Low-temperature stress is a critical abiotic limiting factor for the geographical distribution and yield of rice (Oryza sativa), while nitrogen is a fundamental element affecting normal crop growth and high productivity. Both factors significantly affect rice production; however, how cold tolerance and nitrogen use efficiency are coordinated, and the genetic basis of post‑cold recovery, remain poorly understood. A recent study by Academician Kang Chong’s team revealed that the R1‑type MYB transcription factor CHPO targets OsTCP19 and OsNRT2.4, thereby coupling the regulation of the low‑temperature stress-nitrogen metabolism-tiller recovery” cascade. This study reveals the interconnections among post‑cold recovery, nitrogen use efficiency, and tiller regrowth, providing insightful perspectives for the formation of chilling resilience in rice and related breeding strategies. In light of this, this article provides a hotspot commentary on this cutting‑edge achievement. We review the research progress on rice cold tolerance, nitrogen‑mediated recovery after low‑temperature stress, and the regulation of tiller development. We analyze the innovations and current scientific limitations of this study, with a focus on the role of the CHPO regulatory module in stress response and developmental remodeling. This commentary aims to shift the focus of rice stress research from stress survival to post‑chilling recovery, and to propose new considerations for breeding varieties that combine superior cold tolerance, recovery capacity, and high nitrogen use efficiency.

Research on the proteomics of bryophytes is of great significance for revealing the molecular mechanisms of plant adaptation to terrestrial environments. As one of the earliest plants to colonize land, bryophytes provide a unique model system for studying how plants adapt to changes in terrestrial environments. Through proteomic approaches, we can gain in-depth insights into the molecular mechanisms underlying stress responses, growth and development, and evolutionary processes in bryophytes. This is of considerable value for elucidating the evolutionary history and mechanisms of plant adaptability. Furthermore, studies on bryophyte proteomics offer new perspectives and tools for plant science, aiding our understanding of plant responses and adaptive strategies to environmental changes. This review summarizes recent advances in bryophyte proteomics, focusing on abiotic stress responses, growth and developmental regulation, protein modifications, organelle functions, metabolic processes, and evolutionary adaptations. It further highlights the significance of these findings in understanding plant adaptation mechanisms.

Pre-harvest sprouting (PHS) refers to the phenomenon in which cereal crop seeds germinate on the mother plant under high humidity conditions prior to harvest, representing a major constraint on crop yield and seed production. In recent years, with the increasing frequency of extreme weather events, PHS has become more common, resulting in substantial economic losses in agriculture. The PHS process primarily involves two key physiological stages: the release of seed dormancy and the initiation of germination. This paper provides a systematic review of research advances in the mechanisms underlying rice seed dormancy and germination over the past two decades. Furthermore, a chromosomal distribution map of cloned rice genes associated with seed dormancy and germination has been constructed, offering precise genetic information for PHS resistance breeding in rice. Additionally, this paper summarizes the regulatory roles of epigenetic factors and non-coding RNAs involved in rice seed dormancy and germination. It further discusses the molecular mechanisms through which environmental factors such as light, temperature, and water influence these processes, and proposes potential research directions concerning the interaction between environmental cues and plant hormones in the regulation of PHS. Drawing on recent application cases of PHS-resistant genes, this study also outlines future breeding strategies for improving PHS resistance in rice.

INTRODUCTION: Sorghum (Sorghum bicolor) is an ideal crop for marginal land improvement due to its well-developed root system and high water-use efficiency. ASR proteins serve as key nodes that integrate plant growth and development with stress responses, playing important roles in fruit ripening and stress tolerance, particularly drought and salt tolerance. However, functional studies on the ASR family genes in sorghum regarding salt stress tolerance remain relatively limited, and their underlying molecular mechanisms require further investigation. 

RATIONALE: To identify salt-responsive SbASR genes in sorghum, we analyzed the expression patterns of ASR family members under salt stress and water deficit. To elucidate its biological role in salt acclimation, we further cloned and functionally characterized SbASR4 through subcellular localization and transcriptional activity assays, protein stability assessment, heterologous expression in Arabidopsis, gene silencing in sorghum, transcriptional analysis of stress-re­sponsive marker genes, and protein-protein interaction experiments.

