Subcellular Localization Research Articles

Overexpression of OsSTP1 increases grain yield via enhancing carbohydrate metabolism and transport in rice.

Overexpression of OsSTP1 enhances the non-structural carbohydrate remobilization in the source, starch accumulation in grains, and the transportation of carbohydrates from source to sink during the filling stage. The sugar transporter protein (STP) is the best-characterized subfamily of the monosaccharide transporter (MST) family and plays critical roles in regulating plant stress tolerance, growth, and development. However, the role of STPs in regulating rice yield is poorly understood. In this study, we report that compared with Taipei 309, overexpression of OsSTP1 can achieve higher rice yield. We demonstrate that OsSTP1 mRNA levels are higher than those of the other seven STPs in mixed samples of leaf sheaths, stems, and nodes at 12days after pollination (DAP). OsSTP1 is prominently expressed in the leaf sheaths, stems, and nodes at the grain filling stage. Subcellular localization analysis revealed that OsSTP1 is localized in the plasma membrane. Overexpression of OsSTP1 increased the activities of amylase (AMY) and sucrose phosphate synthase (SPS) in mixed samples of leaf sheaths, stems, and nodes at 12 DAP, the sucrose content of the phloem exudate, and accumulation of soluble sugars and starch in panicles, ultimately increasing seed-setting rates and grain yields in the Taipei 309 cultivar. These findings indicate that overexpression of OsSTP1 can improve grain yield by synergistically promoting non-structural carbohydrate (NSC) remobilization and transportation.

VOZ‐dependent priming of salicylic acid‐dependent defense against Rhizopus stolonifer by β‐aminobutyric acid requires the TCP protein TCP2 in peach fruit

SUMMARYVascular plant one‐zinc finger (VOZ) transcription factors (TFs) play crucial roles in plant immunity. Nevertheless, how VOZs modulate defense signaling in response to elicitor‐induced resistance is not fully understood. Here, the defense elicitor β‐aminobutyric acid (BABA) resulted in the visible suppression of Rhizopus rot disease of peach fruit caused by Rhizopus stolonifer. Defense priming by BABA was notably associated with increased levels of salicylic acid (SA) and SA‐dependent gene expression. Data‐independent acquisition proteomic analysis revealed that two VOZ proteins (PpVOZ1 and PpVOZ2) were substantially upregulated in BABA‐induced resistance (BABA‐IR). Furthermore, the interaction of PpVOZ1 and PpVOZ2 and their potential target of the TEOSINTE‐BRANCHED1/CYCLOIDEA/PCF (TCP)‐family protein PpTCP2 screened from protein–protein interaction networks was confirmed by yeast two‐hybrid (Y2H), luciferase complementation imaging and glutathione S‐transferase pull‐down assays. Furthermore, subcellular localization, yeast one‐hybrid, electrophoretic mobility shift assay and dual‐luciferase reporter assays demonstrated that nuclear localization of both PpVOZ1 and PpVOZ2 was critical for their contribution to BABA‐IR, as these proteins potentiated the PpTCP2‐mediated transcriptional activation of isochorismate synthase genes (ICS1/2). The overexpression of both PpVOZ1 and PpVOZ2 could activate the transcription of SA‐dependent genes and provide disease resistance in transgenic Arabidopsis. In contrast, the ppvoz1cas9 and ppvoz2cas9 loss‐of‐function mutations and the voz1cas9 voz2cas9 double mutation attenuated BABA‐IR against R. stolonifer. Therefore, the three identified positive TFs, PpVOZ1, PpVOZ2, and PpTCP2, synergistically contribute to the BABA‐activated priming of systemic acquired resistance in postharvest peach fruit by a VOZ‐TCP‐ICS regulatory module.

BEACH domain proteins function as cargo-sorting adaptors in secretory and endocytic pathways.

