The bHLH transcription factor AhbHLH112 improves the drought tolerance of peanut

Abstract

Background: Basic helix-loop-helix (bHLH) transcription factors (TFs) are one of the largest gene families in plants. They regulate gene expression through interactions with specific motifs in target genes. bHLH TFs are not only universally involved in plant growth but also play an important role in plant responses to abiotic stress. However, most members of this family have not been functionally characterized.

Results: Here, we characterized the function of a bHLH TF in the peanut, AhHLH112, in response to drought stress. AhHLH112 is localized in the nucleus and it was induced by drought stress. The overexpression of this gene improves the drought tolerance of transgenic plants both in seedling and adult stages. Compared to wild-type plants, the transgenic plants accumulated less reactive oxygen species (ROS), accompanied by increased activity and transcript levels of antioxidant enzymes (superoxide dismutase, peroxidase and catalase). In addition, the WT plants demonstrated higher MDA concentration levels and higher water loss rate than the transgenic plants under drought treatment. The Yeast one-hybrid result also demonstrates that AhbHLH112 directly and specifically binds to and activates the promoter of the peroxidase (POD) gene. Besides, overexpression of AhHLH112 improved ABA level under drought condition, and elevated the expression of genes associated with ABA biosynthesis and ABA responding, including AtNCED3 and AtRD29A.

Conclusions: Drawing on the results of our experiments, we propose that, by improving ROS-scavenging ability, at least in part through the regulation of POD -mediated H₂O₂ homeostasis, and possibly participates in ABA-dependent stress-responding pathway, AhbHLH112 acts as a positive factor in drought stress tolerance.

Keywords: Basic helix–loop–helix transcription factors; Drought stress; Peanut; ROS homeostasis; Transcriptional regulation.

Fig. 1

Expression analyse of AhbHLH112 in different tissues under normal and drought stress conditions. Actin11 was used as an internal reference control, and transcript levels of the tested genes were calculated using the 2 ^−∆∆CT method. Error bars represent SDs for three independent replicates. a: Detection of AhbHLH112 transcript in different tissues of peanut plant under normal condition. Asterisks indicate difference (*P < 0.05) and significant difference (**P < 0.01) comparing to relative expression in leaves; (b): Expression of AhbHLH112 in different tissues in response to drought. Asterisks indicate difference (*P < 0.05) and significant difference (**P < 0.01) comparing torelative expression at 0 h

Fig. 2

Subcellular localization and transcriptional activity of AhbHLH112. a: Subcellular localization of AhbHLH112 protein. CK: pCAMBIA2300-GFP; Bar = 25 μM. b: Growth and X-α-GAL staining assay of transformant colonies on SD/−Trp and SD/−Trp/−His/−Ade medium. The pGBKT7–53 vector was used as a positive control

Fig. 3

Analyse of drought a tolerance at the seedling stage. a: Morphology of transgenic and WT seedlings growing for nine days on 1/2 MS medium with mannitol. b: The analysis of root length of transgenic and WT plants. Y-ray is the root length (cm). Data are presented as means and SDs of three independent experiments. Asterisks indicate difference (*P < 0.05) and significant difference (**P < 0.01) comparing to WT.

Fig. 4

Analyse of drought tolerance in the adult stage. WT: wild-type plants; OE-1 and OE-2: transgenic lines. WT and transgenic plants were grown in soil with sufficient water for one month before water was withheld for 15 days, followed by recovery

Fig. 5

Detection of ROS scavenging capability, reactive oxygen species (ROS) accumulation, MDA and ABA concentrations under control and drought stress conditions. a-c: Activity levels of CAT, SOD and POD, respectively; (d): H₂O₂ concentration; (e): MDA concentration; (f): ABA content. WT: wild-type plants. OE-1 and OE-2: transgenic lines. Error bars represent SDs for three independent replicates. Asterisks indicate difference (*P < 0.05) and significant difference (**P < 0.01) comapring to WT and ()

Fig. 6

AhbHLH112 binds to and activates the promoter of AhPOD. a: Schematic diagrams of the promoters of AhPOD, in which the G-box elements are denoted using purple triangles, E-box elements are denoted using yellow circles. The segments marked with P1/2 represent the promoter fragments used in the yeast one-hybrid (Y1H) assay. b: Growth of yeast cells of positive control (p53-AbAi+pGAD-p53), negative control (bait+pGADT7), and co-transformants (bait+prey) on SD/−Leu/−Ura medium supplemented without (middle panel) or with (right panel) AbA

Fig. 7

Relative expression levels of ABA-biosynthesis gene and ABA stress-responsive gene. Plants treated by normal condition and drought stress. a: Relative expression of AtNCED3 (ABA-biosynthesis gene); b: Relative expression of AtRD29A (ABA stress-responsive gene). WT: wild-type plants. OE-1 and OE-2: transgenic lines. Data were presented as mean and SD values of three independent experiments. Asterisks indicated significant difference (** P < 0.01) comparing to WT.