996 VOLUME 33 NUMBER 9 SEPTEMBER 2015 nature biotechnology

l e t t e r s

The detrimental effects of global warming on crop productivity threaten to reduce the world’s food supply1–3. Although plant responses to changes in temperature have been studied4, genetic modification of crops to improve thermotolerance has had little success to date. Here we demonstrate that overexpression of the Arabidopsis thaliana receptor-like kinase ERECTA (ER) in Arabidopsis, rice and tomato confers thermotolerance independent of water loss and that Arabidopsis er mutants are hypersensitive to heat. A loss-of-function mutation of a rice ER homolog and reduced expression of a tomato ER allele decreased thermotolerance of both species. Transgenic tomato and rice lines overexpressing Arabidopsis ER showed improved heat tolerance in the greenhouse and in field tests at multiple locations in China during several seasons. Moreover, ER-overexpressing transgenic Arabidopsis, tomato and rice plants had increased biomass. Our findings could contribute to engineering or breeding thermotolerant crops with no growth penalty.

The temperature increases associated with global warming reduce plant growth and crop productivity3. In particular, a harmattan climate, which brings desert-like conditions, causes immediate death of plants or their tissues. It has been estimated that rice grain yield declines by 10% for each 1 °C increase in local night temperature in the dry season1 and that global warming has a negative impact on the yield of other major grain crops including wheat, maize and barley2. The heat wave during the 2013 summer in eastern China had a dev-astating impact on crops. Plants respond to temperature changes by reprogramming their growth and development4–8, and progress has been made towards genetic modification of plants to increase their tolerance to heat stress. However, these studies have mainly been restricted to the model plant Arabidopsis9–14. Many studies have shown that expression of heat shock proteins (HSPs) can improve the tolerance of transgenic plants to heat shock (short exposure to high temperatures)15. In particular, HSP101 is required for thermo-tolerance in plants and has potential as a tool for crop improvements.

Overexpression of HSP101 increased tolerance to heat shock or prolonged heat stress in transgenic Arabidopsis, tobacco and cot-ton9,14, although transgenic cotton produced fewer bolls and seeds than control cotton plants under normal growth conditions14. On the other hand, engineering to increase the tolerance of plants to high temperatures during multiple hot summer seasons at different locations has not been reported. In particular, there are few reports of engineering or breeding of thermotolerant staple crop species, except for a recent study, which reported that natural alleles of a gene encoding proteasome α2 subunit from African rice contribute to thermotolerance15. Therefore, genes other than HSP101 are critical to thermotolerance improvement in crops16.

The model plant Arabidopsis is sensitive to high temperatures, probably owing to local adaptation17,18, and cannot survive prolonged heat stress19. Following our previous studies of Arabidopsis heat and harmattan responses19–21, we report in this study that two ecotypes, Columbia-0 (Col-0) and Landsberg erecta (Ler), were substantially dif-ferent from each other in their tolerance to prolonged extreme heat in greenhouse conditions (40 °C; Fig. 1a). Ler plants began wilting 12 h after transfer from 22 °C to 40 °C and completely wilted or died 48 h after heat treatment. Col-0 plants, however, only began wilting 36 h after heat treatment, and a fraction (~50%) of their leaves retained osmotic pressure 48 h after heat treatment, resulting in a significantly higher survival rate (47.2%) compared with Ler (7.8%) 48 h after heat treatment (Supplementary Fig. 1a). This difference in heat tolerance indicates that the Col-0 strain contains one or more gene(s) for heat tolerance (Hat). To map Hat loci in Col-0, we first screened a set of recombinant inbred lines (RILs) derived from a cross of Col and Ler. These lines were classified into three groups based on survival rates: lines with high tolerance (similar to Col-0), lines with high sensitivity (similar to Ler) and lines with intermediate properties (Supplementary Fig. 1a). Based on the RIL phenotype, two major quantitative trait loci (QTL) were detected: qHat2-1, with a single peak having an LOD score of 6.6 and 30% contribution on chromosome 2, and qHat2-2, with three small peaks with an LOD score of 5.0–6.9 and 23–28% contribution on chromosome 4 (Supplementary Fig. 1b

Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomatoHui Shen1,2,9, Xiangbin Zhong1,9, Fangfang Zhao1, Yanmei Wang1, Bingxiao Yan1, Qun Li1, Genyun Chen1, Bizeng Mao3, Jianjun Wang4, Yangsheng Li5, Guoying Xiao6, Yuke He1, Han Xiao1, Jianming Li7 & Zuhua He1,8

1National Key Laboratory of Plant Molecular Genetics and National Center of Plant Gene Research, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 2Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, Shanghai, China. 3College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China. 4Zhejiang Academy of Agricultural Sciences, Hangzhou, China. 5College of Life Science, Wuhan University, Hubei, China. 6Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China. 7Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 8Collaborative Innovation Center of Genetics and Development, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China. 9These authors contributed equally to this work. Correspondence should be addressed to Z.H. (zhhe@sibs.ac.cn).

Received 10 February; accepted 21 July; published online 17 August 2015; doi:10.1038/nbt.3321

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

http://www.nature.com/doifinder/10.1038/nbt.3321http://www.nature.com/naturebiotechnology/

nature biotechnology VOLUME 33 NUMBER 9 SEPTEMBER 2015 997

l e t t e r s

and Supplementary Table 1). We decided to fine-map the qHat2-1 locus by constructing chromosome segment substitution lines (CSSLs), with Ler as the recurrent parent, that cover chromosome 2 with five CSSLs (Supplementary Fig. 1c). The line CSSL-2-3 was associated with a considerably higher survival ratio compared with other CSSLs (Supplementary Fig. 1d), indicating that CSSL-2-3 contains the qHat2-1 locus for heat tolerance.

The line CSSL-2-3 covers a 493-kb region of chromosome 2, and the middle of this segment contains the ER gene (Supplementary Fig. 1d), which encodes a receptor-like kinase that was mutated in Ler and has been implicated in diverse growth and development processes22–28, including warmth-induced abnormal adaxial-abaxial polarity in leaf patterning29 and photosynthetic capacity30. To evaluate

whether ER is the qHat2-1 locus, we first measured the heat sensitivity of er mutants in Col-0 and observed that a null er mutant, er-105 (ref. 22), was more sensitive to heat than the wild-type Col-0 (Supplementary Fig. 2a,b). A similar difference in heat sensitivity was also observed between Ler and its wild-type Landsberg (Lan) (Supplementary Fig. 2c,d). We next transformed er-105 with the Col-0 wild-type ER gene driven by its native promoter (pER::ER) and found that the pER::ER transgene completely restored heat tolerance of the null mutant (Supplementary Fig. 2a,b). Similar results were obtained by complementation with the same pER::ER transgene in Ler lines (Supplementary Fig. 2c,d). Thus, we conclude that ER is the functional gene for qHat2-1, and that er loss-of-function mutations greatly reduce heat tolerance.

GenotypeGenotypeGenotype

22 °C **

30 °C

22 °C

** **

*

*

*

22 °C

Inst

anta

neou

s W

UE

(µm

ol/m

mol

)

WU

E (

mg/

g)

Sto

mat

al c

ondu

ctan

ce

(mol

/m2 /

s)

5 3.5

3.0

2.5

2.0

1.5

1.0

0.5

0

4

3

2

1

0

0.20

0.15

0.10

0.05

0

Col-0

L7-1

er-105

Col-0

L7-1

er-105

Col-0

L7-1

Col-0

L7-1

Col-0

40 °C 48 h

Col-0 L7-1L2-3 L7-1er-105

30 °C 30 d

Col-0 L7-1er-105

0 h 12 h

Col

-0Ler

24 h 36 h 48 h

Heat treatment (40 °C)a

Col-0

L2-3

L7-1

er-105

35S::E

R

b

c

170 kDaM

arke

r

er-105

Col-0LM

13LM

17LM

10

130 kDa

100 kDa

70 kDa

55 kDaActin

ER-MH(α-MYC)

d

Genotype

0

5

10

15

Inflo

resc

ence

num

ber

per

plan

t 20

25 22 °C

**

**

Col-0

L7-1

er-105

e

Genotype

0.5

0.4

0.3

0.2

0.1

0

22 °C**

**

*

See

d w

eigh

t per

plan

t (g)

Col-0

L7-1

L2-3

er-105

f

Genotype

25

20

15

10

5

0

22 °C

**

****

Sho

ot d

ry w

eigh

tpe

r pl

ant (

g)

