:31“.
. \ u x

as :
AV

. hark, m mu:
fig“ 3%...

ans .
..:..,m..,.......ax 3 .n

s
O
a I.‘\q 1 5f.

:1:
. 3.1.1..

I! .i‘!
in)
. . an”... .13»: .0:
Mt, 13:1!- .3:
2 {a . ..
o‘.‘ 11‘ .iIHIItan

.troctsllnuu

5v}...- inhalant.

.Ya‘tnI. If...
. Iv 4 .

 

 

 

“4:23 M.Lrl1BRAFlY
Ll. lC igan State
20% University

 

 

This is to certify that the
dissertation entitled

FUNCTIONAL AND EVOLUTIONARY CHARACTERIZATION
OF ARABIDOPSIS CAROTENOID HYDROXYLASES

presented by
JOONYUL KIM

has been accepted towards fulfillment
of the requirements for the

Ph. D degree in Biochemistry and Molecular
Biology

7 Major Professor’s Signature
MMw/kf / Z, at 363?

Date

 

MSU is an affirmative-action, equal-opportunity employer

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K:lProj/Acc&Pres/CIRC/DateDue indd

 

FUNCTIONAL AND EVOLUTIONARY CHARACTERIZATION OF
ARABIDOPSIS CAROTENOID HYDROXYLASES

By

Joonyul Kim

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry and
Molecular Biology

2008

ABSTRACT

FUNCTIONAL AND EVOLUTIONARY CHARACTERIZATION OF
ARABIDOPSIS CAROTENOID HYDROXYLASES

By

Joonyul Kim

Xanthophylls are a group of more than 500 different oxygenated carotenes that
serve a variety of functions in procaryotes and eucaryotes. Xanthophyll composition is
highly conserved in photosynthetic tissues of higher plants but how changes in
xanthophyll composition occurred in ancestral photosynthetic organisms and why
specific changes have been retained in lineages leading to higher plants remain open
questions. To study on evolution of the xanthophyll biosynthetic pathway, I focused on
the molecular evolution of four Arabidopsis carotenoid hydroxylases (CYP97A3,
CYP97C1, CRTR-Bl and CRTR-BZ) that catalyze key reactions in xanthophyll synthesis.

The protein encoded by CYP97A3 was identified as the primary (it-carotene [3-
ring hydroxylase (Chapter 2). Mutation of C YP97A3 (lut5 locus) caused accumulation of
or-carotene but not a-cryptoxanthin and the major route for lutein synthesis to be
determined. Lutein synthesis occurred by two successive B- and a-ring hydroxylation

from (Jr-carotene give actual sequence of reaction. The susceptibility of a lut5 null mutant

to photooxidation under high light stress is likely due to the massive accumulation of or-

carotene in this mutant.

In Chapter 3, functional divergence of the four carotenoid hydroxylases

(CYP97A3, CYP97C1, CRTR-Bl and CRTR-BZ) were assessed based on their different

in planta substrate specificities and gene expression profile. Generation of a quadruple

mutant in which all four genes are inactive showed these carotenoid hydroxylases

represent the full enzymatic complement in Arabidopsis. Phylogenetic analyses suggested

that the C YP97A3 and C YP97C1 genes were duplicated before the speciation of

Arabidopsis and green algae (of. Chlamydomonas reinhardtii and Ostreococcus tauri)

while duplication of the CRTR-B genes was more recent, after the Arabidopsis/A.

palaestina split. Although the four enzymes exhibit some overlap in activities, most

notably in hydroxylation of the B-ring of a-carotene, the mode of functional divergence

in the two gene pairs appears to be distinct. C YP97 duplicates are strongly coexpressed

but the encoded proteins have distinct in planta substrates, likely due to divergence in

their putative substrate recognition/binding regions. In contrast, the CRT R-B duplicates

are isozymes that show significant expression divergence in reproductive organs.

ACKNOWLEDGMENTS

I am happy to have an opportunity to call over the names of the people one after another
who helped me to continue my Ph. D. Although, only my name is shown on the cover of
the dissertation, I would say that it is impossible to complete my Ph. D. without their
supports.

I deeply thank to my Ph. D. advisor Dr. Dean DellaPenna for his supports and
encouragements during my graduate study. I was lucky to have him who taught me the
way of thinking which was the most critical to continue my career as a researcher. I also
appreciate him to give me a lifelong word, “Hope for the best, prepare for the worst!” I
would also thank to my committee members, Drs. James J. Smith, Kenneth Keegstra,
Shelagh Ferguson-Miller and Gregory Zeikus for their advices both humanly and
scientifically.

Some former and current members of the DellaPenna lab should be
acknowledged. They are Tian Li, Laura Ullrich, Maria Magallanes, Hiroshi Maeda, Dana
Chris, Naoko Kobayashi, and Wan Song. I also thank to Dr. Jongmin Nam in CALTECH,
a lifelong fiiend and a collaborator to let me jump into a study on molecular evolution.

And also many thanks go to several faculty members in the Biochemistry and Molecular

Biology (BMB) and Plant Biology departments for their advices and supports. They are
Drs. Daniel Jones, Shin-Han Shiu, Kaillathe Padmanabhan, Michael Feig, and Robert
Last. I also appreciate to my graduate fellows in the BMB department and Plant Research
Labs for their supports and friendship, especially Hoo-Sun Chung and Colleen Doherty.
Many Korean friends helped me to overcome homesickness. They are Namjoon Kim,
Seunghoon Song, Sangheuk Park, Seunghwan Yang, Abe K00, and Lace Choi.

Most importantly, I have to give a special mention for the support given by my
family. Especially, I would like to express my gratitude to my wife, Sangeun for her love

and patience.

E’éP—I EEMIM are %-?_43l°i 2.51s! $E‘elP—l 7t; ERIE, JEII'.

EEJOlI 74W 9’38- mafi'alflll élMSll Dl%§ ESL—IQ.

TABLE OF CONTENTS

 

 

 

 

 

 

 

LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xii
CHAPTER 1: LITERATURE REVIEW 1
1.1 General property and biological function of carotenoids 2
1.1.1 Mechanic rigidity 2
1.1.1.1 Membrane fluidity 2

 

 

 

 

 

1.1.2 The unique electron cloud of the carotenoid polyene chain -------- 3
1.1.2.1 Light absorption 3
1.1.2.2 Energy transfer between carotenoids and chlorophylls -------- 3

1.1.3 Versatility in carotenoid oxidative cleavage reactions --------- 4
1.1.3.1 Chromophores 4
1.1.3.2 Aroma and flavors 4
1.1.3.3 Hormones 5
1.1.3.4 Fungal growth regulators 5

1.2 Xanthophylls, structural and functional components of light-harvesting

 

 

 

 

 

 

complexes (LHC) in photosynthetic eucaryotes 6

1.3 The xanthophyll biosynthetic pathway in higher plants 8

1.3.1 From colorless to colorful carotenoids 9
1.3.2 From acyclic to cyclic carotenoids 10
1.3.3 From nonoxygenated to oxygenated carotenoids 10
1.3.4 Molecular characterization of carotenoid hydroxylases 11

1.4 Carotenoid hydroxylases as tools to understand evolution of the

 

xanthophyll biosynthetic pathway 13
1.5 General characteristics of P450 and non-heme type carotenoid hydro-

 

xylase gene families 14
1.5.1 Heme-containing cytochrome P450 monooxygenase genes (P450)-- 15

 

1.5.2 Non-heme di-iron monooxygenases (non-heme) 16

 

1.6 Goals of my research 17

vi

CHAPTER 2: DEFINING THE PRIMARY ROUTE FOR LUTEIN SYNTHESIS
IN PLANTS: THE ROLE OF ARABIDOPSIS CAROTENOID B-RING

 

 

 

 

 

HYDROXYLASE CYP97A3 21
2.1 Summary 22
2.2 Introduction 22
2.3 Results 24
2.3.1 The lut5 mutation is in Arabidopsis CYP97A3 and causes
accumulation of high levels of a-carotene 24
2.3.2 a-carotene is incorporated into lut5-1 photosystems ---------- 25
2.3.3 The impact of mutating CYP97-type carotenoid hydroxylases on
the expression of other carotenoid biosynthetic enzymes ------------ 26
2.4 Discussion 27

 

2.4.1 At least four enzymes are involved in carotenoid hydroxylation --- 27
2.4.2 CYP97A3, the new type of B-ring hydroxylase in Arabidopsis ---- 28

 

 

 

 

2.4.3 A model for lutein biosynthesis and regulation in plants ---------- 29

2.5 Material and Methods 34

2.5.1 Plant materials 34

2.5.2 TaqMan Real-Time PCR Assays 34
2.5.3 Isolation of thylakoid membranes and nondenaturing polyacryl-

amide gel electrophoresis (PAGE) 35

2.5.4 Structural determination of an unknown monohydroxy or-carotene-35

CHAPTER 3: MOLECULAR EVOLUTION OF CAROTENOID HYDRO-

 

 

 

 

 

 

 

 

XYLASES IN ARABIDOPSIS 46
3.1 Summary 47
3.2 Introduction 48
3.3 Results 49

3.3.1 Defining the full complement of caroteonid hydroxylases in
Arabidopsis 49
3.3.2 Gene duplication of the C YP97 and CRT R-B genes 50
3.3.3 Functional divergence of Arabidopsis CYP97 and CRTR-B
enzyme pairs 51
3.3.3.1 Substrate divergence 51
3.3.3.2 Expression divergence 55

 

3.3.4 The impact of functional divergence on plant adaptation under

high light 55

 

vii

 

3.4 Discussion 57

3.4.1 Gene duplication and in vivo activity of carotenoid hydroxylase

 

 

 

 

 

 

 

 

 

 

genes 57
3.4.2 The evolution of [Fl-xanthophyll synthesis 59
3.4.3 The evolution of a-xanthophyll synthesis 60
3.5 Material and methods 66
3.5.1 The CYP97 and CR TR-B genes used for phylogenetic analyses ----66
3.5.2 Sequence alignments and computational analyses 66
3.5.3 Plant material and pigment analyses 68
3.5.4 Expression data analyses 68
3.5.5 Colinearity of chromosomal segments 69
3.5.6 Measurement of in vivo chlorophyll fluorescence and C02
fixation rates 69
CHAPTER 4: FUTURE WORK 99
4.1 Fitness test of lut5-1 (a3) mutant under low and light condition ---------- 100
4.2 Identification of P450-type carotenoid hydroxylase in C. reinhardtii -------- 100
4.3 In-depth phenotypic analyses of crtr-bI and crtr-b2 mutants ---------- 101

4.4 Identification of the carotenoid cleavage enzymes recognizing the e-ring -- 102

 

 

APPENDIX 103
Supplementary data 104
BIBLIOGRAPHY 126

 

viii

LIST OF TABLES

Table 1 Pigment composition in the indicated lineages of photosynthetic eukaryote

 

lineages 1 8

 

Table 2 Leaf tissue carotenoid composition in the indicated genotypes 36

Table 3 Carotenoid composition in photosystems (PS I holocomplex and PS II core com-

 

plex) of the indicated genotypes 38

Table 4 Carotenoid composition in the green and white-colored seedlings which are the
progeny from two different parental genotypes, b1b1b2b2c1c1A3a3 and
b1b1b2b2C1c1a3a3, respectively 70

 

Table 5 List of CYP97 homologs used for constructing a Neighbor-joining tree ------- 71

Table 6 List of CRTR-B homologs used for constructing a Neighbor-joining tree ----- 73

Table 7 Carotenoid composition in the leaves of the two wild types (Col-O and Ws) and

 

seven informative genotypes 75

Table 8 Carotenoid composition in the seeds of the two wild types (Col-0 and W3) and

 

seven informative genotypes 77

Supplementary data Table 1 Synonymous substitutions per site (Ks), nonsynonymous

 

substitutions per site (Ka) and corresponding confidence intervals (CL) 122

LIST OF FIGURES

 

Fig. l. Xanthophyll synthesis in Arabidopsis thaliana 20

Fig. 2. Pathway showing all possible routes to major xanthophyll syntheses from

 

lycopene in Arabidopsis 40

Fig. 3. HPLC chromatograms of carotenoids extracted from leaves of the indicated
mutant genotypes (left panel) and comparison of UV-visible absorption spectra of

 

unknown peaks to those of zeinoxanthin and a-carotene 42

Fig. 4. Non-denaturing gel electrophoretic separation of pigmentzprotein complexes from

 

thylakoid membranes of the indicated genotypes 44

Fig. 5. Expression of carotenoid biosynthetic genes in lutI-4, lut5-I and lut5-Ilut1-4
relative to WT 45

 

Fig. 6. Neighbor-joining trees of the C YP97 (A) and CRT R-B (B) gene families ----- 79

Fig. 7. The exon-intron structures of CRTR-B genes (A) and the colinearity of chro-
mosomal segments including CR TR-B genes (B) in Arabidopsis, poplar and rice ---- 82

Fig. 8 Sliding window analyses of CYP97 (A) and CRTR-B pairs (B) and the site-
specific profile of cluster-specific functional divergence (Type II) between the CYP97A
and CYP97C clades (C) 86

 

Fig. 9 Two homology models (open and closed conformations) of CYP97A3 -------- 92

Fig. 10 Expression divergence of duplicates in each C YP97 and CRT R-B gene pair in (A)

 

the indicated tissues and (B) stress conditions in leaf 94

Fig. 11 Non-photochemical quenching (N PO) and maximum photosynthetic efficiency of
PSII (Fv/Fm) in the indicated genotypes 95

 

Fig. 12 CO; fixation rates of WT and a3 before and after ten hour high light
exposure 97

 

Fig. 13 Two possible scenarios for the evolution of (it-xanthophyll biosynthetic

 

pathway 98
Supplementary data Fig. 1 HPLC chromatograms of the indicated mass ions ------- 104

Supplementary data Fig. 2 Whole-plant phenotype of WT and lut5-I under high light
exposure 106

 

Supplementary data Fig. 3 Amino acid alignments of (A) CYP97 homologs and CRT R-

 

B homologs used for neighbor-joining tree construction 107

Supplementary data Fig. 4 C02 fixation rates of WT and a3 mature leaves as a function
of a light level 124

 

Supplementary data Fig. 5 Signal intensity of CRTR-BI and CRTR-BZ obtained from

microarray data obtained from developing seeds 125

 

xi

ABA
a—carotene
a—xanthophylls
B-xanthophylls
[LB-carotenoids
B,e-carotenoids
B-carotene
B-cyclase
e-cyclase

CCD

Chl a

Chl b

GGPP

LHC

MRCA
Non-heme
P450

PC

Polyene
Xanthophyll
WT

LIST OF ABBREVIATIONS

Abscisic acid

B,e-carotene

or-carotene derived xanthophylls
B-carotene derived xanthophylls
B-carotene derived carotenoids
a-carotene derived carotenoids
[3,B-carotene

lycopene B-ring cyclase
lycopene e-ring cyclase
Carotenoid-cleavage dioxygenase
Chlorophyll a

Chlorophyll b

Geranylgeranyl pyrophosphates
Li ght-harvesting complex

Most recent common ancestor
Non-heme di-iron

Cytochrome P450

Photosystem core complex
Poly-unsaturated organic compound
Oxidized carotene

Wild type

xii

CHAPTER 1

LITERATURE REVIEW

1.1. General property and biological function of carotenoids

Carotenoids are a group of more than 700 red, yellow and orange colored
pigments and are some of the most widespread of all natural products (Straub, 1987; Kull
and Pfander, 1995). These features are achieved by Nature’s adoptation of two simple and
economical strategies, making use of a universal pathway, the isoprenoid pathway, for
carotenoic synthesis and elaborating various downstream modification steps in a lineage-
specific fashion (Umeno et al., 2005). The carotenoid backbone is a hydrocarbon chain
with conjugated double bonds, resulting from a series of condensations of five carbon
isoprene building blocks. Most of carotenoids have a C40 backbone resulting from the
condensation of two geranylgeranyl pyrophosphates (GGPP, C20) and only a few
eubacteria produce Cgo-based carotenoids (Armstrong, 1997; Umeno et al., 2005).
Chemical modifications leading to the diversity of carotenoids in nature include different
types of ring cyclizations, oxygenations, glycosylations, prenylations and oxidative
cleavages. This tremendous structural diversity that is widely distributed in nature has
presumably evolved in relation to a number of independent and interdependent
carotenoid biological functions (Vershinin, 1999). In this chapter I summarize the
biological functions of carotenoids derived from their general mechanical and chemical

properties.

1.1.1. Mechanic rigidity
1.1.1.1. Membrane fluidity The polyene structure of carotenoids provides an extremely
rigid backbone in a lipid-soluble environment. This mechanic rigidity may be the basis of

carotenoid functions in controlling membrane fluidity (Rohmer et al., 1979). The effects

of carotenoids on the physical properties of saturated phosphatidylcholine membranes
were also studied with an electron paramagnetic resonance spin-labeling method
(Wisniewska et al., 2006). These effects were monitored at the membrane center as a
function of the amount of the carotenoid added to the sample and as a ftmction of the
critical temperature for fluid-phase transition. This study showd that carotenoids,
especially the dipolar, terminally dihydroxylated carotenoid lutein decreased membrane
fluidity and increased the hydrophobicity of the membrane interior.

1.1.2. The unique electron cloud of the carotenoid polyene chain

1.1.2.1. Light absorption Oxygenic photosynthesis is an ancient mechanism in
cyanobacteria, red algae, green algae, and terrestrial plants that uses light and molecular
oxygen to produce ATP and reducing power to drive carbon fixation (Blankenship, 1992;
Nelson and Ben-Shem, 2004). The first step in oxygenic photosynthesis is absorption of
light by pigments (e. g. chlorophylls and carotenoids) in the photosynthetic complexes. In
the light-harvesting complexes (LHCs) and photosystem core complex (PCs) of
photosynthetic organisms, carotenoids have slightly different absorption spectra from
chlorophylls, the major light-harvesting pigments. This allows the photosynthetic
apparatus to maximize light absorption in a gap of the chlorophyll absorption spectra
(420600 nm) thus increasing the active spectral range of photosynthesis. In
nonphotosynthetic tissues of plants and animals, this feature provides coloration for
sexual displays, pollinator attraction and seed dispersal. Many yellow and orange flowers
and fruits of plants (Howitt and Pogson, 2006) and various red and orange coloration of
male fish and birds are prominent examples (Olson and Owen, 1998).

1.1.2.2. Energy transfer between carotenoids and chlorophylls The unique

arrangement of n-electrons in the carotenoid polyene chain allows bidirectional excitation
energy transfer between carotenoids and chlorophylls. This property is particularly
important for photosynthesis and allows the photosynthetic apparatus to be optimized for
light usage: (1) the captured light energy in LHCs is channeled to PCs by virtue of the
slightly higher excited state (81) of carotenoids compared to chlorophylls in the PCs
(Cogdell and Gardiner, 1993; Liu et al., 2004) and, (2) carotenoids protect chlorophylls
from excessive light energy by quenching the excited triplet of chlrophylls directly (Ma et
al., 2003; Holt et al., 2005). Many photosynthetic eukaryotes developed these
photoprotection mechanisms using carotenoids via xanthophyll cycles (Young and Frank,
1996)

1.1.3. Versatility in carotenoid oxidative cleavage reactions The long polyene chain
of carotenoids can be cleaved enzymatically to produce a diverse range of oxidative
cleavage products.These carotenoid-derived molecules, so called apocarotenoids, have
been identified as bioactive molecules in plants, fungi and animals (Auldridge et al.,
2006a).

1.1.3.1. Chromophores In animals, the chromophore of rhodopsins, retinal, is a
cleavage product of B-carotene and provide the basis for visual reception. The light-
dependent cis-trans isomerization of retinal induces a conformational changes in
rhodopsin which is transmited to the nerve cell (Palczewski, 2006). Bixin in bixa seed
and crocin in saffron stigma are the cleavage products of lycopene and zeaxanthin,
respectively, which are extensively used as food and cosmetic additives for coloration
(Bouvier et al., 2003; Castillo et al., 2005).

1.1.3.2. Aroma and flavors B-ionone, B-cyclocitral, damascenones, and theaspirone

contribute to the flavor and aroma of flowers and foods. For example, both B-ionone and
damascenone are the key fragrant-contributing compounds in flowers (Demole et al.,
1970). In fact, the sweet floral smells present in black tea, aged tobacco, grape, and many
fruits are due to in large part to apocarotenoids (Auldridge et al., 2006a).

1.1.3.3. Hormones Oxidaized derivatives of retinal are involved in vertebrate
morphogenesis, growth, cellular differentiation, and tissue homeostasis by receptor-
mediated signal transduction pathways (Mark et al., 2006). In plants, abscisic acid (ABA)
is the best studied carotenoid—derived phytohormone, having vital functions in plant
adaptation to stressful environments by regulating stomatal aperture and the expression of
stress responsive genes, and in plant development such as seed maturation, germination
and seedling growth (Leung and Giraudat, 1998). In addition, lateral branching
phenotypes shown in two Arabidopsis mutants (max3 and max4) which are dirsupted in
specific carotenoid-cleavage dioxygenases (CCDs), suggest a new class of carotenoid-
derived mobile signaling molecules involving in controlling branching. Orthologous
mutants in pea, petunia and rice exhibit increased branching and some can be restored to
wild-type branching if the mutant is grafted to wild-type tissues (Auldridge et al., 2006a),
indicating that this carotenoid derived signaling molecule is conserved through
angiosperrn.

1.1.3.4. Fungal growth regulators Arbuscular mycorrhizae represent the most
widespread symbiosis on the earth and form in association with the roots of more than
80% of land plants. Arbuscular mycorrhizae (AM) fungi facilitate the uptake of
phosphate, by plants, and in return obtain carbohydrates from their hosts. Massive

accumulation of apocarotenoids including mycorradicin and cyclohexenone derivatives is

initiated during root colonization by AM fungi. For instance, strigolactone, previously
isolated as a seed-germination stimulant for root parasitic weeds, acts as a chemical
signal for arbuscular mycorrhizal fungi during presymbiotic stages. The significance of
apocarotenoids in AM symbiosis has been shown by experiments with maize mutants
deficient in carotenoid biosynthesis. The maize y9 mutant in which a carotenoid
isomerase is disrupted showed remarkable decrease in the mycorrhizal colonization rate

and in exudation of strigolactones (Matusova et al., 2005; Akiyama, 2007).

1.2. Xanthophylls, structural and functional components of light-

harvesting complexes (LHC) in photosynthetic eukaryotes

The more than 700 structurally distinct carotenoid compounds are classified into
two subgroups; the carotenes, acyclic or cyclic hydrocarbons and the xanthophylls,
oxygenated derivatives of carotenes. The introduction of oxygen functional groups into
carotenes seems to be a very ancient event that has been under strong selective pressure
during evolutionary time because photosynthetic eukaryotes produce xanthophylls but no
organism has been identified that only synthesizes carotenes (Table 1). One important
role of xanthophylls is their association with LHC apoproteins to form fimctional light-
harvesting complexes. In vitro LHC reconstitution studies with various carotenes and
xanthophylls revealed that xanthophyll oxygen as a hydroxyl or epoxi is critical for LHC
structure and function (Ruban et al., 1999; Bassi and Cafl‘arri, 2000; Phillip et al., 2002).
For example, lutein, the predominant xanthophyll in higher plants has been shown not
only to induce correct folding of the LHC II apoprotein (Paulsen et al., 1993) but also to

be involved in stability of LHC II trimer (Lokstein et al., 2002). Interestingly, the

function of xanthophylls in LHC assembly and function is conserved in photosynthetic
eucaryotes but significant structural diversity still exists and fulfills their function in
different organisms. For example, red and green algae have a variety of xanthophylls in
which the backbone is or-carotene or B-carotene (Table 1), but most brown algae such as
chromophytes and dinophytes only produce B-carotene derived xanthophylls (Alberte and
Andersen, 1986; Britton, 1998; Pascal et al., 1998). Although LHC apoproteins are one of
the highly structurally conserved proteins between taxa with having a flexibility to
functionally bind xanthophylls (Bassi and Caffarri, 2000; Grabowski et al., 2001),
different binding affinities for each xanthophyll in each LHC binding site suggests that
the diversity of xanthophyll is somehow related and perhaps correlated to the evolution of
LHC apoproteins.

LHC 11 associated with three different xanthophylls (lutein, violaxanthin and
neoxanthin) is the most abundant integral membrane protein in higher plant chloroplasts
and exists as a trimer which binds half of the chlorophyll molecules in the plastids.
Beside light-harvesting, LHC II has also been shown to function in the non-radiative
dissipation of excess excitation energy under high light stress. Recently, the crystal
structure of spinach LHC II was determined with high enough resolution (2.72 A) to
assign all pigments including chlorophylls and xanthophylls at the atomic level (Liu et al.,
2004). In the three dimensional structure, the position of pigments provide a rationale for
how the rate of singlet excitation energy transfer between xanthophylls and chlorophylls
is correlated: two luteins are found to be in favorable orientations and distances to Chl a
for efficient singlet energy transfer fiom lutein to Chl a. One neoxanthin is found to

transfer its energy mostly towards Chl b. Therefore, lutein and neoxanthin may fimction

as effective accessory light-harvesting antennae, absorbing light in the blue-green spectral
region as a complement to Chl a/b absorbing in the red region. The photoprotective role
by the xanthophyll cycle could be also deduced by pr0posing an efficient non-

photochemical energy —transfer pathway.

1.3. The xanthophyll biosynthetic pathway in higher plants

The C5 building blocks for carotenoid synthesis are isopentenyl diphosphate (IPP)
and its isomer, dimethylallyl diphosphate (DMAPP). In higher plants, two independent
pathways to synthesize these molecules are located in separate intracellular compartments,
the cytosol and the plastid. In the cytosol, IPP is derived from the mevalonic acid
pathway that starts from the condensation of acetyl-CoA and is used for the biosynthesis
of sterols, sesquiterpenes and triterpenoids (Qureshi and Porter, 1981) with few
exceptions (Dudareva et al., 2005). In plastids, IPP is formed from pyruvate and
glyceraldehyde 3-phosphate via the 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway
and utilized for the synthesis of carotenoids (Lichtenthaler, 1999; Rohmer, 1999). In
nature, more than two third of carotenoids are xanthophylls (>500) but the initial steps of
xanthophyll biosynthesis leading to B—carotene are believed to have been established in
very ancient organisms based on the widespread distribution of these compounds in both
eubacteria and eukaryotes.

In higher plants, xanthophyll composition is highly conserved, generally consisting
of three predominant xanthophylls, lutein, violaxanthin and neoxanthin. How changes in
xanthophyll composition occurred in ancestral photosynthetic organisms and why

specific changes have been retained in lineages leading to higher plants remain open

questions. In practical terms, understanding synthesis of the major xanthophylls in plants
is an essential component for increasing xanthophyll production or creating new
xanthophyll molecules by modifying the pathway (Britton, 1998). The aim of the
following section of this literature review is to provide an overview of the current state of
knowledge about xanthophyll biosynthesis in higher plants. Fig. l is a schematic diagram
to illustrate the synthesis of major xanthophylls in higher plants.

1.3.1. From colorless to colorful carotenoids All C40 backbone carotenoids are derived
from phytoene, a colorless carotenoid. The formation of a phytoene is achieved from the
condensation of two GGPP molecules catalyzed by phytoene synthase, a 40 kDa protein
encoded by PSY in plants and crtB in bacteria. There is significant amino acid identity
between PSY and crtB, suggesting the functional conservation of phytoene synthase was
established in a very early ancestor. Phytoene synthase in pepper could be partially
purified fi'om a multiprotein complex containing other biosynthetic enzymes required for
the preparation of GGPP, the substrate for phytoene synthase (Dogbo et al., 1988),
indicating substrate channeling for phytoene synthesis in the multiprotein complex.
Phytoene is oxidized to the red colored compound lycopene by the introduction of four
symmetric double bonds in its polyene chain. Each double bond is introduced in the cis
configuration and isomerized to the trans configuration by a pair of carotenoid isomerase
(Isaacson et al., 2002; Park et al., 2002) to yield all trans lycopene, which is the prefered
substrate for the next reaction step, lycopene cyclization. In higher plants, the two distinct
reaction steps from phytoene leading to lycopene, desaturation and isomerazation are
catalyzed by two desaturases encoded by PDS and ZDS, and two isomerases encoded by

Z-ISO and CRTISO. Interestingly, PDS and ZDS are unrelated to the bacterial desaturase,

but CRTISO appear to arise from a progenitor bacterial desaturase (Giuliano et al., 2002;
Isaacson et al., 2002; Park et al., 2002; Li et al., 2007).

