BACKGROUND
carotenoids are a group of c <dig> pigments that contain a conjugated double-bond system, leading to strong absorption of visible light and antioxidant properties.
they are widely distributed among taxa, ranging from cyanobacteria and fungi to red and green algae and land plants  <cit> .
xanthophylls are oxygenated carotenoids that play a crucial role in the photosynthetic apparatus of higher plants  <cit> .
their composition in plants is remarkably conserved and consists of five major xanthophylls, the most abundant being the β-ε-xanthophyll lutein, and the four β-β-xanthophylls violaxanthin, neoxanthin, antheraxanthin and zeaxanthin  <cit> .
xanthophylls act both as photoreceptors, absorbing light energy which is used in photosynthetic electron transport, and as photoprotectants of the photosynthetic apparatus from excess light and from the reactive oxygen species  generated during photosynthesis  <cit> .
moreover, they are structural elements of the photosynthetic apparatus: lhcii, the major light-harvesting complex  of photosystem  ii, binds lutein, violaxanthin and neoxanthin at four distinct binding sites called respectively l <dig>  l <dig>  n <dig> and v <dig>  <cit> ; the occupancy of l <dig> site was shown to be essential for protein folding  <cit> .
xanthophyll biosynthesis in plants is divided in two distinct branches: the α branch leads to the formation of the ε-β-hydroxylated xanthophyll lutein from α-carotene, while the β branch leads to the production of β-β-hydroxylated xanthophylls  from β-carotene .
recent studies on carotenoid biosynthetic mutants of arabidopsis thaliana have improved our understanding on xanthophyll accumulation at the molecular level.
the first steps in plant xanthophyll biosynthesis are the hydroxylation of α- and β-carotene.
two different classes of enzymes are involved: the ferredoxin-dependent di-iron oxygenases  which are active in β-ring hydroxylation, and the cytochromes p <dig>   <cit>  which are active in hydroxylation of both the ε-ring and β-ring of α-carotene, although the activity of lut <dig> on ε-rings is low  <cit> .
it has been suggested that a third chloroplast-targeted member of the cyp <dig> family, cyp97b <dig> might have a role in carotenoid biosynthesis  <cit> .
this hypothesis is however in contrast with the complete lack of xanthophylls in the quadruple chy1chy2lut1lut <dig> mutant  <cit> , suggesting that chy <dig>  chy <dig>  lut1/cyp97c <dig> and lut5/cyp97a <dig> are the complete complement of carotene hydroxylases in a. thaliana.
the strong phenotypes of mutants with altered xanthophyll composition imply that the presence and relative amounts of these pigments have a key role for plant fitness.
the lut <dig> mutant, affected in ε-ring formation, lacks lutein  <cit>  and shows a complex phenotype with reduced antenna size, photodamage in high light  due to impaired chlorophyll triplet quenching  <cit>  and decrease of non-photochemical quenching   <cit> .
additional features include over-accumulation of zeaxanthin in hl with respect to wild-type and monomerization of lhcii.
lack of both lutein and zeaxanthin further decreases the capacity for photoprotection in both arabidopsis and chlamydomonas  <cit> .
the double chy1chy <dig> mutant, in which the two non-heme β-hydroxylases are deleted, shows reduced levels of β-β-xanthophylls and decreased resistance to photooxidation  <cit> .
introduction of the lut <dig> mutation in the chy1chy <dig> background leads to the almost complete disappearance of β-xanthophylls and strong photosensitivity  <cit> .
neoxanthin preserves psii from photoinactivation by superoxide anions  <cit>  while violaxanthin and zeaxanthin show enhanced activity in singlet oxygen scavenging  <cit> .
in order to further detail the effects of altered xanthophyll composition on the organization of photosynthetic complexes and gain understanding on the regulatory events controlling xanthophyll biosynthesis in arabidopsis, we have introduced the lut <dig> mutation in the semi-lethal chy1chy2lut <dig> background.
surprisingly, the chy1chy2lut2lut <dig> mutant shows increased presence of β-β-xanthophylls with respect to chy1chy2lut <dig>  the psi/psii ratio in this mutant is severely decreased as well as the level of total xanthophyll accumulation, suggesting that the latter have a key role, beside photoprotection, in regulating photosystem stoichiometry.
RESULTS
construction of the chy1chy2lut2lut <dig> quadruple mutant
to generate the quadruple mutant chy1chy2lut2lut <dig>  the homozygous triple mutant chy1chy2lut <dig> was crossed with the homozygous single mutant lut <dig>  <cit> .
all t-dna insertions were in the columbia background and appropriate oligonucleotides were used to confirm the presence of the insertions and their homozygous vs. heterozygous state   <cit> .
the mutant was maintained as a triple homozygous, single heterozygous stock: two different parental genotypes were used, heterozygous for either lut5/cyp97a <dig> or chy <dig>  with similar results .
the lut <dig> and chy1chy2lut <dig> lines were included in this characterization, representing respectively the lutein-less and β-xanthophyll-less controls.
when selfed, the progeny of each single heterozygous stock, as well as wild-type, chy1chy2lut <dig> and lut <dig> seeds, were grown under low light conditions  both in agar plates containing sucrose  and in soil.
no quadruple homozygous mutants were recovered in soil after  <dig> week of growth, while the progeny segregated in a 1: <dig> ratio for white: green seedlings in agar plates.
wild type and lut <dig> plants did not display a visible phenotype after  <dig> weeks of growth in agar plates, whereas mutants chy1chy2lut <dig> and chy1chy2lut2lut <dig> showed, respectively, reduced growth and paler leaves .
pigment composition
we analyzed by hplc-dad-ms the pigment content of six-week-old leaves of wild type, lut <dig>  chy1chy2lut <dig> and chy1chy2lut2lut <dig> plants grown on agar plates .
pigments were resolved on a c- <dig> column, able to separate cis- from trans-carotenoids and their identity was confirmed by co-migration with authentic standards and high resolution ms .
chy1chy2lut <dig> and chy1chy2lut2lut <dig> plants showed an increase in chlorophyll a/b ratio, as well as a reduced chlorophyll  and carotenoids  content per fresh weight, with respect to both wild-type and lut2; the effects were more severe in the quadruple mutant, that showed a significant reduction of chl/car ratio  with respect to the other genotypes .
wild-type leaves accumulate four major carotenoids  and trace amounts of α-carotene.
mutants show distinct composition of the xanthophyll fractions: lutein represents > 98% of total xanthophylls in chy1chy2lut <dig> plants; lut <dig> and chy1chy2lut2lut <dig> do not contain lutein and accumulate violaxanthin, antheraxanthin, zeaxanthin and neoxanthin; chy1chy2lut2lut <dig> shows a higher content of β-β-xanthophylls  with respect to chy1chy2lut <dig> .
β-carotene content is strongly increased in chy1chy2lut2lut <dig> with respect to the wild-type and the other mutants .
as a result, the xanthophyll/carotene ratio changes dramatically, ranging from  <dig>  ±  <dig>  in wild-type to  <dig>  ±  <dig>  in chy1chy2lut2lut <dig>
pigment composition was quantified via lc-dad-ms in dark-adapted leaves from 6-weeks-old plants.
data are expressed as mean ± sd .
fw, fresh weight.
values marked with the same letters are not significantly different from each other within a column .
plants grown at  <dig> μmol photons m- <dig> s- <dig> for  <dig> weeks were dark-adapted, then carotenoids were extracted and quantified via lc-dad-ms. data are expressed as mean ± sd .
nd, not detectable; fw, fresh weight.
values marked with the same letters are not significantly different from each other within a column .