Col-0

L7-1

L2-3

er-105

g

h i

Genotype

22 °C

* *** **

****

**

40 °C 40 °C

30 °C

Sur

viva

l rat

e (%

)0

20

40

60

80

100

Col-0

L2-3L7

-1

er-105

Col-0

L2-3L7

-1

er-105

Col-0

Col-0

LM13LM

17LM

10L7

-1

er-105

j

k l m

Figure 1 ER plays an important role in heat tolerance in Arabidopsis. (a) Pictures of 2-week-old Arabidopsis plants grown in a 40 °C growth chamber for 0, 12, 24, 36 and 48 h. Scale bar, 1 cm. (b) Pictures of 4-week-old plants grown at 22 °C (left panels) of the wild-type Col-0, er-105 and two ER-OE lines (L2-3 and L7-1) and their corresponding individual leaves of different developmental stages (right panels). Scale bars, 1 cm. (c) Mature plants of Col-0, er-105 and L7-1 grown at 22 °C. Scale bar, 1 cm. (d) Immunoblot analysis of the ER-MH abundance in wild-type Col-0, er-105 and three selected p35S::ER-MH er1-05 transgenic lines, LM13 (wild-type-looking), and L10 and L17 (bigger than Col-0) (see Supplementary Fig. 4 for pictures of plants and their leaves). Total proteins extracted from 2-week-old plants grown at 22 °C were separated by SDS-PAGE and analyzed by immunoblotting using anti-myc and anti-actin (for loading control) antibodies. (e–g) Average numbers of inflorescent stems (e), average seed production (f) per plant after maturation and average shoot dry weight (g) of 4-week-old wild-type Col-0, er-105 and two ER-OE lines (L2-3 and L7-1) grown at 22 °C, as shown in box plots (n =30). *P < 0.05 or **P < 0.01, Student’s t-test and Bonferroni correction for multiple tests (two comparisons for e, three comparisons for f,g). (h) Pictures of 2-week-old plants of the wild-type Col-0, er-105 and the two ER-OE lines after 2 d treatment at 40 °C. Scale bar, 1 cm. (i) Pictures of mature plants of the wild-type Col-0 and L7-1 grown at 30 °C for 30 d. (j) Survival rates of the wild-type Col-0, er-105 and the ER-OE lines after exposure to a hot (40 °C, 2 d) (h) and a long warm temperature (30 °C, 30 d) (i), as shown with dots of three replicates (30 plants each). *P < 0.05 or **P < 0.01, by Student’s t-test Bonferroni correction for multiple tests in the independent temperature experiments, with three comparisons for Col-0, er-105, L2-3 and L2-7 at 40 °C, and four comparisons for Col-0, er-105, LM10, LM13 and LM7 at 40 °C. (k) Stomatal conductance of 4-week-old wild-type Col-0 and L7-1 grown at 22 °C, as shown with dot plots (n ≥ 8). **P < 0.01, Student’s t-test. (l) Quantitative analysis of instantaneous water use efficiency of 4-week-old plants of the wild-type Col-0 and L7-1 grown at 22 °C, as shown with dot plots (n ≥ 8). **P < 0.01, Student’s t-test. (m) Quantitative measurement of water use efficiency of 4-week-old plants of the wild-type Col-0, er-105 and L7-1 grown at 22 °C or 30 °C, shown as box plots (upper and lower quartiles and median; n = 25). *P < 0.05 or **P < 0.01, by Student’s t-test and Bonferroni correction for multiple (two comparisons) tests in the independent temperature experiments. (See source data.)

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

998 VOLUME 33 NUMBER 9 SEPTEMBER 2015 nature biotechnology

l e t t e r s

We next investigated whether overexpressing the ER gene could improve heat tolerance of wild-type plants. We used the strong and constitutively active 35S promoter from cauliflower mosaic virus with two copies of an enhancer element to drive the expression of a promot-erless ER transgene in wild-type Col-0 plants. Out of p35S::ER trans-genic lines, we identified nine lines with increased biomass as measured by rosette leaf sizes, inflorescence numbers (Fig. 1e), shoot weight and seed production (Fig. 1b,c,e–g and Supplementary Fig. 3c,d). Quantitative real-time RT-PCR (qPCR) analysis showed that ER transcript abundance was stably increased by ~20- to 90-fold in rosette leaves of nine 2-week-old transgenic lines (referred to as ER-OE, hereafter) compared with wild-type Col-0 (Supplementary Fig. 3a). qPCR analysis using RNAs isolated from different tissues of the wild-type Col-0 and L7-1, one of the ER-OE lines, revealed that the ER transcript accumulated to very high levels (Supplementary Fig. 3) in both rosette and cauline leaves (Supplementary Fig. 3b). Further, elevated ER expression had no detectable effect on the transcript levels of two ER homologs, ERL1 and ERL2, at different developmen-tal stages of 2-week-old and 6-week-old plants or in different tissues, such as rosette and cauline leaves (Supplementary Fig. 3e). Despite numerous attempts, we failed to obtain an anti-ER antibody that would allow us to directly test if increased levels of the ER transcript led to increased accumulation of the ER protein. Instead, we added a myc-His (MH) tag to the p35S::ER transgene, transformed er-105 with the resulting p35S::ER-MH transgene and found that although

eight of ten p35S::ER-MH er-105 transgenic lines were phenotypically rescued, two lines (LM10 and LM17) that accumulated higher levels of the transgene transcripts and ER-MH protein grew bigger and had a higher thermotolerance than the wild-type control (Fig. 1d,j and Supplementary Fig. 4). This suggests that the observed growth and physiological phenotypes of ER-OE plants were most likely caused by increased abundance of the ER protein. Whereas er mutations decrease the size of leaf epidermal cells and increase the stomatal density without affecting the stomatal index (the ratio of number of stomata to total number of leaf epidermal cells)28,30, our ER-OE plants had larger leaf epidermal cells and decreased stomatal density but there was no change in their stomatal index (Supplementary Fig. 5), providing additional support for the hypothesis that ER abundance was increased in the ER-OE lines and for a role for ER in regulating cell growth and stomata development.

To directly test the effect of ER overexpression on heat tolerance, we subjected 2-week-old Arabidopsis plants to a 2-d 40 °C treatment in a growth chamber and observed (P < 0.05 or P < 0.01) that the ER-OE plants grew much better (Fig. 1h), with significantly higher survival rates (65–75%) compared with wild-type Col-0 plants (~48%)(Fig. 1h,j). We tested plant tolerance to prolonged exposure to a moderate increase in ambient temperature ( from 22 °C to 30 °C). After 30-d growth at 30 °C the leaves of wild-type Col-0 became yellowed, and most plants (~85%) wilted and died. In contrast, most leaves on the L7-1 line grown at 30 °C for 30 d remained green (Fig. 1i), and a

80

Sur

viva

l rat

e (%

)

40 °C30–40% RH

40 °C90–95% RH

60

* *

*

*

40

20

0

Genotype

Col-0

er-105

L7-1

Col-0

er-105

L7-1

a

**100

Rel

ativ

e w

ater

con

tent

(%

)

90

80

70

60

Col-0er-105

Time (h)

0 12 24

b

lon

leak

age

(%)

100

80

60

40

20

0

Col-0er-105L2-3L7-1

Time (h)

0 12 24 36 48

c40 °C, 0 h

v

v

v

v

v

v

vv

v

v

v

v

v

vv

v

pm

pm pm

pm

pmpmpm

pm

pm pmcw

cwcw

cw

cwcwcwcw

cw cw

40 °C, 12 h 40 °C, 24 hC

ol-0

er-105

L7-1

d

*80

100

Pla

sma

mem

bran

edi

srup

tion

(%)

60

40

20

0

Genotype

Col-0

er-105

L7-1

e

40 °

C, 0

h40

°C

, 48

h

Col-0 er-105 L7-1f 40 °C, 0 h 40 °C, 24 h 40 °C, 48 h

Col

-0er-105

L7-1

g

20

25

30

Rel

ativ

e ex

pres

sion

leve

ls o

f HSFA1a

15

10

5

0

Col-0er-105

L7-1

0 h

40 m

in 2 h

4 h

8 h

12 h

24 h

36 h

h

Rel

ativ

e ex

pres

sion

leve

ls o

f Bl1 Col-0

er-105

L7-120

25

30

15

10

5

0

0 h

40 m

in 2 h 4

h8

h12

h24

h36

h

i

Figure 2 Analysis of leaf water content and heat-induced cell damages. (a) Quantification of survival rates of 2-week-old heat-treated (40 °C for 2 d) plants of the wild-type Col-0, er-105 and L7-1, grown under low (30–40%) and high (90–95%) RH conditions, shown in dots of three replicates (30 plants each). *P < 0.05, by Student’s t-test and Bonferroni correction for multiple tests with two comparisons in the independent RH experiments. (b) Relative water content of 2-week-old wild-type Col-0 and er-105 plants grown at 22 °C treated with a 40 °C growth condition for 0 h, 12 h and 24 h, shown as dots (n = 12). No difference in water content was detected between heat-treated er-105 and wild-type Col-0 but a statistically significant difference was detected between non-heat-treated plants of the wild-type Col-0 and er-105 by Student’s t-test (**P < 0.01). (c) Measurements of cellular ion leakage over a time course of 48 h under heat treatment (40 °C). Leaves of 2-week-old plants grown at 22 °C of the wild-type Col-0, er-105 and the ER-OE lines L2-3 and L7-1 treated with or without 48 h growth at 40 °C were measured. Values are means ± s.d. (n = 15). (d) TEM subcellular observation of leaf cell collapse during the 40 °C heat treatment for 0–24 h. The plasma membrane (pm) became blebbed and collapsed more quickly in the er-105 cells than in the Col-0 cells; blebbing and collapsing of the plasma membrane were observed less often in the ER overexpression L7-1 cells. Red arrowheads indicate pm stacking and twinning, and red arrows indicate PM blebbing in er-105. Scale bars, 500 nm. (e) Quantification of the occurrence of plasma membrane disruption (blebbing and collapsing) in leaf cells after 24 h growth at 40 °C, shown as dots of three replicates (≥50 cells observed each). *P < 0.05, by Student’s t-test and Bonferroni correction for two comparisons. (f) Visualization of cell death using trypan blue staining of rosette leaf cells of 2-week-old plants grown at 22 °C and treated with heat (40 °C) for 0 and 48 h. Scale bars, 2 mm. (g) H2O2 accumulation, detected by the DAB reaction, was more obvious in er-105 than in Col-0. Scale bars, 2 mm. (h,i) qPCR analysis of the heat shock factor gene HSFA1a (h) and the cell death regulator gene BI1 (i) with total RNAs isolated from leaves of 2-week-old plants grown at 22 °C over the course of 36 h heat (40 °C) treatment. ACTIN2 was used as a control to normalize expression levels. Values are means ± s.d. (n = 3). (a,b,e). cw, cell wall; pm, plasma membrane; v, vacuole. (See source data.)

