letters

Natural alleles of a proteasome α2 subunit gene contribute

to thermotolerance and adaptation of African rice
Xin-Min Li1, Dai-Yin Chao1, Yuan Wu1, Xuehui Huang2, Ke Chen1, Long-Gang Cui1, Lei Su1, Wang-Wei Ye1,
Hao Chen1, Hua-Chang Chen1, Nai-Qian Dong1, Tao Guo1, Min Shi1, Qi Feng2, Peng Zhang1, Bin Han2,3,
Jun-Xiang Shan1, Ji-Ping Gao1 & Hong-Xuan Lin1,3

Global warming threatens many aspects of human life1,2,   parent16. We found that CG14 contained at least five QTLs contrib-
for example, by reducing crop yields3–5. Breeding heat-tolerant uting to thermotolerance (Fig. 1a). Among these, we selected for
© 2015 Nature America, Inc. All rights reserved.

crops using genes conferring thermotolerance is a fundamental
way to help deal with this challenge4–9. Here we identify a
major quantitative trait locus (QTL) for thermotolerance in
African rice (Oryza glaberrima), Thermo-tolerance 1 (TT1),
which encodes an α2 subunit of the 26S proteasome involved in
the degradation of ubiquitinated proteins. Ubiquitylome analysis
indicated that OgTT1 protects cells from heat stress through
more efficient elimination of cytotoxic denatured proteins and
more effective maintenance of heat-response processes than
achieved with OsTT1. Variation in TT1 has been selected for on
the basis of climatic temperature and has had an important role
in local adaptation during rice evolution. In addition, we found
that overexpression of OgTT1 was associated with markedly
enhanced thermotolerance in rice, Arabidopsis and Festuca
elata. This discovery may lead to an increase in crop security in
the face of the ongoing threat of global warming.
npg
further characterization a major QTL on chromosome 3, which was
designated TT1 (Fig. 1a).
To clone the causal gene for TT1, we backcrossed SG42 (a thermotol­
erant line carrying a segment of TT1 from CG14) with WYJ to
construct the mapping population BC4F2. Linkage analysis showed
that all progenies containing a homozygous target CG14 segment were
more thermotolerant than the controls (Fig. 1a and Supplementary
Fig. 1b–d). Moreover, ~3/4 of the progenies from heterozygous lines
survived after heat treatment. Genotyping of the surviving progenies
showed that 1/3 of the individuals were homozygous and 2/3
were heterozygous for the target CG14 segment (Supplementary
Fig. 1b–d), indicating that the CG14 TT1 allele is dominant. We also
constructed a nearly isogenic line (NIL) of TT1, NIL(CG14), and its
isogenic control, NIL(WYJ), by means of repetitive backcrossing and
marker-assisted selection. NIL(CG14) exhibited the same appearance
as NIL(WYJ) under normal conditions but was more thermotolerant
at the seedling and adult stages (Fig. 1a and Supplementary Fig. 2).

Global warming has the potential to dramatically reduce agricultural
harvests, resulting in widespread risk of food insecurity and social
problems1,4–7. Heat stress is reported to lead to severe crop-yield
losses and reduced milling quality3,5,6,10 and is predicted to result in
food crises in the future4,6,11,12. African rice (O. glaberrima), a recently
domesticated tropical crop that has developed delicate mechanisms
for adapting to high temperature, represents a valuable gene resource
for breeding heat-tolerant crops13–15. To improve thermotolerance in
crops using this unexploited resource, it is essential to understand the
molecular basis of adaptational thermotolerance in O. glaberrima.
We found that the O. glaberrima accession CG14 is much more
tolerant to heat stress than are Asian rice varieties such as Wuyunjing
(WYJ; Oryza sativa ssp. japonica) (Fig. 1a and Supplementary Fig. 1a).
To map the causal QTLs underlying this trait, we analyzed the
thermotolerance of a set of chromosome segment substitution lines
(CSSLs) with CG14 as the donor parent and WYJ as the recurrent
To isolate TT1, we carried out high-resolution mapping using 6,721
BC4F2 individuals and delimited the candidate gene within a 12.69-kb
region (Fig. 1b). In that region, two genes, encoding intron-binding
protein AQUARIUS (Os03g0387000) and the 20S proteasome α2
subunit (Os03g0387100, also known as OsPAB1), respectively,
were predicted (Fig. 1b).
Expression analysis showed that Os03g0387100 was differentially
expressed in NIL(CG14) and NIL(WYJ) and that high expression
was induced by heat stress (Fig. 1c and Supplementary Fig. 3).
Moreover, it is established that the proteasome has an important role
in plant stress responses as well as in development17–27. Therefore,
we proposed that this gene was the best candidate gene for TT1. To
confirm this, we overexpressed the coding region of Os03g0387100
from CG14 in WYJ, as this gene is expressed throughout all rice
tissues (Fig. 1c and Supplementary Fig. 3). Phenotyping analysis
showed that all positive transgenic lines were more thermotolerant

1National Key Laboratory of Plant Molecular Genetics, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai, China. 2National Center for Gene Research, Shanghai Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological
Sciences, Chinese Academy of Sciences, Shanghai, China. 3Collaborative Innovation Center of Genetics and Development, Shanghai Institute of Plant Physiology
and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. Correspondence should be addressed to
H.-X.L. (hxlin@sibs.ac.cn), J.-P.G. (jpgao@sibs.ac.cn) or J.-X.S. (jxshan@sibs.ac.cn).

Received 1 August 2014; accepted 22 April 2015; published online 18 May 2015; doi:10.1038/ng.3305

Nature Genetics  VOLUME 47 | NUMBER 7 | JULY 2015 827

 letters

than control plants (Fig. 1d and Supplementary Fig. 4a,b). In addi- than were the controls (Fig. 1e and Supplementary Fig. 4c). These
tion, when Os03g0387100 was silenced in NIL(CG14) by artificial results establish that Os03g0387100 is the causal gene for TT1. We
microRNA, the knockdown lines were more sensitive to heat stress compared sequences of the parental alleles and found that three SNPs

a 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 9 11 1 3 5 7 9 11

TT1

WYJ CG14 SG42 NIL(CG14)

45 °C 52 h
RH >80%

28 °C 12 d 28 °C 7 d
RH 60% RH 60% 7
WYJ CG14 WYJ SG42 NIL(WYJ) NIL(CG14)

b TT1 locus
c 7
© 2015 Nature America, Inc. All rights reserved.

