Identification of birch lncRNAs and mRNAs responding to salt stress and characterization of functions of lncRNA

Abstract

Long noncoding RNAs (lncRNAs) are important in abiotic stress tolerance. Here, we identified salt-responsive genes and lncRNAs in the roots and leaves of Betula platyphylla Suk. (birch), and characterized their lncRNAs functions. In total, 2660 mRNAs and 539 lncRNAs responding to salt treatment were identified using RNA-seq. The salt-responsive genes were substantially enriched in ‘cell wall biogenesis’ and ‘wood development’ in the roots and were enriched in ‘photosynthesis’ and ‘response to stimulus’ in the leaves. Meanwhile, the potential target genes of the salt-responsive lncRNAs in roots and leaves were both enriched in ‘nitrogen compound metabolic process’ and ‘response to stimulus’. We further built a method for quickly identifying abiotic stress tolerance of lncRNAs, which employed transient transformation for overexpression and knock-down of the lncRNA, enabling gain- and loss-of-function analysis. Using this method, 11 randomly selected salt-responsive lncRNAs were characterized. Among them, six lncRNAs confer salt tolerance, two lncRNAs confer salt sensitivity, and the other three lncRNAs are not involved in salt tolerance. In addition, a lncRNA, LncY1, was further characterized, which improves salt tolerance by regulating two transcription factors, BpMYB96 and BpCDF3. Taken together, our results suggested that lncRNAs play important roles in the salt response of birch plants.

Acknowledgements

This work was supported by the Xingliao Talent Plan Project XLYC1902007, and Funds for Guiding Local Scientific and Technological Development by the Central Government 202JH6/10500071. We thank Northeast Forestry University, State Key Laboratory of Tree Genetics and Breeding for Arid Areas’ shared instrument platform.

Author contributions

Y.W. conceived the original screening and research plans; H.Z. designed the experiments and analysed the data; Y.J. and Y.N. provided technical assistance to H.Z.; Y.W. and Y.J. conceived the project and wrote the article with the contributions of all the authors.

Data availability

All the data used to support the findings of this study are available from the corresponding author upon reasonable request.

Conflict of interest

The authors declare no competing interests.

References

[1] Benny J, Pisciotta A, Caruso T et al. Identification of key genes and its chromosome regions linked to drought responses in leaves across different crops through meta-analysis of RNA-Seq data. BMC Plant Biol. 2019; 19: 194.

[2] Kindgren P, Ard R, Ivanov M et al. Transcriptional read-through of the long non-coding RNA SVALKA governs plant cold acclimation. Nat Commun. 2019; 10: 5141.

[3] Borah P, Das A, Milner M et al. Long non-coding RNAs as endogenous target mimics and exploration of their role in low nutrient stress tolerance in plants. Genes (Basel). 2018; 9: 459.

[4] Zhao T, Tao X, Feng S et al. LncRNAs in polyploid cotton interspecific hybrids are derived from transposon neofunctionalization. Genome Biol. 2018; 19: 195.

[5] Zhang Z, Zheng Y, Ham BK et al. Plant lncRNAs are enriched in and move systemically through the phloem in response to phosphate deficiency. J Integr Plant Biol. 2019; 61: 492-508.

[6] Washietl S, Kellis M, Garber M . Evolutionary dynamics and tissue specificity of human long noncoding RNAs in six mammals. Genome Res. 2014; 24: 616-28.

[7] Wang X, Ai G, Zhang C et al. Expression and diversification analysis reveals transposable elements play important roles in the origin of Lycopersicon-specific lncRNAs in tomato. New Phytol. 2016; 209: 1442-55.

[8] Chekanova JA . Long non-coding RNAs and their functions in plants. Curr Opin Plant Biol. 2015; 27: 207-16.

[9] Chen Y, Li X, Su L et al. Genome-wide identification and characterization of long non-coding RNAs involved in the early somatic embryogenesis in Dimocarpus longan Lour. BMC Genomics. 2018; 19: 805.

[10] Wu HJ, Wang ZM, Wang M et al. Widespread long noncoding RNAs as endogenous target mimics for MicroRNAs in plants. Plant Physiol. 2013; 161: 1875-84.

