Growth regulatory factor (GRF) genes play a crucial role in plant growth and development, reproduction, metabolism, and stress resistance. In this study, we conducted a genome-wide integrated analysis of transcriptome and miRNA expression profiles in Paulownia fortunei challenged by phytoplasma infection, with a specific focus on elucidating the functional landscape of the Pf GRF transcription factor (TF) family. A comprehensive investigation was conducted on the Pf GRF TF family. A total of 16 Pf GRF genes were identified in this study, among which 13 were located on the chromosomes of P. fortunei. They were divided into six groups based on amino acid sequences. Notably, proteins within the same subgroup exhibited remarkable structural conservation, whereas significant inter-subgroup divergence was observed, suggesting functional specialization. Evolutionary expansion of the Pf GRF family was primarily driven by segmental duplication events, highlighting a key mechanism underlying genetic redundancy and functional diversification in this lineage. Segmental duplication was the main mechanism of Pf GRF family expansion. Cis-acting elements responsive to phytohormones and abiotic stresses were detected in the promoter regions of the PfGRFs. Yeast two-hybrid and bimolecular fluorescence complementation technology confirmed the interaction between PfGRF14 and PfGIPa. This work lays a foundation for future research into the functions of the Pf GRF TF family, and provides a reference for studies of the mechanism of Paulownia Witches' broom (PaWB) development.

Major latex-like proteins (MLPs) play crucial regulatory roles in mediating plant responses to both abiotic and biotic stressors. Although MLPs have been identified in diverse plants, the genome-wide characteristics of MLP-encoding genes and their roles in pathogen defense in Paulownia (Paulownia fortunei) remain largely unexplored. P. fortunei is a fast-growing perennial tree widely cultivated across multiple regions of Asia due to its substantial economic and ecological value. Here, we identified 49 PfMLP genes from the P. fortunei genome and systematically characterized their sequence architectures and phylogenetic relationships. Transcriptome analyses revealed 16 PfMLPs that are responsive to Witches' Broom (PaWB) phytoplasma infection. Notably, we focused on the functional characterization of PfMLP25, whose expression was induced by PaWB infection. Heterogenous overexpression of PfMLP25 in transgenic poplars enhanced pathogen resistance, confirming its critical role in anti-pathogen defense. Additionally, we demonstrated that the PfMYB100 transcription factor regulates the transcription of PfMLP25 and identified its interaction with two key immune system-essential proteins, PfCDPKa and PfRODa. Furthermore, we validated the interaction between PfMLP25 and the PfPUBa protein, indicating a potential role for PfMLP25 in the ubiquitination pathway during PaWB phytoplasma invasion. Taken together, our findings suggest that PfMLP25 functions by mediating immune system activation and participating in ubiquitination processes during PaWB phytoplasma invasion. This study presents new insights into the role of PfMLP25 and its underlying molecular mechanisms in plant pathogen defense.

Protein-protein interaction (PPI) serves as a fundamental regulatory mechanism that mediates diverse biological processes to govern cellular activities. The identification of protein-protein interactions is a pivotal technique for accurately analyzing protein function and systematically elucidating its regulatory mechanisms. Immunoprecipitation-mass spectrometry (IP-MS) represents a commonly employed approach for identifying protein-protein interactions in cells. Mass spectrometry enables the verification of both known protein interactions and the discovery of unknown proteins interacting with target proteins, providing not only information on the presence of proteins in samples but also facilitating comparative analysis of their relative abundance across different samples. In this chapter, we present a method for immunoprecipitating protein complexes from total plant protein extracts, followed by mass spectrometry (IP-MS), to identify the interaction between proteins in strawberry fruit.

Sugars are not only essential energy sources but also central signaling molecules that orchestrate the entire plant life cycle. This review comprehensively outlines the signal transduction pathways mediated by core sugar sensors, including Hexokinase 1 (HXK1), Target of Rapamycin (TOR), and Sucrose Non-Fermenting 1-Related Protein Kinase 1 α (SnRK1α), highlighting the diverse perception mechanisms of glucose, sucrose, and trehalose-6-phosphate. We synthesize how sugar signals interact with phytohormones and environmental factors to form extensive regulatory networks controlling key developmental processes from seed germination to leaf senescence. By systematically integrating these mechanisms across the plant life cycle, this review provides a holistic conceptual framework and mechanistic insights specifically for horticultural crop research. We further identify key knowledge gaps, such as the characterization of sugar sensor interactors and signal integration mechanisms, offering novel targets for future research aimed at improving horticultural crop quality, yield, and stress resistance.

