Whole-genome duplication (WGD) is a recurrent feature of plant evolution and crop improvement, yet its direct contribution to abiotic stress tolerance remains difficult to disentangle from hybridization effects. Using full-sibling diploid (2x) and neotetraploid (4x) Medicago sativa genotypes generated through bilateral sexual polyploidization, we investigated how WGD alone reshapes physiological performance and gene-miRNA regulatory networks under salt stress. Neotetraploid plants displayed enhanced tolerance to salinity, maintaining higher chlorophyll content, stomatal conductance, and shoot integrity, alongside reduced visual damage compared with diploid siblings. Transcriptome profiling revealed that ploidy level, rather than salt treatment per se, was the dominant driver of gene expression variation. Indeed, WGD resulted in extensive and largely constitutive transcriptional reprogramming, with more than 2,000 differentially expressed genes (DEGs) distinguishing 4x from 2x plants under both control and saline conditions. By contrast, under salt stress, 705 DEGs specifically differentiated tetraploids from diploids and were enriched in receptor-like kinases, phosphorylation pathways, ubiquitin-mediated proteolysis, flavonoid biosynthesis, and WRKY- and MYB-centered regulatory modules. Co-expression network analysis identified salt-associated modules linking signaling and antioxidant pathways with stress-related physiological traits. Small RNA sequencing further showed that WGD reshapes post-transcriptional regulation, with limited salt-induced miRNA changes but the emergence of ploidy-dependent miRNA hubs, including miR2593e and a novel miRNA, targeting signaling and defense-related genes. We hypothesize that WGD establishes a stress-ready regulatory architecture in which coordinated transcriptional and post-transcriptional network rebalancing acts as a buffering system that improves alfalfa physiological performance under salinity, without strong induction of canonical salt-responsive genes.

Lateral root organogenesis underlies root branching and plays a key role in plant root system architecture. While the transcriptional and hormonal control of lateral root development has been extensively studied over the past decades, an increasing number of studies highlight the importance of specific metabolic pathways contributing to the lateral root regulatory network. The AP2/ERF transcription factor PUCHI has recently been shown to orchestrate the induction of very-long-chain fatty acid biosynthesis in developing lateral root primordia. In parallel, a root cap cuticle, reminiscent of the very-long-chain fatty acid-rich cuticle covering the emerging primary root, was shown to cover developing lateral root primordia and facilitate their emergence. Here, we demonstrate that PUCHI plays an essential role in lateral root cap cuticle formation. We identified the transcription factors MYB94 and MYB96 as key intermediates in this PUCHI-dependent, cuticle regulatory network. Similar to PUCHI, MYB94 and MYB96 induce the expression of a set of very-long-chain fatty acid- and cutin-related genes in lateral root primordia. The loss of function of either PUCHI or that of MYB94 and MYB96 compromises lateral root cuticle deposition, delays lateral root emergence, and alters the expression pattern of lateral root meristematic genes. These results shed light on a PUCHI- and MYB94 and MYB96-dependent regulatory network controlling the lateral root cap cuticle formation and influencing lateral root primordium development and emergence.

Kiwifruit has become an important horticultural crop widely planted in temperate and subtropical regions. However, most commercially cultivated kiwifruit, particularly those of Actinidia chinensis, are highly sensitive to cold stress. To overcome this industrial bottleneck, it is crucial to reveal the molecular mechanisms behind cold stress responses, ultimately providing new insights into the breeding of cold-tolerant kiwifruit varieties. Here, we found that EIL1 negatively regulates cold tolerance of A. chinensis by directly binding to the promoters of CBF1 and CBF2 to inhibit their expression. Besides, we found that EIL1 interacted and synergized with MYB78 to negatively regulate cold tolerance in A. chinensis. Further evidence suggested that the interaction between EIL1 and MYB78 enhanced the transcriptional repression activity of both proteins on CBF1 and CBF2. Therefore, we conclude that the EIL1-MYB78 module negatively regulates cold tolerance by directly inhibiting the expression of CBF1 and CBF2 genes in A. chinensis.

