BACKGROUND
light energy is essential for growth of photosynthetic organisms but it can also harm them.
excess light can lead to the increased production of reactive oxygen species  which can damage cellular components such as lipids, proteins and dna.
mainly at photosystem  i but also at psii, electron transfer reactions to molecular oxygen causes the production of superoxide anion radicals , hydrogen peroxide  and hydroxyl radicals   <cit> .
at psii, triplet chlorophyll formation and the interaction with molecular oxygen stimulates the formation of singlet oxygen   <cit> .
singlet oxygen was shown to contribute significantly to the ros-induced cellular damage during high light stress  <cit>  and consequently plant and algae have evolved efficient protection mechanisms to prevent the formation of this ros.
some of these protection mechanisms can be detected as non-photochemical quenching  of maximal chlorophyll fluorescence  <cit> .
short and long term acclimation processes like state transition  or adjustment of ps stoichiometry help to prevent overreduction of the photosynthetic electron transport chain  <cit> .
the energy-dependent quenching  of excess light involves a Δph-induced activation of the xanthophyll cycle in which a violaxanthin de-epoxidase converts violaxanthin  into antheraxanthin  and zeaxanthin   <cit> .
increased levels of these xanthophylls together with the protonation of specific pigment-binding antenna proteins cause a conformational change of psii into a high quenching state where excess light energy is dissipated as heat  <cit> .
additionally, zeaxanthin is an efficient 1o <dig> quencher and increased levels of this xanthophyll after exposure to high light conditions might reduce damage to membrane lipids  <cit> .
recently, two lhcsr <dig> genes have been found to be involved in npq in chlamydomonas reinhardtii indicating that other unidentified components might function in photoprotection and prevention of 1o <dig> formation in photosynthetic organisms  <cit> .
singlet oxygen can damage the cell but it has also been found to play an important role in retrograde signaling through the specific activation of nuclear genes by plastid signals.
singlet oxygen produced in the chloroplast of the conditional fluorescent  mutant was shown to stimulate the expression of a set of genes which was different from h2o <dig> induced genes  <cit> .
furthermore, o <dig> -/h2o <dig> exhibited an antagonizing effect on 1o2-induced gene expression in flu  <cit> .
in a suppressor screen for the 1o2-induced programmed cell death response in flu mutants, two thylakoid-localized proteins, executer <dig>  and executer <dig> , were identified which are involved in the regulation of the 1o2-mediated genetic response  <cit> .
in c. reinhardtii, the response to 1o <dig> has been studied using either specific exogenous photosensitizers like rose bengal  or neutral red   <cit>  or in strains lacking some 1o <dig> protective mechanisms like the xanthophyll-deficient mutant npq <dig> lor <dig>  <cit> .
as found in a. thaliana, the response of the chlamydomonas hsp70a gene to 1o <dig> could be distinguished from the response to h2o <dig> by different reporter constructs and was attributed to separate promoter regions  <cit> .
furthermore, the glutathione peroxidase homologous gene gpxh/gpx <dig> of c. reinhardtii was strongly induced by 1o <dig> but to a much lower extent by other ros  <cit> .
during high light stress, gpxh expression is strongly induced by 1o <dig> by transcriptional activation  <cit>  and various regulatory elements in the promoter were required for induction by 1o <dig>  <cit> .
the gpxh protein is predicted to be dual-targeted to the cytoplasm and the chloroplast, and its peroxidase activity with plastidial thioredoxin indicates a role in oxidative stress response of the chloroplast  <cit> .
even though 1o <dig> can function as a signal to activate nuclear gene expression, our knowledge of how the formation of 1o <dig> is controlled in photosynthetic organisms and which components are involved in the signal transduction from the plastid to the nucleus is still far from complete.
membrane lipids are primary targets of 1o <dig>  and oxidized fatty acids could function as signaling intermediates  <cit> .
however, experiments with carotenoid-depleted cultures indicated that in c. reinhardtii the sensor for 1o <dig> is not a lipophilic compound in the thylakoid membrane but probably is located in the aqueous phase of the chloroplast  <cit> .
in an effort to identify components that affect the 1o <dig> induced genetic response in c. reinhardtii, we performed a mutant screen using a 1o2-specific gpxh reporter construct.
mutants with altered gpxh expression were isolated and characterized genetically and physiologically.
RESULTS
isolation of mutants with altered gpxh expression
the expression of the gpxh gene is strongly induced by the increased production of 1o <dig> in the chloroplast.
to identify components affecting 1o2-induced gene expression in c. reinhardtii, a mutant screen was performed using the gpxh-arylsulfatase  reporter gene construct pysn <dig> to search for clones with altered gpxh response  <cit> .
the wild-type strain 4a+ transformed with pysn <dig> was uv-mutagenized and colonies were grown on tap plates in the dark.
then, a total number of  <dig> clones were analyzed for their gpxh-ars expression under medium light  condition of  <dig> μmol photons m- <dig> s- <dig> in the presence or absence of  <dig> μm nr.
average gpxh-ars expression was induced  <dig>  ±  <dig>  fold by nr treatment.
a cutoff of  <dig> -fold induction by nr and  <dig>  fold higher expression was used to select for clones with reduced induction or increased basal expression, resulting in  <dig> gpxh-ars induction deficient  and  <dig> gpxh-ars overexpressor  mutants .
however, after retesting these clones, only six gid and  <dig> gox mutants could be confirmed.
altered response of the reporter enzyme in 4a+ pysn <dig> can result from mutations affecting the cellular ars activity, the production of 1o <dig> or the signal transduction of 1o2-induced gene expression.
to identify the first class of mutants, the induction of the endogenous gpxh gene was measured in the six gid mutants exposed to nr.
unfortunately, all gid mutants retained full induction of the gpxh wild-type gene .
for the  <dig> gox mutants, a secondary screen was performed to reduce the number of strains for gpxh expression analysis.
based on the knowledge that gpxh overexpression increases the resistance of c. reinhardtii to chemicals enhancing ros production  <cit> , the mutants were exposed to the 1o2-producing photosensitizers rb and nr at ml conditions or the o <dig> --producing chemicals metronidazole  and methyl viologen  and the organic tert-butylhydroperoxide  under low light intensity .
one group of mutants with  <dig> members was more resistant to t-booh compared to wild-type, and many but not all of these mutants were also resistant to nr and rb .
