Cold storage of amber-fleshed black plums triggers anthocyanin biosynthesis and accumulation in fruit flesh, resulting in flesh reddening under low-temperature conditions. bHLH transcription factors have potential functions in response to cold induction and anthocyanin biosynthesis. To investigate the role of bHLH genes, two amber-fleshed and blackskinned cultivars (Prunus salicina Lindl. ‘Friar' and ‘Angeleno') were selected as fruit materials to conduct transcriptomic and bioinformatics analyses. Differentially expressed bHLH genes were identified by RNA-seq and validated using qRTPCR. Ten bHLH genes (five upregulated, five downregulated) showed the most significant expression changes during cold- triggered flesh reddening. We characterized the protein structures of these 10 bHLH members and constructed a phylogenetic tree. Among them, the gene evm.TU.Chr8.2504 (the corresponding protein ID is evm.model.Chr8.2504), named PsbHLH3, was cold-responsive and contains MYB-binding sites. Further experiments involving transient overexpression were performed to validate the function of PsbHLH3 in facilitating anthocyanin biosynthesis. Yeast twohybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that PsbHLH3 interacts with PsMYB10.1. Dual-luciferase assay showed that PsbHLH3 promoted the activation of PsMYB10.1 on the promoter of PsUFGT, and consequently augmented the transcription of structural genes related to anthocyanin biosynthesis. The study reveals that cold storage upregulates PsbHLH3, which acts as a molecular switch governing cold-triggered flesh reddening in plum fruit. These findings clarify the regulatory function of PsbHLH3 in cold-triggered anthocyanin biosynthesis in postharvest plums.

Gibberellin 2-oxidases (GA2ox) play an important role in regulating the balance of bioactive gibberellins in plants, while their role in the drought response mechanism of grapes remains unclear. In this study, the subcellular localization analysis revealed that the VvGA2ox5 protein was predominantly localized to the cytoplasm and nucleus. Transient transformation experiments on ‘Pinot noir' grape leaves showed that overexpression of VvGA2ox5 reduced relative electrical conductivity (REC) and malondialdehyde (MDA) levels and increased proline content, antioxidant enzyme activity, and expression of drought-responsive genes. In contrast, virus-induced gene silencing (VIGS) silenced leaves showed the opposite results. Additionally, the overexpression of VvGA2ox5 in ‘Pinot noir' grape calli and Arabidopsis thaliana (Arabidopsis) further validated its positive function. In CRISPR-Cas9-edited grape calli, the results were opposite to those of overexpression lines. Meanwhile, yeast two-hybrid (Y2H) screening identified a drought-responsive protein, dehydration-induced 19 homolog 3 (VvDEH). RNA-seq analyses showed that overexpression of VvGA2ox5 significantly participates in the hormone signaling pathway. Accordingly, VvGA2ox5 is a crucial regulatory gene in enhancing drought tolerance in grapes and serves as a potential candidate gene for improving drought tolerance in plants. This finding offers significant theoretical support for drought tolerance breeding in grapes.

The tea plant (Camellia sinensis) is an economically important leaf crop in which the flowering process consumes substantial nutrients, thereby negatively impacting tea yield and quality. Therefore, deciphering the molecular basis of floral transition is essential for enhancing tea cultivars and optimizing plantation management. The non-flowering tea plant line ‘Ziyang 1' (ZY1H), which has failed to flower for several consecutive years, and the normal-flowering cultivar ‘Ziyang' (ZYQT) were used to study the molecular mechanisms underlying the non-flowering phenotype in ZY1H. Phenotypically, ZY1H exhibited shortened internodes, prolonged vegetative growth, and failure to develop floral meristems. Chromosomal analysis confirmed that ZY1H maintains a normal diploid chromosome number (2n=30), excluding triploidy as a causal factor for its non-flowering trait. Transcriptome analysis revealed an impaired vegetative-to-reproductive transition in ZY1H, manifesting as low expression of SPL genes and sustained high expression of flower-repressive AP2-like genes. Additionally, elevated expression of the flowering repressor SVP and reduced expression of the floral integrator FT further disrupted the convergence of floral inductive signals. Notably, brassinosteroid (BR) levels and CsBZR2 expression were elevated in ZY1H. Functional assays showed that CsBZR2 directly binds the CsFLC promoter and suppresses its expression, thereby blocking the flowering process. Collectively, these results demonstrate that disrupted BR signaling and sustained vegetative growth jointly trigger the floral transition defect in ZY1H. This work provides novel insights into the molecular mechanisms of flowering regulation in tea plants and valuable theoretical support for cultivar improvement.

