Plants activate osmotic regulation, ion and reactive oxygen species (ROS) homeostasis modulation, and plant hormone abscisic acid (ABA)-mediated stomatal movement regulation for survival under salt stress. However, it is unknown if and how a single molecule coordinates multiple responses. Here we characterized the functions of three closest homologs, cyclic nucleotide-gated channel 14, 15, and 16 (OsCNGC14/15/16), in regulating multiple signaling pathways in response to salt. Mutants of each of these three genes had reduced salt tolerance compared to the wild type. These mutants also had slower stomatal closure, lower Ca2+ influx, higher Na+/K+ ratio, and ROS accumulation under salt treatment. Conversely, overexpression of OsCNGC16 led to enhanced salt tolerance, increased Ca2+ influx, lower Na+/K+ ratio, and ROS accumulation in response to salt. OsCNGC14/15/16 also positively regulate shoot growth under ABA treatment. Exogenous ABA treatment rescued the defects in salt tolerance and stomatal closure of the OsCNGC16 mutant. And treatment of ROS scavenger dimethylthiourea (DMTU) partially rescued the salt susceptibility of the OsCNGC16 mutant. In addition, OsCNGC16 can interact with and be phosphorylated by Ca2+-dependent protein kinases 5 and 13 (CPK5/13). This study unveils multiple roles of OsCNGC14/15/16 proteins in salt tolerance and identifies common regulators of stomatal closure, Ca2+ influx, Na+/K+ homeostasis, ROS scavenging, and ABA signaling in response to salt stress.
Salinity induces the accumulation of reactive oxygen species (ROS) in plants, thereby regulating plant growth and development, including seed germination. However, the molecular mechanisms underlying the regulation of ROS homeostasis in response to salt stress remain largely unknown. Here, we showed that an leucine-rich-repeat receptor-like kinase, REK1, regulates ROS production by phosphorylating the NADPH oxidase respiratory burst oxidase homolog D (RBOHD) in Arabidopsis in response to salt stress. Salt-induced ROS accumulates extensively in rek1 mutants, causing stress that reduces the seed germination rate of rek1 below that of the wild-type (Col-0), resulting in the salt-sensitive phenotype. Furthermore, REK1 interacts with RBOHD and phosphorylates it at Ser163, thereby regulating its NADPH oxidase activity and influencing ROS production under salt stress. Genetic data revealed that the salt-sensitive phenotype of rek1 is rescued in rek1 rbohd mutants. In summary, our study revealed a salt stress regulation mechanism that modulates ROS production in Arabidopsis through the REK1-RBOHD pathway.
Agrobacterium transfers DNA into plant cells, leading to tumors, hairy roots (HR), and natural genetically modified organisms (nGMOs). Transferred DNAs (T-DNAs) from agrobacteria and T-DNA-derived cellular T-DNAs (cT-DNAs) from nGMOs vary considerably and may carry up to 15 different genes. Among these, opine synthase (ops) genes encode the synthesis of opines used as nutrients by the agrobacteria. Earlier studies predicted large numbers of naturally transformed plant species, but only few have been identified and studied so far. We therefore developed a general method to detect cT-DNAs in all publicly available whole genome sequences (WGS) and Sequence Read Archive (SRA) data from land plants. To avoid false positives, we only retained DNA sequences coding for T-DNA proteins. A total of 2614 nGMO species were identified, most are eudicots. However, cT-DNAs were also found in 82 mosses and 75 ferns, showing that Agrobacterium can also generate natural transformants among the early land plants. Analysis of 149 cT-DNA maps revealed different types of T-DNAs. Most notably, these included small T-DNAs (mini T-DNAs) with a single opine synthase gene. Mini T-DNAs are not expected to induce tumors or HRs. The predominance of mini cT-DNAs in mosses and ferns, and the presence of more complex cT-DNAs in spermatophytes, indicate that mini T-DNAs represent the earliest types of T-DNA. Our study also detected unusual T-DNA integration patterns, with multiple copies spread out over several hundreds of kilobases.
