(Received for publication, July 28, 1995; and in revised form, August 31, 1995)
From the
To isolate new plant genes involved in the defense against
oxidative stress, an Arabidopsis cDNA library in a yeast
expression vector was transformed into a yeast strain deficient in the YAP1 gene, which encodes a b-Zip transcription factor and
regulates general stress response in yeasts. Cells from 10
primary transformants were subjected to a tolerance screen toward
the thiol-oxidizing drug diamide, which depletes the reduced
glutathione in the cell. Four types of Arabidopsis cDNAs were
isolated. Three of these cDNAs (P1, P2, and P4) belong to a plant
-crystallin family and P3 is an Arabidopsis homolog of
isoflavonoid reductases. As such, all four isolated cDNAs are
homologous to NADPH oxidoreductases. P1, P2, and P3 steady-state mRNAs
accumulated rapidly in Arabidopsis plants under various
oxidative stress conditions, such as treatment with paraquat, t-butylhydroperoxide, diamide, and menadione. The data
suggested that proteins encoded by the isolated cDNAs play a distinct
role in plant antioxidant defense and are possibly involved in
NAD(P)/NAD(P)H homeostasis.
The formation of oxygen radicals by partial reduction of
molecular oxygen is an unfortunate consequence of aerobic life. Active
oxygen species (AOS), ()such as superoxide anion
(O
), hydrogen peroxide
(H
O
), and hydroxyl radicals (OH
) are
natural by-products of metabolism and also result from exposure to free
radical-generating compounds (natural quinones, xenobiotics, and
pollutants)(1) . AOS are highly reactive and damage DNA,
proteins, lipids, and carbohydrates(1, 2) .
Aerobic
organisms have evolved a number of enzymic and nonenzymic antioxidant
defense mechanisms, which counteract the harmful effects of AOS and
maintain the cellular steady-state of pro-oxidants and
antioxidants(1, 3) . Nonenzymic antioxidants, such as
ascorbic acid, glutathione, carotenoids, and -tocopherol scavenge
AOS directly through chemical mechanisms. The major enzymes of
importance in O
scavenging are
superoxide dismutases, which disproportionate superoxide anions to
dioxygen and hydrogen peroxide, and the latter being detoxified by
catalases, glutathione peroxidases, and ascorbate
peroxidases(1, 3, 4, 5) .
Adverse environmental conditions often lead to the disruption of the pro-oxidant/antioxidant homeostasis. A disbalance in favor of pro-oxidants, potentially leading to cell damage, has been defined as oxidative stress(3) . The activity of antioxidant defenses often correlates positively with aging or senescence, disease development, or ability to adapt to changing environment(4) . Furthermore, hereditary defects in antioxidant protection have multiple pleiotropic effects both in prokaryotic and eukaryotic organisms. For example, mutations in the gene coding for the copper/zinc superoxide dismutase are associated with familiar amyotrophic lateral sclerosis in humans (6) and yeast mutants in mitochondrial manganese superoxide dismutase are hypersensitive to oxygen(7) . The increase in activity of AOS-scavenging enzymes by gene engineering, for instance, prolongs the life span of Drosophila(8) and improves stress tolerance of plants(5) .
The importance of the antioxidant defense mechanisms is mirrored by their complexity, and new antioxidant proteins are continuously described. The reversion of mutant phenotypes associated with defects in antioxidant defense by screening for extragenic suppressors or by gene overexpression has been found to be a suitable approach toward better understanding pro-oxidant/antioxidant homeostasis(9, 10, 11, 12) .
Following such studies in yeasts, we became interested in examining whether the random overexpression of plant cDNAs in yeast cells would allow by-pass of phenotypes associated with a deficiency of antioxidant defense mechanisms. This approach allows the characterization of hitherto unidentified genes, the isolation of which would be not amenable by conventional methods.
The yeast mutant deficient in YAP1
was chosen in the present study. The YAP1 gene encodes a
transcription activator of the b-Zip family of DNA-binding proteins and
was originally isolated as a yeast homolog of mammalian jun(13) . In later studies, however, the YAP1 gene was isolated by different research groups as a gene, which
when overexpressed on multicopy plasmids, confers tolerance to
different toxic compounds(14, 15, 16) .
