Abstract
Aims
Deep root traits are important in rice (Oryza sativa L.) because of their implications for better ability to reach water and nutrients from deep soil. Analysis of gene expression and alternative splicing (AS) will help speed gene identification and understand the role of AS in deep rooting.
Methods
Rice varieties displaying extreme ratio of deep rooting (RDR) were grown in nutrient solutions. RNA-seq analysis was conducted using StringTie, SUPPA, and 3D RNA-seq pipeline. Functional validation of AS was performed by transgenic overexpression of OsPIN1.
Results
The reference-based transcriptome assembly transcripts represented an overall portrait of AS in rice roots. A percentage (76.91%) of assembled genes contained at least one predicted intron. Principal component analysis of single-nucleotide polymorphisms from RNA-seq indicated that genetic differentiation generated abundant genetic diversity during adaptive domestication of high RDR varieties. Totals obtained were: 4392 differentially expressed genes, 3053 differentially alternatively spliced genes, 5508 differential transcript usage transcripts, and 1068 differential AS event transcripts. Functional categories of these genes and AS regulation transcripts were involved in DNA metabolic processes, stress responses and cell part. Transgenic overexpression of OsPIN1 containing retained intron events improved RDR in rice.
Conclusions
AS increased the complexity of gene expression for environmental adaptation while the gene expression level did not change significantly. Our findings provide new perspectives for enhancing drought avoidance.
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References
Azhiri-Sigari T, Yamauchi A, Kamoshita A, Wade LJ (2000) Genotypic variation in response of rainfed lowland rice to drought and rewatering. Plant Prod Sci 3:180–188
Champoux MC, Wang G, Sarkarung S, Mackill DJ, O'Toole JC, Huang N, McCouch SR (1995) Locating genes associated with root morphology and drought avoidance in rice via linkage to molecular markers. Theor Appl Genet 90:969–981
Chen L, Bush SJ, Tovar-Corona JM, Castillo-Morales A, Urrutia AO (2014) Correcting for differential transcript coverage reveals a strong relationship between alternative splicing and organism complexity. Mol Biol Evol 31:1402–1413
Chen W, Wang W, Peng M, Gong L, Gao Y, Wan J, Wang S, Shi L, Zhou B, Li Z, Peng X, Yang C, Qu L, Liu X, Luo J (2016) Comparative and parallel genome-wide association studies for metabolic and agronomic traits in cereals. Nat Commun 7:12767
Courtois B, Ahmadi N, Khowaja F, Price AH, Rami JF, Frouin J, Hamelin C, Ruiz M (2009) Rice root genetic architecture: meta-analysis from a drought QTL database. Rice (N Y) 2:115–128
Dolan L, Roberts K (1995) Plant development: pulled up by the roots. Curr Opin Genet Dev 5:432–438
Filichkin SA, Priest HD, Givan SA, Shen R, Bryant DW, Fox SE, Wong WK, Mockler TC (2010) Genome-wide mapping of alternative splicing in Arabidopsis thaliana. Genome Res 20:45–58
Fukai S, Cooper M (1995) Development of drought-resistant cultivars using physiomorphological traits in rice. Field Crops Res 40:67–86
Gowda VRP, Henry A, Yamauchi A, Shashidhar HE, Serraj R (2011) Root biology and genetic improvement for drought avoidance in rice. Field Crop Res 122:1–13
Guo W, Tzioutziou N, Stephen G, Milne I, Calixto C, Waugh R, Brown J W, Zhang R (2019) 3D RNA-seq - a powerful and flexible tool for rapid and accurate differential expression and alternative splicing analysis of RNA-seq data for biologists. bioRxiv 656686
