Heterotrimeric G-proteins, comprising Gα, Gβ, and Gγ subunits, act as crucial molecular switches for signaling transduction in all eukaryotic organisms. Through precise modulation of specific receptors or effectors coupled with heterotrimeric G-proteins in signaling cascades, plants have the capability to activate or suppress unique signaling pathways necessary for plant growth, development, and stress responses. This review provides an overview of the heterotrimeric G-proteins signaling pathway obtained to date, and highlights novel areas for future exploration and agricultural application based on the emerging significance and potential of heterotrimeric G proteins in regulating plant development and responses to abiotic stress.

Mercury (Hg), arsenic (As), cadmium (Cd), lead (Pb) and other toxic heavy metals (HM) pose significant risks to the environment, negatively impacting the morpho-physiological and biological traits of plants. At present, toxic elements constitute a significant proportion of the food chain, exerting an impact on human health due to their mobility and biomagnification. The metal exclusion biological technique stands out for its robust performance, even when dealing with extremely low metal concentrations. Its eco-friendly nature and cost-effectiveness further enhance its value. Due to the exponential growth pattern of bacteria, these exhibit high metal persistence and are recommended for metal exclusion processes. Moreover, vacuoles like vesicles present in mycorrhizal fungi can hold extremely high levels of HM. Microbe-assisted phytoremediation primarily occurs through two mechanisms: through the direct provision of the essential nutrients and phytohormones, such as plant growth regulators, siderophores, enzymes, and mineral; or indirectly by modulating the metal detoxification process. This indirect mechanism involves microbes aiding in the accumulation and sequestration of metals in plants through the secretion of specific extracellular substances like organic acids, biosurfactants, and chelators. Moreover, the metal bioavailability and translocation in the rhizosphere are also altered via various mechanisms like acidification, precipitation, complexation or redox reactions. The understanding of the molecular and physiological processes underpinning the functions of arbuscular mycorrhizal fungi (AMF) in reducing HM toxicity, improving plant performance by procuring nutrients under HM-toxicity has significantly improved in recent years. In this review, adaptive and persistent methods related to physiological and cross-protective mechanisms in bacteria and mycorrhizal fungi (MF) resulting from the evolutionary consequences of dealing with HM toxicity have been addressed. Furthermore, the article offers details on the physiological and molecular reactions of host plants with fungi, and bacteria to HM stress, which may be useful for unveiling new knowledge about the strategies of HMs remediation.

Abscisic acid (ABA), a pivotal plant hormone once primarily associated with stress response, is now increasingly acknowledged for its indispensable role in plant development. This comprehensive review delves into the multifaceted functions of ABA in regulating various aspects of plant growth and development. From inhibiting germination to orchestrating seedling establishment, flowering time, and dormancy induction, ABA emerges as a central player in shaping plant developmental transitions. Unraveling the intricate regulatory mechanisms governing the ABA signaling pathway provides valuable insights into how plants adapt to environmental challenges while effectively managing their growth and reproductive strategies. This expanding knowledge not only highlights the significance of ABA in plant biology but also has profound implications for enhancing agricultural practices.

Juglans regia, an important economic tree species, is planted all over the world, and drought is one of the crucial factors limiting its growth and development. The various polyphenol content in walnut plants constitutes one of the material bases for the differences in stress resistance among various germplasms. However, the molecular mechanism underlying stress response mediated by polyphenol -dependent pathways remains unclear. v-Myb avian myeloblastosis viral oncogene homolog (MYB) protein of transcription factors play important regulatory roles in the process of plant stress responses. Previously, we identified JrMYB44 could be involved in osmotic stress response in walnut. In this study, we confirmed that the drought resistance of four walnut cultivars (‘Chandler’, ‘Xiangling’, ‘Xilin2’ and ‘Xifu1’) is positively correlated with the accumulation of polyphenols. The content and component changes of polyphenols in JrMYB44 overexpression (OE) and suppression (SE) lines in both walnut and Arabidopsis thaliana demonstrated that JrMYB44 positively regulated polyphenols accumulation. The variation of JrMYB44 expression and polyphenol levels under drought treatment indicated significant correlation between JrMYB44-induced drought tolerance and polyphenol accumulation, which was involved in reactive oxidative species (ROS) balance. The differentially expressed genes (DEGs) between OE and WT implied that JrMYB44 could positively activate downstream genes to participate in the drought stress response. Yeast one-hybrid (Y1H), transient GUS expression assay and dual-luciferase reporter assay (DLR) confirmed that JrMYB44 could recognize downstream JrWRKY7 and JrDREB2A, two transcription factors previously reported to be involved in drought response. Meanwhile, it was confirmed by Y2H, GST-pull down and luciferase complementation imaging assay (LCI) that JrMYB44 could interact with JrMYC2 and JrDof1, another two previously reported potential drought response regulators. Collectively, these results indicated that JrMYB44 could activate JrWRKY7, JrDREB2A and interact with JrMYC2 and JrDof1 to promote walnut polyphenol accumulation and improve drought resistance in a ROS dependent manner.

Nitrogen (N), phosphorus (P) or potassium (K) deficiency in plants can lead to a decrease in amino acid and protein synthesis. However, it is unknown how protein translation gets repressed during macronutrient deficiencies. Previous research has shown that general control non-depressible 1 (GCN1) cooperate with GCN2 to phosphorylate the alpha subunit of eukaryotic translation initiation factor (eIF2α). In this study, we observed phosphorylation of eIF2α under N, P, and K deficiencies, which was found to be lost in gcn1. Mutant gcn1 displayed higher sensitivity to macronutrient deficiencies compared to the wild-type (WT). The evidence of in situ reactive oxygen species (ROS) accumulation in leaves indicated that macronutrient starvation triggers ROS production. Treatment with Dimethylthiourea (DMTU), a ROS scavenger, eliminated ROS and reversed eIF2α phosphorylation induced by nutrient deficiency. Moreover, it was discovered that protein translation was reduced under N or K deficiency in the WT but not in gcn1, whereas under P deprivation, protein translation was reduced in both the WT and gcn1. We additionally found that DMTU can partially recover translation inhibition under N or K deprivation. Taken together, it is concluded that GCN1-GCN2-eIF2α pathway is regulated by ROS and is essential for plant survival under macronutrient starvation conditions.