We identify BEACH domain-containing proteins (BDCPs) as novel membrane coat proteins involved in the sorting of transmembrane proteins (TMPs) on the trans-Golgi network and tubular sorting endosomes. The seven typical mammalian BDCPs share a predicted alpha-solenoid-beta propeller structure, suggesting they have a protocoatomer origin and function. We map the subcellular localization of seven BDCPs based on their dynamic colocalization with RAB and ARF small GTPases and identify five typical BDCPs on subdomains of dynamic tubular-vesicular compartments on the intersection of endocytic recycling and post-Golgi secretory pathways. We demonstrate that BDCPs interact directly with the cytosolic tails of selected TMPs and identify a subset of TMPs, whose trafficking to the plasma membrane is affected in cells lacking BDCP. We propose that the competitive binding of BDCPs and clathrin coat adaptors to the cytosolic tails of TMPs, followed by their clustering to distinct subdomains of secretory/recycling tubules function as a mechanism for sorting of TMPs in pleomorphic tubular-vesicular compartments that lack a clathrin coat.

Ubiquitin Ligase U-Box51 Positively Regulates Drought Stress in Potato (Solanum tuberosum L.)

The ubiquitin-proteasome system (UPS) is a key protein degradation pathway in eukaryotes, in which E3 ubiquitin ligases mediate protein ubiquitination, directly or indirectly targeting substrate proteins to regulate various biological processes, including plant growth, hormone signaling, immune responses, and adaptation to abiotic stress. In this study, we identified plant U-box protein 51 in Solanum tuberosum (StPUB51) as an E3 ubiquitin ligase through transcriptomic analysis, and used it as a candidate gene for gene-function analysis. Quantitative real-time PCR (qRT-PCR) was used to examine StPUB51 expression across different tissues, and its expression patterns under simulated drought stress induced by polyethylene glycol (PEG 6000) were assessed. Transgenic plants overexpressing StPUB51 and plants with down-regulated StPUB51 expression were generated to evaluate drought tolerance. The activities of key antioxidant enzymes-superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) as well as malondialdehyde (MDA) content in transgenic plants’ leaves were measured under drought conditions. Protein–protein interactions involving StPUB51 were explored via yeast two-hybrid (Y2H) screening, with interaction verification by bimolecular fluorescence complementation (BiFC). StPUB51 was predominantly expressed in stems, with lower expression observed in tubers, and its expression was significantly upregulated in response to 20% PEG-6000 simulated drought. Subcellular localization assays revealed nuclear localization of the StPUB51 protein. Under drought stress, StPUB51-overexpressing plants exhibited enhanced SOD, POD, and CAT activities and reduced MDA levels, in contrast to plants with suppressed StPUB51 expression. Y2H and BiFC analyses identified two interacting proteins, StSKP2A and StGATA1, which may be functionally linked to StPUB51. Collectively, these findings suggest that StPUB51 plays a positive regulatory role in drought tolerance, enhancing resilience in potato growth and stress adaptation.

Feedback control of the heat shock response by spatiotemporal regulation of Hsp70.

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity.

AttentionEP: Predicting Essential Proteins via Fusion of Multiscale Features by Attention Mechanisms

Identifying essential proteins is of utmost importance in the field of biomedical research due to their essential functions in cellular activities and their involvement in mechanisms related to diseases. In this research, a novel approach called AttentionEP for predicting essential proteins (EP) is introduced by attention mechanisms. This method leverages both cross-attention and self-attention frameworks, focusing on enhancing prediction accuracy through the integration of features across diverse scales. Spatial characteristics of proteins are obtained from the protein-protein interaction (PPI) network by employing Graph Convolutional Networks (GCN) and Graph Attention Networks (GAT). Following this, Bidirectional Long Short-Term Memory networks (BiLSTM) are employed to derive temporal features from gene expression datasets. Furthermore, spatial characteristics are derived by integrating data on subcellular localization with the application of Deep Neural Networks (DNN). In order to effectively integrate features across multiple scales, initial steps involve the application of self-attention techniques to derive essential insights from each unique data set. Following this, mechanisms involving self-attention and cross-attention are employed to enhance the interaction between diverse information sources. To identify essential proteins, a classifier based on the ResNet architecture is developed. The findings from the experiments indicate that the method introduced here shows superior performance in identifying essential proteins, recording an Area Under the Curve (AUC) value of 0.9433. This approach shows a considerable advantage over established techniques. The findings of this study provide a significant advancement in the comprehension of critical proteins, revealing promising potential for applications in the development of therapeutics and addressing various diseases.