1.3.2. From acyclic to cyclic carotenoids Cyclization of lycopene is a key step in
generating carotenoid diversity as it marks a branch point to two major cyclic carotenoid
groups: the B,B- and [3,e-carotenoids. [3,[3-carotenoids (ii-carotene derived carotenoids)
contain two identical B-rings formed by the symmetrical action of the B-ring cyclase (B-
cyclase) whereas [Le-carotenoids (or-carotene derived carotenoids) contain two different
ring structures ([3 and a) formed by the action of the B-cyclase and e-cyclase. B-rings
contain a double bond in conjugation with the polyene chain which results in a rigid ring
structure with only one conformation. In contrast, the a-ring double bond is not in
conjugation and thus has relatively free rotation around the C6'—C7' carbon. Unlike the
ubiquitous B-carotene derived carotenoids which occur in archaea, eubacteria and plants,
(Jr-carotene derived carotenoids are found exclusively in the green plant lineage (Adams
et al., 1993; Schagerl and Pichler, 2000; Yoshii et al., 2004), red algae (Marquardt and
Hanelt, 2004b), and one extant prochlorophyte (a cyanobacterium) (Partensky et al.,
1993; Stickforth et al., 2003). Therefore, from an evolutionary perspective, e-ring
formation and modifications in the (it-carotene derived branch should postdate those of [3-
rings in the B-carotene derived branch in evolutionary time and would be expected to
have evolved only in this subgroup of s-ring carotenoid containing organisms. Plant 13-
and e-cyclases are the archetypal monomeric lycopene cyclases encoded by LYCB and
LYCE, respectively. Both enzymes show high similarities in their amino acid sequence

and it is very likely they originated from duplication of a common ancestral gene

(Krubasik and Sandmann, 2000).

1.3.3. From nonoxygenated to oxygenated carotenoidsThe most common and varied
carotenoids among the hundreds of carotenoids are those that have cyclic end groups
containing at least one oxygen function. The most commonly encountered oxygen
function is a hydroxyl group at C3 (Britton, 1998) because hydroxylation at this position
is the initial step in converting carotenes to xanthophylls in eubacteria, fungi and plants.
Hydroxylation of the rings of a-carotene and B-carotene is catalyzed by a class of
carotenoid hydroxylases. Production of B-carotene derived xanthophylls (B-xanthophylls)
require two B-ring hydroxylations while or-carotene derived xanthophylls (or-
xanthophylls) requires one [3- and one e-ring hydroxylation.

Two successive ring hydroxylations of B-carotene and (it-carotene give rise to the
dihydroxyl carotenoids lutein and zeaxanthin, respectively. Lutein is the most abundant
carotenoid in plants, accounting for over 50% of the carotenoids in Arabidopsis leaves
while zeaxanthin is only accumulated transiently under stress conditions. The major B-
xanthophylls are violaxanthin and neoxanthin resulting from firrther ring modifications of
zeaxanthin such as epoxidation and formation of allene group. These account for
approximately 30% of total carotenoids in Arabidopsis. Experiments with Arabidopsis
carotenoid biosynthetic mutants that have altered xanthophyll compositions make clear
this conserved composition of lutein, violaxanthin and neoxanthin is the most
functionally adaptive xanthophyll combination (Lokstein et al., 2002; Tian et al., 2003;
Tian et al., 2004a; Dall'Osto et al., 2006), however, how this specific composition was
established and maintained over evolutionary time remains an open question.

1.3.4. Molecular characterization of carotenoid hydroxylases Many carotenoid

hydroxylases have been identified across phyla and all of them showed strong B-ring

ll

hydroxylase activity with the exception of two e-ring hydroxylases identified in
Arabidopsis and rice. With very few exceptions (Blasco et al., 2004; Alvarez et al., 2006),
these B-ring hydroxylases have been invariably characterized as members of the non-
heme di-iron (non-heme) type carotenoid hydroxylases. Non-heme B-ring hydroxylases
are further categorized into at least three subgroups (plant and green algal, non-
photosynthetic bacterial, cyanobacterial groups) based on their primary structures. The
catalytic mechanism using iron coordinated by histidine residues is the same in all three
groups (Tian and DellaPenna, 2004), suggesting the existence of the common ancestral
enzyme before the eubacteria and eucaryote split.

Molecular characterizations of two e-ring hydroxylases proved that cytochrome
P450 (P450) type enzymes are another class of carotenoid hydroxylase in higher plants.
Tian, et. a1 (2004) determined the LUTl locus responsible for e-ring hydroxylation by
positional cloning. The encoded protein was a P450 type carotenoid hydroxylase
(CYP97C1) and suggested that the mechanism of s-ring hydroxylatibn evolved
independently from that of non-heme type enzymes (Tian et al., 2004b). In rice,
CYP97C2, an ortholog of Arabidopsis CYP97C1, has also shown to be an e-ring
hydroxylase (Quinlan et al., 2007).

Although three carotenoid hydroxylases had been identified in Arabidopsis (two
non heme oxygenases and CYP97C1) it could not be concluded that these three genes
represent the full complement of carotenoid hydroxylases in Arabidopsis. When a null
LUTI mutant allele (lull-3) was introduced into a Arabidopsis mutant background also
disrupted for the two non-heme enzymes, carotenoid hydroxylation activity was still

present, suggesting that at least one additional unknown carotenoid hydroxylase activity

12

existed in vivo (Tian et al., 2004b). The isolation and characterization of this fourth
carotenoid hydroxylase activity became the major focus of my thesis research.

The fact that two P450 types B-ring hydroxylases had been identified in
eubacteria and fungi (Blasco et al., 2004; Alvarez et al., 2006) suggested that cytochrome
P450 (P450) type [ii-ring hydroxylase might also exist in higher plants. Such a B-ring
hydroxylase activity structurally unrelated to non-heme type enzyme had been suggested
based on molecular genetic studies (Tian et al., 2003; Kim and DellaPenna, 2006): an
Arabidopsis plant in which all non-heme type B-ring hydroxylases are disrupted (crtr-
b1 crtr-bZ hearafier b1 b2) still produced B-xanthophylls up to 20% of the wild type (WT)

leveL

1.4. Carotenoid hydroxylases as tools to understand evolution of the

xanthophyll biosynthetic pathway

The ftmctional diversity of xanthophylls produced by plants has presumably
evolved in relation to evolution of the xanthophyll biosynthetic pathway. Xanthophylls
accumulate in nearly all types of plastids, and are thus found in most plant organs and
tissues where they perform several independent and interdependent functions.
Xanthophylls are predominant in photosynthetic tissues, accounting for about 80% of
total carotenoids in an Arabidopsis leaf. Studying the evolutionary history of carotenoid
hydroxylases is one of the most appropriate approaches to understand how xanthophyll
biosynthesis and diversity was established because the reaction catalyzed by this enzyme
is the first and key step in xanthophyll biosynthesis (Fig. 1). Work to date, including my

Ph. D work demonstrated that four carotenoid hydroxylase genes are present in

13

Arabidopsis: two P450-type (e.g. C YP97A3 and CYP97C1) and two non-heme type (e.g.
CRT R-BI and CRTR-BZ) genes (Tian and DellaPenna, 2004; Kim and DellaPenna, 2006).
The two carotenoid hydroxylases in each class have significant amino acid identity to
each other, implying each gene family member occurred by gene duplication and
subsequent functional divergence. In addition to Arabidopsis, the rice orthologs
CYP97A4 and CYP97C2, have 49% amino acid identity (Quinlan et al., 2007) and two
non-heme type enzymes in tomato (75%), pepper (71%) and saffron (77%) were also
functionally demonstrated to be carotenoid hydroxylases (Bouvier et al., 1998; Castillo et
al., 2005; Galpaz et al., 2006). With these experimental data, firrther molecular
evolutionary analyses such as detailed gene phylogeny and inference of an ancestral
enzyme may provide insight into how the function of each gene evolved. Specifically,
comparison of substrate specificity and gene expression between homologous genes
make it possible to delineate the mode of functional divergence from a common ancestral

gene.

1.5. General characteristics of P450 and non-heme type carotenoid

hydroxylase gene families

Gene families arise through a process of duplication of an ancestral gene
followed by fimctional divergence (e.g. subfunctionalization or neofunctionalization) or
pseudogenization (e.g. nonfunctionalization). Gene families are a basis for classifying
proteins based on amino acid similarity, but their broader biological significance is less
clear. Our growing knowledge of complete genome sequences and molecular genetics in

several organisms now allow us to understand the evolutionary history of gene families

14

such as gene birth-and-death and functional divergence (Zhang, 2003; Taylor and Raes,
2004; Nei and Rooney, 2005).

Carotenoid hydroxylase genes can be used as a test case to attempt to reach some
general conclusions about the evolution of multigene families because the four known
carotenoid hydroxylases show functional conservation and divergence of their activities
in viva (Tian et al., 2003). Here, I describe the general molecular characteristics of P450
and non-heme type enzymes as a preface for understanding the molecular evolution of
carotenoid hydroxylase genes which will be discussed in more detail in Chapter 3.

1.5.1. Heme-containing cytochrome P450 monooxygenase genes (P450) The
cytochrome P450 monooxygenase (P450) gene family is the one of the largest
superfarnilies of divergent genes encoding ~1% of the gene complement in Arabidopsis
and rice. (Werck-Reichhart et al., 2002; Nelson et al., 2004). Proteins encoded by P450
genes are heme—thiolate enzymes that catalyze monooxygenation of a variety of
hydrophobic substrates. Catalysis is based on the activation of molecular oxygen with

insertion of one of its atoms into the substrate and reduction of the other to form water.

RH + 02 + NADPH+H+ H ROH+H20+ NADP+

(R= substrate, ROH= product)

The substrates of plant P4503 are diverse, including the precursors of membrane
sterols, structural polymers and bioactive secondary metabolites such as pigments,
antioxidants and defense compounds. P4503 are also involved in biosynthesis and

catabolism of hormones and signaling molecules, thus contribute to the control of

hormone homeostasis. In addition to their physiological substrates, exogenous molecules
such as pesticides and pollutants are usually detoxified in an organism by P4503 (Werck-
Reichhart et al., 2002).

Interestingly, known substrates are generally classified along with gene phylogeny
of P4503, suggesting the possibility to use P4503 as markers. The CYP97 subfamily,
which includes carotenoid hydroxylase genes, is a deep branch of the P450 gene
phylogeny in Arabidopsis (Werck-Reichhart et al., 2002), indicating divergence of the
CYP97 clade is relatively ancient compared to other P450 clades. Although CYP97
orthologs in other organisms such as nonvascular plants and green algae have not yet
been functionally characterized, the function of CYP97 genes are believed to be
conserved through plants based on the ubiquitous distribution of their product, lutein. At
minimum, the function of C YP97 is very likely to be conserved between monocotyledon
and dicotyledon because the rice orthologs, CYP97A4 and CYP97C2 showed the same
enzymatic activities as Arabidopsis CYP97A3 and CYP97C1 (Quinlan et al., 2007).
1.5.2. Non-heme di-iron monooxygenases (non-heme) With few exceptional cases
(Blasco et al., 2004; Alvarez et al., 2006), all B-carotene B-ring hydroxylases in
eubacteria and plants were characterized as a non-heme di-iron monooxygenase (non-
heme type), having conserved histidine signature histidine motifs originally identified in
membrane fatty acid desaturases. These enzymes require iron, ferredoxin, and ferredoxin
oxidoreductase for activity and all ten of the conserved iron-coordinating histidines are
required for activity (Tian and DellaPenna, 2004). Arabidopsis, pepper, tomato and
saffron contain more than one member of the non-heme CRTR-B family that generally

have identical substrates (ii-carotene) but differ in their gene expression patterns. In

16

Adonis aestivalis, two of three CRTR-B homologs have diverged in enzymatic activity
and are ketolases that are preferentially expressed in flowers (Cunningham and Gantt,

2005)
1.6. Goals of my research

Quantitative measurement to determine how molecular evolution of each
carotenoid hydroxylase gene family member contributed to the evolution of xanthophyll
biosynthetic pathway in plants is still a difficult task. My research aim was to develop a
comprehensive understanding of the mode of functional divergence in each carotenoid
hydroxylase gene pair and its biological impact on Arabidopsis. To reach this goal,
determining the full complement of carotenoid hydroxylases in Arabidopsis was a
prerequisite. Chapter 2 describes the identification of the primary or-carotene B-ring
hydroxylase, which represents the last uncharacterized carotenoid hydroxylase in
Arabidopsis. Chapter 2 also provided data for the primary route and metabolic channel
for lutein biosynthesis to be proposed. In Chapter 3, the function and molecular evolution
of each carotenoid hydroxylase gene in Arabidopsis is investigated. In Chapter 4, I
summarize future work that could provide additional insight into the evolution of

xanthophyll synthesis in photosynthetic eucaryotes.

Table 1 Pigment composition in the indicated lineages of photosynthetic eucaryotes.
Lutein derivatives include prasinoxanthin, loroxanthin, siphonaxanthin derivatives and
lutein 5,6-epoxide etc. The presence and absence of a pigment in the indicated lineage is
indicated as Y and hyphen, respectively. Superscript numbers indicate to the reference
describing pigment compositions of the corresponding lineages. 1, (Johnson and
Schroeder, 1996; Miller et al., 2005); 2, (Partensky et al., 1993; Tomitani et al., 1999;
Hess et al., 2001; Chen et al., 2005); 3, (Marquardt and Hanelt, 2004a; Schubert et al.,

2006); 4, (Yoshii, 2006); 5, (Thayer and Bjorkman, 1990; Koniger et al., 1995).

 

 

 

 

 

 

 

 

> > - - - Ecmeomc

> > > - - :_£cmxm_o_> vo>cmvcc29moé

> > > > - 55:983. .

> > > > > acouoamod pogo—.9080
> > - - - wo>zm>zoné§2

> > > - - £05. vo>tmvbc99moé

> > > > - 82288

- - - > > u __ c 925

- - - > - o __Eaeo_;o

> > - > - a 38530 2358530
> > > > > m __Eaeo_;o

”09cm. new;

«mm _m coma mom _m com No 2 222091 .mtofimnocm o

 

 

o9: EoEmE

 

 

l9

Fig. 1 Xanthophyll synthesis in ArabidOpsis thaliana. The genes are bold italicized.
Carotenoids present at less than 1% of total carotenoids in unstressed WT leaf tissue are

shown in gray.

R

7',9'-crs-lycopene —> MMM/YWVY

CRT’SO trans-lycopene LYCB
ZDS \
9‘—cis-neurosporene wit/SE?

I CRTISO B-carotene (22%)

LYCB 8. CRTR-B1/B2

7,9,9'—cr’s-neurosporene LYCE

H
\ R
T208 CYP97C1,? @ ‘ :g

B—cryptoxanthin

9,9'—di-c/s—t’,-carotene l CRTR-B1/BZ
0H
T2430 WEI/W” if \ .RA”
- - ” lutein (51°/ ) “0 h'
9,15,9'-tn-crs-:-carotene ° zeaxant rn

T... ll

antheraxanthin
9.15—c/s-phytofluene lT

violaxanthin(12%)
p03 Biosynthesis of
abscisic acid

\ neoxanthin(13%)

15-cis-phytoene

T PSY

2 X geranylgeranyl
diphosphate (GGPP)

20

CHAPTER 2

DEFINING THE PRIMARY ROUTE FOR LUTEIN

SYNTHESIS IN PLANTS:
THE ROLE OF ARABIDOPSIS CAROTENOID B-RING

HYDROXYLASE CYP97A31

 

1 This chapter was published in “ Kim, J. & DellaPenna, D. (2006) Proceedings of the National Academy
of Sciences of the United States of America 103, 3474—3479”.

21

2.1. Summary

Lutein, a dihydroxy derivative of or-carotene (Bx-carotene), is the most abundant
carotenoid in photosynthetic plant tissues where it plays important roles in LHCII
structure and function. The synthesis of lutein from lycopene requires at least four
distinct enzymatic reactions: 13- and s-ring cyclizations and hydroxylation of each ring at
the C-3 position. Three carotenoid hydroxylases have already been identified in
Arabidopsis, two non-heme di-iron B-ring monooxygenases (the CRTR-BI and CRTR-BZ
loci) that primarily catalyze hydroxylation of the B-ring of B,B-carotenoids and one heme-
containing cytochrome P450 monooxygenase (CYP97C1, the LUTI locus) that catalyzes
hydroxylation of the s-ring of B,e-carotenoids. In this study I demonstrate that
Arabidopsis CYP97A3 (the LUT 5 locus) encodes a fourth carotenoid hydroxylase with a
major in vivo activity toward the B-ring of or-carotene and a minor activity on B-rings of
B-carotene. A cyp97a3 null allele, lut5-1, causes an accumulation of (Jr-carotene at a level
equivalent to B-carotene in wild type, which is stably incorporated into photosystems and
a 35% reduction in B—xanthophylls. That lut5-1 still produces 80% of wild type lutein
levels indicating at least one of the other carotene hydroxylases can partially compensate
for the loss of CYP97A3 activity. From these data I propose a model for the preferred
pathway for lutein synthesis in plants: ring cyclizations to form or-carotene, B-ring
hydroxylation of or-carotene by CYP97A3 to produce zeinoxanthin, followed by e-ring

hydroxylation of zeinoxanthin by CYP97C] to produce lutein.

2.2. Introduction

22

Numerous studies have shown that a class of non-heme type carotenoid hydroxylases
that are present in most carotenoid containing organisms (Tian and DellaPenna, 2004)
can efficiently hydroxylate the B-rings of several carotenoid substrates (Sun et al., 1996;
Tian and DellaPenna, 2001). Arabidopsis contains two genes encoding non-heme type [3-
ring hydroxylases (the CRTR-BI and CRTR-BZ loci) (Fig. 1). Their primary in planta
substrates are B-rings' of B-carotene, because a crtr-bIcrtr-bZ (b1 b2) double null mutant
reduced B-xanthophyll levels 80% but the level of lutein was slightly increased relative to
wild type (Tian et al., 2003).

Recently, several P450 type enzymes have been identified as new classes of
carotenoid hydroxylases. The archetypal members are the Arabidopsis s-ring hydroxylase
(CYP97C1) encoded by the LUTI locus (Tian et al., 2004b) and the B-carotene
hydroxylase (CYP175A1) of the eubacteria, Thermus thermophilus (Blasco et al., 2004).
Expression of T thermophilus CYP175A1 in E. coli engineered to produce B-carotene
resulted in the production of zeaxanthin, a dihydroxy B-carotene, and is a clear example
of convergent evolution in B-ring hydroxylases (the non-heme and P450 type). In
Arabidopsis, a T-DNA insertion mutant in the CYP97C] (IutI-3) gene accumulates
zeinoxanthin (or-carotene with a hydroxylated B-ring) in place of lutein, consistent with
CYP97C] being the primary enzyme responsible for s-ring hydroxylation in Arabidopsis
(Tian et al., 2004b).

In the triple carotenoid hydroxylase mutant, b1b21ut1-3, B-xanthophylls are reduced
80% relative to WT but zeinoxanthin is still accumulated to high levels suggesting at
least one additional carotene hydroxylase must exist in Arabidopsis with activity toward

the B-rings of B- and or-carotene (Tian et al., 2003). Here, I report that a second member

23

of the Arabidopsis CYP97 family, CYP97A3, encodes a B-ring hydroxylase with a major

activity toward the [3-ring of or-carotene.

2.3. Results

2.3.1 The lut5 mutation is in Arabidopsis CYP97A3 and causes accumulation of high
levels of a-carotene My initial search for a fourth Arabidopsis carotenoid hydroxylase
focused on members of the CYP97 clade because it already contains one carotenoid
hydroxylase (CYP97C1, the LUTI locus). Although all three Arabidopsis CYP97 clade
members are predicted to be targeted to the chloroplast by ChloroP 1.1 (Emanuelsson et
al., 1999), CYP97A3 (At1g31800) was selected as it has the highest (52%) amino acid
identity with CYP97C1. To determine whether loss of CYP97A3 activity affected the
carotenoid composition in Arabidopsis leaf tissue, five-week old leaves of two
independent cyp97a3 mutant alleles were analyzed. lut5-1 contains a T-DNA insertion in
the third exon of CYP97A3 while lut5-2 contains a single amino acid change (E283K).
Fig. 3 shows the HPLC profiles of leaf extracts from WT, b1 b2, lut1-4 (a cyp97c1 null
mutant), and lut5-1. All lines are in the Col-O background except b1b21ut1-3, which are
Wassilewskija (Ws). Individual and total carotenoid levels in Col-O and W3 were not
significantly different (data not shown) and only Col-0 is shown for WT. WT
accumulates four major carotenoids: three xanthophylls (neoxanthin, violaxanthin, and
lutein) and one carotene (ii-carotene). Though the total carotenoid level is not
significantly different between mutants and their respective WT, the carotenoid
composition of each mutant genotype dramatically differs from WT (Table 2). As

previously reported (Tian et al., 2003), the b1b2 mutant has lower levels of B-

24

xanthophylls and increased B-carotene and lutein. IutI-4 (a null lutI allele) virtually lacks
lutein and accumulates a high level of zeinoxanthin, or-carotene with a hydroxylated [3-
ring, and elevated levels of B-xanthophylls (zeaxanthin, antheraxanthin, violaxanthin and
neoxanthin) (Pogson et al., 1996; Tian et al., 2004b).

lut5-1 has a 18% reduction in lutein and contains two novel peaks (minor and major)
relative to WT with retention times (24.3 and 32.7 min), mass and absorption spectra that
are consistent with those of monohydroxy or-carotene derivatives (zeinoxanthin and/or or-
cryptoxanthin, see Fig. 2) and or-carotene, respectively (Fig. 3). Zeinoxanthin and or-
cryptoxanthin cannot be distinguished based on HPLC retention time or spectra. However,
carotenoids that have an allylic hydroxyl group, for example at C-3’ of an a-ring, readily
eliminate water when positively ionized, while a non allylic hydroxyl group, such as at
the C-3 of a B-ring, does not (Pogson et al., 1996). The major ion of the 24.3 min
unknown was [MH+-H20] (see Supplementary data Fig. l) and based on this
comparatively easy loss of water I can conclude its single hydroxyl group is on the e—ring
of a-carotene and hence it is or-cryptoxanthin. Accumulation of high and low levels of or-
carotene and or-cryptoxanthin, respectively, occurs in both lut5 alleles, is accompanied by
a reduction in total [LB-carotenoids and an increase in total or-carotenoids (Table 2). The
lut5-1 phenotype is generally more severe than lut5-2, consistent with lut5-2 being a
weaker allele. The lut5 and [at] mutations are additive: the lut5-[lutI-4 double mutant
accumulates or-carotene and zeinoxanthin at levels nearly identical to lut5-I and lull-4,
respectively.

2.3.2 (it-carotene is incorporated into lut5-I photosystems To determine the location of

(Jr-carotene in lut5 I purified thylakoid membranes from WT, Iut1-4 and lut5-I, separated

25

the pigmentzprotein complexes by non-denaturing gel electrophoresis (Fig. 4) and
analyzed their pigment compositions (Table 3). Fig. 4 shows that the photosystem I
holocomplex and photosystem 11 core complex co-migrated and are well separated from
LHC monomers and trimers in all genotypes (Lee and Thornber, 1995; Lokstein et al.,
2002). The photosystems are the most likely place for or-carotene incorporation, as they
contain B-carotene, a structural isomer of or-carotene (Alfonso et al., 1994; Lee and
Thornber, 1995; Croce et al., 2002; Klimmek et al., 2005). Like B-carotene in WT, or-
carotene in lut5-1 was localized almost exclusively in photosystems, accounting for 39%
of photosystem carotenoids (Table 3). In terms of carotenoid stoichiometry in IutS-I
photosystems, the increase in or-carotene was mirrored by a corresponding decrease in [3-
carotene. Fig. 4 also shows lutI-4 had a higher level of LHC monomers and lower level
of LHC trimers, consistent with a previous report for this mutant showing a key role for
lutein in LHC trimerization (Lokstein et al., 2002). lut5-1 also shows an increase in LHC
monomers, suggesting the 18% reduction in lutein in the mutant also impacts LHC trimer
stability. The faint bands migrating between the photosystem and LHC trimer bands in
Iutl and lut5 were variable in occurrence and levels between experiments and presumable
reflect reduced photosystem stability in the mutants.

2.3.3 The impact of mutating CYP97-type carotenoid hydroxylase on the expression
of other carotenoid biosynthetic enzymes As shown in Table 2, the ratio of total [3,8-
carotenoids to [LB-carotenoids in lut5-1 was over twice that of WT, suggesting that [3-
and/or s-cyclase activities might be affected in the mutant. Because it is not possible to
directly assay carotenoid cyclase or hydroxylase activities in Arabidopsis leaf extracts, 1

determined the steady-state transcript levels of these genes to indirectly assess the impact

26

of the lut5 and [w] mutations on the pathway. As shown in Fig. 5, both cyclase mRNAs
are modestly increased in lut1-4 and lut5-I relative to WT, the B-cyclase more so than the
a—cyclase, but this increase appears unrelated to changes in the [3,8- to [Mi-carotenoid
ratios in the mutants. I also quantified mRNAs for the four known carotenoid
hydroxylase genes (CRTR-BI, CRT R-BZ, CYP97C1, and CYP97A3) to determine whether
altered gene expression plays a role in compensating for the absence of P450-type
carotenoid hydroxylases in the mutants. The detection of each CYP97 gene transcript in
the corresponding gene knockout mutant was possible because the real-time probe is
positioned upstream of each T-DNA insertion site. With the exception of a slight increase
in C YP97A3 mRNA levels, the expression of other carotene hydroxylases is not impacted
in the Iut1-4 mutant. In contrast, BI, 82 and CYP97C] mRNAs were all up-regulated in
lut5-1 and lut5-11utI-4, most notably CYP97C] mRNA which was more than 4-fold

higher than WT.

2.4. Discussion

2.4.1. At least four enzymes are involved in carotenoid hydroxylation The current
study and prior work (Pogson et al., 1996; Sun et al., 1996; Tian and DellaPenna, 2001)
have defined a minimum of four carotenoid hydroxylase genes involved in xanthophyll
biosynthesis in Arabidopsis: two non-heme type B-ring hydroxylases encoded by the
CRTR-BI and CRTR-BZ genes, a P450-type s-ring hydroxylase encoded by the CYP97C]
(a LUTI locus) and now a P450-type B-ring hydroxylase encoded by the CYP97A3 (a

LUT 5 locus). Analyses of mutants defective in one or more carotenoid hydroxylase

activities (Table 2) (Tian et al., 2003) provide insight into the specific and overlapping

27

activities of each enzyme in viva. All mutant genotypes exhibit specific alterations in
xanthophyll and carotene compositions (Table 2) (Tian et al., 2003), indicating the loss of
any single activity cannot be fully compensated by the remaining activities. The degree of
compensation observed in a given mutant genotype reflects the preferential activity of the
missing enzyme(s) and the regulation and substrate specificities of the remaining active
enzymes in the mutant. For example, CYP97C] (LUTl) is the primary s-ring
hydroxylase activity in Arabidopsis as lutein synthesis is nearly completely blocked in the
null lutI-3 (Tian et al., 2004b) and lut1-4 (Table 2) alleles and the presumed CYP97C]
monohydroxy substrate, zeinoxanthin, accumulates. Similarly, disruption of both non-
heme type B-ring hydroxylases in the b1 b2 double null mutant reduces B-xanthophylls
76% without affecting B-ring hydroxylation in lutein synthesis, suggesting that the B-
rings of B,|3-carotenoids are the preferred in planta substrates for CRTR-Bl and CRTR-
BZ.

