Differentially evolved glucosyltransferases determine natural variation of rice flavone accumulation and UV-tolerance

Article abstract of DOI:10.1038/s41467-017-02168-x

Decoration of phytochemicals contributes to the majority of metabolic diversity in nature, whereas how this process alters the biological functions of their precursor molecules remains to be investigated. Flavones, an important yet overlooked subclass of flavonoids, are most commonly conjugated with sugar moieties by UDP-dependent glycosyltransferases (UGTs). Here, we report that the natural variation of rice flavones is mainly determined by OsUGT706D1 (flavone 7-O-glucosyltransferase) and OsUGT707A2 (flavone 5-O-glucosyltransferase). UV-B exposure and transgenic evaluation demonstrate that their allelic variation contributes to UV-B tolerance in nature. Biochemical characterization of over 40 flavonoid UGTs reveals their differential evolution in angiosperms. These combined data provide biochemical insight and genetic regulation into flavone biosynthesis and additionally suggest that adoption of the positive alleles of these genes into breeding programs will likely represent a potential strategy aimed at producing stress-tolerant plants.

Full text of DOI:10.1038/s41467-017-02168-x

A R T I C L E
DOI: 10.1038/s41467-017-02168-x
OPEN
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1 National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research (Wuhan), Huazhong Agricultural University, Wuhan
430070, China. 2 Desert Research Center, Genetics Resource Department, Egyptian Deserts Gene Bank, Cairo 11735, Egypt. 3 Graduate School of Biological
Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan. 4 Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1,
Potsdam-Golm 14476, Germany. 5 Institute of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan 572208, China. Meng Peng, Raheel
Shahzad, Ambreen Gul and Hizar Subthain contributed equally to this work. Correspondence and requests for materials should be addressed to
J.L. (email: jie.luo@mail.hzau.edu.cn)
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A R T I C L E
N A T U R E C O M MU N IC A T IO N S | D O I: 1 0 . 1 0 3 8 /s 4 1 4 6 7 - 0 1 7 -0 21 6 8 -x
lants as sessile species produce hundreds of thousands of and the in planta biological function of glycosylated flavones are
different phytochemicals, especially secondary or specia- largely unknown.
P
lized metabolites, which play vital roles in the adaptation of
We describe here the genetic and biochemical dissection of
plants to the changing environments1,2. Although we know that natural variation in the major rice flavones and the character-
the majority of their chemodiversity comes from decoration of a ization of UGTs in a new clade of flavonoid glucosyltranferases
much smaller number of core skeleton precursor molecules, our including OsUGT706D1 (F7GlcT) and OsUGT707A2 (F5GlcT).
understanding of the genetic factors that shape this chemodi- We show that F7GlcT and F5GlcT enzymes evolved indepen-
versity remains limited3. Flavonoids are a group of plant sec- dently in plants, and furthermore, provide evidence that func-
ondary metabolites playing important roles in a wide range of tional allelic variation in these genes corresponds to the level of
physiological processes4–6 including ultraviolet-B (UV-B, UV exposure and that lines overexpressing OsUGT706D1 and
280–315 nm) protection7,8 which is becoming increasingly OsUGT707A2 have enhanced UV tolerance, indicating that tap-
important for yield stability in the face of present levels of ping the natural variation inherent in these genes may represent a
environmental deterioration9,10. In addition, their beneficial useful route to provide food security.
effects on human health as a dietary source of antioxidants attract
increasing attention11,12. Flavonoids consist of a common
C6–C3–C6 structure with two benzene rings (A ring and B ring)
interconnected by a three-carbon heterocyclic pyran ring (C ring)
and can be subdivided into six major groups, namely flavones,
flavanones, flavonols, flavanols, anthocyanins, and isoflavones
according to structural considerations (Supplementary Fig. 1).
Widely distributed in the plant kingdom, flavonoids usually
exist in their decorated forms catalyzed by glycosyltransferases,
acyltransferases, hydroxylases, and methyltransferases13,14. Gly-
cosylation of flavonoids confers structural complexity, as well as
molecular solubility and stability, subcellular transportability, and
biological activity. For example, glycosylation of anthocyanins has
been demonstrated to play pivotal roles in both the solubility and
stability of these pigments in a range of flowers15. Zhao et al.
reported that multidrug and toxin extrusion transporter (MATE)
1 and 2 show higher transport capacity for epicatechin 3′-glyco-
side and anthocyanin glycosides, respectively16,17. Furthermore,
the bitterness in Citrus species is caused by regiospecific flava-
none glycosylation18, while flavonol 3-O-glycosides have been
demonstrated to impart the astringent taste sensation in infusions
of black tea19.
Glycosylation of flavonoids is usually catalyzed by UDP-
dependent glycosyltransferases (UGTs)20 although exceptions
have also been reported in carnation (Dianthus caryophyllus)21,
rice (Oryza sativa)22, and Arabidopsis (Arabidopsis thaliana)23.
UGTs typically transferring sugars onto small-molecule acceptors
including plant secondary metabolites belong to Family 1 of the
glycosyltransferases super-family (CAZy database; http://www.
cazy.org/GlycosylTransferases.html). They are largely character-
ized by a C-terminus signature motif compromising 44 amino
acid residues (Plant Secondary Product Glycosyltransferase
(PSPG) box)24. A number of flavonoid UGTs have been char-
acterized in a wide range of plant species, including crops,
ornamental and medical plants14. These UGTs include flavonoid
3-O-glycosyltransferases (3GTs), flavonoid 5-O-glycosyl-
transferases (5GTs), flavonoid 7-O-glycosyltransferases (7GTs),
flavonoid 3′-O-glycosyltransferases (3′GTs), flavonoid glycoside
glycosyltransferases (GGTs) and flavonoid C-glycosyltransferases
(CGTs). Phylogenetic analyses of all characterized flavonoid
UGTs have suggested that they can be divided into group of
enzymes with different sugar-attachment specificities25,26, How-
ever, it worth noting that this scenario is based on results
exclusively from UGTs with flavonol and/or anthocyanin sub-
strate preferences.
Results
Flavonoid profiling among species. Application of a widely-
targeted liquid chromatography-mass spectrometry (LC-MS)
method34 resulted in the comprehensive profiling of flavonoids in
14 plant species spanning the pre-angiosperm, monocot and
eudicot clades, namely fern (Nephrolepis auriculata), peat moss
(Sphagnum palustre), citrus (Citrus sinensis), populus (Populus
deltoides), potato (Solanum tuberosum), tobacco (Nicotiana
tabacum), Arabidopsis, palm (Petiolus trachycarpi), maize (Zea
mays), bamboo (Phyllostachys pubescens), wheat (Triticum aesti-
vum), hulless barley (Hordeum vulgare), rice and Brachypodium
(Brachypodium distachyon). A total of 85 flavonoids belonging to
the three subclasses—flavone, flavonol, and flavanone—were
detected in our study (Supplementary Data 1) and their profiles
were visualized by hierarchical cluster analysis (HCA, Fig. 1a). On
the basis of their plant-specific accumulation patterns, flavonoids
could be clearly grouped into six subclusters, reflecting three
common modifications (Fig. 1a). Most flavonoids could be gly-
cosylated and their glycosides were grouped into distinct clades
depends on the core flavonoid skeletons. Flavonoids in cluster 1
accumulate most prominently in monocot plants and are mainly
represented by tricin O-glycosides, including tricin 5-O-hexoside,
tricin 5-O-hexosyl-O-hexoside, tricin 7-O-hexoside and tricin
7-O-rutinoside (Supplementary Data 2). Flavonoids in cluster 2
are mainly represented by flavone O-glycosides, such as apigenin
7-O-glucoside, apigenin 5-O-glucoside, luteolin 7-O-glucoside,
chrysoeriol 7-O-hexoside, with the highest levels detected in
monocots followed by citrus, populus and peat moss. By contrast,
flavonoids in cluster 3 are mainly composed of flavonol-O-gly-
cosides, including quercetin 7-O-hexoside, quercetin 3-O-gluco-
side-7-O-glucoside and quercetin 5-O-hexoside and were
commonly distributed in all species studied here. Flavonoid O-
glycosides could be further modified by acylation and acylated
flavonoid O-glycosides show distinct accumulation patterns
(clusters 4 and 5, Supplementary Data 2). Most flavonoids in
cluster 4 are malonyl flavonoid glycosides, which accumulated in
citrus, populus, and peat moss, whereas hydroxycinnamoyl fla-
vonoid glycosides, as representative flavonoids of cluster 5, are
cereal-specific. Additionally, flavonoids in cluster 6 are generally
tricin-lignans which accumulated to high levels in monocots
(Supplementary Data 2), where they have been demonstrated to
be involved in lignification35. Despite their low-level accumula-
tion, we managed to detect five flavones in Arabidopsis, including
two chrysoeriol derivatives, two tricin-glycosides and one tricin-
lignan (Supplementary Fig. 2). We subsequently choose rice, one
of the main model monocots, which has the highest accumulation
of most flavones, and is of massive importance to the human diet,
for assessing the natural variation in flavone content and
dissecting its genetic and biochemical bases.
