Abstract
Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca2+/H+ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.
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Acknowledgements
We appreciate Dr. Wen-Hui Shen (Institut de Biologie Mole´culaire des Plantes (IBMP), Strasbourg Cedex, France) for supplying the seeds of ubc1ubc2 ko Arabidopsis.
Funding
This work was supported by the Bio-industry Technology Development Program funded by the Ministry for Food, Agriculture, Forestry and Fisheries [312033-5] and the Civil research projects for solving social problems through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning [NRF-2015M3C8A6A06014500], Korea.
Author contributions
S.H. designed and supervised whole research, and wrote the article with contributions of all the authors; R.B. and D.K. equally performed most of the experiments; B.L. performed an initial experiment.
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Ramin Bahmani and DongGwan Kim have contributed equally to this work.
Accession numbers: Sequence data from this article can be found in the NCBI (http://www.ncbi.nlm.nih.gov) data libraries under the following accession numbers: U60495 (NtActin), EB428626 (NtCAX3), HF675181 (NtHMA-A), HF937054 (NtHMA-B), AB263746 (NtIRT1), AB201240 (NtMTP1a), AB201241 (NtMTP1b), AB505625 (NtNRAMP1), AB505626 (NtZIP1), AB026055.1 (NtUBC1).
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Bahmani, R., Kim, D., Lee, B.D. et al. Over-expression of tobacco UBC1 encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (Nicotiana tabacum). Plant Mol Biol 94, 433–451 (2017). https://doi.org/10.1007/s11103-017-0616-6
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DOI: https://doi.org/10.1007/s11103-017-0616-6