The microRNA-encoded peptide miPEP398b regulates heat tolerance in grapevine

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MicroRNAs (miRNAs) are noncoding RNAs consisting of 20 to 24 nt transcribed from primary miRNAs (pri-miRNAs). Recent studies indicate that short open reading frames (sORFs) within pri-miRNAs can encode small peptides known as microRNA-encoded peptides (miPEPs). Both miPEPs and their corresponding miRNAs play important roles in plant growth, development, and responses to abiotic stress (Lauressergues et al. 2015; Chen et al. 2020, 2022; Sharma et al. 2020; Ormancey et al. 2021; Badola et al. 2022; Kumar et al. 2023; Ormancey et al. 2023). Global warming leads to significant reductions in agricultural production (Kan et al. 2023). Grapevine (Vitis vinifera L.) is one of the most widely cultivated fruit species globally, but its growth and development are negatively impacted by heat stress. In this study, we report the essential role of miPEPs in improving heat stress resistance in plants.

To investigate the heat stress response profile of miRNAs, we performed small RNA transcriptome sequencing on grapevine plantlets and found that vvi-miR398 expression was significantly induced (Zhang et al. 2023). The vvi-miR398 family includes 3 precursors, with pre-miR398b and pre-miR398c showing higher expression in the root, stem, and leaf (Supplementary Fig. S1, A and B). More importantly, pre-miR398b exhibited the highest induction under heat stress, suggesting its potential role in grapevine heat tolerance (Supplementary Fig. S1C). We validated 4 target genes of vvi-miR398b in grapevine using RLM-5′ RACE and additional experiments. The identified genes are COPPER/ZINCSUPEROXIDE DISMUTASE 1 (VvCSD1), COPPER/ZINC SUPEROXIDE DISMUTASE 2(VvCSD2), COPPER CHAPERONE FOR SUPEROXIDE DISMUTASE (VvCCS), and CYTOCHROME COXIDASE SUBUNIT 5b (VvCOX5b). (Supplementary Fig. S1, D to F).

To explore whether pri-miR398b can encode miPEP and participate in heat stress response, we predicted potential open reading frames (ORFs) within the 600 bp upstream of pre-miR398b (Fig. 1A, Supplementary Fig. S2A). To evaluate the activity of these ORFs, the start codons (ATG1, ATG2, and ATG3) and the upstream promoter region were fusion-expressed with the β-glucuronidase reporter gene GUS. GUS activity was observed only when fused with ATG1, indicating that ATG2 and ATG3 may lack transcriptional activity (Fig. 1A). Moreover, GUS activity was detected when the complete ORF1 (no termination codon) was fused to the GUS gene (Fig. 1A). We also generated a construct by fusing a mutated GFP (GFPmut; where the start codon ATGGTG is mutated to ATTGTT) to the C-terminal of ORF1 (no termination codon) (Fig. 1B). Fluorescence was absent in GFPmut but was present in ORF-GFPmut (Fig. 1B). These results indicate that ORF1 has translational activity and encodes miPEP398b (Fig. 1C). To confirm the presence of endogenous miPEP398b, we extracted total proteins from grapevine leaves and used a 10 kDa ultrafiltration tube to isolate small peptides (Wang et al. 2020). By LC-MS/MS analysis, we identified the miPEP398b fragment (Supplementary Fig. S2B). Since miPEPs are suggested to enhance the transcription of their corresponding pri-miRNAs, we hypothesized that miPEP398b application might influence pri-miR398b expression. We firstly observed a concentration-dependent increase in pre-miR398b expression in plantlets supplemented with exogenous miPEP398b (0.25 to 1 μm) (Fig. 1D). Subsequently, we selected the 0.5 μm concentration of miPEP398b to investigate its role in heat stress resistance. Supplementation with miPEP398b led to increased expression of mature vvi-miR398 and decreased expression of its target genes (Fig. 1, F and G). Under heat stress, miPEP398b-treated plantlets exhibited mild leaf damage and higher Fv/Fm compared to the control group (Mock) (Fig. 1, E and H). Additionally, these plantlets showed significantly higher expression of heat shock transcription factors (HSFs) and heat shock proteins (HSPs) genes (Fig. 1I). Similar results were observed in grapevine callus overexpressing miPEP398b (Supplementary Fig. S3). These findings suggest that miPEP398b may enhance heat tolerance by promoting the accumulation of vvi-miR398.