To date, knowledge on flavonoid UGTs comes almost exclu-
sively from researches on flavonols which accumulate to rea-
sonable abundance in Arabidopsis27–31, while the biochemical
aspects of glycosylated flavones, ubiquitously distributed in grass
crops but almost absent in Brassicaceae32,33, remain largely
unknown. For example, few flavone-active UGTs have been
characterized and the natural genetic variation of rice flavones
2
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N A T UR E C O M M UN I CA T I O NS | D OI : 1 0. 1 0 3 8 / s 4 1 46 7 - 01 7 - 02 16 8 - x
A
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a
b
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10
5
L1
Glycosides
0
Acylated-
glycosides
Chr.
1
2
3
4
5
6
7
8
9
10 11 12
OsUGT706C1
OsUGT706B1 OsUGT706D1
OsUGT706F1
OsUGT88C2
OsUGT706E1 OsUGT88C3
c
Tricin-lignans
Others
L2
15
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10 15 20 25
log2RA
0
100
OsUGT706C3
OsUGT706C4
OsUGT706C1
OsUGT706D1
OsUGT706D3
OsUGT706D2
OsUGT706B1
OsUGT706E1
Chr.
1
2
3
4
5
6
7
8
9
10 11 12
99
f
OsUGT705A3
78
71
100
OsUGT705A1 OsUGT705A2
100
100
92
d
OsUGT706E2
OsUGT706F1
25
20
15
10
5
This study
92
L3
OsUGT706J1
86
100
OsUGT706G1
OsUGT88C3
OsUGT707A4
OsUGT707A5
OsUGT707A3
OsUGT707A2
100
87
96
100
100
OsCGT
100
ZmCGT
100 FeCGTa
FeCGTb
CGT
0
2
3
4
5
6
7
8
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OsUGT706E2
1
Gt3′GlcT
Chr.
OsUGT706D3
OsUGT706D2 OsUGT706C4
NtIS5s
94
100
Sb7GlcT
OsUGT706C3
OsUGT706J1
DbB5GlcT
72
99
7GT, 3′GT
At7GlcT
At7RhaT
OsUGT706G1
L4
OsUGT705A1
OsUGT705A3
100
99
100
This study
e
OsUGT705A2
30
Pf5GlcT
Vh5GlcT
Ph5GlcT
At5GlcT
Hv3GlcT
80
5GT
3GT
100
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83
100
Zm3GlcT
Mt3GlcT
Ph3GalT
Vv3GlcT
At3RhaT
At3GlcT
At3AraT
CrF3G6″GlcT
99
83
100
100
97
74
Chr.
1
2
3
4
5
6
7
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100
CmF7G2″RhaT
BpA3G2″GlcAT
CsiF7G6″RhaT
PhA3G6″RhaT
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100
OsUGT707A4
OsUGT707A3
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100
100
OsUGT707A5
OsUGT707A2
IpA3G2″GlcT
100
0.1
Fig. 1 I d en ti fic a t i o n a n d p h y l o ge n et i c a n a l y s i s o f fla v o n o i d U G T s i n r i c e. a L C - M S p r ofil i n g o f fla v o n o i d s i n 1 4 s p e c i e s a c r o s s t h e p l a n t k i n g d o m . H e a t m a p
v i s u a l i z a t i o n o f r el a t i v e d i f f e r en ce s o f fla v o n o i d s b y t h e a v e r a ge o f a l l v a l u e s i n t w o b i o l o g i c a l r e p l i c a t e s . S c a t t e r p l o t s o f a s s o c i a t i o n r e s u l t s f o r 4 fla v on oi d
t ra i ts a c r o s s t h e 1 2 r i c e c h r o m o s o m es . T h e s t r e n g t h o f a s s o c i a t i o n i s i n d i c a t e d a s t h e n e g a t i v e l o g a r i t h m o f t h e P v a l u e f o r t h e l i n e a r m i x e d m o de l. A l l
8
m e ta b o l i te - SN P a s s o c i a ti o n s w it h P v a l u e s b e l o w 6 . 6 × 1 0 – ( h o r iz o n t a l d a s h e d l i n e s i n a l l M a n h a t t a n p l o ts) a r e p l o tt e d a g a i n s t g e n o m e l o c a t i o n i n i nt e r v a ls
o f 1 M b . T r a i ts a r e a s f o l l o w s : a p ig e n i n 7 - O- gl u c o s i d e i n l e a f ( b) , t r i ci n O- h e x o s i d e -O- h e x o s i d e i n s e e d (c) , c h r y s o e r i o l O- f e r u l o y lh e x o s i d e -O- he x os id e i n
l e a f ( d) , a p i g e n i n 5 -O- g l u c o s i de i n l e a f ( e) . T h e M a n h a t t a n p l o t s f r o m s i x i n d i v i d u a l r e p l i c a t e ( t w o r e p l i c a t e s i n l e a f a n d f o u r r e p l i c a t e s i n s e e d) f or e a c h
l o c us a r e p r o vi d e d i n S u p p l e m en ta r y F i g. 3. C a n d i d a t e g ly c o s y l t r a n sf e r a s e s w i t h i n e a c h l o c u s a r e i n p u r p l e . f A n u n r o o t e d p h y l o g e n e tic t r e e w a s
c o ns t r u c te d a s d e s c r i b ed i n M et h o d s . O s U G T 8 8C 2 , w h ic h s h o w s 8 9 % i d e n t i t i e s t o O s U G T8 8C 3 , w a s n o t s h o w n i n t h i s t r e e a s i t s s h o r t p r ot e in l e n g t h.
B o o t s t r ap v a l u e s >7 0 % ( b a s e d o n 1 00 0 r e p l ic a t i o n s) a r e i n d i c a t e d a t e a c h n o d e ( b a r : 0 .1 a m i n o a c i d s u b s t i t u t i o ns p e r s i t e) . R A , r e l a t i v e p e a k a r e a
Genetic variation of rice flavonoids. To investigate the genetic Data 3). In searching for candidate genes underlying these loci,
control of natural variation in the major flavonoids in rice, the we found tandem annotated UDP-dependent glycosyltransferase
metabolite profiling data and high-quality SNPs with minor allele encoding genes within the confidence intervals of each locus
frequency (MAF) ≥5% were obtained from previous studies using (Fig. 1b–e). For example, four UGT-encoding genes locate on
a worldwide collection of 529 rice accessions36,37 and a chromosome 7 (locus 4) are significantly associated with the
metabolite-based genome-wide association study (mGWAS) was levels of various flavone 5-O-hexosides such as apigenin 5-O-
performed by a gene-based analysis38. The association results glucoside, chrysoeriol 5-O-hexoside, and tricin 5-O-hexoside
showed that natural variation in rice flavonoids was mainly (Fig. 1e, Supplementary Fig. 3a, b and Supplementary Data 3).
controlled by four prominent loci (Fig. 1b–e; Supplementary Therefore, we posit that these four UGTs are putative flavone
N
A
T
U
R
E
C
OM
M
U
N
I
C
A
T
I
O
N
S
8
:
1
9
7
5
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
46
7
-
0
1
7
-
0
2
1
6
8
-
x
w
ww
.
n
a
t
u
r
e
.
