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 longer led to the death of the root tip. The necrosis extended into the root axis during post-anoxic treatment, leading to the mortality of 30-50% of the seedlings. Using zymography, protease profiles in the root tissues of anoxic seedlings were studied. O₂ deprivation for 24 h or longer repressed pre-existing protease activities and induced a novel soluble enzyme in the roots. The anoxia-induced protease (ALP) activity was predominant in the root apex at 24 h of anoxia and, subsequently, became the most abundant soluble activity in the root axis as well. The induction of AIP and its in vitro renaturation were Ca²⁺-dependent. Inhibitor sensitivity studies indicated that AIP is a cysteine protease. In SDS-acrylamide gels, the enzyme activity migrated as a 23.5 kDa polypeptide. The anoxic induction of the activity was repressed 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. De-tipped seedlings showed lesser root damage and an increased production of lateral roots compared to intact seedlings. Furthermore, the superior anoxia tolerance of de-tipped seedlings was associated with a decreased AIP activity. Thus, the appearance of AIP activity in the root tip at 24 h of anoxia was spatially and temporally associated with the root tissue death. These studies further indicate that the root tip elimination early during anoxia may provide an adaptive advantage.