RESULTS: SbASR4 was significantly downregulated under salt stress and natural water deficit. SbASR4 localized to the nucleus and plasma membrane and underwent rapid degradation under salt stress. Overexpression of SbASR4 significantly exacerbated the sensitivity of Arabidopsis to salt stress, as evidenced by reduced germination rate, inhibited root elongation, increased Na⁺ accumulation, and elevated H₂O₂ levels. In contrast, silencing SbASR4 in sorghum markedly enhanced plant salt tolerance. Under salt stress, SbASR4-silenced lines exhibited significant upregulation of SOS pathway-related genes, CAT, and SnRK1.1, along with notable changes in PP2C family gene expression. Protein-protein interaction experiments confirmed that SbASR4 interacts with SbSOS2.

CONCLUSION: Collectively, SbASR4 negatively regulates plant salt tolerance, potentially through modulating the antioxidant system, ABA signaling pathway, and Na⁺ homeostasis. This study provides new insights into the molecular mechanisms underlying salt tolerance in sorghum and identifies a potential genetic target for salt-tolerant sorghum breeding.

Effects of SbASR4 silencing on salt stress tolerance in sorghum (A) Phenotypes of control and SbASR4-silenced plants treated with 300 mmol·L^–1 NaCl for 7 and 10 days (bars=7 cm) (B) Relative expression level of SbASR4 in SbASR4-silenced lines. Comparison of physiological parameters between control and SbASR4-silenced plants under salt stress: (C) Survival rate (D) Relative fresh weight (E) Relative plant height (F) Leaf Na⁺/K⁺ ratio (G) Electrical conductivity (H) Leaf Na⁺ content (I) H₂O₂ content. In the figure, pTRV:00 represents the empty vector control, and pTRV:SbASR4 represents the SbASR4-silenced lines. Asterisks indicate significant differences between SbASR4-silenced lines and the control (*P<0.05, **P<0.01, ***P<0.001).

INTRODUCTION: The global area of saline-alkali land is approximately 932 million hectares. The problem of soil salinization caused by seawater intrusion in China’s coastal areas is intensifying. Salt stress is a major abiotic stress that affects plant growth and development. Throughout the long-term evolutionary process, plants have developed complex adaptation mechanisms to cope with the stress, in which gene expression regulation playing a central role. Among the numerous salt stress-responsive genes, the DEAD-box RNA helicase gene family has garnered significant attention due to its crucial roles in plant growth, development, and abiotic stress responses. Studies have confirmed that members of this family are involved in regulating of salt stress in a variety of plants. Therefore, the DEAD-box RNA helicase gene family is an important object for studying the molecular mechanism of the plant salt stress response.
RATIONALE: Scaevola taccada is a semi-mangrove plant widely distributed in tropical and subtropical coastal areas. It exhibits excellent salt tolerance, drought resistance, and resilience to wind and tidal waves. This plant holds significant ecological value for coastal sand stabilization and ecological restoration. Although the DEAD-box RNA helicase gene has been extensively studied in model plants, the genome-wide identification, chromosomal distribution, and salt stress response mechanism of this gene family remain unclear in S. taccada. Therefore, based on the genome data of S. taccada, the members of the DEAD-box gene family (StRHs) were systematically identified, and the tissue expression specificity of the StRH10 gene and its response pattern under salt stress were analyzed, aiming to provide a preliminary scientific basis for the molecular breeding of high-salt tolerant S. taccada seedlings.
RESULTS: Fifty StRH genes were identified from the genome of S. taccada. Most StRH proteins contain 10 types of conserved motif, while a few contain 8 or 9. The StRH proteins comprise 18 conserved domains, primarily SrmB and DEAD. Seven StRH genes lack introns. The promoter region of the StRH genes is rich in cis-acting elements associated the stress response. S. taccada and Arabidopsis thaliana have 20 pairs of collinear StRH genes, and S. taccada and Helianthus annuus have 39 pairs. The StRH10 protein is most closely related to the chloroplast-localized DEAD-box RNA helicase AtRH03 protein in A. thaliana. The subcellular localization prediction indicates that the StRH10 protein is localized to the chloroplasts. The StRH10 gene was the most expressed in leaves. Under salt stress treatment, the expression level of the StRH10 gene did not show a significant difference with increasing NaCl concentration but was significantly up-regulated when the NaCl concentration reached 400 mmol·L^-1.
CONCLUSION: The 50 members of the DEAD-box gene family identified in the genome of S. taccada in this study were unevenly distributed across the chromosomes. Most StRH proteins contain 10 conserved motifs and are clustered within the same evolutionary branch. The promoter region of StRH genes is rich in stress-responsive cis-acting elements such as ABRE, TC-rich repeats, MBS, LTR, WUN-motif, and ARE. Collinearity analysis showed that there were 39 pairs of collinearity genes in S. taccada and H. annuus. The StRH10 gene, which encodes a protein predicted to be located in the chloroplast, exhibits high expression in leaves. Under salt stress, the expression of StRH10 gene was significantly up-regulated only when the NaCl concentration reached 400 mmol·L^-1, indicating that this gene responds to salt stress with a certain concentration threshold.

