Abstract
Dunaliella salina is a classic halophilic alga. However, its molecular mechanisms in response to high salinity at the post transcriptional level remain unknown. A unique halophilic alga strain, DS-CN1, was screened from four D. salina strains via cell biological, physiological, and biochemical methods. High-throughput sequencing of small RNAs (sRNAs) of DS-CN1 in culture medium containing 3.42-mol/L NaCl (SS group) or 0.05-mol/L NaCl (CO group) was performed on the BGISEQ-500 platform. The annotation and sequences of D. salina sRNAs were profiled. Altogether, 44 novel salt stress-responsive microRNAs (miRNAs) with a relatively high C content, with the majority of them being 24 nt in length, were identified and characterized in DS-CN1. Twenty-one differentially expressed miRNAs (DEMs) in SS and CO were screened via bioinformatic analysis. A total of 319 putative salt stress-related genes targeted (104 overlapping genes) by novel miRNAs in this alga were screened based on our previous transcriptome sequencing research. Furthermore, these target genes were classified and enriched by GO and KEGG pathway analysis. Moreover, 5 novel DEMs (dsa-mir3, dsa-mir16, dsa-mir17, and dsa-mir26 were significantly upregulated, and dsa-mir40 was significantly downregulated) and their corresponding 10 target genes involved in the 6 significantly enriched metabolic pathways were verified by quantitative real-time PCR. Next, their regulatory relationships were comprehensively analyzed. Lastly, a unique salt stress response metabolic network was constructed based on the novel DEM-target gene pairs. Taken together, our results suggest that 44 novel salt stress-responsive microRNAs were identified, and 4 of them might play important roles in D. salina upon salinity stress and contribute to clarify its distinctive halophilic feature. Our study will shed light on the regulatory mechanisms of salt stress responses.
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Data Availability Statement
The raw sRNA-seq datasets generated in this study are available from the NCBI SRA database under the accession numbers SRR8389145, SRR8389147, SRR8389148, SRR8389149, SRR8389150, and SRR8389153 and the CNCB GSA database under the accession numbers CRA006740, CRA006741, CRA006742, CRA006743, CRA006744, and CRA006745. The authors declare that all the other data supporting the findings of this study are available within the article and its supplementary files.
References
Alzahrani S M, Alaraidh I A, Khan M A et al. 2019. Identification and characterization of salt-responsive microRNAs in Vicia faba by high-throughput sequencing. Genes, 10(4): 303, https://doi.org/10.3390/genes10040303.
Ametaj B N, Bobe G, Lu Y et al. 2003. Effect of sample preparation, length of time, and sample size on quantification of total lipids from bovine liver. Journal of Agricultural and Food Chemistry, 51(8): 2105–2110, https://doi.org/10.1021/jf0259011.
Lokhande H A. 2023. Bioinformatics analysis of miRNA sequencing data. Methods in Molecular Biology, 2595: 225–237. https://doi.org/10.1007/978-1-0716-2823-2_16
Axtell M J. 2013. Classification and comparison of small RNAs from plants. Annual Review of Plant Biology, 64: 137–159, https://doi.org/10.1146/annurev-arplant-050312-120043.
Axtell M J, Meyers B C. 2018. Revisiting criteria for plant microRNA annotation in the era of big data. The Plant Cell, 30(2): 272–284, https://doi.org/10.1105/tpc.17.00851.
Barozai M Y K, Qasim M, Din M et al. 2018. An update on the microRNAs and their targets in unicellular red alga Porphyridium cruentum. Pakistan Journal of Botany, 50(2): 817–825.
Billoud B, Nehr Z, Le Bail A et al. 2014. Computational prediction and experimental validation of microRNAs in the brown alga Ectocarpus siliculosus. Nucleic Acids Research, 42(1): 417–429, https://doi.org/10.1093/nar/gkt856.
Chen Y X, Bi C B, Zhang J et al. 2020. Astaxanthin biosynthesis in transgenic Dunaliella salina (Chlorophyceae) enhanced tolerance to high irradiation stress. South African Journal of Botany, 133: 132–138, https://doi.org/10.1016/j.sajb.2020.07.008.
Chen Y X, Chen Y S, Shi C M et al. 2018. SOAPnuke: a MapReduce acceleration-supported software for integrated quality control and preprocessing of high-throughput sequencing data. Gigascience, 7(1): gix120, https://doi.org/10.1093/gigascience/gix120.
