MiR156 regulates anthocyanin biosynthesis through SPL targets and other microRNAs in poplar

Abstract

Anthocyanins biosynthesized from the flavonoid pathway are types of pigments that are involved in the protection of poplar from biotic and abiotic stresses. Previous researchers studying anthocyanin-related transcription factors and structural genes in poplar have made significant discoveries. However, little is known about the regulatory role of microRNAs in anthocyanin biosynthesis in poplar. Here, we overexpressed miR156 in poplar to study the comprehensive effects of the miR156-SPL module on the biosynthesis of anthocyanins. Small RNA sequencing analysis revealed 228 microRNAs differentially expressed in transgenic poplar plants with dramatically increased miR156 levels. Furthermore, integrated microRNAomic and transcriptomic analysis suggested that two microRNAs, miR160h, and miR858, have the potential to affect anthocyanin accumulation in poplar by regulating auxin response factors and MYB transcription factors, respectively. Additionally, the accumulation of miR160h and miR858 displayed a positive correlation with miR156 levels, suggesting a possible interaction between the miR156-SPL module and these microRNAs in poplar. Last, metabolomics analysis revealed that the levels of anthocyanins, flavones, and flavonols were substantially elevated in transgenic poplar plants overexpressing miR156 compared with the wild type, whereas the total lignin content was reduced in the transgenic plants. Taken together, our results indicate that miR156 can fine tune the anthocyanin biosynthetic pathway via multiple factors, including microRNAs, transcription factors, and the levels of structural genes, in poplar. This provides additional clues for understanding the complex regulatory network of anthocyanin biosynthesis in woody plants.

References

[1] Fraser, C. M. & Chapple, C. The phenylpropanoid pathway in Arabidopsis. Arabidopsis Book 9, e0152 (2011).

[2] Quideau, S. Flavonoids: chemistry, biochemistry and applications. Angew. Chem. Int. Ed. Engl. 45, 6786-6787 (2006).

[3] Chaves-Silva, S. et al. Understanding the genetic regulation of anthocyanin biosynthesis in plants-tools for breeding purple varieties of fruits and vegetables. Phytochemistry 153, 11-27 (2018).

[4] Xu, W. J., Dubos, C. & Lepiniec, L. Transcriptional control of flavonoid biosynthesis by MYB-bHLH-WDR complexes. Trends Plant Sci. 20, 176-185 (2015).

[5] Albert, N. W. et al. A conserved network of transcriptional activators and regulates anthocyanin pigmentation in eudicots. Plant Cell 26, 962-980 (2014).

[6] Qi, T. C. et al. The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accumulation and trichome initiation in Arabidopsis thaliana. Plant Cell 23, 1795-1814 (2011).

[7] Maier, A. et al. Light and the E3 ubiquitin ligase COP1/SPA control the protein stability of the MYB transcription factors PAP1 and PAP2 involved in anthocyanin accumulation in Arabidopsis. Plant J. 74, 638-651 (2013).

[8] Gou, J. Y. et al. Negative regulation of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor. Plant Cell 23, 1512-1522 (2011).

[9] Bartel, D. P. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116, 281-297 (2004).

[10] Song, X. W., Li, Y., Cao, X. F. & Qi, Y. J. MicroRNAs and their regulatory roles in plant-environment interactions. Annu. Rev. Plant Biol. 70, 489-525 (2019).

[11] Yang, F. X., Cai, J., Yang, Y. & Liu, Z. B. Overexpression of microRNA828 reduces anthocyanin accumulation in Arabidopsis. Plant Cell Tissue Organ Cult. 115, 159-167 (2013).

[12] Tirumalai, V. et al. miR828 and miR858 regulate VvMYB114 to promote anthocyanin and flavonol accumulation in grapes. J. Exp. Bot. 70, 4775-4792 (2019).

[13] He, L. H. et al. Uncovering anthocyanin biosynthesis related microRNAs and their target genes by small RNA and degradome sequencing in tuberous roots of sweetpotato. BMC Plant Biol. 19, 232 (2019).

[14] Wu, G. et al. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 13, 750-759 (2009).

[15] Gao, R. M., Austin, R. S., Amyot, L. & Hannoufa, A. Comparative transcriptome investigation of global gene expression changes caused by miR156 overexpression in Medicago sativa. BMC Genomics 17, 658 (2016).