Kato Y, Abe J, Kamoshita A, Yamagishi J (2006) Genotypic variation in root growth angle in rice (Oryza sativa L.) and its association with deep root development in upland fields with different water regimes. Plant Soil 287:117–129
Kim JY, Kim WY, Kwak KJ, Oh SH, Han YS, Kang H (2010) Glycine-rich RNA-binding proteins are functionally conserved in Arabidopsis thaliana and Oryza sativa during cold adaptation process. J Exp Bot 61:2317–2325
Kim D, Paggi JM, Park C, Bennett C, Salzberg SL (2019) Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype. Nat Biotechnol 37:907–915
Kitomi Y, Ito H, Hobo T, Aya K, Kitano H, Inukai Y (2011) The auxin responsive AP2/ERF transcription factor CROWN ROOTLESS5 is involved in crown root initiation in rice through the induction of OsRR1, a type-a response regulator of cytokinin signaling. Plant J 67:472–484
Kitomi Y, Kanno N, Kawai S, Mizubayashi T, Fukuoka S, Uga Y (2015) QTLs underlying natural variation of root growth angle among rice cultivars with the same functional allele of DEEPER ROOTING 1. Rice (N Y) 8:16
Lee J, Jiang W, Qiao Y, Cho YI, Woo MO, Chin JH, Kwon SW, Hong SS, Choi IY, Koh HJ (2011) Shotgun proteomic analysis for detecting differentially expressed proteins in the reduced culm number rice. Proteomics 11:455–468
Li Q, Xiao G, Zhu YX (2014) Single-nucleotide resolution mapping of the Gossypium raimondii transcriptome reveals a new mechanism for alternative splicing of introns. Mol Plant 7:829–840
Li S, Yamada M, Han X, Ohler U, Benfey PN (2016) High-resolution expression map of the Arabidopsis root reveals alternative splicing and lincRNA regulation. Dev Cell 39:508–522
Li X, Guo Z, Lv Y, Cen X, Ding X, Wu H, Huang J, Xiong L (2017) Genetic control of the root system in rice under normal and drought stress conditions by genome-wide association study. PLoS Genet 13:e1006889
Lou Q, Chen L, Mei H, Wei H, Feng F, Wang P, Xia H, Li T, Luo L (2015) Quantitative trait locus mapping of deep rooting by linkage and association analysis in rice. J Exp Bot 66:4749–4757
Lou Q, Chen L, Mei H, Xu K, Wei H, Feng F, Li T, Pang X, Shi C, Luo L, Zhong Y (2017) Root transcriptomic analysis revealing the importance of energy metabolism to the development of deep roots in rice (Oryza sativa L.). front. Plant Sci 8:1314
Luo LJ (2010) Breeding for water-saving and drought-resistance rice (WDR) in China. J Exp Bot 61:3509–3517
Luo R, Wei H, Ye L, Wang K, Chen F, Luo L, Liu L, Li Y, Crabbe MJ, Jin L, Zhong Y (2009) Photosynthetic metabolism of C3 plants shows highly cooperative regulation under changing environments: a systems biological analysis. Proc Natl Acad Sci U S A 106:847–852
Moumeni A, Satoh K, Kondoh H, Asano T, Hosaka A, Venuprasad R, Serraj R, Kumar A, Leung H, Kikuchi S (2011) Comparative analysis of root transcriptome profiles of two pairs of drought-tolerant and susceptible rice near-isogenic lines under different drought stress. BMC Plant Biol 11:174
Mujahid H, Tan F, Zhang J, Nallamilli BR, Pendarvis K, Peng Z (2013) Nuclear proteome response to cell wall removal in rice (Oryza sativa). Proteome Sci 11:26
Norton GJ, Aitkenhead MJ, Khowaja FS, Whalley WR, Price AH (2008) A bioinformatic and transcriptomic approach to identifying positional candidate genes without fine mapping: an example using rice root-growth QTLs. Genomics 92:344–352
Patro R, Duggal G, Love MI, Irizarry RA, Kingsford C (2017) Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods 14:417–419
Pertea M, Pertea GM, Antonescu CM, Chang TC, Mendell JT, Salzberg SL (2015) StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat Biotechnol 33:290–295
