OsmiR528 Enhances Cold Stress Tolerance by Repressing Expression of Stress Response-related Transcription Factor Genes in Plant Cells

Author(s): Wei Tang*, Wells A. Thompson .

Journal Name: Current Genomics

Volume 20 , Issue 2 , 2019

Submit Manuscript
Submit Proposal

Graphical Abstract:


Background: MicroRNAs participate in many molecular mechanisms and signaling transduction pathways that are associated with plant stress tolerance by repressing expression of their target genes. However, how microRNAs enhance tolerance to low temperature stress in plant cells remains elusive.

Objective: In this investigation, we demonstrated that overexpression of the rice microRNA528 (OsmiR528) increases cell viability, growth rate, antioxidants content, ascorbate peroxidase (APOX) activity, and superoxide dismutase (SOD) activity and decreases ion leakage rate and thiobarbituric acid reactive substances (TBARS) under low temperature stress in Arabidopsis (Arabidopsis thaliana), pine (Pinus elliottii), and rice (Oryza sativa).

Methods: To investigate the potential mechanism of OsmiR528 in increasing cold stress tolerance, we examined expression of stress-associated MYB transcription factors OsGAMYB-like1, OsMYBS3, OsMYB4, OsMYB3R-2, OsMYB5, OsMYB59, OsMYB30, OsMYB1R, and OsMYB20 in rice cells by qRT-PCR.

Results: Our experiments demonstrated that OsmiR528 decreases expression of transcription factor OsMYB30 by targeting a F-box domain containing protein gene (Os06g06050), which is a positive regulator of OsMYB30. In OsmiR528 transgenic rice, reduced OsMYB30 expression results in increased expression of BMY genes OsBMY2, OsBMY6, and OsBMY10. The transcript levels of the OsBMY2, OsBMY6, and OsBMY10 were elevated by OsMYB30 knockdown, but decreased by Os- MYB30 overexpression in OsmiR528 transgenic cell lines, suggesting that OsmiR528 increases low temperature tolerance by modulating expression of stress response-related transcription factor.

Conclusion: Our experiments provide novel information in increasing our understanding in molecular mechanisms of microRNAs-associated low temperature tolerance and are valuable in plant molecular breeding from monocotyledonous, dicotyledonous, and gymnosperm plants.

Keywords: Cold stress, Gene expression, microRNAs, Molecular breeding, Pinus, Transcription factor.

