Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes

Abstract

The cells of the endosperm of castor bean seeds (Ricinus communis) undergo programmed cell death during germination, after their oil and protein reserves have been mobilized. Nuclear DNA fragmentation first was observed at day 3 in the endosperm cells immediately adjacent to the cotyledons and progressed across to the outermost cell layers by day 5. We also detected the accumulation of small organelles known as ricinosomes, by using an antibody against a cysteine endoprotease. By the time the nuclear DNA was susceptible to heavy label by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, the ricinosomes had released into the cytoplasm their content of cysteine endoprotease, which became activated because of the cleavage of its propeptide. The cysteine endoprotease is distinguished by a Cterminal KDEL sequence, although it is not retained in the lumen of the endoplasmic reticulum and is a marker for ricinosomes. Homologous proteases are found in the senescing tissues of other plants, including the petals of the daylily. Ricinosomes were identified in this tissue by electron microscopy and immunocytochemistry. It seems that ricinosomes are not unique to Ricinus and play an important role in the degradation of plant cell contents during programmed cell death.Ricinus communis ͉ Hemerocallis sp. ͉ papain-type KDEL peptidase

Programmed cell death eliminates all but one embryo in a polyembryonic plant seed

“…Noteworthy, PCD in a gymnosperm female gametophyte has not previously been identified, whereas angiosperm endosperm has been shown to initiate suicide program either before or during seed germination. 19,20 Indeed, we did find a highly organized pattern of progression of nuclear DNA fragmentation (one of the well-known hallmarks of endosperm PCD 19 ) throughout the female gametophyte ( Figure 5A). At the time when the dominant embryo has been selected and started active growth, all the cells in the female gametophyte seem to be viable ( Figure 5A; 3 waf, upper row), except for a narrow, unicellular layer that lines the corrosion cavity ( Figure 5A; 3 waf, second row).…”

The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum

“…Ricinosomes were isolated from 5-day-old germinating castor bean endosperm (11) because, at this stage of development, the yield is highest and protein bodies are almost completely degraded (5). Ricinosomes isolated on a discontinuous sucrose gradient (10% to 57% sucrose) copurify with glyoxysomes at the interface between the 50 and 57% sucrose steps (cf.…”

“…4). Plant cells undergoing senescence by PCD express papain-type cysteine endoproteases with a C-terminal KDEL sequence (5). They attract attention because they are present in the senescing endosperm cells of castor bean (Ricinus communis) as relatively inactive 45-kDa proenzymes until the final stages of PCD, at which time they are processed into the 35-kDa mature form with a 50-100 times higher activity (6).…”

“…In castor bean, daylily, and mung bean, the KDEL-tailed propeptidase is stored in a special organelle, called ricinosome (5,6) or KDEL-tailed cysteine proteinaseaccumulating vesicles (KV) (7). Ricinosomes were first described and ultrastructurally characterized in the endosperm of R. communis (8,9), whereas KV were described as cytoplasmic ''foci'' in Vigna radiata (10).…”

“…Intact ricinosomes accumulate the 45-kDa proCysEP as shown by Western blot analysis and by in situ fluorescence labeling with antibodies directed against the propeptide. Disintegration of ricinosomes during the final cell collapse releases the mature enzyme, giving a diffuse labeling of collapsed cells near the cotyledons (5).…”