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Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomato

Nature Biotechnology volume 33, pages 996–1003 (2015)Cite this article

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Abstract

The detrimental effects of global warming on crop productivity threaten to reduce the world's food supply1,2,3. 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.

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Figure 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.

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Acknowledgements

We 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.).

Author information

Author notes

  1. Hui Shen and Xiangbin Zhong: These authors contributed equally to this work.

Authors and Affiliations

  1. National 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

    Hui Shen, Xiangbin Zhong, Fangfang Zhao, Yanmei Wang, Bingxiao Yan, Qun Li, Genyun Chen, Yuke He, Han Xiao & Zuhua He

  2. Shanghai Chenshan Plant Science Research Center, Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, Shanghai, China

    Hui Shen

  3. College of Agriculture and Biotechnology, Zhejiang University, Hangzhou, China

    Bizeng Mao

  4. Zhejiang Academy of Agricultural Sciences, Hangzhou, China

    Jianjun Wang

  5. College of Life Science, Wuhan University, Hubei, China

    Yangsheng Li

  6. Institute of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, China

    Guoying Xiao

  7. Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Jianming Li

  8. Collaborative Innovation Center of Genetics and Development, Institute of Plant Physiology & Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Zuhua He

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  1. Hui Shen
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Contributions

H.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.

Corresponding author

Correspondence to Zuhua He.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 QTL mapping for heat tolerance

In the preliminary QTL mapping, the RILs derived from the cross of Col-0 and Ler were classified into 3 groups according to their heat tolerance phenotypes after 40°C treatment for 2 d: lines with high tolerance similar to Col-0 (survival rate 30.8-42.4%), lines with high sensitivity similar to Ler (survival rate 7.5-12.4%), and lines with intermediate properties (survival rate 14.7-28.4%). Values are means ± SD (n = 3). (b) QTL distribution in 5 chromosomes. Note that chromosome 2 has a major contribution peak. The horizontal lines plotted for chromosomes 2 and 4 indicated the QTL LOD threshold. (c) Construction of CSSLs that cover chromosome 2. (d) Fine mapping of qHat2-1, which is located at CSSL2-3 that contains the mutated ERECTA (er) gene in BAC T1D16 in the Ler mutant (red arrow). Survival rates were measured for each CSSL2 lines, shown as dots of 3 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 multiple (6) tests (h,i).

Supplementary Figure 2 Genetic complementation of the ER gene

(a) Heat tolerance of 2-week-old wild-type Col-0, er-105, and complementation pER::ER/er-105 plants treated at 40°C for 48 h. (b) Survival rates of the Col-0, er-105, and complementation plants as shown in dots of 3 replicates (30 plants each). (c) Heat tolerance of two-week-old wild-type Lan, Ler, and complementation pER::ER/Ler plants treated with 40°C for 48 h. (d) Survival rates of Lan, Ler mutants, and complementation plants as shown in dots of 3 replicates (30 plants each). Asterisks indicate a significant difference in comparison with wild-type Col-0 at 40°C, at P < 0.01 (**), by Student’s t-test and Bonferroni correction for multiple (2) tests in the independent temperature experiments (b,d). Bar = 1 cm (a,c).

Supplementary Figure 3 Expression levels of ER and ER homologous genes and leaf sizes in 35S::ER-overexpressing plants

(a) Expression levels of ER in rosette leaves of 2-week-old grown at 22°C wild-type Col-0 and ER-OE lines (T3) and in two continuous homozygous generations (T3 and T4, line L7-1). Values are means ± SD (n = 3). (b) Expression levels of ER in rosette, cauline leaves and inflorescence organs of 6-week-old grown at 22°C wild-type Col-0 and the ER-OE line (L7-1, T3) detected by qPCR. ACTIN2 was used as control to normalise expression levels. Note that ER was stably overexpressed in different tissues of the transgenic plants. Number (2.79) above L7-1 (flower) indicates fold relative to expression level (1) in the control Col-0. Values are means ± SD (n = 3). (c) Ten rosette leaf sizes of 4-week-old ER-OE, Col-0 plants and er-105 plants grown at 22°C. Values are means ± SD (n = 10). (d) Rosette leaf numbers of ER-OE, Col-0 and er-105 plants at rosette (4-week-old) and flowering (5-week-old) stages, grown at 22°C. Values are means ± SD (n = 30). (e) The expression of the homologous ERL1 and ERL2 in rosette, cauline leaves and inflorescence organs of 2- and 6-week-old wild-type Col-0 and ER-OE line (line 7-1, T3) grown at 22°C was not affected by ER overexpression as detected by qPCR. ACTIN2 was used as control to normalise expression levels. Values are means ± SD (n = 3). Asterisks indicate statistically significant differences as determined by Student’s t-test (*, P< 0.05; **, P < 0.01) (c,d).