Chr.3
NIL(WYJ)
6 **
NIL(CG14)
RM

RM

Relative expression of TT1

RM

RM

5
15

15
15

15

10 Kb
08

08
07

08

H9
H1

H6
1

7
7

4
17 13 8 2 1 7 14 n = 6,721 ** ** **
3
** ** **
12.69 kb
2 **
1

0
C I L P R SH SD UBI
ATG TAA
20
WYJ T G Arg T
** **
Relative expression of TT1

CG14 C A His C 16 ** ** ** ** **
** **

90 100 110 12 **
NIL(WYJ)
OsTT 1 Oryza sativa 86 112
OgTT 1 Oryza glaberrima 86 112 NIL(CG14)
OsTT 1-LIKE Oryza sativa 86 112 8
*
npg

M7ZS81 Triticum urartu 86 112

B4FFX7 Zea mays 86 112
C5XY69 Sorghum bicolor 86 112 4 **
O23708 Arabidopsis thaliana 86 112 ** **
M4CW05 Brassica rapa 86 112
Q38M52 Solanum tuberosum 86 112 0
I3SFE3 Medicago truncatula 86 112
I1J717 Glycine max 86 112 0 1 3 5 8 10 12 15 18 21 24 32 40 48
A9PAG0 Populus trichocarpa 86 112 Time after 45 °C treatment (h)

d e
Before treatment 45 °C, 56 h, recovered 7 d Before treatment 45 °C, 45 h, recovered 7 d

CK HB-4 HB-7 HB-9 CK HB-4 HB-7 HB-9 CK MiT-1 MiT-2 MiT-5 CK MiT-1 MiT-2 MiT-5
OE-TT1CG14 OE-TT1CG14 Knockdown-TT1CG14 Knockdown-TT1CG14

Figure 1  Identification of TT1. (a) Characterization of TT1. Purple outlines in CG14 and green regions in SG42 and NIL(CG14) represent segments from
CG14 conferring thermotolerance. RH, relative humidity. (b) Cloning of TT1. Top, high-resolution mapping localized TT1 to a region between H1 and
H6. Structural and natural variations of TT1 are shown. Bottom, alignment of TT1 sequences from various plants. The purple inverted triangle marks a
substitution site (H99 in O. glaberrima). (c) Expression pattern of TT1. C, culm; I, internode; L, leaf blade; P, panicle; R, root; SH, leaf sheath;
SD, seedling; UBI, unelongated basal internode. Data represent mean ± s.d. (n = 3). Significant differences were determined by Student’s t-test
(*P < 0.05, **P < 0.01). (d,e) TT1 overexpression (d) and knockdown (e) analysis. Phenotypes of different transgenic lines before and after treatment
are shown. OE, overexpression; HB, transgene-positive lines with enhanced TT1CG14 expression; MiT, transgene-positive lines with reduced TT1CG14
expression; CK, transgene-negative controls. Scale bars in a, d and e, 5 cm.

828 VOLUME 47 | NUMBER 7 | JULY 2015  Nature Genetics

 letters

a b
135 101

GG
GG
Ub 46
Ub

GG

GG
Ub GG-modified

GG
peptides: 1,504 27

GG
P(interaction) < 0.05
Peptides: 580 248
NIL(WYJ) upregulation
Sample Total protein Trypsin Anti-KGG LC-ESI-MS/MS Sequence assignment NIL(CG14) upregulation
preparation extraction digestion enrichment and data analysis NIL(WYJ) downregulation
NIL(CG14) downregulation
16

c d e Response to heat Protein metabolic process

K

T
K

)-C

)-H
-C

-H

(GO:0009408) (GO:0019538)
14

14
)

)
YJ

YJ

G
(W

(W

(C

(C
IL

IL

IL

IL
N

Enrichment plot: heat-response processes

0.4

0.3 NES = 1.32

Enrichment score

FDR < 0.05

0.2

0.1

–0.1
© 2015 Nature America, Inc. All rights reserved.

–0.720
–0.617
–0.514
–0.411
–0.309
–0.206
–0.103
0.103
0.206
0.309
0.411
0.514
0.617
0.720
0

T
)-H

)-H

)-H

)-H
)-C

)-C

)-C

)-C
YJ

14

YJ

14
YJ

14

YJ

14
G

G
(W

(W

G
(W

(W
(C

(C
(C

(C
IL

IL
IL

IL
IL

IL
IL

IL
N

N
N

N
N

N
N

N
4 8
0 10 10
Figure 2  Role of TT1 in the heat response. (a) Schematic overview of ubiquitin
(Ub)-modified proteome characterization protocol (IP-MS/MS). ESI, electrospray Normalized abundance

ionization. (b) Venn diagram of differentially accumulated (two-way ANOVA,

P(interaction) < 0.05) ubiquitinated sites. (c) Heat map of overlap between upregulated f 30,000
**
sites in both NIL(WYJ) and NIL(CG14) (248 in b). Red, high abundance; blue, low 25,000
Proteasome activity (RFU)

abundance. (d) Gene set enrichment analysis (GSEA) of differentially accumulated

ubiquitinated proteins, including GOs shown in Supplementary Figure 9e. Green 20,000

curve, running enrichment score for the gene set; black columns, enriched proteins **
15,000
located by their correlation with heat treatment; red, positive correlation with
heat treatment; blue, negative correlation with heat treatment; NES, normalized
10,000
enrichment score; FDR, false discovery rate. (e) Heat map of core enriched proteins
of indicated GOs as determined by GSEA. Colors indicate the range of ubiquitination 5,000
npg

levels (low to high as defined in key). CK, control (before treatment); HT, heat
treatment (45 °C for 30 h). (f) Activity analysis of 20S proteasome in different 0
genotypes using Suc-LLVY-AMC substrate. RFU, relative fluorescence units. Samples
G )
)

G )
)

