Abstract
Somatic embryogenesis (SE) is the capacity of plant somatic cells to form embryos through a process resembling zygotic embryogenesis. This process is used as a model system to study zygotic embryo development. miRNAs play a crucial role in regulating the occurrence and development of plant SE. In our previous study, miR171 showed differential expression in the various developmental stages of SE in Lilium pumilum DC. Fisch. and Lilium davidii var. unicolor. However, little is known about the regulatory mechanism of miR171 and its target genes during Lilium SE. Here, the mature sequences of lpu-miR171a, lpu-miR171b, lda-miR171a and lda-miR171c were cloned using stem-loop RT-PCR. Furthermore, SCL6 transcription factors were verified to be the targets of miR171 using RLM-RACE. In addition, the full-length open reading frames for three target genes were cloned. The qRT-PCR results indicate that expression profiles differ among members of the miR171 family. lpu-miR171a expresses at high levels in the embryonic callus stage, while lda-miR171a, lda-miR171c and lpu-miR171b have the highest expression levels in the torpedo-shaped embryo stage. The expression levels of targets were negatively correlated with those of lpu-miR171a and lpu-miR171b; however, the levels of lda-miR171a and lda-miR171c were positively correlated with those of their target genes. The developmental stage-specific expression profiles of miR171 and SCL6 genes during SE implied that they have different regulatory functions during lily SE. According to the relationships between the expression files of miR171 and its target, the miR171 family functions in the regulation of SE, especially in the development of torpedo-shaped embryos.
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References
Bakhshaie M, Babalar M, Mirmasoumi M, Khalighi A (2010) Somatic embryogenesis and plant regeneration of Lilium ledebourii (Baker) Boiss., an endangered species. Plant Cell Tissue Organ Cult 102:229–235. doi:10.1007/s11240-010-9726-4
Bakhshaie M, Khosravi S, Azadi P, Bagheri H, van Tuyl JM (2016) Biotechnological advances in Lilium. Plant Cell Rep 35:1799–1826. doi:10.1007/s00299-016-2017-8
Bassuner BM, Lam R, Lukowitz W, Yeung EC (2007) Auxin and root initiation in somatic embryos of Arabidopsis. Plant Cell Rep 26:1–11. doi:10.1007/s00299-006-0207-5
Bi WL, Yin ZF, Guo L, Chen L, Pan C, Wang QC (2015) Plant regeneration from shoot regrowth and de novo embryo-like structures from cryopreserved shoot tips of Lilium spp. In vitro Cell Dev Biol 51:390–398. doi:10.1007/s11627-015-9696-7
Bolle C (2004) The role of GRAS proteins in plant signal transduction and development. Planta 218:683–692. doi:10.1007/s00425-004-1203-z
Bolle C, Koncz C, Chua NH (2000) PAT1, a new member of the GRAS family, is involved in phytochrome a signal transduction. Genes Dev 14:1269–1278
Chávez-Hernández EC, Alejandri-Ramírez ND, Juárez-González VT, Dinkova TD (2015) Maize miRNA and target regulation in response to hormone depletion and light exposure during somatic embryogenesis. Front Plant Sci 6:555. doi:10.3389/fpls.2015.00555
Curaba J, Talbot M, Li Z, Helliwell C (2013) Over-expression of microRNA171 affects phase transitions and floral meristem determinancy in barley. BMC Plant Biol 13:6. doi:10.1186/1471-2229-13-6
Di Laurenzio L, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann KA, Benfey PN (1996) The scarecrow gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 86:423–433. doi:10.1016/S0092-8674(00)80115-4