Devi, S.J.; Madhav, M.S.; Kumar, G.R.; Goel, A.K.; Umakanth, B.; Jahnavi, B.; Viraktamath, B.C. Identification of abiotic stress miRNA Transcription Factor Binding Motifs (TFBMs) in rice. Gene, 2013, 531(1), 15-22.
Yang, Y.; Zhang, X.; Chen, Y.; Guo, J.; Ling, H.; Gao, S.; Su, Y.; Que, Y.; Xu, L. Selection of reference genes for normalization of microRNA expression by RT-qPCR in sugarcane buds under cold stress. Frontiers. Plant Sci., 2016, 7(1), 86.
Shu, Y.; Liu, Y.; Li, W.; Song, L.; Zhang, J.; Guo, C. Genomewide investigation of microRNAs and their targets in response to freezing stress in Medicago sativa L., based on high-throughput sequencing. G3 Bethseda, 2016, 6(1), 755-765.
Maeda, S.; Sakazono, S.; Masuko-Suzuki, H.; Taguchi, M.; Yamamura, K.; Nagano, K.; Endo, T.; Saeki, K.; Osaka, M.; Nabemoto, M.; Ito, K.; Kudo, T.; Kobayashi, M.; Kawagishi, M.; Fujita, K.; Nanjo, H.; Shindo, T.; Yano, K.; Suzuki, G.; Suwabe, K.; Watanabe, M. Comparative analysis of microRNA profiles of rice anthers between cool-sensitive and cool-tolerant cultivars under cool-temperature stress. Genes Genet. Syst., 2016, 91(1), 97-109.
Bredow, M.; Vanderbeld, B.; Walker, V.K. Knockdown of ice-binding proteins in Brachypodium distachyon demonstrates their role in freeze protection. PLoS One, 2016, 11, e0167941.
Kim, J.J.; Lee, J.H.; Kim, W.; Jung, H.S.; Huijser, P.; Ahn, J.H. The microRNA156-squamosa promoter binding protein-like3 module regulates ambient temperature-responsive flowering via flowering locus T in Arabidopsis. Plant Physiol., 2012, 159(1), 461-478.
Gebelin, V.; Argout, X.; Engchuan, W.; Pitollat, B.; Duan, C.; Montoro, P.; Leclercq, J. Identification of novel microRNAs in Hevea brasiliensis and computational prediction of their targets. BMC Plant Biol., 2012, 12(1), 18.
Kim, J.Y.; Kwak, K.J.; Jung, H.J.; Lee, H.J.; Kang, H. MicroRNA402 affects seed germination of Arabidopsis thaliana under stress conditions via targeting DEMETER-LIKE Protein3 mRNA. Plant Cell Physiol., 2010, 51(1), 1079-1083.
Kamthan, A.; Chaudhuri, A.; Kamthan, M.; Datta, A. Small RNAs in plants: recent development and application for crop improvement. Front. Plant Sci., 2015, 6(1), 208.
Chen, H.; Chen, X.; Chai, X.; Qiu, Y.; Gong, C.; Zhang, Z.; Wang, T.; Zhang, Y.; Li, J.; Wang, A. Effects of low temperature on mRNA and small RNA transcriptomes in Solanum lycopersicoides leaf revealed by RNA-Seq. Biochem. Biophys. Res. Commun., 2015, 464(3), 768-773.
Zeng, C.; Chen, Z.; Xia, J.; Zhang, K.; Chen, X.; Zhou, Y.; Bo, W.; Song, S.; Deng, D.; Guo, X.; Wang, B.; Zhou, J.; Peng, H.; Wang, W.; Peng, M.; Zhang, W. Chilling acclimation provides immunity to stress by altering regulatory networks and inducing genes with protective functions in cassava. BMC Plant Biol., 2014, 14(207)
Sun, X.; Fan, G.; Su, L.; Wang, W.; Liang, Z.; Li, S.; Xin, H. Identification of cold-inducible microRNAs in grapevine. Frontiers. Plant Sci., 2015, 6(1), 595.
Ma, C.; Burd, S.; Lers, A. miR408 is involved in abiotic stress responses in Arabidopsis. Plant J., 2015, 84(1), 169-187.