Direct expression analysis and DNA binding studies have shown that YAP1 plays an important role in stress-induced transcriptional
activation of many yeast genes, and more specifically of antioxidant
genes, including those encoding thioredoxin, TRX2(17) , and -glutamylcysteine synthase, GSH1(18) . The levels of antioxidant defense enzymes,
such as superoxide dismutase, glutathione peroxidase, and
glucose-6-phosphate dehydrogenase are lower in yap1 mutants (16) . The role of YAP1 in regulation of the antioxidant
defense is reflected in the increased sensitivity of yap1 mutants to hydroperoxides, thiol oxidants, and redox-cycling
drugs(17) .
In this paper, we present experiments aimed to isolate Arabidopsis cDNAs, of which the overexpression improves the survival of yeast cells deficient in YAP1 function in the presence of the thiol-oxidizing drug diamide (azodicarboxylic acid bis[dimethylamide]), which depletes intracellular glutathione in vivo(1) and oxidizes cysteine residues in proteins in vitro(19) . The expression of antioxidant genes, such as the thioredoxin gene TRX2 in yeasts and manganese superoxide dismutase in Escherichia is strongly activated in diamide-treated cells(17, 20) . In yeast, diamide-stimulated up-regulation of the TRX2 gene is mediated through YAP1 activation(17) . Furthermore, the YAP1 gene, when present on a high copy number plasmid, confers high tolerance of yeast cells to diamide(17) . In Escherichia, the diamide-induced manganese superoxide dismutase gene activation is mediated through the superoxide anion sensor soxRS(20) .
Escherichia coli strain XLI (Stratagene, La Jolla, CA) was used for plasmid manipulations and was grown on standard LB medium supplemented with antibiotics when required.
For drug treatments, 3-week-old plants grown in
vitro on a Murashige and Skoog medium (Life Technologies, Inc.,
Gaithersburg, MD) were used. Complete plants were collected and
different compounds were infiltrated into the tissues under vacuum in
Erlenmeyer flasks. Vacuum was released the moment air bubbles started
appearing on the leaf surface. The following drugs were used: 5
10
M paraquat (methyl viologen), 5
10
M menadione, 1 mM diamide, 0.5
mMt-butylhydroperoxide (t-BOOH), 40 mM hydrogen peroxide, and 10 mM dithiothreitol. The
infiltrated plants were transferred to Petri dishes and RNA was
extracted at different time points. Plants infiltrated with paraquat
were exposed to a 40-watt bulb from 10 cm distance to facilitate the
light-dependent redox cycling of the drug.
Partial DNA sequencing of the rescued plasmids was used to identify the clones. Four types of clones, designated P1, P2, P3, and P4 were found. Fourteen cDNA clones of eight different lengths contained the P1-type cDNA. The P2 (4 clones), P3 (3 clones), and P4 (2 clones) were represented each as two different length cDNAs. This shows the consistency in the recovery of Arabidopsis cDNAs in the described screen.
The longest
cDNAs of each type were retransformed into the yap1 mutant strain, WYT, and the diamide
tolerance of two independent transformants for each cDNA was assessed
in a semi-quantitative dilution series test, as shown in Fig. 1.
WYT(pFL61) cells from stationary phase culture are sensitive to 1
mM diamide and little growth was observed at the 1/10
dilution. Similar suppression of growth for strains containing P1, P2,
P3, and P4 was observed only at a dilution of 1/250. Logarithmic phase
WYT(pFL61) cells appeared to be more tolerant to diamide, although the
strains containing the isolated cDNAs were still able to grow at the
higher dilution (compare L lanes on Fig. 1). The
isolated cDNAs were not able to improve the survival of yeast cells on
plates supplemented with t-BOOH, cycloheximide, and cadmium
(data not shown).
Figure 1: Diamide tolerance of yeast WYT cells. WYT strains containing either control plasmid pFL61 or pFL61 with Arabidopsis cDNAs P1, P2, P3, or P4 were tested for diamide tolerance in a semi-quantitative dilution test. Cultures of strains were grown to stationary or logarithmic phases, indicated as S and L to the right of the figures. Cell suspensions were serially diluted in 0.3% NaCl (fold of dilutions are indicated above the figures) and spotted as 10-µl aliquots on plates without and with 1 mM diamide. Pictures of plates were taken after 3 days of incubation at 30 °C. Two individual clones of yeast transformants with pFL61, pP1, pP2, pP3, and pP4 were analyzed.
