Salt stress is a major environmental factor limiting plant growth and productivity. We recently discovered an important new salt tolerance pathway, where the cell wall leucine-rich repeat extensins LRX3/4/5, the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23 and receptor-like kinase FERONIA (FER) function as a module to simultaneously regulate plant growth and salt stress tolerance. However, the intracellular signaling pathways that are regulated by the extracellular LRX3/4/5-RALF22/23-FER module to coordinate growth, cell wall integrity and salt stress responses are still unknown. Here, we report that the LRX3/4/5-RALF22/23-FER module negatively regulates the levels of jasmonic acid (JA), salicylic acid (SA) and abscisic acid (ABA). Blocking JA pathway rescues the dwarf phenotype of the lrx345 and fer-4 mutants, while disruption of ABA biosynthesis suppresses the salt-hypersensitivity of these mutants. Many salt stress-responsive genes display abnormal expression patterns in the lrx345 and fer-4 mutants, as well as in the wild type plants treated with epigallocatechin gallate (EGCG), an inhibitor of pectin methylesterases, suggesting cell wall integrity as a critical factor that determines the expression pattern of stress-responsive genes. Production of reactive oxygen species (ROS) is constitutively increased in the lrx345 and fer-4 mutants, and inhibition of ROS accumulation suppresses the salt-hypersensitivity of these mutants. Together, our work provides strong evidence that the LRX3/4/5-RALF22/23-FER module controls plant growth and salt stress responses by regulating hormonal homeostasis and ROS accumulation.

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.

Significance: The histone 3 lysine 9 demethylase Increase in BONSAI Methylation 1 (IBM1) is critical for preventing CHG (H=A, T, or C) methylation in plant genes. We found that the putative chromatin regulator Enhanced Downy Mildew 2 (EDM2) controls genome CHG methylation and transgene silencing by regulating alternative polyadenylation of IBM1 . EDM2 contains a composite plant homeo domain that simultaneously recognizes both repressive and active histone marks, allowing it to associate specifically with the intronic heterochromatin of IBM1 and other plant genes so that it may affect polyadenylation by methylating the RNA through an N6-adenine methyltransferase-like activity. This work significantly advances our understanding of the regulation of DNA methylome and mechanisms of antisilencing and alternative polyadenylation of intronic heterochromatin-containing genes.