A Brachypodium distachyon MAPKK Gene BdMKK6.2 Negatively Regulates Drought Stress Tolerance in Transgenic Tobacco Plants

Plant MAPK cascades have been implicated in various developmental processes and in response to stresses. MAPKKs, a convergent linking point between MAPKKKs and MAPKs, play pivotal roles in MAPK cascades to regulate various stress responses in plants. Few MAPKKs have been functionally characterized in Brachypodium distachyon, a new monocot model plant. In this study, we cloned and characterized a group A MAPKK gene, designated as BdMKK6.2, from B. distachyon. Quantitative RT-PCR analysis revealed that BdMKK6.2 expression was upregulated by cold, heat, and H2O2 treatments but downregulated by PEG and ABA treatments. Transient expression of BdMKK6.2 in onion epidermal cells suggested that it was exclusively localized in the cytoplasm. Overexpression of BdMKK6.2 in transgenic tobacco plants increased their sensitivity to osmotic stress during seed germination. Moreover, BdMKK6.2 overexpression resulted in reduced tolerance to drought stress. Physiological-biochemical analyses showed that BdMKK6.2-overexpressing plants had decreased survival rates but increased water loss rates, ion leakage, malondialdehyde content, and reactive oxygen species accumulation under drought condition. In addition, the transcriptional levels of two ROS-producing genes NtRbohD and NtRbohF were upregulated, but a stress-responsive gene NtNCED1 was downregulated in BdMKK6.2-overexpressing plants compared to wild type plants. These results indicate that BdMKK6.2 is a negative regulator of drought stress response through influencing ROS homeostasis and transcription of stress-responsive gene.