the implication of vacuolar or peroxisomal activities in hydrolysis of proteins from other organelles seems
doubtful, at least throughout the first stages of senescence, when the compartmental integrity of the cell
has been firmly established. Nevertheless, these compartmental barriers may be progressively overridden
throughout senescence. For example, it has been shown in apoptotic animal cells that the activity of 15-
lipoxygenase renders the membranes of organelles leaky to proteins, thus allowing a certain degree of
protease exchange between cellular compartments [127]. In any case, the fusion of organelles with vac-
uoles during the final phases of cell death [56] surely results in those activities gaining access to the re-
maining substrates and producing the ultimate breakdown of the organellar structure. Similar compart-
mental restrictions may limit the activity of the proteasome, which is present only in nucleus and
cytoplasm of intact plant cells. Although some evidence of proteasome processing of ubiquitin-labeled
proteins during plant senescence has been gathered (reviewed in Ref. 128), expression of the proteasome
-type subunit has been shown to decrease during flower and leaf senescence in tobacco, suggesting that
the plant proteasome is likely to play a regulatory role in developing tissues rather than be involved in
massive senescent degradation [129].
In instances in which no correlation between senescence and increased proteolytic activities has been
found [112], the loss of protein during senescence may be due to natural turnover after arrest of synthe-
sis or to modification of the proteins that may label them for proteolysis. In this regard, the case of the
chloroplastic CO 2 -fixing enzyme, Rubisco, appears to be paradigmatic. In some species, such as corn and
wheat, the enzyme seems to be degraded as a result of natural turnover [2,130]. However, in most species
the turnover rate of Rubisco is negligible under nonsenescent conditions [131,132] but changes dramati-
cally with the onset of senescence. It has been shown that the susceptibility of Rubisco to proteases in-
creases markedly through oxidation of sulfhydryl groups belonging to critical cysteine residues of the en-
zyme [133–135]. This suggests that Rubisco degradation may be induced by the oxidative conditions
developed inside the functionally impaired chloroplast during senescence [136]. Evidence for in vivo ox-
idation of Rubisco has been found in different organisms under stress-induced senescence [137–139].
Moreover, a chloroplastic proteolytic activity that is activated by oxidative conditions has been described
[140]. This suggests that alteration of the redox state of the chloroplast may provide a general mechanism
for triggering a selective protein degradation during senescence or other processes that arrest chloroplast
function.
Certain enzymatic activities other than hydrolases are also known to be enhanced during senescence.
For example, transition of leaf peroxisomes to glyoxisomes is a well-characterized phenomenon associ-
ated with senescence. Accordingly, enzymatic activities of markers of the glyoxylate cycle have been
shown to increase dramatically during darkness-induced senescence of spinach leaves [141] and pump-
kin cotyledons [142]. Cytosolic glutamine synthetase also increases about fourfold during senescence of
rice leaves [143]. This rise surely facilitates the mobilization of nitrogen by enhancing the synthesis of
the major transported amino acid (glutamine) [144]. Interestingly, transgenic overexpression of glutamine
synthetase leads to accelerated development and early senescence of plants grown in ammonium-rich
medium [145]. In addition, different isozymes of threonine dehydratase, an enzyme that probably plays a
role in nitrogen remobilization, are specifically synthesized in senescing tomato leaves [146].
- Free Radicals and Antioxidant Enzymes
The relationship between the internal production of free radicals and senescence is well established [147].
Free radicals derived from oxygen such as superoxide, hydroxyl, peroxyl, and alkoxy radicals, as well as
other molecular forms of incompletely reduced oxygen, are known as reactive oxygen intermediates
(ROIs). ROIs are considered both as primary mediators of oxidative damage during senescence and as
signals that trigger cellular defensive responses. In particular, superoxide has been shown to mediate the
spreading of the hypersensitive response (leading to cell death) in mutants of Arabidopsis thalianathat
exhibit spontaneous lesions [148]. ROIs may be generated as by-products of some enzymatic reactions,
but plants specifically produce these radicals as a consequence of photosynthesis [149]. Production of
ROIs is enhanced during late stages of senescence because of the impairment of the electron flow between
the two photosystems, which limits the availability of photosynthetic power [150]. Free radicals are
known to induce the breakdown of nucleic acids, polysaccharides, and proteins [151] and enhance the
ethylene production pathway [152]. In addition, ROIs and other free radicals may initiate a chain reaction
in membranes leading to extensive lipid peroxidation and subsequent alterations of fluidity and perme-
ability [147]. Under normal conditions, oxygen-detoxifying enzymes, such as superoxide dismutases,
SENESCENCE IN PLANTS AND CROPS 189