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Primary transcript of miR858 encodes regulatory peptide and controls flavonoid biosynthesis and development in Arabidopsis

Nature Plants volume 6, pages 1262–1274 (2020)Cite this article

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Abstract

MicroRNAs (miRNAs) are processed products of primary miRNAs (pri-miRNAs) and regulate the target gene expression. Though the regulatory roles of the several mature plant miRNAs have been studied in detail, the functions of other regions of the pri-miRNAs are still unrecognized. Recent studies suggest that a few pri-miRNAs may encode small peptides, miRNA-encoded peptides (miPEPs); however, the functions of these peptides have not been studied in detail. We report that the pri-miR858a of Arabidopsis thaliana encodes a small peptide, miPEP858a, which regulates the expression of pri-miR858a and associated target genes. miPEP858a-edited and miPEP858a-overexpressing lines showed altered plant development and accumulated modulated levels of flavonoids due to changes in the expression of genes associated with the phenylpropanoid pathway and auxin signalling. The exogenous treatment of the miPEP858a-edited plants with synthetic miPEP858a complemented the phenotypes and the gene function. This study suggests the importance of miPEP858a in exerting control over plant development and the phenylpropanoid pathway.

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Fig. 1: Identification of miPEP858a.
Fig. 2: Effect of miPEP858a on Arabidopsis seedlings.
Fig. 3: Transcriptional regulation of miR858a is dependent on miPEP858a peptide.
Fig. 4: Overexpression of miPEP858a affects plant development and altered expression of flavonoid biosynthesis genes and proteins.
Fig. 5: CRISPR–Cas9-derived knockout mutants show altered phenotypes and gene expression.
Fig. 6: Editing of miR858 leads to differential accumulation of metabolite levels.
Fig. 7: Complementation with miPEP858a synthetic peptide leads to restoration of function of CRISPR–Cas9-edited miPEPCR lines.
Fig. 8: Proposed model for the phenylpropanoid pathway via miR858a and its upstream regulator, miPEP858a.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request.

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Acknowledgements

We thank Q.-J. Chen at China Agriculture University for the pHSE401 vector. This research was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi, in the form of NCP project no. MLP026. P.K.T. also acknowledges the Department of Biotechnology, New Delhi, for financial support in the form of projects on pathway engineering, genome editing and a TATA Innovation Fellowship. A.S., P.K.B., D.S. and C.B. acknowledge the Council of Scientific and Industrial Research, New Delhi, and the University Grants Commission (UGC) for a Senior Research Fellowship, respectively. CSIR-NBRI manuscript number: MS/2020/07/04.

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  1. Deepika Sharma

    Present address: National Institute of Plant Genome Research, New Delhi, India

  2. Prabodh Kumar Trivedi

    Present address: Central Institute of Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, India

Authors and Affiliations

  1. CSIR-National Botanical Research Institute, Council of Scientific and Industrial Research (CSIR-NBRI), Lucknow, India

    Ashish Sharma, Poorwa Kamal Badola, Chitra Bhatia, Deepika Sharma & Prabodh Kumar Trivedi

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

    Ashish Sharma, Poorwa Kamal Badola, Chitra Bhatia, Deepika Sharma & Prabodh Kumar Trivedi

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Contributions

A.S. and P.K.T. conceived and designed the study. A.S., P.K.B. and C.B. participated in the execution of all the experiments. A.S., P.K.B., C.B., D.S. and P.K.T. interpreted and discussed the data. A.S., P.K.B., C.B., D.S. and P.K.T. wrote the manuscript.

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Extended data

Extended Data Fig. 1 Overexpression of miPEP858a alters metabolite levels as compared to WT.

a-b, Quantification of total anthocyanin and flavonols in 10-day old seedlings of WT and OX miPEP lines. c, Quantification of lignin in 35-day old stem of WT and OX miPEP plants. For (a-c), the experiments were repeated three times independently with similar results. Small open circles represent individual values. d, Representative images of transverse sections of stem of 35-day old WT and OX miPEP plants stained with phluoroglucinol showing changes in lignin content. The experiment was repeated three times with n= 5 biologically independent replicates with similar results. e-f, Length of vascular bundles and interfascicular fiber of 35-day old stem of WT and OX miPEP plants, n=15 (small open circles represent individual values). These experiments were repeated three times independently with similar results. g, Expression analysis of lignin biosynthesis genes in 35-day old stem of WT and OX miPEP plants. The experiments were repeated three times independently with similar results. Statistical analysis was performed using two-tailed Student’s t-test. Data are plotted as means ±SD. Error bars represent standard deviation. Asterisks indicate a significant difference, *P < 0.1, **P < 0.01, ***P < 0.001).