Pertea M, Kim D, Pertea GM, Leek JT, Salzberg SL (2016) Transcript-level expression analysis of RNA-seq experiments with HISAT, StringTie and Ballgown. Nat Protoc 11:1650–1667
Pierret A, Maeght JL, Clement C, Montoroi JP, Hartmann C, Gonkhamdee S (2016) Understanding deep roots and their functions in ecosystems: an advocacy for more unconventional research. Ann Bot 118:621–635
Price AH, Courtois B (1999) Mapping QTLs associated with drought resistance in rice: Progress, problems and prospects. Plant Growth Regul 1-2:123–133
Price AH, Cairns JE, Horton P, Jones HG, Griffiths H (2002a) Linking drought-resistance mechanisms to drought avoidance in upland rice using a QTL approach: progress and new opportunities to integrate stomatal and mesophyll responses. J Exp Bot 53:989–1004
Price AH, Townend J, Jones MP, Audebert A, Courtois B (2002b) Mapping QTLs associated with drought avoidance in upland rice grown in the Philippines and West Africa. Plant Mol Biol 48:683–695
Ren Z, Qi D, Pugh N, Li K, Wen B, Zhou R, Xu S, Liu S, Jones AR (2018) Improvements to the Rice genome annotation through large-scale analysis of RNA-Seq and proteomics data sets. Mol Cell Proteomics 18:86–98
Rogers ED, Benfey PN (2015) Regulation of plant root system architecture: implications for crop advancement. Curr Opin Biotechnol 32:93–98
Rosloski SM, Singh A, Jali SS, Balasubramanian S, Weigel D, Grbic V (2013) Functional analysis of splice variant expression of MADS AFFECTING FLOWERING 2 of Arabidopsis thaliana. Plant Mol Biol 81:57–69
Sano N, Takebayashi Y, To A, Mhiri C, Rajjou LC, Nakagami H, Kanekatsu M (2019) Shotgun proteomic analysis highlights the roles of long-lived mRNAs and De novo transcribed mRNAs in Rice seeds upon imbibition. Plant Cell Physiol 60:2584–2596
Sato EM, Hijazi H, Bennett MJ, Vissenberg K, Swarup R (2015) New insights into root gravitropic signalling. J Exp Bot 66:2155–2165
Shankar R, Bhattacharjee A, Jain M (2016) Transcriptome analysis in different rice cultivars provides novel insights into desiccation and salinity stress responses. Sci Rep 6:23719
Shen Y, Zhou Z, Wang Z, Li W, Fang C, Wu M, Ma Y, Liu T, Kong LA, Peng DL, Tian Z (2014) Global dissection of alternative splicing in paleopolyploid soybean. Plant Cell 26:996–1008
Singh SK, Roy S, Choudhury SR, Sengupta DN (2010) DNA repair and recombination in higher plants: insights from comparative genomics of Arabidopsis and rice. BMC Genomics 11:443
Swarup R, Kramer EM, Perry P, Knox K, Leyser HM, Haseloff J, Beemster GT, Bhalerao R, Bennett MJ (2005) Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal. Nat Cell Biol 7:1057–1065
Thatcher SR, Zhou W, Leonard A, Wang BB, Beatty M, Zastrow-Hayes G, Zhao X, Baumgarten A, Li B (2014) Genome-wide analysis of alternative splicing in Zea mays: landscape and genetic regulation. Plant Cell 26:3472–3487
Trincado JL, Entizne JC, Hysenaj G, Singh B, Skalic M, Elliott DJ, Eyras E (2018) SUPPA2: fast, accurate, and uncertainty-aware differential splicing analysis across multiple conditions. Genome Biol 19:40
Uga Y, Okuno K, Yano M (2011) Dro1, a major QTL involved in deep rooting of rice under upland field conditions. J Exp Bot 62:2485–2494
Uga Y, Sugimoto K, Ogawa S, Rane J, Ishitani M, Hara N, Kitomi Y, Inukai Y, Ono K, Kanno N, Inoue H, Takehisa H, Motoyama R, Nagamura Y, Wu J, Matsumoto T, Takai T, Okuno K, Yano M (2013a) Control of root system architecture by DEEPER ROOTING 1 increases rice yield under drought conditions. Nat Genet 45:1097–1102
Uga Y, Yamamoto E, Kanno N, Kawai S, Mizubayashi T, Fukuoka S (2013b) A major QTL controlling deep rooting on rice chromosome 4. Sci Rep 3:3040