Elbashir SM, Martinez J, Patkaniowska A, Lendeckel W, Tuschl T (2001) Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate. EMBO J 20:6877–6888. doi:10.1093/emboj/20.23.6877
Fan T, Li X, Yang W, Xia K, Ouyang J, Zhang M (2015) Rice osa-miR171c mediates phase change from vegetative to reproductive development and shoot apical meristem maintenance by repressing four OsHAM transcription factors. PLoS ONE 10:e0125833. doi:10.1371/journal.pone.0125833
Guo R, Chen X, Lin Y, Xu X, Thu MK, Lai Z (2015) Identification of novel and conserved miRNAs in leaves of in vitro grown Citrus reticulata “lugan” plantlets by solexa sequencing. Front Plant Sci 6:1212. doi:10.3389/fpls.2015.01212
Haensch KT (1996) Plant regeneration through somatic embryogenesis in different genotypes of Lilium hybrids. Gartenbauwissenschaft 61:214–218
Ho CW, Jian WT, Lai HC (2006) Plant regeneration via somatic embryogenesis from suspension cell cultures of Lilium × formolongi hort. Using a bioreactor system. In Vitro Cell Dev Biol 42:240–246. doi:10.1079/IVP2006756
Hoshi Y, Kondo M, Mori S, Adachi Y, Nakano M, Kobayashi H (2004) Production of transgenic lily plants by Agrobacterium-mediated transformation. Plant Cell Rep 22:359–364. doi:10.1007/s00299-003-0700-z
Huang W, Xian Z, Kang X, Tang N, Li Z (2015) Genome-wide identification, phylogeny and expression analysis of GRAS gene family in tomato. BMC Plant Biol 15:209. doi:10.1186/s12870-015-0590-6
Hwang EW, Shin SJ, Yu BK, Byun MO, Kwon HB (2011) miR171 family members are involved in drought response in Solanum tuberosum. J Plant Biol 54:43–48. doi:10.1007/s12374-010-9141-8
Kasschau KD, Xie Z, Allen E, Llave C, Chapman EJ, Krizan KA, Carrington JC (2003) P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev Cell 4:205–217. doi:10.1016/S1534-5807(03)00025-X
Kedra M, Bach A (2005) Morphogenesis of Lilium martagon L. Explants in callus culture. Acta Biol Cracov 47:65–73
Komai F, Morohashi H, Horita M (2006) Application of nurse culture for plant regeneration from protoplasts of Lilium Japonicum Thunb. In Vitro Cell Dev Biol 42:252–255. doi:10.1079/IVP2006755
Li T, Chen J, Qiu S, Zhang Y, Wang P, Yang L, Lu Y, Shi J (2012) Deep sequencing and microarray hybridization identify conserved and species-specific microRNAs during somatic embryogenesis in hybrid yellow poplar. PLoS ONE 7:e43451. doi:10.1371/journal.pone.0043451
Li WF, Zhang SG, Han SY, Wu T, Zhang JH, Qi LW (2013) Regulation of LaMYB33 by miR159 during maintenance of embryogenic potential and somatic embryo maturation in Larix kaempferi (Lamb.) Carr. Plant Cell Tissue Organ Cult 113:131–136. doi:10.1007/s11240-012-0233-7
Li WF, Zhang SG, Han SY, Wu T, Zhang JH, Qi LW (2014) The post-transcriptional regulation of LaSCL6 by miR171 during maintenance of embryogenic potential in Larix kaempferi (Lamb.) Carr. Tree Genet Genomes 10:223–229. doi:10.1007/s11295-013-0668-y
Li ZX, Li SG, Zhang LF, Han SY, Li WF, Xu HY, Yang WH, Liu YL, Fan YR, Qi LW (2016) Over-expression of miR166a inhibits cotyledon formation in somatic embryos and promotes lateral root development in seedlings of Larix leptolepis. Plant Cell Tissue Organ Cult 127:461–473. doi:10.1007/s11240-016-1071-9
Li ZX, Zhang LF, Li WF, Qi LW, Han SY (2017) MIR166a affects the germination of somatic Embryos in Larixleptolepis by modulating IAA biosynthesis and signaling genes. J Plant Growth Regul 9:1–8. doi:10.1007/s00344-017-9693-7
