of Arabidopsis.

Plant genomes contain large numbers of duplicated genes that contribute to the evolution of new functions. Following duplication, genes can exhibit divergence in their coding sequence and their expression patterns. Changes in the cis-regulatory element landscape can result in changes in gene expression patterns. High-throughput methods developed recently can identify potential cis-regulatory elements on a genome-wide scale. Here, we use a recent comprehensive data set of DNase I sequencing-identified cis-regulatory binding sites (footprints) at single-base-pair resolution to compare binding sites and network connectivity in duplicated gene pairs in Arabidopsis (Arabidopsis thaliana). We found that duplicated gene pairs vary greatly in their cis-regulatory element architecture, resulting in changes in regulatory network connectivity. Whole-genome duplicates (WGDs) have approximately twice as many footprints in their promoters left by potential regulatory proteins than do tandem duplicates (TDs). The WGDs have a greater average number of footprint differences between paralogs than TDs. The footprints, in turn, result in more regulatory network connections between WGDs and other genes, forming denser, more complex regulatory networks than shown by TDs. When comparing regulatory connections between duplicates, WGDs had more pairs in which the two genes are either partially or fully diverged in their network connections, but fewer genes with no network connections than the TDs. There is evidence of younger TDs and WGDs having fewer unique connections compared with older duplicates. This study provides insights into cis-regulatory element evolution and network divergence in duplicated genes.

MUM ENHANCERS are important for seed coat mucilage production and mucilage secretory cell differentiation in Arabidopsis thaliana.

Pollination triggers not only embryo development but also the differentiation of the ovule integuments to form a specialized seed coat. The mucilage secretory cells of the Arabidopsis thaliana seed coat undergo a complex differentiation process in which cell growth is followed by the synthesis and secretion of pectinaceous mucilage. A number of genes have been identified affecting mucilage secretory cell differentiation, including MUCILAGE-MODIFIED4 (MUM4). mum4 mutants produce a reduced amount of mucilage and cloning of MUM4 revealed that it encodes a UDP-L-rhamnose synthase that is developmentally up-regulated to provide rhamnose for mucilage pectin synthesis. To identify additional genes acting in mucilage synthesis and secretion, a screen for enhancers of the mum4 phenotype was performed. Eight mum enhancers (men) have been identified, two of which result from defects in known mucilage secretory cell genes (MUM2 and MYB61). Our results show that, in a mum4 background, mutations in MEN1, MEN4, and MEN5 lead to further reductions in mucilage compared to mum4 single mutants, suggesting that they are involved in mucilage synthesis or secretion. Conversely, mutations in MEN2 and MEN6 appear to affect mucilage release rather than quantity. With the exception of men4, whose single mutant exhibits reduced mucilage, none of these genes have a single mutant phenotype, suggesting that they would not have been identified outside the compromised mum4 background.

Mapping and Dynamics of Regulatory DNA in Maturing Arabidopsis thaliana Siliques.

The genome is reprogrammed during development to produce diverse cell types, largely through altered expression and activity of key transcription factors. The accessibility and critical functions of epidermal cells have made them a model for connecting transcriptional events to development in a range of model systems. In Arabidopsis thaliana and many other plants, fertilization triggers differentiation of specialized epidermal seed coat cells that have a unique morphology caused by large extracellular deposits of polysaccharides. Here, we used DNase I-seq to generate regulatory landscapes of A. thaliana seeds at two critical time points in seed coat maturation (4 and 7 DPA), enriching for seed coat cells with the INTACT method. We found over 3,000 developmentally dynamic regulatory DNA elements and explored their relationship with nearby gene expression. The dynamic regulatory elements were enriched for motifs for several transcription factors families; most notably the TCP family at the earlier time point and the MYB family at the later one. To assess the extent to which the observed regulatory sites in seeds added to previously known regulatory sites in A. thaliana, we compared our data to 11 other data sets generated with 7-day-old seedlings for diverse tissues and conditions. Surprisingly, over a quarter of the regulatory, i.e. accessible, bases observed in seeds were novel. Notably, plant regulatory landscapes from different tissues, cell types, or developmental stages were more dynamic than those generated from bulk tissue in response to environmental perturbations, highlighting the importance of extending studies of regulatory DNA to single tissues and cell types during development.