2.4.2. CYP97A3, the new type of B-ring hydroxylase in Arabidopsis CYP97A3
(LUT5) is a P450 type B-ring hydroxylase with activity toward the B-rings of both or-
carotene and B-carotene in vivo. The major impact of the null lut5-1 mutation is an
accumulation of (it-carotene at a level equivalent to B-carotene in WT, consistent with the
B-ring of (Jr-carotene being a preferred CYP97A3 substrate in planta. Because lutein is
only reduced 18% in lut5-I relative to WT, at least one of the other carotene hydroxylases
must also be able to catalyze hydroxylation of the B-ring of or-carotene, though less
efficiently than CYP97A3. CRTR-Bl and CRTR-BZ may be able to hydroxylate the [3-
ring of a-carotene as in the Iut5-11ut1-4 double mutant zeinoxanthin is still present at

levels similar to lut1-4. CYP97C1 (LUTl) may also have some level of B-ring

28

hydroxylase activity in viva as CYP97C] expression is strongly up-regulated in the lut5-1
background, as one might expect for a compensating enzyme (Fig. 5), and the level of
total hydroxylated B-rings is further reduced in the bIbZIutl-3 mutant relative to the b1b2
mutant (Table 2) (Tian et al., 2004b).

CYP97A3 also appears to have a minor in viva activity toward the B-rings of B-

carotene as B-xanthophylls (primarily violaxanthin and neoxanthin) are reduced 35% in
lut5-1, versus a 76% reduction in the b1 b2 genotype (Table 2). While CYP97A3 is likely
to be responsible for synthesis of at least a portion of the B-xanthophylls present in b1 b2,
contributions from CYP97C1, an additional uncharacterized hydroxylase activity or
indirect impacts resulting from the elevated levels of or-carotene produced in lut5-1 can
also not be excluded. Unfortunately, attempts to assay CYP97C1 and CYP97A3 in vitro
by heterologous expression in Saccharamyces cerevisiae and Escherichia coli (E. coli)
and in viva in E. coli engineered to accumulate carotenoid substrates have not been
successful (data not shown) and I therefore cannot directly assay potential substrates for
the two enzymes. The generation of triple and quadruple mutant genotypes remains
would be informative in this regard.

2.4.3. A model for lutein biosynthesis and regulation in plants As shown in Fig. 2,
there are several potential biosynthetic routes leading from lycopene to lutein that have
only partially been delineated by prior genetic studies in Arabidopsis (Cunningham et al.,
1996; Pogson et al., 1996; Pogson and Rissler, 2000). The reactions leading to lutein must
be highly efficient or tightly associated as many of the potential pathway intermediates in
WT Arabidopsis leaf tissue are near or below the one ng carotenoid HPLC detection limit

(Table 2). The activities of Arabidopsis 13- and s-cyclases expressed in lycopene

29

producing E. coli suggested the preferential substrate for the a-cyclase is lycopene rather
than y-carotene (Cunningham et al., 1996). Consistent with this, mutation of the
Arabidopsis LYCE, s-cyclase encoded by LUT 2 locus eliminates lutein synthesis without
causing accumulation of the pathway intermediates rubixanthin or y-carotene (Pogson et
al., 1996). These data indicate that e-ring cyclization of lycopene to produce 8—carotene is
the first step in lutein synthesis. 8-Carotene is undetectable in WT Arabidopsis leaf tissue
but oc-carotene is detectable suggesting the 6-carotene produced by LUT2 is efficiently
utilized by the B-cyclase. The isolation and characterization of mutations in the C YP97A3
and C YP97C1 genes now allow us to infer the primary reaction sequence for lutein
synthesis in plant tissues from among the remaining possible steps in Fig. 2.

The pathway shown in Fig. 2 has two possible routes leading to lutein from or-
carotene: B-ring hydroxylation to zeinoxanthin followed by s-ring hydroxylation to lutein
or s-ring hydroxylation to a-cryptoxanthin followed by B-ring hydroxylation to lutein.
Whether one pathway is favored or both occur depends on the substrate specificities and
regulation of the hydroxylases involved, CYP97A3 and CYP97C1. Accumulation of
zeinoxanthin in [m] while the or-carotene level remains identical to that in WT is
consistent with zeinoxanthin, rather than or-carotene, being a preferred substrate for a-
ring hydroxylation by LUTl. If a—carotene were a preferred CYP97C1 substrate the lut5
mutants (which still has CYP97C1 activity) would be expected to accumulate significant
amounts of or-cryptoxanthin, which it does not. lut5 instead accumulates an or-carotene
level ten times that of or-cryptoxanthin, which is consistent with or-carotene being a

preferred substrate for B-ring hydroxylation by CYP97A3 and CYP97C1 having at best a

30

minor activity toward the s-ring of or-carotene. Biochemical regulation (e.g. feedback
inhibition by a-cryptoxanthin) may also contribute to the lut5-1 phenotype but this seems
less likely as the Iut5-11ut1-4 double mutant, that cannot synthesize or-cryptoxanthin, has
a phenotype that is essentially the combination of the 1m] and lut5 single mutants. If or-
cryptoxanthin played a regulatory role one would expect a different phenotype when the
compound were removed in the double mutant. Together, the data presented are
consistent with the preferred pathway for lutein synthesis being two ring cyclizations to
yield or-carotene and then proceeding to zeinoxanthin and lutein by the sequential action
of CYP97A3 and CYP97C1.

The general reaction sequence for lutein synthesis, ring cyclizations followed by
hydroxylations, is the same as that of its closest structural isomer, zeaxanthin, a
dihydroxy B-carotene derivative. However, zeaxanthin is produced primarily by the
action of the non-heme type B-ring hydroxylases (CRTR-Bl and CRTR-B2) while lutein
is produced primarily by the action of P450-mediated or-carotene ring hydroxylases and
the regulatory mechanisms of these two types of enzymes appear to differ significantly.
In lutein synthesis the bicyclic intermediate or-carotene is barely detectable in WT
whereas the corresponding intermediate in zeaxanthin synthesis, B-carotene, is 22% of
total WT leaf carotenoids. Accumulation of high levels of or-carotene and zeinoxanthin in
the lut5 and [w] mutants excludes the possibility these intermediates do not accumulate
in WT simply because they are unstable in viva. The high level of or-carotene in Iut5 is
especially intriguing as it suggests the presence of CYP97A3 is specifically required for
efficient lutein synthesis and that the two non-heme B-ring hydroxylases, CRTR-BI and

CRTR-BZ, cannot substitute for CYP97A3 in this regard.

31

Why would two members of the P450 type carotenoid hydroxylases be
specifically required for efficient lutein biosynthesis in viva? I propose that in WT the
reaction sequence from lycopene to lutein is catalyzed by a protein complex composed of
two lycopene cyclases (the B and s-cyclases) and two P450 type hydroxylases (CYP97A3
and CYP97C1) that allows channeling of substrates between reactions. There is precedent
for this as cytochrome P450 enzymes are known to form dimers (Scott et al., 2003;
Schoch et al., 2004), functionally interact with other cytochrome P450 enzymes
(Karninsky and Guengerich, 1985; Dutton et al., 1987; Kelley et al., 2005) or act as
anchors for soluble and membrane biosynthetic complexes (Burbulis and Winkel-Shirley,
1999) in other systems. Given the clear importance of lutein in LHC structure and
photosystem function (Lokstein et al., 2002; Liu et al., 2004; Wentworth et al., 2004), it is
relatively easy to rationalize a strong selective pressure for the evolution of e-ring
cyclization and hydroxylation activities for lutein synthesis. However, the forces driving
the evolution of different biochemical machineries (P450 and non-heme carotenoid
hydroxylases) for hydroxylation of or-carotene and B-carotene are less obvious. Perhaps
the answer lies in the need to efficiently synthesize lutein for LHC structure and function
while simultaneously tightly controlling or-carotene production, due to the potential
negative consequences of producing (it-carotene containing photosystems.

Plants produce B-carotene containing photosystems almost exclusively under
most environmental conditions (e.g., high light). or-Carotene containing photosystems are
also produced in many plant genera but generally only in shade-grown or low light
adapted plants, where or-carotene can be present in excess of B-carotene (Thayer and

Bjorkman, 1990; Demming-Adams and Adams III, 1992; Koniger et al., 1995).

32

Presumably or-carotene containing photosystems provide a competitive advantage under
low light conditions but at higher light levels such photosystems show increased
photooxidation, or-carotene levels decrease and B-carotene containing photosystems
predominate (Barth et al., 2001). These data suggest tight control of the OMB-carotene
ratio is an important adaptive response to low and high light (Krause et al., 2001; Krause
et al., 2004). Consistent with this hypothesis, the constitutive production of or-carotene in
lut5 renders the mutant much more sensitive than WT to high light exposure (see
Supplementary data Fig. 2). CYP97A3 clearly plays a key role in carotenoid synthesis by
allowing efficient lutein production while limiting or-carotene accumulation and provides
a straightforward biochemical mechanism for producing (rt-carotene and lutein when both
are needed under low light conditions: by regulation of CYP97A3.

When taken together, the current genetic data in Arabidopsis are consistent with in
viva synthesis of the two major groups of xanthophylls in plants being preferentially
catalyzed by two different classes of carotene hydroxylases: P450-type enzymes for
synthesis of lutein and non-heme type enzymes for synthesis of B-xanthophylls. The
evolution of what at first appearance seems to be an unnecessarily complex system can be
understood in considering the contrasting needs of plants in response to changing light
conditions. In high (normal) light it is competitively advantageous to produce lutein
without or-carotene while under low light conditions it is advantageous to produce both
lutein and or-carotene. B-Carotene and B-xanthophylls are needed at different levels and
ratios under these conditions. Thus the P450 type carotenoid hydroxylases may be best
suited to fulfilling these contrasting demands by allowing formation of a separate

biosynthetic complex for efficient metabolic channeling to lutein while providing a

33

regulatory mechanism for or-carotene production that is independent of the synthesis and
regulation of B-carotene and B-xanthophylls. Such independent regulation of the two

branches of the carotenoid pathway allows plants to respond efficiently, effectively and

adaptively to ever changing light conditions.

2.5. Material and Methods

2.5.1. Plant materials Plants were grown under a 12 h photoperiod (100-120 umol rn‘2
s", 22 °C) and 18 °C at night. lut1-4 and lut5-1 are T-DNA knockout mutant alleles of
CYP97C1 (At3g53130) and C YP97A3 (At1g31800), respectively. lut5—2 was identified
by HPLC screening of nine missense mutant alleles isolated by TILLING screening (Till
et al., 2003). The lut5-11ut1-4 double mutant was selected by PCR screening of F2
progeny from a cross of lut5-I and lutI-4. HPLC separation, identification and
quantification by spectra and retention time were performed as previously described
(Tian and DellaPenna, 2001) except quantification of monohydroxy or-carotenes was
performed at 475 nm.

2.5.2. TaqMan Real-Time PCR Assays The transcript levels of six different carotenoid

biosynthetic genes (,B-cyclase, a-cyclase, CRT R-BI , CRTR-BZ, C YP97C1 and C YP97A3)

were quantified by TaqMan real-time PCR using elongation factor 10. mRNA levels for
normalization. The CYP97A3 primers and TaqMan probe are: 5'-
GTTTGATTGGACTGGTTCTGACC-3' (forward primer), 5'-
TTCCGGACCGCCTGAAT—3' (reverse primer), 5'-
ACCCCAAGGTTCCTGAGGCTAAAGGCT—3' (TaqMan probe). Primers and probes for

other genes are as described (Tian et al., 2003). The relative quantity of transcripts was

34

calculated using the comparative threshold cycle (CT) method (Livak, 1997).

2.5.3. Isolation of thylakoid membranes and nondenaturing polyacrylamide gel
electrophoresis (PAGE) Thylakoid membranes from Columbia-0 (Col-0), lut1-4 and
lut5-1 were isolated, and the photosystems and peripheral LHCs were separated by non-
denaturing PAGE as described (Lokstein et al., 2002). Individual pigment containing
bands were excised and homogenized in gel running buffer (Lee and Thornber, 1995) and
pigments extracted and analyzed as described (Tian and DellaPenna, 2001).

2.5.4. Structural determination of unknown monohydroxy or—carotene TLC
separation on Si250F0PA silica plates (Mallinckrodt Baker, Phillipsburg, NJ) with
hexanezisopropanol solvent (9:1) was used to enrich the unknown monohydroxy a-
carotene from lut5-1 saponified leaf extracts (Pogson et al., 1996). A band showing the
same Rf value as that of zeinoxanthin was isolated and subjected to further mass analysis.
The elutant from HPLC was chemically ionized by atmospheric pressure chemical
ionization (APCI) and subsequently analyzed by MS. Lutein was used as a control to
show water loss from a hydroxylated a-ring when ionized, while zeaxanthin and
zeinoxanthin were used to show no water loss from hydroxylated B-rings when ionized

(Pogson et al., 1996).

35

Table 2 Leaf tissue carotenoid composition of the indicated genotypes. Carotenoids are
expressed as mmol pigment mol'l chlorophyll a + b, with the relative molar percentage of
each carotenoid given in parentheses. Each value is the mean result of four experiments i
SD. Student's t test for two samples; *, P<0.05. or-crypto, or-cryptoxanthin; VAZ, the sum
of violaxanthin, antheraxanthin, and zeaxanthin; neo, neoxanthin; HB, the moles of
hydroxylated B-rings; B,a/B,B, the molar ratio of total [Ls-carotenoids to total [3,13-
carotenoids. 3 indicates a monohydroxy or-carotene derivative that could not be identified

because of the low levels present. n.d., not detectable.

36

 

 

. as 88 6: 38 Q8 . . 3.8
m o Sag a an” . . . . . . . . . . a a . . 13:85
seas .e as S .m 3 S. ._ was 9 .m 3w mm .235
. 2: a: 8: a: . . 8: as
a _ .38 sham . . . . . . . . e a . . . . was
.4 E E .m :4 mm .8 3a mm .w $6 3 .s on m .w :2:
. as a: a: as . . as as
m N .838 888 . . . . . . . . e a . . . . 7?:
.s .3 m. .m as R .w Nam R .5 one a. .s cam m .4 ma 8.
. 8.8 a: as :8 as . . 3.8
w o .35 same. . . . . . . . . . . . a a . . 2323
.a of _ .8 3. mm .m 3. 3 m can _ .v is 2: . m one o
. 2 8 $8 $8 3.8 68 . . 2.8
e o ~8me a in . . . . . . . . . . e a . . 3.5
.w :a on .12 a S”: a mos: .w 3 8 .213
. 8.: 5 $8 6.8 8.8 as
a _ .38 mas...” . . . . . . . . . . . . 22
._ :3 hrs: .2138 moi: .mofi: 83.22
. a: a: as 6.8 88 A _ 8
_ _ swam area . . . . . . . . . . . . E:
m mam mm _ a: mm o is m m can _ .m one a _ a3 5
0:0 0.30 0.3 6.30 E sax
adbd n: .532 8: N<> u a .5 Sarge 53:.
-m so 653

 

37

Table 3 Carotenoid composition in photosystems (PS I holocomplex and PS II
core complex) of the indicated genotypes. The amount of carotenoid is expressed
as mmol pigment mol'l chlorophyll a. Each value is the mean result from three
experiments i SD, with the relative molar percentage of each carotenoid given in

parentheses. Student's t test for two samples; *, P<0.05. n.d., not detectable.

38

 

Gas 2.8 88 as . G8 88

. . . . . . . ea . . . . was
Same is : .EA 5. .w 3a.? In: 38%
Gas 8: $8 3 as .. Gs
. . . . . . . . . . a: . . 33
_ 3.3 N we a .a N“: ow moan: .a is E .e 112
8.8 as as E . . . . as

w: w: E
233 22.3. Eras: 3am. 888.3

 

9:383 2.39:8 55:3

cared 53:.
-n Id 553

G0: N<>

 

39

Fig. 2 Pathway showing all possible routes to xanthophyll synthesis in Arabidopsis.
Enzymatic reactions are indicated by numbers: 1, s-cyclization; 2, B—cyclization; 3, B-ring
hydroxylation of [5,8- and [3,w- carotenoids; 4, s-ring hydroxylation; 5, B-ring
hydroxylation of [LB-carotenoids. Reactions blocked by mutation of the indicated loci are
shown: cyp97c1 (lutl, s-ring hydroxylase), lyce (lut2, e-cyclase), cyp97a3 (lut5, B-ring
hydroxylase), crtr-bI, and crtr-b2 (two B-ring hydroxylases). Solid arrows indicate a
reaction sequence that is supported by mutant phenotypes and/or enzyme activity assays
in E. coli while dashed arrows are not. Black arrows, compounds and mutant genes

indicate major biosynthetic routes.

40

55:38:
55:88.03
:_£:mxm:m£:m

:_£:mxmmN

«~33? lfil 0
3.53.. cite
«Rod»?
Set"... cite 0

 

59:.

as.

4.. . Mum-.393

9 “can? G:

:EmeoEmN 55:98“an :

”wt. .Lfl Dix/me

a

mob ........ .
.gfisomquo

mcmEmolmv 38:9:

\ox mmsquo

 

 
 

o e
mcoSLmore p
mmkquo ; /
.. ....1- ® :39?
AW a ..-.-.-. muks / G
®\_/\/\r\/\ (mg gag
/ m /
/ / / / /

m

41

Fig. 3 Left panel: HPLC analyses (440 nm absorption) of leaf extracts of the
indicated genotypes. N, neoxanthin; V, violaxanthin; A, antheraxanthin; L, lutein;
Z, zeaxanthin; chl a, chlorophyll a; chl b, chlorophyll b; zei, zeinoxanthin; B-car,
B-carotene. Right panel: an overlay of sections of the lut5-I (black) and Iut1-4
(gray) HPLC chromatograms containing unknown peaks 1 and 2 and UV-visible

absorption spectra of zeinoxanthin, oc-carotene and unknown peaks 1 and 2.

42

 

 

E:
can c3. 23 omn

 

Tut: I.
.125 I

 

odN

 

o.mN

m .5

 

oéN

EN

 

 

 

 

SE SE

 

an an «N on 2‘ up a an on E“ on or Nw a
{d-

_ ‘ > 2
QEo
‘—

a .5 Nave

Z
>
0.50 .—

ES

5
é. j

 

 

 

 

43

Fig. 4 Non-denaturing gel electrophoretic separation of pigmentzprotein complexes from
thylakoid membranes of the indicated genotypes. PS, photosystem I holocomplex and
photosystem II core complex; LHCT, trimeric form of LHC; LHCM, monomeric form of

LHC; FP, free pigment zone.

Col-0 Iut1-4 lut5-1

PS

LHCT
LHCM

FP

 

44

Fig. 5 Expression of carotenoid biosynthetic genes in lut1-4, lut5-1 and lut5-11ut1-4
relative to WT. The dotted line refers to the expression level of each gene in WT.

Transcripts were quantified by real time PCR using elongation factor 10L as a reference

 

control.
5
3
E
2 3
“5
g 1| I I I
(B ..................................
5 ,. I
2 5.
a Iut5-1Iut1-4

(.0

_X.

I... Incl-II ...... II llllll t'ootullu nnnnnnn I".

p-cvc s-CYC 3;“ 332m CYP97C1 CYP97A3

45

CHAPTER 3

MOLECULAR EVOLUTION OF ARABIDOPSIS

CAROTENOID HYDROXYLASESZ

 

2 The part of this chapter was submitted to Plant Physiology.

46

3.1. Summary

Xanthophylls are a group of more than 500 different oxygenated carotenes that
serve a variety of functions in prokaryotes and eucaryotes. Xanthophyll composition is
highly conserved in photosynthetic tissues of higher plants but how changes in
xanthophyll composition occurred in ancestral photosynthetic organisms and why
specific changes have been retained in lineages leading to higher plants remain open
questions. To gain insight into the evolution of xanthophyll synthesis, we analyzed two
pairs of duplicated enzymes catalyzing key carotenoid hydroxylation steps in Arabidopsis
thaliana. Recent work has suggested that a-carotene hydroxylation is catalyzed primarily
by a pair of cytochrome P450 enzymes, CYP97A3 and CYP97C1, while B-carotene
hydroxylation is catalyzed primarily by two non-heme di-iron enzymes, CRTR-Bl and
CRTR-BZ. We have used a series mutant genotypes null for one to four of these enzymes
to demonstrate they represent the full complement of carotenoid hydroxylases in
Arabidopsis and to infer the activity of each enzyme in vivo. Phylogenetic analyses
suggested that the CYP97A3 and CYP97C1 genes were duplicated before the speciation
of Arabidopsis and green algae (cf. Chlamydomonas reinhardtii and Ostreococcus taun)
while duplication of the CRT R-B genes was more recent, after the Arabidopsis/poplar
split. Although the four enzymes exhibit some overlap in activities, most notably in
hydroxylation of the B-ring of a-carotene, the mode of functional divergence in the
CYP97 and CRTR-B gene pairs appears to be distinct. C YP97 duplicates are strongly
coexpressed but the encoded enzymes have distinct in vivo substrates likely due to
divergence in their putative substrate recognition/binding regions. In contrast, the CRTR-

B duplicates are isozymes that show significant expression divergence in reproductive

47

organs. By integrating the evolutionary history and substrate specificities of each extant
enzyme with the phenotypic responses of mutant genotypes to high light stress we

propose likely scenarios for the evolution of xanthophylls biosynthesis in Arabidopsis.

3.2. Introduction

Prior studies of xanthophyll biosynthetic mutants in Arabidopsis coupled with the
genome sequence and associated wealth of gene expression data in this organism have
advanced understanding of xanthophyll synthesis at the molecular level. Two classes of
structurally unrelated enzymes catalyze ring hydroxylations of a- and B-carotene; P450
type carotenoid hydroxylase (CYP97A3 and CYP97C1) (Tian et al., 2004b; Kim and
DellaPenna, 2006) and non-heme type carotenoid hydroxylases (CRTR-Bl and CRTR-
BZ) (Tian and DellaPenna, 2001; Tian et al., 2003). Mutation of one or more of these
enzymes has shown that CYP97A3 and CYP97C1 are primarily responsible for
catalyzing hydroxylation of the B-ring and 8-ring of a-carotene, respectively, for a-
xanthophyll synthesis while CRTR-Bl and CRTR-B2 primarily catalyze the two B-ring
hydroxylations of B-carotene in B-xanthophyll synthesis.

The fact that ring hydroxylations in each branch of xanthophyll synthesis are
primarily catalyzed by one of two structurally unrelated enzyme groups (CYP97 and
CRTR-B) in viva raises interesting questions about the evolution of the two pathway
branches and their associated enzymes. Additionally, that the two successive ring
hydroxylations are catalyzed by homologous enzymes in each pathway branch suggests
that duplication and subsequent functional diversification of each gene pair has been

important for xanthophyll pathway evolution. Therefore, comparing and contrasting the

48

evolution of the CYP97 and CRT R-B genes may provide important insight into the

evolution of xanthophyll biosynthesis in higher plants.

3.3. Results

3.3.1. Defining the full complement of caroteonid hydroxylases in Arabidopsis To
understand evolution of the xanthophyll synthetic pathway in photosynthetic organisms, I
focused on the molecular evolution of carotenoid hydroxylases because the reactions
catalyzed by these enzymes are key determinants for xanthophyll synthesis. I selected
Arabidopsis enzymes as our model system because four carotenoid hydroxylase genes
(C YP97A3, CYP97C1, CRT R-BI and CRTR-BZ) had been previously identified and
individually studied in detail (Sun et al., 1996; Tian and DellaPenna, 2001; Tian et al.,
2004b; Kim and DellaPenna, 2006). A prerequisite for my study was to first determine
whether these four genes represent the full complement of carotenoid hydroxylases or
whether additional hydroxylase activities are present as had been previously suggested
(Tian et al., 2003; Tian eta1., 2004b; Kim and DellaPenna, 2006). To address this issue, I
created and analyzed a mutant genotype that was null for the four known carotenoid
hydroxylases. Two different parental genotypes were generated that were homozygous
for knockouts in three of the Arabidopsis carotenoid hydroxylase genes and heterozygous
for the fourth (i.e., CYP97A3 or CYP97C1). When selfed, the progeny of each line
segregated in a 3:1 ratio for green:white seedlings with x2 p-values of 0.50 and 0.26.
White seedlings were lethal in soil but viable in tissue culture if supplied with a carbon
source (cf. 1.5 % sucrose). HPLC analysis showed that white seedlings from both crosses

contained trace amounts of oc- and B-carotenes and lacked all xanthophylls (Table 4).

49

3.3.2 Gene duplication of the CYP97 and CRTR-B genes Having established that
CYP97A3, CYP97C1, CRTR-Bl and CRTR-B2 are the full complement of carotenoid
hydroxylases in Arabidopsis, the molecular evolution of each gene was assessed. A
catalogue of CYP97 and CRT R-B genes in photosynthetic eukaryotes that synthesize both
a- and B-xanthophylls (Six et al., 2005; Yoshii, 2006) was produced based on blast
searches against publicly available databases (NCBI, TIGR and JGI) using the three
CYP97 genes (CYP97A3, CYP97B3 and CYP97C1) and two CRTR-B genes (CRTR-BI
and CRT R-B2) of Arabidopsis as queries (Table 5 and 6). tBlastn searches (Altschul et al.,
1997) against the full or nearly full genome sequences of Populus trichocarpa (poplar),
Oryza sativa (rice) and two green algaes, Chlamydomonas reinhardtii (C. reinhardtii) and
Ostreococcus tauri (0. tauri) identified the number of CYP97 and CRTR-B homologs in
each organism which allowed the pattern of gene duplication in each organism to be
deduced.

All three CYP97 gene trees (NJ, MP and ML) support the hypothesis that two
consecutive duplications of CYP97 ancestral genes occurred before the higher plant/green
algae split with further lineage-specific gene duplications occurring in C. reinhardtii and
0. tauri. All three CYP97 gene trees show one to one orthology among Arabidopsis,
poplar and rice in each CYP97A, CYP97B and CYP97C clade, and two CYP97A and
CYP97B genes in C. reinhardtii and 0. tauri, respectively (Fig. 6A). In contrast, the
CRT R-B gene tree (Fig. 68) indicates that duplication of CRTR-B genes in higher plants
occured in a lineage-specific fashion with three of four monocots and six of thirteen

dicots in the analysis having more than one CRTR-B gene. Interestingly, these gene

50

duplications appear to have occurred relatively recently, after the monocot/dicot and stem
eudicot/core eudicot splits, respectively. To assess the mechanisms of these recent
duplications I examined the exon-intron structure of each CRT R-B gene and homology
between pairs of chromosomal segments encoding CRT R-B genes in the fully sequenced
genomes of Arabidopsis, poplar and rice (Fig. 7). In all cases, a significant degree of
conservation in CRTR-B exon-intron structure and colinearity in adjacent chromosomal
segments was observed, suggesting that duplication likely resulted from whole or
segmental genome duplication. The CRTR-B duplication time infered from tree topology
(Fig. 6B) and the Ks value (0.45) for Arabidopsis (See Supplementary data Table 1)
suggest duplication likely occur 24~40 million years ago (Blane et al., 2003; Wang et al.,
2006).

3.3.3 Functional divergence of Arabidopsis CYP97 and CRTR-B enzyme pairs
3.3.3.1 Substrate divergence Having defined four genes as the full complement of
carotenoid hydroxylases in Arabidopsis allowed us to use the leaf and seed xanthophyll
compositions of various multiple mutant combinations to unambiguously deduce the in
vivo activity of the individual carotenoid hydroxylases. The leaf carotenoid compositions
of seven such informative mutant genotypes in reference to wild type (Col-0 or Ws) are
shown in Table 7. The phenotype of the b1 b2 mutant demonstrated that CYP97A3 and/or
CYP97C1 could hydroxylate the B-rings of B-carotene, though at lower efficiency than
the CRTR-B enzymes. Likewise, the a3c1 double mutants (homozygous for the
cyp97a3cyp97c1 mutations) showed that the two CRTR-B isozymes could hydroxylate
the B-ring of a-carotene, though again with lower efficiency than the CYP97 enzymes.

The activities of the CYP97A3 and CYP97C1 enzymes were further clarified by two

51

triple gene knockouts: the b1b2c1 (in which only CYP97A3 is functional) and b1b2a3
mutants (in which only CYP97C1 is functional). B—xanthophylls are produced at a much
higher level in b1b201 than in b1 b2, indicating the increased activity of CYP97A3 toward
the B-rings of B-carotene in the absence of CYP97C1. In contrast, b1 bZa3 only contains
2% of the WT B-xanthophyll level, indicating that CYP97C1 has almost no in vivo
activity toward the B-rings of B-carotene. However, lutein levels in b1b2a3 are 74% of
WT, indicating that in addition to e-ring hydroxylation activity CYP97C1 also has strong
activity toward B-ring of (it-carotene.