Plants, as sessile organisms, must adapt to a range of abiotic stresses, including drought, salinity, heat, and cold, which are increasingly exacerbated by climate change. These stresses significantly impact crop productivity, posing challenges for sustainable agriculture and food security. Recent advances in omics studies and genetics have shed light on molecular mechanisms underlying plant stress responses, including the role of calcium (Ca²⁺) signaling, liquid–liquid phase separation (LLPS), and cell wall-associated sensors in detecting and responding to environmental changes. However, gaps remain in understanding how rapid stress signaling is integrated with slower, adaptive processes. Emerging evidence also highlights crosstalk between abiotic stress responses, plant immunity, and growth regulation, mediated by key components such as RAF-SnRK2 kinase cascades, DELLA proteins, etc. Strategies to enhance crop stress resistance without compromising yield include introducing beneficial alleles, spatiotemporal optimization of stress responses, and decoupling stress signaling from growth inhibition. This review emphasizes the importance of interdisciplinary approaches and innovative technologies to bridge fundamental research and practical agricultural applications, aiming to develop resilient crops for sustainable food production in an era of escalating environmental challenges.

Plants are engaged in a constant battle for survival against pathogens, which triggers a multifaceted immune response characterized by pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) to prevent infection. These two immune responses operate synergistically to enhance plant immunity. PTI is considered the first line of defense involving the recognition of pathogen-associated molecular patterns (PAMPs) by specific receptors in host cells known as pattern recognition receptors (PRRs), which initiate defense signaling. However, many pathogens often overcome the first line of defense (PTI) and successfully deploy effector proteins to promote virulence and subvert plant immunity, leading to host susceptibility. In the counter-defense, the ETI defense mechanism is activated by triggering resistance (R) genes in plants that usually encode nucleotide-binding-leucine-rich-containing (NLR) proteins. During plant-pathogen interactions, transcriptional reprogramming of defense-related genes such as pathogenesis-related proteins and generation of reactive oxygen species (ROS) are essential for facilitating programmed cell death at the infected location to inhibit pathogen proliferation. While ROS and PR protein are critical in plant-pathogen interaction, they are not universally required or effective against all pathogens. Hence, plants’ multilayer immune layer is encrypted with the compensatory activation of ETI defense response towards the failure of one component of the defense system to maintain robust immunity.

Reverse genetics research in complex hexaploid wheat often encounters challenges in determining the priority of gene functional characterization. This study aims to systematically analyze the wheat (Triticum aestivum) receptor-like kinase (TaRLK) gene family and develop an effective strategy to identify key candidate genes for further investigation. We identified 3,424 TaRLKs using bioinformatics methods and analyzed the diverse and conserved evolutionary relationships of RLKs among Arabidopsis, rice and wheat. Based on publicly available and our laboratory’s transcriptome data, we comprehensively analyzed the transcriptional expression patterns of TaRLKs in response to various stresses, particularly Puccinia striiformis f. sp. tritici (Pst). The TaCrRLK1L16, which is upregulated during Pst infection and triggered cell death in Nicotiana benthamiana, has been identified as a key candidate gene for further functional characterization. Furthermore, our results suggested that the transgenic wheat overexpressing TaCrRLK1L16 significantly enhanced resistance to Pst. This study will provide valuable insights into understanding the evolutionary characteristics and expression patterns of TaRLKs while offering a novel strategy for determining the priority of key candidate TaRLKs.

Litchi, a fruit that is highly sought-after worldwide, faces significant yield challenges due to litchi downy blight, primarily caused by Phytophthora litchii. Fluopicolide has exhibited remarkable efficacy in inhibiting this pathogen and is utilized for the management of litchi downy blight. Although understanding the resistance of P. litchii to fluopicolide is critical, studies on its risk and mechanisms remain limited. In this study, we determined the sensitivity of 125 P. litchii isolates to fluopicolide, revealing an average EC₅₀ value of 0.131 ± 0.037 μg/mL. Through fungicide adaptation, four resistant mutants were obtained with resistance factors exceeding 600, indicating that these strains exhibited high levels of resistance. A compound fitness index analysis demonstrated that the survival fitness of resistant mutants was significantly lower than that of their parental strains. Cross-resistance assays revealed no cross-resistance between fluopicolide and other fungicides with different modes of action. However, positive cross-resistance was observed with fluopimomide. A comprehensive evaluation suggested a moderate risk of P. litchii developing resistance to fluopicolide. PlVHA-a^N771S and PlVHA-a^N846S point mutations in resistant mutants were identified by gene sequencing analyses. These two point mutations were validated as contributors to resistance in P. litchii through genetic transformation and molecular docking.

InDel markers are commonly used to assess genetic relationships among populations. In this study, we employed a whole-genome sequence comparison method to identify and develop InDel markers for the rice blast fungus Pyricularia oryzae. We analyzed 152 whole-genome sequences of P. oryzae isolates from diverse global regions, including Brazil, Burundi, China, Colombia, Côte d'Ivoire, France, Ghana, Hungary, India, Japan, Korea, Laos, Madagascar, Mali, Morocco, Nepal, the Philippines, Portugal, Spain, Suriname, Thailand, the UK, the USA, and Zambia. Our analysis identified a total of 233,595 InDel loci distributed across the seven chromosomes of P. oryzae. From these, 82 loci were selected based on their high polymorphism across the 152 genome sequences. The effectiveness of these 82 loci was assessed by analyzing the genetic diversity of 47 Thai rice blast isolates alongside two reference isolates, GUY11 (France) and KJ201 (Korea). Of the 82 InDel loci, 33 exhibited polymorphisms, with 2–4 alleles per locus and polymorphic information content (PIC) scores ranging from 0.04 to 0.67. Principal coordinate and structure analyses revealed two genetic subgroups among the Thai rice blast isolates, categorized according to host specificity. Genetic relationships highlighted disparities among rice blast populations based on their respective hosts: rice and grassy weeds. This finding suggests a correlation between genetic relatedness and the plant hosts susceptible to rice blast disease. The newly developed InDel markers provide a valuable resource for future research in this field.

