Changes in Gene Expression in Space and Time Orchestrate Environmentally Mediated Shaping of Root Architecture.

Shaping of root architecture is a quintessential developmental response that involves the concerted action of many different cell types, is highly dynamic, and underpins root plasticity. To determine to what extent the environmental regulation of lateral root development is a product of cell-type preferential activities, we tracked transcriptomic responses to two different treatments that both change root development in Arabidopsis thaliana at an unprecedented level of temporal detail. We found that individual transcripts are expressed with a very high degree of temporal and spatial specificity, yet biological processes are commonly regulated, in a mechanism we term response nonredundancy. Using causative gene network inference to compare the genes regulated in different cell types and during responses to nitrogen and a biotic interaction, we found that common transcriptional modules often regulate the same gene families but control different individual members of these families, specific to response and cell type. This reinforces that the activity of a gene cannot be defined simply as molecular function; rather, it is a consequence of spatial location, expression timing, and environmental responsiveness.

The transcript elongation factor FACT affects Arabidopsis vegetative and reproductive development and genetically interacts with HUB1/2.

The facilitates chromatin transcription (FACT) complex, consisting of the SSRP1 and SPT16 proteins, is a histone chaperone that assists the progression of transcribing RNA polymerase on chromatin templates by destabilizing nucleosomes. Here, we examined plants that harbour mutations in the genes encoding the subunits of Arabidopsis FACT. These experiments revealed that (i) SSRP1 is critical for plant viability, and (ii) plants with reduced amounts of SSRP1 and SPT16 display various defects in vegetative and reproductive development. Thus, mutant plants display an increased number of leaves and inflorescences, show early bolting, have abnormal flower and leaf architecture, and their seed production is severely affected. The early flowering of the mutant plants is associated with reduced expression of the floral repressor FLC in ssrp1 and spt16 plants. Compared to control plants, reduced amounts of FACT in mutant plants are detected at the FLC locus as well as at the locations of housekeeping genes (whose expression is not affected in the mutants), suggesting that expression of FLC is particularly sensitive to reduced FACT activity. Analysis of double mutants that are affected in the expression of both FACT subunits and factors catalysing the mono-ubiquitination of histone H2B (HUB1/2) demonstrates that they genetically interact to regulate various developmental processes (i.e. branching, leaf venation pattern, silique development) but independently regulate the growth of leaves and the induction of flowering.

Probing the reproducibility of leaf growth and molecular phenotypes: a comparison of three Arabidopsis accessions cultivated in ten laboratories.

A major goal of the life sciences is to understand how molecular processes control phenotypes. Because understanding biological systems relies on the work of multiple laboratories, biologists implicitly assume that organisms with the same genotype will display similar phenotypes when grown in comparable conditions. We investigated to what extent this holds true for leaf growth variables and metabolite and transcriptome profiles of three Arabidopsis (Arabidopsis thaliana) genotypes grown in 10 laboratories using a standardized and detailed protocol. A core group of four laboratories generated similar leaf growth phenotypes, demonstrating that standardization is possible. But some laboratories presented significant differences in some leaf growth variables, sometimes changing the genotype ranking. Metabolite profiles derived from the same leaf displayed a strong genotype x environment (laboratory) component. Genotypes could be separated on the basis of their metabolic signature, but only when the analysis was limited to samples derived from one laboratory. Transcriptome data revealed considerable plant-to-plant variation, but the standardization ensured that interlaboratory variation was not considerably larger than intralaboratory variation. The different impacts of the standardization on phenotypes and molecular profiles could result from differences of temporal scale between processes involved at these organizational levels. Our findings underscore the challenge of describing, monitoring, and precisely controlling environmental conditions but also demonstrate that dedicated efforts can result in reproducible data across multiple laboratories. Finally, our comparative analysis revealed that small variations in growing conditions (light quality principally) and handling of plants can account for significant differences in phenotypes and molecular profiles obtained in independent laboratories.

The Arabidopsis Histone Chaperone FACT: Role of the HMG-Box Domain of SSRP1.

Histone chaperones play critical roles in regulated structural transitions of chromatin in eukaryotic cells that involve nucleosome disassembly and reassembly. The histone chaperone FACT is a heterodimeric complex consisting in plants and metazoa of SSRP1/SPT16 and is involved in dynamic nucleosome reorganization during various DNA-dependent processes including transcription, replication and repair. The C-terminal HMG-box domain of the SSRP1 subunit mediates interactions with DNA and nucleosomes in vitro, but its relevance in vivo is unclear. Here, we demonstrate that Arabidopsis ssrp1-2 mutant plants express a C-terminally truncated SSRP1 protein. Although the structure of the truncated HMG-box domain is distinctly disturbed, it still exhibits residual DNA-binding activity, but has lost DNA-bending activity. Since ssrp1-2 plants are phenotypically affected but viable, the HMG-box domain may be functionally non-essential. To examine this possibility, SSRP1∆HMG completely lacking the HMG-box domain was studied. SSRP1∆HMG in vitro did not bind to DNA and its interactions with nucleosomes were severely reduced. Nevertheless, the protein showed a nuclear mobility and protein interactions similar to SSRP1. Interestingly, expression of SSRP1∆HMG is almost as efficient as that of full-length SSRP1 in supporting normal growth and development of the otherwise non-viable Arabidopsis ssrp1-1 mutant. SSRP1∆HMG is structurally similar to the fungal ortholog termed Pob3 that shares clear similarity with SSRP1, but it lacks the C-terminal HMG-box. Therefore, our findings indicate that the HMG-box domain conserved among SSRP1 proteins is not critical in Arabidopsis, and thus, the functionality of SSRP1/SPT16 in plants/metazoa and Pob3/Spt16 in fungi is perhaps more similar than anticipated.