G )
)
(C YJ
14

(C J
14

(C J
14
IL Y

IL Y
were incubated for 2 h at 37 °C or 45 °C; lactacystin was used as an inhibitor of the
N IL(W

N IL(W

N IL(W
IL
N

20S proteasome. Data represent mean ± s.d. (n = 3). Significant differences were
+Lactacystin 37 °C 45 °C
determined by Student’s t-test (**P < 0.01). 37 °C

were localized in exons, one of which led to an arginine (R99; WYJ)-
to-histidine (H99; CG14) substitution (Fig. 1b). As the CG14 allele of
TT1 (TT1CG14) was found to be dominant, we wondered whether the
WYJ allele of TT1 (TT1WYJ) was a weak or null allele. To address this
question, we overexpressed the TT1WYJ coding region in WYJ using
the construct for TT1CG14 overexpression mentioned above. Lines
overexpressing TT1WYJ exhibited enhanced thermotolerance, but they
were obviously less thermotolerant than TT1CG14-expressing lines
with equivalent or even lower expression of TT1 (Supplementary
Fig. 5). These data indicate that TT1WYJ is a weakly functioning
recessive allele. Taken together, these results confirm that we success-
fully cloned TT1, a QTL for rice thermotolerance.
Sequence analysis showed that TT1 is a highly conserved α2
subunit of the 26S proteasome, sharing 82% similarity with a homol-
ogous protein from mice and 70% similarity with one from yeast
(Supplementary Figs. 6 and 7)28,29, and that H99 is specific to CG14
(O. glaberrima; Fig. 1b and Supplementary Fig. 6). As the 26S protea-
some functions primarily in degrading ubiquitin-modified proteins
and α subunits have important regulatory roles in this process30–33,
we reasoned that CG14 TT1 might contribute to thermotolerance by
enhancing the elimination and recycling of ubiquitinated proteins
that were denatured because of heat stress, and that the H99 sub-
stitution might have a role in promoting degradation efficiency
(Supplementary Fig. 8). To confirm this, we conducted a ubiquity-
lome study of NIL(CG14) and NIL(WYJ) before and after 30 h of
heat treatment using immunoprecipitation coupled with tandem mass
spectrometry (IP-MS/MS) (Fig. 2a and Supplementary Fig. 9a)34,35.
As expected, we observed markedly increased protein ubiquitination
after heat treatment for both total and differentially identified proteins
(two-way analysis of variance (ANOVA), P(interaction) < 0.05; Fig. 2b,
Supplementary Fig. 9b and Supplementary Table 1). The levels of
ubiquitinated proteins that accumulated during heat stress were much

Nature Genetics  VOLUME 47 | NUMBER 7 | JULY 2015 829

 letters

a b 3.0

2.5
2
O. R = 0.74

(relative to WYJ)
TT1 expression
glaberrima 2.0 P < 0.001
Chr.3: 15972765C
1.5
Chr.3: 15972765T
IRGC102635 IRGC102580 IRGC100127 IRGC102370 IRGC102239 ACC. 102265 1.0

0.5

0
0 20 40 60 80 100
O. sativa Survival rate (%)
japonica
** **
c 1.2
**
**
90
**
**
1.0

expression of TT1

Survival rate (%)

Koshihikari Kongyu131 Nippobare Zhonghua11 Jiahua1 Xiushui09 Chr.3: 15972765C
0.8 60

Relative
Chr.3: 15972765T
0.6
0.4 30
O. sativa 0.2
indica
0 0
Teqing SW30 SW31 SW32 Teqing SW30 SW31 SW32

**
Minghui63 Feng’aizhan Guanglu’ai4 Teqing Huanghuazhan IR29 ** 60 **
4
© 2015 Nature America, Inc. All rights reserved.

**

Survival rate (%)

expression of TT1
3
40

Relative
African O. 2
sativa
20
1

0 0
IRAT109 IRAT261 IRAT104 ITA304 ITA182 ITA117 Koshihikari SN32 SN33 Koshihikari SN32 SN33

d Chr.3: 15971486(T:G) Chr.3: 15972765(T:C) e

Chr.3: 15972765(T:C) Chr.3: 15974353(C:A)
Chr.3: 15974353(C:A) Chr.3: 15975546(C:T) 0.18
Ind Ind

100 Trj
Frequency of non-reference

80 100
Frequency of non-reference

80
allele (%)

60
allele (%)

40 60
0.52 0.52 0.49 0.52
20 40
npg

0 20
0)

0)
(4

0
8)
10

7)
a

28

)
ic

°(

42
15
°(
n

)
25

24
po

)
°(
°(

)
30

11
17
N

Trj Tej
°(
40
34
ja

Tej
N

(1
°–

(3
48
°–

N
N
al

18

e
al
°–
°–
ic

25

t
ra
ic
N
op

34

°–
30
N

op

pe

0.24
40
N
Tr

Tr

m
N

Te

O. sativa O. rufipogon Genome-wide mean TT1 locus

Figure 3  TT1 is involved in the local adaptation of rice. (a) Differential performance of O. glaberrima and O. sativa after long-term heat treatment
(45 °C for 72 h). Scale bars, 5 cm. (b) Linear regression of survival rates under short-term heat stress (45 °C for 45 h) and relative expression levels
of TT1 in 22 O. sativa lines with different TT1 haplotypes. (c) Expression of TT1 and thermotolerance analysis in haplotypically substituted SW
and SN lines and recurrent parents. For quantitative PCR, n = 3 replicates; for survival analysis, n = 96 plants (b,c). Data represent mean ± s.d.;
significant differences were determined by Student’s t-test (**P < 0.01). Heat treatments lasted 45 h. (d) Frequency of haplotypical SNPs in various
agroclimatological zones for cultivar groups (left) and wild rice (right). Alleles in brackets represent the Nipponbare reference allele (before colon)
and the non-reference allele (after colon). N, northern latitude (negative correlation with climatic temperature). The number of varieties per group is
shown in brackets. (e) Levels of genetic differentiation (FST) of different groups. Variations around TT1 and the genome-wide mean are indicated.
Tej, temperate japonica; Trj, tropical japonica; Ind, indica.