furthermore, all strains were tested for their tolerance to high light intensities  resulting in the identification of  <dig> hl-sensitive clones.
this phenotype was often combined with sensitivity to rb, nr, mz or mv.
a third group of mutants  showed no or only very weak changes in tolerance to oxidative stress.
since for this subset of mutants a relatively low gpxh-ars overexpression was determined, they were excluded from further analysis.
abbreviations: hl: high light of  <dig> μmol photons m- <dig> s- <dig> par, t-booh: tert-butylhydroperoxide, nr: neutral red, rb: rose bengal, mz: metronidazole, mv: methyl viologen.
tolerance was classified in five different categories compared to the wild-type strain: s: very sensitive, s: sensitive, n: no difference from wild-type, r: resistant, r: very resistant.
mutants could be divided into three different groups: hl sensitive mutants, t-booh resistant mutants and mutants with no or only minor changes in tolerance .
the remaining  <dig> mutants, being either hl-sensitive and/or t-booh resistant, were then tested for the expression of the endogenous gpxh wild-type gene by qpcr.
a significantly  stimulated expression compared to the corresponding wild-type strain could be detected in seven clones ranging from  <dig> - to  <dig> -fold overexpression .
even though there was a clear correlation  between gpxh expression and the expression of the reporter construct, all  <dig> mutants had a stronger overexpression of gpxh-ars measured by enzyme activity than gpxh expression determined by qpcr, which might be the consequence of individual mrna or the reporter enzyme stability .
high doses of uv radiation as applied in this experiment induce multiple point mutations in the genome.
to analyze whether defects in multiple genes might be responsible for the phenotype, we performed tetrad analysis of selected gox mutants by crossing them back to the strain 4a- pys <dig>  twelve independent tetrads were tested for segregation of high gpxh-ars expression in each mutant.
a clear 2: <dig> segregation of the wild-type and mutant phenotypes was found in strains 35h <dig> and 18f <dig> , indicating that mutations in a single nuclear gene is responsible for the increased gpxh expression in each case.
the same was true for 21b <dig> except for one tetrad where one high, two medium and one low expressing progeny were found, suggesting that two closely linked mutations might be responsible for the high ars activity phenotype.
no consistent 2: <dig> segregation was found in backcrosses of strain 22d <dig> and 14a <dig>  whereas for 22d <dig> at least six tetrads resulted in either 3: <dig> or 1:2: <dig> segregations, for 14a <dig> the pattern of three tetrads differed from standard single allele segregation.
gpxh overexpression in gox mutants due to increased singlet oxygen production
stimulated gpxh expression might either be due to a stimulated production of 1o <dig> or a constitutively active signaling pathway under ll condition.
sensitivity to hl intensity of several mutants indicates that the former might be the reason for high gpxh expression in some of the gox mutants.
the formation of 1o <dig> was therefore measured with the fluorescent dye singlet oxygen sensor green  allowing the specific quantification of 1o <dig>  <cit> .
in order to detect significant amounts of 1o <dig> with the membrane impermeable sosg in the wild-type strain, the cells had to be broken by freezing, and exposed to hl intensity  for a period of  <dig> min.
increased 1o <dig> formation compared to wild-type  was detected in all but one of the hl-sensitive mutants .
surprisingly, several mutants with normal resistance to hl also showed a stimulated 1o <dig> production even though the difference to wild-type was not always significant .
comparing 1o <dig> formation with gpxh overexpression in gox mutants, no direct correlation  between the two parameters could be found .
however, since the formation of 1o <dig> is a light intensity-dependent photoreaction and gpxh expression and 1o <dig> formation were quantified at different light intensities , it is difficult to directly compare these parameters.
we therefore measured gpxh expression in all  <dig> gox mutants at the highest possible light intensity at which the mutants could still survive for at least  <dig> h  and which thus corresponds to hl conditions.
indeed, a much stronger stimulation of gpxh expression compared to wild-type could be detected in more than half of the mutants, resulting in a stronger correlation  between 1o <dig> formation and gpxh expression .
increased levels of 1o <dig> in gox mutants are a consequence of either increased generation or lowered detoxification of 1o <dig>  the former might result from a deficient photoprotection mechanism, such as a reduced capacity of npq.
therefore, npq was measured in the gox mutants acclimated to either ll  or hl  for at least  <dig> h. four mutants  had significantly reduced npq under both ll and hl conditions .
four other strains  only had lower npq under hl but not ll conditions, suggesting a light intensity dependent effect, and for three mutants  a stimulation of npq even at ll conditions was found.
energy-dependent quenching  is one component of npq that requires synthesis of the xanthophylls, zeaxathin and antheraxanthin.
however, these and other carotenoids as well as α-tocopherol are also important antioxidants involved in detoxification of 1o <dig>  pigment analysis of the  <dig> gox mutants acclimated to the same ll or hl intensity as for npq measurements revealed only small changes in pigment contents of few strains.
lutein was not altered in ll and only slightly higher in three mutants in hl conditions compared to the wild-type strain .
similarly, α-tocopherol was not strongly affected in ll condition, except for 14h <dig>  but was significantly reduced in  <dig> mutants after exposure to hl for  <dig> h .
for only one strain, 18f <dig>  higher levels of α-tocopherol than in wild-type were detected under hl conditions, and this correlated with increased lutein and zeaxanthin levels in this mutant.
despite the important role of zeaxanthin and antheraxanthin in photoprotection and prevention of 1o <dig> generation, only moderate changes of these xanthophylls were detected in the mutants compared to the wild-type.
only one mutant  had a significantly reduced de-epoxidation of xanthophylls during hl exposure showing that this process seems still to be functional in all mutants .
nevertheless, when grown in ll, five mutants had significantly reduced de-epoxidation states whereas other mutants had rather increased levels of zeaxanthin and antheraxanthin .
gpxh expression and singlet oxygen production negatively correlate with the xanthophyll de-epoxidation state
in order to analyze the relationship of all the parameters measured in the  <dig> gox mutants, linear correlation factors were calculated for every possible combination of parameters .
not surprisingly, a strong positive correlation between antheraxanthin and zeaxanthin levels was found, which negatively correlated with violaxanthin under hl conditions, as expected from operation of the xanthophyll cycle.
as already shown in figure 3b, 1o <dig>  formation correlated with gpxh expression under hl conditions.
