Arabidopsis ubiquitin-specific proteases UBP12 and UBP13 shape ORE1 levels during leaf senescence induced by nitrogen deficiency

Nitrogen deficiency (-N) in plants triggers leaf senescence which is regulated by the transcription factor ORE1. Little is known about post-translational regulation of ORE1 in this process. Here, we show that UBP12/UBP13 (ubiquitin-specific protease 12/13) antagonize the action of NLA (nitrogen limitation adaptation) E3 ligase to maintain ORE1 homeostasis. In vitro pull-down and in vivo co-immunoprecipitation assays demonstrated specific binding between UBP12/UBP13 and ORE1. We further analyzed in various genotypes total Chl content and expression levels of senescence-related genes under -N conditions. We found that UBP12/UBP13 can deubiquitinate polyubiquitinated ORE1 in vitro and increase the stability of ORE1 in vivo in MG132/cycloheximide-chase experiments. Plants overexpressing UBP12/UBP13 display accelerated leaf senescence which is reversed by the ore1 mutation. By contrast, the senescence phenotype of plants overexpressing ORE1 is exacerbated by UBP12/UBP13 overexpression. The expression of senescence-related genes tracks the senescence phenotype. ORE1 protein levels can be elevated by UBP12/UBP13 overexpression but decreased in ubp12-2w/13-3. In conclusion, UBP12/UBP13 deubiquitinate ORE1 to stabilize this transcription factor and promote its activity as a positive regulator for leaf senescence under -N conditions. Our study shows that UBP12/UBP13 counteracts the effect of NLA E3 ligase to accelerate leaf senescence under nitrogen starvation.