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TT2 controls rice thermotolerance through SCT1-dependent alteration of wax biosynthesis

Nature Plants volume 8, pages 53–67 (2022)Cite this article

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Abstract

Global warming threatens crop production. G proteins mediate plant responses to multiple abiotic stresses. Here we identified a natural quantitative trait locus, TT2 (THERMOTOLERANCE 2), encoding a Gγ subunit, that confers thermotolerance in rice during both vegetative and reproductive growth without a yield penalty. A natural allele with loss of TT2 function was associated with greater retention of wax at high temperatures and increased thermotolerance. Mechanistically, we found that a transcription factor, SCT1 (Sensing Ca2+ Transcription factor 1), functions to decode Ca2+ through Ca2+-enhanced interaction with calmodulin and acts as a negative regulator of its target genes (for example, Wax Synthesis Regulatory 2 (OsWR2)). The calmodulin–SCT1 interaction was attenuated by reduced heat-triggered Ca2+ caused by disrupted TT2, thus explaining the observed heat-induced changes in wax content. Beyond establishing a bridge linking G protein, Ca2+ sensing and wax metabolism, our study illustrates innovative approaches for developing potentially yield-penalty-free thermotolerant crop varieties.

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Fig. 1: TT2 is essential for thermotolerance.
Fig. 2: TT2HPS32 protects rice yield from heat-stress-related losses.
Fig. 3: TT2HPS32 attenuates heat-triggered reduction in cuticular wax.
Fig. 4: SCT1 directly binds to the promoter of OsWR2 to negatively regulate thermotolerance.
Fig. 5: TT2 is essential for cytosolic Ca2+ elevation upon heat treatment.
Fig. 6: CaM interacts with and suppresses SCT1 in a Ca2+-dependent manner.

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Data availability

Intact RNA-seq datasets were deposited in the NCBI Gene Expression Omnibus (GEO) under accession number GSE168650. The Rice SNP-Seek Database is accessible at https://snp-seek.irri.org/; the EnsemblPlants database is accessible at http://plants.ensembl.org/index.html. Source data are provided with this paper.

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Acknowledgements

We thank Dr G. Zhang (South China Agricultural University) for providing CSSL plant materials; Drs J. Kudla (University of Heidelberg, Germany), K. Chong and X. Guo (Institute of Botany, CAS, China) for providing YC3.6 vectors and technical support; Drs Y. Guo and L. Ma (China Agriculture University, China) for providing aequorin vectors and technical support; Drs X. Li, M. Shi, W. Cai, S. Yin, Z. Zhang, X. Gao, J. Li and L. Xu (CAS Centre for Excellence in Molecular Plant Sciences, Shanghai Institute of Plant Physiology and Ecology, CAS) for technical support; Dr Y. Liu (South China Agriculture University) for donating CRISPR-Cas9 plasmids. This work was supported by grants from the National Natural Science Foundation of China (31788103, 31630052), the Chinese Academy of Sciences (XDB27010104, QYZDY-SSW-SMC023, 159231KYSB20200008), the Laboratory of Lingnan Modern Agriculture Project (NT2021002), CAS–Croucher Funding Scheme for Joint Laboratories and National Key Laboratory of Plant Molecular Genetics to H.-X.L.

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Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, CAS Centre for Excellence in Molecular Plant Sciences and Collaborative Innovation Center of Genetics and Development, Shanghai Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China

    Yi Kan, Xiao-Rui Mu, Hai Zhang, Jin Gao, Jun-Xiang Shan, Wang-Wei Ye & Hong-Xuan Lin

  2. University of the Chinese Academy of Sciences, Beijing, China

    Yi Kan, Xiao-Rui Mu, Jin Gao & Hong-Xuan Lin

  3. Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, China

    Yi Kan, Jun-Xiang Shan, Wang-Wei Ye & Hong-Xuan Lin

  4. School of Life Science and Technology, ShanghaiTech University, Shanghai, China

    Hai Zhang & Hong-Xuan Lin

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Contributions

H.-X.L. conceived and supervised the project; H.-X.L. and Y.K. designed the experiments; Y.K. performed most of the experiments; X.-R.M., H.Z., J.G., W.-W.Y., J.-X.S. and H.-X.L. performed some of the experiments; Y.K. and H.-X.L. analysed data and wrote the manuscript.

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Correspondence to Hong-Xuan Lin.