both parameters also negatively correlated with antheraxanthin and zeaxanthin levels, especially in ll-grown cultures.
thus, when comparing 1o <dig> production at hl and xanthophyll levels at ll it was striking that all the mutants but one  had a high de-epoxidation state of xanthophylls or increased 1o <dig> production compared to wild-type .
this effect was less pronounced when 1o <dig> production was compared with de-epoxidation at hl because strongly stimulated de-epoxidation reduced the relative differences between the clones.
finally, antheraxanthin and zeaxanthin levels of hl-grown cultures weakly correlated with α-tocopherol and lutein contents under this light condition.
hierachical clustering of the mutants with all measured parameters was performed to test whether there are groups of mutants with similar phenotypic pattern.
these analyses revealed that one mutant  behaved differently from all other mutants .
even though 14h <dig> was originally screened for high gpxh-ars expression and was found to be hl sensitive, it did not show any stimulated gpxh expression or 1o <dig> production at hl intensities.
all other mutants were divided into two major groups  mainly based on the de-epoxidation state of the xanthophyll pool.
group i had lower levels of antheraxanthin and zeaxanthin at ll than wild-type and most of these mutants showed high 1o <dig> production and increased gpxh expression.
they could be further subdivided into two groups, where strain 18f <dig> and 18g <dig> belonged to one group  with stimulated npq in ll conditions and no changes in α-tocopherol levels under any light intensity.
mutants of the other subgroup , on the other hand, had strongly reduced α-tocopherol levels, especially in hl grown cultures, and either lowered or not changed npq compared to the wild-type strain.
the second group of mutants  with similar or slightly increased xanthophyll levels to wild-type had generally a much lower stimulation of 1o <dig> production and gpxh expression than mutants of group i. exceptions were clones 14a <dig> and 21b <dig> from subgroup iia with strong gpxh overexpression and stimulated 1o <dig> production, but these mutants showed strongly reduced npq under both ll and hl intensity that was probably responsible for these phenotypes.
in the other subgroup  only two mutants  had significant gpxh overexpression and higher 1o <dig> production and except for lower α-tocopherol levels in hl for some mutants, little differences compared to wild-type were detected for most parameters.
discussion
no mutants with deficient gpxh induction
screening for mutants with altered gpxh expression resulted in the isolation of several high gpxh expression mutants but no strain with deficient or strongly reduced gpxh induction by 1o <dig> could be isolated.
with a total number of  <dig> uv-mutagenized clones tested we assume that the coverage of mutations in the genome should be high enough to hit at least one component of a putative 1o <dig> signaling cascade with a loss-of-function mutation.
the relatively high uv dose resulting in the survival of only  <dig>  to 2% of the cells and the fact that during segregation analyses at least two out of five gox mutants contained two mutations affecting the phenotype suggested a high mutation density in the screened population.
still, a second mutant screen for suppressor mutants of gpxh overexpression in strain 21b <dig> was performed using an additional plasmid containing the gpxh promoter in front of the nitrate reductase gene .
this enabled a direct selection for reduced expression under low light conditions by selecting for chlorate-sensitive clones.
out of a total number of  <dig> ×  <dig> cells surviving uv mutagenesis,  <dig> clones were chlorate resistant, and of those,  <dig> clones also had low induction  of the gpxh-ars reporter construct by nr.
however, all of these clones showed normal induction of the wild-type gpxh gene, indicating that under these screening conditions no gid mutants can be isolated.
the conclusion that in strain 21b <dig> a constant increased 1o <dig> production is responsible for high gpxh expression might explain why no gpxh induction deficient mutants could be isolated, because gpxh might be essential for defense against 1o2-induced damage.
this can be excluded for the original mutant screen because selection against light-sensitive mutants during the recovery phase after mutagenesis was prevented by growing the  <dig> clones in the dark.
furthermore, none of the clones was light sensitive under ml conditions during the induction tests.
thus, even though steps were taken to minimize a negative selection against gid mutants, we cannot exclude that a functional 1o <dig> response of the gpxh and maybe other genes is essential for c. reinhardtii.
on the other hand, it is also possible that several redundant signaling pathways form a complex network to activate gpxh expression, thereby hampering the isolation of gid mutants.
finally, it still might be that more dark-grown mutagenized clones had to be tested to find the desired mutants.
a similar screen to isolate 1o <dig> responsive mutants in a. thaliana resulted in the identification of at least three mutants deficient in the upregulation of different 1o2-induced genes in the conditional flu mutant  <cit> .
however, 1o2-signaling seems to have different cellular functions in a. thaliana, at least in seedlings, where it is part of a programmed cell death response and in c. reinhardtii where it seems to be involved in response to cytotoxic environmental stresses  <cit> .
gpxh overexpression and correlation with singlet oxygen production, npq and pigment levels
in contrast to the lack of gid mutants, many gox mutants with a stimulated expression of the gpxh wild-type gene, especially under hl intensities, could be isolated .
the light intensity-dependent increase of gpxh overexpression and the sensitivity to hl conditions indicated that in several mutants 1o <dig> formation might be enhanced and cause a photooxidative stress.
indeed, increased 1o <dig> formation was measured in all but one hl-sensitive as well as many hl-resistant gox mutants , which nicely correlated with gpxh expression under hl conditions .
this indicates that in most or even all gox mutants, gpxh overexpression seems not to be caused by a constitutively active signal transduction pathway but by the increased production of 1o <dig> under the light conditions tested.
the poor correlation between 1o <dig> production and gpxh expression under ml , the clustering in at least five phenotypic distinct groups  and the fact that three of the mutants  have been mapped to different linkage groups  indicates that mutations in different nuclear genes are responsible for the increased 1o <dig> production in the various mutants.
singlet oxygen formation in photosynthetic organisms is caused by the conversion of excited chlorophylls into the triplet state and the reaction with molecular oxygen  <cit> .
the organisms try to minimize this process by regulating the excitation pressure on the psii reaction center chlorophylls, optimizing the electron flow in the photosynthetic electron transport chain and quenching chlorophyll triplet states and 1o <dig>  thus, 1o <dig> accumulation can result from either stimulated production, e.g.
due to enhanced triplet chlorophyll formation, or lowered detoxification of the ros due to defects in some light-induced protection mechanisms.
such a defect in protection mechanisms could be the reason for high 1o <dig> formation in most of the group i mutants of cluster analysis  having reduced zeaxanthin and antheraxanthin levels which correlates with high 1o <dig> formation .
