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Overexpression of PtrABF gene, a bZIP transcription factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance in tobacco via scavenging ROS and modulating expression of stress-responsive genes

BMC Plant Biology (2010) 10
DOI: 10.1186/1471-2229-10-230
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Abstract

Background: Drought is one of the major abiotic stresses affecting plant growth, development and crop productivity. ABA responsive element binding factor (ABF) plays an important role in stress responses via regulating the expression of stress-responsive genes.Results: In this study, a gene coding for ABF (PtrABF) was isolated from Poncirus trifoliata (L.) Raf. PtrABF had a complete open reading frame of 1347 bp, encoding a 448 amino acid peptide, and shared high sequence identities with ABFs from other plants. PtrABF was subcellularly targeted to the nucleus, exhibited transactivation activity in yeast cell and could bind to ABRE, supporting its role as a transcription factor. Expression levels of PtrABF were induced by treatments with dehydration, low temperature and ABA. Ectopic expression of PtrABF under the control of a CaMV 35S promoter in transgenic tobacco plants enhanced tolerance to both dehydration and drought. Under dehydration and drought conditions, the transgenic plants accumulated lower levels of reactive oxygen species compared with wild type, accompanied by higher activities and expression levels of three antioxidant enzymes. In addition, steady-state mRNA levels of nine stress-responsive genes coding for either functional or regulatory proteins were induced to higher levels in the transgenic lines with or without drought stress.Conclusions: PtrABF is a bZIP transcription factor and functions in positive modulation of drought stress tolerance. It may be an important candidate gene for molecular breeding of drought- tolerant plants. © 2010 Huang et al; licensee BioMed Central Ltd.

Figures

  • Figure 1 Multiple alignments of the deduced amino acid sequence of PtrABF and those of Arabidopsis thaliana (AtABF1, NP_564551.1; AtABF3, NP_567949.1), Catharanthus roseus (CrABF1, AF329450_1), Populus suaveolens (PsABF2, ABF29696.1), Populus trichocarpa (PtABF, XP_002313119.1), Ricinus communis (RcABF, XP_002518757.1), Solanum lycopersicum (SlABF, AAS20434.1) and Populus trichocarpa (PtABF2, ABN58425.1). Black and gray shaded backgrounds indicated that the amino acids were identical or similar to PtrABF, respectively. The basic region and the three heptad leucine repeats, two important bZIP signatures, are shown by double line and arrows, respectively. C1, C2, C3 and C4 are conserved regions containing residues of serine (’•’) and threonine (’▼’).
  • Figure 2 A phylogenetic tree constructed based on the amino acid sequences of PtrABF (underlined) and other 12 ABFs, including AtABF1, AtABF3, PtABF, PsABF2, SlABF, RcABF, PtABF2, CrABF (AF329450_1), VvABF (CAN64991.1), CkABF (ABG90380), AtABF2 (NP_849777), AtABF4 (NP_566629). The numbers beside the branches represent bootstrap values based on 1000 replications, and the relative amount of change along branches is indicated by the scale bar.
  • Figure 3 Time-course expression levels of PtrABF in trifoliate orange shoots under different treatments with dehydration (A), low temperature (B), salt (C) and ABA (D). Total RNA extracted from leaves of the treated shoots sampled at the indicated time was reversetranscribed to synthesize cDNAs, which were used for quantitative real time PCR analysis. For each treatment, the expression level at time point 0 was defined as 1.0, and data represented means ± SE of three replicates.
  • Figure 4 Subcellular localization of PtrABF via Agrobacterium-mediated transformation of onion epidermis. Fusion construct (PtrABF: GFP) and control plasmid (GFP) were separately transformed into onion epidermal cells via Agrobacterium infection. Bright-field images (A, D), fluorescence images (B, E), and the merged images (C, F) of representative cells expressing GFP (A, B and C) or PtrABF: GFP fusion protein (D, E and F) are shown.
  • Figure 5 Analysis of transactivation activity and ABRE binding of PtrABF. A. Growth of yeast cells transformed with either control vector (pGBKT7) or the fusion vector (pGBKT7: PtrABF) on SD/-Leu/-Trp, SD/-Ade/-His/-Leu/-Trp supplemented with 3-AT (0, 5 and 15 mM). B. Growth of the yeast cells on SD/-Ade/-His/-Leu/-Trp added with 15 mM 3-AT and 20 mM X-a-gal. C. Schematic diagrams of effector plasmids (E1 and E2) and reporter plasmids (R1 and R2) used for yeast one hybrid. D. Growth of yeast cells on SD/-His/-Leu/-Trp supplemented with (60 mM) or without 3-AT. Y1, Y2 and Y3 represent yeast cells transformed with E1 and R1, E2 and R1, E1 and R2, respectively.
  • Figure 6 Phenotype and dehydration tolerance of wild type (WT) and transgenic lines (#4 and #19). (A) Time-course fresh water loss of WT, #4 and #19 during a 90-min dehydration. Aerial parts of 35-d-old in vitro seedlings were dehydrated at ambient environment, and the fresh weight (FW) was measured at the indicated time. Water loss was calculated by the decrease of FW relative to that at time 0. (B) Electrolyte leakage of WT, #4 and #19 after dehydration for 90 min. * and ** indicate that values of the two transgenic lines were significantly different from those of WT at P < 0.05 and p < 0.01, respectively. (C) Representative photographs of dehydrated WT (upper panel), #4 (middle panel) and #19 (lower panel).
  • Figure 7 Phenotype and drought tolerance of wild type (WT) and transgenic lines (#4 and #19). (A-B) Representative photographs of potted plants of WT, #4 and #19 that have been exposed to water stress for 7 d (A) and 21 d (B). (C-D) Electrolyte leakage (C) and total chlorophyll content (D) of WT, #4 and #19 after 7 d of water stress. * and ** indicate that values of the two transgenic lines were significantly different from those of WT at P < 0.05 and P < 0.01, respectively.
  • Figure 9 Activity and expression of antioxidant enzymes (genes) in wild type (WT), transgenic lines (#4 and #19) before and after drought treatment. (A-C) Activity of SOD (A), POD (B) and CAT (C) of WT, #4 and #19 plants before and after 7-d of drought treatment. Enzymes were extracted and assayed as described in ‘Materials and methods’. ** indicates that the values of the two transgenic lines are significantly different those of WT (P < 0.01). Expression of the genes encoding the enzymes was examined by RT-PCR and was shown below each graph.

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Huang, X. S., Liu, J. H., & Chen, X. J. (2010). Overexpression of PtrABF gene, a bZIP transcription factor isolated from Poncirus trifoliata, enhances dehydration and drought tolerance in tobacco via scavenging ROS and modulating expression of stress-responsive genes. BMC Plant Biology, 10. https://doi.org/10.1186/1471-2229-10-230

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