Lin Y, Lai Z (2013) Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan. (Dimocarpus longan Lour.). PLoS ONE 8:e60337. doi:10.1371/journal.pone.0060337
Lin Y, Lai Z, Lin L, Lai R, Tian Q, Ye W, Zhang D, Yang M, Chen Y, Zhang Z (2015a) Endogenous target mimics, microRNA167, and its targets ARF6 and ARF8 during somatic embryo development in Dimocarpus longan Lour. Mol Breed 35:227. doi:10.1007/s11032-015-0420-4
Lin Y, Lai Z, Tian Q, Lin L, Lai R, Yang M, Zhang D, Chen Y, Zhang Z (2015b) Endogenous target mimics down-regulate miR160 mediation of ARF10, -16, and -17 cleavage during somatic embryogenesis in Dimocarpus longan Lour. Front Plant Sci 6:956. doi:10.3389/fpls.2015.00956
Llave C, Xie Z, Kasschau KD, Carrington JC (2002) Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297:2053–2056. doi:10.1126/science.1076311
Luo YC, Zhou H, Li Y, Chen JY, Yang JH, Chen YQ, Qu LH (2006) Rice embryogenic calli express a unique set of microRNAs, suggesting regulatory roles of microRNAs in plant post-embryogenic development. FEBS Lett 580:5111–5116. doi:10.1016/j.febslet.2006.08.046
Ma HS, Liang D, Shuai P, Xia XL, Yin WL (2010) The salt- and drought-inducible poplar GRAS protein SCL7 confers salt and drought tolerance in Arabidopsis thaliana. J Exp Bot 61:4011–4019. doi:10.1093/jxb/erq217
Mercuri A, de Benedetti L, Bruna S, Bregliano R, Bianchini C, Foglia G, Schiva T (2003) Agrobacterium-mediated transformation with rol genes of Lilium longiflorum Thunb. Acta Hortic 612:129–136. doi:10.17660/ActaHortic.2003.612.16
Moxon S, Jing R, Szittya G, Schwach F, Pilcher RL, Moulton V, Dalmay T (2008) Deep sequencing of tomato short RNAs identifies microRNAs targeting genes involved in fruit ripening. Genome Res 18:1602–1609. doi:10.1101/gr.080127.108
Nhut DT, Hanh NTM, Tuan PQ, Nguyet LTM, Tram NTH, Chinh NC, Nguyen NH, Vinh DN (2006) Liquid culture as a positive condition to induce and enhance quality and quantity of somatic embryogenesis of Lilium longiflorum. Sci Hortic 110:93–97. doi:10.1016/j.scienta.2006.05.015
Qiu Z-B, Yuan M-M, Hai B-Z, Wang L, Zhang L (2016) Characterization and expression analysis of conserved miRNAs and their targets in Pinus densata. Biol Plant 60:427–434. doi:10.1007/s10535-016-0617-3
Sha A, Chen Y, Ba H, Shan Z, Zhang X, Wu X, Qiu D, Chen S, Zhou X (2012) Identification of Glycine max microRNAs in response to phosphorus deficiency. J Plant Biol 55:268–280. doi:10.1007/s12374-011-0255-4
Siré C, Moreno AB, Garcia-Chapa M, López-Moya JJ, Segundo BS (2009) Diurnal oscillation in the accumulation of Arabidopsis microRNAs, miR167, miR168, miR171 and miR398. FEBS Lett 583:1039–1044. doi:10.1016/j.febslet.2009.02.024
Sternes PR, Moyle RL (2015) Deep sequencing reveals divergent expression patterns within the small RNA transcriptomes of cultured and vegetative tissues of sugarcane. Plant Mol Biol Rep 33:931–951. doi:10.1007/s11105-014-0787-0
Varkonyi-Gasic E, Gould N, Sandanayaka M, Sutherland P, MacDiarmid RM (2010) Characterisation of microRNAs from apple (Malus domestica ‘Royal Gala’) vascular tissue and phloem sap. BMC Plant Biol 10:159. doi:10.1186/1471-2229-10-159
Wan P, Wu J, Zhou Y, Xiao J, Feng J, Zhao W, Xiang S, Jiang G, Chen JY (2011) Computational analysis of drought stress-associated miRNAs and miRNA co-regulation network in Physcomitrella patens. Genomics Proteomics Bioinform 9:37–44. doi:10.1016/S1672-0229(11)60006-5
Wang L, Mai YX, Zhang YC, Luo Q, Yang HQ (2010) MicroRNA171c-targeted SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis. Mol Plant 3:794–806. doi:10.1093/mp/ssq042.
