Skip to main content
SpringerLink
Log in

Small RNA, transcriptome, and degradome sequencing to identify salinity stress responsive miRNAs and target genes in Dunaliella salina

  • Published:
Journal of Applied Phycology Aims and scope Submit manuscript

Abstract

Dunaliella salina is known as the most salinity-tolerant unicellular eukaryote. To explore its molecular response mechanisms to high salinity concentrations, D. salina transcriptomes, small RNA groups and degradomes were analyzed under salinity stress conditions, by high throughput sequencing. A total of 1008 microRNA (miRNA) sequences were identified, including 998 known conserved miRNAs and 10 novel miRNAs. Further analysis of miRNA expression in D. salina under salinity stress found that 49miRNAs showed significant differences in expression. For the first time in D. salina, 745 target genes, regulated by 194 miRNAs, were validated by degradome sequencing. Gene ontology (GO) enrichment analysis and KEGG analysis showed that these miRNA target genes are involved in a variety of molecular biological regulation processes, such as signal transduction, material transport, transcriptional regulation and protein processing. In combination with transcriptome sequencing results, 14 differentially expressed miRNAs and 87 differentially expressed target genes were found to negatively correlate in expression. Further analysis showed that mmu-miR-466, dme-miR-2493, mmu-mir-669h, dre-mir-29a and dme-mir-9388 play an important role in osmoregulation in response to high salinity stress in D. salina. These results enrich existing hypotheses, while providing new insights into the molecular mechanism of salinity tolerance in D. salina.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Addoquaye C, Miller W, Axtell MJ (2009) CleaveLand: a pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 25:130–131

    Article  CAS  Google Scholar 

  • Belmans D, Van Laere A (2010) Glycerol cycle enzymes and intermediates during adaption of Dunaliella teriolecta cells to hyperosmotic stress. Plant Cell Environ 10:185–190

    Google Scholar 

  • Bočvar DA (2008) Regulation of salt-induced starch degradation in Dunaliella tertiolecta. J Plant Physiol 127:77–96

    Google Scholar 

  • Borowitzka MA (2013) Dunaliella: biology, production, and markets. In: Richmond A, Hu Q (eds) Handbook of microalgal culture. John Wiley & Sons, Ltd, London, pp 359–368

    Chapter  Google Scholar 

  • Borowitzka MA (2018) The ‘stress’ concept in microalgal biology—homeostasis, acclimation and adaptation. J Appl Phycol. https://doi.org/10.1007/s10811-018-1399-0

  • Chen Z, Jiao XZ, Liu H (1991) Role of the plasma membrane H+-ATPase during the osmoregulation of the alga Dunaliella salina under hypertonic stress. Plant Physiol J 17:333–341

  • Chen H, Lao YM, Jiang JG (2011) Effects of salinities on the gene expression of a (NAD+)-dependent glycerol-3-phosphate dehydrogenase in Dunaliella salina. Sci Total Environ 409:1291–1297

    Article  CAS  PubMed  Google Scholar 

  • Degani H, Sussman I, Peschek GA, Avron M (1985) 13C- and 1H-NMR studies of osmoregulation in Dunaliella. Biochim Biophys Acta Mol Cell Res 846:313–323

    Article  CAS  Google Scholar 

  • Emmer E, Rood RP, Wesolek JH, Cohen ME, Braithwaite RS, Sharp GWG, Murer H, Donowitz M (1989) Role of calcium and calmodulin in the regulation of the rabbit ileal brush-border membrane Na+ /H+ antiporter. J Membr Biol 108:207–215

    Article  CAS  PubMed  Google Scholar 

  • Gao P, Bai X, Yang L, Lv D, Li Y, Cai H, Ji W, Guo D, Zhu Y (2010) Over-expression of Osa-MIR396c decreases salt and alkali stress tolerance. Planta 231:991–1001

    Article  CAS  PubMed  Google Scholar 

  • Gao P, Bai X, Yang L, Lv D, Pan X, Li Y, Cai H, Ji W, Chen Q, Zhu Y (2011) Osa-MIR393: a salinity- and alkaline stress-related microRNA gene. Mol Biol Rep 38:237–242

    Article  CAS  PubMed  Google Scholar 

  • Goyal A (2007a) Osmoregulation in Dunaliella, part I: effects of osmotic stress on photosynthesis, dark respiration and glycerol metabolism in Dunaliella tertiolecta and its salt-sensitive mutant (HL 25/8). Plant Physiol Biochem 45:696–704

