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A nitric oxide burst at the shoot apex triggers a heat-responsive pathway in Arabidopsis

Nature Plants volume 8, pages 434–450 (2022)Cite this article

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Abstract

When confronted with heat stress, plants depend on the timely activation of cellular defences to survive by perceiving the rising temperature. However, how plants sense heat at the whole-plant level has remained unanswered. Here we demonstrate that shoot apical nitric oxide (NO) bursting under heat stress as a signal triggers cellular heat responses at the whole-plant level on the basis of our studies mainly using live-imaging of transgenic plants harbouring pHsfA2::LUC, micrografting, NO accumulation mutants and liquid chromatography–tandem mass spectrometry analysis in Arabidopsis. Furthermore, we validate that S-nitrosylation of the trihelix transcription factor GT-1 by S-nitrosoglutathione promotes its binding to NO-responsive elements in the HsfA2 promoter and that loss of function of GT-1 disrupts the activation of HsfA2 and heat tolerance, revealing that GT-1 is the long-sought mediator linking signal perception to the activation of cellular heat responses. These findings uncover a heat-responsive mechanism that determines the timing and execution of cellular heat responses at the whole-plant level.

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Fig. 1: Heat triggers transcriptional activation of HsfA2 at the shoot apex.
Fig. 2: An unknown shoot-to-root mobile signal triggers the whole-plant expression of HsfA2 in response to heat stress.
Fig. 3: NO mediates transcriptional activation of HsfA2 in response to heat stress.
Fig. 4: Heat-induced NO burst in the inflorescence apex and determination of GSNO in bleeding sap from shoots with floral buds.
Fig. 5: GT-1 regulates heat-responsive expression of HsfA2 by binding to NO-responsive elements.
Fig. 6: S-Nitrosylation of GT-1 by GSNO promotes its binding to the promoter of HsfA2 in response to heat stress.
Fig. 7: S-Nitrosylation of GT-1 is required for thermotolerance.

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Data availability

All data generated or analysed during this study are included in the published article and the Supplementary Information. All Arabidopsis genes involved in this study can be found at TAIR (www.arabidopsis.org), with the following accession numbers: GT-1 (AT1G13450), HsfA2 (AT2G26150), Hsp101 (AT1G74310), Hsp70 (AT3G12580), Hsp25.3 (AT4G27670), Hsp18.1 (AT5G59720), Hsp17.7 (AT5G12030), APX2 (AT3G09640) and GolS1 (AT2G47180). Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the Chinese Academy of Sciences (the Strategic Priority Research Program, no. XDB27040105), the Ministry of Science and Technology of China (National Key R&D Program of China, no. 2020YFA0907604) and the National Natural Science Foundation of China (nos U1812401, 31770314 and 31570260). We thank N. Crawford (University of California at San Diego, USA), Z.-M. Pei (Duke University, USA), S. Xu (Northwest A&F University, China) and E. Vierling (University of Massachusetts, USA) for providing plant materials; J.-K. Zhu (Shanghai Center for Plant Stress Biology, CAS, China) and Y.-G. Liu (South China Agricultural University, China) for providing the plasmids of the CRISPR–Cas9 gene-editing system; and the CEMPS Core Facility for technical assistance.

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Authors and Affiliations

  1. National Key Laboratory of Plant Molecular Genetics, CAS Center for Excellence in Molecular Plant Sciences, Institute of Plant Physiology & Ecology, Chinese Academy of Sciences, Shanghai, China

    Ning-Yu He, Li-Sha Chen, Ai-Zhen Sun, Yao Zhao, Shui-Ning Yin & Fang-Qing Guo

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

    Yao Zhao

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  1. Ning-Yu He
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Contributions

F.-Q.G. conceived the project. F.-Q.G. and N.-Y.H. designed the experiments. N.-Y.H. performed most of the experimental work with assistance from A.-Z.S., Y.Z. and S.-N.Y. L.-S.C. contributed to the in situ hybridization. N.-Y.H., F.-Q.G. and L.-S.C. analysed the data. F.-Q.G. and N.-Y.H. wrote the paper.

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Correspondence to Fang-Qing Guo.