lower in NIL(CG14) than in NIL(WYJ) regardless of the protein spe- protein state were notably enriched after heat treatment (Fig. 2d
cies or ubiquitination level (Fig. 2b,c and Supplementary Fig. 9c), and Supplementary Fig. 9e), suggesting that the heat treatment
indicating that CG14 TT1 is more effective in the regulation of protein had caused a reduction in cell-protective abilities. Furthermore, the
homoeostasis under heat stress than WYJ TT1. Immunoblot analysis ubiquitination of these proteins was lower in NIL(CG14) than in
of crude extracts confirmed these results (Supplementary Fig. 9d). NIL(WYJ) (Fig. 2e and Supplementary Fig. 9f), indicating that these
Interestingly, among the differentially ubiquitinated proteins, those proteins remained more active in NIL(CG14). Additionally, enzymatic
in gene ontology (GO) categories related to heat response and assays of the 20S proteasome showed that CG14 TT1–containing core

830 VOLUME 47 | NUMBER 7 | JULY 2015  Nature Genetics

 letters

Figure 4  Applicability of TT1 to breeding.

(a) Yield characteristics of NILs after heat
a 25 50 21

Seed-setting rate on main

** **

Grain yield per plant (g)

18

1,000-grain weight (g)

treatment (12 h 38 °C/12 h 35 °C for 5 d) 20 40
15
during flowering. Panicles from representative

panicle (%)
15 30 12
plants are shown (left). Scale bar, 5 cm.
(b) Performance of NILs after heat treatment (12 h 10 20 9
38 °C/12 h 35 °C for 12 d) at the filling stage. 6
5 10
Harvested grains from representative plants (50 3
from each plant) are shown (left). CK, control 0 0 0
(normal) conditions; HT, heat treatment. n = 6 NIL NIL NIL NIL NIL NIL
NIL(WYJ) NIL (CG14) (WYJ) (CG14) (WYJ) (CG14) (WYJ) (CG14)
plants; for 1,000-grain weight, n = ~100. Scale
bar, 1 cm. (c,d) Heterologous overexpression of b 25 50
** 21
**

Seed-setting rate on main

TT1CG14 enhanced thermotolerance of Festuca **

Grain yield per plant (g)

18

1,000-grain weight (g)

20 40
arundinacea Schreb. (c) and Arabidopsis (d). 15

panicle (%)
HDF, transgene-positive tall fescue expressing 15 30 12
TT1CG14; B, transgene-positive Arabidopsis
NIL(WYJ)/CK NIL(WYJ)/HT 10 20 9
expressing TT1CG14; CK, transgene-negative
6
control. Scale bars: c, 10 cm; d, 2 cm. Data 5 10
3
represent mean ± s.d. Significant differences
0 0 0
were determined by Student’s t-test (**P < 0.01).
NIL(CG14)/CK NIL(CG14)/HT NIL NIL NIL NIL NIL NIL
(WYJ) (CG14) (WYJ) (CG14) (WYJ) (CG14)

particles showed more efficient degradation c d

than those harboring WYJ TT1, especially
© 2015 Nature America, Inc. All rights reserved.

B-3 B-4
under high temperatures (Fig. 2f). In conclu- Anti-FLAG Anti-FLAG
CK B-5
sion, we propose that TT1 protects plant cells
from heat stress by removing cytotoxic pro- Rubisco Rubisco

teins denatured by heat stress and balancing

K

5
F-

F-

F-

3
4

5
C

B-
B-

B-
C
a series of protective responses. On the basis
D

D
H

CK HDF-1 HDF-3 HDF-5

of our results, CG14 TT1 seems to function
more efficiently than WYJ TT1 during these
processes (Supplementary Fig. 10).
To gain insight into the allelic differentiation of TT1 function, we
extended our study from analysis of the diallele TT1 in CG14 and WYJ
to analysis of a wide range of rice varieties. A survey of African and
Asian rice showed that the functional SNP H99 contributes to a hap-
lotype specific to African rice (O. glaberrima), conferring distinctly
greater tolerance to long-term heat stress than observed in Asian rice
(O. sativa) (Fig. 3a). These results are consistent with the fact that
O. glaberrima grows under higher temperatures than O. sativa and
has evolved independently of that species14,15,36. O. sativa varieties
having two different haplotypes of TT1 could be distinguished by
npg
OE-TT1CG14 OE-TT1CG14
more TT1 transcript and exhibited markedly enhanced thermotoler-
ance (Fig. 3c) relative to Koshihikari. Together with the findings that
overexpression of TT1WYJ was also conducive to rice thermotolerance
(although less so than TT1CG14 overexpression) and different lines
expressing distinct levels of TT1CG14 exhibited different degrees of
thermotolerance (Supplementary Fig. 5), the results suggest that
allelic differentiation of TT1 has had two different effects: coarse-
tuning to alter protein characteristics (O. glaberrima), and fine-tuning
to adjust expression levels. These results also suggest roles for TT1
in local adaptation among varieties. To test this notion, we inves-
tigated the geographical distribution of three haplotypical SNPs in