[11] Seo JS, Sun H, Park BS et al. ELF18-INDUCED LONG-NONCODING RNA associates with mediator to enhance expression of innate immune response genes in Arabidopsis. Plant Cell. 2017; 29: 1024-38.

[12] Yatusevich R, Fedak H, Ciesielski A et al. Antisense transcription represses Arabidopsis seed dormancy QTL DOG1 to regulate drought tolerance. EMBO Rep. 2017; 18: 2186-96.

[13] Qin T, Zhao H, Cui P et al. A nucleus-localized long non-coding RNA enhances drought and salt stress tolerance. Plant Physiol. 2017; 175: 1321-36.

[14] Li S, Cheng Z, Dong S et al. Global identification of full-length cassava lncRNAs unveils the role of cold-responsive intergenic lncRNA 1 in cold stress response. Plant Cell Environ. 2022; 45: 412-26.

[15] Cao Z, Zhao T, Wang L et al. The lincRNA XH123 is involved in cotton cold-stress regulation. Plant Mol Biol. 2021; 106: 521-31.

[16] Chen S, Wang Y, Yu L et al. Genome sequence and evolution of Betula platyphylla. Hortic Res. 2021; 8: 37.

[17] Huanca-Mamani W, Arias-Carrasco R, Cárdenas-Ninasivincha S et al. Long non-coding RNAs responsive to salt and boron stress in the hyper-arid Lluteño maize from Atacama Desert. Genes. 2018; 9: 170.

[18] Yuan J, Li J, Yang Y et al. Stress-responsive regulation of long non-coding RNA polyadenylation in Oryza sativa. Plant J. 2018; 93: 814-27.

[19] Shumayla, Sharma S, Taneja M et al. Survey of high throughput RNA-Seq data reveals potential roles for lncRNAs during development and stress response in bread wheat. Front Plant Sci. 2017; 8: 1019.

[20] Wang D, Qu Z, Yang L et al. Transposable elements (TEs) contribute to stress-related long intergenic noncoding RNAs in plants. Plant J. 2017; 90: 133-46.

[21] Song Y, Ci D, Tian M et al. Stable methylation of a non-coding RNA gene regulates gene expression in response to abiotic stress in Populus simonii. J Exp Bot. 2016; 67: 1477-92.

[22] Ma J, Bai X, Luo W et al. Genome-wide identification of long noncoding RNAs and their responses to salt stress in two closely related poplars. Front Genet. 2019; 10: 777.

[23] Ye X, Wang S, Zhao X et al. Role of lncRNAs in cis- and trans-regulatory responses to salt in Populus trichocarpa. Plant J. 2022; 110: 978-93.

[24] Zang D, Wang L, Zhang Y et al. ThDof1.4 and ThZFP1 constitute a transcriptional regulatory cascade involved in salt or osmotic stress in Tamarix hispida. Plant Mol Biol. 2017; 94: 495-507.

[25] Ji X, Nie X, Liu Y et al. A bHLH gene from Tamarix hispida improves abiotic stress tolerance by enhancing osmotic potential and decreasing reactive oxygen species accumulation. Tree Physiol. 2016; 6: 193-207.

[26] Byrt CS, Munns R, Burton RA et al. Root cell wall solutions for crop plants in saline soils. Plant Sci. 2018; 269: 47-55.

[27] Pillai SE, Kumar C, Patel HK et al. Overexpression of a cell wall damage induced transcription factor, OsWRKY42, leads to enhanced callose deposition and tolerance to salt stress but does not enhance tolerance to bacterial infection. BMC Plant Bio. 2018; 18: 177.

[28] Fang C, Li K, Wu Y et al. OsTSD2-mediated cell wall modification affects ion homeostasis and salt tolerance. Plant Cell Environ. 2019; 42: 1503-12.

[29] Zhao S, Zhang Q, Liu M et al. Regulation of plant responses to salt stress. Int J Mol Sci. 2021; 22: 4609.

[30] Zhu X, Hong X, Liu X et al. Calcium-dependent protein kinase 32 gene maintains photosynthesis and tolerance of potato in response to salt stress. Sci Hortic. 2021; 285: 110179.