INTRODUCTION: Waxes are protective substances found on the surface of plants, especially on fruits and leaves, which help prevent non-stomatal water loss from plant tissues. Lycium barbarum is a characteristic economic forest species of Ningxia. Its fruit is a berry with a high-water content, and the surface of the fruit skin is covered with wax, which hinders the normal loss of moisture during the drying process. Alkanes are the main components of the wax on L. barbarum, and the CER3 gene is a key gene involved in alkane biosynthesis. Therefore, an in-depth study of the function of CER3 in L. barbarum provides a theoretical basis for revealing the metabolic synthesis of wax in goji fruits and the molecular mechanism of LbCER3.
RATIONALE: Research on the waxy coating of L. barbarum fruit skin has mainly focused on the structure and composition of the epicuticular wax, with relatively little attention given to its molecular mechanisms. Using the main cultivated varieties of L. barbarum, Ningqi No.1 and Ningqi No.5, as test materials, this study employed methods such as GC-MS, transcriptomics, and molecular biology to investigate the changes in the content and composition of cuticular waxes on the outer and inner layers of the fruit at different developmental stages. It also involved the screening, cloning, and functional validation of key genes related to fruit wax.
RESULTS: During the development and maturation of the fruit, the content of inner and outer cuticular waxes in Ningqi No.1 and Ningqi No.5 gradually decreased, with the total wax content in Ningqi No.1 being significantly higher than that in Ningqi No.5. The outer and inner wax components of both varieties were the same, primarily consisting of alkanes, esters, and acids. Transcriptomic analysis and weighted gene co-expression network analysis (WGCNA) identified LbCER3 as a key gene. The full-length CDS of the LbCER3 gene is 1 884 bp, encoding 628 amino acids, and contains a C-terminal WAX2 domain (Wax2_C) and a fatty acid hydroxylase domain (FA_hydroxylase). An LbCER3 overexpression vector was constructed and successfully transformed into tomato, resulting in a significant reduction in chlorophyll leaching rate in the T₁ generation transgenic lines, along with a notable increase in leaf wax content and significant increases in the wax components of alkanes and acids.
CONCLUSION: These findings indicate that LbCER3 is a key gene in the synthesis of wax in L. barbarum.

