Zinc is an essential micronutrient for all living organisms. When facing a shortage in zinc supply, plants adapt by enhancing the zinc uptake capacity. The molecular regulators controlling this adaptation are not known. We present the identification of two closely related members of the Arabidopsis thaliana basic-region leucinezipper (bZIP) transcription factor gene family, bZIP19 and bZIP23, that regulate the adaptation to low zinc supply. They were identified, in a yeast-one-hybrid screening, to associate to promoter regions of the zinc deficiency-induced ZIP4 gene of the Zrt-and Irtrelated protein (ZIP) family of metal transporters. Although mutation of only one of the bZIP genes hardly affects plants, we show that the bzip19 bzip23 double mutant is hypersensitive to zinc deficiency. Unlike the wild type, the bzip19 bzip23 mutant is unable to induce the expression of a small set of genes that constitutes the primary response to zinc deficiency, comprising additional ZIP metal transporter genes. This set of target genes is characterized by the presence of one or more copies of a 10-bp imperfect palindrome in their promoter region, to which both bZIP proteins can bind. The bZIP19 and bZIP23 transcription factors, their target genes, and the characteristic cis zinc deficiency response elements they can bind to are conserved in higher plants. These findings are a significant step forward to unravel the molecular mechanism of zinc homeostasis in plants, allowing the improvement of zinc bio-fortification to alleviate human nutrition problems and phytoremediation strategies to clean contaminated soils.biofortification | zinc homeostasis regulation | plant nutrition | abiotic stress | adaptation Z inc is an essential cofactor for many transcription factors, protein interaction domains, and enzymes in plants (1). Plants are thought to control zinc homeostasis by using a tightly regulated network of zinc status sensors and signal transducers controlling the coordinated expression of proteins involved in zinc acquisition from soil, mobilization between organs and tissues, and sequestration within cellular compartments (2). Although candidate genes for the required proteins such as zinc transporters and chelator biosynthesizing enzymes are found, no regulator of such network was ever identified in plants.Zinc influx facilitators, members of the ZIP family of metal transporters, are thought to play a major role in zinc uptake in plants (3). In Arabidopsis there are 15 ZIP genes (4), with ZIP1, ZIP2, ZIP3, and IRT3 functionally characterized as zinc uptake transporters (3, 5). Gene expression analysis has shown that approximately half of the ZIP genes are induced in response to zinc deficiency (3,(5)(6)(7)(8). The ZIP4 gene in particular is strongly induced upon shortage in zinc supply (3,(6)(7)(8).We focused on the promoter of this zinc-deficiency-responsive gene as the starting point for unraveling the regulation of the zinc homeostasis network in plants. By using DNA fragments of the zincdeficiency-responsive Arabidopsi...

The plant circadian clock is proposed to be a network of several interconnected feedback loops, and loss of any component leads to changes in oscillator speed. We previously reported that Arabidopsis thaliana EARLY FLOWERING4 (ELF4) is required to sustain this oscillator and that the elf4 mutant is arrhythmic. This phenotype is shared with both elf3 and lux. Here, we show that overexpression of either ELF3 or LUX ARRHYTHMO (LUX) complements the elf4 mutant phenotype. Furthermore, ELF4 causes ELF3 to form foci in the nucleus. We used expression data to direct a mathematical position of ELF3 in the clock network. This revealed direct effects on the morning clock gene PRR9, and we determined association of ELF3 to a conserved region of the PRR9 promoter. A cis-element in this region was suggestive of ELF3 recruitment by the transcription factor LUX, consistent with both ELF3 and LUX acting genetically downstream of ELF4. Taken together, using integrated approaches, we identified ELF4/ELF3 together with LUX to be pivotal for sustenance of plant circadian rhythms.