Low temperature is involved in regulating plant growth, development, and quality formation.  The mechanism by which low temperature affects sucrose accumulation in oriental melon fruit is currently unclear.  Here, high-sucrose (HS) melons were used as the research materials and subjected to two temperature regimes of 30°C/18°C (day/night) and 22°C/10°C (day/night) at the onset of ethylene production.  Low temperature significantly inhibited ethylene release and sucrose accumulation in melon fruit, while ethephon treatment at low temperature partially restored the ethylene production and sucrose content.  Using yeast one-hybrid (Y1H), GUS activity analysis, and luciferase assay, we found that the transcription factor CmCBF3 could bind to CmACO1 (ACC oxidase 1) promoter and inhibit its activity, thereby suppressing ethylene production.  Overexpression of CmCBF3 under low temperature significantly inhibited the synthesis of ethylene and sucrose.  Further research showed that low temperature promoted CmERFV-2 expression, and CmERFV-2 could bind to CmCBF3 promoter to further inhibit ethylene synthesis.  In addition, CmMYB44, a transcription factor that negatively regulated fruit ethylene production and sucrose accumulation, repressed the expression of CmACO1 and CmSPS1 (sucrose phosphate synthase 1).  CmERFV-2 further affected the expression of CmACO1 and CmSPS1 by binding to CmMYB44 promoter, thereby regulating ethylene and sucrose content at low temperature.  In summary, this study revealed the mechanism by which CmERFV-2 affects ethylene release and sucrose accumulation in oriental melon fruit, laying a foundation for breeding high-quality melon varieties suitable for low-temperature cultivation.

Colletotrichum higginsianum, a plant pathogenic fungus threatening cruciferous crops, necessitates advanced genetic tools to study its pathogenesis and host interactions. However, selection markers available for fungal genome editing are often limited by scarcity and variable efficacy across species. Acetolactate synthase (ALS), a key enzyme in branched- chain amino acid (BCAA) biosynthesis, is the target of the herbicide chlorimuron ethyl (CE). Here, we utilized AlphaFold3- guided structural prediction to engineer ALS in C. higginsianum into a dual-functional endogenous selection marker. AlphaFold3-predicted ChALS structures revealed conserved catalytic regions and identified hydrophobic residues (V191, A200, F201) potentially critical for CE binding. Notably, introducing a single A200D substitution conferred the resistance of C. higginsianum to CE without impairing fungal growth, conidiation, or pathogenicity. Molecular docking simulations confirmed that the introduced aspartate side chain causes steric clashes that destabilize CE binding. Leveraging this mutation, we developed a homology-directed integration system enabling precise genetic tagging at the endogenous ChALS locus using the A200D allele for selection. Colletotrichum higginsianum strains expressing fluorescent proteins targeted to the nucleus, mitochondria, peroxisomes, or cytoplasm exhibited stable expression and unaltered infection dynamics in Chinese flowering cabbage (Brassica parachinensis). This work presents a dual-functional marker that addresses limitations in fungal genome editing tools. Moreover, the facility with which the A200D mutation confers resistance serves as a cautionary note on the potential evolutionary instability of single-target enzyme inhibitors in pathogen management.