Sugar content is a key determinant of fruit quality, and sugars also act as signalling molecules that regulate ripening processes, including anthocyanin accumulation. However, the molecular mechanisms underlying sugar accumulation and sugar signal-mediated ripening remain incompletely understood. In this study, we identify FvMAPK6 as an important phosphorylation hub that coordinates both sugar and anthocyanin accumulation in strawberry fruit. FvMAPK6 forms a phosphorylation cascade with FvMAPKK4, which directly phosphorylates the transcription factors FvMYB44.1 and FvMYB44.2. This phosphorylation reduces the stability and transcriptional activity of these proteins, attenuates their repression of downstream target genes such as FvCHI, FvSPS3 and FvSWEET1, thereby coordinating anthocyanin and sugar accumulation. Furthermore, FvMAPK6 increases the protein abundance of the hexose transporter FvSWEET1 in strawberry fruits and alters its transport activity through phosphorylation. We demonstrate that sucrose treatment activates FvMAPK6, reinforcing its regulation of FvMYB44s and FvSWEET1 and thus amplifying sugar and anthocyanin accumulation. These findings establish FvMAPK6 as a key regulator that integrates both sugar accumulation and signalling at both transcriptional and post-transcriptional levels. Although FvMAPK6 promotes sugar accumulation, it significantly reduces fruit yield and vegetative growth. To overcome this limitation, we screen for downstream targets of FvMAPK6 and identify FvSPS3 as a promising breeding target: modulating FvSPS3 improves fruit quality without compromising vegetative growth or yield. Collectively, our findings reveal novel regulatory pathways modulating sugar accumulation and signalling in strawberry while providing a valuable molecular target for the simultaneous improvement of fruit quality and agricultural productivity.

BACKGROUND: Witches’ broom is an important disease of the Paulownia fortunei. Understanding the pathogenesis of witches’ broom is a prerequisite for its prevention and control. Phytoplasma is the pathogen of Paulownia witches’ broom. RESULTS: We investigated the changes in chromatin accessibility before and after phytoplasma infection in Paulownia fortunei by analyzing the DNA accessibility (ATAC-seq). In phytoplasma-infected P. fortunei (PFI) compared to healthy samples (PF), the closed regions of chromatin(1187 regions) were three times more than the open regions (352 regions). Fifty one percent of the accessible chromatin regions were overlapped with either H3K27ac or H3K9ac peaks. The closed regions were enriched in the conserved motif TGGGC[CT] that is recognized by the TCP transcription factor family. The closed regions in PFI are intersected with ARF family gene locus. The gene PfARF3 was verified to interact with the PfTCP23 transcription factor. The PfTCP23 was predicted to be interacted with the effector pawb44 in the pathogen of phytoplasma. CONCLUSIONS: The phytoplasma infection in P. fortunei is involved in the chromatin changes of the DNA accessibility and histone modification. The binding regions of TCP23 were found to be changed mostly in the accessibility between PFI and PF. The TCP-ARF module was found to be the possible regulatory module inducing the crinkled leaf trait.