β-carotene accumulation is expected in chy1chy2lut2lut <dig>  in which the biosynthetic flux is diverted towards the β-β-branch by the lack of three out of four β-carotene hydroxylases  ; however, β-β-xanthophyll accumulation in this mutant suggests that the fourth hydroxylase  is more active toward β-carotene in this background than in the chy1chy2lut <dig> parent, resulting in 28-fold higher levels of β-β-xanthophylls.
gene expression
we measured the lut <dig>  lut <dig>  chy <dig> and chy <dig> mrna levels by real time pcr in the different mutants .
all mrnas are almost completely absent in the corresponding mutants; lut <dig>  chy <dig> and chy <dig> are induced in the lut <dig> mutant, that accumulates higher levels of β-carotene; lut <dig> is induced in the chy1chy2lut <dig> and, even more, in the chy1chy2lut2lut <dig> mutant, which shows drastically reduced xanthophyll/carotene ratios; however, the increase of lut <dig> levels between the two mutants is only  <dig> -fold, while the increase of β-β-xanthophylls is 28-fold.
photosynthesis-related functions: psii quantum efficiency and non-photochemical quenching of chlorophyll fluorescence
the impact of xanthophyll depletion on photosynthesis was investigated by room temperature chlorophyll fluorescence measurements .
the variable/maximum fluorescence yield  of dark-adapted leaves reflects changes in psii photochemical efficiency  <cit> .
lut <dig> had the same fv/fm ratio as wild-type , while the triple chy1chy2lut <dig> and quadruple chy1chy2lut2lut <dig> mutants scored, respectively, values of  <dig>  and  <dig>  .
the efficiency of psii photochemistry  gives a measure of the rate of linear electron transport, an indication of the photosynthetic activity  <cit> .
chlorophyll fluorometry revealed a significant reduction in Φpsii in both chy1chy2lut <dig> and chy1chy2lut2lut <dig> , confirming that efficient light use is compromised by β-β-xanthophyll depletion.
detached leaves of wild-type and mutant plants grown at  <dig> μmol photons m- <dig> s- <dig> for  <dig> weeks were given  <dig> min of illumination , then photosynthetic parameters were provided by analysis of rt chlorophyll fluorescence: maximum quantum yield of psii  and efficiency of psii photochemistry .
data are expressed as mean ± sd .
values marked with the same letters are not significantly different from each other within a column .
non-photochemical quenching  of chlorophyll fluorescence is the fastest photoprotective mechanism in the chloroplast: thermal dissipation is activated within few seconds upon exposure to excess light and it protects photosynthesis by decreasing the lifetime of singlet chlorophylls  <cit>  in order to minimize generation of ros in the psii  <cit> .
npq was measured on detached leaves, in saturating co <dig> .
wild-type plants, upon short illumination at saturating light intensity , showed a rapid rise of npq, reaching a maximum value of  <dig> .
most of this npq relaxed rapidly in the dark, thus reflecting the Δph-dependent de-excitation of excess energy measured as qe, the rapidly-reversible component of npq.
lut <dig> showed npq kinetics in agreement with published results  <cit> , with lower amplitude and slower rise than wild-type plants .
the other mutants showed a strong reduction in npq, scoring  <dig>  in chy1chy2lut <dig> and  <dig>  in chy1chy2lut2lut5; furthermore, upon correction for residual quenching after dark relaxation , both mutants showed very little recovery , suggesting that the measured fluorescence quenching was mainly due to photoinhibition, and the capacity for qe was strongly reduced in mutant leaves .
furthermore, npq kinetics were measured during steady-state photosynthesis, upon a prolonged illumination with increasing light intensities .
all genotypes showed chlorophyll fluorescence quenching, whose magnitude increased with irradiance.
however, fluorescence quenching in wild-type and lut <dig> leaves was mainly due to the qe-type of npq , while in both chy1chy2lut <dig> and chy1chy2lut2lut <dig> the main component of npq was qi-type, irreversible quenching .
these data confirm that both the reduction of β-β-xanthophylls and the lack of lutein are responsible for impaired npq kinetics.
photosensitivity under short-term stress conditions
when photosynthetic organisms are exposed to light in excess, photo-oxidative stress occurs within the chloroplast, with production of ros such as singlet oxygen , leading to oxidative damages to a large variety of biomolecules.
the β-xanthophyll-depleted mutants chy1chy2lut <dig> and chy1chy2lut2lut <dig>  even upon growth in low light , showed signs of photooxidation: lower chlorophyll content and psii quantum yield, retarded growth and paler leaves with respect to wild-type and lut <dig> plants .
therefore, in order to assess whether lower xanthophyll levels affect the capacity to prevent chloroplast photooxidation, leaves from wild-type and mutant plants grown in low light were transferred to strong light  and low temperature  for  <dig>  h; the combination of low temperature and high light intensity is known to enhance the induction of both psii photoinhibition and membrane photooxidation in leaves, since the enzymes of the calvin cycle are slowed down and the light harvested by photosystems rapidly exceed the capacity of plants to use this energy.
thus, the treatment produces a photooxidative stress, which can be measured as a decrease in the chlorophyll content and an increase in oxidation of membrane lipids.
hl treatment was effective in producing higher pigment bleaching in chy1chy2lut <dig>  and chy1chy2lut2lut <dig> , while wild-type and lut <dig> leaves were less affected, loosing around 25% of their chlorophyll content .
to investigate the level of membrane lipid peroxidation, the same leaves were analyzed for mda content : chy1chy2lut2lut <dig> and chy1chy2lut <dig> leaves showed higher accumulation of mda upon stress treatment , thus a far higher level of lipid peroxidation with respect to wild-type and lut <dig> plants ; chy1chy2lut2lut <dig> plants showed a far higher photosensitivity in high-light than chy1chy2lut <dig> ; the latter was the xanthophyll mutant with the highest light sensitivity described so far  <cit> .
results clearly show an unprecedented level of photosensitivity in chy1chy2lut2lut <dig> plants, thus implying a severe impairment of the photoprotection mechanisms in this xanthophyll-depleted mutant.
organization and stoichiometry of pigment binding complexes
the extreme sensitivity to photo-oxidative stress of the chy1chy2lut2lut <dig> mutant could be due to altered pigment composition, to altered protein composition of photosystems, or to both.
this mutant showed the highest chl a/b ratio, and the lowest chlorophyll content and xanthophyll/carotene ratio of all analyzed genotypes .
since both chl b and xanthophylls are associated with lhc, their decrease suggests a decrease in antenna size.
we investigated the organization of pigment-protein complexes in thylakoids by non-denaturing deriphat-page and by sucrose density gradient fractionation of solubilized thylakoids, followed by sds-page of the fractions .
seven major green bands were resolved upon solubilization of wild-type thylakoid membranes with  <dig> % dodecyl-α-d-maltoside  on deriphat-page  <cit> .
the psi-lhci complex was the major band  in the upper part of the gel, while the components of the psii-lhcii complex migrated as multiple bands, namely the psii core  and the antenna moieties, including the cp29-cp24-lhcii-m supercomplex   <cit> , lhcii trimer  and monomeric lhcb .
bands with high apparent masses were detected in the upper part of the gel  containing non-dissociated psii supercomplexes.
the chy1chy2lut <dig> pattern was very similar to that of wild-type, showing no major qualitative changes in the organization of the photosynthetic apparatus: the main differences consisted in a higher psii core/lhcb ratio and a lower content in psii supercomplexes   <cit> .
in the genotypes lacking lutein, namely lut <dig> and chy1chy2lut2lut <dig>  the trimeric organization of lhcii was disrupted, as previously described  <cit> .
however, thylakoid membranes isolated from chy1chy2lut2lut <dig> plants showed additional features, namely the complete absence of bands  <dig>   <dig> and 7; bands  <dig> and  <dig> were much less represented than in wild-type, while psii core complex was the most abundant among pigment-protein complexes .
in order to obtain sufficient amounts of pigment-protein complexes for further analysis, solubilized thylakoids from wild type and mutants were fractionated by sucrose gradient ultracentrifugation ; the results confirmed that trimeric lhcii band, as well as the cp29-cp24-lhcii supercomplex completely disappeared in the chy1chy2lut2lut <dig> mutant; further differences consisted into reduced levels of monomeric lhcb  and a much higher psii/psi ratio  with respect to the other genotypes.
