Programmed Cell Death during Plant Senescence - Postharvest Biology and Technology of Fruits, Vegetables, and Flowers - page 96

process for the release of proteolytic activities, which resembles the regulation of PCD
during apoptosis in animal cells (Gietl and Schmid, 2001).
Also in the extracellular matrix, a metalloproteinase (CSL-MMP) induced late in the
senescence process of Cucumis cotyledons (along with DNA laddering) suggests that these
protease activities may help to eliminate the later cell remnants, as has been described for
its orthodoxy in animals (Delorme et al., 2000).
Lately, many of the components of the autophagy molecular machinery in Arabidop-
sis have been identified based on their homology to the genes identified in Saccha-
romyces cerevisiae (Hanaoka et al., 2002), which makes possible to study the contribu-
tion of this pathway to senescence. Recent evidence indicates that the APG pathway is
upregulated during senescence, although compared to SEN1 mRNA (a vacuolar cysteine
endopeptidase), accumulation of APG7 and APG8 mRNAs occurs rather late, suggest-
ing that the APG system may function later than other molecular responses induced during
senescence (Doelling et al., 2002). Disruption of either of two APG genes ( APG9 or APG7 )in
Arabidopsis produces an accelerated senescence phenotype (both natural and dark-induced)
and starvation-induced chlorosis. This indicates that the APG pathway is required for effi-
cient nutrient recycling and senescence in Arabidopsis (Doelling et al., 2002; Hanaoka et al.,
2002).
It is tempting to speculate that, in addition to their role in N-mobilization, some plant
proteolysis activities (Delorme et al., 2000) could be involved in a cascade of proteolysis
activation regulating PCD event. Elucidation of the substrates for these activities is needed
to clarify their role during senescence.
In addition to substrate and proteases being differently compartmentalized and only
activated and released at the right time, substrate susceptibility for proteolysis appears to
be important in senescence and in other stress-related processes (Pefiarrubia and Moreno,
1990; Cotelle et al., 2000). This is best exemplified for chlorophyll a

b binding proteins
(Cab proteins) that remain stable as far as they are complexes with chlorophyll. Removal
of chlorophyll during senescence increases dramatically Cab susceptibility to proteolysis.
Inhibition of chlorophyll degradation, as in stay-green mutants, increases notably the sta-
bility of Cab, while the rest of the senescence proteolysis and other senescence processes
continue as normal (Thomas and Howarth, 2000). Not only structural changes in substrate
susceptibility are important for proteolysis: binding of 14-3-3s proteins to a wide range of
plant proteins can also induce changes in their stability (Cotelle et al., 2000). The extent
to which this mechanism is important for senescence is indicated by the stay-green phe-
notype and delayed leaf senescence of transgenic potatoes overexpressing 14-3-3s proteins
(Markiewicz et al., 1996). This suggests that proteolysis of the 14-3-3s targets participates
in the progression of senescence. The regulation of 14-3-3s levels could function as a mech-
anism to modulate senescence, probably through the protection exerted by 14-3-3s proteins
on their targets. Conversely, transgenic plants with diminished levels of 14-3-3s proteins
showed an early senescence phenotype (Wilczynski et al., 1998), thus closing the circle and
further supporting the relevance this mechanism may have during senescence.
The nature of SAGs-encoding activities, which are involved in nutrient salvage program,
ranges from glyoxysomal enzymes involved in fatty acid mobilization, like 3-ketoacyl-CoA
thiolase in pumpkin (Kato et al., 1996) or malate synthase gene in cucumber (Graham et al.,
1992; Buchanan-Wollaston, 1997) to cell wall-metabolizing enzymes (Lee et al., 2001)
such as

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β -glucosidases (Callard et al., 1996). Nucleases probably involved in phosphate