Ribosome assembly factor Adenylate Kinase 6 maintains cell proliferation and cell size homeostasis during root growth.

From the cellular perspective, organ growth is determined by production and growth of cells. Uncovering how these two processes are coordinated is essential for understanding organogenesis and regulation of organ growth. We utilized phenotypic and genetic variation of 252 natural accessions of Arabidopsis thaliana to conduct genome-wide association studies (GWAS) for identifying genes underlying root growth variation; using a T-DNA line candidate approach, we identified one gene involved in root growth control and characterized its function using microscopy, root growth kinematics, G2/M phase cell count, ploidy levels and ribosome polysome profiles. We identified a factor contributing to root growth control: Arabidopsis Adenylate Kinase 6 (AAK6). AAK6 is required for normal cell production and normal cell elongation, and its natural genetic variation is involved in determining root growth differences between Arabidopsis accessions. A lack of AAK6 reduces cell production in the aak6 root apex, but this is partially compensated for by longer mature root cells. Thereby, aak6 mutants exhibit compensatory cell enlargement, a phenomenon unexpected in roots. Moreover, aak6 plants accumulate 80S ribosomes while the polysome profile remains unchanged, consistent with a phenotype of perturbed ribosome biogenesis. In conclusion, AAK6 impacts ribosome abundance, cell production and thereby root growth.

The rate and potential relevance of new mutations in a colonizing plant lineage.

By following the evolution of populations that are initially genetically homogeneous, much can be learned about core biological principles. For example, it allows for detailed studies of the rate of emergence of de novo mutations and their change in frequency due to drift and selection. Unfortunately, in multicellular organisms with generation times of months or years, it is difficult to set up and carry out such experiments over many generations. An alternative is provided by "natural evolution experiments" that started from colonizations or invasions of new habitats by selfing lineages. With limited or missing gene flow from other lineages, new mutations and their effects can be easily detected. North America has been colonized in historic times by the plant Arabidopsis thaliana, and although multiple intercrossing lineages are found today, many of the individuals belong to a single lineage, HPG1. To determine in this lineage the rate of substitutions-the subset of mutations that survived natural selection and drift-, we have sequenced genomes from plants collected between 1863 and 2006. We identified 73 modern and 27 herbarium specimens that belonged to HPG1. Using the estimated substitution rate, we infer that the last common HPG1 ancestor lived in the early 17th century, when it was most likely introduced by chance from Europe. Mutations in coding regions are depleted in frequency compared to those in other portions of the genome, consistent with purifying selection. Nevertheless, a handful of mutations is found at high frequency in present-day populations. We link these to detectable phenotypic variance in traits of known ecological importance, life history and growth, which could reflect their adaptive value. Our work showcases how, by applying genomics methods to a combination of modern and historic samples from colonizing lineages, we can directly study new mutations and their potential evolutionary relevance.

Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD.

Previously, we showed that a major in vitro and in vivo mechanism of resistance to pyrazinoic acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Binding was abolished for mutant PanD proteins. Taken together, these findings support a mechanism of action of POA in which the bioactive component of PZA inhibits coenzyme A biosynthesis via binding to aspartate decarboxylase PanD. Together with previous works, these results establish PanD as a genetically, metabolically, and biophysically validated target of PZA.

Natural allelic variation of FRO2 modulates Arabidopsis root growth under iron deficiency.

Low availability of Fe significantly limits crop yields in many parts of the world. However, it is largely unknown which genes and alleles adjust plant growth in Fe limited environments. Using natural variation of a geographically restricted panel of Arabidopsis thaliana accessions, we identify allelic variation at the FRO2 locus associated with root length under iron deficiency. We show that non-coding sequence variation at the FRO2 locus leads to variation of FRO2 transcript levels, as well as ferric chelate reductase activity, and is causal for a portion of the observed root length variation. These FRO2 allele dependent differences are coupled with altered seedling phenotypes grown on iron-limited soil. Overall, we show that these natural genetic variants of FRO2 tune its expression. These variants might be useful for improvement of agronomically relevant species under specific environmental conditions, such as in podzols or calcareous soils.

Interaction of HIF and USF signaling pathways in human genes flanked by hypoxia-response elements and E-box palindromes.

Rampant activity of the hypoxia-inducible factor (HIF)-1 in cancer is frequently associated with the malignant progression into a harder-to-treat, increasingly aggressive phenotype. Clearly, anti-HIF strategies in cancer cells are of considerable clinical interest. One way to fine-tune, or inhibit, HIF's transcriptional outflow independently of hydroxylase activities could be through competing transcription factors. A CACGTG-binding activity in human hepatoma cells was previously found to restrict HIF's access to hypoxia response cis-elements (HRE) in a Daphnia globin gene promoter construct (phb2). The CACGTG factor, and its impact on hypoxia-responsive human genes, was analyzed in this study by genome-wide computational scans as well as gene-specific quantitative PCR, reporter and DNA-binding assays in hepatoma (Hep3B), cervical carcinoma (HeLa), and breast carcinoma (MCF7) cells. Among six basic helix-loop-helix transcription factors known to target CACGTG palindromes, we identified upstream stimulatory factor (USF)-1/2 as predominant phb2 CACGTG constituents in Hep3B, HeLa, and MCF7 cells. Human genes with adjacent or overlapping HRE and CACGTG motifs included with lactate dehydrogenase A (LDHA) and Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3) hypoxia-induced HIF-1 targets. Parallel recruitment of HIF-1α and USF1/2a to the respective promoter chromatin was verified for all cell lines investigated. Mutual complementing (LDHA) or moderating (BNIP3) cross-talk was seen upon overexpression or silencing of HIF-1α and USF1/2a. Distinct (LDHA) or overlapping (BNIP3) promoter-binding sites for HIF-1 and USFs were subsequently characterized. We propose that, depending on abundance or activity of its protein constituents, O(2)-independent USF signaling can function to fine-tune or interfere with HIF-mediated transcription in cancer cells.