a well characterized mutants with reduced xanthophyll levels and defects in photoprotection is the npq <dig> lor <dig> double mutant lacking the carotenoids zeaxanthin, antheraxanthin and lutein.
similar to many mutants of group i, this mutant exhibits increased ros production, gpxh expression and sensitivity upon hl but not ll illumination  <cit> .
however, comparison of npq <dig> and npq <dig> lor <dig> showed that reduced levels of zeaxanthin and antheraxanthin but normal levels of other carotenoids like lutein would probably not cause a very strong phenotype  <cit> .
thus, reduced efficiency of more than one protection mechanism might be required to achieve increased 1o <dig> accumulation.
indeed, five of the group i mutants with reduced xanthophyll levels  also showed lowered α-tocopherol levels under ll and/or hl conditions .
furthermore, levels of other components of the thylakoid membrane with 1o2-quenching capacity like plastoquinone and α-tocopherolquinone  <cit>  were not quantified but might also be affected in some of the mutants.
thus, reduced photoprotection by the combination of lower xanthophylls, α-tocopherol and maybe other deficiencies might be the cause for increased 1o <dig> production and gpxh expression in these mutants.
this could either result from mutations in multiple loci, as found for clone 22d <dig> , or caused by mutations in a single gene encoding for a global regulator of cellular photoprotection mechanisms.
even though deepoxidation of xanthophylls is involved in npq  <cit> , these parameters did not correlate in our mutants  indicating that other factors also play an important role for npq.
for example, two strains of group i  showed rather increased npq levels under ll conditions even though their xanthophyll levels were reduced, clustering them in a separate subgroup  of group i .
very high gpxh expression in 18f <dig> and 18g <dig> under ml conditions indicates stimulated 1o <dig> generation already at ll.
we speculate that these mutants might have increased 1o <dig> production due to an enhanced energy transfer from the psii reaction center to molecular oxygen.
by this, quenching of excitation energy by molecular oxygen would increase npq but reduce the photosynthetic electron transport rate required for building up the proton gradient and activating the xanthophyll cycle.
thus, reduced xanthophylls would not be the cause but the consequence of increased 1o <dig> production.
however, other effects of the mutations cannot be excluded which might also explain these phenotypes.
contrary to 18f <dig> and 18g <dig>  two mutants  had strongly reduced npq both under ll and hl conditions but similar or rather increased xanthophyll levels compared to wild-type showing that defects in qe-independent mechanisms seem to affect npq in these mutants.
photoinhibition  stimulated by excess light should not be relevant under ll conditions, and a deficiency in state transition  should not strongly affect npq at hl intensities.
on the other hand, a qe-independent effect was also found in a c. reinhardtii mutant defective in two linked lhcsr <dig> genes  <cit> .
however, segregation analysis of backcrosses of 14a <dig> and 21b <dig> with a wild-type strain revealed that both mutants have probably mutations in two different genes affecting gpxh expression  indicating that the phenotypes of these mutants could be caused by the combination of different defects.
in mutants with functional detoxification mechanisms, strong 1o <dig> accumulation after stimulated production of the ros might be prevented by the induction of these detoxification mechanisms.
this is supported by the negative correlation of 1o <dig> formation and deepoxidation of xanthophylls, where increased levels of zeaxanthin and antheraxanthin correlate with a low stimulation of 1o <dig> production .
mutants with increased xanthophyll levels are mainly represented in the group iib mutants of cluster analysis with rather few and weak phenotypic changes .
a general increase in antioxidant levels including zeaxanthin, antheraxanthin, lutein and α-tocopherol and a significant rise in npq was detected in strain 14h <dig>  where no stimulated 1o <dig> production and gpxh expression could be measured any more.
this shows that the various protection mechanisms can compensate each other and thus control the production of deleterious ros.
this is in agreement with data of various mutants lacking specific antioxidants: α-tocopherol-deficient strains of c. reinhardtii, synechocystis sp.
pcc <dig> and a. thaliana all were very tolerant to photooxidative stress during hl conditions, and only under extreme conditions such as a combination of very hl and low temperature or chemical treatment a phenotype became visible  <cit> .
it was suggested that the presence of other antioxidants such as zeaxanthin or increased levels of β-tocopherol can compensate for α-tocopherol deficiency  <cit> .
conversely, the a. thaliana npq <dig> mutant, lacking zeaxanthin and antheraxanthin, accumulates higher amounts of α-tocopherol  <cit> .
thus, zeaxanthin, α-tocopherol and plastoquinol have overlapping functions in photoprotection and together prevent the formation of deleterious 1o <dig> under natural conditions  <cit> .
however, when these protection mechanisms are overwhelmed, 1o <dig> starts to accumulate and damage cellular components.
this is when defense genes like gpxh, which repair and remove damaged biomolecules, are required to survive the oxidative stress.
activation of genetic stress response without altering antioxidant levels by an acclimation to increased 1o <dig> production or the direct overexpression of the gpxh gene in c. reinhardtii were shown to increased resistance to oxidative stress by rb, nr and t-booh  <cit> .
increased tolerance against t-booh was also found for  <dig> of the  <dig> gox mutants tested including all the group iib mutants as well as strains 14b <dig>  14c <dig> and 35h <dig> .
thus, increased expression of stress response genes like gpxh might explain the hl resistance of these mutants and shows the important role of gpxh and other defense genes in the photooxidative stress response of photosynthetic organisms.
CONCLUSIONS
the failure to isolate 1o <dig> signal transduction mutants indicates that several redundant signaling pathways might be involved in the gpxh response.
this is supported by the identification of multiple regulatory elements in the gpxh promoter being required for induction of the gene  <cit> .
singlet oxygen generation, on the other hand, was altered in several mutants resulting in higher expression of the gpxh gene.
increased oxidative stress resistance of many of these mutants confirms the importance of the 1o2-induced genetic response in the defense against ros-induced damage.
furthermore, isolation of phenotypic different groups of 1o2-overproducing mutants indicates that mutations in different photoprotective mechanisms might be responsible for higher 1o <dig> levels in various gox mutants which most seem to be, based on pigment analysis, different from known photoprotective mutants like npq <dig> lor <dig>  the comparison of their phenotypes suggests that in several gox mutants multiple defense processes might be affected what might be due to, among other things, mutations in a global regulator of cellular photoprotection mechanisms.
thus, the isolation of these mutants might allow identifying new components involved in the control of 1o <dig> formation by different cellular protection mechanisms.