c
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3
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A R T I C L E
N A T U R E C O M MU N IC A T IO N S | D O I: 1 0 . 1 0 3 8 /s 4 1 4 6 7 - 0 1 7 -0 21 6 8 -x
a
b
40
30
20
10
0
OsUGT706D1+Kae
OsUGT706D1+Api
OsUGT707A2+Api
OsUGT707A2+Kae
Standard
Api 7-O-Glc
Api 5-O-Glc
Api
Kae
Kae 3-O-Glc
6 7
Standard
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
Retention time(min)
5
8
9
10 11 12 13 14 15 16 17 18 19 20
Retention time(min)
H3
C
c
d
5
40
30
20
10
0
O
O H
O
330.7 [Y0]+
4
3
2
1
0
O
O
H O
H3
C
Tri 7-O-Glc
O
+
HO
HO
OsUGT706D1-Tri
314.7
269.8
O
HO
Tri 5-O-Glc
Y0
HO
[M+H]+
492.9
OsUGT707A2-Tri
Standard
Tri
- glu
400
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20
Retention time(min)
100
200
300
500
600
m/z, Da
e
f
500
400
300
200
100
0
250
*
*
*
WT
WT
OsUGT707A2-OE
OsUGT706D1-OE
*
*
*
200
150
100
50
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
0
Hex
Hex
Hex
Hex
-Glc
-Glc
-Glc
-Glc
-Rut
-Rut
-Rut
-Hex
-Hex
-Hex
O
O
O
O
O
O
O
O
O-
O-
O-
O-
O
O
Tri7-
Lut7-
Api5-
Tri5-
Api7-
Tri7-
Hex-
Api7-
Chr7-
Sel5-
Hes5-
Chr5-
Chr7-
Tce5-
O-
Tri5-
Fig. 2 F u n c t i o n al a n a l ys i s o f OsUGT706D1 a n d OsUGT707A2. H PL C c h r o m a t o gra m s o f t h e r e a ct i o n o f O s U G T 7 0 6D 1 a n d O s U G T 7 0 7 A2 w i t h U D P - g lu c os e
a n d d i f f e r en t s u b st r a t e s , k a e m p f e r o l ( a) , a p ig e n i n (b) a n d t r ic i n (c) . C h r o m a t o g r a m s s h o w t h e a b s o r p t i o n a t 3 3 0 n m . d T h e M S s p e c t r u m a n d c h e m i c a l
s tr u c tu r e o f t h e p r o d u c t i n t h e r e a c t io n o f O s U G T 7 07 A2 w it h U D P - g l u cos e a n d t r i ci n . B a r p l o t s f o r t h e c o n t e n t s o f fla v o n e 7 -O- g l y co s i d es a n d fla v on oi d
5 -O- g l yc o s i d es i n o v e re xp r es s i o n l in e s o f O s U G T 7 07A 2 ( e) a n d O s U G T 7 0 6D 1 (f) . T h e d a t a a r e p r es e n t e d a s m e a n S D , n = 3 . *P < 0 .0 5 , * * P < 0 . 0 1 , * **P
< 0 . 0 0 1 , S tu d e n t’s t t es t s . Api a p ig e n i n ; Kae k a e m pf er o l ; Tri t r ic i n ; S e l s e l g i n ; Tce 3′, 4 ′, 5′- t r i c e t i n ; Glc g l u c o s i d e ; Hex h e x o s i d e ; Rut r u t i n o s i d e , Chr c h r y s oe r i ol ;
Hes h e s p e r eti n ; WT t h e t r a n s ge n i c b a c k gr o u n d v a r i e t y Z H 11
5-O-glucosyltransferases underlying this locus. We also found Characterization of putative flavonoid UGTs. Previous phylo-
strong association between flavone 7-O-glucosides and genetic analyses have categorized flavonoid UGTs into unique
OsUGT88C3 (Os01g53370) for locus 1 (Fig. 1b, Supplementary clusters based on their regiospecifity (i.e., the position of glyco-
Fig. 3a, b and Supplementary Data 3). Similarly, significant sylation) for sugar acceptors, including 3GT, 5GT, 7GT, 3′GT and
association between seven UGTs encoding genes in locus 3 and GGT subfamilies14. To figure out which group our putative UGTs
the amount of tricin O-hexoside-O-glucoside (Student’s t tests, P belong to, we generated a phylogenetic tree by neighbor-joining
< 1.53 × 10–15; Fig. 1d, Supplementary Fig. 3b and Supplementary algorithm using amino acid sequences of the 29 previously
Data 3) and the association between three tandem UGT-encoding reported flavonoid UGTs and the candidates identified in this
genes and the levels of chrysoeriol O-feruloylhexosyl-O-hexoside study (Fig. 1f). We found that three of the putative UGTs,
in locus 2 suggest these UGTs are candidates for the corre- OsUGT705A1, OsUGT705A2, and OsUGT705A3, grouped into
sponding GWAS hits (Fig. 1c, Supplementary Fig. 3a and Sup- the 7GT/3′GT clade mainly formed by previously characterized
plementary Data 3). In total, 21 putative UGT-encoding genes flavonoid 7-O-glycosyltransferases and flavonoid 3′-O-glycosyl-
were assigned to be the candidates underlying four identified loci transferases (Fig. 1f). However, the remaining 18 putative UGTs
located on chromosomes 1, 5 and 7.
formed a distinct clade comprising of no characterized UGTs
4
N
A
TU
R
E
C
O
M
M
UN
I
C
A
T
I
O
N
S
8
:
1
9
7
5
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
7
-
0
2
1
6
8
-
x
w
w
w
.
n
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u
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c
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N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
7
-
0
2
1
6
8
-
x
A
R
T
I
C
L
E
a
b
c
n=112/414
***
10
8
OsUGT707A2
y = 0.0004x–0.0056
r = 0.4958
P = 0.0013
600
400
200
6
ATG
TAG
4
45
44 46
2
1
0
1 3
8 10
19
25
34
0
16
18
20
22
24
26
C
T
Relative content (log2)
sf0719059405
r2
Apigenin-5-O-glucoside
1
0.8
0.6
0.4
0.2
0
d
e
vf0719059054
5
4
3
2
1
0
C153
C196
C005
Nip
Frame
shift
f
Os UGT706D1
OsUGT707A2 CDS
ATG
TGA
43 47
50
40
57
63
62
g
67
38
1.2
0.8
0.4
0
41
Allele I
Allele II
45
h
Specific activity (Kae)
Allele I
16.8 1.3
0.4 0.01
0.024
Allele II
Km (μM)
94.3 11.8
0.03 0.01
kcat (s–1
kcat /Km
)
r2
*
*
1
0.8
0.6
0.4
0.2
0
0.00032
Api
Kae
T
i
N
P
A
V
A
M
V
V
A
L
V
S
T
Allele I
Allele II
position
S
L
G
A
V
M
G
R
6
83
125 155
196 221 255 274
402 415
467
Fig. 3 A n a l y s e s o f f u n c ti o n al S NP s w it h i n OsUGT707A2 a n d OsUGT706D1. A r e p r e s e nt a t i o n o f t h e p a i r w i s e r2 v a l u e ( a m e a s u r e o f l i n k a g e d i s e q ui l ibr i um )
a m o n g p o l y m o rp h i c s i t e s i n OsUGT707A2 ( a) a n d OsUGT706D1 (f) , w h e r e t h e d a r k n e s s o f t h e b o x c o r r e s p on d s t o t h e r2 v a l u e a c c o r d i n g t o t h e l e g e n d .