Wang L, Zhao J, Luo K, Cui J, He Q, Xia X, Lu Z, Li W, Jin B (2016) Deep sequencing discovery and profiling of conserved and novel miRNAs in the ovule of Ginkgo biloba. Trees 30:1557–1567. doi:10.1007/s00468-016-1389-2
Wu G, Park MY, Conway SR, Wang JW, Weigel D, Poethig RS (2009) The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell 138:750–759. doi:10.1016/j.cell.2009.06.031
Wu XM, Liu MY, Ge XX, Xu Q, Guo WW (2011) Stage and tissue-specific modulation of ten conserved miRNAs and their targets during somatic embryogenesis of Valencia sweet orange. Planta 233:495–505. doi:10.1007/s00425-010-1312-9
Wu XM, Kou SJ, Liu YL, Fang YN, Xu Q, Guo WW (2015) Genomewide analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA- and siRNA-mediated transcript cleavage involved in somatic embryogenesis. Plant Biotechnol J 13:383–394. doi:10.1111/pbi.12317
Xue LJ, Zhang JJ, Xue HW (2009) Characterization and expression profiles of miRNAs in rice seeds. Nucleic Acids Res 37:916–930. doi:10.1093/nar/gkn998
Yang X, Wang L, Yuan D, Lindsey K, Zhang X (2013) Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis. J Exp Bot 64:1521–1536. doi:10.1093/jxb/ert013
Zhai L, Xu L, Wang Y, Huang D, Yu R, Limera C, Gong Y, Liu L (2014) Genome-wide identification of embryogenesis-associated microRNAs in radish (Raphanus sativus L.) by high-throughput sequencing. Plant Mol Biol Rep 32:900–915. doi:10.1007/s11105-014-0700-x
Zhang B, Pan X, Cannon CH, Cobb GP, Anderson TA (2006) Conservation and divergence of plant microRNA genes. Plant J 46:243–259. doi:10.1111/j.1365-313X.2006.02697.x
Zhang JY, Xu Y, Huan Q, Chong K (2009) Deep sequencing of Brachypodium small RNAs at the global genome level identifies microRNAs involved in cold stress response. BMC Genomics 10:449. doi:10.1186/1471-2164-10-449
Zhang S, Zhou J, Han S, Yang W, Li W, Wei H, Li X, Qi L (2010) Four abiotic stress-induced miRNA families differentially regulated in the embryogenic and non-embryogenic callus tissues of Larix leptolepis. Biochem Biophys Res Commun 398:355–360. doi:10.1016/j.bbrc.2010.06.056
Zhang JH, Zhang SG, Han SY, Wu T, Li XM, Li W, Qi L (2012) Genome-wide identification of microRNAs in larch and stage-specific modulation of 11 conserved microRNAs and their targets during somatic embryogenesis. Planta 236:647–657. doi:10.1007/s00425-012-1643-9
Zhang JH, Zhang SG, Li SG, Han SY, Li WF, Li XM, Qi LW (2014) Regulation of synchronism by abscisic-acid-responsive small noncoding RNAs during somatic embryogenesis in larch (Larix leptolepis). Plant Cell Tissue Organ Cult 116:361–370. doi:10.1007/s11240-013-0412-1
Zhang J, Gai M, Li X, Li T, Sun H (2016a) Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant. Biosci Biotech Biochem 80:1898–1906. doi:10.1080/09168451.2016.1194178.
Zhang J, Gai M, Xue B, Jia N, Wang C, Wang J, Sun H (2016b) The use of miRNAs as reference genes for miRNA expression normalization during Lilium somatic embryogenesis by real-time reverse transcription PCR analysis. Plant Cell Tissue Organ Cult 129:105–118. doi:10.1007/s11240-016-1160-9
Zhang J, Xue BY, Gai MZ, Song SL, Jia NN, Sun HM (2017) Small RNA and transcriptome sequencing reveal a potential miRNA-mediated interaction network that functions during somatic embryogenesis in Lilium pumilum DC. Fisch. Front Plant Sci 8:566. doi:10.3389/fpls.2017.00566
Acknowledgements
This project was supported by the National Natural Science Foundation of China (Grant No. 31672179).
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JZ and HS conceived and designed the experiments. HL, JZ and NJ performed the experiments. HL, JZ, YY, and NJ analysed the data. CW and HS contributed reagents and materials. HL, JZ and HS wrote and revised the manuscript. All authors read and approved the final manuscript.
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HongYu Li and Jing Zhang have contributed equally to this work.
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Li, H., Zhang, J., Yang, Y. et al. miR171 and its target gene SCL6 contribute to embryogenic callus induction and torpedo-shaped embryo formation during somatic embryogenesis in two lily species. Plant Cell Tiss Organ Cult 130, 591–600 (2017). https://doi.org/10.1007/s11240-017-1249-9
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DOI: https://doi.org/10.1007/s11240-017-1249-9