the reduction of monomeric lhcbs in the chy1chy2lut2lut <dig> mutant occurs in spite of the presence of the lut <dig> mutation, which favors lhcii monomerization .
fractions collected from the sucrose gradients of wild-type and chy1chy2lut2lut <dig>  were further characterized by sds-page using two buffer systems  and by absorption spectroscopy .
according to previous results with α-dm, sds-page analyses of wild-type fractions showed that band  <dig> contained the minor antennae cp <dig>  cp <dig> and cp <dig> as well as components of monomerized lhcii, while band  <dig> contained lhcb1- <dig> polypeptides only   <cit> .
band  <dig> was enriched in psii core complex   <cit> , nevertheless the chl b absorption  suggests it retains lhcb proteins; band  <dig> contained almost exclusively the psi-lhci complex   <cit> .
band  <dig> from chy1chy2lut2lut <dig> contained the same polypeptides as the corresponding band from wild-type, although the relative amounts of the lhcb1- <dig> polypeptides and cp <dig> were decreased .
the data confirm that lhcii is present in the mutant, however in far lower amounts than in wild-type, and is in its monomeric aggregation state.
band  <dig> from the mutant contained almost exclusively psii core complex polypeptides , band  <dig> from the mutant contained psi-lhci.
the levels of selected proteins in wild-type and chy1chy2lut2lut <dig> thylakoids were determined by quantitative western-blot analysis using psbb  as internal control : all lhcb subunits were reduced in chy1chy2lut2lut <dig> with respect to wild-type thylakoids; psba , oec- <dig> and psbs, subunits of the psii core complex, were present in the same level in both genotypes, while cytochrome f and atpase β-subunit were in higher amounts in chy1chy2lut2lut <dig>  immunoblotting using psaa as internal standard showed that each lhca protein was present in wild-type amounts, thus suggesting that the lhca/psi ratio is conserved in the mutant.
in contrast, the psi/psii ratio was extremely low in chy1chy2lut2lut <dig>  reaching approximately 22% of wild-type value .
the data shown above indicate that xanthophyll depletion in the chy1chy2lut2lut <dig> plants causes a strong reduction in the amount of lhcb proteins per psii reaction center, and has a negative impact on the total amount of the psi-lhci supercomplex.
the latter result is unexpected, since xanthophylls are mainly bound to lhc complexes while core complexes of both photosystems only bind carotenes, implying that xanthophyll abundance should not affect their folding or stability.
a key question is whether the lower psi/psii ratio found in chy1chy2lut2lut <dig> thylakoids is peculiar to this genotype or is a general consequence of altered xanthophyll content.
to answer this question, the abundance of lhcii and psi core with respect to cp <dig> was assessed by quantitative immunotitration, in thylakoids isolated from  <dig> arabidopsis mutants with altered xanthophyll content  <cit> .
figure  <dig> shows the distribution of lhcii  and psi core  amounts, relative to cp <dig>  in the different mutants with different xanthophyll/carotenoid ratios: both distributions show an increase of lhcii/psii and psi/psii at increasing xanthophyll/carotenoid ratios, and the data can be fitted with an exponential function .
the results display a clear correlation between parameters .
furthermore, quantitative data for the individual pigment-protein complexes accumulation level per fresh weight  were plotted vs. xanthophyll/carotenoid content in the different mutants .
the distributions show a lower lhcii content per fresh weight at decreasing xanthophyll/carotenoid ratios , while psii core  content per fresh weight was essentially unaffected by xanthophylls depletion .
unlike psii, the psi core  content decreases at decreasing xanthophyll/carotenoid ratios .
these results suggest that xanthophyll depletion not only causes a marked depletion in lhc proteins, as expected from the xanthophylls being ligands of lhcs, but also modulates the ratio between psi/psii, despite β-carotene rather than xanthophylls is the ligand for psi and psii core complexes.
discussion
in this work, we analyzed the modifications of the photosynthetic apparatus in the arabidopsis mutant chy1chy2lut2lut <dig>  that accumulates β-xanthophylls despite disruption of the three chy <dig>  chy <dig> and lut <dig> genes encoding carotene hydroxylases.
in this mutant the only carotene hydroxylase activity is provided by the lut <dig> gene product, which allows for accumulation of only 20% of the wild-type xanthophylls.
in these conditions, biogenesis of the photosynthetic apparatus was strongly affected yielding into a reduction of psii antenna size, a decrease of psi/psii ratio and an increased photosensitivity.
lut <dig> as a β-carotene hydroxylase
lut <dig> was originally reported to be only gene product required for the ε-ring hydroxylation of α-carotene  <cit> .
the first indication of a possible involvement of lut <dig> in β-ring hydroxylation came from the analysis of the chy1chy2lut <dig> triple mutant, which accumulates lutein , providing genetic evidence that lut <dig> must be also active in the hydroxylation of the β-ring of α-carotene  <cit> .
introduction of the lut <dig> mutation in the chy1chy2lut <dig> triple mutant completely abolished xanthophyll biosynthesis, indicating that chy <dig>  chy <dig>  lut <dig> and lut <dig> constitute the full complement of carotenoid hydroxylases in arabidopsis  <cit> .
a number of studies  <cit>  have shown a functional redundancy amongst the carotene hydroxylase enzymes.
however, the extent of recovery in xanthophyll content by mutants carrying a single carotene hydroxylase activity gave insights on the substrate preference of residual activity.
as an example, trace amounts of β-β-xanthophylls, present in the chy1chy2lut <dig> triple mutant and abolished by the introduction of the lut <dig> mutation  <cit> , led to the conclusion that lut <dig> has a low level of activity toward the β-rings of β-carotene.
overall, in vivo analysis clearly showed that chy <dig> and chy <dig> are most active in β-carotene hydroxylation, while lut <dig> and lut <dig> enzymes catalyze preferentially the biosynthesis of α-xanthophylls  <cit> .
here we show that, in the absence of α-carotene , lut <dig> shows a major β-carotene hydroxylase activity, leading to the accumulation of substantial amounts of β-β-xanthophylls.
indeed, when considering the moles of hydroxylated β-rings accumulated in chy1chy2lut <dig> and chy1chy2lut2lut <dig> mutants, total amount is maintained upon introduction of the lut <dig> mutation into the chy1chy1lut <dig> background: ~ <dig>  μmol of hydroxylated β-rings/gfw in chy1chy2lut <dig> vs. ~ <dig>  μmol/gfw in chy1chy2lut2lut <dig> .
however, the total amount of xanthophylls per fresh weight is reduced significantly in chy1chy2lut2lut <dig> , while the carotene/xanthophyll ratio is increased .
the most likely scenario is that lut <dig> has a higher affinity towards ε-rings vs. β-rings and that, when α-carotene is available, lut <dig> performs mainly ε-ring hydroxylation; however, since no ε-ring-substrates are available in the chy1chy2lut2lut <dig>  the β-ring substrates are processed, thus bringing out this minor activity of the enzyme.
conversely, in the absence of the main hydroxylases for β-rings chy <dig>  chy <dig> and lut <dig>  all β-rings become available for lut <dig> activity in chy1chy2lut2lut <dig>  a consequence of lut <dig> operation on a less preferred substrate  is its reduced overall catalytic rate that limits the level of xanthophylls accumulated in the chy1chy2lut2lut <dig> genotype.
it is worth noting that the β-hydroxylase activity does not discriminate between β-rings of α- and β-carotene; indeed, the total amount of hydroxylated β-rings per fresh weight is the same in both chy1chy2lut <dig> and chy1chy2lut2lut <dig>  this evidence updates our knowledge on the molecular details of carotenoid hydroxylases, with respect to the view that lut <dig> has only a low level of in vivo hydroxylase activity toward the β-carotene and a stronger activity towards α-carotene  <cit> .