    Article  CAS  PubMed  Google Scholar 

  • Goyal A (2007b) Osmoregulation in Dunaliella, part II: photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta. Plant Physiol Biochem 45:705–710

    Article  CAS  PubMed  Google Scholar 

  • Han X, Yin H, Song X, Zhang Y, Liu M, Sang J, Jiang J, Li J, Zhuo R (2016) Integration of small RNAs, degradome and transcriptome sequencing in hyperaccumulator Sedum alfredii uncovers a complex regulatory network and provides insights into cadmium phytoremediation. Plant Biotechnol J 14:1470–1483

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Harmon AC (2000) CDPKs-a kinase for every Ca2+ signal? Trends Plant Sci 5:154–159

    Article  CAS  PubMed  Google Scholar 

  • He Q, Qiao D, Bai L, Zhang Q, Yang W, Li Q, Cao Y (2007) Cloning and characterization of a plastidic glycerol 3-phosphate dehydrogenase cDNA from Dunaliella salina. J Plant Physiol 164:214–220

    Article  CAS  PubMed  Google Scholar 

  • Issa AA (1996) The role of calcium in the stress response of the halotolerant green alga Dunaliella bardawil Ben-Amotz et Avron. Phyton 36:295–302

    CAS  Google Scholar 

  • Jagadeeswaran G, Saini A, Sunkar R (2009) Biotic and abiotic stress down-regulate miR398 expression in Arabidopsis. Planta 229:1009–1014

    Article  CAS  PubMed  Google Scholar 

  • Jia Y, Xue L, Liu H, Li J (2009) Characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from the halotolerant alga Dunaliella salina and inhibition of its expression by RNAi. Curr Microbiol 58:426–431

    Article  CAS  PubMed  Google Scholar 

  • Jones AK, Sattelle DB (2008) The cys-loop ligand-gated ion channel gene superfamily of the nematode, Caenorhabditis elegans. Invertebr Neurosci 8:41–47

    Article  CAS  Google Scholar 

  • Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAS and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53

    Article  CAS  PubMed  Google Scholar 

  • Katz A, Pick U (2001) Plasma membrane electron transport coupled to Na+ extrusion in the halotolerant alga Dunaliella. Biochim Biophys Acta Bioenerg 1504:423–431

    Article  CAS  Google Scholar 

  • Katz A, Waridel P, Shevchenko A, Pick U (2007) Salt-induced changes in the plasma membrane proteome of the halotolerant alga Dunaliella salina as revealed by blue native gel electrophoresis and nano-LC-MS/MS analysis. Mol Cell Proteomics 6:1459–1472

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Meng X, Liu C, Yu L, Chen X (2006) Effects of osmotic stress on intracellular glycerol content and enzyme activity in Dunaliella salina. Chinese Bull Bot 23:145–151

    Google Scholar 

  • Li B, Qin Y, Hui D, Yin W, Xia X (2011) Genome-wide characterization of new and drought stress responsive microRNAs in Populus euphratica. J Exp Bot 62:3765–3779

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li SP, Dong HX, Yang G, Wu Y, Su SZ, Shan XH, Liu HK, Han JY, Liu JB, Yuan YP (2016) Identification of microRNAs involved in chilling response of maize by high-throughput sequencing. Biol Plant 60:251–260

    Article  CAS  Google Scholar 

  • Liska AJ, Shevchenko A, Pick U, Katz A (2004) Enhanced photosynthesis and redox energy production contribute to salinity tolerance in Dunaliella as revealed by homology-based proteomics. Plant Physiol 136:2806–2817

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Liu HH, Tian X, Li YJ, Wu CA, Zheng CC (2008) Microarray-based analysis of stress-regulated microRNAs in Arabidopsis thaliana. RNA 14:836–843

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Means AR (1994) Calcium, calmodulin and cell cycle regulation. FEBS Lett 347:1–4

    Article  CAS  PubMed  Google Scholar 

  • Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, Witman GB, Terry A, Salamov A, Fritz-Laylin LK, Maréchal-Drouard L (2007) The Chlamydomonas genome reveals the evolution of key animal and plant functions. Science 318:245–250

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mukherjee A (2015) Computational analysis of a cys-loop ligand gated ion channel from the green alga Chlamydomonas reinhardtii. Mol Biol 49:742–754

  • Pantaleo V, Szittya G, Moxon S, Miozzi L, Moulton V, Dalmay T, Burgyan J (2010) Identification of grapevine microRNAs and their targets using high-throughput sequencing and degradome analysis. Plant J 62:960–976