Sosa-Valencia, G.; Palomar, M.; Covarrubias, A.A. Reyes, J.L. The legume miR1514a modulates a NAC transcription factor transcript to trigger phasiRNA formation in response to drought. J. Exp. Bot., 2017, 68(8), 2013-2026.
Shen, X.; Guo, X.; Guo, X.; Zhao, D.; Zhao, W.; Chen, J.; Li, T. PacMYBA, a sweet cherry R2R3-MYB transcription factor, is a positive regulator of salt stress tolerance and pathogen resistance. Plant Physiol. Biochem., 2017, 112(1), 302-331.
Agarwal, M.; Hao, Y.; Kapoor, A.; Dong, C.H.; Fujii, H.; Zheng, X.; Zhu, J.K.A. R2R3 type MYB transcription factor is involved in the cold regulation of CBF genes and in acquired freezing tolerance. J. Biol. Chem., 2006, 281(49), 37636-37645.
Al-Attala, M.N.; Wang, X.; Abou-Attia, M.A.; Duan, X.; Kang, Z. A novel TaMYB4 transcription factor involved in the defence response against Puccinia striiformis f. sp. tritici and abiotic stresses. Plant Mol. Biol., 2014, 84(4-5), 589-603.
Bai, B.; Wu, J.; Sheng, W.T.; Zhou, B.; Zhou, L.J.; Zhuang, W.; Yao, D.P.; Deng, Q.Y. Comparative analysis of anther transcriptome profiles of two different rice male sterile lines genotypes under cold stress. Internat. J. Mol. Sci., 2015, 16(5), 11398-11416.
Bedon, F.; Bomal, C.; Caron, S.; Levasseur, C.; Boyle, B.; Mansfield, S.D.; A., Schmidt; J., Gershenzon; Grima-Pettenati, J.; A., Seguin; MacKay, J. Subgroup 4 R2R3-MYBs in conifer trees: gene family expansion and contribution to the isoprenoid- and flavonoid-oriented responses. J. Exp. Bot., 2010, 61(14), 3847-3864.
Bonthala, V.S.; Mayes, K.; Moreton, J.; Blythe, M.; Wright, V.; May, S.T.; Massawe, F.; Mayes, S.; Twycross, J. Identification of gene modules associated with low temperatures response in Bambara Groundnut by network-based analysis. PloS One, 2016, 11(2), e0148771.
Butelli, E.; Licciardello, C.; Zhang, Y.; Liu, J.; Mackay, S.; Bailey, P.; Reforgiato-Recupero, G.; Martin, C. Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. Plant Cell, 2012, 24(3), 1242-1255.
Butt, H.I.; Yang, Z.; Chen, E.; Zhao, G.; Gong, Q.; Yang, Z.; Zhang, X.; Li, F. Functional characterization of cotton GaMYB62L, a novel R2R3 TF in transgenic Arabidopsis. PloS One, 2017, 12(1), e0170578.
Davey, M.W.; Graham, N.S.; Vanholme, B.; Swennen, R.; May, S.T.; Keulemans, J. Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa. BMC Genom, 2009, 10(1), 436.
Hichri, I.; Barrieu, F.; Bogs, J.; Kappel, C.; Delrot, S.; Lauvergeat, V. Recent advances in the transcriptional regulation of the flavonoid biosynthetic pathway. J. Exp. Bot., 2011, 62(8), 2465-2483.
Imtiaz, M.; Yang, Y.; Liu, R.; Xu, Y.; Khan, M.A.; Wei, Q.; Gao, J.; Hong, B. Identification and functional characterization of the BBX24 promoter and gene from chrysanthemum in Arabidopsis. Plant Mol. Biol., 2015, 89(1), 1-19.
Kiferle, C.; Fantini, E.; Bassolino, L.; Povero, G.; Spelt, C.; Buti, S.; Giuliano, G.; Quattrocchio, F.; Koes, R.; Perata, P.; Gonzali, S. Tomato R2R3-MYB proteins SlANT1 and SlAN2: same protein activity, different roles. PloS One, 2015, 10(8), e0136365.
Lippold, F.; Sanchez, D.H.; Musialak, M.; Schlereth, A.; Scheible, W.R.; Hincha, D.K.; Udvardi, M.K. AtMyb41 regulates transcriptional and metabolic responses to osmotic stress in Arabidopsis. Plant Physiol., 2009, 149(4), 1761-1772.