Promyelocytic leukemia protein (PML) knockout increases mitochondrial Ca2+ uptake in HeLa cells

The multifunctional promyelocytic leukemia protein (PML) is involved in the regulation of various cellular processes in both physiological and pathological conditions. Specifically, PML is one of the inositol-1,4,5-trisphosphate receptors (IP3Rs) activity regulators and can influence Ca2+ transport from the endoplasmic reticulum (ER) to mitochondria. In this work, the effects of PML knockout on calcium homeostasis in the cytosol, ER, and mitochondria of HeLa cells were studied upon stimulation with histamine, which induces Ca2+ mobilization from the ER via IP3Rs. We utilized calcium indicators with different subcellular localizations, including synthetic dyes Fura-2 (cytosolic), Xrhod-5F (mitochondrial), and protein sensor R-CEPIAer (ER), as well as mitochondrial potential-sensitive probes Rh123 and TMRM. Our results show that PML knockout induced changes in HeLa cell and mitochondrial morphology, slightly decreased basal and integral Ca2+ levels, enhanced mitochondrial Ca2+ uptake from the cytoplasm, and maintained residual mitochondrial potential after depolarization. Additionally, it reduced the Ca2+ pool in ER membranes not associated with histamine receptor activation and, consequently, IP3Rs. These findings suggest that changes in calcium ion transport due to PML knockout in HeLa cells affect mitochondrial activity.

Evolutionary Insights and Expression Dynamics of the Glutathione Peroxidase (GPX) Gene Family in Water lily (Nymphaea colorata) in Response to Multiple Abiotic Stresses

The glutathione peroxidase (GPX) family in plants contains various isoenzymes, each with distinct subcellular localizations, tissue-specific expression patterns, and responses to environmental stress. In current study, we conducted genome-wide bioinformatics analyses of the GPX gene family in Nymphaea colorata, identifying five putative NcGPX genes dispersed across five of the fourteen chromosomes. Examination of the protein sequences revealed that NcGPXs comprehend three conserved cysteine residues, with predicted in-silico subcellular localizations in chloroplast, extracellular, or cytoplasm. We reported that NcGPX genes protein lengths and molecular weight (MW) ranged from 171-248 amino acids (aa) and 18.798-27.076 kDa, respectively. A comparative neighbor-joining phylogenetic study revealed that GPX genes were clustered into four main groups (Group A– D) from water lily and two closely related plant species, alongside with Arabidopsis thaliana. The universal analysis showed that segmental duplications occurred within the NcGPX gene family. Investigation of gene structure and motifs suggested that most NcGPX genes have relatively conserved exon-intron structures and motif arrangements. Promoter region exploration of NcGPX genes revealed numerous cis-acting regulatory elements associated with development, stress, and hormone responses. The identified 3D protein structures showed that water lily GPXs form conserved dimeric protein signatures. Finally, gene expression and enzymes accumulation of NcGPXs in response to different abiotic stresses, in leaves of N. colorata were analysed using real-time RT-qPCR and spectrophotometry. The results indicate that different members of the GPX gene family are co-ordinately regulated under specific environmental stress conditions, establishing a foundation for studying their gene functions and improving abiotic stress tolerance in water lily genetic breeding.

GhBZR15 improved the tolerance of cotton to cold and salt stress revealed by genome-wide characterization of BZR genes family

Brassinazole resistant (BZR) transcription factors play important roles in brassinosteroids (BRs) signaling pathway of plants, which are involved in many developmental processes and the responses to abiotic stress by regulating the expression of related genes. In order to explore the molecular mechanism of BZR transcription factors in cotton in response to abiotic stress, the BZR transcription factors in four cotton genomes were identified and analyzed. A total of 67 BZR transcription factors were identified and divided into three subfamilies according to their genetic relationship. The expression levels of BZR under cold, heat, salt, and drought treatments were analyzed to predict their function under abiotic stress. Promoter analysis of BZR proteins showed that light signaling can enhance the BZR transcriptional activity. The subcellular localization experiment showed that GhBZR15 protein was mainly distributed in the nucleus. Arabidopsis thaliana L. overexpressing GhBZR15 gene showed stronger cold tolerance and salt tolerance than wild type. These results, combined with the GhBZR protein-interaction network, suggest that the interaction of GhBZR with phytochrome interacting factors (PIF4) plays an important role in abiotic stress responses. These findings provide a basis for further understanding the function of BZR in cotton.