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature biotechnology VOLUME 33 NUMBER 9 SEPTEMBER 2015 999

l e t t e r s

Figure 3 ER confers heat tolerance in tomato. (a) qPCR analysis of the transgenic ER transcript in selected p35S::ER transgenic tomato lines (4-week-old, grown at 22 °C). The tomato SleIF4a6 gene was used as a control to normalize expression levels. Values are means ± s.d. (n = 3). (b) Pictures of mature branches from 6-week-old plants grown at 22 °C of the vector control and p35S::ER transgenic tomato. Scale bar, 2 cm. (c) Pictures of 6-week-old transgenic tomato plants grown at 22 °C (upper panels) and subsequently for 10 d under a 40 °C (38–42 °C recorded due to daily fluctuation)/28 °C (day/night) cycle (middle panels), followed by 2-d recovery growth at 22 °C (lower panels). (d) Survival rates of transgenic tomato lines after heat treatment in growth chamber (c), shown as dots of three replicates (~30 plants each). **P < 0.01, by Student’s t-test and Bonferroni correction for three comparisons. (e) Pictures of post-summer mature tomato plants (~10-week-old after the summer) grown at the Shanghai experimental station in 2012. (f) Field-grown transgenic tomato plants (6 weeks old) in the summers (July–August) of 2013 and 2014 at the Shanghai station and the 2014 summer at the Wuhan station. (g) Post-summer survival rates of the field-grown transgenic tomato lines tested at Shanghai (2013 and 2014) and Wuhan (2014), shown as dots of replicates (30–50 plants each). **P < 0.01, by Student’s t-test and Bonferroni correction for three comparisons in the independent summer field tests. (h) SlER transcript levels in selected tomato accessions (4 weeks old, grown at 28 °C), as shown in dots of three replicates. The tomato SleIF4a6 gene was used as control to normalize expression levels. (i) Seedling survival rates of the six chosen tomato accessions plus LA1589 after 3-d heat treatment (40 °C/28 °C (day/night)) and 2-d 22 °C recovery growth, shown as dots of three replicates (30 plants each). Different letters at the top of dots indicate a significant difference at P < 0.05, by Welch’s ANOVA and Bonferroni correction for six tests (h,i). Survival rates for normal growth temperature (22 °C) controls are 100% (d,i). (See source data.)

higher proportion of the L7-1 plants (~43%) survived after recovery under 22 °C compared with the wild-type control (~13%) (Fig. 1j). Similarly, the two ER-MH lines (LM10 and LM17) with higher ER-MH abundance were more thermotolerant than the wild-type control (Fig. 1j). Taken together, these experiments show that ER overexpres-sion substantially increased heat tolerance in Arabidopsis.

The ER locus is known to be a determinant of transpiration efficiency (the rate of carbon fixation to water loss) or water use efficiency30. Loss-of-function er mutations have decreased transpira-tion efficiency, likely owing to increased leaf porosity and decreased photosynthetic capacity30. Consistent with this, ER overexpression in Arabidopsis lines L2-3 and L7-1 decreased stomatal density and enlarged epidermal cells (Supplementary Fig. 5), therefore decreas-ing stomatal conductance overall (Fig. 1k). Importantly, we found that ER overexpression increased both instantaneous and integrated water-use efficiency relative to the wild-type Col-0 (Fig. 1l,m). In addition, ER-OE lines were more tolerant to drought than the wild-type (Supplementary Fig. 6). The fewer stomata and lower transpiration rate might result in increased drought tolerance in the ER-OE plants. However, an alternate explanation is that the ER-mediated thermotolerance mechanism occurs to affect drought tolerance. To better understand how ER improves heat tolerance, we first deter-mined whether the ER-mediated thermotolerance was simply caused

by reduced transpiration. We undertook these experiments because ER-OE lines had lower leaf conductance compared with the wild type, which could counteract the effect of higher temperatures on transpiration rates. We carried out 40 °C heat treatments for 48 h for er-105, L7-1 and the wild-type Col-0 under low (30–40%) and high or near-saturated (90–95%) relative humidity (RH) because humidity is known to be inversely related to transpiration; we then measured plant survival rates. Counterintuitively, the survival rate of all three genotypes was higher (31–71%) under low RH than under high RH (16–63%; Fig. 2a), possibly due to a faster transpiration rate, result-ing in lower leaf surface temperature at low RH. No difference in water content between er-105 and wild-type Col-0 incubated at 40 °C for 24 h was detected (Fig. 2b). Notably, the difference in relative survival rate of L7-1/the wild type/er-105 was more significant under high RH (predicted to cause slower transpiration rate with higher leaf surface temperature) (~4:2:1) than under low RH (~2:1.6:1) (Fig. 2a), indicating that ER had a stronger impact on thermotolerance under high RH than under low RH. Taken together, our data indicate that ER enhanced plant thermotolerance through an unknown cellular mechanism that is independent of its effect on leaf transpiration30. Supporting an independent role for ER in thermotolerance, an earlier study showed that ER is also required for thermoprotection in the leaf AS1/AS2 pathway for adaxial-abaxial polarity formation29.

L20 L22 L25

Pre

-tre

atm

ent

40 °

C/2

8 °C

10

days

Rec

over

y

Vector

10,0008,000

Rel

ativ

e ex

pres

sion

6,0004,000

Vecto

rL2

0L2

2L2

5

1000

35S::ER

L20

12

3

Vecto

rL22 L25

2012 summer, ShangahiL20 L22 L25Vector

a b e

c f

g

a a a

b

c cd**

****

d ccbb

aaa

10012

9

6

3

0 Sur

viva

l rat

e (%

)

Sur

viva

l rat

e (%

)

Rel

ativ

e ex

pres

sion

leve

ls o

f S/ER

806040200

80

60

40

20

0

Vecto

rL2

0L2

2L2

5LA

1589

LA17

81

LA15

80M

82

Djen

a go

ld gir

l

Borg

o ce

llano

Heinz

1706

LA15

89

LA17

81

LA15

80M

82

Djen

a go

ld gir

l

Borg

o ce

llano

Heinz

1706

d h i

100

80

Sur

viva

l rat

e (%

)

60

40

20

Vecto

r

Vecto

rL2

0L2

0L2

0L2

2L2

2L2

2L2

5L2

5L2

5

Vecto

r

2013 summer,Shanghai

2014 summer,Shanghai

2014 summer,Wuhan

****

**

******

****

**

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

1000 VOLUME 33 NUMBER 9 SEPTEMBER 2015 nature biotechnology

l e t t e r s

To better understand how ER enhances plant thermotolerance at the cellular level, we analyzed the cellular responses of er-105, ER-OE and the wild-type Col-0 to 40 °C heat stress. We found that the surfaces of wild-type Col-0 and er-105 leaves were similar at normal temperatures but differed when lines were shifted to 40 °C (Supplementary Fig. 7). 12 h after heat treatment, the entire leaf surface of er-105 was wrinkled and rough, whereas the leaf surface of Col-0 was only just starting to become wrinkled, which suggests that leaf cells in er mutants are more easily damaged or killed under heat stress. It is already known that the stability of the cell membrane is essential for plant thermotolerance and that high temperatures dis-rupt cell membranes. We found that er-105 leaf cells had more ion leakage than those in the wild-type Col-0 (82% vs. 60%) (Fig. 2c). Transmission electron microscopy (TEM) revealed that the plasma membrane seemed to be blebbing and broken in er-105 cells 12 h after heat treatment, whereas wrinkled and broken plasma membrane was only detected in WT Col-0 cells 24 h after heat treatment (Fig. 2d and Supplementary Fig. 8), with a higher occurrence of plasma membrane disruption in er-105 than in Col-0 (71% vs. 46%) 24 h after heat treatment) (Fig. 2e). Plasma membrane disruption was often associ-ated with plasmolysis in Col-0; however, the plasma membrane in

er-105 was rapidly broken without typical plasmolysis (Fig. 2d and Supplementary Fig. 8). Earlier and more severe membrane blebbing and collapsing were also detected in the chloroplasts and mitochon-dria of er-105 cells subjected to heat stress (Supplementary Fig. 8). Consistent with the observed membrane damage, trypan blue stain-ing revealed increased cell death in er-105 (Fig. 2f); we also observed increased accumulation of H2O2, which is associated with cell damage, relative to the wild-type control (Fig. 2g). By contrast, cells in the ER-OE plants were resistant to heat-induced cellular damage and remained healthy 24 h after heat treatment, with less ion leakage and cell damage compared with wild type (Fig. 2c–f and Supplementary Fig. 8). These data suggested the hypothesis that ER overexpression might protect membrane integrity during heat stress. Transcript lev-els of HSFA1a31 and BI1 (ref. 32), both of which are involved in heat response and cell death inhibition, increased in L7-1 and decreased in er-105 (Fig. 2h,i). Interestingly, heat stress suppressed transcription of ER (Supplementary Fig. 9). Taken together, these results might indicate an important role for ER in protecting plant cells from heat-induced cellular damage and death.