The isolated Arabidopsis cDNAs were then
tested for their ability to improve the diamide tolerance of the nearly
isogenic to WYT, YAP1 strain DY. As shown in Fig. 2A,
YAP1 strain DY showed better survival on 1.5 mM diamide, when
it expressed P1, P2, P3, and P4 cDNAs. However, P1, P2, P3, and P4
clones were less efficient in rescuing cells from diamide toxicity as
compared to the clone D3, which was isolated in a similar screening
with a DY strain. ()
Figure 2:
Diamide tolerance of DY strains and
analysis of putative additive effects of P clones. A, the DY
strain was transformed with pFL61, pP1, pP2, pP3, and pP4. The diamide
tolerance of stationary phase cells was tested as described in the
legend to Fig. 1. As an additional control, the DY strain
containing the pD3 cDNA isolated in a different screening was used. B, to analyze the possible additive effects of
P clones for diamide tolerance, expression cassettes containing P1 and
P3 cDNA were recloned in another vector pGAD424, which provide LEU2 selection in yeasts. Double transformants of WYT yeasts were
selected on plates without uracil and leucine. The diamide tolerance of
stationary phase cells was tested as described in the legend to Fig. 1.
If the mechanisms of protection of yeast cells from diamide toxicity by the isolated clones were different, we may expect additive effects for the diamide tolerance, when several cDNAs are co-expressed. To test this hypothesis, we recloned the P1 and P3 expression cassettes into another yeast-Escherichia shuttle vector, which has a LEU2 gene as a selectable marker. Yeast strains containing the two plasmids were selected on SD medium plates lacking uracil and leucine. When assessed in a dilution test, none of the tested combinations showed strong additive effects, although slightly better growth on diamide plates may be seen for the strains, which combine P1 and P3, and P4 and P3 (Fig. 2B).
Figure 3:
Analyses of gene copy number and
tissue-specific expression. Genomic DNA of Arabidopsis was
extracted from two different isolates, Landsberg erecta (L) and Columbia (C). DNA samples (1 µg)
were digested with DraI, SacI, EcoRV, PstI, and HindIII, electrophoresed on 0.8% agarose
gel, and blotted to nylon membranes. After hybridization with P-labeled DNA probes for P1 (A), P2 (B),
P3 (C), and P4 (D) membranes were exposed to x-ray
film.
S-Labeled HindIII DNA fragments of phage
were used as a molecular weight markers (MW), their
positions on the membranes are indicated to the left, numbers correspond to molecular masses expressed in thousands of base
pairs. For the determination of sizes and tissue distribution of the
mRNAs, total RNA was extracted from roots (R), leaves (L), stems (S), and flowers (F) of Arabidopsis plants. RNA gel blots were as described under
``Materials and Methods.''
Fig. 4shows that a number of proteins identified in the EMBL
data base have strong amino acid sequence similarity to P1 and P2. The
best scores (Table 1) were found to the rabbit protein AdRab-F,
which is expressed only in the intestine of adult rabbits and not in
newborn animals. Lower homology was found to -crystallins from
guinea pigs, trypanosome Leishmania amazonensis, and to the
plant cDNA TED2, originally isolated as a transcript up-regulated
during in vitro xylem differentiation of zinnia mesophyll
cells (Fig. 4). The actual function of these proteins in
vivo is not well defined, although lens
-crystallin have been
shown to possess NADPH:quinone oxidoreductase activity in
vitro(35) .
Figure 4:
The P1, P2, and P4 cDNA are homologous to
the -crystallin-like proteins. The amino acid sequences of the ORF
encoded by the P1, P2, and P4 cDNAs were aligned to the sequences of
protein AdRab-F from rabbit intestine (L.c.) (Boll and Mantei,
PIR accession number S47093),
-crystallin-like protein from
trypanosome L. amazonensis (L.a.)(32) , guinea pig
-crystallin (zeta) (33) , and zinnia cDNA TED2 (TED2)(34) . The amino acids, which are identical
in P1 ORF compared with other amino acid sequences are indicated by dots and boxed. In addition, amino acids identical to
P4 ORF are bold and shaded. Dashes represent
gaps introduced for the better alignment.
The two isolated P4 cDNAs, of which the longest was 2175 bp, were different in one base pair at the 5` end and 12 bp at the 3`-nontranslated regions (not counting the polyadenylation tail). A 2.4-kilobase transcript was detected by RNA gel blot analysis using P4 as a probe (Fig. 3D). The P4-encoded polypeptide of 630 amino acids is preceded by an in-frame stop codon.