Overlapping expression patterns among the genes encoding Arabidopsis chromosomal high mobility group (HMG) proteins.

High mobility group (HMG) proteins are usually considered ubiquitous components of the eukaryotic chromatin. Using HMG gene promoter-GUS reporter gene fusions we have examined the expression of the reporter gene in transgenic Arabidopsis plants. These experiments have revealed that the different HMGA and HMGB promoters display overlapping patterns of activity, but they also show tissue- and developmental stage-specific differences. Moreover, leader introns that are present in some of the HMGB genes can modulate reporter gene expression. The differential HMG gene expression supports the view that the various HMG proteins serve partially different architectural functions in plant chromatin.

Cell specific analysis of Arabidopsis leaves using fluorescence activated cell sorting.

After initiation of the leaf primordium, biomass accumulation is controlled mainly by cell proliferation and expansion in the leaves(1). However, the Arabidopsis leaf is a complex organ made up of many different cell types and several structures. At the same time, the growing leaf contains cells at different stages of development, with the cells furthest from the petiole being the first to stop expanding and undergo senescence(1). Different cells within the leaf are therefore dividing, elongating or differentiating; active, stressed or dead; and/or responding to stimuli in sub-sets of their cellular type at any one time. This makes genomic study of the leaf challenging: for example when analyzing expression data from whole leaves, signals from genetic networks operating in distinct cellular response zones or cell types will be confounded, resulting in an inaccurate profile being generated. To address this, several methods have been described which enable studies of cell specific gene expression. These include laser-capture microdissection (LCM)(2) or GFP expressing plants used for protoplast generation and subsequent fluorescence activated cell sorting (FACS)(3,4), the recently described INTACT system for nuclear precipitation(5) and immunoprecipitation of polysomes(6). FACS has been successfully used for a number of studies, including showing that the cell identity and distance from the root tip had a significant effect on the expression profiles of a large number of genes(3,7). FACS of GFP lines have also been used to demonstrate cell-specific transcriptional regulation during root nitrogen responses and lateral root development(8), salt stress(9) auxin distribution in the root(10) and to create a gene expression map of the Arabidopsis shoot apical meristem(11). Although FACS has previously been used to sort Arabidopsis leaf derived protoplasts based on autofluorescence(12,13), so far the use of FACS on Arabidopsis lines expressing GFP in the leaves has been very limited(4). In the following protocol we describe a method for obtaining Arabidopsis leaf protoplasts that are compatible with FACS while minimizing the impact of the protoplast generation regime. We demonstrate the method using the KC464 Arabidopsis line, which express GFP in the adaxial epidermis(14), the KC274 line, which express GFP in the vascular tissue(14) and the TP382 Arabidopsis line, which express a double GFP construct linked to a nuclear localization signal in the guard cells (data not shown; Figure 2). We are currently using this method to study both cell-type specific expression during development and stress, as well as heterogeneous cell populations at various stages of senescence.

Functionality of the beta/six site-specific recombination system in tobacco and Arabidopsis: a novel tool for genetic engineering of plant genomes.

The beta recombinase is a member of the prokaryotic site-specific serine recombinases (invertase/resolvase family), which in the presence of a DNA bending cofactor can catalyse DNA deletions between two directly oriented 90-bp six recombination sites. We have examined here whether the beta recombinase can be expressed in plants and whether it displays in planta its specific catalytic activity excising DNA sequences that are flanked by six sites. In plant protoplasts, the enzyme could be expressed as a GFP-beta recombinase fusion which can localise to the cell nucleus. Beta recombinase stably expressed in tobacco plants can catalyse deletion of a spacer region that is flanked by directly oriented six sites and has been placed between promoter and a GUS reporter gene (preventing GUS expression). In transient transformation experiments, beta recombinase-mediated elimination of the spacer results in transcriptional induction of the GUS gene. Similarly, beta recombinase in stably double-transformed Arabidopsis plants deletes specifically the spacer region of a reporter construct that has been incorporated into the genome. In the segregating T1 generation, plants were identified that contain exclusively the recombined reporter construct. In summary, our results demonstrate that functional / recombinase can be expressed in plants and that the enzyme is suitable to precisely eliminate undesired sequences from plant genomes. Therefore, the beta/six recombination system (and presumably related recombinases) may become an attractive tool for plant genetic engineering.