Next, we evaluated whether ER could be used to improve heat tol-erance in crops. We transformed tomato (dicot) and rice (monocot)

Wild type Oser1

42 °C

h25

2014summer,Shanghai

2013summer,Shanghai

2013summer,Wuhan

2013summer,

Changsha

**

******

****

*

20

Gra

in y

ield

per

pla

ntin

�el

d (g

)

15

10

5

0

Vecto

r

Vecto

rL4

0L3

6L3

8

Vecto

rL4

0L3

6L3

8L3

8

Vecto

rL3

8

g

8,000

Rel

ativ

e ex

pres

sion

7,000

6,000

5,000

4,000

3,000

2,000

1,000

0

Vecto

rL4

0L3

6L3

8

a Vector

28 °

C42

°C

L40 L36 L38dVector

Vector

42 °C

L40 L36

35S::ER

35S::ER L36

L38b

c

100

See

d se

tting

(%

)

80

60

40

20

0

Vecto

rL4

0L3

6L3

8

Vecto

rL4

0L3

6L3

8

****

**

2013summer,Shanghai

2014 summer,Shanghai

2013summer,Wuhan

2013summer,

Changsha

*

* **

****

100

See

d se

tting

in �

eld

(%)

80

60

40

20

0

Vecto

r

Vecto

rL4

0L3

6L3

8L3

8

Vecto

rL3

8L3

8L3

6

Vecto

rL4

0

e

f

28 °C 42 °C

See

d se

tting

(%

)

100

**

80

60

40

20

0

Wild

type

Wild

type

Oser

1

Oser

1

i

42 °C

28 °C

Figure 4 ER enhances heat tolerance in rice. (a) qPCR analysis of the transgenic ER expression in 4-week-old transgenic rice grown at 28 °C. The rice ACTIN1 gene was used as control to normalize expression levels. Values are means ± s.d. (n = 3). (b) Pictures of 1-week-old seedlings of transgenic rice plants grown at 28 °C. (c) Pictures of mature transgenic rice plants after 10-d growth with a 42 °C (40–43 °C recorded due to daily fluctuation)/35°C (day/night) cycle and 1-week recovery growth at 28 °C. (d) Shown here are panicles and seeds collected from transgenic plants grown at constant 28 °C or subjected to the 10-d growth with a 42 °C/35 °C (day/night) cycle while flowering (c). Note that aberrant spikelets (white) were greatly decreased in the 35S::ER lines. (e) Quantification of seed setting after heat treatment (c) or grown under normal conditions (28 °C) in growth chambers as shown in box plots (n = 15). **P < 0.01, by Student’s t-test and Bonferroni correction for three comparisons’ test at 42 °C. (f) Quantification of seed-setting rates of transgenic rice plants in field tests at three different locations in the summer of 2013 (Shanghai, Wuhan and Changsha), and in the summer of 2014 at the Shanghai station, shown as box plots (n ≥ 30). (g) Analysis of grain yield of transgenic rice plants tested in the listed four field experiments, shown as box plots (n ≥ 30). Filled grains were weighed for each plant. **P < 0.01, by Student’s t-test and Bonferroni correction for three comparisons in the Shanghai (2013 and 2014) summer field test, or *P < 0.05, by Student’s t-test in the Wuhan and Changsha summer field tests (f,g). (h) Shown here are panicles of the flowering wild-type Dongjin and OsER1 mutant plants that were grown for 10 d at 42 °C/35 °C (day/night) in the growth chamber. Note that spikelets of the OsER1 mutant plants were almost dead. (i) Quantification of seed setting of the OsER1 mutant and wild-type plants after heat treatment (h) or grown under normal conditions (28 °C) in growth chambers, shown as box plots (n = 15). **P < 0.01, by Student’s t-test (**P < 0.01). (See source data.)

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature biotechnology VOLUME 33 NUMBER 9 SEPTEMBER 2015 1001

l e t t e r s

with the p35S::ER genomic transgene and analyzed the growth phenotypes and heat tolerance of the resulting transgenic crop lines in both laboratory and field conditions. We used the small red-fruit tomato species, Solanum pimpinellifolium (accession LA1589, the closest wild relative of the cultivated tomato), which is known to be hypersensitive to chilling, high temperature and high humid-ity, compared with modern tomato varieties. Similar to Arabidopsis, the ER-OE transgenic tomato lines were also bigger than the wild type, with larger leaves (nearly twice as large as wild type, likely resulting from enlarged cells) (Fig. 3a,b and Supplementary Fig. 10), decreased stomatal density, decreased stomatal conductance and increased transpiration efficiency (Supplementary Figs. 10 and 11). This result is consistent with an earlier finding of reduced vegeta-tive growth by dominant-negative disruption of the ER-mediated signaling pathway in transgenic tomatoes33. Importantly, the ER-OE transgenic tomato plants had significantly (P < 0.01) increased heat tolerance as measured by survival rates (Fig. 3c,d) when grown under heat stress at 40 °C/28 °C (day/night) for 10 d in the growth chamber, and also decreased cell death under heat treatment for 48 h (Supplementary Fig. 11).

Field experiments in three summer seasons (2012, 2013 and 2014) and at three planting locations (Shanghai, Wuhan and Hainan) of the transgenic lines L20, L22 and L23 confirmed that ER overexpression improved heat tolerance in tomato. We found that >50% of transgenic tomato plants survived the summer (July and August) heat waves in 2012 and 2013 at our experimental station in Shanghai (30–50 plants each line, three replicates), where the daily high temperature of the summer often surpassed 36 °C (Supplementary Data Set 1), whereas >70% of control transgenic lines carrying the empty vector died or almost died (Fig. 3e–g and Supplementary Fig. 12a). Similar results were observed in the 2014 summer at the Shanghai station (30–50 plants each line, three replicates) (Fig. 3f,g), whereas no significant difference in the survival rate between the two groups was observed in the early summer of 2014 when the growth temperature was normal (early June to early July) (Supplementary Fig. 12d). The increased thermotolerance of transgenic tomato was also observed for two sum-mer field tests in 2013 and 2014 in a mid-eastern China city Wuhan (one of the seven hottest cities, which includes Shanghai, widely known as the ‘seven furnaces’ in China) (Fig. 3f,g and Supplementary Fig. 12b), and an early-summer field test in 2013 on Hainan Island, the southernmost province in China (Supplementary Fig. 12c). The tomato species used in this study produced only a few small fruits that often shriveled under our growth conditions. We could obtain only enough fruits and seeds for our experiments by hand pollination and with extreme care. Thus, we were not able to accurately measure yields of tomato fruit of the wild-type control and transgenic lines. Under normal growth conditions in our greenhouse (22 °C), we observed no difference in the flower number or weight of fruits (produced by hand pollination) between the ER-OE lines and the wild-type plants (Supplementary Fig. 13).

As the parental strain LA1589 is not a widely cultivated crop, we searched a set of modern tomato germplasm and identified four tomato (Solanum lycopersicum) varieties, M82, Djena gold girl, Borgo Cellano and Heinz 1706 (the genome-sequenced cultivated tomato), that carry high-expression alleles (more than fourfold higher compared to LA1589) of SlER, the tomato gene orthologous to ER, and two others (LA1580 and LA1781) with expression levels of SlER similar to that of LA1589 (Fig. 3h). Heat treatment showed that the four varieties with higher SlER expression levels were more heat tolerant as measured by survival rates, whereas the two low-SlER-expression accessions were more susceptible to high temperatures

(Fig. 3i). It therefore seems likely that ER overexpression could greatly improve thermotolerance in commercially relevant tomato lines.

We also introduced the p35S:ER transgene into the model rice vari-ety Nipponbare (Oryza sativa L. japonica) and identified three lines that accumulated high levels of transgenic ER transcripts (Fig. 4a). These ER-OE transgenic rice seedlings developed longer roots and bigger leaves at the seedling stage compared to the control transgenic rice plants carrying the empty vector (Fig. 4b and Supplementary Fig. 14). The ER-OE transgenic rice plants also had increased heat tolerance. After 10 d of growth at 42°C/35°C (day/night) followed by 1-week recovery growth at 28 °C, many leaves and tillers of the ER-OE transgenic plants remained green and alive, whereas those of the vector control lines were dried and almost dead (Fig. 4c). Because pollination, and hence spikelet fertility and seed setting, is the most heat-sensitive trait of rice, we measured seed-setting rates after the prolonged heat treatment. ER-OE transgenic rice plants had significantly higher seed setting (55–70%) than the control transgenic line (~35%), after heat stress (Fig. 4d,e).

