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 H 2 O 2 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.