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Extended data

Extended Data Fig. 1 Thermotolerance phenotypes of CSSLs and various cultivars.

a, Phenotypic response to heat treatment of CSSL (HPS32) and huajingxian (HJX) as a negative control. Scale bars, 4 cm. b, The survival rates of CSSLs and the recurrent parent (HJX). Values are shown as mean ± s.d. (n = 3 samples, each sample represents 24 individual plants). P values according to two-tailed Student’s t-tests are shown. c, Analysis of the sequence identity of the HPS32 substitution segment across different cultivated accessions (chr3:16662761–17212811, 550 kb). Data were acquired from the genomes of 3010 Asian cultivated accessions (Rice SNP-Seek Database, https://snp-seek.irri.org/). We used the frequency of each SNP to evaluate the possible origination of the HPS32 target segment among different subpopulations. d, Distribution of the crucial SNP (C165A) in different subpopulations of rice. The allele frequencies in different subpopulations were calculated to make this histogram. e, Phenotypic response to heat treatment of 9 tropical japonica varieties (GP5, 7, 86, 112, 536, 629, 640, 641, and 642). Scale bars, 4 cm. f, Phenotypic response to heat treatment of six temperate japonica varieties (WYJ: Wuyunjing 7; KY131: Kongyu131; ZH11: Zhonghua11; Jiahua1; NIP: Nipponpare; and Xiushui09). Scale bars, 4 cm.

Source data

Extended Data Fig. 2 Molecular identification and phenotypic analysis of TT2 transgenic lines.

a, Schematic map of the sgRNA target sites in TT2, and a schematic showing vector construction and sequence alignment of the TT2-knockout transgenic lines with wild-type HJX. b, Relative expression level of TT2 in NIL-TT2HPS32 and pUbi:TT2HJX/NIL-TT2HPS32. P = 5.28 × 10−10 by one-way ANOVA (n = 6). c, Grain length in HJX, TT2KO/HJX, NIL-TT2HPS32, pTT2-gTT2HJX/NIL-TT2HPS32, and pUbi-TT2HJX/NIL-TT2HPS32. Scale bar, 1 cm. d, Survival rate of TT2 transgenic lines. The survival rate was determined after heat treatment (42 °C, RH > 90%) for 24 hr and subsequent recovery at 28 °C for 7 days. For box-and-whisker plots, the central line, box and whiskers indicate the median, IQR and 1.5 times the IQR, respectively. n = 6 samples, 24 individual plants per sample. TT2KO/HJX, P = 1.00 × 10−27; pUbi-TT2HJX/NIL-TT2HPS32, P = 1.09 × 10−24; pTT2-gTT2HJX/NIL-TT2HPS32, P = 2.58 × 10−32 by one-way ANOVA. The same letters indicate no significant difference at P > 0.05 as determined by Tukey’s HSD test (b, d).

Source data

Extended Data Fig. 3 Agronomic traits of NILs.

a, Pollen fertility of NILs under heat stress. Pollen grains were stained with KI-I2. Scale bars, 1 mm (left); 1000 μm (right). b, NILs at early filling stage. Scale bars, 10 cm. c, Grain length (n = 15), grain width (n = 15), grain number per panicle (n = 24), panicle number per plant (n = 30), panicle length (n = 30), and plant height (n = 48) of NILs. For box-and-whisker plots, the central line, box and whiskers indicate the median, IQR and 1.5 times the IQR, respectively. P values according to two-tailed Student’s t-tests are shown (c).

Source data

Extended Data Fig. 4 Transcriptomic analysis of DEGs between the two NILs.

a, Venn diagram and volcano plot of DEGs between the NILs under different treatments. Criteria for defining differentially expressed genes (DEGs) are |log2(Fold Change)| > 0 and P value < 0.05, n = 3 samples. H, heat treatment; C, control treatment. b, Genes clustered by expression level Cluster analysis for the two NILs under normal conditions or heat treatment. FKPM value was adjusted by log2(fpkm+1). The adjusted FKPM of every gene in a subcluster is shown as a gray line. A blue line represents the mean adjusted FKPM of the subcluster. c, Results of KEGG enrichment analysis of subcluster 2 and subcluster 3 are shown. Padj, adjusted P value; n = 3 samples. d, Heat map showing the expression patterns of wax-related DEGs in the two NILs under heat treatment. P < 0.05, n = 3 samples. Each sample represents 24 individual plants (a-d). P value was obtained by wald test using a model based on the negative binomial distribution (a,d), P value in KEGG obtained based on hypergeometric distribution model was adjusted to Padj using the Benjamini & Hochberg method (c).

Source data

Extended Data Fig. 5 Characteristics of OsWR2-knockout transgenic lines in NIL-TT2HPS32 backgrounds.

a, Sequence alignments of OsWR2 in wr2/NIL-TT2HPS32 with their wild-type counterparts. b, Toluidine blue (TB) staining assay for wr2/NIL-TT2HPS32 and NIL-TT2HPS32 after a 1-hr and 12-hr heat treatment. Heavier staining in leaves indicates higher permeability of the cuticle layer. Scale bars, 1 cm. Leaves are sampled from different individual plants. c, SEM images of cuticle wax crystal patterns on the surfaces of leaves in NIL-TT2HPS32 and wr2/NIL-TT2HPS32 plants under normal conditions. Images were obtained from three individual plants, respectively. Scale bars, 1 μm.