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Extended data

Extended Data Fig. 1 Heat stress triggers the transcriptional activation of HsfA2 firstly at shoot apical region, subsequently at young rosette leaves and old leaves.

Intensity and developing patterns of LUC-signals were shown in the transgenic Arabidopsis seedlings (20-d-old) harboring the pHsfA2::LUC construct in which a luciferase reporter gene (LUC) was driven by the HsfA2 promoter under control condition (a) or subjected to heat stress (38 °C, 30 min) (b). Under heat stress conditions, time-lapse imaging showed rapid systemic signal transduction from shoot apical region to young rosette leaves and then old leaves via vascular system (leaf veins). Bars = 1 cm.

Extended Data Fig. 2 Transcriptional activation of HsfA2 is severely inhibited in wild-type flowering plants with shoot tip removed in response to heat stress.

The expression level of HsfA2 was analyzed by qRT-PCR in the rosette leaves of wild-type flowering plants or wild-type flowering plants with inflorescence shoot tip removed when subjected to heat stress (38 °C) for the indicated times. Individual values are means of three independent biological replicates. Bars indicate standard deviation (SD) (*P value<0.05, **P value<0.01, ***P value<0.001, two-sided Student’s t test). AtACTIN2 was used as the internal control.

Source data

Extended Data Fig. 3 Heat stress induces burst of nitric oxide (NO) at shoot apex and accumulation in phloem in Arabidopsis seedlings.

a, Detection of endogenous nitric oxide (NO) at shoot apex by the specific probe 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) in 5-day-old wild-type seedlings grown under control conditions (Control) or subjected to heat treatment (38 °C, 5 min) (Heat) by a confocal microscopy. YL, young leaf; SAM, shoot apical meristem. Bars = 20 µm. Three times these experiments were repeated independently with similar results. b, Detection of NO by DAF-FM DA using a fluorescence microscope in hand-made stem cross-sections taken from the inflorescence stems of 23-d-old Arabidopsis plants grown under control conditions (Control) or subjected to heat treatment (38 °C, 30 min) (Heat). Bars = 100 µm. Three times these experiments were repeated independently with similar results. c, Close-up view of the image of DAF-FM fluorescence signals in (b). ep, epidermis; co, cortex; ph, phloem; xylem, xy. Stem-cross sections were also stained with propidium iodide (PI). Bar = 100 µm.

Extended Data Fig. 4 The transcriptional activation of HsfA2 was severely inhibited in the NO-deficient mutant noa1 in response to heat stress.

a, Time-lapse imaging of systemic signal transduction from shoot to root to initiate transcriptional activation of a luciferase reporter gene (LUC) driven by the HsfA2 promoter (pHsfA2::LUC) in the transgenic Arabidopsis plants of wild-type (upper panels) and noa1 (lower panels) under control conditions. b, Time-lapse imaging of systemic signal transduction from shoot to root to initiate rapid transcriptional activation of a luciferase reporter gene (LUC) driven by the HsfA2 promoter (pHsfA2::LUC) in the transgenic Arabidopsis plant of wild-type (upper panels), but relatively slower and weaker transcriptional activation in noa1 (lower panels) in response to heat stress (38 °C, 30 min) (b). Bars = 1 cm.

Extended Data Fig. 5 Micrografting with the heat-stressed wild-type or noa1 shoots as scion and the transgenic wild-type or noa1 roots containing pHsfA2::LUC as rootstocks.

a, Time-lapse imaging showed systemic signal transduction from shoot to root when a control wild-type shoot (upper panels and lower panels) or a control NO-deficient mutant noa1 shoot (middle panels) was grafted onto the rootstock taken from the transgenic plant of wild-type or noa1 harboring the pHsfA2::LUC construct. White arrowheads indicate grafting joints. b, Time-lapse imaging showed rapid systemic signal transduction from shoot to root when a heat-treated wild-type shoot (upper panels and lower panels), rather than the heat-treated noa1 shoot (middle panels), was grafted onto the rootstock taken from the transgenic plant of wild-type or noa1 harboring the pHsfA2::LUC construct. White arrowheads indicate grafting joints. Bars = 1 cm.