Duché O, Trémoulet F, Namane A et al. 2002. A proteomic analysis of the salt stress response of Listeria monocytogenes. FEMS Microbiology Letters, 215(2): 183–188, https://doi.org/10.1111/j.1574-6968.2002.tb11389.x.
Evers M, Huttner M, Dueck A et al. 2015. miRA: adaptable novel miRNA identification in plants using small RNA sequencing data. BMC Bioinformatics, 16: 370, https://doi.org/10.1186/s12859-015-0798-3.
Feng B H, Li G Y, Islam M et al. 2020a. Strengthened antioxidant capacity improves photosynthesis by regulating stomatal aperture and ribulose-1,5-bisphosphate carboxylase/oxygenase activity. Plant Science, 290: 110245, https://doi.org/10.1016/j.plantsci.2019.110245.
Feng K W, Nie X J, Cui L C et al. 2017. Genome-wide identification and characterization of salinity stress-responsive miRNAs in wild emmer wheat (Triticum turgidum ssp. dicoccoides). Genes (Basel), 8(6): 156, https://doi.org/10.3390/genes8060156.
Feng S Y, Hu L, Zhang Q H et al. 2020b. CRISPR/Cas technology promotes the various application of Dunaliella salina system. Applied Microbiology and Biotechnology, 104(20): 8621–8630, https://doi.org/10.1007/s00253-020-10892-6.
Folch J, Lees M, Stanley G H S. 1957. A simple method for the isolation and purification of total lipides from animal tissues. Journal of Biological Chemistry, 226(1): 497–509, https://doi.org/10.1016/S0021-9258(18)64849-5.
Frukh A, Siddiqi T O, Khan M I R et al. 2020. Modulation in growth, biochemical attributes and proteome profile of rice cultivars under salt stress. Plant Physiology and Biochemistry, 146: 55–70, https://doi.org/10.1016/j.plaphy.2019.11.011.
Gao F, Nan F R, Feng J et al. 2021. Transcriptome profile of Dunaliella salina in Yuncheng Salt Lake reveals salt-stress-related genes under different salinity stresses. Journal of Oceanology and Limnology, 39(6): 2336–2362, https://doi.org/10.1007/s00343-021-0164-4.
Gao F, Nan F R, Feng J et al. 2022. Comparative morphological, physiological, biochemical and genomic studies reveal novel genes of Dunaliella bioculata and D. quartolecta in response to salt stress. Molecular Biology Reports, 49(3): 1749–1761, https://doi.org/10.1007/s11033-021-06984-9.
Gao X, Zhang F G, Hu J L et al. 2016. MicroRNAs modulate adaption to multiple abiotic stresses in Chlamydomonas reinhardtii. Scientific Reports, 6(1): 38228, https://doi.org/10.1038/srep38228.
Garcia-Seco D, Zhang Y, Gutierrez-Mañero F J et al. 2015. RNA-Seq analysis and transcriptome assembly for blackberry (Rubus sp. var. Lochness) fruit. BMC Genomics, 16(1): 5, https://doi.org/10.1186/s12864-014-1198-1.
Jin H C, Sun Y, Yang Q C et al. 2010. Screening of genes induced by salt stress from Alfalfa. Molecular Biology Reports, 37(2): 745–753, https://doi.org/10.1007/s11033-009-9590-7.
Joshi G A N, Chauhan C, Das S. 2021. Sequence and functional analysis of MIR319 promoter homologs from Brassica juncea reveals regulatory diversification and altered expression under stress. Molecular Genetics and Genomics, 296(3): 731–749, https://doi.org/10.1007/s00438-021-01778-x.
Kalvari I, Argasinska J, Quinones-Olvera N et al. 2018. Rfam 13.0: shifting to a genome-centric resource for non-coding RNA families. Nucleic Acids Research, 46(D1): D335–D342, https://doi.org/10.1093/nar/gkx1038.
Kanesaki Y, Suzuki I, Allakhverdiev S I et al. 2002. Salt stress and hyperosmotic stress regulate the expression of different sets of genes in Synechocystis sp. PCC 6803. Biochemical and Biophysical Research Communications, 290(1): 339–348, https://doi.org/10.1006/bbrc.2001.6201.
Langdon W B. 2015. Performance of genetic programming optimised Bowtie2 on genome comparison and analytic testing (GCAT) benchmarks. BioData Mining, 8(1): 1, https://doi.org/10.1186/s13040-014-0034-0.