Leaf senescence is a tightly regulated developmental process orchestrated by multiple transcription factor (TF) families. Although C2H2-type zinc finger proteins are known to participate in various aspects of plant growth and abiotic stress responses, their specific role in regulating leaf senescence remains poorly understood. Here, we show that knockout mutation of the C2H2-type zinc finger TF gene ZOS202 (LOC_Os02g02424) resulted in delayed leaf senescence under both dark-induced and natural conditions, while overexpression lines exhibit accelerated leaf senescence, as indicated by reactive oxygen species (ROS) overaccumulation, chloroplast degradation, and leaf cell death. As a dual-function transcription factor whose expression peaks at senescence onset, ZOS202 directly activates senescence-associated TF genes OsWRKY42, OsWRKY53, and ONAC096, while repressing peroxidase genes OsPRX113, OsPRX114, and OsPRX122, thereby disrupting ROS homeostasis and promoting senescence. Collectively, our findings uncover ZOS202 as a positive regulator of leaf senescence that acts by coordinating chlorophyll breakdown and ROS accumulation, providing promising strategies for fine-tuning leaf senescence in rice breeding.
Whole-genome duplication is an important evolutionary mechanism in plants. Although numerous studies have morphologically characterized the polyploid plants, such as increased grain yield and enhanced resistance to pests and diseases, few studies are involved in the response of mitochondria and chloroplasts to the nuclear genome shock to reconcile the nucleo-cytoplasmic interactions in tetraploid rice. In this study, we employed the diploid rice cultivars Huang-Jin-Ke (HJK) and 032 and the artificially synthesized autotetraploid rice lines HJK-4n and 032-4n with normal fertility, to investigate their response of mitochondria and chloroplast genomes to the duplication of nuclear genome. Results revealed that, compared with diploid rice, autotetraploid rice exhibited extensive mitochondrial genome rearrangement and recombination, which led to significantly high expression of mitochondrial genes, and activity of mitochondrial respiratory chain complexes. Meanwhile, autotetraploid rice possessed a greater number of chloroplasts than diploid rice, leading to a significant increase in photosynthetic efficiency and biomass. Correspondingly, the nuclear genes associated with mitochondrial genome rearrangement and plastid duplication exhibited markedly elevated expression and high levels of H3K4me3 and H3K9me2 enrichment. This discovery reveals that the plastid and mitochondria reconcile the nucleo-cytoplasmic interaction in different ways in autotetraploid rice.
Stem strength is a key agronomic trait that influences planting adaptability and mechanical harvesting efficiency. Pepper is the most widely cultivated vegetable crop in China, and full mechanization of its production is crucial for enhancing quality and productivity. In the preliminary stage of this study, transcriptomic analysis was performed on pepper accessions exhibiting substantial differences in stem strength at various developmental stages. Through weighted gene co-expression network analysis focused on genes involved in cell wall biosynthesis and transport, a candidate gene potentially regulating stem strength, designated CaDUF642.01, was identified. The CaDUF642.01 protein contains two DUF642 domains and localizes to the cell wall, plasma membrane, and nucleus. Silencing CaDUF642.01 led to a 42.17% decrease in soluble pectin content, a 1.63% reduction in water content, a 43.18% shortening of internode length, and more compact cell arrangement. Furthermore, CaDUF642.01 was shown to directly interact with pectin methylesterase 2 (CaPME2), forming a protein complex that modulates pectin methylation levels via regulation of random PME demethylesterification, thereby influencing stem strength. Our results demonstrate that the CaDUF642.01-CaPME2 complex attenuates stem strength by altering pectin composition through PME activity, which also increases stem water content and promotes looser parenchyma cell packing. This study reveals a novel regulatory mechanism of stem strength in pepper, providing a theoretical basis for manipulating plant architecture and breeding mechanization-adapted pepper varieties.
