At the beginning of the life cycle of plants propagated by seed, cells die in the process of germination. For example, aleurone cells in cereal grain seeds facilitate germination; however, they also die in the process. Their death fits the general definition of PCD, and attention has been turned to the death process. GA specifically accelerated cell death in isolated aleurone cells and protoplasts, and the GA-signaling inhibitor LY83583 prevented DNA degradation and cell death (Kuo et al., 1996; Bethke et al., 1999). ABA delayed cell death and overcame the hastening effect of GA. During germination aleurone cells expressed characteristics of apoptosis including positive TUNEL staining and DNA cleavage into internudeosomal fragments (Wang et al, 1996c). Subsequently these findings were shown as likely to be technical artifacts due to nucleases in the aleurone cells and in the enzymes used to prepare protoplasts (Fath et al., 1999). Other symptoms of PCD in protoplasts included increased vacuolation, abrupt loss of plasma membrane integrity and then rapid shrinkage of the remainder of the protoplast (Bethke et al., 1999). While DNA hydrolysis began before death, it did not result in accumulation of uniform, low molecular weight fragments.
Some of the proteases and nucleases induced during germination may be specifically involved in PCD of barley aleurone cells. GA promotes PCD and up regulates proteases and nucleases while ABA prevents cell death and inhibits synthesis of these enzymes (Jones and Jacobsen, 1991; Kuo et al, 1996; Wang et al, 1996c; Fath et al., 1999). Some of the proteases are secreted into the endosperm for germination-linked hydrolysis. Other proteases accumulate in vacuoles in the aleurone cells (Bethke et al., 1996) and are presumed to be active in the death of these cells. Secretion of a-amylase from aleurone cells begins 4 to 6 h after GA treatment, while nuclease secretion begins 24 to 36 h after GA treatment (Brown and Ho, 1986). This timing is consistent with involvement of the nuclease(s) in cell death; three nucleases are present (Fath et al., 1999).
Receptors for GA and ABA appear to be on the plasma membrane, as assessed mainly by a-amylase induction, but there is also evidence for a cytoplasmic receptor for the ABA-regulated Em gene (Gilroy, 1996). GA was shown to stimulate transport of Ca2+ into ER isolated from aleurone cells (Bush et al., 1989); GA-enhanced synthesis of a-amylase had previously been shown to require millimolar concentrations of calcium (Bush et al., 1986). In contrast, ABA decreased Ca2+ concentrations in the cytoplasm. Okadaic acid blocked Ca2+ changes, gene expression and cell death induced by GA (Kuo et al., 1996), and it had a similar but less pronounced effect on ABA-induced responses. This suggests that serine/threonine protein phosphatases, which okadaic acid inhibits, are essential for the action of GA on aleurone cells, including its promotion of PCD. Protein kinase, G-protein and lipase inhibitors were ineffective on the wheat aleurone layers studied. As noted earlier, ABA inhibited PCD in barley aleurone, and this ABA effect was promoted by okadaic acid (Wang et al., 1996c). Further, ABA induced a rapid and transient rise in mitogen-activated protein (MAP) kinase activity in barley aleurone protoplasts (Knetsch et al., 1996). ABA also increased the enzyme activity of phospholipase D and increased its product, phosphatidic acid (PPA). Application of PPA caused ABA-like inhibition of a-amylase, and inhibition of phospholipase D also inhibited ABA-inducible responses. Taken together, the evidence is consistent with existence of independent signal transduction pathways for GA and ABA which eventually meet at a common point related to a-amylase production and, presumably, PCD.
Reactive oxygen species (ROS) have been added to the proposed signal transduction pathway for PCD in aleurone cells (Fath et al., 2001). Hydrogen peroxide promoted PCD, and in GA-promoted PCD hydrogen peroxide scavenging enzymes and mRNA for catalase 2 were down regulated. ABA, on the other hand, maintained RSO scavenging enzyme activity and delayed cell death.
The scutellum of the barley embryo also dies during germination. An animal cell death suppressor gene named dad (defender against apoptotic cell death) (Gallois et al., 1997; Tanaka et al., 1997) has been studied in scutellum (Lindholm et al., 2000). Mammalian dadl encodes a subunit of an enzyme for N-linked glycosylation located on the ER (Kelleher and Gilmore, 1997). During PCD of scutellum cells, expression of dadl declined before DNA fragmentation occurred while dad2 and ostl, which encodes another subunit of the transferase complex, did not decline. The complex glycosylates a subunit of phytepsin, a vacuolar proteinase, and the failure of this step may be involved in the onset of PCD. Dad has also been studied in PCD of root cortical cells (see later).
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