Cold stress limits plant growth, development and yields, and the C-repeat binding factors (CBFs) function in the cold resistance in plants. However, how pomegranate CBF transcription factors respond to cold signal remains unclear. Considering the significantly up-regulated expression of PgCBF3 and PgCBF7 in cold-tolerant Punica granatum ‘Yudazi’ in comparison with cold-sensitive ‘Tunisia’ under 4 °C, the present study focused on the two CBF genes. PgCBF3 was localized in the nucleus, while PgCBF7 was localized in the cell membrane, cytoplasm, and nucleus, both owning transcriptional activation activity in yeast. Yeast one-hybrid and dual-luciferase reporter assay further confirmed that PgICE1 could specifically bind to and significantly enhance the activation activity of the promoters of PgCBF3 and PgCBF7. Compared with the wild-type plants, the PgCBF3 and PgCBF7 transgenic Arabidopsis thaliana lines had the higher survival rate after cold treatment; exhibited increased the contents of soluble sugar and proline, while lower electrolyte leakage, malondialdehyde content, and reactive oxygen species production, accompanying with elevated enzyme activity of catalase, peroxidase, and superoxide dismutase; and upregulated the expression of AtCOR15A, AtCOR47, AtRD29A, and AtKIN1. Collectively, PgCBFs were positively regulated by the upstream PgICE1 and mediated the downstream COR genes expression, thereby enhancing freezing tolerance.

Peach is an important fruit tree worldwide. Sucrose transporters (SUTs) have been extensively studied in relation to plant growth, development, and stress responses. In this study, we cloned a putative SUT, PpSUT2, from the peach cultivar ‘New Kawanakajima.’ Bioinformatics analysis predicted that the protein had functional domains typical of the GPH_sucrose family. Phylogenetic analysis revealed high sequence similarity between PpSUT2 and SUTs from other plant species. The qRT-PCR suggested that PpSUT2 was expressed concurrently in the petals, sepals, pistils, stamen, phloem, and leaves. Different trends and different relative expression levels were observed during the flowering and fruit development periods. Western blot analysis produced similar results. The heterologous expression of SUTs in the defective yeast SUSY7/ura3 showed that PpSUT2 had a complementary function on SD sucrose medium. This indicated that it had sucrose transport activity and might be a signal protein. Then, 11PpSUT2 transgenic tobacco plants were obtained.After exposure to low temperature and drought stress conditions, the transgenic overexpression plants exhibited increased proline and soluble sugar content, reduced malondialdehyde (MDA) content and reduced relative conductivity, and less membrane injury. These results suggest that the overexpression of PpSUT2 in tobacco promoted greater resistance to cold and drought.

Walnut is an important nut with a rich nutritional profile and associated health benefits for the human body. B-box (BBX) proteins containing one or two BBX motifs play pivotal roles in plant growth and developmental processes; nevertheless, the functions of JrBBXs in walnut anthocyanin biosynthesis remain inadequately understood. In this study, 39 JrBBXs in red walnut ‘RW-1’ were identified, with phylogenetic analysis suggesting that they were divided into six classes based on the distribution of conserved domains and unevenly distributed on 14 chromosomes. Promoter analysis demonstrated that JrBBX promoters possessed an abundance of light responsiveness elements, ABA responsiveness elements, MYB binding sites and MYC binding sites. The transcriptome analysis results demonstrated that eight JrBBXs were differently expressed in normal green walnut ‘Zhonglin 1’ and red walnut ‘RW-1’ seed coats. Furthermore, qRT-PCR (quantitative real-time polymerase chain reaction) analysis showed that JrBBX3 exhibited lower expression during seed coat development in ‘RW-1’. Y1H (Yeast One-Hybrid) and LUC (dual-luciferase reporter) assays revealed that JrBBX3 directly inhibited the expression of JrUFGT5, considered a key anthocyanin biosynthesis structural gene in research. Subcellular localization analysis indicated both cytoplasmic and nuclear localization of JrBBX3. Transient overexpression of JrBBX3 in walnut leaves resulted in reduced JrUFGT5 expression and anthocyanin accumulation. Collectively, these findings revealed the negative regulation of JrBBX3 in red walnut anthocyanin biosynthesis, and provided a basis to further study the anthocyanin biosynthesis mechanism of red walnut.