Extended Data Fig. 2 Exogenous application of miPEP858a does not restore the function of miR858 in miR858CR plants.

a-c, Representative images of 10 days old seedlings of WT and CRISPR/cas9 edited miR858CR lines grown on 1/2 strength Murashige and Skoog (MS) medium supplemented with water (control), 0.25 μM miPEP858a and 0.25 μM NSP (scale bars, 1 cm). d, Root length of 10 days old seedlings of WT and CRISPR/cas9 edited miR858CR lines grown on 1/2 strength Murashige and Skoog (MS) medium supplemented with water (control), 0.25 μM miPEP858a and 0.25 μM NSP, (n=30 independent seedlings, small open circles). The experiment was repeated three times independently with similar results. Statistical analysis was performed using two-tailed Student’s t-test. Data are plotted as means ±SD. Error bars represent standard deviation Asterisks indicate a significant difference, *P < 0.1, **P < 0.01, ***P < 0.001, ns represents no significant difference.

Extended Data Fig. 3 Exogenous application of miPEP858a complements function of the peptide in miPEPCR plants.

a, Representative image showing altered bolting in WT and CRISPR edited miPEP lines after spraying with water and miPEP858a. b, Change in bolting time of WT and CRISPR edited miPEP lines after spraying with water and 0.25 μM miPEP858a, n=10 independent plants (small open circles). c, Representative image showing change in plant height of WT and CRISPR edited miPEP lines after spraying with water and 0.25 μM miPEP858a. d, Change in plant height (cm) of WT and CRISPR edited miPEP lines after spraying with water and 0.25 μM miPEP858a, n=10 independent plants (small open circles). e, Representative images of transverse sections of stem of 35-day old WT and CRISPR edited miPEP plants sprayed with water and 0.25 μM miPEP858a, stained with phluoroglucinol showing changes in lignin content. f-g, Length of interfascicular fiber and vascular bundles of 35-day old stem of WT and CRISPR edited miPEP plants sprayed with water and 0.25 μM miPEP858a, n=10 (small open circles represent individual values). Statistical analysis was performed using two-tailed Student’s t-test. Data are plotted as means ±SD. Error bars represent standard deviation Asterisks indicate a significant difference, *P < 0.1, **P < 0.01, ***P < 0.001.

Extended Data Fig. 4 miPEP858a and miR858a regulate plant growth and development via auxin transport.

a, Representative image of 10-day old WT and CRISPR/Cas9 edited miR858aCR, miR858bCR, miR858abCR, pre-miR858aCR, miPEP858aCR, OX miPEP858a and OX miR858a seedling grown on 1/2 strength Murashige and Skoog (MS) medium supplemented with ethanol (control) and 25 μM TIBA (scale bars, 1 cm). b, c, Root length of 10-day old WT and CRISPR/Cas9 edited miR858aCR, miR858bCR, miR858abCR, pre-miR858aCR, miPEP858aCR, OX miPEP858a and OX miR858a seedling lines grown on 1/2 strength Murashige and Skoog (MS) medium supplemented with ethanol (control) and 25 μM TIBA (n=30 independent seedlings, small open circles). d, e, Quantification of AUX1, PIN1, ABCB19 and YUC1 in 30-day old rosette of WT and CRISPR/Cas9 edited miR858aCR, miR858bCR, miR858abCR, pre-miR858aCR, miPEP858aCR, OX miPEP858a and OX miR858a. Statistical analysis was performed using two-tailed Student’s t-test. Data are plotted as means ±SD. Error bars represent standard deviation Asterisks indicate a significant difference between control and treatment, *P < 0.1, **P < 0.01, ***P < 0.001.

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Sharma, A., Badola, P.K., Bhatia, C. et al. Primary transcript of miR858 encodes regulatory peptide and controls flavonoid biosynthesis and development in Arabidopsis. Nat. Plants 6, 1262–1274 (2020). https://doi.org/10.1038/s41477-020-00769-x

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