Zinc finger proteins are widely involved and play an important role in plant growth and abiotic stress. In this research, MdZAT5, a gene encoding C2H2-type zinc finger protein, was cloned and investigated. The MdZAT5 was highly expressed in flower tissues by qRT-PCR analyses and GUS staining. Promoter analysis showed that MdZAT5 contained multiple response elements, and the expression levels of MdZAT5 were induced by various abiotic stress treatments. Overexpression of MdZAT5 in apple calli positively regulated anthocyanin accumulation by activating the expressions of anthocyanin biosynthesis-related genes. Overexpression of MdZAT5 in Arabidopsis also enhanced the accumulation of anthocyanin. In addition, MdZAT5 increased the sensitivity to salt stress in apple calli. Ectopic expression of MdZAT5 in Arabidopsis reduced the expression of salt-stress-related genes (AtNHX1 and AtABI1) and improved the sensitivity to salt stress. In conclusion, these results suggest that MdZAT5 plays a positive regulatory role in anthocyanin accumulation and negatively regulates salt resistance.

Phytochrome-interacting factors (PIFs), members of the basic helix-loop-helix transcription factor family that have been extensively investigated in Arabidopsis thaliana, play essential roles in plant growth and development. However, PIF members have not been systematically investigated in apples, a worldwide perennial woody crop of economic importance. Here, seven PIF genes were identified from the Malus × domestica reference genome. Chromosomal locations, gene structures, and phylogenetic relationships of these members were analyzed. Analysis of cis-acting elements in promoter regions of MdPIF genes indicated that various elements were related to light, abiotic stress, and plant hormone responsiveness. Subsequently, subcellular localization and transcriptional activity analysis revealed that MdPIFs were typical nuclear transcription factors with transcriptional activation ability. Expression analysis demonstrated that MdPIF genes had different gene expression patterns for various abiotic factors. Moreover, overexpressed MdPIF4 reduced the sensitivity of apple calluses to abscisic acid (ABA). Our work lays foundations for further investigation of PIF functions in plant growth and development in apples.

Malus baccata, a valuable germplasm resource in the genus Malus, is indigenous to China and widely distributed. However, little is known about the lineage composition and genetic basis of ‘ZA’, a mutant type of M. baccata. In this study, we compared the differences between ‘ZA’ and wild type from the perspective of morphology and ultrastructure and analyzed their chloroplast pigment content based on biochemical methods. Further, the complete mitogenome of M. baccata ‘ZA’ was assembled and obtained by next-generation sequencing. Subsequently, its molecular characteristics were analyzed using Geneious, MISA-web, and CodonW toolkits. Furthermore, by examining 106 Malus germplasms and 42 Rosaceae species, we deduced and elucidated the evolutionary position of M. baccata ‘ZA’, as well as interspecific variations among different individuals. In comparison, the total length of the ‘ZA’ mitogenome (GC content: 45.4%) is 374,023 bp, which is approximately 2.33 times larger than the size (160,202 bp) of the plastome (GC: 36.5%). The collinear analysis results revealed abundant repeats and genome rearrangements occurring between different Malus species. Additionally, we identified 14 plastid-driven fragment transfer events. A total of 54 genes have been annotated in the ‘ZA’ mitogenome, including 35 protein-coding genes, 16 tRNAs, and three rRNAs. By calculating nucleotide polymorphisms and selection pressure for 24 shared core mitochondrial CDSs from 42 Rosaceae species (including ‘ZA’), we observed that the nad3 gene exhibited minimal variation, while nad4L appeared to be evolving rapidly. Population genetics analysis detected a total of 1578 high-quality variants (1424 SNPs, 60 insertions, and 94 deletions; variation rate: 1/237) among samples from 106 Malus individuals. Furthermore, by constructing phylogenetic trees based on both Malus and Rosaceae taxa datasets, it was preliminarily demonstrated that ‘ZA’ is closely related to M. baccata, M. sieversii, and other proximate species in terms of evolution. The sequencing data obtained in this study, along with our findings, contribute to expanding the mitogenomic resources available for Rosaceae research. They also hold reference significance for molecular identification studies as well as conservation and breeding efforts focused on excellent germplasms.

The genus Malus is rich in species and many of its plastid genomes have been released. However, limited resources and few markers are not conducive to the comparison of differences among species and resource identification and evaluation. In this study, the complete chloroplast genome of Malus zhaojiaoensis was studied by NGS sequencing, with a total length of 159998 bp. It consists of four regions, LSC (88,070 bp), IRB (26,359 bp), SSC (19,210 bp) and IRA (26,359 bp). M. zhaojiaoensis cp genome contained a total of 111 genes made up of three classes: 78 coding sequences, 29 tRNA genes, and four rRNA genes. In addition, a total of 91 SSRs and 43 INEs were found in the M. zhaojiaoensis cp genome, which was slightly different from M. baccata and M. hupehensis in number. The analysis of codon usage and RNA editing showed that high-frequency codons tended to end at A/U bases and RNA editing mainly occurred at the second codon. Comparative genome analysis suggested that the cp genomes of eight Malus species had higher overall similarity, but there were more variation hotspots (rps16_trnK-UUU, trnG-UCC_atpA, atpH_atpF, trnT-GGU_psbD, etc.) in the LSC region. By building evolutionary trees, it can be clearly observed that M. zhaojiaoensis formed a large group with eight species of Malus, but was relatively independent in differentiation. In conclusion, this study provides high-quality chloroplast genome resources of M. zhaojiaoensis and discusses the genetic variation characteristics of Malus genus. The findings of this study will provide a good reference for plastid genome assembly and interspecific comparison in the future.