 light energy is essential for growth of photosynthetic organisms but it can also harm them. excess light can lead to the increased production of reactive oxygen species which can damage cellular components such as lipids, proteins and dna. mainly at photosystem i but also at psii, electron transfer reactions to molecular oxygen causes the production of superoxide anion radicals, hydrogen peroxide and hydroxyl radicals. at psii, triplet chlorophyll formation and the interaction with molecular oxygen stimulates the formation of singlet oxygen. singlet oxygen was shown to contribute significantly to the ros-induced cellular damage during high light stress and consequently plant and algae have evolved efficient protection mechanisms to prevent the formation of this ros. some of these protection mechanisms can be detected as non-photochemical quenching of maximal chlorophyll fluorescence. short and long term acclimation processes like state transition or adjustment of ps stoichiometry help to prevent overreduction of the photosynthetic electron transport chain. the energy-dependent quenching of excess light involves a Δph-induced activation of the xanthophyll cycle in which a violaxanthin de-epoxidase converts violaxanthin into antheraxanthin and zeaxanthin. increased levels of these xanthophylls together with the protonation of specific pigment-binding antenna proteins cause a conformational change of psii into a high quenching state where excess light energy is dissipated as heat. additionally, zeaxanthin is an efficient 1o quencher and increased levels of this xanthophyll after exposure to high light conditions might reduce damage to membrane lipids. recently, two lhcsr genes have been found to be involved in npq in chlamydomonas reinhardtii indicating that other unidentified components might function in photoprotection and prevention of 1o formation in photosynthetic organisms. singlet oxygen can damage the cell but it has also been found to play an important role in retrograde signaling through the specific activation of nuclear genes by plastid signals. singlet oxygen produced in the chloroplast of the conditional fluorescent mutant was shown to stimulate the expression of a set of genes which was different from h2o induced genes. furthermore, o -/h2o exhibited an antagonizing effect on 1o2-induced gene expression in flu. in a suppressor screen for the 1o2-induced programmed cell death response in flu mutants, two thylakoid-localized proteins, executer and executer, were identified which are involved in the regulation of the 1o2-mediated genetic response. in c. reinhardtii, the response to 1o has been studied using either specific exogenous photosensitizers like rose bengal or neutral red or in strains lacking some 1o protective mechanisms like the xanthophyll-deficient mutant npq lor. as found in a. thaliana, the response of the chlamydomonas hsp70a gene to 1o could be distinguished from the response to h2o by different reporter constructs and was attributed to separate promoter regions. furthermore, the glutathione peroxidase homologous gene gpxh/gpx of c. reinhardtii was strongly induced by 1o but to a much lower extent by other ros. during high light stress, gpxh expression is strongly induced by 1o by transcriptional activation and various regulatory elements in the promoter were required for induction by 1o. the gpxh protein is predicted to be dual-targeted to the cytoplasm and the chloroplast, and its peroxidase activity with plastidial thioredoxin indicates a role in oxidative stress response of the chloroplast. even though 1o can function as a signal to activate nuclear gene expression, our knowledge of how the formation of 1o is controlled in photosynthetic organisms and which components are involved in the signal transduction from the plastid to the nucleus is still far from complete. membrane lipids are primary targets of 1o and oxidized fatty acids could function as signaling intermediates. however, experiments with carotenoid-depleted cultures indicated that in c. reinhardtii the sensor for 1o is not a lipophilic compound in the thylakoid membrane but probably is located in the aqueous phase of the chloroplast. in an effort to identify components that affect the 1o induced genetic response in c. reinhardtii, we performed a mutant screen using a 1o2-specific gpxh reporter construct. mutants with altered gpxh expression were isolated and characterized genetically and physiologically.  isolation of mutants with altered gpxh expression the expression of the gpxh gene is strongly induced by the increased production of 1o in the chloroplast. to identify components affecting 1o2-induced gene expression in c. reinhardtii, a mutant screen was performed using the gpxh-arylsulfatase reporter gene construct pysn to search for clones with altered gpxh response. the wild-type strain 4a+ transformed with pysn was uv-mutagenized and colonies were grown on tap plates in the dark. then, a total number of clones were analyzed for their gpxh-ars expression under medium light condition of μmol photons m- s- in the presence or absence of μm nr. average gpxh-ars expression was induced ± fold by nr treatment. a cutoff of -fold induction by nr and fold higher expression was used to select for clones with reduced induction or increased basal expression, resulting in gpxh-ars induction deficient and gpxh-ars overexpressor mutants. however, after retesting these clones, only six gid and gox mutants could be confirmed. altered response of the reporter enzyme in 4a+ pysn can result from mutations affecting the cellular ars activity, the production of 1o or the signal transduction of 1o2-induced gene expression. to identify the first class of mutants, the induction of the endogenous gpxh gene was measured in the six gid mutants exposed to nr. unfortunately, all gid mutants retained full induction of the gpxh wild-type gene. for the gox mutants, a secondary screen was performed to reduce the number of strains for gpxh expression analysis. based on the knowledge that gpxh overexpression increases the resistance of c. reinhardtii to chemicals enhancing ros production, the mutants were exposed to the 1o2-producing photosensitizers rb and nr at ml conditions or the o --producing chemicals metronidazole and methyl viologen and the organic tert-butylhydroperoxide under low light intensity. one group of mutants with members was more resistant to t-booh compared to wild-type, and many but not all of these mutants were also resistant to nr and rb. furthermore, all strains were tested for their tolerance to high light intensities resulting in the identification of hl-sensitive clones. this phenotype was often combined with sensitivity to rb, nr, mz or mv. a third group of mutants showed no or only very weak changes in tolerance to oxidative stress. since for this subset of mutants a relatively low gpxh-ars overexpression was determined, they were excluded from further analysis. abbreviations: hl: high light of μmol photons m- s- par, t-booh: tert-butylhydroperoxide, nr: neutral red, rb: rose bengal, mz: metronidazole, mv: methyl viologen. tolerance was classified in five different categories compared to the wild-type strain: s: very sensitive, s: sensitive, n: no difference from wild-type, r: resistant, r: very resistant. mutants could be divided into three different groups: hl sensitive mutants, t-booh resistant mutants and mutants with no or only minor changes in tolerance. the remaining mutants, being either hl-sensitive and/or t-booh resistant, were then tested for the expression of the endogenous gpxh wild-type gene by qpcr. a significantly stimulated expression compared to the corresponding wild-type strain could be detected in seven clones ranging from - to -fold overexpression. even though there was a clear correlation between gpxh expression and the expression of the