INTRODUCTION: Tomato wilt disease, caused by Fusarium oxysporum f. sp. lycopersici (Fol), is a soil borne fungal disease that causes significant economic losses to global tomato production. The molecular mechanism of pathogen sRNA cross-border regulation of host DNA methylation mediated tomato resistance to wilt disease is still unclear. Here, we illustrate that SlyFRG5 methylation level activated by Fol-milR1 mediates immune response against tomato wilt disease.
RATIONALE: The latest research shows that sRNA derived from pathogens can serve as novel effectors for cross-border participation in host-pathogen interactions. Our previous research documented that Fol-milR1 was transferred into host cells as an effector. Fol-milR1 repressed the expression of a resistant gene SlyFRG4 in tomato, as well as binded to SlyAGO4a leading to interfere with the host immune system to achieve successful infection. Therefore, this research further determines whether the transmission of Fol-milR1 into tomato causes DNA methylation in the tomato genome and elucidates other aspects of the molecular mechanism involving the action of Fol-milR1.
RESULTS: The results indicate that SlyAGO4a negatively regulates the resistance to tomato wilt disease. The methylation level of Solyc02g081370 (SlyFRG5) was identified to be directly associated with Fol-milR1 using whole genome methylation sequencing and molecular biology methods. SlyFRG5 loss-of-function alleles created by CRISPR/Cas9 in resistant tomato cultivar Motelle exhibited enhanced disease susceptibility to Fol, further supporting the idea that SlyFRG4 is essential for tomato wilt disease resistance. SlyFRG5 contributed wilt disease resistance by regulating the accumulation of host reactive oxygen species (ROS) in tomato.
CONCLUSION: Combining previous research, we concluded that during the infection Fol, Fol-milR1 was transferred into host cells to hijack SlyAGO4a which leads to methylation a Fusarium disease resistant gene SlyFRG5. This study analyzed how the Fol-milR1-SlyAGO4a-SlyFRG5 functional module mediated tomato resistance to wilt disease, which will provide a new approach for exploring the improvement of tomato resistance to Fusarium disease.

INTRODUCTION: Autophagy is an evolutionarily conserved catabolic process that degrade damaged or unwanted cellular materials including organelles, macromolecules and even pathogens. AUTOPHAGY RELATED GENE 8 (ATG8) proteins play critical roles in autophagy. There are 9 ATG8 isoforms in Arabidopsis that are grouped into two clades. ATG8h and ATG8i are categorized into clade II because of their unique features. However, their functions in resistance against biotrophic and necrotrophic pathogens are not fully understood.
RATIONALE: Our previous study revealed that simultaneous knockout of ATG8h and ATG8i enhanced Arabidopsis resistance to the biotrophic fungal pathogen Golovinomyces cichoracearum. However, neither the role of this gene in resistance to biotrophic bacterial pathogen and necrotrophic fungal pathogen nor the effect of its overexpression on disease resistance has been investigated previously.
RESULTS: To further understand the roles of ATG8i in resistance against both biotrophic bacterial pathogen and necrotrophic fungal pathogens, the ATG8i overexpressing Arabidopsis lines driven by 35S promoter were generated. The generated ATG8i overexpressing lines exhibited an elevated resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (PstDC3000), but displayed an increased susceptibility to a toxin secreted by necrotrophic fungal pathogen. The enhanced resistance to the biotrophic pathogen was associated with the increased expression of Pathogenesis-related (PR) gene, elevated accumulation of H₂O₂ and enhanced callose deposition, whereas the increased sensitivity to the necrotrophic fungal toxin was associated with the significantly reduced expression of the genes in jasmonic acid (JA) pathway.
CONCLUSION: Based on the results reported here together with our previously published results, we conclude that either overexpression or loss of ATG8i function can activate resistance to biotrophic pathogens, but reduce the resistance to necrotrophic pathogens. Overexpression of ATG8i appears to activate salicylic acid (SA)-mediated resistance but suppress JA-mediated resistance.