Aphids are highly destructive agricultural pests characterized by complex life cycles and phenotypic variability, facilitating their adaptation to diverse climates and host plants. Their feeding behavior leads to plant deformation, wilting, stunted growth, disease transmission, and significant yield losses. Given the economic risks aphids pose, regular updates on their seasonal behaviors, adaptive mechanisms, and destructive activities are critical for improving management strategies to mitigate crop losses. This review comprehensively synthesizes recent studies on aphids as plant pests, the extrinsic factors influencing their life cycles, and the intricate interactions between aphids and their hosts. It also highlights recent advancements in biological control measures, including natural enemies, antibiosis, and antixenosis. Additionally, we explore plant defense mechanisms against aphids, focusing on the roles of cell wall components such as lignin, pectin and callose deposition and the genetic regulations underlying these defenses. Aphids, however, can evolve specialized strategies to overcome general plant defenses, prompting the development of targeted mechanisms in plants, such as the use of resistance (R) genes against specific aphid species. Additionally, plant pattern recognition receptors (PRRs) recognize compounds in aphid saliva, which triggers enhanced phloem sealing and more focused immune responses. This work enhances understanding of aphid–plant interaction and plant resistance and identifies key research gaps for future studies.

The fungus Puccinia striiformis f. sp. tritici (Pst) is the causal agent of wheat stripe rust which constitutes a major limitation to wheat production. Cloning and applying disease-resistant genes are considered as an effective solution. Chinese wheat cultivar Xingzi 9104 (XZ9104) has exhibited durable resistance across multiple environments since its release. Through quantitative trait loci (QTL) analysis, eight QTL were found on chromosome arms 1BS, 1BL, 2AL, 2BL, 3BS, 4BL, 5BL and 7BL. YrXZ identified as 1RS.1BL translocation conferred race-specific all-stage resistance to Pst race CYR23. QYrxz.nwafu-1BL.6 and QYrxz.nwafu-3BS.7 were considered as the adult plant resistance genes Yr29 and Yr30, respectively. Notably, QYrxz.nwafu-2BL.5 accounted for 15.75–47.63% of the phenotypic variation across diverse environments and its pyramiding with Yr29 and Yr30 can confer high level of resistance. Other QTL were environment-dependent with minor effects. To clone the above resistance genes, we created a population of over 2,000 M₅ mutants in XZ9104 using ethylmethane sulfonate (EMS) mutagenesis and screened various types of susceptible mutants. Using the MutIsoseq approach with five mutant lines susceptible to race CYR23, we rapid isolated a candidate gene for YrXZ encoding coiled-coil nucleotide-binding site leucine-rich repeat (CC-NBS-LRR) protein. Integrating cytological analysis, gene-based association analysis, transcriptomic profiling and virus-induced gene silencing (VIGS), we confirmed that the causal gene for YrXZ was indeed Yr9. This study demonstrated that multiple QTL with different effects contributed to the durable resistance in XZ9104. Understanding the molecular mechanisms and pathways involved in plant defense can inform future strategies for deploying resistance gene and engineering of genetic resistance against evolving diseases.

The fungicide metconazole, which acts as a sterol 14α-demethylation inhibitor (DMI), can exhibit strong inhibitory effects on Fusarium pseudograminearum. However, the resistance mechanism as well as the risk that F. pseudograminearum develops resistance to metconazole is yet to be fully assessed. In this study, metconazole displayed a mean EC₅₀ value of 0.0559 μg/mL against 105 F. pseudograminearum isolates. Ten sensitive parental isolates were then subjected to fungicide adaptation to generate resistant mutants, with in vitro experiments subsequently highlighting the inferior fitness of the mutants. In addition, metconazole exhibited positive cross-resistance with both mefentrifluconazole and tebuconazole. Altogether, the results confirmed the low risk that F. pseudograminearum develops resistance to metconazole. Finally, a mutation genotype (M151T) was identified in FpCYP51B, with the mutants also overexpressing the FpCYP51 genes. Subsequent molecular docking and transformation-based experiments indicated that M151T substitution and overexpression in FpCYP51 genes conferred resistance to metconazole in F. pseudograminearum.

The Cucurbitaceae family includes a wide range of economically important fruits and vegetables; however, the laborious and highly inefficient genetic transformation efficacy of cucurbits has hindered the exploration of their gene functions. Virus-induced gene silencing (VIGS) technology, employed from the antiviral RNA silencing defense, has emerged as a viable alternative for high-throughput study of plant gene function. In this study, we successfully established a VIGS system utilizing Trichosanthes mottle mosaic virus (TrMMV), a new member of the genus Tobamovirus. We demonstrated the high efficacy and durability of gene silencing mediated by the TrMMV-VIGS vector in Nicotiana benthamiana, as well as in several cucurbit species, including Cucurbita pepo, Cucumis sativus, C. lanatus, and C. melo. The insertion of 90–400 bp fragments into the vector led to effective silencing of the target gene in both C. sativus and C. melo, with a notably higher silencing efficiency observed in C. melo. Furthermore, the TrMMV-VIGS vector induced a pronounced photobleaching phenotype in the flowers of C. melo, underscoring its potential application in functional genomic research concerning floral traits in this particular species. Taken together, the TrMMV-VIGS system developed herein will facilitate rapid and high-throughput identification of gene functions in cucurbit crops.

Plant peptides play crucial roles in various biological processes, including stress responses. This study investigates the functions of plant peptides in response to different adversity stresses, focusing on drought, salt, high temperature, and other environmental challenges. In drought conditions, specific peptides such as CLE25 and CLE9 were found to regulate stomatal closure and root architecture to enhance the efficiency of water utilization. Salt stress induces the expression of CAPE1 and CEP3, which are involved in ion homeostasis and osmoregulation, thereby contributing to salt tolerance in plants. Heat stress triggers the expression of peptides such as CEL45, which contributes to the heat tolerance of cells. Besides, we have also verified a new class of non-conventional peptides, and a large number of non-conventional peptides have been identified in rice seedlings. Understanding the origin and functions of these peptides presents both challenges and opportunities for developing stress-resistant crops. Future research should focus on elucidating the precise molecular mechanisms of peptide-mediated stress responses and exploring their potential applications in agriculture and biotechnology.

Wheat stripe rust, caused by an obligate biotrophic pathogen Puccinia striiformis f. sp. tritici (Pst) seriously threatens wheat production. Discovering and utilizing of wheat resistance genes is the most effective and economical method to control diseases. The G-type lectin receptor-like kinase (LecRLKs) involved in biotic stress perception, while their roles in wheat resistance to Pst remain elusive. In our study, we identified 398 G-type LecRKs in wheat through BLAST and HMM profiling. The transcript level of 16 random selected G-type LecRKs from each subfamily were analyzed and found TaSRLK is highly induced by avirulent Pst CYR23 infection. TaSRLK-silenced wheat plants showed reduced resistance to Pst with increased hyphal length and decreased H₂O₂ accumulation. Surprisingly, TaSRLK was localized to the chloroplast and can induce cell death in Nicotiana benthamiana. Further, TaSRLK was shown to interact with and phosphorylate a peroxidase TaPrx1. Importantly, TaPrx1 involved in wheat resistance to Pst through regulating reactive oxygen species (ROS) production. Together these findings demonstrate that TaSRLK positively modulates ROS-associated wheat resistance by binding with TaPrx1.