Field tests of transgenic rice plants in the Shanghai and Wuhan stations during the summer heat wave of 2013 showed that ER-OE transgenic rice had higher seed-setting rates and yield potential than the control transgenic plants (Fig. 4f,g and Supplementary Fig. 15). Similar results were also observed in the summer of 2014 at the Shanghai station (Fig. 4f,g). We also observed significantly higher (~49%; P < 0.05) seed setting of ER-OE transgenic rice than the control line (~45%) in a field test during the 2013 summer in Changsha (another ‘furnace’ city in China); however, the grain yield of the ER-OE line was not significantly higher than the vector control’s, probably because more seeds developed on the ER-OE plants in this location and thus the ER-OE line produced grains that were slightly smaller than those of the vector control (Fig. 4f,g and Supplementary Fig. 15). When grown in the autumn of 2013 (flowered in early October) with normal growth temperatures in the Shanghai station, the ER-OE transgenic lines had the same seed-setting rates as those of the control line (Supplementary Fig. 15). Therefore, ER could increase rice toler-ance to heat. Additional genetic support for a role of ER in heat toler-ance came from our studies of two rice T-DNA insertional mutants, one carrying a T-DNA null allele in Os06g0203800 (referred to as OsER1) (Supplementary Fig. 16a) and the other with a T-DNA insertion in Os06g0130100 (referred to as OsER2) (Supplementary Fig. 16b). Consistent with our overexpression data, the panicles of the OsER1 plants died quickly when grown at high temperatures in a growth chamber, leading to significantly (P < 0.01) decreased seed setting (Fig. 4h,i). By contrast, no difference in heat tolerance was detected between OsER2 and its wild-type control (Supplementary Fig. 16c), suggesting that OsER2 is a different ER gene that does not function in heat tolerance in rice. Taken together, our transgenic and genetic results clearly demonstrate that ER is also an important thermotolerance regulator in rice.

We have shown that ER is a major QTL for heat tolerance, thereby revealing a new function for this well-studied receptor-like kinase in the stress response25. It is intriguing that no difference was observed in water content between 40 °C-treated er-105 and its wild-type control, despite the fact that ER positively regulates transpiration efficiency, indicating that ER-mediated thermotolerance is likely unrelated to plant water status. Further support for a water loss–independent, ER-mediated thermotolerance mechanism came from our experiment showing that the relative heat tolerance of the ER-OE plants in com-parison with the wild-type Col-0 plants was higher when grown under high relative humidity, which should reduce plant transpiration. ER likely confers heat tolerance by protecting cells from heat-induced

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

http://www.ncbi.nlm.nih.gov/gds/?term=LA1589

1002 VOLUME 33 NUMBER 9 SEPTEMBER 2015 nature biotechnology

l e t t e r s

AUTHOR CONTRIBUTIONSH.S., X.Z., F.Z., Y.W., G.C., Y.H., H.X., J.L. and Z.H. conceived the research project, designed experiments and analyzed the data. H.S., X.Z., F.Z., Y.W., B.Y., Q.L., G.C., B.M., J.W., Y.L. and G.X. conducted the experiments. Z.H. and J.L. oversaw the entire study and wrote the manuscript.

COMPETING FINANCIAL INTERESTSThe authors declare no competing financial interests.

reprints and permissions information is available online at http://www.nature.com/reprints/index.html.

1. Peng, S. et al. Rice yields decline with higher night temperature from global warming. Proc. Natl. Acad. Sci. USA 101, 9971–9975 (2004).

2. Lobell, D.B. & Field, C.B. Global scale climate-crop yield relationships and the impacts of recent warming. Environ. Res. Lett. 2, 014002 (2007).

3. Long, S.P. & Ort, D.R. More than taking the heat: crops and global change. Curr. Opin. Plant Biol. 13, 241–248 (2010).

4. Penfield, S. Temperature perception and signal transduction in plants. New Phytol. 179, 615–628 (2008).

5. Fitter, A.H. & Fitter, R.S. Rapid changes in flowering time in British plants. Science 296, 1689–1691 (2002).

6. Samach, A. & Wigge, P.A. Ambient temperature perception in plants. Curr. Opin. Plant Biol. 8, 483–486 (2005).

7. Kumar, S.V. & Wigge, P.A. H2A.Z-containing nucleosomes mediate the thermosensory response in Arabidopsis. Cell 140, 136–147 (2010).

8. Posé, D.S.H. et al. Temperature-dependent regulation of flowering by antagonistic FLM variants. Nature 503, 414–417 (2013).

9. Queitsch, C., Hong, S.W., Vierling, E. & Lindquist, S. Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12, 479–492 (2000).

10. Finka, A., Cuendet, A.F.H., Maathuis, F.J.M. & Saidi, Y. Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance. Plant Cell 24, 3333–3348 (2012).

11. Iba, K. Acclimative response to temperature stress in higher plants: approaches of gene engineering for temperature tolerance. Annu. Rev. Plant Biol. 53, 225–245 (2002).

12. Kim, M., Lee, U., Small, I., des Francs-Small, C.C. & Vierling, E. Mutations in an Arabidopsis mitochondrial transcription termination factor-related protein enhance thermotolerance in the absence of the major molecular chaperone HSP101. Plant Cell 24, 3349–3365 (2012).

13. Guan, Q., Yue, X., Zeng, H. & Zhu, J. The protein phosphatase RCF2 and its interacting partner NAC019 are critical for heat stress–responsive gene regulation and thermotolerance in Arabidopsis. Plant Cell 26, 438–453 (2014).

14. Burke, J.J. & Chen, J. Enhancement of reproductive heat tolerance in plants. PLoS ONE 10, e0122933 (2015).

15. Li, X. et al. Natural alleles of a proteasome α2 subunit gene contribute to thermotolerance and adaptation of African rice. Nat. Genet. 47, 827–833 (2015).

16. Grover, A., Mittal, D., Negi, M. & Lavania, D. Generating high temperature tolerant transgenic plants: Achievements and challenges. Plant Sci. 205-206, 38–47 (2013).

17. Hancock, A.M. et al. Adaptation to climate across Arabidopsis thaliana genome. Science 334, 83–86 (2011).

18. Fournier-Level, A. et al. A map of local adaptation in Arabidopsis thaliana. Science 334, 86–89 (2011).

19. Yan, C., Shen, H., Li, Q. & He, Z. A novel ABA-hypersensitive mutant in Arabidopsis defines a genetic locus that confers tolerance to xerothermic stress. Planta 224, 889–899 (2006).

20. Lin, L., Zhong, S.H., Cui, X.F., Li, J. & He, Z.H. Characterization of temperature- sensitive mutants reveals a role for receptor-like kinase CRAMBLED/STRUBBELIG in coordinating cell proliferation and differentiation during Arabidopsis leaf development. Plant J. 72, 707–720 (2012).

21. Zhong, S. et al. Warm temperatures induce transgenerational epigenetic release of RNA silencing by inhibiting siRNA biogenesis in Arabidopsis. Proc. Natl. Acad. Sci. USA 110, 9171–9176 (2013).

22. Torii, K.U. et al. The Arabidopsis ERECTA gene encodes a putative receptorprotein kinase with extracellular leucine-rich repeats. Plant Cell 8, 735–746 (1996).

23. Shpak, E.D., Berthiaume, C.T., Hill, E.J. & Torii, K.U. Synergistic interaction of three ERECTA-family receptor-like kinases controls Arabidopsis organ growth and flower development by promoting cell proliferation. Development 131, 1491–1501 (2004).

24. Shpak, E.D., McAbee, J.M., Pillitteri, L.J. & Torii, K.U. Stomatal patterning and differentiation by synergistic interactions of receptor kinases. Science 309, 290–293 (2005).

25. van Zanten, M., Snoek, L.B., Proveniers, M.C. & Peeters, A.J. The many functions of ERECTA. Trends Plant Sci. 14, 214–218 (2009).

26. Lee, J.S. et al. Direct interaction of ligand-receptor pairs specifying stomatal patterning. Genes Dev. 26, 126–136 (2012).

27. Uchida, N. et al. Regulation of inflorescence architecture by intertissue layer ligand-receptor communication between endodermis and phloem. Proc. Natl. Acad. Sci. USA 109, 6337–6342 (2012).

cellular damage and cell death. This hypothesis is consistent with the reported effects of ER on chloroplast electron transport30. Further detailed research should shed light on a better mechanistic understanding of the ER-mediated, water loss–independent, ther-motolerance process.

Given that crop production is often hard hit during hot summers1–3, engineering heat tolerance in plants could prove important. However, until now engineering approaches have not been particularly successful, mainly because either increased stress tolerance has been accompa-nied by a growth and/or yield penalty, or because thermotolerance of engineered crops has not translated from the greenhouse to the field at multiple locations.