Citrus Huanglongbing (HLB) has caused extensive damage to the global citrus industry. ‘Candidatus Liberibacter asiaticus' (CLas), the primary causal agent of HLB, utilizes effectors to modulate host defense responses, though the mechanisms of these effectors remain unclear. This study demonstrates that the citrus armadillo (ARM)-repeated protein CsARM26 interacted with CLIBASIA_00185 (CLas0185) in vivo and in vitro. CLas0185 enhanced the abundance of CsARM26, whereas CsARM26 destabilized the effector. Additionally, the transient co-expression of CLas0185 and CsARM26 facilitated infection by Xanthomonas citri subsp. citri. Moreover, transgenic CsARM26 citrus plants suppressed the accumulation of free salicylic acid (SA) and the expression of SA-associated genes. This study reveals that an ARM-repeated protein plays a role in the immune response to the CLas–citrus interaction, establishing a foundation for further investigation of the molecular mechanisms of CLas infection.

The oriental fruit moth, Grapholita molesta  Busck, is a major pest of fruit trees worldwide, it is necessary to develop green control technology instead of the traditional chemical pesticide. Although the sterile insect technique (SIT) is a safe and effective method for controlling G . molesta , the high cost of mass rearing irradiated pupae hinders its widespread adoption. In this study, we identified the optimal sub-sterilizing X-ray dose for G . molesta  adults and evaluated its effects on the mating competitiveness of males, growth and reproduction of the F 1   generation population, and the sterility rate of field- caught males. The results showed that the sub-sterilizing dose of 1-day-old male was 130 Gy, which rendered most males sterile (76.34%) and females nearly sterile (97.41%). Moreover, the sterile trait of the male parents that had been irradiated with 130 Gy of X-rays was successfully transmitted to the F 1   generation, whose sterility rate (84.13–93.61%) exceeded that of F 0   males. The F 1   generation population exhibited significantly lower values for the net reproductive rate (R 0 ), intrinsic rate of increase (r ), and finite rate of increase (λ ) compared to the non-irradiated group. Furthermore, the competitive mating index and sterility rate of irradiated males were the highest when they were paired with unirradiated males and females at a release ratio of 20:1:1. The irradiating wild males captured by sex pheromones in peach orchards with the sub-sterilizing dose achieved a 75.69% sterility rate, comparable to the laboratory-reared population. This study introduces a novel X-ray irradiation method for adult G . molesta  captured in the orchard field and lays a theoretical foundation for promoting the wide application of the SIT.

The lack of knowledge regarding how the genome is regulated during skeletal myogenesis in pigs hinders the identification of genetic variants for meat traits. Here, we systematically characterized the cis-regulatory elements (CREs, including promoters, enhancers, and others) by using Hi-C coupled chromatin cleavage and tagmentation (HiCuT, H3K27ac), assay for transposase-accessible chromatin using sequencing (ATAC-seq), and RNA-seq to analyze primary myoblasts and in vitro well-differentiated myotubes, and generated an atlas of promoter-enhancer interactions (PEIs) during myogenic differentiation. In total, 179,895 and 159,255 loops were identified in myoblasts and myotubes, respectively, which could be grouped into 3 categories: the interactions between promoter and enhancer (P–E), between promoter and promoter (P–P), and between other genomic regions (O–O). Furthermore, 22,645 cis-expression quantitative trait loci (eQTLs) were pinpointed by integrating public genome-wide association studies (GWAS) and eQTL datasets. Notably, novel promoter-like enhancers were verified, and cis-eQTLs in promoter-like enhancers might influence gene expression over long ranges. In addition, 39,069 structural variants (SVs) within the CREs were identified. A transcription factor (TF)-promoter/enhancer regulatory network for myogenesis was generated, revealing several novel TFs relevant to myogenic differentiation. This work provides a valuable resource for understanding genomic regulation in pig muscles and filtering potential variants to develop breeds with desirable traits.

