A Ca2+-dependent cysteine protease is associated with anoxia-induced root tip death in maize

Imposition of anoxia on maize (Zea mays cv, B73) seedlings for 48 h or long er led to the death of the root tip. The necrosis extended into the root ax is during postanoxic treatment, leading to the mortality of 30-50% of the s eedlings. Using zymography, protease profiles in the root tissues of anoxic seedlings were studied. O-2 deprivation for 24 h or longer repressed pre-e xisting protease activities and induced a novel soluble enzyme in the roots . The anoxia-induced protease (AIP) activity was predominant in the root ap ex at 24 h of anoxia and, subsequently, became the most abundant soluble ac tivity in the root axis as well. The induction of AIP and its in vitro rena turation were Ca2+-dependent. Inhibitor sensitivity studies indicated that AIP is a cysteine protease, In SDS-acrylamide gels, the enzyme activity mig rated as a 23.5 kDa polypeptide, The anoxic induction of the activity was r epressed by cycloheximide treatment, suggesting that new protein synthesis was required for the AIP appearance. Excision of the root tip (de-tipping) before anoxia led to a superior recovery of seedlings from stress injury. D e-tipped seedlings showed lesser root damage and an increased production of lateral roots compared to intact seedlings. Furthermore, the superior anox ia tolerance of de-tipped seedlings was associated with a decreased AIP act ivity. Thus, the appearance of AIP activity in the root tip at 24 h of anox ia was spatially and temporally associated with the root tissue death. Thes e studies further indicate that the root tip elimination early during anoxi a may provide an adaptive advantage.