Extended Data Fig. 6 The homologs of the CaM-binding transcription factor SCT1.

a, The maximum-likelihood tree based on protein sequences of 42 SCT1 homologs. The amino acid sequences were obtained from a EnsemblPlants database search. The sequences were aligned using MEGA6.0. Scale bar represents 0.2 substitutions per site. Yellow, Teiticum aestivum; black, O. sativa; blue, Zea mays; red, Arabidopsis thaliana, green, Glycine max. b, Alignment of the DNA binding domain and CaMBD sequences of SCT1 homologs. The sequences were aligned using MEGA6.0. Hydrophobic amino acid residues corresponding to the CaM motif are marked with red arrows. c, Electrophoretic mobility shift assay (EMSA) of SCT2-NT binding to CG1-like cis-elements. The competition assay was performed with unlabeled wild-type oligonucleotides in 5-, 20-, and 50-fold excess of the wild-type probes. Experiments were independently conducted for at least twice (c).

Source data

Extended Data Fig. 7 Molecular identification and RNA-profiling of the sct1/sct2 mutant.

a, Sequence alignments of SCT1/SCT2 in sct1/sct2 with their wild-type counterparts. b, Results of KEGG enrichment analysis of DEGs which was more highly expressed in sct1/sct2 than in wild-type plants under normal conditions. c, The expression pattern of TT1 in sct1/sct2 and ZH11 under normal conditions and heat treatment. TT1 was used as a negative control. Values are shown as the mean ± s.d., Pinteraction = 0.690 by two-way ANOVA, n = 3 samples. d, Thirty one wax-related marker genes were differentially expressed in sct1/sct2 mutant plants as compared with ZH11 under normal conditions and heat treatment, based on RNA-seq profiling (n = 3 samples). Each sample represents 24 individual plants (b-d).

Source data

Extended Data Fig. 8 Calcium imaging based on NES-YC3.6 and aequorin assays.

a, The ratiometric image (cpVenus/ECFP) in living roots of NIL-TT2HJX and NIL-TT2HPS32 based on NES-YC3.6 before and after heat stimulation, scale bar, 20 μm. b, Images of heat-induced Ca2+ increases scaled by a pseudocolored reference in aequorin-expressing NIL-TT2HJX and NIL-TT2HPS32 seedlings treated with heat stress (left) and discharge buffer (right, recording total remaining luminescence).

Extended Data Fig. 9 CaM-SCT1 interaction is dependent on Ca2+.

a, Subcellular localization of SCT1, SCT2, and CaM in tobacco. Scale bars, 20 μm. b, Gel mobility shift assay with the CaMBD of SCT1. The amino acid sequences of the peptide correspond to amino acids 897–917 of SCT1. CaM was incubated with increasing amounts of the peptide (peptide: CaM molar ratios are indicated) in the presence of 0.1 mM CaCl2 or 2 mM EGTA. Samples were separated by non-denaturing PAGE and stained with Coomassie Brilliant Blue. In the mutant peptides, valine (Val900) and tryptophan (Trp907) were changed to arginine (Arg). c, Protein alignment of CaM1234 with CaM; four conserved Glu residues in the EF-hand motif were mutated to Arg. d, The TT1 mRNA levels under normal condition and heat treatment in coordination with normal, MgCl2, or LaCl3 conditions. P = 3.83 × 10−3 by one-way ANOVA (n = 3 to 4 biological replicates). e, Dual-luciferase assays performed upon blocking of Ca2+ channels by La3+ treatment in rice protoplasts. The expression of luciferase was driven by the 7xUAS reporter system. Mg2+ was used as a negative control. P = 2.31 × 10−10 by one-way ANOVA (n = 3 biological replicates). Values are shown as the mean ± s.d. One-way ANOVA being represented by Tukey’s HSD test using the same letters at P > 0.05 (d, e). Experiments were independently conducted for at least twice (a, b).

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Extended Data Fig. 10 Calculation of KD value between SCT1 and CaM.

Three concentrations of CaM were used to calculate KD value between SCT1 and CaM fitted to one-to-one evaluation type at the presence of CaCl2 or EGTA. Three lines are fitted to one curve and the KD value was calculated. The signal after blocking the SCT1-CaMBD-immobilized chip was set to 0.

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Supplementary information

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Supplementary Tables 1–6

Detailed GSEA data, descriptions of wax/cutin-related genes, sequences of oligonucleotides (including primers, transgenic constructs, and so on), antibodies and strains.

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Kan, Y., Mu, XR., Zhang, H. et al. TT2 controls rice thermotolerance through SCT1-dependent alteration of wax biosynthesis. Nat. Plants 8, 53–67 (2022). https://doi.org/10.1038/s41477-021-01039-0

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