their tolerance to short-term heat stress. Interestingly, the varieties
with different haplotypes generally exhibited distinct levels of thermo-
tolerance. Moreover, the thermotolerance of these varieties was highly
correlated with their expression of TT1 (Fig. 3b and Supplementary
Table 2). These results suggest that TT1 might be a major determinant
of natural variation in O. sativa thermotolerance as well. To explore
this, we first surveyed the 2.5-kb promoter region of TT1 in more
than 150 varieties divided into different groups, and we identified
several possible sites of variation in TT1 expression among the
different groups (Supplementary Fig. 11a,b). Phylogenetic analysis
also showed that the TT1 promoter has evidently diverged among
different groups, corresponding to their geographical distribution
(Supplementary Fig. 11c). We also examined the thermotoler-
ance of two additional sets of CSSLs containing TT1 fragments with
different haplotypes. SW30, SW31 and SW33 are genotypically
identical to Teqing (O. sativa ssp. indica) except for the presence of a
TT1-containing fragment from wild rice (Oryza rufipogon)
(Supplementary Fig. 12a)37. These lines contained lower levels of TT1
transcripts, and consequently were less thermotolerant, than Teqing
(Fig. 3c). Conversely, SN32 and SN33, carrying the TT1 locus of Nona
Bokra (O. sativa ssp. indica) in a Koshihikari (O. sativa ssp. japonica)
genetic background (Supplementary Fig. 12b)38, accumulated
the TT1 region. We found that the frequency of haplotypical SNPs
(non-reference) decreased as the climatic temperature decreased
for both cultivated varieties (O. sativa) and wild rice (O. rufipogon)
(Fig. 3d), suggesting that thermotolerant alleles of the TT1 locus
were selected for by environmental pressure, mainly that related to
temperature. Moreover, we examined the role of TT1 in O. sativa
evolution by estimating the level of population differentiation (FST)
between different subspecies. Compared to the genome-wide mean,
higher differentiation levels were found between tropical japonica
and temperate japonica at the TT1 locus (Fig. 3e). By contrast,
the FST was sharply lower than the genome-wide mean between
tropical japonica and indica at the TT1 locus (Fig. 3e). Considering
the fact that both tropical japonica and indica grow in climates with
high temperatures, we propose that the TT1 locus was specifically
selected (during rice expansion) for local adaptation to regions with
distinct climatic temperatures39.
Notably, the more thermotolerant TT1CG14-harboring plants
(NIL(CG14) and overexpressers) exhibited no changes in morphology
or yield compared with control plants when grown under normal
conditions (Fig. 1d and Supplementary Figs. 2c and 13), suggesting
that TT1CG14 has great potential for thermotolerance breeding. To
evaluate this proposal, we first examined the yield traits of NIL(CG14)

