The histone variant H2A.Z has been implicated in numerous chromatin-mediated processes, including transcriptional activation, euchromatin maintenance, and heterochromatin formation. In yeast and humans, H2A.Z is deposited into chromatin by a conserved protein complex known as SWR1 and SRCAP, respectively. Here, we show that mutations in the Arabidopsis thaliana homologs of two components of this complex, ACTIN-RELATED PROTEIN6 (ARP6) and PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1), produce similar developmental phenotypes and result in the misregulation of a common set of genes. Using H2A.Z-specific antibodies, we demonstrate that ARP6 and PIE1 are required for the deposition of H2A.Z at multiple loci, including the FLOWERING LOCUS C (FLC) gene, a central repressor of the transition to flowering. Loss of H2A.Z from chromatin in arp6 and pie1 mutants results in reduced FLC expression and premature flowering, indicating that this histone variant is required for high-level expression of FLC. In addition to defining a novel mechanism for the regulation of FLC expression, these results support the existence of a SWR1-like complex in Arabidopsis and show that H2A.Z can potentiate transcriptional activation in plants. The finding that H2A.Z remains associated with chromatin throughout mitosis suggests that it may serve an epigenetic memory function by marking active genes and poising silenced genes for reactivation.

Phytochelatin synthase (PCS) catalyzes the final step in the biosynthesis of phytochelatins, which are a family of cysteine-rich thiol-reactive peptides believed to play important roles in processing many thiol-reactive toxicants. A modified Arabidopsisthaliana PCS sequence (AtPCS1) was active in Escherichia coli. When AtPCS1 was overexpressed in Arabidopsis from a strong constitutive Arabidopsis actin regulatory sequence (A2), the A2::AtPCS1 plants were highly resistant to arsenic, accumulating 20–100 times more biomass on 250 and 300 µM arsenate than wild type (WT); however, they were hypersensitive to Cd(II). After exposure to cadmium and arsenic, the overall accumulation of thiol-peptides increased to 10-fold higher levels in the A2::AtPCS1 plants compared with WT, as determined by fluorescent HPLC. Whereas cadmium induced greater increases in traditional PCs (PC₂, PC₃, PC₄), arsenic exposure resulted in the expression of many unknown thiol products. Unexpectedly, after arsenate or cadmium exposure, levels of the dipeptide substrate for PC synthesis, γ-glutamyl cysteine (γ-EC), were also dramatically increased. Despite these high thiol-peptide concentrations, there were no significant increases in concentrations of arsenic and cadmium in above-ground tissues in the AtPCS1 plants relative to WT plants. The potential for AtPCS1 overexpression to be useful in strategies for phytoremediating arsenic and to compound the negative effects of cadmium are discussed.

Endogenous plant arsenate reductase (ACR) activity converts arsenate to arsenite in roots, immobilizing arsenic below ground. By blocking this activity, we hoped to construct plants that would mobilize more arsenate aboveground. We have identified a single gene in the Arabidopsis thaliana genome, ACR2 , with moderate sequence homology to yeast arsenate reductase. Expression of ACR2 cDNA in Escherichia coli complemented the arsenate-resistant and arsenate-sensitive phenotypes of various bacterial ars operon mutants. RNA interference reduced ACR2 protein expression in Arabidopsis to as low as 2% of wild-type levels. The various knockdown plant lines were more sensitive to high concentrations of arsenate, but not arsenite, than wild type. The knockdown lines accumulated 10- to 16-fold more arsenic in shoots (350–500 ppm) and retained less arsenic in roots than wild type, when grown on arsenate medium with <8 ppm arsenic. Reducing expression of ACR2 homologs in tree, shrub, and grass species should play a vital role in the phytoremediation of environmental arsenic contamination.

Phosphate (Pi) availability is a major constraint to plant growth. Consequently, plants have evolved complex adaptations to tolerate low Pi conditions. Numerous genes implicated in these adaptations have been identified, but their chromatin-level regulation has not been investigated. The nuclear actin-related protein ARP6 is conserved among all eukaryotes and is an essential component of the SWR1 chromatin remodeling complex, which regulates transcription via deposition of the H2A.Z histone variant into chromatin. Here, we demonstrate that ARP6 is required for proper H2A.Z deposition at a number of Pi starvation response (PSR) genes in Arabidopsis (Arabidopsis thaliana). The loss of H2A.Z at these target loci results in their derepression in arp6 mutants and correlates with the presence of multiple Pi-starvation-related phenotypes, including shortened primary roots and increases in the number and length of root hairs, as well as increased starch accumulation and phosphatase activity in shoots. Our data suggest a model for chromatin-level control of Pi starvation responses in which ARP6-dependent H2A.Z deposition modulates the transcription of a suite of PSR genes.

Actin-related proteins (ARPs) are found in the nuclei of all eukaryotic cells, but their functions are generally understood only in the context of their presence in various yeast and animal chromatin-modifying complexes. Arabidopsis thaliana ARP6 is a clear homolog of other eukaryotic ARP6s, including Saccharomyces cerevisiae ARP6, which was identified as a component of the SWR1 chromatin remodeling complex. We examined the subcellular localization, expression patterns, and loss-of-function phenotypes for this protein and found that Arabidopsis ARP6 is localized to the nucleus during interphase but dispersed away from the chromosomes during cell division. ARP6 expression was observed in all vegetative tissues as well as in a subset of reproductive tissues. Null mutations in ARP6 caused numerous defects, including altered development of the leaf, inflorescence, and flower as well as reduced female fertility and early flowering in both long- and short-day photoperiods. The early flowering of arp6 mutants was associated with reduced expression of the central floral repressor gene FLOWERING LOCUS C (FLC) as well as MADS AFFECTING FLOWERING 4 (MAF4) and MAF5. In addition, arp6 mutations suppress the FLC-mediated late flowering of a FRIGIDA-expressing line, indicating that ARP6 is required for the activation of FLC expression to levels that inhibit flowering. These results indicate that ARP6 acts in the nucleus to regulate plant development, and we propose that it does so through modulation of chromatin structure and the control of gene expression.