Cloning, sequencing, and expression analysis of 32 NAC transcription factors (MdNAC) in apple

NAC transcription factors play important roles in the regulation of plant growth, development, abiotic and biotic stress responses. The transcriptional level of MdNACs in different tissues and under various biotic and abiotic stress treatments was determined to provide a solid foundation for studying the function of MdNACs.

Thirty-two full-length cDNA sequences of Md NACs were isolated by homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. The prediction of subcellular locations of MdNAC proteins was performed using CELLO v.2.5, PSORT, and SoftBerry ProtComp 9.0. Expression levels of MdNACs were detected in 16 different tissues using an array. Expression patterns of MdNACs were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq, and the expression of MdNACs was analyzed under NaCl and mannitol treatments using RT-qPCR.

The sequencing results produced 32 cDNAs (designated as MdNAC24-39, MdNAC54-65, and MdNAC67-70 with GenBank accession No. MG099861-MG099876, MG099891-MG099902, and MG099904-MG099907, respectively). Phylogenetic analysis revealed that MdNAC34 belonged to the ATAF group, MdNAC63 belonged to the AtNAC3 group, MdNAC24, MdNAC26-30, MdNAC32-33, MdNAC35, MdNAC37-39, MdNAC56-57, MdNAC59-62, MdNAC64-65, and MdNAC67-70 belonged to the NAM group, and MdNAC25, MdNAC36, MdNAC54-55, and MdNAC58 belonged to the VND group. Predictions of subcellular localization showed that MdNAC24-27, MdNAC29-30, MdNAC33-37, MdNAC39, MdNAC54-65, and MdNAC67-70 proteins were located in the nucleus, MdNAC28 proteins were located in the cytoplasm, MdNAC31-32 proteins were located in the nucleus and cytoplasm, and MdNAC38 proteins were located in the nucleus and plasma membrane. Array results indicated that 32 MdNACs were expressed in all examined tissues at various expression levels. RNA-seq results showed that expression levels of MdNAC26-28, MdNAC33-34, MdNAC60, MdNAC62-65, and MdNAC68 were induced, but MdNAC24, MdNAC32, and MdNAC58 were down-regulated in response to AAAP infection. Under salt treatment, MdNAC24, MdNAC27, MdNAC29, MdNAC34, MdNAC37, MdNAC39, MdNAC54, MdNAC59, and MdNAC63 transcription levels were induced. Under mannitol treatment, MdNAC32 and MdNAC54 transcription levels were induced, but MdNAC24, MdNAC28, MdNAC30, MdNAC33, MdNAC35, MdNAC37, MdNAC55, MdNAC56, MdNAC58, and MdNAC59 were down-regulated. Taken together, the results indicated that the cloned MdNAC genes were expressed constitutively in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol, which suggested they may be involved in the regulation of growth, development, and stress response in apple.