Mapping and dynamics of regulatory DNA and transcription factor networks in A. thaliana.

Our understanding of gene regulation in plants is constrained by our limited knowledge of plant cis-regulatory DNA and its dynamics. We mapped DNase I hypersensitive sites (DHSs) in A. thaliana seedlings and used genomic footprinting to delineate ∼ 700,000 sites of in vivo transcription factor (TF) occupancy at nucleotide resolution. We show that variation associated with 72 diverse quantitative phenotypes localizes within DHSs. TF footprints encode an extensive cis-regulatory lexicon subject to recent evolutionary pressures, and widespread TF binding within exons may have shaped codon usage patterns. The architecture of A. thaliana TF regulatory networks is strikingly similar to that of animals in spite of diverged regulatory repertoires. We analyzed regulatory landscape dynamics during heat shock and photomorphogenesis, disclosing thousands of environmentally sensitive elements and enabling mapping of key TF regulatory circuits underlying these fundamental responses. Our results provide an extensive resource for the study of A. thaliana gene regulation and functional biology.

Photomorphogenesis.

As photoautotrophs, plants are exquisitely sensitive to their light environment. Light affects many developmental and physiological responses throughout plants' life histories. The focus of this chapter is on light effects during the crucial period of time between seed germination and the development of the first true leaves. During this time, the seedling must determine the appropriate mode of action to best achieve photosynthetic and eventual reproductive success. Light exposure triggers several major developmental and physiological events. These include: growth inhibition and differentiation of the embryonic stem (hypocotyl); maturation of the embryonic leaves (cotyledons); and establishment and activation of the stem cell population in the shoot and root apical meristems. Recent studies have linked a number of photoreceptors, transcription factors, and phytohormones to each of these events.

Seed coat mucilage cells of Arabidopsis thaliana as a model for plant cell wall research.

Plant cells are encased within a complex polysaccharide wall that strengthens the cell and has key roles in all aspects of plant cell growth, differentiation, and interaction with the environment. This dynamic structure is under continual modification during plant development, and its synthesis and modification require the activity of a myriad of enzymes. The mucilage secretory cells (MSCs) of the Arabidopsis thaliana seed coat provide a model for the discovery of novel genes involved in the synthesis, secretion and modification of cell wall components, particularly pectin. These cells synthesize copious amounts of pectinaceous mucilage during development and, upon hydration of the desiccated seed, the mucilage rapidly swells, bursts from the MSCs and surrounds the seed in a gelatinous capsule. Several genes affecting MSC differentiation, pectin synthesis, and mucilage release have been identified and additional genes involved in these and related processes including pectin secretion and the mechanical alteration of cell walls await to be discovered.

AtBXL1 encodes a bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells.

Following pollination, the epidermal cells of the Arabidopsis (Arabidopsis thaliana) ovule undergo a complex differentiation process that includes the synthesis and polar secretion of pectinaceous mucilage followed by the production of a secondary cell wall. Wetting of mature seeds leads to the rapid bursting of these mucilage secretory cells to release a hydrophilic gel that surrounds the seed and is believed to aid in seed hydration and germination. A novel mutant is identified where mucilage release is both patchy and slow and whose seeds display delayed germination. While developmental analysis of mutant seeds reveals no change in mucilage secretory cell morphology, changes in monosaccharide quantities are detected, suggesting the mucilage release defect results from altered mucilage composition. Plasmid rescue and cloning of the mutant locus revealed a T-DNA insertion in AtBXL1, which encodes a putative bifunctional beta-d-xylosidase/alpha-l-arabinofuranosidase that has been implicated as a beta-d-xylosidase acting during vascular development. Chemical and immunological analyses of mucilage extracted from bxl1 mutant seeds and antibody staining of developing seed coats reveal an increase in (1-->5)-linked arabinans, suggesting that BXL1 is acting as an alpha-l-arabinofuranosidase in the seed coat. This implication is supported by the ability to rescue mucilage release through treatment of bxl1 seeds with exogenous alpha-l-arabinofuranosidases. Together, these results suggest that trimming of rhamnogalacturonan I arabinan side chains is required for correct mucilage release and reveal a new role for BXL1 as an alpha-l-arabinofuranosidase acting in seed coat development.