Traditional genotype-to-phenotype models depend heavily on direct mappings that achieve only modest accuracy, forcing breeders to conduct large, costly field trials to maintain or marginally improve genetic gain. Models that incorporate intermediate molecular phenotypes can achieve higher predictive fit, but remain impractical since such data are unavailable at deployment or design time. Biology-informed neural networks (BINNs) overcome this limitation by encoding pathway-level inductive biases and leveraging multi-omics data only during training, while using genotype data alone during inference. Here, we extend BINNs for genomic prediction and selection in crops by integrating thousands of single-nucleotide polymorphisms with multi-omics measurements and prior biological knowledge. By directly embedding omics-derived priors, BINN outperforms conventional models in low-data (n < p) regimes and enables sensitivity analyses that expose biologically meaningful traits. Applied to maize gene expression and multi-environment field trial data, BINN improves rank correlation accuracy within and across most subpopulations under sparse data conditions and nonlinearly identifies genes that GWAS/transcriptome-wide association studies may fail to uncover. With complete domain knowledge for a synthetic metabolomics benchmark, BINN substantially reduces prediction error relative to conventional neural nets and correctly identifies the most important nonlinear pathway. Importantly, both cases show that highly sensitive BINN latent variables correlate with the experimental quantities they represent, despite not being trained on them. This suggests that BINNs learn biologically relevant representations, nonlinear or linear, from genotype to phenotype. Together, BINNs establish a framework for improved genomic prediction accuracy and biological discovery that can guide genomic selection, candidate gene selection, pathway enrichment, and gene-editing prioritization.
Rice (Oryza sativa L.) is a staple cereal crop whose grain size substantially influences yield. The kinase OsGSK3, a known negative regulator of brassinosteroid (BR) signaling, was previously shown to modulate grain length. Here, we characterize the functional divergence between its two major haplotypes, OsGSK3Hap3 and OsGSK3Hap5. A single-nucleotide polymorphism in OsGSK3Hap5 extends the OsGSK3 open reading frame, adding 13 C-terminal amino acids and correlating with longer grains, whereas OsGSK3Hap3 is associated with shorter grains. Consistently, overexpression of OsGSK3Hap3 reduced grain size, while overexpression of OsGSK3Hap5 or knockout of OsGSK3Hap3 increased it. Both haplotypes physically interact with OsRanBP1, a positive regulator of grain length. Biochemical analyses demonstrated that OsGSK3Hap3 possesses stronger kinase activity than OsGSK3Hap5 and more efficiently phosphorylates OsRanBP1, triggering its degradation and thereby suppressing grain length. Conversely, the weaker kinase activity of OsGSK3Hap5 results in attenuated OsRanBP1 phosphorylation and accumulation, promoting longer grains. Genetic and physiological evidence further demonstrates that OsGSK3 regulates rice grain development within the BR signaling pathway in an OsRanBP1-dependent manner. Our findings reveal that natural variation in OsGSK3 differentially fine-tunes grain size by modulating OsRanBP1 protein stability via phosphorylation, thereby providing a molecular mechanism underlying grain size diversity in rice.
Despite the high importance of Brassica species as crops, their complex genomic relationships and their small, poorly differentiated chromosomes are challenging for analysis. Brassica diploids have AA, BB, and CC genomes, whereas allopolyploids can occur in different subgenome combinations. During interspecific hybridization, frequently used for crop improvement, chromosome fragments are often exchanged, potentially impacting crop traits. Hence, the ability to produce chromosome karyotypes for individual plants is highly desirable. Here, we developed molecular cytogenetics probes designed from unique regions identified from genomic sequence data to differentiate each chromosome pair from A, B, and C subgenomes, generating two complementary oligo libraries, a density- and a synthesis-optimized library. Specifically, we tested the highly similar (homoeologous) A01 and C1 chromosomes, which undergo frequent exchanges and translocation events in Brassica hybrids. Unique signals for one single chromosome pair were observed for the A01 painting probe in B. rapa and B. napus species and for the C1 painting probe in B. oleracea and B. napus. In addition, one A01/C1 translocation in B. napus Surpass400_024DH and a translocation of an A01 chromosome fragment into a C1 chromosome in an allohexaploid Brassica (B. carinata × B. rapa) were identified. Our subgenome chromosome-specific probes may help to uncover rearranged fragments and nonhomologous chromosome pairing involving the Brassica A, B, and C genomes. In summary, we present a subgenome-specific and chromosome-specific cytogenetic tool to unravel the complex dynamics between the highly agronomically relevant Brassica crop genomes.
