Identification and characterization of two waterlogging responsive alcohol dehydrogenase genes (AdADH1 and AdADH2) in Actinidia deliciosa

Abstract

Previous study showed that two alcohol dehydrogenase genes (ADHs) were significantly upregulated in roots of Actinidia deliciosa after treatment with waterlogging using Illumina sequencing technology. Those two ADH genes, named AdADH1 and AdADH2, were isolated from A. deliciosa in this study. QRT-PCR analysis showed that AdADH1 and AdADH2 expression levels were significantly increased in A. deliciosa after treatment with waterlogging, NaCl, 4 °C, and heat stresses, but not ABA and drought stresses. The changes of AdADH1 and AdADH2 expression levels during waterlogging were much higher than those during other stresses. We examined the response to abiotic stresses in transgenic Arabidopsis lines in which the A. deliciosa AdADH1 and AdADH2 genes were introduced under the control of a constitutive promoter, respectively. Overexpression of A. deliciosa AdADH1 or AdADH2 in Arabidopsis could enhance waterlogging tolerance at five week old seedlings. However, the function of AdADH1 for waterlogging resistance was much better than that of AdADH2. Arabidopsis overexpressing AdADH1 gene could enhance the resistance to cold stress at five week old seedlings, but AdADH2 could not. Overexpression of AdADH1 gene enhances the resistance to salt stress at the stage of seed germination and in seedlings, and overexpression of AdADH2 gene enhances the resistance to salinity stress at the stage of seed germination but not in seedlings. However, transgenic Arabidopsis overexpressing AdADH1 or AdADH2 could not enhance to the osmotic stress at the stage of seed germination and in seedlings. These results indicated that A. deliciosa AdADH1 plays an important role in resistance to waterlogging, salinity, and cold stresses, but not drought stress, and AdADH2 plays an important role in resistance to waterlogging and salinity stresses, but not cold and drought stresses.

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Abbreviations

ABA:

Abscisic acid

ADH:

Alcohol dehydrogenase

CaMV:

Cauliflower mosaic virus

HRE1/2 :

Hypoxia responsive ERF1/2

IBA:

Indole-3-butytric acid

MS:

Murashige and Skoog

PDC:

Pyruvate decarboxylase

qRT-PCR:

Real time quantitative RT-PCR

WT:

Wild type

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Acknowledgements

This study was supported by the National Natural Science Foundation of China (NSFC) (31401854) and grants from the Natural Science Foundation of Jiangsu Province (G rants No BK20140760).

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Correspondence to Zhong-Ren Guo.

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Electronic supplementary material

Supplemental Table 1

Primers used for this paper (DOCX 19 kb)

Supplemental Fig. 1

Derived amino acid sequences alignment of AdADH1 and AdADH2 with proteins from Oryza sativa (OsADH1, Genbank accession no. AAF34414; OsADH2, Genbank accession no. AAF34412) and Vitis vinifera (VvADH1, Genbank accession no. NP_001268079; VvADH2, Genbank accession no. NP_001268083). Dots indicate that amino acids sequences are conserved. The major functional domains are indicated, along with the binding sites of catalytic (solid triangle) and structural (solid diamond) Zn ions. (GIF 55 kb)

High Resolution Image (TIFF 3541 kb)

Supplemental Fig. 2

Phylogenetic analyses of ADH proteins. The deduced amino acids sequences were obtained from the NCBI, the species and the accession numbers are indicated. The phylogenetic tree was conducted using the MEGA5 based on the full-length amino acid sequences. Bootstrap analysis with 1000 replicates was performed to obtain confidence levels for the branches of the tree. (GIF 30 kb)

High Resolution Image (TIFF 3045 kb)

