Investigations On The Molecular Mechanisms For Lateral Root Development Regulated By Atrbohd And Atrbohf In Arabidospsis

Reactive oxygen species （ROS） have been considered to play essential roles in root development inplants. However, the molecular mechanisms for which ROS regulate lateral root formation are largelyunknown. Here, we report that AtrbohD and AtrbohF, two ROS-related protein subunits, modulate lateralroot development in Arabidopsis.In the previous study, we found that mutations in both AtrbohD and AtrbohF led to the promotion oflateral root formation. Phenotype analysis suggested that the LR density in the double mutant atrbohD1/F1and atrbohD2/F2was significantly higher than that in wild type （WT）, atrbohD1and atrbohF1plants.Moreover, LRs in the two double mutants emerged remarkably earlier than those of WT, and the singlemutants. These results strongly suggest that disruptions in both AtrbohD and AtrbohF accelerate the LRPdevelopment and the LR emergence in Arabidopsis.Both AtrbohD and AtrbohF can catalyze the production of H₂O₂and O₂˙, which are proposed tofunction in plant root development. Therefore, the levels of H₂O₂and O₂˙in the different regions ofprimary roots from WT and the double mutant seedlings were monitored. Results showed that the levels ofH₂O₂in the primary root tip （including the root cap, meristemic zone and elongation zone） and thematuration zone in WT were significantly higher than those in the double mutants. Surprisingly, the levelsof O₂˙in the primary root maturation zone in WT were clearly lower than those in the double mutants roottip although O₂˙production in the root tip of WT is greater than that of the mutants. To determine ifperoxidase is involved in the O₂˙generation in the double mutant plants, peroxidase activity was assayed.Staining experiments revealed that the peroxidase activity in the roots of atrbohD1/F1andatrbohD2/F2was markedly higher than that of WT. These results imply that AtrbohD and AtrbohF may negatively modulate lateral root development by changing the distribution of superoxide, which is mediatedbyperoxidase.In addition, GUS stainings in the transgenic plants with AtrbohD promoter or AtrbohF promoter wereanalyzed. Both AtrbohD and AtrbohF are strong expressed in the primary root vascular cylinder and theLRP. The former includes the pericycle where Arabidopsis lateral roots originate.Experiments with auxin and an auxin inhibitor N-1-naphthylphthalamic acid （NPA） revealed that NPAinhibited the lateral root development of the double mutants; and the mutants are more sensitive to NPAthan WT. The expression of some auxin related genes in the double mutants were remarkedly upregulatedin comparison with that in WT. These results suggest that auxin signaling is involved in lateral rootformation regulated by AtrbohD and AtrbohF in Arabidopsis.