Hydroxylase-oriented mining and functional characterization of camptothecin 10-hydroxylase from Camptotheca acuminata Decne

The regiospecific C-10 hydroxylation of camptothecin (CPT) is challenging from a chemical perspective. This step bridges the natural-sourced CPT to an important industrial material, 10-hydroxycamptothecin (10HCPT). 10HCPT is naturally generated in a medicinal plant, Camptotheca acuminata Decne. Functionally active hydroxylase responsible for regiospecific C-10 hydroxylation of CPT is undoubtedly existed in this plant. Therefore, the multi-omics database of C. acuminata was utilized to perform hydroxylase-oriented mining and screening. Three CYP81 genes were identified and two of them, including CYP81BQ18 and CYP81B256 catalyzed hydroxylation of CPT at the C-10 position. CYP81B256 and CYP81BN24 displayed quercetin C-5’ hydroxylase activity. CYP81B256 exhibited better catalytic performance for CPT than CYP81BQ18 and CPT10H in vitro. Furthermore, CYP81BQ18 and CYP81B256 displayed CPT 10-hydroxylase activity in tobacco. Functional verification in planta suggested that the newly observed CYP81BQ18 and CYP81B256 were involved in 10HCPT biosynthesis in C. acuminata. Subcellular localization analysis revealed that these CYP81 genes were located on the endoplasmic reticulum. Evolutionary analysis indicated that CYP81B256 and CYP81BN24 probably evolved from a same ancestral gene via gene duplication, while CYP81BQ18 evolved independently and acted as a specie-specific hydroxylase. CYP81B256 acted as an evolutionary intermediate gene bridging the alkaloid and flavonoid metabolism in C. acuminata. This research provides valuable insights into the function and evolutionary origin of CYP81s in the regiospecific hydroxylation step for 10HCPT biosynthesis.

Genome-wide identification of SWEET gene family and the sugar transport function of three candidate genes during female flower bud induction stage of Juglans sigillata Dode

Sugar Will Eventually be Exported Transporter (SWEET) transports sugar to sink organs and regulates intercellular sugar transport to provide energy for plant growth and development. In this study, twenty-two SWEET genes were identified and distributed on eleven chromosomes. Phylogenetic tree analysis showed that these genes could be divided into four subfamilies. Metabolism and transcriptome analysis showed that sucrose and fructose were accumulated in female flower buds at physiological differentiation stage (PDS). The third branch of JsSWEET1 and the fourth branch of JsSWEET9 and JsSWEET17 were highly expressed in female flower buds at undifferentiated stage (UDS) and PDS, which promoted sugar accumulation in female flower bud differentiation, so these three candidate SWEET genes were considered to be involved in the accumulation of sugar in the flower bud differentiation of Juglans sigillata. The subcellular localization showed that all three candidate genes were located on the cell membrane, and JsSWEET17 was also expressed and functioned in the vacuolar membrane. Through overexpression in callus and silencing in female flower buds at UDS and PDS, it was found that JsSWEET1 positively regulated the accumulation of sucrose in female flower buds, and JsSWEET9 and JsSWEET17 are involved in the transport and accumulation of fructose during flower bud differentiation. These results could provide a comprehensive understanding of the JsSWEETs gene family and provide theoretical guidance for further study of the function of SWEET-induced sugar accumulation in plant flower development.

Rbm24-mediated post-transcriptional regulation of skeletal and cardiac muscle development, function and regeneration.

