AtrbohD and AtrbohF negatively regulate lateral root development by changing the localized accumulation of superoxide in primary roots of Arabidopsis.

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CONCLUSION: NADPH oxidase AtrbohD an d AtrbohF negatively modulate lateral root development by changing the peroxidase activity and increasing the local generation of superoxide in primary roots of Arabidopsis in an auxin-independent manner. NADPH oxidase subunits AtrbohD and AtrbohF play pivotal roles in regulating growth, development and stress responses in Arabidopsis. However, whether they modulate lateral root (LR) formation has not yet been addressed, and the detailed mechanisms underlying the process remain unanswered. Here, we show that two null double mutants atrbohD1/F1 and atrbohD2/F2, in which both AtrbohD and AtrbohF genes are disrupted, had remarkably higher LR density than wild-type (WT), or the single mutant atrbohD1 and atrbohF1. Compared to WT, the double mutants exhibited early emerged LRs and enhanced density of lateral root primordia (LRP). Unexpectedly, the production of superoxide (O2 (-)), but not hydrogen peroxide, in the mature area of the primary root containing LRs significantly increased in the double mutants relative to that in WT. Further experiments revealed that the local accumulation of O2 (-) led to the enhancement of LR density in the double mutants. Moreover, the deficiency of AtrbohD and AtrbohF caused a marked increase in peroxidase activity in the mature root zone, which contributed to the localized accumulation of O2 (-) and the elevated LR density in the double mutants. Furthermore, the double mutants were not sensitive to exogenous auxin naphthalene acetic acid or auxin transport inhibitor 1-N-naphthylphthalamic acid in terms of LR formation. The auxin response of LRP in vivo in atrbohD1/F1 was also similar to that in WT. Taken together, these results suggest that AtrbohD and AtrbohF negatively modulate LR development by controlling the local generation of superoxide in an auxin-independent manner. These findings provide new insights into the mechanisms of NADPH oxidase-mediated regulation of LR branching in Arabidopsis.