[31] Wang P, Shen L, Guo J et al. Phosphatidic acid directly regulates PINOID-dependent phosphorylation and activation of the PIN-FORMED2 auxin efflux transporter in response to salt stress. Plant Cell. 2019; 31: 250-71.

[32] Nounjan N, Chansongkrow P, Charoensawan V et al. High performance of photosynthesis and osmotic adjustment are associated with salt tolerance ability in Rice carrying drought tolerance QTL: physiological and co-expression network analysis. Front Plant Sci. 2018; 9: 1135.

[33] Zang D, Wang J, Zhang X et al. Arabidopsis heat shock transcription factor HSFA7b positively mediates salt stress tolerance by binding to an E-box-like motif to regulate gene expression. J Exp Bot. 2019; 70: 5355-74.

[34] Ji XY, Zheng L, Liu YJ et al. A transient transformation system for the functional characterization of genes involved in stress response. Plant Mol Biol Rep. 2014; 32: 732-9.

[35] Mansour MMF . Nitrogen containing compounds and adaptation of plants to salinity stress. Bio Plant. 2000; 43: 491-500.

[36] Kornienko AE, Guenz PM, Barlow DP et al. Gene regulation by the act of long non-coding RNA transcription. BMC Biol. 2013; 11: 59.

[37] Wang Z, He Z, Xu X et al. Revealing salt tolerance mechanism of Tamarix hispida by large scale identification of genes conferring salt tolerance. Tree Physiol. 2021; 41: 2153-70.

[38] Kim D, Langmead B, Salzberg SL . HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12: 357-60.

[39] Trapnell C, Williams BA, Pertea G et al. Transcript assembly and quantification by RNA Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28: 511-5.

[40] Kelley D, Rinn J . Transposable elements reveal a stem cell-specific class of long noncoding RNAs. Genome Biol. 2012; 13: R107.

[41] Kong L, Zhang Y, Ye ZQ et al. CPC: assess the protein-coding potential of transcripts using sequence features and support vector machine. Nucleic Acids Res. 2007; 35: W345-9.

[42] Sun L, Luo H, Bu D et al. Utilizing sequence intrinsic composition to classify protein-coding and long non-coding transcripts. Nucleic Acids Res. 2013; 41: e166.

[43] Wang L, Park HJ, Dasari S et al. CPAT: coding-potential assessment tool using an alignment-free logistic regression model. Nucleic Acids Res. 2013; 41: e74.

[44] Finn RD, Bateman A, Clements J et al. Pfam: the protein families database. Nucleic Acids Res. 2014; 42: D222-30.

[45] Hanano S, Goto K . Arabidopsis TERMINAL FLOWER1 is involved in the regulation of flowering time and inflorescence development through transcriptional repression. Plant Cell. 2011; 23: 3172-84.

[46] Li J, Ma W, Zeng P et al. LncTar: a tool for predicting the RNA targets of long noncoding RNAs. Brief Bioinform. 2015; 16: 806-12.

[47] Yang G, Wang Y, Xia D et al. Overexpression of a GST gene (ThGSTZ1) from Tamarix hispida improves drought and salinity tolerance by enhancing the ability to scavenge reactive oxygen species. Plant Cell Tiss Org. 2014; 117: 99-112.

[48] Dionisio-Sese ML, Tobita S . Antioxidant responses of rice seedlings to salinity stress. Plant Sci. 1998; 135: 1-9.

[49] Madhava Rao KV, Sresty TVS . Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000; 157: 113-28.

[50] Han Y, Zhang J, Chen X et al. Carbon monoxide alleviates cadmium-induced oxidative damage by modulating glutathione metabolism in the roots of medicago sativa. New Phytol. 2008; 177: 155-66.

[51] Bates LS, Waldren RP, Teare ID . Rapid determination of free proline for water-stress studies. Plant Soil. 1973; 39: 205-7.

[52] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001; 25: 402-8.

[53] Chu C, Quinn J, Chang HY . Chromatin isolation by RNA purification (ChIRP). J Vis Exp. 2012; 61: 3912.