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Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants

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Abstract

Resurrection plant Ramonda serbica is a suitable model to investigate mechanisms of desiccation tolerance, while variegated Pelargonium zonale has been proven to serve as an excellent model for the metabolite allocation between sink tissue and source tissue within the same organ. However, the genomes of these plants are still not sequenced, limiting their application in molecular studies. To investigate the transcript abundance by next-generation sequencing, high-quality RNA input is required. Leaves of both P. zonale and R. serbica are rich in polyphenols that interfere with high-quality RNA extraction by common protocols. Moreover, low water content and high amount of sugars and other osmoprotectants in desiccated R. serbica leaves present the additional challenge in total RNA extraction. Here, we evaluated and compared several already established TRIzol- and CTAB-based protocols aiming to develop the efficient, simple and low-cost methods for the extraction of the satisfactory yield RNA of great purity and integrity, required for the construction of high-quality cDNA libraries. Our results show that the CTAB-based protocol (i.e. CTAB 1b) enabled the extraction of high-quality RNA from photosynthetically active and non-photosynthetically active leaf sectors of P. zonale, with high RIN values. On the other hand, TRIzol-based protocol provided a high RNA yield with low contamination and high RNA integrity even in desiccated leaves of R. serbica. We envisage that the proposed protocol would be suitable for the RNA extractions from other desiccated organs (e.g. seeds, grains, pollen grains).

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Abbreviations

βME:

β-Mercaptoethanol

CIA:

Chloroform-isoamyl alcohol

CTAB:

Cethyl-trimethylammoniumbromide

DEPC:

Diethylpyrocarbonate

EDTA:

Ethylenediaminetetraacetic acid

NGS:

Next-generation sequencing

PVP:

Water-soluble polyvinylpyrrolidone

RT:

Room temperature

RWC:

Relative water content

PCR:

Polymerase chain reaction

References

  • Abadie C, Lamothe M, Mauve C, Gilard F, Tcherkez G (2015) Leaf green-white variegation is advantageous under N deprivation in Pelargonium × hortorum. Funct Plant Biol 42:543–551

    Article  CAS  Google Scholar 

  • Bieri P, Leibundgut M, Saurer M, Boehringer D, Ban N (2017) The complete structure of the chloroplast 70S ribosome in complex with translation factor pY. EMBO J 36:475–486

    Article  CAS  Google Scholar 

  • Bogdanović MD, Todorović SI, Banjanac T, Dragićević MB, Verstappen FW, Bouwmeester HJ, Simonović AD (2014) Production of guaianolides in Agrobacterium rhizogenes-transformed chicory regenerants flowering in vitro. Ind Crop Prod 60:52–59

    Article  Google Scholar 

  • Brescia P, Banks P (2009) Multi-volume analysis of nucleic acids using the Epoch/Take3 spectrophotometer system. BioTek Instruments, Inc, Winooski, Application note

  • Chen X, Zhang M, Tan J, Huang S, Wang C, Zhang H, Tan T (2017) Comparative transcriptome analysis provides insights into molecular mechanisms for parthenocarpic fruit development in eggplant (Solanum melongena L.). PloS One 12:6

    CAS  Google Scholar 

  • Dunford R, Walden RM (1991) Plastid genome structure and plastid-related transcript levels in albino barley plants derived from anther culture. Curr Genet 20:339–347

    Article  CAS  Google Scholar 

  • Farrant JM, Moore JP (2011) Programming desiccation-tolerance: from plants to seeds to resurrection plants. Curr Opin Plant Biol 14:340–345

    Article  CAS  Google Scholar 

  • Fleming MB, Richards CM, Walters C (2017) Decline in RNA integrity of dry-stored soybean seeds correlates with loss of germination potential. J Exp Bot 68:2219–2230

    Article  CAS  Google Scholar 

  • Gallagher SR, Desjardins PR (2008) Quantitation of DNA and RNA with absorption and fluorescence spectroscopy. Curr Protoc Protein Sci 52:4K

    Google Scholar 

  • Gallego Romero I, Pai AA, Tung J, Gilad Y (2014) RNA-seq: impact of RNA degradation on transcript quantification. BMC Biol 12:42