3
3
1
9
S ta r s r e p r e s e nt t h e s i g n ific a n t s i t e s i d e n t ifie d i n UGT707A2 (P < 8 × 1 0– ) a n d UGT706D1 (P < 2 × 1 0– ) , a n d s o l i d s t a r s i n d i c a t e t h e f u n c t i o n a l S N P s . T he
P v a l u e i s c a l c u l a t e d u s i n g t h e S t u d e n t’s t t e s t s . b C o r r e l a t i o n b e t w e e n r e l a t i v e c o n t e n t s o f a p i ge n i n 5 -O- g l u c o s e a n d t r a n s c r i pt l e v e l s o f O s U G T 7 0 7 A2 i n
3 9 r i ce v a ri e ti es . c B o x p l o t f o r t h e c o n t e n t o f a p ig e n i n 5 - O- gl u c o s i d e; p l o tt e d a s a n a s s o ci a te d s i t e a t C h r o m o s o m e 7 . s f 0 7 1 9 0 5 9 40 5 . * * * P < 0 . 0 0 1,
S tu d e n t’s t t es t s . d A l i g n me n t o f p a r ti a l n u c l eo t id e s e q u en c e s o f OsUGT707A2 f r o m d i f f e r e n t r i c e v a r i e t i e s . C 1 5 3 , C 1 9 6 a n d C 0 0 5 i n d i c a t e t h r e e r i c e
v a r i e t i es t h a t o r i g i n a t ed i n c h in a , r e s p e c t iv e l y. e C o m p a ri s o n o f t h e c o n t e n t o f a p i ge n i n 5 - O- g l u co s i d e i n d i f f e r e n t r i c e v a r i e t i e s . T h e d a t a a r e p r e s e n t e d a s
m e an S D , n = 3 . g E n z y m a t i c a c t i vi t y o f O s U G T 7 06D 1 f ro m t w o a l l e l e s . A l l e l e I i n d i c a t e s t h e h i g h p r o d u c er g e n o t y p e w h e r e a s t h e a l l e l e I I i n di c a t e s t h e
l o w p r o d u c e r g e n o t y p e . A s s a y w a s r e p e a t e d t h r e e t i m es a n d b a r s i n d i c a t e m e a n S D , n = 3 . * *P < 0 .0 1 , S t u d e n t’s t t e s t s . h K i n e ti c p a r a m e t e r s o f
O s U G T 7 0 6D 1 f r o m t w o a l le l e s . T h e d a t a a r e p r e s e n t e d a s m e a n S D f r o m t w o r e p l i c a t e i n d ep en d e nt a s s a y s . i A l i g n m e n t o f f u l l a m i n o a c i d s e q ue nce s o f
O s U G T 7 0 6D 1 f r o m t w o a l l e le s . B l a c k b l o c k i n d i c a t e s t h e c o n s e r v e d P S P G b o x d o m a i n . Nip n i p p o n b a re , ND n o t d e t e c te d
(Supplementary Fig. 4), suggesting that it is highly likely that (Supplementary Fig. 5 and Supplementary Table 1). Five UGTs
these putative flavonoid UGTs display novel specificities.
were identified to be active on both flavone (apigenin) and
In order to functionally characterize these putative UGTs, their flavonols. OsUGT706E1 displayed higher activities using kaemp-
open reading frames were successfully amplified and cloned into ferol than apigenin as substrate, although its kcat/Km value for
expression vectors with glutathione S-transferase (GST) fused to quercetin was with 10-fold lower (Supplementary Table 2). Similar
the N-terminus. In all, 20 recombinant UGTs that were successfully results were obtained for OsUGT705A2 and OsUGT706C4
expressed in E. coil were purified for enzymatic assays. Two sugar (Supplementary Table 2). By contrast, recombinant OsUGT706D1
donors (UDP-glucose and UDP-galactose) and six typical and OsUGT707A2 exhibited higher activities with flavones than
flavonoid aglycones, including flavones (apigenin and luteolin), flavonols as substrates albeit with different regio-selectivity. For
flavonols (kaempferol and quercetin), and flavanones (naringenin instance, both OsUGT706D1 and OsUGT707A2 reaction products
and eriodictyol) were tested as potential substrates. In term of gave a single peak that had the same retention time (RT; 9.4 min)
sugar acceptor, nine of these UGTs exhibit specific activity on and UV-spectra as kaempferol 3-O-glucoside when using kaemp-
flavonol aglycones. Among them, OsUGT706D2 showed the ferol as a substrate (Fig. 2a). However, OsUGT706D1 reaction
highest catalytic efficiency exhibiting the highest kcat/Km value, product generated a peak different from that of OsUGT707A2 with
followed by OsUGT706B1, OsUGT707A3 and OsUGT706C3 flavone (apigenin) as substrate. The former product eluted at
(Supplementary Table 1). Both OsUGT706C1 and OsUGT706A5 9.3 min had the same RT, fragmentation pattern and UV-spectra
exhibited low activities on kaempferol, whereas OsUGT706F1 with an authentic apigenin 7-O-glucoside standard. The latter,
could utilize both kaempferol and quercetin as substrates however, eluted earlier than apigenin 7-O-glucoside and was
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
ON
S
8
:
1
9
7
5
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
46
7
-
0
1
7
-
0
2
1
6
8
-
x
w
w
w
.n
a
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u
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e
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c
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5
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A R T I C L E
N
A
T
U
R
E
C
O
M
M
U
N
I
C
A
T
I
O
N
S
|
D
O
I
:
1
0
.
1
0
3
8
/
s
4
1
4
6
7
-
0
1
7
-
0
2
1
6
8
-
x
a
b
c
OsUGT706D1 OsUGT707A2
OE OE
OsUGT706D1 OsUGT707A2
OE OE
ZH11
ZH11
d
ZH11
OsUGT706D1-OE
OsUGT707A2-OE
1.2
ZH11
OsUGT706D1 OsUGT707A2
OE OE
ZH11
OsUGT706D1 OsUGT707A2
OE
OE
*
1.0
0.8
0.6
0.4
0.2
0
* *
e
f
WT
OE-3
OE-1
OE-7
Before UV treatment
* *
*
*
*
*
*
*
*
*
*
*
*
3.0
2.5
2.0
1.5
1.0
0.5
0
* *
* *
After UV treatment
0
2.5
5
7.5
10
ZH11
OsUGT706D1
OE
OsUGT707A2
OE
UV irradiation time (h)
Hainan
Hubei
(N)
50°
1.00
1.2
1.0
0.8
0.6
0.4
0.2
0
50
40
30
20
10
0
AA
AR
RR
g
i
h
j
0.75
40°
30°
20°
0.50
0.25
y = 0.0892x - 0.8755
r = 0.8048
P < 0.0001
AA
AR
RR
8
10 12 14 16 18 20 22
Global UV-B irradiance (W h m–2
8
10 12 14 16 18 20 22
Global UV-B irradiance (W h m–2
0.00
9 ~ 14
14 ~ 19
>19
)
)
Global UV-B irradiance (W h m–2
)
80° 90° 100° 110° 120° 130°
(E)
Fig. 4 F u n c t i o n a l c h a r a c t e r iz at i o n o f t h e r o l e o f fla v o n e g ly c o s id e s a n d OsUGT706D1 a n d OsUGT707A2 i n p r o t ec t i o n a g a i n s t U V - B i r r a d i a t i o n i n r i c e . a
F l a v o n o i d a g l y c o n e a n d g l y c o s i d e s u n d e r w h it e l ig h t ( u p p e r ) a n d U V - B ( 3 0 2 n m , l o w e r ) . C o n c e n t r a ti o n o f fla v o n o i d s i n e a c h w e l l w a s 2 μM . P h e n o t ype s o f
2
r i ce s e ed l i n g s ( 4- w ee k o l d ) b e f o r e U V -B t r e a t m e n t ( 1 0 h , 2 0 W m − ) ( b) a n d a f t e r U V - B t r e a tm e n t ( c) . d E ff e c t o f U V - B i r r a d i a t i o n o n g r o w t h o f r i c e
s e e d l i n g s . E x p e r i me n t w a s r e p e a t e d t h r e e t i m es w it h 2 0 i n d i v id u a ls i n e a c h g r o u p . B a r s i n d i c a t e m e a n s S D (n = 3 ) . *P < 0 .0 5 , * *P < 0 .0 1 , S t ud e n t’s t
t e st s . e N B T s t a i n i n g o f r i c e s e e d l i n g s b e f o r e a n d a f t e r U V -B t r e a tm en t . A 3 –4 c m p o r t i o n o f t h e l o n ge s t l e a f w a s c a p t u r e d f o r c o m p a r i s o n . f P l ot s f or
h u nd re d -g r a i n w e i g h t o f OsUGT707A2 o v e r e xp r es s io n p l a n ts a n d W T . S e e d s w e r e c o l l ec t e d a n d m e a s u r ed o n t h r e e i n d i v i d u a l p l a n t s p e r l i n e . T he d at a a r e
p re s e n t e d a s m e a n S D (n = 3 ) . * * * P < 0 . 0 01 , S t u d e n t’s t t e s t s . g G e o g r a p h i c al d i s t r i b u t i on o f t h e t w o - ge n e c o m b i n a t i o n s i n 1 7 7 r i c e v a r i e t i e s i n C hi na . T he
d i f f e re n t c o l o r s r ep r es e n t d i f f e r en t c o m b i n a t i o n s o f OsUGT707A2 a n d OsUGT706D1. R i s t h e r e f e r e n c e a l l e l e , a n d A i s t h e a l t er n a t e a l l e l e . h P l ot f or
r e l a ti o n s h i p b e t w e e n t h e a v e r a ge o f g lo b a l U V -B i r r a d i a n c e m e a s u r e d i n 1 9 9 0 ( S o d a ( h t t p : / / w w w . s od a - i s . co m) ) a n d l a t i t u d e i n C h i n a . i A l l e l e d is t r ib ut i on
o f v a r i et i es c o n t ai ni n g R o r A a l l e le a s s o c i a t e d w it h t h e a v e r a ge o f d a i l y U V - B i r r a d i a n c e m e a s u r e d a t 1 9 9 0. j C o r r e l a t i o n f o r A r a t i o a n d t h e a v e r ag e o f d ai l y
U V - B i r r a d i a n ce . T h e r v a l u e i s b a s e d o n t h e P e a r s o n c o r r el a t io n c o e f fic i e n t . T h e P v a l u e i s c a l c u l a t e d u s i n g t h e S t u d en t ’s t t e s t s
identified as apigenin 5-O-glucoside using an authentic standard observations indicate that OsUGT706D1 and OsUGT707A2 are
(Fig. 2b). These observations indicated that OsUGT706D1 the two major flavone UGTs responsible for glucosylation 7-OH
catalyzed the glycosylation of the 7-OH group of flavone, whereas and 5-OH groups of rice flavones, respectively. No activities were
OsUGT707A2 transfers a glucose group onto the 5-OH group of detected for the remaining UGTs with any of the substrates tested.