Anthocyanins critically determine fruit color, nutrition, and stress resilience in cultivated strawberry (Fragaria × ananassa), directly influencing consumer preference. Despite complex genetic and environmental regulation of their biosynthesis, the basis for tissue-specific pigmentation, notably the widespread occurrence of red skin and pale flesh, remains poorly understood. We integrated genomic, transcriptomic, and functional analyses across 200 cultivars to dissect receptacle pigmentation regulation. Approaches included FaMYB10-2 allele mining, promoter structural variant (SV) identification, expression profiling, regulatory interaction assays, and characterization of upstream light-responsive factors. FaMYB10-2 was identified as the key R2R3-MYB regulator of fruit anthocyanin biosynthesis. Alleles FaMYB10-2.2 and FaMYB10-2.3 encode truncated proteins retaining bHLH-binding capacity but lacking activation domains, functioning as dominant-negative repressors. A promoter SV 986 bp upstream of FaMYB10-2 was associated with reduced pale fruit due to cis-regulatory divergence. The SV (Alt) allele is prevalent in Asian cultivars, while the Ref allele is enriched in Western germplasm. Crucially, a light-responsive FaHYH-FaWRKY71 cascade activates FaMYB10-2 and structural genes haplotype-dependently, compensating for weak MYB activity in the skin. Our findings reveal a multilayered regulatory system integrating allelic variation, cis-regulatory divergence, and environmental signals, advancing anthocyanin understanding and providing engineering targets for polyploid crop color improvement.

Fruit ripening is a highly coordinated developmental process that transforms immature fruits into edible organs adapted for seed dispersal and human consumption. Although transcriptional regulation has long been acknowledged as a fundamental mechanism underlying ripening control, accumulating evidence now indicates that post-translational modifications (PTMs) function as master regulatory switches that precisely control protein activity, stability, and interactions. PTMs such as phosphorylation, ubiquitination, acetylation, redox modifications, and methylation establish dynamic regulatory networks that integrate hormonal signals, metabolic fluxes, and environmental signals to control the complex biochemical and physiological changes during fruit ripening. This review summarizes current understanding of PTM-mediated regulation in both climacteric and nonclimacteric fruits, emphasizing how modification cascades control key processes including ethylene signaling, cell wall remodeling, pigment accumulation, and stress responses. We explore emerging crosstalk networks in which multiple PTMs target important proteins to form complex molecular switches and discuss recent methodological advances that facilitate systems-level analysis of PTM. Integrating PTM research with precision agriculture and biotechnology offers promising approaches for improving fruit quality, extending shelf-life, and enhancing stress tolerance in the context of global climate change.

We revealed essential roles of GmCDC7 in modulating seed size/weight and seed protein/oil content in soybean, presenting potential new targets for improving yield and quality of soybean and other crops. Seed size/weight is a critical factor determining crop yield; however, a limited number of genes regulating this trait have been characterized in soybean. In this study, we identified a Glycine max CELL DIVISION CYCLE 7 (GmCDC7) and revealed its essential roles in seed development. The putative GmCDC7 was highly conserved in both sequences and structure across various species. GmCDC7 transcripts were detectable in multiple tissues, with peak expression occurring during early seed development, while the GmCDC7 proteins were predominantly localized within the nucleus. CRISPR/Cas9-mediated knockout of GmCDC7 led to a significant increase in seed size and 100-seed weight, while overexpression of this gene resulted in a reduction in both seed size and weight. Further cytological analysis demonstrated that GmCDC7 promoted cell expansion and inhibited cell proliferation in seeds. Notably, the gene-edited gmcdc7 mutants showed a substantial increase in protein content alongside a reduction in oil content in seeds. Correspondingly, transcriptomic analyses revealed that GmCDC7 may significantly influence multifaceted regulatory pathways related to cell cycle-related activities, storage protein accumulation, and lipid transport and metabolism during seed development. These findings suggest that GmCDC7 plays pivotal roles in modulating seed size/weight and quality, offering new gene resources and insights into biotechnological strategies for soybean breeding.

