Identification of ANS from Malus halliana reveal flavonoid metabolic pathway involved in response to saline–alkali stress

Soil salinization, as one of the major abiotic stress, restricts the development of global agriculture. Transcription factors always act on the upstream of signal transduction to control a wide range of downstream genes, thus effectively participating in the regulation of various abiotic stress tolerance. In this research, based on transcriptome of Malus halliana, we screened out the transcription factor Anthocyanin synthase (ANS, LOC103437326) in flavonoid metabolic pathway which was significantly induced by saline-alkali stress. Quantitative real-time PCR confirmed that the expression of MhANS in the leaves was 17.15% of that of the control (0 h) under12 h of stress. However, the mechanism of its regulation of saline-alkali stress is unclear. Subsequently, the MhANS gene was isolated and its functional characteristics were further studied in Arabidopsis thaliana, tobacco and apple calli. MhANS contains a complete open reading frame with a length of 1074 bp and encodes 357 amino acids with an isoelectric point of 5.82. Phylogenetic tree analysis showed that MhANS had the highest homology and the closest genetic relationship with Pyrus × bretschneideri. In addition, overexpression of MhANS reduced the tolerance of Arabidopsis thaliana, tobacco and apple calli to saline-alkali stress, and caused a variety of biochemical changes: the contents of chla and chlb of transgenic Arabidopsis thaliana and tobacco were significantly lower than those of wild type, while the electrical conductivity was higher than that of wild type. The enzyme activities (SOD, POD, CAT) of transgenic Arabidopsis, tobacco and overexpressed apple calli decreased significantly, while the contents of malondialdehyde and proline increased. Additionally, the growth of overexpressed apple calli was retarded under saline-alkali stress. All results indicated that resistance to saline-alkali stress was weaked due to the overexpression of MhANS. In conclusion, the ANS gene of flavonoid metabolic pathway respond to saline-alkali stress and play a down-regulation role, providing gene for the regulatory network of saline-alkali stress response in apple, aiming to excavate the excellent resistance genes of apple and provide a theoretical basis for the breeding of apple varieties with strong saline-alkali resistance.