Lutein accumulation in the absence of zeaxanthin restores nonphotochemical quenching in the Arabidopsis thaliana npq1 mutant.
ABSTRACT Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high transthylakoid DeltapH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they accumulate more lutein and alpha-carotene than the wild type. szl1 contains a point mutation in the lycopene beta-cyclase (LCYB) gene. Based on the pigment analysis, LCYB appears to be the major lycopene beta-cyclase and is not involved in neoxanthin synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type, whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.
- SourceAvailable from: Robert Jennings[Show abstract] [Hide abstract]
ABSTRACT: Oxygenic photosynthesis is indispensable both for the development and maintenance of life on earth by converting light energy into chemical energy and by producing molecular oxygen and consuming carbon dioxide. This latter process has been responsible for reducing the CO2 from its very high levels in the primitive atmosphere to the present low levels and thus reducing global temperatures to levels conducive to the development of life. Photosystem I and photosystem II are the two multi-protein complexes that contain the pigments necessary to harvest photons and use light energy to catalyse the primary photosynthetic endergonic reactions producing high energy compounds. Both photosystems are highly organised membrane supercomplexes composed of a core complex, containing the reaction centre where electron transport is initiated, and of a peripheral antenna system, which is important for light harvesting and photosynthetic activity regulation. If on the one hand both the chemical reactions catalysed by the two photosystems and their detailed structure are different, on the other hand they share many similarities. In this review we discuss and compare various aspects of the organisation, functioning and regulation of plant photosystems by comparing them for similarities and differences as obtained by structural, biochemical and spectroscopic investigations.Current Protein and Peptide Science 03/2014; · 2.33 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Under high-irradiance conditions, plants must efficiently protect photosystem II (PSII) from damage. In this study, we demonstrate that the chloroplast protein HYPERSENSITIVE TO HIGH LIGHT1 (HHL1) is expressed in response to high light and functions in protecting PSII against photodamage. Arabidopsis thaliana hhl1 mutants show hypersensitivity to high light, drastically decreased PSII photosynthetic activity, higher nonphotochemical quenching activity, a faster xanthophyll cycle, and increased accumulation of reactive oxygen species following high-light exposure. Moreover, HHL1 deficiency accelerated the degradation of PSII core subunits under high light, decreasing the accumulation of PSII core subunits and PSII-light-harvesting complex II supercomplex. HHL1 primarily localizes in the stroma-exposed thylakoid membranes and associates with the PSII core monomer complex through direct interaction with PSII core proteins CP43 and CP47. Interestingly, HHL1 also directly interacts, in vivo and in vitro, with LOW QUANTUM YIELD OF PHOTOSYSTEM II1 (LQY1), which functions in the repair and reassembly of PSII. Furthermore, the hhl1 lqy1 double mutants show increased photosensitivity compared with single mutants. Taken together, these results suggest that HHL1 forms a complex with LQY1 and participates in photodamage repair of PSII under high light.The Plant Cell 03/2014; · 9.25 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: This is a tale of a career in plant physiological ecology that enjoyed the freedom to address photosynthetic physiology and biochemistry in leaves of plants from diverse environments. It was supported by block funding (now sadly a thing of the past) for research at the Australian National University, by grants during appointments in the United States and in Germany, and by Columbia University. It became a "career experiment" in which long-term, high-trust support for curiosity-driven plant biology in Australia, and at times in the United States, led to surprisingly innovative results. Although the rich diversity of short-term competitive grant opportunities in the United States sustained ongoing research, it proved difficult to mobilize support for more risky long-term projects. A decade after the closure of the Biosphere 2 Laboratory, this article highlights the achievements of colleagues in experimental climate change research from 1998 to 2003.Annual Review of Plant Biology 04/2014; 65:1-32. · 18.71 Impact Factor
Lutein Accumulation in the Absence of Zeaxanthin
Restores Nonphotochemical Quenching in the
Arabidopsis thaliana npq1 Mutant
Zhirong Li,a,bTae Kyu Ahn,b,cThomas J. Avenson,a,c,1Matteo Ballottari,dJeffrey A. Cruz,eDavid M. Kramer,e
Roberto Bassi,dGraham R. Fleming,b,cJay D. Keasling,b,fand Krishna K. Niyogia,b,2
aDepartment of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102
bPhysical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720
cDepartment of Chemistry, University of California, Berkeley, California 94720-1460
dDipartimento Scientifico e Tecnologico, Universita ` di Verona, I-37134 Verona, Italy
eInstitute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340
fDepartment of Chemical Engineering, University of California, Berkeley, California 94720
Plants protect themselves from excess absorbed light energy through thermal dissipation, which is measured as
nonphotochemical quenching of chlorophyll fluorescence (NPQ). The major component of NPQ, qE, is induced by high
transthylakoid DpH in excess light and depends on the xanthophyll cycle, in which violaxanthin and antheraxanthin are
deepoxidized to form zeaxanthin. To investigate the xanthophyll dependence of qE, we identified suppressor of zeaxanthin-
less1 (szl1) as a suppressor of the Arabidopsis thaliana npq1 mutant, which lacks zeaxanthin. szl1 npq1 plants have a
partially restored qE but lack zeaxanthin and have low levels of violaxanthin, antheraxanthin, and neoxanthin. However, they
accumulate more lutein and a-carotene than the wild type. szl1 contains a point mutation in the lycopene b-cyclase (LCYB)
gene. Based on the pigment analysis, LCYB appears to be the major lycopene b-cyclase and is not involved in neoxanthin
synthesis. The Lhcb4 (CP29) and Lhcb5 (CP26) protein levels are reduced by 50% in szl1 npq1 relative to the wild type,
whereas other Lhcb proteins are present at wild-type levels. Analysis of carotenoid radical cation formation and leaf
absorbance changes strongly suggest that the higher amount of lutein substitutes for zeaxanthin in qE, implying a direct
role in qE, as well as a mechanism that is weakly sensitive to carotenoid structural properties.
Light is required for photosynthesis in plants, but the quantity of
light in natural environments is highly variable. Within a certain
range of relatively low incident light intensities, photosynthetic
carbon fixation increases linearly with increases in photon flux
density. However, above a certain threshold, photosynthetic
capacity is saturated, and a plant absorbs more light than it can
actually use. Absorption of excess light can lead to overexcita-
tion of chlorophyll and overreduction of the electron transport
chain, which result in increased generation of reactive interme-
diates and harmful byproducts of photosynthesis (Niyogi, 1999).
For example, overexcitation of chlorophyll would result in an
increase in the lifetime of singlet-excited chlorophyll (1Chl*),
which consequently increases the production of triplet-excited
Chl (3Chl*) via intersystem crossing.3Chl* interacts with molec-
ular oxygen to generate singlet O2(1O2*), which can damage
proteins, pigments, and lipids in the photosynthetic apparatus
(Niyogi, 1999; Asada, 2006).
Photosynthetic organisms have evolved a suite of short-term
and long-term photoprotective mechanisms to cope with the
absorption of excessive light and its consequences. Among
these mechanisms, the thermal dissipation of excess absorbed
light energy in photosystem II (PSII), which is commonly mea-
sured and referred to as nonphotochemical quenching (NPQ), is
believed to play a key role in regulating light harvesting and
preventing photooxidative damage to the photosynthetic appa-
ratus. NPQ can be induced or disengaged in response to
changes in light intensity on a time scale of seconds to minutes.
Although there are several components of NPQ in higher plants,
pH-dependent energy dissipation (also called qE) accounts for
the major part of NPQ and results in deexcitation of1Chl*and the
thermal dissipation of excess absorbed light energy in the light-
harvesting antenna of PSII (Mu ¨ller et al., 2001). Because it
involves the deexcitation of1Chl*, qE can be easily measured
as a decrease in the maximum yield of chlorophyll fluorescence
in intact leaves or isolated chloroplast membranes (Mu ¨ller et al.,
qE is induced by a low thylakoid lumen pH (i.e., a high DpH)
during illumination with excess light (Demmig-Adams and
Adams, 1992; Horton et al., 1996; Mu ¨ller et al., 2001). The low
1Current address: Monsanto, 62 Maritime Drive, Mystic, CT 06355.
2Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Krishna K. Niyogi
WOnline version contains Web-only data.
OAOpen access articles can be viewed online without a subscription.
The Plant Cell, Vol. 21: 1798–1812, June 2009, www.plantcell.org ã 2009 American Society of Plant Biologists
thylakoid lumen pH plays dual roles, one of which is to activate
the violaxanthin deepoxidase (VDE) enzyme, which converts
violaxanthin into antheraxanthin and then zeaxanthin as part of a
xanthophyll cycle (Figure 1) (Yamamoto et al., 1999; Jahns et al.,
2009). The other role of the low thylakoid lumen pH is to
protonate one or more PSII proteins that are involved in qE
(Horton and Ruban, 1992). A light-induced absorbance change
at 535 nm (DA535) is linearly correlated with qE (Ruban et al.,
1993; Bilger and Bjo ¨rkman, 1994; Li et al., 2004). DA535depends
on both zeaxanthin and protonation and is thought to be due to a
change in the absorption spectrum of zeaxanthin (Ruban et al.,
Analysis of Arabidopsis thaliana mutants that lack qE has
been a very useful approach to define factors that are necessary
for qE, including xanthophylls, the PsbS protein, and light-
harvesting complex (LHC) proteins (Niyogi, 2000). qE-deficient
mutants were identified in forward genetics screens by video
imaging of chlorophyll fluorescence yield during exposure of
mutagenized Arabidopsis seedlings to excess light (Niyogi et al.,
1998; Li et al., 2000). The nonphotochemical quenching1 (npq1)
and lutein-deficient2 (lut2) mutants exhibit diminished levels of qE
is completely defective in qE and DA535(Li et al., 2000; Peterson
and Havir, 2000). The npq1 mutant is defective in VDE and
therefore lacks zeaxanthin. Characterization of the npq1 mutant
showed that zeaxanthin is necessary for most of the qE in vivo in
Arabidopsis leaves (Niyogi et al., 1998). The lut2 mutant affects
lycopene «-cyclase (LCYE) activity (Pogson et al., 1998), so it is
unable to synthesize either lutein or a-carotene (Figure 1). The
npq1 lut2 double mutant is totally devoid of any qE, suggesting a
possible role for lutein in qE (Niyogi et al., 2001). Complementary
evidence was reported with LCYE-overexpressing transgenic
qE (Pogson and Rissler, 2000). It has been proposed that lutein
might have a direct role in qE or, alternatively, that the change
of lutein content could indirectly affect qE by disturbing the
assembly and structure of the PSII antenna (Niyogi et al., 1997,
2001; Pogson et al., 1998; Lokstein et al., 2002).