This study has shown, to our knowledge for the first time, that overexpressing ER significantly increased the tolerance of plants to heat and prolonged hot temperatures, not only in the model plant Arabidopsis but also in two important food crop species (or crop relative) with no growth and/or yield penalty. Consistent with our transgenic studies, low expression SlER alleles in tomato and a loss-of- function rice ER gene were associated with reduced thermotoler-ance. Importantly, many of the ER-OE transgenic plants also had increased biomass and enhanced water use efficiency, which further substantiates an earlier claim that ER is a major locus for transpiration efficiency30. This is especially important because global warming is causing a worldwide water deficiency.

Our findings indicate that overexpression of ER is likely superior to using HSPs to improve thermotolerance9,14. Plants engineered to produce HSP101 have not been tested in multiple locations or through several growing seasons and HSP overexpression in model plants has been associated with growth penalties under nonstress conditions34. Finally, HSP genes are often functionally redundant, and no single HSP locus has been reported as a major QTL in the gene-to-field test for crop thermotolerance improvement34.

Our study indicates that a single ER gene-mediated heat tolerance pathway is likely to be conserved in higher plants, which is consistent with a recent finding that a poplar ER homolog could increase biomass and water use efficiency in transgenic Arabidopsis35. ER-like genes are widely distributed in plants36, which is a good basis for improving crop thermotolerance through identification of ER alleles with higher expression levels or higher activity from available crop germplasm collections. Future work will include introgression of identified elite ER alleles into elite crop varieties through marker-assisted selection to improve thermotolerance of major crops.

METHOdSMethods and any associated references are available in the online version of the paper.

Note: Any Supplementary Information and Source Data files are available in the online version of the paper.

ACkNOWLEdGMENTSWe thank J.-S. Jeon and C.Y. Wu for the rice T-DNA mutants, X.L. Wang for the er-105 line, X.G. Zhu, Y.J. Zhang and M.Z. Lv for help in statistical analysis, D.Y. Sun and H.X. Lin for helpful discussions, and L. Lin for help in experiments. This work was supported by the National Key Basic Research and Development Program (2011CB100700 to H.Z.), the National Natural Science Foundation of China (3130061 to H.Z.), the Ministry of Science and Technology of China (2012AA10A302 to Z.H.), the National GMO project (2013ZX08009-003-001 to Z.H.), the National High Technology Research and Development Program of China (2012AA100104-6 to H.X.), the Chinese Academy of Sciences (KSCX2-EW-N-01 to H.Z. and 2009OHTP07 to H.X.), the Shanghai Committee of Science and Technology (11PJ1410900 to H.X.), and the National Key Basic Research and Development Program (2015CB150104 to G.C.).

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

http://www.nature.com/reprints/index.htmlhttp://www.nature.com/reprints/index.htmlhttp://www.nature.com/doifinder/10.1038/nbt.3321http://www.nature.com/doifinder/10.1038/nbt.3321http://www.nature.com/doifinder/10.1038/nbt.3321

nature biotechnology VOLUME 33 NUMBER 9 SEPTEMBER 2015 1003

l e t t e r s

28. Tisné, S. et al. Combined genetic and modeling approaches reveal that epidermal cell area and number in leaves are controlled by leaf and plant developmental processes in Arabidopsis. Plant Physiol. 148, 1117–1127 (2008).

29. Qi, Y., Sun, Y., Xu, L., Xu, Y. & Huang, H. ERECTA is required for protection against heat-stress in the AS1/AS2 pathway to regulate adaxial-abaxial leaf polarity in Arabidopsis. Planta 219, 270–276 (2004).

30. Masle, J., Gilmore, S.R. & Farquha, G.D. The ERECTA gene regulates plant transpiration efficiency in Arabidopsis. Nature 436, 866–870 (2005).

31. Li, S. et al. HEAT-INDUCED TAS1 TARGET1 mediates thermotolerance via HEAT STRESS TRANSCRIPTION FACTOR A1a–directed pathways in Arabidopsis. Plant Cell 26, 1764–1780 (2014).

32. Ishikawa, T., Uchimiya, H. & Kawai-Yamada, M. The role of plant Bax inhibitor-1 in suppressing H2O2-induced cell death. Methods Enzymol. 527, 239–256 (2013).

33. Villagarcia, H., Morin, A.C., Shpaket, E.D. & Khodakovskaya, M.V. Modification of tomato growth by expression of truncated ERECTA protein from Arabidopsis thaliana. J. Exp. Bot. 63, 6493–6504 (2012).

34. Mickelbart, M.V., Hasegawa, P.M. & Bailey-Serres, J. Genetic mechanisms of abiotic stress tolerance that translate to crop yield stability. Nat. Rev. Genet. 16, 237–251 (2015).

35. Xing, H.T., Guo, P., Xia, X.L. & Yin, W.L. PdERECTA, a leucine-rich repeat receptor-like kinase of poplar, confers enhanced water use efficiency in Arabidopsis. Planta 234, 229–241 (2011).

36. Shiu, S.H. et al. Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 16, 1220–1234 (2004).

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

nature biotechnology doi:10.1038/nbt.3321

ONLINE METHOdSPlant materials and treatment. Seeds of the Arabidopsis ecotypes Col-0, Ler and Lan were stratified at 4 °C for 3 d and germinated at 22 °C for 1 week on 1/2× MS medium. Seedlings were transplanted into soil and grown at 22 °C in a growth room with a 16 h/8 h (day/night) photoperiod and ~ 65% relative humidity (RH). For the heat treatment experiment, 2-week-old plants grown at 22 °C were transferred into a growth chamber with 80% RH and temperatures gradually increasing from 22–30 °C for 12 h and then 36 °C for 12 h, followed by 40 °C for 48 h, or simply grown at 30 °C as previously reported18,19, for survival rate analysis. Two-week-old plants were also grown at 40 °C for 48 h under low RH (30–40%) or high RH (90–95%, near saturated with water vapor) to determine the effect of transpiration on heat tolerance. Plants were watered every day (40 °C) or every 2 d (30 °C) to keep the soil wet during heat treat-ment. Each experiment was conducted with 30 plants each line, three replicates (30 × 3), total 90 plants each line. Plants grown at 22 °C were used as heat treatment controls. For drought treatment, 4-week-old plants were subjected to gradual drought stress until er-105 or Ler was dry dead, and the plants were then rewatered with the same amount of water to permit recovery; 30 plants each line, three replicates (30 × 3), total 90 plants each line were analyzed to determine the survival rate.

For rice heat treatments in growth chambers, heading plants were grown in the growth chamber with a 16 h/8 h photoperiod, a 42 °C (40–43 °C recorded due to daily fluctuation)/35 °C (day/night) temperature cycle and 80–90% RH for 10 d, with the control experiments performed at 28 °C. The plants were then allowed to recover at 28 °C in the greenhouse for one week. Spikelet fertility, known to be sensitive to heat stress37, was statistically analyzed with 30 panicles from 15 individual plants for each line with three replicates. For tomato heat treatments in growth chamber, 6-week-old plants or cutting-propagated plants were grown in the growth chamber with a 16 h /8 h photoperiod, a 40 °C (38–42 °C recorded because of daily temperature fluctuation)/28 °C (day/night) tem-perature cycle and 80–90% RH for 10 d followed by 2-d recovery growth at 22 °C, with the control experiments performed at 22 °C. For testing the heat tolerance of tomato SlER alleles, 3-week-old seedlings were grown at 40 °C for 3 d followed by 2-d recovery growth at 22 °C to determine survival rates. About 30 plants each line, three replicates (30 × 3), total 90 plants each line were treated, and survival rate was measured for tomato heat tolerance. Rice and tomato plants were watered daily to keep the soil wet during heat treatment. Flower numbers per inflorescence were recorded from 10 plants (3 inflorescences per plant), and fruit weights were measured for 20 fruits produced by hand pollination, with well maintenance in the plants grown in the greenhouse (22 °C).

Field experiments. Field tests in multiple growing seasons and at different locations for rice and tomato were carried out during 2012–2014. Plants were maintained under the same field conditions and were well irrigated and pro-tected from pests to ensure meaningful field tests. For the rice field tests, rice plants were grown in the summer to flower at mid-August to early September (2013) at three well-controlled biological stations located in Shanghai (eastern China), Wuhan (mid-eastern China) and Changsha (southeastern China), widely known as furnace cities in China because of their hot summer weather, where the recorded daily temperatures during the hot summer often reach 36–40 °C. Rice plants were also tested in the summer of 2014 in the Shanghai station. Daily records of high, low and average temperatures and relative humidity (RH) during the experimental seasons of 2012–2014 were obtained from the local weather stations (Supplementary Data Set 1). Rice was also grown to flower in the autumn (early October) of 2013 at our Shanghai experimental station to test performance under normal growth temperatures. 50–60 plants each line, three replicates (50–60 × 3) were grown. qPCR was used to check ER expression in all transgenic plants grown. About 30–50 plants each line, three replicates (30–50 × 3) were analyzed for agronomic traits. Due to government regulations we could test line 38 and the control vector line only in net-protected fields in Wuhan and Changsha (2013). Seed-setting rate and other important agronomic traits including grain (full-filled) yield per plant were quantified during these field trials (see source data associated with Fig. 4, and Supplementary Figs. 14 and 15).

