Isolation, sequencing, and expression analysis of 30 AP2/ERF transcription factors in apple

Huifeng Li; Qinglong Dong; Qiang Zhao; Song Shi; Kun Ran

doi:10.7717/peerj.8391

Isolation, sequencing, and expression analysis of 30 AP2/ERF transcription factors in apple

PeerJ. 2020 Jan 17:8:e8391. doi: 10.7717/peerj.8391. eCollection 2020.

Authors

Huifeng Li^#¹, Qinglong Dong^#², Qiang Zhao³, Song Shi⁴, Kun Ran¹

Affiliations

¹ Shandong Institute of Pomology, Tai'an, China.
² College of Horticulture, Northwest A and F University, Yangling, China.
³ College of Horticulture, Qingdao Agricultural University, Qingdao, China.
⁴ Nanjing Agricultural University, Nanjing, China.

^# Contributed equally.

Abstract

Background: AP2/ERF transcription factors are involved in the regulation of plant growth, development, and stress responses. Our research objective was to characterize novel apple (Malus × domestica Borkh.) genes encoding AP2/ERF transcription factors involved in regulation of plant growth, development, and stress response. The transcriptional level of apple AP2/ERF genes in different tissues and under various biotic and abiotic stress was determined to provide valuable insights into the function of AP2/ERF transcription factors in apple.

Methods: Thirty full-length cDNA sequences of apple AP2/ERF genes were isolated from 'Zihong Fuji' apple (Malus × domestica cv. Zihong Fuji) via homologous comparison and RT-PCR confirmation, and the obtained cDNA sequences and the deduced amino acid sequences were analyzed with bioinformatics methods. Expression levels of apple AP2/ERF genes were detected in 16 different tissues using a known array. Expression patterns of apple AP2/ERF genes were detected in response to Alternaria alternata apple pathotype (AAAP) infection using RNA-seq with existing data, and the expression of apple AP2/ERF genes was analyzed under NaCl and mannitol treatments using qRT-PCR.

Results: The sequencing results produced 30 cDNAs (designated as MdERF3-8, MdERF11, MdERF16-19, MdERF22-28, MdERF31-35, MdERF39, MdAP2D60, MdAP2D62-65, and MdRAV2). Phylogenetic analysis revealed that MdERF11/16, MdERF33/35, MdERF34/39, and MdERF18/23 belonged to groups A-2, A-4, A-5, and A-6 of the DREB subfamily, respectively; MdERF31, MdERF19, MdERF4/25/28/32, MdERF24, MdERF5/6/27, and MdERF3/7/8/17/22/26 belonged to groups B-1, B-2, B-3, B-4, B-5, and B-6 of the ERF subfamily, respectively; MdAP2D60 and MdAP2D62/63/64/65 belonged to the AP2 subfamily; and MdRAV2 belonged to the RAV subfamily. Array results indicated that 30 apple AP2/ERF genes were expressed in all examined tissues to different degrees. RNA-seq results using previously reported data showed that many members of the apple ERF and DREB subfamilies were induced by Alternaria alternate apple pathotype (AAAP) infection. Under salt treatment, many members in the apple ERF and DREB subfamilies were transcriptionally up or down-regulated. Under mannitol treatment, many members of the apple ERF, DREB, and AP2 subfamilies were induced at the transcriptional level. Taken together, the results indicated that the cloned apple AP2/ERF genes were expressed in all examined tissues. These genes were up-regulated or down-regulated in response to AAAP infection and to salt or mannitol treatment, which suggested they may be involved in regulating growth, development, and stress response in apple.

Keywords: AP2/ERF transcription factors; Abiotic stress; Apple; Biotic stress; Expression analysis; Sequencing.

Grants and funding

This work was supported by Shandong Agricultural Good Cultivar Project (Grant No. 2016LZGC034), the National Natural Science Foundation of China (Grant No. 31501742), the Natural Science Foundation of Shandong Province (Grant No. ZR2019MC071) and the Agricultural Science and Technology Innovation Project of SAAS (CXGC2016A03 and CXGC2018F03). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.