the concentration of β-carotene increased in the quadruple chy1chy2lut2lut <dig> mutant to a level higher than in the wild type .
this is the result of the combination of several factors: first, the lut <dig> mutation redirects the flux in the pathway towards the β-β-branch; this mutation alone is sufficient to cause a slight increase in β-carotene .
moreover, the reduced hydroxylase activity due to the chy <dig>  chy <dig> and lut <dig> mutations, reduces the rate of β-carotene processing into downstream xanthophylls , favoring its accumulation.
the almost 2-fold increase of the lut <dig> transcript in the quadruple mutant with respect to the wild type  is insufficient to fully compensate for the disappearance of the chy <dig>  chy <dig> and lut <dig> hydroxylases, likely due to the low catalytic efficiency of lut <dig> for β-rings.
reduced xanthophyll content negatively affects energy-dependent quenching qe and photoprotection
the excess energy dissipation into heat  is strongly depleted in the chy1chy2lut2lut <dig> mutant, its amplitude being close to zero .
since the level of psbs, the ph sensitive trigger for npq  <cit>  is similar to wild-type, this effect can be attributed to the low level of the interacting partners of psbs where the actual quenching occurs, i.e.
the lhcb proteins  <cit> , and to the lack of lutein which limit npq  <cit> .
all together these results support the correlation between xanthophyll content and amplitude of qe, previously indicated on the basis of antisense inhibition of beta hydroxylation  <cit> .
however, the high photosensitivity of chy1chy2lut2lut <dig> plants is likely to be caused by the failure of additional photoprotection mechanisms, since the npq <dig> mutant, although depleted in npq, only showed minor increase in photosensitivity  <cit> .
the fast leaf chlorophyll bleaching  and high levels of lipid peroxidation  in chy1chy2lut2lut <dig> with respect to wild-type, lut <dig> and chy1chy2lut <dig> are likely due to the strong depletion in lhcb proteins .
xanthophylls are needed for folding of lhc proteins in vitro  <cit> , thus it is not surprising that a strong decrease in their availability leads to a decreased content in lhc.
however, only lhcb proteins are affected, while lhca proteins are maintained with the same stoichiometry with respect to psi reaction center, as shown by the identical migration rate of psi-lhci supercomplexes in sucrose gradients and native gels  and by quantitative immunotitration of lhca versus psaa content .
as a result of the higher stability of lhca vs. lhcb proteins, psi antenna function is maintained, while psii antenna function is impaired.
we conclude that in vivo carotenes cannot replace xanthophylls in stabilizing lhcb proteins, thus leading to their strong reduction.
furthermore, this implies that functional lhcb proteins are essential for photoprotection, in agreement with a previous report  <cit> .
extreme reduction in lhc proteins is obtained with the ch <dig> mutation in arabidopsis, that impairs chl b synthesis and prevents assembly of functional lhc  <cit> .
although this mutant undergoes severe photoinhibition in high light, it can grow on soil and is not photoinhibited in moderate light  <cit> .
thus ch <dig>  with a psii antenna size even smaller than that of chy1chy2lut2lut <dig>  can survive in the absence of a reduced carbon source, a lethal condition for chy1chy2lut2lut <dig>  we conclude that reduced lhc content, although likely contributing to sensitivity, cannot be the only reason for the extreme phenotype of chy1chy2lut2lut <dig>
limitation in total xanthophyll availability affects light-harvesting complex content and psi/psii ratio
the analysis of the pigment-protein complexes in the chy1chy2lut2lut <dig> mutant shows that lhcb proteins are strongly decreased with respect to psii, while lhca assembly into psi-lhci are much less, or not at all, affected .
a reduction in lhcb proteins is also observed in the chy1chy2lut <dig> mutant, albeit to a lesser extent than in chy1chy2lut2lut <dig>  <cit> .
this effect is likely due to the incapacity of lhcb proteins to fold in the absence of xanthophylls  <cit> , while lhca proteins can also bind small amounts of β-carotene  <cit> .
instead, there is no evident reason for the 5-fold decrease in psi/psii ratio : since lhca proteins are maintained with the same stoichiometry with respect to psi reaction center and thus likely contribute to the complex stability , such a strong decrease of psi is not expected.
the dependence of psi/psii ratio on the xanthophyll/carotenoid ratio of different genotypes contrasts with the fact that both psi core and psii core complexes bind β-carotene  <cit>  which is fully available in chy1chy2lut2lut <dig>  as well as in the other genotypes carrying mutations in xanthophyll biosynthesis .
alternatively, it can be hypothesized that psi level might be limited by the amount of lhci available.
however, three lines of evidence are against the hypothesis that psi depletion is a secondary effect of a limitation in lhci:
1) several arabidopsis mutants showing a strong depletion of lhci, including lhca antisense lines  <cit>  or the ch <dig> mutant  <cit>  still accumulate a functional psi core complex in moderate light  <cit> .
2) p700+ is not such a strong oxidizer as p680+, therefore photooxidative damages to psi require very strong irradiance  <cit> .
we have grown chy1chy2lut2lut <dig> and other genotypes  under moderate light, a condition that did not affect psi activity in ch <dig> mutants  <cit> .
thus it is unlikely that a fraction of assembled psi core complexes are destroyed due to lack of the lhci moiety.
3) the capacity of lhci to fold by binding both xanthophylls and β-carotene  <cit>  makes these subunits less limited in their possibility to fold into pigment-protein complexes than lhcb proteins.
we conclude that a tight correlation exists in plant thylakoids between psi accumulation and xanthophyll availability which is not due to either direct stabilization of the complex by xanthophylls or by photoxidative stress.
psi and psii core complex steady state level could be limited by chlorophyll availability, while a co-regulation of chlorophyll and carotenoid accumulation has been reported  <cit> .
nevertheless, the reduced amount of chls in carotenoid biosynthesis mutants appears to be mainly due to de-stabilization of the carotenoid/chlorophyll-binding proteins  <cit> .
analysis of chlorophyll biosynthesis mutants  <cit>  showed that psi accumulation is less reduced than psii accumulation, suggesting that the strong effect on psi we observed in chy1chy2lut2lut <dig> is not due to limitation in chls supply.
the effect of norflurazon treatment, which shows a preferential effect on psii activity  <cit> , further suggests that the phenotype we observe is specific for psi core and is specifically caused by xanthophyll depletion.
the lack of xanthophylls in psi core, however, suggests this specific effect must be indirect.
one possibility is that xanthophylls, or their metabolites, control either psi synthesis or degradation.
a number of factors are involved in the synthesis of psi and psii subunits, either bound to the thylakoid membrane or soluble in the chloroplast stroma, that could be considered as tentative targets of regulation, including srp and ftsy  <cit>  or atab <dig> protein  <cit> .
alternatively, carotenoid catabolites with regulatory roles  <cit>  could be responsible for this effect.
however, while the identification of the mechanisms underlying the down-regulation of psi synthesis under limiting xanthophylls is beyond the scope of this manuscript, it is interesting to consider the implications that such a regulation would have on the function of the photosynthetic apparatus:
a) the regulation of xanthophyll/carotene level in high-light would reflect into a modulation of psi level  <cit> , thus alleviating pq over-reduction and protecting from photoinhibition;
b) while psii reaction centers are subjected to rapid turn-over and their level readily adjusted to environmental conditions, psi is much more stable, thus requiring specific mechanisms for its down-regulation in limiting light.
lhcbs bind large amounts of xanthophylls and are strongly regulated depending on light intensity.
coupling psi to xanthophyll levels would provide a mechanism for coordinated regulation of psii antenna size and psi/psii ratio, a phenomenon observed in many species  <cit> .