Saha, G.; Park, J.I.; Ahmed, N.U.; Kayum, M.A.; Kang, K.K.; Nou, I.S. Characterization and expression profiling of MYB transcription factors against stresses and during male organ development in Chinese cabbage (Brassica rapa ssp. pekinensis). Plant Physiol. Biochem., 2016, 104(1), 200-215.
Tombuloglu, H.; Kekec, G.; Sakcali, M.S.; Unver, T. Transcriptome-wide identification of R2R3-MYB transcription factors in barley with their boron responsive expression analysis. Mol. Genet. Genom, 2013, 288(3-4), 141-155.
Chen, Y.; Chen, Z.; Kang, J.; Kang, D.; Gu, H.; Qin, G. AtMYB14 regulates cold tolerance in arabidopsis. Plant Mol. Biol. Rep., 2013, 31(1), 87-97.
Li, H.; Dong, Y.; Chang, J.; He, J.; Chen, H.; Liu, Q.; Wei, C.; Ma, J.; Zhang, Y.; Yang, J.; Zhang, X. High-throughput microRNA and mRNA sequencing reveals that microRNAs may be involved in melatonin-mediated cold tolerance in Citrullus lanatus L. Front. Plant Sci., 2016, 7(1), 1231.
Song, G.; Zhang, R.; Zhang, S.; Li, Y.; Gao, J.; Han, X.; Chen, M.; Wang, J.; Li, W.; Li, G. Response of microRNAs to cold treatment in the young spikes of common wheat. BMC Genom, 2017, 18(1), 212.
Yuan, S.; Li, Z.; Li, D.; Yuan, N.; Hu, Q.; Luo, H. Constitutive expression of rice MicroRNA528 alters plant development and enhances tolerance to salinity stress and nitrogen starvation in creeping bentgrass. Plant Physiol., 2015, 169(1), 576-593.
Chavez-Hernandez, E.C.; Alejandri-Ramirez, N.D.; Juarez-Gonzalez, V.T.; Dinkova, T.D. Maize miRNA and target regulation in response to hormone depletion and light exposure during somatic embryogenesis. Front. Plant Sci., 2015, 6(1), 555.
Cheah, B.H.; Nadarajah, K.; Divate, M.D.; Wickneswari, R. Identification of four functionally important microRNA families with contrasting differential expression profiles between drought-tolerant and susceptible rice leaf at vegetative stage. BMC Genom, 2015, 16(1), 692.
Ragupathy, R.; Ravichandran, S.; Mahdi, M.S.; Huang, D.; Reimer, E.; Domaratzki, M.; Cloutier, S. Deep sequencing of wheat sRNA transcriptome reveals distinct temporal expression pattern of miRNAs in response to heat, light and UV. Scientific. Rep., 2016, 6, 39373.
Thiebaut, F.; Rojas, C.A.; Grativol, C.; Motta, M.R.; Vieira, T.; Regulski, M.; Martienssen, R.A.; Farinelli, L.; Hemerly, A.S.; Ferreira, P.C. Genome-wide identification of microRNA and siRNA responsive to endophytic beneficial diazotrophic bacteria in maize. BMC Genomics, 2014, 15, 766.
Lv, Y.; Yang, M.; Hu, D.; Yang, Z.; Ma, S.; Li, X.; Xiong, L. The OsMYB30 transcription factor suppresses cold tolerance by interacting with a jaz protein and suppressing beta-amylase expression. Plant Physiol., 2017, 173(2), 1475-1491.
Tang, W.; Page, M. Transcription factor AtbZIP60 regulates expression of Ca2+ -dependent protein kinase genes in transgenic cells. Mol. Biol. Rep., 2013, 40(1), 2723-2732.
Shimono, M.; Sugano, S.; Nakayama, A.; Jiang, C.J.; Ono, K.; Toki, S.; Takatsuji, H. Rice WRKY45 plays a crucial role in benzothiadiazole-inducible blast resistance. Plant Cell, 2007, 19(6), 2064-2076.
