Postharvest Biology and Technology of Fruits, Vegetables, and Flowers

PROGRAMMED CELL DEATH DURING PLANT SENESCENCE 111
posttranscriptional mechanisms that function during plant PCD, a proteomic analysis of
changes in total cellular protein content was performed during both heat- and senescenceinduced
PCD in an Arabidopsis cell culture. Both PCD systems were accompanied by a
decreased protein content and an increased proteolytic activity. Analysis of two-dimensional
gel electrophoresis displays of proteins revealed 11 proteins whose abundance (relative to
total protein) increased following both treatments. The relative increase of these proteins in
both heat- and senescence-induced PCD system suggests that they may play a general role
in the plant cell death program (Swidzinski et al., 2002; Fig. 5.4).
5.16.1 Proteomic analysis of heat- and senescence-induced PCD
To identify proteins that are important in the PCD pathway, Swidzinski et al. (2002) fractionated
equal amounts of protein from control cells, from heat-treated cells, and from senescent
cells using two-dimensional gel electrophoresis. Sets of protein spots that increased in relative
abundance (PCD/control) of at least twofold in comparison to the control in three
replicate gels were identified. The increase in these proteins was statistically significant.
From these sets, a subset of proteins that increased in abundance in both the heat-treated
cells and the senescent cells was identified. Twelve protein spots were commonly increased
in relative abundance in both treatments relative to the control, healthy cell cultures. These
spots were excised from the gel, digested with trypsin, and the proteins identified using
tandem MS/MS mass spectrometry. Four of these spots are isoforms of catalase, while
several, including lipoamide dehydrogenase, the voltage-dependent anion channel protein
Hsr2, and MnSOD are mitochondrial proteins. In addition to an EP1-like glycoprotein and
a protein of unknown function, they also identified an aconitase protein that has previously
been demonstrated to be present in Arabidopsis mitochondria but may also be present in
the cytosol (Millar et al., 2001). The aconitase spot was increased in relative abundance by
a factor of 2.9 in the senescence-induced PCD cells. In addition, the appearance of multiple
spots that are the product of the same gene, suggesting that posttranslational modification
of these proteins had occurred. Spots 4 and 5 both are encoded by the same catalase gene,
At1g20620, and spots 10 and 11 are both products of the same gene, At3g10920, encoding
MnSOD.
5.16.2 Relative increases in antioxidant enzymes are
associated with plant PCD
The increased relative abundance of four catalase isoforms and two forms of mitochondrial
MnSOD in both heat- and senescence-induced PCD is consistent with the observation that
oxidative stress is implicated in the induction/execution of PCD (Swidzinski et al., 2002;
Hildeman et al., 2003). Previous studies have shown that transgenic tobacco plants with
reduced catalase levels show increased susceptibility to stress conditions (Willekens et al.,
1997) and are hyperresponsive to pathogen attack (Mittler et al., 1999), indicating that this
enzyme plays a central role in antioxidant defense. The identification of two isoforms of
the same protein suggests that posttranslational modification of MnSOD may be important
during plant PCD, and that perhaps such modifications occur only under severe conditions
of oxidative stress, that is, those sufficient to cause PCD. This may be particularly important
in preventing widespread mitochondrial damage during the initiation and execution of PCD,