reporter construct, all mutants had a stronger overexpression of gpxh-ars measured by enzyme activity than gpxh expression determined by qpcr, which might be the consequence of individual mrna or the reporter enzyme stability. high doses of uv radiation as applied in this experiment induce multiple point mutations in the genome. to analyze whether defects in multiple genes might be responsible for the phenotype, we performed tetrad analysis of selected gox mutants by crossing them back to the strain 4a- pys twelve independent tetrads were tested for segregation of high gpxh-ars expression in each mutant. a clear 2: segregation of the wild-type and mutant phenotypes was found in strains 35h and 18f, indicating that mutations in a single nuclear gene is responsible for the increased gpxh expression in each case. the same was true for 21b except for one tetrad where one high, two medium and one low expressing progeny were found, suggesting that two closely linked mutations might be responsible for the high ars activity phenotype. no consistent 2: segregation was found in backcrosses of strain 22d and 14a whereas for 22d at least six tetrads resulted in either 3: or 1:2: segregations, for 14a the pattern of three tetrads differed from standard single allele segregation. gpxh overexpression in gox mutants due to increased singlet oxygen production stimulated gpxh expression might either be due to a stimulated production of 1o or a constitutively active signaling pathway under ll condition. sensitivity to hl intensity of several mutants indicates that the former might be the reason for high gpxh expression in some of the gox mutants. the formation of 1o was therefore measured with the fluorescent dye singlet oxygen sensor green allowing the specific quantification of 1o. in order to detect significant amounts of 1o with the membrane impermeable sosg in the wild-type strain, the cells had to be broken by freezing, and exposed to hl intensity for a period of min. increased 1o formation compared to wild-type was detected in all but one of the hl-sensitive mutants. surprisingly, several mutants with normal resistance to hl also showed a stimulated 1o production even though the difference to wild-type was not always significant. comparing 1o formation with gpxh overexpression in gox mutants, no direct correlation between the two parameters could be found. however, since the formation of 1o is a light intensity-dependent photoreaction and gpxh expression and 1o formation were quantified at different light intensities, it is difficult to directly compare these parameters. we therefore measured gpxh expression in all gox mutants at the highest possible light intensity at which the mutants could still survive for at least h and which thus corresponds to hl conditions. indeed, a much stronger stimulation of gpxh expression compared to wild-type could be detected in more than half of the mutants, resulting in a stronger correlation between 1o formation and gpxh expression. increased levels of 1o in gox mutants are a consequence of either increased generation or lowered detoxification of 1o the former might result from a deficient photoprotection mechanism, such as a reduced capacity of npq. therefore, npq was measured in the gox mutants acclimated to either ll or hl for at least h. four mutants had significantly reduced npq under both ll and hl conditions. four other strains only had lower npq under hl but not ll conditions, suggesting a light intensity dependent effect, and for three mutants a stimulation of npq even at ll conditions was found. energy-dependent quenching is one component of npq that requires synthesis of the xanthophylls, zeaxathin and antheraxanthin. however, these and other carotenoids as well as α-tocopherol are also important antioxidants involved in detoxification of 1o pigment analysis of the gox mutants acclimated to the same ll or hl intensity as for npq measurements revealed only small changes in pigment contents of few strains. lutein was not altered in ll and only slightly higher in three mutants in hl conditions compared to the wild-type strain. similarly, α-tocopherol was not strongly affected in ll condition, except for 14h but was significantly reduced in mutants after exposure to hl for h. for only one strain, 18f higher levels of α-tocopherol than in wild-type were detected under hl conditions, and this correlated with increased lutein and zeaxanthin levels in this mutant. despite the important role of zeaxanthin and antheraxanthin in photoprotection and prevention of 1o generation, only moderate changes of these xanthophylls were detected in the mutants compared to the wild-type. only one mutant had a significantly reduced de-epoxidation of xanthophylls during hl exposure showing that this process seems still to be functional in all mutants. nevertheless, when grown in ll, five mutants had significantly reduced de-epoxidation states whereas other mutants had rather increased levels of zeaxanthin and antheraxanthin. gpxh expression and singlet oxygen production negatively correlate with the xanthophyll de-epoxidation state in order to analyze the relationship of all the parameters measured in the gox mutants, linear correlation factors were calculated for every possible combination of parameters. not surprisingly, a strong positive correlation between antheraxanthin and zeaxanthin levels was found, which negatively correlated with violaxanthin under hl conditions, as expected from operation of the xanthophyll cycle. as already shown in figure 3b, 1o formation correlated with gpxh expression under hl conditions. both parameters also negatively correlated with antheraxanthin and zeaxanthin levels, especially in ll-grown cultures. thus, when comparing 1o production at hl and xanthophyll levels at ll it was striking that all the mutants but one had a high de-epoxidation state of xanthophylls or increased 1o production compared to wild-type. this effect was less pronounced when 1o production was compared with de-epoxidation at hl because strongly stimulated de-epoxidation reduced the relative differences between the clones. finally, antheraxanthin and zeaxanthin levels of hl-grown cultures weakly correlated with α-tocopherol and lutein contents under this light condition. hierachical clustering of the mutants with all measured parameters was performed to test whether there are groups of mutants with similar phenotypic pattern. these analyses revealed that one mutant behaved differently from all other mutants. even though 14h was originally screened for high gpxh-ars expression and was found to be hl sensitive, it did not show any stimulated gpxh expression or 1o production at hl intensities. all other mutants were divided into two major groups mainly based on the de-epoxidation state of the xanthophyll pool. group i had lower levels of antheraxanthin and zeaxanthin at ll than wild-type and most of these mutants showed high 1o production and increased gpxh expression. they could be further subdivided into two groups, where strain 18f and 18g belonged to one group with stimulated npq in ll conditions and no changes in α-tocopherol levels under any light intensity. mutants of the other subgroup, on the other hand, had strongly reduced α-tocopherol levels, especially in hl grown cultures, and either lowered or not changed npq compared to the wild-type strain. the second group of mutants with similar or slightly increased xanthophyll levels to wild-type had generally a much lower stimulation of 1o production and gpxh expression than mutants of group i. exceptions were clones 14a and 21b from subgroup iia with strong gpxh overexpression and stimulated 1o production, but these mutants showed strongly reduced npq under both ll and hl intensity that was probably responsible for these phenotypes. in the other subgroup only two mutants had significant gpxh