Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 May 31;41(5):413-422.
doi: 10.14348/molcells.2018.2254. Epub 2018 May 10.

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean

Affiliations
Free PMC article

Confirmation of Drought Tolerance of Ectopically Expressed AtABF3 Gene in Soybean

Hye Jeong Kim et al. Mol Cells. .
Free PMC article

Erratum in

Abstract

Soybean transgenic plants with ectopically expressed AtABF3 were produced by Agrobacterium-mediated transformation and investigated the effects of AtABF3 expression on drought and salt tolerance. Stable Agrobacterium-mediated soybean transformation was carried based on the half-seed method (Paz et al. 2006). The integration of the transgene was confirmed from the genomic DNA of transformed soybean plants using PCR and the copy number of transgene was determined by Southern blotting using leaf samples from T2 seedlings. In addition to genomic integration, the expression of the transgenes was analyzed by RT-PCR and most of the transgenic lines expressed the transgenes introduced. The chosen two transgenic lines (line #2 and #9) for further experiment showed the substantial drought stress tolerance by surviving even at the end of the 20-day of drought treatment. And the positive relationship between the levels of AtABF3 gene expression and drought-tolerance was confirmed by qRT-PCR and drought tolerance test. The stronger drought tolerance of transgenic lines seemed to be resulted from physiological changes. Transgenic lines #2 and #9 showed ion leakage at a significantly lower level (P < 0.01) than non-transgenic (NT) control. In addition, the chlorophyll contents of the leaves of transgenic lines were significantly higher (P < 0.01). The results indicated that their enhanced drought tolerance was due to the prevention of cell membrane damage and maintenance of chlorophyll content. Water loss by transpiration also slowly proceeded in transgenic plants. In microscopic observation, higher stomata closure was confirmed in transgenic lines. Especially, line #9 had 56% of completely closed stomata whereas only 16% were completely open. In subsequent salt tolerance test, the apparently enhanced salt tolerance of transgenic lines was measured in ion leakage rate and chlorophyll contents. Finally, the agronomic characteristics of ectopically expressed AtABF3 transgenic plants (T2) compared to NT plants under regular watering (every 4 days) or low rate of watering condition (every 10 days) was investigated. When watered regularly, the plant height of drought-tolerant line (#9) was shorter than NT plants. However, under the drought condition, total seed weight of line #9 was significantly higher than in NT plants (P < 0.01). Moreover, the pods of NT plants showed severe withering, and most of the pods failed to set normal seeds. All the evidences in the study clearly suggested that overexpression of the AtABF3 gene conferred drought and salt tolerance in major crop soybean, especially under the growth condition of low watering.

Keywords: Agrobacterium-mediated transformation; AtABF3; drought tolerance; soybean; stomatal closure.