Figure 1. Carotenoid Biosynthetic Pathway in Plants.
The block in xanthophyll metabolism in the npq1 mutant, which lacks zeaxanthin due to a defect in the violaxanthin deepoxidase gene, is indicated by
the symbol “npq1.”
Lutein Can Replace Zeaxanthin in qE 1799
Despite these advances in identifying components of qE, the
actual biophysical mechanismof1Chldeexcitation inqEremains
controversial. Two mechanisms, which are not mutually exclu-
sive, have been proposed recently based on ultrafast transient
absorption (TA) spectroscopy experiments (Holt et al., 2005;
Ruban et al., 2007). Both mechanisms include a role for PsbS as
a sensor of lumen pH that triggers conformational changes in the
PSII antenna that result in efficient deexcitation of1Chl* (Ahn
et al., 2008; Avenson et al., 2008; Horton et al., 2008). In one
model, zeaxanthin has a direct role in quenching1Chl* through a
charge-transfer (CT) mechanism (Holt et al., 2005) in the minor
LHCs associated with PSII, CP29 (Lhcb4), CP26 (Lhcb5), and
CP24 (Lhcb6) (Ahn et al., 2008; Avenson et al., 2008, 2009). The
CT quenching mechanism involves energy transfer from chloro-
phyll to closely coupled chlorophyll and zeaxanthin molecules,
followed by charge separation that transiently produces a zea-
xanthin radical cation and chlorophyll radical anion. Subsequent
charge recombination dissipates the excitation energy as heat
(Holt et al., 2005). The second model proposes that qE occurs in
2005), and its molecular mechanism involves energy transfer
(Ruban et al., 2007). According to this model, zeaxanthin is not
required for qE but functions indirectly as an allosteric regulator
(Crouchman et al., 2006) by increasing the pH sensitivity of qE
(Noctor et al., 1991).
To investigate the xanthophyll dependence of qE and, possibly
a screen for suppressors of the npq1 mutation that exhibit higher
qE despite the absence of zeaxanthin. We isolated a suppressor
that accumulates more lutein but has a very small xanthophyll
cycle pool size. Molecular,physiological, and biophysicalanalysis
of this mutant suggest that the higher amount of lutein can
substitute for zeaxanthin to act directly as a quencher in qE.
Isolation of Suppressors of npq1
We used a chlorophyll fluorescence video imaging system
(Niyogi et al., 1997) to isolate suppressors of the npq1 mutant.
Ten potential suppressors that showed much higher NPQ level
compared with that of the npq1 parent were isolated out of
15,000 M2seedlings (Figure 2),and six suppressors were shown
to lack zeaxanthin during the secondary screen by pigment
analysis. One of these six suppressors with partially restored
NPQ had very low levels of violaxanthin and neoxanthin but
backcrossed to the npq1 parent three times and then crossed to
the wild type to isolate an szl1 single mutant, which was able to
synthesize zeaxanthin upon exposure to high light (Figure 3B).
Pigment Content and NPQ in the Suppressor
The wild-type, npq1, szl1, and szl1 npq1 plants were grown
under low light (LL) conditions (150 mmol photons m22s21). In
these growth conditions, the suppressor szl1 npq1 and single
mutant szl1 exhibited a very similar whole-plant phenotype to
that of the wild-type and npq1 plants, with the major difference
being slightly smaller sizes (Figure 4A).The pigment composition
and content of the four genotypes were measured before and
after a short treatment of LL-grown plants with high light (HL;
1000 mmol photons m22s21). All four genotypes had the same
total chlorophyll content, chlorophyll a/b ratio, and total carot-
enoids under both conditions, although szl1 and szl1 npq1 had
lower total carotenes and correspondingly higher total xantho-
phylls (Table 1). Figure 4B compares the relative content of
and a-carotene–derived lutein in wild-type and mutant plants
after HL treatment. Xanthophyll cycle pigment pool size (the sum
of violaxanthin, antheraxanthin, and zeaxanthin) was the same in
szl1 npq1. Because the npq1 mutation affects the VDE gene, no
zeaxanthin was detected in either npq1 or szl1 npq1. The
concentration of lutein in the szl1 and szl1 npq1 mutants was
nearly two times higher than in the wild type and npq1 mutant.
The smaller xanthophyll cycle pool size and the greater lutein
concentration in the suppressor szl1 npq1 and the szl1 single
Figure 2. Screening for Suppressors of npq1 by Video Imaging of
Chlorophyll Fluorescence Quenching.
Mutagenized npq1 plants (M2 generation) on agar medium were ex-
posed to 800 mmol photons m?2s?1for 1 min. In this false-color image of
NPQ, the wild type appears red and an npq1 mutant appears blue,
whereas the szl1 npq1 suppressor appears greenish red.
1800The Plant Cell
mutant (relative to those in the wild type) indicated that the
b-carotene branch of the carotenoid biosynthetic pathway is
affected, and metabolic flux is redirected into the a-carotene
branch from which lutein is synthesized.
The NPQ induction curves of the four genotypes plus the
heterozygous szl1/SZL1 npq1/npq1 mutant were compared
(Figure 5). When illuminated with 1200 mmol photons m22s21,
the wild type showed a rapid establishment of NPQ to a value of
2.2 within 5 min, whereas the npq1 mutant showed a slower rate
of NPQ induction and an NPQ value of <1.2. In the szl1 npq1
plants, the NPQ induction had an even more rapid rise in the first
30 scompared with the wild type, which was probably due to the
which was substantially higher compared with the npq1 mutant.
By subtracting the residual NPQ in each genotype after relaxa-
tion in the dark, we estimated the qE component after 5 min of
light induction to be 1.7, 0.8, and 1.4 in the wild type, npq1, and
szl1 npq1, respectively. The szl1 single mutant showed the same
NPQ induction as that of the suppressor szl1 npq1. The hetero-
zygous szl1/SZL1 npq1/npq1 mutant had NPQ levels identical
to the npq1 mutant, indicating that the szl1 mutation is recessive
for the NPQ phenotype.
The SZL1 Gene Encodes LCYB
The pigment phenotype of the suppressor szl1 npq1 suggested
that the activity of a carotenoid biosynthetic enzyme is impaired.
Since szl1 npq1 had a very small xanthophyll cycle pool size but
twice as much lutein, the most likely candidate for the enzyme
affected by szl1 mutation was LCYB, which functions at the
branch point in the carotenoid biosynthetic pathway (Figure 1).
In Arabidopsis, LCYB is encoded by a single-copy gene
(Cunningham etal., 1996).The LCYB genewas amplified directly
from the genomic DNAs of the wild type, npq1, and szl1 npq1.
Sequence analysis revealed that LCYB from szl1 npq1 carries a
point mutation from Gto A atposition 1352, which translates into
Figure 3. HPLC Analysis of Pigments in the Wild Type, npq1, szl1, and
(A) Comparison of pigment profile of the wild type, npq1, and szl1 npq1
after exposure to high light (1000 mmol photons m?2s?1) for 30 min.
(B) Overlay of HPLC traces of a szl1 single mutant before (LL) and after
(HL) treatment with high light. Neo, neoxanthin; Vio, violaxanthin; An,
antheraxanthin; Zea, zeaxanthin; Chl a, chlorophyll a; Chl b, chlorophyll
b; a-car, a-carotene; b-car, b-carotene.
Figure 4. Characteristics of Wild-Type, npq1, szl1, and szl1 npq1 Plants.
(A) Growth of the four Arabidopsis strains. Plants were grown in LL (150
mmol photons m?2s?1) with a short-day photoperiod (10 h light and 14 h
dark). Plants were photographed at an age of 4 weeks.
(B) Xanthophyll cycle pigment pool size (V+A+Z) and lutein levels of the
four Arabidopsis strains. Plants were grown as described in (A). At the
end of the dark period, whole plants were exposed to HL (2000 mmol
photons m?2s?1) for 30 min, and leaf samples were taken and analyzed
by HPLC. Data were normalized to chlorophyll a and shown as the
means 6 SD (n = 9).
Lutein Can Replace Zeaxanthin in qE1801
a change at conserved residue 451 from Gly to Glu (Figure 6A).
This point mutation creates an EcoRI restriction site, which can
be used as a polymorphism marker for the szl1 allele.
To determine whether the szl1 mutation in the LCYB gene is
genetically linked with the NPQ suppressor phenotype, the szl1
npq1 double mutant was backcrossed to the npq1 mutant, and
the NPQ phenotypes were measured in the resulting F2 gener-
ation. When scoring F2 plants for the polymorphism marker and
NPQ suppressor phenotype, the szl1 allele cosegregated with
the NPQ suppressor phenotype in all of the progeny tested
(Figure 6B). Out of 48 F2 plants, 10 were identified as homozy-
gous szl1/szl1 npq1/npq1 mutants, which is not significantly
single recessive nuclear mutation.
