Abstract Nitrate functions both as a nutrient and a signaling molecule, but how nitrate is perceived and translated into developmental outputs within woody tissues is unknown. Here, we show that developing xylem in hybrid aspen (Populus tremula × P. tremuloides) mounts a rapid, cell-type-specific transcriptional response to nitrate that promotes xylem cell expansion. To isolate local nitrate signaling in wood-forming tissues, we delivered nitrate directly into the xylem sap and profiled transcriptomic responses from 2 to 48 h after treatment. Bulk RNA sequencing revealed a transient response peaking within 2 to 4 h. Single-cell RNA sequencing at the peak response resolved nitrate signaling across developing xylem cell types and showed that nitrate induced cell-type-specific programs linked to nitrogen transport, cell wall remodeling, and developmental progression, while repressing lignin-biosynthetic genes. These data indicate that nitrate acts transiently in wood-forming tissues to fine-tune early xylem development. Among nitrate-responsive transcription factors, CYTOKININ RESPONSE FACTOR 4 (CRF4) emerged as a candidate regulator of this response. CRF4 overexpression in hybrid aspen stimulated xylem cell expansion, whereas simultaneous knockout of CRF4 and its paralog, CRF3, significantly attenuated the nitrate-induced expansion. Together, our findings identify a local nitrate signaling module in developing wood and establish CRF4 as a component linking nitrate sensing to xylem cell expansion.
Abstract Arsenic is a toxic, carcinogenic metalloid that threatens human health through contaminated water and food. Arsenite [As(III)] efflux mediated by ACR3 transporters is an ancient detoxification mechanism conserved across prokaryotes, fungi, algae, and non-angiosperm land plants, yet plant ACR3 proteins remain poorly characterized. Here, we show that MpACR3, the ACR3 ortholog from the model liverwort Marchantia polymorpha, contains an arsenic-responsive N-terminal domain that regulates intracellular trafficking. Under nonstress conditions, MpACR3 is detected in Golgi bodies as well as at the plasma membrane (PM) in plant cells, whereas As(III) promotes its relocalization to the PM. As(III) interacts with a cysteine-rich N-terminal region and stabilizes this domain, as shown by arsenite-biotin competition and increased protease resistance. Together with molecular dynamics simulations, which predict As(III)-dependent changes in secondary structure and surface properties, these findings support a model in which ligand-induced remodeling of the N-terminus modulates MpACR3 retention and trafficking. We further identify a di-arginine-containing LRCRF motif in which leucine and arginine residues are required for intracellular retention, whereas cysteine and phenylalanine residues are required for As(III)-induced relocalization to the PM. The broad conservation of related N-terminal domains in plant ACR3 transporters suggests that arsenic-regulated trafficking is a conserved plant adaptation to arsenic toxicity.
Abstract Transcription activator-like effectors (TALEs) are major virulence factors of Xanthomonas oryzae, the causal agent of bacterial blight in rice. The BED-domain nucleotide-binding leucine-rich repeat receptor Xa1 confers resistance by recognizing TALEs, but the mechanisms underlying Xa1 activation remain unclear. Here we show that structurally conserved canonical TALEs differ in their ability to activate Xa1, revealing unexpected specificity in effector recognition. Using transient expression assays, endogenous epitope tagging, and prime editing of the native Xa1 locus, we demonstrate that nuclear localization is essential for Xa1-mediated immunity. We identified three functional nuclear localization signals (NLSs) within the Xa1 N-terminal region. Prime-edited rice lines carrying mutations in these motifs exhibited compromised resistance, with mutation of the first NLS causing the strongest susceptibility phenotype. Disruption of the NLS motifs in either Xa1 or TALEs did not prevent their physical interaction, indicating that effector binding can occur independently of nuclear localization. However, only nuclear-localized Xa1 triggered cytosolic Ca2+ influx, a hallmark of immune activation. Together, our results establish nuclear localization as a prerequisite for Xa1 immune function and reveal a nuclear surveillance mechanism for BED-NLR–mediated immunity in plants.