    CAS  PubMed  Google Scholar 

  • Park MY, Wu G, Gonzalez-Sulser A, Vaucheret H, Poethig RS (2005) Nuclear processing and export of microRNAs in Arabidopsis. Proc Natl Acad Sci U S A 102:3691–3696

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pick U, Avron M (1992) Modulation of Na+/H+ antiporter activity by extreme pH and salt in the halotolerant alga Dunaliella salina. Plant Physiol 100:1224–1229

    Article  PubMed  PubMed Central  Google Scholar 

  • Popova LG, Shumkova GA, Andreev IM, Balnokin YV (2005) Functional identification of electrogenic Na+-translocating ATPase in the plasma membrane of the halotolerant microalga Dunaliella maritima. FEBS Lett 57922:5002

    Article  CAS  Google Scholar 

  • Qin Z, Chen J, Jin L, Duns GJ, Ouyang P (2015) Differential expression of miRNAs under salt stress in Spartina alterniflora leaf tissues. J Nanosci Nanotechnol 15:1554–1561

    Article  CAS  PubMed  Google Scholar 

  • Saito H, Posas F (2012) Response to hyperosmotic stress. Genetics 192:289–318

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sun XF, Huang F, Liang X, Zhang FW, Yang WG, Bai LH, Qiao DR, Cao Y (2007) Expression of GPD gene from Dunaliella salina treated with different stress and glycerol synthesis of the cells. J Sichuan Univ 44:433–438

  • Sunkar R, Zhu JK (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Walne PR (1966) Experiments in the large scale culture of the larvae of Ostrea edulis. Fish Investig 2:1–53

    Google Scholar 

  • Wang Y, Li L, Tang S, Liu J, Zhang H, Zhi H, Jia G, Diao X (2016) Combined small RNA and degradome sequencing to identify miRNAs and their targets in response to drought in foxtail millet. BMC Genet 17:57

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Weiss M, Pick U (1990) Transient Na+ flux following hyperosmotic shock in the halotolerant alga Dunaliella salina : a response to intracellular pH changes. J Plant Physiol 136:429–438

    Article  CAS  Google Scholar 

  • Yu Y, Wu G, Yuan H, Cheng L, Zhao D, Huang W, Zhang S, Zhang L, Chen H, Zhang J (2016) Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses. BMC Plant Biol 16:124

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang XL (2013) Cloning and bioinformatic analysis of CDPK gene of Dunaliella salina. J Nuclear Agric Sci 27:418–424

    CAS  Google Scholar 

  • Zhao T, Li G, Mi S, Li S, Hannon GJ, Wang XJ, Qi Y (2007) A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii. Genes Dev 21:1190–1203

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhao B, Ge L, Liang R, Li W, Ruan K, Lin H, Jin Y (2009) Members of miR-169 family are induced by high salinity and transiently inhibit the NF-YA transcription factor. BMC Mol Biol 10:1–10

    Article  CAS  Google Scholar 

  • Zhao LN, Gong WF, Chen XW, Chen DF (2013) Characterization of genes and enzymes in Dunaliella salina involved in glycerol metabolism in response to salt changes. Phycol Res 61:37–45

    Article  CAS  Google Scholar 

  • Zhao R, Ng DHP, Fang L, Chow YYS, Yuan KL (2015) MAPK in Dunaliella tertiolecta regulates glycerol production in response to osmotic shock. Eur J Phycol 18:243–248

    Google Scholar 

Download references

Acknowledgments

This work was supported by the National Natural Science Foundation of China (No. 31472260). D. salina cells were provided by the Aquatic Biology Laboratory of Dalian Ocean University, Dalian, China.

Author information

Author notes

  1. Xiangnan Gao and Yuting Cong contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Hydrobiology in Liaoning Province, Dalian Ocean University, 52 Heishijiao Street, Dalian, 116023, Liaoning Province, China

    Xiangnan Gao, Yuting Cong, Jinrong Yue, Zhenyu Xing, Yuan Wang & Xiaojie Chai

Authors
  1. Xiangnan Gao
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yuting Cong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jinrong Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhenyu Xing
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Yuan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Xiaojie Chai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Xiaojie Chai.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gao, X., Cong, Y., Yue, J. et al. Small RNA, transcriptome, and degradome sequencing to identify salinity stress responsive miRNAs and target genes in Dunaliella salina. J Appl Phycol 31, 1175–1183 (2019). https://doi.org/10.1007/s10811-018-1612-1

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10811-018-1612-1

Keywords

Search

Navigation