Tomato plants were transplanted at our experimental stations during the summers (July–August) of 2012–2014 in Shanghai and 2013, 2014 in Wuhan

and early summer (late April–early June) on Hainan Island for field tests of heat tolerance. Tomato plants were also grown in the early summer of 2014 (early June–early July) at the Shanghai station under normal temperature conditions. In 2013 and 2014 in the Shanghai station, 30–50 6-week-old plants of each line with three biological repeats (total 30–50 × 3 plants each line) were grown for about 1 month, by which time the control plants were almost dead. Survival rates (%) were measured (alive plants/total plants as for Arabidopsis) Because of the strict governmental administration on plant numbers of transgenic plant test, the additional field tests conducted in 2012 (Shanghai) and 2013 (Hainan, Wuhan) used ~50 plants/line for direct survival rate measurement. Because there were no replicates, we did not perform statistical significance analysis in these additional field tests.

QTL mapping and plant transformation. A set of 30 recombinant inbred lines (RILs) derived from a cross of Col and Ler38 obtained from the Nottingham Arabidopsis Stock Centre (http://Arabidopsis.info/) was screened for heat tol-erance. Thirty plants per line with three biological replicates (for each line, there were three sets of 30 plants) were tested, divided by phenotype into three groups, and genotyped with PCR-based indel makers. QTLs were detected using Windows QTL Cartographer version 2.5 and a LOD threshold of 2.5 (α = 0.05). Based on RIL screening, CSSLs from the BC3F2 populations of Col-0 × Ler with Ler as the recurrent parent were developed and screened for qHat loci, and subsequent fine mapping of qHat2-1 was performed using the selected CSSL and its progeny.

Generation of transgene constructs and plant transformation. A 7,832-bp genomic fragment including the entire wild-type ER coding region and its native promoter (1,802-bp) and 3′-noncoding region (501 bp) was isolated from BAC T1D16 by digestion using EcoRI and SnaBI and inserted into the EcoRI and SmaI sites of the cloning vector pBluescript II SK(−). The fragment was then released from SK using KpnI and BamHI digestion and inserted into the binary vector pCAMBIA1301 to generate the pER::ER genomic transgene. This plasmid was used to transform Ler or er-105 to generate >10 independent complementation lines using Agrobacterium tumefaciens-mediated transfor-mation with floral dipping39. To generate the p35S::ER construct, a 412-bp 5′ ER fragment with a 42-bp 5′ nontranslation region was PCR-amplified from BAC T1D16 using the primers 35S-ER-5′-F and 35S-ER-5″-R (Supplementary Table 2), digested with ClaI/SphI to release a 140-bp fragment with the 42-bp 5′ nontranslation region, which was cloned into the ClaI/SphI-digested pER::ER plasmid. The resulting plasmid was sequenced to ensure there was no PCR-introduced error and digested with ClaI and BamHI to release the promoter-less ER genomic fragment, which was subsequently cloned into a overexpression vector pCAMBIA1301-35SN (35S- C1301) that contains the 35S promoter of the cauliflower mosaic virus with two copies of an enhancer element to drive the expression of the transgenes40, to generate the p35S::ER overexpression transgene. The plasmid p35S::ER was introduced into Col-0, and the resulting transgenic T1 lines were screened by qPCR for ER- overexpressing lines. Homozygous ER-overexpressing T3-T4 lines were used for phenotypic analyses. All transgene constructs were fully sequenced to ensure no PCR/cloning-introduced error.

The p35S::ER construct was used to transform tomato germplasm (Solanum pimpinellifolium LA1589)41 that was previously shown to be sensitive to temperature stress42, to generate >15 independent transgenic lines (T0) by Agrobacterium tumefaciens-mediated cotyledon transformation. The resulting transgenic lines were screened by qPCR to identify ER-overexpressing lines whose subsequent homozygous progeny (T1–T5) were used for heat tolerance studies and microscopic analyses. The p35S::ER transgene was also introduced into the rice variety Nipponbare to generate >20 lines (T0) by Agrobacterium tumefaciens-mediated transformation, and the resulting transgenic lines were screened by qPCR to find ER-overexpression lines. The T1–T5 homozygous progeny plants of the selected ER-overexpressing lines were assayed for heat tolerance and agronomic traits.

To generate an epitope-tagged ER transgene, the p35S::ER was fused in-frame with a 7× myc–6× His (MH) fragment between the last codon and the stop codon to create the p35S::ER-MH plasmid by PCR fragment fusion using the primers (Supplementary Table 2), which was introduced into er-105 to generate >30 lines. The resulting transgenic lines were screened

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

http://Arabidopsis.info/http://www.ncbi.nlm.nih.gov/gds/?term=LA1589

nature biotechnologydoi:10.1038/nbt.3321

morphologically to identify lines with increased biomass and qPCR for ER-MH overexpression lines, and by immunoblotting to identify lines with higher ER-MH levels. Thermotolerance of the homozygous progeny (T2 and T3) of the chosen lines with increased biomass were compared with wild-type Col-0 as described above.

Identification of rice T-DNA mutants and tomato SlER alleles. A homozygous T-DNA knockout mutant of the rice ER homolog gene (Os06g0203800, OsER1) in Dongjin (japonica) background was obtained from the Korean rice T-DNA mutant center (the National Crop Experiment Station, RDA, Suwon, Korea) whereas a homozygous T-DNA insertional mutant in the second rice ER homolog (Os06g013010, OsER2) in Zhonghua 11 background (japonica) was generously provided by the Chinese rice T-DNA mutant center (the Huazhong rice mutant center, http://rmd.ncpgr.cn/). The molecular effect of the T-DNA insertions on the two ER homologs was analyzed by qPCR using the OsER1-RT8F/R and OsER2-RT3F/R primer sets (Supplementary Table 2). The confirmed homozygous OsER1 and OsER2 mutants and the corresponding wild-type rice plants (~15) were heat-treated in the growth chamber, and seed-setting rates were measured and statistically analyzed with three biological replicates, as described above.

A set of tomato germplasm including >100 modern varieties/inbreed lines were screened for transcript abundance of the tomato ER orthologous gene (SlER, accession no. LOC101266437) by qPCR with the SlER-RT3F/R primer set (Supplementary Table 2). The qPCR-based screen identified four varieties (M82, Djena gold girl, Borgo Cellano and the sequenced reference variety Heinz 1706, ref. 41) with increased transcription levels of SlER in comparison with LA1589 used for generating transgenic tomato plants, and two accessions (LA1580 and LA1781) accumulating similar levels of SlER as LA1589. Seedlings of the six varieties together with LA1589 were heat-treated in the growth chamber, and survival rates (used for measurement of heat tolerance) were calculated with 30 plants and three biological replicates, as described above.

Microscopy and histology. Scanning electron microscopy (SEM) and the preparation of resin-embedded thin sections were performed, as previously described20. In brief, leaf tissues of 2-week-old Arabidopsis and 6-week-old tomato plants with or without heat treatment were fixed in FAA (50% ethanol/acetic acid/formaldehyde = 9:0.5:0.5). The samples were dehydrated in an ethanol series and then dried by supercritical fluid drying with CO2. The dried samples were mounted on copper supports and sputter-coated with gold, and then observed under SEM (JEOL, JSM-6360LV). The stomatal density was determined using SEM images of the abaxial surfaces of expended rosette leaves, with at least five leaves per sample with three biological replicates, and the stomatal index was calculated from the total cells in the images of the leaf abaxial surfaces. Cell size (cell length and width) was statistically analyzed by measuring 30 cells of the same-age leaves from each line with three biological replicates.

For observation by TEM, new mature leaves were cut into pieces, fixed in 2.5% glutaraldehyde and then fixed in 1% osmic acid overnight at 4 °C. After rinsing with phosphate buffer (pH 7.2) three times, the samples were dehy-drated in an ethanol series, treated with propylene oxide, and then infiltrated and embedded with Epon812 resin. The resin sections were stained with ura-nyl acetate and lead citrate and then observed under a transmission electron microscope (CM120, Phillips). The disruption of cells including blebbing and broken plasma membrane, plasmolysis, and the collapse of chloroplasts and mitochondria were recorded and statistically analyzed by observing at least 50 cells with three biological section replicates.

Cell death was observed using trypan blue staining by boiling the leaves for 1 min in a lactophenol solution (lactic acid:phenol:glycerol = 1:1:1) containing 100 µg/ml trypan blue, followed by tissue clearing in chlo-ral hydrate solution (2.5 g chloral hydrate dissolved in 1 ml H2O) over-night at room temperature and mounting in 70% glycerol for microscopic inspection43. The accumulation of reactive oxygen species (H2O2) was visualized by 3,3′-diaminobenzidine (DAB) staining as previously described38. Leaves from plants grown at 22 °C immediately before moving to heat treatment were used as the heat treatment controls (0 h) in these microscopic experiments.