The R1R2R3-MYB (3R-MYB) transcription factor subfamily is associated with stress tolerance; however, the underlying mechanisms in crops remain poorly understood. This study investigates the function of maize MYB3R in regulating seedling drought tolerance. We characterised MYB3R overexpression lines and CRISPR-Cas9 loss-of-function mutants in maize and rice using physiological assays and transcriptome profiling. DNA affinity purification sequencing (DAP-seq) and molecular interaction assays were employed to identify direct downstream targets. MYB3R overexpression enhanced drought tolerance by promoting root development, stomatal closure and antioxidant defence, whereas mutants displayed hypersensitivity. MYB3R binds the mitosis-specific activator (MSA) motif to directly transactivate the B-type cyclin gene CYCB1;2, and cycb1;2 mutants phenocopied the myb3r drought defects. These findings establish that the MYB3R-CYCB1;2 module positively regulates maize drought tolerance by coordinating developmental and physiological adaptations. This pathway provides a valuable molecular target for breeding drought-resilient crops.
Light induces blush sweet cherry fruit coloration. We determined that the phytohormone jasmonic acid (JA) is important for fruit coloration under light conditions. However, the genes and molecular mechanisms underlying the integrated regulatory effects of light and JA signaling pathways on anthocyanin biosynthesis in blush sweet cherry fruits remain unknown. On the basis of whole genome re-sequencing and comparative transcriptome analyses, we identified PavMYB75-like (PavMYB75L) as a candidate gene encoding an R2R3-MYB transcription factor that regulates light-dependent fruit coloration. PavMYB75L overexpression in sweet cherry calli, Nicotiana tabacum, and Fragaria vesca enhanced anthocyanin accumulation and fruit coloration, whereas silencing PavMYB75L in sweet cherry fruits suppressed coloration. PavMYB75L was revealed to bind directly to the promoters of anthocyanin biosynthesis-related genes, leading to increased expression. An E3 ubiquitin ligase PavCOP1 was identified by IP-MS. Notably, PavCOP1 interacts directly with PavMYB75L and ubiquitinates PavMYB75L, targeting it for degradation through the ubiquitin/26S proteasome pathway, ultimately downregulating anthocyanin accumulation in darkness. We observed that PavCOP1 regulates fruit coloration by decreasing PavMYB75L abundance and stability. Light-responsive factor PavHYH and JA-responsive TF PavMYC2, which were identified by yeast one-hybrid screening, interact directly with the PavMYB75L promoter and activate expression, thereby promoting fruit coloration. Furthermore, phytochrome-interacting factor PavPIF3 interacts with PavHYH and PavMYC2 to accelerate the activation of PavMYB75L expression. Thus, PavPIF3/PavMYC2-PavMYB75L/PavCOP1 is a regulatory module that integrates light and JA signaling to promote blush sweet cherry fruit coloration, providing new insights into the molecular basis of anthocyanin accumulation.
Root endosymbiosis is one of the most intimate types of plant-microbe interactions in which a symbiont is hosted within a living plant cell. In this symbiosis, nitrogen-fixing bacteria and arbuscular mycorrhizal fungi support plant nutrient provisioning in exchange for carbohydrates and lipids. To establish these mutually beneficial interactions, plants have evolved complex surveillance mechanisms. These largely rely on the perception of microbe-derived N-acetylglucosamine derivatives by cell-surface receptors that feature a lysine-motif (LysM) ectodomain. Because these derivatives are not exclusive to symbiotic microbes, understanding how plants specifically recognise microsymbionts from the diverse array of soil rhizosphere microbiota has driven genetic, molecular and structural studies in recent years. In this review, we will summarise the current state of the art in molecular recognition by LysM receptors, provide an updated inventory of LysM receptors in land plants with annotated functionalities and highlight key aspects of signal transduction that lead to symbiont accommodation in living plant cells.
