Genome-wide identification and expression profile analysis of the NAC transcription factor family during abiotic and biotic stress in woodland strawberry

Abstract

The NAC transcription factors involved plant development and response to various stress stimuli. However, little information is available concerning the NAC family in the woodland strawberry. Herein, 37 NAC genes were identified from the woodland strawberry genome and were classified into 13 groups based on phylogenetic analysis. And further analyses of gene structure and conserved motifs showed closer relationship of them in every subgroup. Quantitative real-time PCR evaluation different tissues revealed distinct spatial expression profiles of the FvNAC genes. The comprehensive expression of FvNAC genes revealed under abiotic stress (cold, heat, drought, salt), signal molecule treatments (H2O2, ABA, melatonin, rapamycin), biotic stress (Colletotrichum gloeosporioides and Ralstonia solanacearum). Expression profiles derived from quantitative real-time PCR suggested that 5 FvNAC genes responded dramatically to the various abiotic and biotic stresses, indicating their contribution to abiotic and biotic stresses resistance in woodland strawberry. Interestingly, FvNAC genes showed greater extent responded to the cold treatment than other abiotic stress, and H2O2 exhibited a greater response than ABA, melatonin, and rapamycin. For biotic stresses, 3 FvNAC genes were up-regulated during infection with C. gloeosporioides, while 6 FvNAC genes were down-regulated during infection with R. solanacearum. In conclusion, this study identified candidate FvNAC genes to be used for the genetic improvement of abiotic and biotic stress tolerance in woodland strawberry.

Fig 1. Phylogenetic analysis of NAC proteins from woodland strawberry, rice, and Arabidopsis.

The full-length amino acid sequences of NAC genes from woodland strawberry (FvNACs), Arabidopsis (ANACs), and rice (ONACs) were aligned using ClustalX 2.0, and the phylogenetic tree was constructed using the neighbor-joining method with 1000 bootstrap replicates with MEGA 6.06.

Fig 2. Phylogenetic relationships, exon-intron structure, and motif compositions of FvNAC genes.

(a) The unrooted phylogenetic tree was constructed using full-length protein sequences of 37 FvNAC genes by the neighbor-joining method using 1000 bootstrap replicates. (b) Exon-intron structure analyses of FvNAC genes were performed by using the online tool GSDS 2.0. Lengths of exons and introns of each FvNAC genes were exhibited proportionally. (c) The motif composition related to each FvNAC protein. The motifs numbered 1–17 are displayed in different colored boxes. The sequence information for each motif is provided in S1 Fig.

Fig 3. Expression profiles of FvNAC genes in different organs of the woodland strawberry.

The expression profiles were generated by qRT-PCR and visualized as heat map. The heat map was constructed using HemI 1.0 software. The color scale represents log2 expression values, with green indicates low expression and red indicates high expression.

Fig 4. Expression profiles of FvNAC genes in leaves under cold stress.

The cold stress treatment was performed by transferring the plants to a low temperature (4°C) for 48 h following recovery. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 5. Expression profiles of FvNAC genes in leaves under heat stress.

The heat stress treatment was performed by transferring the plants to a high temperature (40°C) for 4 h following recovery. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 6. Expression profiles of FvNAC genes in leaves under drought stress.

The drought stress treatment was simulated by irrigating potted woodland strawberry plants with 200 mM mannitol. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 7. Expression profiles of FvNAC genes in leaves under salt stress.

The salt stress treatment was simulated by irrigating potted woodland strawberry plants with 100 mM NaCl. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 8. Expression profiles of FvNAC genes in leaves under H₂O₂ stress.

The H₂O₂ stress treatment was performed by spraying the woodland strawberry leaves with a solution containing 10 mM H₂O₂. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 9. Expression profiles of FvNAC genes in leaves under ABA stress.

The ABA stress treatment was performed by spraying the woodland strawberry leaves with a solution containing 0.1 mM ABA. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 10. Expression profiles of FvNAC genes in leaves under melatonin stress.

The melatonin stress treatment was performed by spraying the woodland strawberry leaves with a solution containing 0.5 mM melatonin. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 11. Expression profiles of FvNAC genes in leaves under rapamycin stress.

The rapamycin stress treatment was performed by spraying the woodland strawberry leaves with a solution containing 0.01 mM rapamycin. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 12. Expression profiles of FvNAC genes in leaves under C. gloeosporioides infection.

The C. gloeosporioides infection stress was performed by spraying the conidiospores (1×106 conidiospores/mL) to woodland strawberry leaves surface. Log2 based values from cold stress of qRT-PCR data were used to create the heat map.

Fig 13. Expression profiles of FvNAC genes in leaves under R. solanacearum infection.

The R. solanacearum infection stress was irrigating pathogenic bacteria suspension (1×10⁸ CFU) woodland strawberry seedlings.