Figures

Fig. 1
Fig. 1. Production of soybean transgenic plants with At-ABF3 gene using Agrobacterium-mediated transformation of half-seed explants
(A) Vector used for soybean transformation. Amplified AtABF3 (1,365 bp size) was sub-cloned into pB2GW7.0 vector for soybean transformation. LB/RB, left/right T-DNA border; p35S/T35S, CaMV (cauliflower mosaic virus) 35S promoter/terminator; Bar, coding region of the DL-phosphinothricin resistance gene. Probe used for Southern blot analysis is also indicated. SacI and EcoRI restriction enzyme sites are marked. (B) Production of AtABF3 transgenic soybean plants. (a) Co-cultivation of half-seed explants after inoculation (left) and at 5 days after inoculation (right). (b) Shoot induction on SIM without PPT for 14 days. (c) Shoot induction on SIM including 10 mg l−1 PPT for another 14 days. (d) Shoot elongation on SEM including 5 mg l−1 PPT. (e) Root formation. (f) Acclimation of putative transgenic plant in a small pot. (g) Transgenic plant (T0) grown in a large pot in the greenhouse. (h) Leaf painting with herbicide (100 mg l−1 PPT) showing sensitivity in NT plant (left) and resistance in transgenic plant (right). (C) Genomic Southern blot analysis of AtABF3 transgenic soybean. Ten micrograms of genomic DNAs were digested with EcoRI and hybridized with probe AtABF3. The approximate DNA size markers are indicated on the right. NT, non-transgenic; EV, transformed with empty vector carrying only Bar; #2, #3, #9 and #10, AtABF3 transgenic lines (T2).
Fig. 2
Fig. 2. Drought tolerance of AtABF3 transgenic plants (T2)
(A) Drought tolerance analysis of AtABF3 transgenic plants compared with NT and EV plants. Plants were grown on soil until leaves were fully expanded on 2 nodes, withheld from water for 20 days, and then rewatered (n = 12 each). The photographs were taken 15 and 20 days after drought treatment and 3 days after re-watering. (B) AtABF3 gene expression (bottom) with detached leaves of 20 days after drought treatment (top) using quantitative real-time PCR (qRT-PCR). (C, D) Ion leakage and chlorophyll content were measured at the indicated days after drought treatment from 2-node leaves of NT, EV and transgenic plants (n = 6 each). NT, non-transgenic; EV, transformed with empty vector carrying only Bar; #2 and #9, transgenic lines (T2). Error bars indicate mean ± standard deviation. Asterisks indicate significant changes compared with NT (*P < 0.05; **P < 0.01).
Fig. 3
Fig. 3. Transpiration rate of AtABF3 transgenic plants (T2)
Plants were grown on soil until leaves were fully expanded on 2 nodes, leaves were detached (n = 6) and weighed at the indicated times (A, B). The photographs were taken at the indicated times after drought treatment. NT, non-transgenic; EV, transformed with empty vector carrying only Bar; #2 and #9, transgenic lines (T2). Error bars indicate mean ± standard deviation. Asterisks indicate significant changes compared with NT (*P < 0.05; **P < 0.01).
Fig. 4
Fig. 4. Stomatal aperture of AtABF3 transgenic plants (T2)
Stomatal guard cells were observed using a microscope at the indicated days after drought treatment (DAT) from 2-node leaves of NT, EV and AtABF3 transgenic plants (n = 6 each). The ratios of stomatal closure of NT, EV and AtABF3 transgenic plants were measured. The stomata were assessed as completely open, partially open, and completely closed (n = 90 each). NT, non-transgenic; EV, transformed with empty vector carrying only Bar; #2 and #9, transgenic lines (T2). Arrows indicate guard cells. Error bars indicate mean ± standard deviation.
Fig. 5
Fig. 5. Salt tolerance of AtABF3 transgenic plants (T2)
(A, B) Salt tolerance analysis of AtABF3 transgenic plants compared with NT and EV. Plants were grown on wetted rock wool until leaves were fully expanded on 2 nodes, and then soaked in 200 mM NaCl solution for 11 days (n = 12 each). The photographs were taken 7 and 11 days after salt treatment. (C, D) Ion leakage and chlorophyll content were measured at the indicated days after salt treatment from leaves of NT, EV and transgenic plants (n = 6 each). NT, non-transgenic; EV, transformed with empty vector carrying only Bar; #2 and #9, transgenic lines (T2). Error bars indicate mean ± standard deviation. Asterisks indicate significant changes compared with NT (*P < 0.05; **P < 0.01).
Fig. 6
Fig. 6. Agronomic characters of non-transgenic (NT) and transgenic plants (T2) grown in the greenhouse
NT and T2 plants were grown in the greenhouse, and agronomic characters were investigated in regularly watered conditions (n = 8 each) (A) and in rarely watered conditions (n = 8 each) (B). NT, non-transgenic; #9, transgenic (T2) plant. Error bars indicate mean ± standard deviation. Asterisks indicate significant changes compared with NT (*P < 0.05; **P < 0.01).

Similar articles

See all similar articles

Cited by 4 articles

References

    1. Abdeen A., Schnell J., Miki B. Transcriptome analysis reveals absence of unintended effects in drought-tolerant transgenic plants overexpressing the transcription factor ABF3. BMC Genomics. 2010;11:69. - PMC - PubMed
    1. Bruce W.B., Edmeades G.O., Barker T.C. Molecular and physiological approaches to maize improvement for drought tolerance. J Exp Bot. 2002;53:13–25. - PubMed
    1. Choi H.I., Hong J.H., Ha J.O., Kang J.Y., Kim S.Y. ABFs, a family of ABA-responsive element binding factors. J Biol Chem. 2000;275:1723–1730. - PubMed
    1. Choi Y.S., Kim Y.M., Hwang O.J., Han Yj, Kim S.Y., Kim J.I. Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass. Plant Biotechnol Rep. 2013;7:165–173.
    1. Dan Y. Biological functions of antioxidants in plant transformation. In Vitro Cell Dev Biol Plant. 2008;44:149–161.

MeSH terms

Feedback