Seed dormancy and germination are tightly regulated by complex signaling networks that integrate internal and external cues, including the endogenous phytohormone abscisic acid (ABA). ABA HYPERSENSITIVE GERMINATION 1 (AHG1), a group A type 2C protein phosphatase (PP2C), is thought to modulate the activity of transcription factors such as ABA INSENSITIVE 5 (ABI5) in seeds and during germination. AHG1 is regulated by DELAY OF GERMINATION 1 (DOG1), a key regulator of seed dormancy, through physical interaction. We previously reported that AHG1 also interacts with ABI FIVE BINDING PROTEIN 2 (AFP2), a member of the AFP family; however, the molecular basis of AHG1-AFP coordination has remained unclear. In this study, we show that AHG1 interacts with all AFP family members and that AFP3 binds AHG1 and ABI5 through adjacent but distinct amino acid residues within its C-domain, allowing simultaneous association with both proteins. In addition, AHG1 modulates the phosphorylation status of AFP3 at Ser60 in a DOG1-dependent manner, suggesting that DOG1-AHG1 regulates AFP3 post-translationally. Transcriptomic analyses of AHG1- or AFP3-overexpressing lines revealed that these factors are associated with the regulation of a shared set of ABA-responsive genes, including AFPs, and that AFP3 overexpression is predominantly associated with altered expression of genes involved in transcriptional regulation. Large-scale protein interaction analyses showed that AFPs interact with multiple classes of transcription factors, suggesting their involvement in diverse regulatory pathways, including ABA signaling. Together, these findings demonstrate that DOG1 regulates ABI5 function and modulates ABA responses, at least in part, by controlling AHG1-mediated dephosphorylation of AFPs.
Coat protein (CP) is widely viewed as essential for systemic infection of tobacco mosaic virus (TMV), yet the mechanistic basis of this requirement remains incompletely resolved. In this study, we show that TMV can still achieve long-distance vascular transport in the absence of CP, but systemic infection becomes intermittent because the processes underlying "systemic movement" can fail at multiple, separable steps. By resolving movement into phloem loading, vascular translocation, and phloem exit, we show that CP deficiency does not completely block entry into or transport within the vasculature. Instead, CP loss imposes a strong, route-specific defect in bicollateral phloem, disproportionately reducing use of the adaxial/internal pathway associated with efficient upward spread, while downward movement through abaxial/external phloem remains comparatively permissive. In systemic leaves, CP-deficient TMV frequently produces vein/phloem-associated reporter patterns rather than broad lamina invasion, indicating that phloem exit/unloading is an additional, independent bottleneck. Enhancing RNA silencing suppression markedly enhances systemic viral accumulation and symptoms. However, it does not proportionally increase lamina invasion. This finding further supports the idea that exit and establishment are the limiting steps. Finally, among several helper viruses tested, only cucumber mosaic virus consistently alleviates this exit barrier, enabling CP-deficient TMV to escape the vasculature and establish lamina infection.
Phycobilisomes (PBSs) are supramolecular pigment-protein complexes that function as the principal light-harvesting antennae in cyanobacteria, red algae (Rhodophyta), and glaucophytes, and to a lesser extent in cryptophytes. Because the intrinsic chlorophyll-based antennae of the photosystems absorb poorly in the green region of the spectrum, PBSs employ specialized bilin chromophores to capture complementary wavelengths that chlorophyll cannot efficiently harvest. Thus, PBSs broaden the photosynthetic active spectrum of oxygenic phototrophs and thereby increase their photosynthetic capacity. Structural studies using cryogenic electron microscopy and advanced spectroscopic techniques have revealed increasingly detailed insights into excitation energy-transfer pathways and the bilin microenvironments that support highly efficient energy funneling with minimal loss. These approaches have also uncovered previously unrecognized conformational dynamics, although the functional significance of these motions remains unknown. Together, these findings portray PBSs as both ancient and highly adaptable molecular machines, central to the ecological success of cyanobacteria and other phycobiliprotein-containing phototrophs, and still yielding new surprises as analytical techniques continue to improve. This review integrates foundational and recent advances in the structural organization, functional mechanisms, and emerging applications of PBSs. We examine PBS architectures and their distribution across lineages, energy-transfer and photoprotective processes, structural dynamics, evolutionary trajectories, and the expanding landscape of biotechnological uses. These insights establish a foundation for future efforts to link PBS structure with ecological function and biotechnological innovation.