overexpression and higher 1o production and except for lower α-tocopherol levels in hl for some mutants, little differences compared to wild-type were detected for most parameters. discussion no mutants with deficient gpxh induction screening for mutants with altered gpxh expression resulted in the isolation of several high gpxh expression mutants but no strain with deficient or strongly reduced gpxh induction by 1o could be isolated. with a total number of uv-mutagenized clones tested we assume that the coverage of mutations in the genome should be high enough to hit at least one component of a putative 1o signaling cascade with a loss-of-function mutation. the relatively high uv dose resulting in the survival of only to 2% of the cells and the fact that during segregation analyses at least two out of five gox mutants contained two mutations affecting the phenotype suggested a high mutation density in the screened population. still, a second mutant screen for suppressor mutants of gpxh overexpression in strain 21b was performed using an additional plasmid containing the gpxh promoter in front of the nitrate reductase gene. this enabled a direct selection for reduced expression under low light conditions by selecting for chlorate-sensitive clones. out of a total number of × cells surviving uv mutagenesis, clones were chlorate resistant, and of those, clones also had low induction of the gpxh-ars reporter construct by nr. however, all of these clones showed normal induction of the wild-type gpxh gene, indicating that under these screening conditions no gid mutants can be isolated. the conclusion that in strain 21b a constant increased 1o production is responsible for high gpxh expression might explain why no gpxh induction deficient mutants could be isolated, because gpxh might be essential for defense against 1o2-induced damage. this can be excluded for the original mutant screen because selection against light-sensitive mutants during the recovery phase after mutagenesis was prevented by growing the clones in the dark. furthermore, none of the clones was light sensitive under ml conditions during the induction tests. thus, even though steps were taken to minimize a negative selection against gid mutants, we cannot exclude that a functional 1o response of the gpxh and maybe other genes is essential for c. reinhardtii. on the other hand, it is also possible that several redundant signaling pathways form a complex network to activate gpxh expression, thereby hampering the isolation of gid mutants. finally, it still might be that more dark-grown mutagenized clones had to be tested to find the desired mutants. a similar screen to isolate 1o responsive mutants in a. thaliana resulted in the identification of at least three mutants deficient in the upregulation of different 1o2-induced genes in the conditional flu mutant. however, 1o2-signaling seems to have different cellular functions in a. thaliana, at least in seedlings, where it is part of a programmed cell death response and in c. reinhardtii where it seems to be involved in response to cytotoxic environmental stresses. gpxh overexpression and correlation with singlet oxygen production, npq and pigment levels in contrast to the lack of gid mutants, many gox mutants with a stimulated expression of the gpxh wild-type gene, especially under hl intensities, could be isolated. the light intensity-dependent increase of gpxh overexpression and the sensitivity to hl conditions indicated that in several mutants 1o formation might be enhanced and cause a photooxidative stress. indeed, increased 1o formation was measured in all but one hl-sensitive as well as many hl-resistant gox mutants, which nicely correlated with gpxh expression under hl conditions. this indicates that in most or even all gox mutants, gpxh overexpression seems not to be caused by a constitutively active signal transduction pathway but by the increased production of 1o under the light conditions tested. the poor correlation between 1o production and gpxh expression under ml, the clustering in at least five phenotypic distinct groups and the fact that three of the mutants have been mapped to different linkage groups indicates that mutations in different nuclear genes are responsible for the increased 1o production in the various mutants. singlet oxygen formation in photosynthetic organisms is caused by the conversion of excited chlorophylls into the triplet state and the reaction with molecular oxygen. the organisms try to minimize this process by regulating the excitation pressure on the psii reaction center chlorophylls, optimizing the electron flow in the photosynthetic electron transport chain and quenching chlorophyll triplet states and 1o thus, 1o accumulation can result from either stimulated production, e.g. due to enhanced triplet chlorophyll formation, or lowered detoxification of the ros due to defects in some light-induced protection mechanisms. such a defect in protection mechanisms could be the reason for high 1o formation in most of the group i mutants of cluster analysis having reduced zeaxanthin and antheraxanthin levels which correlates with high 1o formation. a well characterized mutants with reduced xanthophyll levels and defects in photoprotection is the npq lor double mutant lacking the carotenoids zeaxanthin, antheraxanthin and lutein. similar to many mutants of group i, this mutant exhibits increased ros production, gpxh expression and sensitivity upon hl but not ll illumination. however, comparison of npq and npq lor showed that reduced levels of zeaxanthin and antheraxanthin but normal levels of other carotenoids like lutein would probably not cause a very strong phenotype. thus, reduced efficiency of more than one protection mechanism might be required to achieve increased 1o accumulation. indeed, five of the group i mutants with reduced xanthophyll levels also showed lowered α-tocopherol levels under ll and/or hl conditions. furthermore, levels of other components of the thylakoid membrane with 1o2-quenching capacity like plastoquinone and α-tocopherolquinone were not quantified but might also be affected in some of the mutants. thus, reduced photoprotection by the combination of lower xanthophylls, α-tocopherol and maybe other deficiencies might be the cause for increased 1o production and gpxh expression in these mutants. this could either result from mutations in multiple loci, as found for clone 22d, or caused by mutations in a single gene encoding for a global regulator of cellular photoprotection mechanisms. even though deepoxidation of xanthophylls is involved in npq, these parameters did not correlate in our mutants indicating that other factors also play an important role for npq. for example, two strains of group i showed rather increased npq levels under ll conditions even though their xanthophyll levels were reduced, clustering them in a separate subgroup of group i. very high gpxh expression in 18f and 18g under ml conditions indicates stimulated 1o generation already at ll. we speculate that these mutants might have increased 1o production due to an enhanced energy transfer from the psii reaction center to molecular oxygen. by this, quenching of excitation energy by molecular oxygen would increase npq but reduce the photosynthetic electron transport rate required for building up the proton gradient and activating the xanthophyll cycle. thus, reduced xanthophylls would not be the cause but the consequence of increased 1o production. however, other effects of the mutations cannot be excluded which might also explain these phenotypes. contrary to 18f and 18g two mutants had strongly reduced npq both under ll and hl conditions but similar or rather increased xanthophyll levels compared to wild-type showing that defects in qe-independent mechanisms seem to affect npq in these mutants. photoinhibition stimulated