Nature Genetics  VOLUME 47 | NUMBER 7 | JULY 2015 831

 letters
and NIL(WYJ) subjected to heat stress at the flowering and filling
periods, two stages in which rice yield is strongly affected by heat
stress8. Although both NILs had reduced yields under heat stress,
NIL(CG14) always outperformed NIL(WYJ) at both stages (Fig. 4a,b
and Supplementary Fig. 14). These results confirm the feasibility of
breeding thermotolerant rice cultivars by introgressing TT1CG14 into
other varieties by means of marker-assisted selection. Moreover, the
yields of plants overexpressing TT1CG14 exceeded control levels under
heat stress at these stages (Supplementary Fig. 15). These findings,
combined with the strong conservation of TT1 among higher plants,
suggest that TT1CG14 would have a dramatic effect on thermotol-
erance if bred into other crops. Consequently, we overexpressed
TT1CG14 in Arabidopsis and tall fescue (Festuca arundinacea Schreb.)
and found that transgene-positive plants of both species possessed
substantially more thermotolerance than the controls (Fig. 4c,d).
Therefore, we conclude that TT1 has great potential for breeding more
thermotolerant cultivars of cruciferous vegetables and graminaceous
crops such as wheat.
In this study, we identified what to our knowledge is the first QTL
(TT1) for crop thermotolerance and obtained evidence that plant
thermotolerance involves the modulation of protein homeostasis by
© 2015 Nature America, Inc. All rights reserved.
the proteasome. Previous studies have established that aggregated
protein refolding mediated by heat shock proteins is important
for plant thermotolerance40,41. However, our data indicate that the
removal of these inherently toxic proteins might be more critical
than recovery of their activity, especially when cells rapidly accu-
mulate large amounts of aggregated proteins. Although research-
ers have been endeavoring to identify the rice proteasome for a
long time42,43, the precise function of each rice proteasome subunit
remains largely unknown. The rice α2 subunit of the 20S protea-
some is encoded by two paralogous genes, TT1 (PAB1) and PAB2,
and TT1 was selected in the evolutionary adaptation of rice; PAB2
was not selected for reasons related to protein properties and expres-
sion levels (Supplementary Fig. 16a,b). Thermotolerance analysis of
PAB2-containing CSSLs further verified this concept (Supplementary
Fig. 16c). TT1CG14 from O. glaberrima is a more efficient allele
for thermotolerance and is expected to considerably enhance crop
productivity under global warming conditions.
npg
URLs. Comparative genomics analysis of O. sativa ssp. japonica
(Gramene), http://plants.ensembl.org/Oryza_sativa/Info/Index; the
Rice Annotation Project (RAP), http://rapdb.dna.affrc.go.jp/; Rice
Haplotype Map Project Database (RiceHap3), http://202.127.18.221/
RiceHap3/; Protein Data Bank (PDB), http://www.rcsb.org/pdb/
home/home.do.
Methods
Methods and any associated references are available in the online
version of the paper.
Accession codes. The CDS sequences of OgTT1 have been deposited
in GenBank and are available under accession KR054757.
Note: Any Supplementary Information and Source Data files are available in the
online version of the paper.
Acknowledgments
We thank G.Q. Zhang and the National Mid-term Genebank for Rice of China
National Rice Research Institute for kindly providing O. glaberrima and O. sativa
from Africa, respectively. We thank Y. Li for help with bioinformatics analysis.
This work was supported by grants from the Ministry of Science and Technology
of China (2012AA10A302, 2012CB944800), the National Natural Science
832 VOLUME 47 | NUMBER 7 | JULY 2015  Nature Genetics
Foundation of China (31421093, 31101128), the CAS-Croucher Funding Scheme
for Joint Laboratories, the China Postdoctoral Science Foundation, and the
Research Grants Council of Hong Kong (CUHK2/CRF/11G and AoE/M-05/12).
AUTHOR CONTRIBUTIONS
H.-X.L. conceived and supervised the project. H.-X.L., J.-P.G., J.-X.S. and X.-M.L.
designed the experiments. X.-M.L. carried out most of the experiments. D.-Y.C.,
Y.W., X.H., K.C., L.-G.C., L.S., W.-W.Y., H.C., H.-C.C., N.-Q.D., T.G., M.S., Q.F.,
P.Z., B.H., J.-X.S., J.-P.G. and H.-X.L. carried out some of the experiments. X.-M.L.,
D.-Y.C. and H.-X.L. analyzed data and wrote the manuscript.
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
Reprints and permissions information is available online at http://www.nature.com/
reprints/index.html.
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 ONLINE METHODS
Plant materials. Thermotolerance screening was done using a set of CSSLs
(designated SG and BC3F6) constructed with African cultivated rice CG14
(O. glaberrima), a variety showing high thermotolerance, as the donor parent
and the heat-susceptible japonica variety Wuyunjing (WYJ) as the recurrent par-
ent16. The set included 183 lines carrying overlapping chromosome segments of
the entire CG14 genome. SG42 and SG43 are independent lines with TT1CG14-
containing segments from CG14 that show more thermotolerance than the
recurrent parent; SG42 was selected for TT1 cloning. SG31 and SG32 are inde-
pendent lines harboring OgPAB2 and showing thermotolerance similar to that
of WYJ. Two additional sets of CSSLs were employed for TT1 functional differ-
entiation analysis. SW30, SW31 and SW33 carried TT1-containing fragments
from common wild rice (O. rufipogon) with thermotolerant Teqing (indica)
as the genetic background37. To construct SN32 and SN33, we introduced the
TT1 locus from Nona Bokra (indica) into a Koshihikari (japonica) genetic
background38. Rice accessions used for the thermotolerance identification
shown in Figure 3a,b were as follows: Jiahua1, Kongyu131, Koshihikari,
Nippobare, Xiushui09, Zhonghua11, Balila, Lemont, and Wuyunjing
(japonica); Feng’aizhan, Guanglu’ai4, Huanghuazhan, IR29, Minghui63, and
Teqing (indica); Kasalath (aus); IRAT104, IRAT109, IRAT261, ITA117, ITA182,
and ITA304 (O. sativa introduced to Africa); and ACC.102265, IRGC100127,
IRGC102239, IRGC102370, IRGC102580, and IRGC102635 (O. glaberrima).
The tall fescue (Festuca arundinacea Schreb.) variety Houndog5 was used for
© 2015 Nature America, Inc. All rights reserved.
thermotolerance engineering, and the Arabidopsis ecotype Columbia was used
for transformation.
Positional mapping and NIL development. The approximate location of
TT1 was determined by linkage analysis using BC4F2 populations, which were
derived by self-pollination of BC4F1 heterozygotes produced by backcrossing
SG42 with WYJ. Segregates containing a target segment from CG14 were more
thermotolerant than controls without segment substitution. These data support
the concept that TT1 was mapped to the target segment on chromosome 3.
To map the TT1 locus precisely, we carried out fine mapping using BC4F2
populations, which showed that the TT1 locus was localized to the interval
between two simple sequence repeat markers, RM15077 and RM15087. At
the same time, an NIL of TT1, NIL(CG14), and its isogenic control NIL(WYJ)
were constructed by means of several backcrosses (BC5F2) and marker-assisted
selection. NIL(CG14) harbored an approximately 200-kb TT1-containing
chromosomal segment from CG14 in the genetic background of WYJ. High-
resolution mapping of TT1 was performed using 6,721 BC4F2 plants and mark-
ers newly developed on the basis of the O. sativa ssp. japonica (Gramene)
and O. glaberrima genome sequences14,44. Homozygous recombinant plants
npg
(BC4F4) were selected for use in phenotypical thermotolerance identification
