Drought is a major environmental factor that significantly limits crop yield and quality worldwide. Basic helix-loop-helix (bHLH) transcription factors have been reported to participate in the regulation of various abiotic stresses. In this study, a bHLH transcription factor in apple, MdbHLH130, which contains a highly conserved bHLH domain, was isolated and characterized. qRT-PCR and P_MdbHLH130::GUS analyses showed that MdbHLH130 was notably induced in response to dehydration stress. Compared with the wild-type (WT), transgenic apple calli overexpressing MdbHLH130 displayed greater resistance to PEG6000 treatment. In contrast, the MdbHLH130-Anti lines were more sensitive to PEG6000 treatment than WT. Moreover, ectopic expression of MdbHLH130 in tobacco improved tolerance to water deficit stress, and plants exhibited higher germination rates and survival rates, longer roots, and lower ABA-induced stomatal closure and leaf water loss than the WT control. Furthermore, overexpression of MdbHLH130 in tobacco also led to lower electrolyte leakage, malondialdehyde contents, and reactive oxygen species (ROS) accumulation and upregulation of the expression of some ROS-scavenging and stress-responsive genes under water deficit stress. In addition, MdbHLH130 transgenic tobacco plants exhibited improved tolerance to oxidative stress compared with WT. In conclusion, these results indicate that MdbHLH130 acts as a positive regulator of water stress responses through modulating stomatal closure and ROS-scavenging in tobacco.

Deciphering the mechanism of malate accumulation in apple (Malus domestica Borkh.) fruits can help to improve their flavor quality and enhance their benefits for human health. Here, we analyzed malate content as a quantitative trait that is determined mainly by genetic effects. In a previous study, we identified an R2R3−MYB transcription factor named MdMYB44 that was a candidate gene in qtl08.1 (quantitative trait locus mapped to chromosome 8) of fruit malate content. In the present study, we established that MdMYB44 negatively regulates fruit malate accumulation by repressing the promoter activity of the malate-associated genes Ma1 (Al-Activated Malate Transporter 9), Ma10 (P-type ATPase 10), MdVHA-A3 (V-type ATPase A3), and MdVHA-D2 (V-type ATPase D2). Two single-nucleotide polymorphisms (SNPs) in the MdMYB44 promoter, SNP A/G and SNP T/−, were experimentally shown to associate with fruit malate content. The TATA-box in the MdMYB44 promoter in the presence of SNP A enhances the basal activity of the MdMYB44 promoter. The binding of a basic-helix–loop–helix transcription factor MdbHLH49 to the MdMYB44 promoter was enhanced by the presence of SNP T, leading to increased MdMYB44 transcript levels and reduced malate accumulation. Furthermore, MdbHLH49 interacts with MdMYB44 and enhances MdMYB44 activity. The two SNPs could be used in combination to select for sour or non-sour apples, providing a valuable tool for the selection of fruit acidity by the apple breeding industry. This research is important for understanding the complex molecular mechanisms of fruit malate accumulation and accelerating the development of germplasm innovation in apple species and cultivars.

Salinity is a severe abiotic stress that limits plant survival, growth, and development. 14-3-3 proteins are phosphopeptide-binding proteins that are involved in numerous signaling pathways, such as metabolism, development, and stress responses. However, their roles in salt tolerance are unclear in woody plants. Here, we characterized an apple (Malus domestica) 14-3-3 gene, GENERAL REGULATORY FACTOR 8 (MdGRF8), the product of which promotes salinity tolerance. MdGRF8 overexpression improved salt tolerance in apple plants, whereas MdGRF8-RNA interference weakened it. Yeast two-hybrid, bimolecular fluorescence complementation, pull-down, and co-immunoprecipitation assays revealed that MdGRF8 interacts with the transcription factor MdWRKY18. As with MdGRF8, overexpressing MdWRKY18 enhanced salt tolerance in apple plants, whereas silencing MdWRKY18 had the opposite effect. We also determined that MdWRKY18 binds to the promoters of the salt-related genes SALT OVERLY SENSITIVE 2 (MdSOS2) and MdSOS3. Moreover, we showed that the 14-3-3 protein MdGRF8 binds to the phosphorylated form of MdWRKY18, enhancing its stability and transcriptional activation activity. Our findings reveal a regulatory mechanism by the MdGRF8–MdWRKY18 module for promoting the salinity stress response in apple.

The most direct technique for studying calcium, which is an essential element for pollen tube growth, is Ca²⁺ imaging. Because membranes are relatively impermeable, the loading of fluorescent Ca²⁺ probes into plant cells is a challenging task. Thus, we have developed a new method of loading fluo-4 acetoxymethyl ester into cells that uses a cell lysis solution to improve the introduction of this fluorescent dye into pollen tubes. Using this method, the loading times were reduced to 15 min. Furthermore, loading did not have to be performed at low (4°C) temperatures and was successful at room temperature, and pluronic F-127 was not required, which would theoretically allow for the loading of an unlimited number of cells. Moreover, the method can also be used to fluorescently stain root hairs.

The 14-3-3 (GRF, general regulatory factor) regulatory proteins are highly conserved and are widely distributed throughout the eukaryotes. They are involved in the growth and development of organisms via target protein interactions. Although many plant 14-3-3 proteins were identified in response to stresses, little is known about their involvement in salt tolerance in apples. In our study, nineteen apple 14-3-3 proteins were cloned and identified. The transcript levels of Md14-3-3 genes were either up or down-regulated in response to salinity treatments. Specifically, the transcript level of MdGRF6 (a member of the Md14-3-3 genes family) decreased due to salt stress treatment. The phenotypes of transgenic tobacco lines and wild-type (WT) did not affect plant growth under normal conditions. However, the germination rate and salt tolerance of transgenic tobacco was lower compared to the WT. Transgenic tobacco demonstrated decreased salt tolerance. The transgenic apple calli overexpressing MdGRF6 exhibited greater sensitivity to salt stress compared to the WT plants, whereas the MdGRF6-RNAi transgenic apple calli improved salt stress tolerance. Moreover, the salt stress-related genes (MdSOS2, MdSOS3, MdNHX1, MdATK2/3, MdCBL-1, MdMYB46, MdWRKY30, and MdHB-7) were more strongly down-regulated in MdGRF6-OE transgenic apple calli lines than in the WT when subjected to salt stress treatment. Taken together, these results provide new insights into the roles of 14-3-3 protein MdGRF6 in modulating salt responses in plants.