flavone, which is consistent with the prediction from the With regard to the sugar donor, catalytic activities were only
association mapping. To investigate this further, another observed with UDP-glucose but not with UDP-galactose.
O-methylflavone, tricin, was tested. Each reaction gave a single
Given that flavones constitute the majority flavonoids in rice
peak with different retention time (Fig. 2c). LC-MS analysis (Fig. 1a), we next investigated the activities of flavone UGTs in
showed that the OsUGT707A2 reaction product gave rise to a planta by overexpressing them independently in ZH11, a widely
molecular ion at mass to charge ratio of 492.9[M + H]+, which was used host cultivar for rice transformation. Flavonoid-targeted
consistent with the mass calculation of tricin glucoside (C23H24O12, profiling was subsequently performed by LC-MS to determine the
Fig. 2d). The MS2 spectra displayed an identical fragmentation differences in flavonoid profile between the wild type and
profile to authentic tricin 5-O-glucoside, in which the Y0+ ion was overexpression lines (Supplementary Fig. 6). Despite being able
observed at m/z 330.7 due to the losses of the glucose moieties from to utilize both flavones and flavonols in vitro, only flavone over-
O-glycosylation of the phenolic hydroxyl (−162, Fig. 2d). These accumulation was observed in the UGT transgenic lines. Levels of
6
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-
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A R T I C L E
100
a
b
100
98
Populus trichocarpa (5)
99
Sp1410s0060
87
Me14G020800
Eucalyptus grandis (4)
MgF00274
Vv01028807001
Dc020278
Kmp-3-OG
Kmp-3-OG
Api-7-OG
55
41
100
Api
Api
Kae
68
95
Citrus sinensis (2)
ZmUGT88C10
BdUGT88C8
OsUGT88C3
OsUGT88C2
BdUGT706C7
OsUGT706C1
ZmUGT706C9
ZmUGT706C10
BdUGT706C8
99
100
65
100
Api-7-OG
Kae
Api-5-OG
79
99
100
EgUGT707C3
EgUGT707C2
EgUGT707C3
EgUGT707C2
51
60
99
OsUGT706D1
OsUGT706E1
BdUGT706E4
100
67
100
ZmUGT706F5
ZmUGT706F4
BdUGT706F2
OsUGT706F1
ZmUGT706B4
OsUGT706B1
BdUGT706B2
BdUGT706B3
84
Kae
Api
Kae-3-OG
Kae-3-OG
Kae-3-OG
Api-5-OG
Api-7-OG
80
OsUGT707A2
ZmUGT707A8
BdUGT707A15
100
OsUGT707A2
90
100
ZmUGT707A8
BdUGT707A15
60
90
99
100
EgUGT707C2
EgUGT707C1
Api
Kae
EgUGT707C3
OsUGT706A2
BdUGT706A15
ZmUGT707A8
OsUGT707A3
ZmUGT706A19
BdUGT706A16
OsUGT707A5
ZmUGT707A17
OsUGT707A4
BdUGT707A14
95
OsUGT707A3
ZmUGT707A19
BdUGT707A16
OsUGT707A3
ZmUGT707A19
BdUGT707A16
73
89
100
100
100
98
100
59
Kae
Api
71
No activity
100
OsUGT707A5
ZmUGT707A17
OsUGT707A5
ZmUGT707A18
VvGT1
73
ZmUGT707A17
At3GT
98
Ph3GT
Os3GT
Zm3GT
Bd3GT
100
6
8
10
12
14
16
6
8
10
12
14
16
0.1
100
90
4
Retention time (min)
Retention time (min)
c
5
3
1
2
d
e
2
EgChr. 1
EgChr. 1
3
1
4
12
11
10
5
6
7
9
8
BdChr. 1
OsChr. 7
BdChr. 2
7
6
8
5
9
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2
1
10
11
12
13
14
10
15
16
OsChr. 1
9
1
8
2
7
3
6
4
5
ZmChr. 7
ZmChr. 3
ZmChr. 8
F5GT syntenic region
F7GT syntenic region
g
f
EgUGT707C1 EgUGT707C2 EgUGT707C3
P. equestris
E. guineensis
Chr. 1
F7GlcT activity
F7GlcT/F5GlcT activity
E. guineensis
T
D
C
UGT707A14
UGT707A4
UGT707A16
UGT707A15
B. distachyon
Chr.1
B. distachyon
O. sativa
D
UGT707A5
UGT707A3
UGT707A2
O.sativa
T
Chr.7
UGT707A18
UGT707A17
UGT707A19
UGT707A20
Z. mays
P
Z.mays
Chr.7
C
Chromosomal recombination
Tandem gene duplication
Gene deletion
Transposition
D
P
No activity
Fl3GlcT
activity
F7GlcT
activity
UGT707A8
Z.mays
T
Chr.9
F5GlcT activity
Fig. 5 G e n o m i c s t ru c t u r e a n d f u n c t i o n a n a l y s is o f t h e F7GlcT a n d F5GlcT g e n e c l u s t e r r e g i o n i n p l a n t s . a P h y l o ge n e ti c a n a l y s i s u s i n g a m i n o a c i d s e qu e n c e o f
U G T g e n e s o b t a i n e d f r o m d i f f e r en t p l a n t s p ec i e s . S c a le i m p l ie s a m i n o a c i d s u b s t i t u t i o ns p e r s i t e. b I n v i t r o b i o ch e m i ca l a s s a y s o f p r o t ei n s f r o m p op ul us ,
p a l m , m a i z e a n d B r a c h y p o d iu m. c G e n e s y n t e n i c r e gi o n s f o u n d i n p l a n t s p e c i e s . S o l i d l i n e i n d i c a t e s F5GlcT s y n te n i c r e g i o n s a n d d a s h e d l i n e i n d i c a t e s F7GlcT
s y nt e n i c r eg i o n s. S y n t e n i c g en e l in k a ge b e t w ee n p l a n t s p ec i e s f o r F5GlcT (d) a n d F7GlcT ( e) . f G r a p h i c a l m a p o f F5GlcT c l u s t e r f o u n d a m o n g
C o m m el i n ac e a e s p e c i es . EgUGT707C1 w a s n o t s u c c e s s f u l ly c l o n e d . g H y p o t h e t i c a l s c h e m e o f e v o lu t i o n a l e v e n ts i n F5GlcT g e n e c l u s t e r r e g i on a m on g
m o n oc o t p l a n t s
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flavone 5-O-glucosides were dramatically increased in significant associated with the content of apigenin 7-O-glucoside
OsUGT707A2 overexpression lines, suggesting OsUGT707A2 act (Student’s t tests, P < 10–19, Fig. 3i and Supplementary Data 5),
as a flavone 5-O-glucosyltransferase in vivo (Fig. 2e). Similarly, implying that they could be functional polymorphisms respon-
metabolic profiling revealed an increased accumulation of flavone sible for natural variation of flavone 7-O-glucoside (apigenin 7-O-
7-O-glucosides and flavone 7-O-rutinoside in OsUGT706D1-over- glucoside). Protein modeling showed that 14 of these amino acid
expression plants (Fig. 2f), mirroring its in vitro activity. Analyzing residues are distant from the active site, and the remaining one,
the genes at the level of expression, we found OsUGT707A2 and Leu83Pro, is located in the putative acceptor binding pocket that
OsUGT706D1 were more highly expressed than the other recognizes flavonoid (Supplementary Fig. 9). Similar results
duplicated UGTs (Supplementary Fig. 7). Taken together, these suggesting the impact of distal mutations on protein catalytic
results allowed us to conclude that OsUGT707A2 (termed flavone properties have been reported previously40,41
5-O-glucosyltransferase, F5GlcT) and OsUGT706D1 (termed
.
flavone 7-O-glucosyltransferase, F7GlcT) are the major enzymes
controlling the natural variation of flavones in rice.