Trehalose-6-phosphate (T6P), an intermediate in trehalose metabolic pathways, is ubiquitously present in nearly all cellular organisms except vertebrates. The most well-characterized metabolic route involves its synthesis by trehalose-6-phosphate synthase (TPS) and dephosphorylation to trehalose by trehalose-6-phosphate phosphatase (TPP) in the TPS/TPP pathway. Besides, alternative trehalose metabolic pathways aslo exist. In addition to being the precursor of trehalose synthesis, T6P functions as a signal molecule regulating various biological processes. In plants, T6P inhibits SnRK1 (Sucrose-nonfermenting 1 Related Kinase 1), while in fungi, T6P primarily inhibits hexokinase and regulates glycolysis. Notably, TPS and TPP themselves also have some regulatory functions. Genetic studies reveal that deletion of TPS or TPP usually causes developmental and virulence defects in fungi, bacteria and invertebrates. Given that TPS and TPP have important biological functions in pathogenic fungi but are absent in humans and vertebrates, they are ideal targets for fungicide development. This review summarizes trehalose metabolic pathways and the multifaceted roles of T6P in plants, fungi and invertebrates, providing a comprehensive overview of its biological functions. Additionally, it discusses some reported TPS/TPP inhibitor to offer insights for pathogen control strategies.

Drought is a common environmental condition that significantly impairs plant growth. In response to drought, plants close their stomata to minimize transpiration and meanwhile activate many stress-responsive genes to mitigate damage. These stress-related mRNA transcripts require the assistance of RNA-binding proteins throughout their metabolic process, culminating in protein synthesis in the cytoplasm. In this study, we identified HLN1 (Hyaluronan 1), an RNA-binding protein with similarity to the animal hyaluronan-binding protein 4 / serpin mRNA binding protein 1 (HABP4/SERBP1), as crucial for plant drought tolerance. The hln1 loss-of-function mutant exhibited higher transpiration rates due to impaired stomatal closure, making it highly susceptible to drought. Drought stress increased HLN1 expression, and the protein underwent liquid–liquid phase separation (LLPS) to form mRNA-ribonucleoprotein (mRNP) condensates in the cytoplasm under osmotic stress. We identified GAD2 as a potential mRNA target of HLN1. GAD2 encodes the predominant glutamate decarboxylase synthesizing γ‐aminobutyric acid (GABA), a non-proteinogenic amino acid that modulates stomatal movement. RIP-qPCR and EMSA showed that HLN1 binds GAD2 mRNA, which promotes HLN1 condensate formation. In hln1 mutants, GAD2 transcripts were less stable, reducing steady-state mRNA levels. As a result, hln1 accumulated less GABA and exhibited impaired stomatal closure under drought. Conversely, HLN1 overexpression stabilized GAD2 mRNA, increased GABA levels, and enhanced drought tolerance in transgenic plants. GAD2 overexpression in hln1 mutants also rescued the drought-sensitive phenotypes. Overall, our study reveals a mechanism whereby HLN1 stabilizes GAD2 mRNA to enhance GABA production and drought tolerance. These findings provide novel strategies for engineering drought-resistant crops.

Trail development is more prevalent as tourism develops globally. The depth effect of trail development on plant diversity and native species’ stress response via tuning flavonoids in natural ecosystems remain relatively poorly understood. We investigated the depth effects by comparing plant species diversity and flavonoid contents (of six common native species) in sampling plots plots (Rabbit Mountain Open Space, Boulder County, CO, USA) with varying distances away from trail. We found plant diversity to be lowest in plots immediately proximal to trails and highest in intermediate plots. We also found the concentrations of total flavonoids to vary significantly between plots closer and away from trails. Specifically, we found the concentrations of isoorientin and myricetin higher in plots closer to trails. On the contrary, the concentrations of vitexin and kaempferol were higher in plots away from trails. Quercetin was higher in the intermediate plots. Overall, trail development negatively impacted herbaceous plant diversity, which was evident as depth effects. The plant species responded to environmental stresses imposed by trail development through fine-tuned flavonoid accumulation.

During interactions, pathogenic fungi are subjected to endoplasmic reticulum (ER) stress from the host plants, resulting in the activation of the unfolded protein response (UPR) pathway. We identified the bZIP transcription factor CfHac1 in C. fructicola, which is a pathogenic organism implicated in a variety of plant diseases, and we found it to be crucial for the ER stress response and pathogenicity. However, the role of CfHac1 in regulating the degradation of ER-associated misfolded proteins remains unclear. In this study, we discovered that the CfHAC1 gene regulates conidial production, appressorium formation, response to ER stress, and pathogenicity through unconventional splicing. Further research revealed that the CfHAC1 gene also affects the ubiquitination of ER-associated misfolded proteins and mediates their degradation. We further identified two ubiquitin ligase genes, CfHRD1 and CfHRD3, that exhibit significant down-regulation in the ΔCfhac1 mutant strain. Subsequent investigations revealed that the CfHAC1 gene affects CfHRD1 and CfHRD3 expression through unconventional splicing, with both genes managing the degradation of ER-associated misfolded proteins via ubiquitination and influencing C. fructicola pathogenicity. Taken together, our results reveal a mechanism by which the transcription factor CfHac1 affects the expression of the ubiquitin ligase genes CfHRD1 and CfHRD3, leading to the ubiquitination and degradation of ER-associated misfolded proteins and pathogenicity. This provides a theoretical basis for the development of novel agents targeting key genes within this pathway.