Extended Data Fig. 6 Characterization of the T-DNA insertion mutant gt-1-1 and heat-sensitive phenotypes of two allelic mutants gt-1-2 and gt-1-3 generated by the CRISPR-cas9 technique.

a, Schematic diagram of the GT-1 gene showing the T-DNA insertion site for gt-1-1 and the editing sites for two allelic mutants gt-1-2 (“TCACAA” inserted at 149 bp from ATG) and gt-1-3 (“TA” deleted at 302 bp from ATG) generated by the CRISPR-cas9 technique. Closed box indicates ORF. Exons (boxes) and introns (lines) were determined by a comparison of the genomic and cDNA sequences. The T-DNA insertion site (931 bp from ATG) and position of the start codon are indicated. b, The specific primers (LP + RP and LBb1.3 + RP), derived from the left (LP) and right (RP) borders of T-DNA insertion and the T-DNA vector sequence (LBb1.3) respectively, were used to amplify the indicated fragments in wild-type and gt-1-1 mutant plants. The amplified fragments were confirmed by sequencing. c, The protein abundance of GT-1 in leaves taken from the plants of wild-type and the gt-1-1 mutant was analyzed by western blots with a polyclonal antibody against GT-1. Ponceau S staining was used as an internal loading control. d, e Expression analysis of GT-1 in the detached fully-expanded leaves of wild-type, the gt-1-1 mutant and the complemented gt-1-1 mutant (gt-1-1 comp) with pGT-1::GT-1 cDNA-GFP (d), and in the detached fully-expanded leaves of wild-type and the CRISPR-cas9-generated gt-1-2 and gt-1-3 mutants (d) by qRT-PCR. It should be noted that around 20% expression level in relative to wild-type was detected in gt-1-2, but the resulted transcript with frameshift mutations had nothing to do with GT-1 when the resulted transcript was sequenced. In gt-1-2, as shown in (a), “TCACAA” was inserted at 149 bp from ATG and this kind of insertion led to a frameshift mutation. No signal was detected in gt-1-3. Individual values are means of three independent biological replicates. Bars indicate standard deviation (SD). ND, non-detectable. ACTIN2 (At3g18780) was used as the internal control. f, g, Phenotypes of the wild-type, gt-1-2 and gt-1-3 as examined with detached fully-expanded leaves (f) and whole plants (g) challenged with heat treatments for the indicated time in dark. Bars = 1 cm in (f) or 2 cm in (g).

Source data

Extended Data Fig. 7 Knockdown of GT-1 inhibits transcriptional activation of the HsfA2-target genes in response to heat stress.

Expression analysis of the representative downstream target genes of HsfA2 including Hsp17.7II, Hsp18.1, GolS1, Hsp70, Hsp25.3, APX2 and Hsp101 in detached fully-expanded leaves of the wild-type and gt-1-1 mutant at the indicated time when subjected to 2-h-heat treatment (38 °C) in dark. Individual values are means of three independent biological replicates. Bars indicate standard deviation (SD). AtACTIN2 was used as the internal control.

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Extended Data Fig. 8 Effects of S-nitrosylation of GT-1 by GSNO on its binding activities to the promoter of HsfA2.

a, b, Recombinant TF-GT-1 was incubated with the indicated concentrations of GSNO and subsequently subjected to the EMSA assay for examining the binding activity of TF-GT-1 to the cis-element 1 (cis1) (a) and the cis-element 2 (cis2) (b) identified in the HsfA2 promoter. c, Single residue mutagenesis of Cys324 or Cys347 of GT-1 led to partial losses of its GSNO-dependent binding activity to the cis-element 1 in the promoter of HsfA2. EMSA assay was performed to examine the binding activity of TF-GT-1 and the mutated TF-GT-1 proteins with the site-directed mutagenesis of Cys324 and Cys347 into Ser residues in single residue mutagenesis (GT-1C324S or GT-1C347S), treated with GSNO (10 μM) or plus cPTIO (200 μM). d, In vitro S-nitrosylation of TF-GT-1C324/347 S containing double-residue mutations of Cys324 and Cys347 into Ser residues (only Cys135 residue is available for S-nitrosylation) with GSNO treatment. The purified recombinant TF-GT-1C324/347 S was incubated with the indicated concentrations of GSNO and then subjected to the TMT-switch assay. TMT is referred to as “tandem mass tags“. The S-nitrosylation levels of resulting proteins were examined by western blot analysis using an anti-TMT antibody. Ponceau S staining was used as an internal loading control. e, EMSA assay was performed to examine the binding activity of TF-GT-1C324/347 S with the site-directed mutagenesis of Cys324 and Cys347 into Ser residues, treated with different concentrations of GSNO (5-200 μM). Three times these experiments were repeated independently with similar results as shown in (a-e).