Fig. 5presents the sequence of the amino terminus of the P4 polypeptide. In this sequence, 250 amino acids are similar to those of a family of short-chain dehydrogenases. It shares, for example, 58.9% similarity and 38% identity with human NAD-dependent 15-hydroxyprostaglandin dehydrogenase, 53% similarity and 28% identity with NADP-dependent glucose dehydrogenase from Bacillus megaterium, and 50% similarity and 30% identity with the Arabidopsis 3-oxoacyl (acyl carrier protein) reductase. The amino acid residues, which are important for the enzymic activity of short-chain dehydrogenases(39, 40) , are well conserved in the P4 polypeptide (Fig. 5).
Figure 5:
The amino-terminal part of the P4 cDNA is
homologous to short-chain dehydrogenases. The amino-terminal part of
the ORF encoded by the P4 cDNA (A.t. P4) was aligned to the
amino acid sequences of human 15-hydroxyprostaglandin dehydrogenase (15prost)(36) , Arabidopsis 3-oxoacyl (acyl
carrier protein) reductase (oxoacyl)(37) , and glucose
dehydrogenase from B. megaterium (glucose)(38) . The amino acids, which are
identical in P4 and other short-chain dehydrogenases, are indicated by dots and boxed. The amino-terminal part of the
oxoacyl dehydrogenase is a chloroplast transit peptide. The amino acid
residues, important for the enzymic activity of the short-chain
dehydrogenases are indicated with lozenges. The arrowhead above the P4 sequence shows the beginning of the P4 domain with
homology to the -crystallins. To improve alignment, gaps indicated
by dashes have been introduced.
The
carboxyl-terminal part of the P4 polypeptide, starting from the 284th
amino acid, shows similarity with the P1 polypeptide (Table 1).
The highest similarity can be found to the L. amazonensis -crystallin, which is followed by the TED2 cDNA, the
guinea pig lens
-crystallin, and the rabbit protein AdRab-F ( Fig. 4and Table 1). Therefore, P4 is most probably a
two-domain protein. Specific peptide sequences for targeting to
different cell compartments could not be identified in P1, P2, or P4,
suggesting that the proteins reside in the cytosol.
The gene copy numbers for P1, P2, and P4 were estimated on DNA gel blots of Arabidopsis genomic DNA digested with DraI, SacI, EcoRV, PstI, and HindIII. DNA extracted from two different land races of Arabidopsis was compared to find RFLP. The pattern of bands hybridizing with the P1 and P2 probes is highly similar, as predicted from the high DNA sequence identity of these two cDNAs (Fig. 3, A and B). Since single hybridizing bands of the same molecular weight were detected with P1 and P2 probes in DNA digested with SacI and PstI, these two genes are likely to be tightly linked on the chromosome. Additionally, weakly hybridizing bands were also detected that may indicate the presence of divergent genes in the Arabidopsis genome. The P4 gene is apparently unique in the Arabidopsis genome, because a simple hybridization pattern was observed (Fig. 3D). The P4 locus is polymorphic and an RFLP polymorphism was found, with four out of the five used restriction endonucleases tested, between Arabidopsis ecotypes Columbia and Landsberg erecta. Using the RFLP polymorphism obtained with DraI and DNA extracted from 98 Arabidopsis inbred recombinant lines(23) , the P4 gene was mapped in the middle of chromosome 1 close to the RFLP marker m213 (mapping data are available from the laboratory of Dr. C. Dean, John Innes Institute, Norwich, UK).
The highest levels of P1, P2, and P4 steady-state
mRNAs were detected in leaves and less in stems. Weak expression
occurred in flowers and very weak expression in roots of Arabidopsis plants (Fig. 3, A, B, and D). From the sequence comparison, it was clear that the cDNAs
P1, P2, and P4 corresponded to a rather divergent family of plant
proteins, which were tentatively named together with zinnia TED2, plant -crystallins.
Figure 6: The amino acid sequence comparison of the P3-encoded ORF. A.t. P3 corresponds to the ORF encoded by cDNA P3. Other sequences are tobacco isoflavonoid reductase, such as cDNA A622 (N.t. A622)(41) , isoflavonoid reductases from chickpea (C.a. IFR)(42) , and alfalfa (M.s. IFR)(43) . Amino acids, which are identical between P3 ORF and other plant proteins, are indicated by dots and boxed. Gaps introduced to optimize the alignment are shown by dashes.