Colletotrichum spp., hemibiotrophic fungal pathogens, threaten global strawberry production. Jasmonate (JA) regulates plant-Colletotrichum interactions, but its mechanisms remain unclear. Here we demonstrate that both exogenous methyl jasmonate (MeJA) treatment and elevated endogenous MeJA levels increase strawberry susceptibility to anthracnose. Two key JA biosynthesis genes, FveAOS2 and FveAOC3, were identified as contributors to Colletotrichum-induced susceptibility. Further analysis revealed that the FveSnRK2.1-FveWRKY50 phosphorylation module functions as an important molecular switch in regulating disease susceptibility. Specifically, Colletotrichum infection or MeJA application activates FveSnRK2.1, which phosphorylates FveWRKY50 at serine residue 88 (S88). This phosphorylation enhances the stability and transcriptional activity of FveWRKY50, leading to increased expression of FveAOS2 and FveAOC3, higher MeJA accumulation and enhanced susceptibility. Notably, the strawberry JASMONATE-ZIM DOMAIN (JAZ) protein FveJAZ5 suppresses susceptibility by directly interacting with FveWRKY50, thereby preventing its interaction with FveSnRK2.1 and inhibiting the activation of FveAOS2 and FveAOC3. Upon pathogen attack or MeJA signalling, FveJAZ5 is degraded, thereby releasing FveWRKY50 from suppression. The study elucidates a Colletotrichum-induced 'JA signaling - JA biosynthesis' positive feedback loop that drives strawberry susceptibility. Knocking out FveWRKY50 and overexpressing FveJAZ5 generated anthracnose-resistant germplasms. These findings deepen understanding of plant-Colletotrichum interactions and provide genes for resistant strawberry breeding.

Seed weight is a key component of crop yield. However, molecular mechanisms underlying soybean seed weight variation remain largely elusive. Here, we identify a major seed weight determining gene Glycine max SMALL SEED 6 (GmSMS6) that encodes a 14-3-3 protein. GmSMS6 physically and genetically interacts with the transcription factor GmbZIP151 and the RING-type E3 ligase GmUBQ1. GmSMS6 acts as a regulatory hub switch that coordinates the transcriptional activation activity and GmUBQ1-mediated stability of GmbZIP151, primarily repressing cellular expansion of soybean cotyledons. Knocking out GmSMS6 increases seed weight and protein content but decreases oil accumulation in multiple soybean genetic backgrounds. A loss-of-function allele of GmSMS6 is absent in available soybean resources, while the weakly expressed haplotype associated with heavy seed weight has undergone selection in G. max. These results provide insights into mechanisms underlying soybean domestication and highlight the importance of gene loss for improving crop yield and quality.

Pod dehiscence facilitates seed dispersal in wild legumes while indehiscence is a key domestication trait in cultivated ones. However, the evolutionary genetic mechanisms underlying its diversity are largely unclear. In this study, we compared transcriptomes of two warm-season (Glycine spp. and Phaseolus spp.) and two cool-season (Pisum spp. and Medicago ruthenica) legumes in analysis of dehiscent and indehiscent pod genotypes. Differentially expressed genes in AP2/ERF-like transcription factors and seven structural gene families, including lactoperoxidase, laccase, and cellulose synthase-interactive proteins, which are involved in secondary cell wall component accumulation, were identified to exert key roles in pod dehiscence variation. In accordance with this, higher lignin and cellulose contents were observed in pod secondary cell wall of dehiscent accessions of soybean and pea; however, the variation patterns of lignin polymers in soybean (accumulation) and pea (proportion) differed between dehiscent and indehiscent pods. Moreover, genome-wide comparative analysis revealed that orthogroups represented <1% of all identified differentially expressed genes could be traced among the four genera of legumes, while recruiting paralogous members may constitute the genetic robustness of legume pod dehiscence. This study compared the genetic mechanism among several legumes in pod dehiscence formation and revealed a compensating role of paralogous redundancy of involved gene families in seed dispersal, which can guide crop breeding.

The typical symptom of Paulownia witches’ broom (PaWB), caused by phytoplasma infection, is excessive branching, which is mainly triggered by auxin metabolism disorder. Aux/IAA is the early auxin-responsive gene that participates in regulating plant morphogenesis such as apical dominance, stem elongation, lateral branch development, and lateral root formation. However, no studies have investigated the response of the Aux/IAA gene family to phytoplasma infection in Paulownia fortunei. In this study, a total of 62 Aux/IAA genes were found in the genome. Phylogenetic analysis showed that PfAux/IAA genes could be divided into eight subgroups, which were formed by tandem duplication and fragment replication. Most of them had a simple gene structure, and several members lacked one or two conserved domains. By combining the expression of PfAux/IAA genes under phytoplasma stress and SA-treated phytoplasma-infected seedlings, we found that PfAux/IAA13/33/45 may play a vital role in the occurrence of PaWB. Functional analysis based on homologous relationships showed a strong correlation between PfAux/IAA45 and branching. Protein–protein interaction prediction showed that PfARF might be the binding partner of PfAux/IAA, and the yeast two-hybrid assay and bimolecular fluorescent complementary assay confirmed the interaction of PfAux/IAA45 and PfARF13. This study provides a theoretical basis for further understanding the function of the PfAux/IAA gene family and exploring the regulatory mechanism of branching symptoms caused by PaWB.