CONCLUSIONS
one of the most noticeable results of recent work on the plant carotenoid biosynthesis pathway is the high level of redundancy in carotene hydroxylation, which is found to be catalyzed by  <dig> different enzymes.
here we show that the lut <dig> protein, previously reported to act in α-carotene hydroxylation, has a major β-carotene hydroxylation activity, which is evidenced in the α-carotene-less genetic background of the chy1chy2lut2lut <dig>  surprisingly, in this mutant lhci proteins are maintained with the same stoichiometry with respect to psi reaction center.
unexpectedly, in spite of its correct folding, psi reaction center is drastically reduced in chy1chy2lut2lut <dig> with respect to wild type, a condition that cannot be explained by a limitation in the availability of its lhci moiety.
upon analysis of genotypes having different xanthophyll/carotenoid ratios, we show that xanthophyll availability correlates with psi/psii ratio within a wide range.
the molecular mechanism underlying regulation of both psii antenna size and psi/psii ratio, alleviating pq over-reduction during acclimation to excess light conditions, are being investigated.

 carotenoids are a group of c pigments that contain a conjugated double-bond system, leading to strong absorption of visible light and antioxidant properties. they are widely distributed among taxa, ranging from cyanobacteria and fungi to red and green algae and land plants. xanthophylls are oxygenated carotenoids that play a crucial role in the photosynthetic apparatus of higher plants. their composition in plants is remarkably conserved and consists of five major xanthophylls, the most abundant being the β-ε-xanthophyll lutein, and the four β-β-xanthophylls violaxanthin, neoxanthin, antheraxanthin and zeaxanthin. xanthophylls act both as photoreceptors, absorbing light energy which is used in photosynthetic electron transport, and as photoprotectants of the photosynthetic apparatus from excess light and from the reactive oxygen species generated during photosynthesis. moreover, they are structural elements of the photosynthetic apparatus: lhcii, the major light-harvesting complex of photosystem ii, binds lutein, violaxanthin and neoxanthin at four distinct binding sites called respectively l l n and v; the occupancy of l site was shown to be essential for protein folding. xanthophyll biosynthesis in plants is divided in two distinct branches: the α branch leads to the formation of the ε-β-hydroxylated xanthophyll lutein from α-carotene, while the β branch leads to the production of β-β-hydroxylated xanthophylls from β-carotene. recent studies on carotenoid biosynthetic mutants of arabidopsis thaliana have improved our understanding on xanthophyll accumulation at the molecular level. the first steps in plant xanthophyll biosynthesis are the hydroxylation of α- and β-carotene. two different classes of enzymes are involved: the ferredoxin-dependent di-iron oxygenases which are active in β-ring hydroxylation, and the cytochromes p which are active in hydroxylation of both the ε-ring and β-ring of α-carotene, although the activity of lut on ε-rings is low. it has been suggested that a third chloroplast-targeted member of the cyp family, cyp97b might have a role in carotenoid biosynthesis. this hypothesis is however in contrast with the complete lack of xanthophylls in the quadruple chy1chy2lut1lut mutant, suggesting that chy chy lut1/cyp97c and lut5/cyp97a are the complete complement of carotene hydroxylases in a. thaliana. the strong phenotypes of mutants with altered xanthophyll composition imply that the presence and relative amounts of these pigments have a key role for plant fitness. the lut mutant, affected in ε-ring formation, lacks lutein and shows a complex phenotype with reduced antenna size, photodamage in high light due to impaired chlorophyll triplet quenching and decrease of non-photochemical quenching. additional features include over-accumulation of zeaxanthin in hl with respect to wild-type and monomerization of lhcii. lack of both lutein and zeaxanthin further decreases the capacity for photoprotection in both arabidopsis and chlamydomonas. the double chy1chy mutant, in which the two non-heme β-hydroxylases are deleted, shows reduced levels of β-β-xanthophylls and decreased resistance to photooxidation. introduction of the lut mutation in the chy1chy background leads to the almost complete disappearance of β-xanthophylls and strong photosensitivity. neoxanthin preserves psii from photoinactivation by superoxide anions while violaxanthin and zeaxanthin show enhanced activity in singlet oxygen scavenging. in order to further detail the effects of altered xanthophyll composition on the organization of photosynthetic complexes and gain understanding on the regulatory events controlling xanthophyll biosynthesis in arabidopsis, we have introduced the lut mutation in the semi-lethal chy1chy2lut background. surprisingly, the chy1chy2lut2lut mutant shows increased presence of β-β-xanthophylls with respect to chy1chy2lut the psi/psii ratio in this mutant is severely decreased as well as the level of total xanthophyll accumulation, suggesting that the latter have a key role, beside photoprotection, in regulating photosystem stoichiometry.  construction of the chy1chy2lut2lut quadruple mutant to generate the quadruple mutant chy1chy2lut2lut the homozygous triple mutant chy1chy2lut was crossed with the homozygous single mutant lut. all t-dna insertions were in the columbia background and appropriate oligonucleotides were used to confirm the presence of the insertions and their homozygous vs. heterozygous state. the mutant was maintained as a triple homozygous, single heterozygous stock: two different parental genotypes were used, heterozygous for either lut5/cyp97a or chy with similar results. the lut and chy1chy2lut lines were included in this characterization, representing respectively the lutein-less and β-xanthophyll-less controls. when selfed, the progeny of each single heterozygous stock, as well as wild-type, chy1chy2lut and lut seeds, were grown under low light conditions both in agar plates containing sucrose and in soil. no quadruple homozygous mutants were recovered in soil after week of growth, while the progeny segregated in a 1: ratio for white: green seedlings in agar plates. wild type and lut plants did not display a visible phenotype after weeks of growth in agar plates, whereas mutants chy1chy2lut and chy1chy2lut2lut showed, respectively, reduced growth and paler leaves. pigment composition we analyzed by hplc-dad-ms the pigment content of six-week-old leaves of wild type, lut chy1chy2lut and chy1chy2lut2lut plants grown on agar plates. pigments were resolved on a c- column, able to separate cis- from trans-carotenoids and their identity was confirmed by co-migration with authentic standards and high resolution ms. chy1chy2lut and chy1chy2lut2lut plants showed an increase in chlorophyll a/b ratio, as well as a reduced chlorophyll and carotenoids content per fresh weight, with respect to both wild-type and lut2; the effects were more severe in the quadruple mutant, that showed a significant reduction of chl/car ratio with respect to the other genotypes. wild-type leaves accumulate four major carotenoids and trace amounts of α-carotene. mutants show distinct composition of the xanthophyll fractions: lutein represents > 98% of total xanthophylls in chy1chy2lut plants; lut and chy1chy2lut2lut do not contain lutein and accumulate violaxanthin, antheraxanthin, zeaxanthin and neoxanthin; chy1chy2lut2lut shows a higher content of β-β-xanthophylls with respect to chy1chy2lut. β-carotene content is strongly increased in chy1chy2lut2lut with respect to the wild-type and the other mutants. as a result, the xanthophyll/carotene ratio changes dramatically, ranging from ± in wild-type to ± in chy1chy2lut2lut pigment composition was quantified via lc-dad-ms in dark-adapted leaves from 6-weeks-old plants. data are expressed as mean ± sd. fw, fresh weight. values marked with the same letters are not significantly different from each other within a column. plants grown at μmol photons m- s- for weeks were dark-adapted, then carotenoids were extracted and quantified via lc-dad-ms. data are expressed as mean ± sd. nd, not detectable; fw, fresh weight. values marked with the same letters are not significantly different from each other within a column. β-carotene accumulation is expected in chy1chy2lut2lut in which the biosynthetic flux is diverted towards the β-β-branch by the lack of three out of four β-carotene hydroxylases; however, β-β-xanthophyll accumulation in this mutant suggests that the fourth hydroxylase is more active toward β-carotene in this background than in the chy1chy2lut parent, resulting in 28-fold higher levels of β-β-xanthophylls. gene expression we measured the lut lut chy and chy mrna levels by real time pcr in the different mutants. all mrnas are almost completely absent in the corresponding mutants; lut chy and chy are induced in the lut mutant, that accumulates higher