RNA-binding proteins are critically involved in the post-transcriptional control of gene expression during embryonic development and in adult life, contributing to regulating cell differentiation and maintaining tissue homeostasis. Compared to the relatively well documented functions of transcription factors, the regulatory roles of RNA-binding proteins in muscle development and function remain largely elusive. However, deficiency of many RNA-binding proteins has been associated with muscular defects, neuromuscular disorders and heart diseases, such as myotonic dystrophy, amyotrophic lateral sclerosis, and cardiomyopathy. Rbm24 is highly conserved among vertebrates and is one of the best characterized RNA-binding proteins with crucial implication in the myogenic and cardiomyogenic programs. It presents the distinctive particularity of displaying highly restricted expression in both skeletal and cardiac muscles, with changes in subcellular localization during the process of differentiation. Functional analyses using different vertebrate models have clearly demonstrated its requirement for skeletal muscle differentiation and regeneration as well as for myocardium organization and cardiac function, by regulating the expression of both common and distinct target genes in these tissues. The challenge remains to decipher the dynamic feature of post-transcriptional circuits regulated by Rbm24 during skeletal myogenesis, cardiomyogenesis, and muscle repair. This review discusses current understanding of its function in striated muscles and its possible implication in human disease, with the aim of identifying research gaps for future investigation.

Genome-Wide Identification of FCS-Like Zinc Finger (FLZ) Family Genes in Three Brassica Plant Species and Functional Characterization of BolFLZs in Chinese Kale Under Abiotic Stresses

FCS-like zinc finger (FLZ) proteins are plant-specific regulatory proteins, which contain a highly conserved FLZ domain, and they play critical roles in plant growth and stress responses. Although the FLZ family has been systematically characterized in certain plants, it remains underexplored in Brassica species, which are vital sources of vegetables, edible oils, and condiments for human consumption and are highly sensitive to various abiotic stresses. Following the whole-genome triplication events (WGT) in Brassica, elucidating how the FLZ genes have expanded, differentiated, and responded to abiotic stresses is valuable for uncovering the genetic basis and functionality of these genes. In this study, we identified a total of 113 FLZ genes from three diploid Brassica species and classified them into four groups on the basis of their amino acid sequences. Additionally, we identified 109 collinear gene pairs across these Brassica species, which are dispersed among different chromosomes, suggesting that whole-genome duplication (WGD) has significantly contributed to the expansion of the FLZ family. Subcellular localization revealed that six representative BolFLZ proteins are located in the nucleus and cytoplasm. Yeast two-hybrid assays revealed that 13 selected BolFLZs interact with BolSnRK1α1 and BolSnRK1α2, confirming the conservation of the SnRK1α-FLZ module in Brassica species. Expression profile analysis revealed differential expression patterns of BolFLZ across various tissues. Notably, the expression levels of seven BolFLZ genes out of the fifteen genes analyzed changed significantly following treatment with various abiotic stressors, indicating that the BolFLZ genes play distinct physiological roles and respond uniquely to abiotic stresses in Brassica species. Together, our results provide a comprehensive overview of the FLZ gene family in Brassica species and insights into their potential applications for enhancing stress tolerance and growth in Chinese kale.

Proteomics and transcriptomic analyses provide new insights into the pectin polysaccharide biosynthesis in Premna puberula Pamp

Premna puberula Pamp. is a plant with similarities to both food and medicine, which has attracted the attention of researchers because of the rich pectin in its leaves. However, the mechanism of efficient pectin accumulation remains unclear. Through transcriptomic and proteomic analyses, this study investigated the differentially expressed genes and proteins associated with pectin during various developmental stages of P. puberula. The combined omics approach revealed that the majority of differential genes are mainly involved in the anabolic metabolism of primary and secondary metabolites. Notable differential pathways include starch and sucrose metabolism, amino acid sugar metabolism, and nucleotide sugar metabolism. The SWEET gene family was identified and analyzed, leading to the cloning of PpSWEET15 and subcellular localization. Overexpression of PpSWEET15 resulted in a significant decrease in sucrose content in leaves from 134.44 μg·g−1 to <10 μg·g−1. Glucose content decreased from 407.75 μg·g−1 to 318.38 μg·g−1, while fructose levels showed no significant difference between the two groups of leaves. This comprehensive transcriptomic and proteomic analysis provides valuable insights into the molecular mechanisms underlying the efficient accumulation of pectin in P. puberula.