    Article  Google Scholar 

  • Gašić K, Hernandez A, Korban SS (2004) RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol Biol Rep 22:437–438

    Article  Google Scholar 

  • Gray JC (1978) Absorption of polyphenols by polyvinylpyrrolidone and polystyrene resins. Phytochemistry 17:495–497

    Article  CAS  Google Scholar 

  • Hua L, Hibberd JM (2019) An optimized protocol for isolation of high-quality RNA through laser capture microdissection of leaf material. Plant Direct 3:e00156

    Article  Google Scholar 

  • Ingram J, Bartels D (1995) The molecular basis of dehydration tolerance in plants. Ann Rev Plant Physiol Plant Mol Biol 47:377–403

    Article  Google Scholar 

  • Japelaghi RH, Haddad R, Garoosi G-A (2011) Rapid and efficient isolation of high quality nucleic acids from plant tissues rich in polyphenols and polysaccharides. Mol Biotechnol 49:129–137

    Article  CAS  Google Scholar 

  • Liu J, Zhou W, Liu G, Yang C, Sun Y, Wu W, Cao S, Wang C, Hai G, Wang Z, Bock R, Huang J, Cheng Y (2015) The conserved endoribonuclease YbeY is required for chloroplast ribosomal RNA processing in Arabidopsis. Plant Physiol 168:205–221

    Article  CAS  Google Scholar 

  • Liu J, Moyankova D, Lin CT, Mladenov P, Sun RZ, Djilianov D, Deng X (2018) Transcriptome reprogramming during severe dehydration contributes to physiological and metabolic changes in the resurrection plant Haberlea rhodopensis. BMC Plant Biol 18:351

    Article  CAS  Google Scholar 

  • Manderville RA (2005) Ambident reactivity of phenoxyl radicals in DNA adduction. Can J Chem 83:1261–1267

    Article  CAS  Google Scholar 

  • Meng L, Feldman L (2010) A rapid TRIzol-based two-step method for DNA-free RNA extraction from Arabidopsis siliques and dry seeds. Biotechnol J 5:183–186

    Article  CAS  Google Scholar 

  • Ong PW, Chan PL, Ooi LCL, Singh R (2019) Isolation of high quality total RNA from various tissues of oil palm (Elaeis guineensis) for reverse transcription quantitative real-time PCR (RT-qPCR). J Oil Palm Res 31:195–203

    CAS  Google Scholar 

  • Reck M, Tomasch J, Deng Z, Jarek M, Husemann P, Wagner Döbler I (2015) Stool metatranscriptomics: a technical guideline for mRNA stabilisation and isolation. BMC Genom 16:1

    Article  CAS  Google Scholar 

  • Rubio-Piña JA, Zapata-Pérez O (2011) Isolation of total RNA from tissues rich in polyphenols and polysaccharides of mangrove plants. Electron J Biotechnol 14:11

    Google Scholar 

  • Schroeder A, Mueller O, Stocker S, Salowsky R, Leiber M, Gassmann M, Lightfoot S, Menzel W, Martin G, Ragg T (2006) The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol 7:3

    Article  Google Scholar 

  • Sgherri C, Stevanović B, Navari-Izzo F (2004) Role of phenolics in the antioxidative status of the resurrection plant Ramonda serbica during dehydration and rehydration. Physiol Plant 122:478–485

    Article  CAS  Google Scholar 

  • Souza-Perera R, Torres-Hernández N, Zúñiga-Aguilar JJ (2016) Extraction of high quality RNA from hard tissues of adult coconut palms. Afr J Biotechnol 17:263–268

    Google Scholar 

  • Tcherkez G, Guérard F, Gilard F, Lamothe M, Mauve C, Gou E, Blign R (2012) Metabolomic characterisation of the functional division of nitrogen metabolism in variegated leaves. Funct Plant Biol 39:959–967

    Article  CAS  Google Scholar 

  • Valenzuela-Avendaño JP, Mota IAE, Uc GL, Perera RS, Valenzuela-Soto EM, Aguilar JJZ (2005) Use of a simple method to isolate intact RNA from partially hydrated Selaginella lepidophylla plants. Plant Mol Biol Rep 23:199–200