Cellulose is synthesized by cellulose synthases (CESAs) in plasma membrane-localized complexes, which act as a central component of the cell wall and influence plant growth and defense responses. Puccinia striiformis f. sp. tritici (Pst) is an airborne fungus that causes stripe rust to seriously endanger wheat production. In this study, a CESA gene, TaCESA7, was identified to be significantly up-regulated during Pst infection in wheat (Triticum aestivum L.). TaCESA7 was localized on the plasma membrane in dimeric form, and the dimers interact to assemble into CESA complexes. Stable overexpression of TaCESA7 weakened the resistance of wheat to Pst. Knockdown of TaCESA7 by RNA interference (RNAi) and virus-induced gene silencing led to restricted hyphal spread, increased necrotic area, and simultaneously promotes reactive oxygen species (ROS) accumulation and the expression of pathogenesis-related (PR) genes. Transcriptome analysis of TaCESA7-RNAi plants revealed that the up-regulated genes were significantly enriched in the phenylpropanoid biosynthesis and plant-pathogen interaction pathways. Moreover, silencing TaCESA7 promoted the deposition of lignin and the expression of genes related to lignin synthesis. CRISPR-Cas9-mediated inactivation of TaCESA7 in wheat could confer broad-spectrum resistance against Pst without affecting agronomic traits. These findings provide valuable candidate gene resources and guidance for molecular breeding to improve the resistance of wheat to fungal disease.

Receptor-like cytoplasmic kinases (RLCKs) function as a central player in plant receptor kinases-mediated signaling, which regulate various aspects of plant immunity and growth. RLCKs receive signals from pattern recognition receptors (PRRs) to activate pattern-triggered immunity (PTI), including reactive oxygen species (ROS) production, Ca²⁺ influx, mitogen-activated protein kinase (MAPK) cascades, cellulose synthesis, phosphatidic acid (PA) production, hormone synthesis and signaling, and transcriptional remodeling. Besides, RLCK also participate in effector-triggered immunity (ETI) and the interplay between ETI and PTI. Increasing evidences show that much more RLCKs are involved in plant immune responses and form an intertwined signaling network. This review summarizes the recent findings about RLCKs-mediated signaling in plant immune responses and emphasizes signal convergence and divergence involved which provides new insights into the RLCKs signaling network in diverse biological processes.

Soil salinization and alkalization have become an increasingly severe global issues, significantly limiting both the yield and quality of apples (Malus ×  domestica). M9-T337 is a widely used apple dwarfing rootstock; however, it is sensitive to saline-alkali stress. Therefore, developing saline-alkali tolerant apple rootstocks is essential. In this study, we utilized RNAi (RNA interference) technology to knock down GH3 genes in the M9-T337 background, aiming to engineer a dwarfing and stress-tolerant apple rootstock. We found that MdGH3 RNAi plants exhibited superior morphology compared to M9-T337 under saline-alkali stress conditions, characterized by more robust root systems, increased plant height, a lower Na⁺/K⁺ ratio, and enhanced photosynthetic and antioxidant capacities. Moreover, when MdGH3 RNAi plants were used as rootstocks, the GL-3/MdGH3 RNAi plants also displayed greater plant height, root vitality, photosynthetic ability, and antioxidant capacity compared to GL-3 grafted onto M9-T337 rootstock. Taken together, our study constructed a saline-alkali-tolerant apple rootstock by knocking down MdGH3 genes.

Heat stress (HS) is a major environmental stress that inhibits plant growth and development. Plants have evolved various mechanisms to cope with heat stress, a key one being the HSF-HSP (Heat stress transcription factor-Heat shock protein) signaling pathway. HSFs can be divided into three classes: A, B, and C. In this study, we report the identification and functional characterization of a specific B2 member LdHSFB2a in Lilium davidii var. unicolor. RT-qPCR (Real-time Quantitative Polymerase Chain Reaction) analyses indicated that LdHSFB2a was highly expressed in HS-exposed leaves. LdHSFB2a was localized in the nucleus, consistent with the characterization of transcription factors. In contrast to other HSFBs, LdHSFB2a did not contain the typical B3 repression domain but exhibited transcriptional repression activity in yeast and plant cells. Transient overexpression and virus-induced gene silencing (VIGS) of LdHSFB2a in lily petals suggested that LdHSFB2a functions positively in lily thermotolerance. Consistent with the implication of LdHSFB2a function in thermotolerance, further analysis revealed that the expression levels of HSFA1, HSFA2, and MBF1c were increased as LdHSFB2a was overexpressed but reduced as LdHSFB2a was silenced. Furthermore, LdHSFB2a bound to the promoters of HSFA3 A, WRKY33, CAT2, and GLOS1. And LdHSFB2a overexpression and silencing enhanced and reduced their expressions, respectively. Therefore, we speculated that LdHSFB2a may be a coactivator that interacts with transcriptional activators to promote thermotolerance in lily by enhancing the expression of heat-responsive genes such as HSFA3 A, WRKY33, CAT2, and GLOS1.

Understanding the genetic mechanism of cold adaptation in cashmere goats and dairy goats is very important to improve their production performance. The purpose of this study was to comprehensively analyze the genetic basis of goat adaptation to cold environments, clarify the impact of environmental factors on genome diversity, and lay the foundation for breeding goat breeds to adapt to climate change. A total of 240 dairy goats were subjected to genome resequencing, and the whole genome sequencing data of 57 individuals from 6 published breeds were incorporated. By integrating multiple approaches such as phylogenetic analysis, population structure analysis, gene flow and population history exploration, selection signal analysis, and genome-environment association analysis, an in-depth investigation was carried out. Phylogenetic analysis unraveled the genetic relationships and differentiation patterns among dairy goats and other goat breeds. Through signal analysis (θπ, FST, XP-CLR), we identified numerous candidate genes associated with cold adaptation in dairy goats (STRIP1, ALX3, HTR4, NTRK2, MRPL11, PELI3, DPP3, BBS1) and cashmere goats (MED12L, MARC2, MARC1, DSG3, C6H4orf22, CHD7, MYPN, KIAA0825, MITF). Genome-environment association (GEA) analysis confirmed the link between these genes and environmental factors. Moreover, a detailed analysis of the critical genes C6H4orf22 and STRIP1 demonstrated their significant roles in the geographical variations of cold adaptation and allele frequency differences among different breeds. This study contributes to understanding the genetic basis of cold adaptation, providing crucial theoretical support for precision breeding programs aimed at improving production performance in cold regions by leveraging adaptive alleles, thereby ensuring sustainable animal husbandry.