Supplementary Figure 4 Development of 35S::ER-MH/er105 plants

(a) Transcription levels of ER-MH detected by qPCR, ACTIN2 was used as control to normalise expression levels. Values are means ± SD (n = 3). Number (2.64) above L13 indicates fold relative to expression level (1) in the control Col-0. (b) Plants and leaves of 2-week-old 35S::ER-MH/er105 (LM10 and LM17) lines grown at 22°C in comparison with wild-type Col-0 and er-105, which accumulated high levels of the ER-MH fusion protein and showed increased thermotolerance (Fig. 1d,j). Bar = 1 cm.

Supplementary Figure 5 Cell morphology of 2-week-old Col-0, er-105 and ER-OE plants grown at 22°C

(a) Leaf cells of Col-0 and ER-OE plants (L7-1). Note that the mesophyll cells are larger in L7-1 than in Col-0. (b) SEM images of leaf epidermal cells and stomata of Col-0, er-105 and 35S::ER plants. (c) Palisade cell width of Col-0, er-105 and 35S::ER plants, as shown with boxes (n = 30). Asterisks indicate statistically significant differences as determined by Student’s t-test (*, P < 0.05). (d) Palisade cell length of Col-0, er-105 and ER-OE plants, shown as boxes (n = 30). Asterisks indicate statistically significant differences as determined by Student’s t-test (*, P < 0.05). (e) Stomatal density was significantly increased and decreased in er-105 and ER-OE plants compared with Col-0 plants, respectively. As shown with boxes (n = 25). Asterisks indicate a significant difference in comparison with wild-type Col-0, at P < 0.01, by Student’s t-test and Bonferroni correction for multiple (3) tests. (f) Stomatal index was not changed in er-105 and ER-OE plants, as shown with boxes (n = 25). Bar = 50 μm (a,b).

Supplementary Figure 6 Increased drought tolerance of ER-OE plants

(a) Four-week-old Col-0, er-105, and ER-OE (L7-1) plants grown at 22°C were subjected to drought treatment until the er-105 plants died and then were re-watered to recovery for 2 d. (b) Survival rates of Col-0, er-105 mutant, and L7-1plants with or without (control) drought treatment, as shown in dots of 3 replicates (30 plants each). Asterisks indicate a significant difference in comparison with wild-type Col-0, at P < 0.05 (*) or P < 0.01(**), by Student’s t-test and Bonferroni correction for multiple (2) tests for drought treatment.

Supplementary Figure 7 Leaf epidermal morphology of two-week-old Col-0 and er-105 under heat stress

Leaf epidermal cell morphology of Col-0 and er-105 plants under normal (40°C, 0 h) and heat (40°C, 12 h) treatments. Severely wrinkled leaf epiderm was observed in er-105 under heat stress. Bar = 50 μm.

Supplementary Figure 8 Subcellular observation by TEM during heat treatment

(a) TEM observation of leaf cell collapse of the two-week-old er-105 and Col-0 plants treated by 40ºC for 0 and 24 h. Red arrow indicates disrupted plasma membrane in er-105, and arrowhead indicates plasma membrane collapsing in Col-0. Bar = 10 μm. (b-e) An earlier and more severe collapse of the chloroplast (b,d) and mitochondria (c,e), with formation of spherical bodies, occurred in the er-105 cells than in the Col-0 cells under heat, which was less or delayed in the L7-1 cells compared with Col-0 cells. Asterisks indicate a significant difference in comparison with wild-type Col-0, at P < 0.05 (*) or P < 0.01 (**), by Student’s t-test and Bonferroni correction for multiple (2) tests, as shown in dots of 3 replicates (d,e). Bar = 1 μm (b) or 100 nm (c). cw, cell wall; v, vacuole; sp, spherical bodies; c, chloroplast; m, mitochondria; s, starch granule.

Supplementary Figure 9 Expression inhibition of ER at high temperature (40°C)

Relative expression levels of ER were determined by qPCR in two-week-old Col-0 plants treated by heat for 0h (control) to 24 h. ACTIN2 was used as control to normalise expression levels. Values are means ± SD (n = 3).

Supplementary Figure 10 Leaf size and cell morphology of transgenic tomato plants

(a) Three leaf sizes of 6-week-old 35S::ER transgenic tomato lines and the empty-vector transgenic control grown at 22°C. Values are means ± SD (n = 10). Asterisks indicate statistically significant differences as determined by Student’s t-test (**, P < 0.01). (b) Enlarged epidermal leaf cells of the 35S::ER transgenic tomato plants in comparison with the vector control grown at 22°C. Bar = 20 μm. (c) Decreased stomatal density in the transgenic tomato plants, as shown with boxes (n = 25). Asterisks indicate a significant difference in comparison with vector control, at P < 0.01, by Student’s t-test and Bonferroni correction for multiple (3) tests. (d) No change in the stomatal index was observed in the transgenic tomato plants, as shown with boxes (n = 25).