[16] Xie, K. B. et al. Gradual increase of miR156 regulates temporal expression changes of numerous genes during leaf development in rice. Plant Physiol. 158, 1382-1394 (2012).

[17] Fu, C. X. et al. Overexpression of miR156 in switchgrass (Panicum virgatum L.) results in various morphological alterations and leads to improved biomass production. Plant Biotechnol. J. 10, 443-452 (2012).

[18] Wu, Z. Y. et al. Switchgrass SBP-box transcription factors PvSPL1 and 2 function redundantly to initiate side tillers and affect biomass yield of energy crop. Biotechnol. Biofuels 9, 101 (2016).

[19] Taylor, G. et al. Sustainable bioenergy for climate mitigation: developing drought-tolerant trees and grasses. Ann. Bot. 124, 513-520 (2019).

[20] Chen, S. B., Songkumarn, P., Liu, J. L. & Wang, G. L. A versatile zero background T-vector system for gene cloning and functional genomics. Plant Physiol. 150, 1111-1121 (2009).

[21] Chen, H., Nelson, R. S. & Sherwood, J. L. Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16, 8664-8670 (1994).

[22] Jia, Z. C. et al. Enhanced resistance to fungal pathogens in transgenic Populus tomentosa Carr. by overexpression of an nsLTP-like antimicrobial protein gene from motherwort (Leonurus japonicus). Tree Physiol. 30, 1599-1605 (2010).

[23] Doyle, J. J. & Doyle, J. L. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytoch. Bull 19, 11-15 (1987).

[24] Varkonyi-Gasic, E., Wu, R. M., Wood, M., Walton, E. F. & Hellens, R. P. Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs. Plant Methods 3, 12 (2007).

[25] Allen, E., Xie, Z. X., Gustafson, A. M. & Carrington, J. C. MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207-221 (2005).

[26] Schwab, R. et al. Specific effects of microRNAs on the plant transcriptome. Dev. Cell 8, 517-527 (2005).

[27] Mao, X. Z., Cai, T., Olyarchuk, J. G. & Wei, L. P. Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary. Bioinformatics 21, 3787-3793 (2005).

[28] Chen, W. et al. A novel integrated method for large-scale detection, identification, and quantification of widely targeted metabolites: application in the study of rice metabolomics. Mol. Plant 6, 1769-1780 (2013).

[29] Fraga, C. G. et al. Signature-discovery approach for sample matching of a nerve-agent precursor using liquid chromatography-mass spectrometry, XCMS, and chemometrics. Anal. Chem. 82, 4165-4173 (2010).

[30] Mita, S., Murano, N., Akaike, M. & Nakamura, K. Mutants of Arabidopsis thaliana with pleiotropic effects on the expression of the gene for β-amylase and on the accumulation of anthocyanin that are inducible by sugars. Plant J. 11, 841-851 (1997).

[31] Chen, F. & Dixon, R. A. Lignin modification improves fermentable sugar yields for biofuel production. Nat. Biotechnol. 25, 759-761 (2007).

[32] Hatfield, R. D., Grabber, J., Ralph, J. & Brei, K. Using the acetyl bromide assay to determine lignin concentrations in herbaceous plants: some cautionary notes. J. Agric. Food Chem. 47, 628-632 (1999).

[33] Jiang, Y. J., Liang, G., Yang, S. Z. & Yu, D. Q. Arabidopsis WRKY57 functions as a node of convergence for jasmonic acid and auxin-mediated signaling in jasmonic acid-induced leaf senescence. Plant Cell 26, 230-245 (2014).

[34] Wang, L. et al. miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis. J. Exp. Bot. 62, 761-773 (2011).

[35] Tang, M. Y. et al. miR172 regulates both vegetative and reproductive development in the perennial woody plant Jatropha curcas. Plant Cell Physiol. 59, 2549-2563 (2018).

[36] Wang, Y. C. et al. Auxin regulates anthocyanin biosynthesis through the Aux/IAA-ARF signaling pathway in apple. Hortic. Res. 5, 59 (2018).

[37] Wang, Y. L., Wang, Y. Q., Song, Z. & Zhang, H. Repression on of MYBL2 by both microRNA858a and HY5 leads to the activation of anthocyanin biosynthetic pathway in Arabidopsis. Mol. Plant 9, 1395-1405 (2016).