Global proteomics indicates subcellular-specific anti-ferroptotic responses to ionizing radiation

Cells have many protective mechanisms against background levels of ionizing radiation (IR) orchestrated by molecular changes in expression, post-translational modifications and subcellular localization. Radiotherapeutic treatment in oncology attempts to overwhelm such mechanisms, but radio-resistance is an ongoing challenge. Here, global subcellular proteomics combined with Bayesian modeling identified 544 differentially localized proteins in A549 cells upon 6 Gy x-ray exposure, revealing subcellular-specific changes of proteins involved in ferroptosis, an iron-dependent cell death, suggestive of potential radio-resistance mechanisms. These observations were independent of expression changes, emphasizing the utility of global subcellular proteomics and the promising prospect of ferroptosis-inducing therapies for combatting radioresistance.

Ultrastructure and cardiac impulse propagation: scaling up from microscopic to macroscopic conduction.

The standard conception of cardiac conduction is based on the cable theory of nerve conduction, which treats cardiac tissue as a continuous syncytium described by the Hodgkin-Huxley equations. However, cardiac tissue is composed of discretized cells with microscopic and macroscopic heterogeneities and discontinuities, such as subcellular localizations of sodium channels and connexins. In addition to this, there are heterogeneities in the distribution of sympathetic and parasympathetic nerves, which powerfully regulate impulse propagation. In the continuous models, the ultrastructural details, i.e. the microscopic heterogeneities and discontinuities, are ignored by 'coarse graining' or 'smoothing'. However, these ultrastructural components may play crucial roles in cardiac conduction and arrhythmogenesis, particularly in disease states. We discuss the current progress of modelling the effects of ultrastructural components on electrical conduction, the issues and challenges faced by the cardiac modelling community, and how to scale up conduction properties at the subcellular (microscopic) scale to the tissue and whole-heart (macroscopic) scale in future modelling and experimental studies, i.e. how to link the ultrastructure at different scales to impulse conduction and arrhythmogenesis in the heart.

Small-Molecule Benzo-Phenoselenazine Derivatives for Multi-Subcellular Biomolecule Profiling.

Elucidating the subcellular localization of RNAs and proteins is fundamental to understanding their biological functions. Genetically encoded proteins/enzymes provide an attractive approach to target many proteins of interest, but are limited to specific cell lines. Although small-molecule-based methods have been explored, a comprehensive system for profiling multiple locations in living cells, comparable to fusion-protein techniques, is yet to be established. In this study, we introduce a novel proximity labeling strategy employing a suite of small molecules derived from benzophenoselenazine (e.g., selenium-containing Nile Blue [SeNB]), which achieves proximity labeling through singlet oxygen generation upon near-infrared light activation in the presence of propargylamine. These SeNB compounds allow forselective labeling of RNAs and proteins within living cells, exhibiting a distinct preference for organelle membranes, which are systematically investigated via in vitro, computational, and in cellulo examinations. Our findings highlight the capabilities of SeNB derivatives as wash-free and genetics-free approaches to illuminate the subcellular localization of biological molecules with deep penetration and high spatial resolution. Moreover, SeNB derivatives are capable of elucidating inter-organelle interactions at the molecular level, as evidenced by proteomic and transcriptomic analyses, thus holding significant potential for advancing our understanding of cellular processes related to disease progression and therapeutic development.

Rational design of potent phosphopeptide binders to endocrine Snk PBD domain by integrating machine learning optimization, molecular dynamics simulation, binding energetics rescoring, and in vitro affinity assay.

Human Snk is an evolutionarily conserved serine/threonine kinase essential for the maintenance of endocrine stability. The protein consists of a N-terminal catalytic domain and a C-terminal polo-box domain (PBD) that determines subcellular localization and substrate specificity. Here, an integrated strategy is described to explore the vast structural diversity space of Snk PBD-binding phosphopeptides at a molecular level using machine learning modeling, annealing optimization, dynamics simulation, and energetics rescoring, focusing on the recognition specificity and motif preference of the Snk PBD domain. We further performed a systematic rational design of potent phosphopeptide ligands for the domain based on the harvested knowledge, from which a few potent binders were also confirmed by fluorescence-based assays. A phosphopeptide PP17 was designed as a good binder with affinity improvement by 6.7-fold relative to the control PP0, while the other three designed phosphopeptides PP7, PP13, and PP15 exhibit a comparable potency with PP0. In addition, a basic recognition motif that divides potent Snk PBD-binding sequences into four residue blocks was defined, namely [Χ-5Χ-4]block1-[Ω-3Ω-2Ω-1]block2-[pS0/pT0]block3-[Ψ+1]block4, where the X represents any amino acid, Ω indicates polar amino acid, Ψ denotes hydrophobic amino acid, and pS0/pT0 is the anchor phosphoserine/phosphothreonine at reference residue position 0.