Oxidative stress was imposed by the following
treatments: the hydroperoxides, hydrogen peroxide
(HO
), and t-BOOH were used to induce
direct oxidation of cell constituents. Treatment with diamide was used
to induce pro-oxidant conditions through depletion of intracellular
glutathione. The herbicide paraquat (methyl viologen) and the quinone,
menadione, are redox-cycling drugs. In the cell they serve as efficient
electron acceptors from electron transport chains. Reduced paraquat and
semi-quinones are readily oxidized by oxygen, leading to the formation
of O
anions. Then, the cycle of
reduction-oxidation is repeated, a process called redox
cycling(1) . Furthermore, potent antioxidants such as reduced
glutathione, dithiothreitol (DTT), and N-acetylcysteine, may
cause the activation of plant oxidative stress defense
genes(46) . Therefore, we analyzed the expression of the
isolated genes after the infiltration of plants with DTT.
The chemicals mentioned above were applied by infiltration into Arabidopsis plants. Although infiltration by itself caused activation of apx, P1, P2, and P4 genes, this method was used to eliminate problems of drug uptake through roots or epidermis.
As
shown in Fig. 7, apx mRNA levels steadily increased up
to 8 h after infiltration of water. The strongest response was observed
in response to paraquat treatment; accumulation of mRNA was detected
after 15 min of treatment and reached a plateau after 30 min, which
lasted at least 8 h. Transiently higher steady-state RNA levels
(compared to control) were observed in plants treated with t-BOOH, diamide, and menadione, with the highest mRNA levels
approximately 1 h after infiltration. The apx gene was also
induced by DTT and levels of mRNA detected in
HO-infiltrated plants after 8 h were already observed after
15 min following DTT infiltration. No differences were observed between
control and H
O
-infiltrated plants.
Figure 7:
Analysis of the mRNA levels in Arabidopsis plants under oxidative stress conditions.
Three-week-old in vitro grown Arabidopsis plants were
infiltrated with water (HO) alone, or water solutions of
hydrogen peroxide (H
O
), t-butyl
hydroperoxide (t-BOOH), diamide, menadione, methyl viologen,
or DTT at the indicated concentrations. RNA was extracted from treated
plants at different time points, as indicated above the lanes either in
minutes (') or in hours. To check equal RNA loading, membranes
were stained with methylene blue, the staining of the 28 S rRNA is
shown in the column rRNA. The levels of mRNAs homologous to probes made
from P1, P2, P3, P4, and apx cDNAs are shown in vertical
columns. For each treatment, the same membrane was re-hybridized with
different probes.
P1 and P2
genes responded similarly, although, considering the high sequence
homology, the detected hybridization signals might be derived from P1
and P2 mRNA together. Like apx, P1 and P2 mRNAs were induced
by water infiltration. Rapid induction of these genes was found after
menadione treatment, with detectable increases over the control after
15 min and a plateau of expression after 30 min. The response to
another redox-cycling drug methyl viologen (paraquat) was detectable
after 15 min and the accumulation of P1 and P2 mRNAs after 8 h was the
highest detected (10-fold). The accumulation of P1 and P2 mRNAs in t-BOOH-treated and diamide-treated plants was faster as
compared to the control, however, expression was lower after 8 h.
Hydrogen peroxide slightly induced P1 and P2 genes after 8 h. DTT
repressed accumulation of P1 and P2 mRNAs.
The P3 mRNA levels were
unresponsive to water infiltration, whereas expression weakened in t-BOOH-treated and DTT-treated plants. The P3 gene was slowly
induced by paraquat, hydrogen peroxide, diamide, and an 3-fold
increase in mRNA levels is detected after 8 h. Menadione did not affect
P3 mRNA levels.
The P4 gene was induced in the control infiltration
with water. HO
, t-BOOH, and DTT
prevented this water-induced mRNA accumulation, whereas diamide,
menadione, and methyl viologen had little effect on P4 gene expression.
The same membranes were also hybridized with a tobacco cDNA probe of the chlorophyll a/b-binding protein (cab). All of treatments lowered the cab gene expression (data not shown).
In this work we have isolated four Arabidopsis cDNAs
which improve the tolerance of yeast cells to the thiol oxidizing drug
diamide. Plant -crystallins and IFR-like cDNAs were consistently
recovered in our screening for diamide tolerance of the yap1
yeast mutant. Furthermore, we have
found that the cDNAs isolated also improved the survival of YAP1 yeast
strain, DY which has a normal response to oxidative stress. The
mechanism of protection is apparently rather specific for diamide.