Uga Y, Kitomi Y, Yamamoto E, Kanno N, Kawai S, Mizubayashi T, Fukuoka S (2015) A QTL for root growth angle on rice chromosome 7 is involved in the genetic pathway of DEEPER ROOTING 1. Rice (N Y) 8:8
Wang YJ, Zhang ZG, He XJ, Zhou HL, Wen YX, Dai JX, Zhang JS, Chen SY (2003) A rice transcription factor OsbHLH1 is involved in cold stress response. Theor Appl Genet 107:1402–1409
Wang ET, Sandberg R, Luo S, Khrebtukova I, Zhang L, Mayr C, Kingsmore SF, Schroth GP, Burge CB (2008) Alternative isoform regulation in human tissue transcriptomes. Nature 456:470–476
Wang Y, Liu J, Huang BO, Xu YM, Li J, Huang LF, Lin J, Zhang J, Min QH, Yang WM, Wang XZ (2015) Mechanism of alternative splicing and its regulation. Biomed Rep 3:152–158
Wei H, Feng F, Lou Q, Xia H, Ma X, Liu Y, Xu K, Yu X, Mei H, Luo L (2016) Genetic determination of the enhanced drought resistance of rice maintainer HuHan2B by pedigree breeding. Sci Rep 6:37302
Wei H, Lou Q, Xu K, Yan M, Xia H, Ma X, Yu X, Luo L (2017) Alternative splicing complexity contributes to genetic improvement of drought resistance in the rice maintainer HuHan2B. Sci Rep 7:11686
Wu J, Feng F, Lian X, Teng X, Wei H, Yu H, Xie W, Yan M, Fan P, Li Y, Ma X, Liu H, Yu S, Wang G, Zhou F, Luo L, Mei H (2015) Genome-wide association study (GWAS) of mesocotyl elongation based on re-sequencing approach in rice. BMC Plant Biol 15:218
Xu M, Zhu L, Shou H, Wu P (2005) A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant Cell Physiol 46:1674–1681
Xu H, Mo Y, Wang W, Wang H, Wang Z (2014) OsPIN1a gene participates in regulating negative phototropism of rice roots. Rice Sci 21:83–89
Zhang H, Feng X, Larssen T, Qiu G, Vogt RD (2010) In inland China, rice, rather than fish, is the major pathway for methylmercury exposure. Environ Health Perspect 118:1183–1188
Zou Y, Liu X, Wang Q, Chen Y, Liu C, Qiu Y, Zhang W (2014) OsRPK1, a novel leucine-rich repeat receptor-like kinase, negatively regulates polar auxin transport and root development in rice. Biochim Biophys Acta 1840:1676–1685
Acknowledgements
This work was supported by grants from National Natural Science Foundation of China (31971926), Natural Science Foundation of Shanghai (19ZR1446800, 19ZR1446900, 17ZR1425600), Shanghai Agriculture Applied Technology Development Program (2017-02-08-00-08-F00071, G2015060101), Academic Specialty Development Project of Shanghai Academy of Agricultural Sciences, Shared Platform of Crop Germplasm Resources in Shanghai (18DZ2293700), Young talent growth plan of Shanghai Municipal Agricultural Commission (2017-1-32), and Platform of National Crop Germplasm Resources (NICGR2019-021). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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H.W., L.C and L.L. conceived the study and designed the experiments. Q.L. and K.X. planted and collected the leaf tissue samples. L.Z. and S.C. isolated RNA and carried out quantitative validation. H.W. and Q.L. performed RNA-seq data analysis. H.W. was mainly involved in preparation of manuscript. All authors provided input during the writing of the manuscript. All authors reviewed and approved the final manuscript.
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Wei, H., Lou, Q., Xu, K. et al. Pattern of alternative splicing different associated with difference in rooting depth in rice. Plant Soil 449, 233–248 (2020). https://doi.org/10.1007/s11104-020-04451-1
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DOI: https://doi.org/10.1007/s11104-020-04451-1