89As experimental evidence supporting above suggestion of H 2 O 2 as a main type
of ROS, we investigated the expression modes and activity dynamics of mito-
chondrial signatures including antioxidant enzymes. In ME yeast cells incubated
for 12 h, CR increases both Cu/Zn-SOD and Mn-SOD activities. In contrast, in
PME yeast cells incubated for several days, CR decreases both Cu/Zn-SOD and
Mn-SOD activities. So CR-based “dual-phase responses” can be typically distin-
guished by the “up-and-down” modes of antioxidant responses.
Additionally, an elevation of the expression level of peroxisome genes in all
treatment groups seemed to support above deduction that H 2 O 2 has become the
major kinds of ROS, which can be thoroughly scavenged by the cooperation of
CAT, GSH-POX, and peroxisomal enzymes. More importantly, upregulation of
peroxisomal β-oxidation enzymes contributes to energy generation by the oxida-
tion of fatty acids released by the degraded storage lipids and recycled cellular
components during carbon starvation.
6.2.2.7 Differential Regulation of Protein Kinase Genes
in Logarithmic and Postlogarithmic Stages
It is noted that a repression of the respiratory activity should lead to the decrease
of ATP and NADH, i.e., the elevation of AMP/ATP and NAD+/NADH ratios,
which can in turn activate AMPK and SIRT1 separately. In postlogarithmic yeast
cells, only a slight upregulation of Snf1 (coding for a yeast homolog of AMPK)
and Sir2 (coding for a yeast homolog of SIRT1) was observed in CR treatment,
but no significant changes of those genes were noticed in mimetic treatment.
Additionally, Sch9, encoding a yeast homolog of mammalian ribosomal S6
kinase 1 (S6K1), was also found to be weakly upregulated in the CR group but
unchanged in mimetic groups.
It was shown that mitogen-activated protein kinase (MAPK) pathway genes
(Kss1 and Fus3 are yeast homologs of MAPK) are mostly downregulated by CR
and mimetics, indicating a common modulation pattern in the PE phase. As a
critical gene controlling cell fate and determining lifespan, To r is upregulated in
either treatment group, in which Tor1 is considerably upregulated for many folds.
Because TOR is activated via phosphorylation, we do not expect that TOR activity
is correlated with its transcript abundance. Nevertheless, it seemed that the inhibi-
tion of TOR activity may induce Tor expression perhaps in a feedback manner.
We further quantified the induced expression levels of Snf1, Tor1, and Kss1
in logarithmic yeast among treatment groups. It is very clear that their expres-
sion patterns are distinct in the logarithmic stage from those in the postlogarith-
mic stage. For example, Tor1 exhibits downregulation in the logarithmic stage, but
shows upregulation in the postlogarithmic stage. In the logarithmic yeast, the over-
all expression levels of Snf1 and Kss1 that are induced by CR, ART, or H 2 O 2 are
typically lower than those in calorie nonrestriction. These results further strength-
ened that protein kinase genes are regulated in a dual-phase mode.
6.2 ART Extends Yeast Lifespan via NO Signaling