Fruit softening, an irreversible ripening process that causes postharvest losses, is mainly attributed to cell wall disassembly, rendering cell-wall-modifying genes critical targets for genetic improvement. However, the molecular mechanism by which expansins loosen the cell wall via the nonenzymatic mechanism, thereby affecting fruit softening, remains largely unknown. In this study, HSM (melting peach, MF) fruits exhibited a rapid decline in firmness, accompanied by more extensive cell wall disassembly and larger intercellular spaces compared with CN14 (non-melting peach, NMF) during the fruit development process. The expression of PpEXPA13, an expansin gene, was significantly higher in HSM than in CN14 during fruit softening. Virus-induced gene silencing in peach delayed firmness loss, while the overexpression of PpEXPA13 in tomato accelerated it. PpEXPA13-OE fruits displayed enlarged intercellular spaces and upregulated expression of multiple cell-wall-modifying genes. Furthermore, a yeast one-hybrid assay identified the transcription factor PpMYC2 as an upstream regulator of PpEXPA13. PpMYC2 specifically binds to the MYC box (CACGTG) in the PpEXPA13 promoter. These findings reveal that PpMYC2 activates PpEXPA13 expression, which might lead to cell wall disassembly and promote peach fruit softening.

The characteristic red walnut germplasm ‘RW-1′ (Juglans regia L.) exhibits a stable red seed coat phenotype due to the abundant accumulation of anthocyanins and proanthocyanidins (PAs). The regulatory mechanisms underlying PA biosynthesis in red walnut remain poorly studied, which hinders the improvement of walnut color quality. Leucoanthocyanidin reductase (LAR) is a key enzyme in the PA metabolic pathway, while its function in red walnut remains unclear. Here, the leucoanthocyanidin reductase gene JrLAR1, whose expression pattern is consistent with the accumulation trend of PAs, was cloned from the seed coats of red walnut ‘RW-1′, and its function in PA biosynthesis was verified via heterologous overexpression in Arabidopsis thaliana, a well-recognized cross-species LAR functional validation system free of endogenous LAR interference due to absent native homologs. The results showed that the full-length coding sequence (CDS) of JrLAR1 gene is 1104 bp, encoding a 367-amino-acid protein belonging to the NADB_Rossmann superfamily, and the protein shares an extremely high sequence similarity with grape VvLAR2. Heterologous overexpression of JrLAR1 significantly increased total PA content in the leaves and seeds of A. thaliana. Integrated transcriptomic and metabolomic analyses further revealed that JrLAR1 overexpression markedly upregulated the core genes involved in PA metabolism and the transcription factor GL3 in A. thaliana, specifically induced (+)-catechin synthesis, and ultimately promoted the significant accumulation of procyanidin B3. In addition, a set of antioxidant enzyme-encoding genes were substantially upregulated in JrLAR1-overexpressing A. thaliana lines. Yeast one-hybrid and dual-luciferase reporter assays demonstrated that JrEGL1b, a homolog of A. thaliana GL3, can bind to the promoter region of JrLAR1 gene and significantly enhance its transcriptional activity. Transient overexpression of JrLAR1 or JrEGL1b in red walnut leaves significantly promoted PA accumulation, and JrEGL1b overexpression notably upregulated JrLAR1 expression. In conclusion, JrLAR1 plays a crucial role in PA biosynthesis in red walnut and is positively regulated by the transcription factor JrEGL1b. These findings improve the molecular regulatory network of pigment metabolism in red walnut and provide valuable molecular targets for the quality improvement and directional breeding of walnuts.