Aspartic proteases (APs) represent the second largest class of plant proteases after serine proteases. Members of the pepsin-like family are widely distributed in plants but, somehow unexpectedly, this family of proteases has been much less studied than other classes of proteases. Strikingly, whereas mammals contain fewer APs coded in their genomes (Homo sapiens has 17), a large representation of AP genes is found in land plants: 70 homologues in Arabidopsis thaliana and 166 in Oryza sativa genome. This clearly suggests that the overrepresentation of APs in plants may represent an important role in diversification of protein functions. Indeed, some functions are starting to be uncovered, with proposed roles in highly regulated processes like resistance to biotic/abiotic stresses, PCD, plastid homeostasis, and reproduction, clearly suggesting functional specialization of plant APs and tight activity regulation.
In this work, two Arabidopsis AP genes were characterized. Phenotypic analysis of T-DNA mutants for each gene revealed significant reductions in primary root growth length and in lateral root number. These phenotypes were evaluated under drought and nutrient limitation, with results suggesting that these genes may be involved in two different regulatory mechanisms in lateral root formation. Therefore, these genes were designated RLR1 and RLR2 (Regulator of Lateral Root). RLR1 gene product was produced using the innovative plant-based expression platform magnICON® in Nicotiana benthamiana leaves and biochemically characterized. RLR1 was shown to be glycosylated, active at acidic pHs, not completely inhibited by pepstatin, and with a distinct specificity pattern determined by PICS. Redox agents have a significant inhibitory effect on RLR1 activity. Identification of putative substrates of RLR1 and RLR2 using iTRAQ analysis is currently ongoing.
Our results unveil a new role for APs in the regulation and adaptation of root development in Arabidopsis under different growth conditions, namely under abiotic stresses.