Figure 1.

miPEP398b confers heat tolerance in grapevine by regulating vvi-miR398b. A) GUS histochemical staining of Nicotiana benthamiana and grapevine leaves transiently expressing empty vector (EV), ProATG1/2/3::GUS and ProORF1::GUS. Images were digitally extracted for comparison. Scale bar: 1 cm. B) GFP fluorescence observation of N. benthamiana leaves transiently expressing GFPwt, GFPmut, and ORF1-GFPmut. Scale bar: 50 μm. C) miPEP398b sequence is encoded by pri-miR398b. D) The expression of pre-miR398b in grapevine plantlets treated with water (Mock) and different concentrations of synthetic miPEP398b. E) The phenotypes of grapevine plantlets treated with synthetic miPEP398b under control and heat stress. Scale bar: 1 cm. F, G) The expression of mature vvi-miR398b and its target genes in grapevine plantlets treated with water (Mock) and synthetic miPEP398b (0.5 μm). In D, F, and G) data are shown as mean ± standard deviation (Sd) of 3 independent replicates. The asterisks indicate significant differences (*P < 0.05; **P < 0.01, ***P < 0.001 by Student's t-test). H) The changes of Fv/Fm in grapevine leaves treated with synthetic miPEP398b under control and heat stress. Center line: median; Box limits: Upper and lower quartiles; Whiskers:1.5 × interquartile range. The data analysis was performed with 3 independent experiments (n = 18 samples in each replicate). The different letters represent significant differences at P < 0.05 (1-way ANOVA and Tukey's multiple comparison test). I) The expression of VvHSFs and VvHSPs in grapevine plantlets treated with synthetic miPEP398b. Data are shown as mean ± standard deviation (Sd) of 3 independent replicates. The different letters indicate significant differences at P < 0.05 (1-way ANOVA and Tukey's multiple comparison test).

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Then, we knocked down (miR398-KD) and overexpressed (miR398-OE) vvi-miR398b in grapevine plantlets (Fig. 2, A to C). Under heat stress, miR398b-KD plantlets displayed severe leaf damage and lower Fv/Fm compared to the empty vector (EV) control, while miR398-OE plantlets showed enhanced heat tolerance (Fig. 2, A and D). Vvi-miR398b also increased the expression of VvHSFs and VvHSPs under heat stress (Fig. 2F). Similar heat tolerance patterns were observed in transgenic grapevine callus and Arabidopsis (Arabidopsis thaliana) (Supplementary Figs. S4 and S5). Previous studies indicate that hydrogen peroxide (H₂O₂) regulates gene expression networks in plants (Volkov et al. 2006; Zhuang et al. 2021). We hypothesized that H₂O₂ influences VvHSFs and VvHSPs expression. To test this, we treated grapevine plantlets with H₂O₂ and found that low concentrations of H₂O₂ induced their expression (Supplementary Fig. S6A). VvHSFs and VvHSPs were also induced under heat stress, but their expression significantly decreased after treatment with N,N″-dimethylthiourea, a reactive oxygen species scavenger (Supplementary Fig. S6B). More importantly, H₂O₂ content was lower in miR398b-KD plantlets compared to the EV under heat stress, while it was higher in miR398b-OE plantlets (Fig. 2E). Furthermore, similar to the other 3 target genes, the newly identified target gene VvCOX5b in grapevine exhibited a phenotype opposite to vvi-miR398b and reduced H₂O₂ content under heat stress (Supplementary Fig. S7) (Guan et al. 2013; Han et al. 2021). Therefore, we hypothesized that vvi-miR398b increases H₂O₂ accumulation through its target genes under heat stress, H₂O₂ may function as a signaling molecule to promote VvHSFs and VvHSPs expression. As one of the key downstream genes, VvHSFA7a exhibited improved heat tolerance (Supplementary Fig. S8).

Figure 2.