Toki, S.; Hara, N.; Ono, K.; Onodera, H.; Tagiri, A.; Oka, S.; Tanaka, H. Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice. Plant J., 2006, 47(6), 969-976.
Tang, W.; Newton, R.J.; Weidner, D.A. Genetic transformation and gene silencing mediated by multiple copies of a transgene in eastern white pine. J. Exp. Bot., 2007, 58(3), 545-554.
Sung, Z.R. Turbidimetric measurement of plant cell culture growth. Plant Physiol., 1976, 57, 460-462.
Becana, M.; Aparicio-Tejo, P.; Irigoyen, J.J.; Sanchez-Diaz, M. Some enzymes of hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa. Plant Physiol., 1986, 82(4), 1169-1171.
Tang, W.; Newton, R.J. Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos. Plant Physiol. Biochem., 2005, 43(8), 760-769.
Tang, W.; Charles, T.M.; Newton, R.J. Overexpression of the pepper transcription factor CaPF1 in transgenic Virginia pine (Pinus Virginiana Mill.) confers multiple stress tolerance and enhances organ growth. Plant Mol. Biol., 2005, 59(4), 603-617.
Tang, W.; Newton, R.J.; Li, C.; Charles, T.M. Enhanced stress tolerance in transgenic pine expressing the pepper CaPF1 gene is associated with the polyamine biosynthesis. Plant Cell Rep., 2007, 26(1), 115-124.
Abe, H.; Yamaguchi-Shinozaki, K.; Urao, T.; Iwasaki, T.; Hosokawa, D.; Shinozaki, K. Role of arabidopsis MYC and MYB homologs in drought- and abscisic acid-regulated gene expression. Plant Cell, 1997, 9(10), 1859-1868.
Raffaele, S.; Vailleau, F.; Leger, A.; Joubes, J.; Miersch, O.; Huard, C.; Blee, E.; Mongrand, S.; Domergue, F.; Roby, D. A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis. Plant Cell, 2008, 20(3), 752-767.
Almeida, T.; Pinto, G.; Correia, B.; Santos, C.; Goncalves, S. QsMYB1 expression is modulated in response to heat and drought stresses and during plant recovery in Quercus suber. Plant Physiol. Biochem., 2013, 73(1), 274-281.
Bergonzi, S.; Albani, M.C.; Ver Loren Van Themaat, E.; Nordstrom, K.J.; Wang, R.; Schneeberger, K.; Moerland, P.D.; Coupland, G. Mechanisms of age-dependent response to winter temperature in perennial flowering of Arabis alpina. Science, 2013, 340(6136), 1094-1097.
Cheng, L.; Li, X.; Huang, X.; Ma, T.; Liang, Y.; Ma, X.; Peng, X.; Jia, J.; Chen, S.; Chen, Y.; Deng, B.; Liu, G. Overexpression of sheepgrass R1-MYB transcription factor LcMYB1 confers salt tolerance in transgenic Arabidopsis. Plant Physiol. Biochem., 2013, 70(1), 252-260.
Lee, H.G.; Seo, P.J. The MYB96-HHP module integrates cold and abscisic acid signaling to activate the CBF-COR pathway in Arabidopsis. Plant J., 2015, 82(6), 962-977.
He, Q.; Jones, D.C.; Li, W.; Xie, F.; Ma, J.; Sun, R.; Wang, Q.; Zhu, S.; Zhang, B. Genome-wide identification of R2R3-MYB genes and expression analyses during abiotic stress in Gossypium raimondii. Sci. Rep., 2016, 6(22980)
Huang, Q.X.; Cheng, X.Y.; Mao, Z.C.; Wang, Y.S.; Zhao, L.L.; Yan, X.; Ferris, V.R.; Xu, R.M.; Xie, B.Y. MicroRNA discovery and analysis of pinewood nematode Bursaphelenchus xylophilus by deep sequencing. PLoS One, 2010, 5(10), e13271.