The study explores Paulownia flowers as an emerging resource in medicine, food, health supplements, cosmetics, and animal feed, despite a lack of characterization of their secondary metabolite accumulation mechanisms. We conducted a comprehensive investigation into the biological activities, metabolite profiles, and gene expression patterns of flavonoids in Paulownia flowers. Our results, obtained using ABTS/FRAP/DPPH assays, MTT assays, and colorimetric analyses, demonstrate the potent antioxidant, antibacterial, hypolipidemic, and antiproliferative activities of Paulownia flower flavonoid (PFF) extracts. Notably, the Paulownia elongata tetraploid (M4) exhibited particularly notable effects. HPLC fingerprinting, molecular docking, and density functional theory calculations led to the identification of rhoifolin and luteolin as key bioactive flavonoids. UV-Vis, fluorescence, and circular dichroism spectroscopy confirmed PFF's role as a reversible and mixed inhibitor of xanthine oxidase (XOD). Transcriptome and metabolomic analyses further revealed substantial enrichment in phenylpropanoid, flavonol, and flavonoid biosynthetic pathways, with PfPP2C, PfPYR, PfPYL, PfNPR1, PfMYBs, and PfbHLHs identified as crucial regulators. This work provides a foundational understanding of flavonoid biosynthesis in Paulownia flowers, paving the way for targeted genetic modification and biotechnological interventions aimed at enhancing flavonoid accumulation for industrial applications.

Leaf color mutants are common in higher plants that can be used as markers in crop breeding and are important tools in understanding regulatory mechanisms of chlorophyll biosynthesis and chloroplast development. Genetic analysis was performed by evaluating F-1, F-2 and BC1 populations derived from two parental lines (Charleston gray with green leaf color and Houlv with delayed green leaf color), suggesting that a single recessive gene controls the delayed green leaf color. In this study, the delayed green mutant showed a conditional pale green leaf color at the early leaf development but turned to green as the leaf development progressed. Delayed green leaf plants showed reduced pigment content, photosynthetic, chlorophyll fluorescence parameters, and impaired chloroplast development compared with green leaf plants. The delayed green (dg) locus was mapped to 7.48 Mb on chromosome 3 through bulk segregant analysis approach, and the gene controlling delayed green leaf color was narrowed to 53.54 kb between SNP130 and SNP135 markers containing three candidate genes. Sequence alignment of the three genes indicated that there was a single SNP mutation (G/A) in the coding region of ClCG03G010030 in the Houlv parent, which causes an amino acid change from Arginine to Lysine. The ClCG03G010030 gene encoded FtsH extracellular protease protein family is involved in early delayed green leaf development. The expression level of ClCG03G010030 was significantly reduced in delayed green leaf plants than in green leaf plants. These results indicated that the ClCG03G010030 might control watermelon green leaf color and the single SNP variation in ClCG03G010030 may result in early delayed green leaf color development during evolutionary process.

The resultant DNA from loss-of-function mutation can be recruited in biological evolution and development. Here, we present such a rare and potential case of "to gain by loss" as a neomorphic mutation during soybean domestication for increasing seed weight. Using a population derived from a chromosome segment substitution line of Glycine max (SN14) and Glycine soja (ZYD06), a quantitative trait locus (QTL) of 100-seed weight (qHSW) was mapped on chromosome 11, corresponding to a truncated β-1, 3-glucosidase (βGlu) gene. The novel gene hsw results from a 14-bp deletion, causing a frameshift mutation and a premature stop codon in the βGlu. In contrast to HSW, the hsw completely lost βGlu activity and function but acquired a novel function to promote cell expansion, thus increasing seed weight. Overexpressing hsw instead of HSW produced large soybean seeds, and surprisingly, truncating hsw via gene editing further increased the seed size. We further found that the core 21-aa peptide of hsw and its variants acted as a promoter of seed size. Transcriptomic variation in these transgenic soybean lines substantiated the integration hsw into cell and seed size control. Moreover, the hsw allele underwent selection and expansion during soybean domestication and improvement. Our work cloned a likely domesticated QTL controlling soybean seed weight, revealed a novel genetic variation and mechanism in soybean domestication, and provided new insight into crop domestication and breeding, and plant evolution.