VvHSFA2a/VvHSFA7a and vvi-miR398b form a circular regulatory pathway in grapevine heat stress resistance. A) The phenotypes of EV, miR398b-KD and miR398b-OE plantlets after heat treatment. Images were digitally extracted for comparison. Scale bar: 1 cm. B and C) The expression of mature vvi-miR398 and its target genes in EV, miR398b-KD, and miR398b-OE plantlets. D) The changes of Fv/Fm in EV, miR398b-KD, and miR398b-OE plantlets. Center line: median; Box limits: Upper and lower quartiles; Whiskers: 1.5 × interquartile range. The data analysis was performed with 3 independent experiments (n = 18 samples in each replicate). The different letters represent significant differences at P < 0.05 (1-way ANOVA and Tukey's multiple comparison test). E) The H₂O₂ content in EV, miR398b-KD and miR398b-OE plantlets. F) The expression of VvHSFs and VvHSPs in EV, miR398b-KD and miR398b-OE plantlets. G) Subcellular localization of VvHSFA2a and VvHSFA7a. Scale bar: 50 μm. H) The expression of pre-miR398b in EV (35S:: GFP), VvHSFA2a-GFP, and VvHSFA7a-GFP callus. I) ChIP-qPCR showing VvHSFA2a and VvHSFA7a bind to the VvMIR398b promoter. J) Y1H assay showing that VvHSFA2a and VvHSFA7a bind to the VvMIR398b promoter. K) Dual luciferase assay indicating that VvHSFA2a and VvHSFA7a promote the transcription of VvMIR398b.  L) A working model for the vvi-miR398b and miPEP398b modulate heat tolerance in grapevine. In B, C, and H) data are shown as mean ± standard deviation (Sd) of 3 independent replicates. The asterisks indicate significant differences (*P < 0.05; **P < 0.01, ***P < 0.001 by Student's t-test). In E, F, I, and K) data are shown as mean ± standard deviation (Sd) of 3 independent replicates. The different letters indicate significant differences at P < 0.05 (1-way ANOVA and Tukey's multiple comparison test).

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Given that vvi-miR398b is rapidly induced under heat stress, we analyzed the promoter region of VvMIR398b and identified 2 heat shock element (HSE) motifs (Supplementary Fig. S9A). Class A HSFs typically bind to HSE motifs and activate transcription (Liu et al. 2023). Therefore, we selected VvHSFA2a and VvHSFA7a as candidate transcription factors because they are the most highly induced class A VvHSFs genes under heat stress (Supplementary Fig. S9B). Both VvHSFA2a-GFP and VvHSFA7a-GFP proteins localized to the nucleus, and pre-miR398b expression was significantly increased in grapevine callus overexpressing them (Fig. 2, G and H). In addition, chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and yeast 1-hybrid (Y1H) assays confirmed that VvHSFA2a and VvHSFA7a bind to the VvMIR398b promoter (Fig. 2, I and J). Dual luciferase assays also indicated that VvHSFA2a and VvHSFA7a enhance the VvMIR398b transcription (Fig. 2K).

Taken together, we discovered that a microRNA-encoded peptide (miPEP398b) modulates heat tolerance. MiPEP398b influences the expression of vvi-miR398 and its target genes. Vvi-miR398 regulates the accumulation of H₂O₂, which enhances heat tolerance by modulating the VvHSFs and VvHSPs pathways. Meanwhile, VvHSFA2a and VvHSFA7a bind to the VvMIR398b promoter, enhancing its transcription and forming a circular regulatory pathway (Fig. 2L). These findings also have significant implications for plant cultivation under abiotic stress conditions.

Acknowledgments

We thank the lab members for help in this study.

Author contributions

C.M. and D.F. designed the project and wrote the paper. D.F., J.L., Y.R., and Z.Z. contributed to experiments design and data analysis. Y.X., Y.S., J.L., M.L., L.W., and J.H. provided important suggestions. All authors reviewed the manuscript.

Supplementary data

The following materials are available in the online version of this article.

Supplementary Figure S1. The vvi-miR398 family and its target genes in grapevine.

Supplementary Figure S2. Identification of miPEP398b.

Supplementary Figure S3. miPEP398b positively regulates heat tolerance.

Supplementary Figure S4. miR398b enhances heat tolerance in grapevine callus.

Supplementary Figure S5. miR398b enhances heat tolerance in Arabidopsis.

Supplementary Figure S6. H₂O₂ promotes the accumulation of VvHSFs and VvHSPs.

Supplementary Figure S7. Function of VvCOX5b under heat stress.

Supplementary Figure S8. VvHSFA7a positively regulates heat tolerance.

Supplementary Figure S9. VvHSFA2a and VvHSFA7a regulate the transcription of VvMIR398b.

Supplementary Table S1. The primers used in this study.

Funding

This research was financially supported by the National Natural Science Foundation of China (32341041, 32122076, 32372673), and the earmarked fund for CARS-29.

Data availability

The data supporting the findings of this study have been provided in the text and in the Supplementary data files and are available upon request.

Dive Curated Terms

The following phenotypic, genotypic, and functional terms are of significance to the work described in this paper:

• superoxide CHEBI: CHEBI:18421

References

Author notes