Lin Y S, Wang B, Wang N H et al. 2019. Transcriptome analysis of rare minnow (Gobiocypris rarus) infected by the grass carp reovirus. Fish & Shellfish Immunology, 89: 337–344, https://doi.org/10.1016/j.fsi.2019.04.013.
Liu Y, Lv J P, Feng J et al. 2019. Treatment of real aquaculture wastewater from a fishery utilizing phytoremediation with microalgae. Journal of Chemical Technology & Biotechnology, 94(3): 900–910, https://doi.org/10.1002/jctb.5837.
Love M I, Huber W, Anders S. 2014. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12): 550, https://doi.org/10.1186/s13059-014-0550-8.
Ma K, Bao Q W, Wu Y et al. 2020. Evaluation of microalgae as immunostimulants and recombinant vaccines for diseases prevention and control in aquaculture. Frontiers in Bioengineering and Biotechnology, 8: 590431, https://doi.org/10.3389/fbioe.2020.590431.
Mera R, Torres E, Abalde J. 2016. Effects of sodium sulfate on the freshwater microalga Chlamydomonas moewusii: implications for the optimization of algal culture media. Journal of Phycology, 52(1): 75–88, https://doi.org/10.1111/jpy.12367.
Mishra A, Jha B. 2009. Isolation and characterization of extracellular polymeric substances from micro-algae Dunaliella salina under salt stress. Bioresource Technology, 100(13): 3382–3386, https://doi.org/10.1016/j.biortech.2009.02.006.
Moayedi A, Yargholi B, Pazira E et al. 2019. Investigated of desalination of saline waters by using Dunaliella salina algae and its effect on water ions. Civil Engineering Journal, 5(11): 2450–2460, https://doi.org/10.28991/cej-2019-03091423.
Naik H K, Varadahalli R D. 2020. Genomic identification of salt induced microRNAs in Niger (Guizotia abyssinica Cass.). Plant Gene, 23: 100242, https://doi.org/10.1016/j.plgene.2020.100242.
Omidbakhshfard M A, Omranian N, Ahmadi F S et al. 2012. Effect of salt stress on genes encoding translation-associated proteins in Arabidopsis thaliana. Plant Signaling & Behavior, 7(9): 1095–1102, https://doi.org/10.4161/psb.21218.
Panahi B, Frahadian M, Dums J T et al. 2019. Integration of cross species RNA-Seq meta-analysis and machine-learning models identifies the most important salt stress—responsive pathways in microalga Dunaliella. Frontiers in Genetics, 10: 752, https://doi.org/10.3389/fgene.2019.00752.
Pandit A, Rai V, Sharma T R et al. 2011. Differentially expressed genes in sensitive and tolerant rice varieties in response to salt-stress. Journal of Plant Biochemistry and Biotechnology, 20(2): 149–154, https://doi.org/10.1007/s13562-010-0022-5.
Rammuni M N, Ariyadasa T U, Nimarshana P H V et al. 2019. Comparative assessment on the extraction of carotenoids from microalgal sources: astaxanthin from H. pluvialis and β-carotene from D. salina. Food Chemistry, 277: 128–134, https://doi.org/10.1016/jfoodchem.2018.10.066.
Rasouli M, Ostovar-Ravari A, Shokri-Afra H. 2014. Characterization and improvement of phenol-sulfuric acid microassay for glucose-based glycogen. European Review for Medical and Pharmacological Sciences, 18(14): 2020–2024.
Saeed A I, Sharov V, White J et al. 2003. TM4: a free, open-source system for microarray data management and analysis. Biotechniques, 34(2): 374–378, https://doi.org/10.2144/03342mt01.
Sewe S O, Silva G, Sicat P et al. 2022. Trimming and validation of Illumina short reads using Trimmomatic, Trinity assembly, and assessment of RNA-Seq data. In: Edwards D ed. Plant Bioinformatics. Humana, New York. p.211–232, https://doi.org/10.1007/978-1-0716-2067-0_11.
Sun X M, Ren L J, Zhao Q Y et al. 2018. Microalgae for the production of lipid and carotenoids: a review with focus on stress regulation and adaptation. Biotechnology for Biofuels, 11(1): 272, https://doi.org/10.1186/s13068-018-1275-9.
Tafreshi A H, Shariati M. 2009. Dunaliella biotechnology: methods and applications. Journal of Applied Microbiology, 107(1): 14–35, https://doi.org/10.1111/j.1365-2672.2009.04153.x.