The carotenoid composition in WT mature seeds differs significantly from that in
leaf tissue (Tian et al., 2003) (Table 8). Xanthophylls account for 99% of the seed
carotenoids with lutein being the most abundant at 80% of total followed by zeaxanthin.
B-carotene accounts for less than 1% of the total seed carotenoids. Despite the differing
carotenoid compositions of leaves and seeds, the activities inferred from carotenoid
hydroxylase mutant xanthophyll composition in the two tissues are generally in
agreement. For example, the B-xanthophyll levels in the b1b2 double mutant (deficient in
both CRTR-B genes) are reduced to 50% that of wild type (WT) but lutein is unaffected.
Similarly, the cyp97c1 mutation (c1) severely impacts lutein levels without affecting [3—
xanthophyll levels while in the cyp97a3 mutant (a3) lutein is unaffected but [3-
xanthophyll levels decrease approximately 40%. In the a3c1 double mutant lutein is still
severely reduced but B-xanthophylls return to WT levels.

In an attempt to delineate any domains/residues in the CYP97A and CYP97C
enzymes that may have contributed to their functional divergence at the protein level, I

performed two different types of molecular evolutionary analyses. First, I scanned the

52

aligned amino acid sequences of each paralogous CYP97A/C pair in Arabidopsis, rice, C.
reinhardtii and 0. tauri using a statistical method that calculates the Z-score from the null
hypothesis that the evolutionary rates are the same between two sequences in a sliding
window (window size = 30 a.a.) (Nam et al., 2005). In this analysis, it is assumed that
diversification of protein function is reflected by the relaxation or intensification of
functional constraints at the protein level (Li, 1983). Because the activities of C YP97A
and CYP97C are conserved between Arabidopsis and rice (Tian et al., 2004b; Kim and
DellaPenna, 2006; Quinlan et al., 2007), one would expect that protein regions important
for differentiating CYP97A and CYP97C substrates would occur consistently in
Arabidopsis and rice. These regions would likely be shared as well in C. reinhardtii and
0. tauri CYP97A/CYP97C pairs if firnctional divergence were initiated before the
speciation of higher plants and green algae. I found only one region (from column 160 to
199) that showed a consistent evolutionary rate difference between CYP97A and
CYP97C in all four organisms (Z >1.0, p <0.l6). Fig. 8A shows the average of the four
Z-scores of this region is 1.57 (p =0.06). When the same analysis was applied to the three
CRT R-B gene pairs from Arabidopsis, rice and pine (Fig. SB), no consistent pattern was
observed and the maximum Z-score was 0.94 (p=0. l 7) for any gene pair.

In a second approach, I estimated the posterior probability for cluster-specific
functional divergence in a given residue (Gu, 2006) in order to identify those that might
be critical for functional divergence of CYP97A and CYP97C. Fig. 8C illustrates the site-
specific profile calculated from comparison between the CYP97A clade including
Arabidopsis, Medicago truncatula, tomato, rice and barley genes, and the C YP97C clade

including Arabidopsis, Medicago truncatula, tomato, carrot and rice genes. The estimated

53

coefficient of cluster-specific functional divergence (0”) is 0.29:0.04, indicating the
analysis is statistically meaningful. Twenty-nine residues were identified with a 7.1 %
prediction error (false-positive rate) and six of these were also conserved in C. reinhardtii
and 0. tauri.

Based on the structural conservation of the cytochrome P450 superfarnily across
phyla (Graham and Peterson, 2002; Schoch et al., 2003; Mestres, 2005), I developed two
CYP97A3 structural models (i.e. closed and open conformation) and mapped the single
domain and six residues identified from these two approaches onto the three-dimensional
structures (Fig. 9). As templates for homology modeling, I used the crystal structures of
human microsomal P450 3A4 (ltan) and mammalian cytochrome P450 2B4 (lpoSA)
which had the best 3D-jury scores (333.71 and 311.71) in the protein data bank (PDB) for
closed and open conformations (Ginalski et al., 2003). The domain identified by sliding
window analysis includes the B—strand that sits on the putative substrate access channel
(blue in Fig. 9) (Graham and Peterson, 2002). Similarly, one of the six conserved amino
acids identified from the second approach was mapped onto the middle of the I helix, (red
in Fig. 9, alanine in CYP97A and serine in CYP97C) which has been shown to be
important for positioning of substrate aromatic rings in the cytochrome P45 0 active site
(Rupasinghe et al., 2003; Schoch et al., 2003). Identified domains and residues are also
shown in amino acid alignment which is used for NJ tree construction (Supplementary
data 3A).
3.3.3.2 Expression divergence To assess any divergence in gene expression between the
CYP97 and CRTR-B duplicates, I determined Pearson’s correlation coefficient (r) as an

index of expression similarity for each gene pair retrieved from the publicly available

54

DNA microarray datasets (Fig. 10) (Wagner, 2000). Only datasets in which at least one of
the two copies for each family was expressed were selected for analysis (Makova and Li,
2003). Because carotenoid hydroxylases are primarily involved in producing pigments
for LHCs, I compared gene expression in photosynthetic tissues (shoot apex, leaf and
green seedling) and in reproductive organs (carpel, sepal, stamen, petal and seed). r-
Values for the CYP97 gene pair are high in nearly all tissues surveyed, indicating that
expression of the genes are relatively highly correlated. r-Values for the CRTR-B gene
pair are also high in unstressed and stressed photosynthetic tissues, with the exception of
cold stress and high light stress, but much lower in all reproductive organs.

3.3.4 The impact of functional divergence under high light To assess the biological
implications of carotenoid hydroxylase diversification we examined the biochemical and
physiological consequence of various knockout genotypes to infer the impact of the
missing enzyme(s) on fitness of the organism. Because xanthophylls play many important
structural and functional roles in photosynthesis we measured the response of mutant
genotypes to high light stress using non-invasive in vivo chlorophyll fluorescence. Non-
photochemical quenching (NPQ) and the maximum photosynthetic efficiency of
photosystem II (F v/Fm) are two fluorescence-derived parameters that are routinely used
as quantitative measures of photosystem adaptation and response to short-term and long-
terrn high light stress, respectively (Maxwell and Johnson, 2000; Holt et al., 2004). NPQ
is rapidly induced (< 1 min) in response to high light and increases non-radiative energy
(e.g. heat) dissipation within the photosystems. The kinetics of NPQ induction and the
maximal NPQ level reflect the coordinate induction of complex structural and functional

changes in the photosystems. Fv/Fm is a measure of the maximal efficiency of

55

photosystem II (PS II) and changes to F v/Fm generally occur over a longer time frame
than NPQ (hours versus minutes), respectively. The Fv/Fm of healthy nonstressed tissues
is 0.8 and approaches zero as PS 11 is progressively damaged.

Fig. 11A and 11B show changes in NPQ and Fv/Fm in the indicated genotypes as a
function of time. The absence of either class of carotenoid hydroxylases (a3c1 and b1 b2)
significantly reduced NPQ and Fv/Fm relative to WT level after high light stress,
indicating both the CYP97 and CRTR-B type enzymes are required for high light
adaptation. However, within an enzyme class, differing degrees of functional
complementation were observed for individual class members. Impairment of NPQ and
Fv/Fm in b1 b2 was partially complemented in the b1 or b2 single mutants, consistent
with a prior study suggesting functional redundancy (Tian et al., 2003). In contrast,
impairment of NPQ and Fv/Fm in the a3c1 mutant is due largely to the cyp97c1 (c1) and
the cyp97a3 (a3) mutations, respectively. Fv/F m in the 0] mutant was indistinguishable
from WT during a 400 min high light treatment (Fig. 11B) but the kinetics of the NPQ
rise and maximal NPQ level was slower and lower, respectively, than WT (Fig. 11A). The
impact of the a3 mutation on NPQ and Fv/Fm was opposite to that of the c] mutation.
The kinetics of the rise in NPQ in a3 was somewhat slower and more variable than WT
but the maximal NPQ achieved after 260 sec was not significantly difl‘erent (Fig. 11A).
However, Fv/Fm in the 03 mutant was strongly negatively impacted relative to WT after
only 80 min of high light treatment (Fig. 11B). The a3c1 double mutant had significantly
slower NPQ induction kinetics, lower maximal NPQ and lower Fv/Fm and is essentially

an additive phenotype of the two single mutations. After 10 hours illumination at

16004800 umol-m'Z-s", the a3 mutant had irreversibly reduced C02 fixation rate under

56

100 umol-m'2°s" (Fig. 12).

3.4. Discussion

3.4.1. Gene duplication and in viva activity of carotenoid hydroxylase genes
Duplication and subsequent functional divergence of genes are being increasingly
recognized as important mechanisms of evolution (Ohno, 1970; Lynch and Conery, 2000;
Moore and Purugganan, 2005). Functional divergence can occur in protein coding regions,
gene expression patterns or both. Here, I compared the evolutionary histories and in viva
firnction of two duplicate gene pairs involved in carotenoid hydroxylation in Arabidopsis,
CYP97A3/C1 and CRT R-BI/BZ which together account for the full complement of
carotenoid hydroxylases in this organism (Table 4). Our phylogenetic analyses (Fig. 6)
showed that the CYP9 7A3/CI genes appear to be the result of an ancient duplication with
no further recent duplication (Fig. 6A) whereas the CRT R-BI /BZ gene pair results from a
relatively recent gene dupication. The in vitro activities of individual hydroxylases
against [3- or s-rings have been studied by heterologous expression in E. coli engineered
to accumulate B-, 8- or a-carotenes (Sun et al., 1996; Tian and DellaPenna, 2001; Quinlan
et al., 2007). While this approach has been quite informative in delineating the possible in
vitro substrate(s) for each enzyme, the approach has inherent limitations. The substrates
tested may not occur in viva and the enzymes are produced and assayed in isolation from
the other pathway enzymes. Hence, the in viva activity, regulation and role of each
hydroxylase in this complex biochemical pathway may not accurately be reflected in the
E. cali assay system. In order to better understand molecular evolution of the four extant

carotenoid hydroxylases, whether they have distinct or overlapping enzymatic activities

57

in viva and what the forces may have driven their evolution, selection and maintenance in
plants we have generated a series of mutant genotypes for one or more of the four genes
and assessed their consequences in viva.

The two classes of carotenoid hydroxylases share significant overlap in substrate
specificities, i.e. all four carotenoid hydroxylases have the ability to hydroxylate the [3-
ring of at-carotene, at least to some degree. The CRTR-B enzymes have been previously
shown to be isozymes with indistinguishable activities toward B-carotene when expressed
in E. coli. In viva analysis indicates the CRTR-B enzymes are most active in the synthesis
of B-xanthophylls, but that they also have significant activity toward the B-ring of at-
carotene. In contrast, the CYP97 enzymes have evolved to preferentially function in at-
xanthophyll synthesis and show substantial divergence in their preferred in viva
substrates (Table 7) likely due to changes in substrate recognition and binding (Fig. 8 and
9). CYP97A3 has high activity toward the B-rings of B-carotene and (at-carotene but no
activity toward the e-ring of a-carotene. CYP97C1 has high activity toward e-rings (Tian
et al., 2004b) and the B-ring of or-carotene but almost no activity toward the B-rings of B-
carotene.

The activities of the individual carotenoid hydroxylases deduced from
xanthophyll accumulation data in seed are consistent with that in leaves with one major
discrepancy: there is a virtual absence of zeinoxanthin and a-carotene in seed of all C]
and 03 containing genotypes, respectively, while the same genotypes accumulate high
levels of these carotenoids in leaves. Rather than postulating a fundamental difference in
carotenoid hydroxylase activities in the two tissues it is more likely that this discrepancy

is due to differing stability of zeinoxanthin and a-carotene in leaves and seed. The fact

58

that total carotenoid levels in the leaves of nine genotypes shown in Table 7 do not
significantly differ while total seed carotenoid levels of the same genotypes are decreased
by as much as 85% is consistent with differential carotenoid turnover in seed but not
leaves. This is likely due to a combination of carotenoid associations with proteins in
leaves but not seed (e.g. LHCs) and enzyme mediated carotenoid degradation. Indeed, a
null mutant for carotenoid cleavage dioxygenase 1 has been shown to have significantly
increase seed carotenoid levels while leaf carotenoids are unaffected (Auldridge et al.,
2006b).
3.4.2. The evolution of B-xanthophyll synthesis B-xanthophylls and CRTR-B-type
enzymes are widely distributed in nature and found in all photosynthetic eukaryotes and
many photosynthetic and non-photosynthetic prokaryotes. The Arabidopsis CRTR-Bl
and CRTR-B2 are isozymes (Tian and DellaPenna, 2001; Tian et al., 2003) and relatively
strong expression correlation in photosynthetic tissues (Fig. 10), suggesting their most
recent common ancestor (MRCA) had a similar biochemical activities and was expressed
in photosynthetic tissues. The b1b2 mutant has negatively impacted NPQ and F v/F m (Fig.
11) suggesting that photoprotection was likely an important function of the CRTR-B
ancestor and a strong selective pressure for retention of this activity during evolution. The
necessity of B-xanthophylls as substrates for abscisic acid (ABA) synthesis may be an
additional selective pressure for retention of CRTR-B activity as the b1 b2 mutant also has
insufficient ABA production under drought conditions (Tian et al., 2004a).

The retention of CRTR-B paralogs appears to be widespread in higher plants
whether underlying selective pressures are common. In Arabidopsis, after duplication of

the ancestral CRTR-B gene, most likely by whole genome or segmental genome

59

duplication 24~48 million years ago (Fig. 7 and Supplementary data Table l), the
duplicates seem to have diverged primarily at the gene expression level (Fig. 10). Our
carotenoid analyses suggest CRTR-B2 is more actively involved in B-xanthophyll
synthesis in seeds than in leaves, consistent with the rapidly induced CR TR-BZ gene
during seed development (Supplementary material 5). Expression divergence of CRTR-B
genes is not restricted to Arabidopsis, as one of the two CRTR-B members in bell pepper,
tomato and saffron (C. sativus) also shows preferential expression in flower or during
fruit development (Bouvier et al., 1998; Castillo et al., 2005; Galpaz et al., 2006). Unlike
Arabidopsis, CRTR-B expression divergence is strongly associated with tissue specific
functional divergence in these other organisms. In tomato crtr-b2 mutant results in a
colorless petal phenotype with no impact on B-xanthophyll synthesis in leaves (Galpaz et
al., 2006). The massive accumulation of [i-xanthophylls during maturation of the saffron
stigma was correlated with high expression of a single CRTR-B gene (C. sativus CI in
Fig. 6B) (Castillo et al., 2005). Flower development involves the transformation of
chloroplasts to chromoplasts (Whatley and Whatley, 1987) and it is likely that the
expression divergence of the CRTR-B genes in reproductive organs (e. g. chromoplast-
specific expression) provides the biochemical flexibility to differentially regulate B-
xanthophyll synthesis in these tissues.

3.4.3. The evolution of a—xanthophyll synthesis Unlike B-xanthophyll synthesis,
which is widespread in nature, the synthesis of or-xanthophylls occurs in only a few
lineages of photosynthetic eucaryotes, some red algae and all green algae and plants. The
prevalence of a-xanthophylls in both green algae and plants suggests that their MRCA

synthesized a-xanthophylls and that strong selective pressure has maintained this trait.

60

Four reaction steps are required for the synthesis of a dihydroxy (it-xanthophyll (i.e.,
lutein): B- and e-ring formations from lycopene by B- and e- cyclase followed by
hydroxylation of each ring by B- and a-ring hydroxylases (Fig. 2). Like B-xanthophylls,
B-cyclases are widespread in nature and have been recruited for use in at-xanthophyll
synthesis. e-Cyclases have only been identified in green algae, plants and
Prachlaracaccus (a cyanobacterium) (Partensky et al., 1993; Krubasik and Sandmann,
2000; Hess et al., 2001; Cunningham et al., 2007) and appear to have arisen from B-
cyclases by gene duplication and subsequent functional divergence before the green algae
and plant split (Krubasik and Sandmann, 2000; Cunningham et al., 2007). They
phylogeny of CYP97 B- and s-ring hydroxylases similarly shows that duplication of the
MRCA also occurred before he speciation of green algae and higher plants and that the
CYP97A and CYP97C genes have been under purifying selection (Fig. 6A). Though these
phylogenetic inferences provide insight into when the genetic materials for tit-xanthophyll
synthesis were generated they cannot answer when the function of each gene evolved and
how each function has been maintained during evolutionary time. However, the
numerous mutant genotypes affecting specific carotenoid biosynthetic enzymes in
Arabidopsis provide important insights into these questions.

The conservation of (it-xanthophyll synthesis during the evolution of green algae
and plants suggests strong selective pressures were involved in the generation and
maintenance of the necessary enzymatic activites, the e-ring cyclase, CYP97A3 and
CYP97C1. To assess these functional constraints, we used several informative carotenoid
biosynthetic mutant genotypes to infer the impact that various pathway intermediates

have when accumulated by a hypothetical ancestral organism (i.e., B-xanthophyll

6l

producing organism) as it acquired one or more of the three necessary enzymatic
activities. The Iut2 mutant, which is defective in e-ring cyclase activity, cannot synthesize
a-xanthophylls (Pogson et al., 1996) and is a reasonable approximation of a hypothetical
B-xanthophyll accumulating ancestral organism. The absence of lutein in lut2 results in
elevated levels of B-xanthophylls, partial impairment of NPQ, a smaller photosystem
cross sectional area and lower LHC trimer stability . While mature lut2 plants grow as
well as wild type under moderate light conditions they are slightly less resistant to high

light stress (Pogson et al., 1998; Niyogi et al., 2001; Dall'Osto et al., 2006).

The consequences of the hypothetical ancestral organism first acquiring e—ring
cyclase activityis approximated by the a3c1 genotype, which has a functional a-ring
cyclase, can synthesize at-carotene and has CRTR-B enzyme activity but lacks both
CYP97A and CYP97C1 activities. In addition to B-carotene and B-xanthophylls the aid
mutant produces a-carotene and zeinoxanthin in approximately equal molar ratios (Table
7), due to inefficient hydroxylation of the [3-ring of the oc-carotene by the extant CRTR-B
enzymes present in the mutant background. The a3cI mutant is highly susceptible to
photooxidation in high light, more so than any other single xanthophyll biosynthetic
mutant. The high light susceptibility of a3c1 is due to the presence of a—carotene rather
than zeinoxanthin as the CI single mutant, which accumulates an equivalent amount of
zeinoxanthin as a3c1 but lacks tit-carotene, does not exhibit such photosensitivity (Table
7 and F ig. 11). The phentoype of the a3c1 mutant suggests that acquisition of e-cyclase
activity by the ancestral organism would have been detrimental under full sunlight and

would have set in place a situation that would strongly select for the evolution of

enzymes that could efficiently hydroxylate a-carotene.

62

Given the strong selection pressure imposed by (it-carotene accumulation and the
presence of CRTR-B-type enzymes in the ancestral organism, it is surprising that the
extant CRTR-B-type enzymes have not evolved to more efficiently hydroxylate the [5-
ring of (it-carotene. This suggests that there is a structural constraint on the CRTR-B class
of enzymes that makes the efficient hydroxylation of the B-rings of both a-carotene
and B-carotene impossible. In this light it is possible to understand why and how a
separate class of carotene hydroxylases, the CYP97 family, evolved and was selected for
in (it-xanthophyll synthesis (Fig. 13). The original CYP97 enzyme acquired by the
ancestral organism likely had at-carotene B-ring hydroxylation activity (CYP97A-like
activity) as both the extant CYP97A3 and CYP97C1 have this activity. The a3 mutant
phenotype suggests the evolution of an eflicient CYP97A-like a—carotene B-ring
hydroxylation activity would have been strongly selected for as it would have alleviated
a-carotene-dependent photooxidation.

Duplication of the ancestral CYP97A-like enzyme and evolution of a CYP97C-
like (e-ring hydroxylase) activity would have allowed efficient synthesis of lutein without
accumulation of (it-carotene or zeinoxanthin. The driving force for selection of a
CYP97C-like activity is efficient photosystem structure and function, which is less
intuitive than the a-carotene-dependent photooxidation driving selection of CYP97A-like
activity, but no less important. In plants, lutein is essential for the assembly and stability
of large light harvesting photosystems and for optimal NPQ kinetics and maximal NPQ.
While the impact of xanthophyll alterations on NPQ is most readily quantified under
experimental constant high light conditions, such as those used in Fig. 11A, the impact of

altering NPQ on plant fitness is most apparent under natural light conditions. Kulheim et.

63

al (2002) showed that NPQ deficiency had no discemable impact on plants grown under
constant light in growth chamber conditions but when grown in natural lighting, which
has large swings in light intensity occurring on the order of seconds to minutes, the
fitness of NPQ deficient plants was severely impaired and the mutants produced 30-50%
fewer seed per generation than wild type (Kulheim et al., 2002). Given this fitness impact
it is clear how evolution of a CYP97C-like activity would be selected for and maintained.

The scenario we have described above (the sequential acquisition of e-cyclase,
CYP97A and CYP97C activities in Fig. 13) is not the only possible sequence of events in
the evolution of at-xanthophyll synthesis based on the data provided. It is equally
probable that a CYP97A-like enzyme was already present in the ancestral organism and
along with a CRTR-B type enzyme was involved in [ii-xanthophyll synthesis. In this case,
and assuming the CYP97A-like enzyme had at least some endogenous a-carotene B-ring
hydroxylation activity, the acquisition of a-cyclase activity would have resulted in
monohydroxy a-carotene (zeinoxanthin) production. If the enzyme was inefficient and
some a-carotene was produced, this would still provide strong selection for improvement
of oc-carotene B-ring hydroxylation activity and against loss of the CYP97A-like gene
(which would lead to a-carotene-dependent photooxxidation). Duplication of the
CYP97A-like gene and evolution of CYP97C-like activity would then occur as described.
Regardless of the exact sequence of events, the phenotypes of mutants defective in extant
carotenoid hydroxylases highlight the strong selective pressures that likely operated in
the evolution and selection of particular genes and activities that have led to the current
biosynthetic pathway found in Arabidopsis. These same strong selective pressures

continue to operate today to maintain the suite of carotenoid biosynthetic enzymes that

64

have been shown to be optimal for light harvesting, photosystem structure, NPQ and

adaptation of plants to the ever changing light conditions in nature.

65

3.5. Materials and methods

3.5.] The CYP97 and CRTR-B genes used for phylogenetic analyses To search for
CYP97 and CRTR-B homologs in photosynthetic eucaryotes, I performed tblastn search
using three Arabidopsis CYP97 genes (CYP97A3, CYP97B3 and CYP97C1) and two
CRTR-B genes (CRT R-BI and CRTR-BZ) as queries in publicly available databases
(NCBI, TIGR and J GI) (E value cutoff = 10'”). To assist in genome sequence annotation,
I experimentally determined the full length sequences for two C. reinhardtii homologs
(CYP97A5, EF587911 and CYP97C3, EF587910) amplified from cDNA pool as for the
CYP97 family members. All sequences used were summarized in Supplementary data 1.

3.5.2. Sequence alignments and computational analyses 27 CYP97 and 33 CRTR-B
sequences were aligned by the computer program ClustalX 1.81 with default parameters
(Thompson et al., 1997) and alignments further refined manually (Supplementary data
Fig. 3A and 3B). Protein sequence rather than DNA sequence was used for alignments, as
protein sequence is more suitable for long-term evolution studies (Hashimoto et al., 1994;
Russo et al., 1996; Glazko and Nei, 2003; Nam et al., 2003). In the CYP97 sequence
alignment (Supplementary data 3A), an alignment block corresponding to columns 160 to
705 was selected for phylogenetic analyses, sliding window analyses and the estimation
of cluster-specific functional divergence. In the CRTR-B alignment (Supplementary data
3B), 3 block corresponding to column 1 to 414 was used for phylogenetic analyses and
column 134 to 403 for sliding window analyses. For phylogenetic analyses, three
different methods were applied: Neighbor-Joining (NJ), Maximum Parsimony (MP) and
Maximum Likelihood (ML). To construct NJ tree, I used Poisson correction (PC)

distance method with pairwise deletion of gaps was used with the MEGA 3.1 program

66

(Kumar et al., 2004). Each MP tree of C YP97 and CRT R-B gene was generated with the
JTT model (Jones et al., 1992), afier removing uninformative residues in the computer
program PAUP* 4.0 beta 10 (Swofford, 2003). The ML tree was constructed using the
PhyML algorithm with gaps treated as unknown characters (Guindon and Gascuel, 2003).
For all trees, branch support was assessed by bootstrapping (500 replicates). The
CYP86A1 and C. reinhardtii CRT R-B genes were selected as outgroups for each tree,
respectively. C YP86A1 was selected because its substrates, fatty acids with chain lengths
from C12 to C18 (Benveniste et al., 1998), are the most similar to carotenoids and the
CYP86 clade is the most closely related to CYP97 clade
(http://www.p450.kvl.dk/cvp_allsubfam_NJil02103.pdf). All P450 nomenclature
follows convention (http://dmelson.utmem.edu/CytochromeP450.html).

For sliding window analysis, seven taxa in each alignment block were selected
and gaps were eliminated. Rates of amino acid substitutions (p-distance) in each sliding
window of two aligned protein sequences with one outgroup (i.e. CYP86A1 in
CYP97A/C and C. reinhardtii C1 in CRTR-B comparisons) since their most recent
common ancestor (MRCA) were estimated using least-square method and compared
using a program developed by Nam et. al (Nam et al., 2005). Because C. reinhardtii and
rice have multiple C YP97A and CRTR-B paralogs, respectively, CYP97A5 and CYP97C3
were used for the C. reinhardtii CYP97A/C comparison, and 0. sativa C2 and C3 were
selected for the rice CRT R-B comparison. The posterior probability to lead cluster-
specific functional divergence was estimated for each residue of CYP97 aligmnent by the
DIVERGE 2 program (Gu, 2006) with ten taxa in the angiosperrn lineage. Ka, Ks and

confidence interval (CI) were calculated by K-estimator 6.0 software (Comeron, 1999)

67

from the CRTR—B alignment block used for the sliding window analysis. C.I was
retrieved fiom 500 replicates.The templates for CYP97A3 homology modeling were
retrieved from Metaserver {http://metabioinfo.pl/submit wizard.pl) and the configuration
of side chains were determined by the computer program maxsprout (Holm and Sander,
1991) (http://www.ebi.ac.uk/maxsprout/) and scrawl 3.0 (Canutescu et al., 2003). The
CYP97A3 homology model was visualized with VMD software available in
(http://www.fiks.uiuc.edu/Research/vmd/).

3.5.3 Plant materials and pigment analyses The leaves of four-week old Ws
background and five-week old Col-0 background genotypes grown under 12 h
photoperiod (100 umol m'Z-sec'l, 22/18 °C) were used for pigment analyses and high light
stress experiment. High light was subjected to plants in the chamber which was set to
1600~1800 umol-m'z-sec", 50% humidity and 22 °C. The seeds were harvested and
stored in the box containing a desiccator for one month before pigment analysis (Tian et
al., 2003; Auldridge et al., 2006b). HPLC separation and quantification by spectra and
retention time were performed as described in (Tian and DellaPenna, 2001; Kim and
DellaPenna, 2006).

3.5.4. Expression data analyses AtGenExpress database was the source of expression
data for the CYP97 and CRT R-B gene pairs
(hinzl/wwwarabidopsis.or2/info/expression/ATGenExpress.isp) with the exception that
the data obtained fi'om high-light stressed leaf tissue was provided by Dr. Dirk Inzé
(Vanderauwera et al., 2005). Used eight tissue types and stress conditions were
independent and nonredundant.

3.5.5. Colinearity of chromosomal segments Protein sequence in Arabidopsis and rice

68

chromosomal segments was obtained from publicly available annotated sequence data.
The putative open reading frames in poplar genome sequence were deduced by
FGENESH software available in SoftBerry (www.softberrv.com). The proteins in
chromosomal segments were compared in a pairwise fashion using bl2seq software
available in NCBI. Cutoff for homologous pair was an e-value less than 10'").