levels of β-carotene; lut is induced in the chy1chy2lut and, even more, in the chy1chy2lut2lut mutant, which shows drastically reduced xanthophyll/carotene ratios; however, the increase of lut levels between the two mutants is only -fold, while the increase of β-β-xanthophylls is 28-fold. photosynthesis-related functions: psii quantum efficiency and non-photochemical quenching of chlorophyll fluorescence the impact of xanthophyll depletion on photosynthesis was investigated by room temperature chlorophyll fluorescence measurements. the variable/maximum fluorescence yield of dark-adapted leaves reflects changes in psii photochemical efficiency. lut had the same fv/fm ratio as wild-type, while the triple chy1chy2lut and quadruple chy1chy2lut2lut mutants scored, respectively, values of and. the efficiency of psii photochemistry gives a measure of the rate of linear electron transport, an indication of the photosynthetic activity. chlorophyll fluorometry revealed a significant reduction in Φpsii in both chy1chy2lut and chy1chy2lut2lut, confirming that efficient light use is compromised by β-β-xanthophyll depletion. detached leaves of wild-type and mutant plants grown at μmol photons m- s- for weeks were given min of illumination, then photosynthetic parameters were provided by analysis of rt chlorophyll fluorescence: maximum quantum yield of psii and efficiency of psii photochemistry. data are expressed as mean ± sd. values marked with the same letters are not significantly different from each other within a column. non-photochemical quenching of chlorophyll fluorescence is the fastest photoprotective mechanism in the chloroplast: thermal dissipation is activated within few seconds upon exposure to excess light and it protects photosynthesis by decreasing the lifetime of singlet chlorophylls in order to minimize generation of ros in the psii. npq was measured on detached leaves, in saturating co. wild-type plants, upon short illumination at saturating light intensity, showed a rapid rise of npq, reaching a maximum value of. most of this npq relaxed rapidly in the dark, thus reflecting the Δph-dependent de-excitation of excess energy measured as qe, the rapidly-reversible component of npq. lut showed npq kinetics in agreement with published results, with lower amplitude and slower rise than wild-type plants. the other mutants showed a strong reduction in npq, scoring in chy1chy2lut and in chy1chy2lut2lut5; furthermore, upon correction for residual quenching after dark relaxation, both mutants showed very little recovery, suggesting that the measured fluorescence quenching was mainly due to photoinhibition, and the capacity for qe was strongly reduced in mutant leaves. furthermore, npq kinetics were measured during steady-state photosynthesis, upon a prolonged illumination with increasing light intensities. all genotypes showed chlorophyll fluorescence quenching, whose magnitude increased with irradiance. however, fluorescence quenching in wild-type and lut leaves was mainly due to the qe-type of npq, while in both chy1chy2lut and chy1chy2lut2lut the main component of npq was qi-type, irreversible quenching. these data confirm that both the reduction of β-β-xanthophylls and the lack of lutein are responsible for impaired npq kinetics. photosensitivity under short-term stress conditions when photosynthetic organisms are exposed to light in excess, photo-oxidative stress occurs within the chloroplast, with production of ros such as singlet oxygen, leading to oxidative damages to a large variety of biomolecules. the β-xanthophyll-depleted mutants chy1chy2lut and chy1chy2lut2lut even upon growth in low light, showed signs of photooxidation: lower chlorophyll content and psii quantum yield, retarded growth and paler leaves with respect to wild-type and lut plants. therefore, in order to assess whether lower xanthophyll levels affect the capacity to prevent chloroplast photooxidation, leaves from wild-type and mutant plants grown in low light were transferred to strong light and low temperature for h; the combination of low temperature and high light intensity is known to enhance the induction of both psii photoinhibition and membrane photooxidation in leaves, since the enzymes of the calvin cycle are slowed down and the light harvested by photosystems rapidly exceed the capacity of plants to use this energy. thus, the treatment produces a photooxidative stress, which can be measured as a decrease in the chlorophyll content and an increase in oxidation of membrane lipids. hl treatment was effective in producing higher pigment bleaching in chy1chy2lut and chy1chy2lut2lut, while wild-type and lut leaves were less affected, loosing around 25% of their chlorophyll content. to investigate the level of membrane lipid peroxidation, the same leaves were analyzed for mda content: chy1chy2lut2lut and chy1chy2lut leaves showed higher accumulation of mda upon stress treatment, thus a far higher level of lipid peroxidation with respect to wild-type and lut plants; chy1chy2lut2lut plants showed a far higher photosensitivity in high-light than chy1chy2lut; the latter was the xanthophyll mutant with the highest light sensitivity described so far. results clearly show an unprecedented level of photosensitivity in chy1chy2lut2lut plants, thus implying a severe impairment of the photoprotection mechanisms in this xanthophyll-depleted mutant. organization and stoichiometry of pigment binding complexes the extreme sensitivity to photo-oxidative stress of the chy1chy2lut2lut mutant could be due to altered pigment composition, to altered protein composition of photosystems, or to both. this mutant showed the highest chl a/b ratio, and the lowest chlorophyll content and xanthophyll/carotene ratio of all analyzed genotypes. since both chl b and xanthophylls are associated with lhc, their decrease suggests a decrease in antenna size. we investigated the organization of pigment-protein complexes in thylakoids by non-denaturing deriphat-page and by sucrose density gradient fractionation of solubilized thylakoids, followed by sds-page of the fractions. seven major green bands were resolved upon solubilization of wild-type thylakoid membranes with % dodecyl-α-d-maltoside on deriphat-page. the psi-lhci complex was the major band in the upper part of the gel, while the components of the psii-lhcii complex migrated as multiple bands, namely the psii core and the antenna moieties, including the cp29-cp24-lhcii-m supercomplex, lhcii trimer and monomeric lhcb. bands with high apparent masses were detected in the upper part of the gel containing non-dissociated psii supercomplexes. the chy1chy2lut pattern was very similar to that of wild-type, showing no major qualitative changes in the organization of the photosynthetic apparatus: the main differences consisted in a higher psii core/lhcb ratio and a lower content in psii supercomplexes. in the genotypes lacking lutein, namely lut and chy1chy2lut2lut the trimeric organization of lhcii was disrupted, as previously described. however, thylakoid membranes isolated from chy1chy2lut2lut plants showed additional features, namely the complete absence of bands and 7; bands and were much less represented than in wild-type, while psii core complex was the most abundant among pigment-protein complexes. in order to obtain sufficient amounts of pigment-protein complexes for further analysis, solubilized thylakoids from wild type and mutants were fractionated by sucrose gradient ultracentrifugation; the results confirmed that trimeric lhcii band, as well as the cp29-cp24-lhcii supercomplex completely disappeared in the chy1chy2lut2lut mutant; further differences consisted into reduced levels of monomeric lhcb and a much higher psii/psi ratio with respect to the other genotypes. the reduction of monomeric lhcbs in the chy1chy2lut2lut mutant occurs in spite of the presence of the lut mutation, which favors lhcii monomerization. fractions collected from the sucrose gradients of wild-type and chy1chy2lut2lut were further characterized by sds-page using two buffer systems and by absorption spectroscopy. according to previous results with α-dm, sds-page analyses of wild-type fractions showed that band contained the minor antennae cp cp and cp as well as components of monomerized lhcii, while band contained lhcb1- polypeptides only. band was enriched in psii core complex, nevertheless the chl b absorption suggests it retains lhcb proteins; band contained almost exclusively the psi-lhci complex. band from chy1chy2lut2lut contained the same polypeptides as the corresponding band from wild-type, although the relative amounts of the lhcb1- polypeptides and cp were decreased. the data confirm that lhcii is present in the mutant, however in far lower amounts than in wild-type, and is in its monomeric aggregation state. band from the mutant contained almost exclusively psii core complex polypeptides, band from the mutant contained psi-lhci. the levels of selected proteins in wild-type and chy1chy2lut2lut thylakoids were determined by quantitative western-blot analysis using