Debat, H.J.; Grabiele, M.; Aguilera, P.M.; Bubillo, R.E.; Otegui, M.B.; Ducasse, D.A.; Zapata, P.D.; Marti, D.A. Exploring the genes of yerba mate (Ilex paraguariensis A. St.-Hil.) by NGS and de novo transcriptome assembly. PLoS One, 2014, 9(10), e109835.
Mathieu, J.; Yant, L.J.; Murdter, F.; Kuttner, F.; Schmid, M. Repression of flowering by the miR172 target SMZ. PLoS Biol., 2009, 7, e1000148.
Yang, C.; Li, D.; Mao, D.; Liu, X.; Ji, C.; Li, X.; Zhao, X.; Cheng, Z.; Chen, C.; Zhu, L. Overexpression of microRNA319 impacts leaf morphogenesis and leads to enhanced cold tolerance in rice (Oryza sativa L.). Plant Cell Environ., 2013, 36(12), 2207-2218.
Peng, X.; Wu, Q.; Teng, L.; Tang, F.; Pi, Z.; Shen, S. Transcriptional regulation of the paper mulberry under cold stress as revealed by a comprehensive analysis of transcription factors. BMC Plant Biol., 2015, 15, 108.
Yan, Y.; Shen, L.; Chen, Y.; Bao, S.; Thong, Z. Yu, H. A MYB-domain protein EFM mediates flowering responses to environmental cues in Arabidopsis. Dev. Cell, 2014, 30(4), 437-448.
Wang, R.K.; Cao, Z.H.; Hao, Y.J. Overexpression of a R2R3 MYB gene MdSIMYB1 increases tolerance to multiple stresses in transgenic tobacco and apples. Physiol. Plant., 2014, 150(1), 76-87.
Chiba, Y.; Mineta, K.; Hirai, M.Y.; Suzuki, Y.; Kanaya, S.; Takahashi, H.; Onouchi, H.; Yamaguchi, J.; Naito, S. Changes in mRNA stability associated with cold stress in Arabidopsis cells. Plant & Cell Physiol., 2013, 54(2), 180-194.
Yang, A.; Dai, X.; Zhang, W.H.A. R2R3-type MYB gene, OsMYB2, is involved in salt, cold, and dehydration tolerance in rice. J. Exp. Bot., 2012, 63(7), 2541-2556.
Samad, A.F.A.; Sajad, M.; Nazaruddin, N.; Fauzi, I.A.; Murad, A.M.A.; Zainal, Z.; Ismail, I. MicroRNA and transcription factor: key players in plant regulatory network. Front. Plant Sci., 2017, 8, 565.
Candar-Cakir, B.; Arican, E.; Zhang, B. Small RNA and degradome deep sequencing reveals drought-and tissue-specific micrornas and their important roles in drought-sensitive and drought-tolerant tomato genotypes. Plant Biotech. J., 2016, 14(8), 1727-1746.
Reyes, J.L.; Chua, N.H. ABA induction of miR159 controls transcript levels of two MYB factors during Arabidopsis seed germination. Plant J., 2007, 49(4), 592-606.
Din, M.; Barozai, M.Y. Profiling microRNAs and their targets in an important fleshy fruit: Tomato (Solanum lycopersicum). Gene, 2014, 535(2), 198-203.
Xie, F.; Wang, Q.; Sun, R.; Zhang, B. Deep sequencing reveals important roles of microRNAs in response to drought and salinity stress in cotton. J. Exp. Bot., 2015, 66(3), 789-804.
Wu, J.; Yang, Z.; Yang, S.; Yao, S.; Zhao, Y.; Wang, P.; Li, X.; Song, L.; Jin, T.; Zhou, Y.; Lan, L.; Xie, X.; Zhou, C.; Chu, Y.; Qi, X.; Cao, Y.; Li, Y. ROS accumulation and antiviral defence control by microRNA528 in rice. Nat. Plants, 2017, 3, 16203.
Sun, Q.; Liu, X.; Yang, J.; Liu, W.; Du, Q.; Wang, H.; Fu, C.; Li, W-X. MicroRNA528 affects lodging resistance of maize by regulating lignin biosynthesis under nitrogen-luxury conditions. Mol. Plant, 2018, 11(6), 806-814.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [100 - 114]
Pages: 15
DOI: 10.2174/1389202920666190129145439
Price: $58

Article Metrics

PDF: 26