Flavone O-glucoside content correlates with UV-B tolerance.
Although flavonols have long been reported to involve in UV-
protection in many plant species by absorbing UV light in the
Polymorphisms underlying variation of major rice flavonoids. 280–350 nm wavelength, it remains unclear whether flavones
To investigate functional variations of OsUGT707A2 and confer UV resistance in plants32,42. To this end, we measured the
OsUGT706D1, we searched for possible function polymorphism absorption of UV-VIS spectra by apigenin aglycone and its gly-
(s) within these two genes, including their promoter regions and cosides (Supplementary Fig. 10a). We found all of them gave
UTRs using the RiceVarMap database39. We discovered 47 SNPs similar λmax as those of flavonols7,43. Interestingly, however, we
and 9 InDels (MAF ≥ 5%) in OsUGT707A2 (Supplementary observed that apigenin 5-O-glucoside emitted a bright yellow
Data 4), and 10 SNPs were significantly associated with the fluorescence under UV-B light (302 nm) whereas the other fla-
content of apigenin 5-O-glucoside (Student’s t tests, P < 8 × 10–33
;
vones do not (Fig. 4a). With the purpose of testing the UV-
Fig. 3a). We next tested whether the variation of their expression protective function of flavone O-glucosides experimentally, we
(SNPs in promoters) and/or coding variation contribute to the grew the overexpression lines of OsUGT707A2 and OsUGT706D1
differences in rice flavonoid levels. For OsUGT707A2, we first alongside the wild-type plants under UV-B light. As shown in
measured the transcription levels of OsUGT707A2, as quantified Fig. 4b, all plants grew normally and no significant difference was
by real-time quantitative reverse transcription PCR (qRT-PCR) observed between the transgenic and the wild-type lines before
across 39 rice varieties. As shown in Fig. 3b, correlation tests UV treatment. However, most of the OsUGT707A2 and
revealed that the expression level of OsUGT707A2 is significantly OsUGT706D1 overexpression plants survived with green leaves,
(Student’s t tests, P < 0.01) correlated with the content of api- while the wild-type plants became dramatically withered after
genin 5-O-glucoside. Further investigation revealed that the SNP, UV-B irradiation although a similar amount of total flavonoids
sf0719059405, 400 bp upstream of OsUGT707A2 coding region, was detected in the transgenic and WT plants (Fig. 4c and Sup-
was strongly associated with the content of apigenin 5-O-gluco- plementary Fig. 10b). In addition, OsUGT707A2 overexpression
side (Fig. 3c) and therefore could represent the functional poly- lines exhibited significantly higher survival rate than that of
morphisms underlying this locus. However, even though strong OsUGT706D1 overexpression plants (Fig. 4d), suggesting that
associations between eight SNPs in the coding region and the plants overaccumulating flavone 5-O-glucosides displayed
level of apigenin 5-O-glucoside were observed (Supplementary enhanced UV-B tolerance of treatment than that of the plants
Data 4), when the enzymatic activities of the two allelic recom- which accumulated only flavone 7-O-glucosides. We then per-
binant proteins were compared, similar kinetic parameters were formed nitroblue tetrazolium (NBT) staining to detect superoxide
obtained (Supplementary Table 3). In addition, we found an low- anion radicals in the leaves of these plants and found significantly
frequency InDel (MAF = 0.629%, confirmed by re-sequence) in more blue precipitate in the wild-type seedlings than their
the coding sequence of UGT707A2, vf0719060607 (a C deletion, transgenic counterparts and that the OsUGT707A2 over-
corresponding to position 773 bp), caused a frameshift and expressing plants produced less precipitate than OsUGT706D1
resulted in a truncated protein lacking the conserved UDP- (Fig. 4e). Furthermore, when WT and OsUGT707A2 over-
glucose binding domain (Fig. 3d) in the three varieties, which expressing lines were grown in Hainan Island (20.2°N, 110.3°E)
display the lowest levels of apigenin 5-O-glucose (Fig. 3e). Taken and Wuhan (30.3°N, 114.2°E), OsUGT707A2 overexpression lines
together, results from qRT-PCR, kinetics and sequencing studies showed significantly higher hundred-grain weight than those of
suggest that the SNP, sf0719059405, and InDel, vf0719060607, WT plants in Hainan Island (which receives relatively high levels
could be the functional polymorphisms responsible for the var- of UV-B irradiance), while no significant difference was observed
iation of apigenin 5-O-glucoside levels in rice. However, the for plants grown in Wuhan (which receives relatively low levels of
effects of unidentified polymorphisms cannot be ruled out, nor UV-B irradiance) (Fig. 4f). Taken together, these results suggested
can the possible roles of the “silent” ones which may affect RNA that O-glycosylated flavones play a positive role in plant UV-B
levels or splicing.
protection and flavone 5-O-glucosides are more effective UV-B
Similar analysis was also performed for OsUGT706D1. We protectants than flavone 7-O-glucosides. These results are inter-
found 74 SNPs and 7 InDels (MAF ≥ 5%) in OsUGT706D1 esting in light of a recent study in Arabidopsis which revealed that
(Supplementary Data 5) and 60 SNPs were significantly phenylacylation of flavonols catalyzed by a recently evolved
associated with the content of apigenin 7-O-glucoside (Student’s phenylacyltransferase also rendered them as more effective UV-B
t tests, P < 2 × 10–19; Fig. 3f). We observed no significant protectants44.
correlation between the transcript level of OsUGT706D1 and
To see whether role of flavone glycosides play similar role in
the accumulation of apigenin 7-O-glucoside (Supplementary natural conditions, the high and low producers, based on the
Fig. 8). However, when the two alleles of OsUGT706D1 were genotypes of selected SNP combinations within OsUGT707A2
cloned and expressed, the resultant recombinant proteins (sf0719059405) and OsUGT706D1 (sf0130711475), were marked
exhibited distinct catalyst efficiency, with a massive 100 fold on a map of China according to their site of origin (Fig. 4g). In
difference in their kcat/Km values (Fig. 3g, h). We identified 14 general, the different combinations were found to be associated
non-synonymous SNPs in the coding sequence, 11 of which were with the pattern of UV irradiation. Most of the accessions
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A R T I C L E
deriving from high irradiation areas such as south china (latitude glycosylation activities with OsUGT707A2 (Fig. 5b). Similarly, in
20°~25°) carry the AA combination (higher flavone glucoside accordance with OsUGT707A4 from rice (Fig. 5b) neither
levels) (Fig. 4g, h). By contrast, the RR combination (less flavone ZmUGT707A18 nor BdUGT707A14 is able to utilize flavonoids
glucosides) accessions were mainly present in low irradiation as substrates. By contrast, EgUGT707C1, EgUGT707C2 and
habitats such as the north of China (latitude 35°–46°) (Fig. 4g, h). EgUGT707C3 from palm mainly exhibit flavone 7-O-glucosyl-
This is consistent with the significant correlation value between transferase activities (Fig. 5b), showing different specificities from
UV-B intensity and A/R ratio (Fig. 4i, j). The similar trend was their Gramineae counterparts. When taken together, these data
also observed when a larger population of 345 cultivated allow us to postulate the hypothetical evolutionary framework for
accessions was examined in Europe and Asia (Supplementary genes in this cluster in the form of the speciation tree based on
Fig. 10c, d). Taken together, the abundance of the flavone O- the NCBI taxonomy database, presented in Fig. 5g.
glucosides appears to be related to adaptation to UV-B irradiance.