Supplementary Figure 11 Stomatal conductance, transpiration efficiency and cell death of transgenic tomato plants

(a) Stomatal conductance of 35S::ER transgenic tomato and the empty-vector control plants (6-week-old) under normal growth conditions (22°C), shown as dots (n ≥ 8). (b) Increased transpiration efficiency (instantaneous WUE) in 35S::ER transgenic tomato relative to the empty-vector transgenic control under normal growth conditions (22°C), shown as dots (n ≥ 8). (c) Decreased cell death in 35S::ER transgenic tomato relative to the empty-vector transgenic control under heat treatment. Bar = 1mm. Asterisks indicate a significant difference in comparison with vector control, at P < 0.05 (*) or P < 0.01 (**), by Student’s t-test and Bonferroni correction for multiple (2) tests (a,b).

Supplementary Figure 12 Field tests of transgenic tomato in different locations and seasons

Survival tests in Shanghai (summer, 2012) (a), Wuhan (summer, 2013) (b), and in Hainan (early summer, 2013) (c). Fifty plants of each line were directly compared for survival/death rates. Tomato plants were also grown in early summer (early June-early July) of 2014 in the Shanghai station as normal growth conditions with 3 biological replicates, no statistical difference in survival rate was observed in the test, as shown with dots of 3 replicates (d). The local temperature and humidity conditions for these field tests were shown in Data Set 1.

Supplementary Figure 13 Flower number and fruit weight of tomato

Plants were grown at the greenhouse (22°C) for 10 weeks, flower number was counted (3 inflorescences each plant, 10 plants) (a), and fruit weight (b) was measured for fruits produced by hand-pollination (c), shown as boxes (n = 30 for a, n = 20 for b). Bar = 1 cm (c).

Supplementary Figure 14 Agronomic traits of transgenic rice plants under normal temperature conditions

(a) Field-grown mature plants of transgenic rice plants compared with the empty-vector control line (T1 generation). (b) No difference was observed in plant height of adult transgenic plants relative to the control transgenic line after heading, as shown with boxes (n = 20). (c,d) One-week-old transgenic rice seedlings were significantly larger than the control, with a larger leaf width (c) and whole plant height (from root tip to leaf tip) (d), as shown with boxes (n = 20); asterisks indicate a significant difference in comparison with vector control, at P < 0.05 (*) or P < 0.01 (**), by Student’s t-test and Bonferroni correction for multiple (2) tests (c,d).

Supplementary Figure 15 Yield traits in the field tests of rice in different locations and seasons

(a-c) Yield performance of the transgenic lines grown to flower in the autumn of 2013 in the Shanghai station as the normal temperature growth conditions as shown in boxes (n ≥ 30), showing no difference in seed setting (a) and panicle number (c). Lines 36 and 38 displayed significant higher grain yield potential in comparison with the vector control (b), asterisks indicate a significant difference in comparison with vector control, at P < 0.05, by Student’s t-test. (d) Panicle numbers per plant flowering in the summer of 2013 at the Shanghai, Wuhan and Changsha stations, shown as boxes (n ≥ 30). Asterisks indicate a significant difference in comparison with vector control, at P < 0.05 (*) or P < 0.01 (**), by Student’s t-test and Bonferroni correction for multiple (3) tests in the Shanghai (2013, 2014) station, or at P < 0.05, by Student’s t-test in the Changsha station. (e) Average grain number per panicle in the summers of 2013 and 2014, shown as boxes (n ≥ 30). (f) Grain weight measurements in the field tests of the 2013 and 2014 summers as shown with boxes (n = 5).

Supplementary Figure 16 Characterization of the rice T-DNA insertion mutants Oser1 and Oser2

(a) Identification of the T-DNA insertion mutant Oser1 that contains a T-DNA insert between exon 11 and 12, abolishing the expression of the OsER1gene detected by qPCR. Values are means ± SD (n = 3). (b) The T-DNA inserted into exon 24 of the rice ER homolog gene OsER2, resulting into a null mutant that abolishes OsER2 expression detected by qPCR. Values are means ± SD (n = 3). The rice ACTIN1 gene was used as control to normalise expression levels (a,b). (c) No difference was observed in thermotolerance in the mutant in comparison with the wild-type control during heat treatment (42°C). Pictures were taken after a one-week recovery period following 10-d treatment at 42°C/35°C (day/night) in growth chamber.

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Supplementary Figures 1–16; Supplementary Tables 1–2 (PDF 2069 kb)

Supplementary Dataset 1

Local temperature and humidity records of field tests (XLSX 182 kb)

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Shen, H., Zhong, X., Zhao, F. et al. Overexpression of receptor-like kinase ERECTA improves thermotolerance in rice and tomato. Nat Biotechnol 33, 996–1003 (2015). https://doi.org/10.1038/nbt.3321

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