Reconstitution of the szl1 Mutant Pigment Phenotype in
To confirm that the LCYB point mutation found in the szl1 npq1
mutant was responsible for the mutant pigment phenotype (low
These plasmids contain bacterial genes for lycopene synthesis
plus the wild-type or szl1 allele of LCYB, respectively. The com-
position of pigments accumulated by E. coli strains containing
either of these two plasmids with or without an additional plasmid
expressing the Arabidopsis LCYE gene was compared.
We first examined the products formed when either the wild-
type or mutant copy of Arabidopsis LCYB was present in the
lycopene-accumulating strain of E. coli. Figure 7A shows an
HPLC profile of the carotenoid pigments accumulated in E. coli
cells containing plasmid pAC-BETA-At. As expected from the
previous work by Cunningham et al. (1996), these cells accumu-
lated predominantly b-carotene and formed yellow colonies.
When the wild-type LCYB gene was replaced by the szl1 mutant
Table 1. Pigment Content of the Wild Type, npq1, szl1, and szl1 npq1
Wild Typenpq1 szl1 szl1 npq1
LL HLLL HLLLHL LL HL
Total chlorophyll (nmol cm?2)
(mmol/mol Chl a)
VAZ pool size
(mmol/mol Chl a)
(mmol/mol Chl a)
(mmol/mol Chl a)
(mmol/mol Chl a)
(mmol/mol Chl a)
(mmol/mol Chl a)
(mmol/mol Chl a)
(mmol/mol Chl a)
21.9 6 0.5
3.31 6 0.02
293.1 6 11.6 287.9 6 4.6
21.3 6 1.5
3.29 6 0.04
22.5 6 0.7
3.46 6 0.07
273.6 6 6.8
22.8 6 1.2
3.33 6 0.01
277.5 6 6.4
21.9 6 2.0
3.37 6 0.09
264.8 6 18.8 277.3 6 5.3
22.3 6 1.6
3.36 6 0.07
20.9 6 1.3
3.35 6 0.03
280.6 6 2.9
21.2 6 0.8
3.40 6 0.06
281.6 6 8.0
48.4 6 3.349.6 6 2.7 41.6 6 0.844.7 6 2.49.4 6 0.210.2 6 0.211.7 6 0.712.5 6 0.2
46.2 6 3.710.6 6 0.239.6 6 0.743.4 6 1.37.2 6 0.5 3.9 6 0.0 9.7 6 0.910.4 6 0.1
2.3 6 0.4 12.6 6 0.9 1.8 6 0.11.9 6 0.2 2.1 6 0.42.5 6 0.32.0 6 0.22.1 6 0.2
026.3 6 3.10003.8 6 0.200
123.2 6 1.7125.8 6 2.0127.5 6 4.5123.8 6 2.0200.2 6 15.0 211.7 6 5.1210.7 6 3.0212.1 6 5.7
32.3 6 3.1 32.7 6 2.7 28.0 6 0.929.5 6 1.06.7 6 0.77.8 6 0.38.5 6 1.19.7 6 0.7
76.5 6 4.377.7 6 3.174.7 6 1.177.4 6 3.631.9 6 2.631.9 6 1.232.9 6 1.732.2 6 1.5
2.6 6 0.92.2 6 0.31.9 6 0.11.9 6 0.016.8 6 0.915.7 6 0.616.9 6 0.515.1 6 0.2
1.16 6 0.051.16 6 0.021.11 6 0.021.20 6 0.010.22 6 0.000.20 6 0.000.23 6 0.000.24 6 0.00
Pigment measurements were performed before and after exposure of LL-grown plants to HL (2000 mmol photons m?2s?1) for 30 min. Data are
presented as the means 6 SD (n = 9). Chl a/b, chlorophyll a/b; Chl a, chlorophyll a.
Figure 5. NPQ Induction Curves in the Wild Type, Homozygous npq1,
szl1, and szl1 npq1, and Heterozygous szl1/SZL1 npq1/npq1.
NPQ was measured during 5 min of illumination with HL (1200 mmol
photons m?2s?1), followed by relaxation in the dark for 5 min. Data are
presented as the means 6 SD (n = 4).
1802The Plant Cell
allele of LCYB, E. coli cells containing plasmid pAC-BETA-At-
szl1 accumulated approximately one-third as much b-carotene
and were paler yellow in color compared with cells containing
plasmid pAC-BETA-At (Figure 7B). The lower level of b-carotene
accumulation suggests that the LCYB gene from szl1 encodes a
still functional but less active b-cyclase relative to the wild-type
The addition of the Arabidopsis LCYE gene to cells containing
pAC-BETA-At resulted in the production of both a- and b-caro-
tene, and the molar ratio of a-carotene to b-carotene was 2:3
(Figure 7C), which mirrors the accumulation of more b-branch
and npq1 Arabidopsis (Table 1). When the same LCYE gene was
change reflects the significant decrease of b-branch carotenoids
and increase of a-branch carotenoids observed in both the szl1
single mutant and szl1 npq1 double mutant.
Effect of Pigment Alteration on the Composition of PSII
and Photosystem I
To investigate how accumulation of lutein in the suppressor szl1
npq1 suppresses the npq1 mutation, we first tested whether the
pigment alteration in szl1 npq1 might indirectly suppress the low
NPQ phenotype of npq1 by affecting the composition of the PSII
antenna. Immunoblot analysis showed that the Lhcb4 (CP29)
and Lhcb5 (CP26) protein levels were reduced in both szl1 npq1
and szl1 compared with wild type and npq1, whereas the
amounts of other LHC proteins were unchanged (Figure 8A).
There were no differences in the levels of the PsbS protein and
the PSII reaction center protein, D1 (Figure 8A). Further quanti-
tative analysis showed that Lhcb4 and Lhcb5 protein levels were
PsaF protein levels were slightly reduced in both szl1 and szl1
npq1 (Figures 8B and 8C). Because the pigment alteration in the
suppressor affected only the levels of the Lhcb4 and Lhcb5
proteins, and it was reported that a relatively minor effect on qE
was observed in antisense or knockout plants that lack detect-
able Lhcb4 or Lhcb5 (Andersson et al., 2001; Betterle et al.,
2009), we then hypothesized that the accumulation of lutein in
szl1 directly suppresses the npq1 mutation by replacing the role
of zeaxanthin in qE at the molecular level.
TA Spectroscopy of szl1 npq1 Thylakoids
formation of a carotenoid radical cation, was restored in szl1
Figure 6. Molecular Genetic Analysis of szl1.
(A) Sequence and position of the szl1 allele of the LCYB gene. The szl1 npq1 suppressor has a single base substitution from G to A, which changes the
Gly residue (position 451) in a predicted transmembrane helix (black box) to a Glu. This Gly is invariant in the seven available plant LCYB enzymes,
including Arabidopsis, maize, rice (Oryza sativa), tobacco (Nicotiana tabacum), tomato, citrus (Citrus sinensis), and papaya (Carica papaya). Dots in the
szl1 npq1 sequence indicate identity to the wild-type sequence.
(B) Cosegregation analysisof szl1 and NPQ phenotype. The szl1 npq1 suppressor was backcrossed to the npq1 parent to obtain F2 progeny. Lanes 1 to
16 are the first 16 plants among the total of 48 F2 progeny that were tested. The PCR fragments amplified from the genomic DNA of F2 progeny were
digested with EcoRI and separated on agarose gel.
Lutein Can Replace Zeaxanthin in qE1803
npq1, we measured ultrafast time-resolved TA spectra and
kinetics in isolated thylakoids in the presence or absence of
light-induced qE. Figure 9A shows the near infrared (NIR) TA
spectra of thylakoid membranes of the szl1 npq1 double mutant
at 15 ps delay between pump and probe laser pulses. The TA
signal in the qE state was measured every 20 nm from 880 to
1040 nm under an actinic light (;600 mmol photons m22s21)
(red line). The spectrum without qE (black line) was measured
after the sample was darkened for 10 min to relax the light-
induced DpH. Because chlorophylls and carotenoids have no
ground-state absorbance or emission in this NIR region, we
selectively measured excited-state absorbance of transient
species. The spectrum without qE exhibits a gradual increase
with wavelength mainly due to chlorophyll excited-state absor-
bance (Polivka et al., 2002; Holt et al., 2005). The qE spectrum
Figure 7. HPLC Analysis of Products Formed from Lycopene in E. coli
Expressing the Arabidopsis Wild-Type e-cyclase and Wild-Type or
Carotenoid pigment composition was examined in cultures of E. coli
containing the plasmids and genes indicated above and to the left. The
Arabidopsis wild-type and mutant copy of b-cyclase were cloned directly
in the pAC-LYC plasmid to give the plasmid pAC-BETA-At and pAC-
BETA-At-szl1, respectively (see Methods). Carotenoids were extracted
with acetone from equal numbers of cells (based on A600), and pigments
were separated by HPLC and detected by absorbance at 445 nm. a-car,
a-carotene; b-car, b-carotene.
(A) pAC-LYC plus the Arabidopsis wild-type b-cyclase.
(B) pAC-LYC plus the Arabidopsis mutant b-cyclase.
(C) pAC-LYC plus the Arabidopsis wild-type b-cyclase and e-cyclase.
(D) pAC-LYC plus the Arabidopsis mutant b-cyclase and wild-type
Figure 8. PSII and PSI Protein Levels in LL-Grown Wild Type, npq1, szl1,
and szl1 npq1.
