Salt stress has a major effect on the quality and yield of crops, and many transcription factors (TFs), such as WRKY, NAC, and ERF TFs have been shown to participate in the regulation of salt stress. Squamosa promoter binding protein-like (SPL) TFs play a role in plant floral organ development, metal ion responses and disease resistance. However, the precise function of SPL TFs in regulating the salt stress in plants remains unclear. In this study, we investigated the mechanism by which SPL TFs regulate salt stress in apple (Malus domestica). Overexpression of MdSPL13B in apple calli revealed that it positively regulated salt stress. The heterologous transformation of MdSPL3B also resulted in significant salt tolerance in Arabidopsis and tomato. Subsequently, a yeast two-hybrid assay showed that MdSPL13B could interact with MdNAC29 and MdPYL4, which was further confirmed by the bimolecular fluorescence complementation assay. Additionally, overexpression of MdNAC29 and MdPYL4 in apple calli, Arabidopsis and Nicotiana benthamiana plants indicated that MdNAC29 negatively regulated salt tolerance and that MdPYL4 positively regulated salt tolerance. Furthermore, the yeast one-hybrid, electrophoretic mobility shift assay and dual-luciferase assay showed that MdSPL13B could enhance salt tolerance by activating MdWRKY100 in apple. In conclusion, our findings enhance the understanding of how MdSPL3B and its target gene regulate salt tolerance in apple, and have implications for the breeding of salt-tolerant apple.

The CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptides are critical for stem cell homeostasis in plant shoot and root apical meristem. Although CLE genes have been reported in numerous plants, there is limited information for apple. Here, twenty-five MdCLE genes were identified from apple genome (Apple Genome V1.0 predicted peptides). Analysis of chromosomal location showed that the 25 MdCLE genes were located on 12 of 17 apple chromosomes. Genetic structure analysis showed that 21 of 25 the MdCLE genes were intron-free. Expression patterns showed that most of the MdCLE genes showed higher expression levels in leaves and root compared with the other tissues. In addition, expression analysis demonstrated that MdCLE genes had different gene expression patterns for abiotic stress treatment, suggesting their potential roles in acclimation of apple to adverse environments. The 25 MdCLE genes encoded 19 different CLE peptides, and they were divided into two groups depending on their effects on the inhibition of Arabidopsis root growth. Our results suggest that MdCLE genes may have an important but redundant function in regulating plant growth and development, and this work provides valuable references for further investigation of the biological functions of MdCLE genes.