Tammam A A, Fakhry E M, El-Sheekh M. 2011. Effect of salt stress on antioxidant system and the metabolism of the reactive oxygen species in Dunaliella salina and Dunaliella tertiolecta. African Journal of Biotechnology, 10(19): 3795–3808.
Tian Y H, Tian Y M, Luo X J et al. 2014. Identification and characterization of microRNAs related to salt stress in broccoli, using high-throughput sequencing and bioinformatics analysis. BMC Plant Biology, 14: 226, https://doi.org/10.1186/s12870-014-0226-2.
Wu Y, Guo J, Cai Y M et al. 2016. Genome-wide identification and characterization of Eutrema salsugineum microRNAs for salt tolerance. Physiologia Plantarum, 157(4): 453–468, https://doi.org/10.1111/ppl.12419.
Xu L, Gao F, Feng J et al. 2022. Relationship between β-carotene accumulation and geranylgeranyl pyrophosphate synthase in different species of Dunaliella. Plants, 11: 27, https://doi.org/10.3390/plants11010027.
Yang H J, Hu C X. 2020. Regulation and remodeling of intermediate metabolite and membrane lipid during NaCl-induced stress in freshwater microalga Micractinium sp. XJ-2 for biofuel production. Biotechnology and Bioengineering, 117(12): 3727–3738, https://doi.org/10.1002/bit.27528.
Yang Y Q, Guo Y. 2018. Elucidating the molecular mechanisms mediating plant salt-stress responses. New Phytologist, 217(2): 523–539, https://doi.org/10.1111/nph.14920.
Yin Z J, Li Y, Zhu W D et al. 2018. Identification, characterization, and expression patterns of TCP genes and microRNA319 in cotton. International Journal of Molecular Sciences, 19(11): 3655, https://doi.org/10.3390/ijms19113655.
Yu Y, Wu G W, Yuan H M et al. 2016. Identification and characterization of miRNAs and targets in flax (Linum usitatissimum) under saline, alkaline, and saline-alkaline stresses. BMC Plant Biology, 16(1): 124, https://doi.org/10.1186/s12870-016-0808-2.
Zavřel T, Očenášová P, Sinetova M et al. 2018. Determination of storage (starch/glycogen) and total saccharides content in algae and cyanobacteria by a phenol-sulfuric acid method. Bio-Protocol, 8(15): e2966, https://doi.org/10.21769/BioProtoc.2966.
Zhang X Z, Tan G R, Huang Y J et al. 1989. Plant Physiological Experimental Technique. Liaoning Science and Technology Press, Shenyang. (in Chinese)
Zhu F Y, Chen M X, Ye N H et al. 2018. Comparative performance of the BGISEQ-500 and Illumina HiSeq4000 sequencing platforms for transcriptome analysis in plants. Plant Methods, 14: 69, https://doi.org/10.1186/s13007-018-0337-0.
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Fan GAO performed the experiments, analyzed the data, and co-wrote the manuscript; Fangru NAN analyzed the data and co-wrote the manuscript; Jia FENG, Junping LÜ, Qi LIU, and Xudong LIU analyzed the data; and Shulian XIE supervised the project and revised the manuscript. All the authors read and approved the final manuscript.
All the samples were obtained from the well-known algae culture and collection, and the sampling procedures in this experiment were performed in accordance with the guidelines for the care and use of laboratory plants of Shanxi University and were approved by the Wildlife Care and Use Committee of Taiyuan Forestry Bureau, Shanxi Province, China.
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Supported by the National Natural Science Foundation of China (No. 32170204), Science and Technology Strategy Research Special Project of Shanxi Province of China (No. 202204031401051), the Basic Research Programs of Shanxi Province of China (No. 202103021224009), the Teaching Reform and Innovation Project of Colleges and Universities in Shanxi of China (No. J20220046), and the Shanxi “1331 Project”. The corresponding author, Shulian XIE, was supported by the National Natural Science Foundation of China and the Shanxi “1331 Project”. The first author, Fan GAO, was supported by the Science and Technology Strategy Research Special Project of Shanxi Province of China, the Teaching Reform and Innovation Project of College and Universities in Shanxi of China, and the Basic Research Programs of Shanxi Province of China
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Gao, F., Nan, F., Feng, J. et al. Identification of novel salt stress-responsive microRNAs through sequencing and bioinformatic analysis in a unique halophilic Dunaliella salina strain. J. Ocean. Limnol. 41, 1558–1574 (2023). https://doi.org/10.1007/s00343-022-2130-1
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DOI: https://doi.org/10.1007/s00343-022-2130-1