Coloration (carotenoids) and aroma (volatile terpenoids) are major determinants of citrus fruit quality, but their coordinated improvement is challenging because carotenoid and volatile-terpenoid biosynthesis draw on a shared isoprenoid precursor network. Here, we used chimeric fruits of the sweet orange (Citrus sinensis L.) bud mutant "Zongcheng" to investigate this metabolic coordination. Metabolomic analyses revealed coordinated accumulation of carotenoids and volatile terpenoids in the brown peel sectors. We systematically characterized the sweet orange trans-prenyltransferase (TPT) family and identified CsGGPPS4, CsGGPPS6, and CsGGPPS8 as plastid-localized enzymes catalyze geranylgeranyl diphosphate (GGPP) biosynthesis. Comparative transcript profiling, biochemical assays, and in vivo functional analyses revealed distinct functions among these enzymes. Expression profiling and in vivo experiments demonstrated that CsGGPPS6 and CsGGPPS8 promote carotenoid accumulation, whereas CsGGPPS4 exerts only minor effects under the tested conditions. Notably, CsGGPPS6 exhibited substrate-ratio-dependent product specificity, producing predominantly farnesyl diphosphate (FPP) under equimolar substrate conditions but both FPP and GGPP when isopentenyl diphosphate (IPP) was enriched; its manipulation in vivo also altered sesquiterpenoids accumulation. CsSSU I directly interacted with CsGGPPS6 and CsGGPPS8 and differentially modulated their catalytic properties: association with CsGGPPS6 favored FPP formation and restricted further elongation to GGPP, whilst association with CsGGPPS8 enabled geranyl diphosphate (GPP) production and enhances GGPP production. Manipulation of CsSSU I in vivo coordinately affected monoterpenoids, sesquiterpenoids, and carotenoids accumulation. Collectively, these results establish that CsSSU I acts as a protein-binding modulator that remodels product output of plastid-localized TPT enzymes, and indicate that this regulatory network orchestrates terpenoid-derived pigmentation and aroma traits in citrus.

Stomata are the gatekeepers to leaf gas exchange and as such are essential for CO2 uptake for photosynthesis, water loss for evaporative cooling and ultimately plant growth and productivity. Research has long focused on understanding the mechanisms that govern stomatal aperture adjustments, in particular how changes in turgor pressure in GCs are achieved and how these cause the reversible cell shape distortion that leads to pore opening and closing. Here we discuss the immense impact the paper by Franks and Farquar (2007) has had on this research field, both in the past and more recently, by stimulating new angles of exploration of stomatal mechanics. As new techniques have emerged, the impact of the mechanical advantage of epidermal/subsidiary cells and how this is overcome by GCs has inspired novel studies into the importance of subsidiary to stomatal function, the effect of different cell shapes (dumbbell vs kidney shaped) on stomatal kinetics, the significance of cell geometries and cell complexes, as well as the underlying biochemical cell wall composition responsible for GC deformation. Whilst many questions remain unanswered, we summarize the take-home messages from the key literature on stomatal biomechanics.

The development of perpetual flowering (PF) cultivars revolutionized octoploid strawberry (Fragaria × ananassa) cultivation by enabling nearly year-round fruit production and doubling fruit yields compared to historic seasonal flowering (SF) cultivars, which flower in spring. Most commercial PF cultivars result from the introgression of a dominant PERPETUAL FLOWERING (PF) allele at the PERPETUAL FLOWERING RUNNERING (PFRU) locus. PFRU has been genetically and physically mapped to chromosome 4B, but the causal gene(s) remain unknown. Here we used the haplotype-phased "UCD Royal Royce" (PFpf) genome, next-generation sequencing-based k-mer genome-wide association study (k-mer GWAS), and RNAseq to investigate PFRU. We narrowed the locus down to a precise physical address and revealed abundant genetic and transcriptomic differences in the locus and associated with flowering. Outside the PFRU locus, flowering regulation diverges at expression of TERMINAL FLOWER 1 (TFL1) in all 4 subgenomes, analogous to diploid Fragaria vesca. This critical step of the flowering pathway is altered by PF in diverse cultivars, and gene editing knockout of TFL1 is sufficient to induce flowering under long days in a seasonal flowering cultivar. Together, these results shed light on the mechanism of flowering regulation by the PF locus and expand biotechnological approaches to achieve extended flowering in any strawberry cultivar.

Tension wood (TW) is a specialized xylem tissue formed in angiosperm trees to counteract mechanical stresses. Increased phytohormones, particularly gibberellins (GAs), and decreased lignin deposition are two well-established features of TW, yet the molecular mechanisms linking GA signaling to lignin repression remain largely unknown. Here we revealed that TW formation in Populus trichocarpa elevates GA accumulation, which activates the expression of PtrLBD39, the most strongly induced transcription factor and a repressor of lignin biosynthesis in TW development. Inhibition of GA biosynthesis attenuates PtrLBD39 induction and restores lignin deposition in TW development. Consistently, CRISPR PtrLBD39 knockout increased lignin content and endonuclease-deficient Cas9-mediated loci-specific PtrLBD39 activation reduced lignin in TW-forming stems. ChIP-seq analysis and EMSA experiments demonstrated that PtrLBD39 binds directly to the promoters of two monolignol biosynthetic genes, PtrHCT1 and PtrHCT6, via a novel TCACGB motif, thereby repressing their transcription. The PtrLBD39-DNA binding allowed PtrLBD39 to recruit two histone deacetylases, PtrHDA6-A and PtrHDA6-B, from 16 known P. trichocarpa HDACs screened and validated by Y2H, BiFC and in vitro pull-down assays, and the specific recruitments to PtrHCT1 and PtrHCT6 were confirmed during TW development by ChIP-qPCR. PtrLBD39-PtrHDA6-A or PtrLBD39-PtrHDA6-B dimers were functional for deacetylating H3K9ac and H3K14ac on PtrHCT1 and PtrHCT6 leading to transcriptional repression and lignin reduction, as demonstrated in PtrHDA6-A overexpression lines under TW development. Consistently, PtrLBD39 overexpression lines under TW development also affected PtrHCT deacetylation for lignin reduction. Thus, we suggest that mechanical stresses transduce a GA-mediated LBD39-HDA6s-HCTs epigenetic regulatory path for effective causative regulations for TW specialization.