3.5.6 Measurement of in viva chlorophyll fluorescence and CO2 fixation rates To
obtain two photosynthetic parameters (NPQ and F v/F m), in viva chlorophyll fluorescence
was measured from the dark—adapted intact leaves for five minutes by applying a
saturation pulse with and without actinic light (530 i.Lmol-m’2-sec'I ) using the Imaging-
PAM Chlorophyll F luorometer (Walz, Germany) (Berger et al., 2004). C02 fixation rate
was measured after five minute in Arabidopsis chamber of LI-COR 6400 (Li-COR
Biosciences), a gas exchanging system for CO2 and H20 analyses. Reference C02
concentration was set to 400 pmolsec'l (slightly above ambient pressure) by the CO2 .
mixer and the temperature in the block was 22 °C. Air flow was set to 100 umol-sec'l.
Before C02 fixation rate measurements, 10 h- treated plants were put in dark for twelve-

hours, followed by 100 umohm’z-sec'l illumination for seven hours.

69

Table 4 Carotenoid composition (nmol-g'l fresh weight) of green and albino progeny

from the indicated genotypes.

 

 

 

Green plantsa Albino plantsa Green plantsb Albino plantsb
1.3-Xanthophyllsc 1.00i0. 10 n.d. 0- 10:0-01 n.d.
Xanthophylls Lutein 0-61i0-02 n.d. 10130-17 n.d.
a-Car-OH 7.18:0.34 n.d. 0.43:0.00 n_d,
memo-em, 0.23:0.02 0.72:0.19 3.84:0.67 05320.21
Carotenes
pewtm 6.84:0.28 0.171003 1.691024 0.171003

 

a progeny from b1b1b2b2c1c1A3a3 parent.

b progeny from b1b1b2b2C1c1a3a3 parent.

° Sum of zeaxanthin, antheraxanthin, violaxanthin and neoxanthin.

NOTE.- n.d., below the HPLC detection limit (0.5 ng); a-Car—OH, monohydroxy 0t-

carotene. The values shown are the mean of at least two biological replicates analyzed in

triplicate :1: standard deviation.

70

Table 5 List of CYP97 homologs used for constructing a neighbor-joining tree.

 

Organism

Gene name

Accession #Ilocus name!

TC number/JGI annotation #

 

Salanum esculentum
Populus tn'chacarpa
Arabidopsis thaliana
Medicaga truncatula
Hadeum vulgare

Oryza sativa

Ostreocaccus taun'
Chlamydomanas reinhardtii
Chlamydomanas reinhardtii
Ostreacaccus tauri
Ostreacaccus taun‘
Skeletanema castatum
Chlamydomanas reinhardtii
Ginka bilaba

Oryza sativa

Arabidopsis thaliana
Populus tn'chacama
Medicaga iruncatula
Ostreacaccus taun'
Chlamydomanas reinhardtii
Oryza sativa

Arabidopsis thaliana
Daucus carata

Salanum esculentum

S. esculentum CYP9 7A
CYP97A7

C YP9 7A3

M. truncatula CYP9 7A
H. vulgare CYP97A
CYP9 7A4

CYP9 7A 1 1

CYP97A5

CYP97A6

CYP97B14
CYP97B15

CYP97E1

CYP9786

G. bilaba CYP9 78

C YP97B4

C YP97B3

CYP97B7

M. truncatula CYP9 7B
CYP97C12

CYP97C3

CYP97CZ

CYP97C1

D.carata CYP97C

S. esculentum CYP9 7C

71

TC126862

gw1 87.99. 1
At1931 800
ABD28565

TC76166

AK068163

Ott 3902550
EF58791 1

DNE_DNE_e _wa.42.59. 1
Ot01905440
Chr15.00010014
AF459441
DNE_e_wa.1.53.1

AY601 887
TC299269

AT4G1 51 10

fgenesh1_pg.C_LG_V|000069

ABE94036

0t09902560
EF587910
AK065689
At39531 30
AB 852076

SGN E542349 and E346934

Table 5 can’t

Populus tn'chocarpa CYP97C4 eugene3.00280258

Medicago truncatula CYP97C10 DQ335801

 

72

Table 6 List of CR T R-B homologs used for constructing a neighbor-joining tree.

 

Organism

Accession #Ilacus name!
Gene name
TC number/JGI annotation #

 

Solanum esculentum
Capsicum annuum
Solanum esculentum
Capsicum annuum
Gentiana lutea
Medicago truncatula
Citrus unshiu

Caffea arabica
Tagetes erecta
Arabidopsis thaliana
Arabidopsis thaliana
Brassica rapa
Daucus carota
Daucus carota

Vitis vinifera

Pop/us trichocarpa
Pap/us trichocarpa
Adonis pa/aestina
Adonis palaestina
Adonis palaestina

Crocus sativus

S. esculentum CRTR—B1 CA855625
C. annuum CRTR-B2 CAA70888
S. esculentum CRTR-BZ CA855626
C. annuum CRTR-B1 CAA70427
G. Iutea CRTR-B homolog 1 A8027187
M. truncatula CRTR-B homolag 1 ABE85312
C. unshiu CRTR-B homolog 1 AAG33636
C. arabica CRTR-B1 DQ157169
T. erecta CRTR-B homolog 1 AAG10430
A. thaliana CRTR-BZ AT5652570
A. thaliana CRTR-B1 AT4GZS7OO
B. rapa CRTR-B homolog 1 DQ156907
D. carota CRTR-B homo/0g 1 A8852074
D. carota CRTR-B homolog 2 ABBSZO75
V. vinifera CRTR-B1 AAM77007

P. trichocarpa CRTR-B homo/0g 1 estExt_fgenesh1_pg_v1.C_440227

P. trichocarpa CRTR-B homo/og 2 estExt_fgenesh1_pg_v1.C_LG_IVOO7O

A. palaestina CRTR~B1 A3193208
A. palaestina AdkeiaZ AY644758
A. palaestina AdKeto1 AY644757
C. sativus CRTR-32 CAC9513O

73

Crocus sativus

Narcissus pseudonarcissus
Oryza sativa

Oryza sativa

Oryza sativa

Zea mays

Zea mays

Zea mays

Pinus taeda

Pinus iaeda

Ostreococcus taun'
Chlamydomanas reinhardtii

Haematococcus pluvialis

C. sativus CRTR—B1

N. pseudonarcissus CRTR-B homo/cg 1
O. sativa CRTR-B homo/cg 1

O. sativa CRTR-B homo/cg 2

O. sativa C CRTR-B homo/cg 3

Z. ma ys CRTR-B homo/cg 1

2. ma ys CRTR-B hamolog 2

Z. mays CRTR—B homo/cg 3

P. taeda CRTR-B hamolog 1

P. taeda CRTR-B homo/cg 2

O. iauri CRTR-B homo/cg 1

C. reinhardtii CRTR-B homo/cg 1

H. pluvialis CR TR-B1

Table 6 can’t
AAT84408
CAC0671 2 .1
080390125100
031090533500
030490578400
AY844956
AY844958
BQ6 l 95 75
TC67291
T067290
gw1.10.00.289.1
AAX54907
AAD54243

 

74

Table 7 Carotenoid composition in the leaves of the two wild types (Col-0 and Ws) and
seven informative genotypes. Carotenoid levels are expressed as mmol pigment mol'1 of
chlorophylls. Each value is the mean :t SD of at least three biological replicates. The

numbers in parenthesis indicate the percentage of total carotenoids

75

 

 

 

 

 

 

3: FdHNd mama“:

«.63
“one“:
00mm
Neva
mm
B
03
0.60

 

808m 300.0 §E.o0mmcmdm0$ 3:302 8:308 6.30m... 6:2. 83500.0

20% 30mm 630085.308 $0.00»... 345003 @802. 6:... $303. $2.03
0.0048. 3.09 35.408 3303 3:905— ES02 $2.03 5302. $3803 $302
205.. $08 6.500883308 A-:~.m0$ 32.03 8:408 €303 @309 5:00;.
:03 0.6.08 383053303 8:303 690.908. @303 6:... 8560: $30:
205 30:. 5:338:03? 50.00% 84:803. @302 8:... 3303 3:303
2008 20m: $2.403 $803 88305 $2.03. 3:308 8:30; 8:308 633.0%
20me 308 630.003. 6.3603 30:39.0 c.0309. 8:308 8:0... £94503 3:303
803... $08 3.0.5.008 €0,000... 8:303 £32080 53.0% 8:... 5303 3:303
.33. 2.29.8-5 10.508 522 555362. 555.83 sficmxflosfi 553x903

 

 

 

 

76

Table 8 Carotenoid composition in the seeds of the two wild types (Col-0 and Ws) and
seven informative genotypes. Carotenoid levels are expressed as nmol pigment-g“l of dry

weight. Each value is the mean i SD of at least three biological replicates. The numbers

in parenthesis indicate the percentage of total carotenoids.

77

 

 

 

303 8:303 8: 303 E 303 .3 3:308 E 303 Q 303 @303 @7303 3.843
3088 8:303 8:303 E303 .3 888803 8:30: 8:303 8:303 @303 3.3
303 898.803 88.803 .3 88:30? 8:30: 8: 303 83303 8:303 8:303 :32
302 89303 .3 8:303 8:303 8:303 €303 8:303 8:303 8:303 83
308 8: 303 83303 .3 .3 88303 83303 $30: 8: 303 83308... was
30% 83303 8: 303 83803 .3 888.8088 8: 303 $303 8:303 8:30: 3
303 883803 E 303 .3 @303 8: 303 8:303 8:303 883803 3:303 B
308 883.803 83.33 .3 .3 888.003 8: 303 8:303 $303 @303 3
308 883303 8: 303 .3 3 8:30; 8:303 8:303 8:303 8:303 38
. 3228.5 55:96ch 55333 cEmefloficm 55:88.03

 

 

78

Fig. 6A Neighbor-joining tree of CYP97 sequences. Scale bar = 0.2 (Poisson distance).

Numbers on branches represent neighbor-joining, maximum parsimony and maximum

likelihood bootstrap support, respectively (NJ/MP/ML); a hyphen indicates support of

less than 50%. Bootstrap values are omitted from branches where support was less than

50% in all three analyses.

7» Solanum esculentum
Populus trichocarpa CYP97A7
~—_— Arabidopsis thaliana CYP97A3

Medicago truncatu/a

68/-/- "

    
 

95/87/-

100/100/ 100
r.— Hodeum vulgare

100/89/100L— Oryza sativa CYP97A4

87/-/~[ I Ostreococcus tauri CYP97A 11

Chlamydomanas reinhardtii CYP9 7A5

Chlamydomanas reinhardtii CYP97A6

82/60/94 ' Ostreococcus taun' CYP97BT4

F—fi 31/483 4461 Ostreococcus tauri CYP97B15

Skeletonema costatum C YP97E 1

89,7}%:{l _______ Chlamydomanas reinhardtii CYP97B6

i
58H-%
1

 

 

1183/53/-

 

 

 

 

 

r——-————— Ginko bilaba
Oryza sativa CYP97B4

71/-/100

 

 

 

 
 
  
  

100/99/92

 

94/8 0/_ _____ Arabidopsis thaliana CYP97B3

69/-/55 Populus trlchocarpa CYP97B7
97/74/‘ l— Medicago truncatula

Ostreococcus tauri CYP97C12

.. _--- i h:— .0— —-—-v—— Chlamydomanas reinhardtii CYP97C3

88513! ~——— Oryza sativa CYP97C2

 

 

 

 

81/53/-
“4‘ ____ Arabidopsis thaliana CYP97C1
100/100/100Lll 0
r—— aucus carota
98/62/- .

58/-/- l” So/anum esculentum

0 { d_ Populus trichocarpa CYP97C4
0'2 55/'/'L—— Medicago truncatula CYP97C10
CYP86A1

 

79

 

CYP97A

CYP97B

CYP97C

Fig. 6B Neighbor-joining tree of CRTR-B sequences. Scale bar = 0.1 (Poisson distance).
CRTR-Bl, CRTR-BZ indicates paralogs in an organism that have been demonstrated to
have [i-ring hydroxylase activity by assay in carotenoid containing E. coli lines. CRT R-B
homolog 1, CRTR-B homolog 2, CRTR-B homolog 3 indicate CRTR-B paralogs in an
organism whose functions have not yet been experimentally defined. Numbers on
branches represent neighbor-joining, maximum parsimony and maximum likelihood
bootstrap support, respectively (NJ/MP/ML); a hyphen indicates support of less than 50%.
Bootstrap values are omitted from branches where support was less than 50% in all three

analyses.

80

0090 :090

.anwocE>O

8.08069:

 

 

E-Em0 £0.23: 0880233: 3:8 i3
0 022:0: 9E5 33:52 0803:0330

     

0 83:6: mith :35 03080300
8 003.3: 9:50 :83 was: 1 8:80:80
0 30:6: miEo «88 3s: U

N 020:6: mihmo 0:9: mmN 3.823
0 uoBEo: mihmo 30E mmN
m 5.2050: 9510 95:0 «Eco
50955. N 020:6: 98.56 95:0 :50
0 83:8: minke 85:0 330
0 33:6: minke 38.930380 028.902
5-”: «0 32:00 300:0
«91.55 03:00 300:0

029.3: 3:030: £33: :8 :02 _
8293 053:8 :83 328 8:2
Bihmo 0.50020: mEnfi<
0 39:2 35:0 88 8.685 2859 _

.356 30:05 0.38.30; $38..

«95.20 30:05 m..mQ0:.5E< 8:42
Sigma Sgt: 0:3
m 029:0: mismo 5900 30:3
“005 0 33:6: m-Em0 $98 0:33
0 @286: mikmo 8080 90:30:.
.0 020:6: 0.55 2:80:95 33%: ilu 8:8 39
F 02050: MAS—to 09:00:95 33:00: _
0 3203: 3:3 3:23 25.6 i
0 00350: SEED 038:3: 0:00.80 ll
Bikmo 8.50:0 00:00]:
5:50 $3: $038
“mihmo Ezzccm anfiqwo
«Quito E55380 EzcmBm
mm-m.Eo E3230 E32300
Bibs E55380 EzcmBm 8:00:09

         

      

I

                    

-Iwn

 

81

Fig. 7A Exon-intron structures of CRT R-B genes in Arabidopsis, poplar and rice. Exons

and introns are represented as boxes and lines in scale, respectively.

82

a 33:6: 0.5mm 8;.

 

 

 

 

N 020:6: m.E.mo 8E ........... ! ..--.---.-I- ...... F '1'

. 022:0: 95:0 8E “AM llIF-Hl ..... ,w l- l Ill
N 022.3: 0.55 Egon. Illllllllllllll
. 83:6: miEo .28: llllnllllllllll

 

«9&th 33003.2( .. l "
$.55 £38324: Ililllllllll l.- l _
:05 0’2

83

Fig. 7B Colinearity of chromosomal segments containing parologous CRT R—B genes in
Arabidopsis, poplar and rice. Syntenic gene pairs (e-value < 10’'0 for amino acid
identities) are represented as thick solid bars on the side of chromosomal regions with
syntenic partners between regions matched with double-headed arrows. Split arrows
indicate instances where apparent tandem duplications have occurred for a syntenic
partner. Numbers on the sides of the chromosomes refer to e-value exponents between
two syntenic proteins. Note that for clarity annotated genes without syntenic partners are
omitted from each chromosomal region. The CRTR-B genes in each chromosomal

segment are marked with asterisks.

84

ego:
tn§22oxm<<u~z
i.euuocszmsq ~z

 

 

 

 

 

 

 

3an 3.69 :32: 3%»:
.v :0 or :0 n :0 3 2230mm
. _ t.
a _ a.
an. :llldlnulllilii
_ 2.. hr. .
. _
2- . 2:
ed _ _
3- _ _
mu- .
:94..- .l l. ..
«.mm- .
a..- _
_ mu...
2.-
. g-
_ _ _ 0.0
2... l1 l: a...
$2”. guaca—

3.3:
m :0

ad _

k 2....
5n.

8|
NM 9.
on-
no...

:-
3-

 

£3339:

/§.....
v :0

 

 

85

Fig. 8A Averaged Z-scores from each paralogous CYP97A/C pair from Arabidopsis, rice,
C. reinhardtii and 0. tauri. The identified region is indicated with an arrow. The numbers
on the X-axis represents column positions in the CYP97 alignment. Note that the length

of the CYP97 protein used for the alignment is 436 amino acid residues.

86

one 3m v3 v3 Em SN mmu ht-

Eoficmza 05 E
:OEmoa 5:200

.55 .0 0:0 50:350.: .0 .02.. dine—030.3 E9: com-.03: I

Q: .

md

mé

aJoos-z

87

Fig. 8B Z-scores from the two CRTR-B pairs of each Arabidopsis, rice and pine. The
numbers on the X-axis represents column positions in the CRTR-B alignment. Note that

the length of the CRTR-B protein used for the alignment is 214 amino acid residues.

88

on”

22:29.: 05 E
cognac 5:200

HP ,-

 

 

 

om

N

mmN

NE

 

 

 

 

L .

 

 

2E l
8E l
«88324: I

5

 

a.aoos-z

89

Fig. 8C Posterior probability of cluster-specific functional divergence in each amino acid
residue. Among the twenty-nine residues having the highest score the six indicated by
arrows are also conserved when both C. reinhardtii and 0. tauri are included in the clades.

The number on X-axis represents amino acid position in CYP97 alignment.

90

ls! l'

 

 

 

 

6 533

¥¥

 

 

473 498 51
J

I!

 

 

 

399
J

 

 

 

 

 

 

 

 

 

 

 

 

161

,X . . . .

3 fi 3 co «3 v N o
aouafiiangp wuogoun;

unloads-193mm go one: Jog-Iaisod

0

91

676
Column position
in the alignment

407 544

272

160

Fig. 9 Two homology models (open and closed conformations) of CYP97A3. The single
domain (blue) and six amino acid residues (red and pink) identified in Fig. 4 are mapped
onto the model. The red indicates the amino acid residue in the middle of I helix which
may bind to the substrate ring in the active site. The image in this dissertation is

presented in color.

92

cos—«8.6200 coao

 

:ozafihecoo 330.0

93

Fig. 10 Expression divergence of C YP97 and CRT R-B gene pairs in the indicated tissues
(A) and stress conditions in leaf (B). The expression correlation coefficient (r) of the
CYP97 and CRT R-B gene pairs is represented as white and black, respectively. The
number of independent microarray datasets used to test for correlation in each condition

is indicated in parenthesis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 _ __ p“. __ El CYP97
0,3 - I CRTR-B
0.6 h
0.4 -
g 0.2 ~
g o 1_ 1 1 1 l 7 I ,
" -o.2 _ I
-0.4 ~
-0.6 ~
-0 8 _ shoot leaf green carpel sepal stamen petal seed
' apex (12) (36) seedling (6) (6) (6) (6) (6) (15)
B D CYP97
1.0 — I CRTR-B
g 0.8 -
g 0.6 ~
0.4~
0.2 -
0
oxidative wound osmotic heat drought salt cold highlight
(12) (14) (12) (16) (14) (12) (12) (6)

94

Fig. 11A Non-photochemical quenching (NPQ) kinetics under 530 |.Lmol-m'2's'l . The

values are fi‘om three biological replicates. For clarity, error bars are omitted having less

than10%error.
A
20* Ws, b2
1.6— b1
0
0.1.2 b1b2
z

0.8 —
0.4-
O l

 

 

 

 

 

95

Fig. 113 Kinetics of maximum photosynthetic efficiency of PSII (Fv/Fm) under 1600~
1800 umol‘m’zs'l. The values are from three biological replicates. For clarity, error bars

are omitted having less than 10% error.

Ws
bngbZ

b1b2

 

 

 

 

 

o 1100 2100 3100 400

0.8 A
E 0.6 -

E 0.4

 

 

 

 

 

0 100 200 300 "300
min

96

Fig. 12 C02 fixation rates of WT and a3 before and afier ten hour high light exposure.
Data are means i SD (n = 6; one leaf of six independents for C02 fixation measurement).

* p < 0.05, by Student's t test of mutant leaves relative to corresponding WT leaves. BHL,

efore high light; AHL, after high light.

I dark
D 100 mmol m'2 a"

."’
0|

 

 

mmol (:02 m'2 sec"

-2.5

 

 

a3

 

I
or
{II

BHL AHL

97

Fig. 13 Two different scenarios for evolution of the a-xanthophyll biosynthetic pathway

in Arabidopsis from a hypothetical B-xanthophyll accumulating ancestral organism. The

acquisition of newly evolved enzymatic activities in each scenario is indicated in bold.

 

Hypothetical .
ancestral organisms

 

V

Extant
Arabidopsis

Scenario 1

B-Xanthophylls

 

 
 
   

B-Xanthophylls
+

a-Carotene
+
Zeinoxanthin

 

      

[3- Xanthophylls
+

Zeinoxanthin

 

 

 

CYP97C-like

Scenario 2

B-Xanthophylls

CYP97A-like

.44?

B-Xanthophylls

s-Cyclase

e-Cyclase

.,

 

CYP97A-like

 

       

B-Xanthophylls
+
Zeinoxanthin

CYP97C-like

;

 

 

B- Xanthophylls
+

Lutein

 

 

B- Xanthophylls
+

 

Lutein

 

 

 

 

98

CHAPTER 4

FUTURE WORK

99

4.1. Fitness test of lut5-1 (a3) mutant under low light condition

In contrast to the undetectable level of (in-carotene in canopy plants, the ratio of on-
/[3-carotene in understory and gap plants is elevated. One hypothesis is that these
differences are dependent on light-intensity and that a-carotene accumulation has an
advantage to adapt under low light, although it clearly has a negative impact on fitness
under high light. The null mutant allele of CYP97A3, lut5-1, in which a-carotene
constitutively accumulates can be used to test this hypothesis. My preliminary data
(Supplementary data Fig. 4) showed that CO2 fixation rate was not significantly different

between WT and lut5-1 mature leaves under light intensities ranging from 0 to 1300
umol-m'zsec'l. This indicates that at least the short term carbon fixation efficiency of
plants with a-carotene containing photosystem is indistinguishable to that of B-carotene
containing (wild type) photosystems. However, it is still open question whether or-

carotene accumulation is beneficial to long term adaption under low light condition or at

specific developmental stages such as young seedlings.

4.2. Identification of P450-type carotenoid hydroxylase in C.

reinhardtii

The computational analyses with CYP97 amino acid sequences of green algae
and higher plants only allowed prediction of domains and residues in which functional
divergence may have occurred before the speciation of higher plants and green algae.
These predictions are made with the assumption that CYP97 enzymatic activities are

conserved at some extents in both green algae and higher plants. However, experimental

100

data to support this assumption has not yet been reported because the enzymatic activities
of CYP97 enzymes have only been determined in Arabidopsis and rice. In this regard, the
functional characterization of C. reinhardtii CYP97 orthologs could be quite insightful.
Examining the activities of CYP97A5 and CYP97A6, which are orthologous to
Arabidopsis CYP97A3, to determine whether these two enzymatic activities have “true”
CYP97A3 activity and/or whether one or both have evolved new enzymatic activities (i.e.
hydroxylation of lutein at C-19 to produce loroxanthin in C. reinhardtii). The amount of

loroxanthin in C. reinhardtii is approximately 60 mmol-mol'l chlorophyll a, accounting

for the half of the amount of lutein (N iyogi et al., 1997).

4.3. In-depth phenotypic analyses of crtr-bl and crtr-b2 mutants

NPQ and Fv/Fm measurement under high light stress with a series of mutants
showed that the three enzymatic activities (CYP97A3, CYP97C1 and either CRTR-Bl or
CRTR-BZ) have the minimal complement to adapt under high light although all four
enzymes have the full complement of carotenoid hydroxylases. Although the distinct
phenotype in both crtr-bl and crtr-b2 was not observed in the study, expression
divergence observed in reproductive organs and stressed leaves (e.g. cold and high light
stress) still remains as interesting subject to be answered in relation to their functional
divergence. Therefore, future study should be focused on the phenotypic difference
between crtr-b] and crtr-b2 mutants. One possible role of CRTR-BZ is to produce B-
xanthophylls for ABA synthesis. Highly induced its mRNA during seed development
(Supplementary Fig. 5) and almost fully recovered B-xanthophyll level by sole activity of

CRTR-B2 in dry seeds (Table 8) suggest that highly expressed CRT R-B2 is related to B-

101

xanthophyll level. In accordance with highly accumulation of ABA, the increasing
mRNA level of CRT R-BZ gene during seed development is somewhat related with ABA
biosynthesis because B-xanthophylls (neoxanthin and violaxanthin) are the precursor for

ABA. In silica analyses to find cis-acting elements also supports this idea, detecting the
ABA-responsive element (CACGTGGC) which is located on -291 upstream of CRT R-BZ

gene but not on CR T R-BI gene.

4.4. Identification of carotenoid cleavage enzymes recognizing the 8-
ring

The major difference in carotenoid composition in dried seeds is the elevated
mol % of total xanthophylls compared to leaves. During de-greening of Arabidopsis
silique, proteolysis of photosynthetic machineries allows their pigments (chlorophylls and
carotenoids) to be exposed without an association with LHC. Seeds contain trace levels
of oc- and B-carotene regardless of mutant genotypes and the level of zeinoxanthin in CI-
containing mutant seeds was also decreased. These results illustrate that nonhydroxylated
[3- and/or e-rings are unstable in seeds. One of possible reason may be that
nonhydroxylated ring (B- and s-rings) containing carotenoids are targets for carotenoid
cleavage enzymes (CCDs) in seeds. Some Arabidopsis CCDs had already been shown to
have activity toward B-carotene by functional complementation in B-carotene producing
E. coli system (Schwartz et al., 2004). Considering the elevated mol % of total
xanthophylls in seeds than in leaf tissues, the low level of zeinoxanthin in cyp97c1 (c1)
mutant seed is noteworthy, suggesting the existence of CCDs in seed that are highly

reactive toward nonhydroxylated e- or B—rings.

102

APPENDIX

Supplementary data

103

Supplementary data Fig. 1 HPLC chromatograms of the indicated mass ions. A and
B, Mass traces corresponding to major quasimolecular ions for a-cryptoxanthin (A)
and zeinoxanthin (B) in the TLC- purified unknown monohydroxy a-carotene
derivative from lut5-1 saponified leaf extracts. C-F, Mass traces corresponding to
major quasimolecular ions for a-cryptoxanthin (C), zeinoxanthin (D), lutein (E) and
zeaxanthin (F) in a mixture of lutI-4 and abaI-5 saponified leaf extracts. Arrows
indicate the position of the respective carotenoid in each mass trace. Note that the
lut5-I trace in (B) lacks a zeinoxanthin peak and that the Iut1-4 + abaI-5 trace in (D)
lacks a a-cryptoxanthin peak. Zeinoxanthin would be readily detectable if present at
a level > 1% of the alpha-cryptoxanthin peak in lut5-1. The mass of quasimolecular
ions ([MH+] and [MW-H20]) of the indicated carotenoids are below. a-
cryptoxanthin (535.5), zeinoxanthin (553.5), lutein (551.5), zeaxanthin (569.5). Both
A and B were separated as described (Tian and DellaPenna, 2001) except that the
following gradient was initiated after sample injection: 0—13.0 min, 0% to 66.6%;

13.0—13.2 min, 66.6% to 100%.

104

com 00.3. cow— aadF com mm o no v. Dad.

UPC—IF._.__...._...._...._I.PFF_.r......._...._F........_...._.....-.._...P—. .._... ./._... J
.34:

 

<1 «(ii-14:31.. n\—.\u 1<<< {4t {4‘0ch . C
8: 5.13389 3.38.: 5.8 88F BJFNFF/meé 388:8 FF8 8.8 «~28 NE Gm 33/ 8F 23 s
2,2. F EE NFF: 55:92.8 n. 2.
Down
+Q(CMQm .

 

37E}...
u .v 329 8.2.

mEFmEF 89898.9 Wt: 8.2

 

 

 

  

 

 

 

 

i.
as... as 3:: Ed: 52:. m a.
8 F8 . .
+u<cmum
—.>b.1—.>..-.-.-—L.-._...._..... .........
. . 88 A . m. $.33 Fm...
N52. rNN— mmfip Qme kmvw ONMV 8.0—. Nam de mmfi h—hhmw
momma + C_£quXOC._.®N 08 BF
+n_<:m.u.m.
_ ..—....—........._...._...._F.»._...._....—...._.p..—.-.._...._.-.._......LF-FLI...
Fm: .ENRE $588 $2 89 EFFMMFF 88F Em 8F 88 km Fm: 8n T
WW...“ :EmeoEboé o a.
+Q<Cmu.m
. F..r-Ll> <$1...._>:1.LL._.L/PF1_...._....—.>F»—LLL|F<—Pkrp_pn...l.P..«FF...—. ....._. .L
. / e . mvd .
8&me FZFJMF 8.9 .8..2.F. 8.9 2.98.9 3: mm a: 8F. 88 8.8.. PT
82.. .
2...... is E 8: 55:92.8. m g.
+n~<CNuW
....—. ...<:-..p~....—..F.—.-.._....—....—P-+LIE..—...._.-..—.... ....—..Ll._FF.
.JV 41.421”. .2 / / . . . 4. ll. . . 2.
8: .8 NF 98F 28F 8.me VF8FRNFFFNF :9 88 8F 33 em 8: 3m 2F RWMT
83..”