Thylakoid protein samples were loaded on the basis of total protein (5 mg
lane?1), and immunoblot analysis was performed with polyclonal anti-
bodies directed against each of the indicated proteins. D1 is a PSII
reaction center protein; PsbS is a PSII protein that is essential for qE;
Lhcb1, Lhbc2, and Lhcb3 are components of LHCII trimers; Lhcb4,
Lhcb5, and Lhcb6 (also called CP29, CP26, and CP24, respectively) are
monomeric, minor antenna proteins of PSII; PsaF is a PSI reaction center
protein; Lhca1 is a PSI antenna protein. For comparison to mutant
samples, dilutions were made from wild-type samples.
(A) Immunoblot analysis of D1, PsbS, and Lhcb protein levels in the four
(B) Quantification of Lhcb4, Lhcb5, and Lhcb6 protein levels in szl1 and
(C) Quantification of PsaF and Lhca1 protein levels in szl1 and szl1 npq1.
1804The Plant Cell
880 to 960 nm. To remove the chlorophyll excited-state absorp-
tion and to emphasize the difference, we subtracted the black
trace under darkness from the red one under actinic light,
resulting in the blue reconstructed spectrum (Figure 9A). The
ing in szl1 npq1, but the spectrum was maximized at ;920 nm,
which is substantially blue-shifted relative to the spectrum of a
b-carotene cation radical (blue dotted line) or a zeaxanthin
radical cation with a broad spectrum centered ;980 to 1000
nm (Holt et al., 2005; Amarie et al., 2007). Instead, the spectrum
observed in szl1 npq1 was consistent with the reported absorp-
tion spectrum of a lutein radical cation, which was centered at
;920 to 950 nm depending on the solvent used (Mortensen and
Skibsted, 1997; Edge et al., 1998; Galinato et al., 2007).
Individual TA kinetic traces of the szl1 npq1 thylakoids at 950
and 1000 nm are shown in Figures 9B and 9C, respectively. The
qE trace under actinic light at 950 nm (red line in Figure 9B)
revealed distinctly slower kinetics than the trace without qE
similar (Figure 9C). Thus, unlike the wild type, which has a
difference in kinetics at 1000 nm but not 950 nm (Holt et al.,
2005), the different kinetics in the szl1 npq1 thylakoids showed
(blue line in Figure 9B) but not at 1000 nm (blue line in Figure 9C).
TA Spectroscopy of Isolated LHC Complexes
To determine if the NIR absorption changes detected in szl1 npq1
thylakoids can be associated with LHC complexes, as in the case
of zeaxanthin radical cation formation (Ahn et al., 2008; Avenson
et al., 2008), we investigated the effect of substituting b-xantho-
phylls by lutein, as observed in the szl1 npq1 mutant, in recon-
refolded in vitro from apoproteins expressed in E. coli and chlo-
rophylls a and b plus lutein only or a total carotenoid mix. The
resulting complexes were characterized (Table 2) by having lutein
in both carotenoid binding sites L1 and L2 (LL complexes),
whereas in the control complexes, the L2 binding site could be
occupied by lutein, violaxanthin, or neoxanthin (LNV complexes)
(Pagano et al., 1998; Ruban et al., 1999; Ballottari et al., 2009),
except for CP24, which cannot bind neoxanthin either in vivo or in
type or the chy1 chy2 lut5 mutant, which has lutein as the only
xanthophyll (Fiore et al., 2006). Both LHCII samples have lutein in
L1 and L2 xanthophyll binding sites; however, LHCII trimers from
chy1 chy2 lut5 have lutein instead of violaxanthin bound in the
external V1 binding sites, while site N1 remained empty (Liu et al.,
2004; Mozzo et al., 2008). All these complexes were analyzed by
NIR TA spectroscopy. LHCII-LL trimers did not show any TA
difference compared with LHCII-LNV complexes; the TA kinetics
in the lutein radical cation absorption region were characterized
1 online). This is similar to the case of CP26-LL, which has been
previously analyzed (Avenson et al., 2009). As shown in Figure 10,
CP24 and CP29 kinetics at 920 nm were instead characterized by
the presence of an additional rise component only in the CP24-LL
and CP29-LL complexes. The difference kinetics reported in
Figures 10C and 10D clearly show the formation of a lutein radical
cation in CP24 and CP29 binding lutein as the only xanthophyll,
with a rise time of 5 ps and main decay of ;50 to 60 ps. Lutein
radical cation formation in CP24 and CP29 complexes was
confirmed by the reconstructed NIR TA spectra reported in
Figures 10A and 10B, showing a peak at 920 nm, consistent
with result obtained for szl1 npq1 thylakoids upon qE induction
Light-Induced Absorbance Changes
A slower light-induced absorbance change, DA535, depends on
both zeaxanthin and the DpH and is closely associated with the
Figure 9. TA Spectroscopy of szl1 npq1 Thylakoids.
(A) TA spectrum from 880 to 1040 nm of szl1 npq1 mutant thylakoids with
qE (red line) or without qE (black line) at 15 ps delay after pump pulse.
The blue line shows the difference kinetics between the red and the black
lines, and the blue dotted line is the spectrum of a b-carotene radical
cation (b-Car+d) for comparison (Tracewell and Brudvig, 2003). Data are
presented as the means 6 SE (n = 5).
(B) TA kinetics probed at 950 nm.
(C) TA kinetics probed at 1000 nm.
Lutein Can Replace Zeaxanthin in qE 1805
induction and relaxation of qE (Ruban et al., 1993; Bilger and
Bjo ¨rkman, 1994; Li et al., 2004). Figure 11 shows a typical qE
spectrum for wild-type Arabidopsis, taken as the difference in
absorbance changes between 10 and 60 s after illumination to
eliminate contributions from the electrochromic shift (see Bilger
and Bjo ¨rkman, 1994). The peak absorbance change occurred at
;530 to 535 nm. By contrast, peaks observed in the szl1, szl1
npq1, and npq1 mutants were blue-shifted by ;5 to 10 nm to
;525 to 530 nm (Figure 11). In each strain, the qE absorbance
signals decayed in the dark with half times of 5 to 10 s,
suggesting that all signals reflected similar processes.
Isolation of the suppressor mutant szl1 npq1, which contains a
point mutation in the LCYB gene and thus results in a dramatic
change in pigment profile and qEcapacity, has provided insights
into carotenoid biosynthesis and the role of lutein in qE.
Regulation of the Carotenoid Biosynthetic Pathway at the
The plant carotenoid biosynthetic pathway branches at the
cyclization reactions to produce carotenoids with either two
b-rings or one b- and one «-ring. In brief, lycopene is either
cyclized twice by LCYB to produce b-carotene and derivatives
thereof or once each by LCYB and LCYE to produce a-carotene
that is the precursor to lutein (Figure 1). It has been hypothesized
that partition of flux into the b- and a-branches of the path-
way is controlled by the relative activities of LCYB and LCYE
Indeed, transgenic manipulations to increase or decrease LCYE
180% of the wild type (Pogson and Rissler, 2000). Similar results
were reported recently with LCYE transgenic potato (Solanum
tuberosum) and Brassica plants (Diretto et al., 2006; Yu et al.,
2007). It has also been shown thatnatural genetic variation at the
LCYE locus in maize (Zea mays) changes the ratio of a- versus
b-branches of the carotenoid pathway (Harjes et al., 2008).
Here, by affecting LCYB instead of LCYE, we provide
complementary experimental evidence to support the above
hypothesis. The suppressor szl1 npq1 has very low levels of
violaxanthin, antheraxanthin,and neoxanthin,butitaccumulates
nearly twice as much lutein and approximately eight times more
a-carotene compared with the wild type (Table 1). Molecular
genetic analysis demonstrated that the szl1 mutation affects the
structural gene encoding LCYB by changing a Gly residue in a
highlyconserved predicted transmembranehelix toaGlu(Figure
6A). It is clear that szl1 is not a complete loss-of-function
but detectable levels of b-carotene when the szl1 allele of LCYB
was expressed in a lycopene-accumulating strain of E. coli
(Figure 7B). In E. coli cells that express both the szl1 allele of
LCYB and the wild-type LCYE, the molar ratio of a-carotene to
b-carotene increased to 4:1 (Figure 7D), which is in agreement
with the high levels of a-branch carotenoids and low levels of
b-branch carotenoids accumulated in the suppressor szl1 npq1.
LCYB Is the Principal Lycopene b-Cyclase in Arabidopsis
The enzyme encoded by the Arabidopsis LCYB gene belongs to
the CrtL protein family, which includes the b- and «-cyclases in
some cyanobacteria and plants (Cunningham et al., 1994, 1996;
Stickforth et al., 2003). Many bacteria, including the green sulfur
bacterium Chlorobium tepidum and cyanobacteria, have an
LCYB that is different from that of plants. The first member of
complementation assay with a lycopene-accumulating strain of
E. coli (Maresca et al., 2007). Two homologs of CruA, denoted
CruA and CruP, were found and characterized in the cyanobac-
that Arabidopsis contains a CruP homolog, which is encoded by
At2g32640 (Maresca et al., 2007). Whether the CruP homolog
also has lycopene b-cyclase activity in Arabidopsis is not yet
known, but the dramatic change of the pigment profile observed
in the suppressor szl1 npq1 suggests that LCYB is the major
lycopene b-cyclase in Arabidopsis.