Supplemental Fig. 3

Comparison of germination rates and root length between AdADH1 transgenic (ADH1–1, ADH1–2, ADH1–3) and wild-type (WT) lines under NaCl stresses. A photograph was taken 10 d after seed germination of WT and transgenic Arabidopsis plants in MS medium containing various concentrations of NaCl (a). Germination rates (c) in WT and transgenic plants grown in MS (control) or MS supplemented with various concentrations of NaCl for 7 d. Germination rates results are presented as mean ± SD from three independent experiments (at least 50 seeds of each line were grown for each experiment). Four day old seedlings of WT and transgenic seedlings which were germinated just from MS agar medium were transferred to MS medium containing different concentrations of NaCl. The phenotypic (b) and root length (c) were measured after 7 days. The experiments were carried out on three replicates of 30 seedlings. The results from one set of experiments are shown. Double asterisks indicate significant differences compared with WT lines values (P < 0.01). (GIF 207 kb)

High Resolution Image (TIFF 12277 kb)

Supplemental Fig. 4

Comparison of germination rates and root length between AdADH2 transgenic (ADH2–1, ADH2–2, ADH2–3) and wild-type (WT) lines under NaCl stresses. A photograph was taken 10 d after seed germination of WT and transgenic Arabidopsis plants in MS medium containing various concentrations of NaCl (a). Germination rates (b) in WT and transgenic plants grown in MS (control) or MS supplemented with various concentrations of NaCl for 7 d. Germination rates results are presented as mean ± SD from three independent experiments (at least 50 seeds of each line were grown for each experiment). Four day old seedlings of WT and transgenic seedlings which were germinated just from MS agar medium were transferred to MS medium containing different concentration of NaCl. The phenotypic (c) and rooting length (d) were measured after 7 days. The experiments were carried out on three replicates of 30 seedlings. The results from one set of experiments are shown. Double asterisks indicate significant differences compared with WT lines values (P < 0.01). (GIF 200 kb)

High Resolution Image (TIFF 13390 kb)

Supplemental Fig. 5

Comparison of germination rates and root length between AdADH1 transgenic (ADH1–1, ADH1–2, ADH1–3) and wild-type (WT) lines under mannitol stresses. A photograph was taken after 10 d of seed germination of WT and transgenic Arabidopsis plants in MS medium containing various concentrations of mannitol (a). Germination rates (b) in WT and transgenic plants grown in MS (control) or MS supplemented with various concentrations of mannitol for 7 d. Germination rates results are presented as mean ± SD from three independent experiments (at least 50 seeds of each line were grown for each experiment). Four day old seedlings of WT and transgenic seedlings which were germinated just from MS agar medium were transferred to MS medium containing 0.15 M mannitol. The phenotypic (c) and rooting length (d) were measured after 7 days. The experiments were carried out on three replicates of 30 seedlings. The results from one set of experiments are shown. (GIF 119 kb)

High Resolution Image (TIFF 4292 kb)

Supplemental Fig. 6

Comparison of germination rates and root length between AdADH2 transgenic (ADH2–1, ADH2–2, ADH2–3) and wild-type (WT) lines under mannitol stresses. A photograph was taken after 10 d of seed germination of WT and transgenic Arabidopsis plants in MS medium containing various concentrations of mannitol (a). Germination rates (c) in WT and transgenic plants grown in MS (control) or MS supplemented with various concentrations of mannitol for 7 d. Germination rates results are presented as mean ± SD from three independent experiments (at least 50 seeds of each line were grown for each experiment). Four day old seedlings of WT and transgenic seedlings which were germinated just from MS agar medium were transferred to MS medium containing 0.15 M mannitol. The phenotypic (b) and rooting length (d) were measured after 7 days. The experiments were carried out on three replicates of 30 seedlings. The results from one set of experiments are shown. (GIF 115 kb)

High Resolution Image (TIFF 4080 kb)

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Zhang, JY., Huang, SN., Chen, YH. et al. Identification and characterization of two waterlogging responsive alcohol dehydrogenase genes (AdADH1 and AdADH2) in Actinidia deliciosa . Mol Breeding 37, 52 (2017). https://doi.org/10.1007/s11032-017-0653-5

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Keywords

  • Abiotic stresses
  • Actinidia deliciosa
  • AdADH1
  • AdADH2
  • Waterlogging
  • Transgenic Arabidopsis