by excess light should not be relevant under ll conditions, and a deficiency in state transition should not strongly affect npq at hl intensities. on the other hand, a qe-independent effect was also found in a c. reinhardtii mutant defective in two linked lhcsr genes. however, segregation analysis of backcrosses of 14a and 21b with a wild-type strain revealed that both mutants have probably mutations in two different genes affecting gpxh expression indicating that the phenotypes of these mutants could be caused by the combination of different defects. in mutants with functional detoxification mechanisms, strong 1o accumulation after stimulated production of the ros might be prevented by the induction of these detoxification mechanisms. this is supported by the negative correlation of 1o formation and deepoxidation of xanthophylls, where increased levels of zeaxanthin and antheraxanthin correlate with a low stimulation of 1o production. mutants with increased xanthophyll levels are mainly represented in the group iib mutants of cluster analysis with rather few and weak phenotypic changes. a general increase in antioxidant levels including zeaxanthin, antheraxanthin, lutein and α-tocopherol and a significant rise in npq was detected in strain 14h where no stimulated 1o production and gpxh expression could be measured any more. this shows that the various protection mechanisms can compensate each other and thus control the production of deleterious ros. this is in agreement with data of various mutants lacking specific antioxidants: α-tocopherol-deficient strains of c. reinhardtii, synechocystis sp. pcc and a. thaliana all were very tolerant to photooxidative stress during hl conditions, and only under extreme conditions such as a combination of very hl and low temperature or chemical treatment a phenotype became visible. it was suggested that the presence of other antioxidants such as zeaxanthin or increased levels of β-tocopherol can compensate for α-tocopherol deficiency. conversely, the a. thaliana npq mutant, lacking zeaxanthin and antheraxanthin, accumulates higher amounts of α-tocopherol. thus, zeaxanthin, α-tocopherol and plastoquinol have overlapping functions in photoprotection and together prevent the formation of deleterious 1o under natural conditions. however, when these protection mechanisms are overwhelmed, 1o starts to accumulate and damage cellular components. this is when defense genes like gpxh, which repair and remove damaged biomolecules, are required to survive the oxidative stress. activation of genetic stress response without altering antioxidant levels by an acclimation to increased 1o production or the direct overexpression of the gpxh gene in c. reinhardtii were shown to increased resistance to oxidative stress by rb, nr and t-booh. increased tolerance against t-booh was also found for of the gox mutants tested including all the group iib mutants as well as strains 14b 14c and 35h. thus, increased expression of stress response genes like gpxh might explain the hl resistance of these mutants and shows the important role of gpxh and other defense genes in the photooxidative stress response of photosynthetic organisms.  the failure to isolate 1o signal transduction mutants indicates that several redundant signaling pathways might be involved in the gpxh response. this is supported by the identification of multiple regulatory elements in the gpxh promoter being required for induction of the gene. singlet oxygen generation, on the other hand, was altered in several mutants resulting in higher expression of the gpxh gene. increased oxidative stress resistance of many of these mutants confirms the importance of the 1o2-induced genetic response in the defense against ros-induced damage. furthermore, isolation of phenotypic different groups of 1o2-overproducing mutants indicates that mutations in different photoprotective mechanisms might be responsible for higher 1o levels in various gox mutants which most seem to be, based on pigment analysis, different from known photoprotective mutants like npq lor the comparison of their phenotypes suggests that in several gox mutants multiple defense processes might be affected what might be due to, among other things, mutations in a global regulator of cellular photoprotection mechanisms. thus, the isolation of these mutants might allow identifying new components involved in the control of 1o formation by different cellular protection mechanisms. 
furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and gpxh expression, which could either result from mutations in multiple loci or in a single gene encoding for a global regulator of cellular photoprotection mechanisms. furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants. to investigate components affecting singlet oxygen signaling in c. reinhardtii, a mutant screen was performed. when photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. many of these gpxh overexpressor mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. mutants with altered gpxh response were isolated from uv-mutagenized cells containing a gpxh-arylsulfatase reporter gene construct. out of clones tested, no mutant deficient in gpxh induction was isolated, whereas several clones showed constitutive high gpxh expression under normal light conditions. interestingly, most gox mutants produced increased singlet oxygen levels correlating with high gpxh expression. screening for mutants with altered gpxh expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ros in photosynthetic organisms. phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. in chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene gpxh/gpx during high light stress, but little is known about the cellular mechanisms involved in this response.
 when photosynthetic organisms are exposed to harsh environmental conditions such as high light intensities or cold stress, the production of reactive oxygen species like singlet oxygen is stimulated in the chloroplast. in chlamydomonas reinhardtii singlet oxygen was shown to act as a specific signal inducing the expression of the nuclear glutathione peroxidase gene gpxh/gpx during high light stress, but little is known about the cellular mechanisms involved in this response. to investigate components affecting singlet oxygen signaling in c. reinhardtii, a mutant screen was performed.  mutants with altered gpxh response were isolated from uv-mutagenized cells containing a gpxh-arylsulfatase reporter gene construct. out of clones tested, no mutant deficient in gpxh induction was isolated, whereas several clones showed constitutive high gpxh expression under normal light conditions. many of these gpxh overexpressor mutants exhibited higher resistance to oxidative stress conditions whereas others were sensitive to high light intensities. interestingly, most gox mutants produced increased singlet oxygen levels correlating with high gpxh expression. furthermore, different patterns of altered photoprotective parameters like non-photochemical quenching, carotenoid contents and α-tocopherol levels were detected in the various gox mutants.  screening for mutants with altered gpxh expression resulted in the isolation of many gox mutants with increased singlet oxygen production, showing the relevance of controlling the production of this ros in photosynthetic organisms. phenotypic characterization of these gox mutants indicated that the mutations might lead to either stimulated triplet chlorophyll and singlet oxygen formation or reduced detoxification of singlet oxygen in the chloroplast. furthermore, changes in multiple protection mechanisms might be responsible for high singlet oxygen formation and gpxh expression, which could either result from mutations in multiple loci or in a single gene encoding for a global regulator of cellular photoprotection mechanisms. 