0F

82...... 2F 3.: 38: 55226520-... < o

105

Supplementary data Fig. 2 Whole-plant phenotype of WT and lut5-1 under normal light
level (upper panel, 100 umol m'zs‘l) and two days of high light exposure (lower panel,
1700 umol m'zs'] for 8 hrs, followed by a 12hr dark period and an additional 12 hr of

high light). The image in this dissertation is presented in color.

WT lut5-1

 

106

Supplementary data Fig. 3A Amino acid alignment of CYP97 homologs which is used
for NJ -tree construction. A domain detected by sliding window analysis is boxed and six

residues identified from estimation of type II functional divergence are pointed with

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

arrows .

60
CYP97C4 MSLTFSVSS----LHPLYKYKP--IST
CYP97C10 MPSCSCSCSC----SLPLSHLSLSSFSK
D.carota CYP97C MPYHSISSLS----LLPIPIRQN--LSK
S. esculentum CYP97C MPVSVTISSFS----LLTDTHHRTTVLRP
CYP97C1 MESSLFSPSSSS----YSSLFTAKPTRLLS
CYP97C2 LPTAHLVSSLSPPNPATPIPQNPSRIPSS
CYP97C3 MMLSNRTSGRPTVGSRSSSSARRPALFVP
CYP97C12 MCNRARVAT ----- VVRALEEPETFTD
H. vulgare CYP97A
CYP97A4 GVLAMSSATSVSAFAMAATSSAAAAAPPPCRLLGSGQAHLRLPPSAAAAAASARRRLLLR
S. esculentum CYP97A QFPTHHYSKSR----LTLSPKFKGSVSNFT
CYP97A7 SSIL
M. truncatula CYP97A MASHLTLLHAPPPLSLQTKTFHSKYITIKPLKPTTTFSSSCSLFPCSLKTSHRGSCSSFI
CYP97A3 MAMAFPLSYTP---TITVKPVTYSRRSNFV
CYP97A11 MGTRERARVGADGASRARTRWSRRVVSPA
CYP97A5 MQTQRPLASPGRQASIPARRAYSLRPPLTQ
CYP97A6 MSPALFNPYVAPNRIAPGPRCRMLQRGAAGRGTA
CYP97B14 MVARARVHAS---
CYP97E1_S.costatum MASYESDLLSTWDEDPSLQKGFDWEIEKLRRYFAGLR
CYP97BIS
CYP97B7 MSLTATSTSPLQLPF
M. truncatula CYP97B MVAVAIST -------
CYP97BB MVAAMAFPAAATYPTHFQG
CYP97B4 MAITAATA ------ A
G.biloba CYP97B MITVRSIPCSFSKDTV
CYP97B6
CYP86A1

120
CYP97C4 TTL PPK ----- PRFLS ----- IKSS--LDD K --------
CYP97C10 TPL PQKRYPLHPRILT ----- KSSTN-KNP K --------
D.carota CYP97C HHP PFHQPPHSLPLSI ----- KSSLD-NKPPKSN--------
S. esculentum CYP97C VP LQNR----SQLTI ----- KSSIDNKKP --------
CYP97C1 PKP KFTFS---IRSSI ----- EKPKPKLETN;SK --------
CYP97C2 RAASAMAAAAAAAVPCVPFLCPPPPPLVSPRLRR-----GHVRLRLRPPRSSGGGGGGGA
CYP97C3 VKH VSRVAPLRAQNED-----DEPSTFGKNIDSKG -------
CYP97C12 DDLDN IVFAD ----- LSEEAAISN AG-------
H. vulgare CYP97A G -------
CYP97A4 CAASGGNGKG--GGGDGSGSDP---VLEERRRRR--QAELAARIASGEF t\QGP--AWIA

S. esculentum CYP97A IRCSNSNGKQP---ESVDEG-VKKVEKLLDEKRR---AELSARIASGEF EQ“-SGFP

107

Supplementary data Fig. 3A con’t

CYP97A7 ACASSSNGREP---ESVDNG-VKKVDKlLEQKRR---AELSARIASG QQ‘—-SGFP
M. truncatula CYP97A ACSS-SNGRSPN--DSVDDGVVKSADQLLEEKRR---AELSAKIASGE QE--SGLP
CYP97A3 VFSSSSNGRDPLEENSVPNG-VKSLEKLQEEKRR---AELSARIASG ---SSFP
CYP97A11 RAWEGTGGVETLGTVTTRRGDRANGTRAATGKDADGGKTLEERIASGE QAK--TGPY
CYP97A5 RQLPIARAEPPQTEEIKLFGIIPTAPRSSKLGEN----—LEDRIQSGE SGSTKEKLT
CYP97A6 RGVAATTHAARPYVPRWSRAAAARVVRAAAPSPPPAQDTARGTEAAGEAVFQSSSKKKRR
CYP97Bl4 RGVDARRVRARGRARVDVIARAVKEPSSAPEEAL ----- PDENFKPE -------------

CYP97EI_S.costatum QTPDGRWVRKSTLFEFLVTNSPSKVVGVGPDGER ----- YESPPKPVNIFDVGVLVGKNT
CYP97BIS

 

 

 

 

CYP97B7 TTSNGNYLQRNDFGAVGISRFLSSKTKGSPLlRC--—-—QSTSTEEPK -----------
M. truncatula CYP97B VTLTGVNLHTR---FHSSRFSSHSKRSSSTIRC----—QAVNGDKKKQES ---------
CYP97B3 GALHLGRTDHCLFGFYPQTISSVNSRRASVSIKC-----QSTEPKTNG ------------
CYP97B4 AAATPHPWQADASPRRHAACPALRGRRRLPVVRC—--‘QSSSVDDKP ---------
G.biloba CYP97B QFIGFRKVLTVRNVVSCGVSSSFRLPGSKFFPRC ----- ESSSTERA ---------
CYP97B6

CYP86A1 MEALNSILTE

CYP97C4

CYP97C10

D.carota CYP97C

S. esculentum CYP97C

CYP97C12

H. vulgare CYP97A
CYP97A4

S. esculentum CYP97A
CYP97A7

M. truncatula CYP97A
CYP97A3

CYP97A11

CYP97A5

CYP97A6

CYP97B14
CYP97E1_S.costatum
CYP97B15

CYP97B7

M. truncatula CYP97B
CYP97B3

CYP97B4

G.biloba CYP97B
CYP97BG

CYP86A1 ————YAVAAIsIYAmelserPKVLPFVGflY SRImImNIRAm—G

 

 

 

 

 

108

D.carota CYP97C

S. esculentum CYP97C
CYP97CI

CYP97CZ

CYP97C3

- CY P97C12

H. vulgare CYP97A
CYP97A4

S. esculentum CYP97A
CYP97A7

M. truncatula CYP97A

CYP97BI4
CYP97E1_S.costatum
CYP97BIS

P11h1‘”

11W11M

H1H1
111¥1111111’1\
1'11\1 1 (11’1\
111\1.1111’1\
11' 1\1 l 1(11’1\
11\1u1(11’1\
MHI’N
‘11 111 @1111 11
H11111L111’1\
1‘11“! \11\1 1\1' (11’1\

5 ‘\\_\1)1

’1\\Sl)1)1'fl1
“ 11\.\'1)[H1

711151)1’H’111\11\‘1.1€l

5 1\S1)1

'111'1511
g1fl11fl11111 .

" 1‘11111‘1‘1 11‘.)
\1\11\11'J
1111111111

1\'\ SM V \RIIVI 1’

1 \1\1)1 ’M \1\11\1

\\SH 1

“ .1‘151)1’||\ \1\11111131)\

5 1\>11)1"
7 1\5111’51

£11111111111:
1151 111111

 

\1\111111’1)\\
\1\1111111),\ ~~-— -

\1\1111 111)).5 ‘
51\51)’1g111111113>\\--- ‘

Supplementary data Fig. 3A con’t
240

‘\1\ 1W1h111

1_\\1!111111
E

cling-i191!

:1111.
1111111
111111“

1111111111
1111111111

11511111

111111

lira-3;

‘

27-2;

1‘ 1

13131.2, "

1 1:1
“1E11M l1
11,.‘.1.‘)111 l
1

 

I
CYP97B7 7 , 1.! m: 1H.
M. truncatula CYP97B .{1! 11111111: --—F 1ghn\1 H011 “111 .H.
CYP97B3..11F111\'1.‘1[(1I’11- -' 1II111’ \1'11..]lLi1"11-1a1
CYP97B4 * !\11\1.‘\1(11‘1\‘ "1101 (1 :1 ’11 11ldi1‘111fi11
G.biloba CYP97B h~\1klm\1 ‘ --—F:1wknl‘11)ll |' 10k.
CYP97B6 51 7 .111111111111111.
CYP86A1 I'= - ‘ ..M
CYP97C4 FAI \ll1ll ‘1 ‘11‘11’11’\\‘ 1 ‘1 1'11’1111 1\1 '1\‘1'" 1111211
CYP97C10 FAI \1211.|'1 ‘ 11’11’11‘ \1\1’ ’i1 lllx1€11 ‘11).II '111 1H1-711‘ .
D.carota CYP97C FM ‘\1.( M ‘11’11’11’,1’1\1 \1,111\1\) 1,5 ‘11\.ll '11‘15 LHEH
S. esculentum CYP97C FAl\Ehil1 ‘RRR\Y\P¢LHKR)1 1\051 11HHH,
CYP97C1 FAI\1 :1” 11 ‘11’11’11’ \\’\1’~1,11RR‘11. 1‘1l '1‘\“1-' LHHH.
CYP97C2 FAI \F( 1 1‘11 11’11‘11’§"1\ 1’.~1 11111111 \1\10 '1.‘15 1‘1311’.‘
CYP97C3 FAIS 1.117111%111111119121111111111 11111-111111 :51
CYP97C12 1 1911:1111-411 1|11111111111 E1111!” 111111 -‘11111Ro1-211 10111

H. vulgare CYP97A
CYP97A4

S. esculentum CYP97A
CYP97A7

M. truncatula CYP97A
CYP97A3

CYP97A11

CYP97A5

CYP97A6

111’ \1)(1:1\1‘ 11 11’11’1’11\1’.-\1 11'1111
111’\l)(11i1111 11’11’|\’\1 \1’\.‘1| 1E.1\\\
1 11’\1)(111:liulx:\.1\’1\’\1\1"\|.11:1\)\M‘111

11111111112111111111.1111'1111-1111M111—1.11.1 -

11V\D11L111\ HRHflH

1118.111": 11;

1 11’.\1

1’\1 11| 1\1' ‘
11>1.:\111111111 111111111111111111'11-‘11
“111111111 11§1 11111111

111131111 1111 \1\1’.\[.IIR11)\ .1111.
1 1.1-1111111511111 1 - 11111111011111 51111111"

 
 

109

  

’11\'111(1IM
111-11101.“ ‘

  
   

‘11 11111 $11111.
“1118 1.11. ‘81)11’1.
1l1| ‘1‘11111

CYP97Bl4
CYP97E1_S.costatum
CYP97BlS

CYP97B7

M. truncatula CYP97B

CYP97B3
CYP97B4
G.biloba CYP97B
CYP9736
CYP86A1

CYP97C4

CYP97C10

D. carota CYP97C

S. esculentum CYP97C
CYP97C1

CYP97C2

CYP97C3

CYP97C12

H. vulgare CYP97A
CYP97A4

S. esculentum CYP97A
CYP97A7

M. truncatula CYP97A
CYP97A3

CYP97A11

CYP97A5

CYP97A6

CYP97Bl4
CYP97E1_S.costatum
CYP97815

CYP97B7

M. truncatula CYP97B
CYP97B3

CYP97B4

G.biloba CYP97B
CYP97B6

CYP86A1

CYP97C4
CYP97C10
D.carota CYP97C

S. esculentum CYP97C

CYP97C1
CYP97C2

   
    

 

Supplementary data Fig. 3A con’t

‘14"; 1\“I.1\’1\
‘11’\I1| ‘11'11’ ’11’1’
.11’11‘fl11 ‘1’1‘
.,11"1111

 

11w l\!lllh\111511 $111"-
1111 n1\.111111..§\1\1¥11s111
"111\

"1‘1.K‘11 111911 1511\1'
.111H‘1xg1111111jgm111113 1151».
1N11.11.1 1u11n"1w\1\1111 11 --w
----E|\l11 1»n111h11.11 IM\111H»1
—-——En1111. 1>1111n11w.1111\.n1h>1

:11. 1sm111n11uH11111n1na11¥
:11. 1.3111H11.31"1\1111»11‘w
————En1111 E>H111h111111 1‘1w1h»1\‘w
":11. r>m111n11uk1

"21' M111111 H1 11m111

|
1\111.911111(11“‘1\1\‘111111\1'#.‘1

GGGAA‘1DWI. 1.\W1\11)11h1“. 1111D11a:

5. M111 1“DIP»1H

111HIIMN111\111951

:1n'111h!11|\111' '
‘171111

""‘S‘I ‘ 159.1 11.1)‘111‘11 11.1111511u115'
“‘“S‘1 M1 5:1111 11“111 1151.1» "“$1 11511 EN
-—-- ‘11M1J9‘1111‘111

11111111151.

1i11‘1 ‘11

1911111
’

 

H1

_\.|5.'-
1 M1111. 1» 111
- 11111 §"11

111131‘"11.§‘"‘
11111.1)"
11011.1."1"

M11\131‘1_'1‘1

in“ ” ”" .
11' 11 111 a:
£1.11I111111

1:11111‘1‘. 11111

'11

110

3A Acon’t

     
   
  
     
     
  

Supplementar data Fi_.
CYP97C3 ’ N
CYP97C12
H. vulgare CYP97A
CYP97A4
S. esculentum CYP97A
CYP97A7

———————————— .I’IWKII 1111 '11
1111111111511 1111 JI\I\

  

   
   

 
 

I I.\IIII DUI I
M. truncatula CYP97A ------------ .I’IW IIEI’I . #:1111111 [MII‘
CYP97A3 ----------- I.\I)I I,I)I)I-I ‘
CYP97A11 ----------- I I ’III IR. \I |~I21~I‘\I)II. III‘II.
CYP97A5 -----------
CYP97A6 ------------ IJI’
CYP97Bl4 ————————————

CYP97E1_S. cost atum
CYP97B15

     
    
 
  
 

 

 

 

   

     
    

  
   

     
  
  
   
     

   
   
    
   
  

 
        
  

 
 
    
  

CYP97B7 ————————————
M. truncatula CYP97B 1 ' ------------ ,
CYP97133 ———————————— ’1 ’1 ' 11511.1 111-...I1i111e1 [IN
CYP97B4 ———————————— I
G.biloba CYP97B ————————————
CYP9786 ——————————— . .121)
CYP86A1 1111112 ———————————— IQKAM 1SEI1I1KKWTYMNEA||DAR®13PSMIIISRF
I 480
CYP97C4---~—:nII.’11\‘I) 11115111115111 SI'IEIIISBVI‘II.) .111I1 I I 51 II 1 11 ;111: 111. 11 11‘ II 11
CYP97C10 —————— 111111 1151111111 .1>11I131M111I11111511.111;;1111111511 11.11.11
D.carota CYP97C 31111\1 I.)I’SII.I\’III 1Hx11111xx 111111111151111'511111 111; 111.1 1311
s. esculentum CYP97C —————— 1151111111111 11511. 1111 1'51- -E\S\ 11.1I1111IIS111 1.111 11;1.1'111511111111l11
CYP97C1 111111 111).\‘11.1I111.1 11.115151“ .1I1111 1 15111 1' 11 ;111: '11 1:11 "".1111111
CYP97C2 —————— 1312111191 1111\11111-11s111~1rxu111I11 1311111111111 1111 1111
CYP97C3 SAAAAA 111-11 1111\111I1111 '1 151111 11I11111511111111 111%.11111
CYP97C12 ”1111151111“ .1111. 1.1 '55111 ssu1111111111-311111111111. 1. 1.1"
H. vulgare CYP97A ——————+1 111\_1 11.115111'11 1.1 1'111111151111 1 1I11 111.111111- 1.11.111; 151111 11111 11
CYP97A4 ——————— 151£.111\111111{fl11*111.1 1'1111111sx11111. 1111111. 111111 111;111 131111 11111 11.
s. esculentum CYP97A ——————+.1 5111\Bi1111511i1111. 1'11111.13\K1111I1111 11.1 111 1.11. 1111151111. 11111 11
CYP97A7 ——————— .iIIIIerDI’sIII 111-1.1 111113111111 1I111 1.111..11111;111£:1$.1,11'1.'1‘111111.
M. truncatula CYP97A —————— 1111\FI111NII 1111. 1::1; 111111511111. 1I11111 111111 1.11;111~:'15.1.11‘1.11111 11
CYP97A3 —————- ~:1:1.11\B-I1111\‘11+1111.1Is1;_11111s\111111I111 111I1111'111"'R111'1 '11 1311
CYP97A11 —————— ‘51111\ 1111 91111111II1.111111\1I1111I11 1111.1. 1.11;111£11.311111 1111111
CYP97A5 -—~~—— 1£1£1£ £11111511¥1 1.1 1 s1.£111 153111 1.11I11 111. 111.111 111;111-:";.111111.1 1111.11.
CYP97A6 ——————— GGGGGSS 11111.1 111; 51.11115 1114111. 1I11111 .111 1.11 11111; 11 .1.1M1.1I1H1.m1
CYP97B14 QN ————— KE111E111I111111111111 .111‘1'1'111'1'111H1 ‘
CYP97E1_S.costatum BE ----- '1hEI)IT\‘l\'Ml.II’I.)I)I .\II\II. I \( III I I. \\III.I
CYP97815 ER ————— ‘ {IIIHEIII 1I11111 1.11.111 1.11;1112'1'13I11-1111‘ 1'111
CYP97B7 QQ ————— 1H;11111111111.11I1 1111 111111.1.;-.11111"1"11111'.'11111M111.
M. truncatula CYP97B QQ ----- ' 1‘. 1\1’ I.
CYP97B3 QE —————
CYP97B4 QQ ----- 831111 .'

ll]

G.biloba CYP97B

 

 

 

 

CYP97BS

cvpsem 1.x ——————————————————— 1RD wmkxmmvwm
T 540

CYP97C411.1\||1 1\‘11 ‘ 2‘. . ’

CYP97C10 ““““““““ GRR 1:111 ;HVJ1D1K1

D.carota CYP97C

S. esculentum CYP97C
CYP97C1 “
CYP97C2 IL 9"11\hh\1!
CYP97C3 1 I
CYP97ClZ

H. vulgare CYP97A

1.
‘ I§=f Ir

I
11‘1‘1 1\11a1I

11\

——-—GRS 11 1w11fl1111fl1

“i11111\1a 1111
i, II
; 1.1,,I .‘ 1a

. “1'
1'1 1)1‘1\1\11\1m1\

111.11\1\K11\1

 

 

1
CYP97A4 . 1 Us:
S. esculentum CYP97A .Q‘ 1Di
CYP97A7 .N 1.1
M. truncatula CYP97A ,T 1m 1EH1
CYP97A3 ‘ 115111511
CYP97A11
CYP97A5
CYP97A6
CYP97Bl4
CYP97E1_S.costatum
CYP97315 1
CYP97B7 .‘ .' 1111-1111
M. truncatula CYP97B .‘ “I“! l\151
CYP97B3 . .1‘ l ”11H“
CYP97B4 ‘- v N: H W111§\15
G.biloba CYP97B ‘- ' ““111 L
CYP97BG
CYP86A1
CYP97C41\\\\
CYP97C10 :1\\1

D.carota CYP97C

S. esculentum CYP97C1
CYP97C1

CYP97C2

CYP97C3

CYP97C12

H. vulgare CYP97A
CYP97A4

S. esculentum CYP97A
CYP97A7

M. truncatula CYP97A

    
 
   

1’111‘1‘\1.11\’1\‘s‘11 311111

   

  

111’1’\1.11111e1,1 91111
PNPP\11HH~1

 

112

'11'1111¥11\\‘1"

uliilfl .

‘1\\1‘1111
‘)1¥:\\1‘111ll\53'\...
1..)1.11¥1\H\\1 1111\\ 311151

1\\1‘1D\1 1‘ ‘
11.-.111 11W‘1‘ 11'IW1H111 D “1‘1

-111
911111;1W\1HN0P1 ‘

.11

.h51111\\“ 1H101~+
11911 11 1\111 111111311

1115111119111

11an111>\1

 

CYP97Bl4
CYP97E1_S.costatum
CYP97BIS

CYP97B7

M. truncatula CYP97B
CYP97B3

CYP97B4

G.biloba CYP97B
CYP97B6

CYP86A1

CYP97C4

CYP97C10

D.carota CYP97C

S. esculentum CYP97C
CYP97C1

CYP97C2

CYP9703

CYP97C12

H. vulgare CYP97A
CYP97A4

S. esculentum CYP97A
CYP97A7

M. truncatula CYP97A
CYP97A3

CYP97A11

CYP97A5

CYP97A6

CYP97BI4
CYP97E1_S.costatum
CYP97BIS

CYP97B7

M.
CYP97B3

CYP97B4
G.biloba CYP97B
CYP9786

CYP86A1

CYP97C4
CYP97C10

truncatula CYP97B

 

1111111111.
1i)”i i)\i
1111.111111
111111111
113111111
1119:1111.
11111111,
1111111111. ‘
1111111111,
1111111111.
1111111111.
1111111111.

11111_‘
1111111 1'1

, 71.5111

iiii“.

.1 '1 1.1111141
' 1.1.11. ‘1 1.1 11.11
11.1111 111.111
1.1..1'1111111111

in I iiii\:.i

in
q

IG—LDGPV ———————————————————— 1 11 .1Ill

LDGPV ——————————————————— 1 111

1113011 ———————————————————— 11 1

 

“(I
1‘ i!
i‘1'\i' .1

W1
HKiI
3211
("i
v.1

ESIEG-WAGFDPDRS
NKGIEG-WAGFDPYRSQG‘-AL
SNPDFGGKWAGYRPDAVTGGAAL

IC—LDGPV ———————————————————— 1 1

1.. TADGER ———————————————————— mp n '11 q

 
 
     
   
 

11:1

i'i.‘i1
1.!

5"1

11111-1111
1 111.511.111.1-
1111111 1. 11111.11

i i\'i\(\.(1i)(.ii"

1i i\’i\(\(1il((i’.‘1.
1i’ix’ i\( 'ii iii 1 1

zifii'x'i" ‘

iiii i

.1

*‘i i in 11i'|\i\i\i11(¥i"".
iiii'Iii1(1:ii\i\i\(:ii¥i"'i

[H

(i_.
11

'. i'sii,’7(ii1(1i1i‘i\'i\..((\1i

. Q‘Iiii 1’ii(i\(i(1i‘1i-\
‘1 “‘“i! MIIIH '1i11’ix’i1kiiiiitiW

TnWIH‘iiII1uIWu1i1HHF

.- 1. .‘1.
i!iii'iiii1i151(i\iiiIii'.ii' ..i

111111111~1111111y
‘1 iii (11,111.11
ii“i( 1'\(i-'('i\ii(1i)(}i1
‘ 1’i( (1(1( .1111 ‘1
i“i(1hmiM|k(

(iii'iil

(\ii\

‘i'jjilsi
Ifli
.iii
11)
L‘ iii'1i(1(( 3i\"((11ii(.1i'
1:) 'iii(((ii\11(1(111(i
111H111\11u1
iiiifl'11111'

15”..

  

Suu lementary data Fi.
D12 i

(1
(1
1
(PHA(\hhwl.
1
(i
1
\1(

MM” "
((1111111111111w11.
iiiiixiliii'mii Iiiiii"' ".

:iiil

3A con’t

i i(i\(.i.(1i)(ii5 i ' 1

EFFZ'?¥!§'53¥3'?JF?

-—31—3~1c}21—‘a

 

 

ll3

D.carota CYP97C u .‘.32
S. esculentum CYP97C 9‘. I
CYP97C1 “ ‘
CYP97C2 , q ~
CYP97C3 ‘. ‘ ..I Q. 'IIIIIIIIIII I
CYP97C12 i- ‘ ‘I‘IIIIIIIII ‘IIII
H. vulgare CYP97A ' ‘
CYP97A4

S. esculentum CYP97A

 

 

 

 

 

 

 

 

 

 

 

CYP97A7 “‘.EI‘II ‘II'II’II .I
M. truncatula CYP97A \IIII\HII ”QL'
CYP97A3 IIIII iIIIIIIr IIIIIIIIIIIIEIII I
CYP97A11 \§| \‘.I| ‘.IIII IIIII.ESKHIIPDGE O‘IIII t:':IIII||II (I|l\.:'I, I‘
CYP97A5 I {I
CYP97A6 ‘ ‘- ,IIIIIEwIIIII.
CYP97Bl4 f P we'IIEIIIIIIIflIIW' ‘
CYP97E1_S.COStatum t-i " 7' INIIIIIIIIIII In.
CYP97315 I ml '. IMIII
CYP97B7
M. truncatula CYP97B \I
CYP97B3
CYP97B4
G.biloba CYP97B
CYP97B6
CYP86A1
780
CYP97C4
CYP97C10 LTSTFFSHRWQNLLANNYQQD
D. carota CYP97C SAAVSSISR
S. esculentum CYP97C SVL‘“n
CYP97C1
CYP97C2 REPDFALSGSR
CYP97C3 ASGSSGVAGGKQL““
CYP97C12 EVPEAERVDWANLMPAKELGGDEWYAPWNQPAPAASGKCPMGH -----------------
H. vulgare CYP97A KPPVIPNLEMKIVS ----- DPEGSTSSTASVAVSTASIASGEGQQVEVSTSQV--—--—-
CYP97A4 KPPVIPNLEMKVIS ----- DSPENMSTTTSMPVSAASIASGEDQQGQVSATRI -------
S. esculentum CYP97A RPPIVPNLEMATLE V
CYP97A7 RPPIMPKL
M. truncatula CYP97A KPPIVPSLQMSTLE ----- VDPSVSISDKTEEIGQKDQVYQAQKS ---------------
CYP97A3 KPLDIPSVPILPMD--—--TSRDEVSSALS
CYP97A11 ALTELDDVDVVRGS ----- IDAPTASADEVDVLIAEDIDAGTVEKIQRATKIIEEEEARA
CYP97A5 VPPTSSGVAETVSTGYAFACGPAVMPVASAEVVAAPATAAGGGCPFHTAAGAAVPAATMS
CYP97A6 VPPPAPRAPAAAAG ----- AAAGSCPHAAAAAATAAAAAAVGCPHAAAAATSGAPAGVTP
CYP97Bl4 QPVTTATAAAV

 

 

CYP97E1_S.COStatum TNPIPGTNEWWTKQHLMRGLSSTGRPYTSDEDAAWTTSANGMRP ———————————————
CYP97BlS

 

114

Supplementary data Fig. 3A con’t

 

 

 

 

 

 

 

CYP97B7 VH

M. truncatula CYP97B AH

CYP97B3

CYP97B4

G.biloba CYP97B YSESLQ

CYP9786 SGGSGSGAPGAAAKVPATV
CYP86A1 VLA

CYP97C4 ---------

CYP97C10 ---------

D.carota CYP97C ---------
S. esculentum CYP97C —————————
CYP97C1 _________
CYP97C2 _________
CYP97C3 _________
CYP97C12 _________
H. vulgare CYP97A —————————
CYP97A4 _________
S. esculentum CYP97A —————————
CYP97A7 _________
M. truncatula CYP97A ---------
CYP97A3 _________
CYP97A11 R ________
CYP97A5 LRPTGPPSA
CYP97A6 Q ________
CYP97Bl4 _________
CYP97E1_S.costatum —————————
CYP97815 _________
CYP97B7 _________
M. truncatula CYP97B ---------
CYP9783 _________
CYP97B4 _________
G.biloba CYP97B —————————
CYP97B6 _________
CYP86A1 _________

115

Supplementary data Fig. 3B Amino acid alignment of CRTR-B homolog genes used for

NJ tree construction.