using BC4F3 progeny derived from corresponding recombinants. The TT1
locus was ultimately determined to be in a 12.69-kb region between markers
H1 and H6. The genomic DNA of the candidate gene was cloned via five
overlapping fragments, which were sequenced and compared. Primers used
for mapping and DNA cloning are shown in Supplementary Table 3.
Plant culture and thermotolerance identification. Rice seedlings used in this
study were cultivated by hydroponic culture in Yoshida solution (pH 5.8). Seeds
were incubated at 42 °C for approximately 15 d to break any possible dormancy,
soaked in tap water at 25 °C for 2 d and transferred to 35 °C tap water for ger-
mination. Water was changed daily. Uniformly germinated seeds were planted
in 96-well plates with the bottoms removed. The plates were placed on scaffolds
in a container of water so that the seeds were semi-immersed in the water and
left for 1 d at 35 °C in the dark to encourage root growth. Rooted seeds were
transferred to Yoshida solution and cultured at 28 °C, 50% relative humidity
under a 13-h light/11-h dark photoperiod. The Yoshida solution was changed
every 2 d. For genetic analysis of rice thermotolerance, it is necessary to
develop an efficient, effective approach with high feasibility and repeatability.
For heat treatment, high relative humidity (>80%) and low light intensity
(50–80 µM m−2 s−1) were used to minimize the effects of high light stress and
hydrophobic stress, which are often accompanied by high temperature stress41.
For genetic mapping (Fig. 1a) and linkage analysis (Supplementary Figs. 1b–d
and 16), 12-d-old (two-leaf stage) seedlings grown in hydroponic culture solu-
tion were incubated at 45 °C for 52 h, after which they were returned to normal
Nature Genetics
conditions (28 °C) for 1 week for recovery (Fig. 1a). A seedling’s survival state
was evaluated to determine its thermotolerance. For thermotolerance analysis
of transgenic plants at the seedling stage, 12-d-old seedlings were subjected to
45 °C treatment for the periods shown in Figures 1d,e and Supplementary
Figure 5. The thermotolerance of NILs and transgenic rice was also verified in
plants at the adult stage as described in Supplementary Figures 2b and 4b. To
determine the thermotolerance of cultivated varieties (Fig. 3a), we incubated
12-d-old seedlings at 45 °C for 72 h and then allowed them to recover for 2
weeks (28 °C). For survival-rate analysis of cultivars and CSSLs (Fig. 3b,c),
12-d-old seedlings were treated at 45 °C for 45 h and recovered at 28 °C for
2 weeks, and the number of each line and its corresponding survival were
recorded and used to calculate survival rates. To evaluate genotype-specific
performance during heat stress at the flowering and filling periods (Fig. 4a,b
and Supplementary Figs. 14 and 15), we transplanted rice plants at relevant
stages from the field (Shanghai (31°N, 121°E)) to a growth chamber under
a 12-h light (38 °C)/12-h dark (35 °C) photoperiod for 5 (flowering period)
or 12 (filling period) d, after which plants recovered at 28 °C until the grains
were ripe. Festuca arundinacea transgenic lines were grown in soil in pots at
18 °C under a 16-h light/8-h dark photoperiod. One-month-old, uniform
plants were treated at 42 °C for 48 h and recovered at 18 °C for 2 weeks.
Homozygous transgenic Arabidopsis and control plants were plated together
on SM plates and grown under a 16-h/8-h light/dark cycle at 22 °C/18 °C
in a growth chamber. For thermotolerance analysis, 11-d-old seedlings were
treated at 45 °C for 3 h and recovered under normal growth conditions for 1
week. After heat treatment of rice, tall fescue and Arabidopsis, viability was
assessed daily during recovery, and the results were recorded photographically
at the appropriate times.
Plasmid construction and transformation. For construction of the TT1
overexpression plasmid, the 708-bp full-length coding sequence of TT1 was
amplified from cDNA using primers TTOXU and TTOXL (Supplementary
Table 3), digested with SacI and XbaI, and cloned into binary vector pCAMBI-
A1301SN with the constitutive CaMV 35S promoter or pHB under the control
of two tandem CaMV 35S promoters45. This yielded TT1 fused to a FLAG
tag (ADYKDDDDK) with a GGGS linker. To construct the TT1 knockdown
plasmid, we generated the precursor of an artificial microRNA targeting
TT1 by overlapping PCR using MT-1, MT-2, MT-3, MT-4, FmiR and RmiR
(Supplementary Table 3) as described previously46, digested it with BamHI
and KpnI, and cloned it into pCAMBIA1301 under the control of the CaMV
35S promoter. To analyze the histological expression patterns of TT1CG14, we
amplified a 2,019-bp promoter fragment from NIL(CG14) genomic DNA using
prTTU and prTTL (Supplementary Table 3) and prepared the promoter region
by double digestion with KpnI-HindIII. The products were fused to the GUS
reporter gene with the nopaline synthase terminator and cloned into binary
vector pCAMBIA1300. All constructs were confirmed by sequencing and intro-
duced into Agrobacterium tumefaciens strain EHA105 or GV3101. Rice and
Festuca arundinacea were transformed using EHA105-mediated methods47,48.
The TT1 overexpression vector was also transformed into Arabidopsis by
the floral dip method mediated by GV3101 (ref. 49). Transgenic plants were
screened using primers HYGU and HYGL (Supplementary Table 3) and con-
firmed by quantitative PCR (qPCR) and protein blot analysis. Homozygous T3
transgene-positive rice or Arabidopsis and corresponding transgene-negative
controls were selected for thermotolerance analysis. Transgenic Festuca arund-
inacea plants shown in Figure 4c were from the first (T0) generation.
Expression analysis. To explore the expression of TT1 and PAB2 at the
mRNA level, we performed real-time qPCR using primers qTTU-qTTL
and qPAB2U-qPAB2L, respectively. OsACTIN, detected using primers
OsACTINU-OsACTINL, was used as an internal control (Supplementary
Table 3). Standard curve analysis revealed that the primer efficiency was as fol-
lows: qTTU-qTTL, 99.8%; qPAB2U-qPAB2L, 96.9%; OsACTINU-OsACTINL,
98.3%. For heat-induced expression analysis, 12-d-old seedlings were treated
at 45 °C, >80% relative humidity in the dark for the indicated periods (Fig. 1c),
and whole plants were sampled. For tissue-specific expression analysis (Fig. 1c),
uniform plants at the heading stage were used for tissue sampling. For geno-
type- or variety-specific expression analysis (Fig. 3b,c and Supplementary
Figs. 4a,c and 5a), 12-d-old seedlings (whole plants) were sampled before heat
doi:10.1038/ng.3305
 treatment. Total RNAs from various tissues were extracted using an RNeasy
Plant Mini Kit with DNaseI digestion on columns according to the user manual
(Qiagen). First-strand cDNA was synthesized using a PrimeScript RT Reagent
Kit (TaKaRa). qPCR was performed on an ABI7300 system using the SYBR
Green method (SYBR Premix Ex Taq (Perfect Real Time), DRR041A, TaKaRa).
The cycling conditions consisted of 40 cycles of amplification (95 °C for
5 s and 60 °C for 31 s), with 95 °C pre-degeneration for 30 s. To confirm the
histological expression patterns of GUS driven by the TT1CG14 promoter, we
performed GUS histochemical staining as described previously50. To verify
the accumulation of the TT1-FLAG protein in planta, we extracted total
proteins from whole transgenic plants in extraction buffer (50 mM sodium
phosphate buffer, pH 7.0, 150 mM NaCl, 5% SDS, 0.1% Tween-20, 50 mM
MG132, 1× complete protease inhibitor cocktail) and boiled them for 5 min,
after which they were centrifuged at 12,000 rpm for 10 min. The supernatant