Sheath blight (ShB), caused by the necrotrophic fungus Rhizoctonia solani, is a globally destructive rice disease responsible for significant yield losses. However, the absence of characterized genes conferring high potential resistance to sheath blight within natural rice germplasm constrains resistance breeding. A recent study published in Nature Genetics uncovered the ShB resistance receptor-like kinase 1 (SBRR1) as a key gene associated with disease resistance. SBRR1-R, an elite resistance allele mainly presented in indica rice and distinguished by a 256-bp promoter insertion, confers strong resistance without obvious yield penalty. SBRR1 is the first gene with major effects underlying natural variation in sheath blight resistance, offering significant potential for rice breeding. Furthermore, the discovery of the “bHLH57—SBRR1-R—SIP1—Chit3/4” defense module provides fundamental insights into rice immunity and a molecular module with substantial breeding potential.

As a barrier between the cell and its environment, the plant cell wall provides structural support during development and stress response. Plants are able to sense their surroundings and adjust their activities accordingly. A crucial mechanism involved in these adaptive changes is the cell wall integrity (CWI) maintenance mechanism, which monitors and maintains the integrity of cell walls via changes in cell and cell wall metabolism without destroying cell wall organization. Different abiotic stresses and changes in plant developmental phases disrupt CWI. However, emerging evidence has demonstrated the initiation of CWI signaling mechanisms as key in promoting plant growth in complex situations. This review discusses recent advances in the Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) protein function in plant cell wall signaling during adaptation to changing environments and development. We conclude by highlighting how current spatially resolved transcriptomics may be used to advance the role of CrRLK1L members in plant cell wall signaling during development and stress response.

Gayal (Bos frontalis) an endangered bovine species inhabitingChina, India, Bangladesh, Myanmar and Bhutan, has a mysterious evolutionary origin. Shaped by natural selection, its unique traits make it a valuable genetic resource; however, its populations are rapidly declining. In this study, comprehensive whole-genome resequencing of fifty-eight samples of Gayal from China, India, Myanmar and Bangladesh was performed. We identified over 44 million SNPs across four Gayal populations. Nucleotide diversity analysis revealed variations in genetic diversity, with the lowest occurring in India and the highest occurring in China. Phylogenetic tree analysis revealed three distinct clades representing China, India and Bangladesh-Myanmar, which were further confirmed by principal component and admixture analyses. The genetic exchanges between Gayal and other bovine species indicate limited influence from domestic cattle in both the Chinese and Bangladeshi Gayal populations. Mitochondrial DNA sequences and a phylogenetic tree highlighted the unique mitochondrial genome of Gayal. Genome-wide selection signals pinpointed candidate genes linked to mitochondrial function, immunity, musculoskeletal development, reproduction and growth performance. Distinct haplotype patterns emerged for the CCDC157, KIAA0753 and MTFP1 genes in the Chinese and Bangladesh-Myanmar Gayal populations, indicating artificial selection in the Chinese population. KEGG pathway and gene ontology enrichment analyses provided insights into processes related to neurodevelopment, cardiac function, tissue growth, immunity and metabolism. In summary, our study enhances our understanding of Gayal genetics, population structure and selection signals across four countries. This knowledge is crucial for conserving this endangered species amid its rapid decline.

Modification of proteins by ubiquitin is a dynamic and reversible process. It is unclear whether rice stripe virus (RSV) can modulate the plant deubiquitination pathway. In this study, we found that RSV infection can specifically upregulate the expression of the deubiquitinase NbUBP16. Further analysis revealed that NbUBP16 stabilizes serine hydroxymethyltrasferase (SHMT1) by binding to NbSHMT1 and removing its polyubiquitination modification mediated by E3 ligase MEL, which inhibits downstream SHMT1-mediated ROS accumulation and thereby facilitates RSV infection. Our findings provide new insights into the molecular arms race between pathogens and plants, demonstrating how a plant virus can undermine plant defenses by hijacking host deubiquitination pathways.

It remains a great challenge to control weeds in rapeseed fields in China. Breeding herbicide-resistant rapeseed varieties and using corresponding herbicide formulations has become the most economical and effective way to control weeds in rapeseed field. Characterization of more herbicide-resistant genetic resources will provide opportunities for breeders to develop rapeseed herbicide-resistant varieties with good agronomic performance. Previously, we obtained the tribenuron methyl (TBM)-resistant mutant K4 from ZS9 (Brassica napus L.) through ethyl methyl sulfonate mutagenesis and TBM foliar-spray screening. In this study, the inheritance and molecular characterization of the mutant K4 are carried out. Genetic investigation indicated that the herbicide-resistance of the K4 was controlled by one dominant allele at a single nuclear gene locus. Molecular characterization showed that a single point substitution at position 535 from C to T in BnAHAS3 (BnAHAS3^535T), which resulted in a mutation at point 179 in BnAHAS3. The K4 showed a certain degree of resistance to TBM, bensulfuron methyl, and monosulfon sodium, which were 50, 30, and 5 times that of ZS9, respectively. AHAS enzyme assay, structural analysis of AHAS proteins, affinity detection between TBM and BnAHAS3 by surface plasmon resonance analysis, and the transgenic experiment in Arabidopsis using BnAHAS3^535T confirmed that BnAHAS3^535T endow the K4 with herbicides resistance. In addition, an allele-specific marker was developed to quickly distinguish the heterozygous and homozygous mutated alleles BnAHAS3^535T. In conclusion, our research identified and characterized one novel mutative AHAS allele in B. napus and enriched genetic resource for developing herbicide-resistant rapeseed cultivars.

Given that lactoferrin (LF) exerts an excellent protection of intestinal homeostasis, the underlying mechanisms, especially epigenetic regulations, are still unknown. This study aimed to investigate the effects of dietary LF epigenetically modulates the oxidative genes by histone modifications to ameliorate ileum inflammation of mice exposed to DON contaminated diet. As expected, we found in the morphology analysis that DON exposure increased ileum crypt depth (CD) and villus width (VW) but reduced villus height (VH) and VH: CD ratio compared to those of the vehicle group. Consistently, the elevated ROS and MDA, along with the decreased ATP, SOD, CAT, GSH, and complex I, III, V were observed in the DON-exposed mice ileum. In contrast, LF markedly ameliorated the impairments of morphological and biochemical indexes. Next, we conducted transcriptome analysis to explore the changed signaling pathways using the ileum RNA of the mice treated with DON or LF. Firstly, the cell cycle pathway genes were significantly downregulated in the DON-exposed mice, and LF improved the cell cycle profile. Again, gene ontology analysis showed that inflammation and oxidative stress were significantly activated by DON exposure, and these were recovered when the DON-exposed mice were supplemented with an LF diet. Consistent with these findings, the signaling pathways of the reduced oxidative phosphorylation and elevated TNFα were also observed to be ameliorated by LF treatment. Importantly, histone modifications, including acetylation, methylation, and lactylation were suggested to be the vital players involved in the DON or LF treatment, in which LF significantly increased the loss of histone modifications on these genes. With a bioinformatics analysis and validation by qRT-PCR, the nuclear receptor NR5A2 was selected as a key master in the ileum of mice stimulated by DON. LF performed the benefit function on the NR5A2-mediated oxidative stress genes Ncoa4 and Prdx3 in the DON-exposed mice. Moreover, a ChIP-qPCR was used to verify that histone marks involving H3K9ac, H3K18ac, H3k27ac, H3K4me1, H3K9la, and H3K18la facilitated the epigenetic regulation of NR5A2-modulated actions. We conclude that dietary LF effectively ameliorated ileum lesions induced by DON in mice by modulating oxidative genes Ncoa4 and Prdx3 through histone modifications. 

