Enhancers located in introns are abundant and play a major role in the regulation of gene expression in mammalian species. By contrast, the functions of intronic enhancers in plants have largely been unexplored and only a handful of plant intronic enhancers have been reported. We performed a genome-wide prediction of intronic enhancers in Arabidopsis thaliana using open chromatin signatures based on DNase I sequencing. We identified 941 candidate intronic enhancers associated with 806 genes in seedling tissue and 1,271 intronic enhancers associated with 1,069 genes in floral tissue. We validated the function of 15 of 21 (71%) of the predicted intronic enhancers in transgenic assays using a reporter gene. We also created deletion lines of three intronic enhancers associated with two different genes using CRISPR/Cas. Deletion of these enhancers, which span key transcription factor binding sites, did not abolish gene expression but caused varying levels of transcriptional repression of their cognate genes. Remarkably, the transcriptional repression of the deletion lines occurred at specific developmental stages and resulted in distinct phenotypic effects on plant morphology and development. Clearly, these three intronic enhancers are important in fine-tuning tissue- and development-specific expression of their cognate genes.

Soybean is a major source of plant-based protein and vegetable oil, but its productivity is severely limited by soil salinity. Transcription factors including NAC family play pivotal roles in regulating stress-responsive pathways. Here, we identified and characterized a salt-induced NAC transcription factor, GmNAC03, in soybean. Overexpression of GmNAC03 significantly improved salt tolerance at both the germination and seedling stages. Physiological analyses revealed that antioxidant enzyme activities, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), were elevated in GmNAC03 transgenic lines, accompanied by reduced malondialdehyde (MDA) accumulation, indicating enhanced oxidative stress resistance. To further explore its regulatory mechanisms, RNA-seq analysis was performed, which showed that GmNAC03 overexpression affected pathways related to amino acid metabolism, particularly glutamine and aspartate family amino acid biosynthesis, as well as phenylpropanoid biosynthesis. Differentially expressed genes were enriched in alanine, aspartate, and glutamate metabolism, suggesting a role for GmNAC03 in metabolic reprogramming under salt stress. Together, these findings demonstrate that GmNAC03 functions as a positive regulator of salt tolerance in soybean by modulating antioxidant defense and amino acid metabolic pathways. This work provides new insights into the molecular basis of NAC-mediated stress adaptation and offers a potential target for breeding soybean varieties with enhanced salinity resistance.

Nearly all eukaryotic proteins are turned over by the ubiquitin (Ub)-26S proteasome system (UPS). Despite its broad cellular roles, only a handful of UPS members, particularly the Ub E3 ligases that specifically recognize a protein for ubiquitylation, have been characterized in plants to date. The challenge arises from the transient recognition and rapid degradation of ubiquitylation substrates by the UPS. To tackle this challenge, the emerging biotinylation-based proximity labeling (PL) offers an exciting tool for enriching transient interactors of Ub E3 ligases. In this study, we examined the efficacy of TurboID in identifying substrates of Arabidopsis Skp1-cullin1-F-box (SCF) ligases. We demonstrate that the Arabidopsis Skp1 Like (ASK)1-TurboID is not fully functioning in planta, which led us to discover a novel antagonism between biotinylation and ubiquitylation in regulating protein stability in vivo. This discovery lowers the effectiveness of PL in ubiquitylome studies. However, using one long-known SCF substrate, phytochrome A, we succeeded to apply its TurboID fusion for complementing the far-red-light response of the phyA-211 null mutant allele, suggesting an efficacy of PL in characterizing single ubiquitylation pathways. This study highlighted a limitation of PL in ubiquitylome studies, discovered a new antagonistic pathway of biotinylation, and developed a theoretical guidance for future PL-based characterization of ubiquitylation pathways.

The ubiquitin-26S proteasome system provides a key mechanism for regulating protein turnover in plants and contributes to the control of diverse developmental and stress-related processes. Within this system, Skp1-Cullin1-F-box (SCF) E3 ligases rely on F-box proteins to confer substrate specificity, enabling selective and dynamic regulation of target protein stability. The large size and structural diversity of the F-box protein family in plants suggest extensive functional specialization, although many members remain poorly characterized. Here, we review recent advances in the understanding of F-box protein function, with a focus on their roles in plant development, stress adaptation, and immunity. Specifically, this review integrates findings across development, abiotic stresses, and immunity to highlight shared and diverging regulatory nodes and critically assesses the strength of substrate evidence to distinguish bona fide from putative F-box targets. We highlight how F-box proteins modulate key regulatory pathways, including phytohormone signaling, reproductive development, root architecture, and secondary metabolism, as well as responses to abiotic and biotic stresses. Emerging evidence indicates that F-box-mediated proteolysis acts as an important layer of control linking environmental signals to downstream transcriptional and physiological outputs. A better understanding of F-box protein substrates and regulatory networks is important for dissecting plant adaptive mechanisms and may provide molecular targets for future crop improvement strategies.