INTRODUCTION: Continuously increasing atmospheric CO₂ concentrations affects plant stomatal movement and water use efficiency. Unveiling the molecular mechanisms underlying CO₂-regulated stomatal closure is of great significance for understanding plant adaptation to high CO₂ environments and ensuring agricultural productivity. Previous studies have indicated that the Gα protein, carbonic anhydrase (CA), and nitric oxide (NO) are all involved in high CO₂-induced stomatal closure, but the signaling relationships among these three components remain unclear.
RATIONALE: Gα mediates stomatal movement in response to various stimuli by regulating NO generation in guard cells; CA is a key enzyme for CO₂ perception in guard cells and participates in early CO₂ signal transduction; NO acts as a gaseous signaling molecule crucial for stomatal movement, with its synthesis primarily dependent on nitrate reductase NIA1. Previous research suggests that Gα may interact with CA, and both likely function upstream of NO to jointly regulate the CO₂ response.
RESULTS: (1) Pertussis toxin (PTX, Gα inhibitor) and gpα1 mutants impaired NaHCO₃-induced stomatal closure and NO production in guard cells of wild-type plants, but not in cGα and wGα lines. Sodium nitroprusside (SNP, NO donor) eliminated the blocking effects of PTX and gpα1 on NaHCO₃-induced stomatal closure. c-PTIO (NO scavenger), Na₂WO₄ (nitrate reductase inhibitor), and the nia1-2 mutant all disrupted NaHCO₃-induced stomatal closure and guard cell NO generation, effects that were not reversed by cholera toxin (CTX, Gα activator). Furthermore, NaHCO₃ failed to induce stomatal closure and NO production in guard cells of cGα/nia1-2 and wGα/nia1-2. These results indicate that Gα is located upstream of NO. (2) The CA inhibitors acetazolamide (AZ) and ethoxzolamide (EZ), as well as the ca1ca4 mutant, disrupted NaHCO₃-induced stomatal closure and guard cell NO generation, while SNP restored NaHCO₃-induced stomatal closure in these samples. These results demonstrate that CA is also situated upstream of NO. (3) The failure of NaHCO₃ to induce stomatal closure and guard cell NO generation in wGα/ca1ca4 and cGα/ca1ca4 was consistent with the phenotype of ca1ca4 but opposite to that of wGα and cGα. This indicates that CA is genetically located downstream of Gα.
CONCLUSION: This study elucidates a sequential signaling pathway of Gα-CA-NO that collectively regulates high CO₂-induced stomatal closure in Arabidopsis. These findings deepen our understanding of CO₂ signal transduction in guard cells and provide a theoretical basis for modulating crop adaptation to future high-CO₂ environments.

INTRODUCTION: The superhydrophobic structures on plant leaves are crucial for self-cleaning and anti-microbial functions, acting as both a core functional mechanism and a biomimetic blueprint for advanced materials design. Ferns, as the earliest vascular plants that transitioned from aquatic to terrestrial habitats, have evolved a remarkable diversity of these water-repellent structures, offering unique insights into their evolutionary adaptations. However, systematic studies on the hydrophobic mechanisms and adaptive significance of ferns remain relatively scarce.
RATIONALE: To elucidate the structural basis, ecological relevance, and evolutionary trajectory of superhydrophobicity in ferns, we combined literature review and preliminary observations to select 25 fern species for detailed analysis of leaf epidermal micromorphology. Among these, 20 species exhibiting notable hydrophobic properties were further investigated to quantify their surface wettability and correlate microstructure with function. We integrated habitat preference data and phylogenetic reconstructions based on chloroplast genomes to examine the potential influence of environmental factors and evolutionary history on the development of hydrophobic traits.
RESULTS: Our investigations revealed that leaf hydrophobicity in ferns is closely associated with surface microstructure. Most hydrophobic species displayed hierarchical structures comprising polygonal protrusions and epicuticular wax crystals, with a contact angle exceeding 130° serving as a reliable indicator of significant hydrophobicity. Habitat correlation analysis indicated that species endemic to humid or water-rich environments exhibited more pronounced hydrophobic properties. Furthermore, phylogenetic reconstruction demonstrated that superhydrophobicity evolved independently multiple times during fern diversification, suggesting convergent adaptation to specific ecological conditions.
CONCLUSION: Leaf hydrophobicity in ferns correlates directly with surface microstructure complexity, exhibiting unique multi-scale hierarchical organizations. Notably, a contact angle exceeding 130° already induces distinct hydrophobicity in ferns-lower than the conventional 140° threshold for superhydrophobicity. Species from humid habitats show enhanced water repellency, supporting superhydrophobicity as an evolutionary adaptation to environmental constraints. These findings advance the theoretical framework for plant surface hydrophobicity and provide biomimetic design principles for functional materials. Future studies should examine wax chemistry, ecological factors, and soral structures to further elucidate hydrophobic mechanisms.

