Functional analysis of transcription factor FaNAC56 in strawberry fruit ripening

Abstract:【Objective】Strawberry is a very valuable horticultural crop, and its fruit ripening is regulated by a complex process. To find the important regulatory factor involved in strawberry fruit ripening, the proteome of Fragaria × ananassa‘Benihoppe’was analyzed around the onset of fruit ripening. The da- ta have showed that a NAC transcription factor has high expression level during fruit ripening, which is named as FaNAC56. NAC is one of the largest families of plant-specific transcription factors, which play an important role in plant growth and development. In order to determine the function of Fa- NAC56 in strawberry fruit ripening, we first cloned the FaNAC56 gene and then investigated its tissue expression pattern and subcellular localization.【Methods】Firstly, we screened differentially expressed proteins around the onset of fruit ripening on the basis of our proteome, and found a NAC transcription factor that increased rapidly during strawberry ripening. According to phylogenetic analysis, this tran- scription factor was named as FaNAC56. The full-length ORF sequence of the FaNAC56 gene was ob- tained by RT-PCR. After sequencing the amino acid sequence of the FaNAC56 gene based on NCBI da- tabase, these genes with high homology were screened and downloaded. MEGA software was used to construct the phylogenetic tree. The molecular weight, isoelectric point, liposoluble index of encoding protein were analyzed by the Expasy website. The secondary structure of its protein, cis-acting elements in the promoter region and downstream target genes were predicted through bioinformatics. Secondly,we used real-time PCR to detect the expression level of FaNAC56 in strawberry including various or- gans and developmental fruits, including the root, stem, leaf, flower, seed, small green fruit, large green fruit, de-greening fruit, white fruit, initial red fruit, partial red fruit, and full red fruit. Meanwhile, the white fruit was made into cylinder-shaped cut with 8-mm diameter. We treated the fruits with the solu- tion containing 100 mmol·L-1 ABA, 500 mmol·L-1 NAA, 50 mg×L-1 GA, and 100 mmol·L-1 ethylene, re- spectively and then used the RT-qPCR to analyze the expression level of FaNAC56 gene after phytohor- mone treatment. Thirdly, the fusion protein of FaNAC56-GFP and the control GFP vector were trans- formed into tobacco (Nicotiana tabacum) leaf cells by Agrobacterium tumefaciens mediated-infiltration. After 48 h infiltration, the subcellular localization of FaNAC56 was observed.【Results】The cloning and sequencing results showed that the FaNAC56 gene contained 1035 bp of open reading frame that encoded 344 amino acids residues with a molecular mass of 38.3 kDa and had typical NAM domain be- longing to the NAC transcription factor. Its GenBank accession number is XP_004291668.1. The re- sults of physical and chemical properties of the FaNAC56 protein showed that the isoelectric point was 5.03 and average hydrophilicity was 0.904, indicating that it was a hydrophobic protein. The results of protein secondary structure prediction showed that the FaNAC56 protein mainly contained a- helix, ex- tended strand, β-corner and random coil with the proportion of 12.21%, 19.48%, 3.78% and 64.53%, re- spectively. The phylogenetic analysis revealed that it had the highest similarity with Rose chinensisNAC56 protein. The results of RT-qPCR showed that the FaNAC56 gene had tissue expression specifici- ty, with higher abundance expression in the fruits than in the roots, stems, leaves, flowers and seeds, suggesting that this gene was mainly expressed in the fruits. And the expression level of FaNAC56 in- creased during fruit ripening and reached the peak at red fruit stage. The cis-acting elements of the Fa- NAC56 promoter revealed that the promoter of FaNAC56 contained multiple hormone responsive ele- ments: ABA-responsive elements (ABRE), ethylene-responsive elements (ERE), auxin-responsive ele- ments (AuxRR-core), Methyl jasmonate-responsive elements (CGTCA-motif, TGACG-motif) and gib- berellin-responsive element (TCA-element). In addition, the promoter contained stress responsive mo- tif, such as MBS site, a MYB binding site involved in drought-inducibility. These results indicated thatFaNAC56 was implicated in a wide range of plant processes. The expression level of FaNAC56 was up- regulated by ABA, GA, NAA or ethylene. These results showed that FaNAC56 was indeed induced by a variety of hormones and the transcription factor may play an important regulatory role in fruit ripening. We used PlantPan website to predict the target genes of FaNAC56. The results showed that FaNAC56 had multiple potential target genes including auxin-induced protein 15A-like, ethylene-responsive tran- scription factor ERF071, UDP- glycosyltransferase 91C1- like, and serine/threonine- protein kinase Nek5. We used FaNAC56-GFP fusion protein for subcellular localization analysis and DAPI as nucleus marker in Nicotiana tabacum leaves. The result demonstrated that the FaNAC56-GFP fusion protein was accumulated in the nucleus.【Conclusion】In this study, FaNAC56 was cloned and its function of regulating strawberry ripening was explored preliminarily. Its secondary structure and cis-acting ele- ments were predicted. RT- qPCR was used to determine its expression pattern and response to ABA, GA, NAA, and ethylene treatments. FaNAC56 was an organ-specific expressed gene. In addition, ABA, GA, NAA and ethylene induced FaNAC56 expression. Prediction target gene analysis showed that many genes related to fruit ripening were the target of FaNAC56. These results revealed that FaNAC56may take part in the regulation of strawberry fruit development and ripening through a variety of hor- mones.