Physiological assay. Gas exchange parameters including the net photosyn-thetic rate, the transpiration rate and the stomatal conductance of Arabidopsis (4-week-old) and tomato (6-week-old) plants grown at 22 °C were measured in situ using a LI-6400 gas exchange system (LI-COR Biosciences). Newly fully expanded leaves at the same development stage of each genotype (eight to ten plants per genotype, three biological replicates) were measured at the conditions of 300 µmol photon/m/s and 400 µmol/mol ambient CO2 concen-tration. The temperature of the leaf chamber was maintained at 22 °C. The leaf to air vapor difference was kept at about 1.0 KPa during measurement. The instantaneous water-use efficiency was calculated as the ratio of net photosyn-thetic rate to transpiration rate. Relative water content (RWC) was measured as previously described44. In brief, 2-week-old Arabidopsis plants grown at 22 °C were heat-treated (40 °C) in the growth chamber. Twelve mature rosette leaves from 12 individuals (1 leaf /plant) were sampled at 0, 12, 24, 36 and 48 h with three biological replicates. Fresh weight (FW) was measured immediately after sampling. Leaves were immersed in deionized water for 4 h, and then blotted dry and weighted (turgid weight, TW). Leaves were then oven-dried at 80 °C for 48 h and weighed (dry weight, DW). RWC was calculated with the follow-ing formula: RWC = (FW – DW) /(TW – DW) × 100%.

WUE was calculated by using a gravimetric method45. Four Arabidopsis seedlings of each line were grown in one container supplemented with 350 ± 5 g soil (wet weight) with a total of 25 containers and three biological replicates. After 1 week, two of the four plants were oven-dried at 65 °C for 1 week and weighed to obtain the initial dry weight (IDW). Then, the containers with the other two plants were grown at 22 °C or 30 °C and covered with plastic wrap to prevent evaporation from the soil surface. Each container was weighed before and after watering every 6 d to determine moisture loss, and the difference in weight was corrected by adding water to the same weight. The total water loss (TWL) was summed over a period of 3 weeks. In addition, the control water loss (CWL) of the same container without plants was measured to calculate the water loss by evaporation. At the end of the experiment, plants were oven-dried and weighed to obtain the terminal dry weight (TDW). The con-tainers with dead plants during 30 °C growth were removed from calculation. The water use efficiency was calculated as increased dry weight divided by cumulative water loss with the following formula: water use efficiency = (TDW – IDW)/(TWL – CWL).

Ion leakage was measured as previously described46. In brief, 15 leaves with three biological replicates of 2-week-old Arabidopsis plants grown at 22 °C with heat treatment (40 °C ) for 0, 12, 24, 36 and 48 h from each line were cut into two pieces from the midrib, and the leaf tissues were incubated in ten tubes (three leaf pieces/tube) containing 3 ml deionized water overnight with shaking at 28 °C. The initial ion leakage (ILi) was determined using a conductivity meter (FE30, Mettler Toledo). The samples were boiled for 10 min under high pressure (0.21 Mpa) and then cooled to room temperature to measure the total ion leakage (ILt). The relative ion leakage was expressed as a percentage of the total electrolytes.

Arabidopsis plants were grown at 22 °C to mature, leaf and branch number, seed weight per plant were analyzed with 30 individual plants per line. Thirty plants of 4-week-old plants grown at 22 °C were measured for aboveground biomass (shoot dry weight). Leaf sizes (in cm2) were determined for ten rosette leaves of 4-week-old Arabidopsis plants grown at 22 °C or for three same-aged full-expended leaves of 6-week-old tomato plants grown at 22 °C with ten individual plants/line.

Analysis of gene expression and protein accumulation. Total RNAs were isolated from leaves or different tissues of Arabidopsis, rice and tomato plants using TRIzol reagent according to the manufacturer’s instructions (Invitrogen). For ER, ERL1 and ERL2 developmental expression, rosette leaves of 2-week-old plants, and rosette and cauline leaves and inflorescence organs of 6-week-old plants (flowering stage) grown at 22 °C were collected for RNA preparation. For tomato and rice ER gene expression, RNA was prepared from leaves of 4-week-old rice and tomato seedlings grown at 28 °C in greenhouses. Total RNAs were also prepared from rosette levels of 2-week-old Col-0 plants treated by heat (40 °C) for a time course of 24 h for analysis of ER expression in response to heat stress. For qPCR analysis of gene expression, 2 µg of total RNAs were converted into cDNA using the SuperScript III First-Strand cDNA Synthesis kit (Invitrogen), and 2 µl of the resulting cDNA was used as

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

http://rmd.ncpgr.cn/

nature biotechnology doi:10.1038/nbt.3321

a template for qPCR analysis using the primer sets listed in Supplementary Table 2. The qPCR reactions were performed in triplicate using a Rotor- Gene 2000 fluorometric thermal cycler (Corbett Research) with SYBR Green real-time PCR master mix (Toyobo). For investigating the molecular mechanism of the ERECTA-mediated thermotolerance, the marker genes, HSFA1a and BI1, functioning in heat tolerance and cell death, respectively30,31, were analyzed for their expression in 2-week-old Arabidopsis plants during heat treatment (40 °C). The relative expression levels were calculated from three replicate qPCR experiments and normalized using the signal derived from ACTIN2 (At03g8780) in Arabidopsis, SleIF4a6 in tomato47 and ACTIN1 in rice.

For immunoblotting detection of the ER-MH fusion protein, total proteins extracted from entire rosettes of 2-week-old homozygous T2/T3 plants were separated by 7.5% SDS-PAGE (for ER-MH) or 12% SDS-PAGE (for Actin) and analyzed by immunoblotting using anti-myc antibody (Millipore, #05-724) or anti-Actin antibody (ABclonal Biotechnology, #AC009).

Statistical analysis. Statistical analysis was performed by Student’s t-test fol-lowed by the most conserved Bonferroni correction that adjusts alpha level based on comparisons for testing multiple hypotheses. We also used Welch’s ANOVA followed by Bonferroni correction for the SlER gene expression and survival rate analysis of the seven tomato accessions (varieties) and the Arabidopsis CSSL lines.

37. Jagadish, S.V. et al. Physiological and proteomic approaches to address heat tolerance during anthesis in rice (Oryza sativa L.). J. Exp. Bot. 61, 143–156 (2010).

38. Lister, C. & Dean, C. Recombinant inbred lines for mapping RFLP and phenotypic markers in Arabidopsis thaliana. Plant J. 4, 745–750 (1993).

39. Clough, S.J. & Bent, A.F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

40. Wang, E.T. et al. Control of rice grain-filling and yield by a gene with a potential signature of domestication. Nat. Genet. 40, 1370–1374 (2008).

41. The Tomato Genome Consortium. The tomato genome sequence provides insights into fleshy fruit evolution. Nature 485, 635–641 (2012).

42. Pennycooke, J.C. et al. The low temperature-responsive, Solanum CBF1 genes maintain high identity in their upstream regions in a genomic environment undergoing gene duplications, deletions, and rearrangements. Plant Mol. Biol. 67, 483–497 (2008).

43. Zhang, H., Zhang, X., Mao, B., Li, Q. & He, Z. Alpha-picolinic acid, a fungal toxin and mammal apoptosis-inducing agent, elicits hypersensitive-like response and enhances disease resistance in rice. Cell Res. 14, 27–33 (2004).

44. Parida, A.K., Dasgaonkar, V.S., Phalak, M.S., Umalkar, G.V. & Aurangabadkar, L.P. Alterations in photosynthetic pigments, protein, and osmotic components in cotton genotypes subjected to short-term drought stress followed by recovery. Plant Biotechnol. Rep. 1, 37–48 (2007).

45. Karaba, A. et al. Improvement of water use efficiency in rice by expression of HARDY, an Arabidopsis drought and salt tolerance gene. Proc. Natl. Acad. Sci. USA 104, 15270–15275 (2007).

46. Charng, Y.-Y. et al. A heat-inducible transcription factor, HsfA2, is required for extension of acquired thermotolerance in Arabidopsis. Plant Physiol. 143, 251–262 (2007).

47. Weng, L. et al. The zinc finger transcription factor SlZFP2negatively regulates abscisic acid biosynthesis and fruit ripening in tomato. Plant Physiol. 167, 931–949 (2015).

npg

© 2

015

Nat

ure

Am

eric

a, In

c. A

ll rig

hts

rese

rved

.

Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomatoMethodsONLINE METHODSPlant materials and treatment.Field experiments.QTL mapping and plant transformation.Generation of transgene constructs and plant transformation.Identification of rice T-DNA mutants and tomato SlER alleles.Microscopy and histology.Physiological assay.Analysis of gene expression and protein accumulation.Statistical analysis.

AcknowledgmentsAUTHOR CONTRIBUTIONSCOMPETING FINANCIAL INTERESTSReferencesFigure 1 ER plays an important role in heat tolerance in Arabidopsis.Figure 2 Analysis of leaf water content and heat-induced cell damages.Figure 3 ER confers heat tolerance in tomato.Figure 4 ER enhances heat tolerance in rice.

Button 1: Page 1: Off