INTRODUCTION: The distribution and concentration of nutrient elements within fruits directly influence their yield and quality. Conventional analytical methods primarily rely on digestion to quantify nutrient content in different fruit tissues. However, as fruits comprise multiple tissue structures, basic quantification techniques fail to precisely resolve nutrient distribution across these microstructural compartments. Consequently, establishing an in-situ method enabling visualization of spatial nutrient concentration profiles in fruit tissues is imperative.
RATIONALE: Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS), as an advanced micro-analytical technique, enables effective visualization of in situ elemental distribution within plant tissues. LA-ICP-MS uses a femtosecond laser (wave-length 343 nm) to scan the fruit sample and the standard sample according to the line of the laser beam during the measurement of the femtosecond laser (wave-length 343 nm). The burned sample material is sent to the ICP with the carrier gas Ar, and the ionic strength of P³¹, K³⁹, Ca⁴⁴, Mg²⁴, Fe⁵⁷, Mn⁵⁵, Cu⁶³, Zn⁶⁶, S³⁴, Al²⁷, Cr⁵², Mo⁹⁵, Pb²⁰⁸, Sr⁸⁸ and Rb⁸⁵ in different parts of the fruit is detected. Visualize the nutrient distribution between different tissues.
RESULTS: Through LA-ICP-MS analysis, we performed in situ, spatially-resolved and semi-quantitative analysis of nutritional element distributions across distinct tissue structures in pomelo fruit during maturation. Significant tissue-specific heterogeneity was observed in elemental distributions among fruit compartments. Three structures demonstrated consistent nutrient enrichment: cuticle of flavedo, vascular bundle-like structures in albedo, and epidermal cells of juice sacs. The concentrations of potassium, nitrogen, calcium, magnesium and phosphorus were higher in the fruit of Wendan pomelo, which were mainly concentrated in the cuticle of flavedo, vascular bundle tissue of albedo and epidermal tissue of juice sac. Among them, the relative average concentration of calcium in the flavedo was as high as (516.21±28.96) mg·L^-1, and the relative average concentration of zinc in the cuticle of the flavedo was (410.22±16.43) mg·L^-1. The relative concentration of the two in the epidermal cells of the juice sac was the highest, suggesting that calcium and zinc play an important nutritional role in the fruit of Wendan pomelo.
CONCLUSION: This study established a novel LA-ICP-MS methodology for in-situ, visual and semi-quantitative analysis for nutrient distribution in different tissue structures of fruits, which was used to establish the spatial relationship of nutrient distribution, filling the gap in the visualization of fruit nutrient distribution. The relative concentration of different elements in each tissue was analyzed, which provided scientific theory for the subsequent guidance of Wendan pomelo farming measures.

INTRODUCTION: Ajania alabasica is a wild species of the genus Ajania in the Asteraceae that is characterized by its remarkable drought resistance and ecological adaptability, making it a valuable genetic resource for breeding. Nevertheless, the absence of an effective in vitro regeneration system has impeded the advancement of rapid propagation and genetic transformation studies on this species.
RATIONALE: The utilization of appropriate explants constitutes a critical strategy for plant regeneration. The core of this methodology lies in optimizing the combinations of plant growth regulators (PGRs) and additives to balance differentiation and physiological disorders such as hyperhydricity. This study was conceived with the objective of systematically determining the effects of different explant types (leaves vs. stem segments) and PGRs, with a view to establishing a robust regeneration protocol for A. alabasica.
RESULTS: This study found that leaf explants exhibited significantly higher regeneration potential than stem segments. The results demonstrated that the optimal medium for callus induction and adventitious bud differentiation from leaves was MS basal medium supplemented with 1.0 mg∙L^-1 6-BA and 1.0 mg∙L^-1 NAA. Following the culture period, this resulted in a callus induction rate of 100% (at 15 days) and an adventitious bud differentiation rate of 92.22% with a multiplication coefficient of 6.93 (at 30 days). To address hyperhydricity, the optimal proliferation medium was identified as containing 0.3 mg∙L^-1 6-BA and 0.05 mg∙L^-1 NAA, with 5 µmol∙L^-1 AgNO₃ proving effective for alleviation. For the rooting phase, the optimal medium was identified as 1/2MS medium supplemented with 0.2 mg∙L^-1 NAA, which resulted in a rooting rate of 100% and a transplant survival rate of 88%.
CONCLUSION: This study successfully established for the first time a highly efficient and stable regeneration system for A. alabasica using leaf explants. The developed system, characterized by its high differentiation and survival rates, provides crucial technical support for subsequent genetic transformation and gene function analysis.