However, we should stress at this point that many of the results
reported here were from greenhouse grown plants and recent Discussion
studies9,45 have shown that translation of such results into a field Unraveling the natural variation of plant metabolites and the
environment is often not absolute.
genetic basis underlying this has received increasing interest in
recent years23,46,47. Analysis of the natural occurring allelic var-
iance not only provides a better understanding of gene function
Comparative genomic signatures in UGT regions. To better within both ecological and evolutionary contexts48, but also
understand the evolutionary context of the UGTs at loci 1, 3 and represents an effective way to determine the function of genes
4 (F5GlcT and F7GlcT), which cluster distinctively from other such that they can subsequently be used in gain-of-function
reported UGTs (Fig. 1f and Supplementary Fig. 4), we performed breeding approaches. Natural variation of metabolites has been
an extensive BLAST search for homologies in Phytozome and reported to be regulated at transcriptional, translational and
NCBI databases using OsUGT707A2 and OsUGT706D1 as baits, posttranslational levels47,49. Here we showed that variation of
respectively. For F5GlcT, we found at least three candidates (also major rice flavonoids is determined by UGT707A2 and
in clusters) exist in oil palm (Elaeis guineensis) and eight in UGT706D1, and is regulated at RNA transcript and enzyme
Gramineae plants. By contrast, numerous F7GlcT homologs were activity level, respectively, thus providing an example of multi-
obtained among both monocots and eudicots such as rice, maize, layered regulation of natural variation within a single biosynthetic
oil palm, orange, populus, and grape (Vitis vinifera). We subse- pathway. In addition, the rare allelic variation of the loss-of-
quently performed a protein phylogeny reconstruction based on a function mutation is caused by an InDel that resulted in a fra-
collection of these homologs and additional flavonoid 3-O-gly- meshift (Fig. 3e, f), compared to less severe variation (which is
cosyltransferases (F3GTs) sequences from Arabidopsis, grape and more common in nature). Consistent with this, close scrutiny
monocots to improve the strength of the sequence clustering revealed that the allelic variation that contributes to the impaired
(Fig. 5a). The F7GlcT clade was formed by widely-distributed mutant is mostly occurring outside of the enzyme active site
F7GlcT proteins found in monocots and eudicots (Fig. 5a) and at region, except one mutant near the flavonoid binding pocket
least 5 UGTs were biochemically proven to show F7GlcT activ- (Supplementary Fig. 9). This mutation (Pro to Leu, no change in
ities (Fig. 5b), suggesting that F7GlcT function is conserved in the amino acid polarity) lies at the turning of a loop into a helix and
Angiosperm. By contrast, F5GlcT genes formed a strongly clus- no difference was observed between the structures harboring
tered clade unique to the Commelinids and independent to the either Pro or Leu at this position (Supplementary Fig. 9), sug-
F3GTs and F7GlcTs (Fig. 5a), confirming the exclusive identifi- gesting P83L is not crucial for enzyme activity, although this
cation of F5GlcT orthologs. Furthermore, this similarity based notion remains to be further examined experimentally. Thus, our
tree reflect s an APGIII taxonomic relationship, suggesting early study is similar to other reported examples41,42 concerning the
appearance of F5GlcT in a common monocot ancestor, in cumulative effect of multiple minor mutations outside of the
accordance with the apparent absence of F5GlcT in Gymnos- enzyme active site region.
perms and core Eudicotyledons. However, the LC-MS profile of
Given the multi-step nature of flavonoid biosynthesis, it is
leaves of Chinese cymbidium (Cymbidium sinense) did not allow interesting that their variation is mainly determined by tailoring
identification of flavone 5-O-glucosides (Supplementary Fig. 11), enzymes that catalyze the final step of their biosynthesis. Similar
suggesting that F5GlcT evolved after speciation between Orchi- observations were made regarding the genetic architecture of other
daceae and Commelinaceae. Additionally, three tandem UGTs plant metabolite such as glucosinolates50–52 and flavonols23,44 in
harbored in Elaeis guineensis belong to a single clade, separated Arabidopsis, α-tocopherol in tomato47 and rice secondary
from those found in the Gramineae (Fig. 5a). These observations metabolism53–55. It thus appears likely that decorating enzymes,
suggest F5GlcT genes evolved independently after speciation which are responsible for the final steps of metabolite synthesis
between Palmaceae and Gramineae and are not duplicated in the generally make a greater contribution to the natural variation of
same manner in Commelinids. To confirm this hypothesis, we metabolite abundance than early pathway enzymes. One possible
took advantage of the available genome data and performed co- explanation of it could be that variation in the activities of these
linear genomic analyses among Elaeis guineensis, Oryza sativa, proteins is of lesser consequence to central metabolism—a fact that
Zea mays and Brachypodium distachyon (Fig. 5c). F5GlcT syn- may benefit the overall fitness of the organism. With this in mind,
tenic blocks were found on OsChr7, ZmChr7, BdChr2 and it is worth noting that tailoring enzymes and/or their products may
EgChr2, whereas F7GlcT syntenic blocks were observed on have quantitative, feedback inhibition, on the upstream biosyn-
OsCh5, ZmChr3, ZmChr8, BdChr1 and EgChr2 (Fig. 5d, e). thetic steps27,29,30,56,57, providing a biochemical as well as a genetic
Closer scrutiny revealed that F5GlcTs found in EgChr1 were explanation for our results.
arranged in a head-to-tail manner, whereas their paralogues on
UV irradiance has a high mutagenic potential given that it is
OsChr7, ZmChr7 and BdChr2 were all separated by putative damaging to DNA and RNA58. Increasing evidence demonstrates
transposons (Fig. 5f). Further enzyme assays using recombinant that elevated UV-B radiation is decreasing the yield of many
proteins also support independent duplication events (Fig. 5f). crops45,59,60, including some cultivars of rice9,61,62. To adapt to
For instance, both ZmUGT707A8 (from maize) and climatic changes of light quality, plants produce UV-protective
BdUGT707A15 (from Brachypodium) display similar flavonoids phytochemicals. Generally, three types of phytochemicals have
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been considered in response to UV-B: ascorbate, hydro- (http://rice.plantbiology.msu.edu/index.shtml), Phytozome 10.3 database (http://
phytozome.jgi.doe.gov/pz/portal.html) and Genomsawit Website (http://
xycinnamates and flavonoids, the role of these phytoprotectants
genomsawit.mpob.gov.my/). The amino acid sequences were aligned using the
being confirmed by studies using knockout mutants deficient in
their biosynthesis10. Flavonoids are widely distributed in the plant
kingdom and form an important group of plant secondary meta-
bolites it being estimated that flux through the pathway constitutes
approximately 20% of the total carbon flux63. Our data suggested
that OsUGT706D1 and OsUGT707A2 are responsible for the bio-
synthesis of flavone O-glucosides, which confer enhanced UV-B
tolerance in rice. A recent study in Arabidopsis showed that phe-
nylacylated-flavonols, collectively named saiginols, function as UV-
B protectants44. Collectively and considering the effect of flavo-
noids on other stresses such as enhanced drought tolerance64, these
two studies thus suggest that increased levels of flavonoids may
represent a strategy for stabilizing crop yield under UV-B, drought,
or even against the combination of multiple stresses, which fre-
quently occur in the field.
ClustalW bundled in MEGA 567. The neighbor-joining trees were constructed
using MEGA 5 software with all default parameters. The reliability of reconstructed
tree was evaluated by bootstrap test with 1000 replicates.
Recombinant protein analysis and enzyme assay. The full cDNA of UGTs from
Nipponbare (O. sativa L. spp. japonica) were cloned into the pGEX-6p-1 expres-
sion vector (Novagen) with a glutathione S-transferase tag. Recombinant proteins
were expressed in BL21 (DE3) cells (Novagen) following induction by addition of
0.1 mM isopropy-β-D-thiogalactoside (IPTG) and growing continually for 16 h at
20 °C. Cells were collected and pellets were resuspended in lysis buffer (50 mM
Tris-HCl, pH 8.0, 400 mM NaCl). The cells were disrupted by the high pressure
cracker and cell debris was removed by centrifugation (14000 g, 1 h). Glutathione
Sepharose 4B agarose (GE Healthcare) was added to the supernatant containing the
target proteins. After incubation for 1 h, the mixture was transferred into a dis-
posable column and washed extensively with lysis buffer (5 column volumes).
Target proteins in collections were confirmed by SDS-PAGE and purified
recombinant proteins were selected for enzyme assays and kinetics determination.
