Active DNA demethylation is critical for controlling the DNA methylomes in plants and mammals. However, little is known about how DNA demethylases are recruited to target loci, and the involvement of chromatin marks in this process. Here, we identify 2 components of the SWR1 chromatin-remodeling complex, PIE1 and ARP6, as required for ROS1-mediated DNA demethylation, and discover 2 SWR1-associated bromodomain-containing proteins, AtMBD9 and nuclear protein X1 (NPX1). AtMBD9 and NPX1 recognize histone acetylation marks established by increased DNA methylation 1 (IDM1), a known regulator of DNA demethylation, redundantly facilitating H2A.Z deposition at IDM1 target loci. We show that at some genomic regions, H2A.Z and DNA methylation marks coexist, and H2A.Z physically interacts with ROS1 to regulate DNA demethylation and antisilencing. Our results unveil a mechanism through which DNA demethylases can be recruited to specific target loci exhibiting particular histone marks, providing a conceptual framework to understand how chromatin marks regulate DNA demethylation. Significance Transgenes and some endogenous genes are protected from silencing by active DNA demethylation. How the demethylase ROS1 targets these specific genomic regions is poorly understood. Here, we show that H2A.Z and components of the conserved SWR1 complex are required for antisilencing. We found that ROS1 is recruited to some of its target regions by H2A.Z deposited by the SWR1 complex. Two bromodomain-containing proteins, AtMBD9 and NPX1, are critical for the recruitment of SWR1 complex to chromatin through recognition of histone acetylation marks created by the increased DNA methylation (IDM) complex, which is known to have a regulatory role in active DNA demethylation. Thus, we have identified a complete regulatory pathway initiated by the IDM complex in active DNA demethylation in Arabidopsis.

Heavy metal (HM) pollution, specifically cadmium (Cd) contamination, is a worldwide concern for its consequences for plant health and ecosystem stability. This review sheds light on the intricate mechanisms underlying Cd toxicity in plants and the various strategies employed by these organisms to mitigate its adverse effects. From molecular responses to physiological adaptations, plants have evolved sophisticated defense mechanisms to counteract Cd stress. We highlighted the role of phytochelatins (PC_n) in plant detoxification, which chelate and sequester Cd ions to prevent their accumulation and minimize toxicity. Additionally, we explored the involvement of glutathione (GSH) in mitigating oxidative damage caused by Cd exposure and discussed the regulatory mechanisms governing GSH biosynthesis. We highlighted the role of transporter proteins, such as ATP-binding cassette transporters (ABCs) and heavy metal ATPases (HMAs), in mediating the uptake, sequestration, and detoxification of Cd in plants. Overall, this work offered valuable insights into the physiological, molecular, and biochemical mechanisms underlying plant responses to Cd stress, providing a basis for strategies to alleviate the unfavorable effects of HM pollution on plant health and ecosystem resilience.

Zinc (Zn) is an essential element for plant growth and development, but at high levels this metal can become toxic. Hyperaccumulator species are often not suitable for phytoremediation technologies because they need to be fast growing and have high biomass production, such as those of the Populus genus. Comparative genomics studies of poplars subjected to stress conditions such as heavy metal contamination have generated resources useful for improving the annotation of genes and have provided novel insights in the defense/tolerance mechanisms governing adaptation in non-hyperaccumulator plants. Using a microarray-based comparative analysis, we identified functional gene sets that are differentially regulated in the leaves of Populus × euramericana clone I-214 subjected to an excess but sub-lethal dose of Zn (1 mM). Eco-physiological and chemical analyses confirmed the results obtained in previous similar experiments. A total of 3861 expressed sequence tags (ESTs) were differentially expressed and grouped into two distinct libraries of up-regulated (40%) and down-regulated (60%) putative genes. The annotation of genes and gene products according to the Gene Ontology vocabularies was performed using Blast2GO software. The two transcriptome data sets were used to query all known Kyoto Encyclopedia of Genes and Genomes (KEGG) biosynthetic pathways of the genes identified in this study. The most represented molecular functions and biological processes were nucleotide binding and transcription, transport and response to stress and abiotic and biotic stimuli. The chloroplast, mitochondrion and their membrane systems were the cellular components most affected by excess Zn, as well as the photosynthetic, defense, sulfur and glutathione (GSH) metabolic pathways. The most up-regulated genes encoded electron carriers associated with ferrodoxin, the small subunit of ribulose-bisphosphate carboxylase oxygenase, and enzymes involved in GSH metabolism. This study is the most in-depth transcriptome and gene-annotation analysis of a hybrid poplar to date. The results are presented and critically discussed in terms of poplar response/tolerance to Zn stress for the characterization of non-hyperaccumulator phenotypes and the identification of candidate genes in perennial plants. These genetic findings provide useful information on tree species' adaptation to metal stress and provide powerful tools for the selection and/or genetic manipulation of stress-tolerant poplar clones.

The enzyme phytochelatin synthase (PCS) has long been studied with regard to its role in metal(loid) detoxification in several organisms, i.e., plants, yeasts, and nematodes. It is in fact widely recognized that PCS detoxifies a number of heavy metals by catalyzing the formation of thiol-rich oligomers, namely phytochelatins, from glutathione and related peptides. However, recent investigations have highlighted other possible roles played by the PCS enzyme in the plant cell, e.g., the control of pathogen-triggered callose deposition. In order to examine novel aspects of Arabidopsis thaliana PCS1 (AtPCS1) functions and to elucidate its possible roles in the secondary metabolism, metabolomic data of A. thaliana wild-type and cad1-3 mutant were compared, the latter lacking AtPCS1. HPLC-ESI-MS analysis showed differences in the relative levels of metabolites from the glucosinolate and phenylpropanoid pathways between cad1-3 and wild-type plants. Specifically, in control (Cd-untreated) plants, higher levels of 4-methoxy-indol-3-ylmethylglucosinolate were found in cad1-3 plants vs. wild-type. Moreover, the cad1-3 mutant showed to be impaired in the deposit of callose after Cd exposure, suggesting that AtPCS1 protects the plant against the toxicity of heavy metals not only by synthesizing PCs, but also by contributing to callose deposition. In line with the contribution of callose in counteracting Cd toxicity, we found that another callose-defective mutant, pen2-1, was more sensitive to high concentrations of Cd than wild-type plants. Moreover, cad1-3 plants were more susceptible than wild-type to the hemibiotrophic bacterial pathogen Pseudomonas syringae. The metabolome also revealed differences in the relative levels of hydroxycinnamic acids and flavonols, with consequences on cell wall properties and auxin content, respectively. First, increased lignification in the cad1-3 stems was found, probably aimed at counteracting the entry of Cd into the inner tissues. Second, in cad1-3 shoots, increased relative levels of kaempferol 3,7 dirhamnoside and quercetin hexoside rhamnoside were detected. These flavonols are endogenous inhibitors of auxin transport in planta; auxin levels in both roots and shoots of the cad1-3 mutant were in fact lower than those of the wild-type. Overall, our data highlight novel aspects of AtPCS1 functions in A. thaliana.