INTRODUCTION: Ilex cornuta ‘Fortunei’ is widely used in landscape gardening and is a common sculptured tree species. An efficient and simple protoplast preparation and separation system is of great value for breeding and genetic research on woody plants. However, there are no relevant reports on the protoplast isolation and transformation system of I. cornuta ‘Fortunei’ thus far, making it necessary to carry out this study.
RATIONALE: Woody plants face challenges in protoplast isolation and transformation due to characteristics such as perennial stem lignification and leaf leatheriness. Differences in cell wall composition and tissue structure among species require the optimization of protoplast systems for specific species. To establish an efficient protoplast preparation and transient transformation system for I. cornuta ‘Fortunei’, orthogonal experiments and gradient experiments were used to explore factors affecting protoplast separation, and the PEG-mediated method was applied for transient transformation.
RESULTS: Compared with other enzyme combinations, the orthogonal test showed that the optimal enzyme concentration combination was 2.5% (w/v) cellulase, 1.0% (w/v) macerozyme, 0.5% (w/v) pectinase, and 0.4% (w/v) snailase. After 3.5 h of enzymolysis, the protoplast yield was the highest (1.18×10⁷∙g^-1 FW), with a viability of 68.30%. The suitable mannitol concentration for osmotic regulation was 0.30 mol·L^-1, and dark pretreatment of leaves for 12 h improved the effect. For transient transformation, 40% (w/v) PEG-4000 mediated the transformation of the pORE_R4-IcFPS2-GFP plasmid into protoplasts, and green fluorescent signals in the nucleus overlapping with DAPI blue fluorescent signals were observed under a laser confocal microscope, indicating successful transformation.
CONCLUSION: In conclusion, this study successfully established an efficient protoplast isolation system for I. cornuta ‘Fortunei’ with optimal conditions including specific enzyme combinations, enzymolysis times, osmotic regulators, and dark pretreatment. The PEG-mediated transient transformation system was also successfully constructed. These results provide a foundation for future protoplast fusion breeding and gene function research on I. cornuta.

Clathrin-mediated endocytosis (CME) is one of the primary pathways for the internalization of plasma membrane proteins in animal and plant cells. Clathrin and its adaptor protein complexes play crucial roles in plant morphogenesis, root gravitropic growth, halotropism response, immune response and nutrient elements transport. This review begins with an overview of clathrin and its mediated endocytosis in animal cells, briefly summarizing the composition, localization, and biological functions of plant clathrin and its adaptor protein complexes. It focuses on the regulatory mechanisms and biological roles of the CME pathway in plants, while also summarizing and prospecting the critical contributions of CME to plant growth, development, and responses to biotic and abiotic stresses. Furthermore, future research directions are also discussed, providing valuable references for in-depth exploration of the molecular mechanisms underlying the plant CME pathway.

CRISPR/Cas9 technology, as an efficient genome editing tool, has gained increasingly widespread application in the life sciences. Incorporating this technology into university experimental teaching enables students to deepen their understanding of cutting-edge research advances while enhancing their innovative research capabilities and practical skills. In this paper, we have designed an innovative teaching experiment, “Using CRISPR/Cas9 technology to study the effect of knocking out rice SSA1 (seedling stage albino1) gene on phenotype”. This experiment helps students to deepen their understanding of the principles and experimental procedures of CRISPR/Cas9 technology and to recognize the important role played by the SSA1 gene in rice growth and development. The experiment can also improve students’ ability of experimental operation and rigorous scientific research thinking, and cultivate students’ exploratory skills and innovative spirit.