Arabidopsis thaliana EARLY FLOWERING3 (ELF3) is essential for the generation of circadian rhythms. ELF3 has been proposed to restrict light signals to the oscillator through phytochrome photoreceptors, but that has not been explicitly shown. Furthermore, the genetic action of ELF3 within the clock had remained elusive. Here, we report a functional characterization of ELF3 through the analysis of the elf3-12 allele, which encodes an amino acid replacement in a conserved domain. Circadian oscillations persisted, and unlike elf3 null alleles, elf3-12 resulted in a short circadian period only under ambient light. The period shortening effect of elf3-12 was enhanced by the overexpression of phytochromes phyA and phyB. We found that elf3-12 was only modestly perturbed in resetting of the oscillator and in gating light-regulated gene expression. Furthermore, elf3-12 essentially displayed wild-type development. We identified targets of ELF3 transcriptional repression in the oscillator, highlighting the action at the morning gene PSEUDO-RESPONSE REGULATOR9. Taken together, we identified two separable roles for ELF3, one affecting the circadian network and the other affecting light input to the oscillator. This is consistent with a dual function of ELF3 as both an integrator of phytochrome signals and a repressor component of the core oscillator.

Natural selection of variants within the Arabidopsis thaliana circadian clock can be attributed to adaptation to varying environments. To define a basis for such variation, we examined clock speed in a reporter-modified Bay-0 x Shakdara recombinant inbred line and localized heritable variation. Extensive variation led us to identify EARLY FLOWERING3 (ELF3) as a major quantitative trait locus (QTL). The causal nucleotide polymorphism caused a short-period phenotype under light and severely dampened rhythm generation in darkness, and entrainment alterations resulted. We found that ELF3-Sha protein failed to properly localize to the nucleus, and its ability to accumulate in darkness was compromised. Evidence was provided that the ELF3-Sha allele originated in Central Asia. Collectively, we showed that ELF3 protein plays a vital role in defining its light-repressor action in the circadian clock and that its functional abilities are largely dependent on its cellular localization.DOI: http://dx.doi.org/10.7554/eLife.02206.001

Circadian clocks mediate adaptation to the 24-h world. In Arabidopsis, most circadian-clock components act in the nucleus as transcriptional regulators and generate rhythmic oscillations of transcript accumulation. In this review, we focus on post-transcriptional events that modulate the activity of circadian-clock components, such as phosphorylation, ubiquitination and proteasome-mediated degradation, changes in cellular localization, and protein-protein interactions. These processes have been found to be essential for circadian function, not only in plants, but also in other circadian systems. Moreover, light and clock signaling networks are highly interconnected. In the nucleus, light and clock components work together to generate transcriptional rhythms, leading to a general control of the timing of plant physiological processes.

The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene, MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.

The polarized partitioning of proteins in cells underlies asymmetric cell division, which is an important driver of development and cellular diversity. The budding yeast Saccharomyces cerevisiae divides asymmetrically, like many other cells, to generate two distinct progeny cells. A well-known example of an asymmetric protein is the transcription factor Ace2, which localizes specifically to the daughter nucleus, where it drives a daughter-specific transcriptional network. We screened a collection of essential genes to analyze the effect of core cellular processes in asymmetric cell division based on Ace2 localization. This screen identified mutations that affect progression through the cell cycle, suggesting that cell cycle delay is sufficient to disrupt Ace2 asymmetry. To test this model, we blocked cells from progressing through mitosis and found that prolonged metaphase delay is sufficient to disrupt Ace2 asymmetry after release, and that Ace2 asymmetry is restored after cytokinesis. We also demonstrate that members of the evolutionarily conserved FACT chromatin-reorganizing complex are required for both asymmetric and cell cycle-regulated localization of Ace2 and for localization of the RAM network components.

Kinetochores serve both a structural role linking chromosomes to the mitotic spindle and a regulatory role, controlling the timing of mitosis via the spindle assembly checkpoint. To identify proteins that regulate the kinetochore we used a genome-wide fluorescence microscopy approach. We combined an array of mutants of either non-essential gene deletions or essential temperature-sensitive alleles with fluorescently tagged spindle pole bodies (centrosome) and outer kinetochores. Quantitative and qualitative analysis revealed mutants that affect the levels and distribution of kinetochores respectively. These mutants are enriched for those involved in mRNA processing, chromatin organization, DNA replication/repair and mitosis. Our data show that the Pkc1 kinase maintains the kinetochore focus via its ability to prevent cell stress and this phenotype is rescued by an osmotic stabilizer. These data support the notion that kinetochore and microtubule homeostasis are perturbed by the stress response pathways. Hence this observation provides a candidate mechanism for extracellular stress leading to chromosome segregation defects.