Proteases associated with programmed cell death of megagametophyte cells after germination of white spruce (Picea glauca) seeds

During post-germinative seedling growth, the major storage organ of the white spruce (Picea glauca) seed, the megagametophyte, undergoes programmed cell death (PCD). Protease activities in megagametophyte cells that arise post-germinatively were investigated. The accumulation of protease activities can be divided into two phases: the first phase correlated with degradation of storage proteins while the second phase was temporally associated with cell death, although some of the early proteases were also active during the later phase. Proteases induced during PCD were mainly serine and cysteine proteases. One of the PCD-associated cysteine proteases had homology to Cys-EP, a PCD-related cysteine protease of the castor bean endosperm. Transcripts encoding a Cys-EP-related protein were not present in megagametophytes when seeds were imbibed, nor were they present during germination and early post-germinative growth (radicle length ca. 2-5 mm). At a later post-germinative stage (i.e when the seed's radicle was ca. 15 mm), the Cys-EP-related transcripts (ca. 1.3 kb) became abundant and, at this time, the 48 kDa proform of the enzyme first appeared. The mature form of the Cys-EP (ca. 38 kDa) was predominant at a very late stage of post-germinative growth. Immunocytochemistry showed that the Cys-EP-related protein was localized to spherical organelles (ca. 2 mum) that may be equivalent to the 'ricinosomes' of castor bean endosperm cells. Caspase-like protease (CLP) activities were first detected 3 days after germination with the caspase-specific substrate Ac-DEVD-AMC; maximum activities occurred when the seed's radicle was ca. 20-25 mm. When germinated seeds were treated with a caspase-3 inhibitor, both the peak of CLP activities and the death of megagametophyte cells were delayed. We propose that the Cys-EP-related protein and CLP activity are involved in PCD of white spruce megagametophyte cells.