The NAC transcription factor ripening inducing factor (RIF) was previously reported to be necessary for the ripening of octoploid strawberry (Fragaria × ananassa) fruit, but the mechanistic basis of RIF-mediated transcriptional regulation and how RIF activity is modulated remains elusive. Here, we show that FvRIF in diploid strawberry, Fragaria vesca, is a key regulator in the control of fruit ripening and that knockout mutations of FvRIF result in a complete block of fruit ripening. DNA affinity purification sequencing coupled with transcriptome deep sequencing suggests that 2,080 genes are direct targets of FvRIF-mediated regulation, including those related to various aspects of fruit ripening. We provide evidence that FvRIF modulates anthocyanin biosynthesis and fruit softening by directly regulating the related core genes. Moreover, we demonstrate that FvRIF interacts with and serves as a substrate of MAP kinase 6 (FvMAPK6), which regulates the transcriptional activation function of FvRIF by phosphorylating FvRIF at Thr-310. Our findings uncover the FvRIF-mediated transcriptional regulatory network in controlling strawberry fruit ripening and highlight the physiological significance of phosphorylation modification on FvRIF activity in ripening.

Pod dehiscence facilitates seed dispersal in wild legumes but results in yield loss in cultivated legumes. The evolutionary genetics of the legume pod dehiscence trait remain largely elusive.We characterized the pod dehiscence of chromosome segment substitution lines of Glycine max crossed with Glycine soja and found that the gene underlying the predominant quantitative trait locus (QTL) of soybean pod-shattering trait was Pod dehiscence 1 (Pdh1). A few rare loss-of-function (LoF) Pdh1 alleles were identified in G. soja, while only an allele featuring a premature stop codon was selected for pod indehiscence in cultivated soybean and spread to low-precipitation regions with increased frequency. Moreover, correlated interactions among Pdh1's haplotype, gene expression, and environmental changes for the developmental plasticity of the pod dehiscence trait were revealed in G. max.We found that orthologous Pdh1 genes specifically originated in warm-season legumes and their LoF alleles were then parallel-selected during the domestication of legume crops.Our results provide insights into the convergent evolution of pod dehiscence in warm-season legumes, facilitate an understanding of the intricate interactions between genetic robustness and environmental adaptation for developmental plasticity, and guide the breeding of new legume varieties with pod indehiscence.

Paulownia witches’ broom (PaWB), caused by phytoplasmas, is the most devastating infectious disease of Paulownia. R2R3-MYB transcription factors (TF) have been reported to be involved in the plant’s response to infections caused by these pathogens, but a comprehensive study of the R2R3-MYB genes in Paulownia has not been reported. In this study, we identified 138 R2R3-MYB genes distributed on 20 chromosomes of Paulownia fortunei. These genes were classified into 27 subfamilies based on their gene structures and phylogenetic relationships, which indicated that they have various evolutionary relationships and have undergone rich segmental replication events. We determined the expression patterns of the 138 R2R3-MYB genes of P. fortunei by analyzing the RNA sequencing data and found that PfR2R3-MYB15 was significantly up-regulated in P. fortunei in response to phytoplasma infections. PfR2R3-MYB15 was cloned and overexpressed in Populus trichocarpa. The results show that its overexpression induced branching symptoms. Subsequently, the subcellular localization results showed that PfR2R3-MYB15 was located in the nucleus. Yeast two-hybrid and bimolecular fluorescence complementation experiments showed that PfR2R3-MYB15 interacted with PfTAB2. The analysis of the PfR2R3-MYB15 gene showed that it not only played an important role in plant branching, but also might participate in the biosynthesis of photosystem elements. Our results will provide a foundation for future studies of the R2R3-MYB TF family in Paulownia and other plants.