Does LCYB Have Neoxanthin Synthase Activity
Conversion of violaxanthin to neoxanthin is catalyzed by the
enzyme neoxanthin synthase (NSY) (Figure 1). Genes encoding
NSY activity have not yet been identified conclusively in Arabi-
dopsis. Two homologous NSY genes have been cloned from
either tomato (Solanum lycopersicum) or potato based on their
ability to convert all-trans-violaxanthin to all-trans-neoxanthin in
vitro or in transient expression systems (Al-Babili et al., 2000;
Bouvier et al., 2000). Polypeptides encoded by NSY genes
are homologous to LCYB, and the tomato NSY gene product
has b-cyclase activity and accounts for the fruit-specific high
Table 2. Pigment Composition of the CP24 and CP29 Recombinant Proteins
Chl aChl bChl a/b Chl/CarCar, No.NVLZ
6.07 6 0.05
5.99 6 0.10
6.01 6 0.01
5.58 6 0.06
3.93 6 0.05
1.99 6 0.01
4.01 6 0.10
2.42 6 0.06
1.54 6 0.03
1.50 6 0.06
3.02 6 0.02
2.31 6 0.08
4.76 6 0.09
4.74 6 0.10
4.20 6 0.10
3.69 6 0.17
2.08 6 0.04
2.11 6 0.04
1.90 6 0.04
2.17 6 0.11
0.37 6 0.05
0.90 6 0.08
0.61 6 0.04
2.08 6 0.03
1.21 6 0.06
1.90 6 0.02
1.19 6 0.01
Pigments of the different complexes were normalized to 10 chlorophylls (a+b) per CP24 and 8 chlorophylls per CP29 (Bassi and Dainese, 1992). Chl a,
chlorophyll a; Chl b, chlorophyll b; Chl/Car, ratio of total chlorophylls to total carotenoids; Car, number of carotenoids per polypeptide; N, neoxanthin;
V, violaxanthin; L, lutein; Z, zeaxanthin; ND, not detectable. Data are presented as the means 6 SD (n = 3).
1806The Plant Cell
b-carotene content of the tomato B mutant (Ronen et al., 2000).
Based on these findings, it has been proposed that NSY has a
dual function in converting both violaxanthin to neoxanthin and
lycopene to b-carotene (Hirschberg, 2001). Because Arabidop-
sis LCYB is encoded by a single-copy gene and shows signif-
icant identity to tomato and potato NSYs, it is possible that
Arabidopsis LCYB is also a bifunctional enzyme that has both
LCYB and NSY activity. Isolation of the szl1 npq1 and szl1
mutants provides an opportunity to test this hypothesis. If LCYB
does function as NSY, the szl1 mutation that impairs LCYB
activity might also decrease NSY activity; thus, we would expect
a preferential reduction of neoxanthin content compared with
that of violaxanthin in the szl1 npq1 or szl1 mutant. On the other
hand, if LCYB does not have NSY activity, then we would expect
to observe a proportional reduction of neoxanthin and violaxan-
plants showed that the molar ratio of neoxanthin to violaxanthin
in szl1 npq1 and szl1 was the same as that of npq1 (Table 1),
indicating that LCYB does not have NSY activity. It is more likely
that in Arabidopsis a novel type of NSY is responsible for the
conversion of violaxanthin to neoxanthin, and the recently iden-
tified ABA4 protein would be a very good candidate (North et al.,
A Direct Role of Lutein in qE
How does the increased lutein content caused by the szl1
mutation restore qE in the npq1 mutant that lacks zeaxanthin?
Based on the very similar chemical structures of lutein and
zeaxanthin, an analogy has previously been drawn with zeaxan-
thin when speculating about the functions of lutein in qE, and a
possible role of luteinin qE has been proposed. Indeed, aneffect
of lutein on qE has been demonstrated in several mutants and
Figure 10. TA Spectroscopy of LHC Complexes.
(A) Difference NIR-TA spectrum (from 880 to 1040 nm) between CP24
complexes binding lutein in both L1 and L2 sites (CP24-LL) and CP24
with violaxanthin in site L2 (CP24-LV). Each point represents the differ-
ence between the DA value obtained at 20 ps delay after pump pulse in
CP24-LL and the corresponding value in CP24-LV. Data are presented
as the means 6 SE (n = 5).
(B) Difference NIR-TA spectrum (from 880 to 1040 nm) between CP29
complexes binding lutein in both L1 and L2 sites (CP29-LL) and CP29
with violaxanthin or neoxanthin in site L2 (CP29-LNV) . Each point
represents the difference between the DA value obtained at 20 ps delay
after pump pulse in CP29-LL and the corresponding value in CP29-LNV.
Data are presented as the means 6 SE (n = 5).
(C) TA kinetics probed at 920 nm of CP24-LL (red trace) and CP24-LV
(black trace). Difference kinetic trace is reported in blue with rise and
decay times indicated.
(D) TA kinetics probed at 920 nm of CP29-LL (red trace) and CP29-LNV
(black trace). Difference kinetic trace is reported in blue with rise and
decay times indicated.
Figure 11. Light-Induced Spectral Absorbance Changes in Leaves.
Intact leaves of wild type (squares), szl1 (circles), szl1 npq1 (triangles),
and npq1 (inverted triangles) were illuminated with;1150 mmol photons
m?2s?1red light for 10 min to induce qE, and absorbance changes (from
460 to 563 nm) were measured during a 1-min dark interval. The qE
spectrum was calculated as the difference in absorbance between 10
and 60 s after illumination to eliminate contributions from the electro-
chromic shift. The dashed lines indicate the peak positions of ;530 to
535 nm in the wild type and ;525 to 530 nm in the szl1, szl1 npq1, and
Lutein Can Replace Zeaxanthin in qE1807
transgenic plants with altered lutein levels (Niyogi et al., 1997,
2001; Pogson et al., 1998; Pogson and Rissler, 2000; Lokstein
et al., 2002), but whether and how lutein plays a direct or an
indirect role in qE has been unclear.
Previously characterized mutants that accumulate extra lutein
have only been obtained together with either the total depletion
of b-xanthophylls (Dall’Osto et al., 2007), which strongly affects
antenna protein composition, or the normal accumulation of
zeaxanthin (Pogson and Rissler, 2000), thus obscuring the spe-
cific lutein-associated qE phenotype. The szl1 npq1 mutant
accumulates nearly double the wild-type amount of lutein,
has lower total carotenes (increased a-carotene but lower
b-carotene), retains low levels of violaxanthin, antheraxanthin,
and neoxanthin, and lacks zeaxanthin (Figure 4), but the total
carotenoid (and chlorophyll) content does not change (Table 1).
Thus, the increase in a-carotenoids occurs at the expense of
b-carotenoids, and the extra lutein and a-carotene are likely
bound at sites that are normally occupied by b-carotenoids.
In principle, the enhancement of qE in the absence of zeaxan-
for b-xanthophylls on the composition and/or structure of the
PSII antenna (Lokstein et al., 2002) or by a direct role of lutein in
We did detect a 50% decrease in the levels of Lhcb4 and Lhcb5
the increased level of qE because only a limited decrease of the
qE amplitude was observed when a complete absence of Lhcb4
or Lhcb5 was induced in a wild-type background by antisense or
knockout (Andersson et al., 2001; Betterle et al., 2009). There-
fore, we considered the possibility of a direct role of lutein in
enhancing a qE mechanism. At present, it is not possible to test
directly for the occurrence of the S1 quenching mechanism in
isolated thylakoids (Ruban et al., 2007), but we were able to
investigate formation of a carotenoid radical cation, a key mo-
by TA spectroscopy.
We hypothesized that lutein might be able to take the place of
zeaxanthin in directly quenching1Chl* during qE through the CT
szl1 npq1 suppressor. Indeed, NIR TA traces revealed a carot-
but not at 1000 nm (Figure 9C), in contrast with wild-type
thylakoids (Holt et al., 2005). The TA spectrum of the suppressor
thylakoids showed a maximum at;920 nm (Figure 9A), which is
blue-shifted relative to the spectrum of a b-carotene or zeaxan-
thin radical cation and is in agreement with the reported spec-
trum of a lutein radical cation (Mortensen and Skibsted, 1997;
Edge et al., 1998; Galinato et al., 2007). Similarly, the DA535leaf
absorbance change associated with zeaxanthin-dependent qE
was missing in npq1 (Niyogi et al., 1998) and replaced with a
blue-shifted absorbance change in szl1 npq1 (Figure 11A). Thus,
(Figure 11) experiments strongly suggest that lutein can substi-
tute for zeaxanthin in qE.
We suspect that the high amount of lutein accumulated in the
suppressor probably magnifies a TA signal that is normally
present at a relatively low level and is obscured by the stronger
zeaxanthin radical cation signal in the wild type. Similarly, we
propose that the residual qE in the npq1 mutant is lutein depen-
dent, but the lutein radical cation signal is likely below the
detection limit of the TA experiment. We are currently trying to
improve the sensitivity and signal-to-noise ratio of our TA setup
to test this idea. A basal level of CT quenching by lutein (that
could be enhanced by PsbS overexpression) could explain
previous observations of zeaxanthin-independent qE, which
indicated that zeaxanthin (and antheraxanthin) is not required
for qE (Crouchman et al., 2006). Indeed, a blue-shifted absor-
detected in npq1 leaves (Johnson et al., 2009) and in wild-type
leaves treated with DTT to phenocopy the npq1 mutation by
inhibition of VDE activity (Crouchman et al., 2006).
