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. 2020 Sep 9;11:543747. doi: 10.3389/fpls.2020.543747

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Copyright © 2020 Khaksar and Sirikantaramas

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Fold changes in expression levels of DzARF2A and ethylene biosynthetic genes in Nicotiana benthamiana leaves transiently expressing DzARF2A and in vivo transcriptional activity of DzARF2A in N. benthamiana leaves. (A) The relative expression levels of DzARF2A and ethylene biosynthetic genes from N. benthamiana; ACC synthase (NbACS) and ACC oxidase (NbACO) were measured in N. benthamiana leaves infiltrated with pGWB2-DzARF2A (treatment) and pGWB2-GFP (control) at three and five days after infiltration by using the 2^-ΔΔCt method. The levels were then normalized by the geometric mean of reference genes and the pGWB2-GFP as control. Three independent biological replicates were used. An asterisk (*) above the bars indicates a significant difference (P < 0.05). In the DzARF2A-infiltrated leaves, comparisons were also made between day 3 and day 5. An asterisk (*) above the bars indicates a significant difference between day 3 and day 5 (P < 0.05). (B) After three days of infiltration, firefly luciferase (LUC) and Renilla luciferase (REN) activities were measured and presented as relative LUC/REN ratio. The LUC/REN ratio in the control leaves co-infiltrated with the pGWB2-GFP and pGreenII-proDzACS or pGreenII-proDzACO was set as 1 and was used as a calibrator. Error bars represent means ± standard deviations (SD) among four independent biological replicates. An asterisk (*) above the bars indicates a significant difference compared to the control (Student’s t-test, P < 0.05).