Nucleotide-binding leucine-rich repeat (NLR) proteins assemble into genetically linked pairs to mediate effector-triggered immunity (ETI) in plants. Here, we characterize the paired NLRs NRCX and NARY (NRCX adjacent resistance gene Y) in Nicotiana benthamiana. CRISPR/Cas9 knockout of NRCX caused severe dwarfism and constitutively activated immunity, marked by PR1 upregulation and enhanced resistance to Phytophthora capsici. Co-silencing or double knockout of the adjacent NLR NARY partially rescued the nrcx phenotype, revealing NARY as a compensatory regulator that modulates growth and immunity. Structural analysis revealed that NARY harbors non-canonical Walker B and MHD motifs, which lack autoactivation capacity despite their divergence from canonical NLR executors. Split-luciferase and co-immunoprecipitation assays showed that NRCX and NARY interact exclusively through their CC domains, forming a non-canonical regulatory complex. Notably, simultaneous silencing of NRC2/3 and NARY incompletely restored growth in nrcx mutants, implicating additional factors in immune modulation. Our findings establish NARY as a compensatory NLR partner of NRCX that fine-tunes immunity without triggering cell death, revealing a novel mechanism for balancing growth and defense in Solanaceae.

Fusarium head blight (FHB, also known as wheat scab or ear blight), caused primarily by the Fusarium graminearum, is a worldwide disease of wheat (Triticum aestivum L.). Studying the pathogen expansion patterns and molecular mechanisms of disease resistance in resistant wheat varieties is crucial for advancing wheat disease management strategies. Here, we found a significant difference between two wheat cultivars with different resistances, and it was revealed that they exhibited divergent pathogen infestation process. The susceptible cultivar showed extensive pathogen in the spike rachis, while resistant varieties only had limited pathogen spread and colonization. Meanwhile, wheat resistance to FHB was positively correlated with transcriptional reprogramming in the early stages, with higher expression of genes responding to plant defense related genes and phenylpropanoid pathway genes in the early stages of disease resistant variety. Weighted gene co-expression network analysis (WGCNA) of differential expression genes (DEGs) analysis led to the construction of a network modules associated with resistance genes, and an important role of heavy metal-associated (HMA) domain protein in plant defense was identified in the tan module. RNA-induced gene silencing preliminarily identified two key genes that resistance to FHB in wheat: a cytochrome P450 (CYP) gene involved in the flavonoid biosynthesis within the phenylpropanoid pathway and HMA gene. This study provides an in-depth analysis of the infection mechanisms of wheat by F. graminearum and elucidates the key molecular mechanisms involved, while being useful for advancing the breeding of wheat varieties resistant to FHB.

Heat stress (HS) disrupts intestinal homeostasis and hepatic lipid metabolism in poultry, yet effective interventions remain limited. We investigate the protective effects of dietary glycerol monolaurate (GML) supplementation in laying hens under HS conditions. In a 10-week trial, 504 Hy-Line Brown hens were assigned to four groups (control and GML at 65, 195, and 325 mg/kg) with six replicates per group. Hens receiving 325 mg/kg GML exhibited significantly higher egg production and egg weight (P < 0.05), alongside improved egg quality metrics, including increased shell strength and Haugh units by week 8 (P < 0.05). Histological analysis revealed that GML (325 mg/kg) improved duodenal and ileal villus height and duodenal villus-to-crypt ratios while reducing duodenal crypt depth (P < 0.05), thereby restoring gut barrier integrity. These findings were supported by reduced plasma D-lactate (D-LA) levels and upregulated expression of tight-junction proteins ZO-1 and Occludin in the ileum and jejunum (P < 0.05). In the liver, GML supplementation alleviated HS-induced steatosis, reducing lipid droplet accumulation (P < 0.05), plasma low-density lipoprotein cholesterol (LDL-C), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) levels, and hepatic triglyceride content, while elevating high density lipoprotein cholesterol (HDL-C). Integrated plasma metabolomics and hepatic transcriptomics identified 36 differential metabolites (enriched in sphingolipid metabolism) and 1,176 differentially expressed genes (enriched in PPAR signaling and Fatty acid degradation), with ACSL1 as a central regulatory gene. Key genes (ACSL1, CPT1 A) and metabolites correlated positively with production performance and gut-liver health, while SCD and Probucol showed negative associations. These findings indicate that GML supplementation enhances intestinal barrier function, promotes hepatic fatty acid β-oxidation, and reinforces sphingolipid metabolism, thereby mitigating HS-induced oxidative stress and lipid dysregulation. Our results identify 325 mg/kg GML as the optimal dosage, proposing a practical strategy to enhance poultry resilience during heat stress.