Efforts to find cross-tolerance to other drugs were not successful,
however, the choice of stresses as well as possible growth conditions
were not exhaustively tested in this study. This observation is not in
disagreement with the data obtained by multicopy plasmid screening in
yeasts. Significant cross-tolerance was usually observed when genes
encoding regulatory proteins, like YAP1 or YAP2 were
overexpressed(17, 47, 48) . The tolerance
provided by enzymes is rather limited to the particular stress
agent(17, 48) .
Although we cannot entirely exclude
the direct detoxification of diamide by -crystallins or IFR, our
expression analysis suggest that the isolated cDNAs encode enzymes with
a distinct role in stress response. The genes encoding P1 and P2 were
rapidly induced by drugs which induce redox cycling, and in general
their expression is very similar to the expression of the well
characterized plant antioxidant protein, ascorbate
peroxidase(45) .
The original interest in -crystallin
stems from the fact that a mutation in the
-crystallin gene is
associated with formation of an autosomal dominant congenital cataract
in guinea pigs(49) . Since cataract development is thought to
result from oxidative damage of lens proteins(1) , it was
logical to presume that high levels of
-crystallin is a
species-specific adaptation to control the formation of active oxygen
species. It was further found that detoxification of hydrogen peroxide
is more efficient in lens of guinea pigs compared to rat lens, which
have only small amounts of
-crystallin(49) . The
expression of
-crystallins is also induced in rat lens under
oxidative stress conditions(50) .
-Crystallin
specifically binds NADP(H) and is a novel NADPH:quinone
oxidoreductase(35, 49) . Based on biochemical data,
Rao and Zigler (49) hypothesized that one of the important
functions of
-crystallins is an initiation of NADPH/NADP redox
cycling, which in turn is coupled to the up-regulation of the hexose
monophosphate shunt. Hexose monophosphate shunt is the principal source
of NADPH in cells and one of the components of antioxidant defense,
because NADPH is needed for the reduction of oxidized glutathione and
ascorbic acid, whose formation greatly increases under oxidative
stress(51, 52, 53) .
The P3 gene was
slower in response to paraquat and HO
and was
not induced by water infiltration. Considering, the importance of
H
O
metabolism in processes of cell wall
cross-linking and lignin biosynthesis (54) and possible
xylem-specific expression of the P3 gene, its product may play a role
in AOS homeostasis in specific cell types. The P3 clone is highly
homologous to isoflavonoid reductases. IFRs are believed to be
specifically involved in the biosynthesis of isoflavonoid phytoalexins
in Leguminosae(55) . However, IFR cDNA homologs have
been isolated from several species, which do not synthesize
isoflavonoid phytoalexins and the tobacco IFR-like protein expressed in E. coli did not show any enzymatic activity (41, 56) . The IFR activity of legume proteins
suggests that a whole IFR-like family are oxidoreductases utilizing
NAD(P)H as a co-factor and the use of isoflavonoids as electron
acceptors is likely to be an acquired enzymatic activity in legumes.
The expression of the P4 gene was only elevated in control
infiltration with water; furthermore, its expression was suppressed by
diamide and t-BOOH. Apparently, P4 protein, which is composed
of two domains has a rather specific function in plant cells. Although
it was isolated in our screening most likely due to the presence of the
carboxyl-terminal -crystallin domain, it also possesses an
amino-terminal domain which shows homology to the short-chain
dehydrogenases. The arrangement of the two domains in the putative P4
protein is quite interesting. If enzymatically active, the
amino-terminal dehydrogenase domain could, through the oxidation of a
substrate molecule, generate NADPH, from which electrons may then be
transferred to another acceptor (quinones?) by the
-crystallin
domain. It is tempting to propose that, short
-crystallins encoded
by the P1 and P2 may in fact compete with P4 for the interaction with
some plant protein(s), and in this way are lowering P4 activity.
In
this work, we have identified 4 new Arabidopsis cDNAs, which,
when expressed under control of a yeast promoter on a multicopy plasmid
provide better tolerance of yeasts to thiol oxidizing drug diamide. It
might not be coincidental that plant -crystallin homologs were
isolated by such a screen. Loss of
-crystallin activity in guinea
pigs leads to cataract formation and this effect can be mimicked by
diamide(57) . The putative NAD(P)H oxidoreductase activities of
proteins encoded by the isolated cDNAs suggest the importance of
NAD(P)H/NAD(P) homeostasis in plant cell antioxidant defense. The use
of other drugs and chemicals in similar functional screening of plant
cDNA libraries may provide an important tool for the identification of
plant genes involved in stress response and defense, which would not be
amenable to isolation by conventional screening.