 
 
  
  
  
 
  
  
 
  
  
   
 
 
  
 
 
 
 
  
 
   
   

6O
- sativus Cl ------------ 1.1 *\ PSATTLAAS PSGARIILLSSLPVRRPVE-———RRIR
- sativus CZ ------------ ” \ .~ PAATTLAA PAGARVILF SVRRVVD————LWPS
. Pseudonarcissus C1 ------------ ‘ '2". AAPPALAISS--APRIRRVILF HSRQIG ________ w

. sativa C1 ------------ ALLRLA-—---FA
. sativa C2 ———————————— 'x AVPRLR _______ p1
. sativa C3 ———————————— \u ; SGRALP _______ FS
mays C1 ----------- AGRALP ----- FS
mays C2 ------------- \Xl - AGRALP ------- FS
palaestina C2 ---------------- ‘ * ILKPP-—-CLLFSPV
palaestina C3 ---------------- Ki” ----- LLLHSKQDILNRP---CLLFSPV
. palaestina C1 ------ --MLA fl‘ ----- LFL IRNPP—--CLLFSPL
. trichocarpa C1 ----------- ”K “TVPKPFRYNSV----SHLLP AASLF--FPPIRHQ
. trichocarpa C2 ------------- ' J;'|TVSKPSGYIFT ----- SHLL ITTTSLS--LPFIRHQ
. vinifera C1 ------------ ‘ ‘ u ————— SFTA SVISLS—-SFLTPVT
. thaliana C1 ---------- - ’...‘~ i ----- SFSSSSTDFRLRLP --------
rapa C1 ------------- ".‘ ,
. thaliana C2 ------------ iXT..~ P ----- SFS

  
 
  
  

  

ISTAVFP —————————

 

   
 
 
 

 

 

  

. carota C1 ------------ .1 \J‘ ; IWLFAP-----SVRKL
. carota C2 ------------ TE~‘L' f
. erecta C1 ----------- n\ " i. ITCH----FPFSF
. esculentum Cl ——————————— . s STl‘SH--VSPISPFS
annuum C1 ——--TTGRYHYQ ‘x-' > i' PS--VYPITPFS
. esculentum C2 ------------ 11 i“ W --‘-PFLSPKSASTAPP--VLFFSPLT
annuum C2 ------------ T. ‘1 ‘ \ I
. lutea C1 ----------- .
. truncatula (:1 ————————— — ' A'I'ILKPYNLLQPSTS—SPSPSPK’ILFFTP—---LRSFPHS
unshiu c1 — ———————— :' MIWIQFCLLTMQPSSLLmLFAP ----- m—m
. arabica c1 —— ————————— n, -VAAGA(II'VCFRVN-----SFL LVADS--LTLSPLA
taeda c1 —-MWSNSSPOGL . DS’ILSPFLAP'I‘KAAGPPPPPGRTASRYYVAFARASSVNRNGLSSG
taeda (:2 MGLRSVSSPYGLSKCDSISPPLSS’IKPAAAP"LGRAVYCYYLALARASSVNRNGFRSA
. reinhardtii c1 MMLASRPAVALGAR-"AQPQVLR
. pluvialis C1 ——————— TF HKPMALPHIGPPPHLHESFAA’I'IMSKLQSISVKARRVELARDITR
. tauri C1
120
c. sativus C1 PP-LL ---TA . AEEKTI‘PFLDD ——————————— VEEEKSIAPSN —————
C. sativus C2 ALGQ “ EA -------------------- EKRMAPSN -----
N. pseudonarcissus C1 PPIRNRRKRS----KST 1‘ASDVDVGKSNGGDG--IVDKIERLKKQEQLMISKS -----
o. sativa c1 PLPARRLAVP ————— :- ———————————————————— DARR ————————
o. sativa c2 VAGRRAVAAP---—-TRA ‘. z AGV ———————————————— DAWAWEED---
o. satlva 03 mumsi . ESQQAPAPSPPPTVPVPVPS EAAAAAAR -----
z. mays c1 PLTTARAP 'n EH—--PAAAPAPVAPVPET EARAAAAR -----

116

Z.
A.
A.
. palaestina C1
. trichocarpa C1
. trichocarpa CZ
. vinifera C1

. thaliana C1

9=0ww°9FPowpwfiPP?w><vv>

mays C2
palaestina C2
palaestina C3

rapa C1
thaliana C2
carota C1
carota C2
erecta C1
esculentum C1
annuum C1

. esculentum C2

annuum C2
lutea C1
truncatula C1
unshiu C1

. arabica C1

taeda C1
taeda C2

. reinhardtii C1
. pluvialis C1

tauri C1

. sativus C1
. sativus C2
. pseudonarcissus C1

sativa C1
sativa C2
sativa C3
mays C1

mays C2
palaestina C2
palaestina C3

. palaestina C1
. trichocarpa C1
. trichocarpa C2
. vinifera C1

. thaliana C1

rapa C1

. thaliana C2
. carota C1
. carota C2
. erecta C1

   
    
 
   
  
  
   
 
 
 

Supplementa data Fig. 33 can’t
PLASTRAP IIXPQDTAA --------- PAAPVP-- EARAAAAR -----
VIMS GD'OIIE ‘GRTRNLDIPQIEEEEEN ------ LIEQTDSDIV---
OIIEII." LIEQTDSGII---
" VFEQMNSASV---

_ , IEDG---IEIE—DD— ————— SPESSN ----------
‘ RRQNSPIENDERPES----TSSTNAIDAEYLAL--—-
- QRQSSPVDNDERPE —————— RTNVIDPELLAL-———

  
 
    
 

"“ RNDSSGKPENNADRD——---EVSREEIEAGSCS---—
‘ " NESPVAAAEAEESSR ----- EVEKQI IESFTVAGG—
“‘GGDSNSNSNNNSDSNSN ----- NPGLD-LNPAVMNRN--
IEEQILAT----
IEEQISAT----
RIQVEINEEKSLA

   

PTLVPRPGMVSNLRLQPVKIAIPIVASETSQVMEAP —————— I
PKVCLHAQRCSLVRLR-VAAPQTEEALGTVQAAGAG _______

 

 

180
- II~ II.1R‘\[\’[\€>ILII‘ I'Il lfl"\\\1>\~|3ql ln‘u} .. III .II‘- III'III. ------------
— II“.- I» IIIIIIIII N11)! I I\“I\ I IIfi I..II Irg-‘III. ————————————
" KmI‘. it??? ' —————————————
-— I ‘l ‘93: .‘II GNTPSPNSRSHLT

——- .I‘ ‘ 1\I\\II\|1|II\\‘\‘1\;IArIN~KI ' ,. ————————————
— EI‘I\1H*\I\KI‘I-1IEIIII‘ \\‘I\‘11In15” ’

\KKYIIKHIII\II\I\ Ihled ‘ g .;'{n ------------

—— I I/eII I"'IIII IE1“ “I 5 ’l ‘I :1 ————————————
‘— HI Ibhhil .“:I\gl I“ . .‘ 1 H ‘ 1 ------------
- i\'\.\lrl \1\MI \HI‘II 1111““ ‘.1 liIi‘I- ------------

K\WII mKIh1[1“1III\\II\ II II‘I‘I.\\IIK‘iuiLhL ------------
‘ EHII\II‘HIIIIIII\\\I\|‘|!”\IIIIIHI limb ------------
- I" \Kld\" l\ I I1‘ \ J 1I] I\ II II \\II|\\ 'Hah ------------
—-KL aKKK41K§IIII\'”' \i§ .3"f ‘rT‘FEJ.EfI ------------
- _ ‘I " IIIIII‘Isllfin-J ------------
— Mi K‘zKIIIwHI‘I {II I\\\ [I W 1%:\.'\‘I“Ii\'i \“5- W: ------------
— ’ I \NKKIIKt[Il‘\“l\ IIIlli' ‘ " i i” ------------

117

. esculentum C1

annuum C1

. esculentum C2

annuum C2

. lutea C1

. truncatula C1
. unshiu C1

. arabica C1

. taeda C1

. taeda C2

. reinhardtii C1
. pluvialis C1

. tauri C1

. sativus C1

. sativus C2

. pseudonarcissus C1
. sativa C1

. sativa C2

. sativa C3

mays C1
mays C2
palaestina C2
palaestina C3

. palaestina C1
. trichocarpa C1
. trichocarpa C2
. vinifera C1

. thaliana C1

rapa C1

. thaliana C2

. carota C1

. carota C2

. erecta C1

. esculentum C1
. annuum C1

. esculentum C2

annuum C2

. lutea C1

. truncatula C1
. unshiu C1

. arabica C1

. taeda C1

. taeda C2

. reinhardtii C1
. pluvialis C1

  

— m \1Kl1Rkks1Rl11|V\\§15<I3115
1\1n1\kkk\lhllI1\\\\d\\fiu IIIII.II\III
II- .‘hkk\lhll\l\\\‘1\fl

hfi\kkk\1hllIl\\\\wal Ilwl\\15\IIRls HIM

& \KKleHIIIl\\\\Iw\i

1\\‘\II\II‘ISI\III\IIK

V:\1AI IRththlII\\1\IS i.ll\d\\i\\ I.II

\\H>\mIllwm

\\3n\\\(lnw‘d

\\I"\\\(

. i;

"1‘. ”n1 \1\1\1\‘EN

"1\11;§\1 \IIII1€\1'II#‘II.\"I
11' ‘ ‘.[l

118

 

Su I I lementa

data Fig. 33 can’t
HI\H\\IIKI~IUHMM ------------
i H ————————————
IIxHI'1\I\IR1 WIIHI ————————————

lwl\I'EIIIhl IHIIH

[::‘:‘«‘I()I[‘(I
HJII:
‘.IIhI a HlKh
W'1\IIKI¥
IIIIfl-L III

IIIJII \I‘ ".III
I-\EIII\:§I‘1iIII_‘HIIIII’-

-\\0u‘1\\\l I I1KIL'9\1

"’..S\U\\\IW1 I R.1\HH\11H\

. " ZIa .I III: 1KI

\ S\h\\\(U11\ N.\\HW%\1IH
\[S\h\\\(1111\HI\HI
“‘rIIsIM \\h111‘\P \HI

”7\I\\h\‘\hlll“\"\Hhst
”rI l\\(\\\hIl[“P”\H1I
"9115\C1\\IHMWI IIIIII'”

.

:I \. "'- I

rIii \I. \("lngl ¥ !
ijINIIII IuIII e unIgI.

CHIPV’ 1‘ fIIquIIII IIIIIIIII
uI II: := '5\l\\I\I\IdlII\P“\hK IIHIs
.flIgfii IIsIIIIIuIII IIIIHIfiyInIs
ILl”\l 3118\h1\\h‘ IIIRIIHIIMH
u11§ 7\L%\h\\\thFfi\R1\HR\11H\>
IuII‘I 'IIIsIm\IIuIIIIIRIIHHIIIHIs
.I\§III "7118\h\\\(IHV"1R11HH111HEw
hLlP .. .q" ’\]\\I\\\hWII‘ IIIHEIIIHIs
iE\Pl\lHI\llelfli IIInIII IHIIHEIIIHE~
nIII LMJII[I\lSII1\\IAII\I\RM'\HQ\1%H\S
IIIIIIIIEI'I '\]5\h III WIIJIR‘HHII‘WIV
hh\PrSEHFh1-\1$\hI\\h1111\[ IHKI11H\I
uh\PISEH!\IIEIRFMI\\MHFIIII IHKIIIHI>
JIEPJSEHIEIFII;3}I\I'III"MIR"HNIIIHIs
‘ IIIIn' IIIIIIIIIIIIIIIIxHfiIIIHI»
HFCII\LS\h\1\IIHLIW1\R1IHEELIHIb
HIC1F\I\\U\\\CHEI”W\I IIIIIIIHI.
: II. 5118\u\\\hUFIM‘ ‘1‘ILI1‘HII
EWIhlIgln\hIfi\hWEIR‘IIIXHRIIAHX\
I="' K‘I IHLI (W1111h1\HR\IfiHII
$§.II3 II ;‘\K‘\NRH'”‘
. quII M1.I\K\\lk

I
I
E
I

”h\>

‘ll\>

\ 5

O

panvoozmowowaPP>w><vv>>?wwpppzpn

.tauri C1

sativus C1
sativus C2
pseudonarcissus C1
sativa C1
sativa C2
sativa C3
mays C1
mays C2
palaestina C2
palaestina C3
palaestina C1
trichocarpa C1
trichocarpa C2
vinifera C1
thaliana C1
rapa C1
thaliana C2
carota C1
carota C2
erecta C1
esculentum C1
annuum C1
esculentum C2
annuum C2
lutea C1
truncatula C1
unshiu C1
arabica C1
taeda C1
taeda C2
reinhardtii C1
pluvialis C1
tauri C1

sativus C1

sativus C2
pseudonarcissus C1
sativa C1

sativa C2

sativa C3

mays C1

mays C2
palaestina C2

38 can’t
P IIIIIHIIII IIHI.IIKIHIHIG$

Supplementar data Fi

———————————————————— PWWMMNEI

   
   

300

IIHIIIIIIIISIIIIRI’R IIGI’I II ,\I)\I7IIII\I.\'PII \I IN; ‘I7I7II1\GI l I’GI LII II.GI III'H
- IHMHRSHP IH SS \D\III IVI\PIIIII IHRGILPIIIIIIGIGIII
I IHIHHZSIRIKIII ' 5 |I\IRI III' I\I\IJII ‘II iIIIII RI I I'IIIIWII (IJIIIIII'II

IMIHWHSWHPR AI\I\\HH\ NIH PPHPIHIIIIIP
IVINIIII NPIIEIIEI GIII‘GIGIGI P-

PHRHI75HHRPR “ 7
" |IV)\I\IE\I\R\I5II IIIIHRII I\’GIII GIGIGIH

IIPPPI5PPPNPIIPPP
IIVDIIII\\\\’II IPIIIPIP \RGIIIIII PIP
P)i \PIIII I\INP I§II MIPIIWPP II’GIIIIIGIGIH

        

 
  
 

   
        
 

  
    
    

  
 
 
   
 

    
 

  

   
  

    

It.

II II

IPPPI5PPPPPIIPPI

MHPHISHHRP

EIHESRHREI IIVDIIIIIVILPIIIIII aPIIP<anII WI GEI

II 1PI5PPPEP I:N\WPIII1\IIPI1\IIR . IIL’GEIIIE UIEI\
I GPI IIVDIHIII VIIPII II IIIHRM IIIIIIIIII IH

PPPP5PPPPP )(PI II\D\I\IE\I\P ”E IGIII (IIIPIHIIIGIII 1H

NI HI5RHR’PEDGPI PIPIHII 1\IIPI1 ‘II IGIIgiIIVRGIIIGIGIGIII

HRHIHHHI’R

I“

I

.IIII‘iIIIIISIIII IR’R .GI’I I,_'71I\I)\I IIIVIII’IIEII gIIIIIII RGIVIIIICIGII III III'I'I
,II

I

III

   
  

IHISHHRRREIG’I IIVIIIIIVV IEI’IIGII¢IGII RIII’GILIIIGIGII\

RHISHHRRR IGI’I
I ‘II IInHHnIR PI] HIIIIE\‘\RIIGII"III RGIVRI IIIIIIIIII‘

IIIIIIIIISIIIIIII’IIEIGa IVDIIII V\IPIIRI III'WIRRGni IIIIIII
III 53m

I7I.\I\I II II.\I\I’IIGII IXGII IIGIII’IIILIIIIGI I'III‘

III
II\IIII\IIII'\I’I\' II\I)\I‘II\I\I‘ IIII II IIIRIIIj II7IIIGIGI.I GII‘
IMNWMWMRIGHEIHMI VUPHRI MIIPHIHRIMI
\IIMIISHHKPR GP]! VDIIIIEV I‘IIIIIIIIIIIHRGIIHGIIIIIGIGIII

IIIIIISIIIIRI’R‘IIII .VI)\I7. II.\\\I’IIIII IIIGIIIIIxIIII’GIIIII I,IGII\

IIIIVIJII IIEI I\I’II GII I gIGII‘IIII'I IIII’G I III IGI (II I\'

IPPPI5PPPPPI
MPHMHISHHRPR
PPPP15PPPPP
11u1a5IPP1P
PPPPI5PPP c
WIPPPI5PPPPP
IIIHPHICSHHRPR
1H.IPPP5PPPPP
.E.H15RHWPR
INIPN 5MP PP

IGRI

   

II\DIIIII\I\ IIINEEIPINPPP IPGIHIIRIIII‘
II\HII\ 1\IIPI1 IIIIMPI IPGIIIGIGIIIII

I

I

:II‘I7I’I

IP (In II\I\IIII\I\PIIIIIE H-NPPIIII IIIIIIPPIIIII
wl

I

WIN

     
        
    
   
  
 

    
    
 

(III I‘IIRIIIIIEIGIIHRIIVRGII7"‘I IIII

I
ilxn\1 IVIIPIIIII IGIIHRIIIRGIIIIII IIH
”I
”I
II

I
GH
I

PPI IIVHIIIIIVIIPI "IIIIPPINIPPII IGIGII IIII
\IIIIII\IIP\IIIM ‘

IIEIIVP‘RR
“HRIIIIVG IPPRI

IHIIIM‘ GEIRP H

-II IIIPIIINGIII IPPIPII
IFPIIIIIP.I11P
IIHIHCIGIGIGII.
I JHPIIIIRHIHII
360

IIIIIIP HIIPPPPPIPP IPIPIPIPIIIIPIIPP5PPPN IPIPIPPPPPPPPPI
IILIIPIIPPPIIPPPIPIIIIIPIPIIIPIIIIPPIPH5LPIQPIIII IPPPPIIIPP
_P5IIPI\PPIIIPPP \PIIIPIPIIPPIIIIP1IPPIIPIIIIPIPIII PPHIII
(PIIPIIPIPI\PPPIPIPPIIIIPIIPPIIIIPPIPPNPPIN,IPI IIIPIPIIII
[(PIIPIIPPPIIHPPIPIIIII\IPIIPPI\IIPPIPPIIPHaIIPIPIIIPPPHIII
IPPIIPI %\ID PIHRRIPIGI’ III\\PIIPP\IIIPNI IPPTPPI NI;\PIP. w IIPIPIIII
I PPIIP IPPPIIPPPIPIPP INIfIPIIRRIIIIH IPPNPPHH \PPPII IPIPIIII
IIPIIPIIPPIIIPPPIPIPP INIPIIPPIIIEINIPIInPIeIIPI IIIIIRIIII‘
«PHIHPNIEHPHP IIHPHIIIIPHHPKWPPMI IHIIMIHI

   

’H

 
 
 

   

          

       
  
    
     
     
         

119

A.
A
P
P
V
A
B
A
D
D
T
S
C.
S
C
G
M
C
C
F
P
C
H
O

palaestina C3

. palaestina C1
. trichocarpa C1
. trichocarpa C2
. vinifera C1

. thaliana C1

. rapa C1

. thaliana C2

. carota C1

. carota C2

. erecta C1

. esculentum C1

annuum C1

. esculentum C2
. annuum C2

. lutea C1

. truncatula C1
. unshiu C1

. arabica C1

. taeda C1

. taeda C2

. reinhardtii Cl
. pluvialis C1
. tauri C1

. sativus C1
. sativus C2
. pseudonarcissus C1

sativa C1
sativa C2
sativa C3
mays C1

mays C2
palaestina C2
palaestina C3

. palaestina C1
. trichocarpa C1
. trichocarpa C2
. vinifera C1

. thaliana C1

rapa C1

. thaliana C2

. carota C1

. carota C2

. erecta C1

. esculentum C1
. annuum C1

 

Sundemenm dMth.3Bcomt
MiHA\jFLHNGL_HRRFPVGLIAXVP\FNKLXXXHQIHHSHKFQHVPEMLFLGPghLEE\
FGMAYMFVHDGLVHRRFPVCPI\XVP\FRKVAXKHQIHHTDAFQHVP\ULFLUPKELEE\
FGLKXMFVHDGLVHKRFPVGPIAW\P\FRR\\A\HEIHHSDKFNM\P\CLFLMVREIEE\
I"(}_\I.\\I\Il5\ III)(}L\’IllgRH’VUP I .\|.|‘\'l’\ I-‘fi|\’\l\.\.\ll()rl l IllSEIx‘l-‘NUVM (iLFUfiEili I I-II-L\
FGHAYMFVHDGLVHRRFPVGPI\\\P\URKV\§\HQLHHSDKFNUVP\GLFLMPMELEE\
1%;L\YMF\HDGLVHKRFP\GPIXW\P\IRK\\AXHQLHHTDKFNHVP\ULFLUPKELEH\
l-‘(}I.\\'.\I["\lll)(il.\'[lKIx’l-‘PVUPl.\|.|‘\I’\II[\’K\L\A\.\IIQHlllTDIx’I-‘WH'MULI’LMI’KICI lil-Z‘x
F(BL\\MF\1HXH.VHKRFIWTH’I\\\1’\!RK\I\\\lkM.HHTWHHJ«,\[W(;LFIIH4\EYHE\
I-‘(i<l;.\\fl\lf5\lll)(}11\'ll}\'l\’13fl\'(}l’I \EVMI<‘l\’1\'\'.\.\.\lIQIJIIIEEM‘EM["1(H.151.(11’I\'[i\"lil)\
IKLHXYMF\1HXHI\HERFPVCPE\\VP\WRENAXXIUMJHUQEKFNM\IW(HJWJLPEELEE\
[KLHXYMF\1HXH2VHRRFE\IH’L\X\P\'RR\I\\\HNIJHYU£KFNH\IW(JJWIJ’KELEE\
l“(i\l.\\'Xll’\'lllXiLVl lKRI’l’VUI ’k’,\.\\ M Iix’K\I\.\C\I 151,111 ISI‘IKFAXA H (M’fit 3i ’linl.I<ll~L\
FGMAYMFVHUGLVHKRFPVGPEX\VP\lRKVAAXHELHHSEKFNU\PXGLFLGPKELEE\
l’(i.\l.\\fl\ll’\'lll)(}l.\'llKIx’I-‘l’\ UP I .\\\ H'FRRV \_\.\llQl.llIISI)I\'l-‘I'I'(i\’l ") (H.151.(xl’lx'Iil.!{l-L\
1“(i.\l.\\'F.II?\'IIDGLVIIKRI-‘H (;PHI’AVH I5El\’\l\.\.\l INLHHSDKIWA lr’\'(}l.l3l ,(il’lx'lil‘lim
F(HL\\RH7VIHXiL\IIKRlH’\(fl)L\u\1WIW€R\I\\\HEIJHISIHUiNH\1W(iLFIIH’KPJJiE\
FG:\\MFVHDGLVHKRFPVGPI\X\P\FfiRV\\\HQLHHSDAFEM\P\hLFLUPKLYkE\
F(AL\\\H’\IHX}L\IIKRIA’V(J)L\E\IWI’RR\I\\\IHJL1HlSIHfiFh}i\P\(HJ:LCIHU{LEIA
l5UNL\Y\H3VHIXiL\1[KRIJ’V(H’IA\\\}’\IW<R\I\\A1H)LIHISIHU:N(AI’E(HJ5L(HW§EIJifi\
I“(L\I,\\I\II"\'I II)(il.\ lllx‘lx’H’VGPl ,\|7\'P\ ifl ‘\ \ \I [(21,] H IM1)1\'I“N( }\ I’\ 01.1"] AH’Ix’liLIiI-Ll

   
 

I"(i_\l.\\\Il“\'lII)(}L\'IIIx’lx’l“P\'(i}’I;\!.l“\'l’\I K‘\'.\;\.\I[QLIII];I)I\‘I5N(;\M(il.l’l,(;l’l\[{l:H
[fulfikllfilII)(;L\’HI\’I\’l-‘I’\'(}P I .\\'H’\ w I" \m 1 ll] I'l‘l‘klitfi I)1~‘(;3\11"1.(;E1c1 .1sz
'mmMMmw'l[WWWHF. 'm. \IMlnrem-gmvu'umflm1.1-11.51”qu

GPLG J1 ‘2' ":[JMA[“(AFLMIEH.“

 

YNNSAEIKT----

IH
‘11

INSRSLDVK -----
ETLDAIQ ————
EAMDAIR -----

 

 

 

 

RPVVCVVRTSV CQSVPNEF -------

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

120

OIO'U'UCOEOOU)

. esculentum C2
. annuum C2

. lutea C1

. truncatula C1
. unshiu C1

. arabica C1

. taeda C1

. taeda C2

. reinhardtii C1
. pluvialis C1
. tauri C1

 

 

 

 

 

 

 

ISKGLL ————————

 

 

 

121

Supplementary data Table 1. Non-synonymous substitutions per site (Ka) and
synonymous substitutions per site (Ks), and corresponding confidence intervals (C.I.) in
CRTR-B genes of the indicated organisms. a Number of nucleotide base pairs used. b

Comparison between CRTR-B homolog 1 and 2 ° Comparison between CRTR-B homolog

1and3.

122

 

 

 

oz: 33 55 $3 $3 33 mm 98a
32c 23 32 23 83 $3 .3 nag
83 ES 83 23 $3. 33 Ra 338
$3 32 :32 £3 a; 83 8a $8835
522 22 ca: 52

sduflo Q Sdufio 5 an .02

 

123

Supplementary data Fig. 4 C02 fixation rates of WT and (13 mature leaves in a light

dependent manner. Data are means i SD from three biological replicates.

>
4.;
ON
0.
9)
«>3
1

rem-l

 

mmol C02 m'2 s'1
o N -hr 0, m

 

 

{a
_2 j 500 1000 1500
_4 nmol m'2 s’1
B 2
‘- l
'm 1 l 5
‘7' l w
E ‘ l i i J
5' 0 “ TL 2.
2 ; 9
o
E '1 c: 2e _
E 2 T L 20 40 60
" nmol m'2 s'1
-3 —

124

Supplementary data Fig. 5 Signal intensity of CRTR-BI and CRTR-B2 obtained from
microarray data obtained from developing seeds. Stage 6, mid torpedo to late torpedo;
Stage 7, late torpedo to early walking-stick; Stage 8, walking-stick to early curled

cotyledon; Stage 9, curled cotyledon to early green cotyledon; Stage 10, green cotyledons.

8000? + CRTR-Bf

 

i
3' a 4— TR-az
E aooog CR
l
2 mol
'2 i
.9 §
0 2000
0L. .P

125

BIBLIOGRAPHY

126

Adams, W.W., Demmig-Adams, B., and Lange, O.L. (1993). Carotenoid Composition
and Metabolism in Green and Blue-Green-Algal Lichens in the Field. Oecologia
94, 576-584.

Akiyama, K. (2007). Chemical identification and functional analysis of apocarotenoids
involved in the development of arbuscular mycorrhizal symbiosis. Bioscience,
biotechnology, and biochemistry 71, 1405-1414.

Alberte, R.S., and Andersen, R.A. (1986). Antheraxanthin, a Light Harvesting
Carotenoid Found in a Chromophyte Alga. Plant Physiol 80, 583-587.

Alfonso, M., Montoya, G., Cases, R., Rodriguez, R., and Picorel, R. (1994). Core
antenna complexes, CP43 and CP47, of higher plant photosystem II. Spectral
properties, pigment stoichiometry, and amino acid composition. Biochemistry 33,
10494-10500.

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, L, Miller, W., and
Lipman, DJ. (1997). Gapped BLAST and PSI-BLAST: a new generation of
protein database search programs. Nucleic acids research 25, 3389-3402.

Alvarez, V., Rodriguez-Sail, M., de la Fuente, J.L., Gudina, E.J., Godio, R.P.,
Martin, J.F., and Barredo, J.L. (2006). The crtS gene of Xanthophyllomyces
dendrorhous encodes a novel cytochrome-P450 hydroxylase involved in the
conversion of beta-carotene into astaxanthin and other xanthophylls. Fungal
Genet Biol 43, 261-272.

Armstrong, GA. (1997). Genetics of eubacteria] carotenoid biosynthesis: A colorful tale.
Annu. Rev. Microbiol. 51, 629-659.

Auldridge, M.E., McCarty, D.R., and Klee, H.J. (2006a). Plant carotenoid cleavage
oxygenases and their apocarotenoid products. Current opinion in plant biology 9,
315-321.