The spectroscopic signature of CT quenching has been
detected recently in recombinant CP29 (Lhcb4), CP26 (Lhcb5),
and CP24 (Lhcb6) (Ahn et al., 2008; Avenson et al., 2008, 2009).
coupled chlorophyll dimer. A lutein radical cation was so far
detected only in CP26, but only when zeaxanthin is also present
(Avenson et al., 2009). Because zeaxanthin is lacking in szl1
npq1, it is unlikely that lutein radical cation formation of the type
observed in CP26 in vitro (Avenson et al., 2009) could explain the
TA signal detected in szl1 npq1 thylakoids (Figure 9). Instead, we
complexes reconstituted with only lutein (Figure 10). Our results
in vitro and in vivo thus highlight the possibility of lutein to
substitute for zeaxanthin as a quencher of excess excitation
energy through formation of radical cations. The fraction of
lutein-only CP24 and CP29 complexes undergoing CT quench-
ing in vitro, however, is rather low (;1%), as in the case of
zeaxanthin binding complexes (Avenson et al., 2008), indicating
the fundamental role of other factors, such as PsbS and DpH, for
the induction of qE in vivo. In wild-type plants, CP24, CP26, and
CP29 bind lutein and violaxanthin/zeaxanthin in carotenoid
binding sites named L1 and L2, respectively. An increase in the
lutein content of these complexes, substituting for violaxanthin/
zeaxanthin in the L2 site as in the case of the szl1 npq1 mutant,
was previously reported to induce a decrease of fluorescence
quantum yield (Formaggio et al., 2001). Although we favor the
hypothesis that the extra lutein bound in the L2 site of LHC
complexes in the szl1 npq1 mutant is directly involved in CT
quenching, we cannot at this point exclude the possibility that it
acts as an allosteric factor, similar to zeaxanthin in CP26
(Avenson et al., 2009), inducing lutein radical cation formation
in site L1. It is worth noting that carotenoid radical cation
reactions have distinct patterns and dependence on xanthophyll
composition in each Lhcb protein, thus contributing to explain
the existence of multiple, conserved gene products in the plant
Plant Material and Growth Conditions
All Arabidopsis thaliana plantswere oftheecotype Columbia-0. The npq1
mutant is affected in VDE and lacks zeaxanthin under high light (Niyogi
1808The Plant Cell
et al., 1998). The szl1 npq1 double mutant was crossed with the wild type,
on Sunshine Mix 4 potting mix (Sun Gro Horticulture Distribution) in
controlled conditions of 10 h light, 228C/14 h dark, 238C, with a light
intensity of150 mmol photonsm22s21. For physiological studies, plants at
the age of 5 to 6 weeks (prior to bolting) were used. For mutant screening
and cosegregation analysis, plants were grown for 2 weeks on minimal
plant nutrient agar medium (Haughn and Somerville, 1986) at 80 mmol
photons m22s21(continuous light) at 238C and then transferred to soil.
Isolation of Suppressors of the npq1 Mutant
by HPLC for pigment composition after exposure to high light.
Chlorophyll Fluorescence Measurement
Chlorophyll fluorescence was measured at room temperature from
attached, fully expanded rosette leaves using an FMS2 fluorometer
(Hansatech). After an overnight dark period, leaves were exposed to
actinic light (1250 mmol photons m22s21) for 5 min followed by 5 min of
darkness. The maximum fluorescence levels after dark adaptation (Fm)
and in the light-adapted condition (Fm’) were recorded after applying a
saturating pulse of light. NPQ was calculated as (Fm2 Fm’)/Fm’.
HPLC analysis of carotenoids and chlorophylls was done as previously
described (Mu ¨ller-Moule ´ et al., 2002). A total of nine samples (three
independently grown sets of plants with three samples each) were
measured. Carotenoids were quantified using standard curves ofpurified
pigments (VKI) and normalized to chlorophyll a.
Genetic Mapping and Cosegregation Analysis
The szl1 mutation was mapped by candidate gene approach. The LCYB
gene was amplified by PCR from genomic DNA of the wild type, npq1,
and szl1 npq1 mutants using primers LBC1 (59-CCATTTTCTCA-
ATCCCTCTGGT-39) and LBC2 (59-AGTATATCCGCATTGCAAGTC-39),
and 1.7-kb PCR products were used for DNA sequencing.
Cosegregation of the szl1 mutation with the mutant (high NPQ)
phenotype was determined by backcrossing the homozygous szl1
npq1 double mutant to the npq1 mutant. For cosegregation analysis,
a 1.5-kb PCR product was amplified from the genomic DNA of the
segregating F2 plants using the forward primer ZL49 (59-GCAGGTTTCT-
GAAGCTGGAC-39) and the reverse primer ZL50 (59-TGAACTTGATG-
TTGGCTTGC-39). The PCR products were digested overnight with
EcoRI whose site is present only in the sequence from the szl1 mutant,
giving two smaller fragments.
Plasmids pAC-LYC, y2, and y8 were kindly provided by Francis X.
Cunningham (University of Maryland). Cells of Escherichia coli con-
taining plasmid pAC-LYC accumulate lycopene and form pink colonies
(Cunningham et al., 1994). Plasmids y2 and y8 are Arabidopsis cDNA
library plasmids that contain functional genes for Arabidopsis LCYE and
LCYB, respectively (Cunningham et al., 1996).
Plasmid y8-szl1 was constructed by introducing a point mutation
G1352A into LCYB of y8 plasmid according to the QuickChange site-
directed mutagenesis kit guidelines (Stratagene). The sequence of the
forward primer was (mutagenic positions underlined) ZL61, 59-ATCTG-
the reverse primer ZL62 was complementary to the forward primer.
Plasmid pAC-BETA-At and plasmid pAC-BETA-At-szl1 were con-
structed by cloning a 1.8-kb XbaI fragment (partially filled recessed
termini with T and C) from plasmids y8 and y8-szl1, containing the
HindIII site (partially filled recessed termini with A and G) of pAC-LYC.
SDS-PAGE and Immunoblot Analysis
To prepare thylakoid membranes, rosette leaves were harvested, frozen
immediately in liquid nitrogen, and stored at 2808C. Thylakoid mem-
branes were prepared and analyzed by SDS-PAGE and immunoblotting
as previously described (Li et al., 2002). Thylakoid protein samples
containing equal amounts of total protein (5 mg) wereloaded in each lane.
The D1 antibody was kindly provided by Anastasios Melis (University of
and Lhca1 antibodies were kindly provided by Stefan Jansson (Umea ˚
University), and the PsaF antibody was kindly provided by Anna Haldrup
(The Royal Veterinary and Agricultural University, Copenhagen, Den-
mark). Proteins were blotted onto Protran nitrocellulose membranes
(BA83; Whatman) and probed with antibodies. After incubation with
peroxidase-conjugated secondary antibodies, the chemiluminescence
signal (ECL; Amersham Pharmacia) was detected with Kodak BioMax
Light film with exposure times in the linear range.
NIR TA Spectroscopy
Thylakoids for the NIR TA measurements were prepared as previously
described (Gilmore et al., 1998). The NIR TA laser system has been
previously described (Holt et al., 2005; Avenson et al., 2008). Briefly, the
repetition rate was 250 kHz, and the pump pulses were tuned to;650 nm
(i.e., the chlorophyll b Qy transition). The maximum pump energy and full
;40 fs, respectively. We chose 650 nm as our excitation wavelength
because the output power of our optical parametric amplifier was higher
than that at 680 nm, yielding higher signal-to-noise ratios. White light
correlation function of the pump and probe overlap was ;85 fs. The
diameters of the pump and probe beams at the sample holder were
estimated to be 146 and 83 mm, respectively. The mutual polarizations of
the pump and probe beams were set to the magic angle (54.78). The time
point (210 to 60 ps), and 5 ps/point (65 to 600 ps). A monochromator
(Spectra Pro 300i; Acton Research) with a spectral resolution of 2.7 nm, a
InGaAs photodiode (DET410; Thorlabs), and a chopper-associated lock-in
amplifier (SR830; Stanford Research Systems) were used to monitor
transmission. The path length of the cuvette used for isolated thylakoids
was 2 mm and was continuously translated during experiments to avoid
sample degradation. NIR TA kinetic profiles were obtained during steady
state actinic illumination and 10 min following a light-to-dark transition by
We obtained transient traces (210 to 20 ps) at every 20 nm (880 to 1040
nm), averaged five times, and plotted the TA spectrum at 15 ps with
Leaf Absorbance Measurements
Absorbance spectra were measured using the diffused optics flash
spectrophotometer (Kramer and Sacksteder, 1998) and the appropriate
narrow band-pass interference filters (Omega Optics). Detached leaves
were gently clamped into the spectrophotometer and illuminated with
;1150 mmol photons m22s21red light, supplied by a high-flux, red light
Lutein Can Replace Zeaxanthin in qE1809
dark interval. Within this interval, spectral contributions from the xantho-
phyll cycle are negligible, since the conversion of zeaxanthin to viola-
xanthin is relatively slow. Typically, spectral changes attributable to the
electrochromic shift (which peak at 518 to 520 nm) are most prominent
during the first second of the dark interval, whereas beyond this window
qE spectral contributions (which peak at ;535 nm) dominate. The qE
absorbance spectra were partially corrected for the contributions of the
residual fast electrochromic shift by subtraction of the 10-ms spectrum,
which contains only electrochromic shift changes.
In parallel experiments, chlorophyll fluorescence was measured
in a modified laboratory-built nonfocusing optics spectrophotometer
(NoFOSpec) (Sacksteder et al., 2001), which used laboratory-built, small
aperture compound parabolic concentrators (CPCs; both solid, clear-
cast acrylic and hollow aluminum) designed specifically for work with
intact Arabidopsis plants. Leaves from intact plants were gently clamped
between the CPCs with the upper surface of the leaf against the solid
acrylic CPC and thelowersurface against the hollow CPC, through which
water saturated air was pumped. The modified design uses two separate
detectors to collect fluorescence and absorbance data. Surface fluores-
a hot mirror (NT43-453; Edmund Optics), through a far-red cut-off filter
(RG-710; Schott) onto the fluorescence detector. High-flux, red LEDs
were used with collimating optics (Polymer Optics) for actinic and
saturating light sources, and measuring pulses were supplied by a green
LED (NSPG500S; Nichia). Maximum fluorescence values obtained from
saturating flashes (1 s, >16,000 mmol photons m22s21red light) were
measured at steady state (Fm’) (with actinic illumination of ;1150 mmol
photons m22s21red light for 10 min) and during a 10-min dark recovery
(Fm”). qE was calculated as (Fm’’ 2 Fm’)/Fm’’.