was mixed with equal amounts of 2× SDS loading buffer and subjected to
SDS-PAGE and subsequent immunoblotting with anti-FLAG antibodies
(Sigma, A8592). The 55-kDa Rubisco large subunit stained with Coomassie
Blue was used as a loading control.
Assessment of the ubiquitin-modified proteome. Ubiquitinated peptides
were detected using an IP-MS/MS strategy as described schematically in
Figure 2a. Fifty representative 12-d-old NIL(WYJ) or NIL(CG14) seedlings
from each of four groups were sampled using the cluster sampling method
© 2015 Nature America, Inc. All rights reserved.
before (CK) or after (HT) 45 °C treatment for 30 h and were then frozen in
liquid nitrogen. Frozen samples were pooled and ground into a powder (1 g per
sample) for total protein extraction using the TCA-acetone method. Proteins
were dissolved in SDT buffer (4% SDS, 100 mM DTT, 150 mM Tris-HCl, pH
8.0), boiled (5 min) and subjected to ultrasonication, and undissolved debris
was removed by centrifugation at 14,000 rpm for 15 min. The supernatants
were collected and quantified with a BCA Protein Assay Kit (Bio-Rad). Proteins
(250 µg per reaction, 60 reactions (15 mg) in total per replicate (three replicates
per sample)) were digested by filter-aided sample preparation51; ubiquitin tags
were digested to yield diGly linked to Lys. Lysate peptides were purified by
Sep-Pak C18 (Waters) after acidification using trifluoroacetic acid (TFA; final
concentration, 1%) according to the manufacturer’s protocol. Immunoaffinity
purification (IPA) of K-GG–modified peptides was performed using a
PTMScan Ubiquitin Remnant Motif (K-ε-GG) Kit (Cell Signaling Technology).
Briefly, lyophilized peptides were redissolved in IPA buffer and incubated
with K-GG motif antibody conjugated to beads for 2 h at 4 °C. IPA buffer and
beads (supplied in the kit) were washed four times in PBS buffer before incu-
bation. After affinity incubation, the beads were washed sequentially, twice
with IPA buffer and three times with ultrapure water. The K-GG–modified
npg
peptides were then eluted twice from the beads in 0.15% (vol/vol) TFA. The
purified peptides were desalted on C18 Cartridges (Empore SPE Cartridges
C18), concentrated by vacuum centrifugation and reconstituted in 40 µl 0.1%
TFA. MS experiments were performed on a Q Exactive Mass Spectrometer
coupled to Easy nLC (Thermo Fisher Scientific, formerly Proxeon Biosystems).
Then, 5-µg peptide samples were loaded onto a C18 reversed-phase column
(Thermo Scientific Easy Column, 10 cm long, 75-µm inner diameter, 3 µm
resin) in buffer A (2% acetonitrile and 0.1% formic acid) and separated with
a linear gradient of buffer B (80% acetonitrile and 0.1% formic acid) at a flow
rate of 250 nL/min controlled by IntelliFlow technology over 120 min. MS data
were acquired using the data-dependent top-10 method, dynamically choos-
ing the most abundant precursor ions from the survey scan (300–1,800 m/z)
for HCD fragmentation. Determination of the target value was based on pre-
dictive automatic gain control. The MS data were analyzed using MaxQuant
software version 1.3.0.5 and searched against the UniProtKB Oryza sativa
database. An initial search used a precursor mass window of 6 ppm. The search
followed the enzymatic cleavage rule for trypsin/P and allowed a maximum
of two missed cleavage sites and a mass tolerance of 20 ppm for fragment
ions. Carbamidomethylation of cysteines was defined as a fixed modification,
and protein N-terminal acetylation, methionine oxidation and GlyGly (K)
ubiquitination were defined as variable modifications for database searching.
The cutoff for the global false discovery rate for peptides was set at 0.01. The
intensity of the monoisotopic parent ion (precursor) was used to quantify
each individual peptide (peptide intensity from MaxQuant software).
Quantification was based on spectral characteristics (retention time, m/z ratio
doi:10.1038/ng.3305
and peak intensity) determined by comparing the direct mass spectrometric
signal intensities for given peptides. Correlation coefficients for all quantified
peptides were calculated as described34 (Supplementary Fig. 9a). Two-way
ANOVA was used to select the differentially accumulated K-GG–modified
peptides (P(interaction) < 0.05; Supplementary Table 1). The heat map was
drawn with Cluster 3.0 and Treeview using the Euclidean distance complete-
linkage method. Gene set enrichment analysis was performed as described
previously52,53. The enrichment score, indicating the degree to which a gene set
is overrepresented at the termini of all differentially accumulated ubiquitinated
proteins, was calculated via the running-sum statistical method. The false
discovery rate was calculated to control the proportion of false positives. To
verify the ubiquitylome patterns of different samples, we carried out biochemi-
cal immunoblotting. Twelve-day-old seedlings (NIL(WYJ) and NIL(CG14))
were treated at 45 °C for 0, 30 or 45 h, and whole plants were then sampled.
Total proteins were extracted as mentioned above, separated by SDS-PAGE,
transferred to a polyvinylidene difluoride membrane and immunoblotted
with anti-polyubiquitinated-conjugate antibody (Biomol, Cay14219, clone
FK1; Supplementary Fig. 9d).
Proteasome activity assay. For active proteasome extraction, 12-d-old seed-
lings (NIL(WYJ) and NIL(CG14)) were sampled, frozen in liquid nitrogen
and ground into a powder. Lysis buffer (500 µl for 300 mg powder, 50 mM
HEPES, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 15% glycerol)
was added to the powder, and the solution was mixed gently at 4 °C for 20 min
and then centrifuged at 12,000 rpm at 4 °C for 10 min. The supernatants were
used for the proteasome activity assay. In brief, 10 µl extracted active proteins
was added to the reaction buffer (final concentration: 25 mM HEPES, pH
7.5, 0.5 mM EDTA, 0.05% NP-40 and 0.001% SDS (wt/vol)), and then fluoro-
genic peptide substrate Suc-LLVY-AMC (Merck Millipore; reconstituted with
dimethyl sulfoxide) was added to a final concentration of 1 mM. The reaction
(total volume of 100 µl) was conducted in a 96-well fluorometer plate for 2 h
at 37 °C or 45 °C. Fluorescence data were collected at 360 nm excitation and
460 nm emission. For the inhibition assay, 25 µM lactacysin was preincubated
with proteasome extract for 15 min at room temperature, after which Suc-
LLVY-AMC substrate was added.
Population genetic analysis. We obtained information about haplotypical
SNPs at the TT1 locus by surveying 651 cultivated varieties (O. sativa) and
428 wild rice accessions (O. rufipogon) from the RiceHap3 Database (Fig. 3d).
Population-differentiation statistics (FST) were calculated in a TT1-containing
100-kb window as described previously54. Pairwise comparisons among the
indica, tropical japonica and temperate japonica landraces were performed.
FST values were calculated using the whole genome (genome-wide mean) as
a background control (Fig. 3e). DNA sequences of TT1 promoters in more
than 150 rice accessions belonging to different groups were cloned by over-
lapping PCR using primers UU-2, UL-2, DU-1 and DL-1 (Supplementary
Table 3). The clones were sequenced and subjected to haplotype analysis
(Supplementary Fig. 11). PAB2 coding sequences were cloned from 10 typical
O. sativa lines (Koshihikari, Nippobare, Wuyunjing, Zhonghua11, Guanglu’ai4,
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sentative O. glaberrima lines (CG14, ACC.102265, IRGC100127, IRGC102370,
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for further alignment analysis (Supplementary Fig. 16).
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