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Extended Data Fig. 9 Quantification of NO levels in wild-type and the NO-related mutants such as noa1, hot5-2/gsnor1 and nox1 under control or heat stress conditions.

The levels of NO were detected by the specific probe 4-amino-5-methylamino-2’,7’-difluorofluorescein diacetate (DAF-FM DA) at shoot apical regions of 5-day-old wild-type seedlings and the NO-related mutants such as noa1, hot5-2/gsnor1 and nox1 under control or subjected to heat treatment (38oC, 5 min) (Heat) or subjected to heat treatment (38 °C, 5 min) plus the NO scavenger cPTIO treatment (100 μM) (Heat + cPTIO) by a confocal microscopy (LSM880, Zeiss), respectively. Seventeen individual seedlings were examined for each genotype or treatment. The signal intensity of DAF-FM fluorescence in each image was quantified using ZEN software (Zeiss). Data were shown as mean of the signal intensities from seventeen individual seedlings (n = 17). Error bars indicate SD (***P value<0.001, two-sided Student’s t test).

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Extended Data Fig. 10 Relative abundance of the S-nitrosylated peptides of GT-1 identified with Cys135, Cys324 or Cys347 as the S-nitrosylated residues respectively in wild-type and hot5-2 under heat stress.

a, b, c, For quantification of the S-nitrosylated peptides of GT-1 identified with Cys135 (a), Cys324 (b) or Cys347 (c) as the S-nitrosylated residues respectively in 5-d-old Arabidopsis seedlings of wild-type and hot5-2 were subjected to heat treatment (38 °C) and harvested at the indicated time points by the MS/MS-based analysis strategy using isotope-coded cysteine thiol-reactive multiplex regent, referred to as “tandem mass tags” (TMTs). The total proteins extracted from Arabidopsis seedlings were first labeled with TMT. The GT-1 protein was immunoprecipitated with anti-GT-1 antibody. The resultant samples were separated on 10% SDS–PAGE. After slicing the protein band corresponding to GT-1 and destaining, the gel slices were reduced with DTT, alkylated with iodoacetamide, and digested with trypsin (V5280, Promega) at 37 °C overnight. The sample was then purified with a C-18 spin column (PepClean C-18 Spin Columns, Thermo), and the resultant peptides were examined by LC–MS/MS. The MS data were initially searched against the NCBI database with the aid of the Sequest search engine. The resultant MS spectra were used to determine the peptide identity and abundance of each peptide in the same spectrum. Relative abundance of peptide was calculated by comparing the intensity of the corresponding tag. The absolute quantification was calculated by comparing with the internal control peptides, and the sum of the S-nitrosylated and non-nitrosylated peptides for each pair was set to 100%. Data are presented as mean values + /- SD (n = 3 independent biological replicates).

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13 (Figs. 11–13 contain only the full scans for Figs. 7b, 9 and 10) and Table 1.

Reporting Summary

Supplementary Data 1

Statistical source data for Supplementary Fig. 3.

Supplementary Data 2

Statistical source data for Supplementary Fig. 8a,b.

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Unprocessed gels.

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Unprocessed western blots/gels.

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Unprocessed gels/western blots.

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Statistical source data.

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Unprocessed western blots.

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Statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

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He, NY., Chen, LS., Sun, AZ. et al. A nitric oxide burst at the shoot apex triggers a heat-responsive pathway in Arabidopsis. Nat. Plants 8, 434–450 (2022). https://doi.org/10.1038/s41477-022-01135-9

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