Isolation and Reconstitution of LHC Complexes
Native LHCII trimers were isolated from the wild type and chy1 chy2 lut5
mutant (Fiore et al., 2006) as previously described (Caffarri et al., 2001).
Recombinant apoproteins CP24 and CP29 were refolded in vitro in the
presence of chlorophylls a and b and total carotenoids, or lutein as the
et al., 2003).
Sequence data from this article can be found in the Arabidopsis Genome
Initiative or GenBank/EMBL databases under accession number U50739
The following material is available in the online version of this article.
Supplemental Figure 1. Transient Absorption Spectroscopy of LHCII
We thank Xiao-Ping Li for generous assistance during the backcrossing
of szl1 npq1, Francis Cunningham for providing plasmids, Anastasios
Melis, Stefan Jansson, and Anna Haldrup for providing antibodies, and
Luca Dall’Osto for chy1 chy2 lut5 plants. We also thank Graham Peers
for critical reading of the manuscript. This work was supported by a
grant from the Office of Basic Energy Sciences, Chemical Sciences
Division, U.S. Department of Energy (Contract DE-AC03-76SF000098)
to G.R.F. and K.K.N. a grant from the National Institutes of Health
(GM058799) to K.K.N., a grant from the U.S. Department of Energy
(Contract DE-FG02-04ER15559) to D.M.K., a grant from Fondo Integra-
tivo Ricerca Basa RBLA0345SF002 (Solanaceae) and Fondo Integrativo
Speciale Ricerca IDROBIO to R.B., and a grant from the Bill and Melinda
Gates Foundation to J.D.K.
Received February 25, 2009; revised May 6, 2009; accepted June 4,
2009; published June 26, 2009.
Ahn, T.K., Avenson, T.J., Ballottari, M., Cheng, Y.C., Niyogi, K.K.,
Bassi, R., and Fleming, G.R. (2008). Architecture of a charge-transfer
state regulating light harvesting in a plant antenna protein. Science
Al-Babili, S., Hugueney, P., Schledz, M., Welsch, R., Frohnmeyer, H.,
Laule, O., and Beyer, P. (2000). Identification of a novel gene coding
for neoxanthin synthase from Solanum tuberosum. FEBS Lett. 485:
Amarie, S., Standfuss, J., Barros, T., Kuhlbrandt, W., Dreuw, A., and
Wachtveitl, J. (2007). Carotenoid radical cations as a probe for the
molecular mechanism of nonphotochemical quenching in oxygenic
photosynthesis. J. Phys. Chem. 111: 3481–3487.
Andersson, J., Walters, R.G., Horton, P., and Jansson, S. (2001).
Antisense inhibition of the photosynthetic antenna proteins CP29 and
CP26: Implications for the mechanism of protective energy dissipa-
tion. Plant Cell 13: 1193–1204.
Asada, K. (2006). Production and scavenging of reactive oxygen
species in chloroplasts and their functions. Plant Physiol. 141:
Avenson, T.J., Ahn, T.K., Niyogi, K.K., Ballottari, M., Bassi, R., and
Fleming, G.R. (2009). Lutein can act as a switchable charge-transfer
quencher in the CP26 light-harvesting complex. J. Biol. Chem. 284:
Avenson, T.J., Ahn, T.K., Zigmantas, D., Niyogi, K.K., Li, Z.,
Ballottari, M., Bassi, R., and Fleming, G.R. (2008). Zeaxanthin
radical cation formation in minor light-harvesting complexes of higher
plant antenna. J. Biol. Chem. 283: 3550–3558.
Ballottari, M., Mozzo, M., Croce, R., Morosinotto, T., and Bassi, R.
(2009). Occupancy and functional architecture of the pigment binding
sites of photosystem II antenna complex Lhcb5. J. Biol. Chem. 284:
Bassi, R., and Dainese, P. (1992). A supramolecular light-harvesting
complex from chloroplast photosystem-II membranes. Eur. J. Bio-
chem. 204: 317–326.
Betterle, N., Ballottari, M., Zorzan, S., de Bianchi, S., Cazzaniga, S.,
Dall’Osto, L., Morosinotto, T., and Bassi, R. (2009). Light induced
dissociation of an antenna hetero-oligomer is needed for non-photo-
chemical quenching induction. J. Biol. Chem. 284: 15255–15266.
Bilger, W., and Bjo ¨rkman, O. (1994). Relationships among violaxanthin
deepoxidation, thylakoid membrane conformation, and nonphoto-
chemical chlorophyll fluorescence quenching in leaves of cotton
(Gossypium hirsutum L). Planta 193: 238–246.
Bouvier, F., D’Harlingue, A., Backhaus, R.A., Kumagai, M.H., and
Camara, B. (2000). Identification of neoxanthin synthase as a carot-
enoid cyclase paralog. Eur. J. Biochem. 267: 6346–6352.
Caffarri, S., Croce, R., Breton, J., and Bassi, R. (2001). The major
antenna complex of photosystem II has a xanthophyll binding site not
involved in light harvesting. J. Biol. Chem. 276: 35924–35933.
Crouchman, S., Ruban, A., and Horton, P. (2006). PsbS enhances
nonphotochemical fluorescence quenching in the absence of zea-
xanthin. FEBS Lett. 580: 2053–2058.
1810The Plant Cell
Cunningham, F.X., Sun, Z.R., Chamovitz, D., Hirschberg, J., and
Gantt, E. (1994). Molecular-structure and enzymatic function of
lycopene cyclase from the cyanobacterium Synechococcus sp strain
PCC7942. Plant Cell 6: 1107–1121.
Cunningham, F.X., Jr., Pogson, B., Sun, Z., McDonald, K.A., Della-
Penna, D., and Gantt, E. (1996). Functional analysis of the beta and
epsilon lycopene cyclase enzymes of Arabidopsis reveals a mecha-
nism for control of cyclic carotenoid formation. Plant Cell 8: 1613–
Dall’Osto, L., Fiore, A., Cazzaniga, S., Giuliano, G., and Bassi, R.
(2007). Different roles of alpha- and beta-branch xanthophylls in
photosystem assembly and photoprotection. J. Biol. Chem. 282:
Demmig-Adams, B., and Adams, W.W. (1992). Photoprotection and
other responses of plants to high light stress. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 43: 599–626.
Diretto, G., Tavazza, R., Welsch, R., Pizzichini, D., Mourgues, F.,
Papacchioli, V., Beyer, P., and Giuliano, G. (2006). Metabolic
engineering of potato tuber carotenoids through tuber-specific si-
lencing of lycopene epsilon cyclase. BMC Plant Biol. 6: 13.
Edge, R., Land, E.J., McGarvey, D., Mulroy, L., and Truscott, T.G.
(1998). Relative one-electron reduction potentials of carotenoid rad-
ical cations and the interactions of carotenoids with the vitamin E
radical cation. J. Am. Chem. Soc. 120: 4087–4090.
Fiore, A., Dall’Osto, L., Fraser, P.D., Bassi, R., and Giuliano, G.
(2006). Elucidation of the beta-carotene hydroxylation pathway in
Arabidopsis thaliana. FEBS Lett. 580: 4718–4722.
Formaggio, E., Cinque, G., and Bassi, R. (2001). Functional architec-
ture of the major light-harvesting complex from higher plants. J. Mol.
Biol. 314: 1157–1166.
Galinato, M.G.I., Niedzwiedzki, D., Deal, C., Birge, R.R., and Frank,
H.A. (2007). Cation radicals of xanthophylls. Photosynth. Res. 94:
Gastaldelli, M., Canino, G., Croce, R., and Bassi, R. (2003). Xantho-
phyll binding sites of the CP29 (Lhcb4) subunit of higher plant
photosystem II investigated by domain swapping and mutation anal-
ysis. J. Biol. Chem. 278: 19190–19198.
Gilmore, A.M., Shinkarev, V.P., Hazlett, T.L., and Govindjee, G.
(1998). Quantitative analysis of the effects of intrathylakoid pH and
xanathophyll cycle pigments on chlorophyll a fluorescence lifetime dis-
tributions and intensity in thylakoids. Biochemistry 37: 13582–13593.
Harjes, C.E., Rocheford, T.R., Bai, L., Brutnell, T.P., Kandianis, C.B.,
Sowinski, S.G., Stapleton, A.E., Vallabhaneni, R., Williams, M.,
Wurtzel, E.T., Yan, J.B., and Buckler, E.S. (2008). Natural genetic
variation in lycopene epsilon cyclase tapped for maize biofortification.
Science 319: 330–333.
Haughn, G.W., and Somerville, C. (1986). Sulfonylurea-resistant mu-
tants of Arabidopsis thaliana. Mol. Gen. Genet. 204: 430–434.
Hirschberg, J. (2001). Carotenoid biosynthesis in flowering plants. Curr.
Opin. Plant Biol. 4: 210–218.
Holt, N.E., Zigmantas, D., Valkunas, L., Li, X.P., Niyogi, K.K., and
Fleming, G.R. (2005). Carotenoid cation formation and the regulation
of photosynthetic light harvesting. Science 307: 433–436.
Horton, P., Johnson, M.P., Pe ´rez-Bueno, M.L., Kiss, A.Z., and
Ruban, A.V. (2008). Photosynthetic acclimation: Does the dynamic
structure and macro-organisation of photosystem II in higher plant
grana membranes regulate light harvesting states? FEBS J. 275:
Horton, P., and Ruban, A.V. (1992). Regulation of photosystem II.