    Article  Google Scholar 

  • Veljović Jovanović S, Kukavica B, Stevanović B, Navari-Izzo F (2006) Senescence-and drought-related changes in peroxidase and superoxide dismutase isoforms in leaves of Ramonda serbica. J Exp Bot 57:1759–1768

    Article  Google Scholar 

  • Veljović Jovanović S, Kukavica B, Navari-Izzo F (2008) Characterization of polyphenol oxidase changes induced by desiccation of Ramonda serbica leaves. Physiol Plant 132:407–416

    Article  Google Scholar 

  • Vidović M, Morina F, Milić S, Albert A, Zechmann B, Tosti T, Winkler JB, Veljović Jovanović S (2015) Carbon allocation from source to sink leaf tissue in relation to flavonoid biosynthesis in variegated Pelargonium zonale under UV-B radiation and high PAR intensity. Plant Physiol Biochem 93:44–55

    Article  Google Scholar 

  • Vidović M, Morina F, Milić-Komić S, Vuleta A, Zechmann B, Prokić L, Veljović Jovanović S (2016a) Characterisation of antioxidants in photosynthetic and non-photosynthetic leaf tissues of variegated Pelargonium zonale plants. Plant Biol 18:669–680

    Article  Google Scholar 

  • Vidović M, Morina F, Prokić L, Milić-Komić S, Živanović B, Veljović Jovanović S (2016b) Antioxidative response in variegated Pelargonium zonale leaves and generation of extracellular H2O2 in (peri) vascular tissue induced by sunlight and paraquat. J Plant Physiol 206:25–39

    Article  Google Scholar 

  • Vidović M, Morina F, Veljović Jovanović S (2017) Stimulation of various phenolics in plants under ambient UV-B radiation. In: Singh VP, Singh S, Prasad SM, Parihar P (eds) UV-B radiation: from environmental stressor to regulator of plant growth. Wiley-Blackwell, Chichester

    Google Scholar 

  • Wang L, Stegemann JP (2010) Extraction of high quality RNA from polysaccharide matrices using cetlytrimethylammonium bromide. Biomaterials 31:1612–1618

    Article  CAS  Google Scholar 

  • Wurtmann EJ, Wolin SL (2009) RNA under attack: cellular handling of RNA damage. Crit Rev Biochem Mol Biol 44:34–49

    Article  CAS  Google Scholar 

  • Yockteng R, Almeida AM, Yee S, Andre T, Hill C, Specht CD (2013) A method for extracting high-quality RNA from diverse plants for next-generation sequencing and gene expression analyses. Appl Plant Sci 1:1300070

    Article  Google Scholar 

  • Zhu Y, Wang B, Phillips J, Zhang ZN, Du H, Xu T, Huang L-C, Zhang X-F, Xu G-H, Li W-L, Wang Z, Wang L, Liu Y-X, Deng X (2015) Global transcriptome analysis reveals acclimation-primed processes involved in the acquisition of desiccation tolerance in Boea hygrometrica. Plant Cell Physiol 56:1429–1441

    Article  CAS  Google Scholar 

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Acknowledgements

The authors would like to acknowledge Dr Milica Bogdanović (IBISS) for useful discussion regarding technical issues.

Funding

This work was funded by the Ministry of Education, Science and Technological Development, Republic of Serbia (Contract No. 451-03-68/2020-14/200053 and Bilateral project no. 451-03-01963/2017-09/09). M.V. wishes to acknowledge the financial support of Centre for Green Technologies, University of Belgrade.

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Authors and Affiliations

  1. Department of Life Science, University of Belgrade, Institute for Multidisciplinary Research, Kneza Višeslava 1, Belgrade, 11030, Serbia

    Marija Vidović

  2. Department of Plant Physiology, Institute for Biological Research “Siniša Stanković”-National Institute of Republic of Serbia, University of Belgrade, Belgrade, Serbia

    Katarina Ćuković

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  1. Marija Vidović
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  2. Katarina Ćuković
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Correspondence to Marija Vidović.

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Vidović, M., Ćuković, K. Isolation of high-quality RNA from recalcitrant leaves of variegated and resurrection plants. 3 Biotech 10, 286 (2020). https://doi.org/10.1007/s13205-020-02279-1

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