Involvement of active MKK9-MAPK3/MAPK6 in increasing respiration in salt-treated Arabidopsis callus

Mitogen-activated protein kinase kinase 9 (MKK9) is an upstream activator of mitogen-activated protein kinase 3 (MAPK3) and MAPK6 in planta. To investigate MKK9 roles in mitochondrial respiration in Arabidopsis, MKK9^DD, the active allele with mutations of Thr-201 and Ser-205 to Asp, and MKK9^KR, the allele lacking MKK9 activity with a mutation of Lys-76 to Arg, were used. Results showed that the total respiratory rate (V_t), alternative pathway capacity (V_alt) and cytochrome pathway capacity (V_cyt) increased under 0-100 mM NaCl treatments but decreased under 150-300 mM NaCl treatments in Col-0 callus. However, the activation of MKK9 by dexamethasone (DEX) increased V_t, V_alt and V_cyt under 200 mM NaCl treatment; moreover, V_alt showed more increase than V_cyt. The activation of MKK9 in MKK9^DD callus sharply increased AOX protein expression under normal and NaCl conditions, but the increase was not observed in MKK9^KR callus. Further results indicated that MAPK3 and MAPK6 were involved in the MKK9-induced increase of AOX protein levels. qRT-PCR results showed that MKK9-MAPK3/MAPK6 was involved in the NaCl-induced AOX1b and AOX1d expression, but only MKK9-MAPK3 was necessary for AOX2 expression; in addition, MAPK3 regulated the AOX1a transcription in an MKK9-independent manner. MKK9 positively regulated SOD and CAT activities by affecting MAPK3 and MAPK6 and negatively regulated APX and POD activities by affecting MAPK3. Moreover, MKK9 functions as a positive factor in H₂O₂ accumulation under salt stress. The regulation of ethylene on alternative respiration was also associated with MKK9 under salt stress. Taken together, the MKK9-MAPK3/MAPK6 pathway plays a pivotal role in increasing alternative respiration in the salt-treated Arabidopsis callus.