Understanding the genetic changes behind the phenotypic variation of Chinese donkeys is helpful to the genetic improvement and breeding of donkeys. However, the population structure and novel genes associated with morphological (coat color and body size) and adaptive (high-altitude adaptation) traits of Chinese donkeys remain largely unknown. Here, we analyzed 391 whole-genome sequencing (WGS) data of Chinese donkeys. Population genomic analyses showed that Chinese native donkey breeds mainly consist of three distinct populations (Southwest plateau, North plain, and Guanzhong plain), and a newly discovered population (Guanzhong plain) was identified. Moreover, we characterized a high-confidence list of 127, 117, and 169 selective signal genes for coat color, body size, and high-altitude adaptation, respectively. We discovered ARID3B gene with strong signals of selection, which may account for coat color in Chinese donkeys. Our study identified EPAS1 as a high-altitude adaptive gene. However, the FAM184B gene shows a stronger signal in response to high-altitude environments in Chinese donkeys. The selective sweep and GWAS analysis showed that LCORL and TMEM154 genes are potentially associated with body size in Chinese donkeys. Utilizing PacBio HiFi sequencing data, this study presents 15,954 highly reliable structural variations (SVs) between large-sized and small-sized donkeys. Utilizing SV data and a graph-based method, we identified an 880-bp deletion in the TMEM154 gene in Sichuan donkeys (small-sized) compared to Guanzhong donkeys (large-sized), which was verified by PCR and is a candidate SV related to body size. Transcriptome sequencing data showed that the TMEM154 gene is highly expressed in the muscle of Guanzhong donkeys (large-sized) compared to Sichuan donkeys (small-sized). Multi-species alignment analysis revealed that the region surrounding the 880-bp deletion in the TMEM154 gene region is conserved in horse, zebra, kiang, as well as two large-sized donkey breeds (Dezhou and Guanzhong), except in in the small-sized Sichuan donkey. Furthermore, after the 880 bp deletion was transfected into 3T3-L1 and HEK293T cells, it was demonstrated that the relative luciferase activity of the mutation was markedly decreased in comparison with that of the wild type. These results suggest that this 880-bp deletion in the TMEM154 gene may play an important role in body size trait of donkey. This study provides valuable genome resources for donkey breeding and sheds light on the domestication history of Chinese donkeys.

Grassland resources are the foundation of the sheep industry, and the development of artificial pastures provides a solution for alleviating the livestock-carrying pressure on natural grasslands. Based on the impact of lipid metabolism on muscle fatty acid composition, studying the differences in rumen and liver lipid metabolism between grazing and indoor feeding lambs helps to analyze the causes of high-quality grazing lamb meat and to improve the productivity of artificial pasture-based grazing systems. A total of 22 weaned lambs with similar weights were assigned to two groups: one fed pellets and hay in separate pens (AF) and the other grazing exclusively on artificial pasture (AG) for 90 d. The results showed significantly higher serum glucose and triglyceride concentrations and significantly lower serum lipase activity in the AF vs. AG group (P<0.05). The metabolome revealed that crucial differential metabolites (DFMs) participating in lipid metabolism, oleic acid and palmitic acid, were down-regulated in the rumen of the AG group. In the liver, oleic acid, α-linolenic acid, and stearic acid were down-regulated in the AG group, while linoleic acid was up-regulated. Meanwhile, rumen hydroxypropanoate and liver succinic acid, enriched in propionate metabolism processes, were down-regulated in grazing lambs. Subsequently, liver transcriptome sequencing revealed that grazing inhibited fatty acid oxidation and lipid synthesis by down-regulating Malic enzyme 1 (ME1), fatty acid-binding protein 1 (FABP1), hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA), Perilipin-2 (PLIN2), and acetyl CoA acyltransferase 2 (ACAA2). The significant correlation between CYP4A6 and propionate metabolites suggested a potential regulatory role of propionate metabolism in liver lipid metabolism. Moreover, oleic acid, palmitic acid, and stearic acid in muscle, rumen, and liver were closely correlated, revealing an essential contribution of rumen and liver lipid metabolism to muscle fatty acid deposition. Through the multi-omics assessment of the rumen and liver, this study systematically revealed the lipid metabolism mechanisms in lambs under different feeding patterns, which are influenced by comprehensive factors such as diet and environment. These findings contribute to the goals of high quality grazing lamb production and efficient grassland productivity.