The enzyme reactions in vitro assay for glycosyltransferases were performed in
a total volume of 100 μl containing 200 μM flavonoid substrates, 1.5 mM UDP-
glucose, 5 mM MgCl2 and totally 500 ng purified protein in Tris-HCl buffer
Interestingly, the combination of UGT707A2 and UGT706D1
alleles (RR and AA) is reflected in the geographical distribution of
rice cultivars in China, the rest of Asia and even in Europe (100 mM, pH 7.4) was incubated at 37 °C. After incubating for 20 min, the reaction
was stopped by adding 300 μl of ice-cold methanol. The reaction mixture was then
(Fig. 4g, h and Supplementary Fig. 10d). We found that most
cultivars overaccumulating flavone O-glycoside (AA genotype)
filtered through a 0.2 μm filter (Millipore) before being used for LC-MS analysis.
HPLC conditions for the analysis of flavonoids were as follows: column, shim-pack
VP-ODS (150 L × 4.6); flow rate, 0.8 mL min−1; solvent A, 0.04% (by volume) acetic
acid in water; solvent B, 0.04% acetic acid in acetonitrile. After injection (40 μL)
into a column that had been equilibrated with 5% solvent B (by volume), the
column was initially developed isocratically with 5% solvent B, followed by a linear
gradient from 5 to 95% solvent B for 20 min. The column was then washed
isocratically with 95% solvent B for 2 min, followed by a linear gradient from 95 to
5% solvent B for 0.1 min. The column was isocratically with 5% solvent B for 5 min.
The chromatograms were obtained with detection at 330 nm. Peak identification of
each component was confirmed using authentic samples and post-run by LC-MS/
MS analysis. The amounts of flavonoids were determined from peak integration
using authentic samples for calibration.
stem from high irradiance habitats. However, the weak alleles (RR
genotype) were also observed in elite cultivars from these regions
(Supplementary Data 6). Incorporation of strong alleles (AA
genotype) into these cultivars could, therefore, potentially
increase their yields. They would additionally be more beneficial
in coping with the increased levels of abiotic stress that future
crops are anticipated to be exposed to. However, considering the
laboratory-UV assay cannot precisely mirror what happens in the
field, the true biological gain of the introduction of the AA allele
into the RR background still remains to be established.
Enzyme kinetics. To determine the kinetic constants of UGTs for flavonoid
acceptors, their activities were determined using 0 to 400 μM different flavonoids at
a fixed concentration of 1.5 mM UDP-glucose. Flavonoids were purchased from
BioBioPha Co., Ltd (www.biobiopha.com/) and UDP-sugars were purchased from
Sigma-Aldrich, USA. All Kinetic parameters were calculated using Michaelis-
Menten model (Sigma Plot, version 12.5). All the reactions were run in duplicate,
and each experiment was repeated twice.
Methods
Plant materials and metabolite profiling. All plants used in this study were
grown in Huazhong Agricultural University, Wuhan. Fern and peat moss were
collected on campus and identified according to FRPS (http://frps.eflora.cn/).
Potato, citrus, palm, populous and bamboo were obtained from college of Horti-
culture and Forestry Sciences. Arabidopsis, tobacco and crops were grown in
greenhouse. Thirteen plant species leaf samples and tuber of potato were collected
using liquid nitrogen with two biological replicate sets. The freeze-dried samples
were crushed using a mix mill (MM 400, Ratsch) with a zirconia bead for 1 min at
30 Hz, 100 mg dried power were weighted and extracted overnight at 4 °C with
1.0 mL 70% aqueous methanol containing 0.1 mg L−1 lidocaine (internal standard)
before analysis using an LC-ESI-MS/MS system34. Qualification of metabolites was
carried out using a scheduled multiple reaction monitoring method34. The relative
signal intensities of flavonoids were standardized by firstly dividing them by the
intensities of internal standard and then log 2 transforming them to generate the
final data matrix. Flavonoids were quantified based on comparison with standards
of apigenin, tricin, apigenin 5-O-glucoside and apigenin 7-O-glucoside.
Rice transformation and qRT-PCR analysis. An overexpression vector (pJC034)
for rice was constructed from the gateway overexpression vector pH2GW7, 35 s
promoter of pH2GW7 was replaced by maize ubiquitin promoter. The over-
expression constructs of UGT705A2, UGT706C4, UGT706D1, UGT706F1and
UGT707A2, were generated by directionally inserting the full cDNAs first into the
entry vector pDONR207 and then into the destination vector pJC034 using the
Gateway recombination reaction (Invitrogen). The constructs were introduced into
Agrobacterium strain EHA105 and then transferred into japonica ZH11. Gene
expression for each line was quantified by qRT-PCRusing the relative quantification
method. The primers used in this study are presented in Supplementary Data 8. For
each construct, at least three independent overexpression plants were selected for
the targeted metabolite analyses. The freeze-dried samples were extracted overnight
at 4 °C with 1.0 mL 70% aqueous methanol containing 0.1 mgL−1 lidocaine
Nomenclature of UGT-encoding genes. The nomenclature of full-length UGT-
encoding genes that had signature PSPG motifs were obtained (Supplementary
Data 7) with the help of the UGT nomenclature committee (http://prime.vetmed.
wsu.edu/resources/udp-glucuronsyltransferase-homepage).
(internal standard) before analysis using an LC-ESI-MS/MS system34
.
UV treatment and NBT staining. All of the selected flavonoids were prepared in
2 mM concentration and UV irradiation for fluorescence was performed in DNA
gel image system (Bio-Rad, 302 nm). All of the seedlings that were used in the UV
treatment were grown in the same condition. Four-week old seedlings
(OsUGT707A2 overexpression plants, OsUGT706D1 overexpression plants and
ZH11, 24 plants each, and three repeats) were treated with UV-B radiation for 10 h,
and the survival rates were calculated. The plants with normal green and
unwithered leaves were considered as survived plants. For NBT staining, leaves of
survived plants were harvested after 5 h UV-B radiation and vacuum infiltrated
with 0.1% nitroblue, 10 mM sodium azide in 10 mM phosphate buffer, pH 7.6.
Samples were incubated for 1 h at room temperature in the light. To remove
chlorophylls, the stained samples were transferred to 95% ethanol and incubated at
80 °C for 15 min.
Genome-wide association mapping by gene-based analysis. Only SNPs with
Minor Allele Frequency (MAF) ≥ 0.05 and the number of varieties with the minor
allele ≥6 in a (sub) population were used to carry out GWAS. Population structure
was modeled as a random effect in LMM using the kinship (K) matrix. We per-
formed GWAS using LMM provided by FaST-LMM program65. Instead of cal-
culating the P value for the large number of SNPs, a test of association between the
trait and each of the available SNPs within a gene was carried out and the resulting
P values and pair-wise correlation coefficients γ for all the SNPs are obtained.
GATES, a modified of Simes test, was used to combine these available P value to
give a gene-base P value as described38. The genome-wide significance threshold
(6.6 × 10–8) of the gene-based GWAS were determined following Bonferroni
correction66
.
Phylogenetic analysis. The amino acid sequences of reported UGTs were
obtained from NCBI (http://www.ncbi.nlm.nih.gov/). The amino acid sequences of
rice UGTs and the related UGTs in this study were extracted from the TIGR
Agriculture phenotype. OsUGT707A2 overexpression plants and WT (ZH11
variety) were grown in Wuhan, Hubei province during May to August at 2015.
These plants were also grown in Haikou, Hainan province during December to
10
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N A T UR E C O M M UN I CA T I O NS | D OI : 1 0. 1 0 3 8 / s 4 1 46 7 - 01 7 - 02 16 8 - x
A R T I C L E
March from 2015 and 2016. Harvested paddy rice was dried and stored at room
temperature for at least 3 months before measurement. 100 fully filled grains were
randomly selected and weight within each line with three repeats. The values were
averaged and used as the measurements for hundred-grain weight.
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Data availability. The authors declare that all data supporting the findings of this
study are available within the manuscript and its supplementary information files
or are available from the corresponding author upon request.
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Author contributions
J.L. conceived the project and supervised this study. M.P., R.S., A.G. and H.S. performed
most of the experiments; S.S., L.L., D.L., E.N. and X.L. participated in the material
preparation; M.P., S.S., J.Z., S.W. and J.L. carried out the metabolite analyses; Z. Z
performed the GWAS analysis. M.P., T.T., A.F. and J.L. analyzed the data; M.P., T.T., A.
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Acknowledgements
This work was supported by the National Science Fund for Distinguished Young
Scholars (No. 31625021), the Ministry of Science and Technology of China
© The Author(s) 2017
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