psbb as internal control: all lhcb subunits were reduced in chy1chy2lut2lut with respect to wild-type thylakoids; psba, oec- and psbs, subunits of the psii core complex, were present in the same level in both genotypes, while cytochrome f and atpase β-subunit were in higher amounts in chy1chy2lut2lut immunoblotting using psaa as internal standard showed that each lhca protein was present in wild-type amounts, thus suggesting that the lhca/psi ratio is conserved in the mutant. in contrast, the psi/psii ratio was extremely low in chy1chy2lut2lut reaching approximately 22% of wild-type value. the data shown above indicate that xanthophyll depletion in the chy1chy2lut2lut plants causes a strong reduction in the amount of lhcb proteins per psii reaction center, and has a negative impact on the total amount of the psi-lhci supercomplex. the latter result is unexpected, since xanthophylls are mainly bound to lhc complexes while core complexes of both photosystems only bind carotenes, implying that xanthophyll abundance should not affect their folding or stability. a key question is whether the lower psi/psii ratio found in chy1chy2lut2lut thylakoids is peculiar to this genotype or is a general consequence of altered xanthophyll content. to answer this question, the abundance of lhcii and psi core with respect to cp was assessed by quantitative immunotitration, in thylakoids isolated from arabidopsis mutants with altered xanthophyll content. figure shows the distribution of lhcii and psi core amounts, relative to cp in the different mutants with different xanthophyll/carotenoid ratios: both distributions show an increase of lhcii/psii and psi/psii at increasing xanthophyll/carotenoid ratios, and the data can be fitted with an exponential function. the results display a clear correlation between parameters. furthermore, quantitative data for the individual pigment-protein complexes accumulation level per fresh weight were plotted vs. xanthophyll/carotenoid content in the different mutants. the distributions show a lower lhcii content per fresh weight at decreasing xanthophyll/carotenoid ratios, while psii core content per fresh weight was essentially unaffected by xanthophylls depletion. unlike psii, the psi core content decreases at decreasing xanthophyll/carotenoid ratios. these results suggest that xanthophyll depletion not only causes a marked depletion in lhc proteins, as expected from the xanthophylls being ligands of lhcs, but also modulates the ratio between psi/psii, despite β-carotene rather than xanthophylls is the ligand for psi and psii core complexes. discussion in this work, we analyzed the modifications of the photosynthetic apparatus in the arabidopsis mutant chy1chy2lut2lut that accumulates β-xanthophylls despite disruption of the three chy chy and lut genes encoding carotene hydroxylases. in this mutant the only carotene hydroxylase activity is provided by the lut gene product, which allows for accumulation of only 20% of the wild-type xanthophylls. in these conditions, biogenesis of the photosynthetic apparatus was strongly affected yielding into a reduction of psii antenna size, a decrease of psi/psii ratio and an increased photosensitivity. lut as a β-carotene hydroxylase lut was originally reported to be only gene product required for the ε-ring hydroxylation of α-carotene. the first indication of a possible involvement of lut in β-ring hydroxylation came from the analysis of the chy1chy2lut triple mutant, which accumulates lutein, providing genetic evidence that lut must be also active in the hydroxylation of the β-ring of α-carotene. introduction of the lut mutation in the chy1chy2lut triple mutant completely abolished xanthophyll biosynthesis, indicating that chy chy lut and lut constitute the full complement of carotenoid hydroxylases in arabidopsis. a number of studies have shown a functional redundancy amongst the carotene hydroxylase enzymes. however, the extent of recovery in xanthophyll content by mutants carrying a single carotene hydroxylase activity gave insights on the substrate preference of residual activity. as an example, trace amounts of β-β-xanthophylls, present in the chy1chy2lut triple mutant and abolished by the introduction of the lut mutation, led to the conclusion that lut has a low level of activity toward the β-rings of β-carotene. overall, in vivo analysis clearly showed that chy and chy are most active in β-carotene hydroxylation, while lut and lut enzymes catalyze preferentially the biosynthesis of α-xanthophylls. here we show that, in the absence of α-carotene, lut shows a major β-carotene hydroxylase activity, leading to the accumulation of substantial amounts of β-β-xanthophylls. indeed, when considering the moles of hydroxylated β-rings accumulated in chy1chy2lut and chy1chy2lut2lut mutants, total amount is maintained upon introduction of the lut mutation into the chy1chy1lut background: ~ μmol of hydroxylated β-rings/gfw in chy1chy2lut vs. ~ μmol/gfw in chy1chy2lut2lut. however, the total amount of xanthophylls per fresh weight is reduced significantly in chy1chy2lut2lut, while the carotene/xanthophyll ratio is increased. the most likely scenario is that lut has a higher affinity towards ε-rings vs. β-rings and that, when α-carotene is available, lut performs mainly ε-ring hydroxylation; however, since no ε-ring-substrates are available in the chy1chy2lut2lut the β-ring substrates are processed, thus bringing out this minor activity of the enzyme. conversely, in the absence of the main hydroxylases for β-rings chy chy and lut all β-rings become available for lut activity in chy1chy2lut2lut a consequence of lut operation on a less preferred substrate is its reduced overall catalytic rate that limits the level of xanthophylls accumulated in the chy1chy2lut2lut genotype. it is worth noting that the β-hydroxylase activity does not discriminate between β-rings of α- and β-carotene; indeed, the total amount of hydroxylated β-rings per fresh weight is the same in both chy1chy2lut and chy1chy2lut2lut this evidence updates our knowledge on the molecular details of carotenoid hydroxylases, with respect to the view that lut has only a low level of in vivo hydroxylase activity toward the β-carotene and a stronger activity towards α-carotene. the concentration of β-carotene increased in the quadruple chy1chy2lut2lut mutant to a level higher than in the wild type. this is the result of the combination of several factors: first, the lut mutation redirects the flux in the pathway towards the β-β-branch; this mutation alone is sufficient to cause a slight increase in β-carotene. moreover, the reduced hydroxylase activity due to the chy chy and lut mutations, reduces the rate of β-carotene processing into downstream xanthophylls, favoring its accumulation. the almost 2-fold increase of the lut transcript in the quadruple mutant with respect to the wild type is insufficient to fully compensate for the disappearance of the chy chy and lut hydroxylases, likely due to the low catalytic efficiency of lut for β-rings. reduced xanthophyll content negatively affects energy-dependent quenching qe and photoprotection the excess energy dissipation into heat is strongly depleted in the chy1chy2lut2lut mutant, its amplitude being close to zero. since the level of psbs, the ph sensitive trigger for npq is similar to wild-type, this effect can be attributed to the low level of the interacting partners of psbs where the actual quenching occurs, i.e. the lhcb proteins, and to the lack of lutein which limit npq. all together these results support the correlation between xanthophyll content and amplitude of qe, previously indicated on the basis of antisense inhibition of beta hydroxylation. however, the high photosensitivity of chy1chy2lut2lut plants is likely to be caused by the failure of additional photoprotection mechanisms, since the npq mutant, although depleted in npq, only showed minor increase in photosensitivity. the fast leaf chlorophyll bleaching and high levels of lipid peroxidation in chy1chy2lut2lut with respect to wild-type, lut and chy1chy2lut are likely due to the strong depletion in lhcb proteins. xanthophylls are needed for folding of lhc proteins in vitro, thus it is not surprising that a strong decrease in their availability leads to a decreased content in lhc. however, only lhcb proteins are affected, while lhca proteins are maintained with the same stoichiometry with respect to psi reaction center, as shown by the identical migration rate of psi-lhci supercomplexes in sucrose gradients and native gels and by quantitative immunotitration of lhca versus psaa content. as a result of the higher stability of lhca vs. lhcb proteins, psi antenna function is maintained, while psii antenna function is impaired. we conclude that in vivo carotenes cannot replace xanthophylls in stabilizing lhcb proteins, thus leading to their strong reduction. furthermore, this implies that functional lhcb proteins are essential for photoprotection, in agreement with a previous report. extreme reduction in lhc proteins is obtained with the ch