Beech leaf disease (BLD) is an epidemic spreading among American beech (Fagus grandifolia) populations, with characteristic symptoms including dark green, yellow, or brown bands of thickened leaf tissue between secondary veins. Because near infrared (NIR) light is reflected by leaf tissue structure, we used multispectral NIR imaging reflectance to distinguish symptomatic from asymptomatic leaves amongst affected trees, as well as to distinguish between different expressions of symptoms (dark green, yellow, and brown bands) within symptomatic leaves and symptom severity (low, moderate high) at the whole leaf scale. Key findings include significantly higher NIR reflectance in dark green bands of symptomatic tissue compared to asymptomatic tissue amongst affected trees (λ = 840, 860, 900, 940, and 980 nm; ∼26.2%, 25.9%, 35.0%, 35.3%, and 32.5% increase respectively). A partial least squares regression model predicted 97% of variation in symptomatic vs. asymptomatic NIR reflectance was attributed primarily to the increase in spongy mesophyll thickness and overall leaf thickness in symptomatic tissue. Whole leaf symptom severity (asymptomatic, moderate, severe) was most clearly diagnosed at λ = 860 nm. Water infiltration of the intercellular airspaces in symptomatic dark green bands and asymptomatic leaves removed differences in NIR reflectance, supporting a mechanistic link between increased NIR reflectance and the foliar symptoms of BLD. Our results highlight the interaction of leaf internal architecture with NIR wavelengths and inform the development of targeted remote sensing tools.

Plant-virus protein-protein interactions (PPIs) play crucial roles in viral infection and host immune responses. However, large-scale experimental characterization of plant-virus PPIs is costly and labor-intensive, suggesting the need to develop computational methods. Recent advances in biological sequence language models are offering unprecedented opportunities to develop machine learning-based plant-virus PPI predictors. In this study, we collected publicly available PPI data between Arabidopsis thaliana (A. thaliana) and viruses, establishing a robust dataset for method development. Based on this dataset, we developed AraVirusPPI, a machine learning framework for predicting A. thaliana-virus PPIs. AraVirusPPI employs the protein language model ESM Cambrian (ESMC) to encode sequence features and combines these representations with Extreme Gradient Boosting (XGBoost) to build the prediction model. Furthermore, we systematically evaluated multiple protein and genomic language models combined with various classification algorithms, thereby justifying the optimal model architecture adopted in AraVirusPPI. We applied AraVirusPPI to predict PPIs in the A. thaliana-Cabbage leaf curl virus (CabLCV) system. Predicted viral target proteins were compared with experimental datasets from A. thaliana-Turnip mosaic virus (TuMV) and A. thaliana-Pseudomonas syringae (Psy) systems. Network topology and functional enrichment analyses showed that the predicted CabLCV targets in A. thaliana were more similar to TuMV than Psy targets. These results suggest potential shared and distinct host-targeting patterns among pathogens and provide computational evidence supporting the biological significance of the proposed AraVirusPPI predictor.

Floral organ identity specification and subsequent organ differentiation are generally regarded as distinct developmental processes, yet the mechanisms connecting these programs remain poorly understood. In orchids, the P code explains how higher-order MADS-box complexes specify sepal, petal, and lip identities; however, how these complexes coordinate downstream developmental outputs remains unknown. Here, we identify an ARF-MADS regulatory module that integrates auxin signaling into the orchid floral identity network. Simultaneous silencing of PaARF6/8/19 causes defects in perianth identity establishment, organ growth, pigmentation, and pedicel abscission that closely resemble those resulting from disruption of P-code complexes. Among these ARFs, PaARF6 functions as the predominant regulator, whereas PaARF8 and PaARF19 provide partially redundant contributions. Mechanistically, PaARF proteins physically interact in vivo with the P-code A/E-class MADS-box proteins OAGL6-1, OAGL6-2, PaSEP1, and PaSEP3. Both genetic and biochemical evidence support a hierarchical organization of this module, with PaARF6 and OAGL6 proteins constituting its principal regulatory axis. Furthermore, conserved interactions between Arabidopsis ARFs and SEP proteins, together with petal identity defects in arf mutants, suggest evolutionary conservation of this mechanism. These findings establish the ARF-MADS module as a mechanistic bridge linking floral organ identity specification with differentiation and maturation.