Auldridge, M.E., Block, A., Vogel, J.T., Dabney-Smith, C., Mila, 1., Bouzayen, M.,
Magallanes-Lundback, M., DellaPenna, D., McCarty, D.R., and Klee, H.J.
(2006b). Characterization of three members of the Arabidopsis carotenoid
cleavage dioxygenase family demonstrates the divergent roles of this
multifunctional enzyme family. Plant J 45, 982-993.

Barth, C., Krause, GH., and Winter, K. (2001). Responses of photosystem I compared
with photosystem II to high-light stress in tropical shade and sun leaves. Plant
Cell and Environment 24, 163-176.

Bassi, R., and Caffarri, S. (2000). Lhc proteins and the regulation of photosynthetic
light harvesting function by xanthophylls. Photosynthesis research 64, 243-256.

127

 

 

Benveniste, 1., Tijet, N., Adas, F., Philipps, G, Salaun, J.P., and Durst, F. (1998).
CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty
acid omega-hydroxylase. Biochemical and biophysical research communications
243, 688-693.

Berger, 8., Papadopoulos, M., Schreiber, U., Kaiser, W., and Roitsch, T. (2004).
Complex regulation of gene expression, photosynthesis and sugar levels by
pathogen infection in tomato Physiologia Plantarum 122, 419-428.

Blanc, G, Hokamp, K., and Wolfe, K.H. (2003). A recent polyploidy superimposed on
older large-scale duplications in the Arabidopsis genome. Genome research 13,
137-144.

Blankenship, RE. (1992). Origin and early evolution of photosynthesis. Photosynthesis
research 33, 91-111.

Blasco, F., Kauffmann, 1., and Schmid, RD. (2004). CYP175A1 from Thermus
thermophilus H827, the first beta-carotene hydroxylase of the P450 superfamily.
Applied microbiology and biotechnology 64, 671-674.

Bouvier, F., Dogbo, 0., and Camara, B. (2003). Biosynthesis of the food and cosmetic
plant pigment bixin (annatto). Science (New York, NY 300, 2089-2091.

Bouvier, F., Keller, Y., d'Harlingue, A., and Camara, B. (1998). Xanthophyll
biosynthesis: molecular and functional characterization of carotenoid
hydroxylases from pepper fruits (Capsicum annuum L.). Biochimica et biophysica
acta 1391, 320-328.

Britton, G (1998). Overview of Carotenoid Biosynthesis. In Carotenoids, G Britton, S.
Liaaen-Jensen, and H. Pfander, eds (Basel: Birkhéiuser Verlag), pp. 30-83.

Burbulis, LE, and Winkel-Shirley, B. (1999). Interactions among enzymes of the
Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci U S A 96, 12929-
12934.

Canutescu, A.A., Shelenkov, A.A., and Dunbrack, R.L., Jr. (2003). A graph-theory
algorithm for rapid protein side-chain prediction. Protein Sci 12, 2001-2014.

Castillo, R., Fernandez, J.A., and Gomez-Gomez, L. (2005). Implications of
carotenoid biosynthetic genes in apocarotenoid formation during the stigma
development of Crocus sativus and its closer relatives. Plant Physiol 139, 674-689.

Chen, M., Bibby, T.S., Nield, J., Larkum, A.W., and Barber, J. (2005). Structure of a
large photosystem II supercomplex from Acaryochloris marina. FEBS letters 579,
1306-1310.

128

Cogdell, R.J., and Gardiner, A.T. (1993). Functions of carotenoids in photosynthesis.

Cameron, J.M. (1999). K-Estimator: calculation of the number of nucleotide
substitutions per site and the confidence intervals. Bioinforrnatics (Oxford,
England) 15, 763-764.

Croce, R., Morosinotto, T., Castelletti, S., Breton, J., and Bassi, R. (2002). The Lhca
antenna complexes of higher plants photosystem 1. Biochim Biophys Acta 1556,
29-40.

Cunningham, F.X., Jr., and Gantt, E. (2005). A study in scarlet: enzymes of
ketocarotenoid biosynthesis in the flowers of Adonis aestivalis. Plant J 41, 478-
492.

Cunningham, F.X., Jr., Lee, H., and Gantt, E. (2007). Carotenoid biosynthesis in the
primitive red alga Cyanidioschyzon merolae. Eukaryotic cell 6, 533-545.

Cunningham, F.X., Jr., Pogson, B., Sun, Z., McDonald, K.A., DellaPenna, D., and
Gantt, E. (1996). Functional analysis of the beta and epsilon lycopene cyclase
enzymes of Arabidopsis reveals a mechanism for control of cyclic carotenoid
formation. Plant Cell 8, 1613-1626.

Dall'Osto, L., Lico, C., Alric, J., Giuliano, G, Havaux, M., and Bassi, R. (2006).
Lutein is needed for efficient chlorophyll triplet quenching in the major LHCII
antenna complex of higher plants and effective photoprotection in vivo under
strong light. BMC plant biology 6, 32.

Demming-Adams, B., and Adams 111, W. (1992). Carotenoid composition in sun ad
shade leaves of plants with different life forms. Plant Cell and Environment 15,
411-419.

Demole, E., Enggist, P., Sauberli, U., and Stoll, M. (1970). Structure and synthesis of
damascenone [2,6,6-trimethyl-l -trans-crotonyl-1 ,3-cyclohexadiene], odorous
constituent in rose oil (Rosa damascena Mill.). Helv. Chim. Acta 53, 541-551.

Dogbo, 0., Laferriere, A., D'Harlingue, A., and Camara, B. (1988). Carotenoid
biosynthesis: Isolation and characterization of a bifunctional enzyme catalyzing

the synthesis of phytoene. Proceedings of the National Academy of Sciences of
the United States of America 85, 7054-7058.

Dudareva, N., Andersson, S., Orlova, 1., Gatto, N., Reichelt, M., Rhodes, 0., Boland,
W., and Gershenzon, J. (2005). The nonmevalonate pathway supports both
monoterpene and sesquiterpene formation in snapdragon flowers. Proceedings of
the National Academy of Sciences of the United States of America 102, 933-938.

Dutton, D.R., McMillen, S.K., Sonderfan, A.J., Thomas, RE, and Parkinson, A.

129

(1987). Studies on the rate-determining factor in testosterone hydroxylation by rat
liver microsomal cytochrome P-450: evidence against cytochrome P-450
isozymezisozyme interactions. Arch Biochem Biophys 255, 316-328.

Emanuelsson, 0., Nielsen, H., and von Heijne, G (1999). ChloroP, a neural network-
based method for predicting chloroplast transit peptides and their cleavage sites.
Protein Sci 8, 978-984.

Galpaz, N., Ronen, G, Khalfa, Z., Zamir, D., and Hirschberg, J. (2006). A
chromoplast-specific carotenoid biosynthesis pathway is revealed by cloning of
the tomato white-flower locus. The Plant cell 18, 1947-1960.

Ginalski, K., Elofsson, A., Fischer, D., and Rychlewski, L. (2003). 3D-Jury: a simple
approach to improve protein structure predictions. Bioinformatics (Oxford,
England) 19, 1015-1018.

Giuliano, G, Giliberto, L., and Rosati, C. (2002). Carotenoid isomerase: a tale of light
and isomers. Trends in plant science 7, 427-429.

Glazko, GV., and Nei, M. (2003). Estimation of divergence times for major lineages of
primate species. Molecular biology and evolution 20, 424-434.

Grabowski, B., Cunningham, F.X., Jr., and Gantt, E. (2001). Chlorophyll and
carotenoid binding in a simple red algal light-harvesting complex crosses
phylogenetic lines. Proceedings of the National Academy of Sciences of the
United States of America 98, 2911-2916.

Graham, S.E., and Peterson, J.A. (2002). Cytochrome P450. In Methods in
Enzymology, E.F. Johnson and MR. Waterman, eds (San Diego: Academic Press),
pp. 15-28.

Gu, X. (2006). A simple statistical method for estimating type-ll (cluster-specific)
functional divergence of protein sequences. Molecular biology and evolution 23,
1937-1945.

Guindon, S., and Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate
large phylogenies by maximum likelihood. Systematic biology 52, 696-704.

Hashimoto, T., Nakamura, Y., Nakamura, F., Shirakura, T., Adachi, J., Goto, N.,
Okamoto, K., and Hasegawa, M. (1994). Protein phylogeny gives a robust
estimation for early divergences of eukaryotes: phylogenetic place of a
mitochondria-lacking protozoan, Giardia lamblia. Molecular biology and
evolution 11, 65-71.

Hess, W.R., Rocap, G, Ting, C.S., Larimer, F., Stilwagen, S., Lamerdin, J., and
Chisholm, S.W. (2001). The photosynthetic apparatus of Prochlorococcus:

130

Insights through comparative genomics. Photosynthesis research 70, 53-71.

Holm, L., and Sander, C. (1991). Database algorithm for generating protein backbone
and side-chain co-ordinates from a C alpha trace application to model building
and detection of co-ordinate errors. Journal of molecular biology 218, 183-194.

Holt, N.E., Fleming, GR., and Niyogi, K.K. (2004). Toward an understanding of the
mechanism of nonphotochemical quenching in green plants. Biochemistry 43,
8281-8289.

Holt, N.E., Zigmantas, D., Valkunas, L., Li, X.P., Niyogi, K.K., and Fleming, GR.
(2005). Carotenoid cation formation and the regulation of photosynthetic light
harvesting. Science (New York, NY 307, 433-436.

Howitt, C.A., and Pogson, B.J. (2006). Carotenoid accumulation and function in seeds
and non-green tissues. Plant Cell Environ 29, 43 5-445.

Isaacson, T., Ronen, G, Zamir, D., and Hirschberg, J. (2002). Cloning of tangerine
from tomato reveals a carotenoid isomerase essential for the production of beta-
carotene and xanthophylls in plants. The Plant cell 14, 333-342.

Johnson, E.A., and Schroeder, WA. (1996). Microbial carotenoids. Advances in
biochemical engineering/biotechnology 53, 119-178.

Jones, D.T., Taylor, W.R., and Thornton, J.M. (1992). The rapid generation of
mutation data matrices fi'om protein sequences. Comput Appl Biosci 8, 275-282.

Kaminsky, L.S., and Guengerich, RP. (1985). Cytochrome P-450 isozyme/isozyme
functional interactions and NADPH-cytochrome P-450 reductase concentrations
as factors in microsomal metabolism of warfarin. Eur J Biochem 149, 479-489.

Kelley, R.W., Reed, J.R., and Backes, W.L. (2005). Effects of ionic strength on the
functional interactions between CYP2B4 and CYP1A2. Biochemistry 44, 2632-
2641.

Kim, J., and DellaPenna, D. (2006). Defining the primary route for lutein synthesis in
plants: the role of Arabidopsis carotenoid beta-ring hydroxylase CYP97A3.
Proceedings of the National Academy of Sciences of the United States of America
103, 3474-3479.

Klimmek, F., Ganeteg, U., Ihalainen, J.A., van Roon, H., Jensen, P.E., Scheller, H.V.,
Dekker, J.P., and Jansson, S. (2005). Structure of the higher plant light
harvesting complex I: in vivo characterization and structural interdependence of
the Lhca proteins. Biochemistry 44, 3065-3073.

Koniger, M., Harris, G, Virgo, A., and Winter, K. (1995). Xanthophyll-cycle pigments

131

and photosynthetic capacity in tropical forest species: a comparative field study
on canopy, gap and understory plants. Oecologia 104, 280-290.

Koniger, M., Harris, GC., Virgo, A., and Winter, K. (1995). Xanthophyll-cycle
pigments and photosynthetic capacity in tropical forest species: a comparative
field study on canopy, gap and understory plants Oecologia 104, 280-290.

Krause, GH., Koroleva, O.Y., Dalling, J.W., and Winter, K. (2001). Acclimation of
tropical tree seedlings to excessive light in simulated tree-fall gaps. Plant Cell and
Environment 24, 1345-13 52.

Krause, GH., Grube, E., Koroleva, O.Y., Barth, C., and Winter, K. (2004). Do
mature shade leaves of tropical tree seedlings acclimate to high sunlight and UV
radiation? Functional Plant Biology 31, 743-756.

Krubasik, P., and Sandmann, G (2000). Molecular evolution of lycopene cyclases
involved in the formation of carotenoids with ionone end groups. Biochemical
Society transactions 28, 806-810.

Kulheim, C., Agren, J., and Jansson, S. (2002). Rapid regulation of light harvesting and
plant fitness in the field. Science (New York, NY 297, 91-93.

Kull, D., and Pfander, H. (1995). Carotenoids. (Birkhauser, Basel).

Kumar, S., Tamura, K., and Nei, M. (2004). MEGA3: Integrated software for
Molecular Evolutionary Genetics Analysis and sequence alignment. Briefings in
bioinforrnatics 5, 1 50-163.

Lee, A.I., and Thornber, J.P. (1995). Analysis of the pigment stoichiometry of pigment-
protein complexes from barley (Hordeum vulgare). The xanthophyll cycle
intermediates occur mainly in the light-harvesting complexes of photosystem I

and photosystem 11. Plant Physiol 107, 565-574.

Leung, J., and Giraudat, J. (1998). Abscisic Acid Signal Transduction. Annu Rev Plant
Physiol Plant Mol Biol 49, 199-222.

Li, R, Murillo, C., and Wurtzel, ET. (2007). Maize Y9 encodes a product essential for
15-cis-zeta—carotene isomerization. Plant Physiol 144, 1181-1 189.

Li, W.H. (1983). Evolution of duplicated genes. In Evolution of genes and proteins, M.
Nei and R.K. Koehn, eds (Sunderland, MA: Sinauer associates), pp. 14-37.

Lichtenthaler, H.K. (1999). The l-deoxy-D-xylulose-5-phosphate pathway of isoprenoid
biosynthesis in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 47-65.

Liu, Z., Yan, H., Wang, K., Kuang, T., Zhang, J., Gui, L., An, X., and Chang, W.

132

(2004). Crystal structure of spinach major light-harvesting complex at 2.72 A
resolution. Nature 428, 287-292.

Livak, K.J. (1997). User Bulletin No. 2: ABI PRISM 7700 Sequence Detection System
(Foster City, CA: PE Applied Biosystems), pp. 11-15.

Lokstein, H., Tian, L., Polle, J.E., 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.

Lynch, M., and Conery, J.S. (2000). The evolutionary fate and consequences of
duplicate genes. Science (New York, NY 290, 1151-1155.

Ma, Y.Z., Holt, N.E., Li, X.P., Niyogi, K.K., and Fleming, GR. (2003). Evidence for
direct carotenoid involvement in the regulation of photosynthetic light harvesting.
Proceedings of the National Academy of Sciences of the United States of America
100, 4377-43 82.

Makova, K.D., and Li, W.H. (2003). Divergence in the spatial pattern of gene
expression between human duplicate genes. Genome research 13, 1638-1645.

Mark, M., Ghyselinck, N.B., and Chambon, P. (2006). Function of retinoid nuclear
receptors: lessons from genetic and pharmacological dissections of the retinoic
acid signaling pathway during mouse embryogenesis. Annual review of
pharmacology and toxicology 46, 451-480.

Marquardt, J., and Hanelt, D. (2004a). Carotenoid composition of Delesseria lancifolia
and other marine red algae from polar and temperate habitats. EUROPEAN
JOURNAL OF PHYCOLOGY 39, 285-292.

Marquardt, J., and Hanelt, D. (2004b). Carotenoid composition of Delesseria lancifolia
and other marine red algae from polar and temperate habitats. European Journal
ofPhycology
39, 285-292.

Matusova, R., Rani, K., Verstappen, F.W., Franssen, M.C., Beale, M.E., and
Bouwmeester, H.J. (2005). The strigolactone germination stimulants of the plant-
parasitic Striga and Orobanche spp. are derived from the carotenoid pathway.
Plant Physiol 139, 920-934.

Maxwell, K., and Johnson, GN. (2000). Chlorophyll fluorescence-~a practical guide.
Journal of experimental botany 51, 659-668.

Mestres, J. (2005). Structure conservation in cytochromes P450. Proteins 58, 596-609.

133

Miller, S.R., Augustine, 8., Olson, T.L., Blankenship, R.E., Selker, J., and Wood, A.M.
(2005). Discovery of a free-living chlorophyll d-producing cyanobacterium with a
hybrid proteobacterial/cyanobacterial small-subunit rRNA gene. Proceedings of
the National Academy of Sciences of the United States of America 102, 850-855.

Moore, RC, and Purugganan, MD. (2005). The evolutionary dynamics of plant
duplicate genes. Current opinion in plant biology 8, 122-128.

Nam, J., dePamphilis, C.W., Ma, H., and Nei, M. (2003). Antiquity and evolution of
the MADS-box gene family controlling flower development in plants. Molecular
biology and evolution 20, 1435-1447.

Nam, J., Kaufmann, K., Theissen, G, and Nei, M. (2005). A simple method for
predicting the functional differentiation of duplicate genes and its application to
MIKC-type MADS-box genes. Nucleic acids research 33, e12.

Nei, M., and Rooney, A.P. (2005). Concerted and birth-and-death evolution of multi gene
families. Annual review of genetics 39, 121-152.

Nelson, D.R., Zeldin, D.C., Hoffman, S.M., Maltais, L.J., Wain, H.M., and Nebert,
D.W. (2004). Comparison of cytochrome P450 (CYP) genes from the mouse and
human genomes, including nomenclature recommendations for genes,
pseudogenes and alternative-splice variants. Pharmacogenetics 14, 1-18.

Nelson, N., and Ben-Shem, A. (2004). The complex architecture of oxygenic
photosynthesis. Nat Rev Mol Cell Biol 5, 971-982.

Niyogi, K.K., Bjorkman, 0., and Grossman, AR. (1997). The roles of specific
xanthophylls in photoprotection. Proceedings of the National Academy of
Sciences of the United States of America 94, 14162-14167.

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. Photosynthesis research 67, 139-145.

Ohno, S. (1970). Evolution by gene duplication. (Springer-Verlag).

Olson, V.A., and Owen, I.P.F. (1998). Costly sexual signals: are carotenoids rare, risky
or required? Trends in Ecology and Evolution 13, 510-514.

Palczewski, K. (2006). G protein-coupled receptor rhodopsin. Annual review of
biochemistry 75, 743-767.

Park, H., Kreunen, S.S., Cuttriss, A.J., DellaPenna, D., and Pogson, B.J. (2002).
Identification of the carotenoid isomerase provides insight into carotenoid
biosynthesis, prolamellar body formation, and photomorphogenesis. The Plant
cell 14, 321-332.

134

Partensky, F., Hoepffner, N., Li, W., Ulloa, 0., and Vaulot, D. (1993).
Photoacclimation of Prochlorococcus sp. (Prochlorophyta) Strains Isolated from
the North Atlantic and the Mediterranean Sea. Plant Physiol 101, 285-296.

Pascal, A.A., Caron, L., Rousseau, B., Lapouge, K., Duval, J., and Robert, B. (1998).
Resonance Raman spectroscopy of a light-harvesting protein from the brown alga
Larninaria saccharina. Biochemistry 37, 2450-2457.

Paulsen, H., Finkenzeller, B., and Kuhlein, N. (1993). Pigments induce folding of light-
harvesting chlorophyll a/b-binding protein. European journal of biochemistry /
FEBS 215, 809-816.

Phillip, D., Hobe, S., Paulsen, H., Molnar, P., Hashimoto, H., and Young, A.J. (2002).
The binding of Xanthophylls to the bulk light-harvesting complex of photosystem
II of higher plants. A specific requirement for carotenoids with a 3-hydroxy-beta-
end group. The Journal of biological chemistry 277, 25160-25169.

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, 1627-1639.

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.

Pogson, B.J., Niyogi, K.K., Bjorkman, 0., and DellaPenna, D. (1998). Altered
xanthophyll compositions adversely affect chlorophyll accumulation and
nonphotochemical quenching in Arabidopsis mutants. Proceedings of the National
Academy of Sciences of the United States of America 95, 13324-13329.

Quinlan, R.F., Jaradat, T.T., and Wurtzel, E.T. (2007). Escherichia coli as a platform
for functional expression of plant P450 carotene hydroxylases. Archives of
biochemistry and biophysics 458, 146-157.

Qureshi, N., and Porter, W. (1981). Biosynthesis of isoprenoid compounds. (New York:
Wiley).

Rohmer, M. (1999). Comprehensive natural product chemistry. (Pergamon: Oxford).

Rohmer, M., Bouvier, P., and Ourisson, G (1979). Molecular evolution of
biomembranes: structural equivalents and phylogenetic precursors of sterols.
Proceedings of the National Academy of Sciences of the United States of America

76, 847-851.

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

135

photosystem I] light harvesting complexes. The Journal of biological chemistry
274, 10458-10465.

Rupasinghe, S., Baudry, J., and Schuler, M.A. (2003). Common active site architecture
and binding strategy of four phenylpropanoid P450s from Arabidopsis thaliana as
revealed by molecular modeling. Protein engineering 16, 721-731.

Russo, C.A., Takezaki, N., and Nei, M. (1996). Efficiencies of different genes and
different tree-building methods in recovering a known vertebrate phylogeny.
Molecular biology and evolution 13, 525-536.

Schagerl, M., and Pichler, C. (2000). Pigment composition of freshwater charophyceae.
Aquatic Botany 67, 117-129.

Schoch, GA., Attias, R., Le Ret, M., and Werck-Reichhart, D. (2003). Key substrate
recognition residues in the active site of a plant cytochrome P450, CYP73A1.
Homology guided site-directed mutagenesis. European journal of biochemistry /
FEBS 270, 3684-3695.

Schoch, GA., Yano, J.K., Wester, M.R., Griffin, K.J., Stout, C.D., and Johnson, ER
(2004). Structure of human microsomal cytochrome P450 2C8. Evidence for a
peripheral fatty acid binding site. J Biol Chem 279, 9497-9503.

Schubert, N., Garcr'a-Mendoza, E., and Pacheco-Ruiz, I. (2006). CAROTENOID
COMPOSITION OF MARINE RED ALGAE. J Phycol. 42, 1208-1216.

Schwartz, S.H., Qin, X., and Loewen, M.C. (2004). The biochemical characterization
of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-
derived compound inhibits lateral branching. The Journal of biological chemistry
279, 46940-46945.

Scott, E.E., He, Y.A., Wester, M.R., White, M.A., Chin, C.C., Halpert, J.R., Johnson,
ER, and Stout, GD. (2003). An open conformation of mammalian cytochrome
P450 234 at 1.6-A resolution. Proc Natl Acad Sci U S A 100, 13196-13201.

Six, C., Worden, A.Z., Rodriguez, F., Moreau, H., and Partensky, F. (2005). New
insights into the nature and phylogeny of prasinophyte antenna proteins:
Ostreococcus tauri, a case study. Molecular biology and evolution 22, 2217-2230.

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.

Straub, O. (1987). Key to Carotenoids. (Birkhauser, Basel).

Sun, Z., Gantt, E., and Cunningham, F.X., Jr. (1996). Cloning and functional analysis
of the beta-carotene hydroxylase of Arabidopsis thaliana. J Biol Chem 271,

136

24349-24352.

Swofford, D.L. (2003). PAUP*. Phylogenetic Analysis Using Parsimony (*and Other
Methods). version 4. (Sunderland, Massachusetts: Sinauer Associates).

Taylor, J.S., and Raes, J. (2004). Duplication and divergence: the evolution of new
genes and old ideas. Annual review of genetics 38, 615-643.

Thayer, S., and Bjorkman, O. (1990). Leaf xanthophyll content and composition in sun
and shade determined by HPLC. Photosynthesis research 23, 331-343.

Thompson, J .D., Gibson, T.J., Plewniak, F., Jeanmougin, F., and Higgins, D.G
(1997). The ClustalX windows interface: flexible strategies for multiple sequence
aligmnent aided by quality analysis tools. Nucleic acids research 24, 4876-4882.

Tian, L., and DellaPenna, D. (2001). Characterization of a second carotenoid beta-
hydroxylase gene from Arabidopsis and its relationship to the LUTl locus. Plant
Mol Biol 47, 379-388.

Tian, L., and DellaPenna, D. (2004). Progress in understanding the origin and functions
of carotenoid hydroxylases in plants. Archives of biochemistry and biophysics
430, 22-29.

Tian, L., DellaPenna, D., and Zeevaart, J.A.D. (2004a). Effect of hydroxylated
carotenoid deficiency on ABA accumulation in Arabidopsis Physiologia
Plantarum 122, 314-320.

Tian, L., Magallanes-Lundback, M., Musetti, V., and DellaPenna, D. (2003).
Functional analysis of beta- and epsilon-ring carotenoid hydroxylases in
Arabidopsis. Plant Cell 15, 1320-1332.

Tian, L., Musetti, V., Kim, J., Magallanes-Lundback, M., and DellaPenna, D.
(2004b). The Arabidopsis LUTl locus encodes a member of the cytochrome p450
family that is required for carotenoid epsilon-ring hydroxylation activity.
Proceedings of the National Academy of Sciences of the United States of America
101, 402-407.

Till, B.J., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E.,
Burtner, C., Odden, A.R., Young, K., Taylor, N.E., Henikoff, J.G, Comai, L.,

and Henikoff, S. (2003). Large-scale discovery of induced point mutations with
high-throughput TILLING Genome Res 13, 524-530.

Tomitani, A., Okada, K., Miyashita, H., Matthijs, H.C., Ohno, T., and Tanaka, A.

(1999). Chlorophyll b and phycobilins in the common ancestor of cyanobacteria
and chloroplasts. Nature 400, 159-162.

137

Umeno, D., Tobias, A.V., and Arnold, EH. (2005). Diversifying carotenoid biosynthetic
pathways by directed evolution. Microbiol Mol Biol Rev 69, 51-78.

Vanderauwera, S., Zimmermann, P., Rombauts, S., Vandenabeele, S., Langebartels,
C., Gruissem, W., Inze, D., and Van Breusegem, F. (2005). Genome-wide
analysis of hydrogen peroxide-regulated gene expression in Arabidopsis reveals a
high light-induced transcriptional cluster involved in anthocyanin biosynthesis.
Plant Physiol 139, 806-821.

Vershinin, A. (1999). Biological functions of carotenoids--diversity and evolution.
BioFactors (Oxford, England) 10, 99-104.

Wagner, A. (2000). Decoupled evolution of coding region and mRNA expression
patterns after gene duplication: implications for the neutralist-selectionist debate.
Proceedings of the National Academy of Sciences of the United States of America
97, 6579-6584.

Wang, X., Shi, X., Li, Z., Zhu, Q., Kong, L., Tang, W., Ge, 8., and Luo, J. (2006).
Statistical inference of chromosomal homology based on gene colinearity and
applications to Arabidopsis and rice. BMC bioinformatics 7, 447.

Wentworth, M., Ruban, A.V., and Horton, P. (2004). The functional significance of the
monomeric and trimeric states of the photosystem 11 light harvesting complexes.
Biochemistry 43, 501-509.

Werck-Reichhart, D., Bak, S., and Paquette, S.M. (2002). Cytochromes P450.
(Rockville, MD: American Society of Plant Biologists).

Whatley, J.M., and Whatley, ER. (1987). When is a chromoplast? New Phytologist 106,
667-678.

Wisniewska, A., Widomska, J., and Subczynski, W.K. (2006). Carotenoid-membrane
interactions in liposomes: effect of dipolar, monopolar, and nonpolar carotenoids.
Acta biochimica Polonica 53, 475-484.

Yoshii, Y. (2006). Diversity and evolution of photosynthetic antenna systems in green
plants. PHYCOLOGICAL RESEARCH 54, 220-229.

Yoshii, Y., Hanyuda, T., Wakana, 1., Miyaji, K., Arai, S., Ueda, K., and Inouye, I.
(2004). Carotenoid compositions of Cladophora balls (Aegagropila linnaei) and
some members of the Cladophorales (Ulvophyceae, Chlorophyta): Their
taxonomic and evolutionary implication. Journal of Phycology 40, 1170-1177.

Young, A.J., and Frank, H.A. (1996). Energy transfer reactions involving carotenoids:
quenching of chlorophyll fluorescence. Journal of photochemistry and
photobiology 36, 3-15.

138

Zhang, J. (2003). Evolution by gene duplication: an update. Trends in Ecology and
Evolution 18, 292-297.

139

------

  

IIIIII

1]]

I]1]]1111111111]I]]]]]]]]]

1293 02956 5714