Photosynth. Res. 34: 375–385.
Horton, P., Ruban, A.V., and Walters, R.G. (1996). Regulation of light
harvesting in green plants. Annu. Rev. Plant Physiol. Plant Mol. Biol.
Jahns, P., Latowski, D., and Strzalka, K. (2009). Mechanism and
regulation of the violaxanthin cycle: The role of antenna proteins and
membrane lipids. Biochim. Biophys. Acta 1787: 3–14.
Johnson, M.P., Pe ´rez-Bueno, M.L., Zia, A., Horton, P., and Ruban,
A.V. (2009). The zeaxanthin-independent and zeaxanthin-dependent
qE components of non-photochemical quenching involve a common
conformational change within the photosystem II antenna in Arabi-
dopsis thaliana. Plant Physiol. 149: 1061–1075.
Kramer, D.M., and Sacksteder, C.A. (1998). A diffused-optics flash
kinetic spectrophotometer (DOFS) for measurements of absorbance
changes in intact plants in the steady-state. Photosynth. Res. 56:
Li, X.P., Bjorkman, O., Shih, C., Grossman, A.R., Rosenquist, M.,
Jansson, S., and Niyogi, K.K. (2000). A pigment-binding protein
essential for regulation of photosynthetic light harvesting. Nature 403:
Li, X.P., Gilmore, A.M., Caffarri, S., Bassi, R., Golan, T., Kramer, D.,
and Niyogi, K.K. (2004). Regulation of photosynthetic light harvesting
involves intrathylakoid lumen pH sensing by the PsbS protein. J. Biol.
Chem. 279: 22866–22874.
Li, X.-P., Phippard, A., Pasari, J., and Niyogi, K.K. (2002). Structure-
function analysis of photosystem II subunit S (PsbS). Funct. Plant Biol.
Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J.P., Gui, L., An, X., and
Chang, W. (2004). Crystal structure of spinach major light-harvesting
complex at 2.72 A˚resolution. Nature 428: 287–292.
Lokstein, H., Tian, L., Polle, J.E.W., and DellaPenna, D. (2002).
Xanthophyll biosynthetic mutants of Arabidopsis thaliana: Altered
nonphotochemical quenching of chlorophyll fluorescence is due to
changes in photosystem II antenna size and stability. Biochim.
Biophys. Acta 1553: 309–319.
Maresca, J.A., Graham, J.E., Wu, M., Eisen, J.A., and Bryant, D.A.
(2007). Identification of a fourth family of lycopene cyclases in pho-
tosynthetic bacteria. Proc. Natl. Acad. Sci. USA 104: 11784–11789.
Mortensen, A., and Skibsted, L.H. (1997). Importance of carotenoid
structure in radical-scavenging reactions. J. Agricult. Food Chem. 45:
Mozzo, M., Dall’Osto, L., Hienerwadel, R., Bassi, R., and Croce, R.
(2008). Photoprotection in the antenna complexes of photosystem II.
Role of individual xanthophylls in chlorophyll triplet quenching. J. Biol.
Chem. 283: 6184–6192.
Mu ¨ller, P., Li, X.P., and Niyogi, K.K. (2001). Non-photochemical
quenching. A response to excess light energy. Plant Physiol. 125:
Mu ¨ller-Moule ´, P., Conklin, P.L., and Niyogi, K.K. (2002). Ascorbate
deficiency can limit violaxanthin de-epoxidase activity in vivo. Plant
Physiol. 128: 970–977.
Niyogi, K.K. (1999). Photoprotection revisited: Genetic and molecular
approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 333–359.
Niyogi, K.K. (2000). Safety valves for photosynthesis. Curr. Opin. Plant
Biol. 3: 455–460.
Niyogi, K.K., Bjorkman, O., and Grossman, A.R. (1997). The roles of
specific xanthophylls in photoprotection. Proc. Natl. Acad. Sci. USA
Niyogi, K.K., Grossman, A.R., and Bjorkman, O. (1998). Arabidopsis
mutants define a central role for the xanthophyll cycle in the regulation
of photosynthetic energy conversion. Plant Cell 10: 1121–1134.
Niyogi, K.K., Shih, C., Soon Chow, W., Pogson, B.J., Dellapenna, D.,
and Bjorkman, O. (2001). Photoprotection in a zeaxanthin- and
lutein-deficient double mutant of Arabidopsis. Photosynth. Res. 67:
Noctor, G., Rees, D., Young, A., and Horton, P. (1991). The relation-
ship between zeaxanthin, energy-dependent quenching of chlorophyll
Lutein Can Replace Zeaxanthin in qE1811
fluorescence, and trans-thylakoid pH gradient in isolated chloro-
plasts. Biochim. Biophys. Acta 1057: 320–330.
North, H.M., De Almeida, A., Boutin, J.P., Frey, A., To, A., Botran, L.,
Sotta, B., and Marion-Poll, A. (2007). The Arabidopsis ABA-deficient
mutant aba4 demonstrates that the major route for stress-induced
ABA accumulation is via neoxanthin isomers. Plant J. 50: 810–824.
Pascal, A.A., Liu, Z., Broess, K., van Oort, B., van Amerongen, H.,
Wang, C., Horton, P., Robert, B., Chang, W., and Ruban, A. (2005).
Molecular basis of photoprotection and control of photosynthetic
light-harvesting. Nature 436: 134–137.
Pagano, A., Cinque, G., and Bassi, R. (1998). In vitro reconstitution of
the recombinant photosystem II light-harvesting complex CP24 and
its spectroscopic characterization. J. Biol. Chem. 273: 17154–17165.
Pecker, I., Gabbay, R., Cunningham, F.X., and Hirschberg, J. (1996).
Cloning and characterization of the cDNA for lycopene beta-cyclase
from tomato reveals decrease in its expression during fruit ripening.
Plant Mol. Biol. 30: 807–819.
deficient mutant of Arabidopsis thaliana possessing normal pigment
composition and xanthophyll-cycle activity. Planta 210: 205–214.
Pogson, B., McDonald, K.A., Truong, M., Britton, G., and DellaPenna,
D. (1996). Arabidopsis carotenoid mutants demonstrate that lutein
is not essential for photosynthesis in higher plants. Plant Cell 8:
Pogson, B.J., Niyogi, K.K., Bjorkman, O., and DellaPenna, D. (1998).
Altered xanthophyll compositions adversely affect chlorophyll accu-
mulation and nonphotochemical quenching in Arabidopsis mutants.
Proc. Natl. Acad. Sci. USA 95: 13324–13329.
Pogson, B.J., and Rissler, H.M. (2000). Genetic manipulation of
carotenoid biosynthesis and photoprotection. Philos. Trans. R. Soc.
Lond. B Biol. Sci. 355: 1395–1403.
Polivka, T., Zigmantas, D., Sundstrom, V., Formaggio, E., Cinque,
G., and Bassi, R. (2002). Carotenoid S-1 state in a recombinant light-
harvesting complex of photosystem II. Biochemistry 41: 439–450.
Ronen, G., Carmel-Goren, L., Zamir, D., and Hirschberg, J. (2000).
An alternative pathway to beta-carotene formation in plant chromo-
plasts discovered by map-based cloning of beta and old-gold color
mutations in tomato. Proc. Natl. Acad. Sci. USA 97: 11102–11107.
Ruban, A.V., Berera, R., Ilioaia, C., van Stokkum, I.H.M., Kennis, J.T.
M., Pascal, A.A., van Amerongen, H., Robert, B., Horton, P., and
van Grondelle, R. (2007). Identification of a mechanism of photo-
protective energy dissipation in higher plants. Nature 450: 575–578.
Ruban, A.V., Lee, P.J., Wentworth, M., Young, A.J., and Horton, P.
(1999). Determination of the stoichiometry and strength of binding of
xanthophylls to the photosystem II light harvesting complexes. J. Biol.
Chem. 274: 10458–10465.
Ruban, A.V., Pascal, A.A., Robert, B., and Horton, P. (2002). Activa-
tion of zeaxanthin is an obligatory event in the regulation of photo-
synthetic light harvesting. J. Biol. Chem. 277: 7785–7789.
Ruban, A.V., Young, A.J., and Horton, P. (1993). Induction of non-
photochemical energy-dissipation and absorbance changes in leaves:
Evidence for changes in the state of the light-harvesting system of
photosystem II in vivo. Plant Physiol. 102: 741–750.
Sacksteder, C.A., Jacoby, M.E., and Kramer, D.M. (2001). A portable,
non-focusing optics spectrophotometer (NoFOSpec) for measure-
ments of steady-state absorbance changes in intact plants. Photo-
synth. Res. 70: 231–240.
Stickforth, P., Steiger, S., Hess, W.R., and Sandmann, G. (2003). A
novel type of lycopene epsilon-cyclase in the marine cyanobacterium
Prochlorococcus marinus MED4. Arch. Microbiol. 179: 409–415.
Tracewell, C.A., and Brudvig, G.W. (2003). Two redox-active beta-
carotene molecules in photosystem II. Biochemistry 42: 9127–9136.
Yamamoto, H.Y., Bugos, R.C., and Hieber, A.D. (1999). Biochemistry
and molecular biology of the xanthophyll cycle. In The Photochemistry
of Carotenoids, H.A. Frank, A.J. Young, G. Britton, and R.J. Cogdell,
eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp.
Yu, B.Y., Lydiate, D.J., Schafer, U.A., and Hannoufa, A. (2007).
Characterization of a beta-carotene hydroxylase of Adonis aestivalis
and its expression in Arabidopsis thaliana. Planta 226: 181–192.
1812 The Plant Cell