Higher plants and mammals exist in similar microbial environments and face similar challenges from microbial pathogens. Both taxonomic groups encode proteins capable of recognizing and triggering responses to pathogens; however, their recognitional capacities differ substantially. The mammalian innate immune system encodes several dozen receptors capable of recognizing a variety of pathogen-associated molecular patterns and, in addition, has an adaptive immune system with a vast recognitional capacity based on the potential production of up to approximately 1016-1018 distinct antibodies through somatic recombination in specialized immune cells. By contrast, plants possess only innate immune receptors. The ability of fast-evolving microbial pathogens to evade the recognitional capabilities of plant innate immune receptors has been clearly demonstrated. Under these circumstances, how do plants, especially species with lifespans of hundreds of years, successfully counteract their populations of rapidly evolving pathogens? Here I discuss the ways that plants navigate their relationship with the microbial world without an adaptive immune system. I suggest that the importance of plant innate immune receptors in resisting infection, though crucial, may often have been overemphasized in the literature. I propose a view of plant immunity which highlights the ability of the plant to suppress, contain and tolerate infections and ways in which this perspective might influence our future actions.
Phosphorus (P) is an essential macronutrient for plant growth and development. Root hairs enhance P acquisition as inorganic phosphate (Pi) from soil by expanding the root surface area, and their elongation is a key adaptive response to low Pi availability. However, the transcriptional regulators that couple Pi starvation signaling to root hair elongation remain largely unknown. Here, we demonstrate that PHOSPHATE STARVATION RESPONSE1 (PHR1), the central transcription factor of the Pi starvation response, positively regulates Pi deficiency-induced root hair elongation in Arabidopsis. RNA-seq analysis of root tips identified ROOT HAIR DEFECTIVE 6-LIKE 2 (RSL2), a bHLH transcription factor governing root hair elongation, as a prominent PHR1-regulated target. We show that PHR1 binds to the promoter of RSL2 to activate its expression, and genetic analysis confirms that RSL2 acts downstream of PHR1. Further RNA-seq analysis revealed that RSL2 regulates cell wall remodeling genes, among which XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE 26 (XTH26) was identified as a key target. RSL2 binds to the promoter of XTH26 to upregulate its transcription, and XTH26 overexpression partially rescues the reduced root hair length of both phr1 and rsl2. Collectively, our findings delineate a PHR1-RSL2 transcriptional module that orchestrates root hair elongation under Pi deficiency, thereby contributing to enhanced Pi acquisition.
Trichomes are crucial for plant resistance to biotic and abiotic stresses. In cucumber, the density and morphology of fruit spines directly influence fruit appearance and market value, making them a key agronomic trait. Nevertheless, the genetic regulators governing trichome initiation and development remain poorly understood. In this study, the AP2/ERF transcription factor ENHANCER OF SHOOT REGENERATION 1 (CsESR1) was identified and shown to be predominantly expressed in the epidermis of stems, leaves, flower buds, and ovaries, as confirmed by quantitative reverse transcriptase-polymerase chain reaction and GUS staining analyses. CRISPR/Cas9-mediated knockout of CsESR1 resulted in a marked decrease in spine density, altered spine morphology, and a glabrous phenotype in vegetative tissues including stems, leaves, and tendrils. Furthermore, the defective development of bloom trichomes in the Csesr1 mutant resulted in increased glossiness of the fruit peel. Protein interaction assays revealed that CsESR1 physically associates with key trichome regulators, CsTOE3, CsGL1, and CsTRY. Moreover, Csesr1 plants exhibited reduced aphid resistance, concomitant with altered expression of defense-related genes, phytohormone levels, and antioxidant enzyme activities. Collectively, this study establishes CsESR1 as a key regulator of trichome formation in cucumber and uncovers its role in plant defense, providing novel insights into the molecular networks coordinating epidermal differentiation and stress adaptation.