Comprehensive analysis of the PbrTBL gene family and functional analysis of PbrTBL43 under Botryosphaeria dothidea infection in Pyrus bretschneideri

The TBL (Trichome Birefringence-Like) gene family, which participates in the initiation of trichomes and the acetylation of xylan in a variety of plant species, plays a significant role in plant biology. However, there is little information regarding TBL family members in pear (Pyrus bretschneideri Rehd). Here, 65 PbrTBL genes were identified in Pyrus bretschneideri genome. Phylogenetic, gene structure, expression pattern and cis-element of promoter analysis were performed and compared. Expression profiling across different tissues and in response to Botryosphaeria dothidea (B. dothidea) infection highlighted the dynamic and coordinated response of PbrTBL genes, with PbrTBL43 showing significant upregulation. Subcellular localization of PbrTBL43 to the plasma membrane and the enhanced susceptibility to B. dothidea infection upon PbrTBL43 silencing further support its role in pathogen resistance. This study enhances our understanding of the PbrTBL gene family's multifaceted involvement in pear biology and provides a foundation for future research aimed at improving pear resistance to diseases and environmental challenges.

MYB transcription factors in Peucedanum Praeruptorum Dunn: the diverse roles of the R2R3-MYB subfamily in mediating coumarin biosynthesis

BackgroundThe MYB superfamily (v-myb avian myeloblastosis viral oncogene homolog) plays a role in plant growth and development, environmental stress defense, and synthesis of secondary metabolites. Little is known about the regulatory function of MYB genes in Peucedanum praeruptorum Dunn, although many MYB family members, especially R2R3-MYB genes, have been extensively studied in model plants.ResultsA total of 157 R2R3-MYB transcription factors from P. praeruptorum were identified using bioinformatics analysis. Comprehensive analyses including chromosome location, microsynteny, gene structure, conserved motif, phylogenetic tree, and conserved domain were further performed. The length of the 157 transcription factors ranged from 120 to 1,688 amino acids (molecular weight between 14.21 and 182.69 kDa). All proteins were hydrophilic. Subcellular localization predictions showed that 155 PpMYB proteins were localized in the nucleus, with PpMYB12 and PpMYB157 localized in the chloroplasts and mitochondria, respectively. Ten conserved motifs were identified in the PpMYBs, all of which contained typical MYB domains. Transcriptome analysis identified 47,902 unigenes. Kyoto Encyclopedia of Genes and Genomes analysis revealed 136 pathways, of which 524 genes were associated with the phenylpropanoid pathway. Differential expressed genes (DEGs) before and after bolting showed that 11 genes were enriched in the phenylpropanoid pathway. Moreover, the expression patterns of transcription genes were further verified by qRT-PCR. With high-performance liquid chromatography (HPLC), 8 coumarins were quantified from the root, stem, and leaf tissue samples of P. praeruptorum at different stages. Praeruptorin A was found in both roots and leaves before bolting, whereas praeruptorin B was mainly concentrated in the roots, and the content of both decreased in the roots and stems after bolting. Praeruptorin E content was highest in the leaves and increased with plant growth. The correlation analysis between transcription factors and coumarin content showed that the expression patterns of PpMYB3 and PpMYB103 in roots align with the accumulation trends of praeruptorin A, praeruptorin B, praeruptorin E, scopoletin, and isoscopoletin, which declined in content after bolting, suggesting that these genes may positively regulate the biosynthesis of coumarins. Eleven distinct metabolites and 48 DEGs were identified. Correlation analysis revealed that the expression of all DEGs were significantly related to the accumulation of coumarin metabolites, indicating that these genes are involved in the regulation of coumarin biosynthesis.ConclusionsR2R3-MYB transcription factors may be involved in the synthesis of coumarin. Our findings provide basic data and a rationale for future an in-depth studies on the role of R2R3-MYB transcription factors in the growth and regulation of coumarin synthesis.