Apple replant disease (ARD) poses a serious threat to apple cultivation, primarily caused by the accumulation of Fusarium species. Bacillus species have demonstrated significant potential as microbial agents, with capabilities in promoting plant growth, suppressing soil-borne pathogens, and improving soil quality. Here in this study, strain LRB-5 was isolated from a healthy apple root system and identified as Bacillus vallismortis based on physiological and biochemical characterization and molecular sequencing analysis. It exhibited broad-spectrum antifungal activity against various Fusarium species, including F. oxysporum, F. moniliforme, F. proliferatum, and F. solani, with inhibition rates exceeding 65%. LRB-5 extracellular metabolites significantly inhibited Fusarium mycelial growth and spore germination. Greenhouse experiments demonstrated that LRB-5 reduced ARD disease severity by more than 50%. The volatile organic compounds produced by LRB-5 exhibited both antimicrobial activity and growth-promoting properties. Further assays revealed LRB-5 can secrete various cell wall-degrading enzymes and possesses plant growth-promoting capabilities. Pot experiments showed LRB-5 had excellent colonization ability in the rhizosphere of Malus hupehensis Rehd. seedlings, significantly increasing seedling biomass, soil bacterial and actinomycete populations, and the activity of root protective enzymes. Moreover, LRB-5 significantly enhanced the activity of soil enzymes while reducing the contents of phlorizin, benzoic acid, and p-hydroxybenzoic acid in the rhizosphere soil. Terminal restriction fragment length polymorphism and quantitative real-time PCR analyses revealed that LRB-5 improved bacterial carbon utilization, increased microbial diversity indices, reduced the abundance of Fusarium spp., and altered the structure of soil microbial communities. Collectively, these rusults suggest that LRB-5 effectively alleviated ARD by protecting apple roots from Fusarium infection and phenolic acid toxicity, optimizing soil microbial communities, and promoting plant growth. Future research should explore the combined application of LRB-5 with other control measures, thereby promoting its practical implementation.

Wall-associated receptor kinases (WAKs) and WAK-likes (WAKLs) play pivotal roles in regulating plant immunity, through multiple downstream signaling components. However, knowledge of WAKs/WAKLs in wheat immune responses to rust diseases remain limited. In this study, we identified and characterized a wheat WAKL, TaWAKL8-2B, which is upregulated during wheat resistance to both Puccinia striiformis f. sp. tritici (Pst) and Puccinia triticina (Ptt), indicating its role in wheat resistance to these two rust fungi. Transgenic wheat plants overexpressing TaWAKL8-2B exhibited enhanced resistance to stripe rust and leaf rust, accompanied by increased reactive oxygen species (ROS) production and up-regulated defense-related gene expression. Whereas, knockout TaWAKL8-2B reduced resistance to Pst and Ptt with less ROS accumulation, highlighting its positive role in wheat resistance. RNA-seq analysis revealed that 33 genes encoding ROS-scavenging enzymes were upregulated in TaWAKL8-2B-KO plants, explaining the reduced ROS. KEGG analysis enriched the monoterpenoid pathway, particularly the linalool biosynthesis pathway, with linalool synthases significantly downregulated in TaWAKL8-2B-KO plants. Correspondingly, linalool synthase content and linalool content decreased in knockout plants. Collectively, our findings uncover a novel mechanism by which TaWAKL8-2B positively modulates wheat rust resistance through modulating linalool biosynthesis and peroxidase activity. These results enhance our understanding of TaWAKL8-2B mediated immune signaling and offer a promising gene for improving wheat broad-spectrum resistance to rust diseases.

Environmental stress adaptation is crucial for the survival and pathogenicity of plant fungal pathogens. In this study, we identified a transcription factor FgMsn2 in Fusarium graminearum, an ortholog of Msn2 in budding yeast. Structural analysis showed that the C2H2 zinc-finger domain is highly conserved across fungi, while other regions are less conserved, suggesting that FgMsn2 may have species-specific functions. Subsequently, we revealed that FgMsn2 is critical for vegetative growth, and conidiogenesis. Deletion of FgMSN2 severely reduced the deoxynivalenol (DON) production and pathogenicity, while enhancing tolerance to oxidative, osmotic, cell wall and membrane stresses. Furthermore, our RNA-seq analysis revealed that FgMsn2 regulates genes involved in energy metabolism, lipid metabolism and stress responses, emphasizing its role in maintaining metabolic balance and stress adaptability. Notably, FgMsn2 influences mitochondrial morphology, as the Fgmsn2 mutant exhibited disrupted mitochondrial structures and reduced ATP production. The Fgmsn2 mutant also showed increased lipid droplet accumulation, indicating the FgMsn2’s role in lipid metabolism. Taken together, the FgMsn2 serves as a key regulator in fungal development, plant infection, stress responses, and metabolism. Our study provides valuable insights into the molecular mechanisms of fungal stress adaptation and pathogenicity, suggesting a potential target for the development of more effective fungicides and disease management strategies.

Cucumber target spot, a major disease that threatens cucumber production, is caused by Corynespora cassiicola. Cyclobutrifluram, a novel succinate dehydrogenase inhibitor (SDHI) developed by Syngenta, has demonstrated strong inhibitory activity against various plant pathogenic fungi and nematodes. However, its antifungal spectrum, resistance risk as well as underlying mechanisms of resistance in C. cassiicola remain poorly understood. In this study, cyclobutrifluram exhibited potent inhibitory activity against anamorphic fungi and selected ascomycetes, with the mean sensitivity of C. cassiicola isolates to the fungicide being 0.98 ± 1.26 μg/mL. Additionally, five laboratory-derived cyclobutrifluram-resistant mutants showed comparable or lower biological fitness than their respective parental isolates. The resistant mutants and field isolates were also found to possess nine distinct point mutations in the CcSdhB, CcSdhC or CcSdhD genes. Finally, cyclobutrifluram exhibited positive cross-resistance with other SDHIs, with the resistance levels varying depending on the specific mutations present. In conclusion, cyclobutrifluram was found to be effective against anamorphic fungi and selected ascomycetes. C. cassiicola’s risk of resistance development to cyclobutrifluram was assessed as moderate to high and was primarily associated with mutations in CcSdh genes.

Plant cells exhibit an extraordinary regenerative potential, achieving cellular totipotency by dedifferentiating to form new tissues. While significant progress has been made in understanding cell fate mechanisms, the regulatory networks governing callus cell development remain insufficiently explored, particularly regarding cell classification, morphology, and regulatory processes. This study provides a detailed investigation into the developmental dynamics and transcriptomic profiles of callus cells in Arabidopsis at key stages: initiation, proliferation, and greening. Employing single-cell RNA sequencing and UMAP-based clustering, we annotated cell clusters based on highly enriched gene expressions. Developmental trajectories were further mapped through pseudotime analysis, revealing distinct transcription factor networks. Additionally, functional analysis of key regulatory genes was conducted using mutant and overexpression lines, affirming their roles in callus development. Gene Ontology analysis highlighted the involvement of environmental factors—low oxygen and salinity promoted callus formation, while light inhibited it, though essential for greening. These findings shed light on the complex regulatory landscape of plant tissue regeneration and guide future research avenues.