mutation in arabidopsis, that impairs chl b synthesis and prevents assembly of functional lhc. although this mutant undergoes severe photoinhibition in high light, it can grow on soil and is not photoinhibited in moderate light. thus ch with a psii antenna size even smaller than that of chy1chy2lut2lut can survive in the absence of a reduced carbon source, a lethal condition for chy1chy2lut2lut we conclude that reduced lhc content, although likely contributing to sensitivity, cannot be the only reason for the extreme phenotype of chy1chy2lut2lut limitation in total xanthophyll availability affects light-harvesting complex content and psi/psii ratio the analysis of the pigment-protein complexes in the chy1chy2lut2lut mutant shows that lhcb proteins are strongly decreased with respect to psii, while lhca assembly into psi-lhci are much less, or not at all, affected. a reduction in lhcb proteins is also observed in the chy1chy2lut mutant, albeit to a lesser extent than in chy1chy2lut2lut. this effect is likely due to the incapacity of lhcb proteins to fold in the absence of xanthophylls, while lhca proteins can also bind small amounts of β-carotene. instead, there is no evident reason for the 5-fold decrease in psi/psii ratio: since lhca proteins are maintained with the same stoichiometry with respect to psi reaction center and thus likely contribute to the complex stability, such a strong decrease of psi is not expected. the dependence of psi/psii ratio on the xanthophyll/carotenoid ratio of different genotypes contrasts with the fact that both psi core and psii core complexes bind β-carotene which is fully available in chy1chy2lut2lut as well as in the other genotypes carrying mutations in xanthophyll biosynthesis. alternatively, it can be hypothesized that psi level might be limited by the amount of lhci available. however, three lines of evidence are against the hypothesis that psi depletion is a secondary effect of a limitation in lhci: 1) several arabidopsis mutants showing a strong depletion of lhci, including lhca antisense lines or the ch mutant still accumulate a functional psi core complex in moderate light. 2) p700+ is not such a strong oxidizer as p680+, therefore photooxidative damages to psi require very strong irradiance. we have grown chy1chy2lut2lut and other genotypes under moderate light, a condition that did not affect psi activity in ch mutants. thus it is unlikely that a fraction of assembled psi core complexes are destroyed due to lack of the lhci moiety. 3) the capacity of lhci to fold by binding both xanthophylls and β-carotene makes these subunits less limited in their possibility to fold into pigment-protein complexes than lhcb proteins. we conclude that a tight correlation exists in plant thylakoids between psi accumulation and xanthophyll availability which is not due to either direct stabilization of the complex by xanthophylls or by photoxidative stress. psi and psii core complex steady state level could be limited by chlorophyll availability, while a co-regulation of chlorophyll and carotenoid accumulation has been reported. nevertheless, the reduced amount of chls in carotenoid biosynthesis mutants appears to be mainly due to de-stabilization of the carotenoid/chlorophyll-binding proteins. analysis of chlorophyll biosynthesis mutants showed that psi accumulation is less reduced than psii accumulation, suggesting that the strong effect on psi we observed in chy1chy2lut2lut is not due to limitation in chls supply. the effect of norflurazon treatment, which shows a preferential effect on psii activity, further suggests that the phenotype we observe is specific for psi core and is specifically caused by xanthophyll depletion. the lack of xanthophylls in psi core, however, suggests this specific effect must be indirect. one possibility is that xanthophylls, or their metabolites, control either psi synthesis or degradation. a number of factors are involved in the synthesis of psi and psii subunits, either bound to the thylakoid membrane or soluble in the chloroplast stroma, that could be considered as tentative targets of regulation, including srp and ftsy or atab protein. alternatively, carotenoid catabolites with regulatory roles could be responsible for this effect. however, while the identification of the mechanisms underlying the down-regulation of psi synthesis under limiting xanthophylls is beyond the scope of this manuscript, it is interesting to consider the implications that such a regulation would have on the function of the photosynthetic apparatus: a) the regulation of xanthophyll/carotene level in high-light would reflect into a modulation of psi level, thus alleviating pq over-reduction and protecting from photoinhibition; b) while psii reaction centers are subjected to rapid turn-over and their level readily adjusted to environmental conditions, psi is much more stable, thus requiring specific mechanisms for its down-regulation in limiting light. lhcbs bind large amounts of xanthophylls and are strongly regulated depending on light intensity. coupling psi to xanthophyll levels would provide a mechanism for coordinated regulation of psii antenna size and psi/psii ratio, a phenomenon observed in many species.  one of the most noticeable results of recent work on the plant carotenoid biosynthesis pathway is the high level of redundancy in carotene hydroxylation, which is found to be catalyzed by different enzymes. here we show that the lut protein, previously reported to act in α-carotene hydroxylation, has a major β-carotene hydroxylation activity, which is evidenced in the α-carotene-less genetic background of the chy1chy2lut2lut surprisingly, in this mutant lhci proteins are maintained with the same stoichiometry with respect to psi reaction center. unexpectedly, in spite of its correct folding, psi reaction center is drastically reduced in chy1chy2lut2lut with respect to wild type, a condition that cannot be explained by a limitation in the availability of its lhci moiety. upon analysis of genotypes having different xanthophyll/carotenoid ratios, we show that xanthophyll availability correlates with psi/psii ratio within a wide range. the molecular mechanism underlying regulation of both psii antenna size and psi/psii ratio, alleviating pq over-reduction during acclimation to excess light conditions, are being investigated. 
xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. moreover, by analyzing a series of single, double, triple and quadruple arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the psi-psii ratio. mutant plants show an even stronger photosensitivity than chy1chy2lut a complete lack of qe, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. the first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases and p cytochromes. biochemical analysis reveals that the chy1chy2lut2lut mutant is depleted in lhcb subunits and is specifically affected in photosystem i function, showing a deficiency in psi-lhci supercomplexes. the physiological and biochemical phenotype of the chy1chy2lut2lut mutant shows that lut1/cyp97c protein reveals a major β-carotene hydroxylase activity in vivo when depleted in its preferred substrate α-carotene; xanthophylls are needed for normal level of photosystem i and lhcii accumulation.
 xanthophylls are oxygenated carotenoids playing an essential role as structural components of the photosynthetic apparatus. xanthophylls contribute to the assembly and stability of light-harvesting complex, to light absorbance and to photoprotection. the first step in xanthophyll biosynthesis from α- and β-carotene is the hydroxylation of ε- and β-rings, performed by both non-heme iron oxygenases and p cytochromes. the arabidopsis triple chy1chy2lut mutant is almost completely depleted in β-xanthophylls.  here we report on the quadruple chy1chy2lut2lut mutant, additionally carrying the lut mutation. this genotype lacks lutein and yet it shows a compensatory increase in β-xanthophylls with respect to chy1chy2lut mutant. mutant plants show an even stronger photosensitivity than chy1chy2lut a complete lack of qe, the rapidly reversible component of non-photochemical quenching, and a peculiar organization of the pigment binding complexes into thylakoids. biochemical analysis reveals that the chy1chy2lut2lut mutant is depleted in lhcb subunits and is specifically affected in photosystem i function, showing a deficiency in psi-lhci supercomplexes. moreover, by analyzing a series of single, double, triple and quadruple arabidopsis mutants in xanthophyll biosynthesis, we show a hitherto undescribed correlation between xanthophyll levels and the psi-psii ratio. the decrease in the xanthophyll/carotenoid ratio causes a proportional decrease in the lhcii and psi core levels with respect to psii.  the physiological and biochemical phenotype of the chy1chy2lut2lut mutant shows that lut1/cyp97c protein reveals a major β-carotene hydroxylase activity in vivo when depleted in its preferred substrate α-carotene; xanthophylls are needed for normal level of photosystem i and lhcii accumulation. 