Overexpression of a Malus baccata NAC Transcription Factor Gene MbNAC25 Increases Cold and Salinity Tolerance in Arabidopsis

Abstract

NAC (no apical meristem (NAM), Arabidopsis thaliana transcription activation factor (ATAF1/2) and cup shaped cotyledon (CUC2)) transcription factors play crucial roles in plant development and stress responses. Nevertheless, to date, only a few reports regarding stress-related NAC genes are available in Malus baccata (L.) Borkh. In this study, the transcription factor MbNAC25 in M. baccata was isolated as a member of the plant-specific NAC family that regulates stress responses. Expression of MbNAC25 was induced by abiotic stresses such as drought, cold, high salinity and heat. The ORF of MbNAC25 is 1122 bp, encodes 373 amino acids and subcellular localization showed that MbNAC25 protein was localized in the nucleus. In addition, MbNAC25 was highly expressed in new leaves and stems using real-time PCR. To analyze the function of MbNAC25 in plants, we generated transgenic Arabidopsis plants that overexpressed MbNAC25. Under low-temperature stress (4 °C) and high-salt stress (200 mM NaCl), plants overexpressing MbNAC25 enhanced tolerance against cold and drought salinity conferring a higher survival rate than that of wild-type (WT). Correspondingly, the chlorophyll content, proline content, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) were significantly increased, while malondialdehyde (MDA) content was lower. These results indicated that the overexpression of MbNAC25 in Arabidopsis plants improved the tolerance to cold and salinity stress via enhanced scavenging capability of reactive oxygen species (ROS).

Keywords: Malus baccata (L.) Borkh; MbNAC25; cold stress; salt stress.

Figure 1

Comparison and phylogenetic relationship of MbNAC25 with other plant NAC25 transcription factors. (A). Homologous comparison of MbNAC25 protein with NAC25 proteins from other plant species. The sequence in the red frame is the conserved amino acid sequence. (B). Evolutionary tree analysis of MbNAC25 (indicated by a red line) and other plant NAC25 proteins. The accession numbers are as follows: MdNAC25-like (Malus domestica, NP_001280970.1), PaNAC25 (Prunus avium, XP_021830786.1), PyNAC25 (Prunus yedoensis var nudiflora, PQQ02263.1), PmNAC25 (Prunus mume, XP_008224995.1), PpNAC25 (Prunus persica, XP_007211453.1), RcNAC25 (Ricinus communis, ASU89555.1), QsNAC25-like (Quercus suber, XP_023921724.1), JrNAC25 (Juglans regia, XP_018807000.1), CcNAC25 (Citrus clementina, XP_006449287.1), CsNAC25 (Citruus sinensis, XP_006467845.1), DzNAC25 (Durio zibethinus, XP_022777366.1), TcNAC25 (Theobroma cacao, XP_007025712.1) and HuNAC56 (Herrania umbratica, XP_021294335.1).

Figure 2

Subcellular localization of MbNAC25 protein. The 35S-GFP and 35S-MbNAC25-GFP translational products were expressed in onion epidermal cells and visualized by fluorescence microscopy in bright light (A,D); in dark field for GFP (B,E) and DAPI staining images (C,F). Scale bar corresponds to 5 μm.

Figure 3

Expression of the MbNAC25 gene in different tissues and organs of Malus baccata. (A) Expression of the MbNAC25 gene in different organs under control condition. Asterisks above columns indicate significant difference compared to that in the new leaf (* p ≤ 0.05). (B) Expression of the MbNAC25 gene in the new leaf and (C) in the root under control condition (CK), low temperature, high salt, drought and high temperature. Data represent means and standard errors of three replicates. Asterisks above columns indicate significant difference compared to that in control condition (* p ≤ 0.05).

Figure 4

Overexpression of MbNAC25 in Arabidopsis improved cold tolerance. (A) Expression levels of MbNAC25 in wild-type (WT) and T₂ transgenic Arabidopsis lines visualized by semi-quantitative RT-PCR using MbNAC25 specific primer (MbNAC25+) and MbNAC25 non-specific primer (MbNAC25-). Actin was used as control. (B) Survival rates of WT and transgenic lines after recovery in control condition (CK) and in cold for 12 h. The number of surviving plants was counted. Three independent experiments were performed, each with about 25 plants. Data represent means and standard errors of three replicates. Asterisks above columns indicate significant difference compared to WT (** p ≤ 0.01). (C) Phenotypes of MbNAC25 transgenic Arabidopsis lines (S3, S6, S10) and WT under low-temperature stress and recovery. Scale bar corresponds to 1 cm.

Figure 5

Physiological Indexes of Arabidopsis overexpressing of MbNAC25 under cold stresses. (A) Chlorophyll content; (B) proline content; (C) superoxide dismutase (SOD) activity; (D) peroxidase (POD) activity; (E) catalase (CAT) activity; (F) malondialdehyde (MDA) content. Data represent means and standard errors of three replicates. Asterisks above columns indicate significant difference compared to WT (** p ≤ 0.01).

Figure 6

Overexpression of MbNAC25 in Arabidopsis improved salt tolerance. (A) Phenotypes of MbNAC25 transgenic Arabidopsis lines (S3, S6, S10) and WT under salt stress and recovery. Scale bar corresponds to 1 cm. (B) Survival rates of seedlings in WT and transgenic lines after recovery. Data represent means and standard errors of three replicates. Asterisks above columns indicate significant difference compared to WT (** p ≤ 0.01).

Figure 7

Physiological Indexes of Arabidopsis overexpressing of MbNAC25 under salt stresses. (A) Chlorophyll content; (B) proline content; (C) SOD activity; (D) POD activity; (E) CAT activity; (F) MDA content. Data represent means and standard errors of three replicates. Asterisks above columns indicate significant difference compared to WT (** p ≤ 0.01).