MdbHLH130, an Apple bHLH Transcription Factor, Confers Water Stress Resistance by Regulating Stomatal Closure and ROS Homeostasis in Transgenic Tobacco

Abstract

Drought is a major environmental factor that significantly limits crop yield and quality worldwide. Basic helix-loop-helix (bHLH) transcription factors have been reported to participate in the regulation of various abiotic stresses. In this study, a bHLH transcription factor in apple, MdbHLH130, which contains a highly conserved bHLH domain, was isolated and characterized. qRT-PCR and P_MdbHLH130::GUS analyses showed that MdbHLH130 was notably induced in response to dehydration stress. Compared with the wild-type (WT), transgenic apple calli overexpressing MdbHLH130 displayed greater resistance to PEG6000 treatment. In contrast, the MdbHLH130-Anti lines were more sensitive to PEG6000 treatment than WT. Moreover, ectopic expression of MdbHLH130 in tobacco improved tolerance to water deficit stress, and plants exhibited higher germination rates and survival rates, longer roots, and lower ABA-induced stomatal closure and leaf water loss than the WT control. Furthermore, overexpression of MdbHLH130 in tobacco also led to lower electrolyte leakage, malondialdehyde contents, and reactive oxygen species (ROS) accumulation and upregulation of the expression of some ROS-scavenging and stress-responsive genes under water deficit stress. In addition, MdbHLH130 transgenic tobacco plants exhibited improved tolerance to oxidative stress compared with WT. In conclusion, these results indicate that MdbHLH130 acts as a positive regulator of water stress responses through modulating stomatal closure and ROS-scavenging in tobacco.

Keywords: MdbHLH130; ROS-scavenging; apple; bHLH transcription factor; stomatal closure; water stress.

Figure 1

Phylogenetic tree, sequence alignment and transcript levels of the MdbHLH130. (A) Phylogenetic analysis of bHLH proteins between 166 AtbHLH proteins and the MdbHLH130 protein via MEGA4.0 using the neighbor-joining method. MdbHLH130 is in the red box. (B) Multiple sequence alignment of the MdbHLH130 protein with its known Arabidopsis homologs. Identical amino acids are shaded in black. The conserved bHLH motif is indicated with a red line. (C) qRT-PCR analysis of MdbHLH130 observed in 30-day-old apple seedlings after the dehydration treatment applied for 0, 1, 3, 6, 12, and 24 h. Data are the means ± SD of three independent biological replicates. (D) and (E) GUS staining and activity analysis of P_MdbHLH130::GUS transgenic apple calli after the dehydration treatment. Error bars indicate the means ± SD from three independent biological replicates. Asterisks indicate significant differences relative to the control (*P < 0.01).

Figure 2

Subcellular localization and transcriptional activity of the MdbHLH130 protein. (A) Subcellular localization of MdbHLH130. The 35S::MdbHLH130-GFP fusion proteins were transiently transferred into epidermal cells of N. benthamiana leaves. Green fluorescence was visualized using confocal microscopy. Scale bar, 10 μm. (B) Transactivation assay of MdbHLH130 in yeast. The yeast cells transformed with different constructs on SD/-Trp or SD/-Trp/-His/-Ade medium for 3–5 days.

Figure 3

Effect of MdbHLH130 on PEG6000 tolerance in transgenic apple calli. (A) Expression analysis of MdbHLH130 in WT, MdbHLH130-ox and MdbHLH130-Anti transgenic calli by RT-PCR. The results were normalized using the internal control MdActin. Data are expressed as the mean ± SD as determined from three independent biological replicates. (B) Growth phenotypes of WT, MdbHLH130-ox and MdbHLH130-Anti transgenic apple calli. The WT and transgenic apple calli were grown on medium at 23°C for 7 days and then treated with 6% PEG6000 for another 20 days. (C, D) Fresh weights and MDA contents of WT, MdbHLH130-ox and MdbHLH130-Anti transgenic apple calli under the control or PEG6000 treatment conditions for another 20 days. Error bars represent the means ± SD taken from three independent biological replicates. Asterisks indicate significant differences relative to the WT (*P < 0.01).

Figure 4

Water deficit stress tolerance to tobacco plants overexpressing MdbHLH130. (A, B) Primary root growth of the WT and MdbHLH130-ox (L1, L2, and L3) seedlings before and after 10% PEG6000 treatment. The seedlings were grown on 1/2 MS medium supplemented with 10% PEG6000 and placed upright in the chamber for 8 days. (C) Water deficit stress phenotypes of the WT and MdbHLH130 transgenic lines. The WT and three transgenic tobacco lines (L1, L2, and L3) were grown at 24°C and then withheld water for 15 days, and photos were taken 7 days after the plants were re-watered. (D, E) Statistical analysis of the survival rates (D), electrolyte leakage (E) and MDA contents (F) in the WT and transgenic lines after water deficit treatment. FW, Fresh weight. Error bars represent the means ± SD of more than 30 plants from three independent biological replicates. Asterisks indicate significant differences relative to the WT (*P < 0.01).

Figure 5

Water loss rate and stomatal aperture ratios from WT and MdbHLH130-ox plants after treatments. (A) Water loss of the detached leaves from the WT and MdbHLH130-ox plants. (B, C) Images of stomata and measurement of stomatal aperture ratios (width/length ratio) of the WT and MdbHLH130-ox plant leaves in response to dehydration or 10 μM ABA. Scale bar, 10 μm. Error bars represent the means ± SD of 100 stomata from three independent biological replicates. Asterisks indicate significant differences between MdbHLH130-ox plants and WT (*P < 0.01).

Figure 6

ROS accumulation and activity of antioxidant enzymes in MdbHLH130-ox plants and WT after water deficit treatment. (A) Fluorescence detection and quantification of ROS by H2DCFDA staining in MdbHLH130-ox plants and WT leaves after water deficit treatment. Bar = 250 µm. (B–E) Statistical analysis of the H₂O₂ content (B), SOD activity (C), POD activity (D), and CAT activity (E) in the WT and three MdbHLH130-ox plants after water deficit treatment. Error bars represent the means ± SD taken from three independent biological replicates. Asterisks indicate significant differences relative to the WT (*P < 0.01).

Figure 7

Expression of the genes involved in ROS scavenging and stress responses in the WT and transgenic plants. The expression levels were analyzed by qRT-PCR in tobacco seedlings under the control or water deficit treatment conditions. Error bars represent the means ± SD taken from three independent biological replicates. Asterisks indicate significant differences from the WT (*P < 0.01).

Figure 8

Oxidative stress tolerance to MdbHLH130-ox plants. (A) Phenotype of the leaf discs from the WT and three MdbHLH130-ox plants after incubation with or without 100 μM MV solution for 48 h. (B) Relative total chlorophyll content in the leaf discs after 100 μM MV treatment. Three independent biological replicates were performed. Vertical bars refer to means ± SD of at least three independent biological replicates. Asterisks indicate a significant difference between the WT and MdbHLH130-ox lines (*P < 0.01).