Carotenoid cleavage dioxygenase (CCD) is the key enzyme for carotenoid cleavage, and the products of carotenoid cleavage regulate the ability of plants to stress. In this paper, six CCD genes were obtained from Morus notabilis (Mn) by reverse transcription-polymerase chain reaction (RT-PCR) and we classified them into three subgroups based on gene structures and phylogenetic analysis. The CDS (coding sequence) regions of the six MnCCD genes were 1617, 1620, 1635, 1713, 1746, and 1791bp in full length, encoding 538, 539, 544, 570, 581, and 596 amino acids, respectively. Then, PcoldTF-MnCCD plasmids were constructed and independently transferred into E. coli BL21 (DE3), and the MnCCD proteins were successfully expressed by prokaryotic expression with an expected molecular weight of recombinant proteins (120kDa) and high solubility. These results will lay a foundation for the identification of mulberry carotenoid products.

As an economically important tree species, mulberry (Morus spp.) has exhibited a remarkable tolerance for salinity, drought and heavy metals. However, the precise mechanism of metabolome‐mediated drought adaptation is unclear. In this study, two new mulberry varieties—‘drought‐sensitive guisangyou62 (GSY62) and highly drought‐tolerant guiyou2024 (GY2024)’—after three days (62F or 2024F) and six days (62B or 2024B) of drought–stress conditions were subjected to transcriptome and metabolome analyses. The enrichment analysis demonstrated that the differentially expressed genes (DEGs) were mainly enriched in carbohydrate metabolism, amino acid metabolism, energy metabolism and secondary metabolite biosynthesis under drought–stress conditions. Notably, compared with the CK group (without drought treatment), 60 and 70 DEGs in GY2024 and GSY62 were involved in sucrose and starch biosynthesis, respectively. The genes encoding sucrose phosphate synthase 2 and 4 were downregulated in GY2024, with a lower expression. The genes encoding key enzymes in starch biosynthesis were upregulated in GY2024 and the transcriptional abundance was significantly higher than in GSY62. These results indicated that drought stress reduced sucrose synthesis but accelerated starch synthesis in mulberry.

There are abundant mulberry germplasm resources in China. Generally, in order to better utilize these resources, a comprehensive evaluation is conducted, among which the evaluation of photosynthetic capacity is an important aspect. To evaluate the photosynthetic characteristics of mulberry trees with different ploidy levels, which determine the number of chromosome sets in a cell or organism, we compared the microstructural features and transcriptomes of triploid and diploid mulberry trees. Haploid (n) means having one set of chromosomes, diploid (2n) has two sets, triploid (3n) has three sets, and so on. We compared the microstructural features and transcriptomes of triploid and diploid mulberry trees. In this study, the photosynthetic rates (P_n) of ‘guisang6hao’ (hereinafter referred to as GS6, 2n = 3x = 42) were 1.2 times that of ‘guisangyou12’(hereinafter referred to as GSY12, 2n = 2x = 28). Here, GS6 and GSY12 are abbreviations for the two mulberry varieties. The leaf thickness, main vein thickness, epidermal thickness, palisade tissue thickness, spongy tissue thickness and lower epidermal stomatal density of GS6 were greater than those of GSY12. Additionally, the transcriptome characteristics of GS6 and GSY12 were characterised by Illumina Novaseq 6000 and the differentially expressed genes (DEGs) were significantly enriched in photosynthesis. Four differentially expressed metabolites (DEMs) related to photosynthesis were identified, playing key roles in chlorophyll metabolism and carbon fixation in photosynthetic organisms. The above results indicated that an increase in the ploidy level enhanced the photosynthetic capacity and utilisation of light energy, as well as the accumulation of chlorophyll content, of triploid mulberry trees. This study provides a technical and theoretical basis for germplasm innovation and the genetic improvement of mulberry.

Our previous experiments confirmed that two mulberry spermidine synthase (SPDS) genes (MnSPDS1 and MnSPDS2) that encode functional proteins are highly expressed under drought stress. In this study, the functions of MnSPDS1/MnSPDS2 in the drought stress response were further explored by silencing and overexpressing these genes in mulberry and tobacco, respectively. Compared with the wild-type (WT) plants, the MnSPDS1/MnSPDS2-overexpression tobacco plants were more tolerant to drought stress and showed a higher spermidine content (P < 0.05). Moreover, overexpression of MnSPDS1/MnSPDS2 at the physiological level alleviated membrane damage caused by drought and improved osmotic regulation and antioxidant capacity. In addition, correlation analysis showed that the content of spermidine was positively correlated with the expression levels of MnSPDS1 and MnSPDS2, with correlation coefficients of 0.762 and 0.715, respectively. Moreover, drought injury was more serious in the MnSPDS-silenced seedlings than in the WT seedlings after drought treatment. These results suggest that MnSPDS genes play important roles in the drought stress response and are valuable for molecular breeding to enhance the drought tolerance of mulberry.

Introduction: Soil salinization severely threatens crop productivity and agricultural sustainability. Mulberry ( Morus notabilis ) is an economically important woody crop, but the molecular mechanisms of its salt tolerance remain unclear. The conserved Salt Overly Sensitive 3 ( SOS3 ) gene regulates ion homeostasis under salt stress, making it a key target for exploring mulberry’s salt adaptation. Methods: We cloned MnSOS3 from M. notabilis , constructed a prokaryotic expression vector (pCold-TF- MnSOS3 ) for transformation into Escherichia coli BL21, and generated MnSOS3 -overexpressing transgenic tobacco ( Nicotiana benthamiana ) via Agrobacterium -mediated transformation. Salt tolerance was evaluated in E. coli (0.6 M NaCl) and tobacco (300 mM NaCl), with Na⁺/K⁺ content quantification in tobacco leaves (250 mM NaCl, 24 h) and verification of MnSOS3-MnSOS2 interaction via yeast two-hybrid assay. Results and discussion: Recombinant E. coli expressing MnSOS3 showed enhanced growth (maximum OD₆₀₀ = 0.338) vs. near-zero growth in the control. Transgenic tobacco line MnSOS3 -2 (highest expression) exhibited 33.3% survival under 300 mM NaCl, 2.5-fold higher than wild-type (WT, 13.3%). Transgenic leaves accumulated 54.8% less Na⁺ (0.95 ± 0.02 vs. 2.10 ± 0.07 mmol/g FW) and 45.8% more K⁺ (1.75 ± 0.02 vs. 1.20 ± 0.02 mmol/g FW) than WT, leading to a 67.8% lower Na⁺/K⁺ ratio (0.55 ± 0.01 vs. 1.71 ± 0.08). The yeast two-hybrid assay confirmed MnSOS3-MnSOS2 interaction. These findings show MnSOS3 boosts salt tolerance via Na⁺/K⁺ homeostasis and SOS pathway activation, a key resource for salt-tolerant mulberry breeding.