A Member of the 14-3-3 Gene Family in Brachypodium distachyon, BdGF14d, Confers Salt Tolerance in Transgenic Tobacco Plants

Abstract

Plant 14-3-3 proteins are involved in diverse biological processes, but for the model monocotyledonous species, Brachypodium distachyon, their roles in abiotic stress tolerance are not well understood. In this study, a total of eight Bd14-3-3 genes were identified from B. distachyon and these were designated respectively as BdGF14a-BdGF14g. The qRT-PCR analyses of 3-month-old plants of B. distachyon showed that these genes were all expressed in the stems, leaves, and spikelets. By contrast, most of the plants had relatively lower transcriptional levels in their roots, except for the BdGF14g gene. The different expression profiles of the Bd14-3-3s under various stress treatments, and the diverse interaction patterns between Bd14-3-3s and BdAREB/ABFs, suggested that these gene products probably had a range of functions in the stress responses. The NaCl-induced Bd14-3-3 gene, BdGF14d, was selected for overexpression in tobacco. BdGF14d was found to be localized throughout the cell and it conferred enhanced tolerance to salt in the transgenic plants. Lowered contents of malondialdehyde, H₂O₂, and Na⁺, and lower relative electronic conductance (Rec%), yet greater activities of catalase and peroxidase, were observed in the overexpressing plants. Higher photosynthetic rate, transpiration rate, stomatal conductance, and water use efficiency were measured in the transgenic lines. Following abscisic acid (ABA) or NaCl treatment, stomatal aperture in leaves of the BdGF14d-overexpression plants was significantly lower than in leaves of the wild type (WT) controls. The stress-related marker genes involved in the ABA signaling pathway, the reactive oxygen species (ROS)-scavenging system, and the ion transporters were all up-regulated in the BdGF14d-overexpressing plants as compared with WT. Taken together, these results demonstrate that the Bd14-3-3 genes play important roles in abiotic stress tolerance. The ABA signaling pathway, the ROS-scavenging system, and ion transporters were all involved in enhancing the tolerance to salt stress in the BdGF14d-overexpression plants.

Keywords: ABA signaling; B. distachyon; Bd14-3-3s; BdGF14d; ROS-scavenging system; salt tolerance.

FIGURE 1

Phylogenetic and gene structure analyses of the Bd14-3-3s. (A) Phylogenetic tree of 14-3-3s from B. distachyon, rice, and Arabidopsis plants constructed using MAGA6. (B) Gene structures of Bd14-3-3s. Solid blue bars and strings represent the exons and introns, respectively; green and red triangles represent the translation starts and stops, respectively. Arrows indicate transcriptional directions on the chromosomes. (C) Amino acid sequence alignments of the Bd14-3-3 family.

FIGURE 2

Yeast two-hybrid analyses between the Bd14-3-3s and the BdAREB/ABF TFs. The yeast strain AH109, transferred along with the described plasmid combinations, was spotted onto Trp-/Leu-/His-/Ade-medium containing x-α-gal. The Bd14-3-3s were cloned into pGBKT7, and the BdAREB/ABFs were cloned into pGADT7 vectors. Transformants containing pGADT7-T and pGBKT7-53 vs. pGBKT7-Lam represent the positive vs. negative controls, respectively.

FIGURE 3

Expression of the Bd14-3-3s in different plant organs. Roots, stems, leaves, and spikelets of 3-month-old plants of B. distachyon were sampled to extract RNA for expression analysis. The y-axis represents the relative expression of the genes. All bars are means (±SE) calculated from three replicates; asterisks denote significant differences between leaves and the other organs (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 4

Expression profiles of Bd14-3-3s under applied stress treatments. Leaves of B. distachyon were collected at 0, 1, 3, 6, 12, and 24 h after being treated with 200 mM NaCl (A), 20% PEG (B), 10 mM H₂O₂ (C), and 100 μM ABA (D), respectively, and RNA was extract for expression analyses. Mock indicates the control (no treatment applied). Bars are means (±SE), all the experiments were repeated three times. Asterisks denote significant differences between 0 h and different time points (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 5

Improved tolerance to salt in the BdGF14d-overexpression plants. (A) The relative expression levels of BdGF14d in transgenic tobacco plants. The Ntubiquitin was used as internal reference. (B) Seven-day-old seedlings of WT, VC, and transgenic lines germinated on MS were transferred to 1/2 MS containing 100 mM and 150 mM NaCl to grow for a further 2 weeks. (C) The root lengths were measured and statistically analyzed. (D) Two-week-old seedlings growing on MS medium were planted in soil to grow under normal condition for 3 weeks and 500 mM NaCl solution was used to treat the seedlings for a further 35 days. (E) The respective survival rates were calculated. All the experiments were repeated three times. Bars are means (±SE); asterisks denote significant differences between WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 6

Plant physiological indices of transgenic lines and WT. Two-week-old WT and transgenic lines geminated on MS were planted in soil to grow for 3 weeks; all seedlings were treated with a 500 mM NaCl solution every 2 weeks for a further 20 days. Leaves of the five lines with or without salt applied were sampled for measurements of MDA (A), Rec% (B), H₂O₂ (C), CAT (G), POD (H), photosynthetic rate (I), transpiration rate (J), stomatal conductance (K), WUE (L), and for their contents of Na⁺ (D) and K⁺ (E). The ratio of K⁺/Na⁺ was also calculated (F). Bars are means (±SE); and asterisks denote significant differences between WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01). All the experiments were repeated three times.

FIGURE 7

Analysis of stomatal aperture in transgenic lines and WT plants under the NaCl and ABA treatments. Detached leaves of WT and transgenic lines were dipped in stomatal opening buffer under light for 6 h. A total of 200 mM NaCl and 50 μM ABA were added to treat the leaves for 1 and 2 h, respectively (A) and the stomatal apertures were calculated (B). Bars are means (±SE); asterisks denote significant differences between WT and the transgenic lines (^∗∗P < 0.01).

FIGURE 8

The qRT-PCR analyses of related marker genes of the transgenic lines and WT under salt stress. Two-week-old seedlings that germinated on MS medium were transferred to MS containing 200 mM NaCl to grow for 7 days. Entire seedlings of the five lines with or without NaCl applied were sampled to extract RNA for expression analyses. Bars are means (±SE); asterisks denote significant differences between WT and the transgenic lines (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 9

Subcellular localization of the BdGF14d protein. BdGF14d and GFP were inserted into the pBI121 vector, and the recombinant plasmid pBI121-BdGF14d-GFP and vector control pBI121 were introduced into A. tumefaciens for injection into the epidermal cells of tobacco leaves.