A 14-3-3 Protein-Encoding Gene, BdGF14g, Confers Better Drought Tolerance by Regulating ABA Biosynthesis and Signaling

Abstract

Abscisic acid (ABA), a phytohormone, enacts a cardinal function in coping with abiotic stress. 14-3-3 proteins can interact with ABA-responsive-element-binding transcription factors (ABFs), a chief constituent of ABA signaling, and play critical roles in the dehydration response involving ABA signaling. Meanwhile, whether and how 14-3-3 proteins regulate ABA signaling to respond to aridity stress is yet to be fully investigated. Herein, BdGF14g, a 14-3-3 gene induced by ABA, H₂O₂, and PEG treatments, was identified in Brachypodium distachyon (B. distachyon). Overexpression of BdGF14g improved drought stress tolerance in tobacco plants, with a higher survival rate, longer root length, enhanced cell membrane stability, and increased antioxidase activity compared with non-transgenic controls in coping with dehydration. Both drought and exogenous ABA treatments resulted in smaller stomatal apertures in BdGF14g-transgenic lines. Additionally, when an ABA biosynthesis inhibitor was added, the better growth statuses, less H₂O₂ accumulation, and higher activities of catalase and superoxide dismutase under mannitol stress disappeared. Moreover, BdGF14g interacted with NtABF2, upregulated the endogenous ABA content, and enhanced the transcription of ABA-related genes, including NtNCED1, a crucial ABA biosynthesis gene, under drought conditions. In conclusion, BdGF14g acts as a positive factor in the water deficiency response by affecting ABA biosynthesis and signaling in tobacco plants.

Keywords: BdGF14g; ROS-scavenging system; abscisic acid; drought stress.

Figure 1

Subcellular localization of BdGF14g::GFP fusion protein and GFP in tobacco epidermal cells. The plasmids of pBI121-BdGF14g-GFP and PBI121-GFP were introduced into EHA105, an Agrobacterium strain, and then injected into tobacco cells via Agrobacterium transformation. After incubating for 48 h, photos were taken under an inverted fluorescence microscope.

Figure 2

Response of BdGF14g transgenic tobacco plants to drought stress. (A) The seedlings of wild type (WT), vacant vector (VC) controls, and three BdGF14g-overexpressing lines after seven days of germination were planted on 1/2 MS with 250 mM or 350 mM of mannitol. After vertical cultivation for two weeks, the root lengths of at least nine seedlings per line were detected and statistically analyzed (B). (C) The two-week-old seedlings growing on MS were transplanted into pots filled with soil for three weeks under normal conditions. After that, watering was stopped for 25 days, and then they were rewatered for a further 7 days. (D) The respective survival rates of BdGF14g-expressing tobacco plants and controls suffering from water withholding were measured based on at least 35 seedlings per line in each replicate. Error bars were calculated from three independent experiments. Asterisks indicate marked differences in statistics (** p < 0.01).

Figure 3

The physiological index analyses of controls and BdGF14g transgenic plants in response to water withholding. The two-week-old seedlings growing on MS solid medium were grown in potting soil for three weeks under normal conditions, and no water was supplied to treat the seedlings for a further fifteen days. Leaves, 0.2 g in weight, from at least three tobacco plants of test and control groups were sampled to measure the (A) relative water content (RWC), (B) malondialdehyde (MDA) content, (C) hydrogen peroxide (H₂O₂) content, (D) ion leakage (IL), the enzyme activities of (E) catalase (CAT), (F) peroxidase (POD), (G) superoxide dismutase (SOD), and (H) total antioxidant capacity (T-AOC). Error bars were calculated from three independent experiments. Asterisks indicate marked differences in statistics (* p < 0.05; ** p < 0.01).

Figure 4

The stomatal movement of WT and BdGF14g-expressing line (OE1) under dehydration and ABA treatments, and abscisic acid (ABA) content under drought stress. (A) The stomatal movements of potted WT and OE1 lines for six weeks were detected in normal, 40 min dehydration, and 50 μM 2 h ABA conditions. Images were captured under a fluorescence microscope in a bright field. (B) The stomatal aperture of at least four leaves per line was examined under dehydration, ABA treatments, and control conditions. (C) The endogenous ABA content under normal and water-withholding conditions. The error bars were calculated based on three independent replicates. Asterisks indicate marked differences in statistics (* p < 0.05).

Figure 5

The BdGF14g-overexpressing lines lost the resistance to drought after adding an endogenous ABA inhibitor, sodium tungstate (Tu). Ten OE1, OE2, and WT tobacco plants of two-week-old seedlings were grown in 1/2 MS containing 350 mM of mannitol or 350 mM of mannitol + 1 mM Tu for two weeks, and then photographs were taken (A). Young seedlings (0.2 g, besides roots) of the transgenic line (OE1) and WT without or with Tu treatment were harvested for measurements of (B) H₂O₂, (C) CAT, and (D) SOD. Error bars were calculated from three independent experiments. Asterisks indicate marked differences in statistics (* p < 0.05).

Figure 6

BdGF14g interacted with NtABF2 in the yeast two-hybrid assay and transcript expression analyses of the ABA signaling pathway. (A) BdGF14g was transferred into the pGBKT7 vector, and NtABF2 was cloned into the pGADT7 vector, followed by their co-transfection into the AH109 strain, and spotted onto the nutritional selective solid media SD/-Trp-Leu, SD/-Trp-Leu-Ade, and SD/-Trp-Leu-His-Ade. The transformants pGADT7-T, pGBKT7-p53, and pGBKT7-LaminC represent the controls. (B) The transcript expression levels of ABA-related genes NtABF2, NtNCED1, and NtERD10C. The error bars were calculated based on three independent replicates. Asterisks indicate marked differences in statistics (* p < 0.05; ** p < 0.01).

Figure 7

A proposed mechanism of BdGF14g involved in regulating ABA-signaling-mediated drought tolerance. BdGF14g interacts with NtABF2, the transcription and activation of which can be enhanced by drought-induced ABA accumulation mediated by ABA-signaling genes, including an ABA-synthesis-related gene, NtNECD1, resulting in increased ABA production, which, in turn, regulates the ABA signaling pathway via a feedback mechanism. BdGF14g interacts with NtABF2 and is involved in upregulating ABA-related genes to enhance drought resistance.